CA1127633A - Cephalosporin antibiotics - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

Cephalosporin antibiotics of the general formula (I) (wherein Ra and Rb, which may be the same or different, each represent a C1-4 alkyl group or Ra and Rb 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 Rb do not each represent a methyl group when R4 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

~l'Z7633 This invention is concerned with cephalosporin compounds possessing valuable antibiotic properties.
The cephalosporin compounds in this specification are named with reference to "cepham" after J.Amer.Chem.
Soc., 1962, 84, 3400, the term "cephem" referring to the basic cepham structure with one double bond.
Cephalosporin antibiotics are widely used in the treatment of diseases caused by pathogenic bacteria in human beings and animals, and are especially useful in the treatment of diseases caused by bacteria which are resistant to other antibiotics such as penicillin compounds, and in the treatment of penicillin-sensitive patients. In many instances it is desirable to employ a cephalospori antibiotic which exhibits activity against both gram-- 15 positive and ~sram-neg~tive 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~ -etherified oximino)-acylamido group, the oximino group having the syn configuration. This class of antibiotic compounds il27633 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 Br~tish Patent Specification No.1,496,757, we describe cephalosporin antibiotics containing a 7~-acylamido group of the formula R.C.CO.N~- RA (A) N
~ O.(CH2)m C (CH2)nCOOH
RB

(wherein R is a thienylor furyl group; RA and ~ may vary widely and may, for example,be Cl 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 l such that the sum of m and n is 0 or 1), the compounds being sYn isomers or mixtures of sYn and anti isomers containing at least 90% of the syn isomer. The 3-position of the cephalosporin molecule may be unsubstituted or may contain one of a wide variety of possible substituents. These compounds have been found to have particularly good activity against gram-negative organismsO

~27~;33 Furthermore, in our British Patent Specification No.1,522,140 we describe cephalosporin antibiotics of the formula H H
~l.C CO ~ CH2 ~ R3 (R) (wherein R represents a furyl or thienyl group; R2 represents a Cl-C4 alkyl group, a C3-C7 cyc]oalkyl group, a furylmethyl or thienylmethyl group; and R represents a hydrogen atom or a carbamoyl, carboxy, carboxymethyl, sulpho or methyl group)~ the compounds being syn isomers or existing as mixtures of svn and anti isomers containing at least 90% of the svn isomer. These compounds exhibit high antibacterial activity against a broad range of gram-positive and gram-negative organisms. The compounds .
also possess high stability to ~-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 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 ~27633 cephalosporin antibiotic compounds falling within the general scope of our British Patent Specification No.
1,399,086, and wherein the group R in formula (A) above may be replaced by a variety of different organic groups, including 2-aminothiazol-4-yl, and the oxygen atom in the oxyimino group is attached to an aliphatic hydrocarbon group which may itself be substituted by, for example, carboxy. In such compounds, the substituent at the 3-position is an acyloxymethyl, hydroxymethyl, formyl 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, B 2-aminothiazol-4-yl, and the oxyimino group is a ~
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 ltself be optionally substituted by any of a large number oE 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 mentioned as intermediates for the preparation of antibiotics described in that Specification.

~27633 Belgian Patent Specification No. 853,545 describes cephalosporin antibiotics wherein the 7~-acylamido side chain is primarily a 2-(2-amlnothiazol-4-yl)-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 include compounds in which the 3-position is substituted by a pyridiniummethyl or 4-carbamoylpyridiniummethyl group.

We have now discovered that by an appropriate selection of a small number of particular groups at the 7~-position in combination with either a pyridiniummethyl or a 3- or 4-carbamoylpyridiniummethyl group at the 3-position, cephalosporin compounds h~ving particularly advantageous activity (described in more detail below) against a wide range of commonly encountered pathogenic organisms may be obtained.

The present invention is concerned with cephalosporin antibiotics of the general formula:

S ~ H H
.CO.NH _ j ~ ~
Rb ~ -CH2 ~ R4 (I) (wherein Ra and Rb, which may be the same or different each represent a Cl 4 alkyl group (preferably a 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 Rb together with the carbon atom to which they are attached form a C3 7 cycloalkylidene group, preferably a C3 5 cycloalkylidene group; and R represents hydrogen or a 3- or 4-carbamoyl group with the proviso that Ra and Rb do not each represent a methyl group when R4 represents hydrogen) and non-toxic salts and non-toxic metabolically labile esters thereof.
The compounds prepared accordiny to the invention are syn isomers. The syn isomeric form is defined by the configuration of the group ,Ra O.C COOH

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

llZ'7633 C.CO.N~

N \ ~Ra O.C.COOH
Rb It will be understood that since the compounds prepared according to the invention are yeometric isomers, some admixture with the corresponding anti isomer may occur.
The invention also includes within its scope the preparation of solvates (especially the hydrates) of the compounds of formula (I). It also includes withLn its scope the preparation of salts of esters of compounds of formula (I).
The compounds prepared according to the present invention may exist in tautomeric forms (for example in respect of the 2-aminothiazolyl group) and it will be understood that such tautomeric forms, e.g. the 2-iminothiazolinyl form, are included within the scope of the invention. Moreover, the compounds of formula (I) depicted above may also exist in alternative zwitterionic forms, for example wherein the 4-carboxyl gxoup is protonated and the carboxyl group in the 7-side chain is deprotonated, which alter-native forms are included within the scope of the present invention.
It will also be appreciated that when Ra and Rb in the above formula represent different Cl 4 alkyl groups, the carbon atom to which they are attached will comprise a centre of asymmetry.
~`

