NO152751B - ANALOGY PROCEDURE FOR THE PREPARATION OF A THERAPEUTIC EFFECTIVE CEPHALOSPORIN DERIVATIVE - Google Patents

Description

The present invention relates to the production of an antibiotic of the cephalosporin series.

The cephalosporin compound in the present description is named with reference to "cepham" according to J.Amer.Chem. Soc. 1962, 84, 3400, the term "cephem" referring to the basic cepham structure with a double bond.

Many cephalosporin compounds exhibiting a certain degree of antibacterial activity are known in the art, and these compounds exhibit a β-ring and are usually substituted in the 3-position with a methyl or substituted methyl group and in the 7(3-position with an acylamido group. It is now well known that the antibiotic properties of a particular ceph-3-em-4-carboxylic acid are essentially determined by the type of both the 7(3-acylamido group and the substituent in the 3-position which the compound carries. Considerable research has been carried out for to find combinations of such groups that will give antibiotics with special properties.

Cephalosporin antibiotics have a wide application in the treatment of diseases caused by pathogenic bacteria in both animals and humans, e.g. in the treatment of diseases caused by bacteria that are resistant to other antibiotics such as penicillin compounds, as well as in the treatment of penicillin-sensitive patients. In many applications, it is desirable to use a cephalosporin antibiotic that exhibits activity with regard to both gram-positive and gram-negative microorganisms, and significant research has been undertaken to develop improved broad-spectrum cephalosporin antibiotics.

The practical utilization of a significant number of known commercial and experimental cephalosporin antibiotics is limited due to their relatively high susceptibility to (3-lactarases, which are produced by many bacteria. A desirable characteristic of a widely used cephalosporin antibiotic is therefore that it must exhibit a significant resistance to β-lactamases, including those produced by gram-negative microorganisms.

A further difficulty with many cephalosporin antibiotics intended for therapeutic use is that they are subject to degradation in vivo. It has thus been found that a significant number of known cephalosporin antibiotics suffer from the disadvantage that after administration they are deactivated, often very quickly, by enzymes (eg esterases) found in the body.

A new cephalosporin compound has now been found with a special combination of the 7(?-acylamido group and a substituent in the 3-position, which gives the compound good broad-spectrum activity combined with the desired goals described above with regard to high (3-lactamase stability and good stability in vivo. This compound is characterized in that the 73-acylamido group is a 2-(fur-2-yl)-2-methoxyiminoacetoamido group, which is essentially in the syn configuration (as defined below) and where the 3-stub substituent is a carbamoyloxymethyl group.

Related compounds are known from Norwegian application no. 1705/72, which will be discussed later, and whose compounds will be compared with the compound produced according to the present invention.

The compound produced according to the present invention thus has the formula

as well as non-toxic salts or biologically acceptable esters of this acid.

This compound is either syn isomer or exists as a mixture of syn and anti isomers containing at least 90% of the syn isomer. More preferably, the compound is syn-isomer, substantially free of the corresponding anti-isomer.

The compound produced according to the present invention is defined to have the syn- (cis-) isomeric form with regard to the configuration of the group OCH^ in relation to the carboxamido group. In this description, the syn configuration is specified as follows:

The Syn configuration is given on the basis of the work according to

Ahnad and Spenser (Can. J. Chem. 1961, 39_, 1*340) .

The term "non-toxic" applied to salts in

according to the present invention means those salts which are physiologically acceptable in the doses in which they are administered.

The expression "biologically acceptable esters" indicates esters of the active compound I which are metabolically labile, i.e. upon administration they are converted in vivo into the antibiotic compound (I).

Salts which can be formed from the compound of formula I include inorganic salts such as of alkali metal (eg sodium and potassium), of alkaline earth metal (eg calcium) and of an organic base (eg procaine, phenylethylbenzylamine, di-benzylethylenediamine , ethanolamine, dreethanolamine, triethanolamine and N-methylglucosamine). These salts can also include resinates formed, for example, with a polystyrene resin or a cross-linked polystyrene divinylbenzene copolymer resin, which contain amino or quaternary amino groups.

As indicated above, the compound produced according to the invention exhibits a particularly valuable combination of properties, namely high antibacterial activity against a wide number of gram-positive and gram-negative organisms. The breadth of the activity spectrum is combined with the particularly high stability of the compound against 3-lactanases produced by various gram-negative organisms. The compound also exhibits the advantageous property of good stability in vivo, especially towards esterases.

The properties exhibited by the compound make it useful

in the treatment of a number of diseases caused by pathogenic bacteria in animals and humans.

The compound of formula I is thus (6R,7R)-3-carbamoyloxymethyl-7-[2-(fur-2-yl)-2-methoxyiminoacetamido]ceph-3-em-4-carboxylic acid (syn isomer), and may suitably be in the form of an alkali metal salt,

especially the sodium salt. This compound is active against a wide number of gram-positive and gram-negative microorganisms, for example Staphylococci including Staphylococcus aurens} Streptococcus pyogenes and Streptococcus viridans, Diplococcus pneumoniae, Haemophilus influenzae, Neisseria and Clostridia species, Escherichia coli, Klebsiella, Proteus and Enterobacter species, which has been proven in experiments both in vitro and in vivo. The compound exhibits good in vitro activity at inoculation levels as high as 10 7 organisms/ml and exhibits particularly high in vitro activity against strains of Haemophilus influenzae, Neisseria gonorrhoeae and Neisseria meningitidis. The compound exhibits high stability against (3-lactamases produced by a number of gram-negative organisms, as demonstrated for example by their in vitro activity against various (3-lactamase-producing strains of Escherichia, Enterobacter and Klebsiella species. The compound is resistant to the action of mammalian esterases and is thus stable in both human and animal bodies, which is demonstrated by the high recovery level of unchanged compound in the urine. Furthermore, the compound gives high serum levels after parenteral administration to both animals and humans and at the same time shows low serum binding.

The use of highly soluble salts, for example alkali metal salts such as the sodium salt, of the compound is very advantageous in therapeutic use due to their rapid distribution in the body when administered by injection?

It has been found that sodium (6R,7R)-3-carbamoyloxymethyl-7-[2-(fur-2-yl)-2-methoxyiminoacetamido]ceph-3-em-4-carboxylate

(syn isomer) exists in a number of different crystal forms

more, including solvates.

The sodium salt is most easily prepared by dissolving the compound (I) in a polar organic solvent (e.g. dimethylacetamide), a mixture of such solvents (e.g. dimethylacetamide/acetone or dimethylformamiH/methanol-denatured ethanol) or an aqueous polar organic solvent system (e.g. aqueous acetone) in contact with a small molar excess of sodium 2-ethyl hexanoate dissolved in a suitable organic solvent (e.g. an alkanol such as ethanol, a ketone such as acetone or a chlorinated hydrocarbon such as methylene chloride, an ester such as ethyl acetate, an ether such as dioxane) suitably at room temperature and then collect the precipitated salt optionally after cooling the solution (eg to 4°C).

When essentially anhydrous solvents are used in this process, form I of sodium (6R,7R)-3-carbamoyloxymethyl-7-[2-(fur-2-yl)-2-methoxyiminoacetamido]ceph-3-em- 4-carboxylate (syn isomer), and this material contains approx. 1.5% water. When the solvent system contains more than approx. 2% water is obtained, however, the form II salt, which normally contains approx. 2% water. When the solvent system contains more than approx. 60% dioxane is normally obtained form III salt, and this material comprises a dioxane solvate containing approx. 1 mole of dioxane. The Form II salt can be obtained if a water-moistened solvent system is used at an elevated temperature (e.g. 60-80°C). Crystallization of amorphous, freeze-dried sodium (6R, 7R)-3-carbamoyloxymethyl-7-[2-(fur-2-yl)-2-methoxyiminoacetamido]ceph-3-em-4-carboxylate (syn isomer) affords. from suitable dry, water-containing or dioxane-rich solvent systems respectively the form I, form II or form III salt.

If the form I salt is exposed to water vapor (e.g. at 75% relative humidity) this will cause the salt to absorb further water and undergo a change in crystal form which generally leads to the form IV salt. The material formed contains approx. 4% water (i.e. approx. 1 mol) and it is assumed that this is a hydrate. This change is reversible, so that the form IV salt can be converted into the form I salt, for example by drying in vacuum over phosphorus pentoxide. The Form II salt does not absorb additional water when exposed to water vapor, but it can be converted to the Form I salt by heating (eg for about 5 min) a suspension of the Form II material in methanol near its boiling point.

