Chemical-enzyme process synthesis method of mirabegron

Abstract

The invention discloses a chemical-enzyme process synthesis method of mirabegron, which comprises the following steps: by taking benzaldehyde and formaldehyde as raw materials and a phosphoric acid buffer salt solution as a solvent, carrying out benzoin condensation reaction under the condition of taking benzaldehyde lyase as a biocatalyst to obtain an intermediate (5), carrying out carbonyl reductase asymmetric catalytic reaction on the intermediate (5) to obtain a key intermediate (4), and carrying out chemical-enzyme process synthesis on the key intermediate (4) to obtain the mirabegron. And then carrying out substitution reaction on the key intermediate (4) and paratoluensulfonyl chloride, and sequentially carrying out deprotection reaction and condensation reaction to obtain the target compound mirabegron. The synthesis method adopts a two-step enzymatic catalysis method to efficiently synthesize the chiral key intermediate, has the advantages of high yield, simplicity in operation, no pollution, low production cost and environmental friendliness, and is easy for industrial application.

Description

Technical Field

[0001] The invention belongs to the technical field of drug synthesis, and particularly relates to a synthesis process method of mirabegron. Background Art

[0002] Mirabegron (generic name: Mirabegron, trade name: Betanli) English alias: 2-Amino-N-[4-[2-[[(2R)-2-hydroxy-2-phenyl-ethyl]amino]-ethyl]phenyl]-4-thiazolea cetamide. The structural formula is shown in formula (1).

[0003]

[0004] Mirabegron is the first β3 adrenergic receptor agonist drug used to treat overactive bladder. It is a drug developed by Japan's Astellas Pharmaceuticals. It has made a great contribution to the treatment of overactive bladder and provided a new treatment option for patients with overactive bladder. The drug is in the form of tablets, which are easy to take, with a small dosage, significant efficacy and few side effects. It can significantly reduce the pain of patients with overactive bladder and is a blessing for patients.

[0005] At present, the synthesis process of mirabegron is to use (R)-styrene oxide and p-nitrophenylethylamine as raw materials, condense to obtain amino alcohol, and then protect the amino group, reduce the nitro group, condense with 2-aminothiazole-4-acetic acid, and remove the protecting group to obtain mirabegron. The (R)-styrene oxide used in this route is a medium-toxic chemical and is relatively expensive; in addition, the use of hydrogen reduction has high requirements for equipment and environment, and is not suitable for large-scale production. Summary of the invention

[0006] The object of the present invention is to provide a new chemical enzyme process for synthesizing mirabegron (1) with readily available raw materials, simple process, mild reaction conditions, green and environmentally friendly properties and suitable for industrialization.

[0007] The technical solution adopted by the present invention is as follows:

[0008] A chemical-enzymatic synthesis method of mirabegron comprises the following steps:

[0009] 1) Compound (7) and compound (6) react with benzoin catalyzed by benzaldehyde lyase to obtain compound (5);

[0010] 2) Compound (5) is subjected to carbonyl reduction reaction catalyzed by carbonyl reductase to obtain compound (4);

[0011] 3) Compound (4) undergoes substitution reaction with p-toluenesulfonyl chloride to obtain compound (3);

[0012] 4) Compound (3) undergoes a deprotection reaction with p-aminophenylethylamine to obtain compound (2);

[0013] 5) Compound (2) undergoes condensation reaction with 2-aminothiazole-4-acetic acid to obtain compound (1), thereby obtaining the target compound mirabegron;

[0014] The reaction formula is as follows:

[0015]

[0016] Furthermore, in step 1), the reaction solvent is a phosphate buffered saline solution, the pH is 7.4±0.2, the reaction temperature is room temperature, the molar ratio of compound (6) to compound (7) is 1:5-1:50, preferably 1:15-1:30. The concentration of compound (6) in the reaction solvent is 10-100 mmol / L, preferably 50-60 mmol / L.

[0017] Furthermore, the enzyme used in the step 1) reaction is benzaldehyde lyase (BAL), whose amino acid sequence is shown in SEQ ID NO.1, and the concentration of wet cells expressing BAL enzyme in the reaction solvent is 10-15 g / L.

