NO148376B - ANALOGY PROCEDURE FOR THE PREPARATION OF THERAPEUTIC ACTIVITY 7-ACYLAMINO-3-HALOGEN-CEPHALOSPORINE COMPOUNDS - Google Patents

Description

The present invention relates to the production of therapeutically effective 7-acylamino-3-halocephalosporin compounds with the formula:

where R is thenyl or α-hydroxybenzyl, and X is chlorine or bromine, as well as non-toxic, pharmaceutically acceptable salts thereof.

Compounds of formula I have unique structural properties in that a halogen atom is directly bonded to the carbon atom in the 3-position of the dihydrothiazine ring. According to the cepham nomenclature system, the above-mentioned compounds can be designated as 7-acylamino-3-halo-3-cephem-4-carboxylic acid.

3-bromomethyl-3-cephem-4-carboxylic acid esters and 3-bromomethyl-2-cephem-4-carboxylic acid esters have previously been described. These known 3-bromomethyl compounds are described as valuable intermediates for the production of cephalosporin antibiotics. In contrast, the 3-halo-3-cephem-4-carboxylic acids of formula I are particularly valuable antibiotics. The 3-methyl analogue of the compounds of formula I has also been previously described, namely cephalexin, see e.g. US Patent 3,507,861. The compounds of formula I are more active than cephalexin.

The compounds of formula I are prepared according to the present invention by reacting a 3-hydroxycephalosporin compound with the formula:

wherein R<1> is thenyl, and R2 is a carboxylic acid protecting ester forming group, with oxalyl chloride, thionyl chloride, phosphorus trichloride, phosphorus oxychloride, oxalyl bromide, thionyl bromide, phosphorus tribromide or phosphorus oxybromide, in dimethylformamide; optionally cleaves the 7-thienylacetamido side chain and reacylates the thus obtained 7-amino-3-halocephalosporin compound with an acid of the formula Cgl^CH(OH)-COOH or a reactive derivative thereof, after which the carboxylic acid-protecting ester-forming group is removed, and, if desired , converts a compound thus obtained into a pharmaceutically acceptable salt thereof.

The thus prepared compounds represent a new group of cephalosporin antibiotics, namely 7-acylamino-3-halo-3-cephem-4-carboxylic acid antibiotics.

The chlorination and bromination of a 3-hydroxy cephemester is carried out by using dry DMF (dimethylformamide) as solvent. DMF is preferably dried over a molecular sieve before use. A co-solvent can be used together with an excess of DMF, although this is not necessary. One can e.g. as cosolvent use tetrahydrofuran, dioxane, methylene chloride, dimethylacetamide or dimethylsulfoxide. The brominating or chlorinating agent is preferably used in an amount corresponding to two equivalents of the amount of 3-hydroxy-cephemester used. The reaction is carried out by adding the halogenating agent to a solution of 3-hydroxycephemester in dry DMF which is kept at a temperature of 5°-15°C, after which the reaction mixture is left at room temperature for 4-8 hours or longer. The reaction is initially exothermic and the reaction vessel should preferably be kept in an ice-water bath so that the temperature is kept below approx. 25°C during the first part of the reaction. The reaction mixture can then be allowed to stand at or above room temperature for the remainder of the reaction. How far the reaction has progressed can be determined by thin-layer chromatography.

Alternatively, the chlorination or bromination can be carried out by first preparing a mixture of the halogenating agent in DMF to form the halogeniminium halide beforehand, after which a solution of the 3-hydroxy-3-cephemester in DMF, a mixture of DMF and a co-solvent is added to this mixture or in a solvent such as dimethylacetamide or tetrahydrofuran.

The 3-chloro- or 3-bromo-3-cephemesters are recovered from the reaction mixture by pouring it into a water-ethyl acetate mixture, after which the organic phase containing the product is separated. Said organic phase is washed, dried and evaporated, whereby 3-halo-3-cephemester is obtained as an amorphous residue. The product can be obtained in crystalline form by treating the residue with ether or with n-hexane.

The preferred chlorinating and brominating agents are phosphorus trichloride and phosphorus tribromide.

The 7-amino-3-halo-3-cephem intermediate in the process according to the invention can best be prepared by cleaving the 7-thienylacetamido side chain from a 7-thienylacetamido-3-halo-3-cephem-4-carboxylic acid ester by a well-known cleavage ninase reaction tion using phosphorus pentachloride. When e.g. The 7-[2-(2-thienyl)-acetamido]-3-chloro-3-cephem-4-carboxylic acid p-nitrobenzyl ester is reacted with phosphorus pentachloride in methylene chloride in the presence of pyridine, then an imino chloride derivative of in this connection. This intermediate is reacted with an alcohol, e.g. methanol or isobutanol, whereby one

gives the corresponding iminoether derivative. A hydrolysis of the imino ether gives p-nitrobenzyl-7-amino-3-chloro-3-cephem-4-carboxylate hydrochloride.

The starting compounds that are used in the preparation of compounds of formula I can be prepared by reacting a 7-acylamino-3-exomethylene cepham-4-carboxylic acid ester or a 7-amino-3-exomethylene cepham-4-carboxylic acid ester with ozone in an inert solvent with temperature between -80 and 0°C, whereby the ozonide derivative is formed at the 3-exomethylene double bond. This ozonide product which is not isolated can be decomposed by reacting it in situ with a mild reducing agent such as sodium bisulphite or preferably sulfur dioxide, whereby the corresponding 3-hydroxy-3-cephem-4-carboxylic acid ester is obtained.

The ozonolysis of 7-amino-3-exomethylene cepham-4-carboxylic acid ester or of 7-acylamino-3-exomethylene cepham-4-carboxylic acid ester of formula V is carried out by passing ozone through a solution of 3-exomethylene cepham ester in an inert solvent medium with temperatures between -80 and 0°C. The exomethylene double bond reacts with ozone to form in situ an intermediate ozonide which decomposes as described below to form a 3-hydroxy-3-cephemester of formula VI:

In the above-mentioned formulas, R1 and other have the above-mentioned meanings.

Although 3-exomethylene cephalosporins can also undergo oxidation with ozone to form the sulfoxide, the exo double bond under the described ozonation conditions will preferentially react with ozone to form the ozonide. The formation of the sulfoxide occurs as a result of overoxidation. While the exo double bond reacts rapidly with ozone, the reaction at the sulfur atom in the dihydrothiazine ring to form sulfoxide occurs at a much slower rate.

However, the following overoxidation products can be formed by ozonolysis reaction.

