GB1593275A - Clavam derivatives - Google Patents

Description

(54) CLARAM DERIVATIVES (71) We, GLAXO LABORATORIES LIMITED, a British company of Green ford, Middlesex, England do hereby declare the invention for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:- This invention relates to novel antibiotics and p-lactamase inhibitors and to a process for their production.

In German OLS No. 2,604,697 we have described the isolation, from fermentations of Streptomyces clavuligerus, of clavulanic acid and salts thereof in pure form.

The present invention relates to analogues of clavulanic acid and its salts and esters which carry a carbon-attached substituent in place of the hydroxyl group thereof. These are of use, as detailed below, as antibiotics or as p-lactamase inhibitors.

The compounds in this specification are named with reference to "clavam"; the name given to the parent heterocycle of formula A

by analogy with the term "cepham" used in the naming of cephalosporin compounds in J.Amer.Chem.Soc.,l962,84,3400.

Accordingly we provide compounds of the formula (I)

wherein R represents a carboxyl or esterifled carboxyl group and R' is the residue of a carbon nucleophile or a product thereof formed by cleavage of any other esterified carboxyl groups present therein, together, in the case where a carboxyl group is present, with salts thereof.

The residue of the carbon nucleophile will most desirably have the formula (II)

wherein X and Y, which may be the same or different, represent groups capable of producing a stabilised carbanion, e.g.

electron-withdrawing groups, and Z represents an alkyl, alkenyl, alknyl, cycloalkyl, aralkyl or aryl group or a hydrogen atom.

Thus, suitable groups which are capable of stabilising or inducing a negative charge will include esterified carboxyl or cyano groups or acyl groups R2CO-, wherein R2 may represent an alkyl, cycloalkyl, aralkyl or aryl group, which may optionally be substituted.

Suitable alkyl, alkenyl or alkynyl groups in Z or R2 mentioned above may be straight or branched unsubstituted or substituted groups having from 1-8 carbon atoms, for example a methyl ethyl, propyl or isopropyl or butyl, sec-butyl, tert-butyl, allyl or propargyl group, desirable substituents being, for example, alkoxy, e.g.

methoxy; halogen, e.g. fluorine, cyano; acyloxy, e.g. alkanoyloxy, such as acetoxy; and alkoxycarbonyl, e.g. ethoxycarbonyl.

Suitable cycloalkyl groups in Z or R2 may have 3-8, preferably 3-6, carbon atoms, e.g. cyclopentyl or cyclohexyl.

Suitable aryl groups in Z or R2 may have up to 12 carbon atoms, e.g. a phenyl or substituted phenyl group, suitable substituents being halo, e.g. chloro; nitro; cyano; alkyl, e.g. methyl; or alkoxy, e.g.

methoxy.

Suitable aralkyl groups in Z or R2 may have up to 10 carbon atoms, especially arylmethyl groups, e.g. a benzyl group, or a phenethyl group, suitable ring substituents being as indicated above for aryl groups.

In general, it is preferred that any alkyl, alkoxy, alkanoyloxy and alkoxycarbonyl groups represented in the group R' have 1-6 carbon atoms.

Preferred residues of carbon nucleophiles of formula (II) are those which are derived from p-diketones or ketoesters, i.e. those wherein both of X and Y represent an acyl group R2CO as defined above, or those wherein one of X and Y represents an acyl group R2CO as defined above and the other represents an esterified carboxyl group.

It is preferred for Z to be a hydrogen' atom. When X or Y represents an acyl group R2CO or esterified carboxyl grou as described above, it is preferred that R or the hydrocarbyl portion of the esterified carboxyl group be a C14 alkyl group.

Particularly preferred are compounds wherein the groupCXYZ represents a pentan-2,4-dion-3-yl group.

It will be appreciated that when the group -CXYZ contains an esterified carboxyl group and the product is submitted to cleavage, R' in the product according to the invention will carry one or more carboxyl groups, which may be in salt form.

The compounds of formula (I) wherein R represents an esterified carboxyl group may in general be represented as compounds of formula (I) in which R is a group COOR3 wherein OR3 represents an organic group which is conveniently derived from an alcohol (aliphatic or araliphatic), a phenol, or a stannanol. Such an alcohol, phenol, or stannanol used to esterify the carboxyl group preferably contains not more than 24 carbon atoms.

Thus, the group R3 may represent a straight or branched unsubstituted or substituted alkyl or alkenyl group, preferably having from 1-8 carbon atoms, for example a methyl, ethyl, propyl, or isopropyl, butyl, sec-butyl, tert-butyl or allyl group, desirable substituents being for example, alkoxy, e.g. methoxy; halogen, i.e.

fluorine, chlorine, bromine, or iodine, cyano, acyloxy, e.g. alkanoyloxy, such as acetoxy or pivaloyloxy or alkoxycarbonyloxy, e.g. ethoxy carbonyloxy; acyl, e.g. p - bromo benzoyl; and alkoxycarbonyl, e.g. ethoxy carbonyl; an aralkyl group having up to 20 carbon atoms, especially an arylmethyl group, e.g.

a benzyl or substituted benzyl group, suitable substituents being either halo, e.g.

chloro; nitro, e.g. o- or p-nitro; cyano; alkoxy, e.g. p-methoxy or alkyl, e.g. pmethyl groups; a diphenylmethyl or triphenylmethyl group or a fur-2-ylmethyl, thien-2-ylmethyl or pyrid-4-ylmethyl group, the heterocyclic groups of which may also be substituted, e.g. by a C14 alkyl group, preferably methyl; an aryl group having up to 12 carbon atoms, e.g. a phenyl or substituted phenyl group, suitable substituents being either halo, e.g. chloro; nitro, e.g. o- or p-nitro; cyano; alkoxy, e.g. p-methoxy or alkyl, e.g.

p-methyl groups; a cycloalkyl group containing not more than 12 carbon atoms, e.g. adamantyl; a heterocyclic group containing not more than -12 carbon atoms, the hetero atom being, for example, oxygen, as in the tetrahydropyranyl or phthalidyl group; or a stannyl group having up to 24 carbon atoms for example a stannyl group carrying three substituents which may be the same or different selected from alkyl, alkenyl, aryl, aralkyl, cycloalkyl, alkoxy, aryloxy or aralkoxy groups. Such groups will include, methyl, ethyl, propyl, n-butyl, phenyl and benzyl groups.

