Biosynthesis method of idoxaban tosylate chiral intermediate

Abstract

The invention provides a biosynthesis method of an esdoxaban tosylate chiral intermediate. According to the preparation method, 3-cyclohexene-1-carboxylic acid methyl ester is taken as a starting raw material, and a target intermediate is finally obtained through carboxylesterase resolution, hydrolysis, hydroxylation, dehydrogenation oxidation, transamination reaction, amidation and Boc2O reaction in sequence. According to the method, high-selectivity carboxylesterase is used for realizing chiral resolution; p450 oxidase with excellent regioselectivity and stereoselectivity is utilized to realize specific hydroxylation of a C-H bond under a mild condition, and a traditional process depending on a highly toxic chemical oxidant is overturned; then, clean oxidation is realized through alcohol dehydrogenase, and a way is laid for subsequent reaction; most importantly, the transaminase is adopted to directly construct the chiral amine, so that the use of a high-risk reagent sodium azide is thoroughly avoided, and major potential safety hazards are eliminated from the source; and finally, green amidation is realized by virtue of lipase, and an acyl chloride method for generating corrosive wastewater is replaced.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical synthesis technology, specifically to a method for the biosynthesis of a chiral intermediate of edoxaban tosylate. Background Technology

[0002] Edoxaban tosylate is an anticoagulant that works by inhibiting thrombin activity, thereby preventing thrombosis. It is indicated for adult patients with nonvalvular atrial fibrillation who have one or more risk factors (e.g., congestive heart failure, hypertension, age over 75 years, diabetes, history of stroke or transient ischemic attack) to prevent stroke and systemic embolism. Edoxaban tosylate is also used to treat deep vein thrombosis (DVT) and pulmonary embolism (PE) in adults and to prevent their recurrence.

[0003] In the synthetic pathway of edoxaban tosylate, [( 1R, 2S, 5S 2-Amino-5-[(dimethylamino)carbonyl]cyclohexyl]tert-butyl carbamate can undergo condensation with both side chains, which is the core step in constructing the molecular structure of edoxaban tosylate. Currently, the key intermediate used to prepare edoxaban tosylate—[( 1R, 2S, 5S The general synthetic route for tert-butyl carbamate (2-amino-5-[(dimethylamino)carbonyl]cyclohexyl]carbamate is as follows.

[0004] Synthetic Route 1: This synthetic route is based on ( S Starting with 3-cyclohexenecarboxylic acid, intermediate VI was obtained through a six-step reaction. Subsequently, it underwent methanesulfonation, azidation, hydrolysis, amidation, and hydrogenation amination, totaling 11 steps, to finally obtain the key intermediate of edoxaban. The specific reaction equations are shown below. This route suffers from low yield (around 30%) and poor diastereoselectivity during the azidation of methanesulfonate, and the process is lengthy, resulting in low yield and high cost.

[0005]

[0006] Synthesis Route 1 Synthetic Route Two: The core steps of the synthetic method disclosed in patent CN 111606826A are as follows: First, compound II (thiophene derivative) undergoes a Diels-Alder diene synthesis reaction with acrylic acid; then, the resulting product is chirally resolved to obtain chiral compound IV; subsequently, this intermediate undergoes an amidation reaction with dimethylamine hydrochloride to obtain compound V; compound V then reacts with di-tert-butyl dicarbonate under a hydrogen atmosphere to generate compound VI and its isomer VI'; finally, through amino deprotection and chiral resolution, the final product compound I is obtained, which is the key intermediate of edoxaban tosylate. Although this route is simple in steps, the raw materials and reagents used are expensive, and it involves a less efficient chiral resolution step, resulting in a low overall yield and ultimately making the production cost uncompetitive.

[0007]

[0008] Synthetic Route 2 In summary, existing synthetic processes for edoxaban tosylate intermediates have significant limitations: lengthy procedures, complex technology, high raw material costs, and high risks. Therefore, developing a new synthetic route that is simple in steps, yields ideal results, is safe and environmentally friendly, and is easy to scale up industrially is particularly urgent and important. Summary of the Invention

[0009] To address the aforementioned problems, this invention provides a biosynthetic method for edoxaban tosylate chiral intermediates. Using methyl 3-cyclohexene-1-carboxylate as the starting material, the method sequentially undergoes carboxylesterase resolution, hydrolysis, hydroxylation, dehydrogenation, transamine reaction, amidation, and reaction with Boc₂O to finally obtain the target intermediate. Its core innovation lies in constructing a precise, efficient, and intrinsically safe green synthesis platform through five functionally complementary biocatalysts.

