WO1998023769A1 - Process for the preparation of n-benzyl-3-pyrrolidinol - Google Patents

Abstract

A process for preparing optically active N-benzyl-3-pyrrolidinol efficiently by reducing N-benzyl-3-pyrrolidinone stereoselectively through an enzymatic reaction. The process is one which comprises reducing N-benzyl-3-pyrrolidinone stereoselectively through an enzymatic reaction conducted in a two-layer system composed of an aqueous layer containing an enzyme and an organic layer capable of forming a two-layer system together with the aqueous layer.

Description

 Specification

Method for producing N-benzyl-1 3-pyrrolidinol

 The present invention relates to a method for producing optically active N-benzyl-3-pyrrolidinol, which is useful as an intermediate for the synthesis of pharmaceuticals such as / -lactam antibiotics and dihydropyridine compounds. Background art

 Optically active N-benzyl 3-pyrrolidinol is useful as a synthetic intermediate for pharmaceuticals. Known methods for producing optically active N-benzyl-3-pyrrolidinol include a method of synthesizing from an optically active compound, a method of asymmetric synthesis starting from a prochiral compound, and a method of optical resolution. As such a method, Japanese Patent Application Laid-Open No. 6-141876 discloses that N-benzyl-3 is used in the presence of an enzyme having an activity of stereoselectively reducing N-benzyl-3-pyrrolidinone. A method for producing optically active N-benzyl-3-pinolidinol by stereoselectively reducing pyrrolidinone has been disclosed. However, this method was not practical for practical use because of its low substrate charge concentration and low conversion rate from substrate to product. Summary of the Invention

 In view of the above, an object of the present invention is to provide a more efficient method for producing optically active N-benzyl-3-pyrrolidinol by an enzymatic reaction for stereoselectively reducing N-benzyl-3-pyrrolidinone. is there.

The present invention relates to a method for producing optically active N-benzyl-3-pyrrolidinol by stereoselective reduction of N-benzyl-3-pyrrolidinone by an enzymatic reaction, wherein the enzymatic reaction is carried out using an enzyme-containing water. A method for producing optically active N-benzyl-3-pyrrolidinol in a two-layer system comprising a layer and an organic solvent layer forming a two-layer with the aqueous layer. Replacement form (Rule 26) The present inventors have investigated the stability of N-benzyl-3-pyrrolidinone, which is a substrate for the enzymatic reaction, in the enzymatic reaction conditions, that is, in water, and found that it is very unstable. This means that the substrate is decomposed even during the enzymatic reaction, and as a result, the conversion rate to the optically active N-benzyl-3-pyrrolidinol decreases. In addition, the same investigation was conducted on the product N-benzyl-3-pyrrolidinol. The results showed that the enzyme was stable under the conditions of the enzyme reaction, that is, even in water. N-benzyl-3-pyrrolidinone was found to be stable in organic solvents, and in a two-layer system in which an aqueous layer and an organic solvent layer coexist, as a result of intensive studies on the stabilization method for It was found to be stable. This phenomenon is due to the fact that N-benzyl-3-pyrrolidinone is mostly dissolved in the organic solvent layer in a two-layer system consisting of an aqueous layer and an organic solvent layer. It is explained that the chances of contact with people are reduced, and as a result they can exist more stably.

 Similarly, N-benzyl-3-pyrrolidinol, a product of the enzymatic reaction, is mostly present in the organic solvent layer in the two-layer system consisting of the aqueous layer and the organic solvent layer. On the other hand, enzymes and various components required for the expression of enzyme activity, such as coenzymes and energy sources, are generally water-soluble compounds, and thus can be expected to be mostly present in the aqueous layer. Therefore, since the chance of contact with the substrate or product is reduced, inhibition of the enzyme activity by the substrate or product generally caused by the enzymatic reaction can be expected, so that a low substrate concentration and a decrease in the conversion rate of the enzymatic reaction can be expected. .

 Furthermore, the present inventors provide a process for producing optically active N-benzyl-3-pyrrolidinol using an enzyme that stereoselectively reduces N-benzyl-3-pyrrolidinone. Layer and an organic solvent layer that forms two layers with this aqueous layer, the resulting N-benzyl-13-pyrrolidinol can be compared with the case where the reaction is performed only in water containing the enzyme. It has also been found that the optical purity of the product is improved. Detailed Disclosure of the Invention

Hereinafter, the present invention will be described in detail. Replacement form (Rule 26) In the present invention, N-benzyl-3-pyrrolidinone as a substrate is stereoselectively reduced by an enzymatic reaction.

