JPH0656774A - Optically active @(3754/24)+)-4,4,4-trifluoro-3-@(3754/24)indole-3-) butyric acid and its production - Google Patents

Abstract

PURPOSE:To provide this novel compound having a rooting-stimulating action and a root growth-stimulating action and useful for plant growth regulators, etc. CONSTITUTION:The optically active (+)-4,4,4-trifluoro-3-(indole-3-) butyric acid of the formula I (* is asymmetric carbon). The compound of formula I is obtained by reacting a compound of formula II with ethyl alcohol and subsequently treating the produced ester with an enzyme having an esterase activity.

Description

Detailed Description of the Invention

[0001]

The present invention relates to an optically active (+)-4,
It relates to 4,4-trifluoro-3- (indole-3-) butyric acid and a method for producing the same. More specifically, the novel optically active (+)-4,4,4-trifluoro-3- (indole-3-) butyric acid and the substance are converted into racemic 4,
It relates to a method for producing 4,4-trifluoro-3- (indole-3-) butyric acid by enzymatic stereoselective hydrolysis.

Optically active (+)-4,4,4-trifluoro-3- (indole-3-) butyric acid (hereinafter referred to as optically active (+)-TFIBA) is a compound of fluorine-containing β-indole butyric acid. It has a rooting action for plants and an action for promoting root elongation.

Thus, the optically active (+)-TFIBA
Is an important substance having an action as a plant hormone, and is widely applicable to agriculture such as increased crop yield and fruit tree cultivation and horticulture.

[0004]

2. Description of the Related Art Indole-3-acetic acid (IA) is used as a so-called auxin which is a substance that promotes plant growth.
A) and its derivatives (indole-3-methyl acetate,
Indole-3-acetamide, etc.) has been known for a long time.

Recently, compounds of fluorine-containing β-indole butyric acids have been developed, and those compounds, namely, 4,
4,4-trifluoro-3- (indole-3-) butyric acid (hereinafter referred to as "TFIBA"), 4,4,4-trifluoro-2-hydroxy-3- (indole-3-) butyric acid (hereinafter referred to as "TFIHBA") , And 4,4,4-trifluoro-3- (indole-3-) butyronitrile (hereinafter referred to as "TFIBN") are known to have strong plant root elongation promoting action [phytochemistry]. Regulation Society, 1990 Conference, Research Presentations, page 31, published by the Ministry of Agriculture, Forestry and Fisheries].

[0006]

The conventional plant regulator IAA having auxin activity and its derivative have insufficient root-elongating action of plants and can be applied to agricultural fields such as increase of crop yield. There wasn't. On the other hand, recently developed compounds of fluorine-containing β-indole butyric acid, that is, TFIBA, TFIHBA and TFIBN are
Compared with AA and its derivatives, it has a considerably stronger plant root elongation action. However, a substance having a stronger plant root elongation action has been desired for practical use such as increase in crop yield.

[0007]

[Means for Solving the Problems] Therefore, as a result of intensive investigations by the present inventors for a more potent plant root promoting and root elongation promoting substance, the inventors have focused on optically active substances and Attention was paid to TFIBA from various known compounds. With respect to TFIBA, an attempt was made to newly develop a method for selectively obtaining an optically active substance in a high yield and confirming its action. As a result, we succeeded in selectively obtaining optically active (+)-TFIBA selectively and in high yield.

That is, first, racemic TFIBA and ethyl alcohol were allowed to act to synthesize ethyl ester of racemic TFIBA, and then an enzyme having esterase activity was allowed to act on the ester to asymmetrically hydrolyze the ester. The present invention has been completed by knowing that optically active (+)-TFIBA can be selectively obtained, and that the obtained optically active substance has a strong rooting promoting action and root elongation promoting action. .

In obtaining the optically active (+)-TFIBA of the present invention by asymmetric hydrolysis with an enzyme, the racemic TFIBA as a starting material is synthesized as follows.

First, trifluoroacetaldehyde ethyl hemiacetal (trifluoroacetald)
Indium-trifluoroethanol (Indo-Ethyl Hemiacetal, hereinafter referred to as TFAE) is subjected to a coupling reaction with an indole.
letrifluoroethanol) (Y. Make, H. Kimoto, S. Fuji).
i, M.I. Senga and L.M. A. Cohen,
J. Fluorine Chem. , 3 (9) 47
-59 (1988)), and then indoletrifluoroethanol was coupled with sodium salt of diethyl malonate in an organic solvent and further hydrolyzed with alkali to obtain racemic TFIBA. Record of Research Presentations, page 31, published by the Ministry of Agriculture, Forestry and Fisheries]

To obtain optically active (+)-TFIBA from racemic TFIBA, asymmetric hydrolysis with an enzyme is carried out as follows. That is, racemic TFIBA is reacted with an alcohol such as ethyl alcohol to synthesize an ester such as ethyl ester of racemic TFIBA (TFEE), and then an enzyme having esterase activity is allowed to act to selectively hydrolyze the ester. Then, optically active (+)-TFIBA is obtained.

