KR100722155B1 - The method of making optically active 2-chloromandelic acid esters and 2-chloromandelic acids by enzymatic method - Google Patents

Abstract

The present invention provides an optical activity represented by the general formula (2) used as a chiral intermediate from the racemic 2-chloromandelic acid ester represented by the general formula (1) in the following [Scheme 1] A method for producing 2-chloromandelic acid ester and an acid represented by the general formula (3). More specifically, the reaction of the racemic 2-chloromandelic acid ester, which is a reactant in an aqueous solution or an aqueous solution containing a solvent, by stereoselective hydrolysis using a hydrolase or a strain comprising the optically active 2-chloroman The present invention relates to a process for preparing a delic acid ester and an optically active 2-chloromandelic acid.

The present invention presents a novel method for the preparation of the above-mentioned optically active 2-chloromandelic acid esters and acids, and is believed to be useful for the actual process due to its high optical purity and ease of preparation and separation of the product.

[Scheme 1]

(Substituent R of the compound is substituted or unsubstituted alkyl group or alkenyl group having 1 to 8 carbon atoms, benzyl group, cycloalkyl group having 3 to 6 carbon atoms, substituted or unsubstituted arylalkyl group, and substituted or unsubstituted heteroaryl It is selected from the group consisting of alkyl groups.)

2-chloromandelic acid, lipase, optical activity, hydrolysis reaction, clopidogrel

Description

The method of making optically active 2-chloromandelic acid esters and 2-chloromandelic acids by enzymatic method}

The present invention relates to an optically active 2-chloromandelic acid ester by an enzyme or a microorganism and a method for preparing an acid thereof. More specifically, the racemic 2-chloromandelic acid ester represented by the general formula (1) in [Scheme 1] using a microorganism having a hydrolase or a hydrolase production capacity is represented by the general formula (2). The present invention relates to a method of hydrolyzing an optically active 2-chloromandelic acid ester and 2-chloromandelic acid represented by the general formula (3).

(Substituent R of the compound is substituted or unsubstituted alkyl group or alkenyl group having 1 to 8 carbon atoms, benzyl group, cycloalkyl group having 3 to 6 carbon atoms, substituted or unsubstituted arylalkyl group, and substituted or unsubstituted heteroaryl Is selected from the group consisting of alkyl groups.)

The above-mentioned optically active 2-chloromandelic acid ester is widely used as a chiral intermediate required for the synthesis of clopidogrel bisulfate ((S) -Clopidogrel bisulfate). The tosylated or mesylated compounds can be epoxidized under inorganic salt conditions to be converted into optically active 2-chlorostyrene oxide, which is an important pharmaceutical intermediate applied to new drugs in various fields (Republic of Korea). 2005-0035057).

Noda Hirobumi et al. (Japanese Patent No. 0072644) use diastereomers from racemic 2-chloromandelic acid using optically active N-benzyl-1-phenylbutylamine as a resolution agent. Isomer salts were prepared and acidified to afford (R) -2-chloromandelic acid. This process has the disadvantage of requiring a recrystallization process. In a similar manner, Hyoda Toshiharu et al. (Japanese Patent 0114737) have obtained the material using optically active alanine as a splitting agent.

North michael et al. (International Patent No. 00210095) use a chiral ligand and a chiral catalyst to undergo cyanation and acetylation of 2-chlorobenzaldehyde to obtain (R) -O -acetyl-cyanohydrin. Obtained and hydrolyzed to obtain (R) -2-chloromandelic acid. This method is difficult to carry out the reaction and requires several recrystallization processes.

On the other hand, there are the following methods using the enzyme and strain.

Kim, Jong-Keun et al. (Korean Patent Laid-Open Publication No. 10-2004-0063264) prepared (R) -2-chloromandelic acid and ester derivatives thereof by using yeast (Baker's yeast). A 2-chloromandelate alkyl ester compound was prepared from 2-chloromandelic acid and synthesized again with alkyl 2-chloro-benzoylformate, followed by asymmetric reduction using yeast. reduction). In the case of methyl 2-chloro-benzoylformate, ethyl 2-chloro-benzoylformate, and butyl 2-chloro-benzoylformate, the conversion was 99.9%, 88.5%, and 99.7% after 4 hours, respectively, and the optical purity was 98.45 ee%, respectively. , 98.5 ee%, 98.4 ee%. This process has the disadvantage of undergoing several synthetic steps before the reaction with yeast.