Esterified carboxyl groups in the grouping of formula (II) may also be represented by -COOR4 where R4 is as defined for R3 other than stannyl.

In some cases the compounds of formula (I) may exist in diastereoisomeric forms and the present invention includes these separately or in combination. It will be apparent that some of the compounds of formula (I) will be capable of keto-enol tautomerism and may exist in the individual forms or as mixtures thereof.

The salts according to the invention include salts with inorganic bases, such as alkali metal salts, e.g. sodium potassium and lithium salts; alkaline earth metal salts, e.g. calcium and magnesium salts; and ammonium salts, as well as salts with organic bases, for example amine salts.

Where more than one acidic group is present, mono-, di- or even tri-salts are possible.

For use in pharmaceutical compositions, the salts should be physiologically acceptable.

The compounds of the invention generally exhibit p-lactamase inhibitory activity, and are of use in the protection of p-lactam antibiotics susceptible to p-lactamase hydrolysis, e.g. antibiotics such as ampicillin or cephalexin.

The compounds of the invention in general also show antibiotic activity against gram-positive organisms and in some cases against gram-negative organisms. Where the 3-carboxylate ester is metabolically labile, the parent acid will be generated in vivo. Examples of such esters include the acyloxymethyl esters, e.g. the acetoxymethyl and pivaloyloxymethyl esters, and a-alkoxycarbonyloxyalkyl esters such as l-ethoxycarbonyloxyethyl esters.

Where the 3-carboxylic ester grouping is readily cleaved, e.g. by hydrolysis or reduction, without significant degradation of the remainder of the molecule, the esters are especially useful as carboxyl-protected derivatives of the parent acids. Certain 3carboxylate esters may be of use either in the purification or characterisation of the acids according to the invention or as carboxyl-protected intermediates for use in preparing further derivatives.

Esters which serve particularly well as carboxyl-protected intermediates and which are primarily of use in this connection include the arylmethyl esters as detailed above, especially the benzyl, pnitrobenzyl, benzhydryl and trityl esters, as well as stannyl, e.g. tri - butyl - stannyl, esters.

The compounds according to the invention for which we have demonstrated antibiotic activity have been active against a range of gram-positive microorganisms, for example, against strains of Staphylococcus aureus, and also against strains of certain gram-negative organisms such as Haemophilus influenzae. Certain of the compounds, namely those wherein R' in formula I represents a pentan - 2,4 dion - 3- yl group or a 1 - carboxy propan - 2- on - 1 - yl group and R represents a carboxyl group in salt form, have been shown to be active against gramnegative organisms including strains of Escherichia coli, Salmonella typhimurium, Shigella sonnei, Enterobacter cloacae, Klebsiella aerogenes, Proteus mirabilis, Proteus organic and Proteus vulgaris.

The new acids and their salts and esters are stable to the action of p-lactamases produced by gram-positive organisms, for example those produced by Staphylococcus aureus, and to p-lactamases produced by gram-negative organisms.

As indicated above, the new acids and their salts and esters generally also possess the ability to inhibit p-lactamase enzymes, for example those produced by grampositive organisms, e.g. those produced by strains of Staphylococcus aureus and the enzymes from gram-negative bacteria produced, for example, by strains of Proteus mirabilis, Escherichia coli, Neisseria gonorrhoeae, Klebsiella aerogenes, Salmonella typhimurium and Haemophilus influenzae and are thus useful in combination with p-lactam antibiotics, e.g.

penicillin antibiotics such as ampicillin and amoxycillin.

Compounds of the invention, including those compounds of formula (I) wherein R is a carboxyl group (in salt form) and R' represents a pentan - 2,4 - dion - 3 - yl group, 1 - ethoxycarbonyl - propan - 2 on 1 I - yl group, 1 - benzoyl - 1 - ethoxycarbonyl - methyl group or 1 - methoxycarbonyl - propan - 2 - on - I - yl group are also absorbed when administered orally as shown by studies in mice. These compounds are of interest for use in conjunction with both injectible and orally absorbed - lactam antibiotics which show susceptibility to - lactamases from grampositive and/or gram-negative organisms.

The new antibiotic acids and their salts and esters may thus be used alone in conjunction with a p-lactam antibiotic, preferably a broad spectrum p-lactam anitbiotic, which may be of a type conventionally administered by the oral or parenteral route. Use of the antibiotic acids and their salts is especially preferred.

Examples of orally absorbed broad spectrum p-lactam antibiotics include cephalexin, cephaloglycin, ampicillin and amoxycillin and their orally absorbed esters, e.g. the acyloxymethyl and phthalidyl esters, and the orally absorbed esters of carbenicillin and ticarcillin and mecillinam, e.g. the indanyl, phenyl and pivaloyloxymethyl esters. Broad spectrum p-lactam antibiotics which are not orally absorbed include carbencillin, ticarcillin, mecillinam, cephalothin, cephaloridine, cefazolin, cephacetrile and cephapirin.

