GB1601688A

GB1601688A - 6-(1-hydroxyethyl)-7-oxo 1-aza bicyclo (3,2,0)-hept-2-ene-2-carboxylic acid derivatives and their use in pharmaceutical compositions

Info

Publication number: GB1601688A
Application number: GB9366/77A
Authority: GB
Original assignee: Beecham Group PLC
Current assignee: Beecham Group PLC
Priority date: 1977-03-05
Filing date: 1977-03-05
Publication date: 1981-11-04
Also published as: ZA781051B; BE864570A

Description

(54) 6-( 1 -HYDROXYETHYL)-7-OXO-1-AZA BICYCLO [3,2()]-HEPT-2-ENE-2-CARBOXYLIC ACID DERIVATIVES AND THEIR USE IN PHARMACEUTICAL COMPOSITIONS (71) We, BEECHAM GROUP LIMITED, of Beecham House, Great West Road, Brentford, Middlesex, England. do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: British Patent Specifications Nos. 1467413, 1489235 and 1483142 discloses that fermentation of Streptomyces olivaceus can lead to the preparation of antibiotics named MM4550, MM13902 and MM17880 which have the formula (III), (IV) and (V) respectively.

None of these British Specifications contained any suggestion that a further antibiotic could be obtained from the fermentation broth of Streptomyces olivaceus. Further antibiotics have now been found.

The present invention provides the compounds of the formulae (I) and (II):

and their salts.

Most suitably the compounds of the formulae (I) and (II) are in the form of a salt since it appears that salts of the compounds of formulae (I) and (II) are more stable than the parent acids.

Suitable salts of the compounds of the formula (I) and (II) include the pharmaceutically acceptable alkali and alkaline earth metal salts such as the sodium, potassium and calcium salts and pharmaceutically acceptable addition salts with nitrogenous bases such as the ammonium, trimethylamine, dimethylamine and pyrrolidine salts.

Particularly suitable salts of the compounds of the formulae (I) and (II) include their sodium and potassium salts.

A preferred compound of this invention is the sodium salt of a compound of the formula (I). A second preferred compound of this invention is the sodium salt of a compound of the formula (II).

Since the compounds of the formulae (I) and (II) and their salts are intended for use in pharmaceutical compositions it will readily be understood that they are each provided in substantially pure form, for example at least 50% pure, more suitably at least 75% pure and preferably at least 90% pure and yet more preferably at least 95% pure. Impure preparations of the compounds of the formulae (I) and (II) and their salts may be used for preparing the more pure forms used in the pharmaceutical compositions, these less pure preparations of the compounds of the formulae (I) and (II) and their salts should contain at least 1%, more suitably at least 5% and preferably from 10 to 49% of a compound of the formula (I) or (II) or their salts. These less pure preparations most usefully comprise a salt of the compound of the formula (I) and a salt of the compound of the formula (II). (% are on a wt/wt basis.) It is generally preferred that the substantially pure salts of the compound of the formula (I) or the formula (II), are not contaminated by substantial amounts of other antibacterial agents such as salts of the compounds of the formula (III), (IV) and (V), derived from the fermentation broth.

The compounds of the formulae (I) and (II) exist as cis- and trans-forms about the p-lactam ring. These forms may be represented by formulae (Ia), (Ib), (Ila) and (IIb) thus:

It will be realised that the preceding compounds may be named as follows: Ia (SR.6R)-3-(2-acetamidoethylthio)-6-[(S)-1-hydroxyethyl]-7-oxo-1- azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid.

Ib (5R,6S)-3-(2-acetamidoethylthio)-6-[(S)-1-ltydroxyethyl]-7-oxo-1- azabicyclo[3.2.0lhept-2-ene-2-carboxylic acrid IIa (5R,6R)-3-[(E)-2-acetamidoethenylthio]-6-[(S)-1-hydroxyethyl]-7-oXo-1- 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-ene-2-carboxylic acid Both cis- and trans- isomers of the compounds of the formulae (I) and (II) have useful antibacterial and B-lactamase inhibiting properties and so this invention extends to the isolated compounds of the formulae (Ia) and (Ib) as well as to mixtures thereof and to the isolated compounds of the formulae (IIa) and (IIb) as well as mixtures thereof.

Naturally isolated cis- and trans- forms will most suitably be in the forms of substantially pure pharmaceutically acceptable salts as described above: that is they should be at least 50% pure, more suitably at least 75% pure, preferably 90% pure and most preferably at least 95% pure. Furthermore it is preferred to use one of the aforementioned compounds when substantially free of its 6-position isomer [that is (Ia) free of (Ib), (IIa) free of (IIb), (Ib) free of (lea) or (lIb) free of (Ib)j. In general such compounds should not contain more than 5% of its 6-position isomer and preferably not more than 1% of its 6-position isomer.

(High pressure liquid chromatography may be used to monitor purities.) A favoured aspect of this invention provides an alkali metal salt of the compound of the formula (lea) having a molar extinction coefficient (in water at neutral pH) of not less than 7700 (preferably not less than 7900) (for UV absorption maximum at about 298 nm).

A favoured aspect of this invention provides an alkali metal salt of the compound of the formula (Ib) having a molar extinction coefficient (in water at neutral pH) of not less than 7700 (preferably not less than 7900) (for UV absorption maximum at about 301 nm).

A favoured aspect of this invention provides an alkali metal salt of the compound of the formula (Ila) having a molar extinction coefficient (in water at neutral pH) of not less than 13000 (preferably not less than 13500) (for UV absorption maximum at about 308 nm).

A favoured aspect of this invention provides an alkali metal salt of the compound of the formula (IIb) having a molar extinction coefficient (in water at neutral pH) of not less than 13000 (preferably not less than 13500) (for UV absorption on maximum at about 308 nm).

Preferably the preceding alkali metal salts are the sodium salts.

The present invention also provides a pharmaceutical composition which comprises a compound of the formula (I) or (II) or a salt thereof and a pharmaceutically acceptable carrier.

The compositions of this invention will generally utilise a pharmaceutically acceptable salt of a compound of the formula (I) or (II), for example a sodium or potassium salt.

The pharmaceutical compositions of this invention may be adapted for oral or parenteral administration. Suitably the compositions are provded as dosage forms which contain from 50 to 500 mg of a compound of the formula (I) or (II) or its salt, for example about 100, 150, 200 or 250 mgs.

Most usually the composition will be adapted for administration by injection.

These compositions may contain diluents, binders disintegrants, lubricants or other conventional excipient and may be fabricated by conventional methods of mixing and filling.

The compositions may take the form of tablets, capsules or vials.

If desired the composition may advantageously contain a penicillin or cephalosporin. In such instances the ratio of synergyst (i.e. the compound of the invention) (preferably as a salt) to penicillin or cephalosporin is usually from 2:1 to 1:12, more usually from 1:1 to 1:5, for example 1:2, 1:3, or 1:4 wt/wt.

Particularly suitable penicillins for inclusion in the compositions include ampicillin, amoxycillin, carbenicillin, ticarcillin and their pro-drugs. When adapted for injection such compounds are generally presented in the form of their sodium salts.

Particularly suitable cephalosporins for inclusions in the compositions include cephaloridin and cephazolin.

Preferred penicillins for inclusion in the compositions include ampicillin trihydrate, amoxycillin trihydrate, sodium ampicillin and sodium amoxycillin.

Preferred cephalosporins for inclusion in the compositions of this invention include cephaloridin and sodium cephazolin.

The compound of the formula (I) or (II) or a salt thereof may be an isolated compound of the formula (lea), (Ib) or (Ila). (IIb) or their salts or mixtures of the compounds of the formula (Ia) and (Ib) or (via) and (lIb) or their salts. However it is preferred to use a compound of one of the preceding formulae free from its isomer. Said compound is generally in the form of a pharmaceutically acceptable salt such as the sodium salt.

The present invention also provides a process for the preparation of a compound of the formula (I) or (II) or a salt thereof which process comprises cultivating a producing strain of Streptomyces olivaceus or Streptomyces gedanesis until a substantial quanity of a compound of the formula (I) or (II) or its salt is produced and thereafter recovering a compound of the formula (I) or (II) or its salt from the cultivation medium.

When used herein, the term "Streptomyces olivaceus" is defined according to the classification of Hütter R (in Systematic der Streptolyceten, S. Korger, Basle, Pages 8-32).

Note that on this definition Streptomyces fitlvovoridis, Streptomyces flavus and Streptomyces flavovirens may be regarded as being synonymous with Streptomyces olivaceus.

Suitable strains include those described in British Patent Specification No. 1467413.

A preferred organism for use in this process is Streptomyces olivaceus ATCC 31126 or a high yielding mutant thereof.

A further preferred organism for use in this process is Streptomyces olivaceus ATCC 31365 or a high yielding mutant thereof.

As previously indicated the recovered material should be at least 1% pure, more suitably 5% pure, yet more suitably at least 50% pure, preferably at least 75% pure and more preferably at least 90% pure, for example at least 95% pure.

