NO782001L - NEW CEPHALOSPORINE DERIVATIVES, MANUFACTURING PROCEDURES AND COMPOSITIONS CONTAINING THE SAME - Google Patents

Abstract

Novel cephalosporin derivatives, process for their preparation and compositions containing the same.

Description

The present invention relates to new cephalosporin derivatives with the general formula

as well as addition salts thereof with acids and possibly metal salts thereof and addition salts thereof with nitrogen-containing organic bases. The invention also relates to the production of the new cephalosporin derivatives as well as pharmaceutical preparations containing these.

In the general formula I, R denotes a carboxy group

(either in free form or in the form of a metal salt or addition

salt with an organic base) or a group of the general formula

where R^ denotes a hydrogen atom or an alkyl group containing 1 to 4 carbon atoms, and R 2 denotes a straight or branched alkyl group containing 1 to ^ carbon atoms or a cyclohexyl group, whereby the group

is a group that is easy to remove enzymatically such as

pivaloyloxymethyl.

The compound of formula I derives from the formulas D, L and DL of an amino acid of the formula

The invention relates to both the diastereoisomeric forms of the compound of formula I as well as mixtures thereof.

According to the invention, the new compounds of formula I can be prepared by reacting the acid of formula IV, which exists in racemic form or in optically active form and whose amino group is protected in advance, or a reactive derivative of this acid, with a cephalosporin with the general formula

where R has the above meaning.

a) When using the acid of formula IV, the amino group can be protected in any known way to block an amino group without affecting the rest of the molecule. It is necessary to use a group which is easy to remove such as tertiary butoxycarbonyl or 2,2,2-trichloroethoxycarbonyl. It is particularly appropriate to use the group tert-butoxycarbonyl which can be introduced by reaction with di-tert-butyl dicarbonate, tert-butyl azidoformate, tert-butyl chloroformate or tert-butyl 1-p-nitrophenylcarbonate (mixed carbonate).

It is also possible to protect the amino group. in the form of an enamine of the general formula where R, denotes an alkyl group containing 1 to 4 carbon atoms and R^ denotes an alkyl group containing 1 to 4 carbon atoms,

an alkoxy group whose alkyl part contains 1 to 4 carbon atoms or a phenyl group.

The enamine of the general formula VI can be prepared according to the method described by E. Dane et al., "Chem. Ber.", 98, 789 (1965).

a) When R denotes a carboxy group, condensation usually

show the compound of formula IV, whose acid group is free and

whose amino group is pre-protected, with 7_amino-3-des-acetoxycephalosporanic acid whose acid group is pre-protected with a readily removable group such as benzhydryl, tert-butyl or 2,2,2-trichloroethy1.

Generally, the condensation is carried out in an organic solvent such as dimethylformamide, acetonitrile, tetrahydrofuran or chloroform in the presence of a condensing agent such as a carbodiimide (e.g. dicyclohexylcarbodiimide) or N,N'-carbonyldimidazole, at a temperature between 4 and 40° C.

The protective group for the amino group and the acid group is then removed.

Depending on the nature of the protecting groups, this may

removal is carried out in one or two steps.

When the protecting group for the amino group is a tertiary butyloxycarbonyl group, the removal is carried out in one of the following ways, depending on the nature of protecting groups for the acid group. 1) When the acid group is protected by means of a tertiary butyl group or a benzhydryl group, the process is carried out in a single step by treatment in an acidic medium. One preferably uses trifluoroacetic acid and works at a temperature between 0 and 20°C (and in the presence of anisole when the protecting group is a benzhydryl group). Alternatively, paratoluene sulphonic acid is used. Under these conditions, the connection is achieved with formula I in the form of trifluoroacetate or paratoluenesulfonate,

whose amino group can be liberated according to any known method for the preparation of an amine starting from a salt thereof without affecting the rest of the molecule. One can in particular bring the salt into contact with an ion exchanger (e.g. a polystyrene amino ion exchanger such as "Amberlite IR-45") or in

contact with an organic base.

2) When the acid group is protected with a 2,2,2-trichloroethyl group, treatment with zinc in acetic acid is first carried out, after which the tertiary butyloxycarbonyl group is removed by treatment in an acidic medium. The treatment in an acidic medium is carried out using trifluoroacetic acid. Hereby, the compound of formula I is obtained in the form of trifluoroacetate and the amine can be released from its salt in the manner described above.

When the protecting group for the amino group is a 2,2,2-trichloroethoxycarbonyl group, the removal of the protecting group is carried out in one of the following ways, depending on the nature of the protecting group for the acid group. 1) When the protecting group for the acid group is a tertiary butyl group or a benzhydryl group, treatment with zinc in acetic acid is carried out first and then treatment in an acidic medium, preferably using trifluoroacetic acid. 2) When the protecting group for the acid group is a 2,2,2-trichloroethyl group, treatment with zinc in acetic acid is carried out.

When the amino group is protected in the form of an enamine, removal of the protecting groups is carried out in one of the following ways, depending on the nature of the protecting group for the acid group. 1) When the protecting group for the acid group is a tertiary butyl group or a benzhydryl group, hydrolysis is first carried out in a dilute acidic medium, e.g. in the presence of hydrochloric acid, and then treatment with trifluoroacetic acid. 2) When the protecting group for the acid group is a 2,2,2-trichloroethyl group, hydrolysis is first carried out in an acidic medium, e.g. in the presence of hydrochloric acid, and then treatment with zinc in acetic acid.

When R represents a group of the general formula II, the acid of formula IV is usually condensed with the cephalosporin derivative of formula V in an organic solvent such as dimethylformamide or chloroform in the presence of a condensing agent such as a carbodiimide (e.g. dicyclohexylcarbodiimide) or N,N'-carbonyldiimidazole, at a temperature between 0 and 40°C, after which the protecting group for the amino group is removed under the above conditions.

b) When using a derivative of the acid of formula IV, it is appropriate to use an anhydride, a mixed anhydride or a reactive ester of the general formula

where R 1 - denotes a succinimido group, a benzotriazol-1-yl group or a 2,4-dinitrophenyl group, and whose amino group is protected in advance. It is also appropriate to use acid chlorides whereby the hydrochloride of the acid chloride of the acid of formula IV is reacted with the cephalosporin of formula V. It is also possible to use Leuch's anhydride. When using the anhydride, a mixed anhydride, Leuch's anhydride or the acid chloride, which can be prepared in situ, the condensation is carried out in an organic solvent such as tetrahydrofuran, chloroform or methylene chloride, in the presence of an acid acceptor such as a nitrogenous organic base, eg pyridine or triethylamine, or in an aqueous organic medium in the presence of a alkaline condensing agent such as sodium dicarbonate, and one works at a temperature between -40 and +40° C. One then optionally replaces the protecting group for the amino group with a hydrogen atom. When R denotes a carboxy group, it is not necessary to protect the acid group.

