Cephalosporin sulfonate esters

© Beginning of patent period: 1975 07 15

Longest possible duration:

© Issued on: 1976 04 26 © inventor:

© dependence:

© Pamphlets considered to delineate the prior art:

OE 329182

No. 329182

The invention relates to a process for the preparation of novel 7-acylamido-3-alkylsulfonyloxy (and

phenyl or substituted phenylsulfonyloxy) -3-oephem-4-carboxylic acids and their esters by sulfonation of 3-hydroxy-3-cephem-estem. The 3-cephem-4-sulfonate esters are interesting antibacterial compounds.

The 7-acylamido-3-hydroxy-3-cephem-4-earbonic esters are reacted in an aprotic solvent with alkylsulfonyl halides having 1 to 6 carbon atoms or with phenyl or substituted phenylsulfonyl halides to give the corresponding 3-alkylsulfonyloxy or phenyl and 3-alkylsulfonyloxy substituted phenylsulfonyloxy-3-cephem-4-carboxylic acid ester. The ester group located in position 4 is then split off, and 7-acylamido-3- (C_zC₆) -alkylsulfonyloxy (or -phenyl10 and substituted -phenylsulfonyloxy -) - 3-cephem-4-carboxylic acid as an antibiotic compound.

The novel cephalosporin-3-sulfonate esters obtainable according to the invention have the following general formula

RC -N (I)

OSR.

C = O

OR wherein R is alkyl having 1 to 6 carbon atoms, haloalkyl having 1 to 3 carbon atoms, cyanoalkyl 15 having 1 to 3 carbon atoms, phenyl, methylphenyl, hydroxyphenyl, halophenyl, nitrophenyl, aminophenyl, methoxyphenyl, 5-amino-5-carboxybutyl or a 5-substituted amino-5-carboxybutyl ester group of the formula

AOC-CH- (CH) "- CH- ι '2' 2 2

NH

A 'is where A is diphenylmethyl, p-nitrohenzyl, benzyl, 2,2,2-trichloroethyl, t-butyl or p-methoxy-20-enyl, and A' is alkanoyl having 2 to 4 carbon atoms, haloalkanoyl having 2 to 4 carbon atoms,

Benzoyl, halobenzoyl, 2,4-dinitrophenyl or phthaloyl, or wherein R is a group of formula a

(Z) -CH - τη 2 a ', in which a and a' independently of one another are hydrogen, alkyl having 1 to 4 carbon atoms, alkoxy having 1 to 4 carbon atoms, halogen, hydroxyl, nitro, amino or carboxy, Z is oxygen or

Is sulfur and m is 0 or 1, or wherein R is a group of the formula

P-CH -

Q is where P is 2-thienyl, 3-thienyl, phenyl or substituted phenyl of the formula

No. 329182

V _ /, wherein a and a 'have the above meaning, Q is hydroxy, formyloxy, acetoxy, carboxy, sulfo, amino or protected amino, or wherein R is a group of the formula

R 'is -CH, wherein R' is 2-thienyl, 3-thienyl, 2-furyl, 2-oxazyl, 2-thiazyl or 1-tetrazyl, R₂ R 1 is hydrogen, benzyl, 4-methoxybenzyl, 4-nitrobenzyl, diphenylmethyl, 2,2,2-trichloroethyl or t-butyl, and R 2 is C 1 -C 6 -alkyl, phenyl, halophenyl, C 1 -C 6 -alkylphenyl or nitrophenyl; if R_t is hydrogen, the pharmaceutically acceptable non-toxic salts hie10 of.

In the above definition of compounds obtainable according to the invention, the term alkyl having 1 to 6 carbon atoms refers to straight-chain or branched-chain alkyl hydrocarbons, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec. Butyl, n-amyl, isoamyl or n-hexyl. Cyanoalkyl having 1 to 3 carbon atoms is understood as meaning groups such as cyanomethyl, 2-cyanoethyl, 3-cyanopropyl 15 or 2-cyanopropyl. The term haloalkanoyl of 2 to 4 carbon atoms refers to chloroacetyl, bromoacetyl, 2-chloropropionyl or 3-bromobutyryl. Alkyl having 1 to 4 carbon atoms is understood as meaning straight-chain or branched-chain alkyl hydrocarbons, such as methyl, ethyl, n-propyl, isopropyl, n-bulyl or t-butyl. Alkoxy of 1 to 4 carbon atoms means methoxy, ethoxy, isopropoxy or n-butoxy. The term halogen is fluorine, bromine or iodine. By halobenzoyl is meant the 20 chlorine- or bromine-substituted benzoyl groups, such as 4-chlorobenzoyl, 4-bromobenzoyl or 2,4-dichlorobenzoyl. Alkanoyl of 2 to 4 carbon atoms refers to acetyl, propionyl or butyryl.

