Derivatives of clavulanic acid, their preparation and their use

Tetrazolyl derivatives of clavulanic acid are described, having the formula: ##STR1## wherein X is an optionally substituted tetrazolyl group attached via a nitrogen atom. These compounds are useful as antibiotics and .beta.-lactamase inhibitors.

This invention relates to derivatives of clavulanic acid and in particular 
to 9-N-tetrazolyldeoxyclavulanate derivatives. These are of use as 
antibiotics and as .beta.-lactamase inhibitors. 
Clavulanic acid and salts and esters thereof are described in U.K. Pat. No. 
1,508,977; clavulanic acid has the formula (I): 
##STR2## 
The present invention relates to compounds wherein the hydroxy group at C-9 
position is replaced by a tetrazole group. 
Accordingly the present invention provides a compound of formula (II) or a 
salt or ester thereof: 
##STR3## 
wherein X is an optionally substituted tetrazolyl group attached via a 
nitrogen atom. 
Suitably X represents a group of sub-formula (i): 
##STR4## 
wherein R is hydrogen; esterified or salified carboxy, optionally 
substituted C.sub.1-6 alkoxy, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, or aryl; 
azido, isocyano, cyano, nitro, bromo, chloro, or is a group of the 
sub-formula (a): 
EQU --(CO).sub.n --NR.sup.1 R.sup.2 (a) 
wherein n is zero or one; R.sup.1 is hydrogen or optionally substituted 
C.sub.1-6 alkyl, C.sub.1-6 alkanoyl, or arylcarbonyl; and R.sup.2 is 
hydrogen, C.sub.1-6 alkyl or C.sub.1-6 alkanoyl; or R.sup.1 and R.sup.2 
may be joined to form with the nitrogen atom to which they are attached, 
an optionally substituted 4, 5 or 6-membered ring. 
In one aspect suitably X represents a group of sub-formula (i): 
##STR5## 
wherein R is hydrogen; optionally substituted C.sub.1-6 alkoxy, C.sub.1-6 
alkyl, C.sub.2-6 alkenyl, or aryl; azido, isocyano, cyano, nitro, bromo, 
chloro, or is a group of the sub-formula (aa): 
EQU --NR.sup.1 R.sup.2 (aa) 
wherein R.sup.1 is hydrogen or optionally substituted C.sub.1-6 alkyl, 
C.sub.1-6 alkanoyl, or arylcarbonyl; and R.sup.2 is hydrogen, C.sub.1-6 
alkyl or C.sub.1-6 alkanoyl; or R.sup.1 and R.sup.2 may be joined to form 
with the nitrogen atom to which they are attached, an optionally 
substituted 4, 5 or 6-membered ring. 
The compounds of the formula (II) may be presented in the form of the 
carboxylic acid at the C-3 position. Alternatively the compounds of the 
formula (II) may be in the form of a pharmaceutically acceptable salt. 
Suitable pharmaceutically acceptable salts of the compounds of formula 
(II) include metal salts, for example aluminium, alkali metal salts such 
as sodium or potassium, alkaline earth metal salts such as calcium or 
magnesium and ammonium or substituted ammonium salts, for example those 
with lower alkylamines such as triethylamine, hydroxy-lower alkylamines 
such as 2-hydroxyethylamine, bis-(2-hydroxyethyl)-amine or 
tri-(2-hydroxyethyl)-amine, cycloalkylamines such as bicyclohexylamine, or 
with procaine, dibenzylamine, N,N-dibenzylethylenediamine, 1-ephenamine, 
N-ethylpiperidine, N-benzyl-.beta.-phenethylamine, dehydroabietylamine or 
N,N-bisdehydroabietylamine. 
Compounds of the formula (II) when in the form of nonpharmaceutically 
acceptable salts, for example the lithium salt or the silver salt are also 
of use for example as intermediates in preparing pharmaceutically 
acceptable salts. 
The compounds of the formula (II) alternatively may be provided as in vivo 
hydrolysable esters. Such esters are those which hydrolyse in the human 
body to produce the parent acid or salt thereof. Suitable in vivo 
hydrolysable esters include those of the sub-formulae (b) and (c): 
EQU --CO--O--CHR.sup.3 --O--CO--Rhu 4 (b) 
EQU --CO--O--R.sup.5 --NR.sup.6 R.sup.7 (c) 
wherein R.sup.3 is a hydrogen atom or a methyl or phenyl group; R.sup.4 is 
C.sub.1-6 alkyl, phenyl, phenyl (C.sub.1-3) alkyl C.sub.1-6 alkoxy, 
phenoxy, phenyl (C.sub.1-3) alkoxy; or R.sup.3 and R.sup.4 are joined to 
form a 1,2-diphenylene or 4,5-dimethoxy-1,2-diphenylene group: R.sup.5 is 
a divalent methylene or ethylene radical, R.sup.6 and R.sup.7 are 
independently methyl or ethyl groups. 
Favourably R.sup.3 is a hydrogen atom. 
When R.sup.3 is a hydrogen atom suitably R.sup.4 is selected from methyl, 
ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, phenyl, benzyl, methoxy, 
ethoxy, n-propyloxy and isopropyloxy. Preferably R.sup.4 is tert-butyl. 
Favourably R.sup.3 and R.sup.4 are joined so that the ester is a phthalidyl 
or 3,4-dimethoxyphthalidyl ester. 
Of these, the preferred esters are the acetoxymethyl, 
.alpha.-ethoxycarbonyloxyethyl, pivaloyloxymethyl and phthalidyl esters, 
of which the phthalidyl is favoured. 
The in vivo hydrolysable nature of the ester may be confirmed by 
administration to an animal such as a mouse or rat and determination of 
the presence of a compound of the formula (II) or salt thereof in the body 
fluids of the animal, for example the blood or urine. Alternatively 
hydrolysis in human blood or serum may be determined. 
Suitably group R may be optionally substituted by one or more groups of 
atoms selected from hydroxy, halo, aryl, carboxy, C.sub.1-6 alkanoyl, 
C.sub.1-6 alkanoyloxy, C.sub.1-6 alkoxy, C.sub.1-6 alkoxycarbonyl, aryl 
(C.sub.1-6) alkoxy, arylcarbonyl, C.sub.1-6 alkylthio, arylthio, amino, 
azido, C.sub.1-6 alkylamino or di(C.sub.1-6) alkylamino. 
Suitably R.sup.1 when it is not methyl may optionally be substituted by one 
or more groups selected from hydroxy, halo, carboxy, C.sub.1-6 alkanoyl, 
C.sub.1-6 alkanoyloxy, C.sub.1-6 alkoxy, C.sub.1-6 alkoxycarbonyl, 
C.sub.1-10 aralkoxy, arylcarbonyl, C.sub.1-6 alkylthio, arylthio, amino, 
C.sub.1-6 alkylamino or di(C.sub.1-6) alkylamino. 
Suitably R.sup.2 when it is not methyl may optionally be substituted by one 
or more groups selected from hydroxy, halo, carboxy, C.sub.1-6 alkanoyl, 
C.sub.1-6 alkanoyloxy, C.sub.1-6 alkoxy, C.sub.1-6 alkoxycarbonyl, aryl 
(C.sub.1-6) alkoxy, arylcarbonyl, C.sub.1-6 alkylthio, arylthio, amino, 
C.sub.1-6 alkylamino or di(C.sub.1-6) alkylamino. 
When R.sup.1 is methyl suitably it may optionally be substituted by 
carboxy, C.sub.1-6 alkanoyl, C.sub.1-6 alkanoyloxy, C.sub.1-6 
alkoxycarbonyl or arylcarbonyl. 
When R.sup.2 is methyl suitably it may optionally be substituted by 
carboxy, C.sub.1-6 alkanoyl, C.sub.1-6 alkanoyloxy, C.sub.1-6 
alkoxycarbonyl or arylcarbonyl. 
When used herein the term "aryl" includes phenyl, pyrrolyl, furyl, thienyl, 
indolyl, benzofuryl, thionaphthyl, and any of such groups being optionally 
substituted. 
Suitably R is a hydrogen atom. Suitably also R is a C.sub.1-6 alkyl, 
C.sub.2-6 alkenyl, C.sub.1-10 aralkyl or aryl group, any of such groups 
being optionally substituted. More suitably R is methyl, ethyl, t-butyl, 
phenyl, benzyl or carboxymethyl. 
Suitably when R is salified carboxy, the carboxy group is salified with a 
pharmaceutically acceptable salt, suitable salts being as described in 
relation to the C-3 carboxy. Suitably when R is esterified carboxy the 
esterifying group is an in vivo hydrolysable ester of sub-formula (b) or 
(c) as hereinbefore defined, or alternatively an ester of the sub-formulae 
(d) or (e): 
EQU CO--OA.sup.1 (d) 
EQU CO--O--CHA.sup.2 A.sup.3 (d) 
wherein A.sup.1 is C.sub.1-6 alkyl optionally substituted by C.sub.1-7 
alkoxy; A.sup.2 is C.sub.2-5 alkenyl optionally substituted by phenyl or 
is a phenyl group optionally substituted by one or more atoms or groups 
selected from fluorine, chlorine, bromine, nitro, C.sub.1-4 alkyl or 
C.sub.1-4 alkoxy; and A.sup.3 is hydrogen, C.sub.1-4 alkyl or a phenyl 
group optionally substituted by fluorine, chlorine, bromine, nitro, 
C.sub.1-4 alkyl or C.sub.1-4 alkoxy. Most suitably A.sup.1 is a C.sub.1-6 
alkyl group such as methyl or propyl; or CHA.sup.2 A.sup.3 is a benzyl or 
substituted benzyl group. 
Suitably R is a group of the sub-formula --CONR.sup.1 R.sup.2 as 
hereinbefore defined. More suitably R.sup.1 is a hydrogen atom or 
optionally substituted C.sub.1-6 alkyl group. More suitably R.sup.2 is a 
hydrogen atom. 
Suitably R is a group of sub-formula --NR.sup.1 R.sup.2 as hereinbefore 
defined. Most suitably R.sup.1 is a hydrogen atom, C.sub.1-6 alkyl, 
C.sub.1-6 alkanoyl or C.sub.1-10 aralkanoyl, any of such groups being 
optionally substituted. More suitably R.sup.2 is a hydrogen atom. 
Favourably when R.sup.2 is a hydrogen atom R.sup.1 is a hydrogen atom or a 
C.sub.1-6 alkanoyl group such as acetyl. 
