CA1131618A - Cephalosporin compounds - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

Cephalosporin antibiotics of general formula

Description

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.
S ., 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 com-pounds, and in the treatment of penicillin-sensitive patients. In many instances it is desirable to employ a cephalosporin antibiotic which e~hibits activity against both gram-positive and gram-negative microorganisms, and a significant amount of research has been directed to the development of various types of broad spectrum cephalosporin an~ibiokics.
Thus, for example, in our British Patent Specifica-20 tion No. 1,399,0867 we describe a novel class of cephalo-sporin antibioticscontaining a 7~ -etherified oxyimino)-acylamido group, the oxyimino group having the ~y~ config-uration. T~is class of antibiotic compounds is character-ised by high antibacterial activity against a range of ., -. :~ . . , ' . ~ :

~ ~ 3~
gram-positive and gram-~egative 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. 1,496,757, we describe cephalosporin antibiotics containing a 7~-acylamido group of the formula R.C.CO.NEI-N RA (A) O.(CH2)m C (CH2) COOH
R
(wherein R is a thienyl or furyl group; RA and RB may vary widely and may, for example, be Cl ~ 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 O

2~ or 1 such that the sum of m and n is O or 1), thecompounds 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 unsub- -stituted or may contain one of a wide variety of possible substituents These compounds have been found to have particularly good activity against gram-negative organisms.
Other compounds of similar structure have been developed from these compounds in further attempts to find antibiotics having improved broad spectrum antibiotic ~.~ 3 i~ ~
activity and/or high activity against gram-negative organisms. Such developments have involved variations in not only the 7~-acylamido group of formula (A) but also the introduction of particular groups in the 3-position o~ the cephalosporin molecule~
Thus, for example, South African Patent Specifica-tion 78/1870 discloses cephalosporin antibiotics wherein the 7~-acylamido side chain is inter alia a 2-(2-amino-thiazol-4-yl)-2-(optionally substituted alkoxyimino)-acetamido group and the 3-position may be substituted, for example, by the group -CH2Y in which Y represents the residue of a nucleophile. The Specification contains, among numerous other examples, references to compounds in which the above-mentioned optionally substituted alkoxy-imino group is a carboxyalkoxyimino or carboxycycloalkoxy-imino group. With regard to the 3-position, mono- and di-alkylaminomethyl substituents arereferred to, among numerous other possibilities. South African Patent Specification 78/2168 discloses in broad terms sulphoxide compounds corresponding to the sulphides described in the last-mentioned Specification.
~ urthermore, Belgian Patent Specification No.
836,813 describes cephalosporin compounds wherein the group R in formula (A) above may be replaced by, ~or example, 2-aminothia2O1-4 yl, and the oxyimino group is a hydroxyimino or blocked hydroxyimino group, e4g, 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 ~ 1 3~
a large number of residues of nucleophilic compounds therein described. N-Alkylaminomethyl groups are mentioned as possible substituents in the 3-position but only mono- and di-alkylaminomethyl groups are specifically identified. 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.

~ ~ 3~
We have now discovered that by an appropriate selection of a small number of particular groups at the 7~-position in combination with a trialkylammoniomethyl group at the 3-position, cephalosporin compounds having particularly advantageous activity (described in more detail below) against a wide range of commonly encountered pathogenic organisms may be obtained.
~ le present invention provides cephalosporin anti-biotics of the general formula:

S N , , 1 C.CO.NH - ~ S ~ R
~ 32 (I) N IR O ~ N ~ CH2l-R
O.C.COOH I a 3 Ib COO R
(wherein R 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 , R and R , which may be the same or different, each represents a Cl 4 alkyl group, e.g~ a methyl group)and non-toxic salts and non-toxic metabolically labile esters thereof.
The compounds according to the invention are synisomers. The syn isomeric form is defined by the configuration of the group . . .

' .

- O.C.COOH
Ib with respect to the carboxamido group. In this Specifi-cation the ~y~ configuration is denoted structurally as C.CO~NH
N Ra O.C.COOH
Ib It will be understood that since the compounds according to the invention are geometric isomers, some admixture with the corresponding anti isomer may occur.
The invention also includes within its scope the solvates (especially the hydrates) of the compounds of formula (I). It also includes within its scope salts of esters of compounds of formula (I).
The compounds according to the present invention may exist in tautomeric forms (for example in respect of the 2-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 group is protonated and the carboxyl group in the 7-side chain is deprotonated. These alternative forms, as well as mixtures of zwitterionic forms1 are ~ 6~
included within the scope of the present invention.
It will also be appreciated that when R and R in the above formula represent different Cl 4 alkyl groups, the carbon atom to which they are attached will comprise a centre of asymmetry. A centre of asymmetry will also be present when Rl, R2 and R3 all represent different alkyl groups. Such compounds are diastereoisomeric and the present invention embraces individual diastereoisomers of these compounds as well as mixtures thereof.
The compounds according to the invention exhibit broad spectrum antibiotic activity. Against gram-nega-tive organisms the activity is unusNally high. This high activity extends to many ~-lactamase-producing gram-negative strainsO The compounds also possess high stability to ~-lactamases produced by a range of gram-negative and gram-positive organisms.
Compounds according to the invention have been found to exhibit unusually high activity against strains of Pseudomonas organisms, e.g. strains of Pseudomonas aeru~inosa as well as high activity against various members of the Enterobacteriaceae (e.g. strains of Escherichia c _ , Klebsiella pneumoniae, Salmonella typhimurium9 Shi~ella sonnei, Enterobacter cloacae, Serratia marcescens, Providence species, Proteus mirabilis, and especialIy indole-positive Proteus organisms such as Proteus vul~aris and Proteus morganii) and strains of Haemophilus influenzae.
~ .
The antibiotic properties of the compounds accord-ing to the invention compare very favourably with those of 3~
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 commercially available anti-biotic compounds. Unlike the aminoglycosides, cephalo-sporin antibiotics normally exhibit low toxicity in man.
The use of aminoglycosides in human therapy tends to be limited or complicated by the relatively high toxicity of these antibiotics. The cephalosporin antibiotics of the present invention thus possess potentially great advantages over the aminoglycosides.
Non-toxic salt derivatives which may be formed by reaction of either or both of the carboxyl groups present in the compounds of general formula (I) include inorganic base salts such as alkali metal salts (e.g. sodium and potassium salts) and alkaline earth metal salts (e.g.
calcium salts); amino acid salts (e.g. lysine and arginine salts); organic base salts (e.g. procaine, phenylethylbenzylamine, dibenzylethylenediamine, ethanol-amine, diethanolamine and N-methylglycosamine salts).
Other non-toxic salt derivatives include acid addition saltsg e.g. fonmed with hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, formic and trifluoroacetic acids. The salts may also be in the form of resinates fnrmed with, for example, a polystyrene resin or cross-linked polystyrene divinylbenzene copolymer resin con-taining amino or quaternary amino groups or sulphonic acid groupsy or with a resin containin~ carboxyl groups, e.g. a polyacrylic acid resin. Soluble base salts (e.g.

