DE2163791A1 - Process for the preparation of 7-amino-3-methyl-3-cephem-4-carboxylic acid and its esters - Google Patents

Description

Takeda Chemical Industries, Ltd.,

27 Doshomachi 2-chome, Higashi-ku, Osaka (Japan) .

Process for the production of 7-cephem- · *! - carboxylic acid and their esters

The invention relates to the production of 7-amino-3-methylcephem compounds, in particular a process for the production of 7-amino-3-methyl-3-cephem-4 "carbonic acid of formula (I) or its esters from 7-acylamido ~ 3-methyl-3-cephem-4-carboxylic acid or their esters by enzymatic H-deacylation.

Me desired 7-amino-3-methyl-3-caphem-4-carboxylic acid as the product the formula

Γτ

ÖOOH

or their best at the 4-carboxyl group is important Intermediate product for the synthesis of cephalosporins with a broad antibiotic spectrum, e.g. cephalexin, known. '

It is known that the compound (I) or its ester . clurqh. Hydrolysis of 7 ~ phenoxyaoetaraido-3 ~

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methyl ~ 3-cephem-4-carboxylic acid ester or by reducing Elimination of the acetoxy group from 7-aminocephalosporanic acid or its ester. However, difficulties arise with the first-mentioned method specifically to cleave off the terminal acid amide bond at the 7-position without damaging other parts of the molecule affect. In particular, in cases in which the compound (i) is desired, the ester radical must be completely be a special radical that is easily hydrolyzable. Furthermore, the yield of the desired compound varies even with a slight deviation from the ■ reaction conditions. In the latter process, the yield is even lower than in the first-mentioned procedure. None of the known processes is therefore suitable for large-scale production suitable.

The first-mentioned method has been expanded by the applicant, but with the help of completely different ways an enzyme system of microorganisms, whereby the development of an industrially feasible process succeeded, in the case of the T-acylamido ^ -methyl-J-cephem - ^ - carboxylic acid or its ester a cleavage of the acid amide bond at the 7-position is carried out, and when the ester is used as a substrate, the ester bond at the 4 position can be further split, with the connection (I) is obtained.

Investigations of the most varied of microorganisms have shown that microorganisms which produce the desired 7-amino- 3-methyl-3-cephem-4-carboxylic acid or its ester from 7-acylaraido-3-methyl-3-cepheni-4-carboxylic acid or its ester are able to form, occur very frequently in nature or in cultural deposit sites among the small mushrooms, yeasts and bacteria including the Actinomycetes s ^ kr. ·

It has also been found that this conversion is due to the Ability of the microorganisms to

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eventually hydrolyze the amide bond, though 4-carboxylic acid is used as substrate, and either only the acidic bond or both the acid amide bond as well as hydrolyze the carboxylic acid ester bond when the ^ -carboxylic acid ester is the substrate.

Investigations by the applicant have confirmed that these microorganisms, for example, occur very frequently in the following genera: Aureobasidium, Anthracobia, Ascotricha, Acremonium, Actinomucor, Alternaria, Aegerita, Beauveria, Botryotinia, Byssochlamys, Botryosphaeria, Corynespora, Cunninghamella, Cylindrocarpon, Cylindrocladium, Colletotrichum, Cylindrocephalum, Cercospora, Ceratocystis, Cephalosporium, Cladosporium, Cöniochaeta, Chaetonium, Chrysosporium, Cephaloascus, Diaporthe, Daedaleopsis ,. Däctylaria, Emericellopsis, Pusariura, Griphosphaeria, Gibberella, Glomerella, Isaria, Microascus, Neurospora, Pestalotiopsis, Pyrenophora, Pseudoplea, Papularia, Pleospora, Pellucularia, Rosellinia, Septoria, Sclerotium, Sartorya, Trichometasphaeria, Taphrina, TaIaromyees, Verticillium, Nocardia, Streptomyces, Escherichia, Bacillus, Xanthomonas, Pseudomonas, Protaminobacter, Mycoplana, Aeromouas, Acetpbacter, Rhodotorula, Cryptococcus, Nadsonia, Saccharomycodes and Saccharomyces. With regard to the effectiveness of the conversion activity, i.e. the rate of reaction, preferred bacteria are those of the genera Escherichia, Bacillus, Xanthomonas, Pseudomonas, Protaminobacter, Mycoplana and Aeromonas. Particularly beneficial.

