CA1130279A - Cephalosporin antibiotics - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
Cephalosporin antibiotics of general formula

Description

2~

This invention is concerned with cephalosporin compounds possessing valuable antibiotic propertles.
The cephalosporin compouncLs in this specification are named with reference to "cepham" after J. Amer Chem. Soc., 1962, 84~ 3400, the term "cephem"
reerring to the basi~ cepham structure with one doubIe bond.
Cephalosporin an~ibiotics 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 coumpounds, and in the treat~ent o~ penicillin-sensitive patients,`
In many instances it i9 desirable to employ a cephalosporin antibiotic which exhibits activity against both gram-positive and gram-negative microorganlsms, and a signiicant ~mo~mt of research has been directed to the development o various types of broad spectr~m cephalosporin antibiotics.
Thus, for example7 in our British Patent Specification No. 1,399yO869 we describe a novel class of Gephalosporin antibiotics containlng a 7~-(a-etherified oximino)-acylamido group, the oximino group having the ~y~

. ~ ~ ; . . , -, . . . : ' '~ , ~ :

tl~

configuration~ This class o~ antibi.otic compounds is characterised by high antibacterial. activity against a range of gram-positive and gram-negative organisms coupled wi.th particularly high stability to ~-lactamases produced by various gram-negative organismsO
The disoovery of this class of coumpounds 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 NoOl,496,757, we describe cephalosporin antibiotics containing a 7~-acylamido group of the formula R~CoCO"NH~ A
" R
~O,(CH2) C (CH2)nCOOH (A) R

(wherein R is a thienyl or furyl group; RA and RB may vary widely and mayjfor example~be Cl_4 alkyl groups or together with ~he carbon atom to which they are attached form a C3 7 cycloalkylidene group, and m and n are each 0 or 1 such that the sum of m and n is 0 or 1), the compounds being syn isomers or mixtures of ~y~ and anti isomers containing at least 90% of the ~y~ isomer. The

3 position of the cephalosporin molecule may be unsubstituted or may contain one of a wide variety of possi~le substikuents. These compounds have been found to have particularly good activity against gram-negative 1~3~

organisms.
Other compounds of similar structure have been developed from these compounds in further attempts to find antibiotics having improved broad spectrum antibiotic activity a~d/o~ hlgh activity against gram-negative organismsO Such developments have involved variations in not only the 7~-acylamido group in the above fomula but also the introduction of particular groups in the 3-position of the cephalosporin molecule.
Thus, for example, in Belgian Patent Speci-fication No.852,427, there are described cephalosporin antibiotic compounds ~alling within the general scope of our British Patent Specification No. 19399,086, and where~n ~he group R in the above formula (A) m~y be replaced by a variety of diferent organic groups~
including 2-aminothiazol-4-yl~ and the oxygen atom in the oxyim;no group is attached to an aliphatic hydrocarbon group which may itself be substituted by, for example7 carboxy. In such compounds~ the substituent at khe 3~position is con~ined to an acyloxymethyl, hydroxymethyl, formyl or optionally substituted heterocyclic-thiomethyl group ~, ~ urthermore, Belgian Patent Specification No~
~36,813 describes cephalosporin compounds wherein the group R in formula (A) above may be replaced by~ for example, 2-aminothiazol-4-yl~ and the oxyimino group is a hydroxyim;no or blocked hydroxyimino group, e~gO a methoxyimino group. In such compounds, the 3-position of the cephalosporin molecule is substituted by a methyl group ~hich may itself be optionally substituted by residues of a large number of nucleophilic compo~mds therein described~ 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 i~ the SpecificationO
Belgian Patent Specification No,853,545 descri~es cephalosporin antibiotics w~erein the 7~-acylamido side chain is primarily a 2-~2~aminothiazol-4-yl)-2-( ~ -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.
We have now discovered that by an appropriate selection of a small number of particular groups at the 7~-position in combination with a methyl group at the 3-position, cephalosporin compounds having particularly advantageous activity (described in more detail bel~w) against a wide range of commonly encountered pathogenic organisms may be obtainedO
The present invention provides cephalosporin antibiotics of the general formula ~ H 11 S N
\ - / C.C~ S
N ~a ~ N ~ ~113 (-~
\ O~C COOH COOH
1b (wherein Ra and R~, which may be the s~me or difer~nt, I each represents a Gl_4alkyl group (preferably a straight ~3~

chain alkyl group i,eO a methyl, ethyl, n-propyl or n-butyl group and particularly a methyl or ethyl group) or Raand Rbtogether with the carbon atom to ~hich they are attached form a C3 7 cycloalkylidene group~ pref~rably a G3 5 cycloalkylidene group) and non-toxic salts and non~
toxic metabolically labile esters ~hereof.
The compounds according to the in~ention are ~X~
is~mer~ The ~y~ isomeric form is defined by the configuxa-tion of the group Ra - O.C~COOH
Ib with respect to the carboxamido group. In this specification the sYn configuration is denoted structurally as ,~12 S N

