WO1995034567A1

WO1995034567A1 - Cyclohexanocephalosporins and their preparation procedure

Info

Publication number: WO1995034567A1
Application number: PCT/EP1995/002276
Authority: WO
Inventors: Gianfranco Cainelli; Achille Umani Ronchi; Sergio Sandri; Michele Contento; Irene Resta; Marco Da Col; Leone Dall'asta
Original assignee: Biochimica Opos Spa
Priority date: 1994-06-14
Filing date: 1995-06-13
Publication date: 1995-12-21
Also published as: AU2881495A

Abstract

New 9,12-epoxycyclohexano[3,4-c]cephalosporins of formula (I) are prepared starting from the corresponding ester obtained reacting a 7-phenylacetamido-3-cephem-4-carboxylic acid having in position 3 a leaving group, with furan, optionally 2-substituted or 2,5-disubstituted in the presence of a base, subsequent deacylation and possible reacylation in 7, elimination of the ester group of the obtained product and possibly salification or esterification, giving a pharmaceutically acceptable compound. The new products thus obtained are antibiotics with an excellent activity.

Description

"CYCLOHEXANOCEPHALOSPORINS AND THEIR PREPARATION PROCEDURE"

The present invention refers to new cyclohexanocephalosporins. More particularly, the invention concerns 9,12-epoxycyclohexano[3,4- c]cephalosporins of formula I

in which X represents hydrogen or an acyl group Ac; R represents hydrogen or a carboxy protecting group; X' and X", identical or different, represent independently hydrogen, alkyl, hydroxyalkyl, acyloxyalkyl, alcoxyalkyl, aryloxyalkyl, arylalcoxyalkyl, cycloalkyl. oxyalkyl, cycloalkylalcoxyalkyl, alcoxyalcoxyalkyl, alkyloxycarbonyl, alkyl, aryloxycarbonylalkyl, aralkyloxycarbonylalkyl, aminocarbonyl, oxyalkyl, alkylaminocarbonyloxyalkyl, aralkylaminocarbonyloxyalkyl, aminoalkyl or acylamido alkyl; the corresponding pharmaceutically acceptable esters, salts and solvates.

Cycloaddition products of cephalosporins with furane are described by R. L. Elliott et al. in J. Org. Chem. 1994, 59, 1606-1607, but that reaction always leads to compounds having an insaturation in the cyclohexane ring.

It has been found that, starting from an ester of the 7-phenylace tamido-3-cephem-4-carboxylic acid, substituted in position 3 with a leaving group, by reaction with a furan optionally 2-substituted or 2,5-disubstituted in the presence of an organic base, the corresponding ester of the 7-phenylacetamido-9,12-epoxycyclohexeno[3, 4-c]ceph-2-em-4-carboxylic acid can be obtained, optionally 9- substituted or 9,12-disubstituted which, by means of 7-deacylation and, if necessary, reacylation, may be reconverted into its 7-amino derivative and into the corresponding 7-acyl derivatives. In this way the compounds of formula I, which in their turn may be converted into their pharmaceutically acceptable esters, salts and solvated, may be obtained. Moreover it has been discovered that the compounds of formula I, as such or as their salts or derivatives, are endowed with an antibacterial activity higher than that of the insaturated product described in literature, at least on most of the microorganisms.

The term "carboxy protecting group", used in the following text describing the invention, indicates one of the groups currently used in organic chemistry, for example methyl, benzyl, benzhydryl and silyl esters. They are illustrated for example by T.W. Greene and P.G.M. Wuts in "Protective Groups in Organic Synthesis", 2nd Edition, 1991, John Wiley & Sons, Inc., pages 224-276. In relation to the meanings of X' and X", such a protecting group may be removed under mild conditions, as in the case of silyl or tetrahydropyranyl ester, or under less mild conditions, for example by basic or acidic hydrolysis or by means of catalytic hydrogenation. According to this invention, as a carboxy protecting group, an ester to be reconverted into the acid by catalytic hydrogenation, is preferably used, for example the benzyloxymethyl, fenacyl, benzyl acid optionally substituted, preferably 4-nitrobenzyl or diphenylmethyl.

Among the meanings of X, in formula I, hydrogen, an Ac radical chosen among the N-protective acyl groups such as formyl, benzyloxy carbonyl, t-butoxycarbonyl, trichloroacetyl, phenylacetyl and some other acyl groups like (Z)-2-(2-aminothiazol-4-yl)-2-methoxyimino acetyl, (Z)-2-(2-aminothiazol-4-yl)-2-(carboxymethoxyimino)acetyl and (Z)-2-(2-aminothiazol-4-yl)-2-(carboxy-1-methylethoxyimino)acetyl are preferred.

The salts of the compounds of formula I are those obtained from mineral or organic bases, such as salts of alkaline or alkaline-earth metals, for example the sodium or calcium salt or salts with organic bases like triethylamine, dimethylaminopyridine and the like or, when for example in formula I X represents hydrogen, the addition salts with mineral or organic acids, for example the hydrochloride, the methansulfonate, the p-toluensulfonate or the picrate.

The esters of derivatives of formula I may be aliphatic, aryl, aliphatic, aromatic, heteroarylaliphatic, heteroaromatic, cycloali phatic, heterocycloaliphatic, cycloalkylaliphatic, heterocycloalkyl aliphatic, hemiacetalic.

The aliphatic esters include methyl, ethyl, propyl, isopropyl, t.butyl, 2,2,2-trichloroethyl, 2-methoxyethyl, 2-dimethylaminoethyl and trimethylsilyl esters.

The arylaliphatic esters include benzyl esters, for example the benzyl ester optionally substituted like the 4-methoxybenzyl, 4- nitrobenzyl, 2-chlorobenzyl esters; the diphenylmethyl esters like benzydryl, α-phenyl-4-methoxybenzyl ester, 4,4'-dimethoxybenzydryl esters; the triphenylmethyl esters like trityl, α,α-diphenyl-4- methoxybenzyl esters; phenylalkylic esters like phenethyl, α-methyl. benzhydryl and phenylpropyl esters

The aromatic esters include phenyl, 4-nitrophenyl, pentafluoro phenyl, pentachlorophenyl, 1-naphthyl, 2-naphthyl and 5,6,7,8-tetrahy dro-1-naphthyl esters.

The heteroarylaliphatic esters include 2-pyridylmethyl, 3-pyridyl methyl, furfuryl and 2-thienylmethyl esters.

The heteroaromatic esters include 2-pyridyl, 3-pyridyl and 3- indolyl esters.

The cycloaliphatic esters include cyclopropyl, cyclopentyl and cyclohexyl esters.

The heterocycloaliphatic esters include particularly tetrahydro pyranyl ester.

The cycloalkylaliphatic esters include cyclopropylmethyl, cyclo pentylmethyl, cyclohexylmethyl and cyclohexylethyl esters.

The heterocycloalkylaliphatic esters include 2-tetrahydrofuryl methyl, 2-tetrahydrothienylmethyl and 2-tetrahydrothienylethyl esters.

The hemiacetal esters include pivaloyloxymethyl, 1-pivaloyloxy. ethyl, 1-ethoxycarbonyloxyethyl, 1-acetoxyethyl, 1-cyclohexyloxycar bonyloxyethyl, 1-isopropoxycarbonyloxyethyl and 4-methyl-2-oxo-1,3- dioxol-S-ylmethyl esters.

Among the compounds described in this invention, a subclass with great advantages is represented by those of formula I, where X is hydrogen, by the corresponding esters, salts and solvates. Among these ones, the compound of formula I, where X, X' and X" are hydrogen, the corresponding esters, salts and solvates are endowed with particular advantages.

Another subclass with great advantages is represented by compounds of formula I, wherein X is an acyl group Ac, as esters, salts and solvates. Among these, the compounds of formula I, where X is an acyl group Ac, while X' and X" are identical or different and represent, independently, hydrogen, methyl, hydroxymethyl, (C₁-C₄)alkanoyloxγ methyl, aminocarbonyloxymethyl, aminomethyl, acylamidomethyl, as well as the corresponding esters, salts and solvates, are of particularly great advantage.

Cyclohexanocephalosporins of greatest interest are those compounds of formula I, wherein X is an acyl group selected from the group consisting of D(-)α-aminophenylacetyl, (Z)-2-(2-aminothiazol-4-yl)-2- methoxyiminoacetyl, (Z)-2-(2-aminothiazol-4-yl)-2-(carboxymethoxyimino)acetyl and (Z)-2-(2-aminothiazol-4-yl)-2-(carboxy-1-methylethoxy imino)acetyl.

