JPH07250697A - New production of cephalosporin derivative having optically active side chain ester - Google Patents

Abstract

PURPOSE:To obtain in an industrially advantageous way the subject compound useful as an antibacterial agent, etc., by subjecting a diastereomer mixture of the 4-site ester of a specific 3-cephem-4-carboxylic acid derivative to an enzyme capable of asymmetrically hydrolyzing the mixture in an aqueous medium. CONSTITUTION:This compound of formula II (* denotes an asymmetric carbon atom) useful as e.g. an antibacterial agent can be easily and efficiently obtained by subjecting a diastereomer mixture of the 4-site ester of a 3-cephem-4- carboxylic acid derivative of formula I [R<1> is H or an acyl; R<2> is a lower alkyl; R<3> is a lower alkyl, a cycloalkyl, an aralkyl or a (substituted) phenyl; X is 0 or a single bond; Y is a group inert to the reaction] to an enzyme (or a material containing the same) capable of asymmetrically hydrolyzing said mixture in an aqueous medium.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a process for producing a cephalosporin derivative having an optically active side chain ester at the 4-position, which is useful as an antibacterial agent by an enzymatic asymmetric hydrolysis reaction.

[0002]

As a method for obtaining a cephalosporin derivative having an optically active side chain ester from a diastereomeric mixture of 4-position esters of a cephalosporin derivative,
Physicochemical separation techniques such as column chromatography or crystallization of a mixture of diastereomers are known (Japanese Patent Laid-Open No. 178861/1993).

[0003]

However, in the above-mentioned known method, there are problems in operability of reaction, yield and economical efficiency when performing large-scale synthesis, and a more efficient and convenient method for production. Is required.

[0004]

Therefore, the present inventors have
In recent years, many attempts have been made to utilize reactions utilizing microorganisms or enzymes for organic synthesis, especially when it is necessary to obtain optically active compounds with complicated chemical structures such as pharmaceuticals and their synthetic intermediates. Paying attention to the fact that the method utilizing the high stereoselectivity of the enzyme is a very effective method, and as a result of continuing various studies to solve the above-mentioned drawbacks, a specific enzyme source was It was found that the compound represented by (2) has an action of asymmetrically hydrolyzing a diastereomeric mixture of 4-position esters of the compound,
The present invention has been completed.

That is, the present invention has the general formula (2)

[0006]

[Chemical 3]

(Wherein R ¹ is a hydrogen atom or an acyl group,
R ² is a lower alkyl group, R ³ is a lower alkyl group, a cycloalkyl group, an aralkyl group or an optionally substituted phenyl group, X is an oxygen atom or a single bond, and Y is
Represents a group not involved in the reaction), and the diastereomer mixture of the 4-position ester of the compound represented by the general formula (2) is asymmetrically hydrolyzed to the diastereomer mixture of the 4-position ester of the compound represented by the general formula (2). A general formula (1) characterized in that an enzyme having the ability to decompose or a substance containing the same is allowed to act.

[0008]

[Chemical 4]

(In the formula, * C represents an asymmetric carbon atom,
R ¹ , R ² , R ³ , X and Y have the same meanings as described above), and a method for producing a cephalosporin derivative having an optically active side chain ester represented by the above.

As the group R ¹ defined in the general formula (2), a hydrogen atom or an acyl group is shown. Examples of this acyl group include acyl groups such as aliphatic acyl, aromatic acyl and heterocyclic acyl. Examples of the aliphatic acyl group include alkanoyl groups such as formyl, acetyl, succinyl, hexanoyl, heptanoyl and stearoyl groups, methoxycarbonyl, ethoxycarbonyl and t-.
Butoxycarbonyl, t-pentyloxycarbonyl,
Examples thereof include an alkoxycarbonyl group such as a heptyloxycarbonyl group and an alkanesulfonyl group such as methanesulfonyl, ethanesulfonyl, and heptanesulfonyl group.

Examples of the aromatic acyl group include aroyl groups such as benzoyl, toluoyl and naphthoyl, aryl (lower) alkanoyl groups such as phenylacetyl and phenylpropionyl groups, aryloxycarbonyl groups such as phenoxycarbonyl and naphthoxycarbonyl groups, Aryloxy (lower) alkanoyl group such as phenoxyacetyl, phenoxypropionyl group, arylglyoxyloyl group such as phenylglyoxyloyl, naphthylglyoxyloyl group, aryl (lower) alkoxycarbonyl such as benzyloxycarbonyl, diphenylmethoxycarbonyl group And benzenesulfonyl groups such as benzenesulfonyl and p-toluenesulfonyl groups.

