JP2003180390A - Method for producing optically active phenoxypropane derivative - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for easily and efficiently producing an optically active intermediate useful for producing antimicrobial compounds, and to provide an improved method for producing levofloxacin. <P>SOLUTION: This method for producing the corresponding optically active phenoxypropane derivative in high chemical and optical purity, high optical yield and conversion comprises treating a phenoxyacetone derivative with cells of microorganisms selected from the group consisting of those belonging to the genera Pseudomonas, Kocuria, Rhodococcus, Bevundimonas, Aeromonas, Methylobacterium, Candida, Yarrowia, Sporothrix, Mycobacterium, Leifsonia and Stenotrophomonas, a cultured product thereof or extracted fraction thereof, thus providing the improved method for producing levofloxacin. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel method for producing an optically active intermediate useful for producing an antibacterial compound.

[0002]

Prior Art S-(-)-9-Fluoro-3-methyl-10- (4-methyl
-1-Piperazinyl) -7-oxo-2,3-dihydro-7H-pyrido
[1,2,3-de] [1,4] benzoxazine-6-carboxylic acid (European Patent Application Publication No. 206283 [Patent Document 1] and JP-A-62-252790 [Patent Document 2]) ) Is known as the superior synthetic antimicrobial agent levofloxacin (LVFX). As the method for producing the synthetic antibacterial agent LVFX, JP-A-2-31695 [Patent Document 3], JP-A-2-732 [Patent Document 4], and JP-A-63-264468 [Patent Document 5 ], There is a method described in (S)-(-)-7,8-difluoro-3-methyl-3,4-dihydro-2H- [1,4] benzoxazine derivative. It is used as a production intermediate.

An important production intermediate (S)-(-)-7,8-difluoro-3-methyl-3,4-dihydro-2H- [1,4] benzoxazine derivative in the production of the above synthetic antibacterial agent LVFX For efficient production, development of a production method for producing an optically active phenoxypropane derivative, which is a useful production intermediate in the production, simply and efficiently is required. Also, it is possible to use inexpensively available starting materials,
There is a demand for a method for producing an optically active phenoxypropane derivative that is easy to process and operate, produces few by-products, and has high chemical, high optical purity and optical yield. The phenoxyacetone derivative has been attracting attention as an inexpensive starting material, and a method for producing an optically active phenoxypropane derivative from the phenoxyacetone derivative is
It has already been reported that some methods, such as those using microorganisms, do not rely on complicated and difficult optical resolution, asymmetric synthesis using expensive catalysts, etc., and are simpler than chemical synthesis methods. It is expected that various treatments and operations will be possible, and economic advantages are also expected.

However, when baker's yeast or the like is used as the microorganism, S-form is only preferentially produced, and LVFX
There is a problem in obtaining an R-form which is useful as a production intermediate of (Acta. Chem. Scand., 48 (6): 506 (1994) [Non-patent document 1]). Thus, there are very few examples of microorganisms that have the ability to act on phenoxyacetone derivatives to produce optically active phenoxypropane derivatives of R-form, and in particular, microorganisms that have the ability to strictly reduce R-selectively have not yet been found. It has been pointed out that this is not the case (JP 20
No. 00-175693 [Patent Document 6]). For example, a method for R-selective reduction of 2-acetonyloxy-3,4-difluoronitrobenzene using a filamentous fungus (JP-A-3-8343).
89 (Patent Document 7)), a method for R-selective reduction of 2-acetonyloxy-3,4-difluoronitrobenzene using a microorganism belonging to the genus Corynebacterium (JP-A-5-68577). (Patent Document 8)), a method for R-selective reduction of phenoxyacetone using a microorganism belonging to the genus Thermoan Aerobactor (JP-A-8-2662).
No. 92 [Patent Document 9]) and the like are known, but a more efficient method is demanded because the optical purity of the product is low.

In addition, a method for reducing 2-acetonyloxy-3,4-difluoronitrobenzene using a microorganism belonging to the genus Candida is also known, but the microorganism used here is Candida. Magnolie IFO 0
Recombinant Escherichia coli transformed with a plasmid having a DNA encoding a carbonyl reductase derived from 705, which is not necessarily produced by a natural (native or non-recombinant) microorganism, and which is not necessarily satisfactory in R selective production. The product cannot be obtained (Japanese Patent Laid-Open No. 2000-175693 [Patent Document 10]). When microbial cells are used, the optical purity of the product may be lowered due to the coexistence of the S-selective carbonyl reductase in the cells. Therefore, using purified R-selective alcohol dehydrogenase, coenzyme, and an enzyme necessary for regenerating the coenzyme, phenoxyacetone or the like is converted into R
A method aiming at selective reduction is also known, but optical purity is not sufficiently high, and an expensive purified enzyme is used, so it is hard to say that it is a realistic production method (US Pat.
5,833 (Patent Document 11)).

[0006]

[Patent Document 1] European Patent Application Publication No. 206283

[Patent Document 2] JP-A-62-252790

[Patent Document 3] JP-A-2-31695

[Patent Document 4] Japanese Patent Laid-Open No. 2-732

[Patent Document 5] JP-A-63-264468

[Patent Document 6] Japanese Patent Laid-Open No. 2000-175693

[Patent Document 7] Japanese Patent Laid-Open No. 3-183489

[Patent Document 8] Japanese Patent Laid-Open No. 5-68577

[Patent Document 9] Japanese Unexamined Patent Publication No. 8-266292

[Patent Document 10] Japanese Patent Laid-Open No. 2000-175693

[Patent Document 11] US Pat. No. 5,385,833

[Non-Patent Document 1] Acta. Chem. Scand., 48 (6): 506 (19
94)

[0007]

There is a demand for the development of a production method for producing an optically active phenoxypropane derivative, which is a useful intermediate in the production of the above synthetic antibacterial agent LVFX, simply and efficiently. In addition, it is possible to use starting materials that are available at low cost, easy to process and operate,
There is a need for a method for producing an optically active phenoxypropane derivative with high chemical yield, high optical purity, and optical yield with little generation of by-products. However, in the conventional methods using the above microorganisms, etc., 1) it is not easy to obtain a product with high optical purity required as a drug raw material,
2) Low productivity due to low substrate concentration, 3) use of expensive purified enzyme, 4) unsatisfactory in terms of conversion rate to desired product and optical purity of desired product. However, as an industrial manufacturing method, there are points to be improved.

[0008]

The present inventors have found a new method for producing a useful optically active intermediate for the production of this synthetic antibacterial agent, and completed the present invention.