Eom, Ki-Nam et al. (Republic of Korea Patent Publication No. 10-2005-0035057) prepared 2-chloromandelic acid ester and then (R) -2-chloromandelic acid ester (B) by ester reaction using a hydrolytic enzyme in an organic solvent. 100 ee%) and (S) -2-chloromandelic diester were optically divided. However, this process has a disadvantage in that it is not easy to separate each product after the reaction.

Van Langen et al. (Organic Process Research & Development, 2003, 7, 828-831) use 2- (2-chlorophenyl) -2- using oxynitrilase from 2-chlorobenzaldehyde. Hydroxyacetonitrile (2- (2-chlorophenyl) 2-hydroxyacetonitrile) was prepared, and hydrolyzed and recrystallized under acidic conditions to prepare (R) -2-chloromandelic acid having an optical purity of 99 ee%.

Thus, the present inventors have attempted to develop a process in which the reaction is simple and the product is easy to recover instead of the previously reported manufacturing method. The production method according to the present invention hydrolyzes racemic 2-chloromandelic acid esters using a hydrolase or a strain comprising the same, thereby optically active 2-chloromandelic acid esters and optically active 2-chloromandelic acid It is a new process that has not been reported so far as a method for preparing. In addition, 2-chloromandelic acid esters and acids thereof prepared according to the present invention can be advantageously used in practical processes because they are easy to separate and recover.

Looking at the present invention in more detail as follows. As described above, the present invention is stereoselectively hydrolyzed by using a hydrolase or a strain comprising the same from racemic 2-chloromandelic acid ester, thereby optically active 2-chloromandelic acid ester and optically active 2-chloroman. To a process for preparing delic acid. In addition, the optically active 2-chloromandelic acid obtained through the reaction can be converted to the optically active 2-chloromandelic acid by hydrolysis using sodium hydroxide (NaOH) and the like.

Enzymes used in the present invention include CAL A (novozyme 735), immobilized lipase CAL B (Novozyme 435, Novozyme) and alcalase, Protease A or these hydrolytic enzymes. There is a strain that is not limited thereto.

In the present invention, the reactants and products used in the reaction were analyzed under the following conditions.

The racemic 2-chloromandelic acid esters were analyzed using gas chromatography (Donan Instruments, Model DS6200) equipped with a capillary column HP-FFAP (Agilent). After heating at 100 ° C. for 5 minutes, the temperature was raised to 220 ° C. by 20 ° C. per minute, and maintained at 220 ° C. for 10 minutes. Helium gas was used as the carrier, and the column head pressure was maintained at 6 psi. Detected. Under these conditions, methyl 2-chloromandelate was detected at 15.31 minutes, ethyl 2-chloromandelate at 14.89 minutes and butyl 2-chloromandelate at 17.28 minutes, respectively.

Optically active 2-chloromandelic acid esters were analyzed using HPLC (Lab Aliance, Model 201) equipped with chiral column AD-H (Daicel). Hexane and isopropanol were mixed at a ratio of 90:10 and flowed at 0.7 ml per minute, and the UV absorbance was analyzed at 220 nm. At this time, (S) and (R) -methyl 2-chloromandelate were detected at 12.71 and 13.93 minutes, respectively, and (S) and (R) -ethyl 2-chloromandelate were detected at 12.00 and 13.95 minutes, respectively. (S) and (R) -butyl 2-chloromandelate were detected at 10.36 and 12.06 minutes, respectively.

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 Racemic Alkyl 2-chloromandelate

2 g of racemic 2-chloromandelic acid was added to 40 ml of methanol, and a 35% hydrochloric acid solution was added thereto and reacted at 75 ° C. for 3 hours. The product was neutralized and extracted with an organic layer, followed by removal of the solvent through distillation under reduced pressure. Racemic ethyl 2-chloromandelate and butyl 2-chloromandelate were prepared after the reaction under the above conditions using ethanol and butanol instead of methanol, respectively. The product thus prepared was purified on silica gel and confirmed to be racemic 2-chloromandelic acid ester by nuclear magnetic resonance (Burker, model DRX300). The analysis results are as follows.

