CA1074325A - Clavulanic acid and its salts - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
A new antibacterially active agent has been isolated from Streptomyces clavuligerus. This new compound which we designate clavulanic acid has the formula (I):

Description

~ 107~3'~

Tllis application is directed to the preparation of clavulanic acid and its salts by deesterification and is a divisional of S.N. 224,970 filed 18 April 1975. The parent application is directed to clavulanic acid and its pr~paration by cultivation of Streptomyces clavuligerus and conversion to salts as required. Other divisional applications S.N. 314,882 and S.N.
314,880 filed 30 October 1978 are directed respectively to the preparation of esters of clavulanic acid and synergistic mixtures of clavulanic acid or its pharmaceutically acceptable salts or esters with a ~ -lactam antibiotic.
This invention relates to a new agent having antibacterial activity. More particu]arly it relates to a new antibacterial agent ob-tainable from Streptomyces clavuligerus, designated as clavulanir acid, and salts and esters of the agent.
BACKGROUND TO THE INVENTION
a. Streptomyces clavuligerus has been described in detail by Higgens et al, Int. J. Systematic Bacteriology, 21, 326 (1971).
This s~reptomycete was of interest because it produced certain ~ -lactam antibiotics such as penicillin N, 7-(5-amino-5-carboxy-valeramido)-3-carbamoyloxymethyl-3-cephem-4-carboxylic acid and 7-(5-amino-5-carboxyvaleramido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylic acid. The streptomycete has been deposited in the Agricultural Research Service Collection as NRRL 3585 and in the American Type Culture Collection as ATCC 27064 Streptomyces clavuligerus has also been referred to in United States Patent No 3,770,590 and also by Nagarajan et al, J. Amer.
Chem, Soc., 93, 2308 (1971), ~rannon et al, Antimicrob. Agents Chemother,, 1, 237 (1972) and Antimicrob. Agents Chemother, 1, 247 (1972) and ~iggens et al, J. Antibi~tics, 27, 298 (1974).

I)~

~07432~

~ -].actamases are enzymes which open the ~ -lactam ring of penicillins and cephalosporins to give products which are devoid of antibacteria]. activity. These enzymes are 4 produced by many la.

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bacteria, notably species or strains of Escherichia, Klebsiella, Proteus, Pseudomonas, Enterobacter and Staphylococcus and are in many instances the explanation for the resistance of certain strains of such organisms to some penicillins and cephalosporins.
The importance of ~-lactamase production may be understood when it is realised that a high proportion of clinically isolated organisms produce ~-lactamases rsee, for example, M. I~ilson and I.A. Freeman, Bacteriological Proceedings, 80 (1969) where in a paper entitled 'Penicillin Inactivation by Gram-negative Bacilli' they showed that 84% of the gram-negative organisms isolated in an American hGspital produeed ~-lactamaseJ. In many cases, some penicillins or cephalosporins are ineffective in treating diseases ascribed to non ~-lactamase-producing organisms because of the common occurance of co-infection by a ~-lactamase producer [see, for example, R. May et al; Brit. J.Dis.Chest., 66, 185 (1972)] .
Combination of a ~-lactamase inhibiting substance with a penicillin or cephalosporin might be expected to protect the latter from degradation by baeterial ~-lactamase and thereby enhance their antibaeterial activity against many infective organisms. This process of enhancement of the antibacterial activity is called synergism when the antibacterial activity of the combination is well in excess of the simple addition of the activities of the two separate substances. The ~-lactamase inhibLtlng component of the mixture is referred to as a synergist and such substances are valuable for increasing the antibaeterial activity of penicillins and cephalosporins against resistant o~garllsms.
It is one of the objects of this invention to provide such synergists.
c. Examples of the use of certain ~-lactamase resistant semi-synthetic penicillins and cephalosporins as ~-lactamase inhibitors and synergists for penici~lins and cephalosporins have already been described in the literature, see for example, Sutherland et al., Nature, 20I, 868 (1964); Sabath et al., Nature, 204, 1066 (1964);
O'Callaghan et al., Antimicrob. Agents and Chemotherapy, 1968, 67 (1969). ~lowever, none of these known agents have a dramatic effect on the spectrum of the other antibiotic present in the mixture.
d. Certain actinomycete cultures have been described as producing ~-lactamase inhibiting substances which act synergistically with penicillins or cephalosporins, for example, those cultures disclosed in British Patent No. 1,363,075 and those described by llata et al, J. Antibiotics, 25, 473 (1972) and Umezawa et al, J. Antibiotics, 26, 51 (1973). None of these ~-lactamase inhibitors of actinomycetal origin have yet been found to be of use in the clinic. Particularly noteworthy features which ! distinguish clavulanic acid from other ~-lactamase $nhibitors of actinomycetal origin are its extractability into organic solvents from culture ~iltrate at pH2, its high stability in human blood and its brosd spectrum of anti-bacterial and ~-lactamase inhibiting activity, its low molecular weight and its high Rf values on paper chromatography using a variety of solvent systems.
DESCRIPTION OF TIIE INVENTION
We have discovered that the aerobic cultivation of StrePtomyces clavuli~erus ln conventional nutrient media at about 25-30C under rougllly neutral conditions produces a ~-lactamase inhibitory substancc which also possesses antibacterial activity. We have designated this new material 'clavulanic acid'.
Clavulanic acid has the following properties:
(a) It is a carboxylic acid.
~b) It forms a sodium salt which has a characteristic infra-red spectrum substantially as shown in Figure 1.

~074325 (c) It is able to inhibit the growth of strains of Staphylococcus aureus.
(d) It is able to synergyse the antibacterial effect of ampicillin against ~-lactamase producing strains of Escherichia coli, Klebsiella aerogenes and Staphylococcus aureus.
(e) It is able to synergyse the antibacterial effect of cephaloridine against the ~-lactamase producing strains of Proteus mirabilis and Staphylococcus aureus.

(f) It forms a methyl ester which has a molecular weight (by mass spectroscopy) of 213.0635 which corresponds to the formula CgUl1N05.
Thus clavulanic acid may be regarded as a ~onobasic carboxylic acid of the formula C8~l9N05 which in the form of its sodium salt has a characteristic infra-red absorption spectrum substantially as shown in Fig. 1.
The compound produced by Streptomyces clavuligerus which has the above properties has the formula ~
~O\ =/c~l ~o~

~ < (II) C02~1 Thus cla w lanic acid may be named 3- ~-hydroxyethylidene)-7-oxo-4-oxa-1-azabicyclol3,2,~ heptane-2-carboxylic acid.
The stereochemistry at C5 and C2 of the clavulanic acid is the same as that found in naturally occurring penicillins and cephalosporins so that clavulanic acid may be represented by the structural formula (I):
r ~
// ~ (I) C02~1 ~1)743'~

Thus a fuller chemical name for clavulanic acid is Z-(2R,5R)-3- ~ hydroxyethylidene)-7-oxo-4-oxa-1-azabicyclo~3,2,0~ heptane-2-carboxylic acid~
The great use~ulness of clavulanic acid may be readily appreciated when it is realised that certain strains of Klebsiella aerogenes A, the growth of which is not inhibited by the presence of 125f~g/ml. of ampicillin, amoxycillin, carbenicillin or benzyl penicillin or by the presence of 10 ~g/ml. of claw lanic acid, are inhibited by the presence of less than 12.5~g/ml. of the previously mentioned penicillins when 5J(g/ml. of clavulanic acid is also present. Similar results have been observed for combinations containing various esters of claw lanic acid. For example, strains of Klebsiella aerogenes A, the growth of which is not inhibited by 125~-g/ml. of ampicillin, or by lO~Ig/ml of clavulanic acid methyl ester are inhibited by ]ess than 12.5/lg/ml. of ampicillin in the presence of 5~g/ml. of the clavulanic acid methyl ester. It has also been found that strains of ococcus aureu= Rus~ell, the growth of which is not inhibited by the pr~ c~ o 100J/g/ml. of amp;cillin or by 5~g/ml of clav~llanic acid, are 1nh:lbited by the presence of less than 10 J~g/ml. of ampicil]in in the presence of 1JIg/ml. of clavulanic acid. In tests on female mice, it has been found that blood and tissue levels of clavulanic acid considerably in excess of 5)lg/ml. can readily be achieved hy subcutaneous administration of 100 mg/kg of the sodium salt of clavulanic acid and that useful levels of clavulanic acid can be obtained after oral administration of 100 mg/kg of the sodium salt of clavulanic acid.
Accordingly, the present invention provides clavulanic acid as hereinbefore described and its salts and esters.
Most suitably the salts of cla w lanic acid will be pharma-ceutically acceptable salts such as the sodium, potassium, calcium, magnesium, aluminium, a~monium and substituted ammonium salts sucl! as the trimethyl-ammonium, benzathine, procaine and like salts conventional]y formed with ~0743Z5 penicillins or eephalosporins. Non-pharmaceutically acceptable salts of claw lanic acid are also included within the scope of this invention as they are useful intermediates in the preparation of esters of clavulanic acid, or example, the lithium or silver salts of claw lanic acid ~ay be reacted with benzyl bromide to form the useful benzyl ester of clavulanic acid.
Salts of claw lanic acid tend to be more stable than the parent acid per se and thus form a favoured aspect of this invention.
Particularly suitable salts of clavulanic acid include the sodium and potassium salts whieh have the formulae (III) and (IV) respectively:

2 J I ~ ~ 2 N O
~02Na C02K

(III) (IV) Crystalline forms of such salts may contain water of hydration.
Suitable esters of clavulanic acid include those notionally derived rom alcohols sueh as methanol, ethanol, propanol, butanol, 2,2,2-triehloroethanol, 2,2,2-trifluoroethanol, benzyl alcohol, p-nitroben~yl aleohol, phenol, acetoxymethanol, pivaloyloxymethanol, 2-dimethylaminoethanol and other eonventional aleohols. ~arious esters of elaw lanic aeid are useful intermediates in certain processes for the purification of clavulanie aeid.
Many elavulanic acid esters are useful synergistic compounds. The activity of such esters might be due to hydrolysis of the ester to the parent acid.
When used herein the term ester includes esters notionally derived from an aleohol or thiol of the formula ~OH or RSH where R is an organic residue. Suitable groups ~ include alkyl, alkenyl, alkynyl, aryl, arylalkyl or other similar groups any of which may be substituted if desired.
In order not to increase the molecular weight to an unreasonable extent, groups R do not normally inelude more than 16 carbon atoms, more suitably, ~074325 not more than 12 carbon atoms and most suitably, not more than 8 carbon atoms.
The esters of the clavulanic acid may thus be represented by the formula n~ 2oll N ~

C~lJ--R
wherein W is O or S and R is as defined.
Preferably, the group R is notionally derived from an alcohol ROH or (less favorably) a thiol RSI~ which is pharmaceutically acceptable.
Suitable substituents which may be included in the group R
include halogen atoms and lower alkoxyl, hydroxyl, lower acyloxyl, lower alkylamino, lower dialkylamino and like groups. The term 'lower' means that the group contains up to 6 carbon atoms, and preferably up to 4 carbon atoms.
Thus, for example, R may be a methyl, ethyl, n-propyl, iso-propyl, straight or branched butyl, pentyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, ~lnyl, allyl, butenyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclohexenyl, cyclohexadienyl, methylcyclopentyl, methylcyclo-hexyl, benzyl, benzhydryl, phenylethyl, napthylmethyl, phenyl, napthyl, propynyl, tolyl, 2-chloroethyl, 2,2,2-trichloroethyl, 2,2,2-trifluoroethyl, acetylmethyl, benzoylmethyl, 2-methoxyethyl, 2-dimethylaminoethyl, 2-diethyl-amilloethyl, 2-piperidinoethyl, 2-morpholinoethyl, 3-dimethylaminopropyl, p-chlorobenzyl, p-methoxybenzyl, p-nitrobenzyl, p-bromobenzyl, m-chlorobenzyl, 6-methoxynaplltllyl-2-metllyl, p-chlorophenyl, p-methoxyphenyl or any like grou~ as well as those groups which are known from the penicillin or cephalo-sporin arts to produce esters known to be readily hydrolysed in vivo to the parent antibiotic.
Readily hydrolysable esters include, but are not limited to, those of the formulae (V) and (~

~0743ZS

r--~'\ ~ C~2011 ~,--/ ~--CO ~1 - C - X -CO ~ 3 (V) r ~ N -~
\ (VI) C0 - o - Cll Z
X -- C = Y

wherein Al is a hydrogen ato~l, alkyl, aryl or aralkyl group; A2 is a hydrogen atom or methyl group; A3 is an alkyl, aryl or aralkyl group; X is oxygen or sulphur; Y ls oxygen or sulphur and Z is a divalent organic group. Esters of the formulae (V) and (VI) which fairly readily release the clavulanic acid lnto tt~e blood stream after administration include those wherein Al is a hydrogen atom, A2 is a hydrogen atom or a methyl group and A3 is a methyl, ethyl, propyl, butyl, benzyl, or phenyl group and those wherein X is oxygen, Y is oxygen and Z is -CH2CH2-, -Cll:CH-, ~ ~ ~ C~13 When used in conjunction with the preceding formula the term 'alkyl' i.ncludes alkyl of up to six csrbon atoms; the term 'aryl' includes phenyl, naphthyl or phenyl substituted by an inert substituent such as a fluorine or chlorine atom or a methyl or methoxyl group or the like; when used herein the term 'aralkyl' means an alkyl group substituted by an aryl group.

