CA1132538A - Cephalosporin antibiotics - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
"Cehalosporin Antibiotics"
The cephalosporin antibiotic of the formula (I)

Description

~13Z538 "Cephalosporin Antibiotics"
This invention is concerned with cephalosporin compounds possessing valuable antibiotic properties.
The cephalosporin cornpounds in this specification 5 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 penlcillin-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 signifi-cant amount of research has been directed to the develop-ment 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 7~-(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 ~-lactamases produced by various gram-negative organisms.
The discovery of this class of compounds has stimulated further research in the same area in attempts to find compounds which have improved properties, for example against particular classes of organisms especially gram-negative organisms.
In our British Patent Specification No.1,49~,757, -- we describe cephalosporin antibiotics containing a 7~-acylamido group of the formula R.C.CO.NH- A (A) ~ O.(C~2) C (CH2)nC~0ll RB

(wherein R is a thienylor furyl group; R and R may vary widely and may, for example,be C1 4 alkyl groups or together with the carbon atom to which they are attached form a C3 7 cycloalkylidene 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 ~y~ and anti isomers containing at least 90% of the syn iso~er. The 3-position of the cephalosporin molecule may be unsubstituted or may contain one of a widç variety of possible substituents. These compounds have been found to have particularly good activity against gram-negative organisms~

1~3Z538 Furthermore, in our British Patent Specification No.1,522,140 we describe cephalosporin antibiotics of the formula ~4.C CO NH ~ _C~I2~ ~ R3 (B) (wherein R represents a furyl or thienyl group; R
represents a Cl-C4 alkyl group, a C3-C7 cycloalkyl group, a furylmethyl or thienylmethyl group; and R represents a hydrogen atom or a carbamoyl, carboxy, carboxymethyl, sulpho br 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-nega.tive organisms. The compounds .
also possess high stability to ~-lactamases produced by various gram-negative organisms, as t~ell 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 ~evelopments have involved variations in not only the 7~-acylamido groups in the above formulae but also the intro-duction of particular groups in the 3-position of the cephalosporin molecule. Thus, for example, in Belgian Patent Specification No.852,427~ there are described 113;~538 cephalosporin an-ibiotic 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, 5 including 2-aminoth;azol-4-yl, and the oxygen atom in the oxyimino group is attached to an aliphatic hydrocarbon group which may itself be substituted by, for example, carboxy. In such cornpounds, the substituent at the 3-position is an acyloxymethyl, hydroxyrnethyl, formyl or 10 optionally substituted heterocycllc-thiome~hyl group.

~urthermore, Belgian Patent Specification No.
836,813 describes cephalosporin compounds wherein the A group Rlin formula (A) above may be replaced by, for example,

2-aminothiazo]-4-yl, and the oxyimino group is a hydr-15 oxyimino or blocked hydroxyimino group, e.g. a methoxyimino group. In such compounds, the 3-position of the cephalosporin molecule is substituted by a methyl group which may itself be optionally substituted by any of a large number of residues of nucleophilic compounds 20 therein described, e.g. the pyridinium g~oup which may be substituted, for example by a carbamoyl group. In the above-mentioned Specification no antibiotic activity is ascribed to such compounds wllich are only mentioned as intermediates for the preparation of antibiotics described 25 in that Specification.

~1~2S38 Bel~i.an Patent Specificati.on No, 853, 545 describes cephalosporin antibiotics wherein the 7~-acylamido side chain is primarily a 2-(2-aminothiazol-4-yl)-2-(syn)-metho~yimino-acetamido group and the substituent in the 3-pOSitiOll is broadly clefined in a similar manner to that in the above~rnentioned Belgian Patent Speci.fication No.
X36, 813. Compoullcls speclfically exemp].i.fied in the Specification include compounds in.which the 3-position is substituted by a pyridiniummethyl or 4-_ carbamoylpyridiniummetllyl group.
We have now discovered that by an appropriate selection of a particular group at the 7~ position i.n combination with a pyridiniummethyl group at the 3-position, a cephalosporin compound having particularlyadvantageous activity (described in more detail below) against a wide range of commonly encountered pathogenic organisms may be obtained. -~ ' ': : .~

The present invention is con oe rned with the cephalosporin antibiotic(6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(2-car~oxyprop-2-oxyimino)acetamido] -3-(l-pyridiniummethyl)-oe ph-3-em~4-carkoxylate, the said compound having the formula:

~H2 N H H
\ / C.CO.NH , ~ S (I)

3 o ~ ~ CH2 N
O.C.COOH COO

and non-toxic salts and non-toxic metabolically labile esters thereof.
mese compounds prepared according to the invention are syn isom~ers.
me syn isomeric form is defined by the configuration of the group.

--O. C. COC~

with respect to the carboxamido group. In this Specification the syn configura-tion is denoted structurally as S N
C.CO.NH -O.C.COOH

.~

Z5:~8 It will be understood that since the compounds prepared according to the invention are geometric isomers, some admixture with the correspcnding anti iscmer may occur.
The invention also includes within its scope the preparation of sol-vates (especially the hydrates) of the compound of formLla (I). It also in-cludes within its scope the preparation of salts of esters of the ccmpound of formllla (I).
The compounds prepared according to the present invention may exist in tautomeric forms (for example in respect of the 2-amlnothiazolyl group) and it will be understood that such tautomeric forms, e.g. the 2-iminothiazolinyl form, are include~ within the scGpe of the invention. Moreover, the ccmpound of formula (I) depicted above may also exist in alternative zwitterionic forms, for example wherein the 4-carboxyl 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.
The compounds prepared according to the invention exhibit broad spec-trum antibiotic activity. Against gram-negative organisms the activity is unusually high. This high activity extends to m~ny ~-lactamase-producing gram-negative strains. The ccmpounds also possess high stability to ~-lactamases pro-duced by a range of gram-negative organisms.
Compounds prepared 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 m~mbers of the Enterobacteriaceae (e.g. strains of Escherichia coli, Klebsiella pneumaniae, SalmDnella typhimurium, Shigella sonnei, Enterobacter cloacae, Serratia maroe scens, Providen oe species, Proteus mirabilis, and especially indole-positive Proteus organisms such as Proteus vulgaris and Proteus morgan ) and strains of Haem~philus influenzae.