When used herein the term "cultivation" means the deliberate aerobic growth of an organism in the presence of assimilable sources of carbon, nitrogen, sulphur and mineral salts. Such aerobic growth may take place in a solid or semi-solid medium but in general it is preferable to use a liquid medium. General cultivation conditions for the growth of Streptomyces olivaceus are as described in British Patent Specification No. 1467413.

General conditions for the growth of Streptomyces gedanensis are similar.

The process of this invention may be adapted to provide 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.

Normally the process is adapted to the preparation of a salt rather than the parent acid.

It is preferable that the cultivation medium does not contain added sulphate since this often leads to the preparation of MM4550, MM13902 and MM17880 at the expense of the production of the compounds of the formula (I) and (II) and their salts.

The compounds of the formula (I) and (II) in the form of their salts may be obtained from the culture filtrate by (a) contacting the filtrate with carbon until the antibiotic activity is absorbed thereon, (b) eluting the antibiotic activity from the carbon using aqueous acetone, (c) combining the fractions containing ss-lactamase inhibitory fractions, (d) evaporating the acetone and much of the water to yield a more concentrated aqueous solution, (e) applying the solution to an anion exchange column, and (f) eluting the ss-lactamase inhibitory metabolites therefrom with a solution of an electrolyte buffered to approximate neutrality collecting the fractions containing the compound of the formula (I) or (II) in salt form, (g) applying the resulting solution to a resin which separates the inorganic materials from the compounds (I) and (II) and (h) isolating the solid preparation of the salt of a compound of the formula (I) or (II) from the resulting solution.

The compounds of the formula (I) and (II) in the form of their salts may also be obtained from the culture filtrate by (1) contacting the clarified cultivation broth with a strongly basic acrylic based anion-exchange resin until the antibiotic activity is absorbed thereon, (2) eluting the antibiotic activity from the resin using an aqueous solution of a buffer optionally also containing a salt, (3) combining the fractions with ss-lactamase inhibitory activity, (4) applying the combined fractions to a XAD-4 column, (5) eluting with aqueous isopropanol, (6) combining the fractions with ss-lactamase inhibitory activity, (7) removing the isopropanol and concentrating the solution by evaporation, (8) applying the solution to an anion exchange resin and proceeding as in steps (f), (g) and (h) outlined above.

It is generally preferred to use an acrylic based strongly basic (type 1) anion exchange resin (in the form of an acid addition salt normally the hydrochloride) such as Amberlite IRA 458 (which may be obtained for example from Rohm and Haas for example at Lennig House, 2 Massons Avenue, Croydon, U.K.) ("Amberlite" is a Registered Trade Mark).

An advantage of such a resin is that it allows the salt of the compounds of the formula (I) and (II) to be eluted successively by using an aqueous salt solution, for example a buffered solution of a chloride such as sodium chloride. If the less favoured strongly basic resins having a polystyrene/divinyl benzene matrix are employed it is generally necessary to elute with an aqueous C14 alkanolic solution of a salt (for example a chloride such as sodium chloride) in order to obtain satisfactory recoveries and such solvents can lead to a less pure preparation of the desired materials.

(This process variant differs from the carbon absorption process in that the salts of MM4550, MM13902 and MM17880 are separated from the salts of the compounds of the formula (I) and (II) at the first elution stage.) The process of this invention differs fundamentally from the previously disclosed process in that the fractions selected for further processing at stage (f) are those containing the salt of a compound of the formula (I) and (II) substantially free of other antibiotics.

The free acids of the formulae (I) and (II) may be obtained by careful acidification of a salt of the compound of the formula (I) or (II) respectively followed by rapid extraction into a water immiscible organic solvent followed by recovery of the acid from solution.

In the processes of this invention it is frequently most convenient to work with an alkali metal salt, of the compound of the formulae (I) and/or (II) such as the lithium, sodium or potassium salts and of these the sodium salt is favoured. It is possible to prepare other salts by the extraction process but it is usually more suitable to first form the purified alkali metal salt especially the sodium salt and then convert this to an alternative salt, for example by passing through a bed of cation exchange resin in the form of the alternative salt. Thus in this description other electrolytes (such as the lithium, potassium or other salt) can be substituted for the described sodium salts but in general it is preferred to work with the sodium salt. Similarly salts other than chloride (for example bromide or nitrate) may be employed although in general it is preferred to work with a chloride.

A preferred method of chromatographic purification (steps f and g) uses an aqueous solution of a sodium salt buffered to approximate neutrality in conjunction with a basic ion-exchange resin. Thus an aqueous solution of sodium chloride (or other similar salt) buffered to about pH 7 with a conventional buffer such as a phosphate buffer may be used in conjunction with support resins which contain secondary or tertiary amino groups or quaternary amino groups. Suitable supports include basic ion-exchange celluloses and basic ion-exchange cross-linked dextrans such as DEAE cellulose, DEAE-Sephadex or QAE-Sephadex ("Sephadex" is a Registered Trade Mark).

A related suitable method of chromatographic purification (steps f and g) uses a solvent system comprising a mixture of water and small quantities of a water immiscible organic solvent such as C14 alkanol in conjunction with an inert support material such as silica gel or cellulose. Suitable solvent systems include aqueous isopropanol or aqueous n-butanol.

For example a very roughly 1:4 mixture of water and isopropanol may be used in combination with a cellulose support.

The product of the preceding procedure frequently contains a high proportion of sodium chloride so that it is beneficial to de-salt the pooled solutions. De-salting may be effected by passing the solution through a bed of lipophilic material onto which the antibiotic is adsorbed but which does not absorb the sodium chloride. Suitable materials include polystyrene based polymeric absorbants such as Amerlite XAD-4 and Diaion HP20. The produce of the preceding process may also be desalted by chromatography on suitable gel filtration agents such as cross-linked dextrans such as Sephadex G10 and G15 and polyacrylamide gels such as Biogel P2. The antibiotic may be eluted from such materials using water or aqueous methanol.

The columns are eluted at such a rate as to allow separation of the antibiotics into distinct fractions. In general distinct zones can be eluted from these columns; these contain di-sodium MM4550, di-sodium MM13902, di-sodium MM17880, the sodium salts of the compounds of the formula (I) and the sodium salts of the compounds of the formula (II) eluting close to the sodium salts of the compound of the formula (I). In general the three di-sodium salts are fairly widely separated from the mono-sodium salts on anion exchange resins. If the column is not carefully monitored it may be that the mono-sodium salts are obtained in overlapping fractions. If this is so then either (a) this solution can be freeze dried to yield a useful impure complex containing the antibiotics which can be reworked later or (b) the solution per se can be re-chromatographed with careful monitoring of the eluant to ensure collection of the solution of sodium salt of a compound of the formula (I) free from the sodium salt of a compound of the formula (II) and/or the collection of the solution of the sodium salt of a compound of the formula (II) free from the sodium salt of a compound of the formula (I); these solutions may then be freeze dried or otherwise rendered solvent free.

The fractions chosen for collection will be those which show significant ss-lactamase inhibitory activity or anti-bacterial activity. Suitable methods of detecting -lactamase inhibitory activity include those of the aforementioned British Patents Specifications although any convenient method may be employed.

The following Scheme shows preferred sequence for obtaining the compounds of the formulae (I) and (II) as their sodium salts. The sodium salts obtained in this manner can be further purified if desired by using the chromatographic procedures described hereinbefore.

Trituration of the salts of the compounds of the formulae (I) and (II) under an organic solvent such as moisture containing acetonitrile or acetone can aid in the removal of impurities.

Culture Filtrate t Absorb onto Carbon Elute Elute with 20% aqueous acetone Combine Fractions with ss-lactamase Inhibitory Activity Remove Acetone and concentrate by Evaporation Cellulose DE52 Anion Exchange Column Elute with Phosphate Buffer pH7 , , > , Sodium Salts of Sodium Salts of Di-Sodium Salts of Compounds of Compounds of MM4550, MM13902 and Formula (I) Formula (11) MM17880 t XAD4 XAD4 De-salted Solution De-salted Solution Freeze Dried Solid Freeze Dried Solid

Culture Filtrate f Absorb onto Strongly Basic Acrylic Anion Exchange Resin Elute with Buffer Combine Fractions with earlier eluting ss-lactamase Inhibitory Activity De-salt on XAD 4 Elute with Aqueous Isopropanol a t Combine Fractions with ss-lactamase Inhibitory Activity Remove Isopropanol and concentrate by Evaporation s e Cellulose DE52 Anion Exchange Resin with with Phosphate Buffer pH7 Sodium Salts of Compounds Sodium salts of Compounds of of Salt or Formula (I) Formula (II) XAD4 l XAD4 De-salted Solution De-salted Solution t Freeze Dried Solid Freeze Dried Solid If desired the salts of the compounds of the formula (I) and (II) prepared by the previous methods may be further subjected to chromatographic separation techniques to yield an isolated salt of a compound of the formula (Ia), (Ib), (IIa) or (IIb). Such processes are favoured aspects of this invention. Normally the salt used in such a process will be a monovalent salt such as the ammonium salt or an alkali metal salt such as the sodium or potassium salt.

One suitable form of chromatography for the separation process is high pressure liquid chromatography (hplc), for example using an aqueous ammonium formate buffered solution. Once fractions containing the desired compound are obtained a solid preparation may be obtained by freeze drying.