10743Z~

Particularly suitable esters of the formulae (V) and (VI) include those of the formulae (VII) and (VIII):

\ ~ A
CO - O - Cll - O - CO - A5 (VII) n' >~
~- rl ~- ~ ~ A6 (YIII) 11 `J\~6 ~4 is a hydrogen atom or a methyl group, A5 is a methyl, t-butyl or phenyl group and A6 is a hydrogen atom or a methoxyl group.
Many esters of clavulanic acid differ from analagous ester;
of penicillins or cephalosporins in that they show an enhanced tendency to hydrolyse to clavulanic acid under mild conditions. Thus, for example, simple alkyl esters such as the methyl ester slowly hydrolyse to clavulanic acid in water buffered to pH7. Esters which undergo some hydrolysis under mlld conditions are included within the formula (IX):

0 ~0 ~1 (IX) wherein Rl is a hydrocarbon group of 1-9 carbon atoms optionally substituted by halogen, lower alkoxy, hydroxyl or optionally salted basic groups of the formula NR R wherein R is a hydrogen atom or a lower alkyl group, R is a hydrogen atom or a lower alkyl group or is attached to R2 so that NR2R3 is a 5- or 6- membered ring.
When used with reference to formula (IX) the term 'lower' means that the group contains 1-4 carbon atoms.
Suita~ly grour~ R includo all;yl and arall;yl groups optionally substituted by halogen, methoxyl, hydroxyl or salted NR R3 groups wherein R2 is a methyl or ethyl group and ~3 is a metilyl or ethyl group or is joined to R so tl~at NR2T~3 is a pyrrolidine, piperidine or morpholine group.
Most suitably alkyl groups Rl are straight chain groups of up to 6 carbon atoms optionally substituted by one methoxyl, hydroxyl, salted NR2R3 group or one chlorine, bromine or iodine atom or by a CC13 or CF3 group-The esters of clavulanic acid of particular usefulness assynergists are those which hydrolyse in mammalian tissues, especially human blood, to yield clavulanic acid or a salt thereof because it is belived that clavulanic acid and its salts tend to be somewhat more useful synergistic agets than ti1e esters per se. ~any of the esters of the formulae (~)-(IX~
are useful for this purpose.
A further group of particularly suitable esters of this invention are those useful intermediates which are readily converted to clavulanic acid or a salt thereof by chemical or biochemical techniques which are known ~rom tl1e penicillin or cephalosporin arts to be sufficiently mild rl~t to 1e~ra~e reactive acid-labl]e ~-lactam rings.
Most suitably, the ester is one removable by hydrogenolysis.
Conventional esters for such a process include benzyl, substituted benzyl, benzhydryl, substituted benzhydryl, trityl and the like. T1le benzyl ester has proved particularly useful for this purpose.
Ey and large, the nature of any substituent in the ester moiety is unimportant as long as it does not interferc with the hydrcgenolysis reaction.
Since clav11]anic acid and its salts arc useIul intermediatcs in the preparation of the desirable antibacterially active esters of this invention, this invention also provides clavulanic acid and its salts ~hen used as chemical intermediates.
As has been previously stated, clavulanic acid and its salts and esters have valuable therapeutic properties. Accordingly, in a further ~07432S

aspect, this invention provides a phar~aceutical composition which comprises clavulanic acid or a salt or ester thereof together with a pharmaceutically acceptable carrier.
The compositions of the invention include those in a form adapted for oral, topical or parenteral use and may be used for the treatment of infection in mammals including humans.
Suitable forms of the co~positions of this invention include tablets, capsules, creams, syrups, suspensions, solutions, reconstitutable powders and sterile forms suitable for injection or infusion~ Such compo-sitions may contain conventional pharmaceutically acceptable materials such as diluents, binders, colours, flavours, preservatives, disintegrants and the like in accordance with conventional pharmaceutical practice in the manner well understood by those skilled in the art of formulating antibiotics.
In;ectable or infusable compositions of the clavulanic acid or its salts are particularly suitable as high tissue levels of the compound of clavulanic acid can occur after administration by injection or infusion.
Thus, one preferred composition aspect of this invention comprises clavulanic ncld or a salt thereof in sterile form.
Unit dose compositions comprising clavulanic acid or a salt or ester thereof adapted for oral administration form a further preferred composition aspect of this invention.
Under certain conditions, the effectiveness of oral compo-sitions of clavulanic acid and its salts and esters can be improved if such compofiitions contain a ~uffering agent or an enteric coating agent such that the compounds of the invention do not have prolonged contact with hiKhly acidic gastric juice. Sucl- buffered or enterically coated compositions may be prepared in accordance with conyentional pharmaceutical practice.
The clavulanic acid or its salt or ester may be present in the composition as sole therapeutic agent or it may be prescnt together with other therapeutic agents such as a ~-lactam antibiotic. Suitable ~-lactam antibiotics for inclusion in such synergistic compositions inclu(le not only those ~.nown to be highly susceptible to ~-lactamases but also those which llave a good degree of intrinsic resistance to ~-lactamases. Thus, suitable lactam antiblotics for inclusion in the compositions of this invention include benzylpenicillin, phenoxymethylpenicillin, carbenicillin, methicillin, propicillin, ampicillin, amoxycillin, epicillin, ticarcillin, cyclacillin, 6-aminopenicillanic acid, 7-aminocephalosporanic acid, 7-aminodesacetoxy-cephalosporanic acid, cephaloridine, cephalothin, cefazolin, cephalexin, cefoxitin, cephacetrile, cephamandole, cephapirin, cephradine, cephaloglycine and other well known penicillins and cephalosporins or pro-drugs therefore such as hetacillin, metampicilli.n, the acetoxymethyl, pivaloyloxymethyl or phthalidyl esters of benzylpenicillin, ampicillin, amoxycillin or cephalo-glycine or the phenyl, tolyl or indanyl c~-esters of carbenicillin or ticarcillin or the like.
Naturally if the penicillin or cephalosporin present in the composition is not suitable for oral administration then the composition will be ndaptcd o~ parenteral administration.
When prcsent in a pharmaceutical composition together with a ~-lactam antibiotic, tlle ratio of clavulanic acid or its salt or ester present to ~-lactam antibiotic present may be from, for example, 20:1 to 1:12, more usually 10:1 to 1:10 and advantageously may be from 3:~ to 1:3.
The total quantity of antibacterial agents present in any u~it dosnge forlll will normally ~e betwccn 50 all(l 1500 mg and w~ll us(lnl~y l~e LW~ 1()() <111(1 10()() 111~.
Compositions of this invention may be used for the treatment of infections of inter alia, the respiratory tract, thc urinary tract and soft tissues in humans.
Compositions of this invention may also be used to treat infections of domestic animals such as mastitis in cattle.

~074325 Normally between 50 and 6000 mg of the compositiDns of the invention will be administered each day of treatment but more usually between 500 and 3000 mg of the composition of the invention will be administered per day. However, for the treatment of severe systemic infections or infections of particularly intransigent organisms, higher doses may be used in accor-dance with clinical practice.
The exact form of the compositions of this invention will depend to some extent on the micro-organism which is being treated. For treatment of most infections the compositions of this invention are normally adapted to produce a peak blood level of at least O.l,ug/ml, more suitably at least 0.25 ~g/ml, and preferably at least 1jug/ml. of synergist, for example, 2.5 - 5,ug/ml. of synergist.
The penicillin or cephalosporin in synergistic compositions of this invention will normally be present by up to or at approximately the amount conventionally used when that penicillin or cephalosporin is the sole therapeutic agent used in the treatment of infection.
Particularly fa~oured compositions of this invention will conta.Ln Xrom 150 - 1000 mg o amoxycillin, ampicillin or a pro-drug therefore and ~rom 50 - 500 mg of clavulanic acid or a salt or in-vivo hydrolysable ester thereof and more suitably from 200 - 500 mg of amoxycil]ln, ampicillin or a pro-drug therefore and from 50 - 250 mg of clavulanic acid or a salt or in-vivo l-ydrolyable ester thereof.
The materials present in such compositions may be hydrated if requlred. The weights of the antibiotics in such composition are expressed on the basis of antibiotic theoretically available from the composition and not on the basis of the weight of pro drug.
In a process aspect, the present invention provides a process for the preparation of clavulanic acid and salts and esters thereof which process comprises cultivating a strain of Streptomyces clavuligerus and recovering clavulanic acid or a salt thereof from the culture medium and 10743'~5 thereafter if desi~ed, forming the free acid or a salt or ester by methods known per se.
Preferably, Stre~tomyces clavuligerus ATCC 27064 or a high yielding mutant thereof is used in the process of this invention.
~ ~C
When used herein, the term 'cultivation' means the dclivcrat~
aerobic growt}- of a clavulanic acid producing organism in the presence of assimilable sources of carbon, nitrogen and mineral salts. Such aerobic growth may take place in a solid or semi-solid nutritive medium, or in a liquid medium in which the nutrients are dissolved or suspended. The culti-vation may take place on an aerobic surface or by submerged culture. The nutritive medium may be composed of complex nutrients or may be chemically dePined. In our hands we have found media containing complex nutrients such as yeast extract, soya bean flour and the like to be particularly suitable.
' The nutrient media which may be used for tl~e cultiva~ion of s Streptomyce_ clav_ligerus may contain, in the range 0.1 - ]0% a complex organic nitrogen source such as yeast extract- corn s-eep liquor, vegetable protein, seed protein, hydrolysates of such proteins, milk protein hydrolysates, ~ t and meat ex~racts and hydrolysates such as peptones. Alternatively chemic~lly defined sources of nitrogen may be used such as urea, amicles, single or mixtures of common amino acids such as valine, asparagine, glutamic acid, proline and phenylalanine. Carbohydrate (0.1 - 5%) may be ~ncluded in the nutrient media but glucose in certain media is undesirable having a depressing effect on the yield of the desired clavulanic acid. Starch or starch hydrolysates such as dextrin, sucrose, lactose or other sugars or glycerol or glycerol esters may also be used. The source of carbon may also be derived from vegetable oils or animal fats. Carboxylic acids and their salts can be included as a source of carbon for growth and production of ~-lactamase inhibitors. A particularly suitable low cost medium is one containing soya bean flour (Arkasoy ) plus dried malt distillers solubles (Scotasol ) plus dextrin.

*Tradcmark 107432~

The addition of antifoam agents such as Pluronic L81 may be necessary to control foaming of certain media in fermenters.
Mineral salts such as NaCl, KCl, MgC12, ZnC12, FeC13, Na~S04, FeS04, MgS04 and Na+ or K salts of phosphoric acid may be added to the media described above particularly if chemically defined; CaC03 may be added as a source of Ca ions or ror its buffering action. Salts of trace elements such as nickel, cobalt or manganese may also be included.
Vitamins may be added if desired.
When used herein the term 'mutant' includes any mutant strain which arises spontaneously or through the effect of an external agent whether that agent is applied deliberately or otherwise. Suitable methods of producting mutant strains include those outlined by H.I. Adler in Techniques for the Development of Micro-Organisms in 'Radiation and Radio-isotopes for Industrial Micro-Organisms', Proceedings of a Symposium, Vienna, 1973, page 241, International Atomic Energy Authority and these include:
i. Ionising radiation (such as X- and ~ - rays), uv light, uv llght plus a photosensiti~ing agent (such as 8-methoxypsoralen), nitrous acid, hydroxylamine, pyrimidine base analogues (such as 5-bromouracil), acridines, alkylating agents (such as mustard gas, ethyl-methane sulphonate), hydrogen peroxide, phenols, formaldehyde, heat, and ii. Genetic techniques such as recombination, trans-formation, transduction, lysogenisation, lysogenic conversion and selective techniques for spontaneous mutants.
Cultivation of Streptomyces clavuligerus normally takes place in the temperature range 15-40C, usually 20-35 and preferably, 25-30C and at a pH of between 5 and 8.5, preferably between 6 and 7.5.

