GB2058791A - Cephalosporin antibiotics - Google Patents

Abstract

Cephalosporin antibiotics of general formula <IMAGE> (wherein R<a> and R<b>, which may be the same or different, each represent a C1-4 alkyl group or R<a> and R<b> together with the carbon atom to which they are attached form a C3-7 cycloalkylidene group; and R<4> represents hydrogen or a 3- or 4-carbamoyl group, with the proviso that R<a> and R<b> do not each represent a methyl group when R<4> represents hydrogen) exhibit broad spectrum antibiotic activity, the activity being unusually high against gram-negative organisms such as strains of Pseudomonas organisms. 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 such antibiotics.

Description

W 1 GB 2 058 791 A 1

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 5

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

Thus, for example, in our British Patent Specification No. 1,399,086, we describe a novel class of 15 cephalosporin antibiotics containing a 7P-(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 gramnegative organisms.

The discovery of this class of compounds has stimulated further research in the same area in 20 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 7p-acylamido group of the formula R. C. CO. NH 19 R A N 1 -0.(CH 2)Mic (C171 2) n C0011 1 B R (A) (wherein R is a thienyl or furyl group; R A and R' may vary widely and may, for example, be C,-4 alkyl groups or together with the carbon atom to which they are attached form a C_,-, cycloalkV11dene group, and m and n are each 0 or 1 such that the sum of m and n is 0 or 1), the compounds being syn isomers or mixtures of svn 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 30 substituents. These compounds have been found to have particularly good activity against gram negative organisms.

Furthermore, in our British Patent Specification No. 1,522,140 we describe cephalosporin antibiotics of the formula H H 1 9 1 9 1 9 @," S R. C. CO. NH It 1 N N cif 2 el \ OR 2 0 /i/ coo 0 R 3 (B) (wherein R' represents a furyl or thienyl group; R' represents a C,-C, alkyl group, a C,-C, cycioalkyl group, a furyimethyl or thienyimethyl group; and R 3 represents a hydrogen atom or a carbamoyl, carboxy, carboxymethyl, sulpho or methyl group), the compounds being syn isomers or existing as mixtures of syn and anti isomers containing at least 90% of the syn isomer. These compounds exhibit high antibacterial activity against a broad range of gram-positive and gram-negative organisms. The 40 compounds also possess high stability to P-lactamases produced by various gram-negative organisms, as well as good stability in vivo.

Other compounds of similar structure have been developed from these compounds in further attempts to find antibiotics having improved broad spectrum antibiotic activity and/or high activity against gram-negative organisms. Such developments have involved variations in not only the 7p acylamido groups in the above formulae but also the introduction of particular groups in the 3-position of the cephalosporin molecule. Thus, for example, in Belgian Patent Specification No. 852,427, there are described cephalosporin antibiotic compounds falling within the general scope of our British Patent Specification No. 1,399,086, and wherein the group R in formula (A) above may be replaced by a variety of different organic groups, including 2-afminothiazol-4-yi, and the oxygen atom in the oxyimino 50 2 GB 2 058 791 A 2 group is attached to an aliphatic hydrocarbon group which may itself be substituted by, for example, carboxy. In such compounds, the substituent at the 3-position is an acyloxymethyl, hydroxymethyl, formy] or optionally substituted heterocyclic-thiomethyl group.

Furthermore, Belgian Patent Specification No. 836,813 describes cephalosporin compounds wherein the group R in formula (A) above may be replaced by, for example, 2-aminothlazol-4-yi, and the 5 oxyimino group is a hydroxyimino or blocked hydroxyimino group, e.g. a metfioxyimino 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, e.g. the pyridinium group which may be substituted, for example by a carbamoyl group. In the above-mentioned Specification no antibiotic activity is ascribed to such compounds which are only 10 mentioned as intermediates for the preparation of antibiotics described in that Specification.

Belgian Patent Specification No. 853,545 describes cephalosporin antibiotics wherein the 7pacylamido side chain is primarily a 2-(2aminothiazol-4-yi)-2-(syn)-methoxyimino-acetamido group and the substituent in the 3-position is broadly defined in a similar manner to that in the above-mentioned

Belgian Patent Specification No. 836,813. Compounds specifically exemplified in the Specification 15 include compounds in which the 3-position is substituted by a pyrimidiniummethyl or 4carba moylpyridiniu m methyl group.

We have now discovered that by an appropriate selection of a small number of particular groups at the 7p-position in combination with either a pyridiniummethyl or a 3- or 4-carbamoyl pyridi ni u m methyl group at the 3-position, cephalosporin compounds having particularly advantageous activity (described 20 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:

NU N c.co.mH d N R a 1 0. C. COON Rh H H a S (1) 0 -N 4 111 - CHA COO 9 \==JR (wherein Ril and R b, which may be the same or different each represent a C1-4 alkyl group (preferably a 25 straight chain alkyl group, i.e. a methyl, ethyl, n-propyl or n-butyl group and particularly a methyl or ethyl group) or Ra and R b together with the carbon atom to which theyare attached form a C3-, cycloalkylidene group, preferably a C3-1 CyCloalkylldene group; and R 4 represents hydrogen or a 3- or 4 carbamoyl group, with the proviso that R" and R b do not each represent a methyl group when R 4 represents hydrogen) and non-toxic salts and nontoxic metabolically labile esters thereof.

The compounds according to the invention are syn isomers. The syn isomeric form is defined by the configuration of the group Ra 1 -u.i;.i;uuH 1 h.

as W V with respect to the carboxamido group. In this Specification the syn configuration is denoted structurally

N14 ,I\, N C.CO.M 11 N R a '-,, 1 0. C. COON 1 R b It will be understood that since the compounds according to the invention are geometric isomers, 14 J k 3 GB 2 058 791 A 3 some admixture with the corresponding anti isomer may occur.

1 --- 15 7 The invention also includes within its scope the solvates (especially the hydrates) of the compounds of formula (1). It also includes within its scope salts of esters of compounds of formula (1).

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- 5 iminothiazolinyl form, are included within the scope of the invention. Moreover, the compounds of formula (1) depicted above may also exist in alternative zwitterionic forms, for example wherein the 4carboxyl group is protonated and the carboxyl group in the 7-side chain is deprotonated, which alternative forms are included within the scope of the present invention.

It will also be appreciated that when R' and R' in the above formula represent different C1-4 alkyl 10 groups, the carbon atom to which they are attached will comprise a centre of asymmetry. Such compounds are diastereoisomeric and the present invention embraces individual diastereoisomers of these compounds as well as rhixtures thereof.

