CH617939A5 - Process for the preparation of cephalosporin compounds - Google Patents

Description

The present invention relates to processes for the preparation of a new group of 7a-methoxy-cephalosporin compounds of the following formula:

oh, 3rd

cooh wherein X represents the acetoxy group, the carbamoyloxy group, the hydroxyl group or the hydrogen atom and R the group —COCOOH or the carboxyl group, and a process for the preparation of salts of these compounds.

Compounds of the cephalosporin type have largely been tested as antibiotic substances with antibacterial effects, a number of which have proven themselves in practice. In addition, compounds having a methoxy group in the 7 a position of the cephem nucleus have recently been tested for their excellent antibacterial effects. Derivatives of such 7a-methoxycephalosporin compounds have also been tried extensively. Under these circumstances, starting materials for these 7 a-methoxycephalosporin derivatives were prepared by various methods. For example, a method is known

according to which 3-acetoxymethyl-7a-methoxy-7ß- (5-amino-5-carboxyvaleramido) -3-cephem-4-carboxylic acid is produced by breeding Streptomyces lipmani (U.S. Patent No. 3,719,563). Furthermore, a method has become known, according to which 7β- (5-amino-5-carboxyvaleramido) -3-carbamoyloxymethyl-7 a-methoxy-3-cephem-4-carboxylic acid by cultivating Streptomyces lactamdurans (cf. Japanese Offenlegungsschrift No. 3287/1971) is generated. Other methods have also been disclosed. However, it is extremely difficult to isolate such 7a-methoxycephalosporin compounds obtained from culture broths by a fermentation process of the type mentioned above because a carboxyl group and an amino group are present in the molecule.

In experiments regarding the oxidation of the 5-ammo-5-carboxyvaleramido group present in the 7 a position in order to eliminate the amino group and to isolate the 7a-methoxycephalosporin compounds from the culture broth more easily, it has now been found that the 5-amino-5-carboxyvalale-rylamido group is specific can oxidize to the 4-carboxybutyrylamido or 5-carboxy-5-oxo-valerylamido group by allowing cell masses or enzyme preparations from Trigonopsis variabilis to act. It was also found that the new compounds of the formula I obtainable according to the invention are valuable intermediates for the 7a-methoxycephalosporine derivatives which are active against various infectious diseases.

The aim of the present invention is therefore to create new 7a-methoxycephalosporin compounds of the formula I which are valuable for the synthesis of valuable cephalosporin derivatives.

The present invention is based on the knowledge that the 7a-methoxycephalo-sporine compounds of the formula I can be obtained from compounds of the formula II below by a fermentation process.

According to the methods of the present invention, a new group of 7a-methoxycephalosporin compound

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fertilize formula I and its salts. Among the compounds of the formula I, especially those in which the symbol X denotes the carbamoyloxy group and R denotes the -COCOOH group or the carboxyl group are to be emphasized. As salts of compounds of formula I are alkali metal and alkaline earth metal salts, e.g. the sodium, potassium, calcium or barium salts, and preferably the sodium salts.

The first aim of the present invention is therefore to create a new method for producing a mixture 10 of the compounds of the formulas:

qch3 (ia)

hoocco- (ch2) 3-conh -; -

0 ^ ~ N ^ - CH "X ■

and cooh oh

(ib)

hoog - (ch2) 7 -conh h — f

CH2X 25

cooh or salts thereof, which consists in making a compound of the formula:

och-

00C

+ uh (ch) conh

/ t y n

h3n

(ii)

ch2x cooh

40

wherein X has the above meaning, or a salt thereof, the action of cell masses or enzyme preparations of the yeast Trigonopsis variabilis. 45

According to this process, the mixture of 7 a-methoxycephalosporin compounds of the formula I obtained by fermentation in a culture broth can easily be obtained in good yield.