~ , . CO~NH -Ra O,~,,(,OOH
Rb It will be appreciated that since the compounds according to the invention are geometric isomers, some ad~
mixture wi~h the corresponding antl isomer may occurO
The invention also includes within its scope the solva~es (especially the hydrates) of the compounds of formula (I)o It also includes within its scope salts of es~ers of compo~mds o~ formula (I)o The compounds ac~ording to the present ll~D2'7~

invention may exist in tautomeric forms (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 înventionO
It ~ill also be appreciated that when Ra and R in the above formula represent different Cl 4 alkyl groups, the carbon ~tom to which they are attached will comprise a centre of asymmetry, Such compounds are diastereoisomeric and the present invention embraces individual diastereo;somers of these compounds as well as mixtures thereof~
The compounds according to the invention exhibit broad spectrum antibiotic activity. Against gram-negative organisms the activity is unusually high.
~S This high activity extends to many ~~lactamase~producing gram-negative strainsV The compounds also possess high stability to ~-lactamases produced by a range of gram-negative oxganisms, Compounds according to the invention have been found to exhibit high activity against various members of the Enterobacteriaceae (eOgO strains of F.scherichia_coli9 Klebsiella pneumoniae, Salmonella ~E~ &~ sonnei, F,nteroba~ter cloacae, Serratia marcescens Providence species Proteus mirabilis ~nd especi~lly indole-positive Proteus organisms such as Proteus and Proteus mo~ ), as well as against strains of ~ influenzae and good activity against strains of Pseudomonas organisms eOg~ strains of ~35Ys~5YL~_ ~3~ ~L o This activity against strains of indole-positive Proteus and Pseudomonas organisms is unusual7 bearing in mind the antibacterial activity of known 3-methyl cephalosporin antibioticsO

~L~3~'~

In addition to their high antibiotic activity, the 3-methy~ compounds a~cording to the present invention possess the further advantage that in comparison with analogous compounds possesslng more elaborate 3-substituents, they can be manufactured relatively easily and economically on an industrial scale from readily available s~arting materials~ iOe. penicillin G
or penicillin V.
Non toxic salt derivatives which may be formed by reaction~ofeither or hoth 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); ~mino acid salts (e~g. lysine and arginine salts); organic base salts (e.gO procaine, phenylethylbénz~amlne, diben~yle~hylenediamine, ethanolamlne, diethanolamine and N-methylglucosamine salts30 Other non-toxic salt derivatives include acid addition salts, e.g. ~ormed with hydrochloric, hydrobromic, sulphuric, ~o nitric, phosphoricg formic and trifluoroacetic acids The salts ~ay also be in the form of resinates formed with, for example, a polyst~rene resin or cross-linked polystyrene dlvinylbenzene copolymer resin containing amino or quaternary amino groups, sulphonic acid groups~ or with a resin containing carboxyl groups9 e~g~ a polyacrylic acid resin~ Soluble base salts ~eOg~ alkali metal salts such as the sodium salt) of compounds of formula ~I) may be used in therapeutir applications because of the rapid distribution of such salts in the body upon administrationO
Where, ho~ver, insoluble salts of compounds ~I) are desired ~L3~

in a particular application, e.g. for use in depot preparations, such salts may be formed in conventional m~nner, for example with appropriate or~anic amines.
mese and other salt derivatives such as the salts with toluene-p-sulphonic and methanesulphonic acids may be employed as intermediates un the pre-paration and/or purification of -the present ccmpoullds of formula (I)l for example in the processes described below.
Non-toxic me-tabolically labile ester derivatives which may be formed by esterification of either or both car~oxyl groups in the parent co~pound of formLla (I) include acyloxyalkyl esters, e.g. lcwer alkanoyloxy-methyl or -ethyl esters such as aoetoxy-~ethyl or -ethyl or pivaloyloxymethyl esters. In addi-tion to the above ester derivatives~ the present invention includes within its sccpe oompounds of formula (I) in the form of other physiologically acceptable equlvalents, i.e. physiol~gically acceptable compounds which like the metabolic-ally labile esters, are converted in vivo into the parent antibiotic compound of formula (I).
Preferred compounds according to the present invention include the following co~pounds 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-carbo~yprop-2-oxyimino) acetamido]-3-methyloeph-3-emr4-carboxylic acid;
(6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1-carboxycyclobut-1-oxyimlno) acetamido]-3-methylceph-3-em~4-carboxylic acid; and : '~;`!

;~
.:

(6R,7R)-7-[(Z)-2-(2~aminothiazol-4-yl)-2-(1-carboxycyclopent-1- :~
yloxyimino) acetamido]-3-methylceph-3-em-4-carbcxylic acid. ~;
Other examples of oompounds of form~la ~I) accoxding to the present invention include those wherein the groups Ra and ~ have the meanings given in the following Table TABLE ~:~

Ra Rb ..
a) Alkyl groups : b) Cycloalkylidene groups cyelopropylidene cyelohexylidene The eompounds of formula (I) may be used for treating a variety of - diseases caused by pathogenie bateria in human beings and anim31s, sueh as respiratory tract infections and urinary tract infeetions.
Aeeording to another embodiment of the invention we provide a proaess for the prep#ration of an ~1 ;
:
~- - g _ ,.~ : . .
?:~ , ' : ~