Compounds of the formula (Ia) and (Ib) may be separated by column chromatography on supports such as acetylated cellulose eluting with alcohol/water mixtures. Compounds of the formula (IIa) and (IIb) may also be separated using similar chromatographic techniques.

Once fractions containing the desired compound are obtained a solid preparation may be obtained by freeze drying.

One process for the preparation of a salt of the compound of the formula (Ia) substantially free of the compound of the formula (Ib) comprises subjecting a mixture of said salts to chromatographic separation on Diaion HP20 or a chromatographically equivalent resin.

A second process for the preparation of a salt of the compound of the formula (Ib) substantially free of the compound of the formula (Ia) comprises subjecting a mixture of said salts to chromatographic separation on Diaion HP20 or a chromatographically equivalent resin.

A third process for the preparation of a salt of the compound of the formula (IIa) substantially free of the compound of the formula (IIb) comprises subjecting a mixture of said salts to chromatographic separation on Diaion HP20 or a chromatographically equivalent resin.

A fourth process for the preparation of a salt of the compound of the formula (IIb) substantially free of the compound of the formula (IIa) comprises subjecting a mixture of said salts to chromatographic separation on Diaion HP20 or a chromatographically equivalent resin.

The salts prepared by the preceding processes will normally be monovalent salts such as alkali metal salts, for example the lithium, sodium or potassium salt and will preferably be the sodium salt.

The salts prepared by the preceding process will not normally contain more than about 5% and more suitably not more than about 1% of the desired isomer.

Diaion HP20 (which is a Registered Trade Mark) is a highly porous polymer manufactured by Mitsubishi Chemical Industries. It is not an ion exchange resin but is a synthetic adsorbant which has an extra large active surface area to which organic compounds may be effectively adsorbed.

Diaion HP20 is a styrene divinylbenzene copolymer in bead form having a macroreticular structure with a specific surface area of about 7.8 m2/g and a pore volume of 1.16 ml/g.

Other details of this resin may be found in the Diaion data sheet (HP series, October 1976) (Mitsubishi Chemical Industries Ltd. Offices may be found via 5-2 Marunouchi 2-chome, Chiyoda-ku, Tokyo. Japan; 277 Park Ave., New York, NY 10017 USA; Ratinger Str. 45, 4 Duesseldorf. West Germany).

Resins chromatographically equivalent to Dinion. HP20 will also normally be chemically and physically similar, that is they will generally be macroreticular resins based on styrene divinylbenzene copolymers and free of ionized groups.

Most suitably the mixture of isomers applied to the resin will be of good purity and will be substantially free from other organic impurities although quantities of inorganic impurities (for example an alkali metal salt such as a chloride, for example sodium chloride) may be present.

Suitably the solvent employed will be water or water in admixture with a lower alkanol or similar miscible organic solvent.

Preferably the solvent used is water.

The desired material may now be obtained by removal of the solvent for example by evaporation or freeze drying. Alternatively the solution may be rechromatographed directly on a suitable resin (such as Biogel P2 and/or Diaion HP20) for further purification prior to removal of the solvent.

Water may be removed from aqueous solutions of salts of this invention by such processes as evaporation under vacuum to about 1/10 volume, making up to the original volume with ethanol, reconcentration to about 1/10 volume under vacuum, making up to the original volume by adding toluene and evaporation to dryness in vacuum. Residual solvents may be removed by storing under high vacuum.

Description 1 Preparation of Clarified Broth A spore suspension of Streptomyces olivaceus ATCC 31126 was used to inoculate 100 ml of a seed stage medium contained in a 500 ml Ehrlenemeyer flask closed with a foam plug.

The seed medium was 2% glucose and 1% soya bean flour made up in deionised water.

(The soya bean flour was Arkasoy 50 supplied by the British Arkady Co. Ltd., Old Trafford, Manchester, U.K.) The seed stage medium was grown for 48 hours on a rotary shaker at 26"C. 5 ml portions of the seed stage medium were used to inoculate 100 ml portions of the fermentation medium contained in 500 ml Ehrlenemeyer flasks closed with foam plugs. The fermentation medium which was made up in deionised water had the following composition: Glucose 2.0 Soya bean flour 1.0 CaCO3 0.02 Cowl2 6H20 0.0001 The fermentation flasks were incubated at 260C on a rotary shaker for 72 hours. 20 flasks were harvested and the resultant whole broth centrifuged at 2200 g for 10 minutes.

Use of Streptomyces olivaceus ATCC 31365 in the above process is also favoured.

Description 2 Preparation of Crude Antibiotics in Solution The culture filtrate (1500ml) obtained after centrifugation was submitted to purification using a carbon column as follows: A 2.5 x 37 cm Darco granular carbon column was prepared in deionised water, the column was washed successively with 1 litre 2% NaOH, 1 litre deionised water, 1 litre 1N HCl and 1 litre deionised water all at 15 ml/minute ("Darco" is a Registered Trade Mark).

The column was then washed with 0.05M pH 7 phosphate buffer until the pH of the eluant was 7.0 The culture filtrate (1500 ml) was run onto the carbon column at 15 ml/minute. The column was then eluted with acetone/water 1/4 at 15 ml/minute and 22 ml fractions were collected. Fractions were monitored for their ss-lactamase inhibitory activity against a preparation of RTEM enzyme (supplied by Microbiological Research Establishment, Porton). Fractions showing the greatest activity (8-22) were combined and evaporated under reduced pressure to remove aceto sodium salt of the compound of the formula (II) substantially free of the salts of the di-basic antibiotics.

(Freeze drying combined fractions 13-16 and 19-21 naturally leads to a preparation containing a mixture of the sodium salts of the antibiotics of the formulae (I) and tit). The di-sodium salts of MM 4550, MM 13902 and MM 17880 eluted after the desired salts).

EXAMPLE 2 Partial Purification of the Sodium Salts of the Compounds of the Formula (I) The freeze dried preparation of the salt of the compound of Example 1 was dissolved in deionised water (10 ml) and sodium chloride (1 g) added to the solution. This solution was run onto a 1.5 x 15 cm XAD-4 (supplied by Rohm & Haas) column prepared in deionised water. The column was eluted with water/n-propanol 4/1 at 2 ml/minute and 4 ml fractions were collected. Fractions were monitored for chloride by their reaction with AgNO3 and for their Blactamase inhibitory activity against a preparation of RTEM ss-lactamase. Fractions with the greatest inhibitory activity and giving a negative reaction with silver nitrate (fractions 6-13) were combined and freeze dried to yield an amorphous solid (32.5 mg) containing the sodium salt of the compound of the formula (I).

The properties of this preparation were as follows: (A) Chromatographic Properties (i) Chromatography on Whatman DE81 Ion Exchange Paper (a weakly basic anion exchange paper) ("Whatman" is a Registered Trade Mark).

Eluant Rf of Sodium Salt 1. 0.05M pH 7 phosphate buffer 0.69 2. 0.05M pH 7 phosphate buffer containing 0.2M NaCI 0.80 (ii) Chromatography on Whatman No. 1 Paper: Solvent System Rf of Sodium Salt 1. Butanol:Ethanol:Water Top Phase 4:1:5 0.12 2. Butanol:Pyridine:Water 1:1:1 0.42 (B) High Voltage Paper Electrophoresis The electrophoresis was carried out on No. 20 paper in pyridine/acetic acid buffer pH 5.3 at 5000 volts for 15 minutes. The RM values for the sodium salt taking benzyl penicillins as 1.0 is also 1.0 (C) Antibacterial Activity The antibacterial activity of the preparation using the microtitre method was determined as follows: Organism MIC ( > g/ml) Bacillus subtilis A < 40 Enterobacter cloacae N1 1250 Escherichia coli 10418 150 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 inhibitory activity of the preparation against a series of t3-lactamase preparations is summarised below: ss-Lactamase Preparation % Inhibition Concentration From: at 2001lg/ml giving 50% Inhibition Rg/ml Staph. Aureus Russell 40 E. coli JT4 - 95 Proteus mirabilis C889 - 112 Pseudomonas aeruginosa Dalgleish - 170 Enterobacter cloacae P99 83 Pseudomonas aeruginosa A 30 Klebsiella aerogenes E70 27 (Method of Belgian Patent Specification No. 827926).

EXAMPLE 3 Partial Purification of the Sodium Salt of the Compounds of the Formula (11) The freeze dried preparation of the salt of the compound II of Example 1 was dissolved in denionised water (10 ml) and sodium chloride (1 g) added to the solution. This solution was run onto a 1.5 x 15 cm XAD-4 (supplied by Rohm & Haas) column prepared in deionised water. The column was eluted with water/n-propanol 4/1 at 2 ml/minute and 4 ml fractions were collected. Fractions were monitored for chloride by their reaction with AgNO3 and for their ss-lactamase inhibitory activity against a preparation of RTEM Blactamase. Fractions with the greatest inhibitory activity and giving a negative reaction with silver nitrate (fractions 7 - 13 were combined and freeze dried to yield an amorphous solid (32.5 mg) containing the sodium salt of the compound of the formula (II).

