CH641805A5 - Derivatives of 3-thio-6-(1-hydroxyethyl)-7-oxo-1-azabicyclo-[3.2.0]hept-2-ene-2-carbo xylic acid, process for their preparation and medicaments containing these compounds - Google Patents

Description

The present invention relates to derivatives of

Fermentation fluid from Streptomyces olivaceus a white 3-thio-6- (l-hydroxyethyl) -7-oxo-l-azabicyclo [3,2,0] hept-

teres antibiotic could be isolated. It has now been found that more in the fermentation liquid

2-en-2-carboxylic acid of the formulas I and II below

h S-CH2-CH2-NH-CO-CH3

(I)

CH_ H

y; S-C = Ç-NH-CO-CH3

(II)

h and their salts.

5

The compounds of the formulas I and II are most conveniently in the form of their salts, since it appears that the salts of the compounds of the formulas I and II are more stable than the corresponding free acids.

Suitable salts of compounds of formulas I and II 5 are pharmacologically acceptable alkali and alkaline earth metal salts, such as the sodium, potassium and calcium salts, and pharmacologically acceptable acid addition salts with nitrogen-containing bases, such as the ammonium, trimethylamine, dimethylamine and pyrrolidine salts and the like . 10th

Particularly suitable salts of the compounds of the formulas

I and II are their sodium and potassium salts.

A preferred compound of the present invention is the sodium salt of the compound of formula I. Another preferred compound of the present invention is 15

Sodium salt of the compound of formula II.

Since it is provided, the compounds of formulas I and

II to use in medicines, it is easy to understand that these compounds are largely pure

Form should be present, for example in a purity of 20 at least 50%, but expediently at least 75% and preferably at least 90% and very particularly preferably

h-c h ho

Ì

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increases at least 95%. Impure preparations of compounds of the formulas I and II and their salts can be used to produce purer compounds which can then be used for the preparation of medicaments. These less pure preparations of the compounds of the formulas I and II and their salts should contain at least 1%, advantageously at least 5% and preferably 10 to 49% of a compound of the formula I or II or their salts. These less pure preparations most advantageously consist of a salt of a compound of the formulas I or II, the percentages relating to percentages by weight, based on the weight of the preparation.

In general, it is advantageous that the practically pure salts of a compound of formula I or formula II are not contaminated by substantial amounts of other antibacterial agents, such as salts of the compounds of formulas III, IV and V, which originate from the fermentation liquid.

With regard to the β-lactam ring, the compounds of the formulas I and II exist both as cis and as trans isomers. These isomers can be illustrated by the formulas Ia, Ib, IIa and IIb:

h s-ch "-ch" -nh-co-ch.

(Ia)

(Ib)

ß s-ch2-ch2-nh-co-ch3

s-c = c-nh-co-ch.

H

(IIa)

h, c

3l ho y

H

H

I.

s-c = c-nh-co-ch.

(IIb)

h co2h

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6

These aforementioned compounds can be named as follows:

Ia) (5R, 6R) -3- (2-acetamido-ethylthio) -6 - [(S) -l-hydroxy-ethyl] -7-oxo-1-azabicyclo [3,2,0] hept-2- en-2-carboxylic acid;

Ib) (5R, 6S) -3- (2-acetamido-ethylthio) -6 - [(S) -l-hydroxy-ethyl] -7-oxo-l-azabicyclo [3,2,0] hept-2- en-2-carboxylic acid;

IIa) (5R, 6R) -3 - [(E) -2-acetamido-ethenylthio] -6 - [(S) -1-hydroxyethyl] -7-oxo-l-azabicyclo [3,2,0] hept- 2-ene-2-carboxylic acid;

IIb) (5R, 6S) -3 - [(E) -2-acetamidoethenylthioJ-6 - [(S) -1-hydroxyethyl] -7-oxo-1-azabicyclo [3,2,0] hept-2 -en-2-carboxylic acid.

Both the cis and the trans isomers of the compounds of the formulas I and II have valuable antibacterial and β-lactamase inhibitory properties, and consequently the present invention extends to the isolated compounds of the formulas Ia and Ib and their mixtures as well the isolated compounds of formulas IIa and IIb and their mixtures.

Of course, the isolated cis and trans isomers are particularly suitable in the form of practically pure pharmaceutically acceptable salts as described above, so that they have a degree of purity of at least 50%, expediently at least 75%, preferably 90% and most advantageously have at least 95%. It is further preferred to use one of the aforementioned compounds if it is practically free of its 6-position isomer, i.e. a compound Ia free of the compound Ib, a compound IIa free of the compound IIb, a compound Ib free of the compound Ia or a compound IIb free of the compound IIa. In general, such compounds should contain no more than 5% of their 6-position isomer and preferably no more than 1% of their 6-position isomer. High pressure liquid chromatography can be used to record the purities.

According to a preferred embodiment, the subject of the present invention is an alkali metal salt of a compound of the formula Ia with a molar extinction coefficient of not less than 7700 in water at neutral pH, preferably not less than 7900, with a UV absorption maximum at about 298 nm.

According to a further preferred embodiment, the subject of the present invention is an alkali metal salt of a compound of the formula Ib with a molar extinction coefficient of not less than 7700 in water at neutral pH, preferably not less than 7900, with a UV absorption maximum at about 301 nm.

According to a third preferred embodiment, the subject of the present invention is an alkali metal salt of a compound of formula IIa with a molar extinction coefficient of not less than 13,000 in water at neutral pH, preferably not less than 13,500, with a UV absorption maximum at about 308 nm.

According to a fourth preferred embodiment, the subject of the present invention is an alkali metal salt of a compound of the formula IIb with a molar extinction coefficient of not less than 13,000 in water at neutral pH, preferably not less than 13,500, with a UV absorption maximum at about 308 nm.

The abovementioned alkali metal salts are preferably the sodium salts.

Another object of the present invention are pharmaceuticals which are characterized by a content of a compound of formula I or formula II or their salts. The medicaments can also contain pharmacologically acceptable carrier materials, diluents, excipients and / or other additives.

The pharmacologically acceptable salts of the compounds of the formulas I or II, for example the sodium or potassium salts, are usually suitable in the medicaments of the present invention.

The pharmaceutical compositions of the present invention can be administered orally or parenterally. The medicaments of the present invention expediently contain 50 to 500 mg of a compound of the formulas I or II or their salts, for example about 100, 150, 200 or 250 mg.

Medicines in an injectable form are most suitable.

These drugs can contain diluents, binders, disintegrants, lubricants and other common excipients and can be prepared by conventional methods, e.g. by mixing, filling or the like.

The medicines can be in the form of tablets, capsules, ampoules or similar forms.

If appropriate, the medicaments can advantageously contain a penicillin or cephalosporin as additives. In these cases, the ratio of synergist-i.e. the compound of the present invention, preferably as a salt - to penicillin or cephalosporin, usually from 2: 1 to 1:12, advantageously from 1: 1 to 1: 5, for example 1: 2.1: 3 or 1: 4. All data refer to the weight.

Particularly suitable penicillins for use in the medicaments of the present invention are ampicillin, amoxycillin, carbenicillin, ticarcillin and their precursors. When the medicaments are to be prepared for injections, such compounds are usually in the form of their sodium salts.

Cephalosporins, such as cephalorodin and cephazolin, are particularly suitable for use in the medicaments according to the invention.

Preferred penicillins in the medicaments of the present invention are ampicillin trihydrate, amoxycillin trihydrate, the sodium salt of ampicillin and the sodium salt of amoxycillin.

Preferred cephalosporins in the medicaments according to the invention are cephaloridine and the sodium salt of cephazoline.

The compound of formula I or formula II or their salts can be an isolated compound of formula Ia, formula Ib, formula IIa, formula IIb or their salts or mixtures of compounds of formulas Ia and Ib or of compounds of formulas IIa and IIb or their salts. However, preference is given to using a compound of one of the abovementioned formulas which is free of its isomer. Such a compound is usually in the form of a pharmacologically acceptable salt, such as the sodium salt.

Another object of the present invention is a process for the preparation of a compound of formula I or formula II and its salts, which is characterized in that a strain of Streptomyces olivaceus or Streptomyces gedanensis, which produce these compounds, is grown until a considerable amount on a compound of the formula I or the formula II or salts thereof, and that the compound of the formula I or the formula II or its salt is then isolated from the growth medium.

The term "Streptomyces olivaceus" used here has been defined according to the classification by R. Hütter in the book "Systematik der Streptomyceten", published by S. Karger, Basel, pp. 8-32. It should be noted that the definitions "Streptomyces fulvovoridis", "Streptomyces flavus" and "Streptomyces flavovirens" can be regarded as synonyms for "Streptomyces olivaceus".

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Suitable strains are described in GB-PS 1467413 and DE-OS 2513855.

A preferred organism for use in this method is Streptomyces olivaceus ATCC 31126, or a mutant that allows high yields to be achieved.

Another preferred organism for use in the aforementioned method is Streptomyces olivaceus ATCC 31365 or a mutant which allows high yields to be achieved.

The strains mentioned were deposited with the "American Type Culture Collection", 12301 Parklawn Drive, Rock-ville, Maryland 2085, USA, namely ATCC 31126 on February 18, 1975 and ATCC 31365 on January 26, 1978 and have been open to the public since then .

As has already been described above, the isolated compound should have a degree of purity of at least 1%, suitably 5%, more suitably at least 50%, preferably at least 75% and more preferably at least 90%, for example at least 95%.

The term "breeding" used here means the conscious aerobic growth of an organism in the presence of assimilable sources of carbon, nitrogen and sulfur and mineral salts. Such aerobic growth takes place on a solid or semi-solid medium, but the use of a liquid medium is generally preferred. The general breeding conditions for the growth of Streptomyces olivaceus are described in GB-PS 1467413 and DE-OS 2513855. The general conditions for the growth of Streptomyces geda-nensis are similar.

