IE49171B1 - Cephalosporin antibiotics - Google Patents

Abstract

Cephalosporin antibiotics of the general formula in which R and R, which are identical to or different from one another, each denote a C1-4-alkyl group or R and R, together with the carbon atom to which they are bonded, form a C3-7-cycloalkylidine group, have a good broad spectrum antibiotic activity, this activity being unusually high against gram-negative organisms such as strains of Pseutomonas organisms. The invention also comprises non-toxic salts, non-toxic metabolically labile esters and solvates of the compounds of the formula (I) and also non-toxic salts of the already mentioned esters. Compositions are also described which contain the antibiotics according to the invention, and processes for the preparation of antibiotics of this type either by appropriate 7-N-acylation or by appropriate O-carbamoylation of the hydroxymethyl group in the 3-position.

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.

Soc., 1962, 84> 3400, the term cephem referring to the basic cepham structure with one double bond.

Cephalosporin antibiotics are widely used in the treatment of diseases caused by pathogenic bacteria in human beings and animals, and are especially useful in the treat10 ment of diseases caused by bacteria which are resistant to other antibiotics such as penicillin compounds, and in the treatment of penicillin-sensitive patients. In many instances it is desirable to employ a cephalosporin antibiotic which exhibits activity against both gram-positive and gram-negative microorganisms, and a significant amount of research has been directed to the development of various types of broad spectrum cephalosporin antibiotics.

Thus, for example, in our Patent Specification No. 38172, we describe a novel class of cephalosporin antibiotics containing a 7p-(a-etherified ojyimino)-acylami no group, the oxyimino group having the syn configuration, ibis class of antibiotic compounds is characterised by high antibacterial activity against a range of gram-positive and gram-negative organisms coupled with particularly high stability to β-lactamases produced by various gram-negative organisms. 48171 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 gram5 negative organisms.

For example, in our Patent Specification No. 39764 we describe cephalosporin antibiotics containing a 3-carbamoyloxymethyl group and a 7p-acylamino group of the formula R1.C.CO.NH\ (A) ^\)R2 2 1° (wherein R is a furyl, thienyl or phenyl group and R is a alkyl group, a C^-C? cycloalkyl group or a phenyl group), the compounds being syn isomers or existing as mixtures of syn and anti isomers containing at least 90% of the syn isomer. These compounds exhibit high anti15 bacterial activity against a broad range of gram-positive and gram-negative organisms and particularly high stability to β-lactamases produced by various gram-negative organisms.

Furthermore, in our Patent Specification No. 42144, *e describe cephalosporin antibiotics containing a 7β-acylamino group of the formula R.C.CO.NH«« N ra °- Other compounds of similar structure have been developed from these compounds in further attempts to find antibiotics having improved broad spectrum antibiotic activity and/or high activity against gram-negative organisms.

Such developments have involved variations in not only the 7p-acylamino group in the ahove formulae but also the introduction of particular groups in the 3-position of the cephalosporin molecule.

Thus, for example, in Belgian Patent Specification No. 852,427, there are described cephalosporin antibiotic compounds falling within the general scope of our Patent Specification No. 38172, and wherein the group R in formula (B) ahove may he replaced by a variety of different organic groups, including 2-aminothiazol-4-yl, and the oxygen atom in the oxyimino group is attached to an aliphatic hydrocarbon group which may itself be substituted by, for example, carboxy. In such compounds, the substituent at the 3-position· is· an acyloxy30 methyl (for example, carbamoyloxymethyl), hydroxymethyl, formyl or optionally substituted heterocyclic-thiomethyl group.

Furthermore, Belgian Patent Specification No. 836,813 describes cephalosporin compounds wherein the group R in formula (B) above may be replaced by, for example, 2-aminothiazol-4-yl, and the oxyimino group is a hydroxyimino or blocked hydroxyimino group, e.g. a methoxyimino group .

In such compounds, the 3-position of the cephalosporin molecule is substituted by a methyl group which may itself be optionally substituted by any of a large number of residues of nucleophilic compounds therein described, an example of such a substituted methyl group being given as a carbamoyloxymethyl group which may itself be substituted by an alkyl group, e.g. a methyl group. In the above-mentioned specification no antibiotic activity is ascribed to such compounds which are only mentioned as intermediates for the preparation of antibiotics described in that specification.

Belgian Patent Specification No. 853,545 describes cephalosporin antibiotics wherein the 7p-acylamino side chain is primarily limited to a 2-(2-aminothiazol-4-yl)-2-(syn)methoxyimino-acetamido group and the substituent in the 3position is broadly defined in a similar manner to that in the above-mentioned Belgian Patent Specification No. 836,813. Compounds specifically exemplified in the Specification include compounds in which the 3-position is substituted by a carbamoyloxymethyl radical.