According to another of its aspects, this invention concerns a process for the preparation of compounds of formula I, which comprises:

(a) treating an ester of 7-phenylacetamido-3-cephem-4-carboxylic acid or a mixture of Δ ²+Δ³ isomers of formula II

wherein R is a carboxy protecting group and W is a leaving group, with a furan of formula III

wherein X' and X" are as defined above, in the presence of a base;

(b) subjecting the cyclohexenocephem thus obtained of formula IVa'

to a deacylation in position 7, in order to obtain a cyclohexenoce phem compound of formula IVb'

wherein R, X' ed X" are as defined above and, possibly, after having subjected the compound IVb' to a carboxy deprotection giving rise to compound IVb

or to a reaction with a reactive derivative of an acid of formula AcOH, where Ac has the same meaning than X, but it is different from phenylacetyl, so as to obtain the compound IVc'

the cyclohexenocephem compound thus obtained, where R represents hydrogen or a carboxy protecting group, X, X' and X" are as defined above, is subjected to a deprotection of the carboxylic group, in case this has not yet been performed, in order to obtain a cyclohexenocephemcarboxylic acid of formula IVc

and to a subsequent catalytic hydrogenation to obtain the compound of formula Ic

and, when R is a carboxy protecting group, to be removed by means of reduction, the compound IVc' is subjected to a catalytic hydrogenation and the compound of formula Ic is directly obtained, which can be transformed into the corresponding esters, salts or solvates.

The step (a) is carried out at a temperature between 0ºC and 40ºC, generally at room temperature, in an organic solvent like dioxane or tetrahydrofurane, or a hydrocarbon such as hexane, toluene, dichloromethane, 1,1,1-trichloroethane or acetonitrile.

As organic base a tertiary amine like triethylamine, diisopropyl ethylamine, 4-methylmorpholine, 1-methylpiperidine is used.

The reaction is over after a time from 10 minutes to 6 hours; it can be monitored by thin layer chromatography (TLC) by using a mixture hexane/ethyl acetate (1:1 v/v). The cycloaddition product of formula IVa' is isolated according to the coonventional techniques, for example by eliminating the subproducts by washing with water and evaporating the solvent.

Particularly, the compound of formula IVa' may be N-deacylated by means of a reaction with phosphor pentachloride in the presence of pyridine and subsequent treatment with methanol, according to known techniques, to give the corresponding 7-amino derivative of formula IVb'.

The protected 7-amino-cyclohexenocephem compound IVb' thus obtained may be deprotected in order to give the 7-amino-cyclohexeno- 2-cephem-4-carboxylic acid IVb. The compound thus obtained may be acylated in position 7 by a reaction with a reactive derivative of the acid AcOH, where Ac is an acyl group different from phenylacetyl or it can be subjected to a temporary protection, for example with a silane, of the carboxylic group so as to obtain a 7-acylamido- cyclohexeno-2-cephem-4-carboxylic acid of formula IVc.

As an alternative, the protected 7-amino-cyclohexenocephem compound IVb' may be N-acylated by a reaction with a reactive derivative of the acid AcOH in order to obtain a protected 7- acylamido-cyclohexenocefem acid of formula IVc' and, when the protective group is not removable by catalytic hydrogenation, it can be deprotected according to the usual methods to give the 7- acylamido-cyclohexeno-2-cephem-4-carboxylic acid of formula IVc.

The compound IVc may also be obtained directly from the compound IVb by acylation with a reactive derivative of an acid AcOH described above.

According to another of its aspects, the present invention refers to a process for the preparation of compounds of formula I, whereby as compound of formula II an ester is used, wherein R is a 4-nitro benzyl or diphenylmethyl and W is a mesyloxy, p-tosyloxy or trifluoro methansulfonyloxy and, as starting furan, a compound of formula III, in which one of X' and X" is hydrogen and the other is hydroxyme thyl, (C₁-C₄)alkanoyloxymethyl or aminocarbonyloxymethyl or both X' and X" are hydroxymethyl, (C₁-C₄)alkanoyloxymethyl or aminocarbonyl oxymethyl.

All cyclohexenocephem compounds described above, i.e. compounds IVa', IVb, IVb', IVc and IVc', embraced in formula IV

in which X, X ' and X" are as defined above and R represents hydrogen or a carboxy protecting group, or the corresponding salts or solvates, may be subjected to step (b).

In that case step (b) is carried out by performing a hydrogenation in the presence of a conventional catalyst. The hydrogen pressure may range from 6,9.10 ³ to 6,9.10⁵ pascal, preferably from 1,4.10⁵ to 3,5-10⁵ pascal.

Hydrogenation occurs in an organic inert solvent like methanol, ethanol or isopropanol, dioxane, tetrahydrofurane or saturated hydro carbon like hexane or cyclohexane. The catalysts used are based on palladium, preferably 10% Pd/C.

After 2-6 hours the reduction of the double bond of the product of formula IV' is over and the reaction may be monitored by means of

TLC.

When in the cyclohexenocephem compound of formula IV' R represents a carboxy protecting group, which can be eliminated by catalytic hydrogenation, it is possible to obtain directly the compound of formula I. It is sufficient to monitor the hydrogenation reaction in order to complete the removal of the protecting group as well as the reduction of the double bond.

The product of formula I is thus isolated according to the conventional techniques, by filtering the catalyst, evaporating the solvent and dissolving the residue with another suitable solvent, for example ethyl acetate. From a solution of the compound of formula I contained in it is isolated by means of extraction with an aqueous solution of a mineral base, for example sodium carbonate or hydrogencarbonate and subsequent acidification of the aqueous solution of the salt thus obtained, for example sodium salt, with an acid, for example hydrochloric acid.

The compound of formula I thus prepared may be transformed according to known methods into its salts with alkaline metals like sodium and potassium, with alkaline-earth metals like calcium or magnesium, with organic bases like ethanolamine or tromethamine, or with aminoacids like lysine and arginine.

The esters may be prepared according to the conventional techniques starting from the sodium salt or from a salt with an organic base, for example triethylamine, and a suitable chloride or bromide. The methyl ester may be easily prepared from the free acid by reaction with diazomethane.

The above mentioned reactions may be embraced in the schemes 1 and 2.

In scheme 1 the reactions are shown which do not involve the reduction of the double bond of the epoxycyclohexene ring, while in scheme 2 the reactions are shown which lead to the reduction of the double bond giving rise to the relative cyclohexanocephem compounds.

Particularly, following scheme 2, the catalytic hydrogenation, in step (b), may be carried out on any of the compounds IVa', IVb, IVb', IVc and IVc', summarized in formula IV', thus obtaining the corresponding compounds la, la', lb, lb', Ic and Ic', which can be subjected to the same reactions applied to the compounds from which they derive.

Similarly to what applied to compound IVa', the cyclohexanocephem compound of formula la' may be N-deacylated in order to obtain the protected 7-amino-cyclohexanocephem compound lb'.

The compound lb' may be deprotected and transformed into the corresponding 7-amino-cyclohexanocephem compound lb, by reaction with a reactive derivative of the acid AcOH, under the same conditions applied in the step from IVb to IVc, gives rise to the 7-acylamido- cyclohexanocephem compound Ic.

As an alternative, the protected 7-aminocyclohexanocephem compound lb' may be acylated with a reactive derivative of the acid AcOH, to give the corresponding protected 7-acylamido-cyclohexanocephem of formula Ic' which, by a suitable deprotection, gives the compound Ic.

The compounds la, lb and Ic thus obtained, summarized in formula

I, are isolated as such or as salts or solvates. They may be transformed into other salts or solvates or into the corresponding esters according to the conventional methods.

The compounds of formula IV, in which X, X' and X" are as above described and at least one of the groups among X' and X" is different from hydrogen, the corresponding esters, salts and solvates, represents, when X is a phenylacetyl group, a further aspect of this invention.

The compounds of formula IV, where X is an acyl group Ac and X' and X", independently, are hydrogen, methyl, hydroxymethyl, (C₁-C₄)- alkanoyloxymethyl or aminocarbonyloxymethyl, the corresponding esters, salts and solvates are preferred.

Some compounds of the invention have been the object of a preliminary pharmacological investigation. During "in vitro" tests the compounds of formula la show a significant activity (reported in the following text in brackets as M.I.C. espressed in μg/ml) towards microorganisms like Sarcina lutea (0.78÷6.25), Staphylococcus aureus (0.78÷1.56), Bacillus subtilis (3.12÷12.5), Moraxella catarrhalis {6.25÷50), Streptococcus pyogenes (3.12÷25), Escherichia coli (6,25÷25), Klebsiella pneumoniae (25÷100), Proteus mirabilis (1.56÷6.25).

