WO2002079485A1

WO2002079485A1 - Process for producing (r)-2-hydroxy-1-phenoxypropane derivative while preventing the formation of transfer by- product

Info

Publication number: WO2002079485A1
Application number: PCT/JP2002/003138
Authority: WO
Inventors: Shigeru Kawano; Miho Horikawa; Naoaki Taoka; Yoshihiko Yasohara
Original assignee: Kaneka Corporation; Ueda, Makoto
Priority date: 2001-03-29
Filing date: 2002-03-29
Publication date: 2002-10-10
Also published as: JP2002281991A

Abstract

It is intended to provide a process for efficiently producing an (R)-2-hydroxy-1-phenoxypropane derivative with high qualities, which is highly useful as a drug intermediate, from an inexpensive material. A (R)-2-hydroxy-1-phenoxypropane derivative with high qualities is produced by a reaction of R-selectively reducing a phenoxyacetone derivative, which is an inexpensive material, with the use of a microorganism-origin enzyme or an optionally processed culture of a microorganism capable of producing this enzyme while controlling the reaction pH value, temperature conditions, etc. so as to prevent the formation of an (R)-1-hydroxy-2-phenoxypropane derivative as a by-product.

Description

Specification

Method for producing (R) _2-hydroxy-1-phenoxypropane derivative with suppressed by-product formation

INDUSTRIAL APPLICABILITY The present invention relates to (R) —2-hydroxy-11-phenoxypropane derivative, which is useful as a pharmaceutical intermediate, and in particular, 2,3-difluoro-6-toro-1 [-(R) —, which is useful as an intermediate for a synthetic antibacterial agent. (2-hydroxypropyl)] oxy] benzene. Background art

Conventionally, the following methods are known as a method for reducing a phenoxyacetone derivative to (R) 12-hydroxy-11-phenoxypropane derivative using a microorganism or the like.

1) A method of producing 2,3-difluoro-6-nitro [[(R)-(2-hydroxy ^ ° pyr)] oxy] benzene using a filamentous fungus (Japanese Patent Application Laid-Open No. 3-183489).

2) A method for producing 2,3-difluoro-6-nitro-[[(R)-(2-hydroxypropy] oxy] benzene using a microorganism belonging to the genus Corynebacterium ( JP-A-5-68577).

3) A method for producing (R) —2-hydroxy-11-phenoxypropane derivative using a carbon reductase derived from the genus Candida (Candida) (WO

00/37666).

However, in the methods 1) and 2), the obtained (R) -2-hydroxy-1-phenoxypropane derivative has a low optical purity or a very low substrate concentration during the reaction. There was a problem. On the other hand, we have already reported an efficient production method that can react at a substrate concentration of 10% or more by the method shown in 3) above and can obtain the target product with an optical purity of 99% ee or more. I have. In general, it is well known that the 1-position acyl group of 1,2-diols transfers to the 2-position under basic conditions. (Aust. J. Chem. (1998), 51 (6), 455), but it is known that an optionally substituted phenyl group transfers from the 1-position to the 2-position. I didn't.

However, we established a precise analytical method that can separate (R) -2-hydroxy-11-phenoxypropane derivative and the transfer product (R) -1-hydroxy-2-phenoxypropane derivative. As a result of a detailed study of the above reduction reaction, the transfer reaction of a phenyl group which may have a substituent proceeds under the reduction reaction conditions, and the (R) -1-hydroxy-2-phenoxypropane derivative is obtained. It was confirmed that by-products were produced. (R) — 1-Hydroxy-2-phenoxypropane derivative has a structure similar to that of (R) — 2-hydroxy 1-phenoxypropane derivative, and is very difficult to remove and purify by physical means. It was difficult and had a new problem in producing high quality (R) -2-hydroxy-11-phenoxypropane derivatives desired as pharmaceutical intermediates. Summary of the Invention

An object of the present invention is to provide a (R) -2-hydroxy-1-phenoxypropane derivative useful as an intermediate of a drug, and in particular, a 2,3-difluoro-6-nitro-1-nitro [useful as an intermediate of a synthetic antibacterial agent. (R)-(2-Hydroxypropyl)] oxy] Benzene can be obtained by using an enzyme derived from a microorganism, a culture of a microorganism capable of producing the enzyme, or a processed product thereof, and using the resulting phenoxyacetone as an inexpensive raw material. A method for producing a derivative by R-selective reduction, in which by-products of (R) -1-hydroxy-2-phenoxypropane derivative due to transfer of a fuel group are suppressed, and high-quality (R) 12- An object of the present invention is to provide a method for efficiently producing a hydroxy-11-hydroxypropane derivative.