The Streptomyces clavuligerus may be cultivated in the above media in glass conical flasks aerated by shaking on a rotary shaker or in baffled stainless steel fermenters stirred with vaned disc impellers and aerated wi~h a sparger. The fermentation may also be carried out in a continuous fashion.
The starting pH of the fermentation is typically 7.0 and maximum yield of clavulanic acid is obtained in 2-10 days at 20-35C. In a stirred stainless steel fermenter using the Arkasoy/Scotasol/Dextrin medium referred to above the preferred temperature is 26C and peak yields clavulanic are obtained within 5 days.
Clavulanic acid may be extracted from culture filtrate by a variety of methods. Solvent extraction from cold culture filtrate adjusted to acid pH values and methods based on the anionic nature of the metabolite such as the use of anion exchange resins have been found to be particularly useful.
The cells of the Streptomyces clavuligerus are normally first removed from the fermentation by filtration or centrifugation before such extraction pro-cedures are commenced.
In the solvcnt extraction process, the culture filtrate is chilled and the pU lowercd into the region of pH 2-3 by the addition of acid while thoroughly mixing with a water immiscible organic solvent such as n-butylacetate, methylisobutylketone, n-butanol or ethylacetate. The acid used to lower the pH of the medium is normally a mineral acid such as hydro-chloric, sulphuric, nitric, phosphoric or the like acid. n-Butanol is a particularly suitable solvent for use in the extraction of the acidified culture filtrate. After separation of the phases by centrifugation, the ~ -lactamase inhibiting metabolite is back extracted from the solvent phase into aqueous sodium bicarbonate or potassium hydrogen phosphate buffer, CaC03 suspension or water while maintaining the pH at approximate neutrality, for example, at pll 7Ø This agueous extract after separation of phases may be concentrated under reduced pressure and free7e dried to give a crude preparation of a salt of cla w lanic acid. This preparation is stable ~hen stored as a dry solid at -20 C.
In the anion exchange resin process, the clarified culture filtrate at an approximately neutral or slightly acid pH, for example pH 6-7, is percolated down a column of weak or strong base anion exchange resin such as Amberlite IR4B or Zerolit FFIF respectively until the resin is saturated and the/~-lactamase inhibiting material emerges from the bottom. The colutnn i9 then washed with water and eluted ~ith aqueous sodium chloride. The ~-lactamase inhibiting fractions are collected, bulked, desalted and freeze dried to yield a c~ude solid salt of clavulanic acid.
Amberlite IR 4B is an example of a weakly basic anion exchange resin with polyamine active groups and cross linked polystyrene-divinyl-benzene matrix. Zerolite FFIP is a strongly basic anion exchange resin with quaternary ammonium active groups and a cross linked polyvinyl-* *
divinylbenzene matrix. Resins similar to Zerolite FFIP include Isopor FFIP
and DeAcidite ~IP SRA.64. These resins were supplied by BDIl Chemicals Ltd., Poole, Dorset, U.K.
An alternat~ve form o~ the extraction process is to contact the culture filtrate (usually at approximately neutral pH) containing a salt of clavulanic acid, with an organic phase in which is dissolved a water insoluble amine. Suitable organic solvents include such conventional water immiscible polar solvents as methylisobutylketone~ trichloroethylene and the like. Suitable amines include secondary or tertiary amines in which one of the substituent groups is a long chain aliphatic group, ~or example~ o 12-16 carbon atoms and the other is a tertiaryalkyl group so that the m~lecule is lipophilic. In our hands Amberlite ~2 has proved a successul atDine.
~ormally t~e amine is used as its acid addition salt.
After this extraction process the clavulanic acid is present in the organic phase as the amine salt. The organic phase is then separated from the culture iltrate. The clavu~anic acid may be back extracted into an * Trademark 1~74325 aqueous phasc by bacl~ c~traction ~ith a salt solution, preferably a con-centrated solution of sodium chloride, sodiuw nitrate or the like. The ; crllde s~llt of clclvu]nnic acid may then be obtained by free~e drying or the like.
Other primary methods of isolation which may be used include conventional methods such as adsorption onto carbon, precipitation, salting out and molecular filtration but these methods are not usuall~r as successful as the above described methods which are preferred.
Purther purification of the crude solids obtained by methods described above may be obtained by a variety of methods but ion exchange column chromatography is particularly suitable especially when using Isopor, DeAcidite FFIP SRA64 or DEAE cellulose. The DeAcidite column may be gradient eluted with aqueous solution of a salt such as sodium chloride (0 - 0.5M).
The column of DEAE cellulose in 0.01M phosphate buffer at l)H7 may be eluted with a salt solution, normally a NaCl solution (0 - 0.2M NaCl in 0.01M
phosphate buffer pH7). Acti~e fractions may be detected by their ~ lactamase ~nhlbl~ory ~IC~ivity and their antibacterlal activity against K ebsiella an agar difusion assay, The fractions containing the bulk of this activity are then combined and concentrated to a small volume under vacuum. This crude preparation of the clavulanic acid salt is desalted by percolating down a column of Bio Gel P2 (Bio Gel (trade mark) P2 is an exsmple or a highly lipophilic resin onto which organic materials may be adsorbed but ~rhich does not retain inorganic salts. Bio Gel P2 is a poly-acrylamide gel supplied by Bio Rad, ~2nd and Griffen Avenue, Richmond, Ca 94~04, U.S.A.). ~I-e active desalted material is then concentrated, mixed with ethanol and further chromatographed on a cellulose column using butanol ethanol~water 4~1~5 v¦v top phase, as solvent.
Fractions containing material which inhibit Escherichia coli ~s'lactamase are bulked, evaporated to dryness under vacuum, redissolved in water and freeze dried to give a salt of clavulanic acid as a white solid.

The methods we have found most useful in detecting clavulanic acid in culture filtrates are paper chromatography and a bioautographic detection ~ystem. Clavulanic acid may be assayed by making use of its ~ -lactamase inhibiting activity. Thin layer chromatography may be used to detect cla-vulanic acid in solid preparations. These detection and assay procedures are described hereinafter.
A variation of the process for the preparation of a pure form of clavulanic acid or its salts comprises isolating an impure form of clavulanic acid or salt thereof, forming an ester of clavulanic in conventional manner, purifying the ester and thereafter regenerating clavulanic acid or a salt thereof from the ester.
The impure clavulanic acid or its salts used in this process will normally contain at least 1% by weight of the antibiotic.
Suitable esters for use in this process include those which may be cleaved by hydrogenolysis, enzymatic methods or by hydrolys~s under very mlld conditions as for example the esters of formula (IX).
~ ne suitable group of esters used in this process is that of the formula (X):

o 5 2 I~Y

N ~ ~X) \C0 - O - Cll - A7 wherein A7 is a hydrogen atom or an optionally substituted phenyl group and A8 is an optionally substituted phenyl group.
Most suitably A7 is a hydrogen atom or a phenyl, tolyl, chloro-phenyl, methoxyphenyl or nitrophenyl group and A8 is a phenyl, tolyl, chlorophenyl, methoxyphenyl or nitrophenyl group.
Preferably A7 is a hydrogen atom and A8 is a phenyl group.

~0743Z~

The esters of formula (X) may be cleaved by hydrogenolysis to yield clavulanic acid or a salt tl-ereof.
The esters of formulae (IX) and (X) may be grouped under the formula ~\~
~02X

wherein X is Rl as already defined or -CH-A7 as already defined.

Other groups of esters which may be used in the process include those of formula (V) and (VI) as hereinbefore described. Such esters may be converted to salts of claw lanic acid by mild alkaline hydrolysis, for example, at p~l 7.5.
The impure form of clavulanlc acid or salt thereof which is to be purified in t1tis process may be in the form of a solid or solution whlch wlll usually also contaln considerable quantities of organic or in-organic impurities.
The clavulanic acid or salt thereof may be con~erted into an ester by the esterification reactions referred to hereinaftPr. The preferred method of forming the required ester of clavulanic acid ls by the reaction of a salt of clavulanic acid with an esterifying agent such as a reactive halide, sulphonate ester or the like as hereinafter described. Such reactions are frequently carried out in an organic solvent of high dielectric constant such as dimethylformamide, dimethylformamide/acetone, dimethylsulphoxide, N-methylacetamide, hexamethylphosphoramide and thP like.
If desired the salt of claw lanic acid may be dissolved in the solvent in conventional manner or it may be bound to a polymeric support.
Suitable supports for use in this process include strong base anion exchange ~074325 resins, especially those possessing a macroreticular nature which permits the use of non-aqueous solvent systems. ~le have found Amberlyst ~26 to be suitable for this purpose. The clavulanic acid salt may be adsorbed onto t~e resin from the culture filtrate and the resin then suspended in dimethyl-formamide containing sodium iodide or alternatively eluted columnwise with a solution of sodium iodide in dimethylformamide or in a mi~cture of dimethyl-formamide and acetone.
Once formed, the impure ester of clavulanic acid is normally purified chromatographically. In such procedures the ester is normally diss~lved in an organic solvent sueh as ethylacetate, methylene chloride, ehloroform, cyelohexane or similar solvents. The solid phase uscd in the ehromatographic proeess is normally an inert material such as silica gel or ehromatographically similar materials.
The fractions emerging from the eolumn may be tested for the ! presence of the ela w lanie aeid by making use of its synergistic properties.
Active fractions are normally eombined and the organic solvent evaporated o~f under reduced pre~sure.
The ester resulting from this process is generally of aeceytable purity, but the material may be reehromatographed if desired.
This purified ester of clavulanie acid may be converted to elavulanie aeid or a salt thereof by the before mentioned methods.
A particularly suitable method of obtalnlng clavulanic aeid or its salt is by hydrogenation of a compound of the formula (X) as llerein-before dcscribcd SUC~I reactlons normal]y take placc in tlle presence of a tr~nsition metal catalyst using low or ~edium pressures of hydrogen. The reaction may be earried out at high, ambient or depressed temperatures, for example at O-100C. Particularly suitable reaction conditions for such hydrogenations will use a slightly superatmospheric pressure of hydrogen at an approximately ambient (12-20C) temperature. The reaction may be carried out in conventional solvents such as lower alkanols, for example, ethanol.

* Tradcmarlc ~0743ZS

We have found that a particularly suitable catalyst is palladium on charcoal.
If the hydrogenation is carried out in the presence of a , base then a sa]t of clavulanic acid is produced, for example, tht sodium or - potassium salts result if the reaction is carried out in the presence of sodium or potassium hydrogen car~onate.
The cla w lanic acid or salt thereof resulting from such reactions is generally of good purity.
Esters or claw lanic acid may be prepared by the esterifi-cation of clavulanic acid or a salt thereof by conventional methods.
Suitable methods of ester formation include (a) reaction of a salt of the acid of clavulanic acid with a compound of t]~e formula Q - R
where Q is a readily displaceable group and R is an organic group; (b) the reaction of clavulanic acid with a diazoalkane and (c) the reaction of clavulanic acid with an alcohol ROH in the presence of a condensatio promoting agent such as carbodilmide or the like.
Sultab]e salts of clavulanic acid which may be reactcd with ~, , coml70llnds U - ~ include alkali metal salts such as the sodium or potassium salts or other conventional salts such as the silver salt.
Suitable groups O~ include those atoms or groups known to ~e displaceable by carboxylate anions and include chlorine, brom-;ne and iodine atoms, sulphonic acid esters such as the O.S02CH3 or O.SO2C6}14CH3 groups, activc cster group.s such as the O.CO.II or O.CO.CF3 group and othcr con-ventional groups displaceable by nucleoplliles.
The preceding reaction ;is norma]ly carried out in an orgar~:ic solvent of relatively high dielectric constant such as dimethylformamide, acetone, dioxane, tetrahydrofuran or the like and at a non-extreme temperature such as -5C to 100C, more usually ~5C to 30C, for example at ambient temperature The reaction of clavulanic acid with a diazocompound is a mild method of making alkyl, aralkyl or similar esters. The diazotization reaction may be performed under conventional reaction conditions, for example at a non-extreme temperature and in a conventional solvent. Such reactions are nonnally carried out at between about -5C and 100C, more usually from SaC to 30C, for example at ambient temperature. Suitable solvents for this reaction include lower alkanols such as methanol and ethanol and solvents such as tetrahydrofuran, dioxane and the li~e. Ethanol has proved a parti-cularly useful solvent for this reaction.
The reaction of clavulanic acid with an alcohol or thiol in the presence of a condensation promoting agent will normally take place in an inert organic solvent of relatively high dielectric constant such as acetonitrile. This reaction is usually carried out at an ambient or depressed temperature, for example at -10C to +22C, more usually -5C to +18C, for example initially at 0C and thereafter gradually warming to about 15C.
The condensation promoting agent used is normally one which removes water from the reaction mixture. Suitable agents include carbodiimides, carbodi-imidazoles or equivalent reagents. Dicyclohexylcarbodiimide has proved to De a particularly suitable condensation promoting agent for use in this proc~s. In order to minimise self condensation of the claw lanic acid, tllls reaction is usually carrled out in the presence of a considerable excess of the alcohol or thiol.
Other suitable methods of ester formation include (d) removal of the elements of carbon dioxide from a compound of the formula (XI) (XI ) ~Co-o-co-o-R4 wherein R is an inert organic group; and (e) reaction of a compound of the formula (XI) with an alcohol ~OH (or less favourably with a thiol RSH).

10~43Z5 The elements of carbon dioxide ~ay be removed from the compound of formula (Xl) spontaneously during its preparation or alternatively by heating the compound of the formula (XI) in an inert solvent. Suitable ~ rah~ rOf~ran inert solvents include ether solvents such as diethylether, tctrahyarofcron, dioxanc and the like. In many cases the compound of the formula (XI) de-composes spontaneously eYen at a depressed temperature, for example, at -5C, to yield an ester of the formula 1 ~ f ~ 4 ~o2R
wherein R4 is an inert group ~ithin the definition of R.
~Ihen the compound of the formula (XI) is to be reacted with an alcohol (or less favourably with a thiol) then this reaction is normally carried out in an inert solvent such as an ether solvent in the presence of an excess of the alcohol (or thiol) in order to prevent self-condensation of tlle clavulanic acid derivative.
Such methods of e~terification are not in general as useful a~ thosc involving reaction of a salt of clavulanic acid with R-~ as herein-before described.
The compound of the formula (XI) may be prepared by the reaction of a salt of clavulanic acid with Cl.CO.O.R4 or the chemical equi-valent thereof. Normally this reaction is carried out at a depressed temperature, for example, at a temperature not greater than 5C, and in an inert solvcnt, for example diethylether, tetrahydrofuran, dioxane and the like. Most suitably the salt of clavulanic acid used in this reaction is a lipophilic salt so that it will dissolve in the solvent although if desired the less favourable sodium salt may be employed by suspending it in the reaction medium.

ASS~Y SUITABLE FOR DETECTION OF Cl.AVVLANIC ~CID
_ _ , . . . . . . . . .

