GB1585124A - Clavulanic acid derivatives - Google Patents

Abstract

Clavamic acid derivatives of the formula <IMAGE> in which R is -NH2 and R<3> is -COOH or an esterified carboxyl group, are prepared from corresponding azides, R = -N3 in the above formula, by reduction. The free carboxylic acid can be in the form of a zwitterion or a salt with acids or bases; it can be esterified. The starting material with R = -N3 is advantageously prepared from an ester of the formula II in which R is an exchangeable atom or an exchangeable group by reaction with an azide. The products (II), especially the free carboxylic acid, have antibacterial activity on various Gram-negative and Gram-positive microorganisms and they inhibit beta -lactamase; they can be used alone as antibiotics or in combination with beta -lactam antibiotics for the treatment of infectious diseases.

Description

(54) CLAVULANIC ACID DERIVATIVES (71) We, GLAXO LABORATORIES LIMITED, a British Company of Greenford, Middlesex, 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 to a process for their production.

In our German OLS 2 604 697 we have described the isolation, from fermentations of Streptomyces clavuligerus, of the carboxylic acid having the formula (I) (clavulanic acid)

and salts thereof in pure form.

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.. 1962, 84, 3400. Thus the compound of formula (I) is named (3R,5R,Z)-2-(2-hydroxyethylidene)clavam-3-carboxylic acid.

The present invention relates to analogues of the compound of formula (I) above and its salts and esters which carry an azido group -N3 or an amino group -NH2 in place of the hydroxy group thereof. These are of use, as detailed below, as antibiotics or as ss-lactamase inhibitors or they may be useful as intermediates in the preparation of further active compounds.

Accordingly, we provide a compound of the formula (II)

(where R is an azido group -N3 or an amino group NH2 and R3 is a carboxyl or esterified carboxyl group) together with salts and zwitterionic forms thereof.

The esters according to the invention may in general be represented as compounds of formula (II) in which R3 is a group COOT4 where R4 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 R4 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. ethoxycarbonyloxy; acyl, e.g. p-bromobenzoyl; and alkoxycarbonyl, e.g.

ethoxycarbonyl; 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. p-methyl groups; a diphenylmethyl or triphenylmethyl group or a fur-2-ylmethyl, thien-2-ylmethyl or pyrid-4ylmethyl groups, 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.

Where R represents a carboxyl group and R represents an amino group, the compound of formula (II) may exist as a zwitterion or may form acid addition salts or salts with bases.

Esters of the compounds in which R is an amino group may form acid addition salts and compounds of formula (II) in which R is an azido group and R3 is a carboxyl group may form salts with bases. The salts with bases may be 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; or salts with organic bases, for example amine salts. Acid addition salts according to the invention include salts with inorganic acids, e.g.

hydrochloric, hydrobromic, perchloric, sulphuric or phosphoric acid salts, and salts with organic acids, e.g. acetic, propionic, citric, maleic or toluene-p-sulphonic acid salts.

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

In general, the compounds are stable to the action of ss-lactamases produced by gram-positive organisms, for example those produced by Staphylococcus aureus, and to the ss-lactamases produced by gram-negative organisms.

The free acids of the invention and their salts and metabolically labile esters in general show antibiotic activity. Examples of such esters include the phthalidyl and acyloxymethyl esters, e.g. the acetoxymethyl and pivaloyloxymethyl esters, and a-alkoxycarbonyloxyalkyl esters such as 1-ethoxycarbonyloxyethyl esters.

Where the carboxylic ester grouping is readily cleaved, e.g. by hydrolysis or reduction, without significant degradation of the remainder of the molecule, the esters may be useful as carboxyl-protected derivatives of the parent acids. Certain 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, p-nitrobenzyl, benzhydryl and trityl esters.

Of particular value in this regard are the readily cleaved esters; e.g. the arylmethyl esters, of the azide compound since catalytic reduction of these readily affords the carboxylic acid of the amino compound. The p-nitrobenzyl ester of the azido compound is especially preferred.

A preferred compound of formula (II) for use in medicine is the compound in which R is NH2 and R3 is COOH (i.e. the amine acid), which generally exists in the zwitterionic form.

This compound has been found to exhibit antibacterial activity against a range of gram-negative and gram-positive microorganisms, for example, against strains of Staphylococcus aureus, Escherichia coli, Salmonella typhimurium, Shigella sonnei, Enterobacter cloacae, Klebsiella aerogenes, Proteus mirabilis, Proteus vulgaris and Proteus morganfi.

This compound has the ability to inhibit ss-lactamase enzymes; these include enzymes produced by gram-positive organisms, for example those produced by strains of Staphylococcus aureus. The compound is particularly noteworthy in inhibiting ss-lactamases produced by an unusually wide range of strains of gram-negative bacteria, for example, by strains of Proteus mirabilis, Escherichia coli, Proteus morganii, Klebsiella aerogenes, Salmonella typhimurium, Haemophilus influenzae, Bacteroides fragilis, Proteus vulgaris and Proteus rettgeri.

This compound is orally absorbed as evidenced from studies in mice.

The new compounds are of interest for use in conjunction with p-lactam antibiotics which show susceptibility to p-lactamases from gram-positive and/or gram-negative organisms.

In general, it is preferred to use the compounds of the invention in conjunction with a broad spectrum p-lactam antibiotic, e.g. a cephalosporin or penicillin, which may be of a type conventionally administered by the oral or parenteral route. Use of the amine acid and its salts is preferred and especially preferred is the amine acid in its zwitterionic form.