We have now discovered that by an appropriate selection of a small number of particular groups at the 7β-position in combination with a carbamoyloxymethyl group at the 3position, cephalosporin compounds having particularly advantageous activity (described in more detail below) against a wide raqge of coiranonly encountered pathogenic organisms may be obtained.

The present invention provides cephalosporin antibiotics of the general formula a b (wherein R and R , which may be the same or different, each represents a alkyl group (preferably a straight-chain alkyl group i.e. a methyl, ethyl, n-propyl or n-butyl group, a b and particularly a methyl or ethyl group) or R and R together with the carbon atom to which they are attached form a C^_y cycloalkylidene group, preferably a ¢3.5 cycloalkylidene group) and non-toxic salts and non-toxic metabolically labile esters thereof.

The compounds according to the invention are syn isomers. The syn isomeric form is defined by the configuration of the group R I — O.G.COOH ib with respect to the carboxamido group. In this specification the syn configuration is denoted structurally as ' 49171 \ Γ O.C.COOH Rb It will be understood that .since, the compounds of the inven tion are geometric isomers, some admixture with the corresponding anti isomer may occur.

The compounds according to the present invention may 5 exist in tautomeric forms (in respect of the 2-aminothiazolyl group) and it will be understood that such tautomeric forms, e.g. the 2-aminothiazolinyl form, are included within the scope of the invention. q. b It will also be appreciated that when R and R in 10 the above formula represent different Cj_^ alkyl groups, the carbon atom to which they are attached will comprise a centre of asymmetry. Such compounds are diastereoisomeric and the present invention embraces individual diastereoisomers of these compounds as well as mixtures thereof.

The present 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 invention exhibit good broad spectrum antibiotic activity. Against gram-negative organisms the activity is unusually high. This high activity extends to many β-lactamase producing gram-negative 9171 strains. The compounds also possess high stability to β-lactamases produced by a range of gram-negative organisms Compounds according to the invention have been found to exhibit unusually high activity against strains of Pseudomonas, e.g. strains Of Pseudomonas aeroginosa, as well as ftigh activity against various members of the Enterobacteriaceae (e.g. strains of Escherichia coli, Klebsiella pneumoniae, Salmonella typhimurium, Enterobacter cloacae, Serratia marcescens, Providence species, Proteus mirabilis and especially indole positive Proteus organisms such as Proteus vulgaris and Proteus morganii), as well as strains of Haemophilus influenzae.

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, phenylethyl8 benzylamine, dibenzylethylenediamine, ethanolamine, diethanolamine and N-methylglucosamine salts). Other nontoxic salt derivatives include acid addition salts, e.g. fpxmed with hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, formic and trifluoroacetic acids. The salts may also be in the form of resinates formed with, for example, a polystyrene resin or cross-linked polystyrene divinylbenzene copolymer resin containing amino or quaternary amino groups or sulphonic acid groups or with a resin con10 taining carboxyl groups, e.g. a polyacrylic acid resin. Soluble base salts (e.g, alkali metal salts such as the sodium salt) of compounds of formula (I) may be used In therapeutic applications because of the rapid distribution of such salts in the body upon administration. Where, however, insoluble salts of compounds (I) are desired in a particular application, e.g. for use in depot preparations, such salts may be formed in conventional manner, for example with appropriate organic amines.

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

Non-toxic metabolically labile ester derivatives which may be formed by esterification of either or both carboxyl groups in the parent compound of formula (I) include acyloxyalkyl esters, e.g. lower alkanoyloxy-methyl or -ethyl esters such as acetoxymethyl, acetoxyethyl or pivaloyloxymethyl esters. In addition to the above ester derivatives, the present invention includes within its scope compounds of formula (I) in the form of other physiologically acceptable equivalents, i.e. physiologically 4Si?i acceptable compounds which, like the metabolically labile esters, are converted in vivo into the parent antibiotic compound of formula (l).

Examples of preferred compounds according to the 5 present invention include the following compounds and their non-toxic salts and non-toxic metabolically labile esters, namely:(6R,7R)-7-[(z)-2-(2-aminothiazol-4-yl)-2-(2-carboxyprop 2- oxyimino) acetamido]-3-carbamoyloxymethyl-ceph-3-em-410 carboxylic acid; and (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(l-carboxycyclobut-1-oxyimino) acetamido]-3-carbamoyloxymethyl-ceph3- em-4-carboxylic acid.

Other compounds according to the present invention & 1? include for example those wherein the groups R and R in formula (X) are as follows:10 4-9171 /. - J Ra Rb a) Alkyl groupsC2H5 "C2H5 -ch3 -c2h5 b) Cycloalkylidene groups cyclopropylidene cyclopentylidene cyclohexylidene The compounds of formula (I) may be used for treating a variety of diseases caused by pathogenic bacteria in human beings and animals such as respiratory tract infections and urinary tract infections.