When using a reactive ester of the general formula VII, one usually works in the presence of triethylamine in an organic solvent such as dimethylformamide at a temperature between 0 and 40°C, after which one replaces the protecting group for the amino group with a hydrogen atom. When R denotes a carboxy group, it is not necessary to protect the acid group.

The acid of formula IV can be prepared according to one of the following methods. a) By deformylation of α-formylamino-(1,3_dithiin-5-yl)-acetic acid.

This reaction is usually carried out in an aqueous acidic medium at a temperature between 0 and 100°C. A water solution of a mineral acid such as hydrochloric acid is preferably used at a temperature close to 100°C.

α-formylamino-(1,3_dithiin-5_yl)-acetic acid can be prepared by saponification of the corresponding ester with the general formula

where Rg denotes an alkyl group containing 1 to 4 carbon atoms, whereby one works under conditions which enable the saponification of an ester to the corresponding acid without the rest of the molecule being affected.

The ester to be saponified is usually treated with an alkali metal hydroxide in a water-alcohol medium at a temperature between 0 and 50°C. The methyl ester or ethyl ester is preferably used and the saponification is carried out in a mixture of water and methanol or in a mixture of water and ethanol at a temperature close to 5°C.

The ester with the formula VII can be prepared by et

isocyanoacetate with the formula

CN - CH2 - COORg (IX)

where Rg has the meaning given above, is reacted with 1,3-dithiacyclohexan-5-one.

This reaction is usually carried out in an anhydrous organic solvent such as tetrahydrofuran in the presence of an alkaline condensing agent such as potassium tert-butylate, and at a temperature between -70 and 0°C.

Isocyanoacetate of the formula IX can be prepared according to

the method described by U. Schøllkopf et al.,

in "Chem. Ber.", 108, 1580 (1975).

1,3_dithiacyclohexan-5-one can be prepared according to the method described by E. B. Howard and R. V. Lindsey

• in "J. Amer. Chem. Soc", 82, 158 (1960).

The optically active forms of the acid with formula IV can be prepared by starting from the racemate either enzymatically or by applying physicochemical methods.

b) When saponifying an ester with the general formula

where denotes an alkyl group containing 1 to 4 carbon atoms, after which the protecting group for the amino group is removed.

The saponification is suitably carried out in a water-alkhol medium whereby an alkali metal hydroxide is treated at a temperature between 0 and 50°C.

Sodium hydroxide is preferably used and the methyl ester or ethyl ester is treated in a mixture of water and methanol or in a mixture of water and ethanol at a temperature close to -20°C.

When one wishes to obtain a compound of formula IV whose amino group is free, the protecting group is usually removed by means of trifluoroacetic acid at a temperature between 0 and 30°C.

If one wishes to obtain the D-form or the L-form of the acid with formula IV, it may be appropriate to carry out separation of the optically active forms before removing the protective group for the amino group.

One can e.g. treat the racemic form with quinine in a solvent such as methyl ethyl ketone.

The salts with forms D and L are purified by crystallization. The free acids with the forms D and L are isolated by starting from these salts after which the protecting group for the amino group is removed to form the forms D and L of the amino acid with the formula IV.

The ester with the formula X can be prepared by reacting potassium tert-butylate with a compound of the formula

where R^ has the meaning given above.

One usually works in an organic solvent such as tetrahydrofuran at a temperature between -78 and 0°C.

The compound of formula XI can be prepared by reacting an alkyl isothiocyanatoacetate (e.g. ethyl isothiocyanatoacetate) with 1,3-dithiacyclohexan-5-one in the presence of potassium tert-butylate, after which the resulting product is reacted with di-tert-butyldicarbonate.

One usually works in a solvent such as

tetrahydrofuran at a temperature between -78 and 0°C.

The compound of formula V where R denotes a carboxy group is 7-amino-3-desacetoxycephalosporanic acid (or 7-ADCA). This acid can be prepared either by starting from a penicillin according to the method described in Belgian patent no. 747,382 or by desacetoxylation of 7-aminocephalosporanic acid (or 7-ACA) according to the method described in Belgian patent no. 779 -034.

The compound of formula V in which R denotes a group of formula II in which R 1 and R 2 have the meaning indicated above can be prepared starting from 7-amino-3-desacetoxycephalosporic acid according to any known method for the preparation of an ester by starting from an acid without the rest of the molecule being affected.

An alkali metal salt or a salt with a tertiary amine of 7-amino-3-cesacetoxycephalosporanic acid is usually reacted with a halide of the general formula

where R1 and R2 have the meanings given above and Z denotes a halogen atom, in an inert solvent such as dimethylformamide at a temperature between 0 and 30°C.

The compound with the formula XII can be prepared according to the method described in DE-OS 2,350,230.

The compounds of formula I in which R denotes a group of formula II in which R-^ and Rp have the meaning given above can also be prepared by esterification of a compound of formula I in which R denotes a carboxy group and whose amino group is protected in advance by application of any known method for producing an ester from an acid without affecting the rest of the molecule.

Generally, an alkali metal salt or a salt with a tertiary amine of a compound of formula I in which R denotes a carboxy group and whose amino group is protected in advance, is reacted with a halide of the general formula XII in which R-, , R 2 and Z have the above indicated importance. One preferably works in an inert solvent such as dimethylformamide at a temperature between 0 and 30°C, after which the protective group for the amino group is removed in any known manner.

The diastereoisomeric forms of the compounds of formula

I can also be produced by starting from mixtures of these by applying known methods. It is possible to carry out this separation at various stages of the synthesis. The separation can e.g. is carried out in that a compound of formula I whose amino and acid group is protected is chromatographed on a suitable support. The diastereoisomers of the compound of formula I are then isolated after removal of the protecting groups under usual conditions.

The new compounds of formula I can optionally be purified by applying physical methods such as crystallization or chromatography.

The new compounds of formula I can be converted into addition salts with acids. According to the invention, the compound of formula I is prepared in the form of trifluoroacetate or paratoluenesulfonate. The compounds of formula I which are prepared in the form of these salts can be liberated and converted into salts with other acids according to usual methods.

The compounds of formula I where R denotes a carboxy group can also be converted to metal salts or to addition salts with nitrogen-containing organic bases according to well-known methods. These salts can be prepared by reacting a metal base (eg an alkali metal base or an alkaline earth metal base) or an amine with a compound of formula I in a suitable solvent such as an alcohol, an ether or water. The salts can also be prepared by yield reaction with a salt of an organic acid. The salt formed falls out, possibly after concentration of the solution and is removed by filtration or decantation.