The properties of this preparation were as follows: (A) Chromarographic Properties (i) Using weakly basic anion exchange paper DE81 cellulose (Whatman) the sodium salt has an Rf of 0.54 when eluted with 0.05M pH 7 phosphate buffer.

(ii) Using Whatman No. 1 paper: the sodium salt has an RF or 0.20 when eluted with butanol/ethanol/water 4/1/5 top phase.

(B) High Voltage Paper Electrophoresis The electrophoresis was carried out on Whatman No. 20 paper in pyridine/acetic acid buffer pH 5.3 at 5000 volts for 15 minutes. The RM value for the salt taking benzyl penicillin as 1.0 is 0.95.

(C) Antibacterial Activity The antibacterial activity of the preparation using the microtitre method was determined and the results are tabulated: Organism MIC llg/ml Bacillus subtilis A 250 Enterobacter cloacae Nl > 1000 Escherichia coli 10418 250 Klebsiella aerogenes A 500 Proteus mirabilis C977 500 Pseudomonas aeruginosa A > 1000 Salmonella typhimurium CTl() 250 Serratia marscens US39 > 1000 Staph. aureus Oxford 500 Staph aureus Russell 250 (D) Enzyme Inhibition ss-Lactamase inhibitory activity of the preparation against a range of enzyme preparations have been determined: ss-Lactamase Preparation % Inhibition Concentration From: at 200,ug/ml giving 50% Inhibition llg/ml Staph. aureus Russell - 150 E. coli JT4 72 Proteus mirabilis C889 - 62 Pseudomonas aeruginosa - 53 Dalgleish Enterobacter cloacae P99 - 10 Pseudomonas aeruginosa A 39 Klebsiella aerogenes E70 13 EXAMPLE 4 A Further Purification of the Sodium Salts of the Compounds of Formula (I) The preparation obtained in Example 2 is dissolved in deionised water and loaded onto a QAE Sephadex A25 column (QAE Sephadex A25 is a strongly basic anion exchanger supplied by Pharmacia Ltd.) prepared in deionised water. The column is eluted with a sodium chloride concentration gradient from 0 to 0.18 M NaCI in deionised water.

Fractions from the column are monitored for their ss-lactamase inhibitory activity against an RTEM preparation and those showing the greatest activity are combined. NaCl to a final concentration of at least 5% is added to the combined fractions. The resulting solution is run onto an Amberlite XAD-column (Rohm & Haas Ltd.) and the column eluted with n-propanol/water 1/4. Fractions containing the desired salt as judged by their ss-lactamase inhibitory activity are combined, evaporated under vacuo to remove organic solvent and freeze dried.

EXAMPLE 5 A Further Purification of the Sodium Salts of the Compounds of the Formula (I) The combined fractions from the QAE Sephadex chromatography of Example 4 may be desalted and further purified by chromatography on a Biogel P2 gel column (Bio-Rad Laboratories Ltd., 27 Homesdale Road, Bromley, Kent) as follows: The freeze dried solid from the QAE Sephadex column is dissolved in a small volume of deionised water and run onto a Biogel P2 column. The column is eluted with 1% aqueous butanol. Fractions are monitored for their ss-lactamase inhibitory activity and for reaction with silver nitrate. Those giving a negative reaction to silver nitrate but giving suitable ss-lactamase inhibitory activity are combined and freeze dried.

EXAMPLE 6 A Further Purification of the Sodium Salts of the Compounds of Formula (I) The preparation obtained in Example 2 is dissolved in deionised water and applied to a column of the strongly basic anion exchange resin Amberlite IRA 458 (Rohm & Haas (UK) Ltd., Lenning House, 2 Mason's Ave.. Croydon, U.K.). The column is prepared in 0.05M pH 7 phosphate buffer and eluted with a sodium chloride gradient in phosphate buffer. The elution is from 0.05M pH 7 phosphate to 0.05M pH 7 phosphate containing 1.OM NaCI.

Fractions showing the greatest ss-lactamase inhibitory activity are combined. NaCI is added to the combined fractions to give a concentration of at least 5%. The resulting solution is run onto an Amberlite XAD-4 column prepared in deionised water. The column is eluted with n-propanol/water 1/4. Fractions showing the greatest ss-lactamase inhibitory activity but giving a negative reaction with silver nitrate are combined and freeze dried.

EXAMPLE 7 A Further Purification of the Sodium Salts af the Compounds of Formula (I) The salt of the compound of the formula (I) may be further purified by chromatography on a column of cellulose (Cellulose CC31 Whatman, Springfield Mill, Maidstone, Kent, U.K.) as follows: The impure solid containing the salt of the compound of the formula (I) is dissolved in a minimum of deionised water and n-propanol added to about 50%. The resulting solution is run onto the cellulose column and the column eluted with n-propanol/water 4/1. The resulting fractions after dilution into deionised water are monitored for their ss-lactamase inhibitory activity. Fractions containing the desired salt are combined, evaporated under reduced pressure to remove solvent and freeze dried.

EXAMPLE 8 A Further Purification of the Sodium Salts of the Compounds of-the Formula (II) The process of Example 4 may be used but replacing the starting material with the preparation obtained in Example 3.

EXAMPLE 9 A Further Purification of the Sodium Salts of the Compounds of the Formula (II) The process of Example 5 may be used but replacing the starting material with the product of Example 8.

EXAMPLE 10 A Further Purification of the Sodium Salts of the Compounds of the Formula (II) The process of Example 6 may be used replacing the starting material with the preparation obtained in Example 3.

EXAMPLE 11 A Further Purification of the Sodium Salts of the Compounds of the Formula (II) The process of Example 7 may be used but replacing the starting material with the preparation obtained in Example 3.

EXAMPLE 12 Preparation of Crude Antibiotics from Culture Filtrate Culture filtrate (80 ml) prepared essentially as described in Description 1 was run onto a 1.5 x 15 cm column of Amberlite IRA 458 a strongly basic acrylic based anion exchange resin (Rohm & Haas). The column was eluted with a sodium chloride concentration gradient. The gradient was from 0 to 1.0 M NaCl in 0.05M pH 7 phosphate buffer at a flow rate of 2.5ml/minute and 5 ml fractions collected. Fractions were monitored for their ss-lactamase inhibitory activity against a preparation of RTEM (3-lactamase. Those fractions giving good inhibitory activity and containing the sodium salts of the compounds of the formulae (I) and (II) (4-13) were combined. (The di-sodium salts of MM 4550, MM 13902 and MM 17880 eluted starting at fraction 17).

To the combined fractions was added sodium chloride (3 g), and the resulting solution was run onto a 1.5 x 15 cm Amberlite XAD-4 column (Rohm - & Haas) prepared in deionised water. The column was eluted with n-propanol/water 1/4 at 3 ml/minute and 4 ml fractions were collected. Fractions were monitored for their ss-lactamase inhibitory activity and for reaction with ARNO3 solution. Those fractions giving good inhibitory activity and a negative reaction with silver nitrate were combined and freeze dried to give a partially purified preparation of the sodium salts of the compounds of the formulae (I) and (II). This impure preparation may be further purified by the processes described hereinbefore.

EXAMPLE 13 Preparation of Crude Antibiotics from Culture Filtrate Culture filtrate (305 ml) prepared essentially as described in Description 1 was run onto a 1.5 x 15 cm column of Amberlite IRA 458 a strongly basic acrylic based anion exchange resin (Rohm & Haas). The column was washed with deionised water (100 ml) at 5 ml/minute and eluted with 0.025 M pH 7 phosphate buffer at 5 ml/minute, 10 ml fractions were collected. Fractions were monitored for their ss-lactamase inhibitory activity against a preparation of RTEM (3-lactamase. Those fractions giving good inhibitory activity and containing the sodium salts of the compounds of the formulae (I) and (II) (12-32) were combined. The combined fractions were freeze dried. The freeze dried solid was dissolved in deionised water (20 ml), NaCI (2 g) was added and the resulting solution was run onto a 1.5 x 15 cm Amberlite XAD-4 column (Rohm & Haas) prepared in deionised water. The column was eluted with n-propanol/water 1/4 at 2 ml/minute and 4 ml fractions were collected. Fractions were monitored for their ss-lactamase inhibitory activity and for reaction with AgNO3 solution. Those fractions (7 - 14)) giving good inhibitory activity and a negative reaction with silver nitrate were combined and freeze dried to yield a partially purified preparation of the salts of the compounds of the formulae (I) and (II). This impure preparation may be further purified by the processes described hereinbefore.

EXAMPLE 14 Fermentation Conditions for 300L Fermentation A freeze dried ampoule of Streptomyces olivaceus (ATCC 31365) was resuspended in 10 ml of a sterile solution of the following composition: Glucose 2% Soya bean flour 1% pH 6.5 prepared in deionised water (The soya bean flour is Arkasoy '50' supplied by the British Arkady Co. Ltd., of Old Trafford, Manchester).