Examples of the heterocyclic acyl group include a heterocyclic carbonyl group and a heterocyclic (lower) alkanoyl group. Examples of the heterocyclic moiety include a saturated or unsaturated 3- to 8-membered monocyclic or polycyclic heterocyclic group containing at least one heteroatom selected from nitrogen atom, sulfur atom, oxygen atom and the like. Preferred are, for example, thiazolyl group, isothiazolyl group, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-
Thiadiazolyl, 1,2,5-thiadiazolyl and other thiadiazolyl groups, dihydrothiadiazinyl and other 5 to 6 membered unsaturated monocyclic heterocycles having 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms as heteroatoms 5- to 6-membered unsaturated monocyclic heterocyclic groups having 1-2 sulfur atoms as hetero atoms such as cyclic groups, thienyl groups, dihydrodithynyl groups, dihydrodithionyl groups, furyl groups, pyranyl groups, etc. 5 to 6-membered unsaturated monocyclic heterocyclic group having 1 to 2 oxygen atoms as a heteroatom, pyridyl group, dihydropyridyl group, pyrimidyl group, imidazolyl group, pyrazolyl group, tetrazolyl group, etc. Examples include 5- to 6-membered unsaturated monocyclic heterocyclic groups having 4 to 4 nitrogen atoms. Further, examples of the lower alkanoyl moiety include alkanoyl groups such as acetyl group and propionyl group.

The above-mentioned acyl moiety may be a lower alkyl group such as methyl or ethyl, methoxy, ethoxy,
Lower alkoxy group such as propoxy, lower alkylthio group such as methylthio and ethylthio, halogen atom such as chlorine atom, bromine atom and fluorine atom, amino group, cyano group, nitro group, imino group, oxo group, carboxyl group, formula = N -O
-R ⁴ (In the formula, R ⁴ is a hydrogen atom, a lower alkyl group such as methyl, ethyl, propyl, isopropyl, butyl, pentyl and hexyl, a lower alkenyl group such as vinyl, allyl and 2-butenyl, ethynyl and 2-propynyl. Lower alkynyl groups such as methoxycarbonylmethyl, ethoxycarbonylmethyl, propoxycarbonylmethyl, t-butoxycarbonylmethyl, 1-t-butoxycarbonylethyl, protected alkoxy groups such as lower alkoxycarbonyl (lower) alkyl groups such as lower alkoxycarbonyl (lower) alkyl groups. ) An alkyl group,
It may have an appropriate substituent such as a group represented by carboxy (lower) alkyl group such as carboxymethyl, carboxyethyl and the like.

More preferred examples of the acyl group defined as above include the following formula

[0015]

[Chemical 5]

(In the formula, R ⁴¹ represents a hydrogen atom or a lower alkyl group).

A lower alkyl group is shown as the group R ² defined in the general formula (2). Examples thereof include linear or branched lower alkyl groups such as methyl, ethyl, propyl, isopropyl, butyl and isobutyl.

The group R ³ defined in the general formula (2) is a lower alkyl group, a cycloalkyl group, an aralkyl group or a phenyl group which may have a substituent. Examples of the above lower alkyl group include methyl,
Linear or branched C1-C6 such as ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl
Alkyl groups. Examples of the cycloalkyl group include cycloalkyl groups having 3 to 8 carbon atoms such as cyclopentyl and cyclohexyl groups. Examples of the aralkyl group include benzyl, 2-phenylethyl,
Examples include phenyl-lower alkyl groups having 1 to 4 carbon atoms such as 2-phenylpropyl. As the substituent in the case of a phenyl group which may have a substituent, a chlorine atom,
Halogen atom such as bromine atom, lower alkyl group such as methyl and ethyl, lower alkoxy group such as methoxy and ethoxy,
Examples thereof include a carboxyl group, a lower alkoxycarbonyl group, a cyano group and a nitro group. The above group R ³ is —O— via a group X (wherein X represents an oxygen atom or a single bond).
It binds to CO of CO-.

The group Y defined in the general formula (2) is a group which does not participate in the reaction. The group that does not participate in the reaction means a group that does not participate in the reaction of the present method and does not substantially interfere with the reaction. Examples thereof include a hydrogen atom, a hydroxyl group, a methoxymethyl group, a carbamoyloxymethyl group which may be substituted with a lower alkyl group, and a heterocyclic thiomethyl group which may be substituted with an appropriate substituent.