.~

~i~2S31!3 me antibiotic properties of the compounds prepared according to the invention compare very favourably with those of the aminoglycosides such as amikacin or gentamicin. In particular, this applies to their activity against strains of various Pseudomonas organisms which are not susceptible to the majority of existing ccmmercially 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. m e cephalosporin antibiotics of the present invention thus possess potentially great advantages over the amino-glycosides.
- Non-toxic salt derivatives which may be formed by reaction of either or both of the carboxyl groups present in the compound 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 æ ginine salts); organic base salts (e.g. procaine, phenylethylbenzyl-amine,dibenzylethylenediamine, ethanolamine, diethanolamine and N-methylglucos- -amine salts). Other non-toxic salt derivatives include acid addition salts, e.g.
formed with hydrochloric, hydrobromic, sulphuric, ~, 1~2S38 9 _ nitric, phosplloric, fornlic ancl trifluoroacetic acids. The sa]ts may also be in the ~orm of resinates formed with, for example, a polystyrene resin or cross-linked polystyrene clivinylbcnzene copolymer resin containing amino or quaternary arnino 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 sodiurn salt) of the compound of formula ~I~ may be used in therapeutic applications' because of the rapid clistribution of such salts in the body upon administration ~here, however, insoluble saJts of the compound (I) are desired in a particular application, e.g. for use in depot preparations, such salts may be fol~ned in conventional rn~lner, for example with appropriate organic amines~
These ancl other salt derivatives such as the salts ~ith toluene-p-sulphonic and methaneslllphollic acids may be employed as intermediates in the preparation and/or purification of the present compound of formula (I), for example in the processes described below.
Non-toxic metabolically labile ester derivatives which may be formed by esterification of either or both carboxyl groups in the parent compound of formula (I) include acyloxyalkyl esters e.g. lower alkanoyloxy-methyl or -ethyl esters SUCIl as acetoxy-methyl or -ethy'l or pivaloyloxylnethyl esters. In addition to the above ester derivatives, the present invention inc]udes within its scope the compound of formula (I) in the form of other physiologically acceptable equivalents, i.e. physiologically acceptable cornpounds which, like the metabolica]ly labile esters,are converted in vivo into the parent antibiotic compound of formula (I).

~L~3Z538 The canpound of formula (I), i.e. (6R,7R)-7-[(Z)-2-(2-am m othiazol-4-yl)-2-(2-carboxyprop-2-oxyimino)aoe tamido]-3-(1-pyridiniummethyl)-oe ph-3-em-4-carboxylate, together with its non-toxic salts (e.g. sodium salt) and non-toxic metabolically labile esters, possesses to an outstanding extent the general anti-biotic properties set out abov However one may emphasize its exoe llent activity against strains of Pseudomonas organisms. The ccmpound has exoe llent 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 inhibitory con oentra-tion. It is well distributed in the bodies of small rodents giving usefultherapeutic levels after subcutaneous injection. In primates the ccmpound gives high and long lasting serum levels after intram~scular injection. The ser~m half-life in primates points to the probability of comparatively long half-life in man, with the possibility of less frequent dosages being required for less serious infections. Experimental infections in mice with gram-negative bacteria were sucoe ssfully treated using the compound and, in particular, ex oe llent pro-tection was obtained against strains of Pseudomonas aeruginosa, an organism normally not susceptible to treatment with oe phalosporin antibiotics. This pro-tection was comparable with the treatment with an aminoglycoside such as amikacin. Akute toxicity tests with the compound in mi oe gave LD50 values in exoe ss of l.Og/kg. No nephrotoxicity was observed in rats at dosages of 2.0g/kg.
me compound of formula (I) may be used for treating a variety of diseases caused by pathogenic bacteria in hum2n beings and animals, such as respiratory tract infections and urinary tract infections.
According to the present invention we provide a prooe ss for the pre-p æ ation of the antibiotic compound of formula (I) as hereinbefore defined or a non-toxic salt or non-toxic metabolically labile ester thereof which ccmprises ,;.X

l~Z538 (A) acylating a compound of the formula H H
' ' B

2 9 (II) COO
[wherein B is >S or >S~O (~- or ~-) and the dotted line bridging the 2-, 3-, and 4-positions indicates that the cc~pound is a ceph-2-en or ceph-3-em ccmpound~
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-_-sulphonic acid) or an N-silyl derivative thereof, or a corresponding ccmpound 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 ester-forming aliphatic or araliphatic alcohol or an ester-forming phenol, silanol or stannanol (the said alcohol, phenol, silanol or stannanol preferably containing 1-20 carbon atcms)] and having an associated anion A0 such as a halide, e.g. chloride or bromide, or trifluoroacetate anion, with an acid of formula S N
C.CCOH
N CH3 (III) O.C.CCOR

(wherein R6 represents a carboxyl blocking group, e.g. as described for R5 and ~ , ~
, . . ..

~, 1~3Z53~

R is an amino or protected amino group) or with an acylating agent correspond-ing thereto; or (B) reacting a compound of formula S N , , B
\-/ C.CO.NH ~ j~ ~
N CH3 N ~ L CH2X ( [V) O. C. COOR 8 COOR

(wherein R7, B and the dotted line are as hereinbefore defined; R8 and R8a may independently 1~32538 represent hydrogen or a carboxyl blocking group; and X is a leaving group, e.g. an acetoxy or dichloroacetoxy group or a halogen atom such as chlorine, bromlne or iodine~ or a salt thereof, with pyridine N`~3 (V) 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 ~2-isomer into the desired ~ 3-isomer, ii) - reduction of a compound wherein B is > S-~ O to form a compound wherein B is > S, iii) conversion of a carboxyl group into a non-toxic salt or non-toxic metabolically labile ester function, and iv) removal of any carboxyl blocking and/or N-protecting groups.
In the above-described procesC (A), the starting material of formula (II) is preferably a compound 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 N-(7-aminoceph-3-em-3-ylmethyl)pyridinium-4'-carboxylate dihydrochloride on account of the high purity in whichit can be prepared.
Acylating agents which may be employed in the preparation of the compound 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.

1~3Z538 phosphorus pentachloride, thionyl chloride or oxalyl chloride. ' Acylations ernploying acid halides may be effected in aqueous and non-aqueous reaction media, conveniently at temperatures of from -50 to +50C, preferably -20 to ~30C, if desired in the presence of an acid binding agent. Sui~able 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 ac;d binding agents include tert~iary amines (e.g. triethy]alnine or dimethyla;~iline), inorganic bases (e.g. calcium carbonate or sodium bicarbonate), and oxiranes such as ]ower 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 the compound 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'-dicyclohexyl-carbodiimide or N-ethyl-N'-y-dimethylaminopropyLcarbodiimide;
a carbonyl compound such as carbonyldiimidazole; or an isoxazoliun~ salt such as N-ethyl-5 phenylisoxazolium perchlorate.
Acylation may also be effected with other amide-forming derivatives of acids of formula (III) such as, for example, an activated ester, a symmetrica] anhydride or a mixed anhydride (e.g~ formed with pivalic acid or with a haloforrnate, such as a lower alkylhaloformate).

113;~S38 ~ 15 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 fonned in situ using, for example, l-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-meIltioned amide-forming derivatives are desirably effected in an anhydrous reaction medium, e.g.
methylene chloride, tetrahydroruran, dimethylformamide or -- acetonitr;le.
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 chlor;des may conveniently be employed as their hydrochloride salts, and acid bromides as their hydrobromide salts.
The pyridine may act as a nucleophile to displace a wide variety of substituents X from the cephalosporin of formula (IV). To some extent the facility of the displacement is related to the PKa of the acid HX from which the substituent is derived. Thus atoms or groups X derived from strong acids tend, in general, to be more easily dis-placed than atoms or groups derived from weaker acids.