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, e.g. the indanyl and phenyl esters. Broad spectrum ss-lactam antibiotics which are not orally absorbed include carbenicillin, ticarcillin, cephalothin, cephaloridine, cefazolin, cephacetrile and cephapirin.

Examples of narrow spectrum ss-lactam antibiotics are penicillin G, penicillin V, mecillinam and pivmecillinam.

A combination of the amine acid, for example, with ampicillin shows synergistic activity against ss-lactamase producing organisms including strains of, for example, Staphylococcus aureus, Escherichia coli, Klebsiella aerogenes, Proteus mirabilis, Salmonella typhimurium, Shigella sonnei, Bacteroides fragilis, Proteus morganii and Proteus vulgaris.

Compounds of formula (II) in which Ris < 3 and R3 is COOH and salts thereof have a similar spectrum of activity to that shown by the amine acid of the invention.

Active compounds of the invention may be of use either alone or in combination with a further p-lactam antibiotic in treating a variety of diseases in humans and animals, caused by pathogenic bacteria, such as respiratory tract or urinary tract infections.

According to a further feature of the invention, we provide pharmaceutical compositions (including veterinary compositions) containing at least one of the acids, physiologically acceptable salts or metabolically active esters of the invention. In view of the protective action described above, the compositions can advantageously contain one or more further ss-lactam antibiotics, preferably ones that are orally absorbed. The compositions may also contain a pharmaceutical (including veterinary) carrier or excipient.

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 parenteral administration.The compounds may thus be formulated in ampoules for reconstitution before use, optionally together with a further antibiotic compound.

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

The active compounds of the invention will generally be administered 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 up to 4 times per day. Where the composition contains a further ss-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 up to 4 times a day. 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 used alone and 25 mg to 5 g, preferably 100 mg to 1 g, of total ss-lactam antibiotic where a further antibiotic is present.

The compounds of the invention may be prepared in any convenient way. The compounds of formula (II) in which R is an amino group may be prepared from the compound of formula (II) in which R is an azido group by reduction, for example by catalytic hydrogenation. The hydrogenation catalyst is normally a noble metal catalyst, e.g.

palladium, platinum or rhodium, or another transition metal catalyst such as nickel. The catalyst may be supported, e.g. on charcoal or kieselguhr; the metal catalyst is preferably palladium, e.g. as 10% palladium on charcoal. Hydrogenation will desirably be effected in a solvent for the starting material.

Suitable solvents for the hydrogenation include ethers such as diethyl ether or tetrahydrofuran; esters such as ethyl acetate; ketones such as acetone or methyl ethyl-ketone; chlorinated hydrocarbons such as methylene chloride; amides such as dimethyl-formamide or dimethylacetamide; or alcohols such as ethanol; or mixtures thereof. Such solvents may advantageously be admixed with water or an aqueous buffer. In some combinations a two phase system may result. In cases where the substrate is sufficiently water-soluble, water or an aqueous buffer may be used alone.

The azido group may alternatively be reduced using dissolving metal reducing agents, e.g. zinc and aqueous hydrochloric acid controlled in the pH range 2 to 6, preferably 4 - 4.5.

Suitable solvents include, for example, water-miscible solvents such as ketones, e.g.

acetone, ethers such as tetrahydrofuran and alcohols such as ethanol.

Certain esters used as starting material may be readily cleaved during reduction, e.g.

arylmethyl esters such as the p-nitrobenzyl esters, to yield a free carboxyl group although, as indicated hereinafter, selective reduction of such esters may be effected without significant reduction of the ester grouping.

The acid product may be isolated from solution by, for example, partitioning between the solvent (where water-immiscible) and water and after removal of catalyst, the aqueous phase may be lyophilised to yield the desired acid.

The acid product may also be isolated from solution by precipitation or crystallisation either as its zwitterionic form or as a salt as defined above. Depending on conditions the isolated compound may be in the form of a solvate or hydrate.

Azido compounds according to the invention may be prepared by reaction of a compound of the formula

(where R3 represents an esterified carboxyl group as defined above and X is a readily displaceable atom or group) with an azide, e.g. an alkali metal azide such as sodium azide, or an organic azide, such as a tetraalkylguanidinium azide e.g. tetramethylguanidinium azide or a tetraalkylammonium azide such as tetrabutylammonium azide. The reaction is preferably carried out in a solvent medium for the reactants; the solvent is normally polar, e.g. a substituted amide or imide solvent such as dimethylformamide or dimethylacetamide; a nitrile solvent such as acetonitrile; a sulphoxide such as dimethylsulphoxide; an ester such as ethyl acetate; a cyclic ether such as tetrahydrofuran, a chlorinated hydrocarbon such as methylene chloride; or a ketone such as acetone. Water-miscible solvents, e.g. acetone, may be desirably slightly aqueous to assist solubilisation of the azide. A small quantity of a carboxylic acid such as acetic acid may advantageously be present.

The substituent X may, for example, be a halogen atom (chlorine, bromine or iodine) or an acyloxy group, such as an aliphatic, aromatic or araliphatic carbonyloxy or 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. C, 8) group, which may be substituted by a halogen atom, e.g. fluorine or chlorine, or an aryl (e.g. C6.15) group which may carry substituents, for example, alkyl, e.g. methyl, alkoxy, e.g. methoxy or halogen, e.g. bromine. The aliphatic or aromatic grouping of such a carbonyloxy group may be, for example, an alkyl (e.g. C1.8) group, preferably substituted by one or more halogen atoms, e.g. chlorine or fluorine, or an aryl (e.g. C6. is). group, optionally substituted by, for example, one or more halogen atoms or nitro groups. Such acyloxy groupings may thus include dichloroacetoxy, mesyloxy, fluoromethanesulphonyloxy, tosyloxy or phenylsulphonyloxy.

