JPH06306031A - Cyano compound and hydrolysis thereof - Google Patents

Abstract

PURPOSE:To provide a novel process for the production of pharmaceuticals and an intermediate useful for the production process. CONSTITUTION:This is a trans-4-aminomethylcyclohexanecarboxylic acid 4-(2- cyanoethyl)phenyl ester or its acid addition salt. A process for producing a carboxylic acid derivative by treating the compound with a microorganism, its cultured product or an extracted and fractionated product of the cultured product, thereby hydrolyzing the cyano group of the compound. A microbial strain useful for the above process.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a drug, a compound useful for the method, and a microorganism useful for the method.

[0002]

2. Description of the Related Art Equation (1)

[0003]

[Chemical 1] Trans-4-aminomethylcyclohexanecarboxylic acid 4- (2-carboxyethyl) phenyl ester hydrochloride represented by (hereinafter, referred to as CEP ester hydrochloride)
Is known to have an excellent action as a remedy for gastritis and gastric ulcer and an antiplasmin drug.

Various methods are known for producing the CEP ester hydrochloride. Most of them are a method of condensing trans-4-aminomethylcyclohexane-1-carboxylic acid with a compound in which the carboxyl group of p-hydroxyphenylpropionic acid is protected, and then removing the protecting group of the carboxyl group. As the deprotection method, for example, a method by catalytic reduction (Japanese Patent Publication Nos. 46-19950 and 52-48978)
No.), acid or alkali hydrolysis method
52-17447 and JP-A-53-56642), a method using a Lewis acid and a nucleophile (JP-A-56-167648), a microorganism, an extract thereof, or a method using a commercially available enzyme preparation (JP-A-56-167648). JP-A-62-294091 and JP-A-1-8148) and the like can be mentioned.

However, although these methods are excellent, they involve two steps of protection and deprotection, which are industrially disadvantageous. And, for example, in the method by catalytic reduction, expensive catalyst such as palladium is required,
In the method by hydrolysis, such as the danger of using hydrogen gas and the need for a special device, the ester portion which is not the protecting group of the CEP ester is also hydrolyzed and the yield is lowered. The method of using a Lewis acid has problems such as complicated collection of metals to be used, such as complicated collection.

[0006]

The object of the present invention is to obtain CE.
It is an object of the present invention to provide an industrially advantageous production method capable of safely and inexpensively producing a P-ester hydrochloride or other acid addition salt or free form by a reaction without a protective group.

[0007]

In view of the above situation, the present inventor has conducted diligent research. As a result, a compound having a structure represented by the formula (2) or an acid addition salt thereof, which can be obtained at low cost, is used as a raw material, and a microbial enzyme is allowed to act on this compound, under mild conditions, CEP
The present invention has been completed by finding that an ester or an acid addition salt thereof can be obtained, and a target compound can be obtained by a shorter production process that does not require a protecting group.

That is, the present invention relates to 4- (2-cyanoethyl) phenyl ester of 4-aminomethylcyclohexanecarboxylic acid or an acid addition salt thereof, and further to 4- (2-cyanoethyl) phenyl trans-4-aminomethylcyclohexanecarboxylic acid. It relates to an ester or an acid addition salt thereof.

The present invention also provides trans-4-aminomethylcyclohexanecarboxylic acid 4- (2-cyanoethyl) phenyl ester or an acid addition salt thereof as a microorganism consisting of Klebsiella, Azospirillum, Bacillus, and Nocardia. A trans-4-aminomethylcyclohexanecarboxylic acid 4-, which is treated in the presence of a microorganism, a culture of the microorganism, or an extracted fraction of the culture of the microorganism, selected from the group It relates to a method for producing (2-carboxyethyl) phenyl ester or a salt thereof.

The present invention also relates to Klebsiella sp. AM-1.
Regarding two bacteria.

Further, the present invention provides trans-4-aminomethylcyclohexanecarboxylic acid 4- (2-cyanoethyl) phenyl ester or an acid addition salt thereof with a Klebsiella genus AM-12 bacterium, a Klebsiella genus AM-12 bacterium culture, Or an extract fraction of a culture of Klebsiella genus AM-12, which is treated with trans-4-aminomethylcyclohexanecarboxylic acid 4- (2-carboxyethyl) phenyl ester, or Regarding the manufacturing method of salt.

