JP5010266B2 - Process for producing optically active biphenylalanine compound or salt thereof and ester thereof - Google Patents

Description

  The present invention relates to a method for producing a compound having an optically active biphenylalanine structure that is useful as an intermediate for pharmaceuticals and the like.

  A compound having an optically active biphenylalanine structure is useful as an intermediate for pharmaceuticals such as neutral endopeptidase inhibitors (see Patent Documents 1 and 2).

  As a method for producing optically active biphenylalanine, (1) triflation of D-Boc-tyrosine methyl ester, Suzuki coupling, hydrolysis of ester (see Patent Document 3), (2) asymmetric hydrogenation Synthesis of optically active Boc-biphenylalanine (see Non-Patent Document 1 and Patent Document 2) is known. However, in the method of (1), the triflation reagent is expensive, the catalyst palladium used in the Suzuki coupling is expensive, and it is difficult to remove residual palladium from the product. However, it is not an industrially advantageous method in that D-tyrosine as a raw material is expensive. Further, the method (2) has a problem that the catalyst and the asymmetric source used in the asymmetric hydrogenation are expensive.

  On the other hand, as a method for producing an optically active amino acid derivative, a method of asymmetric hydrolysis of a racemate of N- (2,6-dimethylphenyl) alanine ester using an enzyme is known (see Patent Document 4). ). In addition, it is known that an optically active amino acid or an optically active phenylalanine derivative is produced by asymmetric hydrolysis using an enzyme (see Non-Patent Documents 2 to 5 and Patent Document 5).

  In addition, racemic N-substituted-DL-amino acid ester is hydrolyzed with protease in the presence of a weak base as a pH control reagent to produce N-substituted-L-amino acid and N-substituted-D-amino acid ester. Is known (see Patent Document 6).

JP-A-6-228187 JP 2003-261522 A US Pat. No. 5,217,996 International Publication No. WO2004 / 055195 Pamphlet Spanish Patent Application Publication No. 547913 Japanese Patent Laid-Open No. 9-206089 Chirality, 1996, Vol. 8, No. 2, p. 173-188 Journal of Chemical Technology and Biotechnology (J. Chem. Technol. Biotechnol.), 1994, Vol. 59, No. 1, p. 61-65 ITE Letters on Batteries, ITE Letters on Batteries, New Technologies & Medicine, ITE-Hohwa Inc., 2004 Vol. 5, No. 4, p. 377-380, Enantiomer, 2002, Vol. 7, No. 1, p1-3 Biotechnol. Lett., 1991, Vol. 13, No. 5, p. 317-322

  An object of the present invention is to provide a method capable of producing a compound having an optically active biphenylalanine structure with high optical purity at a low cost by a simple operation.

As a result of intensive studies to solve the above problems, the present inventors have hydrolyzed biphenylalanine ester under specific conditions using a specific enzyme, thereby producing optically active biphenylalanine or a salt thereof. And the present inventors have found that the ester can be produced with high optical purity, low cost and simple operation, and the present invention has been completed.
That is, the present invention is as follows.

[1] Formula (1)

(Where
R ¹ represents an alkyl group, a haloalkyl group, an alkenyl group, a cycloalkyl group, an aryl group which may have a substituent or an aralkyl group which may have a substituent,
R ² represents an amino-protecting group,
R ³ and R ⁴ each independently represent a hydrogen atom, a halogen atom, an alkyl group, a haloalkyl group, a hydroxy group, an alkoxy group, a cyano group or a nitro group. )
In the presence of one or more alkalis selected from (i) alkali metal hydroxides and alkaline earth metal hydroxides (hereinafter referred to as biphenylalanine ester compounds (1)) , (Ii) in the presence of one or more alkalis selected from alkali metal hydroxides and alkaline earth metal hydroxides and amino acids, or (iii) selected from alkali metal hydroxides and alkaline earth metal hydroxides Formula (2) produced by hydrolysis using a protease derived from a microorganism belonging to the genus Bacillus in the presence of one or more alkalis and aminosulfonic acid.

(In the formula, each symbol has the same meaning as described above.)
An optically active biphenylalanine compound represented by the following (hereinafter also referred to as optically active biphenylalanine compound (2)) or a salt thereof and formula (3)

(In the formula, each symbol has the same meaning as described above.)
An optically active biphenylalanine compound (2) or a salt thereof and an optical compound, characterized by separating an unreacted optically active biphenylalanine ester compound (hereinafter also referred to as optically active biphenylalanine ester compound (3)) A method for producing an active biphenylalanine ester compound (3).
[2] The method of the above-mentioned [1], wherein the alkali is an alkali metal hydroxide.
[3] The method of the above-mentioned [1], wherein the amino acid is glycine.
[4] The method of the above-mentioned [1], wherein the aminosulfonic acid is taurine.
[5] The method according to [1] above, wherein the hydrolysis is performed in the presence of an alkali metal hydroxide and an amino acid.
[6] The method of the above-mentioned [1], wherein the hydrolysis is performed in the presence of an alkali metal hydroxide and glycine.
[7] The method of the above-mentioned [1], wherein the hydrolysis is performed in the presence of an alkali metal hydroxide and aminosulfonic acid.
[8] The method according to [1] above, wherein the hydrolysis is performed in the presence of an alkali metal hydroxide and taurine.
[9] The method described in [1] above, wherein the protease is derived from Bacillus licheniformis.
[10] The method of the above-mentioned [1], wherein R ¹ is an alkyl group.
[11] The method of the above-mentioned [1], wherein R ¹ is methyl or ethyl.
[12] The method described in [1] above, wherein R ² is tert-butoxycarbonyl.
[13] The method described in [1] above, wherein R ³ and R ⁴ are hydrogen atoms.
[14] The method described in [1] above, wherein the hydrolysis is performed while maintaining the pH in the range of 6.0 to 13.
[15] The method described in [1] above, wherein the hydrolysis is performed while maintaining the pH in the range of 6.0 to 10.
[16] The method described in [1] above, wherein the hydrolysis is performed in a mixed solvent of an organic solvent and water.
[17] The method described in [16] above, wherein the organic solvent is at least one selected from tert-butyl methyl ether and toluene.
[18] The method described in [16] above, wherein the organic solvent is tert-butyl methyl ether.
[19] The method described in [1] above, wherein the hydrolysis is performed at 30 to 60 ° C.
[20] The method described in [1] above, wherein the hydrolysis is performed at 35 to 55 ° C.
[21] The optically active biphenylalanine compound (2) or a salt thereof is esterified to give the formula (2 ′)

(Where
R ¹ represents an alkyl group, a haloalkyl group, an alkenyl group, a cycloalkyl group, an aryl group which may have a substituent or an aralkyl group which may have a substituent,
R ² , R ³ and R ⁴ are as defined above. )
A step of obtaining an optically active biphenylalanine ester compound (hereinafter also referred to as optically active biphenylalanine ester compound (2 ′)) represented by:
Racemizing the optically active biphenylalanine ester compound (2 ′) to obtain the biphenylalanine ester compound (1);
A process for producing a biphenylalanine ester compound (1), comprising:
[22] The biphenylalanine ester compound (1) is converted into (ii) an alkali metal hydroxide and an alkali in the presence of one or more alkalis selected from (i) an alkali metal hydroxide and an alkaline earth metal hydroxide. In the presence of one or more alkalis and amino acids selected from earth metal hydroxides, or (iii) the presence of one or more alkalis and aminosulfonic acids selected from alkali metal hydroxides and alkaline earth metal hydroxides A step of hydrolyzing using a protease derived from a microorganism belonging to the genus Bacillus to separate the produced optically active biphenylalanine compound (2) or a salt thereof from an unreacted optically active biphenylalanine ester compound (3);
Esterifying the optically active biphenylalanine compound (2) or a salt thereof to obtain an optically active biphenylalanine ester compound (2 ′);
Racemizing the optically active biphenylalanine ester compound (2 ′) to obtain the biphenylalanine ester compound (1);
A method for recovering a biphenylalanine ester compound (1).

