GB2036738A - Cephalosporin antibiotics - Google Patents

Abstract

Cephalosporin antibiotics of general formula <IMAGE> (wherein Y<(+)> represents a C-attached 1-C1-4alkylpyridinium group) exhibit broad spectrum antibiotic activity with unusually high activity against strains of Pseudomonas organisms as well as high activity against various members of the Enterobacteriaceae. The invention also includes the non-toxic salts and non-toxic metabolically labile esters of compounds of formula (I). Also described are compositions containing the antibiotics of the invention and processes for the preparation of the antibiotics.

Description

SPECIFICATION Cephalosporin antibiotics 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 compounds, and in the treatment of penicillin-sensitive patients. In many instances it is desirable to employ a cephalosporin antibiotic which exhibits activity against both gra-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 contaning a 7ss-(a-etherified oxyirnino)-acylamido group, the oxyimino 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 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.

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 vary widely and may, for example, be hydrogen atoms, and m and n are each 0 or 1 such that the sum of m and n isO 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, including, for example, the 1 -methylpyridinium-2-ylthiomethyl substituent. The compounds described in this specification have been found to have 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 gramnegative organisms. Such developments have involved variations in not only the 7ss-acylamido group of formula (A) 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 formula (A) above 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 may vary widely and may be infer alia an optionally substituted heterocyclicthiomethyl group.

May examples of such groups are given in the specification including those in which the heterocyclic moiety of the group is a 3-to 8- membered heterocyclic ring contaning 1 to 4 nitrogen atoms, e.g. a pyridyl group which may be substituted, e.g. by a lower alkyl 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 any of a large number of residues of nucleophilic compounds therein described. Examples of such residues include the mercapto group which may be attached to a 5- or 6-membered heterocyclic ring which may contain 1 to 4 heteroatoms selected from oxygen, sulphur and nitrogen, e.g. a pyridyl ring, which ring may, if desired, be substituted for example by a lower alkyl group.In the above mentioned Specification no antibiotic activity is ascribed to such compounds which are only mentioned as intermediates for the preparation of antiobiotics described in that specification.

South African Patent Specification No. 78/1630 describes 3-acetoxymethyl cephalosporin antibiotics wherein the group R in formula (A) above may be replaced by a 2-aminothiazol-4-yl group and the oxygen atom in the oxyimino group is attached to an alkyl group substituted by a carboxy group (or a salt or C1 3 alkyl ester thereof), a nitrile group or a carbamoyi group.

South African Patent Specification 78/1870 discloses cephalosporin antibiotics wherein the 7P-acylamido side chain is interalia a 2-(2-aminothiazol-4-yl)-2-(optionally substituted alkoxyimino)-acetamido group and the 3-position may be substituted, for example, by the group -CHPZ in which Z represents the residue of a nucleophile, the Specification containing numerous examples of such nucleophiles, including sulphur nucleophiles, e.g. heterocyclicthiomethyl groups. The Specification contains, among numerous other examples, references to compounds in which the above-mentioned optionally substituted alkoxyimino group is a carboxy-alkoxyimino group, e.g. the carboxymethoxyimino group.

We have now discovered that by an appropriate selection of the (Z)-2-(2-aminothiazol-4-yl) -2 (carboxymethoxyimino)-acetamido group at the 7,3-position in combination with a 1alkylpyridiniumthiomethyl group at the 3-position, cephalosporin compounds having particularly advan tageous activity (described in more detail below) against a wide range of commonly encountered pathogenic organisms may be obtained.

The present invention provides cephalosporin antibiotics of the general formula:

(wherein Y(3 represents a C-attached 1-C1.4aIkyl-pyridinium group) and non-toxic salts, non-toxic metabolically labile esters and 1-oxides (preferably the 1 S-oxide) thereof.

It will be appreciated thatthe group +3 may be attached to the sulphur atom at the 2-, 3-or 4- position of the pyridine ring.

The compounds according to the invention are syn isomers. The syn isomeric form is defined by the configuration of the group -OCH2COOH with respect to the carboxyamido 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.

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 (for example in respect of the 2-aminothiazoyl group) and it will be understood that such tautomericforms, e.g. the 2iminothiazolinyl form, are included within the scope of the invention.

Moreover, the compounds of formula (I) depicted above may also exist in alternative zwitterionicforms, for example wherein the 4-carboxyl group is protonated and the carboxyl group in the 7-side chain is deprotonated. These alternative forms, as well as mixtures of such zwitterionic forms, are included within the scope of the present invention.

The compounds according to the invention exhibit broad spectrum antibiotic activity. Against gramnegative organisms the activity is unusually high. This high activity extends to many ss-lactamase-producing gram-negative strains. The compounds also possess high stability to ss-lactamases produced by a range of gram-negative and gram-positive organisms.

Compounds according to the invention have been found to exhibit unusually high activity against strains of Pseudomonas organisms, e.g. strains of Pseudomonas aeruginosa as well as high activity against various members of the Enterobacteriaceae (e.g. strains of Escherichia coli, Klebsiella pneumoniae, Slamonella typhimurium, Shigella sonnei, Enterobacter cloacae, Serratia marcescens, Providence species, Proteus mirabilis, and especially indole-positive Proteus organisms such as Proteus vulgaris and Proteus organic) and strains of Haemophilus influenzae.

