JPS6338195B2 - - Google Patents

Description

[Detailed Description of the Invention] The present invention relates to the general formula () (In the formula, R ₁ is hydrogen, lower alkyl group, hydroxyl group,
Represents a lower alkoxyl group or lower acyloxy group, R ₂ represents hydrogen or a carboxylic acid protecting group, and the hydrogens at the 6th and 7th positions are in a cis relationship)
The present invention relates to a method for producing an optically active cephalosporin analog represented by and salts thereof.

The present inventors previously demonstrated that 4
We succeeded in synthesizing compounds having various substituents at the 5-position, 5-position, or 3-position. Those compounds were patented in 1972.
-34696 (Japanese Unexamined Patent Application Publication No. 128591/1983), 53-
No. 122403 (Japanese Unexamined Patent Publication No. 55-49376), 53-133072
(Japanese Patent Publication No. 55-59186), Japanese Patent Application Publication No. 53-162005, Japanese Patent Application Publication No. 55-87788), and Japanese Patent Application No. 54-8408 (Japanese Patent Publication No. 59-36916). has been done. Furthermore, the present inventors also succeeded in synthesizing an acyl compound having a novel carbacephem nucleus. These acyl forms are novel antibiotics with strong antibacterial effects, and they are also disclosed in Japanese Patent Application Nos. 53-34696 (1985-128591) and 53-122402 (1988-1983). Publication No. 49375), No. 53-127027 (Japanese Patent Application Laid-open No. 1983-
Publication No. 53290), No. 53-133071 (Japanese Unexamined Patent Publication No. 55-59185)
No. 53-162006 (Japanese Patent Publication No. 55-87789), No. 53-162007 (Japanese Patent Publication No. 59-36915),
No. 53-162008 (Special Publication No. 60-58920), 54-
Disclosed in each specification such as No. 8409 (Japanese Patent Publication No. 59-36917), No. 54-92035 (Japanese Patent Publication No. 56-16491), No. 54-116720 (Japanese Patent Publication No. 56-40683), etc. ing.

However, since the carbacefem nuclei described above are produced by synthetic methods using optically inactive raw materials, all of them are optically inactive DL.
body [or expressed as (±) body].

That is, compounds corresponding to the general formula () in which the hydrogens at the 6th and 7th positions are in a cis relationship are the formula (-1) and the formula (-2), which are in an enantiomeric relationship with each other. [In formulas (-1) and (-2), R ₁ and R ₂ have the same meanings as above. ) is present as a mixture of equal amounts of compounds.

The present inventors succeeded in separating and preparing one of the optically active enantiomers of the compounds represented by the general formulas () and () [described below]. The compound () is obtained by optically selectively deacylating the compound represented by the general formula and [described later] in the specification.
and a method for obtaining the compound (), respectively.

(In the formula, R ₃ represents hydrogen or a lower alkyl group, R ₄ has the same meaning as R ₂ , and has the essential structure (6R, 7S).) (In the formula, R ₅ represents hydrogen, a lower alkyl group or a lower acyl group, R ₆ represents the same meaning as R ₂ , (6R,
7S) has an absolute structure. ) (In the formula, R is a substituted or unsubstituted unsaturated carbon 6-membered ring,
A 5-membered heterocycle, X represents hydrogen, an amino group, a hydroxyl group, or a lower alkyl group, and R ₃ and R ₄ have the same meanings as above. ) (In _the formula, R _, (In the formula, R ₁ represents hydrogen, a lower alkyl group, a hydroxyl group, a lower alkoxyl group, or a lower acyloxy group, and R ₂ represents hydrogen or an ester residue,
We have discovered a method to obtain an optically active compound () by optically selectively deacylating a compound represented by (the hydrogens at the 6 and 7 positions are in a cis relationship).

The optically active substance obtained by the present invention has various physical properties as described below, and the fact that its acyl form exhibits stronger antibacterial activity than the corresponding optically inactive form (dl form), and the relationship between the absolute structure and activity of cephalosporins. From this knowledge, it is considered to have the absolute structure of the above general formula (-1). Therefore, the following description uses the general formula (
-1) will be explained using the structural formula. The compounds of Examples are also named as having this presumed absolute structural formula.

The present invention will be explained in detail below.