l~Z7633 Such compounds are diastereoisomeric and the present invention em-braces individual diastereoisomers of these compounds as well as mixtures thereof.
The compounds prepared according to the invention exhibit broad spectrum antibiotic activity. Against gram-ne~ative organisms the activity is unusually high. This high activity extends to many ~-lactamase-producing gram-negative strains. The compounds also possess high stability to ~-lactamases produced by a range of gram-negative organisms.
Compounds prepared according to the invention have been found to exhibit unusually high activity against strains of Pseudomo-nas organismR e.g. strains of Pscudomonas aeruginosa as well as high activity against various members of the Enterobacteriaceae (e.g.
strains of Eseheriehia eoli, Klebsiella pneumoniae, Salmonella . ~
typhimurium, Shigella sonnei, Enterobaeter eloacae, Serratia mar-_eseens, Providenee species, Proteus mirabilis, and especially indol-epositive Proteus organisms sueh as Proteus vulgaris and Proteus morganii) and strains of Haemophilus influenzae.
The antibiotie properties of the eompounds prepared aceor-ding to the invention eompare very favourably with those of the aminoglyeosides sueh as amikaein or gentamiein. In particular, this applies to their activity against strains of various Pseudomonas organisms whieh are not suseeptible to the majority of existing eommereially available antibiotie compounds. Unlike the aminogly-eosides, eephalosporin antibioties normally exhibit low toxieity in man. The use of aminoglyeosides in human therapy tends to be limited or eomplicated by the high toxieity of these antibiotics. The eephalosporin antibiotics of the present invention thus possess potentiallv great advantages over the aminoglycosides.
._~ 8 ~12~633 g NDn-tDxic salt derivati~s which may be formed by reaction of either or both of the carboxyl groups present in the compounds of general for~ula (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 a~d arginine salts); organic base salts (e.g. p~ocaine, phenylethylb~v~yla~n~ dibenzylethylenediamine, ethanolamine, diethanolamine and N-methylglucosamine salts).
Other non-toxic salt derivatives include acid addition sal~8, e.g formed with hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, formic and trifluoroacetic acids. The salts may also be in the form of resinates formed with, for example, a polystyrene resin or cross~linked polystyrene divinylbenzene copolymer resin containing amino or quaternary amino groups or sulphonic acid groups, or with a resin containing carboxyl groups, e g a polyacrylic acid resin.
Soluble base salts (e.g. alkali metal salts such as the sodium salt) of compounds of formula (I) may be used in therapeutic applications because of the rapid distribution of such salts in the body upon administration Where, however, insoluble salts of compounds (I) are desired in a particular application, e.g. for use in depot preparations, such salts may be fo D ed in conventional manner, for example with appropriate organic aminesO
These and other salt derivatives such as the salts with toluene-p-sulphonic and methanesulphonic acids may be employed as intermediates in the preparation and/or purification of the present compounds of formula (I), for example in the prQcesses 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 such as acetoxy-methyl or -ethyl or pivaloyloxymethyl esters. In addition to the above ester derivatives, the present invention includes within its scope compounds of formula (I) in the form of other physiologically acceptable equivalents, i.e. physiologically acceptable compounds which, like the metabolically labile esters, are converted ln vivo into the parent antibiotic compound of formula (I).
A preferred group of compouncls which may be prepared according to the invention by virtue of their high antibiotic activity are those compounds of formula (I) above whereln R4 rep-resents hydrogen, i.e. compounds of the general formula:-~ H H
S N

O ~ ~ 2 ~ (Ia) O.C.COOH
]~bcooe wherein Ra and Rb have the above deEined meanings, and their non-toxic salts and non-toxic metabolically labile esters.
An outstanding compound of formula (Ia) is (6R,7R)-ilZ7633 7- r(Z)-2-(2-aminothiazol-4-yl)-2-(1-carboxycyclobut-1-oxyimino)-acetamido~-3-(1-pyridiniummethyl)-ceph-3-em-4-carboxylate which has the formula S ~ H, H (Ib) \~Lc. co UH CH2N~

together with its non-toxic salts (e.g. sodium salt) and non-toxic metabolically labile esters. The compound of formula tIb) possesses to an outstanding extent the general antibiotic properties set out above for the compounds of general formula (I). However, one may emphasize its excellent activity 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 inhibitory concentration. It is well distributed in the bodies of small rodents giviny useful therapeutic levels after subcutaneous injection. Experimental infections in mice with gram-negative bacteria were successfully treated using the compound, and in ~ I

- llZ7633 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.
Other examples of preferred compounds which may be prepared according to the present invention include the following compounds of formula (I) and their non-toxic salts and non-toxic metabolically labile esters, namely:-(6R,7R)-7- ~(Z)-2-(2-aminothiazol-4-yl)-2-(2-carboxyprop-2-oxyimino)acetamido~ -3-(4-carbamoyl-1-pyridiniummethyl)-ceph-3-em-4-carboxylate;
(6R,7R)-7- ~(Z)-2-(2-aminothiazol-4-yl)-2-tl-carboxycycloprop-l-oxyimino)acetamido~ -3-(1-pyridinium-methyl)-ceph-3-em-4-carboxylate;
(6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1-carboxycyclopent-1-yloxyimino)acetamido]-3-(l-pyridinium-methyl)-ceph-3-em-4-carboxylate; and (6R,7R)--7-[(Z)-2-(2-aminothiazol-4-yl)-2-(l-carboxycyclobut-1-oxy.imino)acetamido]-3-(4-carbamoyl-l-pyridiniummethyl)-ceph-3-em-4-carboxylate.
Other compounds which may be prepared according to the present invention include for example those wherein the groups Ra, Rb and R4 in formula (I) are as follows:-~1 - ~27633 _ R Rb R4 _ . .
a) Alkyl ~roups

2 5 2 5 .--C2H5 1.
-C2H5 ~ ..

3 4 -CONH2 C2H5 ..

. . ._ . . _ ... _.

~ 1 - 1 3 ~lZ7633 .. . ..... . . ._ . . ~ . , Ra C _ Rb R4 ,,_. _,,.,. ..... , , b) Cvcloalkylidene ~roups Cyclobutylidene 3-CONH2 Cyclopentylidene 3-CONH2 .. 4-CONH2 CyclohexylLdene H
" . 3-CONH2 .. 4-CONH2 ~yclopropylidene 3-CONH2 .. 4-CONH2 ~ . I .

l~Z7633 The compounds of formula (I) may be used for treating a variety of diseases caused by pathogenic bacteria in human beings and animals, such as respiratory tract infections and urinary tract infections.
According to the present invention we provide a process for the preparation of an antibiotic compound of general formula (I) E as hereinbefore defined or a non-toxic salt~3f non-toxic metabo~
ieally labile ester thereof which comprises (A) acylating a compound of the formula H H

~2N ~ ~ r ~ 4 Coo~ R

[wherein R is as defined above; B is >S or >S-~O(~- or ~-); and the dotted line bridging the 2-, 3-, and 4-positions indieates that the eompound is a ceph-2-em or eeph-3-em compound] or a salt, e.g.
an acid addition salt (formed with, for example, a mineral acid such as hydrochlorie, hydrobromie, sulphurie, nitric or phosphorie aeid or an organie acid sueh as methanesulphonic or toluene-~-sulphonic aeid) or an N-silyl derivative thereof, or a eorrespond-ing eompound having a group of formula -CooR5 at the 4-position [where R5 is a hydrogen atom or a earboxyl bloeking group, e.g.

the residue of an ester-forming aliphatic or araliphatie aleohol or an ester-forming phenol, silanol or stannanol (the said alcohol, phenol, silanol or stannanol preferably ~ ' -15-containing 1-20 carbon atoms)] and having an associated anion A~ such as a halide, e.g. chloride cr bromide, or trifluoroacetate anion, with an acid of formula S N
/- C.COOH
N Ra O.l.COOR6 (III) Rb ~wherein R~ and Rb are as herelnbefore defined; R6 represents a carboxyl blocking group, e.g. as described for R ; and R is an amino or protected amino group) or with an acylating agent corresponding thereto; or (B) reacting a compound of formula S N H H
C.CO,NH - L ~ B ~
N Ra O N ~ -CH2X
\ o. I CooR8 cooR8 R (IV) Iwherein Ra, Rb, R7, B and the dotted line are as hereinbefore defined; R8 and R8a may independently ~Z76~3 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, bromine or iodine) or a salt thereof, with a pyridine compound of the formula r ~ R4 (V) (wherein R4 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 ~2-isomer into the desired ~3-isomer, ii) reduction of a compound wherein B is > S-tO 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 process (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-e~m-3-ylmethyl)pyridinium-41-carboxylate dihydrochloride on account of the high purity in which it can be prepared.
Acylating agents which may be employed in the preparation of compounds of formula (I) include acid halides, particularly acid chlorides or bromides.
Such acylating agents may be prepared by reacting an acid (III) or a salt thereof with a halogenating agent e.g.