According to another embodiment of the invention we provide a process for the preparation of an antibiotic compound of general formula (l) as hereinbefore defined or a non-toxic salt or non-toxic metabolically labile ester thereof which comprises (A) acylating a compound of the font lla (Π) [wherein Rc is hydrogen or an N-protecting group, e.g. a labile group such as an acyl group,especially a lower alkanoyl group such as acetyl, a halo-substituted lower alkanoyl group such as mono-, di- or tri-chloroacetyl,or a chloro5 sulphonyl or bromosulphonyl group; B is )S or )S-*O (a- or β-); R represents hydrogen or a carboxyl blocking group, e.g. the residue of an ester-forming aliphatic or araliphatic alcohol or an ester-forming phenol, silanol or stannanol (the said alcohol, phenol, silanol or stannanol preferably containing 1-20 carbon atoms) and the dotted line bridging the 2-, 3-, and 4-positions indicates that the compound is a ceph-2-em or ceph-3-em compound] or a salt, e.g. an acid addition salt (formed with, for example, a mineral acid such as hydrochloric, hydrobromic, sulphuric, nitric or phosphoric acid or an organic acid such as methanesulphonic or toluene-£-sulphonic + acid) or a base salt formed with a cation such as Na or K, or an N-silyl derivative thereof, with an acid of formula C.CO0H ti (III) O.C, COOR (wherein Ra and Rb are as hereinbefore defined; R repre3 sents a carboxyl blocking group, e.g. as described for R ; and R^ is an amino or protected amino group) or with an acylating agent corresponding thereto; or (B) reacting a compound of formula (IV) CH2OH (wherein Ra, R , R , B and the dotted line are as defined 3 3a above; and R and R may independently represent hydrogen or a carboxyl blocking group)or a salt thereof, with an acylating agent serving to form a carbamoyloxymethyl or N-protected carbamoyloxymethyl group at the 3-position; whereafter, if necessary and/or desired in each instance, any jf the following reactions (c) in any appropri13 ate sequence, are carried out;2 3 i) conversion <£ a Δ -isomer into the desired Δ -isomer, ii) reduction of a compound wherein B is /S-^0 to form a compound wherein B is )S} iii) conversion of a earboxyl group into a non-toxic salt non-toxic metabolically labile ester function, and iv) removal of any carboxyl blocking and/or N-protecting groups.

In the above described process (A) the starting material of formula (II) is preferably a compound wherein B is )S and the dotted line represents a ceph-3-em compound, Acylating agents which may ue 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. phosphorus 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 +50°C, preferably -20 to +30°C, if desired in the presence of an acid binding agent. Suitable reaction media include aqueous ketones such as aqueous acetone, esters such as ethyl acetate, halogenated hydrocarbons such as methylene chloride, amides such as dimethylacetamide, nitriles such as acetonitrile, or mixtures of two or more such solvents. Suitable acid binding agents include tertiary amines (e.g. triethylamine or dimethylaniline), inorganic bases (e.g. calcium carbonate or sodium bicarbonate), and oxiranes such as lower 1,2-alkylene oxides (e.g. ethylene oxide or propylene oxide) which bind hydrogen halide liberated in the acylation reaction.

Acids of formula (III) may themselves be used as 14 acylating agents in the preparation of compounds of formula (I), Acylations employing acids (III) are desirably conducted in the presence of a condensing agent, for example a carbodiimide such as Ν,Ν’-dicyclohexylcarbodiimide or N-ethyl-N*-y-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 amideforming derivaties 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-£-sulphonic acid). An activated ester may conveniently be formed in situ using, for example, 1-hydroxybenzotriazole in the presence of a condensing agent as set out above. Alternatively, the activated ester may be preformed.

Acylation reactions involving the free acids or their above-mentioned amide forming derivatives are desirably effected in an anhydrous reaction medium, e.g. methylene chloride, tetrahydrofuran, dimethylformamide or acetonitrile.

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

The acids of formula (ill) and acylating agents corresponding thereto may, if desired, be prepared and employed in the form of their acid addition salts. Thus, for example, acid chlorides may conveniently be employed as their hydrochloride salts, and acid bromides as their ν 48171 hydrobromide salts.

Carbamoylation of 3-hydroxymethyl compounds of formula (IV) may be effected by conventional methods using suitable carbamoylating agents, such suitable carbamoylating agents d d include isocyanates of formula Ra.NCO (wherein RQ is a labile substituent group), to give a compound containing a 3-position substituent having the formula -CH„O,CONHR^ (wherein RQ has the above defined meaning). The labile group R^ may subsequently be cleaved, e.g by hydrolysis, to form a 3-carbamoyloxymethyl group. Examples of labile groups which are readily cleavable upon subsequent treatment include those labile groups hereinbefore given as examples of the group Rc, and halogenated lower aikoxycarbonyl groups such as 2,2,2-trichloroethoxycarbonyl.

