IE46736B1 - 6-(1-hydroxyethyl)-7-oxo-1-aza bicyclo (3,2,0)-hept-2-ene-2-carboxylic acid derivatives and their use in pharmaceutical compositions - Google Patents

Abstract

The novel derivatives are compounds of the formulae I and II and their salts. These compounds are prepared by culturing Streptomyces olivaceus or Streptomyces gedanensis and recovering the compounds formed and/or their salts from the culture medium. Microorganisms preferred for the culture are the strains ATCC 31126 and 31365. The novel compounds of the formulae I and II and their salts exhibit antibacterial action and are employed as active component in medicaments, on their own or in combination with other antibiotics, preferably in the form of an individual, isolated salt.

Description

None of these 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 a compound or the formula (II): and its salts.

Most suitably the compound of the formula (II; is in the form of a salt since it appears that salts of the compound of formula (II) are more stable than the parent acid.

Suitable salts of the compound of the formula (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, trtmethylaraine, dimethylamine and pyrrolidine salts.

Particularly suitable salts of the compound of the formula (II) include its sodium and potassium salts.

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

Since the compound of the formula (II) and the salts thereof are intended for use in pharmaceutical compositions it will readily be understood that it is 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. An impure preparation of the compound of the formula (II) and its salts may be used for preparing the more pure forms used in the pharmaceutical compositions, these less pure preparations of the compound.of the formula (II) and'its salts should contain at least 1%, more suitably at least 5% and preferably from 10 to 49% of a compound of the formula (II) or the salts thereof. These less pure preparations most usefully comprise 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 (II), are not contaminated by substantial amounts of other anti-bacterial agents such as salts of the compounds of the formula (Ill), (IV) and (V), derived from the fermenta· The compound of the formula cis- and trans-forms about the β-lactam : may be represented by formulae (Ila) and fcion broth. exists as These forms thus: (II) 'ing. (lib) (na) (lib) 46Γ.'3θ It will be realised that the preceding compounds may be named as follows: i Ila (5R,6R) -3-/7E-2-acetamidoethenylthio)_7-6-/7(S) -1-hydroxy ethyl7-7-oxo-l-azabicyclo/3.2.07hept-2-ene-2-carboxylic acid. lib (5R, 6S) -3-/(E-2-acetamidoethenylthiol7"6_Z"(S) -1-hydroxy ethyl7-7-oxo-l-azabicyclo/3.2.07hept-2-ene-2-carboxylic acid Both cis- and trans- isomers of the compound of 10 the formula (II) have useful antibacterial and 0-lactamase inhibiting properties and so this invention extends to the isolated compounds of the formulae (Ila) and (lib) as well as mixtures thereof.

Naturally isolated cis- and trans- forms will .· 15 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 cf its ό-position isomer /that is (Ila) free of (lib)/ or (lib) free of (lb)/.

In general such compounds should not contain more than 5¾ 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 (Ila) having a molar extinction coefficient (in water at neutral pH) of not less than 13000 (preferably not less than 13500) (for UV c absorption maximum at about 308 nm).

A favoured aspect cf this invention provides an alkali metal salt of the compound of the formula (lib) having a molar extinction coefficient (in water at neutral pH) of not less than 13000 (preferably not less than 13500) (for CV absorption maximum at about 303 nm).

Preferably the preceding alkali metal salts are the sodium salts. 6 7 3 β The present invention also provides a pharmaceutical composition which comprises a compound of the formula (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 (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 provided as dosage forms which contain from 50 to 500 mg of a compound of the formula (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. x^t* U > ώ U Particularly suitable penicillins for inclusion in chi· compositions include ampicillin, amoxycillin, c&rbenicillin, ticarcillin and their pro-drugs. When adapted for injection such compounds are generally preser.teci in the form of their sodium salts.

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

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

Preferred cephalosporins for inclusion ir. the compositions of this invention include cephaloridin and sodium ce-pnazolin.

Tne compound, of the formula (II) or a salt thereof 15 may be an isolated compound of the formula (Ila) or (lib) or their salts or mixtures of the compounds of the formula (Ila) 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. 6 7 3 6 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 compound I and II producing strain of Streptomyces olivaceus or Streptomyces gedanesis until a substantial quantity 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 Hutter R (in Systematic der Streptolyceten, S. Korger, Basle, Pages 8-32).

Note that on this definition Streptomyces fulvovoridls, Streptomyces flavus and Streptomyces flavovirens but not Streptomyces flavogriseus may be regarded as being synonymous with Streptomyces olivaceus.

Suitable strains include those described in Patent Specification No. 40864.

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 examples at least 95% pure in 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 Patent Specification ho. 40864. 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 MM455O, MM139O2 and MM1783O 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 6 7 3 6 the fractions containing β-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 β-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 β-lactamase inhibitory activity, (4) applying the combined fractions, to a XAD-4 column, (5) eluting with aqueous isopropanol, (6) combining the fractions with β-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, o.k.) ("Amberlite is a Registered Trace iiarh)· An advantage of such a resin is that it allows the salt of the compounds of the formula (I) and (I to be eluted successively by using an aqueous salt solution, for example a buffered solution of a chlorica such as sodium chloride. If the less favoured strongly basic resins having a polystyrene/divinvl benzene matrix are employed it is generally necessary to elute with dfi ci’^UeOUS 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 MM455O, MM139O2 and MM1768O 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 •7 673^ obtained by careful acidification of a salt of the compound I 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 lAE-Kephadex (sephadex is a Trade Mark).

A related suitable method of chromatographic purification (steps £ and g) uses a solvent system comprising a mixture of water and small quantities of a water immiscible organic solvent such as a C.^ alkancl 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 maybe effected by passing the solution through a bed of lipophilic material onto which the antibiotic is absorbed but which does r.ot absorb the sodium chloride. Suitable materials include polystyrene based polymeric absorbants such as Amberlite XAD-4 and-biaion KF20. The prouuoe of the preceding process may also be desalted by chromatography or. suitable gel filtration agents such as cross-linked dextrans such as Sephadex GIO and G15 and polyacrylamide gels such as Biogel P2. The antibiotic may be eluted from such materials using water or aqueous methanol. 46^36 The columns are eluted at such a rate as to allow separation of the antibiotics into distinct fractions.

