GB2062624A - Cephalosporin Antibiotics - Google Patents

Abstract

Cephalosporin antibiotics of general formula <IMAGE> (wherein R represents a hydrogen atom or a C1-4 alkyl group) and non- toxic salts and non-toxic metabolically labile esters thereof, exhibit broad spectrum antibiotic activity and can be incorporated into pharmaceutical compositions. Particularly effective compounds of formula (I) are (6R,7R)-7-[(Z)-2-(2- aminothiazol-4-yl)-2- (carboxymethoxyimino)acetamido]-3- (1-pyridiniummethyl)-ceph-3-em-4- carboxylate, (6R,7R)-7-[(Z)-2-(2- aminothiazol-4-yl)-2- (ethoxycarbonylmethoxyimino)- acetamido)-3-(1- pyridiniummethyl)ceph-3-em-4- carboxylate and (6R,7R)-7-[(Z)-2-(2- aminothiazol-4-yl)-2- (methoxycarbonylmethoxyimino)- acetamido]-3-(1-pyridinium- methyl)ceph-3-em-4-carboxylate.

Description

SPECIFICATION Cephalosporin Antibiotics 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 antiobiotics are widely used in the treatment of diseases caused by pathogenic bacteria in human beings and animals, and are especially useful in the treatment of diseases caused by bacteria which are resistant to other antibiotics such as penicillin compounds, and in the treatment of penicillin-sensitive patients. In many instances it is desirable to employ a 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 British Patent Specification No. 1,399,086, we describe a novel class of cephalosporin antibiotics containing a 7P-(a-etherified oxyimino)-acylamido group, the oxyimino group having the syn configuration. This class of antibiotic compounds is characterised by high antibacterial activity against a range of gram-positive and gram-negative organisms coupled with particularly high stability to ss-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 7p-acylamido group of the formula

(wherein RC is a thienyl or furyl group; RA and RB may vary widely and include, for example, hydrogen atoms, 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 of mixtures of syn and anti isomers containing at least 90% of the syn isomer. The 3-position of the cephalosporin molecule may be unsubstituted or may contain one of a wide variety of possible substituents, including, for example, the pyridiniummethyl group. These compounds have been found to have particularly good activity against gram-negative organisms.The Specification also discloses ester-derivatives of the above compounds which are formed by esterification of one or both of the carboxyl groups present in the 4-position and in the above 7-side chain.

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- acylamido group of formula (A) but also the introduction of particular groups in the 3-position of the cephalosporin molecule.

Thus, for example, Belgian Patent Specification No. 836,813 describes cephalosporin compounds wherein the 7p-acylamido side chain is a 2-(2-aminothiazol-4-yl)-2-(hydroxyimino or blocked hydroxyimino) acetamido group. The blocked hydroxyimino group may be for example 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 e.g. the pyridinium 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- acylamido side chain is primarily a 2-(2-aminothiazol-4-yl)-2-(syn)-methoxyiminoacetamido group and the substituent in the 3-position is broadly defined in a similar manner to that in the above-mentioned Belgian Patent Specification No. 836,813. Compounds specifically exemplified in the Specification include a compound in which the 3-position is substituted by a pyridiniummethyl group.

South African Patent Specification 78/1870 discloses cephalosporin antibiotics wherein the 7p- acylamido side chain is inter alia a 2-(2-aminothiazol-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 alkoxyimino group is a carboxyalkoxyimino or analkoxycarbonylalkoxyimino, e.g. a carboxymethoxy-, ethoxycarbonylmethoxyor t-butoxycarbonylmethoxy-imino group. With regard to the 3-position, the pyridiniummethyl substituent is referred to, among numerous other possibilities.South African Patent Specification 78/2168 discloses in broad terms cephalosporin 1-oxides of the sulphides described in the lastmentioned Specification.

South African Patent Specification No. 78/1630 describes 3-acetoxymethyl cephalosporin antibiotics wherein the group RC in formula (A) above may be replaced by a 2-aminothiazol-4-yl group and the oxyimino group is an alkyl group substituted by a carboxy group (or a salt or C13 alkyl ester thereof), a nitrile group or a carbamoyl group. The Specification describes the preparation of these compounds by acylation of a 7-amino cephalosporin compound with an appropriate carboxylic acid (or functional derivative thereof). In the latter materials any carboxy substituent of the alkoxyimino group may be protected by inter alia a group easily removable by acid hydrolysis or by hydrogenolysis.