~lZ76 phosphoruY pentachloride~ thionyl chloride or oxalyl chloride.
Acylations employing acid halides may be effected in aqueous and non-aqueous reaction media, conveniently at temperatures of from -50 to +50C, preferably -20 to +30C, if desired in the presence of an acid binding agent. Suitable reaction media include aqueous ketones such as aqueous acetone, esters such as ethyl acetate, halogenated hydrocarbons such as methylene chloride, amides such as dimethylacetamide, nitriles such as acetonitrile, or mixtures of two or more such solvents.
Suitable acid binding agents include tertiary amines (e.g. triethylamine or dimethylaniline), inorganic bases (e.g. calcium carbonate or sodium bicarbonate), and oxiranes such as lower 1,2-alkylene oxides (e.g. ethylene oxide or propylene oxide) which bind hydrogen halide liberated in the acylation reaction.
Acids of formula (III) may themselves be used as acylating agents in the preparation of compounds of formula (I). Acylations employing acids (III) are desirably conclucted in the presence of a condenslng agent, for example a carbodiimide such as N,N'-dicyclohexyl-carbodiimide or N-ethyl-N'-y-dimethylaminopropylcarbodiimide;
a carbonyl compound such a~ carbonyldiimidazole; or an isoxazolium 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 symmetrical anhydride or a mixed anhydride (e.g. formed with pivalic acid or with a haloformate, such as a lower alkylhalofonmate).

~lZ7~j33 - lg - .

Mixed anhydrides may also be formed with phosphorus acids (for ex~mple phosphoric or phosphorous acids), sulphuric acid or aliphatic or aromatic sulphonic acids (for example toluene-p-sulphonic acid). An activated ester may conveniently ~e formed 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-mentioned amide-forming derivatives are desirably effected in an anhydrous reaction medium, e.g.
methylene chloride, tetrahydrofuran, dimethylformamide or acetonitrile.
If desired, the above acylation reactions may be carried out in the presence of a catalyst such as 4-dimethylaminopyridine.
The a~ids of formula (III) and acylating agents corresponding thereto may, if desired, be prepared and employed in the form of their acid addition salts. Thus, for example, acid chlorides may conveniently be employed as their hydrochloride salts, and acid bromides as their hydrobromide salts.
The pyridine compound of formula (V) 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 i9 related to the PKa of the acid HX from whlch 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 ~2~633 precise character of the substituent R4 in the compound of formula (V).
The displacement of X by the pyridine compound of formula (V) may conveniently be effected by maintaining the reactants in solution or suspension.
The reaction is advantageously effected using from 1 to 10 moles of the pyridine compound.
Nucleophilic displacement reactions may conveniently be carried out on those compounds of formula (IV) wherein the substituent X is a halogen atom or an acyloxy group for example as discussed below.
Acylo y ~roups 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 i8 the residue of a substituted acetic acid e.g.
chloroacetic acid, dichloroacetic acid and trifluoro-acetic acid.
Displacement reactions on compounds (IV)posséssing 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 thls type are described in more detail in British Patent Specifications Nos.
1,132,621 and l,l71,603.
The substituent X may also be derived from formic acid, a haloformic acid such as chloroformic acid, or a carbamic acid.
When using a compound of formula (IV) in which X
represents an acetoxy or substituted acetoxy group, it lZ7633 is generally desirable that the group ~8 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 medium, preferably a~ a pH of 5 ~o 8, particularly 5.5 to 7.
The above-described process employing compounds of formula (IV) in which X is the residue of a substituted acetic acid may be carried out as described in British Patent Specification No. 1,241,657.
When using compounds of formula (IV) in which X
is an acetoxy group, the reaction is conveniently effected at a temperature of 30 to 110C, preferably 50 to 80C.
Halo~ens 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)O When using compounds of formula (IV) in this class, B may represent >S-~ O and R8 may represent a carboxyl blocking group. The reactlon is con~eniently effected in a non-aqueous medium which preerably comprises one or more organic solvents, advantageously of a polar nature, such as ethers, eOg. dioxan or tetrahydrofuran, esters, e.g. ethyl acetate, amides, e.g. formamide and N,N-dimethylformamide, and ketones e.g. acetone. In certain cases the pyridine compound itself may be the solvent. Other suitable organic solvents are described in more detail in British Patent Specification No. 1,326,531. The reaction medium should be neither extremely acidic nor extremely basic. In the ~LlZ~7633 case of reactions carried out on compounds of fonm~la (IV) in which R8 and R8a sre 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 the desired anion.
When using compounds of formula (IV) in which X
is a halogen atom as described above, the reaction is convenien~ly effected at a temperature of -10~ to +50C, preferably +10 to ~30C.
The reaction product may be separated from the reaction mlxture, which may contain, for example, unchanged cephalosporin starting material and other substances, by a variety of processes including recrystallisation, iono-phoresis, column chromatography and use of ion-exchangers (for example by chromatography on ion-exchange resins) or macroreticular resins.
~ 2-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 base such as pyridine or ~iethylamine.
A ceph-2-em reaction product may also be oxidised to yield the corresponding ceph-3-em l-oxide, for example by reaction with a peracid, e.g. peracetic or m-chloroperbenzoic acid; the resulting sulphoxide may, if desired, subsequently be reduced as described hereinafter to yield the corresponding ceph-3-em sulphideO
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 ~lZ7633 in citu by reaction with e.g. acetyl chloride in the case of an acetoxysulphonium salt, reduction being effected by, for example, sodium dithionite or by iodide ion as in a solution of potassium iodide in a water-miscible solvent e.g. acetic acid, acetone, tetrahydrofuran, dioxan, dimethylformamide or dimethylacetamide. The reaction may be effected at a temperature of from -20 to ~50C.
Metabolically labile ester derivatives of the compounds of formula (I) may be prepared by reacting a compound of formula (I) or a salt or protected derivative thereof with an appropriate esterifying agent such as an acyloxyal~yl halide (e.g. iodide) conveniently in an inert organic solvent such as dimethylformamide or acetone, followed, where necessary, by removal of any protecting groups.
Base salts of the compounds of formula (I) may be formed by reacting an acid of formula (I) with the appropriate base. Thus, for example, sodium or potassium salts may be prepared using the respective 2-ethylhexanoate or hydrogen carbonate salt. Acid addition salts may be prepared by reacting a compound of formula ~I) or a metabolically labile ester derivative thereof with the appropriate acid.
Where a compound of formula (I) is obtained as a mixture of isomers, the ~y_ isomer may be obtained by, for example, conventional methods such as crystallisation or chromatography.
For use as starting materials for the preparation of compounds of general formula (I) according to the invention, compounds of general formula (III) and acid halides and anhydrides corresponding thereto in their llZ7633 svn isomeric form or in the fonm of mixtures of the ~y~
isomers ~nd the corresponding anti isomers containing at least 90% of the syn isomer are preferably used.
Acids of formula (III) (provided that Ra and Rb together with the carbon atom to which they are attached do not form a cyclopropylidene group) may be prepared by etherification of a compound of formula S N
\~ C, CoOR9 Il N
OH (VI) (wherein R7 is as hereinbefore defined and R9 represents a carboxyl bIockIng gro~apt, by reaction with a compound of general ormula Ra T.~.COOR6 Rb (VII) (wherein Ra and Rb and R6 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, eOg~
potassium carbonate or sodium hydride, and is preferably conducted in an organic solvent, for example 2~ ~3~3 dimethylsulphoxide, a cyclic ether such as tetrahydrofuran or dioxan, or 2n N,N-disubstituted amide such as dimethylfo~mamide. Under these conditions the configuration of the o~mino group is substantially unchanged by the etherification reaction. The reaction should be effected in the presence of a base if an acid addition salt of a compound of formula (VI) is used. The base should be used in sufficient quantity to neutrali~e rapidly the acid in question.
Acids of gener~l formula (III) may also be prepared by reaction of a compound of formula ~ P 9 ~ ' CO.COOR (VIII) wherein R7 and R9 are as hereinbefore defined) with a compound of formula H2N.O.C.COOR6 Rb (IX) (wherein Ra, Rb and R6 are as defined above), followed by 20 removal of the carboxyl blocking group R9, and where necessary by the separation of svn and anti isomers.
The last-mentioned reaction is particularly applicable to the preparation of acids of formula (III) wherein Ra and Rb together with the carbon atom to which 25 they are a~tached form a cyclopropylidene group. In this case, the relevant compounds of formula (IX) may be prepared in conventional manner, e.g. by means of the synthesis described in Belgian Patent Specification No.
866,422 for the preparation of t-butyl l-amino-oxycyclopropane carboxylate.
The acids of formula (III) may be convexted 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 (IV), ceph-3-em starting compounds may be prepared in conventional manner, e.g. by halogenation of a 7~-protected amino-3-methylceph-3-em-

