GB2123415A - Cephalosporin antibiotics - Google Patents

Abstract

Cephalosporin antibiotics of general formula <IMAGE> (wherein R represents a 3- or 4-carbamoyl group or a hydrogen atom) and non-toxic salts and non-toxic metabolically labile esters thereof. The compounds exhibit broad spectrum antibiotic activity and have high activity against both Gram-positive and Gram-negative organisms, including many b-lactamase producing strains. Especially preferred are those compounds in which R is hydrogen. Acid intermediates of formula <IMAGE> (wherein R<2> is amine or protected amine) are also disclosed.

Description

SPECIFICATION Cephalosporin antibiotics This invention relates to improvements in or relating to cephalosporins. More particularly it relates to new cephalosporin compounds and derivatives thereof having valuable antibiotic activity.

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 Gram-positive and Gram-negative micro-organisms, 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 7ss(a-etherified oximino)-acylamido group, the oximino group having the syn configuration. This class of antibiotic compounds is characterised by high antibacterial activity against a range of Gram-positive and Gram-negative organisms coupled with particularly high stability to 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 British Patent Specification No. 1,604,971 a wide variety of cephalosporin antibiotics are disclosed in which the 7ss-position side-chain may be selected from, inter alia, a 2-(2aminothiazol-4-yl)-2-(etherified oxyimino)acetamido group, in which the etherifying group, amongst very many possible meanings, may be an alkyl group substituted by a cycloalkyl group, although there is no specific exemplification of compounds having such a group. The 3-position group may also be selected from a large number of alternatives and a possible 3-substituent is an optionally substituted pyridiniummethyl group, although again there is no specific exemplification of the preparation of compounds having such a 3-substituent.

Our British Patent Specification No 2025398A discloses, inter alia, the compound (6R,7R)-7 [(Z)-2-(2-aminothiazol-4-yi)-2-(2-carboxyprop-2-oxyimino)acetamido]-3-(1 -pyridiniummethyl)ceph3-em-4-carboxylate, which is now known by the approved name of "ceftazidime". Other cephalosporin compounds possessing a 2-(2-aminothiazol-4-yl)-2-(etherified oxyimino)acetamido group in the 7ss-position are disclosed in, for example, British Patent Specifications Nos.

1536281 and 1576625.

We have now discovered that by the selection of a (Z)-2-(2-aminothiazol-4-yl)-2-cyclopropylme- thoxyiminoacetamido group at the 7ss-position in combination with either a pyridiniummethyl or a 3- or 4-carbamoylpyridiniummethyl group at the 3-position, cephalosporin compounds having particularly advantageous activity (described in more detail below) against a wide range of commonly encountered pathogenic organisms may be obtained.

Accordingly, we provide cephalosporin antibiotics of the general formula (I)

wherein R represents a 3- or 4-carbamoyl group or a hydrogen atom, and non-toxic salts and non-toxic metabolically labile esters thereof.

The compounds according to the invention are syn isomers. The syn isomeric form is defined by the configuration of the

group 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.

The invention also includes within its scope the solvates (especially the hydrates) of the compounds of formula (I) and of their non-toxic salts. It also includes within its scope non-toxic salts and solvates of metabolically labile esters of the compounds of formula (I). It will be appreciated that the solvates should be pharmacologically acceptable.

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

The compounds according to the invention exhibit broad spectrum antibiotic activity both in vitro and in vivo. They have high activity against both Gram-positive and Gram-negative organisms, including many fi-lactamase producing strains. The compounds also possess high stability to fi-lactamases produced by a range of Gram-negative and Gram-positive organisms.

Compounds according to the invention have been found to exhibit high activity against strains of Staphylococcus aureus, Staphylococcus epidermidis and Streptococcus species including penicillinase producing strains of these Gram-positive bacteria. This is coupled with high activity against various members of the Enterobacteriaceae (e.g. strains of Escherichia coli, Klebsiella pneumoniae, Citrobacter diversus, Enterobacter cloacae, Serratia marcescens, Proteus mirabilis and indole-positive Proteus organisms such as Proteus vulgaris, Proteus morganii and Providence species), strains of Haemophilus influenzae, and Acinetobacter calcoaceticus as well as good activity against Pseudomonas species.This combination of high activity against Grampositive organisms with high activity against Gram-negative organisms possessed by the compounds of the invention is particularly unusual.

Non-toxic salt derivatives which may be formed by reaction of the carboxyl group present in the compounds of formula (I) include inorganic base salts such as alkali metal salts (e.g. sodium and potassium salts) and alkaline earth metal salts (e.g. calcium salts); amino acid salts (e.g.

lysine and arginine salts); organic base salts (e.g. procaine, phenylethylbenzylamine, dibenzylethylenediamine, ethanolamine, diethanolamine and N-methylglucosamine salts). Other non-toxic salt derivatives include acid addition salts, e.g. formed with hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, formic and trifluoroacetic acids. The salts 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. a polyacrylic acid resin. Soluble base salts (e.g.

alkali metal salts such as the sodium salt) of the compounds of formula (I) may be used in therapeutic applications because of the rapid distribution of such salts in the body upon administration. Where, however, insoluble salts of compounds (I) are desired in a particular application, e.g. for use in depot preparations, such salts may be formed in conventional manner, for example with appropriate organic amines.