The compounds of the present invention have an excellent antibacterial activity and may therefore be used, also thanks to their low toxicity, for the preparation of pharmaceutical formulations for the treatment of bacterial infections due to Gram- positive and Gram-negative microorganisms and, to that purpose, they are included into formulations with conventional excipients for the preparation of tablets, capsules, granulates for the reconstitution of syrups or drinkable solutions or injectable solutions for intramuscular administration.

Unitary doses for administration range from 1 to 5000 mg, with great advantage from 20 to 3000 mg, preferably from 50 to 2000 mg of active substance and can be administered from 1 to 5 times a day depending on the severity of the infection.

The following examples illustrate the invention without, however, limiting it.

PREPARATION

4-Nitrobenzyl 7-phenylacetamido-3-mesyloxy-3-cephem-4-carboxylate (Δ ²+Δ³ isomers).

To a mixture of 24.87 g (53 mmoles) of 4-nitrobenzyl 7-phenylaceta mido-3-hydroxy-3-cephem-4-carboxylate, 0.75 g of 4-dimethylaminopyri dine and 8.25 ml (59.3 mmoles) of triethylamine in 200 ml of tetrahy drofurane, cooled to a 0°C, 4.58 ml (59,2 mmoles) of methansulfonyl chloride are added in about 30 minutes and the temperature is kept at about 0ºC. The mixture is let under stirring for 3 hours, then it is treated with water, the product is extracted with ethyl acetate, the organic phase is separated and concentrated under vacuum. The residue is treated with 30 ml of acetone and from this solution the final product crystallizes, obtaining a mixture of 4-nitrobenzyl 7-phenyl acetamido-3-mesyloxy-3-cephem-4-carboxylate and of 4-nytrobenzyl 7- phenylacetamido-3-mesyloxy-2-cephem-4-carboxylate, hereinafter desi gned as mixture of Δ ²+Δ³ isomers.

EXAMPLE 1

(a) In a 250 ml-flask 3 g (5 mmoles) of p-nitrobenzyl 7-phenylace tamido-3-trifluoromethansulfonyloxy-3-cephem-4-carboxylate in 50 ml of dichloromethane, at room temperature, in 50 ml of dichloromethane are treated with 1.45 ml (20 mmoles) of furane and 1.02 ml (6 mmoles) of diisopropylethylamine. The reaction, monitored by TLC (eluent: hexane/ethyl acetate = 1:1 v/v), is over in 15 minutes. Then the reaction mixture is diluted with dichloromethane, washed with acidulated water and the organic phase is concentrated and dried with anhydrous sodium sulfate. The product is purified by Silica gel column chromatography (eluent: hexane/ethyl acetate 7:3 v/v). The reaction mixture is diluted with dichloromethane, washed with acidulated water and the organic phase is concentrated and dried with anhydrous sodium sulfate. The product is purified by Silica gel column chromatography (eluent: hexane/ethyl acetate 7:3 v/v). Thus 1.56 g of 4-nitrobenzyl 7-phenylacetamido-9,12-epoxycyclohexeno[3,4- c]ceph-2-em-4-carboxylate are obtained. Yield: 60%.

IR (Nujol): 1762-1660 cm^-1.

1Η-NMR (CDCl₃): 6: 3.6 (m, 2H); 4.9 (dd, 1H, J=l.l e 4 Hz); 5.28 (d, 1H, J=13.5 Hz); 5.34 (m, 1H); 5.36 (d, 1H, J=13.5 Hz); 5.4 (dd, 1H, J=4 e 8.4 Hz); 5.7 (m, 1H); 5.87 (d, 1H, J=8.4 Hz); 6.15 (s, 1H); 6.5 (m, 2H); 7.3 (m, 5H); 7.5 (d, 2H, J=9 Hz); 8.25 (d, 2H, J=9 Hz).

(b) To a solution of 1.4 g (2 mmoles) of the compound obtained at the end of the previous step in 20 ml of ethyl alcohol, 1 g of 10% Pd/C is added, then the mixture is hydrogenated with a hydrogen pressure of 24.10⁴ pascal for 4 hours. When the reaction is over, after a TLC monitoring, the catalyst is filtered, the ethyl alcohol is evaporated off, the residue is treated with ethyl acetate and NaHCO, aqueous solution. The aqueous phase is separated and, after acidification at pH of about 2, is extracted with ethyl acetate. The organic phase is evaporated, after dehydration with anhydrous Na₂SO₄, and 0,7 g of 7-phenylacetamido-9,12-epoxycyclohexano[3,4-c]ceph-2-em- 4-carboxylic acid are obtained. Yield: 92%.

IR (KBr): 1770, 1737 and 1647 cm^-1.

EXAMPLE 2

0,5 g of the compound obtained in Example 1, diluted with 10 ml of methylene chloride, are treated with an etheric solution of diazome thane. After evaporation under vacuum, 0.55 g of methyl 7-phenylaceta mido-9,12-epoxycyclohexano[3,4-c]ceph-2-em-4-carboxylate are obtained. IR (Nujol): 1760 and 1660 cm^-1.

1H-NMR (CDCl₃): δ: 1.6 (m, 1H); 1.9 (m, 3H); 3.65 (m, 2H); 3.8 (s, 3H); 4.9 (d, 1H, J=4.2 Hz); 5.07 (m, 1H); 5.33 (m, 1H); 5.37 (dd, 1H, J=4.2 e 8.3 Hz); 6.1 (d, 1H, J=8.3 Hz); 6.15 (s, 1H); 7.3 (m, 5H). ¹³C-NMR (CDCl₃): 23.9; 35.3; 43.2; 53.3; 54.8; 59.1; 70.6; 80.6; 80.8; 113.3; 127.5; 129; 129.4; 132.4; 133.8; 160.2; 167.4; 170.9.

EXAMPLE 3

(a) To a solution of 20 g (36.5 mmoli) of a mixture of the Δ ²+Δ³ isomers of 4-nitrobenzyl 7-phenylacetamido-3-mesyloxy-3-cephem-4-car boxylate in 200 ml of acetonitrile, obtained according to the Example 1(a), cooled at 0°C, 20 ml of furane and 9.9 ml di N,N-diiso propylethylamine are added, then the temperature is let to reach the room temperature (20÷30°C), the mixture is stirred for 3 hours, then it is treated with water. The pH of the mixture is adjusted to 5 by adding acetic acid and the product is extracted with dichloromethane, concentrated under vacuum, the oily residue is treated with cyclo hexane and the mixture is stirred; thus the 4-nitrobenzyl 7-phenyl acetamido-9,12-epoxycyclohexeno[3,4-c]ceph-2-em-4-carboxylate (IV, R= = 4-nitrobenzyl, X'= X"= H) precipitates, identical to the product of the Example 1(a). Yield: 79%.

(b) By operating as described in the Example 1(b) the 7-phenylace tamido-9,12-epoxycyclohexano(3,4-c]ceph-2-em-4-carboxylic acid is obtained.

EXAMPLE 4

(a) To a mixture of 6.55 g (10 mmoles) of 4-nitrobenzyl 7-phenyl acetamido-3-mesyloxy-3-cephem-4-carboxylate and 70 ml of acetonitri le, 3 ml (31.2 mmoles) of 2,5-dimethylfurane and 3 ml (23.2 mmoles) of N,N-diisopropylethylamine are added. The mixture is stirred for 4÷5 hours, then it is diluted with water and extracted with ethyl acetate. The organic phase is washed with water acidulated with hydrochloric acid, then the obtained product is evaporated and purified by Silica gel column chromatography (eluent: cyclohexane/ ethyl acetate = 1:1 v/v). Thus, the 4-nitrobenzyl 7-phenylacetamido-9, 12-dimethyl-9,12-epoxycyclohexeno[3,4-c]ceph-2-em-4-carboxylate (IV, R = 4-nitrobenzyl; X'= X" = methyl) is isolated. Yield: 80%.

1H-NMR (CDCl₃): δ: 1.65 (s, 3H); 1.7 (s, 3H); 3.6 (m, 2H); 4.9 (dd, 1H, J=1.3 e 3.9 Hz); 5.3 (d, 1H, J=13.7 Hz); 5.35 (d, 1H, J=13.7 Hz); 5.40 (dd, 1H, J=3.9 e 8.4 Hz); 5.85 (d, 1H, J=8.4 Hz); 5.9 (d, 1H, J=1.3 Hz); 6.2 (d, 1H, J=5.25 Hz); 6.4 (d, 1H, J=5.25 Hz); 7.3 (m, 5H); 7.5 (d, 2H, J=8.9 Hz); 8.2 (d, 2H, J=8.9 Hz).

