IE49174B1 - Cephalosporin antibiotics - Google Patents

Abstract

Cephalosporin antibiotics of the general formula in which R and R, which may be identical or different, each denote a C1-4-alkyl group, or R and R, together with the carbon atom to which they are attached, form a C3-7-cycloalkylidene group exhibit a broad spectrum of antibiotic action and have a particularly high activity against members of the Enterobacteriaceae. The invention also comprises the non-toxic salts and non-toxic metabolically labile esters and the solvates of the compounds of the formula (I). Also described are compositions containing the antibiotics of the invention and processes for the preparation of such antibiotics.

Description

This invention is concerned with cephalosporin compounds possessing valuable antibiotic properties.

The cephalosporin compounds in this specification are named with reference to cepham after J, Amer, Chem. Soc,, 1962, 84, 3400, the term cephem referring to the basic cepham structure with one double bond.

Cephalosporin antibiotics are widely used in the treatment of diseases caused by pathogenic bacteria in human beings and animals, and are especially useful in the treatment of diseases caused by bacteria which are resistant to other antibiotics such as penicillin coumpounds, and in the treatment of penicillin-sensitive patients.

In many instances it is desirable to employ a cephalosporin antibiotic which exhibits activity against both gram-positive and gram-negative microorganisms, and a significant amount of research has been directed to the development of various types of broad spectrum cephalosporin antibiotics.

Thus, , for example, in our Patent Specification No. 38172, we describe a novel class of cephalosporin antibiotics containing a 7P-(a-etherified oximino)-acylamino group, the oximino group having the syn configuration. This class of antibiotic compounds is characterised by high antibacterial activity against a range of gram-positive and gram-negative organisms coupled with particularly high stability to β-lactamases produced by various gram-negative organisms.

The discovery of this class of coumpounds has stimulated further research in the same area in attempts to find compounds which have improved properties, for example against particular classes of organisms especially gram-negative organisms.

In our Patent Specification No. 42144, we describe cephalosporin antibiotics containing a 7p-acylamino group of the formula R.C.CO.NH^0. (GH,) C (CH,) COOH i m ι χ n (A) A B (wherein R is a thienyl or furyl group; R and R may vary widely and may, for example, be alkyl groups or together with the carbon atom to which they are attached form a C^ ? cycloalkylidene group, and tn and n are each 0 or 1 such that the sum of m and n is 0 or 1), the compounds being syn isomers or mixtures of syn and anti isomers containing at least 90% of the syn isomer. The 3-position of the cephalosporin molecule may be unsubstituted or may contain one of a wide variety of possible substituents. These compounds have been found to have particularly good activity against gram-negative organisms.

Other compounds of similar structure have been developed from these compounds in further attempts to find antibiotics having improved broad spectrum antibiotic activity and/or high activity against gram-negative organisms. Such developments have involved variations in not only the 7β-acylamino group in the above fotnula but also the introduction of particular groups in the 3-position of the cephalosporin molecule, Thus, for example, in Belgian Patent Specification No.852,427, there are described cephalosporin antibiotic compounds falling within the general scope of our Patent Specification No. 38172, and wherein the group R in the above formula (A) may be replaced by a variety of different organic groups, including 2-aminothiazol-4-yl, and the oxygen atom in the oxyimino group is attached to an aliphatic hydrocarbon group which may itself be substituted by, for example, carboxy. In such compounds, the substituent at the 3-position is confined to an acyloxymethyl, hydroxymethyl, formyl or optionally substituted heterocyclic-thiomethyl group.

Furthermore, Belgian Patent Specification No. 836,813 describes cephalosporin compounds wherein the group R in formula (A) above may be replaced by, for example, 2-aminothiazol-4-yl, and the oxyimino group is a hydroxyimino or blocked hydroxyimino group, e.g. a methoxyimino group. In such compounds, the 3-position of the cephalosporin molecule is substituted by a methyl group which may itself be optionally substituted by residues of a large number of nucleophilic compounds therein described. In the above-mentioned Specification no antibiotic activity is ascribed to such compounds which are only mentioned as intermediates for the preparation of antibiotics described in the Specification.

Belgian Patent Specification No.653,545 describes cephalosporin antibiotics wherein the 7p-acylamino side chain is primarily a 2-(2-aminothiazol-4-yl)-2-(syn)-methoxvimino-acetamido group and the substituent in the 3-position is broadly IQ defined in a similar manner to that in the above-mentioned Belgian Patent Specification No.836,813.

We have now discovered that by an appropriate selection of a small number of particular groups at the 7p-position in combination with a methyl group at the 3-position, cephalosporin compounds having particularly advantageous activity (described in more detail below) against a wide range of commonly encountered pathogenic organisms may be obtained. 2Q The present invention provides cephalosporin antibiotics of the general formula (wherein Ra and r\ which may be the same or different, each represents a C^_^alkyl group (preferably a straight chain alkyl group i.e. a methyl, ethyl, n-propyl or n-butyl group and particularly a methyl or ethyl group) or a b R and R together with the carbon atom to vhich they are attached form a cycloalkylidene group, preferably a cycloalkylidene group) and non-toxic salts and nontoxic metabolically labile esters thereof.