Non-toxic metabolically labile ester derivatives which may be formed by esterification of the carboxyl group in the parent compound of formula (I) include acyloxyalkyl esters, e.g. lower alkanoyloxy-methyl 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 the compounds of formula (I) in the form of other physiologically acceptable equivalents, i.e.

physiologically acceptable compounds which, like the metabolically labile esters, are converted in vivo into the parent antibiotic compound of formula (I).

These and other salt and ester derivatives such as the salts with toluene-p-sulphonic and methanesulphonic acids or the esters with t-butyl or diphenylmethyl esterifying groups 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.

It will be appreciated that the compounds of the invention are usually present in the form of a betaine containing a positively-charged pyridinium group and a carboxylate group, and therefore esters and salts of compounds of formula (I) with bases will be associated with an anion A- to balance the positive charge on the pyridinium ring. Such an anion will also be non-toxic and may be derived from any of the acids described above which will form non-toxic salt derivatives.

Preferred comnounds according to the invention by virtue of their high antibiotic activity are those compounds of formula (I) above wherein R represents a hydrogen atom, i.e. the compound of formula:

and the non-toxic salts and non-toxic metabolically labile esters thereof. The compound of formula (la) possesses the general antibiotic properties set out above for the compounds of general formula (I). In particular, it has been found that the compound exhibits excellent in vitro activity against strains of Staphylococcus aureus and Staphylococcus epidermidis together with high activity against Gram-negative organisms. The in vitro antibacterial properties of the compound are not impaired by human serum and it has also been found that the minimum inhibitory concentrations are generally not affected by increased inocula.The compound is rapidly bactericidal at concentrations close to the minimum inhibitory concentrations. In vivo, experimental infections in mice with Gram-positive organisms, including Staphylococcus aureus, and Gram-negative organisms including Escherichia coli, have been successfully treated with the compound of formula (la). Acute toxicity tests with the compound in mice gave LD50 values in excess of 2.0g/kg when the compound was administered subcutaneously. No evidence of nephrotoxicity was observed in mice at subcutaneous dosages of 2.0g/kg.

The compounds of the invention 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 or nontoxic metabolically labile ester thereof which comprises (A) acylating a compound of the formula

[wherein R is as defined above;B is

(a- or ss-); 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-sylyl derivative thereof, or a corresponding compound having a group of formula -COOR1 at the 4position [where R1 is a hydrogen atom or a carboxyl blocking group, e.g. the residue of an esterforming 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 E- such as a halide, e.g. chloride or bromide, or trifluoroacetate anion, with an

protected amino group), or a salt thereof or with an acylating agent corresponding thereto; or (B) reacting a compound of formula

(wherein R2, B and the dotted line are as hereinbefore defined; R3 represents hydrogen or a carboxyl blocking group; and X is a replaceable residue of a nucleophile, e.g. an acetoxy or dichloroacetoxy group or a halogen atom such as chlorine, bromine or iodine) or a salt thereof, with a pyridine compound of the formula

{wherein R is as defined above); 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

to form a compound wherein B is

iii) conversion of a carboxyl group into a non-toxic metabolically labile ester function, iv) formation of a non-toxic salt function, and v) removal of any carboxyl blocking and/or N-protecting groups.

The above reactions i) to v) may be carried out in conventional manner.

In the above-described process (A), the starting material of formula (II) is preferably a compound wherein B is

and the dotted line represents a ceph-3-em compound. One such starting material which has been found to be particularly suitable for use in process (A) is (6R,7R)-7-amino-3-(1-pyridinium- methyl)-ceph-3-em-4-carboxylate dihydrochloride.

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 + 50"C, preferably - 40 to + 30"C, if desired in the presence of an acid binding agent. Suitable reaction media include aqueous ketones such as aqueous acetone, aqueous alcohols such as aqueous ethanol, esters such as ethyl acetate, halogenated hydrocarbons such as methylene chloride, am ides 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-N1-y-dimethylaminoprnpylcarbodiimide; a carbonyl compound such as carbonyldiimidazole; or an isoxazolium salt such as N-ethyl-5-phenylisoxazolium perchlorate.

Arylaton may also be effected with other amide-forming derivatives of acids of formula (III) such as, for example, an activated ester, a symmetrical anhydride or a mixed anhydride (e.g.

formed with pivalic acid or with a haloformate, such as a lower alkylhaloformate). Mixed anhydrides may also be formed with phosphorus acids (for example phosphoric or phosphorous acids), sulphuric acid or aliphatic or aromatic sulphonic acids (for example toluene-p-sulphonic acid). An activated ester may conveniently be formed in situ using, for example, 1-hydroxybenzotriazole in the presence of a condensing agent as set out above. Alternatively, the activated ester may be preformed.

