DE112008000311T5 - A method of collecting an L-biphenylalanine compound salt and a method of collecting a biphenylalanine ester compound using the same - Google Patents

Abstract

(wobei R₁ ein Alkyl, Halogenalkyl, Alkenyl, Cykloalkyl, eine optional substituierte Aryl- oder optional substituierte Aralkylgruppe wiedergibt, und R₂ eine Schutzgruppe für die Aminogruppe wiedergibt)
in einem gemischten Lösungsmittel, das ein organisches Lösungsmittel und Wasser umfasst, in einem pH-Bereich von 6 bis 13, und Abtrennen der wässrigen Schicht, die das erzeugte durch folgende Formel (2') wiedergegebene L-Biphenylalanin-Verbindungssalz enthält: [Chemische Formel 2]

(worin M ein Alkalimetallatom oder ½ Erdalkalimetallatom wiedergibt und R₂ dieselbe Definition wie oben besitzt),
aus der organischen Schicht, die die nicht umgesetzte durch folgende Formel (3) wiedergegebene D-Biphenylalaninester-Verbindung enthält: [Chemische Formel 3]

(wobei die Symbole dieselben Definitionen wie oben besitzen); und
Zugabe eines anorganischen Salzes und eines organischen Lösungsmittels zu...Recovery process for an L-biphenylalanine compound salt represented by formula (2 '), which comprises:
Use of a protease from a microorganism belonging to the genus Bacillus for the hydrolysis of a biphenylalanine ester compound represented by the following formula (1): [Chemical formula 1]

(wherein R _{1 represents} an alkyl, haloalkyl, alkenyl, cycloalkyl, an optionally substituted aryl or optionally substituted aralkyl group, and R _{2 represents} a protective group for the amino group)
in a mixed solvent comprising an organic solvent and water in a pH range of 6 to 13, and separating the aqueous layer containing the produced by the following formula (2 ') L-biphenylalanine compound salt: [Chemical formula 2]

(wherein M represents an alkali metal atom or ½ alkaline earth metal atom and R _{2 has the} same definition as above),
from the organic layer containing the unreacted D-biphenylalanine ester compound represented by the following formula (3): [Chemical Formula 3]

(where the symbols have the same definitions as above); and
Addition of an inorganic salt and an organic solvent to ...

Description

Technical area

The The present invention relates to a method for obtaining a L-biphenylalanine compound salt used in preparing D-biphenylalanine compounds, as intermediates for drugs and the like are produced, and a method for obtaining Biphenylalanine ester compounds using same.

State of the art

D-biphenylalanine is useful as an intermediate for drugs such as neutral endopeptidase inhibitors (see Patent Document 1). [Patent Document 1] Unexamined Japanese Patent Publication HEI No. 6-228187 ,

Disclosure of the invention

To be solved by the invention issues

method for preparing D-biphenylalanine include hydrolysis methods of biphenylalanine esters using enzymes (hereafter also called enzyme hydrolysis) (preparation of D-biphenylalanine ester and L-biphenylalanine salts). The L-biphenylalanine salts need however, either discarded or as free acids for effective use by a complicated recovery process, that's the separation and crystallization in an acidic environment includes, be recovered.

One The present invention is a recovery process and to provide the effective use of L-biphenylalanine salts, by enzyme hydrolysis of biphenylalanine esters, using a simple process.

Means for releasing of the problem

When Result of careful research that is done with the goal was to solve the above problem, the inventors have this Invention completed after discovering that L-biphenylalanine compound salts in the form of salts a simple process can be recovered the reaction mixture obtained after enzyme hydrolysis of a biphenylalanine ester was obtained in an aqueous and an organic layer is separated, and an inorganic salt and an organic solvent to the separated aqueous layer for extraction be added. The inventors have also found that, when the recovered L-biphenylalanine compound salt esterified and then racemized, this into a Biphenylalaninester can be converted, which makes it possible the starting material enzyme hydrolysis by a simple procedure to win.

• [1] Ein Rückgewinnungsverfahren für ein L-Biphenylalanin-Verbindungssalz (2'), welches folgendes umfasst: Verwenden einer Protease aus einem Mikroorganismus, der zur Gattung Bacillus gehört, für die Hydrolyse einer durch die folgende Formel (1) wiedergegebenen Biphenylalaninester-Verbindung: [Chemische Formel 1]
  
  (wobei R₁ ein Alkyl, Halogenalkyl, Alkenyl, Cycloalkyl, eine optional substituierte Aryl- oder optional substituierte Aralkylgruppe wiedergibt, und R₂ eine Schutzgruppe für die Aminogruppe wiedergibt) (nachfolgend ebenso als „Biphenylalaninester-Verbindung (1)” bezeichnet) in einem gemischten Lösungsmittel, das ein organisches Lösungsmittel und Wasser umfasst, in einem pH-Bereich von 6 bis 13, und Abtrennen der wässrigen Schicht, die das erzeugte durch die folgende Formel (2') wiedergegebene L-Biphenylalanin-Verbindungssalz enthält: [Chemische Formel 2]
  
  (worin M ein Alkalimetallatom oder ½ Erdalkalimetallatom wiedergibt und R₂ dieselbe Definition wie oben besitzt), (nachfolgend ebenso als „L-Biphenylalanin-Verbindungssalz (2')” bezeichnet), aus der organischen Schicht, die die nicht umgesetzte durch die folgende Formel (3) wiedergegebene D-Biphenylalaninester-Verbindung enthält: [Chemische Formel 3]
  
  (wobei die Symbole dieselben Definitionen wie oben besitzen), (nachfolgend ebenso als „D-Biphenylalaninester-Verbindung (3)” bezeichnet); und Zugabe eines anorganischen Salzes und eines organischen Lösungsmittels zu der wässrigen Schicht zum Extrahieren des L-Biphenylalanin-Verbindungssalzes (2') in der wässrigen Schicht.
• [2] Rückgewinnungsverfahren nach obigem [1], wobei das organische Lösungsmittel für die Hydrolyse wenigstens eines ist, ausgewählt aus Methyl-tert-Butylether und Toluol.
• [3] Rückgewinnungsverfahren nach obigem [1], wobei das organische Lösungsmittel für die Extraktion wenigstens eines ist, ausgewählt aus Methyl-tert-Butylether und Toluol.
• [4] Rückgewinnungsverfahren für eine Biphenylalaninester-Verbindung (1), welches folgendes umfasst: Verwenden einer Protease aus einem Mikroorganismus, der zur Gattung Bacillus gehört, für die Hydrolyse einer Biphenylalaninester-Verbindung (1) in einem gemischten Lösungsmittel, das ein organisches Lösungsmittel und Wasser umfasst, in einem pH-Bereich von 6 bis 13, und Abtrennen der wässrigen Schicht, die das erzeugte L-Biphenylalanin-Verbindungssalz (2') enthält, und der organischen Schicht, die die nicht umgesetzte D-Biphenylalaninester-Verbindung (3) enthält; Zugabe eines anorganischen Salzes und eines organischen Lösungsmittels zu der wässrigen Schicht, zum Extrahieren des L-Biphenylalanin-Verbindungssalzes (2') in der wässrigen Schicht; Verestern des extrahierten L-Biphenylalanin-Verbindungssalzes (2') zum Erhalt einer durch die folgende Formel (2) wiedergegebenen L-Biphenylalaninester-Verbindung (nachfolgend ebenso als „L-Biphenylalaninester-Verbindung (2)” bezeichnet): [Chemische Formel 4]
  
  (wobei die Symbole dieselben Definitionen wie oben besitzen); und Razemisieren der L-Biphenylalaninester-Verbindung (2) mit einer Base zum Erhalt der Biphenylalaninester-Verbindung (1).
• [5] Rückgewinnungsverfahren nach obigem [4], wobei das organische Lösungsmittel für die Hydrolyse wenigstens eines ist, ausgewählt aus Methyl-tert-Butylether und Toluol.
• [6] Rückgewinnungsverfahren nach obigem [4], wobei das organische Lösungsmittel für die Extraktion wenigstens eines ist, ausgewählt aus Methyl-tert-Butylether und Toluol.
• [7] Rückgewinnungsverfahren nach obigem [4], wobei das Verestern durch Umsetzen mit dem entsprechenden Schwefelsäureester oder -halid in der Gegenwart einer Base durchgeführt wird.
• [8] Rückgewinnungsverfahren nach obigem [4], wobei R₁ Methyl ist.
• [9] Rückgewinnungsverfahren nach obigem [8], wobei das Verestern durch Umsetzen mit Dimethylschwefelsäure in Gegenwart einer Base durchgeführt wird.
• [10] Rückgewinnungsverfahren nach obigem [4], wobei die für die Razemisierung verwendete Base ausgewählt ist aus Alkalimetall-Carbonaten und Alkalimetall-Alkoholaten.
• [11] Rückgewinnungsverfahren nach obigem [4], wobei die für die Razemisierung verwendete Base ein Alkalimetall-Alkoholat ist.
• [12] Rückgewinnungsverfahren nach obigem [4], wobei die für die Razemisierung verwendete Base Natriummethylat ist.
• [13] Rückgewinnungsverfahren nach obigem [4], wobei R₁ eine Alkylgruppe ist und R₂ eine durch die Formel -CO₂R₃ wiedergegebene Gruppe ist, (wobei R₃ ein Alkyl, eine optional substituierte Aryl-, optional substituierte Aralkyl- oder eine 9-Fluorenmethylgruppe ist).
• [14] Rückgewinnungsverfahren nach obigem [13], wobei die Razemisierung nach Isolieren der L-Biphenylalaninester-Verbindung (2) als Feststoff durchgeführt wird.
• [15] Rückgewinnungsverfahren für DL-N-Boc-Biphenylalaninmethylester (1a). das folgendes umfasst: Verwenden einer Protease aus einem Mikroorganismus, der zur Gattung Bacillus gehört, für die Hydrolyse eines durch die folgende Formel (1a) wiedergegebenen DL-N-Boc-Biphenylalaninmethylesters: [Chemische Formel 5]
  