Compounds of formula (III) in which X is halogen may be obtained by reaction of an ester of the compound of formula (I) with a halogenating agent such as thionyl halide or with a sulphonylating agent, e.g. a mesylating or tosylating agent, in the presence of halide ions. Compounds of formula (III) in which X is an acyloxy group may be prepared by reacting an ester of the compound of formula (I) with an appropriate acylating agent, e.g.

an acyl halide or anhydride.

Where X in the compound of formula III is halogen, it is preferably chlorine or bromine.

If desired, the compound of formula (III) may be reacted with the azide without isolation from the medium.

In the above reactions, where the initial product is an ester 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 4.0 - 5.5, 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.

tetrahydrofuran) followed by hydrolysis either under 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.

When reductive cleavage is used in the deesterification of an ester of formula (II) in which R is N3, the azido group may tend to be reduced simultaneously. The careful use of selective methods does, however, enable the azido acid of formula (II) to be prepared.

Thus, for example, the p-nitrobenzyl ester of the azide acid can be deesterified by using a dissolving metal reducing agent such as zinc and hydrochloric acid with pH control as described above to yield the azide acid or a salt thereof.

The esters of clavulanic acid used as starting materials in the above processes may be prepared from clavulanic acid or a reactive derivative 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, therefore, at temperatures between -70" and +35"C is preferred.

The alkyl, alkoxyalkyl and aralkyl esters may be prepared by reaction of the acid of formula (I) with the appropriate diazoalkane or diazoaralkane, 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 -15" to +15"C.

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 hydrocarbon-sulphonyl derivative such as a mesyl or tosyl ester, with a salt of the 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, such as a triethylammonium salt. This reaction is preferably carried out in a sulphoxide or amide solvent, e.g. dimethyl sulphoxide, dimethylformamide or hexamethylphosphoramide.

Stannyl esters may conveniently be formed by reaction of the 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 availability and low toxicity.

The acids of formula (II) may, if desired, be esterified using the methods described above for preparation of esters of clavulanic acid. In particular the azido acid of formula (II) may, if desired, be re-esterified to introduce an ester group different from that initially present in the azido ester product prepared from the compound of formula (III). It is thus possible to introduce an ester group which is inert to the conditions used in subsequent reduction of the azido group, thereby facilitating production of esters of the amine acid of formula (II) (R = NH2; R3 = COOH). Such ester groups may be of a type which cannot be split by reductive cleavage, e.g. alkyl esters such as the methyl ester, or they may be susceptible to reductive cleavage but be less reactive than the azido group.

In the formation of salts, 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 salts, an alkanoate is a preferred base, e.g. a 2-ethylhexanoate.

Acid-addition salts of compounds of formula (II) wherein R is an amino group can readily be prepared by reaction of the free amino compound of formula (II) with an acid.

The invention will now be further described in the following Preparations and Examples which should not be construed as limiting the invention.

The following Preparations illustrate the means whereby the starting materials for the preparation of the compounds of the invention may be obtained. All temperatures are in "C.

PREPARATION 1 4-nitrobenzyl- (3R, 5R, Z) -2- (2-hydroxyethylidene) -clavam-3-carboxylate A mixture of lithium (3R,5R,Z)-2-(2-hydroxyethylidene)-clavam-3-carboxylate (lOg), 4-nitrobenzyl bromide (9.5g) and hexamethylphosphoramide (65 ml) was stirred for 18 hr.

at room temperature. The suspension was then partitioned between ethyl acetate (800 ml) and 50% saturated aqueous sodium chloride solution (800 ml). The organic layer was separated, washed successively with water, 0.5M aqueous sodium hydrogen carbonate solution and water, dried and concentrated to a slurry and the colourless crystals collected to give the title ester (8.39 g), m.p. 117.2 (Mettler) (Mettler is a registered Trade Mark) kEta H 264nm (E 11,000), Vmax (CHBr3) 1781 (P-lactam), 1738 (ester), 1680cm1(0-C=C), (CDCl3) values include 4.30 (d, J 2Hz, C-5H), 4.61 (s, benzylic protons), 5.09 (t, J 7Hz, =CH-) and 5.78 (d, J 7Hz,-CH2OH).

PREPARATION 2 4-Nitrobenzyl(3R, SR, Z) -2- (2-chloroethylidene) -clavam-3-carboxylate A solution of 4-nitrobenzyl (3R,5R,Z)-2-(2-hydroxyethylidene)-clavam-3-carboxylate (1.0g) in ethyl acetate (20ml) containing pyridine (0.32 ml) was cooled to -60 , stirred and treated with a solution of thionyl chloride (0.26ml) in ether (2.0ml). The mixture was warmed to 100 and stirred for a further 10 min. at 100 to 0 and then diluted with ether (250ml). The mixture was washed successively with 0.5N aqueous hydrochloric acid, water, saturated aqueous sodium hydrogen carbonate solution (until the washings were colourless) and water. The ether layer was dried and concentrated to give a slurry of colourless needles which were collected by filtration, washed with ether and dried to give the chloroester (320mg), [a]D +30 (c 0.49, DMSO XEtoH 264nm (E 10,550), VmaX (CHBr3) 1800 (p- lactam), 1753 (ester) 1692 cm' (O-C=(f)tT(CDCl3) values include 4.25 (d, J2Hz, C-5H), 4.7 (s, benzylic protons), 5.08 (t, J8Hz, =CH-), 5.82 (d, J 8Hz, CH2Cl).