That is, the present invention is [1] General formula (I):

[Chemical 3] (In the formula, Xa and Xb may be the same or different and each is a halogen atom), and a phenoxyacetone derivative represented by
Fungi of microorganisms selected from the group consisting of Rhodococcus, Brevendimonas, Aeromonas, Methylobacterium, Candida, Yarrovia, Sporothrix, Mycobacterium, Leifsonia and Stenotrophomonas. Body, its culture, an optically active general formula (II) characterized in that it is treated in the presence of a substance selected from the group consisting of a treated product and an extract thereof:

[Chemical 4] (In the formula, Xa and Xb have the same meanings as described above.)
A method for producing a phenoxypropane derivative represented by:

[2] Microorganisms include Pseudomonas spp, Koklia spp, Rhodococcus spp, Brevendimonas spp, Methylobacterium spp, Candida spp, Yarovia spp,
The method according to the above [1], which is selected from the group consisting of the genera Sporothrix, Mycobacterium, Leifsonia and Stenotrophomonas; [3] The microorganisms are Pseudomonas and Cochlia. , Rhodococcus spp, Aeromonas spp, Methylobacterium spp, Candida spp and Stenotrophomonas spp. [4] The method according to the above item [1]; Any of the above-mentioned [1] to [3], which is selected from the group consisting of, genus Pseudomonas, genus Kochlia, genus Rhodococcus, genus Methylobacterium, genus Candida and genus Stenotrophomonas. [5] The microorganisms are Pseudomonas hydrogen thermophila, Pseudomonas syringae, and body A
Rosea, Rhodococcus fascias, Brevendimonas diminuta, Aeromonas hydrophila, Methylobacterium rhodesianum, Methylobacterium extorquens, Candida sonorensis, Yalovia lipolytica, Sporothricus sienkii,
Mycobacterium Diernhoferi, Leifsonia
The method according to [1] above, which is selected from the group consisting of Naganoensis and Stenotrophomonas maltophilia;

[6] The microorganisms are Pseudomonas hydrogen thermophila, Pseudomonas syringae, Cochle rosea, Rhodococcus fascias,
Selected from the group consisting of Brevendimonas diminuta, Methylobacterium rodesianum, Methylobacterium extorquens, Candida sonorensis, Yalovia lipolytica, Sporothrix sienkii, Mycobacterium ziernhoferi, Leifsonia naganoensis and Stenotrophomonas maltophilia. [7] The method according to any one of [1], [2] and [5] above; [7] The microorganisms are Pseudomonas hydrogen thermophila, Pseudomonas syringae, Cochlear
Rosea, Rhodococcus fascinus, Aeromonas
Hydrophila, Brevendimonas diminuta, Methylobacterium rhodesianum, Methylobacterium extorquens, Candida sonorensis and Stenotrophomonas maltophilia [1] , [3] and [5], or [8] the microorganism is Pseudomonas hydrogen thermophila, Pseudomonas syringae, Koklia
[1] to [7], which is selected from the group consisting of Rosea, Rhodococcus fascias, Methylobacterium rodesianum, Methylobacterium extorquens, Candida sonorensis and Stenotrophomonas maltophilia. ] The manufacturing method of any one of these.

In another aspect, the invention comprises

[9] Microorganisms are Pseudomonas hydrogen thermophila IFO 14593 strain, Pseudomonas syringae subspecies syringae IFO 12655 strain, and Cochlea rosea IFO
3768 shares, Rhodococcus fascias IFO 12077 shares,
Breven Dimonas Diminuta IFO 12697 shares, IFO
13182 strain and IAM 12557 strain, Aeromonas hydrophila subspecies hydrophila IFO 3820 strain, Methylobacterium rodesianum JCM 2815 strain, JCM 2816 strain and
IFO 15844 strain, Methylobacterium extorquence IFO 3708 strain, JCM 2812 strain, JCM 2813 strain, JC 2813 strain
M 2814, JCM 2817, JCM 2827, JCM 2834
Stock, JCM 2806, JCM 2807, JCM 2808, JC
M 2809 strain, IAM 1081 strain and IAM 12632 strain, Candida sonorensis IFO 10027 strain, Yarovia repolitica IAM 4694 strain, Sporothrix sienkii IFO
5983 strain, Mycobacterium diernhoferi IFO 37
07 shares and IFO 14756 shares, Leifsonia Naganoensis JCM 10592 shares and Stenotrophomonas maltophilia NBRC 13692 shares, NBRC 12020 shares and NBRC 12 shares
692 shares, NBRC 13923 shares, IAM 12424 shares, JCM 1976 shares
Stock, JCM 1978, JCM 1982, JCM 1983, JC
M 1984, JCM 1986, JCM 1987, JCM 3800
Shares, JCM 3802 shares, JCM 3803 shares, JCM 3804 shares, JC shares
M 3805 strain, JCM 3806 strain, and JCM 3807 strain, selected from the group consisting of the above-mentioned [1]; [10] The microorganism is Pseudomonas hydrogen thermophila IFO 14593 strain , Pseudomonas syringae Subspecies syringae IFO 12655 strain, Cochlea rosea IFO
3768 shares, Rhodococcus fascias IFO 12077 shares,
Breven Dimonas Diminuta IFO 12697 shares, IFO
13182 strain, IAM 12557 strain, Methylobacterium extorquens IFO 3708 strain, IAM 1081 strain, JCM
2806 shares, JCM 2807 shares, JCM 2808 shares, JCM 2812 shares,
JCM 2813 shares, JCM 2814 shares, JCM 2817 shares, and JCM
2834 strain, Methylobacterium rhodesianum JCM 2815
Shares, JCM 2816 shares, IFO 15844 shares, Candida
Sonorensis IFO 10027 strain, Yalovia lipolytica
IAM 4694 stock, Sporothrix schienkii IFO5983
Strain, Mycobacterium diernhoferi IFO 3707 strain and IFO 14756 strain, Leifsonia Naganoensis JC
M 10592 strain and Stenotrophomonas maltophilia NBRC 13692 strain, NBRC 12020 strain, NBRC 12692 strain,
IAM 12424, JCM 1976, JCM 1978, JCM
1982, JCM 1983, JCM 1984, JCM 1986,
JCM 3800 shares, JCM 3802 shares, JCM 3803 shares, JCM 38 shares
The above-mentioned [1], which is selected from the group consisting of 04 strains, JCM 3805 strains and JCM 3806 strains.
[2], [5] and

[9] The method according to any one of [9];

[11] The microorganisms are Pseudomonas hydrogen thermophila IFO 14593 strain, Pseudomonas syringae subspecies syringae IFO 12655 strain, Cochlear rosea IFO 3768 strain, Rhodococcus fascias I
FO 12077 strain, Methylobacterium extorquens
JCM 2807 shares, JCM 2808 shares, JCM 2812 shares, JCM 28 shares
13 strains and JCM 2814 strains, Methylobacterium rhodesianum JCM 2815 strains and JCM 2816 strains, Aeromonas hydrophila subspecies hydrophila IFO 3820 strains, Candida sonorensis IFO 10027 strains, and Stenotrophomonas maltophilia NBRC 13692 strains and JCM 19
The above-mentioned [1], [3], [5] and [5], which are selected from the group consisting of 78 strains and JCM 1983 strains.

[9] The method according to any one of [9]; [12] The microorganisms are Pseudomonas hydrogen Thermophila IFO 14593 strain, Pseudomonas syringae subsp. Syringae IFO 12655 strain, Cochlearosea IFO
3768 shares, Rhodococcus fascias IFO 12077 shares,
Methylobacterium extorquens JCM 2812 and JCM 2814 strains, Methylobacterium rhodesianum
Any one of the above [1] to [11], which is selected from the group consisting of JCM 2815 strain, JCM 2816 strain, Candida sonorensis IFO 10027 strain, and Stenotrophomonas maltophilia NBRC 13692 strain. [13] The microorganism is Pseudomonas hydrogen thermophila IFO 14593 strain, Cochlear rosea IFO 3768.
A strain, Candida sonorensis IFO 10027 strain, and Stenotrophomonas maltophilia NBRC 13692 strain are selected from the group consisting of the above [1] to [12]. .