The compounds according to the invention exhibit broad spectrum antibiotic activity. Against gram- negative organisms the activity is unusually high. This high activity extends to many P-lactamase- 15 producing gram-negative strains. The compounds also possess high stability to P-lactamases produced by a range of gram-negative 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 Entero6acteriaceae (e.g. strains of Escherichia coli Klebsielle pneumonlae, Salmonella typhimurium, Shigella sonnei, Enterobacter cloacae, Serratia marcescens, Providence species, Proteus mirabilis, and especially indole-positive Proteus organisms such as Proteus vulgaris and Proteus morganfil 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 25 activity against strains of various Pseudomonas organisms which are not susceptible to the majority of 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 high toxicity of these antibiotics. The cephalosporin antibiotics of the present invention thus possess potentially great advantages over the aminoglycosides. 30 Non-toxic salt derivatives which may be formed by reaction of either or both of the carboxyl groups present in the compounds of genera[ formula (1) 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. procalne, phenyl ethyl benzy] a mine dibenzyiethylenediamine, ethanolamine, diethanolamine, and N- methyiglucosamine salts). Other non- 35 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 divinyibenzene 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 compounds of formula (1) 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 (1) 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.

These and other salt derivatives such as the salts with toluene-psulphonic and methanesulphonic 45 acids may be employed as intermediates in the preparation and/or purification of the present compounds of formula (1), for example in the processes described below.

Non-toxic metabolically labile ester derivatives which may be formed by esterification of either or both carboxyl groups in the parent compound of formula (1) include acyloxyalkyl esters e.g. lower alkanoyloxy-methyl or -ethyl esters such as acetoxy-methyl or -ethyl or pivaloyloxymethyl esters. In 50 addition to the above ester derivatives, the present invention includes within its scope compounds of formula (1) in the form of other physiologially acceptable equivalents, i. e. physiologically acceptable compounds which, like the metabolically labile esters, are converted in vivo into the parent antibiotic compound of formula (1).

A preferred group of compounds according to the invention by virtue of their high antibiotic activity 55 are those compounds of formula (1) above wherein R4 represents hydrogen, i.e. compounds of the general formula:- Gd 2 058 791 A 4 Nil 2 N C. CO. NR 11 N R a -,, 0. k C. C0011 1 b R (Ia) H a S T.-', o C. H 2 C00 wherein Ra and R' have the above defined meanings and their non-toxic salts and non-toxic metabolically labile esters.

An outstanding compound of formula (1a) is (6R,7R)-7-[(Z)-2-(2aminothiazol-4-yi)-2-(2- carboxycyclobutthe formula- NH 2 3 A N \-/ c. CO. NH 11 N --- 0. C. cooll -oxyimino)acetamidol-3-(1 -pyridiniummethyi)-ceph-3-em-4-carboxylate which has 5 (Ib) H H S N cif 2 _N,0\ coo 9 together with its non-toxic salts (e.g. sodium salt) and non-toxic metabolically labile esters. The compound of formula (Ib) possesses to an outstanding extent the general antibiotic properties set out above for the compounds of general formula (1). However one may emphasise its excellent activity 10 against strains of Pseudomonas organisms. The compound has excellent antibacterial properties which are not impaired by human serum, and, moreover, the effect of increased inocula against the compound is low. The compound is rapidly bactericidal at concentrations close to the minimum inhibitor concentration. It is well distributed in the bodies of small rodents.giving useful therapeutic levels after subcutaneous injection. Experimental infections in mice with gram- negative bacteria were successfully 15 treated using the compound and, in particular, excellent protection was obtained against strains of Pseudomonas aeruginosa, an organism normally not susceptible to treatment with cephalosporin antibiotics. This protection was comparable with the treatment with an aminoglycoside such as amikacin. 20 Other examples of preferred compounds according to the present invention include the following 20 compounds of formula (1) and their non-toxic salts and non-toxic metabolically labile esters, namely:(6R,7R)-7-[(Z)-2-(2-aminothiazol-4-V[)2-(1 -carboxyprop-2oxyimino)acetamidol-3-(4-carbamoyi1 -pyridiniummethyi)-ceph-3-em-4carboxylate; (6R,7R)-7-[(Z)-2-(2-aminothlazol-4-yl)-2-(1 carboxycycloprop-1 -oxyimino)acetamidol-3-(1 pyridiniummethyi)-ceph-3-em-4-carboxylate; (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1 -carboxycyclopent-1 Vioxyimino)acetamidol-3-(1 - pyridiniummethyi)-ceph-3-em-4-carboxylate; and (6R,7R)-7-[(Z)-2-aminothiazol-4-yi)-2-(1 -carboxycyclobut-1 oxyimino)acetamidol-3-(4 carbamoyM -pyridini u m m ethyl)-ceph-3-e m-4-ca rboxyl ate.

Other compounds according to the present invention include for example those wherein the 30 groups R3, R b and R 4 in formula (1) are as follows:- 3 A I A, 1 1, K GB 2 058 791 A 5 1 Ra R b R4 a) Alkyl groups -CH -c 2H, H _C2 H, _C2 H, H -CH, -CH, 3-CONH 2 -CH, -C2H, 3-CONH,.

_C2 H, _C2 H, 3-CONH 2 -CH, _C2 H, 4-CONH 2 _C2 H, -C2H, 4-CONH 2 Ra 1 R 4 C - Rb f b) Cycloalkylidene groups Cyclobutylidene 3-CONH 2 Cyclopentylidene 3-CONH2 Cyclopentylidene 4-CONH2 Cyclohexylidene H Cyclohexylidene 3-CONH2 Cyclohexylidene 4-CONH 2 Cyclopropylidene 3-CONH 2 Cyclopropylidene 4-CONH2 The compounds of formula (1) 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 5 antibiotic compound of general formula (1) as hereinbefore defined or a non-toxic salt or non-toxic metabolically labile ester thereof which comprises (A) acylating a compound of the formula H B 11 2 N ---INCH 2 N R 4 POOC) 1wherein R4 is as defined above; B is > S or > S > 0 (a- or P-); 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, 6 GB 2 058 791 A 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 -COOR5 at the 4-position [where R5 is a hydrogen atom or a carboxyl blocking group, e.g. the residue of an esterforming allphatic 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 AS such as a halide, e.g. chloride or bromide, or trifluoroacetate anion, with an acid of the formula 7 1,11 1 k,' S Pj C. GOOH 11 14 1 R a 11 6 O.C. COOR i b R (wherein R' and R' are as hereinbefore defined; R' represents a carboxyl blocking group, e.g. as described for R'; and R 7 is an amino or protected amino group) or with an acylating agent corresponding 10 thereto; or (B) reacting a compound of formula 7. R 1 H H C14 2 X 8 COOR C.CO.NH 11 N R a I 8a " O.C. COOR 1 b R (iv) (wherein Ra, R', R 7, B and the dotted line are as hereinbefore defined; R' and R11a may independently represent 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 15 thereof, with a pyridine compound of the formula / - X\ NUI, R 4 (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 A'-isomer, ii) reduction of a compound wherein B is > S --3, 0 to form a compound wherein B is > S, iii) (V) conversion of a carboxyl group into a non-toxic salt or non-toxic metabolically labile esterfunction, and iv) removal of any carboxyl blocking and/or N-protecting groups.