The Form III salt is obtained by reacting the compound (II) and sodium 2-ethyl hexanoate in dioxane-rich solvent systems as described above and is normally precipitated as a gel which

can be dried in vacuum to give a solid with a very low specific gravity and which exhibits little or no crystallinity. However, crystalline form III can be obtained by treating an aqueous solution of the sodium salt with a considerable excess (e.g., about 8 parts by volume) of dioxane, and if desired together with a small proportion of ethanol, and collecting the white needle-shaped crystals formed, advantageously after cooling to a low temperature (eg 4°C), wash the product obtained with dioxane and then dry the crystals (eg under vacuum at 20°C).

The Form III salt is hygroscopic and when exposed to water vapor (e.g. at 75% relative humidity) it loses all the dioxane present and forms the Form IV material, which can then be dried (e.g. over phosphorus pentoxide) to give form I the salt. If the crystal form III material is treated in this way, the crystal form of the product appears to be retained throughout the transformation sequence. The Form III salt can also be converted to the Form I salt

by heating a suspension of the Form III material in near-boiling methanol. However, this conversion results in losses

of crystallinity if crystalline material of form III is used.

The four forms of sodium (6R,7R)-3-carbamoyloxymethyl-7-[2-(fur-2-yl)-2-methoxyiminoacetamido]ceph-3-em-4-carboxylate (syn isomer) described above are characterized by the following powder X-ray diffraction patterns (d-distances and intensities) and infrared spectroscopic data.-

Powder X-ray diffraction ion monsters

Camera: Debye-Scherrer, radius 114.6 mm

Irradiation: copper K^ = 1.5418 Å

intensities (I) by visual comparison with a calibrated standard.

Form II (continued) Form III

Form IV

Infrared Spectra

Spectrometer: "Perkin-Elmer 521!' range 4000 - 650 cm-''" Spectra recorded for "Nujol" paste plates (bands associated with "Nujol" are omitted)

Form I

Form II

Form III

Form III (continued) Form IV

Legend:

s = strong

sh = shoulder

m = medium

w = weak

<+> indicates bands characteristic of each crystal form.

When insoluble salts of the compound (I) are desired for special applications, for example for use in depot preparations, such salts can be formed in a conventional manner, e.g. with suitable organic amines.

According to the invention, it is provid-

brought a process for the production of an antibiotic compound of formula I (as previously defined) and non-toxic salts and biologically acceptable esters thereof, and the process is characterized by the fact that a compound of the general formula

wherein R"<*>""<*>" is hydrogen or a carboxyl blocking group, 12 and R is hydrogen or an N-protecting group, or an acid addition salt or N-silyl derivative thereof, is reacted with an acid of the general formula

or a reactive derivative thereof, or

reacts a compound with the general formula

wherein R"^ is as defined above, with a carbamoylating agent,

after which any carboxyl-blocking or N-protecting group is removed, if necessary the anti-isomer is separated and eventually the compound of formula I is converted into a biologically acceptable ester or into a non-toxic salt, preferably the sodium salt.

Non-toxic salts of the compound of formula I may be prepared in any suitable manner, e.g. according to methods well known in the art. For example alkali metal salts can be formed by reacting the cephalosporin acid with sodium or potassium 2-ethyl hexanoate. Biologically acceptable ester derivatives can be prepared using conventional esterification agents.

The compound of formula I can be prepared by condensing

a compound of formula II with an acid chloride or acid bromide corresponding to the acid (III). Such acylation can be carried out at temperatures in the range -50 - +50°C, preferably -20 - +30°C. The acylation can be carried out in aqueous or non-aqueous media.

Acylation with an acid halide can be carried out in the presence of an acid scavenging agent (e.g. a tertiary amine such as triethylamine or dimethylaniline, an inorganic base such as calcium carbonate or sodium bicarbonate, or an oxirane, preferably a lower-1,2-alkylene oxide such as ethylene oxide or propylene oxide), which serves to bind the hydrogen halide which is released during the acylation reaction.

The free acid form of the compound of formula III can itself be used as an acylating agent. Such acylation is advantageously carried out in the presence of, for example, a carbodiimide such as N,N<1->diethyl-, dipropyl- or diisopropylcarbodiimide, N,N'-dicyclohexyl-carbodiimide or N-ethyl-N1-y-dimethylaminopropylcarbodiimide, a carbonyl compound such as carbonyldiimidazole, or an isoxazolium salt such as N-ethyl-5-phenylisoxazolium-3'-sulfonate or N-t-butyl-5-methylisoxazolium perchlorate. The condensation reaction is preferably carried out in an anhydrous reaction medium, for example methylene chloride, dimethylformamide or acetonitrile.

Acylation can also be carried out with other amide-forming derivatives of the free acid (III), such as, for example, a symmetrical anhydride or a mixed anhydride, e.g. with pivalic acid or formed with a halogen formate such as a lower alkyl halogen formate. The mixed or symmetrical anhydride can be prepared in situ. Thus, e.g. a mixed anhydride is formed using N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline. Mixed anhydrides can also be formed with phosphoric acids (for example phosphoric acid or phosphoric acid), sulfuric acid or aliphatic or aromatic sulphonic acids (for example p-toluenesulphonic acid).

When a starting material of formula IV is used, suitable carbamoylating agents include isocyanates of the general formula

where R 13 is a labile substituent group, such carbamoylating agents serve to form at the 3-position an N-protected carbamoyloxymethyl group of the formula

where R 13 has the previously indicated meaning, which can be converted into the desired unsubstituted 3-carbamoyloxymethyl group

13

by subsequent cleavage of the R group, e.g. by hydrolysis. Labile groups R 13 which are easily cleaved by subsequent treatment include chlorosulfonyl and bromosulfonyl, aralkyl groups such as benzyl, p-methoxybenzyl and diphenylmethyl: t-butyl; halogenated lower alkanoyl groups such as dichloroacetyl and trichloroacetyl as well as halogenated lower alkoxycarbonyl groups such as 2,2,2-trichloroethoxycarbonyl. R 13 groups of this type, with the exception of aralkyl groups such as diphenylmethyl, can generally be cleaved by acid- or base-catalyzed hydrolysis, (for example base-catalyzed hydrolysis using sodium bicarbonate). Halogenated groups such as chlorosulfonyl, trichloroacetyl and 2,2,2-trichloroethoxycarbonyl can also be removed by reduction, while groups such as chloroacetyl can also be removed

tes by treatment with thioamides such as thiourea. Aralkyl groups such as diphenylmethyl are most easily cleaved by treatment with acid, for example a strong organic acid such as trifluoroacetic acid.

The carbamoylating agent of formula V can advantageously be used

in excess (for example at least 1.1 mol relative to the compound of formula IV). The carbamoylation can be promoted by the presence of a base, for example a tertiary organic base such as a tri(lower alkyl)amine (for example triethylamine) or by using the acid IV in the form of an alkali metal salt (e.g. sodium), although such aids do not need to be necessary in the case of the more active isocyanates, for example

13

compounds (V), in which R is a strong electron-withdrawing group such as chlorosulfonyl or trichloroacetyl group.

Carbamoylations involving reaction of a free acid (IV) with an excess of isocyanate (V), in which R"^ is a group such as chlorosulfonyl or trichloroacetyl, are thus of particular practical use as a result of the simple reaction conditions, as there is no need for a temporary blocking and subsequent unblocking of the carboxyl group in the 4-position in the cephalosporin and then the electron-withdrawing group R 13 in the obtained N-protected 3-carbamoyloxymethyl cephalosporin product is easily removed, for example by hydrolysis in aqueous sodium bicarbonate.

It should be noted that it may be appropriate to retain or also introduce an N-substituent group R 13 during the conversions of the 3-carbamoyloxymethyl intermediates in order to minimize unwanted side reactions involving the carbamoyloxymethyl group.