- 10 _ antibiotic compound of ge~eral foxmula (I) as hereinbefore defined or a non-toxic salt or non-toxic meta~olically labile ester thereof wh;ch cornprises acylating a compound of the formula H H
H2N ~ B ~ (II) ~N ~CH3 1~ 1 COOR

~whereln ~ is ~S or ~S~O ~c~- or ~-); Rl represent:s h~drogen or a carboxyl blocking group, e~g, the r~sLdue of an ester-fo~ning aliphatic or aralipha~ic alcohol or an ester-forming phenol, 5ilanol or stannanol (the said alcohol, phenol~ silanol or stannanol preferably containing 1-~0 carbon atoms) a~d the dotted line bridging the 2~ 9 3-, and 4-pos~tions indicates that the compound is a ceph-2-em or ceph-3-em cornpound~ or a salt, e.gO an acid addition salt (formed with~ for example, a mlneral acid such as hydrochloric~ hydrobromic~ sulphuric~ nitric or phosphoric acid or an organic acid such as methanesul-phonic or toluene-p-sulphonic acid) or an N-silyl derivative thereo~, with an acid of formula ~3~

S N
.COOH
N Ra . (III) OOR

R
.

(wherein Ra and Rb are as hereinbeore de~ined; R2 represents a carbox~l blocking group~ e.g9 as described for R ; and R3 i9 an amino or protected amino group) or with an acylating agen~ corresponding thereto whereafter, if necessary andjor desired in each instance?
any of the followi~g reactionsD in any appropriate sequence, are carried out:-- i) con~ersîon ofa Q2-isomer into the desire~.~3-isomer, - ii) reduction of a compound wherein B is ~S~ O to form a compound wherein B is ~S, iii)conversiorl of a carboxyl group into a non-toxic salt or mekabolically labile ester unction, and iv) removal of any carboxyl blocking and/or N-protecting : . groups~
In the above~described process~ the starting material of formula ~II) is preferably a compound wherein B is ~S and the dotted line represents a ceph-3~em compound.
Acylating agents which may be employed in the preparation of compounds of fo~mllla ~I) include acid .

~3~

halides, particularly acid chloride~ or bromides. Such acylating agents may be prepared by reacting an acid (III) or a salt thereof with a halogenating age.nt e~g. phosphorus pentachloride~ thionyl chloride or oxalyl chloride Acylations employing acid halides may be effected in aqueous an~ non-aqueous reaction media, convenie~tly at temperatures of from ~50 to ~50C9 preferably -20 to ~30C, if desired in the presence of an acid binding agent. Suitable reaction media include aqueous kPtones such as aqueous acetone, esters such as ethyl acetate, halogenated hydrocarbons such as methylene chloride9 amides such as dimethylacetamide, nitriles such as acetonitrlle, or mixtures of two or more such solvents.
Suitable acid b.inding agents incl~lde tertiary amines (eOg~ triethylamine or dimethylaniline)~ i.norganic bases ( eOg~ calci~m carbonate or sodi~n bicarhonat:e) 7 and oxiranes such as lower 1~2~-allcylene oxides (e~gO ethylene oxide or prop~lene oxide3 which bind hydrogen halide liberated in the acylation reaction.
Acids of formula SIII~ may themselves be used as acylating agents in the preparation of compounds of formula (I). Acylations employing acids (III) are desirably conducted .in ~he presence of a condensing agent, for example a carbodiimide such as N,N'-dicyclohexyl-carbodiim1de or N~ethyl~N~-y-dimethylaminopropyl-carbodiimide; a carbonyl compound such ascarbonyldiimidazole; or 2n ~soxazoliu~ salt such as N-ethyl-5-phenylisoxazolium pe-rchlorateO
Acylation may also be effected with other amide-forming derivatives of acids of formula (III~ such as~ for example~ an activated ester9 a symmetrical `s ~3~

anhydride or a mixed anhydride (e.g. forr~3d with pivalic acid or with a halo-formate, such as a lcwer alkylhaloforrnate). Mixed anhydrides may also be forr~d with phosphorus acids (for example phosphoric or phosphorous acids), sulphuric acid or aliphatic or arcmatic sulphonic acids (for example toluene-p-sulphonic acid). An activated ester may conveniently be formed in situ using, for example, l-hydroxybenzotriazole in the presence of a condensing agent as set out above.
- Alte m atively, the activated estex may be preformed.
Acylation reactions involving the free acids or their above-nEntioned amide-forming derivatives are desirably effected in an anhydrous reaction medium, e.g. methylene chloride, tetrahydrofuran, dimethylformamide or aoetonitrile.
If desired, the acylation reactions mc~y be carried out in the presence of a catalyst such as 4~dimethylaminopyridine.
The acids of formula (III) and acylating agents corresponding thereto may, if desired, be prepared and employed in the orm of their acid c~ddition salts. Thus, or example, acid chlorides mc~y conveniently be employed as their hydrochloride salts, and acid bromides as their hydrobromide salts.
The reaction product may be separated from the rec~ction mixture, which may contain, for example, unchanged caphalosporin starting material and other substances, by a variet~ of prccesses incl~lding recrystallisation, ionophoresis, column chrcmatography and use of ion-exchangers (for example by chromatography on ion-exchange resins) or macroreticular resins.