The starting materials in the present process can thus be any compounds of the formula II which can be obtained by synthesis or by fermentation processes. examples for this are

3-acetoxymethyl-7ß- (5-amino-5-carboxyvalerylamido) -7a-methoxy-3-cephem-4-carboxylic acid, ss

7β- (5-amino-5-carboxyvalerylamido) -

3-carbamoyloxymethyl-7a-methoxy-3-cephem-4-carboxylic acid, 7β- (5-amino-5-carboxyvalerylamido) -3-methyl-7a-methoxy-3-cephem-4-carboxylic acid, 7ß- (5th -Amino-5-carboxyvalerylamido) -3- «o hydroxyrnethyl-7a-methoxy-3-cephem-4-carboxylic acid and their salts, for example their alkali metal salts or alkaline earth metal salts. These connections have already been disclosed. 3-Acetoxymethyl-7ß- (5-amino-5-carboxyvaleryla-s mido) -7a-methoxy-3-cephem-4-carboxylic acid (obtainable by cultivation of Streptomyces lipmani, US) can advantageously be used as starting material for the present process Patent No. 3,719,563), 7β- (5-amino-5-carboxyvalerylamido) -3-

carbamoyloxymethyl-7a-methoxy-3-cephem-4-carboxylic acid (obtainable by breeding Streptomyces lactamdurans, Streptomyces clavurigenes, Streptomyces jumonjinensis etc., Japanese Patent Laid-Open Nos. 3286/1971 and 42 8937 1974 and Dutch Patent No. 7 011 805) , 7ß- (5-Amino-5-carb-oxyvalerylamido) -3-methyl-7a-methoxy-3-cephem-4-carboxylic acid (obtainable by breeding Streptomyces strain WS-3442, Japanese Laid-Open Step No. 26,488 / 1974) u. Like. Use.

The oxidation according to the invention of the 7 β- (5-amino-5-carboxyvalerylamido) -3-cephem-4-carboxylic acid of formula II substituted in the 3-position is based on the action of a D-amino acid oxidase which can be produced under aerobic conditions by a microorganism; the amino group component is oxidized and a mixture of the cephalo-sporin compounds of the above formulas IA and IB is obtained.

The yeast Trigonopsis variabilis is publicly available, for example at the Institute of Applied Microbiology of Tokyo University, Japan, as strain IAM 4443.

When carrying out the present method, the above-mentioned Trigonopsis variabilis strain is advantageously activated in a known manner.

In general, growing a microorganism comprises a vaccine culture and a main culture. In the present invention, the inoculum can usually be prepared using a relatively dense culture medium which contains the usual nutrient sources such as carbon source, nitrogen source, inorganic salt and the like. The like, whereupon the main culture is usually prepared using a culture medium containing a carbon source, a nitrogen source, an inorganic salt and a D-amino acid in order to increase the enzymatic activity and to increase the desired response of the microorganism in an effective and efficient manner can.

The seed culture usually comprises, for example, a sugar, e.g. Glucose, sucrose, maltose, glycerin or other non-carbohydrates, e.g. Paraffin, naturally occurring materials and extracts thereof. The nitrogen source can be, for example, an organic nitrogen compound, e.g. Yeast extract, meat extract, malt extract, various amino acids and inorganic nitrogen compounds such as ammonium nitrate, sodium nitrate or ammonium sulfate. If necessary, you can also inorganic salts such as magnesium sulfate, zinc sulfate, potassium chloride and. Like, admit. The Trigonopsis variabilis strain is preferably inoculated into a seed culture medium which contains the above-mentioned components, the cultivation then being more common. is carried out at 15 to 37 ° C. The period for copious growth of the strain is usually 1 to 5 days.

The main cultivation is then expediently carried out using a culture medium which contains sugars, D-amino acids and salts to increase the oxidase activity of the Trigonopsis variabilis strain which has been achieved in the above-mentioned inoculum culture, the 7β- (5- Amino-5-carboxyvalerylamido) -7a-methoxy-3-cephem-4-car-bonic acid is oxidized; as a result, the substituent present in the 7ß position is converted into a 4-carboxybutyrylamido or. 5-carboxy-5-oxovalerylamido group transferred.