The new cephalosporin derivatives according to the invention exhibit very interesting antibacterial properties. They have a remarkable activity both in vivo and in vitro against both Gram-positive and Gram-negative bacteria.

In vitro, the compounds of formula I are active at a concentration of 2 to 15 ug/cm^ against Staphylococcus strains sensitive to penicillin G (Staphylococcus aureus Smith), at a concentration of 10-150 ug/cm^ against Staphylococcus- strains resistant to penicillin G (Staphylococcus aureus MB 9), at a concentration of 0.125-1.5 yg/cm against Streptococcus 'pyogenes Dig' 7, at a concentration of 2-30 yg/cm-' against Escherichia coli strain Monod , in a concentration of 4-30 yg/cm'' against Klebsiella pneumoniae, in a concentration of 1-8 yg/cm^. against Salmonella typhi, in a concentration of 1-8 yg/cir - 7. against Chigella flexneri and in a concentration of 5~20 yg/cm^ against Proteus,mira-bilis.

In vivo, the compounds of formula I are active against experimentally induced bacterial infections in mice. In an oral or subcutaneous dose of 0.05-0.6 mg/kg, the compounds are thus effective against Staphylococcus aureus Smith' (susceptible to penicillin G), and in an oral or subcutaneous dose of 1-6 mg/kg, the compounds are effective against Escherichia coli.

When administered subcutaneously to mice, the compound of formula I is non-toxic in doses of 2.5 g/kg.

Of very particular interest are the compounds of formula I in which R denotes a carboxy group, especially the compound in which the amino acid bound to the cephalosporin nucleus is in the D form.

The invention shall be illustrated by the following non-limiting examples.

Example 1

To a solution of 13 g of D,L-a-tert-butoxycarbonylamino-(1,3_dithiin-5-yl)-acetic acid in 160 cm3 of tetrahydrofuran, 6.25 cm3 of triethylamine is added while stirring. The resulting mixture is cooled to -10°C, after which 5.8 cm^ of isobutylchloroformate is added dropwise. The reaction mixture is stirred for 10 min. at -10°C and then a solution of 9354 g of 7-amino-2-carboxy-3-methyl-8-oxo-5-thia-1-azabicyclo-/4.2.0/-oct-2-ene is added in a mixture of 140 cm^ of tetrahydrofuran,

140 cnr 3 water and 6.25 cm 3 triethylamine. The resulting reaction mixture is stirred at 0°C for 30 min. and then at 20°C

for 90 min. The solvent is evaporated under a reduced pressure of 20 mm Hg at 30°C. 300 cm^ of water and 50 cm^ of a saturated aqueous solution of sodium bicarbonate are then added, after which extraction is carried out with 500 cm^ of ethyl acetate. The water phase is separated and made acidic to pH 2.0 by adding 4N hydrochloric acid in the presence of 500 cm 3 ethyl acetate. The organic phase is separated

and re-extract with 200 cc of ethyl acetate. The two latter organic phases are combined, washed with 150 cm^ of a saturated aqueous solution of sodium chloride, dried over sodium sulfate<p>and filtered in the presence of decolorizing charcoal. Concentrate to dryness

condition under a reduced pressure of 20 mm Hg at 30°C. 18.7 g of 7~Z~D, L-a-tert-butoxycarbonylamino-(1,3-dithiin-5-yl)-acetamido7-2-carboxy-3-methyl-8-oxo-5-thia-1-azabicyclo are obtained in this way -Z7l. 2 .0_7-oct-2-en in the form of a white meringue-like mass.

2.7 g of 1~ IS>S L-a-tert-butoxycarbonylamino-(1,3_dithiin-5_yl)-acetamide Q7-2-carboxy-3-methyl-8'-oxo-5-thia-1-azabicyclo-Z7i • 2.0_7-oct-2-ene in 3 cm^ of anisole and 15 cm^ of trifluoroacetic acid. The obtained solution is stirred at 20°C for 15 min. It is then concentrated to dryness under a reduced pressure of 0.5 mm Hg at 30 o C. The residue is taken up in 15 ml of ethyl acetate and then 100 ml of isopropyl ether is added. This forms a white precipitate which is separated by filtration. 2.4 g of impure trifluoroacetate of 7-[D,L-a-amino-(1,3-dithiin-5-yl)-acetamido_7-2-carboxy-3-methyl-8-oxo-5-thia-1-azabicyclo- Z"4 . 2 . 0_7-oct-2-ene in the form of a white solid.

A solution of 11.9 g of trifluoroacetate of 7~£b ,L-ot-amino-(1,3,dithiin-5-yl)-acetamido7-2-carboxy-3-methyl-8-oxo-5_thia-1 -azabicycld-/f4 . 2. The 0_7-oct-2-ene in 500 cm 3 of distilled water is extracted with 3 times 100 cc of ethyl acetate. The water phase is treated with 50 cm^ of moist ion exchanger "Amberlite IR-45" in OH form until the pH value is stabilized at 5.25 (approx. 1 hour). The ion exchanger is separated by filtration. The water phase is lyophilized. 5.7 g of 7~£D, L-α-amino-(1,3-dithiin-5~yl)-acetamido7-2-carboxy-3-methyl-8-oxo-5-thia-1-azabicyclo- /I4 . 2. 0_7-oct-2-ene with the following properties: a^<0>= + 131.2<1>2° (c - 0.98; water)

Elemental analysis: Calculated, % : C 43.39 H 4.42 N 10.84

0 16.52 S 24.83

Found, % : C 42.6 H 3.8 N 10.7

S 24,6

o IR spectrum: Characteristic bands:

3300-2200 cm"<1>(NH-amide and NH +)

-1

1755 cm. : carbonyl of g-lactam 1685 cm<-1>: -CONH-

1575 cm<-1>: -C00H

D,L-a-tert-butoxycarbonylamino-(1,3-dithiin-5-yl)-acetic acid can be prepared in the following manner. To a suspension of 19.1 g of D,L-α-amino-(1,3_dithiin-5-yl)acetic acid in a mixture of 130 cm^ of dioxane and 130 cm^ of water, first add 10.6 g of sodium carbonate and then a solution of 24 g of di-tert-butyl dicarbonate in 130 cm^ of dioxane. The suspension is stirred for 18 hours at 20°C. The solvent is then evaporated under a reduced pressure of 20 mm Hg at 40°C. 200 cm^ of water and 50 cm^ of a saturated aqueous solution of sodium bicarbonate are added. It is washed 3 times with 300 cc of ethyl acetate. The water phase is acidified to pH 1.5 with 4N hydrochloric acid. It is extracted with 2 times 250 cm^ of ethyl acetate. The combined organic extracts are washed with 150 cc of a saturated sodium chloride solution and dried over sodium sulfate. The solution is treated with decolorizing charcoal after which it is filtered and concentrated to dryness under a reduced pressure of 20 mm Hg at 50°C. 26 g of D,L-α-tert-butoxycarbonylamino-(1,3-dithiin-5-yl)-acetic acid is obtained in the form of a white meringue-like mass.