Xn general distinct zones can be eluted from these columns; these cqntain di-sodium MM455O, di-sodium MM139O2, di-sodium MM1788O, 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 8-lactamase inhibitory activity or antibacterial activity. Suitable methods of detecting 8-lactamase inhibitory activity include those of the aforementioned 6 Patent Specifications although any convenient method may employed. ‘the following Scheme shows preferred sequence ior obtaining the compounds of the formulae (I) and (IX) 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 ill) under an organic solvent such as moisture containing acetonitrile or acetone can aid in th removal of impurities.

V·' Culture Filtrate Absorb onto Carbon Elute with 20% aqueous acetone Combine Fractions with β-lactamase Inhibitory Activity Remove Acetone and concentrate by Evaporation \Z Cellulose DE52 Anion Exchange Column Compounds of Formula (I) Compounds of Formula (II) of MM455O, MM139O2 and MM1788O XAD4 V XAD4 De-salted Solution V Freeze Dried Solid De-salted solution I Freeze Dried Solid Culture Filtrate Absorb onto Strongly Basic Acrylic Anion Exchange Resin ί Elute with Buffer j Combine Fractions with S-lactamase Inhibitor*/ Activity De-salt on ΧΔ3 4 X'/ Elute with Aqueous Isopropanol Combine Fractions with is-lact&mase Inhibitory Activity | Remove Isopropanol j and concentrate by y Evaporation Cellulose CE52 Anion Exchange Resin ί Elute with Phosphate Buffer pH7 Sodium Salts or Compounds of Formula (I) XAD4 De-salted Solution i A Freeze Dried Solid Sodium Salto of Compounds of Formula (II) i i XAD4 'Ψ De-salted Solution i I S? Freeze Dried Solid - 46736 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 (la), (lb): (Ila) or (lib). 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 (la) and (lb) may be separated by column chromatography on supports such as acetylated cellulose eluting with alcohol/water mixtures. Compounds of the formula (Ila) and (lib) 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 cte caspound of the formula (la) substantially free of the compound of the formula (lb) comprises subjecting a mixture of said salts to chromatographic separation on Diaion HP2O or a chromatographicaliy equivalent resin.

One process for the preparation of a salt of the expound of the formula (lb) substantially free of the compound of the forr.ula (Is) comprises subjecting a mixture of said salts to chromatographic separation on Diaion H?20 or a chromatographicaliy equivalent resin.

One process for the preparation of a salt of th® compound of tne formula (Ila) substantially free of the compound of the formula (lib) comprises subjecting a mixture of said salts to chromatographic separation on Diaion HP20 or a chromatographicaliy equivalent resin.

The present invention provides a process for the preparation of a salt of the compound of the formula (lib) substantially free of the compound of the formula (Ila) which comprises subjecting a mixture of said salts to chromatographic separation on Diaion HP2O or a chromatographicaliy 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 ') 1 I. I 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 undesired isomer.

DiaiOn (which is a 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 2 specific surface area of about 7.8 m /g and a pore volume of 1.16 ml/g. Other details of this resin may be found in (HP series, October 1976) the Diaion data sheet /{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 Diaion HP20 be will also normally/chemically and physically similar, that 20 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.

L L ύ 3 6 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 crying. Alternatively the solution.may be rechromatographed directly on a suitable resin fetch as Biogel ?2 and/or □iaion a?20j for further purification prior to removal of the solvent.

Water may be removed from aqueous solutions of salts of this invention by such precesses 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.

T? <. J Pa.scripti.nn I, Preparation of Clarified Broth A spore suspension of Streptomyces olivaeeus ATCC 51126 wa3 used to inoculate 100 ml of a seed stage medium contained in a 500 ml Ehrleneaeyer flask closed with a foam plug. The seed medium was bean 25a glucose and 1% soya/flour made up in deionised water. (The soya bean flour was Arkasoy 50 supplied by the British Arkady Co. Ltd., Old Trafford, Manchester, Π.Κ.) The seed stage medium was grown for ήό 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 CaCOj 0.02 CoCl2 6H20 0.0001 The fermentation flasks were incubated at S6°C on a rotary shaker for 72 hours. 20 flasks were harvested and the resultant whole broth centrifuged at 2200 g for 10 minutes.

Use Streptomyces olivaeeus ATCC 31365 in the above process is also favoured. trf 1? 'file culture :'.. tret·.· (i5 Λ 2.5 x )7 cai farce granular :an;..'. cjiatn war ; ‘...-.re.. m deionioed water·, the ccltatt was washed successively with , litre 2;', HaOH, i litre deionised water, 1 litre If HCI end 1 litre ieianisei water all at 15 ml/nunute ("Daroo is a Eracta Mark) Eks cohsm Was then washed with 0.05’·ί pK 7 phosphate barter until the pH of the eluant was 7.0 fee culture filtrate <1)00 .al; was run onto the carton ccitrti at mr/tinuta. who colucn was then eluted wit.-, cc at r.c/weter iji, as 15 al/ainute and 22 al fractions were ccl.ecte'i. fractions wex.· monitored I’or tneir l-iactamndd inhibitory activity a-.-atnet a preparation of £-. ,.. eazyr-s (etprltei by Hicrotiologic; 1 hectare. Hctuol-chnens, rertch;. frictions shewing the greatest activity .7 - 2.1 > -An ccbiitei and g/operated under reduced pracctra to rc-.r.cv·;· coetehj. ll.c- racultiiv aqueous nulation was stored best ilrosen prior to sulceqn-cnt wera-up.

(The Sjg.. entyue typifies plaosiid coasroilai '--iactanaaec and may io replaced by other ouch S-iactor d-iaetcmced if desired;. , <6736 .

