SE192426C1 - - Google Patents

Description

Inventors: EP Abraham and GGF Newton Priority requested from 2 February and 16 June 1955 (UK) It has already been shown that a nutrient medium, if contaminated with mold of the species of which Cephalosporium IMI 49 137 (American Type Culture Collection No. 11 550) Or a member, father antibiotic activity. The isolation of Lva various antibiotics from the agent has already been described in detail. One of these antibiotics is mainly effective against gram-positive bacteria and cephalosporin P.

British Patent Specification 745,208 discloses a fermentation process for the preparation of cephalosporin N and various processes for the preparation of cephalosporin N in pure form.

The present invention is based on the observation that if the cephalosporin N in the preparation of the said Mune OverfOres is in an inactive form, the cephalosporin N penicillin acid the preparation nevertheless to exhibit antibiotic activity ph due to the presence of a Latin unknown substance. This third antibiotic material, now called cephalosporin C, Or as well as cephalosporin N is active against both gram-positive and gram-negative bacteria but in contrast to cephalosporin N is insensitive to penicillinase produced by B. subtilis. The new material is soluble in water and almost insoluble in ethanol and ether. It can be identified by its ultraviolet absorption spectrum, which, da. the antibiotic is in the form of an aqueous solution of its sodium salt with a specific rotation of [a] r + 103 °, has an ultraviolet absorption maximum at 260 miz.

By the present invention sAlunda Dupl. at 30 h: 2/03 an antibiotic, which in turn is cephalosporin C and can be obtained in admixture with cephalosporin N penillic acid, if a preparation of cephalosporin N is taken at such a pII value that cephalosporin N is converted to cephalosporin N-penillinic acid. This antibiotic, cephalosporin C, has activity against both gram-positive and gram-negative bacteria, is stable at pH 2.5-5, soluble in water and almost insoluble in ethanol and ether. It shows further, dd. it is in the form of an aqueous solution of the sodium salt, cii specific torsional shape of [a] g + 103 ° and an ultraviolet absorption. maximum at 260 mid.

The above-mentioned British patent specification 745 208 comprises i.a. a. a process for purifying cephalosporin N by separating the mycelium from the liquid, subsequent adsorption of cephalosporin N from the clear liquid ph activated carbon and darph following elnation of the antibiotic substance from the adsorbent. The eluate is contacted with alumina and the absorbed antibiotic is then eluted therefrom. It has been found that cephalosporin C is present together with cephalosporin N in the eluate obtained from the alumina. This eluate is derived from a starting material for cephalosporin C. Other raw materials for cephalosporin C consist of the liquid itself or of solutions or preparations obtained therefrom. Those skilled in the art can readily determine during which stage of purification at some particular precursor cephalosporin N can be recovered without eliminating cephalosporin C. A preparation of cephalosporin N can thus be exposed to the action of a suitable penicillin preparation or maintained at a pH of 2.5-5 and then the remaining activity against suitable bacteria, for example B. coli, is tested. 2- - Cephalosporin C is acidic in nature. Both the free acid and its sodium salt are readily soluble in water and insoluble in acetone or ether. An aqueous solution of the sodium salt [ale + 103 ° shows 1 y an absorption at 260 ma with E = 200. 1 cm Cephalosporin C contains carbon, \ rate, oxygen, nitrogen and sulfur but not halogen or phosphorus. Elemental analysis of the sodium salt and the free acid have given the following results: The sodium salt: Found: C, 39.5, 40.0; H, 4.8, 5.2; N, 8.9; S, 6.2; Na, 4.9%. Ekvi. (titration) 480 ± 15. Molecular weight according to X-ray crystallographic analysis 470 1 15.

C161-102N2SNa, 2 H2 O requires C, 40.5; H, 5.1; N, 8.9; S, 6.7; Na 4.9%, molecular weight 473.

Free acid: Found: C, 44.4; H, 5.6; N, 9.8; S, 7.3%. C16112108N2S - H2O collars G, 44.5; H, 5.3; N, 9.7; S, 7.4%.

The assay values show that cephalosporin C may have the gross formula C16H2102N3S. However, this formula is only preliminary and may need others later.

