Antibiotic compounds obtained from streptomyces clavuligerus their preparation and pharmaceutical compositions containig them

antibiotic compounds obtained from streptomyces clavuligerus, their preparation and pharmaceutical compositions containing them

BEECHAM GROUP LIMITED

C. 44911

NOW.,ANTIBIOTIC. FROM STRSPTOMYCBS CLAVULIGERUS

ABSTRACT OF THE INVENTION

A new antibacterially active agent has been isolated from Streptomyces, clavuligerus. This new compound which we designate clavulanic acid has the formula (1):

ch₂0h

<img file="IL47087A_D0001.tif" />

(1)

V

CO_?_H

In addition to being a broad spectrum antibiotic of medium .5 potency, clavulanic acid and its salts and asters’ have the ability to enhance the effectiveness of β-lactam antibiotics against many S-lactamase producing bacteria.

BACKGROUND TO THE INVENTION

a. Streptomyces clavuligerus has been described in detail by Higgens et al, Int.J.Systematic Bacteriology, 21, ?26 (1971). This streptomycete was of interest because it produced certain β-lactam antibiotics such as penicillin N, 7-(5-amino-5-carboxyvaleramido)-

3-carbamoyloxymethyl-5-cephem-4-carboxylic acid and 7-(5-amino-5-carboxyvalera1nido)-3-carbarooylxynethyl7-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. 3770590, in Israel Patent 35065 and also by Nagarajan et al,J.Amer.Chem.Soc., 93, 2308 (1971),

Brannon et al, Antimicrcb. Agents Chemother., 1, 237 (1972) and Antimicrob. Agents Chemother, 1, 247 (1972) and Higgens et al, !.Antibiotics, 27, 298 (1974).

b. β-lactamases are enzymes which open the β-lactam ring of penicillins and cephalosporins to give products which are devoid of antibacterial activity. These enzymes are produced by many bacteria, notably . species or strains of Escherichia, Klebsiella, _?l_ 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 β-lactamase production may be understood when it is realised that a high proportion of clinically isolated organisms produce β-lactamases [see, for example,

M. Wilson 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 hospital produced β-lactamase]. In many cases, some penicillins or cephalosporins are ineffective in treating diseases ascribed to non, β-lactamase-producing organisms because of the common occurance of co-infection by a β-lactamase producer [see, for example, R. May et al; Brit.

J.Dis. Chest., 66, 185 (1972)]. Combination of a β-lactamase inhibiting substance with a penicillin, or cephalosporin might be expected to protect the latter from degradation by bacterial β-lactamase and thereby enhance their antibacterial activity against many infective organisms,. This process of enhancement of tne an’cioaccerium 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 .substan’ces. The β-lactamase inhibiting component cf the mixture is referred to as a synergist and such substances are valuable for increasing the antibacterial activity, of penicillins and cephalosporins against resistant organisms. It is one of the objects of this invention to provide such synergists י

c. Examples of the use of certain β-lactamase resistant semi-synthetic penicillins and cephalosporins as β-lactamase inhibitors and synergists for penicillins and cephalosporins have already been described in the literature, see for example, Sutherland et al.,

Nature, 201. 868 (1964): .Sabath et al., Nature,

204, 1066 (1964); O'Callaghan et al., Antimicrob. Agents and Chemotherapy, 1968 , 67 (1969). However, 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 prociucmg p-xactumase umiuiviiig, subs^a-iivs*□ which act synergistically with penicillins or cephalosporins, for example, those cultures disclosed in British Patent No.1,363,075 and those described by Hat a et al, _J. Aiitibio bics,. 25, 475 (1972) and Umezawa et al,. J. Antibiotics, 26, 51 (1973). None of these β-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 β-lactamase inhibitors of actinomycetal

I origin are its extractability into organic solvents from culture filtrate at pH2, its high stability in human blood and its broad spectrum of anti-bacterial and β-lactamase inhibiting activity, its lovz צ molecular weight and its high R_f. values on paper chromatography using a variety of solvent systems.

״

DESCRIPTION OF THE INVENTION

We have discovered that the aerobic cultivation of Streptomyces clavuligerus in conventional nutrient media at about 2530°*־C under roughly neutral conditions produces a β-lactemase inhibitory substance 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 Fig. 1.

(c) It is able to inhibit the growth of sti’ains of Staphylococcus aureus.

(d) It is able to synergyse the antibacterial effect of ampicillin against S~lactamase producing strains of ?Escherichia coli. Klebsiella aero- genes and Staphylococcus aureus.

(e) It is able to synergyse' the antibacterial effect of cephaloridine against the β-lactamase producing strains of Proteus mirabilis and Staphylococcus aureus.

.» «*«*«*י« * iwn». &lt;« (f) It forms a methyl ester which has a molecular weight (by mass spectroscopy) of 213.0635 which corresponds to the formula C_oH_n N0_r.

y j-1- 0

Thus clavulanic acid may be regarded as a monobasic carboxylic acid of ־the formula C_oH_nN0_c which in the form of its sodium salt has a' characteristic infra-red absorption spectrum substantially as shown in Fig.l.

The compound produced by Streptomyces,elavuligerus which has the above properties has the formula (II):

CHpOH rAJ ־ ^(II)

CO H

Thus clavulanic acid may be named 3~(p-hydroxyethylidene)“7oxo-4~oxa-l“azabicyclo[3»2_>0J heptane-2-carboxylic acid.

The stereochemistry at C_t. and C^ 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. (1):

CH OH aV;² ω

CO₉H

Thus a fuller chemical name for clavulanic acid is Z-(2R,5R)-3~(3־hydroxyethylidene)-7“0xo~4-oxa!5 l-azabicyclo[3_f2,0]heptane-2-carboxylic acid.

Ths great usefulness 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 125 pg/ml. of ampicillin, amoxycillin, carbenicillin or benzyl penicillin or by the presence of 10 ug/rnl. of clavulanic acid, are inhibited by the presence of less than 12.5 pg/ml. of the previously . mentioned penicillins when 5 pg/ml. of clavulanic acid is also present. Similar results have been observed for combinations containing various esters of clavulanic acid. For example, strains of Klebsiella aerogenes A, the growth of which is not inhibited by 125 pg/ml. of ampicillin, or by 10 pg/ml of clavulanic acid methyl ester are inhibited by less than 12.5 ug/ml. of ampicillin in the presence of 5 pg/ml. of the clavulanic acid methyl ester. It has also been found that strains of Staphylococcus aureus Russell, the growth of ^,which is not inhibited by the presence of 100 pg/ml. of ampicillin or by 5 pg/ml of clavulanic acid, are inhibited by the presence of less than 10 pg/ml. of ampicillin in the presence of 1 pg/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 pg/ml. can readily be achieved by 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 clav.j.ianic acid.

Accordingly, the present invention provides clavulanic acid as hereinbefore described and its salts and esters.

Most suitably the salts of clavulanic acid will be 5 pharmaceutically acceptable salts such as the sodium, potassium, calcium, magnesium, aluminium. ammonium and substituted ammonium salts such as the trimethylammonium, benzathine, procaireand like salts conventionally formed with penicillins or cephalosporins. Non-pharmaceutically PO acceptable salts of clavulanic acid are also included within the scope of this invention as they are useful intermediates in the preparation of esters of clavulanic acid, for example, the lithium or silver salts of clavulanic acid may be reacted with benzyl bromide to _ך- form the useful benzyl ester of clavulanic acid.

Salts of clavulanic acid tend to be more stable than the parentacid per se and thus form a favoured aspect of

T '? this invention. Particularly suitable salts of clavulanic

Λ __ ־־־-־ — -־ -־־’ \ acid include the sodium and potassium salts which have the formula (III) and (IV) respectively:

<img file="IL47087A_D0002.tif" />

I.

Crysta.Hine forms of such salts may contain water of hydration.

47087/2

Suitable esters of clavulanic acid include those notionally derived from alcohols such as methanol, ethanol, propanol, butanol, 2,2,2-trichloroethanol, 2.2,2trifluoroethanol, benzyl alcohol, p-nitrobenzyl alcohol, 5 phenol, acetoxymethanol, pivaloyloxymethanol, 2-dimethyl~ aminoethanol and other conventional alcohols. Various esters of clavulanic acid are useful intermediates in certain processes for the purification of clavulanic acid. Many clavulanic 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 alcohol of the formula ROH where R is (i) C₁_g alkyl, optionally substituted by (a) halogen (b) one or more phenyl groups (c) nitrophenyl, (d) lower alkoxyphenyl or trityloxyphenyl, (e) halophenyl, (f) pyridyl, (g) naphthyl optionally substituted by OCH^, (h) anthracenyl, (i) lower alkoxy, halophenoxy, benzyloxy or nitrobenzyloxy, (j) lower alkylthio, (k) lower alkanoyl or benzoyl, (1) amino, optionally substituted by lower alkyl or benzyloxycarbonyl, (m) optionally salted or lower alkyl or benzyl esterified carboxyl, (n) lower alkanoyloxy, (o) phenylsulphonyl_z(p) nitrile^(q). phthalimido (r) N-benzoxazolonyl_׳ (s) lower alkoxycarbonyloxy;

(ii) lower alkenyl or lower alkynyl;

(iii) cycloalkyl;

(iv) phenyl; or (v) phthalidyl, optionally substituted by methoxy.

47Q87/2

In order not to increase the molecular weight to an unreasonable extent, groups R do not normally include more than 16 carbon atoms, more suitably, not more than 12 carbon atoms and most suitably, not more than 8 carbon atoms.

Preferably, the group R is notionally derived from an alcohol ROH which is pharmaceutically acceptable.

The term ^,lower¹ 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, vinyl, allyl, butenyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, benzyl, benzhydryl, phenylethyl, naphthylmethyl, phenyl, propynyl,

2-chloroethyl, 2,2,2-trichloromethyl, 2,2,2-trifluoroethyl , acetylmethyl, benzoylmethyl, 2-methoxyethyl, p-chlorobenzyl, p-methoxybenzyl, p-nitrobenzyl, p-bromobenzyl, m-chlorobenzyl,

6-methoxynaphthyl-2-methyl, p-chlorophenyl or p-methoxvphenyj or any like group as well -s those groups which are known from the penicillin or cephalosporin arts to produce esters known to fee readily hydrolysed in vivo to the parent antibiotic.

Readily hydrolysable esters include, to, those of the formulae (V) and (VI):

but are not limited

<img file="IL47087A_D0003.tif" />

CH₂OH (V) ־ΌΟ-Ο-Ch O-CO-A₃^A2

<img file="IL47087A_D0004.tif" />

(VI) wherein A₁ is a hydrogen atom or an alkyl or phenyl group;

470S7/2

A״ is a hydrogen atom or methyl group; is an alkyl ^>up; and Z is a divalent organic group. Esters of the formulae (V) and (VI) which fairly readily release the clavulanic acid into the blood stream after administration include those wherein A^ is a hydrogen atom, k^ is ^a hydrogen atom or a methyl group and A^ is a methyl, ethyl, propyl or butyl group and those wherein Z is -CH^CH^-, -CH:CH-,

<img file="IL47087A_D0005.tif" />

When used in conjunction with the proceeding formula the term *alkyl’ includes alkyl of up to six carbon atoms.

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

<img file="IL47087A_D0006.tif" />

(VII) (VIII)

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A^ is a hydrogen atom or a methyl group, A^ is a methyl,/^ t-butyl group and Αθ is a hydrogen atom or a methoxyl group□

Many esters of clavulanic acid differ from analagous esters 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 mild conditions are included within the formula (IX);

<img file="IL47087A_D0007.tif" />

wherein R¹ is an alkyl group of 1-9 carbon atoms or a benzyl group, optionally substituted by halogen or lower alkoxy groups.

When used with reference to formula (IX) the term ¹lower¹ means that the group contains 1-4 carbon atoms.

Suitably groups R¹ include alkyl groups optionally substituted by halogen or methoxyl.

.087/2; ;־ ?lost suitably alkyl groups are straight chain alkyl groups of up to 6 carbon atoms optionally subsi plated by one methoxyl group or one chlorine, bromine or iodine atom or by a CCl^ or group.

The esters of clavulanic acid of particular usefulness as synergists are those which hydrolyse in mammalian tissues, especially human blood, to yield clavulanic acid or a salt thereof because it is believed that clavulanic acid and its salts tend to be somewhat more useful _Syn1q ergystic agents than the esters per se. Many of the esters of the formulae (V)-(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 15 chemical or biochemical techniques which are known from־ the penicillin or cephalosporin arts to be sufficiently mild not to degrade reactive acid-labile β-lactam rings.

Most suitably, the ester is one removable by hydrogenolysis. Conventional esters for such a process include benzyl, substituted benzyl, benzhydryl, sub- stituted benzhydryl, trityl and the like. The benzyl ester has proved particularly useful for this purpose.

By and large, the nature of any substituent in the ester moiety is unimportant as long as it does not inter25 fere with the hydrogenolysis reaction.

Since clavulanic acid and its salts are useful intermediates in the preparation of the desirable antibacterially active esters of this invention, this invention also provides clavulanic acid and its salts when used as chemical intermediates.

As has been previously stated., clavulanic acid and its salts and esters have valuable therapeutic properties. Accordingly, in a further aspect, this invention provides a pharmaceutical 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 compositions of this invention include tablets, capsules, creams, syrups, suspensions, solutions, reconstitutable powders and ׳ sterile forms suitable for injection or infusion״ Such compositions 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.

Injectable 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 acid or a salt thereof in sterile form.

Unit dose compositions comprising clavulanic acid or ־ 16 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 compositions of clavulanic acid and its salts and esters can be improved if such compositions contain a buffering agent or an enteric coating agent such that the compounds of the invention do not have prolonged contact with highly acidic gastric juice. Such buffered or enterically coated compositions may be prepared in accordance with conventional pharmaceutical practice.

The clavulanic acid or its salt or ester may be present in the composition as sole therapeutic agent or it may be present together with other therapeutic agents such as a β-lactam antibiotic. Suitable β-lactam antibiotics for inclusion in such synergistic compositions include not only those known to be highly susceptible to B-lactamases,but also those which have a good degree of intrinsic resistance to β-lactamases. Thus, suitable β-lactam antibiotics 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-aminodesacetoxycephalosporanic acid, cephaloridine, cephalothin, cefazolin, cephalexin, cefoxitin, cephacetrile, cephamandole, cephapirin, cephradine, cephaloglycine and other well known penicillins and cephalosporins or pro-drugs therefore guoh as hetacillin, metampicillin, the acetoxymethyl, pivaloyloxymethyl

I >

or phthalidyl esters of benzylpenicillin, ampicillin, amoxycillin or cephaloglycine or the phenyl, toly! or indanyl a-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 adapted for parenteral administration.

When present in a pharmaceutical composition together with a β-lactam antibiotic, the ratio of clavulanic acid or its salt or ester present to 3-lactam antibiotic 20Ϊ1 to 1:12,more־ usually present may be from, for example,/10:1 to 1:10 and advantageously may be from 3:1 to 1:3.

The total quantity of antibacterial agents present !5 in any unit dosage form will normally be between 50 and 1500 mg and will usually be between 100 and 1000 mg.

Compositions of this invention may be used for the treatment of infections of inter alia, the respiratory tract, the 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.

Normally between 50 and 6000 mg of the compositions of the invention will be administered each day of treatment but more usually between 500 and 3000mg of the composition of the invention will be administered per day. However for the treatment cf severe systemic infections or infections of particularly intransigent organisms, higher doses may be used in accordance with clinical practice.

_ 18 _

The exact form of the compositions of this invention will depend to seme extent on the micro-organism which is ImosV being treated. For treatment of; infections the compositions of this invention are normally adapted to produce a peak blood level of at least 0.1 }!g/ml, more suitably at least 0.25 μg/mlJ and preferably at least . t μ2/π!1 of of׳: synergist; if or 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 thetreatment of infection.

Particularly favoured compositions of this invention will contain from 150 - 1000 mg of amoxycillin, ampicillin or a pro-drug therefore and from 50 - 500 mg of clavulanic acid or a salt or in-vivo hydrolysable ester thereof and more suitably from 200 - 500 mg of amoxycillin, ampicillin or a pro-drug therefore and from 50 - 250 mg of clavulanic acid or a salt or in-vivo hydrolyable ester thereof.

The materials present in such compositions may be hydrated if required. 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 ל recovering clavulanic acid or a salt thereof from the culture medium and thereafter if desired, forming the free acid or a salt or ester by methods known per se.

Preferably, Streptomyces clavuligerus ATCC 27064 or a high yielding mutant thereof is used in the 10 process of this invention.

When used herein, the term ’cultivation’ means the deliberate aerobic growth 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 01־ suspended. The cultivation may take place on an aerobic surface or by submerged culture. The nutritive medium may be composed of complex nutrients or may be chemically defined. 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 the cultivation of Streptomyces clavuligerus may contain, in the range 0.1 - 10% a complex organic nitrogen source such as yeast extract, corn steep liquor, vegetable protein, seed protein, hydrolysates of such proteins, milk protein hydrolysates, fish and meat extracts and hydrolysates such as peptones. Alternatively chemically defined sources of nitrogen may be used such as urea, amides, single or mixtures of common amino acids such as valine, asparagine, glutamic acid, proline and phenylalanine. Carbohydrate (0.1 - 5%) may be included in the nutrient media but glucose in certain media is undesirable having a depressing effect on the yield of the desired clavulanic acid. Starch 01י starch hydrolysates such as dextrin, sucrose, lactose or also other sugars or glycerol or glycerol esters may/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 β-lactamase inhibitors. A particularly suitable low cost medium is one containing soya bean flour (Arkasoy) plus dried malt distillers solubles (Scotasol) plus dextrin.

The addition of antifoam agents such as Pluroxiic LSI may be necessary to control foaming of certain media in fermenters.

Mineral salts such as NaCl, KC1, MgCl^» ZnCl^, FeCly Na״SO_z . FeS0&lt; , MgSO׳ and Na⁺ or K⁺ salts of phosphoric acid may be added to the media described above particularly if chemically defined; CaCO^ may be added as a source of Ca⁺⁺ ions or for 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 5 producing mutant strainsinclude those outlined by H.I.

Adler in Techniques for the Development of Micro-Organisms in *Radiation and Radioisotopes for Industrial MicroOrganisms’, Proceedings of a Symposium, Vienna, 1973, page 241, international Atomic Energy Authority and 10 these include:

i. Ionising radiation' (such as X- and $- rays), uv light, uv light plus a photosensitizing 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, transformation, transduction, lysogenisation, lysogenic conversion and selective techniques for spontaneous mutants.

Cultivation of Streptomyces clavuligerus normally takes place in the temperature range 15-40°c, usually 25 20-35°C and preferably, 25~30°C and at a pH of between 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

22' on a rotary .shaker or in baffled stainless steel fermenter stirred with vaned disc impellers and aerated with 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/obtained in 2-10 days at 2O-35°C. In a stirred stainless steel fermenter using the Arkasoy/Scotasol/Dextrin medium referred to above the preferred temperature is 2.6°c 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 procedures are commenced.

In the solvent extraction process, the culture filtrate is chilled and the pH lowered into the region of pH 2-3 by the addition of acid while thoroughly mixing with a water immiscible organic solvent such as nbutylacetate, methylisobutylketone, n-butanol or ethylacetate. The acid used to lower the pH of the medium is normally a mineral acid such as hydrochloric, sulphuric, nitric, phosphoric or the like acid. n-Butanol is .a particularly suitable solvent for use in

־־-23“ the extraction of the acidified culture filtrate. After ׳^:A. separation of the phases by centrifugation, the β-lactamase . i , Inhibiting metabolite' is back extracted from the solvent : : I .־ phase into aqueous sodium bicarbonate or potassium;/ ׳ hydrogen phosphate buffer, CaCO^ suspension or water while maintaining the׳ pH at approximate neutrality, for example,. ¹ ' י I I ' .

at pH 7.0. This aqueous extract. !after separation of j .:',־ phases may be concentrated under reduced pressure and ' ׳i freeze dried to give a crude preparation of a salt of;

^!:z clavulanic acid. This preparation.is staole when stored. r

׳ ' ¹ '0 as a dry solid at -20 ^C.

