KR100651337B1 - Method for preparing an optical active 1,4-benzodioxan-2-carboxylic compounds by enzymatic method - Google Patents

Abstract

The present invention relates to a method for preparing an optically active 1,4-benzodioxane-2-carboxy compound by enzymatic method, more specifically racemic 1,4-benzodioxane-2-carboxypiperazin ( S-1,4-benzodioxane-2-carboxypiperazine and R-1,4-benzo by enzymatic method of selectively hydrolyzing 1,4-benzodioxan-2-carboxypiperazine) compound using protease enzyme A method for producing dioxane-2-carboxylic acid. The production method of the present invention is simple in the production process using the protease enzyme, high optical purity and yield is very useful industrially.

1,4-benzodioxane-2-carboxypiperazin, 1,4-benzodioxane-2-carboxylic acid, racemic body, hydrolysis, protease, aspergillus, streptomyces

Description

Method for preparing an optical active 1,4-benzodioxan-2-carboxylic compounds by enzymatic method}

The present invention relates to a method for preparing an optically active 1,4-benzodioxane-2-carboxy compound by enzymatic method, more specifically racemic 1,4-benzodioxane-2-carboxypiperazin ( 1,4-benzodioxan-2-carboxypiperazine) is dissolved in a buffer solution, and then optically active 1,4-benzodi by an enzymatic method of selectively hydrolyzing one amide by acting a protease derived from a microorganism. It relates to a method for producing oxane-2-carboxypiperazin (1,4-benzodioxan-2-carboxypiperazine) and 1,4-benzodioxane-2-carboxylic acid (1,4-benzodioxan-2-carboxylic acid). .

Optically active 1,4-benzodioxane-2-carboxypiperazin or 1,4-benzodioxane-2-carboxylic acid is used as an intermediate for the synthesis of Cardura, a therapeutic agent for hypertension. Kadura is a hypertension therapeutic released by Pfizer in 1988, and its chemical name is (± -doxazosin mesylate), and is represented by the following Chemical Formula 1, and is currently sold as a racemic body.                         

The production method of (±) -doxazosin mesylate includes the following methods.

U.S. Patent No. 2007656 published by Pfizer in 1977 prepares racemic doxazosin with a combination of 1,4-benzodioxane-2-carboxypiperazin made from catechol and a heteroaromatic compound made from vanillin and again methanesulfonic acid. A method for preparing methanesulfonic acid salt by neutralization with is described.

Recently, it has been confirmed that the doxazosin is effective for the treatment of Benign Prostatic Hyperplasia (BPH). It is confirmed that the effect is large, and recently, a clinical study of (S) -doxazosin is in progress.

The main optically active intermediates for the synthesis of (S) -doxazosin are (S) -1,4-benzodioxane-2-carboxylic acid represented by the following formula (2) and (S) -1,4 represented by the following formula (3) -Benzodioxane-2-carboxypiperazin, the latter can be easily synthesized by reacting the former with piperazine monochloride.                         

As a method for preparing (S) -1,4-benzodioxane-2-carboxylic acid, a method of separating a racemic body with dihydrodiethylamine has been reported, but the method has a yield of about 1%. As such, there is no commercial value. In 2001, Sepracor, USA, used an esterase derived from Serratia marcescens to process (S) -1,4-benzodioxane-2-carboxylic acid of high optical purity (99.8% ee). A method of preparation has been published (Tetrahedron: Asymmetry 12 (2001) 2169-2174). The method can produce (S) -1,4-benzodioxane-2-carboxylic acid of high optical purity, but the overall yield is as low as 21-25%, and then in the process of reacting with piperazine hydrochloride The disadvantage is that about 10% of diamide, an unwanted impurity, is produced.

In order to solve the above problems, Seppraker has developed a method of separating racemic 1,4-benzodioxane-2-carboxypiperazine into D-tartaric acid (Tetrahedron: Asymmetry 12 (2001) 2169-2174). This method can also produce high optical purity (99.3% ee) of (S) -1,4-benzodioxane-2-carboxypiperazine, but the overall yield is low at about 20% and requires several recrystallization processes. There are disadvantages.