Cephalosporin C produces ninhydrin reaction. Electrometric titration shows that the substance is a monoaminodicarboxylic acid with two acid groups with lower pH values of 2.6 and 3.1 and a basic group with a pK value of 9.8. When the substance is subjected to ionophoresis on paper in a collidine acetate buffer at pH 7, it migrates to the anode at approximately the same rate as cephalosporin N.

The infrared spectrum of the sodium salt of cephalosporin C (in paraffin paste) shows bands at the following wavelengths: 2.94 ft, 3.06 au (NH), 5.77 it (ester or lactone grouping), 6.0ti and 6.57 ft (C = O of mono-substituted amide), 6.29, α (-000-), 7.17, α and 7.36 α (isopropyl group). The information in parentheses is a conceivable interpretation. The sodium salt of cephalosporin C also shows a band at 5.61 pt. A band Mom this area is shown by cephalosporin N and by sodium benzylpenicillin and is introduced at the latter compound to the C = O group in the narrow β-lactam thiazolidine ring system (Thompson, Brattain, Randall and Rasmussen, The Chemistry of Penicillin, Chapter 13, Princeton University Press, 1949).

In contrast to cephalosporin N and benzylpenicillin, cephalosporin C is stable in aqueous solution at pH 2.5. However, the substance is rapidly inactivated at pH 12. After having Milts at pH 12 for 2 hours, atertitration showed that two acid groups per mole had formed. Under similar conditions, only one acid group per mole of cephalosporin N and of benzylpenicillin is released. Cephalosporin C is more stable in the presence of certain heavy metal ions, such as Cu ++, Pb ++ and Zn ++, than cephalosporin N or benzylpenicillin.

Upon hydrolysis with acid (1-N HCl for 8 hours at 110 ° C), cephalosporin C as well as cephalosporin N cleave carbon dioxide and an amino acid, which is similar to α-aminoadipic acid on paper chromatograms. The product obtained when cephalosporin C is reacted with 1,2,4-fluoroditrobenzene gives on hydrolysis a product which behaves in the same manner as the dinitrophenyl derivative of α-arninoadipic acid. It therefore appears that the α-amino group of α-amingadipic acid is free in cephalosporin C and that the α-carboxyl group is also free (since this amino group has a pK value of 9.8).

No indications have been obtained for the formation of any greater amount of penicillamine (fl-thiolvaline) on hydrolysis of cephalosporin C. Hydrolysis of the product obtained by the substance by hydrogenolysis with raney nickel, gay amino acids, which behaved as α-aminoadipic acid and valine, respectively. paper chromatogram.

Cephalosporin C exhibits an activity of the same potency against a large number of gram-positive and gram-negative bacteria. Amnet dr less effective an cephalosporin N against Staph. Aureus and Salm. typhi in vitro, as it exhibits an activity of 8-10 units per meg. (the unit, cited by Abraham, Newton and Hale, 1954, Biochem. J. 58, 94). It has about the same activity as cephalosporin N against a strain of Bact. coll.

The toxicity of the substance could not be assessed by complete biological tests but is assumed to be legal, as moss can be given at least 10 mg of the purified material intravenously without appearing to suffer from adverse effects.

Cephalosporin C can be separated & A a mixture of the same with cephalosporin N by subjecting the mixture to distribution between two solvents, both of which were able to dissolve both cephalosporin N and cephalosporin C. which the ram antibiotics are stable. Cephalosporin N is unstable at pH below 5, while cephalosporin C Or is stable even at as low as 2.5. Da man. according to the invention sacrifices the cephalosporin N activity, the distribution can be carried out at the pH value, at which only cephalosporin C Or is stable.

According to the invention, the cephalosporin N in the mixture can thus be transferred to cephalosporin N-penillic acid and the resulting mixture treated for separating cephalosporin C therefrom, for example by solution fractionation, fractional absorption on a solid absorbent, such as alumina or an anion exchanger, that penillic acid is converted to an insoluble derivative, or by any combination of these two methods. The most convenient way is to convert cephalosporin N to its penillic acid Or to dilute the mixture at a pH at which cephalosporin N is unstable. The reaction can thus be effected by simply mixing the mixture at pH 3 for 2 hours at a temperature of 37 ° C.