Of the mixture of diastereomers of 4-position ester of the compound represented by the general formula (2) [hereinafter, simply referred to as "compound (2)"], one example of the optimum compound is (6R, 7R) -7-Amino-3-methoxymethyl-3-cephem-4-carboxylic acid pivaloyloxyethyl ester, 7β- [2- (2-aminothiazole-4
-(Z) -2- (methoxyimino) acetamide] -3-methoxymethyl-3-cephem-4-carboxylic acid isopropoxycarbonyloxyethyl ester,
7-[(Z) -2- (2-furyl) -2-methoxyiminoacetamide] -3-carbamoyloxymethyl-3-
Cephem-4-carboxylic acid acetoxyethyl ester,
7β- [2- (2-Aminothiazol-4-yl) acetamido] -3-[[[1- (2-dimethylaminoethyl) -1H-tetrazol-5-yl] thio] methyl]
-3-Cephem-4-carboxylic acid cyclohexyloxycarbonyloxyethyl ester and the like can be mentioned. These production methods are described in JP-A-58-189186 and JP-A-5-189186.
9-190995, Japanese Patent Application Laid-Open No. 1-249778, and the like.

In the present invention, the compound (2) contains an enzyme having an ability to asymmetrically hydrolyze a diastereomeric mixture of 4-position esters of the compound represented by the general formula (2) in an aqueous medium or a substance containing the enzyme. Acephalosporin having an optically active side chain ester represented by the general formula (1) of interest is obtained by allowing the reaction mixture to undergo an asymmetric hydrolysis reaction and separating and collecting a product and an unreacted product from the reaction mixture. A derivative [hereinafter, simply referred to as "target compound (1)"] is obtained.

As the enzyme source used in the above asymmetric hydrolysis reaction, an enzyme having the ability to asymmetrically hydrolyze a diastereomeric mixture of 4-position ester of the compound represented by the general formula (2) It may be simply referred to as "asymmetric hydrolase"] or its inclusion. The asymmetric hydrolase refers to the enzyme itself and is in a substantially purified form, and examples thereof include enzymes having enzymatic activity such as lipase and esterase. The content of the asymmetric hydrolase may be in any form as long as it has the enzyme activity, for example, a culture of an asymmetric hydrolase-producing bacterium, a culture filtrate, and a crude enzyme separated from the culture filtrate. Examples thereof include agents, bacterial cells, crushed bacterial cells, crude enzyme agents separated and extracted from crushed bacterial cells, immobilized enzymes in which the enzyme is immobilized on a support, and the like.

Specific examples of the asymmetric hydrolase include monoglyceride lipase (derived from Bacillus, manufactured by Asahi Kasei Co., Ltd.) and cholesterol esterase (derived from pancreas, manufactured by Oriental Yeast) as commercially available enzymes.
Also, esterase MS, which is a novel esterase newly discovered by the present inventors, can be mentioned.

Esterase MS has the following enzymatic properties: (1) Action At least as shown in the following reaction, the present enzyme catalyzes the reaction of forming the compound B from the compound A.

[0025]

[Chemical 6]

(In the formula, * C represents an asymmetric carbon atom,
(R) indicates that the absolute configuration is R configuration)

(2) Substrate specificity formula

[0028]

[Chemical 7]

10% methanol containing 5 mM diastereomer mixture of 4-position ester of compound C
Various enzymes were added to 0.1 M phosphate buffer (pH 7.0), the reaction was carried out at 32 ° C. for 2 hours, and the mass of the remaining group was measured by high performance liquid chromatography (HPLC). The HPLC analysis conditions were quantified under the HPLC analysis conditions shown in the following enzyme activity measurement method (flow rate: 1 ml).
The retention time of R body and S body at 16 / min is 16
Minutes, 17.9 minutes). The results are shown in Table 1.

[0030]

[Table 1]

From the above measurement results, esterase MS has a substrate specificity of specifically hydrolyzing only the R isomer from the diastereomeric mixture of compound C to produce the S isomer. Esterase MS also acts on 4-nitrophenyl acetate and α-naphthyl acetate.

(3) Method for measuring enzyme activity 0.4 ml of 0.25M phosphate buffer (pH 8.0) was added to 0.1 ml of a methanol solution containing 20 mM of Compound A, and the mixture was heated to 30 ° C. and then added thereto. 0.5 ml of the enzyme solution is mixed and reacted at 30 ° C. for 30 minutes. The reaction is stopped by adding 1 ml of methanol to the reaction solution, the solid matter is removed by centrifugation and membrane filtration, and the produced compound A is quantified by HPLC to determine the enzyme activity.