From the above TFEE, the optically active (+)-T
The chemical reaction until FIBA is obtained is shown in Chemical Formula 4 below.

[0013]

[Chemical 4]

(Note that * represents an asymmetric carbon.)

As the enzyme having an esterase activity used in the present invention, lipase and / or protease is preferably used. As the lipase, lipase produced by the genus Pseudomonus, for example, lipase PS (trade name: Amano Pharmaceutical Co., Ltd.) ), Lipase AK (trade name: manufactured by Amano Pharmaceutical Co., Ltd.) and lipase produced by the genus Candida (Candida), for example, lipase AY (trade name: manufactured by Amano Pharmaceutical Co., Ltd.) is more preferably used.
A protease produced by the genus Aspergillus, for example, Protease M (trade name: manufactured by Amano Pharmaceutical) is more preferably used. The enzyme used may be a crude product or a purified product. In addition, bacterial cells that produce these enzymes can also be used.

The asymmetric hydrolysis reaction using an enzyme having esterase activity is advantageously carried out in the presence of a suitable buffer solution and / or organic solvent. As the buffer solution in this case, a buffer solution suitable for the working pH of the enzyme used is used, and for example, a phosphate buffer solution, an acetate buffer solution, a glycine buffer solution and the like are used.

As the organic solvent, a commonly used organic solvent can be used as long as it does not inhibit the enzyme action, and particularly n-hexane, t-butyl alcohol, acetone, DMSO (dimethyl sulfoxide), DMF ( Dimethylformamide) and the like can be preferably used.
The concentration of the organic solvent used in the enzyme reaction in the coexistence with the buffer solution is 0.5 to 50%, preferably 5 to 30% with respect to the buffer solution.

The conditions for the enzymatic reaction include a reaction temperature of 0 to
It is 70 ° C., preferably 4 to 60 ° C. The reaction time is 1 to 500 hours, preferably 20 to 250 hours.

The optically active (+)-TFI thus obtained
BA has a strong root elongation effect, and when it is used as a plant growth regulator, it can be used as it is, or it can be used as a pesticide to promote or stabilize its effect. Mixed with commonly used auxiliaries,
It is used in the form of a preparation such as a liquid preparation, powder preparation, granule preparation, wettable powder, flowable preparation or emulsion.

In the actual use, these preparations may be used directly as they are, or may be diluted with water to a predetermined concentration and used.

The compound of formula (I) is usually 1 × 1 /
It can be used in a concentration range of 10 ⁸ to 1 × 1/10 ² M, but is not limited to this range.

The present invention will be described in more detail below with reference to test examples and examples, but the present invention is not limited thereto.

[0023]

[Test example]

[Test Example 1] Racemic TFIBA ethyl ester prepared in (2) of Example 1 described below was mixed with acetic acid buffer (pH 4.5) and t-butyl alcohol (10% concentration based on the buffer). In the coexistence of 30 ° C, 40 or 108
Various enzyme agents under different time conditions, namely lipase PS, lipase AK, lipase AY, pancreatin F (trade name, manufactured by Amano Pharmaceutical Co., Ltd., derived from pig pancreas), phospholipase A (trade name,
Amano Pharmaceutical Co., Ltd.), Protease M, Cellulase A (trade name, manufactured by Amano Pharmaceutical Co., derived from Aspergillus), and cellulase T (trade name, manufactured by Amano Pharmaceutical Co., Ltd., derived from Trichoderma), and acted to produce optically active (+)-TFIBA Examine the generation. The results are shown in Table 1 below.

[0025]

[Table 1]

As is clear from the above results, it was found that, out of the eight kinds of hydrolases, four kinds of lipase PS, lipase AK, lipase AY and protease M can produce optically active (+)-TFIBA, and Of the four enzymes, when using Lipase AK,
It was found that the yield of optically active (+)-TFIBA was the highest.

[Test Example 2] Lipase AK was added to racemic TFIBA ethyl ester prepared in (2) of Example 1 which will be described later, with a phosphate buffer (pH 7.0) and various solvents (10% concentration based on the buffer). 5) of n-hexane, t-butyl alcohol, acetone, DMSO and DMF.
In the presence of each of the types, 30 ℃, 40-132 hours,
By acting, the production of optically active (+)-TFIBA was examined. The results are shown in Table 2 below.

