DK142367B - 7-Acylamidocephalosporan derivatives for use as intermediates in the preparation of 7-aminocephalosporanoic acid or derivatives thereof, and the process for preparing them. - Google Patents

Description

(11) PUBLICATION NOTICE 1 ^ -2367 (W) \ via / DENMARK (5 "" ', CI' c 12 DpB35 / oo § (21) Application No. 4-1 5G / 69 (22) Filed on 1 · 8- Weekly 19 ^ 9 (24) Running Day 1 · Aug. 19 & 9 (44) The application presented and the petition published on · OKU. I The Patent and Trademark System (30) Pinomet begi »the retreat

Aug 2 1968, 37113/68, GB 18 Jul. 1969, 37113/68, GB

(71) GLAXO LABORATORIES LIMITED, Greenford, Middlesex, GB.

(72) Inventor: Benjamin Harry Arnold, Two Pins, Collinswood Road, Farnham Common, Slough, Buckinghamshire, GB: Robert Anthony Fildes, Romanile,

Birch Tree Grove, Ley Hill, Chesham, Buckinghamshire, GB: David Arthur Gilbert, 29, Spring Lane, Farnham Royal, Slough, Buckinghamshire, GB, (74) Plenipotentiary:

Office for Industrial Excellence by Svend Schønning.

(54) 7-Acylamidocephalosporan derivatives for use as intermediates in the preparation of 7-aminocephalosporanoic acid or derivatives thereof, and the process for their preparation.

The present invention relates to novel 7-acylamidocephalosporan derivatives for use as intermediates in the preparation of 7-aminocephalosporanoic acid or derivatives thereof having 3-sled chain of the formula -CH

The cephalosporin compounds mentioned in this specification are named from the substance cepham which has the structure.

o ^ "V

142367 2 (see J.A.C.S. 1962, 84, 3400 and J. Chem. Soc. 1965, 5031).

Cephem denotes the basic cepham structure with a single double bond.

Cephalosporin C, (3-acetoxymethyl-7-3- (D-5-amino-5-5 carboxypentanamido) -ceph-3-em-4-carboxylic acid) can be converted to 3-acetoxymethyl-73-aminoceph-3-em. 4-carboxylic acid (7-amino-cephalosporanoic acid, 7-ACA) and then to 73-acylamido analogues of cephalosporin C with modified antibacterial activity. N-Deacylation of cephalosporin C is difficult, probably due to the structure of the D-5-amino-5-carboxypentanoyl side chain. Although 7-ACA can be prepared from cephalosporin C in good yield by nitrosyl chloride, alternative methods are needed that can offer benefits.

From i.a. French Patent Specification 1,394,820 and its Appendix No. 89,317, it is known to convert cephalosporin C or derivatives thereof with other 3-side chain to 7-ACA or derivatives thereof with 3-side chain as in the starting material by the so-called imide halide method disclosed later in this description. The conversion requires isolation of the starting compound, especially cephalosporan C, from the medium in which it is formed by fermentation, and it is a difficult process because the zwitterion charge on the 7-side chain renders cephalosporin C unsuitable for solvent extraction. In addition, it is usually necessary to modify the extracted cephalosporin C, for example, the protected amino group in the 7-side chain and esterify the 4-carboxyl group before the first step of the iminohalide method, which is most frequently reacted with phosphorus pentachloride.

It has now surprisingly been found that certain cephalosporin derivatives with a particular 7-side chain can be readily isolated from a fermentation medium, readily formed in the fermentation medium by oxidation of cephalosporin C, and after isolation can be readily converted to 7-aminocephalosporanoic acid by the imide halide method; analogous conditions apply to some analogs with the initially mentioned 3-sided chain.

Said 7-side chain is a 4-carboxybutanamido group and the 7-acylamidocephalosporin derivatives of the invention are therefore characterized in that they have the general formula 3

HOOC- (CH2) £ ONH - / N.

J ν ^ - ° Η2Χ

0 T

COOH

wherein X has the meaning given above, or is made up of salts thereof.

The compounds of formula I can be readily converted to 7-ACA 5 by, for example, the imide halide method. It consists in reacting the 7β-acylamido compound with an agent believed to form an imide halide, e.g., phosphorus pentachloride, PCl the reaction product is then converted by reaction with an alcohol to a product believed to be an imino ether and this is cleaved, for example 10 by hydrolysis, to the desired 7-amino compound. The imide halide method is disclosed in British Patent Specification No. 1,041,985, Belgian Patent Nos. 718,824 and 719,712 and Dutch Patent Publication No. 68,12413.