We have now discovered that by an appropriate selection of a small number of particular groups at the 7ss-position in combination with a pyridiniummethyl 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.

The present invention provides cephalosporin antibiotics of the general formula:

[wherein R represents a hydrogen atom or a C14 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)] 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 --O. CH2COOR with the respect to the carboxamido group. In this Specification the syn configuration is denoted structurally as

It will be understood that since the compounds according to the invention are capable of geometric isomerism, some admixture with the corresponding anti isomer may occur.

The invention also includes within its scope the solvates (including 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 2iminothiazolinyl 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, when the group R does not represent a C,, alkyl group, the 4-carboxyl group may be protonated and the carboxyl group in the 7-side chain deprotonated. Such alternative forms are included within the scope of the present invention.

The compounds according to the invention exhibit broad spectrum antibiotic activity. Against gram-negative organisms the activity is unusually high. This high activity extends to many ,B- lactamase-producing gram-negative strains. The compounds also possess high stability to p- lactamases produced by a range of gram-positive and gram-negative organisms.

Compounds according to the invention have been found to exhibit good activity against a wide range of organisms, including various members of the Enterobacteriaceae such as strains of Escherichia coli, Klebsiella pneumoniae, Salmonella typhimurium, Shigella sonnet Enterobacter cloacae, Serratia marcescens, Providence species, Proteus mirabilis and especially indolepositive Proteus organisms such as Proteus vulgaris and Proteus organic. They also have good activity against Haemophllus influenzae and Pseudomonas aeruginosa. Compounds according to the invention wherein R represents a C,, alkyl group also exhibit good activity against Staphylococcus organisms such as strains of Staphylococcus aureus.

Non-toxic base salt derivatives may be formed from compounds of formula (I) by reaction of the carboxyl function at the 4-position and/or that which is present in the 7-side chain in the case when R represents a hydrogen atom. It will be appreciated that when R represents a C14 alkyl group and the compound of formula (I) is in the form of a base salt, the compound of formula (I) will be in association with an appropriate anion. These salt derivatives 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, phenethylbenzylamine, dibenzylethylenediamine, ethanolamine, diethanolamine and N-methylglucosamine salts).Other nontoxic salt derivatives include acid addition salts, e.g. formed with hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, formic and trifluoroacetic acids. The salts may also be in the form of resinates formed with, for example a polystyrene resin or cross-linked polystyrene divinylbenzene copolymer resin containing amino or quaternary amino groups or sulphonic acid groups, or with a resin containing carboxyl groups, e.g. a polyacrylic acid resin. Soluble base salts (e.g. alkali metal salts such as the sodium salt) of compounds of formula (I) may be used in therapeutic applications because of the rapid distribution of such salts in the body upon administration.Where, however, insoluble salts of compounds (I) are desired in a particular application, e.g. for use in depot preparations, such salts may be formed in conventional manner, for example with appropriate organic amines.

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

Non-toxic metabolically labile ester derivatives may be formed from compounds of formula (I) by esterification of the carboxyl function at the 4-position and/or that which is present in the 7-side chain in the case when R is a hydrogen atom. These ester derivatives include acyloxyalkyl esters e.g. lower alkanoyloxy-methyl or -ethyl esters such as acetoxy-methyl or -ethyl or pivaloyloxymethyl esters. In addition to the above ester derivatives, the present invention includes within its scope compounds of formula (I) in the form of other physiologically acceptable equivalents, i.e. physiologically acceptable compounds which, like the metabolically labile esters, are converted in vivo into the parent antibiotic compound.

Preferred compounds according to the present invention include (6R,7R)-7-[(Z)-2-(2aminothiazol-4-yl)-2-(carboxymethoxyimino)acetamido]-3-(1 -pyridiniummethyl)ceph-3-em-4carboxylate, (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(ethoxycarbonylmethoxyimino)acetamido]-3-(1pyridiniu mmethyl)ceph-3-em-4-carboxylate and (6R,7 R)-7-[(Z)-2- (2-aminothiazol-4-yl)-2 (methoxycarbonylmethoxyimino)acetamidoj-3-( 1 -pyridiniummethyl)ceph-3-em-4-carboxylate as well as their non-toxic salts and non-toxic metabolically labile esters.