This invention extends to the compounds of the formula (II) in both the 
structural forms (II) and (IV): 
##STR6## 
That is the C-9 substituent is either a tetrazol-1-yl or tetrazol-2-yl 
group. 
The present invention also provides a pharmaceutical composition which 
comprises a compound of the formula (II) or a pharmaceutically acceptable 
salt or in vivo hydrolysable ester thereof and a pharmaceutically 
acceptable carrier. 
The compositions of the invention include those in a form adapted for oral, 
topical or parenteral use and may be used for the treatment of the 
infection in animals, particularly in mammals including humans. 
Suitable forms of the compositions of this invention include tablets, 
capsules, creams, syrups, suspensions, solutions, reconstitutable powders 
and sterile forms suitable for injection or infusion. Such compositions 
may contain conventional pharmaceutically acceptable materials such as 
diluents, binders, colours, flavours, preservatives, disintegrant and the 
like in accordance with conventional pharmaceutical practice in the manner 
well understood by those skilled in the art of formulating antibiotics. 
Injectable or infusable compositions of a compound of the invention are 
particularly suitable as high blood levels of the compound can occur after 
administration by injection or infusion. Thus, one preferred composition 
aspect of this invention comprises a compound of the invention in sterile 
form and most suitably in sterile crystalline form. 
The injectable solution of the compound of this invention may be made up in 
a sterile pyrogen-free liquid such as water, aqueous ethanol or the like. 
An alternative approach to administering the compounds of this invention is 
to utilise an injectable suspension. Such suspensions may be made up in 
sterile water; sterile saline or the like and may also contain suspending 
agents such as polyvinylpyrrolidone, lecithin or the like. 
Alternatively such compositions may be prepared in an acceptable oil 
suspending agent such as arachis oil or its equivalent. For use in such 
suspensions the compounds of this invention should be in the form of fine 
particles. 
Unit dose compositions comprising a compound of this invention adapted for 
oral administration form a further suitable composition aspect of this 
invention. 
Unit dose compositions comprising a compound of this invention adapted for 
topical administration are also presented by this invention. In this 
instance `topical administration` also includes local administration to 
internal surfaces of mammary glands of cattle, for example during the 
treatment of mastitis by intra-mammary administration. 
The compound of the formula may be present in the composition as sole 
therapeutic agent or it may be present together with other therapeutic 
agents such as a penicillin or cephalosporin. Considerable advantages 
accrue from the inclusion of a pencillin or cephalosporin which shows 
instability to .beta.-lactamases since the resulting composition shows 
enhanced effectiveness (synergy). Suitable penicillins cephalosporins or 
other .beta.-lactam antibiotic for inclusion in such synergistic 
compositions include not only those known to be highly susceptible to 
.beta.-lactamases but also those which have a degree of intrinsic 
resistance to .beta.-lactamases. 
Suitable penicillins for inclusion in the compositions of this invention 
include benzylpenicillin, phenoxymethylpenicillin, carbenicillin, 
azidocillin, propicillin, ampicillin, amoxycillin, epicillin, ticarcillin, 
cyclacillin, pirbenicillin, azlocillin, mezlocillin, sulbenicillin, 
piperacillin, and other known penicillins including pro-drugs therefor 
such as their in vivo hydrolysable esters such as the acetoxymethyl, 
pivaloyloxymethyl, .alpha.-ethoxycarbonyloxyethyl or phthalidyl esters of 
ampicillin, benzylpenicillin or amoxycillin, and aldehyde or ketone 
adducts of pencillins containing a 6-.alpha.-aminoacetamide side chain 
(such as hetacillin, metampicillin and analogous derivatives of 
amoxycillin) or .alpha.-esters of carbenicillin or ticarcillin such as 
their phenyl or indanyl .alpha.-esters. 
Suitable cephalosporins for inclusion in the compositions of this invention 
include cefatrizine, cephaloridine, cephalothin, cefazolin, cephalexin, 
cephacetrile, cephamandole nafate, cephapirin, cephradine, 
4-hydroxycephalexin, cefaparole, cephaloglycin, cefoperazone, and other 
known cephalosporins or prodrugs therefor. 
Such compounds are frequently used in the form of a salt or hydrate of the 
like. 
Naturally if the penicillin or cephalosprin present in the composition is 
not suitable for oral administration then the composition will be adapted 
for parenteral administration. 
Highly favoured penicillins for use in the compositions of this invention 
include ampicillin, amoxycillin, carbenicillin and ticarcillin. Such 
penicillins may be used as a pharmaceutically acceptable salt such as the 
sodium salt. Alternatively the ampicillin or amoxycillin may be used in 
the form of fine particles of the zwitterionic form (generally as 
ampicillin trihydrate or amoxycillin trihydrate) for use in an injectable 
suspension, for example, in the manner hereinbefore described for a 
compound of this invention. 
The preferred penicillin for use in the synergistic composition is 
amoxycillin, for example as its sodium salt or trihydrate. 
Particularly suitable cephalosporins for use in the compositions of this 
invention include cephaloridine and cefazolin which may be in the form of 
a pharmaceutically acceptable salt for example the sodium salt. 
When present together with a cephalosporin or penicillin, the ratio of a 
compound of the invention to the pencillin or cephalosporin agent may vary 
over a wide range of ratios, such as from 10:1 to 1:15 for example 10:1 to 
1:10 such as about 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5 or 1:6 (wt/wt, based 
on pure free antibiotic equivalent). 
The total quantity of a compound of the invention in any unit dosage form 
will normally be between 25 and 1000 mg and will usually be between 50 and 
500 mg, for example about 62.5, 100, 125, 150, 200 or 250 mg. 
Compositions of this invention may be used for the treatment of infections 
of inter alia, the respiratory tract, the urinary tract and soft tissues 
in humans and mastitis in cattle. 
Normally between 50 and 3000 mg of the compounds of the invention will be 
administered each day of treatment but more usually between 100 and 1000 
mg of the compounds of the invention will be administered per day, for 
example at 1-6 doses, more usually as 2, 3 or 4 doses. However for the 
treatment of more severe systemic infections or infections of particularly 
intransigent organisms higher doses may be used in accordance with 
clinical practice. 
The penicillin or cephalosporin in the synergistic composition of this 
invention will normally be present at approximately the amount at which it 
is conventionally used which will usually be expected to be from about 
62.5 to 3000 mg per dose, more usually about 125, 250, 500 or 1000 mg per 
dose. 
One particularly favoured composition of this invention will contain from 
150 to 1000 mg of amoxycillin as the trihydrate or sodium salt and from 25 
to 500 mg of a compound of this invention. 
A further particularly favoured compositon of this invention will contain 
from 150 to 1000 mg of ampicillin or a pro-drug therefor and from 25 to 
500 
Most suitably this form of composition will contain ampicillin trihydrate, 
ampicillin anhydrate, sodium ampicillin, hetacillin, 
pivampicillinhydrochloride, bacampicillin hydrochloride, or talampicillin 
hydrochloride. Most suitably this form of the composition will contain a 
compound of the formula (II) when in crystalline form. 
Most suitably the preceding composition will contain from 200 to 700 mg of 
the penicillin component. Most suitably the preceding composition will 
comprise from 50 to 250 mg of a compound of the formula (II) preferably in 
crystalline form. 
Such compositions may be adapted for oral or parenteral use except when 
containing an in vivo hydrolysable ester of ampicillin or amoxycillin in 
which case the compositions will not be adapted for parenteral 
administration. 
Another particularly favoured composition of this invention will contain 
from 200 to 2000 mg of carbenicillin, ticarcillin or a pro-drug therefore 
and from 50 to 500 mg of a compound of the invention. 
Suitably this form of composition will contain di-sodium carbenicillin. 
Suitably this form of the composition will contain di-sodium ticarcillin. 
More suitably this form of the composition will contain from 75 to 250 mg 
of a compound of the formula (II) preferably in crystalline form. Such 
compositions containing di-salts of carbenicillin and ticarcillin will be 
adapted for parenteral administration. 
The present invention also provides a method of treating bacterial 
infections in animals, particularly humans or domestic mammals, which 
comprises the administration of a composition of this invention. 
Commonly the infection treated will be due to a strain of Staphylococcus 
aureus, Klebsiella aerogenes, Escherichia coli, Proteus sp., Bacteroides 
fragilis or the like. The organisms believed to be most readily treated by 
an antibacterially effective amount of a compound of this invention is 
Staphylococcus aureus. The other organisms named are more readily treated 
by using a synergistically effective amount of the compound of the 
invention and a penicillin or cephalosporin. The administration of the two 
components may take place separately but in general we prefer to use a 
composition containing both the synergist and the penicillin or 
cephalosporin. 
The indications for treatment include respiratory tract and urinary tract 
infections in humans and mastitis in cattle. 
In another aspect the present invention provides a process for the 
preparation of a compound of the formula (II) or a salt or ester thereof 
which process comprises the reaction of a compound of the formula (V): 
##STR7## 
wherein R.sup.8 is an esterifying group, with: 
(i) a compound of the formula (VI): 
EQU H--X (VI) 
wherein X is as defined in relation to formula (II), and is attached via a 
nitrogen atom to the hydrogen atom: 
(ii) a stabilised diazoalkane, or a compound of formula (VII): 
EQU R.sup.9 OCON.dbd.NCOOR.sup.10 (VII) 
wherein R.sup.9 and R.sup.10 are independently C.sub.1-6 alkyl, aryl or 
aryl (C.sub.1-6) alkyl; and 
(iii) a compound of the formula (VIII): 
##STR8## 
wherein l, m and n are independently 0 or 1 and R.sup.11, R.sup.12 and 
R.sup.13 are each independently C.sub.1-6 alkyl, aryl (C.sub.1-6) alkyl or 
aryl; and thereafter if desired: 
(iv) converting any ester group into a carboxylic acid or salt; 
(v) converting a carboxylic acid or salt into an in vivo hydrolysable 
ester. 
(vi) converting a compound of the formula (II) wherein R is esterified or 
salified carboxy into a compound of the formula (II) wherein R is 
hydrogen. 
Suitably compounds of the formula (VI) are of the sub-formula (VIa): 
##STR9## 
wherein R is as defined in relation to formula (II). 
Suitable compounds of the formula (VII) include those wherein R.sup.9 and 
R.sup.10 are independently selected from methyl, ethyl, propyl, butyl, 
phenyl and benzyl groups. It is generally convenient that R.sup.9 and 
R.sup.10 represent the same moiety. Particularly suitable compounds of the 
formula (VII) include those wherein R.sup.9 and R.sup.10 each represent a 
methyl, ethyl, t-butyl or iso-propyl group. 