[0010] To achieve the above objectives, this invention provides a method for the biosynthesis of a chiral intermediate of edoxaban tosylate, the specific synthetic route of which is as follows:

[0011] The specific reaction steps include: 1): First, methyl 3-cyclohexene-1-carboxylate is cleaved by a carboxylesterase, generating intermediates 2 and 3 simultaneously; 2): Intermediate 3 undergoes a hydrolysis reaction to generate intermediate 4; 3): Intermediate 4 is converted into intermediate 5 under the catalysis of P450 oxidase; 4): Intermediate 5 is converted into intermediate 6 under the catalysis of alcohol hydroxyl dehydrogenase; 5): Intermediate 6 is converted into intermediate 7 under the catalysis of transaminase; 6): Intermediate 7 condenses with isopropylamine under the catalysis of lipase, transforming into intermediate 8; 7): Intermediate 8 condenses with Boc2O to generate edoxaban tosylate intermediate.

[0012] Furthermore, in step 1), intermediate 2 is esterified to intermediate 1'; this intermediate 1' can be racemized back into raw material 1, achieving material recycling. Preferably, the esterification reaction is carried out in an alcohol solvent, using potassium bisulfate as a catalyst, at a reaction temperature of 60-70°C. The racemization reaction is carried out under alkaline conditions, using sodium hydroxide, at a reaction temperature of 25-35°C, achieving the recovery and recycling of unreacted isomers.

[0013] Furthermore, in step 1), the reaction temperature for the carboxylesterase resolution is 30~40℃, the pH is maintained at 7.0~7.5, and phosphate buffer is preferably used as the reaction medium.

[0014] Furthermore, in step 2), the hydrolysis reaction is carried out at 35~45℃, using a two-phase solvent system composed of water and ethyl acetate (water to ethyl acetate volume ratio 1:1 ~ 1:1.5), and the molar ratio of intermediate 3 to sodium hydroxide is 1:1.0~1.2.

[0015] Furthermore, in step 3), the hydroxyl addition reaction temperature is 30~35℃, and the reaction solvent used is a phosphate buffer solution with pH 7.5~8.0.

[0016] Furthermore, in step 4), the oxidation reaction temperature is 30~40℃, and the reaction solvent used is a phosphate buffer solution with pH 8.0~8.5.

[0017] Furthermore, in step 5), the transamination reaction temperature is 35~40℃, and the reaction solvent used is a phosphate buffer solution with pH 8.5~9.0.

[0018] Furthermore, in step 6), the condensation reaction temperature is 40~50℃, and the molar ratio of intermediate 7 to isopropylamine is 1:2.0~3.0.

[0019] Furthermore, in step 7), the condensation reaction temperature is 20~30℃, and the molar ratio of intermediate 8 to Boc2O is 1:1.0~1.5.

