CA1076120A - Clavulanic acid esters - Google Patents
Clavulanic acid estersInfo
- Publication number
- CA1076120A CA1076120A CA314,882A CA314882A CA1076120A CA 1076120 A CA1076120 A CA 1076120A CA 314882 A CA314882 A CA 314882A CA 1076120 A CA1076120 A CA 1076120A
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- Prior art keywords
- clavulanate
- clavulanic acid
- ester
- methyl
- recovering
- Prior art date
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Abstract
ABSTRACT OF THE DISCLOSURE
A new antibacterially active agent has been isolated from Streptomyces clavuligerus. This new compound which we designate clavulanic acid has the formula (I):
A new antibacterially active agent has been isolated from Streptomyces clavuligerus. This new compound which we designate clavulanic acid has the formula (I):
Description
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This application is directed to the preparation of esters of clavulanic acid and is a divisional o~ S.N. 224,970 ~iled 18 April 1975. The -parent application is directed to clavulanic acid and its preparation by cultivation of Streptomyces claYuligerus and conversion to salts as required.
Other divisional applications S.N. 314,881 and S.N. 314,880 filed 30 October , 1978 are directed respectively to the preparation of clavulanic acid and its `~ salts by deesterification and synergistic mixtures of clavulanic acid or its pharmaceutically acceptable salts or esters with a ~-lactam antibiotic.
~ This invention relates to a new agent having antibacterial : !
- i 10 ~ activity. More particuIarly it relates to a new antibacterial agent obtainable from Streptomyces clavuligerus,-designated as clavulanic acid, and salts and ,. . .
~i esters of the agent.
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' B GROUND TO THE INVENTION
., ~ a. Streptomyces clavuligerus has been described in detail by : 1 ; Higgens et al, Int. J. Systematic Bacteriology, 21, 326 (1971).
This streptomycete was of interest because it produced certain lactam antibiotics such as penicillin N, 7-~5-amino-5-carboxy-valeramido)-3-carbamoyloxymethyl-3-cephem-4-carboxylic acid and 7-(5-amino-5-carboxyvaleramido)-3-carbamoyloxy-methyl~
7-methoxy-3-cephem-4-carboxylic acid. The streptomycete has ~ -been deposited in the Agricultural Research Service Collection as NRRL 3585 and in the ~merican Type Culture Collection as i ~:
ATCC 27064. Streptomyces clavuligerus has also been reerred : 1 ~ to in United States Patent No. 3,770,590 and also by Nagarajan et al J.Amer.Chem.Soc., 93 2308 (1971), Bxannon et al, ntimicrob.
Agents Chemother., 1, 237 (1972) ~ntibicrob ~gents Chemother, . , .
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,; , , ~- 1, 247 (1972) Higgens et al, 3. ~ntibiotics, 27, 298 (1974).
~ b ~ -lactamases are enzymes which open the ~ ~lactam ring of :~ penicillins and cephalosporins to give products which are I devoid of antibacterial activity. These enzymes are produced by ~any ' ~' . , ' . ' ; ~!
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bacteria, notably species or strains of scherichia, Klebsiella, Proteus, Pseudomonas, Enterobacter and Staphylococcus and are in many . . -instances the explanation for the resistance of certain strains of such organisms to some penicillins and cephalosporins. The importance of ~-lactamase production may be understood when it is realised that a high proportion of clinically isolated organisms produce ~-lactamses ~ [see, for example, M. Wilson and I.~. ~reeman, Bacteriological Proceed-`. ings, 80 (1969) where in a paper entitled 'Penicillin Inactivation by ~,; Gram-negative Bacilli~ they showed that 84% of the gram-negative IO
organisms isolated in an American hospital produced ~-lactamase] . In many cases, some penicillins or cephalosporins are ineffective in ,~ treating diseases ascribed to non ~-lactamase-producing organisms because of the co~mon occurance of co-infection by a ~ lactamase ~ ~ producer ~see, for example, R. May et al; Brit. J.Dis.Chest., 66, 185 `~!
~l ~ (1972)~ . Combination o~ a ~ lactamase inhibiting substance with a ~:
penicillin or cephalosporin might be expected to protect the latter ~ I .
from degradation by bacterial ~-lactamase and thereby enhance their antibacterial activity against many infective organisms. This process of enhancement of the antibacterial activity is called synergism when ' 1 ~ . .
the antibacterial activity of the combination is well in excess of the simpIe addition of the activities of the two separate substances. The ~. :
~-lacta~ase inhibiting component of the mixture is referred to as a synergist and such substances are valuable for increasing the anti-bacterial activity of penicillins and cephalosporins against resistant organisms. It is one of the objects of this invention to provide such synergists.
c. Examples of the use of certain ~ lactamase resistant semi-synthetic ;` penicillins and cephalosporins as ~-lactamase inhibitors and synergists for penicillins and cephalosporins have already been .
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~, 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 à dramatic effect on the spectrum of the other antibiotic present in the ~ ;
."'! mixture.
,, `` d. Certain actinomycete cultures have been described as producing lactamase inhibiting substances which act synergistically with penicillins or cephalosporins, for example, those cultures -~ 10 ~ disclosed in British Patent No. 1,363,075 and those described ,. . . .
by ~lata et al, J. Antibiotics, 25, 473 (19723 and Umezawa et al, J. Antibiotics, 26, 51 (1973). None of these ~-lactamase inhibitors of actinomycetal origin have yet been found to be of use in the clinic. ParticuIarly noteworthy features wh1ch distinguish clavulanic acid from other ~-lactamase inhibitors of actinomycetal 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 ~-lactamase inhibiting . .
activity, its low molecular weight and its high Rf values on `20 paper chromatography using a variety of solvcnt systems.
DESCRIPTION OF THE INVENTION
` We have discovered that the aerobic cultivation of Streptomyces clavuligerus in conventional nutrLent media at about 25-30C under rougllly , ~ neutral conditions produces a ~-lactamase inhibitory-substance which also possesses antibactcrial activity. We have designated this new material clavulanic acid'.
~`~` Clavulanic acid has the followi~g properties: `
~. . , ~ (a) It is a carboxylic acid.
; (b) It forms a sodlum salt whlch has a characteristic infra-red spectrum substantially as shown in Figure 1.
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(c) It is able, to inhibit the growth of stra ms of Staphylococcus aureus.
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,~ ' (d) It is able to synergyse the antibacterial effect of , , , ~ ampicillin against ~-lactamase producing strains of ,' ,~I ` Escherichia coli, Klebsiella aerogenes and Staphylococcus ~'' :' q '.
. I aureus ''`, (e) It is able to synergyse the antibacterial effect of ' cephaloridine against the ~-lactamase producing strains ` : -: of Proteus mirabilis and Staphylococcus aureus. ' ~ ~
10 ~ (f) It forms a methyl ester which has a molecular weight .: -(by mass spectroscopy) of 213.0635 which corresponds -':. ~ ,, to the formula C9UIlNO5. ~ ''~ ~ ~~` ' "''' Thus clavolanic acid may be regarded as a monobasic carboxylic acid of~the formula C8HgN05 which in the form of its sodium salt,has a .
characterlstic infra-red absorption spectrum substantially as shown in Fig. 1. '~
The compound produced by Streptomycee clavuligerus which has ' ~ the~above praperties has the formula (II)~
"`~ / 0 ~ ~ C~120U
20~ ~ - N ~ ~ (II) , , Thus clavulanic acid may be named 3- ~-hydroxyethylidene)-7- ,' ', oxo-4-oxa-1-azabicyclo r3,2,0] heptane-2-carboxyl-lc acld. ~ '' '-The stereochemistry at C5 and C2 of the clayulanic acid is ~' ' the same as that found in naturally occurring penicillins and cephalosporins ' so that clavulanic acid may be represented by the structural fonmula ~
r~~ C~I20~ ~ .
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3- ~-hydroxyethylidene)-7-oxo-4-oxa-1-azabicyclo!3,2,0J heptane-2-carboxylic .
acid.
The 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~g/ml. of ampicillin, amoxycillin, carbenicillin or benzyl penicillin or by the presence of lO~gtml. of claw lanic acid, are inhibited by the presence of less than 12.5/lg/ml. o the previously mentioned penicillins when 5~g/ml. of clavulanic -; -acid is also present. Similar results have been obser~ed for combinations containing various esters of clavulanic acid. ~or example, strains of ;
Klebsiella aerogenes A, the growth of which is not inhibited by 125~lg/ml. of ampicillin, or by lO~lg/ml of clavulanic acid methyl ester are inhibited by less than l2.5~lg/ml. of ampicillin in the presence of 5~g/ml. oE the clavulanic acid methyl ester. It has also been found that strains of Staphylococcus aureus Russell, the growth of wh1ch lS not inhibited by the presence of 100/.~gtml. of ampicillin or by 5~Lglml of clavulanic acid, are inhibited by the presence of less than 10).Igtml. of ampicillin in the presence of l)lgtml. of clavulanic acid. In tests on female mice, it has been found that blood and tissue levels of clavulanic acid considerably in excess o 5)lg/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 administratlon of 100 mgtkg of the sodium salt of clavulanic acid.
,, Accordingly, the present invention provides clavtl1anic acid as hereinbefore described and its salts and esters.
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Most suitably the salts of clavulanic acid will be pharma-ceutically acceptable salts such as the sod:Lum, potassium, ca].cium, magnesi-tm, aluminium, ammonium and substituted ammonium salts such as the trimethyl-ammonium, benzathine, procaine and like salts conventionally formed with ~;` ' . ;~,'"
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penicillins or~cephalosporins. Non-pharmaceutically 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 form the useful benzyl ester of clavulanic acid.
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Salts of claw lanic acid tend to be more stable than the ~' parent acid per se and thus form a favoured aspect of this invention.
Particularly suitable salts of clavulanic acid include the sodium and , potassium salts which have the formulae (III) and (IV) respectively:
,C~20~1 rl~C~20u ~
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02~a C02K -. ~ ~
Crystalline forms of such salts may contain water of hydration. -Suitable esters of clavulanic acid include those notionally derived from alcohols such as methanol, ethanol, propanol, butanol, 2,2,2~
trichloroethanol, 2,2,2-trifluoroethanol, benzyl alcohol, p-nitrobenzyl ~`
20 alcohol, phenol, acetoxymethanol, pivaloyloxymethanol, 2 dimethylaminoethanoland 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 or thiol of the formula ROH or RSH where R is an organic residue. Suitable groups R include alkyl, alkenyl, alkynyl, aryl, ; arylalkyl or other similar groups any o which may be substituted if desired.
In order not to increase the molecular weight to an unreasonable extent, groups R do not normally include more than 16 carhon atoms, more suitably, . ~' ' ' '' '" ' '`' , ' "~ ~ .
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not more t1ul11 l c~rbo11 atoms and P1ost suitably, not more than 8 carbon atoms. '!.', : , ' . " '.' ~
The esters of tlle clavulanic acid may thus be represented . ' . ,~ , . .
by the formula ., . ,, :.. .
fl'2"
:, o \ : ' C~W---R
wherein W is O or S and R is as defined.
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Prefcrably, the group R is notionally derived from an alcohol ROH or (less favorably) a thiol RSH which is pharmaceutically acceptable.
- ,I Suitable substituents Which may be included in the group R
.... ~ - , , include halogen atoms and lower alkoxyl, hydroxyl, lower acyloxyl, lower alkylamino, lower dialkylamino and like groups. The term 'lower' means that . , !
the group contains up to 6 carbon atoms, and preferably up to 4 carbon atoms.
Thus, for example, R may be a methyl, ethyl, n-propyl, iso-propyl, straight or branched butyl, pentyl, heptyl, octyl, nonyl~ decyl, undecyl, dodecyl, ~ .
vinyl, allyl, butenyl, cyclop~ropyl, cyclobutyl, cyclopentyl, cyclohexyl, .: :1 . ... .
cycloheptyl, cyclohexenyl, cyclohexadienyl, methylcyclopentyl, methylcyclo- .20 ~ hexyl, benzyl, benzhydryl, phenylethyl, napthylwethyl, phenyl, napthyl, propynyl, tolyl, 2-chloroethyl, 2,2,2-trichloroethyl, 2,2,2-trifluoroethyl, ~;
!~ ~ acetylmethyl, bénzoylmethyl, 2-methoxyethyl, 2-dimethylaminoethyl, 2-diethyl-aminoethyl, 2-piperidinoethyl, 2-morpholinoethyl~ 3-dimethylaminopropyl, ~ ; p-chlorobenzyl, p-methoxybenzyl, p-nitrobenzyl, p-bromobenzyl, m-chlorobenzyl, ;
`~ 6-mcthoxynapht11yl~2-met11yl, p~chlorophenyl, p-methoxyphenyl or any like ; group as well as those groups Which are known from the penicillin or cephalo- ;
sporin arts to produce esters known to be readily hydrolysed in Vivo to the . parent antibiotic.
.~ - Readily hydrolysable esters include, but are not l.imited to, those of the formulae (V) and (VI):
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~'~ wherein Al is a hydrogen atom, alkyl, aryl or aralkyl group; A2 is a hydxogen . i~ atom or methyl group; A3 is an alkyl, aryl or aralkyl group; X is oxygen or sulphur; Y is oxygen or sulphur and Z is a divalent organic group. Esters :1 ~ of the formulae (V) and (VI) whicll fairly readily release the clavulanic acid ~ ~ j ~ into the blood stream after administration include those wherein Al is a :: .
~ hydrogen atom, A2 is a hydrogen atom or a methyl group and A3 is a methyl, ' l . .. .
ethyl, propyl:, butyl, benzyl, or phenyl group and those wherein X is oxygen, Y is oxygen and Z is -CH2CH2-, -Cll:CH-, ~ ~ or ~
.,~ When used in conjunction with the preceding foxmula the term : 'alkyl~ includes alkyl of up to six carbon atoms; the term 'aryl~ includes : phenyl, naphthyl or phenyl subs.tituted by an inert substituent such as a ~ .
;: fluorine or chlorine atom or a methyl or methoxyl group or the like; when , used herein the term ~aralkyl~ means an alkyl group substituted by an aryl .~ group.
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, Particularly suitable esters of the formulae (V) and (~
include those of the formulae (VII) and (VIII): ~ -: D
A
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; 10 A4 is a hydrogen atom or a methyl group, A5 is a methyl, t-butyl or phenyl group and A6 is a hydrogen atom or a methoxyl group.
Many esters of clavulanic acid differ from analagous 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 p~l7. Es~ers which undergo some hydrolysis under miId conditions are included within the ormuIa (IX): ~ -L~ C~12~
~' wherein Rl is a hydrocarbon group of 1-9 carbon atoms optionally substituted ' ' ~ by halogen, lower alkoxy, hydroxyl or optionally salted basic groups of the '~ ~ formula NR R3 wherein R is a hydrogen atom or a lower alkyl group, R is a ~ hydrogen atom or a lower alkyl group or is attached to R2 so that NR2R3 is a : :: .
, 5~ or 6- membered ring. ' `
When used wlth referencc to formula (IX) the term 'lower~ ~-, . . :~ .
- means that the group contains 1-4 carbon atoms.
Suitably groups Rl include alkyl and aralkyl groups optionally substituted by halogen, methoxyl, hydroxyl or salted NR2R3 groups wherein R2 : .
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is a methyl or ethyl group and R3 i5 a methyl or ethyl group or is joined to R so that NR2R3 is a pyrrolidine, piperidine or morpholine group.
Most suitably alkyl groups Rl are straight chain groups of up to 6 carbon atoms optionally substituted by one methoxyl, hydroxyl, salted NR2R3 group or one chlorine, bromine or iodine atom or by a CCl3 or CF3 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 belived that !
clavulanic acid and its salts tend to be somewhat more useful synergistic agents than the esters per se. Many of the esters of the formulae (~ (IX) are useful for this purpose.
A further group of particularly suitable`èsters of this mvention are those useful intermedlates which are;readily converted to i ~ clavulanic acid or a salt thereof by chemical or biochemical techniques which . ~
are known from the penicillin or cephalosporin arts to be sufficiently mild not to degrade reactive acid-labile ~-lactam rings.
Most suitably, the ester is one removable by hydrogenolysis.
Conventional esters for such a process include benzyl, substituted benzyl, benzhydryl, substituted benzhydryl, trityl and the like. The benzyl ester has proved particularly useful for this purpose.
~,; By and large, the nature of any substituent in the ester moiety is ~mimportant as long as it does not interfere with the hydrogenolysis reaction.
- . , Since clavulanic acid and its salts are useful intermediates ~t~ 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 : " ' , .
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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 or oral, topical or parenteral use and may be used for the treatment of infection in mam~als 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 compo-~ ~ sltions 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 welI understood by those skilled in the art of formulating an~ibiotics.
.: : .:,. , 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 inEusion. ~;
Ti~us, one preferred composition aspect of this invention comprises cLavulanic acid or a salt thereof m -sterile form.
Unit dose compositions comprising clavulanic acid or a salt '-or ester thereof adapted for oral administration form a further preferred c~mposition aspect of this invention.
Under certain conditions, the effectiveness of oral compo-sitions of clavulanic acid and its salts and esters can be improved :Lf such compositions contain a bufferLng agent or an enterlc coating a8ent sucll that the compounds of the invention do not have prolonged contact with highly acidic gastric juice. Such buffered or enterically coated composit;lons may ''"i ~ ' 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 j 30 other therapeutic agents such as a ~-Lactam antibiotic, Su;ltable ~-].actam ., , -, , , -~ 1 1 --, . . .
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antibiotics for inclusion ln sueh syne~g~stic co~poSitions include not only those known to be highly susceptible to ~ lactamases but also those which have a good degree of intrinsic resistance to ~-lactamases. Thus, suitable ~ -lactam antibiotics for inclusion in the compositions of this invention include benzyl-penicillin, phenoxymethylpenicillin, carbenicillin, methicillin, propicillin, ampicillin, amoxycillin, epicillin, ticarcillin, cyclacillin, 6-aminopenicillanic aeid, 7-aminocephalosporanic aeid, 7-aminodesacetoxycephalosporanic aeid, cephaloridine, cephalothin, cefazolin, cephalexin, eefoxitin, cephacetrile, ~ cephamandole, cephapirin, cephradine, cephaloglycine, and other well known ; ;~ 10 penicillins and cephalosporins or pro~drugs there~ore such as hetaeillin, : . , metampicillin~ the acetoxymethyl, pivaloyloxymethyl or phthalidyl esters of benzylpenicillin, ampieillin, amoxyeillin or eephaloglycine or the phenyl, tolyl or indanyl ~-esters of earbenieillin or ticarcillin or the like.
~ Naturally if the penicillin or cephalosporin present in the i ~ 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 -laetam antibiotic, the ratio of clavulanic acid or its salt or ester present to e-laetam antibiotie present m y be from, for example, 20:1 to 1:12, more usually lO:l to 1:10 and advantageously may be from 3:1 to 1:3.
The total quantity of antibacterial agents present 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 infeetions ôf 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 an-lmals such as mastitis in cattle.
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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 3000 mg of the composition of the invention will be administered per ~-day However, for the trea~ment of severe systemic infections or infections ~` of particularly intransigent organisms, higher doses may be used in accor-dance with clinical practice.
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;~ The exact form of the compositions of this invention will depend to some extent on the micro-organism which is being treated. For treatment of most infections the compositions of this invention are normally adapted to produce a peak blood level of at least O.l,ug/ml, more suitably at lcast 0.25 ~lg/l~, and preferably at least 1 ~Ig/ml. of synergist, for - ~ I
example, 2.5 - 5 yg/ml. of synergist. -~
.- : .
The penicillin or cephalosporin in synergistic compositions of thls invention will normally be present by up to or at approximately the ~ amount conventionally used when that penicillin or cephalosporin is the sole ; ~ therapeutic agent used in the treatment of infection.
. :
Particularly 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 materlals present in such compositions may be hy(lrated if required. Tlle weights of the antibiotlcs 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 Streptom~ces clavuli~ md recovering clavulanic acid or a salt thereof from the culture medlum and ~ ' ;. ~ ' ' .. .
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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 process of this invention.
~ /; ~ r When used herein, the term 'cultivation' means the dcliv~retc 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 or suspended. The culti-- ` 10 vation may take place on an aerobic surface or by submerged culture. The nutritive medium may be composed of complex nutrients or may be chemically ; 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 clavuli~erus may contain, in the range 0.1 - 10% a complex organic nitrogen source such as yeast extract- corn steep liquor, vegetable protèin, seed protein, hydrolysates of such proteins, milk protein hydrolysates, ; fish and meat extracts and hydrolysates such as peptones. ~lternatively chemically defined sources of nitrogen may be used such as urea, amides, ~ single or mixtures of common amino acids such as valine, asparagine, glutamic scid, proline and phenylalanine. Carbohydrate (0.1 - 5%) may be included in the nutrient media but glucose in certain media is undesirable having a ;~ depresslng efect on the yield of the desired clavulanic ac:Ld. Starch or . ~. , .
: - starch hydrolysates such as dextrtn, sucrose, lactose or other sugars or `;
' glycerol or glycerol esters may also be used. The source of carbon may also i : ....
be deriyed from vegetable oils or animal fats. Carboxylic acids and their salts can be included as a source of carbon for growth and prod~ction of : ~, ' :, lactamase inhibitors. A particularly suitable low cost medium is one containing soya bean flour (Arkasoy ) plus dried malt distillers solubles ,: ,::
(Scotasol ) plus dextrin *Trademark ' '" ' ., '' '., ,.',' ~, ::
The addition o~ antifoam agents such as Pluronic L81 may be necessary to control foaming of certain media in fermenters.
Mineral salts such as NaCl, KCl, MgC12, ZnC12, FeC~3, Na2S04, FeS04, MgS04 and Na~ or K+ salts of phosphoric acid may be added to the media described - above particularly if chemically defined; CaC03 may be added as a source of Ca ions or for its buffering action. Salts of trace elements such as nickel, cobalt .!.~
or manganese may àlso be included. Vitamins may be added if desired.
When used 1lerein the term ~mutant' includes any mutant strain which arises spontaneously or through the effect of an external agent whether .
~ 10 that agent is applied deliberately or otherwise. Suitable methods of producing ;~, .i , mutant strains include those outlined by H.I. Adler in Techniques for the Development of Micro-Organisms in 'Radiation and Radioisotopes for Industria - Micro-Organisms~, Proceedings of a Symposium, Vienna, 1973, page 241, International Atomic Energy Authority and these include:
i. Ionising radiation (such as X- and ~-rays), uv light, uv light plus a photosensiti~ing agent (such as 8-methoxy-psoralen), 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 40C, usually 20-35C and preferably, 25-30C and at ' ' a pH of between 5 and 8.5, preferably between 6 and 7.5.
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The Streptomyces clavuli~er~ls may be cultivated in the above . media in glass conical flasks aerated by shaking on a rotary shaker or in baffled stainless steel fermenters stirred with vaned disc impellers and aerated 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 is obtained in 2-10 days at 20-35C. In a stirred stainless steel fermenter using the Arkasoy/Scotasol/Dextrin medium referred to above the preferred temperature is 26C and peak yields clavulanic ; 10 are obtained within 5 days.
Clavulanic acid may be extracted from culture flltrate by a variety of methods. Solvent extraction from cold culture filtrate adjusted to acid pH values and methods ~ased 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 normall~ 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 thorougly mixing with a water immiscible organic solvent such as n--,! :
butylacetate,~methylisobutylketone, n-butanol or ethylacetate. The acid used to lower the pH of the medium ls normally a mineral acid such as hydrochloric, sulphuric, nitric, phosphoric or the like acid. n-Butanol is a part;icularly sultable solvent for use in the extraction of the acidified culture filtrate.
After separation of the phases by centrifugation, the ~'-lactamage inhibiting metabolite is back extracted from the solvent phase into aqueous sodium bi-carbonate or potassium hydrogen phosphate bufEer, CaC03 suspension or ~ater while maintaining the p~l at approximate neutrality, for example, at pH 7Ø
. .
This aqueous extract after separation of phases may be concentrated under : - , 30reduced pressure and freeze dried to give a crude preparation of a salt of ':; .~. : . ' '' :
- 16 - ~
. .
~ 1076~2(1 -~ clavulanic acid. This preparation is stal~le ~hen stored as a dry solid at -20 C.
~: In the anion exchange resin process, the c:Larified culture filtrate at an approximately neutral or slightly acid p~1, for example pH 6-7, -; is percolated down a column of weak or strong base anion e2cchange resin such as Amberlite IR4B or Zerolit ~FIF respectively until the resin is saturated -~
. , ~ and the ~-lactamase inhibiting material emerges from the bottom. The column . ,. , - :
is then washed with water and eluted ~ith aqueous sodium chloride. The ~-lactamase inhibiting fractions are collected, bulked, desalted cmd freeze - ~ ~
dried to yield a crude solid salt o~ clavulanic acid. -~ -. . : -, A2nberlite IR 4B is an example of a weakly basic anion exchange resin with polyamine active groups and cross linked polystyrene-divinyl-benzene matrix. Zerolite FFIP is a strongly baslc anion exchange ~ ~ -resin with quaternary ammonium active groups and a cross linked polyvinyl--divinylbenzene matrix. Resins similar to Zerolite FFIP include Isopor FFIP `
and DeAcidite F~IP SRA.64. These resins were supplied by BD~l Chemicals Ltd.~ ~ ;
Poole, Dorset, U.K. -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, trichloroethylene and the like. Suita~le amines include secondary or tertiary amines :in which one of the substituent groups is a long chaln aliphatic group, for example, oE 12-, ~ .
16 carbon atoms and the other is a tertiaryallcyl group so that the molecule is lipophilic. In our hands Amberlite LA2 has proved a successful amine.
.. : .:
Normally tlle 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 the-n separated from the culture ~iltrate. The clavulanic acld may be back extractecl into an * Trademark ~ ;
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~ ~ ~ 1076~20 aqueous phase by back extraction with a salt solution, preferably a con-centrated 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 o~ isolation which may be used include eonventional methods such as adsorption onto carbon, precipitation, salting out and molecular filtration but these methods are not usually as successEul as the above deseribed methods which are preferred.
Further purification of the crude solids obtained by methods ~` lO described above may be obtained by a variety of methods but ion exchange column chromatography is particularly suitable especially when using Isopor, I . .
` DeAcidite FFIP SRA64 or DEAE cellulose. The DeAcidite column may be gradient -~
; ~eluted with aqueous solution of a salt such as sodium chloride (O - 0.5M).
The column of DEAE cellulose in O.OlM phosphate bufier at ~H7 may be eluted with a salt solution, normally a NaCl so]ution (O - 0.2M NaCl in O.OL~
i phosphate buffer pH7~. Act-ive fractions may be detected by their ~-lactamase inhibitory activity and their antibacterial activity against Klebsiella ' ';
aerogenes in an agar diffusion assay. The fractions containing the bulk of thls activity are then combined and concentra-ted to a small volume under -`;
vacuum. This crude preparation of the clavulanic acid salt is desalted by ~-percolating do~m a eolumn of Bio Gel P2 (Bio Gel (trade mark) P2 is an example or a highly lipophilie resin onto which organic materials may be adsorbed but whieh does not retain inorganic salts. Bio Gel P2 is a poly-aerylamide gel supplied by Bio Radj 32nd and Griffen ~venue, Rie11mond, Ca 94804, U.S.A.). The aetive desalted ma-terial is then coneentrated, mixed with ethanol and further ehromatographed on a eellulose column using butanol/
ethanol/water 4/l/5 v/v top phase, as solvent.
. .. `:: . :~ . .
~j,i Fractions containing material whieh inhibit Escherichia eoli `~
: i , ~. . . .
~,~ ~ laetamase are bulked, evaporated to dryness under vacuum, redissolved in water and freeæe dried to give a salt of clavulanic acid as a white solid.
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.". -` The methods ~e have found most useful in detecting cla w lanic acid - in culture ~iltrates are paper chromatography and a bioautographic detection system. Clavulanic acld may be assayed by maklng use of its ~ -lactamase inhibit ing activity. Thin layer chromatography may be used to detect cla w lanic 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 comprlses isolating an impure form of cla w lanic acid or salt thereof, forming an ester of claulanic in conventional manner, ;
`; 10 purifying the es~er and thereafter regenerating clavulanic acid or a salt ; thereof from the ester. - -~ , . , The impure clavulanic acid or its salts used in this process will normally contain at least 1% by weight of the antibiotic.