According to the production method of the present invention, the biphenylalanine ester compound (1) is converted into (i) one or more alkalis selected from alkali metal hydroxides and alkaline earth metal hydroxides (hereinafter also referred to as caustic alkalis). In the presence of (ii) in the presence of caustic and amino acids, or (iii) in the presence of caustic and aminosulfonic acid, by hydrolysis using a protease derived from a microorganism belonging to the genus Bacillus, L-form Is preferentially hydrolyzed, so that the L-form optically active biphenylalanine compound (2) or a salt thereof and the D-form optically active biphenylalanine ester compound (3) are produced with high optical purity by a simple operation. Can do. In particular, it is preferable to use a protease derived from Bacillus licheniformis from the viewpoint of excellent enantioselectivity.
The production method of the present invention is an industrially advantageous production method because it uses the biphenylalanine ester compound (1) that can be produced from an inexpensive raw material.

The definition of the substituent used in this specification is described below.
As the “alkyl group”, a linear or branched alkyl group having 1 to 6 carbon atoms such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, Hexyl etc. are mentioned, Preferably it is methyl or ethyl.

  Examples of the “alkenyl group” include linear or branched alkenyl groups having 2 to 6 carbon atoms, such as vinyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl, 3-butenyl and the like. Preferably, it is vinyl or allyl.

  Examples of the “cycloalkyl group” include cycloalkyl groups having 3 to 8 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like, preferably cyclopentyl or cyclohexyl.

  Examples of the “halogen atom” include a fluorine atom, a chlorine atom, a bromine atom and an iodine atom, preferably a fluorine atom, a chlorine atom or a bromine atom.

  The “haloalkyl group” is an “alkyl group” as defined above substituted with a “halogen atom” as defined above. Although the number of substitution of the halogen atom is not particularly limited, 1 to 3 is preferable. Examples of the “haloalkyl group” include chloromethyl, bromomethyl, fluoromethyl, dichloromethyl, dibromomethyl, difluoromethyl, trichloromethyl, tribromomethyl, trifluoromethyl, 2,2-dichloroethyl, 2,2,2- Trichloroethyl etc. are mentioned, Preferably it is trifluoromethyl.

  “Alkoxy group” means a straight or branched alkoxy group having 1 to 6 carbon atoms, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, isopentyl Examples thereof include oxy, neopentyloxy, hexyloxy and the like, preferably methoxy or ethoxy.

The “aryl group” of the “aryl group optionally having substituent (s)” includes aryl groups having 6 to 14 carbon atoms, such as phenyl, 1-naphthyl, 2-naphthyl, etc., preferably phenyl. is there.
The aryl group may have a substituent at a substitutable position. Examples of such a substituent include a halogen atom (the same as those defined above) and an alkyl group (defined above). Are the same as those defined above), haloalkyl groups (the same as those defined above are exemplified), hydroxy groups, alkoxy groups (the same as those defined above are exemplified), cyano groups And a nitro group. Although the number of the said substituent is not specifically limited, 1-3 are preferable. When the number of substituents is 2 or more, these substituents may be the same or different.

The “aralkyl group” of the “aralkyl group optionally having substituent (s)” is the “alkyl group” defined above substituted with the “aryl group” defined above. The number of substitution of the aryl group is not particularly limited, but 1 to 3 is preferable. Examples of the “aralkyl group” include benzyl, phenethyl, 1-phenylethyl, 1-phenylpropyl, 2-phenylpropyl, 3-phenylpropyl, 1-naphthylmethyl, 2-naphthylmethyl, benzhydryl, trityl and the like. , Preferably benzyl.
The aralkyl group may have a substituent at a substitutable position. Examples of such a substituent include a halogen atom (the same as those defined above), an alkyl group (defined above). Are the same as those defined above), haloalkyl groups (the same as those defined above are exemplified), hydroxy groups, alkoxy groups (the same as those defined above are exemplified), cyano groups And a nitro group. Although the number of the said substituent is not specifically limited, 1-3 are preferable. When the number of substituents is 2 or more, these substituents may be the same or different.

As the “protecting group for amino group” represented by R ² , a protecting group known per se used as a protecting group for amino group can be used without particular limitation. As such a protecting group, —CO ₂ R ⁵ (wherein R ⁵ may have an alkyl group, a haloalkyl group, an alkenyl group, an aryl group which may have a substituent, or a substituent). An aralkyl group or a 9-fluorenylmethyl group.), —COR ⁶ (wherein R ⁶ is a hydrogen atom, an alkyl group, a haloalkyl group, an alkenyl group, an aryl group which may have a substituent or a substituent; An aralkyl group which may have a group), an aralkyl group which may have a substituent, and the like. Specific examples of the “amino-protecting group” include methoxycarbonyl, ethoxycarbonyl, isobutoxycarbonyl, tert-butoxycarbonyl, allyloxycarbonyl, benzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl , 9-fluorenylmethyloxycarbonyl, formyl, acetyl, benzoyl, benzyl, benzhydryl, trityl and the like.

R ¹ is preferably an alkyl group, more preferably methyl or ethyl.
R ² is preferably —CO ₂ R ⁵ (wherein R ⁵ has the same meaning as described above), and more preferably tert-butoxycarbonyl.
R ³ and R ⁴ are preferably both hydrogen atoms.

  The biphenylalanine ester compound (1), which is a raw material used in the production method of the present invention, can be produced, for example, by the following method.

(In the formula, each symbol has the same meaning as described above.)

Process 1
Compound (6) can be obtained by reacting compound (4) and hydantoin (5) in the presence of a base.
The amount of hydantoin (5) to be used is generally 1 to 3 mol, preferably 1.05 to 2 mol, per 1 mol of compound (4).
Examples of the base include ammonium acetate, sodium acetate, piperidine, triethylamine and the like.
The amount of the base to be used is generally 0.1 to 10 mol, preferably 0.5 to 2 mol, per 1 mol of compound (4).
Examples of the reaction solvent include acetic acid, acetic anhydride, N, N-dimethylformamide and the like.
The reaction temperature is usually 30 to 200 ° C, preferably 100 to 200 ° C. The reaction time is usually 1 to 24 hours, preferably 3 to 10 hours.

Process 2
Compound (7) can be obtained by reducing compound (6).
The reduction can preferably be carried out by catalytic hydrogenation.
Examples of the catalyst used for catalytic hydrogenation include palladium-carbon, palladium hydroxide-carbon, platinum-carbon, rhodium-carbon, ruthenium-carbon, developed nickel, and the like.
The amount of the catalyst used is usually 0.0005 to 0.05 g, preferably 0.005 to 0.02 g in the case of a noble metal such as Pd, based on 1 g of compound (6), and developed nickel. In this case, it is usually 0.1 to 2 g, preferably 0.2 to 1 g.
Examples of the reaction solvent include ethers such as tetrahydrofuran, tert-butyl methyl ether, 1,2-dimethoxyethane, and 1,4-dioxane; alcohols such as methanol, ethanol, isopropanol, n-butanol, and tert-butanol; ethyl acetate And esters such as methyl acetate and butyl acetate; organic acids such as acetic acid and propionic acid; water; and mixed solvents thereof.
Catalytic hydrogenation can be carried out under normal pressure or pressurized conditions.
The reaction temperature is usually 20 to 100 ° C, preferably 40 to 70 ° C. The reaction time is usually 1 to 24 hours, preferably 3 to 10 hours.

