Method for asymmetric reduction of carbonyl reductase to prepare (-)-5-azaspiro[2.4]heptan-7-ol

Abstract

The present invention discloses a method for asymmetric reduction of carbonyl reductase to prepare (-)-5-azaspiro[2,4]heptan-7-ol, which comprises two steps: preparation of carbonyl reductase freeze-dried powder and preparation of (-)-5-azaspiro[2,4]heptan-7-ol. The reaction route is short and the operation steps are simple. It has the advantages of cheap and easily available raw materials, high utilization rate, fast reaction time, mild reaction conditions, high chiral purity of the product, and less environmental pollution. The yield of (-)-5-azaspiro[2,4]heptan-7-ol prepared by the method of the present invention is more than 85% (calculated based on 5-azaspiro[2,4]heptan-7-one), and the ee value of the target product is 100%.

Description

Technical Field

[0001] The present invention belongs to the technical field of organic synthesis, and particularly relates to a method for asymmetric reduction of carbonyl reductase to prepare (-)-5-azaspiro[2.4]heptan-7-ol. Background Art

[0002] AL8326 is a new type 1.1 (not yet on the market globally) anti-tumor drug with independent intellectual property rights developed by Nanjing Aidecheng Pharmaceutical Technology Co., Ltd. It is a small molecule oral tablet that inhibits tyrosine kinase FGF family receptors, VEGF family receptors, and Aurora kinase family's Aurora-B. It has a brand-new structure, clear anti-tumor effects, and relatively small toxic reactions. It has broad anti-tumor activities in vitro and in vivo. Animal experiments have proved that it can effectively inhibit a variety of solid tumors.

[0003] Currently, the AL8326 used in pre-clinical studies is a racemic compound, that is, it has two structures, left-handed and right-handed. Only one configuration can truly act on the diseased site after entering the human body. The other configuration has no drug effect and is the root cause of the drug's toxic and side effects. Clinical verification shows that the levorotatory isomer (-)AL8326 can exert better therapeutic effects. (-)-5-azaspiro[2.4]heptan-7-ol, as an important intermediate for the preparation of the levorotatory isomer (-)AL8326, its synthesis will greatly promote the research progress of the chiral drug (-)AL8326, making it possible to separate the drug AL8326 into a single configuration compound. Furthermore, the drug AL8326 can target specific diseased sites, accumulate or release active ingredients at the diseased sites, form a relatively high drug molecule concentration locally at the diseased sites, improve the drug effect while inhibiting toxic and side effects, and also reduce the damage to normal tissue cells, thereby exerting better high-efficiency and low-toxicity effects.

[0004] Currently, the resolution of racemic 5-azaspiro[2,4]heptan-7-ol has been reported. Chinese Patent Application, CN113072480A, gives a preparation method of chiral (-)-5-azaspiro[2.4]heptan-7-ol, and its synthetic route is shown in Formula 1 below:

[0005]

[0006] Although the above synthetic route can obtain the qualified target product chiral (-)-5-azaspiro[2.4]heptan-7-ol, it has the disadvantages of a long synthetic route, cumbersome operation steps, low synthetic efficiency, low utilization rate of the single-time resolution intermediate, and low total yield of the target product.

[0007] The present invention aims to propose a method for asymmetric reduction of carbonyl reductase to prepare (-)-5-azaspiro[2,4]heptan-7-ol in order to solve the above technical problems. Summary of the Invention

[0008] The object of the present invention is to overcome the defects existing in the prior art and provide a method for asymmetric reduction of (-)-5-azaspiro[2,4]heptan-7-ol by carbonyl reductase, which has a short reaction route, simple operation, and has the advantages of cheap and easily available raw materials, high utilization rate, fast reaction time, mild reaction conditions, high chiral purity of the product, and less environmental pollution; the yield of (-)-5-azaspiro[2,4]heptan-7-ol prepared by the method of the present invention is above 85% (calculated based on 5-azaspiro[2,4]heptan-7-one), and the ee value of the target product is 100%.