~13Z538 The displacement of X by the pyridine may conveniently be effected by maintaining the reactants in solution or suspension. The reaction is advantage-ously effected using from 1 to lO moles of the pyridine.
Nucleophilic displacement reactions may conveniently be carried out on those cc~,pounds of formLla (IV) wherein the substituent X is a halogen atam or an acylcxy group for example as discussed below.
Acyloxy groups Compounds of formula (IV) wherein X is an a oe toxy group are convenient starting materials for use in the nucleophilic displaoe ment reaction with the pyridine. Alternative starting materials in this class include compounds of formula (IV) in which X is the residue of a substituted acetic acid e.g. chloro-aoetic acid, dichloroa oetic acid and trifluoroaoe tic acid.
Displaoement reactions on oompounds (IV) possessing X substituents of this class, particularly in the case where X is an acetoxy group, may be facilitated by the presen oe in the reaction medium of iodide or thiocyanate ions.
Reactions of this type are described in more detail in British Patent Specifica-tions Nos. 1,132,621 and 1,171,603.
me 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 (rV) in which X represents an aoetoxy or substituted aoetoxy gro~p, it ~132S38 is generally d`esirable that the group R in formula (IV)should be a hydrogen atom and that B should represent >S.
In this case, the reaction is advantageously effected in an aqueous mediurn, preferably at a p~l of 5 to 8, particularl~ 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 to 110C, preferably 50~ to 80~C.
Halo~ens Compounds of forrnula (IV) in whicll X is a chlorine, bromine or iodine atom can alsa be conveniently used as starting materials in the nucleophilic displacement reaction with the pyridine .
-~len~ using cc~npounds of formul-a (IV) in this class, B may represent >S--~ O and R may represent a carboxyl blocking group. The reaction is conveniently effected in a non-aqueous medium which preferably comlrises one or more organic solvents, advantageou,ly of a polar nature, such as ethers, e~g. dioxan or t~trahydrofuran, esters, e.g. ethyl acetate, amides, e.g. formamide and N,N-dimethylformamide, and ketones e.g. acetone. In certain cases the pyridine itself may be the solvent. Other suitab]e organic solvents are described in more detail in British Patent Specification No~ 1,326,531. The reaction rnedium should be neither extremely acidic nor extremely basic. In the `:

- ' ~

case of reactions carried out on compounds of formula (IV) in which R and R8 are carbo;xyl blocking groups the 3-pyridiniummethyl product will be formed as the corresponding halide salt which may, i desired, be subjected to one or more ion exchange reactions to obtain a sa]t having the desired anion.
~ len using compounds of forrnula ~IV) in which X
is a halogen atom as described above, the re~ction is conveniently effected at a temperature of -10 to +50C, preferably +lO to +30C.
The reaction product may be separated from the reaction mixture, which may contain, for ~xample, unchanged cephalosporin startir-g material and other substances, by a variety of processes including recrystallisation, iono-phoresis, colurml chromatograplly and use of ionexchangers (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 ~3-derivative by, for `
example, treatment of the ~ -ester with a b~se such as pyridine or triethylamine.
A ceph-2-em reaction prod~lct may also be oxidised - to yield the corresponding ceph-3-em l-oxide, for example by reaction with a peracid, e.g. peracetic or m-chloroperbenzoic acid; the resulting sulphoxide may, if desired, subsequently be reduced as described hereinafter to yield the corresponding ceph-3-em sulphide.
I~lere a compound is obtained in which B is - 30 ~S ~ 0 this may be converted to the corresponding sulphide by, for example, reduction of the corresponding acyloxysulphonium or alkoxysulphonium salt prepared ~132S38 in situ by reaction with e.g. acetyl chloride in the case of an acetoxysulphonium salt, reduction being effected by, for example, sodium dithionite oi- by iodide ion as in a so]ution of potassium iodide in a water-miscible solvent e.g. acetic acid, acetone, tetrahydrofuran, dioxan, dimethylformamide or dimethylacetamide. The reaction rnay be effected at a temperature oE frorn -20 to +50~C.
Metabolically labile ester clerivatives of the compound of fonnula (I) may be prepared by reacting the compound of formula (I) or a salt or protected derivative thereof with an appropriate esterifying agent such as an acyloxyalkyl halide (e.g. iodide) conveniently in an inert organic solvent such as dimethylforll~rnide or acetone~ followed, where necessary, by removal of any protecting groups.
~ase salts of the compound of formula (I) may be formed by reactingthe acid of forn-ula (I) with the appropriate base. Thus, for example, sodiurn or potassium salts rnay be prepared using tlle respective 2-ethylhexanoate or hydrogen carbonate salt. Acid addition salts may be prepared by reactingthe cornpound of formula (I) or a metabolically labile ester derivative thereof with the appropriate acid.
Where the compound of formula (I) is obtained as a mixture of isomers, the syn isomer may be obtained by, for exarnple, conventional methods such as crystallisation or chromatography.
For use as starting materials for the preparation of the compound of formula (I) according to the invention, compounds of general formula (III) and acid halides and anhydrides corresponding thereto in their 11;32~38 syn iscmeric form or in the form of mixtures of the syn iscmers and the corres-ponding anti iscmers containing at least 90~ of the syn isomer are preferably used.
A~ids of formula (III) may be prepared by etherification of a compound of formula R

S N
~ C.CCOR9 (VI) N
OH
(wherein R7 is as hereinbefore defined and R9 repre ænts a carboxyl blocking group), by reaction with a compound of general form~la T.C.COOR (VII) (wherein R is as hereinbefore defined and T is halogen such as chloro, brcm~ or iodo; sulphate~ or sulphonate such as tosylate), followed by remDval of the earboxyl blocking gr3up R9. Separation of isomers may be effected either before or after such etherifieation. The etherification reaction is generally carried out in the presence of a base, e.g. potassium earbonate or sodium hydride, and is preferably oon &eted in an organic solvent, for example ~ 20 -113253~3 2]

.

dimethylsulphoxic]e, a cyclic ether such as tetrahydrofuran or dioxan, or an N,N-disubstituted amide such as dimethylformamide. Under these conditions the configuration of the o~yimino group is substantially unchanged by the ether.ification reaction. The reaction should be effected in the presence of a base i.f an acid addition salt of a compouild of formula (VI) :i.s used. Tlle base should be used in sufficient quantity to neutralise rapidly the acid in question.
Acids of general formula (III) may also be -- prepared by reacti.on of a compound of f~rnlul.a J~
s rl CO.COOR (VIII) ~herein R and R are as hereinbefore defined) with a compound of formula H2N.O.C.COOR
3 (IX) (wherein R is as defined above)~ followed by removal of the carboxyl blocking group R9, and where necessary by the separation of ~y~ and anti isomers.