Such labile groups may generally be cleaved by acid or base catalysed hydrolysis (e.g. by base catalysed hydrolysis using sodium bicarbonate).

The above-defined compounds of formula (IV) employed as starting materials in process (B) above are new compounds, and constitute a further feature of the present invention.

In addition to their utility as intermediates for the preparation of cephalosporin compounds having 7-side chains of the same type as those of formula (I) above and having a variety of groups at the 3-position, the compounds also possess useful antibiotic activity similar to that described above for compounds of formula (II). These compounds may be prepared for example by enzymatic hydrolysis (e.g. using Rhodospiridium toruloides or Rhodotorula rubra) of a corresponding 3-acyloxymethyl compound, e.g. the 3-acetoxymethyl compound, for example in an analogous manner to that described in British Patent Specification 1,531,212 and Patent Specification No. 39550. Δ -Cephalosporin ester derivative obtained in accordance with the process of the invention may be converted into the corresponding Δ -derivative by, for example, treatment 2 of the Δ -ester with a base such as pyridine or triethylamine.

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

Where a compound is obtained in which B is )S-^0 this may be converted to the corresponding sulphide by, for example, reduction of the corresponding acyloxysulphonium or alkoxysulphonium salt prepared in situ by reaction with e.g. acetyl chloride in the case of an acetoxysulphonium salt, reduction being effected by, for example, sodium dithionite or by iodide ion as in a solution of potassium iodide in a water-miscible solvent e.g. acetic acid, acetone, tetrahydrofuran, dioxan, dimethylformamide or dimethylacetamide. Hie reaction may be effected at a temperature of from -20° to +50°C.

Metabolically labile ester derivatives of the compounds of formula (I) may be prepared by reacting a compound of formula (I) or a salt or protected derivative thereof with an appropriate esterifying agent such as an acyloxyalkyl halide (e.g. iodide) conveniently in an inert organic solvent such as dimethylformamide or acetone, followed, where necessary, by removal of any protecting groups.

Base salts of the compounds of formula (I) may be formed by reacting an acid of formula (I) with an appropriate base. Thus, for example, sodium or potassium salts may • 49171 be prepared using the respective 2-ethylhexanoate or hydrogen carbonate salt. Acid addition salts may be prepared by reacting a compound of formula (I) or a metabolically labile ester derivative thereof with an 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 10 compounds of general formula (I) according to the invention, compounds of general formula (III) and acid halides and anhydrides corresponding thereto in their syn isomeric form or in the form of mixtures of the syn isomers and the corresponding anti isomers containing at least 90% of the syn isomer are preferably used. a b Acids of formula (III) (provided that R and R 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 \ / CO.COOR6 (V) IV N ‘‘OH s 6 (wherein IT is as hereinbefore defined and R represents a carboxyl blocking group), by reaction with a compound of general formula T.

C.GOOR (VI) a b (wherein R and R and R are as hereinbefore defined and T is halogen such as chloro, bromo or iodo; sulphate; or sulphonate such as tosylate), followed by removal of the carboxyl blocking group r\ Separation of isomers may be effected either before or after such etherification. Ihe 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 tetrahy10 drofuran or dioxan, or an Ν,Ν-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 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 compound of formula R5 S' I—-/ CO.COOR (Vll) (wherein R^ and R& are as hereinbefore defined) with a compound of formula (wherein Ra, and are as defined above), followed by removal of the carboxyl blocking group R , and where necessary by the separation of syn and anti isomers.

The last mentioned reaction is particularly applicable to the preparation of acids of fonnula (III) wherein Ra and R^3 together with the carbon atom to which they are attached form a cyclopropylidene group. In this case, the relevant compounds of formula (VIII) 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 1-amino-oxycyclopropane carboxylate.

The acids of formula (III) may be converted to the corresponding acid halides and anhydrides and acid addition salts by conventional methods, for example as described hereinbefore.

It should be appreciated that in some of the above transformations it may be necessary to protect any sensitive groups in the molecule of the compound in question to avoid undesirable side reactions. For example, during any of the reaction sequences referred to above it may be necessary to protect the NHj group of the aminothiazolyl moiety, for example by tritylation, acylation (e.g. chloroacetylation), protonation or other conventional method. The protecting group may thereafter be removed in any convenient way which does not cause breakdown of the desired compound, e.g. in the case of a trityl group by using an optionally halo25 genated carboxylic acid, e.g. acetic acid, formic acid, chloroacetic 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 treatment with thiourea.