The following procedures for separating cephalosporin C from cephalosporin-N-penillic acid have been found to give good results. An aqueous solution of the mixture was added to a column of an anion-exchange resin, such as "amberlite IR4B (XE-59)", after which the elution was carried out with a dilute acid, some suitable buffer solution, such as an aqueous solution of ammonium formate or ammonium acetate, having a pH of 3.2-5.0, or an aqueous solution of a ter-. tiar base, such as pyridine, partially neutralized with sulfuric acid or oxalic acid. A band consisting of cephalosporin-N-penillic acid emerges from the column of a band consisting of cephalosporin C.

An aqueous solution of the mixture is subjected in a distribution machine to a countercurrent distribution under weakly acidic phytallands in a two-phase system of water and phenol (or any suitable, substituted phenol, such as cresol or any xylenol). The partition coefficients in the system can possibly be modified by adding an appropriate amount of some organic solvent, for example carbon tetrachloride. The pH of the system is installed pH 2-5 by adding acetic acid or ammonium acetate or ammonium formate buffer. Transfers are performed until cephalosporin C and cephalosporin-N-penillic acid have formed separate bands. The conditions are suitably installed so that the cephalosporin C is taken out of the machine during the aqueous phase.

An aqueous solution of the mixture was placed on a column of acid-quenched alumina and the elution was carried out with dilute lye, such as 0.01 N sodium hydroxide solution. Cephalosporin C emerges from the pillar as a solution of its sodium salt for cephalosporin-N-penillic acid.

An aqueous bit of mercuric chloride was added to a solution containing a mixture of cephalosporin C and cephalosporin-N-penillic acid, the latter compound being converted to a penillamine, which precipitated as a mercaptide. The above solution contains steering wheel cephalosporin C.

Solutions of free cephalosporin C, which have been obtained by any of the foregoing procedures, can be forced to dryness in vacuo and the cephalosporin C passed through another anion exchange column for further purification. The purified cephalosporin C can be converted to the sodium salt by dissolving in water and installing pH 6.0 with sodium hydroxide.

Solutions of the sodium salt of cephalosporin G, which have been obtained ph any of these sat, may be decolorized if sh. Or necessary using active trachol, and concentrated in vacuo. The sodium salt of cephalosporin C forms monoclinic crystals containing crystal water. The pet can be recrystallized from aqueous ethanol or propanol. When dried in vacuo at room temperature, the antibiotic loses the crystal water, which is soon taken up again by contact with the laboratory air. The invention is illustrated by the following examples.

Example 1. 6.9 g of steering wheel cephalosporin N obtained by trembling from alumina, as described in British Patent Specification 745,208 with a strength of 20 units / mg, were boxed in 110 ml of water. A sample was taken to be used as a reference sample in subsequent spectroscopic feeds. 10 N hydrochloric acid (usually about 1 ml) was added until the solution reached a pH of 2.7-3.0. The solution was then heated to 37 ° C and samples were logged every half hour. The samples were diluted with water to solutions containing 0.075 mg / ml and the optical density at 240 microns for these solutions was measured in relation to the reference sample, which had been diluted on the same salt. The reaction was assumed to be complete, as further increase in the optical density at 240 microns could not be observed (120,180 min). The increase in density at 240 microns was usually about 0.3 density units, when a 1 cm cell and a concentration of 0.075 mg / ml were used. When the conversion of the cephalosporin N component to pennilic acid was complete, the solution was lyophilized to give 7.0 g of the mixture.