[0018] Furthermore, in step 2), the reaction solvent is a phosphate buffered saline solution with a pH of 7.4±0.2, the reaction temperature is room temperature, and the concentration of compound (5) in the reaction solvent is 10-100 mmol / L, preferably 40-50 mmol / L.

[0019] Furthermore, the enzyme used in the step 2) reaction is carbonyl reductase (KRED), whose amino acid sequence is shown in SEQ ID NO. 2, and the concentration of wet cells expressing KRED enzyme in the reaction solvent is 10-15 g / L.

[0020] Furthermore, after the reaction of step 1) or 2) is completed, ethyl acetate is added to extract the reaction solution, and the solvent is evaporated to obtain compound (5) or compound (4).

[0021] Furthermore, in the process of preparing compound (3) by reacting compound (4) with p-toluenesulfonyl chloride in step 3), the reaction solvent is dichloromethane, the reaction is carried out in the presence of dibutyltin oxide and triethylamine, the molar ratio of p-toluenesulfonyl chloride to compound (4) is 1-1.5:1, preferably 1-1.1:1, the molar ratio of dibutyltin oxide to compound (4) is 0.1-0.5:1, preferably 0.2-0.25:1, the molar ratio of triethylamine to compound (4) is 1-1.5:1, preferably 1-1.1:1, the reaction is carried out at room temperature, and the reaction is terminated when LC-MS monitoring shows that the raw material compound (4) is completely converted.

[0022] Furthermore, after the reaction in step 3) is completed, 0.4-0.8M hydrochloric acid is added to the reaction solution to quench the reaction, and then dichloromethane is added to extract the reaction solution. The organic phase is evaporated to dryness to obtain a white solid, namely compound (3).

[0023] Furthermore, in step 4), p-aminophenylethylamine is reacted with compound (3) at a molar ratio of 0.8-2:1, preferably 0.9-1.5:1, with acetonitrile as solvent, and stirred under reflux at 65-75° C. for overnight reaction, and the reaction time is 8-12 h.

[0024] Furthermore, after the reaction in step 4) is completed, acetonitrile is distilled under reduced pressure, cooled to 0-5°C and then added to n-hexane. A large amount of light yellow precipitate is formed during stirring. The precipitate formed after filtration is washed with 2-5°C cold toluene and then dried to obtain compound (2).

[0025] Further, in step 5), 2-aminothiazole-4-acetic acid reacts with compound (2) in a molar ratio of 1-2:1, preferably 1.0-1.5:1, and the reaction is carried out in the presence of 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate and N,N-diisopropylethylamine, the molar ratio of 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate to compound (3) is 1-2:1, preferably 1.1-1.5:1, and the molar ratio of N,N-diisopropylethylamine to compound (3) is 1-4:1, preferably 2-3:1, and dimethylformamide is used as solvent and the reaction is stirred at room temperature for 10-15 hours.

[0026] Furthermore, step 5) includes the following post-treatment steps after the reaction is completed: adding a saturated sodium chloride aqueous solution to the reaction mixture, then extracting with dichloromethane, washing the organic layer with a saturated sodium chloride aqueous solution, drying with anhydrous sodium sulfate, and concentrating under reduced pressure to obtain a crude product, and then washing the solid with a methanol-water mixed solution with a volume ratio of 2-4:1 and recrystallizing to obtain the target compound.

[0027] The beneficial effects achieved by the present invention are as follows: according to the method for preparing mirabegron provided by the present invention, a key chiral intermediate is constructed by enzyme catalysis using cheap and readily available raw materials, the overall synthetic route has fewer steps, mild reaction conditions, and a simple synthetic process, and can be used for industrial production. DETAILED DESCRIPTION

[0028] In order to enable those skilled in the art to better understand the technical solution of the present invention, some non-limiting implementation cases are further disclosed below to further illustrate the present invention in detail.

[0029] In the embodiment of the present invention, the plasmid of benzaldehyde lyase (BAL) was synthesized by General Biotechnology (Anhui) Co., Ltd., and the amino acid sequence of the enzyme is shown in SEQ ID NO.1.

