GB1604724A - 7-(2-aminothiazol-4-yl)-2-oxymino-acedamido)-cephem derivatives - Google Patents

Description

(54) 7-l2-AMINOTHIAZOL4YL)-2-OXYIMINO- ACETAMIDO]-CEPHEM DERIVATIVES (71) We, GLAXO OPERATIONS UK LIMITED, formerly known as Glaxo Laboratories Limited, a British Company of Greenford, Middlesex, do hereby declare the invention for which we pray that a patent may be performed, to be particularly described in and by the following statement:- 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 cephem structure with one double bond.

Cephalosporin by Cephalosporin antibiotics are widely used in the treatment of diseases caused 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 compounds, and in the treatment of penidilin- 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 British Patent Specification No. 1,399,086, we describe a novel class of cephalosporin antibiotics containing a 7P - z - etherified oximino) - acylamido group, the compounds being syn isomers or mixtures of syn and anti isomers wherein the syn isomer predominates. This class of antibiotic compounds are characterised by high antibacterial activity against a range of gram positive and gram-negative organisms coupled with partlcularly high stability to p- lactamases produced by various gram-negative organisms.

The discovery of this class of compounds 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.

For example, in our British Patent Specification No. 1,496,757, we describe cephalosporin antibiotics containing a 7ss-acylamido group of the formula

(wherein R is a thienyl or furyl group; RA and RB may be inter alia C14 alkyl groups or together with the carbon atom to which they are attached from a C37 cycloalkylidene group; and m and n are each 0 or I such that the sum of m and n is o or 1), the compounds being syn isomers or mixtures of syn and anti isomers containing at least 90% of the syn isomer. 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 aromatic groups (R1 and R) and the oximino etherifying groups in the above formulae 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 British Patent Specification No. 1,399,086 and wherein the group R in the above formula may be replaced by a variety of different organic groups, including 2 - aminothiazol - 4 - yl, and the oximino group is an aliphatic hydrocarbon group which may be substituted by, for example, carboxy, while the 3-position contains certain specific groups.

We have now discovered that by an appropriate selection of groups constituting the 78- and 3-positions compounds having particularly good activity against a wide range of gram-negative organisms may be obtained.

The present invention provides antibiotics of the general formula

(wherein R" and Rb, together with the carbon atom to which they are attached form a C7 7 cycloalkylidene group and Y represents a carbon-attached 5- or 6-membered heterocyclic ring containing at least one nitrogen atom, which ring may also contain one or more sulphur atoms and/or may be substituted by a C14 alkyl group) and non-toxic salts and non-toxic metabolically labile esters thereof.

Examples of heterocyclic rings which may be represented by Y in formula (I) above include pyridyl and tetrazolyl groups. When the heterocyclic ring is substituted by a C, alkyl group this may, for example, be methyl.

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

When the group Y is in a quaternary form, e.g. an N-alkyl pyridinium group.

the compounds of formula (I) may exist in the form of a betaine containing a --COOB group.

The compounds according to the invention exhibit good broad spectrum antibiotic activity. The compounds exhibit activity against microorganisms which produce p-lactames, and also possess very high stability to p-lactamases produced by a range of gram-negative organisms. Furthermore they have been found to exhibit good activity against various members of the Enterobacteriaceae (e.g.

strains of Escherichia coli, Kiebsiella aerogenes and especially Proteus mirabilis, Proteus morgani and Proteus vulgaris), as well as against Pseudomonas organisms e.g. strains of Pseudomonas aeruginosa.

As mentioned above, the compounds of the invention are syn isomers. The svn isomeric form is defined by the configuration of the group

with respect to the carboxamido group. In this specification the syn configuration is denoted structurally as

It will be understood that, since the compounds according to the invention are geometric isomers, some admixture with the corresponding anti isomer may occur.

Non-toxic salt derivatives which may be formed from 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, dlethanolamine and N-methylglucosamine salts); and, where appropriate, acid addition salts, e.g.

with hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, formic, trifluoroacetic, toluene-p-sulphonic and methane-suiphonic acids. The salts may 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, or, where appropriate, sulphonic acid groups, or, again where appropriate, with a resin containing carboxyl groups, e.g. a polyacrylic acid resin. Use of highly soluble base salts (e.g. alkali metal salts such as the sodium salt) of compounds of formula (I) is generally advantageous 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 formed in a conventional manner, for example with appropriate organic amines.