The carbon sources, nitrogen sources and salts which can be used for the cultivation can be the same as in the case of the previously mentioned seed culture. Any D-amino acid such as d-alanine, DL-alanine and the like can be used as the D-amino acid. Like., Use. In particular, D-a-aminoadipic acid will be preferred as a component component of the desired compound.

The Trigonopsis variabilis strain obtained in the inoculum is preferably inoculated into a culture medium,

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which contains the above-mentioned components, whereupon the cultivation is advantageously carried out under aerobic conditions and usually at 15 to 37 ° C. The breeding period is usually about 1 to 7 days. After culturing, the cells produced can be processed in a manner known per se, e.g. by centrifugation. In the present invention, the cells can be used as such. However, activated cells which have been obtained by cell disruption can also be used in order to achieve a more intimate contact between the enzyme and the substrate. Such a digestion method can be, for example, an ultrasound treatment, a treatment with a so-called «French press», a treatment with a surface-active agent, e.g. Cetyltrimethylam monium bromide, sodium deoxycholate, sodium dodecyl sulfate, digitonin, Triton X 100 (trademark of Rohm & Haas Co., USA) and the like. The like, autolysis with an alcohol, e.g. Ethanol, an ester e.g. Ethyl acetate, an organic solvent, e.g. Benzene or toluene, a shock treatment by osmotic pressure, a method by freezing at a temperature between -20 ° C and -50 ° C and thawing and the like. Like. Include. Because of the easy handling, priority will be given to these methods of freezing and thawing, because sufficient enzyme activation and feasibility are also guaranteed at the same time. This freezing and thawing method can be done by heating the cells kept in a freezer in a frozen state after centrifugation, but if possible, an enzyme will be activated by gradually thawing at room temperature.

The process according to the invention can also be carried out in such a way that the activated cells obtained according to the above information are influenced by a 7β- (5-amino-5-carboxyvaleryl-amido) -7a-methoxy-3-cephem- 4-carboxylic acid or a salt thereof. This treatment is usually carried out in a distilled water, physiological saline or a liquid containing a buffer, the pH of which is 6 to 10 and preferably 7.5 to 8.5. The cephalosporin compound used as the starting material is added to the above-mentioned liquid, then the above-mentioned, collected and activated cells are added, and then the reaction mixture is treated under aerobic conditions, usually at 15 to 37 ° C. For this purpose, any synthetic or digestible cephalosporin compound of formula II can be used as the starting material. The enzyme reaction occurring according to the invention consists in that the substrate, namely the 7β- (5-amino-5-carboxyvalerylamido) -7a-methoxy-3-cephem-4-carboxylic acid substituted in the 3-position, is oxidized by the enzyme preparation from Trigonopsis variabilis, whereby the 5-amino-5-carboxyvalerylamido group present in the 7β-position is converted into the 5-carboxy-5-oxovalerylamido group, the desired 7β- (5-carboxy-5-oxovalerylamido) substituted in the 3-position being converted ) -7 a-methoxy ~ 3-cephem-4-carboxylic acid is obtained. The compound thus obtained can be further oxidized by the action of hydrogen peroxide, which is formed in the course of the course of the reaction, with a 7β- (4-carboxybutyrylamido) -7a-methoxy-3-cephem-4-carboxylic acid substituted in the 3-position forms. Thus a mixture of both compounds usually forms. These two compounds can be separated and obtained in a conventional manner in order to isolate the desired compounds in this way.

To achieve a better yield of compound of formula IA (7ß- (5-carboxy-5-oxovalerylamido) -7 a-methoxy-3-cephem-4-carboxylic acid substituted in the 3-position), the effect of the hydrogen peroxide, i. H. the further oxida-

tion of the first compound, inhibited by the given enzyme reaction mechanism of the type described above; this inhibitory effect is achieved through the action of catalase. Catalase is contained in the microorganism used according to the invention, namely Trigonopsis variabilis, but a sufficient amount of catalase will have to be added to bring about the decomposition of the hydrogen peroxide formed in situ by the enzyme reaction and thereby to prevent the action of such hydrogen peroxide.