In another embodiment, the present invention provides [14] a phenoxypropane derivative represented by the general formula (II) (in the formula, Xa) which is optically active by the production method according to any one of [1] to [13] above. And Xb have the same meanings as in the above [1]), and the resulting phenoxypropane derivative (II) is treated according to a known method to give 2,3-difluoro- (2,2- Diethoxycarbonylethenyl) amino-
((R) -2- (methanesulfonyloxy) propyloxy) benzene is produced; [15] A known method is Japanese Patent No. 2612327 (or Japanese Patent Laid-Open No. 2-732). ), The method disclosed in JP-A 1-250369 and JP-A 1-287096, the method according to [14] above; [16] above. 1] to [13], the optically active phenoxypropane derivative represented by the general formula (II) (in the formula, Xa and Xb have the same meaning as in [1] above). The phenoxypropane derivative (II) produced and obtained is subjected to a combination of the following treatments (a) to (d): (a) reduction of nitro group, (b) methylene malonation reaction, (c) sulfonyl. Oxidation reaction, and (d) Removal of protecting group 2,3-difluoro- (2,2-diethoxycarbonylethenyl)
Amino-((R) -2- (methanesulfonyloxy) propyloxy) benzene is obtained above [14] or [1.
[17] The optically active phenoxypropane derivative represented by the general formula (II) is produced by the production method described in any one of the above [1] to [13], and then the following (a) ) To (d) in combination: (a) reduction of nitro group to amino group, (b) methylenemalonation reaction of amino group, (c) conversion of hydroxy group on 2-hydroxypropyloxy group Sulfonylation reaction, and
(d) Removal of protecting group 2,3-difluoro- (2,2-diethoxycarbonylethenyl)
Amino-((R) -2- (methanesulfonyloxy) propyloxy) benzene is obtained above [14] to [1.
6] The production method described above;

[18] A phenoxypropane derivative represented by the general formula (II) which is optically active by the production method according to any one of the above [1] to [13] (in the formula, Xa and Xb are the above [1]. Of the phenoxypropane derivative (II) obtained is treated according to a known method to give S-(-)-9-fluoro-3-methyl-10- (4- Methyl-1
-Piperazinyl) -7-oxo-2,3-dihydro-7H-pyrido
[1,2,3, -de] [1,4] benzoxazine-6-carboxylic acid (levofloxacin) is obtained; [19] A method for producing levofloxacin; [19] A known method is Japanese Patent No. 2612327 (Or Japanese Patent Laid-Open No. 2-732), Japanese Patent Laid-Open No. 1-250369, Japanese Patent Laid-Open No. 1
-287096 and Japanese Patent Application Laid-Open No. 2-218648, which is a method disclosed in the above [18]; [20] The above [1] to [13] ] The optically active phenoxypropane derivative represented by the general formula (II) (in the formula, Xa and Xb have the same meanings as in the above [1]) by the production method according to any one of The phenoxypropane derivative (II) is combined with the following treatments (a) to (g): (a) reduction of nitro group, (b) methylene malonation reaction, (c) sulfonyloxylation reaction, ( d) Oxazine ring formation reaction, (e) Pyridine ring formation reaction (f) N-methylpiperidine introduction reaction, and (g) Removal of protecting group and / or free carboxyl group formation reaction by ester hydrolysis to obtain levofloxacin. [18] or [19] above, characterized by the above; and [2] 1] An optically active phenoxypropane derivative represented by the general formula (II) is produced by the production method described in any one of the above [1] to [13], and then the following treatments (a) to (g) are performed. Performed in combination: (a) reduction of nitro group to amino group, (b) methylene malonation reaction of amino group, (c) sulfonylation reaction of hydroxy group on 2-hydroxypropyloxy group, (d)
Oxazine ring formation by ring-closing reaction between 2-sulfonyloxypropyloxy group on benzene nucleus and amino nitrogen atom, (e) Pyridine ring formation by ring-closing reaction between methylenemalonated amino group and carbon atom on benzene nucleus Reaction (f) N-methylpiperidine introduction reaction, and (g) removal of protecting group and / or hydrolysis of ester to obtain free carboxyl group formation reaction Levofloxacin [18]-
[20] The method according to the description is provided.

Other objects, characteristics, excellences, and aspects of the present invention will be apparent to those skilled in the art from the following description. However, it is understood that the description of the present specification, including the description below and the description of specific examples and the like, shows the preferred embodiments of the present invention and is provided only for the purpose of explanation. I want to. Various changes and / or alterations (or modifications) within the spirit and scope of the invention disclosed herein can be made by those of ordinary skill in the art based on knowledge from the following description and other portions of the specification. Would be readily apparent. All patent and literature references cited in this specification are cited for the purpose of illustration and are to be construed as part of this specification, the contents of which are included herein. is there.

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, a compound represented by the general formula (I) (in the formula, Xa and Xb may be the same or different and each represents a halogen atom), Pseudomonas sp. Genus, Rhodococcus, Brevendimonas, Aeromonas, Methylobacterium,
Candida, Yalobia, Sporothrix, Mycobacterium, Leifsonia and Stenotrophomonas, a microbial cell selected from the group consisting of the microbial culture, a microbial culture thereof, or an extract fraction thereof. General formula
An optically active compound represented by (II) (in the formula, Xa and Xb
Has the same meaning as above. ) Is manufactured. In the above formulas (I) and (II), Xa and Xb
Are preferably fluorine atoms, and 3,4-difluoro-2-((R)-
2-Hydroxypropyloxy) nitrobenzene is preferably obtained. In the present invention, when using the microbial cells or a microbial culture thereof, the treatment can be performed in the presence of an energy source.

The microorganism used in the present invention has the general formula
As long as it is a microorganism capable of acting on the phenoxyacetone derivative of (I) to produce an optically active phenoxypropane derivative represented by the corresponding general formula (II), a wild strain, mutant strain, cell fusion, etc. Any of the strains such as the recombinant strains derived by the cell engineering techniques described above or genetic engineering techniques such as DNA cloning and gene manipulation can be used. In the present invention, microorganisms used in the synthesis of compound (II) include Pseudomonas spp.
genus ocuria, genus Rhodococcus, genus Brevundimonas, Aeromonas
as) genus, Methylobacterium genus, Candida genus, Yarrowia genus, Sporothrix genus, Mycobacterium (Mycob)
Acterium genus, Leifsonia genus, Stenotrophomonas genus (Senotrophomonas) genus and the like. ), Rhodococcus fascias
(Rhodococcus facians), Brevundimonas diminuta (Brevundimonas diminuta), Aeromonas hydrophila (Aeromonas hydrophila), Methylobacterium rhodesianum (Methylobacterium rhodesianum),
Methylobacte
rium extorquens), Candida sonorensis (Cand
ida sonorrensis), Yalovia lipolytica (Yarrowi
a lipolytica), Sporothrix sienkii (Sporoth
rix schenckii), Mycobacterium ziernhoferi (M
ycobacterium diernhoferi), Leifsonia naganoensis, Stenotrophomonas maltophilia
And so on.

The microorganisms that can be used in the present invention can be obtained from various microorganism preservation institutions. For example, National Institute of Advanced Industrial Science and Technology, National Institute for Biological Biology (National)
Institute of Advanced Industrial Science and Tech
nology, International Patent Organism Depositary:
IPOD) (former name: Ministry of Economy, Trade and Industry, National Institute of Advanced Industrial Science and Technology, Institute of Biotechnology (NIBH)), Institute of Applied Microbiology (IAM), University of Tokyo, Faculty of Engineering (HUT), Hiroshima University, RIKEN (JCM),
Faculty of Engineering (OUT), Osaka University, or National Institute for Genetic Resources, National Institute for Product Evaluation Technology
e of Technology and Evaluation, Biological Resourc
e Center: NBRC) (transferring and succeeding biological resources such as microorganisms stored in the Fermentation Research Institute (IFO) in the past), American Type Culture Collection (ATCC) in the United States, etc. Bacteria can be obtained, and their effects can be easily confirmed by ordinary tests with reference to the examples.

