JPS6111600B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION This invention relates to the conversion of cephalosporin compounds, and more particularly to the enzymatic hydrolysis of 3-acyloxymethyl cephalosporins. The cephalosporin compound herein is generally referred to as "cephalosporin" (journal.
of American Chemical Society,
1962, 84, 3400). The name "Cefm" is one 2
It means a cephalic structure with double bonds. Due to the chemical reactivity of its hydroxy group, 3-hydroxymethyl cephalosporin compounds are
It is therefore a valuable intermediate in the synthesis of cephalosporin antibiotics having a substituted methyl group in the 3-position due to the ease of converting the hydroxy group to the desired 3-(substituted methyl) group. Further 7-
Acylamide-3-hydroxymethylcef-3-
Em-4-carboxylic acid has antibiotic properties. Therefore, 3-acyloxymethyl cephalosporins, especially cephalosporin C [(6R/7R)
-3-acetoxymethyl-7-(D-5-amino-5-carboxypentanamide) cef-3-em-4-carboxylic acid] and its derivatives, such as N-coated derivatives, which are produced by fermentation in nature. and D-5-amino-5 by hydrolysis of 3-acetoxymethyl cephalosporin compounds.
- Preparation of 3-hydroxymethyl cephalosporin compounds by hydrolysis of 3-acetoxymethyl cephalosporin compounds, such as compounds in which the carboxypentanoyl group has been converted or removed and optionally substituted with other acyl groups. Very important. 3-acyloxymethyl cef- by chemical method
Hydrolysis of 3-em-4-carboxylic acid to 3-hydroxymethyl analogues results in rapid and irreversible lactonization by reaction of 3-hydroxymethyl with 4-carboxylic groups, and or β-lactam ring formation. It has been found to be generally impractical as it involves decomposition of the system. However, enzyme-catalyzed methods allow 3-acyloxymethyl cef-3 to be synthesized under conditions that substantially or completely avoid lactonization and β-lactam degradation.
It has been found that it is possible to hydrolyze -Em-4-carboxylic acid. Although esterases derived from a range of materials, such as plant materials, can be used in enzyme-catalyzed methods, there are practical difficulties in isolating sufficient quantities of esterases from the materials. Esterases obtained from microorganisms are most advantageous in practice because of the relative ease of culturing microorganisms on a large scale using standard fermentation techniques to rapidly provide esterases. The present invention provides that 3 esterases obtained from certain microorganisms belonging to Basidiomycetes
-Acyloxymethyl cef-3-em-4-carboxylic acids advantageously accelerate the hydrolysis of the corresponding 3-hydroxymethyl analogs. Therefore, the present invention provides 3-acyloxymethylceph-
This invention relates to a method for converting 3-m-4-carboxylic acid into a 3-hydroxymethyl analog by hydrolysis.
The method of the present invention is a hydrolysis method characterized in that hydrolysis is catalyzed with an esterase produced by culturing a microorganism belonging to the basidiomycete, which has the ability to produce the required esterase activity. The 3-acyloxymethylcef-3-em-4-carboxylic acid hydrolyzed according to the present invention has the general formula Compounds represented by: However, during the ceremony
R ¹ is an amino group or a protected amino group (e.g.
_R2 is hydrogen or an alkoxy group having 8 or less carbon atoms, ^R3 - ^CO is a _C2-20 carboxylic acyl group, and B is a sulfur atom. It is. Acylamide group R ¹ present in the compound of formula ()
includes the following: (a) D-5-amino- found in naturally occurring fermentation produced compounds such as cephalosporin C
5-carboxypentanamide group: (b) N-protected derivative of D-5-amino-5-carboxypentanamide group [for example, in which the amino group is
1302015 or 1313207, i.e. lower alkyl groups, allyl lower alkyl groups, allyl groups (for example 2,4 dinitrophenyl) or acyl groups, especially lower alkanoyl groups ( for example acetyl, propionyl or butyryl groups), α-halo- or α·α-dihalo-lower alkanoyl groups (for example chloroacetyl or dichloroacetyl groups), aroyl groups (for example benzoyl, chlorobenzoyl, nitrobenzoyl or tosyl groups), (c) D-5-amino- acylamido groups obtained by conversion of the 5-carboxypentanamide group (e.g. 4-carboxybutanamide groups obtained therefrom by enzymatic oxidation); (d) formamide; (e) groups of the formula R(CH ₂ ) _o .CONH- [wherein R is a carbocyclic or heterocyclic allyl group (e.g. phenyl group; substituted by one or more halo, hydroxy, lower alkyl, nitro, amino, lower alkanoyl, lower alkoxy or lower alkylthio group)] phenyl group: thienyl group or furyl group) or allyloxy group, allylthio group,
Allyl lower alkoxy group or allyl lower alkylthio group (e.g. phenoxy group, phenylthio group, 5-methyl-1,3,4-thiadiazol-2-ylthio group or benzylthio group)
and n is an integer from 1 to 4]; (F) Formula [wherein R ^a is an allyl group (e.g. phenyl group, naphthyl group, or phenyl substituted by one or more halo, hydroxy, lower alkyl, nitro, amino, lower alkanoyl, lower alkoxy or lower alkylthio group)] a monocyclic or bicyclic carbocyclic allyl group), such as a monocyclic or bicyclic carbocyclic allyl group;
and X is an amino group, a protected amino group (e.g., any of the N-protecting groups described above for the D-5-amino-5-carboxypentanamide group, including e.g. or hydroxy group]; or (g) formula a group in which R ^a has the meaning previously defined (e.g. where R ^a is a phenyl, substituted phenyl, naphthyl, thienyl, furyl or pyridyl group) and R ^b is a hydrogen atom, an acyl group (e.g. a lower alkanoyl), lower alkyl (such as methyl, ethyl, propyl or butyl), cycloalkyl (such as cyclopentyl or cyclohexyl),