In order to solve the above-mentioned problems, it is economically superior by using a simple process and high optical purity (S) -1,4-benzodioxane-2-carboxypiperazine or (S) -1,4- There is a need for development of a process that can produce benzodioxane-2-carboxylic acid.

Therefore, the present inventors, unlike the chemical optical splitting method of Seppraker, enzymatic optical splitting method using an enzyme or microorganism that has not yet been reported, more specifically, racemic 1,4-benzodioxane using a protease enzyme 2-carboxypiperazines as optically active 1,4-benzodioxane-2-carboxylate compounds 1,4-benzodioxane-2-carboxypiperazin and 1,4-benzodioxane-2-carboxylic acid The manufacturing method of dividing was developed and the present invention was completed based on this.

It is therefore an object of the present invention to provide a method for producing optically active 1,4-benzodioxane-2-carboxypiperazines from racemic 1,4-benzodioxane-2-carboxypiperazines using protease enzymes. have.

Another object of the present invention is to provide a method for preparing optically active 1,4-benzodioxane-2-carboxylic acid from racemic 1,4-benzodioxane-2-carboxypiperazin using a protease enzyme. have.

The production method of the present invention for achieving the above object is to hydrolyze racemic 1,4-benzodioxane-2-carboxypiperazine using a protease enzyme derived from microorganisms in the presence of a buffer solution of pH 6-8 A step of separating an organic solvent layer and an aqueous solution layer by adding an organic solvent to the reaction solution, and separating (S) -1,4-benzodioxane-2-carboxypiperazine from the organic solvent layer It is composed.

The production method of the present invention for achieving the above another object is to hydrolyze racemic 1,4-benzodioxane-2-carboxypiperazine using a protease enzyme derived from microorganisms in the presence of a buffer solution of pH 6-8 Comprising a step, separating the organic solvent layer and the aqueous solution layer by adding an organic solvent to the reaction solution, and separating (R) -1,4-benzodioxane-2-carboxylic acid from the aqueous solution layer do.

Looking at the present invention in more detail as follows.

As described above, according to the present invention, after dissolving racemic 1,4-benzodioxane-2-carboxypiperazine as a substrate in a buffer solution of pH 6 to 8, the genus Aspergillus or Streptococcus Proteases from the microorganism of the genus Steptomyces are added without further treatment, and then the reaction mixture is stirred at 100-300 rpm at a temperature of 20-40 ° C. to selectively hydrolyze only the (R) -isomer.

The hydrolysis reaction process of the present invention is shown in Scheme 1 below.                     

An organic solvent was added to the compound hydrolyzed in this way, and the organic solvent layer was separated into an aqueous solution layer. From the organic solvent layer, 1,4-benzodioxane-2-carboxypiperazine having optical activity was obtained. 1,4-benzodioxane-2-carboxylic acid having optical activity is obtained.

On the other hand, the protease used in the present invention is obtained from a microorganism of the genus Aspergillus or Streptomyces, the enzyme is immobilized with a powder or liquid or carrier or biological cells containing the enzyme and immobilized Biological cells.

The Aspergillus (Aspergillus) in microorganisms are Aspergillus melre mouse (Aspergillus melleus), Aspergillus material (Aspergillus sojae), Aspergillus duck material (Aspergillus oryzae), etc., and Streptomyces (Streptomyces) in Microorganisms include Streptomyces griceus .

The protease may be used commercially available, or manufactured and used. Commercially available proteases include, for example, Protease P from Amano, Protease type XIV from Sigma, Protease type XIX, Protease type XXIII, Europa (Europa Protease) 6 (Am), etc., but is not limited thereto.

In the present invention, the enzyme reaction temperature is preferably 20 to 40 ℃, if the temperature is less than 20 ℃ there is a disadvantage that the reaction rate is too slow, if the reaction temperature exceeds 40 ℃ there is a problem that the optical purity is lowered. In addition, the pH of the enzyme reaction is in the appropriate range of pH 6 ~ 8, if the outside the range of the reaction rate is slow and there is a problem that the optical purity falls, the ratio of enzyme and substrate in the hydrolysis reaction is 1: 1 by weight ratio. ~ 10: 1 is preferred, the reaction rate is too slow or out of the range or there is a problem that the economical efficiency is too much because the amount of the enzyme is used too much.