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 (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

[wherein B represents > S or > SoO (a- or P-); 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-p-sulphonic acid) or an N-silyl derivative thereof, or a corresponding compound having a group of formula -COOR1 at the 4-position [where R1 represents a hydrogen atom or a carboxyl blocking group, e.g. the residue of an esterforming 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 having an associated anion yo such as a halide, e.g. chloride or bromide, or trifluoroacetate anion, with an acid of formula

(wherein R2 represents a carboxyl blocking group, e.g. as described for R1; and R3 represents an amino or protected amino group) or with an acylating agent corresponding thereto; (B) reacting a compound of formula

(wherein R3, B and the dotted line are as hereinbefore defined; R4 and R4a may independently represent hydrogen or a carboxyl blocking group; and X represents a replaceable residue of a nucleophile, e.g. an acetoxy or dichloroacetoxy group or a halogen atom such as chlorine, bromine or iodine) or a salt thereof, with pyridine; or (C) when a compound of formula (I) in which R represents a C,~4 alkyl group is desired, reacting a compound of formula

(wherein B, R3 and the dotted line are as hereinbefore defined;R5 represents a carboxyl blocking group; and Q represents a carboxyl group or a functional equivalent thereof) with an alkylating agent serving to convert the group Q into a group COOR where R represents a C14 alkyl group; 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 A2-isomer into the desired A3-isomer, ii) reduction of a compound wherein B is > S-tO to form a compound wherein B is > S, iii) conversion of a carboxyl group into a non-toxic salt or non-toxic metabolically labile ester function, and iv) removal of any carboxyl blocking and/or N-protecting groups.

It will be appreciated that the compound of formula (V) above may, in appropriate cases, be employed in association with one or more ions of suitable charge in order to ensure that the molecule as a whole has a balanced charge.

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.

The reaction should be effected in the presence of a base if an acid addition salt of a compound of formula (II) is used.

It will be appreciated that in processes (A) and (B) above the carboxyl blocking groups R2 and R4 respectively may represent C,, alkyl groups, in which case the appropriate group may be retained in the final product when a compound of formula (I) wherein R represents a C14 alkyl group is desired.

Acylating agents which may be employed in the preparation of compounds of formula (I) include acid halides, particularly acid chlorides or bromides. Such acylating agents may be prepared by reacting an acid (III) or a salt thereof with a halogenating agent e.g. 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 +500C, preferably -20 to +300C, 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 dimethylformamide or dimethylacetamide, nitriles such as acetonitrile, or mixtures of two or more such solvents. Suitable acid binding agents include tertiary amines (e.g. triethylamine or dimethylaniline), inorganic bases (e.g. calcium carbonate or sodium bicarbonate), and oxiranes such as lower 1,2-alkylene oxides (e.g. ethylene oxide or propylene oxide) which bind hydrogen halide liberated in the acylation reaction.

Acids of formula (III) may themselves be used as acylating agents in the preparation of compounds of formula (I). Acylations employing acids (III) are desirably conducted in the presence of a condensing agent, for example a carbodiimide such as N,N'-dicyclohexylcarbodiimide or N-ethyl-N'-ydimethylaminopropylcarbodiimide; a carbonyl compound such as carbonyldiimidazole; or an isoxazoiium 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-suiphonic 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, dimethylacetamide 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 (III) and acylating agents corresponding thereto may, if desired, be prepared and employed in the form of their acid addition salts. Thus, for example, acid chlorides may conveniently be employed as their hydrochloride salts, and acid bromides as their hydrobromide salts.

Pyridine may act as a nucleophile to displace a wide variety of substituents X from the cephalosporin of formula (lV). The facility of the displacement is related to the pK, 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 displacement of X by pyridine may conveniently be effected by maintaining the reactants in solution or suspension. The reaction is advantageously effected using from 1 to 10 mole equivalents of pyridine.

Nucleophilic displacement reactions may conveniently be carried out on those compounds of formula (IV) wherein the substituent X is a halogen atom or an acyloxy group for example as discussed below.