The invention also relates to a process for the preparation of the 7-acylamidocephalosporin derivatives of formula I,

wherein X is as defined, or salts thereof, and this process is characterized by subjecting a cephalosporin compound of the general formula II

~ OOCt S

,]> CH (CHJ ^ CONH --- / \ 20 H3N 1 11

/ -V> CH 2 X

COOH

or a salt thereof, wherein X has the aforementioned meaning, action of a fungal D-amino acid oxidase in the absence of catalase or inhibition of any catalase present in the D-amino acid oxidase and then isolating the compound formed.

For example, the fungal D-amino acid oxidase may be derived from species of Aspergillus, e.g., A. flavus, A. parasiticus, A. flaus oryzae, or A. ustus; or from species of Penicillium, e.g., P. chrysogenum or P. roquefort !; or from species of Neurospora. As another option, the D-amino acid oxidase may be derived from a yeast species. A very convenient source of functional 5-gal D-amino acid oxidase according to the invention is the yeast fungus Tri-gonopsis variabilis, this enzyme yielding high yields. In addition to Trigonopsis variabilis, preferred oxidases are also derived from Aspergillus flavus oryzae. Suitable strains of organisms can be obtained from known cultural collections, such as from the 10 Commonwealth Mycological Institute, Kew, Richmond, Surrey,

England ^ the appropriate strains A. ustus IMI16045, A. flavus IMI52104, A. parasiticus IMI 15957, and A. flavus-oryzae IMI 44242; Trigonopsis variabilis is available from the Centraal Bureau voor Schimmelcultur, Baarn, The Netherlands.

Although it is not necessary to induce the formation of D-amino acid oxidase in the fungal cells, it has been found convenient to increase the enzyme yield. Conveniently, according to the invention, the induction may have been effected by the addition of the D-form or the DL racemate of a common amino acid, preferably alanine, to the fermentation in the seed stage. The type and amount of amino acid needed to induce the development of the D-amino acid oxidase in specific fungal cells can be determined by prior tests and experiments. Thus, formation of D-amino acid oxidase can be induced in Aspergillus flavus and parasiticus by DL-valine and DL-alanine; the latter is particularly effective at a concentration of 0.20% (w / v). Furthermore, methionine has been found to effectively promote the formation of D-amino acid oxidase in Trigonopsis variabilis.

The D-amino acid oxidase must be released from the cells before use, as the whole cells are generally inactive. This can be done either by forming a "powder" or by forming cell-free extracts.

In the preparation of "powder", the cells are filtered from the culture medium and a wet cell suspension is prepared. This can be stored if desired. The suspension may be treated with suitable solvents such as acetone to effect the cell wall bursting, followed by filtration of the solids, washing of the solids and drying. In this way, the formation of "powders" is normally carried out with acetone, but it is not essential that the solvent 5 used causes the cell wall to rupture and does not denature the enzyme. Further purification of acetone powders is achieved by grinding.

In the preparation of cell-free extracts, the wet cell suspension is first treated to cause rupture of the cell walls, for example, by treatment with a solvent such as acetone, by alternate freezing and thawing, by autolysis, by degradation by papain, by rupture by suction treatment, or by homogenization. Cell residues are then removed from the resulting material, for example by centrifugation, leaving a residue extract containing D-amino acid oxidase and catalase. Further concentration and purification of the extract can be effected, for example, by gel filtration, as is the case with Aspergillus extracts, or fractionation with ammonium sulfate, which has been found to be most satisfactory to obtain D-amino acid oxidase from Trigonopsis extracts.

The effect of D-amino acid oxidase can be best determined spectrophotometrically by determining the rate of formation of hydrogen peroxide using the hydrogen donor o-dianisidine as indicator. In the presence of peroxidase, o-dianisidine is oxidized by hydrogen peroxide to form a brown dye.

In the reaction with the D-amino acid oxidase, as mentioned, catalase must be absent or inhibited. It is generally not necessary to purify the D-amino oxidase for all traces of catalase, as its effect can be inhibited. Suitable catalase inhibitors are ascorbic acid, 3-amino-1, 2,3-triazole and inorganic azides. Sodium azide is particularly preferred. The sodium triazide concentration may be as low as 1 mM using D-amino acid oxidase extracts, but using acetone powders up to 100 mM or even more may be desirable.