The antibiotic properties of the compounds according to the invention compare very favourably with those of the aminoglycosides such as amikacin or gentamicin. In particular, this applies to their activity against strains of various Pseudomonas organisms which are not susceptible to the many of the existing commercially available antibiotic compounds. Unlike the aminoglycosides, cephalosporin antibiotics normally exhibit low toxicity in man. The use of aminoglycosides in human therapy tends to be limited or complicated by the relatively high toxicity of these antibiotics. The cephalosporin antibiotics of the present ivention thus possess potentially great advantages over the aminoglycosides.

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, phenethylbenzylamine, dibenzylethylenediamine, ethanolamine, diethanolamine and N-methylglycosamine 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 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 sulphonic acid groups, or with a resin containing carboxyl groups, e.g. 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 apalication, e.g. for use in depot preparations, such salts may be formed 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 metabolitically labile ester derivatives which may be formed by esteriiication of either or both carboxyl groups in the parent compound of formula (I) include acyloxyalkyl ester e.g. lower alkanoyloxymethyl or -ethyl esters such as acetoxy-methyl or -ethyl or pivaloyloxymethyl esters. In addition to the above ester derivatives, the present invention includes within its scope compounds of formula (I) in the form of other physiologivcally 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 those compounds of formula (I) wherein the C1.4 alkyl substituent in the group Yogis a methyl group. Preference is also expressed for those compounds wherein the group P is attached to the sulphur atom at the 2- or 4- position of the pyridine ring.

Particularly preferred compounds according to the invention therefore include the following compounds of formula (I) and their non-toxic salts and hon-toxic metabolically labile esters: (6R, 7R) -7-[(Z)-2-(2-aminothioazol-4-yl)-2- (carboxymethoxyimino)acetamidi] -3-(1 -methylpyridinium-2- ylthiomethyl)- ceph-3-em-4-carboxylate; and (6R, 7R)-7-I(Z)-2-(2-amino-thiazol-yl) -2 (carboxymethoxyimino) acetamidoi-3- (1 -methylpyridinium-4-ylthiomethyl)-ceph-3-em.4-carboxylate.

Other compounds according to the present invention include the 3-(1-methylpyridinium-3-ylthiomethyl) analogue of the above two compounds as well as the three corresponding compounds in which the N-substituent on the pyridinium ring is an ethyl 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 infections and urinary tract infections.

According to another 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, non-toxic metabolically labile ester or 1-oxide thereof which comprises (A) acylating a compound of the formula

[wherein YO is as defined above;B is S or > S~0 (a- or 13-); 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, 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, or a corresponding compound having a group of formula - COOR1 at the 4-position [where R1 is a hydrogen atom 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)] and having an associated anion AOsuch as a halide, e.g. chloride or bromide, ortrifluoroacetate anion, with an acid of formula

(wherein R2 represents a carboxyl blocking group, e.g. as described for R1; and R3 is an amino or protected amino group) or with an acylating agent corresponding thereto; (B) reacting a compound of formula

(wherein R3, B and the dotted line are as hereinbefore defined;R4 and R4a may independently represent hydrogen or a carboxyl blocking group; and X is a replaceable residue of a nucleophile, e.g. an acetoxy of dichloroacetoxy group or a halogen atom such as chlorine, bromine or iodine) or a salt thereof with a sulphur nucleophile serving to form the group -CH2-SY'wherein # is as defined above) at the 3-position; or (C) alkylating a compound of formula

(wherein R3, B and the dotted lines are as hereinbefore defined; Y1 is a C-attached pyridyl group; and R4 and R4a in this instance are both carboxyl blocking groups) with a C1.4 alkylating agent serving to introduce a C, 4 alkyl group as a substituent on the nitrogen atom of the pyridine ring; whereafter, if necessary and/or desired in each instance, any of the following reactions (D) in any appropriate sequence, are carried out: i) conversion of a A2-isomer into the desired 3- isomer, ii) reduction of a compound wherein B is > SO to form a compound wherein B is > S, iii) conversion of a carboxyl group into a non-toxic salt or non-toxic metabolically labile ester function, iv) oxidation of a compound wherein B is > S to form a compound wherein B is > S O, and v) removal of any carboxyl blocking and/or N-protecting groups.

In the above-described processes, the cephalosporin starting materials are preferably compounds wherein the dotted line represents ceph-3-em compounds.

The reaction should be effected in the presence of a base if an acid addition salt of the compound of formula (II) is used.

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) our 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 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. 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'-ydimethylaminopropylcarbodiimide; a carbonyl compound such as carbonyldiimidazole; or an isoxazolium salt such as N-ethyl-5-phenylisoxazolium perchlorate.

Acylation may also be effected with other amideforming 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 -hydroxy-benzotriazole 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 acetonitrile.

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

The acids of formula (III) and acylating agents 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.