The raw material compound in the present invention is a compound represented by the general formula (). In the general formula ()
The lower alkyl group represented by R ₁ has 1-5 carbon atoms.
a chain or branched alkyl group, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t
-Butyl group etc. are exemplified. The lower alkoxyl group is represented by _-OR7 , where _R7 is a lower alkyl group having the same definition as _R1 . Further, the lower acyloxy group is represented by _-OCOR8 , and _R8 is also a lower alkyl group with the same definition as _R1 .

The ester group represented by COOR ₂ in the general formula () is a group that is used in the chemistry of penicillins and cephalosporins and is easily derived into COOH.

R ₂ is a straight chain or branched alkyl group having 1 to 5 carbon atoms (e.g. methyl, ethyl, n-
propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl group, etc.), straight chain or branched lower alkoxymethyl group having 1 to 5 carbon atoms (e.g. methoxymethyl, ethoxymethyl group, etc.), carbon number 1 -5 linear or branched halogenated alkyl group (e.g. chloromethyl,
2,2,2-trichloroethyl, 2,2,2-trifluoroethyl, etc.), lower alkylsulfonylethyl groups (for example, methylsulfonylethyl,
(ethylsulfonylethyl group, etc.), carbon number 7-
12 arylmethyl groups (e.g., benzyl, diphenylmethyl, trityl, triphenylmethyl groups, etc.), substituted arylmethyl groups having 7 to 20 carbon atoms (examples include methoxy groups, nitro groups, etc.), and substitution on phenyl groups. base is 1-5) or general formula () (In the formula, R ₉ represents a straight chain or branched lower alkyl group having 1 to 5 carbon atoms, a straight chain or branched lower alkoxy group having 1 to 5 carbon atoms, or a phenyl group, and R ₁₀ represents hydrogen or a straight chain or branched lower alkyl group having 1 to 5 carbon atoms. Examples include ester residues represented by -5 (representing a straight chain or branched lower alkyl group).

The compound represented by the general formula () is JP-A-55-
It can be produced by the method disclosed in Japanese Patent No. 53290. For example, the compound represented by the general formula () can be easily obtained by phenoxyacetylating the optically inactive compound represented by the general formula () described in publications such as JP-A-54-128591 and 55-49376. is synthesized into This reaction can be carried out by methods commonly used in penicillin and cephalosporin chemistry. That is, it is produced by reacting an optically inactive substance represented by the general formula () with phenoxyacetic acid or a reactive derivative thereof, such as an acid halide or an acid anhydride (including mixed acetic anhydride), if necessary in the presence of a base. be able to.

When phenoxyacetic acid is used, it is preferable to add a carbodiimide such as dicyclohexylcarbodiimide to the reaction system.

_The optically active form _or estimated absolute structural formula (− The compound represented by 1), ie, the target compound of the present invention, is a compound represented by general formula () [hereinafter abbreviated as compound ()]. In addition, the following general formula (), general formula ()...
The compounds represented by are abbreviated as compound (), compound (), ...... ] is produced by optically selective deacylation of

An optically selective deacylation reaction of a compound () to an optically active compound () is carried out using a microorganism having the ability to optically deacylate a compound () to produce an optically active compound (); This can be carried out using a cultured product of the microorganism and/or an enzyme produced by the microorganism.

Examples of microorganisms capable of carrying out the above-mentioned optical selective deacylation reaction include microorganisms belonging to the genus Nocardia, Nieurospora, Talaromyces, and Acinetobacter. Specific examples of bacterial strains include the following.

Nocardia globella ATCC21022 Nieurospora sitophylla IFO 4596 Talaromyces luteus IFO 6896 Acinetobacter calcoaceticus
ATCC21288 In addition to using enzymes produced by the above-mentioned microorganisms, for optical selective deacylation, a culture of the above-mentioned microorganisms or a culture-treated product, isolated bacterial bodies [separated from the culture solution by centrifugation, etc. Furthermore, the bacterial cells are soaked in saline solution (usually about 1
%), may be used after washing with buffer, etc.],
or isolated bacterial cell suspension; bacterial cell disruption solution [a liquid obtained by mechanically or chemically disrupting the bacterial cells obtained above]; bacterial cell disruption solution [the bacterial cell residue obtained from the bacterial cell disruption solution obtained above] Purified enzyme solution [After adding ammonium sulfate etc. to the enzyme solution obtained above, preferably the supernatant solution to precipitate the enzyme protein, this is subjected to gel filtration method, ion exchange cellulose column chromatography, ion exchange cellulose column chromatography, etc. An enzyme solution purified by means such as Adex column chromatography] can also be used in the same manner. Furthermore, an immobilized enzyme obtained by immobilizing bacterial cells or an extracted and purified enzyme by a conventional method can also be used.