Carboxyl blocking groups used in the preparation of compounds of formula (i) or in the preparation of necessary 48171 starting materials are desirably groups which may readily be split off at a suitable stage in the reaction sequence, conveniently at the last stage. It may, however, be convenient in some instances to employ non-toxic metabolically labile carboxyl blocking groups such as acyloxyalkyl groups (e.g. 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 Patent Specification No. 38172. Preferred blocked carboxyl groups include aryl lower alkoxycarbonyl groups such as £-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl and diphenylmethoxycarbonyl; lower alkoxycarbonyl groups such as t-butoxycarbonyl; and lowertnloalkoxycarbonyl groups such as 2,2,2-trichloroethoxycarbonyl. Carboxyl blocking groupfe) may subsequently be removed by any of the appropriate methods disclosed in the literature; thus, for example, acid or base catalysed hydrolysis is applicable in many cases, as are enzymically-catalysed hydrolyses.

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

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

If desired, such powder formulations may contain an appropriate non-toxic base in order to improve the water solubility of the active ingredients, and/or to ensure that when the powder is reconstituted with water, the pH of the resulting aqueous formulation is physiologically acceptable. Alternatively, the base may be present in the water with which the powder is reconstituted. The base may be, for example, an inorganic base such as sodium carbonate, sodium bicarbo15 nate or sodium acetate, or an organic base such as lysine or lysine acetate.

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

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

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

The following Examples illustrate the invention. All temperatures are in °C. ’Petrol* means petroleum ether (b.p. 40-60°).

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 10°C. Stirring and cooling were continued for about 30 min., when a solution of potassium chloride (160 g) in water (800 ml) was added. The resulting Iq mixture was stirred for one hour. The lower oily phase was separated and the aqueous phasewas extracted with diethyl ether. The extract was combined with the oil, washed successively with water and saturated brine, dried, and evaporated. The residual oil, which solidified on standing, was washed with petrol and dried in vacuo over potassium hydroxide, giving ethyl (Z)-2-(hydroxyimino)3-oxobutyrate (309 g), A stirred and ice-cooled solution of ethyl (Z)-2-(hydroxyimino)-3-oxobutyrate (150 g) in 2Q dichloromethane (400 ml) was treated dropwise with sulphuryl chloride (140 g). The resulting solution was kept at room temperature for 3 days, then evaporated.

The residue was dissolved in diethyl ether, washed with water until the washing 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 product was collected by filtration, washed with ethanol and dried to give the title compound (73 g); m.p.188° (decomp.).

Preparation 2 Ethyl (Z)-2-hydroxyimino-2-(2-tritylaniinothiazol-4-yl)acetate, hydrochloride Trityl chloride (16.75 g) was added portionwise 5 over 2 hours to a stirred and cooled (-30°) solution of the product of Preparation 1 (12,91 g) in dimethylformamide (28 ml) containing triethylamine (8.4 ml). The mixture was allowed to warm to 15° over one hour, stirred for a further 2 hours and then partitioned between water (500 ml) and ethyl acetate (500 ml). The organic phase was separated, washed with water (2 x 500 ml) and then shaken with IN HCl (500 ml). The precipitate was collected, ✓ washed successively with water (100 ml), ethyl acetate (200 ml) and ether (200 ml) and dried in vacuo to provide the title compound as a white solid (16.4 g); m.p, 184 to 186° (decomp).

Preparation 3 Ethyl (Z)-2-(2-t-butoxycarbonylprop-2-oxyimino)-2-(2-tritylamino thiazol- 4-yl) acetate Potassium carbonate (34.6 g) and t-butyl 2-bromo-2-methylpropionate (24.5 g) in dimethylsulphoxide (25 ml) were added to a stirred solution under nitrogen of the product of Preparation 2 (49.4 g) in dimethylsulphoxide (200 ml) and the mixture was stirred at room temperature for 6 hours. The mixture was poured into water (21), 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 from 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-Butoxycarbonylprop-2-oxyimino)-2-(2-tritylamino thiazol-4-yl)acetic acid The product of Preparation 3 (2 g) was dissolved in methanol (20 ml) and 2N sodium hydroxide (3.3 ml) was added. The mixture was refluxed for 1.5 hours and then concentrated. The residue was taken up in a mixture of water (50 ml), 2N hydrochloric acid 10 (7 ml), and ethyl acetate (50 ml). The organic phase was separated, and the aqueous phase extracted with ethyl acetate. The organic solutions were combined, washed successively with water and saturated brine, dried, and evaporated. The residue was recrystallised from a mixture of carbon tetrachloride and petrol to give the title compound (1 g), m.p. 152 to 156° (decomp). Preparation 5 Ethyl (Z)-2-(2-tritylafflinothia2ol-4-yl)"2-(l-t-butoxycarbonylcyclobut-l-oxyimino) acetate. 2θ 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 room temperature. After 30 minutes, t-butyl 1-hromocyclobutane 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 1-bromocyclobutane carboxylate (3.45 g) was added after 3 days. After 4 days in all, the mixture was poured into ice-water (ca. litres) and the solid was collected by filtration and 48171 washed well with water and petrol. The solid was dissolved in ethyl acetate and the solution washed with brine (twice), dried with magnesium sulphate and evaporated to a foam. This foam was dissolved in ethyl acetate-petrol(l:2) and filtered through silica gel (500 g).