The enzyme activity is expressed by using 1 unit of the amount of enzyme that produces 1 μM of compound B in 1 minute under the above-mentioned enzyme reaction conditions. The HPLC analysis conditions are as follows. Column; inert sill ODS-2 4.6 x 150 mm
(GL Science) Moving layer: 0.02 M sodium phosphate / methanol containing 5 mM tetrabutylammonium bromide = 4
0:60 mixed solution Detection wavelength: 254 nm

(4) Optimum pH As a result of examining the effect of pH on the activity of the present enzyme using the following buffer solutions having various pH as the buffer for the activity measurement, the optimum pH of the present enzyme is around pH 8. Was recognized. pH 3 to 6; citrate buffer pH 6 to 8; phosphate buffer pH 7 to 9; Tris-hydrochloric acid buffer pH 9 to 10; glycine buffer

(5) pH stability After heating at 50 ° C. for 30 minutes in the above-mentioned buffer solutions having various pH values, the activity of the present enzyme was measured according to the above-mentioned activity measuring method, and p
As a result of examining the effect of H, this enzyme is stable at pH 6 to 8.

(6) Optimum temperature As a result of investigating the influence at various temperatures using the reaction solution and method in the above-mentioned activity measuring method with 0.1 M phosphate buffer (pH 8.0), the optimum temperature of this enzyme was found. Is recognized to be around 40 ° C.

(7) Thermostability The enzyme solution (pH 8) was treated at each temperature of 5 to 70 ° C. for 1 hour, and its residual activity was measured according to the above-mentioned enzyme activity measuring method. 45 ° C at pH 8
It was confirmed to be stable up to the vicinity.

[0038] (8) the influence the enzymatic activity assay of metal ions, various metal salts was added 10 mM, the results of measurement of the enzyme activity, the enzyme Cu ^-, Fe
^- , Fe ^--- shows 50% or more inhibition, but M
^{n -, Mg -, Zn -} , Ni -, Ca -, Ba - and Co ^- At least 80% of the activity remained.

(9) Molecular weight of about 50,000 daltons (measured by gel filtration method using Protein Pack 300 (manufactured by Waters))

From the observation of the above-mentioned enzymatic properties, esterase MS has a very specific property as compared with known esterases and lipases, in particular, only the R-form is specifically hydrolyzed from the diastereomeric mixture of compound C. It is a novel enzyme that has the unique property of producing S-form.

To produce the esterase MS of the present invention, an esterase MS-producing bacterium belonging to the genus Sphingomonas may be cultured in a medium and the esterase MS may be collected from the culture. Examples of the esterase MS-producing bacterium include, for example, Sphingomonas sp. Which was newly separated from soil by the present inventors.
No. It is preferred to use AS-013. The mycological properties of this strain are as follows.

(A) Morphology The cells cultured in the broth agar medium are 0.5 to 1.0 × 3 to 5
It is a micron bacillus that moves with periflagellated flagella. It does not produce spores and does not show polymorphism. Both Gram stainability and acid resistance are negative.

(B) Cultural properties 1) Meat broth agar culture: Cells form cream-colored round colonies. No migration is observed and no diffusible dye is produced. 2) Meat culture: The entire medium grows turbid. No pellicle formation was observed on the liquid surface.

3) Meat broth gelatin puncture culture; liquefaction of gelatin is not observed. 4) Litmus milk culture; no significant change was observed.

(C) Physiological properties 1) Reduction of nitrates; Negative 2) Denitrification reaction; Negative 3) MR test; Negative 4) VP test; Negative 5) Indole formation; Negative 6) Hydrogen sulfide formation; Negative 7) Starch hydrolysis; Positive 8) Utilization of citric acid; Negative 9) Utilization of inorganic nitrogen source; Utilization of ammonium salt as sole nitrogen source 10) Dye formation; None 11) Urease; Positive 12) Oxidase; Positive 13) Catalase; Positive 14) Growth temperature; Grows well at 15 to 37 ° C, but 42
Does not grow at ℃ 15) Growth pH; grows well at pH 6-8, but pH
It does not grow in 4.5 16) Growth under anaerobic condition; not observed 17) OF test (Hurreyson method); Negative 18) Acid production from sugar (indicator phenol red) galactose + glucose + arabinose + fructose + xylose + rhamnose - sucrose + L-arabinose + melibiose + trehalose + lactose + raffinose + mannose + maltose + cellobiose + melezitose - adonitol - inositol - mannitol + dulcitol - sorbitol - erythritol - salicin + glycerol + starch + 19) beta- Galactosidase; Positive 20) Degradation of esculin; Positive 21) Degradation of arginine; Negative 22) Decarboxylation of lysine; Negative 23) Decarboxylation of ornithine; Negative 24) Utilization of malonic acid; Negative 25) Hydrolysis of Tween80; Positive 26) MacConkey agar growth; None 27) Hemolytic; None 28) Polymyxin B growth; None (3
00 μg / ml) 29) Lysis with 3% KOH; positive