[0028]

[Table 2]

As is clear from the above results, lipase AK produced an optically active (+)-TFI regardless of the solvent used.
BA is produced in good yield, but when t-butyl alcohol is used as a solvent, the reaction can be completed in a short time, which is advantageous.

[Test Example 3] Lipase AK was added to racemic TFIBA ethyl ester prepared in (2) of Example 1 to be described later in various buffer solutions, that is, acetate buffer solution (pH 4.
5), in the presence of a combination of phosphate buffer (pH 7.0) and glycine buffer (pH 8.0) and solvent t-butyl alcohol (10% concentration used in the buffer),
The production of optically active (+)-TFIBA was examined by allowing it to act at 4-55 ° C for 36-230 hours. The results are shown in Table 3 below.

[0031]

[Table 3]

As is clear from the above results, lipase A
In the case of asymmetric decomposition with K, even if t-butyl alcohol as a solvent and any buffer are combined, the optically active (+)-T
FIBA can be produced in good yield. However, when combined with an acetate buffer, the reaction time is short even at a relatively low temperature, and the optically active (+)-TFIBA can be obtained with an ee of almost 100%, which is advantageous. .

[Test Example 4] Biological test using black mappe

The black mappe was thoroughly washed with tap water, rinsed with distilled water, sown with distilled water in a petri dish on fully dampened absorbent cotton, and placed at 25 ° C. for about 24 hours. 10 seeds with a slightly rooted state were placed in a petri dish with a diameter of 6 cm, and 4 ml of a chemical solution having a concentration of 1/10 ⁴ (M) of each optically active (+)-, optically active (-)-and racemic TFIBA. It was placed on a filter paper (diameter: 5.5 cm) in which was soaked with and was incubated for 3 days in the dark, and then the root length was measured. The results are shown in Table 4 below.

[0035]

[Table 4]

As is clear from the above results, optically active (+)-TFIBA has a root-extending action about 2.4 times that of racemic TFIBA. And this means that when using an optically active substance as a plant regulator,
It shows that the same effect is obtained by using less than half of the racemate. Therefore, the optically active substance can be expected as a plant regulator with less environmental pollution.

[0037]

【Example】

[Example 1]

(1) Racemic TFIBA (4, 4, 4-
Preparation of trifluoro-3- (indole-3-) butyric acid)

Diethyl malonate (16.0 g, 100 m
(mol) in 50 ml of toluene solution was added with metallic sodium (2.3 g) and heated under reflux for 1.5 hours.
2,2-Trifluoro-1-indole-3-) ethanol (4.3 g, 20 mmol) is added. After heating under reflux again for 4 hours, water is carefully added, and the mixture is extracted 3 times with ethyl acetate. The ethyl acetate layer is washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a crude dicarboxylic acid diester solution in toluene. After this solution was partitioned with hexane-acetonitrile, the acetonitrile layer was concentrated under reduced pressure. The crude diester obtained was converted to methanol 1
After dissolving in 00 ml, an aqueous solution (80 ml) of potassium carbonate (25.7 g) is added, and the mixture is heated under reflux for 90 hours.

After neutralization with hydrochloric acid, the mixture is concentrated under reduced pressure to remove methanol, made alkaline with a 4N sodium hydroxide aqueous solution, and then extracted with ethyl acetate. The aqueous layer is acidified with hydrochloric acid and extracted with ethyl acetate. The ethyl acetate layer is washed with water and saturated brine and dried over anhydrous sodium sulfate. The crude carboxylic acid obtained after concentration under reduced pressure was purified by silica gel column chromatography to obtain racemic TFIBA (4, 4, 4
-Trifluoro-3- (indole-3-) butyric acid)
(4.2 g, 82.0%) was obtained. The physicochemical properties are shown in Table 5 below.

[0042]

[Table 5]

(2) Racemic TFIBA ethyl ester (4,4,4-trifluoro-3- (indole-3
-) Production of butyric acid ethyl ester)

Blowing into a solution of 4,4,4-trifluoro-3- (indole-3-) butyric acid (5.14 g, 20 mmol) in ethanol (99.8%, 300 ml) until dry hydrogen chloride was saturated. Absorbed. After heating and refluxing for 12 hours, the solvent and the like were distilled off under reduced pressure to concentrate the total amount to 30 ml. This solution was poured into ice water (200 ml) and, when cold, neutralized with a 5N sodium hydroxide aqueous solution. The mixture was extracted twice with ethyl acetate (200 ml), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The residue was recrystallized from n-hexane to give racemic TFIBA ethyl ester (5.47 g, yield 95.9%) as colorless columnar crystals (mp. 57 ° C). The physicochemical properties are shown in Table 6 below.