PREPARATION 3 Methyl (3R, SR, Z)-2- (2-hydroxyethylidene)clavam-3-carboxylate A suspension of lithium (3R,5R ,Z)-2-(2-hydroxyethylidene)-clavam-3-carboxylate (4.00g) in a mixture of brine (80 ml) and ethyl acetate (200 ml) was acidified with 2N hydrochloric acid (20ml) and shaken. The separated aqueous phase was further extracted with ethyl acetate (200 ml) and the combined organic solutions were dried, filtered and concentrated under reduced pressure to ca. 200 ml. The resulting ethyl acetate solution of the free acid was stirred, cooled to less than 5" and treated with excess ethereal diazomethane. The solvents were removed under reduced pressure to leave an oil which was chromatographed on a column of silica gel. Elution with ethyl acetate - petroleum (bp.

40-60 ) (3:1) gave the crude title ester as yellow cyrstals (2.32g, 56%).

A portion of this product (350mg) was dissolved in a mixture of ether (30ml) and petroleum (bp 40-60 ) (20ml) and the solution treated with charcoal and filtered through kieselguhr. The filtrate was concentrated to ca. 20 ml and set aside at 0 to crystallise. The resulting colourless needles were collected, washed with petroleum (bp. 40-60 ) and dried in vacuo to give the title ester (312mg), m.p. 63.5 (Mettler), [a]D + 76" (c 1.03, DMSO), Xmax (0.1 N NaOH) 258nm (E 18,300), vmax (CHBr3) 3590 (OH), 1798 ( -lactam), 1748 (CO2R), 1694 cm" (O-C=C), T (CDCl3) values include 4.29 (d, J 2.5 Hz, C-S H), 5.77 (d, J 7 Hz, CH2OH), 6.21 (s, CH3).

PREPARATION 4 Benzyl (3R, SR, Z) -2 - (2-hydroxyethyliden e) clavam 3-carboxylate A mixture of lithium (3R, 5R, Z)-2-(2-hydroxyethylidene) clavam-3-carboxylate (10.25 g) and benzyl bromide (8.55 g) in hexamethylphosphoramide (50 ml) was stirred at room temperature for 22 hrs. The mixture was then diluted with ethyl acetate (1 1.) and washed successively with 50% saturated brine (1 1.), water (2 x 500 ml) 5M NaHCO3 (500 ml) and brine (3 x 250 ml). The organic layer was dried (Na2SO4) and concentrated in vacuo to an oil, which was chromatographed on a column of silica gel (150 g) and eluted with chloroform, followed by ethyl acetate. The appropriate fractions were combined and concentrated in vacuo t6 yield the title ester (8.8 g) as an oil, vmax (CHBr) 3590 (-OH), 1786 (p-lactam), 1732 (ester), 1684 (-O-C=CH), 734 cm~1 (phenyl). T (CDCl3) 2.68 (s, phenyl), 4.38 (d, J 3 Hz, C-S H), 4.85 (s, CH~,Ph), 4.95 (s, C-3 H), 5.16 (t, J 7 Hz, CH), 5.85 (d, J 7 HZ, -CH2 OH), 6.55 (dd, J 17 and 3 Hz, C-6 a H), 6.98 (d, J 17 Hz, C-6 p H), 8.29 (s, -CH2OH).

EXAMPLE 1 4-Nitrobenzyl (3R, 5R, Z) -2- (2-azidoethylidene) clavam-3-carboxylate A mixture of 4-nitrobenzyl (3R,SR Z)-2-(2-chloroethylidene) clavam-3-carboxylate (0.5 g), sodium azide (0.092 g), acetone (15 ml), water (1.5 ml) and acetic acid (ca.1.Oml) was stirred at ambient temperature for 15 minutes and then partitioned between ether and water. The organic phase was washed with water, dried over sodium sulphate and concentrated to ca 10 ml. The residue was loaded onto a dry column of silica gel which was then eluted with ether. Fractions were collected and combined on the basis of thin layer chromatographic examination and the solution concentrated. On standing at 0 the solution deposited pale yellow crystals. The crystals were collected, washed with ether and dried in vacuo to afford the title ester (0.078 g), m.p. 63-64", [a]D + 38 (c 0.9; EtOAc), (D2O) 4.20 (d, J 3Hz, C-S H), 4.98 (s, C-3 H), 5.14 (t, J 8 Hz, =CH-), 6.30 (d, J 8Hz, CH2NH2), 6.39 and 6.86 (dd, J 17 and 3Hz; d, J 17Hz, C-6 protons).

EXAMPLE 5 Sodium (3R,5R, Z) -2- (2-azidoethylidene) clavam-3-carboxylate Zinc dust (1.9 g) was added in small portions over a 2 hour period to a stirred solution of 4-nitrobenzyl (3R,5R,Z)-2(2-azidoethylidene)clavam-3-carboxylate (1.5 g) in tetrahydrofuran (80 ml) and water (50 ml) which was maintained at 0 by cooling and at pH 4.3 by the dropwise addition of N hydrochloric acid. The resulting mixture was stirred for a further 1l/2 hours, filtered and partitioned between ethyl acetate (150 ml) and water (100 ml).

Hydrogen sulphide was passed through the two phase mixture and the pH of the aqueous phase maintained between 2.5 and 4.5 by the addition of N sodium hydroxide solution.