Solutions containing clavulanic acid (culture filtrate, samples from isolation procedure and the li~e) are incubated for 15 minutes with a ~-lactamase preparation in 0.05M phosphate ~uffer at pH 7 and 37 C.
During this time, enzyme 1nhibition or inactivation occurs. Substrate (benzylpenicillin) is then added and incubation continued for 30 minutes at 37 C. The amount of enzymic degradation of the substrate to penicilloic acid ls determined by ~he hydroxylamine assay for penicillin. The amount of p-lactamase used is sucll as to give 75% llydrolysis of tlle ~enzylpenici11i in 30 minutes at 37C.
The extent of hydrolysis is a reflection of the amount of enzyme remaining uninhibited. The results are expressed as per cent inhi~ltion of the enzyme activity by a given dilution of the clavulanic acid -contalning solution (e.g. cultuxe filtrate) or the concentration of clavulanic acid ~lg/ml) giving 50X inhibition of the enzyme under the above stated conditlons (I50).
~-lactamase Enzyme The ~-lactamase produced by Escherichia coli JT4 is used as an enzyme. This culture is an ampicillin resistant strain and owes its resi.stance to the production of an R-factor controlled ~-lactamase. Other similar R-f~ctor controlled l-lactamases may be used if desired.
Thc culture maintained on nutrient agar s3Opes, is i~loculated into 400 ml. of sterile Tryptone medium contained in a 2 ~iter conical flask.
This medium has the following composition Tryptone (Oxoid) 32 g~1, yeast extract (Oxoid) 20 g/l, ~aC1 5 g~1 and CaC~2~H2O 2.2 g/l. The final pH was adjusted to 7.4 with dilute NaOH. The flas~ is shaken at 25C for 20 hours on a rotary shaler at 240 r.p.m.

The bacterial cells are collected by centrifugation, washed with 0.05M phosphate buffer pH 7 (resuspended and centrifuged) and resuspended in water to give cell concentration 25 times that in the cultivation medium.
This cell suspension was then disrupted in a MSE ultrasonic disintegrator at 4 C. The cell debris was removed by centrifugation and aliquots of the supernatant stored deep frozen. For use in the assay procedure, the super-natant is diluted in 0.005~ phosphate buffer until it gives about 75% hydro-lysis of a 1 mg/ml. solution of benzylpenicillin in 30 minutes at 37C.
Assay Procedure Sùitable dilutions of the inhibitor preparation and fl -lactamase solution are mixed and incubated at 37C for 15 minutes (Test). A control with buffer in place of inhibitor preparation is also incubated. Benzylpenicillin solution (substrate) is then added to test and control mixtures, incubation continued for a further 30 minutes at 37C. The residual benzylpenicillin in each mixture is then estimated using the hydroxylamine assay as described by Batchelor et al, Proc. ~oy. Soc., B 154, 498 (1961). 6 ml, of hydroxylamine rea~ent are added to all tests, controls and blanks and are allowed to react ~or 10 minutes at room temperature prior to the addition of 2 ml. of ferric ammonium sulphate reagent. The absorption of the final solutions is measured in an E.~.L. Colorimeter or a Spectrophotometer at 490 nm against the reagent blank The composition of the reactions, tests and blanks prior to the hydroxylamine assay are as follows:

Components TestBenzyl- ~eagent (all dissolved in or diluted penicill~nControl Blan~
bitfhf OjO05M p~ 7 phosphate Blank ml. ml.

Escherichia coli ~ -lactamase solution l.g 0.0 1.9 1.9 Tnh~bitor solution 0.1 0.0 0.0 0.0 Benzylpenicillin 5mg/ml. 0.5 0.5 0.5 0.0 0.005M pH 7 phosphate buffer 0.0 2.0 0.1 0.6 ~074325 Calculation of Rcsults The percentage inhibition of the ~'lacta~ase is calculated as fo~]ows -~bsorption of benzylpenicillin blank minus absorption of control (uninhibited reaction) e x ~bsorption of test (inhibited reaction) minus absorption of control (uninhibited reaction) = y % inhibition ~ - x 100 To obtai.n the I50 value, the inhibitor preparation is diluted until 50%
inhibition of the ~-lactamasc inactivation of benzylpenicillin is obtained in the above procedure.

PAPER CIIRO~ATOGRAP~IIC DI:TECTION OF CL~VULANIC ACID
Culture filtrate and a reference solution of clavulanic acid (250Jug/ml partially purified preparation), are spotted (20 ~l/origin) onto Whatman No. 1 paper strips 1 cm. wide. The chromatograms are run by de~cendlng cllromatogr~phy for 16 hours at 5C using n-butanol/isopropanoll water, 7/7/6 v/v as solvent. The strips are dried at 40C and laid on agar plates containing 6Jug/ml benzylpenicillin and seeded with a/~--lactamase producing strain of Klebsiella aerogenes (synergism syste~). The pl~tes are incubated oyernight at 30C and clavulanic acid revealed as a zone of inhibited growth. The Rf value of the zone was 0.46. Tl-e 6)lg/ml benzyl~
penicillin alone is below the concentration required to kill the ~lebsiella aerogenes but in the presence of a /Llactamase inhibitor, this concentration becomes toxic, that is to say there is synergism.
Use of tlle above synergism system enables cla~llanic acid to be detected at concentrations below those at which it shows antibacterial activity.

_ 27 -T~IIN LAYER CUROMATOGRAPHIC DETECTION OF CLAVULANIC ACID SODIUM SALT
Solutions of clavulanic acid sodium salt preparations are spotted (5 ~l of lmg/ml) onto glass plates coated with a 0.25 mm layer of silica gcl (F254) as supplied by E. Merck, Darmstadt, Germany. Tlle chromatograms are run at 22C using the top phase of the mixture n-butanol/ethanol/water 4/1/5 v/v. The chromatogram plates are dried at 40C and clavulanic acid sodium salt located by bioautography on agar plates containing 6)ug/ml.
benzylpenicillin and seeded with Klebsiella aerogenes (synergism system -see section on paper chromatography above). The agar surface is covered by a ~ine filter cloth before laying the TLC plate onto it. After allowing 15-30 minutes for wetting and diffusion, the TLC plate is lifted off with the aid of the filter cloth and the agar plate incubated overnight at 30C
to reveal the zones of inhibited growtll. The Rf value of clavulanic acid sodium salt in the above solvent is approximately 0.37. Two spray reagents, s Rhrlich and tripllenyltetrazolium chloride are also used to reveal the clavulanic acid sodium salt zone. The former reagent conslsts of 300 mg o~ p dlmethylnminobenzaldehyde dissolved in 9 ml. of ethyl alcohol, 54 ml.
o~ n-butanol and 9 ml of concentrated ~ICl. On heating t1,e sprayed TLC plate at 120C for 1-2 minutes, clavulanic acid sodium salt appears as a pink spot.
The triphenyltetrazolium chloride reagent consists of a mixture of 1 volume of a 4% solution of tllis compound in metl2anol with 1 volume o~ metllanolic sodium hydroxide. After spraying, the TLC ylates are heated at 80C.
Clavulanic acid sodium salt appears as a red spot on a white bac~ground.

CULTIyATION OF STREPTOMYCES CL~VULIGERUS
Streptomyces clavuligerus was cultivated at 26C on agar slopes containing 1% Yeatex (yeast extract), 1% glucose and 2~o Oxoid agar No. 3, pH 6.8. A
sterile loop was used to transfer mycelium and spores from the slop into 100 m:L of a liquid medium in a 500 ml Ellrlenmeyer ilask.

The liquid medium had the following composition:-nxoid Malt Extract 10 g/l ; Oxoid Bacterlological Peptone 10 g/l Glycerol 20 g/l Tap water 1 liter The medium was adjusted to pH 7.0 with sodium hydroxide solution and 100 ml. volumes dispensed into flasks which were closed with foam plugs prior to autoclaving at 15 lb/sq.in. for 20 minutes. An inoculated seed flask was shaken for 3 days at 26C on a rotary shaker with 2 inch throw and a speed of 240 r.p.m. Production stage flasks containing the liquid medium descr:lbed above were inoculated with 5~ vegetative inoculum and grown under tlle same conditions as the seed flask. Samples of culture filtrate were assayed for inhibitor action against the~-lactamase of Escherichia coli JT4, Optimum activity was obtained after 3 days. The results are shown in Table 1. A zone of clavulanic acid at Rf 0.46 was seen wllen the cul~ure filtrate was examined by the paper chromatographic method previously described.
The increase in size of the zone paralleled the increase in the ~ lactamase lnhibltor assay.
Stre~tomyces clavuligerus was also cultivated in 2 litre shaken flasks containing 400 mls. of medium (Production stage) using the same medium and cultural conditions as described earlier in this Example. fn these larger vessels, growth of the organism was slower and optimumjJ!lactamase inhibitory activi.ty was achieved 7-9 days afterinoculation with the vegetative seed.
The results are also shown in Table 1.

* Trademark ' TABLE 1 /~-Lactamase Inhibiting Actlvity of Streptomyces clavuligerus Grown -in S00 ml. and 2000 ml. Flasks _, , , ,, , _ , ,, . , _ . . _ .. .. _ .. . _ % Inhibition of Escherichia coli /~-lactamase at a final dilution of Fermentation 1/2500 of culture filtrate Time (Days) 500 ml. Shaken Flask 2G00 ml. Shaken Flask . .

3 55

4 50 10 7 _ 51 8 _ 53 9 _ _ E _ CULTIVATION OF S~'REPTOMYCES CLAVULIGERUS
A seed flask prepared as in E~ample 1 was used to inoculate 500 ml. conical flasks containing 100 ml. aliquots of the following rnediun in deionised water:-Soluble Starch 2% w/v Glycerol 0.3~ w/v Scotasol 0.1% w/v Arkasoy 1% w/v Feso4~7}~2o 0,01% w/v The medium was sterilized by autoclaving at 15 p.s.i. for 20 minutes and inoculated by the addition of the 5% vegetative seed stage.
The flasks were shaken at 26C on a rotary shakcr as in ~xample 1, * Trademark Optimum titre of clavulanic acid was achieved between 3-5 days. A dilution of 1/2500 of the culture filtrate gave 60% inhibition in the ~ -lactamase inhibition assay. A zone of clavulanic acid was seen at Rf 0.46 when using the paper chromatograpl,ic (bioautographic) method previously described.
This zone increased in size in parallel with the increase of the activity in the ~ -lactamase inhibitor assay.
[Soluble starch supplied by British Drug Houses Ltd., Poole, U.K.;
Scotasol i5 dried distillers solubles supplied by Thomas Borthwich Ltd., 60 Wellington Street, Glasgow, U.K.;
Arkasoy is soya bean flour supplied by British Arkady Co., Old Trafford, Manchester, U.K.].

CULTIVATION OF STREPTOMYCES CLA W LIGERUS
A seed flask as produced in Example 1 was used to inoculate 500 ml.
conical flasks containing 100 ml,aliquots of the following medium prepared in deio~s~d water and sterilised as previously described. The inoculum level was 5~.
Dextrin 2% w/v Arkasoy 1% w/v Scotasol0.1% w/v 4 20.01% w/v The inoculated flasks were shaken at 26C. Optimum ~ -lactamase inhibitory activity was achieved between 3-5 days. The activity was similar to that achieved in Example 2.
~Dextrin is supplied by C P C (UK) Ltd., Trafford Park, Manchester, U.K.]

*Trade Mark CULTIVATION OF STREPTOMYCES CLA W LIGER~S
The seed stage as described in Example 1 was used to inoculate 500 ml. conical flasks containing the following medium prepared in deionised water.
Dextrose 1% w/v Soyabean Meal1% w/v Scotasol0,05% w/v CaC03 1% w/v These flasks were treated exactly as in previous Examples and cultured under identical conditions. ~ -lactamase inhibitory activity was produced be-tween 3-5 days. Culture filtrate at a final dilution of 1/2500 gave 35-45% inhibition in the ~ -lactamase inhibition assay.

CVLTIVATION OF STREPTOMYCES CLAVVLIGERUS
~ -lactamase inhibitory activity attributable to clavulanic acid was produced u~ing the following medium with identical seed stage and cultivation conditions to Example 1.
Glycerol 2% w/v Soyabean Meal 1.5% w/v Mg SO4 0.1% w/v K2HPO4 0.1% w/v Medium prepared in deionised water ~ -lactamase inhibitory act~vity reached a maxim~m level between 3-5 days and was of a similar order to that produced in Example 4.
EXP~PLE 6 CULTIVATIO~ OF STREPTOMY OE S CLA W LIGERUS
The following medium produced clavulanic acid when using the con-ditions and vegetative seed inoculum as described in Example 1.

~)74325 Glucose 2%
Lab Lemco (Oxoid) 1%

Oxoid Yeast Extract 0.3%

CaC03 0-3%
Medium prepared in deionised water Optimum titres were achieved in 3-5 days and a 1/2500 dilution of the culture filtrate gave 35-45% inhibition in the ~ -lactamase enzyme in-hibition assay.

CULTIVATION OF STREPTOMYCES CLAVULIGERUS
As in Examples 4, 5 and 6 the following medium produced 35-45%
inhibition (1/2500 dilution) in the ~ -lactamase assay at the optimum titre which is reached 3-5 days after inoculation. All conditions were as pre-viously described.
Glucose2% w/v Arkasoy1% w/v CaC030.02% w/v CoC12.6 20.0001% w/v Medlum prepared in deionised water EXAMPLE 8C~LTIVATION OF STREPTOMYCES CLAVULICER~S
The ~ollowing production stage medium when used under standard cul-tivation conditions as described in previous Examples produced 20-30% inhi-bition at 1/2500 dilution in the ~ -lactamase assay between 3-5 days after inoculation. Using the paper chromatographic method previously described, a zone of clavulanic acid was seen at Rf 0.46 when culture filtrate was examined.

Scotasol 2%
Oxoid Yeast Extract 1 Medium prepared in tap water Final pH 7.0 EX~MPLE 9 CUJTIVATION OF STR~PTOMYCES C~AVULIG~RUS
Undcr standard cultlvation conditions, the following medium produced clavulanic acid 3-5 days after inoculation with the vegetative seed.
A 1/2500 dilution of the culture gave 20-30% inhibition in the ~-lactamase inhibition assay.

g/l Glycerol 15 Sucrose ~0 Proline 2.5 Monosodium Glutamate 1.5 NaC1 5.0 K2HP04 2.0 CaC12 0 4 24H2 O.1 36~l20 0.1 ænC12 0 05 MgS47H2 1 . 0 Medium prepared in deionised water Final pH 7.1 CULTIVATION OF STREPTOMYCES CLAVULIGERUS
A stoclc Yeatex /glucose agar slope was used to inocu~ate a Yeatex /glucose agar slope in a Roux bottle by ma~ing a myceliumlspore suspension in sterile water. The Roux bottle slop was incubated at 26 C
for lU days. To this slope 100 mls. of sterile water was added and a mycelial suspension prepared. This was used to inoculate 50 litre of steam sterilised seed medium of the following composition in tap water.