Acyloxy Groups Compounds of formula (IV) wherein X is an acetoxy group are convenient starting materials for use in the nucleophilic displacement reaction with pyridine. Alternative starting materials in this class include compounds of formula (IV) in which X is the residue of a substituted acetic acid e.g.

chloroacetic acid, dichloroacetic acid and trifluoroacetic acid.

Displacement reactions on compounds (IV) possessing X substituents of this class, particularly in the case where X is an acetoxy group, may be facilitated by the presence in the reaction medium of iodide or thiocyanate ions. Reactions of this type are described in more detail in British Patent Specifications Nos. 1,132,621 and 1,171,603.

The substituent X may also be derived from formic acid, a haloformic acid such as chloroformic acid, or a carbamic acid.

When using a compound of formula (IV) in which X represents an acetoxy or substituted acetoxy group, it is generally desirable that the group R4a in formula (IV) should be a hydrogen atom and that B should represent > S, In this case, the reaction is advantageously effected in an aqueous medium, preferably at a pH of 5 to 8, particularly 5.5 to 7.

The above-described process employing compounds of formula (IV) in which X is the residue of a substituted acetic acid may be carried out as described in British Patent Specification No. 1,241,657.

When using compounds of formula (IV) in which X is an acetoxy group, the reaction is conveniently effected at a temperature of 300 to 1 1 OOC, preferably 500 to 900C.

Halogens Compounds of formula (IV) in which Xis a chlorine, bromine or iodine atom can also be conveniently used as starting materials in the nucleophilic displacement reaction with pyridine. When using compounds of formula (IV) in this class, R48 preferably represents a carboxyl blocking group. The reaction is conveniently effected in a non-aqueous medium which preferably comprises one or more organic solvents, advantageously of a polar nature, such as halogenated hydrocarbons, e.g.

dichloromethane or dichloroethane, ethers, e.g. dioxan or tetrahydrofuran, esters, e.g. ethyl acetate, nitriles, e.g. acetonitrile, amides, e.g. formamide, dimethylformamide or dimethylacetamide and ketones e.g. acetone. If desired pyridine itself may be the solvent. Other suitable organic solvents are described in more detail in British Patent Specification No. 1,326,531. The reaction medium should be neither extremely acidic nor extremely basic. In the case of reactions carried out on compounds of formula (IV) in which R4 and R4a are carboxyl blocking groups the 3-pyridiniummethyl product may 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 Xis a halogen atom as described above, the reaction is conveniently effected at a temperature of 100 to +600C, preferably + 10 to +300 C.

In process (C) above, the alkylating agent may be for example a diazo-(C14) alkane such as diazomethane, a C, 4 alkanol or a reactive derivative thereof e.g. a C14 alkyl halide such as methyl or ethyl iodide. When an alkyl halide is employed, the reaction is preferably effected in the presence of a base, e.g. a tertiary amine such as triethylamine or an inorganic base such as potassium carbonate. Q preferably represents the group COOH.

Where a C14 alkanol is used, this may be reacted with an acid (i.e. where Q represents COOH) of formula (V) in the presence of a coupling agent, e.g. dicyclohexylcarbodiimide, preferably in the presence of a catalyst such as 4-dimethylaminopyridine.

The alkylation reaction is conveniently effected in a solvent medium comprising for example an amide solvent such as dimethylformamide or dimethylacetamide, ketones such as acetone, esters such as ethyl acetate, halogenated hydrocarbons such as dichloromethane or dichloroethane, nitriles such as acetonitrile, ethers such as dioxan, tetrahydrofuran or diethyl ether, or mixtures thereof, preferably at a temperature in the range 0 to +500C.

The reaction product may be separated from the reaction mixture, which may contain, for example, unchanged cephalosporin starting material and other substances, by a variety of processes including recrystallisation, ionophoresis, column chromatography and use of ion-exchangers (for example by chromatography on ion-exchange resins) or macroreticular resins.

A2-Cephalosporin ester derivatives obtained in accordance with the processes of the invention may be converted into the corresponding A3-derivatives by, for example treatment of the ,\2ester 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 1oxide, for example by reaction with a peracid, e.g. peracetic or m-chloroperbenzoic acid; the resulting sulphoxide may, if desired, subsequently be reduced as described hereinafter to yield the corresponding ceph-3-em sulphide.