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

An alternative method of activation is, for example, by reacting an acid of formula (III) with a solution or suspension preformed by adding a carbonyl halide, in particular oxalyl chloride or phosgene, or a phosphoryl halide such as phosphorus oxychloride to a solvent such as a halogenated hydrocarbon, for example methylene chloride, containing a lower acyl tertiary amide such as N,N-dimethylformamide. The activated form of the acid of formula (III) may then be reacted with a 7-amino compound of formula (II) in a suitable solvent or mixture of solvents for example an alkanol such as an alcohol, e.g. aqueous ethanol or aqueous industrial methylated spirts.The acylation reaction may conveniently be effected at temperatures of from - 50" to + 50"C, preferably - 40" to + 30"C, if desired in the presence of an acid binding agent, for example as described above (e.g. triethylamine).

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.

A pyridine compound of formula (V) may act as a nucleophile to displace a wide variety of substituents X from a cephalosporin of formula (IV). To some extent the facility of the displacement is related to the pK, 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 character of the substituent R in the compound of formula (V).

The displacement of X by the pyridine compound of formula (V) may conveniently be effected by maintaining the reactants in solution or suspension. The reaction is advantageously effected using from 1 to 10 moles of the pyridine compound.

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 below.

Acyloxy groups Compounds of formula (IV) wherein X is an acetoxy group are convenient starting materials for use in the nucleophilic displacement reaction with the pyridine compound of formula (V).

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 (IV) 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. Reactions of this type are described in more detail in British Patent Specifications Nos. 1,132,621 and 1,171,603.

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

When using a compound of formula (IV) in which X represents an acetoxy or substituted acetoxy group, it is generally desirable that the group R3 in formula (IV) should be a hydrogen atom and that B should represent

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.

The above-described process employing compounds of formula (IV) 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 X is an acetoxy group, the reaction is conveniently effected at a temperature of 30"C to 110"C, preferably 50 to 80"C.

Halogens Compounds of formula (IV) in which X is a chlorine, bromine or iodine atom can also be conveniently used as starting materials in the nucleophilic displacement reaction with the pyridine compound of formula (V). When using compounds of formula (IV) in this class, B may represent

and R3 may represent a carboxyl blocking group. The reaction is conveniently effected in a nonaqueous 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 and N,N-dimethylformamide, and ketones e.g. acetone. In certain cases the pyridine compound itseif may be the solvent.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 R3 is a carboxyl blocking group the 3-pyridiniummethyl product will be formed as the corresponding halide salt which may, if desired, be subjected to one or more ion exchange reactions to obtain a salt having the desired anion.

When using compounds of formula (IV) in which X is a halogen atom as described above, the reaction is conveniently effected at a temperature of - 1 0, to + 50 , preferably + 10 to + 30"C.

The reaction product may be separated from the reaction mixture, which may contain, for example, unchanged cephalosporin starting material and other substances, by a variety of processes including recrystallisation, ionophoresis, column chromatography and use of ionexchangers (for example by chromatography 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 desired 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 also be oxidised to yield the corresponding ceph-3-em 1oxide, for example by reaction with a peracid, e.g. peracetic or mchloroperbenzoic acid; the resulting sulphoxide may subsequently be reduced as described hereinafter to yield the corresponding desired ceph-3-em sulphide.

Where a compound is obtained in which B is

this may be converted into the corresponding sulphide by, for example, reduction of the corresponding acyloxysulphonium or alkoxysulphonium salt prepared in situ by reaction with e.g. acetyl chloride in the case of an acetoxysulphonium salt, reduction being effected by, for example, sodium dithionite or by iodide ion as in a solution of potassium iodide in a watermiscible solvent e.g. acetic acid, acetone, tetrahydrofuran, dioxan, dimethylformamide or dimethylacetamide. The reaction may be effected at a temperature of from - 20" to + 50"C.

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

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

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

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

Acids of formula (III) and their derivatives are themselves novel compounds and form a further feature of the present invention. They may be prepared by etherification of a compound of formula

(wherein R2 is as hereinbefore defined and R4 represents hydrogen or a carboxyl blocking group) or a salt thereof, by selective reaction with a compound of general formula

(wherein T is halogen, such as chloro, bromo or iodo; sulphate; or sulphonate, such as tosylate), followed by removal of any carboxyl blocking group R4. Separation of isomers may be effected either before or after such etherification.The etherification reaction is conveniently carried out in the presence of a base, e.g. potassium carbonate or sodium hydride, and is preferably conducted in an organic solvent, for example dimethylsulphoxide, a cyclic ether such as tetrahydrofuran or dioxan, or an N,N-disubstituted amide such as dimethylformamide. Under these conditions the configuration of the oxyimino group is substantially unchanged by the etherification reaction. When the compound of formula (VI) is employed in the form of a free acid or a salt with a base, the etherification reaction is generally carried out in the presence of a strong base, e.g., potassium t-butoxide, sufficient base being added to form a dianion.

