KR20040063264A - Method for preparing optically active (R)-2-chloromandelic acid and its ester derivatives using Baker's Yeast - Google Patents

Abstract

PURPOSE: A method for preparing optically active (R)-2-chloromandelic acid and ester derivatives thereof using Baker's yeast is provided, thereby reducing the preparation costs by using yeast which is cheap and easily obtained, simplifying the method, and improving optical purity and preparation yield. CONSTITUTION: The method for preparing optically active (R)-2-chloromandelic acid and ester derivatives thereof comprises selectively reducing alkyl 2-chloro-benzoyl formate such as methyl 2-chloro-benzoyl formate in the presence of phosphate buffer solution using Baker's yeast, Saccharomyces cerevisiae type II at 20 to 37 deg. C and pH 6.0 to 8.0, wherein the weight ratio of the yeast and substrate is 1:0.1 to 1:10.

Description

Method for preparing optically active (R) -2-chloromandelic acid and its ester derivatives using Baker's Yeast}

The present invention relates to a method for producing optically active (R) -2-chloromandelic acid ((R) -2-chloromandelic acid) and its ester using yeast, and more specifically, alkyl 2-chloro-benzoylform After dissolving the mate (Alkyl 2-Chloro-benzoylformate) in a buffer solution, the yeast (Baker's yeast) used to make bread is acted to asymmetrically reduce the ketone group to an alcohol of (R) -form. The present invention relates to a method for producing optically active (R) -2-chloromandelic acid and esters thereof by asymmetric reduction.

The (R) -2-chloromandelic acid and ester compounds thereof are used as intermediates for the synthesis of Flavix, which is used as an antithrombotic agent. Flavix is an antithrombotic agent released by Sanofi in 1998 and its chemical name is (S) -clopidogrel bisulfate, and is represented by the following Chemical Formula 1.

According to the manufacturing method of (S) -clopidogrel bisulfite (US Patent No. 4,847,265) published by Sanofi in 1989, Sanofi was treated with tetrahydro in 2-chloromandelic acid of racemic body. After the introduction of tetrahydrothieno [3,2-c] pyridine, the chemical activity of camphorsulfonic acid was used to determine the optical activity of (S)-. Clopidogrel bisulfite was prepared. This method has a disadvantage in that yield and economical efficiency are reduced by performing chemical division at the rear end of the reaction.

Methods for obtaining optically active (R) -2-chloromandelic acid as an intermediate include chemical cleavage, asymmetric catalysis or enzymatic resolution. In the presentation, such as the 2001 Noda Hirobumi (NODA hirobumi) JP-A No. 2001-072644 (R) - N - benzyl-1-phenylethylamine ((R) - N - benzyl -1-phenylethylamine) dividing the was used as a resolving agent to produce diastereomeric salts of racemic 2-chloromandelic acid, which were recrystallized to 99.0% pure salts with diastereomeric excess. . Yield at this time was 90.4%. The diastereomeric salts thus prepared were made into (R) -2-chloromandelic acid by acidification, with optical purity of 100% ee and yield of 90%. However, this method has the disadvantage that theoretical yield does not exceed 50% and requires recrystallization process.

In 2002, North Michael et al. Published a method for synthesizing optically active (R) -2-chloromandelic acid using a catalyst having a chiral ligand in WO 02-10095. It was. This method uses cyanation and acetylation of 2-chlorobenzaldehyde using salen-type chiral ligands and Ti. Was obtained in a yield of 88.6% (R) - O - acetyl - cyanohydrin ((R) - O -acetyl- cyanohydrin) is I have the ee of 88.3%, through the hydrolysis thereof, (R) -2- chloro-only Prepare delic acid. The recrystallization process using (R) -2-chloromandelic acid thus obtained as a seed (seed) to obtain 99% ee (R) -2-chloromandelic acid. This method has a higher theoretical yield than the chemical splitting method, but has a disadvantage in that the reaction temperature for asymmetric reaction must be maintained at -42 ° C and the recrystallization process must be repeated several times.

Cyanohydrin is also used in enzymatic reactions. In 2001, Diva (Co.) used cyanohydrin using nitrileases in European Patent No. 1252589 and WO 01-48175. A method for preparing (R) -2-chloromandelic acid at 98% ee was disclosed through hydrolysis and continuous racemization of. The nitrile used at this time is an enzyme made using Diver's Direct evolution technology. This method also has higher yield than the chemical splitting method, but there are many problems to be solved such as mass production of newly produced enzyme. It has a hard disadvantage.

Therefore, it is necessary to develop a process that can produce economically superior (R) -2-chloromandelic acid or ester derivatives thereof having excellent optical purity and high optical purity by using a simple process for the same reason.

In order to solve the problems described above, the present inventors use alkyl 2-chloro-benzoylformate as a starting material to develop an economical manufacturing process, and use cheap and easily obtained yeast. A method for preparing (R) -2-chloromandelic acid and its ester compound has been developed by a simple manufacturing process called asymmetric reduction of α-keto ester, and the present invention has been completed based on this. Since the asymmetric reduction reaction yields a theoretical yield of 100%, it is possible to obtain significantly higher productivity compared to enzyme or chemical splitting (50% of theoretical yield), and because yeast (Baker's Yeast) is not only easily available but also very cheap. It is possible to develop an economical manufacturing process compared to the existing method.