Ozone gas is produced using an ozone generator of the type that is usually used in synthetic and analytical chemical work for the production of ozone, and where the ozone is produced by an electrical discharge in oxygen. The ozone is developed in a stream of oxygen which is then fed directly into the reaction vessel. The percentage content of ozone in the oxygen stream can be varied as desired, e.g. by varying the flow rate of the oxygen through the ozone generator, in addition to varying the intensity of the electrical discharge. The percentage content of ozone in the oxygen stream can be determined iodometrically by titrating with sodium thiosulphate the amount of iodine released from a standard solution of potassium iodide with ozone from the generator. The percentage content of ozone in the oxygen stream is not critical, but a determination of the amount of ozone that flows into the reaction mixture will, in the present method, make it possible to determine the time when the desired reaction is complete, whereby a minimal formation of overoxidation products is obtained.

Alternatively, the ozonolysis reaction can be followed chromatographically. E.g. a small part of the reaction mixture can be taken out, the ozonide is decomposed and the amount of unreacted starting material and possibly the 3-hydroxy-3-cephem product in the sample can be examined using thin-layer chromatography,

where one compares with a known amount of starting material and the 3-hydroxy-3-cephem compound.

Inert solvents that can be used in the ozonolysis are those solvents in which the 3-exomethylene-cepham esters are at least partially soluble and which are unreactive with ozone under the described conditions. Generally, satisfactory results will be achieved with organic solvents such as methanol, ethanol, ethyl acetate, methyl acetate and methylene chloride.

The concentration of the starting material in the inert solvent is not critical and it is preferred to use a sufficient amount to form a complete solution. The preferred temperature during the ozonolysis reaction is between -80 and -50°C.

When ozonide formation is complete as determined

in one of the above methods, excess ozone can be driven out of the reaction mixture by bubbling nitrogen or oxygen through the mixture.

After any excess ozone has been removed, the ozonide can be decomposed by adding a mild reducing agent selected from the group consisting of sodium bisulphite, sulfur dioxide and trimethylphosphite to the reaction mixture, whereby the 3-hydroxy-3-cephem-4-carboxylic acid ester is obtained. The decomposition is carried out by adding an excess of the reducing agent and stirring the reaction mixture at a temperature from -80 to 0°C, until the reaction mixture is negative in the potassium iodide starch test.

A preferred agent for decomposition of the intermediate ozonide is gaseous sulfur dioxide. This reagent is preferred because it can be completely expelled from the reaction mixture during the subsequent work-up and thus does not complicate the recovery of the reaction product.

The 7-acylamino-3-hydroxy-3-cephem-4-carboxylic acid esters produced can be recovered from the reaction mixture by first evaporating the mixture to dryness and then extracting the product from the residue. Alternatively, N-acylated 3-hydroxy-3-cephemesters can be recovered from the organic liquid phase in the decomposition mixture by separating the liquid phase from insoluble solids, and after washing and drying, the organic layer can be evaporated, whereby 3-hydroxy esters are obtained.

Said 3-hydroxy core ester, i.e. a 7-amino-3-hydroxy-3-cephem-4-carboxylic acid ester, can best be isolated in the form of a salt, e.g. in the form of a hydrochloride or hydrobromide.

When an ester of 7-amino-3-exomethylene cepham-4-carboxyl-

acid is ozonized, it is preferred to use a salt of this core, e.g. the hydrochloride or the p-toluenesulfonate salt.

In a specific example of the preparation of a 3-hydroxy-3-cephem ester, p-methoxybenzyl-7-phenoxyacetamido-3-exomethylene cepham-4-carboxylate was dissolved in ethyl acetate and reacted with ozone at a temperature of approximately -78°C. The excess of ozone was driven out by bubbling through oxygen in the cold solution. The ozonide was decomposed by adding an excess of sodium bisulfite at 0°C with stirring. The organic layer was separated from the insoluble solids and washed, dried and evaporated to give p-methoxybenzyl-7-phenoxyacetamido-3-hydroxy-3-cephem-4-carboxylate.

In another example, p-nitrobenzyl-7-amino-3-methylene-cepham-4-carboxylate hydrochloride was dissolved in methanol and ozone bubbled through the solution at temperatures of about -78°C. Excess ozone was expelled from the mixture with nitrogen, and the ozonide was decomposed by bubbling through sulfur dioxide. The reaction mixture was evaporated to dryness and the residue, i.e. p-nitrobenzyl 7-amino-3-hydroxy-3-cephem-4-carboxylate, was obtained in the form of the hydrochloride salt.

The starting compounds for the production of the 3-exomethylene cephamesters can be prepared by reacting a 7-acylamino-cephalosporanic acid with a sulfur-containing nucleophilic compound using known methods, whereby a nucleophilic displacement of the acetoxy group in the cephalosporanic acid is obtained, and a 7-acylamino- 3-Thiosubstituted-methyl-3-cephem-4-carboxylic acid. This 3-thiosubstituted cephem product is then reduced with hydrogen in the presence of Raney nickel or with zinc/formic acid in the presence of dimethylformamide, whereby 3-exomethylene cephem acid is produced. Thus, e.g. 7-phenylacetamido-cephalosporanic acid is reacted with potassium methylxanthate, whereby 7-phenylacetamido-3-ethoxythiono-carbonylthiomethyl-3-cephem-4-carboxylic acid is obtained which, on reduction with zinc/formic acid in the presence of DMF, gives 7-phenylacetaraido-3-exomethylenecepham -4-carboxylic acid with the formula: 3-exomethylene cepham nucleus with the formula:

is prepared by reacting a 7-acylamino-3-exomethylene cepham-4-carboxylic acid ester with phosphorus pentachloride (PCl^) in methylene chloride in the presence of pyridine, whereby the intermediate imino chloride was obtained. This was reacted with methanol in the cold, so that the iminoether was obtained. The imino ether can easily be subjected to hydrolysis, whereby the 7-amino-3-exomethylene cepham-4-carboxylic acid ester hydrochloride is obtained. The ester group can then be removed, whereby 3-exomethylene cepham cores are obtained.

The specified 7-acylamino-3-halo-3-cephem-4-carboxylic acids of formula I are useful antibiotic compounds for combating infections caused by gram-positive and gram-negative organisms. The compounds can be administered by injection (subcutaneous or intramuscular) in the free acid form or in the form of a pharmaceutically acceptable, non-toxic salt. Salts formed with the free acids and inorganic bases such as sodium bicarbonate, potassium carbonate, sodium hydroxide and calcium hydroxide give sodium, potassium and calcium salts respectively, which can be worked up for supply, e.g. in the form of isotonic solutions or as liquid suspensions.