[0009] To achieve the above object, the technical solution of the present invention is to design a method for asymmetric reduction of (-)-5-azaspiro[2,4]heptan-7-ol by carbonyl reductase, which includes the following steps:

[0010] S1: Preparation of carbonyl reductase lyophilized powder. Inoculate the ATCC 7330 strain onto Luria-Bertani liquid medium with an inoculation amount of 1%, the chloramphenicol content in the medium is 100 μg / mL, the culture temperature is 25 °C, and culture at 220 rpm for 24 h. When the measured OD 600 concentration is 0.6, add isopropyl-β-D-thiogalactoside and make its concentration 0.4 mM, induce at 25 °C for 16 h. After the induction ends, centrifuge to collect the cells, and after washing the cells, resuspending, and ultrasonic disruption, freeze-dry to obtain the carbonyl reductase lyophilized powder;

[0011] S2: Mix the carbonyl reductase lyophilized powder, coenzyme, coenzyme recycling enzyme, buffer, and solvent prepared in step S1 to prepare a reaction system solution, add 5-azaspiro[2,4]heptan-7-one to the reaction system solution, and under the catalysis of carbonyl reductase, 5-azaspiro[2,4]heptan-7-one undergoes an asymmetric reduction reaction to generate (-)-5-azaspiro[2,4]heptan-7-ol.

[0012] The preferred technical solution is that in step S2, the solvent is one or more of methanol, ethanol, n-propanol, butanol, isopropanol, dimethyl sulfoxide, and water.

[0013] A further preferred technical solution is that in step S2, the buffer is formate buffer with a solute molar concentration of 0.05 mol / L.

[0014] A further preferred technical solution is that in step S2, the formate buffer is an aqueous solution of HCOOH-HCOONa, and the volume feeding ratio of the solvent to the formate buffer is 1:1 to 30.

[0015] A further preferred technical solution is that in step S2, the solvent is water, and the volume ratio of water to formate buffer is 1:6.

[0016] Another preferred technical solution is that in step S2, the mass concentration ratio of 5-azaspiro[2,4]heptane-7-one and carbonyl reductase in the reaction system solution is 1:0.1-1, wherein the mass concentration of 5-azaspiro[2,4]heptane-7-one is 3.5%.

[0017] Another preferred technical solution is that in step S2, the coenzyme is NADPH or NADH, and the mass feeding ratio of the coenzyme to the carbonyl reductase lyophilized powder is 1:20-100; the coenzyme cycle enzyme is formate dehydrogenase, and the mass feeding ratio of formate dehydrogenase to the carbonyl reductase lyophilized powder is 1:1.25.

[0018] A further preferred technical solution is that in step S2, the coenzyme is NADPH, and the mass ratio of the coenzyme to the carbonyl reductase freeze-dried powder is 1:50.

[0019] Another preferred technical solution is that in step S2, the catalytic reaction temperature of the carbonyl reductase is 10-50° C., the reaction time is 1-2 h, and the pH of the reaction system solution is 6.5-8.0.

[0020] A further preferred technical solution is that in step S2, the catalytic reaction temperature of the carbonyl reductase is 20-30°C.

[0021] The advantages and beneficial effects of the present invention are:

[0022] 1. The method of the present invention for preparing (-)-5-azaspiro[2,4]heptane-7-ol by asymmetric reduction of carbonyl reductase has the advantages of a short reaction route, simple operation, cheap and readily available raw materials, high utilization rate, fast reaction time, mild reaction conditions, high chiral purity of the product, and less environmental pollution.