Non-toxic metabolically labile ester derivatives which may be formed from the compounds of general formula (I) and which on administration are converted In vivo into the antibiotic compound of formula (I), include, for example, acyloxymethyl esters, e.g. (C16 alkanoyl)oxymethyl esters such as acetoxyrnethyl, acetoxyethyl and pivaloyloxymethyl esters.

Preferred compounds according t9 the present invention by virtue of their high activity against Pseudomonas organisms, e.g. strains of Pseudomonas aeruginosa include those compounds of formula (I) wherein Re and Rb together with the carbon atom to which they are attached form a cyclobutylidene grou and Y represents a 1 - methylpyridinium - 2 - yl or 1 - methyltetrazol - 5 - yi group.

The compounds of formula (I) may be used for treating a variety of diseases caused by pathogenic bacteria in human beings and animals, such as respiratory tract and urinary tract infections.

The compounds according to the invention may be prepared by any convenient method, for example by techniques analogous to those described in British Patent Specification No. 1,399,086.

Thus according to one embodiment of the invention we provide a process for the preparation of an antibiotic compound of general formula I as hereinbefore defined or a non-toxic salt or non-toxic metabolically labile esters thereof which comprises either (A) condensing a compound of the formula

[wherein Y is as defined above; B is > S or > S < O (cr- or ,B-)- R3 represents hydrogen or a carboxyl blocking group, e.g. the residue of an ester-forming aliphatic or araliphatic alcohol or an ester-forming phenol, silanol or stannanol (the said alcohol, phenol, silanol or stannanol preferably containing 1-20 carbon atoms) or a symmetrical or mixed anhydride group derived from an appropriate acid; and the dotted line bridging the 2-, 3- and 4-positions indicates that the compound is a ceph - 2 - em or ceph - 3 - em compound], or salt, e.g. an acid addition salt such as a hydrochloride, hydrobromide, sulphate, nitrate, phosphate, methanesulphinate or tosylate, or an N-silylated derivative thereof with an acid of formula

(wherein Ra and Rb are as defined above; and R4 represents a carboxyl blocking group, e.g. as defined above for R4 and Rs is an amino or protected amino group) or with an acylating agent corresponding thereto; or (B) reacting a compound of formula

(wherein R", Rb, R5, B and the dotted line are as defined above; R3 and R38 may independently represent hydrogen or a carboxyl blocking group; and X is a replacable residue of a nucleophile, e.g. an acetoxy or dichloroacetoxy group or a halogen atom such as chlorine, bromine or iodine) with a sulphur nucleophile serving to form a group of formula CH2SY (wherein Y is as defined above) at the 3-position; whereafter, if necessary and/or desired in each instance, any of the following reactions (C) in any appropriate sequence, are carried out: (i) conversion of a A2-isomer into the desired A3-isomer, (ii) reduction of a compound wherein B is > S~O to form a compound where B is > S, (iii) conversion of a carboxyl group into a non-toxic metabolically labile ester function, and (iv) removal of any carboxyl blocking and/or N-protecting groups: and finally (D) recovering the desired compound of formula (I) or a non-toxic salt or non-toxic metabolically labile ester thereof, if necessary after separation of isomers.

Base salts of compounds of formula (I) may be formed by reaction of the cephalosporin acid with, for example, sodium 2-ethylhexanoate or potassium 2 ethylhexanoate. Biologically acceptable ester derivatives may be formed using conventional esterifying agents.

Acylating agents which may be employed in the preparation of compounds of formula (I) include acid 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 +500C, preferably -20 to +300C, if desired in the 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.

tFiethylamine or dimethylaniline), inorganic bases (e.g. calcium carbonate or uum bicarbonate0, and oxiranes such as C2~ 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'-diethyl-, dipropyl- or diisopropylcarbodiimide, N,N'dicyclohexylcarbodiimide or N - ethyl - N' - y dimethylaminopropylcarbodiimide; a carbonyl compound such as carbonyldiimidazole; or an isoxazolinium salt such as N - ethyl - 5 - phenylisoxaxolinium 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. with pivalic acid or formed with a haloformate such as a C18 alkylhaloformate). An activated ester may conveniently be formed in situ using, for example, 1 - hydroxy - benzotriazole in the presence of a condensing agent as set out above.

Mixed anhydrides may be formed with phosphorus acids (for example phosphoric or phosphorous acids), sulphuric acid or aliphatic or aromatic sulphonic acids (for example p-toluene sulphonic acid).