In view of the above, the present inventors have conducted intensive studies and, as a result, have found that the transfer reaction can be suppressed by controlling the pH, temperature conditions, and the like during the reduction reaction, and have completed the present invention.

That is, the present invention provides a compound represented by the general formula (1):

(Wherein, R represents a phenyl group which may have a substituent) or an enzyme derived from a microorganism having the ability to R-selectively reduce the carboxy group of a phenoxyaceton derivative represented by the formula: The R-selective reduction of the phenoxyacetone derivative represented by the above formula (1) using a culture of a microorganism capable of producing an enzyme or a treated product thereof yields the general formula (2);

OH

(Wherein R represents the same group as described above). A method for producing a (R) -2-hydroxy-1-phenoxypropane derivative represented by the following general formula (3):

Ηα people (3)

(Wherein, R represents the same group as described above) (wherein the by-product amount of the ί-1-hydroxy-12-phenoxypropane derivative is 2% or less as a ratio in the product. R) — 2-Hydroxy-1-phenyl; related to the production of nonoxypropane derivatives.

Further, the present invention provides the above-mentioned production method, wherein the by-product amount of the (R) -1-hydroxy-2-phenoxypropane derivative is 1% or less in the product; (R) -1-hydroxy-2- The above-mentioned production method, wherein the by-produced amount of the phenoxypropane derivative is 0.5% or less in the product; the above-mentioned production method, wherein R is a 2,3-difluoro-6-nitrophenyl group.

Further, the present invention provides the above-mentioned production method wherein the reduction is carried out under the condition of ρΗ2 to 7; the production method wherein the reduction is carried out under the condition of ρΗ2 to 6.5; and adjusting the ρΗ by using a weak base. The above method, wherein the weak base is ammonia, carbonate or phosphate; The above-mentioned production method, which is ammonium carbonate, sodium carbonate, calcium carbonate, disodium hydrogen phosphate or dihydrogen hydrogen phosphate; the above-mentioned production method in which the reduction is carried out at 10 to 36 ° C; the substrate concentration is 5% ( w / v) The above-mentioned production method. In addition, the present invention provides the above-mentioned production method, wherein the enzyme is derived from Candida maris (C andida maris); the above-mentioned production method, wherein the enzyme is derived from Candida maris (C andida maris I FO 10003); The present invention relates to the above-mentioned production method, wherein the microorganism capable of producing the enzyme is Escherichiacoli HB101 (NT FPG) accession number FERM BP-71117. Detailed Disclosure of the Invention

Hereinafter, the present invention will be described in detail.

In the above formulas (1), (2) and (3), R represents a phenyl group which may have a substituent. Examples of the substituent include a halogen atom such as a fluorine atom, a chlorine atom, a bromine atom and an iodine atom, a nitro group, a nitroso group, a cyano group, an amino group, a hydroxyamino group, an alkylamino group having 1 to 10 carbon atoms, and a carbon number of 2 ~ 10 dialkylamino groups, N-protected amino groups, azide groups, trifluoromethyl groups, carboxynore groups, honoleminolele groups, acetinole groups, benzoyl groups, hydroxynole groups, carbon number :! An alkyloxy group having 1 to 10 carbon atoms, an alkoxy group having 1 to 10 carbon atoms, an alkylthio group having 1 to 10 carbon atoms, and the like.