-7 carbon atoms), an allyl group (e.g. a carbocyclic allyl group such as a phenyl group) or an allyl lower alkyl group (e.g. a benzyl or phenethyl group). Examples of R ¹ groups included in compounds of formula D-5-
Benzoylamino-5-carboxypentanamide, phenylacetamide, thienylacetamide,
2-Hydroxy-2-phenylacetamide, 2-
Included are t-butoxycarbonylamino-2-phenylacetamide and syn-2-furyl-2-methoxyiminoacetamide. The acyl groups R ³ .CO present in compounds of the formula include various aliphaateic, alarifateic and aromateic groups, including lower alkanoyl groups (e.g. acetyl, propionyl and butyryl groups); lower alkenoyl groups (e.g. crotonoyl group); Included are allyl lower alkanoyl groups (eg, phenylacetyl groups); and aroyl groups (eg, benzoyl groups). As previously indicated, the method of the present invention is particularly useful for the hydrolysis of cephalosporin C and its derivatives, ie, compounds of formula in which R ³ .CO is an acetyl group. R ² and B in the formula preferably represent hydrogen and >S, respectively. The strength of the desired esterase activity produced by a particular microorganism belonging to the basidiomycete can be easily determined in a preliminary small test. The produced 3-hydroxy Determine the amount of methylcef-3-em-4-carboxylic acid. Examples of methods available for yeast include:
The yeast to be tested is, for example, D-glucose (2
%), yeast extract (1%), peptone (1%), potassium dihydrogen phosphate (0.5%) and agar powder (2%).
%) on a conventional nutrient medium such as slant agar, and after primary culture at 25° C. for 2-4 days, transfer from the slant to a small flask using a sterile loop. The contents of this flask include medium (3 ml) containing D-glucose (2.7%), yeast extract (1.3%), oxoid peptone (1.3%), potassium dihydrogen phosphate (0.7%) and cephalosporin C (potassium salt). This is an 8% solution (1 ml) of An uninoculated flask was used as a control and cultured with shaking at 25°C for 3 days, followed by assay using thin layer chromatography. The contents of the flask (1 μ) and standard cephalosporin C and its 3-hydroxymethyl analog (1 μ of 20 mg/ml) are spotted onto a cellulose plate. After buffering with M/15 phosphate buffer, the plates are developed with 70% n-propanol in water. The degree of deacetylation of cephalosporin C is qualitatively assayed by exposing the dried plate to ultraviolet light and comparing spots between the starting material and the 3-hydroxymethyl product obtained from the contents of the flask. The following slightly modified method can be used for microorganisms other than yeast. The agar medium used for the microorganisms tested contains maltose (4%) and peptone (1%).
%), malt extract (2.4%) and agar powder (2.5
%) and adjust the pH to 7.5 before sterilization. After culturing the slants at 25°C for 7 days, each slant was transferred to the first liquid medium (40 ml). This medium contains glucose (2
%), yeast extract (1%), peptone (1%) and potassium dihydrogen phosphate (0.5%), adjusted to pH 5.8. After being cultured with shaking at 25°C for 10 days, the cells are cultured in a second liquid medium for usually 3 to 8 days depending on the degree of growth, and then harvested. The second medium is the same as the first liquid medium and contains cephalosporin C.
(0.1%). Each culture is harvested and mixed vigorously in a mixer to obtain an even suspension. Prepare the reaction mixture and add an equal cell suspension (2 ml), 0.5M
It contains a potassium phosphate buffer (PH6.0, 0; 2 ml) and a cephalosporin C solution (3.33%; 6 ml), and is cultured with shaking at 25°C for 5 days. To test each sample by thin layer chromatography, PH
A plate coated with cellulose buffered to 5.8−6.0 and an aqueous n-propanol solution (70V/V
%) is used. The presence of the 3-hydroxymethyl-cephalosporin product can be confirmed by exposing the plate to ultraviolet light. (6R・7R)-7
-(D-5-amino-5-carboxypentanamide)-3-hydroxymethylcef-3-em-
Elute a portion of the sample equivalent to a standard of 4-carboxylic acid (DAC) and determine the optical density (260 nm).
Determine the amount of DAC by measuring . Microorganisms without esterase activity do not produce spots corresponding to 3-hydroxymethyl compounds. The microorganism of the Basidiomycete family used as the source of the desired esterase activity is Ustila genera.
genales) or the family Sporobolomycetaceae. Yeasts of the order Ustylagenales are microorganisms of the genera Leucosporidium and Rhodosporidium, such as Leucosporidium scottei (L.
scotti), Rhodosporidium, R. toru-loides and Rhodosporidium
Contains strains of R. sphaerocarpum. Yeasts of the family Sporopolomycetaceae include the genera Bullera, Sporidiobolus and Sporobolomyces, such as B.alba, B.tsugae, Sporidiobolus S.johnsonil,
S. ruinenii, s. roseus and S. salmonicol.
salmonicolor). Among microorganisms other than yeasts of the Basidiomycete family, those exhibiting the required esterase activity can also be used. Typical examples of this kind of microorganisms include microorganisms of the order Ustilagenarales, such as smut fungi, such as Ustilago maydis, and microorganisms of the order Agaricales, such as Schizophyllum colmiune.
microorganisms of the order Polyporales, such as Polyporus commune) and Coprininus Comatus;
They are Polyporus dichrous, P.versicolor, and P.monticola. When using microorganisms other than yeast, it is best to choose microorganisms that can be cultured deep. Yeasts of the genus Rhodosporidium, such as Rhodosporidium toruloides CBS14 or
Rhodosporidium toruloides species such as CBS349 are particularly suitable for the method of the invention due to the high titer of the final product. Generally, microorganisms that can be used as a source of esterase activity are placed in a sealed glass tube at a concentration of 2%.