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, dimethylformamide or acetonitrile.

The acids of formula (III) may, if desired, be employed in the form of their acid addition salts e.g. their hydrochlorides.

In addition to the general methods described in British Patent Specification No. 1,399,086 referred to above, compounds of formula I may also be prepared as the reaction of a corresponding 3-acetoxymethyl cephalosporin compound with an appropriate sulphur nucleophile, for example, in an analogous manner to that described in British Patent Specification Nows. 1,012,943 and 1,206,305.

Compounds of formula I may also be prepared by the reaction of a corresponding 3-halomethylcephalosporin with an appropriate sulphur nucleophile disclosed in the above references, such a process being described in British Patent Specification No. 1,241,657, or by the reaction of a 3-halomethylcephalosporin sulphoxide with any appropriate sulphur nucleophile disclosed in the above references, such a process being described in British Patent Specification No.

1,326,531.

Appropriate sulphur nucleophiles include those of formula YSH in which Y is as defined above. However, when Y is a 1 - methylpyridinium - 2 - yl group, the corresponding N - methylpyrid - 2 - thione is preferably employed, the reaction being advantageously effected in the presence of sodium iodize.

A2-Cephalosporin ester derivatives obtained in accordance with the process of the invention may be converted into the corresponding A3 derivative by, for example, treatment of the 2 ester with a base.

A ceDh-2-em reaction product may also be oxidised to yield the corresponding ceph-3-em l-oxide, for example by reaction with a peracid, e.g.

peracetic acid; the resulting sulphoxide may, if desired, subsequently be reduced as described hereinafter to yield the corresponding ceph-3-em sulphide.

Where a compound is obtained in which B is > SO this may be converted to the corresponding sulphide by, for example, reduction of the corresponding acyloxysulphonium or alkyloxysulphonium 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 200 to +50"C.

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.

Where a compound of formula I is obtained as a mixture of isomers, the svn isomer may be obtained by, for example, conventional methods such as crystallisation or chromatography. Syn and anti isomers may be distinguished by appropriate techniques, e.g. by their ultraviolet spectra, by thin layer, paper or high pressure liquid chromatography or by their proton magnetic resonance spectra.

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 least90% of the syn isomer are preferably used.

Acids of formula (III) provided that R" and Rb 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

(wherein R5 is as hereinbefore defined and R6 represents hydrogen or a carboxyl blocking group), by reaction with a compound of general formula

[wherein Ra, Rb and R4 are as hereinbefore defined (provided that Ra and Rb together with the carbon atom to which they are attached do not form a cyclo ropylidene group) and T is halogen such as chloro, bromo or iodo; sulphate; or sulphonate such as tosylate], followed by removal of any carboxyl blocking group R6. Separation of isomers may be effected either before or after such etherification. The etherification reaction is desirably 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 dimethylsul hoxide, a cyclic ether such as tetrahydrofuran or dioxan, or an N,N-disubstituted amide such as dimethylformamide. Under these conditions the configuration of the oximino group is 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 rapidly neutralise the acid in question.

Acids of general formula (III) may also be prepared by reaction of a compound or formula

(wherein R3 and R4 are as hereinbefore defined) with a compound of formula

(wherein Ra, Rb and R4 are as defined for formula (III), followed by removal of any carboxyl blocking group R6.

The last-mentioned reaction is particularly applicable for the preparation of compounds of formula (III) wherein Ra and Rb together with the carbon atom to which they are attached form a cyclopropylidene group; in this case, the compounds of formula (VIII) may be prepared in an analogous manner to that described in British Patent Application No. 17679/77, (Serial No. 1602725).

Reaction of (VII) with (VIII) may be followed where necessary by the .separation of syn and anti isomers.

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

During any reaction sequences referred to above, it may be necessary to protect the NH2 group of the aminothiazolyl moiety for example by tritylation, acylation, e.g. chloracetylation or t-butoxycarbonylation, 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 aqueous solution of an optionally halogenated carboxylic acid, e.g. acetic acid, formic acid, chloroacetic acid or trifluoroacetic acid.