Alternatively the compound of the formula (VII) may be replaced by a 
stabilised diazoalkane, such as diphenyldiazomethane or ethyl 
diazoacetate. This aspect is not preferred when the compound of the 
formula (VI) is strongly acidic. 
Suitable compounds of the formula (VIII) include those wherein the 
R.sup.11, R.sup.12 and R.sup.13 groups are selected from methyl, ethyl, 
n-propyl, n-butyl, benzyl, phenyl and methoxyphenyl groups. It is 
generally convenient that R.sup.11, R.sup.12 and R.sup.13 each represent 
the same moiety. Favoured compounds of the formula (VIII) include 
tri-arylphosphines and tri-alkylphosphites. Particularly suitable 
compounds of the formula (VIII) include triphenylphosphine, 
trimethylphosphite, tri-ethylphosphite, tri-p-methoxyphenylphosphine and 
tri-n-butylphosphine. 
Any convenient ester of clavulanic acid may be used in this reaction (i.e. 
the compound of the formula (V) but in general it is most suitable to use 
an in-vivo hydrolysable ester of sub-formulae (b) or (c) as hereinbefore 
defined, or alternatively an ester of sub-formulae (d) or (e) may be used 
as such esters are readily converted to the parent acid or its salt by 
methods appropriate to the particular ester, for example basic hydrolysis, 
enzymatically-catalysed hydrolysis hydrogenolysis, electrolysis or 
photolysis. 
EQU CO--O--A.sup.1 (d) 
EQU CO--O--CHA.sup.2 A.sup.3 (e) 
wherein A.sup.1 is C.sub.1-6 alkyl optionally substituted by C.sub.1-7 
alkoxy; A.sup.2 is C.sub.2-5 alkenyl optionally substituted by phenyl or 
is a phenyl group optionally substituted by one or more atoms or groups 
selected from fluorine, chlorine, bromine, nitro, C.sub.1-4 alkyl or 
C.sub.1-4 alkoxy; and A.sup.3 is hydrogen, C.sub.1-4 alkyl or a phenyl 
group optionally substituted by fluroine, chlorine, bromine, nitro, 
C.sub.1-4 alkyl or C.sub.1-4 alkoxy. 
Further suitable ester-forming groups are those which may also be removed 
under conventional conditions by methods appropriate to the particular 
ester, for example basic hydrolysis, enzymatically-catalysed hydrolysis or 
hydrogenolysis, for example benzoylmethyl, pyridylmethyl, 
2,2,2-trichloroethyl, 2,2,2-tribromoethyl, triphenylmethyl, 
2-benzyloxyphenyl, 4-methoxycarbonylbenzyl, 4-methylthiophenyl, or a 
silyl, stannyl or pentavalent phosphorus-containing group. 
Particularly suitable esters of clavulanic acid for use in this process 
include methoxymethyl clavulanate, benzyl clavulanate, p-nitrobenzyl 
clavulanate, p-methoxybenzyl clavulanate, methyl clavulanate, and silyl 
esters such as tri-(C.sub.1-6)alkylsilyl clavulanates and 
di-(C.sub.1-6)alkyl-phenyl silyl clavulanates, for example 
tri-isopropylsilyl clavulanate and di-tert-butyl-phenyl silyl clavulanate. 
In general it has been found suitable to use one more of an ester of 
clavulanic acid, a slight molar excess of both of the compounds of the 
formulae (VII) and (VIII), and two molar equivalents of the tetrazole of 
the formula (VI). 
The reaction is performed in an inert organic solvent. The solvent used 
should be aprotic and substantially unreactive towards the reagents 
involved. Suitable solvents include tetrahydrofuran, dioxan, ethyl 
acetate, benzene, toluene and chlorobenzene. Of these tetrahydrofuran is 
referred. On occasion it is necessary to have a small proportion of 
dimethylformamide in the reaction solvent to aid solubility of the 
compound of the formula (VI). 
The reaction is normally carried out at a nonextreme temperature such as 
-20.degree. C. to +100.degree. C., more usually from about 5.degree. C. to 
50.degree. C. and conveniently at ambient temperature (approximately 
15.degree. C. to 25.degree. C.). 
Once the reaction is complete de-esterfication may be performed in 
conventional manner to afford the carboxylic acid or salt thereof. The 
lithium salts of the compounds of the formula (II) may be formed first and 
then converted to a different salt, for example by ion-exchange. The salts 
and free acids of the compounds of the formula (II) may be converted to 
esters of the compounds of the formula (II) in conventional manner, for 
example by reaction with one equivalent of a reactive halide in a solvent 
such as dimethylformamide. 
The foregoing process provides the compounds of the formula (II) and salts 
and esters thereof when in structural formula (III) or (IV). 
A compound of the formula (II) wherein R is esterified carboxy may be 
converted into a compound of the formula (II) wherein R is salified 
carboxy in conventional manner, for example, the methods for 
de-esterification of the C-3 ester of clavulanic acid may be used where 
appropriate, in particular basic hydrolysis of alkyl esters is useful. 
Conversion of a compound of the formula (II) wherein R is salified carboxy 
to a compound of the formula (II) wherein R is hydrogen may be in 
conventional manner for example by acidification, extraction into organic 
solvent and treatment. 
In another aspect the present invention provides a process for the 
preparation of a compound of the formula (III) or salt or ester thereof 
which process comprises the reaction of azide ion with an ester of a 
compound of the formula (IX): 
##STR10## 
wherein R is as defined in relation to formula (II) and R.sup.14 is a 
bromine or chlorine atom, and thereafter if desired: 
(i) converting any ester group into a carboxylic acid or salt; 
(ii) converting a carboxylic acid or salt into an in vivo hydrolysable 
ester. 
(iii) converting a compound of the formula (III) wherein R is esterified or 
salified carboxy into a compound of the formula (III) wherein R is 
hydrogen. 
The reaction is suitably performed in an inert organic solvent such as 
dichloromethane or chloroform. 
The reaction may be carried out at a non-extreme temperature such as 
-60.degree. C. to +60.degree. C., preferably from -20.degree. C. to 
40.degree. C., more preferably from 0.degree. C. to +30.degree. C. and 
conveniently at ambient temperature. 
The azide ion may be introduced to the reaction as an inorganic azide for 
example sodium azide, or as an organiz azide such as 
tetramethylguanidinium azide. Care must be exercised when selecting an 
appropriate azide as many azides, particularly heavy metal azides, are 
explosive. 
This method is not preferred for compounds of the formula (I) wherein R is 
--CONR.sup.1 R.sup.2. 
The compound of the formula (IX) may be prepared by a process which 
comprises the reaction of a corresponding ester of a compound of the 
formula (X): 
##STR11## 
wherein R is as defined in relation to formula (II), with an 
imino-halogenating agent and a base. 
The imino-halogenating agent used is one which will convert an amide group 
--NH--CO-- to an imino-halide --N.dbd.CR.sup.14 -- wherein R.sup.14 is as 
hereinbefore defined, for example, phosphorus pentachloride, phosphorus 
pentabromide, thionyl chloride, thionyl bromide, phosgene, phosphorus 
trichloride, phosphorus tribromide, phosphorus oxychloride or phosphorus 
oxybromide. 
The solvent used is suitably an inert organic solvent such as 
dichloromethane, chloroform, carbon tetrachloride, dichloroethane, 
tetrahydrofuran or dioxan. 
The reaction is performed in the presence of a base. Suitably the base is 
an organic base for example a tertiary amine such as triethylamine, 
trimethylamine or N-alkylmorpholine, and pyridine. Most suitably the 
organic base is N-methylmorpholine. 
It is also possible to use triphenylphosphine or 
tir-p-methoxyphenylphosphine with carbon tetrahalide as an 
imino-halogenating agent, for example triphenylphosphine in carbon 
tetrachloride or triphenylphosphine and carbon tetrabromide in an organic 
solvent, for example dichloromethane, chloroform or benzene. 
The compounds of the formula (X) may be prepared by the methods of Belgian 
Pat. Nos. 860042 and 866496. 
In a further aspect the present invention provides a process for the 
preparation of a compound of the formula (XI): 
##STR12## 
or salt or ester thereof wherein R.sup.15 is an esterified or salified 
carboxy or a group CONR.sup.1 R.sup.2 as hereinbefore defined, which 
process comprises the reaction of a compound of the formula (XII): 
##STR13## 
wherein R.sup.8 is as hereinbefore defined, with a reactive nitrile of the 
formula (XIII): 
EQU R.sup.15 --C.tbd.N (XIII) 
and thereafter if necessary: 
(i) converting any ester group into a carboxylic acid or salt; 
(ii) converting a carboxylic acid or salt into an in-vivo hydrolysable 
ester. 
(iii) converting a compound of the formula (II) wherein R.sup.. is 
esterified or salified carboxy into a compound of the formula (II) wherein 
R.sup.15 is hydrogen. 
Suitably R.sup.15 is a conjugating group, for example an esterified carboxy 
group of the sub-formula (ii): 
EQU R.sup.16 O.sub.2 C-- (ii) 
wherein R.sup.16 is an esterifying group. Suitably R.sup.16 is an ester of 
the sub-formulae (b), (c), (d) or (e). Preferably R.sup.16 is optionally 
substituted C.sub.1-6 alkyl or optionally substituted benzyl, for example 
methyl, ethyl or benzyl. 
Suitably the reaction is performed at a temperature between 0.degree. C. 
and 120.degree. C., preferably between 50.degree. C. and 100.degree. C. 
The reaction may be performed in the absence of solvent (if the compound 
of the formula (XIII) is a liquid) or alternatively in the presence of 
solvent, suitably an organic solvent. Suitable solvents include inert 
organic solvents such as tetrahydrofuran and chloroform. 
The compounds of the formula (XII) may be prepared by the methods of 
Belgian Pat. No. 855,375. 
The following Examples illustrate the invention.

EXAMPLE 1 
Benzyl 9-tetrazol-2-yldeoxyclavulanate 
To a solution of benzyl clavulanate (2.89 g) in tetrahydrofuran (50 ml), 
triphenylphosphine (3.14 g) and tetrazole (1.4 g) were added. The mixture 
was stirred until all was in solution, cooled to a 2.degree.-5.degree. C. 
in an ice-bath and diethyl azodicarboxylate (2.1 ml) added all at once. 
