JP2005204644A - Method for producing (-)-(2s,3r)-2-amino-3-hydroxy-3-phenylpropionic acid derivative - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing L-threo-3-(3,4-dihydroxyphenyl)serine in a high yield and in a high purity. <P>SOLUTION: This method for producing L-threo-3-(3,4-dihydroxyphenyl)serine is to enzymatically hydrolyzing racemic threo-3-(3,4-methylenedioxyphenyl)serine (in which, the amino acid group is protected with a phenylacetyl group substituted optionally at its benzene ring) by using a penicillinacylase. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to the preparation of L-threo-3- (3,4-dihydroxyphenyl) serine by the enzymatic route. More particularly, the present invention relates to DL-threo optionally protected at a catechol hydroxyl group and N-substituted with a 2-substituted acetyl group, preferably with an optionally substituted phenylacetyl group. It relates to the enzymatic hydrolysis of -3- (3,4-dihydroxyphenyl) serine.

で表されるL-トレオ-3-(3,4-ジヒドロキシフェニル)セリン、すなわち、(-)-(2S,3R)-2-アミノ-3-ヒドロキシ-3-(3,4-ジヒドロキシフェニル)プロピオン酸（国際一般的名称「ドロキシドパ」として、よく知られている）は、起立性低血圧症の治療において使用され、また、パーキンソン病の治療に関しても提案されている、ノルアドレナリン（「ノルエピネフリン」としても知られている）の前駆体である合成アミノ酸である。 Formula (A)

L-threo-3- (3,4-dihydroxyphenyl) serine represented by: (-)-(2S, 3R) -2-amino-3-hydroxy-3- (3,4-dihydroxyphenyl) Propionic acid (well known as the international generic name “droxidopa”) is used in the treatment of orthostatic hypotension and has also been proposed for the treatment of Parkinson's disease as noradrenaline (“norepinephrine”) Is also a synthetic amino acid that is a precursor.

  Droxidopa is prepared, for example, by the chemical synthesis disclosed in US Pat. No. 4,480,109. This chemical synthesis is made from piperonal and is treated with glycine followed by the benzyloxycarbonyl group (carbobenzoyl, CBZ or simply Z) of the amino group under conditions that allow isolation of the DL-threo form only. ) To produce racemic threo-N-benzyloxycarbonyl-3- (3,4-methylenedioxyphenyl) serine, which is converted to the salt of an optically active base such as ephedrine. Isolated by formation, the L-threo-N-benzyloxycarbonyl-3- (3,4-methylenedioxyphenyl) serine thus obtained is N, O-deprotected and droxidopa is isolated. However, this synthesis method has the disadvantage that the yield is low.

  The inventors have identified racemic threo-3- (3,4-methylenedioxyphenyl) serine, ie DL-threo-3- (3,4-methylenedioxyphenyl) serine (wherein the amino group is L-threo-3- (3,4-methylenedioxyphenyl) serine is treated with penicillin G acylase (PGA) by treatment with an optionally substituted phenylacetyl group on the benzene ring. The knowledge that it was obtained with good yield and high purity was obtained. Removal of the methylene group with aluminum trichloride, optionally in the presence of an alkyl mercaptan, produces droxidopa.

  The inventors first introduced the methylene group of DL-threo-3- (3,4-methylenedioxyphenyl) serine, where the amino group is protected by an optionally substituted phenylacetyl group. , Removed with aluminum trichloride, followed by DL-threo-3- (3,4-dihydroxyphenyl) serine (where the amino group is protected by an optionally substituted phenylacetyl group) ) Was subjected to enzymatic hydrolysis with PGA, and it was also found that droxidopa was obtained.

  Finally, we have DL-threo-3- (3,4-dihydroxyphenyl) serine, where the catechol function is free or diprotected and the amino group is 2-substituted It was found that (protected by the acetyl group) can be subjected to enzymatic hydrolysis in the presence of penicillin acylase.

（式中、２つのＸ'は、共に水素であるか、又は、共にカテコール官能基の保護基である）で表される(-)-(2S,3R)-2-アミノ-3-ヒドロキシ-3-フェニルプロピオン酸の製法であって、一般式(Ｂ')

（式中、Ｘは、ベンゼン環において任意に置換されたフェニルアセチル基、ベンゼン環において任意に置換されたフェノキシアセチル基、ベンゼン環において任意に置換されたマンデロイル基、ベンゼン環において任意に置換されたα-アミノフェニルアセチル基、チオフェン環において任意に置換された2-(2-チエニル)アセチル基、チオフェン環において任意に置換された2-(3-チエニル)アセチル基、フラン環において任意に置換された2-(2-フリル)アセチル基、フラン環において任意に置換された2-(3-フリル)アセチル基、又は2-(5-テトラゾリル)アセチル基であり；２つのＸ'は前記の定義のとおりである）で表されるラセミのトレオ形化合物又はその塩を、ペニシリンアシラーゼの存在下における酵素加水分解に供することを特徴とする製法を提供する。 Thus, according to the first aspect, the present invention provides a general formula (B)

(Wherein the two X ′ are both hydrogen or both are protecting groups for the catechol functional group) (−)-(2S, 3R) -2-amino-3-hydroxy- A process for producing 3-phenylpropionic acid, which is represented by the general formula (B ′)

Wherein X is a phenylacetyl group optionally substituted on the benzene ring, a phenoxyacetyl group optionally substituted on the benzene ring, a manderoyl group optionally substituted on the benzene ring, and optionally substituted on the benzene ring α-aminophenylacetyl group, 2- (2-thienyl) acetyl group optionally substituted on the thiophene ring, 2- (3-thienyl) acetyl group optionally substituted on the thiophene ring, optionally substituted on the furan ring A 2- (2-furyl) acetyl group, a 2- (3-furyl) acetyl group optionally substituted on the furan ring, or a 2- (5-tetrazolyl) acetyl group; two X ′ are as defined above A racemic threo-form compound represented by the formula (1) or a salt thereof is subjected to enzymatic hydrolysis in the presence of penicillin acylase. To do.

  The above-mentioned compounds represented by the general formula (B ′) and salts thereof are novel intermediates, which constitute another aspect of the present invention.