Process 3
The compound (8) can be obtained by hydrolyzing the compound (7).
Hydrolysis is usually performed by treating with a base in a solvent.
Examples of the base include sodium hydroxide, potassium hydroxide, lithium hydroxide, calcium hydroxide, barium hydroxide, potassium carbonate, sodium carbonate and the like.
The amount of the base to be used is generally 2 to 10 mol, preferably 3 to 8 mol, per 1 mol of compound (7).
Examples of the reaction solvent include alcohols such as methanol, ethanol and ethylene glycol; ethers such as tetrahydrofuran, tert-butyl methyl ether, 1,2-dimethoxyethane and 1,4-dioxane; water; mixed solvents thereof and the like. It is done.
The reaction temperature is usually 80 to 200 ° C, preferably 100 to 170 ° C. The reaction time is usually 1 to 24 hours, preferably 3 to 10 hours.

Process 4
Biphenylalanine ester compound (1) can be obtained by subjecting compound (8) to amino group protection and carboxyl group esterification reaction. Protection of the amino group and esterification of the carboxyl group can be performed according to conventional methods. The order of protecting the amino group and esterifying the carboxyl group is not particularly limited, and can be performed in any order.

  The biphenylalanine ester compound (1) has two optical isomers (L-form and D-form) having an asymmetric carbon atom at the α-position, and the biphenylalanine ester compound used in the method of the present invention. (1) may be a racemate containing an equal amount of these optical isomers, or a mixture containing one optical isomer in excess (in any proportion). A racemate is preferable.

  The biphenylalanine ester compound (1) thus obtained is used in the present invention in the presence of (i) caustic alkali, (ii) caustic and amino acid, or (iii) caustic and amino. Hydrolysis is performed using a protease derived from a microorganism belonging to the genus Bacillus in the presence of sulfonic acid. When hydrolyzed using the protease, the L form is preferentially hydrolyzed.

  As a protease derived from a microorganism belonging to the genus Bacillus, a protease derived from Bacillus licheniformis is preferable because of its excellent enantioselectivity. Specific examples of the protease derived from Bacillus licheniformis include a protease derived from Bacillus licheniformis containing subtilisin, preferably Alcalase (Novozyme), particularly preferably Alcalase 2.4L (Novozyme).

  The purity or form of the protease is not particularly limited, and the protease can be used in various forms such as a purified enzyme, a crude enzyme, a microorganism culture, a microbial cell, and a processed product thereof. Here, the treated product refers to, for example, freeze-dried microbial cells, microbial cell lysates, microbial cell extracts, and the like. Further, enzymes having various purities or forms as described above immobilized on, for example, an inorganic carrier such as silica gel or ceramics, cellulose, ion exchange resin, or the like may be used.

  Although the usage-amount of the said protease is not specifically limited, It is 0.001-0.5g normally in conversion of a purified enzyme with respect to 1g of biphenylalanine ester compounds (1), Preferably it is 0.001-0.1g. is there.

  The hydrolysis with the protease is preferably carried out while maintaining the pH at 6.0 to 13, more preferably at pH 6.0 to 10, and even more preferably at 6.0 to 9.5, depending on the type of protease. . By carrying out hydrolysis in the above pH range, a product with high optical purity can be obtained.

  The hydrolysis is usually performed in water or a mixed solvent of an organic solvent and water, and a product with high optical purity is obtained. Therefore, the hydrolysis is preferably performed in a mixed solvent of an organic solvent and water. .

  Examples of the method for adjusting the pH to the above range include caustic aqueous solution; phosphate buffer (disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate), and acetate buffer ( Buffer solutions such as sodium acetate and potassium acetate); caustic aqueous solutions in the presence of buffers; caustic aqueous solutions in the presence of amino acids; caustic aqueous solutions in the presence of aminosulfonic acid; and the like. Alkaline aqueous solutions and buffers also serve as solvents.

  In the present invention, an amino acid is an organic compound having both an amino group (including a substituted amino group and a substituted amino group having a cyclic structure) and a carboxyl group in one molecule (the hydrogen atom of the amino group is a side chain moiety). In addition to α-amino acids, β-amino acids, γ-amino acids, δ-amino acids and the like are also included. Specific examples of α-amino acids include glycine, alanine, α-aminobutyric acid, leucine, isoleucine, valine, phenylalanine, tryptophan, tyrosine, methionine, cysteine, threonine, serine, proline, hydroxyproline, asparagine, glutamine, etc. Amino acids; acidic amino acids such as aspartic acid and glutamic acid; basic amino acids such as lysine, tryptophan, arginine, ornithine, histidine, and hydroxylysine. Specific examples of β-amino acids include β-alanine and β-aminobutyric acid. Specific examples of γ-amino acids include γ-aminobutyric acid. Specific examples of δ-amino acids include 5-aminovaleric acid.

  In the present invention, aminosulfonic acid is an organic compound having a sulfo group in place of the carboxyl group in the amino acid, and specific examples thereof include taurine, N-methyltaurine, 2- (4-morpholinyl) ethanesulfonic acid, and the like. Is mentioned.

  In the present invention, as a method of adjusting the pH to the above range, from the viewpoint of economical and environmentally undesirable waste reduction, (i) a method using a caustic aqueous solution, (ii) using a caustic aqueous solution in the presence of an amino acid. Or (iii) a method using an aqueous caustic solution in the presence of aminosulfonic acid. In the methods (ii) and (iii), amino acid or aminosulfonic acid serves as a buffer. In the present invention, the methods (ii) and (iii) are particularly preferred.

  In the above method, the caustic is preferably an alkali metal hydroxide, particularly preferably potassium hydroxide or sodium hydroxide. The amino acid is preferably glycine, and the aminosulfonic acid is preferably taurine.

  The concentration of the caustic aqueous solution is not particularly limited, but is usually 2 to 30%, preferably 3 to 15%, more preferably 3 to 13%. By setting the concentration of the alkaline aqueous solution in the above range, a product with high optical purity can be obtained more easily.

  The concentration of the buffer is usually 0.05 to 0.8M, preferably 0.1 to 0.8M, more preferably 0.1 to 0.5M. By setting the concentration of the buffer in the above range, a product with high optical purity can be obtained.

  Although the amount of the buffer used depends on the concentration, it is usually 2 to 100 mL, preferably 2 to 10 mL, with respect to 1 g of the biphenylalanine ester (1). If the amount of the buffer used is outside this range, the salt produced by hydrolysis may precipitate, the reaction may become difficult to control, or the productivity may increase due to an increase in the solvent.

  The usage-amount of caustic aqueous solution is 0.45-0.7 equivalent normally with respect to biphenylalanine ester (1), Preferably it is 0.48-0.65 equivalent. If the amount of alkali used is outside this range, it will be difficult to maintain an appropriate pH range, the reaction may be stopped, and natural hydrolysis with alkali may proceed.

  The amount of amino acid or aminosulfonic acid used is usually 0.05 to 1.0 mol, preferably 0.1 to 0.5 mol, more preferably 0.1 to 1 mol per 1 mol of biphenylalanine ester (1). 0.3 mole. By making the usage-amount of an amino acid or aminosulfonic acid into said range, the maintenance of the pH range required for an enzyme reaction becomes easy, and it becomes possible to advance reaction smoothly.

Examples of the organic solvent include a hydrophobic organic solvent and a hydrophilic organic solvent.
Examples of the hydrophobic organic solvent include ethers such as tert-butyl methyl ether and diisopropyl ether; hydrocarbons such as toluene, hexane, cyclohexane and heptane. Examples of hydrophilic organic solvents include ethers such as tetrahydrofuran; alcohols such as methanol, ethanol, isopropanol, n-butanol and tert-butanol; sulfoxides such as dimethyl sulfoxide; ketones such as acetone; nitriles such as acetonitrile; Is mentioned. These organic solvents may be used alone or in combination of two or more.
The organic solvent is preferably tert-butyl methyl ether or toluene, particularly preferably tert-butyl methyl ether.

  The usage-amount of an organic solvent is 1-50 mL normally with respect to 1 g of biphenylalanine ester compounds (1), Preferably it is 1-5 mL. When the amount of the organic solvent used is outside this range, the raw material and the product may be precipitated, resulting in a decrease in reaction rate or a decrease in productivity.