[0020] The specific steps of this invention are as follows: 1): 3-Cyclohexene-1-carboxylic acid methyl ester was dissolved in phosphate buffer at pH 7.0~7.5, carboxylesterase was added, and the temperature was controlled at 30~40℃ for the resolution reaction. After the reaction was completed, intermediate 2 and intermediate 3 were obtained by extraction and separation. 2): Intermediate 3 was placed in a two-phase solvent system consisting of water and ethyl acetate, sodium hydroxide was added, and the reaction was carried out by stirring at a temperature of 35~45℃. After the reaction was completed, intermediate 4 was obtained through post-processing. 3): Dissolve intermediate 4 in phosphate buffer at pH 7.5-8.0, add whole cells of P450 oxidase and nicotinamide adenine dinucleotide phosphate (NADPH), and carry out the reaction at 30-35℃; after the reaction is completed, post-processing is performed to obtain intermediate 5; 4): Dissolve intermediate 5 in phosphate buffer at pH 8.0-8.5, add whole cells of alcohol hydroxyl dehydrogenase, and carry out the oxidation reaction at a controlled temperature of 30-40℃; after the reaction is completed, post-processing is performed to obtain intermediate 6; 5): Dissolve intermediate 6 in phosphate buffer solution at pH 8.0-8.5, add pyridoxal phosphate (PLP) and transaminase, control the temperature at 35-40℃, and stir for 8-12 hours; after the reaction is completed, the intermediate 7 is obtained through post-processing. 6): Dissolve intermediate 7 and isopropylamine in methyl tert-butyl ether, add lipase and molecular sieve, and carry out condensation reaction at 40~50℃. During the reaction, monitor the reaction by TLC until the raw material is completely converted. After the reaction, filter out the molecular sieve and immobilized lipase. The organic phase is concentrated under reduced pressure to obtain crude edoxaban tosylate intermediate, which can be directly used for subsequent purification or reaction. 7): Dissolve intermediate 8 in dichloromethane, add Boc2O, and stir at 25~30℃ for 4~6 hours; after the reaction is completed, post-processing is performed to obtain edoxaban tosylate intermediate.

[0021] Further, in step 1), intermediate 2 is dissolved in an alcohol solvent, potassium bisulfate is added as a catalyst, and esterification is carried out at 60~70℃ to generate intermediate 1′; intermediate 1′ is racemized under alkaline conditions with sodium hydroxide as an alkali at 25~35℃ to regenerate raw material 1, thus realizing the recycling of unreacted isomers.

[0022] Further, the post-treatment in step 2) is as follows: after the reaction is complete, the mixture is allowed to stand and separate into layers. The pH of the aqueous phase is adjusted to 2-3 with hydrochloric acid, and the solid is precipitated. After filtration, washing and drying, intermediate 4 is obtained.

[0023] Further, the post-treatment in step 3) is as follows: after the reaction is completed, the pH is adjusted to acidic, and the mixture is extracted with ethyl acetate. The organic phase is washed with water, dried, and concentrated to obtain intermediate 5, which is directly used in the next reaction.

[0024] Further, the post-treatment in step 4) is as follows: after the reaction is completed, the pH is adjusted to 3-4, extracted with ethyl acetate, and the organic phase is washed with water, dried and concentrated to obtain intermediate 6.

[0025] Further, the post-treatment in step 5) is as follows: after the reaction is completed, the reaction solution is extracted with ethyl acetate, the organic phases are combined, dried with anhydrous sodium sulfate and concentrated, and the resulting intermediate 7 is directly used in the next reaction.

[0026] The technical principle of this invention is as follows: This route achieves chiral resolution using highly selective carboxylesterases, and can overcome theoretical yield limitations through dynamic kinetic resolution; it utilizes P450 oxidases with excellent regio and stereoselectivity to achieve specific hydroxylation of CH bonds under mild conditions, overturning the traditional process that relies on highly toxic chemical oxidants; subsequently, clean oxidation is achieved through alcohol dehydrogenases, paving the way for subsequent reactions; most importantly, chiral amines are directly constructed using transaminases, completely avoiding the use of the highly dangerous reagent sodium azide, eliminating major safety hazards at the source; finally, green amidation is achieved with the help of lipases, replacing the acyl chloride process that produces corrosive wastewater.

[0027] Compared with existing technologies, the beneficial effects of this invention are as follows: The preparation method provided by this invention exhibits significant advantages in multiple dimensions: Firstly, the process route is simple, the reaction conditions are mild, and the post-processing is convenient; secondly, experimental verification shows that the product yield is high and the purity is good, ensuring the reliability of quality. Compared with existing technologies, this invention effectively overcomes industrialization bottlenecks such as high cost and complex processes, has excellent overall economic efficiency, conforms to the concept of green chemistry, and has broad prospects for industrial application. Attached Figure Description

[0028] Figure 1 The chiral detection spectrum of the chiral intermediate of edoxaban tosylate in Example 2 is shown. Figure 2 The hydrogen spectrum of the chiral intermediate of toluenesulfonic acid edoxaban in Example 2 is shown. Detailed Implementation

[0029] The technical solutions of the present invention will be described in detail below with reference to the embodiments and accompanying drawings. These embodiments are merely examples and do not exhaustively represent all possible implementations of the present invention. All other technical solutions obtained by those skilled in the art based on the embodiments of the present invention without creative effort fall within the protection scope of the present invention.