4-carboxyllc acid ester l~-oxide, removal of the 7~-protecting group, acylation of the resulting 7~-amino compound to form the desired 7~-acylamido group, e.g. in an analogous manner to process (A) above, followed by reduction of the l~-oxide group later in the sequence.
This is described in British Patent No. 1,326,531O The corresponding ceph-2-em compounds may be prepared by the method of Dutch published Patent Application No.
6,902,013 by reaction of a 3-methylceph-2-em compound with N-bromosuccinimide to yleld the corresponding 3-bromomethylceph-2-em-compound.
Where X in formula (IV) is an acetoxy group, such starting materials may be prepared for example by acylation of 7-aminocephalosporanic acid, e.g. in an analogous manner to process (A) above~ Compounds of formula (IV) in which X represents other acyloxy groups can be prepared by acylation of the corresponding 3-hydroxymethyl compounds which may be prepared for example by hydrolysis of the appropriate 3-acetoxymethyl 1~2~633 compounds, e.g. as described in British Patent Specifications Nos. 1,474,519 and 1,531,212.
The starting materials of formula (II) may also be prepared in conventional manner, for example, by nucleophilic displacement of the corresponding 3-acetoxymethyl compound with the appropriate nucleophile, e.g.
as described in British Patent Specification no. 1,028,563.
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 that in some of the above transformations it may be necessary to protect any sensitive groups in the molecule of the compound in question to avoid undesirable side reactions. For example, during any of the reaction sequences referred to above it may be necessary to protect the NH2 group of the aminothiazolyl moiety, for example by tritylation, acylation (e.g. chloroacetylation), protonation or other conventional method.
The protecting group may thereafter be removed in any convenient way which does not cause breakdown of the desired compound, e.g. in the case of a trityl group by using an optionally halogenated carboxylic acid, e.g. acetic acid, formic acid, chloroacetic acid or trifluoroacetic acid or using a mineral acid, e.g. hydrochloric acid or mixtures o such acids, prefcrably 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 oE compounds of formula (I) or in the preparation of necessary starting materials are desirably groups which may readily be split off at a suitable stage in the reaction sequence, " -27-conveniently at the last stage. It may, however, be convenient in some instances to employ non-toxic metabolically labile carboxvl blocking groups such as acyloxy-methyl or -ethyl groups (e.g. ace-toxy-methyl or -ethyl or pivaloyloxymethyl) and retain these in the final product to give an appropriate ester derivative of a compound of formula (I).
Suitable carboxyl blocking groups are well known in the art, a list of representative blocked carboxyl groups being included in British Patent No. 1,399,086. Preferred blocked carboxyl groups include aryl lower alkoxycarbonyl groups such as _-methoxybenzyloxy-carbonyl, p-nitrobenzyloxycarbonyl and diphenylmethoxycarbonyl;
lower alkoxycarbonyl groups such as _-butoxycarbonyl; and lower haloalkoxycarbonyl groups such as 2,2,2-trichloroethoxycarbonyl.
Carboxyl blocking group(s) may subsequently be removed by any of the appropriate methods disclosed in the literature; thus, for example, acid or base catalysed hydrolysis is applicable in many cases, as are enzymically-catalysed hydrolyses.
The antibiotic compounds prepared according to the invention may be formulated for administration in any convenient way, by analogy with other antibiotics. Such pharmaceutical compositions comprising an antibiotic compound prepared in accordance with the invention and adapted for use in human or veterinary medicine may be presented for use in conventional manner with the aid of any necessary pharmaceutical carriers or excipients.
The antibiotic compounds prepared according to the inven-tion 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 llZ7633 compositions may also take such forms as suspensions, solutions, oremulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilising and/ordispersingagents.
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 formula-tion 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 inorgan_c 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 preparation~ in either long acting or quick-release bases.
The compositions may contain from 0.1~ upwards, e.g. 0.1-99%, of the active material, depending on the method of administration. When the compositions comprise dosage units, each unit will preferably contain 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 fre-quency of administration. For example, in adult human treatment '~ -29-~, - 1127~33 1000 to 3000 mg per day administered intravenously or intra-muscularly will normally suffice. In treating Pseudomonas infections higher daily doses may be required.
The 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 following Examples 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 constants, J, are in Hz, the signs not being determined; s = singlet, d = doublet, m = multiplet and ABq = AB quartet.