 The above N-benzyl-3-pyrrolidinone can be synthesized by the method disclosed in Japanese Patent Application Laid-Open No. 54-166666. That is, a 3-ethylalanine derivative obtained by Michael addition of benzylamine and ethyl acrylate is reacted with ethyl ethyl acetate in the presence of a base. The obtained compound is cyclized in the presence of sodium metal to obtain N-benzyl-4-carboetkin-13-pyrrolidone. This is decarboxylated with hydrochloric acid to give N-benzyl-1-pyrrolidinone.

 In the present invention, the enzymatic reaction is carried out in a two-layer system comprising an aqueous layer containing the enzyme and an organic solvent layer forming a two-layer with the aqueous layer.

 The enzyme is not particularly limited as long as it has an ability to stereoselectively reduce the N-benzyl-3-pyrrolidinone. For example, the enzyme is exemplified in Japanese Patent Application Laid-Open No. 6-141876. Enzymes and the like. In addition, microbial cells, cultures, or processed products thereof having the ability to stereoselectively reduce N-benzyl-3-pyrrolidinone can also be used.

The microorganism is not particularly limited. For example, the genus Depodascus (Dep0 dascus), the genus Debaryomyces, the genus Cryptoococcus, the genus Pichia, the genus Richidosporium (Rh genus odsporidi um, genus Trichosporon, genus Komagataera (K 0 maga t_ ae 11a), genus Og ataea, genus Zyg'o sacchar omy es, genus E scherichia), Micrococcus (W), Genus Pseud omo nas, C andida, C. saccharomycopsis, Pasizolene (P achys) 0 1 en) Microorganisms and the like belonging to the genus. Such microorganisms can generally be obtained from a stock that is easily available or purchased. It can also be separated from nature. In addition, it is necessary to obtain mutations in these microorganisms to obtain strains having more advantageous properties for this reaction. Replacement paper (Rule 26) Can also. Moreover, those derived from these microorganisms by recombinant DNA, genetic engineering such as cell fusion, or biotechnological techniques may be used.

 The treated product of the above cells is not particularly limited, and examples thereof include a dried product of the cells, a surfactant or an organic solvent-treated product, a lysed enzyme-treated product, an immobilized cell or an enzyme extract extracted from the cells, and the like. Can be mentioned.

 The processed product of the culture is not particularly limited, and examples thereof include a concentrate, a dried product, a processed product of a surfactant or an organic solvent, a processed product of a lytic enzyme, and the like. Further, the enzyme may be purified from the cultured cells or culture and used.

 As the organic solvent constituting the organic solvent layer, an aqueous layer and a two-layer are formed, the N-benzyl-3-pyrrolidinone and the product N-benzyl-3-pyrrolidinol are dissolved, and the activity of the enzyme is reduced. There is no particular limitation as long as it is not allowed to be produced. Examples thereof include esters such as acetate and butyrate; alcohols such as 1-butanol and 1-octanol; aromatics such as benzene and toluene;

Ethers such as diisopropyl ether, diisopropyl ether, etc .; hydrogenated hydrocarbons such as chloroform and methylene chloride; aliphatic hydrocarbons such as n-hexane and n-decane; ketones such as methyl ethyl ketone and methyl isobutyl ketone And the like. These organic solvents are appropriately selected and used in consideration of the partition ratio and stability depending on reaction conditions such as temperature and pH.

 The ratio between the aqueous layer and the organic solvent layer in the two-layer system is not particularly limited, but is preferably in the range of 95/5 to 5Z95 on a weight basis.

 The stereoselective reduction of N-benzyl-3-pyrrolidinone by the enzymatic reaction is specifically performed by mixing an aqueous medium containing the enzyme with the organic solvent and stirring or shaking the mixture. Can be.

The enzyme reaction requires reduced nicotinamide * adenine dinucleotide (NADH) and reduced nicotinamide / adenine dinucleotide phosphate (NADPH) as coenzymes in addition to the above enzymes. It is necessary to add these or add a reaction system that produces ADH, NADPH, etc. to the reaction system. For example, a reaction in which formate dehydrogenase produces NADH from NAD when producing carbon dioxide and water from formic acid, and a method in which glucose dehydrogenase replaces glucose with glucono (Rule 26) When producing lactones, a reaction for producing NADH from NAD or NADPH from NADP can be used. In addition, a coenzyme generation system that the microorganisms originally have in their own cells can be used as it is.