In the above-described process (A), the starting material of formula (11) is preferably a compound 25 wherein B is > S 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 W(7-aminoceph-3-em-3 yimethyl)pyridinium-4'-carboxylate dihydrochloride on account of the high purity in which lt can be prepared.

Acylating agents which may be employed in the preparation of compounds of formula (1) include 30 -acid halides, particularly acid chlorides or bromides. Such acylating agents may be prepared by reacting an acid (111) 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 nonaqueous reaction media, c z W v 7 GB 2 058 791 A 7 conveniently at temperatures of from -50 to +501C, preferably -20 to + 301C, 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 di methylani line), 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 (111) may themselves be used as acylating agents in the preparation of compounds of formula (1). Acylations employing acids (111) are desirably conducted in the presence of a condensing 10 agent, for example a carbodlimide such as N,N-dicyclohexyicarbodiimide or Wethyl-N'-V7 dimethylaminopropylcarbodiimide; a carbonyl compound such as carbonyidilmidazole; or an isoxazolium salt such as Wethyl5phenylisoxazolium perchlorate.

Acylation may also be effected with other amide-io-ming derivatives of acids of formula (111) 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, tetra hydrofura n, dimethylformamide or acetonitrile.

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

The acids of formula (111) 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.

The pyridine compound of formula (V) may act as a nucleophile to displace a wide variety of substituents X from the cephalosporin of formula OV). To some extent the facility of the displacement is 30 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 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 35 maintaining the reactants in solution or suspension. The reaction is advantageously effected using from 1 to 10 moles of the pyridine compound.

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

Acyloxygroups 1 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 50 acid, or a carbamic acid.

When using a compound of formula (M in which X represents an acetoxy or substituted acetoxy group, it is generally desirable that the group R' in formula (M should 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.

The above-described process employing compounds of formula OV) 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 300 to 11 OOC, preferably 500 to 801C.

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 > S---+ 0 8 GB 2 058 791 A and R8 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 and N,N-dimethyiformamide, and ketones e.g. acetone. In certain cases the pyridine compound itself may be 5 the solvent. Other suitable organic solvents are described in more detail in British Patent Specification No. 1,326,53 1. 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 R' and R" are carboxyl blocking groups 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 thedesired anion.

When using compounds of formula (IV) in which X is a halogen atom as described above, the 10 reaction is conveniently effected at a temperature of -101 to +501C, preferably + 10 to +300C.

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 ion-exchangers (for example by chromatography on ion-exchange resins) or macroreticular resins.

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

A ceph-2-em reaction product may also be oxidised to yield the corresponding ceph-3-em 1 - oxide, for example by reaction with a peracid, e.g. peracetic or m- chloroperbenzoic acid; the resulting 20 sulphoxide may, of desired, subsequently be reduced as described hereinafter to yield the corresponding ceph-3-em sulphide.

Where a compound is obtained in which B is > S -+ 0 this may be converted to the corresponding sulphide by, for example, reduction of the corresponding acyloxysulphonium or alkoxysulphonium salt prepared in situ by reaction with e.g. acetyl chloride in the case of an acetoxysulphonium salt, reduction 25 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, tetra hyd rofu ran, dioxan, dimethylformamide or dimethylacetamide. The reaction may be effected at a temperature of from -200 to +501C.

Metabolically labile ester derivatives of the compounds of formula (1) may be prepared by reacting a compound of formula (1) or a salt or protected derivative thereof with an appropriate esterifying agent 30 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 (1) may be formed by reacting an acid of the for Mula (J) with the appropriate base. Thus, for example, sodium or potassium salts may be prepared using the respective 2-ethylhexanoate or hydrogen carbonate salt. Acid addition salts may be prepared by reacting a compound of formula (1) or a metabolically labile ester derivative thereof with the appropriate acid.

Where a comnound of formula (1) is obtained as a mixture of isomers, the syn isomer may be obtained by, for example, conventional methods such as crystallisation or chromatography. 40 For use as starting materials for the preparation of compound of general formula (1) according to 40 theinvention, compounds of general formula (111) and acid halides and anhydrides corresponding thereto in their syn isomeric forms 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 (111) (provided that Ra and R' together with the carbon atom to which they are attached do not form a cyclopropylidene group) maybe prepared by etherification of a compound of formula 7 R 11 k, S N C. COOR 9 it N "..1 011 (wherein R' is as hereinbefore defined and R9 represents a carboxyl blocking group), by reaction with a compound of general formula (V) ra 1 I.C.COUR6 (V10-50 & i 1 9 GB 2 058 791 A 9 (wherein R' and R' and R' are as hereinbefore defined and T is halogen such as chloro, bromo or iodo; sulphate; or sulphonate such as tosylate), followed by removal of the carboxyl blocking group R'. Separation of isomers may be effected either before or after such etherification. The etherification reaction is generally carried out in the presence of a base, e.g. potassium carbonate or sodium hydride, and is preferably conducted in an organic solvent, for example dimethyisulphoxide, 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. The reaction should be effected in the presence of a base if an acid addition salt of a compound of formu [a (V1) is used. The base should be used in sufficient quantity to neutralise rapidly the 10 acid in question.

Acids of general formula (111) may also be prepared by reaction of a compound of formula 7 v R A.

S CO.COOR 9 wherein R and R' are as hereinbefore defined) with a compound of formula Ra 1 H,N.O.C.COOR 6 1 RD (VIII) OX) (wherein R', R b and R6 areas defined above), followed by removal of the carboxyl blocking group R', and 15 where necessary by the separation of syn and anti isomers.

The last-mentioned reaction is particularly applicable to the preparation of acids of formula (Ill) wherein Ra and Rb together with the carbon atom to which they are attached form a cyclopropylidene group. In this case, the relevant compounds of formula OX) may be prepared in conventional manner, e.g. by means of the synthesis described in Belgian Patent Specification No. 866,422 for the 20 preparation of t-butyl 1 -aminooxycyclopropane carboxylate.

The acids of formula (111) may be converted to 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 (R), ceph-3-em starting compounds may be prepared in conventional manner, e.g. by halogenation of a 7p-protected amino3methylceph-3-em-4-carboxylic acid ester 'I p-oxide, removal of the 7pprotecting group, acylation of the resulting 7p-amino compound to form the desired 7p-acylamido group, e.g. in an analogous manner to process (A) above, followed by reduction of the 1 P-oxide group later in the sequence. This is described in British Patent No. 1,326,53 1. 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 30 compound with N-bromosuccinimide to yield the corresponding 3- bromomethylceph-2-em-compound.

Where X in formula (R) 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 35 the appropriate 3-acetoxymethyl compounds, e.g. as described in British Patent Specification Nos.

1,474,519 and 1,531,212.

The starting materials of formula (11) 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.