Furthermore, the reaction should be effected in the presence of a base if an acid addition salt of a compound of formula (VI) is used, the amount of base being sufficient to neutralise rapidly the acid in question.

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

(wherein R2 and R4 are as herein before defined) with a compound of formula

followed by removal of any carboxyl blocking group R4, and where necessary the separation of syn and anti isomers.

The acids of formula (III) may be converted into the corresponding acid halides and anhydrides and acid addition salts by conventional methods, for example as described hereinabove.

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 protected amino-3-methyl-ceph-3-em-4-carboxylic acid ester 1ss-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 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. 6,902,01 3 by reaction of a 3-methylceph-2-em compound with Nbromosuccinimide to yield the corresponding 3-bromomethylceph-2-em-compound.

Where X is formula (IV) is an acetoxy group, such starting materials may be prepared, for example, by acylation of 7-aminocephalosporanic acid, e.g. in an analogous manner to process (A) above. Compounds of formula (IV) in which X represents other acyloxy groups 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 for example in British Patent Specifications Nos. 1,474,519 and 1,531,212.

The starting materials of formula (II) may also be prepared in conventional manner, for example, by nucleophilic displacement of the corresponding 3-acetoxymethyl compound with the appropriate nucleophile, e.g. as described in British Patent Specification No. 1,028, 563, or by the method described in British Patent Specification No. 2052490A.

A further method for the preparation of the starting materials of formula (II) comprises deprotecting a corresponding protected 7amino compound in conventional manner, e.g. using PCl5.

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 or formylation), protonation or other conventional method.The protecting group may thereafter be removed in any convenient way which does not cause breakdown of the desired compound, e.g. in the case of a trityl group by using an optionally halogenated carboxylic acid, e.g. acetic acid, formic acid, chloroacetic acid or trifluoroacetic acid or using a mineral acid, e.g. hydrochloric acid or mixtures of such acids, preferably in the presence of a protic solvent such as water, or, in the case of a chloroacetyl group, by treatment with thiourea.

Carboxyl blocking groups used in the preparation of compounds of formula (I) or in the preparation of necessary starting materials are desirably groups which may readily be 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-methyl 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 (1).

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 pmethoxybenzyloxycarbonyl, pnitrobenzyloxycarbonyl and diphenylmethoxycarbonyl; lower alkoxycarbonyl groups such as tbutoxycarbonyl; and lower haloalkoxycarbonyl groups such as 2,2,2-trichloroethoxycarbonyl.

The carboxyl blocking group 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, for example, be formulated for injection and may be presented in unit dose form, in ampoules, or in multi-dose containers, if necessary with an added preservative. The compositions may also take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilising and/or dispersing agents. Alternatively the active ingredient may be in powder form for reconstitution with a suitable vehicle, e.g. sterile, pyrogenfree water, before use.

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

The antibiotic compounds may also, for example, be formulated as suppositories e.g.

containing conventional suppository bases such as cocoa butter or other glycerides.

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

The compositions may contain from 0. 1% upwards, e.g. 0.1 -99% of the active material, depending on the method of administration. When the compositions comprise dosage units, each unit will preferably contain 100-3000 mg of the active ingredient e.g. 200-2000 mg.

The daily dosage for adult human treatment will preferably range from 200 to 1 2000 mg e.g.

1000-9000 mg per day, depending inter alia on the nature of the infection and the route and frequency of administration. In general, intravenous or intramuscular administration will be employed, for example using 400 to 4000 mg per day of the active ingredient in adult human treatment. In treating Pseudomonas infections higher daily doses may be required. It will be appreciated that in some circumstances, for example, in the treatment of neonates, smaller dosage units and daily dosages may be desirable.

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

The following Examples illustrate the invention. All temperatures are in C. DMSO is dimethylsulphoxide. Sorbsil U30 is silica gel manufactured by Joseph Crosfield and Son of Warrington, Lancashire, England. Amberlite LA-2 is a weakly basic liquid anion exchange resin; Amberlite XAD-2 resin is a non-functional macroreticular resin, both manufactured by Rohm and Haas of Philadelphia, USA.