Other useful carbamoylating agents are cyanic acid, which is suitably formed in situ for example from an alkali metal cyanate, such as sodium cyanate, the reaction being promoted by the presence of an acid, for example a strong organic acid such as trifluoroacetic acid. Cyanic acid corresponds to the compound of formula V in which R 13 is hydrogen and therefore converts compounds of formula IV directly to their 3-carbamoyloxymethyl analogues.

The 3-hydroxymethyl starting material for use in the method according to this embodiment of the invention can, for example, be produced by the methods described in British patent no. 1,121,308 and Belgian patent no. 783,449.

Any blocking group on the 4-carboxy group in compounds of formula II or IV is preferably a group which can be easily cleaved off in a later step in the reaction sequence and is advantageously a group containing 1-20 carbon atoms. Suitable carboxyl blocking groups are well known in the art and a list of representative groups is shown in Belgian Patent No. 783,449. Preferred groups include aryl-lower alkoxycarbonyl groups such as p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl and diphenylmethoxycarbonyl; lower alkoxycarbonyl groups, such as t-butoxycarbonyl, and lower halogenoalkoxycarbonyl groups, such as 2,2,2-trichloroethoxycarbonyl. The carboxyl-blocking group can then be removed by any of the suitable methods indicated in the literature, for example: acid- or base-catalyzed hydrolysis is applicable in many cases, as are enzyme-catalyzed hydrolyses.

The antibiotic compound prepared according to the present invention can be formulated for administration in any suitable manner in the same manner as other antibiotics.

The antibiotic compound produced according to the invention can be formulated for injection and can be found in unit doses in ampoules, or in multi-dose containers with added preservative. The compound can be used in the form of suspensions, solutions and emulsions in oily or aqueous carriers, and can contain auxiliaries such as suspending agents, stabilizers and/or dispersing agents. Alternatively, the active ingredient can be in powder form for processing with a suitable carrier, for example sterile, pyrogen-free water, before use.

In general, pharmaceutical mixtures may contain from 0.1%

and upwards, for example 0.1 - 99%, preferably from 10-60%, of the active compound, depending on the administration method. When the mixtures comprise dose units, each dose will preferably contain 50 - 1500 mg of the active ingredient. The dose used in the treatment of adults is preferably in the range of 500 - 4000 mg per day, depending on the route of administration and frequency of administration.

The compound can be administered in combination with other compatible therapeutic agents such as antibiotics, for example penicillins, or other cephalosporins or tetracyclines.

The following novel compounds are useful as intermediates in the preparation of the antibiotic compound of general formula I: diphenylmethyl (6R,7R)-7-amino-3-trichloroacetyl-carbamoyloxymethyl-ceph-3-em-4-carboxylate and its toluene- p-sulfonic acid salt, diphenylmethyl (6R,7R)-7-amino-3-carbamoyloxymethyl-ceph-3-em-4-carboxylate-toluene-p-sulfonic acid salt,

t-butyl (6R,7R)-7-[2-(fur-2-yl)-2-methoxyiminoacetamido]-3-hydroxymethyl-ceph-3-em-4-carboxylate (syn isomer)

(6R,7R)-7-amino-3-chloroacetylcarbamoyloxymethyl-ceph-3-em-4-carboxylic acid, and

(6R,7R)-7-amino-3-trichloroacetylcarbamoyloxymethyl-ceph-3-em-4-carboxylic acid.

The following examples illustrate the invention and the production of starting materials. Melting points were determined on a "Kofler" block.

A) MANUFACTURE OF STARTING MATERIALS

Production 1

a) Diphenylmethyl-(6R.7R)-7-(thien-2-vlacetamido)-3-trichloro-acetylcarbamoyloxymethyl- ceph-3-em-4-carboxylate.

Trichloroacetyl isocyanate (13.2 g, 70 mmol) was added to a stirred suspension of diphenylmethyl-(6R,7R)-3-hydroxymethyl-7-(thien-2-ylacetami^o)ceph-3-em-4-carboxylate ( 26.0 g, 50 mmol)

in anhydrous acetone (600 ml) at 20°C. The solid dissolved quickly and after the mixture had been stirred at 2o°C for 1

hr, it was cooled for 1 hr and a resulting solid was filtered off and washed with ether to give the desired compound (33.1 g, 93%), m.p. 183-184°CJ [a]<21> + 24° (c 0.95 in DMS0)J *inf<H> 235 nm (£l4»50°) °9 X infH 256 1101 (t 8, 82°) -IR, NMR and microanalytical data confirmed that the structure was

the one for the desired connection.

b) Diphenylmethyl-(6R, 7R)- 7- amino- 3- trichloroacetyl- carbamoyloxymethyl- ceph- 3- em- 4- carboxylate- toluene- p- sulfonic acid-

s everything.

Anhydrous pyridine (31 mL, 0.384 mol) was added to a solution of phosphorus pentachloride (20 g, 96 mmol) in dry dichloromethane

(300 ml) at 3°C. The suspension was stirred for 10 minutes at

3°C and diphenylmethyl-(6R,7R)-7-(thien-2-ylacetamido)-3-trichloro-acetylcarbamoyloxymethyl ceph-3-em-4-carboxylate (22.5 g, 32

mmol) was added. It was stirred by; about. 2°C for 1 hour.

The dark solution was slowly poured into a cold (0°C) water

free mixture of methanol (80 ml) and dichloromethane (200 ml) with maintaining the temperature below 5°C. The temperature of the solution was then allowed to rise to 23°C and after stirring the solution at this temperature for 1 hour, water (200 ml) was added. The organic layer was separated and washed with 2N sulfuric acid, water, sodium bicarbonate solution and water, dried over magnesium sulfate and evaporated in vacuo. The resulting oil was dissolved in ethyl acetate and a solution of toluene-p-sulfonic acid monohydrate (6.0 g, 31.5 mmol) in ethyl acetate was added. The combined solutions (ca. 350 mL) were poured into diethyl ether (ca. 1 L) and the resulting solid was filtered off and dried in vacuo to give the desired compound (17.2 g, 72%), m.p.

o

150 - 153°C; [α]p + 7.5° (c 0.82 in DMSO); 263 nm (£ 7.600) and A ^OH 267 nm ( t 7.350).

IR, NMR and microanalytical data confirmed that the structure was

the one for the desired connection.

Evaporation of the filtrate and trituration of the residue with ethanol gave unchanged starting material (3.2 g, 14.2%). c) Diphenylmethyl-(6R,7R)-7-amino-3-carbamoyloxymethyl 1- ceph- 3- em- 4- carboxylate- toluene- p- sulfonic acid salt. The toluene-p-sulfonic acid salt of diphenylmethyl-(6R,7R)-7-amino-3-trichloroacetylcarbamoyloxymethyl ceph-3-em-4-carboxylate (17.2 g, 22.7 mmol) was dissolved in a mixture of anhydrous methanol (900 ml) and acetyl chloride (45 ml) and allowed to stand at 20°C for 5 hours. Removal of the solvent under reduced pressure gave an oil which was dissolved in dichloromethane. This solution was shaken with aqueous sodium bicarbonate solution and then washed with water. The toluene-p-sulfonic acid monohydrate (4.3 g, 22.7 mmol) was added and the solvent was evaporated in vacuo. The residue was dissolved in hot isopropanol (approx. 150 ml) and the solution was poured into diisopropyl ether (approx. 600 ml). The precipitated solid was filtered off and dried in vacuo to give the desired compound (8.9 g, 64%), m.p. 110 - 112°C; [a] 21

-14° (c, 1.0 in CHCl.J; A E!;°H 259 nm (£6.120) and

>. FtoH. J majcs

X rjjr 227nm (£15,800) .

IR, NMR and microanalytical data confirmed the structure as that of the desired compound.