From the above-mentioned mycological characteristics, this strain is a gram-negative aerobic bacillus, and has a morphological characteristic that it does not form spores but moves by pericardium. In addition, this strain is negative in both oxidation and fermentation in the OF test by the usual Hureyfson test method, but when cresol red, phenol red or the like is used as an indicator, production of acid is recognized from many carbohydrates. . Further, this strain has a weak acid-producing ability from a carbohydrate. Therefore,
Based on the physiological properties of this strain, Bergy's Manual
l of Systemic Bacteria
y, Volume 1 (1986), Cowan and Ste.
el's Manual for the Identity
information of Medical Bact
Searching for taxonomic position based on eria, 3rd edition (1993), this strain was found to be Sphingomonas adhaesi (Sphingomonas adhaesi).
va), but there was no matching strain, so this strain was selected as Sphingomonas sp. No. AS-0
It was named 13. This strain has been deposited with the Institute of Microbial Science and Technology as Microorganism Research Institute No. 14118 (FERM P-14118).

As the esterase MS-producing bacterium belonging to the genus Sphingomonas used for the production of esterase MS, the above-mentioned Sphingomonas sp. AS-0
13 is an example thereof, and is not limited to this strain, but any microorganism belonging to the genus Sphingomonas and producing esterase MS can be induced by a wild strain, a mutant strain, or a genetic method such as cell fusion or gene manipulation method. Any strain can be used, including recombinant strains.

To produce esterase MS, the esterase MS-producing bacterium is first cultivated in an appropriate medium. Regarding the form of culture, it is usually desirable to carry out aeration stirring culture in a liquid medium. As the nutrient source of the medium, those usually used for culturing microorganisms can be widely used. The carbon source may be any assimilable carbon compound, for example, maltose, saccharides such as lactose, a nitrogen compound that can be used as the nitrogen source, such as peptone, meat extract, yeast extract, protein hydrolyzate, Ammonium sulfate and the like can be used, and other inorganic salts, trace metal salts, vitamins and the like can be used as needed.

Cultivation of the above microorganisms may be carried out by a conventional method,
The esterase MS producing bacterium grows, and the esterase MS is produced.
Can be appropriately changed within the production range, but usually 20 to 32
It may be aerobically cultured for 1 to 3 days at a temperature of 6 ° C and a pH of 6 to 8.

The esterase MS of the present invention is collected from the thus obtained culture.
Usually, since it is contained in the microbial cells, the microbial cells collected from the obtained culture by means such as filtration or centrifugation are subjected to microbial treatment by means such as mechanical disruption means or enzymatic destruction means such as lysozyme. An enzyme preparation of the target esterase MS at various purification stages can be obtained by using a crushed body and using known means for separating and purifying a known enzyme.

The asymmetric hydrolysis reaction of the present invention is carried out by mixing the compound (2) with an asymmetric hydrolase or a substance containing the same in an aqueous medium and stirring or shaking. Examples of the aqueous medium include water, and a mixture of water and an organic solvent which is freely miscible with water, such as methanol, ethanol and acetonitrile. The asymmetric hydrolysis reaction is carried out at a reaction temperature of 10 to 40 ° C., preferably 20 to 30 ° C., and a reaction pH of 7 to 8. The reaction time depends on the substrate concentration, the concentration of the asymmetric hydrolase or the content thereof, and other reaction conditions, but the reaction progress can be traced by high performance liquid chromatography (HPLC), etc. The reaction may be appropriately terminated after waiting until the maximum is produced, but usually 1-2
It is preferable to set the reaction conditions so that the reaction is completed in about time.

In the above asymmetric hydrolysis reaction, the general formula (3) produced by asymmetric hydrolysis is used.