For a better yield of 7 β- (4-carboxybutyrylamido) -7 a-methoxy-3-cephem-4-car-bonic acid substituted in the 3-position, it is necessary to determine the oxidative effect of the hydrogen peroxide formed in situ during the enzyme reaction on the first to form 7ß- (5-carboxy-5-oxovalerylamido) -7 a-methoxy-3-cephem-4-carboxylic acid substituted in the 3-position. It is therefore necessary to inactivate the catalase in order to enrich it with hydrogen peroxide. The inactivation of the catalase can be effected by treating a suspension of the cells in question with an inactivator or by adding an inactivator to the enzyme reaction mixture. Catalase inactivators include inorganic azides such as sodium azide, ascorbic acid, 3-amino-l, 2,3-triazole, etc. Sodium azide is preferred for practical reasons.

The enzyme reaction in question can be carried out under aerobic conditions at 15 to 37 ° C. The course of the reaction can be prevented by the disappearance of the antibacterial effect of the substrate or by a temporary test by thin layer chromatography of a sample of the new reaction product formed, e.g. 7 ß- (4-carboxybutyrylamino) cephalosporin or 7 ß- (5-carboxy-5-oxovalerylamido) cephalosporin, which can be used to determine the completion of the reaction. To assess the antibacterial effect, any conventional methods can be used, such as are usually used to find an antibiotic substance. For example, if a cephalosporin compound, such as 7β- (5-amino-5-carboxyvalerylamido) -3-carbamoyloxymethyl-7a-methoxy-3-cephem-4-carboxylic acid, is used as the substrate, gram-positive bacteria, e.g. those of the genus Staphylococcus or Bacillus, and gram-negative bacteria, e.g. Use coliform bacilli or those of the genus Proteus. The plate method is usually used as a simple test method.

When the reaction is complete, the cells can be removed immediately under cool conditions by centrifugation, after which the desired products are generally isolated and purified. The isolation and cleaning can be carried out by various combinations of known measures, as mentioned in the examples.

As can be seen from the above, according to the invention, those cephalosporin compounds are obtained which correspond to formula I above. Examples include 7β- (5-carboxy-5-oxovalerylamido) -3-carbamoyloxymethyl-7a-methoxy-3-cephem-4-carboxylic acid, 3-acetoxymethyl-7β- (5-carboxy-5-oxovalerylamido) -7 a-methoxy-3-cephem-4-carboxylic acid, 7β- (5-carboxy-5-oxovalerylamido) -7 a-methoxy-3-methyl-3-cephem-4-carboxylic acid, 7ß- (5-carboxy-5 -oxovalerylamido) -3-hydroxymethyl-7a-methoxy-3-cephem-4-carboxylic acid, 7ß- (4-carboxybutyrylamido) -3-carbamoyloxymethyl-7a-methoxy-3-cephem-4-carboxylic acid, 3-acetoxymethyl-7ß- (4-carboxybutyryl-amido) -7a-methoxy-3-cephem-4-carboxylic acid,

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7β- (4-carboxybutyrylamido) -7 a-methoxy-3-methyl-3-cephem-4-carboxylic acid,

7β- (4-carboxybutyrylamido) -3-hydroxymethyl-7 a-methoxy-3-cephem-4-carboxylic acid,

and their salts with alkali metals or alkaline earth metals.

The 7a-methoxycephalosporin compounds of the formula I are valuable as intermediates for the synthesis of various 7a-methoxycephalosporin compounds which have antibacterial effects. For example, a compound of formula I can easily be reacted with thienylacetyl chloride in cefoxitin, i.e. H. Transfer 7ß- (2-thienylacet-amido) -3-carbamoyloxymethyl-7a-methoxy-3-cephem-4-car-bonic acid, as described in Japanese Patent Application Laid-Open No. 931/1972. It is also easy to convert the compounds of the formula I into the cephalosporin derivatives described in Japanese Patent Application Laid-Open No. 83 386/1975 by the aforementioned method. Examples of valuable compounds obtained in this way are