.
j ~ Suitable esters for use in this process include those which may ~ be cleaved by hydrogenolysis, enæymatic methods or by hydrolysis under very `~ mild conditions as for example the esters of formula (IX).
:;, ~ One suitable group of esters used in this process is that of the ~, . .
formula (X): o ~ ;2~0~ ~ off~ /
~ ~ CO - O ~ ,CH -A7 (X) - ~ wherein A7 is a hydrogen atom or an optiona ~ y substituted phenyl group and A8 ; is an optionally substituted phenyl group.
Most suitably A7 is a hydrogen atom or a phenyl, tolyl, chloro phenyl, methoxyphenyl or nltrophenyl group and A8 is a phenyl, tolyl, chloro-phenyl, methoxyphenyl or nitrophenyl group.
.
' ~ Preferably A7 is a hydrogen atom and A8 is a phenyl g~oup.
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- The esters of formul~ (X) may ~e eleaved by hydrogenolysis - to yield clavulanic aeid or a salt thereof.
The esters o formulae (IX) and (X) may be grouped under the formula \ / 2 oJ
.~ CO X
wherein X lS Rl as already defined or -CH-~7 as already defined~
-~ ~ Other groups of esters which may be used in the process nclude those of formula (V) and (VI) as hereinbefore described. Such esters may be converted to salts of clavulanic acid by mild alkaline hydrolysis, `
~ I ~r~
~ for example, at pH 7.5.
. ~, :
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 in-organic impurities.
~ `20 The clavulanie aeid or salt thereof may be conyerted into an `,.t~ ester by the esterification reaetions referred to hereinafter. The preferred ~ method of forming the required ester of clavulanic acid is by the reaction~of ::;, . . . .
,j` a salt of elavulanic aeid with an esterifying agent such as a reactive halide, ~ -~ sulphonate ester or the like as hereinafter deseribed. Sueh reaetions are ,'~ frequently earried out in an organic solve~t of high dieleetric eonstant , ~ sueh as dimethylformamide, dimethylformamide/aeetone, dimethylsulphoxide, `t , ~ :' .
N-methylaeetamide, hexamethylphosphoramide and the like.
'~ If desired the salt of elavulanie acid may be dissolved in the solvent in eonventional manner or it may be bound to a polymerie support. ; ` `
Sultable supports for use in this proeess inelude strong base anion exehange '~ ,;,. ' ', ~.. - . , .
- 20 - ` ` ~
: . , ~::
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~esins, especially those possessing a ~acroreticular nature which permits the use of non-aqueous solvent systems. ~e have found Amberlyst AZ6 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 contain-ing sodium iodlde or alternatively eluted columnwise with a solution of sodium iodide in dimethylformamide or ln a mixture of dimethylformamide and acetone.
Once formed, the lmpure 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 chromatographic 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 properties.
Active fractions are normally combined and the organic solvent e~aporated off under reduced pressure.
.
The ester resulting from this process is generally of acceptable purity, but the material may be rechromatographed 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 hydrogenatlon of a compound of the formula (X) as hereinbefore . , .
described. Such reactions normally take place ln 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 0-100C.
Particularly suitable reaction conditions for such hydrogenations will use a slightly superatmospheric pressure of hydrogen at an approximately ambient (12-20C) temperature. The reaction may be carried out in conventlonal solvents such as lower alkanols, for example, ethanol.
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~L07GlZ0 ; ;~ We have found that a particularly sui~able catalyst is palladium on charcoal.
If the hydrogenation is carried out in the presence of a base then a salt of clavulanlc 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 carbo-nate.
..
The clavulanic acid or salt thereof resulting from such reactions is generally of good purity.
Esters 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 dis-placeable 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 clavllanic 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 , 20 atoms, sulphonic acid esters such as the O.SO2CU3 or O.SO2C6H4CH3 groups, ' ~ active ester groups such as the O.CO.~ or O.CO.CF3 group and other conventional j~ groups displaceable by nucleophiles.
The preceding reaction is normally carried out in an organic - solvent of relatively high dielectric constant such as dimethylformamide, - acetone, dioxane, tetrahydrofuran or the like and at a non-extreme temperature ~ -such as -5 C to 100 C, more usually +5 C to 30 C, for example at ambient temperature.
The reaction of clavulanic acid with a diazocompound is a mild ~ method of making alkyl, aralkyl or similar esters. The dlazotization ; - 22 -76~2~
~ reaction may be performed under conventional reaction conditions, for example ; at a non-extreme temperature and in a convcntional solvent. Such reactions are normally carried out at between about ~5C and 100C, more usually from 1 5 C to 30C, for example at ambient temperature. Suitable solvents for this ~ ~ .".:
reaction include lower alkanols such as methanol and ethanol and solvents such as tetrahydrofuran, dioxane and the like. Ethanol has proved a parti-.. . , . .
~j cularly useful solvent for this reaction.
i, :
The reaction of clavulanic acid with an alcohol or thiol in ; the presence of a condensation promoting agent ~ill normally take place in an inert organic solvent of relatively high dielectric constant such as acetonitrile. This rcaction is usually carried out at an ambient or depressed temperature, for example at -10C to +22C, more usually 5C to ~18C, for example initially at 0C and thereafter gradually warming to about 15C. ;
The condensation promoting agent used is normally one which removes water ~`~ from the reaction mixture. Suitable agents include carbodiimides, carbodi-~ ., , -, .
imidazoles or equivalent reagents. Dicyclohexylcarbodiimide has proved to be a particularly suitable condensation promoting agent for use in this process. In order to minimise self condensation of the cla w lanic 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) .:' !
~ 201 -N ~ (XI) o-o-co-o,R4 " ' :
~herein R is an inert organic group~ and (e) reaction of a compound of the formula (XI) with an alcohol ROH (or less favourably with a thiol RSH).
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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 fonnula (XI) in an inert solvent. Suitable ~ 4~ yd~og~ r~
inert solvents include ether solvents such as diethylether, Le~Y~ r~fcron, dioxane and the like. In many cases the compound of the formula (XI) de-co~poses spontaneously even at a depressed temperature, for example, at -5C, to yield an ester of the formula : ': ' . ' ' \ f H2011 ~ - - M y ` ~ wherein R is an inert group witllin the definition of R. ~ -When the compound of the formula (XI) is to be reacted with an alcohol (or less favourably with a thiol) then this reaction is normally .: ,.;, : :
~ carried out in an inert solvent such as an ether solvent in the presence of... .... . .
;, an excess of the alcohol (or thiol) in order to prevent self-condensation of -~
the cla w lanic acid derivative. -Such methods of esterification are not in general as useful :: ~ ,.:: : ~.
as those involving reaction of a salt of clavulanic acid with R-~ as herein-` ~ 20 before described.
j~ The compound of the formula ~XI) may be prepared by the .~ ~:: ':: ' .
reaction of a salt of cla w lanic acid with Cl.CO.O.R4 or the chemical equi-valent thereof, Normally this reaction is carried out at a depressed ~ temperature, for example, at a temperature not greater than 5C, and in an - ~ inert solvent, for example diethylether, tetrahydrofuran, dioxane and the like. Most suitably the salt of clavulanic acid used in this reaction is a lipophilic salt so that it will dissolve in the solvent although if desired the less favourable sodium salt may be employed by suspending it in the reaction medium.
~: ' ;,~,':' :` , ' ' ':, ' '' ' '' ' ' , ,.. .. , . , , . . . . . ~
- ~ 1076~ZI) ~ DESCRIPTION 1 .~ ~ ,. .
: 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 -lactamase preparation in 0.05M phosphate buffer at pH 7 and 37 C. During-this time, enzyme inhibition or inactiyation occurs. Substrate ~benzylpenicillin) is then added and incubation continued for 30 minutes at 37C. The amount of , enzymic degradation of the substrate to penicilloic acid is determined by the hydroxylamine assay for penicillin. The amount of ~ -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 enzy;me - :
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 ~ug/ml) giving 50% inhibition of the enzyme under the above stated conditions (I50).
~ ~ -lactamase nzyme ..
: ~ The~ lactamase produced by Escherichia coli JT4 is used as an ~ enzyme. This culture is an ampicillin resistant strain and owes its resistance .: `
- ; 20 to the production of an R-factor controlled ~-lactamase. Other similar R-factor controlled ~-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/l, yeast extract (Oxoid) 20 g/1, NaC1 5 g/1 and CaC126H20 2.2 g/1. The final pH was adjusted to 7.4 with dilute NaOH. The flask is shaken at 25C for 20 hours on a rotary . ~.~ , .
shaker at 240 r.p.m.
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The bacterlal cells are collected by centri~ugation, washed with 0.05M phosphate buffer pH 7 (resuspended and centrifuged) and resuspended in water to give cell concentration 25 times that in t~e cultivation medium. This cell suspension was then disrupted in an ~SE ultrason~c disintegrator at 4C. - -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 a 1 mg/ml.
solution of benzylpenicillin in 30 minutes at 37C.
Assay Procedure , 10 Suitable dilutions of the inhibitor preparation and ~ lactamase solution are mixed and incubated at 37C for 15 minutes ~Test). A control with buffer in . :
place of inhibitor preparation is also incubated. Ben~ylpenicillin solution (substrate) is then added to test and control mixtures, incubation continued for a further 30 minutes at 37C. The residual ben~ylpenicillin 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 mlnutes at room temperature prior to the addition of 2 ml of ferric ammonium sulphate reagent. The absorption of the final solutions is measured in an E.E.L. Colori-meter or a Spectrophotomerer at 490 nm against the reagent blank. The compositionof the reactions, tests and blanks prior to the hydroxylamine assay are as follows:
~ . _ . .
, ComponentsTest Benzyl- Reagent (all dissolved in or diluted penicillin Control Blank diluted with 0.005M Blank ml. ml pH 7 phosphate buffer) ml __ ... _ Escherichia Coli ~ -lactamase solution 1.9 0.0 1.9 1.9 Inhibitor solution 0.1 0 0 0.0 0.0 BenYiylpenicillin 5mg/ml. 0.5 0.5 0.5 0.0 0.005M pH 7 phosphate buffer 0.0 2.0 0.1 0.6 , :
/
: ' :
': ~
:: 107~20 , - , ~i Calculation of ~esults . ~ . .
The percentage inhibition of the ~-lacta~ase is calculated as follo~s Absorption of benzylpenicillin blank minus absorption of control (uninhibited reaction) = x Absorption of test (inhibited reaction) minus absorption of control (uninhibited reaction) = y S~
% inhibition = - x 100 To obtain the I50 value, the inhibitor preparation is diluted until 50%
inhibition of the ~ lactamase inactivation of benzylpenicillin is obtained in the above procedure.
PAPER CHROMATOGRAPHIC DETECTION O~ CLAW LANIC ACID
- - -- , . . .
Culture filtrate and a reference solution of claw lanic acid (250~ug/ml partially purified preparatlon), are spotted (20Ju1/origin) onto ,3~~ Whatman No. l paper strips l cm. wide. The chromatograms are run by descending chromatography for 16 hours at 5C using n butanol/isopropanollwater, 7/7/6 v/v as solvent. The strips are dried at 40C and laid on agar plates containing 6~ug/ml benzylpenicillin and seeded with a ~ lactamase producing strain of Klebsiella aerogenes (synergism system). The plates are incubated overnight at 30G and clavulanic acid revealed as a zone of inhibited growth.
The Rf value of the zone was 0.46. The 6Jug/ml benzylpenicillln alone is below the concentration required to kill the Klebsiella aerogenes but in the ;~presence of a ~-lactamase lnhibitor, thls concentration becomes toxi.c, 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.
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1076~Z(~
. ' TIIIN L~Y~R Cl1ROMATOGRAPilIC D~TECTION OF CLAVUL~NIC ACID SOOIUM SALT .
Solutions of clavulanic acid sodium saLt preparations are spotted -;
(5Jul of lmg/ml) onto glass plates coated with a 0.25 mm layer of silica gel (F254) as supplied by E. Mcrck, Darmstadt, Germany. The chromatograms are run at 22C using the top phase of the mixture n-butanol/ethanol/water 4/1/5 v/v. The chromatogram plates are dried at 40C and clavulanic acid sodium salt located by bioautography on agar plates containing 6)ug/ml.
. .
benzylpenicillin and seeded with Klebsiella aerogenes (synergism system -.. . I .
~ ~ l0 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 ~ , ~ ~ : 15-30 minutes for wetting and diffusion, the TLC plate is lifted off with -;,, the aid of the filter cloth and~the agar plate incubated overnight at 30 C
.. ~ . .
., to reveal the zones of inhibited gro~th. The Rf value of claw lanic acid sodium salt in the above solvent is app~oximately 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, 54 ml.
. ~ .
of n-butanol and 9 ml of concentrated HCl. On heating the sprayed TLC plate at 120C for 1-2 minutes, clavulanic acid sodlum salt appears as a pink spot.
The triphenyltetrazolium chloride reagent consists of a mixture of 1 volume of a 4% solution of this compound in methanol with l volume of methanolic ~,-,. . . , :
sodlum hydroxide. After spraying, the TLC plates are heated at 80 C.
Clavulanic acid sodium salt appears as a red spot on a white background.
~, EX~MPLE 1 ::
;j .
CULTIVATION OF STREPTOMYCES CLAVULIGERUS
Streptomyces clavuligerus was cultivated at 26C on agar slopes containing ., ,: .
1% Yeatex (yeast extract), 1% glucose and 2% Oxold agar No. 3, pH 6.8. A
sterile loop wa6 used to transfer mycelium and spores from the slop into :' ~ -;ii 30 100 ml of a liquid medium in a 500 ml Ehrlenmeyer flask.
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The liquid medium had the following composition:-Oxoid Malt Extract 10 g/1 . . * . .
: Oxoid Bacteriological Peptone 10 g/l Glycerol 20 g/l Tap water 1 liter The medium was ad~usted to pli 7.0 with sodium hydroxide ~: solution and 100 ml. volumes dispensed into flasks which were closed with ' foam plugs prior to autoclaving at 15 lb/sq.in. for 20 minutes. An inoculated ,! seed flask was shaken for 3 days at 26C on a rotary shaker with 2 inch throw , 10 and a speed of 240 r.R.m. Production stage flasks containing the liquid I medium described above were inoculated with 5~O vegetative inoculum and grown under the same conditions as the seed flask. Samples of culture filtrate , ~ ~ were assayed for inhibitor action against the ~-lactamase of Escherichia coli JT4. Optimum activity was obtained after 3 days, The results are shown in . , .
~ Table 1. A zone of clavulanic acid at ~f 0.46 was seen when the culture .~ : :
filtrate was examined by the paper chromatographic method previously described.
æ
The increase in size of the zone paralleled the increase in the ~ lactamase - inhibitor assay, ; ~
Streptomyces clavuligerus was also cultivated in 2 litre shaken flasks containing 400 mls, of medium ~Production stage) using the same medium and cultural conditions as described earlier in this Example, In these larger vessels, growth of the organism was slower and optimum ~ lactamase inhibitory activity was achieved 7-9 days afterlnoculation with the vegetative seed.
The results are also shown in Table l, ' . , . . ' ~ ', . ~' ' ' ' .
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1076~LZ0 Lactamase Inhibiting Activity of Streptomyces cla w ligerus Gr~m in 500 ml. and 2000 ml. Flasks _ , . __. ._. _ _ . . ---- ---- : , . . .
% Inhibition of Escherichia coli ~' ~-lactamase at a final dilution of Fermentation 1/2500 of culture filtrate Time (Days) 500 ml. Shaken Flask 2000 ml. Shaken Flask ;i~ ..... 'v,,.
This application is directed to the preparation of esters of clavulanic acid and is a divisional o~ S.N. 224,970 ~iled 18 April 1975. The -parent application is directed to clavulanic acid and its preparation by cultivation of Streptomyces claYuligerus and conversion to salts as required.
Other divisional applications S.N. 314,881 and S.N. 314,880 filed 30 October , 1978 are directed respectively to the preparation of clavulanic acid and its `~ salts by deesterification and synergistic mixtures of clavulanic acid or its pharmaceutically acceptable salts or esters with a ~-lactam antibiotic.
~ This invention relates to a new agent having antibacterial : !
- i 10 ~ activity. More particuIarly it relates to a new antibacterial agent obtainable from Streptomyces clavuligerus,-designated as clavulanic acid, and salts and ,. . .
~i esters of the agent.
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' B GROUND TO THE INVENTION
., ~ a. Streptomyces clavuligerus has been described in detail by : 1 ; Higgens et al, Int. J. Systematic Bacteriology, 21, 326 (1971).
This streptomycete was of interest because it produced certain lactam antibiotics such as penicillin N, 7-~5-amino-5-carboxy-valeramido)-3-carbamoyloxymethyl-3-cephem-4-carboxylic acid and 7-(5-amino-5-carboxyvaleramido)-3-carbamoyloxy-methyl~
7-methoxy-3-cephem-4-carboxylic acid. The streptomycete has ~ -been deposited in the Agricultural Research Service Collection as NRRL 3585 and in the ~merican Type Culture Collection as i ~:
ATCC 27064. Streptomyces clavuligerus has also been reerred : 1 ~ to in United States Patent No. 3,770,590 and also by Nagarajan et al J.Amer.Chem.Soc., 93 2308 (1971), Bxannon et al, ntimicrob.
Agents Chemother., 1, 237 (1972) ~ntibicrob ~gents Chemother, . , .
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,; , , ~- 1, 247 (1972) Higgens et al, 3. ~ntibiotics, 27, 298 (1974).
~ b ~ -lactamases are enzymes which open the ~ ~lactam ring of :~ penicillins and cephalosporins to give products which are I devoid of antibacterial activity. These enzymes are produced by ~any ' ~' . , ' . ' ; ~!
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bacteria, notably species or strains of scherichia, Klebsiella, Proteus, Pseudomonas, Enterobacter and Staphylococcus and are in many . . -instances the explanation for the resistance of certain strains of such organisms to some penicillins and cephalosporins. The importance of ~-lactamase production may be understood when it is realised that a high proportion of clinically isolated organisms produce ~-lactamses ~ [see, for example, M. Wilson and I.~. ~reeman, Bacteriological Proceed-`. ings, 80 (1969) where in a paper entitled 'Penicillin Inactivation by ~,; Gram-negative Bacilli~ they showed that 84% of the gram-negative IO
organisms isolated in an American hospital produced ~-lactamase] . In many cases, some penicillins or cephalosporins are ineffective in ,~ treating diseases ascribed to non ~-lactamase-producing organisms because of the co~mon occurance of co-infection by a ~ lactamase ~ ~ producer ~see, for example, R. May et al; Brit. J.Dis.Chest., 66, 185 `~!
~l ~ (1972)~ . Combination o~ a ~ lactamase inhibiting substance with a ~:
penicillin or cephalosporin might be expected to protect the latter ~ I .
from degradation by bacterial ~-lactamase and thereby enhance their antibacterial activity against many infective organisms. This process of enhancement of the antibacterial activity is called synergism when ' 1 ~ . .
the antibacterial activity of the combination is well in excess of the simpIe addition of the activities of the two separate substances. The ~. :
~-lacta~ase inhibiting component of the mixture is referred to as a synergist and such substances are valuable for increasing the anti-bacterial activity of penicillins and cephalosporins against resistant organisms. It is one of the objects of this invention to provide such synergists.
c. Examples of the use of certain ~ lactamase resistant semi-synthetic ;` penicillins and cephalosporins as ~-lactamase inhibitors and synergists for penicillins and cephalosporins have already been .
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~, 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 à dramatic effect on the spectrum of the other antibiotic present in the ~ ;
."'! mixture.
,, `` d. Certain actinomycete cultures have been described as producing lactamase inhibiting substances which act synergistically with penicillins or cephalosporins, for example, those cultures -~ 10 ~ disclosed in British Patent No. 1,363,075 and those described ,. . . .
by ~lata et al, J. Antibiotics, 25, 473 (19723 and Umezawa et al, J. Antibiotics, 26, 51 (1973). None of these ~-lactamase inhibitors of actinomycetal origin have yet been found to be of use in the clinic. ParticuIarly noteworthy features wh1ch distinguish clavulanic acid from other ~-lactamase inhibitors of actinomycetal 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 ~-lactamase inhibiting . .
activity, its low molecular weight and its high Rf values on `20 paper chromatography using a variety of solvcnt systems.
DESCRIPTION OF THE INVENTION
` We have discovered that the aerobic cultivation of Streptomyces clavuligerus in conventional nutrLent media at about 25-30C under rougllly , ~ neutral conditions produces a ~-lactamase inhibitory-substance which also possesses antibactcrial activity. We have designated this new material clavulanic acid'.
~`~` Clavulanic acid has the followi~g properties: `
~. . , ~ (a) It is a carboxylic acid.
; (b) It forms a sodlum salt whlch has a characteristic infra-red spectrum substantially as shown in Figure 1.
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(c) It is able, to inhibit the growth of stra ms of Staphylococcus aureus.
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,~ ' (d) It is able to synergyse the antibacterial effect of , , , ~ ampicillin against ~-lactamase producing strains of ,' ,~I ` Escherichia coli, Klebsiella aerogenes and Staphylococcus ~'' :' q '.
. I aureus ''`, (e) It is able to synergyse the antibacterial effect of ' cephaloridine against the ~-lactamase producing strains ` : -: of Proteus mirabilis and Staphylococcus aureus. ' ~ ~
10 ~ (f) It forms a methyl ester which has a molecular weight .: -(by mass spectroscopy) of 213.0635 which corresponds -':. ~ ,, to the formula C9UIlNO5. ~ ''~ ~ ~~` ' "''' Thus clavolanic acid may be regarded as a monobasic carboxylic acid of~the formula C8HgN05 which in the form of its sodium salt,has a .
characterlstic infra-red absorption spectrum substantially as shown in Fig. 1. '~
The compound produced by Streptomycee clavuligerus which has ' ~ the~above praperties has the formula (II)~
"`~ / 0 ~ ~ C~120U
20~ ~ - N ~ ~ (II) , , Thus clavulanic acid may be named 3- ~-hydroxyethylidene)-7- ,' ', oxo-4-oxa-1-azabicyclo r3,2,0] heptane-2-carboxyl-lc acld. ~ '' '-The stereochemistry at C5 and C2 of the clayulanic acid is ~' ' the same as that found in naturally occurring penicillins and cephalosporins ' so that clavulanic acid may be represented by the structural fonmula ~
r~~ C~I20~ ~ .
~i~ C0 ~ 30 .,.~ , :
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.' ' f--~ ~~r ' ' ~` ~076~Z0 ~ Thus a fuller chemical name for clavulanic acid is Z-(2R,5R)-;^ .- - :.
3- ~-hydroxyethylidene)-7-oxo-4-oxa-1-azabicyclo!3,2,0J heptane-2-carboxylic .
acid.
The 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~g/ml. of ampicillin, amoxycillin, carbenicillin or benzyl penicillin or by the presence of lO~gtml. of claw lanic acid, are inhibited by the presence of less than 12.5/lg/ml. o the previously mentioned penicillins when 5~g/ml. of clavulanic -; -acid is also present. Similar results have been obser~ed for combinations containing various esters of clavulanic acid. ~or example, strains of ;
Klebsiella aerogenes A, the growth of which is not inhibited by 125~lg/ml. of ampicillin, or by lO~lg/ml of clavulanic acid methyl ester are inhibited by less than l2.5~lg/ml. of ampicillin in the presence of 5~g/ml. oE the clavulanic acid methyl ester. It has also been found that strains of Staphylococcus aureus Russell, the growth of wh1ch lS not inhibited by the presence of 100/.~gtml. of ampicillin or by 5~Lglml of clavulanic acid, are inhibited by the presence of less than 10).Igtml. of ampicillin in the presence of l)lgtml. of clavulanic acid. In tests on female mice, it has been found that blood and tissue levels of clavulanic acid considerably in excess o 5)lg/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 administratlon of 100 mgtkg of the sodium salt of clavulanic acid.
,, Accordingly, the present invention provides clavtl1anic acid as hereinbefore described and its salts and esters.
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Most suitably the salts of clavulanic acid will be pharma-ceutically acceptable salts such as the sod:Lum, potassium, ca].cium, magnesi-tm, aluminium, ammonium and substituted ammonium salts such as the trimethyl-ammonium, benzathine, procaine and like salts conventionally formed with ~;` ' . ;~,'"
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penicillins or~cephalosporins. Non-pharmaceutically 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 form the useful benzyl ester of clavulanic acid.
: I :
Salts of claw lanic acid tend to be more stable than the ~' parent acid per se and thus form a favoured aspect of this invention.
Particularly suitable salts of clavulanic acid include the sodium and , potassium salts which have the formulae (III) and (IV) respectively:
,C~20~1 rl~C~20u ~
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02~a C02K -. ~ ~
Crystalline forms of such salts may contain water of hydration. -Suitable esters of clavulanic acid include those notionally derived from alcohols such as methanol, ethanol, propanol, butanol, 2,2,2~
trichloroethanol, 2,2,2-trifluoroethanol, benzyl alcohol, p-nitrobenzyl ~`
20 alcohol, phenol, acetoxymethanol, pivaloyloxymethanol, 2 dimethylaminoethanoland 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 or thiol of the formula ROH or RSH where R is an organic residue. Suitable groups R include alkyl, alkenyl, alkynyl, aryl, ; arylalkyl or other similar groups any o which may be substituted if desired.
In order not to increase the molecular weight to an unreasonable extent, groups R do not normally include more than 16 carhon atoms, more suitably, . ~' ' ' '' '" ' '`' , ' "~ ~ .
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not more t1ul11 l c~rbo11 atoms and P1ost suitably, not more than 8 carbon atoms. '!.', : , ' . " '.' ~
The esters of tlle clavulanic acid may thus be represented . ' . ,~ , . .
by the formula ., . ,, :.. .
fl'2"
:, o \ : ' C~W---R
wherein W is O or S and R is as defined.
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Prefcrably, the group R is notionally derived from an alcohol ROH or (less favorably) a thiol RSH which is pharmaceutically acceptable.
- ,I Suitable substituents Which may be included in the group R
.... ~ - , , include halogen atoms and lower alkoxyl, hydroxyl, lower acyloxyl, lower alkylamino, lower dialkylamino and like groups. The term 'lower' means that . , !
the group contains up to 6 carbon atoms, and preferably up to 4 carbon atoms.
Thus, for example, R may be a methyl, ethyl, n-propyl, iso-propyl, straight or branched butyl, pentyl, heptyl, octyl, nonyl~ decyl, undecyl, dodecyl, ~ .
vinyl, allyl, butenyl, cyclop~ropyl, cyclobutyl, cyclopentyl, cyclohexyl, .: :1 . ... .
cycloheptyl, cyclohexenyl, cyclohexadienyl, methylcyclopentyl, methylcyclo- .20 ~ hexyl, benzyl, benzhydryl, phenylethyl, napthylwethyl, phenyl, napthyl, propynyl, tolyl, 2-chloroethyl, 2,2,2-trichloroethyl, 2,2,2-trifluoroethyl, ~;
!~ ~ acetylmethyl, bénzoylmethyl, 2-methoxyethyl, 2-dimethylaminoethyl, 2-diethyl-aminoethyl, 2-piperidinoethyl, 2-morpholinoethyl~ 3-dimethylaminopropyl, ~ ; p-chlorobenzyl, p-methoxybenzyl, p-nitrobenzyl, p-bromobenzyl, m-chlorobenzyl, ;
`~ 6-mcthoxynapht11yl~2-met11yl, p~chlorophenyl, p-methoxyphenyl or any like ; group as well as those groups Which are known from the penicillin or cephalo- ;
sporin arts to produce esters known to be readily hydrolysed in Vivo to the . parent antibiotic.
.~ - Readily hydrolysable esters include, but are not l.imited to, those of the formulae (V) and (VI):
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~'~ wherein Al is a hydrogen atom, alkyl, aryl or aralkyl group; A2 is a hydxogen . i~ atom or methyl group; A3 is an alkyl, aryl or aralkyl group; X is oxygen or sulphur; Y is oxygen or sulphur and Z is a divalent organic group. Esters :1 ~ of the formulae (V) and (VI) whicll fairly readily release the clavulanic acid ~ ~ j ~ into the blood stream after administration include those wherein Al is a :: .