  (nachfolgend ebenso als „DL-N-Boc-Biphenylalaninmethylester (1a)” bezeichnet) in einem gemischten Lösungsmittel, das Methyl-tert-Butylether und Wasser umfasst, in einem pH-Bereich von 6 bis 13 mit einer wässrigen Lösung aus Taurin und Kaliumhydroxid, und Abtrennen der wässrigen Schicht, die das erzeugte durch die folgende Formel (2'a) wiedergegebene L-N-Boc-Biphenylalaninkaliumsalz enthält: [Chemische Formel 6]
  
  (nachfolgend ebenso als „L-N-Boc-Biphenylalaninkaliumsalz (2'a)” bezeichnet) und der organischen Schicht, die den nicht umgesetzten durch die folgende Formel (3a) wiedergegebenen D-N-Boc-Biphenylalaninmethylester enthält: [Chemische Formel 7]
  
  (nachfolgend ebenso als „D-N-Boc-Biphenylalaninmethylester (3a)” bezeichnet); Zugabe eines anorganischen Salzes und Methyl-tert-Butylether zu der wässrigen Schicht zum Extrahieren des L-N-Boc-Biphenylalaninkaliumsalzes (2'a) in der wässrigen Schicht; Verestern des extrahierten L-N-Boc-Biphenylalaninkaliumsalzes (2'a) mit Dimethylschwefelsäure in der Gegenwart einer Base, zum Erhalt des durch die folgende Formel (2a) wiedergegebenen L-N-Boc-Biphenylalaninmethylesters: [Chemische Formel 8]
  
  (nachfolgend ebenso als „L-N-Boc-Biphenylalaninmethylester (2a)” bezeichnet); und Razemisieren des L-N-Boc-Biphenylalaninmethylesters (2a) mit Natriummethylat nach dessen Isolierung als Feststoff, zum Erhalt des DL-N-Boc-Biphenylalaninmethylesters (1a).
• [16] Verfahren zum Herstellen einer D-Biphenylalaninester-Verbindung (3), welches folgendes umfasst: Verwenden einer Protease aus einem Mikroorganismus, der zu der Gattung Bacillus gehört, für die Hydrolyse einer Biphenylalaninester-Verbindung (1), die durch ein Rückgewinnungsverfahren gemäß obigem [4] in einem gemischten Lösungsmittel erhalten wurde, welches ein organisches Lösungsmittel und Wasser umfasst, in einem pH-Bereich von 6 bis 13, und Abtrennen der organischen Schicht, die die nicht umgesetzte D-Biphenylalaninester-Verbindung (3) enthält; und Reinigen der D-Biphenylalaninester-Verbindung (3).
• [17] Verfahren zum Herstellen von D-N-Boc-Biphenylalaninmethylester (3a), welches folgendes umfasst: Verwenden einer Protease aus einem Mikroorganismus, der zur Gattung Bacillus gehört, für die Hydrolyse von DL-N-Boc-Biphenylalaninmethylester (1a), der durch ein Rückgewinnungsverfahren gemäß obigem [15] in einem gemischten Lösungsmittel erhalten wird, das ein organisches Lösungsmittel und Wasser umfasst, in einem pH-Bereich von 6 bis 13, und Abtrennen der organischen Schicht, die den nicht umgesetzten D-N-Boc-Biphenylalaninmethylester (3a) enthält; und Reinigen des D-N-Boc-Biphenylalaninmethylesters (3a).

In particular, the invention provides the following.

• [1] A recovery method of an L-biphenylalanine compound salt (2 ') comprising: using a protease from a microorganism belonging to the genus Bacillus for the hydrolysis of a biphenylalanine ester compound represented by the following formula (1): [Chemical Formula 1]
  
  (wherein R ₁ represents an alkyl, haloalkyl, alkenyl, cycloalkyl, an optionally substituted aryl or optionally substituted aralkyl group, and R ₂ is a protective group for the amino group represents) (hereinafter also referred to as "Biphenylalaninester compound (1)") in a mixed solvent comprising an organic solvent and water in a pH range of 6 to 13, and Ab separating the aqueous layer containing the produced L-biphenylalanine compound salt represented by the following formula (2 '): [Chemical Formula 2]
  
  (wherein M represents an alkali metal atom or ½ alkaline earth metal atom and R _{2 has the} same definition as above), (hereinafter also referred to as "L-biphenylalanine compound salt (2 ')"), from the organic layer, those unreacted by the following formula (3) D-biphenylalanine ester compound shown contains: [Chemical Formula 3]
  
  (wherein the symbols have the same definitions as above), (hereinafter also referred to as "D-biphenylalanine ester compound (3)"); and adding an inorganic salt and an organic solvent to the aqueous layer to extract the L-biphenylalanine compound salt (2 ') in the aqueous layer.
• [2] Recovery method according to [1] above, wherein the organic solvent for the hydrolysis is at least one selected from methyl tert-butyl ether and toluene.
• [3] Recovery method according to [1] above, wherein the organic solvent for extraction is at least one selected from methyl tert-butyl ether and toluene.
• [4] The recovery method of a biphenylalanine ester compound (1) which comprises using a protease from a microorganism belonging to the genus Bacillus for the hydrolysis of a biphenylalanine ester compound (1) in a mixed solvent containing an organic solvent and Water, in a pH range of 6 to 13, and separating the aqueous layer containing the produced L-biphenylalanine compound salt (2 ') and the organic layer containing the unreacted D-biphenylalanine ester compound (3) contains; Adding an inorganic salt and an organic solvent to the aqueous layer, extracting the L-biphenylalanine compound salt (2 ') in the aqueous layer; Esterifying the extracted L-biphenylalanine compound salt (2 ') to obtain an L-biphenylalanine ester compound represented by the following formula (2) (hereinafter also referred to as "L-biphenylalanine ester compound (2)"): [Chemical Formula 4]
  
  (where the symbols have the same definitions as above); and racemizing the L-biphenylalanine ester compound (2) with a base to obtain the biphenylalanine ester compound (1).
• [5] recovery method according to [4] above, wherein the organic solvent for the hydrolysis we at least one is selected from methyl tert-butyl ether and toluene.
• [6] Recovery method according to [4] above, wherein the organic solvent for extraction is at least one selected from methyl tert-butyl ether and toluene.
• [7] Recovery method according to [4] above, wherein the esterification is carried out by reacting with the corresponding sulfuric acid ester or halide in the presence of a base.
• [8] Recovery method according to [4] above, wherein R _{1 is} methyl.
• [9] Recovery method according to [8] above, wherein the esterification is carried out by reacting with dimethylsulfuric acid in the presence of a base.
• [10] Recovery method according to [4] above, wherein the base used for the racemization is selected from alkali metal carbonates and alkali metal alcoholates.
• [11] Recovery method according to [4] above, wherein the base used for the racemization is an alkali metal alkoxide.
• [12] Recovery method according to [4] above, wherein the base used for the racemization is sodium methylate.
• [13] Recovery method according to [4] above, wherein R _{1 is} an alkyl group and R _{2 is} a group represented by the formula -CO ₂ R ₃ (wherein R _{3 is} an alkyl, an optionally substituted aryl, optionally substituted aralkyl or a 9-fluorenemethyl group).
• [14] The recovery method of [13] above, wherein the racemization is carried out after isolating the L-biphenylalanine ester compound (2) as a solid.
• [15] Recovery procedure for DL-N-Boc-biphenylalanine methyl ester (1a). which comprises: using a protease from a microorganism belonging to the genus Bacillus for the hydrolysis of a DL-N-Boc-biphenylalanine methyl ester represented by the following formula (1a): [Chemical Formula 5]
  
  (hereinafter also referred to as "DL-N-Boc-biphenylalanine methyl ester (1a)") in a mixed solvent comprising methyl tert-butyl ether and water in a pH range of 6 to 13 with an aqueous solution of taurine and potassium hydroxide and separating the aqueous layer containing the LN-Boc-biphenylalanine potassium salt produced by the following formula (2'a): [Chemical Formula 6]
  
