JP2009114065A - Method for producing (2r,3r) and (2s,3s)-3-phenylisoserine derivative - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing (2R,3R) and (2S,3S)-3-phenylisoserine derivatives useful as intermediates of medicines, simply and industrially advantageously. <P>SOLUTION: It is possible to remove regioisomers as byproducts efficiently by reacting a (2R,3S) or a (2S,3R)-3-phenylglycidic acid salt with ammonia to make a (2R, 3R) or a (2S,3S)-3-phenylisoserine salt, or further neutralizing to the (2R,3R) or (2S,3S)-3-phenylisoserine, and then performing purification by crystallization. Also, it is possible to remove the regioisomers and other impurities efficiently by esterifying the above compound either treated or not treated with the crystallization, by a normal method and then purifying by crystallization. By the above methods, it is possible to produce the (2R,3R) and (2S,3S)-3-phenylisoserine derivatives simply and also industrially advantageously. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a process for producing (2R, 3R) and (2S, 3S) -3-phenylisoserine derivatives useful as pharmaceutical intermediates.

  As a method for producing (2R, 3R) and (2S, 3S) -3-phenylisoserine derivatives, for example, a 3-phenylglycidate salt in which a phenyl group and a carboxyl group are in a trans position is reacted with ammonia. In addition, there is known a method (Patent Document 1) in which erythro-3-phenylisoserine salt is esterified and then resolved using optically active tartaric acid.

  However, since the above method is a split method, the maximum yield is 50%, and it is difficult to say that it is a practical and economical method for industrial production.

  If the optically active (2R, 3R) and (2S, 3S) -3-phenylglycidate are reacted with ammonia, (2R, 3R) and (2S, 3S) -3-phenylisoserine will not be resolved. Salts can be obtained and (2R, 3R) and (2S, 3S) -3-phenylisoserine can be obtained by neutralization, and (2R, 3R) and (2S, 3S) -3 by further esterification. -Phenylisoserine ester can be obtained. As such a method, although a method of ring-opening an optically active 3-phenylglycidic acid derivative obtained by asymmetric epoxidation of a cinnamic acid derivative is known, a specific method is not disclosed, and industrial methods are not disclosed. It is unclear whether it can be implemented (Patent Document 2).

  In addition, although many examples of methods for obtaining optically active 3-phenylglycidate by asymmetric epoxidation of cinnamic acid derivatives have been reported (for example, Non-Patent Documents 1 to 3), all of them are expensive. In addition, it is necessary to use a reagent or a catalyst that is concerned about toxicity, and there are many points to be improved in order to obtain an industrially advantageous production method.

  On the other hand, the optically active 3-phenylglycidate is an optically active 2-chloro-3-hydroxy-3-acid obtained by asymmetric reduction of 2-chloro-3-oxo-3-phenylpropionic acid ester. Phenylpropionic acid ester is intramolecularly ring-closed in a suitable solvent (eg, lower alkanol) in the presence of a base (eg, alkali metal alkoxide) at −10 to 50 ° C., particularly 0 ° C. to room temperature, and water is added thereto. It can also be obtained by adding and hydrolyzing the ester group.

  As a method for producing an optically active 2-chloro-3-hydroxy-3-phenylpropionic acid ester derivative, asymmetric reduction of methyl 2-chloro-3-oxo-3- (p-methoxyphenyl) propionate using a microorganism Thus, a method for producing methyl 3R or 3S-2-chloro-3-hydroxy-3- (p-methoxyphenyl) propionate is known (Patent Document 3). However, the feed concentration (0.2%) and the yield (1-60%) are low, and it is difficult to say that it is a practical production method. Moreover, Arthrobacter protoformier, Streptomyces olivochromogenes, etc. have been reported as microorganisms that produce anti-isomers, particularly (2S, 3S) isomers, with a yield of 5%. It is as follows.

  Even if optically active 3-phenylglycidate is obtained, it is difficult to avoid by-production of 3-phenylserine salt, which is a regioisomer, in the reaction with ammonia. The removal method is not known.

  As an isolation and purification method, an isolation and purification method by crystallization of (2R, 3S) -3-phenylisoserine salt, which is a diastereomer of the compound, is known (Patent Document 4). It is unclear whether (2R, 3R) and (2S, 3S) -3-phenylisoserine salts will be isolated.

Therefore, development of a practical and economical production method of (2R, 3R) and (2S, 3S) -3-phenylisoserine derivatives useful as pharmaceutical intermediates is strongly desired.
WO2003 / 003804 WO2005 / 058893 JP 7-79706 6-504549 Tetrahedron 1994, 50, 4323 J. et al. Am. Chem. Soc. 2002, 124, 14544 J. et al. Org. Chem. 2004, 69, 4217

  In view of the above, an object of the present invention is to provide a practical method by which (2R, 3R) and (2S, 3S) -3-phenylisoserine derivatives can be produced conveniently and industrially advantageously.

  As a result of intensive studies in view of the above, the present inventors have reacted (2S, 3S) or (2R, 3R) -3-phenylglycidic acid or a salt thereof with ammonia (2R, 3R) or (2S, 3S) -3-Phenylisoserine or a salt thereof, and then esterified by a conventional method (2R, 3R) or (2S, 3S) -3-phenylisoserine alkyl ester or a salt thereof in the process of (2R) , 3S) or (2R, 3S) -3-phenylglycidic acid or its alkali salt and (2S, 3S) or (2R, 3R) -3-phenylserine or its salt produced as a by-product in the reaction of ammonia (2S, 3S) or (2R, 3R) -3-phenylserine ester or a salt mixed as an impurity in esterification is efficiently removed by crystallization. It found that can be, and have completed the present invention.

  That is, the present invention relates to the general formula (1);

(In the formula, R ¹ represents a phenyl group which may have a substituent, R ² represents a hydrogen atom, an alkali metal, an alkaline earth metal, or a nitrogenous base, and * ₁ is an asymmetric carbon atom. The steric configuration is represented by (2R, 3S) or (2S, 3R)) and a compound represented by the general formula (2) obtained by reaction of ammonia;

(Wherein R ¹ and R ² represent the same meaning as described above, * ₂ represents an asymmetric carbon atom, and the configuration thereof represents (2R, 3R) or (2S, 3S)). Further, the present invention relates to a method for producing a compound represented by the formula (2), which is further subjected to a crystallization step.

  Moreover, this invention makes it the compound represented by the said Formula (2) by reaction with the compound represented by the said Formula (1), and ammonia, and is then esterified, General formula (3) obtained;

(Wherein R ¹ and * represent the same meaning as described above, and R ³ represents a C _{1 to} C ₄ alkyl group), and further subjected to a crystallization step. The present invention relates to a method for producing a compound represented by formula (3).

    Further, the present invention provides a general formula (5);

(Wherein R ¹ and R ³ represent the same meaning as described above, X represents a halogen atom) an enzyme source having the ability to stereoselectively reduce the carbonyl group of the compound is acted on General formula (7) by

(Wherein R ¹ , R ³ and * ₂ represent the same meaning as described above).

  According to the present invention, (2R, 3R) and (2S, 3S) -3-phenylisoserine derivatives useful as pharmaceutical intermediates can be conveniently and industrially advantageously produced.