[0053]

[Chemical 8]

The carboxylic acid represented by the formula (wherein R ¹ and Y have the same meanings as described above) is neutralized, and the pH of the reaction solution is adjusted.
It is desirable to use a buffer solution of an inorganic salt such as sodium phosphate or potassium phosphate or to add a base such as sodium hydroxide or potassium hydroxide in order to keep the concentration constant.

The substrate concentration in the reaction solution is not particularly limited, but is usually preferably about 0.1 to 10 (W / V)%. The asymmetric hydrolase or its content is usually used in an amount of about 0.1 to 50 (W)% with respect to the weight of the substrate. At this time, in order to disperse the compound (2) as a substrate in the reaction solution well, a solvent mixed with water, for example, methanol, ethanol, acetonitrile or the like may be added.

In the present invention, the selectivity of asymmetric hydrolysis differs depending on the type of asymmetric hydrolase used or the content thereof. Therefore, when the desired compound (1) is to be obtained as a desired optically active substance, The asymmetric hydrolase to be used or its inclusion may be selected.

The target compound (1) thus produced in the reaction mixture is separated and purified from the reaction mixture, which is the product carboxylic acid (3) and the unreacted target compound (1). As a result, the objective compound (1) which is the objective optically active substance can be obtained. As a method for separating and purifying, known methods used in cephalosporin chemistry can be used. For example, centrifugation of the asymmetric hydrolase or its contents from the reaction mixture,
After separation by filtration or the like, the reaction filtrate is purified by a suitable solvent, concentrated under reduced pressure, crystallized, freeze-dried, recrystallized, or subjected to a method such as chromatography using an anion or cation exchange resin, macroporous nonionic adsorption resin, or the like. Is mentioned.

As a result of the above-mentioned asymmetric hydrolysis reaction, the target compound (1) obtained is at most half the amount of the compound (2) which is the substrate, but corresponds to the carboxylic acid (3) which is the product. Since the compound (2), which is a mixture of diastereomers, can be converted to an ester by esterification, the target compound (1) can be efficiently obtained by repeating this operation without discarding the carboxylic acid (3). be able to.

[0059]

INDUSTRIAL APPLICABILITY According to the method of the present invention, which is an enzymatic asymmetric hydrolysis method using an asymmetric hydrolase or a substance containing the same, the optically active side chain represented by the general formula (1) is better than the conventional method. Cephalosporin derivative with ester is simple,
And it can be obtained efficiently.

[0060]

EXAMPLES Next, the production examples of the present invention will be specifically described with reference to examples and reference examples, but the present invention is not limited thereby.

[0061]

Example 1 (6R, 7R) -7-Amino-3-methoxymethyl-3
AS-0 of a cefem-4-carboxylic acid pivaloyloxyethyl ester diastereomeric mixture (compound C)
Asymmetric hydrolysis by suspension of 13 strains Peptone 0.5%, meat extract 0.3%, yeast extract 0.
10 L of a liquid medium (pH 7.0) containing 1% and 0.01% of magnesium sulfate heptahydrate was placed in a 20 L jar fermenter, sterilized by heating, and then cultured in the same composition medium as above for Sphingomonas sp. AS. 500 ml of a culture solution of the -013 strain was inoculated, and cultured by aeration and stirring at 32 ° C for 48 hours. The cells were separated from the culture by centrifugation, and the cells were suspended in 100 ml of water.

On the other hand, (6R, 7R) -7-amino-3-
P of methoxymethyl-3-cephem-4-carboxylic acid pivaloyloxyethyl ester diastereomeric mixture
-Toluene sulfonate 10g methanol 120ml
The solution dissolved in 580 ml of water was added to 580 ml of water, the pH was adjusted to 8.0 with an aqueous sodium hydroxide solution, 100 ml of the bacterial cell suspension was added to this solution, and the pH was adjusted to 2 at 25 ° C while maintaining the pH at 8.0. Reacted for hours. After completion of the reaction, bacterial cells were separated, and the reaction filtrate was extracted with 800 ml of ethyl acetate. The organic layer was separated and concentrated under reduced pressure. A solution of 0.94 g of p-toluenesulfonic acid monohydrate dissolved in 8 ml of ethyl acetate was added dropwise thereto, and the mixture was stirred for 10 minutes. After 6 ml of diethyl ether was added to the reaction product and the mixture was stirred for 30 minutes, the generated crystals were collected by filtration. The obtained crystals were washed with 6 ml of ethyl acetate and 6 ml of diethyl ether in this order, and dried under reduced pressure to obtain 3.2 g of white powder (yield 32%). This white powder was dissolved in methanol and analyzed by HPLC. As a result, the obtained product had the same retention time as the S-form and had the following physical properties (1S)-(2,2-dimethylpropionyloxy). Ethyl-7-amino-3-methoxymethyl-3
-Cephem-4-carboxylate Identified as p-toluenesulfonate.