[0030] The plasmid of carbonyl reductase (KRED) was synthesized by Jiangsu Saisuofei Biotechnology Co., Ltd., and the amino acid sequence of the enzyme is shown in SEQ ID NO.2.

[0031] The pET 28a plasmid (pET 28a plasmid purchased from General Biotechnology (Anhui) Co., Ltd.) with correct sequencing (gene sequence is SEQ ID NO.1) was transformed into Escherichia coli BL21 (DE3) competent cells, spread on LB plates containing 50ug / mL kanamycin and cultured overnight at 37°C, and a single transformed bacterium was picked and inoculated into 3mL of LB liquid culture medium containing 50ug / mL kanamycin. The culture was shaken at 37°C for 6-8h until the bacterial solution became slightly turbid. The bacterial solution was then inoculated into 1L of LB liquid culture medium containing 50ug / mL kanamycin at a volume ratio of 1:10 for culture, and cultured on a shaking table at 37°C for 3-5h. After the bacterial solution became turbid, 1mL of 0.5M IPTG solution was added, and the solution was placed in a 16°C shaker for 16h of shaking culture. After centrifugation in a centrifuge at 6000rpm for 20min, a wet cell precipitate of benzaldehyde lyase (BAL) was obtained.

[0032] The pET 28a plasmid (pET 28a plasmid purchased from Jiangsu Saisuofei Biotechnology Co., Ltd.) with correct sequencing (gene sequence is SEQ ID NO.2) was transformed into Escherichia coli BL21 (DE3) competent cells, spread on LB plates containing 50ug / mL kanamycin and cultured overnight at 37°C, and a single transformed bacterium was picked and inoculated into 3mL of LB liquid culture medium containing 50ug / mL kanamycin. The culture was shaken at 37°C for 6-8h until the bacterial solution became slightly turbid. The bacterial solution was then inoculated into 1L of LB liquid culture medium containing 50ug / mL kanamycin at a volume ratio of 1:10 for culture, and cultured on a shaker at 37°C for 3-5h. After the bacterial solution became turbid, 1mL of 0.5M IPTG solution was added, and the solution was placed in a 16°C shaker for 16h of shaking culture. After centrifugation in a centrifuge at 6000rpm for 20min, a wet cell precipitate of carbonyl reductase (KRED) was obtained.

[0033] Example 1 Preparation of Compound (5)

[0034]

[0035] In a 3000 mL shaking flask, 50 mmol of compound (6), 1000 mmol of compound (7) and 10 g of benzaldehyde lyase BAL wet cell precipitate were added to 1000 mL of phosphate buffered saline solution (pH 7.4), and the reaction was carried out at room temperature for 10 h. After the reaction was completed by TLC monitoring, ethyl acetate was added to extract the reaction solution, and the solvent was evaporated to obtain compound (5) with a yield of 85% and a purity of 98% as determined by HPLC.

[0036] 1 H-NMR (DMSO-d 6 , 400MHz): δ (ppm) 7.92 (d, 2H, J = 7.8Hz); 7.64 (t, 1H); 7.52 (t, 2H); 5.11 (s, 1H); 4.80 (d, 2H, J = 0.2Hz).

[0037] ESI-MS [M+H] + =137.

[0038] Preparation of compound (4)

[0039]

[0040] In a 3000 mL shaking flask, 50 mmol of compound (5) and 10 g of carbonyl reductase KRED wet cells were added to 1000 mL of phosphate buffered saline (pH 7.4), and the reaction was carried out at room temperature for 10 h. After the reaction was completed by TLC monitoring, ethyl acetate was added to extract the reaction solution, and the solvent was evaporated to obtain compound (4) with a yield of 82% and a purity of 99% as determined by HPLC.

[0041] 1 H-NMR (CD 3 OD, 400MHz): δ (ppm) 7.37-7.21 (m, 5H); 4.79-4.76 (m, 1H); 3.73-3.60 (m, 2H).

[0042] ESI-MS [M+H] + =139.