; . ' י . ’ .

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 ' >1 .-⁵ resin is saturated and the β-lactamase inhibiting . ; ( ' .

material emerges from the bottom. The column is then ׳ . p ij washed with water and eluted with aqueous sodium chloride. ! .!

The β-lactamase inhibiting fractions are collected, bulked, . : desalted and .freeze dried to yield a crude solid salt of ¹.:.

clavulanic acid. >;

.Amberlite IR 4B is an example of a weakly basic .'. ’ .;־

׳.' . .I . י anion exchange resin with pplyamine active groups and cross!’ linked.polystyrene-divinyl-benzene matrix. Zerolite FFIP is )־.T / a strongly basic anion exchange resin with quaternary' !( ammonium active groups and a cross link’ed polyvinyl-divinyl-:.

benzene matrix. Resins similar to Zerolite FFIP include Isopor • FFIP and DeAcidite FFIP SRA.64. These resins were supplied ,.

by BDH Chemicals ltd., Poole, Dorset, U.K,.

:. I:

An alternative form of 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,

י׳ 24a ״ trichloroethylene and the like. Suitable amines include secondary׳. or tertiary amines in which one of the substituent groups is a long chain aliphatic group, for example, of 1216 carbon atoms and the other is a tertiaryalkyl. group so that the molecule is lipophilic. In cur hands Amberlite LA2 has proved a successful amine. Normally the 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 filtrate. The clavulanic acid may be back extracted into an aqueous phase by back extraction with a salt solution, preferably a concentrated solution of sodium.chloride, sodium nitrate or the like. The crude salt of clavulanic acid may then be obtained by. freeze drying or the like.

Other primary methods of isolation which may be used include conventional methods such as adsorption onto carbon, .!rcrfr'gair eiEtro. 04110*1, precipitation, salting out and molecular filtration but these methods are not usually as successful as the above described methods which are preferred.

Further 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 BEAE cellulose. The DeAcidite column may be gradient eluted with aqueous solution of a^alt such as sodium chloride (0 - 0.5M). The column of DEAE cellulose in 0.OIK phosphate buffer at pH7 may be eluted with a salt solution, normally a NaCl solution (0 -0.2M NaCl in 0.01M phosphate buffer pH7). Active fractions may be detected by their β-lactamase inhibitory activity and their antibacterial activity against Klebsiella aerogenes in an agar diffusion 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 P2 is an example or a highly lipophilic resin onto which organic materials may be adsorbed but which does not retain inorganic salts. Bio Gel P2 is a polyacrylamide gel supplied by Bio Rad, 32nd and Griffen Avenue, Richmond, Ga. 94804, U.S.A. The 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 coll β-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 system. Clavulanic acid may be assayed by making use of its β-lactamase inhibiting activity. Thin layer chromatography may be used to detect clavulanic 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.

־ 2.7 -

* ־

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 5 which may be cleaved by hydrogenolysis, enzymatic methods or by hydrolysis under very mild conditions.

One suitable group of esters used in this process is that of the formula ( xj:

<img file="IL47087A_D0008.tif" />

ch₂0h (X *

'C0-0-CH-A7 ^A8 wherein Ay is a hydrogen atom or a phenyl group optionally substituted by nitro, lower alkoxy, trityloxy or halogen and A_n is an optionally substituted phenyl group as o defined for ft hereinabove.

Most suitably A ך is a hydrogen atom or a phenyl, tolyl, chlorophenyl or methoxyphenyl group and A gis a phenyl, tolyl, chlorpphenyl or methcxyphenyl group.

Preferably Ay is a hydrogen atom and A gis a phenyl group.

The esters of formula ( X ) may. be cleaved by hydrogenolysis to yield clavulanic acid or a salt thereof.

Other groups of esters which may be used in this process include those of formulae (V) and (VI) as hereinbefore described. Such esters may be converted to salts of clavulanic acid by mild alkaline hydrolysis, for example, at pH 7.5.

־ 28 “

The impure form of clavulanic acid or salt thereof which is to be purified in this process may be in the form of a solid or solution which will usually also contain considerable quantities of organic or inorganic impurities.

The clavulanic acid or salt thereof may be converted into an ester by the esterification reactions referred to hereinafter. The preferred method of forming the required ester of clavulanic acid is 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 ditnethylformamide, dimethylformamide/acetone, dimethylsulphoxide, N-methylacetamide, hexamethylphosphoramide and the like.

If desired the salt of clavulanic 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 resins, especially those possessing a macroreticular nature which permits the use . of non-aqueous solvent systems. We have found Amerblyst A26 to be suitable for this purpose. The clavulanic acid salt may be adsorbed onto the resin from the culture filtrate and the resin then suspended in dimethylformamide containing sodium iodide or alternatively eluted columnwise with a solution of sodium iodide in dimethylformamide or in a mixture of dimethylformamide and acetone.

Once formed, the impure ester of clavulanic acid is normally purified chromatographically. In such procedures the ester is normally dissolved in an organic solvent such as ethylacetate, methylene chloride, chloroform, cyclohexane or similar solvents. The solid phase used in the chromatogi-aphic process is normally an inert material such as silica gel or chromatographically similar materials.

The fractions emerging from the column may be tested for the presence of the clavulanic acid by making use of its synergistic propei'ties. Active fractions are normally combined and the organic solvent evaporated off under reduced pressure.

The ester resulting from this process is generally of acceptable purity, but the material may be re- chromatographed if desired.

This purified ester of clavulanic acid may be converted to clavulanic acid or a salt thereof by the’ before ,mentioned methods.

A particularly suitable method of obtaining clavulanic acid or its salt is by hydrogenation of a compound of the formula ( X) as hereinbeforedescribed. \

Such reactions normally take place in the presence of a transition metal catalyst using low or medium pressures of hydrogen. The reaction may be carried out at high, ambient or depressed temperatures, for example at 0-100°C. Particularly suitable reaction conditions for such hydrogenations will use a slightly superatmospheric pressure ~

of hydrogen at an approximately ambient (12-20°C) . temperature. The reaction may be carried out in conventional solvents such as lower alkanols, for example, ethanol. 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 salt of clavulanic acid is produced, for example, the sodium or potassium salts result if the reaction is carried out in the presence of sodium or potassium hydrogen carbonate.

The clavulanic acid or salt thereof resulting from such reactions is generally of good purity.

Ssters or clavulanic acid may be prepared by the esterification 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 the 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 condensation promoting agent such as carbodiimide or the like.

Suitable salts of. clavul-anic acid which may be reacted with compounds R - Q include alkali metal salts such as the sodium or potassium salts or other conventional salts such as the silver salt

Suitable groups Q include those atoms or groups known to be displaceable by carboxylate anions and include chlorine, bromine and iodine atoms, sulphonic acid esters such as the O.SC^CH^ or: 0.SC^C^H^CH^ groups, . > active ester groups such as the O.CO.H or O.CO.CF^ group and other conventional groups displaceable by nucleophiles.

The preceding reaction is normally carried out in an organic solvent of relatively high dielectric constant such as dimethylfomamide, acetone, dioxane, tetrahydrofuran or the like and at a non-extreme temperature such as -5°C to 100°C, more usually ־ 52 +5°C to 30°C, for example at ambient temperature., ־

The reaction of clavulanic acid with a cffiaKoaodpuxaid 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 normally carried out at between about -5°C and 100°C, more usually from 5°C to 30°C, 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 like. Ethanol has proved a particularly 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 -10°C to +22°C, more usually -5°C to +1S°C, for example initially at 0°C and thereafter gradually warming to about 15°C. The condensation promoting agent used is normally one which removes water from the reaction mixture. Suitable agents include carbodiinides, carbodiimidazoles or equivalent reagents. Dicyclohexylcarbodiimide has proved to be a particularly suitable condensation promoting agent for use in this process. In order to

“ 35 ־ minimise self condensation of the clavulanic acid, this reaction is usually carried 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)

<img file="IL47087A_D0009.tif" />

wherein R^ is an inert organic group within the definition of R hereinabove; and (e) reaction of a compound of the formula (XI) with an alcohol ROH־

The elements of carbon dioxide may be removed from the compound of formula (XI) spontaneously during its preparation or alternatively by heating the compound of the formula (XI) in an inert solvent. Suitable inert solvents include ether solvents such as diethylether, tetrahydroferon, dioxane and the like. In many cases the compound of the formula (XI) decomposes spontaneously even at a depressed temperature, for example, at -5°C, to yield an ester of the formula

<img file="IL47087A_D0010.tif" />

wherein is an inert group within, the definition of R.

י 34 -

ץ

When the compound of the formula (XI) is to be . _ reacted with an alcohol 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 in order to prevent self-condensation of the clavulanic acid derivative.

Such methods of esterification are not in general as useful as those involving reaction of a salt of clavulanic acid with R-Q as hereinbefore described.

The compound of the formula (XI) may be prepared by the reaction of a salt of clavulanic acid with Cl.CO.O.R or the chemical equivalent thereof. Normally this reaction is carried out at a depressed temperature, for example, at a temperature not greater than 5°C, and in an inert !5 solvent, for example diethylether, tet!־ahydrofuran, 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.

DESCRIPTION

ASSAY SUITABLE FOR DETECTION OF CLAVULANIC ACID

Principle of.the Assay

Solutions containing clavulanic acid (culture filtrate, samples from isolation procedure and the like) are) incubated for 15 minutes with a β-lactamase preparation in 0.05M phosphate buffer at pH 7 and 37°C. During this time, enzyme inhibition 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 is determined by . the hydroxylamine assay for penicillin. The amount of β-lactamase used is such as to give 75% hydrolysis of the benzylpenicillin in 30 minutes at 37°C.

The extent of hydrolysis is a reflection of the amount of enzyme remaining uninhibited. The results are expressed as per cent inhibition of the enzyme activity by a given dilution of the clavulanic acid - containing solution (e.g. culture filtrate) or the concentration of clavulanic acid (dg/ml) giving 50% inhibition of the enzyme under the above stated conditions (I^_Q).

β-lactamase Enzyme

The β-lactamase produced by Escherichia coli JT4 is used as an enzyme. This culture is an ampicillin resistant 25 strain and owes its resistance to the production of an ί

R-factor controlled β-lactamase. Other similar R-factor controlled β-lactamases may be used if desired.

The culture maintained on nutrient agar slopes, is inoculated into 400 ml. of sterile Tryptone medium contained in a 2 liter conical flask. This medium has the following composition Tryptone (Oxoid) 32 g/1, yeast extract (Oxoid) 20 g/1, NaCl 5 g/1 and CaCl₂6H₂0 2.2 g/1. The final pH was adjusted to 7.4 with dilute NaOH. The flask is shaken at 25°C for 20 hours on a rotary shaker 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 an 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 supernatant is diluted in 0.005M phosphate buffer until it gives about 75% hydrolysis of $ 1 mg/ml. solution of benzylpenicillin in 30 minutes at 37 C.

Assay Procedure

Suitable dilutions of the inhibitor preparation and β-lactamase solution are mixed and incubated at 37°C 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 37°C. The.residual benzylpenicillin in each mixture is then estimated using the hydroxylamine assay as described by Batchelor et al, Proc. Roy. Soc., ^B 154, 498 (1961). 6 ml. of hydroxylamine reagent are added to all tests, controls and blanks and are allowed to react for 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 id an

E.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:

<td> Components (all dissolved in or diluted with 0.005M pH 7 phosphate buffer)</td><td> Test</td><td> Benzyl-־ penicillin Blank ml.</td><td> Control ml.</td><td> Reagent Blank ml.</td>

<td> Escherichia coli β-lactamase solution</td><td> 1.9</td><td> 0.0</td><td> 1.9</td><td> 1.9</td>

<td> Inhibitor solution</td><td> 0.1</td><td> 0.0</td><td> 0.0</td><td> 0.0</td>

<td> Benzylpenicillin 5mg/ml.</td><td> 0.5</td><td> 0.5</td><td> 0.5</td><td> 0.0</td>

<td> 0.005M pH 7 phosphate buffer</td><td> 0.0</td><td> 2.0</td><td> 0.1</td><td> 0.6</td>

V ? ./

Calculation of Results

The percentage inhibition of the β-lactamase is calculated as follows Absorption of benzylpenicillin blank minus absorption 5 of control (uninhibited reaction) = x

Absorption of test (inhibited reaction) minus absorption of control (uninhibited reaction) = y ^y% inhibition ־־ - x 100 x

To obtain the I^_o value, the inhibitor preparation is diluted until 50% inhibition of the β-lactamase inactivation of benzylpenicillin is obtained in the above procedure.

DESCRIPTION

PAPER CHROMATOGRAPHIC DETECTION OF CLAVULANIC ACID

Culture filtrate and a reference solution of clavulanic acid (250 pg/ml partially purified preparation), ל are spotted (20 μΐ/origin) onto Whatman No.l paper strips cm. wide. The chromatograms are run by descending chromatography for 16 hours at 5°C using n-butanol/isopropanol/water, 7/7/6 v/v as solvent. The strips are dried at 40°C and laid on agar plates containing

6 pg/ml benzylpenicillin and seeded with a (3-lactamase producing strain of Klebsiella aerogenes (synergism system). The plates are incubated overnight at 30°C and clavulanic acid revealed as a zone of inhibited growth.

The Rf value of the zone was 0.46. The 6 pg/ml benzyl15 penicillin alone is below the concentration required to kill the Klebsiella aerogenes but in the presence of a β-lactamase inhibitor, this concentration becomes toxic, that is to say there is synergism.

Use of the above synergism system enables clavulanic acid to be detected at concentrations below those at which it shows antibacterial activity.

DESCRIPTION

THIN LAYER CHROMATOGRAPHIC DETECTION OF CIAVULANIC ACID

SODIUM SALT

Solutions of clavulanic acid sodium salt preparations are spotted (5 μΐ of Img/ml) onto glass plates coated with a 0.25 mm layer of silica gel (F25A) as supplied by E. Merck, Darmstadt, Germany. The chromatograms are run at 22°C using the top phase of the mixture n-butanol/ethanol/water 4/1/5 v/v. The chromatogram plates are dried at 40°C and clavulanic acid sodium salt located by bioautography on agar plates containing 6 pg/ml. benzylpenicillin and seeded with Klebsiella aerogenes (synergism system - see section on paper chromatography above). The agar surface is covered by a fine filter cloth before laying the TLC plate onto it. After allowing 1550־ 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 30°C to reveal the zones of inhibited growth. The R_fvalue of clavulanic acid sodium sa?»t in the above solvent is approximately 0.37. Two spray reagents, Ehrlich and triphenyltetrazolium chloride are also used to reveal the clavulanic acid sodium salt zone. The former reagent consists of 300 mg of p-dimethylaminobenzaldehyde dissolved in 9 ml. of ethyl alcohol, 5-+ ml. of n-butanol and 9 ml of concentrated HC1. On heating the sprayed TLC plate at 120°C 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

ץ ' λ^ζ

4% solution of this compound in methanol with 1 volume of methanolic sodium hydroxide. After spraying, the TLC plates are heated at 80°C. Clavulanic acid sodium salt appears as a red spot on a white background.

EXAMPLE 7

CULTIVATION OF STREPTOMYCES CLAVULIGERUS

Streptomyces clavuligerus was cultivated at 26°C on agar slopes containing 1% Yeatex (yeast extract), 1% glucose and 2$ Oxoid agar No.3, pH 6.8. A sterile loop was used to transfer mycelium and spores from the slope into 100 nil of a liquid medium in a 500 ml Ehrlenmeyer flask. The liquid medium had the following composition:Oxoid Malt Extract 10g/1

Oxoid Bacteriological Peptone 10g/1

Glycerol 20g/1

Tap water 1 liter

Tha medium was adjusted to pH 7.0 with sodium hydroxide solution and 100 ml. volumes dispensed into !5 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 26°C on a rotary shaker with 2 inch throw and a speed of 240 r.p.in. Production stage flasks containing the liquid medium described above were inoculated with 5% vegetative inoculum and grown under the same conditions as. the seed flask. Samples of culture filtrate were assayed for inhibitor action against the β-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 R^ 0.46 was seen when the X culture filtrate was examined by the paper chromatographic . ' \ b i method previously described. The increase in size of the zone paralleled the increase in the β-lactamase inhibitor assay. Streptomyces clavuligerus was also cultivated in 2 ־ 5 lit 1׳e shaken flasks containing 400 mis. of medium (Production stage) using the same medium and cultural conditions as described earlier in this Example. In ׳ these larger vessels, growth of the organism was slower and optimum β-lactamase inhibitory activity was achieved 10 7-9 days after inoculation with the vegetative seed.

The results are also shown in Table 1.

TABLE

P-Lactamase Inhibiting Activity of Streptomyces clavuligerus Grown in 500 ml. and 2000ml. Flasks

<td></td><td> % Inhibition of</td><td> Escherichia coli</td>

<td rowspan="2"> Fermentation Time (Days)</td><td colspan="2"> β-lactamase at a final dilution of 1/2500 of culture filtrate</td>

<td> 500 ml. Shaken Flask</td><td> 2000 ml. Shaken Flask</td>

<td> 1</td><td> 15</td><td> —</td>

<td> 2</td><td> 30</td><td> —</td>

<td> 3</td><td> 55</td><td> —</td>

<td> 4</td><td> 50</td><td> 10.</td>

<td> 5</td><td></td><td> 21</td>

<td> 6</td><td> 57</td><td> 36</td>

<td> 7</td><td> —</td><td> 51</td>

<td> 8</td><td> ״־*</td><td> 53</td>

<td> 9</td><td> —</td><td> 50 !----------—----״—</td>

<img file="IL47087A_D0011.tif" />

EXAMPLE

CULTIVATION OF STREPTOMYCES CLAVULIGERUS

A seed flask prepared as in Example 1 was used to inoculate 500 ml. conical flasks containing 100 ml.

aliquots of the following medium in deionised water:Soluble Starch 2%w/v

Glycerol 0.3%w/v

Scotasol 0.1%w/v

Arkasoy 1%w/v

FeS0_z,.7H₂0 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 26°C on a rotary shaker as in Example 1. Optimum titre of !5 clavulanic acid was achieved between 3-5 days. A dilution of 1,/2500 of the culture filtrate gave 60% inhibition in the β-lactamase inhibition assay. A zone of clavulanic acid was seen at R^ 0.46 when using the paper chromatographic (bioautographic) method previously 20 described. This zone increased in size in parallel with the increase of the activity in the β-lactamase inhibitor assay.

[Soluble starch supplied by British Drug Houses Ltd., Poole, U.K.;

Scotasol is dried distillers solubles supplied by Thomas Borthwich Ltd., 60 Wellington Street, Glasgow, UKJ Ai'kasoy is soya bean flour supplied by British Arkady Co., Old Trafford, Manchester, UK ].

EXAMPLE

CULTIVATION OF STREPTOMYCES CLAVULIGERUS

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 deionised water and sterilised as previously described. The inoculum level was 5%.

Dextrin

Arkasoy

Scotasol

FeSO^ 7H_?0

2% w/v

1% w/v

0.1% w/v

0.01% w/v

The inoculated flasks were shaken at 26°C. Optimum β-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, UK J

״ 45 _

EXAMPLE 4

CULTIVATION OF STREPTOMYCES CLAVULIGERUS

The seed stage as described inExample 1 was used to inoculate 500 ml. conical flasks containing the following medium prepared in deionised water.

Dextrose 1%w/v

Soyabean Meal 1$w/v

Scotasol . 0.05%w/v

CaC0₇ 1%w/v

These flasks were treated exactly as in previous Examples and cultured under identical conditions. β-lactamase inhibitory aetr/ity was produced between

3-5 days. Culture filtrate at a final dilution of 1/2500 gave 35-45% inl.1ibit.ion in the β-lactamase inhibition assay.

