NO744621L - - Google Patents

Abstract

ANTIBIOTICS

Description

The present invention relates to improvements with regard to

to cephalosporin compounds, and relates more particularly to a new class of cephalosporin compounds having valuable antibiotic properties.

The cephalosporin compounds according to the present description

is named in reference to "cepham", according to J.Amer. Chem.Soc, 1962, 84, 3400, whereby the term "cephem

refers to the basic cepham structure with a double bond.

Cephalosporin antibiotics are widely used in the treatment of diseases caused by pathogenic bacteria in men-

pets and animals, e.g. in the treatment of diseases caused by bacteria that are resistant to other antibiotics, such as e.g. penicillin compounds, and by treating

dling of patients sensitive to penicillin.

In many cases, it is desirable to use a cephalosporin antibiotic that exhibits activity against both gram-

positive and gram-negative microorganisms, and a significant number of investigations have been directed at the development of different types of broad-spectrum cephalosporin antibiotics.

Considerable interest has continued to be directed at the development

of broad-spectrum cephalosporin antibiotics, which have high activity against gram-negative organisms. Existing, commercially available, (3-lactam antibiotics have

tend to show relatively low activity against certain Gram-negative organisms, such as Proteus organisms, which are increasingly a common source of human infection, and are generally inactive against Pseudomonas organisms. Several Pseudomonas organisms are resistant to the majority of existing commercially available antibiotic compounds, and the practical therapeutic applications of amino-glycoside antibiotics such as gentamicin that exhibit Pseudomonas activity tend to be limited or complicated by the high toxicity of these antibiotics. It is well known that cephalosporin antibiotics normally exhibit low toxicity in humans, so that the development of broad-spectrum cephalosporin antibiotics with high activity against gram-negative organisms, such as strains of Proteus and Pseudomonas, covers a significant need in chemotherapy.

The present invention provides 7(3-acylamidocep-3-em-4-carboxylic acid antibiotics and non-toxic derivatives thereof, which are characteristic in that said acylamido portion has the formula

where R means a thienyl or furyl group,

R 3 and R<b> , which may be the same or different, are

each selected from hydrogen, C^_^alkyl (e.g. methyl, ethyl, n-propyl, isopropyl or butyl), C 2_^alkenyl (e.g. vinyl or allyl), C 3_7 cycloalkyl (e.g. cyclopropyl, cyclobutyl , cyclopentyl or cyclohexyl), phenyl, naphthyl, thienyl, furyl, carboxy, C2-5 alkoxycarbonyl (e.g. ethoxycarbonyl) and cyano, or Ra and R*3 form together with the carbon atom, to which

they are linked, a C₁₆Cycloalkylidene or cycloalkenylidene group (eg, a cyclobutylidene, cyclopentylidene or cyclohexylidene group), and m and n are 0 or 1, such that the sum of m and n is 0 or 1,

and wherein the compounds are cis isomers or occur as mixtures of cis and trans isomers, containing at least 90% cis isomer.

These compounds exhibit broad-spectrum antibiotic activity, characterized by particularly high activity against gram-negative microorganisms, including those that produce (3-lactamases), and they also have very high stability against (3-lactamases produced by a number of gram-negative organisms.

A characteristic feature of the compounds is their high activity in vitro against gram-negative organisms, such as Enterobacter clocae, Serratia marcescens and Klebsiella

aerogenes. The compounds have particularly high activity against strains of Escherichia coli, Haemophilus influenzae and Proteus organisms, e.g. strains of Proteus morganii and Proteus mirabilis.

Compounds where at least one of Ra and R° is different from I hydrogen have also shown unusually high activity against Pseudomonas organisms, e.g. strains of Pseudomonas aeruginosa.

The compounds according to the invention have cis-isomeric form as regards the configuration of the group

with respect to the carboxamido group. In the present description, the cis configuration is indicated structurally as

This configuration has been established on the basis of the work of Ahmad and Spenser, reported in Can. J. Chem, 1961, 39, 1340. As indicated above, the compounds may occur in mixtures

of cis- and trans-isomers, provided that such mixtures contain at least 90% of the cis-isomer. However, it is preferred that the compounds are cis isomers which are substantially free of the corresponding trans isomer.

The antibiotic compounds according to the invention therefore consist of compounds with the general formula where R, Ra, R<*3>, m and n have the previously stated meanings and

Z is a group in which 2 carbon atoms connect the core sulfur atom and the 4-position carbon atom, as

that the compound has A3 -olefinic unsaturation),

and non-toxic derivatives thereof.

By "non-toxic derivatives" is meant those derivatives which are physiologically acceptable in the dose in which they are administered. Such derivatives may include e.g. salts, biologically acceptable esters, 1-oxides and solutions (especially hydrates). It is preferred that derivatives, such as salts and esters, can be formed by reaction of one or both carboxyl groups present in the compounds of formula I.

Non-toxic salt derivatives which can be formed from compounds of the 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)$ organic base salts (e.g. procaine-, f-enylethylbenzylamine-^ dibenzylethylenedianine-, ethanolamine-j diethanolamine-5 triethanolamine- and N-methylglucosamine salts)5 and, where appropriate, acid addition salts, e.g.

with hydrochloric acids, hydrobromic acids, sulfuric acids, nitric acids,

phosphoric acids, trifluoroacetic acids, toluene-p-sulphonic acids and methane-sulphonic acids. The salts can also be in the form of resinates, e.g. formed with a polystyrene resin or cross-linked polystyrene-jdivinylbenzene copolymer resin, containing amino- or

Quaternary amino groups, or if appropriate, sulfonic acid groups, or, again if appropriate, a resin-

containing a carboxyl group, e.g. a polyacrylic acid resin. Use of easily soluble base salts (e.g. alkali metal salts, such as sodium salt) of compounds of formula I

is usually advantageous in therapeutic use, due to the rapid distribution of such salts in the body after administration. However, where insoluble salts of compounds (I) are desired in a particular application, e.g. for use in depot preparations, these salts can be formed in the usual way, e.g. with suitable organic amines.

Biologically acceptable, metabolically unstable ester derivatives,

which can be formed from compounds of formula I include e.g. acyloxymethyl esters, e.g. lower alkanoyloxymethyl esters,

such as acetoxymethyl or pivaloyloxymethyl esters.

When the group R in the above formula is a furyl group,

it can be fur-2-yl or fur-3-yl and when it is a thienyl group it can be thien-2-yl or thien-3-yl.

When Ra and R*3 in the above formula are different, it will be advantageous for the carbon atom to which they are attached to comprise an asymmetric center. Compounds according to the present invention, where R and R are different can thus be diastereoisomers. The invention encompasses the individual diastereoisomers of such compounds, as well as mixtures thereof.

Cephalosporin antibiotics according to the present invention can

be unsubstituted in the 3-position or can carry in this position any of the large range of substituents described in the literature relating to cephalosporin compounds, whereby the characteristic feature according to the invention is the properties of the 7(3-acylamido group. The scope of the invention thus includes compounds with the general formula

in

!

9 b

where R, R , R , m and n have the above meaning and

P means a hydrogen atom; a halogen atom, such as fluorine, chlorine or bromine, or an organic group, e.g. a saturated or unsaturated, substituted or unsubstituted, organic group, containing 1-20 carbon atoms, and non-toxic derivatives thereof.

When P is an unsaturated, organic group, it can e.g. be a group with the formula

1 2

wherein R and R , which may be the same or different, are each selected from hydrogen, carboxy, cyano, C 2-7 alkoxycarbonyl (e.g. methoxycarbonyl or ethoxycarbonyl), and substituted or unsubstituted aliphatic (e.g. alkyl, preferably C 1 - C^alkyl, such as methyl, ethyl, isopropyl or n-propyl), C^-C^cycloaliphatic (e.g. Cc_7 cycloalkyl, such as cyclopentyl or cyclohexyl), C7~C^0 araliphatic (e.g. phenyl C 1 -alkyl, such as benzyl or phenylethyl) and C 8 -C 1 -aromatic (e.g. mono- or bicyclic carbocyclic

aryl, such as phenyl, nitrophenyl, tolyl or naphthyl) groups. Specifically substituted vinyl groups with it

the above formula comprises 2-carboxyvinyl, 2-

methoxycarbonylvinyl, 2-ethoxycarbonylvinyl and 2-cyanovinyl.

P can also be a substituted methyl group which can be visualized with the formula

where Y means an atom or a group, e.g. the residue of a nucleophile or a derivative of a residue of a nucleophile.

Y can thus e.g. be derived from the wide range of nucleophiles characterized by having a nucleophilic nitrogen, carbon, sulfur or oxygen atom, which is carefully described in previous patents and literature relating to cephalosporin chemistry. Examples of

such nucleophiles include:

Nitrogen nucleophiles

Examples of nitrogen nucleophiles are tertiary, aliphatic, aromatic, araliphatic and cyclic amines, e.g. tri(C 1-6 alkyl) amines, such as triethylamine and heterocyclic, tertiary amines.

The heterocyclic tertiary amines may, if desired, contain one or more additional heteroatoms in addition to the basic nitrogen atom, and may be substituted or unsubstituted. The heterocyclic tertiary amine can thus e.g. be a pyridine, pyrimidine, pyridazine, pyrazine, pyrazole, imidazole, triazole or thiazole; fused bi- or poly-cyclic analogues of any of these heterocycles, e.g.

purine or benzotriazole5 and any of the above amines, substituted with one or more aliphatic (e.g. lower alkyl, such as methyl, ethyl, n-propyl or isopropyl), aryl- (e.g. C^_^2mono- or bicyclic carbocyclic aryl, such as phenyl or naphthyl), araliphatic (eg, phenyl-lower alkyl, such as benzyl or phenylethyl), lower alkoxymethyl- (eg, methoxymethyl, ethoxymethyl, n-propoxymethyl or isopropoxymethyl), acyloxy-!methyl- ( eg lower a1kanoyloxymethyl, such as acetoxymethyl), !

[formyl-, acyloxy- (e.g. lower alkanoyloxy, such as acetoxy)<!>, carboxy-, esterified carboxy- (e.g. lower alkoxycarbonyl, •such as methoxycarbonyl), carboxy-lower-alkyl- (e.g. . carboxymethyl), sulfo-, lower alkoxy- (e.g. methoxy, ethoxy, n-propoxy or iso-propoxy), aryloxy- (e.g. phenoxy), aralkyl- (e.g. benzyloxy), alkylthio- (e.g. methylthio or ethylthio), arylthio-, aralkylthio-, cyano-, hydroxy-, carbamoyl-, N-monolower alkylcarbamoyl- (e.g. N-methylcarbamoyl or N-ethylcarbamoyl), N,N-dilower alkylcarbamoyl - (e.g. N,N-dimethylcarbamoyl or N,N-diethylcarbamoyl), N-(hydroxylavereal-cyl)carbamoyl- (e.g. N-(hydroxymethyl)carbamoyl or N-(hydroxyethyl)-carbamoyl) , or carbamoyl-lower alkyl (eg carbamoylmethyl or carbamoylethyl) groups. Examples of Y groups which can be obtained from heterocyclic tertiary amine nucleophiles of the above type include pyridine, 3- and 4-carbamoylpyridine, 3-carboxymethylpyridine, 3-sulfopyridine, thiazol-3-yl, pyrazol-1-yl, pyridazine, and benzotriazol-1-yl.

Another class of nitrogen nucleophiles consists of azides, e.g. alkali metal azides, such as sodium azide.

When the group Y is a derivative of a residue of a nitrogen nucleophile, it can e.g. be an amino group or an acylamido group. Compounds in which Y is amino can be derived from the corresponding compound in which Y is azido by reduction, e.g. by catalytic hydrogenation of the azide using a noble metal catalyst such as palladium or platinum. Compounds in which Y is an acylamido group can be derived by acylation of a compound in which Y is amino, e.g. by any method suitable for acylating an aminocephalosporin, e.g. reaction of the amino compound with an acid chloride, acid anhydride or mixed anhydride of an acid, corresponding to the desired acyl group and another acid.

Compounds in which Y is amino can also be reacted with a substituted isocyanate or isothiocyanate to give urea or thio-urea derivatives.

i Other compounds, wherein Y is a derivative of a residue of a

Nitrogen nucleophile can be obtained by reacting a compound, ■ in which Y is azido, with a dipolarophile. Examples of such dipolarophiles include acetylenic, ethylenic and cyano-dipolarophiles.

Acetylenic dipolarophiles can be shown using the structure

3 4

wherein R and R , which may be the same or different, are atoms or groups.

Usually it is preferred that R 3 and preferably also R<4>

is of an electro-negative nature. Examples of such groups

5 5 5

is cyano, CO 2 R , COR (where R is e.g. hydrogen, lower alkyl, aryl or lower aralkyl), and trihalomethyl, e.g. trifluoromethyl.

R^ and also R^ may, however, preferably also be electro-positive, e.g. alkoxy or alkylamino.

3 4

R and R can together form a ring system with the acetylene group

like for example. in an aryn.

When R 3 and R 4 are separate atoms or groups which are identical, the result will be a single compound upon reaction with azido-cephalosporin. If they are different, a mixture of positional isomers will usually be obtained.

Ethylene dipolarophiles can be shown with the structure

6 7 8 9

where R , R , R and R , which can be the same or different

6 7 different, are atoms or groups. Although R , R , 8 9

R and R are all hydrogen, in and of themselves ethylene as well as acetylene then the compound reacts slowly with

6 8

azido groups. R and R can together form a cyclic structure, e.g. a carbocyclic structure with the ethenoid group, so that the double bond is straight chain. Examples of ethylenic dipolarophiles containing straight-chain double bonds include norbornenes, trans-cycloalkenes and acenaphthalene.

Additional ethylenic dipolarophiles that may be used include

6 7 8 9

compounds of formula R . R C = CR . R , where at least one of

6 7 8

R , R , R and R are an electronegative group. R and R can thus be identical electronegative groups, R and R^ are other desired groups. R 7 and R 9 can thus form together

a ring system. Examples of such dipolarophiles include benzoquinone and core-substituted benzoquinones and maleimide.

6 7 8 9

Again, all of R , R , R and R can be identical electro-negative groups. Electronegative groups that can be used include those listed under the section on acetylenic dipolar philes and examples of such compounds thus include dicyanoethylene and lower mono- and di- alkoxycarbonyl ethylenes.

6 7 8 9

One or more of R , R , R and R may, if desired, be electro-positive.

Cyano compounds, especially those activated by electronegative groups can function as cyano-dipolarophiles. Examples of such dipolarophiles include lower alkoxycarbonyl cyanides and cyanogen.

I ■ ' !

IN

In Carbon nucleophiles

Examples of carbon nucleophiles are inorganic cyanides, pyrroles and substituted pyrroles, e.g. indoles, and compounds stabilized carbanions, e.g. acetylenes and compounds having (3-diketone groups, e.g. acetoacetic acid and malonic acid esters and cyclohexane-1,3-diones or enamines, ynamines or enols.

The carbon nucleophile thus causes the formation of cephalosporin compounds which are characterized by having a substituent in the 3-position, in which a carbonyl group is connected to the cephalosporin nuclei through two carbon atoms.