Hydrolysis is performed by mixing a biphenylalanine ester compound (1), a protease derived from a microorganism belonging to the genus Bacillus, caustic and a solvent, and if necessary, an amino acid or aminosulfonic acid.
These addition orders are not particularly limited. For example,
(i) A biphenylalanine ester compound (1) dissolved in an organic solvent is added to a caustic aqueous solution or buffer, and then the protease is added.
(ii) adding a caustic aqueous solution or buffer to the biphenylalanine ester compound (1) dissolved in an organic solvent, and then adding the protease.
(iii) adding the protease (and water if necessary) to the biphenylalanine ester compound (1) dissolved in an organic solvent, and then adding (preferably dropwise) caustic;
(iv) To the biphenylalanine ester compound (1) dissolved in an organic solvent, the protease and an amino acid or aminosulfonic acid (and water if necessary) are added, and then an aqueous caustic solution is added (preferably dropwise).
And the like.

The protease may be added during the reaction if necessary. Further, when the pH of the reaction solution falls below the above range during the reaction, the pH of the reaction solution is maintained within the above range using an appropriate pH adjuster.
Examples of the pH adjusting agent include caustic alkalis such as sodium hydroxide, potassium hydroxide, lithium hydroxide, calcium hydroxide, magnesium hydroxide, barium hydroxide, or aqueous solutions thereof.

The reaction temperature for the hydrolysis is preferably 30 to 60 ° C, more preferably 35 to 55 ° C, and particularly preferably 40 to 45 ° C. If the reaction temperature is outside the above range, the stability of the enzyme may decrease or the reaction rate may decrease.
The reaction time for the hydrolysis is usually 3 to 24 hours, preferably 4 to 15 hours.

By separating the reaction mixture after the reaction into an aqueous layer and an organic layer, a salt of the optically active biphenylalanine compound (2) (L form) produced by hydrolysis and an unreacted optically active biphenylalanine ester compound (3) (D-form) can be separated. At this time, a salt of the optically active biphenylalanine compound (2) is contained in the aqueous layer, and an optically active biphenylalanine ester compound (3) is contained in the organic layer.
In the hydrolysis, when a mixed solvent of a hydrophobic organic solvent and water is used, the obtained reaction mixture may be separated as it is.
In the hydrolysis, when a hydrophobic organic solvent is not used, or when separation is not possible due to a small amount of the hydrophobic organic solvent and / or water, an appropriate amount of the hydrophobic organic solvent and / or water is added. The liquid may be separated after the addition.
Examples of hydrophobic organic solvents include ethers such as tert-butyl methyl ether and diisopropyl ether; hydrocarbons such as toluene, hexane, cyclohexane and heptane; halogenated hydrocarbons such as dichloromethane, 1,2-dichloroethane, chloroform and chlorobenzene And esters such as ethyl acetate, methyl acetate, and butyl acetate.
During the liquid separation operation, the pH of the aqueous layer is usually adjusted to 6.5 to 12, preferably 7.5 to 11.

From the obtained aqueous layer, the optically active biphenylalanine compound (2) or a salt thereof can be obtained by the following method.
1) A crude product of a salt of the optically active biphenylalanine compound (2) (alkali metal salt, alkaline earth metal salt, etc.) can be obtained by distilling off the solvent from the aqueous layer.

2) The salt of the optically active biphenylalanine compound (2) can be isolated by adding an inorganic salt and an organic solvent to the aqueous layer for extraction, and distilling off the organic solvent.
Here, examples of the inorganic salt include sodium chloride, potassium chloride, ammonium chloride, and the like, and potassium chloride or sodium chloride is preferable from the viewpoint of economy and the solubility of the salt of the optically active biphenylalanine compound (2). The amount of the inorganic salt used is preferably 30 g to 100 g with respect to 100 g of the salt of the optically active biphenylalanine compound (2) contained in the aqueous layer from the viewpoint of extraction rate and the like.
Examples of the organic solvent include solvents such as toluene, tert-butyl methyl ether, and ethyl acetate. Of these, toluene and tert-butyl methyl ether are preferable.
The usage-amount of an organic solvent is 60g-130g normally with respect to 100g of salts of optically active biphenylalanine compound (2) contained in a water layer, Preferably it is 80g-120g.
If extraction is not sufficient, an acid may be added to adjust the pH to neutral or slightly acidic.
Extraction is usually performed at 10 to 50 ° C.

3) The optically active biphenylalanine compound (2) is isolated by making the aqueous layer acidic (usually pH 1-7, preferably pH 1-4), then extracting with an organic solvent, and distilling off the organic solvent. Can do. Examples of the organic solvent include the same organic solvents used in the above extraction.

  The obtained optically active biphenylalanine compound (2) or a salt thereof can be purified by a general method such as recrystallization or column chromatography. In addition, you may convert the salt of an optically active biphenylalanine compound (2) into an optically active biphenylalanine compound (2) with an acid as needed.

On the other hand, the optically active biphenylalanine ester compound (3) can be isolated by distilling off the solvent from the organic layer. Furthermore, the optically active biphenylalanine ester compound (3) in the aqueous layer can be recovered by extracting the aqueous layer separated by the liquid separation operation with an organic solvent. The pH of the aqueous layer in this extraction operation is usually 6.5 to 12, preferably 7.5 to 11. As the organic solvent, those exemplified as the hydrophobic organic solvent can be used.
The obtained optically active biphenylalanine ester compound (3) can be purified by a general method such as recrystallization or column chromatography.

The isolated optically active biphenylalanine ester compound (3) is hydrolyzed according to a conventional method, whereby R ¹ is a hydrogen atom (optical isomer (optical form of optically active biphenylalanine compound (2) (D-form)) or The salt can be produced.

Moreover, R ² is a hydrogen atom by deprotecting the protecting group of the amino group of the isolated optically active biphenylalanine compound (2) or a salt thereof and the optically active biphenylalanine ester compound (3) according to a conventional method. Each compound can be produced.

  The optically active biphenylalanine compound (2) or a salt thereof and the optically active biphenylalanine ester compound (3) produced by the method of the present invention are useful as intermediates for producing a pharmaceutical such as a neutral endopeptidase inhibitor. is there. For example, using the method described in JP-A-6-228187, the optically active biphenylalanine compound (2) or a salt thereof or the optically active biphenylalanine ester compound (3) is used for the N-phosphonomethyl- Biaryl substituted dipeptide derivatives can be prepared.

  When the optically active biphenylalanine ester compound (3) is a desired compound, the optically active biphenylalanine compound (2) or a salt thereof is esterified to obtain the optically active biphenylalanine ester compound (2 ′), and then racemized. By converting to biphenylalanine compound (1), it can be reused for the above hydrolysis. The method will be described below.

(In the formula, each symbol has the same meaning as described above.)

The esterified optically active biphenylalanine compound (2) or a salt thereof is esterified to obtain the optically active biphenylalanine ester compound (2 ′).
In this esterification, the solution of the optically active biphenylalanine compound (2) or the salt thereof obtained in the extraction after the hydrolysis is used as it is.
Esterification can be performed according to a conventional method, for example, a method using alcohols such as methanol or ethanol and an acid; a method using a sulfate ester such as dimethyl sulfate or diethyl sulfate and a base; a method using a condensing agent such as R ¹ OH and DCC. A method using an alkyl halide such as methyl iodide, bromoethane, benzyl chloride and a base; among them, a method using a sulfate ester and a base is preferable from the viewpoint of by-products and convenience.