  The protecting group represented by the substituent X ′ in the above general formulas (B) and (B ′) is the stability of (−)-(2S, 3R) -2-amino-3-hydroxy-3-phenylpropionic acid. Is a group that can protect the phenol and catechol functional groups and is easily removed in the presence of an amide group. Suitable protecting groups are disclosed in the literature, T.W. Greene and P.G.M. Wutz, "Protecting groups in organic synthesis", 3rd edition, 1999, John Wiley & Sons, Inc., p246-292.

  Among the protecting groups X ′ of the phenol functionality, benzyl groups that are unsubstituted or substituted on the benzene ring (for example p-chlorobenzyl group), or alkenyl groups with 3 or 4 carbon atoms (for example allyl groups and 3 -Butenyl group) can also be used (also disclosed in EP 375574).

  The catechol functional group is also protected by a substituent that blocks both catechol hydroxy groups simultaneously. Examples of this type of protecting group include methylene, 2,2,2-trimethylethylidene, isopropylidene, cyclohexylidene, diphenylmethylene and ethoxymethylene. The introduction and removal thereof are disclosed in p287-291 of the above-mentioned document. Preferably, two X ′ together form a methylene group.

（式中、２つのＲ'は、共に水素であるか、又は、一緒になって、メチレン基を形成し；Ｘは上記の定義のとおりである）で表される］又はその塩は、本発明のさらに他の態様を構成する。 Advantageous compounds according to this aspect of the invention are those in which in the general formula (B ′) two X ′ are both hydrogen or together form a methylene group. These compounds [general formula (BB ')

Wherein two R ′ are both hydrogen or taken together to form a methylene group; X is as defined above] or a salt thereof It constitutes yet another aspect of the invention.

  Preferred salts are salts with alkali or alkaline earth metals, organic tertiary bases and ammonium salts.

The compound represented by the general formula (BB ′) is DL-threo-3- (3,4-methylenedioxyphenyl) serine or DL-threo-3- (3,4-dihydroxyphenyl) serine (which itself is Prepared as disclosed in US Pat. No. 4,480,109)
X-COOH
(Wherein X is as defined above) and is prepared by treatment with an acid chloride, for example. DL-threo-3- (3,4-dihydroxyphenyl) serine is also DL-threo-N-benzyloxycarbonyl-3- (3,4-dibenzyloxyphenyl) serine (US Pat. No. 4,319,040 or B. Hegedus Et al., Helv. Chim. Acta, 1975, 58, 147-162), and is prepared by catalytic hydrogenation with simultaneous removal of benzyl and benzyloxycarbonyl groups.

  Penicillin acylase used as a catalyst in enzymatic hydrolysis is one of the acylases that can hydrolyze the 6- (substituted) acetamide group of penicillin to 6-aminopenicillanic acid. In fact, penicillin G acylase is a phenylacetyl group and a manderoyl group optionally substituted in the benzene ring and a 2- (2-thienyl) acetyl group, 2- (3-thienyl) optionally substituted in the thiophene ring or furan ring. ) Acetyl group, 2- (2-furyl) acetyl group, 2- (3-furyl) acetyl group, α-amino-2-phenylacetyl group and (5-tetrazolyl) acetyl group optionally substituted on the benzene ring. Can be hydrolyzed. Penicillin V acylase can hydrolyze phenoxyacetyl groups optionally substituted on the benzene ring. The α-amino-2-phenylacetyl group optionally substituted on the benzene ring is hydrolyzed in the presence of α-amino acid ester hydrolase (included in the penicillin acylase in the present description).

  According to a preferred embodiment, the general formula (BB ′), wherein X is phenylacetyl optionally substituted by a substituent that is inert under the conditions of enzymatic hydrolysis and deprotection of the methylenedioxy group Or a salt thereof is used as a raw material, and enzymatic hydrolysis is performed in the presence of penicillin G acylase.

（ここで、Ｒ'は、共に水素であるか、又は、一緒になって、メチレン基を形成する）で表される(-)-(2S,3R)-2-アミノ-3-ヒドロキシ-3-フェニルプロピオン酸の製法であって、一般式(Ｉ)

（ここで、Ｒは、水素又は酵素加水分解及びメチレン基の除去の条件下において不活性である置換基であり；及び２つのＲ'は、前記の定義のとおりである）で表されるラセミのトレオ形化合物又はその塩を、ペニシリンＧアシラーゼの存在下における酵素加水分解に供することを特徴とする製法を提供する。 Thus, in particular, the present invention provides a general formula (AA ′)

(Wherein R 'are both hydrogen or together form a methylene group) (-)-(2S, 3R) -2-amino-3-hydroxy-3 -Phenylpropionic acid production process comprising the general formula (I)

Wherein R is a hydrogen or a substituent that is inert under the conditions of enzymatic hydrolysis and removal of the methylene group; and the two R ′ are as defined above. A threo-form compound or a salt thereof is subjected to enzymatic hydrolysis in the presence of penicillin G acylase.

  Penicillin G acylase (PGA) used in connection with stereoselective hydrolysis is, for example, on an epoxy group on a resin matrix containing functional groups (preferably epoxy groups) that are present in solution or capable of binding penicillin G acylase. On styrene and divinylbenzene resins containing for example Lewaqtit® R259K or R260K, or Diaion® HP-40, or on polyacrylic resins containing epoxy groups (eg FP 4000) Or a penicillin G acylase obtained from a microorganism covalently immobilized on a polymethacrylic resin containing epoxy groups (eg Sepabeads® FP-EP or Eupergit® C) Or commercially available penicillin G reeds from Escherichia coli or Arthrobacter viscosus It is over zero.

  Enzymatic hydrolysis uses an appropriate amount of a water-miscible solvent (eg, methanol, ethanol or acetonitrile), optionally in the presence of a buffer (eg, carbonate or phosphate buffer) in an aqueous solvent. Implemented. The pH of the reaction is 6.0 to 10, advantageously 6.5 to 9.5, preferably 6.5 to 9. The temperature of the enzyme reaction can vary in the range of 4-30 ° C.

  Typically, a calculated amount of enzyme (optionally immobilized as described above) is added to a buffer (eg, phosphate buffer) solution of a compound of general formula (I) The above pH and temperature are maintained and the hydrolysis process is added while being controlled by HPLC at 220-280 nm. In general, reaction control by HPLC is performed at a temperature of 25-30 ° C. using a suitably sized column and a phosphate buffer containing about 10% acetonitrile as the eluent.