Manufacturing 2

Piphenylmethyl-(6R, 7R)- 7- amino- 3- carbamoyloxymethyl ceph- 3-em- 4- carboxy1at- toluene- p- sulfonic acid salt

A stirred suspension of phosphorus pentachloride (156 g, 0.75 mol)

in dry dichloromethane (1.5 L) was cooled in an ice bath and treated with pyridine (60.5 mL, 0.75 mol) at such a rate that the temperature of the mixture remained at ca. 20 - 25°C. The mixture was stirred and cooled to 8°C and Hiphenylmethyl -(6R,7R)-7-thien-2-yl)acetamido-3-trichloroacetylcarbamoyloxymethylceph-3-em-

4-carboxylate (354.5 g, 0.5 mol) was added in portions over 10 minutes. The mixture was stirred at approx. 8°C for 1.75 hours and then added over 10 minutes to a stirred mixture of butane-1,3-diol (225 mL, 2.5 mol) and dichloromethane (500 mL), precooled to -20°C /so that the temperature of the mixture was kept within the range -15 - -20°C. The dressing bath was removed and the mixture stirred at approx. -10°C for 20 minutes. Water (1

1) was added and the two-phase mixture was stirred for 30 minutes. The aqueous phase was extracted with dichloromethane (2 x 500 ml), and the organic phases were washed successively with 2N hydrochloric acid (1 L), combined and evaporated to a brown gum. The gum was dissolved in methanol (3.6 liters) and this solution was stirred and treated with saturated aqueous sodium bicarbonate solution (1.2 L) over a period of 10 minutes. The mixture

was reshuffled at approx. 20°C for 1.5 hours and a small amount of brown solid was removed by filtration. The yellow filtrate was concentrated in vacuo (bath temperature not above 40°C) to approx.

1.5 liters and water (1.5 1) was added. The resulting suspension was cooled for 1 hour and the yellow solid was filtered off, washed well with water, sucked as dry as possible and dried in vacuo at 40°C for 24 hours. The oily solid thus obtained, followed by toluene p-sulfonic acid monohydrate (81 g, 0.425 mol) was added to stirred chloroform (2 L). After several minutes, the toluene-p-sulfonic acid salt began to crystallize. Stirring was continued for an additional 30 minutes, after which the water was removed azeotropically in vacuo under continuous replacement of chloroform to maintain a volume of 2 liters. The suspension was cooled overnight and the product was filtered off, the slurry washed with chloroform (2 x 250 ml), filtered again, washed by replacing the chloroform used (250 ml) and dried in vacuo at 40°C to give the desired compound as an off-white crystalline solid (237.8 g, 74.1%); Amak

(EtOH) 262 nm (t 7.250); The NMR spectrum (Me 2 SO-d 6 ) indicated the presence of 0.25 mol chloroform.

Manufacturing 3

( 6R, 7R)- 7- amino- 3- carbamoyloxymethylceph- 3- em- 4- carboxylic acid Diphenylmethyl-(6R, 7R)-7- amino-3- carbamoyloxymethylceph-3-em-4-

carboxylate-toluene-p-sulfonic acid salt (300.0 g, 0.44 mol), solvated with approx. 0.6 mole of chloroform, was added in portions over 30 minutes to a stirred mixture of trifluoroacetic acid (300 ml) and anisole (300 ml) immersed in a water bath at 20°C. The temperature of the mixture rose from 23 to 28°C during the first 20 minutes but fell back to 26°C at the end of the addition. The golden-yellow solution was stirred for 1 hour as the temperature dropped to 21°C, and was then added to a stirred mixture of ethyl acetate (1.5 L) and water (1.5 L) immersed in an ice bath. The pH of the stirred mixture was adjusted to 3.8 over 10 minutes with ammonia solution (specific gravity 0.880) as the temperature rose to 38°C. The suspension was stirred and cooled to 10°C over 1.25 hours and filtered. The creamy solid was washed with water (750 mL) and ethyl acetate (4 x 200 mL) and dried in vacuo to give the desired compound (115.6 g, 96.2%); Amakg (pH6 phosphate) 265 nm ( E. 7.7 50),- purity by HPLC (high pressure liquid chromatography) 99.7%.

Microanalytical data confirmed that the structure was 'Jen for the desired compound.

Manufacturing 4

a) ( 6R, 7R )- 7-( R- 5- benzoylamino- 5- carboxypentanamido)- 3- hydroxymethyl c eph- 3- em- 4- carboxylic acid

A stirred solution of (6R,7R)-7-(R-5-amino-5-carboxypentane-amido)-3-hydroxymethylceph-3-em-4-carboxylic acid monopotassium salt (ca. 67% pure) (62.00 g, ca. 100 mmol) in water (300 mL) was cooled to +5° and treated with a solution of benzoyl chloride (17.4 mL, 150 mmol) in acetone (200 mL) over 25 minutes. The pH of the reaction mixture was maintained at 8.2 - 8.5 by controlled addition of 30% (w/v) aqueous tripotassium orthophosphate. The mixture was stirred for a further 10 minutes and then covered with ethyl acetate (140 ml) and the pH was lowered to 5.6 with orthophosphoric acid. The layers were separated and the aqueous part washed with more ethyl acetate (2 x 400 ml). The aqueous portion was diluted with water (21), covered with ethyl acetate (2 L), and the pH of the stirred mixture brought to 2.0 with orthophosphoric acid. The layers were separated and the aqueous layer extracted with additional ethyl acetate

(3 x 1500 ml). The combined extracts were washed with brine (800 mL), dried and concentrated in vacuo to a volume of 300-400 mL. The resulting slurry was stirred with ether (2 L) for 20 minutes and then filtered. The collected solid was washed with ether (2 x 250 mL) and dried in vacuo (1 mm) to give the desired compound as a white solid (54.95 g, 88.6% v/v), [a ]D° + 74° (c 1.00, dioxane); A (pH 6 buffer) 231 nm (E m 275), 266 nm (inflection, E m 145). The NMR spectrum (Me2SO-d6) showed the presence of approx. 20% lactone impurity and ethyl acetate (approx. 0.4 mol).

b) 6r, 7R)- 7-(R- 5- benzoylamino- 5- carboxypentanamido)- 3-chloroacetylcarbamoyloxymethylceph- 3- em- 4- carboxylic acid monosodium salt

The product from (a) above (25.46 g) was treated with a solution of chloroacetyl isocyanate (9.00 g, 75 mmol) in dry acetone (92 ml). The resulting solution was stirred for 25 minutes at approx. 20°C, then cooled to approx. 5°C over 5 minutes and treated with a solution of sodium 2-ethyl hexanoate (8.47 g, 51 mmol) in acetone (51 mL). The crystalline suspension was stirred at approx. 5°C for 5 minutes, and the solid was collected by filtration, washed with acetone (80 mL) and ether (250 mL), then dried in vacuo (1 mm) to give the desired compound (27.23 g , 107.0% v/v), [et]<20> + 72.0° (c, 1.00, 3% aqueous NaHCO 3 ); Amax (pH 6 buffer) 227 nm (E*^ 249) 261 nm (inflection, e}^ 0 105). NMR spectrum (Me~S0-dc) showed the presence of approx.

' limb £ b

35% lactone impurity and chloroacetamide (about 1.0 mol).

c) ( 6R, 7R)- 7- amino- 3- chloroacetylcarbamoyloxymethylceph- 3-em- 4- carboxylic acid

A suspension of the product from (b) above (24.77 g) in dry methylene chloride (3 20 ml) under nitrogen was cooled to ca. 10°C with stirring. Pyridine (17.60 mL, 218.0 mmol), then dichlorodimethylsilane (16.80 mL, 139.2 mmol) was added and the slightly brown suspension was stirred at ca. 20°C for 20 minutes and was then cooled to -17°C. Phosphorus pentachloride (104 g, 52.0 mmol) was added and the mixture was stirred at -17° - -23°C for 2 hours. Phyridin (6.48 mL, 80.4 mmol) was added and the mixture was added to methanol (104 mL + 20 mL washes, precooled to

-35°C at such a rate that the temperature of the stirred solution did not exceed -10°C. The stirred solution got

reach +2°C over 25 minutes, then water (88 ml) was added and the pH of the mixture was adjusted from 0.6 to 3.8 with aqueous ammonia solution (specific gravity 0.880). The solid was cooled for 1 hour and then filtered. The solid was washed successively with 50% v/v aqueous methanol (100 mL), methanol (80 mL), and methylene chloride (40 mL), then dried in vacuo (1 mm) to give the desired compound as a creamy powder (6.86 g, 27.7% v/v), [α]£<9> + 48° (c, 1.04, Me 2 SO );

<X>max (pH 6 Puffer) 237 >5 nm (Elcm 149) ' 261'5 (E15n 145) ••

B) EXAMPLES

Example 1

a) piphenylmethyl-(6R,7R)-3-carbamoyloxymethyl-7-f 2-(fur-2-yl)-2- methoxyiminoacetamido | ceph- 3- em- 4- carboxylate

(syn isomer)

Method (i)

Crude diphenylmethyl-(6R,7R)-7-amino-3-carbamoyloxymethylceph-3-em-4-carboxylate-toluene-p-sulfonic acid salt obtained from the corresponding 3-trichloroacetylcarbamoyloxymethyl compound (25.0 g, 0.33 mol) was dissolved in a mixture of ethyl acetate and aqueous sodium bicarbonate solution. The • organic layer was separated, washed with water, dried over magnesium sulfate and evaporated on a rotary evaporator to give diphenylmethyl-(6R,7R)-7-amino-3-carbamoyloxymethyl ceph-3-em-4-carboxylate (11.5 g, 0.262 mol, 77%) as a foam.