As a typical strain, for example, Pseudomonas hydrogen thermophila IFO 14593 (Pseu
domonas hydrogenthermophila IFO 14593) strain, Pseudomonas syringae subspecies syringae IFO 12655 (Ps
eudomonas syringae subsp.syringae IFO 12655) strain,
Kocuria rosea IFO 3768
Strain, Rhodococcus fascias IFO 12077 (Rhodococ
cus facians IFO 12077) strain, Brevundimonas diminuta IFO 1269
7) strain, IFO 13182 strain, IAM 12557 strain, Aeromonas hydrophila subspecies hydrophila IFO 3820 (Aerom
onas hydrophila subsp.hydrophila IFO 3820) strain, Methylobacterium rodesianum JCM 2815 (Methyloba
cterium rhodesianum JCM 2815) strain, JCM 2816 strain, I
FO 15844 strain, Methylobacterium extorquens JCM 2812
Shares, JCM 2813 shares, JCM 2814 shares, JCM 2817 shares, JC shares
M 2827, JCM 2834, JCM 2806, JCM 2807
Stock, JCM 2808, JCM 2809, IFO 3708, IA
M 1081 strain, IAM 12632 strain, Candida sonorrensis IFO 10027 strain, Yalovia lipolytica IAM 4694 (Yarrowialipolytic)
a IAM 4694) strain, Sporothrix sienkii IFO 5983
(Sporothrixschenckii IFO 5983) strain, Mycobacterium diernhoferi IFO 3707 (Mycobacterium diern
hoferi IFO 3707), IFO 14756, Leifsonia
Naganoensis JCM 10592 (Leifsonia naganoensis JCM
10592) Stock, Stenotrophomonas maltophilia
NBRC 13692 (Stenotrophomonas maltophilia NBRC 1369
2) Shares, NBRC 12020 shares, NBRC 12692 shares, NBRC 13923 shares
Shares, IAM 12424 shares, JCM 1976 shares, JCM 1978 shares,
JCM 1982, JCM 1983, JCM 1984, JCM 1986
Stock, JCM 1987, JCM 3800, JCM 3802, JC
M 3803, JCM 3804, JCM 3805, JCM 3806
Shares, JCM 3807 shares, etc.

Preference is given to Pseudomonas
Hydrogen Thermophila IFO 14593 strain, Pseudomonas syringae subspecies syringae IFO 12655 strain, Cochlea rosea IFO 3768 strain, Rhodococcus fascias IFO 12077 strain, Breven Dimonas diminuta I strain
FO 12697 strain, IFO 13182 strain and IAM 12557 strain, Methylobacterium extorcence IFO 3708 strain,
IAM 1081 strain, JCM 2806 strain, JCM 2807 strain, JCM 2808 strain
Shares, same JCM 2812 shares, same JCM 2813 shares, same JCM 2814 shares, same JC
M 2817 strain and JCM 2834 strain, Methylobacterium rhodesianum JCM 2815 strain, JCM 2816 strain and IFO 15844 strain
Strains, Candida sonorensis IFO 10027 strains, Yarovia lipolytica IAM 4694 strains, Sporothrix schienkii IFO 5983 strains, Mycobacterium diernhoferi IFO 3707 strains and IFO 14756 strains, Leifsonia strains
Naganoensis JCM 10592 strain, Stenotrophomonas
Marutophilia NBRC 13692 shares, NBRC 12020 shares,
NBRC 12692 shares, IAM 12424 shares, JCM 1976 shares, JCM shares
1978, JCM 1982, JCM 1983, JCM 1984,
JCM 1986 shares, JCM 3800 shares, JCM 3802 shares, JCM 38 shares
03 shares, JCM 3804 shares, JCM 3805 shares, JCM 3806 shares and the like. In one embodiment, Pseudomonas hydrogen thermophila I is preferable.
FO 14593 strain, Pseudomonas syringae subspecies syringae IFO 12655 strain, Cochlea rosea IFO 3768 strain,
Rhodococcus fascias IFO 12077 strain, Methylobacterium extorcens JCM 2807 strain, JCM 28
08 shares, JCM 2812 shares, JCM 2813 shares and JCM 2814 shares,
Methylobacterium rhodesianum JCM 2815 strain and
JCM 2816 strain, Aeromonas hydrophila subsp. Hydrophila IFO 3820 strain, Candida sonorensis I
FO 10027 strain, Stenotrophomonas maltophilia
Examples include NBRC 13692 strain, JCM 1978 strain, JCM 1983 strain, and the like. Moreover, as an even more preferable one,
Pseudomonas Hydrogen Thermophile IFO 145
93 strains, Pseudomonas syringae Subspecies syringae
IFO 12655 strain, Cochlea rosea IFO 3768 strain, Rhodococcus fascias IFO 12077 strain, Methylobacterium extorcence JCM 2812 strain and JCM 2814 strain
Strain, Methylobacterium rhodesianum JCM 2815 strain and JCM 2816 strain, Candida sonorensis IFO 10027
Stock, Stenotrophomonas maltophilia NBRC136
92 strains and the like, and particularly preferable one is Pseudomonas hydrogen thermophila IFO 14593.
Strains, Cochlea Rosea IFO 3768 strains, Candida sonorensis IFO 10027 strains, Stenotrophomonas maltophilia NBRC 13692 strains and the like.

Examples of the bacterial cells include those obtained by harvesting the above-mentioned microorganism from the culture solution, those obtained by collecting and washing the microorganism from the culture solution, and those obtained by drying or treating with acetone powder. The cells can be used as they are or in a fixed form. 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 entrapping method, etc.).
The immobilization carrier may be any commonly used carrier, for example, cellulose, agarose, dextran, κ-carrageenan, alginic acid, gelatin,
Polysaccharides such as cellulose acetate; natural polymers such as gluten; inorganic substances such as activated carbon, glass, clay, kaolinite, alumina, silica gel, bentonite, hydroxyapatite, calcium phosphate; polyacrylamide, polyvinyl alcohol, polypropylene glycol, urethane, etc. The synthetic polymer and the like. Also,
The bacterial cells can be used in the form of being encapsulated in microcapsules, and can be appropriately selected and used from methods known in the art.

Examples of the culture include those obtained by culturing the above-mentioned microorganism in an appropriate medium. As the treated products and extracts of the above-mentioned microorganisms, bacterial cells or cultures are, if necessary, suspended in a buffer solution and obtained by autolyzing the resulting suspension, or French press, Sonic waves, a physical method such as a homogenizer, may further refer to a disrupted cell obtained by crushing in combination with an enzymatic method such as lysozyme, from the product thus obtained, water Alternatively, the product extracted with an appropriate buffer, the precipitate obtained by adding ammonium sulfate or alcohol to the extract and the extract are separated by ultrafiltration, gel filtration, hydrophobic chromatography, ion exchange chromatography, etc. Some of them can be mentioned. Further, the treated product and extract of the microorganism may be, for example, heat-treated bacterial cells or culture, or a product obtained by subjecting the heat-treated product to the above treatment. The heat treatment can be performed by a method known in the art, and specific conditions can be appropriately determined by experiments or the like according to the purpose. The temperature of the heat treatment includes a temperature of about 37 ° C or higher, for example, about 40 to 70 ° C, preferably about 45 ° C.
~ 60 ° C. The heat treatment time depends on the treatment temperature, but is, for example, about 5 minutes to about 24 hours, preferably about 30 minutes to 10 hours, more preferably about 1 to 5 hours. The treatment is performed at about 45 ° C, about 50 ° C or about 55 ° C for about 2 to 4 hours, preferably about 45 to
It is treated at 55 ° C for about 3 hours. By using the heat-treated product, good results including selectivity, conversion rate, etc. can be obtained.