1 ml of this suspension was used to inoculate 100 ml of medium of the same composition contained in a 500 ml Ehrlenemeyer flask closed with a foam plug. After inoculation the flask was incubated on a rotary shaker at 28"C for 30 hours, 5 ml portions of this seed culture was used to inoculate solid agar slants in Roux bottles of the following composition: V8 Vegetable juice 20.0% Bacto agar (Difco) 2.5% pH 6.0 prepared in deionised water (The V8 vegetable juice is supplied by Campbell's Soups Ltd., Kings Lynn, Norfolk, England and Bacto agar is supplied by Difco Laboratories, Detroit, Michigan, U.S.A.) ("Difco" is a Registered Trade Mark).

Each Roux bottle was incubated at 280C for 1 week. After that time 100 ml sterile deionised water containing 0.1% :Triton X (surfactant - Registered Trade Mark) was added to one Roux bottle culture and the spores suspended by shaking. This spore suspension was added as inoculum to 75 L of sterilised seed stage medium in a stainless steel baffled fermenter. The composition of the medium was: Soya bean flour (Arkasoy 50) 1% Glucose 2% Pluronic L81 antifoam 0.03% Prepared in distilled water (Pluronic L81 was supplied by Ugine Kuhlmann Chemicals Ltd.) ["Pluronic" is a Registered Trade Mark].

The medium was steam sterilised in the fermenter for 20 minutes at 1200C. The seed stage culture was stirred at 140 rpm with a 7.5 inch vaned disc agitator and supplied with sterile air at 75L/minute through an open ended sparger. The temperature was controlled at 28"C and after incubation under these conditions for 48 hours 7.5L of this seed culture was added as inoculum to 150L sterile fermentation medium in a 300L stainless steel fully baffled fermenter. The fermentation medium had the following composition: Soya bean flour (Arkasoy 50) 0.9% Glucose 2.0% Chalk 0.02% CoCI2 6H2O 0.0001% Pluronic L81 antifoam 0.2% pH 6.0 before sterilisation Prepared in distilled water The fermentation stage medium was stirred at 340 rpm with an 8.5 inch turbine disc impeller. The temperature was controlled at 29"C, air was supplied at the rate of SOL/minute and the pH maintained at 6.5 - 7.0.

Fermentations as above may be harvested at times ranging between 48 and 54 hours.

EXAMPLE 15 Fermentation Conditions for 2000L Fermentation The fermentation conditions up to and including the seed stage were essentially as described in Example 14. 75L of this seed stage was used to inoculate 1500L of sterile fermentation medium contained in a 2000L fully baffled stainless steel fermenter. The fermentation medium was the same as that described in Example 14.

The fermentation was stirred with two 19" diameter turbine disc impellers at 106 rpm and air was supplied at a rate of 400L/minute. The temperature was maintained at 29"C and the pH at 6.5 - 7.0. the fermenter was harvested at 48 hours.

EXAMPLE 16 Isolation Procedure for the Preparation of Substantially Pure Sodium Salts of the Compounds of the Formulae (I) and (11) 150L whole broth prepared essentially as described in Example 14 were clarified on a continuous flow centnfuge (Sharples Super Centrifuge) at approx. 2.4L/minute. 120L of the clarified culture filtrate was percolated onto a 6" diameter column of the strongly basic anion exchange resin Amberlite IRA 458 (in chloride form) supplied by Rohm & Haas Co., Philadelphia, Pa.. U.S.A.) with a bed volume of 9.6L at a rate of 400ml/minute. (The Amberlite IRA 458 column had been previously prepared in deionised water). After percolation of the culture filtrate the column was washed with a bed volume of water, then eluted with ().2M NaCl in 0.05M pH 7 sodium phosphate buffer at a rate of 230ml/minute. 2L fractions were collected and those showing good inhibitory activity to a preparation of an RTEM mediated ss-lactamase preparation (fractions 2-11) were combined.

Sodium chloride was added to the combined fractions (46.75 g/L) to make the solution 1M in respect to NaCl. The resulting solution was run onto a 4" diameter column of Amberlite XAD-4 (supplied by Rohm & Haas Co.) with a bed volume of 4L and equilibrated in 1M NaCL. The percolation rate was 200ml/minute. The column was eluted with 10L deionised water then water/isopropanol (4/1) both at 100ml/minute. Fractions showing good ss-lactamase inhibitory activity against an RTEM enzyme preparation (3-10) and 13-17) were combined, evaporated at reduced pressure to remove isopropanol and freeze dried.

A 3.8 x 30 cm column of the weakly basic anion exchange Cellulose DE52 (in chloride form) (supplied by Whatman Ltd.. Springfield Mill, Maidstone, Kent) was prepared in deionised water. The freeze dried solid from the desalting stage was dissolved in 300 ml deionised water and run onto the DE52 cellulose column at 6ml/minute. The column was washed with deionised water (200 ml) and eluted with 0.025M pH 7 potassium phosphate buffer at 2.5ml/minute, 10 ml fractions were collected. Fractions were monitored for their ss-lactamase inhibitory activity and the first two major peaks of activity, fractions 30-85 (containing the sodium salt of the compound of the formula (I)) and fractions 86-130 (containing the sodium salt of the compound of the formula (II)) were combined separately.

Combined fractions (30-85) were run onto a 3.8 x 29 cm QAE Sephadex A25 (in chloride form) (supplied by Pharmacia Ltd., Uppsala, Swedeli) column prepared in deionised water.

The column was eluted with 0.1M NaCI at 3 ml/minute and 18 ml fractions were collected.

Fractions showing good RTEM ss-lactamase inhibitory activity (58-68) were combined. To this solution (180 ml) was added 9 g sodium chloride and the resulting solution was run onto a 1.5 x 15 cm Amberlite XAD-4 column. The column was eluted with deionised water (135 ml) then water/isopropanol (4/1) at 3ml/minute and 4.5 ml fractions were collected.

Fractions with UV spectra eharacteristic of the partially purified sodium salts of the compounds of the formula (I) (UV maximum approx 297 mll) (8-16 and 34-40) were combined and freeze dried to yield solids (53 mg and 42 mg respectively) with characteristic properties of the substantially pure sodium salts of the compounds of the formula (I).

Fractions 86-130 from the cellulose DE52 column were run onto a 3.8 x 29 cm, QAE Sephadex A25 (in chloride form) column and the column eluted with 0.1M NaCl at 3ml/minute, 18 ml fractions were collected. Fraction were monitored for their RTEM ss-lactamase inhibitory activity and those showing good activity (94-100) were combined (120 ml). Sodium chloride (6 g) was added to the combined fractions and the resulting solution run onto a 1.5 x 15 cm Amberlite XAD-4 column. The column was eluted with deionised water (90 ml) then water/isopropanol (4/1) both at 3 ml/minute, and 4 ml fractions were collected. Fractions with characteristic spectra of the impure sodium salts of the compounds of the formula (II) (maximum approx 307 m) (10-16 and 26-32) were combined and freeze dried to yield solids (9.7mg and 21.7mg respectively). These solids had properties consistent with substantially pure sodium salts of the compounds of the formula (if).

EXAMPLE 17 Alternative Isolation Procedure for the Sodium Salts of the Compounds of the Formulae (I) and (II) Culture filtrate (120 L) containing the sodium salts of the compounds of the formulae (I) and (II) was processed by chromatography on Amberlite IRA 458 (in chloride form) and desalting on Amberlite XAD-4 essentially as described in Example 16 above.

The freeze dried solid from the desalting stage was dissolved in 300 ml deionised water and run onto a 3.8 x 25 cm cellulose DE52 column (in chloride form). The column was washed with deionised water (200 ml) and eluted with 0.025M pH 7 potassium phosphate buffer at 6ml/minute and 20 ml fractions were collected. Fractions showing good RTEM ss-lactamase inhibitory activity (350 ml) were combined and run onto a 3.8 x 30 cm QAE Sephadex A25 column. The column was eluted with 0.18M NaCI at 3ml/minute and 20 ml fractions were collected. Fractions were monitored for their UV spectra and those showing characteristic adsorption of the sodium salts of the compounds of the formula (I) (50-56) and characteristic adsorption of the sodium salt of the compound of the formula (II) (88-97) were combined separately.

To the bulked fractions (50-56) was added sodium chloride (15 g) and the resulting solution run onto a 1.5 x 15 cm Amberlite XAD-4 column. The column was washed with deionised water (15 ml) and eluted with water/n-propanol (4/1) at 3ml/minute. 3 ml fractions were collected. Fractions with UV spectra characteristic of the purified sodium salts of the compounds of the formula (I) (9-15) were combined, evaporated under reduced pressure to remove propanol and freeze dried to yield a solid (35 mg) with properties consistent with the substantially pure sodium salts of the compounds of the formula (I).

To the bulked fractions (88-97) from the QAE Sephadex column, sodium chloride (24 g) was added and the solution percolated through an Amberlite XAD-4 column (1.5 x 15 cm).

The column was eluted with water/n-propanol (411) after washing with deionised water (15 ml). Fractions with UV spectra characteristic of the substantially pure sodium salts of the compounds of the formula (II) were combined. The combined fractions were evaporated under reduced pressure to remove propanol and freeze dried to yield the substantially pure sodium salts of the compounds of the formula (II) (34 mg).