2-(Fur-2-yl)-2-methoxyiminoacetic acid (syn isomer) (5.32 g, 0.312 mol) in dry dichloromethane (100 mL) was added to a solution of this amine in dichloromethane (50 mL) cooled to 3°C, followed 10 minutes later by a solution of DL-dicyclohexylcarbodiimide (6.5 g, 0.312 mol) in dichloromethane (30 mL). The reaction mixture was stirred in an ice bath for 45 minutes, during which time a solid (presumably N,N'-dicyclohexyl urea) crystallized out. This was filtered off and discarded, and the filtrate was washed with aqueous sodium bicarbonate solution and water, dried. over magnesium sulfate and evaporated to dryness. The residue was triturated with ethanol to give a crude product (10.6 g) which was purified by chromatography on silica gel (1 kg). Elution with 10% acetone in dichloromethane removed non-polar impurities and fractions eluted with 20% acetone in dichloromethane gave the desired compound (4.8 g, 31%), m.p. 199 - 202°c$ UJ^<1>+ 14° (c, 1.0 in DMSO); A EtOH 2?? nm (£ 18}600) and \EtOH 270 nm (£i7 9oo).

IR, NMR and microanalytical data confirmed the structure to be that of the desired compound.

Method (ii)

Triethylamine (1.86 g, 18.4 mmol) was added to a dichloromethane solution (35 mL) of 2-(fur-2-yl)-2-methoxyiminoacetic acid (syn isomer) (3.1 g, 18.4 mmol). After cooling this solution in an ice bath for 5 minutes, oxalyl chloride (1.57 mL, 18.4 mmol) and one drop of N,N-dimethylformamide were added. After 1/2 hour, the solvent was removed under reduced pressure and the solid residue was dried for 1 hour in vacuo. Anhydrous ether (150 mL) was added to dissolve the acid chloride that had formed,

and the insoluble triethylamine hydrochloride (2.5 g) was filtered off. The ether was evaporated on a rotary evaporator and the oily residue was redissolved in dichloromethane.

Diphenylmethyl-(6R,7R)-7-amino-3-carbamoyloxymethylceph-3-em-4-carboxylate-toluene-p-sulfonic acid salt (8.9 g, 14.7 mmol) was dissolved in anhydrous dichloromethane. This solution was shaken with aqueous sodium bicarbonate solution, washed with water and dried over magnesium sulfate. To this solution of the free amine was added the dichloromethane solution of 2-(fur-2-yl)-2-methoxyiminoacetyl chloride (syn isomer) and propylene oxide (5 ml). After 10 minutes, a crystalline solid (1.1 g) was filtered off, which was subsequently identified as diphenylmethyl-(6R,7R)-7-amino-3-carbamoyloxymethyl ceph-3-em-4-carboxylate hydrochloric acid salt. The filtrate was washed with 2N sulfuric acid, water, aqueous sodium bicarbonate solution and water and was dried over magnesium sulfate and evaporated to dryness to give the desired compound (2.5 g, 30.5%), which was similar in physical properties to the product according to method (i) in front.

b) Sodium-(6R.7R)-3-carbamoyloxymethyl-7-f 2-(fur-2-vl)-2-methoxyiminoacetamido| ceph-3-em-4-carboxylate (syn isomer)

Trifluoroacetic acid (20 mL) was slowly added to a mixture of anisole (5 mL) and diphenylmethyl-(6R,7R)-3-carbamoyloxymethyl-7-[2-(fur-2-yl)-2-methoxyiminoacetamido]eeph-3 -em-4-carboxylate (syn isomer) (4.7 g, 8 mmol) which had been cooled in an ice bath. The flask was shaken occasionally over the course of the next 10 minutes to ensure complete dissolution of the solid.

It was then taken up in the ice bath and excess trifluoroacetic acid was removed on a rotary evaporator. Trituration of the residue with ethyl acetate (5 mL) gave (6R,7R)-3-carbamoyloxymethyl-7-[2-(fur-2-yl)-2-methoxyiminoacetamido]ceph-3-em-4-carboxylic acid (syn isomer ) (3.3 g, 94%) as a solid, which was filtered off and washed with diethyl ether.

The free acid was dissolved in acetone and a slight excess of sodium 2-ethyl hexanaphthoate in acetone (8.0 ml of a molar solution) was added. After the reaction mixture was stirred at 0°C for 2 hours, the desired salt (2.3 g, 73%) was filtered off. This was combined with another batch of the desired salt (0.8 g) and purified by washing an aqueous solution (250

ml) with ether (2 x 100 ml, 1 x 50 ml). The aqueous solution was freeze-dried to give sodium 6R,7R)-3-carbamoyloxymethyl-7-(fur-2-yl)-2-methoxyiminoacetamidoJceph-3-em-4-carboxylate (syn isomer) (2.66 g). , 1752 (azetidin-2-one) and 1652 and 1600 cm<-1 >(carboxylate)5 r(Me2S0-d6) 0.24 (d,J8Hz, CONH), 2.12 (d,j2Hz, furyl C5- H), 3.25 and 3.30 (m, furyl C3-H and C4-H), 3.44 (broad s, C0NH2), 4.34 (dd, J 5 and 8Hz, C7-H), 4 .92 (d, J4.5HZ, C6-H), 5.15 (q, Jl3Hz C3-CH2) , 6.07 (s, N0CH3) and 6.58 (q, J 18Hz, <C>2-< H>2)

(found: C 42.0$ H 3.8; N 12.1; S 7.2.

C16H15N4Na08S•°"5H2° (455>37) calculated

C 42.2,- H 3.35$ N 12.3$ S 7.0%) .

Example 2

(6R,7R)-3-carbamoyloxymethyl-7-[2-fur-2-yl)-2-methoxyimino-acetamido]ceph-3-. em- 4- carboxy 1 acid ( syn- isomer)

A stirred mixture of N,N-dimethylacetamide (75 mL), acetonitrile (75 mL), triethylamine (42 mL, 0.3 mol) and (6R,7R)-7-amino-3-carbamoyloxymethylceph-3-em-4 -carboxylic acid (16.4 g, 0.06 mol) was immersed in an ice bath and water (10 mL) was added. The mixture was stirred at 0-2°C for 45 minutes, the solid slowly dissolving to give a yellow solution.

Meanwhile, a stirred suspension of phosphorus pentachloride (14.99 g, 0.072 mol) in dry dichloromethane (150 mL) was cooled to 0°C and N,N-dimethylacetamide (27.5 mL) was added. The resulting solution was recooled to -10°C and 2-(fur-2-yl)-2-methoxyiminoacetic acid (syn isomer) (12.17 g, 0.072 mol) was added. The mixture was stirred at -10°C for 15 minutes and crushed ice (35 g) was added. The mixture was stirred at 0°C for 10 minutes, after which the lower dichloromethane phase was added over 10 minutes to the cephalosporin solution prepared above, cooled to -10°C so that the reaction temperature rose steadily to 0°C. The mixture was stirred at 0 - 2°C for 1 hour, after which the cooling bath was removed and the reaction temperature allowed to rise to 20°C

in the course of 1 hour. The reaction mixture was then added slowly to 2N hydrochloric acid (100 ml) diluted with cold water (1.15 L) at 5°C. The pH of the two-phase mixture was adjusted below 2 with 2N hydrochloric acid (10 mL), and the mixture was stirred and recooled to 5°C. The precipitated solid was filtered, washed with dichloromethane (100 mL) and water (250 mL), and dried in vacuo at 40°C overnight to give the desired compound (22.04 g, 86.6%). [a]<20> + 58° (c, 1.08, Me2S0) 5 (PH 6 phosphate buffer) 274 nm (£.17,500)* \aks (<N>ujol) 3480, 3440, 3367,

3255 and 3133 (bound NH and NH2) , 2725 and 2 5 90 (CO^) , 1760 (azetidin-2-one) , 1728, 1712 and 1698 (0C0NH2 and CO^) , 1655 and 1530 cm"<1> ( C0NH)5 T(Me2SO-d6) 0.25 (d, J 8 Hzj CONH) , 2^.8

(s, furyl C5-H), 3.28 and 3.4 (m, furyl C4~H and C3-H), 3.42

(s, C0NH2, 4.19 (dd, J 8 and 5 Hz$ C7-H), 4.80 (d, J 5 Hzj C6-H) , 5.06 and 5.39 (q, J 13 Hzj C3 ~CH2) , 6.09 (s; NOCH3) ,

6.44 (collapsed q; C2-H2) and 7.99 (0.03 mol CH3CON(CH3)2).