In process (B) the sulphur nucleophile may serve to displace a wide variety of substituents X from the cephalosporin of formula (IV). To some extent the facility of the displacement is related to the pKa of the acid HX from which the substituent is derived. Thus atoms or groups X derived from strong acids tend, in general, to be more easily displaced than atoms or groups derived from weaker acids. The facility of the displacement is also related, to some extent, to the precise, identity of the sulphur nucleophile. The latter nucleophile may be employed for example in the form of the appropriate thione or thiolate.

The displacement of X by the sulphur nucieophile may conveniently be effected by maintaining the reactants in solution or suspension. The reaction is advantageously effected using from 1 to 10, preferably 1 to 4, mole equivalents of the nucleophile.

Nucleophilic displacement reactions may conveniently be carried out on those compounds of formula (IV) wherein the substituent X is a halogen atom or an acyloxy group for example as discussed belovv.

Acyloxy groups Compounds of formula (IV) wherein Xis an acetoxy group are convenient starting materials for use in the nucleophilic displacement reaction with the sulphur nucleophile. Alternative starting materials in this class include compounds of formula (IV) in which X is the residue of a substituted acetic acid e.g. chloroacetic acid, dichloroacetic acid and trifluoroacetic acid.

Displacement reactions on compounds (lav) possessing X substituents of this class, particularly in the case where X is an acetoxy group, may be facilitated by the presence in the reaction medium of iodide or thiocyanate ions.

The substituent X may aiso be derived from formic acid, 1 haloformic acid such as chloroformic acid, or a carbamic acid.

When using a compound of formuia (lav) in which X etresen.s an acetoxy or substituted acetoxy group, it is generally desirable that the group R4 in formula (lV) sl:isuld be a hydrogen atom and that B should represent > S. In this case, the reaction is advantageously effected in an aqueous medium, preferably at a pH of 5 to 8, particularly 5.5 to 7. These pH ranges may be achiaved, if necessary by the addition of a base for example an alkali metal or alkaline earth metal hydroxide a: bicarbonate such as sodium hydroxide or bicarbonate. A base is generally used when the compound of formula (IV) is in the form of an acid addition salt.

The above-described process employing compounds of formula (,V) in which X is the residue of a substituted acetic acid may be carried out as described in British Patent Specification No. 1,241,657.

When using compounds of formula (IV) in which Xis an acetoxy group, the reaction is conveniently effected at a temperature of 30 to 1 10", preferably 50 to SC C.

Halogens Compounds of formula (IV) in which Xis a chlorine, bromine or iodine atom can also be conveniently used as starting materials in the nucleophilic displacement relation with the sulphur nucleophile. When using compounds of formula (IV) in this class, B may represent > < O abd R4 may represent a carboxyl blocking group. The reaction is conveniently effected in a non-aqueous medium which preferably comprises one or more organic solvents, advantageously of a polar nature, such as ethers, e.g. dioxan or tetrahydrofuran, esters, e.g. ethyl acetate, amides, e.g. formamide or N,N-dimethylformamide, or ketones, e.g. acetone. Other suitable organic solvents are described in more detail in British Patent Specification No. 1,326,531. The reaction medium should be neither extremely acidic nor extremely basic.In the case of reactions carried out on compounds of formula (IV) in which R4 and R4a are carboxyl blocking groups the 3-(1 -methyl-pyridibium) thiomethyl product will be formed as the correspondina halide salt which may, if desired, be subjected to one or more ion exchange reactions to obtain a saint having the desired anion.

When using compounds of formula (lav) in which X is a halogen atom as described above, the reaction is conveniently effected atatemperature of-200to +50'C, preferalily0 to +35 C.

In process (C) above, the compound of formula (V; is advantageously reacted with a C, 4 alkylating agent of the formula RC Z' wherein Rc is a C14 alkyl group and i' is a leaving group such as a halogen atom (e.g.

iodine, chlorine or bromine) or a hydrocarbylsuiphonate (e.g. mesylate or tosylate) group. Aklternatively, a di-(C1.4) alkylsulphate, e.g. dimethylsuiphate, may be employed as the alkylating agent. The alkylation reaction is preferably carried out at a temperature in the range of 0 to 60 C, advantageously 20 to 300C. The reaction may be conveniently effected in an inert solvent such as an ether e.g., tetrahydrofuran, an amide, e.g. dimethylformamide, or a halogenated hydrocarbon, e.g. dichloromethane. Alternatively, where the alkylating agent is liquid under the reaction conditions, this agent can itself serve as a solvent. lodomethane is a preferred alkylating agent.

The compound of formula (V) used as starting mater a) in process (C) may be prepared for example by reaction of a compound of formula (IV) (as defined above) with an appropriate sulplur nucleophile in an analogous manner to the nucleophile displacement reaction described with respect to process (B). This reaction is generally carried out in the presence of an acid scavenging agent, e.g. a basaas described above in relation to process (B).

The reaction product may be separated from the reaction mixture, which may contain, for example, unchanged cephalosporin starting material and other substances, by variety of processes including recrystallisation, ionophoresis, column chromatography and use of ion-exchangers (for example by chromatograhy on ion-exchange resins) or macroreticular resins.