Hereinafter, a method using a base and dimethyl sulfate will be described.
Examples of the base include sodium hydrogen carbonate, potassium hydrogen carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, diisopropylethylamine, 2,6-dimethylpyridine, triethylamine, pyridine and the like. Among them, sodium hydrogencarbonate is preferable.
The usage-amount of a base is 0.3-1.2 times mole amount normally with respect to 1 mol of optically active biphenylalanine compounds (2) or its salt in a solution.
As the sulfate ester, dimethyl sulfate is preferable from the viewpoint of reactivity and post-treatment.
The amount of sulfate used is usually 1.2 to 2.5 times the molar amount, preferably 1.6 to 2.0 times the molar amount of the optically active biphenylalanine compound (2) or salt thereof in the solution. Amount.
As a procedure for esterification, from the viewpoint of reactivity, it is preferable to add a sulfate ester dropwise to a solution containing a base and an optically active biphenylalanine compound (2) or a salt thereof.
The esterification temperature is usually 30 to 50 ° C. The reaction time is usually 1 to 10 hours, although it depends on the reagent amount, reaction temperature, and the like. Completion of the reaction can be confirmed by HPLC analysis.

After completion of the reaction, an amine such as triethylamine is added, and the remaining sulfuric acid ester is decomposed by stirring at a temperature of 30 to 50 ° C. for 2 to 5 hours. The amount of amine used may be about 10 mol% with respect to the sulfate ester used.
Next, the reaction solution is separated, and the organic layer is dehydrated. For dehydration, a dehydrating agent such as anhydrous magnesium sulfate, anhydrous sodium sulfate, or molecular sieves may be used, but from the viewpoint of operability, a method of azeotropic dehydration with a solvent azeotropic with water (for example, toluene) is preferable. .
The amount of the azeotropic solvent used may be an amount that can be sufficiently dehydrated, but is usually 50 to 100% by weight with respect to the amount of the solution.
For the next racemization, the water content in the optically active biphenylalanine ester compound (2 ′) solution is preferably 500 ppm or less, and therefore, it is preferable to sufficiently perform dehydration.
The solution of the optically active biphenylalanine ester compound (2 ′) thus obtained can be used as it is for the next racemization. Alternatively, the optically active biphenylalanine ester compound (2 ′) may be isolated and used by a general method.

The optically active biphenylalanine ester compound (2 ′) obtained by racemization esterification is racemized and converted to the biphenylalanine compound (1).
Racemization is usually performed using a base in a solvent.
As a solvent, it is preferable to add alcohols such as methanol, ethanol and isopropanol to the solution of the optically active biphenylalanine ester compound (2 ′) obtained by esterification from the viewpoint of promoting racemization.
The amount of alcohol used is usually 80 to 120% by weight with respect to the weight of the optically active biphenylalanine ester compound (2 ′).
Examples of the base include alkali metal alcoholates such as sodium methoxide, sodium ethoxide and potassium tert-butoxide; alkali metal hydrides such as sodium hydride and potassium hydride. Among them, from the viewpoint of handling, alkali metal alcoholates From the viewpoint of economy, sodium methoxide is particularly preferable.
The usage-amount of a base is 80-120 mol% with respect to an optically active biphenylalanine ester compound (2 ').
The temperature of racemization is usually 30-50 ° C, preferably 35-45 ° C. The reaction time is usually 10 minutes to 6 hours, although depending on the amount of reagent used and the temperature. Completion of the racemization reaction can be confirmed by HPLC.

After completion of racemization, it is preferable to add an acid such as acetic acid to the reaction solution to suppress the decomposition of the ester. Here, the usage-amount of an acid is 1.1-1.3 times mole amount normally with respect to the base used for the racemization.
The post-treatment after the completion of racemization is performed according to a conventional method.

  The solution of the biphenylalanine ester compound (1) thus obtained may be directly subjected to hydrolysis by an enzyme, or after concentration, dissolved in another organic solvent such as tert-butyl methyl ether and hydrolyzed. You may use for.

When the optically active biphenylalanine compound (2) or a salt thereof is a desired compound, the optically active biphenylalanine ester compound (3) is racemized and converted to the biphenylalanine compound (1) to Can be reused for hydrolysis.
This racemization can be performed by a method similar to the racemization method described above, using a solution of an optically active biphenylalanine ester compound (3) obtained by extraction after completion of hydrolysis in an organic solvent.

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

In the following Examples, pH 7.0 phosphate buffer and pH 8.0 phosphate buffer prepared by the following method were used.
Phosphate buffer solution with pH 7.0 Dissolved 17.25 g (0.099 mol) of dipotassium hydrogen phosphate in 900 mL of water, adjusted to pH 7.0 with phosphoric acid, and made up to a total volume of 1 L with water
pH 8.0 phosphate buffer Dissolve 7.38 g (0.052 mol) of disodium hydrogen phosphate and 5.46 g (0.035 mol) of sodium dihydrogen phosphate in 195 mL of water, and use 20% aqueous sodium hydroxide solution. Aqueous solution adjusted to pH 8.0

The optical purity (enantiomeric excess) of the optically active substance was determined by high performance liquid chromatography (HPLC) analysis.
HPLC analysis column: Chiralpak AD-RH (Daicel Chemical Industries, Ltd.) (4.5 mmφ x 15 cm, 5 mm)
Moving bed; Liquid A; 0.1% phosphoric acid aqueous solution
Liquid B; acetonitrile elution conditions; Liquid B 40% (15 minutes)-30 minutes-80% (0 minutes) Gradient column temperature; 40 ° C
Flow rate; 1.0 mL / min detector; UV (254 nm)
Retention time; LN-Boc-biphenylalanine; 10 minutes
DN-Boc-biphenylalanine; 13 minutes
LN-Boc-biphenylalanine methyl ester; 27 minutes
DN-Boc-biphenylalanine methyl ester; 30 minutes

Production Example 1
DL-biphenylalanine (12)
4-biphenylaldehyde (9) (100.0 g, 0.549 mol), hydantoin (82.4 g, 0.823 mol) and ammonium acetate (63.5 g, 0.824 mol) were heated to reflux in acetic acid (360 mL) for 5 hours. After adding water (360 mL), the mixture was cooled to room temperature, and the crystals were collected by filtration and washed with isopropanol-water (1: 1, 400 mL) to give hydantoin compound (10) (143.14 g, yield 98.7% )

  To a mixture of hydantoin compound (10) (60.2 g), tetrahydrofuran (THF) (540 mL) and water (60 mL), 5% palladium-carbon (50% water content, 2.7 g) was added. Stir at 60 ° C. for 3 hours. After removing the catalyst by filtration, the filtrate was concentrated to obtain a reduced product (11) (60.63 g, yield 100%).

  Sodium hydroxide (36.65 g) was added to a mixture of the reductant (11) (59.7 g, 0.224 mol), ethylene glycol (300 mL) and water (10 mL), and the mixture was stirred at 130-140 ° C. for 5 hours. After cooling to room temperature, water (130 mL) was added, and an aqueous hydrochloric acid solution consisting of concentrated hydrochloric acid (85 g) and water (99 g) was added to adjust the pH of the mixture to 6.9. The obtained crystals were collected by filtration, washed with water (300 mL), washed with methanol (300 mL), and dried to obtain DL-biphenylalanine (12) (53.25 g, yield 98.4%). .