EXAMPLE

CULTIVATION OF STREPTOMYCES CLAVULIGERUS β-lactamase inhibitory activity attributable to clavulanic acid was produced using the following medium with identical seed stage and cultivation conditions to Example 1.

Glycerol 2% w/v

Soyabean Meal 1.5% w/v

Mg SO^ 0.1% w/v ^k2^HP04 °.1% ^w/^v

Medium prepared in deionised water β-lactamase inhibitory activity reached a maximum level between 3-5 days and was of a similar order to that produced in Example4 ־,

EXAMPLE

CULTIVATION OF STREPTOMYCES CLAVULIGERUS

-an _ ־ 111 11———— ־ ' ——

The following medium produced clavulanic acid when using the conditions and vegetative seed inoculum as described in Example 1.

Glucose 2%

Lab Lemco (Oxoid) 1%

Oxoid Yeast Extract 0.3%

CaCO₇ 0.3%

ח

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 β-lactamase enzyme. inhibition assay.

״

EXAMPLE 7

CULTIVATION OF STREPTOMYCES CLAVULIGERUS

As in Examples 4, 5 and 6 the following medium produced 35-45% inhibition (1/2500 dilution) in the β-lactamase assay at the optimum titre which is reached 3-5 days after inoculation. All conditions were as previously described.

Glucose 2%w/v

Arkascy 1%w/v

CaCO^ 0.02%w/v

CoC1₂.6H₂0 0.0001%w/v

Medium prepared in deionised water

EXAMPLE 8

CULTIVATION OF STREPTOMYCES CLAVULIGERUS

The following production stage medium when used under standard cultivation conditions as described in previous Examples produced 20-J0% inhibition at 1/2500 dilution in the β-lactamase assay between 3-5 days after inoculation. Using the paper chromatographic method previously described, a zone of clavulanic acid was seen at R^. 0,46 when culture filtrate was examined.

Scotasol 2%

Oxoid Yeast Extract 1%

Medium prepared in tap water Final pH 7.0

EXAMPLE

CULTIVATION OF STREPTOMYCES CLAVULIGERUS

Under standard cultivation 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 β-lactamase inhibition assay.

g/1

Glycerol15

Sucrose20

Proline2.5

Monosodium Glutamate1.5

NaCl5;0

Κ₂ΗΡ0^2.0

CaCl₂0.4MnCl₂4H₂00.1

FeCl,6H_o00.1 ^ά

ZnCl₂0.05

MgS0₄7H₂01.0

Medium prepared in deionised water

Final pH 7.1

EXAMPLE

CULTIVATION OF STREPTOMYCES CLAVULIGERUS

A stock Yeatex/glucose agar slope was used to inoculate a Yeatex/glucose agar slope in a Roux bottle by making a mycelium/spore suspension in sterile ^,water. The Roux bottle slope was incubated at 26°C for 10 days. To this slope 100 mis. 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.

<td> Oxoid Malt Extract</td><td> 1% w/v</td>

<td> Oxoid Bacteriological Peptone</td><td> 1% w/v</td>

<td> Glycerol</td><td> 1% w/v</td>

<td> 10% Pluronic LSI Antifoam in Soyabean Oil</td><td> 0.05% w/v</td>

<td> [Pluronic supplied by Jacobs and</td><td> Van den Berg UK</td>

<td> Ltd., 231 The Vale, London. W3 &lt;</td><td> containing a poly</td>

propylene-polyethylene block polymer, and Soyabean Oil supplied by British Oil and Cake Mills Ltd., Stoneferry Road, Hull, U.K. ]. The medium was contained in a 90 litre stainless steel baffled fermenter, agitated by a 5״ vaned disc impeller at 240 r.p.m. Sterile air was supplied at 50 1 /min and the tank incubated at 26°C.

After 72 hours, the seed fermenter was used to inoculate 150 litre of the same medium using a 5% v/v

־

I 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 !50 1/min. The fermentation was maintained at 26°C. Antifoam was added when required in 10 ml. shots (10% Pluronic L81 in soyabean oil). Samples were removed for 6-lactamase inhibition assay at regular intervals. The fermenter was harvested between 4-5 days at the optimum level of .β-lactamase inhibitory activity (Table 2).

TABLE 2 β-Lactamase Inhibitory Activity of Samples of Culture Filtrate taken from a 300 litre Fermentation of Streptomyces Clavuligerus .

<td> Fermentation Time . (days)</td><td> % Inhibition in β-lactamase Inhibition Assay at a Final Dilution of 1/200ע</td>

<td> 1.0</td><td> 12</td>

<td> 1.5</td><td> 20</td>

<td> 2.0</td><td> 3!</td>

<td> 2.5</td><td> 36</td>

<td> 3.0</td><td> 50</td>

<td> 3.5</td><td> 54</td>

<td> 4.0</td><td> 51</td>

<td> 4.5</td><td> 56</td>

<td> 5.0</td><td> 55</td>

חי .

EXAMPLE

CULTIVATION OF STREPTOMYCES CLAVULIGERUS

The seed fermenter was run exactly as described in Example 10 using the same medium.

After 72 hours, the seed fermenter was used to give a 5% v/v vegetative inoculum into a 300 litre stainless steel fully baffled fermenter 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 !θ 150' Ι/min. The fermentation was maintained at 26°C.

Antifoam was added when required in 10 ml. shots (10% Pluronic LSI in soya bean oil).

The medium used in the production stage was as described in Example 3 with the addition of 0.05% v/v 15 of 10% Pluronic L81/soyabean oil antifoam prior to sterilisation.

The β-lactamase inhibitory activity of fermentation samples was similar to those of Example 10 (see Table 2). Paper chromatographic examination revealed a zone of 20 clavulanic acid at R_f 0.46 using the bioautographic (synergism) method previously described. The size of the clavulanic acid zone increased in parallel with the increase in the β-lactamase inhibitor assay.

EXAMPLE 12

CULTIVATION OF STREPTYMYCES CLAVULIGERUS

100 mis of sterile water was added to a sporing culture which had been grown on Bennetts agar in a Roux bottle for 10 days at 26°C. A mycelium/spore suspension was produced and used to inoculate 75 litres of steam sterilised medium of the following composition in tap water.

<td> Dextrin</td><td> 2%</td><td> W/V</td>

<td> Arkasoy ’50’</td><td> 1%</td><td> W/V</td>

<td> 10% Pluronic L81</td><td> 0.03%</td><td> V/V</td>

in soyabean cil

The pH of the medium was adjusted to 7.0

The medium was contained in a ICO litre stainless steel baffled fermenter, agitated by a 7i vaned disc impeller at 140 rpm. Sterile air was supplied at .75 1 /minute and the tank incubated for 72 hours at 26°C.

The contents of the seed fermenter were used to inoculate 1500 litres cf steam sterilised medium of the following composition in tap water.

Arkasoy ’50’ 1.5% W/V

Glycerol 1.0%, י W/V kh₂po₄ 0.1% 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 added in 25 ml amounts as required.

(10% Pluronic LSI in soyabean oil). The fermentation was controlled at 26°C until a maximum yield of clavulanic acid was obtained between 3-5 days when 200 - 300 με/πιΐ of clavulanic acid were produced.

־

EXAMPLE

CULTIVATION OF STREPTOMYCES CLAVULIC-ERUS

Inoculum was produced in a seed flask as previously described, but using the medium described in Example 3 (with pH of the medium adjusted to 7.0). This was used to inoculate 500 ml conical flasks containing 100 ml aliquots of the following medium prepared in deionised water and sterilised ׳. The inoculum level was 5%.

Prichem P224 1% W/V . Arkasoy ’50 1.5% ׳ W/V KH₀PO. 0.1% W/V

The pH of the medium was adjusted to ,7.0 The inoculated flasks were shaken at 26°C and ^Ύ optimum β-lactamase inhibitory activity was achieved between 3-5 days. Levels of 300 - 500 p,g/ml of clavulanic 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.

EXAMPLE 14

ISOLATION OF CRUDE CLAVULANIC ACID SODIUM SALT

Harvested culture liquor produced as described in

Example 10 was clarified by continuous flow centrifugation and the mycelium discarded. From 150 litre of fermentation liquor 120 litre of clarified culture fluid was obtained. This filtrate gave 58% inhibition in the β-lactamase inhibition assay at 1/2500. The filtrate was chilled to 5°0 and 40 litre of n-butanol added. The mixture was stirred and 25% H^SO^ added until the pH was 2.0. The acidified mixture was stirred for a further mins, before separating the phases by centrifugation.

The aqueous phase was discarded.

extract 0.5% of Norit CSX carbon mixture stirred for 15 minutes.

To the n-butanol was added and the

The carbon was discarded after removal by filtration using a diatomaceus earth as a filter aid. To the n-butanol a | volume of deionised water was added and the mixture stirred while adding 20% NaOH solution until the pH had equilibated t 7.0. The phases were separated by centrifugation and the n-butanol phase discarded. The aqueous phase was concentrated under reduced vacuum to 800 ml. and then freeze dried. This yielded 35g. of a preparation of clavulanic acid with an crude solid

I of 1.3 gg/ml 50

This solid in the β-lactamase inhibition, assay, preparation was stored dry at -20°C while awaiting further purification.

EXAMPLE 15

ISOLATION OF CRUDE CLAVULANIC ACID SODIUM SALT

One litre of culture filtrate giving 55% inhibition at 1/2500 in the β-lactamase inhibition assay and obtained as described .in Example 12 was percolated down a 1 inch diameter x 6 inch column of Permutit Isopore resin FF IP (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 wat^r to wash the column. Elution of the active β-lactaraase inhibitor was achieved with 0.2M NaCl solution. Fractions (20 ml.) were collected and assayed at a 1/2500 final dilution in the β-lactamase inhibition assay. Active fractions were combined and concentrated 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 Laboratories, 32nd and Griffin Ave., Richmond, California, U.S.A.]. The active fractions, as determined by the β-lactamase inhibition assay, were combined. Sodium chloride eluted after clavulanic 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 0.45g. of a crude solid preparation of clavulanic acid having an _of 0,92 pg/ml.

This solid was stored at -20°c while awaiting further purification. - 60 EXAMPLE 16

ISOLATION OF CRUDE CLAVULANIC SODIUM SALT

Culture filtrate containing 300 ng/ml of clavulanic acid is acidified using an in-line mixer system, extracted 5 with n-butanol and clavulanic acid is back extracted into water at neutral pH.

Chilled culture filtrate (5 - 10°C) was pumped to an in-line mixer at the inlet of which, enough 6% (v/v) nitric acid was added to maintain an outlet pH of 2.0 0.1 ״. The 10 acidified filtrate was passed at 4.1/min 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 passing into a three stage counter current sepai'ator (Westfalia Separator Ltd., Model EG 1006).

Chilled water saturated n-butanol (at about 5°C) was pumped at 3 ]./min into the counter current separator.

The aqueous outlet from the counter current separator was run to waste. Entrained water was removed from the butanol outflow of the counter current separator using a 20 liqaid/liquid centrifugal separator. (Alfa Laval Ltd. Model 3024X - G). The butanol was collected in a stainless steel vessel fitted with a cooling jacket in which it was stored at abou€ 5°C.

From the vessel, 40 1 aliquots were removed and 25 thoroughly mixed with 2 1 of chilled water (5°C), saturated with n-butanol. The pH of this mixture was adjusted to pH 6.8 - 0.1 using 20% sodium hydroxide solution.

This aqueous extract/butanol mixture was fed to a liquid/liquid centrifugal separator (Sharpies Centrifuge ל Ltd. Model M35PY '5 ־־ PH) at a pumped rate of 2 1/min.

From 1800 1 of cultui'e filtrate, 90 1 of aqueous phase was recovered, containing 39% of the clavulanic acid present in the culture filtrate.

1 of the aqueous extract was adjusted from 2%, to 8%, total solids by ths addition of 60 g sodium chloride per litre, and spray dried (Anhydro, Copenhagen, Type Lab S 1). The conditions used were: Feed rate 2 1/hr Atomizer voltage 170 v; Heater setting 6—7;

Inlet terap 150°C; Outlet temp 80°C.

The dried product, total weight 1 kg., contained

62% of the clavulanic acid present in the feedstock.

The remaining 75 1 of aqueous extract was concentrated by ultrafiltration (De Danske Sukkerfabrikker. Laboratory Module, Membrane Type 900). The operating procedure was to re-circulate the retentate from a stainless steel tank, fitted with a cooling system, with the outlet valve set 50 as to give a differential pressure across the 40 membranes of 25 atmospheres. The temperature was maintained at 2 - 5°C and the pH at 6.8 i 0.1 by addition of 2N hydrochloric acid, as necessary. The volume was reduced to 34 1 which contained 72% of the clavulanic acid present in thevfeedstock. v.

The aqueous concentrate was stored at about 5°’C,' 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.

EXAMPLE

PARTIAL PURIFICATION OF CRUDE CLAVULANIC 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 value of l.J pg/ml (final concentration) were dissolved in 25 ml. of distilled water and applied to a 1-2 x 16 bed of Permutit FF IP (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 ths chromatographic column. 10 ml. cuts were collected and β-lactamase inhibitory activity assayed using a 1/2500 dilution of the fi’actions. Activity was eluted after a main band of colour between fractions 24 and 30. The active fractions were combined and concentrated 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 β-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. β-lactamase inhibitory activity correlated with the pink or. red spots respectively produced by these reagents. Active cuts were combined, excluding those containing sodium chloride and concentrated under vacuum to dryness. This yielded 20ל rag. of partially purified clavulanic acid sodium salt with an of 0.2 pg/ml in the standard 3-lactamase inhibitor assay.

Thin layer chromatography (silica gel) of this clavulanic acid preparation gave the following values: n-butanol/ethanol/vzater 4:1:5 v/v top phase FL. 0.57;

n-butanol/acetic acid/water 12:3:5 v/v R_f 0.44;

isopropanol/water 7:3 v/v 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 R^ values of 0.38; 0.39 and 0.77 15 respectively.

־ 65 EXAMPLE 18

PARTIAL PURIFICATION OF CLAVULANIC ACID SODIUM SALT

Culture filtrate produced as described in .י’ ־ Example 12 was solvent extracted as in Example 14 to give a solid preparation which was further purified by ion exchange chromatography using Whatman diethylaminoethyl cellulose DE 52. This solid (10g.) was dissolved in 20 ml. of distilled water and applied to a It x 20^,!column of DE 52 cellulose previously equilibrated with 0.01M sodium phosphate buffer pH 7*5. The column was eluted with a NaCl gradient. 0.1M NaCl in 0.01M sodium phosphate buffer pH 7-5 was fed into a mixing chamber containing 1 litre of 0.01M phosphate buffer pH 7.5 which in turn was connected to the column. Fractions (10 ml.) were collected and these were assayed for β-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 β-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.

EXAMPLE 19

ISOLATION OF SOLID CLAVULANIC AciD SODIUM SALT

A partially purified solid preparation of clavulanic acid (500 mg) prepared as in Example ^7 was loaded onto a Whatman microcrysialhiieCC 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 wa.s run at 4°C and 4 ml. fractions collected. Fractions were tested for the presence of clavulanic 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 β-lactamase inhibition assays at a 1/1250 dilution. Active fractions were combined and dried under vacuum on a 1’otary evapoi’ator. The solid v/as dissolved in a small volume of distilled water and freeze dried. A white solid preparation of the sodium . salt of clavulanic acid was obtained (40 mg) having an I θ of 0.08 pg/ml in the β-lactamase inhibition assay.

י EXAMPLE

ISOLATION OF SOLID CLAVULANIC ACID SODIUM SALT

Concentre.ted back extract (6 1) (from ultrafiltration in Example 16 ) containing 10 g of clavulanic acid as determined by the β-lactamase inhibition assay of Description 1. This was percolated at 1 1/hr onto a 2 x 24” column of Permutit Zerolit FF 1 P SRA 62 anion exchange 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 reservour containing 4 1 of 1.4 m NaCl feeding a stirred reservoir containing 4 1 of 0.7'NaCl which in ־^urn was connected to a stirred reservoir containing 4 1 of deionized water which was connected via a pump ־co the column. The column was eluted at

2.5 ml/min and 25 ml fractions collected. Fractions were assayed by the β-lactamase inhibition assay .

• Active fractions (nos. 140-230) were combined and vacuum evaporated to near di'yness.

Ethanol (500 mis) was then added and the solid filtered off after vigorous shaking. The ethanol extract was then vacuum evaporated to dryness on a rotary avaporator and redissolved in deionized water (40 mis). This was loaded onto a 4” x 24” column of Biorad Biogel2?־ ^and eluted with a 1% n-butanol solution. Fractions were collected.(25 ml) and assayed for β-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 clavulanic acid free of sodium chloride were 5 combined, concentrated by evaporation of the solvent under reduced pressure to 20 mis and then freeze dried. This yielded 4.8 g of the sodium salt.of clavulanic acid. (I about 0 06 μ-g/ml)

EXAMPLE 21

PREPARATION OF AN ESTER OF CLAVULANIC ACID (METHYL ESTER)

<img file="IL47087A_D0012.tif" />

19.8 mg. of the sodium salt of clavulanic acid was dissolved in 0.5 ml. dry dimethylformamide and treated wirh 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. cn silica gel (Kieselgel 60F254 supplied by Ξ. Merck, Darmstadt, Germany) eluting with ethyl acetate to give clavulanic acid methyl ester as a colourless oil (R^ 0-38; red colour with triphenyltetrazolium chloride spray) which had the following properties:

Analysis: Found C 50.49 H 5.43 N 6.29

C Η_ηΝ0 Requires C 50.70 H 5.20 N 6.57 )imax (Methanol): no absorption ?215

V׳max (Film): 3300 - 3600 (Broad), 1800, 1750, 1695 cm ־“־ Approximate 1st order N.M.R. (CDCl^): 2.49 (broad S, 1, exchanged with D₂0), 3.05 ״ 70 (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, J_? = 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 C'rf״NO_r : 213.0637

11 5 .

Thin ].ayer chromatography of the methyl ester showed a single zone in each of the following solvent systems; butanol/ethanol/water 4:1:5 v/v top phase 0.75; isopropanol/water, 7:3 v/v R_f 0.95; ethylacetate/ ethylalcohol 8:2 v/v R^ 0.87. The zones were detected by oicartography using Klebsiella aerogenes with added benzylpenicillin (synergism system).

i ) י EXAMPLE 22

PREPARATION OF AN ESTER OF CLAVULANIC ΑΓΙΡ (p-nitrobenzyl estejg)

<img file="IL47087A_D0013.tif" />

<img file="IL47087A_D0014.tif" />

Treatment of the sodium salt of clavulanic acid with p-nitrobenzyl bromide in dry DMF gave, after P.L.C., a colourless oil. which crystallised from chloroform ether to give co p-nitrobenzyl ester of clavulanic acid as white feathery needles, m.p. Ill - 112 C, which on recrystallisation had a mp of 117.5 - 118°C.

EXAMPLE 2צ־

PREPARATION OF AN ESTER OF CLAVULANIC ACID (BENZYL ESTER)

<img file="IL47087A_D0015.tif" />

Impure 5״3)־hydroxyethylidine)-7-oxo-4-oxa-r-azabicyclo [3,2,0]heptane-2-ca.rboxylic acid sodium salt (thought 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.

17-18°C) 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-(3”hydroxyethylidine)־־7-־oxo-4-oxa-l׳־azabicyclo [3,2.0]heptane-2-’Carboxylic acid 63 mg.) as a colourless oil. i.r. (film) 1800, 1745, 1695 0111^-¾ n.m.r. (CDCl^), 2.25 (s,l, exchangeable with D₂0), 3.05 (d,l,J = 17Hz), 3.51 (dd.l .!17־־ Hz, J_o=2.5Hz), 4.24 (d,2,J7.5־Hz), 4.92 (at, l,J=7.5Hz, J₂=1.5Hz), 5.15 (d,l,J1.5־־Hz), 5.24 (s,2), 5-71 (d,l,J=2.5 Hz), 7.45 £ (s,5).