Accordingly, an object of the present invention is to use an optical microorganism (R) -2-chloro from an alkyl 2-chloro-benzoylformate, which is a relatively simple manufacturing process using a microorganism called yeast, which is an environmentally friendly process and an hourly production efficiency is increased. It is to provide a method for producing mandelic acid and ester compounds thereof.

The production method of the present invention for achieving the above object consists in selectively reducing the alkyl 2-chloro-benzoylformate to yeast in the presence of a phosphate buffer solution.

Looking at the present invention in more detail as follows.

As described above, in the present invention, alkyl 2-chloro-benzoylformate was used as a substrate for asymmetric reduction reaction by yeast, and this compound used various alcohols using racemic 2-chloromandelic acid as a solvent in the presence of HCl. It is prepared by racemic alkyl 2-chloromandelate, which is prepared by oxidation using a phase transfer catalyst such as NaOCl and benzyltriethylammonium chloride.

The alkyl 2-chloro-benzoylformate thus prepared was added to a phosphate buffer solution at pH 6-8, and yeast was added without a separate treatment step, and the reaction mixture was stirred at 100-300 rpm at a temperature of 20-37 ° C. By selectively reducing the α-keto group, an alkyl (R) -2-chloromandelate having optical activity is prepared.

The asymmetric reduction process of the present invention is shown in Scheme 1 below.

Here, R is a lower alkyl group, preferably a methyl group, an ethyl group or a butyl group, more preferably a methyl group.

Preferred yeasts used in the present invention may be directly cultured as Saccharomyces cerevisiae Type II, and may be purchased and used as a product from a reagent company such as Sigma or many yeast production companies.

In the present invention, the enzyme reaction temperature is preferably 20 ~ 37 ℃, the lower the reaction temperature, the lower the reaction rate, the higher the reaction temperature, the lower the optical purity of the product was found to be about 30 ℃. In addition, the pH of the enzyme reaction was found to be in the proper range of pH 6 ~ 8, when the pH is less than 6.0, the reaction did not occur, when the pH exceeds 8.0 there is a problem that the optical purity falls, the optimum pH is 7.0 appear.

In addition, the ratio of the yeast and the substrate is preferably 1: 0.1 to 10 by weight ratio, but when the amount of the substrate is out of the range, the reaction efficiency tends to be inferior.

The optically active alkyl 2-chloromandelate prepared according to the present invention can be easily analyzed for R-form and S-form in HPLC using Chiralcel OD-H column, which is a chiral column of Daicel. Can be. In this case, for analysis by HPLC, a mobile phase containing n-hexane and isopropyl alcohol in a weight ratio of 97: 3 was flowed at a rate of 0.5 ml / min, and the absorbance was set at 254 nm, and the retention time of each product ( retention time) is shown in Table 1 below.

product Retention time Methyl (S) -2-chloromandelate 29.588 Methyl (R) -2-chloromandelate 36.258 Ethyl (S) -2-chloromandelate 24.723 Ethyl (R) -2-chloromandelate 29.713 Butyl (S) -2-chloromandelate 19.523 Butyl (R) -2-chloromandelate 23.038

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

Example 1

Preparation of Methyl 2-chloromandelate

2-chloromandelic acid (33 g, 0.18 mol) is dissolved in 100 ml of methanol, and then 2 ml of concentrated hydrochloric acid is added. The reaction is heated to 80 ° C. with stirring for 12 h. The reaction is cooled to room temperature and extracted using CH ₂ Cl ₂ . The extract is washed with saturated NaHCO ₃ solution and then water is removed with MgSO ₄ . The solvent CH ₂ Cl ₂ was removed and methyl 2-chloromandelate (32 g, 0.16 mol) was obtained through distillation.

Example 2

Preparation of Methyl 2-chloro-benzoylformate

Methyl 2-chloromandelate (32 g, 0.16 mol) obtained in Example 1 was dissolved in 50 mL of EtOAc, and aqueous NaOCl (13%, 81 mL, 0.22 mol) solution was added. Benzyltriethylammonium chloride (0.7 g, 3.2 mmol) was added thereto as a phase transfer catalyst, and the reaction was stirred at room temperature for 6 hours. After extracting the organic layer using EtOAc, water is removed with MgSO ₄ . The solvent was removed and distillation gave methyl 2-chloro-benzoylformate (29 g, 0.14 mol).

Example 3

Asymmetric Reduction of Methyl 2-Chloro-benzoyl Formate Using Yeast

In 150 ml of 100 mM phosphoric acid solution (pH 7.0), 1.5 g of methyl 2-chloro-benzoylformate, 7.5 g of yeast ( Saccharomyces cerevisiae Type II, 4.5 g of glucose) were added, and the reaction was performed at 30 ° C. During the reaction, the pH was maintained between 6.5 and 7.5 using 20% sodium hydroxide solution, and reaction samples were taken at regular intervals to analyze the progress of the reaction. % Was recorded and the optical purity of the resulting product, methyl (R) -2-chloromandelate, was 98.5% ee.