  (hereinafter also referred to as "LN-Boc-biphenylalanine potassium salt (2'a)") and the organic layer containing the unreacted DN-Boc-biphenylalanine methyl ester represented by the following formula (3a): [Chemical Formula 7]
  
  (hereinafter also referred to as "DN-Boc-biphenylalanine methyl ester (3a)"); Adding an inorganic salt and methyl tert-butyl ether to the aqueous layer for extraction the LN-Boc-biphenylalanine potassium salt (2'a) in the aqueous layer; Esterifying the extracted LN-Boc-biphenylalanine potassium salt (2'a) with dimethylsulfuric acid in the presence of a base to obtain the LN-Boc-biphenylalanine methyl ester represented by the following formula (2a): [Chemical formula 8]
  
  (hereinafter also referred to as "LN-Boc-biphenylalanine methyl ester (2a)"); and racemizing the LN-Boc-biphenylalanine methyl ester (2a) with sodium methylate after isolation thereof as a solid to obtain the DL-N-Boc-biphenylalanine methyl ester (1a).
• [16] A method for producing a D-biphenylalanine ester compound (3), which comprises using a protease from a microorganism belonging to the genus Bacillus for the hydrolysis of a biphenylalanine ester compound (1) produced by a recovery method according to above [4] in a mixed solvent comprising an organic solvent and water in a pH range of 6 to 13, and separating the organic layer containing the unreacted D-biphenylalanine ester compound (3); and purifying the D-biphenylalanine ester compound (3).
• [17] A process for producing DN-Boc-biphenylalanine methyl ester (3a) which comprises using a protease from a microorganism belonging to the genus Bacillus for the hydrolysis of DL-N-Boc-biphenylalanine methyl ester (1a), which is characterized by a recovery process according to [15] above is obtained in a mixed solvent comprising an organic solvent and water in a pH range of 6 to 13, and separating the organic layer containing the unreacted DN-Boc-biphenylalanine methyl ester (3a) contains; and purifying the DN-Boc-biphenylalanine methyl ester (3a).

Effect of the invention

According to the Process of the invention it is possible to L-biphenylalanine compound salts in the form of salts from reaction mixtures back to after enzyme hydrolysis of biphenylalanine ester compounds were obtained using a simple method. additionally can, since the conversion to Biphenylalaninester connections by esterification and racemization of the recovered L-biphenylalanine compound salts possible, the starting materials of the enzyme hydrolysis be recovered by a simple process. Therefore may be the L-biphenylalanine compounds that are not the target Enzyme hydrolysis are effective for a more efficient Preparation of D-biphenylalanine ester compounds can be used and therefore the process of the invention is economically advantageous.

Best way to run the invention

The Definitions of the substituents used in the present specification will be explained now.

As "alkyl groups" may be mentioned C ₁ -C ₆ straight or branched chain alkyl groups such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl and hexyl, with methyl or ethyl preferred and methyl is more preferred.

As "alkenyl groups" may be mentioned C ₂ -C ₆ straight or branched alkenyl groups such as allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl and 3-butenyl, with allyl being preferred.

As "cycloalkyl groups" may be mentioned C ₃ -C ₈ cycloalkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl, with cyclopentyl or cyclohexyl being preferred.

As "halogen atoms" can Fluorine, chlorine, bromine and iodine, with fluorine, Chlorine or bromine are preferred.

As "haloalkyl groups" may be mentioned "alkyl groups" according to the aforementioned definition which are substituted with halogen atoms. The number of halogen atom substituents is not particularly limited, but is preferably 1 to 3. As examples of "haloalkyl groups", chloromethyl, bromomethyl, fluoromethyl, dichloromethyl, dibromomethyl, difluoromethyl, trichloromethyl, tribromomethyl, trifluoromethyl, 2,2-dichloroethyl and 2 , 2,2-trichloroethyl, with trifluoromethyl being preferred.

As "alkoxy groups" may be mentioned C ₁ -C ₆ straight or branched alkoxy groups such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, isopentyloxy, neopentyloxy and hexyloxy, with methoxy or ethoxy are preferred.

As "aryl groups" for the "optionally substituted aryl group" may be mentioned C ₆ -C ₁₄ aryl groups, such as phenyl, 1-naphthyl and 2-naphthyl, with phenyl being preferred.

Such Aryl groups may have substituents at substitutable positions in which the substituents are halogen atoms (examples thereof are mentioned above), alkyl groups (examples thereof are above mentioned), haloalkyl groups (examples of which are mentioned above), hydroxy, alkoxy groups (examples thereof mentioned above), cyano, nitro and the like. The number of substituents is not particularly limited but is preferably 1 to 3. When the number of substituents is two or more, the substituents may be the same or be different.

The "aralkyl groups" for the "optionally substituted aralkyl group" be the aforementioned "alkyl groups", the substituted with the aforementioned "aryl groups" are. The number of aryl group substituents is not particular limited, but is preferably 1 to 3. Als Examples of "aralkyl groups" may be Benzyl, phenethyl, 1-phenylethyl, 1-phenylpropyl, 2-phenylpropyl, 3-phenylpropyl, 1-naphthylmethyl, 2-naphthylmethyl, benzhydrile and Trityl, with benzyl being preferred.

Such Aralkyl groups may have substituents on substitutable Have positions at which the substituents halogen atoms (examples the same are mentioned above), alkyl groups (examples thereof are mentioned above), haloalkyl groups (examples thereof are mentioned above), hydroxy, alkoxy groups (examples the same are mentioned above), cyano, nitro and the like be. The number of substituents is not particularly limited however, is preferably 1 to 3. When the number of substituents is two or more, the substituents may be the same or to be different.

The "protective group for the amino group" represented by R ₂ may be any protecting group known as a protective group for amino groups, without any particular limitations. As such protecting groups, -CO ₂ R ₃ (wherein R _{3 represents} an alkyl, an optionally substituted aryl, optionally substituted aralkyl or a 9-fluorenemethyl group), -COR ₄ (wherein R _{4 is} an alkyl, haloalkyl, alkenyl, an optional substituted aryl group or optionally substituted aralkyl group), and optionally substituted aralkyl groups. As examples of the "protective group for the amino group", there can be mentioned methoxycarbonyl, etoxycarbonyl, isobutoxycarbonyl, tert-butoxycarbonyl (Boc), allyloxycarbonyl, benzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 9-fluorene-methyloxycarbonyl, benzoyl, benzyl, benzhydril and trityl become.

R ₁ is preferably an alkyl group, more preferably a C ₁ -C ₄ alkyl group, even more preferably methyl or ethyl, and most preferably methyl. R ₂ is preferably -CO ₂ R ₃ (wherein R _{3 has the} same definition as above), and more preferably tert-butoxycarbonyl (Boc).

As "alkali metal atoms" can Potassium, sodium and lithium are listed.

As "alkaline earth metal atoms" can Calcium and magnesium are given.

M is preferably an alkali metal atom, more preferred Potassium or sodium, and most preferably potassium.

The biphenylalanine ester compound (1) used as a starting material in the process of the invention can be produced, for example, by the following method. [Chemical formula 9]

(The Symbols in the formulas have the same definitions as above).

Step 1
The compound (4) and hydantoin can be reacted in the presence of a base to obtain the compound (5).

step 2
The compound (5) can be reduced to obtain the compound (6). The reduction is preferably carried out by catalytic hydrogenation.

step 3
The compound (6) can be hydrolyzed to obtain the compound (7).

Step 4
The compound (7) may be supplied to protect the amino group and the esterification reaction with the carboxyl group to obtain the biphenylalanine ester compound (1). Protecting the amino group and esterifying the carboxyl group can be carried out by conventional methods. There is no particular restriction on the order of protecting the amino group and esterifying with the carboxyl group, and these may be carried out in any desired order.

The Biphenylalanine ester compound (1) includes two optical Isomers (L and D isomers) with an asymmetric center on the carbon atom at the α-position, and those used in the process of the invention Biphenylalanine ester compound (1) may be a racemic mixture containing equal amounts of optical isomers, or a mixture containing one of the two optical isomers in excess (in any desired proportion). It is preferably a racemic mixture.

The obtained in the manner described above Biphenylalaninester compound (1) using a protease from a microorganism, which belongs to the genus Bacillus, hydrolyzed. Using the protease for hydrolysis results in a preferred Hydrolysis of the L-isomer.

The Proteases from the microorganisms belonging to the genus Bacillus, are preferably proteases from Bacillus lichenformis, due to their excellent enantioselectivity. As specific examples of Proteases from Bacillus licheniformis can be a protease from Bacillus licheniformis containing subtilisin under ALCALASE (registered trademark of Novozymes) is preferred, and ALCALASE 2.4 L (registered trademark of Novozymes) is particularly preferred.

It There are no special restrictions regarding the purity and shape of the protease, and various forms including purified enzymes, crude enzymes, microbial cultures, cells or treated products of the foregoing. A treated product may be, for example, lyophilized cells, crushed cells or a cell extract. An enzyme with any Purity and in any of the various above-mentioned Forms on an inorganic support material, such as Silica gel or ceramic or on cellulose, an ion exchange resin or the like is immobilized, may be used.