Examples of the alkylamino group having 1 to 10 carbon atoms as the substituent, an alkyloxy group having 1 to 10 carbon atoms, and an alkyl moiety having 1 to 10 carbon atoms in the alkylthio group having 1 to 10 carbon atoms include, for example, methyl and ethyl. , N-propyl, isopropyl, n-butyl, isobutynole, sec-butyl, tert-butylinole, pentyl, n-hexyl, heptyl, octyl, noel, decyl and the like. In the dialkylamino group having 2 to 10 carbon atoms, the alkyl group may be selected so that the total carbon number of the two alkyl groups is 2 to 10. Further, examples of the C 1-10 acryloxy group include a formyloxy group, an acetyloxy group, a propionyloxy group, a ptyryloxy group, a valeryloxy group, a bivaloyloxy group and a hexanoloxy group. Examples of protecting groups for N-protected amino groups include, for example, Protective 'Groups'in' Organic Synthesis, 2nd edition (Protective Group Organic Synthesis, 2nd Ed.) And Ao dora double. Protective groups described in Theodora W. Green, published by John Wiley & Sons, 1990, pp. 309-384, 1990. Specifically, aralkyl-type protecting groups such as a benzyl group, a phenethyl group, and a triphenylmethyl group; a methanol phenol group, a trifluoromethyl phenol group, a benzene phenol group, a benzene phenol group, and a phenol group. o-Nitrobenzenesnolephoninole group, m-2-nitrobenzens / lefonyl group, s-lefonyl-type protecting group such as benzenesulfo- / le-group; methoxycarbonyl group, ethoxycarbol Protective groups such as phthaloyl, acetyl, chloroacetyl, trifluoroacetyl, bivaloyl, benzoyl and the like; and the like. Can be Preferably, it is a protecting group such as a methoxycaponyl group, an ethoxycarbonyl group, a tert-butoxycarbonyl group, or a benzyloxycarbonyl group, and more preferably a benzyloxycarbonyl group.

The substituent is preferably a halogen such as a fluorine atom, a nitro group, a cyano group, or the like, and more preferably a fluorine atom, a nitro group, or the like. In addition, the number of the substituents is 0 to 3, preferably 3.

Accordingly, the phenyl group which may have a substituent is preferably, for example, a phenyl group, a 2-cyanophen group, a 2-ethrophen // group, a 4-trofenynole group, a 2, 3 —Difluoro-6-nitrophenyl group and the like, and particularly preferably 2,3 difluoro-6-nitrophenyl group and the like.

The phenoxyacetone derivative represented by the general formula (1), which is a raw material of the present invention, is, for example, 2-acetoeroxy-1,3,4-difluorobenzene, and 2,3-difluoro-6-nitro. It is known that phenol can be easily synthesized by reacting with chloroacetone in the presence of a strong base (JP-A-61-246151). The (R) -1-hydroxy-2-phenoxypropane derivative represented by the general formula (3) by-produced by the rearrangement reaction is, for example, 2,3-difluoro-6-nitro [[(R ) — (1—Hydroxyisopropyl)] benzene] In the case of benzene, 2,3,4-trifluoroetrobenzene is reacted under basic conditions with (R) — 1,2 monopropanediol It is known that synthesis is possible by such a method (Japanese Patent Application Laid-Open No. 2-178287).

Examples of the enzyme derived from a microorganism having the ability to R-selectively reduce the hepatic group used in the present invention include an enzyme derived from a microorganism belonging to the genus Candida, preferably Candida maris. (C andida maris), and more preferably an enzyme derived from Candida's Maris (C andida maris I FO 1 0003), in view of the strength of its reducing ability to the phenoxyacetone derivative (1). It is.

In addition, the microorganism capable of producing the enzyme may be any of a wild strain or a mutant strain. Alternatively, a microorganism induced by a genetic technique such as cell fusion or genetic manipulation can also be used. A microorganism capable of producing the above-mentioned enzyme, which has been genetically engineered, may be, for example, a step of isolating and / or purifying the enzyme and determining a part or all of the amino acid sequence of the enzyme, based on the amino acid sequence. It can be obtained by a method comprising a step of obtaining a DNA sequence encoding an enzyme, a step of introducing this DNA into another microorganism to obtain a recombinant microorganism, and a step of culturing the recombinant microorganism to obtain the present enzyme ( WOO 1/05 996).

Examples of the microorganism capable of producing the enzyme include, for example, a transformant transformed with a plasmid having DNA encoding the enzyme and a DNA encoding an enzyme capable of regenerating a coenzyme on which the enzyme depends. Cells and the like. Preferably, the above-mentioned transformed cell, wherein the enzyme having the ability to regenerate the coenzyme is glucose dehydrogenase, wherein the glucose dehydrogenase is derived from Bacillus megaterium (Bacillus sme gaterium). The above-mentioned transformed cells, the above-mentioned transformed cells wherein the plasmid is pNTFPG, the above-mentioned transformed cells wherein the transformed cells are Escherichia coli, and the like. More preferably, Escherichiaco 1 i HB101 (pNTFPG) accession number FERM BP-71 17 is mentioned. This is located at the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary at the National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1, Tsukuba-Higashi, Ibaraki, Japan, on April 11, 2012. Deposited internationally under a treaty.