~1~2S38 The acids of formula (III) may be con~erted 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) atorn in formula (IV), ceph-3-em starting cornpounds may be prepared in conveintional manner, e.g. by halogenation of a 7~3-protected amino-3-rnet11ylceph-3-em-

4-carboxylic acid ester l~-oxide, removal of the 7~-protecting group, acylation of the resulting 7~-amino compound to foL~l the desired 7~-acylamido. group, e.g. in an analogous manner to process tA) above, followed by reduction of the l~-oxide group la~er in the sequence.
This is described in British Patent No. 1,326,531. The corresponcling ceph-2-em compounds may be prepared by the method of Dutch published Patent Application No.
6,902,013 by reaction of a 3-methylceph-2-em compound with N-bromosuccinimide to yield the corresponding 3-bromomethylceph-2-em-compound.
Where X in formula (IV) is an acetoxy group, such starting materials may be prepared for exanlple by acylation of 7-aminocephalosporanic acid, e.g. in an analogous manner to process (A) above. Compounds of for~nula (IV) in which X represents o~her 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 in British Patent Specifications Nos. 1,474,519 and 1,531,212.

1~3ZS38 The starting materials of formula (II) may also be prepared in conventional manner, for example, by nucleophilic displacement of the corresponding 3-acetoxymetllyl compound with the appropriate nucleophile, e.g. as describe~ in ~ritish Patent Specific~tion No. 1,028,563.
A further method for the preparation of the starting materials of formula (II) comprises deprotecting a corresponding protected 7~-amino compound in conventional manner e.g. using PC15.
It should be appreciated thclt in some of the above transformat;ons it may be necessary to p~o~ect any ~~ sensitive groups in the molecul~ of the compound in question to avoid undesirable sicle reactiorls. ~or exampl~, c]uring any of the reaction se~uences referred to above it may be necessary to protect the Nli2 group of the aminothia~olyl 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 breakdoi~l c~ the desired compound, erg. 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 minera] acicl, e.g. hydrochloric acid or mixtures of such acids, preferably in the presence of a protic solvent such as water or~ tne case o~ a chloroacetyl gr~up, by treatment with thiourea.
Carboxyl blocking groups used in the preparation of the compound of formula (I) or in the preparation of necessary starting materials are desirably groups which niay readily be split off at a suitable stage in the eaction sequence, conveniently at tlle last stage. It may, however, be convenient in scme in-stances to employ non-toxic metabolically labile carboxyl blocking groups such as acyloxy-methyl or -ethyl groups (e.g. acetoxy-methyl or -ethyl or pivaloyloxy-methyl) and retain these in the final product to give an appropriate ester derivative of the co~pound of formula (I).
Suitable carboxyl blocking groups are well known in the art, a list of representative blocked carboxyl groups being included in British Patent No.
1,399,086. Preferred blocked carboxyl groups include aryl lower aIkoxycarbonyl groups such as _-methoxybenzyloxycarbonyl, p-nitr~benzyloxycarbonyl and diphenyl-methoxycarbonyl; lower alkoxycarbonyl groups such as _-butoxycarbonyl; and lower haloaLkoxycarbonyl gr~ups such as 2,2,2-trichloroethoxycarbonyl. Carboxyl block-ing group(s) may subsequently be removed by any of the appropriate methods dis-closed in the literature; thus, for example, acid or base catalysed hydrolysis is applicable in many cases, as are enzymically-catalysed hydrolyses.
me antibiotic compounds prepared according to the invention may be formulated for administration in any convenient way, by analogy with other anti-biotics. Such pharmaoe utical co~positions comprising an antibiotic co~pound in accordance with the invention and adapted for use in human or veterinary medicine may be presented ~or use in conventional manner with the aid of any neoe ssary ph~rmaceutical carriers or excipients.
me antibiotic compounds prepared according to the invention may be formLlated for injection and may be presented in unit dose form in ampoules, or in multi-dose containers, if necessary with an added preservative. The composi-tions 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 p~wder 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 acc~ptable. Alternatively, the base may be present in the water with which the pcwder is reconstituted. me 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 aoe tate.
me antibiotic campound may also be form~lated as suppositories, e.g.
containing conventional suppository bases such as cocoa butter or other gly-oe rides.
Cc~positions for veterinary medicine may, for example, be formulated as intramammary preparations in either long acting or quick-release bases.
The ccmpositions may contain from 0.1% upwards, e.g. 0.1-99%, of the active material, depending on the method of administration. When the composi-tions camprise dosage units, each unit will preferably cantain 50-1500 mg of the active ingredient. The dosage as employed for adult human treatment will prefer-ably range from 500 to 6000 mg per day, depending on the route and frequency of administration. For example, in adNlt human treatment 1000 to 3000 mg per day 1~3ZS38 administered intravenously or intramuscularly will normally suffice. In treat-ing Pseudamonas infections higher daily doses may be required.
m e antibiotic compounds prepared according to the invention may be administered in combination with other therapeutic agents such as antibiotics, for example penicillins or other cephalosporins.
The follcwing Exa~ples illustrate the invention All temperatures are in C. 'Petrol' means petroleum ether (b.p. 40-60).
Proton magnetic resonance (p.m.r.) spectra were determined at 100 MHz.
The integrals are in agreement with the assignments, coupling oonstants, J, are in Hz, the signs not being determined; s = singlet, d = doublet, dd = double doublet, m = multiplet, ABq = AB quartet and t = triplet.

ll~Z538 Preparation 1 Ethyl (Z)-2-(2-amanothiazol-4-yl)-2-(hydroxyimino)acetate To a stirred and ice-cooled solution of ethyl aoe toacetate (292 g) in glacial acetic acid (296 ml) was added a solution of sodium nitrite (180 g) in water (400 ml) at such a rate that the reaction temperature was maintained below lo&. Stirring and cooling were oontinued for about 30 min., when a solution of potassium chloride (160 g) in water (800 ml) was added. me resulting mixture was stirred for one hour. m e lower oily phase was separated and the aqueous phase was extracted with diethyl ether. me extract was combined with the oil, washed sucoessively with water and saturated brine, dried, and evaporated. m e residual oil, which solidified on standing, was washed with petrol and dried in vacuo over potassium hydroxide, giving ethyl (Z)-2-(hydroxyLmino)-3-oxobutyrate (309 g)-A stirred and ioe -oooled solution of ethyl (Z)-2-(hydroxyimino)-3-oxcbutyrate (150 g) in dichlorcmethane (400 ml) was treated dropwise with sulphuryl chloride (140 g). The resulting solution was kept at room temperature for 3 days, then evaporated. me residue was dissolved in diethyl ether, washed with water until the washings were almDst neutral, dried, and evaporated. The residual oil (177 g) was dissolved in ethanol (500 ml) and dimethylanLline (77 ml) and thiourea (42 g) were added with stirring. After two hours, the product was collected by filtration, washed with ethanol and dried to give the title com-E~ (73 g); m-p. 188 (decomp.).