(b) A mixture of 4 g of the product obtained at the end of the step (a) in 25 ml of tetrahydrofurane and 15 ml of methanol is hydrogenated in the presence of 4 g of 5% Pd/C at a pressure of 3.0*10 pascal. After 3 hours, the product is filtered on Celite ^® , concentrated and treated with ethyl acetate. The undissolved part is further eliminated by filtration, the solution is extracted with water and potassium hydrogencarbonate. The mother liquors are treated with hydrochloric acid (1:1 v/v) and ice to reach an acid pH value and extracted with ethyl acetate. After evaporation of the solvent, the 7-phenylacetamido-9,12-dimethyl-9,12-epoxycyclohexano[3,4-c]ceph- 2-em-4-carboxylic acid is obtained with a quantitative yield.

1H-NMR (CDCl₃) δ: 1.65 (s, 3H); 1.8 (s, 3H); 1.85 (m, 3H); 2.4 (m, 1H); 3.65 (m, 2H); 5.25 (dd, 1H, J=4.2 e 7.8 Hz); 5.40 (d, 1H, J=4.2 Hz); 5.95 (d, 1H, J=7.8 Hz); 6.15 (s, 1H); 7.35 (m, 5H).

EXAMPLE 5

(a) To a solution of 6.55 g (10 mmoles) of 4-nitrobenzyl 7-phenyj. acetamido-3-mesyloxy-3-cephem-4-carboxylate in 70 ml of acetonitrile, 3 ml (30.6 mmoles) of 2-hydroxymethylfurane and 3 ml (23.2 mmoles) of N,N-diisopropylethylamine are added. After 4÷5 hour-stirring at room temperature, the reaction mixture is diluted with water and the product is extracted with ethyl acetate. The organic phase is washed with acidulated water, then concentrated and the purification of the resulting product is carried by Silica gel column chromatography (eluent: cyclohexane/ethyl acetate = 1:1 v/v). The 4-nitrobenzyl 7- phenylacetamido-9-hydroxymethyl-9,12-epoxycyclohexeno[3,4-c]ceph-2-em- 4-carboxylate (IV, R = 4-nitrobenzyl; X'= hydroxymethyl; X" = H). Yield: 80%.

1H-NMR (CDCl₃): δ: 3.6 (m, 2H); 4.25 (d, 1H, J=15.3 Hz); 4.5 (d, 1H , J=15.3 Hz); 4.9 (dd, 1H, J=1.3 e 3.9 Hz); 5.3 (m, 2H); 5.35 (dd, 1H, J=3.9 e 8.3 Hz); 5.45 (m, 1H); 5.80 (d, 1H, J=8.3 Hz); 6.0 (d, 1H, J=1.3 Hz); 6.5 (m, 2H); 7.3 (m, 5H); 7.5 (d, 2H, J=8.9 Hz); 8.2 (d, 2H, J=8.9 Hz).

(b) By hydrogenation, according to the procedure described in the Example 4, the 7-phenylacetamido-9-hydroxymethyl-9,12-epoxycyclohexa no[3,4-c]ceph-2-em-4-carboxylic acid is obtained in a quantitative yield.

1H-NMR (D₂O/NaOH): δ: 1.75 (m, 1H); 1.9 (m, 1H); 2.0 (m, 2H); 3.70 (m, 2H); 4.02 (d, 1H, J=12.9 Hz); 4.18 (d, 1H, J=12.9 Hz); 4.9 (dd, 1H, J=1.2 e 3.9 Hz); 5.0 (d, 1H, J=3.9 Hz); 5.1 (m, 1H); 6.1 (d, 1H, J=1.2 Hz); 7.35 (m, 5H).

By operating as described in the Examples 4 and 5, by reaction of 4-nitrobenzyl 7-phenylacetamido-3-mesyloxy-3-cephem-4-carboxylate with 2,5-bis(hydroxymethyl)furane, 2-acetoxymethylfurane, 2,5-bis (acetoxymethyl)furane, 2-aminocarbonyloxymethylfurane, 5-aminocarbo nyloxymethylfurane, 2,5-bis(aminocarbonyloxymethyl)furane, 2,5-bis (methoxymethyl)furane, 2,5-bis(butirryloxymethyl)furane, 2,5-bis(esa noyloxymethyl)furane and 2,5-bis(pivaloyloxymethyl)furane, the products indicated in the examples from 6 to 15 are respectively obtained.

EXAMPLE 6

(a) 4-Nitrobenzyl 7-phenylacetamido-9,12-bis(hydroxymethyl)-9,12- epoxycyclohexeno[3,4-c]ceph-2-em-4-carboxylate. ¹H-NMR (CDCl₃): δ: 3.6 (m, 2H); 4.1 (d, 1H, J=13 Hz); 4.15 (m,

2H); 4.25 (d, 1H, J=13 Hz); 4.95 (dd, 1H, J=1.2 e 3.9 Hz); 5.3 (m,

2H); 5.35 (dd, 1H, J=3.9 e 8.2 Hz); 5.95 (d, 1H, J=8.2 Hz); 6.05 (d, 1H, J=1.2 Hz); 6.4 (d, 1H, J=5.5 Hz); 6.5 (d, 1H, J=5.5 Hz); 7.35 (m, 5H); 7.5 (d, 2H, J=8.9 Hz); 8.2 (d, 2H, J=8.9 Hz).

(b) 7-Phenylacetamido-9,12-bis(hydroxymethyl)-9,12-epoxyciclohexa no[3,4-c]ceph-2-em-4-carboxylic acid.

¹H-NMR (D₂O/NaOH): δ: 1.65 (m, 1H); 1.85 (m, 1H); 2.05 (m, 2H);

3.60 (m, 2H); 3.95 (d, 1H, J=12.9 Hz); 3.98 (d, 1H, J=12.9 Hz); 4.05 (d, 1H, J=12.9 Hz); 4.15 (d, 1H, J=12.9 Hz); 4.82 (dd, 1H, J=1.2 e

3.9 Hz); 4.95 (d, 1H, J=3.9 Hz); 6.1 (d, 1H, J=1.2 Hz); 7.35 (m, 5H).

EXAMPLE 7

(a) 4-Nitrobenzyl 7-phenylacetamido-9-acetoxymethyl-9,12-epoxy cyclohexeno[3,4-c]ceph-2-em-4-carboxylate .

1H-NMR (CDCl₃): δ: 2.05 (s, 3H); 3.6 (m, 2H); 4.55 (d, 1H, J=12.9

Hz); 4.7 (d, 1H, J=12.9 Hz); 4.9 (dd, 1H, J=1.2 e 4 Hz); 5.33 (m, 2H); 5.40 (dd, 1H, J=4 e 8.2 Hz); 5.45 (m, 1H); 5.80 (d, 1H, J=8.2 Hz); 6.0 (d, 1H, J=1.2 Hz); 6.5 (m, 2H); 7.3 (m, 5H); 7.5 (d, 2H, J=8.9 Hz); 8.2 (d, 2H, J=8.9 Hz).

( b ) 7-Phenylacetamido-9-acetoxymethyl-9,12-epoxycyclohexano[3,4-c] ceph-2-em-4-carboxylic acid.

1H-NMR (CDCl₃): δ: 1.85 (m, 2H); 2.10 (s, 3H); 2.2 (m, 1H); 2.35 (m, 1H); 3.65 (m, 2H); 4.73 (d, 1H, J=13.3 Hz); 4.8 (d, 1H, J=13.3 Hz); 5.18 (dd, 1H, J=4.2 e 7.7 Hz); 5.25 (m, 1H); 5.38 (d, 1H, J=4.2 Hz); 6.0 (d, 1H, J=7.7 Hz); 6.2 (s, 1H); 7.3 (m, 5H).

EXAMPLE 8

(a) 4-Nitrobenzyl 7-phenylacetamido-9,12-bis(acetoxymethyl)-9,12- epoxycyclohexeno[3,4-c]ceph-2-em-4-carboxylate.

1H-NMR (CDCl₃): δ: 2.01 (s, 3H); 2.03 (s, 3H); 3.6 (m, 2H); 4.6 (d, 1H, J=12.9 Hz); 4.65 (d, 1H, J=12.9 Hz); 4.7 (m, 2H); 4.9 (dd, 1H, J=1.2 e 3.9 Hz); 5.3 (d, 1H, J=13.5 Hz); 5.36 (d, 1H, J=13.5 Hz); 5.37 (dd, 1H, J=3.9 e 8.3 Hz); 5.8 (d, 1H, J=8.3 Hz); 6.1 (d, 1H, J=1.2 Hz); 6.4 (d, 1H, J=5.5 Hz); 6.57 (d, 1H, J=5.5 Hz); 7.3 (m, 5H); 7.5 (d, 2H, J=8.9 Hz); 8.2 (d, 2H, J=8.9 Hz).