In the method according to the invention ■ 7-phenyl-acetamido- or T-phenoxyacetamido-i-methyl - ^ - cephem- ^ carboxylic acid or their esters with a culture broth of the microorganism or a processed or treated ingredient the culture broth combined in an aqueous medium.

As an output bond, which serves as a substrate, is thus 7-PhenylGcutamido-3-methyl-3-cGph (5m-4-carbonic acid,

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an ester of this acid is used .. The HersteHuing diieseor Output connections are made for example; allowed Wmwauad— from the corresponding 6-AGylami.äöpaniG, iHan; säuire> « esters, via their S-oxides. as in <- der WoS ^ Ao-PatentscftElft 3,275,626 and in Belgian patent specification; W7 Ϊ1; © ' described ,,

The starting compound can be in the form of the free; Acid or. as sodium salt, potassium salt, ammonium salt, antioxidant salt, or Another salt used Werdieno examples of: "Esters which are suitable as: starting compounds' ,, areJ Alkyl esters (e.g. methyl esters, ethyl esters, butyl esters and Hexyl esters), substituted alkyl esters (e.g. methylthi-e- methyl ester, methylsulfenyl methyl ester ,. Methylsulfonyl- methyles.ter, chloromethyl ester, bromomethyl ester and: trichloroethyl ester, "in which the substituent is an alkyl- thlorest, alkylsulfenylrest, alkylsulfonylrest, halogen atom etc. Phenylethyl ester).

The microorganism can be obtained from the collected cultures that are available in public cultural collection institutesi, . 'selected or from the ground, from sewage, seawater ,, "After rivers, fruits, air or other sources known methods can be isolated. According to the experience the applicant is this selection, for example 'after d; en The following procedures are easily possible

Procedure A

The strains to be examined are in 20 ml of an aqueous Medium cultivated aerobically under the conditions mentioned in Example 1, depending on whether the Strains of bacteria, actinomycetes, small fungi or yeasts are, are different "

SAD ORiGfNAt

Microorganism

Table 1

Composition of the culture medium

cultivation

bacteria

1 $ meat extract, 1 $ peptone, 0.5 $ monosodium L-glutamate, 0.05 $ phenylacetic acid (possibly also phenoxyacetic acid), P _H 7.2

1-2 days at 28-37 ⁰ C

Fungi or actinomycetes

3 $ sucrose, 0.2 $ NaNO ,, 0.1 $ K ₂ HPO ₄ , 0.05 $ KCl, 0.05 $ MgSO ₄ .7H ₂ O, 0.01 $ 'PeSO, .7H ₂ O, 0 , 5 $ yeast extract, 0.5 $ peptone, 0.5 $ corn steep liquor, 0.05 $ phenoxyacetic acid (or phenylacetic acid, if applicable), ρ-ΰ 6.0

4 days at
28 ⁰ C

Yeasts

5 $ sucrose, 0.05 $ ₂ 0.1 $ CaNO ₃ , 0.025 $ MgSO ₄ 7E ₂ O, 0.012 $ KCl, p _H 6.0

2 days at 28 ° C

A solution of 20 mg of a substrate (methyl 7-phenylacetamido- or 7-phenoxyacetamido-3-methyl.3-cephem-4-carboxylate) in 0.4 ml of acetone is added to the culture. The mixture is then shaken for 16 hours at 28 ⁰ C ■> A very small proportion (for example, 0.01 ml) of the mixture obtained is subjected to the Dünnsci.ichtchromatographie on silica gel with methanol-acetone (volume ratio 1: 2) up to a length developed by 10 cm. The chromatogranim is irradiated with ultraviolet light to find the important spots. Under the special conditions mentioned above, the respective Rf values of the substrate and the products can be determined beforehand, for example, as follows:

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0.86 for methyl-7-phenylacetamido- or 7-phenoxyacet-

amido 3-methyl-3-cephem-4-carboxylate

0.65 for methyl ^ -amino ^ -methyl ^ -cephem ^ -carboxylate 0.42 for 7-amino-3-ciethyl-3-cephera-4-carboxylic acid

In the above example, the reaction mixture is chosen that one Q of several spots "at Rf 0.65 and / or Rf 0.42. This G-emic becomes through a pillar of resin beads made from styrene-divinylbenzene copolymer. (e.g. the commercially available product "Amberlite XAD-2",

»\ Manufacturer Rohm & Haas Co ₀ , USA) with a diameter of about 10 mm and a length of 100 mm. The fractions eluted with 40 ml v / water are collected and the total yield of products is measured by UY absorption at a wavelength of 260 μm. If the measurement shows that the yield is $ 10 or higher, based on the amount of substrate used, the microorganism investigated used to form the reaction mixture is considered suitable for the process according to the invention.