It can be advantageously preserved by freeze-drying as a suspension in skim milk or by subculturing the microorganism on a solid medium, such as yeast extract/peptone/agar medium. Freeze-drying is a good way to preserve cultured yeast, as sterile water can be added to the freeze-dried medium. To culture the resuspended microorganisms, e.g. glucose (2%), yeast extract (1%),
Peptone (1%), potassium dihydrogen phosphate (0.5%)
Agar (2%) (all percentages are W/V)
%) and cultured at 25°C until the surface culture grows and covers the agar, then it can be stored for several months at a low temperature, e.g. 5°C. can do. A sample of the surface culture can also be transferred to a liquid nutrient medium containing carbon and hydrogen sources and small amounts of minerals and cultured in depth, for example at a temperature of 25°C. An example of a suitable nutrient medium for this purpose is the surface culture medium described above, minus the agar. It is sometimes advantageous to preserve liquid cultures for subsequent use in liquid cultures, such as large-scale liquid cultures. In this case, many surface cultures do not have to be replanted for liquid culture, and even larger quantities can be transplanted. After culturing the above-mentioned second liquid culture for an appropriate period of time, for example, about 3 months, practically at a temperature of 25°C, the resulting microbial suspension is used as an esterase source, and 3-acyloxymethylceph-3- M-
The 4-carboxylic acid starting material can be deacylated. However, for example, Leukosporidium
In the case of certain microorganisms, such as S. scotti, lower culture temperatures, such as about 17° C., are even more advantageous. Esterase formation is enhanced in some cases by adding an inducing agent to the liquid medium. Suitable inducers include cephalosporin C, cephalothin [(6R・7R)-3-acetoxymethyl-7-(thien-2-yl)acetamidocef-3-em-4-
carboxylic acids] and their salts (for example, alkali metal salts such as potassium salts). Inducers such as cephalosporin C (potassium salt) are 0.1%
(W/V) or less was found to be effective. The inducing agent is preferably added to the liquid medium after sterilization. The inducing agent can be sterilized by passing through a sterile filter, while the medium can be sterilized, for example, at a temperature of about 121°C.
Can be autoclaved for 15 minutes at a pressure of Kg/ ^m2 . Esterases are used in a variety of different forms to hydrolyze the starting material 3-acyloxymethylcef-3-em-4-carboxylic acid. For example, the liquid culture itself can be used as a source of esterase, if desired after disruption of the bacterial cells by conventional methods such as, for example, sonication or treatment with degrading enzymes. An aqueous extract of the suspension resulting from such destruction of bacterial cells can also be used. Alternatively, it is also possible to use the entire bacterial cells filtered from the liquid culture in the same way as the culture filtrate. If a filtrate is used, all the bacterial cells may be destroyed again, for example, as described above, before filtration. The advantage of using whole cells is that they can be easily separated from a liquid medium, e.g. as a cell suspension, or as a dried or frozen product, which can be added directly to the hydrolysis reaction mixture, for example. After hydrolysis, all bacterial cells can be easily removed from the reaction mixture by, for example, filtration. The bacterial cells thus isolated can be reused, for example, after washing with water, in order to hydrolyze the next starting material, 3-acyloxymethylcef-3-em-4-carboxylic acid.
In this case, sufficient esterase activity is retained to produce cephalosporin hydrolysates of satisfactory quality in consistent yields during several cell reuses. If desired, the cells may be coated with a chemically stable material or matrix (polymer or (membrane) can be used for passivation. For this purpose, it is passivated, for example, by covalent bonding to inorganic or organic polymers, by entrapment in fibers (eg cellulose triacetate) or by encasing in small granules. One suitable material for passivating bacterial cells is polyacrylamide gel. Esterase can also be used in a form that does not contain bacterial cells, for example by precipitating it from the filtrate of a liquid culture or a bacterial cell extract using a suitable protein precipitant such as a salt or a solvent as described above. The precipitated esterase free of bacterial cells is added, for example, directly to the hydrolysis reaction system, or dissolved in water and added as an aqueous solution. Esterases can be prepared in a suitable passive form, such as those described in British Patent No. 1224947 and Belgian Patent No. 782646, for example by entrapment with an inert material or by insolubilization. used in Esterases obtained, for example, from extracts of liquid cultures or by redissolution of precipitated esterases, may be covalently bonded to inert inorganic or organic polymers, or may be covalently bonded to fibers (e.g. cellulose triacetate). fibrous polymers), or with membranes or polymers such as polyacrylamide gels, or adsorbed on ion exchange resins, or in encapsulates such as small particles. be trapped in These types of passivated esterases can be reused in a reusable batch process. That is, it is effectively used to hydrolyze a continuous stream of 3-acyloxymethylceph-3-em-4-carboxylic acid passing through a column containing passivated esterase. The hydrolysis reaction is preferably carried out by combining an esterase with an aqueous medium containing 3-acyloxymethylcef-3-em-4-carboxylic acid or a salt thereof (e.g. an alkali metal salt such as a sodium or potassium salt) and preferably a 0.5- This is done by contacting with a medium containing 10% (W/V) of a cephalosporin compound. Although the medium may be sterilized prior to hydrolysis if desired, we have found that the reaction need not be carried out under sterile conditions and that the hydrolysis can therefore advantageously be carried out under non-sterile conditions. 3-acyloxymethylcef-3-em-4-
When the carboxylic acid is a fermentation-produced cephalosporin, such as in the case of cephalosporin C, the fermentation solution obtained by culturing cephalosporin-producing bacteria (e.g., Cephalosporium spp.) is directly treated with esterase to avoid intermediate separation of the cephalosporin compound. can do. If desired, the mycelia are removed from the culture before processing, but in many cases it is more suitable to use the entire culture directly. Generally, a pH of 4 to 8 is advantageous for hydrolysis.