The process of the present invention is based on the preparation of a compound of the formula I or its salt, a compound of the formula II or its salt or a compound of the formula I or its salt together with a compound of the formula II or its salt.

Typically, the process is geared toward salt production rather than free acid production.

The growth medium preferably does not contain any added sulfate, since this often leads to the formation of the compounds MM 4550, MM 13902 and MM 17880 at the expense of the production of the compounds of the formula I and the formula II and their salts.

The compounds of the formula I and the formula II in the form of their salts can be obtained from the filtrate of the culture liquid by a) contacting the filtrate with activated carbon until the antibiotic activity is adsorbed thereon,

b) eluting the antibiotic activity from the activated carbon using aqueous acetone,

c) pooling the fractions containing the β-lactamase inhibiting fractions,

d) evaporating off the acetone and most of the water to give a more concentrated aqueous solution,

e) applying the solution to an anion exchange column,

f) eluting the β-lactamase inhibiting metabolites with a solution of an electrolyte buffered to approximately the neutral point and collecting the fractions containing the compounds of formula I or formula II in salt form,

g) applying the solution obtained to a resin which separates the inorganic substances from the compounds of the formula I and the formula II and h) isolating the solid salt preparation of a compound of the formula I or the formula II from the solution obtained.

The compounds of formula I and formula II in

Form of their salts can also be obtained from the filtrate of the culture liquid

1. contacting the clarified growth liquid with a strongly basic anion exchange resin based on acrylic acid until the antibiotic activity is adsorbed thereon,

2. Elution of the antibiotic activity from the resin using an aqueous buffer solution, which may optionally also contain a salt,

3. pooling the fractions with a β-lactamase inhibitory activity,

4. application of the combined fractions to a column with an exchange resin based on phenol-formaldehyde condensation products which are specially adjusted for water-soluble organic compounds (“Amberlite XAD-4”),

5. eluting with aqueous isopropanol,

6. pooling the fractions with a β-lactamase inhibitory activity,

7. removing the isopropanol and concentrating the solution by evaporation,

8. Apply the solution to an anion exchange resin and continue as above for steps (f), (g) and (h).

Strongly basic anion exchange resins based on acrylic acid are preferably used in the form of their acid addition salts of the hydrochloride, such as the “Amberlite IRA 458” commercially available from Rohm & Haas. An advantage of such a resin is that when using an aqueous salt solution, for example a buffered solution of a chloride such as sodium chloride or the like, it allows the salts of the compounds of formula I and formula II to be eluted one after the other. In general, when somewhat less advantageous, strongly basic, poly-styrene / divinylbenzene based resins are used, it is necessary to mix with an aqueous solution of a salt containing a lower alcohol, e.g. of a chloride, such as sodium chloride, to obtain satisfactory yields. It should be noted, however, that such solvents can lead to less pure products on the desired compounds.

This process variant differs from the adsorption process on coal in that the salts of the compounds MM 4550, MM 13902 and MM 17880 are separated from the salts of the compounds of the formulas I and II according to the invention in the first elution stage.

The process of the present invention differs fundamentally from the process described earlier in that the fractions selected for further processing in step (f) are those which contain the salt of a compound of the formulas I and II which are practically free from other antibiotics .

The free acids of the formulas I and II can be obtained by carefully acidifying the solution of a salt of a compound of the formulas I or II or by subsequent rapid extraction into a water-miscible organic solvent with subsequent recovery of the acid from the solution.

In the processes of the present invention, it is often particularly expedient to work with an alkali metal salt of the compounds of the formulas I and / or II, such as the lithium, sodium or potassium salts, with the sodium salt being preferred. It is possible to prepare other salts using the extraction process, but it is usually more convenient to first form the purified alkali metal salt, especially the sodium salt, and then convert it to another salt, for example by passing the solution through a cation exchange bed in the form of the other salt conducts. Thus, according to this description

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other electrolytes, such as lithium, potassium or other salts, can be used instead of the sodium salts described, but it is generally preferred to work with the sodium salt. In the same way, salts other than the chlorides, namely for example bromides, nitrates or the like, can be used, although it is generally preferred to work with the chloride.

In a preferred chromatography process for purification according to steps (f) and (g), an aqueous solution of the sodium salt, which is buffered up to approximately the neutral point, is used in conjunction with a basic ion exchange resin. Accordingly, an aqueous solution of sodium chloride (or other similar salt) that has been buffered to about pH 7 with a suitable buffer such as a phosphate buffer can be used in conjunction with a carrier resin containing secondary or tertiary amino groups or quaternary amino groups . Suitable carriers include basic ion exchangers based on cellulose or from crosslinked dextrans, as are known as DEAE cellulose, DEAE Sephadex, QAE Sephadex and the like.

A related suitable chromatography process for purification according to steps (f) and (g) uses a solvent system consisting of a mixture of water and small amounts of a water-miscible organic solvent, such as a lower alcohol, e.g. with 1 to 4 carbon atoms, in connection with an inert carrier material such as silica gel or cellulose. Examples of suitable solvent systems are aqueous isopropanol, aqueous n-butanol and the like. For example, a mixture of roughly approximately 1 part water and 4 parts isopropanol can be used in conjunction with a cellulose carrier material.

The product from the aforementioned process stages often contains a high proportion of sodium chloride, so that it is advantageous to desalt the combined solutions. Desalting can be accomplished by passing the solution through a bed of lipophilic substance to which the antibiotic but not the sodium chloride is adsorbed. Suitable substances are polystyrene-based polymer absorbents, such as “Amberlite XAD-4”, “Diaion HP20” and the like. The product from the abovementioned process can also be desalted by means of chromatography on suitable gel filtration media, such as on crosslinked dextrans, for example “Sephadex G10” and “Sephadex G15”, and on polyacrylamide gels, such as “Biogel P2”. The antibiotic can be eluted from such substances using water, aqueous methanol or the like.

The columns are eluted at such a rate that the antibiotics can be separated into different fractions. In general, different zones can be eluted from these columns, s These contain the disodium salt of the compound MM 4550, the disodium salt of the compound MM 13902, the disodium salt of the compound MM 17880, the sodium salts of the compounds of the formula I and the sodium salts of the compounds of the formula II , the latter being eluted immediately after the 10 sodium salts of the compounds of formula I. Usually the three disodium salts are quite apart from the monosodium salts on the anion exchange resins. If the column is not carefully monitored, the monosodium salts may be obtained in 15 overlapping fractions. If this happens, one can either (a) freeze dry this solution to obtain a useful impure complex containing the antibiotics which can then be worked up later, or (b) the solution per se with a carefully calculated amount of eluent chromatograph again for an enrichment in the solution of the sodium salt of a compound of the formula I which is free of the sodium salt of a compound of the formula II and / or an enrichment in a solution of the sodium salt of a compound of the formula II which is free of the sodium salt of a compound of formula I. The solutions obtained can then be freeze-dried or made solvent-free in some other way.

The fractions selected for enrichment are 30 which show remarkable β-lactamase inhibitory activity or antibacterial activity. Suitable methods for determining a β-lactamase inhibitory activity are described in the British patents and German laid-open publications mentioned at the outset, 35 although other suitable methods can also be used.

The following diagrams show the preferred process steps in order to obtain the compounds of the formulas I and II in the form of their sodium salts. The sodium salts obtained in this way can optionally be further purified using the chromatographic methods described above.

Trituration of the salts of the compounds of formulas I and II in the presence of an organic solvent such as acetonitrile or acetone containing water can provide valuable support for removing impurities.

9

641

Filtrate of the culture liquid

1

Activated carbon absorption

Elute with 20 percent aqueous acetone

V

Pooling the fractions with ß-lactamase inhibitory activity

\ /

Evaporation of the acetone and concentration by evaporation

Cellulose DE52 anion exchange resin

Elute with pH 7 phosphate buffer solution

V

Sodium salts of the compounds of formula I.

V

Sodium salts of the compounds of formula II

Disodium salts of the substances MM4550, MM 15902 and MM 17880

V

desalinated solution

Chromatography on "Amberlite XAD 4"

/

desalinated solution

V

freeze-dried solid compound

V

freeze-dried solid compound

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Filtrate of the culture liquid

\! /

Absorb on a strongly basic acrylic-based exchange resin

V

Elute with buffer solution

Pool the fractions with the first eluted β-lactamase inhibitory activity

Desalt on Amberlite XAD 1

Elute with aqueous. Isopropanol

\ /

Pool the fractions with β-lactamase inhibitory activity

Evaporation of the isopropanol and concentration by evaporation

V

Cellulose DE52 anion exchange resin

Elute with pH 7 phosphate buffer solution

Sodium salts of the compounds of formula I.

Sodium salts of the compounds of formula II

£ - Chromatography on "Amberlite XAD4"

->

v Desalted solution

> /

desalinated solution

freeze-dried test compound

!

freeze-dried solid compound

11

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If appropriate, the salts of the compounds of the formulas I and II which have been prepared by the above processes can be subjected to further chromatographic separation processes in order to obtain the isolated respective salts of the compounds of the formulas Ia, Ib, IIa or IIb. Such methods are preferred embodiments of the present invention. Usually the salt used in such a process is a monovalent salt such as the ammonium salt or an alkali metal salt such as the sodium or potassium salt.

One of the suitable chromatographic separation methods is high pressure liquid chromatography, for example using an aqueous solution buffered with ammonium formate. As soon as the fractions containing the desired compound have been obtained, they can be obtained therefrom in solid form by freeze-drying or the like.