~ ~ 3~
alkali rnetal 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 appli-cation, e.g. for use in depot preparations, such salts may be formed in conventional manner, for example with appropriate organic amines.
These and other salt derivatives such as the salts with toluene-p-sulp'honic and methanesulphonic acids may be employed as intermediates in the preparation and/or purification of the present compounds of formula (I), for example in the processes described below~
Non-toxic metabolically labile ester derivatives which may be formed by esterification of either or both car'boxyl 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 pivaloyloxymet'hyl esters. In addition to the above ester derivatives 9 the present invention includes within its scope compounds of formula (I) in the form of other physiologically acceptable equivalents, i.e. physiologic-ally acceptable compounds which, like the metabolically labile esters are converted in vivo into the parent anti-biotic compound of formula (I).
Preferred compounds according to the presentinvention include those compounds of formula (I) wherein Rl, R and R all represent methyl groups. Preference is also expressed for those compounds wherein R and R~
both represent methyl groups or together with the carbon ' ' ' , ' ' ~1~3~B
atom to which they are attached form a cyclobutylidene group. (6R, 7R)-7-~(Z)-2-(2-Aminothiazol-4-yl)-2-(1-carboxycyclobut-l-oxyimino)acetamido] 3-trimethylammonio-methyl-ceph-3-em-4-carboxylate ~nd its non-toxic salts and non-toxic metabolically labile esters are particularly preferred compounds according to the present invention.
Other preferred compounds include (6R, 7R)-7-[(Z)-2-(2-aminothiazol 4-yl)-2-(2-carboxyprop-2-oxyimino)acetamido~-3-trimethylammoniomethyl-ceph-3-em-4-carbOxylate acid and its non-toxic salts and non-toxic metabolically labile esters.
Other compounds according to the present invention inclùde those for example wherein the groups Ra, Rb, Rl, R2 and R3 in formula (I) are as follows:-Ra - Rb - R3 a) Alkyl ~roups . -C2H5 C2H5 CH3 CH3 CH3 - r b . _ - R3 R - ~ - R R
b) Cycloalkylidene grou*s cyclopropylidene -CH3 -CH3 -CH3 cyclopentylidene _CH3 CH3 -CH3 cyclopropylidene 2 5 -CH3 -CH3 cyclobutylidene 2 5 -CH3 -CH3 cyclopentylidene 2H5 -CH3 -CH3 cyclopropylidene 2H5 -C2H5 -CH3 cyclobutylidene 2 5 -C2H5 -CH3 cyclopentylidene 2H5 -C2H5 -CH3 cyclopropylidene 2 5 -C2H5 -C2H5 cyclobutylidene C2H5 -C2H5 -C2H5 cyclopentylidene -C2H5 -C2H5 -C2H5 The compounds of formula (I) may be used for treat-ing a variety of diseases caused by pathogenic bacteria in human beings and animals, such as respiratory tract infections and urinary tract infections.
According to another embodiment of the invention we provide a process for the preparation of an antibiotic compound of general formula (I) as hereinbefore defined or a non-toxic salt or non-toxic metabolically labile ester thereof which comprises (A) acylating a compound of the formula H H
2 ~ B ~ R
~ CH2N - R2 (II) coo~3 R3 [wherein R , R and R are as defined above; B is ,S or ~S ~3 0 (a- or ~-); and the dotted line bridging the 2-, 3- and 4-positions indicates that the compound is a ceph-2-em or ceph-3-em compound] or a salt, e.g. an acid addi-tion salt (formed with, for example, a mineral acid such as hydrochloric, hydrobromic, sulphuric, nitric or phos-phoric acid or an organic acid such as methanesulphonic or toluene-p-sulphonic acid) or an N~Silyl derivative thereof, or a corresponding compo~nd having a group of formula - CoOR4 at the 4-position [where R4 is a hydrogen atom or a carboxyl blocking group, e.g. the residue of an es~er-forming aliphatic or araliphatic alcohol or an ester-forming phenol, silanol or stannanol (the said alcohol, phenol, silanol or stannanol preferably ~3~6~

containing 1-20 carbon atoms)] and having an associated anion A~
such as a halide, e.g. chloride or bromide, or trifluoroacetate anion, with an acid of formula S ~ N

C.COOH

R lIII) O.C.COOR
Rb (wherein Ra and Rb are as hereinbefore defined; R5 represents a carboxyl blocking group, e.g. as described for R4; and R6 is an amino or protected amino group) or with an acylating agent corresponding theret~; (B) reacting a compound of formula S ~ N H H
\ / C.CO.NH ~ L~ ~

N R O ~ C~2X
O.C.COOR a 1 7 (IV) Rb COOR

(wherein Ra, Rb, R6, ~ and the dotted line are as hereinbefore defined; R7 and R7a may independently represent hydrogen or a carboxyl blocking group; and X is a replaceable residue of a nucleophile (i.e. a leaving group), e.g. an acyloxy group such as a dichloroacetoxy group or a halogen atom such as , _ lL _ chlorine7 bromine or iodine) or a salt thereof, with a tertiary amine of the formula IRl 2 N - R (V) (wherein Rl, R2 and R are as defined above);
or (C) alkylating a compound of the formula R

~ H H
\ 7, ~ CH2N

O.C.COOR COOR R
Ib (VI) (wherein Ra, Rb, Rl, R2, R6, B and the dotted line are as hereinbefore defined; and R7 and R7a both represent car-boxyl blocking groups) with an alkylating agent serving to form a group of formula Rl -CH N6~- R

at the 3-position;
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 ~ -isomer into the desired ~ -isomerS
ii) reduction of a compound wherein B is ~S-~ O to ~ ~ 3 form a compound wherein ~ is ~S~
iii) conversion o a carboxyl group into a non-toxic salt or non-toxic metabolically labile ester ~unction, 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 preEerably a compound wherein the dotted line represents a ceph-3-em compound.
Acylating agents ~hich 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. phos-phorus 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 ~50Ct preferably -20 to +30~C, if desired in the presence of an acid binding agent. Sui-table reaction media include aqueous ketones such as aqueous acetone, esters such as ethyl acetate, halogenated hydrocarbons su~h 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 - 16 ~

propylene oxide) which bind hydrogen halide liberat~d 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 desir-ably conducted in the presence of a condensing agent, for example a carbodiimide such as N,N'-dicyclohexylcarbodi-imide or N-ethyl-N'-~-dimethylaminopropylcarbodiimide; a carbonyl compound such as 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 alkylhaloformate). Mixed anhydrides may also be formed with phosphorus acids (for example phosphoric or phosphorous acids), sulphuric acid or aliphatic or aromatic sulphonic acids (for example toluene-p-sulphonic acid). An activated ester may con-veniently be formed in situ using, for example, l-hydroxy-benzotriazole in the presence of a condensing ~gent 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.