The PH is maintained at eg PH60 during the reaction by using a buffer such as a phosphate buffer. However, it may be necessary to add a small amount of strong base. The temperature of hydrolysis is preferably room temperature, i.e. 25°C, with stirring and/or aeration of the reaction mixture being beneficial. The amount of esterase or esterase-containing material required for a given reaction can be estimated in advance by small-scale tests. 3-acyloxymethylcef-3-em-4-
The time required for complete deacylation of carboxylic acid is
Depends on starting material, esterase and reaction conditions.
The progress of the reaction can be advantageously followed by product separation by thin layer or paper chromatography using suitable support and solvent system combinations and densitometric assay. Starting material 3-acyloxymethylcef-3-
When the em-4-carboxylic acid has a D-5-amino-5-carboxypentanamide group at the 7-position as in the case of cephalosporin C, the hydrolytic deacylation method of the present invention Can be carried out in combination with enzymatic oxidative deamination,
For example, in the case of 4-carboxybutanamide group, filamentous fungal oxidase, especially British Patent No. 1272769
and Belgian Patent No. 782393 with oxidase from the yeast Trigonopsis variabilis. Enzymatic conversion of the groups at positions 3 and 7 may be carried out either sequentially or simultaneously. The method for isolating the product 3-hydroxymethylcef-3-em-4-carboxylic acid depends on the nature of the product and the reaction system, but generally conventional methods are used. For example, if whole cells are used as the esterase source, they are removed by filtration or centrifugation (optionally reused) and the liquid is further clarified, for example by passing through a bed of diatomaceous earth. When 3-hydroxymethylcef-3-em-4-carboxylic acid is desacetyl cephalosporin C, for example, after adsorption with charcoal powder,
The solution was desalted by water-acetone elution to separate desacetyl cephalosporin C. In addition, anion exchange resins (e.g. acetic acid type Amberlite)
IRA-68), eluted from the resin with potassium acetate solution, and purified by precipitation with acetone. Other methods used to isolate different cephalosporin products include solvent extraction, acid precipitation and isoelectric focusing [(6R·7R)-7-amino-3-hydroxymethylcef-3-em-4-carboxylic acid] There is a case of amphibious ion production such as The invention will be explained in detail by the following examples.
There temperatures are given at 0° C. and the nature and purity of the products were determined by thin layer chromatography, ultraviolet spectroscopy, infrared spectroscopy, magnetic nuclear resonance and liquid high pressure chromatography. The tooth stocks listed in the specification with CBS numbers or IMI numbers are available from the respective depositary institutions. Reference Example 1 Production of (6R・7R)-7-(D-5-amino-5-carboxypentanamide)-3-hydroxymethylcef-3-em-4-carmenic acid (6R・7R)-3-acetoxy Methyl-7-(D
-5-amino-5-carboxypentanamide)
After dissolving the potassium salt of cef-3-m-4-carboxylic acid (4 g) in demineralized water (80 ml),
Cell suspension (20 ml) was treated with 0.5 M potassium phosphate buffer (PH6). The culture (40 ml) of Rhodosporidium toruloides CBS14 from which these cells were obtained was obtained by culturing in the above-mentioned yeast extract/peptone liquid medium for 72 hours, and then the cells were collected by centrifugation. Washed with water.
The resulting mixture was kept at a temperature of 25°C under shaking.
The course of the reaction was monitored by thin layer chromatography, which indicated that the reaction was complete after 42 hours. The reaction mixture was centrifuged to remove solids and the resulting solution was treated with 5 volumes of acetone. The generated precipitate was collected by centrifugation, dried at room temperature under reduced pressure, and the target compound (4.56
g) was obtained. 70% with cellulose coated board
This compound was assayed by thin layer chromatography using a V/V aqueous n-propanol solvent system. Purity approximately 53.5% as determined by comparison with standard samples and densitometer
The total yield was 100%. Example 1 Production of (6R・7R)-7-(D-5-amino-5-carboxypentanamide)-3-hydroxymethylcef-3-M-4-carboxylic acid By culturing Sporidioborus jillonsonii CBS5470 Using the obtained cell suspension and carrying out enzyme-catalyzed deacetylation for 114 hours, the above compound was prepared in the same manner as in Reference Example 1.