[0023] 2. A method for preparing (-)-5-azaspiro[2,4]heptane-7-ol by asymmetric reduction of carbonyl reductase according to the present invention, wherein the yield of the prepared (-)-5-azaspiro[2,4]heptane-7-ol is above 85% (based on 5-azaspiro[2,4]heptane-7-one) and the ee value of the target product is 100%. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a reaction formula for a method for preparing (-)-5-azaspiro[2,4]heptane-7-ol by asymmetric reduction using a carbonyl reductase according to the present invention;

[0025] Figure 2Gas-phase purity detection chromatogram of (-)-5-azaspiro[2,4]heptan-7-ol (code JD0338);

[0026] Figure 3 Chiral purity detection chromatogram of (-)-5-azaspiro[2,4]heptan-7-ol. Detailed implementation manners

[0027] The following combines the accompanying drawings and examples to further describe the detailed implementation manners of the present invention. The following examples are only used to more clearly illustrate the technical solutions of the present invention and cannot be used to limit the protection scope of the present invention.

[0028] Example 1

[0029] Using the method of the present invention to prepare (-)-5-azaspiro[2,4]heptan-7-ol, the reaction formula is shown in the attached Figure 1 , including the following operation steps:

[0030] S1: Preparation of freeze-dried carbonyl reductase powder. Inoculate the ATCC 7330 strain onto Luria-Bertani liquid medium with an inoculation amount of 1%, the chloramphenicol content in the medium is 100 μg / mL, the culture temperature is 25°C, and culture at 220 rpm for 24 h. When the measured OD 600 concentration is 0.6, add isopropyl-β-D-thiogalactoside (IPTG) and make its concentration 0.4 mM, induce at 25°C for 16 h. After the induction is completed, centrifuge to collect the bacterial cells, wash the cells, resuspend, ultrasonically disrupt and then freeze-dry to obtain the freeze-dried carbonyl reductase powder;

[0031] S2: Add 5-azaspiro[2,4]heptan-7-one (5 g), freeze-dried carbonyl reductase powder (2.5 g), formate dehydrogenase (2 g), NADPH (50 mg), 0.05 mol / L HCOOH-HCOONa buffer solution (120 mL), and water (20 mL) into a 250 mL reaction flask in sequence, stir at 250 rpm, control the reaction pH at 6.5 - 8.0, react at 25°C for 2 h. When gas chromatography detects no residue of 5-azaspiro[2,4]heptan-7-one, terminate the reaction; filter the reaction solution with diatomaceous earth, extract the filtrate with dichloromethane six times, combine the organic phases, and concentrate under reduced pressure to dryness to obtain 4.25 g of the product (-)-5-azaspiro[2,4]heptan-7-ol, with a yield of 85% (calculated based on 5-azaspiro[2,4]heptan-7-one); the Figure 2 gas-phase purity detection chromatogram shows that the purity of the target product is 94.44%; the Figure 3 chiral purity detection chromatogram shows that the ee value of the target product is 100%.

[0032] Example 2

[0033] The method for preparing (-)-5-azaspiro[2.4]heptan-7-ol by using the method of the present invention comprises the following operation steps:

[0034] S1: Preparation of freeze-dried carbonyl reductase powder, which is the same as step S1 in Example 1;

[0035] S2: Add 5-azaspiro[2.4]heptan-7-one (5 g), freeze-dried carbonyl reductase powder (0.5 g), formate dehydrogenase (2 g), NADPH (5 mg), 0.05 mol / L HCOOH-HCOONa buffer solution (20 mL), and water (20 mL) into a 250 mL reaction flask in sequence, stir at 250 rpm, control the reaction pH at 6.5 - 8.0, react at 10 °C for 2 h, terminate the reaction when gas chromatography detects no residue of 5-azaspiro[2.4]heptan-7-one; filter the reaction solution with diatomaceous earth, extract the filtrate with dichloromethane six times, combine the organic phases, and concentrate under reduced pressure to dryness to obtain 4.0 g of the product (-)-5-azaspiro[2.4]heptan-7-ol, with a yield of 80% (calculated based on 5-azaspiro[2.4]heptan-7-one); the gas chromatographic purity detection spectrum shows that the purity of the target product is 89.05%; the chiral purity detection spectrum shows that the ee value of the target product is 100%.