A further method for the preparation of the starting materials of formula (11) comprises deprotecting a corresponding protected 7p-amino compound in conventional manner, e.g. using PCl., 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 45 NH, 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 optimally halogenated carboxylic acid, e.g. acetic acid, formic acid, chloroacetic acid ortrifluoroacetlic acid or using a mineral acid, e.g. hydrochloric acid or mixtures of such acids, preferably 50 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 (1) or in the preparation GB 2 058 791 A 10 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 pivaloyloxym ethyl) and retain these in the final product to give 5 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 p-methoxybenzy[oxycarbonyl, pnitrobenzyloxycarbonyl and diphenyimethoxycarbonyi; lower alkoxycarbonyl groups such as t- butoxycarbonyl; and lower haloalkoxycarbonyl groups such as 2,2,2- trichloroethoxycarbony]. Carboxyl 10 blocking group(s) may subsequently be removed by any of the appropriate methods disclosed in the literature; thus, for example, acid or base catalysed hydrolysis is applicable in many cases, as are enzymically- catalysed hydrolyses.

The antibiotic compounds of the invention may be formulated, for administration in any convenient way, by analogy with other antibiotics and the invention therefore includes within its scope pharmaceutical compositions comprising an antibiotic compound in accordance with the invention adapted for use in human or veterinary medicine. Such compositions may be presented for use in conventional manner with the aid of any necessary pharmaceutical carriers or excipients.

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

If desired, such powder formulations may contain an appropriate non-toxic base in order to improve the water-solubility of the active ingredient and/or to ensure that when the powder is reconstituted with water, the pH of the resulting aqueous formulation is physiologically acceptable.

Alternatively, the base may be present in the water with which the powder is reconstituted. The base may be, for example, an inorganic base such as sodium carbonate, sodium bicarbonate or sodium acetate, or an organic base such as lysine or lysine acetate.

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

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

The compositions may contain from 0.1 % upwards, e.g. 0.1-99%, of the active material, 35 depending on the method of administration. When the compositions comprise dosage units, each unit will preferably contain 50-1500 mg of the active ingredient. The dosage as employed for adult human treatment will preferably range from 500 to 6000 mg per day, depending on the route and frequency of administration. For example, in adult human treatment 1000 to 3000 mg per day administered intravenously or intramuscularly will normally suffice. In treating Pseudomonas infections higher daily 40 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 penicillins or other cephalosporins.

The following Examples illustrate the invention. All temperatures are in 'C. 'Petrol' means petroleum ether (b.p. 40-600).

Proton magnetic resonance (p.m.r.) spectra were determined at 100 MHz. The integrals are in agreement with the assignments, coupling constants, J, are in Hz, the signs not being determined; s = singlet, d = doublet, m = multiplet and ABq = AB quartet.

PREPARATION 1 Ethyl (Z)-2-(2-aminothiazol-4-yi)-2-(hydroxyimino)acetate t, To a stirred and ice-cooled solution of ethyl acetoacetate (292 9) in glacial acetic acid (296 mi) was added a solution of sodium nitrite (180 g) in water (400 mi) at such a rate that the reaction temperature was maintained below 1 01C. Stirring and cooling were continued for about 30 min., when a solution of potassium chloride (160 g) in water (800 m]) was added. The resulting mixture was stirred for one hour. The lower oily phase was separated and the aqueous phase was extracted with diethyl ether. The extract was combined with the oil, washed successively with water and saturated brine, dried, and evaporated. The residual oil, which solidified on standing, was washed with petrol and dried in vacuo over potassium hydroxide, giving ethyl (Z)-2-(hydroxyimino)-3- oxobutyrate (309 g).

A stirred and ice-cooled solution of ethyl (Z)-2-(hydroxyimino)-3oxobutyrate (150 g) in dichloromethane (400 mi) was treated dropwise with sulphuryl chloride (140 g). The resulting solution 60 was kept at room temperature for 3 days, then evaporated. The residue was dissolved in diethyl ether, washed with water until the washings were almost neutral, dried, and evaporated. The residual oil (177 g) was dissolved in ethanol (500 mi) and dimethylaniline (77 m]) and thiourea (42 g) were added with stirring. After two hours, the product was collected by filtration, washed with ethanol and dried to give ^it 4 11 GB 2 058 791 A the title compound (73 g); m.p. 1881 (dedomp.).

PREPARATION 2 Ethyl (Z)-2-hydroxyimino-2-(2-tritylaminothiazol-4-yi)acetate, hydrochloride, Trityl chloride (16.75 g) was added portionwise over 2 hours to a stirred and cooled (-300) solution of the product of Preparation 1 (12.91 g) in dimethylformamide (28 mi) containing triethylamine (8.4 mi). The mixture was allowed to warm to 150 over one hour, stirred for a further 2 hours and then partitioned between water (500 mi) and ethyl acetate (500 m]). The organic phase was separated, washed with water (2 x 500 mi) and then shaken with 1 N HCI (500 m]). The precipitate was collected, washed successively with water (100 m]), ethyl acetate (200 mi) and ether (200mi) and dried in vacuo to provide the title compound as a white solid (16.4 g); m.p. 184 to 1861 (decomp).

PREPARATION 3 Ethyl (Z)-2-(2-t-butoxycarbonylprop-2-oxyimino)-2-(2-tritylaminothiazol-4yi)aceta te Potassium carbonate (34.6 g) and t-butyl 2-bromo-2-m ethyl propionate (24. 5 g) in dimethyisulphoxide (25 m]) were added to a stirred solution under nitrogen of the product of Preparation 2 (49.4 g) in dimethyisulphoxide (200 mi) and the mixture was stirred at room temperature 15 for 6 hours. The mixture was poured into water (2 1), stirred for 10 mins. , and filtered. The solid was washed with water and dissolved in ethyl acetate (600 mi). The solution was washed successively with water, 2N hydrochloric acid, water, and saturated brine, dried, and evaporated. The residue was recrystallised from petroleum ether (b.p. 60-801) to give the title compound (34 g), m.p. 123.5 to 1250 PREPARATION 4 (Z)-2-(2-t-Butoxycarbonylprop-2-oxyimino)-2-(2-tritylaminothiazol-4-yi)acet ic acid The product of Preparation 3 (2 9) was dissolved in methanol (20 m]) and 2N sodium hydroxide (3.3 mO was added. The mixture was refluxed for 1.5 hours and then concentrated. The residue was taken up in a mixture of water (50 mi), 2N hydrochloric acid (7 mi), and ethyl acetate (50 mi). The organic phase was separated, and the aqueous phase extracted with ethyl acetate. The organic solutions were combined, washed successively with water and saturated brine, dried, and evaporated. The residue was recrystallised from a mixture of carbon tetrachloride and petrol to give the title compound (1 g), m.p. 152 to 1560 (decomp).

PREPARATION 5 Ethyl (Z)-2-(2-tritylaminothiazol-4-yi)-2-(1 -tbutoxycarbonylcyclobut- 1 -oxyimino) acetate.

