EP0127636A1 - Compounds containing beta-lactams - Google Patents

Abstract

Compound of formula (I) or its salt or its ester in which A represents -CH2- or -CH2-CH2-.

Description

COMPOUNDS CONTAINING β-LACTAMS

This invention relates to a class of novel β-lactam compounds, to the process for their preparation and to pharmaceutical compositions containing them.

U.K. Patent Nos. 1,508,977 and 1,508,978 disclose inter alia clavulanic acid and its salts and esters. Clavulanic acid has the formula (A):

We have now found a novel class of compounds containing a cyanide group, which have anti-bacterial and β-lactamase inhibitory activity.

Accordingly, the present invention provides a compound of formula (I) or a salt or ester thereof :

wherein A represents -CH₂- or -CH₂-CH₂-. The major utility of the compound of formula (I) is as a pharmaceutical and, accordingly, the salts and esters of the compound of formula (I) are preferably pharmaceutically acceptable. The compound of formula (I) may also be used as an anti-bacterial or β-lactamase inhibitor in non-pharmaceutical uses such as, for example, as a disinfectant or paint additive; those salts and esters which are not normally considered to be pharmaceutically acceptable are suitable for this application.

Suitably A represents -CH₂-.

Suitably A represents -CH₂-CH₂-.

Suitable pharmaceutically acceptable esters of the compound of formula (I) include in vivo hydrolysable esters,

Suitable pharmaceutically acceptable salts of the compound of formula (I) include metal salts, eg 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, cycloalkylamines such as bicyclohexylamine, or with dibenzylamine, N,N-dibenzylethylenediamine, 1-ephenamine, N-ethylpiperidine, or N-benzyl-β-phenethy¬- amine.

Particularly suitable pharmaceutically acceptable salts of the compound of formula (I) are the alkali metal salts such as lithium, sodium and potassium. The preferred pharmaceutically acceptable salts are the sodium and potassium salts. Examples of suitable pharmaceutically acceptable in vivo hydrolysable ester (groups include those which break down readily in the human body to leave the parent acid or its salt, for example acyloxyalkyl groups such as acetoxymethyl, pivaloyloxymethyl, α-acetoxyethyl and α-pivaloyloxmethyl groups; alkoxycarbonyloxyalkyl groups, such as ethoxycarbonyloxymethyl and α-ethoxycarbonyloxyethyl; dialkylamino alkyl groups such as dimethylaminoethyl, dimethylaminoethyl, diethylaminomethyl or diethylaminoethyl and lactone groups such as phthalidyl or dimethoxyphthalidyl.

The present invention also provides a pharmaceutical composition which comprises a compound of this invention 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 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 anti-biotics.

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 invent! on 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 penicillin or cephalosporin which shows instability to β-lactamases since the resulting composition shows enhanced effectiveness (synergy). Suitable penicillins, cephalosporins or other β-lactam anti-biotics for inclusion in such synergistic compositions include not only those known to be highly susceptible to β-lactamases but also those which have a degree of intrinsic resistance to β-lactamases.

Suitable penicillins for inclusion in the compositions of this invention include benzylpenicillin, phenoxymethyl-penicillin, carbenicillin, azidocillin, propicillin, ampicillin, amoxycillin, epicillin, ticarcillin, cyclacillin, pirbenicillin, azlocillin, mezlocillin, sulbenicillin, pipericillin, and other known penicillins including pro-drugs therefor such as their in vivo hydrolysable esters such as the acetoxymethyl, pivaloyloxymethyl, α-ethoxycarbonyloxyethyl or phthalidyl esters of ampicillin, benzylpenicillin or amoxycillin, and aldehyde or ketone adducts of penicillins containing a 6-α-aminoacetamide side chain (such as hetacillin, metampicillin and analogous derivatives of amoxycillin) or α-esters of carbenicillin or ticarcillin such as their phenyl or indanyl α-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 pro-drugs thereof.

Such compounds are frequently used in the form of a salt or hydrate of the like. Naturally if the penicillin or cephalosporin present in the composition is not suitable for oral administration then the composition will be adapted for parenteral administration.

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 find 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 penicillin or cephalosporin agent may vary over a wide range of ratios , such as from 10:1 to 1:10 for example about 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5 or 1:6, (wt/wt, based on pure free anti-biotic equivalent). Orally administrable compositions containing a compound of the invention will normally contain relatively more synergist than corresponding injectable compositions. 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 for adult human treatment 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 composition of this invention will contain from 150 to 1000 mg of ampicillin or a pro-drug therefor and from 25 to 500 mg of a compound of this invention.