Examples of narrow spectrum p-lactam antibiotics are penicillin G and penicillin V.

Combinations of the compounds according to the invention with, for example, ampicillin generally show synergistic activity against -lactamase producing strains of Staphylococcus aureus.

In general the compounds, when in combination with -lactamase susceptible p-lactam antibiotics, e.g. penicillins or cephalosporins, generally show synergistic activity against p-lactamase producing strains of Klebsiella aerogenes, Proteus mirabilis, Proteus rettgeri and Proteus vulgaris.

According to a further feature of the invention, we provide pharmaceutical compositions (including veterinary compositions) containing at least one acid, physiologically acceptable salt or metabolically labile ester of the invention.

In view of the protective action described above, the compositions can advantageously contain further -lactam antibiotics. The compositions will normally also contain a pharmaceutical (including veterinary) carrier or excipient.

The compositions of the invention include those in a form adapted for oral or parental use.

The compositions may, for example, take the form of powders, tablets, capsules, lozenges, solutions and syrups suitable for oral administration, and may include, for example, starch, lactose, talc, magnesium stearate, gelatin, distilled water and suspending, dispersing, emulsifying.

flavouring or colouring agents.

The compounds may further be formulated in rectal compositions such as suppositories or retention enemas.

The compounds of the invention may be formulated for administration by injection.

The compounds may thus be formulated in ampoules for reconstitution before use.

The active compounds of the invention will generally be administered to humans at a total daily dosage level of 50 mg to 20 g, preferably from 100 mg to 10 g, which may be in divided doses given 1-4 times per day.

Where the composition contains a further p-lactam antibiotic the total quantity of p-lactam antibiotic will desirably be from 100 mg to 20 g, which may be given in divided doses from 14 times a day. In general, the total dosage of p-lactam antibiotic when the active compound of the invention is used either alone or in combination with a further - lactam antibiotic will advantageously be from 250 mg to 5 g. Dosage units will in general contain 12.5 mg to 5 g, preferably 50 mg to 1 g, of active compound according to the invention, when this is employed alone and from 25 mg to 5 g, preferably 100 mg to 1 g, of total p-lactam antibiotic when a compound of the invention and a further p-lactam antibiotic are present.

In general, the weight ratio of the compound of the invention to a p-lactam antibiotic to be protected will be in the range of 10:1 to 1:10, more preferably 5:1 to 1:5, especially 2:1 to 1:2.

Active compounds of the invention may be of use in treating a variety of diseases in humans and animals, caused by pathogenic bacteria, such as respiratory tract or urinary tract infections.

The compounds of the invention may be prepared by any convenient method. Thus, in one aspect of the invention, we provide a process for the preparation of a compound of formula (I) wherein a compound of formula (III),

(wherein R represents an esterified carboxyl group and E is a readily displaceable substituent), or a compound of formula (IV)

(wherein R represents an esterified carboxyl group) is reacted with a compound of formula R'H in the presence of a base or with a salt of the compound of formula R'H, R' being the residue of a carbon nucleophile. The cation in said salt may, for example, be a monovalent metal ion or an ammonium ion. Suitable cations will be alkali metal, tetra-alkylammonium or thallous ions. Thallous salts are particularly preferred where the starting material is a compound of formula (III).

The readily displaceable substituent E may, for example, be a halogen (e.g.

chlorine, bromine or iodine) atom or an aromatic or aliphatic sulphonyloxy group containing, for example, 1--20 carbon atoms. The aliphatic or aromatic grouping of such a sulphonyloxy group may, for example, be an alkyl (e.g. C1~8) group, which may be substituted by a halogen atom, e.g. fluoring or chlorine, or an aryl (e.g. C,,,) group which may carry alkyl, e.g. methyl, alkoxy, e.g. methoxy, or halogen, e.g. bromine, substituents.

Preferred sulphonyloxy groups include methanesulphonyloxy, tosyloxy or phenylsulphonyloxy. The substituent E is preferably chlorine or bromine.

Reaction with the compound of formula (III) will desirably be effected in an aprotic organic solvent at or below ambient temperature, e.g. from -800C to +400C.

Ether solvents, e.g. 1,2 - dimethoxy ethane or tetrahydrofuran, have been found convenient. Other suitable solvents include amides such as dimethyl - formamide or hexamethylphosphoramide, sulphoxides, e.g. dimethyl sulphoxide, and sulphones, e.g. sulpholan. Where tetra alkylammonium salts are used, halogenated hydrocarbon solvents such as methylene chloride may be suitable.

In some solvents, particularly in dipolar aprotic solvents such as dim ethyl sulphoxide, and in the case where Z represents a hydrogen atom, some further alkylation may occur to provide material containing two residues of the clavulanic acid or derivative thereof attached to the residue of the carbon nucleophile.

Reaction of a compound of the formula (III) or the diene compound of formula (IV) with the compound of formula R'H may be effected in the presence of an inorganic base and a crown ether in a suitable aprotic liquid medium, e.g. a halogenated hydrocarbon solvent. This is a preferred procedure when the starting material is a diene of formula (IV).

A crown ether is a macrocyclic'- polyether, e.g. a number of ethyleneoxy units joined to form a ring, the internal diameter of the ring being approximately that of a particular metal ion, e.g. sodium or potassium, which is to be used in the reaction. In general 6 ethyleneoxy units (18 - crown - 6) are suitable to entrain a potassium ion while 5 ethyleneoxy units (15 - crown - 5) are suitable for sodium.

Some of the ethylene groups in such crown ethers may be replaced by other divalent units such as o - phenylene or 1,2 cyclohexylene units.

The inorganic base may, for example, be an alkali or alkaline earth metal hydroxide, carbonate or bicarbonate, e.g. potassium carbonate.