Carboxyl blocking groups used in te preparation of compounds of formula (I) or in the preparation of necessary starting materials are desirably groups which may readily be split off at a suitable stage in the reaction sequence, conveniently as the last stage. It may, however, be convenient in some instances to employ nontoxic, metabolically labile carboxyl blocking groups such as acyloxymethyl groups (e.g. acetoxymethyl, acetoxyethyl and pivaloyloxymethyl groups) and retain these in the final product to give a non-toxic 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 British Patent Specification No. 1,399,086. Preferred blocked carboxyl groups include aryl C26 akoxycarbonyl groups such as p-methoxybenzyloxycarbonyl, pnitrobenzyloxycarbonyl and diphenylmethoxycarbonyl; C26 alkoxycarbonyl groups such as t-butoxycarbonyl; and C 6 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 multidose containers with an added preservative. The compositions may 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.

The antibiotic compounds may also be presented in a form suitable for absorption by the gastro-intestinal tract, e.g. as tablets or capsules. The antibiotic compounds may also be formulated as suppositories, e.g. containing conventional suppository bases such as cocoa butter or other glycerides.

Compositions for verterinary 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%, preferably from 1060% of the active material, depending on the method of administration.

When the compositions comprise dosage units, each unit will preferably contain 501500 mg of the active ingredient. The dosage as employed for adult human treatment will preferably range from 250 to 6000 mg per day, depending on the route and frequency of administration, although in treating Pseudomonas infections higher daily doses may be required.

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

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-(hydroxyimino)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 phase was 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 stand in, 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 sulphur 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) was 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 (Zk2-hydroxyimino-242-tritylaminothiazol-4-yl)- acetate, hydrochloride Trityl chloride (16.75 g) was added portionwise 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 150 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 (2x500 ml) and then shaken with IN HC1 (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 1869 (decomp).

Preparation 3 Ethyl (Z)-242-TritylaminothiazolXyl)-241-t-butoxy- carbonylcyclobut- 1 -oxyimino) acetate The product of Preparation 2 (59.8 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 l-bromocyclobutane carboxylate (29.2 g) was added. After 8 hours further potassium carbonate (31.2 g) was added. More potassium carbonate (6x 16 g portions) was added during the next three days and further t-butyl l-bromocyclobutane carboxylate (3.45 g) was added after 3 days.

After 4 days in all, the mixture was poured into ice-water (ca. 3 litres) and the solid was collected by filtration and washed well with water and petrol (b.p. 4060").

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 (1:2) and filtered through silica gel (500 g).

Evaporation gave the title compound (60 g) as a yellow foam, Ymax (CHBr3) 3400 (NH) and 1730 cm~l (ester). was added l-hydroxybenzotriazole hydrate (1.18 g), followed by dicyclohexylcarbodiimide (2.39 g). The resulting suspension was treated with diphenylmethyl 7 - amino - 3 - ( I - methyltetrazol - 5 - ylthiomethyl)ceph - 3 - em - 4 - carboxylate (3.82 g) and stirred at 25 for 24 h. The mixture was filtered and evaporated, and the residue was chromatographed over silica gel, using ethyl acetatepetro (1 ;2 to 1:1) as the eluant. The product (4.52 g) was crystallised from ether to give the title compound, m.p. 1390 (decomp.); [a]0 (DMSO)-680; Amex (EtOH) 260 nm (inf.) (E 22,000).

(b) (6R,7R)- 7 - [Z - 2 - (1 - Carboxycyclobut - 1 - oxyimino) - 2 - (2 - tritylaminothiazol - 4 - yl)acetamido] - 3 - (1 - methyltetrazol - 5 ylthiomethyl)ceph - 3 - em - 4 - carboxylic acid The product of stage (a) 2.445 g) in anisole (10 ml) was treated with trifluoroacetic acid (20 ml), and the mixture was stirred at 250 for I h. Most of the volatile solvent was removed in vacuo and the residue was dissolved in ethyl acetate, washed thoroughly with water, dried, and evaporated. The residual oil was treated with petrol and the resulting solid was collected, and dissolved in ethyl acetate. The solution was washed thoroughly with water, dried, and evaporated to give the title compound, 1.21 g), m.p. 143 (decomp.) Ax max (pH 6 buffer) 258 (inf) (E 20.530), 305 nm (inf) (E 6,450).

c) (6R,7R) - 7 - [(Z) - 2 - (2 - Aminothiazol - 4 - yl) - 2 - (1 - carboxycyclobut - oxyimino)acetamido] - 3 - ( I - methyltetrazol - 5 - ylthio methyl)ceph - 3 - em - 4 - carboxylic acid The product from stage (b) (1.155 g) was dissolved in 90 /O formic acid (15 ml) and treated with water (4 ml). The mixture was stirred at 250 for 1 h, then poured into water and filtered. The filtrate was extracted with dichloromethane, and the aqueous phase was concentrated under reduced pressure. The resulting solid was collected and dried to give the title compound (407 mg), [a]0 (DMSO)-65", Amax (pH 6 buffer) 240 (E 18,350), 252 (inf.) (E 18,050), 302.5 nm (inf) (E 7,600).