The compounds according to the invention are syn isomers. ' The syn isomeric form is defined by the configuration of the group I - O.C.COOH Rb with respect to the carboxamido group. In this specification the syn configuration is denoted structurally as NHq A2 s N 2θ \=J— c.CO.NH H pa N R O.C.COOH Rb It will be appreciated that since the compounds 25 according to the invention are geometric isomers, some admixture with the corresponding anti isomer may occur.

The invention also includes within its scope the solvates (especially the hydrates) of the compounds of formula (I). It also includes within its scope salts of esters of compounds of formula (I).

The compounds according to the present invention may exist in tautomeric forms (in respect of the 2-aminothiazolyl group) and it will be understood that such tautomeric forms, e.g, the 2-iminothiazolinyl form, are included within the scope of the invention.

It will also be appreciated that when R and Rb in the above formula represent different Cj_^ alkyl groups, the carbon atom to which they are attached will comprise a centre of asymmetry. Such compounds are diastereoisomeric and the present invention embraces individual diastereoisomers of these compounds as well as mixtures thereof.

The compounds according to the invention exhibit broad spectrum antibiotic activity. Against gram-negative organisms the activity is unusually high.

This high activity extends to many β-lactamase-producing gram-negative strains. The compounds also possess high stability to β-lactamases produced by a range of gram-negative organisms.

Compounds according to the invention have been found to exhibit high activity against various members of the Enterobacterlaceae (e.g. strains of Escherichia coli, Klebsiella pneumoniae. Salmonella typhimurium, Shigella sonnei. Enterobacter cloacae.

Serratia marcescens,Providence species, Proteus mirabilis and especially indole-positive Proteus organisms such as Proteus vulgaris and Proteus morganil). as well as against strains of Haemophilus influenzae and good activity against strains of Pseudomonas organisms e.g, strains of Pseudomonas aeruginosa. This activity against strains of indole-positive Proteus and Pseudomonas organisms is unusual, bearing in mind the antibacterial activity of known 3-methyl cephalosporin antibiotics.

In addition to their high antibiotic activity, the 3-methyl compounds according to the present invention possess the further advantage that in comparison with analogous compounds possessing more elaborate 3-substituents, they can be manufactured relatively easily and economically on an industrial scale from readily available starting materials, i.e. penicillin G or penicillin V.

Non-toxic salt derivatives which may be formed by reaction of either or both of the carboxyl groups present in the compounds of general formula (I) include inorganic base salts such as alkali metal salts (e.g. sodium and potassium salts) and alkaline earth metal salts (e.g. calcium salts); amino acid salts (e.g. lysine and arginine salts); organic base salts (e.g. procaine, phenylethylbenzylamine, dibenzylethylenediamine, ethanolamine, diethanolamine and N-methylglucosamine salts). Other non-toxic salt derivatives include acid addition salts, e.g. formed with hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, formic and trifluoroacetic acids. The salts $ay also be in the form of resinates formed with, for example, a polystyrene resin or cross-linked polystyrene divinylbenzene copolymer resin containing amino or quaternary amino groups, sulphonic acid groups, or with a resin containing carboxyl groups, e.g. a polyacrylic acid resin. Soluble base salts (e.g. alkali metal salts such as the sodium salt) of compounds of formula (I) may be used in therapeutic applications because of the rapid distribution of such salts in the body upon administration. Where, however, insoluble salts of compounds (I) are desired in a particular application, e.g. for use in depot preparations, such salts may be fonned in conventional manner, for example with appropriate organic amines.

These and other salt derivatives such as the salts with toluene-p-sulphonic and methanesulphonic acids may be employed as intermediates in the preparation and/or purification of the present compounds of formula (I), for example in the processes described below.

Non-toxic metabolically labile ester derivatives which may be formed by esterification of either or both carboxyl groups in the parent compound of formula (I) include acyloxyalkyl esters, e.g» lower alkanoyloxy-methyl or -ethyl. esters such as acetoxymethyl or -ethyl or pivaloyloxymethyl esters. In addition to the above ester derivatives, the compounds . of the present invention may be used in the form of other physiologically acceptable equivalents, i.e. physiologically acceptable compounds which, like the metabolically labile esters, are converted in vivo into the parent antibiotic compound of formula (I).

Preferred compounds according to the present invention include the following compounds of formula (I) and their non-toxic salts and non;·toxic metabolically labile esters, namely:(6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(2carboxyprop-2-oxyimino) acetamido]-3-methylcepb-3-em-4carboxylic acid; (6K,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1carboxycyclobut-1-oxyimino) acetamido]-3-methylceph-39 em-4-carboxylic acid; and (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(lcarboxycyclopent-1-yloxyimino) acetamido]-3-methylceph-3em-4-carboxylic acid.

Other examples of compounds of formula (I) according to the present invention include those wherein θ b the groups R and R have the meanings given in the following Table TABLE a) Alkyl groups CH., c2h5C2H5 b) Cycloalkylidene groups cyclopropylidene cyclohexylidene The compounds of formula (I) may be used for treating a variety of diseases caused by pathogenic bateria in human beings and animals, such as respiratory tract infections and urinary tract infections.