[0043] Preparation of compound (3)

[0044]

[0045] In a 250 mL single-necked round-bottom flask, 40 mmol of compound (4), 8.3 mmol of dibutyltin oxide and 40 mmol of triethylamine were added to 50 mL of dichloromethane and cooled to 0°C in an ice bath. 41 mmol of p-toluenesulfonyl chloride was dissolved in 60 mL of dichloromethane and added dropwise to the round-bottom flask. The mixture was stirred at room temperature for 3 h. After the reaction was completed under TLC monitoring, 0.5 M hydrochloric acid was added to quench the reaction. Dichloromethane was then added to extract the reaction solution. The solvent was evaporated to obtain a white solid, i.e., compound (3). The yield was 93% and the purity was 99% as determined by HPLC.

[0046] 1 H NMR (DMSO-d 6 , 400MHz): δ7.73(d,J=8.2Hz,2H); 7.10–6.49(m,7H,); 4.85(dd,J 1 =8.4Hz,J 2 =3.1Hz,1H);4.23(dd,J 1 =10.1Hz,J 2 =3.1Hz,1H);4.14(dd,J 1 =10.2Hz,J 2 =8.3 Hz, 1H); 2.26 (s, 3H).

[0047] ESI-MS [M+H] + =293.

[0048] Preparation of compound (2)

[0049]

[0050] In a 250 mL single-necked round-bottom flask, 35 mmol of compound (3) and 38.5 mmol of p-aminophenylethylamine were added to 100 mL of acetonitrile, heated to 70°C, stirred and refluxed overnight, and reacted for 10 hours. After the reaction was completed by TLC monitoring, acetonitrile was distilled under reduced pressure at 55°C, cooled to 5°C, and 45 mL of n-hexane was added. A large amount of light yellow precipitate was formed during the stirring process, which was washed and dried with 4°C cold toluene to obtain compound (2) with a yield of 86%.

[0051] 1 H NMR (DMSO-d 6 , 400MHz): δ7.30–7.22(m,5H); 6.86(d,J=12.0Hz,2H); 6.45(d,J=8.0Hz,2H); 5.23 (s,1H);4.81(s,2H);4.60(t,1H,J=8.0Hz);2.71–2.55(m,4H);2.51–2.50(m,2H).

[0052] ESI-MS [M+H] + =257.

[0053] Preparation of compound (1)

[0054]

[0055] In a 1L round-bottom flask, 30mmol of compound (2), 30.6mmol of 2-aminothiazole-4-acetic acid, 45mmol of 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, 90mmol of N,N-diisopropylethylamine and 300mL of dimethylformamide were added and stirred at room temperature for 13h. After completion, a saturated sodium chloride aqueous solution was added to the reaction mixture, and then the mixture was extracted with dichloromethane. The organic layer was washed with a saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product. A white solid was obtained by washing and recrystallization with a methanol: water = 3:1 (v / v) mixed solution, which was compound (1) with a yield of 84% and an HPLC purity of 92%.

[0056] 1 H NMR (DMSO-d 6,400MHz):δ7.50(d,J=8.0Hz,2H);7.29(t,J=8.0Hz,4H);7.21(t,J=8.0Hz,1H);7.11(d,J=12.0Hz,2H);6.93(s,2H);6.30(s,1H);5.26(s,1H);4.60(s,1H);3.45(s,2H);2.75–2.71(m,2H);2.65(d,J=4.0Hz,4H).

[0057] ESI-MS[M+H] + =397.

Claims

1. A chemical-enzymatic synthesis method of mirabegron, characterized in that: The following steps are involved: 1) Compound (7) and compound (6) react with benzoin catalyzed by benzaldehyde lyase to obtain compound (5); 2) Compound (5) is subjected to carbonyl reduction reaction catalyzed by carbonyl reductase to obtain compound (4); 3) Compound (4) undergoes substitution reaction with p-toluenesulfonyl chloride to obtain compound (3); 4) Compound (3) undergoes a deprotection reaction with p-aminophenylethylamine to obtain compound (2); 5) Compound (2) undergoes condensation reaction with 2-aminothiazole-4-acetic acid to obtain compound (1), thereby obtaining the target compound mirabegron; The reaction formula is as follows:

2. A chemical-enzymatic synthesis method of mirabegron according to claim 1, characterized in that: Step 1) In the reaction, the reaction solvent is a phosphate buffered saline solution, the pH is 7.4±0.2, the reaction temperature is room temperature, the molar ratio of compound (6) to compound (7) is 1:5-1:50, preferably 1:15-1:30, and the concentration of compound (6) in the reaction solvent is 10-100 mmol / L, preferably 50-60 mmol / L; The enzyme used in the reaction is benzaldehyde lyase BAL, whose amino acid sequence is shown in SEQ ID NO.