Oxoid Malt Extract 1% w/v Oxoid Bacteriological Peptone 1~ wtv * Trademark ~07432S

Glycerol 1% w/v 10% Pluronic L81 Antifoam in Soyabean Oil 0.05% w/v [Pluronic supplied by Jacobs and Van den Berg UK Ltd., 231 The Vale, London, W3 containing a poly-propylene-polyethylene block polymer, and Soyabean Oil supplied by British Oil and Cake Mills Ltd., Stoneferry Road, Hull, U.K.3. Trade Mark The medium was contained in a 90 litre stainless steel baffled fer-menter, agitated by a 5`' vaned disc impeller at 240 r.p.m. Sterile air was supplied at 50 /min and the tank incubated at 26C.
After 72 hours, the seed fermenter was used to inoculate 150 litre of the same medium using a 5% v/v addition by sterile transfer. This production stage medium was contained in a 300 L stainless steel, fully baffled fermenter agitated by a 8'~" vaned disc impeller at 210 r.p.m.
Sterile air was supplied at 150 l/min. The fermentation was maintained at 26 C. ~nti~oam was added when required in 10 ml. shots (10% Pluronic L81 in soyabean oil). Samples were removed for ~ -lactamase in~)ibition assay at re~ular intervals. The fermenter was harvested between 4-5 days at the optimum level o~ ~ -lactamase inhibitory activity (Table 2).

~ -35-~Lactamase Inhibitory Activity of Samples of Culture Filtrate taken from a 300 litre Fermentation of Str~ptomyces Clavuligerus Fermentation% Inhibition in ~ -lactamase Time Inhibition Assay at a Final (days) Dilution of 1/2500 1.0 12 1.5 20 2.0 ~1 2.5 36 3.0 50 3.5 54 4.0 51 4.5 56

5.0 SS

-35a-CULTIVATION OF STREPTOMXCES CL~VULIGERUS
.
The seed fermenter was run exactly as described in Example 10 using the same medium.
Afer 72 hours, the seed fermenter was used to give a 5% v/v vegetative inoculum into a 300 litre stainless steel fully baffled farmenter containing 150 litre of steam sterilised medium agitated by an 8~ inch vaned disc impeller at 210 r.p.m. Sterile air was supplied at 150 1/min. The fermentation was maintained at 26C. Antifoam was added when required in 10 ml. shots (10% Pluronic L81 in soya bean oil).

The medium used in the pr~duction stage was as described in Example 3 with the addition of 0.05% v/v of 10% Pluronic L81/soyabean oil antifoam prior to sterilisation.
The ~-lactamase inhibitory activity of fermen~ation samples was simllar to those of Example 10 (see Table 2). Paper chromatograpllic examination revealed a zone of clavulanic acid at Rf 0.46,using the bioauto-~rapllic (synergism) method previously described. The size of the clavulal1ic ~ld zon~ increased in parallel with the increase in the ~-lactamase inhibitor assay .

CULTIVA'rION OF STREPTYM~CES CLAVULIGERUS
100 mls of sterile water was added to a sporing culture which had been grown on Bennetts agar in a Roux bottle for 10 days at 26C. A
myceliumlspore suspension was produced and used to inoculate 75 litres of steam sterllised medium of the following composition in tap water.
Dextrin 2% W/V
Arkasoy t 501 ~% ~/V

10% Pluronic L81 0.03% V/V
in soybean oil The pH oE the medium was adjusted to 7.0 The medium was contained in a 100 litre stainless steel baffled fermenter, agitated by a 7'z" vaned disc impeller at 140 rpm. Sterile air was eupplied at 75 l/minute and the tank incubated for 72 hours at 26 C.
The contents of the seed fermenter were used to inoculate 1500 litres of steam sterilised medium of the following composition in tap water.
Arkasoy '50' 1.5% W/V
Glycerol 1.0% W/V

KH2P4 O. 1% W/V .
10% Pluronic L81 0.2% V/V
in soyabean oil The pH of the medium was adjusted to 7.0 The medium was contained in a 2000 litre stainless steel fully baffled fermenter agitated by two 19" vaned disc impellers at 106 r.p.m.
Sterile air was supplied at 1200 litres per minute. Antifoam was adde,d in 25 ml amounts as required. (10% Pluronic L81 in soyabean oil).
The fermentation was control,led at 26C until a maximum yield of cla~ulanic acid w~ obtained between 3 - 5 days when 200 - 300~ug/ml of clavulanic acid were p~oduced.

.
CULTIVATION OF STREPTOMYCES CLAVULIGERUS
Inoculum was produced in a seed flask as previously described, but using the medium described in Example 3 (with pll of the medium acljusted to 7.0). This was used to inoculate 500 ml conical flasks containing 100 ml aliquots of the following medium prepared in deionised wa~er and sterilised.
The inoculum level was 5%.
Prichem P224 1% W/V
Arkasoy '50~ 1.5% W/V
KH2P04 0.1% W/V

The p}l of t]~e medium was adjusted to 7.0 - :37 -10743;~5~

The inoculated flasks were shaken at 26C and optimum /~-]actamasc inhibitory activity was achieved betwcen 3 - 5 day;. Levcls of 30~ - 500 ~g/ml of claw lanic acid were achieved.
Prichem P224 is a triglyceride supplied by Prices Limited, Bromborough, Bebington, Wirral, Cheshire, U.K. Prichem P224 is based on oleic acid (65%), palmitic acid (11%) and other similar acids.
~XAMPLE 14 (~L~ L~/JIC
R ISOLATION OF CRUDE CL~UL~7IC ~CID SODIUM SALT

~larvested culture liquor produced as described in Example 10 was clarified by continuous flow centrifugation and the mycelium discarded.

lrom lSO lltre of fermellta~ion liquor 120 litre of clarified culture fluid was obtained. This filtrate gave 58% inhibition in the ~-lactamase ir,hibition assay at 1/2500. The filtrate was chilled to 5C and 40 litre of n-butanol added. The mixture was stirred and 25% H2S04 added until the p~ was 2Ø
The acidified mixture was stirred for a further lO mins. before separating the phase~ by centrifugatlon. The aqueous phase was discarded. To the n-butanol extract 0.5% o Norit GSX carbon was added and the mixture stirred for lS mlnu~e~. The cart~on was discarded aft~r removal by filtration using a dia~omaceus earth as a filter aid. To the n-butanol a ~ volume of deionised water was added and the mixture stirred while adding 20% NaO}I solution until the pH had equilibated at 7Ø The phases were separated by c~ntrift~gation and the n-butanol phase discarded. The aqueous phase was concentrated under reduced vacuum to 800 ml. and tllen freeze dried This yielded 35g. of a crude solid preparation of clavulanic acid ~it~ an I50 of ] 3 ~gtml in tll~
~-lactamase inhibition assay. This solid preparation was stored dry at -20 C

while awaiting further purification.

ISOLATION OF CRUDE CLA W LANIC ~CID SODIUM SALT

One litre of culture filtrate giving 53~ inhibition at 1/2500 in the ~-lactamase inhibition assay and obtained as described in Example 12 * Trademark - 3~ -was percolated down a 1 inch diameter x 6 inch column of Permutit Isopore resin FF lP (SRA 62) in the Cl form [supplied by Permutit Co. Ltd., 632-652 London Road, Isleworth, Middlesex, U.K.]. The culture filtrate was followed by 300 ml. of distilled water to wash the column. Elution of the active ~ -lactamase inhibitor was achieved with 0.2M NaCl solution. Fractions t20 ml-) were collected and assayed at a 1/2500 final dilution in the ~ -lactamase inhibition assay. Active fractions were combined and concen-trated under vacuum to 20 ml. This solution was desalted by gel exclusion chromatography on a Biorad Biogel P2 column 1~ inches in diameter with a gel bed of 16 inches and eluted with 1% n-butanol in water. [Biogel P2 is supplied by Bio Rad J,aboratories, 32nd and Griffin Ave., Richmond, California, U.S.A.]. The active fractions, as determiend by the ~ -lacta-mase inhibition assay, were combined. Sodium chloride eluted after clavu-lanic acid and was detected using silver nitrate solution. The combined active fractions were concentrated and freeze dried.
One litre of culture filtrate after the above treatment yielded 0-45 8- of a crude solid preparation of clavulanic acid having an I50 f 0.92 ~g/ml.
This solid was stored at -20C while awaiting further purification.

ISOLATION OF C~UDE CLAVULANIC SODIUM SALT
Culture filtrate containing 300 ug/ml of clau~anic aid is acidified using an in-line mixer system, extracted with n-butanol and clavu-lanic acid is back extracted into water at neutral pH.
Chilled culture filtrate (5 - 10C) was pumped to an in-line mixer at the inlet of which, enough 6% (v/v) nitric acid was added tD maintain an outlet pH of 2.0+ 0.1. The acidified filtrate was passed at 4.1Jmin.
through a glycol cooled plate heat exchanger (A.P.V. Ltd.) to maintain a temperature between 2 - 5 . The pH was monitored in a flow cell before *Trade Mark passing into a three stage counter current separator (l~cstfalia Separator Ltd., Model EG 1006).
Chilled water saturated n-butanol (at about 5C) was pumped at 3 l/min into the counter current separator.
The aqueous outlet fro~ the counter current separator was run to waste. Entrained water was removed from the butanol outflow of the counter current separator using a liquid/liquid centrifugal separator.
~Alfa Laval Ltd. Model 3024X - G). The butanol was collected in a stainless steel vessel fitted with a cooling jacket jn which it was stored at about 5 C.
From the vessel, 40 1 aliquots were removed and thoroughly mlxed with 2 1 of chilled water (5C), saturated with n-butanol. The pll of this mixture was adjusted to pH 6.~ ~ 0.1 using 20~ sodium hydroxide solution.
This aqueous extract/butanol mixture was fed to a liquid/
liquid centrifugal separator (Sharples Centrifuge Ltd. Model M35PY - 5PH) at a pumped rate of 2 l/min.
From 1800 1 of culture filtrate, 90 1 of aqueous phase was recovered, containing 39~ of the clavulanic acid present in the culture ~ rate, 15 1 of the aqueous extract was adjusted from 2%, to 8%, total solids by the addition of 60 g sodium chloride per litre, and spray dried (Anhydro, Copenha~en, Type Lab S 1). The conditions used were: Feed rate 2 l/hr Atomizer voltage 170 v; Heater sctting 6 - 7; Inlet temp 150 C;
Outlet temp 80C.
Tl~e dried product, total wcight 1 kg , contained 62% of the c.lavulallic aci(l prcscnt ;n tl-c fcedstock The remaining 75 1 of aqueous extract was concen~rated by ultrafiltration (De Danske Sukkerfabrikker. Laboratory Module, ~embrane Type 900). The operating procedure was to re-circulate the retentate from a stainless steel tank, ~itted with a cooling system, witn the outlet valve set so as to give a differentlal pressure across the 40 mcmb~aIIes of .

25 atmospheres. The temperature was maintained at 2 - 5 C and the pH at

6.8+ 0.1 by addition of 2N hydrochloric acid, as necessary. The volume was reduced ~o 34 1 which contained 72% of the clavulanic acid present in the feedstock.
The aqueous concentrate was st~red at about 5C, adjusted to 8%
solids, and spray dried as above. The dried material contained 75% of the clavulanic acid present in the feedstock to the spray drier.
The total spray dried product, from the 90 1 of aqueous extract contained 69.4 g of clavulanic acid which was 72% of the clavulanic acid in the spray drying feedstock and 21% of the clavulanic acid present in the 1800 1 of culture filtrate.
EXAMPJ,E 17 PARTIAL PURIFICATION OF CRUDE CLAW LANIC ACID
Crude clavulanic acid preparations obtained as described in Example 15 were purified by ion exchange chromatography. Eighteen grams of material prepared as described in Example 15 having an I50 value of 1.3 yg/ml ~{inal concentration) were dissolved in 25 ml. of distilled water and applied to a 1~," x 16" bcd of Permutit FF lP (SRA 62) resin in the chloride form.
The column was eluted with a sodium chloride gradient formed by gravity feeding 0.5M sodium chloride into a mixing reservoir containing 1 litre of distilled water which in turn fed the chromatographic column. 10 ml. cuts were collected and ~ -lactamase inhibitory activity assayed using a 1/2500 dilution of the fractions. Activity was eluted after a main band of colour between fractions 24 and 30. The active fractions were combined and concen-trated to 30 ml.
This solution was desalted using a 2" x 18" bed of Biorad Biogel P2 and eluting with 1% n-butanol in water. The 20 ml. fractions were assayed for clavulanic acid content using the ~ -lactamase inhibition assay.
The fractions were also spotted onto paper strips and sprayed with either the Ehrlich or the triphenyltetrazolium spray reagents described in Description 3.

10743,'~

~ -lactamase inhibitory activity correlated with the pink or red spots res-pectively produced by these reagents. Active cuts were combined, excluding those containing sodium chloride and concentrated under vacuum to dryness.
This yielded 520 mg. of partially purified clavulanic acid sodium salt with an I50 of 0,2 ~g/ml in the standard ~ -lactamase inhibitor assay.
Thin layer chromatography (silica gel) of this clavulanic acid preparation gave the following Rf values: n-butanol/ethanol/water 4:1:5 v/v top phase Rf 0.37; n-butanol/acetic acid/water 12:3:5 v/v Rf 0,44; isopro-panol/water 7:3 v/v Rf 0.78, The zones were detected by spraying with Ehrlich's reagent. 6-Aminopenicillanic run as a marker and detected with the same spray had Rf values of 0.38; 0.39 and 0.77 respectively.

