JPH06141876A - Production of optically active n-benzyl-3-pyrrolidinol - Google Patents

Abstract

PURPOSE:To provide method for producing optically active N-benzyl-3- pyrrolidinol which is an important intermediate for optically active compounds useful as a medicine. CONSTITUTION:This method for producing optically active N-benzyl-3- pyrrolidinol is characterized by stereoselectively reducing N-benzyl-3- pyrrolidinone in the presence of an enzymic source having the activity of stereoselectively reducing the compound.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a process for producing optically active N-benzyl-3-pyrrolidinol, which is an important intermediate of optically active compounds useful as pharmaceuticals such as β-lactam antibiotics and dihydropyridine compounds.

[0002]

2. Description of the Related Art Conventionally, methods for producing optically active 3-pyrrolidinol derivatives are roughly classified into (1) a method of synthesizing a chiral compound as a starting material and (2) an asymmetric synthesis using a prochiral compound as a starting material. Alternatively, two methods of synthesizing an optically active substance by resolution are known.

Regarding (1), a method of synthesizing from hydroxyproline (JP-A-60-23328) and a method of synthesizing from D-malic acid [Heterocycles
s), 24 , 1331 (1986)], a method of synthesizing from glutamic acid [Synth. Comms (Synth. Comm.
un.), 16 , 1815 (1986)], a method of synthesizing from aspartic acid [Heterocycles, 24 , 1331 (198).
6)], a method of synthesizing from glutamine [Bulletin of the Chemical Society of Japan (Bul
l. Chem. Sco. Jpn.), 51 , 3296 (1978)] and the like.

Regarding (2), 4-hydroxy-2-
Method of synthesizing from pyrrolidone (JP-A-1-207266), method of synthesizing from aminobutanol derivative (JP-A-3-176462), method of synthesizing from sulfonic acid ester of 4-halo-3-hydroxybutanenitrile ( JP-A-3-176463) and the like are known.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide a novel process for producing optically active N-benzyl-3-pyrrolidinol.

[0006]

According to the present invention, N-benzyl-3-pyrrolidinone is stereoselectively reduced in the presence of an enzyme source having the activity of stereoselectively reducing the compound. And a method for producing optically active N-benzyl-3-pyrrolidinol. Less than,
The present invention will be described in detail. As the enzyme source used in the reduction reaction of the present invention, any microorganism cells, cultures or processed products thereof having the activity of stereoselectively reducing N-benzyl-3-pyrrolidinone may be used. it can.

Microorganisms having such activity include:
For example, genus Acetobacter, genus Corynebacterium, Gluconobacter
onobacter genus, Pseudomonas genus, Bacillus genus, Escherichia genus, Brevibacterium genus, Aspergillus
(Aspergillus) genus, Sierravia (Thielavia) genus, Saccharomycopsis (Saccharomycopsis) genus, Saccharomyces
(Saccharomyces) genus, Pachysolen genus, Rhodotorula genus, Candida genus, Mycobacterium genus (Mycobacterium) genus, Rhodococcus (Rh
genus odococcus), genus Actinoplanes,
The genus Amycolata, Kitasato
sporia genus, Citrobacter genus, Amycolatopsis genus, Vibrio genus, Fellomyces genus, Nocardiopsis (Noc
Microorganisms belonging to the genus Ardiopsis) and the like. Such microorganisms can generally be obtained from stock strains that are easy to obtain or purchase. It can also be separated from nature. It is also possible to mutate these microorganisms to obtain highly productive strains.

[0008] Examples of the treated product of bacterial cells obtained by culturing the above-mentioned microorganisms include dried bacterial cells, additives of surfactants or organic solvents, treated products of lytic enzymes, immobilized bacterial cells or enzyme extracted from bacterial cells. Goods are given. Examples of the processed product of the culture include a concentrated product, a dried product, a surfactant or an organic solvent additive, and a lytic enzyme-processed product. Furthermore, the enzyme may be purified from the cultured bacterial cell or culture and used.