Compounds of the formulas Ia and Ib can be separated by column chromatography on a support material, such as acetylated cellulose, and eluting with mixtures of water and alcohol. The compounds of the formulas IIa and IIb can also be separated using the same chromatographic methods.

When the fractions containing the desired compound have been obtained, the compounds can be obtained in solid form by freeze-drying or the like.

The present invention also relates to a process for the preparation of a salt of the compound of the formula Ia which is practically free of the compound of the formula Ib, with the characteristic feature that a mixture of these salts is chromatographically separated on a crosslinked divinylbenzene resin (Diaion HP20 ») Or an equivalent exchange resin.

The present invention furthermore relates to a process for the preparation of a salt of the compound of the formula Ib which is virtually free from a compound of the formula Ia, with the characteristic feature that a mixture of these salts is chromatographically separated on a crosslinked divinylbenzene resin (" Diaion HP20 ») or an equivalent exchange resin.

The present invention furthermore relates to a process for the preparation of a salt of the compound of the formula IIa which is virtually free from a compound of the formula IIb, with the characteristic feature that a mixture of these salts is chromatographically separated on a crosslinked divinylbenzene resin (" Diaion HP20 ») or an equivalent exchange resin.

Finally, an object of the present invention is a process for the preparation of a salt of the compound of formula IIb, which is practically free of a compound of formula IIa, with the characteristic feature that a mixture of these salts is chromatographically separated on a crosslinked divinylbenzene resin ("Diaion HP 20 ») or an equivalent exchange resin.

The salts produced by the processes described above are usually monovalent salts, such as alkali metal salts, for example the lithium, sodium or potassium salt, preferably the sodium salt.

The salts prepared by the methods described above usually contain no more than about 5%, and more preferably no more than about 1%, of the undesired isomer.

The adsorption resins (“diaion”) used in the abovementioned processes are highly porous polymers which are produced by the Japanese company Mitsubishi Chemical Industries and are not actually ion exchange resins, but are synthetic

Adsorbents that have a particularly large active surface area on which organic compounds can be effectively adsorbed.

The specifically named adsorbent “Diaion HP20” is a styrene-divinylbenzene copolymer in pearl form with a macro-crosslinked structure and a specific surface area of approximately 7.8 mVg and a pore volume of 1.16 ml / g. Further details of this resin can be found in the "Diaion HP-Series" brochure from the company mentioned in October 1976.

The resins for chromatography purposes which are equivalent to the abovementioned adsorbent are usually chemically and physically similar, because they are generally macro-crosslinked resins based on styrene-divinylbenzene copolymers and contain no ionizable groups.

Most conveniently the mixture of isomers applied to the column is of good purity and practically free of other organic contaminants, although inorganic contaminants, e.g. Alkali metal salts such as a chloride e.g. Sodium chloride, may be present.

The solvent used is expediently water or water in a mixture with a lower alkanol or a similar miscible organic solvent. However, the solvent is preferably water.

The desired substances can then be obtained by removing the solvent, for example by evaporation, freeze-drying and the like. If necessary, the solution can be applied directly to a suitable resin, e.g. "Biogel P2" and / or "Diaion HP20" should be chromatographed again for further cleaning before removing the solvent.

The water can be removed from the aqueous solutions of the salts according to the invention, for example by evaporation under reduced pressure to about one tenth of the original volume, filling up with ethanol to the original volume, re-evaporation under reduced pressure to about one tenth of the original volume, refilling to the original volume Add toluene and then evaporate to dryness under reduced pressure. Traces of solvent can be removed by storing the substances under high vacuum.

Description No. 1

Production of a clarified breeding liquid

A spore suspension of Streptomyces olivaceus ATCC 31126 is used and inoculated into 100 ml of growth medium in a 500 ml Erlenmeyer flask which is closed with a foam stopper. The culture medium contains 2% glucose and 1% soybean meal in deionized water.

The inoculated growth medium is allowed to grow on a rotary shaker at 26 ° C for 48 hours. Then 5 ml portions of the culture medium are used and inoculated onto 100 ml portions of a fermentation medium in 500 ml Erlenmeyer flasks which are sealed with foam stoppers. The fermentation medium that has been prepared with the fully demineralized water contains the following components:

Glucose 2.0%

Soybean meal 1.0%

CaCOj 0.02%

CoCh-óHzO 0.0001%

The fermentation bottles are incubated for 72 hours at 26 ° C on a rotary shaker. Then 20 bottles are harvested, and all the culture fluid obtained is

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Centrifuged at 2200 g for 10 minutes.

It is also advantageous to use Streptomyces olivaceus ATCC 31365 in the aforementioned method.

Description No. 2

Manufacture of tube antibiotics in solution

1500 ml of the culture liquid filtrate obtained after centrifugation are subjected to purification using a column with activated carbon as follows: A column with granulated activated carbon with a length of 37 cm and a diameter of 2.5 cm is prepared with deionized water. The column is then washed successively with 1 liter of a 2 percent sodium hydroxide solution, with 1 liter of fully deionized water, with 1 liter of 1N hydrochloric acid and finally with 1 liter of fully deionized water, with 15 ml per minute. The column is then washed with a 0.05 m phosphate buffer solution of pH 7 until the pH of the eluate is 7.0.

1500 ml of the culture liquid filtrate is passed through the activated carbon column at a rate of 15 ml per minute. The column is then eluted with a mixture of 1 part acetone and 4 parts water at a rate of 15 ml per minute and 22 ml fractions are collected. The fractions are assayed for their beta-lactamase inhibitory effects against an Rtem enzyme preparation supplied by the Microbiological Research Establishment, Porton. The fractions showing the greatest activity, namely fractions 8 to 22, are combined and evaporated under reduced pressure to remove the acetone. The aqueous solution obtained is stored frozen before the subsequent workup.

(The RTEM enzyme is typical of plasma-influenced ß-lactamases and can be replaced by other R-factor ß-lactamases if necessary.)

The examples illustrate the invention.

example 1

Isolation of the compounds of the formulas I and II in the form of their sodium salts which are practically free from salts of the compounds MM 4550, MM 13902 and MM 17880

The crude liquid obtained according to description No. 2 is evaporated under reduced pressure to about 20 ml and onto a 3.8 x 27 cm column with a weakly basic anion exchanger "DEAE-Cellulose", which is dipped with a 0.025 m phosphate buffer solution pH 7 has been raised. Then the column is eluted with a 0.025 m phosphate buffer solution of pH 7 at a rate of 8 ml per minute. 25 ml fractions are collected. The fractions are examined for their ß-lactamase inhibitory activity against a Rtem-ß-lactamase preparation. The fractions with the first two maxima for inhibitory activity are retained. Fractions 13 to 16 with the first maximum containing the salt of the compound of formula I are combined and freeze-dried to give a solid substance containing the sodium salt of the compound of formula I which is practically free of salts of is dibasic antibiotics. Fractions 19 to 21 with the second maximum containing the salt of the compound of formula II are combined and freeze-dried to give a solid substance containing the sodium salt of the compound of formula II which is practically free of salts of is dibasic antibiotics.

(Freeze-drying of the combined fractions 13 to 16 and 19 to 21 naturally leads to a substance which is a mixture of the sodium salts of the antibiotics of the formulas I and

11 contains. The disodium salts of the compounds MM 4550,

MM 13902 and MM 17880 are eluted after the desired salts.)

Example 2

Partial purification of the sodium salts of the compounds of formula I.

The freeze-dried substance with the salt of the compound of Example 1 is dissolved in 10 ml of deionized water and the solution is mixed with 1 g of sodium chloride. This solution is placed on a 1.5 x 15 cm column with «Amberlite XAD-4», which has been drawn up with fully demineralized water. The column is eluted with a mixture of 4 parts water and 1 part n-propanol at a rate of 2 ml per minute, and 4 ml fractions are collected. The fractions are tested for chloride by addition of silver nitrate and further against an RTEM-β-lactamase preparation for their β-lactamase inhibitory activity. The fractions with the greatest inhibitory activity and a negative silver nitrate reaction, namely fractions 6 to 13, are combined and freeze-dried to give 32.5 mg of an amorphous solid containing the sodium salt of the compound of formula I.

The properties of this substance are as follows:

A. Chromatographic properties

I. Chromatography on a weakly basic ion exchange paper ("Whatman DE81"):

Eluent

Rf value of the

Sodium salt

1. 0.05 molar phosphate buffer solution

from pH 7

0.69

2. 0.05 molar phosphate buffer solution

of pH 7 with a content of

0.2-m NaCl

0.80

II. Chromatography on “Whatman No. 1 Paper”

Solvent system

RrValue of

Sodium salt

1. Butanohethanol: water upper

Phase 4: 1: 5

0.12

2. ButanoL pyridine: water 1: 1: 1

0.42

B. High voltage paper electrophoresis

Electrophoresis is performed on paper # 20 using a pH 5.3 pyridine / acetic acid buffer solution as the eluent at 5000 V for 15 minutes. The RM values for the sodium salt are 1.0, as for the comparison compound benzylpenicillin.

C. Antibacterial effectiveness

The antibacterial activity of the substance using the microtiter method was determined as follows:

Organism minimum inhibitory concentration

tion (| ig / ml)

Bacillus subtilis A <40

Enterobacter cloacae N1 1250

Escherichia coli 10418 150

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organism

Minimum inhibitory

trations (} ig / m!)