The present invention also relates to a method for converting a cyano compound into a carboxyl compound by treating it with Klebsiella genus AM-12, and to a carboxyl compound obtained by treating the cyano compound with Klebsiella genus AM-12. Is.

4-Aminomethylcyclohexanecarboxylic acid 4- (2-cyanoethyl) used in the method of the present invention
Phenyl ester, especially trans-4-aminomethylcyclohexanecarboxylic acid 4- (2-cyanoethyl) phenyl ester has a structure represented by the formula (2) shown in Chemical formula 2 (hereinafter, referred to as compound (2) That.). Here, the bond between the aminomethyl group and the carboxyl group moiety may be either equatorial or axial with respect to the cyclohexane ring.

[0014]

[Chemical 2]

The compound (2) may be an acid addition salt. This acid may be either an organic acid or an inorganic acid. Examples of inorganic acids include hydrochloric acid, sulfuric acid, nitric acid and phosphoric acid. Also, as the organic acid, acetic acid,
Examples thereof include acids having a carboxyl group such as butyric acid, oxalic acid, succinic acid, glutaric acid, citric acid, lactic acid, tartaric acid, and sulfonic acids such as paratoluenesulfonic acid and methanesulfonic acid.

As shown in Chemical formula 3, this compound (2) can be obtained by reacting (4-hydroxyphenyl) propionitrile with the acid chloride of trans-4-aminomethylcyclohexanecarboxylic acid. . Further, this (4-hydroxyphenyl) propionitrile can be produced from phenol and acrylonitrile by a known method.

[0017]

[Chemical 3]

In the method of the present invention, the compound (2) or its acid addition salt may be treated with a microorganism, a microorganism culture, or an extract fraction of the microorganism culture. Here, treating with a microorganism means adding a qualified microorganism to a solution or suspension of the compound (2) or an acid addition salt thereof, and culturing the same under conditions suitable for the microorganism to be treated. In addition, the treatment with a microorganism culture means that a microorganism is first cultured, and then a culture solution containing the cultured cells is added to a solution or suspension of the compound (2) or an acid addition salt thereof,
It indicates that further culturing is performed. Further, treating with an extract fraction of a microbial culture means culturing a microorganism first, separating the components necessary for the treatment from the bacterial cells in the culture, and adding the compound (2) or its acid addition by this isolate. Indicates to treat salt.

Examples of bacteria that can be used in the method of the present invention include bacteria belonging to the genus Klebsiella, the genus Azospirillum, the genus Bacillus and the genus Nocardia. Among these microorganisms, Klebsiella sp. AM-1
The method using two bacteria is typical of the method of the present invention. This AM-12 bacterium is a bacterium belonging to the genus Klebsiella newly discovered by the present inventors in the soil, and is named Klebsiella genus AM-12. Although this bacterium has the following mycological properties, FERM P-13565 at the Institute of Biotechnology, Institute of Biotechnology, AIST.
It is preserved as a deposit.

A) Morphology 1) Shape and size of cells Short bacilli (0.5 x
1.0 μm) 2) Presence or absence of cell polymorphism 3) Presence or absence of motility 4) Presence or absence of spores 5) Gram stain Negative

[0021]

[0022]

Among the microorganisms that can be used in the present invention,
Preferred species of the genus Azospirillum include Azospirillum brasilence, Azospirillum lipoferrum, and the like.
Examples of the genus Bacillus include Bacillus laterosporus, and examples of the genus Nocardia include Nocardia parafinica. These are commercially available and can be easily obtained.

Cultivation of the microorganism used may be carried out under aerobic conditions. The culture temperature may be in the range of 25 to 35 ° C and the culture pH may be in the range of 6.5 to 7.5. The culturing time is usually one day.