[0030] Example 1: Preparation of various enzymes 1. Preparation of carboxylesterase The nucleotide sequence of the carboxylesterase described in this invention is shown in SEQ ID NO: 1, and its encoded amino acid sequence is shown in SEQ ID NO: 6. The synthesized gene was amplified by PCR and cloned into the EcoRI and HindIII restriction sites of the pET-28a plasmid to construct a recombinant expression vector. The recombinant plasmid was transformed into *E. coli* BL21 (DE3) competent cells, and positive clones were obtained by antibiotic resistance plate selection, named... Escherichia coli T-001. The obtained engineered bacteria were inoculated into LB liquid medium at an inoculum rate of 1% (v / v) and cultured overnight with shaking at 37 ℃ and 220 rpm to prepare the seed culture. The seed culture was then transferred to LB medium for fermentation culture and cultured at 37 ℃ and 220 rpm until OD... 600 When the concentration reached 0.6-0.8, IPTG was added to a final concentration of 0.5 mM, and expression was induced at 28 ℃ for 12 h. After induction, the fermentation broth was centrifuged at 4 ℃ and 12000 rpm for 5 min to obtain whole-cell wet cells of carboxylesterase.

[0031] 2. Preparation of P450 oxidase The nucleotide sequence of the P450 oxidase described in this invention is shown in SEQ ID NO: 2, and its encoded amino acid sequence is shown in SEQ ID NO: 7. The synthesized gene was amplified by PCR and cloned into the EcoRI and HindIII restriction sites of the pET-28a plasmid to construct a recombinant expression vector. The recombinant plasmid was transformed into *E. coli* BL21 (DE3) competent cells, and positive clones were obtained by antibiotic resistance plate selection, named... Escherichia coli T-002. The obtained engineered bacteria were inoculated into LB liquid medium at an inoculum rate of 1% (v / v) and cultured overnight with shaking at 37 ℃ and 220 rpm to prepare the seed culture. The seed culture was then transferred to LB medium for fermentation culture and cultured at 37 ℃ and 220 rpm until OD... 600 When the concentration reached 0.6-0.8, IPTG was added to a final concentration of 0.5 mM, and expression was induced at 28 ℃ for 12 h. After induction, the fermentation broth was centrifuged at 4 ℃ and 12000 rpm for 5 min to obtain whole-cell wet cells of P450 oxidase.

[0032] 3. Preparation of alcohol hydroxyl dehydrogenase The nucleotide sequence of the alcohol hydroxyl dehydrogenase described in this invention is shown in SEQ ID NO: 3, and its encoded amino acid sequence is shown in SEQ ID NO: 8. The synthesized gene was amplified by PCR and cloned into the EcoRI and HindIII restriction sites of the pET-28a plasmid to construct a recombinant expression vector. The recombinant plasmid was transformed into *E. coli* BL21 (DE3) competent cells, and positive clones were obtained by antibiotic resistance plate selection, named... Escherichia coli T-003. The obtained engineered bacteria were inoculated into LB liquid medium at an inoculum rate of 1% (v / v) and cultured overnight with shaking at 37 ℃ and 220 rpm to prepare the seed culture. The seed culture was then transferred to LB medium for fermentation culture and cultured at 37 ℃ and 220 rpm until OD... 600 When the concentration reached 0.6-0.8, IPTG was added to a final concentration of 0.5 mM, and expression was induced at 28 ℃ for 12 h. After induction, the fermentation broth was centrifuged at 4 ℃ and 12000 rpm for 5 min to obtain whole-cell wet cells of alcohol hydroxyl dehydrogenase.