Evaporation gave the title compound (60 g) as a foam, v (CHBr_) 3400 (NH) and 1730 cm (ester).

ITiaX O Preparation 6 (Z)-2-(l-t-Butoxycarbonylcyclobut-l-oxyimino)-2-(2-tritylaminothiazol-4-yl) acetic acid.

A mixture of the product of Preparation 5 (3.2g) and potassium carbonate (1.65 g) was refluxed in methanol (180 ml) and water (20 ml) for 9 hours and the mixture was cooled to room temperature,, The mixture was concentrated and the residue partitioned between ethyl acetate and water, to which was added 2N HCl (12.2 ml).

The organic phase was separated and the aqueous phase extracted with ethyl acetate. The combined organic extracts were washed with saturated brine, dried and evaporated to give the title compound (2.3 g); λ η o< max (ethanol) 265 nm (E^^ 243).

Example 1 a) Diphenylmethyl (6R,7R)-7-[(Z)-2-(2-t-butoxycarbonylprop2-oxyimino)-2-(2-tritylaminothiazol-4-yl)acetamido]-3carbamoyloxymethyl-ceph-3-em-4-carboxylate.

A stirred solution of the product of Preparation 4 (2.86 g) and diphenylmethyl (6R,7R)-7-aitiino-3-carbamoyloxymethyl-ceph-3-em-4-carboxylate (2.64 g) in dimethylformamide (50 ml) was cooled to 0°, and 1-hydroxybenzotriazole (745 mg) added, followed by dicyclohexylcarbodiimide (1.14 g). 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 diluted with water (200 ml) and extracted with ethyl acetate. The organic extracts were combined, washed· successively with 2N hydrochloric acid, water, sodium bicarbonate solution water, and saturated brine, dried, and evaporated. The residue was eluted through a silica column with ethyl acetate The appropriate fractions of the eluate were collected and concentrated to give the title compound (3.6 g)»^^^ (ethanol) 237.5 nm (eJ 270),^^256 nm (eJ 207), [α]ρθ + 150 Cc 1.0, DMSO). b) (6R,7R)-7-[(z)-2-(2-Aminothiazol-4-yl)-2-(2-carboxyprop2-oxyimino)acetamido]-3-carbamoyloxymethyl~ceph-3-em-4carboxylic acid.

Trifluoroacetic acid (20 ml) was added to a solution of the product of stage (a) (3 g) in anisole (20 ml) at 0°. The mixture was stirred at room temperature for 2% hours md concentrated. The residue was dissolved in ether and re-evaporated. The residue was dissolved in ethyl acetate and extracted with saturated sodium bicarbo28 * 49171 nate solution. The pH of the aqueous extracts was adjusted to 6, and the solution washed with ethyl acetate. The aqueous phase was acidified to pH 1.5 under ethyl acetate, and extracted with ethyl acetate. The combined organic extracts were washed with water and saturated brine, dried, and evaporated. The residue was dissolved in warm 75% 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, re10 filtered, and lyophilized to give the title compound (450 mg),\. ~ (pH 6 buffer) 236 nm (E^53), 257 nm , xnr . Jem , (ET 224), 295 nm (E. 104), y (flujol) 1540,1670 cm x J* .. 7 φ3Χ I n (CONH), 1680 cm A (OCONHp, 1720 cm x (CO2H), 1780 cm (β-lactara).

Example 2 a) Diphenylmethyl (6R,7R)-7-[(Z)-2-(l-t-butoxycarbonylcyclobut-l-oxyimino^-te-tritylaminothiazol^-yOacetainidol3-carbamoyloxymethvl-ceph-3-em-4-carboxylate A stirred solution of the product of preparation 6 and diphenylmethyl (6R,7R)-7-amino-3-carbamoyloxymethylceph-3-em-4-carboxylate (0.88 g) in dimethylformamide (20 ml) was cooled to 0°, treated with 1-hydroxybenzotriazole (0.3 g) followed by dicyclohexyicarbodiimide (0.45 g) and the product isolated substantially according to the method of Example la) to give the title compound (1.6 g), \ (ethanol) 240 nm (E^ 242),^. F 305 nm (e| 58), ΤΪ13Χ X*i ΧΩΣ -ι X V (CHBrJ '1522,1688 cm1 (CONH), 1685 cio (OCONHj, IPciX 7 1 ~ 1730 ent (CO2R), 1790 cm (β-lactam). b) (6R;7R)-7-[(Z)-2-(2-Aminothiazol-4-yl)-2-(l-carboxycyclobut-l-oxyimino)acetamidoJ-3-carbamoyloxymethyl-ceph3-em-4-carboxylic acid Trifluoroacetic acid (8 ml) was added to a solution of the product of stage (a) (2.3 g) in anisole (2 ml) at 0°. The mixture was treated substantially as described in Example lb) to give the title compound (285 mg), \ (pH 6 buffer) 239.5 nm (E* 194), \inf 292.5 nm (E* 129), [a]20 + 32.5° (c 1.0, DMSO).