Most suitably this form of composition will contain ampicillin trihydrate, ampicillin anhydrate, sodium ampicillin, hetacillin, pivampicillinhydrochloride, bacampicillin hydrochloride, or talarapicillin hydrochloride. Most suitably this form of the composition will contain a compound of the formula (I) 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 (I) 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 therefor 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 (I) 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 humans or animals including 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 aeroqenes, Escherichia coli, Proteus sp. , Bacteroides fraqilis or the like. The organisms believed to be most readily treated by an anti-bacterially 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.

The present invention also provides a process for the preparation of a compound of formula (I) or a salt or ester thereof, which process comprises mildly oxidising a compound of formula (II):

wherein R^x is a carboxy protecting group and Z is S-A-CN or SO-A-CN wherein A is as hereinbefore defined

and thereafter where necessary carrying out one or more of the following steps :

(a) removing the carboxy protecting group R^x

(b) converting a salt to the free carboxylic acid or to an ester, or to a different salt.

The oxidation, may take place at an ambient or depressed temperature, for example at -20°C to +20°C, more suitably at -12°C to +5°C, for example at about 0°C.

The oxidation is best brought about using an organic per-acid as the oxidizing agent. Suitable acids include m-chloroperbenzoic acid arid equivalent reagents. Use of one equivalent of the oxidizing agent when Z is S-A-CN leads to a compound of the formula (II) wherein Z is SO-A-CN whereas the use of an excess of the oxidising agent leads to a compound of the formula

(I); Similarly, use of one equivalent of the oxidising agent when Z is SO-A-CN leads to a compound of formula

(I). lt is normal to carry out the oxidation in an inert solvent such as methylene chloride or the like.

The compounds of formula (II) wherein Z is S-A-CN may be prepared by reactirig a compound of formula (III) with a thiol of formula (IV) or a salt thereof in the presence of a Lewis acid catalyst.

HS- A-CN (IV)

In formula (III), R^x is as defined for formula (II)

Suitable Lewis acid catalysts include boron trifluoride or its equivalent such as a boron trifluoride etherate, for example BF₃.O(C₂H₅)₂.

The preceding reaction normally takes place in a solvent inert under the reaction conditions such as chloroform, dichloromethane, tetrahydrofuran or dioxane or may be carried out using the thiol reagent as solvent. The reaction is generally carried out at a depressed or non-elevated temperature for example -80ºC to +30ºC, and preferably at a depressed temperature, for example -20ºC to 0ºC, and conveniently at about

-10°C. The compounds of formula (II) wherein Z is S-A-CN may also be prepared by the reaction of a compound (V) with a thiolate of a thiol of formula (IV).

In formula (V) R^x is as defined for formula (II) and Z¹ is a displaceable atom or group.

Suitable moieties Z¹ displaceable by the thiolate include conventional displaceable groups such as sulphonate or carboxylate esters such as OSO₂R^y, OCOR^y or OCO₂R^y where R^y is an inert organic group such as a lower alkyl group (such as a methyl, ethyl, propyl or butyl group) optionally substituted by one or more halogen atoms, a phenyl, benzyl, methoxyphenyl, methylphenyl, halophenyl, nitrophenyl or like group.

Other suitable moieties Z¹ displaceable by the thiolate are halogen atoms, for example a chlorine or bromine atom.

Preferred values of Z¹ are a chlorine or bromine atom, or a dichloroacetoxy, mesyloxy, tosyloxy or phenylsulphonyloxy group. Particularly preferred values of

Z¹ are chlorine and dichloroacetoxy. The thioetherification reaction may be carried out at a non-extreme temperature such as -60ºC to +60ºC, preferably from -30°C. The thioetherification reaction may be performed in an inert non-hydroxylic solvent such as dimethylformamide.

Suitable carboxy protecting groups for the group -CO₂R^x in formula (II) include ester derivatives of the carboxylic acid. The derivative is preferably one which may readily be cleaved at a later stage of the reaction.