Example 3

a) ( 6R, 7R)- 7-( R- 5- benzoylamino- 5- carb o xypentanamido)- 3- hydroxymethylceph- 3- em- 4- carboxylacid monoquinoline- s alt monohydrate

A stirred solution of (6R,7R)-7-(R-5-amino-5-carboxypentane-amido)-3-hydroxymethylceph-3-em-4-carboxylic acid, monopotassium salt (18.45 g, 30 mmol) in water (93 mL) was cooled to 0-5°C (ice/water bath) and treated with a solution of benzoyl chloride (5.19 mL, 45 mmol) in acetone (63 mL) over 25 minutes.

The pH of the reaction mixture was maintained at pH 8.5 0.1) by controlled addition of 30% (w/v) aqueous tri-potassium orthophosphate (approx. 100 ml). The mixture was stirred for an additional 5 minutes, covered with ethyl acetate (150 mL) and the pH was then lowered to 5.6 with orthophosphoric acid. The layers were separated and the aqueous portion washed with additional ethyl acetate (2 x 300 mL).

The combined washings were extracted with water (200 mL). The combined aqueous portion and washings were diluted with water (600 ml), covered with ethyl acetate (600 ml), and the pH of the stirred mixture was adjusted to 2.0 with orthophosphoric acid. The organic layer was separated and quinoline (10.64 mL, 45 mmol) in ethyl acetate (25 mL) was added with stirring to give a white precipitate. The aqueous portion was extracted with additional ethyl acetate (3 x 300 mL) and these were added to the suspension containing quinoline. The mixture was stirred for 1 hour at approx. 18°C and then concentrated in vacuum to approx. 500 ml. Ether (900 mL) was added with stirring and after 30 min the solid was collected by filtration, washed with ether (5 x 200 mL) and dried in vacuo (1 mm) to give the desired compound as a white powder ( 19.20 g, 104.1% w/w ), [oc]i8 + 78° (c1, °/1.00, dioxane) , ^ ks (pH 6 buffer) 258 nm (inflection, E^/0cm 185 ). IR and NMR data confirmed that the structure was that of the desired compound containing approx. 15% lactone contamination and trace amounts of ether and ethyl acetate.

b) ( 6R, 7R)- 7-( R- 5- benzoylamino- 5- carboxypentanamido)- 3-trichloroacetviccarbamoyloxymethyl ceph- 3- em- 4- carboxylic acid- monoquinoline salt

The product from (a) above (4.24 g, equivalent to 7 mmol) was treated with dry dioxane (100 ml) in which it partially dissolved. To the stirred mixture was added trichloroacetyl isocyanate (2.90 mL, 24.5 mmol). The resulting solution was stirred for 30 minutes and then cleared by filtration and evaporated in vacuo to give a yellow foam. This was dissolved in acetone (approx. 10 ml) and poured into stirred isopropyl ether (approx. 100 ml). The resulting white precipitate was collected by filtration and dried in vacuo (1 mm) to give the desired compound as a white powder (6.26 g, 147.8% v/v). NMR data confirmed the structure to be that of the desired compound and also showed the presence of lactone (ca. 22%), isopropyl ether

(0.75 mol), dioxane (0.2 mol) and a small amount of acetone.

c) ( 6R, 7R)- 7- amino- 3- trichloroacetylcarbamoyloxymethyl- ceph- 3- em- 4- carboxylic acid

A solution of the product from (b) above (4.77 g, equivalent to 6 mmol) in dry methylene chloride (40 ml) under nitrogen was cooled to ca. 10°C with stirring. Pyridine (2.20 mL, 27.3 mmol) and then dichlorodimethylsilane (2.10 mL, 17.4 mmol) were added and the brown solution was stirred at ca. 17°C for 20 minutes and then cooled to -17°C. Phosphorus pentachloride (1.355 g, 6.5 mmol) was added and the mixture was stirred at ca. -16°C for 2 hours. Pyridine (0.81 mL, 10 mmol) was added and the mixture was added to methanol (13 mL + 2.5 mL washes, pre-cooled to -35°C) at such a rate that the temperature of the stirred solution did not exceed -10°C. The stirred solution now reached +9°C over the course of 25 minutes, water (11 ml) was then added and the pH of the mixture was brought from 0.3 to 3.8 with ammonia solution (specific gravity 0.880). The resulting two-phase mixture containing a precipitated solid was stirred for 1 hour and then filtered. The solid was washed successively with 50% v/v aqueous methanol (12 mL), methanol (10 mL) and methylene chloride (5 mL) and then dried in vacuo (1 mm) to give the desired compound as a creamy powder ( 1.22 g, 25.6% v/v) , [cc]D +44° (c, 1.02, M<e>2SO); max (pH ^ buffer) 240 nm (E^<%> 133), 263 nm (E<1%> 140). NMR data confirmed that the structure was that of the desired compound.

d) (6R,7R)-3-carbamoyloxymethyl-7-[2-(fur-2-yl)-2-methoxy-iminoacetamido | ceph- 3- em- 4- carboxylic acid ( syn isomer)

Phosphorus pentachloride (4.5 g, 21.5 mmol) was dissolved in dry methylene chloride (90 mL) and cooled with stirring to -15°C. N,N-dimethylacetamide (9 mL) was added slowly, keeping the temperature below -10°C, and the mixture was stirred for 10 minutes. 2-(fur-2-yl)-2-methoxyiminoacetic acid (syn isomer) (3.66 g,

21.5 mmol) was added and the mixture stirred at -15°C for 15 minutes. Crushed ice (18 g) was carefully added so that the temperature of the mixture did not exceed -7°C. The mixture was stirred for 10 minutes, and the organic portion was separated and added dropwise to a solution of (6R,7R)-7-amino-3-

trichloroacetylcarbamoyloxymethylceph-3-em-4-carboxylic acid (7.52 g, 18 mmol) in dry methylene chloride (90 mL) and triethylamine (5.5 mL, 40 mmol), pre-cooled to -10°C. acid chloride solution

ning was added over the course of 20 minutes, the temperature of the reaction mixture being maintained between -10° and -8°C. The mixture was then stirred for 80 minutes, during which time the temperature was allowed to rise to +3°C, and methanol (6 mL) was added. After a further 5 minutes of stirring, the solution was extracted with 3% w/v aqueous sodium bicarbonate (2 x 120 ml) and water (150 ml). The combined extracts were allowed to stand at approx. 2o°C for 3.5 hours, and was then washed with ethyl acetate (100 ml) and acidified to pH 1.5 with concentrated hydrochloric acid. The resulting deposited oil was extracted into ethyl acetate (2 x 300 mL). The combined organics were washed with water (2 x 100 mL), dried (MgSO 4 ) and evaporated in vacuo to give a yellow solid (7.1 g) which was triturated with ether (150 mL), filtered and dried in vacuo (1 mm) to give the desired compound as a pale yellow solid (5.20 g, 68.2% of theory), \iaks pH 6 Buffer) 275 nm (Ei<%>cm 385) * IR and NMR data confirmed that the structure was that of the desired compound containing traces of ether.