When the pH had become steady at 4.5, the mixture was filtered through kieselguhr and the separated organic layer extracted with 0.5N aqueous sodium hydrogen carbonate solution ( x 3). These extracts were combined with the separated aqueous layer, washed with ethyl acetate, acidified to pH2 with 2N hydrochloric acid and extracted with ethyl acetate. The organic extract was dried over magnesium sulphate, filtered and treated with a solution of sodium 2-ethylhexanoate (0.6 g) in ethyl acetate. The resulting solution was concentrated to ca, 2 ml and slowly diluted with ether. The precipitate was collected, washed with ether and dried to afford the title salt (0.45g), [aJ20 + 51 (c0.95; H2O), Xmax (0.1 N NaOHaq) 258 nm (E 14,100), Vrnax (Nujol) 2100 (azide), 1782 (p-lactam), 1688 (enol ether) and 1614 cm-l (carboxylate); t (D20) 4.19 (d, J 3Hz, C-SH), 4.92 (s, C-3 H), 5.01 (t, J 8 Hz, =CH-), 6.03 (d, J 9Hz, -CH2N3), 6.36 and 6.84 (dd, J 17 and 3 Hz, d, J 17Hz, C-6 protons).

EXAMPLE 6 Methyl (3R,5R, Z) -2- (2-azidoethylidene) clavam-3carboxylate An aqueous solution of sodium (3R ,SR,Z)-2-(2-azidoethylidene)clavam-3-carboxylate (0.2 g) was acidified with 2N hydrochloric acid and extracted with ethyl acetate. The organic solution was dried over magnesium sulphate and treated at 0 with an excess of ethereal diazomethane. The solution was purged with a stream of nitrogen for 10 minutes and then evaporated to give the title ester as an oil (0.16 g), [a]20 + 66" (c 0.85; CHC13), Xmax (0.1 N NaOHaq) 258nm (E 15,700), vmax (CHBr3) 2100 (azide), 1798 (p-lactam), 1748 (ester) and 1690 cm- (enol ether); x(CDCl3) 4.27 (d, J 3Hz, C-S H), 4.88 (s, C-3H), 5.15 (t, J 8Hz, = CH-), 6.13 (d, J 8Hz, -CH2N3), 6.20 (s, CH3), 6.49 and 6.92 (dd, J 17 and 3Hz, d, J 17Hz, C-6 protons).

EXAMPLE 7 Methyl (3R,SR, Z) -2- (2-azidoethylidene)clavam-3-carboxylate A stirred solution of methyl (3R,SR,Z)-2-(2-hydroxyethylidene)clavam-3-carboxylate (2.0 g) under nitrogen was cooled to50 and treated with pyridine (2.17 ml), followed by thionyl bromide (0.92 ml) in ethyl acetate (10 ml). The resulting mixture was stirred for 10 minutes at -45" and then poured into water (80 ml). The separated organic phase was added to a stirred suspension of sodium azide (0.71 g) in a mixture of water (8 ml) and acetone (55 ml) at -5". Acetic acid (0.2 ml) was added dropwise and the mixture allowed to warm to room temperature. After 15 minutes the resulting solution was partitioned between water (150 ml) and ether (100 ml). The separated organic layer was washed with water and with saturated brine. The solution was dried over magnesium sulphate and evaporated to give the title ester as an oil (0.79 g), [a]20 + 63" (c 1.1; CHCl3), Xmax (0.1 N NaOH aq) 258 mm (E 15,260). The n.m.r. and i.r. spectra were similar to those described in Example 6.

EXAMPLE 8 Benzyl (3R,5R, Z) -2- (2-azidoethylid ene) clavam-3-carboxylate A stirred solution of benzyl (3R,SR,Z)-2-(2-hydroxyethylidene)clavam-3-carboxylate (1.Og) in ether (20 ml) under nitrogen was cooled to -55" and treated with pyridine (0.8 ml), followed by thionyl bromide (0.34 ml) in ether (5 ml). The resulting mixture was stirred for 10 minutes at ca - 45" and was then poured into water. The organic phase was washed with water and was then treated with a solution of sodium azide (0.26 g) in a mixture of water (3 ml) and acetone (20 ml). The resulting mixture was concentrated by evaporation until homogeneous, treated with acetic acid (ca 0.2 ml) and stood at room temperature for 10 minutes. The solution was partitioned between ether and water and the organic layer was washed with water and brine. The dried solution was concentrated to ca 10 ml and chromatographed on a dry column of silica gel with ether as eluant. Appropriate fractions were combined and evaporated to an oil. The oil was redissolved in chloroform and evaporated to give the title ester as an oil (0.395 g), vrnax (CHBr3) 2100 (azide), 1792 (p-lactam), 1744 (ester) and 1688 cm-l (enol ether); T (CDCl3) 2.62 (s, aromatic protons), 4.29 (d, J 3Hz, C-S H), 4.79 (s, CH2 Ph), 4.86 (s, C-3 H), 5.20 (t, J 8Hz, = CH-), 6.17 (d, J 8Hz, -CH2N3), 6.50 and 6.96 (dd, J 17 and 3 Hz, d, J 17Hz, C-6 protons).

EXAMPLE 9 (3R,5R, Z)-2- (2-aminoethylidene)clavam-3-carboxylic acid Zinc powder (10.0 g) was added in small portions over a period of a hour to a stirred solution of 4-nitrobenzyl (3R,5R,Z)-2-(2-azidoethylidene)clavam-3-carboxylate (2.5g) in a mixture of tetrahydrofuran (130 ml) and water (80 ml) which was maintained at 0 by cooling and at pH 4.5 by dropwise addition of N hydrochloric acid. The reaction mixture was stirred for a further 21/2 hours and was then partitioned between water (60 ml) and ethyl acetate (60 ml). Hydrogen sulphide was passed through the mixture and the pH of the aqueous phase was maintained at ca 3 by the addition of N sodium hydroxide solution.