~i~2538 Preparation 2 Ethyl (Z)-2-hydroxyimino-2-(2-tritylaminothiazol-4-yl)acetate! hydrochloride Trityl chloride (16.75 g) was added portionwise over 2 hours to a stirred and cooled (-30) solution of the product of Preparation 1 (12.91 g) in dimethylformamide (28 ml) containing triethylamine (8.4 ml). me mixture was allowed to warm to 15 over one hour, stirred for a further 2 hours and then partitioned between water (500 ml) and ethyl acetate (500 ml). m e organic phase was separated, washed with water (2 x 500 ml) and then shaken with IN HCl (500 ml). The precipitate was collected, washed suc oe ssively wi~h water tlOO ml), ethyl acetate (200 ml) and ether (200 ml) and dried in vacuo to pro-vide the title compound as a white solid (16.4 g); m.p. 184 to 186 (decomp.).
Preparation 3 Ethyl (Z)-2-(2-t-butoxycarbonylprop-2-oxyimino)-2-(2-tritylaminothiazol-4-yl)-aoe tate Potassium carbonate (34.6 g) and t-butyl 2-bromL-2-methylpropionate (24.5 gj in dimethylsulphoxide (25 ml) were added to a stirred solution under nitrogen of the product of Preparation 2 (49.4 g) Ln dimethylsulphoxide (200 ml) and the mLxture was stirred at room temperature for 6 hours. me mixture was poured into water (2 1), stirred for 10 mins., and filtered. me solid was washed with water and dissolved in ethyl aoetate (600 ml). me solution was washed sucoessively with water, 2~ hydrochloric acid, water, and saturated brine, dried, and evaporated. me residue was recrystallised from petroleum ether (b.p. 60-80) to give the title compound (34 g), m.p. 123.5 to 125.

. ,~, .. .
.~ ,., 11~2538 Preparation 4 (Z)-2-(2-t-Butoxycarbonylprop-2-oxyimino)-2-(2-tritylaminothiazol-4-yl)acetic acid me product of Preparation 3 ~2 g) was dissolved in methanol (20 ml) and 2N sodium hydroxide (3.3 ml~ was added. The mixture was refluxed for 1.5 hours and then concentrated. The residue was taken up in a mixture of water (50 ml), 2N hydrochloric acid (7 ml), and ethyl acetate (50 ml). The organic phase was separated, and the aqueous phase extracted with ethyl acetate. m e organic solutions were combined, washed sucoe ssively with water and saturated brine, dried, and evaporated. The residue was recrystallised from a mixture of carbon tetrachloride and pe~rol to give the title compound (1 g), m.p. 152 to 156 (deccmp.).
Preparation S
(6R,7R)-7-amino-3-(1-pyridiniummethyl? eph-3-en-4-c rboxylic acid dihydrochloride (a) A stirred suspension of (6R,7R)-7-(2-thienylacetamido)-3-(1-pyridinium-methyl)ceph-3-em-4-carboxylate (4.15 g) in dichloromethane (30 ml~ was treated with N,N-dimethylaniline (5.09 ml) and chlorotrimethylsilane (2.52 ml). This mixture was stirred at 30-35 for one hour and then cooled to -28 and treated with phosphorus pentachloride (4.16 g), stirred at -25 to -30 for another hour and then poured into a stirred cooled (-20) solution of butane-1,3-diol (8.1 ml) and dichloromethane (20 ml). me solution was allowed to attain 0 temperature over 30 minutes, and the precipitated solid (A) was filtered, washed 1~3ZS38 with dic~llo~methane and dried m vacuo. It was redissolved in methanol (17.5 ml), stirred and diluted with dichloromethane (87.5 ml) and the preeipi-tated solid filtered off, washed with dichloromethane and dried in vacuo to yield the title compound as a white solid (3.2 g), ~ max (pH 6 buffer) 258 nm (ElCm 318); T (D20) values inelude 0.95, 1.32 and 1.84 (pyridinium protons), 4.10 to 4.46 (ABq, J 16 Hz, 3-CH2-), 4.56 (d, J 5 Hz 7-H), 4.70 (d, J 5 Hz, 6-H), 6.14 to 6.50 (AEq, J 17 Hz, C2-H).
(b) Solid (A) prepared as in stage (a) above (8 g) was dissolved in IN
hydroehlorie acid (25 ml). Addition of isoprGpanol (95 ml) precipitated the erystalline title eompound as a dihydrate (4.95 g). ~ (D20) values inelude 1.02, 1.36 and 1.87 (pyridinium protons); 4.2 + 4.55 ~A~q, J = 14 HZ, 3-CH2-);
4.62 (d, J = 5 Hz, C7-H); 47.4 (d, J = 5 Hz, & -H); 6.19 + 6.38 (AEq, J = 18 Hz, C2-H). Water content by Karl Fiseher method : 9.4%.