The reaction is carried out in an inert solvent that does not affect the reaction.
C., preferably 20-45.degree. C., and pH 4-10. The most preferred solvent is water, but organic solvents (eg, acetone, methanol, ethanol, N,N-dimethylformamide, dimethyl sulfoxide, etc.) can also be used to dissolve the cephalosporin analog substrate. In order to control the PH during the reaction,
It is also effective to add phosphate buffers, veronal buffers, and citrate buffers. The reaction time depends on the type of enzyme, concentration, type of substrate, reaction temperature, and reaction time.
It usually takes 30 minutes to 24 hours, although it depends on pH etc. Most preferably, the reaction is terminated when the reaction yield reaches a maximum value.

The bacterial cell concentration used is dry weight, 1 ml of reaction solution
1 mg to 50 mg per serving is preferred. When a purified enzyme is used, its activity is used in an amount corresponding to the amount of dry bacterial cells. The substrate concentration used is 0.5 mg to 50 mg of compound () per ml of reaction solution.
mg is preferred.

In addition, if the bacterial cells used contain enzyme activities that interfere with this reaction, such as β-lactamase or esterase, the bacterial cells are subjected to mutation treatment to eliminate this enzyme activity, and then the bacterial cells are removed. This bacterial cell can also be used. Furthermore, the reaction yield can be improved by adding an inhibitor of these interfering enzymes to the reaction system.

After completion of the reaction, the target product is generally isolated by adsorption using various carriers, ion exchange chromatography,
Alternatively, gel filtration, liquid-liquid extraction, etc. may be used.

Furthermore, among the compounds represented by the general formula (), the general formula (-3) (In the formula, R ₁ represents the same meaning as above, R′ ₂ represents a carboxylic acid protecting group, and the hydrogens at the 6th and 7th positions are in a cis relationship). General formula (-4) obtained as follows (In the formula, R ₁ represents the same meaning as above, and the hydrogens at the 6th and 7th positions are in a cis relationship.) It can also be obtained by esterifying an optically active form of a cephalosporin analog represented by It will be done.

The optically active compound of the present invention, compound (), is itself a useful compound that can be expected to have antibacterial activity, but the compound obtained by acylating the optically active compound () has the effect of acylating the corresponding inactive compound (). It is a useful compound that exhibits extremely strong antibacterial activity compared to other compounds.

Examples of these compounds and their antibacterial activity are shown in the Examples.

Examples of the salt of the compound () of the present invention include hydrochloride, sulfate, phosphate, formate, malate, sodium salt, potassium salt, calcium salt, and organic amine salt.

Examples are shown below.

Example 1 (6R,7S)-7-amino-1-azabicyclo[4,2,0]oct-2-en-8-one-
Production of 2-carboxylic acid 1-1 Preparation of bacterial cell suspension Acinetobacter calcoaceticus as a seed culture
ATCC21288 [Mycological description is Bergey's Manual
of Determinative Bacteriology 8th Edition
p.436-438 (1974)].

As a seed medium, a 5N-aqueous solution containing 1% polypeptone, 1% yeast extract, 0.5% meat extract, 0.5% monosodium glutamate, and 0.25% salt was used as a seed medium.
A solution adjusted to pH 7.0 with NaOH was used. A loopful of seed culture 1 was inoculated into 10 ml of seed medium in a 50 ml wide test tube, and cultured with shaking at 28°C for 24 hours. The entire amount of this seed medium was inoculated into 300 ml of the main medium placed in the pleated Erlenmeyer flask No. 2.
Culture with shaking at 28℃. The fermentation medium used had the same composition as the seed medium. After 24 hours of the main culture, the bacterial cells are removed from the culture medium by centrifugation and washed twice with 50 ml of 0.9% saline. The cells are suspended in 1/30M phosphate buffer (PH7.5). The bacterial cell concentration was 40 mg/ml, and the bacterial weight was calculated in terms of dry bacterial cells.