More of the title compound was obtained by evaporation of the mother liquors, and by evaporation of the aqueous washings from stage (b) above, Example 2 a) t-Butyl (6R,7R) - 3 - Acetoxymethyl - 7 - [(Z) - 2 - (1 - t - butoxy- carbonylcyclobut - 1 - oxyimino) - 2 - (2 - tritylaminothiazol - 4 yl)acetamido]ceph - 3 - em - 4 - carboxylate A stirred solution of the product of Preparation 4 (24.2 g) and t-butyl (6R,7R) - 3 - acetoxymethyl - 7 - aminoceph - 3 - em - 4 - carboxylate (13.6 g) in dimethylformamide (300 ml) was cooled to 0 , and l-hydroxybenzotriazole monohydrate (4.5 g) added, followed by dicyclohexylcarbodiimide (6.4 g). The mixture was warmed to room temperature and stirred overnight. The mixture was filtered, and the white solid washed with a little ether. The filtrate and washings were diluted with water (1.51) and extracted with ethyl acetate. The organic extracts were combined, washed successively with water and saturated brine, dried, and evaporated. The residue was taken up in ether, filtered, and re-evaporated.

The required product was isolated after elution through two silica columns with ether and concentrating the appropriate fractions. The residues were recrystallised from di-isopropyl ether to give the title compound (12.8 g), m.p. 113.5 to 116.5 , [a]n20+15.0C (e 1.0, DMSO).

b) (6R,7R) - 3 - Acetoxymethyl - 7 - [(Z) - 2 - (2 - aminothiazol - 4 - yl) - 2 (I - carboxycyclobut - 1 - oxyimino)acetamido]ceph - 3 - em - 4 - carboxylic acid Trifluoroacetic acid (100 ml) was added to a mixture of the product of Stage a) (12.5 g) and anisole (5 ml) at 00. The mixture was stirred at room temperature for 1 hour and concentrated. The residue was dissolved in ethyl acetate and reconcentrated. The residue was dissolved in ethyl acetate and extracted with saturated sodium bicarbonate solution. The aqueous extracts, at pH 7 to 7.$, were washed with ethyl acetate, acidified to pH 1.5, and extracted with ethyl acetate.

The combined organic extracts were washed with saturated brine, dried, and evaporated. The residue was dissolved in formic acid (70 ml), water (18 ml) added, and the mixture stirred at room temperature for 2 hours. The mixture was diluted with water (300 ml) and filtered. The filtrate was concentrated. The residue was taken up in water (400 ml), refiltered, and lyophilized to give the title compound (4 g), Amax (pH 6 buffer) 246 nm (E:r,264), Af 295 nm (E1Sbml 18), [r]D +27.3 (c 1.0, DMSO).

c) (6R,7R) - 7 - [Z - 2 - (2 - Aminothiazol - 4 - yl) - 2 - (1 - carboxycyclobut 1 - yloxyimino)acetamido] - 3- (1 - methylpyridinium - 2 ylthiomethyl)ceph - 3 - em - 4 - carboxylate mono-sodium salt (6R,7R) - 3 - Acetoxymethyl - 7 - [Z - 2 - (2 - aminothiazol - 4 - yl) - 2 (1 - carboxycyclobut - I - yloxyimino)acetamido]ceph - 3 - em - 4 - carboxylic acid (1.60 g) and sodium hydrogen carbonate (0.50 g) were warmed with water (2 ml) and to the suspension was added 1 - methylpyrid - 2 - thione (564 mg) and then more sodium hydrogen carbonate (ca 350 mg) to give a mixture of pH ca 6.5.

Sodium iodide (2.7 g) was added and the slightly cloudy solution was heated at 65" for 5 hours and then cooled and applied to a column of Amberlite XAD-2 (100 g).