Where a compound is obtained in which B is > SoO this may be converted into 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. The reaction may be effected at a temperature of from -200 to +500 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 the appropriate base. Thus, for example, sodium or potassium salts may be prepared using the respective 2-ethylhexanoate, acetate 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 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.

Acids of formula (III) may be prepared by etherification of a compound of formula

(wherein R3 is as hereinbefore defined) or a salt thereof, by reaction with a compound of general formula T. CH2COORZ (Vll) (wherein R2 is as hereinbefore defined and T represents a halogen atom such as chlorine, bromine or iodine; a sulphate group; or a sulphonate group such as a tosylate). Separation of isomers may be effected either before or after such etherification. The etherification reaction is generally carried out in the presence of a base, e.g. potassium tert-butoxide and is preferably conducted in an organic solvent, for example dimethylsulphoxide, a cyclic ether such as tetrahydrofuran or dioxan, or an N,Ndisubstituted amide such as dimethylformamide, sufficient base being added to form a dianion.Under these conditions the configuration of the oxyimino group is substantially unchanged by the etherification reaction.

Acids of general formula (III) may also be prepared by reaction of a compound of formula

(wherein R3 is as hereinbefore defined and Rs represents a hydrogen atom or a carboxyl blocking group) with a compound of formula H2NOCH2.COOR2 (IX) (wherein R2 is defined above), followed by removal of any carboxyl blocking group R6, and where necessary by the separation of syn and anti isomers.

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 hereinabove.

Where X represents halogen (i.e. chlorine, bromine or iodine) atom in formula (IV), ceph-3-em starting compounds may be prepared in conventional manner, e.g. by halogenation of a 7-prntected amino-3-methyl-ceph-3-em-4-carboxylic acid ester 1 oxide, removal of the 7-prntecting group, acylation of the resulting 7amino compound to form the desired 7P-acylamido group. e.g. in an analogous manner to process (A) above, followed by reduction of the 1 oxide group later in the sequence. This is described in British Patent Specification No.1,326,531.The corresponding ceph-2em compounds may be prepared by acylation of the corresponding 7amino compound.

Where X in formula (IV) represents an acetoxy group, such starting materials may be prepared for example by acylation of 7-aminocephalosporanic acid, e.g. in an analogous manner to process (A) above. Compounds of formula (IV) in which X represents other acyloxy groups can be prepared by acylation of the corresponding 3-hydroxymethyl compounds which may be prepared for example by hydrolysis of the appropriate 3-acetoxymethyl compounds, e.g. as described in British Patent Specifications Nos. 1,474,519 and 1,531,212.

The starting materials of formula (II) may also be prepared in conventional manner, for example, by nucleophilic displacement of the corresponding 3-acetoxymethyl compound with pyridine e.g. as described in the British Patent Specification No. 1 ,028,563.

A further method for the preparation of the starting materials of formula (II) comprises deprotecting a corresponding protected 7p-amino compound in conventional manner e.g. using PCI5.

It should be appreciated that in some of the above transformations it may be necessary to protect any sensitive groups in the molecule of the compound in question to avoid undesirable side reactions.

For example, during any of the reaction sequences referred to above it may be necessary to protect the NH2 group of the aminothiazolyl moiety, for example by tritylation, acylation (e.g chloroacetylation), protonation or other conventional method. The protecting group may thereafter be removed in any convenient way which does not cause breakdown of the desired compound, e.g. in the case of a trityl group by using an optionally halogenated carboxylic acid, e.g. acetic acid, formic acid, chloroacetic acid or trifluoroacetic acid or using a mineral acid, e.g. hydrochloric acid or mixtures 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 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 acyloxymethyl or -ethyl groups (e.g. acetoxymethyl or -ethyl or pivaloyloxymethyl groups) and retain these in the final product to give an appropriate ester derivative of a compound of formula (I). Other examples of blocking groups which may be retained in the final product include, of course, C,, alkyl groups esterifying the free carboxyl function in the 7-side chain.