4.03g was obtained. The purity of the product was approximately 37.5% and the total yield was 63%. Reference example 2 (6R・7R)-3-hydroxymethyl-7-(thien-2-ylacetamido)cef-3-M-
Production of 4-carboxylic acid (6R・7R)-3-acetoxymethyl-7-(thien-2-ylacetamido)cef-3-m-
The sodium salt of 4-carboxylic acid (4 g) was dissolved in demineralized water (80 ml). Rhodosporidium
The above solution was treated with a cell suspension obtained by culturing S. toruloides CBS14. Completion of deacetylation was confirmed by thin layer chromatography after 42 hours. The reaction mixture was then centrifuged to remove solids and the resulting solution was mixed vigorously with butyl acetate (200ml). The pH was adjusted from 2.2 to 2.5 by adding concentrated phosphoric acid dropwise. Separate the aqueous layer and add another butyl layer. Extract with acetate (20 ml) and add a few drops of
Gemex surfactant was added. The butyl acetate fractions were collected and a technical grade methyl alcohol solution (approximately 8 ml) of potassium acetate (12.5% w/v) was slowly added until no precipitate formed. Next, the precipitate was filtered, washed with a small amount of acetone, and dried at room temperature under reduced pressure to obtain the target compound (2.87 g). The compound was identified by thin layer chromatography using cellulose-coated plates and a solvent system of ethyl acetate: n-butanol: sodium acetate in comparison to standards. The purity of this product as determined by densitometer was approximately 75%, and the total yield was 65%. Example 2 (6R・7R)-3-hydroxymethyl-7-(thien-2-ylacetamido)cef-3-M-
Production of 4-carboxylic acid A cell suspension obtained by culturing Sporidiobolus jionsonii OBS5470 was used. In addition to performing enzyme-catalyzed deacetylation for 114 hours, the same method as in Reference Example 2 was repeated to obtain the target compound (1.99
g) was obtained. Its purity is about 77% and the total yield is 46%
It was hot. Examples 3-6 and Reference Example 3 below illustrate methods used to measure desired esterase activity obtained by specific microorganisms. Reference Example 3 (a) Add 2% W/V of (6R・7R)-3-acetoxymethyl-7-(D-5-amino-5-carboxybentanamide) Cef- to the above yeast extract/peptone liquid nutrient medium. Addition of 3-M-4-carboxylic acid (potassium salt) was used to plant Rhodosporidium toruloides CBS14. This strain was previously cultured on yeast extract/peptone/agar slants at 25°C for 4 days.
A sample obtained by culturing the liquid medium with shaking at 25°C for 3 days was assayed by thin layer chromatography.
Here, a plate coated with cellulose was used, the pH was adjusted to 5.8-6.0 with phosphate buffer (0.067M), and aqueous n-propanol (70% V/V) was used as the solvent system. When each plate was irradiated with ultraviolet rays, it corresponded to the standard product (6R・7R)-7-(D-5-amino-5-carboxypentanamide)-3-hydroxymethylcef-3-em-4-carboxylic acid. A spot was recognized. (b) (6R・7R)-3-acetoxymethyl-7-
(D-5-amino-5-carboxypentanamide) cef-3-em-4-carboxylic acid (kariur salt) was replaced with (6R・7R)-3-acetoxymethyl-7-[2-(fur- 2-yl)-2
-Methoxyiminoacetamide] Cef-3-em-4-carboxylic acid (syn isomer) was used, and the same method as in (a) above was used. When the product of the liquid medium was assayed by thin layer chromatography, it was found that (6R・7R)-7-[2-(fur-2-yl)-2-methoxyiminoacetamide]-3-hydroxymethylceph- The presence of 3-m-4-carboxylic acid (syn isomer) was observed. Example 3 In addition to replacing Rhodosporidium toruloides CBS14 with one of the following strains, Reference Example 3(a) and
When the same method as (b) was repeated, (6R・7R)-7-(D-5-amino-5-carboxypentanamide)-3-hydroxymethylcef-3-em-4- Carboxylic acid or (6R・7R)-7-[2-(fur-2-yl)-2-methoxyiminoacetamide]-3-hydroxymethylcef-3-em-4-carboxylic acid (syn isomer) existence was confirmed. [Table] Example 4 Sporidioborus ruinedonii CBS5001 was added to the above yeast extract/peptone/agar slant medium at 25°C.
After culturing for 4 days, yeast extract (0.3%W/V),
Malt extract (0.3%W/V), peptone (0.5%W/
V) and D-glucose (1.0% W/V), cultured at 25°C for 3 days, and then a portion of the culture (1 ml) was inoculated with yeast extract (0.23% W/V). /V), malt extract (0.23%W/V), peptone (0.38%W/V), D-glucose (0.75%W/V)
V) and (6R・7R)-3-acetoxymethyl-
Transplanted into an aqueous medium (53 ml) containing potassium salt of 7-(D-5-amino-5-carboxypentanamide)cef-3-M-4-carboxylic acid (1.96% W/V) and incubated at 25°C for 5 days. Cultured with shaking for 1 day. The obtained culture solution was assayed by thin layer chromatography in the same manner as described in Reference Example 3, and (6R・7R)-7-(D-
5-amino-5-carboxypentanamide)-
The presence of 3-hydroxymethylcef-3-em-4-carboxylic acid was observed. Example 5 In the same manner as described in Example 4, Burrella
After culturing Tsugae CBS5038, (6R・7R)-7-(D-5-amino-5-carboxypentanamide)-3-hydroxymethylcef-3-M-4-carvone was determined by thin layer chromatography. The presence of acid was recognized. Example 6 Ustilago mayzis IMI103761, which had been cultured on a modified Sabouraud agar slant at 25°C for 72 hours, was transferred to the yeast extract/peptone liquid nutrient medium described above.