Cultivation of the microorganism used in the present invention is generally carried out under aerobic conditions such as shaking culture or aeration-agitation deep culture. As the medium, either a synthetic medium or a natural medium can be used, so long as it contains a carbon source, a nitrogen source, an inorganic salt and a trace amount of organic nutrient source which can be assimilated by the strain used.

As the carbon source, carbohydrates such as glucose, maltose, starch hydrolyzate and molasses can be used. As the nitrogen source, various inorganic and organic ammonium salts such as ammonia, ammonium sulfate and ammonium chloride, or nitrogen-containing organic substances such as meat extract, yeast extract, malt extract, peptone, polypeptone, corn steep liquor and casein hydrolyzate. Etc. can be used.

As the inorganic salt, magnesium sulfate, ferrous sulfate, manganese sulfate, zinc chloride, potassium chloride, cobalt chloride and the like can be used. In addition, salts such as molybdenum, tungsten and strontium may be added as trace elements. Culture temperature is 20-37 ° C, culture pH is 6-9.
Incubate for 1 to 14 days.

The substrate of the present invention, N-benzyl-3-pyrrolidinone, can be synthesized according to the method described in JP-A-54-16466. The stereoselective reduction of N-benzyl-3-pyrrolidinone of the present invention is carried out by mixing N-benzyl-3-pyrrolidinone with the microbial cells, culture or treated product thereof in an aqueous medium and stirring or It is performed by shaking.

As the aqueous medium, water or a mixed solvent of water and ethanol, acetone, dioxane, tetrahydrofuran, dimethylformamide or the like is used. The substrate, N-benzyl-3-pyrrolidinone, is used in an amount of 0.1 to 50% by weight based on the reaction solution, and the treated product of microbial cells is used in an amount of 1 to 500% by weight based on the substrate. At this time,
In order to improve the dispersibility of N-benzyl-3-pyrrolidinone, which is a substrate, a surfactant such as nonion (Nippon Oil & Fats Co., Ltd.), Span (Kanto Chemical Co., Inc.), Triton (Hanai Chemical Co., Ltd.) may be added. it can. Furthermore, in order to keep the pH of the reaction solution constant, a buffer solution of an inorganic acid salt such as sodium phosphate or potassium phosphate or a buffer solution of an organic acid salt such as sodium acetate or sodium citrate can be used. If necessary, a base such as sodium hydroxide can be added.

The reaction temperature is 0 to 70 ° C., preferably 20 to
It is carried out under the conditions of 50 ° C. and pH 4-9. The reaction time varies depending on the substrate concentration used, the amount of microbial cells, the reaction temperature, etc., but it is usually completed in 1 to 100 hours. To isolate and purify the desired product from the reaction solution, after removing microbial cells,
It can be performed by solvent extraction, washing, concentration, silica gel column chromatography and the like.

N-benzyl-3 thus obtained
-Pyrrolidinol is (5R, 6S) -2-[(S)-
1- (acetimidoyl) pyrrolidin-3-ylthio]
-6-[(R) -1-hydroxyethyl] carbapene-
Β-lactam antibiotics such as 2-em-3-carboxylic acid (JP-A-60-19764) and (3S) -1-benzyl-3-pyrrolidinylmethyl (4S) -2,6- Dimethyl-4- (m-nitrophenyl) -1,4-dihydropyridine-3,5-dicarboxylate [Compound (I);
Journal of Medicinal Chemistry (J. Me
d. Chem.), 29 , 2504 (1986)] and the like, which are useful as synthetic intermediates for dihydropyridine compounds. For example, compound (I) is prepared from optically active N-benzyl-3-pyrrolidinol by Journal of Medicinal Chemistry.
, 29 , 2504 (1986).

The following examples illustrate aspects of the present invention.