Example 1 Isolation of the Compounds oi' Formulae (l) and (11) an their Ooiiium Salta Substantially Free of Salts of. MM 4559, MM 13902 and 134 17880 The crude liquor.obtained in Inscription 2 were evaporated 5 ‘ under reduced pressure to approximately 20 ml mid. loaded onto a 3·θ x 27 cm DEAE cellulose weakly basic anion exchange column (the DEAE cellulos> was DE52 cellulose supplied by Whatman Ltd., Springfield Mill, Maidstone, Kent) prepared in 0.025 M pH 7 phosphate buffer. The column was eluted with 0.025 M pH 7 phosphate buffer at a rate of 8 ml/minute and 25 al fractions were collected. The fractions were monitored for their . β-lactamase inhibitory activity against a preparation of β-lactamase. The first two peaks of inhibitory activity were retained.

The first band fractions (13 - 16) containing the salt of the compound of formula (i) were combined and freeze dried to yield a solid preparation containing the sodium-salt of the compound of formula (i) substantially free of the salts of the di-basic antibiotics.

The second band fractions (19 - 21) containing the salt of the compound of formula (II) were combined and freeze dried to yield a solid preparation containing the sodium salt of the compound of the formula (ii) substantially free of the salts of the di-ba3ic 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 (ii). The di-sodium salts of MM 4550» MM 13902 and MM 17880 eluted after the desired .salts). ‘. ϊ . ζ ν·If: ρgfif.k·;.....ΡΡ-ΑΙΙ· .·--, -.1-11 5, (tng--Hhxt^ih.

The freeze dried preparation of the suit the ;:op:ai of Exacpie 1 ae linsoivei in deionised xator (1- m:, neaiuc chloric..:· (1 c.'i aided to tne solution, -his solution was run .r.t1.5 :c 15 ca XAD-4 (supplied by Scha ά Seas) wiaa ,-r-.;ax·..: i.-. udjiwoi waior. The colunn w3s olutc-i wit? water/h-prog ano.. ,;/.. :r. :2 xl/ainute &..1 al fractions vena s ?..... :. .. Ira:; :1:.,3 -./ ..-:. :..:.,1:::::1 I:.:: chloride bv their reaction wit?, A--H5. and for -lactamase ' j jg inhibitory activity against a preparation of ?..,... l-itotansre. fractions with the greatest inhibitory activity ani giving a n.>tive reaction with silver nitrate (fractions ό - 15; wore o-ofoinec as; freeos dried to yield an acorphous solid (32.5 s.p- containing the coiinn salt of the coapound of the fonsula (i)< lj Tin- properties of this pre: era,ton were nt fe — owss (A) Ch.--pm.itoflra.phic Fropertie.? (i) Chromatography on Vnataan 13-11. Ion ititoi.;-! lager ..a veably oasis anion exchange paper) (Whatman .is a itegistered Trade Mark) Eluant : H.. of Sodium Salt 1 ' i 1. 0.05M pH 7 phosphate buffer , C.65 j 2. 0.05K ?H 7 phosphate buffer containing 0.211 MaCl ί ' ' j ?7 Λ 6 ‘7 3 6 (ii) Chromatography on Whatman ilo. 1 Paper: Solvent System 11^ of Sodium Salt 1. Butanol:Ethanol:Water 0.12 'Top Phase 4’1:5 2. Butanol:Pyridine:Water 1:1:1 ' 0.42 (b) Hi/gh Voltare Paner Electrophoresis The electrophoresis was carried out on Ko. 29 paper in pyridine/acetic acid buffer pH 5-3 at 5°00 volts for 15 minutes.

The R,, values for the sodium salt taking bensyl penicillino 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 (pg/ml) Bacillus subtilis A <4° Enterobacter cloacae Hl 1250 Escherichia coli 10413 150 E. coli JT 410 625 . 20 Klebsiella aerogenes A 312 Proteus mirabilis 0977 625 Pseudomonas aeruginosa A > 2500 Salmonella typhimurium CT10 312 Serratia marcescens USJ9 625 25 Staph, aureus Russell 150 6 7 3 6 (ΐ>) Εηζντν Iiiiii iritjon The enzyme inhibitory activity of the preparation against a scries of (3-lactamaae preparations is summarised below: d-Lactsmase Preparation From: 1 1 A Inhibition | at 200ug/ml 1 ι Concentration i giving 5'Ζό j Inhibition 1 ug/ml Staph. Aureus Russell 40 i E. ooli J1'4 95 10 Proteus mirabilis C3 ‘.J - 112 Pseudomonas aeruginosa Dalgleish 170 Enterobacter cloacae P99 85 Pseudomonas aeruginosa A 50 15 Klebsiella aerogsnos Ξ70 27 (Method of Belgian Patent Specification No. 827926). 2? 6 7 3 6 E?:nrmle 5 Partial Purification of the Sodium Salt of the Compounds of the Formula (XI) 'i'll;.· freeze dried preparation of the salt of the compound II of Example 1 was dinsolved in deionised water (lo ml) and sodium 5 chloride (l 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 AgNO^ and for their p-lactemase inhibitory activity against a preparation of R^j p-lactamase. Fractions with the greatest inhibitory activity and giving a negative reaction with silver nitrate (fractions 7 - 15) were combined and freeze dried to yield an amorphous solid (52.5 mg) containing the sodium salt of the compound of the formula (ii).

The properties of this preparation were as follows: (A) Chromatographic Properties (i) Using weakly basic anion exchange paper DE31 cellulose (Whatman) the sodium Salt has an R^ of 0.54 when eluted with 0.05H pH 7 phosphate buffer. (ii) Using Whatman Ko. 1 paper: the sodium salt has an Rf or 0.20 when eluted with butanol/ethanol/water 4/1/5 top phase. 3-3 (H) High Vnitug- Γλ;>ι·γ· The electrophoresis was carried out on Whatman Ko. 2u paper in pyridinc/acetic acid buffer pH 5·5 at 5000 volts for 15 minutes.