The cells were cultured with shaking at 25°C for 72 hours. Next, a portion (1 ml) of the culture solution was added to 0.1 M phosphate buffer (8 ml) and 10%
W/V (6R・7R)-3-acetoxymethyl-7
-(D-5-amino-5-carboxypentanamide)cef-3-em-4-carboxylic acid (1 ml)
The mixture was incubated with shaking at 25°C for 48 hours. Samples of the cultures were assayed by thin layer chromatography using cellulose-coated plates and an aqueous n-butanol (70% V/V) solvent system. When each board was irradiated with ultraviolet rays, the standard product (6R/7R)-7-(D-5
-amino-5-carboxypentanamide)-3
The presence of spots corresponding to -hydroxymethylceph-3-em-4-carboxylic acid was observed. The concentration of this product was 1.7 mg/ml and the concentration of the non-microbial control was 0.5 mg/ml. Reference example 4 (6R・7R)-7-[2-(fur-2-yl)-2-
Production of methoxyiminoacetamide]-3-hydroxymethylcef-3-em-4-carboxylic acid (syn isomer) (a) A culture (100 ml) of Rhodosporidium toruloides CBS14 using a liquid medium was Monohydrate glucose (2.2%), oxoid yeast extract (1
%), oxoid peptone (1%), potassium dihydrogen phosphate (1.5%), polypropylene:white mineral oil (1:1) (0.1%) and cephalosporin C
(potassium salt, 0.1%) and cultured at 25°C for 32 hours with agitation (3 l/min). The product was collected by centrifugation, the supernatant was decanted, and demineralized water was added. was added to obtain a fluid slurry. (b) (6R・7R)-7-[2-(Flu-2-yl)-
2-methoxyiminoacetamide]-3-acetoxymethylceph-3-em-4-carboxylic acid (syn isomer) (8.0 g), caustic soda (2.6 g), demineralized salt water (120 ml), orthophosphoric acid (2.2
ml) and the slurry of Rhodosporidium toruloides CBS14 obtained by (a) above (18
ml) was shaken at a temperature of 25°C. After 20 hours, completion of the reaction was determined by thin layer chromatography (using silica gel plates and 0.5M sodium chloride solution). The reaction mixture was centrifuged and the supernatant decanted and filtered through a bed of asbestos. Methyl, isobutyl,
Ketone (14 ml) was added, the mixture was stirred magnetically and orthophosphoric acid solution (20%) was added dropwise until the pH was 2.0. The mixture was then allowed to stand, the lower aqueous layer was removed by a pipette, the solvent layer containing a large amount of white precipitate was filtered under reduced pressure, and the precipitate was dissolved in methyl isobutyl ketone (10 ml) and ice-cold water (2 x 10 ml). Washed with. The product was aspirated and dried under reduced pressure at room temperature to give the native compound (5.01 g). This compound was fixed in infrared spectrum and magnetic nuclear resonance spectrum. High pressure liquid chromatography (hpl)
The purity determined in c) was 92.2%. Reference example 5 Production of (6R・7R)-7-amino-3-hydroxymethylcef-3-em-4-carboxylic acid (6R・7R)-7-amino-3-acetoxymethylcef-3-em-4 -Toluene of carboxylic acid-
p-sulfonate (6.4 g), demineralized brine (120
ml), a sufficient amount of ammonia water (specific gravity 0.880) to dissolve cephalosporin, and Rhodosporidium obtained by (a) of Reference Example 4.
The reaction mixture containing a slurry (12 ml) of Truroides CBS14 was adjusted to pH 6.5 with 20% orthophosphoric acid. The mixture was stirred at 25°C and the reaction was determined to be complete after 20 hours by thin layer chromatography (using silica gel plates and 0.5M sodium chloride solution). The reaction mixture was centrifuged and the supernatant was decanted and filtered through a bed of stone silk. Then 20% under stirring into the solution
Orthophosphoric acid was added dropwise to lower the pH to 3.6. The precipitate formed was filtered off and mixed with ice-cold water (5 ml) and acetone (10 ml).
ml). A second precipitate, obtained by adding an equal volume of acetone to the filtrate, was filtered off and washed with water (10 ml) and acetone (20 ml). The two solids were dried separately under reduced pressure at room temperature and then combined to give the desired compound (1.99 g). It was confirmed to be the target compound by magnetic nuclear resonance spectrum and infrared spectrum. The purity of the product determined by gradient elution method is
It was 98.1%. Reference example 6 (6R・7R)-7-(D-5-benzoylamino-
Deacetylation of (5-carboxypentanamide)-3-acetoxymethylceph-3-em-4-carboxylic acid Cells remaining by centrifugation of the reaction mixture according to Reference Example 4(b) were washed and diluted with demineralized brine. was added to obtain a fluid slurry. (6R・7R)-7-(D-5-
Benzoylamino-5-carboxybentanamide)-3-acetoxymethylcef-3-em-4
-Carboxylic acid (8g), caustic soda (2.5g),
The above slurry (18 ml) was added to a reaction mixture (adjusted to pH 6.0) consisting of demineralized water (120 ml) and orthophosphoric acid (2.2 ml) with a specific gravity of 1.75, and after shaking and incubating at 25°C for 21 hours, it was subjected to thin layer chromatography. Completion of the reaction was confirmed by graphie. At that time, a silica gel plate and a solvent system of chloroform:methanol:formic acid (48:8:2) were used. After centrifuging the reaction mixture and decanting the supernatant, the product reacts with diphenyldiazomethane to form diphenylmethyl-
(6R・7R)-(D-5-benzoylamino-5-diphenylmethoxy-carbonylpentanamide)
-3-Hydroxymethylcef-3-em-4-carboxylate (8.5 g) was produced.