~ I - 30 _ 31 -Preparation 1 Ethyl (Z)-2-(2-aminothi~ y~)-2-~h~ L___ o)acetate To a stirred and ice-cooled solution of ethyl aceto~cetate (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 10C. Stirring and cooling were continued for about 30 min., when a solution of potassium chloride (160 g) in water (800 ml) was sdded. The resulting mixture was stirred for one hour. The lower oily phase was separated and the aqueous phasewas 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)-3-oxobutyrate (150 g) in dichloromethame (400 ml) was treated dropwise with sulphuryl chl~Dride (140 g). The resulting solution was kept at room temperature for 3 days, then evaporated.
The residue was dissolved in diethyl ether, washed with water until the washingg were almost neutral, dried, and evaporated. The residual oil (177 g) was dissolved in ethanol (500 ml) and dimethyl~niline (77 ml) ~nd 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 compound (73 g);
m.p.188 (decomp.), ~7633 Preparation 2 Ethyl (Z)-2-hydroxyimino-2-(2-tritvlaminoth~ DI g~yl) acetate hydrochloride, Trityl chloride (16.75 g) was added portionwise over 2 hours to a stirred and cooled (-30) solution of the product of Preparation 1 (12.91 g) in dimethylformamide (28 ml) containing triethylamine (8.4 ml). The mixture was allowed to warm to 15 over one hour, stirred for a further 2 hours and then partitioned between water (500 ml) and ethyl acetate (500 ml). The organic phase was separated, washed with water (2 x 500 ml) and then shaken with lN HCl (500 ml), The precipitate was collected, washed successively with water (100 ml), ethyl acetate (200 ml) and ether (200 ml) and dried in vacuo to provide the title compound as a white solid (16.4 g); m.p. 184 to 186 (decomp).
Preparation 3 Ethyl (Z)-2-(2-t-butoxycarbonYlprop-2-oxyimino)-2-(2-trityl-minothiazol-4-vl)acetate ~Potassium carbonate (34,6 g) and t-bu~yl 2-bromo-2-methylpropionate (24,5 g) in dimethylsulphoxide (25 ml) were added to a stirred solution under nitrogen of the product of Preparation 2 (49.4 g) in dimethylsulphoxide (200 ml) and the mixture was stirred at room temperature for 6 hours. ~he 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 ml)~ The solution was washed successively with water, 2N hydrochloric acid, water, and saturated brine, dri~d, and evaporated. The residue was recrystallised from petroleum ether (b.p~ 60-80) to give the title ~27~33 compound ~34 g), m.p. 123.5 to 125 Preparation 4 (Z)-2 (2-t-Butoxycarbonylprop-2-oxyimino~-2-(2-tritylamino-thiazol-4-yl)acetic acid The 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 hydro-chloric acid (7 ml), and ethyl acetate (50 ml). 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 156 (decomp).
Preparation 5 Eth ~ ritylaminothiazol-4-Yl)-2-(1-t-butoxycarbonyl-cyclobut-l-oxYimino) acetate.
The product of Preparation 2 (55.8 g) was stirred under nitrogen in dimethylsulphoxide (400 ml) wi-th potassi.um carbonate (finely ground, 31.2 g) at room temperature. After 30 minutes, t-butyl l-bromocyclobutane carboxylate (29.2 g) was added. After 8 hours further potassium carbonate (31.2 g) was added. More potassium carbonate (6 x 16 g portions) was added during the next three days and further t-butyl l-bromocyclobutane carboxylate (3.45 g) was added after 3 days. After 4 days in all, the mixture was poured into ice-water (ca. 3 litres) and the solid was collected by filtration and 6~3 washed well with water and petrol. The solid was dissolved in ethyl acetate and the solution washed with brine (twice), dried withmagnesium sulphate and evaporated to a foam. This foam was dissolved in ethyl acetate-petrol(1:2) and filtered through silica gel (500 g). Evaporation gave the title compound (60 g) as a foam, ~~ max (CHBr3) 3400 (NH) and 1730 cm 1 (ester).
Preparatlon 6 (Z)-2-(1-t-Butoxycarbonylcyclobut-l-oxyimino)-2-(2-trityl-aminothiazol-4-yl) acetic acid.
A mixture of the product of Preparation 5 (3.2g) and potassium carbonate (1.65 g) was refluxed in methanol (180 ml) and water (20 ml) for 9 hours and the mixture was cooled to room temperature. The mixture was concentrated and the residue partitioned between ethyl acetate and water, to which was added 2N HCl (12.2 ml). 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); ~max (ethanol) 265 nm (ElCm 243).

Preparation 7 (Z)-2-(l-t-Butoxycarbonylcycloprop-l-oxyimino)-2-(2-trl~y~
aminothiazol-4-yl)acetic acid.
A solution of hydrazine hydrate (0.20 g) in methanol (0.4 ml) was added to a solution of l-t-butoxycarbonylcyclo-~ -34-2'~63 3 prop-l-oxyphthalimide (0.61 g; prepared as described in Belgian Patent No. 866,422) in dichloromethane (7 ml). The mixture was stirred at room temperature for 1 hour, and treated with 5N aqueous ~mmonia solution (7 ml). The organic phase w&sseparated and the aqueous 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 ml) and ethyl acetate (5 ml). 2-Tritylaminothiazol-4-ylglyoxylic acid (0~73 g;
prepared as described in Belgian Patent No. 864,828) was added.
The mixture was~tirred at room temperature overnight and then filtered. The solid was washed with a little ether and dried in vacuo to give the title com~ound (0.5 g), m.pO 156~8 -157.2-; vmax (CHBr3) 2300-3500 (O-H, N-H); 1750 (t-butyl ester);
1690 cm (acid).

Prepara ion 8 E _ tritylaminothiazol ~
The product of Preparation 2 (10 g) was stirred with t-butyl 2-bromo-cyclopentanecarboxylate(7g) in dimethyl-sulphoxide( 40ml) containing potassium carbonate (lOg) under nitrogen at21- for 21 hours. The mixture was poured into ice-water (500ml) and the grey solid was collected by filtra-tion, washed with water and air driedO
Recrystallisation of this solid from methanol (500ml) ~27633 gave the title compound (11~7g), m.p. 179-180C~ v (CHBr3) 3410 (NH), 1735 (ester), 1275 (ester) and 755 cm (phenyl).

Preparation 9 (22-2-(1-t-Butoxyca ~ _ o)-2-(2-trityl-S aminothiazol-4-vl)acetic ~cid The product of PreparatiQn 8 (625mg) was refluxed with 2N sodium hydroxide solution (0.5ml) and water (lml) in methanol (12ml) for seven hours. The mixture was left to cool overnight. After dilution with water, orthophosphoric acid was added to adju~t the solution to pH 2. The precipi-tate 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 (493mg). Recrystal-lisation from di-isopropyl ether gave the title compound (356mg) m.p. 171-173-, vmax (CHBr3) 2500-3500 (OH and NH), 1755 (ester), 1692 (acid) and 755 and 770 cm (phenyl).

~0 (6 ~ -3-(l-pyridiniummethyl)ceph-3-em-4--carboxyl-ic ~ loride 20 ~ A stirred suspension of t6R~7R)-7-~2-thienylacetamido)-3-(1-pyridiniummethyl)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), stirredat -25- to -30- for another hour and then poured into a stirred cooled (-20-) solution of butane-1,3-diol (8.1 ml) and dic-loromethane (20 ml). The solution was allowed to attain 0- temperature over 30 minutes 9 and the precipitated l~Z7633 solid (A) was fil-tered, washed with dichloromethane and dried _ vacuo. It was redissolved in methanol (17.5 ml), stirred and diluted with dichloromethane (87.5 ml) and the precipitated solid filtered off, washed with dichloromethane and dried 1n vacuo to yield the title _ompound as a white solid (3.2 g), ~ max (pH 6 buffer) 258 nm (~lcm 318); ~ (D20) values include 0.95, 1.32 and 1.84 (pyridinium protons), 4.10 to 4.46 (ABq, J 16 Hz, 3-CH2-), 4056 (d, J 5 Hz 7-H), 4.70 (d, J 5 Hz, 6-EI), 6.14 to 6.50 (ABq, J 17 ~z, C2-~l).