Such compounds can thus contain, as the 3-substituent,

a group with formula

wherein R"<*>"<0>and R"^, which may be the same or different, are selected from hydrogen, cyano, lower alkyl, eg methyl or ethyl, phenyl, phenyl substituted with eg halogen , lower alkyl, lower alkoxy, nitro, amino or lower alkylamino, carboxy, lower alkoxycarbonyl, mono- or di-aryl lower alkoxycarbonyl, lower alkylcarbonyl, aryl lower alkyl or C,- or C ft cycloalkyl and

12

R is selected from hydrogen, lower alkyl, e.g. methyl or ethyl, phenyl, phenyl substituted with, e.g. halogen, lower alkyl, lower alkoxy, nitro, amino or lower alkylamino, aryl lower alkyl or C 1 - or C 1 -cycloalkyl.

Sulfur nucleophiles

Examples of sulfur nucleophiles include thioureas, including aliphatic, aromatic, araliphatic, alicyclic and heterocyclic substituted thioureas, dithiocarbamates, aromatic, j aliphatic and cyclic thioamides, e.g. thioacetamide and thiosemia;-

Icarbazid, thiosulphates, thiols, thiophenols, thioacids, e.g. thiobenzoic acid or thiopicolinic acid, and dithio acids.

A class of sulfur nucleophiles includes those compounds with

13 13

mel R .S(0)nH, where R is an aliphatic group, e.g.

lower alkyl, such as methyl, ethyl or n-propyl, an alicyclic group, e.g. lower cycloalkyl, such as cyclohexyl or cyclopentyl, an aromatic group, e.g. C 1-12 mono- or bicyclic aryl, such as phenyl or naphthyl, an araliphatic group, e.g. phenyl-lower (eg, C^_^) alkyl, such as benzyl, or a heterocyclic group, and n is 0, 1 or 2.

A preferred class of nucleophiles falling within it

14

the above formula is that of the general formula R SH,

14

where R is aliphatic group, e.g. lower alkyl, such as methyl, ethyl or n-propyl or lower alkanoyl, such as acetyl, araliphatic group, e.g. phenyl lower alkyl, such as benzyl or phenethyl or substituted phenyl lower alkyl, alicyclic group, e.g. cycloalkyl, such as cyclopentyl or cyclohexyl^aromatic group, e.g. phenyl, substituted phenyl or a heterocyclic group, containing at least one 5- or 6-membered ring and which .

has one or more heteroatoms selected from 0, N and S. Such

14

heterocyclic groups R may be substituted, and examples of suitable heterocyclic groups include thiadiazolyl, e.g. 5-methyl-1,3,4-thiadiazol-2-yl, diazolyl, triazolyl, e.g. triazol-4-yl, tetrazolyl, e.g. 1-methyltetrazol-5-yl, 1-ethyl-tetrazol-5-yl or 1-phenyltetrazol-5-yl, thiazolyl, thiatria-

zolyl, oxazolyl, oxadiazolyl, e.g. 2-phenyl-1,3,4-oxadiazol-5-yl, pyridyl, e.g. N-methylpyrid-2-yl, pyrimidy1, fused heterocyclic ring systems, such as benzimidazolyl, benzoxazolyl, benzothiazolyl, such as benzothiazol-2-yl, triazolopyridyl or purinyl, and substituted types of such fused ring systems, e.g. nitrobenzothiazol-2-yl,

such as 5- or 6-nitrobenzothiazol-2-yl.

Oxygen nucleophiles

Examples of oxygen nucleophiles include water, alcohols,

e.g. alkanols, such as methanol, ethanol, propanol and butanol,

|and lower alkano and alkeno acids.

I The expression "oxygen-nucleophile" thus includes compounds with the following formula:

15

where the group R can be lower alkyl (e.g. methyl, ethyl, n-propyl, isopropyl, n-butyl or isobutyl), lower alkenyl (e.g. allyl), lower alkynyl (e.g. propynyl), lower cycloalkyl (e.g. cyclopropyl, cyclopentyl or cyclohexyl), lower cycloalkyl lower alkyl (e.g. cyclopropylmethyl, cyclopentylmethyl or cyclohexylethyl), aryl (e.g. phenyl or naphthyl), aryl lower alkyl (e.g. benzyl), heterocyclic groups (e.g. a heterocyclic group as indicated for R 14 such as N-methylpyrid-2-yl), heterocyclic lower alkyl (e.g. furfuryl), or any of these groups substituted with e.g. . one or more lower alkoxy- (e.g. methoxy or ethoxy), lower alkylthio- (e.g. methylthio or ethylthio), halogen- (chloro, bromo, iodo or fluorine), lower alkyl-(e.g. methyl or ethyl), nitro, hydroxy, acyloxy, carboxy, caralkyloxy, lower alkylcarbonyl, lower alkylsulfonyl, lower alkoxysulfonyl, amino, lower alkylamino or acylamino groups.

In the case where water is the nucleophile, 3-hydroxymethyl-cephalosporin compounds will be obtained. Such 3-hydroxymethyl compounds and non-toxic derivatives thereof may show antibacterial activity and it must be noted that there may be products obtained by metabolism of compounds of the general formula II, where P means acetoxymethyl. 3-Hydroxymethyl-cephalosporins can be acylated to form derivatives such as

if\

are characterized by having the group 3-CK2.O.CO.R

or 3-CH„.O.CO.AR<17>, where A means 0, S or NH,R<16>er

17

an organic group and R is hydrogen or an organic group.

1f\ 17

The group R CO- or R A.CO- may be selected from the large class of such groups described in the literature and they may have '

carbon atoms.<16><17>

in up to 20 carbon atoms. R and optionally R can thus each be a hydrocarbon group or such a group bearing one or more substituent atoms or groups, and they can thus be selected from the following list, which is not intended to be comprehensive:

(i) C H_ ,,, where n is an integer from 1-7, e.g.

n 2n+l' '

1-4. The group can be straight chain or branched and,

if desired, it can be interrupted by an oxygen or sulfur atom or an imino group or substituted with cyano, carboxy, lower carboxycarbonyl, hydroxy, carboxycarbonyl..

(H00C.C0), halogen (eg chlorine, bromine or iodine) or amino. Examples of such groups are methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, sec-butyl and 2-chloroethyl.

(ii) C H~ , , where n is an integer from 2-7. The group n 2n-1' ^ may be straight chain or branched and, if desired, interrupted by an oxygen or sulfur atom or an imino group. Examples of such groups are vinyl and propenyl. (iii) R 18 , where R 18 is carbocyclic aryl (e.g. c^_^ 2 mono- or bicyclic carbocyclic aryl), heterocyclic aryl (e.g. consisting of a 5- or 6-membered ring, containing at least one of O, N and S), lower cycloalkyl, substituted aryl and substituted cycloalkyl. Examples of this group are phenyl, substituted phenyl, e.g. hydroxyphenyl, chlorophenyl, fluorophenyl, tolyl, nitrophenyl, aminophenyl, methoxyphenyl or methylthiophenyl, thien-2- and -3-yl, pyridyl, cyclohexyl, cyclopentyl, cyclopropyl, sydnon, naphthyl and substituted naphthyl, e.g. 2-ethoxynaphthyl. (iv) R 18 (CH 2 )m, where R 18 has the above meaning under (iii), and m is an integer from 1-4. Examples of this group are methyl, ethyl or butyl substituted with the various specific R 18 groups listed under (iii), e.g. lower cycloalkyl C^_^-alkyl and carbocyclic or heterocyclic aryl, C, _. alkyl, such as benzyl and the appropriate substituted benzyl groups.

The I I 3-position substituent of the aforementioned type thus comprises lower alkanoyloxymethyl groups, such as acetoxymethyl and isobutyryloxymethyl, lower alkenoyloxymethyl groups, such as crotonyloxymethyl, aroyloxymethyl groups, such as benzoyloxymethyl, carbamoyloxymethyl, N-(lower alkyl)carbamoyloxymethyl, such as N-methylcarbamoyloxymethyl, and N-(haloalkyl)carbamoyloxymethyl, such as N-(2-chloroethyl)carbamoyloxymethyl.

A further important class of cephalosporin compounds is

those containing the group 3-CH2Hal?in which Hal means chlorine, bromine or iodine. Such compounds are primarily of value as intermediates for use in the production of active cephalosporin compounds, where the halogen atom is replaced by a nucleophile, e.g. a nitrogen, oxygen or sulfur containing nucleophile, which are described below.

The term "lower" used in the present description and in

the. subsequent requirements for determining the aliphatic groups indicate, unless otherwise noted, that said group may contain up to 6 carbon atoms. "Lower" used to designate cycloaliphatic groups indicates that the group may contain 3-7 (eg 5-7) carbon atoms.

A particularly interesting class of cephalosporin antibiotics according to the present invention comprises compounds of the general formula where R has the above meaning,

R° means methyl, ethyl, propyl, allyl or phenyl and R^ means hydrogen, carboxy or preferably a

c cd group as indicated for R , or R and R together with the carbon atom to which they are attached form a cyclobutylidene, cyclopentylidene or cyclohexylidene group, and

W is selected from

i) hydrogen,

ii) acetoxymethyl,

iii) benzoyloxymethyl,

iv) carbamoyloxymethyl,

v) N-methylcarbamoyloxymethyl,

vi) a group with the formula

where R<z>means cyano, carboxy or a C2-5al-olcsy-carbonyl group, such as methoxycarbonyl or ethoxycarbonyl, vii)'the group -CH,,^, where G means the residue of a nitrogen nucleophile, selected from compounds with the general formula

where R^ means hydrogen, carbamoyl, carboxymethyl or sulfo,

and pyridazine,

viii) azidomethyl, and

ix) the group -CK2SR w , where R w is selected from pyridyl,

diazolyl, triazolyl, tetrazolyl, thiazolyl, thiadiazolyl, oxadiazolyl, and substituted (eg lower alkyl- or phenyl-substituted) types of these groups,

such as N-methylpyrid-2-yl, 1-methyltetrazol-5-yl, 1-phenyltetrazol-5-yl, 5-methyl-1,3,4-thiadiazol-2-yl and

I 5-phenyl-1,3,4-oxadiazol-2-yl,<;>

and non-toxic derivatives thereof.

These compounds exhibit broad-spectrum antibiotic activity (including very high activity against strains of Haemophilus influenzae and Proteus organisms) and high (3-lactamase stability and are further characterized by particularly high in vitro activity against Pseudomonas organisms, such as strains of Pseudomonas aeruginosa.

Particularly preferred compounds of the above type in effect

of their particularly high level of activity against Proteus and Pseudomonas organisms, include the following:

(6R,7R)- 7-[2-(2-carboxyprop-2-yloxyimino)-2-(fur-2-yl)acetamido]-3-pyridinium-methylceph-3-em-4-carboxylic acid (cis isomer ). (6R,7R)-7-[2-(2-carboxyprop-2-yloxyimino)-2-(fur-2-yl)acetamido]-3-(5-methyl-1,3,4-thiadiazol-2- ylthiomethyl)ceph-3-em-4-carboxylic acid (cis-isomer), (6R,,7R)-3-carbamoyloxymethyl-7-[ 2-(2-carboxyprop-2-yloxyimino)-2-(fur-2- yl)acetamido]ceph-3-em-4-carboxylic acid (cis isomer), (6R,7R)-7-[2-(2-carboxyprop-2-yloxyimino)-2-(fur-2-yl)acetamido ] -3-(trans-2-methoxycarbonylvinyl)ceph-3-em-4-carboxylic acid (cis isomer),

(6R,7R)-7-[2-(2-carboxyprop-2-yloxyimino)-2-(fur-2-yl)acetamido]-3-pyridaziniummethylceph-3-em-4-carboxylic acid (cis isomer) ,

(6R,7R)-7-[2-(2-Carboxyprop-2-yloxyimino)-2-(fur-2-yl)acetamido]ceph-3-em-4-carboxylic acid (cis isomer), (6R, 7R)-3-acetoxymethyl-7-[2-(1-carboxycyclopent-1-yloxyimino)-2-(fur-2-yl)acetamido]ceph-3-em-4-carboxylic acid (cis-isomer),

(6R,7R)-7-[2-(1-Carboxycyclopent-1-yloxyimino)-2-(fur-2-yl)acetamido]-3-pyridiniummethylceph-3-em-4-carboxylic acid (cis-isomer),

(6R,7R)-7-[2-(1-carboxycyclopent-1-yloxyimino)-2-(fur-2-yl)acetamido]-3-(trans-2-carboxyvinyl)ceph-3-em-4- carboxylic acid (ci s-i somer), j

I (6R,7R)-7-[2-(1-carboxycyclopent-1-yloxyimino)-2-(fur-2-yl)-''acetamido]-3-(1-methyltetrazol-5-ylthiomethyl)ceph- 3-em-4-carboxylic acid (cis-isomer),

(6R,7R)-3-carbamoyloxymethyl-7-[2-(1-carboxycyclopent-1-yloxyimino)-2-(fur-2-yl)acetamido]ceph-3-em-4-carboxylic acid (cis-isomer) ,

(6R, 7R) - 7- [2-(1-Carboxycyclopent-1-yloxyimino)- 2-(fur-2-yl)-acetamido] ceph-3-em-4-carboxylic acid (cis isomer), (6R ,7R)-3-acetoxymethyl-7-[2-(1-carboxybut-3-enyloxyimino)-2-(fur-2-yl)acetamido]ceph-3-em-4-carboxylic acid (cis isomer), ( 6R,7R)-3-acetoxymethyl-7-[2-(1-carboxycyclobut-1-yloxyimino)-2-(fur-2-yl)acetamido]ceph-3-em-4-carboxylic acid (cis-isomer), (6R,7R)-7-[2-(1-Carboxycyclobut-1-yloxyimino)-2-(fur-2-yl)-acetamido]-3-pyridiniummethylceph-3-em-4-carboxylic acid (cis-isomer) , (6R,7R)-7-[2-(1-carboxycyclobut-1-yloxyimino)-2-(fur-2-yl)-acetamido]-3-(1-methyltetrazol-5-ylthiomethyl)ceph-3- em-4-carboxylic acid (cis-isomer),

(6R,7R)-3-carbamoyloxymethyl-7-[2-(1-carboxycyclobut-1-yloxyimino)-2-(fur-2-yl)acetamido]ceph-3-em-4-carboxylic acid (cis-isomer) ,

(6R,7R)-3-acetoxymethyl-7-[2-(1-carboxypropoxyimino)- 2-(fur-2-yl)acetamido]ceph-3-em-4-carboxylic acid (cis isomer), (6R, 7R)-3-acetoxymethyl-7-[2-(3-carboxypent-3-ylpoxyimino)-2-(fur-2-yl)acetamido]ceph-3-em-4-carboxylic acid (cis isomer), (6R ,7R)-3-acetoxymethyl-7-[2-(2-carboxyprop-2-yloxyimino)-2-(thien-2-yl)acetamido]ceph-3-em-4-carboxylic acid (cis isomer),

and non-toxic derivatives thereof, e.g. alkali metal salts, such as sodium or potassium salts.

A further interesting class of cephalosporin antibiotics according to the invention comprises compounds of the general formula

i i ! in

where R and W have the above meaning and

p is 1 or 2,

and non-toxic derivatives thereof.

These compounds exhibit broad-spectrum antibiotic activity combined with high β-lactamase stability. A characteristic feature of the compounds is their high activity against strains of Haemophilus influenzae together with their particularly high activity against strains of Escherichia coli and Proteus organisms.