Procedure B (simplified procedure) '

A culture of each strain to be tested is set up in the manner described above. To 5 ml of the culture broth * A solution of 50 mg of a substrate (for example, T-Phenylacetamiäo ^ -methyl ^ -cephem ^ -carboxylic acid) in 1 ml of a 0.25 molar phosphate buffer (p _H 7.0). The mixture is then shaken ^{at 28 ° C. for 16 hours.} The mixture obtained is subjected to the following three tests:

1) By suitable modifications of the paper disk method by Hi £ gins and co-workers, "Antibiotic and Chemotherapy", 2 »50-54 (1953), and Loo et al, Journal of Bacteriology, 50, 701-709 (1945) determined whether the antibacterial effect of the obtained Mixture obtained Bacillus subtilis PCI-219 80? Ö of effectiveness before the reaction or below has fallen

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Z) By a suitable modification of the method; von Bätehelor and: employees ^ "Eature" · ,, ta? - _t: 257-258 Ci ^; 95-9 -) - 5, it is investigated whether the: ant! Bacterial effect of the mixture obtained by marriage / nylaeetylation ■ with phenylacetyl chloride:

5); It is: fiastgesteliltt, © ΐ), äüfe Extinkiri ^ rl ies EeakiJxoGS'gemisGteea "at e-inex Weliierrliänge v: on 260 by at least 2Qtfo d: er Exifcinikrtiön de: s _; Semis oh es before the reaction"beirragi;"

The Tes-tmikroorganisnien ,, For which: all three tests one jibe positive result; can apply for the procedure according to of the invention can be used.

According to the applicant's experience, the procedure Ä and method B: substantially the same judgment the usefulness of the same test microorganism.

The contact of the substrate with the Kuiturteühe · or their processed ingredients kan ^r ni sö effected in that the microorganism in a Kulturmediium kmLtiviesrfc is ,, · containing the substrate, if the microorganism is resistant to the substrate. In general, however, it is operated so that the microorganism is appropriately cultivated and then the obtained culture broth or its processed ingredients are used. The cultivation can be carried out with stirring with aeration, with shaking or stationary, but an aerobic cultivation is generally preferred. The culture media are prepared by selecting the components alone or in combination as necessary from the following substances: meat extract, yeast extract, peptone, casein hydrolyzate, corn steep liquor, potato juice or any other commonly used natural substances, carbon compounds, e.g. sugar, organic acids or n- Paraffins, the most diverse inorganic UMCl or / in-nia-ehon compounds that form nitrogen in:

ZMW §31 / iffi. bath

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Contain amino nitrogen or nitrogen nitrogen, phosphates, magnesium salts, table salt or other metal ions and a wide variety of vitamins. The addition of phenoxyacetic acid or phenylacetic acid, which corresponds to the acyl radical of the substrate to be used, to the culture medium in a concentration of 0.05 "to 1 $ can have the result that the activity of the culture broth obtained for the formation of the desired product from the substrate is increased The suitable p n value of the culture medium and the cultivation temperature vary depending on the type of microorganism 9 for bacteria or actinomycetes. The temperature is generally in the range of about 10 to 40 ⁰ G. The time at which the activity of the culture broth reaches the maximum differs depending on the type of microorganism used, so that the optimal cultivation time is determined appropriately for each strain r the production of T-amino - ^ - raethyl ^ -cephem- ^ carboxylic acid or its ester used. Anything can be used as a processed component of the culture broth

t what has been converted by suitable treatments to increase or concentrate the conversion activity so that it is advantageous for the preparation of the desired compound. If, for example, the activity is mainly intracellular, it is expedient to use 1) cell suspensions which contain various concentrations of the separated cells in a buffer solution, 2) cell-free extracts of the cells that have been obtained by conventional methods, or 3) enzyme solutions that are obtained by conventional methods have been purified or partially purified from the cell-free extracts. For example, if the activity is mainly concentrated extracellularly, 1) supernatant solutions containing