The reaction mixture was stirred for 30 mins. at ambient temperature them 
evaporated to small volume. Ethyl acetate and cyclohexane (50 ml, about 
1:1) were added and the colourless crystalline precipitate filtered off; 
the filtrate was re-evaporated and subjected to gradient elution 
chromatography on silica gel using ethyl acetate and cyclohexane graded 
from 3:1 to 1:1 ratio. The second major .beta.-lactam-containing component 
was separated, and fractions containing it were combined and evaporated 
under reduced pressure on a rotary evaporator. The residue (which 
contained some diethyl hydrazodicarboxylate) was treated with CCl.sub.4 
(ca. 1 ml), filtered and the filtrate evaporated, to yield the title 
product as an oil, 0.24 g, still containing a little solvent. 
Infra-red (film) 1805 (.beta.-lactam C.dbd.O), 1755 (ester C.dbd.O) and 
1705 cm.sup.-1 (C.dbd.C) 
EXAMPLES 1 (a)-(f) 
(a) Diethylazodicarboxylate may be replaced by di-tert-butyl 
azodicarboxylate to yield the title compound (0.07 g). 
(b) Diethylazodicarboxylate may be replaced by di-isopropyl 
azodicarboxylate to yield the title compound (0.1 g). 
(c) Diethylazodicarboxylate may be replaced by dimethyl azodicarboxylate (2 
ml) to yield the title compound (0.19 g). 
(d) Diethylazodicarboxylate may be replaced by ethyl diazoacetate (1.4 ml), 
the reaction being stirred at 2.degree.--3.degree. C. for 2 days to yield 
the title compound (0.05 g). 
(e) Diethylazodicarboxylate may be replaced by diphenyl diazomethane, in 
excess, the reaction being warmed to 50.degree. C. for one hour, the title 
compound being detected by t.l.c. 
(f) Triphenyl phosphine may be replaced by tributylphosphine (.about.2 g) t 
yield the title compound (0.1 g). 
EXAMPLE 2 
Methoxymethyl 9-tetrazol-2-yldeoxyclavulanate 
In a method analogous to that of Example 1, substituting methoxymethyl 
clavulanate (5.1 g) for benzyl clavulanate, with the same ratios of 
reagents the title compound was obtained as a pale yellow oil (0.1 g), 
i.r. (liq film) 1795 (.beta.-lactam), 1745 (ester), 1700 (C.dbd.C) 
cm.sup.-1. 
EXAMPLE 3 
Methyl 9-tetrazol-2-yldeoxyclavulanate 
In a method analogous to that of Example 1, substituting methyl clavulanate 
(2.12 g) for benzyl clavulanate with the same ratios of reagents, the 
title compound was obtained (0.25 g). i.r. (liq film) 1795 
(.beta.-lactam), 1740 (ester), 1695 (C.dbd.C) cm.sup.-1. 
EXAMPLE 4 
4-Bromobenzyl 9-tetrazol-2yldeoxyclavulanate 
In a method analogous to that of Example 1 substituting 4-bromobenzyl 
clavulanate (3.7 g) for benzyl clavulanate, with the same ratios of 
reagents the title compound was obtained (0.24 g). i.r. (liq. film) 1807 
(.beta.-lactam), 1755 (ester), 1707 (C.dbd.C) cm.sup.-1. 
EXAMPLE 5 
4-Nitrobenzyl 9-tetrazol-2-yldeoxyclavulanate 
In a method analogous to that of Example 1 substituting 4-nitrobenzyl 
clavulanate (8.4 g) for benzyl clavulanate, with the same ratios of 
reagents the title compound was obtained (1.0 g), i.r. (liq. film) 1800 
(.beta.-lactam), 1750 (ester), 1695 (C.dbd.C) cm.sup.-1. 
EXAMPLE 6 
Benzyl 9-(tetrazol-2-yl)deoxyclavulanate and Benzyl 9-(tetrazol-1-yl) 
deoxyclavulanate 
To a stirred solution of benzyl clavulanate (11.8 g) in dry redistilled 
tetrahydrofuran (175 ml) was added tetrazole (5.0 g) and 
triphenylphosphine (12.3 g). When the reactants had dissolved, the mixture 
was cooled to 2.degree.-3.degree. C. in an ice-bath, then diethyl 
azodicarboxylate (9 ml) was added rapidly from a pipette. The mixture was 
stirred at ambient temperature for 30 mins, at which time tlc showed a 
number of KMnO.sub.4 -positive zones. The reaction mixture was evaporated 
under reduced pressure to about 70 ml, then re-evaporated with two 
successive portions of 50 ml of toluene until reduced to a syrup 
containing crystalline material. 100 ml of toluene was added, cooled to 
2.degree.-3.degree. C., filtered off insolubles and the filtrate 
re-evaporated. The mixture was subjected to gradient elution 
chromatography on silica gel, using a short wide column, eluted with ethyl 
acetate and n-hexane graded from 1:2 to 2:1 ratio. After an initial 
fractionation into more polar and less polar products, the latter were 
combined and rechromatographed on a similar column. Fractions containing 
benzyl 9-(tetrazol-2-yl) deoxyclavulanate were collected and combined to 
yield O. 9 g of an oil; I.r. (film) 1805(.beta.-lactam C.dbd.O) 1755 
(ester C.dbd.O) and 1705 cm.sup.-1 (C.dbd.C); nmr; .delta. (CDCl.sub.3) 
3.09 (1H,d, J 17Hz, 6-.beta.-CH) 3.52 (1H,dd, J17 and 3Hz, 6-.alpha.-CH), 
4.9-5.4 (6H,m, including s. for PhCH.sub.2 at 5.16, PhCH.sub.2, 3-CH, 
8-CH, 9-CH.sub.2), 5.77 (1H,d, J 3Hz, 5-CH), 7.30(5H, Ph), 8.47 (1H, s, 
tetrazole CH). The more polar prducts were rechromatographed using a 
reverse gradient on silica gel, the solvent being graded from ethyl 
acetate to 4:1 ethyl acetate:cyclohexane. The fractions containing benzyl 
9-(tetrazol-1-yl)deoxyclavulanate were collected and combined to yield 
0.15 g of a slightly impure oil; i.r. (film) 3140 (tetrazole CH), 
1802(.beta.-lactam C.dbd.O), 1748 (ester C.dbd.O) and 1700 cm.sup.-1 
(C.dbd.C). 
EXAMPLE 7 
Lithium 9-tetrazol-2-yldoexyclavulanate 
A solution containing benzyl 9-tetrazol-2-yldeoxyclavulanate (0.48 g, 
containing a little inert solvent) in redistilled tetrahydrofuran (30 ml) 
and water (ldrop) was hydrogenated at ambient temperature and pressure 
over 10% palladised charcoal for 2 hours, when the uptake of hydrogen had 
ceased. The catalyst was removed by filtration, washed with a little 
tetrahydrofuran, the filtrate diluted with water (100 ml) and titrated 
with 1.0M aqueous lithium hydroxide to pH 7.5. The mixture was evaporated 
under reduced pressure to small volume (&gt;1 ml) diluted with 1-propanol (10 
ml) and re-evaporated to dryness to leave a pale buff crystalline solid. 
This was triturated with acetone and collected by filtration, washed with 
ether and dried in vacuo, to yield 0.19 g of the title compound. 
I.R. spectrum (nujol) 1785(.beta.-lactam C.dbd.O), 1700(C.dbd.C) and 1620 
cm.sup.-1 (CO.sub.2.sup.-). 
This compound may be hydrated. 
EXAMPLE 8 
Lithium 9-(tetrazol-2-yl)deoxyclavulanate 
Methyl 9-tetrazol-2-yldeoxyclavulanate (250 mg) in THF-water was maintained 
at pH 9.5 at the pH-Stat by the addition of 1M lithium hydroxide. When 
uptake became slow the reaction was stopped, and the mixture evaporated to 
a small volume under reduced pressure. The residue was triturated under 
acetone and the lithium salt filtered off, washed with acetone and dried 
to afford the title compound. Ir (film of DMSO solution) 1778 cm.sup.-1 
(.beta.-lactam C.dbd.O). 
EXAMPLE 9 
Benzyl 9-(5-aminotetrazolyl)deoxyclavulanate 
5-aminotetrazole monohydrate was dehydrated by heating in a Dean and Starke 
apparatus using toluene to oo-distil the water. When no further water 
distilled, the suspension was cooled to room temperature, decanted off the 
toluene, added dry ether and decanted again, finally dried in vacuo over 
P.sub.2 O.sub.5 : 2.3 g of this material was dissolved in about 8 ml of 
warm dry N,N-dimethylformamide. 
A solution of benzyl olavulanate (2.89 g) in dry tetrahydrofuran (70 ml) 
containing triphenylphosphine (3.14 g) was cooled in ice-water and 
stirred. To this solution were added successively the 5-aminotetrazole 
solution and then diethyl azodicarborylate (2.1 ml). The mixture was 
stirred for 30 mins, then evaporated under reduced pressure. Most of the 
dimethylformamide was then removed by evaporation at 0.2 mm pressure. The 
residue was diluted with ethyl acetate-cyclohexane (1:1, about 50 ml), and 
the precipitated solids removed by filtration. The filtrate was 
re-evaporated, and subjected to gradient elution chromatography on silica 
gel using ethyl acetate and cyclohexane mixtures, granded from 1:2 ratio 
through 2:1 ratio to pure ethyl acetate, as elution solvent. The most 
polar .beta.-lactam-containing product (as determined by tlc (SiO.sub.2) 
in 2:1 ethyl acetate-cyclohexane) was collected, and fractions containing 
it were combined and evaporated, to yield 140 mg of an oil containing some 
N,N-dimethylformamide. This was not further purified. 
Infra-red spectrum (film) 3600-2800 (broad, with fine structure, NH.sub.2), 
1805 (.beta.-lactam C.dbd.O), 1760 (ester C.dbd.O), 1705 cm.sup.-1 
(C.dbd.C). 