~ 1 3~
If desired, the above acylation reactions may be carried out in the presence of a catalyst such as 4-di-methylaminopyridine.
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, Eor example, acid chlorides may conveniently be employed as their hydrochloride salts, and acid bromides as their hydrobromide salts.
The amine compound of formula (V) ~ay 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 displaced than atoms or groups derived from weaker acids. The facility of the dis-placement is also related 9 to some exten-t, to the precise identities of the alkyl groups in the compound of formula (V).
The displacement of X by the amine of formula (V) may conveniently be effected by maintaining the reactants in solution or suspensionO The reaction is advantageously effected using from 1 to 20, preferably 1 to 4, moles of the amine (V).
Nucleophilic displacement reactions may conven-iently 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.

~Y~Y~ 3~
Convenient starting materials for use in the nucleophilic displacement reaction with the amine of formula (V) include compounds of formula (IV) in which X
is the residue of a substituted acetic acid e.g. chloro-acetic acid, dichloroacetic acid and trifluoroacetic acid.
The substituent X may also be derived from formic acid7 a haloformic acid such as chloroformic acid, or a carbamic acid.
When ~sing a compound of formula (IV) in which X
represents a substituted acetoxy group, it is generally desirable that the group R in formula (IV) should be a hydrogen atom and that B should represent >S. In this case, t~e reaction is advantageously e~fected in an aqueous medium, preferably at a pH of 5 to 8, particu-larly 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. 19241,657.
When using compounds of formula (IV) in which X
is an acyloxy group~ the reaction is conveniently effected at a temperature of -20 to +80C, preferably 0 to ~50C.

Halo~ens Compounds o formula (IV) in which X is a chlorine, bromine or iodine atom can advantageously be used as starting materials in the nucleophilic displacement reac-tion with the amine of formula (V). Wh~n using compoundsof formula (IV) in this class, B may represent~ S ~ O and R7 may represent a carboxyl blocking group. The reaction is conveniently effected in a non-aqueous medium which preferably comprises one or more organic solvents, advantageously ethers, e.g. dioxan or tetrahydrofuran, esters, e.g. ethyl acetate, amides, e.g. formamide and N~N-dimethylformamide, and ketones~ e.g. acetone.
Other suitable organic solvents are described in more detail in Rritish Patent Specification No. 1,326,531.
The reaction medium should be neither extremely acidic nor extremely basic. In the case of reactions carried out on compounds of formula (IV) in which R7 and R7a are carboxyl blocking groups the 3-trialkylammoniomethyl product will be formed as the corresponding halide salt which may, if desired, be subjected to one or more ion exchange reactions to obtain a salt having the desired anion.
When using compounds of formula (IV) in which X is a halogen atom as described~above, the reaction is conveniently effected at a temperature of -10 to ~50C, '- ~ ' . . - ~ .

.
. .

preferably +10 to +30C.
In process (C) above, the 3-di-C1 4alkylaminomethyl compound of formula (VI) is advantageously reacted with a Cl 4 alkylating agent of the formula R Y wherein R is as defined above and Y is a leaving group such as a halogen atom (e.g. iodine, chlorine or bromine) or a hydrocarbyl-sulphonate (e.g. mesylate or tosylate) group, or R3Y
represents dimethyl sulphate. The alkylation reaction is preferably carried out at a temperature in the range of 0 to 60C, advantageously 20 to 30C. The reaction may be conveniently effected in an inert solvent such as an ether e.g. tetrahydrofuran, an amide, e.g. dimethylformamide, or a halogenated hydrocarbon, e.g. dichloromethane. Alter-natively, where the alkylating agent is liquid under the reaction conditions, this agent can itself serve as a solvent.
The compound of formula (VI) used as starting material in process (C) may be prepared for example by reaction of a compound of formula (IV) (as defined above) with a secondary amine of formula ~Rl HN (VII) \ R2 (wherein R and R are as defined above) in an analogous manner to the nucleophilic displacement reaction described with respect to process (B). This reaction is preferably carried out in the presence of an acid scavenging agent.
The amine itself may act as an acid scavenging agent.
The reaction product may be separated from the 1 1 3~
reaction mixture, which may contain, for example, un-changed cephalosporin starting material and other sub-stances, by a variety of processes including recrystallisa-tion, ionophoresis, column chromatography and use of ion-exchangers (for example by chromatography on ion-exchange resins~ or macroreticular resins.
L~2-Cephalosporin ester derivatives obtained in accordance with the process of the invention may be con-verted into the corresponding L~3-derivative by, for example, treatment of thel~2-ester with a base such as pyridine or triethylamine.
A ceph-2-em reaction product may also be oxidised to yield the corresponding ceph-3-em l-oxide, for e~ample by reaction with a peracid, e.g. peracetic or m-chloro-pe~benzoic acid; the resulting sulphoxide may, ifdesired, subsequently be reduced as described hereinafter to yield the corresponding ceph-3-em sulphide.
Where a compound is obtained in which B is >S -~ O
this may be converted to the corresponding sulphlde by, for example, reduction of the corresponding acyloxy~
sulphonium or alkoxysulphonium salt prepared in situ by reaction with e.gO acetyl chloride in the case of an acetoxysulphonium salt, reduction being effected by, for example, sodium dithionite or by iodide ion as in a solution of potassium iodide in a water-miscible solvent e.g. acetic acid~ acetone, tetrahydrofuran, dioxan, di-methylformamide or dimethylacetamide. The reaction may be effected at a temperature of from -20 to ~50C.
Metabolically labile ester derivatives of the ~ ~ 3~
compounds of formula (I~ may be prepar~d by reacting a compound of formula (I) or a salt or protected derivative thereof with an appropriate esterifying agent such as an acyloxyalkyl halide (e.g. iodide) conveniently in an inert organic solvent such as dimethylformamide or acetone, followed, where necessary, by removal of any protecting groups.
Base salts of the compo~mds of formula (I) may be formed by reacting an acid of Eormula (I) with the appropriate base. Thus, or example, sodium or potassium salts may be ~repared using the respective 2-ethyl-hexanoate or hydrogen carbonate salt. Acid addition salts may be prepared by reacting a compound of formula (I) or a metabolically labile ester derivative thereof with the appropriate acid.
Where a compound of formula (I~ is obtained as a mixture of isomers, the syn isomer may be obtained by, for example, conventional methods such as crystallisation or chromatography.
For use as starting materials for the preparation of compounds of general formula (I) according to the invention, compounds of general formula (III) and acid halides and anhydrides corresponding thereto in their ~_ isomeric form or in the~form of mixtures of the ~X~
isomers and the corresponding anti isomers containing at least 90% of the syn isomer are pxefera~ly 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
5C.COOR8 (VIII) N