Typical examples of groups of the general formula

<img file="AT329182B_D0001.tif" />

a 'are, in the case of m is 0, phenylacetyl, 4-methylphenylacetyl, 3-ethylphenylacetyl, 4-isopropylphenyl-25-acetyl, 2-methylphenylacetyl, 4-chlorophenylacetyl, 4-nitrophenylacetyl, 4-bromophenylacetyl, 2,4-dichlorophenylacetyl, 3 Bromophenylacetyl, 4-iodophenylacetyl, 2-fluorophenylacetyl, 3,4-dihydroxyphenylacetyl, 4-hydroxyphenylacetyl, 3-hydroxyphenylacetyl, 2,6-dimethoxyphenylacetyl, 3-carboxyphenylacetyl, 4-aminophenylacetyl, 3-ethoxyphenylacetylene, 4-methoxyphenylacetyl, 3,4-dimethoxyphenylacetyl, 4-t-butoxyphenylacetyl or 3-nitrophenylacetyl. In the above formula, when the symbol m is 1 and Z is oxygen, typical examples for this formula are phenoxyacetyl, 4-hydroxyphenoxyacetyl, 3-hydroxyphenoxyacetyl, 4-chlorophenoxyacetyl, 3-bromophenoxyacetyl, 3-ethylphenoxyacetyl, 4-methylphenoxyacetyl, 3 Hydroxy-3-methylphenoxyacefyl, 4-aminophenoxyacetyl, 3-nitrophenoxyacetyl, 2-carboxyphenoxyacetyl, 2-chlorophenoxyacetyl, 4-t-butylphenoxyacetyl, 4-methoxyphenoxyacetyl, 3,4-dimethoxyphenoxyacetyl, 2-aminophenoxyacetyl, 4-isopropoxyphenoxyaceiyl or 4-nitrophenoxyacetyl. Is in the above formula that

Symbol m for 1 and Z is sulfur, typical examples are phenylmercaptoacetyl, 4-chlorophenylmercaptoacetyl, 3-hydroxyphenylmercaptoacetyl, 3,4-dimethylphenylmercaptoacetyl, 4-aminophenylmercaptoaclyne, 3,4-dichlorophenylmercaptoacetyl, 3-bromophenylmercaptoacetyl, 4-fluorophenylmercaptoacetyl, 2, 6-Difluorophenylmercaptoacetyl, 4-nitrophenylmercaptoacetyl or 3-fluorophenylmercaptoacetyl.

In the case of the above formula (I), if the substituent R is a 5-substituted amino-5-carboxybutyl group, then

RC = 0 representative of esterified amino-protected adipoyl groups, wherein the ester group is diphenylmethyl,

Nr.3291

4-p-nitrobenzyl, benzyl, p-methoxybenzyl, 2,2,2-trichloroethyl or t-butyl, and the substituted amino groups acetamido, propionamido, chloroacetamido, benzamido, 2,4-di-chlorobenzamido, 4-bromobenzamido, phthalimido or 2, 4-DinitroaniIino can be.

In the above formula (I), the substituent R is a group of the formula

P-CHI

Q then typical acyl groups R - C = Ο, the mandeloyl group of the formula

CH-C - a '

OH, its O-formyl derivative of the formula

<img file="AT329182B_D0002.tif" />

the o! -Carboxyphenylacetylgruppe of the formula a '- CH - C • I c = o I

OH is the α-sulfophenylacetyl group of the formula - CH - C - a '

SO ^ the phenylglyeyl group of the formula - CH-C a '

NH.

and those 2-thienyl and 3-thienylacyl groups in which in the above formula the phenyl group is replaced by a 2-thienyl or a 3-thienyl ring.