DAL-α-amino-(1,3-dithiin-5-yl)-acetic acid can be prepared in the following way. A suspension of 63 g of D,L-α-formylamino-(1,3-dithiin-5-yl)-acetic acid in 320 cm 2 4N hydrochloric acid is heated to 90°C until complete dissolution. It is then cooled to 20°C and treated with decolorizing charcoal. The solution is filtered, after which the pH value is adjusted to 4.5 by adding 4N sodium hydroxide solution. The precipitate formed is separated by filtration. This gives 41 g of D,L-α-amino-(1,3-dithiin-5-yl)-acetic acid in the form of a white solid which melts at 270°C during cleavage.

D,L-α-formylamino-(1,3-dithiin-5-yl)-acetic acid can be prepared in the following way. A suspension of 72 g of an equilibrium mixture of ethyl-α-formylamino-(4,5-dihydro-1,3-dithiin-5-ylidene)-acetate and ethyl-D,L-α-formylamino-(1,3- dithiin-5-yl)-acetate in 720 cm^ of ethanol and 200 cm^ of water is cooled to 5°C. 80 cm^ of 4N sodium hydroxide solution is added and it is stirred. resulting reaction mixture at 5°C for 90 min. One then concentrates to a. volume of 100 cm 3 under a reduced pressure of 20 mm Hg. The obtained solution is cooled in an ice bath and acidified to pH 2.0 by the addition of 4N hydrochloric acid. The precipitate formed is separated by filtration. 67 g of impure D,L-a-formylamino-(1,3-dithiin-5-yl)-acetic acid are thereby obtained in the form of a white solid.

The mixture of ethyl α-formylamino-(4,5-dihydro-1,3-dithiin-5-ylidene)-acetate and ethyl D,L-α-formylamino-(1,3-dithiin-5-yl)-acetate can is produced in the following way. A solution of 27.2 g of potassium tert-butylate in 200 cirr 3 of tetrahydrofuran is cooled to 0°C. A solution of 25 g of ethyl isocyanoacetate in 150 cm² of tetrahydrofuran is added dropwise. The reaction mixture is stirred for 1 hour at 0°C and then a solution of 29.6 g of 1,3-dithiacyclohexan-5-one in 375 cm 3 of tetrahydrofuran is added. The reaction mixture is stirred at 0°C for 90 min. 50 cm 3 of acetic acid is then added and the precipitate formed is separated by filtration. The solvent is evaporated under a reduced pressure of 20 mm Hg at 30°C. The residue is taken up in 2500 cm^ of methylene chloride. It is extracted with a mixture of 500 cm^ of water and 300 cm^ of a saturated aqueous solution of sodium bicarbonate. The organic phase is washed with 500 cm 3 of water and dried over sodium sulfate. The solution is treated with decolorizing charcoal, filtered and concentrated to dryness under a reduced pressure of 20 mmHg at 30 o C. The residue is vigorously stirred with 400 cirr3 of ethyl ether. The precipitate obtained is separated by filtration. 18.5 g of ethyl-α-formylamino-(4,5-dihydro-1,3-dithiin-5-ylidene)-acetate are thereby obtained. The filtrate is concentrated to dryness under a reduced pressure of 20 mm Hg at 20°C. The residue is purified by chromatography on 250 g of silicon dioxide in a column with a diameter of 3.7 cm and a height of 47 cm. One elutes with 2 liters of a mixture of methylene chloride and ethyl acetate in a volume ratio of 85:15* 4.4 g of a mixture of ethyl-a-formylamino-(4,5-dihydro-1,3-dithiin-5) is thereby obtained -ylidene)-acetate and ethyl-α-formylamino-(1,3-dithiin-5-yl)-acetate.

Ethyl isocyanoacetate can be prepared according to the method described by U. Schøllkopf, D. Hoppe and R. Jentsch, "Chem. Ber," 108, 1580 (1975).

1,3-dithiacyclohexan-5-one can be prepared according to it

method described by E. G. Howard and R. V. Lindsey, "J. Amer. Chem. Soc", 82, 158 (1960).

Example 2

To a stirred solution of 102 g of D-α-tert-butoxycarbonylamino-(1,3-dithiin-5-yl)-acetic acid in 1330 cm 3 of tetrahydrofuran is added 49 cm 3 of triethylamine. The reaction mixture is cooled to -10°C and 48.4 cm 3 of isobutyl chloroformate is added dropwise. The reaction mixture is stirred for 15 min. at -10°C

and then a solution of 75 g of 7-amino-2-carboxy-3-methyl-8-oxo-5-thia-1-azabicyclo-Z1t is added. 2. 0_7-oct-2-ene in a mixture of 1190 cm 3 of tetrahydrofuran and 49 cm 3 of triethylamine. The reaction mixture is stirred at 0°C for 1 hour and then at 20°C for 2 hours. The solvent is evaporated under a reduced pressure of 20 mm Hg at 40°C. 1000 cm<5> of water and 200 cm<5> are added

of a saturated aqueous solution of sodium bicarbonate, after which extraction is carried out with 1000 cm^ of ethyl acetate. The water phase is acidified to

pH 2 by addition of 4N hydrochloric acid in the presence of 1000 cirr ethyl acetate. The organic phase is separated and extracted a further 2 times with 500 ml of ethyl acetate. The organic phases are combined, washed with 1000 cm 3 of a saturated aqueous solution of sodium chloride, dried over magnesium sulfate, filtered and concentrated to dryness under a reduced pressure of 20 mm Hg at 30°C. The residue is treated with 1500 cc of cyclohexane. The insoluble substance is filtered off and dried. 158.5 g of 7-/- b-a-tert-butoxycarbonylamino-(1,3-dithiin-5-yl)-acetamido7-2-carboxy-3-niethyl-8-oxo-5-thia-1 are thereby obtained -azabicyclo- Z"4 . 2 . 07-oct-2-ene in the form of an impure white powder.

