DE2163791C2 - Process for the preparation of 7-amino-3-methyl-3-cephem-4-carboxylic acid and its esters - Google Patents

Description

The invention relates to a process for the preparation of 7-amino-3-methyl-3-cephem-4-carboxylic acid of the formula (I) or its esters from certain 7-acylamido-3-methyl-3-cephem-4-carboxylic acids or their Esters by enzymatic N-deacylation.
The desired product 7-amino-3-methyl-3-cephem-4-carboxylic acid of the formula

" ² ^ ~ 1 I 12

COOH

or its ester at the 4-carboxyl group is an important intermediate for the syntheses of cephalosporins with a broad spectrum of antibiotics, e.g. B. Cefalexin, known.
It is known that the compound (I) or its ester can be prepared chemically by hydrolysis of 7-phenoxyacetamido-3-methyl-S-cephem ^ -carboxylic acid ester or by reducing elimination of the acetoxy group from 7-aminocephalosporanic acid or its ester. In the first-mentioned process, however, difficulties arise in specifically cleaving off the terminal acid amide bond at the 7-position without affecting other parts of the molecule. In particular, in cases where the compound (I) is desired, the ester residue must be a very special radical which is easily hydrolyzable. Furthermore, the yield of the desired compound fluctuates even with a slight deviation from the reaction conditions. In the latter process, the yield is even lower than in the former process.

The first-mentioned method was expanded by the applicant, but in a completely different way The help of an enzyme system of microorganisms, whereby the formation of an industrially feasible Method succeeded as it is characterized by the claim.

It has been found that this conversion is due to the ability of the microorganisms to hydrolyze the amide bond only when 4-carboxylic acid is used as the substrate, and to hydrolyze either the amide bond only or both the amide bond and the carboxylic acid ester bond when the 4- Carboxylic acid ester is the substrate.
In the method according to the invention, 7-phenyl-acetamido- or 7-phenoxyacetamido-3-methyl-3-cephem-4-carboxylic acid or its esters are combined with a culture broth of the microorganism or a processed or treated component of the culture broth in an aqueous medium.

The starting compounds are prepared, for example, by conversion from the corresponding 6-acylamidopenicillanic acid esters via their S-oxides, as in US-PS 32 75 626 and in BE-PS 747118 described.

The starting compound can be in the form of the free acid or as the sodium salt, potassium salt, ammonium salt, Amine salt or another salt can be used. Examples of esters that are used as starting compounds Suitable are sird ~ alkyl esters (e.g. methyl esters, ethyl esters, butyl esters and hexyl esters), substituted alkyl esters (e.g. methylthiomethyl ester, methylsulfenylmethyl ester, methylsulfonylmethyl ester, chloromethyl ester, Bromomethyl ester and trichloroethyl ester, in which the substituent is an alkylthio radical, alkylsulfenyl radical, Alkylsulfonyl radical, halogen atom, etc.) and aralkyl esters (e.g. benzyl esters or phenylethyl esters).

The contact of the substrate with the culture broth or its processed ingredients can be such that the microorganism is cultured in a culture medium containing the substrate when the microorganism is resistant to the substrate. In general, however, the procedure is that the microorganism cultivated in a suitable manner and then the culture broth obtained or its processed components are processed

w) must be turned. The cultivation can be carried out with stirring with aeration, with shaking or stationary, however, organic cultivation is generally preferred. The culture media are prepared by the Components alone or in combination can be selected from the following materials as required: Hay extract, yeast extract, peptone, casein hydrolyzate, corn steep liquor, potato juice or any other commonly used natural substances, carbon compounds, e.g. B. sugar, organic acids or n-

(i5 Paralfins, the most diverse inorganic and organic compounds, the nitrogen form of amino stick, or Contains nitrate nitrogen, phosphates, magnesium salts, common salt or other metal ions and the most varied of vitamins. The addition of phenoxyacetic acid or phenylacetic acid, which is the acyl radical of the substrate to be used corresponds to the culture medium in a concentration of 0.05 to 1%

The consequence is that the activity of the culture broth obtained for the formation of the desired product from the substrate is increased. The appropriate Pn value of the culture medium and the cultivation temperature vary depending on the kind of the microorganism. However, a Pn value in the range of Pn 6 to 9 is usually suitable. The temperature is generally in the range of about 10 to 40 ^° C. The time at which the activity of the culture broth reaches the maximum differs depending on the Kind of the microorganism used so that the optimal cultivation time is determined appropriately for each strain. The culture broth obtained in this way or a processed component of this broth is used for the preparation of 7-amino-3-methyl-3-cephem-4-carboxylic acid or its ester. Anything that has been converted by suitable treatments to increase or concentrate the conversion activity in such a way that it is advantageous for the production of the desired compound can be used as the processed component of the culture broth. For example, if the activity is mainly intracellular, it is expedient to use 1) cell suspensions which contain different 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 have been purified or partially purified from the cell-free extracts by conventional methods. When the activity is mainly extracellularly concentrated, for example 1) supernatant solutions obtained by removing cells from the culture broth, or 2) enzyme solutions purified or partially purified from the supernatant solutions by per se known enzyme purification methods are to be used.