3-carbamoyloxymethyl-7ß-cyanomethylthioacetamido

7a-methoxy-3-cephem-4-carboxylic acid,

7β-azidomethylthioacetamido-3-carbamoyloxymethyl-

7 <x-methoxy-3-cephem-4-carboxylic acid,

3-carbamoyloxymethyl-7ß-propargyl-

thioacetamido-7a-methoxy-3-cephem-4-carboxylic acid,

3-carbamoyloxymethyl-7ß- (3-isoxazolyloxy) -acetamido-

7 a-methoxy-3-cephem-4-carboxylic acid,

3-carbamoyloxymethyl-7ß-2- (l, 3,4-thia-

diazolyl) thioacetamido-7a-methoxy-3-cephem-4-carboxylic acid, etc.

The 2,4-dinitrophenylhydrazone compound of 7 a-methoxycephalosporin, which bears the 5-carboxy-5-oxovaIerylamido group in the 7 ß-position, shows a 2 to 3 times stronger antibacterial activity against gram-positive bacteria, like Staphylococcus aureus, than the 5-amino-5-carboxyvalerylamido compound used as the starting material.

The present invention also has the advantage that 7a-methoxycephalosporin compounds, whose isolation and purification from culture broths were previously difficult, can now be easily isolated and obtained in a high degree of purity with a good yield.

The invention is illustrated by the following examples.

example 1

(1) Trigonopsis variabilis (SANK 59 963 = IAM 4443) was inoculated in a medium in a test tube on an inclined plate, which was obtained by adding 15 g of agar to a medium which was extracted from 1 liter of beet juice, 20 g of glucose , 1 g of ammonium sulfate and 5 g of potassium dihydrogen phosphate were added, followed by cultivation at 30 ° C for 7 days. A medium was separately prepared which contained 20 g of glucose, 5 g of yeast extract, 4 g of potassium dihydrogen phosphate, 1 g of magnesium sulfate • 7 H20.2 g of ammonium sulfate, 25 mg of iron sulfate • 7 H2O, 500 mg / liter of calcium chloride, the pH being up 6.0 was set. Then 100 ml portions of this medium were placed in 5 flasks with a capacity of 500 ml, followed by sterilization under pressure at 121 ° C (15 lbs.) For 20 minutes. A small amount of the above-mentioned slant culture medium was added as a vaccine to the medium just mentioned, and the culture was shaken for 3 days at 28 ° C and at 120 revolutions per minute to obtain a seed culture broth.

5 liters of a medium containing 20 g glucose, 3 g DL-alanine, 4 g potassium dihydrogen phosphate, 1 g magnesium sulfate • 7 H20.0.5 g calcium chloride, 0.1 g boric acid, 40 mg

Ammonium molybdate, 40 mg manganese sulfate • 7 H20.40 mg zinc sulfate ■ 7 H20.45 mg copper sulfate • 5 H2O, 25 mg iron sulfate • 7 H2O, 20 µg biotin, 100 (ig / liter thiamine hydrochloride (pH = 6.0)) Poured 100 ml portions into 50 flasks, and the contents of the flask were sterilized under pressure (15 lbs.) for 20 minutes at 121 ° C. After cooling, 3 ml of the above-mentioned inoculum culture broth was inoculated and the culture broth for 3 days shaken at 28 ° C. and at 120 revolutions per minute, after culturing, the culture broth was collected from the 50 flasks, cooled to 0 to 5 ° C. and centrifuged at 10,000 revolutions per minute. The cells thus collected were washed twice with water, whereby 100 g of moist cells were obtained. These cells were immediately frozen in a freezer at -20 ° C., whereby a frozen cell mass was obtained. The cells frozen and thawed in this way showed a D-amino acid oxidase activity of 400 b is 600 units per mg protein.