  When 50% hydrolysis is observed, the reaction is stopped by filtering the catalyst (if the reaction is carried out at high pH) or by increasing the pH value and filtering off the catalyst. Hydrolysis control is preferred when the enantioselectivity is unknown, but is very high by proper selection of the source of the enzyme and, optionally, a suitable support for immobilization. Enantioselectivity can be obtained, whereby the enzymatic reaction is stopped when the desired L-threo enantiomer is hydrolyzed.

  From the remaining solution, the L-threo isomer with enantiomeric purity 95-99% is filtered according to known methods, eg acidified, non-hydrolyzed enantiomer (consisting of D-threo isomer) Removal by hydrolysis, extraction of the phenylacetic acid formed during hydrolysis with a suitable solvent (eg ethyl acetate or dichloromethane) and precipitation by cold acidification of the L-threo derivative represented by the general formula (AA ') Isolate by

According to another preferred embodiment, the present invention relates to DL-threo-3- (3,4-methylenedioxyphenyl) serine in which the amino group is protected with an optionally substituted phenylacetyl group or The salts, in order or in any order,
(a) subject to enzymatic hydrolysis by penicillin G acylase; and
(b) Provided is a method for producing droxidopa, which is characterized by being subjected to removal of a methylene group by a Lewis acid.

  The expression “in order or in any order” refers to DL-threo-3- (3,4-methylenedioxyphenyl) serine in which the amino group is protected by an optionally substituted phenylacetyl group. (a)-(b) or (b)-(a) means that two steps are performed alternately.

  The process according to this preferred embodiment is represented by Scheme 1.

ここで、Ｒは、水素又は酵素加水分解及びメチレン基の除去の条件下において不活性である置換基であり、化合物(I')及び(I''）はラセミのトレオ形である。 Scheme 1

Where R is a substituent that is inert under the conditions of hydrogen or enzymatic hydrolysis and removal of the methylene group, and compounds (I ′) and (I ″) are in the racemic threo form.

  When the process is carried out in the direction (a)-(b), in step (a), the removal of the optionally substituted phenylacetyl group by enzymatic hydrolysis with PGA results in L-threo-3- (3,4-Methylenedioxyphenyl) serine is provided. The compound thus obtained is subjected to removal of the methylene group by treatment with aluminum trichloride to produce droxidopa directly.

  When the process is carried out in the direction (b)-(a), in step (b), DL-threo-3- (3,4-dihydroxyphenyl) is obtained by removal of the methylene group by treatment with aluminum trichloride. ) Serine, where the amino group is protected by an optionally substituted phenylacetyl group. The product thus obtained is subjected to enzymatic hydrolysis with PGA, which directly produces droxidopa.

  Step (a) of the process of the invention (ie enzymatic hydrolysis) is present in solution or preferably immobilized on an inert support (eg Eupergit C) PGA (> 99% Of enantiomers of N- (optionally substituted) phenylacetyl-DL-threo-3- (3,4-methylene) Dioxyphenyl) serine or N- (optionally substituted) phenylacetyl-DL-threo-3- (3,4-dihydroxyphenyl) serine.

  Typically, a buffer (eg, phosphate buffer) solution of a compound represented by the general formula (I ′) or (I ″) is added to a calculated amount of enzyme (optionally immobilized or as described above). To be treated). The product thus obtained (consisting of L-threo-3- (3,4-methylenedioxyphenyl) serine or L-threo-3- (3,4-dihydroxyphenyl) serine) was prepared by a conventional method. Isolate according to

  Step (b) (i.e. removal of the methylene group) comprises N-phenylacetyl-DL-threo-3- (3,4-methylenedioxyphenyl) serine (general formula (I ')) or L-threo-3 For-(3,4-methylenedioxyphenyl) serine (general formula (A ′)), as described in US Pat. No. 4,480,109, these materials are advantageously converted to alkyl mercaptans (preferably carbon Lewis acids (e.g., trichloride or aluminum tribromide) in the presence of thiophenol in the presence of thiophenol or in a halogenated solvent (e.g., dichloromethane) or in a hydrocarbon solvent (e.g., toluene). ). At the end of the deprotection reaction, DL-threo-N-phenylacetyl-3- (3,4-dihydroxyphenyl) serine (general formula (I ″)) is isolated as the free acid or sodium salt according to conventional techniques. Release. In practice, the reaction mixture is acidified in an aqueous or aqueous-organic medium, and DL-threo-N-phenylacetyl-3- (from the aqueous phase is suitably adjusted to a pH of 2 to 5.5. Recover 3,4-dihydroxyphenyl) serine. By performing step (b) on DL-threo-N-phenylacetyl-3- (3,4-methylenedioxyphenyl) serine (general formula (I ′)), aluminum trichloride in the presence of alkyl mercaptan and After completion of the deprotection by the reaction of N-phenylacetyl-DL in pure form by subjecting the product obtained at the end of step (b) to purification using a preparative column containing a hydrophobic resin. -Threo-3- (3,4-dihydroxyphenyl) serine (general formula (I '')) is isolated. The hydrophobic resin used is a styrene divinylbenzene, polyacrylic or polymethacrylic type polymer or copolymer. Purification by one of the above resins involves DL-threo-N-phenylacetyl-3- (3) present in solution at the end of the deprotection reaction during step (b) in sequence (b)-(a). 1,4-dihydroxyphenyl) serine is carried out without isolation and the solution containing the substantially pure product is directly subjected to enzymatic hydrolysis.

  DL-threo-3- (3,4-dihydroxyphenyl) serine used as a raw material (wherein the amino group is protected by an optionally substituted phenylacetyl group) (general formula (I ′)) Alternatively, a salt thereof is described in, for example, US Pat. No. 4,480,109 using free DL-threo-3- (3,4-methylenedioxyphenyl) serine (which is itself prepared according to a known method) as a raw material. As prepared by removal of the benzyloxycarbonyl group of DL-threo-N-benzyloxycarbonyl-3- (3,4-methylenedioxyphenyl) serine by Pd / C-catalyzed hydrogenolysis in methanol . The racemic threo- (3,4-methylenedioxyphenyl) serine thus obtained is treated with a functional derivative of phenylacetic acid optionally substituted on the benzene ring to produce the corresponding N-protected product. Get things.