Particularly preferred compounds of the above type, by virtue of their particularly high level of activity against Escherichia coli and Proteus organisms, include the following: (6R,7R)-3-acetoxymethyl-7-[2-carboxymethoxyimino-2-(fur-2- yl) acetamido]ceph-3-em-4-carboxylic acid (cis-isomer), (6R,7R)-3-azidomethyl-7-[2-carboxymethoxyimino-2-(fur-2-yl)-acetamido]ceph- 3-em-4-carboxylic acid (cis isomer), (6R,7R)-7-[2-carboxymethoxyimino-2-(fur-2-yl)acetamido]-3-(1-methyltetrazol-5-ylthiomethyl)ceph -3-ene-4-carboxylic acid (cis-isomer),

(6R,7R)-3-carbamoyloxymethyl-7-[2-carboxymethoxyimino-2-(fur-2-yl)acetamido]-ceph-3-em-4-carboxylic acid (cis isomer), (6R,7R) -7-[2-carboxymethoxyimino-2-|fur-2-yl)acetamido]ceph-3-em-4-carboxylic acid (cis-isomer),

and non-toxic derivatives thereof, e.g. alkali metal salts, such as sodium or potassium salts.

in i

The compounds according to the invention can be prepared in a conventional manner, e.g. using techniques analogous to those described in Belgian Patent No. 783,449.

According to one embodiment of the invention, a method for producing an antibiotic compound with the general formula I as previously described, or a non-toxic derivative thereof, is thus provided, which comprises either

(A) condensation of a compound of formula

in which B is >S or >S — > 0 (a- or (3-)

19

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

(said alcohol, phenol, silanol or stannanol preferably contains 1-20 carbon atoms) or a symmetrical or mixed anhydride group, derived from a suitable acid, and

Z' is a group in which 2 carbon atoms connect the core sulfur atom and the 4-position carbon atom

so that the compound has ZA or Z\ unsaturation, or a salt, e.g. an acid addition salt, such as a hydrochloride, hydrobromide, sulfate, nitrate, phosphate, methanesulfonate or tosylate, or an N-silylated derivative thereof with an acylating agent corresponding to an acid of the formula

9 b

where R, R , R , m and n have the previously stated meanings and

20

R means a carboxyl-blocking group, e.g. a group previously described in connection with or

(B) where Z in formula I is the group

where Y has the previously indicated meaning, reaction of a compound of formula

9 b

where B, R, R , R , m and n have the above

meaning, each of

j R"^ can independently represent hydrogen !

or a carboxyl-blocking group,

Y' is a replaceable residue of a nucleophile, e.g.

an acetoxy or dichloroacetoxy group or a halogen atom, such as chlorine, bromine or iodine, and the dashed line crossing the 2-, 3- and 4-positions indicates that the compound is a ceph-2-em or ceph- 3-em connection,

with a nucleophile, after which, if necessary and/or desired,

in each case, any of the following reactions (C) in any suitable order, is carried out:

i) conversion of a Z1^-isomer to the desired A^-isomer,

ii) reduction of a compound wherein B is ^> S—>0 to form a compound wherein B is >S;

iii) reduction of a 3-azidomethyl compound to form a 3-aminomethyl compound;

iv) acylation of a 3-aminomethyl compound to form a 3-acylaminomethyl compound;

v) reaction of a 3-azidomethyl compound with a dipolarophile to form a compound having a polyazole ring attached to the 3-position carbon atom by a methylene group;

vi) deacylation of a 3-acyloxymethyl compound to form a 3-hydroxymethyl compound;

vii) acylation of a 3-hydroxymethyl compound to form a 3-acyloxymethyl compound;

viii) carbamoylation of a 3-hydroxymethyl compound to form an unsubstituted or substituted 3-carbamoyloxymethyl compound, and

ix) removal of carboxyl-blocking groups;

log finally (D) recovery of the desired compound with

In formula I or a non-toxic derivative thereof, if necessary, after separation of the isomers.

Non-toxic derivatives of compounds of formula I can

is formed in a conventional way, e.g. according to methods which are well known in the state of the art. Thus, e.g. base salts are formed by reaction of the cephalosporin acid with sodium 2-ethylhexanoate or potassium 2-ethylhexanoate. Biologically acceptable ester derivatives can be formed using conventional esterification agents. 1-oxides can be formed by treating the corresponding cephalosporin sulfide with a suitable oxidizing agent, e.g. with a peracid, such as metaperiodic acid, peracetic acid, monoperphthalic acid or m-chloroperbenzoic acid, or with t-butyl hypochlorite. This last reagent is usually used in the presence of a weak base,

such as pyridine.

Acylating agents which can be used in the preparation of compounds of formula I include acid halides, especially acid chlorides or bromides. Such acylating agents can be prepared by reacting an acid (VI) or a salt thereof with a halogenating agent, e.g. phosphorus pentachloride, thionyl chloride or oxalyl chloride. Treatment of the sodium, potassium or triethylammonium salt of the acid (VI) with oxalyl chloride is advantageous in that the isomerization is minimal under these conditions.

Acylations using acid halides can be carried out in aqueous

and non-aqueous reaction media, usually at a temperature 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 binders include tertiary amines (eg triethylamine or dimethylaniline), inorganic bases (eg calcium carbonate or sodium bicarbonate) and oxides such as lower-1,2-alkylene oxides (eg ethylene oxide or propylene oxide)

Which bind the hydrogen halide that was released at

in

the acylation reaction.

Acids of formula VI can themselves be used as acylating agents in the preparation of compounds of formula I. Acylations using acids (VI) are preferably carried out in the presence of a condensing agent, e.g. a carbodiimide, such as

■ N,N'-diethyl, dipropyl or diisopropylcarbodiimide, N,N'-dicyclohexylcarbodiimide or N-ethyl-N'-y-dimethylaminopropylcarbodiimide, a carbonyl compound, such as carbonyldiimidazole, or an isoxazolinium salt, such as N-ethyl -5-phenylisoxazolinium perchlorate. Acylation reactions of this type are preferably carried out in an anhydrous reaction medium, e.g. methylene chloride, dimethylformamide or acetonitrile.

Acylation can also be carried out with other amide-forming derivatives

of acids with formula VI, such as e.g. a symmetrical anhydride or a mixed anhydride (eg with pivalic acid or formed with a halogen formate, such as a lower alkyl halogen formate). The mixed or symmetrical anhydride may be produced

in situ. Thus, e.g. a mixed anhydride is provided using N-ethoxycarbonyl-2-ethoxy-1,2-dihydro-quinoline. Mixed anhydrides can also be formed with phosphorus-

acids (e.g. phosphoric acids or phosphoric acids), sulfuric acid or aliphatic or aromatic sulphonic acids (e.g. p-toluene sulphonic acid).

One is aware that in methods for the production of pre-

9 b

bonds of formula I, in which R or R represents carboxy, it will in many cases be necessary to protect the carboxy group, e.g. by substitution with a carboxyl-blocking group, e.g. a group described in it

19

subsequently in connection with R

Any transfer of the substituents in the 3-position, which

may be necessary in the preparation of special compounds with formula I can e.g. is carried out using methods described in the literature.

^ i

In Thus, e.g. compounds which are substituted in the 3-position by a group,

"wherein Y is an ether or thioether group or a halogen atom,

is produced as described in British Patent Nos. 1,241,656, 1,241,657, 1,277,415 and 1,279,402. Compounds where Y is the residue of a nucleophile can also be prepared by reaction of a 3-acetoxymethyl-cephalosporin compound with a nucleophile, e.g. pyridine or another tertiary amine, as described in British Patent No. 912,541, a sulfur-binding, nitrogen-

binding or, inorganic nucleophile, as described in British Patent No. 1,012,943, a sulfur-binding nucleophile which bes-

claimed in British Patent Nos. 1,059,562, 1,101,423 and 1,206,305, or a nitrogen-bonding nucleophile as described in British Patents Nos. 1,030,630, 1,082,943 and 1,082,962. Compounds in which Y is a derivative of a residue of a nucleophile, e.g. where

Y is an amino or acylamido group, derived from an azido group can be prepared as described in British Patent Nos. 1,057,883 and 1,211,694. These patents further describe the reaction of compounds wherein Y is azido with a dipolarophile. Compounds in which Y is the residue of a nucleophile can also be prepared by: reaction of a 3-halomethylcephalosporin with any of the nucleophiles listed in the above references,

such a method is otherwise described in British patent no. 1,241,657, or by the reaction of a 3-halomethyl-cephalosporin sulphoxide. with any of the nucleophiles listed in the above references, and such a method is described in British Patent No. 1,326,531. The contents of the above-mentioned British patents are incorporated in this description by reference.

In that case, a 3-halomethylcephalosporin sulfide or

-sulfoxide ester is reacted with a tertiary nitrogen nucleophile,

like for example. pyridine, according to the method according to the British patent

No. 1,241,657 or 1,326,531, then the reaction product will usually be obtained, e.g. in the form of the corresponding 3-pyridinium-I

IN

methyl halide. It has been observed that the esterification of compounds of this type by treatment with trifluoroacetic acid tends to accelerate isomerization of the oxyimino moiety of the 7(3-acylamido side chain. Such isomerization is clearly undesirable if a product containing at least 90% of cis The -isomer is to be obtained without the necessity of a subsequent isomer separation step.

However, it has been observed that the tendency to isomerization is significantly reduced if the 3-pyridinium methyl halide is converted to the 3-pyridinium methyl salt by a non-hydrohalide

acid (e.g. trifluoroacetic acid, acetic acid, formic acid, sulfuric acid, nitric acid or phosphoric acid) for stripping. Conversion of the halide salt to a non-hydrohalic acid salt is conveniently carried out by means of an anion exchanger. It can be provided by e.g. use of a suitable anion-exchange resin, e.g. in trifluoroacetate form. Where an anion-exchange resin is used, the 3-pyridinium methyl halide may pass through a column of resin for removal. It may be advantageous to use an inert organic solvent system (ie one that has no detrimental effect on the resin) for

to ensure adequate solubility of the cephalosporin compound. Organic solvent systems, which can be used are

lower alkanols such as ethanol, ketones such as acetone and nitriles such as acetonitrile.

Compounds containing a 3-substituent

wherein Y means a hydroxy group,

can be prepared by the methods described in British Patent No. 1,121,308 and Belgian Patent No. 841,937.

If Y represents a halogen atom (ie chlorine, bromine or iodine), the starting ceph-3 compounds can be prepared by halogenation of the 7-(3-acylamido-3-methylceph-3-em-4-carboxylic acid ester-1(3-oxide followed by reduction of the 1(3-oxide group in a later step, as described in British Patent No. 1,326,531. The corresponding ceph-2-lem compounds can be prepared by the method according to Dutch Patent Application No. 6,902,013 by reaction of a 3-methylceph-2-em compd. with N-bromosuccinimide to give

the corresponding 3-bromo-methylceph-2-em compound.

Carbamoylation of the 3-hydroxymethyl compounds can be carried out

by conventional methods. Thus, e.g. 3-Hydroxymethyl-cephalosporin reacts with an isocyanate of the formula R.NCO (wherein R means a labile substituent group or an alkyl group) to give a compound containing a 3-position substituent of the formula -CH20.CONHR<e >(where Re has the previously defined meaning). Where Re means a labile substituent, this substituent can, if desired, subsequently be cleaved, e.g. on hydrolysis, to form a 3-carbamoyloxymethyl group. Labile groups Re that are easily cleaved by subsequent treatment include chlorosulfonyl and bromosulfonyl, halogenated lower alkanoyl groups, such as dichloroacetyl and triacetyl, and halogenated lower alkoxycarbonyl groups , such as 2,2,2-trichloroethoxycarbonyl. These labile Re groups can generally be cleaved by acid- or base-catalyzed hydrolysis (eg, by base-catalyzed hydrolysis using sodium bicarbonate).

Another carbamoylating agent for use in the carbamoylation gene of 3-hydroxymethyl-cephalosporins is cyanic acid, which is conveniently developed in situ from e.g. an alkali metal cyanate, such as sodium cyanate, whereby the reaction is facilitated in the presence of an acid, e.g. a strong organic acid, such as trifluoroacetic acid. Cyanic acid actually corresponds to a compound of formula R<e>.NC0, where Re means hydrogen, and therefore converts the 3-hydroxymethyl-cephalosporin compounds directly into their 3-carbamoyloxymethyl analogues.

3-Hydroxymethyl-cephalosporins for use in the above carbamoylation reactions can e.g. is produced by methods described in British Patent No. 1,121,308 and Belgian Patent No. 783,449 and 841,937.

The cephalosporin compounds which have an acyloxymethyl group as a b-position substituent can e.g. is prepared from a cephalosporin compound having a -CT^X group (where X = OH or the residue of an acid H X, having a pKa of not more than 4.0, preferably not more than 3.5, as measured in water at 25°C) in the 3 position. X can thus e.g. be chlorine, bromine, iodine, formyloxy, an acetoxy group with at least one electron-withdrawing ("electron-withdrawing") substituent on the α-carbon atom, or

a nuclear substituted benzoyloxy group (the nuclear substituent being of the electron-withdrawing type as described in British Patent No. 1,241,657), and the nucleophilic displacement reaction, to form the desired 3-position acyloxymethyl can be carried out as described in previously mentioned British Patent No. 1,241,657.

Alternatively, if X is hydroxy, a 3-acyloxymethyl-cephalosporin can be obtained by acylation analogous to that described in British Patent No. 1,141,293, i.e. by aralkylation of the 4-carboxy group, acylation of the 3-hydroxymethyl group by the protected compound, and subsequent removal of the aralkyl group.

Compounds having a vinyl or substituted vinyl group

in the 3-position can be obtained by the method described in Belgian patent no. 761,897.

The A^-cephalosporin ester derivatives obtained in accordance with the method according to the invention can be converted into the corresponding A3 derivative by e.g. treatment of the sterene with a base.

The ceph-2-em reaction products can also be oxidized to give the corresponding ceph-3-em-1-oxide, e.g. by reaction with a peracid as previously mentioned. The resulting sulfoxide may, if desired, then be reduced, as described hereinafter, to give the corresponding ceph-3-em sulfide.

When a compound is obtained, in which B is ^ >S — >0, this can be converted into the corresponding sulphide by e.g. reduction of i in the corresponding acyloxysulfonium or alkyloxysulfonium salt^ produced in situ by reaction with e.g. acetyl chloride in the case of an acetoxysulfonium salt, whereby the reduction e.g. is carried out by a sodium dithionite or by iodide ion as in a solution of potassium iodide in a water-miscible solvent, e.g. acetic acid, tetrahydrofuran, dioxane, dimethylformamide or dimethylacetamide. The reaction can be carried out at a temperature of -20° to +50°C.

Where a compound of formula I is obtained as a mixture of isomers, the cis-isomer can be obtained by e.g. conventional methods, such as crystallization or chromatography. Cis- and trans-isomers can be distinguished by suitable methods, e.g. by ultraviolet spectra, by thin layer or paper chromatography or by their proton magnetic resonance spectra. Thus shows e.g. p.m.r. spectra of DMSO-dg solutions of cis compounds of formula I doublet for the amide NH in a lower field than corresponding solutions of the corresponding trans isomers do. These factors can be used in control reactions.

Starting materials of formula V, wherein Z' is the group

can e.g. produced by methods according to Belgian patent no. 774,480 and French patent no. 2,165,834. The starting materials of formula V, wherein Z' is a group of formula

where Hal means a halogen atom, such as fluorine, chlorine

or bromine,

can e.g. is produced as described in DOS no. 2,408,686.

in I

I Acids (VI) can be obtained by reacting a glyoxylic acid with the j formula

where R has the above meaning,

or an ester thereof with a hydroxylamine derivative of the formula

where Ra, R*3, R^°, m and n have the previously stated meanings.

The resulting acid or ester can be separated into its

cis- and trans-isomers by e.g. crystallisation, chromatography or distillation, after which the ester derivatives can be hydrolysed to give the corresponding acid.