Example 4

Preparation of (R) -2-chloromandelic acid

NaOH (0.6 g, 0.015 mol) is dissolved in 20 ml water and methyl 2-chloromandelate (2.2 g, 0.011 mol) of 98.5% ee optical purity obtained in Example 3 is added. After stirring for 6 hours at room temperature, the mixture was extracted with EtOAc under acidic conditions. The solvent was removed and the product obtained was recrystallized from toluene / hexane to give pure (R) -2-chloromandelic acid (1.5 g, 0.008 mol, ee> 99%). ([a] _d = -131.94, c = 3, H ₂ O, 30.6 ° C).

Example 5

Effect of temperature on asymmetric reduction

150 g of 100 mM phosphoric acid solution (pH 7.0) was added 1.5 g of methyl 2-chloro-benzoylformate, 7.5 g of Saccharomyces cerevisiae Type II and 4.5 g of glucose, followed by temperature 20, 30 and 37. The reaction was carried out by changing to ℃. The pH during the reaction was kept between 6.5 and 7.5 using 20% sodium hydroxide solution. Reaction samples were taken at regular intervals to analyze the reaction progress. The reaction results according to the reaction temperature are shown in Table 2 below. As a result of the reaction, the reaction rate was increased at 37 ° C., but the optical purity of the product was lowered.

Effect of temperature on asymmetric reduction Reaction temperature (℃) Reaction time (hr) GC conversion rate (%) Optical purity (% ee) 20 7 99.8 98.2 30 4 99.9 98.5 37 2 99.8 93.4

However, even when the optical purity is poor in the reaction, when the hydrolysis and recrystallization reaction is carried out under the same conditions as in Example 4, the optical purity is 99% e.e. The above (R) -2-chloromandelic acid could be manufactured.

Example 6

Effect of pH on Asymmetric Reduction

In the reaction conditions of Example 5, the reaction temperature was maintained at 30 ° C., and the other conditions were the same, and the influence of pH was measured under conditions such that the reaction pH was maintained between pH 6, pH 6.5 to 7.5, and pH 8 and above. As a result, the reaction did not occur below pH 6, and the conversion rate was maintained at 99% or more when the pH was exceeded 8, but the optical purity of the product was about 93.5% ee, compared to the case where the pH was maintained between 6.5 and 7.5. fell. Thus, the optimum pH of the reaction was found to be 7.0.

However, even when the optical purity is poor in the reaction, when the hydrolysis and recrystallization reaction is carried out under the same conditions as in Example 4, the optical purity is 99% e.e. The above (R) -2-chloromandelic acid could be manufactured.

Example 7

Effect of Substrate on Asymmetric Reduction

In order to investigate the effect of the substrate on the asymmetric reduction reaction using yeast, the experiment was changed to another ester. At this time, the reaction conditions are the same as in Example 3. Experimental results showed that all other ester compounds showed good results in terms of optical purity, and methyl 2-chloro-benzoylformate showed the best results in terms of reaction time and conversion rate. Comparative experiment results are shown in Table 3 below.

Effect of Substrate on Asymmetric Reduction Substrate Response time (hr) GC conversion rate (%) Optical purity (% ee) Methyl 2-chloro-benzoylformate 4 99.9 98.4 Ethyl 2-chloro-benzoylformate 4 88.5 98.5 Butyl 2-Chloro-benzoylformate 5 99.7 98.4

As described above, the method of the present invention has the effect of producing a high optical purity optically active (R) -2-chloromandelic acid and its ester compound by a simple method using yeast. Compared with the conventional manufacturing method, the production process is relatively simple and the price is very low, and by using yeast which can be easily obtained, it is an eco-friendly process and the production efficiency per hour is increased, so it is economical and easy for mass production. to be. In particular, when methyl 2-chloro-benzoylformate is used as a reaction substrate, optical purity of methyl (R) -2-chloromandelate, which is used as an intermediate of Plavix ((S) -clopidogrel bisulfite), a major thrombotic drug 98% ee It can be made into the above products, there is an advantage that does not require additional hydrolysis and recrystallization process.

Claims (6)

Optically active (R) -2-chloromandelic acid and its esters, wherein the alkyl 2-chloro-benzoylformate is selectively reduced in the presence of a phosphate buffer solution using a yeast (Baker's Yeast). Process for the preparation of derivatives. The method of claim 1 wherein the alkyl 2-chloro-benzoylformate is methyl 2-chloro-benzoylformate. The method of claim 1, wherein the yeast is Saccharomyces cerevisiae Type II. The method of claim 1, wherein the temperature of the selective reduction reaction is 20 to 37 ℃, pH is 6.0 to 8.0. The method of claim 4, wherein the temperature of the selective reduction reaction is 30 ° C. and the pH is 7.0. The method of claim 1, wherein the ratio of the yeast and the substrate in the selective reduction reaction is 1: 0.1 to 10 by weight ratio.