The above reaction was repeated on a larger scale, using benzyl clavulanate 
(11.56 g) and the same ratio of reactants, to afford the title compound as 
a crude product (0.7 g). This crystallised on standing, and after 
trituration with carbon tetrachloride, the product was collected by 
filtration, washed with a little further carbon tetrachloride and dried in 
vacuo to yield the title product (0.49 g); i.r. (Nujol mull) 
3335,3260(NH.sub.2), 1800(.beta.-lactam C.dbd.O), 1753 (ester C.dbd.O), 
1712cm.sup.-1 (C.dbd.C). This compound is believed to be the 
EXAMPLE 10 
Lithium 9-(5-aminotetrazolyl)deoxyclavulanate 
The partially purified benzyl ester of example 9 (140 mg) was dissolved in 
redistilled tetrahydrofuran (10 ml) containing water (1 drop) and 10% 
palladised charcoal (70 mg). It was hydrogenated at ambient temperature 
and pressure for about 1 hour. The catalyst was removed by filtration, 
washed with water and the filtrate and washings diluted with water and 
titrated to pH 7.3 with lithium hydroxide solution. The solution was 
evaporated to near dryness. 1-propanol added and re-evaporated to dryness. 
The colourless crystalline solid was triturated with acetone filtered off, 
washed with acetone and dried in vacuo, to yield the title compound (30 
mg). 
Infra-red spectrum (mull) 1790(.beta.-lactam C.dbd.O), 1705 (C.dbd.C) and 
1640 (broad, CO.sub.2.sup.-). 
EXAMPLE 11 
Benzyl 9-(5-acetamidotetrazolyl)deoxyclavulanate 
To a solution of banzyl olavulanate (2.89 g) and triphenylphosphine (3.14 
g) in dry redistilled tetrahydrofuran (50 ml), stirred and cooled in ice 
+water, were added a solution of 5-aoetamidotetrazole (2.5 g) in 
N,N-dimethylformamide (20 ml) and immediately diethyl azodicarboxylate 
(2.2 ml). Allowed to stir for 30 mins, evaporated to low volume. Ethyl 
acetate and cyclohexane (60 ml, 1:1) were added, then washed with water) 
2.times.60 ml portions), to remove DMF and other water-soluble materials. 
The solvent layer was dried over sodium sulphate, filtered, evaporated to 
a syrup and subjected to gradient elution chromatography on silica gel 
using ethyl acetate and cyclohexane, graded from 2:1 to neat ethyl 
acetate, as eluents. Fractions containing the most polar 
.beta.-lactam-containing component were collected and combined, to yield 
the title compound as an oil (100 mg). 
Infra-red spectrum (film) 1800 (.beta.-lactam C.dbd.O), 1745 (ester 
C.dbd.O), 1700 (C.dbd.C). 
EXAMPLE 12 
Sodium 9-(5-acetamidotetrazolyl)deoxyclavulanate 
A solution of the benzyl ester of example 11 (0.1 g) in redistilled 
tetrahydrofuran (15 ml) was hydrogenated at ambient temperature and 
pressure over 10% palladised charcoal (50 mg) for about 1 hour (tlc at 
this time showed that some ester remained, but the reaction was worked up 
notwithstanding). The catalyst was removed by filtration, the filtrate 
diluted with water (50 ml) and titrated to pH 7.3 with 0.5 M NaOH 
solution. The solution was evaporated to dryness, and triturated with 
acetone. The colourless crystalline product was filtered off, crushed 
quickly with acetone and dried in vacuo. It was somewhat hygroscopic. 
Yield 25 mg. 
Infra-red spectrum (mull) 1780 (.beta.-lactam C.dbd.O), 1700 (shoulder, 
C.dbd.C), 1690 (NHCO), and 1620 cm.sup.-1 (CO.sub.2.sup.-). 
EXAMPLE 13 
Benzyl 9-(5-ethoxycarbonyltetrazol-l-yl)deoxyclavulanate 
To benzyl 9-azidodeoxyclavulanate (4 g) was added ethyl cyanoformate (10 
ml). The mixture was heated under reflux in an oil-bath at 
80.degree.-100.degree. for 11/2 days. The tlc (SiO.sub.2, 1:1 hexane-ethyl 
acetate) of the mixture at this stage showed some unreacted 9 -azido 
compound. The excess ethyl cyanoformate was evaporated in vacuo, and the 
residue subjected to gradient elution chromatography on silica gel, using 
ethyl acetate and hexane (or cyclohexane) graded from 1:2 to 2:1 ratio. 
The unreacted azide eluted first. Those fractions which contained mainly 
the 1-tetrazole were combined, diluted with approximately an equal volume 
of cyclohexane and cooled to 2.degree.-3.degree. C. The compound 
crystallised and it was collected by filtration, washed with ether and 
dried in vacuo. The yield of benzyl 9-(5-ethoxycarbonyltetrazole-1-yl) 
deoxyclavulanate was 1.3 g; mp 100.degree. C.; Ir (mull) 1798, 
(.beta.-lactam C.dbd.O) 1740 (esters) 1688 cm.sup.-1 (C.dbd.C). Nmr 
(CD.sub.3 COCD.sub.3 ;CDCl.sub. 3 was unsuitable) .delta.1.39 (3H,t,J 7Hz, 
CH.sub.3 CH.sub.2), 3.10 (1H,d,J 17Hz, 6-.beta.-CH), 3.64 (1H,dd,J 17 and 
3Hz, 6-.alpha. CH), 4.47 (2H,q,J 7Hz, CH.sub.3 CH.sub.2), 5.05 (1H,t,J 
8Hz, 8-CH.dbd.), 5.18 (2H,s, PhCH.sub.2), 5.23 (1H,s, 3-CH), 5.44 (2H,d, J 
8Hz, 9-CH.sub.2) 5.81 (1H,d, J 3Hz, 5-CH) and 7.33 (5H,s, Ph). 
EXAMPLE 14 
Lithium 9-(5-ethoxycarbonyltetrazol-1-yl) deoxyclavulanate and sodium 
9-(5-ethoxycarbonyltetrazol-1-yl) deoxyclavulanate 
A solution of benzyl 9-(5-ethoxycarbonyltetrazol-1-yl)deoxyclavulanate (1 
g) in redistilled tetrahydrofuran (25 ml) containing water (.about.0.1 ml) 
and 10% palladised charcoal (0.5 g) was hydrogenated at ambient 
temperature and pressure. Uptake of hydrogen was very rapid (about 1 
minute). The catalyst was removed by filtration, then the filtrate was 
diluted with water and titrated to pH 7.3 with IM lithium hydroxide 
solution. The solvents and water were removed under reduced pressure until 
a syrup remained; this began to crystallise. It was diluted slowly with 
acetone, filtered off, washed with acetone and dried in vacuo. (A small 
sample was air-dried for Karl Fischer water determination; it contained 
10%, equivalent to a dihydrate). Yield 0.76 g. 
Nmr (D.sub.2 O) .delta. : 1.33 (3H,t, J 7Hz, CH.sub.3 CH.sub.2) 2.12 
(acetone) 2.98 (1H,d, J 17Hz, 6-.beta.-CH) 3.47 (1H,dd, J 17 and 3Hz, 
6-.alpha.-CH) 4.47 (partially obscured by HOD,q, J 7Hz, CH.sub.3 
CH.sub.2), 4.90 (1H,s, 3-CH) 8-CH probably about 5.18, 5.4 (2H,d, J 8Hz, 
9-CH.sub.2) and 5.66 (1H,d, J 3Hz, 5-CH). Ir. (mull): 1782 (.beta.-lactam 
C.dbd.O) 1735 (ester C.dbd.O) 1690 (C.dbd.C) and 1625 cm.sup.-1 
(CO.sub.2.sup.-). 
The sodium salt was obtained analogously be neutralisation of the filtered 
hydrogenation solution using aqueous sodium hydroxide solution. It was 
also a crystalline solid, and hydrated. I.r. (mull) 1785 (.beta.-lactam 
C.dbd.O) 1735 (ester C.dbd.O) 1690 (C.dbd.C) 1620 cm.sup.-1 
(CO.sub.2.sup.-). 
EXAMPLE 15 
Disodium 9-(5-carboxylatotetrazol-1-yl) deoxyclavulanate 
A solution of 0.1 g of sodium 5-ethoxycarbonyltetrazol-1-yldeoxyclavulanate 
in 10 ml of water was maintained at pH 9.5 on a pH--stat by the addition 
of 0.05 M aqueous sodium hydroxide solution, until the uptake of alkali 
became very slow. The solution was evaporated under reduced pressure, the 
residue triturated with acetone and then filtered off, washed with ether 
and dried. I.r. spectrum (mull) 1755 (.beta.-lactam C.dbd.O) 1690 (sh) 
(C.dbd.C) 1655 and 1605 cm.sup.-1 (CO.sub.2.sup.-). Nmr (D.sub.2 O) 
.delta. 2.98 (1H,d, J 17Hz, 6-.beta.-CH), 3.45 (1H,dd, J 17 and 3Hz, 
6-.alpha.-CH), 4.75-5.4 (5H,m, 3-CH9CH.sub.2, 8-CH), and 5.63 (1H,d, J 
3Hz, 5-CH). 
EXAMPLE 16 
Benzyl 9-(tetrazol-1-yl) deoxyclavulanate 
A solution of benzyl 9-(5-ethoxycarbonyltetrazol-1-yl) deoxyclavulanate (1 
g) in redistilled tetrahydrofuran (20 ml) was diluted with water until the 
first permanent turbidity appeared. The solution was then stirred and 
maintained at pH 9-9.5 at a pH--Stat by the addition of IM lithium 
hydroxide (about 2 ml). The mixture was diluted with water (100 ml) 
extracted with several portions of ethyl ecetate (each 50 ml), then 
dichloromethane (100 ml) was added and the mixture acidified to pH1 by the 
addition of 2.5M H.sub.2 SO.sub.4. The aqueous layer was re-extracted with 
ethyl acetate, then the solvent layers combined, dried (Na.sub.2 SO.sub.4) 
and evaporated. The residue was subjected to gradient elution 
chromatography on silica gel, using ethyl acetate and cyclohexane graded 
from 1 : 1 ratio to neat ethyl acetate as eluents. The product was the 
most polar .beta.-lactam-containing component, having an R.sub.f (1:1 
ethyl acetate-hexane)&gt;0.1. About 50 mg of the pure ester was obtained 
after evaporation of solvent, though some further fractions contained the 
compound in a less pure state. It was an oil, Ir (film): 3140 (tetrazole 
C-H stretch), 1807 (.beta.-lactam C.dbd.O), 1755 (ester C.dbd.O) and 1705 
cm.sup.-1 (C.dbd.C). Nmr (CDCl.sub.3) .delta. :3.07 (1H,d, J 17Hz, 
6-.beta.-CH), 3.53 (1H,dd, J 17 and 3Hz, 6-.alpha.-CH), 4.8-5.4 (6H,m, 
9-CH.sub.2, 3-CH, 8-CH,PhCH.sub.2) 5.76 (1H,d, J 3Hz, 5-CH), 7.33 (5H,s, 
C.sub.6 H.sub.5) and 8.38 (1H,d, tetrazole CH; the tetrazole 5-H signal 
shifts to about .delta. 9.2 D.sub.6 -acetone. 