An anion exchange resin (Amberlite XE 59) buffered with 0.2 M ammonium formate at pH 3.0-3.2 was prepared in the salt described by Hirs, Moore and Stein (1952, J. Biol. Chem., 195, 699). . A pillar of 59 x 4 cm was packed with the buff-. the resin, after which 500 ml of 0.2 M ammonium formate containing 0.5% thiodiglycol was allowed to flow through the columns. 3.5 g of the mixture of cephalosporin C and cephalosporin-N-penillic acid was dissolved in 30 ml of 0.2 M buffer and the pH of the solution was adjusted so that it was 0.2 pH units higher than the buffer in the column. This solution is halided ph pillar in 3 portions of 10 ml. The flow rate of the column was adjusted so that each 10 ml portion needed 10-15 minutes to sink into the column. The duct was then flushed into the column with 2 x 10 ml of 0.2 M buffer containing 0.5 ° A, thiodiglycol. The column was then filled with the same buffer solution and connected to a constant pressure container. The flow rate of the column was adjusted so that a fraction of 20 ml could be collected every thirty minutes.

Sample frail was the fourth fraction logos out and was diluted 10 times with water. The optical density of the diluted solutions was measured at 240 m, u, and 260 microns in relation to a similarly diluted sample of the buffer solution. Cephalosporin C was found mainly in fractions Nos. 96-118.

Fractions 96-118 were combined and concentrated to about 60 ml in a rotary vacuum evaporator at a temperature below 25 ° C. The concentrate was then concentrated to a thick syrup in a lyophilizer. The syrup was washed twice with acetone to remove the thiodiglycol. A mixture of ammonium formate and the ammonium salt of cephalosporin C remained. The ammonium formate could be removed by either A) sublimation at Mgt vacuum or B) electrophoresis.

A) The solid mass which remained after washing with acetone was dissolved in a few ml of water and transferred to a sublimation apparatus. The sublimation was performed in the manner described by Hirs, Moore and Stein (1952, J. Biol. Chem. 195, 699). The residue from the sublimation consisted mainly of the ammonium salt of cephalosporin C. The salt was dissolved in 2-3 ml of water and the solution was allowed to flow through a small column (0.5 x x 0.5 cm) of activated carbon to remove the dyes. The leachate seemed to evaporate slowly, crystallizing the product.

B) The solid mass remaining after washing with acetone was dissolved in 25 ml of water and introduced into the neutral chamber of a four-chamber cell (Synge, 1951, Biochem. J. 49, 642). The cell was equipped with a cooling loop, through which alcohol at - 5 ° C was pumped. The acetic acid and ammonia chambers were changed every three hours. The desalination was usually completed in 12-16 hours, (Tipping voltages up to 500 V were used. The solutions in the attic acid chambers were combined and lyophilized. a small amount of water, finding with a large excess of acetone and stirring the precipitate with dry acetone, the free acid of cephalosporin C was then dissolved in water and neutralized with 2N NaOH (0.3-0.2 ml). The crystals were liberated from uncrystallized slime by washing with 70% ethanol and then recrystallized from aqueous alcohol. The yield of recrystallized material was 100 .mu.g / mg. Steering wheel cephalosporin N (4 mg containing 22 units / mg) was dissolved in 30 ml of water and the pH of the solution was adjusted to 3.0 with 2N HCl, the solution was kept at 37 ° C for 2.5 hours and then led to give nom a 14 cm long pillar containing 10 g alumina (Savory & Moore Ltd., London. For Chromatographic Analysis). The alumina had previously been stirred with a sufficient amount of dilute hydrochloric acid to bring the pH of the supernatant to equilibrium at pH 4.8.

When all the solution had been added to the alumina, a thin, brown band appeared from the upper spirit of the column and a faint yellow band stretched down from this to about 2/3 of the length of the column. Only traces of ninhydrin-positive material appeared in the leachate. The column was washed with 20 ml of water, after which the elution started with 0.01N NaOH. The eluate was collected in 5 ml fractions. The flow rate was 1 ml / min. The leaching in these fractions at 240 my and 260 my was measured in a Beckmans spectrophotometer. As of fraction 13, the absorption at ram vaginal lengths began to rise rapidly. From fractions 13-17, which contained cephalosporin C, the absorption was greater than 260 microns at 240 microns. At fraction 18, a sudden increase in relative absorption occurred at 240 microns pa. due to the appearance in the eluate of cephalosporin-N-penillic acid. At fraction 21, a yellow pigment appeared in the eluate.