In the present invention, the culture medium for the microorganism capable of producing the enzyme is not particularly limited as long as the microorganism can grow. Examples of carbon sources include sugars such as glucose and sucrose, alcohols such as ethanol and glycerol, fatty acids and esters thereof such as oleic acid and stearic acid, and oils such as rapeseed oil and soybean oil. Ammonium sulfate, sodium nitrate, peptone, strength zamino acid, corn steep liquor, bran, yeast extract, etc .; inorganic salts such as magnesium sulfate, sodium chloride, calcium carbonate, sodium hydrogen phosphate, potassium dihydrogen phosphate And the like; a normal liquid medium containing malt extract, meat extract and the like as other nutrient sources can be used. The cultivation is performed aerobically, and can usually be performed at a culturing time of about 5 to 120 hours, a pH of the medium of 3 to 9, and a culturing temperature of 10 to 50 ° C.

The reduction reaction of the present invention is carried out by adding a substrate, a phenoxyacetone derivative, a coenzyme NAD, a culture of the above microorganism or a processed product thereof, to an appropriate solvent, and stirring the mixture under pH adjustment. obtain. Here, the term “culture of microorganisms” means a culture solution or cultured cells containing cells, and the “treated product” includes, for example, crude extract, freeze-dried microorganisms, and acetone-dried microorganisms. Or a crushed product of the cells. Furthermore, the culture of the microorganism and the processed product thereof can be used by immobilizing the enzyme itself or the bacterial cells by known means. The immobilization can be performed by a method well known to those skilled in the art (for example, a crosslinking method, a physical adsorption method, an entrapment method, etc.). Examples of the suitable solvent include water or a mixed solvent of water and an organic solvent such as toluene, ethyl acetate, and hexane, and preferably water.

In the above reduction reaction, since a transfer reaction of a phenyl group which may have a substituent is very likely to occur, a (R) -1-hydroxy-2-phenoxypropane derivative tends to be by-produced. This (R) -1-hydroxy-1-phenoxypropane derivative has a very similar structure to the target product, (R) -2-hydroxy-11-phenoxypropane derivative. Is a difficult compound. Therefore, the high-quality (R) -2-hydroxy-11-phenoxypro desired as a pharmaceutical intermediate In order to obtain a pan derivative, it is necessary to minimize the by-product of the trans (R) -1-hydroxy-2-phenoxypropane derivative.

Here, the acceptable amount of (R) -1-hydroxy-2-phenoxypropane derivative as a by-product is the ratio in the product, that is, the amount of transfer product Z (the amount of target product + the amount of transfer product) (Amount) X 100% is 2% or less, preferably 1% or less, more preferably 0.5% or less (wherein, the transition form is (R) -1-hydroxy-2-phenoxypropane). Represents a derivative, and the intended substance is (R) -2-hydroxy-11-phenoxypropane derivative).

The reaction conditions for suppressing the above by-products are as follows: to avoid contact with the base as much as possible, adjust the pH from acidic to near neutral using a weak base, and react at a relatively low temperature. Is desirable. In addition, it is desirable to shorten the reaction time as much as possible, for example, by using a substrate concentration as high as possible or using an enzyme having a strong reducing ability. Further, it is desirable to gradually add a base (to avoid local strong alkaline conditions). These reaction conditions may be used alone as long as the by-product amount of the (R) -1-hydroxy-2-phenoxypropane derivative is 2% or less in the product. , Or two or more of them may be used in combination.

The pH in the above reduction reaction is usually 2 to 8, preferably 2 to 7, more preferably 2 to 6.5, and still more preferably 5.0 to 6.5.