According to another embodiment of the invention we provide a process for the preparation of an 48174 antibiotic compound of general formula (I) as hereinbefore defined or a non-toxic salt or non-toxic metabolically labile ester thereof which comprises acylating a compound of the formula hydrogen or a carboxyl blocking group, e.g, the residue of an ester-forming aliphatic or araliphatic alcohol or 15 an ester-forming phenol, silanol or stannanol (the said alcohol, phenol, silanol or stannanol preferably containing 1- 20 carbon atoms) and the dotted line bridging the 2-, 3-, and 4-positions indicates that the compound is a ceph2- em or ceph-3-em compound] or a salt, e.g, an acid addition salt (formed with, for example, a mineral acid such as hydrochloric, hydrobromic, sulphuric, nitric or phosphoric acid or an organic acid such as methanesulphonic or toluene-p-sulphonic acid) or an N-silyl derivative thereof, with an acid of formula 25 R3 A S N — C.COOH R \ 1 2 O.C.COOR (III) a b 2 (wherein R and R are as hereinbefore defined; R represents a carboxyl blocking group, e.g. as described 1 3 for R ; and R is an amino or protected amino group) or with an acylating agent corresponding thereto whereafter, if necessary and/or desired in each instance, any of the following reactions, in any appropriate sequence, are carried outj2 3 i) conversion of a Δ -isomer into the desire^ Δ -isomer, ii) reduction of a compound wherein B is /S-^0 to form a compound wherein B is /S, iii) conversion of a carboxyl group into a non-toxic salt or metabolically labile ester function, and iv) removal of any carboxyl blocking and/or N-protecting · groups.

In the above-described process, the starting material of formula (II) is preferably a compound wherein B is and the dotted line represents a ceph-3-em compound.

Acylating agents which may be employed in the preparation of compounds of formula (I) include acid 12 4-817 4 halides, particularly acid chlorides or bromides. Such acylating agents may be prepared by reacting an acid (III) or a salt thereof with a halogenating agent e.g. phosphorus pentachloride, thionyl chloride or oxalyl chloride.

Acylations employing acid halides may be effected in aqueous and non-aqueous reaction media, conveniently at temperatures of from -50 to +50°C, preferably -20 to +30°C, if desired in tbe presence of an acid binding agent. Suitable reaction media include aqueous ketones such as aqueous acetone, esters such as ethyl acetate, halogenated hydrocarbons such as methylene chloride, amides such as dimethylacetamide, nitriles such as acetonitrile, or mixtures of two or more such solvents. Suitable acid binding agents include tertiary amines (e.g. triethylamine or dimethylaniline), inorganic bases ( e.g. calcium carbonate or sodium bicarbonate), and oxiranes such as lower 1,2-alkylene oxides (e.g. ethylene oxide or propylene oxide) which bind hydrogen halide liberated in the acylation reaction.

Acids of formula (III) may themselves be used as acylating agents in the preparation of compounds of formula (I). Acylations employing acids (III) are desirably conducted in the presence of a condensing agent, for example a carbodiimide such as N,N’-dicyclohexylcarbodiimide or N-ethyl-N'-y-dimethylaminopropylcarbodiimide; a carbonyl compound such as carbonyldiimidazole; or an isoxazolium salt such as N-ethyl-5-phenylisoxazolium perchlorate, Acylation may also be effected with other amide-forming derivatives of acids of formula (III) such as, for example, an activated ester, a symmetrical anhydride or a mixed anhydride (e.g. formed with pivalic acid or with a haloformate, such as a lower alkylhaloformate). Mixed anhydrides may also be formed with phosphorus acids (for example phosphoric or phosphorous acids), sulphuric acid or aliphatic or aromatic sulphonic acids (for example toluene-p-sulphonic acid). An activated ester may conveniently be formed in situ using, for example, 1-hydroxybenzotriazole in the presence of a condensing agent as set out above.

Alternatively, the activated ester may be preformed.

Acylation reactions involving the free acids or their above-mentioned amide-forming derivatives are desirably effected in an anhydrous reaction medium, e.g. methylene chloride, tetrahydrofuran, dimethylformamide or acetonitrile.

If desired, the acylation reactions may be carried out in the presence of a catalyst such as 4dimethylaminopyridine.

The acids of formula (ill) and acylating agents 20 corresponding thereto may, if desired, be prepared and employed in the form of their acid addition salts. Thus, for example, acid chlorides may conveniently be employed as their hydrochloride salts, and acid bromides as their hydrobromide salts.

The reaction product may be separated from the reaction mixture, which may contain, for example, unchanged cephalosporin starting material and other substances, by a variety of processes including recrystallisationf ionophoresis, column chromatography and use of ion-exchangers (for example by chromatograptyon ion-exchange resins) or macroreticular resins. Δ -Cephalosporin ester derivatives obtained in accordance with the process of the invention may be converted into the corresponding Δ -derivative? by, for 2 example, treatment of the Δ -ester with a base, such as pyridine or triethylamine.

A ceph-2-em reaction product may also be oxidised to yield the corresponding ceph-3-em 1-oxide, for example by reaction with a peracid, e.g. peracetic or m-chloroperbenzoic acid; the resulting sulphoxide may subsequently be reduced as described hereinafter to yield the corresponding ceph-3-em sulphide.

Where a compound is obtained in which B is ^S->0 this may be converted to the corresponding sulphide by, for example, reduction of the corresponding acyloxysulphonium or alkoxysulphonium salt prepared in situ by reaction with e.g. acetyl chloride in the case of an acetoxysulphonium salt, reduction being effected by, for example, sodium dithionite or by iodide ion as in a solution of potassium iodide in a water-miscible solvent e.g. acetic acid, acetone, tetrahydrofuran, dioxan, dimethylformamide or dimethylacetamide. The reaction may be effected at a temperature of from -20° to +50°C.