Various conditions in the method for culturing the above-mentioned microorganism are as follows:
There is no particular limitation, it is carried out by a commonly used method, bacteria,
It is carried out in a medium suitable for each of the fungus and yeast. Usually, a liquid medium containing a carbon source, a nitrogen source, and other nutrients is used. As the carbon source of the medium, any one can be used as long as the above microorganism can be used. Specific examples include assimilating ones, for example, glucose, fructose, sucrose, dextrin, starch, sugars such as sorbitol, methanol, ethanol, alcohols such as glycerol, fumaric acid, citric acid, acetic acid, Organic acids such as propionic acid and salts thereof, hydrocarbons such as paraffin, molasses, or a mixture thereof can be used. As the nitrogen source, any source can be used as long as the above microorganism can be used. Specific examples include assimilating ones, for example, ammonium salts of inorganic acids such as ammonium chloride, ammonium sulfate, ammonium nitrate and ammonium phosphate, ammonium fumarate,
Ammonium salts of organic acids such as ammonium citrate,
A nitrate such as sodium nitrate or potassium nitrate, a meat extract, a yeast extract, a malt extract, peptone, corn steep liquor, an inorganic or organic nitrogen-containing compound such as soybean protein hydrolyzate, or a mixture thereof can be used. In addition, nutrients such as inorganic salts such as potassium phosphate, iron sulfate, zinc sulfate, and manganese sulfate, trace metal salts, vitamins and the like used in ordinary culture may be appropriately added to the medium. If necessary, the medium may be supplemented with substances that induce the activity of microorganisms, buffer substances effective for maintaining the pH of the medium, antifoaming agents, and further silicone, adecanol, pluronic and the like.

Cultivation of the microorganism can be carried out under conditions suitable for growth. Specifically, it can be carried out at a pH of the medium of 3 to 10, preferably 4 to 9, and a temperature of 0 to 50 ° C, preferably 20 to 40 ° C. Culturing of microorganisms can be performed under aerobic or anaerobic conditions. Culture time is 1 to 300 hours,
It is more preferably 10 to 300 hours, but it should be appropriately determined depending on each microorganism. As the reaction method in the production of the phenoxypropane derivative (II) according to the present invention, the phenoxyacetone derivative represented by the general formula (I) is a microorganism, or a culture thereof, a treated product and an extract thereof. There is no particular limitation as long as it is a method in which the selected one acts to generate the optically active phenoxypropane derivative represented by the corresponding general formula (II), and the aqueous solution of the raw material compound is washed with a buffer solution or water. The reaction is initiated by mixing the selected bacterial cells, or a culture of the microorganism, a treated product thereof, and an extract. The reaction can usually be carried out in water or a liquid two-phase system of an organic solvent substantially insoluble or hardly soluble in water and water, but it is generally preferred to carry out the reaction in an aqueous system. Further, the phenoxyacetone derivative represented by the general formula (I) is, if necessary, dissolved in a suitable organic solvent such as ethanol, methanol, dioxane or dimethylsulfoxide, and then the solution is used as an aqueous solution. You can also

The reaction conditions can be selected within a range that does not impair the production of the optically active phenoxypropane derivative represented by the general formula (II). Typically, the amount of cells is preferably 1/100 to the compound of the formula (I) as dry cells.
It is 1000 times, more preferably 1/10 to 100 times. Also, the concentration of the compound of formula (I) that is the substrate is preferably 0.
It is from 01 to 20%, more preferably from 0.1 to 10%. further,
The pH of the reaction solution is preferably 5-9, more preferably 6-
8 and the reaction temperature is preferably 10 to 50 ° C, more preferably 20 to 40 ° C. Buffers can also be used to stabilize the pH. In addition, to adjust the pH,
It can also be adjusted using an acid or a base. The reaction time is 1 to 200 hours, preferably 5 to 150 hours, but it should be appropriately determined depending on each microorganism.

In order to make the reaction proceed more efficiently, sugars such as glucose, organic acids such as acetic acid, and energetic substances such as ethanol and glycerol can be added. Each of these may be used alone or in the form of a mixture thereof. The addition amount is preferably 1/100 to 1/100 times the amount of the substrate. Moreover, a coenzyme or the like can be added. Examples of coenzymes include nicotinamide adenine dinucleotide (NAD), reduced nicotinamide adenine dinucleotide (NADH), nicotinamide adenine dinucleotide phosphate (NADP), reduced nicotinamide adenine dinucleotide phosphate (NADPH), etc. They may be used alone or in the form of a mixture thereof, and the addition amount thereof is preferably 1/1000 to 1/5 times the amount of the substrate compound. . In addition to these coenzymes, a coenzyme regenerating enzyme, for example, glucose dehydrogenase can be added, and the addition amount is preferably 1/1000 to 1/5 times the substrate compound. Is. It is also possible to add a substrate for the coenzyme-regenerating enzyme, for example glucose, and the addition amount is preferably 10 with respect to the substrate compound.
It is one-tenth to one-tenth the amount. Further, sugars such as glucose, organic acids such as acetic acid, energetic substances such as glycerol, coenzymes, coenzyme regenerating enzymes, and substrates for coenzyme regenerating enzymes may be used in combination. They are,
Although it is originally accumulated in the bacterial cells, it may be possible to increase the reaction rate, yield, etc. by adding these substances as necessary, and it can be appropriately selected.

If necessary, a substance selected from the group consisting of a substrate, a microbial cell, a culture thereof, a treated product and an extract thereof, and other substances are sequentially added to the reaction system. It is also possible to add continuously. By carrying out the reaction while continuously taking out the product,
It is also possible to increase the reaction rate. The optically active phenoxypropane derivative (II) produced by the reaction can be isolated and purified by a conventional separation and purification means. For example, directly or after separating cells from the reaction solution, membrane separation, extraction with an organic solvent (eg, toluene, chloroform, etc.),
It can be subjected to usual purification methods such as column chromatography, concentration under reduced pressure, distillation, crystallization and recrystallization. For example, after completion of the reaction, butyl acetate, ethyl acetate, toluene,
The crude product can be obtained by extracting the product from the reaction solution with an organic solvent such as chloroform and distilling off the solvent. The crude product may be used as it is in the next step, but if necessary, after purification by means such as silica gel column chromatography, high performance liquid chromatography using an optically active carrier such as a cellulose derivative, etc. You may refine | purify by the means of.

The phenoxyacetone derivative of the general formula (I) used in the present invention can be obtained by a known method. For example, 2-acetonyloxy-3,4-difluoronitrobenzene can be obtained by Chem. Pharm. Bull., 32 (12): 4907-491
3, 1984, etc., and it is also known that 2,3-difluoro-6-nitrophenol can be easily synthesized by reacting it with chloroacetone in the presence of a strong base (JP-A-61-246151). No.). The optically active phenoxypropane derivative (II) obtained in the present invention is obtained by a reaction step which is obvious to those skilled in the art, for example, Japanese Patent No. 2612327, JP-A 1-250369, JP-A 1-287096, and Kaihei 2-21
It can be converted to levofloxacin by the method disclosed in Japanese Patent No. 8648. The optically active phenoxypropane derivative (II) obtained in the present invention can be obtained by a reaction step obvious to those skilled in the art, for example, in the case of 3,4-difluoro-2-((R) -2-hydroxypropyloxy) nitrobenzene, S)-(-)-7,
It can be converted to 8-difluoro-3-methyl-3,4-dihydro-2H- [1,4] benzoxazine derivatives.