EXAMPLE 4

(6R.7R)-3-carbamoyloxymethyl-7-\2-(fur-2-yl)-2-methoxyimino-acetamido| ceph-3-em-4-carboxylic acid (syn isomer) N,N-dimethylacetamide (1.5 mL) was added to a solution of phosphorus pentachloride (750 mg, 3.6 mmol) in anhydrous dichloromethane (15 mL) cooled to -10°C. After 10 minutes, 2-(fur-2-yl)-2-methoxyiminoacetic acid (syn isomer) (612 mg, 3.6 mmol) was added to the resulting suspension which soon gave a clear solution and was stirred at -10°C for 15 minutes. Ice (3 g) was added and after 10 minutes the layers were allowed to separate in a dropping funnel. The organic phase was slowly (over the course of 5 minutes) poured into a cooled (-10°C) solution of (6R,7R)-7-amino-3-chloroacetylcarbamoyloxymethylceph-3-em-4-carboxylic acid (1.05 g, 3 mmol) in dichloromethane (15 mL) containing triethylamine (0.9 mL, 6.5 mmol). Methanol (1 mL) was added after 40 min, and 5 min later the reaction mixture was extracted

twice with 3% w/v aqueous sodium bicarbonate solution (150 ml). The aqueous extract was washed with ethyl acetate (25 ml) and allowed to stand at 20°C for 4 hours, the solution was washed twice with ethyl acetate, acidified with 2N hydrochloric acid and extracted 3 times with ethyl acetate. The combined organic layers were dried over magnesium sulfate, decolorized with activated charcoal, and evaporated in vacuo to give a pale yellow solid (1.15 g, 87%). This was washed with diethyl ether and filtered off to

afford the desired compound (0.91 g, 71%) ,• ApH 6 274 nm (£.17,300). The IR and NMR spectra of the product matched those of an authentic sample.

Example 5

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

Method (i)

Acetone (750 mL) was cooled to 0°C and treated with trichloroacetyl isocyanate (28.8 mL, 240 mmol) and the solution recooled to 0°C. (6R,7R)-7-[2-(fur-2-yl)-2-methoxyiminoacetamido]-3-hydroxymethylceph-3-em-4-carboxylic acid (syn-i some)

(45.6 g, 120 mmol) was added to the stirred isocyanate solution in portions over 5 minutes so that the reaction temperature did not exceed 6°C. The yellow solution was stirred for an additional 15 minutes and methanol (4.5 mL) was added. The solution was concentrated to 60 ml and the concentrate dissolved in methanol (750 ml). Sodium bicarbonate (45.3 g, 540 mmol) in water (600 mL) was added, followed by activated charcoal (4.5 g), and the resulting suspension was stirred at room temperature for 2 hours. The charcoal was removed by filtration through diatomaceous earth

and the pale yellow filtrate was adjusted to pH 4.5 by the addition of dilute hydrochloric acid, the solution was concentrated to half volume under reduced pressure and an equal volume of water was added. The pH was adjusted to 2.0 with dilute hydrochloric acid and the product was isolated by filtration, washed with water (3 x 150 mL) and dried at 40°C for 16 h in vacuo to give the desired compound (37.46 g, 73 .5%) ,- [e<t]20><+> 63.7° (c 1.0; 0.2 M pH 7 phosphate buffer)<;> Amak (pH 6 phosphate buffer) 274 nm (£ 17,600).

IR, NMR and microanalytical data confirmed the structure to be that of the desired compound.

Method (ii)

A slurry of (6R,7R)-7-[2-(fur-2-yl)-2-methoxyiminoacetamido J-3-hydroxymethylceph-3-em-4-carboxylic acid (syn isomer)

(3.81 g, 9.55 mmol) in dichloromethane (70 mL)/tetrahydrofuran (25 mL) at 5°C was treated with dichloroacetyl isocyanate (2.6 mL, 25 mmol). The reaction mixture was then processed in the same way

manner as in method (i) to give the desired compound (3.36 g, 83.0%); [a]^<0> + 63°-, Amax 273.5 nm (£ 17,800) j with similar IR and NMR spectra as the product from method (i).

Method (iii)

A slurry of (6R, 7R)-7-[ 2-(fur-2-yl)-2-methoxyiminoacetami-do]-3-hydroxymethylceph-3-em-4-carboxylic acid (syn-isome) (19 .05 g, 50 mmol) in dry acetonitrile (250 ml) was treated at between 5 and 10°C with chlorosulfonyl isocyanate (6.33 ml, 75 mmol) in acetonitrile (80 ml).

The reaction was stirred at between 0 and 5°C for 10 minutes and water (50 mL) was added. The mixture was stirred at approx. 2o°C, and after 20 minutes a white crystalline solid separated. Evaporation and filtration afforded the desired compound (18.17 g, 85.7%); [o]q° + 62.5°; ^max 273.5 nm (£.17,820); with similar IR and NMR spectra as the product under method (i). Another yield (1.88 g, 8.6%) of the product, with similar constants, was obtained by evaporation of the mother liquors.

Example 6

Form I sodium-(6r,7R)-3-carbamoyloxymethyl 1-7- i 2-(fur-2-yl)-2- methoxyiminoacetamido lceph-3-em-4- carboxylate (syn isomer)

Method (i)

(6R,7R)-3-carbamoyloxymethyl-7-[2-(fur-2-yl)-2-methoxyimino-acetamido]ceph-3-em-4-carboxylic acid (syn isomer) (100 g) in N, N-dimethylacetamide (400 mL)/acetone (1 L) was treated with sodium 2-ethyl hexanoate (40 g) in acetone (200 mL). The mixture

was inoculated and stirred at room temperature for 1 1/4 hours. the product was filtered off, washed with acetone (500 mL) and then slurried with acetone (3 x 300 mL) and thoroughly slurried with ether to give the desired compound (101.4 g, 92.5%) containing (after equilibrium in the atmosphere) 0.65 mol equivalents of water. The product has [a]D + 61° (c 0.5, pH 4.5 phosphate buffer) and <A>max <2>73 nm Ef°cm 412 <H2°> '

IR and NMR data confirmed that the structure was the same as that of the title compound, with the IR spectrum indicating that the compound was in the salt form.

Method (ii)

The procedure of method (i) was repeated except that the cephalosporin acid was first dissolved in N,N-dimethylformamide/methanoate denatured ethanol instead of N,N-dimethylacetamide/acetone to give the desired compound (80%)

which in terms of properties is similar to the product under method (i). The IR spectrum indicated that the compound was the form I salt.

Method (iii)

(6R,7R)-3-Carbamoyloxymethyl-7-[2-(fur-2-yl)-2-methoxyimino-acetamidojceph-3-em-4-carboxylic acid (syn isomer) (4.24 g, 10 mmol) was dissolved in N,N-dimethylacetamide (20 ml) which had been dried over molecular sieves (Linde 4A) for 24 hours. To this was added a solution of sodium 2-ethyl hexanoate (2.0 g, 12 mmol - recrystallized from dioxane and dried over phosphorus pentoxide) in ethyl acetate (80 ml) which had been dried over molecular sieves (Linde 4A) for 24 hours. The solution was stirred in a closed vessel for approx. 15 minutes until crystallization began and then cooled to 4°C for 1 hour. The product was filtered, washed with dry ethyl acetate (^100 mL) and, while still moist from this solvent, transferred to an oven and dried at 20°C in vacuo over phosphorus pentoxide overnight to give the desired compound (3.89 g, 87%).

The IR and NMR spectra of the product agreed with those of an authentic sample.

Example <7>

Form II sodium-(6R,7R)-3-carbamoyloxymethyl-7-f2-(fur-2-yl)-2-methoxyiminoacetamido | ceph-3-em-4-carboxylate (syn isomer)

Method (i)

Charcoal (0.2 g) was added to a solution of (6R,7R)-3-carbamoyloxymethyl-7-[2-(fur-2-yl)-2-methoxyiminoacetamido]-ceph-3-em-4-carboxylic acid (syn isomer) (4.00 g, 9.42 mmol) in a mixture of acetone (132 mL) and water (1.33 mL). The suspension was stirred for 30 minutes and filtered through a layer of diatomaceous earth, the filter layer being washed with acetone (10 ml). A filtered solution of sodium 2-ethyl hexanoate (1.66 g, 10 mmol) in acetone (20 mL) was added over 1 hour to the stirred filtrate. The resulting suspension was stirred for an additional 10 min and the white solid was filtered off, washed with acetone (2 x 25 mL) and dried in vacuo to give the desired compound (4.06 g, 93.0%). - [ a + 60° (c, 0.91, H20)"> Anax (H20) 274 ^ (É-17-4^0)?

