Method for the preparation of penicillanic-.beta.-lactamase inhibitors

In vivo hydrolyzable esters of 6.beta.-bromopenicillanic acid when substantially free from the corresponding in vivo hydrolyzable ester of 6.alpha.-bromopenicillanic acid is useful for their .beta.-lactamase inhibitory activity and may be administered either per se or in combination with a penicillin or cephalosporin.

This invention relates to penicillanic acid derivatives and in particular 
to a class of 6 .beta.-bromopenicillanic acid derivatives. 
It is known that 6-.beta.-bromopenicillanic acid is a .beta.-lactamase 
inhibitor [see Loosemore et al., J. Org. Chem., 43, 3611 (1978); Pratt et 
al., Proc. Natl. Acad. Sci. U.S.A., 75, 4145 (1978); Knott-Hunziker et 
al., F.E.B.S. Letters, 59, (1979); and Knott-Hunziker et al., Biochem J., 
177, 365 (1979)]. However in the past 6-.beta.-bromopenicillanic acid has 
always been described as a minor component of a mixture together with 
6-.alpha.-bromopenicillanic acid. No active esters of 
6-.beta.-bromopenicillanic acid have yet been described whether pure or in 
admixture with the corresponding 6-.alpha.-compound. The 
6-.beta.-bromopenicillanic acid tends to be unstable and is apparently 
readily isomerised to a mixture with the 6-.alpha.-compound. It would be 
desirable to provide a compound that released 6-.beta.-bromopenicillanic 
acid in-vivo without producing beforehand dominating quantities of the 
6-.alpha.-compounds. Such desirable compounds have now been produced. 
The present invention provides in-vivo hydrolysable esters of 
6-.beta.-bromopenicillanic acid when substantially free from the 
corresponding in-vivo hydrolysable ester of 6-.alpha.-bromopenicillanic 
acid. 
When use of herein the term "substantially free from" means "not 
contaminated by more than 15% w/w". 
More suitably the 6-.beta.-compound of this invention is not contaminated 
by more than 10% w/w of the corresponding 6-.alpha.-compound, favourably 
is not contaminated by more than 5% w/w of the corresponding 
6-.alpha.-compound, preferably less than 590 w/w, and preferably is not 
contaminated by more than 2% w/w w/w of the corresponding 
6-.alpha.-compound. 
The presence of any contaminating 6-.alpha.-compound may be estimated by 
standard analytical techniques such as n.m.r. spectroscopy or h.p.l.c. 
In-vivo esters are those which hydrolyse in the human body to produce the 
parent acid. 
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 6-bromopenicillanate in the blood or urine of the animal. 
Alternatively hydrolysis in human blood or serum may be determined. 
Suitable in-vivo hydrolysable ester groups are those derivatives of a 
penicillin (i.e. a 6-.beta.-acylamino penicillanic acid) which are 
hydrolysable in-vivo. 
In-vivo hydrolysable esters of this invention include those of the formula 
(I): 
##STR1## 
wherein R.sub.1 is a hydrogen atom or a methyl group; R.sub.2 is an alkyl 
group of 1 to 6 carbon atoms, a phenyl group, an alkyl group of 1 to 3 
carbon atoms substituted by a phenyl group, an alkoxyl group of 1 to 6 
carbon atoms, a phenoxyl group, or an alkoxyl group of 1 to 3 carbon atoms 
substituted by a phenyl group; or R.sub.1 is attached to R.sub.2 to form a 
1,2-diphenylene or 4,5-dimethoxy-1,2-diphenylene group. 
Favourably R.sub.1 is hydrogen. 
When R.sub.1 is hydrogen suitably R.sub.2 is selected from methyl, ethyl, 
n-propyl, iso-propyl, n-butyl, tert-butyl, phenyl, benzyl, methoxy, 
ethoxy, n-propyloxyl and iso-propyloxy. Preferably R.sub.2 is tert-butyl. 
Favourably R.sub.1 and R.sub.2 are joined so that the ester is a phthalidyl 
or 3,4-dimethoxyphthalidyl ester. 
Thus preferred in-vivo hydrolysable ester groups include acetoxymethyl, 
pivaloyloxymethyl, .alpha.-acetoxyethyl, .alpha.-acetoxybenzyl, 
.alpha.-pivaloyloxyethyl, ethoxycarbonyloxymethyl, 
.alpha.-ethoxycarbonylethyl, phthalidyl and 5,6-dimethoxyphthalidyl. 
Other suitable in-vivo hydrolysable groups include dialkylamino alkyl 
groups such as dimethylaminomethyl, dimethylaminoethyl, diethylaminomethyl 
and diethylaminoethyl; and N-phthalimindomethyl and methoxymethyl groups. 