(b) 7-Phenylacetamido-9,12-bis(acetoxymethyl)-9,12-epoxyciclohexa no[3,4-c]ceph-2-em-4-carboxylic acid.

¹H-NMR (CDCl₃): δ: 1.9 (m, 3H); 2.10 (s, 3H); 2.12 (s, 3H); 2.5

(m, 1H); 3.65 (m, 2H); 4.3 (d, 1H, J=12.9 Hz); 4.7 (d, 1H, J=13.3

Hz); 4.8 (d, 1H, J=13.3 Hz); 4.95 (d, 1H, J=12.9 Hz); 5.1 (dd, 1H, J=4 e 7.6 Hz); 5.32 (d, 1H, J=4 Hz); 5.9 (d, 1H, J=7.6 Hz); 6.3 (s, 1H); 7.3 (m, 5H).

EXAMPLE 9

(a) 4-Nitrobenzyl 7-phenylacetamido-9-aminocarbonyloxymethyl-9,12- epoxycyclohexeno[3,4-c]ceph-2-em-4-carboxylate.

1H-NMR (CDCl₃): δ: 3.6 (m, 2H); 4.6 (ABq, 2H, J=12.6 Hz); 4.9 (dd, 1H, J=1.2 e 4 Hz); 5.33 (ABq, 2H, J=13.5 Hz); 5.35 (dd, 1H, J=4 e 8 Hz); 5.45 (d, 1H, J=1.5 Hz); 5.85 (d, 1H, J=8 Hz); 6.0 (d, 1H, J=1.2 Hz); 6.52 (dd, 1H, J=1.5 e 5.4 Hz); 6.57 (d, 1H, J=5.4 Hz); 7.3 (m, 5H); 7.5 (d, 2H, J=8 Hz); 8.2 (d, 2H, J=8 Hz).

(b) 7-Phenylacetamido-9-aminocarboniloxymethyl-9,12-epoxycyclohexa no[3,4-c]ceph-2-em-4-carboxylic acid.

1H-NMR (DMSO-d₆): δ: 1.5-1.9 (m, 4H); 3.55 (m, 2H); 4.55 (ABq, 2H, J=12.6 Hz); 4.8 (d, 1H, J=4 Hz); 4.92 (m, 1H); 5.0 (dd, 1H, J=4 e 7.7 Hz); 6.05 (s, 1H); 6.5 (m, 2H); 7.3 (m, 5H); 9.1 (d, 1H, J=7.7 Hz).

EXAMPLE 10

(a) 4-Nitrobenzyl 7-phenylacetamido-12-aminocarbonyloxymethyl-9, 12-epoxycyclohexeno[3,4-c]ceph-2-em-4-carboxylate.

¹H-NMR (CDCl₃): δ: 3.6 (m, 2H); 4.65 (ABq, 2H, J=13 Hz); 4.7 (bs,

2H); 4.9 (dd, 1H, J=l e 4 Hz); 5.3 (m, 2H); 5.4 (dd, 1H, J=4 e 8.5 Hz); 5.7 (d, 1H, J=1.6 Hz); 5.95 (d, 1H, J=8.5 Hz); 6.2 (d, 1H, J=l

Hz); 6.4 (d, 1H, J=5.6 Hz); 6.55 (dd, 1H, J=1.6 e 5.6 Hz); 7.3 (m,

5H); 7.5 (d, 2H,_., J=8 Hz); 8.22 (d, 2H, J=8 Hz).

(b) 7-Phenylacetamido-12-aminocarbonyloxymethyl-9,12-epoxycyclo hexano[3,4-c]ceph-2-em-4-carboxylic acid.

1H-NMR (DMSO-d₆): δ: 1.5-2 (m, 4H); 3.55 (m, 2H); 4.35 (ABq, 2H, J=13.3 Hz); 4.9 (d, 1H, J=3.9 Hz); 5.1 (d, 1H, J=5.4 Hz); 5.2 (dd, 1H, J=3.9 e 7.8 Hz); 6.6 (s, 1H); 7.3 (m, 5H); 9.1 (d, 1H, J=7.8 Hz).

EXAMPLE 11

(a) 4-Nitrobenzyl 7-phenylacetamido-9,12-bis(aminocarbonyloxyme thyl)-9,12-epoxycyclohexeno[3,4-c]ceph-2-em-4-carboxylate.

1H-NMR (CDCl₃): δ: 3.6 (m, 2H); 4.53 (d, 1H, J=12.8 Hz); 4.6 (d, 1H, J=13.2 Hz); 4.7 (d, 1H, J=12.8 Hz); 4.76 (d, 1H, J=13.2 Hz); 4.8 (bs, 2H); 4.85 (dd, 1H, J=1.2 e 4 Hz); 4.9 (bs, 2H); 5.3 (m, 2H); 5.35 (dd, 1H, J=4 e 8.4 Hz); 6 (d, 1H, J=8.4 Hz); 6.1 (d, 1H, J=1.2 Hz); 6.4 (d, 1H, J=5 Hz); 6.55 (d, 1H, J=5 Hz); 7.3 (m, 5H); 7.5 (d, 2H, J=8 Hz); 8.25 (d, 2H, J=8 Hz).

(b) 7-Phenylacetamido-9,12-bis(aminocarbonyloxymethyl)-9,12-epoxy cyclohexano[3,4-c]ceph-2-em-4-carboxylic acid.

1H-NMR (DMSO-d₆): δ: 1.5-2 (m, 4H); 3.4 (m, 2H); 3.6 (m, 2H); 4.3 (d, 1H, J=12.2 Hz); 4.4 (d, 1H, J=12.9 Hz); 4.48 (d, 1H, J=12.2 Hz); 4.55 (d, 1H, J=12.9 Hz); 4.8 (d, 1H, J=4 Hz); 5.15 (dd, 1H, J=4 e 8.3 Hz); 6.5 (s, 1H); 6.6 (m, 2H); 7.3 (m, 5H); 9.12 (d, 1H, J=8.3 Hz ) .

EXAMPLE 12

(a) 4-Nitrobenzyl 7-phenylacetamido-9,12-bis(methoxymethyl)-9,12- epoxycyclohexeno[3,4-c)ceph-2-em-4-carboxylate.

1H-NMR (CDCl₃): δ: 3.3 (s, 3H); 3.4 (s, 3H); 3.6 (m, 2H); 3.9 (d, 1H, J=11.6 Hz); 3.95 (ABq, 2H, J=10.9 Hz); 4.1 (d, 1H, J=11.6 Hz);

4.9 (dd, 1H, J=1.2 e 4 Hz); 5.25 (bs, 2H); 5.35 (dd, 1H, J=4 e 8.4 Hz); 5.8 (d, 1H, J=8.4 Hz); 6.1 (d, 1H, J=1.2 Hz); 6.45 (bs, 2H); 7.3

(m, 5H); 7.55 (d, 2H, J=8 Hz); 8.2 (d, 2H, J=8 Hz).

(b) 7-Phenylacetamido-9,12-bis(methoxymethyl)-9,12-epoxycyclohexa no[3,4-c]ceph-2-em-4-carboxylic acid.

1H-NMR (CDCl₃): δ: 1.7-1.9 (m, 2H); 2-2.25 (m, 2H); 3.4 (s, 3H); 3.45 (s, 3H); 3.65 (bs, 2H); 3.9 (ABq, 2H, J=10.8 Hz); 4.0 (bs, 2H); 5.18 (d, 1H, J=4.2 Hz); 5.3 (dd, 1H, J=4.2 e 8 Hz); 5.95 (d, 1H, J=8 Hz); 6.2 (s, 1H); 7.3 (m, 5H).

EXAMPLE 13

(a) 4-Nitrobenzyl 7-phenylacetamido-9,12-bis(butyryloxymethyl)-9, 12-epoxycyclohexeno[3,4-c]ceph-2-em-4-carboxylate.

1H-NMR (CDCl₃): δ: 0.9 (m, 6H); 1.6 (m, 4H); 2.2 (m, 4H); 3.6 (m,

2H); 4.62 (ABq, 2H, J=12.9 Hz); 4.7 (s, 2H); 4.9 (dd, 1H, J=1.2 e 4

Hz); 5.3 (ABq, 2H, J=13.8 Hz); 5.40 (dd, 1H, J=4 e 7.7 Hz); 5.8 (d, 1H, J=7.7 Hz); 6.1 (d, 1H, J=1.2 Hz); 6.36 (d, 1H, J=5.5 Hz); 6.55 (d, 1H, J=5.5 Hz); 7.3 (m, 5H); 7.55 (d, 2H, J=8 Hz); 8.25 (d, 2H, J=8 Hz).