1-2 Preparation of substrate (a) Production of (±)-cis-7β-phenoxyacetamide-1-azabicyclo[4,2,0]oct-2-en-8-one-2-carboxylic acid sodium salt (Here, cis refers to the stereochemistry of hydrogen at the 6th and 7th positions. The same applies below.) JP-A-55-49376, (±)-cis-7β-amino-1-azabicyclo[4,2,0]-oct-2-en-8-one-2-carvone obtained in the same manner as Example 10 Dissolve 300 mg (1.01 mmole) of the trifluoroacetate of the acid in 5 ml of 50% tetrahydrofuran-water and add 720 μ (5.0 mmole) of triethylamine. Add phenoxyacetyl chloride to this while stirring on ice.
Add 206 μ (1.5 mmole). After stirring for another hour at the same temperature, 0.5N-hydrochloric acid was added to adjust the pH to 3-4, and the mixture was diluted with 20 ml of ethyl acetate.
Extract times. The ethyl acetate solution was washed with brine, dried with mirabilite, and concentrated under reduced pressure to obtain 600 mg of powder. Dissolve this in 3 ml of saturated sodium bicarbonate aqueous solution, and add Diaion HP-10 100
Charge the column packed with ml. Elute with water and concentrate the eluate under reduced pressure to 160 mg (47%)
Obtained the objective.

IR (KBr) νcm ^-1 max: 3330, 1780, 1545,
1240, 760 Pmr (CDCl ₃ + CD ₃ OD) δ (ppm): 7.6 ~ 6.6
(5H, m) 6.11 (1H, t, J = 4.4Hz) 5.37
(1H, d, J=5.0Hz) 4.54 (2H, s) 2.6
~1.1 (4H, m) (b) 40 mg of the compound obtained in (a) was taken and dissolved in 1 ml of M/30 phosphate buffer, pH 7.5, to prepare a substrate solution.

1-3 Enzyme reaction and isolation/purification of target product Add 1 ml of the above bacterial cell suspension to 1 ml of substrate solution,
Perform the enzymatic reaction at 25°C for 20 hours. Cells are removed by centrifugation, and the supernatant is adjusted to pH 3.0 with 2N hydrochloric acid. Next, the reaction solution was poured into a column (column diameter 2.14 cm, height
27cm). When eluted with deionized water, the target product was eluted in a fraction of 87 ml to 153 ml. After concentrating this under reduced pressure, it was freeze-dried and a small amount of water-methanol (50:50, volume ratio,
A column (column diameter: 0.9 cm,
The sample was charged to a height of 70 cm) and elution was performed with a water-methanol (50:50) mixture. The target product was eluted in a fraction of 24 ml to 30 ml. This fraction was concentrated under reduced pressure to remove methanol, and the residual liquid was then freeze-dried to obtain 8 mg of white powder.
The physical properties of this product are as follows.

IR (KBr) νcm ^-1 max: 1800, 1790, 1775, 1640,
1620 NMR (100M, _D2O −DSS) δ (ppm): 6.44
(1H, dd, J = 3.5, 4.7Hz), 4.86 (1H, d,
J = 5.2) 4.04 (1H, m) 2.5 to 1.5 (4H, m) Elemental analysis revealed that this compound had one molecule each of hydrochloric acid and water. Optical rotation is [α] ²⁰ ° _D
= +47.6° (c = 0.11 PH7.0 in 1M phosphate buffer). This value is the optical rotation of the optically active substance determined by Reference Example 1 described later: [α] ¹⁵ ° _D = +48.5° (c
= 0.5 in 1M phosphate buffer, pH 7.0).

Example 2 (4S, 6R, 7S)-7-amino-4-hydroxy-1-azabicyclo[4,2,0]octo-2
Production of -en-8-one-2-carboxylic acid 2-1 Preparation of bacterial cell suspension The same procedure as in 1-1 was carried out.