The substrate is combined with the culture broth prepared in this way or its processed constituents in water or any other aqueous medium. The _pH value of the reaction media is advantageously set to a value between 4 and 9, especially 6 to 8 The appropriate reaction time is influenced by the concentrations of the substrates, the activity of the culture broth or its processed ingredients for conversion, the reaction temperature, etc., but 1 to 24 hours is generally sufficient. The reaction temperature is between 10 and 50 ^° C., expediently between 15 and 40 ^° C. The concentration of the substrate is mainly determined as a function of the intensity of the conversion activity to form the desired compound, but generally from a range from 0.1 to 5 % (Weight / volume), calculated as the starting compound and based on the medium, selected.

The 7-amino-3-methyl-3-cephem-4-carboxylic acid or its ester can be prepared under mild conditions by a combination of processes known per se, e.g. B. by extraction with solvents or by chromatography, isolated and cleaned. It is particularly recommended to subject the filtrate of the reaction mixture to column chromatography on the ion exchange resin »Amberlile * XAD-2« and to carry out the elution with water and to collect the fractions that contain the desired product.

The invention is further illustrated by the following examples. There are the percentages of the ingredients the culture medium on a weight / volume basis; d. H. given in grams / 100 ml. The remaining quantities are based on weight, unless otherwise stated. Parts by weight relate to space divide as grams to cubic centimeters. In the tables, "-" means that the product was not detectable. The IFO and ATCC numbers added to the names of the respective microorganisms are the Deposit numbers at the Institute for Fermentation, Osaka, Japan and at the American Type Culture Collection, Maryland, USA:

Escherichia coli IFO-3210 ATCC-21758 Escherichia coli IFO-3467 ATCC-21753 Xanthomonas oryzae IFO-3312 ATCC-21754 Protaminobacter alboflavus IFO-13221 ATCC-21755 Mycoplana sp. IFO-13240 ATCC-21756 Aeromonas hydrophila IFO-12634 ATCC-21757

For Mycoplana sp. IFO13240 the species has not yet been established. Hence the characteristics of the tribe indicated below:

Irregular rods from 0.6 x 2 to 0.6 X 6μ, 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. Agar slant culture: thread-like, light gray, smooth, Broth: cloudy,

Gelatin stab culture: no liquefaction. Thin surface growth. Casein, starch and cellulose are not hydrolyzed. <io

No formation of nitrites from nitrates. No hydrogen sulfide formation. No indole formation. Methyl red test: negative

Catalase: positive ,, 5

Litmus milk: unchanged Potato: no growth No growth or only scant growth in carbohydrate media.

Aromatic compounds, ζ. Β. Phenylglycine, were used. Isolated from the ground.

Example !

A 2-day inoculum (5 roughness) of Escherichia coli IFO-3210 was inoculated into 200 parts of a culture medium which had a p "value of 7.2 and 1.0% meat extract, 1.0% peptone, 0, 5%. Sodium glutamate, 0.5% sodium chloride and 0.05% phenylacetic acid. The culture media was incubated with shaking for 48 hours at 37 ° C, whereupon a solution of 0.8 parts by weight of methyl-T-phenyl-acetamido-J-methyl-S- cephem-4-carboxylate in 8 parts by volume of acetone was added together with 50 parts by volume of a 0.25 molar phosphate buffer solution (pH 7.0). The entire mixture was then shaken at 28 ° C for 16 hours, whereupon the methyl 7-pbenylacetamido-3-methyl-3-cephem-4-carboxylate in the mixture completely disappeared and 0.4 Parts by weight of 7-amino-3-methyl-3-cephem-4-carboxylic acid had been formed therein. The reaction mixture was filtered to remove the cells. The filtrate became column chromatography Phy to "Amberlite * XAD-2" using distilled water as the eluent, whereby 0.35 parts by weight of 7-amino-3-methyl-3-cephem-4-carboxylic acid with a melting point of 240-242 ⁰ C (dec .) were obtained.