E. coli JT 410

625

Klebsiella aerogenes A

312

Proteus mirabilis C977

625

Pseudomonas aeruginosa A

> 2500

Salmonella typhimurium CT10

312

Serratia marcescens US39

625

Staph. aureus russell

150

D. Enzyme Inhibition

The enzyme-inhibiting effect of the substance against a number of β-lactamase preparations is summarized as follows:

ß-lactamase preparations from:

% Inhibition

Concentration that

at 200

a 50%

[ig / ml

Inhibition results (Hg / ml)

Staph. aureus russell

40

E. coli JT4

-

95

Proteus mirabilis C889

-

112

Pseudomonas aeruginosa

Dalgleish

-

170

Enterobacter cloacae P99

83

Pseudomonas aeruginosa A

30th

Adhesive aerogenic E70

27th

(Procedure according to BE-PS 827926)

Example 3

Partial purification of the sodium salt of the compounds of formula II

The freeze-dried substance of the salt of the compound II of Example 1 is dissolved in 10 ml of deionized water. 1 g of sodium chloride is added to the solution.

Then the solution is placed on a 1.5 x 15 cm column with «Amberlite XAD-4», which has been drawn up with fully demineralized water. The column is eluted with a mixture of 4 parts water and 1 part n-propanol at a rate of 2 ml per minute. 4 ml fractions are collected. The fractions are examined for chloride using silver nitrate and for an RTEM-β-lactamase preparation for their β-lactamase inhibitory activity. The fractions with the greatest inhibitory activity and a negative reaction to silver nitrate, namely fractions 7 to 13, are combined and freeze-dried to give 32.5 mg of an amorphous solid containing the sodium salt of the compound of formula II.

The properties of this substance are as follows:

A. Chromatographic properties

I. Using a weakly basic anion exchange paper ("DE81 cellulose") the running value of the sodium salt is 0.54 when eluting with a pH 7 solution of 0.05 m phosphate buffer.

II. Using Whatman No. 1 Paper: The sodium salt has a running value of 0.20 when eluted with a 4: 1: 5 mixture of butanol / ethanol / water as the upper phase.

B. High voltage paper electrophoresis

Electrophoresis is performed on Whatman No. 20 paper in

Pyridine / acetic acid buffer solution of pH 5.3 as eluent carried out at 5000 V for 15 minutes. The RM value for the salt is 0.95 compared to benzylpenicillin with the value 1.0.

C. Antibacterial effectiveness

The antibacterial activity of the substance was determined using the microtiter method. The results are as follows:

Organism minimum inhibitory concentration

trations (ng / ml)

Bacillus subtilis A

250

Enterobacter cloacae N1

>

1000

Escherichia coli 10418

250

Klebsiella aerogenes A

500

Proteus mirabilis C? 77

500

Pseudomonas aeruginosa A

>

1000

Salmonella typhimurium CT 10

250

Serratia marscens US39

>

1000

Staph. aureus oxford

500

Staph. aureus russell

250

D. Enzyme Inhibition

The β-lactamase inhibitory activity of the substance against a number of enzyme preparations was determined with the following results:

ß-lactamase preparations from:

% Inhibition

Concentration that

at 200

a 50%

Hg / ml

Inhibition results (ixg / ml)

Staph. aureus russell

150

E. coli JT4

-

72

Proteus mirabilis C889

-

62

Pseudomonas aeruginosa

Dalgleish

-

53

Enterobacter cloacae P99

-

10th

Pseudomonas aeruginosa A

39

Klebsiella aerogenic E70

13

Example 4

Further purification of the sodium salts of the compounds of formula I.

The substance obtained according to Example 2 is dissolved in deionized water and placed on a strongly basic anion exchange column (“QAE-Sephadex A25”), which has been drawn up with deionized water. The column is eluted with deionized water with an increasing sodium chloride gradient from 0 to 0.18-m. The fractions from the column are examined for an ß-lactamase inhibitory activity against an RTEM preparation. The fractions with the greatest activity are pooled. Sodium chloride is added to the combined fractions to a final concentration of at least 5%. The solution obtained is placed on a column of «Amberlite XAD-4». Then the column is eluted with a mixture of 1 part n-propanol and 4 parts water. The fractions containing the desired salt, selected for their β-lactamase inhibitory activity, are combined, evaporated to remove the organic solvent under reduced pressure, and then freeze-dried.

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Example 5

Further purification of the sodium salts of the compounds of formula I.

The combined fractions from the chromatography on “QAE-Sephadex” from Example 4 can be desalted and further purified by chromatography on a column with “Biogel P2” as follows:

The freeze-dried solid from the column with "QAE-Sephadex" is dissolved in a small volume of fully demineralized water and placed on a column with "Biogel P2". The column is eluted with 1 percent aqueous butanol. The fractions are checked for their β-lactamase inhibitory activity and for the reaction with silver nitrate. Those fractions which show a negative reaction to silver nitrate but an adequate β-lactamase inhibitory activity are pooled and freeze-dried.

Example 6

Further purification of the sodium salts of the compounds of formula I.

The substance obtained by the process of Example 2 is dissolved in deionized water and placed on a column with a strongly basic anion exchange resin "Amberlite IRA 458". The column is drawn up with 0.05 m phosphate buffer solution of pH 7 and eluted with phosphate buffer solution with increasing sodium chloride content until the sodium chloride content is 1.0 molar. The fractions with the greatest β-lactamase inhibiting activity are pooled. Then sodium chloride is added to the pooled fractions to a concentration of at least .5%. The solution obtained is placed on a column with “Amberlite XAD-4”, which has been drawn up with deionized water. Then the column is eluted with a mixture of 1 part n-propanol and 4 parts water. The fractions with the greatest β-lactamase inhibiting activity but a negative reaction to silver nitrate are pooled and freeze-dried.

Example 7

Further purification of the sodium salts of the compounds of formula I.

The salt of the compound of the formula I can be purified further by chromatography on a cellulose-filled column (“Cellulose CC31”) as follows:

The impure solid containing the salt of the compound of formula I is dissolved in the minimum amount of deionized water and up to about 50% mixed with n-propanol. The solution obtained is added to the cellulose column. The column is then eluted with a mixture of 4 parts of n-propanol and 1 part of water. The fractions obtained are examined for their β-lactamase inhibitory activity after dilution with fully demineralized water. The fractions containing the desired salt are combined, evaporated to remove the solvent under reduced pressure and then freeze-dried.

Example 8

Further purification of the sodium salts of the compounds of formula II

The method described in Example 4 is used, but the substance obtained by the method of Example 3 is used as the starting substance.

Example 9

Further purification of the sodium salts of the compounds of formula II

The method described in Example 5 is used, but with the compound obtained according to Example 8 as the starting substance.

Example 10

Further purification of the sodium salts of the compounds of formula II

The method described in Example 6 is used, but with the substance obtained according to Example 3 as the starting compound.

Example 11

Further purification of the sodium salts of the compounds of formula II

The method described in Example 7 is used, but with the substance obtained according to Example 3 as the starting compound.

Example 12

Production of raw antibiotics from the culture fluid filtrate

80 ml of the culture liquid filtrate, which was essentially prepared by the process described in description No. 1, are placed on a 1.5 x 15 cm column with a strongly basic anion exchange resin on an acrylic basis ("Amberlite IRA 458") . Then the column is eluted with 0.05 m phosphate buffer solution of pH 7 with a gradually increasing content of 0 to 1.0 m sodium chloride at a flow rate of 2.5 ml per minute. 5 ml fractions are collected. The fractions are examined against an RTEM-β-lactamase preparation for their β-lactamase inhibitory activity. Those fractions with a good inhibitory activity and a content of the sodium salts of the compounds of the formulas I and II, namely fractions 4 to 13, are combined. (The disodium salts of the compounds MM 4550, MM 13902 and MM 17880 are only eluted at the beginning of fraction 17.)

3 g of sodium chloride are added to the combined fractions. The solution obtained is placed on a 1.5 x 15 cm column with «Amberlite XAD-4», which has been drawn up with fully demineralized water. The column is then eluted with a mixture of 1 part n-propanol and 4 parts water at a rate of 3 ml per minute. 4 ml fractions are collected. The fractions are examined for their β-lactamase inhibitory activity and for their reaction with silver nitrate solution. Those fractions with a good inhibitory activity and a negative reaction to silver nitrate are combined and freeze-dried, so that a partially purified preparation of the sodium salts of the compounds of the formulas I and II is obtained. This contaminated preparation is further cleaned using the methods described above.

Example 13

Manufacture of raw antibiotics from the culture fluid filtrate

305 ml of the culture liquid filtrate, which was essentially prepared by the process described in the description No. 1, are placed on a 1.5 × 15 cm column with a strongly basic acrylic-based anion exchange resin (“Amberlite IRA 458”) . The column is washed at a rate of 5 ml per minute with 100 ml of deionized water and then eluted at a rate of 5 ml per minute with a 0.025 m phosphate buffer solution of pH 7. 10 ml fractions are collected. The fractions are examined against an RTEM-β-lactamase preparation for their β-lactamase inhibitory activity. Those fractions with a good inhibitory activity and a content of sodium salts

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the compounds of the formulas I and II, namely fractions 12 to 32, are combined. The combined fractions are freeze-dried. The freeze-dried solid is dissolved in 20 ml of fully demineralized water. The solution is mixed with 2 g of sodium chloride and then placed on a 1.5 x 15 cm column with «Amberlite XAD-4», which has been drawn up with fully demineralized water. The column is eluted at a rate of 2 ml per minute with a mixture of 1 part n-propanol and 4 parts water. 4 ml fractions are collected. The fractions are examined for their β-lactamase inhibitory activity and for the reaction with silver nitrate solution. Those fractions with a good inhibitory activity and a negative reaction with silver nitrate, namely fractions 7 to 14, are combined and freeze-dried, so that a partially purified preparation of the salts of the compounds of the formulas I and II is obtained. This impure preparation can be further purified using the methods described above.

Example 14

Fermentation conditions for a 3000-liter fermentation A freeze-dried ampoule of Streptomyces olivaceus ATCC 31365 is resuspended in 10 ml of a sterile solution of 2% glucose and 1% soybean meal of pH 6.5 in deionized water.