Melting point: 175 ° C. (decomposition) ¹ H-NMR (DMSO-d6) δ; 1.15 (s, 9)
H), 1.48 (d, 3H), 2.29 (s, 3H),
3.21 (s, 3H), 3.69 (ABq, 2H),
4.17 (s, 2H), 5.2-5.3 (m, 2H),
6.88 (q, 1H), 7.11 (d, 2H), 7.4
8 (d, 2H) optical purity; 97% e. e.

HPLC analysis conditions; Column: Inertosyl ODS-2 4.6 × 150 mm
(GL Science) Mobile phase: 0.2M sodium phosphate containing 5 mM tetrabutylammonium bromide / methanol = 40: 6
Mixture of 0 Detection wavelength: 254 nm Flow rate: 1 ml / min Retention time: R form; 16 min, S form; 17.9 min

[0065]

Example 2 (6R, 7R) -7-Amino-3-methoxymethyl-3
-Cephem-4-carboxylic acid pivaloyloxyethyl ester diastereomeric mixture (compound C) asymmetric hydrolysis with monoglyceride lipase (6R, 7R) -7-amino-3-methoxymethyl-3
-Cephem-4-carboxylic acid pivaloyloxyethyl ester diastereomer mixture 0.5 g of p-toluenesulfonate was dissolved in 10 ml of methanol, and 9 ml of 0.1 M potassium phosphate buffer (pH 7.0) was added thereto. And 100 mg of monoglyceride lipase were further added, and the mixture was stirred at 30 ° C. for 2 hours. The reaction solution was analyzed under the HPLC analysis conditions described in Example 1, and as a result, (1R)-
(2,2-Dimethylpropionyloxy) ethyl-7-
Amino-3-methoxymethyl-3-cephem-4-carboxylate yield 45% (50% × 90%), optical purity 99% e. e. Generated by.

[0066]

Example 3 (6R, 7R) -7-Amino-3-methoxymethyl-3
-Cephem-4-carboxylic acid pivaloyloxyethyl ester diastereomeric mixture (compound C) by asymmetric hydrolysis by cholesterol esterase (6R, 7R) -7-amino-3-methoxymethyl-3
-Cephem-4-carboxylic acid pivaloyloxyethyl ester diastereomer mixture 0.5 g of p-toluenesulfonate was dissolved in 10 ml of methanol, and 9 ml of 0.1 M potassium phosphate buffer (pH 7.0) was added thereto. And 10 mg of cholesterol esterase were added, and the mixture was stirred at 32 ° C. for 2 hours. The reaction solution was analyzed under the HPLC analysis conditions described in Example 1, and as a result, (1R)-
(2,2-Dimethylpropionyloxy) ethyl-7-
Amino-3-methoxymethyl-3-cephem-4-carboxylate yield 30% (50% x 60%), optical purity 96% e. e. Generated by.

[0067]

Example 4 7β- [2- (2-aminothiazol-4-yl)-
(Z) -2- (Methoxyimino) acetamide] -3-methoxymethyl-3-cephem-4-carboxylic acid isopropoxycarbonyloxyethyl ester diastereomer mixture as asymmetrically hydrolyzed by AS-013 strain cell suspension Decomposition 7β- [2- (2-aminothiazol-4-yl)-
(Z) -2- (Methoxyimino) acetamide] -3-methoxymethyl-3-cephem-4-carboxylic acid isopropoxycarbonyloxyethyl ester diastereomer mixture 10 mg was dissolved in methanol 2 ml, and 0.5 M phosphorus was added thereto. 5 ml of a potassium acid buffer solution (pH 8.0) was added, 3 ml of a bacterial cell suspension of the AS-013 strain described in Example 1 was further added, and the mixture was stirred at 32 ° C. for 2 hours. As a result of analyzing the reaction solution under the following HPLC analysis conditions, the yield of diastereomer 2 was 42.5% (50% × 80%), and the optical purity was 95% e. e. Obtained in.