P~RTI~L PURIFICATIO~ OF CL~VVLANIC ACID SODIUM SALT
Culture filtrate produced as described in Example 12 was solvent extracted as in Example 14 to give a solid preparation wllich was further purified by ion exchange chromatography using ~1hatman diethylaminoethyl cellulose ~E S2. This solid (lOg) was dissolved in 20 ml. of distilled water and applied to a 1~2~ X 20" column of DE 52 cellulose previously equilibrated with O.OlM sodium phosphate buffer pH 7.5. The column was eluted with a NaCl gradient. O.lM NaCl in O.OlM sodium phosphate buffer pH 7.5 was fed into a mixing chamber containing 1 litre of O.OIM phosphate bufer pH 7.5 which in turn was connected to the column. Fractions (10 ml.) were collected and these were assayed for ~ -lactamase inhibitory activity at a dilution of 1/2500. The fractions were also examined for antibacterial activity by the hole-in-plate assay method using nutrient agar plates seeded with Klebsiella aerogenes. The fractions having the highest ~ -lactamase inhibitory activity and giving zones of inhibition in the hole-in-plate assay were combined, concentrated and then desalted on a Biorad Biogel P2 column. These fractions were shown to contain clavulanic acid by paper and thin layer chromatography.

ISOLATION OF SOLID CLAVULANIC ACID SODIUM SALT
A partially purified solid preparation of clavulanic acid (500 mg) prepared as in Example 17 was loaded onto a Whatman microcrystalline CC 31 cellulose column with 1" x 20" bed size. The chromatographic solvent was n-butanol/ethanol/water 4:1:5 v/v, top phase. The column was run at 4C
and 4 ml. fractions collected. Fractions were tested for the presence of calvulanic acid by spotting onto filter paper and spraying with the Ehrlich (pink spot) or triphenyltetrazolium (red spot) spray reagents. These spot tests were confirmed by ~ -lactamase inhibition assays at a 1/1250 dilution.
Active fractions were combined and dried under vacuum on a rotary evaporator.
The solid was dissolved in a small volume of distilled water and freeze dried. A white solid preparation of the sodium salt of clavulanic acid was obtained t40 mg) having an I50 of 0.08 ~ug/ml in the ~ -lactamase inhibition assay.

ISOI~ATION OF SOLID CLAVULANIC ACID SODIUM SALT
_ Concentrated back extract (6 1 ) (from ultrafiltration in Example 16) containing 10 g of clavulanic acid as determined by the ~ -lactamase inhibition assay of Description 1. This was percolated at 1 lthr onto a 2" x 24" column of Permutit Zerolit FF 1 P SRA 62 anion exchang/ resin in the chloride form. The column was then washed with 2 1 of deionized water prior to elution with a sodium chloride gradient. The gradient was formed by a reservoir containing 4 1 of 1.4 m NaCl feeding a stirred reservoir containing 4 1 of 0.7 NaCl which in turn was connected to a stirred reser-voir containir.g 4 l of deionized water which was connected via a pump to the column. The column was eluted at 2.5 ml/min and 25 ml fractions col-lected. Fractions were assayed by the ~ -lactamase inhibition assay.
Active fractions (nos. 140-230) were combined and vacuum evapor-ated to near dryness. Ethanol (500 mls) was then added and the solid filtered off after vigorous shaking. The ethanol extract was then vacuum evaporated to dryness on a rotary eyaporator and redissolved in deionized watcr (40 mls). This was loadcd onto a 4" x 24" column of Biorad l~;ogel P2 and eluted with a 1% n-butanol solution. Fractlons were collected (25 ml) and assayed for~s-lactamase inhibitory activity at a 1/2500 final dilution.
Tests for sodium chloride content on 1/25 dilutions of the fractions were made using silver nitrate solution. Those fractions containing clayulanic acid free of sodium chloride were combined, concentrated by evaporation of the solvent under reduced pressure to 20 mls and then freeze dried. This yielded 4.8 g of the sodium salt of clavulanic acid. (I50 about 0 06 ~ug/ml) EXA~LE 21 PREPARATION OF AN ESTER OF CLAVllLANIC ACID (METHYL ESTER) r C~O ~O>~CH~
CO2Na J~_N

19,8 mg. of the sodium salt of clawlanic acid was dissolved in 0.5 ml. dry dimethylformamide and treated with 0.25 ml. methyl iodide.
After standing at room temperature for 1,5 hours under anhydrous conditions, the solvents were removed in vacuo. The residue was purified by P.L.C. on silica gel (Kieselgel 60F254 supplied by E. Merck, Darmstadt, Germany) eluting with ethyl acetate to give clavulanic acid methyl ester as a colourless oil (Rf 0.38; red colour with triphenyltetrazolium chloride spray) which had the following properties:
Analysis: Found C 50.49 H 5,43 N 6,29 CgHllNC15 Requires C 50,70 H 5~20 N 6-57 ,~ max (Methanol): no absorption ~215 nm - max (Film): 3300 - 3600 (Broad), 1800, 1750, 1695 cm Approximate 1st order N.M.R. (CDC13): 2,49 ~broad S, 1, exchanged with D20), 3,05 ~0743Z~
(d, 1, J - 17.5 Hz), 3.54 (dd, 1, J = 17.5 Hz, J2 = 2.5 Hz), 3.84 (S, 3) 4,24 (d, 2, J = 7 Hz), 4.93 (dt, 1, J = 7 Hz, J2 ~ 1.5 Hz), 5.07 (d, 1, J - 1.5 Hz), 5.72 (d, 1, J = 2.5 Hz) ; Molecular weight (mass spectrum) : 213.0635.
Calculated for CgH11N05 : 213.0637 Thin layer chromatography of the methyl ester showed a single zone in each of the following solvent systems; butanol/ethanol/water 4:1:5 vtv top phase Rf 0.75; isopropanol/water, 7:3 v/v Rf 0.95; ethylacetate/ethyl-alcohol 8:2 v/v Rf 0.87. The zones were detected by bioautography using Klebsiella aerogenes with added benzylpenicillin (synergism system).

PREPARATION OF AN ESTER OF CLAYUL~NIC ACID (p-nitrobenzyl ester) ~ CH20H

( ~C2Na \ CO2CH2- ~ 2 Treatment of the sodium salt of claw lanic acid with p-nitrobenzyl bromide in dry DMF gave, after P.L.C., a colourless oil which crystallised from chloroform - ether to give to p-nitrobenzyl ester of clavulanic acid as white feathery needles, m.p. 111 - 112C, which on recrystall~sation had a m.p. of 117.5 - 11~C, PREPA~ATION OE AN ESTER OF CLA W LANIC ACI~ (BENZYL ESTER) CH~OH - CH2O~

~ 1 ¦ \E ~ ~ ~\H
N 02Na ~ 2 ~ 2Ph _ 45_ 10743'~S

Impure 3- ~-hydroxyethylidine)-7-oxo~4-oxa-1-azabicyclo ~3,2,0]heptane-2-carboxylic acid sodium salt (thougllt to be roughly 55 mg. of pure material) in dry dimethylformamide (0.64 ml.) was treated with benzyl bromide (0.18 ml.).
The solution was kept at room temperature (approx. 17-18C~ for 3 hours under anhydrous conditions. The reaction mixture was fractionated on silica gel, eluting with ethyl acetate, to give in substantially pure form the benzyl ester of 3- ~-hydroxyethylidine)-7-oxo-4-oxa-1-azabicyclo r3,20J heptane-2-carboxylic acid 63 mg.) as a colourless oil. i.r. (film) 1800, 1745, 1695 cm 1; n.m.r. (CDC13), 2.25 (s,l, exchangeable with D20), 3.05 (d,l,J=
17Hz), 3.51 (dd,1,J=17 l~z, J2=2.5 Hz), 4.24 (d,2,J=7.5Hz), 4.92 (dt,1,J=7.5Hz, J2=1.5Hz), 5.15 (d,1,J-1.51~z), 5.24 (s,2), 5.71 (d,l,J-2.5 ~z), 7.45 ~ (s,5).

PREPARATION OF THE BENZYL ESTER OF CL~VULANIC ACID FROM CRUDE EXTRACTS OF TIIE
FULTURE FILTRATE OF S.CLAVULIGERUS
Culture filtrate 20 ~ obtained as described in Example 10 was vacuum evaporated using a climbing film evaporator to 5 1. The con-centrate was then freeze dried using an Edwards E.F.6 shelf freeze drier manufactured by Edwards High Vacuum Ltd. The 300 g. of solid so obtained contained 3 g. of sodium Clavulanic acid as determiDed by the enzyme inhibition a5say. The solid was suspended in 900 ml. of dry dimethylformamide and 15Q ml. of benzyl bromide was added~ The mixture ~7as stirred for 2 hours at room temperature and then diluted with 1 1. of ethyl acetate. The reaction mixture was filtered and the filtrate concentrated to as low a volume as was possible. The oily residue was extracted with a further 1 1. of ethyl acetate and the extract filtered. The filtrate was again concentrated and the resulting oily residue loaded onto a 3" x 14" silica gel column (Biogel Biosil A 100 mesh) in cyclohexane. The column was eluted with cyclohexane to remove benzyl bromide and the solvent was then changed to ethyl acetate and 20 ml. fractions collected. Fractions were tested for the presence of the benzyl ester of clavulanic acid by spotting onto glass backed silica gel t,l,c, plates (Merck precoated silica gel 60 F 254) and spraying with 2,3,5-triphenyl-tetrazolium chloride (TTC) spray reagent, Fractions giving intense red spots with this reagent were further examined by t.l.c. on silica gel plates using chloroform-ethyl acetate 8:2 as the solvent and spraying the developed plates with TTC spray. The benzyl ester of clavulanic acid runs at Rf 0,31 at 22C, ~ractions containing this ester were combined and concentrated to 15 ml. and this solution was further chromatographed on a 1~" x 16" silica gel column (Merck silica gel H, type 60) with chloroform/ethyl acetate 8:2 as the solvent. 15 ml, fractions were collected and tested for the benzyl ester as described above, Those fractions containing the ester were concentrated to 8 ml. and finally purified by column chromatography on a 1" x 16" silica gel column (Merck silica gel H, type 60) with ethyl acetate cyclohexane 8:2- as the solvent.
Selected fractions were combined and vacuum evaporated to give pure benzyl ester as an oil, 160 mg.

PREPARATION OF CLAVULANIC ACID EENZYL ESTER
Spray dried solid (3,3 tcg) containing 69.4 g of clavulanic acid as determined by enzyme inhibition assay was obtained as described in Example 16. The solid was slurried in 5.5 1. of dimethylformamide and 500 mls.
of benzyl bromide added, After stirring at room temperature for 2 hours, 12 1. of ethyl acetate were added and the solids removed by filtration, The filtrate was vacuum evaporated to an oily residue (212 g), The residue was loaded onto a column containing a 4" x 13" bed of silica gel tHopkins &
Williams MFC) in cyclohexane, The column was eluted with 12 1. of cyclohexane to remove excess benzyl bromide. The-eluent was then changed to ethyl acetate and 500 ml, fractions collected. These were tested for benzyl clavulanate content by spotting onto silica gel t 1 c plates (Merck precoated silica gel 60 F 254) and spraying with 2,3,5 triphenyltetrazolium chloride (TTC) spray reagent. Fractions giving intense red spots were further examined by t 1 c 107~325 on silica gel with chloroformlethyl acetate 8:2 as the solvent and spraying the developed plates with T T C spray. ~ractions 5-13 contained the bulk of : the ester, and these were combined and vacuum concentrated to an oil (79.3 g).
This preparation was then chromatographed on a 4" x 18" column of silica gel (Merck silica gel H type 60) with chloroform/ethyl acetate 8:2 as the solvent.
Fractions were selected as described above and yielded on concentration 45.9g. of oil which was of 62% purity as adjudged by NMR spectroscopy.
This product was finally chromatographed on a 2 3/4" x 18"
column of Sephadex LH 20 in cyclohexane/chloroform 1:1. After selection of fractions and concentration a colourless oil (27.6 g) was obtained which proved to be 95% pure benzyl ester of clavulanic acid as determined by NMR
spectroscopic examination (Sephadex (Trade Mark) LH20 is a hydroxypropyl derivative of Sephadex Q25 supplied by Pharmacia Great Britain, 75 Uxbridge Road, London W5, U.K.).

PREPARATION OF CLAVULANIC ACID BENZYL ESTER
a Culture filtrate (150 1) pH 7.0 contained 16.2 g. of clavulanic acid (sodium salt) as determined by the enzyme inhibition assay. This r filtrate was stirred with 5 kg. of Amberlyst A.26 anion exchange resin in the chloride form (Rohm & Haas Company, Philadelphia, USA) for 1 hour at room temperature. The resin was then filtered and the filtrate reassayed, showing that 6.4 g of clavulanic acid had been removed. The resin was washed with 20 l. of deionised water followed by 20 1. of acetone and 10 l. of dimethyl formamide (DMF~. After refiltering the resin was suspended in 2.3 1. of DMF/0.2 M NaI. To this was added 200 mls. of benzyl bromide and the suspension stirred thoroughly. After standing at room temperature for 16 hours, ethyl acetate (2 1~ was added, and the resin then filtered, further washings (Ethyl acetate) of the resin were combined with the filtrate. The extract was then concentrated to a small volume and chromatographed on 3" x 18" silica gel column (Merck silica gel H type 60) with ethyl a~etate/cyclohexane 8:2 as the solvent. Fractions containing benzyl clavulanate were selected by spotting onto silica gel t 1 c plates and spraying with TTC reagent as described previously (Example 24). Those selected were concentrated to 20 mls and then chromatographed on a 1~" x 18" silica gel column (Merck silica gel H type 60) with chloroform/ethyl acetate 8:2 as the solvent. Selected fractions were combined and evaporated to a colourless oil (440 mgs) which was 90% benzyl clavulanate as deter~ined by NMR spectroscopy.