The in vitro antimicrobial activity of 3-haloencephalosporins is indicated by data in Table I. Table I contains the antimicrobial activity of 7-[2-(2-thienyl)-acetamido]-3-chloro-3-cephem-4-carboxylic acid which was obtained in a standard plate method. The numerical values are the diameter in millimeters of inhibition zones that were observed with the indicated organisms.

The following Table II indicates the minimum inhibitory concentration (MIC) of 7-[2-(2-thienyl)acetamido]-3-chloro-3-cephem-4-carboxylic acid against penicillin-resistant Staphylococcus both in the presence and absence of serum. The MIC values were obtained by the so-called gradient plate technique which was carried out essentially as described by Bryson and Szybalski, Science, 116, 45 (1952).

In table III below, the MIC value for the test compound from tables I and II above for representative gram-negative organisms is indicated. The indicated data were obtained by the gradient plate technique.

In Table IV below, in vitro antimicrobial activity for 7-[2-(2-thienyl)acetamido]-3-bromo-3-cephem-4-carboxylic acid is indicated for several illustrative microorganisms. These data were obtained by a standard plate method, where the numerical values indicate the diameter in millimeters of the inhibition zones that were obtained with the indicated microorganisms.

The following Table V indicates the minimum inhibitory concentration (MIC) of the antibiotic 7-[2-(2-thienyl)acetamido]-3-bromo-3-cephem-4-carboxylic acid against penicillin-resistant strains of Staphylococcus in the absence of serum.

The inhibitory concentrations were obtained by the gradient plate method.

Minimum inhibitory concentrations for 7-[2-(2-thienyl)-acetamido]-3-bromo-3-cephem-4-carboxylic acid against representative Gram-negative bacteria are given in Table VI. These data were obtained by the gradient plate method. The obtained 7-acylamino-3-halo-3-cephem-'4-carboxylic acid esters constitute intermediate products for the preparation of the compounds in the form of the free acids.

Ester groups falling within the definition of R₂ are all well-known groups commonly used to protect the C₂ carboxylic acid group of the cephalosporin molecule while carrying out reactions involving other groups in the molecule. These ester-formed groups can be easily removed, whereby the free acid is obtained by reduction or hydrolysis. Thus, e.g. The p-nitrobenzyl ester group is removed via catalytic hydrogenolysis over palladium and carbon, the diphenylmethyl group (benzhydryl) can be removed with trifluoroacetic acid in anisole at approx. 10°C, the p-methoxybenzyl group can be removed with trifluoroacetic acid at approx. 10°C. (J.Org.Chem. 36, 1259 (1971)), the 2,2,2-trichloroethyl group can be removed with zinc and acid (J.Am.Chem.Soc. 88, 852 (1966)), the benzyl ester group can be removed via catalytic hydrogenolysis over palladium catalyst (J.Org.Chem. 27, 1381 (1962)) and the tertiary butyl group can be removed as described in J.Org.Chem. 31, 444 (1966).

The following examples illustrate the production of starting materials.

Example 1

p-nitrobenzyl-7-amino-3-methylene cep am-4-carboxylate hydrochloride.

To a solution of 965 mg (2 mmol) of p-nitrobenzyl-7-phenoxy-acetamido-3-methylene cepham-4-carboxylate in 10 ml of methylene chloride was added 175 mg of dry pyridine and 460 mg of phosphorus pentachloride, and the mixture was stirred at room temperature for 6 hours . One ml of isobutanol was added to the mixture which was then stored at 0°C overnight. The reaction product p-nitrobenzyl-7-amino-3-methylene cepham-4-carboxylate hydrochloride which had formed as a crystalline precipitate was filtered off and the yield was 430 mg (58% yield).

Elemental analysis for C^j-H^N^Oj-SCl

Theoretical: C 46.69 K 4.18 N 10.89

Found: C 46.40 H 4.20 N 10.62

I.R. (Nujol):

Carbonyl absorption at 5.65 (6-lactam) and 5.75 (ester) microns

N.M.R. signals at 6.34 (2d, 2H, C2~H2), 4.98 (d,

1H, Cg-H), 4.7-4.4 (m, 6H, C^- K, ester CH2, C4"CH2

and C^-H) and 2.4-1.6 (m, 4H, aromatic H) tau

Example 2

p-nitrobenzyl-7-amino-3-methylene cepham-4-carboxylate p-toluenesulfonate salt.

A solution of 965 mg of p-nitrobenzyl-7-phenoxyacetamido-3-methylene cepham-4-carboxylate in 10 ml of methylene chloride was added to 175 mg of dry pyridine and 460 mg of phosphorus pentachloride and the mixture was stirred for 5 hours at room temperature. It was then cooled to 0°C and 50 ml of cold methanol was added. After stirring for one hour at room temperature, the mixture was evaporated in vacuo to remove the solvents, and the remaining reaction product was dissolved in a mixture of ethyl acetate and water. The pH was adjusted to 7 and the ethyl acetate layer was separated and washed with water and dried. An equivalent of p-toluenesulfonic acid was added to the dried solution, and upon cooling, 600 mg of p-nitrobenzyl-7-amino-3-methylene cepham-4-carboxylate p-toluenesulfonate was formed as a crystalline precipitate. The product was purified by recrystallization from a mixture of 12 ml of methanol and 24 ml of ether and 15 ml of petroleum ether.

Elemental analysis for C-^H^N-jOgS-j

Theoretical: C 50.66 H 4.45 N 8.06

Found: C 50.41 H 4.51 N 7.86

I.R. (Nujol):

Carbonyl absorption at 5.65 (Q-lactam) and 5.71 (ester) microns

N.M.R. (DMSO d,)

6

Signals at 7.70 (s, 3H-p-methyl), 6.39 (s, 2H,

C2-H2), 4.93 (d, 1H, Cg-H), 4.7-4.3 (m, 6H, C^-H, ester CH2, C3"CH2, and C^H) and 2, 93-1.68 (m, 8H, aromatic H) tau.

UV (pH 6 buffer)

Maximum at 219 my (e=19,600) and

268 mp (e= 9,400) .

Example 3

p-Methoxybenzyl 7-amino-3-methylene cepham-4-carboxylate hydrochloride

A solution of 4.3 g of p-methoxybenzyl-7-phenoxyacetamido-3-methylene cepham-4-carboxylate in 50 ml of methylene chloride was added

added 880 mg of dry pyridine and 2.3 g of phosphorus pentachloride and the mixture was stirred under reflux for 3 hours. It was then cooled in an ice water bath and 5 ml of isobutanol was added. The mixture was stirred in the cold for several hours and a total of 2.2 g of the reaction product, p-methoxybenzyl-7-amino-3-methylene-cepham-4-carboxylate hydrochloride, precipitated. The product was filtered off and washed with cold methylene chloride and then dried in vacuo.