The product of Preparation 2 (55.8 g) was stirred under nitrogen in dimethyisulphoxide (400 mi) with potassium carbonate (finely ground, 31.2 g) at room temperature. After 30 minutes, t-butyl 1 - bromocyclobutane carboxylate (29.2 g) was added. After 8 hours further potassium carbonate (31.2 g) was added. More potassium carbonate (6 x 16 g portions) was added during the next three days and 35 further t-butl 1 -bromocyclobutane carboxylate (3.45 9) was added after 3 days. After 4 days in all, the mixture was poured into ice-water (ca. 3 litres) and the solid was collected by filtration and washed well with water and petrol. The solid was dissolved in ethyl acetate and the solution washed with brine (twice), dried with magnesium sulphate and evaporated to a foam. This foam was dissolved in ethyl acetate-petrol (12) and filtered through silica gel (500 g). Evaporation gave the title compound (60 9) 40 as a foam, Pn.,, (CHBr3) 3400 (NH) and 1730 cm-1 (ester).

PREPARATION 6 (Z)-2-(1-t-Butoxycarbonylcyclobut-l-oxyimino)-2-(2-tritylaminothiazol-4yi) acetic acid.

A mixture of the product of Preparation 5 (3.2 g) and potassium carbonate (1.65 g) was refluxed in methanol (180 mi) and water (20 mO for 9 hours and the mixture was cooled to room temperature. The 45 mixture was concentrated and the residue partitioned between ethyl acetate and water, to which was added 2N HCI (12.2 mi). The organic phase was separated and the aqueous phase extracted with ethyl acetate. The combined organic extracts were washed with saturated brine, dried and evaporated to give the title compound (2.3 g);.Xm (ethanol) 265 rim (E]%W, 243).

PREPARATION 7 (Z)-2-(1-t-Butoxycarbonylcycloprop-1 -oxyimino)-2-(2tritylaminothiazol-4-yi)acetic acid.

A solution of hydrazine hydrate (0.20 9) in methanol (0.4 mi) was added to a solution of 1 -tbutoxycarbonyleyclo-prop-l-oxyphthalimide (0.61 g; prepared as described in Belgian Patent No. 866,422) in dichloromethane (7 mi). The mixture was stirred at room temperature for 1 hour, and treated with 5N aqueous ammonia solution (7 mi). The organic phase was separated and the aqueous 55 phase was extracted with dichloromethane. The combined organic solutions were washed with water, dried, and evaporated. The oily residue (0.30 g) was dissolved in a mixture of ether (5 mi) and ethyl acetate (5 m]). 2-Tritylaminothiazoi4-ylgiyoxylic acid (0.73 g; prepared as described in Belgian Patent No. 864,828) was added. The mixture was stirred at room temperature overnight and then filtered. The 12 GB 2 068 791 A 12 solid was washed with a little ether and dried in vacuo to give the title compound (0.5 g), m.p. 156. 8-157.21; v a,,. (CHBr, 2300-3500 (O-H, N-H); 1750 (t-butyl ester); 1690 em-' (acid).

PREPARATION 8 Ethyl (Z)-2-(1 -t-butoxycarbonyleyclopent-1 --yioxyi mi no)-2-(2-trityl aminothi azol-4-y1) acetate The product of Preparation 2 (10 g) was stirred with tbutyl 2-bromo- cyclopenta necarboxyl ate (7 5 g) in dimethyisulphoxide (40 mi) containing potassium carbonate (10 g) under nitrogen at 210 for 21 hours. The mixture was poured into ice-water (500 mi) and the grey solid was collected by filtration, washed with water and air dried.

Recrystallisation of this solid from methanol (500 mi) gave the title compound (11.7 g), m.p.

1() 179-1806, V,a, (CHBr3) 3410 (NH), 1735 (ester), 1275 (ester) and 755 em-' (phenyl).

PREPARATION 9 (Z)-2-(1 -t-Butoxycarbonylcyclopentl -Vioxyimino)-2-(2-tritylaminothiazol- 4-yi)acetic acid The product of Preparation 8 (625 mg) was refluxed with 2N sodium hydroxide solution (0.5 m[) and water (1 rril) in methanol (12 mi) for seven hours. The mixture was left to cool overnight. After dilution with water, orthophosphoric acid was added to adjust the solution to pH 2. The precipitate was extracted with ether and the combined extracts were washed with brine. After drying with magnesium sulphate, the solvent was evaporated to give a gum (493 mg). Recrystallisation from di-isopropyl ether gave the title compound (356 mg) m.p. 171-1730, v,,,,,, (CHBr3) 2500- _3500 (OH and NW, 1755 (ester), 1692 (acid) and 755 and 770 em-' (phenyl).

PREPARATION 10 (6R, 7R)-7-amino-3-(1-pyridiniummethyi)ceph-3-em-4carboxylic acid dihydrochloride A stirred suspension of (6R,7R)-7-(2thienylacetamido)-3-(1-pyridiniummethyi)ceph-3-em-4carboxylate (4.15 g) in dichloromethane (30 m]) was treated with N,N-dimethylaniline (5.09 mi) and chlorotrimethyisilane (2.52 mi). This mixture was stirred at 30-351 for one hour and then cooled to -280 and treated with phosphorus pentachloride (4.16 g), stirred at -250 to -300 for another hour and then poured into a stirred cooled (-201) solution of butane1,3-diol (8.1 mi) and dichloromethane (20 mi). The solution was allowed to attain 01 temperature over 30 minutes, and the precipitated solid (A) was filtered, washed with dichloromefhane and dried in vacuo. It was redissolved in methanol (17.5 ml), stirred and diluted with dichloromethane (87.5 mi) and the precipitated solid filtered off, washed with dichloromethane and dried in vacuo to yield the title compound as a white solid (3.2 g), A max (pH 30 6 buffer) 258 rim (Ell%c. 318); r (D,O) values include 0.95,132 and 1.84 (pyridinium protons), 4.10 to 4.46 (ABq, J 16 Hz, 3-CH2--), 4.5 6 (cl, J 5 Hz 7-H), 4.'10 (d, J 5 Hz, 6-H), 6.14 to 6.50 (ABq, J 17 Hz, C27-H).

EXAMPLE 1 a) t-Butyl (6R,7R)-3-Acetoxymethyi-7-[(Z)-2-(2-t-butoxycarbonylprop-2oxyimino)-2-(2- tritylaminothiazol-4-yi)acetamidolceph-3-em-4-carboxylate A stirred solution of the product of Preparation 4 (572 mg) and t-butyl (6R,7R)-3-acetoxymethyl 7-aminoceph-3-em-4-carboxylate (328 mg) in dimethylformamide (10 mO was cooled to 01, and 1 - hydroxybenzotriazole (150 mg) was added, followed by dicyclohexylcarbodiimide (225 mg). The mixture was warmed to room temperature, stirred for 5 hours, and allowed to stand overnight. The 40 mixture was filtered, and the white solid washed with a little ether. The filtrate and washings were diluted with water (50 mi) and extracted with ethyl acetate. The organic extracts were combined; washed successively with water, 2N hydrochloric acid, water, sodium bicarbonate solution, and saturated brine, dried and evaporated. The residue was eluted through a silica column with ether. The prod uct-contianing eluate was collected and concentrated to give the title compound (533 mg). A portion was recrystallised from di-isopropyl ether, m.p. 103 to 113 (decomp.); [a] 20 + 8.50 (c, 1.0, D DMSO).