PREPARATION OF T~E BENZYL ESTER OF CLAVULANIC ACID FROM CRUDE EXTRACTS OF
THE CULTURE FILTRATE OF S. CLAVULIGERUS
i An aliquot of aqueous back extract of the butanol extract of culture filtrate obtained as described in Example 14 was freeze dried using an Edwards chamber drier. A 24 g. portion of the solid obtained contained 0.96 g. of sodium clavulanic acid as determined by the enzyme inhibition assay. This solid was suspended in 75 ml. of dry dimethylformamide and 75 ml.
of benzyl bromide was added. The mixture was stirred for 2 hours at room temperature. The suspension was then diluted with 500 ml. of ethyl acetate and the mixture filtered. The filtrate was concentrated to an oily residue on a vacuum rotary evaporator. This residue was loaded onto a 2" x 14" silica gel column (Biogel Biosil A.IOO mesh) in cyclohexane. Benzyl bromide was eluted from the column and then the solvent was changed to ethyl acetate and 10 ml. fractions was collected. Fractions containing the benzyl ester of clavulanic acid were selected as in Example 24. Further purification was also achieved as described~in Example 24 by column chromatography. This process yielded 220 mg. of pure benzyl ester.

PREPARATION OF CLAVULANIC ACID SODIUM SALT

~ ~ ~ll2~ ~ ~ H20 C02C112C6~15 ~C02 Substantially pure benzyl clavulanate (281 ~g) in ethanol (25 ml.) containing sodium hydrogen carbonate (82 mg.) was hydrogenated over 10~ Pd/C (90 mg.) for 25 minutes at room temperature and atmospheric pressure. The catalyst was filtered off, washed with water and ethanol, and the combined filtrates evaporated under reduced pressure at room temperature. The residual semi-solid was triturated with acetone, filtered and washed with ether to yield sodium cla w lante (135 mg.).

HYDROLYSIS OF CLAVULANIC ACID METHYL ESTER TO CLA~ULANIC ACID
2.17 mg. of clavulanic acid ester was dissolved in 0.1 ml.
. methanol and treated with 0.208 ml. sodium hydroxide solution (0.0482N).
After 1 hour at room temperature, the reaction mixture contained several products. T.L.C. analysis indicated that one of the maior components had ; an Rf identical to that of the sodium salt of clavulanic acid; colour reactions and biological assay were consistent with this component being the sodium salt of clavulanic acid.
Slow conversion of the ester to clavulanic acid was seen when 1 mg/ml. of the compound was incubated at 37C in 0.05M phosphate buffer at pH 7. The reaction was followed by paper chromatography (bioautographic system). Using the butanol/ethanolfwater system to follow the reaction over a period of 2~hours the zone of the methyl ester at Rf 0.79 decreased in size as the zone of clavulanic acid at Rf 0.12 increased.

ANTIBACTERIAL SPECTRUM OF CLAVULANIC ACID
The antibacterial activity of clavulanic acid sodium salt against a range of bacteria was determined using the microtitre method.
Serial dilutions of clavulanic acid sodium salt in Oxoid sensitivity test broth contained in a microtitre plastic tray were inoculated with an over-night broth culture of each organism so that the final dilution of the B inoculum was 0.5 x 10 4. The cultures were incubated overnight and the points O~

`- ` 1074325 bacterial growth recorded next morning by observing the turbidity of the culture. The results, expressed as approximate MIC values (minimum inhibi-tory concentration ~g/ml.) are recorded in Table 3 which shows that the compound has a broad spectrum of antibacterial activity.

ANTIBACTERIAL SPECTRUM OF CLAW LANIC ACID SODIUM SALT

Bacterial Strain Minimum Inhibitory Concentration pglml, 10 Staphylococcus aureus (Oxford H) 7.5 Staphylococcus aureus (Russell) 7.5 Bacillus subtilis 62 Streptococcus faecalis ~ 500 Streptococcus pyogenes CN 10 125 Escherichia coli NCTC 10418 31 Klebsiella aerogenes 31 - 62 Klebsiella oxytocum 62 Enterobacter aerogenes T 624 31 Enterobacter cloacae 62 20 Acinetobacter anitratus 125 - Providentia stuartii 125 Serratia marcescens 125 Proteus mirabilis C977 62 Proteus vulgaris W090 31 Salmonella typhimurium 31 Shigella sonnei 62 Pseudomonas aeruginosa A 500 EXA~PLES OF ~ -LACTAMASE INHIBITION BY CLA W LANIC ACID SODIUM SALT

Clavulanic acid progressively and irreversibly inhibits the ~ -lactamase of Escherichia coli. The method of Description 1 shows that the other -lactamases shown in Table 4 are also inhibited by clavulanic acid.

INHIBITION OF ~ -LACTAMASES BY CLAVULANIC ACID

Source of ~ -lactamase I50 Value Relative to Escherichia coli JT 4 = 1 Staphylococcus aureus (Russell) 1.0 Escherichic coli JT4 1.0 Escherichia coli Bll 2.0 Klebsiella aerogenes A 0.6 Pseudomonas aeruginosa 1~22 (R factor) 5.0 Pseudomonas dalgleish 0.1 _ .
With penicillin G as substrate the I50 of clavulanic acid sodium salt against the ~ -lactamase of Staph. aureus (Russell~ is approximately 0.06 yg/ml.

EXAMPLES OF ACTIVITY OF CLA W LANIC ACID METHYL ESTER
Tests for antibacterial activity in broth showed clavulanic acid methyl ester to have broad spectrum activity but of a lower order than shown by clavulanic acid. It was not clear whether this activity was the activity of the compound itself or of clavulanic acid liberated by slow aqueous hydrolysis of the ester. Clavulanic acid methyl ester showed marked anti-bacterial synergism in combination with ampicillin or cephaloridine against bacteria resistant to these antibiotics. Thus, the minimum inhib~tory con-centration (M.I.C.) for ampicillin against Starhylococcus aureus (Russell)was reduced from 500 yg/ml. to ~ 0.4 in the presence of 1.0 jug/ml. clavulanic 10'-~432S

acid methyl ester. The M.I.C. for cephaloridine was reduced from 1.5~ug/ml.
to < 0.03 ~ug/ml. in the presence of 1)ug-/ml. of claw lanic acid methyl ester.
The M.I.C. for ampicillin against Proteus mirabilis C889 was reduced from 500Jug/ml. to 31 ~g/ml. in the presence of 5 jug/ml. clavulanic acid metllyl ester.
EXA~LE 33 PREPARATION PIVALOYLOXYMETHYL CLAVULANATE
To a stirred solution of bromomethyl pivalate (0.37g~ in dry dimethylformamide (5 ml) was added sodium cla w lanate (0.49g). After 2 hrs.
at room temperature the réaction mixture was treated with ethyl acetate (20 ml), cyclohexane (10 ml) and water (20 ml). The mixture separated into two layers and the non-aqueous layer was separated, washed with water (20 ml) and dried over sodium sulphate. The dried solution was evaporated to leave the required product as a pale yellow oil.
(500 mg). N.m.r. (CDC13), 1.26 (s,9), 3.13 (d,l,J=17 Hz), 3.62 (dd, l,J,=17Hz, J1=2.5Hz, 4.3(d,2,J=7.5Hz), 5.0(dt, 1,J=7.5Hz, J2=1.5Hz), 5.16(d,1,J-1.5Hz), 5.79(d,1,J=2.511z), 5.92~(s,2); i.r.(liquid film), ~ ~'lactam C.O. 1800 cm 1, ester CzO 1760 cm 1 ~REPARATION OF CLAVULANIC ACID PHT~ALIDE ESTERS
To a stirred solution of 3-bromophthalide (0.43g) in dry dimethylformamide (5 ml) was added sodium clavulanate (0.5g) and the solution was left at room temperature for 2 hours. The solution was treated with ethyl acetate (20 ml~, cyclohexane (10 ml) and water (30 ml) and shaken thoroughly. The non-aqueous layer was washed with water (20 ml), dried (Na2SO4) and evaporated to yield a pale yellow gum. The two diastereomeric esters were separated using high pressure liquid chromatography on a 40 cm x l0 mm column of silica gel (Merckosorb SI 60, 5 ~) eluting with ethyl acetate at a flow rate of 3 ml/min.
The first phthalide èster (retention time 7.15 min) crystal-lised from ethyl acetate as needles, mp 102, and had the following i.r.

(nujol mull) ~ -lactam C=O 1790 cm 1 ester C~O 1755 cm 1 n.m.r. (CD3COCD3):
3,14 (d,l,J-17,5Hz) 3.76 (dd,l,J,=17.5Hz, J2F2.5Hz), 4.25(d,2~J=7.5Hz), 5.0 (dt~l~Jl=7.5llz~ J2=1.5Hz), 5.4 (s,l,J-1.5Hz), 5.82 (d,l,J-2.5Hz),

7.7 (s,l), 8.06 ~ (m,4); M.wt (mass spectrometry: 331.0696 corresponds to C16H13NO7 (calc. 331.0692). The second diasterioisomer (retention time

8.85 min) had the following i.r. (CH2C12solution) ~ ~-lactam C=O 1800 cm ester C=O 1780 cm 1; nmr (CDC13) 2.42 (broad S,l, exchangeable with D20), 3.12 (d,l,J=18 ~ Hz), 3.60 (dd,l,Jl=18 Hz, J2= 2.5Hz), 4.30 (d,2,J=7.5Hz), 5.0 (dt,l,Jl-7.5 Hz, J2=1.5 Hz), 5.12 (d,l,J=1.5Hz), 5.76 (d,l,J=2.5 llz), 7.52 (S,l), 7.85 ~ (m,4).
J~ >_/ 2 N
/~ O
O= CO ~ ~ O

~, _ O CH2OH //

O ~ ~ >

C02Na ~N-!
H ~ ~

-/

10~4325 EXA~LE 35 PREPARATION 0~ NONYL CLAVULANATE
Sodium clavulanate (44 mg) in dry dimethylformamide ~2 ml) was treated with nonyl iodide (76 mg) and left at room temperature for 2 hours.
The solution was evaporated and the residue fractionated on silica gel, eluting with ethyl acetate-hexane (2:1) to give the product as an oil; i.r.
(film) 1800, 1745, 1690 cm 1. M.wt. (mass spectrometry) = 325.1890 which corresponds to C17H27NO5. (calc. 325-1889)-PREPARATION OF CLAWLANIG ACID
Benzyl clavulanate (100 mgs) in ethanol (5 ml) was hydrogenated over 10% Pd/C (30 mgs) for 45 minutes at ambient temperature and atmospheric pressure. The catalyst was filtered, washed with ethanol and the combined filtrates were evaporated in vacuo to give clavulanic acid as an unstable, viscous oil (58 mgs). N.m.r. (C5D5N): 3.05~d,1,J=18~1z), 3.60(dd,1,J1=18Hz, J2= 2.5Hz), 4.75(d,2,J=7.5Hz), 5.58(t,1,J=7.5Hz), 5.66 (S,l), 6.0 5 (d,l,J=
2.5Hz).

PREPARATION OF METHYL CLAVULANATE
-Clavulanic acid (130 mgs) in ethanol (10 ml) was treated with excess diazomethane in ether. After 2 minutes at room temperature the reaction was shown (t 1 c) to be complete. The solution was evaporated in vacuo and tlle residue purified by chromatography on silica gel, eluting with ethyl acetate. The fractions containing methyl clavulanate were combined and evaporated to give a clear oil (104 mgs).

PREPARATION OF ~IETHYL CLAYUEANATE
Clavulanic acid (200 mgs) in acetonitrile (5 ml) was cooled and stirred at 0. Methanol (0.5 ml) and then dicyclohexyldicarbodiimide (206 mg.) were added and the reaction mixture was stirred at room temperature overnight. The suspension was filtered and the filtrate eyaporated in vacuo to give crude methyl cla w lanate. The crude product was purified by chroma-tography on silica gel, eluting with ethyl acetate, to give a clear oil (140 mg).

PREPARATION OF PHENYL CLAVULANATE
Claw lanic acid (100 mg) in acetonitrile (5 ml) was cooled and stirred at 0. To the solution was added phenol (0.94g) and dicyclo-hexyldicarbodiimide(100 mg) and the reaction mixture was stirred at room temperature overnight. The suspension was filtered and the filtrate evaporated. -The residue was fractionated on silica gel, eluting with ethyl acetate-hexane (1:1) to give phenyl clavulanate (70 mg). I.r (film) 1800, 1770, 1690 cm N.m.r. (CDC13) 2.18 (broad s,l), 3.06 (dd, 1,J-17Hz,J2=0.911z), 3.54 (dd,l,Jl=17Hz,J2=2.6Hz), 4.29 (d,2,J=7.5Hz), 5.1(dt,1,Jl=7.511z,J2=1.5Hz) 5.29 (d,1,J=1.5Hz), 5.76 (dd,1,Jl=2.6Hz,J2=0.9Hz), 7.35 ~ (m,5). M.wt. (mass spectrometry) = 275.0777 which corresponds to C14H13N05 (calc. 275.0794).