(Found: C 41.0, 41.2: H 3.45, 3.6$ H 12.3 12.4-, Na 5.2$

S 6.6, 6.85; H2O 2.7,

C16H15N4Na°8S' 0-7 H2° (459>°) theoretical: C 41.8; H 3.6;

N 12.2, • Na 5.0; S 7.0; H20 2.7%),-

Purity by HPLC 99.4%. The NMR spectrum of the product resembled that of an authentic sample, and the IR spectrum indicated that the product was the form II salt.

Method (ii)

(6R ,7R )-3-carbamoyloxymethyl-7-[2-fur-2-yl)-2-methoxyiminoacet-amido]ceph-3-em-4-carboxylic acid (syn isomer) (16.98 g, 40 mmol ) was added to a stirred mixture of acetone (333 mL) and water (8.5 mL). After treatment with charcoal and filtering this

solution, sodium 2-ethyl hexanoate (7.32 g, 44 mmol) in acetone (85 mL) was slowly added over 1 hour. The reaction mixture was stirred for 15 minutes, filtered and the product was washed with acetone (.2 x 65 mL) and dried in vacuo at 20°C overnight to give the desired compound (17.95 g, 98.5%) , which contains 0.5 mol H20. The NMR spectrum of the product agreed over-

similar to that of an authentic sample, and the IR spectrum indicated that the product was the form II salt.

Example °

FQrm III sodium-(6R,7R)-3- carbamoyloxymethyl-7- r2-(fur-2-yl)-2- methoxyiminoacetamido| ceph-3-em-4-carboxylate (syn isomer)

Sodium -(6R,7R)-3-carbamoyloxymethyl-7-[2-(fur-2-yl)-2-methoxyiminoacetamido]ceph-3-em-4-carboxylate (syn isomer) (4.0 g ) was dissolved in water (20 mL). Methanol^denatured ethanol (20 mL) and dioxane (160 mL) were added and the solution was filtered and then set aside at 4°C to crystallize. The very white needle-shaped crystals were filtered off, washed with dioxane (100 mL) and, while still moist with dioxane, transferred to an oven and dried at 20°C in vacuo overnight to give the desired compound (3, 96 g, 78.5%). The IR and NMR spectra of the product agreed with those of an authentic sample.

Example 9

Form IV sodium (6R.7R)-3-carbamovloxmethyl-7-f 2-(fur-2-yl)-2- methoxyiminoacetamido| ceph-3-em-4-carboxylate (syn isomer)

Samples of form I and form III sodium 3-carbamoyloxymethyl-7-[2-(fur-2-yl)-2-methoxyiminoacetamido]ceph-3-em-4-carboxylate (syn isomer), prepared according to method (iii ) in example 6 acc. Example 8, was exposed to humidity (75% relative humidity) for 3 days to give the desired compound. The IR and NMR spectra of the products agreed with those of an authentic sample. Karl-Fischer water analysis gave 4.0 and 3.85% respectively (1 mol H20 is equivalent to 3.9%).

The activity of the present compound is compared with the known compounds cephalothin, cephaloridine and cefazolin, which in the following table are marked respectively with A,

B and C.

In the table, compounds D and E indicate closely related syn compounds known from Norwegian patent application no. 1705/72.

The activity for the present connection and the activity for

the compounds shown above appear from the following tables A-H.

Table A shows the in vitro activity of the indicated compounds for a representative selection of test organisms, measured using the agar dilution technique. The results in the table are given as the minimum inhibitory concentration in µg/ml, the organisms were used at a level of 10J organisms/ml.

The organisms examined in Table A were chosen because of the wide spread in sensitivity to cephalosporin antibiotics. Thus, Staph. aureus 663E very sensitive to all active cephalosporins, while strains 1414E and 853E are pene-cillinase-producing and generally more resistant and strain 1613E is methicillin-resistant and usually not very sensitive to cephalosporins. E. coli strains 573E, 851E and 1507E are representative of strains that are normally sensitive to cephalosporins, while strain 1193E carries the transferable resistance factor RTEM which causes the formation of a type 3-lactamase of class III (M.H. Richmond & R.B. Sykes, 19 73, Advances in Microbial Physiology, 9_, 31) . S. typhimurium 804E, Pr. mirabilis 431E and H. influenzae 1184E are sensitive strains for the respective species. E.cloacae 1321E and K. aerogenes 1522E are not (3-lactamase-producing mutants of normally resistant enzyme-producing strains. S.marcescens 1324E, Providence spp. 1497E and H.influenzae 1788E are all ampicillin-resistant, (3-lactamase -producing organisms It will be understood that any new cephalosporins must show activity against normally sensitive organisms, and the more activity they show against the more difficult-to-control, p<->lactamase-producing strains, the greater their therapeutic potential.

It will be seen that the compound produced according to the present invention exhibits an activity comparable to that of the known compounds when it comes to normally sensitive organisms, but is significantly more active in B-lactamase-producing organisms.

Table B shows in vitro results for the compound produced according to the present invention and the compounds A, B and C.

It will, among other things, appear:

(i) S. aureus. These strains were chosen because of their penicillin sensitivity and further to investigate whether any of the compounds exhibited poor activity. All compounds showed a high activity. (ii) Klebsiella. It appears from the results that the compound produced according to the invention is very effective. (iii) Proteus. It will be seen that the compound produced according to the invention exhibits significantly better activity compared to known ones against the (3-lactamase^-producing organisms. (iv) E.coli. It can again be seen that the compound produced according to the invention is the most active against the 3-lactamase-producing strains. (v) Salmonella and Shigella. These are both (3-lactamase-resistant organisms due to the R factor which they carry.

(vi) Citrobacter and Enterobacter. These connections have

a tendency to be highly resistant to known cephalosporins, but as can be seen from Table B, the compound produced according to the present invention is very active.

Table C shows the activity against different strains of Haemophilus influenzae and it will be clear that the activity of the compound produced according to the invention is comparable to the known compounds when it comes to non-β-lactamase-producing strains and significantly more active against 3- lactamase-producing strains.

Table D shows the activity against strains of Neisseria for representative strains and it appears that the compound produced according to the invention exhibits a very high activity against the strains of Neisseria shown.

Tables E and F show the activity of the compound prepared according to the invention compared to known compounds at two levels, namely 10 5 and 10 7 organisms/ml. As can be seen, the activity level for the compound according to the invention varies little with the number of organisms per ml, which is not the case for the comparison compounds. Table F shows the corresponding effect for certain gram-negative organisms.

As previously mentioned, the compound produced according to the invention exhibits the advantageous property that it is not affected by esterases, this in contrast to the compounds known from Norwegian application no. 1705/72. Table G shows the effect of esterases on the compound produced according to the invention compared to compounds D and E, known from the aforementioned Norwegian application. It will appear from the table that the compound produced according to the invention is not affected by esterases, while more than 50% of the known compounds are broken down within 4 hours. As sources for esterase, homogenized rat liver was used.

Finally, table H shows in vivo results for the compound produced according to the present invention and the compound known from example 53 in Norwegian application no. 1705/72.

Claims (1)

1. Analogy procedure for the preparation of a therapeutically effective cephalosporin derivative with the general one formula where the compound is the syn isomer or exists as a mixture of syn and anti isomers containing at least 90% of the syn isomer, as well as non-toxic salts and biologically acceptable esters thereof, characterized by that a compound with the general formula wherein R<11> is hydrogen or a carboxyl blocking group, and R 12 is hydrogen or an N-protecting group, or an acid addition salt or N-silyl derivative thereof, is reacted with an acid with the general fornel or a reactive derivative thereof, or (B) reacts with a compound of the general formula 11 wherein R is as defined above, with a carba-moylating agent, after which any carboxyl-blocking or N-protecting group is removed, if necessary the anti-isomer is separated and optionally the compound of formula I is converted into a biologically acceptable ester or into a non-toxic salt, preferably the sodium salt.