  Hereinafter, the present invention will be described in detail. In the present invention, “may have a substituent” means that it may be substituted with another atom or substituent. The “substituent” is not particularly limited as long as it does not adversely affect the reaction. Specifically, the hydroxyl group, alkyl group, alkoxy group, alkylthio group, nitro group, amino group, cyano group, carboxyl group, A halogen atom etc. are mentioned.

  First, general formula (1);

The production method of the compound represented by In the compound represented by the formula (1) (hereinafter referred to as compound (1)), R ¹ represents a phenyl group which may have a substituent. Examples of the phenyl group that may have a substituent include a 4-hydroxyphenyl group, a 4-methoxyphenyl group, a 4-chlorophenyl group, and a phenyl group, and a phenyl group is preferable. R ² represents a hydrogen atom, an alkali metal, an alkaline earth metal, or a nitrogenous base. Needless to say, compound (1) is in the form of a carboxylate salt with R ^{2 when it} is an alkali metal, alkaline earth metal, or nitrogenous base. Examples of the alkali metal include lithium, sodium, and potassium, and examples of the alkaline earth metal include magnesium, calcium, and barium. Nitrogenous bases include ammonia and primary amines such as methylamine, benzylamine and cyclohexylamine, secondary amines such as dimethylamine, dibenzylamine and dicyclohexylamine, and tertiary amines such as triethylamine and tributylamine. Kind. R ² is preferably an alkali metal such as lithium, potassium, sodium, or hydrogen, more preferably sodium or hydrogen, and particularly preferably sodium. * ₁ is an asymmetric carbon atom, and the configuration thereof is (2R, 3S) or (2S, 3R), preferably (2R, 3S).

  Although it does not restrict | limit especially as a method to obtain a compound (1), The method of obtaining (2R, 3S) -3-phenylglycidic acid salt which can be implemented on an industrial scale is demonstrated below.

  General formula (5);

A carbonyl group of the compound represented by formula (6):

Or general formula (7);

A method obtained by hydrolyzing an ester group after treating a compound represented by the formula with a base and cyclizing is preferable. Here, the compound represented by formula (5) (hereinafter referred to as compound (5)), the compound represented by formula (6) (hereinafter referred to as compound (6)) and the compound represented by formula (7) ( In the following compound (7)), R ¹ is as described above. R ³ represents a C ₁ -C ₄ alkyl group. The alkyl group of C ₁ -C _4, a methyl group, an ethyl group, n- propyl group, an isopropyl group, n- butyl group, isobutyl group, sec- butyl group, and a t- butyl group, preferably an ethyl group It is. X represents a halogen atom, and examples thereof include fluorine, bromine, chlorine, and iodine. Preferred are chlorine and bromine, with chlorine being particularly preferred. * Represents an asymmetric carbon atom, and the configuration is preferably S. * ₂ represents an asymmetric carbon atom, and the configuration thereof is (2R, 3R) or (2S, 3S), preferably (2S, 3S).

  First, selection of the compound (6) and the compound (7) will be described. It is known that when compound (6) is treated with a base and subjected to a cyclization reaction, a trans-type glycidic acid derivative is produced regardless of the configuration at the 2-position (see Tetrahedron 1995, 6, 2211). That is, the compound (1) can be obtained regardless of the stereo at the 2-position, and either may be selected. However, in order to perform the cyclization reaction with higher selectivity, the compound (7) is selected. It is preferable.

  The method of asymmetric reduction is not particularly limited as long as it is a method that gives compound (6) or compound (7) with high selectivity, but a method of reducing using a hydride reducing agent modified with an optically active compound, Examples thereof include a method of hydrogenation in the presence of an asymmetric transition metal catalyst, a method of reduction by a hydrogen transfer type in the presence of an asymmetric transition metal catalyst, or a method of reduction using a microorganism or a microorganism-derived enzyme. For industrial implementation, a reduction method using an enzyme derived from a microorganism is preferred. Hereinafter, for example, a method for obtaining the compound (7) by stereoselectively reducing the compound (5) in the presence of an enzyme source having the ability to stereoselectively reduce the carbonyl group of the compound (5) will be described. .

  Here, the “enzyme source” includes not only the enzyme having the reducing activity itself, but also a culture of microorganisms having the reducing activity and processed products thereof. The “microorganism culture” means a culture solution or culture containing microbial cells, or may be a processed product thereof. The “processed product” means, for example, a crude extract, freeze-dried cells, acetone-dried cells, or a crushed product of these cells. Furthermore, the enzyme source can be immobilized by a known means and used as an immobilized enzyme or an immobilized cell. Immobilization can be performed by methods well known to those skilled in the art (for example, a crosslinking method, a physical adsorption method, a comprehensive method, etc.).

In the present invention, the enzyme source having an ability to reduce the compound carbonyl group (5) (2S, 3S) selectively, Deboshia (Devosia) genus Paenibacillus (Paenibacillus) genus, or belonging to Pseudomonas (Pseudomonas) genus enzyme source derived from a microorganism and the like, preferably, Deboshia-Ribofurabina (Devosia riboflavina), Paenibacillus alvei (Paenibacillus alvei), Pseudomonas putida (Pseudomonas putitda), or Pseudomonas Sututozeri (Pseudomonas stutzeri) microbial enzymes derived from such Sources are mentioned. Examples of the enzyme source having the ability to selectively reduce (2R, 3R) the carbonyl group of the compound (5) include an enzyme source derived from a microorganism belonging to the genus Devosia , preferably, Devosia riboflavina ( Devosia riboflavina ) derived enzyme source.

  The microorganism from which the reductase is derived may be either a wild strain or a mutant strain. Alternatively, microorganisms derived by genetic techniques such as cell fusion or genetic manipulation can also be used. Furthermore, it may be a recombinant microorganism having the ability to produce a reductase derived from these microorganisms. The recombinant microorganism producing the enzyme is, for example, a step of isolating and / or purifying the enzyme to determine part or all of the amino acid sequence of the enzyme, and a DNA sequence encoding the enzyme based on the amino acid sequence. The method can be obtained by a method comprising the steps of: obtaining the recombinant microorganism by introducing the DNA into another microorganism; and culturing the recombinant microorganism to obtain the enzyme (WO 98/35025). reference).

Examples of the recombinant microorganism as described above include a transformed microorganism transformed with a vector having a DNA encoding the reductase. The host microorganism is preferably Escherichia coli . More preferably, the Deboshia-Ribofurabina (Devosia riboflavina) NBRC13584 strain derived from carbonyl reductase gene (WO2004 / 027 055 reference) Escherichia coli HB101 transformed with a vector having (pNTDR) Accession No. FERM BP-08457 (2002 May 29 Tsukuba City East 1-1-1 Tsukuba Center Ibaraki Pref. Central 6th National Institute of Advanced Industrial Science and Technology (AIST).

  The culture medium for the microorganism used as the enzyme source is not particularly limited as long as the microorganism can grow. For example, as a carbon source, sugars such as glucose and sucrose, alcohols such as ethanol and glycerol, fatty acids such as oleic acid and stearic acid and esters thereof, oils such as rapeseed oil and soybean oil, and ammonium sulfate as a nitrogen source , Sodium nitrate, peptone, casamino acid, corn steep liquor, bran, yeast extract, etc. Inorganic salts such as magnesium sulfate, sodium chloride, calcium carbonate, potassium hydrogen phosphate, potassium dihydrogen phosphate, etc. A normal liquid medium containing an extract, meat extract or the like can be used. The culture is performed aerobically. Usually, the culture time is about 1 to 5 days, the pH of the medium is 3 to 9, and the culture temperature is 10 to 50 ° C.