EXAMPLE 17 
Dipotassium 9-(5-carboxytetrazol-1-yl) deoxyclavulanate 
A solution of benzyl 9-(5-ethoxycarbonyltetrazol-1-yl) deoxyclavulanate 
(6.2 g) in redistilled tetrahydrofuran (30 ml) containing water (0.1 ml) 
was hydrogenated at ambient temperature and pressure over 10% palladium on 
charcoal (1.6 g) until uptake of hydrogen became very slow. The catalyst 
was removed by filtration through silica, the filtrate diluted with water 
(100 ml) and titrated to pH7 with IM sodium hydroxide. Most of the 
tetrahydrofuran was evaporated under reduced pressure, and the aqueous 
solution then maintained at pH 9-9.5 by the automatic addition of IM NaOH 
solution until the uptake of alkali became slow. The solution was 
acidified by the addition of pre-washed IR120 (H.sup.+) resin in two 
portions, decanting each time, to give a pH of 1.5-1.7, then neutralised 
to pH 7.2 by the addition of IM KOH. The solution was evaporated to 
crystallisation, isopropanol and acetone added (about 1 Lit. total), the 
solid was filtered off, washed with acetone and dried in vacuo, to yield 
3.6 g of the title compound as a colourless crystalline solid; Ir (Nujol 
mull) 3500, 3400 (broad, H.sub.2 O of crystallisation) 1775 (.beta.-lactam 
C.dbd.O) 1698 (C.dbd.C) 1665 and 1615 cm.sup.-1 (CO.sub.2.sup.-). 
EXAMPLE 18 
Lithium 9-(tetrazol-1-yl) deoxyclavulanate 
A solution of disodium 9-(5-carboxytetrazol-1-yl)deoxyclavulanate (1.0 g) 
in water (50 ml) was layered with ethyl acetate (100 ml) and saturated 
with sodium chloride. The mixture was vigorously stirred and acidified to 
pH 1.0 by the addition of IM hydrochloric acid. The layers were separated 
and the aqueous layer extracted with further 2.times.75 ml portions. The 
ethyl acetate extracts were combined, dried over a little anhydrous sodium 
sulphate, filtered and added to water (50 ml). The mixture was stirred 
vigorously and titrated to pH 7.3 with IM lithium hydroxide solution. The 
layers were separated, the solvent layer washed with a little water and 
the aqueous extracts combined, diluted with an equal volume of 1-propanol 
and evaporated to dryness under reduced pressure. The crystalline pale 
orange residue was triturated with acetone, filtered off, washed and dried 
in vacuo, to yield 0.65 g of the title product as a pale apricot coloured 
crystalline solid; I.R. (Nujol mull) 3400 (very broad, H.sub.2 O), 3120 
(tetrazole CH stretch), 1785 (.beta.-lactam C.dbd.O), 1702 (C.dbd.C) and 
1610 cm.sup.-1 (broad, CO.sub.2.sup.-): Nmr .delta. (D.sub.2 O, CH.sub.3 
CN.dbd.2.0) 3.04 (1H, d, J 3Hz, 6-.beta.-CH), 3.52 (1H,dd, J 3.0 and 17Hz, 
6-.alpha.-CH), 4.97 (1H,s, 3-CH), 5.0-5.25 (3H,m, 8-CHand 9-CH.sub.2), 
5.73 (1H,d, J 3.0 Hz, 5-CH) and 9.09 (1H,s, tetrazole CH). 
EXAMPLE 19 
Sodium 9-(tetrazol-1-yl)deoxyclavulanate 
The procedure of Example 18 was repeated using sodium hydroxide solution in 
place of lithium hydroxide solution. The product was a hygroscopic solid; 
Ir (Nujol mull) 3400 (broad, H.sub.2 O), 3140 (weak, tetrazole CH stretch) 
1785 (.beta.-lactam C.dbd.O) 1698 (C.dbd.C), 1620 and 1580 cm.sup.-1 
(CO.sub.2.sup.-). 
EXAMPLE 20 
Potassium 9-(tetrazol-1-yl)deoxyclavulanate 
the procedure of Example 18 was repeated using potassium hydroxide solution 
in place of lithium hydroxide solution. The product was obtained from 
slightly aqueous acetone or isopropanol. It was very slightly hygroscopic. 
EXAMPLE 21 
Potassium 9-(tetrazol-1-yl)deoxyclavulanate 
A solution of dipotassium 9-(5-carboxytetrazol-1-yl) deoxyclavulanate (3.3 
g) in water (100 ml) was layered with 300 ml of ethyl acetate. To it was 
added sufficient `Amberlite` IR 120 (H.sup.+) resin (prewashed with dilute 
H.sub.2 SO.sub.4 and then washed with water until almost neutral) to give 
pH 1.5-1.8. The mixture was allowed to stir for 1 hour at ambient 
temperature (about 20.degree. C.), then decanted from the resin. The resin 
was washed with a little water, which was added to the solution. The 
mixture was titrated to pH 7.2 with IM KOH solution, the layers separated 
and the aqueous layer evaporated to near dryness, diluted slowly with 
isopropanol-acetone and cooled to 2.degree.-3.degree. C. when 
crystallisation occurred. The compound was filtered off, washed with 
acetone and dried in vacuo, to yield 1.7 g of the potassium salt. 
The compound was further purified by solution in a small volume of water 
and treatment with activated charcoal. After filtration to remove charcoal 
the filtrate was diluted with acetone or isopropanol; after seeding, 
scratching and cooling the pure colourless potassium salt crystallised and 
was collected, washed with acetone and dried, to yield 1.1 g. A further 
0.3 g was obtained from the mother liquor by evaporation and trituration 
with acetone: Infrared spectrum of potassium salt (Nujol mull) 3400 (weak, 
br, H.sub.2 O) 3120 (weak, tetrazole C-H stretch) 1785 (.beta.-lactam 
C.dbd.O) 1695 (C.dbd.C, sharp and rather strong), 1627 and 1605 cm.sup.-1, 
(CO.sub.2.sup.-, strong). Other strong unassigned peaks in the spectrum 
were at 1294, 1172, 1034, 788, 756 and 676 cm.sup.-1 ; numerous other 
sharp peaks. 
EXAMPLE 22 
Sodium 9-(tetrazol-1-yl)deoxyclavulanate 
A solution of mixed lithium and potassium salts (1.2 g, about 2:3) in water 
(10 ml) was passed down a column of IR120 (Na.sup.+ form) that had been 
thoroughly washed with water. The eluate was collected and evaporated to 
near dryness; 1-propanol was added and re-evaporated. The solid residue 
was triturated with acetone-ether, filtered off quickly, washed with dry 
ether and dried in vacuo, to yield 1.0 g of sodium salt. 
EXAMPLE 23 
Benzyl 9-(5'-methyltetrazol-1'-yl) deoxyclavulanate 
(i) Benzyl 9-acetamido-9-deoxyclavulanate 
Benzyl-9-azido-9-deoxyclavulanate (2 g) was dissolved in tetrahydrofuran 
(40 ml)/water (20 ml) and the solution ice cooled and stirred vigorously. 
Zinc powder (5 g) was added in small quantities over 11/2 hours, while 
maintaining the pH of the solution between 3 and 4 by dropwise addition of 
2N HCl. When benzyl 9-azido-9-deoxyclavulanate could no longer be detected 
in the solution (silica tlc, eluent ethyl acetate/petroleum ether 1:2), 
the pH was adjusted to 6 with 1N aqueous sodium bicarbonate and the 
solution filtered. The filtrate was saturated with NaCl and extracted with 
ethyl acetate (3.times.30 ml). The combined ethyl acetate extracts were 
dried over MgSO.sub.4 and evaporated under reduced pressure to ca. 50 ml 
to provide a solution containing benzyl 9-amino-9-deoxyclavulanate. This 
solution was treated with acetic anhydride (1.0 ml) and pyridine (0.52 
ml), stirred 1 hour at room temperature, washed with 0.1N HCl (50 ml), 1N 
aqueous sodium bicarbonate solution (50 ml) and water (50 ml), dried over 
MgSO.sub.4 and evaporated under reduced pressure. 
The residue was chromatographed on silica, eluting with ethyl acetate, to 
provide benzyl 9-acetamido-9-deoxyclavulanate as a white solid (1.47 g). 
This was crystallised from ethyl acetate. Found C 61.8, H 5.5, N 8.5%. 
C.sub.17 H.sub.18 N.sub.2 O.sub.5 requires C 61.8, H 5.5, N 8.5%; 
.nu..sub.max (CHCl.sub.3) 3440,1800, 1750,1690,1660,1505 cm.sup.-1. 
(ii) Benzyl 9-(5'-methyltetrazol-1'-yl)-9-deoxyclavulanate 
Benzyl 9-acetamido-9-deoxyclavulanate (0.40 g, 1.21 mmole) was dissolved in 
dry dichloromethane (7 ml) and ice cooled. This solution was treated with 
pyridine (0.195 ml, 2.42 mmole) and then with a 12.5% solution of phosgene 
in toluene (1.48 ml). After stirring at room temperature for 80 minutes, 
the solution was evaporated under reduced pressure and the residue quickly 
dissolved in dry dichloromethane (10 ml). This solution was ice cooled, 
treated with tetramethylguanidinium azide (0.47 g, 3.02 mmole) and then 
stirred at room temperature for 30 minutes. The resulting solution was 
washed with 0.5N HCl (10 ml) and brine (10 ml), dried over MgSO.sub.4 and 
evaporated under reduced pressure. The residue was chromatographed on 
silica, eluting with ethyl acetate/petroleum ether 3:1, and appropriate 
fractions were combined and evaporated to provide benzyl 
9-(5'methyltetrazol-1'-yl)-9-deoxyclavulanate as a colourless gum (0.286 
). [.alpha.].sub.D.sup.20 +29.8.degree. (c.1.0; CHCl.sub.3);.nu..sub.max 
(CHCl.sub.3) 1805, 1750,1695 cm.sup.-1 ; .delta.(CDCl.sub.3) 2.39 
(3H,s,--CH.sub.3), 3.06 (1H,d, J 17 Hz, 6.beta.--CH), 3.54 (1H, dd, J 17 
and 2.5 Hz, 6.alpha.--CH), 4.7-5.0 (3H,m, 8--CH and 9--CH.sub.2), 5.0-5.3 
(3H,m, 3--CH and --OCH.sub.2 Ph), 5.73 (1H,d, J 2.5 Hz, 5--CH), 7.29 
(5H,s, Ph-H); Found 355.1272: C.sub.17 H.sub.17 N.sub.5 O.sub.4 requires 
355.1280. 