(2) 20 g of a cell paste obtained by freezing and thawing the frozen cells obtained by the above method were added to 500 ml of a 1/10 molar pyrophosphate buffer solution (pH 8.1), which was a 1/10 molar Concentration of sodium azide in solution was added, and the reaction mixture was treated for 60 minutes to inactivate the catalase in the thawed cell suspension. This cell suspension inactivated against catalase was treated with 4 g of cephamycin C, i.e. H. 7ß- (5-Amino-5-carboxyvaleryla-mido) -3-carbamoyloxymethyl-7 a-methoxy-3-cephem-4-car-bonic acid, with a purity of 50%, and the enzymatic reaction was carried out with shaking for 3 hours . New spots were found in thin layer chromatography with the decrease in cephamycin C. After the reaction had ended, the clear part floating on top and the cells were separated from one another immediately by centrifugation. The part flowing on top was adjusted to a pH of 1.5 by adding 2N hydrochloric acid with ice cooling and extracted eight times with equal amounts of ethyl acetate. The ethyl acetate layer was treated with diphenyldiazomethane and the esterified mixture was concentrated under reduced pressure. The concentrate was subjected to thin layer chromatography using a developer consisting of a 3: 2 mixture of benzene and ethyl acetate, and the desired fractions were collected, extracted, and concentrated to give 788 mg of diphenylmethyl-7ß- (4-carboxybutyryl-amido) - 3-carbamoyloxymethyl-7a-methoxy-3-cephem-4-carb-oxylate.

UV spectrum Vax 263 nm

IR spectrum vmax ^ 3 cm_11780 (ß-lactam) N.M.R. spectrum (CDCI3) òppm

1.7-2.7 (6H multiplet)

3.3 (2H AB-quartet, J = 18 Hz)

3.5 (3H singlet)

4.85 (2H AB quartet, J = 12 Hz) 5.05 (1H singlet)

6.86 (1H singlet) 6.94 (1H singlet) 7.35 (20H singlet)

Example 2

In 300 ml 1/10 mol pyrophosphate buffer (pH 8.1) 10 g of the cell paste, which was obtained by freezing and thawing the s obtained in Example 1 (1) above

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frozen cells has been obtained, and 3 g of cephamycin C with a purity of 50% are dissolved and this solution is then mixed with 30,000 units of catalase. The enzyme reaction was continued with shaking at 28 ° C for 3 hours. After the reaction had ended, the top-flowing part and the cells were separated from one another by centrifugation, and the top-flowing part was adjusted to a pH of 1.5 by adding 2N hydrochloric acid with ice-cooling and then extracted in 10 equal portions of ethyl acetate. In this way, the 7 ß- (5-carboxy-5-oxovalerylamido) -3-carbamoyloxymethyl-7a-methoxy-3-ceph-em-4-carboxylic acid was obtained.

In order to confirm the structure of the compound obtained in this way, a concentrated solution of this compound in ethyl acetate was evaporated to dryness and the residue was immediately dissolved in 6 ml of distilled water and this solution with an excess of 0.35% 2,4-dinitrophenylhy- Drazin solution in 2N hydrochloric acid and then the reaction was carried out for 6 hours at room temperature. After the reaction had ended, the reaction mixture was adjusted to a pH of 8.0 by adding 2N sodium hydroxide solution and extracted twice with 100 ml of ethyl acetate in order to remove the excess 2,4-dinitrophenylhydrazine. The aqueous layer was then again adjusted to a pH of 1.5 to 2.0 by adding 2N hydrochloric acid and then extracted four times with 200 ml of ethyl acetate each time. The combined ethyl acetate extracts were concentrated to give 1.38 g of the concentrate.

xMeOH 263 max

UV spectrum

IR spectrum vmax ^ cm-11780 (ß-lactam)

N.M.R. spectrum (acetone-dô) ôppm

1.5-2.6 (6H multiplet)

3.3 (2H AB-quartet, J = 18 Hz)

3.5 (3H singlet)

4.9 (2H AB-quartet, J = 12 Hz)

5.1 (1H singlet)

8.1 (1H AB-quartet, J = 18 Hz)

8.5 (1H AB-quartet, J = 18 Hz, 2Hz)

9.05 (1H doublet, 3 = 2 Hz)

Example 3

A culture broth with a productivity of 750 (xg / ml) cephamycin C, which was obtained by culturing a cephamycin C-producing microorganism (Streptomyces jumonjinensis), was centrifuged to separate the overhead portion from the cells. The top portion was separated into 2 Fractions divided.