For example, an intermediate 2,3-difluoro- (2,2-diethoxycarbonylethenyl) amino-((R) -2- (methanesulfonyloxy) propyloxy) benzene derivative produced from an optically active phenoxypropane derivative As a method for producing, for example, JP-A-1-250369, JP-A-2-218648, etc., the method described in JP-A-2-218648 and the like, for example, amino nitrogen with a dialkylalkoxyethylene malonate such as diethylethoxymethylene malonate (EMME) It can be malonated. The methylene malonated product is converted to an (S)-(-)-7,8-difluoro-3-methyl-3,4-dihydro-2H- [1,4] benzoxazine derivative by ring closure of oxazine with potassium carbonate. be able to. In addition, as a method for producing an intermediate (S)-(-)-7,8-difluoro-3-methyl-3,4-dihydro-2H- [1,4] benzoxazine derivative from an optically active phenoxypropane derivative, JP-A-2-732 (Examples 22, 23, 24, 33, 39, 40, 41 and 42, etc.),
JP-A-1-250369 (Examples 17, 18, 19, 22, 23, 2
4, 25, 26, 27 and 29) and the like, and is produced by combining a sulfonylation reaction, a reduction of a nitro group, and a ring closure reaction. Specifically, for example, JP-A 1-250369. Obtained by a method as described in Examples 22, 23, 26, 27, 29 of the publication, tosylating an optically active phenoxypropane derivative using tosyl chloride in pyridine,
Palladium-carbon is added to ethanol in ethanol and catalytic reduction is carried out at room temperature and atmospheric pressure to reduce the nitro group, and then the product is subjected to a ring-closing reaction. It is carried out by adding potassium and a catalytic amount of 18-crown-6-ether and heating and stirring. Optionally, for a given compound, diethyl ethoxymethylene malonate (EMME)
It is also possible to add a dialkylalkoxymethylene malonate such as, and stir with heating to convert the amino nitrogen into methylene malonate, and subject it to a ring-closing reaction in the next step so that a pyridonecarboxylic acid ester structure can be formed.

In a typical embodiment, 3,4-difluoro-2-((R) -2-hydroxypropoxy) nitrobenzene obtained as described above is prepared by the method described in JP-A-2-732. , For example, by sulfonylating the hydroxyl group using a substituted sulfonyl chloride such as paratoluenesulfonic acid chloride, and then reducing the nitro group, (S)-
(-)-7,8-Difluoro-3-methyl-3,4-dihydro-2H- [1,
4] Can be converted to benzoxazine derivatives.
The obtained (S)-(-)-7,8-difluoro-3-methyl-3,4-dihydro-2H- [1,4] benzoxazine derivative was synthesized as an antibacterial agent LV.
To convert into FX, the method described in JP-A-62-252790 may be used. For example, (S)-(-)-7,8-difluoro
As a method for producing levofloxacin LVFX from a 3-methyl-3,4-dihydro-2H- [1,4] benzoxazine derivative,
The methods described in JP-A-62-252790 and JP-A-1-287086 (Examples 4, 5, and 6) can be mentioned, and specifically, for example, the examples of JP-A-1-287086. 4, 5, 6
As described above, the title benzoxazine derivative is heated under reflux with EMME, if necessary, to give methylene malonate, the methylene malonate product is dissolved in acetic acid, and concentrated hydrochloric acid is added to give the corresponding carboxylic acid. Then, add boron trifluoride diethyl ether complex in diethyl ether and stir. Further in dimethyl sulfoxide,
It can be manufactured by adding triethylamine and N-methylpiperazine and stirring.

Thus, the present invention provides the above-mentioned microbial cells,
The culture, treated in the presence of those selected from the group consisting of processed products and extracts, the phenoxyacetone derivative of the general formula (I) as an optically active phenoxypropane derivative of the general formula (II), The present invention also relates to a process for producing levofloxacin, which comprises treating the obtained phenoxypropane derivative (II) according to a known method to obtain levofloxacin. To produce levofloxacin from the phenoxypropane derivative (II), for example, the following (a)
~ (G) may be combined with each other: (a) reduction of nitro group to amino group, (b) methylene malonate reaction of amino group, (c) hydroxy on 2-hydroxypropyloxy group. Sulfonylation of groups, (d) on the benzene nucleus
Oxazine ring formation reaction by ring closure reaction between 2-sulfonyloxypropyloxy group and amino nitrogen atom, (e) Pyridine ring formation reaction by ring closure reaction between methylenemalonated amino group and carbon atom on benzene nucleus, (f) ) N-methylpiperidine introduction reaction, and (g) removal of protecting group and / or hydrolysis of ester to form free carboxyl group. the above
(a) ~ (g) the reagents and reaction conditions used for the treatment, specific treatment operation, Japanese Patent No. 2612327, JP 1-250
No. 369, Japanese Patent Application Laid-Open No. 1-287096, Japanese Patent Application Laid-Open No. 2-218648, etc., the contents of which are hereby incorporated by reference as a part of this specification. It should be done.

[0033]

EXAMPLES The present invention will be specifically described with reference to examples below, but it is understood that the present invention is not limited to these examples and various embodiments based on the idea of the present specification are possible. It should be. All examples, unless otherwise indicated in detail, are carried out or can be carried out using standard techniques, which are well known and routine to those skilled in the art. ..

Example 1 Pseudomonas hydrogen thermophila (Pseudom)
onas hydrogenthermophila) IFO14593 glucose 1%
5 mL of medium containing 0.5% peptone and 0.3% yeast extract
Was inoculated and shake cultured at 30 ° C. for 72 hours. After obtaining the bacterial cells by centrifuging the culture solution, 2-acetonyloxy-3,4-difluoronitrobenzene 1 mg, ethanol 0.35 mL, glucose 20 mg, 0.2 M sodium phosphate buffer (pH 7.0)
The reaction was carried out by adding 1 mL and shaking at 30 ° C. for 24 hours. After the reaction was completed, the reaction solution was centrifuged to remove the bacterial cells, and the reaction solution was extracted with 1 ml of butyl acetate to obtain the reductant 3,4-difluoro-2.
A solution containing-((R) -2-hydroxypropyloxy) nitrobenzene was obtained. The organic layer was dehydrated by adding magnesium sulfate (anhydrous), centrifuged and the supernatant was subjected to HPLC to measure the conversion rate, absolute configuration and optical purity of the reaction product. The conversion rate is
HPLC analysis [column: YMC-Pack SIL (manufactured by YM), eluent: hexane / tetrahydrofuran = 10/1, flow rate: 0.
When 8 ml / min, temperature: 30 ° C., detection wavelength: 260 nm], the conversion was 78%. The optical purity is HPLC [column: CHIRALCEL OJ (manufactured by Daicel), eluent: hexane / isopropanol / TFA = 100/2 / 0.1, flow rate: 0.8m
L / min, temperature: 40 ° C., detection wavelength: 265 nm], optical purity was 100% ee.

Example 2 Instead of Pseudomonas hydrogenthermophila IFO14593 of Example 1, Aeromonas hydrophila subsp. Hydrophila IFO3820
Was used in the same manner as in Example 1. After completion of the reaction, 3,
4-Difluoro-2-((R) -2-hydroxypropyloxy) nitrobenzene was obtained and analyzed by HPLC to find that the optical purity was 96.9% ee.

Examples 3 to 10 Instead of Pseudomonas hydrogenthermophila IFO14593 of Example 1, the reaction was carried out in the same manner as in Example 1 using the microorganisms of Table 1. After the reaction was completed, the solution containing 3,4-difluoro-2-((R) -2-hydroxypropyloxy) nitrobenzene was analyzed by HPLC, and the conversion rates shown in Table 1 were obtained.