Elemental analysis for C,,HiriSCl

16 19

Theoretical: C 51.82 H 5.16 N 7.55

Found: C 51.65 H 5.04 N 7.72

Example 4

p-Methoxybenzyl 7-amino-3-methylene cepham-4-carboxylate p-toluenesulfonate.

A solution of 937 mg of p-methoxybenzyl-7-phenoxyacetamido-3-methylene cepham-4-carboxylate in 10 ml of methylene chloride was added

added 0.18 ml of dry pyridine and 460 mg of phosphorus pentachloride. The mixture was stirred at room temperature for 2 hours and then cooled to 5°C. The cold mixture was added with 50 ml of cold methanol and allowed to warm to room temperature. The mixture was evaporated in vacuo and the residue was dissolved in a mixture of ethyl acetate and water. The pH of the solution was adjusted to 7 and the ethyl acetate layer was separated, washed with water and dried. The dried ethyl acetate layer was added a

equivalent of p-toluenesulfonic acid. Upon cooling, a crystalline solid precipitated approx. 600 mg of p-methoxybenzyl-7-amino-3-methylene cepham-4-carboxylate p-toluenesulfonate.

Elemental analysis for C23H2g<N>2<0gS>2

Theoretical: C 54.53 H 5.17 N 5.53

Found: C 54.33 H 5.05 N 5.47

I.R. (Nujol):

Carbonyl absorption bands at 5.65 (Ø-lactam) and 5.78 (ester) micron.

N.M.R. (DMSO dg):

Signals at 7.69 )s, 3H, para-methyl), 6.41 (s,

2H, C2-H2), 6.23 (s, 3H, para-methoxy), 5.0 (d, 1H, Cg-H), 4.82 (s, 2H, ester CH2), 4.7-4 .55 (m, 4H, C4-H, C3-CH2 and C7-H), 3.2-2.0 (m, 8H, aromatic

H) rope.

Example 5

p-nitrobenzyl-7-amino-3-hydroxy-3-cephem-4-carboxylate hydrochloride.

A solution of 3.85 g of p-nitrobenzyl-7-amino-3-methylene cepham-4-carboxylate hydrochloride prepared as described in Example 1, in 600 ml of methanol was cooled in an acetone-dry ice bath. Ozone was bubbled through the reaction mixture for approx. 20 minutes,

and the mixture at this point took on a faint blue color. Nitrogen was passed through the reaction mixture to drive off an excess of ozone. The intermediate ozonide was then decomposed by passing sulfur dioxide gas through the reaction mixture until it gave a negative potassium iodide starch test.

The reaction mixture was evaporated in vacuo and the residue was dissolved in 200 ml of 0.1 N hydrogen chloride in methylene chloride. The solution was evaporated to dryness and the remaining reaction product was dissolved in acetone. Upon cooling, 3.15 g of p-nitrobenzyl-7-amino-3-hydroxy-3-cephem-4-carboxylate hydrochloride were precipitated as a crystalline solid.

In .R. (Nujol) :

Carbonyl absorption at 5.55 (3-lactam carbonyl) and 5.02 (ester carbonyl hydrogen bonded to 3 hydroxy) microns.

Electrometric titration (66% DMF) pKa 4.0 and 6.3.

Example 6

p-nitrobenzyl-7-amino-3-hydroxy-3-cephem-4-carboxylate hydrochloride.

A solution of 4 g of p-nitrobenzyl-7-amino-3-methylene-cepham-4-carboxylate hydrochloride in 620 ml of methanol was cooled in a dry ice-acetone bath, and ozone was bubbled through the cold solution for approx. 20 minutes. The reaction mixture was purged of residual ozone by passing nitrogen through the solution, and 10 g of sodium bisulfite was added.

The reaction mixture was stirred for one hour at ice bath temperature,

and the mixture at this time gave a negative potassium iodide-starch test.

The mixture was then evaporated in vacuo to give the reaction product as an amorphous, yellow residue. This was crystallized in acetone, whereby 3.4 g of p-nitrobenzyl-7-amino-3-hydroxy-3-cephem-4-carboxylate hydrochloride was obtained as a crystalline acetone solvate.

I.R. (Nujol):

Carbonyl absorption band at 5.60 (|3-lactam) and

6.04 (ester carbonyl hydrogen bonded to 3-hydroxy)-micron.

N.M.R. (DMSO dg):

Signals at 7.92 (s, 3H, 1/2 mol acetone), 6.22 (2d, 2H, C2-H2), 5.07 (d, 1H, CgH), 4.8-4.5 (m , 3H, ester CH2 and C7H), 2.4-1.6 (m, 4H, aromatic H) tau.

Example 7

p-nitrobenzyl-7-amino-3-hydroxy-3-cephem-4-carboxylate hydrochloride.

Using the ozonation methodology described in Examples 5 and 6, 3.85 g of p-nitrobenzyl-7-amino-3-methylene-cepham-4-carboxylate hydrochloride was ozonized in methanol, and the intermediate ozonide was decomposed at a temperature of

0°C with 3.5 ml of trimethylphosphite. The reaction mixture was evaporated and the residue was dissolved in 100 mL of 0.1 N HCl in methylene chloride. Said solution was then evaporated and the residue was crystallized from acetone, whereby 2.8 was obtained

g p-nitrobenzyl-7-amino-3-hydroxy-3-cephem-4-carboxylate hydrochloride.

Example 8

p-nitrobenzyl-7-amino-3-hydroxy-3-cephem-4-carboxylate.

4 millimoles of p-nitrobenzyl-7-amino-3-hydroxy-3-cephem-4-carboxylate hydrochloride, prepared as described in Example 5, were dissolved in water and ethyl acetate was added to the solution.

The pH of the suspension was adjusted from pH 2.2 to pH 5 with IN sodium hydroxide. The ethyl acetate layer was separated and washed with water and dried over magnesium sulfate. The dried ethyl acetate layer was evaporated to dryness to give 1.2 g of p-nitrobenzyl'-7-amino-3-hydroxy-3-cephem-4-carboxylate as a crystalline residue.

Elemental analysis for C-^H^N^<OgS:>

Theoretical: C 47.86 H 3.73 N 11.96

Found: C 47.87 H 4.00 N 12.11

I.R. (Nujol):

Carbonyl absorption ion at 5.65 (broad, (3-lactam and ester) and 6.0 (amide) microns.