~ hydrogen atom, A2 is a hydrogen atom or a methyl group and A3 is a methyl, ' l . .. .
ethyl, propyl:, butyl, benzyl, or phenyl group and those wherein X is oxygen, Y is oxygen and Z is -CH2CH2-, -Cll:CH-, ~ ~ or ~
.,~ When used in conjunction with the preceding foxmula the term : 'alkyl~ includes alkyl of up to six carbon atoms; the term 'aryl~ includes : phenyl, naphthyl or phenyl subs.tituted by an inert substituent such as a ~ .
;: fluorine or chlorine atom or a methyl or methoxyl group or the like; when , used herein the term ~aralkyl~ means an alkyl group substituted by an aryl .~ group.
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, Particularly suitable esters of the formulae (V) and (~
include those of the formulae (VII) and (VIII): ~ -: D
A
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; 10 A4 is a hydrogen atom or a methyl group, A5 is a methyl, t-butyl or phenyl group and A6 is a hydrogen atom or a methoxyl group.
Many esters of clavulanic acid differ from analagous 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 p~l7. Es~ers which undergo some hydrolysis under miId conditions are included within the ormuIa (IX): ~ -L~ C~12~
~' wherein Rl is a hydrocarbon group of 1-9 carbon atoms optionally substituted ' ' ~ by halogen, lower alkoxy, hydroxyl or optionally salted basic groups of the '~ ~ formula NR R3 wherein R is a hydrogen atom or a lower alkyl group, R is a ~ hydrogen atom or a lower alkyl group or is attached to R2 so that NR2R3 is a : :: .
, 5~ or 6- membered ring. ' `
When used wlth referencc to formula (IX) the term 'lower~ ~-, . . :~ .
- means that the group contains 1-4 carbon atoms.
Suitably groups Rl include alkyl and aralkyl groups optionally substituted by halogen, methoxyl, hydroxyl or salted NR2R3 groups wherein R2 : .
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is a methyl or ethyl group and R3 i5 a methyl or ethyl group or is joined to R so that NR2R3 is a pyrrolidine, piperidine or morpholine group.
Most suitably alkyl groups Rl are straight chain groups of up to 6 carbon atoms optionally substituted by one methoxyl, hydroxyl, salted NR2R3 group or one chlorine, bromine or iodine atom or by a CCl3 or CF3 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 belived that !
clavulanic acid and its salts tend to be somewhat more useful synergistic agents than the esters per se. Many of the esters of the formulae (~ (IX) are useful for this purpose.
A further group of particularly suitable`èsters of this mvention are those useful intermedlates which are;readily converted to i ~ clavulanic acid or a salt thereof by chemical or biochemical techniques which . ~
are known from the penicillin or cephalosporin arts to be sufficiently mild not to degrade reactive acid-labile ~-lactam rings.
Most suitably, the ester is one removable by hydrogenolysis.
Conventional esters for such a process include benzyl, substituted benzyl, benzhydryl, substituted benzhydryl, trityl and the like. The benzyl ester has proved particularly useful for this purpose.
~,; By and large, the nature of any substituent in the ester moiety is ~mimportant as long as it does not interfere with the hydrogenolysis reaction.
- . , Since clavulanic acid and its salts are useful intermediates ~t~ 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 : " ' , .
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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 or oral, topical or parenteral use and may be used for the treatment of infection in mam~als 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 compo-~ ~ sltions 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 welI understood by those skilled in the art of formulating an~ibiotics.
.: : .:,. , 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 inEusion. ~;
Ti~us, one preferred composition aspect of this invention comprises cLavulanic acid or a salt thereof m -sterile form.
Unit dose compositions comprising clavulanic acid or a salt '-or ester thereof adapted for oral administration form a further preferred c~mposition aspect of this invention.
Under certain conditions, the effectiveness of oral compo-sitions of clavulanic acid and its salts and esters can be improved :Lf such compositions contain a bufferLng agent or an enterlc coating a8ent sucll that the compounds of the invention do not have prolonged contact with highly acidic gastric juice. Such buffered or enterically coated composit;lons may ''"i ~ ' 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 j 30 other therapeutic agents such as a ~-Lactam antibiotic, Su;ltable ~-].actam ., , -, , , -~ 1 1 --, . . .
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antibiotics for inclusion ln sueh syne~g~stic co~poSitions include not only those known to be highly susceptible to ~ lactamases but also those which have a good degree of intrinsic resistance to ~-lactamases. Thus, suitable ~ -lactam antibiotics for inclusion in the compositions of this invention include benzyl-penicillin, phenoxymethylpenicillin, carbenicillin, methicillin, propicillin, ampicillin, amoxycillin, epicillin, ticarcillin, cyclacillin, 6-aminopenicillanic aeid, 7-aminocephalosporanic aeid, 7-aminodesacetoxycephalosporanic aeid, cephaloridine, cephalothin, cefazolin, cephalexin, eefoxitin, cephacetrile, ~ cephamandole, cephapirin, cephradine, cephaloglycine, and other well known ; ;~ 10 penicillins and cephalosporins or pro~drugs there~ore such as hetaeillin, : . , metampicillin~ the acetoxymethyl, pivaloyloxymethyl or phthalidyl esters of benzylpenicillin, ampieillin, amoxyeillin or eephaloglycine or the phenyl, tolyl or indanyl ~-esters of earbenieillin or ticarcillin or the like.
~ Naturally if the penicillin or cephalosporin present in the i ~ 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 -laetam antibiotic, the ratio of clavulanic acid or its salt or ester present to e-laetam antibiotie present m y be from, for example, 20:1 to 1:12, more usually lO:l to 1:10 and advantageously may be from 3:1 to 1:3.
The total quantity of antibacterial agents present 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 infeetions ôf 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 an-lmals such as mastitis in cattle.
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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 3000 mg of the composition of the invention will be administered per ~-day However, for the trea~ment of severe systemic infections or infections ~` of particularly intransigent organisms, higher doses may be used in accor-dance with clinical practice.
,.: .
;~ The exact form of the compositions of this invention will depend to some extent on the micro-organism which is being treated. For treatment of most infections the compositions of this invention are normally adapted to produce a peak blood level of at least O.l,ug/ml, more suitably at lcast 0.25 ~lg/l~, and preferably at least 1 ~Ig/ml. of synergist, for - ~ I
example, 2.5 - 5 yg/ml. of synergist. -~
.- : .
The penicillin or cephalosporin in synergistic compositions of thls invention will normally be present by up to or at approximately the ~ amount conventionally used when that penicillin or cephalosporin is the sole ; ~ therapeutic agent used in the treatment of infection.
. :
Particularly 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 materlals present in such compositions may be hy(lrated if required. Tlle weights of the antibiotlcs 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 Streptom~ces clavuli~ md recovering clavulanic acid or a salt thereof from the culture medlum and ~ ' ;. ~ ' ' .. .
~ .
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.
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 process of this invention.
~ /; ~ r When used herein, the term 'cultivation' means the dcliv~retc 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 or suspended. The culti-- ` 10 vation may take place on an aerobic surface or by submerged culture. The nutritive medium may be composed of complex nutrients or may be chemically ; 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 clavuli~erus may contain, in the range 0.1 - 10% a complex organic nitrogen source such as yeast extract- corn steep liquor, vegetable protèin, seed protein, hydrolysates of such proteins, milk protein hydrolysates, ; fish and meat extracts and hydrolysates such as peptones. ~lternatively chemically defined sources of nitrogen may be used such as urea, amides, ~ single or mixtures of common amino acids such as valine, asparagine, glutamic scid, proline and phenylalanine. Carbohydrate (0.1 - 5%) may be included in the nutrient media but glucose in certain media is undesirable having a ;~ depresslng efect on the yield of the desired clavulanic ac:Ld. Starch or . ~. , .
: - starch hydrolysates such as dextrtn, sucrose, lactose or other sugars or `;
' glycerol or glycerol esters may also be used. The source of carbon may also i : ....
be deriyed from vegetable oils or animal fats. Carboxylic acids and their salts can be included as a source of carbon for growth and prod~ction of : ~, ' :, lactamase inhibitors. A particularly suitable low cost medium is one containing soya bean flour (Arkasoy ) plus dried malt distillers solubles ,: ,::
(Scotasol ) plus dextrin *Trademark ' '" ' ., '' '., ,.',' ~, ::
The addition o~ antifoam agents such as Pluronic L81 may be necessary to control foaming of certain media in fermenters.
Mineral salts such as NaCl, KCl, MgC12, ZnC12, FeC~3, Na2S04, FeS04, MgS04 and Na~ or K+ salts of phosphoric acid may be added to the media described - above particularly if chemically defined; CaC03 may be added as a source of Ca ions or for its buffering action. Salts of trace elements such as nickel, cobalt .!.~
or manganese may àlso be included. Vitamins may be added if desired.
When used 1lerein the term ~mutant' includes any mutant strain which arises spontaneously or through the effect of an external agent whether .
~ 10 that agent is applied deliberately or otherwise. Suitable methods of producing ;~, .i , mutant strains include those outlined by H.I. Adler in Techniques for the Development of Micro-Organisms in 'Radiation and Radioisotopes for Industria - Micro-Organisms~, Proceedings of a Symposium, Vienna, 1973, page 241, International Atomic Energy Authority and these include:
i. Ionising radiation (such as X- and ~-rays), uv light, uv light plus a photosensiti~ing agent (such as 8-methoxy-psoralen), 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 40C, usually 20-35C and preferably, 25-30C and at ' ' a pH of between 5 and 8.5, preferably between 6 and 7.5.
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The Streptomyces clavuli~er~ls may be cultivated in the above . media in glass conical flasks aerated by shaking on a rotary shaker or in baffled stainless steel fermenters stirred with vaned disc impellers and aerated 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 is obtained in 2-10 days at 20-35C. In a stirred stainless steel fermenter using the Arkasoy/Scotasol/Dextrin medium referred to above the preferred temperature is 26C and peak yields clavulanic ; 10 are obtained within 5 days.
Clavulanic acid may be extracted from culture flltrate by a variety of methods. Solvent extraction from cold culture filtrate adjusted to acid pH values and methods ~ased 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 normall~ 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 thorougly mixing with a water immiscible organic solvent such as n--,! :
butylacetate,~methylisobutylketone, n-butanol or ethylacetate. The acid used to lower the pH of the medium ls normally a mineral acid such as hydrochloric, sulphuric, nitric, phosphoric or the like acid. n-Butanol is a part;icularly sultable solvent for use in the extraction of the acidified culture filtrate.
After separation of the phases by centrifugation, the ~'-lactamage inhibiting metabolite is back extracted from the solvent phase into aqueous sodium bi-carbonate or potassium hydrogen phosphate bufEer, CaC03 suspension or ~ater while maintaining the p~l at approximate neutrality, for example, at pH 7Ø
. .
This aqueous extract after separation of phases may be concentrated under : - , 30reduced pressure and freeze dried to give a crude preparation of a salt of ':; .~. : . ' '' :
- 16 - ~
. .
~ 1076~2(1 -~ clavulanic acid. This preparation is stal~le ~hen stored as a dry solid at -20 C.
~: In the anion exchange resin process, the c:Larified culture filtrate at an approximately neutral or slightly acid p~1, for example pH 6-7, -; is percolated down a column of weak or strong base anion e2cchange resin such as Amberlite IR4B or Zerolit ~FIF respectively until the resin is saturated -~
. , ~ and the ~-lactamase inhibiting material emerges from the bottom. The column . ,. , - :
is then washed with water and eluted ~ith aqueous sodium chloride. The ~-lactamase inhibiting fractions are collected, bulked, desalted cmd freeze - ~ ~
dried to yield a crude solid salt o~ clavulanic acid. -~ -. . : -, A2nberlite IR 4B is an example of a weakly basic anion exchange resin with polyamine active groups and cross linked polystyrene-divinyl-benzene matrix. Zerolite FFIP is a strongly baslc anion exchange ~ ~ -resin with quaternary ammonium active groups and a cross linked polyvinyl--divinylbenzene matrix. Resins similar to Zerolite FFIP include Isopor FFIP `
and DeAcidite F~IP SRA.64. These resins were supplied by BD~l Chemicals Ltd.~ ~ ;
Poole, Dorset, U.K. -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, trichloroethylene and the like. Suita~le amines include secondary or tertiary amines :in which one of the substituent groups is a long chaln aliphatic group, for example, oE 12-, ~ .
16 carbon atoms and the other is a tertiaryallcyl group so that the molecule is lipophilic. In our hands Amberlite LA2 has proved a successful amine.
.. : .:
Normally tlle 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 the-n separated from the culture ~iltrate. The clavulanic acld may be back extractecl into an * Trademark ~ ;
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~ ~ ~ 1076~20 aqueous phase by back extraction with a salt solution, preferably a con-centrated 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 o~ isolation which may be used include eonventional methods such as adsorption onto carbon, precipitation, salting out and molecular filtration but these methods are not usually as successEul as the above deseribed methods which are preferred.
Further purification of the crude solids obtained by methods ~` lO described above may be obtained by a variety of methods but ion exchange column chromatography is particularly suitable especially when using Isopor, I . .
` DeAcidite FFIP SRA64 or DEAE cellulose. The DeAcidite column may be gradient -~
; ~eluted with aqueous solution of a salt such as sodium chloride (O - 0.5M).
The column of DEAE cellulose in O.OlM phosphate bufier at ~H7 may be eluted with a salt solution, normally a NaCl so]ution (O - 0.2M NaCl in O.OL~
i phosphate buffer pH7~. Act-ive fractions may be detected by their ~-lactamase inhibitory activity and their antibacterial activity against Klebsiella ' ';
aerogenes in an agar diffusion assay. The fractions containing the bulk of thls activity are then combined and concentra-ted to a small volume under -`;
vacuum. This crude preparation of the clavulanic acid salt is desalted by ~-percolating do~m a eolumn of Bio Gel P2 (Bio Gel (trade mark) P2 is an example or a highly lipophilie resin onto which organic materials may be adsorbed but whieh does not retain inorganic salts. Bio Gel P2 is a poly-aerylamide gel supplied by Bio Radj 32nd and Griffen ~venue, Rie11mond, Ca 94804, U.S.A.). The aetive desalted ma-terial is then coneentrated, mixed with ethanol and further ehromatographed on a eellulose column using butanol/
ethanol/water 4/l/5 v/v top phase, as solvent.
. .. `:: . :~ . .
~j,i Fractions containing material whieh inhibit Escherichia eoli `~
: i , ~. . . .
~,~ ~ laetamase are bulked, evaporated to dryness under vacuum, redissolved in water and freeæe dried to give a salt of clavulanic acid as a white solid.
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.". -` The methods ~e have found most useful in detecting cla w lanic acid - in culture ~iltrates are paper chromatography and a bioautographic detection system. Clavulanic acld may be assayed by maklng use of its ~ -lactamase inhibit ing activity. Thin layer chromatography may be used to detect cla w lanic 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 comprlses isolating an impure form of cla w lanic acid or salt thereof, forming an ester of claulanic in conventional manner, ;
`; 10 purifying the es~er and thereafter regenerating clavulanic acid or a salt ; thereof from the ester. - -~ , . , The impure clavulanic acid or its salts used in this process will normally contain at least 1% by weight of the antibiotic.
.
j ~ Suitable esters for use in this process include those which may ~ be cleaved by hydrogenolysis, enæymatic methods or by hydrolysis under very `~ mild conditions as for example the esters of formula (IX).
:;, ~ One suitable group of esters used in this process is that of the ~, . .
formula (X): o ~ ;2~0~ ~ off~ /
~ ~ CO - O ~ ,CH -A7 (X) - ~ wherein A7 is a hydrogen atom or an optiona ~ y substituted phenyl group and A8 ; is an optionally substituted phenyl group.
Most suitably A7 is a hydrogen atom or a phenyl, tolyl, chloro phenyl, methoxyphenyl or nltrophenyl group and A8 is a phenyl, tolyl, chloro-phenyl, methoxyphenyl or nitrophenyl group.
.
' ~ Preferably A7 is a hydrogen atom and A8 is a phenyl g~oup.
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- The esters of formul~ (X) may ~e eleaved by hydrogenolysis - to yield clavulanic aeid or a salt thereof.
The esters o formulae (IX) and (X) may be grouped under the formula \ / 2 oJ
.~ CO X
wherein X lS Rl as already defined or -CH-~7 as already defined~
-~ ~ Other groups of esters which may be used in the process nclude those of formula (V) and (VI) as hereinbefore described. Such esters may be converted to salts of clavulanic acid by mild alkaline hydrolysis, `
~ I ~r~
~ for example, at pH 7.5.
. ~, :
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 in-organic impurities.
~ `20 The clavulanie aeid or salt thereof may be conyerted into an `,.t~ ester by the esterification reaetions referred to hereinafter. The preferred ~ method of forming the required ester of clavulanic acid is by the reaction~of ::;, . . . .
,j` a salt of elavulanic aeid with an esterifying agent such as a reactive halide, ~ -~ sulphonate ester or the like as hereinafter deseribed. Sueh reaetions are ,'~ frequently earried out in an organic solve~t of high dieleetric eonstant , ~ sueh as dimethylformamide, dimethylformamide/aeetone, dimethylsulphoxide, `t , ~ :' .
N-methylaeetamide, hexamethylphosphoramide and the like.
'~ If desired the salt of elavulanie acid may be dissolved in the solvent in eonventional manner or it may be bound to a polymerie support. ; ` `
Sultable supports for use in this proeess inelude strong base anion exehange '~ ,;,. ' ', ~.. - . , .
- 20 - ` ` ~
: . , ~::
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~esins, especially those possessing a ~acroreticular nature which permits the use of non-aqueous solvent systems. ~e have found Amberlyst AZ6 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 contain-ing sodium iodlde or alternatively eluted columnwise with a solution of sodium iodide in dimethylformamide or ln a mixture of dimethylformamide and acetone.
Once formed, the lmpure 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 chromatographic 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 properties.
Active fractions are normally combined and the organic solvent e~aporated off under reduced pressure.
.
The ester resulting from this process is generally of acceptable purity, but the material may be rechromatographed 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 hydrogenatlon of a compound of the formula (X) as hereinbefore . , .
described. Such reactions normally take place ln 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 0-100C.
Particularly suitable reaction conditions for such hydrogenations will use a slightly superatmospheric pressure of hydrogen at an approximately ambient (12-20C) temperature. The reaction may be carried out in conventlonal solvents such as lower alkanols, for example, ethanol.
*Trademark - 21 :'.`; :' '.
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~L07GlZ0 ; ;~ We have found that a particularly sui~able catalyst is palladium on charcoal.
If the hydrogenation is carried out in the presence of a base then a salt of clavulanlc 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 carbo-nate.
..
The clavulanic acid or salt thereof resulting from such reactions is generally of good purity.
Esters 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 dis-placeable 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 clavllanic 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 , 20 atoms, sulphonic acid esters such as the O.SO2CU3 or O.SO2C6H4CH3 groups, ' ~ active ester groups such as the O.CO.~ or O.CO.CF3 group and other conventional j~ groups displaceable by nucleophiles.
The preceding reaction is normally carried out in an organic - solvent of relatively high dielectric constant such as dimethylformamide, - acetone, dioxane, tetrahydrofuran or the like and at a non-extreme temperature ~ -such as -5 C to 100 C, more usually +5 C to 30 C, for example at ambient temperature.
The reaction of clavulanic acid with a diazocompound is a mild ~ method of making alkyl, aralkyl or similar esters. The dlazotization ; - 22 -76~2~
~ reaction may be performed under conventional reaction conditions, for example ; at a non-extreme temperature and in a convcntional solvent. Such reactions are normally carried out at between about ~5C and 100C, more usually from 1 5 C to 30C, for example at ambient temperature. Suitable solvents for this ~ ~ .".:
reaction include lower alkanols such as methanol and ethanol and solvents such as tetrahydrofuran, dioxane and the like. Ethanol has proved a parti-.. . , . .
~j cularly useful solvent for this reaction.
i, :
The reaction of clavulanic acid with an alcohol or thiol in ; the presence of a condensation promoting agent ~ill normally take place in an inert organic solvent of relatively high dielectric constant such as acetonitrile. This rcaction is usually carried out at an ambient or depressed temperature, for example at -10C to +22C, more usually 5C to ~18C, for example initially at 0C and thereafter gradually warming to about 15C. ;
The condensation promoting agent used is normally one which removes water ~`~ from the reaction mixture. Suitable agents include carbodiimides, carbodi-~ ., , -, .
imidazoles or equivalent reagents. Dicyclohexylcarbodiimide has proved to be a particularly suitable condensation promoting agent for use in this process. In order to minimise self condensation of the cla w lanic 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) .:' !
~ 201 -N ~ (XI) o-o-co-o,R4 " ' :
~herein R is an inert organic group~ and (e) reaction of a compound of the formula (XI) with an alcohol ROH (or less favourably with a thiol RSH).
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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 fonnula (XI) in an inert solvent. Suitable ~ 4~ yd~og~ r~
inert solvents include ether solvents such as diethylether, Le~Y~ r~fcron, dioxane and the like. In many cases the compound of the formula (XI) de-co~poses spontaneously even at a depressed temperature, for example, at -5C, to yield an ester of the formula : ': ' . ' ' \ f H2011 ~ - - M y ` ~ wherein R is an inert group witllin the definition of R. ~ -When the compound of the formula (XI) is to be reacted with an alcohol (or less favourably with a thiol) then this reaction is normally .: ,.;, : :
~ carried out in an inert solvent such as an ether solvent in the presence of... .... . .
;, an excess of the alcohol (or thiol) in order to prevent self-condensation of -~
the cla w lanic acid derivative. -Such methods of esterification are not in general as useful :: ~ ,.:: : ~.
as those involving reaction of a salt of clavulanic acid with R-~ as herein-` ~ 20 before described.
j~ The compound of the formula ~XI) may be prepared by the .~ ~:: ':: ' .
reaction of a salt of cla w lanic acid with Cl.CO.O.R4 or the chemical equi-valent thereof, Normally this reaction is carried out at a depressed ~ temperature, for example, at a temperature not greater than 5C, and in an - ~ inert solvent, for example diethylether, tetrahydrofuran, dioxane and the like. Most suitably the salt of clavulanic acid used in this reaction is a lipophilic salt so that it will dissolve in the solvent although if desired the less favourable sodium salt may be employed by suspending it in the reaction medium.
~: ' ;,~,':' :` , ' ' ':, ' '' ' '' ' ' , ,.. .. , . , , . . . . . ~
- ~ 1076~ZI) ~ DESCRIPTION 1 .~ ~ ,. .
: 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 -lactamase preparation in 0.05M phosphate buffer at pH 7 and 37 C. During-this time, enzyme inhibition or inactiyation occurs. Substrate ~benzylpenicillin) is then added and incubation continued for 30 minutes at 37C. The amount of , enzymic degradation of the substrate to penicilloic acid is determined by the hydroxylamine assay for penicillin. The amount of ~ -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 enzy;me - :
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 ~ug/ml) giving 50% inhibition of the enzyme under the above stated conditions (I50).
~ ~ -lactamase nzyme ..
: ~ The~ lactamase produced by Escherichia coli JT4 is used as an ~ enzyme. This culture is an ampicillin resistant strain and owes its resistance .: `
- ; 20 to the production of an R-factor controlled ~-lactamase. Other similar R-factor controlled ~-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/l, yeast extract (Oxoid) 20 g/1, NaC1 5 g/1 and CaC126H20 2.2 g/1. The final pH was adjusted to 7.4 with dilute NaOH. The flask is shaken at 25C for 20 hours on a rotary . ~.~ , .
shaker at 240 r.p.m.
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The bacterlal cells are collected by centri~ugation, washed with 0.05M phosphate buffer pH 7 (resuspended and centrifuged) and resuspended in water to give cell concentration 25 times that in t~e cultivation medium. This cell suspension was then disrupted in an ~SE ultrason~c disintegrator at 4C. - -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 a 1 mg/ml.
solution of benzylpenicillin in 30 minutes at 37C.
Assay Procedure , 10 Suitable dilutions of the inhibitor preparation and ~ lactamase solution are mixed and incubated at 37C for 15 minutes ~Test). A control with buffer in . :
place of inhibitor preparation is also incubated. Ben~ylpenicillin solution (substrate) is then added to test and control mixtures, incubation continued for a further 30 minutes at 37C. The residual ben~ylpenicillin 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 mlnutes at room temperature prior to the addition of 2 ml of ferric ammonium sulphate reagent. The absorption of the final solutions is measured in an E.E.L. Colori-meter or a Spectrophotomerer at 490 nm against the reagent blank. The compositionof the reactions, tests and blanks prior to the hydroxylamine assay are as follows:
~ . _ . .
, ComponentsTest Benzyl- Reagent (all dissolved in or diluted penicillin Control Blank diluted with 0.005M Blank ml. ml pH 7 phosphate buffer) ml __ ... _ Escherichia Coli ~ -lactamase solution 1.9 0.0 1.9 1.9 Inhibitor solution 0.1 0 0 0.0 0.0 BenYiylpenicillin 5mg/ml. 0.5 0.5 0.5 0.0 0.005M pH 7 phosphate buffer 0.0 2.0 0.1 0.6 , :
/
: ' :
': ~
:: 107~20 , - , ~i Calculation of ~esults . ~ . .
The percentage inhibition of the ~-lacta~ase is calculated as follo~s Absorption of benzylpenicillin blank minus absorption of control (uninhibited reaction) = x Absorption of test (inhibited reaction) minus absorption of control (uninhibited reaction) = y S~
% inhibition = - x 100 To obtain the I50 value, the inhibitor preparation is diluted until 50%
inhibition of the ~ lactamase inactivation of benzylpenicillin is obtained in the above procedure.
PAPER CHROMATOGRAPHIC DETECTION O~ CLAW LANIC ACID
- - -- , . . .
Culture filtrate and a reference solution of claw lanic acid (250~ug/ml partially purified preparatlon), are spotted (20Ju1/origin) onto ,3~~ Whatman No. l paper strips l cm. wide. The chromatograms are run by descending chromatography for 16 hours at 5C using n butanol/isopropanollwater, 7/7/6 v/v as solvent. The strips are dried at 40C and laid on agar plates containing 6~ug/ml benzylpenicillin and seeded with a ~ lactamase producing strain of Klebsiella aerogenes (synergism system). The plates are incubated overnight at 30G and clavulanic acid revealed as a zone of inhibited growth.
The Rf value of the zone was 0.46. The 6Jug/ml benzylpenicillln alone is below the concentration required to kill the Klebsiella aerogenes but in the ;~presence of a ~-lactamase lnhibitor, thls concentration becomes toxi.c, 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.
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1076~Z(~
. ' TIIIN L~Y~R Cl1ROMATOGRAPilIC D~TECTION OF CLAVUL~NIC ACID SOOIUM SALT .
Solutions of clavulanic acid sodium saLt preparations are spotted -;
(5Jul of lmg/ml) onto glass plates coated with a 0.25 mm layer of silica gel (F254) as supplied by E. Mcrck, Darmstadt, Germany. The chromatograms are run at 22C using the top phase of the mixture n-butanol/ethanol/water 4/1/5 v/v. The chromatogram plates are dried at 40C and clavulanic acid sodium salt located by bioautography on agar plates containing 6)ug/ml.
. .
benzylpenicillin and seeded with Klebsiella aerogenes (synergism system -.. . I .
~ ~ l0 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 ~ , ~ ~ : 15-30 minutes for wetting and diffusion, the TLC plate is lifted off with -;,, the aid of the filter cloth and~the agar plate incubated overnight at 30 C
.. ~ . .
., to reveal the zones of inhibited gro~th. The Rf value of claw lanic acid sodium salt in the above solvent is app~oximately 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, 54 ml.
. ~ .
of n-butanol and 9 ml of concentrated HCl. On heating the sprayed TLC plate at 120C for 1-2 minutes, clavulanic acid sodlum salt appears as a pink spot.