The reaction may be effected in the temperature range -15 to +500C, advantageously 0 to +350C.

When one or more of the reactants, for example the base, is not soluble in the solvent in which the reaction takes place, the crown ether may effect transfer of the reactant or reactants from the solid phase to the liquid phase or between two liquid phases.

A preferred single - liquid - phase system is dichloromethane with potassium carbonate/18 - crown - 6.

The diene compounds of formula (IV) may be prepared from compounds of the formula (III) by treatment with a tertiary amine such as triethylamine, whereby a compound of formula (IV) is obtained.

Compounds of formula (III) wherein E is a halogen atom may be prepared as described in our German OLS 2657081.

Compounds of formula (III) wherein E is organic sulphonyloxy may be prepared as described in German OLS 2616087.

In the above reactions, where the initial product contains an ester grouping in the group R and/or in the groupCXYZ and the corresponding acid or salt is required, the compound may be subjected to deesterification. For this purpose readily cleavable esters, for example, arylmethyl esters which may be cleaved by reduction, e.g. by hydrogenolysis, are preferred.

Cleavage of an arylmethyl ester, e.g. a p nitrobenzyl ester, may be effected by hydrogenolysis, for example using a metal catalyst, e.g. a noble metal such as platinum, palladium or rhodium. The catalyst may be supported, e.g. on charcoal or kieselguhr. A p - nitrobenzyl group may also be removed by reduction of the nitro group (e.g. using a dissolving metal reducing agent such as zinc in acetic acid, or zinc in aqueous tetrahydrofuran or acetone controlled, for example, in the pH range 3-6, preferably 4.05.5, by the addition of aqueous hydrochloric acid; aluminium amalgam in a moist ether, e.g.

tetrahydrofuran; or iron and ammonium chloride in an aqueous ether, e.g.

tertrahydrofuran) followed by hydrolysis either under the reduction conditions or by subsequent treatment with acid.

Alternatively, a stannyl ester can be cleaved by very mild solvolysis, e.g. by reaction with water, alcohols, phenols or carboxylic acids, e.g. acetic acid.

Where it is desired to produce a salt of the compound of formula (I), an acid initially formed in solution in an appropriate organic solvent may be reacted with an appropriate base, preferably under conditions favouring precipitation of the salt. In the formation of alkali metal salts e.g. sodium or potassium salts, an alkanoate is a preferred base, e.g. a 2 - ethyl hexanoate. Salts may be formed either at the carboxyl group attached to the ring, or any carboxyl group which may be present in Rl.

Where an ester of formula (I) is required which is different from the ester initially produced, the ester-forming group may be removed by methods as described above and the carboxylic acid product reesterified by the methods described hereinafter.

The esters of formula (I) may be prepared from the corresponding acids or reactive derivatives thereof by reaction with an alcohol, phenol or stannanol or a reactive derivative thereof to form the desired ester. Reaction will desirably be effected under mild conditions in order to prevent rupture of the bicyclic nucleus. The use of neutral or mild acidic or basic conditions, at temperatures of, e.g., -70" to +35 is preferred.

The alkyl, alkoxyalkyl and simple aralkyl esters may be prepared by reaction of an acid of formula (I) with the appropriate diazoalkane or diazo - aralkane, e.g.

diazomethane or diphenyldiazomethane.

The reaction will generally be effected in an ether, ester or halohydrocarbon solvent, e.g. diethyl ether, ethyl acetate or dichloromethane. In general, reduced temperatures are preferred, for example from -15" to +150C, desirably around ambient temperature.

The esters derived from alcohols may be produced by reaction of a reactive derivative of the alcohol, for example, a halide.such as the chloride, bromide or iodide, or a hydro - carbonsulphonyl derivative such as a mesyl or tosyl ester with a salt of an acid of formula (I), e.g. an alkali or alkaline earth metal salt such as a lithium, sodium, potassium, calcium or barium salt or an amine salt, e.g.

triethylammonium. This reaction is preferably carried out in a substituted sulphoxide or amide solvent, e.g. dimethyl sulphoxide, dimethylformamide or hexamethylphosphoramide.

Stannyl esters may conveniently be formed by a reaction of a carboxylic acid of formula (I) or a salt thereof with reactive tetravalent tin moieties. Trialkyl tin oxides are preferred for the synthesis of tin compounds in view of their availablity and low toxicity.

In the preparation of compounds of formula (III) from esters of clavulanic acid, some of the corresponding E-isomers may be formed and if desired isolated, which Eisomers, using the methods given above, will provide the E-isomers of the compounds of formula (I). .

The carbanion salts which are of use in the process of the invention have been described in numerous literature sources, and the thallous salts of ethyl acetoacetate, acetylacetone and ethyl benzoylacetate are known from E.C. Taylor, G.H. Hawks and A. Mckillop, J. Amer. Chem Soc. (1968), 20, 2421. The thallous salts of p nitrobenzyl acetoacetate and methyl acetoacetate were prepared following the procedure described in the above reference.

The invention will now be more particularly described in the following nonlimiting Examples.