When the pH had ceased to change, the mixture was filtered through kieselguhr and the separated aqueous phase was washed with ethyl acetate and lyophilised. The residue was stirred with methanol and some insoluble material removed by filtration. The solution was concentrated to ca 25 ml and precipitated sodium chloride removed by filtration. The filstrate was diluted with ether and the resulting precipitate was collected, washed and dried to yield the title acid (0.94 g). The n.m.r. and i.r. spectra were similar to those described in Example 4.

EXAMPLE 10 Benzyl (3R, 5R, Z) -2- (2-aminoethylidene) clavam-3-carboxylate A stirred solution of benzyl (3R,5R,Z)-2-(2-aminoethylidene)clavam-3-carboxylate (0.lg) in tetrahydrofuran (2ml) and water (Iml) was treated with zinc dust (0.2 g) in small portions over a S minute period while the pH was maintained between 2 and 4 by dropwise addition of 2N hydrochloric acid. The resulting mixture was decanted from the zinc residue and partitioned between brine and ethyl acetate. The aqueous phase at pH S was extracted with ethyl acetate. The organic extract was dried over sodium sulphate and evaporated to give a gum. The gum was redissolved in chloroform and evaporated to yield the title ester as a foam (0.092 g), vmax (CHBr3) 3320 (NH2), 1798 (p-lactam), 1740(ester) and 1696 cm-' (enol ether); T (CDCl3) 2.62 (s, aromatic protons), 4.31 (d, J 2Hz, C-S H), 4.80 (s, CH2Ph), 4.86 (s, C-3 H), 5.08 (d, J 7Hz, = CH-), 6.41 (d, J 7Hz, -CH2NH2), 6.58 and 6.80 (dd, J 17 and 2Hz, d, J 17Hz, C-6 protons).

EXAMPLE 11 Methyl (3R,5R, Z) -2- (2-am in oethylidene) clavam-3-carboxylate Zinc powder (1.2 g) was added in portions over a 20 minute period to a stirred solution of methyl (3R,5R,Z)-2-(2-azidoethylidene)clavam-3-carboxylate (0.5 g) in tetrahydrofuran (25 ml) and water (15 ml) which was maintained at 0 by cooling and at pH 4-4.5 by the dropwise addition of N hydrochloric acid. The mixture was stirred for a further 20 minutes and was then filtered and partitioned between saturated brine at pH 7.2 and ethyl acetate.

The separated aqueous phase was extracted with further ethyl acetate and the combined organic extracts were dried over magnesium sulphate and evaporated to yield the title ester as a gum, vmnx (CHBr3) 1790 ( -lactam) and 1750 cm-l (ester); T(DMSO-d6) 4.17 (d. J 2Hz, C-5H), 4.68 (s, C-3 H), 5.16 (t, J 7Hz, = CH-), 6.21 (s, CH3), 6.54 (m, CH2NH2), 6.3-6.9 (m, C-6 protons).

EXAMPLE 12 (3R,5R, Z) -2- (2-ammonioeth vlidene) clavam-3-carboxvlic acid-4-toluenesulphonate A solution of (3R,SR,Z)-2-(2-aminoethylidene)clavam-3-carboxylic acid (100 mg) and 4-toluenesulphonic acid monohydrate (90 mg) in water (10 ml) was lyophilised to afford the title salt, vfT,ax (Nujol) 2700 (NH3+), 1794 (P-lactam), 1730 (carboxyl), 1700 (enol ether) and 1200 cm (sulphonate), T (D20) 2.30 and 2.65 (doublets, J 8Hz. aromatic protons)+4.20 (d. J 3Hz. C-5H), 4.75 (s. C-3 H), 5.08 (t, J 7Hz, = CH-), 6.31 (d. J 7Hz, CH2NH3), 6.39 and 6.82 (dd. J 17 and 3 Hz. d, J 17 Hz, C-6 protons) and 7.61 (CH.).

EXAMPLE 13 (3Ri5R, Z) -2- (2-aminoethylid ene) clavam-3-carboxvlic acid A solution of benzyl (3R.SR,Z)-2-(2-aminoethylidene)clavam-3-carboxylate in ethyl acetate (10 ml) was prepared from benzyl (3R,5R,Z)-2-(2-aminoethylidene)clavam-3- carboxylate (0.49 ) in a manner similar to that described in Example 10. The solution was diluted with ethanol (10 ml) and the mixture hydrogenated over 10% palladium on charcoal (0.35 g) for 20 minutes at room temperature and atmospheric pressure. Water (10 ml) was added and the catalyst removed by filtration. The aqueous phase was washed with ethyl acetate and lyophilised to give the title acid (0.04g) as a foam. The n.m.r. and i.r. spectra resembled those described in Example 3.

EXAMPLES A - C In these Examples, (3R,5R,Z)-2-(2-aminoethylidene)clavam-3-carboxylic acid (as the zwitterion) is used as densified granules containing 1% magnesium stearate, which are prepared as follows: Blend (3R,5R,Z)-2-(2-aminoethylidene)clavam-3-carboxylic acid with 1% magnesium stearate and prepare tablet slugs by direct compression on a tablet machine. Break down the slugs through a series of screens (12 mesh, 16 mesh and 20 mesh) on a rotary granulator to produce free flowing densified granules with an apparent bulk density of about 0.7 gms per ml (BSS method).

Ampicillin trihydrate granules (used in Example B) may be prepared in a similar manner.