[0033] 4. Preparation of transaminases The nucleotide sequence of the transaminase described in this invention is shown in SEQ ID NO: 4, and its encoded amino acid sequence is shown in SEQ ID NO: 9. The synthesized gene was amplified by PCR and cloned into the EcoRI and HindIII restriction sites of the pET-28a plasmid to construct a recombinant expression vector. The recombinant plasmid was transformed into *E. coli* BL21 (DE3) competent cells, and positive clones were obtained by antibiotic resistance plate selection, named... Escherichia coli T-004. The obtained engineered bacteria were inoculated into LB liquid medium at an inoculum rate of 1% (v / v) and cultured overnight with shaking at 37 ℃ and 220 rpm to prepare the seed culture. The seed culture was then transferred to LB medium for fermentation culture and cultured at 37 ℃ and 220 rpm until OD... 600 When the concentration reached 0.6-0.8, IPTG was added to a final concentration of 0.5 mM, and expression was induced at 28 ℃ for 12 h. After induction, the fermentation broth was centrifuged at 4 ℃ and 12000 rpm for 5 min to obtain whole-cell wet transaminase cells.

[0034] 5. Preparation of lipase The nucleotide sequence of the lipase described in this invention is shown in SEQ ID NO: 5, and its encoded amino acid sequence is shown in SEQ ID NO: 10. The synthesized gene was amplified by PCR and cloned into the EcoRI and HindIII restriction sites of the pET-28a plasmid to construct a recombinant expression vector. The recombinant plasmid was transformed into *E. coli* BL21 (DE3) competent cells, and positive clones were obtained by antibiotic resistance plate selection, named... Escherichia coli T-005. The obtained engineered bacteria were inoculated into LB liquid medium at an inoculum rate of 1% (v / v) and cultured overnight with shaking at 37 ℃ and 220 rpm to prepare the seed culture. The seed culture was then transferred to LB medium for fermentation culture and cultured at 37 ℃ and 220 rpm until OD... 600 When the concentration reached 0.6-0.8, IPTG was added to a final concentration of 0.5 mM, and expression was induced at 28 ℃ for 12 h. After induction, the fermentation broth was centrifuged at 4 ℃ and 12000 rpm for 5 min to obtain whole-cell wet lipase cells.