The Amount of protease used is not particularly limited however, it is usually 0.001-0.5 g and preferably at 0.001-0.1 g, such as purified enzyme per one gram the biphenylalanine ester compound (1).

The Hydrolysis with the protease is in a pH range of 6 to 13 carried out, preferably pH 6 to 10, and stronger preferably 6.0 to 9.5, depending on the type of protease. The Hydrolysis within this pH range can be a product with high optical purity.

Of the pH can be adjusted within the aforementioned range by appropriate Use of an alkaline and / or buffering agent are kept.

When Examples of buffering agents may include phosphate buffers, Acetate buffer, amino acid buffer, aminosulfonate buffer and the like.

When Phosphate buffers may include disodium hydrogen phosphate, sodium dihydrogen phosphate, Dipotassium hydrogen phosphate, potassium dihydrogen phosphate and the like be cited.

When Acetate buffers may include sodium acetate, potassium acetate and the like be cited.

The Amino acids in amino acid buffers are organic Compounds containing both an amino group (including substituted amino groups and cyclic substituted amino groups) as well as a carboxyl group in a molecule (these close Imino acids, wherein a hydrogen of the amino group a having cyclic structure with part of the side chain), and they do not just exclude α-amino acids, but also β-amino acids, γ-amino acids and δ-amino acids. As specific examples of α-amino acids can be neutral amino acids, such as glycine, alanine, α-aminobutyric acid, leucine, Isoleucine, valine, phenylalanine, tryptophan, tyrosine, methionine, Cysteine, threonine, serine, proline, hydroxyproline, asparagine and glutamine be cited; acidic amino acids such as aspartic acid and glutamic acid; and basic amino acids like Lysine, tryptophan, arginine, ornithine, histidine and hydroxylysine. As specific examples of β-amino acids may be β-alanine and β-aminobutyric acid be cited. As a specific example of a γ-amino acid can be γ-aminobutyric acid be cited. As a specific example of a δ-amino acid 5-aminovaleric acid can be mentioned. glycine is preferred as the amino acid.

A Aminosulfonic acid for an aminosulfonate buffer is an organic compound that uses a sulfur group instead the carboxyl group in any of the aforementioned amino acids and, as specific examples, taurine, N-methyl taurine and 2- (4-morpholinyl) ethanesulfonic acid with taurine being preferred.

taurine or phosphate buffers are preferred as buffering agents, with taurine is particularly preferred.

The Concentration of the buffering agent is usually 0.05-0.8 M and preferably at 0.1-0.8 M, and more preferably at 0.1-0.5 M. A concentration of the buffer solution Within this range can be a product with high optical purity result.

The Amount of buffering agent used depends on the concentration but is usually 0.1-100 ml and preferably 0.4-10 ml based on 1 g of the biphenylalanine ester compound (1). Using the buffer solution within this area contributes to a uniform implementation at.

When a base is used, it may be used as a solid or as an aqueous solution, although an aqueous solution is preferable for handleability. As aqueous basic solutions, aqueous solutions of alkali metal hydroxide (sodium hydroxide, potassium hydroxide), aqueous solutions of alkaline earth metal hydroxide (calcium hydroxide, magnesium hydroxide), aqueous solutions of alkali metal carbonate (sodium carbonate, potassium carbonate), aqueous solutions of alkaline earth metal carbonate (calcium carbonate, Magnesium carbonate), aqueous solutions of alkali metal hydrogencarbonates (sodium hydrogencarbonate, potassium hydrogencarbonate), aqueous solutions of alkaline earth metal hydrogencarbonate (calcium hydrogencarbonate, magnesium hydrogencarbonate), and the like. Preferred among these are aqueous solutions of sodium hydroxide and potassium hydroxide, with an aqueous solution of potassium hydroxide being particularly preferred.

The Amount of used base can be a lot to adjust the pH to be 6 to 13.

The pH adjustment is preferred using taurine and a aqueous solution of potassium hydroxide or a Phosphate buffer and an aqueous solution of Potassium hydroxide performed, and strongest preferably it is using taurine and an aqueous Solution of potassium hydroxide performed.

The Hydrolysis is carried out in a mixed solvent which includes an organic solvent and water.

When organic solvents for the hydrolysis can hydrophobic organic solvents and hydrophilic organic Solvents are listed.

When hydrophobic organic solvents can be ethers, such as methyl tert-butyl ether (MTBE) and diisopropyl ether and hydrocarbons such as toluene, hexane, cyclohexane and heptane. As hydrophilic organic solvents can Ethers such as tetrahydrofuran (THF); Alcohols such as methanol, ethanol, [sopropanol, n-butanol and tert-butanol; Sulfoxides such as dimethylsulfoxide; Ketones such as acetone; and nitriles such as acetonitrile become. These organic solvents can used alone or in combinations of two or more.

MTBE and toluene are preferred as organic solvents, MTBE being particularly preferred.

The Amount of organic solvent used is usually 1-50 ml and preferably 1-5 ml based on 1 g of Biphenylalaninester compound (1). Using of organic solvent within this range contributes to a uniform implementation at.

The Water in the buffering agent and in the aqueous alkaline Solution can also serve as a solvent.

The Hydrolysis is achieved by mixing the biphenylalanine ester compound (1), the protease from a microorganism belonging to the genus Bacillus belongs, a solvent, the base or their aqueous solution and / or a buffering agent carried out.

• (i) Zugabe der in einem organischen Lösungsmittel gelösten Biphenylalaninester-Verbindung (1) zu einer Base und/oder einem Puffermittel und dann Zugabe der Protease,
• (ii) Zugabe einer Base und/oder eines Pufferungsmittels zu der in einem organischen Lösungsmittel gelösten Biphenylalaninester-Verbindung (1) und dann Zugabe der Protease,
• (iii) Zugabe der Protease (wenn nötig mit Wasser) zu der in einem organischen Lösungsmittel gelösten Biphenylalaninester-Verbindung (1) und dann Zugabe einer Base und/oder eines Pufferungsmittels (bevorzugt durch tropfenweise Zugabe), oder
• (iv) Zugabe der Protease und des Pufferungsmittels (wenn nötig mit Wasser) zu der in einem organischen Lösungsmittel gelösten Biphenylalaninester-Verbindung (1) und dann Zugabe einer Base (bevorzugt durch tropfenweise Zugabe).

There is no particular limitation on the order of addition, and the method may include, for example:

• (i) adding the biphenylalanine ester compound (1) dissolved in an organic solvent to a base and / or a buffering agent and then adding the protease,
• (ii) adding a base and / or a buffering agent to the biphenylalanine ester compound (1) dissolved in an organic solvent and then adding the protease,
• (iii) adding the protease (if necessary with water) to the biphenylalanine ester compound (1) dissolved in an organic solvent and then adding a base and / or a buffering agent (preferably by dropwise addition), or
• (iv) adding the protease and the buffering agent (if necessary with water) to the biphenylalanine ester compound (1) dissolved in an organic solvent and then adding a base (preferably by dropwise addition).

additional Protease may, if necessary, during the reaction be added. When the pH of the reaction mixture during the implementation falls below the aforementioned range, is a base or its aqueous solution for Adjusting the pH of the reaction mixture back in used the area.

The Reaction temperature for the hydrolysis, in terms of enzyme stability and conversion rate, preferably 30-60 ° C, more preferably 35-55 ° C, and most preferably 40-45 ° C.

The Reaction time for the hydrolysis is normally 3-24 hours and preferably 4-15 hours.

The liquid separation of the reaction mixture into an aqueous and an organic layer after hydrolysis allows separation of the L-biphenylalanine compound salt (2 ') produced by the hydrolysis and the unreacted D-biphenylalanine ester compound (3). The L-biphenylalanine compound salt (2 ') is in the aqueous Layer containing, and the D-biphenylalanine ester compound (3) is contained in the organic layer. The D-biphenylalanine ester compound can be purified from the organic layer by a known method become.

According to the Invention are an inorganic salt and an organic solvent to the aqueous layer containing the L-biphenylalanine compound salt (2 ') for extracting the L-biphenylalanine compound salt (2 ') was added with the organic solvent. This allows the L-biphenylalanine compound salt (2 ') recovered from the aqueous layer directly in the form of a salt becomes.

When inorganic salts for this step may be sodium chloride, Potassium chloride, ammonium chloride and the like of which potassium chloride and sodium chloride are from the standpoint the economy and solubility of the L-biphenylalanine compound salt (2 ') are preferred. The amount of inorganic salt used is usually in terms of a satisfactory Extraction rate 6 g or more and preferably 6-9 g, based to 100 g of the L-biphenylalanine compound salt (2 ') containing aqueous solution. The inorganic salt can be the aqueous layer also in a supersaturation amount be added.

The organic solvents for extraction can a solvent such as MTBE, toluene, ethyl acetate or the like of which MTBE or toluene are preferred, and especially MTBE is preferred.