Fractions 13-17 (pH 5.0) were combined and concentrated in vacuo to 2 ml. The pigment was removed from the resulting solution in Iran by passing it through a small column of activated carbon (0.5 cm diameter and 3 cm high), which had previously been washed with water. Concentration of the leachate in vacuo hardened to a crystallized mass of cephalosporin C. The product weighed 33 mg. It contained a small amount of non-crystallized mucus and the feed of its absorption at 260 microns showed the presence of 25 mg of cephalosporin C. The crystals were separated from the mucus by stirring with a. singe mangd 70% ethanol and filtration. The crystallized product was recrystallized from aqueous propanol.

Example 3. Unloading system. 5 l of water matted with phenol, 4.5 l of phenol matted with water and 0.45 l of glacial acetic acid were mixed and lingo reached equilibrium at 20 ° C.

Distribution. 7.3 g of a mixture of cephalosporin N-pennillic acid and cephalosporin C obtained on the same Ott as in Example 1 were dissolved in 80 ml of the top phase and 40 ml of the bottom phase in the solution system. The bottom phase, 20 ml, and the top phase, 40 ml, of this solution were introduced into the 'Oren' 0 »and» 1 »of an all-in-glass machine for countercurrent distribution (Craig and Craig, 1950 Technique & Organic Chemistry, Volume 3, Chapter 4). ). The top layer, 40 ml, was reacted 100 times using the basic procedure, followed by 300 drains of the top layer, 40 ml, from the tube "100".

Analysis of the fractions. A sample of 0.2 ml was taken from Iran every tenth fraction of the drained series, and the fatigue which developed on heating with ninhydrin (0.5 ml) was measured photometrically at the Ott, given by Moore and Stein, 1948, J Biol Chem. 176, 367. When the values of the optical particle obtained in this way were set against the number of the fraction, a band containing cephalosporin-Npenillic acid in fractions 51-120 and a band containing cephalosporin C in fractions 190300 were detected. K = = conc. In the aqueous phase / conc. In the phenol phase) for cephalosporin C, which is calculated from the layer for the top of the band, was 0.2, while the value for cephalosporin-N-penillic acid was 0.63.

The withdrawn fractions 200-300 were combined and concentrated to about 400 ml (400 ml water layer and 100 ml phenol layer) in a rotary vacuum evaporator. The concentrate was then halided in a separatory funnel and shaken with 400 ml of carbon tetrachloride. After the layer had separated, the carbon tetrachloride phase was drained, while the yatten phase was extracted three times with the same volume of benzene. The aqueous residue was concentrated to about 100 ml in a rotary evaporator and then lyophilized. Traces of attic acid were removed from the Iran product by dissolving it in a small amount of water and cephalosporin C (free acid) was precipitated with excess acetone. The precipitate was washed with dry acetone.

The free acid was dissolved in a small amount of water and then neutralized with 2N NaOH (0.5-0.6 ml). The sodium salt of cephalosporin C crystallized, cla the neutralized solution was allowed to evaporate slowly. The crystals were washed with 70% ethanol, whereby non-crystallized material was deposited. Yield 168 mg. This material was recrystallized from aqueous propanol. Example 4. An aqueous solution of ran cephalosporin C, acidified to pH 2.5 and maintained at. 37 ° C for 2 hours. The cephalosporin N was completely inactivated. The solution was freeze-dried, since its pH had been installed at pH 5.0. The product (600 mg with 0.45 units per mg) was added to 3 ml of pyridine acetate buffer pH 5.0, 150 ml of 2 M acetic acid, 250 ml of N-pyridine and 600 ml of water, after which some insoluble material was removed by centrifugation. The resulting clear solution (pH 5.0) was slowly added in two portions to the upper part of a column (1.9 x 20 cm) Amberlite IR4B (grain size corresponding to sieve no. 200-400). The resin was buffered to pH 5.0 with pyridine acetate by washing the acetate form of the resin with pyridine acetate buffer, which had been prepared as above, on a filter, until the supplied and the outgoing buffer had the same pH, i.e. 5.0.