354.5 g of a product formed during the above mentioned

3 o

conditions are dissolved in 1770 errr methanol at 35 C. 145 cm^ dicyclohexylamine is added. and then 1770 cm^ of acetone. The reaction mixture is allowed to stand for 90 min. at 0°C after which the formed crystals are filtered off and dried under a reduced pressure of 1 mm Hg at 20°C. 326.4 g of the dicyclohexylamine salt of 7-[D-α-tert-butoxycarbonylamino-(1,3-dithiin-5-yl)-acetamido7-2-carboxy-3-methyl-8-oxo-5- thia-1-azabicyclo-Z"4.2.0_7-oct-2-ene in the form of white crystals.

■A suspension of 29.5 g of the above-mentioned salt in 300 cm^ of water is acidified to a pH value of 2 by adding 4N hydrochloric acid in the presence of 3-00 cm^ of ethyl acetate. It is separated

organic phase and extract a further 2 times with a total of 150 cm-' of ethyl acetate. The organic phases are combined, washed with 150 cm^ of a saturated aqueous solution of sodium chloride, dried over sodium sulfate, filtered and concentrated to dryness under a reduced pressure of 20 mm Hg at 30°C. 21.5 g of 7- £D-a-tert-butoxycarbonylamino-(1,3-dithiin-5_yl)_acetamido7-2-carboxy-3_methyl-8-oxo-5-thia-1-azabicyclo-D\.2.Q7-oct-2-ene in the form of a white solid.

IR spectrum (KBr): characteristic bands

3320 cm^ (NH amide and carbamate)

1770 cm<-1> (carbonyl of Ø-lactam)

1700 cm ^ (carbonyl of acid and of carbamate)

l680 cm ^ (carbonyl of amide)

1390' and 1365 cm-1 (tert-butyl)

A solution of 20.3 g of 7-ZX,-α-tert-butoxycarbonylamino-(1,3-dithiin-5-yl)-acetamido7-2-carboxy-3-methyl-8-oxo-5-thia-1-azabicyclo- Z7h. 2. Q7-oct-2-ene in 200 cm-5 trifluoroacetic acid is stirred for 20 min. at approx. 20°C. The solution is set within 30 min. and with stirring to 1000 cm-5 ethyl ether at

0°C. The precipitate formed is filtered off and dried under a reduced pressure of 1 mm Hg at 20°C. 15.7 g of trifluoroacetate of 7-β-D-α-amino-(1,3-dithiin-5-yl)-acetamido7-2-carboxy-3-methyl-8-oxo-5-thia-1-azabicyclo are obtained -£4 .2.07-Oct-2-ene which dissolves in 750 cm-<5>acetone. 3.2 cm<5>triethylamine is then added to adjust the pH value. to 4.8. The precipitate formed is filtered off and washed with 400 cm<5>ethyl ether. 10.6 g of 7-[D-a-amino-(1,3-dithiin-5-yl)-acetamido]-2-carboxy-3-methyl-8-oxo-.5-thia-1-azabicyclo-Z^ are thereby obtained. 2. 0_7-Oct-2-en in the form of a beige solid.

14.45 g of 7-Zb-α-aminoj-(1,3-dithiih-5-yl)-acetamido7-2-carboxy-3-methyl-8-oxo-5-thia-1-azabicyclo-Z~ are dissolved 4. 2. Oj7-3 - 3 Oct-2-en 1,750 cm of water, after which 750 cirr of acetonitrile is added. After 1 hour, the formed crystals are filtered off and dried under a reduced pressure of 1 mm Hg at 20°C. 9.8 g of 7~.£D-α-amino-(1,3-dithiin-5-yl)-acetamido-7-2-carboxy-3-methyl-8-oxo-"5-thia-1-azabicyclo-A are thereby obtained. 2. 0.7-oct-2-ene in the form of white crystals.

f a7p°= + 137 ~ 2° (c = I, 0.1 N sodium bicarbonate)

Elemental analysis: Calculated, % : C 43.39 H 4.42 N 10.84

0 16.52 S 24.83

Found, % : C.43.9 H 4.9 N 10.3

016.6 S 23.5 D-a-tert-butoxycarbonylamino-(1,3-dithiin-5-yl)-acetic acid can be prepared in the following way. A solution of 212 g of D,L-a-tert-butoxycarbonylamino-(1,3_dithiin-5-yl)-acetic acid and 236 g of quinine in 4300 cir of methyl ethyl ketone is refluxed. After cooling, the reaction mixture is stirred for 3 hours. 169 g of white crystals are separated by filtration, which are removed. The filtrate is stirred for 16 hours at room temperature. After filtration, 150 g of white crystals are isolated.

A suspension of 375 g of crystals obtained in this way

in 3750 cm<5>methyl ethyl ketone is heated at 80°C until complete dissolution. The resulting solution is cooled, after which the formed crystals are separated by filtration. 187 g of quinine salt of D-a-tert-butoxycarbonylamino-(1,3-dithiin-5-yl)-acetic acid is obtained in the form of white crystals.

98.5 g of the quinine salt of D-α-tert-butoxycarbonylamino-(1,3_dithiin-5-yl)-acetic acid are added to a mixture of 2 liters of water and 2 liters of ethyl ether, whereby the pH value is kept at 11 by the addition of 4N sodium hydroxide solution. After dissolution, the aqueous phase is decanted and acidified to pH 1 by adding 4N hydrochloric acid in the presence of 1 liter of ethyl ether. The organic phase is decanted and extracted with a further 1 liter of ethyl ether. The organic extracts are combined and dried over magnesium sulfate. It is concentrated to dryness under a reduced pressure of 20 mm Hg at 30°C. 41.7 g of D-a-tert-butoxycarbonylamino-(1,3-dithiin-5-yl)-acetic acid are thereby obtained in the form of a yellow oil.

5 g of the acid thus obtained is dissolved in a mixture of

3 3 10 cm of isopropyl ether and 25 cm of cyclohexane under reflux. After cooling, the formed crystals are filtered off and 4 g of D-α-tert-butoxycarbonylamino-(1,3-dithiin-5~yl)-acetic acid is obtained in the form of white crystals with a melting point of 110°C (Kofler).

ra7p°= - 121 - 2° (c = 1, . dimethylformamide)

D,L-α-tert-butoxycarbonylamino-(1,3-dithiin-5-yl)-acetic acid can be prepared in the following way. A suspension of 0.96 g of ethyl α-tert-butoxycarbonylamino-(4,5-dihydro-1,3-dithiin-5-ylidene)-acetate in 7 cm<5>ethanol and 3.3 cm<5>IN sodium hydroxide solution is stirred at 20°C for 40 min. It is then concentrated to a volume of 5 era<5> under a reduced pressure of 20 mm Hg at 30°C. The obtained solution is acidified to pH 2.0 by the addition of IN hydrochloric acid in the presence of 25 cm<5> of ethyl ether. The organic phase is decanted and dried over sodium sulfate. The solution is filtered and concentrated to dryness under a reduced pressure of 20 mm Hg at 30°C. 0.85 g of D,L-α-tert-butoxycarbonylamino-(1,3-dithiin-5-yl)-acetic acid is thereby obtained with the following analysis.