PREPARATION OF 2,2,2-trichloroethyl claw lanate Sodium clavulanate (221 mgs) was suspended in dry tetro-hydrofuran (5 mls) and stirred at 0. Trichloroethylchloroformate (211 mg) in dry tetrohydrofuran (1 ml) was added to the above suspension over 20 minutes. The mixture was allowed to reach room temperature and stirred overnight. The suspension was filtered and the filtrate evaporated in vacuo.
The residue was chromatographed on silica gel eluting with ethyl acetate -hexane (2:1) to give the required ~roduct as an oil. i.r. (film) 1800, 1760, B 1690 cm 1. ~ (CDC13) 1.56 (broad S,l), 3.07 (dd,l,J1=17.5Hz,J2=0.7Hz), 3.56 (dd,1,Jl=17.5Hz,J2z2.5Hz), 4.24 (d,2,J=7.5Hz), 4.69 (d,l,J=121{z), 4.92 (d,l,J=12Hz), 5.02 (dt,l,J1=7.511z, J2z1.3Hz), 5.19 (d,l,J-1.311z), 5.73 ~ (dd,l,J1=2.5Hz, ~2=0.7Hz). M.wt. (~ass spectrometry) = 328.9621 which corresponds to CloHloN05C13 (calculated 328.9625).

=fll20}1 ~ >~ H2011 C2Na CO-O-CO-O-R
ou R=CH2C13 -- ' PREPARATION OF SODIUM CL~VULANATE
Benzyl clavulanate (840 mgs) in ethanol (30 ml) and water (5 ml~ was hydrogenated over 10% Pd/C (267 mgs) and sodium bicarbonate (244 mgs) for 25 minutes at room temperature and atmospheric pressure.
The catalyst was filtered, washed with water and ethanol and the combined filtrates were evaporated in vacuo. The product crystallised from a water-acetone mixture as microneedles (565 mgs). Recrystallisation from water-acetone gave needles which, after drying over P2O5 in vacuo for 24 hours gave the following analysis:
C 41.01, 40.86; 1~ 3.77, 3.64; N 5.68, 5.51; i.r.(KBr disc) 1785, 1700, 1620 cm ; Nmr (D2O) 3.06 (d,1,3=18.5Hz), 3.57 (dd,l,J1=18.5Hz, J2=2.5~z), 4.15 (d,2,J=8Hz), 5.3 (~IOD), 4.9(m), 5.71 (d,1,J=2.5Hz).

ANTIBACTERIAL SYNERGISM BETWEEN AMPICILLIN AND CLA~ULANIC ACID SODIUM SALT
The minimum inhibitory concentration (M.I.C. values) of ampicillin, clavulanic acid sodium salt and ampicillin in the presence of l ~ug/ml. clavulanic acid sodium salt were determined for a range of ~-lactamaseproducing bacteria. The organisms were inoculated into Oxoid sensitivity ` ~0'7432S
r ; test broth located in small wells in a plastic tray and containing separate ~concentration gradients of ampicillin, clavulanic acid sodium salt or . : ampicillin plus 1 ~g/ml. clavulanic acid sodium salt (microtitre method).
The final dilution of the overnight broth inoculum was 0.5 x 10 2. The tray was incubated at 37C overnight and a ~ecord made next morning of the end points of bacterial growth. The M.I.C. values in ~g/ml. are recorded in . Table 5 wl-ich reveals that the synergist at the low concentration of l~ug/ml.
markedly enhances the antibacterial activity of ampicillin against certain gram + vc and gram - ve bacteria. The mechanism of this synergism is li~ely to involve inhibition of ampicillin destroying ~ lactamase enzymes but the existence of other mechanisms cannot be excluded.
Similar results to those shown in Table 5 were obtained when ampicillin was replaced by amoxycillin or by the phthalidyl ester of ampicillin .

10743;~5 . TABLE 5 _TIBACT1;RIAL SYNERG~SM BETI~N AMPICILLIN AND
CLAVULANIC ACID SODIUM SALT
_ _ _ ~ ~inimum Inhibitory Concentrations ~g/ml _ , Bacterial strain Clavulanic Ampicillin in acid~mpicillin1 ,ug/ml clavu-sodium lanic acid salt sodium salt Escherichia coli NCTC 10481 31 1.8 < 0.4 Escheriohia coli .
B 11 62 > 500 125 Klebsiella aerogenes A 31 125 ~ 0.4 Klebsiella sp 62 31 125 ~ 0.4 Enterobacter cloacae 62 250 62 Serratia marcescens 125 ~ 500 62 Staphylococcusl aureus(Russell) 15 500 < 0.4 Staphylococcusl aureDs 62 250 7.5 a methicillin resistant strain ANTI~ACTERIAL SY~ERCYSM BETW~EN CEPHALORIDINE AND C~AW LANIC ACID SODIUM SALT
The minimum inhibitory concentrations of cephaloridine, clavulanic acid sodium salt and cephaloridine in the presence of 5Juglml cla~ulanic acid sodium salt were determined by the method described in Example 42. The results in Table 6 show that synergism can be obtained between cla~ulanic acid sodium salt and cephaloridine particularly for the ~-lactamase producing strain of Staphylo-coccus aureus (Russell).

_ 59 -~i'43;~5 ANTIBACTERIAL SYNERGISM BETWEEN CEP1~LORIDINE
,, AND CLAVULANIC ACID SODIUM SALT

Minimum Inhibitory Concentrations ~g/ml.
Bacterial Strain Clavulanic Cephaloridine in acid presence of sodium Cephaloridine 5 ~g/ml clavulanic salt acid sodium salt Proteus mirabilis 889 1 ~500* 62 7.5 Staphylococcus aureus(Russell 15 3.1 C 0.03+
Staphylococcus aureus 62 15 3.7 a methicillin resistant strain Tailing Point ~, Same value obtained when synergist added at 1 ~g/ml. instead of 5 ~g/ml.

ANTIBACTERIAL SYNERGISM BETWEEN CLAVULANIC ACID SODIUM SALT AND
VARIOUS PENICILLINS
The results presented in Table 7 were obtained by the method described in Example 42.

_ 60 -10743'~5 ANTIBACTERIAL SYNERGISM BETWEEN CLAVULANIC ACID
: . SODIUM SALT AND VARIOUS PENICILLINS AGAINST
STRAINS OF KLEBSIELLA AEROGENES

Amoxycillin Carbenicillin*Benzylpenicillin Strain Alonel +5~g/ml. Alone 1~5~g/ml, Alone~ +5~ug/ml, synergist synergist synergist I l A 500 1 0.97 500 1 7.8250 1 7.8 E 70 500 1 3.9 500 15 500 15.6 62 250 1 15.6 1251 7.8 250 ~ 15.6 *Similar results observed when carbenicillin replaced by carbenicillin phenyl ~-ester or ticarcillin.

ANTIBACTERIAL SYNERGISM BETUEEN AMPICILLIN AND ESTERS OF CLAW LANIC ACID
The results presented in Table 8 were obtained by the method described in Example 42.

ESTERS OF CLAYULANIC ACID AGAINST STRAINS OF
KLEBSIELLA AEROGENES

Ampicillin + Ampicillin . . . 5 ~uglml of 5jug/ml of AmplcllllnMethyl Ester ~enzyl Ester Strain Alone of of clavulanic acid cla w lanic acid 50~ ~9 1.9 500 3.9 3.9 62 500 3.9 3.9 Neither clavulanic acid methyl ester nor clavulanic ac.id benzyl ester inhibited the growth of the test organisms at a c~ncentration o~ 100 ~Ig/ml.

..

~o7~3z5 ~NTIBACTERIAL ACTIVITY O~ CL~VUL~NIC ACID ESTER
Using the method of'Example 30 but using a dilution of 1/100 of overnight broth, the MIC values in Table 9 were obtained for certain esters of cla~ nic acid against a number of organisms:

ANTIBACTERIAL ACTIVITY OF CLAVULANIC ACID ESTERS

MIC*
_ MIC of ~ of 10Organism Benzyl ~ Nonyl Pivaloyloxy- Phthalidyl clavulanic ester ester methyl ester ester salt Bacillus subtilis A 25031 62 125 62 Staph.aureus Oxford 62 31 31 31 15 Staph.aureus 12531 62 15 15 Russell Escherichia 'coli 10418 125~ 500 125 125 125 *

The MIC of clavulanic acid sodium salt is included for comparison; the high MIC values (if compared to those of Example 30) are due to the heavy inocula used.

EXTRACTION OF CLAVULANIC ACID USING LIQUID ION EXCHANGE RESIN
Culture filtr~te (200 ml, obtained in a similar manner to Example 3 bllt using a medium containing 0.1% v/v ~l2PO4 instead of 0.01% FeSO4.7H2O) was extracted with Amberlite LA2 (C1 form, 15% v/v in methylisobutyl ketone, 66 ml) for 30 minutes at 5 C.
The phases were separated by centrifugation (1660 g, 20 minutes). The solvent phase (60 ml) was recovered by pipette and divided into four equal portions. Each portion was extracted by stirring at 5C for 20 minutes 10743'~S

with 1/4 volume (3.75 ml) aqueous extractant as indicated in the table below.
The resulting mixture was centrifuged (1660 g, 15 minutes). 3.6 ml. aqueous phase was recovered from each extraction.

.
Sample 1 concentration Clavulanic (m ) (~ug ml~1 acid (mg) _ ~ _ clarified brew200 128 25.4 extracted brew200 15 3.0 M NaC1 extract3.6 305 1.1 2M NaCl extract 3.6 598 2.5 ~I NaN03 extract 3.6 638 2.3 2M NaNO3 extract 3.6 758 2.73 Tlle result obtained with 2M NaNO3 represents a-recovery of 43% from clarified brew.
Amberlite LA2 is obtainable from Rohm and Haas (UK) Ltd. Croydon.

_ EXTRACTION OF CLAVULANIC ACID USING LIQUID ION EXCHANGE RESIN
Clarified brew (47 litres, obtained as in Example 12) was extracted with Amberlite LA2 (acetate form, 15% v/v in methylisobutyl ketone, 12.5 litres) by stirring for 1 hours at 17C. After adding octan-1-ol-(500 ml) the phases were separated in a continuous flow centrifuge yielding

9.2 litres solvent phase, which was then stirred at 5 C for 1'~ hours with molar sodium nitrate (2.3 litres). The mixture was separated ~y continuous flo~ centr-fugation yielding 2,4 litres aqueous phase (including water used for displacement purposes). Aqueous phase pH (initially 8.0) was adjusted to 7.0 with concentrated hydrochloric acid.

- ~3 -.
clavulanic acid clavulanic : Sample Volume concentrlation ac d clarified brew 47 146 . 6862 extracted brew 47 19 893 M NaNO3 extract 2.4 1638 3931 Extraction efficiency from clarified brew. to sodium nitrate extract is 57%.

!

a,, :

Claims (18)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY
OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process for the preparation of clavulanic acid or a salt thereof which comprises de-esterifying an ester of clavulanic acid of the formula wherein X is R' or , R' being a C1-9 hydrocarbon group substituted with halogen, lower akloxy or hydroxy; A7 being H, phenyl, tolyl, chlorophenyl, methoxyphenyl or nitrophenyl and A8 being phenyl, tolyl, chlorophenyl, methoxyphenyl or nitrophenyl, exclusive of hydrolysis of the ester in the presence of a sodium or potassium base, and recovering the acid or forming a salt thereof and recovering said salt.

2. A process as claimed in claim 1 wherein clavulanic acid or a pharmaceutically acceptable salt is prepared and recovered.

3. Clavulanic acid or a salt thereof when prepared by the process of claim 1 or an obvious chemical equivalent.

4. Clavulanic acid or a pharmaceutically acceptable salt when prepared by the process of claim 2 or an obvious chemical equivalent.

5. A process for the preparation of sodium clavulanate which comprises hydrogenation of benzyl clavulanate in ethanol in the presence of sodium bicarbonate and palladium on carbon catalyst and re-covering the sodium clavulanate.

6. Sodium clavulanate when prepared by the process of claim 5 or an obvious chemical equivalent.

7. A process for the preparation of clavulanic acid which comprises hydrolyzing methyl clavulanate under alkaline conditions and recovering the clavulanic acid.

8. Clavulanic acid when prepared by the process of claim 7 or an obvious chemical equivalent.

9. A process for the preparation of clavulanic acid which comprises hydrogenating benzyl clavulanate in ethanol in the presence of palladium on carbon catalyst and recovering the clavulanic acid.

10. Clavulanic acid when prepared by the process of claim 9 or an obvious chemical equivalent.

11. A process for the preparation of sodium clavulanate which comprises hydrogenating benzyl clavulanate in ethanol and water in the presence of palladium on carbon catalyst and sodium bicarbonate and recovering the sodium clavulanate.

12 . Sodium clavulanate when prepared by the process of claim 11 or an obvious chemical equivalent.

13. A process for the preparation of lithium clavulanate which comprises hydrogenation of benzyl clavulanate in ethanol water in the presence of palladium on charcoal catalyst and lithium bicarbonate and recovering the lithium salt of clavulanic acid.

14. Lithium clavulanate when prepared by the process of claim 13 or an obvious chemical equivalent.

15. A process for the preparation of potassium clavulanate which comprises hydrogenating benzyl clavulanate in ethanol in the presence of palladium on charcoal catalyst and potassium bicarbonate and recovering the potassium clavulanate.

16. Potassium clavulanate when prepared by the process of claim 15 or an obvious chemical equivalent.

17. A process for the preparation of calcium clavulanate dihydrate which comprises hydrogenating benzyl clavulanate in methanol in the presence of palladium on charcoal and calcium carbonate and recovering calcium clavulanate dihydrate.

18. Calcium clavulanate dihydrate when prepared by the process of claim 17 or an obvious chemical equivalent.