\ OH
(wherein R6 is as hereinbefore defined and R8 represents a carbaxyl blocking group), by reaction with a compound of general formula Ra T.C.CooR5 (IX~
Ib (wherein R , Rb and R5 are as hereinbeore deined and T is halogen such as chloro, bromo or iodo; sulphate; or sulphonate such as tosylate), followed by removal of the carboxyl blocking group R8. Separation of isomers may be effec~ed either before or aftPr such etherification. The etherification reaction is generally carried out in the presence of a base, e.g. potassium carbonate or sodium hydride, and is preferably conducted in an organic solvent, for example dimethylsulphoxide, a cyclic ether such as tetrahydrofuran or dioxan, or an N,N-disubstituted amide such as dimethylformamide. Under these conditions the configuration of the oxyimino group is substantially unchanged by the etherification reaction. The reaction should be effected in the presence of a base if an acid addition salt of a compound of Eormula (VIII) is used.

~13~8 The base should be used in sufficient quantity to neutral-ise rapidly the acid in question.
Acids of general fonmula (III) may also be prepared by reaction of a compound of formula R
,~
S ~ .
CO. COOR8 (X) (wherein R and R are as hereinbefore defined) with a compound of formula Ra H2N.o.C.CooR5 (XI) Rb (wherein-Ra, Rb and R5 are as defined above), followed by removal of the carboxyl blocking group R8, and where necessary by the separation of ~y~ and anti iso~ers.
The last-mentioned reaction is particularly applic-able to the preparation of acids of formula (III) wherein R and R together with the carbon atom to which they are attached form a cyclopropylidene group. In this case, the relevant compounds of formula (XI) may be prepared in conventional manner, e.g. by means of the synthesis described in Belgian PatentSpecification No. 866,422 for the preparation of t-butyl l-amino-oxycyclopropane carboxylate.
The acids of formula (III) may be converted to the corresponding acid halides and anhydrides and acid addi- -tion salts by conventional methods, for example as described hereinaboveO

'~ ' 3L~3~
Where X is a halogen (i.eO chlorine, bromine or iodine) atom in formula (IV), ceph-3-em starting compounds may be prepared in conventional manner, e.g. by halogena-tion of a 7~-protected amino-3-methylceph-3-em-4-carboxylic

5 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. 1,326,5310 The corresponding ceph-2-em compounds may be prepared by the method of ~utch 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 example by acyla-tion 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-hydroxy-methyl 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.
~ e starting materials of formula (II) are new compounds. These compounds may be prepared in conventional ~nanner, for example, by deprotecting a corresponding protected 7~-amino compound in converltional manner e g. using PC15.
It should be appreciated that in some of the above transformations it may be necessary to pro~ect 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 (eOg. chloroacetylation), protonation or other conventional method. The protecting group may there-after 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, chloro-acetic acid or trifluoroacetic acid or using a mineral acid, e.g. hydrochloric acid or mixtures of such acids, preferably in the presence of a protic solvent such as water or, in the case of a chloroacetyl group, by treat-ment with thiourea.
Carboxyl blocking groups used in the preparation ofcompounds 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 se~uence, conveniently at the last stage. It may, however, be convenient in some instances to employ non-toxic metabolically labile carboxyl blocking groups such as acyloxy-methyl or -ethyl groups (eOgO acetoxy-methyl or -ethyl or pivaloyloxymethyl) and retain these in the final product to give an appropriate ester derivative of a compound of formula (I).
Suitable carboxyl blocking groups are well known in the art, a list of representative blocked carboxyl groups being included in British Patent No. 1,399,086.
Preferred blocked carboxyl groups include aryl lower alkoxycarbonyl groups such as ~-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl and diphenylmethoxycarbonyl;
lower alkoxycarbonyl groups such as t-butoxycarbonyl; and lower haloalkoxycarbonyl groups such as 2,2,2-trichloro-ethoxycarbonyl. Carboxyl blocking group(s) may subsequently be removed by any of the appropriate methods disclosed in the literature; thus, for example, acid or base catalysed hydrolysis is applicable in many cases, as are enzymically-catalysed hydrolyses.
The following Examples illustrate the invention.
All temperatures are in C. 'Petrol' means petroleum ether (b.p. 40-60)o Preparation 1 Et~ (Z)-2-(2-aminoth_azol-4-vl)~2-(hydrox~imino)acetate To a stirred and ice-cooled solution of ethyl acetoacetate (292 g) in glacial acetic acid (296 ml) was added a solution of sodium nitrite (180 g~ in water (~00 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 (~00 ml) was added. The result-ing mixture was stirred for one hour. The lower oily _~ phase was separa.:ed and the aqueous phase was extracted with diethyl ether. The extract was combined with the - 28 - 1 ~ 3~