The column was eluted with water in ca 150 ml. fractions (1 to 5) and then with water:ethanol=3:l in 150 ml. fractions (6 to 10). Fractions 4 to 7 were combined and the ethanol was removed by evaporation. The yellow solution was washed with ethyl acetate (3x300 ml) and ether (200 ml) and evaporated until the organic solvents had been removed. It was then lyophilised to give a brown foam which was washed with ether and filtered and dried in vacuo to give the title compound (816 mg) as a pale brown foam, [a]+2 10 (H3O:EtOH 1:1; c 0.84%); Amex (pH6 buffer) 248 nm (E1icm294). Amax 305 5 mm (E1 cml91).

WHAT WE CLAIM IS: 1. Cephalosporin antibiotics of the general formula

(where Ra and Rb, together with the carbon atom to which they are attached form a C37 cycloalkylidene group and Y represents a carbon-attached 5- or 6membered heterocyclic ring containing at least one nitrogen atom, which ring may also contain at least one sulphur atom and/or be substituted by a C1-C4 alkyl group) and non-toxic salts and non-toxic metabolically labile esters thereof.

2. Compounds as claimed in claim 1 wherein Ra and Rb together with the carbon atom to which they are attached form a cyclobutylidene group.

3. Compounds as claimed in any one of the preceding claims wherein the said heterocyclic ring in the group Y is a pyridyl or tetrazolyl group.

4. Compounds as claimed in any one o the preceding claims wherein the said heterocyclic ring is substituted by a methyl group.

5. Compounds as claimed in claim 4 wherein Y represents an Nmethylpyridinium group.

6. (6R,7R) - 7 - [(Z) - 2 - (2 - Aminothiazol - 4 - yl) - 2 - (1 - carboxy- cyclobut - 1 - oxyimino)acetamidol - 3 - (1 - methylpyridinium - 2 ylthiomethyl)ceph - 3 - em - 4 - carboxylate and non-toxic salts and non-toxic metabolically labile esters thereof.

7. (6R,7R) - 7 - [(Z) - 2 - (2 - Aminothiazol - 4 - yl) - 2 - (1 - carboxy- cyclobut - 1 - oxyimino)acetamido] - 3 - (1 - methyltetrazol - 5 ylthiomethyl)ceph - 3 - em - 4 - carboxylic acid and non-toxic salts and non-toxic metabolically labile esters thereof.

8. A process for the preparation of a compound of formula I (as defined in claim 1) or a non-toxic salt or non-toxic metabolically labile ester thereof which comprises either (A) condensing a compound of the formula

**WARNING** end of DESC field may overlap start of CLMS **.

Claims (15)

1. **WARNING** start of CLMS field may overlap end of DESC **.

   g), Amax (pH 6 buffer) 246 nm (E:r,264), Af 295 nm (E1Sbml 18), [r]D +27.3 (c 1.0, DMSO).

   c) (6R,7R) - 7 - [Z - 2 - (2 - Aminothiazol - 4 - yl) - 2 - (1 - carboxycyclobut 1 - yloxyimino)acetamido] - 3- (1 - methylpyridinium - 2 ylthiomethyl)ceph - 3 - em - 4 - carboxylate mono-sodium salt (6R,7R) - 3 - Acetoxymethyl - 7 - [Z - 2 - (2 - aminothiazol - 4 - yl) - 2 (1 - carboxycyclobut - I - yloxyimino)acetamido]ceph - 3 - em - 4 - carboxylic acid (1.60 g) and sodium hydrogen carbonate (0.50 g) were warmed with water (2 ml) and to the suspension was added 1 - methylpyrid - 2 - thione (564 mg) and then more sodium hydrogen carbonate (ca 350 mg) to give a mixture of pH ca 6.5.

   Sodium iodide (2.7 g) was added and the slightly cloudy solution was heated at 65" for 5 hours and then cooled and applied to a column of Amberlite XAD-2 (100 g).

   The column was eluted with water in ca 150 ml. fractions (1 to 5) and then with water:ethanol=3:l in 150 ml. fractions (6 to 10). Fractions 4 to 7 were combined and the ethanol was removed by evaporation. The yellow solution was washed with ethyl acetate (3x300 ml) and ether (200 ml) and evaporated until the organic solvents had been removed. It was then lyophilised to give a brown foam which was washed with ether and filtered and dried in vacuo to give the title compound (816 mg) as a pale brown foam, [a]+2 10 (H3O:EtOH 1:1; c 0.84%); Amex (pH6 buffer) 248 nm (E1icm294). Amax 305 5 mm (E1 cml91).