Elemental analysis: Calculated, % : C 45.34 H 5.88 N 4.80

0 21.97 S 22.01

Found, C44.8 H5.7 N4.7

S 21.5 The ethyl α-tert-butoxycarbonylamino-(4,5-dihydro-1,3-dithiin-5-ylidene) acetate can be prepared in the following way. A solution of 5.35 g of potassium tert-butylate in 40 cm<5>tetrahydrofuran is cooled to -70°C. A solution of 9 g of 3-tert-butoxycarbonyl-4-ethoxycarbonyl-2-thioxo-1-oxa-7,9-dithia-3-azaspiro-Z?4 is added dropwise. 57-decane in 40 cm<5>tetrahydrofuran. The reaction mixture is stirred for 3 hours and then 2.9 g of acetic acid is added. The temperature of the reaction mixture is then allowed to rise to room temperature. Tetrahydrofuran is evaporated under a reduced pressure of 20 mm Hg at 30°C. The obtained residue is dissolved in 25 cm<5>ethyl acetate. The resulting solution is washed with 25 cm<5> of water and then dried over sodium sulfate. The solution is filtered and concentrated to dryness under a reduced pressure of 20 mm Hg. A solid residue is obtained which is treated with 100 cm 3 of petroleum ether. The precipitate is filtered off and dried under a reduced pressure. 5.3 g of ethyl-a-tert-butoxycarbonylamino-(4,5-dihydro-1,3-dithiin-5-ylidene)-acetate is thereby obtained in the form of a beige solid with a Kofler melting point of 154°C.

The 3-tert-butoxycarbonyl-4-ethoxycarbonyl-2-thioxo-oxa-7-,9-dithia-3-azaspior-D1.57-decane can be prepared in the following manner. A solution of 4.6 g of potassium tert-butylate in 40 cm<5>tetrahydrofuran is cooled to -70°C. A solution of 5.8 g of ethyl isothiocyanatoacetate and 5.4 g of 1,3-dithia-cyclohexan-5-one in 80 cm<5>tetrahydrofuran is added dropwise. After 40 min. up-

heat the solution to approx. 0°C and a solution of 8.75 g of di-tert-butyl carbonate in 10 cm<5>tetrahydrofuran is added. The resulting mixture is stirred for 12 hours at approx. 20°C after which it is concentrated to dryness under a reduced pressure of 20 mm Hg at 30°C. The rest dissolves in 150 cm? methylene chloride.

3 cm<5> of acetic acid is added and the insoluble substance is separated by filtration. The filtrate is concentrated to dryness under a reduced pressure of 20 mm Hg at 30°C. The residue is crystallized in 100 cm 3 of ethyl ether and the crystals obtained are filtered off and dried under a reduced pressure of 1 mm Hg at 20°C. This gives 9.8 g of 3-tert-butoxycarbonyl-4-ethoxycarbonyl-2-thioxo-1-oxa~7,9-dithia-3-azaspiro-A.57-decane in the form of white crystals with a Kofler melting point of 125°C .

The ethyl isothiocyanatoacetate can be prepared according to the method described by D. Hoppe and R. Follmann in "Chem. Ber.", 109, 3047 (1976).

Example 3

24 g of 7-ZjD-α-tert-butoxycarbonylamino-(1,3-dithiin-5-yl)-acetamido7-2-carboxy-3-methyl-8-oxo-5-thia-1-azabicyclo-A.2. The Q_7-oct-2-ene prepared in the above-mentioned manner is dissolved in 750 cm<5>acetonitrile. 18.7 g of p-toluenesulfonic acid are added and the resulting mixture is allowed to react at 20°C

1 20 hours. You then add 40 cm<5> of water and process

with decolorizing charcoal. The solution is filtered and 17.2 cm 3 of triethylamine is added slowly up to pH 4.8. The crystals are filtered off and dried under a reduced pressure of 1 mm Hg at 20°C. 17.7 g of 7-ΔED-α-amino-(1,3-dithiin-5-yl)-acetamido-2-carboxy-3-methyl-8-oxo-5-thia-1-azabicyclo-A.2.0_7 are thereby obtained - Oct 2. This product has identical properties to the product produced in example 2.

Example 4

18.7 g of 7-Zt,L-α-tert-butoxycarbonyl-amino-(1,3-dithiin-5-yl)-acetamido7-2-carboxy-3-methyl-8-oxo-5-thia-1 are dissolved -azabicyclo-A.2.Q7-oct-2-ene, prepared as in example 1, in 200 cm<5>tetrahydrofuran, after which a solution of 7.45 g of diphenyldiazomethane in 50 cm<5>tetrahydrofuran is added. The reaction mixture is stirred for 16 hours at 20°C and then evaporated to dryness under a reduced pressure of 20 mm Hg at 30°C. The residue is taken up in 350 cm<5>ethyl acetate. The organic layer is washed with 100 cm<5> of a saturated aqueous solution of sodium bicarbonate and then dried over sodium sulfate. The solution is treated with decolorizing charcoal and filtered, after which it is concentrated to dryness under a reduced pressure of 20 mm Hg at 30°C. The residue is purified by chromatography on 400 g of silicon dioxide in a column with a diameter of 4.5 cm and a height of 54 cm. One elutes with mixtures of ethyl acetate and cyclohexane containing increasing levels of ethyl acetate. One thus elutes first with 1000 cm<5>of a mixture of ethyl acetate and cyclohexane in a volume ratio of 10:90, then with 1000 cm<5>of a mixture in a volume ratio of 20:80 and finally with 1000 cm of a mixture with volume ratio 25:70, collecting fractions of 100 cm<5.> Fractions 35-45 are concentrated to dryness under a reduced pressure of 20 mm Hg at 30°C. 10 g of 7-z^Dj L-α-tert-butoxycarbonylamino-(1,3-dithiin-5-yl)-ac et amido_7-2-benes hydroxycarbonyl-3-methyl-8-oxo-5-thia-1 -azabicyclo-A . 2. 0_7-Oct-2-en in the form of a white meringue-like mess see.