Suitable ester-forming carboxyl-blocking groups are those which may be removed under conventional conditions. Such groups for R^x include benzyl, p-methoxybenzyl,p-nitrobenzyl, 2,4,6-trimethylbenzyl, 3,5-di-t-butyl - benzyl, 4-pyridylmethyl, allyl, diphenylmethyl, triphenymethyl, 2-benzyloxphenyl, 4-methylthiophenyl, methoxymethyl, a silyl, or phosphorus-V-containing group, or methyl or ethyl.

The carboxylic group or a salt thereof may be regenerated from any of the above esters by usual methods appropriate to the particular R^x group, for example, base-catalysed hydrolysis, or by enzymically-catalysed hydrolysis, or by hydrogenation, or by dissolving metal reduction.

Acids within formula (I) may also be prepared by the careful acidification of a corresponding salt such as the sodium salt.

Salts within formula (I) may be prepared by treatment of an acid within formula (I) with, for example, sodium ethyl hexanoate or potassium ethyl hexanoate. Salts within formula (I) may also be prepared by salt exchange in conventional manner, for example a solution of the lithium salt in water may be passed through a bed of ion exchange resin in the sodium form (e.g. Amberlite 120; a sodium salt of a sulphonated polystyrene divinyl benzene co-polymer)

in about ten-fold excess until elution is complete; the resulting sodium salt may be obtained by freeze drying or by evaporaition to crystallisation. Similarly, a sodium salt may be converted to a lithium salt or to a potassium salt in similar manner.

The following Examples illustrate the invention.

Preparation 1a 4-Nitrobenzyl (3R,5R,Z)-2-[2-cyanoethylthio-ethylidene]

- clavam-3-carboxylate

p-Nitrobenzyl-9-0-dichloroacetyl clavulanate (10.5g; 0.0234m) was dissolved in dry dimethylformamide (100ml) and 3-mercaptopropionitrile (3.57g; 0.041m) was added. The reaction was cooled to -30° and treated dropwise with a solution of triethylamine(2.3-4g;0.0234m) in dry dimethylformamide (40ml). The mixture was stirred at -30ºfor 0.5 hr. and was then allowed to warm up to 0º . The reaction mixture was then diluted with ethyl acetate and the solution washed thoroughly with water. After washing the organic layer with brine and drying over anhydrous magnesium sulphate the ethylacetate solution was evaporated. Chromatography of the residue on silica using 1:1 ethylacetate-petrol as eluent gave the title compound as a colourless oil(7.17g;76%) [α]_D ²⁰ +18.77[c 1.0;CHCl₃], vmax. (CHCl₃) 2250, 1805, 1755, 1690 and 1610 cm^-1. δ(CDCl₃) 2.60 (4H,s,SCH₂CH₂CN) , 3.07(1H, d, J 17HZ, 6β-CH), 3.28 (2H,9-CH₂), 3.52(1H,dd,J 17 and 3HZ, 6α-CH), 4..71(1H,t,J 7HZ, 8-CH), 5.13(1H,S,3-CH), 5.28 (2H,s, Ar-CH₂), 5.69(1H, d, J 3HZ, 5-CH) ,7.52(2H,d) and 8.23(2H,d) [Aromatic H].

Preparation 1b 4-Nitrobenzyl (3R, 5R,Z)-2-[2-Cyanoethylsulphinylethylidene]-clavam-3-carboxylate

p-Nitrobenzyl(3R,5R,Z)-2-[2-cyanoethylthioethylidene]-clavam-3- carboxylate (172mg) was dissolved in dichlormethane(10ml), ice-cooled with stiring and treated dropwise over 5 mins. with a solution of m-chloroperbenzoic acid (73mg.) in dichloromethane(5ml). After stiring for 1½ hour the solution was washed with sat. aqueous sodium bicarbonate solution and water, dried over anhydrons magnesium sulphate and evaporated. The residue was chromatographed on silica with ethylacetate as eluant to give the title compound as a pale yellow oil (54mg, 30%). [α]_D ²⁰+ 13.5°[c 1.0; CHCl₃]; v max(CHCl₃) 1810, 1760, 1695 and 1610 cm^-1.; δ(CDCl₃) 2.85(4H,brs. , -CH₂CH₂-), 3.10 (1H,d,J17HZ, 6β-CH), 3.47-3.66(3H,m,6αCH and 9-CH₂) , 4.79 (1H,t,J 7HZ, 8-CH), 5.21(1H,S, 3-CH) , 5.28(2H,S, Ar-CH₂), 5.77(1H,5-CH), 7.51 and 8.14(4H,2d,Ar-H) .