1127~i3;~

Example 1 a) t-butyl (6R.7R)-3-Acetoxymethyl-7-r(Z)-2-(2-t-butoxy-c ~ -oxyimino~ 2-(2-trit~laminothiazol-4-yl) acetamido3 ce~h-3-e_ S 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 ml) was cooled to 0-, and l-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 mixture was filtered, and the white solid washed with a little etherO 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, The product-containing eluate was collected and concentrated to give the title compound (533 mg)0 A portion was recrystallised from di-isopropyl ether, m,pO 103 to l13-(decomp ); [~JD + 8.5 (C, 1.0, DMSO).

b)_ _( 6R,7R~-3-Acetoxym~ Lic~ 2-~2~aminothiazol~4-yl)-2-~2-carboxYprop-2-oxyimi-n-o~acetàmidplceph-3--em-4-carboxylic acid_ Trifluoroacetic acid (18 ml) was added to a solution of the product of Stage a) (2.4 g) in anisole (18 ml) at 0-The mixture was stirred at room temperature for 2 hours and concentrated, The residue was dissolved in ethyl acetate and ex racted with saturated sodium bicarbonate solution.

~12~33 Tlle pH 0~ t h~ a~ueous extracts was adjusted to 6; and the SOIutiOll WclSIlCd wi~h ethy] aceta~e The aqueous phase was aciclifie~l to pil ].5 und~r ethyl aceLate, saturated with sodiuln ch]or;d(, ancl c~racted with etlly] acetate. The com-bined orgallic exLracts were washed with saturated brine,dried and ev;lporated. The residue was dissol.ved in warm 50% aqlleous ~ormic acid (20 ml) and alloweci to stand for 2 hours. Thc Inix~ure was clil.u~ed with water (50 ml), and filtered. rhe fil.trate was concentrated. The residue was taken up ;n water (50 ml), reriltered, and lyophilized to give Lhc L i l ]( conll)o~ d (9,70 rng), A (pi~ buffer) 236 nm ~ / 250) ~ 2')5 nlrl (El/ 235), 2'~ nln (E~lcrnlo3); Ca~D
+ 20.0- (c 1.0, DMS0).

c)_ (6R,7R)-7-r(Z)-2-(2-~!n nothiazo'l-4-yl~-?-(2-carboxy ~ p-2_ oxyir_.Lno) ace~arlli(]o~-3-(4-carbclrlloy~ -pyric~ i.unurlethyl)- _ ceph-3-em-4-carl~oxylatet mor~o-sodLulrl sal.t.
IsonLcoti.namide (0.56g) was added to a.stirred solùtion of the product oE Stage b) (0.59g) :i.n waler (0.7 ml) containing su~Liciellt sod:iulrll):icilrl~ollilte to give a ~ al.
pll oE G.5. .So(lilllll io(li(le (2.:lg) was .Id(le(l an(l the m-ixture was stirred at 80~C Eor olle hour; sodi.uln 17icarbonate was added at intervals to maintain a pH in the range 5.5 - 6.5.
Ihe aqueous resklue was diluted with water, methyl 1 butyl ketone (a fcw drops) was added and the solution was 2r~ acidified to pll l with 2N hydrochloric acid. The mixture was filtered and the solid was washed with a little water. The fi].trate and washings were collected and washed with ethyl acetate, and the pll adjusted to 6.0 with 2N

l~Z7633 sodium hydroxide solution. The solution was concentrated and applied to a column of 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 compound (0 09g~ ~ max (pH 6 buffer) 257.5 nm (El% 276), ~i f 291.5 nm (ElCm 125); r(D2O) values include 0.92, 1.70 (4H; pyridinium protons); 3.10 (lH,aminothiazole-5-H);
4.34, 4.64(2H;ABq; 3-CH2-); 8.54 (6H;-CMe2-).
Example 2 a) t-Butyl (6R,7R) 3-Acetoxymeth~ [(Z) 2-(1-t-butoxy-carbonylcyclobut-l-oxy mino)-2-(2-tritylaminothiazol-4-Y])acetamido~ceph-3-em-4-carboxylate A stirred solution of the product of Preparation 6 (24.2g) and t-butyl (6R,7R)-3-acetoxymethyl-7-aminoceph-3-em-4-carboxylate (13.6g) in dimethylformamide (300ml) was cooled ~o O, treated with l-hydroxybenzotriazole monohydrate (4~5g), followed by dicyclohexylcarbodiimide (6.4g) and the product isolated substantially as described in Example l a) to give the title comPound (12.8g), m.p. 113.5 to 116.5 (decomp.); [~D
15.0 (c 1.0, DMSO).

~, 763;~

b) (6R,7R~-3-Acetox~methyl-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(l-carboxycyclobut-l-oxyimino)acetamido]ceph-3~em-4-carboxylic acid Trifluoroacetic acid (100 ml) was added to a mixture of the product of Stage a) (12.5g) and anisole (5 ml) at O. The mixture was treated substantially as described in Example 1 b) to give the title compound (4g)~ Amax (P~ 6 buffer) 246 nm cm inf295 nm (El%mllg); [~]20 + 27 c) (6R,7R)-7-[(Z)-2-(2-Aminot _ zol-4-yl)-2-(1-carb_xycyclobut-1-oxyimino)acetamido]-3-(1-pyridiniummethY1)-ceph-3-em-4-carboxylate, mono-sodium salt Pyridine (4.1 ml) and the product of Stage b) (3.75g) were added to a stirred solution of sodium iodide (14.6g) in water (4.5 ml) at 80C. The solution was stirred at 80C for one hour, cooled and diluted with water. The pH of the solution was ad~usted to 6.0 with 2N sodium hydroxide solution and this solution was concentrated to remove pyridine. The product was isolated substantially as described in Example 1 c) to give the title_compound (1.3g) ~max (~1 6 buffer) 252.5 nm (E1Cm 310), ~inf291 nm (E1C%ml39); [~]D0 + 43.5 (c l.O.DMSO).