1 ml of this suspension is inoculated onto 100 ml of a medium of the same composition, which is contained in a 500 ml Erlenmeyer flask and sealed with a foam stopper. After inoculation, the flask is incubated on a rotary shaker at 28 ° C for 30 hours. Then 5 ml portions of the culture medium are inoculated into Roux bottles to inoculate a solid slant agar. The agar has the following composition:

V8 vegetable juice 20.0%

"Bacto-Agar (Difco)" with pH 6.0 2.5%

The composition is made in demineralized water.

Each Roux bottle is incubated at 28 ° C for a week. 100 ml of sterile, fully demineralized water containing 0.1% of a surface-active compound (“Triton X”) are then added to a Roux bottle culture and the spores are suspended by shaking. The spore suspension is added as a vaccine to 75 liters of sterilized culture medium in a fermenter with stainless baffles. The composition of the medium is

Soybean meal 1%

Glucose 2%

Antifoam (Pluronic L81 ») 0.03% in distilled water

The medium is sterilized with steam in the fermenter at 120 ° C. for 20 minutes. The culture is stirred using a 19.05 cm vane stirrer at 140 rpm and aerated at a rate of 75 liters per minute with sterile air through the open end of the stirrer. Compliance with the temperature of 28 ° C is checked. After 48 hours of incubation under these conditions, 7.5 liters of this culture medium are inoculated as a vaccine for 150 liters of sterile fermentation medium in a 300 liter fermenter which is completely equipped with rustproof baffles. The fermentation medium has the following composition:

Soybean flour 0.9%

Glucose 2.0%

Chalk 0.02%

CoCh-ÓHzO 0.0001%

Antifoam (Pluronic L81) 0.2%

Before sterilization, the pH was 6.0. The composition is made with distilled water.

The fermentation medium is stirred with a 21.59 cm turbine mixer at 340 rpm. Compliance with the temperature at 29 ° C is checked. Air is fed in at a rate of 50 liters per minute and the pH is kept at 6.5 to 7.0.

The fermentation products can be harvested after a period of 48 to 54 hours.

Example 15

Fermentation conditions for a 2000 liter fermentation batch

The fermentation conditions up to and including the cultivation essentially correspond to the conditions described in Example 14. 75 liters of culture are used to inoculate a 1500 liter sterile fermentation medium which is in a 2000 liter stainless fermenter fully equipped with baffles. The fermentation medium is the same as in Example 14.

The fermentation broth is stirred with 2 turbine mixers, each with a diameter of 48.26 cm and at 106 rpm. Air is introduced at a rate of 400 liters per minute. The temperature is maintained at 29 ° C and the pH at 6.5 to 7.0 and the fermenter is harvested after 48 hours.

Example 16

Isolation processes for the preparation of essentially pure sodium salts of the compounds of the formulas I and II 150 liters of the total medium, which has been prepared essentially as described in Example 14, are clarified in a continuously operating centrifuge at approximately 2.4 liters / minute. 120 liters of the clarified filtrate of the fermentation liquid are at a speed on a column with a diameter of 15.24 cm and a bed volume of 9.6 liters, which is filled with a strongly basic anion exchange resin ("Amberlite IRA 458") in the chloride form percolated at 400 ml per minute. The column has previously been drawn up with demineralized water. After percolating the culture liquid filtrate, the column is washed with half the bed volume of water, then eluted at a rate of 230 ml per minute with a pH 7 0.05 m sodium phosphate buffer solution containing 0.2 m sodium chloride. 2 liter fractions are collected. Those fractions with a good inhibitory activity against a Rtem-β-lactamase preparation, namely fractions 2 to 11, are combined. Then 46.75 g / liter of sodium chloride are added to the combined fractions so that they contain a concentration of 1 m NaCl. The solution obtained is placed on a column of 10.16 cm in diameter and a bed volume of 4 liters and a filling of “Amberlite XAD-4” and equilibrated with a 1 m sodium chloride solution.

The percolation rate is 200 ml per minute. Then the column is eluted with 10 liters of deionized water and then with a mixture of 4 parts of water and 1 part of isopropanol each at a rate of 100 ml / minute. The fractions with a good ß-lactamase inhibitory activity against an RTEM enzyme preparation, namely fractions 3 to 10 and 13 to 17, wer5

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16

the combined, evaporated to remove the isopropanol under reduced pressure and freeze-dried.

A 3.8 x 30 cm column with weakly basic anion exchange resin «Cellulose DE52» is prepared in the chloride form with deionized water. The freeze-dried solid from the desalination stage is dissolved in 300 ml of fully demineralized water. The solution is added to the «Cellulose DE52» column at a rate of 6 ml per minute. The column is washed with 200 ml of deionized water and then eluted with 0.025 m potassium phosphate buffer solution of pH 7 at a rate of 2.5 ml per minute. 10 ml fractions are collected. The fractions are examined for their β-lactamase inhibitory activity. The first two main maxima of activity, namely fractions 30 to 85, which contain the sodium salt of the compound of formula I, and fractions 86 to 130, which contain the sodium salt of the compound of formula II, are combined individually.

The combined fractions 30 to 85 are placed on a 3.8 x 29 cm column with "QAE-Sephadex A25" in the chloride form, which has been taken up with fully demineralized water. The column is eluted at a rate of 3 ml per minute with a 0.1 M sodium chloride solution. 18 ml fractions are collected.

The fractions which have a good RTEM-β-lactamase inhibitory activity, namely fractions 58 to 68, are combined. 9 g of sodium chloride are added to this solution (180 ml). The solution obtained is placed on a 1.5 × 15 cm column with “Amberlite XAD-4”. The column is eluted with 135 ml of deionized water and then with a mixture of 4 parts of water and 1 part of isopropanol at a rate of 3 ml per minute. 4.5 ml fractions are collected. The fractions with UV spectra, which are characteristic of the partially purified sodium salts of the compounds of the formula I, namely UV maxima at approximately 297 nm - these are fractions 8 to 16 and 34 to 40 - are combined and freeze-dried, see above that 53 or 42 mg of solids with the characteristic properties of essentially pure sodium salts of the compounds of the formula I are obtained.

Fractions 86 to 130 from the "Cellulose DE52" column are placed on a 3.8 x 29 cm column with "QAE-Sephadex A25" in the chloride form. The column is eluted at a rate of 3 ml per minute with 0.1 M sodium chloride solution. 18 ml fractions are collected. The fractions are examined for their RTEM-ß-lacta-mase inhibitory activity. Those fractions with a good effect, namely fractions 94 to 100, are combined (120 ml). 6 g of sodium chloride are added to the combined fractions. The solution obtained is placed on a 1.5 x 15 cm column with «Amberlite XAD-4». The column is eluted with 90 ml of deionized water and then with a mixture of 4 parts of water and 1 part of isopropanol, each at a rate of 3 ml per minute. 4 ml fractions are collected. The fractions with the characteristic spectra of the impure sodium salts of the compounds of formula II with a maximum at approximately 307 nm, namely fractions 10 to 16 and 26 to 32, are combined and freeze-dried to give 9.7 and 21.7 mg, respectively Solids.

These solids have properties which essentially correspond to the pure antrium salts of the compounds of the formula II.

Example 17

Optionally, another process to be used to obtain the sodium salts of the compounds of the formulas I and II

The filtrate from the culture broth (120 liters), which contains the sodium salts of the compounds of the formulas I and II, is chromatographed on an ion exchange resin in the chloride form (“Amberlite IRA 458”) and desalted with an ion exchange resin (“Amberlite XAD-4 »).

The desalted freeze-dried solid is dissolved in 300 ml of deionized water and applied to a cellulose column in the chloride form (3.8 x 25 cm, "Cellulose DE52"). This column is washed with 200 ml of deionized water and eluted with 0.025 m potassium phosphate buffer, pH 7, at a rate of 6 ml per minute. 20 ml fractions are collected. The fractions which show a good RTEM-ß-lactamase inhibitory activity (350 ml) are combined and applied to a column with crosslinked dextran (3.8 × 30 cm, “QAE-Sephadex A25”). The column is eluted with 0.18 m sodium chloride at a rate of 3 ml / minute. 20 ml fractions are collected and analyzed on the basis of their UV spectrum. Fractions which show the characteristic absorption properties of the sodium salts of the compounds of the formula I (fractions 50 to 56) or for the sodium salt of the compound of the formula II (fractions 88 to 97) are combined separately.

Fractions 50 to 56 are mixed with 15 g of sodium chloride and applied to an ion exchange column (1.5 x 15 cm, «Amberlite XAD-4»). The column is washed with 15 ml deionized water and eluted with water / n-propanol in a 4: 1 ratio at a rate of 3 ml / minute. 3 ml fractions are collected. Fractions which show the characteristic UV spectrum of the purified sodium salt of the compound of the formula I (fractions 9 to 15) are combined, evaporated to remove the propanol under reduced pressure and freeze-dried. 35 mg of a solid are obtained, which shows the properties of the pure sodium salt of the compound of the formula I.

Fractions 88 to 97 are mixed with 24 g of sodium chloride and applied to an ion exchange column (1.5 × 15 cm, “Amberlite XAD-4”). The column is washed with 15 ml of deionized water and eluted with water / n-propanol in a 4: 1 ratio. The fractions which show the characteristic UV spectrum of the pure sodium salt of the compound of formula II are combined, evaporated to remove the propanol under reduced pressure and freeze-dried. 34 mg of the pure sodium salt of the compound of the formula II are obtained.