A A2-cephalosporin ester derivative obtained in accordance with the process of the invention may be converted into the corresponding A3-derivative by. for example, treatment of the A2-ester with a base such as pyridine or triethylamine.

A ceph-2-em reaction product may aiso be oxidisef to fid yield the corresponding ceph-3-em 1-oxide, for example by reaction with a peracid, ezg. peracetic or m-chlorobebenzoic acid; the resulting sulphoxide may, if desired, subsequently be reduced as described heren-.fter to yield the corresponding ceph-3-em sulphide.

Where a compound is obtained in which B is > S~O this may be converted to the corresponding sulphide by, for example, reduction of the corresponding acyloxy-sulphonium 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, di-methylformamide 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 an 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-ethyl-hexanoate 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.

1-Oxides may be prepared by oxidation of the corresponding 1-sulphide, e.g. with a per 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.

The acids of formula (III) and the compounds of formula (IV) wherein X represents an acetody group may be prepared by the methods described in South African Patent Specification No.78/1630.

Where X is a halogen (i.e. chlorine, bromine or iodine) atom in formula (IV), ceph-3-em starting compounds may be prepared in conventional manner, e.g. by halogenation of a 7ss-protected amino-3methylceph-3-em -4-carboxylic acid ester 1P-oxide, removal of the 7ss-protecting group, acylation of the resulting 7amino compound to form the desired 7ss-acylamido group, e.g. in an analogous manner to process (A) above, followed by reduction of the 1P-oxide group later in the sequence. This is described in British Patent No. 1,326,531.The corresponding ceph-2-em compounds may be prepared by the method of Dutch published Patent Application No. 6,902,013 by reaction of a 3-methylceph-2-em compound with N-bromosuccinimide to yield the corresponding 3-bromomethylceph-2-em compound.

Compounds of formula (IV) in which X represents acyloxy groups other than acetoxy can be prepared by acylation of the corresponding 3-hydroxymethyl compounds which may be prepared for example by hydrolysis of the appropriate 3-acetoxymethyl compounds, e.g. as described in British Patent Specifications Nos. 1,474,519 and 1,531,212.

The starting materials of formula (II) may be prepared in conventional manner, for example, by nucleophilic displacement of the corresponding 3-acetoxymethyl compound with the appropriate nucleophile.

A further method for the preparation of the starting materials of formula (II) comprises de-protecting a corresponding protected 7amino compound in conventiona manner e.g. using PCI5.

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 NH2 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 ortrifluoroacetic acid or using a minineral 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 split 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 acyloxy-methyi or-ethyl groups (e.g. acetoxy-methyl or-ethyl or 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 British Patent No. 1,399,086. Preferred blocked carboxyl groups include aryl lower alkoxycarbonyl groups such as p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl and dipheny Imethoxycarbonyl; lower alkoxycarbonyl groups such as t-butoxycarbonyl; and lower haloalkoxycarbonyl groups such as 2,2,2-trichloro-ethoxycarbonyl. Carboxyl blocking group(s) may subsequently be removed by any of the appropriate methods disclosed in the literature; thus, for exaple, acid or base catalysed hydrolysis is applicable in many cases, as are enzymically-catalysed hydrolyses.

The following Examples illustrate the invention. All temperatures are in C.

Proton magnetic resonance (p.m.r.) spectra are inserted where appropriate and were determined at 100 MHz. The integrals are in agreement with the assignments, and the signs of the coupling constants, were not determined; s = singlet, d = doublet and m = multiplet.

Example 1 (6R, 7R)- 7-f(Z)-2-(2-A minothiazol-4-yl)-2-(carboxymeth oxy4mino)acetamido]-3- ('-meth yIp yridinium-2- ylthiomethyl)-ceph-3-em-4-carboxylate, sodium salt 1-Methyl pyrid-2-thione (0.6 g) was added to a stirred solution of (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2- (carboxymethoxyimino acetamido]-3-acetoxymethyl-ceph-3-em-4-carboxylic acid disodium salt (1.7 g) (described in Example 5 of South African Specification No.78/1630) in water (2.1 ml). The pH of the solution was adjusted to about 6.5 with sodium hydrogen carbonate before the addition of sodium iodide (2.9 g). The resulting mixture was stirred and heated, at 650C, for 51/2 hours.When cool, the resulting mixture was added dropwise to acetone (200 ml), giving a solid, which was isoiated and washed with diethyl ether and then applied, as a solution in water, to an Amberlite XAD-2 column, which was prepared in water. Elution was by water and aqueous ethanol. Evaporation of a combination of the appropriate fractions gave, after trituration with diethyl ether, the title compound as a solid (0.7 g), #max (pH 6 buffer) 243 mn (E1% 321), Xmax 308nm (E'% 203); Vmax (Nujol) 2500-3700 (-NH,-NH2), 1770 (13-lactam), 1670,1540 (7-CONH), 1610 cm-1 (-CO2 ).