EXAMPLE 24

PREPARATION OF THE BENZIE ESTER OF CLAVULANIC ACID FROM ^^»»^.^^^,.ר.,. !11׳ III! ι.ι.Μ1ιι&lt;^»»Μ.ι.ίΜ.ιι.1 «— ¹«׳«f י-—— '» w»'¹ !י י ι.μμ

CRUDE EXTRACTS GF THE CULTURE FILTRATE OF S.CLAVULIGERUS

Culture filtrate 20 1. obtained as described in Example 10 was vacuum evaporated using a climbing film evaporator to 5 1. The concentrate was then freeze dried using an Edwards E.F.6 shelf freeze drier manufactured by Edwards High Vacuum Ltd. The 300g. of solid so obtained contained 3 g. of sodium Clavulanic acid as determined by the enzyme inhibition assay. The solid was suspended in 900 ml. of dry dimethylformamide and 150 ml. of benzyl bromide was added. . The mixture .was 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 volimie 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 2j3,5-triphenyl-tetrazoliu1n chloride (TTC) spray reagent.

.-74'י 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 0.31 at 22°0.

Fractions containing this ester were. combined and concentrated to 15 ml. and this solution was further chromatographed on a ZL-g־*’ x 16” silica gel column (Merck silica., gel H, type 60) with chloroform/ethyl acetate 10 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, 15 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.

EXAMPLE 23

PREPARATION CT CLAVULANIC ACID BENZYL ESTER W .־ I. I 11 I 11 1.1 II1 I—»1 WWW-TrrWT»» .II ιυ—I'll

Spray dried solid (5.3 kg) 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 mis. 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. Ths filtrate was vacuum evaporated to an oily residue (212 g). The residue was loaded onto a column containing a 4 x 15” bed of silica gel (Hopkins &amp; 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 benzyj. cj-avulanate convent by spotting 0!!ν0 silica gel tic plates (Merck precoated silica gel

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 on silica gel with chloroform/ethyl acetate 8:2 as the solvent and spraying the developed plates with TTC spray. Fractions 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 ״ 76 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^' x 18 column of Sephadex LH 2.0 ^Pfrurmaoia) in cyclohexane/chloroforrn

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 LH20 is a hydroxypropyl derivative of 'Sephadex Q25 supplied by Pharmacia Great Britain,

Uxbridge Road, Loncon W5, U.K.) ^: EXAMPLE 26

¾.11^1 rur~1r .11 *1

PREPARATION OF CLAVULANIC ACID BENZYL ESTER

Culture filtrate (150 1) pH 7.0 contained 16.2 g. of clavulanic acid (sodium salt) .as determined by the enzyme inhibition assay. This filtrate was stirred with kg. of Amberlyst A.26 anion exchange resin in the chloride- form (Rohm &amp; Hass 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 1. of deionised, water followed by 20 1. ’of . acetone and 10 1. of dimethyl formamide (DMF). After refiltering the rosin was suspended in 2.3 1. of DKF/0.2 M Nal. To ’this was added 200 mis. 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 cn 3” x 13” silica gel column. (Merck silica,gel H type 60) with ethyl acetate/cyclphexane 8:2 as the solvent.' Fractions containing benzyl clavulanate. were selected by spotting onto silica gel tie plates and spraying with TTC reagent as described.

previously (Example 24). Those selec?>od'were concentrated to 20 mis and then . - 78 ' chromatographed on a 1¾ x 18 silica gel column (Merck silica gel H type 60) with chloroform/ethyl acetate8:2 as the solvent. Selected fractions were combined and evaporated to a colourless oil (440 mgs) which was 90% benzyl clavulanate as determined by NMR spectroscopy.

״ 79 ״

EXAMPIE

PREPARATION OF THE BENZYL ESTER OF CLAVULANIC ACID FROM

CRUDE EXTRACTS OF THE CULTURE FILTRATE OF

S. CLAVULIGBRUS

An aliquot, of aqueous back extract of the butanol extract of culture filtrate obtained as described i.1״ 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 acetat and the mixture filtered. The filtrate was concentrated tc an oily residue on a vacuum rotary evaporator. This residue was loaded onto a 2” x 14 silica gel column (Biogel Biosil A.100 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.

EXAMPLE 28

PREPARATION OF CLAVULANIC ACID SODIUM SALT

<img file="IL47087A_D0016.tif" />

co₂ch₂c₆h₅

<img file="IL47087A_D0017.tif" />

Substantially pure benzyl clavulanate (281 mg) 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 clavulanate (135 mg.)

EXAMPLE

HYDROLYSIS OF CLAVULANIC AC IP METHYL ESTER TO

CLAVULANIC ACID

2.17 mg. of clavulanic acid ester was dissolved in 0.1 ml. ל methanol and treated with 0.203 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 major components had an R_f identical to that of the sodium salt of clavulanic acid; colour reactions and biological assay were consistent with this component being tne 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 37°C 15 in 0.0.5M phosphate buffer at pH 7. The reaction was followed by paper chromatography (bioautographic system). Using the butanol/ethanol/water system to follow the reaction over a period of 2 hours the zone of the methyl ester at R_f 0.79 decreased in size as the zone 20 of clavulanic acid at 0.12 increased.

- &lt;32 EXAMPLE 30 'ANTIBACTERIAL SPECTRUM OF CLAVULANIC ACID mm.»«, 1111! 1 11 » ot »&lt;wi mn

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 miorotitre plastic tray were inoculated with an overnight broth culture of each organism so that the final dilution of the inoculum was 0.5 x 10 A The cultures were incubated overnight and the points of bacterial growth recorded next morning by observing the turbidity of the culture. The results, expressed as approximate MIC values (minimum inhibitory concentration p.g/ml.) are recorded in Table 3 which. shows that the compound has a broad spectrum of antibacterial activity.

־ 83 TABLE 3

ANTIBACTERIAL SPECTRUM OF CLAVULANIC ACID SODIUM SALT

<td> Bacterial Strain</td><td> Minimum Inhibitory Concentration pg/ml.</td>

<td> Staphylococcus aureus (Oxford H)</td><td> 7.5</td>

<td> Staphylococcu3 aui'eus (Russell) .</td><td> 5״7</td>

<td> Bacillus subtil is KTWV w me » I«“— .!</td><td> 62</td>

<td> Streptococcus faecal-is</td><td> &gt;500</td>

<td> Streptococcus pyogenes CM 10</td><td> 125</td>

<td> Escherichia coli NCTC .1.0418</td><td> 31</td>

<td> Klebsiella aerogenes</td><td> . 62 ״ 31</td>

<td> Klebsiella oxvtocum</td><td> 62 |</td>

<td> Enterobacter T 624</td><td> 3!</td>

<td> Enterobacter cloacae</td><td> 62</td>

<td> Acinetopacter anitratus</td><td> 125</td>

<td> Providentis s tuartii</td><td> 125</td>

<td> Serratia raarcescens</td><td> 125</td>

<td> Proteus rnirabilis C977</td><td> 62</td>

<td> Proteus vulgaris W090</td><td> 31</td>

<td> Salmonella typhimurium</td><td> 31</td>

<td> Shigella sonnei Π־Ί W—MI 1!IW .I1־t IT</td><td> 62</td>

<td> Pseudomonas aeruginosa A</td><td> 500</td>

<td></td><td></td>

״ 84 ~

EXAMPLE 1י

RXAMPT.ES GF 8-LACTAMASE INHIBITION BY CLAVULANIC ACID

,---------------------------------. . ן ן! ך_.

SODIUM SALT

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

TABLE 4 1 !II 1m! 1—1~1-INHIBITION OF 3-LACTAMASES BY CLAVULANIC ACID nw ►‘י I»»., UW.M. .* ״» ־־w—.-- ־ד r.

<td> Source of β-lactamase</td><td> Approximate I _fי Value Relative to Escherichia coli JT 4=1</td>

<td></td><td> _____ ______.__________________________________________</td>

<td> Staphylococ:׳cus aureus (Russel 1) Escher!chic coli JT4 Escher.i.chia coli Bll Klebsiella asr0genes A Pseudomonas aeruginosa 1822 1R factor) Pseudomonas dalgleish</td><td> 1.0 !.0 2.0 0.6 5.° θ.¹</td>

With penicillin 0 as substrate the of clavulanic acid sodium salt against the β-lactamase of Stanh. aureus I^usselljis approximately 0.06 |1g/ral.

EXAMPLE

EXAMPLES OF ACTIVITY OF CLAVULANIC ACID METHYL ESTER

Tests for antibacterial' activity in broth shewed, clavulanic acid methyl ester to have broad spectrum activity ל 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 antibacterial synergism in combination with ampicillin or cephaloridine against bacteria resistant to these antibiotics. Thus, the minimum inhibitory concentration (M.I.C.) for ampicillin against Staphylococcus aureus (Russell) was reduced from 500 ^ig/ml. to &lt; O.-4׳ in the presence of 1.0 pg/ml.

clavulanic acid methyl ester. The M.I.C. for cephaloridine was reduced from 1.5 gg/ml. to &lt;0.05 ug/ml. in the presence of ?&lt;. gg־/ml. of clavulanic acid, methyl ester.

The M.I.C. for ampicillin against Proteus mirabijis C889 was reduced from 500 pg/ml. to 51 ug/ml. in the presence of 5 !ig/ml. clavulanic acid methyl ester.

EXAMPLE 55

PREPARATION PIVALOYLOXYMETHYL CLAVUI.ANATE

- ~ ' Ur.־:— . — . . ----------------— —

To a stirred solution of bromomethyl pivalate (0.57g) in dry dimethylformamide (5 ml) was added sodium . clavulanate (0.49g). After 2 hrs. at room temperature the reaction mixture was treated with etnyl acetate (20 ml), cyclohexane (10 ml) and water (20 ml). The mixture separated into two layers and the non-aqu&amp;ous 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. (CDCl^), 1.26 (s,9), 3.13 (d,l,J=17 Hz), 3.62 (dd, l,J,-17Hz, J1*2.5I&lt; 4.3(d,2,J=7.5Hz), 5.0 (dt, 1,J7.5־־H2, J₉1.5־Hz), 5.16(d;l;J=1.5Hz), 5.79(d,l,

J2.5־Hz), 5.92£( s;2); i.r. (liquid film), .־v β-lactam 0.0

1800 cm \ ester 0=0 1760 cm \

EXAMPLE

PREPARATION OF CLAVULANIC ACID PHTHALIDE ESTERS

To a stirred solution of 3-bromophthalide (0.43g) in dry dimethylformami.de (5 ml) was added sodium clavulanate (0.5g) and the solution was left at rcom 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 (NagSO^) and evapo10 rated to yield a pale yellow gum. The two diastereomeric esters were separated using high pressure liquid chromatography on a 40 cm x 10 mm column of silica gel (Merckosorb SI 60, 5 μ) eluting with ethyl acetate at a flow rate of 3 ml/min.

The first phthalide ester (retention time 7.15 min) crystallised from ethyl acetate as needles, mp 102°, and had the following i.r. (hujol mull) ׳^׳ β-lactam C=0 1790 cm“¹ ester C1755 0־־ cm ¹ n.m.r. (CD^COCDj):

3.14 (d,l,J=17.5Hz) 3-76 (dd,l,J,17.5־Hz, J2.5=₂־Hz),

4.25(d,2,J=7.5Hz), 5.0 (dt,l,J-,=7.5Hz, ^=1.5Hz), 5.4 (s,l,J1.5־Hz)5.82 (d,l,J=2.5Hz), 7.7 (s,l), 8.06$(m,4);

M.wt (mass spectrometry: 331-0696 corresponds to C₁₆H₁₃NO₇.'fcalc. 331.0692). The second diasterioisomer (retention time 8.85 min) had the followingi.r.(CH₂Cl₉solution) *\5 β25 lactam C=0 1800 cm“¹, ester 01780 0־ cm¹; nmr (CDClj)

2.42 (broad 5,1, exchangeable with D₂0), 3-12 (d,l, 1=18 ;Hz), 3.60 (dd,1,12=18 Hz, J₂=.2.5Hz), A.30 (d,2,J7.5־Hz), 5.0 (dt, 1,12=7.5 Hz., J₂=1.5 Hz), 5.12 (d,1,1=1.5 Hz), 5.76 (d,1,1=2.5 HZ), 7.52 (3,1), 7-85S(m,4).’

<img file="IL47087A_D0018.tif" />

<img file="IL47087A_D0019.tif" />

<img file="IL47087A_D0020.tif" />

EXAMPLE

PREPARATION OF 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 oilj l.r.(film) 1800, 1745, 1690 cm”“. M.wt. (mass spectrometry) = 525-1890 which corresponds to C,״H_o״N0 (calc. 325.1839).

27 5

EXAMPLE

PREPARATION OF CLAVULANIC ACID

Benzyl clavulanate (100 mgs) in ethanol (5 ml) was hydrogenated over 10% Pd/C (30 mgs) for 45 minutes 5 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. (C_cD_cN): 3.05(d,l,J=18Hz). 3.60(dd,l,Jn=18Hz,

5. 5 ' -*.

J₂= 2.5Hz), 4.75(d,2,J«7.5Hzj, 5.58(t,l,J=7.5Hz),5.66 (8,1), 6.0i(d,l,J25־Hz).

EXAMPLE

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 the residue purified by chromatography on silica gel, eluting with ethyl acetate. The fractions containing methyl clavulanate were comoined and evaporated to give a clear oil (104 mgs).

EXAMPLE

PREPARATION OF METHYL CLAVULANATE

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 evaporated in vacuo to give crude methyl clavulanate. The crude product was purified by chromatography on silica gel, eluting with ethyl acetate, to give a clear oil (140 mg).

f

ג

EXAMPLE

PREPARATION OF PHENYL CLAVULANATE

Clavulanic acid (100 mg) in acetonitrile (5 ml) was cooled and stirred, at 0°. To the solution was added phenol (0.94g) and dicyclohexyldicarbodiiEd.de( 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_s eluting with ethyl acetate-hexane (1:1) to give phenyl clavulanate (70 mg). I.r (film) 1800,

1770, 1690 cm¹״. N.m.r. (CDC!₅)2.18 (broad s,l), 3.06 (dd, 1,.Ι17־Ηζ,.Ι₂=0.9Ηζ), 3-34 (dd.l,^=17Ηζ,J₂= 2.6Hz), 4.29 (d,2,J=7.5Hz), 5.1(dt,l, ^=7.5Hz,J₂=1.5Hz) 5.29 (d,l,J=1.5Hz),5.76(dd,l,J₁=2.6Hz,.J₂0.9־Hz), 7.355(m,5).

M.vzt. (mass spectrometry) 0777־275 ־־ which corresponds to ^C14^H13^NO5 (calc, 275.0794).

EXAMPLE

PREPARATION OF 2,2,2-trichioroethyl clavulanate

Sodium clavulanate (221 mgs) was suspended in dry tetrb.hydrofuran (5 mis) and stirred at 0°. Trichloroל ethylchlorofcrmate (211 mg) in dry tetrahydrofuran (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 reauired produc⁴־ as a^r oil. i.r (film) 1800,1760,1690cm¹־. n.m.r. (CDC1J 1.56 (broad S,l), 3.07 (dd,1,J =17.5Hz,J₂=0.7Hz), 3.56 (dd,l,0^17.5Hz, J₂=2.5Hz), 4.24 (d,2,J7.5־Hz), 4.69 (d,l,J12־Hz),

4.92 (d,l,J=12Hz), 5.02 (dt, 1,^=7.51^, J₂=1.3Hz),

5.19(d,l,J=1.3Hz), 5.73 J^- (dd.l,J-j=2.5Hz, J₂=0.7Hz). M.wt. (mass spectrometry) = 328.9621 which corresponds t° ^c1c^h10^NC)5^c15 (calculated 328.9625).

<img file="IL47087A_D0021.tif" />

EXAMPLE

PREPARATION OF SODIUM CLAVULANATE

Benzyl clavulanate (840 mgs) in. ethanol (30 ml) and water (5 ml) was hydrogenated ovei10% ״ Pd/C (267 mgs) and sodium bicarbonate (244^xmgs) 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 P₉0^ vacuo for 24 hours gave the following analysis: C 41.01, 40.86; H 3.77, 3.64; N 5.68, 5.51;

i.r.(KBr disc) 1735, 1700, 1620 cm“¹; Nair (D₂0 ) 3.06 (d,l,J18.5־־Hz), 3-57 (da, 1,^=18.5Hz, J₂=2.5Hz),

4.15 (d,2,J=8Hz), 5.3 (HOD), 4.9(m), 5.71 (d,l,J=2.5Hz)

EXAMPLE 42

ANTIBACTERIAL SYNERGISM BETWEEN AMPICILLIN AND .- ־I-—- «ו ו ־ ־ ¹'‘.‘‘1 -----------------¹ , II II יי—! I Mil

CLAVULANIC ACID SODIUM SALT ; .. '׳L

The minimum inhibitory concentration (M.I.C, values) of ampicillin, clavulanic acid sodium salt find ampicillin in the presence of 1 gg/ml. clavulanic acid sodium salt were determined. for a range of β-lactamase producing bacteria. The organisms were inoculated into Oxoid sensitivity 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 pg/ml. clavulanic acid sodium salt (microtitre method)_c The final dilution of the overnight ”broth inoculum was 0.5 x 10“^. The tray was incubated at 37°C overnight and a record made next morning of the end points of bacterial growth. The M.I.C. values in μβ/πΐ. are recorded in Table 5 which reveals that the synergist at the low concentration of 1 μ6/π!1. markedly enhances the antibacterial activity of ampicillin against certain gram + ve and gram - ve bacteria. The mechanism of this synergism is likely to involve inhibition of ampicillin destroying 3״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.

״

TABLE

ANTIBACTERIAL synergism between ampicillin and

CLAVULANIC ACID SODIUM SALT

<td rowspan="2"> 1 Bacterial strain</td><td colspan="2"> Minimum Inhibitory Cone</td><td rowspan="2"> entrations pg/ml Ampicillin in presence of lpg/ml clavulanic acid sodium salt</td>

<td> Clavulanic acid sodium 1 salt</td><td> 1 Ampicillin</td>

<td> Escherichia coli</td><td></td><td></td><td rowspan="2"> &lt;0.4</td>

<td> NCTC 10481</td><td> ' 31</td><td> 1.8</td>

<td> Escherichia coli</td><td rowspan="2"> 62</td><td rowspan="2"> )500</td><td></td>

<td> B 11</td><td> 125</td>

<td> Klebsiella aerogenes A</td><td> .3!</td><td> 125</td><td> &lt;0.4</td>

<td> Klebsiella s;g 62</td><td> 31</td><td> 125</td><td> ^0.4</td>

<td> | Enterobacter</td><td></td><td></td><td></td>

<td> ן cloacae</td><td> 62</td><td> 250</td><td> 62</td>

<td> 1 Serratia</td><td></td><td></td><td></td>

<td> I marcescens</td><td> li־\ 1H</td><td> &gt;500</td><td> 62</td>

<td> S taphyl0 c 0 c cus</td><td></td><td></td><td rowspan="2"> &lt; 0.4</td>

<td> aureus (:Russell)</td><td> 15</td><td> 500</td>

<td> Staphylococcus I aureus ׳(a methi-</td><td> 62</td><td> 250</td><td> 7.5</td>

<td> 1 cillin resistant li strain) _ ________</td><td> L__-</td><td> L_____</td><td></td>

EXAMPLE

ANTIBACTERIAL SYNERGYSM BETWEEN CEPHALORIDINE AND CLAVULANIC ACID SODIUM SALT . .

The minimum inhibitory concentrations of cephaloridine, clavulanic acid sodium salt and cephaloridine in the presence of 5 pg/ml clavulanic acid sodium salt were determined by the method described in Example 42. The results in Table 6 show that synergism can be obtained between clavulanic acid sodium salt and cephaloridine particularly for the !*״lactamase producing strain of Staphylococcus aureus (Russell).