The present invention also provides a pharmaceutical composition which 
comprises an in-vivo hydrolysable ester of 6-.beta.-bromopenicillanic acid 
and a pharmaceutically acceptable carrier therefor said composition being 
substantially free from the corresponding in-vivo hydrolysable ester of 
6-.beta.-bromopenicillanic acid. 
The composition of this invention may be adopted for administration by 
injection or by the oral route. 
Generally the composition will be presented as a unit dose containing from 
60 to 600 mgs. of the .beta.-lactamase inhibitor, more suitably 100 to 500 
mgs. of the .beta.-lactamase inhibitor and preferably from 125 to 300 mgs. 
of the inhibitor. Such compositions may be administered 2-6 times daily 
and usually 3 or 4 times daily in a manner such that the total daily dose 
for a 70 kg human will be about 200 to 1000 mgs. 
The composition of this invention may be administered concurrently or 
consecutively with a penicillin or cephalosporin. However, it is greatly 
preferred to administer the penicillin or cephalosporin in the same 
composition as the .beta.-lactamase inhibitor of this invention. British 
Patent Specification No. 1,508,978 discloses suitable penicillins and 
cephalosporins for use in such synergistic compositions. The forms of 
compositions, methods of preparation and ratios of components disclosed in 
Specification No. 1,508,978 may be also used with the synergists of this 
invention. The disclosures of Specification 1,508,978 with respect to 
compositions are accordingly incorporated herein by reference. A suitable 
penicillin is amoxycillin as the trihydrate or sodium salt. 
The esters of the present invention may be used in such compositions in 
place of the esters of clavulanic acid. 
The present invention also provides a process for the preparation of an 
in-vivo hydrolysable ester of 6-.beta.-bromopenicillanic acid when 
substantially free from the corresponding in-vivo hydrolysable ester of 
6-.alpha.-bromopenicillanic acid which process comprises preparing a 
mixture of isomeric in-vivo hydrolysable esters of 6.alpha.- and 
6.beta.-bromopenicillanic acids and thereafter chromatographically 
separating the mixture of isomers into fractions and recovering the 
in-vivo hydrolysable ester of 6-.beta.-bromopenicillanic acid from a 
fraction containing said in-vivo hydrolysable ester of 
6-.beta.-bromopenicillanic acid substantially free from the in-vivo 
hydrolysable ester of a 6-.alpha.-bromopenicillanic acid. 
The esterification step is generally effected by reaction of the salts with 
a reactive halide or the like, for example, with a compound of the formula 
(II): 
EQU X--CHR.sub.1 --O--CO--R.sub.2 (II) 
wherein R.sub.1 and R.sub.2 are as defined in relation to formula (I) and X 
is Cl, Br or I. 
Esterification to afford the dialkylaminoalkyl, N-phthalimidomethyl and 
methoxymethyl esters is similarly performed, for example using 
dialkylaminoalkyl halides, N-(chloromethyl)phthalimide and chloromethyl 
methyl ether respectively. 
Alternatively the mixture of isomeric in-vivo hydrolysable esters of 
6.alpha.- and 6.beta.-bromopenicillanic acid may be prepared by the 
treatment of an in-vivo hydrolysable ester of 6,6-dibromopenicillanic acid 
with a strong organic base such as methyl lithium followed by protonation. 
Protonation is most conveniently effected by acetic acid. The treatment 
with brine is carried out in anhydrous conditions at a low temperature, 
for example under nitrogen in a dry solvent at -78.degree. C. In-vivo 
hydrolysable esters of 6,6-dibromopenicillanic acid are prepared by 
conventional methods of esterification. 
Such esterification reactions to prepare a mixture of isomeric in-vivo 
hydrolysable esters of 6.alpha.- and 6.beta.-bromopenicillanic acids will 
be carried out under conditions similar to those known to be suitable for 
the preparation of in-vivo hydrolysable esters of penicillins. For example 
the reaction may be carried out in an organic solvent such as 
dimethylformamide, optionally together with methylene chloride. 
The salt of the mixture of 6-bromopenicillanic acids may be generated in 
situ, for example by using an organic tertiary base such as trimethylamine 
or triethylamine. 
Since known mixtures of 6-bromopenicillanic acids contain only a minor 
proportion (usually about 12% w/w) of the desired 6-.beta.-bromo isomer, 
the resulting mixture of in-vivo hydrolysable esters only contains a minor 
proportion of the desired 6-.beta.-bromo isomer. As will be appreciated by 
the skilled chemist obtaining a reactive minor component from a mixture of 
materials is difficult and not necessarily possible. Fortunately it has 
been found that repeated chromatography of the mixture of in-vivo 
hydrolysable esters can produce the desired in-vivo hydrolysable ester 
6-.beta.-bromo isomer substantially free from the corresponding in-vivo 
hydrolysable ester of the 6-.alpha.-bromo isomer. 