  Raw material, racemic threo- (3,4-methylenedioxyphenyl) serine (general formula (I ′)) N-protected by an optionally substituted phenylacetyl group and the sequence (b)-(a) The racemic threo- (3,4-dihydroxyphenyl) serine and its salt obtained at the end of step (b) by N-protection with an optionally substituted phenylacetyl group are used in the process of the invention. It is a novel compound useful as a key intermediate.

（ここで、Ｒは水素又は酵素加水分解及びメチレンジオキシ基の脱保護の条件下において不活性である置換基であり；及び２つのＲ'は、共に水素であるか、又は、一緒になって、メチレン基を形成する）で表されるDL-トレオ-N-フェニルアセチル-3-(3,4-ジヒドロキシフェニル)セリン誘導体又はその塩が提供される。 Thus, according to another aspect, the present invention provides compounds of the general formula (I)

(Where R is hydrogen or a substituent that is inert under the conditions of enzymatic hydrolysis and deprotection of the methylenedioxy group; and the two R ′ are both hydrogen or together. DL-threo-N-phenylacetyl-3- (3,4-dihydroxyphenyl) serine derivative or a salt thereof represented by the following formula:

  Advantageously, R is hydrogen or an alkyl group containing 1 to 4 carbon atoms, preferably R is hydrogen or a methyl group.

  Suitable compounds include DL-threo-N-phenylacetyl-3- (3,4-methylenedioxyphenyl) serine derivatives or salts thereof, DL-threo-N-phenylacetyl-3- (3,4-dihydroxyphenyl). ) Serine derivatives or salts thereof, in particular sodium salts.

  Preferred salts are salts with alkali or alkaline earth metals, organic tertiary bases and ammonium salts. Sodium, potassium and triethylamine salts are preferred.

（ここで、Ｒは、上記の定義のとおりである）で表される、任意に置換されたフェニル酢酸の官能誘導体にて処理し、このようにして得られたアミドを、常法に従って単離する。 In practice, for the preparation of compounds of the general formula (I), where two R ′ together form a methylene group, DL-threo-3- (3, 4-methylenedioxyphenyl) serine has the general formula (II)

(Wherein R is as defined above) and treated with an optionally substituted functional derivative of phenylacetic acid, and the amide thus obtained is isolated according to conventional methods To do.

  Chloride is used as the functional derivative. In such a case, for example, DL-threo-3- (3,4-methylenedioxyphenyl) serine is reacted with phenylacetyl chloride in a basic medium at a temperature of 2-4 ° C. The N-phenylacetyl-DL-threo-3- (3,4-methylenedioxyphenyl) serine thus obtained in high yield and purity is isolated by acidification of the reaction medium.

According to another aspect of the present invention, the present invention provides a method for producing droxidopa,
(a °) DL-threo-3- (3,4-methylenedioxyphenyl) serine has a substituent in the benzene ring, optionally inert under conditions of enzymatic hydrolysis and removal of the methylene group Treated with a functional derivative of phenylacetic acid; and DL-threo-N-phenylacetyl-3- having substituents that are inert on the benzene ring, optionally under conditions of enzymatic hydrolysis and removal of the methylene group (3,4-methylenedioxyphenyl) serine or a salt thereof in order or in any order,
(a) subject to enzymatic hydrolysis by penicillin G acylase; and
(b) Provided is a production method characterized by subjecting to removal of a methylene group by a Lewis acid.

  According to a preferred embodiment, the process is carried out according to Scheme 2, wherein R is as defined above and compounds I °, I ′ and I ″ are in racemic threo form.

Scheme 2

  In step (a °), a functional derivative of phenylacetic acid having a substituent that is optionally inert on the benzene ring under the conditions of enzymatic hydrolysis and removal of the methylene group is, for example, dicyclohexylcarbodiimide, mercaptobenzo Its acid halides, anhydrides, mixed anhydrides, preferably activated with triazoles or with so-called “BOP reagents”, ie benzotriazol-1-yloxy-tris (dimethylamino) phosphonium hexafluorophosphate, The active ester or the free acid itself. An advantageous functional derivative of optionally substituted phenylacetic acid is obtained by reaction of the free acid with thionyl chloride and directly “in situ” DL-threo-3- (3,4-methylenedioxyphenyl) It is an acid chloride that reacts with serine.

  Phenylacetyl chloride is used as a suitable functional derivative of optionally substituted phenylacetic acid.

  DL-threo-N-phenylacetyl-3- (3,4, which has a substituent which is optionally inert in the benzene ring thus isolated under conditions of enzymatic hydrolysis and removal of the methylene group. -Methylenedioxyphenyl) serine is subjected to steps (a) and (b) in order or in any order as described above. In particular, in step (a), enzymatic hydrolysis is advantageously performed using penicillin G acylase from E. coli or Arthrobacter viscosus present or immobilized in solution. Preferably, the PGA is immobilized on a matrix selected from the group consisting of styrene and divinylbenzene resins containing epoxy groups, polyacrylic resins containing epoxy groups and polymethacrylic resins containing epoxy groups. In step (b), the Lewis acid advantageously used is aluminum trichloride, optionally in the presence of an alkyl mercaptan or thiophenol. Preferably, the alkyl mercaptan contains 6 to 18 carbon atoms.

  The following examples illustrate the invention.

Preparation
DL-threo-3- (3,4-dihydroxyphenyl) serine autoclave (1 L) at room temperature with 100 ml of methanol, DL-threo-N-benzyloxycarbonyl-3- (3,4-dibenzyloxyphenyl) serine (B. Hegedus et al., Helv. Chim. Acta, 1975, 58, 147-172) Introduce 1.5 ml of concentrated HCl diluted with 6.0 g (0.1137 mol) and 3.4 ml of demineralized water, followed by 50% moisture. Of 10% Pd / C was added. After purifying the autoclave twice with hydrogen, hydrogenation was started at 3 bar pressure with stirring (mechanical shaking). After completion of hydrogenation (2 hours), the mixture was filtered, washed with methanol (2 × 15 ml) and the solution concentrated to about 50-60 ml under reduced pressure. The pH of the solution was adjusted to 5.0-5.5 with 2N sodium hydroxide under stirring. The mixture containing the precipitate was left to stand at a temperature of about 4 ° C. for 3 hours, then filtered, washed with methanol (2 × 10 ml) followed by acetone. After drying to a constant weight at 50 ° C. under reduced pressure, 1.9 g of DL-threo-3- (3,4-dihydroxyphenyl) serine identical to the reference sample was obtained.