~ - 13 -~9 3C~79 ~ -Cephalosporin ester derivatives obtained in accordance ~ith the pro-oess of the invention may be converted into the corresponding Q3-derivatives by, for example, treatment of the A -ester with a base, such as pyridine or triethyl-amine.
A ceph-2-em reaction product may also be oxidised to yield the corres-ponding ceph-3-em l-oxide, for example by reaction with a peracid, e~g. per-acetic or m-chloroper~enzoic acid; the resulting sulphoxide may, if desired, sub-sequently be reduoed as described hereinafter -to yield the corresponding ceph-3-em sulphide.
Where a ccmpovnd is obtained in which B is ~O -this may be converted to the corresponding sulphide by, for example, reduction of the corresponding acyloxysulphonium or alkoxysulphonium salt prepared in situ by reactioll with e.g.
aoetyl chloride in the case of an acetoxysulphonium salt, reduction being effected by, Eor example, sodium dithionite or by iodide ion c~s in a solution of potassium iodide m a water-miscible solven-t e.g. acetic acid, acetone, tetra-hydrofuran, dioxan, dimethylformamide or dirnethylacetamide. me reaction may ke effected at a temperature of from -20 to +50 &.
Metabolically labile ester derivatives of the compounds of formwla (I~
rnay be prepared ky reacting a compound of form~la (I) or a salt or protected derivative thereof with the appropriate esterifying agent such as an acyloxy-alkyl halide (e.g. iodide) conveniently in an inert organic solvent such as dimethylformamide or acetone, followed, where necessary, by removal of any pro-tecting groups.
Base salts of the compounds of formula (I) rnay be formed by reacting ~. ., , .
;, .

~L3~ 79 an acid of ormula (I) with the appropriate base. Thus, for exc~mple, 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 camr pound of formula (I) or a metabolically labile ester derivative thereof with the appropriate acid.
: Where a cQmpound of formula (I) is obtained as a mixture of isomers, the syn isomer may be obtained by, for example, corlventional methods such as : crystallisation or chrQmatography.
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 syn isomers form or in the form of mixtures of the syn isomers and the cor:resp~nding anti isQmers containing at least 90% of the _y~ isomer are preferably used.
Acids oE forrnula (III) (provided that Ra and ~ together with the carbon ato.m to which they are attached do not form a cyclopropylidene group) may be prepared by etherification of a compound of formula (IV) S N

.COOR

N\ OH

. ' - ' ~ .:

.

~L~3~'~9 (wherein R3 is as herein~efore defined and R represents a carboxyl blocking group), by reaction with a compound of general formula Ra T.C.COOR (V) R
(wherein Ra and Rb are as hereinbefore defined and T is halogen such as chlor~, bromo or iodo; sulphate; or sulphonate such as tosylate), follcwed 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 m the presence of a base, e.g. potassium carbonate or sodium hydride, and is preferably oonducted 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 oxyinuno groups 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 (IV~ is used. The base should be used in sufficient quantity to neutralise rapidly the acid in question.
Acids of general formula (III) may also be prepared by reaction of a ocmpound of formula ':' ' , : -, R

S N
~ ~ CO.COOR

(wherein R3 and R4 are as hereinbefore defined) wi~h a ccmpound of formula Ra H2N.O.C.COOR (VII) R

(wherein Ra, Rb and R2 are as defined above), followed by removal of ~he carboxyl blocking group R4, and where necessary by the separation of ~y~ and anti isomers.
The last mentioned reaction is particularly applicable to the prepara-tion of acids of formula (III) wherein Ra and Rb together with the carbon atom to which they are attached Eorm a cyclopropylidene group. In this case, the relevant compounds of formula (VII) 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 c æboxylate.
The acids of formula (III) may be converted to the correspo~ding acid , ~

` ~L3 ~'~

halides and anhydrides and acid addition salts by conventional methods.
It should be appreciated that in same of the above transformatians it may be ne oe ssars~ to protect any sensiti~e groups in the molecule of the com-pound in question to avoid undesirable side reactians For example, during any of the reaction sequen oes referred to above it may be ne oessary to protect the NH2 group of the ~ninothiazolyl moietyr for example by tritylation/ acylation (e.g. chloroaoetylation), protonation or other conventional method. The protect-ing group may thereafter be removed in any convenient wa~ which does not cause breakdown of the desired comQound, 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~ hydro-chloric acid or mixtures of such acids, preferably in the presence of a protic solvent such as water, or in the case of a chloroacetyl group, by treatment with thiourea.
Carbo~yl blocking groups used in the preparation of compounds of formula ~I) or in the pr~paration of necessary starting materials are desirably groups which may readily be split off at a suitable stage in the reaction sequence, conveniently at the last stage~ It may, hcwever, be convenient in some instances to e~,ploy non-toxic metabolically labile carboxyl blocking groups such as acyloxy-methyl or -ethyl (e.g. acetoxy-methyl or -ethyl and pivaloyloxy-methyl) and retain these in the final prDduct to give an appropriate ester derivative of a compound of formula (I).