EXAMPLE 24 
Benzyl 9-(5'-methyltetrazol-1'-yl)-9-deoxyclavulanate 
Benzyl 9-acetamido-9-deoxyclavulanate (0.10 g, 0.303 mmole) was dissolved 
in dry dichloromethane (5 ml) and ice cooled. The solution was treated 
with pyridine (0.081 ml) and phosphorus pentachloride (0.066 g, 0.318 
mmole) and stirred 30 minutes at 0.degree.-5.degree. C. 
Tetramethylguanidinium azide (0.156 g, 1 mmole) was then added and the 
solution stirred a further 30 minutes at 0.degree.-5.degree. C., washed 
with 0.5 NHCl (10 ml), dried over MgSO.sub.4 and evaporated under reduced 
pressure. The residue was chromatographed as in example 23 to provide 
benzyl 9-(5'-methyltetrazol-1'-yl)-9-deoxyclavulanate (0.08 g), identical 
to the product of example 23. 
EXAMPLE 25 
Potassium 9-(5'-methyltetrazol-1'-yl)-9-deoxyclavulanate 
Benzyl 9-(5-methyltetrazol)-1-yl)-9-deoxyclavulanate (0.28 g, 0.79 mmole) 
was dissolved in distilled tetrahydrofuran (15 ml), treated with 10% 
palladium/charcoal (90 mg) and hydrogenolysed for 30 minutes at 
atmospheric pressure. The suspension was filtered through celite and the 
filtrate evaporated under reduced pressure to 2 ml. Water (5 ml) was added 
and this solution was brought to pH 7 by dropwise addition of 0.5n aqueous 
KOH, washed with ethyl acetate (2.times.10 ml), concentrated to ca. 5 ml 
under reduced pressure and freeze dried. Potassium 
9-(5'-methyltetrazol-1'-yl)-9-deoxyclavulanate was obtained as a yellow 
solid (0.234 g). .nu..sub.max (KBr) 1780,1690,1610 cm.sup.-1 ; 
.delta.(D.sub.2 O: HOD.tbd.4.06.delta.) 2.48 (3H,s --CH.sub.3), 3.02 
(1H,d, J 17 Hz, 6.beta.--CH), 3.52 (1H,dd, J 17 and 2.5 Hz, 6.alpha. 
--CH), 4.6-5.1 (4H,m, 3--CH, 8--CH and 9--CH.sub.2), 5.70 (1H,d, J 2.5 Hz, 
5--CH). 
EXAMPLE 26 
Benzyl 9-(5'-phenyltetrazol-1'-yl)-9-deoxyclavulanate 
Benzyl 9-benzamido-9-deoxyclavulanate (0.25 g, 0.64 mmole) was dissolved in 
dry dichloromethane (15 ml) and ice cooled. The solution was treated with 
pyridine (0.52 ml) and then with a 12.5 solution of phosgene in toluene 
(5.6 ml). This mixture was stirred at room temperature for 18 hours, 
evaporated under reduced pressure and the residue quickly redissolved in 
dry dichloromethane (15 ml). This solution was ice cooled, stirred and 
treated with tetramethylguanidinium azide (0.203 g, 1.3 mmole). Stirring 
was continued for 11/4 hours at room temperature and the resulting 
solution was washed with 0.5N hydrochloric acid (20 ml) and brine (20 ml), 
dried over MgSO.sub.4 and evaporated under reduced pressure. The residue 
was chromatographed on silica, eluting with ethyl acetate/petroleum ether 
1:2, and appropriate fractions were combined and evaporated to provide 
benzyl 9-(5'-phenyltetrazol-1'-yl)-9-deoxyclavulanate (0.182 g) as a 
colourless gum. [.alpha.].sub.D.sup.20 +21.2.degree. (c. 1.3; 
CHCl.sub.3); .nu..sub.max (CHCl.sub.3) 1805,1750,1695 cm.sup.-1 ; 
.delta.(CDCl.sub.3) 2.95 (1H,d,J 17 Hz, 6.beta.--CH), 3.47 (1H,dd,J 17 and 
2.5 Hz, 6.alpha.--CH) 4.7-4.95 (1H,m, 8--CH), 4.95-5.3 (5H,m,--OCH.sub.2 
Ph, 9--CH.sub.2, 3--CH), 5.63 (1H,d,J 2.5 Hz, 5--CH), 7.25 (5H,s,Ph--H), 
7.3-7.7 (5H,m,Ph--H); Found 417.1456: C.sub.22 H.sub.19 N.sub.5 O.sub.4 
requires 417.1438. 
EXAMPLE 27 
Potassium 9-(5'- henyltetrazol-1'-yl)-9-deoxyclavulanate 
Benzyl 9-(5'-phenyltetrazol-1'-yl)-9-deoxyclavulanate (0.18 g, 0.433 mmole) 
was dissolved in distilled tetrahydrofuran (10 ml), treated with 10% 
palladium/charcoal (50 mg) and hydrogenolysed for 30 minutes at 
atmospheric pressure. The suspension was filtered through celite and the 
filtrate evaporated under reduced pressure to 2 ml. Water (5 ml) was added 
and this solution was brought to pH 7 by dropwise addition of 0.5N aqueous 
KOH, washed with ethyl acetate (2.times.10 ml), concentrated to ca. 5 ml 
under reduced pressure and freeze dried. Potassium 
9-(5'-phenyltetrazol-1'-yl)-9-deoxyclavulanate was obtained as a yellow 
solid (0.11 g). .nu..sub.max (KBr) 1775,1695,1610 cm.sup.-1 ; 
.delta.(D.sub.2 O:HOD=4.60.delta.) 2.80 (1H,d, J 17 Hz, 6.beta.--CH), 3.44 
(1H,dd,J 17 and 2.5 Hz,6.alpha.--CH), 4.6-4.9 (2H,m,3--CH and 8--CH), 
5.0-5.2 (2H,m,9--CH.sub.2), 5.51 (1H,d,J 2.5 Hz, 5--CH), 7.54 
(5H,s,Ph--H). 
EXAMPLE 28 
tert-Butyldiphenylsilyl 9 -(tetrazol-2-yl) deoxyclavulanate 
(i) tert-Butyldiphenylsilyl clavulanate 
A solution of clavulanic acid (1.25 mmole) in distilled tetrahydrofuran (4 
ml) was treated with triethylamine (0.173 ml, 1.25 mmole) and then with 
tert-butyldiphenylsilyl chloride (0.325 ml, 1.25 mmole). After stirring at 
room temperature for 15 minutes, the suspension was filtered and the 
filtrate evaporated under reduced pressure. The residue was 
chromatographed on silica, eluting with ethyl acetate/petroleum ether 1:2, 
and appropriate fractions were combined and evaporated under reduced 
pressure to provide tert-butyldiphenylsilylclavulanate (0.36 g) as a 
colourless gum. .nu..sub.max (CHCl.sub.3) 1800, 1735,1690 cm.sup.-1 ; 
.delta.(CDCl.sub.3) 1.13 (9H,s,tBu) 1.65 (1H,br.s,OH), 3.14 (1H,d,J 17 Hz, 
6.beta.--CH), 3.53 (1H,dd,J 17 and 2.5 Hz, 6.alpha.--CH), 4.24 (2H,d,J 7.5 
Hz, 9--CH.sub.2), 4.8-5.3 (2H,m, 3--CH and 8--CH), 5.70 (1 H,d,J 2.5 Hz, 
5--CH), 7.3-7.9 (10H,m,Ph--H). 
(ii) tert-Butyldiphenylsilyl 9-(tetrazol-2-yl)deoxyclavulanate 
A mixture of tert-butyldiphenylsilylclavulanate (0.437 g, 1 mmole), 
tetrazole (0.14 g, 2 mmole) and triphenylphosphine (0.314 g, 1.2 mmole) 
were dissolved in distilled tetrahydrofuran (5 ml) and the solution cooled 
to -20.degree. under nitrogen. Diethylazodicarboxylate (0.197 ml, 1.2 
mmole) was then added all at once and the mixture stirred while gradually 
warming to room temperature over 30 minutes. The solution was evaporated 
under reduced pressure and the residue chromatographed on silica, eluting 
with ethyl acetate/petroleum ether 1:2. Fractions containing the second 
major .beta.-lactam containing component to be eluted were combined and 
evaporated under reduced pressure to provide tert-butyldiphenylsilyl 
9-(tetrazol-2-yl)-9-deoxyclavulanate (0.06 g) as a colourless gum. 
.nu..sub.max (CHCl.sub.3) 1800,1735,1695 cm.sup.-1 ; .delta.(CDCl.sub.3) 
1.10 (9H,s,tBu), 3.10 (1H,d,J 17 Hz, 6.beta.--CH), 3.57 (1H,dd,J 17 and 
2.5 Hz,6.alpha.--CH), 4.9- 5.6 (4H,m,3--CH, 8--CH and 9--CH.sub.2), 5.80 
(1H,d,J 2.5 Hz,5--CH), 7.0-7.9 (10H,m,Ph--H), 8.50 (1H,s,tetrazol--H). 
EXAMPLE 29 
Lithium 9-(tetrazol-2-yl)-9-deoxyclavulanate 
tert-Butyldiphenylsilyl 9-(tetrazol-2-yl)-9-deoxyclavulanate (30 mg) was 
dissolved in distilled tetrahydrofuran (2.7 ml), ice cooled and treated 
with 1N aqueous hydrochloric acid (0.3 ml). The solution was stirred at 
room temperature for 45 minutes and saturated brine (10 ml) was added. The 
resulting mixture was extracted with ethyl acetate (2.times.10 ml) and the 
ethyl acetate dried over MgSO.sub.4 and evaporated under reduced pressure. 
The residue was taken up in distilled tetrahydrofuran (2 ml)/water (2 ml) 
and the solution brought to pH 7 by dropwise addition of 0.2N LiOH 
solution. A further 5 ml water was added and the mixture washed with ethyl 
acetate (3.times.10 ml) and freeze dried to provide lithium 
9-(tetrazol-2-yl)-9-deoxyclavulanate (16 mg) as a pale yellow solid 
identical with the product of example 7. 