Example 2 a) Diphenylmeth yl (6R, 7R)-3-Bromomethyl-7-[(Z)-2-(t-but-oxycarbonylmethoxyimino) -2- (2- tritylaminothiazol-4-yl)acetamido] ceph-3-em-4-carb oxylate (Z)-2-(t-Butoxycarbonylmethoxyimino)-2-(2-tritylamino-thiazol-4-yI) acetic acid (1.1 g) (described in Example 3 of South African Specification No.78/1630) was added to a stirred solution of phosphorus pentachloride (0.46 g) in dichloromethane (20 ml) at -20 . The solution was kept at -15 + 5" for 40 minutes.

More phosphorus pentachloride (0.05 g) was added after 30 minutes. The solution was cooled to -20 and triethylamine (0.6 ml) was added. The solution was stirred at -20 for 5 minutes and then added to a stirred solution of diphenyl methyl (6R,7R)-7-amino-3-bromomethylceph-3-em-4-carboxylate, hydrochloride salt (1g) in dichloromethane (20 ml) containing triethylamine (0.3 ml) at -20 . The temperature did not exceed -20 during the addition. The mixture was stirred at -20 for 10 minutes and then warmed to 220 over 45 minutes.

The mixture was diluted with water (25 ml) and dichloromethane )20 ml) and shaken. The organic phase was separated and the aqueous layer was backwashed once with dichloromethane. The organic phase was washed with dilute sodium bicarbonate solution and the aqueous phase was backwashed. The combined organic solutions were washed with dilute brine, dried bver sodium sulphate, and evaporated to a foam.This was triturated with di-isopropyl ether, filtered, and dried in vacuo at 60 for 2 hours followed by room temperature overnight to give the title compound, (1.7 g), [a] j21 -28 (c 1.0 CHCI3); XinfX (ethanol) 239 nm, E1C m 252; 260 nm, E1u m 196; 265.5 nm, E1cm 193; 273 nm, E% b) Diphenylmethyl (6R, 7R)-7-[(Z-2-(t-Butoxycarbonyl-methoxyiminol-2-(2-tritylaminothiazol-4-yl) acetamidoj-3-( 1-methylp yridinium-4-ylthiomethyl)ceph-3-em-4-carb oxylate bromide A mixture of the product of stage (a) (1.48g) and N-methylpyrid-4-thione (0.19g) in tetrahydrofuran (15 ml) was stirred at 22 for 3 hours and then stood at 5 for 18 hours. The mixture was diluted with ether (60 ml) with stirring, cooled to 0 and then filtered.The solid was washed with ether, collected, and dried in vacuo at 60 for 1 hour to give the title compound (1.5g), laa1 -56 (c 1.0, CHCl3); "max (ethanol) 306.5 nm, E11230; #inf. 260 nm, E1C' m 173; 235 nm, E%m 281.

c) 66R,7R)-7-[NZ)-2-62-Aminothiazol-4-yl)-2-{carboxymethoxysmino) acetamidol -3-(1-methylpyridinium-4- ylthio methyl) ceph-3-em-4-carboxylate, bishydrochloride salt A solution of the product of stage (b) (1.33g) in a mixture of trifluoroacetic acid (16 ml) and anisole (5 ml) was stirred at 0 for 1 hour. Water (1.5 ml) was added and the mixture was stirred for 15 minutes and then evaporated. The residue was triturated with ether, and the solid was filtered off and dried. This solid (0.959) was dissolved in formic acid (5 ml) and concentrated hydrochloric acid (0.25 ml) was added. The mixture was stirred at 21 for 30 min. and then filtered. The solid was washed with a littie formic acid. The filtrate and washings were combined and evaporated.The residue was triturated with acetone, and the solid was filtered off, washed with a little acetone and dried in vacuo at 60 for 1 hour to give the title compound as a monoacetone solvate, (0.76g), [a]22 -20 (c 0.8, pH 7 buffer); Xmax. (pH 6 buffer) 231.5 nm, E: 326; 253 nm, E1Cm 232; 304.5 nm, E11"'m 378. D 1cm Example 3 a) Diphen ylmeth yl (6R, 7R)-7-amino-3-( 1-methylpyridirnum-4-ylthiomethyl)-ceph-3-em-carboxylate bromide Diphenylmethyl (6R,7R)-7-amino-3-bromomethylceph-3-em-4-carboxylate hydrochloride (500 mg) was suspended in tetrahydrofuran (20 ml) and treated with triethylamine (0.14 ml). After stirring for ca 5 minutes at ca 0 the solution was clarified and treated with N-methylpyrid-4-thione (150 mg). The mixture was stirred for 2 hours at ambient temperature, then stored at 0 for 16 hours. The white solid was filtered off, washed with tetrahydrofuran and diethyl ether and dried in vacuo at ambient temperature for 16 hours to yield the title compound (500 mg), T (DMSO-d6) 1.26 (d, pyridinium protons adjacent to NMe, 2.09 (d,pyridinium protons),3.01 CH.Ph2), (d, (d, J5Hz,7-H), 5.08 (d, J 5Hz, 6-H), 5.77 (s, 3-CH2 and NMe), 6.15 and 6.50 (m, 2-H2).