The Amount of organic solvent used is normally 20-100 g and preferably 25-50 g, based on 100 g of the L-biphenylalanine compound salt (2 ') containing aqueous solution.

The Extraction can be done twice or more if necessary become.

The Extraction usually takes place at 10-50 ° C carried out.

Of the The extract containing L-biphenylalanine compound salt (2 ') can washed with an aqueous alkaline solution become. The aqueous alkaline solution may be a aqueous solution of potassium hydroxide, a aqueous solution of sodium hydroxide or the like, usually at a concentration of 5-15% (w / w). The amount of aqueous alkaline solution used is 0.95-1.01 mol, based on 1 mol of the L-biphenylalanine compound salt (2 '). When the extract with the aqueous alkaline solution can be washed, an inorganic salt, such as potassium chloride, Potassium bromide or sodium chloride to the aqueous solution be added for an improved yield. The amount used inorganic salt may be an amount for a inorganic salt concentration of at least 5% (w / w) in the aqueous be alkaline solution.

Therefore According to the process of the invention, the L-biphenylalanine compound salt (2 ') directly in salt form without conversion to a free acid be recovered.

The recovered L-biphenylalanine compound salt (2 ') esterified and then converted to the biphenylalanine ester compound (1) for the recovery of the starting material the enzyme hydrolysis, be racemized.

On the other hand, the D-biphenylalanine ester compound (3) is useful as an intermediate for producing drugs such as neutral endopeptidase inhibitors. For example, in the Japanese Unexamined Patent Publication HEI No. 6-228187 described methods for preparing the described in the same publication N-phosphonomethylbiphenyl substituted dipeptide derivative from the D-Biphenylalaninester compound (3) can be used.

Now is a method for esterification and racemization of the L-biphenylalanine compound salt (2 '), for conversion to the biphenylalanine ester compound (1).

First is the L-biphenylalanine compound salt (2 ') to obtain a Esterified L-biphenylalanine ester compound (2).

The Esterification usually takes place in a solvent however, the L-biphenylalanine compound salt is preferred (2 ') extract used directly. If necessary can also a solvent may be added. MTBE and toluene are preferred as organic solvents with MTBE being stronger is preferred.

The esterification may be carried out by a conventional method such as a method in which an acid and an alcohol such as methanol or ethanol are used, a method in which a base and a sulfuric acid ester ((R ₁ O) ₂ SO ₂ ) such as dimethylsulfuric acid or diethylsulfuric acid; a method in which R ₁ OH and a condensing agent such as DCC are used; or a method in which a base and a halide (R ₁ X, X: halogen atom) such as methyl iodide, bromoethane or benzyl chloride are used; in these methods, it is preferable to use bases and sulfuric acid esters in terms of smaller by-products and greater convenience.

Now is a process in which a base and a sulfuric acid ester be used explained.

When Bases may be inorganic bases such as sodium bicarbonate, Potassium bicarbonate, potassium carbonate, sodium hydroxide and potassium hydroxide, and organic bases such as diisopropylethylamine, 2,6-dimethylpyridine, Triethylamine and pyridine, with sodium bicarbonate is preferred.

The Amount of base used is usually 0.3-1.2 mol and preferably 0.8-1.2 mol, based to 1 mole of the L-biphenylalanine compound salt (2 ').

Of the Sulfuric acid ester is according to the desired L-biphenylalanine ester compound (2) product selected, however, according to the invention is preferably a methyl ester, and therefore, dimethylsulfuric acid is preferably used.

The Amount of sulfuric acid ester used usually 1.2-2.5 moles and preferably 1.6-2.0 Mole, based on 1 mole of the L-biphenylalanine compound salt (2 ').

Regarding the reactivity closes the process for esterification prefers the dropwise addition of the sulfuric acid ester to a solution containing the base and the L-biphenylalanine compound salt (2 ') contains.

The Temperature for esterification is usually 30-50 ° C. The reaction time varies depending of the amount of reagents and the reaction temperature but usually 1-10 hours. The termination the reaction can be confirmed by HPLC analysis.

To To terminate the reaction, an amine such as triethylamine may be added and the mixture at a temperature of 30-50 ° C stirred for 2 to 5 hours to the remaining Reduce sulfuric acid ester. The amount of used Amine can be about 10 mole% based on the sulfuric acid ester be.

The separated aqueous layer is then by liquid Separation removed and the organic layer is dehydrated. Dehydration may be carried out using a dehydrating agent, such as anhydrous magnesium sulfate, anhydrous sodium sulfate, or molecular sieves are performed, but in terms of Handleability, the method preferably includes the azeotropic Dehydration with a solvent that is mixed with water is azeotrope (for example, toluene).

The Azeotropic solvents can be used in an amount be used, which allows sufficient dehydration, and is usually 50-100% by weight with respect to the solution.

The subsequent racemization may be with the dehydrated solution without isolation of the L-biphenylalanine ester compound (2) or the racemization can be carried out after being first as a solid, as in crystalline Form or as oil was isolated.

in the In the first case, the water content of the solution is preferably not higher than 500 ppm, and therefore, sufficient dehydration prefers.

In the latter case, for example, when the L-biphenylalanine ester compound (2) is an is in which R _{1 is} an alkyl group and R _{2 is} -CO ₂ R ₃ , the dehydrated solution can be concentrated and crystallization from methanol and water can be performed , The isolation in the form of a solid, such as a crystal or an oil in this way, can prevent the consumption of the base by the esterification reaction residue (for example, sulfuric acid ester derived compounds), allowing the racemization to be carried out with a smaller amount of base, as well prevents hydrolysis of the L-biphenylalanine ester compound (2).

One specific preferred example of crystallization from methanol and water will now be described.

The The amount of methanol used is usually 380-520 g and preferably 450-490 g, based on 100 g of L-Biphenylalaninester compound (2).

water is added dropwise to a methanol-dissolved solution at a temperature of about 45-55 ° C added. The amount of water added is normally 160-220 g and preferably 190-210 g, based on 100 g of the L-biphenylalanine ester compound (2). After adding the Water is added to a small amount of a seed crystal and the mixture is stirred at the same temperature. After confirmation the deposition of crystals becomes water at the same temperature added for sufficient crystal deposition. The Amount of water in this step is normally 0-60 g and preferably 40-60 g, based on 100 g of the L-biphenylalanine ester compound (2). The mixture then becomes cooled to about 5 ° C, and the crystals are filtered and with about 70% aqueous Washed methanol solution. The crystals of the L-biphenylalanine ester compound (2) can be obtained by drying under reduced pressure become.

The L-biphenylalanine ester compound obtained by the esterification (2) is then converted to a biphenylalanine ester compound (1) racemized.

In This Razemisierungsschritt can the L-Biphenylalaninester compound (2) directly as a dehydrated solution used in the esterification step , or the L-biphenylalanine ester compound (2) can be isolated first. In the latter case, the L-biphenylalanine ester compound becomes (2) dissolved in a solvent. MTBE and toluene are preferred in this case as a solvent, wherein MTBE is more preferred. The amount of solvent used is usually 150-230 g and preferred 180-220 g, based on 100 g of the L-Biphenylalaninester compound (2).

The Racemization is carried out using a base.

When Bases may include organic bases such as triethylamine become; Alkali metal bicarbonates, such as potassium bicarbonate and sodium bicarbonate; Alkali metal carbonates, such as potassium carbonate and sodium carbonate; Alkali metal alcoholates such as sodium methylate, Sodium ethylate and potassium tert-butyrate; Alkali metal hydrides such as Sodium hydride and potassium hydride; and mixtures of the foregoing, among which are alkali metal alkoxides, alkali metal carbonates and their mixtures are preferred, wherein alkali metal alcoholates in terms handleability are most preferred, and sodium methylate is particularly preferred in terms of economy. The Base may be added dropwise as a solution.

If the L-biphenylalanine ester compound (2) directly as dehydrated Solution obtained from the esterification step, is used, the amount of base used usually 20-120 mole% and preferably 50-100 Mol%, based on the L-Biphenylalaninester compound (2).

If the L-biphenylalanine ester compound (2) is used after it was isolated, the amount is usually 5-50 Mole% and preferably 10-50 mole% for an alkali metal carbonate. For an alkali metal alkoxide, the amount is usually 1-5 mol%, and preferably 2-4 Mol%.

As mentioned above, the use of the isolated L-Biphenylalaninester compound (2) avoid consumption of the base by the esterification residue, which allows the racemization with a lower Amount of the base is carried out. This can also be the Prevent hydrolysis of the L-biphenylalanine ester compound (2).

Under it is the point of view of promoting racism preferably an alcohol such as methanol, ethanol or isopropanol, admit.

The Amount of alcohol used is usually 10-120 Wt .-%, and preferably 10-30 wt .-%, based on the L-Biphenylalaninester compound (2).

The Reaction temperature for the racemization is usually 30-70 ° C, preferably 30-60 ° C and more preferably 30-40 ° C. The reaction time depends on the amount of reagents used and the temperature but is usually 10 minutes to 25 Hours and preferably 10 minutes to 6 hours. The termination of the Racemization reaction can be confirmed by HPLC.