Example 18

The filtrate of the culture broth (105 liters) is worked up according to the procedure of Example 16 with ion exchange columns of the type "Amberlite IRA 458" and "Amberlite XAD-4". The fractions from the “Amberlite-XAD-4” column are concentrated to half the volume to remove the isopropanol and stored at 5 ° C. for about 65 hours. The solution (800 ml) is applied to a column with crosslinked dextran in deionized water (3.8 × 30 cm, “QAE-Sephadex A25”). The column is eluted with 800 ml of 0.05 m sodium chloride, then with 0.1 m sodium chloride at a rate of 4 ml / minute. 20 ml fractions are collected, which are analyzed for their RTEM-β-lactamase inhibitory activity and on the basis of their UV spectra. The fractions which contain the sodium salt of the compound of the formula I (fractions 73 to 80) or the sodium salt of the compound of the formula II (fractions 112 to 125) are combined separately.

Fractions 73 to 80 are concentrated under reduced pressure to about 15 ml and onto a polyacrylamide gel column (3.8 × 30 cm, “Biogel P2”, particle size 0.3 to 0.4

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mm, from Biorad Laboratories, Bromley, Kent), which was grown with water containing 1% butanol. The column is eluted at a rate of 3 ml / minute with deionized water containing 1% butanol. 6 ml fractions are collected and analyzed on the basis of their UV spectrum and the silver nitrate reaction. Fractions containing the sodium salt of the compound of formula I and showing a negative silver nitrate reaction (fractions 29 to 39) are combined and freeze-dried. 72 mg of a solid with the characteristic properties of the purified sodium salt of the compound of the formula I are obtained.

Fractions 112 to 125 are concentrated to about 15 ml under reduced pressure and applied to the polyacrylamide gel column described above. The column is eluted with deionized water containing 1% butanol at a rate of 3 ml / minute. 6 ml fractions are collected. The fractions which show the characteristic UV spectrum of the sodium salt of the compound of the formula II and a negative silver nitrate reaction are pooled (fractions 43 to 48). The combined fractions are freeze-dried, 27 mg of a solid with the characteristic properties of the pure sodium salt of the compound of formula II are obtained.

Example 19

1500 liters of the total broth prepared according to Example 15 are filtered through diatomaceous earth on a rotatable auxiliary layer filter. 1400 liters of filtrate are obtained, which is fed through an ion exchange column in the chloride form (“Amberlite IRA 458”) with a diameter of 30.5 cm and a bed volume of 100 liters with an average flow rate of 4 liters per minute. The column is eluted at a rate of 1.6 liters / minute with 0.2 M sodium chloride in 0.05 M sodium phosphate buffer, pH 6.7. 20 liter fractions are collected. Fractions with a good antibacterial activity against Klebsiella aerogenes A (a variant of NCTC 418) are pooled (fractions 1 to 5). These fractions are mixed with sodium chloride to a final concentration of 1.0 m. The solution is applied to an ion exchange column (“Amberlite XAD-4”) with a diameter of 15.3 cm and a bed volume of 22.4 liters at a rate of 1.2 liters / minute. This column is eluted with 20 liters of deionized water, then with water / isopropanol in a 4: 1 ratio at a rate of 500 ml / minute. 8 liter fractions are collected.

The fractions containing the desired salts (fractions 2 to 4) are combined (24 liters) and evaporated to 11.4 liters. After adjusting the pH to 7.0, the concentrate is stored at 5 ° C. for about 65 hours. The solution is then further concentrated to 3.75 liters and applied to a cellulose column (7.8 x 31 cm, "DE 52") at a rate of 6 ml / minute. The column is eluted with 0.025 m potassium phosphate buffer, pH 7, at a rate of 4 ml / minute. 22 ml fractions are collected and examined for their RTEM-β-lactamase inhibitory effect. Fractions 60 to 135 and 136 to 210 show good inhibitory activity and are combined separately.

The combined fractions 60 to 135 (1580 ml) are applied to a column with crosslinked dextran (4.8 × 25 cm, “QAE-Sephadex A25”) at a rate of 6 ml / minute. This column is eluted with 0.1 M sodium chloride at a rate of 4 ml / minute. 20 ml fractions are collected and analyzed on the basis of their UV spectra. The fractions showing the characteristic spectrum of the sodium salt of the compound of formula I (fractions 50 to 70) are combined.

Fractions 136 to 210 are applied to a column of cross-linked dextran (4.8 x 26 cm) at a flow rate of 6 ml / minute. The column is eluted with 0.1 M sodium chloride at a rate of 4 ml / minute. 20 ml fractions are collected. Fractions with the characteristic spectra of the partially purified salts of the compounds of the formula II (fractions 91 to 104) are combined, mixed with 25 g of sodium chloride and on an ion exchange column (2.4 × 32 cm, “Amberlite XAD-4”) at a rate of 6 ml / minute.

The column is washed with about 50 ml of deionized water and eluted at a rate of 5 ml / minute with water / n-propanol in a 4: 1 ratio. 10 ml fractions are collected. Fractions which give a negative silver nitrate reaction to chloride but which have the characteristic UV spectrum of the sodium salt of the compound of the formula II are pooled (fractions 17 to 30). These fractions are evaporated under reduced pressure to remove the n-propanol and freeze-dried. 585 mg of the pure sodium salt of the compound of the formula II are obtained.

Example 20

The sodium salts of the compounds of the formulas I and II have the following properties:

1. UV spectrum: The mixed sodium salts of the compounds of formula I have a characteristic maximum at about 297 nm.

The mixed sodium salts of the compounds of formula II have a characteristic maximum at about 307 nm (1 of 2).

2. The IR spectrum of the sodium salts shows characteristic ß-lactam carbonyl absorptions at 1750 cm-1.

3. The in vitro antibacterial activity of the compounds described in the previous examples is shown in the table.

organism

Sodium salt of

Sodium salt of

Connection of

Connection of

Formula I;

Formula II;

Minimum inhibition

Minimum inhibition

Concentrations Concentrations

((ig / ml)

(Hg / ml)

Bacillus subtilis A

0.8

0.2

Enterobacter cloacae N1

25.0

25.0

Escherichia coli 10418

1.5

1.5

Klebsiella aerogenes A

12.5

6.25

Proteus mirabilis C977

6.25

6.25

Pseudomonas aeruginosa A

> 100

> 100

Salmonella typhimurium CT 10

3.12

0.8

Serratia marcescens US39

50.0

25.0

Staph. aureus oxford

0.8

1.5

Example 21

The practically pure mixed sodium salts of the compounds of the formulas Ia and Ib are tested by high pressure liquid chroamtography under the following conditions:

Column: 300 x 3.9 mm, filled with a monomolecular octadecylsilane layer on a solid support.

Solvent: 0.05-m ammonium acetate, adjusted to pH 4.5 with acetic acid in 5% acetonitrile / 95% water.

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Flow rate: 2.5 ml / minute.

Detection: UV absorption at 295 nm.

Task: 50 microliters of a solution of 1.6 mg in 0.5 ml of water.

The two sodium salts are dissolved in two peaks of 3 minutes 45 seconds and 4 minutes 45 seconds. The eluates of both peaks are combined separately and neutralized to pH 7 with dilute sodium hydroxide solution. The solutions are evaporated to give the desired solid salts of the compounds of the formulas Ia and Ib.

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Example 22

According to the procedure of Example 21, the mixed sodium salts of the compound of the formulas IIa and IIb are separated by high pressure liquid chromatography with residence times of 6.0 minutes and 7 minutes and 10 seconds, respectively.

Example 2 ~ $

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Filtrate of the culture liquid

Absorption on a strongly basic resin "Amberlite

IRA 458 "

Elute with 0.2 M NaCl in buffer solution

Pooling the fractions containing the compounds of the formulas Ia, Ib, IIa and IIb v

Desalt on "Amberlite XAD-4" elute with isopropanol / water

Evaporation under reduced pressure to remove the isopropanol

Ia, Ib

Desalting on "Amberlite XAD-4"

y

Freeze-drying the fractions containing the compounds Ia, Ib

Chromato-

Chromatography on "QAE-Sephadex

V

IIa, IIb

V

Desalting on "Amberlite XAD-4"

graph to "QAE-Sephadex

Fractions containing the compounds Ia, Ib

Freeze-drying the fractions containing the compounds IIa, IIb

Chromatography on Diaion HP20 "

1/

Chromatography on Diaion HP20 "

vy

Fractions containing compound IIa

Fractions containing compound IIb

Fractions containing compound Ia

Fractions containing the compound Ib ir chromatography - on "Biogel P2"

v /

Fractions containing compound IIa v

Fractions containing compound IIb

Chromatography, on "Biogel P2" ^

v

Fractions containing compound Ia

V

Fractions containing the compound Ib v

IIa

Chromatography on "Diaion HP20" and ^ ^ freeze-dry

\ / IIb

Chromatography on "Diaion HP20" and ^ 1 / freeze-drying

Ia, Ib, IIa and IIb here mean the sodium salts

Yes

Ib

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20th

Streptomyces olivaceus ATCC 31365 spores are grown according to the procedure of Example 14.

The spore suspension from a Roux bottle is used as an inoculum for 75 liters of sterilized nutrient medium in a 100 liter stainless steel fermenter with guide plates. The nutrient medium has the following composition:

Soybean meal 1%

Glucose 2%

Antifoam (Pluronic L81) 0.03% in distilled water

The medium is sterilized with steam in the fermenter at 120 ° C. for 20 minutes. The nutrient solution is stirred at 140 rpm using a 19 cm diameter vane stirrer and supplied with 75 liters / minute of sterile air by an open-ended spray device. The temperature is adjusted to 28 ° C. After 48 hours of incubation under these conditions, 75 liters of this culture solution are used as inoculum for 1500 liters of sterile nutrient medium in a 2000 liter stainless steel fermenter which is provided with baffles. The fermentation medium has the following composition:

Soybean meal 2.0%

Glucose 0.9%

Chalk 0.02%

CoCh • 6H2O 0.0001%

Antifoam (Pluronic L81) 0.2%

in distilled water, adjusted to pH 6 before sterilization

The fermentation medium is stirred with 2 disc turbine mixers with a diameter of 48 cm each at 106 rpm and supplied with air at a rate of 400 liters / minute. The temperature is kept at 29 ° C. and the pH is kept at 6.5 to 7.0. The harvest takes place after 48 hours.