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by removing the cells or mycelium from the culture broth obtained v / ground, or 2) enzyme solutions, which are from the supernatant solutions according to methods known per se Purified or partially purified for the purification of enzymes have been used »

The substrate is with the culture broth prepared in this way or its processed ingredients in V / water or any other aqueous media brought together. The PjT · of the reaction media becomes appropriate set to a value between 4 and 9, in particular between 6 and 8 »The suitable reaction time is from the concentrations of the substrates, the activity of the culture broth or its processed ingredients for the Conversion, the reaction temperature, etc. affects, but generally 1 to 24 hours suffice. The reaction temperature is between 10 and 50 C, expediently between 15 and 40 C. The concentration of the substrate is mainly dependent on the intensity of the conversion activity to form the desired compound determined, but generally from a range from 0.1 to 5 $ (weight / volume), calculated as the starting compound and based on the medium.

The 7-araino-3, -: methyl-'3-cephem-4-carboxylic acid or its ester can be carried out under mild conditions by a combination of methods known per se, e.g. by extraction with solvents or by chromatography, isolated and purified. I particularly recommend the piltrat of the reaction mixture of the column chromatography on the Submit ion exchange resin "Amberlite XAD-2" and to make the elution with water and thereby the .Collect fractions that contain the desired product.

The invention is further illustrated by the following examples. There are the percentages of the components deo culture medium based on weight / volume, i.e.

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given in grams / 100 ml. The remaining quantities are based on weight, unless otherwise stated. Parts by weight relate to parts of space as grams to cubic centimeters. In the tables "-" means that the product was undetectable. The IFO and ATCC numbers, which are added to the names of the respective microorganisms are the deposit numbers at Institute for Fermentation, Osaka, Japan and at the American Type Culture Collection, Maryland, USA. All Strains were available prior to this invention and are included in the lists of cultures produced by the respective Cultural collection institutes are published. The following microorganisms, which are present in the Publications not mentioned, but also in the American Type Culture Collection under the following Access numbers have been stored:

Escherichia coli IFO-3210 ATCC-21753

Escheriohia coli IFO-3467 'ATCC-21753

Xanthomonas oryzae IFO-3312 ATCC-21754

Protaminobacter alboflavus IFO-13221 ΑΤΟΌ-2Ί755

Mycoplana sp. IFO-13240 ATCC-21756

Aeromonas hydrophila IFO-12634 ATCC-217f> 7

For Mycoplana sp.IFO 13240 the species has not yet / not been established. Therefore, the characteristics of the Tribe given below:

Irregular rods from 0.6 χ 2 to 0.6 χ 6 η, sometimes curved and branched at a very young stage. Movable with the help of polar flagella. Gram negative, aerobic.

Agar colonies: circular, light gray, convex, smooth, closed (entire)

Agar slant culture: thread-like, light gray, smooth broth; cloudy.

Gelatine stab culturesi no liquefaction. Thin surface lächenwachatunio ■

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Casein, starch and cellulose are not hydrolyzed.

No formation of nitrites from nitrates <, No hydrogen sulfide formation.

No indole formation.

Methyl red test: negative

Catalase: positive

Litmus milk: unchanged

Potato: no growth

No growth or only sparse growth in carbohydrate media.

Aromatic compounds such as phenylglycine were used.
Isolated from the ground.

example 1

A 2-day inoculum (.5 parts by volume) of Escherichia coli IFO 3210 of a culture medium was inoculated in 200 Raumteiie that had a p _H value of 7.2 ~ 1.0 and $ meat extract, peptone 1.0 $, 0 , 5 $ sodium glutamate, 0.5 $ sodium chloride and 0.05 $ phenylacetic acid. The culture medium was incubated with shaking for 48 hours at 37 ° C., whereupon a solution of 0.8 parts by weight of methyl 7-phenylacetamido-3-methyl-3-cephem-4-carboxylate in 8 parts by volume of acetone together with 50 parts by weight of 0 , 25 molar phosphate buffer solution (Ptt 7jO) was added. The entire mixture was then shaken at 28 ° C. for 16 hours, whereupon the methyl 7-phenylacetamido-3-methyl-3-cephem-4 ⁱ -carboxylate completely disappeared in the mixture and 0.4 part by weight of 7-amino-3 ~ methyl-3-cephem-4-carboxylic acid was formed therein.