Example I 4 - Nitrobenzyl (3R, SR, l'Z) - 2 - (3 acetyl - 4 - oxopentyl idene)clavam - 3 - carboxylate Thallous acetylacetonate (2.7 g.) was added to a suspension of 4 - nitrobenzyl (3R,SR,Z) - 2 - (2 - chloroethylidene) clavam - 3 - carboxylate (3.17 g.) in 1,2 - dimethoxyethane (25 ml.) and the mixture stirred at room temperature for 2 hrs. The reaction mixture was centrifuged and the insoluble thallous salts discarded. The organic liquors were evaporated to dryness and the residue dissolved in ethyl acetate (10 ml.). The solution was diluted with ethyl acetate: petroleum ether (b.p. 60 80 ) (1:1, 50 ml.) and the deposited solid removed by filtration through kieselguhr. The filtrate was evaporated to dryness and the residue chromatographed on silica gel preparative plates, using ethyl acetate: petroleum ether (b.p. 6080 ) (1:1) as eluant, to yield the title ester as an oil (0.85 g.), [sr]D+19 (c1.0; CHCl3) Vmax (CIlBr3) 1794 (p-lactam), 1750 (ester), 1728 (acetyl), 1694 (enol ether), 1526 and 1349 cm-l (NO2) T (CDCl2) for the major (keto) tautomer 1.76 and 2.50 (doublets, j=8 Hz, aromatic protons), 4.32 (s, C-S H), 4.7 (s, -CH2Ar), 4.95 (s, C-3 H), 5.44 (t, J=7 Hz, =CH-), 6.32 (t, J=7Hz, C-3'H) 6.48 and 6.94 (doublets, J=16 Hz, C-6 protons), 7.38 (t, J=7 Hz, C-2' protons) and 7.85 (s, -CH3); resonances due to the minor (enol) tautomer were observed at 6.98 (d, J=8 Hz, C-2' protons) and 7.94 (s, =C.OH.CH3) Example 2 4 - Nitrobenzyl (3R,SR,l'Z,3'RS) - 2 - (3 ethoxycarbonyl - 4 - oxopentyl idene)clavam - 3 - carboxylate The thallium (I) salt of ethyl acetoacetate (2.66 g., 8 mmole) was added to a suspension of 4 - nitrobenzyl (3R,SR,Z) 2 - (2 - chloroethylidene)clavam - 3 carboxylate (3.17 g.) in 1,2 dimethoxyethane (25 ml.) and the mixture stirred at room temperature for I hr. The reaction mixture was centrifuged to remove the insoluble thallium salts. The organic liquors were evaporated to dryness and the residue redissolved in ethyl acetate (5 ml.).

The solution was diluted with ethyl acetate:petroleum ether (b.p. 400) (1:1, 50 ml.) and the precipitated solid discarded.

The organic solution was evaporated to dryness and the residue chromatographed on preparative silica gel plates, using ethyl acetate:petroleum ether (b.p. 40--60")(1:1) as eluant, to yield the title ester as an oil (0.61 g.), Vmax (CHBr3) 1800 (p-lactam), 1746 (ester), 1710 (acetyl), 1528 and 1348 cm-' (NO2), T(CDCI3) 1.75 and 2.48 (doublets, J=8.5 Hz, aromatic protons), 4.33 (d, J=2 Hz, C-S H), 4.73 (s, -CH2Ar), 4.95 (s, C-3 H), 5.38 (t, J=7 Hz, =CH-), 5.83 (q, J=7 Hz -COOCH2CH3), 6.50 and 6.93 (dd, J=2 and 17 Hz; and d, J= 17 Hz, C-6 protons), 6.52 (m, C-3' H), 7.37 (t, J=7 Hz, C-2' protons), 7.80 (s, -COCH3) and 8.75 (q, J=7 Hz; -COOCH2CH3) The title ester was also prepared under similar conditions from the sodium salt of ethyl acetoacetate. The spec C-3 H), 5.29 (t, J=7 Hz, =CH-), 5.63 (t, J=7 Hz, C-3'H), 5.97 (q, J=7 Hz, -COOCH2CH3), 6.54 and 7.01 (multiplets.

C-6 protons), 7.21 (t, J=7 Hz, C-2' protons) and 8.88 (t, J=7 hz, -COOCH2CH3).

* some of the peaks were doubled due to the presence of the two diastereoisomers about C-3'.

Example 4 4 - Nitrobenzyl (3R,5R,1'Z,3'RS) - 2 - (3 methoxycarbonyl - 4 - oxopentyl idene)clavam - 3 - carboxylate In 14% yield, Vmax (CHBr3) 1800 (ss- lactam), 1748 (ester), 1714 (acetyl), 1698 (enol ether), 1522 and 1346 (nitro), T(CDCI3) 1.78 and 2.51 (aromatic protons), 4.35 (d, J=2 Hz, C-S H), 4.76 (s, -CH2Ar), 4.97 (s, C-3 H), 5.40 (t, J=7 Hz =CH-), 6.30 (s, -OCH3), 6.51 (m, C-3' H), 6.52 and 6.96 (dd, J=2 and 17 Hz and d, J=17 Hz, C-6 protons), 7.39 (t, J=7 Hz, C-2' protons) and 7.80 (s, -COCH3) Example 5 4 - Nitrobenzyl (3R,5R,1'Z,3'RS) - 2 - [3 -(4 - nitrobenzyloxycarbonyl) - 4 oxopentylidene] - clavam - 3 carboxylate In 10% yield, smaX (CHBr3) 1800 (X3- lactam), 1750 (ester), 1716 -(acetyl), 1520 and 1346 cm-l (nitro), T(CDCl3) 1.79 and 2.52 (aromatic protons), 4.35 (d, J=2 Hz, C-S proton), 4.73 and 4.75 (singlets, -CH2Ar), 4.95 (s, C-3 proton), 5.38 (t, J=7 Hz, =CH-), 6.38 (m, C-3' proton), 6.48 and 6.95 (dd, J=2 and 17 Hz and d, J=17 Hz, C-6 protons), 7.32 (t, J=7 Hz, C-2' protons) and 7.80 (s, -COCH3).