TUio Hjj value for the salt taking benzyl penicillin as 1.0 is 0-95(c) Antibacterial Activity The antibacterial activity of the preparation using tne micro titre method wa3 determined and the results are tabulated: Orcaninm T MIO ug/ml Bacillus subtilis A J 250 Enterobacter cloacae Ml ( > looo Escherichia coli 10413 250 Klebsiella ccrogenes A 500 Proteus mirabilis C977 500 ! Pseudomonas aeruginosa A > 1000 Salmonella typhimurium CT10 250 Serratia marscens U3J9 > 1000 Staph, aureus Oxford 500 Staph aureus Russell 1 250 6736 (ft) Enzyme Inhibition β-Lactamase inhibitory activity of the preparation against a range of enzyme preparations have been determined: p-Laotaraaso Preparation From: % Inhibition at 20'Jpg/nl ( 1 Concentration giving 5θ'° I Inhibition . Ug/ml j ' j Staph, aureus Russell - 150 ' | E. coll JT4 - 72 j Proteus mirabilis Gii-39 - 62 Pseudomonas aeruginosa j Dalgleish 1 I - 55 Enterobaoter cloacae P99 | 10 Pseudomonas aeruginosa A j59 Klebsiella aerogenes E70 j 13 _ ΐαχ..:·.· λ Α ii.r: ,cr I’-.u-ification of the h'o.iio.Ti Salta of the Compounds of Formula (l) The preparation obtained in Example 2 io dissolved in deionised water and loaded onto a QAE Sephadex A25 column (QAB Sephadex A25 is a strongly basic anion exchanger supplied by fharmacia Ltd·). prepared in deionised water. The column is eluted with a sodium chloride concentration gradient from 0 to 0.13 M Nad in deionised water.

Fractions from the column are monitored for their β-lactamase inhibitory activity against an B_,. preparation and those’showing the greatest lbi'i activity arc combined. NaCl to a final concentration of at least 5% is added to the combined fractions. The resulting solution is run onto an Amberlite XADcoluan (Rohm & Haa3 Ltd.) and the column eluted with n-propanol/water 1/4. Fractions containing the desired salt as judged by their fi-lactamase inhibitory activity are combined, evaporated under vacuo to remove Ip organic solvent and freeze dried. 4673θ ' Exempli.1 5 A Further Purification of the Sodium Salisof the Compounds of the Formula. (l) The combined fractions from the QAE Sephadex chromatography of Example 4 nay be desalted and further purified by chromatography on a Biogel P2 gel column (Bio-Had laboratories Ltd., 27 Homesdale Hoad, Bromley, Kent) a3 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 l;o aqueous butanol. Fractions are monitored for their β-lactamase inhibitory activity and for reaction with silver nitrate. Those giving a negative reaction to silver nitrate but giving suitable β-lactamase inhibitory activity are combined and freeze dried.

A Ihirtlier Purification of the Sodium Sattaol* the G’oiupn'ni'fa'»1' Fonnula (l) The preparation obtained in Example 2 is dissolved in deionised vzatcr and applied to a column of the strongly basic anion exchange recin Amberlite IRA 451 (Rohm & Haas (UK) Ltd., Lonning House, 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 O.'jJM pH 7 phosphate to 0.05M pH 7 phosphate containing 1.0M NaCl. Fractions showing the greatest β-lactamase inhibitory activity are combined. NaCl is added to the combined fractions to give a concentration of at least 5?«. The resulting solution i3 run onto an Amberlite XAD-4 colunri prepared in deionised water. The column is eluted with n-propanol/watcr 1/4, Fractions showing the greatest [i-lactamasc inhibitory activity but giving a negative reaction with silver nitrate are combined and freeze dried. 6 7 3 6 A Further Purification of the Sodium Salteof Lii·· Coii;ioi.:hb of ! .a (l) The salt of the compound of the formula (i) may be further purified by chromatography on a column of cellulose (Cellulose CCJl Whatman, Springfield Mill, Maidstone, Kent, U.K.) a.s 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-propunol/water ?,/l. The resulting fractions after dilution into deionised water are monitored for their |3-lactamase inhibitory activity. Fractions containing the desired salt are combined, evaporated under reduced pressure to remove solvent and freeze dried. 6 7 3 6 iismdbJi A Further Purification of the Sotllnm Saltsof him Comnoimrlsof' :-’.i'· Formula (Ii.) The process of Example 4 may he used hut replacing the starting material with the preparation obtained in Example Example 9 A Further Purification of the Sodium Saliaof the Compounds of the Formula (ii) The process of Example 5 may be used but replacing the starting material with the product of Example '3.

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

Example 11 A Further Purification of the Sodium Saltsof the Compoundsof the Formula (II) The process of Example 7 may be used but replacing the starting material with the preparation obtained in Example J. • «t 6 7 3 6 iilVUiU·; t: 1? Preparation of Crude Antibiotics from Culture Filtrate Culture filtrate (30 ml) prepared essentially as described in Description 1 was run onto a 1.5 x 15 cm column of Amberlite IDA 45>? a strongly basic acrylic based anion exchange resin (Rote & Haas). The column was eluted with a sodium chloride concentration gradient.

The gradient was from 0 to 1.0 M KaCl in 0.05K pH 7 phosphate buffer at a flow rate of 2.5ml/minute and 5 ml fractions collected. Fractions were monitored for their β-lactamase inhibitory activity against a preparation of β-lactamase. Those fractions giving good inhibitory activity and containing the 3odium salts of the omnounds of the formulae (I) and (II) (4 - 13) were combined. (The di-sodium salts of MM 4550, ΙΦΙ 13902 and MM 17830 eluted starting at fraction 17)· To the combined fractions was added sodium chloride (5 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 l/4 at 5 ml/minute and 4 ml fractions were collected. Fractions were monitored for their β-lactamase inhibitory activity and for reaction with AgKO, 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. 6 7 3 6 ProparatJon of Crude Antibiotics from Cult'ii-e FU irate Culture filtrate (305 ml) prepared essentially as described in Description 1 was run onto a 1.5 x 15 chi column of Amberlite iRA 45 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 β-lactamase inhibitory activity against a preparation of |?-lactamase. Those fractions giving good inhibitory activity and containing the sodium salts of the compounds of the formulae (l) and (II) (12 -32) were combined. The combined fractions were freeze dried. The freeze dried solid was dissolved in deionised water (20 ml), HaCl (2 g) was added and the resulting solution was run onto a 1.5 x 15 cm Amberlite ZAD-4 column (Rohm A Haas) prepared in deionised water. The column was eluted with n-propanol/water 1/4 at 2 ml/minute and 4 ml fractions wei'e collected. Fractionc were monitored for their β-lactamase inhibitory activity and for reaction with AgHO^ 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. 6 7 3 6 Example 1·1 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 25 Soya bean flour 15 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)., 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 20.0¾ Bacto agar (Difco) 2.5S 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 Trade Mark). 6 7 3 6 w' Knell ί(υιι.\ bottle was ineuiaiLei, .it ..-1 ι o r ) x. ..