N.M.R. (DMSO d,):

o

Signals at 6.63 (2d, 2H, C2H), 5.31 (d, 1H, CgH), 4.89 (d, 1H, C?H), 4.62 (s, 2H, ester CH2), 4.30 (broad, s, 2H, 7 N-H), 2.51-1.8 (m, 4H, aromatic H) and 1.2 (d, 1H, C30H) tau.

Example 9

p-nitrobenzyl 7-[2-(2-thienyl)acetamido)-3-hydroxy-3-cephem-4-carboxylate.

A solution of 1.55 g of p-nitrobenzyl-7-amino-3-hydroxy-3-cephem-4-carboxylate hydrochloride in 30 ml of acetone containing 364 mg (0.5 ml, 3.6 mmol) of triethylamine was added to 962 mg of urea . While stirring at room temperature, a solution of 730 mg (4.4 mmol) of 2-thiophene-acetyl chloride in 20 ml of acetone was added dropwise to the mixture. After 2.5 hours, the reaction mixture was filtered and evaporated. The residue was dissolved in ethyl acetate and the solution washed successively with water, a 5% solution of sodium bicarbonate, 5% hydrochloric acid and a saturated solution of sodium chloride. The washed solution was dried and concentrated by evaporation in vacuo to give 1.2 g of the reaction product as a crystalline residue. The product was recrystallized from ethyl acetate, whereby pure p-nitrobenzyl 7-[2-(2-thienyl)acetamido)-3-hydroxy-3-cephem-4-carboxylate was obtained with the following spectral properties. I.R. (Nujol): absorption peaks at 3.0 (amide NH), 5.68 (B-lactam carbonyl), and 6.1.

(amide, and ester hydrogen bonded to 3 OH) micron.

N.M.R. (CDC13/DMS0 dg): signals at 6.54 (2d, 2H,

C2H2), 6.16 (s, 2H, side chain CH2), 4.90 (d, CgH, 4.60 (d, 2H, ester CH2), 4.43 (q, 1H, C?H),

3.1-1.6 (m, 7H, aromatic H) and 1.30 (d, 1H, amide NH) tau.

Example 10

p-nitrobenzyl 7-[2-(2-thienyl)acetamido]-3-hydroxy-3-cephem-4-carboxylate.

p-nitrobenzyl-7-amino-3-methylene-encephem-4-carboxylate hydrochloride, 3.85 g, was reacted with ozone in methanol as indicated in Example 5 to prepare the ozonide. This was decomposed with sulfur dioxide, whereby the 3-hydroxy product was obtained which was isolated as a crude product. This crude product was dissolved in 175 ml of tetrahydrofuran and 50 ml of water. 2.1 g of sodium bisulphite was suspended in the solution, after which a solution of 4.8 g of 2-thiophene-acetyl chloride in 200 ml of THF was added dropwise.

The mixture was stirred for two hours at room temperature and then evaporated to an aqueous residue. This was stirred with ethyl acetate, the organic layer separated and washed with 5% hydrochloric acid and with water. The washed layer was dried and evaporated to dryness, yielding the reaction product as a crystalline residue. This residue was treated three times with diethyl ether to remove contamination of 2-thiophenacetic acid, and 2.9 g of purified crystalline product was obtained, i.e. p-nitrobenzyl-7-12-(2-thienyl)acetamido]-3-hydroxy -3-cephem-4-carboxylate.

Electrometric titration (66% aqueous DMF)

pKa 5.9

N.M.R. (CDC13/D20): signals at

6.60 (s, 2H, <C>2H2), 6.13 (s, 2H, side chain CH2), 4.96 (d, 1H, CgH), 4.62 (d, 2H, ester CH2),

4.46 (d, 1H, C?H) and 3.1-1.7 (m, 7H, aromatic H) tau.

The following examples illustrate the invention.

Example 11

Diphenylmethyl 7-[2-(2-thienyl)acetamido]-3-chloro-3-cephem-4-carboxylate.

a) A solution of 34 g (100 mmol) 7-[2-(2-thienyl)acetamido]-3-methylenecepham-4-carboxylic acid in 500 ml methylene chloride

21.4 g (110 mmol) of diphenyldiazomethane was added and the resulting mixture was stirred for 2 hours at room temperature. The solvent was removed under reduced pressure and the residue was dissolved in ethyl acetate. The ethyl acetate solution was washed with 5% sodium bicarbonate solution, then with water and then dried over magnesium sulfate. The dried solution was concentrated to a small volume. On standing, 40 g of diphenylmethyl-7-[2-(2-thienyl)acetamido]-3-methylene cepham-4-carboxylate were separated which melted at 132-133°C as a crystalline solid. I.R. (chloroform): absorption peaks at 2.9 (amide N-H), 5.65, 5.75 and 5.93 (3-lactam, esters and amide carbonyl groups respectively) and 6.62 (amide II) microns.

N.M.R. (CDC13): signals at

6.72 (ABq, 2H, C2~H2), 6.21 (s, 2H, a-CH2>, 4.83-4.65 (m, 4H, C4~H, Cg-H and C3"CH2) ,

4.39 (q, 1H, C7-H), 3.4-2.65 (m, 15H, C7~NH, ester CH and aromatic H) tau.

b) A solution of 8.1 g (16 mmol) of the above ester in 80 ml of methylene chloride was added to 1.57 g (1.6 ml, 19.6 mmol)

dry <p>yridine and 3.8 g (18.1 mmol) phosphorus pentachloride. The reaction mixture was stirred for 2 hours at room temperature and then cooled in an ice-water bath. The cold mixture was treated with 8 ml of isobutanol while stirring. Stirring was continued for 2 hours at the same time that 3 g of diphenyl-methyl-7-amino-3-methylene cepham-4-carboxylate hydrochloride were precipitated as a crystalline precipitate. The product was filtered off and washed with methylene chloride and vacuum dried.

Elemental analysis (%) for C^H^^O-jSCl:

Theoretical: C 60.50 H 5.08 N 6.72 Cl 8.50 Found: C 60.70 H 5.02 N 6.71 Cl 8.80

N.M.R. (DMSO dg): signals at

6.45 (ABq, 2H, C2-H2), 5.00 (d, 1H, Cg-H),

4.68 (d, 1H, C?-H), 4.60 (s, 2H, 3-CH2>, 4.44

(s, 1H, C4-H), 3.10 (s, 1H, ester CH) and 2.61

(s, 10H, aromatic H) tau.

c) 7-amino-3-exomethylene cepham ester hydrochloride salt, 2.1 g (5 mmol) was dissolved in 200 ml of methanol and the solution was

cooled in an acetone-dry ice bath. Ozone was bubbled into the cold solution for 7 minutes to form the intermediate ozonide. This was then decomposed by passing a stream of sulfur dioxide gas through the reaction mixture for 2 minutes. The mixture was then evaporated and the residue treated with diethyl ether to give 1.6 g of diphenylmethyl-7-amino-3-hydroxy-3-cephem-4-carboxylate hydrochloride as a crystalline solid.