Example 6 Sodium (3R,5R,I'Z,3'RS) - 2 - (3 ethoxycarbonyl - 4 - oxopentyl idene)clavam - 3 - carboxylate Palladium on charcoal (10%, 0.65 g.) was added to a solution of 4-nitrobenzyl (3R,5R,1'Z,3'RS) - 2 - (3 ethoxycarbonyl - 4 - oxopentylidene) clavam - 3 - carboxylate (0.67 g.) in ethyl acetate and the mixture shaken on a hydrogenator for several minutes until uptake of hydrogen ceased. The mixture was filtered through kieselguhr and a solution of sodium 2-ethylhexanoate (0.165 g.) in ethyl acetate (5 ml.) added to the filtrate. The filtrate was evaporated to a small volume and the concentrate diluted with diethyl ether (50 ml.). The precipitated solid was filtered off and dried in a vacuum desiccator, yielding 0.28 g. of the title compound. [α]D+13 (c 1.0, H2O) #max (Nujol) (Nujol is a registered trade mark) 1786 (ss-lactam), 1734 (ester), 1708 (acetyl) and 1610 cm-' (-CO2-), T(D2O) 4.28 (d, C-S H), 5.11 (s,C-3 H), 5.79 (q, -COOCH2CH3), 6.12 (t, C-3' H), 6.40 and 6.92 (dd and d, C6 protons), 7.35 (m, C-2' protons), 7.70 (s, -COCH3) and 8.76 (q, -COOCH2CH2).

Prepared in a similar fashion were: Example 7 Sodium (3R,5R,I'Z) - 2 - (3 - acetyl - 4 oxopentylidene)clavam - 3 carboxylate In 48% yield, smaX (nujol) 1780 (,B-lactam), 1720 (acetyl), 1688 (enol ether) and 1600 (-CO2-), T(D2O) 4.32 (d,C-5 H), 5.15 (s, C-3 H), 6.45 and 6.96 (dd and d, C-6 protons), 7.42 (d, J=7 Hz, C-2'protons) and 7.76 (s, -COCH3).

Example 8 Sodium (3R,5R,I'Z,3RS) - 2 - (3 benzoyl - 3 - ethoxycarbonyl propylidene)clavam - 3 - carboxylate In 47% yield, im;X (pH 6 buffer) 250 nm (#=9,900), Omax (Nujol) 1790(ss-lactam), 1732 (ester), 1690 (enol ether) and 1620 cm-' (-CO2-), T (D2O), 2.2.7 (m, aromatic protons), 4.44 (d, C-S H), 5.87 (q, J=7 Hz,-COOCH2CH3), 6.53 and 7.21 (C-6 protons), 7.30 (m, C-2' protons), and 8.86 (q, J=7 Hz, -COOCH2CH3).

Example 9 Sodium (3R,5R,1'Z,3'RS) - 2 - (3 methoxycarbonyl - 4 - oxopentyl idene)clavam - 3 - carboxylate In 48% yield, Vmax (Nujol) 1790 (p-lactam), 1740 (ester), 1710 (acetyl) and 1618 cm-' (CO20-), T (D2O) 4.33 (d, J=2 Hz, C-S proton), 5.16 (s, C-3 proton), 6.26 (s, WCH3), 6.46 and 6.95 (dd, J=2 and 17 Hz and d, J=17 Hz, C-6 protons), 7.39 (m, C-2' protons) and 7.72 (s, -COCH3).

Example 10 4 - Nitrobenzyl (3R,5R,1'Z) - 2 - (3 acetyl - 4 - . oxopentylidene)clavam 3 - carboxylate The tetra n-butylammonium salt of acetylacetone (0.33g) was added to a suspension of 4-nitrobenzyl (3R,5R,Z) - 2 (2-chloroethylidene)clavam - 3 carboxylate (0.35g) in 1,2-dimethoxyethane (5ml). After stirring at room temperature for 20 min, the yellow solution was concentrated in vacuo. The residue was chromatographed on silical gel preparative plates using ethyl acetate:petroleum ether (b.p. 60-80 )(1:1) eluent, to yield 17 mg of the title ester, whose spectral properties resembled those given in Example 1.

Example 11 Sodium (3R,5R,I'Z,3'RS) - 2 - (3 carboxy - 4 - oxopentylidene) clavam - 3 - carboxylate Palladium on carbon (10% 0.70g) was added to a solution of 4 - nitrobenzyl (3R,5R,1'Z,3'RS) - 2 - [3 - (4 nitrobenzyloxycarbonyl) - 4 oxopentylidene]clavam - 3 - carboxylate (0.73g) in ethyl acetate (50ml) and the mixture shaken vigorously on a hydrogenator for 2 min. The mixture was filtered rapidly through Kieselguhr and a solution of sodium 2 - ethylhexanoate (0.14 g) in ethyl acetate (2ml) added to the filtrate. A precipitate formed rapidly and this was filtered off, washed with ether and dried in a desiccator to yield 0.17g of the title compound, Vmax (Nujol) 1786 (p-lactam), 1702 (acetyl) and 1608 cm-l (-CO2-), T(D2O) 4.32(D, C--5H), 5.13(s, C-3H), 6.47 and 6.92(ABq, J=17Hz, C-6 protons) 7.33-7.6(m, C-2' protons) and 7.75(-COCH3).