[0036] Example 3

[0037] The method for preparing (-)-5-azaspiro[2.4]heptan-7-ol by using the method of the present invention comprises the following operation steps:

[0038] S1: Preparation of freeze-dried carbonyl reductase powder, which is the same as step S1 in Example 1;

[0039] S2: Add 5-azaspiro[2.4]heptan-7-one (5 g), freeze-dried carbonyl reductase powder (5 g), formate dehydrogenase (2 g), NADH (250 mg), 0.05 mol / L HCOOH-HCOONa buffer solution (600 mL), and isopropanol (20 mL) into a 250 mL reaction flask in sequence, stir at 250 rpm, control the reaction pH at 6.5 - 8.0, react at 30 °C for 1 h, terminate the reaction when gas chromatography detects no residue of 5-azaspiro[2.4]heptan-7-one; filter the reaction solution with diatomaceous earth, extract the filtrate with dichloromethane six times, combine the organic phases, and concentrate under reduced pressure to dryness to obtain 4.25 g of the product (-)-5-azaspiro[2.4]heptan-7-ol, with a yield of 85% (calculated based on 5-azaspiro[2.4]heptan-7-one); the gas chromatographic purity detection spectrum shows that the purity of the target product is 91.38%; the chiral purity detection spectrum shows that the ee value of the target product is 100%.

[0040] Example 4

[0041] The method for preparing (-)-5-azaspiro[2.4]heptan-7-ol by using the method of the present invention comprises the following operation steps:

[0042] S1: Preparation of freeze-dried carbonyl reductase powder, which is the same as step S1 in Example 1;

[0043] S2: Add 5-azaspiro[2.4]heptan-7-one (5 g), freeze-dried carbonyl reductase powder (0.5 g), formate dehydrogenase (2 g), NADPH (100 mg), 0.05 mol / L HCOOH-HCOONa buffer solution (150 mL), and DMSO (20 mL) into a 250 mL reaction flask in sequence, stir at 250 rpm, control the reaction pH at 6.5 - 8.0, react at 25 °C for 2 h, stop the reaction when no 5-azaspiro[2.4]heptan-7-one residue is detected by gas chromatography; filter the reaction solution with diatomaceous earth, extract the filtrate with dichloromethane six times, combine the organic phases, and concentrate under reduced pressure to dryness to obtain 4.15 g of the product (-)-5-azaspiro[2.4]heptan-7-ol, with a yield of 83% (calculated based on 5-azaspiro[2.4]heptan-7-one); the gas chromatographic purity detection spectrum shows that the purity of the target product is 92.56%; the chiral purity detection spectrum shows that the ee value of the target product is 100%.

[0044] Example 5

[0045] The method for preparing (-)-5-azaspiro[2.4]heptan-7-ol by using the method of the present invention comprises the following operation steps:

[0046] S1: Preparation of freeze-dried carbonyl reductase powder, which is the same as step S1 in Example 1;

[0047] S2: Add 5-azaspiro[2.4]heptan-7-one (5 g), freeze-dried carbonyl reductase powder (3 g), formate dehydrogenase (2 g), NADPH (200 mg), 0.05 mol / L HCOOH-HCOONa buffer solution (400 mL), and ethanol (20 mL) into a 250 mL reaction flask in sequence, stir at 250 rpm, control the reaction pH at 6.5 - 8.0, react at 50 °C for 1 h, stop the reaction when no 5-azaspiro[2.4]heptan-7-one residue is detected by gas chromatography; filter the reaction solution with diatomaceous earth, extract the filtrate with dichloromethane six times, combine the organic phases, and concentrate under reduced pressure to dryness to obtain 4.25 g of the product (-)-5-azaspiro[2.4]heptan-7-ol, with a yield of 85% (calculated based on 5-azaspiro[2.4]heptan-7-one); the gas chromatographic purity detection spectrum shows that the purity of the target product is 88.71%; the chiral purity detection spectrum shows that the ee value of the target product is 100%.

[0048] A method for asymmetric reduction of carbonyl reductase to prepare (-)-5-azaspiro[2,4]heptan-7-ol according to the present invention has only one-step reaction, simple operation, and has the advantages of cheap and easily available raw materials, high utilization rate, fast reaction time, mild reaction conditions, high chiral purity of products, and less environmental pollution.