Metabolically labile ester derivatives of the compounds of formula (I) may be prepared by reacting a compound of formula (I) or a salt or protected derivative thereof with the appropriate esterifying agent such as an acyloxyalkyl halide (e.g. iodide) conveniently in an inert organic solvent such as dimethylformamide or acetone, followed, where necessary, by removal of any protecting groups.

Base salts of the compounds of formula (I) may be formed by reacting an acid of formula (I) with the appropriate base. Thus, for example, sodium or potassium salts may be prepared.using the respective 2-ethylhexanoate or hydrogen carbonate salt. Acid . addition salts may be prepared by reacting a compound of formula (I) or a metabolically labile ester derivative thereof with the appropriate acid.

Where a compound of formula (I) is obtained as a mixture of isomers, the syn isomer may be obtained by, for example, conventional methods such as crystallisation or chromatography.

For use as starting materials for the preparation of compounds of general formula (I) according to the invention, compounds of general formula (III) and acid halides and anhydrides corresponding thereto in their syn isomeric form or in the form of mixtures of the syn isomers and the corresponding anti isomers containing at least 90% of the syn isomer are preferably used.

Acids of formula (ill) (provided that Ra and together with the carbon atom to which they are attached do not form a cyclopropylidene group) may be prepared by etherification of a compound of formula _ C.COOR II OH (IV) (wherein R3 is as hereinbefore defined and R^ represents a carboxyl blocking group), by reaction with a compound of general formula Ra ' 2 T.C.COOR (wherein Ra and Rb are as hereinbefore defined and T is halogen such as chloro, bromo or iodo; sulphate; or sulphonate such as tosylate), followed by removal of the 4 carboxyl blocking group R . Separation of isomers may be effected either before or after such etherification.

The etherification reaction is generally carried out in the presence of a base, e.g. potassium carbonate or sodium hydride, and is preferably conducted in an organic solvent, for example dimethylsulphoxide, a cyclic ether such as tetrahydrofuran or dioxan, or an N,N-disubstituted amide such as dimethylformamide. Under these conditions the configuration of the oxyimino groups ie substantially unchanged by the etherification reaction. The reaction should be effected in the presence of a base if an acid addition salt of a compound of formula (IV) is used. The base should be used in sufficient quantity to neutralise rapidly the acid in question.

Acids of general formula (III) may also be prepared by reaction of a compound of formula (VI) \=/-co. COOR4 (wherein RJ and R* are as hereinbefore defined) with a compound of formula H2N.O.C.COOR2 (VII) (wherein Ra, Rb and R2 are as defined above), followed by 4 removal of the carboxyl blocking group R , and where necessary by the separation of syn and anti isomers.

The last mentioned reaction is particularly applicable to the preparation of acids of foimula (III) jq fo wherein R and R together with the carbon atom to which they are attached form a cyclopropylidene group. In this case, the relevant compounds of formula (VII) may be prepared in conventional manner, e.g. by means of the synthesis described in Belgian Patent Specification No. 866,422 for the preparation of t-butyl 1-amino-oxycyclopropane carboxylate.

The acids of formula (III) may be converted to the corresponding acid halides and anhydrides and acid addition salts by conventional methods.

It should be appreciated that in some of the above transformations it may be necessary to protect any sensitive groups in the molecule of the compound in question to avoid undesirable side reactions. For example, during any of the reaction sequences referred to above it may be necessary to protect the NHj group of the aminothiazolyl moiety, for example by tritylation, acylation (e.g. chloroacetylation), protonation or other conventional method. The protecting group may thereafter be removed in any convenient way which does not cause breakdown of the desired compound, e.g. in the case of a trityl group by using an optionally halogenated carboxylic acid, e.g. acetic acid, formic acid, chloroacetic acid or trifluoroacetic. acid or using a mineral acid, e.g. hydrochloric acid or mixtures of such acids, preferably in the presence of a protic solvent such as water, or in the case of a chloroacetyl group, by treatment with thiourea.

Carboxyl blocking groups used in the preparation of compounds of formula (I) or in the preparation of necessary starting materials are desirably groups which may readily be spilt off at a suitable stage in the reaction sequence, conveniently at the last stage.

It may, however, be convenient in some instances to employ non-toxic metabolically labile carboxyl blocking groups such as acylaxy-methyl or-ethyl'(e.g. acetoxy-methyl or -etlyl and pivaloyloxymethyl) and retain these in the final product to give an appropriate ester derivative of a compound of formula (I).

Suitable carboxyl blocking groups are well known in the art, a list of representative blocked carboxyl groups being included in Patent Specification No. 38172. Preferred blocked carboxyl groups include aryl lower alkoxycarbonyl groups such as p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl and diphenylmethoxycarbonyl; lower alkoxycarbonyl groups such as t-butoxycarbonyl; and lower haloalkoxycarbonyl groups such as 2,2,2-trichloroethoxycarbonyl. Carboxyl blocking group(s) may subsequently be removed by any of the appropriate methods disclosed in the literature; thus, for example, acid or base catalysed hydrolysis is applicable in many cases, as are enzymically-catalysed hydrolyses, The antibiotic compounds of the invention may be formulated for administration in any convenient way, by analogy with other antibiotics and the invention therefore includes within its scope pharmaceutical compositions comprising an antibiotic compound in accordance with the invention adapted for use in human or veterinary medicine. Such compositions may be presented for use in conventional manner with the aid of any necessary pharmaceutical carriers or excipients.