The medium contains a carbon source, a nitrogen source, inorganic salts and trace amounts of nutrients that can be assimilated by microorganisms. That is, the carbon source, glucose, maltose, carbohydrates such as dextrin, as the nitrogen source, yeast extract, corn steep liquor, meat extract, natural organic nitrogen sources such as mixed amino acid powder, or acetonitrile, propionitrile, butyronitrile, Acrylonitrile, organic nitrile compounds such as crotononitrile, or acetamide, propioamide, butyroamide, crotonoamide, etc., organic carboxylic acid amide compounds, ammonia,
Inorganic salts such as ammonium chloride or organic ammonium salts can also be used.

The inorganic salts include carbonates, hydrogencarbonates, hydrochlorides, sulfates, nitrates, phosphates, hydroxides of metals such as magnesium, iron, manganese, potassium and sodium.
Various salts such as organic carboxylic acid salts are appropriately used.

Further, if necessary, various organic substances, inorganic substances, or commonly used defoaming agents necessary for the growth of microorganisms can be added.

The microorganisms used in the reaction may be those obtained by collecting and washing the above-mentioned microorganisms from a culture solution, dried or treated with acetone powder, and the like. Further, the culture may be a culture of the above microorganism in an appropriate medium.

The amount of bacterial cells used is not particularly limited, but as a guide, it is 2 to 10% by weight with respect to the weight of the raw material compound.
The degree may be used, but depending on the reaction conditions, it may be less than this.

The concentration of the compound (2) in the reaction solution is expressed by weight ratio.
A range of 0.1 to 10% is possible, but usually 4 to 5
% Is preferred. The reaction temperature may be in the range of 10 to 50 ° C, but is preferably in the range of 25 to 35 ° C. PH of reaction solution
May be in the range 5 to 8, but is preferably in the range 6 to 7. 50 to 1 to control the pH of the reaction mixture
A buffer solution of 00 mM can be appropriately used. The reaction composition can be quantified by thin layer chromatography (TLC) or high performance liquid chromatography (HPLC).

After the reaction is completed, the product CEP ester usually precipitates as a solid or crystal. This CE
This is a method for isolating P-ester. First, CE was prepared by adding a mixed solution of isopropyl alcohol and hydrochloric acid to the reaction solution.
Dissolve the P-ester. After that, the bacterial cells are removed and the pH of the resulting solution is adjusted to around 7, so that the free form of the CEP ester is precipitated, so the solution is collected by filtration. Then, hydrochloric acid in an amount equivalent to that of the obtained crystals is added, and crystallization is performed using water or the like to obtain a highly pure CEP ester hydrochloride. At this time, if another acid is used, another acid addition salt can be obtained.

On the other hand, there is also a method in which after the reaction, crystals are first collected, and then the crystals are dissolved in a mixed solution of isopropyl alcohol and hydrochloric acid to remove the microbial cells, and then the same treatment as above is performed. The method for removing the microbial cells may be a method using a membrane filter or a method using centrifugation, but a commonly used method may be used. Sopropyl alcohol and hydrochloric acid may usually be used in a volume ratio of 1: 1.

[0033]

EFFECTS OF THE INVENTION By using the production method of the present invention, an excellent drug for treating gastritis / gastric ulcer and an anti-plasmin drug, CEP ester derivative, can be obtained with high purity and high yield in a shorter production process. . Furthermore, the method of the present invention has excellent safety and is advantageous in terms of post-treatment. Therefore, the method of the present invention is industrially very useful.

[0034]

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

In the examples and the specification, trans-4-aminomethylcyclohexanecarboxylic acid 4-
The (2-carboxyethyl) phenyl ester (free form) is referred to as a CEP ester, and its hydrochloride is referred to as a CEP ester hydrochloride.

Reference Example 1 Normal broth medium (meat extract
Klebsiella spp.pre-cultured with 0.5%, peptone 1%, NaCl 0.5%, pH 7) was added to glucose 1%, n-butyroamide 0.5%, mixed amino acid powder 0.5%, dipotassium phosphate 0.75%, phosphate 1%. Medium containing 0.25% potassium, 0.01% magnesium sulfate, and 0.001% yeast extract (pH 7.
5) was inoculated and cultured at 30 ° C for 12 hours.

After completion of the culture, physiological saline was added to the cells separated by centrifugation and the cells were centrifuged again to obtain viable cells. This was dried in vacuum to obtain dried cells.