An impure product obtained by esterification of 13.6 g of 7-ZJ), L-a-ter t-b ut oxycarbonyl lamino-(1, 3_ di ti in- 5-y 1)-acetamidoJ-2-carboxy-3_methyl-8-oxo- 5-thia-1-azabicyclo-£k .2.07-oct-2-ene with diphenyldiazomethane is chromatographed on 1200 g of silica gel in a column with a diameter of 6.6 cm and a height of 73 cm. One elutes with 1500 cm<5> of a mixture of cyclohexane and ethyl acetate in a volume ratio of 90:10, then with 1500 cm<5> of a mixture of cyclohexane and ethyl acetate in the volume ratio 85:15, then with 1500 cm^ of a mixture of cyclohexane and ethyl acetate in the volume ratio 75:25, then with 1500 cm<5> of a mixture of cyclohexane and ethyl acetate in the volume ratio 70:30 and finally with 5000 cm<5> of a mixture of cyclohexane and ethyl acetate in the volume ratio 65 :35. Eluate fractions with a volume of 250 cm<3> are collected.

The fractions 35-37, obtained by elution with the mixture of cyclohexane and ethyl acetate in the volume ratio 65:35, give after evaporation 1.83 g of 7-Æ-a-tert-butoxycarbonylami.no-(1,3-dithiin-5-yl) -acetamide oj-2-benzhydryloxycarbonyl-3-methyl-8-oxo-5-thia-1-azabicyclo-D\ .2. 07-Oct-2-en. After recrystallization of this product from 165 cm<5>methanol, 1.19 g of product are obtained in the form of white crystals. Fractions 38-40 give 3.09 g of a mixture

of forms D and L.

After evaporation, fractions 41-45 yield 3.09 g of a product which is recrystallized in 150 cm 5 of ethanol. 2.01 g of 7-(L-α-tert-butoxycarbonylamino-(1,3-dithiin-5-yl)-acetamido-7~2-benshydryoxycarbonyl-3-methyl-8-oxo-5-thia) is thereby obtained -1-azabicyclo-A . 2. Q_7-oct-2-ene in the form of white crystals.

One dissolves 1.09 g of 7-Z£>-α-tert-butoxycarbonylamino-(1,3-dithiin-5-yl)-acetamido_7-2-bens hy dry lox y carbonyl-3-methyl 1- 8-oxo-5-thia-l-azabicyclo-£4 . 2 .0_7-oct-2-ene in 1.3 cm<3>anisole and 50 .cm 5 trifluoroacetic acid. The obtained solution is stirred for 15 min. at 20°C and then concentrated to dryness under a reduced pressure of 0.5 mm Hg at 30°C. The residue is dissolved in 5 cm 3 of ethyl acetate. The solution obtained is poured into 40 ml of isopropyl ether. The precipitate formed is separated by filtration. This gives 0.94 g of a white powder which is dissolved in 20 cm<5> of water and 10 cm of 0.1N hydrochloric acid. The water phase is washed 2 times with 15 cirr 3 ethyl acetate and then treated with stirring with 15 cirr<3> moist ion exchanger "Amberlite IR-45" in OH form until the pH value is stabilized at 5.0. The ion exchanger is then removed by filtration and the filtrate is lyophilized. 0.33 g of 7-β-α-amino-(1,3-dithiin-5-yl)-acetamido_7-2-carboxy-3-methyl-8-oxo-5-thia-1-azabicyclo-ZT4 is obtained . 2. 07-Oct-2-en .

CoJ^ ° = + 128 - 4° (c = 1.020, water)

Elemental analysis: Calculated, % : C 43.39 H 4.42 N 10.84

0 16.52 S 24.83

Found, % : C 39.7 H 3.5 N 10.0

S 22.1 NMR spectrum: δ (ppm) (CF₂COOH) : 2.40 (s, CH₂ in 3-position, 3H); 3.46

(s, -CH2-S-cefenv 2H); 3.55 (s,-'CH 2 -S-dithionine, 2H); 4.06 (s, -S-CH -S-, 2H); 5.15 (s, -CH-CO, 1H); 5.26 (d, J = 5Hz,

N<

H in 6 position); 5.80 (d, J = 5Hz, H in 7-position); 7.06 (s, -CH=, 1H).

This product has a minimum bacteriostatic concentration of 2 yg/cm<5> against Staphylococcus■aureus Smith and a minimum bacteriostatic concentration of 15 yg/cm<5> against Staphylococcus aureus MB 9, Escherichia coli and Klebsiella pneumoniae.

When working in the same way as above, starting from 1.99 g of 7-.Z~L-α-tert-butoxycarbonyl lamino-(1,3-dithiin-5~yl)-acetamido7-2-benzhydryloxycarbonyl-3-methyl -8-oxo-5-thia-1-azabicyclo-/T4 . 2. 0_7-oct-2-ene, 0.57 g of 7-£T>a-amino-(1,3-dithiin-5-yl)-acetamido7-2-carboxy-3-methyl-8-oxo-5-thia is obtained -1-azabicyclo-Z?4 . 2. Q7-Oct-2-en.

CoJ^ ° =<1>2° (c, 1.012, Elemental analysis: Calculated, % : C 43.39 H 4.42 N 10.84

0 16.52 S 24.83

Found, % : C 42.7 H 3.8 N 11.1

S 24.5 NMR spectrum: δ (ppm) (CF₂COOH); 2.43 (s, CH^- in 3-position, 3H), 3.43

(s, -CH2-S- cephem, 2H); 3.55 (s, -CHg-S-dithiin, 2H), 4.06 (s, -S-CH?-S-, 2H); 5.13 (s, -CH-CO-, 1H); 5.26 (d, J = 5Hz,

N<

H in 6 position), 5.70 (d, J = 5Hz, H in 7 position); 7.03

(s, -CH=, 1H).