b) (6R,7R)-3-Acetoxymethy7-[(Z)-2-(2-aminothiazol-4-yl)-2-(2-carboxyprop2- oxyimino)acetamidolceph-3-em-4-carboxylic acid Trifluoroacetic acid (18 mi) was added to a solution of the product of Stage a) (2.4 g) in anisole 50 (18 mO at 011. The mixture was stirred at room temperature for 2 hours and concentrated. The residue was dissolved in ethyl acetate and extracted with saturated sodium bicarbonate solution. The pH of the aqueous extracts was adjusted to 6, and the solution washed with ethyl acetate. The aqueous phase was acidified to pH 1.5 under ethyl acetate, saturated with sodium chloride, and extracted with ethyl acetate. The combined organic extracts were washed with saturated brine, dried and evaporated. The 55 residue was dissolved in warm 50% aqueous formic acid (20 mi) and allowed to stand for 2 hours. The mixture was diluted with water (50 mi), and filtered. The filtrate was concentrated. The residue was taken up in water (50 mi), refiltered, and Iyophilized to give the title compound (920 mg), A.. i,' (pH 6 [a] 20 buffer) 236 rim (E'I'C,,, 250), Ai,,f 255 rim (E%,,, 235), 296 rim ('1 c D 1 c E' 103); + 20.0. (c 1.0, DMSO).

z 13 GB 2 058 791 A 13 i c) (6R,7R)-7-[(Z)-2-(2-Aminothiazol-4-yi)-2-(2-carboxyprop-2-oxyimino) acetamidol-3-carbamoyl-l pyridiniummethyl)-ceph-3-em-4-carboxylate, monosodium salt.

Isonicotinamide (0.56 g) was added to a stirred solution of the product of Stage b) (0,59 g) in water (0.7 mi) containing sufficient sodium bicarbonate to give a final pH of 6.5. Sodium iodide (2.1 g) was added and the mixture was stirred at 800C for one hour; sodium bicarbonate was added at intervals to maintain a pH in the range 5.5-6.5. The aqueous residue was diluted with water, methyl lsobutyl ketone (a few drops) was added, and the solution was acidified to P.H 1 with 2N hvorochloric acid. The mixture was filtered, and the solid was washed with a little water. The filtrate and washings were collected and washed with ethyl acetate, and the pH adjusted to 6.0 with 2N sodium hydroxide solution. The solution was concentrated and applied to a column of Amberlite XAD-2 resin, using first 10 water and then 20% aqueous ethanol as eluting solvent. The product-containing fractions were concentrated and lyophilized to give the title compound (0.09 g), A (pH 6 buffer) 257. 5 nm (E:,. 276), A1,,f, 291.5 nm (E',%.. 125); T (D,O) values include 0. 92, 1.70 (41-1; pyridinium protons); 3.16"("1-11, aminothiazole-5-H); 4. 34, 4.64 (21-1; ABq; 3-CH2-); 8.54 (6H;-CMe,-).

EXAMPLE 2 t-Butyl (6R,7R) -3-Acetoxymethyi-7-[(Z)-2-(1 -tbutoxycarbonylcyclobut-1 -oxyimino)-2-(2tritylaminothiazol-4yi)acetamidolceph-3-em-4-carboxylate A stirred solution of the product of Preparation 6 (24.2 g) and t-butyl (6R,7R)-3-acetoxymethyi-7aminoceph-3-em-4-carboxylate (13.6 g) in dimethylformamide (300 mi) was cooled to 00, treated with 1 -hyd roxybenzotriazole monohyd rate (4.5 g), followed by dicycl oh exyl ca rbodii m ide (6.4 g) and the product isolated subtantially as described in Example 1 a) to give the title compound (12.8 g), m.p. 113. 5toll6.51(decomp.);[a]20+15.00(cl.O,DMSO).

D b) (6R,7R)-3-Acetoxymethyi-7-[(Z)-2-(2-aminothiazol-4-yi)-2-(1 carboxycyclobut-l oxyimino)acetamido]ceph-3-em-4-carboxylic acid Trifluoroacetic acid (100 m[) was added to a mixture of the product of Stage a) (12.5 g) and 25 anisole (5 mO at 00. The mixture was treated substantially as described in Example 1. b) to give the title compound (4 g) (pH 6 buffer) 246 nm (E ll%cn 264), Ainf295 rim (E I%M118); [Cr]20+27.30(cl.0, IC n DMSO).

c) (6R,7R)-7-[(Z)-2-(2-Aminothiazol-4yl)-2-(1 -carboxycyclobut-1 oxyimino)acetamidol-3-(1 - pyridiniummethyi)-ceph-3-em-4-carboxylate, mono-sodium salt Pyridine (4.1 mi) and the product of Stage b) (3.75 9) were added to a stirred solution of sodium iodide (14.6 g) in water (4.5 mi) at 801C. The solution was stirred at WC for one hour, cooled and diluted with water. The pH of the solution was adjusted to 6.0 with 2N sodium hydroxide solution and this solution was concentrated to remove pyridine. The. product was isolated to give the title comnound(1.3 g) A (pH 6 buffer) 252.5 nm (E '%,n 310), Ain, 291 nm (E 1%, 139); [al 20 +43.50 1 c c D 35 (c 1.0, DMSO).

EXAMPLE 3 (6R,7R)-7-[(Z)-2-Aminothlazol-4-yi)-2-(1-carboxycyclobut-1 oxyimino)acetamido1-3-(4-carbamoyi-l pyridiniummethyi)-ceph-3-em-4-carboxylate, mono-sodium salt Isonicotinamide (1.22 g) was added to a stirred solution of the product of Example 2 b) (1.08 g) in 40 water (1.3 mO containing sufficient sodium bicarbonate to give a final pH of 6.5. Sodium iodide (4 9) was added and the mixture was stirred at 801C for 1 hour; sodium bicarbonate was added at intervals to maintain a pH in the range 6.0-6.5. The product was isolated substantially as described in Example 1 c) to give the title compound, (0. 16 g), [a] 20 D -180 (c 1.08, H20); AMe,. (pH 6 buffer) 256 nm (E'% 298), Ainf 294 nm (E ',%,n 135). - 1 CM c EXAMPLE 4 a) t--Butyl (6R,7R) 3-acetoxymethyi-7-[(Z)-2-(2-tritylaminothiazol-4-yi)2-(1-t-butoxycarbonylcy cloprop- 1 -oxyi m ino)aceta m idolceph-3-e m-4-carboxyl ate 1 -Hydroxybenzotriazole monohydrate (0. 12 g) and dicyclohexylcarbodiimide (0. 16 g) were added 50. to a stirred solution of the product of Preparation 7 (0.34 g) and t- butyl (6R,7R)-3-acetoxymethyi-7- 50 aminoceph-3-em-4-carboxylate (0.25 g) in tetrahydrofuran (6 mi). The mixture was stirred at room temperature overnight and then filtered. The filtrate was evaporated. The residue was dissolved in a little ethyl acetate - Oetroleum ether (bp 60-80') (1:1) and eluted throu - gh a column of neutral alumina (10 g) with the same solvent. The eluate was concentrated to a foam (0.44 g) which was recrystallized from di-isopropi ether (15 mi) to give the title compound, (0.29 g), m.pt.1 150-1191; [a] 20 (c 1.0, DMSO) + 130.