l~ZS38 Example 1 a?_ - t-sutyl (6R,7R)-3-Acetoxymethyl-7-[(Z)-2-(2-t-butoxycarbonylprop-2-oxyimino)-2-(2-tritylaminothiazol-4-yl)acetamido]ceph-3-em-4-carboxylate A stirred solution of the product of Preparation 4 (572 mg) and t-butyl (6R,7R)-3-acetoxy~ethyl-7-aminoceph-3-em-4-carboxylate (328 mg) in di~ethylformamide (10 ml) was cooled to 0, and 1-hydroxybenzotriazole (150 mg) was added, followed by dicyclohexylcarbodiimide (225 mg). me mixtu~e was warmed to room temperature, stirred for 5 hours, and allowed to stand overnight.m e mixture was filtered, and the white solid washed with a little ether. The filtrate and washings were diluted with water (50 ml) 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.m e product-contain~lg 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.); [~]D ~ 8.5 (c, 1.0, DMSO).
b) - (6R,7R)-3-Acetoxymethyl-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(2-carboxy-prop-2-oxyimino)aoetamido]ceph-3-emr4-carboxylic acid _ .
Trifluoroaoe tic acid (18 ml) was added to a solution of the product of Stage a) (2.4 g) in anisole (18 ml) at 0. m e mixture was stirred at room temperature for 2 hours and concentrated. The residue was dissolved in ethyl aoetate and extracted with saturated sodium bicarbonate solution. The pH of the 1~25361 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. m e combined organic ex-tracts were washed with saturated brine, dried and evaporated. The residue was dissolved in warm 50% aqueous formic acid (20 ml) and allowed to stand for 2 hours. The mixture was diluted with water (50 ml), and filtered. 1`he filtrate was concentrated. me residue was taken up in water (50 ml), refiltered, and lyophilized to give the title compound (920 mg), ~max (pH 6 buffer) 236 nm (Elcm 250), ~inf 255 nm (Elcm 235), 296 nm (ElCm 103); [a]D + 20.0 (_ 1.0, 10 nMSO).
c) (6R,7R)-7-[(Z)-2-(2-Aminothiazol-4-yl)-2-(2-carboxyprop-2-oxyimino) acetamido]-3-(1-pyridiniummethyl)-ceph-3-em-4-carboxylate, mono-sodium salt Pyridine (2 ml) and the product of Stage b) (1.8 g) were added to a stirred solution of sodium iodide (7.12 g) in water (2.2 ml) at 80 . The solu-tion was stirred at 80 C for 1 hour, cooled, and diluted to 100 ml 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 aqueous residue was diluted to 100 ml with water, methyl 1 butyl ketone (2 drops) was added, and the solution was acidified to pH 1 with 2N hydrochloric acid. The mixture was filtered, and the solid was washed with a little water. The filtrate and wash-ings were collected and washed with ethyl acetate, and the pH adjusted to 6.0 with 2N sodium hydroxide solution. m e solution was concentrated to 50 ml and applied to a column of 500 g Amberlite XAD-2 resin, using first water and then 20% aqueous ethanol as eluting solvent. The product-containing fractions were concentrated and lyophilized to give the title ccmpound, (0.56 g)~ ~max ( 1 m 307)~ ~inf 282 nm (Elcm 159), 260 nm (El% 295);
[~]D + 24.5 (c 1.0, DMSO).
Example 2 (6R,7R)-7-[(Z)-2-(2-Aminothiazol-4-yl)-2-(2-carboxyprop-2-oxyimino)acetamidol-3-(l-pyridinium~ethyl)-ceph-3-em-4-carboxylate sodium salt (6R,7R)-7-[(Z)-2-(2-Aminothiazol-4-yl)-2-(2-carboxyprop-2-oxyimino)-aoe tamido]-3-(1-pyridiniummethyl)-oeph-3-em-4-carboxylate (2.5 g) was dissolved in water and the solution treated with sodium 2-ethylhexanoate (1.52 g) in -methanol (8 ml).
The miY.ture was added to stirred aoetone over 15 minutes, and the suspension obtained filte~ed, washed and dried to give the title compound (2.5 g); [~]D3 (c 1.0, H20)1 ~max (pH 6 phosphate), 255 (ElCm 327, ~ 18630) with ~infl at 240 (El%m 305, ~ 17,370) and 280 (ElCm 172, ~ 9,800), vm~x (Nujol), 1780 cm (~-lactam); sodium, found : 4.5%; calculated for C22H21o7N6S2 Na 4.04%.

Example ~

a) l.~e~methyl (]S, 6R,7P~)-7~ ~ 2-(2-t-butoxycarbonylprop-2-oxyimino)-2-(2-tritylaminothiazol-4-yl)-acetarnido]-3-bromomethylceph-3-cln 1-oxide-4-carboxylate.

Phosphonls pentachloride (0.75 g) was suspended with stirring in rmethylene dichloride (20 ml). The mixture was cooled to -10 and the product of Preparation 4 (2.0 g) was added. Stirring was continued at -5 to -I0 for 10 minutes.
Triethylamine (0.88 ml) in methylene dichloride (5 ml) at -10, was added, followed after 5 minutes with a suspension of diphenylmethyl (lS,6R,7R)-7-amino-3-bromomethylceph-3-em-1-oxide-4-carboxylate hydrobromide (1.67 g) in methylene dichloride (30 ml) containing triethylamine (0.42 ml), washed in ~ith methylene dichloride (5 ml). The mixture was stirred for 20 minutes at -5 to -10~ then poured into half-saturated a~UeOUS sodium bicarbonate solution (50 ml).
The organic layer was separated, washed with dilute hydro-chloric acid solution (lN, 3 x 30 ml) and brine (2 x 30 ml), and evaporated in vacuo to a foam. The foarn was taken up in ethyl acetate (ca 10 ml) and treated with di-isopropyl ether (100 ml). The precipitated solid was collected by filtration, washed Witil di-isopropyl ether and dried at 40 ;n vacno overnight to give the title colll~ound (2.1 g) ~(CDC13) values include 3.11 (s, -Cll Ph2), 3.37 (s, thiazol-

5-yl proton) 3.88 (dd, J 9Hz and 5Hz, 7-~l), 5.22 + 6.02 (ABq - 3C112), 5.49 (d, 51lz 6-H), 8.46 (s, C~e2).

-~ ~32S3~

b~ (6R,~)-7-~(7~2-(2-Arnlnothia~ 4-yl~-2-~2-carboxyprop-2-oxyimino) acetanli.(lol-3-(1-pyriclir~ rnmethvl),-ceLh-3-em-__ .
4-car~oxylaLe.

The product of Stage a) (I g) was dissolved in acetone (22 ml) and stirred at room temperature. Pyridine (0.08 ml) was added ancl the l-nixture was stirred at room temperature for 3 hours, ~ore pyridine (0.72ml) was adde~ and the mixture was allowed to stand at roorn temperature overnight~ The mix-ture was poured into stirred diethyl ether (75 ml) and the - 10 precipitated solid was collected by filtration, washed with ether and dried at 40 in vacuo~ This solid (0.8 g) was re-dissolved in acetone (22 ml) at -10. Potassium iodide (0.7 g) was added, followed by acetyl chloride (0.17 ml). The mixture was stirred at -10~ for 20 minutes and thell more potassium iodide (0.7 g) and acetyl chloride (0.17 ml) were added. After stirring for a Lurther 20 minutes at -10 the mixture was added to a solution of sodium metabisulphite (0.6 g) in water (60 ml~ and saturated brine.(30 ml), The product was extracted with methylene dichloride (2 x 50 ml) and the extracts were washed with brine, dried over magnesium sulphate and evapora-ted under reduced pressure to a foam. ~lis was dissolved in formic acicl (6.5 ml) and allowed to stancl at room temperature - for 15 minutes. Concen~ ted hydro-chloric acicl (0.25 ml) was added and the mi~ture was allowed to stand ror a further ].25 hour. The solkl precipitate was filtercd ancl washecl with a small qllantity Or formic acid.
The combillecl ~iltrate and wash were po~lred into ethyl acctate (5 ml) and diethyl ether (5 rnl) witll water (10 ml) and acetonitrile (5 ml). More water was added until two distinct ~32538 36 ~

layers ~ere obtained. The lower layer was run off and extrac-',` ted with cli,ethyl ether (14 ml) containing Arnberllte LA2 (7 ml)ancl acetic acid (0.7 ml). The aqueous layer was again sepa-rated and applied to a co],umn of "Zerol.i.t" 225 SRC 15 (H
form 1,5 m].). Tlle col.urnn was washed with water uMtil neutral.
The product was eluted with a 10% solution of pyridine in water. The eluate was evaporated in vacuo to small bulk and treated wi.th ace~one, The mixture was cooled to 0 to 40' overnight, and filterecl. The so].id was washed witll acetone and dried at 40- in vacuo to give the title cornpound (0.25 g).
Tlle nmr spectrum resembled that o~ t.,he compoullcl prepared in -- Exampl.e 2.
max (p~16 phospl~ate) 255.5nm (ElC~rn 374), ~inL. at 23 (ElCm 340) and 290 nm (E]Cm 160).