Examples of the above-mentioned acyl groups are:

4-methylmandeloyl, 4-hydroxymandeloyl, 3-hydroxymandeloyl, 4-aminomandeloyl, 3-bromomandeloyl, 20 4-chloromandeloyl, 3-methyl-4-fluoromandeloyl, 2-fluoromandeoyl, 4-fluoromandeloyl, 4-methoxymandeloyl, 3,4-dimethyl -O-formylmandeloyl, 4-chloro-O-formylmandeloyl, 3-amino-O-formylmandeloyl, 3-bromo-O5

No. 329182

-formylmandeloyl, 3,4-dimethoxy-O-formylmandeloyl, O-acetylmandeloyl, O-acetyl-4-hydroxymandeloyl, α-carboxy-4-methylphenylacetyl, o; -carboxy-3,4-dichlorophenylacetyl, α-carboxy-4- hydroxyphenylacetyl, o; -carboxy-2-methoxyphenylacetyl, a-carboxy-4-isopropoxyphenylacetyl, Q! -carboxy-3-hydroxyphenylacetyl, a-carboxy-4-aminophenylacetyl, a-sulfo-4-methylphenylacetyl, c! sulfo-3 , 4-dichlorophenylacetyl, a-formyloxy-2-thienylacetyl, o'-sulfo-2-thienylacetyl, phenylglycyl, 4-hydroxyphenylglycyl, 3-chlorophenylglycyl, 3-hydroxyphenylglyeyl, 4-methoxyphenylglycyl, Q! -Amino-2-thienylacetyl or o; -amino-2-furylacetyl.

In the above formula, if the substituent R is a group of the formula

R '- CH -, then typical acyl groups of formula (I) are 2-thienylacetyl, 3-thienylacetyl, 2-furylacetyl, oxazyl-2-acetyl, thiazyl-2-acetyl or tetrazyl-1-acetyl.

The novel sulfonate esters are prepared according to the invention by reacting a 7-acylamido-3-hydroxy-3-cephem-4-carboxylic acid ester with a C 2 -C 4 alkylsulfonyl halide, a phenylsulfonyl halide or a phenylsulfonyl halide substituted with 16O, preferably at temperatures between about -5 and 35 ° C in a aprotic solvent, in the presence of a hydrogen halide acceptor. The 7-aeylamido-3-hydroxy-3-cephem-4-carboxylic acid esters used as starting materials have the following formula

Ο H

II II

RCN

\.

-N "(Π)

O'

COOR₁

OH in which R has the same meaning as in the above-mentioned formula (I) and R_t an ester-forming carboxylic acid protecting group.

Typical alkyl or phenylsulfonyl halides are methanesulfonyl chloride, methanesulfonyl bromide, ethanesulfonyl chloride, n-butanesulfonyl chloride, n-hexanesulfonyl bromide, phenylsulfonyl chloride, p-chlorosulfonyl chloride, p-fluorosulfonyl chloride, p-toluenesulfonyl chloride, p-toluenesulfonyl bromide, 3- or 4-nitrobenzenesulfonyl chloride or bromide, 3 Ethylbenzylsulfonyl chloride or 3-bromobenzenesulfonyl chloride or bromide,

Suitable aprotic solvents are the ether solvents such as tetrahydrofuran, dioxane or the dimethyl ether of ethylene glycol. Dimethylacetamide is preferred as a solvent.

The reaction is carried out in the presence of a hydrogen halide acceptor, for example a non-reacting tertiary amine such as triethylamine or pyridine, or an alkylene oxide such as propylene oxide or butylene oxide. Propylene oxide is preferred as the hydrogen halide acceptor. The tertiary amine type acceptors tend to isomerize a 3-cephem to a 2-cephem compound. When using an alkylene oxide, such isomerization is kept to a minimum for most sulfonyl halides.