Reference Example 2 Azospirillum bacteria pre-cultured in the same manner as in Reference Example 1 were used to prepare glucose 1%, acrylonitrile 0.25%, yeast extract 0.5%, dipotassium phosphate.
0.75%, monopotassium phosphate 0.25%, magnesium sulfate 0.
It was inoculated into a medium (pH 7.5) containing 1% and cultured at 30 ° C. for 24 hours.

After the completion of the culture, physiological saline was added to the cells separated by centrifugation, and the cells were centrifuged again to obtain viable cells.

[Reference Example 3] Bacillus bacteria pre-cultured in the same manner as in Reference Example 1 were treated with dextrin 1%, isobutyronitrile 0.5%, yeast extract 0.05%, dipotassium phosphate.
0.75%, monopotassium phosphate 0.25%, magnesium sulfate 0.
It was inoculated into a medium (pH 7.5) containing 1% and cultured at 30 ° C for 24 hours.

After the completion of the culture, physiological saline was added to the separated and separated bacterial cells, which were again centrifuged to obtain viable bacterial cells.

[Reference Example 4] Bacteria of the genus Nocardia pre-cultured in the same manner as in Reference Example 1 were treated with lactose 1%, isobutyronitrile 0.5%, yeast extract 0.05%, dipotassium phosphate 0.7.
5%, monopotassium phosphate 0.25%, magnesium sulfate 0.1%
Was inoculated into a medium (pH 7.5) containing and cultured at 30 ° C. for 24 hours.

After culturing, physiological saline was added to the cells separated by centrifugation, and the cells were centrifuged again to obtain viable cells.

Example 1 314.4 g of trans-4-aminomethylcyclohexanecarboxylic acid was added to 150 parts of ethylene dichloride.
Suspend in 0 ml, gradually add thionyl chloride 249.9 g,
The reaction was carried out at 50 ° C for 4 hours. Next, 323.4 g of p-hydroxyphenyl-propionitrile was added to 350 ml of ethylene dichloride.
The solution dissolved in was added dropwise and further reacted at 50 ° C. for 3 hours.

After completion of the reaction, excess acid gas was removed by passing nitrogen gas through the reaction solution. Then, the reaction liquid was cooled, and the precipitated crystals were collected by filtration and dried to obtain 620 g of trans-4-aminomethylcyclohexanecarboxylic acid 4- (2-cyanoethyl) phenyl ester hydrochloride.

Elemental analysis, as C ₁₇ H ₂₃ ClN ₂ O ₂ (%): Analytical value: C, 63.10; H, 7.22; N, 8.54 Calculated value: C, 63.25; H, 7.18; N, 8.68 IR (KBr ) νmax (cm ^-1 ): 2900, 2220 (CN), 1735, 1500 ¹ H-NMR (D ₂ O) δ: 0.90-3.10 (16H, m), 6.95, 7.23 (4
H, dd).

Example 2 Trans-4-Aminomethylcyclohexanecarboxylic acid 4- (2-cyanoethyl) phenyl ester hydrochloride 323 g, cells obtained in Reference Example 1
9.7 g was suspended in 8000 ml of 50 mM citrate buffer (pH 7) and reacted at 30 ° C for 24 hours. After the reaction was completed, the reaction solution was analyzed by high performance liquid chromatography to find that C
The production rate of EP ester was 99%.

The reaction solution was filtered, the collected crude CEP ester was suspended in a mixed solution of isopropyl alcohol and water (1: 1, volume ratio, the same applies below), and the pH was adjusted to 2.2 with hydrochloric acid. This mixed solution was filtered to completely remove bacterial cells. The clear filtrate was adjusted to pH 7 with an aqueous sodium hydroxide solution and the precipitated crystals were collected by filtration to obtain a purified CEP ester. The obtained CEP
The ester was suspended in a mixture of isopropyl alcohol and water, adjusted to pH 2.2 with hydrochloric acid, and treated with activated carbon. The clear filtrate was concentrated under reduced pressure, and the precipitated crystals were collected by filtration and dried to obtain CEP ester hydrochloride (315 g, yield 92%).