EXAMPLE 18 Alternative Isolation Procedure for the Sodium Salts of the Compounds of the Formulae (I) and (II) Culture filtrate (105 L) was prepared essentially as described in Example 16 and was processed on Amberlite IRA 458 and Amberlite XAD-4 as described in the same example.

The bulked fractions from the Amberlite XAD-4 column were evaporated to about half volume under reduced pressure to remove isopropanol and stored at 50C for approximately 65 hours. The resulting solution (800 ml) was run onto a 3.8 x 30 cm QAE Sephadex A25 column previously prepared in deionised water. The column was eluted with 0.05M NaCI (800ml) then with 0.1M NaCl both at 4ml/minute and 20 ml fractions were collected.

Fractions were monitored for their RTEM ss-lactamase inhibitory activity and by their UV spectra and those containing essentially the sodium salts of the compounds of the formula (I) (73-80) and the sodium salts of the compounds of the formula (II) (112-125) were combined separately.

Combined fractions (73-80) were evaporated under reduced pressure to approximately 15 ml and run onto a 3.8 x 30 cm Biogel P2 (200.400 mesh) (supplied by Bio Rad Laboratories, 27 Homesdale Road, Bromley, Kent) column previously prepared in deionised water containing 1% butanol. The column was eluted at 3ml/minute with deionised water containing 1% butanol and 6ml fractions were collected. Fractions were monitored by their UV spectra and for reaction with silver nitrate. Fractions containing the sodium salts of the compounds of the formula (I) giving a negative reaction with AgNO3 (29-39) were combined and freeze dried to yield a solid (72 mg) with properties characteristic of the purified sodium salt of the compound of the formula (I).

Combined fractions (112-125) were evaporated under reduced pressure to approximately 15 ml volume. This solution was chromatographed on a 3.8 x 30 cm Biogel P2 column (prepared as above). The column was eluted with deionised water containing 1% butanol at 3ml/minute and 6 ml fractions were collected. Fractions giving UV spectra characteristic of the sodium salts of the compounds of the formula (II) but with a negative reaction for chloride were combined (43-48). The combined fractions were freeze dried to yield a solid (27 mg) with properties characteristic of the substantially pure sodium salts of the compounds of the formula (II).

EXAMPLE 19 Alternative Isolation for the Sodium Salts of the Compounds of the Formulae (I) and (II) 1500 L of whole brew prepared essentially as described in Example 15 was clarified by filtration using Dicalite 478 filter aid on a rotary pre-coat filter to yield 1400 L of culture filtrate ("Dicalite" is a Registered Trade Mark . This culture filtrate was percolated through a 12" diameter Amberlite IRA 458 column (chloride form) (100 L bed volume) at an average percolation rate of 4L/minute. The column was eluted at 1.6minute with 0.2M NaCl in 0.05M pH 6.7 sodium phosphate buffer and 20L fractions were collected. Fractions showing good antibacterial activity when tested on Klebsiella aerogenes A (a variant of NCTC 418) (1-5) were combined. Sodium chloride was added to the bulked fractions to a final concentration of 1.OM and the resulting solution run onto a 6" diameter column of Amberlite XAD-4 (22.4 L bed volume) at 1.2 L/minute. The Amberlite XAD-4 column was eluted with 20 L deionised water followed with water/iso-propanol (4/1) at 500 ml/minute and 8 L fractions were collected.

Fractions containing the desired salts (2-4) were combined (24 L) and concentrated to 11.4 L by evaporation. The concentrate was stored for approximately 65 hours at 5"C after adjustment to pH 7.0. This solution was further concentrated to 3.75 L and the resulting solution run onto a 7.8 x 31 cm Cellulose DE52 column at 6 ml/minuted. The column was eluted with 0.025M pH 7 potassium phosphate buffer at 4ml/minute and 22 ml fractions collected. Fractions were monitored for their RTEM -lactamase inhibitory activity, fractions 60-135 and 136-210 showing good activity were combined separately.

The combined fractions 60-135 (1580 ml) were run onto a 4.8 x 25 cm QAE Sephadex A25 column at 6ml/minute. This column was eluted with 0.1M NaCI at 4ml/minute and 20 ml fractions were collected. Fractions were monitored by their UV spectra and those characteristic of the sodium salts of the compounds of the formula (I) (50-70) were combined ready for subsequent processing.

The combined fractions (136-21

3. The in vitro antibacterial activity of material prepared essentially as described hereinbefore was as follows: Sodium Salt of the Sodium Salt of the Organism Compound of the Compound of the Formula (I) Formula (II) MIC (g/ml) MIC (llg/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 CT10 3.12 0.8 Serratia marcescens US39 50.0 25.0 Staphylococcus aureus Oxford 0.8 1.5 Staphylococcus aureus Russell 3.12 1.5 EXAMPLE 21 Separation of the Salts of the Compounds of the Formulae (la) and (lb) A substantially pure preparation of the mixed sodium salts of the compounds of the formulae (Ia) and (Ib) were separated by high pressure liquid chromatography (hplc) by the following procedure: Column: 300 mm x 3.9 mm filled with u bondapack C18 (Waters Associates, Milford, Massachusetts, USA).

Solvent: 0.OSM ammonium acetate, adjusted to pH 4.5 with acetic acid in 5% acetonitrile - 95% water.

Flow rate: 2.5 ml per minute.

Detection: UV absorbance at 295 nm.

Load: 50 ul of a solution of 1.6 mg in 0.5 ml water.

The two sodium salts were resolved into two peaks with retention times of 3.45 and 4.45 minutes. The eluate for each peak was separately combined and neutralised to pH 7 with dilute sodium hydroxide solution.

The separated combined solutions were evaporated to yield the desired solid salts of the compounds of the formulae (Ia) and (Ib).

EXAMPLE 22 Separation of the Salts of the Compounds of the Formulae (Ila) and (lIb) Using the same system as described in Example 21 the mixed sodium salts of the compounds of the formulae (lea) and (IIb) were separated by high pressure liquid chromatography with retention times of 6.0 and 7.1 minutes.

EXAMPLE 23

CULTURE FILTRATE ABSORB ONTO STRONGLY BASIC RESIN AMBERLJTE IRA 458 Elute with 0.2M NaCl in buffer COMBiNE s CONTAINING Ia, Ib, Ila and lib Desalt on XAD-4 Elute with o EVAPORATE UNDER REDUCED o 03: TO REMOVE PROPANOL X H on QAE Sephadex " r Ce on XAD-4 Desalt on 5 ; FREEZE DRY FRACTIONS FREEZE DRY FRACTIONS Cdhl Ia, Ib CONTAINING Ila, JIb Chromatograph on Chromatograph QAE Sephadex on HP20 P," CONTAINING Ia, (d p?l FRACTIONS E Ila CONTAINING lib Chromatograph on Diaion HP20 Y~ S X M d dJWg2 FRACTION U U lG5Ia Biogel P2 -1 CONTAINING Ib CONTAINING Ila CONTAINING JIb W-tEU 3P! on Biogel P2 on HP2O and ;D FRACTIONS Freezer p Z Ia CONTAINING Ib Ila lIb m- on CP and O O Dry Ia Ib n LZ 0D O cee Z - > z Y s c c X Z o Z O Z 2 Gu 5 C g < vC C U C v O v O CXl O U1 a uo (In this Example reference to (Ia), (IIa) (Ib) and (IIb) means reference to their sodium salts.) Spores of Streptomyces olivaceus ATCC 31365 were prepared essentially as described in Example 14.

The spore suspension from one Roux bottle was used as inoculum for 75L of sterilised seed stage medium contained in a 100L stainless steel baffled fermenter. The composition of the medium was: Soya bean flour (Arkasoy 50) 1% Glucose 2% Pluronic L81 antifoam 0.03% Prepared in distilled water.

The medium was steam sterilised in the fermenter for 20 minutes at 1200C. The seed stage culture was stirred at 140 rpm with a 7.5 inch vaned disc agitator and supplied with sterile air at 75 L/minute, (through an open ended sparger). The temperature was controlled at 280 and after incubation for 48 hours, 75L of the seed culture was added as inoculum to 1500L of fermentation medium contained in a 2000L fully baffled stainless steel fermenter.

The composition of the fermentation medium was: Soya bean flour (Arkasoy 50) 2.0% Glucose 0.9% Chalk 0.02% CoCl2 6H2O 0.0001% Pluronic L81 antifoam 0.2% (Medium made up in distilled water, pH adjusted to 6.0 before sterilisation.) The fermentation was stirred with two 19 inch diameter turbine disc impellers at 106 rpm and air was supplied at a rate of 400 1/minute. The temperature was maintained at 290C and the pH at 6.5-7.0. The fermenter was harvested at 48 hours.

Further culture filtrate suitable for the extraction of the compounds Ia, Ib, IIa and IIb was prepared as follows: A spore suspension of S.olivaceus prepared from two Roux bottles as described above was used to inoculate 1501 of sterilised seed stage medium contained in a 300 I fully baffled stainless steel fermenter. The medium and growth conditions of this seed stage were essentially as described above. After 48 hours 150 1 of the seed stage were used to inoculate 3000 l of the fermentation medium contained in a 5000 1 fully baffled stainless steel fermenter. The fermentation stage was continued under essentially similar conditions to that described above except that the fermentation was harvested at 55 hours.