Acids (VI) can also be produced by etherification of an acid

with the formula

where R has the above meaning,

e.g. by reaction with a compound with the general r~ t

r oi nie ±

in which Ra, R°, R^, m and n have the previously stated meaning and .

T represents a halogen, such as chlorine, bromine or iodine;

j sulfate or sulfonate, such as tosylate |

Separation of isomers can be carried out either before or after such etherification. The etherification reaction is preferably carried out in the presence of a base, e.g. potassium t-butoxide or sodium hydride, and is preferably carried through in an organic solvent, e.g. dimethylsulfoxide, a cyclic ether such as tetrahydrofuran or dioxane, or an N,N-disubstituted amide such as dimethylformamide. Under these conditions, the configuration of the oximino group is essentially unchanged by the etherification reaction.

Acids of formula VI and acylating agents derived therefrom (eg acyl halides, such as the chloride) are new and comprise a feature of the present invention.

Derivatives of compounds according to the invention, in which the carboxy substituent of the 7(3-acylamido side chain is substituted with a carboxyl-blocking group) are also new and comprise a feature according to the invention. These monoester derivatives, which can be represented by the general formula

* ab

where R, R , R , Z, m and n they have. formerly stated meanings and

R 20 is a carboxyl-blocking group, such as t-butyl or diphenylmethyl),

are valuable as intermediates in the preparation of antibiotic compounds of the general formula I. The compounds (XII) may themselves exhibit antibiotic activity, although it is generally at a very low level compared to j

In corresponding compounds (I).

Carboxyl-blocking groups R20 and optionally R1, used

in the preparation of compounds of formula I or in the preparation of necessary starting materials, groups which can be easily cleaved off in a suitable step in the reaction sequence are preferably groups, most expediently in the last step.

However, it may be advantageous in some cases to use biologically acceptable, metabolically labile carboxyl blocking groups, such as acyloxymethyl groups (e.g. pivaloyloxymethyl) and retain these in the final product to give a biologically acceptable ester derivative of a compound of formula I.

Suitable carboxyl blocking groups are well known in the art and a list of representative blocked carboxyl groups is found in Belgian Patent No. 783,449. Preferred blocked carboxyl groups include aryl lower alkoxycarbonyl groups such as p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl and diphenylmethoxycarbonyl; lower alkoxycarbonyl groups,

such as t-butoxycarbonyl; and lower haloalkoxycarbonyl groups, such as 2,2,2-trichloroethoxycarbonyl. The carboxyl-blocking group can then be removed by some suitable method described in the literature. Thus, e.g. acid- or base-catalyzed hydrolysis applicable in many cases as well as enzyme-catalyzed hydrolyses.

The antibiotic compounds according to the invention, e.g. compounds of formula I and non-toxic derivatives thereof,

can be formulated for administration in any suitable way, analogously to other antibiotics, and the invention therefore includes within the scope of invention, pharmaceutical compositions, consisting of an antibiotic compound according to the invention, adapted for use in medical science concerning humans and animals. Such compositions may be prepared for use in the usual manner by means of any necessary pharmaceutical carrier material or excipients.

The antibiotic compounds according to the invention can be formulated for injection and can be presented in unit dose form in ampoules,! or in multi-dose containers with an added preservative. Compositions can take the form of suspensions, solutions or emulsions in oily or aqueous solvents, and can contain formulation agents, such as suspension stabilizers and/or dispersants. Alternatively, the active substance can be in powder form for re-dilution with a suitable solvent, e.g. sterile, pyrogen-free water, for use.

The antibiotic compounds can also be prepared in a form suitable for absorption in the gastrointestinal tract. Tablets and capsules for oral administration may be in unit dose presentation forms and may contain conventional excipients, such as binders, e.g. syrup, acacia, gelatin, sorbitol, tragacanth or polyvinyl pyrrolidone; fillers, e.g. lactose, sugar, corn starch, calcium phosphate, sorbitol or glycine; lubricants, e.g. magnesium stearate, talc, polyethylene glycol or silicon; crumbling agents, e.g. potato starch; or acceptable wetting agents, such as sodium lauryl sulfate. The tablets can be coated according to well-known methods according to the state of the art. Oral liquid preparations can e.g. be in the form of e.g. aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or they may be presented as a dry product for reconstitution with water or other suitable solvents for use. Such a liquid preparation may contain conventional additives, such as suspending agents, e.g. sorbitol syrup, methyl cellulose, glucose/sugar syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminum stearate gel or hydrogenated . edible fats, emulsifiers, e.g. lecithin, sorbitan mono-oleate or acacia; non-aqueous solvents (which may include edible oils), e.g. almond oil, fractionated coconut oil, oily esters, propylene glycol or ethyl alcohol; and preservatives, e.g. methyl or propyl p-hydroxybenzoate or 2,4-hexadienoic acid. The antibiotic compounds can also be formulated as suppositories, e.g. containing conventional suppository bases, such as cocoa butter or other glycerides.

Compositions for veterinary medical use can e.g. are formulated as intramammary preparations on either a long-acting or rapid-release basis.

The compositions can contain from 0.1% and upwards, e.g. 0.1 - 99%, preferably from 10 - 60% of the active material, depending on the method of administration. When the compositions consist of dose units, each unit will preferably contain 50 - 1500 mg of the active substance. The dose used for adults will preferably be in the range from 500 to 5000 mg per day, depending on the method and speed of administration, although higher daily doses may be required in the treatment of Pseudomonas.

The antibiotic compounds according to the invention can be administered in combination with other therapeutic agents, such as antibiotics, e.g. penicillins, tetracyclines or other cephalosporins.

The following examples illustrate the invention. All temperatures are indicated in °C. The structure of the products was confirmed by p.m.r. spectroscopy (presentations and examples) and i.r. spectroscopy (examples only).

in

j Preparation 1 2-t-butoxycarbonylmethoxyimino-2-(fur-2-yl)acetic acid (cis-isomer)

The pH of the mixture of fur-2-ylglyoxylic acid (4.2 g), t-butoxy-carbonyl methoxyamine (4.5 g) and water (50 mg) was adjusted to 5.0 with 2 N sodium hydroxide solution. The resulting solution was stirred for 16 hours. The pH of the solution was adjusted to 7.0 and the solution was washed twice with ether.

The aqueous solution was acidified to pH 1.8 under ether,

and further extracted with ether. The combined ether extracts were washed (water, brine), dried and concentrated to give a solid (7.62 g), which was crystallized from carbon tetrachloride to give the title compound (3.67 g, 46 %), m.p. 105.1 - 106.2°; ^-max (pH6 phosphate

buffer) 277.5 nm ( £- 16,300).

Manufacturing 2

2- t -butoxycarbonylmethoxyimino-2-(thien-3-yl)acetic acid

(cis isomer)

Thien-3-ylglyoxylic acid and t-butoxycarbonylmethoxyamine were reacted as described in Preparation 1 to give the title compound, m.p. 102.6 - 104.4° (from carbon tetrachloride);

A- max (pH6 phosphate buffer) 258 nm ( £- 13,700).

EXAMPLE 3

2- RS- g- t- butoxycarbonylbenzyloxyimino- 2-( fur- 2- yl)- acetic acid (cis- isomer)

a) (i) A mixture of N-hydroxyphthalimide (2.45 g), anhydrous potassium carbonate (16.5 g), t-butyl-oc-bromophenyl acetate (41 g)

and dimethyl sulfoxide (225 mg) was stirred for 18 hours and then poured into water (1.2 liters). The precipitated solid was filtered off, washed well with water, dried and crystallized from industrial methylated spirit to give N-[α-(t-butoxycarbonyl)benzyloxy]phthalimide (41 g, 78%), m.p. 120.6 - 121.5°.

(ii) To a solution of the above oxyphthalimide (40 g)

in dichloromethane (500 ml) was added 100% hydrazine hydrate I (11.4 ml). A precipitate was immediately formed. The mixture was

stirred for 1.5 hours, after which sufficient 5 N ammonium hydroxide solution was added to dissolve the precipitate. The two layers were separated and the aqueous layer was extracted once with methylene chloride. The combined organic extracts were washed (water, brine), dried and concentrated to give t-butyl-oc-(aminooxy)phenyl acetate (25.0 g, 98%) as colorless crystals, m.p. 48.2 - 49.6°. b) Fur-2-ylglyoxylic acid and t-butyl-α-(aminooxy)phenylacetate were reacted as described in Preparation 1 to give the title compound in 42% yield, m.p. 97.9 - 98.9° (from carbon tetrachloride) 5<>>^-max (pH6 phosphate buffer) 278 nm (E- 18,400).

Manufacturing 4

2- t-butoxycarbonylmethoxyimino-2-(thien-2-yl)acetic acid (cis-isomer)

To a stirred suspension of sodium hydride (60% dispersion in oil, 0.96 g) in tetrahydrofuran (40 mL) was added 2-hydroxyimino-2-(thien-2-yl)acetic acid (cis isomer) (1.71 g). The mixture was stirred for 30 minutes, after which dimethyl sulfoxide (25 mL) was added and stirring was continued for a further hour. t-Butyl chloroacetate (1.78 g) was added to the mixture, which was stirred for 16 hours and then poured into water (300 ml). After being washed twice with ether, the aqueous phase was acidified to pH 1.7. Extraction with ether and concentration of the washed (water, brine) and dried extracts gave a solid (2.71 g), which was crystallized from carbon tetrachloride to give the title compound (0.952 g, 33%), m.p. . 88.3 - 91.3°;

X max (PH 6 phosphate buffer) 2 70.5 and 288.5 nm ( £L 9.200 and 10.800).

Manufacturing 5

2-( 2- t- butoxycarbonylpropyl- 2- yloxyimino)- 2-( fur- 2- yl) acetic acid (cis- isomer)

A solution of 2-(fur-2-yl)-2-hydroxyiminoacetic acid (cis isomer) (14.1 g) in dimethyl sulfoxide (100 mL) was added all at once to a magnetically stirred solution of potassium t-ibutoxide (22.4 g) in dimethyl sulfoxide (400 mL), whereby the reaction mixture was maintained under an atmosphere of dry nitrogen. A gel was formed which, on stirring, became a finely divided yellow solid. Stirring was continued for one hour and then a solution of t-butyl-2-bromo-2-methylpropionate (24.0 g)

in dimethyl sulfoxide (50 mL) added over one hour to the reaction mixture at room temperature. After the addition was complete, the resulting solution was stirred for an additional hour. The reaction was poured into ice water (1.5 liters) and acidified under ether (500 ml) to pH 1.8 with concentrated hydrochloric acid. The two layers were separated and the aqueous layer was extracted with more ether. The combined ether extracts were washed once with water, then extracted with aqueous sodium bicarbonate solution. The combined alkaline extracts were acidified under ether to pH 1.8 with concentrated hydrochloric acid,

and the acid solution was further extracted with ether.

The combined ether extracts were washed (water, saturated brine), dried and concentrated to a yellow oil which crystallized under high vacuum (22.41g, 83%), X max (<E>tOH) 272.5nm 15.400) .

The above solid (22.4 g) was crystallized from carbon tetrachloride (25 mL) to give the title compound (16.42 g, 61%), m.p. 72.5 - 74.2° (73.0°).

Presentations 6-20

Method A

The dipotassium salt of 2-(fur-2-yl)-2-hydroxyimino-acetic acid

(cis-isomer) was developed under an atmosphere of dry nitrogen and alkylated with the appropriate halo-t-butyl ester as described in Preparation 5. The products were isolated by pouring into water, acidifying and extracting in the conventional manner.

Method B

As method A, but with the use of halogen diphenyl methyl ester. The half-esters prepared according to these methods are listed in

table 1.

Production 21

2-(2-t-butoxycarbonylpropyl-2-yloxyimino)-2-(thien-2-vl)-acetic acid (cis-isomer)

The title compound was prepared from 2-hydroxyimino-2-(thien-2-yl)acetic acid (cis-isomer) and t-butyl-2-bromo-2-methyl-propionate, in the same manner as described for preparation 5,

in 78% yield, as a colorless oil, and was characterized as the N-benzyl-2-phenylethylammonium salt, m.p. 201.3 - 201.9°

(from ethanol).

Production 22

2-(2-t-butoxycarbonyloxyimino)-2-(fur-2-yl)acetic acid

(cis isomer)

To a mixture of methyl acetohydroxy acid [CH3.C (:N0H).0CH3]

(8.9 g) and t-butyl acrylate (12.8 g) was added a solution of potassium t-butoxide (0.1 g) in t-butanol (1 ml). The mixture was kept at 0° for 65 hours, then was washed with water, dried and distilled to give 2-t-butoxycarbonyl-ethyl-methyl-acetohydroxymate (2.37 g, 11%), b.p. 85-87°/l.2 mmHg.

To a solution of fur-2-ylglyoxylic acid (1.26 g) in water

(50 mL) was added 2-t-butoxycarbonylethylmethyl-aceto-hydroxymate (2.15 g) and sufficient methanol to give a homogeneous mixture, which was stirred for 30 minutes at pH

1.5.

IN

The pH was adjusted to 4.5 with 2N sodium hydroxide solution and the mixture was stirred for an additional 16 hours, after which the reaction was almost complete. Methanol was removed under reduced pressure, the pH of the residue was adjusted to 7.0, and the aqueous mixture was washed twice with ether. The aqueous phase was acidified in the presence of dichloromethane to pH 1.7, and the phases

were separated. The aqueous phase was extracted twice more with dichloromethane. The combined dichloromethane extracts were washed with water, dried and concentrated to give a tan solid (1.53 g) (mixture of cis and trans isomers, 85:15), which was crystallized from carbon tetrachloride to give afford the title compound (0.975 g, 34%), m.p. 74.7 - 77.2°;

max ^pH6 buffer) 277 nm (£ 16,500).

Production 23

t-butyl-l-bromocyclopentanecarboxylate

To a mixture of 1-bromocyclopentanecarboxylic acid (36.99g) and anhydrous ether (35ml) in a 500ml pressure flask containing a magnetic stir bar was added concentrated sulfuric acid (3.5ml), followed by precondensed isobutene (150 ml). The bottle was sealed and stirred at ambient temperature for 20 hours. The flask was then opened, excess isobutene was evaporated, and the residue in ether was washed with aqueous sodium bicarbonate solution and water, dried and concentrated. The residue was distilled under reduced pressure to give the title ester

(bp. 66-74°/0.5-2.Omm) (33.6 g, 70%)5^ max (CHBr 3 ) 1702 cm<-1>;

T (CDCl 3 ) 7.78, 8.20 (cyclopentane protons) and 8.54 (C(CH 3 ) 3 ).

Production 24

Diphenylmethyl-g-bromohexanoate

α-bromohexanoic acid (1.95 g) in a petroleum fraction with bp.

40 - 60°C (25 ml) was treated with a stock solution of

diphenyldiazomethane in a petroleum fraction with bp. 40 - 60°C

(ca. 3.8 mmol/10 ml) dropwise with stirring until a faint violet color persisted. The mixture was stirred for 2 hours at room temperature, whereupon the solvent was removed in vacuo. The resulting oil in ethyl acetate was washed with a saturated aqueous solution of sodium bicarbonate, then with water and<I>dried. Removal of the solvent gave the title ester (3.0 g,

90%), /v-max (EtOH) 252, 258, 263.5, 267.5 and 276 nm 1650, 1600, 1350, 1150 and 850).

