JPH0779711B2 - Method for producing indoline-2-carboxylic acid by optical resolution - Google Patents

Description

Detailed Description of the Invention

[0001]

This invention is of general formula I

[0002]

[Chemical 7]

(In the formula, R is a C ₂ -C ₈ aliphatic hydrocarbon group) and (R, S) -indoline-2-carboxylic acid ester is asymmetrically hydrolyzed to give a structural formula ( S)-
II

[0004]

[Chemical 8]

A microorganism belonging to the genus Pseudomonas having a stereoselective esterase activity for producing an optically active (S) -indoline-2-carboxylic acid represented by or belonging to the genus Pseudomonas or Aspergillus. by the action of enzymes derived from microorganisms, the racemic I optically active compounds indoline-2-carboxylic acid (S) - II and general formula (R) - I

[0006]

[Chemical 9]

Optically resolved to an optically active indoline-2-carboxylic acid ester represented by the formula (R is the same as above),
Separating the partial optically active body each, were collected and further optionally, collected (R) - I hydrolyzed to (S) a - antipode II (R) - II to generate, taken And a method for producing an indoline-2-carboxylic acid ester by optical resolution.

In the present invention, (S) -indoline-2-carboxylic acid ester and (R) -indoline-2-carboxylic acid ester can be optionally collected by selecting the stereoselective esterase to be used. These optically active indoline-2-carboxylic acid compounds can be used as raw materials for various medicines. For example, (S) -indoline-2-carboxylic acid is a hypotensive agent effective as an inhibitor of angiotensin I converting enzyme, (S) -1-[(S) -3-mercapto-2-oxopropyl] -indoline. -2-carboxylic acid

[0009]

[Chemical 10]

[Reference J. Med. Chem.,2
6, 394 (1983)].

[0011]

2. Description of the Related Art For the production of these optically active indoline-2-carboxylic acids, a method using an optical resolving agent as shown below is known.

[0012]

[Chemical 11]

[0013]

The optical resolution method using these resolving agents is complicated in operation, and is a simple method suitable for mass production, which is an optically active indoline-2-carboxylic acid or an optically active indoline-2-carboxylic acid. Development of the method of obtaining an acid ester was desired.

[0014]

[Means for Solving the Problems] The inventors of the present invention esterify the carboxylic acid moiety of indoline-2-carboxylic acid with various alcohols, and act microbial cells or enzymes on the ester to asymmetrically hydrolyze the ester. We have conducted repeated studies, thinking that an optically active substance can be obtained by decomposition. As a result, Pseudomonas or Aspergillus (As
Pergillus) reacted with an enzyme derived from a microorganism or microorganism belonging to the genus asymmetrically hydrolyze, (S) - indoline-2-carboxylic acid (S) - II and (R) - indoline-2-carboxylic After the acid ester R- I is formed, (S) -II and (R) can be obtained by separating and extracting with an organic solvent.
- I can respectively collected and further collected (R) - alkaline hydrolysis of I, or by performing the enzymatic degradation (R) - generate II,
They have found that they can be collected and completed the present invention. Hereinafter, the present invention will be described in more detail.

The general formula used as the substrate of the present invention

[0016]

[Chemical 12]

The indoline-2-carboxylic acid ester represented by the formula (1) is a compound in which R is a C ₂ -C ₈ aliphatic hydrocarbon group, and is preferably an ester consisting of ethyl, butyl, amyl and hexyl groups.

The indoline-2-carboxylic acid ester I is obtained as follows. That is, an alcohol serving as a solvent and a reaction reagent is added to (R, S) -indoline-2-carboxylic acid to give a concentration of indoline-2-carboxylic acid of 5 to 20.
% (W / v), the condensation reaction is carried out under strong acidity in the range of 50 ° C. to reflux temperature for 1 to 5 hours. Further, this reaction solution is adjusted to pH 7.0 and then concentrated under reduced pressure to remove excess alcohol. Add water or saturated sodium bicarbonate to the concentrate,
Extraction using a hydrophobic organic solvent such as ethyl acetate or hexane, and further concentration give (R, S) -indoline-2-carboxylic acid ester I of high purity.

Asymmetric hydrolysis of the racemate I (S)
-As a microorganism having a stereoselective esterase that produces II , for example, there is a microorganism belonging to the genus Pseudomonas or the genus Aspurgill, more specifically, Pseudomonas aeruginosa (Pseudomonas aeruginosa) IFO
3080, IFO 13130 and Aspergil Niger
lus niger) There is an IFO 4407. Culture of these microorganisms
Any nutrient medium can be used as long as it can grow microorganisms. For example, a nutrient medium composed of glucose, peptone, yeast extract, meat extract and the like is used. Culture temperature in the medium is 10-40
C., preferably 25-35.degree. C., pH 3-8, preferably 6-7, aerobically cultivated, usually 24-4.
You only have to do it for 8 hours.