(b) 7-Phenylacetamido-9,12-bis(butyryloxymethyl)-9,12-epoxycyclo hexano[3,4-c]ceph-2-em-4-carboxylic acid.

¹H-NMR (CDCl₃): δ: 0.9 (m, 6H); 1.6 (m, 5H); 1.9 (m, 3H); 2.2 (m, 4H); 3.6 (m, 2H); 4.25 (d, 1H, J=12.9 Hz); 4.75 (ABq, 2H, J=13.2 Hz); 4.95 (d, 1H, J=12.9 Hz); 5.1 (dd, 1H, J=4 e 7.6 Hz); 5.3 (d, 1H, J=4 Hz); 5.97 (d, 1H, J=7.6 Hz); 6.25 (s, 1H); 7.3 (m, 5H).

EXAMPLE 14

(a) 4-Nitrobenzyl 7-phenylacetamido-9,12-bis(hexanoyloxymethyl)-9, 12-epoxycyclohexeno[3,4-c]ceph-2-em-4-carboxylate.

¹H-NMR (CDCl₃): δ: 0.9 (m, 6H); 1.25 (m, 8H); 1.6 (m, 4H); 2.25 (m, 4H); 3.6 (ABq, 2H, J=15.7 Hz); 4.62 (ABq, 2H, J=12.9 Hz); 4.68 (ABq, 2H, J=12.9 Hz); 4.9 (dd, 1H, J=1.2 e 4 Hz); 5.34 (ABq, 2H, J=13.5 Hz); 5.40 (dd, 1H, J=4 e 7.1 Hz); 5.8 (d, 1H, J=7.1 Hz); 6.1 (d, 1H, J=1.2 Hz); 6.4 (d, 1H, J=5.6 Hz); 6.6 (d, 1H, J=5.6 Hz); 7.3 (m, 5H); 7.55 (d, 2H, J=8 Hz); 8.25 (d, 2H, J=8 Hz).

(b) 7-Phenylacetamido-9,12-bis(hexanoyloxymethyl)-9,12-epoxycyclo hexano[3,4-c]ceph-2-em-4-carboxylic acid.

1H-NMR (DMSO-d₆): δ: 0.8 (m, 6H); 1.2 (m, 8H); 1.5 (m, 5H); 1.6-2 (m, 3H); 2.3 (m, 4H); 3.5 (m, 2H); 4.4 (ABq, 2H, J=12.4 Hz); 4.65 (ABq, 2H, J=12.5 Hz); 4.7 (d, 1H, J=4 Hz); 5 (dd, 1H, J=4 e 7.7 Hz); 6.2 (s, 1H); 7.3 (m, 5H); 9.1 (d, 1H, J=7.7 Hz).

EXAMPLE 15

(a) 4-Nitrobenzyl 7-phenylacetamido-9,12-bis(pivaloyloxymethyl)-9, 12-epoxycyclohexeno[3,4-c]ceph-2-em-4-carboxylate.

1H-NMR (CDCl₃): δ: 1.12 (s, 9H); 1.15 (s, 9H); 3.55 (m, 2H); 4.5

(ABq, 2H, J=12.8 Hz); 4.6 (ABq, 2H, J=10.8 Hz); 4.8 (dd, 1H, J=1.2 e

4 Hz); 5.28 (s, 2H); 5.34 (dd, 1H, J=4 e 8.3 Hz); 5.9 (d, 1H, J=8.3

Hz); 6.0 (d, 1H, J=1.2 Hz); 6.3 (d, 1H, J=5.5 Hz); 6.55 (d, 1H, J=5.5 Hz); 7.3 (m, 5H); 7.5 (d, 2H, J=8 Hz); 8.2 (d, 2H, J=8 Hz).

(b) 7-Phenylacetamido-9,12-bis(pivaloyloxymethyl)-9,12-epoxycyclo hexano[3,4-c]ceph-2-em-4-carboxylic acid.

¹H-NMR (DMSO-d₆): δ: 1.1 (m, 18H); 1.5 (m, 1H); 1.9 (m, 3H); 3.55

(m, 2H); 4.25 (bs, 2H); 4.52 (d, 1H, J=12.8 Hz); 4.8 (d, 1H, J=4 Hz); 4.9 (d, 1H, J=12.8 Hz); 5.05 (dd, 1H, J=4 e 7.8 Hz); 6.25 (s, 1H); 7.3 (m, 5H); 9. 15 ( d, 1H, J=7.8 Hz).

EXAMPLE 16

(a) To a solution of 11.85 g (22.8 mmoles) of 4-nitrobenzyl 7- phenylacetamido-9,12-epoxycyclohexeno[3,4-c]ceph-2-em-4-carboxylate, obtained as described in the Example 1(a) or in the Example 3(a), in 230 ml of dichloromethane, cooled at -20°C, 11.5 ml (142 mmoles) of pyridine are added and, portionwise, 14.9 g (71.6 mmoles) of phosphorus pentachloride. The mixture is kept under stirring for two hours at a temperature of -10°C, then it is treated with methanol, maintaining the temperature lower than 0ºC. Then it is kept at rest for 30 minutes, water and hydrochloric acid (1:1 v/v) are added to reach a pH value of 3. The organic phase is separated and the aqueous one is extracted with more dichloromethane. The collected organic phases are concentrated, after dehydratation with anhydrous sodium sulfate, to reach an oil, which is treated with hot isopropanol. Thus the 4-nitrobenzyl 7-amino-9,12-epoxy-10-cyclohexeno[3,4-c)ceph-2-em- 4-carboxylate hydrochloride is obtained.

(b) A solution of 23.16 g (53 mmoles) of 4-nitrobenzyl 7-amino-9, 12-epoxycyclohexeno[3,4-c]ceph-2-em-4-carboxylate hydrochloride thus obtained, in 480 ml of acetonitrile and 40 ml of diluted hydrochloric acid (1:1 v/v) is hydrogenated at 25°C at the pressure of 2-10 pascal in the presence of 64 g of 5% Pd/C (wet 50%). After 2½ hours of hydrogenation, the catalyst is filtered and the pH is adjusted to 4.5. The brownish product is eliminated by filtration, the solution is concentrated under vacuum and the residue is treated with a little methanol. The undissolved part is eliminated by filtration, the solution is concentrated under vacuum and the residue is treated with dichloromethane. After filtration the 7-amino-9, 12-epoxycyclohexano [3,4-c]ceph-2-em-4-carboxylic acid is obtained. Starting from a solution of the acid in isopropanol, by adding a 15% solution of HCl in isopropanol, the corresponding hydrochloride is obtained.

EXAMPLES 17 - 23

By operating as described in the Example 16, starting from the cyclohexenocephem compounds obtained in the Examples 4(a), 5(a), 6(a), 7(a) and 8(a), the 7-aminoderivative compounds described in the Examples from 17 to 23 are obtained.

Example 17

(a) 4-Nitrobenzyl 7-amino-9,12-dimethyl-9,12-epoxycyclohexeno[3,4- c]ceph-2-em-4-carboxylate.

(b) 7-Amino-9,12-dimethyl-9,12-epoxycyclohexano[3,4-c]ceph-2-em-4- carboxylic acid and its hydrochloride.

Example 18

(a) 4-Nitrobenzyl 7-amino-9-hydroxymethyl-9,12-epoxycyclohexeno[3, 4-c]ceph-2-em-4-carboxylate.

(b) 7-amino-9-hydroxymethyl-9,12-epoxycyclohexano[3,4-c]ceph-2-em- 4-carboxylic and its hydrochloride.

Example 19

(a) 4-Nitrobenzyl 9-amino-9,12-bis(hydroxymethyl)-9,12-epoxycyclo hexeno[3,4-c]-ceph-2-em-4-carboxylate.

(b) 7-amino-9,12-bis(hydroxymethyl)-9,12-epoxycyclohexano[3,4-c] ceph-2-em-4-carboxylic acid and its hydrochloride.

Example 20

(a) 4-Nitrobenzyl 7-amino-9,12-bis(acetoxymethyl)-9,12-epoxycyclo hexeno[3,4-c]ceph-2-em-4-carboxylate.

(b) 7-amino-9,12-bis(acetoxymethyl)-9,12-epoxycyclohexano[3,4-c] ceph-2-em-4-carboxylic acid and its hydrochloride.

Example 21

(a) 4-Nitrobenzyl 7-amino-9-acetoxymethyl-9,12-epoxycyclohexeno[3, 4-c]ceph-2-em-4-carboxylate.