HPLC analysis conditions; Column: Inertosyl ODS-2 4.6 × 150 mm
(Manufactured by GL Sciences Inc.) Mobile phase: methanol: water = 45:55 Detection wavelength: 254 nm Flow rate: 1.5 ml / min Retention time: diastereomer 1; 16.4 min, diastereomer 2; 21.8 min

[0069]

Example 5 7β- [2- (2-aminothiazol-4-yl)-
Asymmetric hydrolysis of (Z) -2- (methoxyimino) acetamido] -3-methoxymethyl-3-cephem-4-carboxylic acid isopropoxycarbonyloxyethyl ester diastereomer mixture with monoglyceride lipase 7β- [2- ( 2-aminothiazol-4-yl)-
(Z) -2- (Methoxyimino) acetamide] -3-methoxymethyl-3-cephem-4-carboxylic acid isopropoxycarbonyloxyethyl ester diastereomer mixture (10 mg) was dissolved in methanol (1 ml), and 0.1 M phosphorus was added thereto. 9 ml of potassium acid buffer (pH 7.0) was added, 20 mg of monoglyceride lipase was further added, and the mixture was stirred at 32 ° C. for 2 hours. As a result of analyzing the reaction solution under the HPLC analysis conditions described in Example 4, the yield of diastereomer 1 was 46% (50% × 90%), and the optical purity was 98%.
e. e. Obtained in.

[0070]

[Reference Example 1] Production of esterase MS 125 ml of a culture solution of Sphingomonas sp. AS-013 strain obtained by the same method as described in Example 1 was centrifuged, and the obtained wet cells were treated with 0.05 M phosphate. Buffer solution (pH
7.0) Suspended in 30 ml and sonicated at 5 ° C,
The solid matter was removed to prepare a cell-free enzyme solution. This enzyme solution was passed through a column packed with 100 ml of DEAE-Sepharose CL-6B (Pharmacia) equilibrated with 0.05 M phosphate buffer (pH 7.0) in advance, and Example 1
It was confirmed that the enzyme activity was adsorbed by measuring using the substrate described in 1. As an eluent, a 0.05M phosphate buffer solution (pH 7.0) in an amount 6 times the column volume was used, and elution was carried out by a salt gradient elution method in which the salt concentration was linearly increased to a final concentration of 0.5M. as a result,
The desired enzyme activity was eluted at a salt concentration of around 0.42M. This active site fraction was collected, desalted, and lyophilized to obtain esterase MS.

[0071]

Example 6 3-carbamoyloxymethyl-7-[(Z) -2-
(2-Furyl) -2-methoxyiminoacetamide] -3
-Cephem-4-carboxylic acid acetoxyethyl ester diastereomeric mixture asymmetric hydrolysis by esterase MS 3-carbamoyloxymethyl-7-[(Z) -2-
(2-Furyl) -2-methoxyiminoacetamide] -3
-Cephem-4-carboxylic acid acetoxyethyl ester diastereomer mixture 10 mg was dissolved in methanol 1 ml, and 0.1 M potassium phosphate buffer solution (pH was added thereto).
7.0) 8.5 ml was added, and 0.5 ml of the enzyme solution of esterase MS obtained in Reference Example 1 was further added, and the mixture was stirred at 32 ° C. for 3 times.
Stir for 0 minutes. As a result of analyzing the reaction solution under the following HPLC analysis conditions, the yield of diastereomer 2 was 48% (50%
X 96%), optical purity 99% e. e. Obtained in.

HPLC analysis conditions; Column: Inertosyl ODS-2 4.6 × 150 mm
(GL Science) Mobile phase: Methanol: 0.2 M ammonium dihydrogen phosphate aqueous solution = 37.5: 62.5 Detection wavelength: 254 nm Flow rate: 1.5 ml / min Retention time: diastereomer 1; 18.8 Min, diastereomer 2; 22.3 min

Claims (1)

[Claims] 1. A compound represented by the general formula (2): (In the formula, R ¹ is a hydrogen atom or an acyl group, R ² is a lower alkyl group, R ³ is a lower alkyl group, a cycloalkyl group,
An aralkyl group or a phenyl group which may have a substituent, X represents an oxygen atom or a single bond, and Y represents a group which does not participate in the reaction) An enzyme having the ability to asymmetrically hydrolyze a diastereomeric mixture of 4-position esters of a compound represented by the general formula (2) or an inclusion thereof is allowed to act in an aqueous medium. Chemical 2] (In the formula, * C represents an asymmetric carbon atom, and R ¹ , R ² ,
R ³ , X and Y have the same meanings as described above), and a method for producing a cephalosporin derivative having an optically active side chain ester represented by the above formula.