Example 12 Acetoxymethyl (3R,5R,Z) - 2 - (3 acetyl - 4 - oxopentylidene)clavam 3 - carboxylate Chloromethyl acetate (0.43 ml) was added to a solution of sodium iodide (1.44 g) in anhydrous acetone (10 ml) and the mixture stirred at room temperature for 3 hr. The mixture was then concentrated in vacuo and the residue partitioned between water (10 ml) and petroleum ether (bp 680C) (10 ml). The organic extract was dried (Na2SO4) and added to a stirred solution of sodium (3R,5R,Z)- 2 - (3 acetyl - 4- oxopentylidene)clavam - 3 - carboxylate (0.90 g) in dimethylformamide (7 ml). After stirring for 10 min, the mixture was partitioned between brine (50 ml) and ethyl acetate (50 ml). The organic layer was washed with water (3x50 ml), dried (Na2SO4) and concentrated in vacuo. The residue was passed down a column of silica gel (25 g), using ethyl acetate:petroleum ether (bp 6080 ) (2:1) eluent, to give 0.60 g of the title esteras a colourless oil AmaX (0.1 N NaOH) 258 (E 12,800) and 307 nm (E 4000), Vmax (CHBr3) 1795 (p-lactam), 1762 (ester) and 1692 cm-' (enol ether), T CDCI3) 4.2.40 (m,-OCH2O- and C-S H), 5.00 (C-3 H), 5.46 (t, J=7Hz, =CH-), 6.32 (t, J=7Hz, C-3'H), 6.50 and 6.9? (ABq, J=17Hz, C protons), 7.39 (t, J=7Hz, C-2' protons) and 7.8-8.0 (m,methyl protons).

Example 13 4 - Nitrobenzyl (3R,5R,Z) - 2 - (3 acetyl - 4 - oxopentylidene)clavam 3 carboxylate Potassium carbonate (50 mg) and 4nitrobenzyl (3R,5R,Z) - 2 - (2 chloroethylidene) - clavam - 3 carboxylate (0.32 g) were added to a solution of acetylacetone (0.40 ml) and 18 crown - 6 (0.10 g) in dry dichloromethane (5 ml). After stirring for 20 min, the solution had cleared and a further portion of potassium carbonate (90 mg) was added to the mixture. After stirring for a further 15 min, the reaction was diluted with dichloromethane (5 ml) and washed with 2N aqueous sulphuric acid (10 ml). The organic phase was dried (Na2SO4) and chromatographed on preparative silica gel plates, using ethyl acetate:petroleum ether (bp 6080 ) (1:1) as eluent, to yield 80 mg of the title ester spectroscopic properties resembled those given in Example 1.

Example 14 4 - Nitrobenzyl (3R,SRZ) - 2 - (3 - acetyl 4 - oxopentylidene)clavam - 3 carboxylate Potassium carbonate (50 mg) was added to a solution of 4-nitrobenzyl (5R) - 2 vinylclav - 2 - em - 3 - carboxylate (0.20 g), acetylacetone (0.35 g) and 18 - crown 6 (0.10 g) in dry dichloromethane (5 ml).

After stirring for 15 min, the mixture was diluted with dichloromethane (5 ml) and washed with 0.5 N aqueous hydrochloric acid (5 ml). The organic phase was dried (Na2SO4) and chromatographed on silica gel preparative plate using ethyl acetate:petroleum ether (bp 4060 ) (1:1) eluent, yielding 40 mg of an oil whose i.r.

and n.m.r. spectra resembled those given in Example 1.

Example 15 4 - Nitrobenzyl (3R,5R,I'Z,3'RS) - 2 - (3 cyano - 3 - methoxycarbonyl propylidene)clavam - 3 - carboxylate Potassium carbonate (50 mg) was added to a solution of 4 - nitrobenzyl (5R) - 2 vinylclav - 2 - em - 3 - carboxylate (0.20 g), methyl cyanoacetate (0.20 g) and 18 crown - 6 (0.10 g) in dry dichloromethane (5 ml). After stirring for 15 min, the mixture was diluted with dichloromethane (5 ml) and washed with 0.5 N aqueous hydrochloric acid (5 ml). The organic phase was dried (Na2SO4) and chromatographed on silica gel preparative plates using ethyl acetate:petroleum ether (bp 40ǑO) (1:1) as eluent, yielding 35 mg of the title ester as an oil, Vmax (CHBr3) 1796 (p - lactam), 1745 (ester), 1692 (enol ether), 1520 and 1342 cm-' (nitro), T(CDCI3), 1.74 and 2.46 (aromatic protons), 4.28 (m, C 5 H), 4.70 (s, benzyl protons), 4.86 (s, C-3 H), 5.28 (t, J=7Hz, =CH ), 6.20 (s, -OCH3), 6.40 (t, J=7Hz, C 3' proton), 6.46 and 6.88 (m, C-6 protons) and 7.23 (t, J=7Hz, C 2' protons).

Claims (37)

WHAT WE CLAIM IS:

1. Cpmpounds of the formula (I)

wherein R represents a carboxyl or esterified carboxyl group and Rl is the residue of a carbon nucleophile or a product thereof formed by cleavage of any other esterified carboxyl group present therein, together, in the case where a carboxyl group is present, with salts thereof.

2. Compounds as claimed in claim 1 wherein Rl represents a group of formula (II)

wherein X and Y, which may be the same or different, represent groups capable of producing a stabilised carbanion, and Z represents an alkyl, alkenyl, alkynyl, cycloalkyl, aralkyl or aryl group or hydrogen atom.

3. Compounds as claimed in claim 2 wherein the group capable of producing a stabilised carbanion is an esterified carboxyl group, a cyano group or a group R2CO wherein R2 represents an optionally substituted alkyl, cycloalkyl, aralkyl or aryl group.

4. Compounds as claimed in claim 2 or claim 3 wherein Z or R2 represents a C18 alkyl, alkenyl or alkynyl group, optionally substituted by a halogen atom or an alkoxy, cyano, alkanoyloxy or alkoxycarbonyl group; a C28 cycloalkyl group; an aryl group having up to 12 carbon atoms and being optionally substituted by a halogen atom, alkyl group or alkoxy group; or an aralkyl group having up to 10 carbon atoms and being optionally substituted by a halogen atom, alkyl group or alkoxy group.