DL-threo-N-phenylacetyl- (3,4-methylenedioxyphenyl) serine (general formula (I '))
To a solution containing 26.2 g (0.1 mol) of DL-threo-3- (3,4-methylenedioxyphenyl) serine hydrochloride in 250 ml of water, 8.0 g (0.2 mol) of NaOH pellets in 50 ml of water. ) Was added to the resulting solution. Under stirring at a constant temperature of about 20 ° C., 17.0 g (0.11 mol) of phenylacetyl chloride was added while maintaining the pH at 8.5-9.5 by addition of 15% sodium hydroxide. After the addition, the reaction was kept under stirring for 40-50 minutes until a stable pH was achieved. The pH was adjusted to a value of 2.0 ± 0.1 with 15% HCl, thereby precipitating the product. DL-threo-N-phenylacetyl- (3,4-methylenedioxyphenyl) with HPLC purity ≧ 98% after filtration, washing with water, drying under reduced pressure at 55 ° C. until constant weight 24.8 g of serine was obtained. Melting point = 166-168 ° C.
¹ HNMR (DMSO) δ (ppm): 3.41 (AB quartet system, 2H), 4.42 (d, 1H), 5.10 (d, 1H), 5.95 (s, 2H), 6.6-7.4 (aromatic, 8H) , 8.17 (d, 1H)

DL-threo-N-phenylacetyl-3- (3,4-dihydroxyphenyl) serine (general formula (I ''))
Under a nitrogen flow, 5.3 g (0.04 mol) of powdered anhydrous aluminum trichloride and 70 ml of anhydrous dichloromethane were introduced into a completely anhydrous balloon (500 ml). To the mixture cooled to 0 / + 5 ° C., 8.1 g (0.04 mol) of 1-dodecanethiol was added with stirring, and after stirring for 15 minutes, the resulting yellowish solution was added with a temperature of 0 / + 5. At 0 ° C., 3.43 g (0.01 mol) of DL-threo-N-phenylacetyl-3- (3,4-methylenedioxyphenyl) serine obtained as described in Example 1 was added. At the beginning of the addition, the product dissolved in the solution, but a large amount of gel-like precipitate was formed towards the end of the reaction, which reduced the stirring rate. After stirring for 4 hours at 0 / + 5 ° C., 15 ml of ethyl acetate was added to bring the solution into an exothermic reaction, which was controlled by maintaining the temperature below 10 ° C. The resulting solution was poured into a separately prepared mixture consisting of 75 ml water, 3.6 ml 37% HCl, 20 ml acetone and 75 ml ethyl acetate. After stirring for 10 minutes, the phases were separated. The aqueous phase was extracted with 50 ml of ethyl acetate while the organic phase was added to the extract. The collected organic phases were extracted with a solution containing 1.5 g of sodium bicarbonate in 40 ml of water. The pH of the solution was adjusted to a value of 2.0 ± 0.1 with 15% HCl in the presence of 50 ml of ethyl acetate. After separating the phases, the aqueous phase was extracted again with 30 ml of ethyl acetate, while the organic phase was added to the organic phase from this extraction. The collected organic phase was concentrated under reduced pressure to give an oil. The obtained oil (consisting of DL-threo-N-phenylacetyl-3- (3,4-dihydroxyphenyl) serine) was collected in 30 ml of acetone, and 1.5 g of sodium 2-hexanoate was contained in 15 ml of acetone. After adding the solution to be obtained, a white precipitate of sodium salt was obtained, which was allowed to stand for 2 hours and then filtered. After washing with acetone and drying to a constant weight at 55 ° C. under reduced pressure, 1.8 g of DL-threo-N-phenylacetyl-3- (3,4-dihydroxyphenyl) serine sodium having an HPLC purity of 91.6% was obtained. Obtained.
¹ HNMR (D ₂ O) δ (ppm): 2.85 (AB quartet system, 2H), 4.55 (d, 1H), 5.24 (d, 1H), 6.4-7.7 (aromatic, 8H)

  In another preparation method, the oil obtained (consisting of DL-threo-N-phenylacetyl-3- (3,4-dihydroxyphenyl) serine) was dissolved in dichloromethane, ethyl acetate, methanol, water and acetic acid, respectively, 77 , 15, 8, 1.2, and 1 to 1.5 parts (volume) of a solvent mixture, 10 ml, and the resulting solution is silica gel 60, 240-400 using the same solvent mixture as the eluent. Subjected to flash chromatography on a mesh. Fractions showing only one spot in TLC (using the same solvent mixture as eluent) were collected and the solvent was distilled off under reduced pressure. The oil obtained was treated with ethyl ether suitable for crystallization. After standing in a cold place (about 4 ° C.) for 3 to 15 hours, DL-threo-N-phenylacetyl-3- (3,4-dihydroxyphenyl) serine free acid having a purity of 98.76% was filtered and filtered with ether. Isolated by washing and drying.

DL-threo-N-phenylacetyl-3- (3,4-dihydroxyphenyl) serine (general formula (I ''))
A suspension of 40 ml of demineralized water and 1.5 g (0.0074 mol) of DL-threo-3- (3,4-dihydroxyphenyl) serine obtained as described in the section “Preparation” above is added to 3 ml of water. Was added a solution containing 0.285 g NaOH pellets. To the solution thus obtained having a pH of 11.5 to 12.0, 1.2 g (0.0077 mol) of phenylacetyl chloride dissolved in 5 ml of ethyl acetate was added, and the pH was adjusted to 8.0 to 8.0 by addition of 5N sodium hydroxide. The mixture was allowed to react for 1 hour while controlling the reaction with TLC (eluent: 10/10/1 mixture of methanol / ethyl acetate / acetic acid) while maintaining at 8.5. . At the end of the reaction, the mixture was washed with 25 ml of ethyl acetate and the pH of the aqueous solution was adjusted to 1.9 to 2.0 with 2N HCl in the presence of 25 ml of ethyl acetate. After 5 minutes, the phases were separated. The aqueous phase was extracted with 20 ml of ethyl acetate, while the organic phase was added to the organic extract. The collected organic phase was concentrated to 1.8 g of oil, which was suspended in 30 ml of water. The pH of the suspension was adjusted to 7.5 with 10% NaOH under stirring, resulting in a light brown solution. After decolorization with activated carbon, stirring for 15 minutes, filtration and washing with a few ml of water on the filter, the solution is concentrated under reduced pressure to an oily residue which is taken up in absolute ethanol (4 × 15 ml), The water was removed and then collected with 20 ml of ethyl acetate. The solution was allowed to stand overnight at room temperature, then the crystallized product was washed with acetone and dried at 50 ° C. under reduced pressure to a constant weight. In this way, 1.2 g of DL-threo-N-phenylacetyl-3- (3,4-dihydroxyphenyl) serine sodium salt was obtained.