The productions 25 - 34

oc- halogen- substituted carboxylic acid e- ester e

Method A

The appropriate o-halocarboxylic acid was treated with isobutene and concentrated sulfuric acid in a pressure flask at room temperature for 10-40 hours by the procedure described in Preparation 23 to give t-butyl esters listed in Table 2.

Method B

The appropriate o-halocarboxylic acid in a solvent (eg, ether, petroleum, ethyl acetate) was treated with a solution of diphenyldiazomethane until a faint permanent color was obtained. The ester was washed with alkali in the same manner as described in Preparation 24 to give diphenylmethyl esters listed in Table 2.

Production 35

Di-t-butyl-2-bromo-2-methylmalonate

To a stirred suspension of sodium hydride (1.7 g, 80% dispersion in oil) in tetrahydrofuran (60 mL) under an atmosphere of nitrogen was added di-t-butyl methyl malonate (11.52

g). The mixture was stirred at 60-70° for 1.5 hours to

give a clear solution. This solution was cooled to -25° and to it was quickly added a solution of bromine (2.6 ml) in dichloromethane (30 ml). The solution was warmed to ambient temperature and then concentrated. The residue in ether was washed with water, dried and fractionally distilled under reduced pressure to give the title compound, b.p. 78 - 86°/ 1.0 mmHg, (7.56 g, 49%); v max (CHBr 3) 1730 cm"<1>(CO^uS ;

T (CDC13) values include 8.05 (s, C(CH3)3 and 8.53 (2s,

CH3 and C (CH3) 3) .

Production 36

t- butyl- ethyl- 2- bromo- 2- methylmalonate

The title compound was prepared in the same way as the dibutyl ester in preparation 35, in 83% yield, b.p. 64 - 68°/0.03 mmHg.

Production 37

1- bromomethylcyclopropane- 1- carboxylic acid

The bromination of cyclobutane-carboxylic acid leads to the corresponding bromic acid together with the title acid (approx. 15% yield),

j m.p. 83 - 84° (light petrol, bp. 60 - 80°); T (d6-DMS0) values 6.25, 8.65 and 8.90.

In EXAMPLE 1

a) ( 6R, 7R )- 3- acetoxymethyl- 7-[ 2- t- butoxycarbonylmethoxyimino- 2-( fur- 2- yl) acetamido] ceph- 3- em- 4- carboxylic acid

(cis isomer)

Oxalyl chloride (0.45 mL) was added at 5° to a stirred solution of 2-t-butoxycarbonylmethoxyimino-2-(fur-2-yl)acetic acid (cis isomer) (1.35 g) in dry dichloromethane ( 50 ml), containing triethylamine (0.7 ml) and dimethylformamide (1 drop). The solution was stirred at 5° for one hour and then evaporated to dryness at 5°. The residue was suspended in acetone (50 ml) and added over 30 minutes to a stirred, ice-cooled solution of (6R,7R)-3-acetoxymethyl-7-aminoceph-3-em-4-carboxylic acid (1.35 g) in water (100 mL) and acetone (50 mL), containing sodium bicarbonate (1.0 g).The reaction mixture was stirred for one hour, after which acetone

was evaporated under reduced pressure. The residue was acidified to pH 1.8 and the mixture was extracted with ether. The combined extracts were washed (water, brine), dried, and evaporated to give the title compound (2.52 g, 96%) as a yellow foam, [α]D+ 28.5° (c 0.96, DMSO ); > max (pH6 phosphate buffer) 276.5nm (&- 17.900).

b) ( 6R, 7R )- 3- acetoxymethyl- 7-[ 2- carboxymethoxyimino- 2-( fur- 2- yl)- acetamido] ceph- 3- em- 4- carboxylic acid„ disodium salt

( cis- isomer) A solution of (6R,7R)-3-acetoxymethyl-7-[2-t-butoxy-carbonylmethoxyimino-2-(fur-2-yl)acetamido]ceph-3-em-4-carboxylic acid ( cis isomer) (1.422 g) and anisole (0.25 mL) in trifluoroacetic acid (5 mL) was kept at ambient temperature for 5 minutes. The mixture was concentrated under reduced pressure,

ethyl acetate (10 mL) was added and the mixture was re-evaporated. The residue was partitioned between ether and sodium bicarbonate solution. The ether layer was further extracted with sodium bicarbonate solution and the combined alkaline extracts were acidified to pH 1.8 under ether. The acid mixture was extracted with ether and the combined ether extracts were washed

(water, saturated brine), dried and evaporated to give the dicarboxylic acid corresponding to the title compound (942 mg,

] 74%),?"' (dg- DMSO) values include 0.24 (d, J 8Hz, NH) , 4.13

(dd, 7-H) , and 5.31 (s, CH 2 CO 2 H) .

This diacid (900 mg) in acetone (9 ml) was neutralized with

a solution of sodium 2-ethyl hexanoate (700 mg) in acetone (5 ml). The mixture was stirred for 10 min, then the precipitated solid was filtered off, washed with a little acetone and dried to give the title compound (807 mg, 60%), [oc]

+ 15° (c 1.08, DMS<O>); V max (Nujol)1766 cm<-1>((3-lactam) .

EXAMPLES 2-26

General procedure for the preparation of (6R, 7R)-7-(2-aryl-2-carboxy-R^-oximinoacetamido)-3-(substituted) ceph-3-em-4-carboxylic acids (cis-isomers) and/or their salts

Method A

By following the procedure described in example 1

was a solution of a suitable 2-aryl-2-t-butoxycarbonyl-R^-oximinoacetic acid (cis-isomer) (1 eq.) in methylene chloride, optionally containing a few drops of N,N-dimethylformamide and triethylamine (1 eq.) treated with oxalyl chloride (1 eq.) by 0 - 5° for approx. 1 hour. The mixture was then evaporated to dryness. The residue was suspended or dissolved in acetone and added to a stirred, ice-cold solution of (6R,7R)-3-acetoxymethyl-7-aminoceph-3-em-4-carboxylic acid (1-1.2 eq.) in water or a mixture of acetone and water containing sodium bicarbonate (2-2.5 eq.). The reaction mixture was stirred for 0.5-2.5 hours, and the temperature was allowed to rise to room temperature, after which the acetone was removed under reduced pressure. The pH was adjusted to 1.5 - 2.0 and the product extracted into ethyl acetate (alternatively, ether or methylene chloride can be used). The organic layer was washed with water and/or saturated brine, dried and evaporated to give the corresponding (6R,7R)-3-acetoxymethyl-7-(2-aryl-2-t-butoxycarbonyl-R^-oxyiminoacetamido)ceph -3-em-4-carboxylic acid

(cis-isomer), which was characterized by optical rotation and/or by spectroscopy.

The t-butyl esters were deprotected by treatment with trifluoro-

in acetic acid, containing anisole at room temperature for at least 5 min.

I The reaction mixture was evaporated in vacuo and the product 1 was isolated by trituration or by partitioning between ethyl acetate (or ether) and an aqueous solution of sodium hydrogen carbonate, separation of the aqueous extracts, acidification of these extracts under ethyl acetate and isolation of the title the dicarboxylic acid in the usual way. The products are listed in table 3.

Method B

As method A, except that the 2-aryl-2-diphenylmethoxycarbonyl-R 2 -oxyiminoacetic acid (cis-isomer) used was used instead of the t-butyl ester. The products are listed in table 3.

Method C

As method A or B, except that the dicarboxylic acid

was converted to its disodium salt by treating a solution of the acid in acetone with a solution of sodium 2-ethylhexanoate in acetone. The precipitated disodium salt was washed and dried. The products are listed in table 3.

Method D

As method A, except that (6R,7R)-3(substituted methyl)-7-aminoceph-3-em-4-carboxylic acid or its salt was used instead of (6R,7R)-3-acetoxymethyl-7- aminoceph-3-em-4-carboxylic acid. The products are listed in table 4.

EXAMPLE 2 7

( 6R, 7R )- 7-[ 2- carboxymethoxyimino- 2-( fur- 2- yl)- acetamido]-3-( 1- methyltetrazol- 5- ylthiomethyl) ceph- 3- em- 4- carboxylic acid ( cis- isomer )

A solution of 2-t-butoxycarbonylmethoxyimino-2-(fur-2-yl)-acetic acid (cis isomer) (0.97 g) in methylene chloride (20 mL) was added dropwise at room temperature over 15 minutes to a stirred solution of diphenylmethyl (6R,7R)-7-amino-3-(1-methyltetrazol-5-ylthiomethyl)ceph-3-em-4-carboxylate (1.484 g)

and dicyclohexylcarbodiimide (0.743 g) in methylene chloride (45 mL). After stirring for a further 2 hours, the solvent was removed by evaporation and the residue was stirred for 5 minutes with ethyl acetate (50 ml) and filtered. The filtrate was washed with saturated sodium bicarbonate solution, diluted with the same volume of water and then with brine (25 ml of each), dried and evaporated to a foam (2.5 g), which was dissolved in benzene and purified by chromatography on silica gel ( 70g). Elution with benzenemethyl acetate (10:1), combining the fractions used and evaporating to dryness gave a foam

(2.05 g), which was dissolved in ethyl acetate and poured into a petroleum fraction of boiling point 40 - 60°C to give diphenylmethyl (6R,7R)-7-[2-t-butoxycarbonylmethoxyimino-2-(fur-2- yl)-acetamido]-3-(1-methyltetrazol-5-ylthiomethyl)ceph-3-em-4-carboxylate (cis isomer) (2.02 g, 90%) as a white amorphous solid, [a] 23 - 102° (c 0.99, CHCl 3 ); >^max (<E>tOH)278 nm '(.<£>19,800) .

A solution of its diester (1.93 g) in a mixture of trifluoroacetic acid (7.7 ral) and anisole (1.9 ml) was kept at

0° for 10 minutes and then added to a mixture of saturated sodium bicarbonate and water (1:3, 850 ml). After stirring for 10 minutes, the mixture was washed with ethyl acetate, covered

with more ethyl acetate (200 ml) and acidified to pH 2 with concentrated hydrochloric acid. The organic phase was separated, washed with water and brine, dried and evaporated to a foam (1.54 g). Thick layer chromatography indicated that deprotection was incomplete and the product was treated again with trifluoroacetic acid (4.3 ml) and anisole (1.1 ml) at 20° for 15 minutes, after which the product was isolated as a foam (1.3 g) which previously described. This foam in ethyl acetate was poured into a petroleum fraction bp 40 - 60° to give the title dicarboxylic acid (0.8 g, 59%) as a white amorphous solid, [cc]^<3>- 99° (c 1 .05, acetone) 5 >-max (0.1M-pH 6 phosphate buffer) 277 nm (e 21,900); Vmax (Nujol) 1780 cm-1; ^(d6~DMS0) values include 0.19

(d, NH) , 4.14 (dd, 7-H), 5.30 (s, CH 2 CO 2 H) .

EXAMPLE 28

( 6R, 7R)- 3- carbamoyloxymethyl- 7-[ 2- carboxymethoxyimino- 2-( fur- 2- yl) acetamido] ceph- 3- em- 4- carboxylic acid ( cis- isomer) Diphenylmethyl (6R, 7R)-7 -amino-3-carbamoyloxymethyl-ceph-3-em-4-carboxylate-toluene-p-sulfonic acid salt (2.08 g) was dissolved in a mixture of ethyl acetate (60 ml) and saturated aqueous sodium bicarbonate (60 ml) . The ethyl acetate layer was separated, washed with water, dried and evaporated to a foam.

2-t-Butoxycarbonylmethoxyimino-2-(fur-2-yl)acetic acid (cis isomer) (1.0 g) and dicyclohexylcarbodiimide (0.76 g), dissolved in a small volume of anhydrous dichloromethane, were added to a solution of the above amine, dissolved in anhydrous dichloromethane (20 mL). The reaction mixture was stirred at 3° for 35 minutes, during which tin N,N'-dicyclohexylurea crystallized out. This was filtered off and the filtrate was evaporated to an oil which solidified by trituration with diisopropyl ether. The solid product was dissolved in ethanol and decolorized with charcoal. Evaporation of the solution gave diphenylmethyl (6R,7R)-3-carbamoyloxymethyl-7-[2-t-butoxycarbonyl-

|methoxyimino-2-(fur-2-yl)acetamido]ceph-3-em-4-carboxylate (cis-isomer) (2.08 g, 86%); m.p. 97 - 98°-, [a]D+ 5.5°

(c 1.00, DMSO); ^-max (<E>tOH)276.5 nm ( £- 17.250).

Trifluoroacetic acid (6 mL) was added to a slurry of

the above diester (2.08 g) in anisole (6 ml) and the reaction mixture was stirred at 20° for 25 minutes. The solution was poured into a mixture of ethyl acetate and aqueous sodium bicarbonate solution, the aqueous layer was separated and washed with ethyl acetate. Ethyl acetate (100 mL) and 2-N-hydrochloric acid were added to reduce the pH to 2. The organic layer was then separated, washed with water, dried and evaporated to a yellow oil. p.m.r. indicated that this product was (6R,7R)-3-carbamoyloxymethyl-7-[2-t-butoxycarbonylmethoxyimino-2-(fur-2-yl)-acetamido]ceph-3-em-4-carboxylic acid (cis isomer) .

The acid was treated with anisole (4 mL) and trifluoroacetic acid

(4 ml) at 20° for 30 minutes and the reaction mixture was partitioned between ethyl acetate and aqueous sodium bicarbonate solution. The aqueous layer was washed with ethyl acetate and acidified

to pH 2 with 2N hydrochloric acid under ethyl acetate. Evaporation of the ethyl acetate layer gave a solid which was purified by precipitation from ethyl acetate with diisopropyl ether and filtered to give the title dicarboxylic acid (620 mg, 44%); m.p. 159 -

161°; Ta]<21>+ 35° (c 1.0, DMSO): V 1 (pH 6 phosphate buffer)

' L JD ' ' ' max ^ _,

275 nm (€. 15,400); \i max (NuJol) 1778 cm ? T(d 6 -DMSO) values include 0.20 (d,NH) 4.15 (dd, 7-K) 5.32 (s, C(CH 3 ) 2 ).

EXAMPLES 29-42

General procedure for the preparation of (6R,7R)-7-[2-carboxy-R^-oxyimino-2-(fur-2-yl)acetamido]-3-(substituted)-ceph-3-em-4-carboxylic acids (cis-isomers) using dichlorohexylcarbodiimide

(i) To a solution of diphenylmethyl (6R,7R)-7-amino-3-(substituted)'ceph-3-em-4-carboxylate (1 eq.) and dicyclohexylcarbodiimide (1-1.3 eq.) in dry methylene chloride, a solution of the appropriate 2-t-butoxycarbonyl-R,-oxyimino-2-(fur-2-yl)acetic acid (cis-isomer) (1,1,15 equiv.) was added at 0 - 25°. ) i j i Itorr methylene chloride. After stirring for 0.5-5.0 hours, dicyclohexylurea was removed by filtration and the filtrate was evaporated. The residue in ethyl acetate or methylene chloride was washed sequentially with aqueous sodium bicarbonate, water and brine, dried and evaporated. The diester was purified by chromatography on silica or, after decolorization with charcoal,

by trituration or crystallization. The product was characterized by its p.m.r. spectrum and by thin-layer chromatography.

When the 7-amino starting material is available as an acid addition salt, the free base is liberated by shaking with a mixture of ethyl acetate (or methylene chloride) and an excess of an aqueous solution of sodium bicarbonate. After washing with water and brine, the organic layer was evaporated to dryness and the free amine used as described above.