The reaction mixture was filtered to remove the cells. The filtrate was subjected to column chromatography on "Amberlite XAD-2" (see above), using distilled water as the eluant, whereby 0.35 part by weight of 7-amino-3-methyl-3-eephem-4-carboxylic acid was obtained Melting point 240-242 ⁰ C (Zera.) Were obtained.

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• Example 2 ·

In the manner described in Example 1, 2-day cultures of the bacterial starches mentioned in Table 2 were produced. A solution of 800 mg of methyl 7-phenyllactamido-3-methyl-3-cephem-4-carboxylate (substrate 1) in 8 ml of acetone and 50 ml of a 0.25 molar phosphate buffer solution (p _H 7.0) A solution of 800 mg of 7-phenylacetate "amiao;5-me1; hyl-3-cephem-4-carboxylic acid (substrate 2) in 50 ml of a 0.25 molar phosphate buffer solution (p-rr 7.0) added. ^{The mixtures were shaken at 28 °} C. for a further 16 hours in order to allow the reaction to take place. The products were separated from the reaction mixture and purified by column chromatography. The yields were determined after the absorptions at 260 μm. The results are given in Table 2, in which

lat the product A and 7-amino-3-methyl-3-cephem-4-carboxylic acid the product is B.

Tabel Microorganism 1 2 Substrate 1 mg Substrate 2 mg Per
duct A Product 3 ■
i mg; Product B mg Escherichia coli IFO-3467 - 400 mg j 420 mg Xanthomonas oryzae IFO-3312 - 350 .rag i 300 mg Xanthomonas malvacearum
IFO-3383 380 mg 350 mg Bacillus pumilus IFO-12093 - 140 mg 380 mg Bacillus licheniforrnis
IFO-12199 80 mg 390 mg Pseudomonas solanacearum
IFO-12510 ... 110 mg 250 mg Protaminobacter alboflavus
IFO-13221 140 370 mg Mycoplana sp. IFO-13240 - 370 250 Aeromonas hydrophila-
IFO-12634 50 mg - 300

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Example 3

A set of eyes of the bacterial strains mentioned in Table 3 was inoculated in 5 ml of an aqueous medium (p "7.0) containing 0.8 $" Bacto-Nutrient Broth, dehydrated "(commercially available, manufacturer Difco Laboratories, USA) and 0, 3 '/ Phenylacetic acid contained, which was ^{incubated with shaking at 28 0} C for 24 hours. A solution of either 30 mg of 7-phenylacetamido-3-methyl-3-.cephem-4-carboxylic acid (substrate 1) or of 30 mg of 7-phenoxyacetamido-3-methyl-3-eephem-4-carboxylic acid (substrate 2) added in 1 ml of a 0.25 molar phosphate buffer solution (pu 7.0). In the case of substrate 1 ^{, the mixture was then shaken for a further 4 hours, at 28 °} C. and in the case of substrate 2 for a further 24 hours at 28 ^° C., whereupon the reaction mixture showed no antimicrobial activity. This means that the substrates were completely converted. The yield of 7-amino-3-methyl-3-cepbem-4-carboxylic acid was determined with the aid of the restored antimicrobial activity after phenylacetylation with phenylacetyl chloride. The results are given in Table 3.

Table 3

Microorganism Substrate 1 mg Substrate 2 Escherichia coli IFO-3210 18th 15 mg Escherichia coli Vocommunior mg ΙϊΌ-3550 * 17th mg 14 mg Bacillus sp. ATCC-14552 18th mg 14 mg Bacillus pumilus IFO-12093 16 13 mg Example 4

An amount of loops of Escherichia coli IFO-3210 was found in 50 parts of an aqueous culture medium (p "7.0) inoculated, the $ 1 peptone, $ 1 meat extract, $ 0.5 NaCl and $ 0.3 Phenylacetic acid contained, whereupon an inoculum by 24-3 hours of incubation with shaking. The inoculum was inoculated into 500 parts by volume of an aqueous medium of the above-mentioned composition.