1. The concentration of wet cells expressing BAL enzyme in the reaction solvent is 10-15 g / L.

3. A chemical-enzymatic synthesis method of mirabegron according to claim 1, characterized in that: Step 2) In the reaction, the reaction solvent is a phosphate buffered saline solution, the pH is 7.4±0.2, the reaction temperature is room temperature, and the concentration of compound (5) in the reaction solvent is 10-100 mmol / L, preferably 40-50 mmol / L; The enzyme used in the reaction is carbonyl reductase KRED, whose amino acid sequence is shown in SEQ ID NO.

2. The concentration of wet cells expressing KRED enzyme in the reaction solvent is 10-15 g / L.

4. A chemical-enzymatic synthesis method of mirabegron according to claim 2 or 3, characterized in that: After the reaction in step 1) or 2) is completed, ethyl acetate is added to extract the reaction solution, and the solvent is evaporated to obtain compound (5) or compound (4).

5. A chemical-enzymatic synthesis method of mirabegron according to claim 1, characterized in that: In the process of preparing compound (3) by reacting compound (4) with p-toluenesulfonyl chloride in step 3), the reaction solvent is dichloromethane, the reaction is carried out in the presence of dibutyltin oxide and triethylamine, the molar ratio of p-toluenesulfonyl chloride to compound (4) is 1-1.5:1, preferably 1-1.1:1, the molar ratio of dibutyltin oxide to compound (4) is 0.1-0.5:1, preferably 0.2-0.25:1, the molar ratio of triethylamine to compound (4) is 1-1.5:1, preferably 1-1.1:1, the reaction is carried out at room temperature, and the reaction is terminated when LC-MS monitoring shows that the raw material compound (4) is completely converted.

6. A chemical-enzymatic synthesis method of mirabegron according to claim 5, characterized in that: Step 3) After the reaction is completed, 0.4-0.8M hydrochloric acid is added to the reaction solution to quench the reaction, and then dichloromethane is added to extract the reaction solution. The organic phase is evaporated to dryness to obtain a white solid, namely compound (3).

7. A chemical-enzymatic synthesis method of mirabegron according to claim 1, characterized in that: In step 4), p-aminophenylethylamine reacts with compound (3) at a molar ratio of 0.8-2:1, preferably 0.9-1.5:1, with acetonitrile as solvent, stirring and refluxing at 65-75° C. overnight, for 8-12 h.

8. A chemical-enzymatic synthesis method of mirabegron according to claim 7, characterized in that: Step 4) After the reaction is completed, acetonitrile is distilled under reduced pressure, cooled to 0-5°C and then added to n-hexane. A large amount of light yellow precipitate is formed during stirring. The precipitate formed after filtration is washed with 2-5°C cold toluene and then dried to obtain compound (2).

9. A chemical-enzymatic synthesis method of mirabegron according to claim 1, characterized in that: In step 5), 2-aminothiazole-4-acetic acid reacts with compound (2) in a molar ratio of 1-2:1, preferably 1.0-1.5:

1. The reaction is carried out in the presence of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate and N,N-diisopropylethylamine. The molar ratio of 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate to compound (3) is 1-2:1, preferably 1.1-1.5:1, and the molar ratio of N,N-diisopropylethylamine to compound (3) is 1-4:1, preferably 2-3:

1. Dimethylformamide is used as solvent and the reaction is stirred at room temperature for 10-15 hours.

10. A chemical-enzymatic synthesis method of mirabegron according to claim 9, characterized in that: Step 5) After the reaction is completed, the following post-treatment steps are included: saturated sodium chloride aqueous solution is added to the reaction mixture for quenching, then extracted with dichloromethane, the organic layer is washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product, and the solid is then washed with a methanol-water mixed solution with a volume ratio of 2-4:1 and recrystallized to obtain the target compound.

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