Optically active 1,4-benzodioxane-2-carboxypiperazine can be easily analyzed for R-form and S-form using Chiralpak AD-H, a chiral column of Daicel Corporation. The analysis of 4-benzodioxane-2-carboxylic acid can be analyzed using Chiralcel OD-H, which is also Chiral's chiral column.

In the case of optically active 1,4-benzodioxane-2-carboxypiperazine, ethanol containing 0.1% diethylamine was poured at 0.4 ml / min, and the absorbance of HPLC was analyzed at 254 nm. The retention time of (S) -1,4-benzodioxane-2-carboxypiperazine is 14.7 minutes, and the retention time of (R) -1,4-benzodioxane-2-carboxypiperazine is 17.6. In minutes, the two isomers can be easily distinguished.

For optically active 1,4-benzodioxane-2-carboxylic acid, a mixture of nucleic acid, isopropanol and trifluoroacetic acid in a ratio of 95: 5: 0.1, at a rate of 0.5 ml / min When the absorbance of HPLC was 254 nm and analyzed, the retention time of (S) -1,4-benzodioxane-2-carboxylic acid was 42.5 minutes and (R) -1,4-benzodioxane-2. The retention time of the carboxylic acid is 53 minutes, making it easy to distinguish the two isomers.

Hereinafter, the present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples.

Example 1

Into 5 ml of 100 mM phosphoric acid solution (pH 7.0), 0.1 g (2% by weight) of hydrochloric acid of racemic 1,4-benzodioxane-2-carboxypiperazine was added and pH was adjusted to 7.0. 0.1g (2% by weight) of the degrading enzyme was added thereto, and the reaction was performed at 30 ° C. 0.5 ml of the reaction solution was mixed with 10 μl of 10N sodium hydroxide solution, and then the reactant was extracted with 1 ml of ethyl acetate, and analyzed by HPLC using the aforementioned analytical method.

enzyme microbe Optical purity (% ee) Absolute Arrangement of Amide Protease Aspergillus melleus 100 S Streptomyces griceus 96 S Aspergillus oryzae 93.8 S Aspergillus sojae 80.8 S Bacillus polymax 23.1 S Bacillus licheniformis 27.8 S Aspergillus niger 13.5 S Acylase Penicillium sp. 57.5 S

In Table 1, the absolute sequence S of the amide means that the (R) -isomer is selectively hydrolyzed as a result of the enzymatic reaction, so that the (S) -isomer remains relatively more in the reactant, and the optical purity is The higher the enzyme, the higher the selectivity of the (R) -isomer of the substrate, the better the preparation of the (S) -isomer compound.

Experiments were carried out using about 40 hydrolytic enzymes in addition to the enzymes shown in Table 1 above, but most of them did not hydrolyze amide, which is not selective or substrate.

Example 2

Among the enzymes selected in Example 1, the following reaction was carried out using a protease derived from Aspergillus melleus . To 350 ml of 100 mM phosphoric acid solution (pH 7.0) was added 7 g (2% by weight) of racemic 1,4-benzodioxane-2-carboxypiperazine, pH was adjusted to 7.0, and 35 g of the protease was added thereto. The reaction was carried out. The pH during the reaction was maintained at 6.5-7.5 using 20% sodium hydroxide solution. The reaction samples were taken at regular intervals to analyze the reaction situation. When the optical purity of (S) -1,4-benzodioxane-2-carboxypiperazine reached 98.0% ee after 49 hours of reaction, the reaction was stopped and sodium hydroxide solution was added to the reaction solution to adjust the pH to 8.0. After adjusting above, the amide was extracted with ethyl acetate. The organic solvent layer was separated and ethyl acetate was removed by distillation under reduced pressure, whereby 2.8 g of (S) -1,4-benzodioxane-2-carboxypiperazine was recovered. The upper organic solvent layer was separated and hydrochloric acid solution was added to the remaining aqueous solution to lower the pH to 2 or less, and then ethyl acetate was added to the remaining hydrolyzed (R) -1,4-benzodioxane-2-carboxate on the aqueous solution. Acid was extracted. At this time, the optical purity of (R) -1,4-benzodioxane-2-carboxylic acid was 95.1% ee.