~3~;27~

- lg -Suitable carboxyl blocking groups a~e well known in the art, a list of representative blocked carboxyl groups being included in British Patent NoO
1,399,086~ Preferred blocked carboxyl groups include 5 aryl lower alkoxycarbonyl groups such as p methoxybenzyl-oxycarbonyl, p-nitrobenzyloxycarbonyl and diphenyl-methoxycarbonyl; lower alkoxycarbonyl groups such as t-butoxycarbonyl; and lower haloalkoxycarbonyl groups such as 2,2,2-trichloroethoxycarbonyl. Carboxyl blocking group(s) may su~sequently be removed by any of the appropriate methods disclosed in the literature; thus, for example, acid or base catalysed hydrolysis is applicable in manycases, as are enæymically-catalysed hydrolyses, The antibiotic compounds of the invention may be formulated for administration in any convenient way9 by analogy with other antibiotics and the invention therefore includes within its scope pharmaceutical composikions comprising an antibiotic com~ound in accordance with the invention adapted for use in human or veterinary medicineO Such compositions may be presented for use in conventional rnanner with the aid of any necessary pharmaceutical carriers or excipients.
The antibiotic compounds according to the invention ma~ be ormulated for injection and may be presented in unitdoseform in ampoules, or in multi dose containers~ if necessary with an added preservative.
The compositions may also take such forms as suspensions9 solutions, or emulsions in oily or aqueous vehicles9 and may contain formulatory agents such as suspending, . .

stabilising and/or dispersing agen-ts. Alternatively the active ingredient may be in powder form for reconstitution with a sui-table vehicle, e.g. sterile, pyrogen-free water, before use.
If desired, such p~wder fonmulations may contain an appropriate non-toxic base in order to improve the water-solubility o~ -the active ingredient and/or to ensure that when the powder is reconstituted with water, the pH of the resulting aqueous formulation is physiologically acceptable. Alternatively, the base may be present in the water with which the pcwder is reconstituted. The base may be, for example, an inorganic base such as sodium carbonate, sodium bic æbonate 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 oonventional suppository bases such as cocoa butter or other glycerides.
Compositions for veterinary medicine may, for example, be form~lated as intramammary preparations in either long acting or quick-release bases.
The ccmpositions may oontain from 0.1% upwards, e.g. 0.1-99% of the active material, depending on the method of administration. When the composi-tions co~lprise dosage unitsr each unit will preferably contain 50-1500 nxJ of the active ingredient. The dosage as employed for adult human treatment will pre-ferably range from 500 to 6000 mg per day, depending on the route and frequency of administration. For example, in adult human treatment 1000-3000 mg per day administered intravenously or :
~ : .

-21~

intramuscularly will normally suf~iceO In treating infections higher daily doses may be required.
The antibiotic compou~ds according to the invention may be administered in comb:ination 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).

~11 3~ 79 Preparation 1 Ethyl (Z)-2-(2-aminothiazol-4-yl)-2-(hydroxyimino)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 (400 ml) at such a rate that the reaction teMperature was maintained below 10C. Stirring and cooling were continued for abo~lt 30 min., when a solution of potassium chloride (160 g) in water (800 ml) was a~ded. The resulting mLxture was stirred for one hour~ The lower oily phase was separated and the a~ueous phase was extracted with diethyl ether. The extract was ocmbined with the oil, washed successively with water and saturated brine, dried, and evaporated. me residual oil, which solidified on standing, was washed with petrol and dried Ln va over potassium hydro~ide, giving ethyl (Z)-2-(hydroxyimino)-3-oxobutyrate (309 g)-A stirred and ice-oooled solution of ethyl (Z)-2-(hydroxyimino)-3-oxobutyrate (150 g) in dichlorcmethane (400 ml) wa~s ~reated dropwise with sulphuryl chloride (140 g). m e resulting solution was kept at room temperature for 3 days, then evaporated. The residue w~s dissolved in diethyl ether, washed with water until the washings were almost neutral, dried, and evaporated. The residual oil (177 g) was dissolved in ethanol (500 ml) and dimethylaniline (77 ml) and thiourea (42 g) were added with stirring. After two hours, the pro-duct was collected by filtration, washed with ethanol and dried to give the title compound (73 g); m.p. 188 (deoomp.).

-~ .

~L3~

Preparation 2 Ethyl (Z)-2-hydroxyimino-2-(2--tritylaminothiæ ol-4-yl)-acetate, hydrochloride l~ityl 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 triethyL1mine (8.4 ml). me mixture was allcwed to warm to 15 over one hour, stirred for a further 2 hours and then partitioned between water ~500 ml) and ethyl aoetate (500 ml). me 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-oxylmino)-2-(2-tritylaminothiazol-4-yl) acetate Potassium carbonate (34.6 g) and t-butyl 2-brcmo-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. The mixture was poured into water (2 1), stirred for 10 mins., and filtered. The solid was washed with water and dissolved in ethyl acetate (600 ml). The solution was washed successively with water, 2N hydrochloric acid, water, and saturated brine, dried, and evaporated. The residue was recrystallised frcm petroleum ether (b.p~ 60-80) to give the title compound (34 g), m.p. 123.5 to 125.