  In the present invention, the reduction reaction of the carbonyl group of the compound (5) is performed by adding the compound (5) serving as a substrate, the coenzyme NAD (P) H, the culture of the microorganism or a treated product thereof to an appropriate solvent, It can carry out by stirring under pH adjustment.

  The reaction conditions vary depending on the enzyme, microorganism or treated product thereof, substrate concentration, etc., but the substrate concentration is usually about 0.1 to 100% by weight, preferably 1 to 60% by weight, and the coenzyme NAD (P) H Is 0.0001 to 100 mol%, preferably 0.001 to 0.1 mol% with respect to the substrate, the reaction temperature is 10 to 60 ° C, preferably 20 to 50 ° C, and the pH of the reaction is 4 to 9, The reaction time is preferably 5 to 8, and the reaction time can be 1 to 120 hours, preferably 1 to 72 hours. In the reaction, an organic solvent may be mixed and used. Examples of the organic solvent include toluene, ethyl acetate, n-butyl acetate, hexane, isopropanol, methanol, diisopropyl ether, acetone, dimethyl sulfoxide and the like. The substrate can be added by batch or continuous addition. The reaction can be performed in a batch mode or a continuous mode.

  In the reduction step of the present invention, by using a commonly used coenzyme NAD (P) H regeneration system, the amount of expensive coenzyme used can be greatly reduced. A typical NAD (P) H regeneration system includes, for example, a method using glucose dehydrogenase and glucose.

  A transformed microorganism in which a gene of a reductase gene and an enzyme (for example, glucose dehydrogenase) having the ability to regenerate a coenzyme on which the enzyme depends are introduced into the same host microorganism, that is, a DNA encoding the reductase of the present invention And the same as described above, using a culture of a transformed microorganism in which a gene of an enzyme (for example, glucose dehydrogenase) having an ability to regenerate a coenzyme on which the enzyme depends is introduced into the same host microorganism or a processed product thereof. If this reaction is carried out, it is not necessary to separately adjust the enzyme source necessary for the regeneration of the coenzyme, and therefore the compound (7) can be produced at a lower cost.

The transformed microorganism as described above includes a transformed microorganism transformed with a plasmid having both the DNA encoding the reductase and the DNA encoding the enzyme having the ability to regenerate the coenzyme on which the enzyme depends. Can be mentioned. Here, as the enzyme having the ability to regenerate the coenzyme, glucose dehydrogenase is preferable, glucose dehydrogenase derived from Bacillus megaterium is preferable, and Escherichia coli is preferable as the host microorganism. . As such a preferred transforming microorganism, Escherichia coli HB101 (pNTRDG1) accession number FERM BP-08458 (May 29, 2002 Tsukuba City 1-1-1 Tsukuba Center Chuo 6 And deposited at the Patent Biological Depositary Center).

  Culture of transformed microorganisms can be performed using a normal liquid nutrient medium containing a carbon source, a nitrogen source, inorganic salts, organic nutrients and the like as long as they grow. In addition, the activity of an enzyme having a coenzyme regeneration ability in the transformed microorganism can be measured by a conventional method. For example, the activity of glucose dehydrogenase is obtained by adding 100 mM glucose, 2 mM coenzyme NADP or NAD, and an enzyme to 1 M Tris-HCl buffer (pH 8.0) and reacting at 25 ° C. for 1 minute. Of the absorbance at a wavelength of 340 nm.

  When the reduction step of the present invention is carried out in combination with a coenzyme regeneration system, or when a culture of the above-mentioned transformed microorganism or a treated product thereof is used as an enzyme source, a cheaper oxidized form is used as a coenzyme. It is also possible to carry out the reaction by adding NAD (P).

  The compound (7) produced by the reduction reaction can be purified by a conventional method. For example, the reaction solution is subjected to treatment such as centrifugation and filtration to remove suspensions such as cells, and then extracted with an organic solvent such as ethyl acetate and toluene, and the organic solvent is removed under reduced pressure. It can be purified by performing a treatment such as distillation or chromatography.

  The cyclization reaction of compound (7) can be performed by treating with a base in an appropriate solvent. Examples of the base to be used include sodium methoxide, sodium ethoxide, sodium tert-butoxide, lithium methoxide, lithium ethoxide, lithium tert-butoxide, potassium methoxide, potassium ethoxide, potassium tert-butoxide and the like. Alkali metal alkoxide, lithium hydride, sodium hydride, alkali metal hydride such as potassium hydride, alkaline earth metal hydride such as calcium hydride, alkali metal water such as sodium hydroxide, potassium hydroxide, cesium hydroxide Alkali earth metal hydroxides such as oxides, magnesium hydroxide and calcium hydroxide, alkali metal carbonates such as lithium carbonate, potassium carbonate and sodium carbonate, alkali metals such as lithium hydrogen carbonate, potassium hydrogen carbonate and sodium hydrogen carbonate It can be mentioned hydrogen phosphate. Of the above bases, alkali metal alkoxides are preferable, and sodium ethoxide is particularly preferable. The amount of the base used is not particularly limited as long as the reaction proceeds smoothly, but it is preferably 1 to 5 mole times the amount of compound (7), and 1 to 2 mole times from the economical viewpoint. Particularly preferred. The solvent to be used is not particularly limited, and examples thereof include alcohols such as methanol and ethanol, aprotic polar solvents such as dimethyl sulfoxide and dimethylformamide, ethers such as tetrahydrofuran and 1,4-dioxane, and the like. Among them, alcohols are preferable, and ethanol is particularly preferable.

  The subsequent ester hydrolysis may be carried out by a usual method (for example, see Experimental Science Course 22, edited by The Chemical Society of Japan, Maruzen Co., Ltd.), and is not particularly limited. For example, either an acid method or an alkali method is possible, but when excess sodium ethoxide is used in the cyclization reaction of the compound (7), sodium ethoxy remaining in the system by adding water is used. The sodium hydroxide becomes sodium hydroxide and the hydrolysis reaction can be carried out as it is. If the amount of remaining sodium ethoxide is small, a necessary alkali may be added additionally. Examples of the alkali to be used are as described in the cyclization reaction of the compound (7), preferably sodium hydroxide.

  The compound (1) thus obtained is obtained by reacting with ammonia by the general formula (2);

Is converted to a compound represented by the following (hereinafter referred to as compound (2)). Here, R ¹ and R ² are as described above. When R ² is a hydrogen atom, the amino group may form a salt with an appropriate acid. The acid forming the salt is not particularly limited, and examples include formic acid, acetic acid, hydrochloric acid, sulfuric acid, nitric acid, trifluoroacetic acid, methanesulfonic acid, trifluoromethanesulfonic acid, p-toluenesulfonic acid, and the like. Preferably, it is hydrochloric acid, sulfuric acid or methanesulfonic acid. * ₂ is the same as above, but is preferably a (2R, 3R) isomer. When the (2R, 3S) compound (1) is used as a raw material, the (2R, 3R) compound (2) is used, and when the (2S, 3R) compound (1) is used (2S, 3S) 3S) compound (2) is obtained.