As a base to be added for pH adjustment, a strong base such as sodium hydroxide can be used if other conditions (temperature, substrate concentration, enzyme used, etc.) are sufficiently appropriate. For example, ammonia; carbonic acid A weak base such as a carbonate such as ammonium, sodium carbonate and calcium carbonate; a weak base such as a phosphate such as dihydrogen phosphate and sodium phosphate sodium is preferably used, and more preferably ammonium carbonate and the like are used.

The reaction temperature in the above reduction reaction is usually 10 to 36 ° C, preferably 15 to 30 ° C, more preferably 15 to 27 ° C.

The substrate concentration in the above reduction reaction is usually 1% (w / v) as a percentage of the weight (g) of the phenoxyacetone derivative (1) as the substrate with respect to the solvent volume (ml) used at the start of the reaction. ), Preferably at least 5% (w / v), more preferably at least 10% (w / v). Also, the base is adjusted to satisfy the substrate concentration. The quality can be added together or continuously. Examples of the solvent used at the start of the reaction include water, a mixed solvent of water and an organic solvent, and the like. The volume of the culture solution, buffer, etc. shall be included in the solvent volume.

The reaction time in the above-mentioned reduction reaction varies depending on the type and concentration of the enzyme, microorganism or its processed substance, and the substrate used, but it is usually 1 to 96 hours, preferably 1 to 96 hours.

48 hours, more preferably 1-32 hours, and even more preferably 1-24 hours. In addition, the above-described reduction reaction can greatly reduce the amount of expensive coenzyme used by using a commonly used NADH regeneration system in combination. As a typical NADH regeneration system, for example, a method using gnorecose dehydrogenase and glucose can be mentioned.

Further, a culture of a transformed microorganism into which a gene for a carbohydrate reductase gene and a gene for an enzyme (eg, glucose dehydrogenase) having the ability to regenerate a coenzyme dependent on the enzyme are introduced into the same host microorganism; If the same reaction as above is carried out using a product or the like, it is not necessary to separately prepare an enzyme source required for coenzyme regeneration, so that (R) — 2-hydroxyl-one-fuoxy Propane derivatives can be produced.

The (R) -2-hydroxy-11-phenoxypropane derivative generated by the above reduction reaction can be purified by a conventional method. For example, if microorganisms are used, the (R) -2-hydroxy-1-phenoxypropane derivative may be subjected to treatment such as centrifugation or filtration to remove suspended cells such as bacterial cells, if necessary. Then, after extraction with an organic solvent such as ethyl acetate or toluene, the organic solvent is removed under reduced pressure, and further purified by distillation under reduced pressure or a process such as mouth chromatography. BEST MODE FOR CARRYING OUT THE INVENTION

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

(Example 1) Escherichiaco 1 i HB101 (p NTFPG) Accession No. F ERM BP—7117 was sterilized in a 500 ml volume flask with 100 ml medium (tryptone 16 g, yeast extract 10 g, sodium chloride 5 g, water 1 Littoner was inoculated at pH 7.0 before sterilization, and cultured with shaking at 37 ° C for 13 hours. After centrifuging 100 ml of the obtained culture solution, the cells without the supernatant were suspended in 5 ml of 50 mM phosphate buffer (pH 6.5), and then disrupted by sonication. Thus, a cell-free extract was prepared.

0.5 M of this cell-free extract was added to 0.5 M potassium phosphate buffer adjusted to various pH (pH 5.5, 6.0, 6.5, 7.0, 7.5 or 8.0) 1. 9 ml, glucose 23 mg, NAD 0.11 mg and 2-acetonyloxy 3,4-difluoronitrobenzene 2 Omg (substrate concentration 1% (w / v)), 1M K ₂ HPO ₄ aqueous solution The reaction was carried out for 67 hours with stirring at 30 ° C. while adjusting the pH of the solution. At 5 hours, 30 hours and at the end of the reaction, a part of the reaction solution was separated, extracted with ethyl acetate, and concentrated under reduced pressure. The obtained concentrate was analyzed by HP LC (column: CO SMO SIL 5 C8-MS (manufactured by Nacalai Tesque, 0.46 x 25 cm), connected in series, column temperature: 40 ° C, eluent: Acetonitrile, 0.1% aqueous solution of phosphoric acid = 25/75, Flow rate: 1 ml / min, Detection wavelength: 210 nm, Elution time: 2-acetonyloxy-1,3,4-difluoronitrile benzene 64 minutes, 2,3-diphnoleol 6-2 Trow [[(R)-(2-hydroxypropyl)] oxy] benzene 52 minutes, 2,3-difluoro-6-etro-1 [[(R) -1 (1-hydroxypropyl)]] benzene 56 minutes ), Conversion rate, and by-product amount of 2,3-difluoro-6-trow [[(R)-(1-hydroxyisopropyl)] oxy] benzene were calculated according to the following formula. Table 1 shows the results. Conversion rate (%) = (Ap + Ab p) / (A s + Ap + Ab p) X 100