. ~, ., ~ ~ , .

Preparation 4 (Z)-2-(2-t-Butox~carbonylprop-2-oxyimino)-2-(2-tritylaminothiazol-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. me mixture was refluxed for 1.5 hours and then concen-trated. The residue was taken up in a mlxture of water (50 ml), 2N hydrochloric acid (7 ml), and ethyl aoe tate (50 ml). m e organic phase was separated, and the aqueous phase extracted with ethyl acetate. The organic solutions were cambined, washed suc oessively with wa-ter and saturated brine, dried, and evaporated. m e residue was recrystallised frcm a mixture of caxbon tetrachloride and petrol to give the title ccm~ound (1 g), m.p. 152 to 156 (decamp).
Prep æ ation 5 Ethyl (Z)-2-(2-tritylam mothiazol-4-yl)-2-tl-t-butoxycarbonylcyclobut-1-oxyimino) aoetate The product of Preparation 2 (55.8 g) was stirred under nitrogen in dimethylsulphoxide (400 ml) with potassium carbonate (finely ground, 31.2 g) at roam temperature. After 30 minutes, t-butyl l-bramocyclobutane 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-bramocyclobutane 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 washed well with ~3~9~

water and petrol. The solid was dissolved in ethyl acetate and the solution washed with brine (twice), dried with magnesium sulphc~te and evaporated to a foam. mis 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).
Preparation 6 (Z)-2-(1-t-Butoxycar~onylcyclobut-l-ox_1mino)-2-(2-tritylaminothiazol-4-yl) acetic acid :
A mi~ture of the product of Preparation 5 (3.2 g) and potassium carbonate (1.65 g) was refluxed in methanol (180 ml) and water (20 ml) for 0 hours and the mixture was cooled to roam temperature. The mixture was concen-trated 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 wer~ washed with saturated brine, dried and evaporated to give the title compound (2.3 g); ~max (ethanol) 265 nm OElcm 243).
Preparation 7 Ethyl(Z)-2-(1-t-butoxycarbonylcyclopent-1-yloxyimino)-2-(2-tritylam~nothiazol-4-yl)a oe tate me product of Preparation 2 (10 g) was stirred with t-b~Ityl 2-bromo-cylcpentanecar~oxylate (7 g) in dimethylsulphoxide (40 ml) containing potassium carbonate (10 g) under nikrogen ak 21 for 21 hours. The mixture was poured into ice-water (500 ml) and -the grey solid was collected by filtration, washed wikh water and air dried.

~36~27~

2~ -Recrystallisation of this solid from methanol ~500ml~ gave the title ~ (11~7g)~ m.pO 179-~180, vmax ~CHBr3) 3410 (NH), 1735 (ester)~ 1275 (ester) and 755 cm ~phenyl)~
~.
S
~.
The Product rom Preparation 7 (625mg) was refluxed with 2N sodium hydroxide solution (O.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 adjust the solution to ' pH 2O The precipitate was extracted with ether and the combîned extracts were washed with brine~ Ater drying with magnesium sulpha~e, the solven~ was evaporated to g;.ve a gum (493mg), Recrystallisation from di-isopropyl ether gave the title ~ (356mg) mOpO 171-173, ~m~x (CHBr3) 2500-3500 (OH and NH), 1755 (ester), 1692 (acid) and 755 and 770 cm (phenyl)O

:, Example 1 r~ony~prop-2 ox~imino~-2- ~-trit~laminothiazol 4-Yl? acetamido~-3 m ~
A stirred solution of the product of Preparation 4 (2.~6 g) and diphenylmethyl (6R, 7R~-7 amino-3-methyl-ceph-3-em-4-carboxylate (2~09 g) in dimethylformamide (50 ml) was cooled to 0' and treated with hydroxybenzo-triazole ~745 mg) and dicyclohexylcarbodiimide (1.14 g)O
The mixture was warmed to room temperature and stirred overnight. The mixture was filtered, and the white solid washed with a little ethyl acetate. The filtrate and washings were dilutecl with water (100 ml) and extracted with ethyl acetate, The organic extracts were combined, washed successively with 2N hydrochloric acid, water, sodium bicarbonate solution, and saturated brine, dried, and evaporated. The residue was eluted through a silica column with ethyl acetateO The product containing eluate was collected and concentrated to give the_title (3.6 g)~Amax(ethanol) 238 nm (El 283~, A inf 263.5 nm (E1%186); ~ D0 ~ 6.5- (c 1.0, DMSO).
b ~ )-7-~ Z)-2- ~ 4 ~ 2~ 2-carboxyprop-2-oxyimino) acetamido ~ -4-carbox~lic acid _ Trifluoroacetic acid (20 ml) was added to a solution of the product of stage a) (2.8 g) in anisole (20 ml) at 0-.
The mixture was stirred at room temperature for 2 hours and concentrated. The residue was dissolved in ether and re-evaporated, The residue was dissolved in ethyl acetate and exLracted with saturated sodium bicarbonate solution.
:

- 2~ ~

The pH of the aqueous ex~racts wa~s adjuted to 6, and the solut;on washed with ethyl acetate The aqueous phase was acidified to pH 1 .S under ethyl acetate 9 and extracted with ethyl acetate The combined org~nic extracts were washed with saturated briney dried 9 and evaporated The residue was dissolved in warm 75% aqueous formic acid (40 ml) and allowed to stand for 1 hour~ The mixture was diluted with water and filtered, The filtrate was concentrated, The residue was taken up in water, refiltered, and lyophilized to give ~ ~md ; (900 mg), ~. f(pH 6 buffer) 236 nm ~El~ 287)~ 261 nm n ~O/ c (El' 254), 296 nm (ElCmll5); vmax (Nujol~ 1530, 1665 (CONH), 1720 ~C02H), 1765 cm (~-lactam~O
Exc~mple 2 ~L~L~
cyclob t-l-ox~imino)-2-~2-tritylAminothiAzol~4 ~:L~-_ acetamido1-3~ L ~ ~w ~ç~b~x~
A stirred solution of the product of Preparation 6 (2.2 g) and diphenylmethyl ~6R~7R~-7-amino-3-methylceph~
3-em-4~carboxylate (2.2 g) in dimethylformamide (45 ml) was cooled to 0-~ and l-hydroxybenzotriazole (655 mg)was added, followed by dicyclohexylcarbodiimide (982 mg)O
The mixture was warmed to room temperature and sti.rred overnight. The mixture was filtered. The filtrate was diluted with water (300 ml) and extracted with et.hyl acetate.
The organic extracts were combined, washed successively with 2N hydrochloric acid, sodium bicarbonate solution, and saturated brine, dried, and evaporated. The residue was eluted through a silica column with ether-petrol (3-1).
The app~opriate ractions were concen~rated to give ~3~27~

the title compound ~12vjl g~, Ainf (~ElCm238), 302 nm (ElCm 67); v max (CHBr3) 1520, 1682 (CONH), 1722 (C02R), 1788 cm ~ lactam).
b~ ~6R~7~)-7-r~2)-2-(2-Aminothiazol-4-yl?-2~ carboxy ~ lceph-3-em-4-carbox~lic acid Trifluoroacetic acid (8 ml) was added to a solution ~-~ of the product of Stage a)(l.9 g) in anisole (2 rnl) at 0-.
The mixture was stirred for 5 minutes and trifluoroacetic acid (32 ml) was added. Tne mixture was stirred at room temperature for 30 minutes and concentrated. The residue was dissolved in ethyl acetate and re-evaporated. The : residue was dissolved in ethyl acetate ~nd extracted with saturated sodiurn bicarbonate solution, The aqueous extracts, at pH 7 to 7,5, were washed with ethyl acetate, acidified to pH 1.5 under ethyl acetate, and extrac~ed with ethyl acetate. The combined organic extracts were dried and ; evaporated. The residue was dissolved in formic acid (30 ml), water (9 ml) added, and the mixture stirred at room temperature ~or 2 hours. The mixture was diluted with water (200 ml) and filtered. The filtrate was concentrated.
The residue was taken up in water, refiltered, and lyo-philized to give the title ~e~ (730 mg)9 Amax (pH 6 bufer) 241 nm (E~% 272), Ainf252 nm (ElCm 269), 291 nm (ElCm 129); [a~ D ~ 62.5- (c 1 0, DMSO).
E~e~
,, a) Diphenylmethyl ~6R~7R)-7-r~Z)-2-(l-t-butoxycarbonylcyc-lo,Pent-l-yloxyimino~-2-(2~trit~1aminothi.azol 4-yl)acetamido o Th~ product of Preparation ~ (lg) was dissolved in tetra-- hydrofuran ~25ml) and diphenylmethyl (6R,7R)-7 amino-3-~L~3~;~7~

methylceph-3 em-4~carbo~ylate (76~mg~ and l~hydroxybenzo-triazole hydrate (380mg) were added with stirring. When a clear solution had formedg dicyclohexylcarbodiimide (520mg) was added and the mixture was stirred at 21~ for 24 hours. The solution was filtered and the filtrate was concentr~ted, This solution was percolated through a short column of neutral alumina (30g) with elution by ethyl acetate: 60~-80~ petroleum ether (1~3). Ihe product was loaded onto a column of silica ~kieselgel 60G of 15~M
particle size; SOg), This column was eluted with ethyl acetate: 60-80- petroleum ether (1:3) at a pressure of 81b/sq. in. and e~aporation of the appropriate fractions gave the title compound ~SlOmg) as a oam, ~JD
~ 25-2 ( 0~95, CMC13), vmax (CHBr3) 3405,3275 ~NH), 1790 (~-lactam), 1727 (esters), 1683 and LS27 cm~l (amide).
~ othiazol~y~l) ~ rboxy~yc-carboxylic acid,_trifluoroace ate salt.
The product from Stage a) (408mg) was mixed with anisole (0,3ml) and trifluoroacetic acid (2ml~ was added.After 2 hours at 21-, the solution was concentratecl and the residue was poured ;nto water (20ml). This mixture was washecl with ether (three times) each time the ether being back extracted with water, The combined a~ueous layers were evaporated to dryness and the residue was triturated with ether to give the ~L~ (88mg) Am~X ~pH6 buffer) 239,5nm (E %304)~ A, 290 (E V/ol3l~ v (NuJol) 3700-, lcm lnfl J l~m max -1 2100 (NH3, NH and OH), 1770 ~-lactam~ and 7 680 cm (amide and acid)O