  Next, the step of converting to compound (2) by amination reaction with compound (1) and ammonia will be described. The compound (1) used here may be obtained by the above-described method or may be obtained separately.

  In this step, the general formula (8);

Is generated as a by-product. In the compound represented by the formula (8) (hereinafter referred to as compound (8)), R ¹ and R ² are as described above. When R ² is a hydrogen atom, the amino group may form a salt with an appropriate acid. The acid forming the salt is not particularly limited, and examples include formic acid, acetic acid, hydrochloric acid, sulfuric acid, nitric acid, trifluoroacetic acid, methanesulfonic acid, trifluoromethanesulfonic acid, p-toluenesulfonic acid, and the like. Preferably, it is hydrochloric acid, sulfuric acid or methanesulfonic acid. * ₂ is the same as above. As for the configuration of compound (8), the opposite configuration to that preferentially generated in the generated compound (2) is preferentially generated.

  The reaction is carried out in an organic solvent and / or water. Examples of the organic solvent include chloroalkanes such as dichloromethane, chloroform and dichloroethane, substituted benzenes such as benzene and toluene, ethers such as diethyl ether, tetrahydrofuran and 1,4-dioxane. One or more solvents selected from alcohols such as methanol, ethanol, isopropanol, and t-butanol can be used. When using 2 or more types of mixed solvents, the mixing ratio is not specifically limited. In order to obtain the compound (2) with good yield, water or a mixed solvent of water and an organic solvent is preferable as the solvent, and water is more preferable.

  The amount of ammonia used is not particularly limited as long as the reaction proceeds smoothly and the compound (2) can be obtained with good yield, but is preferably 1 to 100 mole times the amount of the compound (1), In order to suppress the by-production of the compound (8) which is a positional isomer and to obtain the compound (2) with good yield, the amount is particularly preferably 5 to 50 mole times. Ammonia may be used as a gas or an aqueous solution or a solution of an appropriate organic solvent.

  If the reaction temperature is too low, it may take a long time to reduce the reaction yield or complete the reaction. Conversely, if the reaction temperature is too high, not only the by-product of the compound (8) will increase, Therefore, it is not realistic in manufacturing on an industrial scale. Preferably it is 0-100 degreeC, Most preferably, it is 20-80 degreeC. The reaction time may be carried out until the disappearance of the compound (1) is confirmed, and is generally about 1 to 36 hours.

  Compound (8) and other impurities can be efficiently removed by subjecting compound (2) obtained in this reaction to a crystallization step. Needless to say, the crystallization step may not be performed according to the required quality of the compound (2).

First, a method for crystallizing the compound (2) obtained after the reaction between the compound (1) and ammonia will be described. Crystallization is performed in the compound (2) in which R ² is an alkali metal, alkaline earth metal, or nitrogenous base and is in the form of a carboxylate, the compound (2) in which R ² is a hydrogen atom, or the compound (2). Although it can be carried out in any of the compounds (2) in which R ² is a hydrogen atom and the amino group forms a salt with an acid, the number of steps is reduced when considering production on an industrial scale. In addition, since R ² is an alkali metal, an alkaline earth metal, a nitrogenous base and is in the form of a carboxylate (2), or a compound (2 in which R ² is a hydrogen atom) ) Is preferable. More preferably, R ² is an alkali metal, alkaline earth metal, or nitrogenous base, and is performed as the compound (2) in the form of a carboxylate. In any crystallization, when the target quality cannot be obtained in one crystallization, the crystallization can be performed twice or more to improve the quality. The same crystallization method may be repeated, or crystallization may be performed in the form of different compounds (2).

  In this crystallization step, the compound represented by compound (8) can be efficiently removed. As a crystallization solvent, chloroalkanes such as dichloromethane, chloroform and dichloroethane, substituted benzenes such as benzene and toluene, ethers such as diethyl ether, methyl t-butyl ether, tetrahydrofuran and 1,4-dioxane, methanol, ethanol One or more solvents selected from the group consisting of alcohols such as isopropanol and t-butanol, alkanes such as hexane and pentane, esters such as ethyl acetate and isopropyl acetate, ketones such as acetone and methyl ethyl ketone, and water An optimum solvent may be selected according to the form of the compound (2) to be crystallized.

Preferably, general formula (4);
R ³ OH (4)
(R ³ is the same as above) and one or more solvents selected from the group consisting of alcohols and water, more preferably water and ethanol.

As an example of a method for crystallization as a compound (2) in which R ² is an alkali metal, an alkaline earth metal, a nitrogenous base and is in the form of a carboxylate after the amination reaction, R ² is sodium. The case will be described.

  As a crystallization solvent in this case, it is preferable to use 2 or more types selected from water and alcohols among the solvent group mentioned above. Examples of the alcohol include alcohols represented by the formula (4), and more specifically, methanol, ethanol, n-butanol, isopropanol, sec-butanol, and t-butanol. Crystallization may be performed with two or more alcohols selected from these groups, or crystallization may be performed with one or more alcohols selected from these groups and water. A combination of water and alcohol is preferable, and water and ethanol are particularly preferable.

Crystallization can be carried out by adding ethanol to an aqueous solution of the compound (2) in which R ² is sodium. When the solvent for the amination reaction in the previous step is water, the reaction solution is removed up to a predetermined amount after removing ammonia. Can be concentrated and ethanol can be added. The method will be described below.

The concentration in the case of removing ammonia after the amination reaction and concentrating with water is preferably such that the concentration of the compound (2) in which R ² is sodium is 5 to 50 wt%, and the R ² has a good crystallization yield. In order to obtain the compound (2) in which is sodium, 10 to 40 wt% is particularly preferable. A predetermined amount of ethanol is added after the completion of concentration, and the amount of ethanol is preferably 1 to 20 times the weight, more preferably 5 to 15 times the weight of water. Crystallization is performed by adding ethanol and then heating to a predetermined temperature, followed by cooling to a predetermined temperature. The temperature range is preferably -20 to 80 ° C, the impurity removal rate and the crystallization yield. In view of the above, the range of −5 to 60 ° C. is more preferable.

Next, a method of performing crystallization as a compound (2) in which R ² is a hydrogen atom after the amination reaction will be described.

  As a crystallization solvent, it is preferable to use 2 or more types selected from water and alcohol among the solvent group mentioned above. Examples of the alcohol include alcohols represented by the formula (4). Crystallization can also be carried out with one or more alcohols selected from these groups and water, or water, preferably water and ethanol or water alone.

  When the solvent of the amination reaction in the previous step is water, crystallization can be carried out by removing the ammonia, concentrating the reaction solution to a predetermined amount, and adjusting the pH to an appropriate pH by adding an acid. The method will be described below.

Preferably from a concentration of 5-50 wt% of R ² is a hydrogen atom (2) as enrichments for performing water concentrate, and R ² is a hydrogen atom with a good crystallization yield (2 ) Is particularly preferably 10 to 40 wt%. After completion of the concentration, crystallization is performed by adjusting to a predetermined pH by adding an appropriate acid. The acid to be added is not particularly limited, and for example, formic acid, hydrochloric acid, sulfuric acid, acetic acid, nitric acid, methanesulfonic acid, trifluoromethanesulfonic acid, p-toluenesulfonic acid and the like can be mentioned, preferably hydrochloric acid and sulfuric acid. . Although it will not specifically limit if it is pH which can obtain a crystal | crystallization with a favorable yield, For example, it is the range of 4-9, Especially preferably, it is the range of 5-8.