Metabolite by-product (%) = Ab p / (Ap + Ab p) X I 00

As: Peak area of 2-acetonyloxy 3,4 difluoro mouth-trobenzene Ap: Peak area of 2,3-difluoro-6-tro [[(R)-(2-hydroxypropynole)] oxy] benzene

Ab p: 2,3-Difluoro-6-nitro _ [[(R) — (1-Hydroxyiso Propyl)] Doxy] Benzene peak area

Anti-J heart pH conversion (%) Metabolite by-product (%)

5hrs 30hrs 67hrs 5hrs 30hrs 67hrs

5.5 83.7 83.7 84.1 0.09 0.25 0.37

6.0 99.6 99.7 99.8 0.14 0.38 0.85

6.5 99.9> 99.9> 99.9 0.30 1.03 2.18

7.0 99.7 99.6 99.8 0.70 1.98 4.86

7.5 99.6 99.9 99.8 1.67 5.42 12.15

8.0 99.8 99.9 99.9 4.20 29.29 34.54

(Example 2)

0.1 ml of the cell-free extract used in Example 1 was added to 1.9 ml of 0.5 M phosphate buffer (pH 6.5), 23 mg of gnorecose, 0.1 mg of NAD and 0.1 mg of NAD. Ninoreokishi 3, was added 4-difluoromethyl O b nitrobenzene 2 Omg (substrate concentration 1% (w / v)) , 1M 1 ^ 2 ^ 1 0 4 1 with water) ^: 6. adjusted to 5 Meanwhile, the reaction was carried out at 27, 30, 33, 36 or 40 ° C. with stirring for 67 hours. At 5 hours, 30 hours, and at the end of the reaction, a part of the reaction solution was collected, extracted with ethyl acetate, and concentrated under reduced pressure. The obtained concentrate was analyzed in the same manner as in Example 1 to determine the conversion and the conversion of the 2,3-difluoro-6-tro [[(R) — (1-hydroxyisopropyl)] oxy] benzene The biomass was calculated. Table 2 shows the results.

Table 2

JR. 'Heart m W Thumb rate (%) Metabolite by-product amount!

(° C) 5hrs 30hrs 67hrs 5hrs 30hrs 67hrs

27 95.6 96.3 98.7 0.26 0.65 1.43

30 99.9> 99.9> 99.9 0.30 1.03 2.18

33 99.8 99.8 99.7 0.71 1.32 3.12

36 99.5 99.6 99.7 0.87 1.96 4.80

40 99.1 99.6 99.8 0.90 3.31 8.09

(Example 3)

0.5 ml of the cell-free extract used in Example 1, 49.5 ml of deionized water, 5.9 g of dalcose, 5.6 mg of NAD, 2-acetonyloxy 3,4-diphnoleole -Add _{5 g} of trobenzene (substrate concentration 10% (w / v)), 30% (w / v ) The reaction was carried out for 32 hours with stirring at 30 ° C while adjusting the pH to 6.5 with an aqueous sodium hydroxide solution. At 6 hours and 20 hours after the start of the reaction, 5.6 mg of NAD was added to the mixture. After completion of the reaction, the reaction solution was extracted with toluene and concentrated under reduced pressure to obtain a yellow oily 2,3-difluoro-6 tro [[(R) _ (2-hydroxypropyl)] oxy] benzene concentrate. When this concentrate was analyzed by the same method as in Example 1, the conversion was 99.9% or more, and the transition product 2,3-difluoro-1-6-2 tro [[(R) — (1-hydroxyisopropyl) [Oxy] Benzene by-product amount was 1.29%. In addition, a part of the above concentrate was purified by column chromatography and dinitrated benzoylated, and then subjected to HP LC analysis (column: Chira 1 ce 1 OD-H (0.46 x 25 cm, manufactured by Daicel Chemical). , Eluent: Hexane Z-isopropanol = 50/50, Flow rate: lm 1Z min, Temperature: 40 ° C, Detection wavelength: 254 nm, Elution time: R-enantiomer 34 min, S-enantiomer 27 min) As a result, the optical purity was 99.9% ee or more. (Example 4)