[0035] Example 2: 1): Dissolve 100 g of methyl 3-cyclohexene-1-carboxylate in 1000 mL of phosphate buffer (pH 7.0-7.5), add 10 g of carboxylesterase (enzyme activity ≥300 U / g), and stir the reaction at 30-40 °C for 6-8 h. After the reaction is complete, extract with ethyl acetate, combine the organic phases, concentrate and dry to obtain intermediates 2 and 3. Intermediate 2 was dissolved in 300 mL of methanol, and 3 g of potassium hydrogen sulfate was added. The mixture was refluxed at 60-70 °C for 3-4 h to generate intermediate 1′. Intermediate 1′ was placed in 200 mL of 10% sodium hydroxide solution and stirred and racemized at 25-35 °C for 2-3 h to regenerate starting material 1. After dynamic kinetic separation, intermediate 3 weighed approximately 81 g, with a purity of 95% and chiral properties. ee Value 99.2%; 2): 60 g of intermediate 3 was placed in a biphase solvent consisting of 400 mL of water and 200 mL of ethyl acetate, and 24 g of sodium hydroxide was added. The mixture was stirred at 35-45 °C for 2 h. After the reaction was completed, the mixture was allowed to stand and separate into layers. The pH of the aqueous phase was adjusted to 2-3 with concentrated hydrochloric acid, and a white solid precipitated out. After filtration, washing with water, and vacuum drying at 50 °C, approximately 51.3 g of intermediate 4 was obtained. 3): Dissolve 50 g of intermediate 4 in 1000 mL of phosphate buffer at pH 7.8, add 25 g of P450 oxidase whole-cell catalyst (enzyme activity ≥500 U / g), and stir the reaction at 30-35 ℃. During the reaction, add glucose-glucose dehydrogenase regeneration system in batches (total glucose 25 g, total glucose dehydrogenase 2500 U, total β-nicotinamide adenine dinucleotide disodium phosphate 100 mg) to maintain the coenzyme cycle. After the reaction, adjust the pH to 3.0-4.0 with 2 M hydrochloric acid, extract with ethyl acetate (500 mL × 3), combine the organic phases, wash with water, dry with anhydrous sodium sulfate, concentrate under reduced pressure, and dry to obtain approximately 52.0 g of intermediate 5, with a purity of 98%. 4): Dissolve 50 g of intermediate 5 in 1000 mL of phosphate buffer (pH 8.0-8.5), add 10 g of whole-cell alcohol hydroxy dehydrogenase (enzyme activity ≥500 U / g), and stir the reaction at 30-40 ℃ for 6-8 h. After the reaction, adjust the pH of the system to 3-4 with dilute hydrochloric acid, extract with ethyl acetate (2 × 500 mL), combine the organic phases, wash with water, dry with anhydrous sodium sulfate, concentrate under reduced pressure, and dry to obtain approximately 47 g of intermediate 6, with a purity of 96%. 5): Dissolve 50 g of intermediate 6 in 1000 mL of phosphate buffer (pH 8.0–8.5), add 1.2 g of pyridoxal phosphate (PLP) and 10 g of transaminase (enzyme activity ≥300 U / g), and add 60 g of isopropylamine hydrochloride. Control the reaction temperature at 35–40 °C and stir for 10–12 h. After the reaction, extract the reaction solution with 2 × 500 mL of ethyl acetate, combine the organic phases, dry with anhydrous sodium sulfate, concentrate under reduced pressure, and dry again. The resulting intermediate 7 is approximately 48 g, and its purity is 98%, with chiral properties. ee Value 99.5%; 6): Dissolve 50 g of intermediate 7 and 25 g of isopropylamine in 600 mL of methyl tert-butyl ether, add 10 g of immobilized lipase (enzyme activity ≥500 U / g) and 20 g of 4Å molecular sieve, and carry out the condensation reaction at 40~50 ℃. Monitor the reaction process by TLC until the raw material is completely converted. After the reaction, filter out the molecular sieve and immobilized lipase (the immobilized enzyme can be recovered and reused). The organic phase is concentrated under reduced pressure and dried to obtain approximately 52 g of intermediate 8 (chemical purity approximately 95.0% as determined by HPLC). 7): 50 g of intermediate 8 and 60 g of Boc₂O were dissolved in 700 mL of dichloromethane, and a condensation reaction was carried out at 20–30 °C. The reaction was monitored by TLC until the starting material was completely converted. After the reaction, the reaction solution was washed successively with saturated sodium bicarbonate solution and brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 58 g of crude edoxaban tosylate intermediate (chemical purity approximately 95.5% as determined by HPLC). Chiral purity was confirmed by chiral HPLC analysis. ee Value > 99.5% (e.g.) Figure 1 (as shown) 1 H-NMR spectrum as shown Figure 2 As shown, the product can be further purified by column chromatography.