EXAMPLE 30 
Triisopropylsilyl 9-(tetrazol-2-yl)-9-deoxyclavulanate 
(i) Triisopropylsilyl clavulanate 
A solution of clavulanic acid (9 mmole) in distilled tetrahydrofuran (40 
ml) was stirred at room temperature and treated with triethylamine (1.25 
ml) and then with triisopropylsilyl chloride (1.74 g). After stirring at 
room temperature for 15 minutes the suspension was filtered and the 
filtrate evaporated under reduced pressure. The residue was 
chromatographed on silica, eluting with ethyl acetate/hexane 2:3, and 
appropriate fractions were combined and evaporated under reduced presssure 
to provide triisopropylsilyl clavulanate (2.47 g) as a colourless oil. 
.nu..sub.max (CHCl.sub.3) 1800,1730,1690 cm.sup.-1 ; .delta.(CDCl.sub.3), 
0.8-2.0 (21H,m,(Pr.sup.i).sub.3), 2.18 (1H,broad s, --OH), 3.03 (1H,d,J 17 
Hz, 6.beta.--CH), 3.47 (1H,dd,J 17 and 2.5 Hz, 6.alpha.--CH), 4.23 
(2H,broad d, J 7 Hz, 9--CH.sub.2), 4.8-5.1 (2H,m,3--CH and 8--CH), 5.67 
(1H,d,J 2.5 Hz, 5--CH). 
(ii) Triisopropylsilyl 9-(tetrazol-2-yl)-9-deoxyclavulanate 
Triisopropylsilylclavulanate (1.20 g, 3.38 mmole), tetrazole (0.5 g, 7.15 
mmole) and triphenylphosphine (1.06 g, 4.05 mmole) were dissolved in 
distilled tetrahydrofuran (20 ml), cooled to -10.degree. C. and treated 
with diethylazodicarboxylate (0.665 ml, 4.05 mmole). The mixture was 
stirred for 15 minutes while warming to about 10.degree. C. and evaporated 
under reduced pressure. The residue was chromatographed on silica, eluting 
with ethyl acetate/hexane 1:2. Fractions containing the second major 
.beta.-lactam containing component to be eluted were combined and 
evaporated under reduced pressure to provide triisopropylsilyl 
9-(tetrazol-2-yl)-9-deoxyclavulanate (190 mg) as a colourless gum. 
.nu..sub.max (CHCl.sub.3) 1800,1730,1700 cm.sup.-1 ; .delta.(CDCl.sub.3) 
0.9-1.6 (21H,m, (Pr.sup.i).sub.3), 3.10 (1H,d,J 17 Hz,6.beta.--CH), 3.54 
(1H,dd,J Hz,6.alpha.--CH), 4.9-5.2 (2H,m,3--CH and 8--CH), 5.3-5.5 
(2H,m,9--CH.sub.2), 5.79 (1H,d, J 2.5 Hz, 5--CH), 8.47 (1H,s,tetrazol--H). 
EXAMPLE 31 
Lithium 9-(tetrazol-2-yl)-9-deoxyclavulanate 
Triisopropylsilyl 9-(tetrazol-2-yl)-9-deoxyclavulanate (90 mg) was 
dissolved in distilled tetrahydrofuran (9 ml), ice cooled and treated with 
1N aqueous hydrochloric acid (1 ml). The solution was stirred 25 minutes 
at 0.degree.-5.degree. C. and saturated brine (20 ml) added. The resulting 
mixture was extracted with ethyl acetate (2.times.30 ml) and the ethyl 
acetate dried over MgSO.sub.4 and evaporated. The residue was taken up in 
distilled tetrahydrofuran (5 ml) and water (5 ml) and the solution brought 
to pH 7 with 1N LiOH solution. A further 10 ml water was added and the 
mixture washed with ethyl acetate (2.times.20 ml) and freeze dried to 
provide lithium 9-(tetrazol-2-yl)-9-deoxyclavulanate (51 mg) as a pale 
yellow solid identical with the product of example 7. 
EXAMPLE 32 
Benzyl 9-(5-tert.butyltetrazol-1-yl)deoxyclavulanate 
A solution of benzyl 9-aminodeoxyclavulanate in ethyl acetate was reacted 
with pivaloyl chloride to afford the pivalamide (0.36 g). The pivalamide 
(0.256 g) in dry dichloromethane (10 ml) was cooled to 0.degree. C. 
Pyridine (0.11 ml) and 12.5% COCl.sub.2 in toluene (1.08 ml) were added, 
stirred for 6 hours, then evaporated to dryness. The residue was taken up 
in dry dichloromethane and stirred at 0.degree. C. Tetramethylguanidinium 
azide (0.286 g) was added, the reaction mixture was stirred for one hour 
at ambient temperature, washed with 0.5M aqueous HCl and dried over 
MgSO.sub.4. The desiccant was filtered off and the filtrate evaporated. 
The residue was subjected to column chromatography on silica gel using 2:1 
ethyl acetate-cyclohexane as eluent. Fractions containing the desired 
product were combined and evaporated, to yield the title product (0.16 g); 
Ir (film) 1800 (.beta.-lactam C.dbd.O) 1745 (ester) 1695 (C.dbd.C). 
EXAMPLE 33 
Potassium 9(5-tert.butyltetrazol-1-yl)deoxyclavulanate 
The benzyl ester (0.237 g) in redistilled tetrahydrofuran (20 ml) was 
hydrogenated over 10% palladised charcoal (monitored by tlc). After 45 
minutes the catalyst was filtered off, the filtrate cooled to 0.degree. C. 
and diluted with water (40 ml). The solution was adjusted to pH 7 with 1M 
potassiumhydroxide solution and evaporated to dryness to yield 0.2 g of 
crude potassium salt. The product was chromatographed on silica gel using 
2:1:1 butanol-ethanol-water as elution solvent. Fractions containing the 
desired product were combined and evaporated, to yield 0.16 g of pure 
product; nmr (D.sub.2 O).delta.;1.46 (9H,s,tert Bu) 3.07 (1H,d,J 17 
Hz,6-.beta.--CH) 3.56 (1H,dd,J 3 and 17 Hz, 6.alpha.--CH) ca 4.95 
(2H,m,3--CH and 8--CH) 5.17-5.37 (2H,m,9--CH.sub.2) and 5.75 (1H,d, J 3 
Hz, 5--CH). 
EXAMPLE 34 
Benzyl 9(5-formamidotetrazoyl) deoxyclavulanate 
(i) 5-Formamidotetrazole 
5-Aminotetrazole monohydrate (10 g) was heated under reflux in anhydrous 
formic acid for 48 hours. The mixture was cooled, and the 
5-formamidotetrazole filtered off, washed with formic acid, ether, and 
dried in vacuo, to yield 8.3 g of the tetrazole: IR(nujol null) C.dbd.O at 
1680 cm.sup.-1. 
(ii) Benzyl 9-(5-formamidotetrazolyl)deoxyclavulanate 
Following the procedure of Example II, the title compound was obtained with 
an Rf (in 1:1 ethyl acetate-cyclohexane) of about 0.4; above 
triphenylphosphine oxide: IR (liquid film) 1800 (.beta.-lactam C.dbd.O), 
1745 (ester C.dbd.O) and 1695 cm.sup.-1 (C.dbd.C and amide C.dbd.O). 
EXAMPLE 35 
Lithium 9-(5-formamidotetrazolyl)deoxyclavulanate 
The product from Example 34 (0.45 g) in tetrahydrofuran was hydrogenated 
over 10% palladised charcoal (0.2 g). When the reaction was complete, the 
catalyst was removed by filtration, water added and the mixture titrated 
to pH 7.0 with 1M LiOH. The solution was evaporated to dryness, triturated 
with acetone and the product collected by filtration and dried: IR (nujol) 
1770 (.beta.-lactam C.dbd.C) 1700 (broad, C.dbd.C and amide C.dbd.O) 1603 
and 1575 cm.sup.-1 (CO.sub.2.sup.-): Ir (KBr disc) 3400 (NH, H.sub.2 O) 
1780 
(.beta.-lactam C.dbd.O) 1700 (C.dbd.C and amide C.dbd.O) 1610 and 1580 
cm.sup.-1 (CO.sub.2.sup.-). 
Demonstration of Effectiveness 
In a standard MIC test the following data were obtained for the synergistic 
activity of tetrazolyl derivatives with amoxycillin. 
______________________________________ 
dose of 
Amoxycillin and 
compound MIC .mu.g/ml amoxycillin 
compound of 
of this St. aureus 
K. aerogenes 
E. coli 
example number 
invention Russell E70 JT39 
______________________________________ 
7 5.0 .mu.g/ml 
0.02 0.4 2.0 
1.0 .mu.g/ml 
0.6 3.1 4.0 
10 5.0 .mu.g/ml 
0.04 0.4 -- 
1.0 .mu.g/ml 
0.3 3.1 4.0 
12 5.0 .mu.g/ml 
0.08 1.5 .ltoreq.0.5 
1.0 .mu.g/ml 
0.3 3.1 4.0 
35 5.0 .mu.g/ml 
0.16 1.6 4.0 
1.0 .mu.g/ml 
0.6 6.2 8.0 
14 5.0 .mu.g/ml 
0.8 3.1 0.5 
1.0 .mu.g/ml 
3.1 6.2 8.0 
15 5.0 .mu.g/ml 
0.3 1.6 4.0 
1.0 .mu.g/ml 
2.5 6.2 31.2 
18 5.0 .mu.g/ml 
0.08 0.4 2.0 
1.0 .mu.g/ml 
0.6 3.1 4.0 
27 5.0 .mu.g/ml 
-- 100 62.5 
1.0 .mu.g/ml 
0.4 &gt;100 500 
25 5.0 .mu.g/ml 
0.16 0.8 -- 
1.0 .mu.g/ml 
0.6 3.1 8.0 
amoxycillinalone 500 1000 2000 
______________________________________ 
In a standard MIC test the following antibacterial activities were 
obtained. 
______________________________________ 
Compound of 
St. aureus K. aerogenes 
E. coli 
example No. 
Russell E70 JT39 
______________________________________ 
7 16.0 16.0 16.0 
10 8.0 31.2 8.0 
12 16.0 31.2 16.0 
35 16.0 62.5 31.2 
14 8.0 31.2 8.0 
15 16.0 31.2 16.0 
18 16.0 62.5 16.0 
27 4.0 &gt;500 125 
25 16.0 62.5 8.0 
______________________________________