The antibiotic compounds according to the invention may be formulated for injection and may be presented in unit dose form in ampoules, or in multi-dose containers, if necessary with an added preservative.

The compositions may also take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilising and/or dispersing agents. Alternatively the active ingredient may be in powder form for reconstitution with a suitable vehicle, e.g. sterile, pyrogen-free water, before use.

If desired, such powder formulations may contain an appropriate non-toxic base in order to improve the water-solubility of the active ingredient and/or to ensure that when the powder is reconstituted with water, the pH of the resulting aqueous formulation is physiologically acceptable. Alternatively, the base may be present in the water with which the powder is reconstituted. The base may be, for example, an inorganic base such as sodium carbonate, sodium bicarbonate or sodium acetate, or an organic base such as lysine or lysine acetate.

The antibiotic compounds may also be formulated as suppositories, e.g. containing conventional suppository bases such as cocoa butter or other glycerides.

Compositions for veterinary medicine may, for example, be formulated as intramammary preparations in either long acting or quick-release bases.

The compositions may contain from 0.1% upwards, e.g. 0,1-99% of the active material, depending on the method of administration. When the compositions comprise dosage units, each unit will preferably contain 50-1500 mg of the active ingredient. The dosage as employed for adult human treatment will preferably range from 500 to 6000 mg per day, depending on the route and frequency of administration. For example, in adult human treatment 1000 - 3000 mg per day administered intravenously or intramuscularly will normally suffice. In treating Pseudomonas infections higher daily doses may be required.

The antibiotic compounds according to the 5 invention may be administered in combination with other therapeutic agents such as antibiotics, for example penicillins or other cephalosporins.

The following Examples illustrate the invention, All temperatures are in °C. ‘Petrol’ means petroleum ether (b.p. 40-60°).

Preparation 1 Ethyl (Z)-2-(2-aminothiazol-4-yl)-2-(hydroxyiniino)acetate To a stirred and ice-cooled solution of ethyl acetoacetate (292 g) in glacial acetic acid (296 ml) was added a solution of sodium nitrite (180 g) in water (400 ml) at such a rate that the reaction temperature was maintained below 10°C. Stirring and cooling were continued for about 30 min., when a solution of potassium chloride (160 g) in water (800 ml) was added. The resulting mixture was stirred for one hour. The lower oily phase was separated and the aqueous phasewas extracted with diethyl ether. The extract was combined with the oil, washed successively with water and saturated brine, dried, and evaporated. The residual oil, which solidified on standing, was washed with petrol and dried in vacuo over potassium hydroxide, giving ethyl (Z)-2-(hydroxyimino)3-oxobutyrate (309 g).

A stirred and ice-cooled solution of ethyl (Z)-2-(hydroxyimino)-3-oxobutyrate (150 g) in dichloromethane (400 ml) was treated dropwise with sulphuryl chloride (140 g). The resulting solution was kept at room temperature for 3 days, then evaporated.

The residue was dissolved in diethyl ether, washed with water until the washings were almost neutral, dried, and evaporated. The residual oil (177 g) was dissolved in ethanol (500 ml) and dimethylaniline (77 ml) and thiourea (42 g) were added with stirring. After two hours, the product was collected by filtration, washed with ethanol and dried to give the title compound (73 g); m.p.188 (decomp.).

Preparation 2 Ethyl (Z)-2-hydroxyimino-2-(2-tritylaminothiazol-4-yl)acetate, hydrochloride.

Trityl chloride (16.75 g) was added portionwise 5 over 2 hours to a stirred and cooled (-30°) solution of the product of Preparation 1 (12.91 g) in dimethylformamide (28 ml) containing triethylamine (8.4 ml). The mixture was allowed to warm to 15° over one hour, stirred for a further 2 hours and then partitioned between water (500 ml) and ethyl acetate (500 ml). The organic phase was separated, washed with water (2 x 500 ml) and then shaken with IN HCl (500 ml). The precipitate was collected, washed successively with water (100 ml), ethyl acetate (200 ml) and ether (200 ml) and dried in vacuo to provide the title compound as a white solid (16.4 g); m.p. 184 to 186° (decomp).