~, -41-~127633 Example 3 ~6R,7R?-7-[(Z~-2-(2-Aminothiazol-4-yl)-2~ carboxycyclobut-l-oxyimino)acetamido]-3-(4-carbamoyl-l-pyridiniummethyl)-ceph-3-em-4-carboxylate, mono-sodium salt Isonicotinamide (1.22g) was added to a stirred solution of the product of Example 2 b) (1.08g) in water (1.3 ml) containing sufficient sodium bicarbonate to give a final p~l of 6.5. Sodium iodide (4g) was added and the mixture was stirred at 80C 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 l c) to give the title compound, (0.16g), 2 ~`max (pH 6 buffer) 256 nm (El% 298) Ainf 294 nm (Elcm 135)-Example 4 a) t-Butyl (6R,7R) 3-acetoxymethYl-7-[(Z)-2-(2-tritylaminothiazol-4-yl)-2-(l-t-butoxycarbonylcycloprop-1-oxyimino)acetamido]ceph-3-em-4-carbox~late l-Hydroxybenzotriazole monohydrate (0.12 g) and dicyclo-hexylcarbodiimide (0.16 g) were added to a stirred solution oE the product of Preparat,ion 7 (0.34 g) and t-buty] (6R,7R)-3-acetoxy-methyl-7-aminoceph--3-em-4-carboxylate (0.25 g) in tetrahydrofuran (6 ml). The mixture was stirred at room temperature overnight and then filtered. The filtrate was evaporated. The residue was dissolved in a little ethyl acetate - petroleum ether (bp 60-80) (1:1) and eluted through a column of neutral a]umina (10 g) with ' ~ -42-,i `

:~127633 the same solvent. The eluate was concentrated to a foam (0.44g) which was recrystallized from di-isopropyl ether (15 ml) to give the title compound, (0.29g), m.pt. 115 - 119; [~]D0 (c 1.0, DMSO) + 13.
b) (6R,7R)-3-Acetoxymethyl-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(l-carboxycycloprop-l-oxyimino)acetamido]ceph-3-em-4-carboxylic acid, ~y~drochlo e salt.
Concentrated hydrochloric acid (0.6 ml) was added to a stirred solution of the product from Stage a) (1.92g) in formic acid (7.5 ml) at 10. 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 (1.16g), [~]D0 (c 1.0, DMSO) + 35 ; AmaX(pH 6 buffer) 239 nm, (ElCm 300) c) (6R,7R)-7-[(Z)-2-(2-Aminothiazol-4-yl)-2-(1-carboxycycloprop-l_oxyimino)acetamido]-3-(1-pYridiniummethyl)ceph-3-em-4-carboxYlate, sodium salt.
A mixture of the product from Stage b) (0.56q), sodium bicarbonate (0.17g), and water (0.5 ml) was warmed to 50. More sodium bicarbonate (0.09g) was added, followed by pyridine (0.2 ml). The solution was warmed to 80 and sodium iodide (2g) was added. The solution was stirred at 80 for 40 minutes, cooled, and diluted with acetone (50 ml). The mixture was filtered, and the solid was washed with acetone and ether to give a solid. This solid was dissolved in water (20 ml) and acidified dropwise ~$~

~27633 with 2N hydrochloric acid until a precipitate formed which did not redissolve on standing. The mixture was stirred with neutral alum-ina (5g) and filtered through a pad of neutral alumina (lOg). The pad was eluted thoroughly with water. The aqueous eluate was con-centrated and the residue was triturated with acetone. The solid was filtered and dried to a solid (0.35g). The solid (0.30g) was dissolved in a little water and eluted through a column of 50g 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, (0.06g);r~] 23 o + 1.5 (_ 0.1, water); ~max (pII 6 buffer) 254 nm, (Æ]cm 340); ~i E 296 nm~ (El~ 125)-Example 5(6R,7R?-7- ~(Z)-2-(2-Aminothiazol-4-yl)-2-(1-carboxycyclopent-1-yl-oxyimino)-acetamido]-3-(1-pyridiniummethyl)ceph-3-em-4-carboxylate, . . ~
dihydrochloride salt.
Phosphorus pentachloride (0.46g) was dissolved in methylene chloride (20 ml) at ambient temperature and the solution was cooled to 10; the product oE Preparation 9 (:I.095 g) was aclded in one charge. I'he mixture warmed to -5 and was stirred for 30 minutes.
The solution was cooled to -10 and triethylamine (0.61 ml) followed by water (6.7 ml) was added with vigorous stirring such -that the water did not freeze yet the temperature did not exceed 0. 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 ~ (0.76 g) in N,N-~127633 dimethylacetamide (10 ml) and acetonitrile (10 ml) containing tri-ethylamine (1.4 ml), which had been precooled to -20 and the add-ition was made such that the temperature did not exceed -10. The mixture was stirred for 45 minutes at -5 to -10 and was then allowed to warm to 21 over one hour. Methanol (0.3 ml) was added and the methylene chloride was evaporated at reduced pressure with a bath temperature of 30. The residue was carefully par-titioned between ethyl acetate (30 ml) and wa-ter (30 ml) and a little sodium chloride added. The organic layer was washed with further water (2 x 30 ml). The combined washings and further added sodium chloride were extracted with e-thyl acetate (20 ml) and the combined oryanic layers were dried with magnesium sulphate.
Evaporation gave a foam (1.79g) and this was triturated with diiso-propyl ether to give a solid (1.35 g).
Most of this solid (1.2 g) was dissolved in Eormic acid (5 ml) and concentrated hydrochloric acid (0.38 ml) was added with vigorous stirring. After one hour at 21, the suspension was filtered and the residue was leached with a little formic acid~
The combined filtrates were concentrated by evaporation and the residue was tritura-ted wlth aCQtOne to give the tl le compound (374 mg) ~c~ D + 8-6 (_ 1.02, H2O) ~max (pH 6 buffer) 255 nm (ElCm infl. 95 (Elcm 273), ~infl 280 (El~ 158).

_ample 6 a) (6R,7R)-7- ~(Z)-2-(2-triphenylmethylaminothiazol-4-yl)-2-(1-t-butoxycarbonylcyclobut-l-oxyimino)acetamldo]-3-(1-pyridiniummethyl)-ceph-3-em-4~carboxylate Phosphorus pentachloride (1.38 g) was dissolved in 60 ml ~1 ,, llZ~7633 of dichloromethane. The solution was cooled to -10 and the product of ~reparation 6 (3.48 g) was added in one charge. The solution was stirred at -5 for 30 minutes. Triethylamine (1.8 ml) was added, followed by water (20 ml). The mixture was stirred at 0C for 3 minutes. I'he lower phase was -then added to a pre-cooled mixture of the product of Preparation 10 (2.18g) in dimethylacetamide (30 ml) and acetonitrile (30 ml) with triethylamine (4.2 ml) added at -10C.
The reaction mixture was stirred for 45 minutes between -5C and -10C. 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 tri-turated with isopropyl ether to yive the _tle compound (3.80 g).
vmax (Nujol) 1780 cm (~-lactam) I(CDCl3) values include 2.74 (s, triphenylmethy]) 8.66 (s, t-butyl) b) (6R,7~)-7- ~(Z)-2-(2-aminothiazol-4-yl)-2-(1-carboxycyclo-but-1-oxyimino)acetamido~ -3-(1-pyridiniummethyl)ceph-3-em-4-carboxy-lic acid dihydrochloride.
The product from Stage a), (2.57 g) was s-tirred at ambient -temperature in a mixture of 98~ formic acid (15 ml), and concentrated hydrochloric acid (0.9 ml) for one hour. The mix-ture was then filtered and the solvent removed under reduced pressure.
The resulting residue was triturated with acetone to produce the title compound (1.79 g).
-~127~i33 vmax ~Nujol) 1785 cm 1 (~-lactam) rvalues (D2O + NaHCO3) include 1.05, 1.42, 1.91 (m, pyridinium protons), 3.01 (s, aminothiazole proton) 4.13 (d, J 5Hz, C7 proton), 4.68 (d, J SHz, C-6 proton) 7.4 - 8.4 (broad m, cyclobutyl protons) Dimethylacetamide (1/3 mole) and acetone (~ mole) by n.m.r.
Water content 7.4% (Karl Fischer method) Chlorine, found 9.2% (C23H24N6O7S2C12 + 1/3 mole dimethylacetamide + ~ mole acetone + 7.4~ water requires Cl, 9.5%).