b) Diphenylmethyl (6R, 7R)-7-[(Z)-2-(t-Butoxycarbon yl-methoxyimino)-2-(24ritylaminothiazol-4-yl) acetamido]-3-r1-methylpyridinium-4ylthiomethyl)ceph-3-em-4-carboxylate halide 2-(t-Butoxycarbonylmethoxyimino)-2-(2-tritylamino-thiazol-4-yI) acetic acid (0.42 g) was added to a stirred solution of phosphorus pentachloride (0.19 g) in dichloromethane (10 ml) at -10". The solution was stirred at -10" for 30 minutes, and triethylamine (0.25 ml) was added. The solution was stirred at -5" for 5 minutes and then added to a stirred mixture of the product of stage (a) (0.45 g) in dichloromethane (5 ml) at 0'. The mixture, which dissolved, was warmed to 21", stirred for 2 hours, and left to stand at 5' for 16 hours.The mixture was partitioned between ethyl acetate and water; the aqueous phase was extracted thoroughly with ethyl acetate. The combined organic phase was washed with brine, dried over sodium sulphate and evaporated. The residue was triturated with ether. The solid was collected by filtration and dried in vacuo at 60 to give the title compound (0.55 g), the spectroscopic and chromatographic properties of which resembled those of the product of Example 2(b). The title compound may be deprotected as described in Example 2 (c) to yield (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(carboxymethoxyimino)acetamido]-3-(1 - methylpyridiniu m -4-ylthiomethyl)ceph-3-em-4-carboxylate, bis hydrochloride salt.

Example 4 a) Diphenylmethyl (iS, 6R, 7R,2'Z)-3-Bromomethyl-7-f2-(butoxycarbonylmethoxyimino)-2-(2- tritylamin othiazol-4-yl)acetamido]ceph-3-em-4-carb oxylate, 1-oxide (Z)-2-(t-Butoxycarbonylmethoxyimino)-2-(2-tritylaminotht zol-4-yl) acetic acid (1.63 g) was added to a stirred solution of phosphorus pentachloride (0.69 g) in dichlo methane (25 ml) at -15 . The solution was kept at -15" t 5" for 35 minutes. The solution was cooled to -20" and triethylamine (0.9 ml) was added.The solution was stirred at -20" for 5 minutes and then added to a stirred solution of diphenylmethyl (1 S,6R,7R)-7-amino-3-bromomethylceph-3-em-4-carboxylate,1 oxide hydrobromide salt (1.7 g) in dichloromethane (25 ml) containing triethylamine (0.5 ml) at -20". The temperature did not exceed -15 during the addition. The mixture was stirred at -15" for 10 minutes and then warmed to 210 over 45 minutes. The mixture was diluted with water (40 ml) and dichloromethane (30 ml) and shaken. The organic phase was separated and the aqueous layer was washed with dichloromethane. The organic phase was washed with dilute sodium bicarbonate solution and the aqueous phase was backwashed.The combined organic solutions were washed with dilute brine, dried over sodium sulphate, and evaporated to a foam. This was gradient eluted through a column of kieselgel 60 silica (ca 100 g) with ethyl acetate - 60 /80 petroleum ether mixtures.

Appropriate fractions were combined and evaporated to foam of the title compound (2.1 g), the spectroscopic and chromatographic properties of which resembled those described below in Example 5a).

b) Diphenylmethyl (IS, 6R, 7R)-7-f(Z)-2-(t-Butoxycarbonyl-methoxyimin o)-2-(2-oitylaminothiazoA4 yl)acetamidoj-3-(1-meth yip yridinium-4-yithiomethyl)ceph-3-em-4-carboxylate bromide, 1-oxide A solution of the product of stage (a) (1.99) in tetrahydrofuran (20 ml) was treated with N-methylpyrid-4thione (0.3 g). The mixture, which solidified, was diluted with tetrahydrofuran (30 ml) and stirred at 21 for 21/2 hours. The mixture was kept at 5" for 17 hours, diluted with ether (50 ml) and filtered. The solid was washed with ether and dried in vacuo to give the title compound (1.96 g), [a]2D -35 (c 1.4, CHCI3); Xmax (etha nol) 308 nm, E1%n 143; kinf 260 nm E1 Km 166.

Icm E1 1cm c) Diphenylmethyl (6R, 7R)-7-J(Z)-2-(t-Butoxycarbonyl-methoxyimino)-2-(tritylaminothiazol-4-yl) acetamido]-3- {1-methylpyridinium-4ylthiomethyl)ceph-3-em-4carboxylate halide A mixture of the product of stage (b) (0.32 g), powdered anhydrous potassium iodide (0.24 g) and acetone (2 ml) was stirred and cooled to -3 . Acetyl chloride (0.1 ml) was added, and the mixture was stirred at 0" for 1 hour and then added to a solution of sodium metabisulphite (0.12 g) in water (15 ml). The mixture was stirred at 21 for 15 minutes and then filtered.The solid was washed with water and ether and dried in vacuo at 45 for 1 hour to give the title compound (0.25 g), the spectroscopic and chromatographic properties of which resembled those of the product of Example 2(b). The title compound may be deprotected as described in Example 2 (c) to yield (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl) -2-(carboxymethoxyimino)acetamido]-3-(1 - methyl pyridiniu m -4-ylthiomethyl )ceph-3-em-4-carboxylate, bis hydrochloride salt.