2-2 Preparation of substrate (a) (±)-cis-7β-(phenoxyacetamide)-4α-hydroxy-1-azabicyclo[4,2,0]oct-2-en-8-one-2- Production method of carboxylic acid (±)-cis-7β-amino-4α-hydroxy-1-azabicyclo[4,2,0]oct-2-en-8-one- obtained in the same manner as in Example 22 of JP-A-54-128591 Trifluoroacetate of 2-carboxylic acid 404 mg (1.29 mmole)
Dissolve in 4 ml of water and 8 ml of acetone. Add sodium bicarbonate under ice-cooling and stirring to adjust the pH to 9, then add 0.196 ml of phenoxyacetyl chloride.
(1.42 mmole) and stirred at 0°C for 1 hour. After the reaction, acetone is removed by concentration under reduced pressure, and 1N hydrochloric acid is added to adjust the pH to 2. After saturating with salt, extract 6 times with 20 ml of ethyl acetate. The extract is washed with saturated saline and dried over anhydrous sodium sulfate. After removing the sodium sulfate, the solution is concentrated under reduced pressure. Add this to methanol 6
ml and add ethyl ether and n-hexane. The precipitate was collected by centrifugation and vacuum dried to yield 83.8mg (0.252mg) of the target substance.
mmole, 20%). The physical properties of this product are as follows: IR (KBr) νcm ^-1 max: 3400, 1770, 1730, 1680 NMR (D ₂ O) δ (ppm): 7.48−6.95 (5H, m)
6.09 (1H, d, J = 5.4Hz) 5.50 (1H, d,
J = 4.9Hz) 4.67 (2H, s) 4.6−4.4 (1H,
m) 4.1-3.8 (1H, m) 1.9-1.2 (2H, m) (b) Take 80 mg of the compound obtained as in (a) above, and add M/15 phosphate buffer (PH7.5) to this. )1
Add ml. Add a small amount of 2NKOH aqueous solution to adjust the pH.
When the pH was adjusted to 7.5, the compound dissolved. Add deionized water to this solution to make 2 ml,
This was used as a substrate solution.

2-3 Enzyme reaction and isolation/purification of target product Add 2 ml of the above bacterial cell suspension to 2 ml of substrate solution.
Carry out the reaction for 20 hours at °C. The bacterial cells were removed by centrifugation, and 2N-hydrochloric acid was added to the supernatant to adjust the pH to 3.5. This is Diaion HP-20AG 100~
200 meshes (column diameter 0.88cm, height 70cm) 43
ml was charged into a column. When eluted with deionized water, the target product was eluted in a fraction of 35 ml to 43 ml. collect this fraction
After adjusting the pH to 7.0 by adding 2N-KOH, it was charged into a column (diameter 2.0 cm, height 9.5 cm) filled with 30 ml of Diaion WA-30-S (manufactured by Mitsubishi Kasei Corporation). The weakly basic anion exchange resin Diamond WA-30-S used here is 2N in advance.
- Pour 60ml of acetic acid aqueous solution, then deionized water
The acetic acid type was used by passing 60 ml of liquid through it. After passing 40 ml of deionized water, a 0.5N acetic acid aqueous solution was passed, and the target product was eluted in a fraction of 52 ml to 80 ml after passing 0.5 N acetic acid. Combine these and freeze-dry to give 18 mg of pale yellow powder.
I got it. The physical properties of this product were as follows, and it was identified as the acetate salt of the listed compound.

IR (KBr) νcm ^-1 max: 3400, 1805, 1760, 1745
(sh), 1600, 1560 (sh), 1410 NMR (100M D ₂ O−DSS) δ (ppm): 6.16
(1H, d, J = 5.4Hz), 4.89 (1H, d, J =
5.4Hz), 4.57 (m, overlaps with H ₂ O signal)
4.08 (1H, m) 2.21 (1H, m) 1.97 (3H, s)
1.78 (1H, m) Optical rotation [α] ²⁰ ° _D = -61.8° (c = 0.18, 1M phosphate buffer PH7.0) Reference example 1 (+)-7β-amino-1-azabicyclo[4,
2,0]-oct-2-ene-8-one-2-
Method for producing carboxylic acid (alternative method) 1-1 Preparation of disrupted bacterial cell solution (a) Cultivation of microorganisms having optical selective deacylation ability Kluyvera citrophila as a seed culture
ATCC21285 [Mycological publication is J.General
Applied Microbiology 3 , 28-31 (1957)
Reference] is used.

As a seed medium, an aqueous solution containing 1% polypeptone, 1% yeast extract, 0.5% meat extract, 0.5% monosodium glutamate, and 0.25% salt was used.
A solution adjusted to pH 7.0 with 5N-NaOH was used. Place 1 platinum loopful of the inoculum into a 50ml thick test tube.
ml of seed medium was inoculated and cultured with shaking at 30°C for 24 hours. The entire volume of this seed medium was placed in a pleated Erlenmeyer flask as shown in No. 2.
Inoculate into 300ml and culture with shaking at 30℃.

The fermentation medium used had the same composition as the seed medium.