oil, washed successlvely with wate~ and saturated brine7 drie~, ~nd evaporated, The residual oil, which solidified on standing, was washed with petrol and dried ~n vacuo over potassium hydroxide, glving ethyl (Z)-2-(hydroxy-S imino)-3-oxobutyrate (309 g).
A stirred and ice-cooled solution of ethyl (Z)-2-(hydroxyimino)-3-oxobutyrate (150 g)-in dichloromethane (400 ml) was treated dropwise with sulphuryl chloride (140 g). Th~ resulting solution was kept at room temp-erature for 3 days, then evaporated. The residue wasdissolved in diethyl ether, washed with water until the washings were almost neutral, dried, and evaporated. The residual oil (177 g) was dissolved in ethanol (500 ml) and dimethylaniline (77 ml) and thiourea (42 g) was added with stirring. After two hours, the mixture was filtered and the residue washed with ethanol and dried to give the title compound (73 g); m.p. 188 (decomp.).
Preparation 2 Ethyl (Z)-2-hydroxyimino-2-(2-trit~L~LL~c ~ gl g yl)-acetate, hydrochloride Trityl chloride (16 75 g) was added portionwiseover 2 hours to a stirred and cooled (-30) solution of the product of Preparation 1 (12.91 g) and triethylamine (8.4 ml) in dimethylformamide (28 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 wlth 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 113~8 provide the title ~ as a white solid (16. 4 g);
m.p. 184 to 186 (decomp.).
Preparation 3 Ethyl (2?-2-(2-t-butoxycarbonyl~rop-2-oxyimino)-2-(2-5 tritylaminothiazol-4-yl)acetate Potassium carbonate (34.6 g) and t-butyl 2-bromo-2-methylpropionate (24.5 g) were added to a stirred solu-tion under nitrogen of the product of Preparation 2 (49. 4 g) in dimethylsulphoxide (200 ml) and the mixture 10 was stirred at room temperature for 6 hours. The mixture was poured into water (2 1), stirred ~or 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 15 saturated brine, dried, and evaporated. The residue was recrystallised from petrol to give the title compound (34 g), m.p. 123. 5 to 125.
Preparation 4 (Z)-2-(2-t-ButoxycarbonYlprop-2-oxyimino~-2-(2-trit~
- 20 aminothiazol-~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 25 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. The organic solutions were combined, washed successively with water and saturated brine, dried and evaporated. The ~3~8 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 Ethyl (Z)-2-(2-tritylaminothiazol-4-yl)-2-(1-t-butoxy-carbonylc~clobut-l-oxyimino) acetat_ The product of Preparation 2 (55.8 g) was stirred under nitrogen in dimethylsulphoxide (400 ml) with potassium carbonate (finel~ ground, 31.2 g) at room temperature. After 30 minutes, t-butyl l-bromocyclo-butanecarboxylate (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 nex-t three days and further t-butyl l-bromocyclo-butanecarboxylate (3.45 g) was added after 3 days. After4 days in all, the mixture was poured into ice-water (caO
3 litres) and the solid was collected by filtration and washed well with water and petrol. The solid was dissolved in ethyl acetate and the solution washed with brine (twice), dried with magnesium sulphate and evapor-ated 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 yellow foam, ~ (CHBr3) 3400 (NH) and 1730 cm (ester).
~
(Z)-2~ t-Butoxycarbonylc clobut-l-o imino)-2 (2-trityl-~ _ _ y_ xy aminothiazol-4-yl) acetic acid A mixture of the product of Preparation 5 ~3.2 g3 and potassium carbonate (1.65 g) was refluxed in methanol , , "

3L3~

(180 ml) and water (20 ml) for 9 hours and the mixture was cooled to room temperature. The mixture was concen-trated and the residue partitioned between ethyl acetate and water, to which was added 2N HCl (1202 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); Amax (ethanol) 265 nm (El/m243).
Example 1 a) Diphenylmethyl (lS,6R,7R~-3-Bromomethyl-7-[(Z)-2-(2-t-butoxycarbonyl~rop-2-oxyimino)-2-(2-tritylaminothiazol-4-yl)acetamido]ce~h-3-em-4-carboxylate, l-Oxide A solution of the product of Preparation 4 (0.526 g) in dry tetrahydrofuran (6 ml) was treated successively with l-hydroxybenztriazole monohydrate (0,141 g) and N,N'-dicyclohexylcarbodiimide (0.198 g) in tetrahydrofuran (4 ml). The developing suspension was stirred for 30 minutes at 23O and then filtered. A solution of diphenyl-methyl (lS,6R,7R) 7-amino-3-bromomethylceph-3-em-4-carboxylate~ 1 oxide (0.427 g) in dichloromethane (260 ml) was treated at 23O with the above filtrate. The solution was stirred for 18 hours at 20O to 25, Qvaporated to dryness, then the residue was dissolved in dichloromethane and washed successively with saturated aqueous sodium bicar-bonate~ water and brine, then dried and evaporated in vacuo to a foam (1.01 g).
This foam was purified by chromatography on preparative silica plates using toluene:ethyl acetate:acetic acid = 190:50:2.5 as eluant. The purified product was isolated as a foam which was dissolved in ethyl acetate (5 ml) and precipitated from petrol (200 ml) to give the title compound (0.69 g) as a colourless powder; ~max (EtOH) 268 nm (ElCml82) with an inflection at 242 nm El 230), i~ vmax(Nujol~ 3375 (NH), 1805 (~-lactam), 1730 (C02R) and 1688 and 1515 cm 1 (CONH).
b) Dipheny methyl (lS?6Rj7R~-7-C(Z)-2-(2-t-butoxycarbonYl-~ 4 y1~ ~cet o ~3-trimethylammoniomethylceph-3-em-4-carboxylate~ l-Oxide, Bromide Salt The product of stage a) (0.154 g), alumina-dried tetrahydrofuran (0.3 ml) and anhydrous trimethylamine in dry tetrahydrofuran (0.155 g of trimethylamine in 1 ml of solution) (0~065 ml) were stirred at 24 for 1 hour. The reaction mixture was added dropwise to well-stirred ether (220 ml) and the resulting suspension was stirred vigorously for 10 minutes. The solid was filtered off, washed with ether and dried in vacuo to give the title compound (0.131 g) m.p. 158 to 178 (decomp.); ~a]D +
11 (c 0.53, CHC13).

I rade /~ k , ~ , .

3~

c) Diphenylmethyl (6R,7R)-7- r ( z ) - 2-(2-t-Butoxycarbonyl-prop-2-oxyimino~-2-~2-tritylaminothiazol-4-vl)acetamido]
3-trimethylammoniomethylceph-3--em-4-carboxylate~ Iodide Salt The product of stage b) (1.87 g) and acetone (4.7 ml) were stirred at -10 as a solution. Dry, powdered potassium iodide (lo 14 g) was added and the mixture was stirred at -10 for 2 minutes. Dry, powdered potassium iodide (1.14 g) followed by acetyl chloride (0.25 ml) were added and the vigorously stirred mixture was allowed to warm to 0 over 20 minutes. The mixture was stirred at 0 to ~2 for 1 hour. The mixture was added dropwise to a stirred solution of sodium metabisulphite (0.850 g) in water (47 ml). The resulting solid was filtered off, washed with water and dried in vacuo over phosphorus pent-oxide to gi~e a solid (1.939 g). The above procedure was repeated using the solid (1.87 g), acetone (4.7 ml), dry powdered potassium iodide (1.14 g), acetyl chloride (0.25 mlj and a reaction time at 0 to +2 of 25 minutes. This gave the title compound (1.951 g) as a solid, m.p. 142 to 176; [a]2-716 (c 0.38, CHC13).
d) Diphenvlmethvl (6R.7R~-7-r(Z~2-(2-t-Butoxycarbony~
prop-2-oxyimino?-2-(2~tritylaminothiazol-4-yl)-acetamido~-acetate Salt The product of stage c) (1.826 g) was dissolved in acetone:ethanol = 9:1 and chromatographed on "Deacidite"l FF SRA 62 ion exchange resin (strong anion exchange resin) trifluoroacetate form. The column was eluted with the - 34 ~