[0036] Example 3: 1): Dissolve 100 g of methyl 3-cyclohexene-1-carboxylate in 800 mL of phosphate buffer at pH 7.0-7.5, add 5 g of carboxylesterase (enzyme activity ≥300 U / g), and stir the reaction at 30-40 ℃ for 6-8 h. After the reaction is complete, extract with ethyl acetate, combine the organic phases, concentrate and dry to obtain intermediate 2 and intermediate 3. Intermediate 2 was dissolved in 300 mL of methanol, and 3 g of potassium hydrogen sulfate was added. The mixture was refluxed at 60-70 °C for 3-4 h to generate intermediate 1′. Intermediate 1′ was placed in 200 mL of 10% sodium hydroxide solution and stirred and racemized at 25-35 °C for 2-3 h to regenerate starting material 1. Total intermediate 3 produced was approximately 78 g, with a purity of 95% and chiral properties. ee Value 90%; 2): 60 g of intermediate 3 was placed in a biphase solvent consisting of 400 mL of water and 200 mL of ethyl acetate, and 23 g of sodium hydroxide was added. The mixture was stirred at 35-45 °C for 2 h. After the reaction was completed, the mixture was allowed to stand and separate into layers. The pH of the aqueous phase was adjusted to 2-3 with concentrated hydrochloric acid, and a white solid precipitated out. After filtration, washing with water, and vacuum drying at 50 °C, approximately 50.0 g of intermediate 4 was obtained. 3): Dissolve 50 g of intermediate 4 in 800 mL of pH 7.8 phosphate buffer, add 20 g of P450 oxidase whole-cell catalyst (enzyme activity ≥500 U / g) and 0.6 g of NADPH (nicotinamide adenine dinucleotide phosphate, reduced form), and stir the reaction at 30-35 ℃. During the reaction, add glucose-glucose dehydrogenase regeneration system (containing 15 g of glucose and 1500 U of glucose dehydrogenase) in batches to maintain the coenzyme cycle. After the reaction is completed, adjust the pH to 3.0-4.0 with 2 M hydrochloric acid, extract with ethyl acetate (500 mL × 3), combine the organic phases, wash with water, dry with anhydrous sodium sulfate, concentrate under reduced pressure, and dry to obtain approximately 51 g of intermediate 5 with a purity of 91%. 4): Dissolve 50 g of intermediate 5 in 800 mL of phosphate buffer (pH 8.0-8.5), add 5 g of whole-cell alcohol hydroxy dehydrogenase (enzyme activity ≥500 U / g), and stir the reaction at 30-40 ℃ for 6-8 h. After the reaction, adjust the pH of the system to 3-4 with dilute hydrochloric acid, extract with ethyl acetate (2 × 500 mL), combine the organic phases, wash with water, dry with anhydrous sodium sulfate, concentrate under reduced pressure, and dry to obtain approximately 46 g of intermediate 6 with a purity of 95%. 5): Dissolve 50 g of intermediate 6 in 800 mL of phosphate buffer (pH 8.0–8.5), add 1.2 g of pyridoxal phosphate (PLP) and 5.0 g of transaminase (enzyme activity ≥300 U / g), and stir the reaction at 35–40 °C for 10–12 h. After the reaction, extract the reaction solution with 2 × 500 mL of ethyl acetate, combine the organic phases, dry with anhydrous sodium sulfate, concentrate under reduced pressure, and dry again to obtain approximately 44 g of intermediate 7. The purity was determined to be 93%, and its chiral purity was confirmed by chiral HPLC analysis. ee Value: 85%.

[0037] 6): Dissolve 50 g of intermediate 7 and 25 g of isopropylamine in 600 mL of methyl tert-butyl ether, add 5 g of immobilized lipase (enzyme activity ≥ 500 U / g) and 20 g of 4Å molecular sieve, and carry out the condensation reaction at 40-50 °C. Monitor the reaction process by TLC until the starting material is completely converted. After the reaction, filter out the molecular sieve and immobilized lipase, and concentrate and dry the organic phase under reduced pressure to obtain intermediate 8 (approximately 50 g, purity approximately 95.0%). 7) 50 g of intermediate 8 and 60 g of Boc₂O were dissolved in 700 mL of dichloromethane, and a condensation reaction was carried out at 20–30 °C. The reaction was monitored by TLC until the starting material was completely converted. After the reaction, the reaction solution was washed successively with saturated sodium bicarbonate solution and brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 57 g of crude edoxaban tosylate intermediate (chemical purity approximately 94.5% as determined by HPLC). Chiral purity was confirmed by chiral HPLC analysis. ee If the concentration is >99.5%, it can be further purified by column chromatography to obtain a pure product.

[0038] The data above demonstrates that this invention has successfully established a superior synthetic route for edoxaban tosylate intermediates. This route not only features concise steps and excellent chirality control, resulting in high yield and low cost, but also demonstrates greater atom economy and intrinsic safety in production by eliminating the highly toxic sodium azide, thus possessing excellent potential for industrial application.

Claims

1. A method for the biosynthesis of a chiral intermediate of edoxaban tosylate characterized in that, The specific reaction steps include: 1) first, 3-cyclohexene-1-carboxylic acid methyl ester is resolved by carboxylic acid esterase, while generating intermediate 2 and intermediate 3; 2) intermediate 3 is subjected to a hydrolysis reaction to generate intermediate 4; 3) intermediate 4 is converted into intermediate 5 under the catalysis of P450 oxidase; 4) intermediate 5 is converted into intermediate 6 under the catalysis of alcohol hydroxyl dehydrogenase; 5) intermediate 6 is converted into intermediate 7 under the catalysis of transaminase; 6) intermediate 7 is condensed with isopropylamine under the catalysis of lipase to generate intermediate 8; 7) intermediate 8 is reacted with Boc2O to generate the intermediate of edoxaban tosylate.