After the discharge of the raft cephalosporin C had flowed down into the column, it was followed by two additions of 2 ml of pyridine acetate buffer, after which the upper ankle of the column was connected to a constant pressure container containing pyridine acetate buffer. The level of the tank was adjusted to obtain the desired flow rate (9 ml / h). Donna flow velocity was achieved, in that the height of the buffer in the container was only a few cm above the bottom plate of the column. The effluent was collected in 3 ml fractions every twenty minutes.

Alternating fractions were analyzed by determining the antibacterial activity against S. typhi pa pray, which was both neutralized with sodium hydroxide solution, and also by a photometric ninhydrin method (Moore and Stein, 1948, J. Biol. Chem. 176, 367). Cephalosporin C was eluted in fractions No. 108. These fractions were combined and concentrated in vacuo (at a bath temperature of 30 ° C) in a rotary evaporator. The residue was dissolved in a small volume of water and transferred to a ski and dried overnight under vacuum over KOH and 1320, after which no odor of pyridine or acetic acid could be detected. The product was dissolved in a small amount of water (0.3 ml) and 10 ml of acetone was added. The precipitate was centrifuged, stirred with a few ml of acetone, centrifuged again and dried in vacuo to give a white powder of the free acid cephalosporin C. Donna free acid was dissolved in 2 ml of water to give a solution of pH 3 .0, after which 0.49 ml of 0.1 N NaOH was carefully added from a burette until the pH had risen to 6.5. The resulting solution of the sodium salt of cephalosporin C was lyophilized. The dried material was crystallized by dissolving the same in a small amount of water and slowly evaporating the solution. The dry crystallized material (39 mg) was freed from uncrystallized compounds by washing with a small amount of 70% (v / v) ethanol.

Example 5. A rhyme of steering wheel cephalosporin C was acidified to pH 2.5. The Mills then at 37 ° C for 2 hours, during which all cephalosporin N activity was increased. The solution was lyophilized after its pH had been set at 5.0. The product. (4 g with 0.08 units per mg) was shaken with 8 ml of pH 5.0 pyridine acetate solution (see Example 7). A few drops of pyridine were added to keep the reaction of the solution at pH 5.0. After about 20 minutes, the solution was centrifuged and the clear liquid was decanted ay. The precipitate was shaken with another 4 ml of buffer and centrifuged again, after which the second batch of clarified liquid (12 ml) in 4 portions of 3 ml was introduced into a 2 x 42 cm column of Amberlite IR4B (79% corresponding to screen no. 40). -60 and 20% towards visibility no. 60-100). The resin had been buffered with pyridine acetate buffer (pH 5.0) as described in Example 7. The solution of raft cephalosporin C was followed by two portions of 3 ml each of pyridine acetate buffer, after which the column was run in the same manner as in Example 7.

Cephalosporin C was found in the effluate volume of 280-500 ml. The 388,443 ml effluent volume was freed from pyridine acetate buffer and precipitated with acetone as described in Example 7 to give 18 mg of the free acid form of cephalosporin C in the crude state. The free acid was dissolved in 2.0 mL of water and 0.14 mL of 0.1 N NaOH was added until the pH stagnated to 6.5. As this liquid had evaporated slowly, some of the material was excreted in crystallized form. The mixture of crystallized and non-crystallized material was washed with 40% ethanol in a filter to give 2 mg of the crystallized sodium salt of cephalosporin C. From the Rr wash using the ethanol, 14 mg of a material of 2.5 units / mg were obtained. The remaining solution from the effluent volume of 280-500 ml was estimated to contain 23 mg of material at 2.5 units / mg.

It should be emphasized that the above-described examples of preferred embodiments have only the task of illustrating the invention, and that various deviations are taken without exceeding the scope of the invention. It may be mentioned in this connection that any of the solution systems which have been used in separating cephalosporin C from cephalosporin-N-penillic acid by countercurrent distribution can be applied to partition chromatography, one of the phases being absorbed on a bar, such as diatomaceous earth, or that they can be used in a continuous extraction pillar, for example the one described by Scheibel & Karr (Ind. Eng. Chem. 42, p. 1048, 1950).