Production Example 2
DL-N-Boc-Biphenylalanine methyl ester (13)
DL-biphenylalanine (12) (20.0 g, 0.0829 mol) was added to a 10% aqueous sodium hydroxide (116 g, 0.29 mol) solution. After adding THF (50 mL), a THF (20 mL) solution of di-tert-butyl dicarbonate (23.5 g, 0.108 mol) was added dropwise at 30 ° C. over 1 hour. Further, tetrabutylammonium bromide (0.20 g, 0.62 mmol) was added, and dimethyl sulfate (12.5 g, 0.099 mol) was added dropwise at 30 ° C. After stirring at room temperature for 16 hours, dimethyl sulfate (5.4 g, 0.0428 mol) was added, and the mixture was stirred at 35 ° C. for 4.5 hours, further dimethyl sulfate (2.93 g, 0.0232 mol) was added, and stirred at 35 ° C. for 2.5 hours.
After adding tert-butyl methyl ether (MTBE) (40 mL) and water (100 mL), the mixture was separated and the aqueous layer was removed to obtain 104.1 g of an MTBE solution of DL-N-Boc-biphenylalanine methyl ester. It was.
As a result of quantification by HPLC, the content of DL-N-Boc-biphenylalanine methyl ester was 29.4 g, and the yield from DL-biphenylalanine was 99.8%.
78.1 g of the MTBE solution of DL-N-Boc-biphenylalanine methyl ester thus obtained was concentrated to dryness, and the residue was recrystallized from isopropanol (9 mL) and heptane (80 mL) to obtain DL-N- Boc-biphenylalanine methyl ester (19.1 g) was obtained as colorless crystals (recrystallization yield 86.6%).
¹ H-NMR (CDCl ₃ ); 1.42 (9H, s), 3.09 (1H, dd, J = 5, 14 Hz), 3.16 (1H, dd, J = 5, 14 Hz), 3.74 (3H, s) , 4.55-4.70 (1H, m), 4.90-5.08 (1H, m), 7.20 (2H, d, J = 8 Hz), 7.33 (1H, t, J = 8 Hz), 7.43 (2H, t, J = 8 Hz), 7.52 (2H, d, J = 8 Hz), 7.57 (2H, d, J = 8 Hz).

Example 1 (potassium hydroxide dropping method)
DL-N-Boc-biphenylalanine methyl ester 221.08g (0.622mol) dissolved in MTBE 467.76g, 99.49g water and Alcalase 2.4L FG (from Bacillus licheniformis) (Novozyme) 55.26g And stirred. The mixture was stirred at 40 ° C. for 22 hours while 369.8 g (0.317 mol) of 5% aqueous potassium hydroxide solution was added dropwise thereto. The pH range of the reaction solution at this time was 6.82 to 9.58.
When the reaction solution was analyzed, the optical purity of DN-Boc-biphenylalanine methyl ester was 99.2% ee.

Example 2 (taurine addition method)
DL-N-Boc-biphenylalanine methyl ester (25.0 g, 70.3 mmol) dissolved in MTBE (41.14 g) was dissolved in 11.25 g of water, 1.76 g (14.1 mmol) of taurine and alcalase 2.4L FG (from Bacillus licheniformis) (Novozyme) ) 4.50 g was added and stirred at 40 ° C. Thereto, 47.28 g (40.67 mmol) of 5% aqueous potassium hydroxide solution was added dropwise and stirred at 40 ° C. for 17 hours. The pH range of the reaction solution at this time was 6.30 to 8.16.
When the reaction solution was analyzed, the optical purity of DN-Boc-biphenylalanine methyl ester was 99.4% ee, and the optical purity of LN-Boc-biphenylalanine was 99.9% ee.
The reaction solution was allowed to stand for 5 minutes and then separated to obtain 35.72 g of an organic layer A and 83.46 g of an aqueous layer A. To the aqueous layer, 37.5 g of toluene was added and stirred at 40 ° C. for 30 minutes. After allowing to stand for 5 minutes, liquid separation was performed to obtain 42.80 g of an organic layer B and 76.80 g of an aqueous layer B. Organic layer A and organic layer B were combined, 58 g of water and 1.49 g of sodium carbonate were added, and the mixture was stirred at 40 ° C. for 30 minutes. After allowing to stand for 5 minutes, liquid separation was performed to obtain 76.07 g of an organic layer C. The obtained organic layer C (76.07 g) was concentrated while being kept at 50 ° C., and 63.2 g was distilled off.
Methanol 75 mL was added to 12.87 g of the concentrated residue, and 18.75 g of water was added while keeping the temperature at 40 ° C. 2 mg of seed crystals of DN-Boc-biphenylalanine methyl ester were added, and the mixture was stirred at 40 ° C. for 30 minutes. 12.5 g of water was added dropwise thereto over 30 minutes, and the mixture was kept at 40 ° C. for 1 hour. It was then cooled to 20 ° C. and filtered. The obtained crystals were washed with a mixed solution of 8.75 g of methanol and 3.75 g of water.
The crystals were dried under reduced pressure to obtain 10.94 g of white crystals of DN-Boc-biphenylalanine methyl ester. The yield of DN-Boc-biphenylalanine methyl ester was 43.8% based on DL-N-Boc-biphenylalanine methyl ester.

Example 3 (Glycine addition method)
DL-N-Boc-biphenylalanine methyl ester 6.38 g (18.0 mmol) dissolved in MTBE 10.50 g in a solution of 2.88 g water, 0.27 g glycine (3.6 mmol) and alcalase 2.4L FG (derived from Bacillus licheniformis) (Novozyme) ) 1.53 g was added and stirred at 40 ° C. The mixture was stirred at 40 ° C. for 18 hours while 11.0 g (9.90 mmol) of 5% potassium hydroxide aqueous solution was added dropwise thereto. The pH range of the reaction solution at this time was 6.53 to 8.90.
When the reaction solution was analyzed, the optical purity of DN-Boc-biphenylalanine methyl ester was 99.5% ee, and the optical purity of LN-Boc-biphenylalanine was 99.4% ee.

Example 4 (Reuse of L-form after enzymatic hydrolysis)
(i) Methylation of LN-Boc-biphenylalanine LN-Boc-biphenylalanine equivalent to 50.0 g (0.146 mol) of LN-Boc-biphenylalanine obtained by the same method as in Example 2 (taurine addition method) 50 g of toluene and 37.5 g of sodium chloride were added to 328.54 g of an aqueous solution containing a potassium salt, and the mixture was stirred at 40 ° C. for 20 minutes. After standing for 5 minutes, liquid separation was performed to obtain 156.23 g of an organic layer. To the organic layer, 12.27 g (0.146 mol) of sodium bicarbonate was added and stirred at 40 ° C. Thereto, 33.15 g (0.263 mol) of dimethyl sulfate was added dropwise over 2 hours, followed by stirring at 40 ° C. for 1 hour. When the reaction solution was analyzed, the remaining LN-Boc-biphenylalanine was below the detection limit in the LC analysis.
To the reaction solution, 2.95 g (0.029 mol) of triethylamine was added and stirred at 40 ° C. for 3 hours. After standing for 5 minutes, liquid separation was performed to obtain 114.04 g of an organic layer. Thereto was added 86.72 g of toluene, and the mixture was concentrated while being kept at 50 ° C., and 57.92 g was distilled off. 7.39 g of toluene was added thereto to obtain 150.23 g of a toluene solution. When the obtained toluene solution was quantified, the yield of LN-Boc-biphenylalanine methyl ester was 96.2% with respect to LN-Boc-biphenylalanine. Further, when the measurement was performed with a Karl Fischer moisture measuring device, the water content of the obtained toluene solution was 204 ppm.

(ii) Racemization of LN-Boc-biphenylalanine methyl ester
50 g of methanol was added to 150.23 g of a toluene solution containing 50 g (0.141 mol) of LN-Boc-biphenylalanine methyl ester, and the mixture was stirred at 40 ° C. Thereto, 27.20 g (0.141 mol) of 28% sodium methylate methanol solution was added dropwise over 1 hour, followed by stirring at 40 ° C. for 1 hour.
When the reaction solution was analyzed, the optical purity of LN-Boc-biphenylalanine methyl ester was 0.02% ee, and the LC area percentage of DL-N-Boc-biphenylalanine methyl ester was 89.3%.
To the reaction solution, 10.16 g (0.169 mol) of acetic acid was added dropwise over 15 minutes and stirred at 40 ° C. for 30 minutes.
Next, 50 g of water was added and stirred at 40 ° C. for 5 minutes. After standing for 5 minutes, liquid separation was performed to obtain 151.90 g of an organic layer. To the organic layer was added 45 g of water and 2.37 g (0.028 mol) of sodium hydrogen carbonate, and the mixture was stirred at 40 ° C. for 50 minutes. The mixture was allowed to stand for 5 minutes and then separated to obtain 147.67 g of an organic layer. When the obtained solution was quantified by LC, the yield of DL-N-Boc-biphenylalanine methyl ester was 89.7% with respect to LN-Boc-biphenylalanine methyl ester.
The solution was concentrated while being kept at 54 to 56 ° C., and 62.67 g was distilled off. MTBE75g was added there, it concentrated, keeping at 52 degreeC, and 74.23g was distilled off. MTBE75g was added there, it concentrated, keeping at 52 degreeC, and 84.71g was distilled off. MTBE51.46g was added there and the solution of 127.52g was obtained. When the obtained solution was quantified by LC, the yield of DL-N-Boc-biphenylalanine methyl ester was 89.5% with respect to LN-Boc-biphenylalanine methyl ester. The toluene content in the MTBE solution was 18.5%.