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 benzy(oxycarbonyl, p-methoxybenzyloxycarbonyl, pnitrobenzyloxycarbonyl and diphenylmethoxycarbonyl; t-butoxycarbonyl; and 2,2,2trichloroethoxycarbonyl. 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 enzyme-catalysed hydrolyses.

Compounds which may be used as intermediates in the preparation of the compounds according to the invention include: (6R,7 R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(benzyloxycarbonylmethoxyimino)acetamidoj-3-( 1 pyridiniummethyl)ceph-3-em-4-carboxylate, hydrochloride salt [ymex (Nujol) 1788 (D-lactam), 1750 (ester) and 1740 cam~' (acid)]; and (6R,7 R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(p-nitrobenzyloxycarbonylmethoxyimino)acetamido]-3 (1-pyridiniummethyl)-ceph-3-em-4-carboxylate, hydrochloride salt [[a]023-46.1 (c 1.0, dimethylsulphoxide)].

The following non-limiting Examples illustrate the invention. All temperature are in OC.'Petrol' means petroleum ether (b.p. 40--600C).

Preparation 1 (Z)-2-Methoxycarbonylmethoxyimino-2-(2-trityla minothiazol-4-yl)acetic Acid A stirred solution of sodium (Z)-2-hydroxyimino-2-(2-tritylaminothiazol-4-yl)acetate (7 g) in dimethylformamide (80 ml) was treated at 0 under nitrogen with sodium hydride (80% dispersion, 463 mg). After 2.5 hours at 00, the solution was cooled to -30 to 400 and methyl bromoacetate (4.74 g) in dimethylformamide (20 ml) was added over 20 minutes. The mixture was then treated at -30 to --400 with 2N hydrochloric acid t7.75 ml), allowed to warm to 200 and partitioned between water and ethyl acetate. The organic phase was dried and evaporated to a syrup, which was dissolved in a little ethyl acetate and added dropwise to petrol.The resulting solid was collected and dried to give the title compound (5.3 g); T (CDl3) 3.36 (thiazole), 5.30 (CH2), 6.28 (CO2Me).

Preparation 2 (6R,7R)-3-Acetoxymethyl-7-[(Z)-Z-(2-aminothiazol-4-yl)-2 (carboxymethoxyimino)acetamido1ceph-3-em-carboxylic Acid Hydrochloride Salt A stirred solution of t-butyl (6R,7R)-3-acetoxymethyl-7- [(Z)-2-(t-butoxycarbonylmethoxyimino)2-(2-tritylaminothiazol-4-yl)acetamido] ceph-3-em-4-carboxylate (described in Example 3 of South African Specification No. 78/1630) (276 mg) in formic acid (0.5 ml) was cooled to 100. A 10% solution of concentrated hydrochloric acid in formic acid (0.9 ml) was added. The mixture was stirred at room temperature for 75 minutes and then filtered The solid was washed with a little formic acid. The filtrate and washings were added to diisopropyl ether (60 ml). The mixture was stirred at room temperature for 1 hour and then filtered.The solid was washed with diisopropyl ether followed by diethyl ether, and then dried in vacuo to give the title compound (1 10 mg); [a] D2+49.0 (c 1.0, DMSO); Ref 0.4 (cellulose, 1 -propanol-water, 7-3).

Example 1 (6R,7R)-7-[(Z)-2-(2-Aminothiazol-4-yl)-2-(carboxymethoxyimino)acetamido]-3-(1 pyridiniummethyl)ceph-3-em-4-carboxylate A mixture of (6R,7 R)-3-acetoxymethyl-7-[(Z)-(2-aminothiazol-4-yI)-2- (carboxymethoxyimino)acetamido]ceph-3-em-4-carboxylic acid, hydrochloride salt (2.01 g), sodium bicarbonate (0.95 g) and water (2.25 ml) was warmed at 500 until dissolution occurred. Pyridine (0.75 ml) was added and the solution was warmed to 800. Sodium iodide (7.5 g) was added and the solution was stirred at 800 for 40 minutes, cooled and diluted with acetone (100 ml). The mixture was filtered and the solid was washed with acetone and ether to give an orange product (2.5 g). This solid was dissolved in water (10 ml) and acidified with glacial acetic acid (10 ml). The mixture was filtered through a pad of Kieselguhr, and the filtrate was concentrated. The residue was triturated with acetone and filtered to give an orange solid (1.7 g). This solid was dissolved in water (25 ml) and eluted through a column of 200 g Amberlite XAD-2 resin, using first water and then 25% ethanol in water as eluting solvent. The product-containing fractions were concentrated and the residue was triturated with acetone to give the title compound, (0.28 g) as a monoacetone solvate; [a]22+2O.O0 (c, 1.0, water); AmaX (ethanol) 236 nm, (E1%m 299, AInf 250 nm, (Ei1cm 286) and illnf 291 nm, (El%m 150).