142367 6

The reaction of the enzyme system with the compound of the general formula II can be carried out at pH 6-9 and preferably approx. 8. Temperatures between ambient and 50 ° C can be used, the optimum temperature is approx. 33 ° C. Do not use pH and temperature resulting in deactivation of the enzyme. An enzyme concentration of not less than 20 units (as defined below) per mg of cephalosporin starting material may be used. At this concentration, a reaction time of at least 3 hours may be necessary. With higher enzyme concentration, eg 10 40-60 units / mg, shorter reaction times such as 2 hours are possible. The oxidation rate of compounds of general formula II is also proportional to the concentration of compound of general formula II in the enzymatic oxidation system. Stirring with gas flow, either with air or oxygen, is desirable. One gas flow rate of 1 volume gas / volume system per per minute gives good results.

As mentioned, cephalosporin C is difficult to extract from the fermentation liquid due to its amphoteric structure and hydrophilic nature. The process of the invention can be carried out in situ, before or after removal of the mycelium, in a cephalosporin C fermentation liquid under appropriate conditions to form the compound of the general formula I wherein X is acetoxy. The process is preferably carried out after 25 proteins have been filtered out. Then, the resulting compound I can be obtained by solvent extraction or by absorption in a column of ion exchanger.

Conversion of cephalosporin C to 3-acetoxymethyl-7-3- (4-carboxybutanamido) -ceph-3-em-4-carboxylic acid in a deprotected cephalosporin liquid requires a significant excess of enzyme compared to what is needed when starting with already isolated cephalosporin C; it is due to the presence of oxidase inhibitors in the fluid.

The compound of general formula I wherein X is an acetate group can be readily extracted from the aqueous solution in which it is prepared, for example, by acidification to pH 1.5 or lower and extraction with a suitable organic solvent such as ethyl acetate or n-butanol. Several ethyl acetate extractions will provide substantially complete extraction, but the efficacy may be increased by saturating or substantially saturating the aqueous phase with a soluble inorganic salt, e.g., sodium chloride. However, this process is not very suitable for extracting the compounds from the fermentation liquid.

In isolating the products from both fermentation liquids and simple aqueous solutions, it has been found according to the invention that a combination of ion exchange and solvent extraction provides particularly good yields and purity. Suitable ion exchangers are the high molecular weight liquid amine anion exchangers sold under the names "Amberlite" ^ LA1, LA2 and LA3; LA1 and LA2 are secondary amines, LA3 a primary amine. Preferred solvents for use with the liquid ion exchangers are n-butanol and butyl acetate.

One system that has been used with particular advantage in extractions from de-proteinized cephalosporin fermentation liquid is "Amberlite" ® LA1 in n-butanol, followed by back extraction with sodium bicarbonate solution and extraction with 20 ethyl acetate. The pH of the liquid is preferably lowered to below 6 and most conveniently to 3-5 before the extraction.

The solid anion exchange resin "Amberlite" yXAD2, which is a macroreticular cross-linked polystyrene polymer, can also be used to extract compounds of the general formula I, wherein X is an acetate group, from purified or deproteinized cephalosporin fermentation liquids. A suitable solvent for eluting the absorbed compound from the resin may be determined by preliminary experiments. In the case of 3-acetoxymethyl 1-7- (3- (4-carboxybutanamido) -ceph-3-em-4-carboxylic acid, a suitable solvent is acetone.

Compounds of general formula I wherein X is a hydroxy group or a hydrogen atom can be recovered from the aqueous media in which they have been prepared by the process already described.

The intermediate product and method of the invention will be elucidated in the following by some experiments and examples, followed by two examples of conversion of the intermediate into 7-ACA.

8 142367

Initial trial

Aspergillus flavus oryzae and Trigonopsis variabilis.

In the initial experiments, both cultures were grown in 250 ml shake flasks containing 50 ml medium. Aspergillus 5 was grown in nutrient medium (2% corn cast water and 2% glucose in water with pH adjusted to 5.0) containing 0.2% alanine at 27 ° C and harvested after 48 hours. Trigonopsis was grown in the medium containing methionine described by Senthe-shanmuganathan and Nickerson, 1962, J. Gen. Microbiol. 27, 10 465, at 27 ° C and harvested after 72 hours.

Preparation of cells.