(a) To 50 ml of the first fraction (pH 7.60), 2 g of the thawed cell paste from Trigonopsis variabilis, obtained according to the above example 1 (1), and 5000 to 10,000 units of excess catalase were added, after which the carried out enzymatic reaction at 28 ° C. for 180 minutes. The final pH was 6.98. In this way, the 7ß- (5-carboxyl-5-oxovaleryl-amido) -3-carbamoyloxymethyl-7a-methoxy-3-cephem-4-car-bonic acid was obtained at a 50% conversion rate.

(b) 50 ml of a 1/10 molar pyrophosphate buffer solution (pH 8.1) were added to 50 ml of the second fraction (pH 7.60) and the resulting mixture was then stirred thoroughly and then mixed with 2 g of a mixture as described in Example 1 above (1 ) Thawed Trigonopsis variabilis cell paste obtained and 5000 to 10,000 units of catalase added. The enzymatic reaction was then carried out, giving 7β- (5-carboxy-5-oxovalerylamido) -3-carbamoyloxymethyl-7a-meth-oxy-3-cephem-4-carboxylic acid at a final pH of

8.1 and a conversion ratio of approximately 70%.

Example 4

s A culture broth (with a cephamycin C productivity of 750 fig / ml) obtained by culturing a cephamycin C-producing microorganism was centrifuged to separate the overhead and the cells. 500 ml of a 1/10 molar pyrophosphate buffer solution (pH 8.1), which contained sodium azide in such a solution, were separated

that the concentration was 1/10 mol, was added to 20 g of the thawed cell paste obtained from the frozen cells according to Example 1 (1) above, and the resulting mixture was allowed to stand at room temperature for 60 minutes to allow the catalase to the thawed cell paste inactivate.

To this paste thus obtained, 500 ml of the cephamycin C solution obtained after adjusting the pH to 8.1 beforehand was added, and then the enzyme reaction was carried out at 28 ° C for 180 minutes to obtain the 7β- ( To obtain 4-carboxybutyrylamido) -3-carba-moyloxymethyl-7 a-methoxy-3-cephem-4-carboxylic acid at an 80% conversion rate.

The enzyme reaction mixture was centrifuged to separate the 25 above-flowing part. The part thus separated and flowing on top was adjusted to a pH of 6.0 and poured over 9 g of activated carbon in order to adsorb the desired product. The product so obtained was eluted with a 30% aqueous Ace-30 ton solution and the eluates passed through 100 ml of a cation exchange resin, namely Dowex 50 W (H + form; trademark of Dow Chemical Co., U.S.A.). The outflowing liquid was freeze-dried, giving 660 mg (E 5 cm = 70; purity = 44%) of the desired 7β- (4-35 carboxybutyrylamido) -3-carbamoyloxymethyl-7a-methoxy-3-cephem-4-carboxylic acid as a pale yellow Powder obtained in a yield of 77%.

The use of a compound of formula I for the preparation of useful cephalosporin compounds according to 40 of this invention is described below.

(1) 7β- (4 / -Benzhydryloxycarbonylbutyramido) -3-carbamoyloxymethyl-7 a-methoxy-3-cephem-4-carboxylic acid benzhydryl ester 45 A solution of 1.5 g diphenyldiazomethane in 10 ml tetrahydrofuran was added to a solution of 1.3 g slightly crude 7β- (4'-carboxybutyramido) -3-carbamoyloxymethyl-7 a-meth-oxy-3-cephem-4-carboxylic acid in 20 ml of tetrahydrofuran added at room temperature. Then the reaction mixture was stirred overnight at room temperature. The solution was evaporated under reduced pressure. The resulting residue was dissolved in benzene and precipitated with n-hexane, whereby 2.1 g of an oily substance was obtained. The oil was purified by silica gel column chromatography to give 1.71 g of 7β- (4 / -Benzhydryloxycarbonylbutyramido) -3-carbamoyloxymethyi-7a-methoxy-3-cephem-4-carboxylic acid benzhydryl ester in the form of a foam. NMR (CDCI3 + D2O):

1.80-2.65 (6H, multiplet),

3.20 and 3.40 (2H, AB-quartet, J = 18 Hz),

3.44 (3H, singlet),

4.87 (2H, broad singlet), 5.02 (1H, singlet),

6.88 (1H, singlet), 6.93 (1H, singlet),

7.10 - 7.50 (20H, multiplet).