[0017]

【Example】

Example 1 Peptone 2%, meat extract 0.7%, yeast extract 0.5%,
30 ml of medium consisting of 0.3% salt (pH 7.2)
In a Erlenmeyer flask with a capacity of 300 ml and after sterilization,
Each of the strains shown in the table was inoculated and shake-cultured at 28 ° C. for 24 hours. After completion of the culture, the culture was centrifuged (10,000 rpm,
After 5 minutes), the bacterial cells were collected and washed with physiological saline. The cells were suspended in 3 ml of 0.1 M phosphate buffer (pH 7) containing 0.5% Triton X100, 0.06 ml of ethanol containing 12 mg of N-benzyl-3-pyrrolidinone was added, and the suspension was added at 30 ° C. for 24 hours. Shake for hours. After the reaction was completed, the reaction solution was extracted with ethyl acetate, and the conversion rate and optical purity of N-benzyl-3-pyrrolidinol produced by high performance liquid chromatography (HPLC) were measured. The HPLC conditions are as follows.

HPLC column Chiralcel OD (manufactured by Daicel), elution solvent hexane: isopropanol:
Diethylamine = 95: 5: 0.1, flow rate 1 ml /
Min., Detection UV254 nm absorption results are shown in Table 1.

[0019]

[Table 1]

Example 2 Malt extract 2%, glucose 2%, peptone 0.1%
30 ml of a medium having a composition of (pH 6) was placed in a 300 ml Erlenmeyer flask, and after sterilization, the strains shown in Table 2 were inoculated and shake culture was performed at 28 ° C. for 4 days. After completion of the culture, the culture was centrifuged (10,000 rpm, 5 minutes) to collect the bacterial cells, which were washed with physiological saline. This bacterium is Triton X1
0.1M phosphate buffer (pH 7) 3m containing 0.5% of 00
12 mg of N-benzyl-3-pyrrolidinone
Add 0.06 ml ethanol containing
Shake for 4 hours. After completion of the reaction, the reaction solution was extracted with ethyl acetate, and the conversion rate and optical purity of N-benzyl-3-pyrrolidinol produced by HPLC were measured. In addition, HPL
The conditions of C are the same as in Example 1. The results are shown in Table 2.

[0021]

[Table 2]

Example 3 Peptone 2%, meat extract 0.7%, yeast extract 0.5%,
30 ml of medium consisting of 0.3% salt (pH 7.2)
Place in a 300 ml Erlenmeyer flask, sterilize, and
Bacteria aceti IFO 3288 was inoculated and shake culture was performed at 28 ° C. for 24 hours. After culturing, the culture is centrifuged (1
Then, the cells were collected and washed with physiological saline. This cell contains 0.1% Triton X100 in 0.1M
Suspended in 3 ml of phosphate buffer (pH 7), added 0.06 ml of ethanol containing 12 mg of N-benzyl-3-pyrrolidinone and 27 mg of glucose, and added thereto at 30 ° C. for 24 hours.
Shake for hours. After completion of the reaction, the reaction solution was extracted with ethyl acetate, and the conversion rate and optical purity of N-benzyl-3-pyrrolidinol produced by HPLC were measured.
The (R) isomer was obtained with 95% and an optical purity of 60% ee. The HPLC conditions are the same as in Example 1.

Example 4 1% glucose, 0.5% peptone, 0.3 yeast extract
%, Malt extract 0.3% (pH 6) in a medium of 30 ml was placed in a 300 ml Erlenmeyer flask, and after sterilization, each strain shown in Table 3 was inoculated and shake culture was carried out at 28 ° C. for 24 hours. It was After culturing, the culture is centrifuged (1
Then, the cells were collected and washed with physiological saline. This cell contains 0.1% Triton X100 in 0.1M
The suspension was suspended in 3 ml of a phosphate buffer (pH 7), 0.06 ml of ethanol containing 12 mg of N-benzyl-3-pyrrolidinone was added, and the mixture was shaken at 30 ° C. for 24 hours. After completion of the reaction, the reaction solution was extracted with ethyl acetate, and the conversion rate and optical purity of N-benzyl-3-pyrrolidinol produced by HPLC were measured. The HPLC conditions are the same as in Example 1. The results are shown in Table 3.