Whole brew from the 2000 l and 5000 1 fermenters prepared as described above gave a combined volume of 4725 l. The brew was clarified by filtration on a rotary pre-coat vacuum filter, the combined filtrates yielding 4200 l of clarified brew. The clarified brew was percolated at 10 I/minute through columns of the strongly basic anion exchanger resin Amberlite IRA458 (chloride form). The resin was washed with 60 l deionised water then eluted with an aqueous solution of 0.2M NaCI with 0.075M sodium phosphate pH 6.7.

Elution of Ia, Ib, IIa and Jib commenced when the conductivity of the eluant had reached that of 0.1M NaCl and had been eluted when 450 1 of eluant had been collected. Where necessary the presence of Ia, Ib, IIa and IIb was determined using an analytical high pressure liquid chromatography (hplc) system based on the preparative method described in Example 21.

To the combined eluates containing Ia. Ib, IIa and IIb from the IRA458 column was added Amberlite XAD-4 (90 kg damp weight). The mixture was adjusted to pH 6.0 using 50% hydrochloric acid and stirred gently at 5"C for 1 hour. The resin was then filtered off and washed with deionised water at 50C until the conductivity of the washings was less than that of a 0.05M NaCI solution. The washed resin was slurried in 42 1 of isopropanol/water (1/1) and 20% w/v NaOH added until the pH was steady at 7.5. The eluant was filtered off and retained. The elution of the resin was repeated with a further portion of isopropanol/water (1/1) and finally the process was repeated using isopropanol/water (1/3).

The eluates were combined (130 l) and evaporated under reduced pressure to remove isopropanol. The resulting solution (67 l) was percolated at 15 I/hour onto a column of QAE Sephadex A25 (15 x 43 cm) pre-equilibrated in 0.1M Nazi. The column was washed with 7.5 l of 0.05M NaCl and eluted with 0.1M NaCI at 7.8 1/hour and 500 ml fractions collected.

Fractions were monitored for the presence of Ia, Ib, IIa and IIb by hplc. Fractions containing Ia and Ib, which were eluted together, were combined (12.6 1) and those containing IIa and IIb which were eluted later were also combined (5.9 1).

To the combined fractions containing Ia and Ib was added 44.16 g/l NaCl. The resulting solution was percolated at 5.8 I/hour through a column of Amberlite XAD-4 (10 x 36 cm).

The column was washed with 3 1 deionised water at 2.9 1/hour then eluted with isopropanol/water 1/9. Eluant containing Ia and/or Ib at suitable levels as judged by hplc was collected after the conductivity of the eluant had fallen to a level equivalent to 0.01M NaCI. The eluate solution (7.7 1) containing Ia and Ib was adjusted to pH 7.0 using 20% w/v NaOH, concentrated under reduced pressure and freeze dried to yield a solid (38.5 g).

The combined eluates from the QAE Sephadex A25 column containing IIa and IIb were treated similarly to yield a solid (13.7 g).

The freeze dried product (38.5 g) from the XAD-4 column containing Ia and Ib was dissolved in deionised water (50 ml) and run onto a QAE Sephadex A25 column (7.8 x 30 cm) prepared in deionised water. The column was washed with 4 1 of 0.05M NaCl then eluted with 0.08M NaCI both at 8 ml/min. Approximately 20 ml fractions were collected from the commencement of the elution. The fractions were monitored for their UV spectra and those with spectra consistent with containing compounds Ia and Ib (130 - 170) were combined (950 ml). The QAE Sephadex chromatography was carried out at 50C.

A portion (450 ml) of the combined fractions containing Ia and Ib from the QAE Sephadex chromatography was taken and NaCI (23.8g) added. The resulting solution was run onto a Diaion HP20 column (4.8 x 62 cm) (Mitsubishi Chemicals Ltd., Agents Nippon Rensui Co.. Fuji Bldg., 2-3 Marunouchi, 3-Chome, Chiyoda - Ku, Tokyo 100, Japan) at 12 ml/min. and approximately 20 ml. fractions were collected. Fractions were monitored by their UV spectra and those containing Ia (58-74) and Ib (78-97) were combined separately, concentrated by evaporation under reduced pressure and freeze dried to yield solids 490 mg and 357 mg respectively.

The remaining combined eluates from the QAE column containing Ia and Ib were processed in a similar manner to yield solids 363 mg and 258 mg containing Ia and Ib respectively.

A portion (538 mg) of solid containing Ia from the above process was dissolved in deionised water (25 ml) and run onto a Biogel P2 column (200-400 mesh) (7.8 x 40 cm).

The column was eluted with deionised water at 3 ml/mins. and 25 ml fractions were collected. The chromatography was carried out at 50C. Fractions were monitored by their UV spectra and those with spectra characteristic of highly purified Ia (37-42) were combined. The combined fractions were concentrated by evaporation under reduced pressure to approximately 10 ml. The resulting solution was run onto a 3.0 x 50 cm Diaion HP20 (chromatographic grade) column. The column was eluted with deionised water at 5 ml/minute and 10 ml fractions collected. Fractions were monitored by their UV spectra and those with spectra characteristic of highly purified Ia (50-62) were combined, concentrated under reduced pressure and freeze dried to yield a solid (40 mg) of Ia.

A portion (5.8 mg) of the solids containing Ib from the HP20 chromatography was dissolved in approx. 25 ml deionised water and the resulting solution run onto a 7.8 x 40 cm Biogel P2 column. The column was eluted with deionised water at 3 ml/mm and 25 ml fractions collected. Fractions containing highly purified Ib (32-37) as judged by their UV spectra were combined. The combined fractions were evaporated under reduced pressure to approximately 10 ml and the resulting solution run onto a 2.4 x 40 cm Diaion HP 20 (chromatographic grade) column. The column was eluted with deionised water at 5 ml/min and 10 ml fractions were collected. Fractions were monitored for their UV spectra and those containing highly purified Ib (35-48) were combined and freeze dried to yield a solid 53 mg of Ib.

A portion of the freeze dried solid (7 g) from the XAD-4 desalting stage containing IIa and Ilb was dissolved in deionised water (50 ml) and run onto a 4.8 x 57 cm Diaion HP20 column. The column was eluted with deionised water at 10 ml/min and approximately 20 ml fractions were collected. Fractions were monitored by their UV spectra and those containing IIa (49-63) and 1Ib (71-110) were combined separately and freeze dried to yield solids 875 mg and 1.47 g respectively.

The remaining solid IIa and IIb from the XAD-4 stage was processed in a similar way to yield solids 860 mg and 1.44 g of IIa and IIb respectively.

A portion (860 mg) of IIa prepared above was dissolved in 25 ml deionised water and run onto a Biogel P2 column (200-400 mesh) (7.8 x 40 cm). The column was eluted with deionised water at 3 ml/min and 25 ml fractions collected. Fractions containing highly purified IIa as judged by their UV spectra (42-50) were combined; The combined fractions were concentrated to approximately 10 ml by evaporation under reduced pressure and loaded onto a 2.8 x 40 cm Diaion HP20 (chromatographic grade) column. The column was eluted with deionised water and fractions were monitored by their UV spectra and those with spectra characteristic of highly purified IIa (52-64) were combined. The combined fractions were concentrated by evaporation and freeze dried to yield a solid (95 mg) of IIa.

A portion (750 mg) of IIb prepared above was dissolved in approximately 25 ml deionised water and run onto a Biogel P2 column (200-400 mesh) (7.8 x 40 cm). The column was eluted with deionised water at 3 ml/min and 25 ml fractions collected. Fractions containing highly purified IIb as judged by their UV spectra (47-55) were combined. The combined fractions were evaporated under reduced pressure to approximately 10 ml and this solution loaded onto a 2.8 x 42 cm Diaion HP20 (chromatographic grade) column. The column was eluted with deionised water at 5 ml/minute. The first 25 fractions were collected as 5 ml and remaining fractions as 10 ml. Fractions were monitored by their UV spectra and those containing highly purified IIB (77-91) were combined and freeze dried to yield a solid (105 mg) of IIb.

EXAMPLE 24 Properties of the Sodium Salts of Compounds of the Formulae Ia, Ib, IIa and IIb.

The materials prepared essentially as described in Example 23 and being essentially pure have the following properties: (1) UV spectra Ia Single maxima 298 nm (molar extinction e=8,131) Ib Single maxima 301 nm (molar extinction E=7,930 IIa 2 maxima 228 and 308-309 (E=13,627) IIb 2 maxima 229 and 308-310 (r=13,933) (2) The antibacterial activity of the sodium salts determined by the microtitre method is demonstrated in Table A.

(3) The Blactamase inhibitory activity of the sodium salts is demonstrated in Table B.

(4) The synergistic activity of the sodium salts is demonstrated in Table C.

(5) The sodium salts did not cause any obvious toxic effects in mice when administered in aqueous solution subcutaneously at 50 mg/kg.

(6) NMR Spectra in D2O (6 values relative to HOD at 64.6) Compound la 1.35 (d, J 6HZ). 1.97 (s), 2.85-3.55 (m), 3.62 (dd, J 6 and 10 Hz), 3.95-4.5 (m).