 The above enzyme reaction is preferably performed at a reaction temperature of 0 to 70 ° C and a pH of 4 to 9. More preferably, the temperature is 20 to 50 ° C. The time for the above enzyme reaction varies depending on the substrate concentration, the amount of the enzyme, the amount of the capture enzyme used, the reaction temperature and the like, but is usually from 1 to 100 hours. Further, it is preferable that the enzymatic reaction is carried out with stirring to such an extent that the organic solvent layer and the aqueous layer are mixed. It is determined as appropriate depending on the ratio, progress of the reaction, and the like.

 After completion of the enzyme reaction, the organic solvent layer is collected, dehydrated, and then the organic solvent is distilled off to obtain the desired optically active N-benzyl 3-pyrrolidinol. Furthermore, high-purity optically active N-benzyl-3-pyrrolidinol can be obtained by purification by silica gel chromatography or distillation. BEST MODE FOR CARRYING OUT THE INVENTION

 Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to only these Examples.

Example 1

10 mg of N-benzyl 3-pyrrolidinone was weighed into a test tube with a stopper, and 100 mM phosphate buffer (pH 6.5) 0.5 ml and the organic solvent shown in Table 1 were added. 5 ml was added and the mixture was stirred well. Three of these were prepared, and one of them was added 4 ml of ethyl acetate and sodium bicarbonate immediately after stirring until the aqueous layer became saturated, and then stirred well. A part of this organic layer was subjected to gas chromatography to analyze the amount of N-benzyl-13-pyrrolidinone. The remaining two tubes were shaken at 30 ° C. for 18 hours and 42 hours, respectively, and then N-benzyl-3-pyrrolidinone was similarly quantified. Table 1 shows the residual ratio of N-benzyl-3-pyrrolidinone after 18 hours and 42 hours when the concentration of N-benzyl-3-pyrrolidinone at 0 hour was 100%. Gas Chromatography-Measurement conditions: Column; Uniport B, 10% PEG—20 M, 4.0 mm ID x 1.0 m, Column temperature; C, Carrier gas replacement paper (Rule 26) ; Nitrogen, detection; FID

table 1

Example 2

 A liquid medium having the following composition was prepared, dispensed into large test tubes in 5 ml increments, and steam sterilized at 20 ° C for 20 minutes.

Medium composition:

Glucose 4%

Yeast extract 0 3%

H ₂ P 0 0 1%

(NH ₄ ) HP 0 0 6 5% Replacement paper (Rule 26) N a C 1 0.1%

M g S 0 ₄・ 7 H 2 0 0.8%

Z n S 0 ₄ 7H ₂ 0 0.06%

F e S 0 ₄ 7H ₂ 0 0 .0 9%

C u S 0 ₄・ 5 H ₂ 0 0 .0 0 5%

Mn S〇 ₄ '4〜6 H ₂ 〇 0.0 1%

Tap water

pH 7.0

 One loopful of the microorganisms shown in Table 2 was inoculated into these liquid media and cultured with shaking at 30 ° C. for 24 to 72 hours. Next, each culture was centrifuged to collect the cells, washed with water, and each cell was suspended in 1 ml of 100 mM phosphate buffer (pH 6.5). Used as an ingredient.

Reaction liquid composition:

 (1) Above cell suspension 0 m 1

 (2) Glucose 4 mg

 (3) Nicotinamide 'adenine dinucleotide phosphate 275 mg (4) Glucose dehydrogenase (manufactured by Amano Pharmaceutical Co., Ltd.) 84 units (5) N-benzyl-1-pyrrolidinone 1 mg

 (6) 0.5 ml of butyl acetate The above (1) to (6) were dispensed into a test tube, mixed, and reacted at 30 ° C. for 20 hours with shaking. After the reaction, 3.5 ml of ethyl acetate was added to each reaction solution, mixed well, centrifuged, and the amount of the product in the organic layer was quantified by the gas chromatography method described in Example 1. The optical purity of the product was measured by HPLC.