The triphenyltetrazolium chloride reagent consists of a mixture of 1 volume of a 4% solution of this compound in methanol with l volume of methanolic ~,-,. . . , :
sodlum hydroxide. After spraying, the TLC plates are heated at 80 C.
Clavulanic acid sodium salt appears as a red spot on a white background.
~, EX~MPLE 1 ::
;j .
CULTIVATION OF STREPTOMYCES CLAVULIGERUS
Streptomyces clavuligerus was cultivated at 26C on agar slopes containing ., ,: .
1% Yeatex (yeast extract), 1% glucose and 2% Oxold agar No. 3, pH 6.8. A
sterile loop wa6 used to transfer mycelium and spores from the slop into :' ~ -;ii 30 100 ml of a liquid medium in a 500 ml Ehrlenmeyer flask.
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The liquid medium had the following composition:-Oxoid Malt Extract 10 g/1 . . * . .
: Oxoid Bacteriological Peptone 10 g/l Glycerol 20 g/l Tap water 1 liter The medium was ad~usted to pli 7.0 with sodium hydroxide ~: solution and 100 ml. volumes dispensed into flasks which were closed with ' foam plugs prior to autoclaving at 15 lb/sq.in. for 20 minutes. An inoculated ,! seed flask was shaken for 3 days at 26C on a rotary shaker with 2 inch throw , 10 and a speed of 240 r.R.m. Production stage flasks containing the liquid I medium described above were inoculated with 5~O vegetative inoculum and grown under the same conditions as the seed flask. Samples of culture filtrate , ~ ~ were assayed for inhibitor action against the ~-lactamase of Escherichia coli JT4. Optimum activity was obtained after 3 days, The results are shown in . , .
~ Table 1. A zone of clavulanic acid at ~f 0.46 was seen when the culture .~ : :
filtrate was examined by the paper chromatographic method previously described.
æ
The increase in size of the zone paralleled the increase in the ~ lactamase - inhibitor assay, ; ~
Streptomyces clavuligerus was also cultivated in 2 litre shaken flasks containing 400 mls, of medium ~Production stage) using the same medium and cultural conditions as described earlier in this Example, In these larger vessels, growth of the organism was slower and optimum ~ lactamase inhibitory activity was achieved 7-9 days afterlnoculation with the vegetative seed.
The results are also shown in Table l, ' . , . . ' ~ ', . ~' ' ' ' .
' ' ' . . .
, * Trademark ~', ... ~ , . . . .
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1076~LZ0 Lactamase Inhibiting Activity of Streptomyces cla w ligerus Gr~m in 500 ml. and 2000 ml. Flasks _ , . __. ._. _ _ . . ---- ---- : , . . .
% Inhibition of Escherichia coli ~' ~-lactamase at a final dilution of Fermentation 1/2500 of culture filtrate Time (Days) 500 ml. Shaken Flask 2000 ml. Shaken Flask ;i~ ..... 'v,,.
2 - 30 '~ 3 ~ 55 _ 4 50 lO
7 _ 51 8 _ 53 9 _ 50 , . . .;, . . .
`i ~ CULTIVATION OF STREPTOMYCES CLAVULIG~RUS
~; Z ~ A seed flask prepared as in Example 1 was used to inoculate 500 ml. conical flasks containing 100 ml. alitluots 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 : . 'i .
FeSO4,7~l2O 0.01% w/v ` ;
The medium was sterilized by autoclaving at 15 p.s.i. for ;~
, 20 ~inutes and inoculated by the addition of the 5% vegetative seed stage.
-~ 30 The ~lasks were shaken at 26C on a rotary s~aker as in Example 1.
'," ' ' , .
* Trademark . .
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`. Optlmum titre o~ clavulanic ac~d waS ach~eved between 3~5 days. A dilution of - 1/2500 of the culture ~iltrate ~ave 60% inhibltion in the ~-lactamase inhibition assay, ~ zone of clavulanic acid was seen at Rf 0.46 when using the paper chromatographic (bioautographic) method previously described. This ~one in-. .
creased in size in parallel with the increase of the activity in the activity in the ~-lactamase inhibitor assay.
[Soluble starch supplied by British Drug Houses Ltd., ~; Poole, U.K.;
` Scotasol is dried distillers solubles supplied by ~ ;10 Thomas Borthwich Ltd., 60 Wellington Street, Glasgow, U.K.;
-~ Arkasoy is soya bean flour supplied by British ~rkady Co., ~ Old Trafford, Manchester, U.K~ .
, ~
~ EXAMPLE 3 ..,: ~
,. .
CULTIVATION OF STREPTOMYCES CLAVULIGERUS
A seed flask as produced in Example l was used to inoculate 500 ml. conical flasks containing lOO ml aliquots of the following medium prepared in deionised water and steri].ised as previously described. The ; inocuIum level was 5%.
Dextrin 2% w/v 20~ ~ Arkasoy 1% w/v Scotasol 0.1% w/v FeS047H20 0.01% w/v The~inoculated flasks were shaken at 26C. Optimum / ~ lactamase inhibitory activity was achieved between 3-5 days. The activity was similar to that achieved in Example 2.
~: .
[Dextrin is supplied by C P C (UK) Ltd., Trafford Park, Manchester, U.K.]
*Trademark .l 31 -. i . .
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~076120 , . .
.
; CULTIVATION OF STREPTOMYCES CLAVULIG~RUS
: - -The seed stage as desc~ibed in E~ample 1 was used to inoculate ~ ~ 500 ml. conical ~lasks containing the following medium prepared in deionised .~ . . .. .
water.
Dextrose 1% w/v ,i Soyabean Meal1% w/V
1 Scotasol 0.05% w/V
'~ CaC03 1% w/v ' ' These flasks were treated exactly as in preVious Examples and cultured under identical conditions. ~-lactamase inhibitory actiVity was produced between
7 _ 51 8 _ 53 9 _ 50 , . . .;, . . .
`i ~ CULTIVATION OF STREPTOMYCES CLAVULIG~RUS
~; Z ~ A seed flask prepared as in Example 1 was used to inoculate 500 ml. conical flasks containing 100 ml. alitluots 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 : . 'i .
FeSO4,7~l2O 0.01% w/v ` ;
The medium was sterilized by autoclaving at 15 p.s.i. for ;~
, 20 ~inutes and inoculated by the addition of the 5% vegetative seed stage.
-~ 30 The ~lasks were shaken at 26C on a rotary s~aker as in Example 1.
'," ' ' , .
* Trademark . .
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,. :
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:, . . , .: .
.' ' ::' . '. ' . ' -, ' , ' '. .' . ~ ' ' ' ' 1~76~0 .
`. Optlmum titre o~ clavulanic ac~d waS ach~eved between 3~5 days. A dilution of - 1/2500 of the culture ~iltrate ~ave 60% inhibltion in the ~-lactamase inhibition assay, ~ zone of clavulanic acid was seen at Rf 0.46 when using the paper chromatographic (bioautographic) method previously described. This ~one in-. .
creased in size in parallel with the increase of the activity in the activity in the ~-lactamase inhibitor assay.
[Soluble starch supplied by British Drug Houses Ltd., ~; Poole, U.K.;
` Scotasol is dried distillers solubles supplied by ~ ;10 Thomas Borthwich Ltd., 60 Wellington Street, Glasgow, U.K.;
-~ Arkasoy is soya bean flour supplied by British ~rkady Co., ~ Old Trafford, Manchester, U.K~ .
, ~
~ EXAMPLE 3 ..,: ~
,. .
CULTIVATION OF STREPTOMYCES CLAVULIGERUS
A seed flask as produced in Example l was used to inoculate 500 ml. conical flasks containing lOO ml aliquots of the following medium prepared in deionised water and steri].ised as previously described. The ; inocuIum level was 5%.
Dextrin 2% w/v 20~ ~ Arkasoy 1% w/v Scotasol 0.1% w/v FeS047H20 0.01% w/v The~inoculated flasks were shaken at 26C. Optimum / ~ lactamase inhibitory activity was achieved between 3-5 days. The activity was similar to that achieved in Example 2.
~: .
[Dextrin is supplied by C P C (UK) Ltd., Trafford Park, Manchester, U.K.]
*Trademark .l 31 -. i . .
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~076120 , . .
.
; CULTIVATION OF STREPTOMYCES CLAVULIG~RUS
: - -The seed stage as desc~ibed in E~ample 1 was used to inoculate ~ ~ 500 ml. conical ~lasks containing the following medium prepared in deionised .~ . . .. .
water.
Dextrose 1% w/v ,i Soyabean Meal1% w/V
1 Scotasol 0.05% w/V
'~ CaC03 1% w/v ' ' These flasks were treated exactly as in preVious Examples and cultured under identical conditions. ~-lactamase inhibitory actiVity was produced between
3-5 days. CuIture filtrate at a final dilution of 1/2500 gaVe 35-45%
nhibition~in the ~-lactamase inhibition assay.
EXAMPLE 5 ~ -CULTIVATION OF STREPTOMYCES CLAVULIGERUS
lactamase inhibitory activity attributable to clavulanic ac1d was produced using the fo~llowing medium with identical seed stage and cultivation conditions to Example 1.
Glycerol ~ 2% w/v 20~ ~ ~ Soyabean Meal1.5% w/v Mg SO4 0.1% w/V ~ -` K2HP04 0.1% w/v Medium prepared in deionised water lactamase inhibitory activity reached a maximum level between 3-5 days and was of a similar order to that produced in Example 4.
CULTIVATION OF STREPTOMYCES CLAVU~IGERUS
, The following medium produced clavulanic acid when using the~''!' conditions and vegetative seed inoculum as described in Example 1.
.,, ~ .
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' ~- "' ' ' ' . ' ' . :: ' : . . : ,: ' ,.' '' . : . ::
~: . ' .' .. . , ' .. ; ~ . . . . ., . :
:1076~Z0 ; Glucose 2%
Lab Lemco (Oxoid) 1%
~. . . .
`~ Oxoid Yeast Extract ~ 0.3% ~ -.. ... . . .
~ ~ CaC03 ~ 0.3%
., , Medium prepared in deionised water.
Optimum titres were achieved in 3 5 days and a 1/2500 dilution of the culture filtrate gave 35-45% inhibition in the ~-lactamase enzy~e ,.. .
, inhibition assay.
:, , ; 10 ; CULTIVATION OF STREPTOMYCES CLAVULIGERUS`
As in Examples 4, 5 and 6 the following medium produced 35-45%
inhibition ~1/2500 dilution) in the ~-lactamase assay at the optimum titre whlch ls reached~3-5 days after inoculation. All condltions were as previously ~ ;~
; described.
Glucose 2% ~/V
- "
Arkasoy - 1% w¦v CaC03 0.02% w/v C12.6H2o 0.001% w/v Medium prepared in deionised water 20 ~ EXAMPLE 8 ~ ~
. `s ~ CULTIVATION OF STREPTOMYCES CLAVULIGERUS
The following production stage medium when used under standard cultivation conditions as described in previous Examples produced 20-30%
inhibition at 1/2500 dilution in the ~-lactamase assay between 3-5 days a~ter inoculation. Using the paper chromatographic method previously described, a zone of clavulanic acid was seen at Rf 0.46 when culture filtrate was examined.
Scotasol 2%
- ~ ~ Oxoid Yeast Extract 1% ~' Medium prépared ln tap water ~ Final pH 7.0 '':':;~:`` , ~ 3,3 -~!. ~ . .
,' , , ,, ; , ,... ` ,' ' ;~ ;$
rr ~ 1a~7~ii LZ(~
~= , v . .. .
' . ' .
-~ EXAMPLE 9 .. . .
CULTIVATION OF STRFPTOMYCES CE~VULIGERUS
Under standard cultivation conditions, the fol]owing medium produced clavulanic acid 3-5 days after inoculation with the vegetatlve seed.
A 1/2500 dilution of the culture gaVe 20-30% inhlbition in the ~-lactamase .
~- inhibition assay. -Glycerol 15 Sucrose ? . . ~ ~ .
.. ~, . .. . .
j 10 ~ Proline 2.5 Monosodium Glutamate 1.5 ` NaC1 5.0 --K2~1PO4 2.0 CaCl 0.4 , -~
2 ' H2o 0.1 ` ~ ~ FeCl36H2O 0,1 Zncl2 MgSC47H2O 1.0 Medium prepared in delonised water 2O~ Final pH 7.1 CULTIVATION OF STREPTO~YCES 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 slop was incubated at 26 C
for 10 days. To this slope 100 mls. o~ sterile water was added and a mycelial suspension prepared. This was used to inoculate 50 lltre o~ steam sterilised seed medium of the following composition in tap water.
, ~ Oxoid ~alt Extract 1% w/v .. -'' .
~ 30 Oxoid Bacteriological Peptone 1% w/V
~ ) . :
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.; ' ' ' ' :
1~61~0 .
Glycerol 1% w/v 10% Pluronic L81 ~ntlfoam ~n ~; Soyabean Oil0.05% w/v ~Pluronic su~plied by Jacobs and Van deD Berg UK
- Ltd., 231 The Vale, London, W3 containing a polypropylene :,~
polyethylene block polymer, and Soyabean Oil supplied by . . ~ .
;~ British Oil and Cake ~ills Ltd., Stoneferry Road, Hull, U.K.J .
.. * . ,.
i~ Trademark The medium was contained in a 90 litre stainless steel baf1ed fermenter, agitated by a 5" vaned disc lmpéller at 240 r.p.m. Sterile air was supplied at 50 l/min and the tank incubated at 26C.
After 72 hours, the seed fermenter was used to inoculate 150 litre of the same medium using a 5% v/v addition by sterile transfer. This production :` ~ stage medium was contained in a 300 L stainless steel, fully baffled fermenter agitated by a 8~" vaned disc impeller at 210 r.p.m. Sterile aire was supplied at~150 1/min. The fermentation was maintained at 26C. ~ntifoam was added .
when requlred in 10 ml. shots (10%~Pluronic L81 in soyabean oil). Sample= were removed for ~ -lactamase inhibition assay at reguIar intervals. The fermenter ~ was~harvested between 4-5 days at the optimum level of ~ -lactamase inhibitory 7~ 20 activity (Table 2).
, `.`,,~:. :
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. .
.
:~ 'L,ABLE_ 2 Lactamase Inhlbîtory Activity o~ Samples of Culture Filtrate .j taken from a 300 litre Fermentation of Streptomyces Claw ligerus . ~. .. ... . .
.':: . ~
Fermentation ¦ % Inhibition in ~ -lactamase ~ Time Inhibition Assay at a Final :~-.~ (days) .Dilution of 1/2500 .~ . .
.~ 1.0 : 12 ~ :
: 1.5 20 :
2.0 ~ 31 .
- '':
2 . 5 ~ ~ 3 6 ' ~ :
i ~ ~ 10 - 3.0 50 .
3 . 5 : 54
nhibition~in the ~-lactamase inhibition assay.
EXAMPLE 5 ~ -CULTIVATION OF STREPTOMYCES CLAVULIGERUS
lactamase inhibitory activity attributable to clavulanic ac1d was produced using the fo~llowing medium with identical seed stage and cultivation conditions to Example 1.
Glycerol ~ 2% w/v 20~ ~ ~ Soyabean Meal1.5% w/v Mg SO4 0.1% w/V ~ -` K2HP04 0.1% w/v Medium prepared in deionised water lactamase inhibitory activity reached a maximum level between 3-5 days and was of a similar order to that produced in Example 4.
CULTIVATION OF STREPTOMYCES CLAVU~IGERUS
, The following medium produced clavulanic acid when using the~''!' conditions and vegetative seed inoculum as described in Example 1.
.,, ~ .
, . . - ' : . . . . . , - , . .
' ~- "' ' ' ' . ' ' . :: ' : . . : ,: ' ,.' '' . : . ::
~: . ' .' .. . , ' .. ; ~ . . . . ., . :
:1076~Z0 ; Glucose 2%
Lab Lemco (Oxoid) 1%
~. . . .
`~ Oxoid Yeast Extract ~ 0.3% ~ -.. ... . . .
~ ~ CaC03 ~ 0.3%
., , Medium prepared in deionised water.
Optimum titres were achieved in 3 5 days and a 1/2500 dilution of the culture filtrate gave 35-45% inhibition in the ~-lactamase enzy~e ,.. .
, inhibition assay.
:, , ; 10 ; CULTIVATION OF STREPTOMYCES CLAVULIGERUS`
As in Examples 4, 5 and 6 the following medium produced 35-45%
inhibition ~1/2500 dilution) in the ~-lactamase assay at the optimum titre whlch ls reached~3-5 days after inoculation. All condltions were as previously ~ ;~
; described.
Glucose 2% ~/V
- "
Arkasoy - 1% w¦v CaC03 0.02% w/v C12.6H2o 0.001% w/v Medium prepared in deionised water 20 ~ EXAMPLE 8 ~ ~
. `s ~ CULTIVATION OF STREPTOMYCES CLAVULIGERUS
The following production stage medium when used under standard cultivation conditions as described in previous Examples produced 20-30%
inhibition at 1/2500 dilution in the ~-lactamase assay between 3-5 days a~ter inoculation. Using the paper chromatographic method previously described, a zone of clavulanic acid was seen at Rf 0.46 when culture filtrate was examined.
Scotasol 2%
- ~ ~ Oxoid Yeast Extract 1% ~' Medium prépared ln tap water ~ Final pH 7.0 '':':;~:`` , ~ 3,3 -~!. ~ . .
,' , , ,, ; , ,... ` ,' ' ;~ ;$
rr ~ 1a~7~ii LZ(~
~= , v . .. .
' . ' .
-~ EXAMPLE 9 .. . .
CULTIVATION OF STRFPTOMYCES CE~VULIGERUS
Under standard cultivation conditions, the fol]owing medium produced clavulanic acid 3-5 days after inoculation with the vegetatlve seed.
A 1/2500 dilution of the culture gaVe 20-30% inhlbition in the ~-lactamase .
~- inhibition assay. -Glycerol 15 Sucrose ? . . ~ ~ .
.. ~, . .. . .
j 10 ~ Proline 2.5 Monosodium Glutamate 1.5 ` NaC1 5.0 --K2~1PO4 2.0 CaCl 0.4 , -~
2 ' H2o 0.1 ` ~ ~ FeCl36H2O 0,1 Zncl2 MgSC47H2O 1.0 Medium prepared in delonised water 2O~ Final pH 7.1 CULTIVATION OF STREPTO~YCES 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 slop was incubated at 26 C
for 10 days. To this slope 100 mls. o~ sterile water was added and a mycelial suspension prepared. This was used to inoculate 50 lltre o~ steam sterilised seed medium of the following composition in tap water.
, ~ Oxoid ~alt Extract 1% w/v .. -'' .
~ 30 Oxoid Bacteriological Peptone 1% w/V
~ ) . :
* Trademark :.~
~; ~ :,,, ~ ~ 34 ~
.; ' ' ' ' :
1~61~0 .
Glycerol 1% w/v 10% Pluronic L81 ~ntlfoam ~n ~; Soyabean Oil0.05% w/v ~Pluronic su~plied by Jacobs and Van deD Berg UK
- Ltd., 231 The Vale, London, W3 containing a polypropylene :,~
polyethylene block polymer, and Soyabean Oil supplied by . . ~ .
;~ British Oil and Cake ~ills Ltd., Stoneferry Road, Hull, U.K.J .
.. * . ,.
i~ Trademark The medium was contained in a 90 litre stainless steel baf1ed fermenter, agitated by a 5" vaned disc lmpéller at 240 r.p.m. Sterile air was supplied at 50 l/min and the tank incubated at 26C.
After 72 hours, the seed fermenter was used to inoculate 150 litre of the same medium using a 5% v/v addition by sterile transfer. This production :` ~ stage medium was contained in a 300 L stainless steel, fully baffled fermenter agitated by a 8~" vaned disc impeller at 210 r.p.m. Sterile aire was supplied at~150 1/min. The fermentation was maintained at 26C. ~ntifoam was added .
when requlred in 10 ml. shots (10%~Pluronic L81 in soyabean oil). Sample= were removed for ~ -lactamase inhibition assay at reguIar intervals. The fermenter ~ was~harvested between 4-5 days at the optimum level of ~ -lactamase inhibitory 7~ 20 activity (Table 2).
, `.`,,~:. :
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: .' , , .,:, .. :,: .,, ,, , ., , .. ,, . . .. . : ,..... ,.. ~ . : .
,,:, . . :, .:, ., - .,, . ,:: , . , . , . .. , , : . .: . : ,. : , ' ~ . , ' ' . ~ ' ' '~' ' ' ' ' . ' " ' , . ' ' . ' ' . ` , ,, . . ' !, . . . . . .
' " ' .'. " ' ' ' "" " " '. " , ' ' " ' '' " ' ',' " ' ' ' . : ' . ' ' .", ' ' : ' "' ., ' ' . ', 107~ 0 . " . . .
. .
.
:~ 'L,ABLE_ 2 Lactamase Inhlbîtory Activity o~ Samples of Culture Filtrate .j taken from a 300 litre Fermentation of Streptomyces Claw ligerus . ~. .. ... . .
.':: . ~
Fermentation ¦ % Inhibition in ~ -lactamase ~ Time Inhibition Assay at a Final :~-.~ (days) .Dilution of 1/2500 .~ . .
.~ 1.0 : 12 ~ :
: 1.5 20 :
2.0 ~ 31 .
- '':
2 . 5 ~ ~ 3 6 ' ~ :
i ~ ~ 10 - 3.0 50 .
3 . 5 : 54
4 . O 51 4.5 56
5 . O ~ 55 : .' : ; ~
~ 20 :~
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.
35a ~
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1076~L20 ~1 EXAMPL~ 11 CULTIV~TION OF STREPTOMXCES CL~VULIGE~IJS
~ The seed fermenter was run exactly as described in Example 10 -_ using the same medium.
Afer 72 hours~ the seed fermenter was used to give a 5% v/v vegetative inoculum into a 300 litre stainless steel fully baffled 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 150 l/min. The fermentation was maintained at 26C. Antifoam was added when required in 10 ml. shots (10% Pluronic L81 in soya bean oil).
The medium used in the production stage was as described in ~; Example 3 with the addition of 0.05% v/v of 10% Pluronic L81/soyabean oil ,i ~ antifoam prior to sterilisation.
The ~-lactamase inhibitory activity of fermentation samples wss similar to those of Example 10 (see Table 2). Paper chromatographic sxamination revealed a zone of clavulanic acid at Rf 0.46 using the bioauto-,~; graphic (synergism) method previously described. The size of the clavulanic acid zone increased in parallel with the increase in the ~~lactamase inhibitor assay.
CULTIVATION 0~ STREPTYMYCES CLA W LIGERUS
; 100 mls of sterile water was added to a sporing culture which had been grown on Bennetts agar in a Roux bottle for 10 days at 26C. A
mycelium/sporc suspension was produced and used to inoculatc 75 litres of ,~ ~ steam sterllised medium of the followlng composition ln tap water. ' Dextrin 2% W/V
Arkasoy ~50~ 1% ~/V
`~, 10% Pluronic L81 0.03% y/v ~ in soybean oil ;~ 30 The pH of the medium was ~d;justcd to 7.0 , .,. " ' " ": :
:"'"' ' '."'' . .
~ - - 36 - ~
10761~0 . :~ . ` . ' ''' ' The medium was contalned in a 100 litre stainlesc: steel --baffled fermenter, agitated by a 1~ vaned dlsc impeller at 140 rpm. Sterile . .
air was supplied at 75 l/minute and the tank incobated for 72 hours at 26 C. ~;
The contents of the seed fermenter were used to inoculate -`:
. .
1500 litres of steam sterilised medium of the following composition in tap -`!
water.
.` ~rkasoy ~50~ 1.5% W/V
: :
Glycerol 1.0% W/V
, ~ , .
K~2P04 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 suppli:ed at 1200 litres per minute. Antifoam was added in 25 ml amounts aS required. (10% Pluronic L81 in soyabean oil).
~"r ~ The~fermentation was controlled at 26 C untll a maximum yield of clavulanic acid was~obtained between 3 - 5 days when 200 - 300Jug/ml of clavulanic acid : , :
were produced.
20~ ~EXAMPLE 13 CULTIVATION OF STREPTOMYCES CLAVULIGERUS
~ Inoculum was produced in a seed flask as previously clescribed, `.' but using the medium described in Example 3 (with pl{ of the medium ad~usted 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 steril:lsed.
,.~ . ~ . .
~:. ~ The inoculum level Wa6 5%.
f.r/~ Prichem P224 1% W/V
- ~ Arkasoy ~50~ 1.5% W/V
KH2PO~ 0,1% W/V
~ 30 Thc pll oE t~le meclllllll was adJuc;ted to 7.0 `, ' ' ~.
:' ' ' ~ '. ' . :
!
.
107Gl'~0 The inoculated flasks were shaken at 26C and optimum ~ lactamase inhibitory activity was achieved between 3 - 5 days. Levels of 300 - 500~ug/ml of clavulanic acid were achieved.
Prichem P224 is a triglyceride supplied by Prices Limited, -Bromborough, Bebington, Wirral, Cheshire, U.K. Prichem P224 ls based on oleic acid (65%), palmitic acid (11%) and other similar acids.
. ISOLATION OF CRUDE CLAW LANIC ACID SODIUM SALT
" - .
; Harvested culture liquor produced as described in ~xample 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 ~ -lactamase inhibition ~ assay at 1/2500. The filtrate was chilled to 5C and 40 litre of n-butanol h, added. The mixture was stirred and 25% H2SO4 added until the pH was 2Ø
~ The acidified mixture was stirred for a further 10 mins. before separating the :,, .
phases by centrifugation. The aqueous phase was discarded. To the n-butanol extract 0.5% of Norit GSX carbon was added and the mixture stirred or 15 minutes. The carbon was discarded after removal by filtration using a dia~
tomaceus 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 at 7Ø The phases were separated by centrifugation and the - : , :
n-butanol phase discarded. The aqueous phase was concentrated under ~educed vacuum to 800 ml. and then freeze dried. This yielded 35g. of a crude solid preparation of clavulanic acid with an I50 of 1.3~ug/ml in the ~ -lactamase inhibieion assay. This solid preparation was stored dry at ~20C while awaiting further purification.
ISOLATION OF CRUDL CLAVULANIC ACID SODIUM SALT
One litre of culture filtrate giVing 53% inhibition at 1/2500 in the ~ -lactamase inhibition assay and obtained as described in Example 12 *Trademark - 38 -. . .
,' , '' , ,:' . ' ,'' , ~ ',, ' '' :': ' . ' ; :: ' '' 1~7~;~L20 ; was percolated down a 1 inch diamete~ x 6 inch column of Permutit Isopore resin FF lP tSRA 62) in the Cl form ~supplled by Permutit Co. Ltd., 632-652 London Road, Isleworth, Middlesex, U.K.]. The culture filtrate was Eollowed by 300 ml. of distilled water to wash the column. Elution of the active ~-lactamase inhibitor was achieved With 0.2~ NaCl solution. Fractions (20 ml.) were collected and assayed at a 1/2500 final dilution in the~-lactamase lnhibition 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. rBiogel P2 is supplied by Bio Rad Laboratories, 32nd and GriEfin Ave., Richmond, California, U.S.A~ . The active fractions, as determined by the ~ lactamase inhibition assay, were combined.
Sodium chloride eluted after clavulanic acid and was det~cted using silver nitrate solution. The combined active fractions were concentrated and freeze : .
dried, One litre of culture filtrate after the above -treatment yielded 0.45 g. of a crude solid Freparation of clavulan1c acid having an 50 of 0.92,ug/ml.
This solid was stored at -20C while awaiting further ~20 purification.
: .. :
~ ISOLATION OF CRUDE CLAYULANIC SODIUM SALT
- - .
Culture filtrate containing 300,ug/ml of claulanic acid is acidified using an in-line mixer system, extracted with n-butanol and clavulanic acid is back extracted into water at neutral p~l.
Chilléd culture filtrate (5 - 10 C) was pumped to an in-line mixer at the inlet of which, enougll 6% (v/v) nitric acid was added to maintain an outlet pH of 2.0 ~ 0.1. The acidified filtrate was passed at 4.1/min.
through a glycol cooled plate heat exchanger tA.P.V. Ltd.) to ~aintain a , temperature between 2 - 5. The pH Was monitored in a flow cell before * Trademark .. . . .