The product was confirmed to be consistent with the standard by infrared and magnetic resonance spectra. Reference example 7 (6R・7R)-7-[2-(fur-2-yl)-2-
Production of methoxyiminoacetamide]-3-hydroxymethylcef-3-em-4-carboxylic acid (syn isomer) Rhodosporidium toruloides CBS349 was mixed with glucose and yeast extract according to the method of Reference Example 4(a). - After 3 days of culture at 25°C in the presence of 0.1% cephalosporin C (potassium salt) using peptone medium, the culture (60 ml) was centrifuged and the cells were resuspended in water (25 ml). This thing (6R・
7R)-7-[2-(Flu-2-yl)-2-methoxyiminoacetamide]-3-acetoxymethylcef-3-em-4-carboxylic acid (syn isomer) (4.0g), caustic soda (1.3 g) and a solution consisting of orthophosphoric acid (1.1 ml) and demineralized water (30 ml), and the reaction mixture was kept shaking at 25° C. for 3 days. Thereafter, completion of the reaction was confirmed by thin layer chromatography. The reaction mixture was then treated in the same manner as in Example 10 to obtain the target compound (2.5 g). The product was identified by thin layer chromatography, infrared spectroscopy and magnetic nuclear resonance spectroscopy. Reference Example 8 Production of (6R・7R)-7-(D-5-amino-5-carboxypentanamide)-3-hydroxymethylcef-3-em-4-carboxylic acid Rhodosporidium, Toruroides CBS349 and
A cell suspension (PH6.0) was prepared by the method of Reference Example 7 using 0.25M phosphate buffer. Cephalosporin C dissolved in demineralized water (60 ml)
(potassium salt; 6.7 g) was added to the yeast suspension, and the mixture was shaken at a temperature of 25° C. for 20 hours, after which completion of the reaction was confirmed by thin layer chromatography. The reaction mixture was worked up according to the method of Example 1 to obtain 4.27 g of the desired compound (λ _nax 260 nm). Two thin layer chromatographies confirmed that the product corresponded to a standard of the target compound. Purity was 45% as determined by UV spectroscopy. Reference example 9 (6R・7R)-3-hydroxymethyl-7-[2-
Methoxyimino-2-(fur-2-yl)-acetamide] cef-3-m-carboxylic acid (syn.
Production of (6R・7R)-3-acetoxymer-7-[2-
Methoxyimino-2-(fur-2-yl)acetamide] cef-3-em-4-carboxylic acid (syn.
isomer) (50 g) was added and the resulting solution had a pH of 6.4. After adding 20% orthophosphoric acid and adjusting the pH to 6.3,
A cell suspension (150 g) of Rhodosporidium toruroides CBS14 was added. The mixture was aerated at 25°C for 8 hours, during which time 1M caustic soda solution (105ml) was added to keep the pH in the range 5.8 to 6.3.
had to be added. Completion of the reaction was confirmed by thin layer chromatography. Next 16 mixtures
After stirring and aerating for an hour, the yeast was centrifuged and washed by suspending in water (200 ml) for 30 min.
Centrifuged again. After adding charcoal (2g) to the main section and stirring, add Hyflo Supercel (Hyflo
Supercel) was filtered through a bed. The bed was washed with the yeast wash solution. Sodium chloride (250
g) and 4-methylpentan-2-one (500ml)
In order to adjust the pH of the filtrate to 6.0, 20% orthophosphoric acid was added over 20 minutes. The mixture was cooled on ice during acidification. Stirring was stopped and the mixture was allowed to stand still.
The clear aqueous layer was removed and the precipitated solid was filtered off and washed with 4-methylpentan-2-one (50ml) and water (3 x 40ml). Product at 40℃ for 24 hours
The mixture was dried under reduced pressure for hours to obtain an off-white target compound (39.39 g). Concentration determined by high pressure liquid chromatography is 99.1% and total impurities determined by thin layer chromatography is 2.5%.