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The device was shaken at 28 C for 24 hours. the Culture broth was filtered to separate the cells. The cells were then washed with water and placed in 50 parts of distilled water suspended »

The cell suspension was made up of 50 parts by volume, one aqueous Solution of 2 parts by weight of T-phenylacetamido - ^ - methyl - ^ - cephem-4-carton acid given. The mixture was 3 hours stirred at 37 ° C., the p ^ value being kept at 7.0 by intermittent addition of 0, In-NaOH. At intervals A small part of the reaction mixture was removed from 30 minutes and its absorbance at 260 mu was measured, The same sample was then subjected to thin layer chromatography on silica gel using a mixture of Ethyl acetate, acetic acid and water (volume ratio 50: 5: 4) as a developer. From the results showed that the substrate (Rf 0.50) in the 3-hour Reaction completely disappeared and 7-amino-3-methyl-3-cephem-4-carboxylic acid (Rf 0.05) was formed. The absorbance of the reaction mixture at 260 μm changed not essential during the reaction.

The reaction mixture was filtered to remove the cells. The piltrate was combined with the cell wash. The combined solution was sold to 'JAmberlite XAD-2 ". The column was eluted with distilled water. The freeze-drying of the eluates yielded 1.0 part by weight of pure 7-amino-3-methyl-3-cephem-4-carboxylic acid as a powder with an authentic sample in terms of melting point, specific rotation, infrared spectrum, nuclear magnetic resonance spectrum and UV spectrum matched well ο

Example · '.5

A loop amount of 7-day slant cultures of the following microorganisms was inoculated into 30 ml of a KuIturraediuniü which had a p _{ , - value of 6 and 3 ^L p

..., - - ^: 5 0 9 8 3 0/0988 _eAD original

Sucrose, $ 0.2 Sodium Nitrate, $ 0.1 Dipotassium Hydrogen Phosphate, $ 0.05 Potassium Chloride, $ 0.05 Magnesium Sulphate, 0.0019b Iron (ll) Sulphate, p, $ 5 6 Yeast Extract, 0.5 ^ 6 Peptone, 0 , $ 5 corn steep liquor and $ 0.05 phenoxyacetic acid. The culture media were incubated for 4 days with shaking "at 28 ⁰ C", whereupon a solution of 100 mg of methyl 7-phenoxyacetamido-3-methyl-3-cephem-4-car'boxylat in 1 ml of acetone together with 7 ml of a 0 , 25-M phosphate buffer solution containing a ert p _H ~ W ~ of 7.0, had been added. The reaction was carried out for 16 hours "at 28 ^° C. with shaking. The product" or the products of each strain are listed in Table 4 below, in which the numbers show the respective conversions to the desired 7-amino-3-methyl-3-cephem-4-carboxylic acid (product i) and / or to the desired methyl-7-amino -3-methyl-3-cephem-4-carboxylate (product II) mean.

Table 4

sales ($) Microorganism Product I. Produlcb II Aureobasidium pullulans ifO-4464 Around 20th Arthrinium phaeospermum IFO-6574 55 Anthracobia melaloma IFO-6985 _ 22nd Ascotricha chartarum IFO-6310 15th Asperflillus parasiticus IFO-4082 10 35 Aspergillus brevipes IFO-5821
Acremonium potronii IFO-5706 64
18th Actinomucor elegans IFO-4022 15th Alternaria lon ^ ipes IFO-6149 46 Ae ^ erita candida IPO-6988 23 Beauveria barjßiana. IFO-4848 '13 Botryotiriia fuckeliana 1ΓΟ-5365 21st