  If the crystal acquisition rate is low even when the pH is adjusted to a predetermined value with a water-only solvent, the crystallization yield can be improved by adding an alcohol such as ethanol. The amount of ethanol to be added is not particularly limited, and may be added until sufficient crystal precipitation is observed. The temperature range for crystallization is preferably in the range of −20 to 80 ° C., and more preferably in the range of −5 to 60 ° C. from the viewpoint of impurity removal rate and crystallization yield.

When it is desired to obtain a compound in which R ² is a hydrogen atom in the formula (2) and the amino group forms a salt with an acid, for example, a compound in which R ² obtained in the above method is a hydrogen atom ( 2) can be obtained by dissolving in a suitable solvent and adding an acid to form a salt.

  Examples of the solvent used here include chloroalkanes such as dichloromethane, chloroform and dichloroethane, substituted benzenes such as benzene and toluene, ethers such as diethyl ether, methyl-t-butyl ether, tetrahydrofuran and 1,4-dioxane, methanol, One or more solvents selected from the group consisting of alcohols such as ethanol, isopropanol and t-butanol, alkanes such as hexane and pentane, esters such as ethyl acetate and isopropyl acetate, ketones such as methyl ethyl ketone, and water. It is sufficient to select a solvent that can be used, and the resulting salt precipitates as crystals in a high yield and that quality improvement is recognized.

  Examples of the acid used include formic acid, hydrochloric acid, sulfuric acid, acetic acid, nitric acid, methanesulfonic acid, trifluoromethanesulfonic acid, p-toluenesulfonic acid, and the like, preferably hydrochloric acid or sulfuric acid.

  Next, the compound (2) is esterified to give a general formula (3);

A method for obtaining a compound represented by the formula: In the compound represented by the formula (3) (hereinafter referred to as compound (3)), R ¹ and R ³ are the same as described above. In the compound (3), the amino group may form a salt with an appropriate acid. The acid that forms the salt of the compound (3) is not particularly limited. For example, formic acid, acetic acid, hydrochloric acid, sulfuric acid, nitric acid, trifluoroacetic acid, methanesulfonic acid, trifluoromethanesulfonic acid, p-toluenesulfone An acid etc. are mentioned, Preferably they are hydrochloric acid, a sulfuric acid, or methanesulfonic acid, and hydrochloric acid is especially preferable. * ₂ is the same as described above, preferably (2R, 3R). When the (2R, 3R) compound (2) is used as a raw material, the (2R, 3R) compound (3) is used, and when the (2S, 3S) compound (2) is used (2S, 3R) 3S) compound (3) is obtained.

  As the compound (2) used here, the compound (2) obtained by the reaction of the compound (1) and ammonia by the above-described method may be used as it is, or a compound subjected to a crystallization step may be used. .

  Esterification may be performed by a usual method (for example, see Experimental Science Course 22, edited by The Chemical Society of Japan, Maruzen Co., Ltd.), and is not particularly limited. For example, in the presence of a suitable acid, the above formula (4 The method performed by reaction with alcohol represented by this is demonstrated. As the alcohol to be used, an alcohol corresponding to the desired ester may be selected, and the amount used is preferably 5 to 200 mol times, particularly preferably 10 to 100 mol times the amount of the compound (2).

  The acid to be used is not particularly limited as long as the reaction proceeds with a good yield. For example, formic acid, hydrochloric acid, sulfuric acid, acetic acid, nitric acid, methanesulfonic acid, trifluoromethanesulfonic acid, p-toluenesulfonic acid, etc. Preferred are hydrochloric acid, sulfuric acid, methanesulfonic acid, and p-toluenesulfonic acid, and particularly preferred is methanesulfonic acid. The usage-amount of an acid is 2-20 mol times amount with respect to a compound (2), Preferably it is 2-10 mol times amount.

  The reaction temperature is not particularly limited, but is preferably in the range from 25 ° C. to the boiling point of the alcohol used, and particularly preferably in the range from 40 ° C. to the boiling point of the alcohol used. The reaction time may be performed until the disappearance of the compound (2) is confirmed, and is not particularly limited, but is generally about 1 to 24 hours.

  The post-treatment after the reaction may be carried out by a usual method and is not particularly limited. For example, water is added after completion of the reaction, neutralized with an appropriate base, alcohol is removed, and the remaining aqueous layer The compound (3) can be efficiently separated from the inorganic salts in the system by making the pH of the solution alkaline, followed by extraction with a suitable organic solvent.

  The base for making the aqueous layer alkaline after neutralization and alcohol removal is not particularly limited. For example, an alkali metal such as sodium hydroxide, potassium hydroxide, cesium hydroxide, magnesium hydroxide, calcium hydroxide, etc. Mention may be made of hydroxides or alkaline earth metal hydroxides, alkali metal carbonates such as lithium carbonate, potassium carbonate and sodium carbonate, and alkali metal hydrogen carbonates such as lithium hydrogen carbonate, potassium hydrogen carbonate and sodium hydrogen carbonate. . The use of alkali metal hydroxides or alkaline earth metal hydroxides is preferred, and sodium hydroxide is particularly preferred because it does not generate gas due to an economic viewpoint and reaction with an acid.

  The extraction solvent is not particularly limited as long as it can efficiently extract the compound (3) from the aqueous layer. For example, chloroalkanes such as dichloromethane, chloroform, dichloroethane, and substituted benzenes such as benzene or toluene. , Ethers such as diethyl ether and methyl tert-butyl ether, alkanes such as hexane and pentane, and esters such as ethyl acetate and isopropyl acetate, preferably dichloromethane, toluene, methyl tert-butyl ether and ethyl acetate. Yes, particularly preferably ethyl acetate.

  The obtained extract can be subjected to a crystallization step as necessary after concentration to obtain a highly pure compound (3). When the compound (2) contains the compound (8) which is a regioisomer as an impurity, the compound (8) is represented by the general formula (9) by an esterification reaction;

(Hereinafter, referred to as compound (9)). In the compound (9), R ¹ and R ³ are as described above. In the compound (9), the amino group may form a salt with an appropriate acid. The acid forming the salt is not particularly limited, and examples include formic acid, acetic acid, hydrochloric acid, sulfuric acid, nitric acid, trifluoroacetic acid, methanesulfonic acid, trifluoromethanesulfonic acid, p-toluenesulfonic acid, and the like. Preferred is hydrochloric acid, sulfuric acid or methanesulfonic acid, with hydrochloric acid being particularly preferred. * ₂ is the same as described above, preferably (2S, 3S).

  Impurity (9) contained as an impurity in compound (3) can be removed by crystallization. Therefore, when obtaining the compound (3) or a salt thereof, the compound (1) is reacted with ammonia, and then the obtained compound (2) is subjected to a crystallization step to obtain the compound (8) and other impurities. Or after esterification without crystallization of compound (2), compound (9) and other impurities are removed by a crystallization step, or after reaction of compound (1) with ammonia There is a method of performing the crystallization step both after the esterification reaction. Which method should be selected may be determined from the required quality. In order to obtain a compound (3) with higher purity, it is preferable to perform crystallization after both steps of the amination reaction and the esterification reaction. Needless to say, crystallization may not be performed according to the required quality of the compound (3).