Suitable methods of chromatography include column chromatography using 
silica gel as stationary phase. Gradient elution using ethyl 
acetate/cyclohexane mixtures has been found suitable. The progress of the 
individual product through the chromatographic system may be monitored by 
thin layer chromatography and developing with Ehrlich's reagent. The 
initial major product is the 6-.alpha.-isomer and the initial minor 
product is the 6-.beta.-isomer so that Rf values and relative positions 
may be known. 
Initial separation of the two isomers is not good so that the leading edge 
of the faster running component or the trailing edge of the slower running 
component is selected for re-chromatography. Two or three repetitions of 
this chromatographic process are normally sufficient to produce the 
desired substantially pure product. In the silica gel ethyl 
acetate/cyclohexane system described in Example 1 herein the 
6-.beta.-isomer is the slower running component so that the trailing edge 
of the slower running component is selected for rechromatography. 
The 6.alpha., .beta. starting material may be obtained as described in the 
Loosemore et al., reference referred to hereinbefore.

The following Examples illustrate the process of this invention: 
EXAMPLE 1 
Preparation of pivaloyloxymethyl 6.beta.-bromopenicillanate (method 1) 
6.alpha./.beta.-Bromopenicillanic acid (10 g) was dissolved/suspended in 
dimethylformamide (600 ml) containing methylene chloride (200 ml) and was 
treated sequentially with triethylamine (12 ml) and bromomethyl pivalate 
(10.4 ml) with stirring at 0.degree. C. The reaction was allowed to warm 
to room temperature and was monitored by thin layer chromatography. After 
2 hours, the reaction mixture was evaporated in vacuo to approx. 100 ml; 
it was diluted with chloroform (400 ml), washed with water (2.times.400 
ml), dried (MgSO.sub.4), and evaporated in vacuo to afford a gum. This gum 
was subjected to column chromatography on silica gel (80 g) using ethyl 
acetate:cyclohexane (1:5.fwdarw.1:7). The appropriate fractions were 
combined and evaporated in vacuo to afford pivaloyloxymethyl 
6.beta.-bromopenicillanate as an impure gum. This was resubjected to 
column chromatography on silica gel (80 g) using ethyl acetate:cyclohexane 
(1:8.fwdarw.1:10) as eluant). The appropriate fractions were combined and 
evaporated in vacuo to afford pivaloyloxymethyl 6.beta.-bromopenicillanate 
(0.23 g), i.r. (liq. film) 1790, 1750 cm.sup.-1, n.m.r. (CDCl.sub.3) 1.20 
(9H, s, C(CH.sub.3).sub.3), 1.48 (3H, s, CH.sub.3), 4.52 (1H, s, H-3), 
5.32 and 5.56 (2H, 2d, J=4 Hz, H-5 and H-6), 5.8 (2H, ABq J=5 Hz, 
--CH.sub.2 --) ppm. 
(Combination and evaporation in vacuo of some earlier fractions afforded a 
mixture of pivaloyloxymethyl 6.alpha.-bromopenicillanate and the title 
compound (1:1) (0.25 g) which could be rechromatographed if desired). 
EXAMPLE 2 
Preparation of pivaloyloxymethyl 6.beta.-bromopenicillanate (method 2) 
Methyllithium (ca 2 M, 4 ml) was added to a solution of pivaloyloxymethyl 
6,6-bromopenicillanate (2.75 g) in toluene (150 ml) at -78.degree. under 
nitrogen. The mixture was stirred at -78.degree. for 10 min. Acetic acid 
(1 ml) in toluene (10 ml) was then added and the mixture was allowed to 
come to room temperature. This was washed with water, dried and evaporated 
to dryness. The residue was chromatographed over silica gel (60 g). 
Elution of the column with light petrol-ethyl acetate (4:1) gave 
pivaloyloxymethyl 6.beta.-bromopenicillanate (0.2 g) identical to an 
authentic sample (t.l.c. and n.m.r. comparisons). 
DEMONSTRATION OF EFFECTIVENESS 
The MIC of ampicillin was determined in the presence of the 
pivaloyloxymethyl ester of 6-.beta.-bromopenicillanic acid (inhibitor). 
The results obtained were as follows: 
______________________________________ 
MIC (.mu.g/ml) of Ampicillin 
Inhibitor Klebsiella 
conc. Staph. aureus 
aerogenes Proteus sp. 
E. coli 
(.mu.g/ml) 
Russell E70 C889 JT. 39 
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20 0.78 6.2 8 2 
5 3.1 12.5 31 4 
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