above solvent mixture. A forerun of 20 ml was discarded and the next 250 ml were evaporated in vacuo immediately to give the title ~E~ (1.595 g) as a red-brown foam.
e) (6R,7R)-7-~(Z)-2-(2-Aminothiazol-4-yl)~2-(2-carboxy-prop-2-oxvimlno)acetamido~-3-trimethylammoniomethylceph~
3-em-4-carboxyl~te The product of stage d) (1.37 g), anisole (1.37 ml) and trlfluoroacetlc acid (5.5 ml) were swirled together at 25 for 1~ minutes, when a solution was formed, and then for a further 1 minute. The volatile material was evap-orated off and the residue was azeotroped with toluene (twice). The gum was dissolved in acetone (10 ml) and preclpitated into petrol (500 ml3. The brown solid was flltered off~ washed wlth petroleum ether and dissolved in acetone. The solution was evaporated to a foam (1.117 g).
The foam (1.117 g), anisole (0.25 ml) and tri- -fluoroacetic acid (5 ml) were swirled together at 28 for 5 minutes. The volatile material was removed and the residue was azeotroped with toluene (twice). The result-ing brown oil was precipitated with acetone (10 ml) and 40 to 60 petroleum ether (500 ml) to give a solid (1.066 g)-The solid (0.2 g) was dissolved in trifluoroa~eticacid:water = 1:1 (2 ml) and the solution was stirred at 28 for 30 minutes. The mixture was evaporated to dry-ness and the resulting gum was dissolved in water (10 ml).
The cloudy solution was filtered, the-residue was washed with water (10 ml, 5 ml) and the filtrate was free~e-dried to give a foam, (0.17 g). The foam was triturated 1 ~ 3 with ether, the solid obtained was filtered rapidly and dried in vacuo to give the title compou~ (0.148g) as a solid associated with 1.8 moles of trifluoroacetic acid;
[~]~ ~ 120 (c 0.3; EtOH:H20 = 1~ inf (pH6 buffer~
230 nm (~ 17,000), ~ inf 260 nm (~ 10,200).
Example ~
a) Diphenylmethyl (lS,6R,7R~-3-Bromomethyl-7- r (z) - 2-(l-t-butoxycarbonylcyclobut-l-oxyimino)-2-(2-tritylc~min tlliazol-4-yi~acetaLllido~-cep;l-~-erll-4-carboxYiate~ 1 Oxiae A stirred solution of the product of Preparation 6 (1.167 g) in tetrahydrofuran (15 ml) was treated successively with l-hydroxybenztriazole hydrate (0.337 g) and N,N'-dicyclohexylcarbodiimide (0.495 g) for 30 minutes at 220.
Filtration afforded a solution of the activated ester which was added to a solution of diphenylmethyl (lS~6R~7R)-7-amino-3-bromomethylceph-3-em-4-carboxylate l-oxide (0.95 g) in dichloromethane (550 ml). The solution was stirred for 16 hours then evaporated to dryness. A
solution of the residue in dichloromethane was washed successively with aqueous sodium bicarbonate, and brine, and then dried and evaporated to a foam (2.2 g) which was purified by preparative thin-layer chromatography ~using toluene;ethyl acetate:acetic acid = 40:10:1 for development) to give the title compound (1.4 g) with ~maX(EtOH) 266 nm (ElCml92) and an inflection at 242.5 nm (ElCm224), vmax(Nujo~ 3360 (NH)~ 1805 (~-lactam), 1730 (C02R) and 1689 and 1520 cm 1 (CONH).
b) Diphenylmethyl (lS,6R,7R)-7-~(Z)-2-(l-t-ButoxYcarbonyl-cyclobut-l-oxy mino)-2-(_-tritylaminothiazol-4-Yl~-acetamido]-3-trimethylammoniomethylceph-3-em-4-carboxyl ate l-Oxide Bromide Salt The product of stage a) (1.2 g), alumina-dried tetrahydrofuran (205 ml) and anhydrous trimethylamine in dry tetrahydrofuran [O. 49 ml of a solution of trimethyl-amine (0.155 g) in tetrahydrofuran (1 ml)~ were stirred at 24 for 30 minutes. The reaction mixture was added drop-wise to stirred ether (900 ml~ and the resulting sus-pension was stirred vigorously for 10 minutes. The solid was filtered off, washed with ether and dried in vacuo to give the title compound (1.16 g), m.p. 156 to 170 (decomp); [a~D ~ 6 (c 0. 48, CHCl3).
c) Diphenylmethyl ~6R~7R~-7-r(z2--2-(l-t-Butoxvcarbon cyclobut-l-oxyimino2~(2-tritylaminothiazol-4-yl)-acetamido~-3-trimethylammoniomethylcep~-em-4-carboxylate, Iodide Salt The product of stage b) (1.05 g) and acetone (2.6 ml3 were stirred as a solution at -10. Dry, powdered potassium iodide (0.625 g) was added and the mixture was stirred at -10 for 2 minutes. Further dry, powdered potassium iodide (0.625 g) das added followed by acetyl chloride (0.14 ml). The stirred mixture was allowed to warm to 0 and it was stirred at 0 to +2 for 3~ hour~

~ ~ 3 The mixture was added dropwise to a stirred solution of sodium metabisulphite (0.465 g) in water (26 ml). The resulting solid was filtered off, washed with water and dried in vacuo over phosphorus pentoxide to give a solid (1.072 g). The above procedure was repeated using the solid (1.072 g), acetone (2.6 ml), dry, powdered potassium iodide (0.625 g), and acetyl chloride (0.14 ml) to give the title compound (1.131 g) as a solid, m.p. 133 to 170 (decomp) [a~D-33 (c 0.6, CHC13).
d) (6R?7R)-7-[~Z)-2-(2-Amin thiazol;4-yl)-2-(1-carbox~-cyc_ but-l-oxYimino)ac_ ~mido~-3-trimeth~Lammoniomethyl--ceph-3-em-4-carboxY~te The product of stage c) (0.2 g) was wetted with anisole (0.2 ml) and trifluoroacetic acid (0.8 ml) was added. An immediate precipitate was formed and the suspension was swirled for 2 minutes at ca 23 when the precipitate became gummyO The mixture was evaporated to dryness and the residue was triturated with ether to give a solid which was wetted with anisole (0.035 ml) and tri-fluoroacetic acid (4 ml) was added. A very fine precipi-tate formed and the suspension was swirled at 23 for 15 minutes. The mixture was evaporated to a gum which was triturated with ether to gi~e the title compound (0.091 g~
as a solid, associated with 1 mole of trifluoroacetic acid and 0.4 mole of hydrogen iodîde, Ea~D ~ 45 (c 0.22, ethanol:water = 1~ inf (pH 6 buffer) 257.5 nm (El/ 240), ~ inf 296 nm (ElCm 115).