[0024]

[Table 3]

Example 5 30 ml of a medium having a composition of 30% diced potato and 2% glucose (pH 6) was placed in an Erlenmeyer flask having a capacity of 300 ml, sterilized, Aspergillus sydowi IFO 4284 was inoculated, and shake culture was performed at 28 ° C. for 8 days. I went. After culturing,
The culture was centrifuged (10,000 rpm, 5 minutes), the bacterial cells were collected and washed with physiological saline. The cells were suspended in 3 ml of 0.1 M phosphate buffer (pH 7) containing 0.5% Triton X100, 0.06 ml of ethanol containing 12 mg of N-benzyl-3-pyrrolidinone was added, and the suspension was added at 30 ° C. for 24 hours. Shake for hours. After completion of the reaction, the reaction solution was extracted with ethyl acetate,
When the conversion rate and optical purity of N-benzyl-3-pyrrolidinol produced by HPLC were measured, the conversion rate was> 95.
%, Optical purity> 95% ee to give the (R) isomer. The HPLC conditions are the same as in Example 1.

Example 6 30 ml of medium consisting of 1% glucose, 2% peptone, 0.7% meat extract, 0.5% yeast extract and 0.3% salt (pH 7.2) was added to a 300 ml Erlenmeyer flask. After sterilization, inoculate the strains shown in Table 4 to 28 ° C.
Shaking culture was carried out for 2 days. After completion of the culture, the culture was centrifuged (10,000 rpm, 5 minutes) to collect the bacterial cells, which were washed with physiological saline. The cells were suspended in 3 ml of 0.1 M phosphate buffer (pH 7) containing 0.5% Triton X100 to give N-.
0.06 m containing 12 mg of benzyl-3-pyrrolidinone
l of ethanol was added, and the mixture was shaken at 30 ° C. for 24 hours.
After completion of the reaction, the reaction solution was extracted with ethyl acetate, and the conversion rate and optical purity of N-benzyl-3-pyrrolidinol produced by HPLC were measured. The HPLC conditions are the same as in Example 1. The results are shown in Table 4.

[0027]

[Table 4]

[0028]

According to the present invention, optically active N-benzyl-3-pyrrolidinol, which is an important intermediate of optically active compounds useful as pharmaceuticals such as β-lactam antibiotics and dihydropyridine compounds, can be obtained in high yield. It can be manufactured.

Continuation of the front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location (C12P 17/10 C12R 1:01) (C12P 17/10 C12R 1:38) (C12P 17/10 C12R 1: 07) (C12P 17/10 C12R 1: 185) (C12P 17/10 C12R 1:13) (C12P 17/10 C12R 1:66) (C12P 17/10 C12R 1: 645) (C12P 17/10 C12R 1: 85) (C12P 17/10 C12R 1:72) (C12P 17/10 C12R 1:32) (C12P 17/10 C12R 1:63) (C12P 17/10 C12R 1: 365)

Claims (2)

[Claims] 1. An optically active N-benzyl-3, which is characterized in that N-benzyl-3-pyrrolidinone is stereoselectively reduced in the presence of an enzyme source having an activity of stereoselectively reducing the compound. -Method for producing pyrrolidinol. 2. The enzyme source is Acetobacter, Corynebacterium, Gluconobacter, Pseudomonas,
Bacillus, Escherichia, Brevibacterium,
Aspergillus, Sierravia, Saccharomycopsis, Saccharomyces, Patissolen, Rhodotorula, Candida, Mycobacterium, Rhodococcus, Actinoplanes, Amycolata, Kitasatosporia, Citrobacter, Amycolatopsis A microbial cell, a culture, or a processed product thereof, which belongs to the genus Genus, the genus Vibrio, the genus Ferromyces, or the genus Nocardiopsis and has the activity of stereoselectively reducing N-benzyl-3-pyrrolidinone. Manufacturing method.