EXAMPLE A Formula per tablet Densified (3R,5R,Z)-2-(2-aminoethylidene) clavam-3-carboxylic acid granules containing 1% magnesium stearate 252.5 mg Sodium lauryl sulphate 1.25 mg Avicel PH 101 grade of microcrystalline cellulose to tablet core weight of 325 mg (Avicel is a registered Trade Mark) Method of preparation Blend sodium lauryl sulphate with an equal weight of Avicel and pass the blend through a 40 mesh sieve. Blend together granules of the amine, the sodium lauryl sulphate-Avicel blend and the balance of the Avicel and compress them on deep concave punches, 9.5 mm in diameter. Take precautions to avoid unnecessary exposure to light and conditions of high humidity.

EXAMPLE B Formula per capsule Densified (3R,5R,Z)-2-(2-aminoethylidene)clavam-3-carboxylic acid granules containing 1% magnesium stearate 252.5 mg sodium lauryl sulphate 1.25 mg Densified ampicillin trihydrate granules containing 1% magnesium stearate equal to 250 mg ampicillin (approx.) 300.0 mg Sodium starch glycollate (Primojel) 11.5 mg Target capsule fill weight 565.25 mg Method of preparation Remove a proportion of fine granules passing 40 mesh equal to the weight of Primojel from the bulk densified granules of the amine and blend with Primojel. Add the sodium lauryl sulphate and blend. Add the blend to the bulk and re-blend. Blend with the ampicillin granules. Fill the composite granules, into size 0 hard gelatin capsules (lock fitting type) on an automatic capsule filling machine. Take precautions to avoid unnecessary exposure to light and conditions of high humidity.

EXAMPLE C Dry powder for injection Fill sterile (3R,5R,Z)-2-(2-aminoethylidene)clavam-3-carboxylic acid (500 mg) into glass vials, carry out the filling aseptically, under a blanket of nitrogen. Close the vials using rubber discs or plugs held in position by aluminium sealing rings, thereby preventing gaseous exchange or ingress of micro-organisms. Constitute the product by dissolving in Water for Injection shortly before administration. Other suitable sterile vehicles can be used in place of Water for Injection.

Claims (61)

WHAT WE CLAIM IS:

1. A compound of the formula (II)

wherein R is an azido group -N3 or an amino group -NH2 and R3 is a carboxyl or esterified carboxyl group, together with salts and zwitterionic forms thereof.

2. A compound of formula (II) as shown in Claim 1 wherein R is an amino group -NH2 and R is a carboxyl group or esterified carboxyl group, together with salts and zwitterionic forms thereof.

3. A compound a formula (1I) as shown in Claim 1, wherein R is an azido group -N3 and R3 is a carboxyl or esterified carboxyl group, together with salts thereof.

4. A compound as claimed in any of claims 1 to 3 wherein R3 represents an esterified carboxyl group derived from an aliphatic or araliphatic alcohol, a phenol or a stannanol.

5. A compound as claimed in claim 4 wherein R3 represents an esterified carboxyl group -COOR4 wherein R4 represents a straight or branched substituted or unsubstituted alkyl or alkenyl group having from 1-8 carbon atoms; an aralkyl group having up to 20 carbon atoms; an aryl group having up to 12 carbon atoms; a cycloalkyl group containing up to 12 carbon atoms, optionally containing one or more heteroatoms in the ring system, and unsaturation optionally being present when a heteroatom is present, or a stannyl group having up to 24 carbon atoms.

6. A compound as claimed in claim 5 wherein -COOR4 is a metabolically labile ester group.

7. A compound as claimed in either Claim 5 or Claim 6, wherein R4 is an acetoxymethyl, phthalidyl, a-ethoxycarbonyloxyethyl or pivaloyloxymethyl group.

8. A compound as claimed in either Claim 5 or Claim 6, wherein R4 is a methyl, methoxymethyl, acetoxyethyl or ethoxycarbonyloxymethyl group.

9. A compound as claimed in claim 5 wherein R4 is a diphenylmethyl group, a benzyl group or a benzyl group substituted by an o- or p-nitro, p-methoxy or p-methyl group.

10. Methyl (3R.5R.Z)-2-(2-aminoethylidene)clavam-3-carboxylate.

11. Benzyl (3R.SR ,Z)-2-(2-aminoethylidene)clavam-3-carboxylate.

12. Methyl (3R,5R.Z)-2-(2-azidoethylidene)clavam-3-carboxylate.

13. Benzyl (3R,5R.Z)-2- (2-azidoethylidene)clavam-3-carboxylate .

14. 4-Nitrobenzvl (3R.5R.Z)-2-(2-azidoethylidene)clavam-3-carboxylate.

15. (3R.SR ,Z)-2-(2-aminoethylidene)clavam-3-carboxylic acid.

16. (3R,5R,Z)-2-(2-aminoethylidene)clavam-3-carboxylic acid in the form of a crystalline solid.

17. Physiologically acceptable salts of (3R,5R,Z)-2-(2-aminoethylidene) clavam-3carboxylic acid.

18. The alkali metal, alkaline earth metal, ammonium or organic base salts of (3R .SR,Z)-2-(2-aminoethylidene)clavam-3-carboxylic acid.

19. The lithium. sodium. potassium, calcium. magnesium or ammonium salt of (3R,SR,Z)-2-(2-aminoethylidene)clavam-3-carboXvlic acid.

20. The acid addition salts of (3R.SR.Z)-2-(2-aminoethylidene)clavam-3-carboxylic acid and its esters.

21 (3R.SR.Z)-2-(2-Azidoethylidene)clavam-3-carboxylic acid and physiologically acceptable salts thereof.