L-threo-3- (3,4-methylenedioxyphenyl) serine (general formula (A '))
1.716 g of DL-threo-N-phenylacetyl-3- (3,4-methylenedioxyphenyl) serine obtained as described in Example 1 in 50 ml of 10 mM phosphate buffer at pH 6.5 4 g of a commercial derivative of PGA from E. coli immobilized on Eupergit C (Recordati), previously washed with water and conditioned with 10 mM phosphate buffer at pH 6.5. (Corresponding to 296 enzyme units) was added. The reaction mixture is allowed to stand at room temperature, with column Waters Symmetry (150 × 4.6 mm; 5 μn) and (A) 90% 10 mM KH ₂ PO ₄ buffer, 10% acetonitrile and (B) 90% acetonitrile, 10% water. Eluent, gradient: 0-2 min = 100% (A); 2-3 min = 95% (A) -5% (B) (step); 3-4 min = 90% (A) -10% (B) (step); 4-9 minutes = 60% (A) -40% (B) (linear); 9-14 minutes = 100% (A) (step), and flux: 1.2 ml / min [DL-threo-N-phenylacetyl-3- (3,4-methylenedioxyphenyl) serine external injection concentration: 2 mM; DL-threo-3- (3,4-methylenedioxyphenyl) serine Retention time: 2 minutes; retention time of phenylacetic acid (220 nm): 9 minutes; retention time of DL-threo-N-phenylacetyl-3- (3,4-methylenedioxyphenyl) serine: 10 minutes] It was monitored by HPLC (λ = 220~280nm) at 30 ° C. for. After 3 hours 30 minutes, the reaction achieved 50% hydrolysis. The reaction was stopped by filtration of the catalyst and washed with basic pH water, and then the filtrate was collected. The efficiency of the catalyst (ie enantioselectivity) was determined by chiral HPLC analysis that can separate the two enantiomers of the material [column: AGP Chromtech (100 × 4 mm; 5 μn); eluent: 10 mM KH ₂ PO ₄ buffer 90% , Isopropanol 10% (pH 4.1); flux: 0.9 ml / min; λ = 280 nm; T = 25 ° C .; L-threo-N-phenylacetyl-3- (3,4-methylenedioxyphenyl) serine Retention time: 8.5 minutes; D-threo-N-phenylacetyl-3- (3,4-methylenedioxyphenyl) serine retention time: 1.5 minutes]. Enantioselectivity (E) of the enzyme was assessed by calculating the enantiomeric excess value (95%) relative to unreacted material and knowing the percent change value (50%) achieved by the reaction (E = 145 ). In order to facilitate the precipitation of unhydrolyzed DL-threo-N-phenylacetyl-3- (3,4-methylenedioxyphenyl) serine, the pH of the filtrate was adjusted to an acidic value (approximately It was adjusted to 1.5). The resulting precipitate was filtered, and the resulting liquid was extracted with dichloromethane while maintaining an acidic pH in order to remove phenylacetic acid generated during the reaction. The aqueous phase containing L-threo-3- (3,4-methylenedioxyphenyl) serine was brought to pH 3.2 in a cold place and precipitated L-threo-3- (3,4-methylenedioxyphenyl). Serine was isolated by filtration. Isolated product mass: 300 mg; purity (evaluated by HPLC): 97%.
¹ HNMR (DMSO in D ₂ O) δ (ppm): 3.4 (d, 1H), 5.0 (d, 1H), 5.9 (s, 2H), 6.8 (s, 2H), 7.0 (s, 1H)

L-threo-3- (3,4-methylenedioxyphenyl) serine (general formula (A '))
340 mg of DL-threo-N-phenylacetyl-3- (3,4-methylenedioxyphenyl) serine obtained as described in Example 1 in 50 ml of 10 mM phosphate buffer pH 6.5 3700 mg of commercial PGA from Arthrobacter viscosus immobilized on Eupergit C, previously washed with water and conditioned with 10 mM phosphate buffer at pH 6.5. -AVE1G derivative: equivalent to 220 enzyme units). Hydrolysis reached 49% after maintaining at room temperature (25 ° C.) for only 30 minutes while monitoring by HPLC as described in Example 4. After 180 minutes, the reaction reached 50% hydrolysis. The reaction was continued for 6 hours, but the hydrolysis rate did not change, indicating that the reaction was stopped because only the desired L-threo isomer was hydrolyzed. After filtration of the catalyst and washing with basic pH water, the filtrate was collected. As a result of chiral HPLC analysis as described in Example 4, the calculated enantiomeric excess (ee _s ) was 99% or higher and the calculated enantioselectivity (E) was 200 or higher. L-threo-3- (3,4-methylenedioxyphenyl) serine was isolated as described in Example 4.