The reaction is carried out by adding the stoichiometric amount of the sulfonyl halide or a slight excess thereof to a solution of the 3-hydroxy-3-cephemester in the aprotic solvent containing at least a stoichiometric amount of the hydrogen halide acceptor. The reaction mixture is preferably stirred at a temperature between 10 and 25 ° C for about 3 to 12 hours. The sulfonate ester thus obtained is isolated from the reaction mixture by extraction with an organic solvent such as ethyl acetate or methylene chloride, and recovered from the extract. The 3-sulfonate esters can be purified by chromatography on silica gel. If the starting material in the 40 7-acylamido side chain contains a functional group capable of reacting with the sulfonyl halide, then this reactive group is protected with a suitable protecting group. For example, the a-amino group of the phenylglycidyl side chain can be protected with a variety of amino protecting groups during the formation of the sulfonyl ester. Suitable examples of these are the urethane protecting groups such as t-butyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, benzyloxyo-carbonyl or p-nitrobenzyloxycarbonyl, the enamine protecting groups formed with ethyl acetoacetate or acetylacetone, the trityl group or other amino-protecting groups. An amino substituent present on the phenyl group in the 7-side chain may be further protected with the same groups. Similarly, a hydroxy group present in the 7-acyl amino side chain, such as the mandeloyl side chain, is protected with a readily cleavable group, for example the formyl group or the trichloroethoxycarbonyl group. After sulfonylation

No. 329182, these protecting groups are removed again. Kerner also removes the carboxy-protecting group present on the C-atom so as to obtain the 3-sulfonyloxy-3-cephem-4-carboxylic acid which is antibiotically active.

The above description of the preparation of the sulfonate esters can be explained by the following reaction scheme:

Ο H

II

RCN

\.

j "

-N.

COOR

OH

X-SO - R 2 2

RCN

\.

z^L

-N.

i

COOR

O-SO-R 2 2

Esterspaltung

H

I

RCN> COOH

O-SO - R "2 2 where R, R ^ and R_ß have the abovementioned meaning.

In one embodiment of the invention, p-nitrobenzyl-7- (nt-butyloxyearbonyl-D-phenylglyeylamido) -3-hydroxy-3-cephem-4-carboxylate is reacted with methanesulfonyl chloride in dimethylacetamide in the presence of propylene oxide at about 5 ° C. to give the 7- (Nt-butyloxycarbonyl-D-phenylglycylamido) -3-methylsulfonyloxy-3-cephem-4-carboxylic acid p-nitrobenzyl ester. The product obtained is hydrogenated over a pre-reduced palladium-on-carbon catalyst in an inert solvent to thereby cleave the p-nitrobenzyl ester group, and the deesterified product is then reacted with p-toluenesulfonic acid in acetonitrile to cleave the p-butyloxycarbonyl group and obtaining the anti-biotic 7- (D-phenylglylamamido) -3-methylsulfonyloxy-3-cephem-4-carboxylic acid.

The starting materials used to prepare the new compounds can be obtained by reacting a 7-acylamido-3-exomethylencepham-4-carboxylic acid ester or a 7-amino-3-exomethylencepham-4-carboxylic acid ester with ozone in an inert solvent at a temperature between -80 and 0 ° C to give the Ozonidderivat the 3-Exomethylendoppelbindung. The ozonide intermediate is decomposed without isolation by reacting the ozonide in situ with a mild reducing agent such as sodium bisulfite or preferably sulfur dioxide to give the corresponding 3-hydroxy-4-cephemcarboxylic acid ester.

The ozonolysis of a 7-amino-3-exomethylencepham-4-carboxylic acid ester or a 7-acylamido-3-exomethylencepham-4-carboxylic acid ester represented by the following formula (HI) is carried out by adding a solution of the 3-exomethylene epham ester in an inert solvent at a temperature between -80 and 0 ° C ozone conducts. The exomethylene double bond reacts with ozone to form, in situ, an intermediate ozonide product which, as described below, forms the 3-hydroxy-3-cephem ester of the

No. 3291 82

Formula (IV) is decomposed.