In the asymmetric hydrolysis reaction of indoline-2-carboxylic acid ester by a microorganism, at the same time as the start of the culture, the substrate and the compound I are added to the medium and the hydrolysis is carried out in parallel with the culture. Compound I is added to the microbial cell-containing culture solution obtained in step 1), or after culturing, the cells obtained by centrifugation or filtration are suspended in a buffer solution to give compound I Although there is a method of contacting and hydrolyzing the cells, it is desirable to concentrate the cells by centrifugation or filtration to obtain a high-concentration cell suspension, and to add the compound I to this suspension after the reaction. Excellent from the standpoint of product recovery.

The solubility of the compound I in water is generally low, but stirring does not hinder the reaction. or,
For example, an organic solvent such as acetone or methanol, a surfactant or the like may be added to such an extent that the reaction is not hindered.

The reaction conditions are a temperature of 10 to 40 ° C., preferably 25 to 35 ° C., a pH of 5 to 8, preferably 6.5 to 7.5, and a reaction time of substrate and bacterial cells. Although it varies depending on the ratio of the amounts, it may be stopped when the unreacted ester and the carboxylic acid as the product reach a molar ratio of 50%. However, from the viewpoint of the reaction activity of the bacterial cells, it is usually desirable to determine the addition amount of the substrate so as to reach 50% in 24 to 72 hours.

As a method using an enzyme, after crushing the microbial cells, crude enzyme obtained by ammonium sulfate fractionation or acetone treatment, or further column chromatography operation is carried out,
The resulting purified enzyme can be used. Examples of commercially available enzymes include lipoprotein lipase (L.P.L.
Amano 3, origin: Pseudomonas aeruginosa, manufactured by Amano Pharmaceutical Co., Ltd., lipase AP-6 (origin: Aspergillus niger, manufactured by Amano Pharmaceutical Co., Ltd.) and the like can also be used.

In the asymmetric hydrolysis reaction, the racemate I of the substrate is suspended in the reaction solution at a concentration of 2 to 80% (w / v), and the enzyme is added in an appropriate amount, for example, the weight ratio of the enzyme to the substrate is 1: 5. To 1: 1000, and the reaction is carried out at a temperature of 10 to 40 ° C., preferably 25 to 35 ° C., and the production amount of carboxylic acid and the reduction amount of carboxylic acid ester are measured by high performance liquid chromatography. in (R) - I and (S) - they become 50 mol% of II may be stopped the reaction. The pH range for hydrolysis is 4-8.5.
However, since the pH of the reaction solution tends to be acidic as the hydrolysis reaction proceeds, it is desirable to maintain the pH at 6 to 7 with a neutralizing agent such as NaOH solution. Furthermore, the above-mentioned asymmetric hydrolysis reaction can be repeated by immobilizing microbial cells or enzymes, for example.

As a method for separating (S) -II and (R) -I in the reaction solution after asymmetric hydrolysis using a microorganism or enzyme, a hydrophobic organic solvent such as hexane, cyclohexane or toluene is used. And hydrophobic optically active indoline-
2-carboxylic acid ester (R) - extracting I only, hydrophilic optically active indoline-2-carboxylic acid (S) - can be readily separated and II.

Optically active indoline-2 obtained by separation
The -carboxylic acid ester can be obtained as an ester with high optical purity by concentrating as it is, and can be further converted into an optically active indoline-2-carboxylic acid as follows.
That is, the optically active indoline-2-carboxylic acid ester (R) -I is added at room temperature at a pH of 10 to 13.5 in the range of 2 to
If alkali hydrolysis is carried out for 5 hours, (R) -II is produced. Furthermore, (R) - an enzyme having the ability to hydrolyze the I, for example, by performing hydrolysis in hydrolytic conditions by the enzyme by the action of steapsin, (R) - can be obtained II.

The hydrolyzed liquid thus obtained has a pH of
Is adjusted to about 4 to 6, preferably about 5.0, extracted with an organic solvent such as methylene chloride and ethyl acetate, concentrated and then crystallized in an organic solvent such as acetone to obtain a high optical purity (R). -II is obtained. On the other hand, during extraction and separation, the optically active indoline-2-carboxylic acid remaining on the aqueous layer side is adjusted to pH 4 to 6, preferably around 5.0, as described above, and then subjected to the same extraction and purification operation. of enantiopure (S) - II can be easily obtained.

In the asymmetric hydrolysis reaction using microbial cells, the cells remain on the water layer side after extraction and separation with an organic solvent, but if the pH is subsequently lowered and the organic solvent extraction operation is performed,
It does not hinder the collection of the target optically active indoline-2-carboxylic acid. After removing the microbial cells by centrifugation or filtration, the indoline-2-carboxylic acid and the indoline-2-carboxylic acid ester can be separated and extracted based on the above method.