Alter that Lime 100 ml sterile doioni:-.v Soya bean flour (Arkasoy 50) 1 '·.

Glucose 2 2 Pluronic L81 antifoam 0.1)32 Prepared in distilled water (Pluronic LSI was supplied by Ugine Kuhlmann Chemicals Ltd.) /*Plurcnic is a Trade Mark/.

The medium was steam sterilised in the fermenter for minutes at 120°C. The seed stage culture ww stirred at 140 rpm with a 7.5 inch vaned disc agitator and supplied with sterile air at 75L/minute through an open endec! sparger. The temperature was controlled at 28°C and after incubation under these conditions for 48 hours 7.5Γ, 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 Hour (Arkasoy ίο) 0.9 . Glucose 2.0 ό Chalk 0.025 CoCl2 6H20 0.00015 Pluronic L81 antifoam 0.25 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 50L/minute and the pH maintained at 6.5 - 7.0.

Fermentations as above may be harvested at times ranging between 48 and 54 hours. 6 3 6 Example 15 ΙΌtmentution Conditions ΐor 20001. ΙΌηΐϋ-ηLa I. ion The fermentation conditions up to and including tin seed stage were essentially as described in Example 14. 75L of this seed stage was used to inoculate 15001, 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. 6 7 3 6 Example 16 Isolation Procedure for the Preparation of Substantially Pure Sodium Salts of the Compounds of the Formulae (I) and_(IIί 150L whole broth prepared essentially as described 5 in Example 14 were clarified on a continuous flow centrifuge (Sharpies 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 rosin 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 -j a bed volume of water, then eluted with 0.2M NaCl in 0.05M pH 7 sodium phosphate buffer nt a rate of 230ml/minute. 2L fractions were collected and those showing good inhibitory activity to a preparation of an Rtem mediated (5-lactamase preparation (fractions 2-il) 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. •i 6 7 3 6 Tin.· percolation rate was 200ml/mi in,ι Ήη ,-- -1.111/.:1 --. eluted with 101, deionised water lin-n w u-.-1-/1.:,-..,,,.- m,,i (·!/«> both at 1.OOml/minute. Fractions showing goon 1-.-lactamase inhibitory activity against an 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/minutc.

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 β-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 (11)) 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, Sweden) column prepared in deionised water. The column was eluted with 0.1M NaCl at 3ml/minute and 18 ml fractions were collected. 6 7 3 G Fraction:; showing good i'.-lactamac.e inhib-i >..·.· x hM 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 characteristic of the partially purified sodium saltsof the compounds of the formula (I) (UV maximum approx 297 ma) (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 saltsof 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, ml fractions were collected. Fractions were monitored for their RTEM 8-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 saltsof the compoundsof the formula (II) (maximum approx 307 mp) (,10-16 and 26-32) were combined and freeze dried to yield solids (9.7mg and 21.7 mg respectively). These solids had properties consistent with substantially pure sodium salts of the compoundsof the formula (II).

V' ί ό 7 3 6 KXuiiij/te I 7 Λ I t ιίίι.ι1 i Vi i .ill 1 ,ιΐ i mi Pi or. -lim . I or in, ρ,,,ίπιηι .-.. ι i : . of tlic Compound» of Lim Forum l.η· (i) and (It) Culture filtrate (120 L) containing the sodium salts 5 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 t:h, do..;., (tin; 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 mi) and eluted with 0.025M pH 7 potassium phosphate buffer at 6ml/winute and 20 ml fractions were collected. Fractions showing good RT£M 6-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 N’aCL at 3ml/minute and 20 ml fractions were collected. Fractions were monitored for their UV spectra and those showing characteristic absorption of the sodium salts of the compounds of the formuia (I) (50-56) and characteristic absorption of the sodium salt of the compound of the formula (II) (88-97) were combined separately.

To the bulked Iruction:. (50-5(>) was .uid.·.. i.odiun 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 5 water/n-propanol (4/1) at 3ml/minute. 3 ml fractions were collected. Fractions with UV spectra characteristic of the purified sodium saltgof the compoundsof 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 compoundsof 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 (4/1) after washing with deionised water (15 ml). Fractions with UV spectra characteristic of the substantially pure sodium salts of the compoundsof 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). t·**: V· Λ1 t.i-πιαtivc Isolation Procedure-_for the dodrimi b...t of the compounds of the Formulae (T)__ίi»d__(£(} Culture filtrate (105 L) was prepared essentially 5 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 λΛΰ-4 column were evaporated to about half volume under reduced pressure to remove isopropanoi and stored at 5°C for approximately 65 hours. The resulting solution (660 mid was run onto a 3.8 x 30 cm QAE Sephndex Λ25 column previous! prepared in deionised water. The column was: eluted :-.:611 0.05M NaCl (800 ml) then with 0.1.'·' flaCi boch at 4ml/minute and 20 nil fractions were collected. Fractions were monitored for their P.T1?M 3-lactaraase inhibitory activity and by their EV spectra and those containing essentially the sodium saltsof the compoundsof the formula (I) (73-80) and the sodium saltsof the compounds of the formula (II) (112-125) were combined separately.

Combined Fractions (73-80) wore pvapmated under reduced pressure to approximately 15 niL 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 1¾ butanol.