Example 3 a) t-Butyl (6R17R)-3-Acetoxymethyl-7-[(Z)-2-(2-t-butoxycarbonylprop-2-oxyimino)-2-(2-tritylaminothiazol-4-yl) acetamido]ceph-3-em-4-carboxylate A stirred solution of the product of Preparation 4 15 (572 mg) and t-butyl (6R,7R)-3-acetoxymethyl-7-aminoceph3-em-4-carboxylate (328 mg) in dimethylformamide (10 ml) was cooled to 0°, treated with 1-hydoxybenzotriazole (150 mg) followed by dicyclohexylcarbodiimide (225 mg) and the product isolated substantially as described in Example 1 a) to give the title compound (533 mg). A portion recrystallised from di-isopropyl ether had m.p. 103 to 113* (decomp.);+ 8.5° (c 1.0, DMSO). b) (6R,7R)-3-Acetoxymethyl-7-[(Z)-2-(2-aminothiazol-4-yl)2-(2-carboxyprop-2-oxyifflino)acetamidojceph-3-em-4-carboxylic 25 acid Trifluoroacetic acid (18 ml) was added to a solution of the product of Stage a) (2.4 g) in anisole (18 ml) at 0°. The mixture was treated substantially as described in Example 1 b) to give the title compound (920 mg), λ. -» -ι ot max (pH 6 buffer) 236 nm (E7 250), λ. f 255 nm (E, 235), 296 nm (E^103); [a]*0 +20.0« (c 1.0, DMSO). c) (6R,7R)-7-[(Z)-2-(2-Aninothia2ol-4-yl)-2-(2-Carboxyprop2- oxyimino)acetamido]-3-hvdroxvmethylceph-3-em-4-carboxylate,di-sodium salt.

The product of stage b) (1 g) was suspended m a mixture of 0.2 molar pH7 phosphate buffer (15 ml) and water (5 ml). The pH was adjusted to 6.8 by addition of dilute sodium hydroxide solution and the resulting solution was treated with Rhodospiridium toruloides (CBS 349) (1.5 ml of a semi frozen suspension) and the mixture was stirred at 22° for 3 hours.

The mixture was filtered and the filtrate was freezedried to give the crude product (1.37 g) as an amorphous solid.

A sample (0.66 g) of the above product in water was applied to a column (33 cm x 2.5 cm) containing Amberlite XAD-2 resin. The column was eluted successively with water followed by 25% ethanol in water. Appropriate fractions were collected and combined and evaporated in vacuo to low volume.

This solution was freeze-dried to give the title salt ,1% 275) with inflec(0.216 g) Xwax (pH6 buffer) 235 nm (Εχ' tions at 251 nm (E, 257) and 290 nm (E7A 130), v cm x cm max (Nujol), 1760 (β-lactam), 1660 and 1534 (CONH) and 1590 cm (CO). 171 Example 4 a) t-Butyl (6Κ,7Κ)-3-Αοβίοχ^6^ν1-7[(Ζ)-2-(1-ί-6πίοχγcarbonylcyclobut-l-oxyimino)-2-(2-tritylaminothiazol-4-yl) acetamido3cep'n-3-em-4-carboxylate A stirred solution of the product of Preparation 6 (24.2 g) and t-butyl (6R,7R)-3-acetoxymethyl-7-aminoceph3-em-4-carboxylate (13.6 g) in dimethylformamide (300 ml) was cooled to 0°, treated with 1-hydroxybenzotriazole monohydrate (4.5 g), followed by dicyclohexylcarbodiimide (6.4 g) and the product isolated substantially as described in Example lb) to give the title compound (12.8 g), m.p. 113.5 to 116.5° (decomp); [α]ρθ + 15.0° (c 1.0, DMSO). b) (6R,7R)-3-Acetoxymethyl-7-[(Z)-2-(2-aminothiazol-4-yl·)2-(l-carboxycyclobut-l-oxyimino)acetamido]ceph-3-em-4carboxylic acid, Trifluoroacetic acid (100 ml) was added to a mixture of the product of Stage a) (12.5 g) and anisole (5 ml) at 0°. The mixture was treated substantially as described in Example 1 b) to give the title compound (4 g), λ. .max (pH 6 buffer) 246 ran (E, 264),λ. , 295 nm (E, 118), 1cm inf 1 cm [D + 27.3° (c 1.0, DMSO). c) (6R,7R)-7-[(z)-2-(Aminothiazol-4-yl)-2-(l-carboxycyclobutl-oxyiminoacetamido]-3-hydroxymethylceph-3-em-4-carboxylic acid, sodium salt The product of Stage b) (1 g) was suspended in 0.2 molar pH 7 buffer (15 ml) and water (5 ml) and treated with a susp nsion of Rhodospiridium toruloides (CBS 349) substantially as described in Example 3 c) to give the title compound (0.756 g), [aL· + (pH6 buffer), 246 nm (Ej^ 294.5 nm (E?% 124).