R

<img file="AT329182B_D0003.tif" />

COOR_i (III) [_Ozonide J

L^HJ

H

RN

S

<img file="AT329182B_D0004.tif" />

COOR (IV)

In the above formulas, the substituent R is hydrogen or an acyl group derived from a carboxylic acid, which acyl group is not oxidizable under the ozonolysis conditions described. The substituent R 1 is an ester-forming group therein, and it is preferably such a group that can be easily split off in the hydrogenolysis or in acidic or basic hydrolysis;

The required ozone gas is produced with an ozone generator commonly used in synthetic and analytical chemistry, after which ozone is obtained by the action of an electrical discharge on oxygen. A suitable ozone generator is made, for example, by the Welsback Corporation. The ozone is formed in an oxygen stream, which is then introduced directly into the reaction vessel. The percentage of ozone present in the oxygen stream may be varied as desired, for example by changing the flow rate of oxygen through the ozonizer or the intensity of the electrical discharge. The percentage of ozone present in the oxygen stream can be determined iodometrically by titrating the amount of iodine released from a standard solution of potassium iodide by the ozone from the generator with sodium thiosulfate. The percentage of ozone present in the oxygen stream is not critical. An approximate value of the amount of ozone flowing into the reaction mixture, however, makes it easier to carry out the ozonolysis process according to the invention, since it is possible to determine the time at which the desired reaction is to be ended, whereby the formation of overoxidation products can be minimized.

Optionally, the ozonolysis can also be monitored by chromatography. For this purpose, for example, a small portion of the reaction mixture is removed, decomposes the ozonide and determines the present in the sample amount of unreacted starting material and formed 3-hydroxy ~ 3-cephem by comparing the Dünnschichtehromatogramms with that of a known amount of starting material and a 3-hydroxy -3-cephem.

Suitable inert solvents for the ozonolysis are those in which the 3-Exomethylencephamester are at least partially soluble and which do not react with ozone under the conditions described. As the solvent, methanol, ethanol, ethyl acetate, methyl acetate and methylene chloride are generally used for this purpose.

The concentration of starting material in the inert solvent is not critical. Preferably, a sufficient amount of solvent is used for complete dissolution.

The ozonolysis is preferably carried out at temperatures between about -80 and -50 ° C.

No. 329182

After completion of ozonide formation, as determined by one of the methods described above, excess ozone present in the reaction mixture is expelled from the reaction mixture by passing nitrogen or oxygen through the mixture.

After removal of the excess of ozone, the ozonide is decomposed by adding to the reaction mixture a mild reagent selected from the group of sodium bisulfide, sulfur dioxide or trimethyl phosphite to give the 3-hydroxy-3-cephem-4-carboxylic acid ester. The decomposition is carried out by adding an excess of the reducing agent and then stirring the reaction mixture at temperatures between about -80 and 0 ° C until it reacts negatively in the potassium iodide-starch test.

For the decomposition of the ozonide intermediate, gaseous sulfur dioxide is preferably used. This agent is preferred because it volatilizes completely out of the reaction mixture during the subsequent work-up and thus does not complicate the recovery of the reaction product.

The 7-aeylamido-3-hydroxy-3-eephem-4-carboxylic acid esters are recovered from the reaction mixture by first evaporating the mixture to dryness and then extracting the desired product from the residue. Optionally, the N-acylated 3-hydroxy-3-cephem esters may also be recovered from the organic liquid phase of the decomposition mixture by separating the liquid phase from insoluble constituents and evaporating the organic layer after washing and drying to give the liquid

3-hydroxyester.

The 3-hydroxy-core ester, namely a 7-amino-3-hydroxy-3-cephem-4-carboxylic acid ester, is best isolated in the form of a salt, for example the hydrochloride or hydrobromide.

If ozonized an ester of 7-amino-3-exomethylencepham-4-carboxylic acid (formula IH, R = H), then it is preferable to use a salt of this nucleus, for example the hydrochloride or p-toluenesulfonate.

The starting products for the preparation of the 3-exomethylene cephalamides are obtained by reacting a 7-acylamidocephalosporanic acid in a known manner with a sulfur-containing nucleophile, 25 thereby causing nucleophilic displacement of the acetoxy group of the cephalosporanic acid to give a 7-acylamido-3-thio-substituted methyl 3-cephem-4-carboxylic acid comes. The 3-thio-substituted cephem product is then reduced with hydrogen in the presence of Raney nickel or with zinc / formic acid in the presence of dimethylformamide to give the 3-exomethyleneacetamic acid. For example, 7-phenylacetamidocephalosporanic acid is reacted with potassium ethylxanthate to give 7-phenyl-30-acetamido-3-ethoxythiocarbonylthiomethyl-3-cephem-4-carboxylic acid, from which the 7-phenylacetamido is obtained by reduction with zinc / formic acid in the presence of dimethylformamide. 3-exomethylencepham-4-carhonic acid of the formula \ = / -CH

Ο H

<img file="AT329182B_D0005.tif" />

COOH receives.