Another culture filtrate suitable for the extraction of the compounds of the formulas Ia, Ib, IIa and IIb is obtained by using the spore suspension of S. olivaceus from 2 Roux bottles as an inoculum for 150 liters of sterile nutrient medium in a 300 liter steel fermenter. The medium and growth conditions are as described above. After 48 hours, 150 liters of this medium are used to inoculate 3000 liters of fermentation broth in a 5000 liter steel fermenter. The fermentation medium is incubated as above, but harvesting takes 55 hours.

The culture broths from the two 2000 and 5000 liter fermenters together give a volume of 4725 liters. They are filtered on a rotating auxiliary layer filter under reduced pressure. 4200 liters of clear broth are obtained, which are applied to columns with strongly basic anion exchange resin in the chloride form (“Amberlite IRA 458”) at a flow rate of 10 liters / minute. The resin is washed with 60 liters of deionized water, then eluted with an aqueous solution of 0.2 M sodium chloride and 0.075 M sodium phosphate, pH 6.7. The elution of the compounds of the formulas Ia, Ib, IIa and IIb begins when the conductivity of the eluent has reached the conductivity of 0.1 M sodium chloride and when 450 liters of eluent have drained away. If necessary, the presence of compounds of the formulas Ia, Ib, IIa and IIb is determined by means of analytical high-pressure liquid chromatography according to Example 21.

The combined eluates, which are the compounds of the formulas

Ia, Ib, IIa and IIb contain 90 kg (wet weight) of ion exchange resin («Amberlite XAD-4»). The mixture is adjusted to pH 6.0 with 50 percent hydrochloric acid and stirred gently at 50 ° C. for 1 hour. The resin is then filtered off and washed with deionized water at 5 ° C until the conductivity of the wash water is below that of 0.05 m sodium chloride. The washed resin is slurried in 42 liters of isopropanol / water in a 1: 1 ratio and mixed with 20 percent sodium hydroxide solution until the pH remains constant at 7.5. The eluent is filtered and collected. The elution of the resin is repeated first with further isopropanol / water in the ratio 1: 1, then with isopropanol / water in the ratio 1: 3. The combined eluates (130 liters) are evaporated under reduced pressure to remove the isopropanol. The solution obtained (67 liters) is placed on a column with cross-linked dextran (15 x 43 cm, “QAE-Sephadex A25”), which has been pretreated with 0.1 m sodium chloride, at a flow rate of 15 liters / hour. given up. The column is washed with 7.5 liters of 0.05 m sodium chloride and eluted with 0.1 m sodium chloride at a rate of 7.8 liters / hour. 500 ml fractions are collected, which are examined by means of high-pressure liquid chromatography for the presence of compounds of the formulas Ia, Ib, IIa and IIb. Fractions containing the compounds of formulas Ia and Ib that were eluted together are pooled (12.6 liters). Fractions containing the compounds of formulas IIa and IIb elute later and are also pooled (5.9 liters).

The fractions containing the compounds of the formulas Ia and Ib are mixed with 44.16 g / liter of sodium chloride and applied to an ion exchange column (10 × 36 cm, “Amberlite XAD-4”) at a flow rate of 5.8 liters / hour. The column is washed with 3 liters of deionized water at a rate of 2.9 liters / hour, then eluted with isopropanol / water in a ratio of 1: 9. The fractions which contain the compounds of the formulas Ia and / or Ib in sufficient amounts (determined by high pressure liquid chromatography) are collected after the conductivity of the eluate corresponds to the conductivity of 0.01-m sodium chloride. The eluted solution (7.7 liters) containing the compounds of the formulas Ia and Ib is adjusted to pH 7.0 with 20 percent sodium hydroxide solution, concentrated under reduced pressure and freeze-dried. 38.5 g of a solid are obtained.

The combined eluates from the column with the crosslinked dextran, which contain the compounds of the formulas IIa and IIb, are treated accordingly. 13.7 g of solid are obtained.

The freeze-dried product (38.5 g) containing the compounds of the formulas Ia and Ib is dissolved in 50 ml of deionized water and applied to a column with crosslinked dextran (7.8 × 30 cm, “QAE-Sephadex A25”) in deionized water . The column is washed at a rate of 8 ml / minute first with 4 liters of 0.05 m sodium chloride, then eluted with 0.08 m sodium chloride. Fractions of about 20 ml are collected and examined for the presence of compounds of the formulas Ia and Ib using their UV spectrum (fractions 130 to 170). These fractions are pooled (950 ml). Chromatography is carried out at 50 ° C.

450 ml of these combined fractions containing the compounds of the formulas Ia and Ib are mixed with 23.8 g of sodium chloride and placed on a column (4.8 × 62 cm, “Diaion HP20” from Mitsubishi Chemicals Ltd., Agents Nippon Rensui Co. , Tokyo, Japan) at a rate of 12 ml / minute. 20 ml fractions are collected and determined on the basis of their UV spectra. The Fractio5

10th

15

20th

25th

30th

35

40

45

50

55

60

o5

21st

641 805

Those containing the compound of the formula Ia (fractions 58 to 74) or the compound of the formula Ib (fractions 78 to 97) are combined separately, evaporated under reduced pressure and freeze-dried. 490 and 357 mg of solid are obtained.

The remaining eluate from the dextran column is worked up accordingly, 363 and 258 mg of solid are obtained.

538 mg of the solid containing the compound of the formula Ia are dissolved in 25 ml of deionized water and applied to a polyacrylamide gel column (7.8 × 40 cm, “Biogel P2”, particle size 0.3 to 0.4 mm). The column is eluted at 5 ° C with deionized water at a rate of 3 ml / minute. 25 ml fractions are collected and analyzed on the basis of their UV septums. Fractions which have a characteristic spectrum for very pure compounds of the formula Ia (fractions 37 to 42) are combined and concentrated to about 10 ml under reduced pressure. This solution is applied to a «Diaion-HP20» column (3.0 x 50 cm) and eluted with deionized water at a rate of 5 ml / minute. 10 ml fractions are collected and analyzed on the basis of their UV spectra. Fractions with a characteristic spectrum for very pure compounds of the formula Ia (fractions 50 to 62) are combined, concentrated under reduced pressure and freeze-dried. 40 mg of the compound of the formula Ia are obtained as a solid.

5.8 mg of the solid containing the compound of the formula Ib are dissolved in about 25 ml of deionized water and applied to a polyacrylamide gel column (7.8 x 40 cm). The column is eluted with deionized water at a rate of 3 ml / minute. 25 ml fractions are collected. Fractions which contain very pure compounds of the formula Ib (fractions 32 to 37, determined on the basis of their UV spectrum) are combined and concentrated to about 10 ml under reduced pressure. The solution is applied to a «Diaion-HP20» column (2.4 x 40 cm) and eluted with deionized water at a rate of 5 ml / minute. 10 ml fractions are collected and analyzed on the basis of their UV spectrum. Fractions containing very pure compounds of the formula Ib (fractions 35 to 48) are combined and freeze-dried. 53 mg of the compound of the formula Ib are obtained as a solid.

7 g of the freeze-dried solid containing the compounds of the formulas IIa and IIb from the desalination treatment carried out with “Amberlite XAD-4” are dissolved in 50 ml of deionized water and applied to a “Diaion-HP20” column (4.8 × 57 cm) . The column is eluted with deionized water at a rate of 10 ml / minute. 20 ml fractions are collected and analyzed on the basis of their UV spectrum. Fractions containing the compound of formula IIa (49 to 63) or IIb (71 to 110) are combined separately and freeze-dried. 875 mg or 1.47 g of solid are obtained.

The remaining solid containing the compounds of the formulas IIa and IIb is worked up accordingly, 860 mg and 1.44 g of compounds of the formulas IIa and IIb are obtained.

860 mg of the compound of the formula IIa are dissolved in 25 ml of deionized water and applied to a polyacrylamide gel column (7.8 × 40 cm, “Biogel P2”, particle size 0.3 to 0.4 mm). The column is eluted with deionized water at a rate of 3 ml / minute. 25 ml fractions are collected. Fractions containing very pure compound of the formula IIa (fractions 42 to 50, determined on the basis of their UV spectrum) are pooled, evaporated to about 10 ml under reduced pressure and placed on a «Diaion-HP20» column (2.8 x 40 cm). The column is eluted with deionized water. The factions

the UV spectrum of which indicates a very pure compound of the formula IIa (fractions 52 to 64), are combined, evaporated and freeze-dried. 95 mg of the compound of the formula IIa are obtained as a solid.

750 mg of the compound of the formula IIb are dissolved in about 25 ml of deionized water and placed on a polyacrylamide gel column (7.8 × 40 cm, “Biogel P2”, particle size 0.3 to 0.4 mm). The column is eluted with deionized water at a rate of 3 ml / minute. 25 ml fractions are collected. Fractions whose UV spectrum indicates a very pure compound of the formula IIb (fractions 47 to 55) are combined, evaporated to about 10 ml under reduced pressure and placed on a «Diaion-HP20» column (2.8 x 42 cm) given up. The column is eluted with deionized water at a rate of 5 ml / minute. The first 25 fractions are collected as 5 ml fractions, the remaining fractions as 10 ml fractions and analyzed on the basis of their UV spectrum. Fractions containing very pure compound of formula IIb (fractions 77 to 91) are combined and freeze-dried. 105 mg of the compound of the formula IIb are obtained as a solid.