[0029]

EXAMPLES The present invention will now be described in detail with reference to examples, but the present invention is not limited to these examples. Reference Example 1 Production of (R, S) -indoline-2-carboxylic acid amyl (R, S) -Indoline-2-carboxylic acid (R, S)-
50 g of II was dissolved in 500 ml of amyl alcohol, 100 ml of concentrated hydrochloric acid was further added, and a condensation reaction was carried out at 95 to 100 ° C. for 3 hours. After the reaction, cool once and
The pH was adjusted to 7.0 with% caustic soda solution. Further, excess amyl alcohol and water were removed by vacuum concentration operation. In the concentrated liquid, the target substance (R, S) -indoline-
2-carboxylic acid amyl - (R, S) - Ia and inorganic salts are included. To this concentrated solution was added 1 liter of ethyl acetate, and twice with 200 ml of saturated sodium bicarbonate water (total 400 ml).
After washing, the ethyl acetate layer was concentrated (R, S) -I
60.8 g of a was obtained in a yield of 85%.

Reference Example 2 In 100 ml of 0.1 M phosphate buffer (pH 7.0), 1.0 g of steapsin and a substrate (R, S) -indoline-2-carboxylate amyl Ia were added.

[0031]

[Chemical 13]

10 g was added and the asymmetric hydrolysis reaction was carried out at 30 ° C. for 24 hours while stirring while adjusting the pH to 7.0 with a 1N NaOH solution. The reaction solution was extracted twice with 100 ml of hexane, and the hexane layer was dehydrated with anhydrous sodium sulfate and concentrated under reduced pressure. The specific optical rotation [α] ²⁵ _D +
5.8 ° (c = 1.0, ethanol) Syrup (S) with - Ia is 4.7 g - obtained ((R, S) 94% yield from Ia). ¹ HNMR (90 MHz) measured values were as follows. ¹ H NMR (CDCl ₃ ) δppm: 0.8 to 1.
8 (9H, m, CH ₃ CH ₂ CH ₂ CH ₂ −), 3.2 to 3.45 (2H,
_{d, - CH 2 O -)} , 4.0~4.4 (4H), 6.45~7.
05 (4H, m, Ar-) resulting (S) - the 4.7g of Ia added to 1NNaOH solution of 25 ml, at room temperature, carried out for approximately 3 hours hydrolysis, the reaction solution with 1N hydrochloric acid pH5.0 After adjusting to 2, the extraction operation was performed 4 times with 50 ml of ethyl acetate. Further, after dehydration treatment with anhydrous sodium sulfate, the mixture was concentrated under reduced pressure, and the dried solid matter was mixed with acetone-hexane (5 ml-
(1ml) reconstituted for specific rotation [α] ²⁵ _D + 32.4 °
(C = 1.0, dimethylformamide) (literature value, J. M.
ed. Chem., 26 , 394 (1983), [α] ²⁵ _D + 34.5 °
(C = 1.0, dimethylformamide) white powder with (S) - indoline-2-carboxylic acid (S) - II is 2.45g ((R, S) - 69% yield from Ia) to give Was given. ¹ H NMR (90 MHz) measured values were as follows. ¹ H NMR (DMSO-d6) δ ppm: 2.85-3.
45 (2H), 4.10 to 4.35 (1H), 6.40
~ 7.05 (4H, m, Aryl), 7.2 to 9.0 (2H, b
road). On the other hand, the aqueous layer was adjusted after hexane extraction to pH5.0 with 1N hydrochloric acid, ethyl acetate with 100ml increments repeated 4 times extraction, following (S) - The same procedure as in II,
(R) - II is 2.6g ((R, S) - yield from Ia 7
4%) was obtained. Specific rotation [α] ²⁵ _D −33.1 ° (c
= 1.0, dimethylformamide).

Examples 1 to 2 The same operation as in Reference Example 2 was carried out by changing the enzyme, and the results shown in Table 1 were obtained.

[0034]

[Table 1]

Example 3 A nutrient liquid medium having the following composition was prepared, dispensed into a 2-liter Sakaguchi flask by 400 ml, and sterilized at 120 ° C. for 15 minutes. [Media composition] Glucose 4%, yeast extract 0.3
%, Meat extract 0.3%, peptone 0.3%, diammonium phosphate 0.2%, monopotassium phosphate 0.1% (pH
6.8)

Separately, 10 ml of a seed culture of Pseudomonas aeruginosa IFO 3080 pre-cultured in a medium having the same composition was inoculated into the culture medium and shaken at 30 ° C. for 24 hours. A total of 5 cultures were performed to obtain a total of 2 liters of culture solution. The culture was centrifuged to collect bacterial cells. The cells were suspended in 200 ml of 0.1 M phosphate buffer (pH 7.0), and 2.0 g of substrate (R, S) -indoline-2-carboxylate amyl Ia was added. While stirring this in a 500 ml container while adjusting the pH to 7.0 with a 1N NaOH solution,
The reaction was carried out at 30 ° C for 18 hours. After the reaction, the supernatant obtained by centrifugation is extracted and separated four times with 200 ml of hexane each time,
Then, the same operation as in Reference Example 2 was performed, and the results shown in Table 2 were obtained.