Preparation 3 Ethyl (Z)-2-(2-t-butoxycarbonylprop-2-oxyimino)-2-(2-tritylaminothiazol-4-yl)acetate Potassium carbonate (34.6 g) and t-butyl 2-brorao-2-methylpropionate (24.5 g) in dimethylsulphoxide (25 ml) were added to a stirred solution under nitrogen of the product of Preparation 2 (49.4 g) in dimethylsulphoxide (200 ml) and the mixture was stirred at room temperature for 6 hours. The mixture was poured into water (2 1), stirred for 10 mins., and filtered. The solid was washed with water and dissolved in ethyl acetate (600 ml). The solution was washed successively with water, 2N hydrochloric acid, water, and saturated brine, dried, and evaporated. The residue was recrystallised from petroleum ether (b.p. 60-80°) to give the title 48174 compound (34 g), m.p. 123.5 to 125° Preparation 4 (Z)-2-(2-t-Butoxycarbonvlprop-2-oxyimino)-2-(2-tritylaminothia2ol-4-yl)acetic acid The product of Preparation 3 (2 g) was dissolved in methanol (20 ml) and 2N sodium hydroxide (3.3 ml) was added. The mixture was refluxed for 1.5 hours and then concentrated. The residue was taken up in a mixture of water (50 ml), 2N hydrochloric acid (7 ml), and ethyl acetate (50 ml). The organic phase was separated, and the aqueous phase extracted with ethyl acetate. The organic solutions were combined, washed successively with water and saturated brine, dried, and evaporated. The residue was recrystallised from a mixture of carbon tetrachloride and petrol to give the title compound (1 g), m.p. 152 to 156° (decomp). Preparation 5 Ethyl (Z)-2-(2-tritylaniinothiazol-4-yl)-2-(l-t-butoxycarbonylcyclobut-l-oxyimino) acetate.

The product of Preparation 2 (55,8 g) was stirred under nitrogen in dimethylsulphoxide (400 ml) with potassium carbonate (finely ground, 31.2 g) at room temperature. After 30 minutes, t-butyl 1-bromocyclobutane carboxylate (29.2 g) was added. After 8 hours further potassium carbonate (31.2 g) was added. More potassium carbonate (6 x 16 g portions) was added during the next three days and further t-butyl 1-bromocyclobutane carboxylate (3.45 g) was added after 3 days. After 4 days in all, the mixture was poured into ice-water (ca. lit~es) and the solid was collected by filtration and . 49174 washed well with water and petrol. The solid was dissolved in ethyl acetate and the solution washed with brine (twice), dried with magnesium sulphate and evaporated to a foam. This foam was dissolved in ethyl acetate-petrol(l:2) and filtered through silica gel (500 g) Evaporation gave the title compound (60 g) as a foam, V (CHBr_) 3400 (NH) and 1730 cm b (ester), max J Preparation 6 (Z)-2-(1-t-Butoxycarbonvlcyclobut-l-oxyimino)-2-(2-trityl·aminothiazol-4-yl) acetic acid.

A mixture of the product of Preparation 5 (3.2g) and potassium carbonate (1.65 g) was refluxed in methanol (180 ml) and water (20 ml) for 9 hours and the mixture was cooled to room temperature. The mixture was concentrated and the residue partitioned between ethyl acetate and water, to which was added 2N HCl (12.2 ml).

The organic phase was separated and the aqueous phase extracted with ethyl acetate. The combined organic extracts were washed with saturated brine, dried and evaporated to give the title compound (2.3 g): λ ——— max (ethanol) 26 5 nm (E^ 243).

Preparation 7 Ethyl(Z)-2-(i-t-butoxycarbonylcvclopent-l-vloxyimino)-2- (2-tritylaminothiazol-4-yl)acetate The product of Preparation 2 (lOg) was stirred with t-butyl 2-bromo-cyclopentanecarboxylate (7g) in dimethylsulphoxide (40ml) containing potassium carbonate (lOg) under nitrogen at 21° for 21 hours. The mixture was poured into ice-water (500ml) and the grey solid was collected by filtration, washed with water and air dried. • 48174 Recrystallisation of this solid from methanol (500ml) gave the title compound (11.7g), m.p. 179-180°, (CHBr^) 3410 (NH), 1735 (ester), 1275 (ester) and 755 cm1 (phenyl). Preparation 8 (Z)-2"(l-t-Butoxycarbonylcyclopent-l‘vloxvin)ino)-2- IZz tritvlaminothiazol-4-vl·) acetic acid The Product from Preparation 7 (625mg) was refluxed with 2N sodium hydroxide solution (0.5ml) and water (lml) in methanol (12ml) for seven hours. The mixture was left to cool overnight. After dilution with water, orthophosphoric acid was added to adjust the solution to pH 2. The precipitate was extracted with ether and the combined extracts were washed with brine. After drying with magnesium sulphate, the solvent was evaporated to give a gum (493mg). Recrystallisation from di-isopropyl ether gave the title compound (356mg) m.p. 171-173°, γ (CHBr_) 2500-3500 (OH and NH), 1755 (ester), 1692 I0SJC j (acid) and 755 and 770 cin (phenyl).

Example 1 a) Diphenylmethyl (6R,7R)-r(Z)-2-(2-t-butoxycarbonylprop2-oxyimino)-2-(2-tritylaminothiazol-4-yl) acetamido]-3methylceph-3-em-4 -carboxylate A stirred solution of the product of Preparation 4 (2.86 g) and diphenylmethyl (6R, 7R)-7-amino-3«methylceph-3-em-4-carboxylate (2.09 g) in dimethylformamide (50 ml) was cooled to 0° and treated with l-hydroxybenzotriazole (745 mg) and dicyclohexyicarbodiimide (1.14 g).