5. Compounds as claimed in any of claim 24 wherein X is an acyl group, R2CO wherein R2 is as defined in claim 3 and Y is an acyl group R2CO or an esterified carboxyl group.

6. Compounds as claimed in claim 1 in which Rl is a group

as defined in claim 2 wherein one or more esterified carboxyl groups therein are replaced by free carboxyl groups.

7. Compounds as claimed in any of claims 1-5 wherein R1 is a pentan - 2,4 - dion 3 - yl group.

8. Compounds as claimed in any of the preceding claims wherein R is a group COOR3 wherein OR3 represents an organic group which is derived from aliphatic or araliphatic alcohol, a phenol or a stannanol.

9. Compounds as claimed in any of the preceding claims wherein other esterified carboxyl groups present in the compound of formula (I) are represented by the formula -COOR4 wherein OR4 is a group derived from an aliphatic or araliphatic alcohol or phenol.

10. A compound as claimed in claim 8 or claim 9 wherein R3 and R4 represent straight or branched substituted or unsubstituted alkyl or alkenyl groups having from 1-8 carbon atoms; aralkyl groups having up to 20 carbon atoms; aryl groups having up to 12 carbon atoms; cycloalkyl groups containing up to 12 carbon atoms, optionally containing one or more heteroatoms in the ring system and unsaturation optionally being present when one or more heteroatoms is present, or R3 represents a stannyl group having up to 24 carbon atoms.

11. A compound as claimed in any of claims 8-10 wherein the esterified carboxyl group is metabolically labile.

12. A compound as claimed in any of claims 8-10 wherein R represents a benzyloxycarbonyl group, optionally substituted by an o- or p- nitro, p- methoxy or p - methyl group.

13. A compound as claimed in any of claims 8-10 and 12 wherein R represents a p - nitrobenzyloxycarbonyl group.

14. An alkali metal, alkaline earth metal, ammonium or organic base salt as claimed in any of claims 1--13.

15. A sodium, potassium lithium, calcium, magnesium or ammonium salt as claimed in any of claims 1--13.

16. A salt as claimed in claim 14 or claim 15 which is physiologically acceptable.

17. Compounds as claimed in any of claims 2-16 in which Z represents a hydrogen atom.

18. (3R,5R,I'Z) - 2 - (3 - Acetyl - 4 oxopentylidene)clavam - 3 - carboxylic acid and physiologically acceptable salts and metabolically labile esters thereof.

19. A pharmaceutical composition (including a veterinary composition) comprising at least one acid, physiologically acceptable salt or metabolically labile ester as claimed in claim 1 in admixture with a pharmaceutical carrier or excipient and/or a further p- lactam antibiotic.

20. A composition as claimed in claim 19 which is in the form of dosage units containing 12.5 mg to 5 g of active compound of the invention when this is employed alone and from 25 mg to 5 g of total p-lactam antibiotic when a compound of the invention and a further p-lactam antibiotic are present.

21. A composition as claimed in claim 19 which contains the compound of formula (I) as claimed in claim I wherein R' represents a pentan - 2,4 - dion - 3 - yl group or a I - carboxy - propan - 2 - on - 1 yl group and R represents a carboxyl group in salt form.

22. A process for the preparation of a compound of formula (I) as claimed in claim 1 which comprises reacting a compound of formula (III)

(wherein R represents an esterified carboxyl group and E is a readily displaceable substituent), or a compound of formula (IV)

(wherein R represents an esterified carboxyl group) with a compound of formula R'H in the presence of a base or with a salt of the compound of formula R'H, R' being the residue of a carbon nucleophile, followed, where an acid of formula (I) is required, by deesterification, and by salt formation where a salt if required.

23. A process as claimed in claim 22 wherein the salt of the compound of formula R!H is an alkali metal, tetraalkylammonium or thallous salt and this is reacted with a compound of formula (III).

24. A process as claimed in claim 23 wherein the readily displaceable substituent E is a halogen atom or an aromatic or aliphatic sulphonyloxy group.

25. A process as claimed in claim 24 wherein E represents a chlorine or bromine atom.

26. A process as claimed in claim 22 wherein a compound of formula R1H is employed and is reacted with a compound of formula (IV) in the presence of an inorganic base and a crown ether in an aprotic liquid medium.

27. A process as claimed in claim 22 wherein a compound of formula R'H is employed and is reacted with a compound of formula (III) in the presence of an inorganic base and a crown ether in an aprotic liquid medium.

28. A process as claimed in claim 26 or claim 26 wherein the medium is a halogenated hydrocarbon solvent.

29. A process as claimed in any of claims 2628 wherein the inorganic base is an alkali metal hydroxide, carbonate or bicarbonate.

30. A process as claimed in any of claims 22-29 wherein any ester grouping in the compound of formula (I) initially formed is cleaved by reduction.

31. A process as claimed in claim 30 wherein reduction is effected by catalytic hydrogenolysis.

32. A process as claimed in claim 30 or claim 31 wherein the ester is ap-nitrobenzyl ester.

33. A process as claimed in any of claims 22-31 wherein an acid product of formula (I) is re-esterified.

34. A process as claimed in claim 22 substantially as hereinbefore described.

35. A process as claimed in claim 22 substantially as hereinbefore described with reference to Examples I11.

36. A process as claimed in claim 22 substantially as hereinbefore described with reference to Examples 13-15.

37. A process as claimed in claim 33 substantially as hereinbefore described with reference to Example 12.