~ 47 ~i~7~33 PHARMACY EXAMPLES
-Example A - Dry Powder for Injection -Formula ~er Vial (6R,7R)-7-[(Z)-2-~-Aminothiazol-4-yl)-2-(1-carboxycyclobllt-1-oxy-imino)acetamido]-3-(1-pyridiniummethyl)ceph-3-em-4-carboxylate. 500mg Lysine Acetate 185mg 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 2 ml Water for Injections.
Example B - Dry Powder for Injection Fill sterile (6R,7R)-7-[~Z)-2-(2-aminothiazol-4-yl)-2-(1-carboxycyclo-but-l-oxyimino)acetamido]-3-(1-pyridiniummethyl)ceph-3-em-4-car-boxylate, monosodium salt into glass vials such that each vial contains an amount equivalent to 1.0 g 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.
Bxample_C - Injection Twin-Pack (a) Fill 500 mg quantities of sterile (6R,7R)-7-[(Z)-2-(2-amino-thiazol-4-yl)-2-(1-carboxycyclobut-1-oxyimino)acetamido]-3-(1-pyridiniummethyl)ceph--3-em-4-carboxylate aseptically into glass vials under a blanket of sterile nitrogen. Close the vials using . . , 112'7633 rubber disks or plugs, held in position by aluminium overseals, thereby preventing gaseous ~xchange or ingress of microorganisms.
(b) Prepare a 3.76%w/v solution of sodium bicarbonate, clarify by filtration and fill 2.15 ml 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 antihiotic shortly before administrationby dissolving in 2.0 ml of the sodium bicarbonate solution.
Example D - Dry Powder for In~ection Formula per Vial (6R,7R)-7-[(Z)-2-(2-Aminothiazol-4-yl)-2-(1-carboxycyclobut-1-oxy-imino)acetamido]-3-(1-pyridiniummethyl)ceph-3-em-4-carboxylate 500mg sodium carbonate, anhydrous 47mg Method The cephalosporin antibiotic was blended with sodium carbonate and filled into a glass vial. The vialheadspace was purged with nitro-gen and a combination seal applied by crimping. The product was dissolved, as for administration by the addition of 2ml Water for Injections.
Example E - ~ tion for Veterinary Use Formula (6R,7R)-7-[(Z)-2-(2-Aminothiazol-4-yl)-2-(1-carboxycyclobut-1-oxy-imino)acetamido]-3-(1-pyridiniummethyl)ceph-3-em-4-carboxylate 10% w/Y

Aluminium distearate2% w/v) ) to 100% w/v Ethyl Oleate to 100% w/v 1~27633 Method Disperse the aluminum distearate in ethyl oleate, heat at 150C 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 position by aluminium over-seals.

i

Claims (14)

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 cephalosporin antibiotics of the general formula (I) (wherein Ra and Rb, which may be the same or different, each represent a C1-4 alkyl group or Ra and Rb together with the carbon atom to which they are attached form a C3-7cycloalkylidene group;
and R4 represents hydrogen or a 3- or 4-carbamoyl group with the proviso that Ra and Rb do not each represent a methyl group when R4 represents hydrogen) and non-toxic salts and non-toxic metabol-ically labile esters thereof, characterised in that (A) a compound of formula (II) (wherein B is >S or ; R4 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 correspond-ing 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 the formula (III) (wherein Ra and Rb are as defined above; R6 represents a carboxyl blocking group; and R7 is an amino or protected amino group) or with an acylating agent corresponding thereto; or (B) a compound of formula (IV) (wherein Ra, Rb, R7, 8 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 a salt thereof, is reacted with a pyridine compound of the formula (V) (wherein R4 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 .DELTA.2-isomer into the desired .DELTA.3-isomer, ii) reduction of a compound wherein B is 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.

2. A process as claimed in claim 1 wherein in the starting materials at least one of Ra and Rb represents a methyl or ethyl group.

3. A process as claimed in claim 1 wherein in the starting materials Ra and Rb together with the carbon atom to which they are attached form a C3-5cycloalkylidene group.

4. A process as claimed in claim 1 wherein in the starting materials Ra and Rb are as defined in claim 1 and R4 is hydrogen, whereby a compound of the formula or a non-toxic salt thereof is recovered.

5. A process as claimed in claim 1 wherein in the starting materials, Ra and Rb together with the carbon atom to which they are attached, form a cyclobutylidene group and R4 represents hydrogen.

6. A process for the preparation of (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1-carboxycyclobut-1-oxyimino)acetamido]-3-(1-pyridiniummethyl)-ceph-3-em-4-carboxylate, mono-sodium salt which comprises reacting (6R,7R)-3-acetoxymethyl-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1-carboxycyclobut-1-oxyimino)acetamido]ceph-3-em-4-carboxylic acid with pyridine in the presence of sodium iodide.

7. A process for the preparation of (6R,7R)-7-[(Z)-2-(2-amino-thiazol-4-yl)-2-(1-carboxycyclobut-1-oxyimino)acetamido]-3-(1-pyridiniummethyl)-ceph-3-em-4-carboxylic acid dihydrochloride which comprises reacting (Z)-2-(1-t-butoxycarbonylcyclobut-1-oxyimino)-2-(2-tritylaminothiazol-4-yl)acetic acid with (6R,7R)-7-amino-3-(1-pyridiniummethyl)ceph-3-em-4-carboxylic acid dihydrochloride and treatment of the product thus obtained with hydrochloride acid.

8. A process as claimed in claim 1, 2 or 3 characterised in that a ceph-3-em compound of formula (II) in which B is >S is employed.

9. A process as claimed in claim 1, 2 or 3 characterised in that a compound of formula (II) is acylated with an acid halide corresponding to the acid of formula (III).

10. A process as claimed in claim 1, 2 or 3 characterised in that a compound of formula (IV) wherein X is an acetoxy group or bromine atom is employed.

11. A cephalosporin antibiotic of the general formula (I) defined in claim 1, and the non-toxic salts and non-toxic metabolically labile esters thereof, when prepared by the process of claim 1 or by an obvious chemical equivalent thereof.

12. (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1-carboxycyclo-but-1-oxyimino)acetamido]-3-(1-pyridiniummethyl)ceph-3-em-4-carboxylic acid and the non-toxic salts and non-toxic metabolically labile esters thereof, when prepared by the process of claim 5 or by an obvious chemical equivalent thereof.

13. (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1-carboxycyclo-but-1-oxyimino)acetamido]-3-(1-pyridiniummethyl)-ceph-3-em-carboxylate, mono-sodium salt, when prepared by the process of claim 6 or by an obvious chemical equivalent thereof.

14. (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1-carboxycyclo-but-1-oxyimino)acetamido]-3-(1-pyridiniummethyl)ceph-3-em-4-carboxylic acid dihydrochloride, when prepared by the process of claim 7 or by an obvious chemical equivalent thereof.