The mixture was warmed to room temperature and stirred overnight. The mixture was filtered, and the white solid washed with a little ethyl acetate. The filtrate and washings were diluted with water (100 ml) and extracted with ethyl acetate. The organic extracts were combined, washed successively with 2N hydrochloric acid, water, sodium bicarbonate solution, and saturated brine, dried, and evaporated. The residue was eluted through a silica column with ethyl acetate. The product-containing eluate was collected and concentrated to give the title compound (3.6 g),X (ethanol) 238 nm (E^’283), λ inf & ’ maxo lcm ’ 263.5 nm (ε]·7ο186); [a] 20 + 6.5· (c 1.0, DMSO). lcm D — ’ b) (6R,7R)-7-f(Z)-2-(2-Aminothiazol-4-yl)-2-(2-carboxyprop2-oxyimino) acetamido]-3-methylceph-3-em-4-carboxylic acid Trifluoroacetic acid (20 ml) was added to a solution of the product of stage a) (2.8 g) in anisole (20 ml) at 0 The mixture was stirred at room temperature for 2 hours and concentrated. The residue was dissolved in ether and re-evaporated. The residue was dissolved in ethyl acetate and excracted with saturated sodium bicarbonate solution.

The pH of the aqueous extracts was adjuted to 6, and the solution washed with ethyl acetate. The aqueous phase was acidified to pH 1.5 under ethyl acetate, and extracted with ethyl acetate. The combined organic extracts were washed with saturated brine, dried, and evaporated.

The residue was dissolved in warm 75% aqueous formic acid (40 ml) and allowed to stand for 1 hour. The mixture was diluted with water and filtered. The filtrate was concentrated. The residue was taken up in water, refiltered, and lyophilized to give the title compound (900 mg), λ£η£(ρΗ 6 buffer) 236 nm (£^287), 261 (En1% 254), 296 nm (e}% 115)j v (Nujol) 1530, 1665 ' lcm ’ lcm (CONH), 1720 (C02H), 1765 cm (β-lactam).

Example 2 a) Diphenylmethyl (6R.7R)-7~r(Z)-2-(l-t-butoxycarbonylcyclobut-l-oxyimino)-2-(2-tritylaminothiazol-4-yl)acetamido1-3-methvlceph-3-em-4-carboxylate A stirred solution of the product of Preparation 6 (2.2 g) and diphenylmethyl (6R,7R)-7-amino-3-methylceph3-em-4-carboxylate (2.2 g) in dimethylformamide (45 ml) was cooled to O’, and 1-hydroxybenzotriazole (655 mg)was added, followed by dicyclohexylcarbodiimide (982 mg).

The mixture was warmed to room temperature and stirred overnight. The mixture was filtered. The filtrate was diluted with water (300 ml) and extracted with ethyl acetate. The organic extracts were combined, washed successively with 2N hydrochloric acid, sodium bicarbonate solution, and saturated brine, dried, and evaporated. The residue was eluted through a silica column with ether-petrol (3-1).

The appropriate fractions were concentrated to give ~ 49174 the title compound (2.1 g), (ethanol) 244 nm (E^m238), 302 nm (E^ &?); v max (CHBrp 1520, 1682 (CONH), 1722 (CO2R), 1788 cnA-lactam). b) (6R,7R)-7~r(2)-2-(2-Aminothiazol-4-vl)-2-(l-carboxy5 eyelobut-1-oxyimino) acetamido]-3-methylceph-3-em-4carboxylic acid Trifluoroacetic acid (8 ml) was added to a solution of the product of Stage a)(1.9 g) in anisole (2 ml) at 0* „ The mixture was stirred for 5 minutes and trifluoroacetic acid (32 ml) was added. The mixture was stirred at room temperature for 30 minutes and concentrated. The residue was dissolved in ethyl acetate and re-evaporated. The residue was dissolved in ethyl acetate and extracted with saturated sodium bicarbonate solution. The aqueous extracts, at pH 7 to 7.5, were washed with ethyl acetate, acidified to pH 1.5 under ethyl acetate, and extracted with ethyl acetate. The combined organic extracts were dried and evaporated. Tlie residue was dissolved in formic acid (30 ml), water (9 ml) added, and the mixture stirred at room temperature for 2 hours. The mixture was diluted with water (200 ml) and filtered. The filtrate was concentrated. The residue was taken up in water, refiltered, and lyophilized to give the title compound (730 mg), Xmax '+ 62.5* (c 1.0, DMSO). (pH 6 buffer) 241 nm (E 291 nm 129); [oc] , Example 3 a) Diphenylmethyl (6R,7R)-7-r(Z)-2-(l-t-butoxycarbonylcyclopent-l-yioxyiniino)-2-(2-tritylaminothiazol-4-yl)acetamido] -3-methylceph-3-em-4-carboxylate.

Th ι product of Preparations (lg) was dissolved in tetra· hydrofuran (25ml) and diphenylmethyl (6R,7R)-7-amino-330 methylceph-3-em-4-carboxylate (760mg) and 1-hydroxybenzotriazole hydrate (380mg) were added with stirring. When a clear solution had formed, dicyclohexylcarbodiimide (520mg) was added and the mixture was stirred at 21* for 24 hours. The solution was filtered and the filtrate was concentrated. This solution was percolated through a short column of neutral alumina (30g) with elution by ethyl acetate: 60*-80· petroleum ether (1:3). The product was loaded onto a column of silica (kieselgel 60G of 15μΜ particle size; 50g). This column was eluted with ethyl acetate: 60-80* petroleum ether (1:3) at a pressure of 81b/sq. in. and evaporation of the appropriate fractions 29 gave the title compound (510mg) as a foam, + 25.2* (C 0.95, CHClj), (CHBr3) 3405,3275 (NH), 1790 (β-lactam), 1727 (esters), 1683 and 1527 cm~l (amide). b) (6R.7R)-7~r(Z)-2-(2-aminothiazol-4-yl)-2-(l-carboxycvclopent-l-yioxviroino^acetamidoj-S-methylceph-S-em-A. carboxylic acid, trifluoroacetate salt.