Example 1

4-Nitrobenzyl (3R, 5R,Z)-2-[2-cyanoethylsulphonylethylidene] - clavam-3- carboxylate

A stired, ice-cooled solution of p-nitrobenzyl-(3R,5R,Z)-2-(2-cyano ethylthioethylidene] - clavam-3-carboxylate (7.10g;0.0177m) in dichloromethane (400ml) was treated dropwise over 15 mins with a solution of m-choroperbenzoic acid (7.63g; 0.044m) in dichloromethane (150ml). The mixture was stirred at 0-5°for 1½ hour and then allowed to warm to room temperature. The mixture was washed with saturatedaqueoussodium bicarbonate, water, and brine, dried over magnesium sulphate and evaporated. Trituration of the solid residue with ether-petrol gave the title compound as a white solid (6.17g; 80%), vmax. (CHCl₃) 1805, 1755, 1690 and 1610 cm^-1. δ(CD₃)₂CO 2.96(2H,t,J7HZ, CH₂CN) , 3.14(1H, d, J 17H2,6β-CH), 3.40(2H,t,J 7HZ, (-SO₂ CH₂ CH₂CN), 3.68(1H,dd,J17 and 3HZ 6α-CH) 3.95(2H,d,J 8HZ, 9-CH₂), 4.93 (1H, t, J 8HZ, 8-CH) , 5.40(2H, s, Ar-CH₂), 5.41(1H,s,3-CH), 5.87 (1H,d,J3HZ, 5-CH) , 7,70 and 8.14 (4H, 2d, Ar-H).

Preparation 2a

Sodium (3R,5R,Z)-2-[2 cyanoethylthioethylidene] - clavam-3-carboxylate

A solution of p-nitrobenzyl (3R,5R,Z)-2-[2-cyanoethylthioethylidene]- clavam-3-carboxylate (0.229) in dry tetrahydrofuran (10 ml) was added to a prehydrogenated suspension of 10% Pd/c (200mg.) and the mixture hydrogenated at 1 atmosphere for 1 hour. After filtration through celite one equivalent of aqueous sodium bicarbonate solution was added and the tetrahydrofuram evaporated. Water (10ml) was added and the aqueons layer washed with ether, the ph adjusted to 7.5 (0.1N HCl) and freeze dried to yield the title salt as an amorphous solid (106 mg;65%); vmax (KBr) 2250, 1785, 1690 and 1620 cm. δ(D₂0) 2.78(4H, br s.,-S CH₂CH₂CN) , 3.07(1H,d,J 17Hz, 6β-CH), 3.34(2H,d,J7HZ,9-CH₂), 3.57(1H,dd,J17 and 3HZ, 6α-CH), 4.79(1H,t J7HZ, 8-CH) , 4.94(1H,S,3-CH) , 5.69 (1H,d,J3HZ, 5-CH). Example 2

Lithium (3R,5R,Z)-2-(2-cvanoethylsulphonylethylidene) clavam-3-carboχylate

p-Nitrobenzyl (3R, 5R, Z)-2-(2-cyanoethylsulphonylethylidene) clavam-3-carboxylate (3.0g) was deprotected as described in preparation 2a (saving the use of lithium carbonate in place of sodium bicarbonate) to afford the title salt (2g;97%); vmax(KBr) 2255, 1770, 1695 and 1620 cm^-1.; δ(D₂O) 3.0(2H,t CH₂CN), 3.11(1H,d,J17HZ, 6β-CH) , 3.43 (2H,t,-SO₂CH₂), 3.60(1H,dd,J 17 and 3HZ,6α-CH), 4.06 (2H,d,J7HZ,9CH₂), 5.03(1H,s,3-CH), 5.77(1H,d,J3KZ,5-CH).

Preparation 3a

p-Nitrobenzyl (3R,5R,Z)-2-(2-cyanoethylthioethylidene) -3-carboxylate

p-Nitrobenzyl clavulanate (1g.) was dissolved in methylene dichloride (30ml) and 3-mercaptopropionitrile (0.26g) was added. The solution was cooled to -10°C and boron trifluoride (0.1ml) was added and the mixture was stirred under nitrogen at -10° for 0.5 hour. The mixture was washed with 3% sodium bicarbonate solution (2x50ml) and water (50ml). The organic phase was dried over anhydrous magnesium sulphate and the solvent evaporated to give an oil, which after chromatography on silica gave the title ester (0.275g;23%) as a colourless oil which was identical in all respects (ir,p.m.r.etc) with the product of preparation 1a. Preparation 4