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Byssochlamys fulya
Botryos ^ liaeria ribis IFO-63G7
IFO-4837
IFO-4445 Cylindroc.ladium peni.cilloides • 1F0-6784
IFO-5995 IFO-8526 13th 17th
17th
■ 50 cliromossena
Cunninglianiella e'chinulata IFO-6703 ■ Cylindrocarpon willkommii Cladosporium cladosporioides IPO-63.71 IFO-6785 - _ Colletotrichum dematiuin IFO-6807
IPO-6711 Coniochaeta'tetraspora IFO-6707 15th
12th 45
23 Cylindrocephalum aureujn
Cercospora kikuchii IFO-7644
IFO-8668 Dactylaria mycophila IFO-4910 33 Cerabocystis piceae
Ceratocystis coerulescens IFO-6615 Diaporthe phaseolorum IFO-4469
IFO-8503 Cephalosporium mycopliilum IFO-6554 Daedaleopsis styracina IFO-8509 10 25th Chaetomium elatum IFO-8389 Pusarium oxysporum
Pusarium roseum IFO-7444 Chaetomium anahelicinum IFO-8279 Fusarium solani IFO-7160 11 18th Chrysosporium verrucosum IFO-7785 Griphosphaeria nivali? I.FO-5257 mm Cephaloascus fragrans IFO-8162 Gibberella zeae 22nd Corynespora smithii Glomcre] la ei n ^ iü.ata «« · 19th
19th mm 20th 18th 65 15th 25th 18th 55 20th 75 _ ■ 25th 65 12th 60 13th

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Isaria kogane Hicroascus desmosporus Neurospora sitophila

Λ I Il Il ■ I Il I I I I Il

Pestalotiopsis funerea Pleospora herbarum Pyrenophora graminea Pseudoplea trifolii Papularia sphaerosperma Pellicularia filamentosa Rosellinla necatrix Septoria ^ lycines Sclerotium delphiriii Sartorya

Trichometaaphaeria turcica Taphrina cerasi Talaromyces luteus Verticillium, theobromae Nocardia mexicana Streptomycea lavendulae IPO-5299 IFO-6761 IFO-4596 IPO-5427 IFO-6125 IPO-7507 IFO-6681 IPO-IFO-6574 IPO-734623 IPO- -5866 IFO-6358 IFO-0675 IFO-6896 IFO-6669 IFO-3927 IFO-3145

18th

45 40 55 52 42 47 21 45 18 20 45 55 50 13 15 15 21 25 12

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Example 6

A 4-day inoculation culture (30 parts by volume) of Griphosphaeria nivalis ΙΙΌ-7436 was inoculated into 1000 parts by volume of a culture medium which had a ρττ value of 6 and 3 $ sucrose, 0.2 $ sodium nitrate, 0.1 $ dipotassium hydrogen phosphate, 0 0.05 $ potassium chloride, 0.05 $ magnesium sulfate, 0.001 $ ferrous sulfate and 0.05 $ phenoxyacetic acid. The culture medium was shaken for 4 days at 28 ⁰ C and a solution of 3.7 parts by weight of methyl 7-phenoxyacetamido-3-methyl-3-cepheiü-4-carboxylate in 20 parts by volume of acetone was added. The mixture was then shaken ^{at 28 °} C. for a further 16 hours.

A portion of the reaction mixture was subjected to thin layer chromatography . using a methanol-acetone mixture (1: 1) as a solvent the substrate was found to be methyl 7-phenoxyacetamido-3-diethyl-3-cephem-4-carboxylate (Rf 0.95) had disappeared, and that instead 7-amino-3-methyl-3-cephera-4-carboxylic acid was formed in almost quantitative yield "

The reaction mixture was filtered to remove the mycelium. The Piltrat was concentrated and purified by column chromatography to give 1.9 parts by weight of product of melting point 240-242 ⁰ C (dec ").

Example 7

An amount of a 4-day agar slant of the in Strains mentioned in Table 5 were inoculated into 30 ml of a culture medium which had a p n value of 6 and was off 5 / £. Sucrose, $ 0.025 Potassium dihydrogen phosphate, $ 0.1 Calcium Nitrate, $ 0.025 Magnesium Sulphate, $ 0.012 Potassium Chloride and tap water. The cultivation was carried out for 48 hours at 28 ° C. with shaking »Zum Culture medium was a solution of 100 mg of methyl 7-phenoxyacetamido-3-methyl-3-cephem ~ 4-carboocylate in 1 ml of acetone

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and 7 ml of (7.0 p _H) of a 0.25 molar phosphate buffer solution was shaken followed by further 16 hours' at 28 ⁰ C, to take place the reaction. The yields obtained with the different strains of 7-Άηι1ηο- 3-methyl-3-cephem-4-cartonic acid (product I) or its methyl ester (product II) are listed in the table below.