0.5 ml of the cell-free extract used in Example 1, 49.5 ml of deionized water, 5.9 g of dalcose, 5.6 mg of NAD, 2-acetonyloxy 3,4-difluoro Add 5 g of nitrobenzene (substrate concentration 10% (w / v)) and 20% (w / v) in aqueous ammonium carbonate solution; adjust the H to 6.5, and react for 32 hours with stirring at 30 ° C Went. At 6 hours and 20 hours after the start of the reaction, 5.6 mg of NAD was added, respectively. After completion of the reaction, the reaction solution was extracted with toluene and concentrated under reduced pressure to obtain a yellow oily 2,3-difluoro-612-tallow [[(R)-(2-hydroxypropyl)] oxy] benzene concentrate. . When this concentrate was analyzed by the same method as in Example 1, the conversion was 99.9% or more, and the transition product 2,3-difluoro-1-6-nitro [[(R) — (1-H The amount of by-products of droxyisopropyl)] oxy] benzene was 0.38%. Further, part of the concentrate was purified by column chromatography one after dinitrobenzoyl I le of was subjected to HPLC analysis (as in Example 3), optical purity 99. 9% _e. E. More Met. (Example 5)

The cell-free extract used in Example 1 and 1.25 mg of deionized water 23.75 m 1 _s glucose 14.6 NAD 2.8 mg were added to 25 ml of 2-acetonyloxyl 3 , 4 difluoronitrobenzene 0.25 g (substrate concentration 1% (w / v)), 1.25 g (substrate concentration 5% (w / v)), 2.5 g (substrate concentration 10% (w / v)) / v)), 5.Og (substrate concentration 20% (w / v)) or 12.5 g (substrate concentration 50% (w / v)) and add 20% (w / v) ammonium carbonate The reaction was carried out for 30 hours with stirring at 30 ° C. while adjusting the pH to 6.5 with an aqueous solution. At 6 hours and 20 hours after the start of the reaction, 2.8 mg of NAD was added, respectively. After the completion of the reaction, the reaction solution was extracted with toluene and concentrated under reduced pressure to obtain a yellow oily concentrate of 2,3-difluoro-6-1-tro [[(R) _ (2-hydroxypropyl)] oxy] benzene. This concentrate was analyzed in the same manner as in Example 1 to obtain the conversion and the by-product of the transition product 2,3-difluoro-6-twotro [[(R) — (1-hydroxyisopropyl)] oxy] benzene. The amount was calculated. A part of the above concentrate was purified by column chromatography, subjected to dinitrobenzoylation, and analyzed by HPLC (same as in Example 3). The optical purity was 99.9% eeK in all cases. Was on. Table 3 shows the results.

Table 3 Substrate filtration conversion rate Transfer by-product amount

(% (w / v)) (%) (%)

1> 99.9 1.27

5> 99.9 0.42

10> 99.9 0.36

20> 99.9 0.28

50 99.9 0.20

(Example 6)

EXAMPLE cell-free extract 2. 5 m 1 used in 1, de Ion water 20. 2 m 1, Dar course 25. 3 g _s NAD 7. 3 mg and 2 § Seto sulfonyl O Kishi 3, 4-diphenyl Add 25 g of roto-torobenzene (substrate concentration 110% (w / v)) and stir at 27 ° C while adjusting the pH to 6.5 with 20% (wZv) aqueous ammonium carbonate solution The reaction was carried out for 21 hours. At 6 hours and 19 hours after the start of the reaction, 7.2 mg of NAD was added, respectively. After completion of the reaction, the reaction solution was extracted with toluene and concentrated under reduced pressure to obtain a yellow oily concentrate of 2,3-difluoro-6-nitro [[(R)-(2-hydroxypropyl)] oxy] benzene. . When this concentrate was analyzed by the same method as in Example 1, the conversion was 99.9% or more, and the transition product 2,3-difluoro-6-twotro [[(R)-(1-hydroxyisopropyl)] oxy ] The amount of benzene by-product was 0.04%. A part of the above concentrate was purified by column chromatography, subjected to dinitrobenzoylation, and subjected to HP LC analysis (same as in Example 3). The optical purity was 99.9% ee. That was all.