p-Nitrobenzyl-9-cyanomethylthio-9-deoxyclavulanate

p-nitrobenzyl-9-0-dichloroacetyl clavulanate (2.7 g, 6 mmol) in dimethylformamide (30 ml) was treated with mercaptoacetonitrile (0.73 g; 10 mmol) and the solution cooled to -35°C. Triethylamine (0.84 ml; 6 mmol) was added and the mixture stirred at -35ºC for 40 minutes and the solution then allowed to warm up to 0°C over 20 minutes. The reaction mixture was poured into water (150 ml) and extracted with ethylacetate (150 ml). The organic solution was washed with water (2x80ml), brine (50ml) and dried over anhydrous magnesium sulphate. Evaporation gave an orange gum and chromatography on silica-gel using ethyl acetate-petrol (bp 60°-80°) 1:1 as eluent gave the title compound (0.42g; 18%); [α]_D ²⁰ + 8.7°[C,0.92 CHCl₃] vmax.(CHCl₃) 2240, 1805, 1760, 1690 and 1520cm^-1. δ(CDCl₃) 2.90-3.70 (6H,m,6.CH₂, 9-CH₂ and SCH₂CN), 4.73(1H,t,J7Hz, 8C-H) , 5.07(1H,S, 3-CH) , 5.28(2H,S,Ar-CH₂), 5.72(1H,d,J2.5HZ, 5-CH) , 7.53 and 8.23(4H,2d, Ar-H)ppm.

Example 4

p-Nitrobenzyl-9-cyanomethylsulphonyl-9-deoxyclavulanate

p-Nitrobenzyl-9-cyanomethyl-9-deoxyclavulanate (0.24g; 0.62 mmol) in dichloromethane (40ml) was cooled to 0° and a solution of m-chloroperbenzoic acid (0.24g; 1.36mmol) in dichloromethane (15ml) was added dropwise to the stirred mixture . After stirring the mixture at 0° for 1 hour it was then washed successively with water (20ml), saturated aqueous sodium bicarbonate (20ml), water (20ml) and finally brine (20ml). After drying over anhydrous magnesium sulphate the solution was evaporated to give a pale yellow residue. Chromatography on silica-gel using ethyl acetate-petrol 1:1 as eluent gave the title product as a crisp white foam (0.13g;50%); -2.9°(C,1.05 CHCl₃), (CHCI₃) 2250, 1805, 1760 and 1690cm^-1; δ(CDCl₃) 3.15(1H,d,J17Hz, 6β-CH), 3.58 ( 1H,dd ,J17 and 2.5Hz, 6α-CH), 3._.90(2H,s,-SO₂CH₂CN), 4.05 ( 2H,m, 9-CH₂) . 4.85(1;H,t,J7Hz, 8-CH) , 5.20-5.45(3H,br.s, 3-CH and Ar-CH₂),

Preparation 5a

p-Nitrobenzyl-9-carbamoylmethylthio-9-deoxyclavulanic acid

p-Nitrobenzyl-9-0-dichloroacetyl clavulanate (2.7g) in dimethylformamide (20 ml.) was treated with thioglycolamide (1g) and the solution cooled to -30º Triethylamine (0.85 ml.) was added and the mixture stirred at -30°C for ½ hour and the mixture allowed to warm up to room temperature over 20 min. The reaction mixture was poured into water (100ml) and extracted with ethyl acetate (100ml). The organic solution was washed thoroughly with water (x4) and dried over magnesium sulphate. Evaporation gave an orange gum and chromatography on silica-gel using ethyl acetate as eluent gave the title compound as a white solid (1.5g; 62%). (CHCl₃) 3500, 3380, 1800, 1760 and 1680 cm^-1. P.m.r. δ(CDCl₃) 3.08 (2H, S, CH₂CONH₂) , 3.15 (1H,d, J17H_z, 6β-CH) 3.28 (2H₂, d, J7Hz, 9-CH₂) , 3.48(1H, dd, J17 and 2.5Hz, 6α-H) 4.71(1H,t,J7Hz, 8-CH) , 5.13(1H,S, 3-CH) , 5.68 (1H,d,J2.5Hz. 5-CH) , 5.9 and 6.65(2H, b.s. NH₂) 5.9 and 6.65 (4H, 2d, Ar-H) . Preparation 5b