Moist cell suspensions. The cells were filtered on No. 1 Whatman filter paper, washed twice with distilled water and stored at -10 ° C. An appropriate amount was thawed if necessary and suspended in a 0.01M buffer at pH 8.1 (sodium pyrophosphate or ethanolamine).

Preparation of acetone powder.

Acetone Powder. The cell suspension was added with stirring to 10 volumes of acetone (w / v). The suspension was thoroughly mixed and then filtered through No. 1 Whatman paper and dried with a little ether.

Preparation of cell-free extracts.

Overlydssønderdeling. The suspension of moist cells was treated with sound at 4 ° C at 20 Kc / sec in a 60 watt super sonic 25 parts.

Homogenization. The wet cell suspension was cooled to about 15 ° C and then passed through an "A.P.V. Manton Gaulin Laboratory and Submicron Disperser" (type 15M-8BA SMD).

After each passage through the machine, the homogenate in an ice bath was cooled.

The extracts prepared by these processes were trifuged at 38,000 G for 15 minutes to remove cell; Distributors residues.

i) i 142367 9

Purification of Amino Acidase (AAO).

Purification of Aspergillus Amino Acidase by Gel Filtration.

An extract from Aspergillus was lyophilized, resuspended in 0.001 M sodium pyrophosphate buffer pH 8.1 (1/10 of the original volume) and clarified by centrifugation at 38,000 G for 15 minutes.

10 ml of concentrated extract was added to a column (100 x 2.5 cm) of Bio-gel P.2 or P.100 and developed at a rate of 50 ral / h with 0.002 M sodium pyrophosphate buffer pH 8.1. The eluate, controlled with a LBK Uvicord at 260 nm, was collected in 10 ml fractions.

Purification of Trigonopsis Amino Acidase by (NHis JSO ^ Fractionation. 15 400 ml of Trigonopsis extract was saturated 30% with (NH ^) at 4 ° C. The solution was carefully mixed to avoid foaming and then left to stand for 30 minutes. The suspension was centrifuged at 23,000 G for 30 minutes at 4 ° C. The supernatant was saturated 50% with 20 (NH 2 SO 2), stirred for 5 minutes and left for a further 30 minutes. The precipitate was collected by centrifugation at 38,000 G for 30 minutes. and resuspended in 0.1M sodium pyrophosphate buffer.

Determination of D-amino acid oxidase activity.

Amino acid oxidase (AAO) was determined spectrophotometrically by following hydrogen peroxide formation rate

The method is based on the coupled reactions shown in Equations 1 and 2.

In AAO) (1) amino acid + H 2 O + O 2 - v α-keto acid + NH

(2) H 2 O 2 + DH 2 -> 2 H 2 O + D

Hydrogen peroxide in the presence of peroxidase (POD) oxidizes hydrogen dono o-dianisidine (DH 2) to a brown dye.

142367 10

The test was performed at 37 ° C in a glass cuvette with a 1 cm light path and the formation of the brown dye was monitored at 420 nm. The reaction mixture consisted of 1.0 ml of 0.1M sodium pyrophosphate buffer pH 8.1, 0.5 ml of o-dianisidine solution (0.04% o-dianisidine HCl in water), 0.3 ml of substrate (1% sodium cephalosporin in sodium pyrophosphate buffer pH 8.1), 0.01 ml of peroxidase (10 mg / ml aqueous solution) and sufficient water until the volume eventually becomes 2.8 ml.

The reaction starts by adding 0.2 ml of enzyme solution to the reaction mixture. The comparison cuvette contained 2.8 ml of water and 0.2 ml of enzyme solution.

The linear increase in optical density at 420 nm for the first 5 minutes was used to measure AAO activity.

An enzyme activity unit is defined as the amount of enzyme 15 which at 37 ° C pH 8.1 produces a change in the optical density of 0.001 / minute.

Horizontal paper electrophoresis.

10 μΐ samples were placed on strips of Whatman 3MM chromatography paper (2 cm x 35 cm) and electrophoresed against the anode 20 for 2 hours at 500 volts in 0.01 M phosphate buffer pH 7.0. The paper was dried at ambient temperature. The compounds were located by ultraviolet light examination. The absorption zones were cut out and soaked in 5% sodium bicarbonate (5 ml) for up to 60 minutes. The absorption of the solution was read at 257 nm and used to calculate the amount of cephalosporin compound present.

Thin-layer chromatography (TLC)

Samples of 5 µl were placed on a cellulose TLC plate and developed with 70% propanol. The compounds are located by examining the plate under ultraviolet light.