The data of thin layer chromatography, high performance liquid chromatography, IR spectrum and ¹ H-NMR spectrum of this product were completely in agreement with those of the standard product. When the purity of this product was examined by high performance liquid chromatography, it was 100%.

Example 3 32.3 g of trans-4-aminomethylcyclohexanecarboxylic acid 4- (2-cyanoethyl) phenyl ester hydrochloride and 3.2 g of viable cells obtained under the culture conditions of Reference Example 1 were dried. Without suspending, it was suspended in 800 ml of 50 mM citrate buffer (pH 7) and reacted at 30 ° C. for 20 hours. After the reaction was completed, the reaction solution was analyzed by high performance liquid chromatography to find that the production rate of CEP ester was 99%.

The obtained reaction liquid was treated in the same manner as in Example 2 to obtain CEP ester hydrochloride (31.4 g, yield 92%). The data of thin-layer chromatography, high performance liquid chromatography, IR spectrum and ¹ H-NMR spectrum of this product were completely consistent with those of the standard product. When the purity of this product was examined by high performance liquid chromatography, it was 100%.

[Example 4] The culture obtained in Reference Example 1
To 800 ml, trans-4-aminomethylcyclohexanecarboxylic acid 4- (2-cyanoethyl) phenyl ester hydrochloride (32.3 g) was added, and the mixture was reacted at 30 ° C for 17 hours. After the reaction was completed, the reaction solution was analyzed by high performance liquid chromatography to find that the production rate of CEP ester was 99%.

The obtained reaction solution was treated in the same manner as in Example 2 to obtain CEP ester hydrochloride (30.8 g, yield 90%).

Example 5 The reaction was carried out under the same conditions except that a phosphate buffer was used instead of the citrate buffer of Example 2. After the reaction was completed, the reaction solution was analyzed by high performance liquid chromatography to find that the production rate of CEP ester was 99%.

The obtained reaction solution was treated in the same manner as in Example 2 to obtain CEP ester hydrochloride (315 g, yield 92%).

[Example 6] The reaction was carried out in the same manner as in Example 3 except that the viable bacterial cells obtained in Reference Examples 2 to 4 were used instead of the viable bacterial cells of Example 3. After the reaction was completed, the reaction solution was analyzed by high performance liquid chromatography, and the results shown in Table 1 were obtained. By treating in the same manner as in Example 2, the CEP ester hydrochloride can be obtained.

[0057]

[Table 1]

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location C12P 13/00 2121-4B // (C12N 1/20 C12R 1:22) (C12P 13/00 C12R 1:22) (C12P 13/00 C12R 1:01) (C12P 13/00 C12R 1:07) (C12P 13/00 C12R 1: 365)

Claims (7)

[Claims] 1. 4-Aminomethylcyclohexanecarboxylic acid 4- (2-cyanoethyl) phenyl ester or acid addition salt thereof 2. Trans-4-aminomethylcyclohexanecarboxylic acid 4- (2-cyanoethyl) phenyl ester or acid addition salt thereof 3. Trans-4-aminomethylcyclohexanecarboxylic acid 4- (2-cyanoethyl) phenyl ester or its acid addition salt is selected from the group of microorganisms consisting of Klebsiella, Azospirillum, Bacillus and Nocardia. A trans-4-aminomethylcyclohexanecarboxylic acid 4- (2-carboxyethyl) phenyl ester, characterized by being treated in the presence of a microorganism, a culture of the microorganism or an extracted fraction of the culture of the microorganism, or How to make that salt 4. Klebsiella genus AM-12 bacterium 5. A trans-4-aminomethylcyclohexanecarboxylic acid 4- (2-cyanoethyl) phenyl ester or an acid addition salt thereof is added to Klebsiella sp.
Trans-4-aminomethylcyclohexanecarboxylic acid 4- (2-, characterized by being treated in the presence of an extract of a culture of Klebsiella genus AM-12 or a culture of Klebsiella genus AM-12. Method for producing carboxyethyl) phenyl ester or its salt 6. A Klebsiella sp. AM-
Method for converting into carboxyl compounds by treatment with 12 bacteria 7. A Klebsiella sp. AM-
Carboxyl compounds obtained by treatment with 12 bacteria