Table 5 Microorganisms Product I Product II

Rhodotorula glutinis IFO-0756 - 4-0 mg Cryptococcus albidus IFO-0940. - 50 mg Nadsonia elongata ΪΪΌ-Ο665 - 10 mg Saccharpmycodes ludwigii IFO-1266 - 10 mg

Example 8

To 30 ml of a 4-day culture broth of the strains mentioned in Table 6, prepared in the manner described in Example 5, 7 ail of a 0.25 molar phosphate buffer solution (p = 7.0) and a solution of 100 mg Benzyl 7-phenoxyacetamido-3-methyl-3-cephem-4-carboxylate was added to 1 ml of acetone. The mixture was shaken for 16 hours at 28 ⁰ C ¹ overall, to take place the reaction. The yield of 7-amino-5-raethyl-3-cephem-4-carboxylic acid (product I) and its benzyl ester (product II) are given as conversions in Table 6.

Table 6 Microorganisms Product I Product II

Fusarium solani IFQ-8509 35 ° / o I67O Diaporthe phaseolorum IFO-6707 45 $ II96

Example 9

To 30 ml of a 4-day culture broth prepared in the manner described in Example 5 as shown in Table 7 Strains were 7 ml of a 0.25 molar phosphate buffer yielded (p ^ 7.0) and a solution of 100 mg of methy 1-

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thiomethyl ^ -phenoxyacetamido ^ -methyl ^ -cephem ^ -carboxylate. The mixture was ^{shaken at 28 °} C. for 16 hours in order to allow the reaction to take place. The yields of 7-amino-3-methyl-3-cephem-4-carboxylic acid (product I) and / or its methylthiomethyl ester (product II) are given as conversions in Table 7.

Table 7 Microorganisms _: Product I Product II

Aspergillus brevipes IFO-5821

) Fusarium oxysporum. IFO-4469

Diaporthe phaseolorum IFO-6707

2 09830/0968

Claims (11)

Claims 1) Process for the preparation of 7-amino-3-methyl-3-cephem-4-carboxylic acid or their esters, characterized in that 7-acylamido ~ 3-methyl-3-cephem-4-carboxylic acid or their esters, the acyl radical of the 7-acylamino group being a phenylacetyl radical or phenoxyacetyl radical is, with a culture broth of a microorganism, the 7-amine-3-methyl-3-cephem-4-carboxylic acid or their esters from 7-acylamido-3-methyl-3-cephem-4-carboxylic acid or yours. Z-stone by hydrolysis exclusively of the acid amide bond in the case of the 4-carboxylic acid or either by hydrolysis of the acid amide bond only or by hydrolysis of both the acid amide bond and the carboxylic acid ester bond in the case of the 4-carboxylic acid ester is able to form in one aqueous medium or with a processed component of this culture broth. 2) The method according to Anspraeh- ^ tT 'characterized in that the merging with a p "value zw-ts-ehe-ft- 4 and 9 and with a:
noramen will. and at a temperature between 10 and 50 C. 3) Method according to claim 1 and 2, characterized in that that the microorganism is a bacterium of the genus Escherichia, Bacillus, Xanthomonas, Pseudomonas, Protaminobacter, Mycoplana and Aeromonas is used. 4) Method according to claim 1 to 3, characterized in that the substrate is combined with a culture broth of the microorganism. ^; 5) Method according to claim 1 to 3, characterized in that that the substrate with a cell suspension of the microorganism " is merged. BAD ORiQlMAL " 6) Method according to claim 1 to 5, characterized in that that JSscherichia coli uses IFO-3210 as a bacterium will. 7) Method according to claim 1 "to 5, characterized in that Escherichia coli IFO-3467 used as a bacterium
will ο 8) Method according to claim 1 to 5, characterized in that that Xanthömonas raalvacaerum uses IFO-3383 as a bacterium will, 9) Method according to claim 1 to 5, characterized in that 'that Bacillus pumilus uses ΙΙΌ-12Ο93 as a bacterium will ο 10) Method according to claim 1 to 5, characterized in that that Bacillus iicheniformis uses ΙΙΌ-12199 as a bacterium will «■ 11) Method according to claim 1 to 5, characterized in that Bacillus sp ₀ ATCC-14552 is used as the bacterium
will ο 209830/0968