The column was eluted at 3ml/minutc with deionised water containing 15'butanol and 6 ml fractions were collected. Fractions were monitored by their UV spectra and for reaction with silver nitrate. Fractions containing the sodium saltsof the compounds of the formula (I) giving a negative reaction with AgNO-j (29-39) were combined and freeze dried to yield a solid (72 mg) with properties characteristic of thepurified 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 15 butanol at 3ml/minute and 6 ml fractions were collected. Fractions giving UV spectra characteristic of the sodium saltsof the compoundsof the formula (II) but with a negative reaction for chloride were combined (43-48). Tne combined fractions were freeze dried to yield a solid (27 mg) with properties characteristic of the substantially pure sodium salts of the compoundsof the formula (II). 6 / j 6 !-·.< nap I r 19 Alternative Isolation for the .-(odium s.·ij tvf tii<.· Compounds of the Formulae (I) and (ij > 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 Trade Mark). This culture filtrate was percolated through a 12 diameter Amberlite IRA 458 column (chloride form) (100 b bed volume) at an average percolation rate of 4i/ninate.

The column was eluted at 1.6L/minute wit.i 0.2M MaCl in 0.05M pH 6.7 sodium phosphate buffer ..nd 201, fruet-icns were collected. Fractions showing good .mt ii,.ictcri-il activity when tested on Klebnie 1.1 a aerogi'nes A (n variant of NCTC 418) (1—5) were combined. Sodium ci.ivri Jc was added to the bulked fractions to a final concentration of l.OM and the resulting solution run onto a 6 diameter column of Amberlite XAD-4 (22.4 L bed volume) at 1.2 L/niinute. The Amberlite XAD-4 column was eluted viith 20 L deionised water followed with water/iso-propanol ¢4/1) at 500 ml/minute and 8 L fractions were collected. £1 6 7 3 6 Fractions containing the desired salts (2-4) were e Fractions were monitored for their RTEM-β-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 NaCl at 4ml/minute and 20 ml fractions were collected. Fractions were monitored by their UV spectra an.d those characteristic of the sodium saltsof the compounds-cf the formula (I) (50-70) were combined ready for subsequent processing.

The combined fractions (136-210) from the Cellulose DE52 column were run onto a 4.8 x 26 cm QAE Sephadex A25 column at 6ml/minute. The column was eluted with 0.1M NaCl at 4ml/minute and 20 ml fractions were collected. Fractions showing characteristic absorption spectra of the partially purified saltsof the compoundsof the formula (II) (91-104) were combined. To the combined fractions was added sodium chloride (25 g) and the resulting solution run onto a 2.4 χ 32 cm Amberlite XAD-4 column at 6ml/n)inute. 6 ϊ ο 0 The column was washed wrtii appro;·, ι..i.n ; - " · ..- u .'. ... water end eluwl with watcr/n-prupunoi (1/. ί at 5uil/minute and 10 mi. fraction:', were c<.. lerii-ii. . r .iCt j ΟΛ: (living a negative sil"cr ni trite et j·: -r cni'a ί d·...· but with characteristic UV spectra of tiie sodin.n salts of the compounds of the formula (II) w ·,ί. .·<·'. owed (17-1() The combined fractions were evaporated under reduced pressure to remove n-propanol and freeze dried to yioid 585 mg of the substantially pure sodium salts of tne compoundsof the formula (II). 6 7 3 6 Example 20 Properties of the Sodium Salta of the Compounds of the Formulae (1) and (11) 1. OV Spectra: mixed The/sodium gaits of the compouncfe of the formula (I) has a characteristic maximum at approximately 297 mu. . mixed The/sodium salts of the compouncfe of the formula (II) has a characteristic maximum at approximately 307 ma (one of two) 2. The sodium salts have characteristic 3-lactam 10 carbonyl absorptions at 1750 cm 1 ip their IR spectra. 3. The in vitro antibacterial activity of material prepared essentially as described hereinbefore was as follows: Organism Sodium Salt of the Compound of the Formula (I) MIC (μα/ml) J Sodium Salt of the Compound of the Formula (II) MTC (pg/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 Separation of the Salta of the Compounds of the f'e-rmtire lUj and fib) Λ substantially pure preparation of tnc mixed sodium suits of Liie compounds of the formulae ί ’-'ο) ..ι.< ί Ibj were separated by high pressure liquid chromatography (hplc) by the following procedure: Column: 300 mm x 3.9 mm filled with ,. bo.'.iiapack dlo (Waters Associates, Milford, Massachusetts, USA).

Solvent: 0.05M ammonium acetate, adjusted to yii 4.5 with acetic acid in 5. acetonitrile - 9 S'- water. l-'iow 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. Tne 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 m, cumpounon of the formulae (la) and (Ila) . 6 7 3 6 |·;χ.ιιιΐ|Η e 22 Separation of the Salts of the Compounds of the Formulae (Ila) and (lib) Using the same system as described in Example 21 5 the mixed sodium salts of the compounds of the formulae (Ila) and (lib) were separated by high pressure liquid chromatography with retention times of 6.0 and 7.1 minutes.

./ EXditi^Xu 2.

CULTURE FILTRATE ABSORB ONTO STRONGLY BASIC RESIN AMBERLITE IRA 458 Elute with O.2M NaCl in buffer Ψ COMBINE FRACS. CONTAINING la, Ih, Ila and lib Desalt on XAD-4 Elute with iscpropanol/water Ψ EVAPORATE UNDER REDUCED PRESSURE TO REMOVE PROPANOL la Chromatograph on QAE Sephadex ί , lb Ila lib Desalt on XAD-4 Ψ FREEZE DRY FRACTIONS CONTAINING la, lb Chromatograph on QAE Sephadex 4/ Desalt on XAD-4 FREEZE DRY FRACTIONS CONTAINING Ila, lib Chromatograph on HP2O FRACTION CONTAINING la, lb Chromatograph on Diaion HP2O FRACTIONS FRACTIONS CONTAINING Ila CONTAINING lib -J FRACTIONS CONTAINING la FRACTIONS CONTAINING lb Chromatograph on Biogel P2 Ψ ' ψ FRACTIONS FRACTIONS CONTAINING Ila CONTAINING lib Chromatograph on Biogel P2 Ψ v FRACTIONS CONTAINING la FRACTIONS CONTAINING lb Chromatograph on HP2O and Freeze Dry Ψ Ψ la lb \|/ Ila Chromatograph on HP2O and Freezer Dry lib 6 7 3 6 (In this Example reference to (la), (Ila), (lb) and (lib) 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 120°C. 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/fainute, (through an open ended sparger). The temperature was controlled at 28° 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 6H20 0.0001% Pluronic 1.81 antifoam 0.2% 0 7 3 6 (Medium made un in distilled water, oil 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 4001/ninute. The temperature was maintained at 29°C 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 la, lb, Ila and lib was prepared as follows: A spore suspension of S,olivaeeus prepared from two Roux bottles as described above was used to inoculate 1501 of sterilised seed stage medium contained in a 300 1 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 1 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 1 and 5000 1 fermenters prepared as described above gave a combined volume of 4725 1 The brew was clarified by filtration on a rotary pre-coat vacuum filter, the combined filtrates yielding 4200 1 of clarified brew. The clarified brew was percolated at 10 1/ininute Amberlite IRA458 through columns of the strongly basic anion exchanger resin / (chloride form). The resin was washed with 60 1 deionised water then eluted with an aqueous solution of O.M NaCl with O.O75M sodium phosphate pH 6.7. Elution of la, lb, Ila and lib commenced when the conductivity of the eluant had reached that of 0.1M NaCl and had been eluted when 450lof eluant had been collected. Where necessary the presence of la, lb, Ila and lib was determined using an analytical high pressure liquid chromatography (hplo) system based on the preparative method described in Example 21.