Example 2 (6R,7 R)-7-[(Z)-2-(2-Aminothiazol-4-yl)-2-(ethoxycarbonylmethoxyimino)aceta mido]-3-(1 pyridiniummethyl)ceph-3-em-4-carboxylate, Dihydrochloride Salt Phosphorus pentachloride (690 mg) was dissolved in methylene chloride (30 ml) and the solution was cooled to -100. (Z)-2-ethoxycarbonylmethoxyimino-2-(2-tritylaminothiazol-4-yl)acetic acid (1 .55 g) was added and the solution was stirred at -5 for 25 minutes. The solution was cooled to 10C and triethylamine (0.92 ml), followed by water (10 ml), was added with stirring. The temperature was maintained at 0 for 5 minutes by cooling.The lower phase was added to (6R,7R)-7-amino-3-(1 pyridiniummethyl)ceph-3-em4-carboxylate dihydrochloride salt (1.09 g) in N,N-dimethylacetamide (15 ml) and acetonitrile (15 ml) containing triethylamine (2.1 ml), precooled to --200. The addition was made such that the temperature of the reaction mixture did not exceed 10a. After 45 minutes at -5 to --100, the mixture was warmed to 21 over one hour. Methanol (1 ml) was added and the methylene chloride was evaporated. The residue was partitioned between ethyl acetate (50 ml) and water (50 ml) containing salt. The ethyl acetate layer was mixed with further water (50 ml) and salt.

The material which separated was collected and triturated with diisopropyl ether and diethyl ether to give a brown solid (1.49 g). This solid (1.40 g) was dissolved in formic acid (5 ml) and aqueous 11 N hydrochloric acid (0.49 ml) was added. After 1.25 hours at 21 0, the solution was filtered and the filter cake was leached with formic acid. The filtrate was evaporated to a gum which was triturated with acetone to give the title compound (970 mg); [a]021-2 130 (c 1.41, dimethylsulphoxide); silmaX pH6 buffer) 253 nm (Ei1cm 251) and iInf 282 nm (E1cm 110).

Example 3 (6R,7R)-7-[(Z)-2-(2-Aminothiazol-4-yl)-2-( methoxy-carbonylmethoxyimino)aceta mido]-3-(1 - pyridiniummethyl)-ceph-3-em-4-carboxylate, Dihydrochloride Salt (Z)-2-Methoxycarbonylmethoxyimino-2-(2-tritylaminothiazol-4-yl)acetic acid (1.55 g) was added to a stirred suspension of phosphorus pentachloride (0.69 g) in dichloromethane (15 ml) at 200. The temperature was maintained at less than --50 for about 20 minutes and then reduced to about 200 when triethylamine (0.8 ml) was added, with vigorous stirring, followed by water (5 ml). After about 2 minutes the phases were allowed to separate.The organic phase was added dropwise to a stirred mixture of (6R,7R)-7-amino-3-( 1 -pyridiniummethyl)-ceph-3-em-4-carboxylic acid dihydrochloride salt (1.13 g), N,N-dimethylacetamide (12 ml) and acetonitrile (12 ml) containing triethylamine (2.1 ml), precooled to about --250. After about 45 minutes the temperature was allowed to rise to about 0 when methanol (1 ml) was added. The resulting mixture was reduced in volume in vacuo and partitioned between ethyl acetate (50 ml) and water (50 ml) The phases were separated and the aqueous phase was washed with ethyl acetate. The material which separated was collected and triturated with diethyl ether. The resulting solid was dissolved in methanol and then precipitated in diisopropyl ether.The solid (1.2 g) was added to stirred and ice-water cooled formic acid (6 ml), followed by 11 N hydrochloric acid (0.42 ml). After 1 hour the temperature had risen to 5 and the resulting suspension was filtered and the filtrate was reduced in volume and added dropwise to stirred diisopropyl ether (80 ml). A gum resulted which was dissolved in methanol and added to stirred diisopropyl ether. The resulting solid was triturated with acetone giving the title compound (0.87 g); [a]20-280 (c 1.3, dimethylsulphoxide); vilnf (pH 6 buffer) 236 nm (E1%m 278). Amx 255 nm (E1%m 286) and 288 nm (El%m 177).