Examp].e 4 a~ (6R,7R~-7-r(Z)-2-(2-Triphen~lmethylaminot}li.azol.-4-yl)-2-(2-t-butoxycarbonylprop-2-ox~itrlino)acet;alrlido 1-3-(1-Pyricli-niummethyl)-ceph-3-em-4-carboxy].ate The procluct of Preparation 4 (3.44 g) was aclcled to a stirred solution of phosphorus pentachlori.de (I.38 g) in methylene chloride (60 ml), cooled to -I.0-. The resul.ting solution was stirred at -5 for 30 minutes, and then cooled to -10-. Triethylamine (1033 g) was aclded, followed by water (20 ml.). The mixture was stirred ~or 3 minutes at 0-, when the lower phase was added over l.0 minutes to a stirred suspension of t,he product of Preparation 5 (a) (2.19 g), in a mixture of N,N-di.methylacetarnide (30 ml)/acetonitrile (30 ml) conLaining triethylamine (3~3 g), cooled to -lO~o ~6 Z5~

The mixture was stirred fo~ 45 minutes at -10' ~o -5-, followed byl hollr without cooling. Methanol (1 ml) was added. Methylene chloride was removed by e~aporation under reduced pressure. The residual solution was added to water (300 ml) witll s~irring to precipitat~ the ti~]e compound (4.89 ~).
T(CDC13) values inclu(le 2.78 (s, - [C6115~3); 3.37 (s -thjazole proton); 0.35, 1.80, 2.12 (pyridilliurn protons);
4.18 (In~ - 7-11); 4.95 (6-H); 8.66 (s -t-butyl); 8 50 (s, -10 C(CH3)2) b) (6R 7R)-7-r(Z)-2-(2-Aminothiazol-4-yl)-2-(2-car~oxyprop-2-oxyimi!lo)acetamiclol-3-(1-pyridiniu~ netll~l)ceph-3-em-4-car~ox~lic acidcli]lyc]rochloride The procluct from Stage (a) (3.38 g) ~ias dissolved in 98% formic acid (20 ml) with stirring~ Concentrated hydroch-loric aci~l (].2 ml) was aclded, and the rnixture was stirred for 1 houl-. The precipitated so]id was -emove(l by vacuum ~i]tration. Solvent was removed from the filtrate by eva-poration under reduced pressure to leave an oil which was triturated with acetone (30 ml) to give tlle title compound (2.20 g).
~(D20/NaHC03) values include 3.08 (s, -thiazole proton);
l~OG, 1.44, 1.93 (pyridinium protons); 4~16 (d, H 5Hz, 7-1-1);
4,74 (d,J5Hz, ~-H); 8.55 (s,-C(CH3)2).
Acetone by n.m.r., l mole.
Water content, 5% (Karl Fischer rnet]lod).
chlOrine, fou~ 1o.l%.(c22H24N6o7s2cl2 + clcetone (1 mole) +
water (5%) reguires Cl, 10.0%).

~7 Z~3E~

~ 3~ ~
~`xalllple 5 a) ~ 7R~-7-~(%)-2-(2-Tripheny]methylarninothiazo]--4-yl)-2-(2-t-butoxycarbonylprop-2-oxyimino)acetamidol-3-(1-pyridinium-methyl)-ceph-3-em-4-cclrboxylate I`lle procluct rrom ~reparation 5b) (2,l8 g), was reacted as in Example 4 (a) I-o give the t;tle compound (4.()3 g), ~hose spectroscopic properties resembled those of tlle product of ~xample 4a) b) (~R,71~)-7-~(Z)-2-(2-Aillinotl~ia7Ol-4-yl)-2-(2-carboxy-prop-2-oxyimillo)acetamido~-3-(l-pyridini~lmmeLhy])cepll-3-ern 4-carboxyLic acid dillydrochloride .
~-~- The product ~rom Stage (a) (3.8 g) was treated as in Example4 (~) to give the title compound (2.17 g) whose spectroscopic properties resemblecl those of the product of Examp~e 4b).

~ .
~a) ~5l~,7~ -Aceto,~yme~ vl-7-[(Y.)- -(2-~minotl~;a~ol-4-yl~-2-_ . . _ . _ _ _ _ _ _ _ _ _ _ _ _ .
(2-carboxyprol)-2-oxyirnino)-acetalrlidol-cepll-3-eln-4 carboxylic Acicl llydl-ocll]oride The prod~lct of Examplel(a) (200 g) was clissolved in formic acid (800 ml) pre-cooled to +10 and concentrated hydrochloric acid (60 n-l]) was added over 5 minutes to the stirred mixture. Stirring was continued at 2(~ to 22 for 14 hours before cooling to +10 and filtering, The bed was washed with formic acid (30 ml).
The combined filtrate and wash were concentrated by evaporation at 20 to a yellow foam which was triturated with ethyl aceta~e (800 ml). The solid which deposited was collecLed by fil.tration, washed with ethyl acetate (200 ml~ ancl dried in vacuo at room terrlperature overnight to give the title compound (124.6 g) ~m~x (ethanol) 234.5 nm, El 311.
(b~ (6R,7R)-7-[(Z)-2-(2-Aminothiazol-4-y])-2-(2-carboxYprop-2 oxyimino)acetamido]-3-(pyridinillm-1-ylmethyl)ceph-3-em-4-carboxylate l-lydrate l`he proclllct from Stage a) (40 g) was added to a stirred mixture of w~ter (40 ml) and pyri.dine (25.6 rnl) followed by sodium iodide (160 g) and the mixture heated at 60 for 3% hours. The hot solution was poured into .
stirred acetone (2 1) and diluted ~itll diettlyl ether (1.2 1). The suspension was cooled to 2 and the crude product callected ~y filtration (50.65 g). This was dissolved in water (480 ml) and stirred wit.ll formic acid (19.3 ml), '~nberlite LA2' (280 ml) in ether (560 ml).
The mixture was separated and the organic layer washed twice with w~tcr (240 ml each-). The aqueous layers were washed with ether (280 ml) and applied to a column of 'Zerolit 225, SRC 15' (200 ml ~l ) followed ~y distilled water until the eluate was neutral. The column was eluted witll 1.0% pyr:i.cl;.ne in water and the eluate passed through a column of neutral alumina (40 g). The eluate was evaporated to a syrup under reduced pressure and the - 25 syrup added dropwi.se to stirred acetone (500 ml). The title compound (13.09 g) was obtained by filtration and e4uilibrati.on in air. Il20, 7.0% (~rl Eischer); ~
255 nm ~Elc 364) ~in~l 243 and 285 nm (ElCm 338 and 171), [a]2-3 (pll 6 phosphate buffer).