"Amberlite" ®ΪΑ1, LA2 and LA3.

15% s (volume / volume) solutions of the liquid amines in n-butanol were prepared and saturated with water. The solvent was regenerated by shaking 3 times with 4N sodium hydroxide saturated with n-butanol and washing with water saturated with n-butanol until the washing solutions were neutral. "Amberlic LA1 can also be recovered by distillation under right pressure, cpt. 210 ° C / 10 mm Hg.

"Amberlite" ® XAD2.

5 20 g and 100 g columns of "Amberlite" ® XAD2 preparation are conducted by suspending the resin in acetone, standing for 15-30 minutes, then washing with water in an amount equal to 20 times the volume of the substance in the column.

Dinitrophenylhydrazine (D.N.P.) sample of amino acid oxidase.

The enzyme activity was measured by following the formation of 3-acetoxymethyl 1-7- (3- (5-carboxy-5-oxopentanamido) -ceph-3-em-4-carboxylic acid columetrically as its phenylhydrazone derivative.

Acetone powder in 5 ml of 0.1 M pyrophosphate buffer pH 8.1 containing 1250 units of catalase (sigma units) and 0.05 mg of 15 flavein adenine dinucleotide were mixed and incubated at 33 ° C.

with 5 ml of 6% s cephalosporin C solution previously adjusted to pH 7.9 with 0.1N sodium hydroxide. Samples of 1 ml were taken at 0 minutes and after 15 minutes incubation, thoroughly mixed with 2 ml of 0.35% s 2,4-dinitrophenylhydrazine in 2N HCl, then extracted with 8 ml of ethyl acetate. 6 ml of the ethyl acetate layer was back extracted with 5 ml of 0.4 m sodium bicarbonate sodium carbonate buffer and the yellow color of 2,4-dinotrophenylhydrazone was measured at 370 nm.

The amount of dinitrophenylhydrazone formed was calculated by a calibration curve prepared with purified 2,4-dinitrophenylhydrazone: 1 enzyme unit is defined as the amount which produces 1 ymol of 3-acetoxymethyl 1-7- (3- (5-carboxy-5) oxopentanamido) -ceph-3-em-4-carboxylic acid per minute under the standard conditions used.

A sigma unit of catalase is defined as the amount of catalase that can decompose 1 µmol of hydrogen peroxide per liter. per minute at pH 7.0 and 25 ° C, while the peroxide concentration drops from 10.3 to 9.2 µmol per minute. ml of reactants.

142367 12

Example 1 5 mg / ml cephalosporin C, 100 mM sodium azide and a freeze-dried cell-free extract (obtained by noise decomposition) of an amino acid oxidase preparation obtained from Aspergillus flavus-5 oryzae (40 units per mg cephalosporin C) was incubated at 30 ° C. , 1M sodium pyrophosphate buffer pH 8.1. 10 µl samples were taken at 30 minute intervals and examined by electrophoresis on paper.

The process was repeated with amino acid oxidase, which was partially purified by gel filtration.

The electrophoresis papers were examined under ultraviolet light. The electrophoresis papers from the experiments using crude amino acid oxidase preparation (freeze-dried cell-free extract) showed a zone of the desired product, 3-acetoxymethyl-7β- (4-carboxybutanamido) -ceph-3-em-4-carboxylic acid, in the incubation sample. was taken after 30 minutes; it increased in strength and was strongest in the samples taken after 2 and 2 1/2 hours. Incubation for another 4 hours neither increased the size nor the strength of the zone.

The electrophoresis results of the method using partially purified amino acid oxidase showed that the cephalosporin C zone disappeared completely after 2.5-3 hours of incubation and that the zone corresponding to the desired product reached a maximum intensity around the same time.

Example 2

Two 100 ml systems containing 5 mg / ml cephalosporin C, 10 mM sodium azide and amino acid oxidase (60 units / mg cephalosporin C), obtained by homogenization of cells of Trigo-nopsis variabilis, were incubated at 33 ° C. One system was gently stirred, the other stirred and aerated with a rotary cross at about 100 ml air / min.

The results on the thin-layer chromatography plates showed that all cephalosporin C in the aerated system had been oxidized after 2 hours of incubation, with 75% of the oxidation product being 3-acetoxymethyl-73- (4-carboxybutanamido) -ceph-3-em-4 carboxylic acid.