` 1~7t;~ZO
passing into a three stage counter current separator (West.falia Separator Ltd., Model ~G 1006).
: Chilled water saturated n-butanol (at about 5C) was pumped at 3 l/min into the counter current separator.
The aqueous outlet from the countér current separator was run to waste. Entrained water was removed from the butanol outflow of the counter current separator using a liquid/liquid centrifugal separator.
~Alfa Laval Ltd. Model 3024X - G). The butanol was collected in a stainless steel vessel fitted with a cooling ~acket in which it was stored at about 5C. :
From the vessel, ~0 1 aliquots were removed and thoroughly mixed with 2 1 of chilled water ~5C), saturated with n-butanol. The pll 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 (Sharples Centrifuge Ltd. Model ~B5PY - 5YH) at . : :
a pumped rate of 2 l/min. ~ .
: ~ From 1800 1 of culture filtrate, 90 1 of aqueous phase was . . : .
reco~ered, containing 39% of the clavulanic acid present in the culture ~. :
filtrate ~ ~: .:. .
15 1 of the aqueous extract was adjusted from 2%, to 8~, total . : :-solids by the addition of 60 g sodium chloride per litre, and spray dried .
(Anhydro, Copenhagen, Type Lab S l). The conditions used were: Feed rate ~ : ~
2 l/hr Atomizer voltage 170 v; ~eater setting 6 - 7; Inlet temp 150C; : .: : .
.~ Outlet temp 80C.
; The dried product, tota.1. we:Lgllt 1 kg " conta;ined 62% of the : :
: clayul.anic acid present ln the feedstock.
The remaining 75 1 of aqueous extract was concentrated by ultrafiltration (De Danske Sukkerfabrikker. Laboratory Modu:le, Membrane Type 900j. The operating procedure Was to re-circulate the retentate from a stainless steel tank, fitted with a coollng system, wlth the outlet valve ~.:
set-so as to give a dlfferential pressure across the ~0 membranes af ' - ~10- ', .' ~'`' "' .
, " , , , ` , ' . ' ` ' ' ' ' ' ' " ' ' ' ' . ' ' ' ' .
076~LZ0 25 atmospheres. The temperature WaS maintained at 2 - 5C and the pH at
~ 20 :~
."
.
35a ~
: . : :.
~, ~ ,............................. . .
,.,, . . . , . . . . , . ,, , . , : . . :. , :, .:
.. ... : , .. ,, . : . . . . . . . : :, ~:.. , ..... , '. ' ' '' : , : , . , :,: : , ' - , . .: ,. . . .
., . ., i ,- ,:. . .. .. .
1076~L20 ~1 EXAMPL~ 11 CULTIV~TION OF STREPTOMXCES CL~VULIGE~IJS
~ The seed fermenter was run exactly as described in Example 10 -_ using the same medium.
Afer 72 hours~ the seed fermenter was used to give a 5% v/v vegetative inoculum into a 300 litre stainless steel fully baffled 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 150 l/min. The fermentation was maintained at 26C. Antifoam was added when required in 10 ml. shots (10% Pluronic L81 in soya bean oil).
The medium used in the production stage was as described in ~; Example 3 with the addition of 0.05% v/v of 10% Pluronic L81/soyabean oil ,i ~ antifoam prior to sterilisation.
The ~-lactamase inhibitory activity of fermentation samples wss similar to those of Example 10 (see Table 2). Paper chromatographic sxamination revealed a zone of clavulanic acid at Rf 0.46 using the bioauto-,~; graphic (synergism) method previously described. The size of the clavulanic acid zone increased in parallel with the increase in the ~~lactamase inhibitor assay.
CULTIVATION 0~ STREPTYMYCES CLA W LIGERUS
; 100 mls of sterile water was added to a sporing culture which had been grown on Bennetts agar in a Roux bottle for 10 days at 26C. A
mycelium/sporc suspension was produced and used to inoculatc 75 litres of ,~ ~ steam sterllised medium of the followlng composition ln tap water. ' Dextrin 2% W/V
Arkasoy ~50~ 1% ~/V
`~, 10% Pluronic L81 0.03% y/v ~ in soybean oil ;~ 30 The pH of the medium was ~d;justcd to 7.0 , .,. " ' " ": :
:"'"' ' '."'' . .
~ - - 36 - ~
10761~0 . :~ . ` . ' ''' ' The medium was contalned in a 100 litre stainlesc: steel --baffled fermenter, agitated by a 1~ vaned dlsc impeller at 140 rpm. Sterile . .
air was supplied at 75 l/minute and the tank incobated for 72 hours at 26 C. ~;
The contents of the seed fermenter were used to inoculate -`:
. .
1500 litres of steam sterilised medium of the following composition in tap -`!
water.
.` ~rkasoy ~50~ 1.5% W/V
: :
Glycerol 1.0% W/V
, ~ , .
K~2P04 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 suppli:ed at 1200 litres per minute. Antifoam was added in 25 ml amounts aS required. (10% Pluronic L81 in soyabean oil).
~"r ~ The~fermentation was controlled at 26 C untll a maximum yield of clavulanic acid was~obtained between 3 - 5 days when 200 - 300Jug/ml of clavulanic acid : , :
were produced.
20~ ~EXAMPLE 13 CULTIVATION OF STREPTOMYCES CLAVULIGERUS
~ Inoculum was produced in a seed flask as previously clescribed, `.' but using the medium described in Example 3 (with pl{ of the medium ad~usted 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 steril:lsed.
,.~ . ~ . .
~:. ~ The inoculum level Wa6 5%.
f.r/~ Prichem P224 1% W/V
- ~ Arkasoy ~50~ 1.5% W/V
KH2PO~ 0,1% W/V
~ 30 Thc pll oE t~le meclllllll was adJuc;ted to 7.0 `, ' ' ~.
:' ' ' ~ '. ' . :
!
.
107Gl'~0 The inoculated flasks were shaken at 26C and optimum ~ lactamase inhibitory activity was achieved between 3 - 5 days. Levels of 300 - 500~ug/ml of clavulanic acid were achieved.
Prichem P224 is a triglyceride supplied by Prices Limited, -Bromborough, Bebington, Wirral, Cheshire, U.K. Prichem P224 ls based on oleic acid (65%), palmitic acid (11%) and other similar acids.
. ISOLATION OF CRUDE CLAW LANIC ACID SODIUM SALT
" - .
; Harvested culture liquor produced as described in ~xample 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 ~ -lactamase inhibition ~ assay at 1/2500. The filtrate was chilled to 5C and 40 litre of n-butanol h, added. The mixture was stirred and 25% H2SO4 added until the pH was 2Ø
~ The acidified mixture was stirred for a further 10 mins. before separating the :,, .
phases by centrifugation. The aqueous phase was discarded. To the n-butanol extract 0.5% of Norit GSX carbon was added and the mixture stirred or 15 minutes. The carbon was discarded after removal by filtration using a dia~
tomaceus 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 at 7Ø The phases were separated by centrifugation and the - : , :
n-butanol phase discarded. The aqueous phase was concentrated under ~educed vacuum to 800 ml. and then freeze dried. This yielded 35g. of a crude solid preparation of clavulanic acid with an I50 of 1.3~ug/ml in the ~ -lactamase inhibieion assay. This solid preparation was stored dry at ~20C while awaiting further purification.
ISOLATION OF CRUDL CLAVULANIC ACID SODIUM SALT
One litre of culture filtrate giVing 53% inhibition at 1/2500 in the ~ -lactamase inhibition assay and obtained as described in Example 12 *Trademark - 38 -. . .
,' , '' , ,:' . ' ,'' , ~ ',, ' '' :': ' . ' ; :: ' '' 1~7~;~L20 ; was percolated down a 1 inch diamete~ x 6 inch column of Permutit Isopore resin FF lP tSRA 62) in the Cl form ~supplled by Permutit Co. Ltd., 632-652 London Road, Isleworth, Middlesex, U.K.]. The culture filtrate was Eollowed by 300 ml. of distilled water to wash the column. Elution of the active ~-lactamase inhibitor was achieved With 0.2~ NaCl solution. Fractions (20 ml.) were collected and assayed at a 1/2500 final dilution in the~-lactamase lnhibition 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. rBiogel P2 is supplied by Bio Rad Laboratories, 32nd and GriEfin Ave., Richmond, California, U.S.A~ . The active fractions, as determined by the ~ lactamase inhibition assay, were combined.
Sodium chloride eluted after clavulanic acid and was det~cted using silver nitrate solution. The combined active fractions were concentrated and freeze : .
dried, One litre of culture filtrate after the above -treatment yielded 0.45 g. of a crude solid Freparation of clavulan1c acid having an 50 of 0.92,ug/ml.
This solid was stored at -20C while awaiting further ~20 purification.
: .. :
~ ISOLATION OF CRUDE CLAYULANIC SODIUM SALT
- - .
Culture filtrate containing 300,ug/ml of claulanic acid is acidified using an in-line mixer system, extracted with n-butanol and clavulanic acid is back extracted into water at neutral p~l.
Chilléd culture filtrate (5 - 10 C) was pumped to an in-line mixer at the inlet of which, enougll 6% (v/v) nitric acid was added to maintain an outlet pH of 2.0 ~ 0.1. The acidified filtrate was passed at 4.1/min.
through a glycol cooled plate heat exchanger tA.P.V. Ltd.) to ~aintain a , temperature between 2 - 5. The pH Was monitored in a flow cell before * Trademark .. . . .
` 1~7t;~ZO
passing into a three stage counter current separator (West.falia Separator Ltd., Model ~G 1006).
: Chilled water saturated n-butanol (at about 5C) was pumped at 3 l/min into the counter current separator.
The aqueous outlet from the countér current separator was run to waste. Entrained water was removed from the butanol outflow of the counter current separator using a liquid/liquid centrifugal separator.
~Alfa Laval Ltd. Model 3024X - G). The butanol was collected in a stainless steel vessel fitted with a cooling ~acket in which it was stored at about 5C. :
From the vessel, ~0 1 aliquots were removed and thoroughly mixed with 2 1 of chilled water ~5C), saturated with n-butanol. The pll 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 (Sharples Centrifuge Ltd. Model ~B5PY - 5YH) at . : :
a pumped rate of 2 l/min. ~ .
: ~ From 1800 1 of culture filtrate, 90 1 of aqueous phase was . . : .
reco~ered, containing 39% of the clavulanic acid present in the culture ~. :
filtrate ~ ~: .:. .
15 1 of the aqueous extract was adjusted from 2%, to 8~, total . : :-solids by the addition of 60 g sodium chloride per litre, and spray dried .
(Anhydro, Copenhagen, Type Lab S l). The conditions used were: Feed rate ~ : ~
2 l/hr Atomizer voltage 170 v; ~eater setting 6 - 7; Inlet temp 150C; : .: : .
.~ Outlet temp 80C.
; The dried product, tota.1. we:Lgllt 1 kg " conta;ined 62% of the : :
: clayul.anic acid present ln the feedstock.
The remaining 75 1 of aqueous extract was concentrated by ultrafiltration (De Danske Sukkerfabrikker. Laboratory Modu:le, Membrane Type 900j. The operating procedure Was to re-circulate the retentate from a stainless steel tank, fitted with a coollng system, wlth the outlet valve ~.:
set-so as to give a dlfferential pressure across the ~0 membranes af ' - ~10- ', .' ~'`' "' .
, " , , , ` , ' . ' ` ' ' ' ' ' ' " ' ' ' ' . ' ' ' ' .
076~LZ0 25 atmospheres. The temperature WaS maintained at 2 - 5C and the pH at
6.8 ~ 0.1 by addlti~n of'2N hydrochloric acid, as necessary. The volume was reduced to 34 1 which contained 72% of the-'clavulanic acid present in the feedstock.
The aqueous concentrate was stored at about 5C, ad~usted 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 c'ulture filtrate.
, PARTIAL P~RIFICATION OF CRUD~ _L~ W LANIc ~CID
Crude clavulanic acid preparations obtained as described in Example 15 were purified by ion exchange chromatography. Eighteen grams of material prepared as described in Ex~mple 15 having an I50 value of 1.3~ug/ml ~'' ' (final concentration) were dissolved in 25 ml. of distilled water and applied to a 1~" x 16" bed of Permutit FF lP (SRA 62) resin in the chloride form.
The column was eluted with a sodium chloride gradient formed by graVity feeding ~20 0.5M sodium chloride into a mixing reservolr containing 1 litre of distilled water which in turn fed the chromatographic column. 10 ml. cuts were collected `and ~ -lactamase inhibitory activity assayed using a l/2500 dilutioD of the fractions. Activity was eluted after'a main band o~ 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 o~ Biorad Biogel P2 and eluting with 1% n-butanol in water. The 20 ml. fractions were assayed ' for clavulanic acid content using the ~ -lactamase inhibition assay. The ractions were also spotted onto paper strips and sprayed with either the ,~ Ehrlich or the triphenyltetrazolium spray reagents described in Description 3.
.
~.. . . . .
,. ., . . , . . . ,, ~ .
~. . ~ ,,,,, ,., .. , : :
': .... ".,.,. ' .". ! ' :.; . ~,:".
.' .. "
.
~' " " '' ' ' " ' ' ' ' ~076~Z0 /~-lactamase inhibitory activity correlated With the pink or red spots respec-tively produced by these reagents. Active Cllts were combined, excluding those containing sodium chloride and concentrated under ~acuum to dryness. This yielded 520 mg. of partially purified clavulanic acid sod:ium salt with an I50 ' of 0.2 yg/ml in the standard f~-lactamase inhibitor assay.
Thin layer chromatography (silica gel) of this clavulanic ' '' ' acid preparation gave the following Rf values:~ n-butanol/ethanol/water 4:1:5 v/v top phase Rf 0.37; n-butanol/acetic acid/water 12:3:5 v/v Rf 0.44;
isopropanol/water 7:3 v/v Rf 0.78. The ~ones were detected by spraying with 10 ~ hrlich~s reagent. 6-A~inopenicillanic run as a marker and detected with the same spray had Rf vaIues oi~ 0.38; 0.39 and 0.77 respectively~
E~AMPLE 18 PARTIAL PURIFICATION OF CLAVULANIC ACID SODIUM SALT :
Culture filtrate produced as described in Example 12 was ' , solvent extracted~as in Example 14 to giVe a solid~preparation which was further purified by ion exchange chromatography using ~Ihatman diethylaminoethyl ~ :
cel1010se DE 52. This solid llOg.~ was dlssolved 1n 2~0 ml. of d1sti11ed ' .'' water and applied to a 1~' x 20" column of DE 52 cellulose previously equili-brated with O.OlM sodium phosphate buffer pll 7.5. The column was eluted with ~-' a NaCl gradient, O.lM NaCl in O.OlM sodium phosphate buffer pH 7.5 was ed into a'mixing chamber containing 1 litre of O.OlM phosphate buffer pll`7.5 ~
which in~turn was connected to the column. Fractions (10 ml.) were collected and these were assayed for ~ lactamase lnhibitory activity at a dilution of 1/2500. The fractions were also exa~ined for antibacteria.L actiVity by the ~' hole-in-plate assay method using nutrient agar plates seeded wlth Lebsiella aero~enes. The fractions having the highest t~lactamase inhibitory actiVity and glving 70nes of inhibition in the hole-in-plate assay were combined, concentrated and then desalted on a ~iorad Biogel P2 column. These fractions ''' were shown to contain clavulanic acid by paper and thin layer chromatography.
' .
:
, - ~2 ~ ,':
. , ' ,':
:
-, . , ... , j - ,. , , .. , ..... ' .
,. . ,' , ~ .. . '.''. ~' "~
~.~76~ O
~ .
EXA~LE 19 ISOLAT~ON OF SOLID CLAV~LANIC ACID SODIUM SALT
,' A partially purified solid preparation of clavulanic acid (500 mg) prepared as in Example 17 was loaded onto a Whatman microcrystalline CC 31 cellulose column with 1" x 20" bed size. The chromatographic solvent was n-butanol/ethanol/water 4:1:5 v/v, top phase. The column was run at 4C
and 4 ml. fractions collected. Fractions were tested for the presence of - 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 ~-lactamase inhibition assays at a l/1250~diLution.
-~ ~ctive fractions were combined an-l dried under vacuum on a rotary~evaporator.
The~solid was dissolved in a small volume of distilled water and freeze dried.
A~white solid preparation of the sodium salt of clavulanic acid was obtained (40~mg) having an I50 of 0.08Jug/ml in the ~-lactamase inhibition assay.
E ~ EXAMPLE 20 ISOLATION OF SOLID CIA~JLANIC ACID SODIUM SALT
Concentrated back extract (6 1) (from ultrafiltration in Example 16) containing 10 g of c1avulanic acid as determined by the ~-lactamase nhibition assay of Description 1. This was percolated at 1 l/hr onLo a 20~ 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 sod1um chloride gradient. The gradient was formed by a reservoir containing 4 1 of 1.4 m NaCl feeding a stirred reservoir containing 4 1 of 0.7 NaC1 which in turn was connected to a stirred reservoir containing 4 1 of deionized water which was connected via a pump to the column, The column was eiuted at 2,5 ml/min and 2S ml fractions collec~ed. Fractions , were assayed by the ~-lactamase inhibition assay.
Active fractions (nos, 140-230) were combined and vacuum evaporated to near dryness. Ethanol (500 mls) was then added and the solid ~ ~ ' filtered off aftec vigorous shaking. The ethanol extract was then vacuum : ' ' ' _, . . . ... .. .
, ', ' ,' ' -' ~ ' , :
, ~`~
~7~
'' ,'.
evaporated to dryness on a rotary eyaporato~ and redissolved in deioni~ed ~
:. . . .
water (40 mls). This Was loaded onto a 4l~ x 24l~ column of Biorad Biogel P2 .and eluted With a 1%.n-butanol solution. Fractions Were collected (25 ml) ~.
and assayed for ~ -lactamase inhibitory actiVity at a 1/2500 final dil~tion. . ~:
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 combined~ concentrated by evaporation of the solvent under reduced pressure to 20 mls and then freeze dried. This ~.
yielded 4.8 g of the sodium salt of clavulanic acid. (I50 about 0 06~ug/ml) .
' -: ~. ,~ ':
PREPARATION OF AN ESTER OF CLAVULANIC ACID (MET~L ESTER) .
~0 ~ CH20H
S~C}120N
~ CO2Na .J ~ N ~ .' .:~ \CO2CM3 . .
19.8 mg. of the sodium salt of clavulanic acid Was dissolved in 0.5 ml. dry dimethylformamide and treated With 0.25 ml. methyl iodide. .~.
After standing~at room temperature ~or 1.5 hours under anhydrous conditions, .
the solvents Were removed in vacuo. The residue Was purified by P.L.C. on sil1ca gel (Kieselgel 60F254 supplied by E. Merck~ Darmstadt~ Germany) eluting Witll ethyl acetate to giVe clavulanic acid methyl ester as a colourless ;:
: oil (Rf 0.38; red colour With triphenyltetrazolium chloride Spray) which had ; ~
the following properties: :
Analysis: Found C 50.49 H 5.43 N 6.29 ; : .
CgHllNO5 Requires C 50.70 H 5.20 N 6.57 max (Methanol): no absorption ~215 nm .
~rmax (Film): 3300 - 3600 (Broad)~ 180Q~ 1750~ 1695 cm ;.
Approximate lSt order N.M.R. (CDC13): 2.49 (broad S~
exchanged with D2O), 3.05 _ 44 -_ .
.: ~"'' '' : ' '. ' ~,, ' ' : 10761Z0 (d, 1, J - 17.5 Hz), 3.54 (dd, 1, J = 17.5 Hz, :. ' :-- .
J2 = 2.5 }Iz), 3.84 (S, 3) ; 4 24 (d, 2, J = 7 }Iz), 4.93 (dt, 1, J = 7 Hz, J2 = 1.5 Hz), 5.07 ~d, 1~ J = 1.5 Hz), 5.72 (d, 1, J = 2.5 Hz) Molecular weight (mass spectrum) : 213.0635. ~-~
Calculated for C9Hl1N05 : 213.0637 Thin layer chromatography of the methyl ester showed a single zone in each of the following solvent systems; butanol/ethanol/water 4:1:5 v/v .
i ~`~ top phase Rf 0.75; isopropanol/water, 7:3 v/v Rf 0.95; ethylacetate/ethyl- ~
~10 alcohol 8:2 v/v Rf 0.87. The zones were detected by bioautography using ~ -ebsiella aerogenes with added benzylpenicillin (synergism system).
PREPARATION OF AN ESTER OF CLA~ULANIC ACID (p-nitrobenzyl ester) ~, ~ , ...
~ : .
l2ou ~ j CU2OH
\C2Na \ O2CU2 ~ -~2 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 to p-nitrobenzyl ester of clavulanic acid as ; white feathery needles, m!p. 111 - 112C, which on recrystallisation had a ~:~
~ ~ m.p. of 117.5 - 118C, ~
:~ .
~ EXAMPLE 23 ~, : , , .
~ PREPARATION OF AN ESTER OF CLAYULANIC ACID (BENZYL ESTER) ::: :
CH20H CH20H : ' ~ ~ o T l -:
~ r 1 1 \H ~ I ~ y H
~ ~ - N ~ C02Na ~ C02C}12Ph ~ , , i _ 45 _ .
., .
,, : , .. .:
~' ' . . , ' - ' ' ', " ' ' "" '' ' ' '' " .
lZO
.
Impure 3- ~ -hydroxyethylidine)-7-oxo-4-oxa-l~azabicyclo ~3,2,0] heptane-2-carboxylic 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. 17-18C) for 3 hours under ~anhydrous conditions. The reaction mixture was fractionated on silica gel, eluting with ethyl acetate, to give in substantially pure form the benzyl ester o 3- ~ -hydroxyethylidine)-7-oxo-4-oxa-1-azabicyclo ~3,20~ heptane-2-carboxylicacid 63 mg.) as a colourless oil. i.r. (film) 1800, 1745, 1695 cm 1; n.m.r.
(CDC13), 2.25 (s,l, exchangeable with D2O), 3.05 (d~l,J=17Hz), 3.51 (dd,l,J=17 Hz, J2=2.5 Hz~, 4.24 (d,2,J=7.5Hz), 4.92 (dt,l,J=7.5Hz, J2=1.5Uz), 5.15 (d,l,J=1.5Hz), .24 (s,2), 5.71 (d,l,J=2.5 Hz), 7.45 ~ (s,5).
PREPARATION OF THE BENZYL ESTER OF CLAVULANIC ~CID FROM CRUDE EXTRACTS OF THE
- FULTURE FILTRATE`OF S.CLAVULIGERUS . ;
Culture filtrate 20 1 obtained as described in Example 10 was vacuum evaporated using a climbing film e~aporator 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 300 g. 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 or 2 hours at room temperature and then ~;~ ` diluted~with 1 1. of ethyl acetate. The reaction mixture was filtered and the filtrate concentrated to as low a volume as was possible. The oily residue was extracted with a further 1 1. of ethyl acetate and the extract filtered. The filtrate was again concentrated and the resulting oily residue loaded onto a ~:
3~' x 14~ silica gel column (Biogel Biosil A 100 mesh) in cyclohexane. The column was eluted with cyclohexane to remove benzyl bromide and the solvent was then changed to ethyl acetate and 20 ml. ractions collected. Fractions were tested for the presence o the benzyl ester of clavulanic acid by spotting onto glass backed silica gel . . '' ': ., ' ~ '. . - ~ '. .. ' ;
~:, '~ ' ":: ,' ' -:' ' . '.': ' ', ' ' ' ' " " ' :
. ~ . ' . ' ~' .' : - . ' , . .. .. .
., . ; . ,,,: , ., . ' '.' ~ .~,.i,., ', ~ ' ' .
.' .' '',' ' '' '' , .' ~ , ', ' ~
t.l,c, plates (Merck precoated silica gel 60 F 254) and spraying with 2,3,5-triphenyl-tetrazolium chloride (TTC) spray reagent, : Fractions giving intense red spots with this reagent were further examined by t.l,c. on silica gel plates using chloroform-ethyl acetate 8:2 as the solvent and spraying the developed plates with TTC spray. The benzyl ester of clawlanic acid runs at Rf 0.31 at 22C. ~ractions containing this ester were combined and concentrated to 15 mL. and this solution was further chromatographed on a 11~" x 16" silica gel column (Merck silica gel H, type 60) with chloroform/ethyl acetate ~3:2 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 Einally purified by column chromatography on a 1" x 16" silica gel column (Merck silica gel H, type 60) with ethyl acetate cyclohexane 8:2 as the solvent.
Selected fractions were combined and vacuum evaporated to give pure benzyl ester as an oil, 160 mg.
EXA~LE 25 ~ .
PREPARATION OF CLAVVL~NIC ACID ,l~ENZYL ESTFU
Spray dried solid (3.3 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 mls.
of benzyl bromide added. After stirring at room temperature for 2 hours, 12 1. of ethyl acetate were added and the solids removed by filtration. The filtrate was vacuum evaporated to an oily residue (212 g). The residue was ~; loaded onto a column containing a 4" x 13" bed of silica gel (Flopkins ~
Williams MFC) in cyclohexane, The column was eluted with 12 1. of cyclohexane to remove excess benzyl bromide. The eluent was then changed to ethyl acetate and 500 ml. fractions collected, These were tested for benzyl clavu:Lanate content by spotting onto silica gel t 1 c plates (~erck precoated sillca gel 60 F 254) and spraying with 2,3,5 triphenyltetrazolium chloride (TTC) spray réagent. Fractions giving intense red spots were further exarllined by t 1 c .
' _ l,7 _ . .
:: , :, . . . . ,, . " .
~076~0 on silica gel with chloroformlethyl acetate $:2 as the solvent and spraying the developed plates with T T C spray~ ~ractions 5-13 contained the bulk of '~ the ester, and these were combined and vacuum concentrated to an oil (79.3 g).
This preparation was then chromatographed on a 4" x 18" column of silica gel (Merck silica gel H type 60) with chloroform/ethyl acetate 8:2 as the solvent.
Fractions were selected as described above and yielded on concentration 45.9g. of oil which was of 62% purity as ad~udged by NMR spectroscopy.
This product was finally chromatographed on a 2 3/4"'x 18" ~ ' column of Sephadex L~l 20 in cyclohexane/chloroform 1:1. After selection of fractions and concentration a colourless oil (27.6 g) was obtained which proved to be 95% pure benzyl ester of clavulanic acid as determilled by NMR ~ '' spectroscopic examination. (Sephadex (Trade Mark) LH20 is a hydroxypropyl derivative of Sephadex Q25 supplied by Pharmacia Great Britain, 75 Uxbridge ~ '~' " ' Road, London W5, U.K.).
-PREPAR~TION 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 5 kg. of Amberlyst A.26 anion exchange resin in the 20 ~ chloride form (Rohm ~ Haas Company, Philadelphia, USA) for 1 hour at room temperature. ~The resin was then filtered and the filtrate reassayèd, 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 L. of dimethyl ~ .
formamide (DMF). After refiltering the resin was suspended in 2.3 l. ofDMF/0.2 M NaI. To this was added 200 mls. of benzyl bromide and the suspension stirred thoroughly. After standing at room temperature for i6 hours~ ethyl acetate (2 1) was added, and the resin then filtered,' further washings (Ethyl acetate) of the resin were comblned wlth the'filtrate. The extract was then concentrated to a small volume and chromatographed on 3'i x 181' silica gel column (Merck silica gel H type 60) with ethyl acetate/cyclohexane 8:2 as the ~ .
' - 48 - -.
' ' ' - ' , ; ' , . ' , ': ' ' , '",: ' ' :
G~2~
solvent, Fractions containing benzyl clavulanate were selected by spotting onto silica gel t l c plates and spraying with TTC reagent as described previously (Example 24). Those selected were concentrated to 20 mls and then chromatographed on a 11~" x 18" silica gel column (Merck silica gel H type 60) with chloroform/ethyl acetate 8:2 as the solvent. Selected fractions were -combined and evaporated to a colourless oil (440 mgs) which was 90% benzyl clavulanate as determined by NMR spectroscopy.