(Comparative Example 1)

Escherichiacoli HB101 (pNTS1G) accession number F ERM BP—5835 (producing reductase from Candida magnoliae) was sterilized in a 500-ml flask with a 100-ml medium (tryptone 16). g, 10 g of yeast extract, 5 g of Shiridani sodium, 1 liter of water, pH 7.0 before sterilization), and cultured with shaking at 37 ° C for 20 hours. To 50 ml of the obtained culture medium, 2.5 g of 2-acetoninoleoxy-1,3,4-difluoronitrobenzene, 2.97 g of gnorecose, and 2.9 mg of NADP were added (substrate concentration 5% (w / v)), while adjusting the pH to 6.5 with a 5 N aqueous sodium hydroxide solution, the reaction was carried out at 30 ° C for 23 hours with stirring. After completion of the reaction, the reaction solution was extracted with toluene and concentrated under reduced pressure to obtain a yellow oily concentrate of 2,3-difluoro-6-etro [[(R)-(2-hydroxypropyl)] oxy] benzene. When this concentrate was analyzed by the same method as in Example 1, the conversion was 80%, and the transition product 2,3-difluoro-6-two-trough [[(R)-(1-hydroxyisopropyl) /]] The amount of benzene by-product was 2.28%. A part of the above concentrate was purified by column chromatography, converted into dutrobenzoyl, and subjected to HPLC analysis (same as in Example 3). The optical purity was 99.0% ee. there were. Industrial applicability By the method of the present invention, a high-quality (R) -2-hydroxy-11-phenoxypropane derivative extremely useful as a pharmaceutical intermediate can be efficiently produced from inexpensive raw materials.

Claims

Claims (1)

(Wherein, R represents a fuel group which may have a substituent) An enzyme derived from a microorganism having the ability to R-selectively reduce the carboxy group of the phenoxyacetone derivative represented by R-selective reduction of the phenoxyacetone derivative represented by the above formula (1) using a culture of the microorganism having the formula (1) or a treated product thereof, whereby the general formula (2);

OH

(Wherein R represents the same group as described above).

'In the method for producing mouth bread derivatives, the general formula (3):

R

0

(Wherein, R represents the same group as described above), wherein the amount of by-product of the (R) -1-hydroxy-12-phenpropane derivative is 2% or less as a ratio in the product. (R) — A method for producing 2-hydroxy-1-phenoxypropane derivatives.

2. The process according to claim 1, wherein the by-product amount of the (R) -11-hydroxy-12-phenoxypropane derivative is i% or less as a ratio in the product.

3. The production method according to claim 2, wherein the by-product amount of the (R) -11-hydroxy-12-phenoxypropane derivative is 0.5% or less as a ratio in the product.

4. The method according to any one of claims 1 to 3, wherein R is a 2,3-difluoro-6-nitrophenyl group.

5. The production method according to any one of claims 1 to 4, wherein the reduction is performed under conditions of pH 2 to 7.

6. The production method according to claim 5, wherein the reduction is carried out under a condition of pH 2 to 6.5.

7. The production method according to any one of claims 1 to 6, wherein pH is adjusted using a weak base.

8. The method according to claim 7, wherein the weak base is ammoair, carbonate or phosphate.

9. The production method according to claim 8, wherein the weak base is ammonium carbonate, sodium carbonate, calcium carbonate, disodium hydrogenphosphate, or dihydrogen phosphate.

10. The production method according to any one of claims 1 to 9, wherein the reduction is performed under a condition of 10 to 36 ° C.

11. The method according to any one of claims 1 to 10, wherein the substrate concentration is 5% (w / v) or more.

12. The production method according to any one of claims 1 to 11, wherein the enzyme is derived from Candida's Maris (Candi damaris).

13. The method according to claim 12, wherein the enzyme is derived from Candida maris IFO 10003.

14. The method according to any one of claims 1 to 13, wherein the microorganism capable of producing the enzyme is EscherichiaciacoliHB101 (pNTF PG) accession number F ERMBP-7117.