It was hot. Reference example 10 (a) (6R・7S)-3-acetoxymethyl-7-methoxy-7-phenyl-acetamidoceph-3-
Preparation of em-4-carboxylic acid t-Butyl (6R 7R)-3-acetoxymethyl-7-phenylacetamidocef-3-em-4 dissolved in dry tetrahydrofuran (10 ml)
-Carboxylic acid salt (1.5g) was cooled (-78
℃). Separately, lithium methoxide (358 mg) was dissolved in dry methanol (8.8 ml) and tetrahydrofuran (50 ml), stirred and cooled (-78
℃). The former was added to the latter over a period of 3 minutes under a nitrogen atmosphere, and after 3 minutes t-butyl-hypochlorite (0.69 ml) was added. Ammonium chloride (1g) and sodium metabisulfite (1g)
Water (150 ml) containing g) was mixed with ethyl acetate (150 ml) under stirring, the yellow solution was added under stirring, and the mixture was further stirred for 5 minutes. The aqueous phase was separated and extracted with ethyl acetate (50 ml), the organic phases were combined, washed with underground brine (50 ml), concentrated to dryness under reduced pressure, and t-butyl (6R・7S)-3-acetyl Methyl-7methoxy-7-phenylacetamide cef-3-em-4-carboxylate (1.6 g) was obtained. This (1.6 g) was dissolved in trifluoroacetic acid (6.8 ml) and anisole (1.7 ml) under stirring, kept at about 23°C, residual trifluoroacetic acid was evaporated off, and then toluene (2 x 15 ml) was dissolved. Conjugate distillation was carried out using The brown residue was dissolved in sodium bicarbonate (25ml) and ethyl acetate (25ml) was added with stirring over 10 minutes. Separate the aqueous phase and adjust the pH to 2 with 2N hydrochloric acid.
Extracted with ethyl acetate (3x25ml). The extract was washed with underground brine and concentrated to dryness to give a yellow solid (1.3 g). Add this to acetone (50
ml), treated with charcoal, the mixture was filtered and the filtrate was concentrated to dryness under reduced pressure. Triturate the residue with ether (25 ml) to obtain the desired acid (500 mg).
I got it. Melting point 167-170℃, [α] ^21D ₊₁₉₀゜( c
O, 28, DMSO), λ _nax (0.1 M -PH6, phosphate buffer) 265 nm ( ^E1 % ₁ cm196). (b) Production of (6R・7S)-3-hydroxymethyl-7-methoxy-7-phenylacetamidoceph-3-em-4-carboxylic acid (sodium salt) (6R・7S)-3-acetoxy Methyl-7-methoxy-7-phenylacetamidoceph-3-em-4-carboxylic acid (200 mg) was suspended in water (3 ml) and an aqueous solution of sodium bicarbonate (saturated) was added to adjust the pH to 6.0. After adjustment, 0.4 M phosphate acid buffer (PH6; 3 ml) was added. A frozen homogeneous suspension (1 g) of Rhodosporidium toruloides CBS14 was added to the resulting solution under stirring at a temperature of about 20°C. After 16 hours, thin layer chromatography (chloroform: methanol: acetic acid =
90:16:5) to confirm the completion of the reaction of the substrate. The mixture was centrifuged, and the resulting supernatant was decanted and freeze-dried. Add ether (10 ml) to the residue and triturate to obtain a colorless solid (400 mg).
I got it. It was confirmed by magnetic nuclear resonance spectroscopy that this product contained the target compound. Example 7 (a) An agar medium containing maltose (4%), peptone (1%), malt extract (2.4%) and agar powder (2.5%) was adjusted to pH 7.5 before sterilization and used to The microorganisms listed in Table 1 were cultured. After 7 days of slant culture at 25°C, glucose (2
%), yeast extract (1%), peptone (1%) and potassium dihydrogen phosphate (0.5%) (PH5.8; 40 ml). After culturing with shaking at 25°C for 10 days, the cells were transplanted to another medium prepared by adding cephalosporin C (0.1%) to the same medium, and further cultured for 3-3 hours depending on the degree of growth.
After culturing for 8 days, the cells were collected. A homogeneous suspension of each culture was obtained by vigorous mixing in a mixer. This suspension (2ml), 0.5M
A reaction mixture containing potassium phosphate buffer (PH6.0; 2 ml) and cephalosporin C (3.33%; 6 ml) was prepared and shaken at 25°C for 5 days. Each sample was assayed by thin layer chromatography similar to that described in Reference Example 3.
Elute the fraction corresponding to the standard sample of (6R・7R)-7-(D-5-amino-5-carboxybentanamide)-3-hydroxymethylcef-3-em-4-carboxylic acid (DAC), The results of quantitative determination using a densitometer (260 nm) are shown in Table 1. (b) Instead of cephalosporin C (6R/7R)-7
-[2-(Flu-2-yl)-2-methoxyiminoacetamide]-3-acetoxymethylcef-
Using a reaction mixture containing 3-m-4-carboxylic acid, and repeating the method described in (a), 3-
A hydroxymethyl compound was obtained. This product was assayed using a method similar to the thin layer chromatography described in Reference Example 4, and the results are shown in Table 1. [Table] [Table] In Table 1, 3-hydroxymethyl compounds
(a) and (b) are (6R・7R)-7-(D-5-amino-5-carboxypentanamide)-3-hydroxymethylcef-3-em-4-carboxylic acid and (6R・7R), respectively. )-7-[2-(fur-2-yl)
-2-methoxyiminoacetamide] -3-hydroxymethylcef-3-em-4-carboxylic acid.

Claims (1)

[Claims] 1. 3-acyloxymethylcef-3-em-4-carboxylic acid represented by the following general formula ()
Ustilago spp., Leucosporidium spp., Burrella spp. capable of producing the required esterase activity in the process of hydrolytically converting to hydroxymethylcef-3-em-4-carboxylic acid;
A conversion method characterized in that hydrolysis is catalyzed by an esterase produced by culturing microorganisms belonging to the genus Sporobolomyces, Sporidiobolus, Polyporus, Poria, Schizophyllum and Coprinus. (In the formula, R ¹ is an amino group or a protected amino group, R ² is hydrogen or a lower alkoxy group,
R ³ is a methyl group and B is a sulfur atom. )