- 38 ~ 8 Example 3 a) Diphenylmethyl (lS~6R,7R~-7-~(Z)-2-(1-t-Butoxycarbonyl-cyclobut-l-oxyimino)-2-(2-tritylaminot ~
acetamido]-3-dimethylaminomethylceph-3-em-4-carboxylate~ 1-Oxide The product o Example 2(a) (0.52 g) in dry tetrahydrofuran (2 ml) was treated with a solution of dimethylamine in ethanol (33% w/w; 0.20 ml). ~fter 15 minutes at 21, the mixture was partitioned between ethyl acetate (25 ml) and water (25 ml). The aqueous layer was extracted with more ethyl acetate (25 ml) and the total organic solution was washed with water (2x50 ml) and dried (Na2S04) and evaporated to a foam (0.498 g). The crude product was purified by preparative thin-layer chroma-tography on silica-gel plates (2mm thick) eluted with ethyl acetate. The main band, Rf 0.4 yielded a foam (0.331 g) which was dissolved in ethyl acetate (2 ml) and added slowly to stirred petrol (50 ml)O The precipitate was filtered off and washed with petrol and dried in vacuo to give the title compound (0.224 g) as a solid, ~]D-21 (c 0-87%~ G~Cl~ inf(EtOH) 245 nm (El/D 225), 260 (El 210 and 305 nm (ElCm57).
b) ~

The product from stage (a) (01201 g) was dissolved in iodomethane (1 ml) and the solution was left to stand ~3~

at 21 for 1~ hours. Diethyl ether (20 ml) was added, and the precipitate was tri~urated and then filtered off and washed with ether and dried in vacuo to give the title ~e~_ d (0.199 g) as a solid, [~]D~10 (c 0.87~J CHC13), ~inf(EtOH) 260 nm (ElCm160), 265 nm (E1%Cml54) and 305 nm (ElC~n64) with a ~ max at 394 nm (ElCm43).
The title compound may be converted into (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2~ carboxycyclobut-1-oxyimino)acetamido]-3-trimethyl-ammoniomethylceph-3-em-4-carboxylate as described in Example 2.
Example 4 a) Diphenylmethyl (lS,6R,7R)-7-~ormamido-3-trimethylammoniomethylceph-3-em-4-carboxylate, l-oxide, Bromide Salt A solution of diphenylmethyl (lS,6R,7X)-3-bromomethyl-7-formamido-ceph-3-em-4-carboxylate, l-oxide (1.01 g) in dry N,N-dimethylformamide (3 ml) was treated with a solution (0.8 ml) of anhydrous trimethylamine in tetrahydro-furan (0.155 g of trimethylamine per ml of solution) and the solution was stirred at 21 for 15 minutes. Ether (10 ml) was added and the supernatant solution was discarded. Trituration of the oily residue with ether (ca. 15 ml) gave a precipitate which was filtered off, washed with ether and dried rapidly in vacuo to give the title compound (1.002 g) as a solid m.p. 140 to 150 (with decomp), Vmax(Nujol) ca. 3400 (NH), 1798 ~B-lactam), 1680 (C=O of HCONH), 1732 (CO2R), and 1035 cm 1 (sulphoxide).
b) Diphenylmethyl (lS,6R,7R)-7-Amino-3-trimethylammoniomethylceph-3-em-4-carboxylate, l-Oxide, Hydrochloride and Bromide Salts A mixture of the product of stage (a) (0.562 g) in methanol (5 ml) was stirred at 0 and treated~ dropwise, with phosphoryl chloride (0.28 ml) over 10 minutes. The mixture was stirred at 0 for 2 hours to precipitate a ;~ -39-buff solid. Ether (15 ml) was added to the stirred mixture then the precipitate was filtered off and washed successively with ether and ethyl acetate and dried in vacuo to give the title compound (0.479 g) as a solid, ~` 5 ~maX(EtOH) 280 nm (ElCmll7), vmax(Nujoi~ 3700 to 2200 (~ 3), 1807 (~-lactam) and 1734 cm 1 ~C02R).
c) Diphenylmethyl (lS,6R~Z~-7-C(7)-2-(2-t-Butoxycarbonyl-prop-2-ox~imino)-2-(2-trit~laminothiazol-4-y~acetamidol 3-trimethylammoniomethy~ceph-3-em-4-carboxylate L 1 - Oxide, Bromide Salt Phosphorus pentachloride (0.11 g), in dry dichloromethane (10 ml) at 0 was treated with the product of Preparation 4 and the solution was stirred at 0 for 35 minutes Triethylamine (0.16 ml) was added and stirring was continued at 0 for 5 minutes. The solution was then added dropwise, over 5 minutes to a vigorously stirred suspension of the product of stage (b) (0.286 g) in dichloromethane (15 ml) at 0O The suspension was stirred at 0 for 15 minutes, 20 for 2 hours and was then allowed to stand at 40 overnight.
The mixture was partitioned between ethyl acetate (100 ml) and water (100 ml) and the emulsion was clarified by filtration. The organic phase was washed with water (100 ml~ then dried over sodium sulphate and evaporated to a foam. A solution of this foam in ethyl acetate (4 ml) was added dropwise to stirred petrol (120 ml). The precipitate was filtered off and washed with petrol and dried in vacuo to give the crude product (0.363 g) as a solid. Some solid remained on the sinter;

~ ~ 3 this was dissolved in ethyl acetate and the solution evaporated to a gum (0.019 g). The above combined products (0033 g) were stirred with ethyl acetate (20 ml) for 15 minutes with trituration of the precipitate. The stirred mixture was slowly diluted with ether (20 ml) and, after a further 10 minutes 3 the solid was filtered off and washed with ether and dried in vacuo to give, the title (O.211 g~ as a solid, ~inf(EtOH) 240 nm (ElCm214), 260 nm (E~ml59), 266 nm (ElCml50), 272.5 nm (ElCml40) and 305 nm (El 68)with ~max at 385 nm (Elcm32)' ~max(CHBr3) 3670 (water), 3600 to 2500 (NH), 1804 (~-lactam), 1730 (C02R)j 1680 and 1513 cm (CONH).
The title compound may be converted into (6R,7R)-7-C(Z)-2-'(2-Aminothiazol-4-yl)-2-(2-carboxyprop-2-oxyimino) acetamido]-3-trimethylammoniomethvIceph-3-em-~4-car~ox~late^
as described in Example 1.