[0049] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the technical principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for the asymmetric reduction of carbonyl reductase to prepare (-)-5-azaspiro[2,4]heptan-7-ol, which is characterized in that, It includes the following steps: S1: Preparation of carbonyl reductase lyophilized powder. Inoculate the strain ATCC 7330 onto Luria-Bertani liquid medium with an inoculation amount of 1%, the chloramphenicol content in the medium is 100 μg / mL, the culture temperature is 25°C, and culture at 220 rpm for 24 h. When the measured OD 600 concentration is 0.6, add isopropyl-β-D-thiogalactoside to make its concentration 0.4 mM, induce at 25°C for 16 h. After the induction ends, centrifuge to collect the thalli, wash the thalli, resuspend, ultrasonically disrupt and then lyophilize to obtain the carbonyl reductase lyophilized powder; S2: Mix the freeze-dried carbonyl reductase prepared in step S1, coenzyme, coenzyme recycling enzyme, buffer solution and solvent to prepare a reaction system solution. Add 5-azaspiro[2,4]heptan-7-one to the reaction system solution. Under the catalysis of carbonyl reductase, 5-azaspiro[2,4]heptan-7-one undergoes an asymmetric reduction reaction to generate (-)-5-azaspiro[2,4]heptan-7-ol. The yield of (-)-5-azaspiro[2,4]heptan-7-ol is above 85% and the ee value is 100%; In the said step S2, the mass concentration ratio of 5-azaspiro[2,4]heptan-7-one to carbonyl reductase in the reaction system solution is 1:0.1 - 1, where the mass concentration of 5-azaspiro[2,4]heptan-7-one is 3.5%; the coenzyme is NADPH or NADH, and the mass feeding ratio of the coenzyme to the freeze-dried carbonyl reductase is 1:20 - 100; the said coenzyme recycling enzyme is formate dehydrogenase, and the mass feeding ratio of formate dehydrogenase to the freeze-dried carbonyl reductase is 1:1.25; in the said step S2, the catalytic reaction temperature of carbonyl reductase is 10 - 50 °C, the reaction time is 1 - 2 h, and the pH of the reaction system solution is 6.5 - 8.

0.

2. The method for asymmetric reduction of carbonyl reductase to prepare (-)-5-azaspiro[2,4]heptan-7-ol according to claim 1, wherein, In the said step S2, the solvent is one or several of methanol, ethanol, n-propanol, butanol, isopropanol, dimethyl sulfoxide, water.

3. The method for asymmetric reduction of carbonyl reductase to prepare (-)-5-azaspiro[2.4]heptan-7-ol according to claim 2, wherein, In the said step S2, the buffer solution is formate buffer solution, and its solute molar concentration is 0.05 mol / L.

4. The method for asymmetric reduction of carbonyl reductase to prepare (-)-5-azaspiro[2.4]heptan-7-ol according to claim 3, characterized in that, In the said step S2, the formate buffer solution is an HCOOH-HCOONa aqueous solution, and the volume feeding ratio of the solvent to the formate buffer solution is 1:1 - 30.

5. The method for asymmetric reduction of carbonyl reductase to prepare (-)-5-azaspiro[2,4]heptan-7-ol according to claim 4, wherein In the said step S2, the solvent is water, and the volume feeding ratio of water to the formate buffer solution is 1:

6.

6. The method for asymmetric reduction of carbonyl reductase to prepare (-)-5-azaspiro[2.4]heptan-7-ol as claimed in claim 1, characterized in that, In the said step S2, the coenzyme is NADPH, and the mass feeding ratio of the coenzyme to the freeze-dried carbonyl reductase is 1:

50.

7. The method for asymmetric reduction of carbonyl reductase to prepare (-)-5-azaspiro[2,4]heptan-7-ol according to claim 1, wherein In the said step S2, the catalytic reaction temperature of carbonyl reductase is 20 - 30 °C.

Images