2. The biosynthetic method of claim 1, wherein, In step 1), intermediate 2 is esterified into intermediate 1'; the intermediate 1' can be reconverted into the raw material 1 through racemization, so as to realize the recycling of the material; 。 3. The biosynthetic method of claim 2, wherein, The esterification reaction is carried out in an alcohol solvent, and potassium bisulfate is used as the catalyst, and the reaction temperature is 60-70°C; the racemization reaction is carried out under alkaline conditions, and the reaction temperature is 25-35°C, so as to realize the recycling and recycling of the unreacted isomers.

4. The biosynthetic method of claim 1 wherein, In step 1), the reaction temperature of the carboxylic acid esterase resolution is 30-40°C, and the pH is maintained at 7.0-7.5, and a phosphate buffer is used as the reaction medium.

5. The biosynthetic method of claim 1 wherein, In step 2), the hydrolysis reaction is carried out at 35-45°C, and a biphasic solvent system composed of water and ethyl acetate is used, and the volume ratio of the water to the ethyl acetate is 1:1-1:1.

5.

6. The biosynthetic method of claim 1 wherein, In step 3), the reaction temperature is 30-35°C, and a phosphate buffer with a pH of 7.5-8.0 is used as the reaction solvent.

7. The biosynthetic method of claim 1 wherein, In step 4), the reaction temperature is 30-40°C, and a phosphate buffer with a pH of 8.0-8.5 is used as the reaction solvent.

8. The biosynthetic method of claim 1 wherein, In step 5), the reaction temperature is 35-40°C, and a phosphate buffer with a pH of 8.5-9.0 is used as the reaction solvent.

9. The biosynthetic method of claim 1 wherein, In step 6), the reaction temperature is 40-50°C, and the molar ratio of intermediate 7 to isopropylamine is 1:2.0-3.

0.

10. The biosynthesis method according to any one of claims 1, 3-9, wherein, 1) 3-cyclohexene-1-carboxylic acid methyl ester is dissolved in a phosphate buffer with a pH of 7.0-7.5, carboxylic acid esterase is added, and the resolution reaction is carried out at a temperature of 30-40°C; after the reaction is completed, intermediate 2 and intermediate 3 are separated by extraction; 2) intermediate 3 is placed in a biphasic solvent system composed of water and ethyl acetate, sodium hydroxide is added, and the hydrolysis reaction is stirred at a temperature of 35-45°C; after the reaction is completed, intermediate 4 is obtained through post-treatment; 3) intermediate 4 is dissolved in a phosphate buffer with a pH of 7.5-8.0, P450 oxidase whole cells and nicotinamide adenine dinucleotide phosphate are added, and the reaction is carried out at a temperature of 30-35°C; after the reaction is completed, intermediate 5 is obtained through post-treatment; 4) intermediate 5 is dissolved in a phosphate buffer with a pH of 8.0-8.5, alcohol hydroxyl dehydrogenase whole cells are added, and the oxidation reaction is carried out at a temperature of 30-40°C; after the reaction is completed, intermediate 6 is obtained through post-treatment; 5): intermediate 6 was dissolved in phosphate buffer with pH 8.0~8.5, pyridoxal phosphate and transaminase were added, the temperature was controlled at 35~40℃, and the reaction was stirred for 8~12 hours; after the reaction was completed, intermediate 7 was obtained through post-treatment; 6): intermediate 7 and isopropylamine were dissolved in methyl tert-butyl ether, lipase and molecular sieve were added, the temperature was controlled at 40~50℃, and the condensation reaction was carried out, the reaction process was monitored by TLC until the raw material was completely converted, after the reaction was completed, the molecular sieve and lipase were filtered out, and the organic phase was concentrated under reduced pressure to obtain intermediate 8; 7): intermediate 8 was dissolved in dichloromethane, Boc2O was added, and the reaction was stirred at 25~30℃ for 4~6 hours; after the reaction was completed, edoxaban tosylate intermediate was obtained through post-treatment.

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