 HP LC analysis conditions: Column; Chiralcel OB (manufactured by Daicel Chemical Industries), eluent: n-hexane / isopropanol Z getylamine = 99/1 Z 0.1, detection: 255 nm, flow rate 1 m 1 / min, elution time; (R) form 6.1 min, (S) form 7.9 min

 Table 2 summarizes the conversion to the product and the optical purity of the product.

Replacement form (Rule 26) Table 2

Comparative Example 1

 A microbial cell suspension was prepared in the same manner as in Example 2, and the reaction was carried out without adding butyl acetate. Table 3 summarizes the results.

Table 3

Example 3

 A liquid medium having the following composition was prepared, dispensed into a large test tube in an amount of 10 ml, and sterilized with steam at 120 ° C for 20 minutes.

Medium composition:

Meat extract 1.0%

Pupton 1.0%

Yeast extract 0.5%

 N a C 1 0.3%

Tap water

pH 7.0 Replacement sheet (Rule 26) One platinum loop was inoculated with Escherichiaco coli (Escherichiaco 1 i) IFO 127 34 into this liquid medium, and cultured with shaking at 30 ° C for 24 hours. Next, the culture was centrifuged to collect the cells, washed with water, and each cell was suspended in 2 ml of 10 OmM phosphate buffer (pH 6.5). Used as an ingredient. After the reaction for 20 hours, the conversion to the product and the optical purity of the product were measured in the same manner as in Example 2. As a result, the conversion was 20% and the optical purity was (S) 59% ee. Comparative Example 2

 A cell suspension of Escherichiaco 1 I FO 127 7 34 was prepared in the same manner as in Example 3, and the reaction was carried out without adding butyl acetate to the reaction. The optical purity was 7% and the optical purity was (S) 58% ee. Example 4

 The same operations as in Example 2 were performed on the microorganisms shown in Table 4, and the results are summarized in Table 4.

Replacement form (Rule 26) Four

Replacement form (Rule 26) Example 5

 The same operation as in Example 3 was performed on the microorganisms shown in Table 5, and the results are summarized in Table 5.

Table 5

Example 6

A liquid medium having the composition shown in Example 3 was prepared, and 25 tubes of 100 ml were poured into a 500 ml volume flask, and sterilized with steam at 120 ° C for 20 minutes. Was performed. Each of them was aseptically inoculated with 2 ml of a culture solution of Pseudomonas diminut-a IF 0 126 997 cultured in the same manner as in Example 3. Shaking culture was performed at 30 ° C for 24 hours. Cells were collected from the resulting culture by centrifugation and suspended in 500 ml of 10 OmM phosphate buffer (pH 6.5). 5 g of N-benzyl-1-pyrrolidinone and 10 g of glucose, reduced nicotinamide dodenine dinucleotide phosphate (produced by Kojin) 275 mg, glucose dehydrogenase (manufactured by Amano Pharmaceutical) 1 420 units and 500 ml of butyl acetate were added, and the mixture was stirred at 30 ° C. for 24 hours to react. The pH of the reaction solution was maintained at 6.5 with a 6N aqueous solution of sodium hydroxide. After the reaction, 2.5 L of ethyl acetate was added to the reaction solution for extraction, and the aqueous layer was further extracted with 1 L of ethyl acetate. The organic layers were combined, dehydrated with sodium sulfate anhydride, and the solvent was distilled off under reduced pressure. The residue was distilled to obtain 1 g of N-benzyl 3-pyrrolidinol. Yield 20%, optical purity 95% ee (R) form, boiling replacement paper (Rule 26) P

1 2 points 13 2 to 13 7 ° C / 3 mmHg, optical rotation [a] D ² . 3.73 ° (CH ₃ 〇H, C = 5) o 'H-NMR 6 (CDC 13): 1.63-1.76 (1H, m), 2.09-9. 2 1 (1 H, m), 2.26-2.37 (1 H, m), 2.5 1-2.64 (2 H, m), 2.75-2.85 (1H, m), 3.38 (1H, brs), 3.61 (2H, s), 4.24-4.33 (1H, m), 7.1