N.M.R. (CDC13): signals at

6.4 (ABq, 2H, <C>2"<H>2), 5.0-4.5 (m, 2H, Cg-H and C?-H), 3.2-2.4 '( m, 11H, ester CH and aromatic H) tau I.R. (chloroform): carbonyl absorption peaks at 5.57 and 5.70 (6-lactam and ester carbonyl respectively) micron.

UV (pH 7 buffer): Amax 275 m , e = 7550.

Electrometric titration (60% aqueous DMF): titratable groups at 4.5 and 6.5.

d) To a solution of 840 mg of diphenylmethyl-7-amino-3-hydroxy-3-cephem-4-carboxylate in 10 ml of water and 10 ml of acetone was added 1 g of sodium bisulphite. The mixture was stirred, while 800 mg of thiophene-2-acetyl chloride in 10 ml of acetone was added dropwise. The mixture was stirred for 4.5 hours at room temperature and then evaporated under reduced pressure. The residue was dissolved in a mixture of ethyl acetate and a 5% aqueous solution of sodium bicarbonate. The ethyl acetate layer was separated, washed with water and dried. The dried solution was evaporated and the residue treated with ether to give 500 mg of diphenylmethyl 7-[2-(2-thienyl)acetamido]-3-hydroxy-3-cephem-4-carboxylate.

N.M.R. (CDC13): signals at

6.79 (s, 2H, C2-H2), 6.16 (s, 2H, α-CH2), 5.0

(d, 1H, C6-H), 4.32 (q, 1H, Cj-H), 3.05-2.46

(m, 15H, C7~NH, ester CH and aromatic H) tau.

I.R. (chloroform): absorption peaks at 2.9 (amide NH), 5.6, 5.73 and 5.93 (3-lactam, ester and amide carbonyl groups respectively) and 6.6 5 (amide II) micron. e) A solution of 4.2 g of diphenylmethyl-7-[2-(2-thienyl)-acetamido]-3-hydroxy-3-cephem-4-carboxylate in 44 ml of dry dimethylformamide was added to 865 mg of phosphorus trichloride. The mixture was stirred for 1.5 hours at room temperature and was then poured into a mixture of ethyl acetate and 5% aqueous hydrochloric acid. The ethyl acetate layer was evaporated, washed with 5% hydrochloric acid, with water and then dried. The dried solution was concentrated in vacuo and the product crystallized. The 3-chloroester was filtered off, washed with cold ethyl acetate and dried, whereby 2.2 g were obtained.

Elemental analysis (%) for C26H21<N>2°4<S>2<C1>:

Theoretical: C 59.48 H 4.03 N 5.34 Cl 6.75 Found: C 59.77 H 4.25 M 5.40 Cl 6.91

N.M.R. (CDC13): signals at

6.49 (ABq, 2H, C2~H2), 6.22 (s, 2H, α-CH2>,

5.08 (d, 1H, Cg-H), 4.19 (q, 1H, C?-H), 3.13-2.5 (m, 15H, C^-NH, ester CH and aromatic H) rope. I.R. (CHCl^): absorption peaks at 2.9 (amide NH), 5.55, 5.72 and 5.90 (α-lactam, ester and amide carbonyl groups) and 6.6 0 (amide II) micron .

UV (dioxane): Amax 275 my, e = 8700.

Example 12

p-nitrobenzyl 7-[2-(2-thienyl)acetamido]-3-chloro-3-cephem-4-carboxylate. (via thionyl chloride).

A solution of 1.9 g (4 mmol) of p-nitrobenzyl-7-[2-(2-thienyl)acetamido]-3-hydroxy-3-cephem-4-carboxylate in 10

ml of DMF (dried over a molecular sieve) was added 950 mg (0.58 ml, 8 mmol) of freshly distilled thionyl chloride. The mixture was stirred at room temperature for 6.5 hours and poured into 100 ml of ethyl acetate. The mixture was extracted three times with 30 ml portions of 5% hydrochloric acid and with a saturated solution of sodium chloride. The washed ethyl acetate solution was filtered and evaporated to dryness in vacuo. The residue was treated with ether, whereby 1.2 g of p-nitrobenzyl-7-[2-(2-thienyl)-acetamido]-3-chloro-3-cephem-4-carboxylate was obtained as a brown crystalline solid, melting point approx. . 164-166°C.

Elemental analysis (%) for C^H^N-^OgS^l:

Theoretical: C 48.63 H 3.27 N 8.51 Cl 7.18 Found: C 48.47 H 3.29 N 8.78 Cl 6.96 I.R. (chloroform) showed absorption bands at 2.9 (amide NH), 5.59 (B-lactam carbonyl), 5.75 (ester carbonyl) and 5.92 microns (amide carbonyl).

UV absorption spectrum (acetonitrile) showed maxima at

'max 235 mp, t: = 12,100

•.max 268 mu, r = 15,800

The mass spectrum of the product showed a molecular ion of 493 m/e.

N.M.R. (CDC13) showed signals at

6.39 (ABq, 2H, C, -H2), 6.17 (s, 2H, a-CH2), 4.99 (d, 1H, Cg-H), 4.64 (s, 2H, ester CH2 ), 4.19 (q, 1H, C?-H), 3.45 (d, 1H, C?-NH), 3.1-1.67 (m, 7H, aromatic H) tau.

Example 13

7- [2-(2-thienyl)acetamido]-3-chloro-3-cephem-4-carboxylic acid

A solution of 995 mg (2 mmol) of p-nitrobenzyl-7-[2-(2-thienyl)-acetamido]-3-chloro-3-cephem-4-carboxylate, prepared as described in Example 12, in 60 ml of tetrahydrofuran and 100 ml of methanol containing 5 drops of 1N hydrochloric acid, 1 g of a 5% palladium-on-carbon catalyst was added. The catalyst was pre-reduced in a suspension of 40 ml of ethanol at room temperature under a hydrogen pressure of 3.5 kg/cm 2 .