  Solvents used for crystallization are, for example, chloroalkanes such as dichloromethane, chloroform, dichloroethane, substituted benzenes such as benzene or toluene, ethers such as diethyl ether, methyl-t-butyl ether, tetrahydrofuran, 1,4-dioxane, One or more solvents from the group consisting of alcohols such as methanol, ethanol, isopropanol, t-butanol, alkanes such as hexane or pentane, esters such as ethyl acetate or isopropyl acetate, ketones such as acetone or methyl ethyl ketone, and water. Can be done. When ethyl acetate is used as the extraction solvent, the extract can be concentrated to a predetermined amount and crystallized from the ethyl acetate solution as it is, or an appropriate solvent selected from the above group can be used as a poor solvent. It can also be used as a solvent.

  The crystallization method using ethyl acetate as the extraction solvent and hexane as the poor solvent after concentrating the extract is described below. The concentration of the extract is preferably in the range of 5 to 50 wt% of the concentration of the compound (3), and particularly preferably 10 to 30 wt% in order to obtain the compound (3) with a good crystallization yield. A predetermined amount of hexane is added after the completion of concentration, and the amount of hexane is preferably 30 to 200 v / v%, particularly preferably 30 to 150 wt%, based on ethyl acetate. Crystallization is performed by concentrating the extract and heating to a predetermined temperature to dissolve the precipitated solid content, adding hexane, and cooling to a predetermined temperature. The temperature range is -20 to 80 ° C. From the viewpoint of the removal rate of impurities and the crystallization yield, it is more preferably in the range of −5 to 60 ° C.

  When it is desired to obtain the compound (3) as a salt with an acid, it can be obtained by adding an appropriate acid to the compound (3) obtained above, and the extract or the extract is concentrated to an appropriate solvent. It is also possible to obtain the salt of the compound (3) directly as crystals by adding an acid after the substitution to.

  Examples of the solvent to be used include chloroalkanes such as dichloromethane, chloroform and dichloroethane, substituted benzenes such as benzene and toluene, ethers such as diethyl ether, methyl-t-butyl ether, tetrahydrofuran and 1,4-dioxane, methanol, Select one or more solvents from the group consisting of alcohols such as ethanol, isopropanol, t-butanol, alkanes such as hexane or pentane, esters such as ethyl acetate or isopropyl acetate, ketones such as acetone or methyl ethyl ketone, and water. An optimum solvent may be selected according to the type of acid used. Moreover, when using 2 or more types of mixed solvents, the mixing ratio is not specifically limited.

  The acid to be used is not particularly limited, and examples thereof include formic acid, hydrochloric acid, sulfuric acid, acetic acid, nitric acid, trifluoroacetic acid, methanesulfonic acid, trifluoromethanesulfonic acid, p-toluenesulfonic acid, and the like. Hydrochloric acid or sulfuric acid, with hydrochloric acid being particularly preferred. The crystallization temperature is preferably in the range of −20 to 80 ° C., and more preferably in the range of −5 to 60 ° C. from the viewpoint of impurity removal rate and crystallization yield.

  In the case of obtaining either the compound (3) or the salt of the compound (3) as a final product, crystallization can be carried out twice or more if the desired quality cannot be obtained by one crystallization. . When crystallization is performed twice or more, it may be repeated with the same solvent or with different solvents.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

Example 1 (2R, 3R) -3-Phenylisoserine sodium salt (2S, 3S) -2-chloro-3-hydroxy-3-phenylpropionic acid ethyl ester (14.33 g, 62.67 mmol), EtOH To a solution consisting of (30 mL) was added 20 wt% NaOEt / EtOH (23.46 g, 68.94 mmol) at room temperature and stirred for 40 minutes. The precipitated inorganic salt was separated by filtration, washed with EtOH (30 mL), and water (30 mL) and a 30 wt% NaOH aqueous solution (9.19 g, 68.94 mmol) were added to the filtrate. After stirring at room temperature for 35 minutes, the content of (2R, 3S) -3-phenylglycidic acid sodium salt in the reaction solution is determined by HPLC analysis using separately synthesized racemic-3-phenylglycidic acid sodium salt as a sample. The yield was calculated to be 98.3%. Under reduced pressure, the reaction solution was concentrated to about 30 wt% before concentration, and 28 wt% aqueous ammonia solution (124.9 mL, 1.85 mol) was added to the obtained orange slurry. The reaction solution was transferred to a pressure resistant reactor, heated to 50 ° C. and stirred for 19 hours, then cooled to room temperature, and ammonia was removed under reduced pressure. When the (2R, 3R) -3-phenylisoserine sodium salt in the obtained reaction solution was quantified by HPLC analysis using a sample, the yield was 68.8%. The yield of (2S, 3S) -3-phenylserine sodium salt, which is a regioisomer, was similarly determined by quantitative analysis by HPLC and found to be 18.4%. Under reduced pressure, (2R, 3R) -3-phenylisoserine sodium salt was concentrated until the content was about 30 wt%, EtOH (255 mL) was added to the resulting slurry, and the mixture was stirred at 50 ° C. for 30 minutes and stirred to room temperature. The mixture was allowed to cool, further cooled to 0 to 1 ° C, and stirred at the same temperature for 30 minutes. The precipitated crystals were collected by filtration, washed with EtOH (80 mL), and dried under reduced pressure at 50 ° C. for about 18 hours to obtain white crystals (8.21 g). From the content of (2R, 3R) -3-phenylisoserine sodium salt and (2S, 3S) -3-phenylserine sodium salt in the filtrate, (2R, 3R) -3-phenylisoserine sodium salt in the crystals The content was 90.4 wt% (crystallization yield 86.2%), and the content of (2S, 3S) -3-phenylserine sodium salt was 1.4 wt% (removal rate 95.2%).

(Yield analysis)
(2R, 3S) -3-phenylglycidic acid sodium salt column: COSMOSIL C18 AR (4.6 mm × 250 mm) manufactured by Nacalai Co., Ltd., eluent: acetonitrile / 0.5 wt% KH ₂ PO ₄ aqueous solution (pH = 3.0) ) = 5/5, flow rate: 1 ml / min, detection: 210 nm, column temperature: 40 ° C., elution time: (2R, 3S) -3-phenylglycidic acid sodium salt 3.4 minutes.

(2R, 3R) -3-Phenylisoserine sodium column: Luna 5μ C18 (2) (4.6 mm × 250 mm) manufactured by Phenomenex, eluent: acetonitrile / 0.5 wt% KH ₂ PO ₄ aqueous solution (pH = 2.0) = 1/9, flow rate: 0.5 ml / min, detection: 210 nm, column temperature: 40 ° C., elution time: (2R, 3R) -3-phenylisoserine sodium salt 5.7 minutes, ( 2S, 3S) -3-Phenylserine sodium salt 6.7 min.