I cm 78.8° 311) (c 1.00, DMSO), with an inflection at PHARMACY EXAMPLES EXAMPLE A - Dry Powder for Injection.

Fill sterile (6R, 7R)-7-[(Z)-2-(2-awinothiazol-4-yl) -2-(2-carboxyprop-2-oxyimino)acetamido]-3-carbamoyloxymethy lceph-3-em-4-carboxylic acid, disodium salt into glass vials, such that each vial contains an amount equivalent to 250mg of the antibiotic acid. Carry out the filling aseptically under a blanket of sterile nitrogen. Close the vials using rubber discs or plugs, held in position by aluminum overseals, thereby preventing gaseous exchange or ingress of micro-organisms. Reconstitute the product by dissolving in Water for Injections or other suitable sterile vehicle shortly before administration.

EXAMPLE B - Dry Powder for Injection Formula Per Vial (6R, 7R)—7—[(2)-2-(2-Aminothiazo1-4-y 1)-2-(1-carboxycyclobut-l-oxyimino)acetamido]-3-carbamoyloxymethylceph-3em-4-carboxylic acid 500mg Lysine Acetate 368mg Method Blend the sterile cephalosporin antibiotic with sterile lysine acetate 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 micro-organisms.

Reconstitute the product by dissolving in Water for Injections or other suitable sterile vehicle shortly before administration.

Claims (5)

1. Cephalosporin antibiotics of the general formula Λ 1? (wherein R and R , which may be the same or different, each represents a alkyl group or R and R together 5 with the carbon atom to whidi they are attached form a cycloalkylidene group) and non-toxic salts and non-toxic metabolically labile esters thereof.

2. Compounds as claimed in claim 1 wherein at least one η H of R and R represents a methyl or ethyl group. a b 1q 3, Compounds as claimed in claim 1 wherein R and R together with the carbon atom to which they are attached form a C3..5 cycloalkjiidene group.

3. 3a above; and R and R may independently represent hydrogen or a carboxyl blocking group), or a salt 5 thereof, with an acylating agent serving to form a carbamoyloxymethyl or N-protected carbamoyloxymethyl group at the 3position; whereafter, if necessary and/or desired in each instance, any of the following reactions (C) in any appropriate sequence, are carried out;2 3 10 i) conversion of a Δ -isomer into the desired Δ -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 15 iv) removal of any carboxyl blocking and/or N-protecting groups. 7. A process as claimed in claim 6 wherein the starting material of formula (II) is a ceph-3-em compound in which B is >S. 20 8. A process as claimed in either of claims 6 and 7 wherein a compound of formula (II) or an acid addition salt or N-silyl derivative thereof is condensed with an acid of formula (III) in the presence of a condensing agent comprising a carbodiimide, carbonyldiimidazole or isoxazolium salt. 9„ A pharmaceutical composition for use in human or veterinary medicine comprising an antibiotic compound as claimed in any of claims 1 to 5 in association with a pharmaceutical carrier or excipient.

4. (6R,7R)-7-[(Z)-2-(2-Aminothiazol-4-yl)-2-(2-carboxyprop-2-oxyimino) acetamido]-3-carbamoyloxymethylceph-315 em-4-carboxylic acid and its non-toxic salts. 5. (6R,7R)-7-[(z)-2-(2-Aminothiazol-4-yl)-2-(1-carboxycyclobut-1-oxyimino) acetamido]-3-carbamoyloxymethylceph3-em-4-carboxylic acid and its non-toxic salts. 6. A process for the preparation of an antibiotic compound 20 of general formula(I)as defined in claim 1 or a non-toxic metabolically labile ester thereof whih comprises (A) acylating a compound of formula 4917 1 (wherein R c is hydrogen or an N-protecting group; B is >S or )>S—5>0j R represents hydrogen or a carboxyl blocking group and the dotted line in formula (Cl) bridging the 2-, 5 3- and 4- positions indicates that the compound is a ceph2-em or ceph-3-em compound, or a salt or N-silyl derivative thereof, with an acid of formula \-Z _C. COOH (III) R 1 4 0. C. COOR* (wherein R a and r' 3 are as defined in claim 1; represents a carboxyl blocking group; and R^ is an amino or protected amino group) or with an acylating agent corresponding thereto; or (B) reacting a compound of formula 48171

5. 10. Compounds of formula(IV) (as defined in claim 6).