?.TABLE 6 nw־.MaM>n .*mk י

ANTIBACTERIAL SYNERGISM BETWEEN CEPHALORIDINE

AND CLAVULANIC ACID SODIUM SALT w» 1^«׳ιπι..1.ι.η

Minimum Inhibitory־Concentrations pg/ml.J

<td> nacvena.L strain</td><td> Clavulanic acid sodium salt</td><td> Cephaloridine</td><td> Cephaloridine in presence of 5μβ/ιπ1 claviianic acid sodium salt</td>

<td> Proteus mirabilis W</td><td> &gt;500*</td><td> 62</td><td> 7.5</td>

<td> Staphylococcus aureus(Russell</td><td> 15</td><td> 3.1</td><td> &lt; 0.05⁺</td>

<td> Staphylococcus aureus( acmethi</td><td></td><td> 15</td><td rowspan="2"> 3.7</td>

<td> pillin’ resistai</td><td> it</td><td></td>

saitrainT * Tailing Point

Same value obtained when synergist added at 1 pg/ml. instead of 5 pg/ml.

EXAMPLE 44

ANTIBACTERIAL SYNERGISM BETWEEN CLAVULANIC ACID SODIUM SALT AW VARIOUS PENICILLINS

The results presented in Table 7 were obtained by the method described in Example 42.

TABLE 7 .ANTIBACTERIAL SYNERGISM BETWEEN CLAVULANIC ACID

SODIUM SALT AND VARIOUS PENICILLINS AGAINST

STRAINS OF KLEBSIELLA AEROGENES

Strain

A

E 70

Amoxycillin

<td> «Will Alone. 1 ^c</td><td> +5^g/m ,ynergi</td>

<td> 500 ¹</td><td> 0.97</td>

<td> 5</td><td></td>

<td> ן 500</td><td> 3.9</td>

<td>^! 250 ⁵ s</td><td> 15.6</td>

<td> ___________________I</td><td></td>

Carbenicillin* | Bengylpenic.il linl

Al one I +5!1&amp;/Jplj * Alone» +5μ£/φ.χ.. ן. synergist! . ' synergist!

<td> --------_r 500 I 1</td><td> jjiiiijn t ׳w 1111111׳ 1 ׳m 1׳ 7.8</td><td> li . יזעק rasfct umari 101 .y_ op I 250,</td><td> 7.8</td><td></td>

<td> 500,</td><td> 15</td><td> 500.</td><td> 15.6</td><td></td>

<td> 1</td><td></td><td> 1 1</td><td></td><td></td>

<td> 125}</td><td> 7.8</td><td> } 250, 1 .</td><td> 15.6</td><td></td>

*Similar results observed when carbenicillin replaced by carbenicillin phenyl a-ester or ticajLillin.

EXAMPLE 45

Γ^.ίΓ-ιΊ.Π1Γ^!.״τ - τ י־

ANTIBACTERIAL SYNERGISK .aETVEEN AMPICILLIN AND _ T* ._L.r *I ־־ ־ , . r 1.. ,. -!II- --- ז״ »IK—*J MMM! *mm»«.

ESTERS OF CLAVULANIC ACID

The results presented in Table 8 were obtained by the method described in Example 42

TABLE 8

A1THBACTERI AL SYNERGISM BETWEEN AMPICILLIN Al© tea 1 - utn — — - -- -י. MMWWU- I I —.«iWTr,« β.!1.ι.....»..&lt;..ι.*Λ.Ι'..*..“*

ESTERS OF CLAVULANIC ACID AGAINST STRAINS OF nil-- 1r—-—.->[ TI—-------- I a air !inr rw^a«—־! jw 1_ *T -1 Tr-“'A»* 14

KLEBSIELLA AEROGENES

<td> Strain</td><td> Ampicillin Alone </td><td> Ampicillin + 5 jig/ml of Methyl Ester of clavulanic acid</td><td> Ampicillin + 5 pg/ml of Benzyl Ester of clavulanic acid</td>

<td> A</td><td>_י 500</td><td> 1.9</td><td> Ϊ 1.9</td>

<td> E 70</td><td> 500</td><td> 3 - 9</td><td> 3.9</td>

<td> 62</td><td> 500 1</td><td> 3.9</td><td> 3.9 . J</td>

Neither clavulanic acid methyl ester nor clavulanic acid benzyl ester inhibited the growth of ־che test organisms at a concentration of 100 pg/ml.

־ 101 EXAMPLE 46

ANTIBACTERIAL ACTIVITY OF CLAVULANIC ACID ESTER W * '&lt;. 11.1.1 iMfc *. IM ־.I * י. ι,.'.ι. I *i וי — . -w. ^11!. toMl.nMI.Mll.MI I II ,|l I. '

Using tfe® 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 clavulanic acid against a number' of organisms:

TABLE

ANTIBACTERIAL ACTIVITY OF CLAVULANIC ACID ESTERS

MIC of Ester of Clavulanic Acid

<td> Organism</td><td> benzyl 1 ester.</td><td> »mi Nonyl ester</td>

<td> Bacillus subtilis</td><td> A | 250 J</td><td> 31</td>

<td> Staph,sure e»*a»uuuuuu£.»k 1 » —— :.-י Oxford</td><td> I d׳11 62 ך</td><td> 31</td>

Pivaloyloxy-IPhtha1idy methvl ester ester M I X wo wwmii 1 .»»—.Μ» .lirir^T ^,tin » 1»'1־־ חדו T1r> u1־t j.־t. — t .

Russell 1

ע12

Escherichia !יי,.! Φ! <rvw1j> J co.Li 10418

5&lt;׳ GO

MIC* of clavulanic acid sodium salt

4.

*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.

102 EXAMPLE 47 —*UA. MW?« » .MW*

EXTRACTION OF CLAVULANIC ACID USING LIQUID ION UjL 1LL1 1 I J ־ -־- ־-־ ~ - I I .ו—— — w,1M.r ar—

EXCHANGE RESIN

Culture filtrate (200 ml, obtained in a similar manner to Example 3 but using a medium containing 0.1% v/v KEL/PO^ instead of 0., 01% FeS0,7׳H₉0) 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

m.inutes). The solvent phase (60 ml) was recovered by pipette and divided into four equal portions. Each portion was extracted by stirring at 5°C for 20 minutes with 1/4 volume (?5.75 ml) aqueous extractant as indicated in ׳the table below. The resulting mixture was centrifuged (1660 g, 1.9 minutes).

J.6 ml. aqueous phase was recovered from each extraction.

<td> Sample</td><td> u ww***w ’j* guMiw a Volume (Λ)</td><td> Clavulanic acid concentration (pg Kl'j</td><td> 1 ClavulaniJ acid(mg) !</td>

<td> clarified brew</td><td> 200</td><td> 123</td><td> 29.4</td>

<td> extracted brew</td><td> 200</td><td> 15</td><td> 3.0</td>

<td> M NaCl extract</td><td> 3.6</td><td> 305</td><td> 1.1</td>

<td> 2M NaCl extract</td><td> 5.6</td><td> 598</td><td> 2-5</td>

<td> M NaNO- extract 9</td><td> 5.6</td><td> 633</td><td> -.2.:3</td>

<td> 2M ?JaNO._z extract . z</td><td> 3.6</td><td> 758</td><td> 2.73</td>

The result obtained with 2M NaNO^ represents a recovery of 43% from clarified brew.

*Amberlite IA2 is obtainable from Rohm and Haas (UK)

Ltd. Croydon.

EXAMPLE

EXTRACTION OF CLAVULANIC ACID USING LIQUID ION

EXCHANGE RESIN J I»—ΜΙΙΙ1.1|

Clarified brevr (47 litres, obtained as in Example 12) was extracted with Amberlite LA2 (acetate form, 15% v/v in methyl,isobutyl ketone, 12.5 litres) by stirring for 1 hour at 17°C. After adding octan-1-01 (500 ml) the phases were separated in a continuous flow centrifuge yielding 9-2 litres solvent phase, which was then stirred at 5^UC for 1.¾ hours with molar sodium nitrate (2.3 litres). The mixture was separated by continuous flow centra.fugation yielding 2.4 litres aqueous phase.(including writer used for displacement purposes). Aqueous phase pH (initially 8.0) was adjusted to 7.0 with concentrated hydrochloric acid.

<td> ------------- -------------------ן 1 | Sample</td><td> Volume (V</td><td> clavulanic acid concentrajion (pg ml )</td><td> 1 clavulanic .acid (mg)</td>

<td> clarified brew</td><td> 47</td><td> 146</td><td> 6862</td>

<td> extracted brew</td><td> 47</td><td> 19</td><td> 893</td>

<td> M Ma Nil &lt;1rrwo+ ....₃.....</td><td> 2.4</td><td> 1633</td><td> .. i to I ί to 1 ____1</td>

Extraction efficiency from clarified brew to sodium nitrate extract is 57%

47ΟΘ7/2

EXAMPLE

Benzyloxycarbonylmethyl clavulanate

<img file="IL47087A_D0022.tif" />

<img file="IL47087A_D0023.tif" />

Sodium clavulanate (0.6 g) and benzyl bromoacetate (Ο.46 ml) were dissolved in Ν,Ν-dimethylformamide (7 nil). After standing for 2 ל hours at room temperature the solvent was evaporated in vacuo.

Diethyl ether (0ל ml), ethyl acetate (2ל ml) and ice-water (0ל ml) were added, shaken and separated. The aqueous layer was washed with a further 0ל ml of the solvent mixture. The combined solvent layers were washed with 10 ml ice-water, dried oved sodium sulphate and evaporated to an oil, which was subjected to chromatography on silica gel using cyclohexane and ethyl acetate as eluents. The fractions containing benzyloxycarbonylmethyl clavulanate were collected and evaporated to a colourless oil.

I.r. (film): 5510 (b, OH), 180ל (β-lactam 0=0), l?60 (br, ester C=O's), 15 1700 cm¹ (0=0).

N.m.r. (CDCip: 1.Θ6 (1H, bs, disappears on deuteration, CH^OH), 3.04 (1H, d, J 18Hz, 6β-Η), 3.46 (1H, dd, J 18Hz, J 2.3Hz, 6a-H),

4.19 (2H, d, J 8Hz, CH₂0H), 4-71 (2H, s, OCH₂C0), 4.97 (1H, t, J 8Hz, =CHCH₂OH), 5.14 (1H, s, 3־CH), 2) 19 .לH, s, PhCHg), 1) 66.לH, d, J 2^Hz, 20 5) 7.34 ,(!0-לH, s, PhCH₂).

47087/2 r

EXAMPLE

Sodium carboxymethyl clavulanate

<img file="IL47087A_D0024.tif" />

CH₂0H 'CO-CH_C0₉CH.C,E_rά &lt;L &lt;!. t. ע ס

<img file="IL47087A_D0025.tif" />

'C0₂CH₂C0₂Na

Benzyloxycarbonylmethyl clavulanate (0.2 g) in redistilled tetrahydro furan (5 1ט) was hydrogenated at 22°C over 5% palladised barium sulphate (0.2 g) for 50 minutes. The catalyst was removed by filtration. To the filtrate was then added water (1.0 ml) and solid sodium hydrogen carbonate (0.11 g). The mixture was cooled to 10°C and stirred for 5 minutes.

The excess NaECO was filtered off and the filtrate evaporated in vacuo to a crisp foam.

I.r. (concentrated BMSO solution): 1801 (β-lactam 0=0), 1755 (ester 0=0), 1700 (C=C), 1650 cm (00₂~).

EXAMPLE

Benzhydryl clavulanate

<img file="IL47087A_D0026.tif" />

<img file="IL47087A_D0027.tif" />

Biphenyldiazomethane (112 mg) in petroleum ether (40° - 60°C) wan added to a solution of clavulanic acid (38 nig) in tetrahydrofaran (4 ml).

The resulting solution was stirred overnight at room temperature and then the solvent removed under reduced pressure to give an oily residue which was chromatographed on silica gel, eluting with an ethyl acetate hexane mixture, to give the title compound (135 mg).

I.r. (CECI,): 3400 (OH), 1805 (β-lactam C=0), 1755 cm^-i(ester C=0),

1700 (c=c).

N.m.r. (CDClj): 2.05 (1H, s, OH), 3.03 (1H, d, J l?Hz, όβ-CH),

3.53 (1Ξ, dd, J l?Hz, J’ 2.5Hz, 6a-CH), 4-22 (2H, d, J 7Hz, =CHCH₂),

4.94 (1H, t, J 7Hz, =CHCH₂), 5.27 (1H, s, 3־CH), 5-76 (1H, d, J 2.5Hz,

5-CH), 7.03 (1H, s, CHPh^), 7.46S (10H, s, aromatic protons).

EXAMPLE

2-Benzyloxycarbonylamino-2-methoxycarbonylethyl clavulanate

<img file="IL47087A_D0028.tif" />

<img file="IL47087A_D0029.tif" />

Benzyloxycarbonyl-L-serine methyl ester (500 mg) and dicyclohexylcarbodiimide (143 mg) were added to a cooled (0°C) and stirred solution of clavulanic acid (158 mg) in tetrahydro furan (4 ml). The reaction mixture was allowed to warm to room temperature and was stirred overnight. The reaction mixture was filtered, washed, dried and the solvent removed under reduced pressure to give a residue which was chromatographed on silica gel, eluting with ethyl acetate - hexane, to give the title compound as a gum (103 mg).

I.r. (CHClj): 3400, (br, OH and NH), 1805 (β-lactam 0=0), 1760 and 1725 (0=0 x 5), 1700 cm (C=C).

N.m.r. (CDC1J: 2.42 (1H, s, OH), 3-08 (1H, d, J 17Hz, 6β-0Η), 3

3.58 (1H, d, J 17Hz, J' 2.5Hz, 6a-CH), 3-88 (3H, s, 00^), 3-98 ־

5.15 (6H, m, =CHCH₂ and CO^CH), 5-2 (1H, m, 3־CH), 5-25 (2H, s,

CH₂Ph), 5.73 (1H, d, J 2.5Hz), 6.01 (1H, m, NH), 7-535^- (5H, s, aromatic protons).

t

EXAMPLE 53 ׳-ΑπΗ no-2-methoxycarbonylethyl clavulanate hydrochloride

<img file="IL47087A_D0030.tif" />

<img file="IL47087A_D0031.tif" />

2-Benzyloxycarbonylamino-2-methoxycarbonylethyl clavulanate (85 ^mg) in distilled tetrahydrofuran (2 ml) containing acetic acid (10 mg) was hydrogenated over 10% Pd/C (40 mg). After 60 minutes thin layer chromatography (solvent - ethyl acetate) showed the formation of one product and some unreacted starting material. The catalyst was filtered off and the filtrate concentrated to give an oil which was chromatographed on an ion exchange column (^,Amberlite’ IRA 4θ1 in Cl form) (eluting with ethyl acetate - hexane). Evaporation of the resulting solution gave the desired material as a viscous gum.

I.r. (liquid film): 1805 (β-lactam), 1760 (ester C0), 1695 cm (C=0).

N.m.r. (D₂0): J.13 (1H, d, J 17Hz, 6β-0Η), 5.57 (1H, dd, J 17Hz, J ?Hz, 6a-CH), ?.8 (JH, s, OCH^), 4-14 (2H, d, J 7Hz, =CHCH₂), !5 5.70$ (1H, d, J JHz, 5־CH), remaining peaks obscured by HOD.

EXAMPLE

2-(2-Phenylsulphonyl)ethyl clavulanate

<img file="IL47087A_D0032.tif" />

<img file="IL47087A_D0033.tif" />

A solution of clavulanic acid (199 ®g) in tetrahydrofuran (8 ml) was cooled to 0°C and treated with dicyclohexylcarbodiimide (206 mg), pyridine (79 mg) and p-(phenylsulphonyl)ethanol (1.0 g). The stirred solution was allowed to reach room temperature and stirred at room temperature overnight. The suspension was filtered and the filtrate concentrated under reduced pressure. The resulting residue was chromatographed on silica gel, eluting with ethyl acetate - hexane mixtures, to yield the title compound as an oil (155 ®g).

I.r. (CHClj)! 3400 (br, OH), 1800 (β-lactam C=0), 1750 (ester 0=0), 1695 (0=0), 1510 and 1150 cm (S0₂);

N.m.r. (CDC1 ): 2.18 (1H, s, OH), 3.08 (1H, d, J 17Hz, 6p-CH),

3.52 (1H, dd, J 17Hz, J' 2.5Hz, 6a-CH), 32) 59יH, t, J 6Hz, CHgSOg), !5 4.25 (2H, d, J 7.5Hz, =CHCH₂), 4.57 (2H, t, J 6Hz, CH^SOg);

4.7 - 5.01 (2H, m, =CHCH₂ and 3־־CH), 5.7 (1H, d, J 2.5Hz, 5־CH),

7.5 5) 8.2$ ־H, m, Ph).

EXAMPLE 55

2-Methylthioethyl clavulanate

<img file="IL47087A_D0034.tif" />

<img file="IL47087A_D0035.tif" />

A solution of clavulanic acid (199 mg) in tetrahydrofuran (8 ml) was cooled to 0°C and treated with dicyclohexylcarbodiimide (200 mg) and p-(methylthio)ethanol (960 mg). The stirred solution was allowed to reach room temperature and stirred at room temperature overnight. The suspension was filtered and the filtrate concentrated under reduced pressure. The resulting residue was chromatographed on silica gel, eluting with ethyl acetate - hexane mixtures, to yield the title compound as an oil (145 nig).

l. r. (CHC1_T): 3400 (0H), 1805 (β-lactam C=0), 1750 (ester 0=0),

1695 cm“¹ (C=C),

N.m.r. (CDCip: 1.72 (1H, s, OH), 2.15 (JH, s, SCH^, 2.85 (2H, t,

J 7Hz, CHgSCH ), 3.05 (1H, dd, J !?Hz, J* 1Hz, 6β-0Η), 3.55 (1H, dd,

J 17Hz, J¹ 2.5Hz, 6a-CH), 4.52 (2H, t, J 7Hz, CH^SCH^, 4-30 (2H, d, J 7.5Hz, =CHCH₂), 4-97 (1H, dt, J 7.5Hz. J' 1.5Hz, =CHCH₂), 5-09 (1H, m, 3-CH), 5.738 (1H, dd, J 2.5Hz, J’ 1Hz, 5־CH).

Ill

EXAMPLE 56

־♦

2-Pyridylethyl clavulanate

<img file="IL47087A_D0036.tif" />

'co₂h

<img file="IL47087A_D0037.tif" />

A solution of clavulanic acid (135 mg) in tetrahydrofuran (5 nil) was cooled to 0°C and treated with 2-(2-hydroxyethyl)pyridine (200 mg) (2.5 equivalents) and dicyclocarbodiimide (145 mg) (1 equivalent). The stirred solution was allowed to reach room temperature and stirred at room temperature overnight. The suspension was filtered and the filtrate was concentrated tinder reduced pressure. The resulting residue was chromatographed on silica gel, eluting with ethyl acetate containing

5% methanol, to yield the title compound as an oil (100 mg).

I.r. (CHC1₅): 5400 (0H), 1805 (β-lactam 0=0), 1750 (ester C=0), 1695 cm (0=0);

N.m.r. (CDClj): 2.20 (1H, s, OH), 2.97 (1H, d, J 17Hz, 6p-CH),

5.11 (2H, t, J 7Hz, CH₂CH₂Py), 5-42 (1H, dd, J 17Hz, J' 2.5Hz, 6a-CH),

4.15 (2H, d, J 7HZ, =CHCH₂), 4.45 5) 9.4 ־H, m, CHgCHgiy and =CHCH₂),

4.97 (1H, m, 3-OH), 5.51 (1H, d, J 2.5Hz, 50־h), 7.0 - 7.4 (2H, m, pyridyl-5-CH and 5-CH), 7-45 1) 7-95 ־H, m, pyridyl-4-CH), 8.35 8.70S (1H, m, pyridyl-6-CH).