(ii) Method A.

The intermediate diesters thus derived were deprotected by dissolving in a mixture of trifluoroacetic acid

(3 - 10 ml/l g of diester) and anisole (0.8 - 12 ml/l g of diester) and allowed to stand between 0° and room temperature for between 5 minutes and 2.5 hours. The mixture was concentrated under reduced pressure and added to a mixture of ethyl acetate or ether and excess aqueous sodium bicarbonate, and the aqueous layer was washed with ethyl acetate. The aqueous phase was converted with ethyl acetate and acidified to pH 1-2 with hydrochloric acid. The organic layer was washed, dried and evaporated to give the required dicarboxylic acid.

(ii) Method B.

In some cases where treatment with trifluoroacetic acid was insufficient to complete deprotection, the intermediate monoester (usually the t-butoxycarbonyl group cleaves more slowly than the diphenylmethoxycarbonyl group) was treated with trifluoroacetic acid and anisole and the diacid was isolated as described above.

(iii) Method C.

The 3-carboxyvinyl derivative as described in example 36 was prepared!

t

I stilted from a 7-amino derivative, in which both carboxy groups were protected as the diphenylmethyl ester. The resulting triester was deprotected as in Method B to provide the required tricarboxylic acid.

The properties of the reaction products are listed in table 5.

I in 1 in

EXAMPLE 43

( 6R, 7R )- 7- f 2-( 1- carboxycyclopent- 1- cycloximino)- 2-( fur- 2-yl) acetamidocep- 3- em- 4- carboxylic acid (cis- isomer)

a) A stirred solution of diphenylmethyl (6R,7R)-7-aminoceph-3-em-4-carboxylate (5.50 g) and propylene oxide (5.23 g)

in dry methylene chloride (100 ml) at -5° was treated dropwise,

over the course of 20 minutes, with a solution of 2-(1-t-butoxy-carbonylcyclopent-1-yloxyimino)-2-(fur-2-yl)-acetyl chloride (cis-isomer) in methylene chloride (see below). The resulting solution was stirred at 0° for 40 minutes and was warmed to 22° with stirring for an additional hour. Methanol (15

ml) was added and stirring was continued for 15 minutes. The solution was washed with 2N hydrochloric acid, saturated aqueous sodium bicarbonate and saturated brine. After drying and clarifying with charcoal, evaporation gave a foam, which was dissolved in ether and added dropwise to a petroleum fraction of boiling point 40 - 60°C to give diphenylmethyl (6R,7R)-7-[2-(1 -t-butoxycarbonylcyclopent-1-yloxyimino)-2-(fur-2-yl)-acetamido]ceph-3-em-4-carboxylate (cis isomer) as a colorless powder (8.3 g, 83% ); [α]D + 43° (c 0.1, CHCl 3 ); ^max (EtOH) 277 nm ( £. 17,300).

(b) The diester prepared according to (a) (6 g) in anisole (30 ml)

at 0° was treated with trifluoroacetic acid (24 mL) and the solution was stirred for 30 min. The reaction mixture was now allowed to reach 22° under stirring for a further 2 hours, and was then concentrated in vacuo to approx. 20 ml. The resulting oil was partitioned between ethyl acetate and saturated aqueous sodium bicarbonate

Bonnet. The organic layer was extracted with bicarbonate solution and the combined alkaline layers were washed with ethyl acetate and ether. The aqueous layer was covered with ethyl acetate and adjusted to pH 1.5 with concentrated hydrochloric acid.

The resulting organic layer was washed with saturated brine, dried and evaporated to a foam containing the title acid.

The foam was dissolved in a buffer containing 0.1 M sodium acetate, 0.1 M acetic acid and 0.2 M sodium chloride (75 ml) using sodium bicarbonate. The solution was treated with Amberlite XAD-8 (50 mL) and adjusted to pH 4.8 with 2N hydrochloric acid. The resulting stirred suspension was treated with Woelm neutral alumina (20 g) while maintaining the pH at 4.8. The suspension was stirred for 20 minutes and poured onto a column of Woelm neutral alumina (25 g). The title acid was eluted with the aforementioned buffer (300 ml) and with 2% aqueous sodium acetate (500 ml). The eluates were freeze-dried and redissolved in a minimum volume of water. After adjusting to pH 1.5 with concentrated hydrochloric acid, the acidic liquids were extracted with ethyl acetate and the combined extracts were dried and evaporated to a foam. Trituration with ethyl acetate, petroleum and ether afforded the title acid as a colorless solid (3.5 g, 88%); [cc]D+ 162° (c 0.01,

5% aqueous NaHCO 3 );<X>max (pH 6 phosphate buffer) 282 nm ( 6- 14,000);

V max (NuJol) 1775 (P-lactam), 1710 (C02H), and 1680 and

1528 cm<-1>(CONH); t"(DMSO-d6) values include 0.37 (d, J

8Hz, CONH), 2.10, 3.24 and 3.31 (m, furyl protons), 4.05 (dd,

J5 and 8 Hz, 7-H), 4.80 (d, J 5 Hz, 6-H), and 7.90 and 8.25 (2 x wide m, cyclopentyl protons). p.m.r. spectrum also showed the presence of 1/2 mole each of ethyl acetate and water.

The acylating agent used in step (a) above was

done in the following way:

A stirred suspension of phosphorus pentachloride (3.75 g) in dry methylene chloride (120 ml) and dimethylacetamide (6.53 g) at -10°

was treated with 2-(1-t-butoxycarbonylcyclopent-1-yloxyimino)-i

2-(fur-2-yl)-acetic acid (cis-isomer) (5.82 g) in small portions

to keep the temperature below -5°. After stirring for 15 minutes, the solution was treated with ice (40 g) and stirred for 20 minutes to give the corresponding acid chloride in the organic layer.

EXAMPLES 44-48

General procedure for the preparation of (6R, 7R)-3-(optionally substituted)-7-|"2-(carboxy-R^-oxyimino)-2-(fur-2-yl)-acetamido]-ceph-3 - em- 4- carboxylic acids ( cis- isomers) by treating an ester of a ( 6R, 7R)- 3-( optionally substituted) 7- aminoceph- 3- em- 4- carboxylic acid with an acid chloride ( cis- isomer) A 2-(t-butoxycarbonyl-R 3 -oxyimino)-2-(fur-2-yl)acetic acid

(cis-isomer) was converted to its acid chloride as described in Examples 2-24. A solution of the acid chloride (1-1.3 eq.) in methylene chloride was added dropwise at -5° to +5°

over a period of 10 - 30 minutes to a solution of diphenylmethyl (6R,7R)-3-(optionally sorb substituted)-7-aminoceph-3-em-4-carboxylate (1 eq.) in dry methylene chloride, containing propylene oxide (5 - 20 eq.). The reaction mixture was stirred for approx. 1 - 3 hours at 0° to room temperature and was then washed sequentially with 2N hydrochloric acid, aqueous sodium bicarbonate, water and/or saline. The dried organic layer was evaporated and the residue was purified by trituration, precipitation, chromatography or crystallization.

The resulting diphenylmethyl (6R,7R)-3-(optionally substituted)-7-[2-(t-butoxycarbonyl-R^-oxyimino)-2-(fur-2-yl)acetamido]-ceph-3-em- 4-carboxylate (cis-isomer) was deprotected as described for diesters described in Examples 29-42, Methods A and B. The products are listed in Table 6.

[ EXAMPLE 49

(a) (6R,7R)- 3- benzoxylmethyl- 7-[ 2-( 2- t- butoxy- carbonylprop-2- yloxyimino)- 2-( fur- 2- yl) acetamido] cep- 3- em- 4 - carboxylic acid

(cis isomer)

A suspension of phosphorus pentachloride (313 mg) in dry dichloromethane (4 mL) at -10° was treated with N,N-dimethylacetamide (0.7 mL) followed by 2-(2-t-butoxy-carbonylprop-2-yloxyimino )-2-(fur-2-yl)-acetic acid (cis isomer) (446 mg) portionwise. The resulting solution was stirred at -10° for 30 minutes, treated with ice (ca. 1 g) and stirred for 15 minutes at a temperature below 0°. The organic phase was added dropwise to a solution of (6R,7R)-7-amino-3-benzoyloxymethylceph-3-em-4-carboxylic acid (502 mg) in N,N-dimethylacetamide (2 ml) and acetonitrile (2 ml ), containing triethylamine (1.09 mg) at below 0°. The solution was stirred at 0° to 5° for 2 hours, methanol (0.3 mL) was added and stirring was continued for 15 minutes. The solution was diluted with dichloromethane (20 mL) and washed sequentially with 2N hydrochloric acid, water and brine, dried and evaporated to a gum (1.02 g). A solution of this gum in ethyl acetate (6 mL) was added dropwise to stirred petroleum ether (b.p. 40 - 60°, 200 mL) to give the title acid as a cream-colored powder (769 mg, 83%); [ct]D+ 26° (c 1.05, acetone); ^-max (pH 6 phosphate buffer) 233.5 nm ( £ 19,200) and 274 nm ( £ 18,600); V ^ (Nujol) 1790cm"<1>((3-lactam) ; 7." (d6-DMS0) values include 0.29 (d, J 8 Hz, NH) , 4.03 (dd, J 8 and J b Hz, 7-H) , 8.50 (s, C(CH3)2 and 8.58 (s, C(CH3)3.

(b) ( 6R, 7R )- 3- benzoyloxymethyl- 7[ 2-( 2- carboxyprop- 2- yloxyimino)- 2-( fur- 2- yl) acetamido] ceph- 3- em- 4- carboxylic acid ( cis- isomer)

A solution of (6R,7R)-3-benzoyloxymethyl-7-[2-(2-t-butoxycarbonylprop-2-yloxyimino)-2-(fur-2-yl)acetamido]-ceph-3-em-4- carboxylic acid (cis isomer) (620 mg) in anisole (0.6 mL) and trifluoroacetic acid (3 mL) was stirred at 20° for 25 minutes. The solution was partitioned between ethyl acetate and aqueous sodium bicarbonate solution and the pH was adjusted to pH 8 by adding sodium bicarbonate to the solid feed. The aqueous phase was separated, washed with ethyl acetate, covered with ethyl acetate, and the pH was adjusted to pH 1.5 by addition of concentrated hydrochloric acid.

'The organic phase was separated, washed sequentially with water and brine, dried and evaporated to an oily foam

(584 mg). A solution of this foam in ethyl acetate was poured dropwise into stirred petroleum ether (b.p. 40-60°) to give the title dicarboxylic acid as an off-white solid (384 mg, 67%); [α] + 30.4° (c 1.0, acetone); ^-max <PH 6 phosphate buffer) 234 nm (£. 19,700) and 273 nm ( €. 18,450); "Vmax(Nujol) 1784 cm"<1>(p-lactam); T (d6-DMS0) values include 0.30 (d, J8 Hz, NH), 4.07 (dd, J8 and J5HZ, 7-H), and 8.50 (s, C(CH3)2).

EXAMPLE 50

Potassium-(6R,7R)-7-f" 2-carboxymethoxyimino-2-(fur-2-yl)acetamido]-3-pyridinium methyl- ceph-3-em-4-carboxylate (cis- isomer)

A mixture of (6R,7R)-3-acetoxymethyl-7-[2-carboxymethoxyimino-2-(fur-2-yl)acetamido]ceph-3-em-4-carboxylate (cis isomer) (3.26 g ), potassium thiocyanate (5.25 g), pyridine (0.72 ml)

and water (2 mL) was stirred and heated for 40 min at 80°. The cooled reaction mixture was diluted with water (5 mL)

and adsorbed on a column of XAD-2 resin (500 g). The components of the reaction mixture were eluted, first with water and then with aqueous ethanol (1:3) and collected using an automatic fraction collector. These fractions having the characteristic ultraviolet absorption pattern of the claimed product were combined and evaporated to dryness in vacuo at < 35°. The crude material (600 mg) was crystallized from aqueous acetone (1:9) to give the title compound (295 mg); X. max

(pH 6 phosphate buffer) 261 nm ( P. 19,000): X „ 275 nm ( £. 18,400).

'7 inri'

EXAMPLE 51

Potassium-(6R,7R) 2-carboxypropyl-2-yloxyimino-2-(fur-2-yl)-acetamido]-3-pyridinium methyl-ceph-3-em-4-carboxylate (cis-isomer)

A mixture of (6R,7R)-3-acetoxymethyl-7-[2-(carboxyprop-2-yloxyimino)-2-(fur-2-yl)acetamido]ceph-3-em-4-carboxylic acid (cis isomer ) (4.0 g), pyridine (4 ml) and water (40 ml) were heated for 1 hour at 80°, after which the mixture was cooled.

The cooled mixture was diluted with water (50 mL) and extracted five times with methylene chloride (25 mL) and the combined organic extracts were washed with water. The combined aqueous phases were evaporated in vacuo at 35° to about 50 mL and acidified to pH 2 with 2N hydrochloric acid. The precipitated solid was removed by filtration, the filtrate was adjusted to pH 6.5 with potassium bicarbonate. and the solution was concentrated in vacuo at <C 35° to about 40 ml. The product was purified on a column of XAD-2 resin (500 g), eluting with water and then aqueous ethanol (1:3). Fractions which had the characteristic ultraviolet absorption of the product was combined and evaporated to dryness in vacuo at <C 35° to give the title compound (880 mg), ^max (<H>2<0>) 261 and 277 nm 17,000

and 16,950).

EXAMPLE 52

Sodium-(6R,7R)-7-[2-(carboxyprop-2-yloxyimino)-2-(fur-2-yl)-acetamido]-3- pyridinium methyl ceph-3-em-4-carboxylate (cis-i isomer )

A mixture of sodium iodide (50.0 g), water (15.5 ml) and pyridine (14 ml) was heated to 80° and stirred vigorously upon addition, over a period of approx. 10 minutes, of (6R,7R)-3-acetoxymethyl-7-[2-(carboxyprop-2-yloxyimino)-2-(fur-2-yl)acetamido]ceph-3-em-4-carboxylate (cis- isomer) (14.4

g). The mixture was stirred at 80° for a total of 55 minutes and was then cooled and diluted to approx. 400 ml with

water. 0.1 N sodium hydroxide was added to adjust the pH

to approx. 6.5 and the solution was concentrated under reduced pressure at <T40° to a volume of approx. 100 ml. The resulting solution was diluted to approx. 400 ml of water, methyl iso-butyl ketone (0.3 ml) was added and the stirred solution was acidified with 2N hydrochloric acid (15 ml) to give a pH of 1 - 2. The solid was collected, washed with water and discarded. The filtrate and washings were treated with more 2N hydrochloric acid (about 10 ml) and extracted with ethyl acetate, the organic layer was re-extracted with a small volume of water. The aqueous phase was adjusted to pH 6 with 1N sodium hydroxide (ca. 43 ml), and evaporated under reduced pressure at <f40° to a volume of ca. 175 ml. This solution was applied to a column of XAD-2 resin (700 g, 42 cm x 5.5 cm) which had previously been washed with water (2 liters). The column was eluted with water, the fractions were collected automatically and controlled by UV spectroscopy.

^Once the inorganic salts and some impurities had been removed' the eluent was changed to a mixture of ethanol and water (1:4). The fractions having the characteristic U.V. absorption of the product were combined, concentrated under reduced pressure at <T40° and then freeze-dried. The product was dried over phosphorus pentoxide in vacuo to give the title salt (4.10 g) 5 [oc]D+ 10.5° (c 1.00, H 2 O) 5 ^ maXs (pH 6 buffer) 261.5 and 2 78 .5 nm (£. 20,100: 19,200): V , (Nujol) 1770 cm"<1>

' ^ ' ' ' ' ' max

((3-lactam) ; l(D20, 100 MHz) values include 1.03, 1.43, 1.91 (pyridine protons) , 4.12 (dd, 7-H) and 8.50 (s, C(CH 3 ) 2 .