[0037]

[Table 1]

Examples 11 to 16 In place of Pseudomonas hydrogen thermophila IFO14593 of Example 1, the microorganisms of Table 2 were used.
The reaction was performed in the same manner as in. After completion of the reaction, 3,4-difluoro-2
When the solution containing-((R) -2-hydroxypropyloxy) nitrobenzene was analyzed by HPLC, the results shown in Table 2 were obtained.

[0039]

[Table 2]

Example 17 Stenotrophomonas Maltophilia
monas maltophilia) NBRC 13692 was inoculated into 5 mL of a medium containing glucose 1%, peptone 0.5%, and yeast extract 0.3%, and shake-cultured at 30 ° C for 72 hours. After the culture solution was centrifuged to obtain bacterial cells, heat treatment was performed at 55 ° C for 3 hours. After heat treatment, 2-acetonyloxy-3,4-difluoronitrobenzene 1 mg, ethanol 0.35 mL, glucose 20 mg, 0.
Add 1 mL of 2M sodium phosphate buffer (pH 7.0) at 30 ℃
The reaction was carried out by shaking for 24 hours. After completion of the reaction, the reaction solution was centrifuged to remove the cells, the reaction solution was extracted with 1 ml of butyl acetate, and the reductant 3,4-difluoro-2-((R) -2-hydroxypropyloxy) was used. A solution containing nitrobenzene was obtained. The organic layer was dehydrated by adding magnesium sulfate (anhydrous), centrifuged and the supernatant was subjected to HPLC to measure the conversion rate, absolute configuration and optical purity of the reaction product. The conversion rate is determined by HPLC analysis [column:
YMC-Pack SIL (manufactured by YM), eluent: hexane / tetrahydrofuran = 10/1, flow rate: 0.8 ml / min, temperature: 30
At a temperature of 260 ° C and a detection wavelength of 260 nm, the conversion rate was 86%. The optical purity is determined by HPLC [column: CHIRALCEL OJ
(Manufactured by Daicel), eluent: hexane / isopropanol / TFA = 100/2 / 0.1, flow rate: 0.8 ml / min, temperature: 40
℃, detection wavelength: 265nm], the optical purity was 10
It was 0% ee.

According to the above embodiment, in the method of the present invention,
R-isomer optically active compound, which is important as an intermediate for LVFX production,
4-Difluoro-2-((R) -2-hydroxypropyloxy) nitrobenzene was used as the starting material 2-acetonyloxy-3,4-
100% conversion rate from difluoronitrobenzene and 100%
It can be seen that it can be obtained with an optical purity of ee. Therefore, the isolation / purification process required when the product is used in the next reaction step can be omitted or extremely simplified, and cost reduction can be expected. For example, it is possible to omit or greatly simplify the treatment by chromatography using an optically active carrier, which is usually used for isolating and purifying an optically active product.

Example 18 2,3-Difluoro-6-nitro-[[(R) -2-hydroxypropyl] oxy] benzene (4.62 g) was dissolved in pyridine (21 ml) and then tosyl chloride (4.53 g) was added. ) Was added, and the mixture was stirred under ice-cooling for 4 hours and stirred at room temperature for 4 hours to tosylate. After the reaction, ethyl acetate and 1N hydrochloric acid were added and shaken, the organic layer was separated, and the organic layer was washed with water and anhydrous magnesium sulfate. Dried in. The solvent was evaporated under reduced pressure and the obtained residue was purified by silica gel column chromatography to give 2,3-difluoro-6-nitro-
[[(R) -2-Tosyloxypropyl] oxy] benzene (6.7
4g) was obtained. The obtained compound (581 mg) was added to ethanol (20 ml).
, 5% palladium-carbon (200 mg) was added, and catalytic reduction was performed at room temperature and atmospheric pressure. After confirming the absorption of the theoretical amount of hydrogen, the catalyst was filtered off and the solvent was distilled off under reduced pressure. The obtained residue was subjected to silica gel column chromatography to give 2,3-difluoro-6-amino-[[(R) -2-tosyloxypropyl] oxy].
Benzene (520 mg) was obtained. The obtained compound (720 mg) was dissolved in anhydrous DMF (5 ml), potassium carbonate (276 mg) and a catalytic amount of 18-crown-6-ether were added, and the mixture was heated with stirring at 80 ° C. overnight. After the reaction, ethyl acetate and water were added and after shaking, the organic layer was separated, and the organic layer was further washed with water. After drying over anhydrous magnesium sulfate, the residue obtained by concentrating the solvent to dryness was subjected to silica gel column chromatography, and (S)-(-)-7.8-difluoro-3,4-dihydro-3-methyl-2H- [ 1,4] Benzoxazine (262 mg) was obtained.

Diethyl ethoxymethylene malonate (EMME, 24.0 mg) was added to the obtained compound (15.8 mg), and the mixture was stirred under reduced pressure at 130 to 140 ° C. for 1 hour. After cooling, it was dissolved in acetic anhydride, and a mixed solution of acetic anhydride-concentrated sulfuric acid (2: 1 v / v) was added dropwise little by little while stirring with ice cooling. After stirring for 1 hour at room temperature, at a bath temperature of 50-60 ° C
Stir for 30 minutes. Ice water was added to the reaction solution, which was then neutralized by adding powdery potassium carbonate, and extracted with chloroform. The extract was washed with saturated aqueous sodium hydrogen carbonate solution and saturated brine, and dried over sodium sulfate. Chloroform was distilled off, diethyl ether was added to the residue, and the crystals were collected by filtration to give (S)-(-)-9,10-difluoro-3-methyl-7-oxo-2,3.
-Dihydro-7H-pyrido [1,2,3-de] [1,4] benzoxazine
-6-carboxylic acid ethyl ester was obtained. The obtained ester compound (19.5 mg) was dissolved in acetic acid, and 0.4 ml of concentrated hydrochloric acid was added to add 3
Reflux for hours. After cooling, the precipitated crystals were collected by filtration, washed successively with water, ethanol and diethyl ether and dried to give the corresponding carboxylic acid. The obtained carboxylic acid (14.3 mg) was suspended in diethyl ether, boron trifluoride diethyl ether complex (70 μl) was added, and the mixture was stirred at room temperature for 5 hr. The supernatant was decanted, diethyl ether was added to the residue, and the residue was collected by filtration, washed with diethyl ether and dried. The product was dissolved in dimethylsulfoxide, triethylamine and N-methylpiperazine (7.3 μl) were added, and the mixture was stirred at room temperature for 18 hours. The solvent was distilled off under reduced pressure, diethyl ether was added to the residue, the yellow powder collected by filtration was suspended in 95% methanol, triethylamine was added, and the mixture was heated under reflux for 25 hours. The solvent was distilled off under reduced pressure, the residue was dissolved in 10% hydrochloric acid, washed three times with chloroform, and washed with 4N aqueous sodium hydroxide solution to pH 11
The pH was adjusted to 7.3 again with 1N hydrochloric acid, extracted with chloroform, and dried over sodium sulfate. Chloroform was distilled off, and the obtained solid was recrystallized from ethanol-diethyl ether to obtain levofloxacin (ofloxacin (S)-
(-)-Body) was obtained. Melting point 226-230 ° C (decomposition)