¹ H NMR (D ₂ O, 400 MHz) δ 7.27-7.38 (m, 5H), 4.18 (d, 1H, J = 4.4 Hz), 4.14 (d, 1H, J = 4. 2Hz)

(Example 2) (2R, 3R) -3-Phenylisoserine ethyl ester White crystals (8.21 g) obtained in Example 1 ((2R, 3R) -3-Phenylisoserine sodium salt: 36. EtOH (200 mL) and MsOH (18.37 g, 191.2 mmol) were added to 54 mmol, (2S, 3S) -3-phenylserine sodium salt: containing 0.54 mmol, and refluxed with heating for 20 hours. After allowing to cool to room temperature, water (200 mL) was added, and the pH was adjusted to 6.67 with 30 wt% NaOH aqueous solution. EtOH was distilled off under reduced pressure, and the pH was adjusted to 11.87 with a 30 wt% NaOH aqueous solution. Extraction with ethyl acetate (3 × 150 mL) was performed, and the extract was washed with saturated brine (200 mL). When the contents of (2R, 3R) -3-phenylisoserine ethyl ester and (2S, 3S) -3-phenylserine ethyl ester in the extract were determined by quantitative analysis by HPLC using a sample, 2R, 3R) -3-phenylisoserine ethyl ester was 32.62 mmol (yield 89.3%), and (2S, 3S) -3-phenylserine ethyl ester was 0.38 mmol. The extract was concentrated until (2R, 3R) -3-phenylisoserine ethyl ester was about 17 wt%, and the resulting slurry was heated to 40 ° C. to obtain a uniform solution. Hexane (25 mL) was added at the same temperature, the mixture was allowed to cool while stirring to room temperature, further cooled to 0 to 2.5 ° C., and stirred at the same temperature for 1 hour. The precipitated crystals were separated by filtration, washed with hexane (30 mL), and then dried at 50 ° C. under reduced pressure for about 12 hours to obtain white crystals (5.82 g). When the contents of (2R, 3R) -3-phenylisoserine ethyl ester and (2S, 3S) -3-phenylserine ethyl ester were determined by HPLC analysis, (2R, 3R) -3-phenylisoserine ethyl ester was obtained. Was> 99.9 wt% (crystallization yield 85.2%), and (2S, 3S) -3-phenylserine ethyl ester was not detected.

(Yield analysis)
Column: COSMOSIL C18 AR (4.6 mm × 250 mm) manufactured by Nakarai Co., Ltd., eluent: acetonitrile / 0.5 wt% KH ₂ PO ₄ aqueous solution (pH = 2.0) = 2/8, flow rate: 1 ml / min, detection: 210 nm, column temperature: 40 ° C., elution time: (2R, 3R) -3-phenylisoserine ethyl ester 3.3 minutes, (2S, 3S) -3-phenylserine ethyl ester 3.9 minutes.

¹ H NMR (CDCl ₃ , 400 MHz) δ 7.23-7.33 (m, 5H), 4.44 (d, 1H, J = 4.1 Hz), 4.30 (d, 1H, J = 3.9 Hz) ) 4.06-4.12 (m, 2H), 1.16 (t, 3H, J = 7.1 Hz)

Example 3 (2R, 3S) -3-Phenylglycidic acid sodium salt (2S, 3S) -2-chloro-3-hydroxy-3-phenylpropionic acid ethyl ester (3.72 g, 16.3 mmol), To a solution consisting of EtOH (10 mL) was added 20 wt% NaOEt / EtOH (12.19 g, 35.86 mmol) at room temperature and stirred for 1 hour. The precipitated inorganic salt was filtered off, washed with EtOH (10 mL), a solution of water (323 mg, 17.93 mmol) in EtOH (5 mL) was added to the filtrate, and the mixture was stirred for 1 hour at room temperature. Was low, water (10 mL) was added, and the precipitated white crystals were filtered off. The crystals were washed with EtOH (20 mL) and vacuum dried for about 12 hours to give white crystals (1.46 g). When this product was analyzed by HPLC, it was found that it contained 84.1 wt% (2R, 3S) -3-phenylglycidic acid sodium salt (yield 37.2%).

(Example 4) Microbial reduction of 2-chlorobenzoylacetic acid ethyl ester A liquid medium (pH 7.0) having a composition of 1.5% glycerin, 1.0% pro-extract and 0.5% yeast extract was prepared. 5 ml aliquots were poured into test tubes and steam sterilized at 120 ° C. for 20 minutes. One platinum ear of each of the microorganisms shown in Table 1 was inoculated into these liquid media, and cultured with shaking at 30 ° C. for 2 to 3 days. The cells were collected from this culture by centrifugation, suspended in 1 ml of 0.1 M phosphate buffer (pH 5.5) containing 0.2% of 2-chlorobenzoyl acetate ethyl ester and 2% of glucose, and stoppered. Shake for 20 hours at 30 ° C. in a test tube. After the reaction, extraction is performed with an equal volume of ethyl acetate, and the product in the extract is analyzed by HPLC, yield (%), diastereomeric ratio ((2R, 3R) / (2S, 3R)). And optical purity (% ee). The results are shown in Table 1.

(Yield analysis)
Column: YMC-Pack Pro C18 AS-303 (4.6 mm × 250 mm) manufactured by YMC, eluent: 0.1% phosphoric acid / acetonitrile = 5/5, flow rate: 1 ml / min, detection: 220 nm, column temperature: 30 ° C., elution time: 2-chloro-3-hydroxy-3-phenylpropionic acid ethyl ester 8.9 minutes, 2-chlorobenzoylacetic acid ethyl ester 13.8 minutes.

[Analysis of diastereomeric ratio and optical purity]
Column: CHIRALCEL OJ-H (4.6 mm × 250 mm) manufactured by Daicel Chemical Industries, Ltd., eluent: hexane / isopropyl alcohol = 9/1, flow rate: 1 ml / min, detection: 220 nm, column temperature: 30 ° C., elution time: (2R, 3R) -2-Chloro-3-hydroxy-3-phenylpropionic acid ethyl ester 14.4 min, (2S, 3S) -2-chloro-3-hydroxy-3-phenylpropionic acid ethyl ester 15.1 Min, (2R, 3S) -2-chloro-3-hydroxy-3-phenylpropionic acid ethyl ester 20.5 min, (2S, 3R) -2-chloro-3-hydroxy-3-phenylpropionic acid ethyl ester 24 .5 minutes.

(Example 5) (2S, 3S) -2-Chloro-3-hydroxy-3-phenylpropionic acid ethyl ester Bacto-tryptone 1.6%, Bacto-yeast extract 1%, Sodium chloride 1% (pH 7.0) After sterilization in a 500 ml Sakaguchi flask containing 50 ml of the culture medium, Escherichia coli HB101 (pNTDRG1) FERM BP-08458 was inoculated and cultured with shaking at 37 ° C. for 24 hours. After completion of the culture, 5 g of 2-chlorobenzoylacetic acid ethyl ester, 4.8 g of glucose and 15 mg of oxidized nicotinamide adenine dinucleotide (NAD ⁺ ) were added, and the reaction was allowed to proceed for 44 hours while maintaining the pH at 6.5 with 30% NaOH. It was. After completion of the reaction, the reaction mixture was extracted twice with 150 ml of ethyl acetate, and the obtained organic phase was concentrated under reduced pressure to obtain 4.8 g of oily 2-chloro-3-hydroxy-3-phenylpropionic acid ethyl ester. When the obtained product was analyzed according to the method described in Example 4, Anti / Syn = 99.2 / 0.8, and the optical purity of the Anti isomer (2S, 3S) was 99.4% e.e. e. Met.