EXAMPLES 53-57

In the same manner as in Example 52, the acetoxy group of (6R,7R)-3-acetoxymethyl-7-[2-(carboxy-R<q->oxyimino)-2-(fur-2-yl)-acetamido]ceph- 3-ém-4-carboxylic acids (cis-isomers) substituted by treatment with pyridine or a substituted pyridine in aqueous sodium iodide solution at 80° for 45 - 60 minutes. The products were purified as the sodium salts by XAD-2 chromatography and their physical properties are listed in Table 7.

IN!

EXAMPLE 58 ( 6R , 7R )- 7- r 2-( 2- carboxylprop- 2- yloxyimino)- 2-( fur- 2- yl)-acetamido]- 3- pyridazinium methyl ceph- 3- em- 4- carboxylic acid trifluoroacetate ( cis - isomer) (a) A suspension of diphenylmethyl (IS,6R,7R)-3-bromo-methyl-7-[2-(2-t-butoxycarbonylprop-2-yloxyimino)-2-(fur-2-yl) -acetamido]ceph-3-em-4-carboxylate-1-oxide (cis-isomer) (1.51 g) in N,N-dimethylformamide (1 ml) was treated with pyridazine (400 mg). The mixture was stirred for 2 hours at 25° to give

a clear solution, which was then diluted with ether (50 ml, added slowly with stirring). The resulting precipitate was filtered off, washed with ether and dried to give diphenylmethyl(IS,6R,7R)-7-[2-(2-t-butoxycarbonylprop-2-yloxyimino)-2-(fur-2-yl) acetamido]-3-pyridazinium methyl ceph-3-em-4-carboxylate-1-oxide bromide (cis isomer as a light red powder (1.59 g, 94%); [oc]D + 13° (c 1.07 , DMSO); . NH) , 3.76 (dd, J 4 and

8 Hz, 7-H), 8.51 (s, C(CH 3 ) 2 ) and 8.61 (s, C(CH 3 ) 3 ).

(b) The product of the above (a) (1.49 g) in N,N-dimethylformamide (5 ml) at -10° was treated with potassium iodide (1.33 g) and then with acetyl chloride (0.28 ml) . The mixture was stirred for 1 hour while the temperature slowly rose to 0°, then was added dropwise to a stirred solution of sodium metabisulfite (1 g) in water (50 mL). The resulting suspension was stirred for 10 minutes and then the solid was filtered off, washed with water and dried over phosphorus pentoxide to give a light brown powder (1.28 g). ;

i This material, in acetone : ethanol =9:1 (20 ml), was rapidly precipitated through a column of Deacidite-FF resin (trifluoroacetate foam, 15 cm x 2.5 cm i.d.) eluted with the same up- the solvent mixture. The eluent fractions, which contained ultraviolet light-absorbing material, were combined and evaporated, and the residue triturated with ether to give diphenylmethyl (6R,7R)-7-[2-(2-t-butoxycarbonyl-prop-2-yloxyimino )-2-[Eur-2-yl]acetamido]-3-pyridazinium-methylceph-3-em-4-carboxylate-trifluoroacetate (cis-isomer), (1.24 g, 82%); [a]D-20°

(c 0.76, DMSO); Kmax (<E>tOH) 278 nm ( £- 18,300); Vmax (Nujol) 1780 cm-1; T (d^-DMSO) values include 0.30 (d,

J 8 HZ, NH) , 3.95 (dd, J 5 and 8 Hz, 7-H) , 8.55 (s, C(CH 3 ) 2 ) and 8.59 (s, C(CH 3 ) 3 ).

(c) The product according to (b) (1.13 g), mixed with anisole (1.5

ml), was treated with trifluoroacetic acid (6 ml) at 5° for 5 minutes, and then at 20° for 55 minutes. The solution was evaporated in vacuo, the residue was stirred with ethyl acetate, and the evaporation was repeated. The resulting gum was triturated

pea with ether to give the crude product as a light brown solid, which was filtered off, washed with ether and dried. This was extracted with water (3 x 150 ml); the extracts were filtered, washed with ethyl acetate and then ether, and finally freeze-dried. The combined residues were triturated with ether to give the title salt as a white powder (586 mg, 72%); [ct]D<+>48°

(c 0.98, DMSO); X ^ (pH 6 phosphate buffer) 277 nm (&_ 18,100);

Vmax(NuJo1) 1776 011-1 (P-Iafctam) ; V (dg-DMSO) 0.37 (d J 8 Hz, NH), 4.09 (dd, J 5 and 8 Hz, 7-H) and 8.53 (s, C(CH 3 ) 2 ).

EXAMPLES 59-64

The trifluoroacetate salts listed in Table 8 were prepared

prepared by reacting the 3-bromomethyl ester (see below) with

the appropriate tertiary base (or quaternary mercaptan for example 61), and by reduction of the sulfoxide and removal of both protecting groups in the same manner as described in Example 58. The starting material was prepared as follows: A solution of phosphorus pentachloride (5.20 g) in dry dichloromethane ! (60 ml) at -10° was treated with N,N-dimethylacetamide (12 ml), in

In and then 2-(2-t-butoxycarbonylprop-2-yloxyimino)-2-(fur-2-yl)acetic acid (cis-isomer) (6.43 g) was added portionwise. The solution was stirred at -10° for 15 minutes and then ice (14 g) was slowly added and the temperature allowed to rise to 0° over the course of 10 minutes. The organic layer was separated and added dropwise to a suspension of diphenylmethyl (IS,6R,7R)-7-amino-3-bromomethylceph-3-em-4-carboxylate-1-oxide hydrobromide (10.62 g) in dichloromethane (80 ml), containing propylene oxide (15 ml) at 0°. The mixture was stirred for 1 hour, during which time the temperature rose to 20° and the suspension became clear. The resulting yellow solution was washed with 2.5% aqueous sodium bicarbonate solution (50 mL) and then 2N hydrochloric acid (50 mL), after which the solution was dried and evaporated to a yellow oil.This material in ethyl acetate (20 mL) was added dropwise to stirred petroleum (bp. 40 - 60°) to give a gummy precipitate. The upper layer was decanted and the gum was chromatographed on a column of silica gel eluting with dichloromethane containing from 0 to 10% acetone. The eluent fractions, containing the major product, were combined and evaporated to a foam. Trituration with cyclohexane gave diphenylmethyl (1S,6R,7R)-3-bromomethyl-7-[2-(2-t-butoxycarbonylprop-2-yloxyimino)-2-(fur-2-yl)-acetamido]ceph-3- εm-4-carboxylate-1-oxide (cis-isomer) as a pale yellow microcrystalline powder (13.61 g, 90%); [<x]D- 22° (c 1.0, DMSO); \max (<E>tOH) 281 nm (e 22,200);

v max (CHBr3) 1800 cm_1 (β-lactam); T( to DMSO) values include 1.26 (d, J 8 Hz, NH), 3.86 (dd, J 4 and 8 Hz, 7-H),

8.51 (s, C(CH3)2) and 8.61 (C(CH3)3)..

EXAMPLE 65

( 6R, 7R )- 3-( benzotriazol-1- ylmethyl)- 7-[ 2-( 2- carboxyprop- 2- yloxyimino)- 2-( fur- 2- yl) acetamido] ceph- 3- em- 4- carboxylic acid

(cis isomer)

Diphenylmethyl (IS,6R,7R)-3-bromomethyl-7-[2-(2-t-butoxycarbonylprop-2-yloxyimino)-2-(fur-2-yl)acetamido]-ceph-3-em-4- carboxylate 1-oxide (cis isomer) (1.51 g) in N,N-dimethylformamide (3 ml) was stirred with benzotriazole (480 mg) for 4 days. The solution was diluted with dichloromethane and then washed twice with 2N hydrochloric acid. The solution was then dried and evaporated, and the major product was isolated from the residue by column chromatography on silica gel, with chloroform containing 0 to 10% by volume for elution. This material (700 mg) in N,N-dimethylformamide (2 ml) with potassium iodide (665 mg) at -10° was treated with acetyl chloride (0.14 ml). The suspension was stirred for 1.25 hours and then slowly warmed to 0°, then added dropwise to water (40 mL) containing sodium metabisulfite (0.5 g). The precipitate was filtered off, washed with water, dried and purified by chromatography on silica gel, eluting with dichloromethane, containing from 0 to 3

volume % acetone.

The diester thus obtained (520 mg), together with anisole

(0.5 ml) was treated with trifluoroacetic acid (2 ml) at 25°

1 1 hours. The solution was then added dropwise below

stirring into saturated aqueous sodium bicarbonate (50 mL) and ice (25 g). The mixture was stirred, after which ethyl acetate was added. The aqueous layer was separated and acidified under ethyl acetate to pH 2. The organic layer was separated and the aqueous layer extracted with more ethyl acetate. The combined extracts were dried and concentrated to an oil, which was added dropwise to stirred petroleum (b.p. 40 - 60°). The white precipitate was filtered off, washed with petroleum and dried to give the title dicarboxylic acid as a white powder (350 mg, 31%); [cc]D + 37° (c 1.02, DMSO); ^max (pH 6 phosphate buffer) 269 nm (£.23,600):s) . (Nujol) 1780 cm"1

' ' max J

((3-lactam) ; X (d^-DMSO) values include 0.40 (d, J 8 Hz, NH) , 4.11 (dd, J 5 and 8 Hz, 7-H) and 8.53 ( s, C(CH3)2).

IN

EXAMPLE 66 The compound listed in Table 9 was prepared from

the nucleophile used, using the method of Example 65.

in

EXAMPLE 67 Pivaloyloxymethyl ( 6R , 7R )- 3- carbamoyloxymethyl- 7-[ 2- carboxymethoxyimino- 2-( fur- 2- yl) acetamido] ceph- 3- em- 4- carboxylate

(cis isomer)

A stirred solution of sodium (6R,7R)-3-carbamoyloxy-methyl-7-[2-t-butoxycarbonylmethoxyimino)-2-(fur-2-yl)-acetamido]-ceph-3-em-4-carboxylate ( cis isomer) (8.50 g) in dimethylformamide (100 mL) was treated with chloromethyl pivalate (4.68 mL). The solution was stirred at room temperature for 25 hours and partitioned between ethyl acetate and water. The aqueous layer was further extracted with ethyl acetate, and the combined extracts were washed sequentially with brine, saturated sodium bicarbonate, and brine, and then dried and evaporated to a small volume. The residue was added slowly to stirred petroleum (b.p. 40 - 60°, 1.5 liters) and the precipitate was collected, washed with a petroleum fraction boiling between 40 and 60° and dried in vacuo to give a mixture of the t-butyl ester of the title compound and its

A 2 analogue (8.32g) , (ratio A<2>: A<3>^3:2 at p.m.r.).

A solution of this mixture of isomers (8.1 g) in dichloromethane (75 ml) was cooled to -40° and stirred during the dropwise addition of m-chloroperbenzoic acid (2.41 g) in dichloromethane (65 ml). The solution was stirred at -40° for 1.25 hours and more m-chloroperbenzoic acid (1.2 g) in dichloromethane was added. After stirring for a further 40 minutes, the solution was evaporated and the residue partitioned between ethyl acetate and saline. The ethyl acetate solution was washed with sodium bicarbonate solution and brine, dried over sodium sulfate and evaporated to a yellow foam (9.77 g). This was triturated with ether and a yellow solid was filtered off, washed with ether and dried in vacuo to give the 1-oxide of the t-butyl ester of the title compound (7.27g).

A solution of this sulfoxide (7.07 g) and potassium iodide

(14.34 g) in dimethylformamide (100 mL) was surrounded by an ice-salt bath and stirred during the addition of acetyl chloride (3.08 mL) in dimethylformamide (15 mL). The solution was stirred

J for 3.5 hours and more acetyl chloride (1.54 mL) in dimethylformamide was added. The solution was stirred for 1 hour and partitioned between sodium metabisulfite solution and ethyl acetate. The organic layer was washed with sodium metabisulfite solution and water, then dried and evaporated to a small volume. The resulting solution was added slowly to excess stirred petroleum (b.p. 40 - 60°) and the resulting precipitate was collected, washed with a ^petroleum fraction bp 40 60° and dried in vacuo to give pivaloyloxymethyl (6R,7R)-3 -carbamoyloxymethyl-7-[2-t-butoxycarbonylmethoxyimino-2-(fur-2-yl)acetamido]ceph-3-em-4-carboxylate (cis isomer) (7 g)5 max (EtOH) 277 ^ (^ -16.540)5 max (Nujol) 1782 ((3-lactam), 1740 and 1720 cm<-1>(C0NH2); T (d6-DMS0) values include 0.25 (d, NH), 3.40 (s , NH2) and 8.8 (s, C(0*3)3).

The above diester (1.0 g) was dissolved in trifluoroacetic acid (5 ml), and this solution was stirred at room temperature for 15 minutes and was then diluted with ether, washed with water and evaporated. The residue was partitioned between ethyl acetate and sodium bicarbonate solution and the organic layer was extracted again with sodium bicarbonate solution. The combined aqueous extracts were washed with ethyl acetate, acidified with 2N hydrochloric acid and extracted with ethyl acetate. The extracts were washed with water, dried and evaporated to give an oil. This was dissolved in a small volume of ethyl acetate and slowly added to an excess of stirred petroleum (b.p. 40 - 60°). The greyish-white precipitate was collected, washed with a petroleum fraction of b.p. at between 40 and 60° and dried in vacuo to give the title ester (0.645 g); X max ^EtOH^ 278 nm (2 16.900); ^ max (Nujol) 1778 cm-1 (p-lactam)5

T (d6-DMS0) values include 0.25 (d, NH), 2.16, 3.26,

3.38 (fur-2-yl protons) and 4.10 (dd, 7-H).

The sodium salt starting material for the above process

was produced as follows:

Triethylamine (2.58 mL) and dimethylformamide (2 drops) were added to a solution of 2-t-butoxycarbonyl-methoxyimino-2-(fur-2-yl)acetic acid (cis isomer) (5 g) in dry dichloromethane

in (200 ml). The stirred solution was cooled to 0° and ok- !

salyl chloride (1.58 mL) was added. The solution was stirred

for 1 hour and then evaporated. The residue was suspended in acetone (150 mL) and added slowly over 0.5 h to a stirred ice-cold solution of (6R,7R)-3-carbamoyloxymethyl-7-aminoceph-3-em-4-carboxylic acid (5.08 g) in acetone (150 ml) and water

(300 ml), containing sodium bicarbonate (3.74 g). The reaction mixture was stirred for 1 hour and evaporated to remove acetone. The aqueous residue was acidified under ether to pH 1.8 with dilute hydrochloric acid and the layers were separated. The aqueous layer was further extracted with ether and the combined extracts were washed with water and brine, dried and evaporated to give (6R,7R)-3-carbamoyloxymethyl-7-[2-t-butoxycarbonylmethoxyimino-2-(fur-2 -yl)acetamido]-ceph-3-em-4-carboxylic acid (cis-isomer); ^max ^PH ^ phosphate buffer) 274.5 nm (£.17.520); vmax (CHBr3) 1785 ((3-lactam) , 1686 and 1530 cm"1 (CONH) ; (d6-DMS0) values include 0.29 (d, NH) , 4.19 (dd, 7-H) and 4 .79 (d, 6-H) .