Example 24

The sodium salts of the compounds of the formulas Ia, Ib, IIa and IIb prepared according to Example 23 are pure and have the following properties:

1. UV spectrum

Ia: a single maximum at 298 nm (molar extinction 6 = 8131) (cf. FIG. 1).

Ib: a single maximum at 301 nm (molar extinction s = 7930) (cf. FIG. 2).

IIa: 2 maxima at 228 and 308-309 nm (s = 13627) (see Fig. 3).

IIb: 2 maxima at 229 and 308-310 nm (s = 13933) (see Fig. 4).

2. The antibacterial activity of the sodium salts is determined by the microtiter method and is given in Table A.

3. The β-lactamase inhibitory effect of the sodium salts is shown in Table B.

4. NMR spectra (each determined in D2O)

Ia: cf. Fig. 5

Ib: cf. Fig. 6

IIa: cf. Fig. 7

IIb: cf. Fig. 8

5. The synergistic activity of the sodium salts is shown in Table C.

6. The sodium salts have no toxic effects in mice after subcutaneous administration in a dose of 50 mg / kg.

Table A Minimum inhibitory concentrations (ng / ml)

Strain Ia Ib IIa IIb Ampicil lin

B. subtilis

0.16

1.2

<0.08 2.5

<3.0

Enterobacter cloacae N.l

2.5

10th

2.5

10th

200

E. coli 10418

0.3

2.5

0.3

5.0

<3.0

E. coli JT39

5.0

2.5

5.0

5.0

800

E. coli JT68

5.0

2.5

10th

5.0

1600

E. coli JT410

0.6

5.0

0.6

5.0

200

Klebsiella A

2.5

5.0

1.2

5.0

100

Klebsiella E70

10th

5.0

10th

10th

400

Klebsiella Ba95

40

10th

40

10th

> 6400

Proteus mirabilis C977

0.6

10th

0.6

10th

<3.0

5

10th

15

20th

25th

30th

35

40

45

50

55

60

DD

641 805

22

Strain la Tb IIa IIb ampicillin

Proteus mirabilis C889 5.0 10 10 20 400 Proteus morganii 1580 2.5 10 2.5 20 100 Proteus vulgaris Q3618 5.0 10 10 20 400 Pseudomonas aeruginosa A> 160> 160> 160> 160 1600

Salmonella CT10 Serratia US39 Staph. Oxford Staph. Russell Staph. Smith Strep. faecalis

0.3 20 1.25 0.6 0.6

2.5 10 1.2 2.5 2.5

1.25 20

0.3 20 0.3 0.3 0.6 1.2

5.0 10 2.5 2.5 2.5 20

<0.3 1600 <3.0 400 <3.0 <3.0

Table B

Iso (jxg / ml)

Connection Enterobacter Ps.aerug. Proteus E. coli Staph.

P99 A C889 JT4 Russell

15

Table C.

Minimum inhibitory concentrations (µg / ml)

connection

Staph. aureus russell

E. coli JT39

Ampicillin alone

1000

2000

+ (Ia) 0.1 µg / ml

> 10

> 500

1 µg / ml

-

500

+ (Ib) 0.1 µg / ml

> 10

500

1 p.g / ml

10th

31.2

+ (IIa) 0.1 µg / ml

10th

> 500

1 jig / ml

-

500

-t- (IIb) 0.1 µg / ml

> 10

500

1 µg / ml

10th

31.2

IIa 0.02

IIb 0.04

Ia 0.02

Ib 0.04

3.0

0.04

4.0

0.4

4.0

0.1

4.0

> 2.0

> 2.0 0.08

0.1> 2.0

> 2.0 3.25

0.28> 2.0

4 sheets of drawings

Claims (31)

641 805 2. Compound according to claim 1 of formula Ia and its salts. 2nd PATENT CLAIMS 1. Derivatives of 3-thio-6- (l-hydroxyethyl) -7-oxo-l-aza-bicyclo [3,2,0] hept-2-ene-2-carboxylic acid of formulas I and II and their salts. 3rd 641 805 h s-c = c-nh-co-ch. H (Hb) and their salts. > 5 // ^ 3 and their salts. 3. A compound according to claim 1 of formula Ib 40 ^ - s-ch2 ~ ch2-nh-co-ch3 and their salts. 4th net by containing a penicillin or cephalosporin as an additive. 42. Medicament according to one of claims 39 to 41, characterized by a content of 50 to 500 mg of the active component. 43. Medicament according to one of claims 39 to 42 in injectable form. In British patents 1467 413, 1489235 and 1483 142, which correspond to German Offenlegungsschriften 2513 855, 2513 854 and 2609766, it is described that during the fermentation of Streptomyces olivaceus antibiotics can be produced which are known as MM 4550, MM 13902 and MM 17880 »and which have the following formulas III, IV and V: H03S0 ho3so C-NH-CO-CH. (III) S-C = C-NH-CO-CH3 (IV) h "c h h 4. A compound according to claim 1 of formula IIa H3C H J I I H - s-c = c-nh-co-ch- 5. A compound according to claim 1 of formula IIb 6. Pharmacologically acceptable salts of the compounds according to one of claims 1 to 5. 7. alkali metal and alkaline earth metal salts of the compounds according to any one of claims 1 to 5. 8. sodium salts of the compounds according to any one of 20 claims 1 to 5. 9. Potassium salts of the compounds according to one of claims 1 to 5. 10. A salt according to any one of claims 6 to 9 with a degree of purity of at least 50%. 25th 11. A salt according to any one of claims 6 to 9 with a purity of at least 75%. 12. A salt according to any one of claims 6 to 9 with a degree of purity of at least 90%. 13. A salt of the compound of formula Ia according to any one of claims 6 to 12, which is free of a salt of the compound of formula Ib. 14. A salt of the compound of formula Ib according to any one of claims 6 to 12, which is free of a salt of the compound of formula Ia. 35 15. A salt of the compound of formula IIa according to any one of claims 6 to 12, which is free of a salt of the compound of formula IIb. 16. A salt of the compound of formula IIb according to any one of claims 6 to 12, which is free of a salt of the compound of formula IIa. 17. An alkali metal salt of the compound of formula Ia according to claim 2 having a molar extinction coefficient in water at a neutral pH of not less than 7700. 18. A salt according to claim 17 having a molar extinction coefficient in water at neutral pH of not less than 7900. 19. Sodium salt according to claim 17 or 18. 20. An alkali metal salt of the compound of formula Ib according to claim 3 with a molar extinction coefficient 50 in water at a neutral pH of not less than 7700. 21. A salt according to claim 20 having a molar extinction coefficient in water at neutral pH not below 7900. 22. Sodium salt according to claim 20 or 21. 55 23. An alkali metal salt of the compound of formula IIa according to claim 4 with a molar extinction coefficient in water at a neutral pH of not less than 13,000. 24. A salt according to claim 20 having a molar extinction coefficient in water at neutral pH of 60 not less than 13500. 25. Sodium salt according to claim 23 or 24. 26. An alkali metal salt of the compound of formula IIB according to claim 5 having a molar extinction coefficient in water at a neutral pH of not less than 13,000. 05 27. A salt according to claim 20 having a molar extinction coefficient of not less than 13500. 28. Sodium salt according to claim 23 or 24. 29. A process for the preparation of compounds according to claim 1, characterized in that one cultivates a strain of Streptomyces olivaceus or Streptomyces gedanensis until a substantial amount of the compounds is produced and then the compounds are obtained from the growth medium. 30. The method according to claim 29, characterized in that Streptomyces olivaceus ATCC 31126 is used. 31. The method according to claim 29, characterized in that one uses Streptomyces olivaceus ATCC 31365. 32. The method according to any one of claims 29-31 for the preparation of a salt of the compound of formula Ia, which is practically free of a compound of formula Ib, characterized in that a mixture of these salts of a chromatographic separation on a polystyrene-based polymer absorbent or subject to an equivalent exchange resin. 33. The method according to any one of claims 29-31 for the preparation of a salt of the compound of formula Ib, which is practically free of a compound of formula Ia, characterized in that a mixture of these salts of a chromatographic separation on a polystyrene-based polymer absorbent or subject to an equivalent exchange resin. 34. The method according to any one of claims 29-31 for the preparation of a salt of the compound of formula IIa, which is practically free of a compound of formula IIb, characterized in that a mixture of these salts of a chromatographic separation on a polystyrene-based polymer absorbent or subject to an equivalent exchange resin. 35. The method according to any one of claims 29-31 for the preparation of a salt of the compound of formula IIb, which is practically free of a compound of formula IIa, characterized in that a mixture of these salts of a chromatographic separation on a polystyrene-based polymer absorbent or subject to an equivalent exchange resin. 36. The method according to any one of claims 32 to 35 for the preparation of an alkali metal salt. 37. The method according to claim 36 for the preparation of the sodium salt. 38. The method according to any one of claims 32 to 37, characterized in that water is used as the solvent. 39. Medicament, characterized in that it contains a derivative of 3-thio-6- (l-hydroxyethyl) -7-oxo-l-azabi-cyclo- [3,2,0] hept-2-en-2-carboxylic acid of formula I or II or their salts as an active component. 40. Medicament according to claim 39, characterized by a content of pharmacologically acceptable carrier materials, diluents, excipients and / or additives. 41. Medicament according to claim 39 or 40, 641 805 31 • i h03s0 S-CH2-CH2-NH-CO-CH3 (V) None of these British patents or German antibiotics have been isolated. There is an indication that there is information from the