(b) 7-amino-9-acetoxymethyl-9,12-epoxycyclohexano[3,4-c]ceph-2-em- 4-carboxylic acid and its hydrocloride.

Example 22

(a) 4-Nitrobenzyl 7-amino-9-aminocarbonyloxymethyl-9,12-epoxycyclo hexeno[3,4-c]ceph-2-em-4-carboxylate.

(b) 7-amino-9-aminocarbonyloxymethyl-9,12-epoxycyclohexano[3,4-c] ceph-2-em-4-carboxylic acid ind its hydrochloride.

Example 23

(a) 4-Nitrobenzyl 7-amino-9,12-bis(aminocarbonyloxymethyl)-9,12- epoxycyclohexeno[3,4-c]ceph-2-em-4-carboxylate.

(b) 7-amino-9,12-bis(aminocarbonyloxymethyl)-9,12-epoxycyclohexano [3,4-c]ceph-2-em-4-carboxylic acid and its hydrocloride.

EXAMPLE 24

(i) To a solution of 41.4 mmoles of ethyl potassium Dane salt of D-phenylglycine in 65 ml of dichlorometane and 12.5 ml of N,N-dime thylformamide, cooled to -40°C, 2 drops of 4-methylmorpholine and 3.8 ml (40,3 mmoles) of ethyl chloroformate are added, then the mixture is stirred at room temperature for 1½ hours. At the same temperature a solution of 12.59 g (28.8 mmoles) of 4-nitrobenzyl 7-amino- 9,12-epoxycyclohexeno[3,4-c]ceph-2-em-4-carboxylate hydrochloride (obtained as described in the Example 9(a)) in 100 ml of dichloro methane and 4.4 ml (31 mmoles) of triethylamine are added. The solution is stirred for 1½ hours at -40°C, acidified with 5 ml of concentrated hydrochloric acid, then the cooling is stopped, 60 ml of water are added and the mixture is kept under stirring for 30 minutes at room temperature. The organic phase is concentrated under vacuum, the residue is taken up with isopropanol, and after stirring the product thus obtained is filtered. Thus the 4-nitrobenzyl 7-(2-amino- 2-phenyl)acetamido-9,12-epoxycyclohexeno(3,4-c]ceph-2-em-4-carboxyla te hydrochloride is obtained.

(ii) A solution of 11.68 g (21 mmoles) of the product obtained as described in the previous step (i) in 40 ml of dimethylformamide is made acid with diluted hydrochloric acid (1:1 v/v) until pH 1, then is hydrogenated at 25ºC and a pressure of 2.10⁵ pascal in the presence of 37 g of 5% Pd/C (50% wet). After 1½. hours the hydrogenation is stopped, the catalyst is removed and the pH of the mixture is adjusted to 4.5 with triethylamine, then is diluted with the same volume of acetonitrile. The product is filtered, treated with water and the suspension is acidified with diluted hydrochloric acid (1:1 v/v). The undissolved part is eliminated by filtration, and the pH of the solution is adjusted to 4.5, then the solution is concentrated under vacuum until a small volume. The crystalline product is the 7-(2-amino-2-phenyl)acetamido-9,12-epoxycyclohexano[3, 4-c]ceph-2-em-4-carboxylic acid having a purity of 93.5%.

EXAMPLES 25 - 31

By operating as described in the Example 24, starting from the products obtained in the Examples 17(a), 18(a), 19(a), 20(a), 21(a), 22(a) and 23(a), by (i)reaction with ethyl potassium Dane salt of D- phenylglycine and (ii) catalytic hydrogenation of cyclohexenocephem thus obtained, the compounds of the Examples from 25 to 31 are prepared.

Example 25

(i) 4-Nitrobenzyl 7-(2-amino-2-phenyl)acetamido-9,12-dimethyl-9, 12-epoxycyclohexeno[3,4-c]ceph-2-em-4-carboxylate hydrochloride.

(ii) 7-(2-amino-2-phenyl)acetamido-9,12-dimethyl-9,12-epoxycyclo hexano[3,4-c]ceph-2-em-4-carboxylie acid.

Example 26

(i) 4-Nitrobenzyl 7-(2-amino-2-phenyl)acetamido-9-hydroxymethyl-9, 12-epoxycyclohexeno[3,4-c]ceph-2-em-4-carboxylate hydrochloride.

(ii) 7-(2-amino-2-phenyl)acetamido-9-hydroxymethyl-9,12-epoxycyclo hexano[3,4-c]ceph-2-em-4-carboxylic acid.

Example 27

(i) 4-Nitrobenzyl 7-(2-amino-2-phenyl)acetamido-9,12-bis(hydroxyme thyl)-9,12-epoxycyclohexeno[3,4-c]ceph-2-em-4-carboxylate hydrochlori de.

(ii) 7-(2-amino-2-phenyl)acetamido-9,12-bis(hydroxymethyl)-9,12- epoxycyclohexano[3,4-c]ceph-2-em-4-carboxylic acid.

Example 28

(i) 4-Nitrobenzyl 7-(2-amino-2-phenyl)acetamido-9,12-bis(acetoxyme thyl)-9,12-epoxycyclohexeno[3,4-c]ceph-2-em-4-carboxylate hydrochlori de.

(ii) 7-(2-amino-2-phenyl)acetamido-9,12-bis(acetoxymethyl)-9,12- epoxycyclohexano[3,4-c]ceph-2-em-4-carboxylic acid.

Example 29

(i) 4-Nitrobenzyl 7-(2-amino-2-phenyl)acetamido-9-acetoxymethyl-9, 12-epoxycyclohexeno[3,4-c]ceph-2-em-4-carboxylate hydrochloride.

(ii) 7-(2-amino-2-phenyl)acetamido-9-acetoxymethyl-9,12-epoxycyclo hexano[3,4-c]ceph-2-em-4-carboxylic acid.

Example 30

(i) 4-Nitrobenzyl 7-(2-amino-2-phenyl)acetamido-9-(aminocarbonyl oxymethyl)-9,12-epoxycyclohexeno[3,4-c]ceph-2-em-4-carboxylate hydro chloride.

Example 31

(i) 4-Nitrobenzyl 7-(2-amino-2-phenyl)acetamido-9,12-bis(aminocar bonyloxymethyl)-9,12-epoxycyclohexeno[3,4-c]ceph-2-em-4-carboxylate hydrochloride.

(ii) 7-(2-amino-2-phenyl)acetamido-9,12-bis(aminocarbonyloxyme thyl-9,12-epoxycyclohexano[3,4-c]ceph-2-em-4-carboxylic acid.

EXAMPLE 32

11.12 g (20 mmoles) of 4-nitrobenzyl 7-(2-amino-2-phenyl)acetami do-9,12-epoxycyclohexeno[3,4-c]ceph-2-em-4-carboxylate hydrochloride, obtained as described in the Example 24(i), are dissolved in 50 ml of dimethylformamide and 50 ml of acetonitrile, then treated with 14,4 ml of concentrated hydrochloric acid and, portionwise, 3.9 g (60 mmoles) of zinc powder. After 30 minute-stirring at room temperature, the mixture is filtered on Dicalite ^®, diluted with 500 ml of acetonitrile and the pH is adjusted to 4.5 with triethylamine. The product thus obtained is filtered, treated with water and the suspension is acidified to pH 1 with hydrochloric acid, the undissolved part is eliminated by filtration. The pH of the solution is adjusted to 4.5 with triethylamine and the water is evaporated off under vacuum to an oil, which is treated with a small volume of a mixture of acetonitrile/methanol (1:1 v/v). After stirring the white crystalline product is filtered and is shown to be 7-(2-amino-2-phe nyl)acetamido-9,12-epoxycyclohexeno[3,4-c]ceph-2-em-4-carboxylic acid having a purity by HPLC of 95%.

EXAMPLE 33

To a suspension of 10.6 mmoles of 7-amino-9,12-epoxycyclohexano[3, 4-c]ceph-2-em-4-carboxylic acid, obtained as described in the Example 16, in 55 ml of dichloromethane, 36 mmoles of N,0-bis(trimethylsilyl) acetamide are added and the mixture is maintained under 30 minute- stirring in order to obtain a solution, which is treated with 11.5 mmoles of 2-[2-(2-aminothiazol-4-yl)-2-methoxyimino]acethylthiobenzo thiazole. The reaction mixture is stirred for 90 minutes and treated with water. The aqueous phase is separated, the pH is adjusted to 4 by adding triethylamine, then is concentrated to an oil, which is treated with acetonitrile. The crystalline product is (Z)-7-[2-(2-aml nothiazol-4-yl)-2-methoxyimino]acetamido-9,12-epoxycyclohexano[3,4-c] ceph-2-em-4-carboxylic acid having a purity by HPLC of 94%.