Methyl 2-chloromandelate

¹ H-NMR (CDCl ₃ , 300 MHz) δ (ppm) = 3.76 (s, 3H), 5.57 (s, 1H), 7.27 (m, 2H), 7.39 (m, 2H)

Ethyl 2-chloromandelate

¹ H-NMR (CDCl ₃ , 300 MHz) δ (ppm) = 1.21 (t, 3H), 4.21 (m, 2H), 5.54 (s, 1H), 7.27 (m, 2H), 7.38 (m, 2H)

Butyl 2-chloromandelate

¹ H-NMR (CDCl ₃ , 300 MHz) δ (ppm) = 0.83 (t, 3H), 1.23 (m, 2H), 1.54 (m, 2H), 4.16 (t, 2H), 5.56 (s, 1H), 7.25 (m, 2H), 7.38 (m, 2H)

Example 2-3. Preparation of Optically Active Methyl 2-chloromandelate Through Hydrolysis

To a vial containing 5 ml of 0.1 M potassium phosphate buffer (pH 7.0), add racemic methyl 2-chloromandelate produced by the method of Example 1 to 1% (w / v) and The reaction was carried out at 30 ° C. using 1% (w / v) of the enzyme described in 1. After a certain reaction, the product was extracted with ethyl acetate and analyzed according to the above analysis method. The results are shown in Table 1.

Example Enzyme Type Response time (hr) % Conversion Optically active methyl 2-chloromandelate (ee%) shape 2 CAL A 67 94.5 > 99 R 3 CAL B 67 79.5 95.9 R

Example 4-7. Preparation of optically active ethyl 2-chloromandelate via hydrolysis reaction

Instead of methyl 2-chloromandelate used as a reactant in Example 2 using ethyl 2-chloromandelate using the enzymes listed in Table 2 and the results are as follows.

Example Enzyme Type Response time (hr) % Conversion Optically active ethyl 2-chloromandelate (ee%) shape 4 Alcalase 2.5L 87 79.2 93.4 S 5 CAL A 46 89.3 99.3 R 6 CAL B 22 63.4 99.8 R 7 Protease A 87 92.4 99 S

Example 8. Preparation of Optically Active Butyl 2-Chloromandelate Through Hydrolysis Reaction

Butyl (R) having an optical purity of 76.0 ee% when the conversion rate was 93.1% using butyl 2-chloromandelate instead of methyl 2-chloromandelate used as a reactant in Example 2 and reacting with CAL B for 48 hours. ) -Chloromandelate was prepared.

Example 9 Preparation of Optically Active (R) -2-chloromandelic Acid

Racemic ethyl 2-chloromandelate was obtained by hydrolysis reaction with CAL B used in Example 6 as ethyl (R) -2-chloromandelate of optical purity 99 ee%. The collected ethyl (R) -2-chloromandelate was dissolved in methanol, 10% (w / v) sodium hydroxide solution was added, and then sufficiently reacted at room temperature. The reaction solution was then adjusted to pH 1 using hydrochloric acid solution. And t-butyl methyl ester (t-BME) to give a white solid as a product. The analysis results confirmed that the optical purity was 99 ee% (R) -2-chloromandelic acid.

As can be seen through Example 2-9, in the method for preparing the optically active 2-chloromandelic acid ester, the hydrolysis reaction according to the present invention is easy to react, it is possible to obtain a product of high optical purity have. In addition, it is easy to recover each optically active ester and 2-chloromandelic acid after the reaction is a suitable manufacturing process for industrial mass production.

Claims (2)

The racemic 2-chloromandelic acid ester represented by the general formula (1) in the following [Scheme 1] is Candida antarctica , Bacillus subtilis or Aspergillus orisase ( Aspergillus oryzae). Lipase derived from a microorganism selected from C.) or a microorganism having a lipase-generating ability selected from Candida antarctica, Bacillus subtilis, or Aspergillus orisase, represented by the general formula (2). Method for producing an optically active 2-chloromandelic acid ester and the optically active 2-chloromandelic acid represented by the general formula (3). Scheme 1

(Substituent R of the compound is substituted or unsubstituted alkyl group or alkenyl group having 1 to 8 carbon atoms, benzyl group, cycloalkyl group having 3 to 6 carbon atoms, substituted or unsubstituted arylalkyl group, and substituted or unsubstituted heteroaryl Is selected from the group consisting of alkyl groups.) delete