(b) Preparation of disrupted bacterial cell solution After 24 hours of the above main culture, the bacterial cells were removed from the fermentation solution by centrifugation, and 50 ml of 0.9% saline was added.
Wash twice with The cells are suspended in 1/30M phosphate buffer. Bacterial cell concentration is 40mg/ml
The bacterial weight is calculated in terms of dry bacterial cells. 10 ml of the above suspension was placed in a 50 ml large test tube, and the cells were crushed by ultrasonication at 200 watts for 2 minutes to prepare a cell suspension. For this process, an ultrasonic generator model manufactured by Tomy Seiko Co., Ltd.
UR200P was used.

1-2 Preparation of substrate solution (+)-cis-7β-[(R)-2-phenyl-2-aminoacetamide]-1-azabicyclo[4,2,0] prepared in the same manner as Reference Example 2 described later
Oct-2-en-8-one-2-carboxylic acid
Dissolve 100 mg in 5 ml of 1/30M phosphate buffer (PH6.5).

1-3 Enzyme reaction Add 5 ml of the above bacterial cell disruption solution to 5 ml of the above substrate solution, and perform an enzyme reaction at 30°C for 24 hours.

1-4 Isolation/purification The same operation as 1-4 of Example 1 was carried out to obtain 46ft of white powder.

The physical properties of this product agree well with those of the compound obtained in Example 1.

[α] ¹⁵ ° _D = +48.5°, [c = 0.5, 1M phosphate buffer (PH7.0)] Reference example 2 (+) and (-)-cis-7-[(R)-2-phenyl -2-aminoacetamide]-1-azabicyclo[4,2,0]oct-2-ene-8
-Production method of -one-2-carboxylic acid This compound is produced in the following two steps.

(2-1)(±)-cis-7-[(R)-2-phenyl-2-t-butyloxycarbonylaminoacetamide]-2-t-butyloxycarbonyl-1-azabicyclo[4,2,0 ]Octo-
Method for producing 2-en-8-one (Method a) (±)-cis-7-amino-2-t- obtained in the same manner as in Example 11 of JP-A-55-49376
Butyloxycarbonyl-1-azabicyclo[4,2,0]oct-2-en-8-one
81 mg (0.34 mmole) and (R)-Nt-butyloxycarbonylphenylglycine 94.0 mg
Dissolve (0.34 mmole) in 2 ml of anhydrous methylene chloride and cool with ice-salt to yield 77 mg (0.34 mmole).
Dicyclohexycarbodiimide is dissolved in 1 ml of anhydrous methylene chloride and added. Ice cooling 2
After reacting for an hour, 2 drops of acetic acid were added, stirred for an additional 20 minutes, and filtered under reduced pressure. Wash the cake with 2.0 ml of ethyl acetate, filter and add 20 ml of ether to the washings.
ml, washed with 1% phosphoric acid aqueous solution and saturated saline in that order, dried with sodium sulfate, and concentrated under reduced pressure to obtain 187 mg.
of crude product is obtained. This was purified by silica gel chromatography (9 g of silica gel, solvent n-hexane: ethyl acetate 1:1) to obtain 104 mg (64.9%) of the desired product as a colorless glass.

IR (KBr) νmax (cm ^-1 ): 1770, 1750, 1720,
1630 NMR: δ (CDCl ₃ ) 7.32 (s, 5H), 6.31 (m,
1H), 5.90 (m, 1H), 2.50-1.70 (m,
4H), 1.50 (s, 9H), 1.40 (s, 9H) (Method b) Dissolve 297.3 mg (1.18 mmole) of (R)-Nt-butyloxycarbonylphenylglycine in 5 ml of anhydrous tetrahydrofuran -30
Add 1.18 ml (1.18 mmole) of 1N-N methylmorpholine-tetrahydrofuran and 1.18 ml (1.18 mmole) of 1N-isobutyl chloroformate at °C.
Stir for 30 minutes. (±)-cis-7-amino-2
-t-butyloxycarbonyl-1-azabicyclo[4,2,0]oct-2-ene-8
234 mg (0.983 mmole) of -one was dissolved in 5 ml of anhydrous methylene chloride and reacted for 45 minutes at -30°C, and further reacted for 4 hours and 15 minutes at 0°C. Diluted with methylene chloride (15 ml), water, 1N HCl,
Washed with water and saturated saline in that order, dried with Glauber's salt and concentrated to obtain 588 mg of crude acyl compound. Purified with silica gel chromatography (28 g of silica gel) according to (method a) to obtain 322 mg of the target product in the form of a colorless glass.
(69.4%) obtained. The IR and NMR spectra of this product matched those obtained by (method a).