U 2367 13

The non-aerated system showed only weak oxidation of cephalosporin C, even after 4 hours of incubation.

Example 3 3-Acetoxymethyl-7β- (4-carboxybutanamido) -ceph-3-em-4-carboxylic acid from cephalosporin C fermentation. 300 ml of an acidified filtrate of cephalosporin C fermentation liquid were adjusted to pH 8.1 by 10 N sodium hydroxide and treated with 32.5 mg ImM sodium azd added 4 x 10 units Trigonopsis variabilis enzyme preparation obtained by homogenization and ammonium sulfate fractionation and pH was again adjusted to 8.1. The mixture was then incubated at 33 ° C and stirred (150 stirring / min), under aeration with moist air at a volume of 1 v / v / min. The foam was checked with "M.S. antifoam R.D." silicone antifoaming agent. Samples were removed at one hour intervals for examination and the reaction was terminated after 4 hours. Quantitative paper electrophoresis tests showed that 95% of the cephalosporin C present was converted to 3-acetoxymethyl-70- (4-carboxybutanamido) -ceph-3-em-4-carboxylic acid.

Example 4

Extraction and purification of 3-acetoxymethyl-73- (4-carboxybutanamido) -ceph-3-em-4-carboxylic acid.

In the first section of Example 2 with aeration, cephalosporin C in solution was converted into 3-acetoxymethyl-78- (4-carboxybutanamido) -ceph-3-em-4-carboxylic acid. 450 ml of the solution was adjusted to pH 2.3 with sulfuric acid and extracted 4 times with 150 ml of 15% v / v "Amberlite" ® LA1 in n-butanol by stirring for 6 minutes with pH meter at pH 4.0 + 0.3, using sulfuric acid 30 to adjust the pH. The solvent extracts were back-extracted with 6 x 75 ml of 1M sodium bicarbonate. The combined aqueous extracts were washed with 2 x 200 ml of ethyl acetate, acidified with sulfuric acid and extracted with ethyl acetate (1 x 150 ml and 5 x 100 ml). The combined solvent extracts were washed with 3 x 20 ml of saturated sodium chloride, dried and concentrated under reduced pressure to 15 ml. Addition of 300 ml of petroleum gave a sticky precipitate which was dissolved in acetone and distilled to dryness leaving 0.897 g of white glass.

5 By direct ultraviolet sample, this solid was found to be 89.5% pure (79% extraction).

Example 5

Extraction and purification of 3-acetoxymethyl-7 (3- (4-carboxybutane-amido) -ceph-3-em-4-carboxylic acid from fermentation liquid using "Amberlite" LA1._ 100 ml of cephalosporin C deproteinized Fermentation liquid containing 0.05 g of 3-acetoxymethy 1-7 (3- (4-carboxybutanamido) -ceph-3-em-4-carboxylic acid was acidified to pH 2.0 with sulfuric acid and extracted twice with 60 ml of 15% vol. The "Am-15 berlite" ® LA1 in n-butanol pH was adjusted to 2.0 before the second extraction. The solvent extracts were combined, washed with 10 ml of water and extracted with 3 x 20 ml of 1M sodium bicarbonate. The combined aqueous extracts were washed with 2 x 20 ml of ethyl acetate, acidified with sulfuric acid and extracted with 3 x 25 ml of ethyl acetate. After washing with 10 ml of water and 2 x 15 ml of saturated sodium chloride and drying, the ethyl acetate extract was stirred for 3 minutes with 0.5 g of SS No. Charcoal, filtered and distilled under reduced pressure to 10 ml. Treatment of the concentrated extracts with 250 ml of petroleum gave a sticky precipitate which was dissolved in acetone and distilled to dryness under reduced pressure, leaving 0.493 g of white glass. By direct ultraviolet sample, 1% found that this solid has E. ° = 185, λ = 260 nm.

limb max (Purity = 80.5%, 70.5% extraction).

Example 6

Extraction and purification of 3-acetoxymethyl-7β- (4-carboxybutanamido) -ceph-3-em-4-carboxylic acid from fermentation using "Amberlite" XAD2. 100 ml of cephalosporin C deproteinized fermentor containing 1423B7 0.397 g of 3-acetoxymethyl-7β- (4-carboxybutanaraido) -ceph-3-em-4-carboxylic acid was adjusted to pH 8.2 with 4N sodium hydroxide. A heavy precipitate which formed was removed by centrifugation and the supernatant was passed through an Amberlite ® XAD2 column of 20 g at 2-3 ml / min. The combined filtrate and wash was adjusted to pH 2.7 with sulfuric acid and passed through another column of "Amber-lite" ® XAD2 of 20 g at 1-2 ml / min. After washing with water, the second column was eluted with 120 ml of acetone. The acetone was distilled off from the eluate under reduced pressure and the aqueous residue was freeze-dried to give 0.595 g of a pale brown solid. By direct ultraviolet test, this solid was found to be 65% pure.