Claims (27)

General formula (1);

(In the formula, R ¹ represents a phenyl group which may have a substituent, R ² represents a hydrogen atom, an alkali metal, an alkaline earth metal, or a nitrogenous base, and * ₁ is an asymmetric carbon atom. The steric configuration is represented by (2R, 3S) or (2S, 3R)) and a compound represented by the general formula (2) obtained by reaction of ammonia;

(Wherein R ¹ and R ² represent the same meaning as described above, * ₂ represents an asymmetric carbon atom, and the configuration thereof represents (2R, 3R) or (2S, 3S)). The method for producing a compound represented by the formula (2), wherein the compound is further subjected to a crystallization step. The compound represented by the formula (2) obtained by the method according to claim 1 is esterified to give a general formula (3);

(Wherein R ¹ and * ₂ represent the same meaning as described above, and R ³ represents a C _{1 to} C ₄ alkyl group). Of the compound to be produced.   A method for producing a compound represented by the formula (3), comprising a crystallization step of the compound represented by the formula (3) obtained by the method according to claim 2.   The crystallization step of the compound represented by the formula (2) is at least one selected from the group consisting of chloroalkanes, substituted benzenes, ethers, alcohols, alkanes, esters, ketones and water. The manufacturing method in any one of Claims 1-3 performed using the solvent of. The crystallization step of the compound represented by the formula (2) is represented by the general formula (4);
R ³ OH (4)
The method according to any one of claims 1 to 4, wherein the reaction is carried out using one or more solvents selected from alcohols represented by the formula (wherein R ³ represents a C _{1 to} C ₄ alkyl group) and water. Manufacturing method.   The method according to claim 4 or 5, wherein the crystallization temperature is -20 to 80 ° C. General formula (1);

(In the formula, R ¹ represents a phenyl group which may have a substituent, R ² represents a hydrogen atom, an alkali metal, an alkaline earth metal, or a nitrogenous base, and * ₁ is an asymmetric carbon atom. The steric configuration is represented by (2R, 3S) or (2S, 3R)) and the reaction of the compound represented by general formula (2);

(Wherein R ¹ and R ² represent the same meaning as described above, * ₂ represents an asymmetric carbon atom, and the configuration thereof represents (2R, 3R) or (2S, 3S)). And then esterified to obtain the general formula (3);

(Wherein R ¹ and * ₂ represent the same meaning as described above, and R ³ represents a C _{1 to} C ₄ alkyl group), and further subjected to a crystallization step. The manufacturing method of the compound represented by said Formula (3).   The crystallization step of the compound represented by the formula (3) is one or more selected from the group consisting of chloroalkanes, substituted benzenes, ethers, alcohols, alkanes, esters, ketones and water. The manufacturing method of Claim 7 performed using the solvent of.   The crystallization method according to claim 8, wherein the crystallization temperature is -20 to 80 ° C.   In the reaction of the compound represented by the formula (1) and ammonia, the amount of ammonia used is 1 to 100 mole times the compound represented by the formula (1). The production method described in 1.   The method according to claim 1, wherein the reaction temperature is 0 to 100 ° C. in the reaction of the compound represented by the formula (1) and ammonia.   The method according to claim 1, wherein the reaction of the compound represented by the formula (1) and ammonia is performed in water or a mixed solvent of water and an organic solvent. The compound represented by the formula (1) is represented by the general formula (5);

(Wherein R ¹ and R ³ represent the same meaning as described above, and X represents a halogen atom) asymmetric reduction of the carbonyl group of the compound represented by formula (6) obtained;

In the formula, R ¹ , R ³ and X represent the same meaning as described above, and * represents an asymmetric carbon atom. After the compound is treated with a base and cyclized, the ester group is hydrolyzed. The production method according to any one of claims 1 to 12, which is obtained by carrying out the process. The compound represented by the formula (1) asymmetrically reduces the carbonyl group of the compound represented by the formula (5), and the resulting general formula (7);

(Wherein R ¹ , R ³ , X and * ₂ represent the same meaning as described above) The compound represented by the above was obtained by treating with a base and cyclizing, and then hydrolyzing the ester group. The production method according to claim 1, which is a product. The asymmetric reduction of the compound represented by the formula (5) is performed by acting an enzyme source capable of (2S, 3S) selective or (2R, 3R) selective reduction of the compound. The manufacturing method of Claim 13 or 14 characterized by these. (2S, 3S) selective enzymatic source, Deboshia (Devosia riboflavina) genus Paenibacillus (Paenibacillus) genus or Pseudomonas (Pseudomonas) method according to claim 15, wherein the enzyme source derived from a microorganism belonging to the genus. (2S, 3S) is selective enzyme source, Deboshia-Ribofurabina (Devosia riboflavina), Paenibacillus alvei (Paenibacillus alvei), is selected from the group consisting of Pseudomonas putida (Pseudomonas putitda), and Pseudomonas Sututozeri (Pseudomonas stutzeri) The production method according to claim 16, which is an enzyme source derived from a microorganism. The (2S, 3S) selective enzyme source is a recombinant Escherichia coli culture transformed with a carbonyl reductase derived from Devosia riboflavina or a processed product thereof. The manufacturing method described. The method according to claim 15, wherein the (2R, 3R) selective enzyme source is an enzyme source derived from a microorganism belonging to the genus Devosia . The production method according to claim 19, wherein the (2R, 3R) selective enzyme source is an enzyme source derived from Devosia riboflavina . General formula (5);

(Wherein R ¹ represents an optionally substituted phenyl group, R ³ represents a C _{1 to} C ₄ alkyl group, and X represents a halogen atom) By acting an enzyme source having the ability to stereoselectively reduce the carbonyl group of the compound, general formula (7);

(Wherein R ¹ and R ³ represent the same meaning as described above, * ₂ represents an asymmetric carbon atom, and the configuration thereof represents (2R, 3R) or (2S, 3S)). a method of manufacturing a that compound, Deboshia (Devosia) genus Paenibacillus (Paenibacillus) genus, or derived from a microorganism belonging to Pseudomonas (Pseudomonas) genus, a compound represented by the formula (5) (2S, 3S ) (2S, 3S) of the compound represented by the formula (7) is produced using an enzyme source having the ability to selectively reduce, or derived from a microorganism belonging to the genus Devosia , Producing an (2R, 3R) isomer of the compound represented by the formula (7) using an enzyme source capable of (2R, 3R) selectively reducing the compound represented by the formula (5). A manufacturing method characterized by (2S, 3S) is selective enzyme source, Deboshia-Ribofurabina (Devosia riboflavina), Paenibacillus alvei (Paenibacillus alvei), is selected from the group consisting of Pseudomonas putida (Pseudomonas putitda), and Pseudomonas Sututozeri (Pseudomonas stutzeri) The production method according to claim 21, which is an enzyme source derived from a microorganism. The production according to claim 21 or 22, wherein the (2S, 3S) selective enzyme source is a recombinant Escherichia coli culture transformed with a carbonyl reductase derived from Devosia riboflavina or a processed product thereof. Law. The production method according to claim 23, wherein the (2R, 3R) selective enzyme source is an enzyme source derived from Devosia riboflavina . The production method according to any one of claims 1 to 24, wherein R ¹ is an unsubstituted phenyl group. Process according to any one of claims 1 to 20 R ² is hydrogen or an alkali metal. The production method according to any one of claims 1 to 20, wherein the configuration of the formula (2) is a (2R, 3R) isomer.