L-threo-3- (3,4-dihydroxyphenyl) serine (droxidopa: general formula (A))
4.7 g of sodium DL-threo-N-phenylacetyl-3- (3,4-dihydroxyphenyl) serine obtained as described in Example 2 in 52.6 ml of 25 mM phosphate buffer at pH 6.5 A commercial derivative of PGA from E. coli immobilized on Eupergit C (Recordati), previously washed with water in a solution containing (13.3 mmol) and conditioned with 25 mM phosphate buffer at pH 6.5. 5 g (corresponding to 680 enzyme units (evaluated at room temperature)) was added. The reaction mixture was kept at room temperature, column Zorbax SB-18 (250 × 4.6 mm; 5 μn) and (A) 10 mM KH ₂ PO ₄ buffer 95%, acetonitrile 5% and (B) acetonitrile 90%, water 10% Eluent, gradient: 0-3 min = 100% (A); 3-6 min = 100% (A) -60% (A) -40% (B) (linear); 6-16 min = 60% (A) -40% (B); 16-28 minutes = 100% (A) (step), and flux: 1 ml / min [DL-threo-N-phenylacetyl-3- (3,4-dihydroxy) Phenyl) serine external reference injection concentration: 1.25 mM; droxidopa retention time: 2.48 minutes; DL-threo-N-phenylacetyl-3- (3,4-dihydroxyphenyl) serine retention time: 12 minutes; Retention time of L-threo-3- (3,4-methylenedioxyphenyl) serine: 4.95 minutes; impure DL-threo-N-phenylacetyl-3- (3,4-methylenedioxyphenyl) serine Down retention time: 16.15 min] was monitored by HPLC (λ = 280nm) at a 25 ° C. to be used. After 17 hours, the reaction achieved 50% hydrolysis. After filtration of the catalyst, the reaction mixture is brought to pH 1.5 and extracted with ethyl acetate (3 × 20 ml), the aqueous phase is freed from traces of organic solvent by evaporation under reduced pressure and then ice-cold. The mixture was cooled at, and treated with 400 mg of activated carbon for 15 minutes for decolorization. After removing the activated carbon by filtration, the pH of the solution was brought to 4.5 by the addition of sodium acetate and kept on ice until the resulting product was completely crystallized. After filtration, washing with water and then with acetone, followed by drying, 1.1 g of droxidopa having a purity of 99% was obtained. Melting point = 225 ° C. Optical rotation (at 25 ° C.): [α] _D = −39.9 ° (c = 1 in 1M HCl).

To a completely anhydrous balloon (100 ml), droxidopa 2.03 g (0.015 mol) anhydrous aluminum trichloride powder and 50 ml anhydrous dichloromethane were introduced under nitrogen flow. To the mixture cooled to 0 / + 5 ° C., 3.05 g (0.015 mol) of 1-dodecanethiol was added with stirring. After stirring for 15 minutes, the resulting yellowish solution was subjected to L-threo-3- (3,4-methylenedioxyphenyl) serine obtained as described in Example 4 at a temperature of 0 / + 5 ° C. 1.08 g (0.01 mol) was added. At the start of the addition, the product dissolved in the solution, but a large amount of gel-like precipitate formed towards the end of the addition. After 2 hours at a temperature of 0 / + 5 ° C., 25 ml of methanol were added, which led the solution to an exothermic reaction, which was controlled by maintaining the temperature below 10 ° C. The solution was concentrated under reduced pressure to obtain an oil, which was then collected in 30 ml of water. The pH was brought to 2 by addition of 3N HCl, followed by extraction with ethyl acetate (3 × 20 ml) and stirring for 10 minutes, then the phases were separated. The aqueous phase was extracted again with 50 ml of ethyl acetate. Sodium acetate trihydrate was added to the aqueous phase until droxidopa precipitated. After drying, 0.51 g of droxidopa having a purity of 97% was obtained. Melting point: 225 ° C .; Optical rotation: [α] _D = −39.9 ° (c = 1 in 1M HCL).

L-threo-3- (3,4-dihydroxyphenyl) serine (droxidopa: general formula (A))
DL-threo-N-phenylacetyl-3- (3,4-dihydroxyphenyl) having a purity of 98.76% obtained as described in Example 2 in 60.5 ml of 25 mM phosphate buffer at pH 6.5. ) PGA from E. coli immobilized on Eupergit C, previously washed with water and conditioned with 25 mM phosphate buffer, pH 6.5, in a solution containing 5 g (15.1 mmol) of serine free acid. 2 g of a commercial derivative (derivative IB-ECE1G; corresponding to 232 enzyme units (evaluated at room temperature)) was added. The reaction mixture was kept at room temperature, column Zorbax SB-18 (250 × 4.6 mm; 5 μn) and (A) 10 mM KH ₂ PO ₄ buffer 90%, acetonitrile 10% (pH 7) and (B) 10 mM KH ₂ PO ₄ Eluent consisting of 70% buffer, 30% acetonitrile (pH 7), gradient: 0-4 minutes = 100% (A); 4-7 minutes = 100% (A) -100% (B) (linear) 7-13 minutes = 100% (B); 13-20 minutes = 100% (A) (step), and flux: 1 ml / min [DL-threo-N-phenylacetyl-3- (3,4-dihydroxy) Phenyl) serine external reference injection concentration: 3.29 mM; droxidopa retention time: 2.27 minutes; DL-threo-N-phenylacetyl-3- (3,4-dihydroxyphenyl) serine retention time: 5.16 minutes; impure Retention time of L-threo-3- (3,4-methylenedioxyphenyl) serine: 3.40 min; impure DL-threo-N-phenylacetyl-3- (3,4-methylenedioxy Phenyl) serine retention time: 11.57 min] was monitored by HPLC (lambda = 254 nm) at a 25 ° C. to be used. After 5 hours, the reaction achieved 45% hydrolysis. The pH of the reaction batch containing the catalyst was adjusted to 9.5 by adding 5 M NaOH to completely dissolve droxidopa. After filtration of the enzyme, the reaction mixture is brought to pH 1.3 by addition of 6N HCl, extracted with ethyl acetate (4 × 50 ml), and the aqueous phase is free from traces of organic solvents by evaporation under reduced pressure. Then, the mixture was cooled to 10 ° C. with ice and decolorized by treatment with 300 mg of activated carbon for 30 minutes while raising the temperature to 25 ° C. After removing the activated carbon by filtration, to the solution was added NaCl and ascorbic acid 50 mg corresponding to 10% mass / volume for the volume of the solution. The solution was then cooled to 4 ° C., its pH was adjusted to 4.5 by the addition of sodium acetate and left on ice until the resulting product had completely crystallized. After filtration, washing with water and then with acetone, followed by drying, 0.93 g of droxidopa having a purity of 99.39% was obtained.