D b) (6R,7R)-3-Acetoxymethy]-7-[(Z)-2-(2-aminothiazol-4-yi)-2-(1 carboxycycloprop-1 - oxyimino)acetamido]ceph-3-em-4-carboxylic acid, hydrochloride salt.

Concentrated hydrochloric acid (0.6 mi) was added to a stirred solution of the product from Stage 14 GB 2 058 791 A 14 a) (1.92 g) in formic acid (7.5 mi) at 101. The mixture was stirred at room temperature for 1.25 hours and then filtered. The filtrate was added to di-isopropyl ether (300 ml), and the mixture was stirred for 1.5 hours. The solid was filtered off, washed with di-isopropyl ether and diethyl ether, and dried in vacuo to give the title compound (1. 16 g), [a] 2 DI (c 1.0, DMSO) + 35; (pH 6 buffer) 239 rim, (E 1'%cn 300).

c) (6R,7R)-7-[(Z)-2-(2-Aminothiazol-4-yi)-2-(1-carboxycycloprop-loxyimino)acet amidol-3-(1- pyridiniummethyi)ceph-3-em-4-carboxylate, sodium salt.

A mixture of the product from Stage b) (0.56 g), sodium bicarbonate (0.17 g), and water (0.5 mi) was warmed to 501. More sodium bicarbonate (0.09 g) was added, followed by pyridine (0.2 m[). The solution was warmed to 801 and sodium iodide (2 9) was added. The solution was stirred at 801 for 40 minutes, cooled, and diluted with acetone (50 mi). The mixture was filtered, and the solid was washed 10 with acetone and ether to give a solid. This solid was dissolved in water (20 mi) and acidified dropwise with 2N hydrochloric acid until a precipitate formed which did not redissolve on standing. The mixture was stirred with neutral alumina (5 g) and filtered through a pad of neutral alumina (10 g). The pad was eluted thoroughly with water. The aqueous eluate was concentrated and the residue was triturated with acetone. The solid was filtered and dried to a solid (0.35 g). This solid (0.30 g) was dissolved in a little water and eluted through a column of 50 g Amberlite XAD-2 resin, using first water and then 20% ethanol in water as eluting solvent. The product-containing fractions were concentrated, and the residue was triturated with acetone to give the title compound, (0.06 g); [a] 23 0 0 1.50 W0.1, water); A a. (pH 6 buffer) 254 rim, (E 11"c1,, 340); Ainf 296 rim, (E '1"'C,,, 125).

EXAMPLE 5 (6R,7R)-7-[(Z)-2-(2-Aminothlazol-4-yi)-2-(1 -carboxycyclopent-1 -yl- oxyimino)-acetamidol-3-(1 - pyridiniummethyl)ceph-3-em-4-carboxylate, dihydrochloride salt.

Phosphorus pentachloride (0.46 g) was dissolved in methylene chloride (20mi) at ambient temperature and the solution was cooled to 101; the product of Preparation 9 (1.095 g) was added in one charge. The mixture warmed to -50 and was stirred for 30 minutes.

The solution was cooled to -101 and triethylamine (0.61 mi) followed by water (6.7 mi) was added with vigorous stirring such that the water did not freeze yet the temperature did not exceed 00.

The two phase mixture was stirred for 3 minutes and transferred to a tap funnel. The lower phase was added to a vigorously stirred suspension of the product of Preparation 10 a) (0.76 9) in KW dimethylacetamide (10 mi) and acetonitrile (10 mi) containing triethylamine (1.4 mi), which had been 30 precooled to -200 and the addition was made such that the temperature did not exceed -101. The mixture was stirred for 45 minutes at -5 to -100 and was then allowed to warm to 210 over one hour.

Methanol (0.3 mi) was added and the methylene chloride was evaporated at reduced pressure with a bath temperature of 301. The residue was carefully partitioned between ethyl acetate (30 mi) and water (30 mi) and a little sodium chloride added. The organic layer was washed with further water (2 x 30 mi). The combined washings and further added sodium chloride were extracted with ethyl acetate (20 mi) and the combined organic layers were dried with magnesium sulphate. Evaporation gave a foam (1.79 g) and this was triturated with diisopropyl ether to give a solid (1.35 g).

Most of this solid (1.2 g) was dissolved in formic acid (5 m!) and concentrated hydrochloric acid (0.38 mO was added with vigorous stirring. After one hour at 210, the suspension was filtered and the 40 residue was leached with a little formic acid. The combined filtrates were concentrated by evaporation and the residue was triturated with acetone to give the title compound (374 mg) [a], + 8.61 (c 1.02, H,O) (pH 6 buffer) 255 rim (E '%,,, 289), Ainfl. 295 (E'l%cn 273), Ainfi. 280 (E 1% 158).

1 c 1 mn EXAMPLE 6 a) (6R,7 M-7-[(Z)-2-(2-tri phenyl methyl a minothi azol-4-yl)2-0 -t- butoxycarbonylcyclobut-1 - oxyimino)acetamidol-3-(1 -pyridiniummethyi)ceph-3-em-4-carboxylate Phosphorus pentachloride (1.38 9) was dissolved in 60 mi of dichloromethane. The solution was cooled to -101 and the product of Preparation 6 (3.48 g) was added in one charge. The solution was stirred at -50 for 30 minutes. Triethylamine (1.8 mi) was added, followed by water (20 mi). The mixture was stirred at OIC for 3 minutes. The lower phase was then added to a pre- cooled mixture of the product of Preparation 10 (2.18 g) in di methyl acetam ide (30 mi) and acetonitrile (30 mi) with triethylamine (4.2 mi) added at - 1 OIC.

The reaction mixture was stirred for 45 minutes between -50C and -1 OIC. Cooling was then removed and the reaction was stirred for a further hour, ambient temperature being attained during this time. The solvent was removed under reduced pressure and the residue partitioned between ethyl acetate and water. The organic phase was washed with brine and the combined aqueous extracts extracted with ethyl acetate. The combined ethyl acetate extracts were dried in the presence of charcoal and the solvent was removed under reduced pressure. The residue was triturated with isopropyl ether to give the title compound (3.80 g).

vmax (Nujol) 1780 em (Mactam) -c(CDCI,) values include 2.74 (s, tri phenyl methyl) 8.66 (s, t-butyl).

%I GB 2 058 791 A 15 b) (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yi)-2-(1-carboxycyclobut-loxyimino)aceta midol-3-(1- pyridiniummethyi)ceph-3-em-4-carboxylic acid dihydrochloride.