Compound Ib 1.30 (d, J 6Hz), 1.97 (s), ca. 2.7-3.4 (m). 3.47 (dd, J 3 and ca. 5 Hz), ca. 4.0-4.3 (m).

Compound Ila 1.28 (d. J 6Hz), 2.01 (s) 3.00 and 3.12 (d, J 3 and 1Hz respectively), 3.55 (dd, J 6 and 10 Hz). 3.9-4.4 (m), 5.98 (d. J 14Hz), 7.08 (d, J 14Hz).

Compound lib 1.23 (d. J 6Hz), 1.99 (s), 2.96 and 3.07 (d. J 2.5 and 2Hz respectively), 3.59 (dd. J 3 and 5 Hz), 3.9-4.2 (m). 5.93 (d, J 14Hz), 7.04 (d, J 14Hz).

TABLE A MIC ( g/ml) Strain la Ib Ila lib Ampi cillin B.subtilis 0.16 1.2 < 0.08 2.5 # 3.0 Enterobacter cloacae N1 2.5 10 2.5 10 200 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 10 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 10 5.0 10 10 400 Klebsiella Ba95 40 10 40 10 > 6400 Proteus mimbilis 0.6 10 0.6 10 #3.0 C977 Proteus mirabilis 5.0 10 10 20 400 C889 Proteus morganii 2.5 10 2.5 20 100 1580 Proteus vulgaris 5.0 10 10 20 400 Q3618 Pseudomonas aeruginosa A > 160 > 160 > 160 > 160 1600 Salmonella CT1() 0.3 2.5 0.3 5.0 60.3 Serratia US39 20 10 20 10 1600 Staph. Oxford 1.25 1.2 0.3 2.5 63.0 Staph. Russell 0.6 2.5 0.3 2.5 400 Staph. Smith 0.6 2.5 0.6 2.5 #3.0 Strep. faecalis 1.25 20 1.2 20 #3.0

Claims

TABLE B I50( g/ml) Compound Entero bacter PS. aerug Proteus E. coli Staph P99 A C889 JT4 Russell lIa 0.02 3.0 0.04 > 2.0 0.08 IIb 0.04 4.0 0.4 0.1 > 2.0 la 0.02 4.0 0.1 > 2.0 3.25 Ib 0.04 4.0 > 2.0 0.28 TABLE C MIC ( g/ml) Compound Staph aureus E.coli JT39 Russell Ampicillin alone 1000 2000 +(Ia) 0.1 g/ml > 10 > 500 1 g/ml - 500 +(Ib) 0.1 g/ml > 10 500 1 ,ug/ml 10 31.2 +(IIa) 0.1 g/ml 10 > 500 1 g/ml - 500 +(IIb) 0.1 g/ml > 10 500

1 g/ml 10 31.2 WHAT WE CLAIM IS:1. A compound of the formulae (I) or (II):

or a salt thereof.

2. A compound of the formula (I) or a salt thereof.

3. A compound of the formula (II) or a salt thereof.

4. A compound of the formula (Ia)

or a salt thereof.

5. A compound of the formula (Ib):

or a salt thereof.

6. A compound of the formula (lea):

or a salt thereof.

7. A compound of the formula (Jib):

or a salt thereof.

8. A pharmaceutically acceptable alkali or alkaline earth metal salt of a compound as claimed in any of claims 1 to 3.

9. A pharmaceutically acceptable alkali or alkaline earth metal salt of a compound as claimed in any of claims 4 to 7.

10. A sodium, potassium or calcium salt as claimed in claim 8.

11. A sodium, potassium or calcium salt as claimed in claim 9.

12. A sodium salt as claimed in claim 10.

13. A sodium salt as claimed in claim 11.

14. A potassium salt as claimed in claim 10.

15. A potassium salt as claimed in claim 11.

16. A compound as claimed in any one of claims 1, 2, 3, 8, 10, 12 or 14 when at least 50cue wt/wt pure.

17. A compound as claimed in any one of claims 4. 5, 6, 7, 9, 11, 13 or 15 when at least 50cue wt/wt pure.

18. A compound as claimed in claim 16 when at least 75% wt/wt pure.

19. A compound as claimed in claim 17 when at least 75% wt/wt pure.

20. A compound as claimed in claim 18 when at least 90% wt/wt pure.

21. A compound as claimed in claim 19 when at least 90% wt/wt pure.

22. A pharmaceutical composition prepared from a compound as claimed in any one of claims 1, 2, 3, 8, 10, 12, 14, 16, 18 or 20 and a pharmaceutically acceptable carrier.

23. A pharmaceutical composition prepared from a compound as claimed in any one of claims 4, 5, 6, 7, 9, 11, 13, 15, 17, 19 or 21 and a pharmaceutically acceptable carrier.

24. A pharmaceutical composition as claimed in claim 22 prepared from 50 to 500 mg of a compound.

25. A pharmaceutical composition as claimed in claim 23 prepared from 50 to 500 mg of a compound.

26. A composition as claimed in either claim 22 or claim 24 which comprises a penicillin or cephalosporin.

27. A composition as claimed in either claim 23 or claim 25 which comprises a penicillin or cephalosporin.

28. A composition as claimed in claim 26 which comprises amoxycillin.

29. A composition as claimed in claim 27 which comprises amoxycillin.

30. A composition as claimed in claim 26 which comprises ticarcillin.

31. A composition as claimed in claim 27 which comprises ticarcillin.

32. A process for the preparation of a compound as claimed in any of claims 1 to 3 or a salt thereof which process comprises recovering the compound or salt from the cultivation medium in which it has been produced.

33. A process for the preparation of a compound acclaimed in any of claims 1 to 3 or a salt thereof which process comprises cultivating a producing strain of Streptomyces olivaceus or Steptomyces gedanesis until a substantial quantity of compound or salt is produced and thereafter recovering said compound or salt from the cultivation medium.

34. A process as clamed in claim 33 which utilises Streptomyces olivaceus ATCC 31126 or a mutant thereof.

35.A process as claimed in any of claims 32 to 34 for the preparation of a salt of a compound as claimed in any of claims 1 to 3 which process comprises contacting the culture filtrate with carbon until the antibiotic activity is absorbed thereon and thereafter eluting the antibiotic activity from the carbon and recovering said salt.

36. A process as claimed in claim 35 wherein the antibiotic activity is eluted from the carbon, the ss-lactamase inhibitory fractions are combined, concentrated, appied to an anion exchange column and eluted therefrom with an electrolyte.

37. A process as claimed in any of claims 32 to 34 for the preparation of a salt of a compound as claimed in any of claims 1 to 3 which process comprises contacting the culture filtrate with a strongly basic acrylic based anion-exchange resin until the antibiotic activity is absorbed thereon and thereafter eluting the antibiotic activity from the resin and recovering said salt from the eluate.

38. A process as claimed in claim 37 wherein the antibiotic activity is eluted from the resin with an aqueous solution of a buffer optionally also containing a salt.

39. A process as claimed in claim 38 wherein the antibiotic activity is eluted from the resin, the ss-lactamase inhibitory fractions are combined and desalted.

40. A process as claimed in any of claims 32 to 39 wherein the salt is a lithium, potassium or sodium salt.

41. A process as claimed in claim 40 wherein the salt is a sodium salt.

42. A process as claimed in claim 40 wherein the salt is a potassium salt.

43. A process for the preparation of a compound as claimed in claim 4 or a salt thereof which process comprises a chromatographic separation technique.

44. A process for the preparation of a compound as claimed in claim 5 or a salt thereof which process comprises a chromatographic separation technique.

45. A process for the preparation of a compound as claimed in claim 6 or a salt thereof which process comprises a chromatographic separation technique.

46. A process for the preparation of a compound as claimed in claim 7 or a salt thereof which process comprises a chromatographic separation technique.

47. A process as claimed in any of claims 43 to 46 wherein high pressure liquid chromatography is used.

48. A process as claimed in claim 47 wherein a aqueous ammonium formate buffered solution is utilised.

49. A process as claimed in any of claims 43 to 46 wherein column chromatography is used.

50. A process as claimed in claim 49 wherein the support system is acetylated cellulose.

51. A process as claimed in claim 49 or claim 50 wherein the eluent is an alcohol/water mixture.

52. A process for the preparation of a solid form of a compound as claimed in any of claims 1 to 3 which comprises freeze-drying.

53. A process for the preparation of a solid form of a compound as claimed in any of claims 4 to 7 which comprises freeze-drying.

54. A process as claimed in claim 33 substantially as described in any one of Examples 1 to 23.

55. A compound as claimed in claim 1 whenever prepared by the process of any one of claims 32 to 54.

56. A process for the preparation of a composition as claimed in claim 22 which comprises bringing into association a compound as claimed in any one of claims 1, 2, 3, 8, 10, 12, 14, 16, 18 or 20 and a pharmaceutically acceptable carrier.

57. A process for the preparation of a composition as claimed in claim 23 which comprises bringing into association a compound as claimed in any one of claims 4, 5, 6, 7, 9, 11, 13, 15, 17, 19 or 21 and a pharmaceutically acceptable carrier.