Examples 4-5 Pseudomonas aeruginosa IFO 13130 of Example 4
Was cultured in the same manner as in Example 4, and the culture of the microorganism of the genus Aspergillus of Example 5 was 8.0% glucose, 1.0% polypeptone, 0.5% yeast extract, 0.2% diammonium phosphate. , Monopotassium phosphate 0.1% (pH
The same procedure as in Example 3 was performed except that the medium of 6.5) was used and the temperature was 28 ° C. After culturing, each strain was subjected to centrifugation for Pseudomonas aeruginosa and filtration for microorganisms in the Aspergillus layer, and the cells were collected and suspended in 0.1 M phosphate buffer pH 7.0. Asymmetric hydrolysis reaction by a microorganism, extraction and purification were carried out, and the results shown in Table 2 were obtained.

[0038]

[Table 2]

Example 6 Using a Pseudomonas aeruginosa IFO 3080, 2 liters of the culture solution obtained in the same manner as in Example 4 was centrifuged to collect the cells. The cells were suspended in 200 ml of 0.1M phosphate buffer (pH 7.0), disrupted with a Brown homogenizer while cooling with ice, and centrifuged to obtain a cell-free extract enzyme solution. To this enzyme solution, 10 g of substrate (R, S) -indoline-2-carboxylate amyl Ia was added, and 1N
While adjusting the pH to 7.0 with NaOH solution, under stirring,
Asymmetric hydrolysis was performed at 48 ° C. for 48 hours. Then, extraction and purification are carried out according to Example 3 to obtain a specific optical rotation [α] ²⁵ _D +22.4.
° (c = 1.0, dimethylformamide) with a (S) - II is 1.9g and a specific rotation of [α] ²⁵ _D -3.3 °
(R) -Ia with (c = 1.0, ethanol) 4.
2 g was obtained.

[0040]

INDUSTRIAL APPLICABILITY According to the present invention, (R, S) -indoline-2-carboxylic acid ester can be obtained by appropriately selecting and using a hydrolase esterase having stereoselectivity or a microorganism having the same hydrolyzing ability. To an optically active (R) form of the ester, or an optically active indoline-
The (R) form or (S) form of 2-carboxylic acid can be obtained.

Front Page Continuation (72) Inventor Takehisa Ohashi 3-9-14 Shinohara Auntanoyama-cho, Nada-ku, Kobe-shi, Hyogo (72) Inventor Kiyoshi Watanabe 5-41 Matsugaoka, Akashi-shi, Hyogo (72) Inventor Kenji Takahara 1-13-2 Miyakodai, Kamiso-cho, Kakogawa-shi, Hyogo

Claims (2)

[Claims] 1. The general formula I : (In the formula, R is a C ₂ -C ₈ aliphatic hydrocarbon group) and (R, S) -indoline-2-carboxylic acid ester is asymmetrically hydrolyzed to give a structural formula (S)- II [Chemical 2] Derived from a microorganism belonging to the genus Pseudomonas or a microorganism belonging to the genus Pseudomonas or Aspergillus having stereoselective esterase activity for producing optically active (S) -indoline-2-carboxylic acid By reacting an enzyme, the racemate I is converted to the optically active compound indoline-2-carboxylic acid (S) -II and the general formula (R) -I. (R is the same as above)
A method for producing indoline-2-carboxylic acid by optical resolution, which comprises optically resolving with a 2-carboxylic acid ester and separating and collecting each optically active substance. 2. The general formula I : (In the formula, R is a C ₂ -C ₈ aliphatic hydrocarbon group) and (R, S) -indoline-2-carboxylic acid ester is asymmetrically hydrolyzed to give a structural formula (S)- II [Chemical 5] Derived from a microorganism belonging to the genus Pseudomonas or a microorganism belonging to the genus Pseudomonas or Aspergillus having stereoselective esterase activity for producing optically active (S) -indoline-2-carboxylic acid By reacting an enzyme, the racemate I is converted into the optically active compound indoline-2-carboxylic acid (S) -II and the general formula (R) -I (R is the same as above)
2-carboxylic acid is optically divided into the ester, separating the optically active substance of each were taken, further (R) - I and hydrolyzed to the compound (S) - optically active indoline a antipode II -
2- carboxylic acid (R) - II to produce a process for producing an optically active indoline-2-carboxylic acid, and recovering.