Preparation 1 Ethyl (Z)-2-Cyclopropylmethoxyimino-2-(2-tritylaminothiazol-4-yl) acetate Ethyl (Z)-2-hydroxyim ino-2-(2-tritylam inoth iazol-4-yl)acetate, hydrochloride salt (309) was stirred with cyclopropylmethyl bromide (1 3.5g) in dimethylsulphoxide (150 ml) containing potassium carbonate (30g) under nitrogen at 21 for 7 hours. The mixture was partitioned between methylene chloride and water. The aqueous layer was extracted with more methylene chloride and the combined organic solutions were washed with water. After drying with magnesium sulphate, the solution was concentrated and loaded onto a column of Sorbsil U30 silica gel (2009). The column was eluted with ethyl acetate (10 to 30%) in petroleum ether (b.p. 40-60 ).Evaporation of appropriate fractions gave the title compound (20.9g); Amax (ethanol) 234.5nm (E1 403); cam Ajn,l 254.5nm (E 1% 302), 1cm 259.5nm (E 1 267), 1cm 265nm (E1% 229), 271.5nm (E1% 190) 1 cm and 294nm 1% (E 1cm 111); #max (CHBr3) 3398 (NH), 1730 (ester), and 1593 and 1491cm-1 (aromatic double bond).

Preparation 2 (Z)-2-Cyclopropylmethoxyimino-2-(2-tritylaminothiazol-4-yl)acetic acid.

The product of Preparation 1 (209) was dissolved in ethanol (200 ml) and sodium hydroxide (3.129) in water (40ml) was added. The mixture was refluxed for 45 minutes during which precipitation occurred. Some of the ethanol (ca 150ml) was distilled off and the residue was cooled. The mixture was partitioned between methylene chloride and water containing 2N hydrochloric acid (70 ml). The organic layer was washed with water, each aqueous layer being back-extracted with more methylene chloride.The combined organic layers were dried with magnesium sulphate and evaporated to give the title compound (209); Nnn (ethanol 234.5nm (E1%/1cm 383) 259.5nm (E 1 cm 242), 266.5nm (E1 22 1cm 6) and 272.5nm (E1cm 217); vmax (Nujol) 3260 (NH) and 1685cm-' (acid).

Preparation 3 Diphenylmethyl (6R, 7R)-3-Bromomethyl- 7-(Z)-2-cyclopropylmethoxyimino-2-(2-tritylaminothiazol- 4-yl)-acetamido]-ceph-3-em-4-carboxylate Oxalyl chloride (0.37 ml) was added to a solution of N,N-dimethylformamide (0.38ml) in methylene chloride (10ml) at - 20" with stirring under nitrogen.The mixture was stirred with ice-water cooling for ten minutes before recooling to - 20". (Z)-2-Cyclopropylmethoxyimino-2 (2-tritylaminothiazol-4-yl)acetic acid (1.949) was added and the solution was stirred with icewater cooling for ten minutes before recooling to - 20". A suspension of diphenylmethyl (6R, 7R)-7-amino-3-bromomethylceph-3-em-4-carboxylate (1.989) in methylene chloride (10ml) containing N,N-dimethylaniline (1.76ml) was added. A clear solution formed as the mixture was allowed to warm to 21 over one hour. The solution was washed with dilute hydrochloric acid and water, each wash being back extracted with more methylene chloride and the combined extracts were dried with magnesium sulphate and evaporated to a small volume.This solution was filtered through Sorbsil U30 (509) in ethyl acetate and the eluate was evaporated. The residue (3.549) was crystallised from diethyl ether-petroleum ether (bp 40 to 60 ) to give the title compound (2.439) mp 135 to 147 ,[&alpha;]D 21 - 11.9' (e 0.6, CHCl3) Example I (6R, 7R)- 7-[(Z)-2-(2-Aminothiazol-4-yl)-2-cyclopropylmethoxysminoacetamido]-3-( 1 -pyridiniumme thyl)ceph-3-em-4-carboxylate, dih ydrochloride salt Oxalyl chloride (0.74 ml) was added to a stirred solution of N,N-dimethylformamide (0.76 ml) in methylene chloride (20 ml) at - 20" under nitrogen.The mixture was stirred with ice-water cooling for 10 minutes and then recooled to - 20". (Z)-2-Cyclopropylmethoxyimino-2-(2tritylaminothiazol-4-yl)acetic acid (3.87 g), was added and the solution was stirred in an ice-bath for 10 minutes. The solution was recooled to - 20"C and added to a solution of (6R,7R)-7 amino-3-( 1 -pyridiniummethyl)ceph-3-em-4-carboxylate dihydrochloride, dihydrate (3.049) in industrial methylated spirits (24ml) and water (6ml) containing triethylamne (4.7ml) at - 7" with vigorous stirring. The solution was allowed to warm to 21 over 15 minutes. It was then washed twice with water, each time back extracting with methylene chloride.The combined organic layers were dried with magnesium sulphate and evaporated to a gummy oil, which was triturated with diethyl ether to give a solid (4.589). This solid (4.319) was dissolved in formic acid and concentrated hydrochloric acid (1.6 ml) was added. After stirring for one hour at 21", the mixture was filtered and the filter cake was leached with formic acid.The combined filtrates were evaporated and the residue was triturated with acetone to give the title compound (3.209); [a]21 38.0' (e 1.26, DMSO); Amax (pH 6 buffer) 255nm (E1 1cm 307); Amf 240nm, 1% (E1 cm 287) and 289.5nm (E11cm 128).