To Termination of racemization is preferred of the reaction mixture Add an acid, such as acetic acid, to the breakdown to minimize the ester. The amount of acid used is usually 1.1-1.3 molar equivalents, based on the base used for the racemization.

The Post-treatment after completion of the racemization can according to conventional Procedures are performed.

The Solution of the biphenylalanine ester compound obtained in the manner described above (1) can be fed directly for enzyme hydrolysis or they can concentrate for enzyme hydrolysis and Dissolve in another organic solvent be supplied.

Examples

The The present invention will now be described in more detail using the following preparation examples and working examples explained provided that the invention does not refer to these examples is limited.

The optical purities (enantiomeric excess rates) and the racemization of the optically active compounds were determined by high performance liquid chromatography (HPLC) analysis.
HPLC analysis conditions:
Column: CHIRALPAK AD-RH (product of Daicel Chemical Industries, Ltd.) (4.5 mmφ x 15 cm, 5 μm)
Mobile phase: Solution A: 0.1% aqueous phosphoric acid solution
Solution B: acetonitrile
Elution conditions: Solution B 40% (15 min.) - 30 min. - 80% (0 min) gradient
Column temperature: 40 ° C
Flow rate: 1.0 ml / min
Detector: UV (254 nm) Retention time: LN-Boc-biphenylalanine: 10 mins DN-Boc-biphenylalanine: 13 minutes LN-Boc-Biphenylalaninmethylester: 27 minutes DN-Boc-Biphenylalaninmethylester: 30 minutes (Boc is the abbreviation for tert-butoxycarbonyl)

The esterification was analyzed under the following conditions.
HPLC analysis conditions:
Column: SUMIPAX A212 ODS (product of Sumika Chemical Analysis Service, Ltd.) (φ: 6 mm × L: 15 cm)
Mobile phase: Solution A: 25 mM aqueous dipotassium hydrogen phosphate solution (prepared with a pH of 6.8 with phosphoric acid)
Solution B: acetonitrile
Elution conditions: Solution B 40% (15 min.) - 20 min. - 80% (5 min) gradient
Column temperature: 40 ° C
Flow rate: 1.0 ml / min
Detector: UV (254 nm) Retention time: LN-Boc-biphenylalanine: 7 minutes LN-Boc-Biphenylalaninmethylester: 24 minutes

[Chemische Formel 10]

[Chemical formula 10]

Production Example 1 (Production of DL-biphenylalanine (7)

A Mixture of 4-biphenyl aldehyde (1) (100.0 g, 0.549 mol), hydantoin (82.4 g, 0.823 mol) and ammonium acetate (63.5 g, 0.824 mol) until reflux for 5 hours in acetic acid (360 ml) heated. After addition of water (360 ml) was added the mixture cooled to room temperature and the precipitated crystals were filtered out and washed with isopropanol-water (1: 1, 400 ml), to obtain the hydantoin compound (5) (143.14 g, 98.7% yield) washed.

To the mixture of the hydantoin compound (5), (60.2 g), THF (540 ml) and water (60 ml) were added 5% palladium-carbon (50% water, 2.7 g) was added and the resulting mixture was at 60 ° C for 3 hours under hydrogen atmosphere Stirred 0.5 MPa. After removal of the catalyst by Filter the filtrate was concentrated to the reduced form (6) (60.63 g, 100% yield).

To the mixture of reduced form (6) (59.7 g, 0.224 mol), ethylene glycol (300 ml) and water (10 ml) was added sodium hydroxide (36.65 g), and the mixture was at 130-140 ° C for Stirred for 5 hours. After cooling to room temperature Water (130 ml) was added, an aqueous hydrochloric acid solution, comprising concentrated hydrochloric acid (85 g) and water (99 g), was further added, and the pH of the mixture was adjusted to 6.9. The precipitated crystals were filtered off and washed with Water (300 ml) and then washed with methanol (300 ml). The crystals were obtained to give DL-biphenylalanine (7) (53.25 g, 98.4% yield). dried.

Production Example 2 (Production of DL-N-Boc-biphenylalanine methyl ester (1a))

DL-biphenylalanine (7) (20.0 g, 0.0829 mol) was added to a 10% aqueous sodium hydroxide solution (116 g, 0.29 mol). After then THF (50 ml) was added, a THF solution (20 ml) containing di-tert-butyl dicarbonate (23.5 ml, 0.108 mol) was added dropwise over a period of one hour at 30 ° C. After further addition of tetrabutylammonium bromide (0.20 g, 0.62 mmol), dimethylsulfuric acid (12.5 g, 0.099 mol) was added dropwise at 30 ° C. The mixture was at room temperature stirred for 16 hours, and then dimethylsulfuric acid (5.4 g, 0.0428 mol) was added and stirring was continued at 35 ° C for 4.5 hours. Dimethylsulfuric acid (2.93 g, 0.0232 mol) was further added and stirring was continued at 35 ° C for 2.5 hours.

After this then MTBE (40 ml) and water (100 ml) were added Mixture separated and the aqueous layer was removed, containing 104.1 g of a DL-N-Boc-Biphenylalaninmethylester (1a) To get MTBE solution.

When The result of the quantification by HPLC was the DL-N-Boc-biphenylalanine methyl ester (1a) content 29.4 g, giving a yield of 99.8% of DL-biphenylalanine (7).

A 78.1 g portion of the MTBE solution of the DL-N-Boc-biphenylalanine methyl ester (1a) thus obtained was concentrated and dried. The residue was recrystallized from isopropanol (9 ml) and heptane (80 ml) to give DL-N-Boc-biphenylalanine methyl ester (1a) (19.1 g) as colorless crystals.
(Recrystallization yield: 86.6%)
¹ H-NMR (DCDl _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 = 8Hz), 7.33 (1H, t, J = 8 Hz), 7.43 (2H, t, J = 8Hz), 7.52 (2H, d, J = 8Hz), 7.57 (2H, d, J = 8Hz).

Production Example 3 (enzyme hydrolysis of DL-N-Boc-biphenylalanine methyl ester (1a))

To a solution of 25.0 g (70.3 mmol) of DL-N-biphenylalanine methyl ester (1a) in 41.14 g of MTBE was added 11.25 g of water, 1.76 g (14.1 mmol) of taurine and 4.50 g ALCALASE 2.4 L FG (from Bacillus licheniformis) (Novozymes) and the mixture was stirred at 40 ° C. The mixture was stirred at 40 ° C for 17 hours, while dropwise 47.28 g (40.67 mmol) of a 5% aqueous potassium hydroxide solution were added. The pH of the reaction mixture was in the range of 6.30-8.16 set.

The Analysis of the reaction mixture gave an optical purity of 99.4%. ee (enantiomeric excess) for the D-N-Boc-biphenylalanine methyl ester (3a) and an optical purity of 99.9% ee for the L-N-Boc-biphenylalanine potassium salt (2'a).

The Reaction mixture was for 5 minutes for liquid separation was left to give 35.72 g of the organic Layer A and 83.46 g of the aqueous layer A to obtain. After adding 37.5 g of toluene to the aqueous layer The mixture was stirred at 40 ° C for 30 minutes. She was then liquid for 5 minutes Separation allowed to stand 42.80 g of the organic layer B and 76.80 g of the aqueous layer B to obtain. The 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 became stirred at 40 ° C for 30 minutes. she got then for 5 minutes for the liquid separation allowed to stand to obtain 76.07 g of the organic layer C. The 76.06 g of organic layer C was concentrated while was heated to 50 ° C, and 63.2 g were distilled off.

To the 12.87 g of the concentration residue became 75 ml of methanol and 18.75 g of water added while at 40 ° C was heated. Next were 2 mg of D-N-Boc-biphenylalanine methyl ester (3a) seed crystals added, and the mixture was at 40 ° C for Stirred for 30 minutes. Then 12.5 g of water were added dropwise a period of 30 minutes was added and the mixture was heated to 40 ° C warmed for an hour. She was then at 20 ° C cooled and filtered. The crystals were mixed with a mixed solution containing 8.75 g of methanol and 3.75 g of water contains, washed.

The Crystals were dried under reduced pressure to give 10.94 g D-N-Boc-biphenylalanine methyl ester (3a) as white crystals to obtain. The D-N-Boc-biphenylalanine methyl ester (3a) yield was 43.8%, based on the DL-N-Boc-Biphenylalaninmethylester (1a).

example 1

(i) Recovery of L-N-Boc-biphenylalanine potassium salt (2'a)

To 328.54 g of an aqueous solution containing 55.4 g (0.146 mol) of LN-Boc-biphenylalanine potassium salt (2'a) obtained in the same manner as in Preparation Example 3 (aqueous layer B) became 50 toluene and 37.5 g of sodium chloride, and the mixture was stirred at 40 ° C for 20 minutes. they WUR then allowed to stand for 5 minutes for the liquid separation to obtain 156.23 g of an organic layer.