A stirred solution of the above acid (9.02 g) in acetone (40 ml) was treated with a solution of sodium 2-ethyl hexanoate (2.86 g) in acetone (40 ml). The resulting solution was added slowly to a petroleum fraction bp 40 - 60° (1600 ml) and the precipitate was collected, washed and dried in vacuo to give sodium (6R,7R)-3-carbamoyloxymethyl-7-[2-t- butoxycarbonyl-methoxyimino-2-(fur-2-yl)acetamido]-ceph-3-em-4-carboxylate (cis isomer) (8.71 g); ^-max (PH ^ buffer) 274.5 nm (£.17.650): V , (Nujol) 1755 (p-lactam)

," ' " max

and 1608 cm (C02~.) ; TT (d^-DMSO) values include 0.4 (d,

NH), 4.4 (dd, 7-H) and 4.98 (d, 6-H).

EXAMPLE 68

( IS, 6R, 7R )- 3- acetoxymethyl- 7- r 2-( 2- carboxyprop- 2- yloxyimino)-2-( fur- 2- yl) acetamido] ceph- 3- em- 4- carboxylic acid- l- oxide (cis-isomer)

(a) A solution of (6R,7R)-3-acetoxymethyl-7-[2-(2-carboxyprop-2-yloxyimino)-2-(fur-2-yl)acetamido]ceph-3-em-4- carboxylic acid, di-t-butyl ester (cis isomer) (1.21 g) in pyridine (25 mL) and water (1 mL) at -45° was treated with t-butyl hypochlorite (0.3 mL) with vigorous stirring. After 2 minutes!

2N sulfuric acid (1 mL) was added to the solution and the mixture was immediately poured into aqueous phosphoric acid (100

ml, 20% by volume. The aqueous solution was extracted with ethyl acetate and the organic extracts were washed with 0.5N hydrochloric acid (50 mL), aqueous sodium bicarbonate solution (50 mL) and water, then dried and concentrated in vacuo. The crude product was chromatographed on silica gel preparative plates, using ethyl acetate:petroleum ether (b.p. 60 - 80°) (4:1) as eluent. The slower running band was extracted with ethyl acetate to give the ditt-butyl ester of the title compound (505mg); v max (<N>ujol)1798 ((3-lactam) , 1738, 1727, 1715 (acetate and CO2.tBu), 1680 and 1542 cm<-1>(CONH); " values (DMS0-d6) include 0 .28 (d, J 8 Hz, NH) , 4.19 (dd, J 5 and 8

Hz, 7-H), 5.02 (d, J 5 Hz, 6-H), 5.71 and 6.38 (ABq, J 18 Hz, 2-H).

The di-t-butyl ester (0.38 g) in trifluoroacetic acid (5 ml, containing a few drops of anisole) was stirred at room temperature for 15 minutes. The solution was concentrated in vacuo to a red oil, diluted with ethyl acetate (2 mL) and added dropwise to vigorously stirred petroleum ether (b.p. 60 - 80° (50 mL). The precipitated solid was collected, washed with ether (5 mL)

and dried to give the title acid (185 mg, 60%); X^a^s(ethanol) 276 nm ( &16,600); V ak (Nujol) 1790 ((3-lactam), 1730 (acetate), 1720 (CO 2 H), 1680 and 1523 (CONH) and 1040 cm"<1>

(S —> 0); Tverdier (DMSO-dg) include 0.21 (d, J 8 Hz, NH), 4.17 (dd, J 5 and 8 Hz, 7-H), 5.01 (d, J 5 Hz, 6-H ), 5.71 and 6.32 (ABq, J 18 Hz, 2-H), 8.49 (s, C(CH 3 ) 2 ).

The starting material in the above oxidation process was prepared as follows: A solution of t-butyl(6R,7R)-7-amino-3-acetoxy-methylceph-3-em-4-carboxylate (1.05 g) in dry dichloromethane (10 ml ) was added to a solution of 2-(2-t-butoxycarbonylprop-2-yloxyimino)-2-(fur-2-yl)acetic acid (cis isomer) (0.99 g) and dicyclohexylcarbodiimide (0.69 g) in dry dichloromethane (10 mL), and the mixture was stirred at room temperature for 1 hour. The solution was filtered and concentrated in vacuo. The crude product was passed through a column of silica gel (MFC, 100 - 200 mesh, 2 x 20 cm) using ethyl acetate:petroleum ether (b.p. 60 - 80°)

(1:1) as eluent. Combination of the appropriate fractions was determined by thin-layer chromatography to give (6R,7R)-3-acetoxymethyl-7-[2-(2-carboxyprop-2-yloxyimino)-2-(fur-2-yl)acetamdio]ceph-3 -em-4-carboxylic acid, di-t-butyl ester (cis isomer) (1.29 g); v max (CHBr3) 1776 (β-lactam), 1725, 1712 (acetate and CO2.tBu), 1678 and 1512 cm<-1>(CONH); T (CDC13) values include 1.90 (d, J 8 Hz, NH), 4.08 (dd, J 5 and 8 Hz, 7-H) and 4.98 (d, J 5 Hz, 6-H) .

EXAMPLE A

This example illustrates the design of a pharmaceutical composition.

Dry powder for injection

Sterile (6R,7R)-3-acetoxymethyl-7-[2-(1-carboxy-cyclopent-1-yloxyimino)-2-(fur-2-yl)acetamido]ceph-3-em-4-carboxylate disodium salt (cis-isomer) was filled in glass bottles, whereby the required content in each bottle was 500 mg or 1.00 g of the desired antibiotic. The filling was carried out aseptically under a blanket of nitrogen. The bottles are closed using rubber discs or plugs, which are held in position by aluminium-sealed rings, thereby preventing gas mixing or the introduction of micro-organisms. The product is intended for dilution with water for injections or other suitable sterile solutions for a short time before administration.

Claims (12)

1. Process for the preparation of an antibiotic compound of the general formula where R means thienyl or'furyl, R a and R , which may be the same or different, are each selected from hydrogen, C 2 -4 alkyl, C 2 _4 alkenyl, C 3 _7 cycloalkyl, phenyl, naphthyl, thienyl, furyl, carboxy, C 0 j -alkoxycarbonyl and cyano, or R and R can, together with the carbon atom to which they are attached, form a C3 _7 cycloalkylidene or cycloalkenylidene group, m and n are each 0 or 1, so that the sum of m and n is 0 or 1, and Z is a group in which 2 carbon atoms connect the core sulfur atom and the 4-position carbon atom so that the compound contains A3 -unsaturation) or non-toxic derivatives thereof, said compound being a cis-isomer or existing as a mixture of cis- and trans-isomers, containing at least 90% of the cis-isomer, characterized by (A ) one condenses a compound with the formula 19 R is hydrogen or a carboxyl blocking group, and Z' means a group in which 2 carbon atoms connect the core sulfur atom and the 4-position carbon atom so that the compound has A2 or A3 unsaturation, or an acid addition salt or an N-silyl derivative thereof with an acylating agent corresponding to an acid of the formula where R, Ra? R13, m and n have the previously stated meaning and 20 R means a carboxyl-blocking group, or (B) where Z in formula I means the group where Y means the residue of a nucleophile or a derivative of the residue of a nucleophile, one reacts a compound with the formula in 9 b where B, R, R , R , m and n have the above meaning, 19 each of R can independently be hydrogen or a carboxyl blocking group, Y' is a replaceable residue of a nucleophile, and the dashed line crossing the 2-, 3-, and 4-positions indicates that the compound is a ceph-2-em or ceph-3-em compound with a nucleophile$ after which, if necessary and/or desired in each case, any of the following reactions (C) in any suitable order, is carried out: i) conversion of a 'isomer to the desired A <3-> isomer, ii) reduction of a compound, wherein B means S —> 0 to form a compound, wherein B is >S, iii) reduction of a 3-azidomethyl compound to form a 3-aminomethyl compound; iv) acylating a 3-aminomethyl compound to form a 3-acylaminomethyl compound; v) reaction of a 3-azidomethyl compound with a dipolarophile to form a compound having a polyazole ring attached to the 3-position carbon atom via a methylene group; vi) deacylation of a 3-acyloxymethyl compound to form a 3-hydroxymethyl compound; vii) acylation of a 3-hydroxymethyl compound to form a 3-acyloxymethyl compound; viii) carbamoylation of a 3-hydroxymethyl compound to form an unsubstituted or substituted 3-carbamoyloxymethyl compound, and ix) removal of carboxyl blocking groups; and finally (D) recovery of the desired compound of formula I or a non-toxic derivative thereof, if necessary, after separation of isomers. 2. Procedure' according to claim 1, charac- characterized by the compound of formula I or a non-toxic derivative thereof being recovered as the cis-isomer which is essentially free of the trans-isomer. 3. Process according to claim 1 or 2, characterized in that a compound (V) or an acid addition salt or N-silyl derivative thereof is condensed with an acid halide corresponding to the acid (VI). 4. Method according to claim 3, characterized in that the condensation is carried out in the presence of an acid binding agent consisting of a tertiary amine, an inorganic base or an oxirane. 5. Process according to claim 1 or 2, characterized in that a compound (V) or an acid addition salt or N-silyl derivative thereof is condensed with an acid (VI) in the presence of a condensation agent consisting of a carbodiimide, carbonyldiimidazole or an isoxazolinium salt . 6. Method according to one of claims 1-5, characterized in that a compound with the formula i j 9 b where R, R , R , m and n have the meaning stated in claim 1 and P is selected from a) hydrogen, b) halogen, c) a group with the formula wherein R 1 and R 2 , which may be the same or different, is selected from hydrogen, carboxy, cyano, C2 _7 alkoxycarbonyl, C1 _6 alkyl, C5 _7 cycloalkyl, phenyl 4 alkyl and C6 _12 mono- or bicyclic carbocyclic aryl, andd) a group of the formula where Y is selected from: d) (i) the residue of a nitrogen nucleophile which is a tri(C 1 -C 3 alkyl)amine or a heterocyclic tertiary amine, d) (ii) azido, d) (iii) amino , d) (iv) acylamido, d)(v) a derivative obtained by reacting a compound, in which Y means azido an acetylenic, ethylenic or cyano-dipolarophy 1, ' d) (vi) a group with the formula wherein R <10> and R <11> , which may be the same or different, are each selected from hydrogen, cyano, lower alkyl, phenyl, phenyl substituted with one or more halogen, lower alkyl, lower alkoxy, nitro, amino or lower alkylamino; and 12 R is selected from hydrogen, lower alkyl, phenyl, phenyl substituted with one or more halogen, lower alkyl, lower alkoxy, nitro, amino or lower alkylamino, aryl-lower-alkyl, and and C 6 cycloalkyl, d) (vii) the residue of a sulfur nucleophile which is a thiourea, dithiocarbamate, thioamide, thiosulfate, thioacid or dithioacid, d)(viii) a group with the formula 13 wherein R is a lower alkyl, lower cycloalkyl, phenyl lower alkyl, C&_12 mono- or bicyclic carbocyclic aryl or heterocyclic group and n is 0, 1 or 2, d) (ix) a group of the formula 15 wherein R means hydrogen, lower alkyl, lower alkenyl, lower alkynyl, lower cycloalkyl, lower cycloalkyl-lower-alkyl, aryl, aryl-lower-alkyl, hetero-j cyclic, heterocyclic-lower-alkyl, or any ' of the preceding groups substituted with one or more lower alkoxy, lower alkylthio, halogen, lower alkyl, nitro, hydroxy, acyloxy, carboxy, lower alkoxycarbonyl, lower alkylcarbonyl, lower alkylsulfonyl, lower alkoxysulfonyl, amino, lower alkylamino and acylamino,d) (x) a group of formula 16 wherein R is selected from C1_7~ alkyl, which may be interrupted by an oxygen or sulfur atom or by an imino group or substituted by cyano, carboxy, lower carboxycarbonyl, hydroxy, carboxycarbonyl, halogen or amino; C^^ alkenyl, which may be interrupted by an oxygen or sulfur atom or by an imino group; lower cycloalkyl; carbocyclic or heterocyclic aryl, which may be substituted with hydroxy, halogen, nitro, amino, lower alkyl or lower alkylthio; lower cycloalkyl ^ alkyl; and carbocyclic or heterocyclic aryl C 1 -C 3 alkyl and d) (xi) a group of the formula 17 wherein R means a hydrogen or a group which previously defined for R <1> ^ and A is >0, >S or >NH, or a non-toxic derivative thereof is recovered. 7. Method according to claim 6, characterized in that P is a group with formula wherein Y means the residue of a nitrogen nucleophile, comprising a pyridine, pyrimidine, pyridazine, pyrazine, pyrazole, imidazole, triazole, thiazole, purine, benzotriazole or any of these compounds substituted with one or more lower alkyl, c§_^ 2 mono~ or bicyclic carbocyclic aryl, phenyl-lower- alkyl, lower alkoxymethyl, acyloxymethyl, formyl, acyloxy, carboxy, esterified carboxy, carboxy-lower-alkyl, sulfo, lower alkoxy, phenoxy, phenyl-lower-alkoxy, lower alkylthio, phenylthio, phenyl-lower-alkylthio, cyano, hydroxy, carbamoyl, N-mono (lower alkyl) carbamoyl, N,N-di (lower alkyl) carbamoyl, N-(hydroxy-lower alkyl) carbamoyl or carbamoyl-lower alkyl groups. 8. Method according to claim 6, characterized in that P is a group with the formula where Y means a group 14 where R is a diazolyl, triazolyl, tetrazolyl, thiazolyl, thiadiazolyl, thiatriazolyl, oxazolyl, oxadiazolyl, pyridyl, pyrimidyl, benzimidazolyl, benzoxazolyl, benzothiazolyl, triazolopyridyl or purinyl group* 9. Process according to claim 6, characterized in that P means an acetoxymethyl or carbamoyloxymethyl group. 10. Method according to one of claims 1-5, characterized in that a compound with the general formula where R has the meaning defined in claim 1, R means methyl, ethyl, propyl, allyl or phenyl and R means hydrogen, carboxy or a group which c c d defined for R or R and R together with car- the bone atom to which they are attached forms a cyclobutylidene, cyclopentylidene or cyclohexylidene group, and W is selected from: i) hydrogen, ii) acetoxymethyl, iii) benzoyloxymethyl, iv) carbamoyloxymethyl, v) N-methylcarbamoyloxymethyl, vi) a group with the formula -CH=CHR<Z> where Z means cyano, carboxy or C 2 -5 alkoxycarbonyl, vii) a group of formula -CH 2 2 G where G means the residue of a nitrogen nucleophile selected from compounds of formula where R means hydrogen, carbamoyl, carboxymethyl or sulfo, and pyridazine, viii) azidomethyl, and ix) a group with the formula wherein R <w> means a pyridyl, diazolyl, triazolyl, tetrazolyl, thiazolyl, thiadiazolyl or oxadiazolyl group or a lower alkyl or phenyl substituted analogue of such a group, or non-toxic derivatives thereof, are recovered. 11. Method according to claim 10, characterized in that W means a group with the formula wherein R w means N-methylpyrid-2-yl, 1-methyltetrazol-5-yl or 5-methyl-1,3,4-thiadiazol-2-yl. 12. Method according to one of claims 1-5, characterized in that a compound with the general formula According to claim 10; and p means 1 or 2, or a non-toxic derivative thereof, is recovered.