Example 2 (a) t-Butyl (6R, 7R)-3-Acetoxymethyl-7-[(Z)-2-(cyclopropylmethoxyimino)-2-(2-tritylaminothiazol-4- yl)acetamido]-ceph-3-em-4-carboxylate Oxalyl chloride (0.74 ml) was added to a solution of dimethylformamide (0.76ml) in methylene chloride (20ml) at - 20" with stirring under nitrogen. After 10 minutes stirring with ice-water cooling, the mixture was recooled to - 20" and (Z)-2-cyclopropylmethoxyimino-2-(2tritylaminothiazol-4-yl)acetic acid (3.88 g) was added. After 10 minutes with water cooling the solution was recooled to - 20" and a solution of t-butyl 3-acetoxymethyl-7-aminoceph-3-em-4carboxylate (3.28g) in methylene chloride (20ml) containing N,N-dimethylaniline (2.52 ml) was added.The solution was stirred at 21 for one hour. The solution was then washed successively with dilute hydrochloric acid and water, each time back extracting with methylene chloride. The combined organic solutions were dried with magnesium sulphate, concentrated, and chromatographed on Sorbsil U30 (150g) in ethyl acetate [10 to 40% in petroleum ether (bp 40-60 )] to give the title compound (5.79g); [a]2Da + 25.3" (c 1.11, CHCI3), VmaX (CHBr3) 3400 (NH), 1790 (p-lactam), 1729 (esters), and 1689 and 1512 cm-' (amide).

(b) (6R, 7R)-3-Acetoxymethyl- 7-[(Z)-2-(2-aminothiazol-4-yl)-2-cyclopropylmethoxyimino]acetamido- ceph-3-em-4-carboxylic acid, hydrochloride salt The product of stage (a) (5.60g) was dissolved in formic acid (22ml) and concentrated hydrochloric acid (1.69ml) was added. The solution was stirred at 21 for one hour and the mixture was filtered. The filter-cake was leached with formic acid and the combined filtrates were added to stirred diisopropyl ether (620ml).The precipitate was collected by filtration, washed with diisopropyl ether and dried to give the title compound (3.37g), [az)2"'+ 37.25 (c 0.59, DMSO), may (Nujol) 3700-2300 (NH3 and OH), 1780 (ss-lactam), 1725 (ester and acid) and 1662 and 1543 cm-1 (amide).

(c) (6R, 7R)- 7-[(Z)-2-(2-Aminothiazol-4-yl)-2-cyclopropylmethoxyiminoacetamido]-3-(4-carbamoyl- 1 -pyridiniummethyl)-ceph-3-em-4-carboxylate The product of stage (b) (1.58g) was mixed with sodium bicarbonate (500mg) and water (2ml) was added slowly with warming to 50 whereupon a syrup formed. Isonicotinamide (1.459) was added followed by further sodium bicarbonate to adjust the pH of the mixture to 7.0. Sodium iodide (6.67g) was added and the solution was heated to 80 with stirring. This solution was heated at 80 for 3.5 hours. The syrup was allowed to cool and diluted with acetone (250ml) with agitation. The solid was collected by filtration and was washed well with acetone and dried in vacuo. The solid was dissolved in water and loaded onto a column of Amberlite XAD-2 resin.The column was eluted with water and then 25% ethanol in water.

Fractions were assayed by examining their UV spectra and appropriate portions of eluate were combined and evaporated to an oil. This oil was triturated with acetone to give the title compound (360mg), Amax (pH6 phosphate buffer) 231 nm (E1cm 338), Nnti 251.5 nm (E 1% 308) 1cm and 292 nm 1% (E1cm 146); #max (Nujol) 1775 (Plactam), 1680 (amide) and 1615 cm-' (carboxylate).

Example 3 (6R,7R)-7-[(Z)-2-(2-Aminothiazol-4-yl)-2-cyclopropylmethoxyiminoacetamido]-3-(1-pyridiniummeth yl)ceph-3-em-4-carboxylate The dihydrochloride salt of the title compound (420 mg) was stirred with water (5ml.) and ethyl acetate (4ml) and Amberlite LA-2 resin (0.6 ml) was added dropwise to adjust the mixture to pH 5.2. The organic layer was extracted with further water and the combined aqueous layers were washed with ethyl acetate. Evaporation gave a gum which was triturated with acetone to give the title compound (270 mg); [a]2 - 69.7% (c 1.14, DMSO); Ama, (pH6 buffer) 256 nm (E1crn 362); Am 241 nm (E1cm 340) and 292 nm 1% (Elcm 145).

Example 4 (a) Diphenylmethyl(1S,6R,7R)-3-Bromomethyl-7-[(Z)-2-cyclopropylmethoxyimino-2-(2-tritylami- nothiazol-4-yl)acetamido]ceph-3-em-4-carboxylate, 1-oxide.