EXAMPLE 57

Phthalimidomethyl clavulanate

<img file="IL47087A_D0038.tif" />

<img file="IL47087A_D0039.tif" />

Phthalimidomethyl chloride (1-95 g) was added to a stirred solution of sodium clavulanate (221 mg) in d3y dimethylformamide (2 ml) and the solution left at room temperature for four hours. The solution was treated with ethyl acetate and water and shaken thoroughly. The non-aqueous layer was separated, dried and concentrated under reduced pressure to give a residue which crystallised from ethyl acetate light petroleum (40° - 60°C) to yield the title compound (130 mg); m.p. 165°C.

I.r. (CHClj): 3400 (hr, OH), 1805 (β-lactam 0=0), 1785, 1760 and 1755 cm ¹ (phthalimidyl 0=0, ester 0=0).

N.m.r. 1.7 (1H, 3, OH), 3.08 (1Ξ, d, J 17Hz, όβ-ΟΗ), 3-56 (1H! dd, J 17Hz, J' 2.5Hz, 6a-CH), 4-25 (2H, d, J 7-OHz, =CHCH₂), 4-95 (1H, dt, J 7.5Hz, J* 1Hz, =CHCH₂), 5.11 (1H, d, J 1Hz, J-CH), 5.75 (1H, d, J 2.5Hz, 5-CH), 5.87 (2H, d, CH^Phth), 7.75 - θ.2ί> (4H, m, Phth).

EXAMPLE

Allyl clavulanate

<img file="IL47087A_D0040.tif" />

<img file="IL47087A_D0041.tif" />

To a stirred solution of sodium clavulanate tetrahydrate (2.05 g) in dry dimethylformamide (9 ml) was added ally bromide (2.44 ml). The 5 mixture was stirred at room temperature for J hours and the solvent then removed under vacuum below JO°C. The residue was partitioned between water and ethyl acetate. The ethyl acetate layer was washed with water, dried over magnesium sulphate and evaporated. The residue was purified by column chromatography (Kieselgel 60/ethyl acetate,

Rf ^v 0.53) to yield the title compound (1.31 g) as an oil.

I.r. (film): 3400, 1805, 1745, 1695.

N.m.r. (CDCl^, 90MH^) £ 1.9S (1H, singlet exchangeable), J.02 (1H, doublet, J l?Hz), 3-46 (1H, double doublet, J 17Hz x JHz), 4-19 (2H, doublet, J 7Ξζ), 4.61 (2H, doublet with finer coupling J 6Hz), 4.θθ (1Ξ, 15 triplet with fine coupling, J 7Hz), 5.θ2 (1H, singlet with fine coupling),

5.29 (2H, triplet with fine coupling, J 8Hz), 5-64 (1H, doublet, J JHz),

5.7 1) 6.1 ־H, multiplet).

EXAMPLE 59

Acetonyl clavulanate

<img file="IL47087A_D0042.tif" />

To a stirred solution of sodium clavulanate tetrahydrate (2.05 s) in dry dimethylformamide (9 ml) was added bromoacetone (2.55 ml). The 5 mixture was stirred overnight at room temperature, and then the solvent was removed under vacuum below 30°C. The residue was partitioned between water and ethyl acetate. The ethyl acetate solution was washed with water, dried over magnesium sulphate and evaporated. The ester was isolated by column chromatography (Kieselgel 60/ethyl acetate eluant;

Rf 0.36) to give the title compound (565 mg).

I.r. (film): 5400, 1800, 1755, 1730.

N.m.r. (CDClj 90MH^)$ 2.14 (3H, singlet), 2.20 (1H, broad exchangeable), 5.04 (1Ξ, doublet, 17Hz), 3.46 (1Ξ, double doublet, J 17Hz x 3Hz),

4.20 (2H, doublet with fine coupling, J 8Hz), 4*7 (2H, singlet), 4.97 (1H, 15 double triplet, 8Hz» J^_Oub 1.5Ηζ), 5.12 (1H, finely coupled singlet), 5-67 (1H, doublet J 3Hz).

Example

Cyclohexyl clavulanate

<img file="IL47087A_D0043.tif" />

A solution of N-carboethoxy-N-nitroso-cyclohexylamine (1-4 5 ־ prepared by the reaction of nitrogen dioxide on N-carboethoxy-cyclohexylamine in CCl^ in the presence of anhydrous sodium acetate) in dichloromethane (25 ml) at 0°C was treated with pyrrolidine (0.62 ml) and stirred at 0°C for 30 minutes. To this solution over 5 minutes was added a solution of clavulanic acid in tetrahydrofuran (prepared by the hydrogenation of benzyl clavulanate [1 g] in tetrahydrofuran [30 ml] over 10% palladium on charcoal). The resulting solution was treated with excess sodium bicarbonate solution. The resulting mixture was extracted with several portions of chloroform and the chloroform solution dried over magnesium sulphate and evaporated. Repeated chromatography (twice with 1:1 ethyl acetate: cyclohexane and once with chloroform on Kieselgel H) gave the title compound (46 mg).

I.r. (film): 34θθ» 1800, 174θ» 1695 cm ¹.

N.m.r. (CDClj): $1.1 - 2.0 (11H, m 1H exchangeable), 5.01 (1H, d, J = 16Hz), 5.45 (1H, dd, J = 16 and 3Ξζ), 4-17 (2H, d, J = 7Hz), 4-5 5.0 (1H, m), 4.97 (1H, t, J = 7Hz). 4-96 (1Ξ, s), 5.63 (1H, d, J - 3Hz).

Example

Prou’irgyl ester

<img file="IL47087A_D0044.tif" />

Sodium clavulanate tetrahydrate (4.4 g, 0.0150 moles) was dissolved in dry DHF (25 ml) and propargyl bromide (1.6 ml, 2.53 gt 0.0212 moles) added slowly with stirring. The mixture was stirred at room temperature for 2 hours and then poured into water (20ל ml) and the product extracted with ethyl acetate (3 x 100 ml). The combined ethyl acetate extracts were washed with water (4 x 100 ml) and dried over magnesium sulphate. The clear, dry.ethyl, acetate solution was evaporated to dryness in vacuo to give a thick oil, which, was shown by tic (silica gel plates/ethyl acetate developing solvent) to contain two components.

The oil was dissolved in chloroform (2 ml) and chromatographed on Sephadex LH20 using a 1:1 mixture of chloroform and cyclohexane as the eluent. Tiie fractions (25 ml) were assayed by tic and those fractions which contained the faster running component in appreciable quantities were bulked and concentrated in vacuo to give a thick oil (2.2 g). This oil solidified on standing overnight. A tic assay showed only one component. The nmr spectrum showed the presence of some chloroform.

The product was ether washed to give a solid (?00 mg, m.p. 65-6°). The low recovery was due to its appreciable solubility in this solvent. The following assay results were obtained:

Elemental analysis: found:

Calculated for C^H^NO^:

nmr spectrum δ (CDClj);

0, 55.55; Η, 4.6δ; N, 6.12%

C, 55.69; II, 4.68; N, 5.91%

2.01 (s 1H), 2.6 (t 1H), 2.95 3.75 ־ (m 2H), 4.29 (d 2H), 4-79 - 5.00 (m 3Ξ), 5.13 (d 1H), 5.75 (d 1H)

DMF = dimethylformamide tic ₌ thin layer chromatography

Example

<img file="IL47087A_D0045.tif" />

<img file="IL47087A_D0046.tif" />

<img file="IL47087A_D0047.tif" />

4-Hothoxybenzyl ester ch₂0h ;ch₂0h ־CO₂Na

CO״CH-C_rH.OCH,

2 0 4 3

Sodium clavulanate (18,2 g, ca 0.07 mole of anhydrous scdium clavulanate, water content 22.8%) was dissolved in dimethyl formamide (100 ml) and treated with a solution of 4-methoxybenzyl bromide (13.5 g, 0.07 mole) in dimethylformamide (25 ml). The clear solution was stirred at room temperature for 80 mins, then diluted with ethyl acetate (3OO ml) and water (200 ml). The aqueous layer was separated off and re-extracted with ethyl acetate (300 ml). The ethyl acetate extracts were combined and cashed with water (200 ml x 6) and dried over I4g3O

־4

Evaporation gave an oil,

19.5 g.

The oil was dissolved in chloroform/cyclohexane (1:1) and chromatographed on a 65 x 400 mm column of Sephadex (500 g, LH20 made up in 1:1 chloroform/cyclohexane mixture). The fractions containing the required ester were bulked and evaporated in vacuo to give a pale yellow oil. Trituration with n-hexane gave a solid powdery product, which was filtered off, washed on the filter pad with fresh n-hexane and dried in vacuo, yield 12.6 g (57%)» m.p. 54-5 - 55.5°.

ir (KBr disc/1515 ,1615 ,1690 ,1735 ,1795 ,2840 ,2950 ,3400 ״, ' ^y max .ן

1305, 1250, 1175, 1005, 890, 825 cm

The same product could be prepared from clavulanic acid in tetrahydro-, furan solution and 4-methoxybenzyl alcohol and dicyclohexylcarbodiimide.

Yield (0,0052 mole scale) 0.3 g (18%)

Example

4-Nitrobenzyl

<img file="IL47087A_D0048.tif" />

<img file="IL47087A_D0049.tif" />

<img file="IL47087A_D0050.tif" />

CH״ OH (K J ^ο₂οη₂ο₆η₄νο₂(ρ)

Sodium clavulanate (2.4 g, ca. 0.01 mole, water content 10%) was dissolved in dimethylformamide (10 ml) and treated with ή-nitrobenzyl bromide. (2.2 g, ca. 0.01 mole) at room temperature for 40 mins. The mixture was diluted with ethyl acetate (100 ml), washed with water (e0 ml x ל) and brine (50 ml), dried over magnesium sulphate, filtered and evaporated to give an oil. The oil was triturated with ״-hexane rapidly.giving rise to a crystalline product. The solidified product was crushed in n-hexane, filtered, washed on the pad with fresh solvent and dried in vacuo.

Yield 2.7 g (81%), m.p. 110 - 112°

Elemental analysis: found: C, 54.07; Η, 4.30; N, 8.32% calculated for C._cH.N0״: C, 53.θ9; Η» 4-22; N, 8,38% 14 לו z / ),

70.ל (d, 1H, C^H), 2 ,פ)’ל2.לH, C0.0.CH₂-),

05.ל (m, 1Π, 4-75 (m, 1H=S1), 4-W (d,

2H, -CH₂0), 3.2 (m, 2H, CH^), 1.95 (2, 1H, OH)

8.10, 7.55, 7.40 (ABq, 4H,

<img file="IL47087A_D0051.tif" />

nmr spectrum (CDCl^): 8.2ל,

Example

6-I4ethoxynRphth-2-ylr!ethyl ester

<img file="IL47087A_D0052.tif" />

A solution of anhydrous sodium clavulanate (2.2 g, 10 m.moles) and 6-methoxy-2-bromomethylnaphthalene (2.51 g, 10 m.mole) in dry DID? (80 ml) was stirred at room temperature. After J hours examination of the reaction mixture by tic showed that no bromomethylnaphthalene was present. The reaction solvent was removed in vacuo at 50° and the residue treated with ethyl acetate (100 ml). The suspension was filtered and the filtrate washed with water (2 x 25 ml), dried over magnesium sulphate, filtered and stripped to afford an orange oil (3.2 g). Chromatography of this product on Sephadex LH20 (100 g) (eluent 0ל:0ל chloroform:cyclohexane, collect 10 ml fractions; fractions 12-20 contained the ::equired ester as determined by tic visualising with TTC spray*) afforded 7. clear oil which solidified on standing. This material was dissolved in dichloromethane and 60-80° petrol added until the solution became cloudy. On standing in an open beaker, a gum formed. The supernatant liquid was allowed to evaporate further and a white crystalline solid formed; 2.52 g, 60Jb, m.p. 89-90°.

Elemental analysis, found: C, 64.91; Η, 5.θθ! Ν» 5.77% calculated for C^H^HO^: C, 65.04; H, 5.15; N, 3-79% nmr (CHC1_?) 6 1.45 (1Π,. bs, €¾011), 2.9 (1H, d, 6(:-11), 5-36 (1H, dd, 6a-H), 3.Θ (JH, s, 0€¾). 4*03 (2H, d, CJI^H), 4-7 (1H, t,־Z^H), 4-93 (1H, s, CHC0₂-), 5.27 (2H, s, -CllgOCO), 5.65. (1Π, d, 5P-H), 7-37 (6H, m, aromatic H's) * 4% triphenyltetrazolium chloride in methanol + equal volume of methanolic N NaOH

Example 6¾

2-Jfethoxyethyl ester /

CH₂OH

1) S0C1״

0Η,00Η_ο0Π0״Π ττ״,. _τ > CH,0CH_oCH_oI ^EOfl1^ —> Γ

2 2 2) lial >22 clavulanate | ₄H0 J—M--^{ά u} 'COnCH^CH^OCli.

ά ά ά 79

2-Methoxyethyl chloride (43 St 0.455 mole) (JACS 193θ» 52 , 653) ^waG added to a solution of sodium iodide (75 g, 0,50 mole) in acetone (500 ml) and the reaction heated under reflux for 34־ hours. Most of the acetone was distilled off at atmospheric pressure, the residue was cooled and diluted out with ether (200 ml). The ethereal solution was filtered free of inorganic salts, washed successively with brine (100 ml), 5% aqueous sodium thiosulphate (100 ml), brine again (100 ml) and dried over magnesium sulphate. After filtration, the ether was removed by distillation at atmospheric pressure and the product isolated by distillation, collecting the fraction boiling between 130° and 140’ (atmospheric pressure). The product, a dense, pale yellow liquid, weighed . 20 g (23% if 100% pure).

To a solution of sodium clavulanate tetrahydrate (6 g, 0.0205 mole) in dimethylformamide (100 ml) was added 2-methoxyethyliodide (5.6 g, 0.05 mole) at room temperature. The reaction was stirred for 5 hours after which time tic (silica plates developed in ethyl acetate, spots visualised with alkaline TTC spray, Rf sodium clavulanate 0.0, Rf ester 0.5) indicated some ester foi’mation. The reaction was diluted out with ethyl acetate (200 ml) and. water (500 ml), the aqueous layer re-extracted with ethyl acetate (100 ml) and the bulked organic extracts washed with water (4 x 100 ml), N sodium bicarbonate (100 ml), and water again (3 x 100 ml), then dried over magnesium sulphate. After filtration the solvent was evaporated in vacuo and the ester thus obtained as a pale yellow oil finally dried under high vacuum to remove the last traces of solvent and unreacted iodo compound. The product obtained was essentially pure by pmr spectrum. ,

Yield: less than 5% pmr (CDCip; δ 1) ל6.לH d): 55.05 (1E s) and 54.9 (1H t) overlapping each other: δ4.2 (4H m): 63.6 (2Π m): 53.35 (?H ^s) overlying 63.3 (1H m): δ2.9 (1H s)

Example 66 !

Preparation of Lithium Salt of Clavulanic Acid J

A solution of benzyl clavulanate (0.84 g) in ethanol (30 ml: was mixed with a solution of lithium hydrogen cabonate (which had been prepared as below) in water (5 ml) . This solution was hydrogenated over 10¾ palladised charcoal (0.27 g) for 25 minutes at ambient temperature (^ 20°C), after which time no starting material was detected by thin layer chromatography (Si0₂־ethyl acetate: KMnO^ spray). The catalyst was filtered, washed with water (5 ml) and with ethanol (5 ml), and the combined filtrates evaporated under reduced pressure. The crystalline residue was triturated with acetone (25 ml) and the solid was filtered, washed with acetone (10 ml) and dried over P₂°5׳ yielding 0.41 g of product. The product was taken up in water (3 ml) and acetone added slowly until crystallisation began. After cooling for 20 minutes at 2-3°C, a crop of crystals was collected by filtration, washed with- a little acetone and dried in vacuo (80 mg). The filtrate was diluted with an equal volume of acetone, and again cooled, depositing a further crop (80 mg). Assays: first crop = 84.3¾ pure lithium clavulanate second crop - 85.1% pure lithium clavulanate (The lithium hydrogen carbonate solution was prepared by suspending lithium carbonate (14 g) in water (480 ml) at 5° - 10°C, and passing a steady stream of carbon dioxide gas through the suspension during 8 hours, by which time a clear solution had been obtained. The volume of the solution was then made up to 500.0 ml with water saturated with CO״. This solution then contained 5.15% w/v LiHCO,).

^J

12?

Example

Preparation of -Lithium Salt of Clavulanic Acid

A solution of clavulanic acid in ethanol (50 ml) and water (80 ml) was titrated to pH 7.5 using 1.0M lithium hydroxide solution. The solution was evaporated under low pressure to low volume (about 2 ml) and the residue triturated with acetone (50 ml). The resulting solid was filtered, washed with acetone and then with diethyl ether and air dried to yield 0.62 g of almost colourless crystals which assayed as 83.2% pure lithium salt. This material was dissolved in water (5 ml) and applied to a cellulose column (50 g cellulose) made up in butanol - ethanol - water 4:1:5 (top phase) and eluted with that solvent. The column was monitored by thin layer chromatography using silica gel and. the same solvent. Fractions containing the compound were combined taking two cuts. The cuts were evaporated in vacuo to dryness, triturated with acetone (10 ml), filtered off and dried in vacuo. The first cut yielded 0.21 g of 98.0% pure lithium salt of clavulanic acid and the second cut yielded 0.12 g of 98.5% pure lithium salt of clavulanic acid.

[The solution to be neutralised was prepared by hydrogenation of benzyl clavulanate (1 g) in ethanol (50 ml) for 30 minutes at ambient temperature and pressure over 10% palladium on charcoal. At the end of this time the catalyst was removed by filtration and the filtrate diluted with water (80 ml)].

Example 68

Preparation of Benzyl Ester of Clavulanic Acid ־

A solution of pure lithium clavulanate (0.55 g). in warm water (3 ml) was added dropwise during 5 minutes to a stirred solution of benzyl bromide (1.8 ml) in dimethylformamide (8 ml). The mixture was stirred at room temperature (20°C) for 2 hours. Thin layer chromatography (ethyl acetate-SiO₂) then showed that a large proportion of the clavulanate salt had been esterified. The reaction mixture was applied to the top of a column of silica gel (200 g) made up in cyclohexane-ethyl acetate (10:1), and eluted with cyclohexane and ethyl acetate graded from 10:1 ratio, through 1:1, then with pure ethyl acetate. Fractions containing benzyl clavulanate were combined and evaporated under reduced pressure to leave an oil, from which the residual solvents were evaporated under high vacuum to yield 0.37 g (47%) of pure benzyl clavulanate.