.
PREPARATION OF TUE BENZYL ESTER OF Cl.A~ULANIC~ACID FROM CRUDE EXTRACTS OF
THE CULTURE FILTRATE OF S. CLAVULIGERUS
An aliquot o~ aqueous back extract of tlle butanol extract of culture filtrate obtained as described in Example 14 was freeze dried using - : :
an Edwards chamber drier. A 24 g. portion of the solid obtained contained ~"`
0.96~g. of sodium clavulanic acid as determined by the enzyme inhibition assay. This solid was suspended in 75 ml. of dry dimethylformamide and 75 ml.
~ .
o~ benzyl bromide was added. The mixture was stirred for 2 hours at room temperature. The suspension was then diluted with 500 ml. of ethyl acetate ;~ ~ and the mixture filtered. The filtrate was concentrated to an oily residue on a vacuum rotary evaporator. This residue was loaded onto a 2" x 14" silica gel column (Biogel Biosil A.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 ben~yl ester of :
clavulanic acid were selected as in Example 24. Further purification was also achieved as described in Example 24 by column chromatography. This - ,:: . :
process yielded 220 mg. of pure benzyl ester.
.... . ..
PREPARATION OF CLAVULANIC ACID SODIUM SALT ~ ~
: . .
:, ~:.::
C~I~OH ~ ~ ~l2 C2C~12C6~5 \CO2Na , ~ 49 ~
.,.: :.: ... :. . . . , ... . . , . ' :. . . :
1076120 ~ ~
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 clavulante (135 mg.).
HYDROLYSIS OF CLAVULANIC ACID ~ETHYL ESTER TO CLAVULANIC ACID
2.17 mg. of clavulanic acid ester was dissolved in 0.1 ml. ,~
methanol and treated with 0.208 ml. sodium hydrPxide 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 Rf identical to that oE the sodium salt of clavulanic acid; colour reactions and biological assay were consistent with this component being the sodium salt of clavulanic acid. -~
Slow conversion of the ester to clavulanic acid was seen when l;mglml. of the compound was incubated at 37C in 0.05M phosphate buffer at pH 7. The reaction was followed by paper chromatography ~bioautographic ~20~ sys~em). Us1ng the butanol/ethanol/water system to follow the react;lon over ~; ~ a period of 2 hours the zone of the methyl ester at Rf 0.79 decreased in size as the zone of clavulanic acid at Rf 0.12 increased.
: EXAMPLE 30 . .
~ ANTIBACTERIAL SPECTRUM OF CLAVULANIC ACID
: ~,~ ,: .. : .
~ The antibacterial activity of clavulanic acid sodium salt , against a rang~ of bacteria was determined uslng the microtitre method.
Serial dilutions of clavulanic acid sodium salt in Oxoid sensitivity test , ~ . . .
broth contained in a microtitre plastic tray were i~oculated with an over-;~ night broth culture of each organism so that the final dilution of the ~30 B inoculum was 0.5 x 10 4. The cultures were incubated overnight and the points ~4 : ~ ' ' . .
' ~ . ... .. .
. . , .: . :
'~ ;~ ,' ' - '. . ' ' :. . , ` ' 1076~;~0 : . .
bacterial growth recorded next morning by observing the turbidit~J of th~
cul.ture. The results, expressed as approxi.mate MIC values (minimum inllibitory :
concentration jug/ml.) are recorded in Table 3 which shows that the compound has a broad spectrum of antibacterial activity.
TABLE 3 -~
ANTIBACTERIAL SPECTRUM O~ CLAVULANIC ACID SODIUM SALT
.
Bacterial Strain Minimum Inhibitory .
ConcentrationJug/ml.
10 ~. Staphylococcus aureus (Oxford H) ~ 7.5 , ~
: Staphylococcus aureus (Russell)~ 7.5 Ba~clllus:subtiIis 62 Streptococcus~faecalis . ~ 500 Streptococcus pyogenes CN 10 125 Escherichia coli NCTC 10418 3.1 ebsiella aerogenes 31 - 62 ebsiella oxytocum 62 Enterobacter aerogenes T 624 31 Enterobacter cloacae 62 .:~zo~ Ac metobacter anitratus 125 Providentia stuartii 125 :Serratia ::rce~cens 125 ; :Proteus mirabilis C977 62 .. .... !" . ' Prot WO90 31 . ` : :: : .:
Salmonell.a typhimurium 31 : Shigella sonnei - 62 Pseudomonas aeruginosa A 500 , :~
; . . ~' :',.
, .
. :
": ' ' .
': ' "::
~ 51 ~
. ' ,~:
,~
.. ._..... . . " . . .. ,- , . . , .... , . : , ~76~L20 -EXA~LES OF ~-LACTA SE INHI~ITION ~ CLAVUL~NIC ~CID SODIUM SALT
Clavulanic acid progressively and irreversibly inhibits the ~-lactamase of Escherichia coli. The method o~ Description 1 shows that the other ~-lactamases shown in Table 4 are also inhibited by clavulanic acid.
INHIBITION OF ~-LACT~MASES BY CLAVULANIC ACID
Approximate Source of ~-lactamase I50 Value Relative to Escherichia coli JT 4 = 1 , 10 _ ~.
Staphylococcus aureus (Russell) l.O
~ ~ .
Escherichic coli JT4 1.0 : : ....
Escherichia coli B11 2.0 Klebsiella aerogenes ~ 0.6 . j .
Pseudomonas aeru~inosa 1822 5.0 (R factor) Pseudomonas dalgleish 0.1 ith~pen1cillin G as sub`strate the I50 of clavulanic acid sodium salt against the ~-lactamase of Staph. aureus (Russel1) is approximately 0.06Jug/ml.
~ ~ ~ EXAMPLE 32 ~ EXAMPLES OF ACTIVITY OF CLAVULANIC ACID METHYL ESTER
Tests for antibacterial activity in broth showed clavulanic acid -:
methyl ester to have broad spectrum activity but of a lower order than shown by clavulanic acid. It was not clear whether this activity was the activity o~ the .
compound itself or of clavulanic acid liberated by slow aqueous hydrolysis of the ester. Clavulanlc acid methyl ester showed marked antibacterial synerglsm in combination with ampicillin or cephaloridlne against bacteria resistant to these antibiotics. Thus, the minimum inhibitory concentration (M.I.C.) or ampicillin against Staphylococcus aureus (Russell) was reduced from 500~ug/ml.
to <0.4 in the presence of l.O~ug/ml. clavulanic '- ' ' ' ~ ' ' ' ' . ~ ~, , ;, ' ' ' ' ' , ' ' , ' ' ' . : . .. , . :. .
' ~ . .
, ~ , .. ~
.
.
: , ,. ,~ , , i - , .
. .
. . . ~ (~761 ZO
, acid methyl ester. The M.I.C. for cephaloridine was reduced from 1.5~ug./ml.
to <0.03Jug/ml. in the presence o~ 1,ug-lml. of clavulanic acid methyl ester.
The M.I.C. for ampiclllin against ~roteus mirabilis C889 was reduced from 500Jug/ml. to 31 ~g/ml. in the presence of 5~ug/ml. clavulanic acid methyl ester.
~XAMiPLE 33 ,:
PREPARATION_PIVALOYLOXYMETHYL CLAVULANATE
To a stirred solution o~ bromomethyl pivalate (0.37g) in dry dimethylformamide (5 ml) was added sodium clavulanate (0.49g). After 2 hrs. at room temperature the reaction mixture was treated with ethyl acetate (20 ml), cyclohexane (10 ml) and water (20 ml). The mixture separated into two layers and the non~aqueous layer was separated, washed with water (20 ml) and dried over sodium sulphate. The dried solution was evaporated to leave the required product as a pale yellow oil.
(500 mg). N.m.r. (CDC13), 1.26 (s,9)3 3.13 (d,1,J=17 H~), 3.62 (dd, 1,J,=17Hz, =2.5Hz, 4.3(d,2,J=7.5Hz), 5.0(dt, 1,J=7.5Hz, J2=1.5Hz), 5.16(d,1,J-1.5Hz), 5.79(d,1,J=2.5Hz), 5.92G(s,2); i.r.(liquid film), ~ -lactam C.O. 1800 om l, ester C=O 1760 cm~l.
EXAMiPLE 34 -PREPA ATION OF CLAVULANIC ACID PHTHALIDE ESTERS
~ To a stirred solution of 3-bromophthalide (0.43g) in dry dimethyl formamide (5 ml) was added sodium clavulanate (0.5g) and the solution was lePt at room temperature for 2 hours. The solution was treated with ethyl acetate (20 ml), cyclohexane (10 ml) and water (30 ml) and shaken thoroughly. The non-aqueous layer was washed with water (20 ml), dried (Na2S04) and evaporated to yield a pale yellow gum. The two diastereomeric esters were separated using high pressure li~uid chromatography on a 40 cm x 10 mm column of silica gel (Merckosorb SI 60, 5JU) eluting with ethyl acetate at a flow rate of 3 ml/min.
The first phthalide ester (retention time 7.15 min) crystallised rom ethyl acetate as needles, mp 102, and had the following i.r.
: ' : .
, , . , , . ", ,, . . .. , , , ,. ; ' , ,' ,' . '.i ,"' , ; : : ' ' ' "', ' ' ' ' :
'. , , '. ' ' ' ' "'' ' . .' ' , .' ': , " ' ': . '' '.'' ' ' '~
~ ` 1076120 (nujol mull~ lactam C~0 1790 cm 1 ester C~0 1755 cm 1 n.m.r. (CD3COCD3):
3.14 (d,l,J=17.5Hz) 3.76 (dd,l,J,=17.5Hz, J2=2.511z), 4.25(d,2,J=7.5Hz), 5.0 (dt~l~Jl=7.5Hz~ J2=1.5Hz), 5.4 (s,l,J-1.5Hz), 5.82 (d,l,J=2.5Hz),
The aqueous concentrate was stored at about 5C, ad~usted 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 c'ulture filtrate.
, PARTIAL P~RIFICATION OF CRUD~ _L~ W LANIc ~CID
Crude clavulanic acid preparations obtained as described in Example 15 were purified by ion exchange chromatography. Eighteen grams of material prepared as described in Ex~mple 15 having an I50 value of 1.3~ug/ml ~'' ' (final concentration) were dissolved in 25 ml. of distilled water and applied to a 1~" x 16" bed of Permutit FF lP (SRA 62) resin in the chloride form.
The column was eluted with a sodium chloride gradient formed by graVity feeding ~20 0.5M sodium chloride into a mixing reservolr containing 1 litre of distilled water which in turn fed the chromatographic column. 10 ml. cuts were collected `and ~ -lactamase inhibitory activity assayed using a l/2500 dilutioD of the fractions. Activity was eluted after'a main band o~ 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 o~ Biorad Biogel P2 and eluting with 1% n-butanol in water. The 20 ml. fractions were assayed ' for clavulanic acid content using the ~ -lactamase inhibition assay. The ractions were also spotted onto paper strips and sprayed with either the ,~ Ehrlich or the triphenyltetrazolium spray reagents described in Description 3.
.
~.. . . . .
,. ., . . , . . . ,, ~ .
~. . ~ ,,,,, ,., .. , : :
': .... ".,.,. ' .". ! ' :.; . ~,:".
.' .. "
.
~' " " '' ' ' " ' ' ' ' ~076~Z0 /~-lactamase inhibitory activity correlated With the pink or red spots respec-tively produced by these reagents. Active Cllts were combined, excluding those containing sodium chloride and concentrated under ~acuum to dryness. This yielded 520 mg. of partially purified clavulanic acid sod:ium salt with an I50 ' of 0.2 yg/ml in the standard f~-lactamase inhibitor assay.
Thin layer chromatography (silica gel) of this clavulanic ' '' ' acid preparation gave the following Rf values:~ n-butanol/ethanol/water 4:1:5 v/v top phase Rf 0.37; n-butanol/acetic acid/water 12:3:5 v/v Rf 0.44;
isopropanol/water 7:3 v/v Rf 0.78. The ~ones were detected by spraying with 10 ~ hrlich~s reagent. 6-A~inopenicillanic run as a marker and detected with the same spray had Rf vaIues oi~ 0.38; 0.39 and 0.77 respectively~
E~AMPLE 18 PARTIAL PURIFICATION OF CLAVULANIC ACID SODIUM SALT :
Culture filtrate produced as described in Example 12 was ' , solvent extracted~as in Example 14 to giVe a solid~preparation which was further purified by ion exchange chromatography using ~Ihatman diethylaminoethyl ~ :
cel1010se DE 52. This solid llOg.~ was dlssolved 1n 2~0 ml. of d1sti11ed ' .'' water and applied to a 1~' x 20" column of DE 52 cellulose previously equili-brated with O.OlM sodium phosphate buffer pll 7.5. The column was eluted with ~-' a NaCl gradient, O.lM NaCl in O.OlM sodium phosphate buffer pH 7.5 was ed into a'mixing chamber containing 1 litre of O.OlM phosphate buffer pll`7.5 ~
which in~turn was connected to the column. Fractions (10 ml.) were collected and these were assayed for ~ lactamase lnhibitory activity at a dilution of 1/2500. The fractions were also exa~ined for antibacteria.L actiVity by the ~' hole-in-plate assay method using nutrient agar plates seeded wlth Lebsiella aero~enes. The fractions having the highest t~lactamase inhibitory actiVity and glving 70nes of inhibition in the hole-in-plate assay were combined, concentrated and then desalted on a ~iorad Biogel P2 column. These fractions ''' were shown to contain clavulanic acid by paper and thin layer chromatography.
' .
:
, - ~2 ~ ,':
. , ' ,':
:
-, . , ... , j - ,. , , .. , ..... ' .
,. . ,' , ~ .. . '.''. ~' "~
~.~76~ O
~ .
EXA~LE 19 ISOLAT~ON OF SOLID CLAV~LANIC ACID SODIUM SALT
,' A partially purified solid preparation of clavulanic acid (500 mg) prepared as in Example 17 was loaded onto a Whatman microcrystalline CC 31 cellulose column with 1" x 20" bed size. The chromatographic solvent was n-butanol/ethanol/water 4:1:5 v/v, top phase. The column was run at 4C
and 4 ml. fractions collected. Fractions were tested for the presence of - 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 ~-lactamase inhibition assays at a l/1250~diLution.
-~ ~ctive fractions were combined an-l dried under vacuum on a rotary~evaporator.
The~solid was dissolved in a small volume of distilled water and freeze dried.
A~white solid preparation of the sodium salt of clavulanic acid was obtained (40~mg) having an I50 of 0.08Jug/ml in the ~-lactamase inhibition assay.
E ~ EXAMPLE 20 ISOLATION OF SOLID CIA~JLANIC ACID SODIUM SALT
Concentrated back extract (6 1) (from ultrafiltration in Example 16) containing 10 g of c1avulanic acid as determined by the ~-lactamase nhibition assay of Description 1. This was percolated at 1 l/hr onLo a 20~ 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 sod1um chloride gradient. The gradient was formed by a reservoir containing 4 1 of 1.4 m NaCl feeding a stirred reservoir containing 4 1 of 0.7 NaC1 which in turn was connected to a stirred reservoir containing 4 1 of deionized water which was connected via a pump to the column, The column was eiuted at 2,5 ml/min and 2S ml fractions collec~ed. Fractions , were assayed by the ~-lactamase inhibition assay.
Active fractions (nos, 140-230) were combined and vacuum evaporated to near dryness. Ethanol (500 mls) was then added and the solid ~ ~ ' filtered off aftec vigorous shaking. The ethanol extract was then vacuum : ' ' ' _, . . . ... .. .
, ', ' ,' ' -' ~ ' , :
, ~`~
~7~
'' ,'.
evaporated to dryness on a rotary eyaporato~ and redissolved in deioni~ed ~
:. . . .
water (40 mls). This Was loaded onto a 4l~ x 24l~ column of Biorad Biogel P2 .and eluted With a 1%.n-butanol solution. Fractions Were collected (25 ml) ~.
and assayed for ~ -lactamase inhibitory actiVity at a 1/2500 final dil~tion. . ~:
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 combined~ concentrated by evaporation of the solvent under reduced pressure to 20 mls and then freeze dried. This ~.
yielded 4.8 g of the sodium salt of clavulanic acid. (I50 about 0 06~ug/ml) .
' -: ~. ,~ ':
PREPARATION OF AN ESTER OF CLAVULANIC ACID (MET~L ESTER) .
~0 ~ CH20H
S~C}120N
~ CO2Na .J ~ N ~ .' .:~ \CO2CM3 . .
19.8 mg. of the sodium salt of clavulanic acid Was dissolved in 0.5 ml. dry dimethylformamide and treated With 0.25 ml. methyl iodide. .~.
After standing~at room temperature ~or 1.5 hours under anhydrous conditions, .
the solvents Were removed in vacuo. The residue Was purified by P.L.C. on sil1ca gel (Kieselgel 60F254 supplied by E. Merck~ Darmstadt~ Germany) eluting Witll ethyl acetate to giVe clavulanic acid methyl ester as a colourless ;:
: oil (Rf 0.38; red colour With triphenyltetrazolium chloride Spray) which had ; ~
the following properties: :
Analysis: Found C 50.49 H 5.43 N 6.29 ; : .
CgHllNO5 Requires C 50.70 H 5.20 N 6.57 max (Methanol): no absorption ~215 nm .
~rmax (Film): 3300 - 3600 (Broad)~ 180Q~ 1750~ 1695 cm ;.
Approximate lSt order N.M.R. (CDC13): 2.49 (broad S~
exchanged with D2O), 3.05 _ 44 -_ .
.: ~"'' '' : ' '. ' ~,, ' ' : 10761Z0 (d, 1, J - 17.5 Hz), 3.54 (dd, 1, J = 17.5 Hz, :. ' :-- .
J2 = 2.5 }Iz), 3.84 (S, 3) ; 4 24 (d, 2, J = 7 }Iz), 4.93 (dt, 1, J = 7 Hz, J2 = 1.5 Hz), 5.07 ~d, 1~ J = 1.5 Hz), 5.72 (d, 1, J = 2.5 Hz) Molecular weight (mass spectrum) : 213.0635. ~-~
Calculated for C9Hl1N05 : 213.0637 Thin layer chromatography of the methyl ester showed a single zone in each of the following solvent systems; butanol/ethanol/water 4:1:5 v/v .
i ~`~ top phase Rf 0.75; isopropanol/water, 7:3 v/v Rf 0.95; ethylacetate/ethyl- ~
~10 alcohol 8:2 v/v Rf 0.87. The zones were detected by bioautography using ~ -ebsiella aerogenes with added benzylpenicillin (synergism system).
PREPARATION OF AN ESTER OF CLA~ULANIC ACID (p-nitrobenzyl ester) ~, ~ , ...
~ : .
l2ou ~ j CU2OH
\C2Na \ O2CU2 ~ -~2 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 to p-nitrobenzyl ester of clavulanic acid as ; white feathery needles, m!p. 111 - 112C, which on recrystallisation had a ~:~
~ ~ m.p. of 117.5 - 118C, ~
:~ .
~ EXAMPLE 23 ~, : , , .
~ PREPARATION OF AN ESTER OF CLAYULANIC ACID (BENZYL ESTER) ::: :
CH20H CH20H : ' ~ ~ o T l -:
~ r 1 1 \H ~ I ~ y H
~ ~ - N ~ C02Na ~ C02C}12Ph ~ , , i _ 45 _ .
., .
,, : , .. .:
~' ' . . , ' - ' ' ', " ' ' "" '' ' ' '' " .
lZO
.
Impure 3- ~ -hydroxyethylidine)-7-oxo-4-oxa-l~azabicyclo ~3,2,0] heptane-2-carboxylic 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. 17-18C) for 3 hours under ~anhydrous conditions. The reaction mixture was fractionated on silica gel, eluting with ethyl acetate, to give in substantially pure form the benzyl ester o 3- ~ -hydroxyethylidine)-7-oxo-4-oxa-1-azabicyclo ~3,20~ heptane-2-carboxylicacid 63 mg.) as a colourless oil. i.r. (film) 1800, 1745, 1695 cm 1; n.m.r.
(CDC13), 2.25 (s,l, exchangeable with D2O), 3.05 (d~l,J=17Hz), 3.51 (dd,l,J=17 Hz, J2=2.5 Hz~, 4.24 (d,2,J=7.5Hz), 4.92 (dt,l,J=7.5Hz, J2=1.5Uz), 5.15 (d,l,J=1.5Hz), .24 (s,2), 5.71 (d,l,J=2.5 Hz), 7.45 ~ (s,5).
PREPARATION OF THE BENZYL ESTER OF CLAVULANIC ~CID FROM CRUDE EXTRACTS OF THE
- FULTURE FILTRATE`OF S.CLAVULIGERUS . ;
Culture filtrate 20 1 obtained as described in Example 10 was vacuum evaporated using a climbing film e~aporator 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 300 g. 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 or 2 hours at room temperature and then ~;~ ` diluted~with 1 1. of ethyl acetate. The reaction mixture was filtered and the filtrate concentrated to as low a volume as was possible. The oily residue was extracted with a further 1 1. of ethyl acetate and the extract filtered. The filtrate was again concentrated and the resulting oily residue loaded onto a ~:
3~' x 14~ silica gel column (Biogel Biosil A 100 mesh) in cyclohexane. The column was eluted with cyclohexane to remove benzyl bromide and the solvent was then changed to ethyl acetate and 20 ml. ractions collected. Fractions were tested for the presence o the benzyl ester of clavulanic acid by spotting onto glass backed silica gel . . '' ': ., ' ~ '. . - ~ '. .. ' ;
~:, '~ ' ":: ,' ' -:' ' . '.': ' ', ' ' ' ' " " ' :
. ~ . ' . ' ~' .' : - . ' , . .. .. .
., . ; . ,,,: , ., . ' '.' ~ .~,.i,., ', ~ ' ' .
.' .' '',' ' '' '' , .' ~ , ', ' ~
t.l,c, plates (Merck precoated silica gel 60 F 254) and spraying with 2,3,5-triphenyl-tetrazolium chloride (TTC) spray reagent, : Fractions giving intense red spots with this reagent were further examined by t.l,c. on silica gel plates using chloroform-ethyl acetate 8:2 as the solvent and spraying the developed plates with TTC spray. The benzyl ester of clawlanic acid runs at Rf 0.31 at 22C. ~ractions containing this ester were combined and concentrated to 15 mL. and this solution was further chromatographed on a 11~" x 16" silica gel column (Merck silica gel H, type 60) with chloroform/ethyl acetate ~3:2 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 Einally purified by column chromatography on a 1" x 16" silica gel column (Merck silica gel H, type 60) with ethyl acetate cyclohexane 8:2 as the solvent.
Selected fractions were combined and vacuum evaporated to give pure benzyl ester as an oil, 160 mg.
EXA~LE 25 ~ .
PREPARATION OF CLAVVL~NIC ACID ,l~ENZYL ESTFU
Spray dried solid (3.3 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 mls.
of benzyl bromide added. After stirring at room temperature for 2 hours, 12 1. of ethyl acetate were added and the solids removed by filtration. The filtrate was vacuum evaporated to an oily residue (212 g). The residue was ~; loaded onto a column containing a 4" x 13" bed of silica gel (Flopkins ~
Williams MFC) in cyclohexane, The column was eluted with 12 1. of cyclohexane to remove excess benzyl bromide. The eluent was then changed to ethyl acetate and 500 ml. fractions collected, These were tested for benzyl clavu:Lanate content by spotting onto silica gel t 1 c plates (~erck precoated sillca gel 60 F 254) and spraying with 2,3,5 triphenyltetrazolium chloride (TTC) spray réagent. Fractions giving intense red spots were further exarllined by t 1 c .
' _ l,7 _ . .
:: , :, . . . . ,, . " .
~076~0 on silica gel with chloroformlethyl acetate $:2 as the solvent and spraying the developed plates with T T C spray~ ~ractions 5-13 contained the bulk of '~ the ester, and these were combined and vacuum concentrated to an oil (79.3 g).
This preparation was then chromatographed on a 4" x 18" column of silica gel (Merck silica gel H type 60) with chloroform/ethyl acetate 8:2 as the solvent.
Fractions were selected as described above and yielded on concentration 45.9g. of oil which was of 62% purity as ad~udged by NMR spectroscopy.
This product was finally chromatographed on a 2 3/4"'x 18" ~ ' column of Sephadex L~l 20 in cyclohexane/chloroform 1:1. After selection of fractions and concentration a colourless oil (27.6 g) was obtained which proved to be 95% pure benzyl ester of clavulanic acid as determilled by NMR ~ '' spectroscopic examination. (Sephadex (Trade Mark) LH20 is a hydroxypropyl derivative of Sephadex Q25 supplied by Pharmacia Great Britain, 75 Uxbridge ~ '~' " ' Road, London W5, U.K.).
-PREPAR~TION 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 5 kg. of Amberlyst A.26 anion exchange resin in the 20 ~ chloride form (Rohm ~ Haas Company, Philadelphia, USA) for 1 hour at room temperature. ~The resin was then filtered and the filtrate reassayèd, 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 L. of dimethyl ~ .
formamide (DMF). After refiltering the resin was suspended in 2.3 l. ofDMF/0.2 M NaI. To this was added 200 mls. of benzyl bromide and the suspension stirred thoroughly. After standing at room temperature for i6 hours~ ethyl acetate (2 1) was added, and the resin then filtered,' further washings (Ethyl acetate) of the resin were comblned wlth the'filtrate. The extract was then concentrated to a small volume and chromatographed on 3'i x 181' silica gel column (Merck silica gel H type 60) with ethyl acetate/cyclohexane 8:2 as the ~ .
' - 48 - -.
' ' ' - ' , ; ' , . ' , ': ' ' , '",: ' ' :
G~2~
solvent, Fractions containing benzyl clavulanate were selected by spotting onto silica gel t l c plates and spraying with TTC reagent as described previously (Example 24). Those selected were concentrated to 20 mls and then chromatographed on a 11~" x 18" silica gel column (Merck silica gel H type 60) with chloroform/ethyl acetate 8:2 as the solvent. Selected fractions were -combined and evaporated to a colourless oil (440 mgs) which was 90% benzyl clavulanate as determined by NMR spectroscopy.
.
PREPARATION OF TUE BENZYL ESTER OF Cl.A~ULANIC~ACID FROM CRUDE EXTRACTS OF
THE CULTURE FILTRATE OF S. CLAVULIGERUS
An aliquot o~ aqueous back extract of tlle butanol extract of culture filtrate obtained as described in Example 14 was freeze dried using - : :
an Edwards chamber drier. A 24 g. portion of the solid obtained contained ~"`
0.96~g. of sodium clavulanic acid as determined by the enzyme inhibition assay. This solid was suspended in 75 ml. of dry dimethylformamide and 75 ml.
~ .
o~ benzyl bromide was added. The mixture was stirred for 2 hours at room temperature. The suspension was then diluted with 500 ml. of ethyl acetate ;~ ~ and the mixture filtered. The filtrate was concentrated to an oily residue on a vacuum rotary evaporator. This residue was loaded onto a 2" x 14" silica gel column (Biogel Biosil A.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 ben~yl ester of :
clavulanic acid were selected as in Example 24. Further purification was also achieved as described in Example 24 by column chromatography. This - ,:: . :
process yielded 220 mg. of pure benzyl ester.
.... . ..
PREPARATION OF CLAVULANIC ACID SODIUM SALT ~ ~
: . .
:, ~:.::
C~I~OH ~ ~ ~l2 C2C~12C6~5 \CO2Na , ~ 49 ~
.,.: :.: ... :. . . . , ... . . , . ' :. . . :
1076120 ~ ~
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 clavulante (135 mg.).
HYDROLYSIS OF CLAVULANIC ACID ~ETHYL ESTER TO CLAVULANIC ACID
2.17 mg. of clavulanic acid ester was dissolved in 0.1 ml. ,~
methanol and treated with 0.208 ml. sodium hydrPxide 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 Rf identical to that oE the sodium salt of clavulanic acid; colour reactions and biological assay were consistent with this component being the sodium salt of clavulanic acid. -~
Slow conversion of the ester to clavulanic acid was seen when l;mglml. of the compound was incubated at 37C in 0.05M phosphate buffer at pH 7. The reaction was followed by paper chromatography ~bioautographic ~20~ sys~em). Us1ng the butanol/ethanol/water system to follow the react;lon over ~; ~ a period of 2 hours the zone of the methyl ester at Rf 0.79 decreased in size as the zone of clavulanic acid at Rf 0.12 increased.
