JP2693536B2 - Process for producing (7R) -cyclopenta [d] pyrimidine derivative - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention has (7R) -4- (4-cyanoanilino) -6,7-dihydro-7-hydroxy-5H having antidepressant action and brain disorder improving action. A novel process for the production of cyclopenta [d] pyrimidine.

(Prior art) 4- (4-cyanoanilino) -6,7-dihydro-7-
Hydroxy-5H-cyclopenta [d] pyrimidine is known as a compound having an antidepressant action and a cerebral disorder improving action (JP-A-62-70 and JP-A-63-183532).

(Problems to be solved by the invention) 4- (4-cyanoanilino) -6,7-dihydro-7-
Hydroxy-5H-cyclopenta [d] pyrimidine can be obtained by the method described in JP-A-62-70. Then, the optically active substance is obtained by optical resolution according to a conventional method. However, the method of optical division is not well established.

The present inventors have asymmetrically hydroxylated 4- (4-cyanoanilino) -6,7-dihydro-5H-cyclopenta [d] pyrimidine using a microorganism to obtain (7R) -4- (4-cyanoanilino)-. The present invention has been completed by finding that 6,7-dihydro-7-hydroxy-5H-cyclopenta [d] pyrimidine can be obtained.

(Means for Solving the Problems) The present invention comprises hydroxylating 4- (4-cyanoanilino) -6,7-dihydro-5H-cyclopenta [d] pyrimidine using a microorganism belonging to the genus Caningamegra, Having (7R) -4- (4-cyanoanilino) -6,7-
This is a method for producing dihydro-7-hydroxy-5H-cyclopenta [d] pyrimidine.

Examples of the microorganism belonging to the genus Cunninghamella used in the present invention include Cunninghamella echinu
lata) IFO 4443 The same IFO 4446 The same IFO 4447 is suitable.

Microorganisms preferably used in the present invention can be obtained from a public bacterial preservation organization (IFO).

It should be noted that the properties of mold are not constant, they are natural,
It is well known that the population can easily be changed, and the strains usable in the present invention belong to the genus Kaningamela, 4-
(4-Cyananilino) -6,7-dihydro-5H-cyclopenta [d] pyrimidine to (7R) -4- (4-cyanoanilino) -6,7-dihydro-7-hydroxy-5H-cyclopenta [d] pyrimidine It includes all strains that can be converted.

In carrying out the method of the present invention, as a method of hydroxylating, a method of culturing a converting bacterium under a culture condition suitable for its growth, and (1) adding a starting compound to a medium and bringing them into contact (2) conversion A method of culturing and collecting bacteria and contacting the obtained converted bacterial cells with the starting compound, and (3) a method of contacting the cell-free extract prepared from the converted bacterial cells with the starting compound are used. .

Culture of the transformed bacteria is usually performed by culturing in a medium containing nutrients that can be used by the microorganism. As a nutrient source, a known nutrient used for culturing a general microorganism can be used.

For example, as a carbon source, glucose, sucrose, maltose, lactose, starch, glycerin, starch syrup, molasses, soybean oil and the like are used. Examples of the nitrogen source include soybean flour, wheat germ, meat flour, fishmeal, meat extract, peptone, corn steep liquor, dried yeast, and ammonium salts such as ammonium nitrate. In addition, if necessary, in addition to inorganic salts such as salt, potassium chloride, calcium carbonate, and phosphate, as well as additives and the like that assist the growth of bacteria and promote the production of the enzyme having the hydroxylation ability, etc. Can be used.

The culture is carried out under aerobic conditions, and the culture temperature is 20-28.
℃, preferably 24-27 ℃.

Next, the method (1) is carried out by adding the starting compound and culturing. The timing of the addition varies depending on the optimal culture conditions of the transformed bacteria used, especially the culture device, medium composition, culture temperature, etc. It is preferred that 1-5 days have elapsed. The amount of the raw material compound added is 0.01 to the medium.
-1.0%, preferably 0.01-0.1%.

The cultivation after the addition of the starting compound is carried out at the above-mentioned culturing temperature under aerobic conditions. The culture period is about 1-10 days after the addition of the raw material compound.

The method (2) is carried out by culturing the transformed bacterium in the presence of a small amount of the starting compound according to the above method (1) and culturing the transformed bacterium until the hydroxylation ability of the transformed bacterium is maximized.

That is, the hydroxylation ability varies depending on the type of medium, temperature, etc., but usually becomes maximum 2-3 days after the start of culture.
At this point, the culture is terminated. Harvesting involves centrifuging the culture,
It is carried out by applying a method such as filtration. It is preferable that the collected transformed cells are usually washed with a physiological saline solution, a buffer solution or the like before use. The thus obtained transformed bacterial cells are brought into contact with the starting compound, usually in an aqueous medium, for example, in a phosphate buffer of pH 5-9. The reaction by contact is usually carried out at 20-40 ° C, preferably at 25-35 ° C. The concentration of the starting compound is usually 0.01-
0.1%. The reaction time depends on the concentration of the raw material compound, the reaction temperature, etc., but is usually about 1-5 days.

The cell-free extract obtained by the method (3) is obtained by applying a physical or chemical technique to the transformed bacterial cells obtained by the above method, for example, by grinding, sonication, etc. Alternatively, it can be obtained as a cell lysate by treatment with an organic solvent, a surfactant, an enzyme, or the like.

The contact of the cell-free extract thus obtained with the starting compound is carried out in the same manner as the above-mentioned method of contacting the cells of the converting bacterium.