It has been mentioned above that cephalosporin C Or is unlikely for penicillinase, i.e. that it remains in 6— 192 926 - essentially unaffected by it. This is a very beneficial property of the new antibiotic, since it is equally valuable against such microorganisms as produce penicillinase and are therefore more or less resistant to penicillin. Cephalosporin C can therefore be used to protect penicillin against inactivation by such microorganisms.

Tests have shown that cephalosporin C remains essentially unaffected by pure penicillinase generated by strains of B. subtilis and B. cereus. The following table shows the amounts of different penicillinase preparations required to achieve a 50% inactivation expressed in the amount required for cephalosporin C divided by the amount required for the same amount of cephalosporin N.

Table 1.

Origin of Penieillinas B. subtilis (sludge 749) B. cereus (NRRL 569) B. cereus (NRRL 569) B. cereus 5 / B adverse effects on cephalosporins C.

The action of cephalosporin C against various microorganisms is shown in the following table, which, however, should not be construed as indicating the full bacteriological spectrum and only illustrates the antibiotic action which has hitherto been established.

Table 6.

Organism Lowest concentration, which prevents growth for 24 hours in pg / m1 Staph. aureus, Heatley strain, penicillin probable 50-100 Staph. aureus, penicillin-resistant Str. pyogenes B. anthracis N. meningilidis 1.6 N. gonorrhoeae 1.6 H. pertussis 1.6 H. influenzae 12, S. typhi S. paratyphi B.

S. typhimurium V. cholerae Bad. friedlanderi Bact. coli 200 The properties of Cephalosporin C are illustrated by the accompanying drawings, where Fig. 1 is the ultraviolet absorption curve and Fig. 2 is the infrared spectrum.

Claims (10)

Patent claim: 1. Introduced a new, saliva against gram-positive and gram-negative bacteria active antibiotic, can be drawn ddrav, that a nutrient substrate, which is fermented with a species of Cephalosporium and which in addition to the known antibiotic substance cephalosporin N contains another, antibiotic active substance, or solutions, which contain these antibiotic substances and which are obtained from such fermented nutrient substrates, are treated for enlargement of cephalosporin N by subjecting the nutrient substrate to the action of penicillinase or ox. Melt at a pH value of 2.5-5.5, after which the new antibiotic substance, which is called cephalosporin C and which can be identified, is isolated from the nutrient medium, partly because its sodium salt in aqueous solution gives a torsional capacity of [a ] g = 103 °, partly because it gives an absorption curve for ultraviolet according to Fig. 1 and for infrared according to Fig. 2 and partly because it is indistinguishable for penicillinase, formed by B. subtilus. 2. according to claim 1, characterized in that the cephalosporin N is enlarged by heating the nutrient medium at a pH value of 2.5-5.0. 3. A kit according to claim 1 or 2, characterized in that cephalosporin C is isolated from the treated nutrient medium by solvent extraction. 4. A kit according to claim 1 or 2, characterized in that cephalosporin C is isolated from the treated nutrient substrate by adsorption on an ion exchange material and subsequent elution from the delta with an aqueous elution solution. 5. A kit according to claim 4, characterized in that the elution is carried out with an aqueous buffer solution buffered to a pH of 2.58.0. 6. A kit according to claim 5, characterized in that the buffer solution is formed by neutralizing a weak, tertiary base. 7. A kit according to claim 6, characterized in that the buffer solution is prepared by neutralizing pyridine. 8. A kit according to claim 4 or 5, characterized in that cephalosporin C is adsorbed on a synthetic resin. 9. A kit according to any one of claims 4-8, characterized in that the eluted solution of cephalosporin G is treated with an alkaline earth metal to form the acid from the buffer solution and the formation of the alkaline earth metal salt of cephalosporin C, which is converted into the sodium salt by contacting the solution with a cation exchange material in the sodium form. Purity Relative amount C / N ra> 100 ra purified crystallized 10000 - —7 10. according to any one of claims 1-9, characterized in that the fermented nutrient medium is first purified by contact with activated carbon, which is eluted, after which the eluate is adsorbed on alumina, which is then eluted to form a purified solution containing the ram types cephalosporins. Request publications: Chemical abstracts 46 (1952), sp. 10295. (Antibiotic production by species of cephalosporium). Nature 175 (1955), pp. 548.