(iii) Production of DN-Boc-biphenylalanine methyl ester The MTBE solution obtained above was subjected to enzymatic hydrolysis under the same conditions as in Example 2 (taurine addition method). The optical purity of Boc-biphenylalanine methyl ester was 99.4%, and the optical purity of LN-Boc-biphenylalanine was 100%.

LC analysis (HPLC) in methylation and racemization was performed under the following conditions.
HPLC analysis condition column; SUMIPAX A212 ODS (Sumitomo Chemical Analysis Center) (φ: 6mm × L: 15cm)
Moving bed; solution A; 25 mM dipotassium hydrogen phosphate aqueous solution (pH adjusted to 6.8 with phosphoric acid)
Liquid B; acetonitrile elution conditions; liquid B 40% (5 minutes)-20 minutes-80% (5 minutes) Gradient column temperature: 40 ° C
Flow rate; 1.0 mL / min detector; UV (254 nm)
Retention time; LN-Boc-biphenylalanine; 7 minutes
LN-Boc-biphenylalanine methyl ester; 24 minutes

Reference example 1
A solution of DL-N-Boc-biphenylalanine methyl ester 118 mg (0.33 mmol) in MTBE 2 mL was added to 10 mL of pH 7.0 0.1 M phosphate buffer. Alcalase 2.4L FG (derived from Bacillus licheniformis) (Novozyme) 0.4 mL was added thereto, and the mixture was stirred at pH 6.82 to 7.0 at 40 ° C. for 8.5 hours.
When the reaction solution was analyzed, the optical purity of DN-Boc-biphenylalanine methyl ester was 99.7% ee, and the optical purity of LN-Boc-biphenylalanine was 100.0% ee.

Reference example 2
A solution prepared by dissolving 0.59 g (1.66 mmol) of DL-N-Boc-biphenylalanine methyl ester in 2 mL of MTBE was added to 5 mL of 0.4 M phosphate buffer at pH 7.0. Alcalase 2.4L FG (Novozyme) 2mL was added there, and it stirred at 40 degreeC at pH6.57-7.0 for 7.5 hours.
When the reaction solution was analyzed, the optical purity of DN-Boc-biphenylalanine methyl ester was 96.7% ee, and the optical purity of LN-Boc-biphenylalanine was 100.0% ee.

Reference example 3
A solution prepared by dissolving 0.59 g (1.66 mmol) of DL-N-Boc-biphenylalanine methyl ester in 2 mL of MTBE was added to 5 mL of 0.4 M phosphate buffer at pH 8.0. 2 mL of Alcalase 2.4L FG (Novozyme) was added thereto and stirred at 40 ° C. for 7.5 hours. At that time, the pH of the aqueous layer was 6.78. After adjusting the pH of the aqueous layer to 8.1 with 20% aqueous sodium hydroxide solution, the mixture was further stirred at 40 ° C. for 4 hours.
When the reaction solution was analyzed, the optical purity of DN-Boc-biphenylalanine methyl ester was 99.8% ee, and the optical purity of LN-Boc-biphenylalanine was 100.0% ee.

Reference example 4
A solution of 3.7 mL of water and 0.59 g (1.66 mmol) of DL-N-Boc-biphenylalanine methyl ester in 2 mL of MTBE was added to 1.3 mL of 0.4 M phosphate buffer at pH 8.0. 2 mL of Alcalase 2.4L FG (Novozyme) was added thereto and stirred at 40 ° C. for 7.5 hours. At that time, the pH of the aqueous layer was 6.50. The pH of the aqueous layer was adjusted to 8.0 with a 20% aqueous sodium hydroxide solution, and further stirred at 40 ° C. for 4 hours.
When the reaction solution was analyzed, the optical purity of DN-Boc-biphenylalanine methyl ester was 99.5% ee, and the optical purity of LN-Boc-biphenylalanine was 100.0% ee.

Reference Example 5
A solution prepared by dissolving 0.59 g (1.66 mmol) of DL-N-Boc-biphenylalanine methyl ester in 2 mL of MTBE was added to 5 mL of 0.4 M phosphate buffer at pH 8.0. 1 mL of Alcalase 2.4L FG (Novozyme) was added thereto and stirred at 40 ° C. for 2.5 hours. At this point, the pH of the aqueous layer was 6.85. After adjusting the pH of the aqueous layer to 8.03 with 20% aqueous sodium hydroxide solution, the mixture was further stirred at 40 ° C. for 2.5 hours.
When the reaction solution was analyzed, the optical purity of DN-Boc-biphenylalanine methyl ester was 99.9% ee, and the optical purity of LN-Boc-biphenylalanine was 100.0% ee.

Reference Example 6
A solution prepared by dissolving 0.59 g (1.66 mmol) of DL-N-Boc-biphenylalanine methyl ester in 2 mL of MTBE was added to 5 mL of 0.4 M phosphate buffer at pH 8.0. Alkalase 2.4L FG (Novozyme) 0.06 mL was added there, and it stirred at 40 degreeC for 2.5 hours. At this point, the pH of the aqueous layer was 6.90. The pH of the aqueous layer was adjusted to 8.0 with a 20% aqueous sodium hydroxide solution, and further stirred at 40 ° C. for 2.5 hours.
When the reaction solution was analyzed, the optical purity of DN-Boc-biphenylalanine methyl ester was 99.8% ee, and the optical purity of LN-Boc-biphenylalanine was 100.0% ee.

Reference Example 7
A solution of 1.0 g (2.81 mmol) of DL-N-Boc-biphenylalanine methyl ester in 2 mL of toluene was added to 8.5 mL of 0.4 M phosphate buffer at pH 8.0. Alcalase 2.4L FG (Novozyme) 0.1 mL was added there, and it stirred at 40 degreeC for 3 hours. At this point, the pH of the aqueous layer was 7.43. After adjusting the pH of the aqueous layer to 8.1 with 20% aqueous sodium hydroxide solution, the mixture was further stirred at 40 ° C. for 3.5 hours. At this point, the pH of the aqueous layer was 7.18. The pH of the aqueous layer was adjusted to 8.0 with a 20% aqueous sodium hydroxide solution, and further stirred at 40 ° C. for 3 hours.
When the reaction solution was analyzed, the optical purity of DN-Boc-biphenylalanine methyl ester was 96.9% ee, and the optical purity of LN-Boc-biphenylalanine was 100.0% ee.