22. The alkali metal. alkaline earth metal. ammonium or organic base salts of (3R.SR.Z)-2-(2-azidoethylidene)clavam-3-carboxylic acid.

23. The lithium. sodium. potassium, calcium, magnesium or ammonium salt of (3R.SR,Z)-2-(2-azidoethylidene)clavam-3-carboxylic acid.

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

25. A composition as claimed in Claim 24. wherein the weight ratio of the compound of formula (II) to the further -lactam antibiotic is 10.1 to 1:10.

26. A composition as claimed in claim 24 which is in the form of dosage units containing 12.5 mg to S g of a compound as claimed in claim 1 when this is employed alone and from 25 mg to 5 g of total ss-lactam antibiotic when a compound as claimed in claim 1 and a further ss-lactam antibiotic are present.

27. A composition as claimed in any of claims 24 to 26 wherein the further ss-lactam antibiotic is a cephalosporin or penicillin.

28. A composition as claimed in Claim 27 wherein the cephalosporin or penicillin antibiotic is cephalexin, cephaloglycin, cephalothin, cephaloridine, cefazolin, cephacetrile, cephapirin, penicillin G, penicillin V, ampicillin, amoxycillin, carbenicillin or ticarcillin or an orally absorbed ester thereof.

29. A composition as claimed in Claim 28 wherein the further p-lactam antibiotic is ampicillin. .

30. A composition as claimed in Claim 28 wherein the further ss-lactam antibiotic is amoxycillin.

31. A composition as claimed in any of claims 24-30 wherein the compound of formula (II) is (3R,5R,Z) -2-(2-aminoethylidene)clavam-3 -carb oxylic acid.

32. A process for the preparation of a compound of formula (II) as claimed in claim 1 wherein R is an amino group (-NH2) wherein a compound of formula (II) as claimed in claim 1 wherein R is an azido group (-N3) is subjected to reduction.

33. A process as claimed in claim 32 wherein reduction is effected by catalytic hydrogenation.

34. A process as claimed in claim 33 wherein hydrogenation is effected using a noble metal catalyst.

35. A process as claimed in claim 34 wherein hydrogenation is effected using a palladium catalyst.

36. A process as claimed in claim 32 in which reduction is effected using a dissolving metal reducing agent.

37. A process as claimed in claim 36 in which reduction is effected using zinc and aqueous hydrochloric acid controlled in the pH range 2-6.

38. A process as claimed in any of claims 32 to 37 in which R3 in the starting compound of formula (II) is a readily cleavable esterified carboxyl group.

39. A process as claimed in claim 38 wherein R3 in the starting compound of formula (II) is a p-nitrobenzyl-oxycarbonyl group.

40. A process as claimed in claim 39 wherein the p-nitrobenzyloxycarbonyl group is cleaved during the reduction so as to provide a compound of formula (Il) wherein R represents an amino group and R3 represents a carboxyl group.

41. A process as claimed in any of claims 32 to 37 in which R3 in the starting compound of formula (II) is an esterified carboxyl group and reduction of the azido group is effected without substantial cleavage of the group R3.

42. A process as claimed in any of claims 32 to 40 wherein R3 in the compound of formula (II) initially produced is a carboxyl group and the said compound is subjected to esterification followed, where an acid addition salt of the ester so formed is required, by reaction thereof with an acid.

43. A process as claimed in any of claims 32-40 wherein a salt of the compound of formula (II) wherein R is an amino group and R3 is a carboxyl group is formed by treatment thereof with an acid or a base.

44. A process for the preparation of a compound of formula (II) wherein R is an azido group (-N3) and R3is an esterified carboxyl group wherein a compound of the formula (III).

(wherein R3 is an esterified carboxyl group and X is a readily displaceable atom or group) is reacted with an azide.

45. A process as claimed in claim 44 wherein X represents a halogen atom or acyloxy group.

46. A process as claimed in claim 45 wherein X represents a chlorine or bromine atom.

47. A process as claimed in claim 45 wherein X represents a mesyloxy or tosyloxy group.

48. A process as claimed in any of claims 44-47 wherein the azide is an alkali metal azide.

49. A process as claimed in any of claims -11-17 wherein the azide is an organic azide.

50. A process as claimed in claim 48 wherein sodium azide is employed.

51. A process as claimed in claim 49 wherein the azide is a tetraalkvlammonium or tetraaikylguanidinium azide.

52. A process as claimed in any of claims 45-51 wherein an azide ester initiallv formed is deesterified to yield the azide acid of formula (II) wherein R is -N3 and R-7 is carboxyl.

53. A process as claimed in claim 52 in which R3 in the said azide ester is a p-nitroberzyloxycarbonyl group and deesterification is effected by selective reduction without substantial reduction of the azide group.

-

54. A process as claimed in claim 52 or claim 53 in which the azide acid produced is subjected to reesterification.

55. A process as claimed in claim 52 or claim 53 in which the azide acid produced is converted to a salt thereof.

56. A process as claimed in claim 32 substantiallv as hereinbefore described.

57. A process as claimed in claim 32 substantially as hereinbefore described xzith reference to Example 3.

58. A process as claimed in claim 3' substantially as hereinbefore described with reference to Examples 4. 9-11 and 13.

59. A process as claimed in claim g substantially as hereinbefore described.

60. A process as claimed in claim u substantially as hereinbefore described with reference to Examples 1 and 2.

61. A process as claimed in claim a substantially as hereinbefore described with reference to either of Examples 7 and 8.