To the combined eluates containing la, lb, Ila and 10 lib 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 5°C until the conductivity of the washings was less than that of a 0.05M NaCl solution. The washed resin was slurried in 421 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 (1301) and evaporated under reduced pressure to remove isopropanol. The resulting solution (671) was percolated at 15 3/hour onto a column of QAE Sephadex A25 (15 x 43 cm) pre-equilibrated in 0.1M NaCl. The column was of washed with 7.51/0.05M NaCl and eluted with 0.1M NaCl at 7.8 1/hour and 500 ml fractions collected. Fractions were β 7 3 6 monitored for the presence of la, lb, Ila and lib by hplc. Fractions containing la and lb, which were eluted together, were combined (12.6 1) and those containing Ila and lib which were eluted later were also combined (5.91).

To the combined fractions containing la and lb was added 44.16 g/1 NaCl, The resulting solution was percolated at 5.8 1/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 la and/or lb at suitable levels as judged by hplc was collected after the conductivity of the eluant had fallen to a level equivalent to 0.01M NaCl. The eluate solution (7.71) containing la and lb 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 Ila and lib were treated similarly to yield a solid (13.7 g).

The freeze dried product (38.5 g) from the XAD-4 column 20 containing la and lb 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 NaCl 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 la and lb (130 - 170) were combined (950 ml). The L·’ 4.6736 QAE Sephadex chromatography was carried out at 5°C.

A portion (450 ml) of the combined fractions containing la and lb from the QAE Sephadex chromatography was taken and NaCl (23.8g) added. The resulting solution was run onto a Diaion HP20 column (4.8 x 62 cm) (Mitsubishi Chemicals Ltd., Agents Nippon Sensui 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 con10 taining la (58-74) and lb (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 la and lb were processed in a similar manner to yield solids 363 mg and 258 mg containing la and lb respectively.

A portion (538 mg) of solid containing la 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 5°C. Fractions were monitored by their UV spectra and those with spectra characteristic of highly purified la (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 χ 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 la (50-62) were combined, concentrated under reduced pressure and freeze dried to yield a solid (40 mg) of la.

A portion (5.8 mg) of the solids containing lb 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 lb(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 freeze dried to yield solids 875 mg and 1.47 g respectively.

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

A portion (860 mg) of Ila 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 Ila 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 Ila (52-64) were combined. The combined fractions were concentrated by evaporation and freeze dried to yield a solid (95 mg) of Ila.

A portion (750 mg) of lib 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 lib 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 6 7 3 6 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 lib (77-91) were combined and freeze dried to yield a solid (105 mg) of lib. 6 7 3 6 Example 24 Properties of the Sodium Salts of Compounds of the Formulae la, lb, Ila and lib, The materials prepared essentially as described in Example 23 and being essentially pure have the following properties: (1) UV spectra la Single maxima 298 nm (molar extinction ε=8,131)(Fig. 1) lb Single maxima 301 nm (molar extinction e=7,93O)(Fig. 2) and Ila 2 maxima 228/308-309 (ε=13,627)(Fig. 3) and lib 2 maxima 229/308-310 (ε=13,933)(Fig. 4) (2) The antibacterial activity of the sodium salts determined by the microtitre method is demonstrated in Table A. (3) The β-lactamase inhibitory activity of the sodium salts is demonstrated in Table B. (4) NMR spectra (6 values are relative to HOI) at 54.6 la See Fig. 5 for spectrum in d20 lb See Fig. 6 for spectrum in d2° Ila ' See Fig. 7 for spectrum in d2o lib See Fig. 8 for spectrum in d2° (5) The synergistic activity of the sodium salts is demonstrated in Table C.

The sodium salts did not cause any obvious toxic effects in mice when administered in aqueous solution subcutaneously at 50 mg/kg. (6) 6 7 3 6 TABLE A MIC (ug/ml) Strain la lb Ila lib I Arnpi- cillin B.subtilis 0.16 1.2 <0.08 2.5 <3. 0 Enterobacter t 2.5 10 2.5 10 200 cloacae NI 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 I 0.6 5.0 0.6 5.0 200 Klebsiella A 2.5 5.0 1.2 5.0 100 Klebsiella E70 10 S.O 10 10 400 Klebsiella Ba95 40 10 40 10 >6400 Proteus mirabilis 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 vulqaris Q3618 Pseudomonas aeruginosa A 5.0 10 10 20 400 >160 >160 >160 >160 1600 Salmonella CTIO 0.3 2.5 0.3 5.0 <0. 3 Serratia US39 ...........- 20 10 20 10 1600 Staph. Oxford 1.25 1.2 0.3 2.5 <3. 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.faecalls 1.25 20 1.2 20 «3 .0 467 36 TABLE B I5O Uig/m.1) Compound Entero- bacter Ps.aerug Proteus E.coli Staph P99 A C889 JT4 Russell Ila 0.02 3.0 0.04 >2.0 0.08 lib 0.04 4.0 0.4 0.1 >2.0 la 0.02 4.0 0.1 >2.0 3.25 lb 0.04 4.0 >2.0 0.28 »2.0 TABLE C Compound MIC (ug/ml) Staph aureus Russell E.coli JT39 Ampicillin alone 1000 2000 , . 0.1 ug/ml + (Ia) 1 Pg/ml >10 >500 - 500 . .. 0.1 pg/ml +(Ib) 1 ug/ml >10 500 10 31.2 +(113)0,1 ug/ml +(IIa) 1 ug/ml 10 >500 500 +(iib)0,1 μ9/πΊ +(IIb) 1 ug/ml >10 10 500 31.2 6736