Reference Example 8
Dissolve disodium hydrogen phosphate 2.5 g (0.0176 mol) and sodium dihydrogen phosphate dihydrate 1.8 g (0.0115 mol) in 65 mL of water, and adjust the pH to 8.12. did.
To this aqueous solution was added 45.4 g of DL-N-Boc-biphenylalanine methyl ester MTBE solution [containing 16.6 g (0.0457 mol) of DL-N-Boc-biphenylalanine methyl ester], and Alcalase 2.4L FG (Novozyme) 2.5 mL was added, and the mixture was stirred at 40 ° C. for 10 hours while maintaining the pH of the aqueous layer at 7.44 to 8.45 with a 20% aqueous sodium hydroxide solution.
The reaction solution was separated, and the obtained aqueous layer was extracted with 30 mL of toluene. The organic layers were combined and washed with 3% aqueous sodium carbonate solution to obtain a toluene solution (69.29 g) of DN-Boc-biphenylalanine methyl ester.
When analyzed by HPLC, the content of DN-Boc-biphenylalanine methyl ester was 8.36 g, and the optical purity was 99.9% ee. The yield based on DL-N-Boc-biphenylalanine methyl ester was 50%.
The content of LN-Boc-biphenylalanine in the aqueous layer was 7.57 g, and the optical purity was 100.0% ee. The yield based on DL-N-Boc-biphenylalanine methyl ester was 47.5%.
DN-Boc-biphenylalanine methyl ester
¹ H-NMR (CDCl ₃ ); 1.42 (9H, s), 3.09 (1H, dd, J = 5, 14 Hz), 3.16 (1H, dd, J = 5, 14 Hz), 3.74 (3H, s) , 4.55-4.70 (1H, m), 4.90-5.08 (1H, m), 7.20 (2H, d, J = 8 Hz), 7.33 (1H, t, J = 8 Hz), 7.43 (2H, t, J = 8 Hz), 7.52 (2H, d, J = 8 Hz), 7.57 (2H, d, J = 8 Hz).
LN-Boc-biphenylalanine
¹ H-NMR (CDCl ₃ ); 1.48 (9H, s), 3.12 (1H, dd, J = 5, 14 Hz), 3.24 (1H, dd, J = 5, 14 Hz), 4.55-4.70 (1H, m), 4.90-4.99 (1H, m), 7.26 (2H, d, J = 8 Hz), 7.33 (1H, t, J = 8 Hz), 7.42 (2H, t, J = 8 Hz), 7.53 ( 2H, d, J = 8 Hz), 7.56 (2H, d, J = 8 Hz).

  In each Example, the absolute configurations of D-form and L-form were confirmed by comparing the retention times with the standard products in HPLC using the above-mentioned chiral column.

  According to the production method of the present invention, a compound having an optically active biphenylalanine structure can be produced from an inexpensive raw material by a simple operation with high optical purity, and thus the production method is very advantageous industrially. It is a simple manufacturing method.

Claims (22)

Formula (1)

(Where
R ¹ represents an alkyl group, a haloalkyl group, an alkenyl group, a cycloalkyl group, an aryl group which may have a substituent or an aralkyl group which may have a substituent,
R ² represents an amino-protecting group,
R ³ and R ⁴ each independently represent a hydrogen atom, a halogen atom, an alkyl group, a haloalkyl group, a hydroxy group, an alkoxy group, a cyano group or a nitro group. )
In the presence of one or more alkalis selected from (i) alkali metal hydroxides and alkaline earth metal hydroxides, (ii) alkali metal hydroxides and alkaline earths In the presence of one or more alkalis and amino acids selected from metal hydroxides, or (iii) in the presence of one or more alkalis selected from alkali metal hydroxides and alkaline earth metal hydroxides and aminosulfonic acids. Formula (2) produced by hydrolysis using a protease derived from a microorganism belonging to the genus Bacillus

(In the formula, each symbol has the same meaning as described above.)
And an optically active biphenylalanine compound represented by formula (3):

(In the formula, each symbol has the same meaning as described above.)
An optically active biphenylalanine compound represented by the formula (2) or a salt thereof and an optically active biphenyl represented by the formula (3) are characterized by separating an unreacted optically active biphenylalanine ester compound represented by: A method for producing a phenylalanine ester compound.   The method according to claim 1, wherein the alkali is an alkali metal hydroxide.   The method of claim 1, wherein the amino acid is glycine.   The method of claim 1 wherein the aminosulfonic acid is taurine.   The process according to claim 1, wherein the hydrolysis is carried out in the presence of an alkali metal hydroxide and an amino acid.   The process according to claim 1, wherein the hydrolysis is carried out in the presence of an alkali metal hydroxide and glycine.   The process according to claim 1, wherein the hydrolysis is carried out in the presence of an alkali metal hydroxide and an aminosulfonic acid.   The process according to claim 1, wherein the hydrolysis is carried out in the presence of an alkali metal hydroxide and taurine.   The method according to claim 1, wherein the protease is derived from Bacillus licheniformis. The method according to claim 1, wherein R ¹ is an alkyl group. The method of claim 1, wherein R ¹ is methyl or ethyl. The method of claim 1, wherein R ² is tert-butoxycarbonyl. The method according to claim 1, wherein R ³ and R ⁴ are hydrogen atoms.   The process according to claim 1, wherein the hydrolysis is carried out while maintaining the pH in the range of 6.0-13.   The process according to claim 1, wherein the hydrolysis is carried out while maintaining the pH in the range of 6.0 to 10.   The method according to claim 1, wherein the hydrolysis is carried out in a mixed solvent of an organic solvent and water.   The method according to claim 16, wherein the organic solvent is at least one selected from tert-butyl methyl ether and toluene.   The process according to claim 16, wherein the organic solvent is tert-butyl methyl ether.   The process according to claim 1, wherein the hydrolysis is carried out at 30-60 ° C.   The process according to claim 1, wherein the hydrolysis is carried out at 35 to 55 ° C. Formula (2)

(Where
R ² represents an amino-protecting group,
R ³ and R ⁴ each independently represent a hydrogen atom, a halogen atom, an alkyl group, a haloalkyl group, a hydroxy group, an alkoxy group, a cyano group or a nitro group. )
Is esterified with an optically active biphenylalanine compound represented by formula (2 ′):

(Where
R ¹ represents an alkyl group, a haloalkyl group, an alkenyl group, a cycloalkyl group, an aryl group which may have a substituent or an aralkyl group which may have a substituent,
R ² , R ³ and R ⁴ are as defined above. )
A step of obtaining an optically active biphenylalanine ester compound represented by:
The optically active biphenylalanine ester compound represented by the formula (2 ′) is racemized to give the formula (1)

(In the formula, each symbol has the same meaning as described above.)
A step of obtaining a biphenylalanine ester compound represented by:
The manufacturing method of the biphenylalanine ester compound represented by Formula (1) characterized by including these. Formula (1)

(Where
R ¹ represents an alkyl group, a haloalkyl group, an alkenyl group, a cycloalkyl group, an aryl group which may have a substituent or an aralkyl group which may have a substituent,
R ² represents an amino-protecting group,
R ³ and R ⁴ each independently represent a hydrogen atom, a halogen atom, an alkyl group, a haloalkyl group, a hydroxy group, an alkoxy group, a cyano group or a nitro group. )
In the presence of one or more alkalis selected from (i) alkali metal hydroxides and alkaline earth metal hydroxides, (ii) alkali metal hydroxides and alkaline earths In the presence of one or more alkalis and amino acids selected from metal hydroxides, or (iii) in the presence of one or more alkalis selected from alkali metal hydroxides and alkaline earth metal hydroxides and aminosulfonic acids. Formula (2) produced by hydrolysis using a protease derived from a microorganism belonging to the genus Bacillus

(In the formula, each symbol has the same meaning as described above.)
And an optically active biphenylalanine compound represented by formula (3):

(In the formula, each symbol has the same meaning as described above.)
Separating the unreacted optically active biphenylalanine ester compound represented by:
An optically active biphenylalanine compound represented by the formula (2) or a salt thereof is esterified to give the formula (2 ′)

(In the formula, each symbol has the same meaning as described above.)
A step of obtaining an optically active biphenylalanine ester compound represented by:
Racemizing the optically active biphenylalanine ester compound represented by the formula (2 ′) to obtain a biphenylalanine ester compound represented by the formula (1);
A method for recovering a biphenylalanine ester compound represented by the formula (1).