Pharmaceutical Formulations 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 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 powder is reconstituted. 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.

Compositions for veterinary medicine may aiso, 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 501500 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 should 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 formulation illustrates how the compounds according to the invention may be made up into pharmaceutical compositions.

Formulation-For Injection Formula per Vial (6R,7R)-7-[(Z)-2-(2-Aminothiazol-4-yl)-2-(carboxymethoxy imino)acetamidoj-3-( 1 -pyridinium methyl)ceph-3 em-4-carboxylate 1.00 g Sodium Carbonate, anhydrous 122 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. Constitute the product by dissolving in Water for Injections or other suitable sterile vehicle shortly before administration.

Claims (9)

Claims

1. Cephalosporin antibiotics of general formula

(wherein R represents a hydrogen atom or a C14 alkyl group) and non-toxic salts and non-toxic metabolically labile esters thereof.

2. Compounds as claimed in claim 1 wherein R represents a straight chain C14 alkyl group.

3. Compounds as claimed in claim 1 wherein R represents a methyl or ethyl group.

4. (6R,7 FI)-7-[(Z)-2-(2-Aminothiazol-4-yl)-2-(carboxymethoxyimino)acetamido]-3-( 1- pyridiniummethyl)-ceph-3-em-4-carboxylate and the non-toxic salts thereof.

5. (6R,7 R)-7-[(Z)-2-(2-Aminothiazol-4-yl)-2-(ethoxywarbonylmethoxyi mino)acetamído]-3- 1 pyridiniummethyl)ceph-3-em-4-carboxylate and the non-toxic salts thereof.

6. (6R,7 R)-7-[(Z)-2-(2-Aminothiazol-4-yI)-2-(methoxycarbonylmethoxyimino)acetamido]-3-( 1 pyridiniummethyl)ceph-3-em-4-carboxylate and the non-toxic salts thereof.

7. A process for the preparation of a compound of formula (I) as defined in claim 1 or a non-toxic salt or non-toxic metabolically labile ester thereof which comprises (A) acylating a compound of the formula

[wherein B represents > S or > SoO (a- or ,B-);; 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 an N-silyl derivative thereof, or a corresponding compound having a group of formula -COOR1 at the 4-position (where R' represents a hydrogen atom or a carboxyl blocking group) and having an associated anion To, with an acid of formula

wherein R2 represents a carboxyl blocking group and R3 represents an amino or protected amino group) or with an acylating agent corresponding thereto; (B) reacting a compound of formula

(wherein R3, B and the dotted line are as defined above;R4 and R4a may independently represent hydrogen or a carboxyl blocking group; and X represents a replaceable residue of a nucleophile) or a salt thereof, with pyridine; or (C) when a compound of formula (I) in which R represents a C14 alkyl group is desired, reacting a compound of formula

(wherein B, R3 and the dotted line are as defined above R5 represents a carboxyl blocking group; and Q represents a carboxyl group or a functional equivalent thereof) with an alkylating agent serving to convert the group Q into a group COOR where R represents a C14 alkyl group; 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 A2-isomer into the desired A3-isomer, ii) reduction of a compound wherein B is > SoO 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.

8. A pharmaceutical composition for use in human or veterinary medicine comprising an antibiotic compound as claimed in any one of claims 1 to 6 in association with a pharmaceutical carrier or excipient.

9. A method of combating a bacterial infection in a warm-blooded animal comprising administering to said animal an antibacterially effective amount of at least one cephalosporin antibiotic as claimed in claim 1. ~~~~~~~~~~~~~~~~~