: EXAMPLE 30 . .
~ ANTIBACTERIAL SPECTRUM OF CLAVULANIC ACID
: ~,~ ,: .. : .
~ The antibacterial activity of clavulanic acid sodium salt , against a rang~ of bacteria was determined uslng the microtitre method.
Serial dilutions of clavulanic acid sodium salt in Oxoid sensitivity test , ~ . . .
broth contained in a microtitre plastic tray were i~oculated with an over-;~ night broth culture of each organism so that the final dilution of the ~30 B inoculum was 0.5 x 10 4. The cultures were incubated overnight and the points ~4 : ~ ' ' . .
' ~ . ... .. .
. . , .: . :
'~ ;~ ,' ' - '. . ' ' :. . , ` ' 1076~;~0 : . .
bacterial growth recorded next morning by observing the turbidit~J of th~
cul.ture. The results, expressed as approxi.mate MIC values (minimum inllibitory :
concentration jug/ml.) are recorded in Table 3 which shows that the compound has a broad spectrum of antibacterial activity.
TABLE 3 -~
ANTIBACTERIAL SPECTRUM O~ CLAVULANIC ACID SODIUM SALT
.
Bacterial Strain Minimum Inhibitory .
ConcentrationJug/ml.
10 ~. Staphylococcus aureus (Oxford H) ~ 7.5 , ~
: Staphylococcus aureus (Russell)~ 7.5 Ba~clllus:subtiIis 62 Streptococcus~faecalis . ~ 500 Streptococcus pyogenes CN 10 125 Escherichia coli NCTC 10418 3.1 ebsiella aerogenes 31 - 62 ebsiella oxytocum 62 Enterobacter aerogenes T 624 31 Enterobacter cloacae 62 .:~zo~ Ac metobacter anitratus 125 Providentia stuartii 125 :Serratia ::rce~cens 125 ; :Proteus mirabilis C977 62 .. .... !" . ' Prot WO90 31 . ` : :: : .:
Salmonell.a typhimurium 31 : Shigella sonnei - 62 Pseudomonas aeruginosa A 500 , :~
; . . ~' :',.
, .
. :
": ' ' .
': ' "::
~ 51 ~
. ' ,~:
,~
.. ._..... . . " . . .. ,- , . . , .... , . : , ~76~L20 -EXA~LES OF ~-LACTA SE INHI~ITION ~ CLAVUL~NIC ~CID SODIUM SALT
Clavulanic acid progressively and irreversibly inhibits the ~-lactamase of Escherichia coli. The method o~ Description 1 shows that the other ~-lactamases shown in Table 4 are also inhibited by clavulanic acid.
INHIBITION OF ~-LACT~MASES BY CLAVULANIC ACID
Approximate Source of ~-lactamase I50 Value Relative to Escherichia coli JT 4 = 1 , 10 _ ~.
Staphylococcus aureus (Russell) l.O
~ ~ .
Escherichic coli JT4 1.0 : : ....
Escherichia coli B11 2.0 Klebsiella aerogenes ~ 0.6 . j .
Pseudomonas aeru~inosa 1822 5.0 (R factor) Pseudomonas dalgleish 0.1 ith~pen1cillin G as sub`strate the I50 of clavulanic acid sodium salt against the ~-lactamase of Staph. aureus (Russel1) is approximately 0.06Jug/ml.
~ ~ ~ EXAMPLE 32 ~ EXAMPLES OF ACTIVITY OF CLAVULANIC ACID METHYL ESTER
Tests for antibacterial activity in broth showed clavulanic acid -:
methyl ester to have broad spectrum activity but of a lower order than shown by clavulanic acid. It was not clear whether this activity was the activity o~ the .
compound itself or of clavulanic acid liberated by slow aqueous hydrolysis of the ester. Clavulanlc acid methyl ester showed marked antibacterial synerglsm in combination with ampicillin or cephaloridlne against bacteria resistant to these antibiotics. Thus, the minimum inhibitory concentration (M.I.C.) or ampicillin against Staphylococcus aureus (Russell) was reduced from 500~ug/ml.
to <0.4 in the presence of l.O~ug/ml. clavulanic '- ' ' ' ~ ' ' ' ' . ~ ~, , ;, ' ' ' ' ' , ' ' , ' ' ' . : . .. , . :. .
' ~ . .
, ~ , .. ~
.
.
: , ,. ,~ , , i - , .
. .
. . . ~ (~761 ZO
, acid methyl ester. The M.I.C. for cephaloridine was reduced from 1.5~ug./ml.
to <0.03Jug/ml. in the presence o~ 1,ug-lml. of clavulanic acid methyl ester.
The M.I.C. for ampiclllin against ~roteus mirabilis C889 was reduced from 500Jug/ml. to 31 ~g/ml. in the presence of 5~ug/ml. clavulanic acid methyl ester.
~XAMiPLE 33 ,:
PREPARATION_PIVALOYLOXYMETHYL CLAVULANATE
To a stirred solution o~ bromomethyl pivalate (0.37g) in dry dimethylformamide (5 ml) was added sodium clavulanate (0.49g). After 2 hrs. at room temperature the reaction mixture was treated with ethyl acetate (20 ml), cyclohexane (10 ml) and water (20 ml). The mixture separated into two layers and the non~aqueous layer was separated, washed with water (20 ml) and dried over sodium sulphate. The dried solution was evaporated to leave the required product as a pale yellow oil.
(500 mg). N.m.r. (CDC13), 1.26 (s,9)3 3.13 (d,1,J=17 H~), 3.62 (dd, 1,J,=17Hz, =2.5Hz, 4.3(d,2,J=7.5Hz), 5.0(dt, 1,J=7.5Hz, J2=1.5Hz), 5.16(d,1,J-1.5Hz), 5.79(d,1,J=2.5Hz), 5.92G(s,2); i.r.(liquid film), ~ -lactam C.O. 1800 om l, ester C=O 1760 cm~l.
EXAMiPLE 34 -PREPA ATION OF CLAVULANIC ACID PHTHALIDE ESTERS
~ To a stirred solution of 3-bromophthalide (0.43g) in dry dimethyl formamide (5 ml) was added sodium clavulanate (0.5g) and the solution was lePt at room temperature for 2 hours. The solution was treated with ethyl acetate (20 ml), cyclohexane (10 ml) and water (30 ml) and shaken thoroughly. The non-aqueous layer was washed with water (20 ml), dried (Na2S04) and evaporated to yield a pale yellow gum. The two diastereomeric esters were separated using high pressure li~uid chromatography on a 40 cm x 10 mm column of silica gel (Merckosorb SI 60, 5JU) eluting with ethyl acetate at a flow rate of 3 ml/min.
The first phthalide ester (retention time 7.15 min) crystallised rom ethyl acetate as needles, mp 102, and had the following i.r.
: ' : .
, , . , , . ", ,, . . .. , , , ,. ; ' , ,' ,' . '.i ,"' , ; : : ' ' ' "', ' ' ' ' :
'. , , '. ' ' ' ' "'' ' . .' ' , .' ': , " ' ': . '' '.'' ' ' '~
~ ` 1076120 (nujol mull~ lactam C~0 1790 cm 1 ester C~0 1755 cm 1 n.m.r. (CD3COCD3):
3.14 (d,l,J=17.5Hz) 3.76 (dd,l,J,=17.5Hz, J2=2.511z), 4.25(d,2,J=7.5Hz), 5.0 (dt~l~Jl=7.5Hz~ J2=1.5Hz), 5.4 (s,l,J-1.5Hz), 5.82 (d,l,J=2.5Hz),
7.7 (s,l), 8.06 ~ (m,4); M.wt (mass spectrometry: 331.0696 corresponds to ;~
C16H13N07 (calc. 331.0692). The second diasterioisomer (retention time
C16H13N07 (calc. 331.0692). The second diasterioisomer (retention time
8.85 min) had the following i.r. (CH2C12solutlon) ~ lactam C=0 1800 cm 1, ester C=0 1780 cm 1; nmr (CDC13) 2,42 (broad S,l, exchangeable with D20), 3.12 (d,l,J=18 ~ llz), 3.60 (dd,l,Jl=18 llz, J2e 2.5Hz), 4.30 (d,2,J=7.5Hz), 5.0 (dt,l,J1~7.5 Hz, J2=1.5 Hz), 5.12 (d,l,J=1.5Hz), 5.76 (d,l-,J=2.5 llz), 10 7.52 (S,l), 7.85 0 (m,4).
~ ~> /C~12~
o '. - ~ .
O=CO~~ >-- ,'' r ~ C02Na > fU2U ~ ~ ~
O--CO ~\ /=o . ~ `
\.----/ ' `' ,~` `.
:
_ 54 -'', ' `
1076~0 EXi~LE 35 PREPARATION O~ NONYL CLAVULA~IATE
.. . .
Sodium clavulanate (44 mg) in dry dimethyl~ormamide (2 ml) was treated with nonyl iodide (76 mg) and left at room temperature for 2 hours.
The solution was evaporated and the residue fractionated on silica gel, eluting with ethyl acetate-hexane (2:1) to give the product as an oil, i.r. ;
(film) 1800, 1745, 1690 cm 1. M.wt. (mass spectrometry) = 325.1890 which corresponds to C17H27NO5. (calc. 325.1889).
EXAMPLE 36 ;
PREPARATION OF CLAVULANIC ACID
' ' .:'. ~ .:
Benzyl clavulanate (100 mgs) in ethanGl (5 ml) was hydrogenated over 10% PdjC (30 mgs) for 45 minutes at ambient temperature and atmospheric ;
pressure. The catalyst was filtered, washed with ethanol and the combined -trates were evaporated in vacuo to give clavulanic acid as an unstable, -, . ,:
viscous oil (58 mgs). N.m.r. (C5D5N): 3.05(d,1,J=18Hz), 3.60(dd,1,Jl=18~z, Jz= 2.5Hz), 4.75(d,2,~=7.5Hz3, 5.58(t,1,Js7.5Hz), 5.66 (S,l), 6.0 5 (d,1,J=
~`~ 2.5Hz)~
:: ~ : ..
~PREPARATION OF METHYL CLA W LANATE
~ Clavulan1c 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 chromatograplly on silica gel, eluting ~; with ethyl acetate. The fractions containing methyl clavulanate were combir.ed and evaporated to give a clear oil (104 mgs).
PREPARATION OF METHYL CLAVULANATE
Clavulanic acid (200 mgs) in acetonitrile (5 ml) was cooled and~stirred at O . Methanol (0.5 ml) and then dicyclohexyldicarbodiimide (206 mg.) were added and the reaction mixture was stirred at room temperature ~`~
:
:
- 55 - ~
.
. . .
, - . . . . . .
1~)76~
~ . .
overnight. The suspcnsion was filtered alld the Eiltrate evaporated in vacuo to give crude methyl clavulanate, The crude product was purified l~y chroma- ;-tography on silica gel, eluting with ethyl acetate, to give a clear oil - i, (140 mg).
EXA~?LE 39 PREPAR~TION OF PHENYL CLAVULANATE
Clavulanic acid (100 mg) in ace-tonitrile (5 ml3 was cooled -~
and stirred at 0. To the solution was added phenol (0.94gj and dicyclo-:. :
hexyldicarbodiimide(100 mg) and the reaction mixture was stirred at room temperature overnight. The suspension was filtered and the filtrate evaporated.
The residue was fractionated on silica gel, eluting with ethyl acetate-hexane 1) to give phenyl clavulanate (70 mg). I.r (film) 1800, 1770, 1690 cm W.m.r. (CDC13) 2.18 (broad s,l), 3.06 (dd, l,J317Hz,J2=0.9Hz), 3.54 (dd,l,Jl=1711z,J2=2.6Hz), 4.29 (d,2,J=7.5Hz), 5.1(dt,1jJl=7.51lz,J2=1.51lz) 5.29 (d,1,J-1.511z), 5.76 (dd,l,Jl=2.6Hz,J2=0.9Hz), 7.35 ~; (m,5). ~I.wt. (mass spectro~etry) = 275.0777 which corresponds to C14H13N05 (calc. 275.0794).
EXAM~LE 40 .: :... ' . .
PREPARATION OF 2,2,2-trichloroethyl clavulanate . . ~ , .
Sodium clavulanate (221 mgs) was suspended in dry tetro-~20 hydrofuran (5 mls) and stirred at 0. Trichloroethylchloroformate (211 mg) !
in dry tetrohydrofuran (1 ml) was added to the above suspension over 20 minutes. The mixture was allowed to reach room temperature and stirred overnight. The suspension was ~iltered and the filtrate evaporated in vacuo.
The residue was chromatographed on silica gel eluting with ethyl acetate -hexane (2:1) to g:ive thc requ:Lred product as an oil. i.r. (fi:Lm) 1800, 1760, 1690 cm 1. n r...~ (CDC13) 1.56 (broad S,l), 3.07 (dd,l,Jl=17.5Hz,J2=0.7Hz), 3.56 (dd,l,J1dl7.5Hz,J2=2.5Hz), 4.24 ~d,2,J=7.5Hz), 4.69 (d,l,J=12Hz), 4.92 (d,l,J-12Hz), 5.02 (dt,l,Jl=7.5Hz, J2=1.3Hz), 5.19 (d,:L,J=1.311z), 5.73 ~ (dd,l,Jl=2.5Hz, J2=0.7Hz). ~I.wt. (mass spectrometry) = 328.9621 which corresponds to C1oHloN05C13 (calculated 328.9625).
- -., , ' - `
.
'~ ' .
1~76~L20 ` ' i ,' :' :
.
.,.: :,:
~ O ~ C112OII _ C11 O~
N ~ > ~ > ~ 2 ;~
CO Na D \ ~CH2OH
.N
.
~10 CO2R
:: , , ;, ~ ,:
~ PREPARATION OF SODIU~I CLAYUL~NATE
; ~ ~ Ben~yl clavulanate (840 mgs~ in ethanol (30 ml) and water (5~ml) was hydrogenated over 10% Pd/C (267 mgs) and sodium bicarbonate (244 mgs) for 25 minutes at room temperature and atmospheric pressure.
.: .
The catalyst was filtered, washed with water and ethanol and the combined ~ :
~- filtrates were evaporated in vacuo. The product crystallised from a water~
~20 acetone mixture as microneedles (565 mgs). Recrystallisation from water-.:: . ...
acetone gave needles which, after drying over P2O5 in vacuo for 24 hours gaVe the following analysis:
C 41.01, 40.86; H 3.77, 3.64; N 5.68, 5.51; i.r.(KBr disc) 1785, 1700, 1620 cm ; Nmr (D2O) 3.06 (d,1,3=18.5Hz), 3.57 (dd,l,J1=18.5Hz, J2~2,5Hz), `
4.15 (d,2,J=8Hz), 5.3 (~IOD), 4.9(m), 5.71 (d,l,J=2.5Hz). -ANTIBACTERIAL SYNERGISM BETWEEN AMPICILLIN AND CLAW LANIC ACID SODIUM SALT
The minimum inhibitory concentration (M.I.C. values) of ampicillin, elavulanie acid sodium salt and ampicillin in the presence of 1 jug/ml. clavulanic acid sodium salt were determined for a range of ~-lactamase ~;
producing bacteria. The organisms were inoeulated into Oxoid sensitivity - 57 - :
.
1076~20 ::
test broth located in small wells in a pl,astic tray and contain~ng separate concentrati,on gradients of ampicillin, clavulan,ic acid sodium salt or : ampicillin plus l~ug/ml. clavulanic acld sodium salt (microtitre method).
The final dilution of the overnight broth inoculum was 0.5 x 10 2, The tray was incubated at 37C overnight and a record made next morning of the end points of bacterial growth. The ~.I.C. values in jug/ml. are recorded in Table 5 which reveals that the synergist at the low concentration of 1 ~g/ml.
markedly enhances the antibacterial activity of ampicillin against certain eram ~ ve and gram - ve bacteria. The mechanism of this synergism is likely 10 to involve inhibition of ampicillin destroying ~-lactamase en~ymes 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 1~ ampicilIin.
: ' 5~ .
, : ' , . ' ~ 20 ;~ .
: . ' .
..
1: ' - 5~ _ .- , . .- . .
', ' ' , ~: :
76i~2~
: TABLE 5 : ~ : . .: . .
ANTIBACTERIAL SYNLRG~SM BETl~EEN ~E'ICILLIN AND
:- : ...
: ; CLAW LANIC AC~D SODIUM SALT
:' . :~ , :
~inimum Inhibitory Concentrations/ug/ml _ Ampicillin in Bacterial strain Clavulanicpresence of acid~mpicillin1,ugtml clavu-.sodium lanic acid salt sodium salt ::.
.: . . .. '~
~:Escherichia coli .
~: 10NCTC 10481 31 1.8 ~0.4 Escheriohia coli . . . :
B 11 : 62 > 500 125 : Klebsiella . - .
aerogenes A 31 125 ~ 0.4 - :
~: Klebs_ella sp 62 31 125 ~ 0.4 . . :
Enterobacter .
cloaca 62 250 62 : - :
: Serratia .
marcescens 125 ~ 500 62 : Staphylococcus aureus(Russell) 15 500 < 0.4 . :~ ;
:Staphylococcusl . . ~ .
20: ~ aureus 62 250 7.5 :
; ' : ~
: . , -a methicillln resistant strain ~ :
: .
. ~ .
. ANTIBACTERIAL SYNERGYSM BETWEEN CEPHALORIDINE AND CLAVULANIC ACID SODIUM SALT
~- The minimum inhibitory concentrationfi o~ cephaloridine, clavulanic acid sodium salt and cephaloridine in the presence of 5~ug/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 pr~ducing strain of ~ : :
coccus aureus (Russell).
- 59 - :
, , " , : . , .:: : . .
. , . : : ,. . . . ., : .: , : . ,, . : . . : : .. :
~ f ~
1076~LZ0 . , .
:..' , ANTIBACTERIAL SYNERGISM BETWEEN CEP1LALORIDIN~
~ AND CLAVULANIC ACID SODIUM SALT
:' ~ . '' - ' '-- . ' :' Minimum Inhibitory Concentrations ,ug/ml.
Bacterial Strain Clavulanic Cephaloridine in ; acid presence of sodium Cephaloridine 5lug/ml clavulanic salt acid sodium salt _ .. . :
Proteus mirabilis 889 ~500* 62 7.5 Staphylococcus1 aureus(Russell 15 3.1 ~' 0.03 Staphylococcus aureus 62 15 - 3.7 . .. _, - ................................ . .
a methicillin resistant strain *
Tailing Point ~s ~ Same value obtained when synergist added at g/ml. instead of 5Jug/ml.
, ~ ' :
~: , ~`~ 20 ANTIBACTERIA SYNERGISM BETWEEN CL~VULANIC ACID SODI~M SALT AND
VARIOUS PENICILLINS
The results presénted in Table 7 were obtained by tlle method described ` ~ in Example 42.
.
~ , , - :
:
. ' ' ' ~ , . . .
1076~Z0 : ~ ~
'TABLE 7 ANTIBACTERIAL'SYNERGISM BE'rWEEN CLAVULANIC ACID
SODIUM SALT AND VARIOUS PENICILLINS AGAINST '~
STRAINS O~ KLEBSIELLA ARROGENES
': -' :
.
~ .: .
, Amox cillin Carbenicillin* Benz 1 enicillin Y Y P
Strain Alonql +51Ug/ml I synergist Alonel Syyergist . I I
' A 500 1 0.97 500 1 7.8 250 1 7.8 .:
,~ ~ ' E 70 500 1 3.9 500 15 . 500 1 15.6 :
10 : 62 ' 250 1 15.6 125 1 7.8 250 1 15.6 ' ~ ' . :
*Similar results observed when carbenicillin replaced : by carbenicillin phenyl ~t~-ester or ticarcillin.
~, :: '''' .
; EXAMPLE ~5 ', ~: ANTIBACTERIAL SYNERGISM BETWERN ~MPICILLIN AND ESTERS OF Cl.AVULANIC ACID ,: ~
a .:
~' ~ The results presented in Table 8 were obtained by the method described in ~: ~ Example 42. '.
~ . .
.
~: ; ANTIBACTERIAL SYNERGISM BETWEEN AMPICILLIN AND
,.
~, , ESTRRS OF CLAVULANIC ACID AGAINST STRAINS OF
:~ : ' KLRBSIRLLA AEROGRNES
. .
Ampicillin + Ampicillin +
.Ampicillin 5 ~g/ml of ~enzyl Ester . '' StrainAlone of '. oE
: : clavulanic acld cla w lanic acid ~ . . . _ _ , :
A 500 1.9 1.9 ' . :
E 70 500 3.9 3,9 :. :
62 _.. _. 3.9 3.9 ~ :
.
Neither clavulanic acid methyl ester nor clavulan`'i.c ac.ld benzyl ester inhibited .' ~he growtll of the tcst organlsllls at'a concelltr;lt:Lon of 100~lg/ml.' .'.
, - 6l -... . .
: . .
,, .
,, . . .. , . , , , ., , . :
' . , .. ' . . ~' :: ':
7~;i20 .. , :
EXAMrLE 46 ~:'.
; ANTIBACTERIAL ACTIVITY OF CLA W LANXC'ACID'ESTER
Using the method of'Example 30 but using a dilution of 1/100 of overnight broth, the MIC values in Table 9 were obtained for certain ~:
esters of clavu.~ nic acid again.st a number of organisms:
ANTIBACTERIAL ACTIVITY OF CLAVULANIC ACID ESTERS
.
MIC*
MIC of Ester of Clavulanic Acid of ~10Organism ben yl Nonyl Pivaloyloxy- ester acid sodium : Bacillus .
s~btiIis A 25031 62 125 62 S~ph.aureus Oxford . 6231. 31 31 15 ~' ~: Staph.aureus12531 62 15 - 15 Russell . :. "~:
Escherichia coli I0418 125~ 500 125 125 125 . , *
The MIC of clavulanic acid sodium salt is included for comparison; the high MIC values (if compared to those of Example 30) are due to the heavy inocula used.
EXTRACTION OF CLAVULANIC ACID USING LIOUID ION EXCI~NGE RESIN
Culture filtratc (200 ml, obtained in a similar manner to Example 3 lut us:Lng a me(l.Lum containing 0.1% v/v'l<ll2P04 instead of 0.01% FeS04.7U20) was extracted with ~mberlite LA2 (C1 form, 15% v/v in methylisobutyl ketone, 66 ml) for 30 minutes at 5C.
The phases were separated by centrifugation (1660 g, 20 minutes). The solvent phase (6.0 ml) was recovered by pipette and divided into four equal portlons. Each portion was extracted by stirring.at 5C for 20 m:Lnutes _ 62 -.. ~ .. . .
, ' , ~ ' .:', ' i 1 076~20 . . .
with 1/4 volume (3.75 ml) a~ueous extractant as indicated :in the table below. ~
~ . .
The resulting mixture was cen~tri~uged (1660 g, 15 minutes). 3.6 ml. aqueous phase was recovered from each extraction : ' ' ; .
Vo.lume Clavulanic acid Sample 1` concentration Clavulanic (m ~ (~g ml~l acid (mg) -clarified brew 200 128 25.4 -extracted brew 200 15 3.0 M NaCl extract 3.6 305 i.l ~
2M NaCl extract 3.6 598 2.5 ~ -M NaNO3 extract 3.6 638 2.3 2M NaNO3 extract 3.6 ~ 758 2.73 The resolt obtained with 2M NaNO3 represents a recovery of 43% from clarified brew.
Amberlite LA2 is obtainable from Rohm and Haas (UK) Ltd. Croydon.
EXTRACTION OF CLA~ULANIC ACID USING LIQUID ION EXCHANGE RESIN
Clarified brew (47 litres, obtained as in Example 12) was extracted with Amberllte LA2 (acetate form, 15% v/v in methylisobutyl ketone, 12.5 1itres) by stirring for 1 hours at 17C. AEter adding Dctan-l-ol (500 ml) the phases were separated in a continuous flow centrifuge yielding
~ ~> /C~12~
o '. - ~ .
O=CO~~ >-- ,'' r ~ C02Na > fU2U ~ ~ ~
O--CO ~\ /=o . ~ `
\.----/ ' `' ,~` `.
:
_ 54 -'', ' `
1076~0 EXi~LE 35 PREPARATION O~ NONYL CLAVULA~IATE
.. . .
Sodium clavulanate (44 mg) in dry dimethyl~ormamide (2 ml) was treated with nonyl iodide (76 mg) and left at room temperature for 2 hours.
The solution was evaporated and the residue fractionated on silica gel, eluting with ethyl acetate-hexane (2:1) to give the product as an oil, i.r. ;
(film) 1800, 1745, 1690 cm 1. M.wt. (mass spectrometry) = 325.1890 which corresponds to C17H27NO5. (calc. 325.1889).
EXAMPLE 36 ;
PREPARATION OF CLAVULANIC ACID
' ' .:'. ~ .:
Benzyl clavulanate (100 mgs) in ethanGl (5 ml) was hydrogenated over 10% PdjC (30 mgs) for 45 minutes at ambient temperature and atmospheric ;
pressure. The catalyst was filtered, washed with ethanol and the combined -trates were evaporated in vacuo to give clavulanic acid as an unstable, -, . ,:
viscous oil (58 mgs). N.m.r. (C5D5N): 3.05(d,1,J=18Hz), 3.60(dd,1,Jl=18~z, Jz= 2.5Hz), 4.75(d,2,~=7.5Hz3, 5.58(t,1,Js7.5Hz), 5.66 (S,l), 6.0 5 (d,1,J=
~`~ 2.5Hz)~
:: ~ : ..
~PREPARATION OF METHYL CLA W LANATE
~ Clavulan1c 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 chromatograplly on silica gel, eluting ~; with ethyl acetate. The fractions containing methyl clavulanate were combir.ed and evaporated to give a clear oil (104 mgs).
PREPARATION OF METHYL CLAVULANATE
Clavulanic acid (200 mgs) in acetonitrile (5 ml) was cooled and~stirred at O . Methanol (0.5 ml) and then dicyclohexyldicarbodiimide (206 mg.) were added and the reaction mixture was stirred at room temperature ~`~
:
:
- 55 - ~
.
. . .
, - . . . . . .
1~)76~
~ . .
overnight. The suspcnsion was filtered alld the Eiltrate evaporated in vacuo to give crude methyl clavulanate, The crude product was purified l~y chroma- ;-tography on silica gel, eluting with ethyl acetate, to give a clear oil - i, (140 mg).
EXA~?LE 39 PREPAR~TION OF PHENYL CLAVULANATE
Clavulanic acid (100 mg) in ace-tonitrile (5 ml3 was cooled -~
and stirred at 0. To the solution was added phenol (0.94gj and dicyclo-:. :
hexyldicarbodiimide(100 mg) and the reaction mixture was stirred at room temperature overnight. The suspension was filtered and the filtrate evaporated.
The residue was fractionated on silica gel, eluting with ethyl acetate-hexane 1) to give phenyl clavulanate (70 mg). I.r (film) 1800, 1770, 1690 cm W.m.r. (CDC13) 2.18 (broad s,l), 3.06 (dd, l,J317Hz,J2=0.9Hz), 3.54 (dd,l,Jl=1711z,J2=2.6Hz), 4.29 (d,2,J=7.5Hz), 5.1(dt,1jJl=7.51lz,J2=1.51lz) 5.29 (d,1,J-1.511z), 5.76 (dd,l,Jl=2.6Hz,J2=0.9Hz), 7.35 ~; (m,5). ~I.wt. (mass spectro~etry) = 275.0777 which corresponds to C14H13N05 (calc. 275.0794).
EXAM~LE 40 .: :... ' . .
PREPARATION OF 2,2,2-trichloroethyl clavulanate . . ~ , .