Diphenylmethyl (6 R,7 R)-3-bromomethyl-7-(Z)-2-cyclopropylmethoxyimino-2-(2-tritylaminothia- zol-4-yl)acetamido]ceph-3-em-4-carboxylate (1.2g) was dissolved in methylene chloride (10ml.) and 3-chloroperbenzoic acid (292 mg) was added with stirring and ice-water cooling. After thirty minutes, the solution was washed with aqueous sodium bicarbonate solution and water (twice).

After drying with magnesium sulphate, the solution ws concentrated and purified on a column of Sorbsil U-30 (50g) which was eluted with ethyl acetate (50 to 80%) in petroleum ether (b.p.

40 to 60 ) to obtain the title compound (1.1 g); [a]2D1+ 14.2" (c 1.23 CHCl3); NnfI 244.5nm (E1 cm 260) 258.5 nm (Eicm 232), 266nm (E1cm 225), and 272 nm 1% (E1cm 214).

(b) Diphenylmethyl (15, 6R, 7R)- 7-Z)-2-Cyclopropylmerhoxyimino-2-(2-tritylaminothiarol-4-yl)ace- tamido]-3-(1-pyridiniummethyl)ceph-3-em-4-carboxylate, 1-oxide, bromide.

The product of stage (a) was dissolved in pyridine (0.2ml) and the solution was stirred at 21" for about 2.5 hours. The solution was diluted with diethyl ether (8ml.) and after stirring for 10 minutes, the precipitate was collected by filtration, washed with diethyl ether and dried to give the title compound (90 mg); [a]21 65.2' (c 1.38, CHCl3); VmaX (Nujol) 3700-3100 (NH and H2O), 1803 (p-lactam), 1727 (ester), 1682 + 1514 (amide) and 1028 cm - ' (sulphoxide).

(c) Diphenylmethyl (6R, 7R)-7-[(Z)-2-Cyclopropylmethoxyimino-2-(2-tritylaminothiazol-4-yl)acetam- ido]-3-( 1 -pyridiniummethyl)ceph-3-em-4-carboxylate, mixed halide Diphenylmethyl (1 S,6 R, 7 R)-7-[(Z)-2-Cyclopropylmethoxyimino-2-(2-tritylaminothiazol-4-yl)ace- tamido]-3-(1-pyridiniummethyl)ceph-3-em-4-carboxylate, 1-oxide, bromide (300mg) was stirred in suspension with potassium iodide (0.249) in acetone (2ml) with ice-water cooling. Acetyl chloride (0.1 ml) was added.After stirring with cooling for ca. 1.5 hours, the solution was poured into sodium metabisulphite (0.129) dissolved in water (15ml). The resulting precipitate was collected by filtration, washed with water and diethyl ether and dried to give the title compound(300 mg); vmax (Nujol) 3700-3100 (NH), 1790 (ss-lactam), 1721 (ester) and 1679 and 1518 cm-1 (amide; T (d6-DMSO) contains 1.03, 1.33 and 1.82 (pyridinium protons), 3.08 (COOCH), 3.23 (thiazole proton), 4.05 (d,d, J 9 and 5HZ; 7-H and 6.08 (d, J 6HZ;NOCH2-) (d) (6R, 7R)- 7[(Z)-2-(2-Aminothiazol-4-yl)-2-cyclopropylmethoxyiminoacetamido]-3-( 1 -pyridin iummethyl)ceph-3-em-4-carboxylate, dihydrochloride salt Diphenylmethyl (6 R, 7 R)-7-[(Z)-2-cyclopropylmethoxyimino-2-(2-tritylaminothiazol-4-yl)acetami- doJ-3-( 1 -pyridiniummethyl)ceph-3-em-4-carboxylate, mixed halide (200 mg) was mixed with formic acid (0.8ml) at 21 and concentrated hydrochloric acid (0.06ml) was added. The mixture was stirred at 21" for two hours and then filtered. The filter cake was leached with formic acid and the combined filtrates were added to acetone (10 ml.) and ether (3ml).The precipitate was collected by filtration, washed and dried to give the title compound {22mg) whose spectroscopic characteristics resembled those of the product of Example 1.

Example 5 (a) Diphen ylmethyl (6R, 7R)- 7-[(Z)-2-Cyclopropylmethoxyimino-2-(2-tritylaminothiazol-4-yl)acetam- idoj-3-( 1 -pyridiniummethyl)-ceph-2-em-4-carboxylate, bromide Diphenylmethyl (6R,7R)-3-bromomethyl-7-[(Z)-2-cyclopropylmethoxyimino-2-(2-tritylaminothia- zol-4-yl)acetamido]ceph-3-em-4-carboxylate (100mg) was dissolved in pyridine (0.2ml) and the solution was stirred at 21' for three hours.The solution was diluted with diethyl ether (8ml) and after ten minutes the precipitate was collected by filtration, washed with diethyl ether and dried to give the title compound (1 00mg); VmaX (Nujol) 3390 (Nh), 1809 (ss-lactam), 1730 (ester) and 1682 and 1513 cm-' (amide); T(d6-DMSO) includes 1.00, 1.42 and 1.93 (pyridinium protons), 3.05 (COOCH), 3.21 (2-H), 3.24 (thiazole proton), 4.42 (d,d, J 9 and 5HZ; 7-H, and 6.12 (d,J 6HZ: NOCH2-).