~3~'æ7~

PHARMACY ~AMPLES

Formula Per Vial (6R, 7~)-7-~(Z)-2-(2-Aminothia~ol 4 yl)-2-(2-carboxyprop 5 -2-oxyimino)acetamido]-3-methylceph 3-em-4-carboxylic acid 500mg Sodium Carbonate3 anhydrous 113mg Method Blend the sterlle cephalosporin antibiotic with sterile 10 sodium carbonate under aseptic conditions. Fill.
aseptical~y 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 micro-organi.sms.
Reconstitute the product by dissolving in Water for Injections or other suitable sterile vehicle shortly before administrat ion .

EXAMPLE B - Dry ~
Fill sterile (6R, 7R)-7-[(~)-2-(2-aminothiazol-4 yl) ~2-(1-carboxycyclob~t-1-oxyimino)acetamido]~3-methylceph-3-em-4-carboxylic acid9 disodium salt into glass vials such that each vial contains an amount equivalent to 500mg of the antibiotic acidO Carry out the filling aseptically under a blanket of sterile nitrogen, Close the vials using rubber discs or plugs, held in position by aluminium overseals 9 thereby :L~3~Z7~

.

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.

Claims (13)

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 C3-7 cycloalkylidene group) and non-toxic salts and non-toxic metabolically labile esters thereof, characterised in that a compound of formula (II) wherein B is >S or >S?O, R1 represents hydrogen or a carboxyl blocking group and the dotted line in formula (II) bridging the 2-, 3- and 4- positions indicates that the compound is a ceph-2-em or ceph-3-em compound, or an acid addition salt or N-silyl derivative thereof, is acylated with an acid of formula (III) (wherein Ra and Rb are as defined above; R2 represents a carboxyl blocking group; and R3 is an amino or protected amino group) or with an acylating agent corresponding thereto, whereafter, where required in each instance, any of the following reactions, are carried out, in any appropriate sequence:-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 characterised in that there is employed a compound of formula (III) in which at least one of Ra and Rb represents a methyl or ethyl group.

3. A process as claimed in claim 1 characterised in that there is employed a compound of formula (III) in which 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 characterised in that there is employed a compound of formula (III) in which both Ra and Rb represent methyl groups.

5. A process for the preparation of (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(2-carboxyprop-2-oxyimino) acetamido]-3-methylceph-3-em-4-carboxylic acid, characterised in that diphenylmethyl (6R,7R)-7-amino-3-methylceph-3-em-4-carboxylate is acylated with (Z)-2-(2-t-butoxycarbonylprop-2-oxyimino)-2-(2-tritylaminothiazol-4-yl)acetic acid in the presence of 1-hydroxybenzotriazole and dicyclohexylcarbodiimide, whereafter the diphenylmethyl, t-butoxy and trityl groups are removed.

6. A process as defined in claim 1 characterised in that there is employed a compound of formula (III) in which Ra and Rb together with the carbon atom to which they are attached form a cyclopentylidene group.

7. A process for the preparation of (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1-carboxycyclopent-1-yloxyimino) acetamido]-3-methylceph-3-em-4-carboxylic acid, characterised in that di-phenylmethyl (6R,7R)-7-amino-3-methylceph-3-em-4-carboxylate is acylated with (z)-2-(l-t-butoxycarbonylcyclopent-l-yloxyimino)-2-(2-tritylaminothiazol-4-yl)acetic acid in the presence of l-hydroxybenzotriazole and dicyclohexylcarbodiimide, whereafter the diphenylmethyl, t-butoxy and trityl groups are removed.

8. A process as defined in claim 1 characterised in that there is employed a compound of formula (III) in which Ra and Rb together with the carbon atom to which they are attached form a cyclobutylidine group.

9. A process for the preparation of (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1-carboxycyclobut-1-oxyiminoo) acetamido]-3-methylceph-3-em-4-carboxylic acid, characterised in that diphenylmethyl (6R,7R)-7-amino-3-methylceph-3-em-4-carboxylate is acylated with (Z)-2-(1-t-butoxycarbonylcyclobut-1-oxyimino)-2-(2-tritylaminothiazol-4-yl)acetic acid in the presence of l-hydroxybenzotriazole and dicyclohexylcarbodiimide, whereafter the diphenylmethyl, t-butoxy and trityl groups are removed.

10. A process as claimed in claim 1, 2 or 3 characterised in that the cephalosporin antibiotic is recovered in the form of a sodium, potassium or calcium base salt or a hydrochloride acid addition salt of a compound of formula (I).

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

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

13. 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.