p-Nitrobenzyl-9-cyanomethylthio-9-deoxyclavulanic acid

p-Nitrobenzyl-9-carbamoylmethylthio-9-deoxyclavulanic acid (1.8g) was dissolved in dry redistilled tetrahydrofuran (40 ml.), the solution cooled in an ice-bath to 0º, and pyridine (0.73ml.) added. Trifluoroacetic anhydride (0.65 ml.) was added and the mixture stirred at 0º for ½ hour. The solution was allowed to warm up to room temperature and evaporated to a small volume. Ethyl acetate (100 ml.) was added and the organic solution washed with water (2 x 50 ml.) and dried over anhydrous magnesium sulphate. Evaporation gave a yellow gum and chromatograghy on silica-gel 1:1 using ethyl acetate-petrol (b.p 60-80°) as eluent gave the title compound as a white solid (1.61g; 93%).

The spectroscopic and analytical details of this product were identical to those shown in preparation 4.

Example 5

Lithium-9-cyanomethylsulphonyl-9-deoxyclavulanate.

p-Nitrobenzyl-9-cyanomethylsulphonyl-9-deoxyclavulanate (0.13g; 0.3mmol) in tetrahydrofuran (20ml) was added to a prehydrogenated suspension of 10%Pd/C(0.13g) in tetrahydrofuran (10ml). The mixture was shaken under 1 atmosphere of hydrogen at room temperature for ¼ hour. After filtration through celite the solution was reduced to 10ml, diluted with water (10ml) and 0.2N lithium hydroxide solution added dropwise to bring the solution to pH 7.0. The aqueous solution was then washed with ethyl acetate (2x50ml), filtered through celite and, freeze dried to give the title compound (0.07g;70%) (KBr) 2260, 1780, 1690 and 1615cm^-1; δ(D₂O) 3.20(1H,d,J17.5Hz, 6β-CH) , 3.62(1H, dd, J17.5 and 2.5Hz, 6α-CH) , 4.10-4.42(2H,m, 9-CH₂), 4.86(1H,t,J7Hz, 8-CH), 5.07 (1H,S,3-CH), 5.83(1H,d,J2.5Hz, 5-CH)ppm. Biological Data

Table 1

Geometric mean MIC's for amoxycillin alone and in combination with compound of Example 2 of the present invention and with the compound of Example

92 (Ex 1579531) of UK Patent 1579531 against amoxycillin-resistant E. coli and amoxycillinresistant K. aeroqenes strains

The Inhibitors alone showed no antibacterial activity at concentrations of 1 ug/ml and 5 μg/ml.

+ MIC value (μg/ml)

* used as the lithium salt rather than as the sodium salt of Example 92 of UK Patent 1579531. Table 2

Geometric mean MIC value (μg/ml) for Amoxycillin alone and in the presence of Inhibitors against Amoxycillin resistant E.coli.

The Inhibitors alone showed no antibacterial activity at concentrations of 1 μg/ml and 5 μg/ml.

Claims

CLAIMS : -

1. A compound of formula (I) or a salt or ester thereof:

wherein A represents -CH₂- or -CH₂-CH₂-.

2. A compound of formula (I) or a pharmaceutically acceptable salt or pharmaceutically acceptable ester thereof.

3. A compound as claimed in claim 1 or claim 2 wherein the salt is an alkali metal salt.

4. The sodium or potassium salt of a compound of formula (I).

5. A compound as claimed in any one of claims 1 to 4 wherein A represents -CH₂-.

6. A compound as claimed in any one of claims 1 to 4 wherein A represents -CH₂-CH--.

7. A pharmaceutical composition which comprises a compound as claimed in claim 2 and a pharmaceutically acceptable carrier.

8. A pharmaceutical composition as claimed in claim7 which further comprises a penicillin or cephalosporin.

9. A process for the preparation of a compound as claimed in claim 1 or a salt or ester thereof, which process comprises mildly oxidising a compound of formula (II) :

wherein R^x is a carboxy protecting group and Z is S-A-CN or SO-A-CN wherein A is as hereinbefore defined, and thereafter where necessary carrying out one or more of the following steps :

(a) removing the carboxy protecting group R^x

(b) converting a salt to the free carboxylic acid or to an ester or to a different salt.

10. A method of treating bacterial infections in humans or animals which comprises the administration an effective amount of a compound according to claim 1.