(b) Diphenylmethyl (1 S, 6R, 7R)- 7-[(Z)-2-Cyclopropylmethoxyimino-2-(2-tritylaminothiazol-4-yl)ace- tamido]-3-(1-pyridiniummethyl)ceph-3-em-4-carboxylate, 1-oxide, bromide.

Diphenylmethyl (6R,7R)-7-[(Z)-2-cyclopropylmethoxyimino-2-(2-tritylaminothiazol-4-yl)acetami- do]-3-(1-pyridiniummethyl)ceph-2-em-4-carboxylate, bromide (300mg) was dissolved in methylene chloride (5ml) with stirring and ice-water cooling. 3-Chloroperbenzoic acid (70mg) was added. After 1.5 hours, the solution was washed with sodium bicarbonate solution and water and dried with magnesium sulphate. The solution was evaporated to a gum which was triturated with diethyl ether to give a solid. This solid was dissolved in methylene chloride (5ml) and further 3-chloroperbenzoic acid (2 X 50 mg) was added at 30 minute intervals. The solution was washed with sodium bicarbonate solution, sodium metabisulphite solution and with water, and then dried with magnesium sulphate.After evaporation, the residue was triturated with diethyl ether to give the crude title compound (230 mg). A sample of the product was purified by chromatography on Sorbsil U30 (5g) in methanol and propanol-water mixture to give the title compound whose spectroscopic characteristics resembled those of the product of Example 4(b).

The product of Example 5(b) may be reduced and deprotected as in Examples 4(c) and 4(d) to give (6 R,7 R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-cyclopropylmethoxyiminoacetamido]-3-(1 -pyridini- ummethyl)ceph-3-em-4-carboxylate dihydrochloride salt.

Example A Dry Powder for Injection Fill sterile (6 R, 7 R)-7-((Z)-2-(2-am inoth iazol-4-yl)-2-(cyclopropylmethoxyimi no)acetamido]-3-( 1- pyridiniummethyl)ceph-3-em-4-carboxylate aseptically into glass vials, the content of each container being equivalent to 500mg of the anhydrous, pure material. Purge the vial headspaces with sterile nitrogen. Close the vials using rubber discs or plugs and metal overseals, applied by crimping, to prevent gaseous exchange or ingress of micro organisms. The product may be constituted by dissolving in Water for Injections or other suitable sterile vehicle shortly before administration.

Claims (8)

1. Cephalosporin antibiotics of the general formula

(wherein R represents a 3- or 4-carbamoyl group or a hydrogen atom) and non-toxic salts and non-toxic metabolically labile esters thereof.

2. A compound according to claim 1 which is (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2 cyclopropylmethoxyiminoacetamido]-3-( 1 -pyridiniummethyl)ceph-3-em-4-carboxylate.

3. Non-toxic salts and non-toxic metabolically labile esters of the compound of claim 2.

4. N-Protected and carboxyl clocked forms of the compounds according to claim 1.

5. A process for the preparation of a compound according to claim 1 which comprises (A) acylating a compound of the formula

(wherein R is as defined in claim 1, B is

and the dotted line bridging the 2-, 3- and 4-positions indicates that the compound is a ceph-2em or ceph-3-em compound) or a salt or 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) and having an associated anion E-, with an acid of formula

(wherein R2 is an amino or protected amino group) or a salt thereof, or with an acylating agent corresponding thereto; or (B) reacting a compound of formula

(wherein R2, B and the dotted line are as defined above;R3 represents hydrogen or a carboxyl blocking group; and X is a replaceable residue of a nucleophile) or a salt thereof, with a pyridine compound of the formula

(wherein R is as defined in claim 1); 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 lv3-isomer, ii) reduction of a compound wherein B is

to form a compound wherein B is

iii) conversion of a carboxyl group into a non-toxic metabolically labile ester function, iv) formation of a non-toxic salt function, and v) removal of any carboxyl blocking and/or N-protecting groups.

6. A process according to claim 5 wherein the compound of formula (II) is (6R,7R)-7-amino 3-( 1 -pyridiniummethyl)-ceph-3-em-4-carboxylate dihydrochloride.

7. A pharmaceutical composition comprising as active ingredient a compound according to claim 1 in association with a pharmaceutical carrier or excipient.

8. An acid of formula (Ill) as defined in claim 5 and salts, esters and reactive derivatives thereof.