Example 2

In the manner described in Example I, 2-day cultures of the bacterial strains listed in Table 2 were prepared. A solution of 800 mg of methyl-phenylacetamido-O-methyl-3-cephem-4-carboxylate (substrate 1) in 8 ml of acetone and 50 ml of a 0.25 molar phosphate buffer solution was added to 200 ml of each culture broth Cpn 7.0) given. A solution of 800 mg of 7-Phe was added to another 200 ml amount of each culture broth nylacetamido-3-methyl-3-cephem-4-cairbonsäure (substrate 2) in 50 ml of a 0.25 molar Phosphatpufferlö solution (p _H 7.0). ^{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 ηημ. The results are given in Table 2, in which methyl 7-amino-3-methyl-3-cephem-4-carboxylate is the product A and 7-amino-3-methyl-3-

30 cephem-4-carboxylic acid is product B.

Table 2 Microorganism substrate 1 substrate 2 Product A Product B Product B

Escherichia coli IFO-3467 Xanthomonas oryzae IFO-3312

⁴⁰ ProtaminobacteralboflavusIFO-13221

Mycoplana sp. IFO-13240 Aeromonas hydrophilic IFO-12634 150 mg

Example 3

An amount of the bacterial strain mentioned in Table 3 was inoculated into 5 ml of an aqueous medium (Ph TO) containing 0.8% "Bacto-Nutrient Broth, dehydrated" (commercially available, manufacturer Difco Laborato ries, USA) and 0.3% phenylacetic acid, followed by incubation with shaking at 28 ° 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-cephem-4-carboxylic acid (substrate 2) was added to the culture broth ) in 1 ml of a 0.25 molar phosphate buffer _{solution (p l (} 7.0) was added. In the case of substrate 1, the mixture was 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 rate mixed showed no antimicrobial activity. This means that the substrates were completely converted. The yield of 7-amino-3-methyl-3-cephem-4-carboxylic acid was determined with the aid of the restored antimicrobial activity after phenylacetylation with phenylacetyl chloride. The results are in the table 3 called.

60 Table 3

400 mg 420 mg 350 mg 300 mg 140 mg 370 mg 370 mg 250 mg - 300 mg

Microorganism substrate 1 substrate 2

Hschcrichia coli IFO-3210 18 mn 15 mg Example 4

A Ösenmenge of Escherichia coli 1FO-3210 was inoculated in 50 parts by volume of an aqueous culture medium _(M p 7.0) containing 1% peptone, 1% meat extract, 0.5% NaCl and 0.3% phenylacetic acid solution, followed by a seed culture Incubation with shaking for 24 hours. The inoculation culture was inoculated into 500 parts by volume of an aqueous medium of the above composition.

The mixture 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 suspended in 50 parts by volume of distilled water.

50 parts by volume of an aqueous solution of 2 parts by weight of 7-phenylacetamido-3-methyl-3cephem-4-carboxylic acid were added to the cell suspension. The mixture was stirred for 3 hours at 37 ° C, the _pH value was maintained by intermittent addition of 0, In-NaOH at 7.0. A small part of the reaction mixture was removed at intervals of 30 minutes and its extinction measured at 260 μm. 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. The results showed that the substrate (Rf 0.50) completely disappeared in the 3-hour reaction and 7-amino-3-ismethyl-S-cephem ^ -carboxylic acid (RfO, O5) was formed. The extinction of the reaction mixture at 260 ΐημ did not change significantly during the reaction.

The reaction mixture was filtered to remove the cells. The filtrate was with the washing liquid for the cells united. The combined solution was chromatographed on "Amberlite * XAD-2". The pillar was eluted with distilled water. Freeze-drying of the eluates gave 1.0 part by weight of pure 7-amino-3-methylO-cephem ^ -carboxylic acid as a powder that is specific with an authentic sample in terms of melting point Rotation, infrared spectrum, nuclear magnetic resonance spectrum and UV spectrum matched well.

Claims (1)

Claim: Process for the preparation of 7-amino-3-methyl-3-cephem-4-carboxylic acid or its esters, thereby ge label «that you can use 7-acylamido-3-methyl-3-cephem-4-carboxylic acid or its esters, the Acylresf. of the 7-acylamido group is phenylacetyl or phenoxyacetyl, with a culture broth of a microorganism from the group Escherichia coli IFO-3210, Escherichia coli IFO-3467, Xanthomonas oryzae IFO-3312, Protaminobacter alboflavus IFO-13 221, Mycoplana sp. IFO-13 240 and Aeromonas hydrophila IFO-12634 in an aqueous medium or with a processed component of this culture broth brings together.