   WHAT WE CLAIM IS: 1. Cephalosporin antibiotics of the general formula

   (where Ra and Rb, together with the carbon atom to which they are attached form a C37 cycloalkylidene group and Y represents a carbon-attached 5- or 6membered heterocyclic ring containing at least one nitrogen atom, which ring may also contain at least one sulphur atom and/or be substituted by a C1-C4 alkyl group) and non-toxic salts and non-toxic metabolically labile esters thereof.
2. 2. Compounds as claimed in claim 1 wherein Ra and Rb together with the carbon atom to which they are attached form a cyclobutylidene group.
3. 3. Compounds as claimed in any one of the preceding claims wherein the said heterocyclic ring in the group Y is a pyridyl or tetrazolyl group.
4. 4. Compounds as claimed in any one o the preceding claims wherein the said heterocyclic ring is substituted by a methyl group.
5. 5. Compounds as claimed in claim 4 wherein Y represents an Nmethylpyridinium group.
6. 6. (6R,7R) - 7 - [(Z) - 2 - (2 - Aminothiazol - 4 - yl) - 2 - (1 - carboxy- cyclobut - 1 - oxyimino)acetamidol - 3 - (1 - methylpyridinium - 2 ylthiomethyl)ceph - 3 - em - 4 - carboxylate and non-toxic salts and non-toxic metabolically labile esters thereof.
7. 7. (6R,7R) - 7 - [(Z) - 2 - (2 - Aminothiazol - 4 - yl) - 2 - (1 - carboxy- cyclobut - 1 - oxyimino)acetamido] - 3 - (1 - methyltetrazol - 5 ylthiomethyl)ceph - 3 - em - 4 - carboxylic acid and non-toxic salts and non-toxic metabolically labile esters thereof.
8. 8. A process for the preparation of a compound of formula I (as defined in claim 1) or a non-toxic salt or non-toxic metabolically labile ester thereof which comprises either (A) condensing a compound of the formula

   [wherein Y is as defined in Claim 1; B is > S or > S < O (a- or p-); R3 represents hydrogen or a carboxyl blocking group; and the dotted line bridging the 2-, 3- and 4- positions indicates that the compound is a ceph-2-em or ceph-3-em compound, or a salt or an N-silylated derivative thereof with an acid of formula

   (wherein Ra and Rb are as defined in claim l; R4 represents a carboxyl blocking group; and R5 is an amino or protected amino group) or with an acylating agent corresponding thereto; or (B) reacting a compound of formula

   (wherein Ra, Rb, R5, B and the dotted line are as defined above; R3 and R38 may independently represent hydrogen or a carboxyl blocking group; and X is a replaceable residue of a nucleophile) or an acid addition salt thereof, with a sulphur nucleophile serving to form a group of formula -CH2SY (wherein Y is as defined in claim 1 at the 3-position; whereafter, if necessary and/or desired in each instance, any of the following reactions (C) in any appropriate sequence, are carried out: (i) conversion of a 52 isomer into the desired 3 isomer, (ii) reduction of a compound wherein B is > S-tO to form a compound where B is > S.

   (iii) conversion of a carboxyl group into a non-toxic metabolically labile ester function, and (iv) removal of any carboxyl blocking and/or N-protecting groups; and finally (D) recovering the desired compound of formula (I) or a non-toxic salt or non-toxic metabolically labile ester thereof, if necessary after separation of isomers.
9. 9. A process as claimed in claim 8 wherein a compound of formula (II) is condensed with an acid halide corresponding to the acid of formula (III).
10. 10. A process as claimed in claim 9 wherein the condensation is effected in the presence of an acid binding agent comprising a tertiary amine, an inorganic base or an oxtrane.
11. 11. A process as claimed in claim 8 wherein a compound of formula (II) is condensed with an acid of formula (III) in the presence of a condensing agent comprising a carbodiimide carbonyldiimidazole or an isoxazolinium salt.
12. 12. A process as claimed in claim 8 substantially as herein described.
13. 13. A process as claimed in claim 8 substantially as herein described with reference to the Examples.
14. 14. Compounds of the general formula I (as defined in claim 1) whenever prepared by a process as claimed in any of claims 8 to 13.
15. 15. Pharmaceutical compositions comprising a compound of formula I (as defined in claim 1) or a non-toxic salt or non-toxic metabolically labile ester thereof in association with a pharmaceutical carrier or excipient.