Example 5 a) Diphenylmethyl {1S,6R,7R)-3-Bromomethyl-7-[rZ)-2-{t-butoxycarbonyl-methoxysmino)-2-62-62- tritylaminothiazol-4-yl) acetamidolceph-3-em-4carboxylate, 1-oxide A solution of 85% metachloroperbenzoic acid (0.23 g) in 1,2-dichloroethane (20 ml) was added to a stirred solution of the product of Example 2 (a) (0.99g) in 1,2-dichloroethane (30 ml) maintained at-10 . The solution was warmed to 20 over 1 hour. More metachloroperbenzoic acid (0.02 g) was added and the solution was stirred at 20 for 1/2 hour. The solution was washed once with aqueous sodium metabisulphite solution and evaporated. The residue was combined with an ethyl acetate extract of the aqueous phase, washed with fresh sodium metabisulphite solution, sodium bicarbonate solution, and brine, and dried over sodium sulphate. The solution was evaporated and the residue was eluted through a column of Kieselgel 60 silica (ca 60g) in ethyl acetate: petroleum ether (60"-80") (1 :1). Appropriate fractions were collected, combined, and evaporated to give a foam, which was triturated with di-isopropyl ether.The solid was collected by filtration and dried in vacuo at50 to give the title compound (0.59), la]2Da -2.2" (c0.9,CHC13); Amax(ethanol) 267.5 nm, E1cm 169; kinf 238 nm, Ei / m 221; kjnf272.5 nm, Ecm 166.

b) Diphenylmethyl (1S,6R, 7R)-7-[NZ)-2-t-Butoxyearbonyl-methoxyimino)-2-62-tritylaminothiazol-4yl) acetamidol-3-Epyrid-4ylthiomethyl)-ceph-3-em-4carboxylate, 1-oxide A mixture of the product of stage 4 (a) (0.36 g), finely divided calcium carbonate (Calofort U, 0.12 g) and 4-mercaptopyridine (0.06 g) in acetone (12 ml) was stirred and refluxed for 90 minutes. The mixture was cooled and filtered. The residue was washed with a little acetone. The filtrate and washings were evaporated to a foam which was partitioned between ethyl acetate and water. The aqueous phase was extracted with ethyl acetate. The combined extracts were washed with water and brine, dried, and evaporated to a foam.

This material was chromotographed on silica gel using chloroform containing ethanol (2%) and added methanol (0%-5%) as eluent. The appropriate fractions were combined and evaporated to give the title compound (0.12 g), [ic22 -2" (c 0.76, chloroform); kmax. (ethanol) 255 nm, E]'ci'rn251; hi,f 265 nm, E3Com 245; hint.

272.5 nm, E1C%m 232.

c) Diphenylmethyl /7S, 6R, 7R)-7-[{Z)-2-rt-Butoxycarbonyl-methoxyimino)-2-r2-tritylaminothiazol-4 yl)acetamidol-3- (1-meth yip yridinium-4-yithiometh yl)ceph-3-em-4-carboxylate iodide, 1-oxide A solution of the product of stage b) (55mg) in iodomethane (0.5ml) was ailowed to stand at 21 for 2 hours. The solution was evaporated and the residue was triturated with ether. The solid was collected by filtration, washed with a little ether, and dried in vacuo at 50 for 1 hour to give the title compound, (43 mg), the spectroscopic and chromatographic properties of which resembled those of the product of Example 4 (b).The title compound may deprotected as described in Example 2 (c) to yield (6R,7R)-7-[(Z)-2-(2-amino thiazol -4-yl)-2-(carboxymethoxyimino)acetamido]-3- (1 -methylpyridinium-4-yl-thiomethyl)ceph-3-em-4carboxylate, bis hydrochioride salt.

Example 6 11S,6R, 7R)-7-[(Z) -2- (2-Amino thiazol-4-yl) -2-{carboxymethoxy-imino)acetamidol -3-ll-methylpyridinium-4- ylthiomethyl) ceph-3-em-4-carboxylate, 1-oxide, dihydrochloride salt.

The Product of Example 4b) (1.13 g) was stirred with anisole (4 ml) and trifluoroacetic acid (15 ml) at 0" for one hour. The mixture was evaporated and the residual oil was triturated with ether. The precipitate was collected by filtration and washed with ether to give a solid (0.8 g). This solid was dissolved in formic acid (4 ml) and concentrated hydrochloric acid (0.2 ml) was added. The mixture was stirred at 21 0for 30 minutes and filtered. The filter cake was leached with formic acid and the combined filtrates were evaporated. The residue was triturated with acetone to give the title compound (535 mg), [a]22 -8 (c 0.83, pH7 buffer); Bmax (pH6 buffer) 230.5 nm (Er 323), 258 nm (E11"m 242), 303.5 nm (E1%m 372).D PHARMACEUTICAL FORMULATIONS 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, stabilizing and/or dispersing agents. Alternatively the active ingredient may be in powder form for constitution 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 constituted 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 constituted. 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. contaning conventional suppository bases such as cocoa butter or other glycerides.