~ o ~3Z538 EXample 7 (a) (6R,7R)-7-[(Z)-2-(2-Tritylaminothiazol-4-yl)-2-(2-t-butoxycarbonyl-prop-2-oxyimino)acetamido]-3~ pyridiniummethyl)ceph-3-em-4-carboxylate N,N-dimethylformamide Solvate Finely powdered product of Example 4(a) was added to stirred N,N-dimethylformamide (15 ml) at 23. The solid dissolved and shortly thereafter crystallisation occurred. The stirred mixture was diluted by dropwise addition of diisopropyl ether (20 ml). The solid was collected by filtration to give the title compound (3.06g) as colourless needles.
N,N-dimethylformamide by nmr = 2~ moles.
(DMS0-d6) : 2.4-3.0 (m, trityl); 3.32 (s, aminothiazole ring proton); 0.47, 1.38, 1.82 (pyridinium protons); 4.34 (m, C-7 proton); 4.92 (d, J-5, C-6 proton);
8.64 ~s, t-butyl protons); 8.62 (s, CH3)2-C ~), [a]D = -27.5 ~C = 1.1 in methanol).
(b) (6R,7R)-7-[(Z)-2-~2-Aminothiazol-4-yl)-2-(2-carboxyprop-2-oxyimino)-acetamido]-3-(1-pyridiniummethyl)ceph-3-em-4-carboxylic Acid Dihydrochloride The product from Stage a) (2.1 g) was dissolved in formic acid (10 ml) at 22. Concentrated hydrochloric acid (0.8 ml) was added and after 75 minutes, the precipitated solid was filtered off. The filtrate was evaporated and industrial methylated spirits (10 ml) was added. The solution was re-evaporated. The residue was dissolved in methanol and the solution added to diisopropyl ether, giving the title compound, (1.35 g) [a3D - 14.7 (c = 0.95 in pH 6 buffer) T (DMS0-d6): 0.28 (d, J 9, -C-NH), 0.77 (d, J 6), 1.25 (t, J 6), 1.70 (t, J 6, pyridinium ring protons); 3.0 (s, aminothiazole ~ -40-protons), 3.99 (d d, J 9.5, 7-H); 4.67 (d, J 5, 6-H); 8.42 (s, -(CH3)2).
PHARMACY EXAMPLES
. ... _ Example A - Dry Powder for Injection Formula Per Vial (6R,7R)-7-[(Z)-2-(2-Aminothiazol-4-yl)-2-(2-carboxyprop-2-oxyimino) acetamido]-3-(1-pyridiniummethyl)ceph-3-em-4-carboxylate. 500mg Lysine Acetate 189mg Method - The cephalosporin antibiotic was blended with lysine acetate 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, by the addition of 2ml Water for Injections.
Example B - Dry Powder for Injection Fill sterile (6R r 7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(2-carboxyprop-2-oxyimino)acetamido]-3-(1-pyridiniummethyl)-ceph-3-em-4-carboxylate, monosodium salt into glass vials such that each vial contains an amount equivalent to l.Og of the antibiotic acid. Carry out the filling aseptically under a blanket of sterile nitrogen.
Close the vials using rubber disks or plugs, held in position by aluminium overseals, thereby preventing gaseous exchange or ingress of micro-organisms. Reconstitute the product by dissolving in Water for Injections or other suitable sterile vehicle shortly before administration.
Example C - Injection Twin-Pack (a) Fill 500mg quantities of sterile (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(2-carboxyprop-2-oxyimino)acetamido]-3-(1-~ ~ -41-113~:538 pridiniummethyl)ceph-3-em-4-carboxylate aseptically into glass vials under a blanket of sterile nitrogen. Close the vials using rubber disks or plugs, held in position by aluminium overseals, thereby preventing gaseous exchange or ingress of microorganisms.
(b) Prepare a 3.84~ w/v solution of sodium bicarbonate, clarify by filtration and fill 2.15ml into clean ampoules. Pass carbon dioxide into the contents of each ampoule for one minute before sealing. Sterilise the ampoules by autoclaving and check for clarity.
(c) Reconstitute the cephalosporin antibiotic shortly before administration by dissolving in 2.Oml of the sodium bicarbonate solution.
Example D - Dry Powder for Injection Formula Per Vial (6R,7R)-7-[(Z)-2-(2-Aminothiazol-4-yl)-2-¦2-carboxyprop-2-oxyimino) acetamido]-3-(1-pyridiniummethyl)ceph-3-em-4-carboxylate 500mg sodium carbonate, anhydrous 48.5mg 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, by the addition of 2ml Water for Injections.

.~

Claims (4)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process for the preparation of the cephalosporin antibiotics (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(2-carboxyprop-2-oxyimino)-acetamido]-3-(1-pyridiniummethyl)ceph-3-em-4-carboxylate and non-toxic salts and non-toxic metabolically labile esters thereof, characterised in that (A) a compound of formula (II) (wherein B is >S or >S?O and the dotted line bridging the 2-,3- and 4-positions indicates that the compound is a ceph-2-em or ceph-3-em compound), or a salt or N-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) and having an associated anion A?, is acylated with an acid of formula (III) (wherein R6 represents a carboxyl blocking group; and R7 is an amino or protected amino group) or with an acylating agent corresponding thereto; or (s) a compound of formula (IV) (wherein R7, B and the dotted line are as hereinbefore defined; R8 and R8a may independently represent hydrogen or a carboxyl blocking group; and X is a leaving group) or salt thereof is reacted with pyridine of the formula (V) 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 .DELTA.2-isomer into the desired .DELTA.3-isomer, ii) reduction of a compound wherein B is >S?O to form a compund wherein B is >S, iii) conversion of a carboxyl group into a non-toxic salt or non-toxic metabolically labile ester function, and iv) removal of any carboxyl blocking and/or N-protecting groups.

2. A process as claimed in claim 1 characterised in that a compound of formula (II) is acylated with an acid halide corresponding to the acid of formula (III).

3. A process as claimed in claim 1 characterised in that a compound of formula (IV) wherein X is an acetoxy group or bromine atom is employed.

4. A cephalosporin antibiotic selected from (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(2-carboxyprop-2-oxyimino)-acetamido]-3-(1-pyridinium-methyl)ceph-3-em-4-carboxylate and the non-toxic salts and non-toxic metabolically labile esters thereof, when prepared by the process of claim 1, 2 or 3 or by an obvious chemical equivalent thereof.