[0045]

[Table 6]

(3) Optical activity (+)-TFIBA (4,
Production of 4,4-trifluoro-3- (indole-3-) butyric acid)

A jar fermenter (MBF, manufactured by Tokyo Rikakikai) was added to an acetate buffer (450 ml) (0.2 N C).
H ₃ COOH solution (205ml), 0.2N CH ₃
COONa aqueous solution (45 ml); pH 4.0: diluted 2-fold at the time of use), auxiliary solvent (t-BuOH: 50 ml) and lipase AK (15 g) were added, and the mixture was stirred at 30 ° C. for 20 minutes. Racemic TFIBA ethyl ester (0.5 g) was added to this suspension, and the mixture was further stirred at 30 ° C. After 60 hours, the reaction was stopped, the pH was adjusted to 2.0 with a 1N aqueous hydrochloric acid solution, and the mixture was extracted 3 times with ethyl acetate (500 ml). The extract was dried over anhydrous magnesium sulfate and the solvent was distilled off. The residue was purified by silica gel chromatography to obtain optically active (+)-TFIBA with a yield of 92%. Optical purity 9
9% ee. At the same time, optically active (-)-TFIBA ethyl ester (4,4,4-trifluoro-3- (indole-3-) butyric acid ethyl ester) was also obtained in a yield of 93 (%).
Got with. Optical purity 99% ee. These physicochemical properties are shown in Table 7 below.

[0048]

[Table 7]

(4) Optical activity (-)-TFIBA (4
Preparation of 4,4-trifluoro-3- (indole-3-) butyric acid):

Optically active (−)-TFIBA ethyl ester (28.5 mg, 0.1 mmol) in methanol (8
9.8%, 3 ml) solution into potassium carbonate (68.9 m
g, 0.5 mmol) aqueous solution 3 ml was added. After heating under reflux at 70 ° C. for 3 hours, methanol was removed under reduced pressure, and the mixture was extracted 3 times with ethyl acetate. The aqueous layer was acidified with 4N hydrochloric acid, extracted with ethyl acetate, the ethyl acetate layer was washed with water and saturated brine, and dried over anhydrous sodium sulfate. After concentration under reduced pressure. The crude carboxylic acid was purified by silica gel column chromatography to obtain optically active (−)-TFIBA (2
0.5 mg, 80%) was obtained. Optical purity 97% ee. The physicochemical properties are shown in Table 8 below.

[0051]

[Table 8]

[0052]

The optically active (+)-T can be obtained only by performing asymmetric hydrolysis using an enzyme according to the present invention.
It has been successful in producing FIBA. This optically active substance has a very strong rooting promoting action and root elongation promoting action, and is not only useful in the technical field of agrochemicals such as plant growth regulators, but also for plant biotechnology such as callus cultivation. Its usefulness is also highly expected in the technical field.

 ─────────────────────────────────────────────────── --- Continuation of the front page (72) Inventor Hiroshi Kimoto 5-7-130 Oyamada, Kuwana City, Mie Prefecture (72) Inventor Sho Fujii 18-52, Kagoden, Chikusa-ku, Nagoya City, Aichi Prefecture (72) Inventor Masato Katayama 1-1603, Shinonokaze, Midori-ku, Nagoya, Aichi (72) Inventor Yoshihiko Hirose, Nishiharu Kasugai-gun, Aichi Kuninotsubo Nishijo Mansion 51, Amano Pharmaceutical Co., Ltd. (72) Inventor, Sasa Seiji Ki Kunitsubo Nishijo Mansion, Nishiharu Town, Nishikasugai-gun, Aichi Prefecture 51, Amano Pharmaceutical Co., Ltd. Central Research Laboratory (72) Inventor Tamase Tamase Nishinoharu Town, Nishikasugai-gun, Aichi Prefecture Kinotsubo Nishijo Mansion 51, Central Research Laboratory, Amano Pharmaceutical Co., Ltd.

Claims (4)

[Claims] 1. A compound represented by the following chemical formula 1 (In the formula, * represents an asymmetric carbon atom) Compound of formula (I), optically active (+)-4,4,4-trifluoro-3- (indole-3-) butyric acid. 2. The following chemical formula 2 is shown below. Ethyl alcohol is allowed to act on the compound of formula (II) to synthesize an ester, and then the ester is allowed to act on an enzyme having an esterase activity, which is represented by the following Chemical formula 3 (In the formula, * represents an asymmetric carbon atom.) An optically active (+)-4,4,4-trifluoro-3, which is characterized in that an optically active substance of the formula (I) is produced and collected. -(Indole-3-) butyric acid production method. 3. The asymmetric hydrolysis with an enzyme is carried out in the presence of an organic solvent and / or a buffer solution.
The manufacturing method described in. 4. The method according to claim 2 or 3, wherein a lipase and / or a protease is used as the enzyme having an esterase activity.