After the completion of the conversion reaction, the target compound can be collected, separated and purified from the product by a known method. For example, the obtained product is filtered, and the obtained filtrate is extracted with an organic solvent that is hardly miscible with water, such as ethyl acetate, and after the solvent is distilled off from the extract, the obtained crude target compound is obtained. Separation and purification can be achieved by column chromatography using silica gel, alumina or the like and eluting with a suitable eluent.

The optically active substance of the present invention exhibits an antidepressant action and a cerebral disorder improving action similarly to the racemic form.

The dosage form and dosage are used according to the disclosures of JP-A Nos. 62-70 and 63-183522.

(Examples) Next, the present invention will be described more specifically with reference to Examples, but the present invention is not limited thereto.

Example 1 Each of the microorganisms shown in the table was inoculated into a 100 ml Erlenmeyer flask containing 20 ml of the A medium having the following composition. 26 this
Culture was carried out at 200 ° C. and 200 rpm for 2 days with rotary shaking. Start of culture 2
Days later 4- (4-cyanoanilino) -6,7-dihydro-5
0.1 ml of 5% dimethylsulfoxide solution of H-cyclopenta [d] pyrimidine was added, and further 7 days, 26 ° C, 200r.
Cultured at pm. After completion of the culture, the culture solution was diluted by adding 4 times the volume of 80% methanol. Then, the diluted solution was subjected to high performance liquid chromatography (column; Yamamura Chemical Laboratory, YM
C-Pack A-312 ODS: eluent; acetonitrile-0.5% ammonium acetate (50:50): flow rate; 1.0 ml / min: detection wavelength; 3
The conversion rate from the starting compound to 4- (4-cyanoanilino) -6,7-dihydro-7-hydroxy-5H-cyclopenta [d] pyrimidine (racemic form) was determined. Furthermore, (7R) -4- (4-cyanoanilino) -6,7
-Dihydro-7-hydroxy-5H-cyclopenta [d]
The optical purity of pyrimidine ((R) form) was measured by high performance liquid chromatography (column; Daicel Chemical Industries, Ltd., CHIR
ALPAK WE: Eluent; acetonitrile-0.25 mM copper sulfate (90:
10): Flow rate; 0.5 ml / min: Detection wavelength; 315 nm), and the retention time was determined by separating the (S) form in 20 minutes and the (R) form in 26 minutes. The results are shown in the table.

A medium composition Glucose 10g Yeast extract 3g Malt extract 3g Polypeptone 5g Ion-exchanged water 1000ml pH unadjusted Example 2 2 containing 500 ml of the same A medium as used in Example 1
Kaningamera Echinurata IFO 4446 in a conical Erlenmeyer flask
Was cultivated and cultured at 210 rpm for 3 days at 210 rpm to obtain a seed culture solution. Next, defoamer (CB 442) on medium A
1% of the seed culture solution was added to a 600 tank containing 300 medium containing 0.01%, and the aeration rate was 0.5 vvm at 26 ° C and 100 to 110 r.p.
Cultured at m. Two days after the start of the culture, 5% dimethyl sulfoxide solution of 4- (4-cyanoanilino) -6,7-dihydro-5H-cyclopenta [d] pyrimidine was added 3 times,
The cells were further cultured for 5 days at 26 ° C. and 100 to 110 rpm. After completion of the culture, the culture solution was applied to a filter press, and the obtained filtrate
330 is extracted 3 times with 150% ethyl acetate and 500 ml of extract
Concentrated. The precipitated crystals were removed by filtration, and ethyl acetate was added to the obtained filtrate, followed by washing with water. Ethyl acetate was distilled off from the obtained mixture under reduced pressure, the precipitated crystals were washed with n-hexane-toluene, and the obtained crystals were subjected to column chromatography using silica gel [eluent: ethyl acetate-methanol ( 40: 1)] and purified (7R)
-4- (4-cyanoanilino) -6,7-dihydro-7-
Pure product of hydroxy-5H-cyclopenta [d] pyrimidine
2.91 g were obtained.

1) Properties: White crystals 2) Melting point: 213-216 ° C 3) Molecular weight: 252.3 6) mass spectrum: (m / z) 252 ( M +), 251 (M + -H), 223 (251-C 2 H 4), 102 7) 1 NMR Spectrum: [delta] ppm heavy dimethylsulfoxide, internal standard Nuclear magnetic resonance spectrum (270M
Hz) is as follows.

1.8 (1H, m), 2.4 (1H, m), 2.7 (1H, m) 2.9 (1H, m),
4.9 (1H, d), 5.5 (1H, d), 7.7 (1H, d), 8.0 (2H, d), 8.
6 (1H, s), 9.2 (1H, s) 8) Specific rotation: ▲ [α] ²⁵ _D ▼ -31.6 ° (C = 0.25, ethanol) 9) Optical purity: 100% ee [High performance liquid chromatography ( Column: Daicel Chemical Industries, Ltd., CHIRALPAK W
E; Eluate; acetonitrile-0.25 mM copper sulfate (90:10):
Flow rate; 0.5 ml / min; elution wavelength; 315 nm: retention time; 26 minutes]

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Takeyuki Kagasaki, 389-4 Ogura, Shimokawa, Izumi-cho, Iwaki-shi, Fukushima Sankyo Co., Ltd. (72) Ryuji Kobayashi, Ube, Yamaguchi Prefecture Usan Laboratory, Kosan Co., Ltd.

Claims (1)

(57) [Claims] 1. A method of hydroxylating 4- (4-cyanoanilino) -6,7-dihydro-5H-cyclopenta [d] pyrimidine using a microorganism belonging to the genus Kaningamela, Having (7R) -4- (4-cyanoanilino) -6,7-
A process for producing a compound having the above formula, which comprises converting to dihydro-7-hydroxy-5H-cyclopenta [d] pyrimidine and collecting the compound having the above formula from the system containing the conversion reaction product.