(ii) Preparation of L-N-Boc-biphenylalanine methyl ester (2a)

To of the aforementioned organic layer was 12.27 g (0.146 mol) Sodium bicarbonate was added, and the mixture was at 40 ° C touched. After dropwise addition of 33.15 g (0.263 mol) Dimethylsulphuric over a period of two hours, the mixture was at 40 ° C for a Hour stirred. After analyzing the reaction mixture by HPLC it was found that the starting materials were under the detection limit lie.

Next, 2.95 g (0.029 mol) of triethylamine was added to the reaction mixture, and the mixture was stirred at 40 ° C for three hours. It was then allowed to stand for 5 minutes, after which the aqueous layer was separated and removed to obtain 114.04 g of an organic layer. To this was added 86.72 g of toluene for concentration while being heated to 50 ° C and 57.92 g of toluene were distilled off. Further, 7.39 g of toluene was added to obtain 150.23 g of a toluene solution. Quantitation of the resulting toluene solution gave an LN-Boc-biphenylalanine methyl ester (2a) yield of 96.2% based on the LN-Boc-biphenylalanine potassium salt (2'a). After measuring with a Karl Fischer moisture meter, the moisture content of the obtained toluene solution was determined to be 204 ppm.
LN-Boc-biphenylalanine methyl ester (2a)
¹ H-NMR (CDCl _3); 1.42 (9H, s), 3.09 (1H, dd, J = 6, 12 Hz), 3.17 (1H, dd, J = 6, 12 Hz), 3.73 (3H, s), 4.60-4.65 (1H, m), 5.01 (1H, d, J = 8Hz), 7.20 (2H, d, J = 8Hz), 7.33 (1H, t, J = 8 Hz), 7.43 (2H, t, J = 8Hz), 7.52 (2H, d, J = 8Hz), 7.57 (2H, d, J = 8Hz).

(iii) racemization of L-N-Boc-biphenylalanine methyl ester (2a)

To 150.23 g of a toluene solution containing 50 g (0.141 mol) of L-N-Boc-biphenylalanine methyl ester (2a), 50 g of methanol were added, and the mixture was stirred at 40 ° C. After further dropwise Add 27.20 g (0.141 mol) of a 28% sodium methylate-methanol solution for a period of one hour, the mixture became 40 ° C stirred for an hour.

The Analysis of the reaction mixture gave an optical purity of 0.02%. ee for the L-N-Boc-biphenylalanine methyl ester (2a) and a HPLC area percentage of 89.3% for the DL-N-Boc-biphenylalanine methyl ester (1a).

To dropwise addition of 10.16 g (0.169 mol) of acetic acid to the reaction mixture over a period of 15 minutes The mixture was stirred at 40 ° C for 30 minutes.

When Next, 50 g of water was added and stirring was continued at 40 ° C for 5 minutes. The Mixture was for 5 minutes for the liquid Separation allowed to stand 151.90 g of an organic layer to obtain. After 45 g of water and 2.37 g (0.028 mol) of sodium bicarbonate were added to the organic layer, the mixture was added 40 ° C stirred for 50 minutes. She was then for 5 minutes for the liquid separation allowed to stand 147.67 g of an organic layer. Quantification of the resulting solution by HPLC revealed a DL-N-Boc-biphenylalanine methyl ester (1a) yield of 89.7%, based on the L-N-Boc-Biphenylalaninmethylester (2a).

The Solution was concentrated while at 54-56 ° C was heated, and 62.67 g were distilled off. Next 75 g of MTBE were added, the mixture was concentrated while It was heated to 52 ° C, and 74.23 g were distilled off. After further addition of 75 g of MTBE, the mixture was concentrated, while it was heated to 52 ° C, and 84.71 g were distilled off. The residue was 51.46 g of MTBE was added to obtain 127.52 g of a solution. Quantification of the resulting solution by HPLC revealed a DL-N-Boc-biphenylalanine methyl ester (1a) yield of 89.5%, based on the L-N-Boc-Biphenylalaninmethylester (2a). The toluene content the MBTE solution was 18.5%.

Example 2

(i) Recovery of L-N-Boc-biphenylalanine potassium salt (2'a)

To 227.19 g of an aqueous solution containing 50.0 g (0.132 mol) of LN-Boc-biphenylalanine potassium salt (2'a) was added 70.0 g of MTBE and 25.0 g of potassium chloride, and the mixture became at 40 ° C for 30 min stirred. The mixture was allowed to stand for 5 minutes for the liquid separation to obtain 204.97 g of an organic layer. After further adding 73.9 g (0.132 mol) of a 10% aqueous potassium hydroxide solution and 10 g of potassium chloride to the organic layer, the mixture was allowed to stand at 40 ° C for 15 minutes. It was then allowed to stand for 5 minutes for the liquid separation to obtain 190.18 g of an organic layer.

(ii) Preparation of L-N-Boc-biphenylalanine ester (2a)

To 95.1 g of the resulting organic layer (equivalent to 22.5 g of L-N-Boc-biphenylalanine potassium salt) then 5.55 g (0.066 mol) of sodium bicarbonate, and the Mixture was stirred at 35 ° C. Next 15.0 g (0.119 mol) of dimethylsulfuric acid were added dropwise a period of 30 minutes was added and the mixture was at 35 ° C stirred for 5 hours. After analysis of the reaction mixture by HPLC, the unreacted L-N-Boc-biphenylalanine potassium salt (2'a) to 0.19%.

When Next, 1.33 g (0.013 mol) of triethylamine and 35 g of water was added to the reaction mixture, and the mixture became stirred at 35 ° C for three hours. she was then for 5 minutes for the liquid separation allowed to stand an aqueous layer (78.16 g) receive. To the organic layer was added 41.9 g (0.010 mol) of a 2.5% by weight aqueous sodium carbonate solution given, and the mixture was for 1 hour and 20 minutes touched. She was then for 5 minutes for the liquid separation was allowed to stand 60.74 g of a to obtain organic layer. The quantification of the organic Layer gave an L-N-Boc-biphenylalanine methyl ester (2a) yield of 92.3% based on the L-N-Boc-biphenylalanine potassium salt (2'a).

(iii) Crystallization of L-N-Boc-biphenylalanine methyl ester (2a)

One 408.12 g portion of the MTBE solution containing the L-N-Boc-diphenylalanine methyl ester (2a) obtained by the method described above was obtained under reduced pressure to obtain 130.3 g of an oily concentrate. To this were 617.4 added methanol and the mixture was stirred at 40 ° C, 747.65 g of a methanol solution (equivalent to 130.3 g of L-N-Boc-biphenylalanine methyl ester (2a)) to obtain.

One 373.82 g proportion of the resulting methanol solution (corresponds 65.1 g of L-N-Boc-biphenylalanine methyl ester (2a)) was heated to 50 ° C heated while 130.3 g of water were added. Next was 1 mg of L-N-Boc-biphenylalanine methyl ester seed crystals was added, and the mixture was stirred for one hour. Then 32.6 g of water were added dropwise over a period of 30 Minutes were added, and the mixture was heated to 50 ° C for warmed for an hour. It was then cooled to 5 ° C and the precipitated crystals were filtered. The Crystals were washed with a mixed solution containing 45.6 g of methanol and 19.5 g of water.

The Crystals were used under reduced pressure to obtain 60.77 g white L-N-Boc-biphenylalanine methyl ester (2a) crystals dried. The L-N-Boc-biphenylalanine methyl ester (2a) yield was 93.3% based on the L-N-Boc-biphenylalanine potassium salt (2'a).

Example 3 (racemization of L-N-Boc-biphenylalanine methyl ester (2a))

20 g (56.3 mmol) of L-N-Boc-biphenylalanine methyl ester (2a) was added to 40 g MTBE dissolved, and the mixture was at 30 ° C heated. This was a mixed solution, the 4.0 g of methanol and 0.22 g (1.1 mmol) of a 28% sodium methylate-methanol solution contains, and the resulting mixture was added 30 ° C for two hours.

The Analysis of the reaction mixture gave an optical purity of 0.44%. ee for the L-N-Boc-biphenylalanine methyl ester (2a) and a HPLC area percentage of 98.9% for the DL-N-Boc-biphenylalanine methyl ester (1a).

After adding 0.07 g (1.2 mmol) of acetic acid to the reaction mixture, the mixture was stirred at 30 ° C for 5 minutes. Next, 20 g of water was added and stirring was continued at 30 ° C for 5 minutes. The mixture was then allowed to stand for 5 minutes for the liquid separation to obtain 61.56 g of an organic layer (upper layer). To this was added 18 g of water and 0.94 g (11.3 mmol) of sodium bicarbonate, and the mixture was stirred at 30 ° C for 30 minutes. The Mi The mixture was then allowed to stand for 5 minutes for the liquid separation to obtain 58.80 g of an organic layer. The quantification of the obtained organic layer by HPLC gave a DL-N-Boc-biphenylalanine methyl ester (2a) yield of 100%, based on the LN-Boc-biphenylalanine methyl ester (1a). The value by HPLC quantification was 101.6%.