Example 7 Conversion of a Compound of the Invention to 7-ACA A suspension of 772 mg (0.002 mol) of 3-acetoxymethyl-7β- (4'-carboxybutanamido) -ceph-3-em-4-carboxylic acid in 7 ml of methylene dichloride was solubilized by addition. of 0.55 ml (0.0039 mol) of triethylamine, and then 0.515 ml of 20 (0.0045 mol) of trimethylchlorosilane was added to the resulting solution.

The reaction mixture was stirred for two hours, with 1.92 ml (0.025 mol) of pyridine and, after cooling to -10 ° C, a solution of 1.246 g (0.006 mol) of phosphorus pentachloride in 12 ml of methylene dichloride was added dropwise. The suspension was stirred at -10 ° C for 35 minutes, 5 ml of ice-cold methanol was added dropwise and the solution was stirred at 0 ° C for an additional 3 hours before pouring into 2 ml of water. The pH was adjusted to 3.5 with 6N sodium hydroxide and after standing at 0 ° C for 16 hours 0.033 g (6%) of 3-acetoxymethyl-7 & -30 amino-ceph-3-em-4 was isolated. carboxylic acid (7-ACA) by filtration, [α] D = + 85.9 ° (c = 0.99 in 0.2N, pH 7.0 phosphate buffer), λ 261 nm (e = ΤΠαΧ · 6,900) , pH 6 phosphate buffer. Electrophoresis at pH 1.9 showed that the sample was identical to authentic material.

Example 8

Converting a Compound According to the invention to 7-ACA.

0.515 ml (0.0045 mol) of trimethylchlorosilane was added to a solution of 777 mg (0.002 mol) of 3-acetoxymethyl-73- (4'-carboxybutanamido) -ceph-3-em-4-carboxylic acid in 7 ml of chloroform containing 0.55 ml (0.0039 mol) of triethylamine and after stirring at room temperature for 2 hours, 0.64 ml (0.008 mol) of pyridine was added and the solution was cooled to -10 ° C and treated with a solution of 456 mg (0.0022 mole) of phosphorus-pentachloride in 10 ml of chloroform over 2 minutes. The reaction mixture was stirred at 0 ° C for 30 min, 5 ml of ice-cold methanol was added dropwise at -10 ° C and the solution was stirred at 0 ° C for an additional 3 h before pouring into 2 ml of water. The pH was adjusted to 3.5 with 6N sodium hydroxide and after standing the two-phase system at 0 ° C for 16 hours, by filtration, 0.105 g, 20%, of the precipitated 3-acetoxymethyl-78-aminoceph-3-em-4-carboxylic acid was isolated. (7-ACA). [<x] p ° = + 92.7 ° (c = 1.11, 0.2M, pH 7.0 phosphate buffer), λ 262 nm (ε = rosx · 6.80.0) in pH 6 phosphate buffer. Electrophoresis at pH 1.9 showed that our sample was identical to authentic material.

Claims (2)

1. 7-Acylamidocephalosporine derivatives for use as intermediates in the preparation of 7-aminocephalosporanoic acid or derivatives thereof having 3-side chain of the formula -CH 2 X, wherein 5. is a hydrogen atom, a hydroxy group or an acyloxy group having a maximum of 3 carbon atoms, characterized in that they have the general formula Hooc- (ch2) 3conh-rvs-o "; ch7x COOH where X has the meaning given above, or is made up of 10 salts thereof. A process for the preparation of the 7-acylamidocephalosporin derivatives of claim 1 of the general formula I wherein X has the meaning, or salts thereof, characterized by subjecting, under aerobic conditions, a cephalosporin compound of the general formula “OOC . S o T ch 2 x COOH or a salt thereof, in which formula X is as defined in claim 1, the action of a fungal D-amino acid oxidase in the absence of catalase or inhibition of any catalase present in the D-amino acid oxidase 20 and then isolates the compound formed .