For medication of the eyes or ears, the preparations may be formulated as individual capsules, in liquid or semi-solid form, or may be used as drops.

Compositions for veterinary medicine may, for exampie, 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 should 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 to 3000 mg per day administered intravenously or intramuscularly should normally suffice. 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 penicilins or other cephalosporins.

The following formulations illustrate how the compounds according to the invention may be made up into pharmaceutical compositions.

A Formulation-Forinjection Formula per Vial (6R,7R)-7-[(Z)-2-(2-Aminothiazol-4-yl)-2-(carboxymethoxyimino)acetamido] -3-(1 -methylpyridinium-4 ylthiomethyl)ceph-3-em-4-carboxylate 1.00 g Sodium Carbonate, anhydrous 112 mg Method Blend the sterile cephaiosporin 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 microorganisms. Constitute the product by dissolving in Water for Injections or other suitable sterile vehicle shortly before administration.

B Formulation forlnjection Fill sterile (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)- 2-(carboxymethoxyimino)acetamido]-3-(1 - methylpyridinium-4-ylthiomethyl) ceph-3-em-4-carboxylate monosodium salt into glass vials, such that each vial contains an amount equivalent to 500 mg 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 preventing gaseous exchange or ingress of microorganisms. Constitute the product by dissolving in Water for Injections or other suitable sterile vehicle shortly before administration.

Claims (10)

1. Cephalosporin antibiotics of general formula

(wherein Y(3 represents a C-attached 1 - C1.4alkylpyridinium group) and non-toxic salts, non-toxic metabolically labile esters and 1-oxides thereof.

2. Compounds as claimed in claim 1 wherein Yrepresents a C-attached 1-methylpyridinium group.

3. Compounds as claimed in claim 1 wherein Yf3 represents a 1-C1.4 alkylpyridinium group attached to the adjacent sulphur atom at the 2- or 4-position of the pyridine ring.

4. (6R,7R)- 7-[(Z)-2-(2-Aminothiazol -4-yl)-2-ca rboxym ethoxyim ino)acetamido] -3-( 1 -methylpyridinium-2- ylthio methyl)ceph-3-em-4-ca rboxylate.

5. The non-toxic salts of the compound of claim 4.

6. (6R,7R)-7-[(Z)-2-(2-Aminothiazol-4-yl) -2-(carboxymethoxyimino)acetamido]-3-(1-methylpyridinium-4- yl-thiomethyi) ceph-3-em-4-carboxylate.

7. The non-toxic salts of the compound of claim 6.

8. (1 S,6R,7R)-7-[(Z)-2-(2-Am inoth iazo14-yl)-2- (carboxymethoxyimino)acetamido]-3-(1- methyl pyridinium-4-ylthiomethyl)ceph-3-em-4-carboxylate, 1-oxide and its non-toxic salts.

9. A process for the preparation of an antibiotic compound of general formula (I) as defined in claim 1 or a non-toxic salt, non-toxic metabolically labile ester or 1-oxide thereof which comprises (A) acylating a compound of formula

(wherein Ys is as defined in claim 1;B is > S or > S < O 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 N-siiyl derivative thereof or a corresponding compound having a group of formula -COOR' at the 4-position (where R1 is a hydrogen atom or a carboxyl blocking group) and having an associated anion Ae, with an acid of formula

(wherein R2 represents a carboxyl blocking group; and R3 is an amino or protected amino group) or with an acylating agent corresponding thereto; (B) reacting a compound of formula

(wherein R3, B and the dotted line are as hereinbefore defined;R4 and R4a may independently represent hydrogen or a carboxyl blocking group; and X is a replaceable residue of a nucleophile) or a salt thereof with a sulphur nucleophile serving to form the group -S-YW(wherein Y'3is as defined above) at the 3-position; or (C) alkylating a compound of the formula

(wherein R3, B and the dotted lines are as hereinbefore defined;R4 and R4a both represent carboxyl blocking groups; and y1 is a C-attached pyridyl group) with a C1 4 alkylating agent serving to introduce a C1.4 alkyl group as a substituent on the nitrogen atom of the pyridine ring; whereafter if necessary and/or desired in each instance, any of the following reactions, 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 wherein B is > S, iii) conversion of a carboxyl group into a non-toxic salt or non-toxic metabolically labile ester function, iv) oxidation of a compound wherein B is > S to form a compound wherein B is > SO, and v) removal of any carboxyl blocking and/or N-protecting groups.

10. A pharmaceutical composition for use in human or veterinary medicine comprising an antibiotic compound as claimed in any of claims 1 to 8 in association with a pharmaceutical carrier or excipient.