Example 4 (racemization of L-N-Boc-biphenylalanine methyl ester (2a))

2.0 g (5.63 mmol, 81.25% ee) L-N-Boc-biphenylalanine methyl ester (2a) were dissolved in 2.0 g of MTBE and 12.0 g of methanol and the Mixture was heated to 60 ° C. For this purpose were Add 0.15 g (1.13 mmol) of potassium carbonate and stir was continued at 60 ° C for 8 hours.

The Analysis of the reaction mixture gave an optical purity of 2.25%. ee for the L-N-Boc-biphenylalanine methyl ester (2a) and a HPLC area percentage of 81.3% for the DL-N-Boc-biphenylalanine methyl ester (1a).

Example 5 (racemization of L-N-Boc-biphenylalanine methyl ester (2a))

2.0 g (5.63 mmol, 81.25% ee) L-N-Boc-biphenylalanine methyl ester (2a) were dissolved in 2.0 g of MTBE and 12.0 g of methanol and the Mixture was heated to 60 ° C. For this purpose were Add 0.12 g (1.13 mmol) of sodium carbonate and stir was continued at 60 ° C for 25 hours.

The Analysis of the reaction mixture gave an optical purity of 6.02%. ee for the L-N-Boc-biphenylalanine methyl ester (2a) and a HPLC area percentage of 90.0% for the DL-N-Boc-biphenylalanine methyl ester (1a).

Industrial applicability

According to the Process of the invention it is possible to L-biphenylalanine compound salts to recover in the form of salts from reaction mixtures, after enzyme hydrolysis of biphenylalanine ester compounds under Using a simple procedure were obtained. additionally can, since the conversion into Biphenylalaninester connections by esterification and racemization of the recovered L-biphenylalanine compound salts possible, the starting materials of the enzyme hydrolysis be recovered. Therefore, L-biphenylalanine compounds, that are not the target of enzyme hydrolysis, are used effectively and the method of the invention is therefore economically advantageous.

Summary

The The invention relates to a recovery process for L-biphenylalanine compound salts (2 ') wherein a biphenyl ester compound (1) using a Protease from a microorganism belonging to the genus Bacillus, in a mixed solvent that is an organic Solvent and water, in a pH range of 6 to 13, the aqueous layer is hydrolyzed containing the L-biphenylalanine compound salt (2 ') and the organic layer containing the unreacted D-Biphenylalaninester compound (3), to be separated, and an inorganic salt and an organic solvent to the aqueous Layer for extracting the L-biphenylalanine compound salt (2 ') in the aqueous layer.

QUOTES INCLUDE IN THE DESCRIPTION

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Cited patent literature

• JP 6-228187 [0002, 0069]

Claims (17)

A recovery method for an L-biphenylalanine compound salt represented by formula (2 '), which comprises using a protease from a microorganism belonging to the genus Bacillus for the hydrolysis of a biphenylalanine ester compound represented by the following formula (1): [Chemical Formula 1]

(wherein R _{1 represents} an alkyl, haloalkyl, alkenyl, cycloalkyl, an optionally substituted aryl or optionally substituted aralkyl group, and R _{2 represents} a protecting group for the amino group) in a mixed solvent comprising an organic solvent and water in a pH Range of 6 to 13, and separating the aqueous layer containing the produced by the following formula (2 ') represented L-biphenylalanine compound salt: [Chemical formula 2]

(wherein M represents an alkali metal atom or ½ alkaline earth metal atom and R _{2 has the} same definition as above) from the organic layer containing the unreacted D-biphenylalanine ester compound represented by the following formula (3): [Chemical Formula 3]

(where the symbols have the same definitions as above); and adding an inorganic salt and an organic solvent to the aqueous layer to extract the L-biphenylalanine compound salt represented by formula (2 ') in the aqueous layer. Recovery method according to claim 1, the organic solvent for the hydrolysis is at least one selected from methyl tert-butyl ether and toluene. Recovery method according to claim 1, the organic solvent for extraction is at least one selected from methyl tert-butyl ether and toluene. A recovery method of a biphenylalanine ester compound represented by formula (1), which comprises using a protease from a microorganism belonging to the genus Bacillus for the hydrolysis of a biphenylalanine ester compound represented by the following formula (1): [Chemical formula 4]

(wherein R _{1 represents} an alkyl, haloalkyl, alkenyl, cycloalkyl, an optionally substituted aryl or optionally substituted aralkyl group, and R _{2 represents} a protective group for the amino group) in a mixed solvent comprising an organic solvent and water in one pH range of 6 to 13, and separating the aqueous layer containing the produced by the following formula (2 ') L-biphenylalanine compound salt: [Chemical formula 5]

(wherein M represents an alkali metal atom or ½ alkaline earth metal atom and R _{2 has the} same definition as above) from the organic layer containing the unreacted D-biphenylalanine ester compound represented by the following formula (3): [Chemical Formula 6]

(where the symbols have the same definitions as above); Adding an inorganic salt and an organic solvent to the aqueous layer, extracting the L-biphenylalanine compound salt represented by formula (2 ') in the aqueous layer; Esterification of the extracted L-biphenylalanine compound salt represented by formula (2 ') to obtain an L-biphenylalanine ester compound represented by the following formula (2): [Chemical Formula 7]

(where the symbols have the same definitions as above); and racemizing the L-biphenylalanine ester compound represented by formula (2) with a base to obtain the biphenylalanine ester compound represented by formula (1). Recovery method according to claim 4, the organic solvent for the hydrolysis is at least one selected from methyl tert-butyl ether and toluene. Recovery method according to claim 4, the organic solvent for extraction is at least one selected from methyl tert-butyl ether and toluene. Recovery method according to claim 4, wherein the esterification by reacting with the corresponding sulfuric acid ester or halide in the presence of a base. The recovery process of claim 4 wherein R _{1 is} methyl. Recovery method according to claim 8, wherein the esterification by reacting with dimethyl sulfuric acid in the presence of a base. Recovery method according to claim 4, wherein the base used for the racemization is selected is from alkali metal carbonates and alkali metal alkoxides. Recovery method according to claim 4, wherein the base used for the racemization is an alkali metal alkoxide is. Recovery method according to claim 4, wherein the base used for the racemization sodium methylate is. A recovery process according to claim 4, wherein R _{1 is} an alkyl group and R _{2 is} a group represented by the formula -CO ₂ R ₃ (wherein R _{3 is} an alkyl, an optionally substituted aryl, optionally substituted aralkyl or a 9-fluorenemethyl group) , Recovery method according to claim 13, the racemization following isolation of those represented by formula (2) L-Biphenylalaninester compound performed as a solid becomes. A recovery method for a DL-N-Boc-biphenylalanine methyl ester represented by formula (1a), comprising: using a protease from a microorganism belonging to the genus Bacillus for the hydrolysis of a DL-N- represented by the following formula (1a) Boc-biphenylalanine methyl ester: [Chemical Formula 8]

in a mixed solvent comprising methyl tert-butyl ether and water in a pH range of 6 to 13 with an aqueous solution of taurine and potassium hydroxide, and separating the aqueous layer which produced by the following formula (2'a ) represented LN-Boc-Biphenylalaninkaliumsalz contains: [Chemical formula 9]

and the organic layer containing the unreacted DN-Boc-biphenylalanine methyl ester represented by the following formula (3a): [Chemical formula 10]

Adding an inorganic salt and methyl tert-butyl ether to the aqueous layer to extract the LN-Boc-biphenylalanine potassium salt represented by the formula (2'a) in the aqueous layer; Esterification of the extracted LN-Boc-biphenylalanine potassium salt represented by the formula (2'a) with dimethylsulfuric acid in the presence of a base to obtain the LN-Boc-biphenylalanine methyl ester represented by the following formula (2a): [Chemical Formula 11]

Racemization of the LN-Boc-biphenylalanine methyl ester represented by the formula (2'a) with sodium methylate after isolation thereof as a solid, to obtain the DL-N-Boc-biphenylalanine methyl ester represented by the formula (1a). A process for producing a D-biphenylalanine ester compound represented by the formula (3), which comprises using a protease from a microorganism belonging to the genus Bacillus for the hydrolysis of a biphenylalanine ester compound represented by the following formula (1) : [Chemical formula 12]

(where the symbols have the same definitions as above); characterized by a recovery process according to claim 4 in a mixed solvent comprising an organic solvent and water in a pH range of 6 to 13, and separating the organic layer containing the unreacted D- represented by the following formula (3) Biphenylalanine ester compound contains: [Chemical Formula 13]

(where the symbols have the same definitions as above); and purifying the D-biphenylalanine ester compound. A process for producing DN-Boc-biphenylalanine methyl ester which comprises using a protease from a microorganism belonging to the genus Bacillus for the hydrolysis of a DL-N-Boc-biphenylalanine methyl ester represented by the following formula (1a): [Chemical formula 14]

obtained by a recovery process according to claim 15 in a mixed solvent comprising an organic solvent and water in a pH range of 6 to 13, and separating the organic layer containing the unreacted DN represented by the following formula (3a) Boc-biphenylalanine methyl ester contains: [Chemical Formula 15]

Purify the DN-Boc-biphenylalanine methyl ester.