Claims (1)

1. 5. A pharmaceutically acceptable alkali or alkaline earth metal salt of a compound as claimed in either of claims 2 or 3. 6. A sodium, potassium or calcium salt as claimed in claim 4. 7. A sodium, .potassium or calcium salt as claimed in claim 5. 8. A sodium salt as claimed in claim 6. 9. A sodium salt as claimed in claim 7. 10. A potassium salt as claimed in claim 6. 11. A potassium salt as claimed in claim 7. 12. A compound as claimed in any one of claims 1,4, 6,8 or 10 when at least 50% wt/wt pure. 13. A compound as claimed in any one of claims 2,3,5, 15 7, 9 Or 11 when at least 50% of wt/wt pure. 14. A compound as claimed in claim 12 when at least 75% wt/wt pure. 15. A compound as claimed in claim 13 when at least 75% wt/wt pure. 70· 16. A compound as claimed in claim 14 when at least 90% wt/wt pure. 17. A compound as claimed in claim 15 when at least 90% wt/wt pure. 2. 18. A pharmaceutical composition prepared from a compound as claimed in any one of claims 4,6,8,10,12,14 or 16 and a pharmaceutically acceptable carrier. 19. A pharmaceutical composition prepared from a compound as claimed in any one of claims 2,3,5,7,9,11, 10 13,15 or 17 and a pharmaceutically acceptable carrier. 20. A pharmaceutical composition as claimed in claim 18 prepared from 50 to 500 mg of a compound. 21. A pharmaceutical composition as claimed in claim 19 prepared from 50 to 500 mg of a compound. 15 22. A composition as claimed in either claim 18 or claim 20 which comprises additionally a penicillin or cephalosporin. 23. A composition as claimed in either claim 14 or claim 21 which comprises additionally a penicillin or cephalosporin. 24. A composition as claimed in claim 22 which comprises 20 amoxycillin. Ί\ 467 3 6 25. A composition as claimed in claim 23 which comprises amoxycillin. 26. A composition as claimed in claim 22 which comprises ticarcillin. ' 3. 5 27. A composition as claimed in claim 23 which comprises ticarcillin. 28. A process for the preparation o£ a compound as claimed in claim 1 or a salt thereof which process comprises recovering the compound or salt from the cultivation medium in which 4. 10 it has been produced. 29. A process for the preparation of a compound of either of the formulae I or-II or a salt thereof which process comprises cultivating a 15 compound I and II producing strain of Streptomyces Ollvaceus or Streptomyces gedanesis until a substantial quantity of compound or salt is produced and thereafter recovering said compound or salt from the cultivation medium. 30. A process as claimed in claim 29 which utilises 20 Streptomyces ollvaceus ATCC 31126 or a mutant thereof. 31. A process as claimed in any of claims 29 and 30 for the preparation of a salt of a compound of the formula I or II given in claim 29 which process comprises contacting the culture filtrate with carbon until the antibiotic activity is .6730 absorbed thereon and thereafter eluting the antibiotic activity from the carbon and recovering said salt. 32. A process as claimed in claim 31 wherein the antibiotic activity is eluted from the carbon, the β-lactamase 5 inhibitory fractions are combined, concentrated, applied to an anion exchange column and eluted therefrom with an electrolyte. 33. A process as claimed in any of claims 29 and 30 for the preparation of a salt of a compound of the formula I or II 10 given in claim 24 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. 5. 15 34. A process as claimed in claim 33 wherein the antibiotic activity is eluted from the resin with an aqueous solution of a buffer optionally also containing a salt. 35. A process as claimed in claim 34 wherein the anti 37. A process as claimed in claim 36 wherein the salt is a sodium salt. 38. A process as claimed in claim 36 wherein the salt is a potassium salt. 5 39. A process for the preparation of a compound as claimed in claim 2 or a salt thereof which process comprises a chromatographic separation technique. 40. A process for the preparation of a compound as claimed in claim 3 or a salt thereof which process comprises 10 a chromatographic separation technique. 41. A process as claimed in any of claims 39 or 40 therein high pressure liquid chromatography is used. 42. A process as claimed in claim 41 wherein an aqueous ammonium formate buffered solution is utilised. 4 6 7 3 6 43. A process as claimed in any of claims 39 or 40 wherein column chromatography is used. 44. A process as claimed in claim 43 wherein the support system is acetylated cellulose. 5 45. A process as claimed in claim 43 or claim 44 wherein the eluent is an alcohol/water mixture. 46. A process for the preparation of a solid form of a compound as claimed in claim 1 which comprises freeze-drying. 10 47. A process for the preparation of a solid form of a compound as claimed in any of claims 2 or 3 which comprises freeze-drying. 48. A process as claimed in claim 29 substantially as described in any one of Examples 1 to 23. 15 49. A compound of formula I or II as defined in claim 29 whenever prepared by the process of any one of claims 29 to 48. 50. A process for the preparation of a composition as claimed in claim 18 which comprises bringing into association a compound as clained in any one of claims 4,6,3,10,12,14 or 16 6. 20 and a pharmaceutically acceptable carrier. 4 6 7 3 6 51. A process for the preparation of a composition as claimed in claim 19 which comprises bringing into association a compound as claimed in any one of claims 2, 3, 5, 7, 9, 11, 13, 15, 17 and a pharmaceutically acceptable carrier. F.R. KELLY & CO., AGENTS FOR THE APPLICANTS.