Example 69

Preparation of Lithium Clavulanate . /

A preparation of lithium clavulanate of poor purity due to the presence of a high level of inorganic material was used in this experiment. A 2 g aliquot was dissolved in 15 nil of deionised water and applied to a 1V x 18 bed of.Biogel P2 and eluted with deionised water containing 1% n-butanol. The column was run at 10°C and 5 ml fractions collected. Samples of fractions were spotted onto filter paper and sprayed with T.T.C. reagent in order to locate the lithium clavulanate. Samples (0.1 ml) were also removed and added to 0.5 ml of silver nitrate solution to test for the presence of chloride. Fractions containing clavulanate which were salt free (25 - 31) were combined concentrated to 10 ml. Lithium clavulanate was then precipitated by addition of acetone. The resultant crystalline precipitate was filtered, washed with acetone and then air dried. This yielded a white solid 0.93 g. Analytical results in comparison with the starting material are given below:

<td></td><td> Starting</td><td> Material</td><td> Final Product</td>

<td> Purity hplc assay</td><td> 68%</td><td> pf a</td><td> 91.4% pfa</td>

<td> Imidazole</td><td> 69.6%</td><td> pf a</td><td> 94.45% pfa</td>

<td> Lithium content</td><td> . 6.43?</td><td> i</td><td> 3.62%</td>

<td> Water content</td><td> 7.9%</td><td></td><td> 1.2%</td>

Example 70

Λ Preparation of Calcium Diclavulanate Dihydrate

A solution of benzyl clavulanate (2.89 g) in ethanol (103 ml) and water (17 ml) was hydrogenated over 10% palladium on charcoal (0.92 g) (Engelhard type 4505) and calcium carbonate (0.55 g, 10% excess) at ambient temperature (circa 20°C) and pressure. The reaction was followed by thin layer chromatography (ethyl acetate/silica gel), and was stopped after 30 minutes when no significant amount of ester could be detected. The mixture was evaporated to about 25 ml under reduced pressure at ambient temperature, diluted with water (50 ml) and the catalyst and excess calcium carbonate removed by filtration through a bed of calcium carbonate. The filtrate was evaporated in vacuo at ambient temperature to a syrup which started to crystallise upon scratching. Most of the remaining water was then removed slowly be evaporation in vacuo, eventually leaving a pasty mass of crystals. This was triturated with acetone (50 ml), the insoluble material filtered off, washed with acetone (25 ml) and ether (25 ml) and air-dried at ambient temperature, to yield 2.0 g of pale apricot coloured crystalline solid. (Purity: approx. 92% as calcium diclavulanate dihydrate. Water:' approx. 8.7% by Karl Fischer).

Example 71

Preparation of Calcium Clavulanate Dihydrate _y

Benzyl clavulanate (1.6 g) in methanol (17 ml) and water (3 ml) was hydrogenated over 5¾ palladium on calcium carbonate (0.8 g) (Engelhard,, code T/CH/898) for 25 minutes at 25°C. The catalyst was removed by filtration, washed with water (2 ml) and the washings and filtrate evaporated in vacuo to a pale straw coloured syrup (&lt;5 ml). This was treated gradually with acetonitrile (100 ml total). The crystalline precipitate was collected by filtration, washed with acetonitrile. (5 ml) and acetone-ether (1:1 mixture, 5 ml) and air-dried, to yield 0.9 g of pale-straw coloured, non-hygroscopic crystalline solid.

Water (by Karl Fischer) approximately 7.4% w/w. Purity (as calcium clavulanate dihydrate) approximately 91%. (Major impurity CaCO^).

Example . י

Preparation of Calcium Diclavulanate Dihydrate

Freeze dried clavulanic acid sodium salt (9g,. purity approximately 60%) was dissolved in de-ionized water (100 ml). This solution was passed through a bed of Permulit Zerolit FF IP SRA-62 resin (310 ml) in the chloride form at a flow rate of 5 ml/minute. (The bed has a diameter of 1.5 inches). After the solution was loaded the column was washed with 1 bed volume of water. The column was then eluted with calcium chloride solution (0.25 M) at a flow rate of 3ml/minute. Fractions (15 ml) were collected and those containing the salt of clavulanic acid were combined. (.The desired salt was detected by spotting small samples onto filter paoer, drying and spraying with Ehlich's reagent). The combined fractions (total volume 480 ml) were treated with calcium chloride to give a final concentration of 0.5M and then percolated through a 1¾¹¹ column containing 1240 ml of XAD-4 resin which had been packed in 0.5M calcium chloride solution. The percolation rate was 10 ml/minute. When all the materials had been loaded onto the column, the bed was washed with calcium chloride solution (0.5M, 100 ml). The column was then eluted with de-ionized water at a flow rate of 10 ml per minute. Fractions (15 ml) were collected and tested for clavulanate (as described) and for chloride (using silver nitrate). Those fractions containing clavulanate free of chloride were combined (total volume 1800 ml) and evaporated in vacuo to a syrup. 1-Butanol (1ml) -/ was added to prevent foaming and the syrup evaporated further to yield a pasty mass. On trituration with acetone (25 ml) a crystalline solid formed which was filtered off, washed with di-ethyl ether (10 ml) and dried in air at room temperature to yield crystalline calcium diclavulanate dihydrate (3.44 g; purity approx. 79%).

A sample of the thus formed material (1.3 g) was dissolved in the minimum quantity of water (~10 ml) and applied to a cellulose column (80 g) made up in n—butanolethanol-water 5:1:5, top phase. The column was eluted with that solvent. The material was detected in the eluate by thin layer chromatography on silica gel using the same solvent, and detection by spraying with 0.02M permanganate solution (an alternative solvent system for thin layer chromatography, 7:7:1 chloroform-ethanol-acetic acid, was also used) . A middle cut was taken, and these fractions were combined and evaporated to dryness in vacuo at ambient temperature. The residue was dissolved in water (70 ml), filtered through a bed of charcoal on Kieselguhr, and the filtrate evaporated slowly in vacuo until crystallisation started. When a pasty mass of crystals remained, it was triturated with acetone (25 ml) and then with ether (25 ml), and dried in air, to yield crystalline calcium diclavulanate dihydrate (0.55 g; purity approx. 94%).

Example 73

Preparation of Calcium Diclavulanate Dihydrate

The combined fractions (480 ml total volume) from a Zerolit FFIP column as described in Additional Example 7 were concentrated to 20 ml by evaporation in vacuo and then loaded onto a 1V x 18 bed of Bio Rad Biogel P2. The column was eluted with de-ionized water and fractions (15 ml) collected. Fractions containing clavulanate were identified by spotting onto filter paper and spraying with Ehlich's reagent. Fractions were also tested for chloride using silver nitrate solution. Those fractions containing clavulanate but not containing chloride were combined. Treatment of this de-salted solution as described in

Example 72 produced crystalline calcium diclavulanate dihydrate similar to that described in Example 72.

Example 74

Preparation of Potassium Salt of Clavulanic Acid

Impure sodium salt of clavulanic acid equivalent to 205 g pure free acid was dissolved in water (20 1) and percolated through a column (6 1) of Zerolit KK ip SRA 61 in Cl” form at 0.2 1/min. After washing the resin with 0.1 M potassium chloride the product was eluted with 0.3 M potassium chloride at 0.14 1/min. Solid potassium chloride was added to the above eluate to increase the KC1 concentration to 1.0 M then the solution was percolated through a column (20 1) of Amberlite XAD-4 pre-equilibrated in 1.0 M KC1 solution. The product was eluted with demineralised water at 0.15 1/min. The eluate (18 1) was contacted with GSX carbon (80 g) for 10 minutes then filtered to remove carbon. The resulting solution was concentrated by reverse osmosis to 2.24 1 and the product was freeze dried to give 167 g solid product. 100 g of this material was dissolved in water (1.2 1) and chilled to 50°C. Chilled acetone was added slowly until precipitation of yellowish oily material was complete. 24 1 acetone was required. The precipitated impurities were filtered off and the filtrate divided into two equal halves.

1 acetone was added to each half. Crystalline potassium salt of clavulanic acid was precipitated and was collected by filtration, washed with acetone and air dried.

Yields: portion 1 24.5 g 98.6% pure portion 2 26.0 g 99.4% pure

Example 75

Preparation of Potassium Salt of Clavulanic Acid

A solution of benzyl clavulanate (of good purity) (2.89 g) in ethanol (100 ml) and water (17 ml) was hydrogenated at ambient temperature and pressure over 10% palladised charcoal (Engelhard 4505, 0.92 g) in the presence of finely divided potassium hydrogen carbonate (1.0 g). After 30 minutes, thin layer chromatography showed that only a trace of benzyl ester remained.

The catalyst was removed by filtration, and washed with water (10 ml) and with ethanol (10 ml). The combined filtrates and washings (which were neutral, ~ pH 7) were evaporated under reduced pressure at ambient temperature to yield a pale yellow syrup. This was dissolved in water (17 ml) and diluted with acetone (450 ml). The turbid mixture was filtered through kieselguhr, and the filtrate diluted to 1.0 litre with acetone. The compound crystallized, and after 30 minutes at room temperature it was collected by filtration, washed with acetone (50 ml) and dried in vacuo, to yield the required potassium clavulanate (0.75 g) as a colourless crystalline solid which was 98.5% pure potassium clavulanate anhydrate (containing about 0.2 - 0.3% water).

Example

Trimethylammonium Clavulanate

-/

Trimethylammonium hydrogen carbonate solution was prepared by dissolving trimethylamine (5 g) in ice-cold water (50 ml) and passing CC>₂ through the solution in a rapid stream until the pH of the solution was circa 8.5, This solution was assayed by titration with 1.0M hydrochloric acid using screened methyl orange as indicator and found to be 0.4M.

To a solution of benzyl clavulanate (2.89 g) in ethanol (100 ml) was added 10% palladised charcoal (0.9 g) and 0.4M trimethylammonium hydrogen carbonate (25 ml). The mixture was hydrogenated for 30 minutes at ambient temperature and pressure; thin layer chromatography showed that no starting material remained. The catalyst was removed by filtration, and washed with ethanol (30 ml) and water (10 ml). The filtrate was evaporated in vacuo to leave a brown oil. The oil was triturated with acetone (50 ml) when it crystallised. The crystals were collected by filtration, washed with acetone (10 ml) and ether (10 ml) and dried in vacuo> to yield 0.84 g of pale brown crystalline trimethylammonium clavulanate. Assay (by high pressure liquid chromatography) 68.7% as free acid; 89.1¾ as trimethylammonium clavulanate. I.r. (KBr) v 3320 (br, with sharp peak), 1775, 1690 , 1590 - 1620 cm ¹ (broad); no deoxy visible in the n.m.r. spectrum.

Example /

Preparation of Aluminium Clavulanate

A solution of benzyl clavulanate (2.89g) and aluminium isopropoxide (0.68g) in dry redistilled tetrahydrofuran (150ml) was hydrogenated over 10% palladised charcoal (0.86g) for 40 minutes (t.l.c. showed no unreacted benzyl clavulanate after this time). The catalyst was filtered off, washed with tetrahydrofuran (20ml) and the filtrate evaporated to dryness in vacuo. The solid residue was triturated with acetone (100ml) and the resulting solid filtered off and dried in a desiccator over phosphorus pentoxide. The aluminium salt was thus obtained as a white solid (0.3g). H.p.l.c. assay gave 48%clavulanate;aluminium content by EDTA titration was 5.76%; Karl Fischer determination gave 5.4% 1^0. Vmax (KBr) 1790, 1695, 1615 cm \

Example

Preparation of Magnesium Clavulanate

A solution containing sodium clavulanate (estimated as about 4.4g) in water (15ml) was loaded onto an ion exchange, column, Zerolit FF(1P), SRA62 and the column washed with one volumn of water. The column was eluted with 0.2m magnesium chloride solution, the fractions collected were z monitored by paper chromatography (detection by Ehrlich’s reagent) and the most intense fractions combined and concentrated to small volume under vacuum.

This crude preparation of the magnesium salt was desalted by percolating down a column of Bio Gel P2 (paper chromatography - Ehrlich's spray).

The desalted material was then concentrated, mixed with ethanol, and chromatographed on a cellulose column using butanol/ethanol/water 4/1/5 v/v top phase, as eluant. Fractions containing the bulk of the material were combined and evaporated to dryness in vacuo. Trituration with acetone followed by filtration gave the desired salt as a white solid (0.58g); h.p.l.c. assay indicated 91% pure magnesium clavulanate; atomic absorption showed 5.13% Mg⁺⁺; Karl Fischer analysis gave 6.8% water; Vmax (KBr) 1780, 1693, 1610 cm.

Example j χ

N, Ν'-Dibenzylethylenediammonium Clavulanate

A solution of benzyl clavulanate (5g) and benzathinecarbonate (2.46) in ethanol (120ml) was hydrogenated over 10% palladised charcoal (1.67g). After 40 minutes, the reaction was virtually complete by t.l.c. The catalyst was filtered off, and the filtrate evaporated in vacuo to a yellow oil (8g). This contained 50% w/w ethanol (by nmr). Hplc analysis indicated purity of 72.4% benzathine diclavulanate on a dry weight basis.

The benzathine-carbonate was prepared as follows:N,Ν'-Dibenzylethylenediammonium acetate (15g) was dissolved in water (100ml) and basified to pH 10 with sodium hydroxide solution. The oily free base was extracted into ether (2x20ml). The ether extracts were dried over sodium sulphate, which was filtered off. The filtrate was treated with solid carbon dioxide to precipitate the internal carbonate of benzathine as a colourless crystalline solid. It was filtered off, washed with ether and allowed to dry in air, to yield 11.2g, 95% yield.

Example /

Magnesium Clavulanate

A solution of benzyl clavulanate (4.34g) and magnesium acetate tetrahydrate (1.61g) in ethanol (100ml) was hydrogenated over 10% palladised charcoal (1.45g) until , no further uptake of hydrogen occurred (circa 20 mins). At this time, t.l.c. showed no unreacted benzyl clavulanate. The catalyst was removed by filtration, washed with ethanol (30ml) and water (10ml). The filtrate was evaporated in vacuo, to yield 5.2g of pale yellow gum, containing some solvent. This was (viturated with acetone (50ml), the resulting solid filtered off, washed with acetone (5ml) and dried in air, to yield 2.4g of colourless solid. H.p.l.c. assay indicates 75% pure magnesium salt.

Example 81

Preparation of Ammonium Clavulanate

Benzyl clavulanate (4.34g) in ethanol (50ml) was treated with ammonium acetate (1.155g; 1 equiv. ) in ethanol (50 ml) and hydrogenated over 10% Pd/C (1.4g) for 10 mins. The catalyst was filtered and the solvent was removed in vacuo to yield a semi-solid. The residue was dissolved in water (5ml) and treated with acetone (75ml). The supernatant was decanted from the originally formed oil and set aside to give needles (.348g); h.p.l.c. assay showed the ammonium clavulanate content was 97.4%; Karl Fischer analysis indicated 0.44% water;Vmax (KBr) 1785, 1700 and 1595 cm.

Example p-BromObenzyl Clavulanate

<img file="IL47087A_D0053.tif" />

Sodium clavulanate (70mg) in dry dimethylformamide (1.0ml) was treated with g-bromobenzylbromide (245mg) and the mixture was left at room temperature for 2 hours. The solvent was evaporated in vacuo and the residue fractionated on silica gel.,-eluting with ethyl acetate-hexane 3:1)־־) to give the product as needles, mp 101-2°; ir(CHCl^), 1805, 1750 and 1695cm“¹; nmr (CDCl^) £.1.94 (1H, S, OH), 3.12 (1H, dd, J

17.5 and 0.8 Hz, ββ-CH), 3-62 (H, dd, J 17.5 and 2.5 Hz, 6aCH), 4.32 (2H, d, J 7.0 Hz, 9-CH^, 4.99 (1H, dt, J 7.0 and

1.5 Hz, = CHCH₂0H), 5.20 (1H, S, 3-CH), 5.28 (2H, S, C0₂CH₂)

5.81 (1H, dd, J 2.5 and 0.8 Hz, 5-CH), 7.45 (2H, d, J 8.5Hz, Aromatic protons) and 7.68 (2H, d, J 8.5 Hz, Aromatic protons).

Example

P-(N,N-diethyl)aminoethyl clavulanate

<img file="IL47087A_D0054.tif" />

<img file="IL47087A_D0055.tif" />

C0₂CH₂CH₂N(Et)

a) Silver Clavulanate

All the following operations were performed in near total darkness.

Sodium clavulanate tetrahydrate (2.93 g) in de-ionised wat&lt; (6 ml) was added to silver nitrate (1.69 g) in de-ionised. water i (2 ml). After 5 minutes the brownish-yellow precipitate was filtered, washed with de-ionised water (2ml) and then acetone (10ml) and dried in vacuo to give silver clavulanate (620mg) as a brownish-yellow solid; V _max (Nujol) 1785, 1695 -1 (shoulder) and 1580 (broad) cm

b) Ester Formation

Silver clavulanate (500mg) was added to 3-(N,N-diethyl)3minoethyl chloride (135mg; 0.6 equivalent) in dimethylformamide (3ml) and the mixture was magnetically stirred in ־the dark for 1 hour at ambient temperature. The suspension was diluted with ethyl acetate (10ml) and filtered. The filtrate was evaporated in vacuo to give a pale yellow oil which gradually -1 darkened on standing; *J (film) 1800, 1750 and 1695cm ;

n.m.r. (CDCl₅)f1.04 (t., J 7.5Hz, N(CH₂CH₃)2), 2.43 - 3-0 (M, -CH₂CH₂N(CH₂^CH3)2), 3.07 (d, J 17Hz, ββ-CH) 3.53 (dd, J 17 and 2.5Hz, 6a-CH), 3-62 (broad s., OH), 4.32 (m, 9-¾ and -C0₂CH₂CH₂N ) 4.98 (br.t., J 7Hz, 8-CH), 5.12 (br.s., 3-CH), 5.76 (d, J 2.5Hz, 5-CH).

Example _£/ ¾/84 (Anthracen-2-yl)methyl clavulanate

<img file="IL47087A_D0056.tif" />

Sodium clavulanate (0.5g) and 9-chloromethylanthracene (1.0g) were stirred in dimethylformamide at room temperature overnight. The reaction mixture was partitioned between ethyl acetate and water and the ethyl acetate layer was concentrated under reduced pressure. Fractionation on silica gel, eluting with ethyl acetate - hexane (1:1) gave the yellow crystalline product (0.5g; 57%), mp 120°; ir (CHCip 1805, 1745 and 1695cm; n.m.r. (CDCl^) ζ 1.35 (1H, br., OH), 2.90 (1H, d, J 17 Hz, 6β-0Η), 3.33 (1H, dd, J 17 and 2.5 Hz, 6a-CH), 3.96 (2H, d, J 7Hz, 9-¾), 4.61 (1H, br.t., J 7 Hz = CH CH₂0H), 4.98 (1H, S, 3-CH), 5.52 (1H, d, J 2.5Hz, 5-CH), 6.13 (2H, S, ¾¾), 7.2 - 8.5 (9H, m, aromatic protons.

Example

N-Benzoxaz olony!methyl clavulanate

<img file="IL47087A_D0057.tif" />

Sodium clavulanate tetrahydrate (0.6g) dissolved in Ν,Ν-dimethylformamide (6ml) was treated with N-chloromethyl־־ benzoxazolone (0.4g) at room temperature with stirring.

After 4 hours, the solvent was evaporated in vacuo and the residue partitioned between ethyl acetate (25ml) and water (25ml) The ethyl acetate layer was dried with sodium sulphate and evapor142 470.87/2 ated to an oil, which was purified by column chromatography on silica gel using cyclohexane and ethyl acetate as eluents; yield 0.5g of a crisp foam after removal of the solvents.

Ir. (film) 3400 (br, OH) 1790 (br, (3-lactam, both ester C=0) 1695cm”'¹ (C=C). N.m.r. 1.6 (1H, bs, exchanges with D₂0, CH₂0H), 3.0 (1H, d, J 17Hz, 6-β-ΟΗ), 3-44 (1H, dd, J17Hz, .

J 3Hz,6-a-CH), 4.08 (2H, d, J 7Hz, CH₂0H), 4.79 (1H, t, J 7Hz, CH=) 5.02 (1H, S, 3-CH), 5.60 (1H, d, J 3Hz, 5-CH), 5.86 (2H, S, CH₂-N), 7.12 (4H, bs, CgH^).

Another sample prepared analogously solidified on standing, it then had mp 95°C.

It had Ir (nujol mull) 3560, 3490, 1795, 1750 and 1690cm \

Example J/ w 86

2-(Benzyloxycarbonyl)-2-(Benzyloxycarbonyl-amino^thyl clavulanate ⁷ i—OH _x0\ ן .