The product from Stage a) (408mg) was mixed with anisole (0.3ml) and trifluoroacetic acid (2ml) was added.

After 2 hours at 21*, the solution was concentrated and the residue was poured into water (20ml). This mixture was washed with ether (three times) each time the ether being back extracted with water. The combined aqueous layers were evaporated to dryness and the residue was triturated with ether to give the title compound (88mg) λ (pH6 buffer) 239.5nm (E,/o304), λ. ., 290 (E,/o131), v (Nujol) 3700, lcm infl. lcm rosx -r 2100 (NH*, NH and OH), 1770 (β-lactam) and 1680 cm (amide and acid). 4917 4 PHARMACY EXAMPLES EXAMPLE A - Dry Powder for Injection Formula Per Vial (6R, 7R)-7-[(Z)-2-(2-Aminothiazol-4-yl)-2-(2-carboxyprop 5 -2-oxyimino)acetamido]-3-methylceph-3-em-4-carboxylic acid 500mg Sodium Carbonate, anhydrous 113mg Method Blend the sterile cephalosporin antibiotic with sterile sodium carbonate under aseptic conditions. Fill aseptically into glass vials under a blanket of sterile nitrogen. Close the vials using rubber discs or plugs, held in position by aluminium overseals, thereby preventing gaseous exchange or ingress of micro-organisms, Reconstitute the product by dissolving in Water for Injections or other suitable sterile vehicle shortly before administration.

EXAMPLE B - Dry Powder for Injection Fill sterile (6R, 7R)-7-[(Z)-2-(2-aminothiazol-4-yl) -2-(1-carboxycyclobut-1-oxyimino)acetamido]-3methylceph-3-em-4-carboxylic acid, disodium salt into glass vials such that each vial contains an amount equivalent to 500mg of the antibiotic acid. Carry out the filling aseptically under a blanket of sterile nitrogen. Close the vials using rubber discs or plugs, held in position by aluminium overseals, thereby 48174 preventing gaseous exchange or ingress of micro-organisms. Reconstitute the product by dissolving in Water for Injections or other suitable sterile vehicle shortly before administration.

Claims (9)

1. Cephalosporin antibiotics of general formula wherein R and R , which may be the same or different, s b 5 each represents a C^_^ alkyl group or R and R together with the carbon atom to which they are attached form a C^ 2 cycloalkylidene group, and non-toxic salts and non-toxic metabolically. labile esters thereof.

2. Compounds as claimed in claim 1 wherein at q b 10 least one of R and R represents a methyl or ethyl group. a b

3. Compounds as claimed in claim 1 wherein R and R together with the carbon atom to which they are attached form a C 3 _ 5 cycloalkylidene group. 15

4. (6R,7R)-7-[(Z)-2-(2-Aminothiazol-4-yl)-2(2-carboxyprop-2-oxyimino) acetamido]-3-methylceph-3em-4-carboxylic acid and its non-toxic salts.

5. (6R,7R)-7-[(Z)-2-(2-Aminothiazol-4-yl)-2(1-carboxycyclobut-l-oxyiraino) acetamido]-3-methylceph3-em-4-carboxylie acid and its non-toxic salts.

6. , (6R, 7R) - 7 - [ (Z ) - 2-(2-Aminothiazo1-4-yl)- 2-(1-carboxy cyclopent-l-yloxyimino) acetamido]-3-methylceph-3-era-4carboxylic acid and its non-toxic salts.

7. A process for the preparation of an antibiotic compound of general formula(l)as defined in claim 1 or a non-toxic salt or non-toxic metabolically labile ester thereof which comprises acyiatilng a 5 compound of formula wherein B is or OS—·*·0, R^ represents hydrogen or a carboxyl blocking group and the dotted line in formula(II)bridging the 2-, 3- and 4- positions indicates 10 that the compound is a ceph-2-em or ceph-3-etn compound, or an acid addition salt or N-silyl derivative thereof, with an acid of formula jq θ £ (wherein R and R are as defined in claim 1; R represents a 3 15 carboxyl blocking group; and R is an amino or protected amino group) or with an acylating agent corresponding thereto, whereafter, if necessary and/or desired in each instance, any of the following reactions, are carried out in any appropriate sequence:35 49 17 4 2 3 i) conversion of a Δ -isomer into the desired Δ -isomer ii) reduction of a compound wherein B is >-S ->0 to form a compound wherein B is >S iii) conversion of a carboxyl group into a non-toxic 5 salt or non-toxic metabolically labile ester function, and iv) removal of any carboxyl blocking and/or N- protecting groups;

8. A process as claimed in claim 7 wherein the 10 starting material of formula (II) is a ceph-3-em compound in which B is ^»S.

9. A pharmaceutical composition for use in human or veterinary medicine comprising an antibiotic compound as claimed in any of claims 1 to 6 in association 15 with a pharmaceutical carrier or excipient.