Claims (25)

General formula (B)

(Wherein the two X ′ are both hydrogen or both are protecting groups for the catechol functional group) (−)-(2S, 3R) -2-amino-3-hydroxy-3 -Phenylpropionic acid derivative production method comprising the general formula (B ′)

Wherein X is a phenylacetyl group optionally substituted on the benzene ring, a phenoxyacetyl group optionally substituted on the benzene ring, a manderoyl group optionally substituted on the benzene ring, and optionally substituted on the benzene ring α-aminophenylacetyl group, 2- (2-thienyl) acetyl group optionally substituted on the thiophene ring, 2- (3-thienyl) acetyl group optionally substituted on the thiophene ring, optionally substituted on the furan ring A 2- (2-furyl) acetyl group, a 2- (3-furyl) acetyl group optionally substituted on the furan ring, or a 2- (5-tetrazolyl) acetyl group; two X ′ are as defined above (-)-(2S, 3R) -2-, characterized in that the racemic threo-form compound represented by (2), or a salt thereof is subjected to enzymatic hydrolysis in the presence of penicillin acylase. Mino-3-hydroxy-3-preparation of phenylpropionic acid derivatives.   The process according to claim 1, wherein two X's in the general formula (B ') of the raw material are taken together to form a methylene group, and X is a phenoxyacetyl group.   The process according to claim 1, wherein the enzymatic hydrolysis is carried out in the presence of penicillin V acylase. General formula (AA ')

(Wherein two R ′ are both hydrogen or together form a methylene group) (−)-(2S, 3R) -2-amino-3-hydroxy In order to produce a -3-phenylpropionic acid derivative,

Wherein R is a substituent which is inert under the conditions of hydrogen or enzymatic hydrolysis and removal of the methylene group; two R ′ are as defined above. The process according to claim 1, wherein the form compound or a salt thereof is subjected to enzymatic hydrolysis in the presence of penicillin G acylase.   The process according to claim 4, wherein the raw material is a compound represented by the general formula (I) (wherein R and both R 'are hydrogen) or a salt thereof.   5. The compound represented by the general formula (I) (wherein R is hydrogen; two R ′ together form a methylene group) or a salt thereof is used as a raw material. The manufacturing method described.   Enzymatic hydrolysis from Escherichia coli or Arthrobacter viscosus covalently immobilized in a resin matrix containing functional groups present in solution or capable of binding penicillin G acylase The manufacturing method of any one of Claims 4-6 performed using penicillin G acylase of this.   The process according to claim 7, wherein the resin matrix is selected from the group consisting of polymethacrylic resins containing epoxy groups. A method for producing droxidopa, wherein DL-threo-3- (3,4-methylenedioxyphenyl) serine or a salt thereof in which an amino group is protected by an optionally substituted phenylacetyl group, in order or in any In order,
(a) subject to enzymatic hydrolysis by penicillin G acylase; and
(b) A method for producing droxidopa, which is used for removal of a methylene group by a Lewis acid.   The process according to claim 9, wherein the amino group is protected by a phenylacetyl group which is unsubstituted or substituted by a methyl group. A method for producing droxidopa,
(a °) Phenyl optionally having a substituent that is inactive on the benzene ring under conditions of enzymatic hydrolysis and removal of the methylene group, DL-threo-3- (3,4-methylenedioxyphenyl) serine Treatment with a functional derivative of acetic acid; and DL-threo-N-phenylacetyl-3- () in the benzene ring, optionally with a substituent that is inert under conditions of enzymatic hydrolysis and removal of the methylene group 3,4-methylenedioxyphenyl) serine or a salt thereof in order or in any order,
(a) subject to enzymatic hydrolysis by penicillin G acylase; and
(b) A method for producing droxidopa, which is used for removal of a methylene group by a Lewis acid.   Activated with dicyclohexylcarbodiimide, mercaptobenzotriazole or benzotriazol-1-yloxy-tris (dimethylamino) phosphonium hexafluorophosphate as a functional derivative of optionally substituted phenylacetic acid in step (a °) 12. The process according to claim 11, wherein the acid halide, anhydride, mixed anhydride, active ester or free acid itself is used.   The process according to claim 11, wherein phenylacetyl chloride is used as a functional derivative of optionally substituted phenylacetic acid.   Penicillin from Escherichia coli or Arthrobacter viscosus, wherein in step (a) the enzyme hydrolysis is covalently immobilized on a resin matrix present in solution or containing a functional group capable of binding penicillin G acylase The manufacturing method of any one of Claims 9-13 performed using G acylase.   The process according to any one of claims 9 to 14, wherein in step (a), penicillin G acylase is covalently immobilized on a matrix selected from the group consisting of polymethacrylic resins containing an epoxy group.   The process according to any one of claims 9 to 15, wherein the Lewis acid used in step (b) is aluminum chloride.   The process according to any one of claims 9 to 16, wherein in step (b), the Lewis acid is used in the presence of an alkyl mercaptan or thiophenol.   The process according to claim 17, wherein the alkyl mercaptan contains 6 to 18 carbon atoms. General formula (B ')

Wherein X is a phenylacetyl group optionally substituted on the benzene ring, a phenoxyacetyl group optionally substituted on the benzene ring, a manderoyl group optionally substituted on the benzene ring, and optionally substituted on the benzene ring α-aminophenylacetyl group, 2- (2-thienyl) acetyl group optionally substituted on the thiophene ring, 2- (3-thienyl) acetyl group optionally substituted on the thiophene ring, optionally substituted on the furan ring A 2- (2-furyl) acetyl group, a 2- (3-furyl) acetyl group optionally substituted in the furan ring, or a 2- (5-tetrazolyl) acetyl group; the two X ′ are both hydrogen Or a catechol functional group protecting group) or a salt thereof.   20. The compound according to claim 19, which is represented by the general formula (B ′) (wherein two X ′ together form a methylene group). Formula (I)

Wherein R is hydrogen or a substituent that is inert under the conditions of enzymatic hydrolysis and deprotection of the methylenedioxy group; the two R ′ are hydrogen or together DL-threo-N-phenylacetyl- (3,4-dihydroxyphenyl) serine derivative or a salt thereof.   The DL-threo-N-phenylacetyl- (3,4-dihydroxyphenyl) serine derivative or a salt thereof according to claim 21, which is represented by the general formula (I) (wherein R is hydrogen or a methylene group).   DL-threo-N-phenylacetyl- (3,4-methylenedioxyphenyl) serine or a salt thereof.   DL-threo-N-phenylacetyl- (3,4-dihydroxyphenyl) serine or a salt thereof.   DL-threo-N-phenylacetyl- (3,4-dihydroxyphenyl) serine sodium.