Example 19 2.17 g of 3,4-difluoro-2-((R) -2-hydroxypropyloxy) nitrobenzene obtained in Example 1 was added to 25 m of ethanol.
Dissolve in 1 l, add 450 mg of 5% Pd-C as a catalyst, and subject to catalytic reduction at atmospheric pressure. After the reaction, the catalyst was filtered off and the solvent was distilled off to give 2,3-
A crude product of difluoro-6-amino-((R) -2-hydroxypropyloxy) benzene is obtained. To this, 2.42 g of diethyl ethoxymethylene malonate (EMME) was added, and at normal pressure for 30 minutes,
The mixture is heated and stirred at 120 ° C for 30 minutes while removing ethanol generated under reduced pressure, and further for 30 minutes at normal pressure. The reaction mixture was subjected to silica gel column chromatography, and 2,3-difluoro- (2,2-diethoxycarbonylethenyl) amino-((R)
-2-Hydroxypropyloxy) benzene was obtained. The obtained compound was dissolved in 3.7 ml of pyridine, 0.74 g of methanesulfonyl chloride was added under stirring with ice cooling, and the mixture was stirred at room temperature for 2 hours. The solvent was distilled off under reduced pressure, and the residue was extracted with 1,2-dichloroethane. The extract was washed with 1N hydrochloric acid, 5% aqueous sodium hydrogen carbonate solution and water, and dried over anhydrous magnesium sulfate. 1.1 g of silica gel was added to the dried organic layer, the mixture was stirred for 30 minutes, and then the insoluble matter was filtered off. The solvent was distilled off under reduced pressure, the residue was crystallized from isopropyl ether and then collected, and 2,3-
Difluoro- (2,2-diethoxycarbonylethenyl) amino-((R) -2- (methanesulfonyloxy) propyloxy)
2.3 g of benzene was obtained. 1.5 g of this compound was added to anhydrous DMF,
It was dissolved in 7.5 ml, 0.46 g of potassium carbonate was added, and the mixture was stirred at 80 ° C for 2 hours. The solvent was evaporated under reduced pressure, the residue was extracted with ethyl acetate, the extract was washed with water and dried over anhydrous magnesium sulfate. The solvent was evaporated under reduced pressure and the obtained residue was subjected to silica gel column chromatography to give (S)-(-)-9,10-difluoro-3-
Methyl-7-oxo-2,3-dihydro-7H-pyrido [1,2,3-de]
[1,4] Benzoxazine-6-carboxylic acid ethyl ester was obtained. Levofloxacin was obtained from this compound according to the method of Example 18. The instrument data matched that of the standard.

[0045]

Industrial Applicability According to the method of the present invention, the production intermediate of the optically active synthetic antibacterial agent useful as a medicine has a high optical purity,
And it is easily manufactured. The method of the present invention, unlike the case of performing reduction by a chemical method, does not require an expensive asymmetric catalyst or a special device, and has an advantage that a desired optically active phenoxypropane derivative can be easily produced. is there. Moreover, there is an advantage that the chemical and optical yields of the obtained optically active phenoxypropane derivative are high. The method of the present invention is an important production intermediate (S)-(-)-7,8-difluoro-3-methyl-3,4-dihydro-2H- [1,4] benzoxazine, which is an important production intermediate in the production of synthetic antibacterial agent LVFX. The present invention provides a method for efficiently producing a derivative, and is useful for easily and efficiently producing an optically active phenoxypropane derivative which is a useful production intermediate in the production. In addition to using inexpensively available starting materials, it is possible to obtain an optically active phenoxypropane derivative with simple processing and operation, little generation of by-products, high chemical efficiency, high optical purity and optical yield. It is possible to manufacture and is excellent as an industrial manufacturing method. Obviously, the present invention may be carried out other than as specifically described in the above description and examples. Many modifications and variations of the present invention are possible in light of the above teachings, and are therefore within the scope of the claims appended hereto.

   ─────────────────────────────────────────────────── ─── Continued front page    F-term (reference) 4B064 AC16 AE58 BC04 CA02 CA06                       CB16 CE08 CE10 CE15 CE16                       DA01                 4C072 AA02 BB02 BB06 CC01 CC11                       EE07 FF07 GG01 GG07 GG09                       HH08 UU01                 4C086 AA04 CB22 ZB35

Claims (9)

[Claims] 1. General formula (I): (In the formula, Xa and Xb may be the same or different and each is a halogen atom), and a phenoxyacetone derivative represented by
Fungi of microorganisms selected from the group consisting of Rhodococcus, Brevendimonas, Aeromonas, Methylobacterium, Candida, Yarrovia, Sporothrix, Mycobacterium, Leifsonia and Stenotrophomonas. The optically active general formula (II), which is characterized in that it is treated in the presence of a body, a culture thereof, a treated product thereof and an extract thereof. (In the formula, Xa and Xb have the same meanings as described above.)
A method for producing a phenoxypropane derivative represented by: 2. The microorganisms are Pseudomonas spp., Cochlear spp., Rhodococcus spp., Brevendimonas spp., Methylobacterium spp., Candida spp., Yarovia spp., Sporolithix spp., Mycobacterium spp., Leifsonia spp. And Stenotrophomonas spp. The method according to claim 1, wherein the method is selected from the group consisting of genera. 3. The microorganism is selected from the group consisting of Pseudomonas, Cochlia, Rhodococcus, Aeromonas, Methylobacterium, Candida and Stenotrophomonas. The manufacturing method according to claim 1. 4. The microorganism is selected from the group consisting of Pseudomonas, Cochlia, Rhodococcus, Methylobacterium, Candida and Stenotrophomonas. [3] The method according to any one of [3] to [3]. 5. The microorganisms are Pseudomonas hydrogen thermophila, Pseudomonas syringae, Cochle rosea, Rhodococcus fascias, Brevendimonas diminuta, Aeromonas hydrophila, Methylobacterium rodesianum, Methylobacterium extorcensis, Candida sonorensis, Jalovia. Lipolitica, Sporothrix sienkii, Mycobacterium Ziernhoferi, Leifsonia Naganoensis and Stenotrophomonas
The method according to claim 1, wherein the method is selected from the group consisting of maltophilia. 6. The microorganisms are Pseudomonas hydrogen thermophila, Pseudomonas syringae, Cochle rosea, Rhodococcus fascias, Brevendimonas diminuta, Methylobacterium rhodesianum, Methylobacterium extorquens, Candida sonorensis, Yarovia lipolytica, Sporothrix. Sienki, Mycobacterium
The process according to any one of claims 1, 2 and 5, characterized in that it is selected from the group consisting of Gjernhoferi, Leifsonia Naganoensis and Stenotrophomonas maltophilia. 7. The microorganisms are Pseudomonas hydrogen thermophila, Pseudomonas syringae, Cochle rosea, Rhodococcus fascias, Aeromonas hydrophila, Brevendimonas diminuta, Methylobacterium rhodesianum, Methylobacterium extorquens, Candida sonorensis and Stenoensis. The method according to any one of claims 1, 3 and 5, wherein the method is selected from the group consisting of Trophomonas maltophilia. 8. The microorganism comprises a group consisting of Pseudomonas hydrogen thermophila, Pseudomonas syringae, Cochle rosea, Rhodococcus fascias, Methylobacterium rodesianum, Methylobacterium extorquens, Candida sonorensis and Stenotrophomonas maltophilia. It is selected, The manufacturing method as described in any one of Claims 1-7 characterized by the above-mentioned. 9. A phenoxypropane derivative represented by the formula (II) which is optically active by the method according to any one of claims 1 to 8 (wherein Xa and Xb have the same meanings as in claim 1). The phenoxypropane derivative (II) obtained is treated with S-(-)-9-
Fluoro-3-methyl-10- (4-methyl-1-piperazinyl)-
A process for producing levofloxacin, which comprises obtaining 7-oxo-2,3-dihydro-7H-pyrido [1,2,3, -de] [1,4] benzoxazine-6-carboxylic acid (levofloxacin).