The antibiotic compounds of the invention may be formulated for administration in any convenient way, by analogy with other antibiotics and the invention therefore includes within its scope pharmaceutical compositions comprising an antibiotic compound in accordance with the invention adapted to use in human or veterinary medicine.
Such compositions may be presented for use in conventional manner with the aid of any necessary pharmaceutical carriers or excipients.
The antibiotic compounds according to the invention may be formulated for injection and may be presented in unit dose form in ampoules, or in multi-dose containers, if necessary with an added preservative. The compositions may also take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilising and/or dispersing agents. Alternatively the active ingredient may be in powder form for reconstitution with a suitable - vehicle, e.g. sterile, pyrogen-free water, before use.
If desired, such powder formulations may contain an appropriate non-toxic base in order to improve the water-solubility of the active ingredient and/or to ensure that when the powder is reconstituted with water, the pH of the resulting aqueous formulation is physio-logically acceptable. Alternatively, the base may be present in the water with which the powder is reconstit-uted. The base may be, for example, an inorganic base such as sodium carbonate, sodium bicarbonate or sodium acetate, or an organic base such as lysine or lysine acetate.
The antibiotic compounds may also be formulated as suppositories, e.g. containing conventional suppository bases such as cocoa butter or other glycerides.
For medication of the eyes or ears, the prepara-tions may be formulated as individual capsules, in liquid or semi~solid form, or may be used as drops.
Compositions for veterinary medicine may, for example, be formulated as intramammary preparations in either long acting or quick-release bases The compositions may contain from 0.1% upwards, e.g. 0.1-99%, of the active material, depending on the method of administration. When the compositions comprise dosage units, each unit should preferably con~ain 50-1500 mg of the active ingredient. The dosage as employed for adult human treatment will preferably range from 500 to 6000 mg per day, depending on the route and frequency of administration. For example, in adult human treatment 1000 to 3000 mg per day administered intravenously or intramuscularly ~hould normally suffice. In treating Pseudomonas infections higher daily doses may be required.
The antibiotic compounds according to the invention may be administered in combination with other therapeutic agents such as antibiotics, for example penicillins or other cephalosporins.

~ ~ 3 The following formulation illustrates how a compound according to the invention may be made up into a pharmaceutical composition.
Formulatin ~
Formula Per Vial (6R,7R)-7- C(2)-2-(2-Aminothiazol-4-yl)-2-(l-earboxycyelo-but-l-oxyimino)-aeetamido~-3-trimethylammoniomethyl-eeph-3-em-4-carboxylate 500 mg Sodium carbonate, anhydrous ~9 mg Method Blend the sterile cephalosporin antibiotic with sterile sodium carbonate under aseptic conditions Fill aseptically into glass vials under a blanket of sterile nitrogen.` Close the vials using rubber discs, or plugs, held in position by aluminium overseals, thereby preventing gaseous exchange or ingress of microorganisms.
Reconstitute the product by dissolving in Water for Injections or other suitable sterile vehicle shortly before administration.

, -. ~
r

Claims (11)

1. A process for the preparation of cephalosporin antibiotics of 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 R1, R2 and R3, which may be the same or different, each represents a C1-4 alkyl group)and non-toxic salts and non-toxic metabolically labile esters thereof, which comprises (A) acylating a compound of formula (II) (wherein R1, R2 and R3 are as defined above; 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 -COOR4 at the 4-position (where R4 is a hydrogen atom or a carboxyl blocking group) and having an associa-ted anion A?, with an acid of formula (III) (wherein Ra and Rb are as defined above; R5 represents a carboxyl blocking group; and R6 is an amino or protected amino group) or with an acylating agent corresponding thereto; (B) reacting a compound of formula (IV) (wherein Ra, Rb, R6, B and the dotted line are as hereinbefore defined; R7 and R7a may independently represent hydrogen or a carboxyl blocking group; and X is a leaving group) or a salt thereof with a tertiary amine of the formula (V) (wherein R1, R2 and R3 are as defined above); or (C) alkylating a compound of formula (VI) (wherein Ra , Rb , R1 , R2 , R6, B and the dotted lines are as hereinbefore defined; and R7 and R7a both represent carboxyl blocking groups to form a group of formula (wherein R1, R2 and R3 are as defined above) at the 3-position;
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 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 for the preparation of compounds of formula (I) wherein at least one of Ra and Rb represents a methyl or ethyl group.

3. A process as claimed in claim 1 for the preparation of compounds of formula (I) wherein Ra and Rb together with the carbon atom to which they are attached form a C3 5 cycloalkylidene group.

4. A process as claimed in claim 1 for the preparation of compounds of formula (I) wherein R , R2 and R3 all represent methyl groups.

5. A process as claimed in claim 1 wherein Ra , Rb, R1 , R2 and R3 all represent methyl groups.

6. A process for the preparation of (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-y1)-2-(2-carboxyprop-2-oxyimino)-acetamido]-3-trimethylammoniomethyl-ceph-3-em-4-carboxylate which comprises reacting diphenylmethyl (1S,6R,7R)-3-bromomethyl-7-[(Z)-2-(2-t-butoxycarbonylprop-2-oxyimino)-2-(2-tritylaminothiazol 4-y1)-acetamido]ceph-3-em-4-carboxylate, 1-oxide with trimethylamine, reducing the 1-oxide function and removing the protective groups.

7. A process as claimed in claim 1 wherein Ra and Rb together with the carbon atom to which they are attached form a cyclobutylidene group, and R1, R2 and R3 are all methyl groups.

8. A process for the preparation of (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-y1)-2-(1-carboxycyclobut-1-oxyimino)-acetamido]-3-trimethylammoniomethyl-ceph-3-em-4-carboxylate which comprises reacting diphenylmethyl (1S,6R,7R)-3-bromomethyl-7-[(Z)-2-(1-t-butoxycarbonylcyclobut-1-oxyimino)-2-(2-tritylaminothiazol-4-y1)-acetamido]-ceph-3-em-4-carboxylate, 1-oxide with trimethylamine, reducing the 1-oxide function and removing the protective groups.

9. A cephalosporin antibiotic of formula (I) defined in claim 1 or a non-toxic salt or non-toxic metabolically labile ester thereof, when prepared by the process of claim 1 or by an obvious chemical equivalent thereof.

10. A process for the preparation of (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-y1)-2-(1-earboxycyclobut-1-oxyimino)acetamido]-3-trimethylammoniomethylceph-3-em-4-carboxylate which comprises reacting diphenylmethyl (1S,6R,7R)-7-[(Z)-2-(1-t-butoxy-carbonylcyclobut-1-oxyimino)-2-(2-tritylaminothiazol-4-y1)-acetamido]-3-dimethylaminomethylceph-3-em-4-carboxylate, 1-oxide, iodide salt with iodomethane, reducing the 1-oxide function and removing the protecting groups.

11. A process for the preparation of (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-y1)-2-(2-carboxyprop-2-oxyimino)acetamido]-3-trimethylammoniomethylceph-3-em-4-carboxylate which comprises reacting diphenylmethyl (1S,6R,7R)-7-amino-3-trimethylammonio-methylceph-3-em-4-carboxylate, 1-oxide, hydrochloride and bromide salts with (Z)-2-(2-t-butoxycarbonylprop-2-oxyimino)-2-(2-trityl-aminothiazol-4-y1)acetic acid, reducing the 1-oxide function and removing the protecting groups.