EXAMPLES 34 - 40

By operating as described in the Example 33, starting from the product obtained in the Examples 17(b), 18(b), 19(b), 20(b), 21(b), 22(b) and 23(b), the compounds of the Examples from 34 to 40 are respectively obtained.

Example 34

(Z)-7-[2-(2-aminothiazol-4-yl)-2-methoxyimino]acetamido-9,12-dime thyl-9,12-epoxycyclohexano[3,4-c]ceph-2-em-4-carboxylic acid .

Example 35

(Z)-7-[2-(2-aminothiazol-4-yl)-2-methoxyimino]acetamido-9,hydroxy methyl-9,12-epoxycyclohexano[3,4-c]ceph-2-em-4-carboxylic acid.

Example 36

(Z)-7-(2-(2-aminothiazol-4-yl)-2-methoxyimino]acetamido-9,12-bis (hydroxymethyl)-9,12-epoxycyclohexano[3,4-c]ceph-2-em-4-carboxylic acid.

Example 37

(Z)-7-[2-(2-aminothiazol-4-yl)-2-methoxyimino]acetamido-9,12-bis

(acetoxymethyl)-9,12-epoxycyclohexano[3,4-c]ceph-2-em-4-carboxylic acid.

Example 38

(Z)-7-[2-(2-aminothiazol-4-yl)-2-methoxyimino]acetamido-9-acetoxy methyl-9,12-epoxycyclohexano[3,4-c]ceph-2-em-4-carboxylic acid.

Example 39

(Z)-7-[2-(2-aminothiazol-4-yl)-2-methoxyimino]acetamido-9-aminocar bonyloxymethyl-9,12-epoxycyclohexano[3,4-c]ceph-2-em-4-carboxylic acid.

Example 40

(Z)-7-[2-(2-aminothiazol-4-yl)-2-methoxyimino]acetamido-9,12-bis (aminocarbonyloxymethyl)-9,12-epoxycyclohexano[3,4-c]ceph-2-em-4- carboxylic acid.

EXAMPLE 41

To a solution of 14.4 g (27 mmoles) of methyl 7-phenylacetamido-3- bromo-3-cephem-4-carboxylate in 200 ml di dichloromethane, 5.9 ml (81 mmoles) of furane and 9.2 ml (54 mmoles) of N,N-diisopropylethyl amine are added and the mixtured is heated to 50°C and is kept at this temperature for 16 hours. After elimination of the impurities by filtration, the solution si washed with acidulated water by hydrochloric acid. The organic phase is dried with anhydrous sodium sulfate, evaporated off to a crude prouct, which is purified by Silica gel column chromatography. Thus, the 4-nitrobenzyl 7-phenylace tamido-9,12-epoxycyclohexeno[3,4-c]ceph-2-em-4-carboxylate, identical to the product of the Example 1(a), is obtained.

EXAMPLE 42

By operating as described in the Example 41, starting from 4-nitro benzyl 7-phenylacetamido-3-iodo-3-cephem-4-carboxylate (obtained by reaction of the corresponding 3-mesyloxy derivative with 2 moles of lithium iodide) obtaining the same final product, identical to the compound of the Example 1(a).

Claims

1. Cyclohexanocephalosporins of formula I

in which X represents hydrogen or an acyl group Ac; R represents hydrogen or a radical of an ester group; X' and X", identical or different, represent independently hydrogen, alkyl, hydroxyalkyl, acyloxyalkyl, alcoxyalkyl, aryloxyalkyl, arylalcoxyalkyl, cycloalkyl oxyalkyl, cycloalkylalcoxyalkyl, alcoxyalcoxyalkyl, alkyloxycarbonyl alkyl, aryloxycarbonylalkyl, aralkyloxycarbonylalkyl, aminocarbonyl oxyalkyl, alkylaminocarbonyloxyalkyl, aralkylaminocarbonyloxyalkyl, aminoalkyl or acylamido alkyl; the corresponding pharmaceutically acceptable esters, salts and solvates.

2. Cyclohexanocephalosporins according to claim 1 of formula I, in which R is as defined in claim 1 and X, X' and X" are hydrogen, and the pharmaceutically acceptable salts and solvates thereof.

3. Cyclohexanocephalosporins according to claim 1 of formula I, in which R is as defined in claim 1, X is an acyl group Ac, X' and X" are identical or different and represent, independently, hydrogen, methyl, hydroxymethyl, (C₁-C₄)alkanoyloxymethyl, aminocarbonyloxyme thyl, aminomethyl, acylamidomethyl, as well as the pharmaceutically acceptable esters, salts and solvates thereof.

4. Cyclohexanocephalosporins according to claim 3, in which X is a protecting acyl group Ac, selected from the group consisting of formyl, benzyloxycarbonyl, t.butoxycarbonyl, trichloroacetyl, phenyl. acetyl.

5. Cyclohexanocephalosporins according to claim 3, in which X is an acyl group Ac selected from the group consisting of D(-)α-amino phenylacetyl, (Z)-2-(2-aminothiazol-4-yl)-2-methoxyiminoacetyl, (Z)- 2-(2-aminothiazol-4-yl)-2-(carboxymethoxyimino)acetyl and (Z)-2-(2- aminothiazol-4-yl)-2-(carboxy-1-methylethoxyimino)acetyl.

6. Cyclohexanocephalosporins of formula I'

wherein R represents hydrogen, a cation or a radical of an ester group and their pharmaceutically acceptable salts and solvates .

7. Cyclohexanocephalosporins of formula la

wherein X' and X" are as defined in claim 1, and their pharmaceutically acceptable esters, salts and solvates.

8. A process for the preparation of cyclohexanocephalosporins according to claim 1, which comprises:

(a) treating an ester of 7-phenylacetamido-3-cephem-4-carboxylic acid or a mixture of Δ ²+Δ ³ isomers of formula II

wherein X' and X" are as defined above, in the presence of a base;

(b) subjecting the cyclohexenocephem thus obtained of formula IVa'

wherein R, X' ed X" are as defined above and, possibly, after having subjected the compound IVb' to a carboxy deprotection giving rise to a compound of formula IVb

and to a subsequent catalytic hydrogenation to obtain the compound of formula lb

9. A process for the preparation of cyclohexanocephalosporins according to claim 1, which comprises:

(a) treating an ester of 7-phenylacetaraido-3-cephem-4-carboxylic acid or a mixture of Δ ²+Δ³ isomers of formula II

wherein X' and X" are as defined above, in the presence of a base;

(b) optionally subjecting the cyclohexenocephem compound thus obtained of formula IVa'

wherein R, X' ed X" are as defined above and, subsequently, to a reaction with a reactive derivative of an acid of formula AcOH, wherein Ac has the meaning defined in claim 1 for X, but is other than phenylacetyl, to obtain the compound IVc'

and then subj ecting the cyclohexenocephem compound thus obtained, wherein R represents hydrogen or a carboxy protecting group, Ac , X ' and X" are as defined above, to a deprotection of the carboxylic group , in case it has not been carried out , to obtain a cyclohexenocephemcarboxylic acid of formula IVc

and, when R is a carboxy protecting group, to be removed by means of reduction, the compound IVc' is subjected to a catalytic hydrogenation to obtain the compound of formula Ic, which is optionally transformed into the corresponding pharmaceutically acceptable esters, salts or solvates thereof.

10. A process according to claims 8 and 9, in which a compound of formula II, wherein R is 4-nitrobenzyl or diphenylmethyl and W is a mesyloxy, p.tosyloxy or trifluoromethanesulfonyloxy group, is used as starting material.

11. A process according to claims 8 and 9, in which a compound of formula III, wherein X' and X", independently, are hydrogen, methyl, hydroxymethyl, (C₁-C₄)alkanoyloxymethyl or aminocarbonyloxymethyl is used as starting material.

12. A process according to claims 8 and 9, in which an organic based is used.

.

13. A process according to claim 12, in which the organic base is a tertiary amine.

14. A process according to claim 13, in which the organic base is triethylamine, diisopropylamine, 4-methylmorpholine, 1-methylpiperidi ne.

15. Cyclohexenocephalosporins of formula IV

wherein X, X' and X" are as defined in claim 1, with the proviso that, when X' and X" are both hydrogen, X is other than phenylacetyl and its pharmaceutically acceptable esters, salts and solvates.

16. Cyclohexenocephalosporins according to claim 15, wherein X is hydrogen, X' and X", independently, are hydrogen, methyl, hydroxy. methyl, (C₁-C₄)alkanoyloxymethyl, aminocarbonyloxymethyl and the corresponding pharmaceutically acceptable esters, salts and solvates thereof.