Sodium clavulanate (221 mgs) was suspended in dry tetro-~20 hydrofuran (5 mls) and stirred at 0. Trichloroethylchloroformate (211 mg) !
in dry tetrohydrofuran (1 ml) was added to the above suspension over 20 minutes. The mixture was allowed to reach room temperature and stirred overnight. The suspension was ~iltered and the filtrate evaporated in vacuo.
The residue was chromatographed on silica gel eluting with ethyl acetate -hexane (2:1) to g:ive thc requ:Lred product as an oil. i.r. (fi:Lm) 1800, 1760, 1690 cm 1. n r...~ (CDC13) 1.56 (broad S,l), 3.07 (dd,l,Jl=17.5Hz,J2=0.7Hz), 3.56 (dd,l,J1dl7.5Hz,J2=2.5Hz), 4.24 ~d,2,J=7.5Hz), 4.69 (d,l,J=12Hz), 4.92 (d,l,J-12Hz), 5.02 (dt,l,Jl=7.5Hz, J2=1.3Hz), 5.19 (d,:L,J=1.311z), 5.73 ~ (dd,l,Jl=2.5Hz, J2=0.7Hz). ~I.wt. (mass spectrometry) = 328.9621 which corresponds to C1oHloN05C13 (calculated 328.9625).
- -., , ' - `
.
'~ ' .
1~76~L20 ` ' i ,' :' :
.
.,.: :,:
~ O ~ C112OII _ C11 O~
N ~ > ~ > ~ 2 ;~
CO Na D \ ~CH2OH
.N
.
~10 CO2R
:: , , ;, ~ ,:
~ PREPARATION OF SODIU~I CLAYUL~NATE
; ~ ~ Ben~yl clavulanate (840 mgs~ in ethanol (30 ml) and water (5~ml) was hydrogenated over 10% Pd/C (267 mgs) and sodium bicarbonate (244 mgs) for 25 minutes at room temperature and atmospheric pressure.
.: .
The catalyst was filtered, washed with water and ethanol and the combined ~ :
~- filtrates were evaporated in vacuo. The product crystallised from a water~
~20 acetone mixture as microneedles (565 mgs). Recrystallisation from water-.:: . ...
acetone gave needles which, after drying over P2O5 in vacuo for 24 hours gaVe the following analysis:
C 41.01, 40.86; H 3.77, 3.64; N 5.68, 5.51; i.r.(KBr disc) 1785, 1700, 1620 cm ; Nmr (D2O) 3.06 (d,1,3=18.5Hz), 3.57 (dd,l,J1=18.5Hz, J2~2,5Hz), `
4.15 (d,2,J=8Hz), 5.3 (~IOD), 4.9(m), 5.71 (d,l,J=2.5Hz). -ANTIBACTERIAL SYNERGISM BETWEEN AMPICILLIN AND CLAW LANIC ACID SODIUM SALT
The minimum inhibitory concentration (M.I.C. values) of ampicillin, elavulanie acid sodium salt and ampicillin in the presence of 1 jug/ml. clavulanic acid sodium salt were determined for a range of ~-lactamase ~;
producing bacteria. The organisms were inoeulated into Oxoid sensitivity - 57 - :
.
1076~20 ::
test broth located in small wells in a pl,astic tray and contain~ng separate concentrati,on gradients of ampicillin, clavulan,ic acid sodium salt or : ampicillin plus l~ug/ml. clavulanic acld sodium salt (microtitre method).
The final dilution of the overnight broth inoculum was 0.5 x 10 2, The tray was incubated at 37C overnight and a record made next morning of the end points of bacterial growth. The ~.I.C. values in jug/ml. are recorded in Table 5 which reveals that the synergist at the low concentration of 1 ~g/ml.
markedly enhances the antibacterial activity of ampicillin against certain eram ~ ve and gram - ve bacteria. The mechanism of this synergism is likely 10 to involve inhibition of ampicillin destroying ~-lactamase en~ymes 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 1~ ampicilIin.
: ' 5~ .
, : ' , . ' ~ 20 ;~ .
: . ' .
..
1: ' - 5~ _ .- , . .- . .
', ' ' , ~: :
76i~2~
: TABLE 5 : ~ : . .: . .
ANTIBACTERIAL SYNLRG~SM BETl~EEN ~E'ICILLIN AND
:- : ...
: ; CLAW LANIC AC~D SODIUM SALT
:' . :~ , :
~inimum Inhibitory Concentrations/ug/ml _ Ampicillin in Bacterial strain Clavulanicpresence of acid~mpicillin1,ugtml clavu-.sodium lanic acid salt sodium salt ::.
.: . . .. '~
~:Escherichia coli .
~: 10NCTC 10481 31 1.8 ~0.4 Escheriohia coli . . . :
B 11 : 62 > 500 125 : Klebsiella . - .
aerogenes A 31 125 ~ 0.4 - :
~: Klebs_ella sp 62 31 125 ~ 0.4 . . :
Enterobacter .
cloaca 62 250 62 : - :
: Serratia .
marcescens 125 ~ 500 62 : Staphylococcus aureus(Russell) 15 500 < 0.4 . :~ ;
:Staphylococcusl . . ~ .
20: ~ aureus 62 250 7.5 :
; ' : ~
: . , -a methicillln resistant strain ~ :
: .
. ~ .
. ANTIBACTERIAL SYNERGYSM BETWEEN CEPHALORIDINE AND CLAVULANIC ACID SODIUM SALT
~- The minimum inhibitory concentrationfi o~ cephaloridine, clavulanic acid sodium salt and cephaloridine in the presence of 5~ug/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 pr~ducing strain of ~ : :
coccus aureus (Russell).
- 59 - :
, , " , : . , .:: : . .
. , . : : ,. . . . ., : .: , : . ,, . : . . : : .. :
~ f ~
1076~LZ0 . , .
:..' , ANTIBACTERIAL SYNERGISM BETWEEN CEP1LALORIDIN~
~ AND CLAVULANIC ACID SODIUM SALT
:' ~ . '' - ' '-- . ' :' Minimum Inhibitory Concentrations ,ug/ml.
Bacterial Strain Clavulanic Cephaloridine in ; acid presence of sodium Cephaloridine 5lug/ml clavulanic salt acid sodium salt _ .. . :
Proteus mirabilis 889 ~500* 62 7.5 Staphylococcus1 aureus(Russell 15 3.1 ~' 0.03 Staphylococcus aureus 62 15 - 3.7 . .. _, - ................................ . .
a methicillin resistant strain *
Tailing Point ~s ~ Same value obtained when synergist added at g/ml. instead of 5Jug/ml.
, ~ ' :
~: , ~`~ 20 ANTIBACTERIA SYNERGISM BETWEEN CL~VULANIC ACID SODI~M SALT AND
VARIOUS PENICILLINS
The results presénted in Table 7 were obtained by tlle method described ` ~ in Example 42.
.
~ , , - :
:
. ' ' ' ~ , . . .
1076~Z0 : ~ ~
'TABLE 7 ANTIBACTERIAL'SYNERGISM BE'rWEEN CLAVULANIC ACID
SODIUM SALT AND VARIOUS PENICILLINS AGAINST '~
STRAINS O~ KLEBSIELLA ARROGENES
': -' :
.
~ .: .
, Amox cillin Carbenicillin* Benz 1 enicillin Y Y P
Strain Alonql +51Ug/ml I synergist Alonel Syyergist . I I
' A 500 1 0.97 500 1 7.8 250 1 7.8 .:
,~ ~ ' E 70 500 1 3.9 500 15 . 500 1 15.6 :
10 : 62 ' 250 1 15.6 125 1 7.8 250 1 15.6 ' ~ ' . :
*Similar results observed when carbenicillin replaced : by carbenicillin phenyl ~t~-ester or ticarcillin.
~, :: '''' .
; EXAMPLE ~5 ', ~: ANTIBACTERIAL SYNERGISM BETWERN ~MPICILLIN AND ESTERS OF Cl.AVULANIC ACID ,: ~
a .:
~' ~ The results presented in Table 8 were obtained by the method described in ~: ~ Example 42. '.
~ . .
.
~: ; ANTIBACTERIAL SYNERGISM BETWEEN AMPICILLIN AND
,.
~, , ESTRRS OF CLAVULANIC ACID AGAINST STRAINS OF
:~ : ' KLRBSIRLLA AEROGRNES
. .
Ampicillin + Ampicillin +
.Ampicillin 5 ~g/ml of ~enzyl Ester . '' StrainAlone of '. oE
: : clavulanic acld cla w lanic acid ~ . . . _ _ , :
A 500 1.9 1.9 ' . :
E 70 500 3.9 3,9 :. :
62 _.. _. 3.9 3.9 ~ :
.
Neither clavulanic acid methyl ester nor clavulan`'i.c ac.ld benzyl ester inhibited .' ~he growtll of the tcst organlsllls at'a concelltr;lt:Lon of 100~lg/ml.' .'.
, - 6l -... . .
: . .
,, .
,, . . .. , . , , , ., , . :
' . , .. ' . . ~' :: ':
7~;i20 .. , :
EXAMrLE 46 ~:'.
; ANTIBACTERIAL ACTIVITY OF CLA W LANXC'ACID'ESTER
Using the method of'Example 30 but using a dilution of 1/100 of overnight broth, the MIC values in Table 9 were obtained for certain ~:
esters of clavu.~ nic acid again.st a number of organisms:
ANTIBACTERIAL ACTIVITY OF CLAVULANIC ACID ESTERS
.
MIC*
MIC of Ester of Clavulanic Acid of ~10Organism ben yl Nonyl Pivaloyloxy- ester acid sodium : Bacillus .
s~btiIis A 25031 62 125 62 S~ph.aureus Oxford . 6231. 31 31 15 ~' ~: Staph.aureus12531 62 15 - 15 Russell . :. "~:
Escherichia coli I0418 125~ 500 125 125 125 . , *
The MIC of clavulanic acid sodium salt is included for comparison; the high MIC values (if compared to those of Example 30) are due to the heavy inocula used.
EXTRACTION OF CLAVULANIC ACID USING LIOUID ION EXCI~NGE RESIN
Culture filtratc (200 ml, obtained in a similar manner to Example 3 lut us:Lng a me(l.Lum containing 0.1% v/v'l<ll2P04 instead of 0.01% FeS04.7U20) was extracted with ~mberlite LA2 (C1 form, 15% v/v in methylisobutyl ketone, 66 ml) for 30 minutes at 5C.
The phases were separated by centrifugation (1660 g, 20 minutes). The solvent phase (6.0 ml) was recovered by pipette and divided into four equal portlons. Each portion was extracted by stirring.at 5C for 20 m:Lnutes _ 62 -.. ~ .. . .
, ' , ~ ' .:', ' i 1 076~20 . . .
with 1/4 volume (3.75 ml) a~ueous extractant as indicated :in the table below. ~
~ . .
The resulting mixture was cen~tri~uged (1660 g, 15 minutes). 3.6 ml. aqueous phase was recovered from each extraction : ' ' ; .
Vo.lume Clavulanic acid Sample 1` concentration Clavulanic (m ~ (~g ml~l acid (mg) -clarified brew 200 128 25.4 -extracted brew 200 15 3.0 M NaCl extract 3.6 305 i.l ~
2M NaCl extract 3.6 598 2.5 ~ -M NaNO3 extract 3.6 638 2.3 2M NaNO3 extract 3.6 ~ 758 2.73 The resolt obtained with 2M NaNO3 represents a recovery of 43% from clarified brew.
Amberlite LA2 is obtainable from Rohm and Haas (UK) Ltd. Croydon.
EXTRACTION OF CLA~ULANIC ACID USING LIQUID ION EXCHANGE RESIN
Clarified brew (47 litres, obtained as in Example 12) was extracted with Amberllte LA2 (acetate form, 15% v/v in methylisobutyl ketone, 12.5 1itres) by stirring for 1 hours at 17C. AEter adding Dctan-l-ol (500 ml) the phases were separated in a continuous flow centrifuge yielding
9.2 litres solvent phase, which was then stirred at 5C for ll~ hours with molar sodium n:itrate (2.3 litres). The mixture was se~Qrated by contimlous flow centrlfugation yieldlng 2.4 litres aqueous pllase (including water used for displacement purposes). Aqueous phase pH (initially 8.0) was ad~usted to 7.0 with concentrated hydrochloric acid.
; ,'.':~
, ,. , ,, , ~
.. . . .
~~ ~ 107~ 0 - -;~
~ Volume clavulanlc acld cla~ l.an:Lc : Sample (1)concentration acid (,~g ml~l) (mg) .,~
~' clarified brew 47 146 6862 ., . , extracted brew 47 19 893 -:
~ .
~ M NaN03 extract 2.41638 3931 , . . ., Extraction efficiency fro~n clarified bre~ to sodium nitrate extract is 57%. ~ :--~ 10 . :,, 1~ , ~1 ` ~ ' . ' :
~: ~ 20~
; : ' , ~ , :
.
., - - . . . . . .. . . .
,: .. ,, , , . : . . .
;: ., ' , , , . ', : :, : - , .. ,, : . .
; ,'.':~
, ,. , ,, , ~
.. . . .
~~ ~ 107~ 0 - -;~
~ Volume clavulanlc acld cla~ l.an:Lc : Sample (1)concentration acid (,~g ml~l) (mg) .,~
~' clarified brew 47 146 6862 ., . , extracted brew 47 19 893 -:
~ .
~ M NaN03 extract 2.41638 3931 , . . ., Extraction efficiency fro~n clarified bre~ to sodium nitrate extract is 57%. ~ :--~ 10 . :,, 1~ , ~1 ` ~ ' . ' :
~: ~ 20~
; : ' , ~ , :
.
., - - . . . . . .. . . .
,: .. ,, , , . : . . .
;: ., ' , , , . ', : :, : - , .. ,, : . .
Claims (33)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A process for the preparation of an ester of clavulanic acid of the formula wherein W is O or S and R is where A4 is H or methyl and A5 is methyl, t-butyl or phenyl;
where A6 is H or methoxyl;
a hydrocarbon group of 1-9 C atoms or such group substituted with halogen, lower alkoxy or hydroxyl; or where A7 is H, phenyl, tolyl, chlorophenyl, methoxyphenyl, or nitrophenyl and A8 is phenyl, tolyl, chlorophenyl, methoxyphenyl or nitrophenyl which comprises:
(a) when W is O, reacting clavulanic acid or salt thereof with a compound of formula Q-R where Q is a group readily displaceable by carboxylate ion and R is as defined, in an organic solvent and recovering the ester of clavulanic acid exclusive of benzyl clavulanate prepared from benzyl bromide and clavulanic acid sodium salt obtained by cultivation of Streptomyces clavuligerus;
(b) when W is o and R is alkyl or aralkyl, reacting clavulanic acid with a diazoalkane of formula R-N=N-R
wherein R is alkyl or aralkyl of not more than 16 carbon atoms in a solvent and recovering the corresponding ester;
(c) reacting clavulanic acid with ROH or RSH wherein R
is as defined in the presence of a carbodiimide or carbo-diimidazole condensing agent which removes water from the reaction mixture, in an inert organic solvent and recovering the corresponding ester;
(d) where W is O, by removal of CO2 from a compound of formula wherein R4 is an inert organic group within the definition of R, in an inert solvent, with heating where required, and recovering the corresponding ester, and (e) reacting a compound of the formula wherein R4 is an inert organic group within the definition of R with an excess of a compound ROH and RSH wherein R
is as previously defined in an inert solvent and recovering the corresponding ester.
where A6 is H or methoxyl;
a hydrocarbon group of 1-9 C atoms or such group substituted with halogen, lower alkoxy or hydroxyl; or where A7 is H, phenyl, tolyl, chlorophenyl, methoxyphenyl, or nitrophenyl and A8 is phenyl, tolyl, chlorophenyl, methoxyphenyl or nitrophenyl which comprises:
(a) when W is O, reacting clavulanic acid or salt thereof with a compound of formula Q-R where Q is a group readily displaceable by carboxylate ion and R is as defined, in an organic solvent and recovering the ester of clavulanic acid exclusive of benzyl clavulanate prepared from benzyl bromide and clavulanic acid sodium salt obtained by cultivation of Streptomyces clavuligerus;
(b) when W is o and R is alkyl or aralkyl, reacting clavulanic acid with a diazoalkane of formula R-N=N-R
wherein R is alkyl or aralkyl of not more than 16 carbon atoms in a solvent and recovering the corresponding ester;
(c) reacting clavulanic acid with ROH or RSH wherein R
is as defined in the presence of a carbodiimide or carbo-diimidazole condensing agent which removes water from the reaction mixture, in an inert organic solvent and recovering the corresponding ester;
(d) where W is O, by removal of CO2 from a compound of formula wherein R4 is an inert organic group within the definition of R, in an inert solvent, with heating where required, and recovering the corresponding ester, and (e) reacting a compound of the formula wherein R4 is an inert organic group within the definition of R with an excess of a compound ROH and RSH wherein R
is as previously defined in an inert solvent and recovering the corresponding ester.
2. An ester of clavulanic acid of the formula wherein W is O or S and R is where A4 is H or methyl and A5 is methyl, t-butyl or phenyl;
where A6 is H or methoxyl;
a hydrocarbon group of 1-9 C atoms or such group substituted with halogen, lower alkoxy or hydroxyl; or where A7 is H, phenyl, tolyl, chlorophenyl, methoxyphenyl or nitrophenyl and A8 is phenyl, tolyl, chlorophenyl, methoxyphenyl or nitrophenyl;
when prepared by the process of claim 1 or an obvious chemical equivalent, exclusive of benzyl clavulanate obtained by reaction of benzyl bromide and sodium clavulanate obtained from cultivation of Streptomyces clavuligerus.
where A6 is H or methoxyl;
a hydrocarbon group of 1-9 C atoms or such group substituted with halogen, lower alkoxy or hydroxyl; or where A7 is H, phenyl, tolyl, chlorophenyl, methoxyphenyl or nitrophenyl and A8 is phenyl, tolyl, chlorophenyl, methoxyphenyl or nitrophenyl;
when prepared by the process of claim 1 or an obvious chemical equivalent, exclusive of benzyl clavulanate obtained by reaction of benzyl bromide and sodium clavulanate obtained from cultivation of Streptomyces clavuligerus.
3. A process for the preparation of an ester of clavulanic acid of the formula where R is where A4 is H or methyl and A5 is methyl, t-butyl or phenyl;
where A6 is H or methoxyl;
a hydrocarbon group of 1-9 C atoms or such group substituted with halogen, lower alkoxy or hydroxyl; or where A7 is H, phenyl, tolyl, chlorophenyl, methoxyphenyl or nitrophenyl and A8 is phenyl, tolyl, chlorophenyl, methoxyphenyl or nitrophenyl;
which comprises reacting clavulanic acid or a salt of clavu-lanic acid with a compound of formula Q-R where Q is a group readily displaceable by carboxylate ion and R is as defined, in an organic solvent of relatively high dielectric constant, and recovering the ester of clavulanic acid, exclusive of benzyl clavulanate obtained by reaction of benzyl bromide and sodium clavulanate obtained by cultivation of Streptomyces clavuligerus.
where A6 is H or methoxyl;
a hydrocarbon group of 1-9 C atoms or such group substituted with halogen, lower alkoxy or hydroxyl; or where A7 is H, phenyl, tolyl, chlorophenyl, methoxyphenyl or nitrophenyl and A8 is phenyl, tolyl, chlorophenyl, methoxyphenyl or nitrophenyl;
which comprises reacting clavulanic acid or a salt of clavu-lanic acid with a compound of formula Q-R where Q is a group readily displaceable by carboxylate ion and R is as defined, in an organic solvent of relatively high dielectric constant, and recovering the ester of clavulanic acid, exclusive of benzyl clavulanate obtained by reaction of benzyl bromide and sodium clavulanate obtained by cultivation of Streptomyces clavuligerus.
4. The process of claim 3 wherein the salt of clavulanic acid is the sodium or potassium salt and Q is one of chlorine, bromine, iodine, a sulfonic acid ester group of O.sO2CH3 and O.sO2C6H4CH3, or an active ester group of O.CO.CH3 or O.CO.CF3.
5. The process of claim 4 wherein the solvent is dimethylformamide acetone, dioxane or tetrahydrofuran.
6. A process for the preparation of an ester of clavu-lanic acid of the formula wherein R is alkyl or aralkyl of not more than 9 carbon atoms which comprises reacting clavulanic acid with a diazoalkane of formula R-N=N-R where R is as defined, in a solvent and recovering the corresponding ester of clavulanic acid.
7. The process of claim 6 wherein the solvent is methanol, ethanol, tetrahydrofuran or dioxane.
8. The process for the preparation of an ester of clavulanic acid of the formula:
where W is O or S and R is where A4 is H or methyl and A5 is methyl, t-butyl or phenyl;
where A6 is H or methoxyl;
a hydrocarbon group of 1-9 C atoms or such group substituted with halogen, lower alkoxy or hydroxyl; or where A7 is H, phenyl, tolyl, chlorophenyl, methoxyphenyl or nitrophenyl and A8 is phenyl, tolyl, chlorophenyl, methoxyphenyl or nitrophenyl;
which comprises reacting clavulanic acid with an excess of ROH
or RSH wherein R is as defined, in the presence of a carbo-diimide or carbodiimidazole condensing agent which removes water from the reaction mixture, in an inert organic solvent and recovering the corresponding ester of clavulanic acid.
where W is O or S and R is where A4 is H or methyl and A5 is methyl, t-butyl or phenyl;
where A6 is H or methoxyl;
a hydrocarbon group of 1-9 C atoms or such group substituted with halogen, lower alkoxy or hydroxyl; or where A7 is H, phenyl, tolyl, chlorophenyl, methoxyphenyl or nitrophenyl and A8 is phenyl, tolyl, chlorophenyl, methoxyphenyl or nitrophenyl;
which comprises reacting clavulanic acid with an excess of ROH
or RSH wherein R is as defined, in the presence of a carbo-diimide or carbodiimidazole condensing agent which removes water from the reaction mixture, in an inert organic solvent and recovering the corresponding ester of clavulanic acid.
9. A process for the preparation of an ester of clavu-lanic acid of the formula wherein R4 is alkyl, alkenyl, alkynyl, aryl or arylalkyl of not more than 9 carbon atoms, which comprises removal of CO2 from a compound of formula:
where R4 is as defined, in an inert solvent with heating where required, and recovering the ester of clavulanic acid.
where R4 is as defined, in an inert solvent with heating where required, and recovering the ester of clavulanic acid.
10. The process of claim 9 wherein the solvent is diethylether, tetrahydrofuran or dioxane.
11. A process for the preparation of an ester of clavulanic acid of the formula:
where W is O or S and R is where A4 is H or methyl and A5 is methyl, t-butyl or phenyl;
where A6 is H or methoxyl;
a hydrocarbon group of 1-9 C atoms or such group substituted with halogen, lower alkoxy or hydroxyl; or where A7 is H, phenyl, tolyl, chlorophenyl, methoxyphenyl or nitrophenyl and A8 is phenyl, tolyl, chlorophenyl, methoxyphenyl or nitrophenyl;
which comprises reacting a compound of formula wherein R4 is alkyl, alkenyl, alkynyl, aryl or arylalkyl of not more than 9 carbon atoms with an excess of a compound ROH or RSH where R is as defined in an inert solvent and recovering the ester of clavulanic acid.
where W is O or S and R is where A4 is H or methyl and A5 is methyl, t-butyl or phenyl;
where A6 is H or methoxyl;
a hydrocarbon group of 1-9 C atoms or such group substituted with halogen, lower alkoxy or hydroxyl; or where A7 is H, phenyl, tolyl, chlorophenyl, methoxyphenyl or nitrophenyl and A8 is phenyl, tolyl, chlorophenyl, methoxyphenyl or nitrophenyl;
which comprises reacting a compound of formula wherein R4 is alkyl, alkenyl, alkynyl, aryl or arylalkyl of not more than 9 carbon atoms with an excess of a compound ROH or RSH where R is as defined in an inert solvent and recovering the ester of clavulanic acid.
12. A process for the preparation of the methyl ester of clavulanic acid which comprises reacting sodium clavulanate with methyl iodide in dimethylformamide and recovering methyl clavulanate.
13. Methyl clavulanate when prepared by the process of claim 12 or an obvious chemical equivalent.
14. A process for the preparation of p-nitrobenzyl clavulanate which comprises reacting sodium clavulanate with p-nitrobenzyl bromide in dimethylformamide and recovering the p-nitrobenzyl clavulanate.
15. p-nitrobenzyl clavulanate when prepared by the process of claim 14 or an obvious chemical equivalent.
16. A process for the preparation of benzyl clavulanate which comprises reacting sodium clavulanate, exclusive of sodium clavulanate obtained by cultivation of Streptomyces clavuligerus.
with benzyl bromide in dimethylformamide and recovering the benzyl clavulanate.
with benzyl bromide in dimethylformamide and recovering the benzyl clavulanate.
17. Benzyl clavulanate when prepared by the process of claim 16 or an obvious chemical equivalent.
18. A process for the preparation of pivaloyloxymethyl clavulanate by reacting sodium clavulanate with bromomethyl pivalate in dimethylformamide and recovering the pivaloyloxymethyl clavulanate.
19. Pivaloyloxymethyl clavulanate when prepared by the process of claim 18 or an obvious chemical equivalent.
20. A process for the preparation of the diastereoisomers of phthalidyl clavulanate with comprises reacting sodium clavu-lanate with 3-bromophthalide in dimethylformamide and recovering the diastereoisomers of the phthalidyl clavulanate as separate compounds.
21. A The diastereolsomers of the phthalidyl clavulanate when prepared by the process of claim 20 or an obvious chemical equivalent.
22. A process for the preparation of nonyl clavulanate which comprises reacting sodium clavulanate with nonyl iodide in dimethylformamide and recovering the nonyl clavulanate.
23. Nonyl clavulanate when prepared by the process of claim 22 or an obvious chemical equivalent.
24. A process for the preparation of methyl clavulanate which comprises reacting clavulanic acid in ethanol with diazo-methane in ether and recovering the methyl clavulanate.
25. Methyl clavulanate when prepared by the process of claim 24 or an obvious chemical equivalent.
26. A process for the preparation of methyl clavulanate which comprises reacting clavulanic acid in acetonitrile with methanol if the presence of dicyclohexyldicarbodiimide and recovering the methyl clavulanate.
27. Methyl clavulanate when prepared by the process of claim 26 or an obvious chemical equivalent.
28. A process for the preparation of phenyl clavulanate which comprises reacting clavulanic acid in acetonitrile with phenol in the presence of dicyclohexyldicarbodiimide and recovering the phenyl clavulanate.
29. Phenyl clavulanate when prepared by the process of claim 28 or an obvious chemical equivalent.
30. A process for the preparation of 2,2,2,-trichloroethyl clavulanate which comprises reacting sodium clavulanate in tetra-hydrofuran with trichloroethylchloroformate and recovering the 2,2,2,-trichloroethyl clavulanate.
31. 2,2,2,-trichloroethyl clavulanate when prepared by the process of claim 30 or an obvious chemical equivalent.
32. A process for the preparation of benzyl clavulanate which comprises reacting lithium clavulanate with benzyl bromide in dimethylformamide and recovering the benzyl clavulanate.
33. Benzyl clavulanate when prepared by the process of claim 32 or an obvious chemical equivalent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA314,882A CA1076120A (en) | 1974-04-20 | 1978-10-30 | Clavulanic acid esters |
Applications Claiming Priority (6)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB17410/74A GB1508977A (en) | 1974-04-20 | 1974-04-20 | Beta-lactam antibiotic from streptomyces clavuligerus |
| GB2771574 | 1974-06-21 | ||
| GB4365174 | 1974-10-09 | ||
| GB5352574 | 1974-12-11 | ||
| CA224,970A CA1059050A (en) | 1974-04-20 | 1975-04-18 | Clavulanic acid from streptomyces clavuligerus |
| CA314,882A CA1076120A (en) | 1974-04-20 | 1978-10-30 | Clavulanic acid esters |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1076120A true CA1076120A (en) | 1980-04-22 |
Family
ID=27543273
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA314,882A Expired CA1076120A (en) | 1974-04-20 | 1978-10-30 | Clavulanic acid esters |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1076120A (en) |
-
1978
- 1978-10-30 CA CA314,882A patent/CA1076120A/en not_active Expired
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