9-1 7.37 (5H, m). Example Ί

 A liquid medium having the composition shown in Example 2 was prepared, and 50 ml of a 500 ml dispensed volume was placed in a 500 ml volumetric flask, and steam sterilized at 120 ° C for 20 minutes. Natsuta. Each of them was aseptically inoculated with 1 ml of a culture solution of Pichiame's membrane fashion (Pichiamembranaefaciens) IFO 0182, and cultured at 30 ° C for 24 hours. The cells were cultured with shaking for hours. The cells were collected from the obtained culture solution by centrifugation, and suspended in 100 ml of 100 mM phosphate buffer (pH 6.5). Add 5 g of N-benzyl-3-pyrrolidinone and glucose

Add 10 g, reduced nicotinamide dodenine dinucleotide phosphate (produced by Kojin Co., Ltd.) 275 mg, glucose dehydrogenase (manufactured by Amano Pharmaceutical Co., Ltd.) 144 units, and add butyl acetate 500 ml The mixture was stirred at 30 ° C. for 48 hours to be reacted. The pH of the reaction solution was kept at 6.5 with a 6N aqueous sodium hydroxide solution. After the reaction, 2.5 L of butyl acetate was added to the reaction solution for extraction, and the aqueous layer was further extracted with 1 L of ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / methanol = 2/1).

(S) 2.5 g of 1 N-benzyl-3-pyrrolidinol was obtained. Yield 49%, optical purity 96% ee, boiling point 132-137 ° CZ 3 mmHg, optical rotation [a] D ² ° —3.73. (CH ₃ 〇H, C = 5). Ή-NMR 5 (CD C 1 3): 1. 6 3-

1.76 (1H, m), 2.09-2.21 (1H, m), 2.26-2.37

(1 H, m), 2.5 1-2.64 (2 H, m), 2.75—2.85 (1 H, m), 3.38 (1 H, brs), 3.6 1 (2 H, s), 4.2 4—4.3 3

(1H, m), 7.19-7.37 (5H, m). Replacement form (Rule 26) Example 8

 A liquid culture medium having the composition shown in Example 2 was prepared, and 50 ml of 500 ml dispensed into E-Saka Lofrasco was prepared, and sterilized by steam at 120 ° C for 20 minutes. Was performed. Each of them was aseptically inoculated with a culture solution lm1 of Trichosporon fermentans ATC C10675, which was cultured in the same manner as in Example 2, and incubated at 30 ° C for 24 hours. The cells were cultured with shaking for an hour. The cells were collected from the resulting culture by centrifugation, and suspended in 100 mM phosphate buffer (pH 6.5) (500 ml). The cells were disrupted with a brown cell disrupter under ice cooling, and the supernatant obtained by centrifugation was used as a cell-free extract and used as the following reaction solution components.

Reaction liquid composition:

 (1) Above cell-free extract 0 m 1

 (2) Glucose 4 mg

 (3) Nicotinamide adenine dinucleotide phosphate 26 mg

 (4) Glucose dehydrogenase (manufactured by Amano Pharmaceutical Co., Ltd.) 84 uunits (5) 1 mg of N-benzyl-1-3-pyrrolidinone

 (6) Butyl acetate 0.5 ml

 The above (1) to (6) were dispensed into a test tube, mixed, and reacted at 30 ° C. for 20 hours with shaking. After the reaction, the conversion to the product and the optical purity of the product were measured in the same manner as in Example 1. As a result, the conversion was 4% and the optical purity was (S) 9.9% ee. Industrial applicability

 Since the method for producing N-benzyl-3-pyrrolidinol of the present invention has the above-mentioned constitution, it is possible to efficiently produce optically active N-benzyl-3-pyrrolidinol on an industrial scale. . The optically active N-benzyl-3-pyrrolidinol obtained by the present invention has a high optical purity and is an important intermediate of a compound useful as a pharmaceutical such as a 3-lactam antibiotic ゃ dihydropyridine compound. is there. Replacement form (Rule 26)

Claims

The scope of the claims 1. A method for producing optically active N-benzyl-3-pyrrolidinol by stereoselectively reducing N-benzyl-3-pyrrolidinone by an enzymatic reaction, wherein the enzymatic reaction is carried out using an aqueous layer containing an enzyme, And in a two-layer system comprising the aqueous layer and an organic solvent layer forming a two-layer. A process for producing optically active N-benzyl-3-pyrrolidinol. 2. The optically active N according to claim 1, wherein the organic solvent layer is composed of esters, alcohols, aromatics, ethers, ketones, aliphatic hydrocarbons, or haematogenic hydrocarbons. —Benzyl—3-Pyrrolidinol production method. Replacement form (Rule 26)