Π NHCO₉ co₂c.h₂c₆h₅

Clavulanic acid (199mg) in dry tetrahydrofuran was cooled to 0°.and treated with N-benzyloxycarbonyl-L-Serine benzyl ester (660mg) and dicyclohexylcarbdiimide. The mixture was allowed to reach room temperature and left overnight (magnetic stirring). Evaporation of the solvent under reduced pressure and fractionation of the residue on silica gel, eluting with ethyl acetate - hexanemixtures, gave the product (108mg) as an oil, infra red (CHCl^) 1805, 1760, 1725cm ¹;

n.m.r. (CDCl^) S 2.2 (1H, br., OH), 2.94 (1H, d, J 17Hz, ββ-CH),

3.35 (׳IH, dd, J 17 and 2.5Hz, 6a-CH), 4.99 (1H, S, 3-CH), 5-10 j (2H, S, C0₂CH₂C₆H ),5.18 (2H, S, C0₂CH₂C₆H₅) 5.45 (IH, d, J 2.5Hz, 5-CH), 5.81 (1H, br.d, J 8Hz, NH), 3-35 (1OH, S,2 _x CgH^.

The remaining protons appeared in the region 4.1-4.9 £ as a complex pattern.

Example /&lt;$ 87

3-cyanopropyl Ester of clavulanic acid | x^>CH. CH₂0H

1- — ^xC0₂Na

CH.CH OH / 2

----->

₀/ ^{N x} C0₂CH₂CH₂CH₂CN

Sodium clavulanate tetrahydrate (2.9g) was dissolved in dimethylformamide (50ml) at room temperature then 4-bromobutyronitrile (2.2g) was added. The reaction was stirred for 3 hours at room temperature when a sample examined by tic (silica plates developed in ethyl acetate) showed appreciable ester formation R_f 0.6. The reaction was worked up in the usual ' way i.e. extraction into ethylacetate followed by several' water washes, drying over MgSO^ resulting,after evaporation of the solvent,in a yellow oil. N.m.r. examination indicated an excess of unreacted nitrile. The oil was dissolved in chloroform and applied to a column of G60 silica (20g) and eluted with chloroform. The product-containing fractions (tic) were collected and evaporated in vacuo to yield the desired product as a pale yellow oil.

Ir and N.m.r. consistent with structure (contained trace solvents).

Nmr (CDC1 ): 2.0? (2H, m, CH₂CH₂CN), 2.50 (2H, t, J 6Hz, CH₂CN), 2.68 (1H, br., OH), 3.05 (1H, d, J 1.7.5 Hz, 6β-0Η),_.

3.55 (1H, dd, J 17*5 and 2.5 Hz, 6a-CH), 4.1-4.5 (4H, m, C0₂CH₂ and CH₂0H), 4.93 (1H, t, J 7.5 Hz, 8-CH), 5.13 (1H, S, 3-CH), 5.72 (1H, d, J, 2.5Hz, 5-CH).

Example p-Tritylether of benzyl clavulanate

0.CH.CH₂0H

OT ^x C0₂Na _/O^CH.CH₂OH

L. k—L 0C — Hi

2 2 Hi

Sodium clavulanate tetrahydrate (17.1g) was dissolved .Ph

Ph

Ph

Hi in dimethylformamide (500ml) at room temperature then the p-tritylether of benzyl bromide (23g very crude material/was added . The solution was stirred for 1 hour at room temperature, then worked up in the usual way by. extraction into ethyl acetate, etc. The product was isolated by column chromat ography on silica G60 (400g)using chloroform: n-hexane 1:1. The product thus obtained still contained some UV-sensitive impurities (not clavulanic impurities), and a further chromatographic separation was carried out on G60 silica (40g) using ethylacetate:n-hexane 1:1 as eluant. This yielded a satisfactory product.

N.m.r. consistent with structure.

Infra red,(nujol) 1805, 1747־ and 1695cm. Nmr (CDCl^)

2.94 (1H, d, J, 17 Hz, 6β-0Η), 3.40 (1H, dd, J 17 and

2.5 Hz, 6a-CH), 4.16 (2H, d, J 6.5 Hz, CH₂0H), 4.83 (1H, t, J 6.5Hz, 8-CH), 4.99 (3H, br.S, C0₂CH₂ and 3-CH), 5.61 (1H, d, J 2.5Hz, 5-CH), 6.6-7.7 (m, aromatic protons).

Example

Ethoxymethyl clavulanate

To a solution of anhydrous sodium clavulanate (5.0g) in dry dimethylformamide (50ml) was added ethoxymethyl chloride (2.14g, 1.5ml) with stirring. After circa 30 mins at ambient temperature the mixture was evaporated in vacuo and water and ethyl acetate added to the residue. The thylacetate extract was dried (Ν3₂80^) and evaporated to an oil, which was subjected to chromatography on silica gel using cyclohexane and ethyl acetate (1:1 ratio) as eluent. The fractions containing the product (by tic) were combined and evaporated under reduced pressure, to yield a very pale yellow oil (2.98g).

Ir (film) *׳max 3400(br), 1800, 1746, 1698cm; SiCDCl.^): 1.22

47087/2 (3H, t, J 7Hz, ¾), 2.94(1H, d, J 17Hz, β-β-CH), 5.13 (1H, s, OH), 3-50 (1H, dd, J 17 and 3Hz, 6-a-CH), 3.68 (2H, q, J 7 Hz, ¾¾), 4.16 (2H, d, J 7Hz, 9-¾), 4.91 (1H, t, J 7Hz, 8-CH), 5.06 (1H, s, 3-CH), 5.29, 5.37 (2H, ABq, J 6Hz, CH₂OEt) and 5.68 (1H, J 3Hz, 5-CH).

Example 1} ^90

2-Bromoethyl clavulanate

<img file="IL47087A_D0058.tif" />

A solution of sodium clavulanate tetrahydrate (2.05g) in dimethylformamide (5ml) was treated with 1,2 - dihromoethane (1ml). The mixture was stirred at room temperature for 7 days, and then most of the solvent was removed under vacuum at 30°C. Water was added to the residue and the mixture extracted with ethyl acetate. Theethyl acetate solution was washed with water, dried over magnesium sulphate and evaporated. The product was isolated by column chromatography (Kiesel.gel, ethyl acetate as eluent). Yield = I68mg .

N.m.r. (CDClj) 2.0 (1H, broad 5> exchangeable), 3.04 (1H, d,J 17Hz), 3.49 (2H, t, J 7Hz), 3.48 (1H, dd, J 3 and 17Hz), 4.18 (2H, d, J 7Hz), 4.43 (2H, t, J 7Hz), 4.93 (1H, t, J 7Hz), 5.05 (1H, s with fine, coupling), 5.67 (1H, d, J 3Hz).

Example 9^91

Phe.nacyl clavulanate

<img file="IL47087A_D0059.tif" />

S0״CH_oC CrH.

2 לס

Sodium clavulanate tetrahydrate (0.5g) in the minimum quantity of dimethylformamide was treated with phenacyl bromide (0.34g) and the mixture stirred at room temperature for 3 hours. The solvent was removed in vacuo and the residue dissolved in ethyl acetate. The ethyl acetate solution was washed with water, dried (MgSO^) and evaporated to give the phenacyl ester (400mg) as a solid; mp 89-90°; infra red (film) 3450, 1800, 1760 and 1700cm; n.m.r. (CDCl^S 3.02 (1H, dd, J 17 Hz and 1Hz, ββ-CH), 3-65 (1H, dd, J 17 and 3Hz, βα-CH),

4.20 (2H, d, J 8Hz, 9-¾), 5.00 (1H, dt, J 8Hz, = CH CH₂0H), 5.23 (1H, br. s., 3-CH), 5-58 (2H, S, ¾000^), 5.74 (1H, dd, J 17 and 1Hz, 5-CH), 7.7 (5H, m, COC^H^); M.Wt. (mass spectrescopy) 317.0894 (observed), 317.0899 (calculated).

Analysis Found C 60.23; H 5-00; N 4.40%

Calculated C 60.57; H. 4.73; N 4.42%

Example JZ. ⁹Λ92

2-Pyridy|nethyl clavulanate

<img file="IL47087A_D0060.tif" />

To a solution of sodium clavulanate tetrahydrate (1.17g) and 2-pyridylmethyl bromide hydrobromide (1.01g) in DMF (8 ml) was added potassium carbonate (552 mg). The mixture was stirred overnight, then diluted with an equal volume of ethyl acetate. The solution was filtered, the filtrate evaporated, and the residue was extracted with ethyl acetate. This procedure was repeated and the product purified by chromatography (Kieselgel, ethyl acetate). Yield 335. mg.׳

I.r (film): 3450, 1805, 1750, 1695cm

N.m.r (CDCl^) £ 2.48 (1H, br., exchangeable) 3-01 (1H, d, J 17Hz) 3.45 (1H, dd, J 17 and 3Hz) 4.19 (2H, d, J 7Hz) 4.91 (1H, t with fine coupling, J 7Hz) 5.1 (1H, s with fine coupling) 5.25 (2H, s) 5.66 (1H, d, J 3Hz) 7.1-7.4 (2H, m) 7.67 (1H, dt, J 8 and 1Hz) 8.52 (1H, d, J 6Hz)

Example ^95

N-Benzyloxycarbonyl-N-methylaminoethyl clavulanate

<img file="IL47087A_D0061.tif" />

<img file="IL47087A_D0062.tif" />

C0_oCH_oC&lt;H.

&lt;L &lt;L 5 ס

Benzyl clavulanate (1g) was hydrogenated over 10% Pd/C (400mg) in THF (30ml) until no ester remained. Thesolution was filtered and used as described below.

1

A solution of N-methyl-N -nitroso-Ν,Ν -dibenzyloxycarbonyl1,2-diaminoethane (3.71g) in (10ml) was cooled to -30 C and treated with pyrrolidine (0.71g). The solution was stirred at -25 to -30°C for 30 mins, then cooled to -35°C. The clavulanic acid solution prepared as described above was then added.to the stirred solution of the thus-prepared diazo compound at such a rate that the temperature did not rise above -30°C. After a further 15 minutes stirring, BF^ etherate (0.5ml) was added, and the solution stirred for.5 minutes. Excess NaHCO-, solution was added, the aqueous layer was

כ separated, and extracted with methylene chloride. The combined methylene chloride solutions were washed with water, dried (MgSO^) and evaporated. Repeated chromatography of the residue (Kieselgel H, ethyl acetate, chloroform and ether as eluents) gave a colourless gum (180mg).

־ 150 — ή ¹

Ir (film): 3450, 1800, 1745, 1600cm

N.m.r. (CDC1_Z): S 2.25 (1H, br. s, exchangeable) 2.93 (3H,s)

¹ כ

3.00 (1H, d) 3.3-3.6 (5H, m) .4.17 (2H, d, J 7Hz) 3.85 (1H, t, J 7Hz with fine coupling) 4.98 (1H, s,) 5.08 (2H,s) 7.28 (5H,s).

Example 0$94.

1-Acetoxyethyl clavulanate

To dry DMF (5ml) stirred at 0° was added sodium clavulanate tetrahydrate (1.025g), followed,. after 10 min by 1-bromoethyl acetate (1ml). The mixture was allowed to stand overnight in a refrigerator, then the solvent was removed in vacuo. Water was added to the residue, and the mixture extracted with ethyl acetate. The organic phase was washed with water, dried (MgSO^) and evaporated. Chromatography of the residue (Kieselgel 60, ethyl acetate) gave the title compound (176 mg) (Rf 0.49).

i

Ir (CHClj): 3350, 1810, 1770, 1695cm¹ ' I

N.m.r. (CDC1₃):S1.47 and 1.49 (3H, two doublets, J 5Hz, mixture of isomers at ethyl C1) 2.04 (3H, s) 3.01 (1H, d, J 17Hz) 3.45 (1H, dd, J 17 and 3Hz) 3.15 (1H, br, exchangeable) 4.17 (2H, d, J 7Hz) 4.80 (1H, m) 4.98 (1H, s with fine coupling) 5.63 (1H, d, J 3Hz) 61) 78.־H, q, J 5 Hz.)

Example $/ W95

-Ethoxycarbonyloxyethyl clavulanate (a) 1-Chloroethyl. chloroformate

Ethyl chlorformate (108.5g) and a-azoisobutyronitrile (0.5g) were stirred and refluxed. Dry chlorine gas was passed through the solution until a weight increase of 34.5g (corresponding to monochlorination) was observed. The liquid was fractionated at atmospheric pressure to give 1-chloroethyl chloroformate (33g) b.pt. 117-118°C.

(b) Ethyl 1-chloroethyl carbonate

The product of (a) above (14.3g) was added with stirring to ethanol (50ml), the temperature rising spontaneously to 62°C. When the temperature had dropped to 30°C, water . (200ml) was added. A heavy oil separated and was collected. The aqueous phase was extracted with dichloromethane . (3 x 50ml), the extracts were combined, dried (MgSO^) and evaporated. The resultant oil.was distilled at atmospheric pressure to give the required compound (12.1g) b.pt. 155157°C. The structure was confirmed by n.m.r. and elemental analysis (found: C 39-5, H 6.2, Cl 23.25%; C^H^CIO^ required C 39.4, H 5.9, Cl 23.2%.

(c) 1-(Ethoxycarbonyloxy)ethyl clavulanate

The product from (b) above (3.04g) in DMF (5ml) was added to a stirred solution of sodium clavulanate (5.86g) and potassium iodide (500mg) in DMF (15ml). After 21 hours, the mixture was diluted with ethyl acetate (250ml), washed with saturated NaHCO^ solution, dried (MgSO^) and evaporated to an oil. The product was purified by chromatography (Sephadex LH2O, chloroform/cyclohexane 1:1), to yield a pale yellow oil (206mg) giving a single spot on tic analysis using chloroform/cyclohexane 1:1. The structure was confirmed by n.m.r. spectroscopy.

Example /796

Methoxymethyl clavulanate

Sodium clavulanate (293־g, 0.01 mol) was dissolved in dimethylformamide (20 ml). Chloro dimethylether (0.8 ml, 0.86g) was added with cooling and stirring. After 2 hours at room temperature, the DMP was evaporated, in vacuo and the residue taken up in ethylacetate (40 ml) and cyclohexane (20 ml), Silica gel (t.l.c. grade 7g) was added, and the insoluble materials filtered off, washed with a little fresh solvent and discarded,. The filtrate was evaporated under reduced pressure to small volume, and subjected to column chromatography on silica gel using ethyl acetate and cyclohexane as eluents.

The product was isolated as a colourless oil (2 g). Ir, !/max (film) 1802 (β-lactam C=0) 1755 (ester C=0) 1695an”^](C=C); _nm_r (CDCL^) 2.3 (lH,b3, OH) 31) 02־H, d, J 17 Hz, ό-β-CH) 33) 42־H, s, OCl^) 347־ (Hi, dd, J 17 Hz, 3Hz, 6 CH) 42) 16־H, d, J 6 Hz, CH^QH) 4.89 (1H, bt, J 6Hz, CH=CH ) 5.03 (HI, bs, 3-CH) 5.21, 5.29 (2H, ABq, J ¹⁵ 5.5hZ, CH₂OCH₃) 5.65 S(1H, d, J 3Hz, 5-CH).

f

Example p-Chlorophenoxymcthyl clavulanate

Λ cooled (ice-water), stirred solution of sodium clavulanate tetrahydrate (6g, 0.02mole) in dimethylformamide (60 ml) was treated with p-chlorophenoxymethyl chloride (3.5g, 0.02mole). After

2I hours at &lt; 10°, the ditnethylformamide was largely evaporated in vacuo at room temperature (20 C). To this syrup was added ethyl acetate (200ml) and ice-cold water (20 ml). The ethyl acetate was dried over sodium sulphate, evaporated to a syrup, and the residue was subjected to column chromatography on silica gel using ethyl acetate and cyclohexane as eluents (graded from 1:2 to 2:1 ratio). A small quantity of the p-chlorophenoxymethyl ester of the diene of clavulanic acid eluted first, followed by the required pure p-chlorophenoxymethyl clavulanate (4.7 g). (This contained a very small quantity of tlie diene, detectable by thin layer, chromatography sufficient to be seen in the n.m.r.) I.r.i 3400 (br, OH) 1805 (βlactam C-0) 1765 (ester C=0) 1695cm ¹ (C=C).

N.m.r. : (CDCl ) 1.5 (0H) 3.00 (1H, d, J 17Hz, 6-P-CH) 3.44 (1H, dd, J 17 and 3Hz,6-a-CH)4.14 (2H, d, J 7Hz, ¾011) 4.77 (1H, bt,

J 7Hz, CH =C11J 5.02 (1H, bs, 3-CH) 5.60 (1H, d, J 3Hz, 5-CH) 5.70.

5.77 (2H, AB([, J 7Hz, OCH 0) 6.89, 7.218 (411, A^q, J 9Hz, 0¾) ״ 155 Example/// S#98

Benzyloxyme thy1clavulanate

Sodium clavulanate tetrahydrate (2.93 g) was dissolved in dimethyl formamide (20 ml) and the resulting cloudy solution filtered. The clear yellow solution was cooled with ice and stirred while benzyl chloromethyl ether (1.57 g, prepared according to the method of Graham and McQuillin. J Chem.

Soc., 1963, 4634) was added dropwise. Stirring and cooling were maintained for 22־ hours. The reaction mixture was evaporated to give an orange coloured oil which was extracted several times with ethyl acetate. The combined ethyl acetate extracts were evaporated to a yellow oil.

Analysis of the oil by thin layer chromatography (on silica gel plates, eluted with methyl acetate: ethyl acetate: methylcyclopentane, 1:1:2, visualised with permanganate spi’ay) showed two zones at Rf 0.21 and 0.55, of approximately equal intensity. The oil was chromatographed on a column packed with silica gel and eluted with a mixture of ethyl acetate and cyclohexane (1:1). The composition of the eluted fractions was analysed by tic and the slow running material collected free of any detectable impurity. Evaporation gave an almost colourless oil (0.7 g).

Nmr (CDCl^) : δ 2.08 (1H, bs) , 3.02 (1H, d, J16IIz) , 3.38 (111, dd, J16Hz, J'3Hz),

4.15 (2H, d, J7Hz). 4.64 (2H, s), 4.87 (1H, bt, J7Hz),

4.99 (1H, bs), 5.34 (2H, s), 5.58 (1H, d, J2Hz), 7.25 (5H, bs).

Example j/ fW99

Preparation of p-nitrobenzyloxymethy!clavulanate

Sodium clavulanate tetrahydrate (2.93 g) was dissolved in dimethylformamide (20 ml) and the resulting cloudy solution filtered. The clear yellow solution was cooled with ice and stirred while chloromethyl p-nitrobenzylether (2.02 g) was added dropwise. (This reagent was prepared by the method used by D N Kursanov and P A Solodkov, J Applied Chem. (USSR) (1943), 16 , pp 351-5). The reaction mixture was stirred for 2 hours and the progress of the reaction followed by tic (silica gel plates, eluted with methyl acetate, ethyl acetate, methylcyclopentane, 1:1:2). At the end of this time the major product was p-nitrobenzyloxymethylclavulanate (Rf 0.18) with a small amount of the ether ester as well (at Rf 0.35). Solvent was removed by evaporation and the crude reaction product purified by column chromatography on silica gel eluted with cyclohexane : ethyl acetate 5 : 4. The product was obtained by evaporation of the appropriate chromatography fractions and was initially a yellow oil, but later crystallized.

Infra-red spectrum attached. X-ray powder diffractogram (CuK radiation) reflections at 2Θ :9.7, 11.65, 14.0, 15.6, 16.6, 18.0. 19.55, 20.4, 21.1, 22.8, 23.5, 24.75, 26.3, 28.05, 29.95,

Nmr (CDC1 ) δ 1.85 (HI, bs), 3.01 (1H, d, J=16Hz), 3.42 (1H, dd, J=16Hz, 3Hz)

4,14 (2H, d, J=7Hz), 4.73 (2H, s), 4.85 (Hi, bt, J=7Hz),

4.97 (1H, s), 5.39 (2H, s), 5.59 (1H, d, J=3Hz),

7.75 (4H, dd, J=62Hz, 8Hz).