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(2) 3-Carbamoyloxymethyl-7cc-methoxy-7β- (2'-thienylacetamido) -3-cephem-4-carboxylic acid benzhydric ester

(Cefoxitin-benzhydryl ester)

A mixture of 300 mg of 7β- (4'-benzhydryloxycarbonylbu-tyramido) -3-carbamoyloxymethyl-7a-methoxy-3-cephem-4-carboxylic acid benzhydryl ester, 300 mg of N-trimethylsilyltrichloroacetamide, 2.3 ml of thienylacetyl chloride and 7 ml of 1, 2-dichloroethane heated to 500 C over 15 hours. The mixture obtained in this way was then mixed with 0.35 ml of methanol at 0 ° C. and then stirred at room temperature for 2 hours. The reaction mixture was then diluted with ethyl acetate and the solid substance was filtered off and washed with methanol. The combined filtrate was washed twice with a sodium bicarbonate solution and then twice with a saturated saline solution. After drying over sodium sulfate, the organic solvents were removed under reduced pressure, giving 576 mg of a

15

Got oil. This crude substance was purified by preparative thin layer chromatography using silica gel plates (20x40 cm, 2 mm thick), the development being carried out with a mixture of chloroform, ether and acetone in a mixing ratio of 2: 2: 1. Band Rf = 0.51 was collected with ethyl acetate to give 83 mg of 3-carbamoyloxymethyl-7a-methoxy-7β- (2'-thienylacet-amido) -3-cephem-4-carboxylic acid benzyl ester in the form of an oil. NMR (CDCI3 + D2O):

3.22 and 3.29 (2H, AB-quartet, J = 18 Hz),

3.40 (3H, singlet),

3.76 (2H, singlet),

approx.4.83 (2H, a partially resolved peak),

4.99 (1H, singlet),

6.80-7.00 (1H + 2H, multiplet),

7.05 - 7.50 (11H, multiplet).

B

Claims (9)

617939 2. The method according to claim 1, characterized in that X represents the carbamoyloxy group. 2nd cooh and och (ib) hooc- (ch2) 3 ~ conh cooh in which X each represents the acetoxy, carbamoyloxy or hydroxyl group or the hydrogen atom, or their salts, characterized in that a compound of the formula: (ii) oh « ooc ch- (cho), - c0nh gooh in which X has the above meaning, or a salt thereof is subjected to the action of a cell mass or an enzyme preparation of the yeast Trigonopsis variabilis. 2nd PATENT CLAIMS 1. Process for making a mixture of compounds of the formulas: (ia) and hoocco- (ch2) 3-conh gh 3. The method according to claim 1, characterized in that one produces an alkali metal or alkaline earth metal salt as salt. 4. A process for the preparation of compounds of the formula IA given and defined in claim 1 or their salts, characterized in that a compound of the above formula II or a salt thereof under the action of a cell mass or an enzyme preparation of the yeast Trigonopsis variabilis in the presence of a subjecting the decomposition of the hydrogen peroxide formed in situ to a sufficient amount of catalase. 5. The method according to claim 4, characterized in that X represents the carbamoyloxy group. 6. The method according to claim 4, characterized in that an alkali metal or alkaline earth metal salt is prepared as the salt. 7. A process for the preparation of compounds of the formula IB given and defined in claim 1 or their salts, characterized in that a compound of formula II above or a salt thereof under the action of a cell mass or an enzyme preparation of the yeast Trigonopsis variabilis in the presence of an InaktivatoTS the catalase submits. 8. The method according to claim 7, characterized in that X represents the carbamoyloxy group. 9. The method according to claim 7, characterized in that one produces an alkali metal or alkaline earth metal salt as salt.