The suspension was hydrogenated at room temperature for 2.5 hours under a hydrogen pressure of 3.5 kg/cm . The catalyst was filtered off and washed on the filter with THF and then with water. The combined filtrate and catalyst washing solutions were evaporated to dryness, and the reaction product residue was dissolved in a mixture of ethyl acetate and water. The pH of the solution was adjusted to 2.5 and the ethyl acetate layer was separated. The acid reaction product was extracted with water from the ethyl acetate solution at pH 7. The aqueous phase was separated, overlaid with ethyl acetate and acidified to pH 2.5. The ethyl acetate layer was separated, washed with water, dried over sodium sulfate and dried in vacuo. The amorphous residue was treated with ether, whereby 165 mg of 7-[2-(2-thienyl)acetamido]-3-chloro-3-cephem-4-carboxylic acid was obtained as a crystalline solid, melting point 114-120°C with decomposition and softening at approx. 110°C.

This reduction product had the following physical characteristics. I.R. (Nujol): showed absorption bands at 3.1 (amide NH), 5.64 and 5.75 (fl-lactam and carboxylic acid carbonyl groups respectively) and 6.1 (amide II)-micron.

UV (acetonitrile): absorption maxima at

Amax 2 35 mp, t = 10,700

Amax 268 mp, f. = 7,200

N.M.R. (CDC1 ) showed signals at

6.38 (ABq, 2H, C2"H2), 6.16 (s, 2H, α-CH2),

4.98 (d, 1H, Cg-H), 4.20 (q, 1H, C?-H) and 3.1-2.5 (m, 4H, aromatic H and C7~NH) tau.

Elemental analysis (%) for C^H^lN^C^SCl:

Theoretical: C 43.52 H 3.09 N 7.81 Cl 9.88 Found: C 43.55 H 3.79 N 7.27 Cl 9.28

Example 14

p-nitrobenzyl-7-[2-(2-thienyl)acetamido1-3-chloro-3-cephem-4-carboxylate (via phosphorus trichloride).

A cooled solution of 439 mg (0.93 mmol) p-nitrobenzyl-1-{2-(2-thienyl)acetamido]-3-hydroxy-3-cephem-4-carboxylate in 4.4 ml DMF was slowly added 85 mg (0.05 ml, 0.63 mmol) of phosphorus trichloride. The reaction mixture was left for 4 hours at room temperature, after which the reaction product mixture was worked up as described in example 12, and a total of 374 mg of p-nitrobenzyl-7-f2-(2-thienyl)acetamido]-3-chloro-3-cephem was obtained -4-carboxylate. The NMR spectrum of the product was consistent with the expected product and with that corresponding to the compound from example 12.

Example 15

p-nitrobenzyl-7-[2-(2-thienyl)acetamido]-3-chloro-3-cephem-4-carboxylate (via phosphorus oxychloride).

A solution of 325 mg (0.7 mmol) of p-nitrobenzyl-7-1.2-(2-thienyl)acetamido]-3-hydroxy-3-cephem-4-carboxylate in 3.3 ml of DMF cooled in an ice-water bath , 212 mg (0.13 mL, 1.4 mmol) of phosphorus oxychloride was slowly added. The mixture was allowed to stand for 4 hours at room temperature and the product totaling 225 mg was recovered using the method described in Example 12. The nuclear magnetic resonance spectrum of the product was consistent with the spectrum of the previously characterized compound.

Example 16

p-nitrobenzyl 7-[2-(2-thienyl)acetamido]-3-chloro-3-cephem-4-carboxylate (via oxalyl chloride).

A solution of 4 39 mg (0.93 mmol) p-nitrobenzyl-7-[2-(2-thienyl)acetamido]-3-hydroxy-3-cephem-4-carboxylate in 4.4 ml DMF cooled in an ice bath 118 mg (0.07 ml, 0.93 mmol) of oxalyl chloride was added dropwise. The reaction mixture was allowed to stand for 4 hours at room temperature and was then poured into a mixture of aqueous 5% hydrochloric acid and ethyl acetate. The organic layer was separated and washed first with 5% hydrochloric acid, then with water and a saturated sodium chloride solution. The washed layer was dried and evaporated to dryness to give the reaction product p-nitrobenzyl-7-[2-(2-thienyl)acetamido]-3-chloro-3-cephem-4-carboxylate as an amorphous solid. The product was obtained in crystalline form by treating the amorphous residue with ether. Yield 360 mg. The infrared spectrum and the NMR spectrum of the crystalline product were consistent with a spectrum from authentic material.

Example 17

7-[2-(2-thienyl)acetamido]-3-bromo-3-cephem-4-carboxylic acid.

A solution of 19 g (40 mmol) of p-nitrobenzyl-7-[2-(2-thienyl)-acetamido]-3-hydroxy-3-cephem-4-carboxylate in 300 ml of dry DMF was added to 15 g (56 mmol ) phosphorus tribromide and the reaction mixture was stirred at room temperature overnight. It was then poured into a mixture of ethyl acetate and water, and the organic phase was separated and washed several times with water and finally dried over magnesium sulfate. The dry organic phase was evaporated in vacuo to dryness. The impure reaction product that weighed approx. 9 g were purified by chromatography over 500 g of silicon dioxide gel, using ethyl acetate-hexane (55:45 v:v) as eluent. The eluate was evaporated to dryness under reduced pressure, and the product p-nitrobenzyl-7-[2-(2-thienyl)acetamido]-3-bromo-3-cephem-4-carboxylate was obtained crystalline by treating the residue with diethyl

ether.

UV (ethanol) Amax 270 mp, (c = 13,300)

and Amax 243 my, (e = 12,700)

Elemental analysis calculated for C„AH,,BrNo0,S»:

z(J 16 362 Theoretical: C 44.61 H 3.00 N 7.81 Br 14.84 Found: C 44.78 H 3.03 N 7.6 5 Br 14.91

Nuclear magnetic resonance spectrum (DMSOdg) showed signals at 6.21 (s, 2H, ct-CH2), 5.98 (ABq, 2H, C2-H2), 4.72 (d, 1H, Cg-H), 4.51 (s, 2H, ester-CH2), 4.20 (q, 1H, C?-H), 3.04-1.74 (m, 7H, aromatic H) and 0.66 (d, 1H, C7" CH) rope.

The above 3-bridged master was deesterified as follows. The ester of 54 5 mg (1.0 mmol) was hydrogenated at room temperature in ethanol in the presence of a pre-reduced 5% palladium-on-carbon catalyst. The catalyst was filtered off, the filtrate was evaporated under reduced pressure to dryness. The remaining product was treated with diethyl ether and 180 mg (44%) of a crystalline product was obtained, i.e. 7-[2-(2-thienyl)-acetamido]-3-bromo-3-cephem-4-carboxylic acid.

Electrometric titration (66% aqueous DMF) showed a pKa of 4.4 and an apparent molecular weight of 393.

Calculated molecular weight = 40 3.