(2-2)(±)-cis-7-[(R)-2-phenyl-2-aminoacetamide]-1-azabicyclo[4,2,0]oct-2-en-8-one-2- Method for producing carboxylic acid trifluoroacetate.

(±)-cis-7- obtained in Reference Example 2-1
[(R)-2-phenyl-2-t-butyloxycarbonylaminoacetamide]-2-t-butyloxycarbonyl-1-azabicyclo[4,2,0]oct-2-en-8-one 280
mg (0.59 mmole) in 2.5 ml anhydrous methylene chloride,
Dissolved in 2.5 ml of anisole, added 5.0 ml of trifluoroacetic acid under ice cooling, left under ice cooling for 4 hours and 50 minutes, concentrated, added 10 ml of ether to the residue, stirred at room temperature for 1 hour, collected the resulting precipitate, and obtained a pale yellow powder. the object
202 mg (70.9%) was obtained.

IR (KBr) νmax (cm ^-1 ) 1765, 1680, 1630 NMR: δ (D ₂ O, using DSS as internal standard) 7.51 (d, 5H), 6.31 (m, 1H), 4.95
(d, 1H), 3.8-3.5 (m, 1H), 2.6-2.9 (m,
4H) (±)-cis-7-[(R)-2-phenyl-2-
Aminoacetamide]-1-azabicyclo[4,
2,0] Separation of diastereoisomers of oct-2-en-8-one-2-carboxylic acid 50 mg of the compound obtained above was dissolved in 150μ of water, and Bondapak C-18 (manufactured by Waters) was used as a carrier. %-methanol-0.2N dipotassium phosphate solvent 8 times. 254n
Two fractions are collected by absorption analysis at m, and after removing methanol under reduced pressure, each fraction is lyophilized. Dissolve in water again and use Diaion HP-10 (manufactured by Mitsubishi Chemical Corporation)
After adsorbing on a 20ml column and washing with 200ml of water,
Elution was carried out with 20% ethanol water, and fractions colored by ninhydrin were collected and lyophilized to obtain white powders containing 14.0 mg of A isomer and 24.6 mg of B isomer, respectively.

A: 14 mg The fraction eluted first in high performance liquid chromatography, namely (-)-cis-7α-[(R)-2-phenyl-2-aminoacetamide]-1-azabicyclo[4,2,0]octo-2 -en-8-one-2-carboxylic acid [α] ²²゜_D (H ₂ O, c=0.5) -74.2゜IR (KBr) νcm ^-1 max: 1750, 1690, 1640 PMR (D ₂ O) δ :7.51 (5H, s), 6.15 (1H, t,
J = 3.9Hz), 5.20 (1H, d, J = 4.9Hz), 5.19
(1H, s), 3.88 (1H, octet, J=8.6, 3.7,
4.9Hz), 2.41-1.41 (4H, m) B: 24.6mg The fraction that comes out later in high performance liquid chromatography, namely (+)-cis-7β-[(R)-2-phenyl-2-aminoacetamide]- 1-Azabicyclo[4,2,0]oct-2-en-8-one-2-carboxylic acid [α] ²² ° _D (H ₂ O, c=0.5) + 57.2°IR (KBr) νcm ^-1 max: 1760, 1690, 1640 PMR (D ₂ O) δ: 7.51 (5H, s), 6.08 (1H, t,
J = 4.2Hz), 5.41 (1H, d, J = 4.9Hz), 3.83
(1H, octet, J=8.6, 3.7, 4.9Hz), 2.28~1.01
(4H, m)

Claims (1)

[Claims] 1 General formula () (In the formula, R ₁ represents hydrogen or a hydroxyl group, R ₂ represents hydrogen or a carboxylic acid protecting group, 6,
The hydrogen at the 7th position is in a cis relationship) is optically selectively deacylated to form the general formula () A microorganism belonging to the genus Acinetobacter and/or a cultured product of the microorganism having the ability to produce an optically active cephalosporin analog represented by the formula (wherein R ₁ and R ₂ have the same meanings as above) is represented by the general formula (). General formula () characterized in that an optically active cephalosporin analog represented by general formula () is produced by acting on a compound
A method for producing an optically active cephalosporin analog represented by