Example 3

The reaction was carried out using a protease derived from Aspergillus oryzae instead of the protease derived from Aspergillus meleus of Example 2. After 47 hours of reaction, the optical purity of (S) -1,4-benzodioxane-2-carboxypiperazine was 89.9% ee, and the optical purity of (R) -1,4-benzodioxane-2-carboxylic acid. Showed 88.9% ee. After recovering (S) -1,4-benzodioxane-2-carboxypiperazine in the same manner as in Example 1, the extraction solvent was removed by distillation under reduced pressure, and again toluene was added thereto to dissolve it and cooled to room temperature. As a result of recrystallization twice by the method described above, 98% ee or more of (S) -1,4-benzodioxane-2-carboxypiperazine was obtained.

Example 4

After recovering (R) -1,4-benzodioxane-2-carboxylic acid having an optical purity of 95.1% obtained in Example 2, the extraction solvent was removed by distillation under reduced pressure and recrystallized again using toluene. (R) -1,4-benzodioxane-2-carboxylic acid with a purity of 98% or more was obtained.

Comparative Example 1

The reaction was performed by changing the temperature to 50 degreeC among the reaction conditions of Example 2. After about 20 hours the reaction did not proceed anymore. At this time, the optical purity of (S) -1,4-benzodioxane-2-carboxypiperazine was 21.6% ee, which was significantly lower in light of Example 2.

Comparative Example 2

The reaction was carried out by changing the pH to 9.0 among the reaction conditions of Example 2. After 22 hours of reaction, the optical purity of (S) -1,4-benzodioxane-2-carboxypiperazine was 9.4% ee, and the optical purity of (R) -1,4-benzodioxane-2-carboxylic acid. Was 72.4% ee, which was significantly lower in optical purity in the case of Example 2.

As described above, the method of the present invention is a high optical purity of (S) -1,4-benzodioxane-2-carboxypiperazine and (R) -1,4 by a simple method using protease enzymes derived from microorganisms. -Benzodioxane-2-carboxylic acid could be prepared.

Claims (8)

In the presence of a buffer solution of pH 6-8, Hydrolyzing racemic 1,4-benzodioxane-2-carboxypiperazin using a microbial protease enzyme, Adding an organic solvent to the reaction solution to separate the organic solvent layer and the aqueous solution layer, and S-1,4-benzodioxane-2-carboxy in an enzymatic method comprising the step of separating (S) -1,4-benzodioxane-2-carboxypiperazine from the organic solvent layer. Process for preparing piperazine. In the presence of a buffer solution of pH 6-8, Hydrolyzing racemic 1,4-benzodioxane-2-carboxypiperazin using a microbial protease enzyme, Adding an organic solvent to the reaction solution to separate the organic solvent layer and the aqueous solution layer, and Preparation of optically active 1,4-benzodioxane-2-carboxylic acid by enzymatic method, characterized by separating (R) -1,4-benzodioxane-2-carboxylic acid from the aqueous solution layer Way. The method according to claim 1 or 2, wherein the protease enzyme is a microbial-derived protease immobilized in powder, liquid or carrier. The method of claim 1 or 2, wherein the microorganism is a microorganism of the genus Aspergillus or a microorganism of the genus Streptomyces. The method of claim 4, wherein the Aspergillus (Aspergillus) in the microorganism, characterized in that Aspergillus melre mouse (Aspergillus melleus), Aspergillus material (Aspergillus sojae), or Aspergillus duck material (Aspergillus oryzae) Way. The method of claim 4, wherein the Streptomyces genus microorganism is Streptomyces griceus . The method of claim 1 or 2, wherein the temperature of the hydrolysis reaction is 20 to 40 ℃. The method according to claim 1 or 2, wherein the ratio of the enzyme and the substrate in the hydrolysis reaction is 1: 1 to 10: 1 by weight.