Elemental analysis calculated for C^H-^BrN-jO^S,"

5 diethyl etherate:

Theoretical: C 40.91 H 3.66 N 6.36 Br 18.15 Found: C 41.29 H 3.20 N 6.29 Br 18.50

Nuclear magnetic resonance spectrum (CDCl^) showed signals at 8.8 (t, diethyl ether-CH3), 6.68-5.86

(m, C2-H2, -~CH2 and diethyl ether-CH2), 4.90 (d, 1H, Cg-H), 3.0-2.63 (m, 3H, aromatic H) and 1.9 (d ,

1H, amide NH) tau.

The examples below illustrate the preparation of intermediate products in the present f remganase method >;■ c<- of H?' 'e

Example 18

p-nitrobenzyl-7-amino-3-chloro-3-cephem-4-carboxylate.

A solution of 500 mg of p-nitrobenzyl-7-[2-(2-thienyl)acetamido]-3-chloro-3-cephem-4-carboxylate in 6 ml of methylene chloride was added to 95 mg of dry pyridine and 237 mg of phosphorus pentachloride. The reaction mixture was stirred at room temperature for 1.5 hours and then cooled in an ice water bath to approx. 5°C, and 0.6 ml of iso-butyl alcohol was added. By continued cooling and stirring, the reaction product crystallized out, p-nitrobenzyl-7-amino-3-chloro-3-cephem-4-carboxylate hydrochloride. This was filtered off, washed with cold methylene chloride and dried, and a total of 200 mg of crystalline product was obtained which melted with decomposition at approx. 168°C.

Percent elemental composition for C-^H^ClN-jO^S-HC1

Theoretical: C 41.39, H 3.20, N 10.34, Cl 17.45 Found: C 41.14, H 3.31, N 10.44, Cl 17.29

I.R. (Nujol)<:>

Shown absorption band at 5.55 (B-lactam carbonyl)

and 5.78 (ester carbonyl) microns.

UV (pH 7 buffer). showed the absorption maximum

Amax 268 my (e=13,800)

N.M.R. (DMSO dg): signals at

5.97 (s, 2H, C2-H2), .8-4.5 (m, 4H, Cg-H, C7~H

and ester CH^), and 2.35-1.6 (q, 4H, aromatic H) tau.

Example 19

Diphenylmethyl 7-amino-3-chloro-cephem-4-carboxylate.

To a solution of 525 mg of diphenylmethyl-7-[2-(2-thienyl)acetamido 1-3-chloro-3-cephem-4-carboxylate in 20 ml of methylene chloride was added 0.1 ml of dry pyridine and 237 mg of phosphorus pentachloride. The reaction mixture was stirred for 2 hours at room temperature and then cooled in an ice-water bath. To the cold mixture was added 0.6 ml of isobutanol and after 30 minutes the reaction mixture was evaporated. The residue was dissolved in ethyl acetate, and the solution washed with 5% sodium bicarbonate and then with water and then dried. The dried solution was evaporated to dryness and the residue treated with ether, whereby 190 mg of 3-chloro-nuclear ester, i.e. diphenylmethyl-7-amino-3-chloro-3-cephem-4-carboxylate, was obtained. I.R.(Nujol): absorption peaks at 5.7 and 5.9 (3- lactam and ester carbonyl) micron.

N.M.R. (CDC13): signals at

6.35 (ABq, 2H, C2"H2), 4.78 (2d, 2H, Cg-H and C7-H), 3.05 (s, 1H, ester CH) and 2.65 (s, 10H, aromatic H).

Example 20

7-(D-mandelamido)-3-chloro-3-cephem-4-carboxylic acid.

A suspension of 812 mg (2 mmol) of p-nitrobenzyl-7-amino-3-chloro-3-cephem-4-carboxylic acid hydrochloride in 40 ml of ethyl acetate was added to a solution of 520 (5 mmol) of sodium bisulfite in 40 ml of water. The mixture was vigorously stirred, while 395 mg (2.2 mmol) of D-mandelic acid-O-carboxylic anhydride was added. The mixture was stirred for 1.5 hours at room temperature, and the aqueous layer was separated from the ethyl acetate layer and washed with ethyl acetate. The ethyl acetate wash solution was combined with the ethyl acetate layer, and these were washed several times with water and then dried and evaporated, whereby the reaction product was obtained as a dry residue. This was treated with ether and 685 mg of p-nitrobenzyl-7-(D-mandelamido)-3-chloro-3-cephem-4-carboxylate was obtained which melts at approx. 158-164°C with decomposition.

Elemental analysis for C22H gN 07SC1:

Theoretical: C 52.44 H 3.60 N 8.34 Cl 7.04% Found: C 52.25 H 3.45 N 8.58 Cl 6.82%

N.M.R. (CDC13): signals at 6.24 (ABq, 2H, C2"H2),

5.04-4.7 (m, 2H, Cg-H and u-H), 4.57 (s, 2H, ester-CH2), 6.23 (q, 1H, C?-H) and 2.8- 1.2 (m, 10H, aromatic H and C7~NH) tau.

UV (acetonitrile: max 265 mj (18,600).

The reaction product of 200 mg was reacted with hydrogen in the presence of 5% palladium-on-carbon to remove the p-nitrobenzyl ester group, and 75 mg of 7-(D-mandelamido)-3-chloro-3-cephem-4-carboxylic acid was obtained. '

N.M.R. (D20 sodium bicarbonate): signals at

6.42 (ABq, 2H, C„-H_), 4.90 (d, 1H, C-H),

4.68 (s, 1H, c1-CH), 4.37 (d, 1H, C7~H) and 2.49 (s, 5H, aromatic H) tau.

Claims (1)

Analogous process for the preparation of therapeutically active 7-acylamino-3-halocephalosporin compounds of the formula where R is thenyl or α-hydroxybenzyl, and X is chlorine or bromine; as well as non-toxic, pharmaceutically acceptable salts thereof, characterized by reacting a 3-hydroxycephalosporin compound with the formula: where R' is thenyl, and is a carboxylic acid protecting ester forming group, with oxalyl chloride, thionyl chloride, phosphorus trichloride, phosphorus oxychloride, oxalyl bromide, thionyl bromide, phosphorus tribromide or phosphorus oxybromide in dimethylformamide; optionally cleaves the 7-thienylacetamido side chain and reacylates the thus obtained 7-amino-3-halocephalosporin compound with an acid of the formula CgH,-CH(OH).COOH or a reactive derivative thereof, after which the carboxylic acid-protecting ester-forming group is removed, and, if desired, converting a compound thus obtained into a pharmaceutically acceptable salt thereof.