The product from Stage a), (2.57 g) was stirred at ambient temperature in a mixture of 98% formic acid (15 mi), and concentrated hydrochloric acid (0.9 m]) for one hour. The mixture was then filtered and the solvent removed under reduced pressure. The resulting residue was triturated with acetone to 5 produce the title compound (1.7 9 g).

vmax (Nujol) 1785 cm (P-Iactam) Tvalues (D20 + NaHC03) include 1.05, 1.42, 1.91 (m, pyridinium protons), 3.01 (s, aminothiazole proton) 4.13 (d, J 5Hz, C, proton), 4.68 (cl, J 5Hz, C-6 proton) 7.4-8.4 (broad m, cyclobutyl protons) 10 Dimethylacetamide (l/3 mole) and acetone (-L mole) by n.m.r. 2 Water content 7.4% (Karl Fischer method) Chlorine, found 9.2% (C231124N607S2C12 + 1/3 mole dimethylacetamide + -1 mole acetone + 7.4% water requires Cl, 9.5%).

PHARMACY EXAMPLES 15 EXAMPLE A - Dry Powder for Injection Fr)rmula Per Vial (6R,7R)-7-[(Z)-2-(2-Aminothiazol-4-yi)-2-(1 carboxycyclobut-l -oxyimino)acetamidol-3-(1 - pyridini u m methyl)ceph-3-e m-4-ca rboxyl ate 500 mg sodium carbonate, anhydrous 47 mg 20 Method The cephalosporin antibiotic was blended with sodium carbonate and filled into a glass vial. The vial headspace was purged with nitrogen and a combination seal applied by crimping. The product was dissolved, as for administration, the addition of 2 mi Water for Injections.

EXAMPLE B -Injection for Veterinary Use Formula (6R,7R)-7-[(Z)-2-(2-Aminothiazol-4-yi)-2-(1 carboxycyclobut-l oxyimino)acetamidol-3-(1 - pyridini u m methyl) ceph-3-e m-4-ca rboxylate 10% W/V 30 Aluminium distearate 2% w/v - Ethyl Oleate to 100% w/v to loo% W/V Method Disperse the aluminum distearate in ethyl oleate, heat at 1 50C for one hour with stirring and cool to room temperature. Add the sterile milled antibiotic aseptically to the vehicle and refine with a high speed mixer. Fill the product aseptically into injection vials and close with rubber seals or plugs held in 35 position by aluminium overseals.

Claims (11)

1. Cephalosporin antibiotics of general formula NR /1.." _ S N \-/ C.CO.NH 11 [1 R a \ 0. c'. COOH b 9 R 0 (1) - 4 N coo (0 (wherein R' and R', which maybe the same or different, each represent a C1-4 alkyl group or Ra and Rb 40 together with the carbon atom to which they are attached form a C3-7 cycloalkylidene group; and R4 represents hydrogen or a 3- or 4-carbamoyl group, with the proviso that Ra and R b do not each represent a methyl group when R 4 represents hydrogen) and non-toxic salts and non-toxic metabolically labile esters thereof.

GB 2 058 791 A 16 group.

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

3. Compounds as claimed in claim 1 wherein Ra and R' together with the carbon atom to which they are attached form a C3-5 cycloalkylidene group.

4. Compounds as claimed in claim 1 of the general formula Nil 2 A N \-' C.CO.NR fl N e k O.C.COOH 1 b R H J4 S 0i MO (1a) wherein Ra and R b have the above defined meanings, and their non-toxic salts.

5. (6R,7R)-7-[(Z)-2-(2-Aminothlazol-4-yi)-2-(1 -carboxycyclobut-1 oxyimino) acetamidol3-0 pyridiniummethyi)-ceph-3-em-4-carboxylate and its non-toxic salts.

6. (6R,7R)-7-[(Z)-2-(2-Aminothiazol-4-yi)-2-(1 -carboxycycloprop-1 oxyimino)acetamidol-3-(1 - pyridiniummethyi)-ceph-3-em-4-carboxylate and its non-toxic salts.

7. (6R,7R)-7-[(Z)-2-(2-Aminothiazol-4-yi)-2-(1 -carboxycyclopent-1 yioxyimino)acetamidol-3-(1 pyridiummethyi)-ceph-3-em-4-carboxyalte and its non-toxic salts.

8. (6R,7R)-7-[(Z)-2-(2-Aminothiazol-4-yi)-2-(2-carboxyprop-2oxyimino)acetamido l-3-(4- carbamoyi-l -pyridi niu m methyl)-ceph-3-em-4-carboxyl ate and its non- toxic salts.

9. (6R,7R)-7-[(Z)-2-(2-Aminothlazol-4-yi)2-(1 -carboxycyclobut-1 oxyimino)acetamidol-3-(4carbamoyi-l-pyridiniu m methyl)-ceph-3-e m-4carboxyl ate and its non-toxic salts.

10. A process for the preparation of an antibiotic compound of general formula (1) as defined in claim 1 or a non-toxic salt or non-toxic metabolically labile ester thereof which comprises (A) acylating a compound of formula 11 H 11 N B 2 N CH N 2 R 4 000 z R (11) (wherein B is >Sor >S-Q; R 4 represents hydrogen or a 3- or 4-carbamoyl group; and the dotted line bridging the 2-, 3- and 4-positions indicates that the compound is a ceph-2-em or ceph-3-em compound), or a salt or N- silyl derivative thereof or a corresponding compound having a group of formula -COORI at the 4-position (where R' is a hydrogen atom or a carboxyl blocking group) and 25 having an associated anion AG, with an acid of formula e 7 R 1 1.1 k, S i \_1 c. COON 11 1N R a I 1 6 O.C. COOR 1 b R (wherein Ra and R' are as defined in claim 1; R' represents a carboxyl blocking group; and R' is an amino or protected amino group) or with an acylating agent corresponding thereto; or (B) reacting a 30 compound of formula (111) 17 GB 2 058 791 A 17 H Ii g C. Co. NH C14,;X (,OOR 8 it N R a 0. COOR Ba lb R OV) (wherein Ra, Rb, R7, B and the dotted line are as hereinbefore defined; R' and R may independently represent hydrogen or a carboxyl blocking group; and X is a replaceable residue of a nucleophile) or salt thereof with a pyridine compound of the formula / X\ 11 U_, R 4 (V) 5 (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, fl) reduction of a compound wherein B is >S--O to form a compound wherein B is >S, 10 iii) conversion of a carboxyl group into a non-toxic salt or non-toxic metabolically labile ester function, 10 and 1 iv) removal of any carboxyl blocking and/or N-protecting groups.

11. A pharmaceutical. composition for use in human or veterinary medicine comprising an antibiotic compound as claimed in any one of claims 1 to 9 in association with a pharmaceutical carrier 1 5 or excipient.

15, Printed for Her Majesty's Stationery Office by the Courier Press, Leamington Spa, 1981. Published by. the Patent Office. 25 Southampton Buildings, London, WC2A IlAY, from which copies may be obtained.