Example 5

To a solution of 10 g of D-a-tert-butoxycarbonylamino-(1,3_dithiin-5~yl)-acetic acid in 100 cm<5>dimethylformamide is added 11.3 g of 7-amino-3_methyl-8-oxo-2-pivaolyloxy- methoxycarbonyl-5-thia-1-azabicyclo-£4 . 2. 0_7-oct-2-ene and 7.1 g of dicyclohexylcarbodiimide. The reaction mixture is stirred for 2 hours and then filtered. The filtrate is diluted with 300 cm<5>ethyl acetate and 500 cm<5>water. The organic phase is decanted, washed with 100 cm<3> of water and then with 100 cm<3> of a saturated aqueous solution of sodium bicarbonate, dried over sodium sulfate and filtered in the presence of decolorizing charcoal. It is then concentrated to dryness under a reduced pressure of 20 mm Hg at 30°C. The residue is chromatographed on 150 g of silicon dioxide in a column with a diameter of 3.5 cm and a height of 35 cm. One elutes with mixtures of ethyl acetate and cyclohexane containing increasing levels of ethyl acetate. One thus elutes first with 500 cm<5> of a mixture of ethyl acetate and cyclohexane in the volume ratio 10:90, then with 500 cm<3> of a mixture with the volume ratio 20:80, then with 500 cm3 of a mixture with the volume ratio 30: 70 and finally with 2000 cm<5> of a mixture with the volume ratio 40:60. Eluate fractions with volumes of 200 cm<5> are collected. Fractions 14-21 are concentrated to dryness under a reduced pressure of 20 mm Hg at 30°C. 8 g of 7-ZT)-a-tert-butoxycarbonylamino-(1,3-dithiin-5-yl)-acetamido7-3-methyl-8-oxo-2-pivaloyloxyrne toxycarbonyl 1-5 - ti a-l.-azab in cy klo- A . 2. 07-Oct-2-en in the form of an orange-coloured meringue-like mass.

The product thus prepared is dissolved in 50 cm<3>trifluoroacetic acid and the resulting solution is stirred at 20°C for 15 min. The solution is then concentrated to dryness under a reduced pressure of 0.5 mm Hg at 30°C. The residue is dissolved in 200 cm 3 of water and the resulting solution is washed with 1■ 00 cm<J>^ of ether. The water phase is stirred with 250 cm<3>ethyl acetate. 100 cm<5> of a saturated aqueous solution of sodium bicarbonate is added while stirring. The organic phase is decanted, dried over sodium sulfate and filtered in the presence of decolorizing charcoal. It is concentrated to 15 cm<5> under a reduced pressure of 20 mm Hg at 30°C. 100 cm<3> of ethyl ether is added and then 10 cm<5> of a 2.7N ether solution of anhydrous hydrochloric acid is added dropwise. Stir for 10 min. and then filters off the precipitate. 4.8 g of hydrochloride of 7-[D-a-amino-(1,3-dithiin-5-yl)-acetamido]-3-methyl-2-pivaloyloxynetoxycarbonyl-8-oxo-5-thia-1-azabicyclo-Z7l are thereby obtained . 2 .0_7-oct-2-en in the form of a white powder.

Elemental analysis: Calculated, % : C 44.64 H 5.24 Cl 6.59

N 7.81 0 17.84 S17.88 Found, % : C45.3 H5.4 Cl 6.7

N 7.9 S 17.7 7-amino-3-methyl-8-oxo-2-pivaloyloxynetoxycarbonyl-5-thia-1-azabicyclo-A . 2. 0_7-oct-2-ene can be prepared according to the method described in German patent document 1,951,012.

The invention also relates to therapeutically usable pharmaceutical preparations which contain at least one compound of the formula I as active ingredient in combination with one or more pharmaceutically acceptable diluents or excipients. These preparations can be used orally, parenterally or rectally.

As solid preparations for oral administration can be used

tablets, pills, powders or granules. In these preparations, the active compound according to the invention is mixed with one or more inert diluents or excipients such as sucrose, lactose or starch. These preparations may also contain substances other than diluents, e.g. lubricants such as magnesium stearate.

As liquid preparations for oral administration, pharmaceutically acceptable emulsions, solutions, suspensions, syrups or elixirs containing inert diluents such as water or paraffin oil can be used. These preparations may also contain substances other than the diluents, e.g. humectants, sweeteners or flavourings.

As preparations for parenteral administration, sterile solutions (both aqueous and non-aqueous) can be used,

suspensions or emulsions. Propylene glycol, polyethylene glycol, vegetable oils, especially olive oil, and injectable organic esters, e.g. ethyl oleate. These preparations may also contain excipients, especially wetting agents, emulsifying agents or

dispersants. Sterilization can be carried out in several ways, e.g. by means of a bacteriological filter, by adding sterilizing substances to the preparation, by irradiation or by heating. It is also possible to prepare solid sterile preparations that can be dissolved in, when used

sterile water or another injectable sterile medium.

Preparations for rectal administration consist of suppositories which, in addition to the active ingredient, may contain a laxative such as cocoa butter or suppository wax.

Within human therapy, the preparations according to the invention can especially be used for the treatment of infections of bacterial origin.

The doctor usually decides which dose should be used with regard to the patient's age and body weight, degree of infection and other factors. The doses are usually between 1 and 12 g of active compound per day by oral, intramuscular or intravenous administration to adults.

The following non-limiting examples illustrate the preparations according to the invention.

Example A

An injectable solution is prepared with the following composition: 7-Z~D-α-amino-(1,3_dithiin-5-yl)-acetamido_7-2-carboxy-3~niety 1-8-oxo-5-thia-1-azabicyk-

Example B

Tablets are usually prepared with the following

composition:

Claims (1)

Process for the preparation of a cephalosporin derivative of the general formula where R denotes a carboxy group or a group of the general formula where R^ denotes a hydrogen atom or an alkyl group containing 1-4 carbon atoms and R^ denotes a straight or branched alkyl- group containing 1-4 carbon atoms or a cyclohexyl group whereby the group is easy to remove enzymatically; in the form of diastereoisomers or mixtures thereof, addition salts thereof with acids and possibly metal salts thereof and addition salts thereof with nitrogen-containing organic bases, characterized in that (a) an acid with the formula which exists in racemic form or in optically active form bg whose amino group is protected in advance, or a reactive derivative of this acid, is reacted with a cephalosporin with it general' formula: where R has the meaning stated above, whereby the acid group is optionally protected, after which the protecting group is removed and the product obtained is optionally converted into an addition salt with an acid or, when R denotes a carboxy group, into a metal salt or an addition salt with a nitrogen-containing organic base, or that (b) a compound of formula I where R denotes a carboxy group and whose amino group is protected in advance is esterified according to known methods for the preparation of an ester by starting from an acid without affecting the rest of the molecules to form a compound of formula I where R denotes a group of the formula where R 1 and R 2 have the meaning given above, after which the protecting group for the amino group is removed and the product obtained is optionally converted into an addition salt with an acid, after which a compound of the formula I is prepared, which contains a mixture of diastereoisomers and whose amino and acid groups optionally is protected, optionally split into its diastereoisomers by the application of physical methods, after which the protecting groups are removed and each diastereoisomer obtained is optionally converted into an addition salt with an acid or, when R denotes a carboxy group, into a metal salt or into an addition salt with a nitrogen-containing organic' base.