In a similar way, the 3-Exomethylencephamkem the formula

HN 2 \

Z '

-N.

CH.

Produce COOH by reacting a 7-acylamido-3-exomethylencepham-4-carboxylic acid ester with phosphorus pentahydoride (PCI) in methylene chloride in the presence of pyridine to obtain the corresponding iminochloride as an intermediate. The iminochloride is then reacted in the cold with methanol to imino ether.

The imino ether hydrolyzes readily, yielding the 7-amino-3-exomethylencepham-4-carboxylic acid ester hydrochloride. Cleavage of the ester group then leads to 3-Exomethylencephamkem.

An ester of 3-Exomethylencephamkems can acyHeren with the desired derivative of a carboxylic acid to 7-acylamido-3-exomethylencepham-4-carboxylic acid ester, and the thus obtained aeylierte ester is then the 3-hydroxy ester, namely the starting material of the formula (H), ozonated. Optionally

No. 329,182 acylate an ester of 3-hydroxy-7-amino-3-cephemkem also under non-anhydrous conditions to 7-acylamido-3-hydroxy-3-cephem-4-carboxylic acid ester. The acylation of this nucleus takes place under the conditions which are also customary for the acylation of 7-aminocephalosporanic acid. However, the acylation of a 7-amino-3-hydroxy-3-ephem ester is preferably carried out in an aqueous medium, for example in aqueous acetone or aqueous acetonitrile.

The 7-acylamido-3-alkyl or phenylsulfonyloxy-3-cephem-4-carboxylic acid esters (formula I, R = esters) are interesting intermediates for the preparation of the antibiotically active free acids. Under the definition of R₁ falling ester-forming groups are all known groups which are normally used to protect the C-carboxylic acid group of the cephalosporin molecule, while the molecule 4

Implements conversions affecting other groups. These ester-forming groups are readily cleaved by known reduction or hydrolysis techniques to form the free acid. For example, the p-nitrobenzyl ester group is removed by catalytic hydrogenolysis over palladium on carbon. The diphenylmethyl (benzhydryl) is removed, for example, with trifluoroacetic acid in anisole at about 10 ° C. The p-methoxybenzyl group is, for example, deprotected with trifluoroacetic acid at about 10 ° C (J. Org. Chem., 36 [1971], p. 1259). The 2,2,2-trichloroethyl group is removed, for example, with zinc and acid (J. Am. Chem. Soc., 88 [1966], p 852). The cleavage of the benzyl ester group is carried out, for example, by catalytic hydrogenolysis over a palladium catalyst (J. Org. Chem., 27 [1962], p. 1381). The cleavage of the tertiary butyl group is described, for example, in J. Org. Chem., 31 [1966], p. 444.

In the preparation of the new 3-sulfonate ester, only a small amount of the corresponding arises

2-cephem isomers. The preparation of the tosylate esters is normally accompanied by a stronger isomerization to the 2-cephem isomer than the preparation of the alkyl sulfonate esters. If a considerable amount of 2-cephem-3-sulfonate ester forms in the sulfonylation, then the mixture of 2- and 3-sulfonate esters can be formed

Separate 3-cephemsulfonates by chromatography on silica gel. The mixture of isomers is preferably oxygenated with a perfluoric acid, for example with m-chloroperbenzoic acid or peracetic acid, to produce the corresponding sulfoxides. The sulfoxide formation of a 2-cephem is known to be accompanied by isomerization to the 3-cephem isomer (J.A. Chem. Soe., 35 [1970], p. 2430). The resulting 3-cephem-3-sulfonate ester sulfoxide is then reduced to the sulfide, which can be done for example with a phosphorus halide or with phosphorus trichloride. Any 2-cephem isomer present in the desired 3-cephemsulfonate 30 ester can therefore be converted to the desired 3-cephem isomer. The 7-acylamido-3-cephem-3-sulfonate esters of formula (I) wherein Rj is hydrogen (namely, the free acids) are valuable antibiotics which are more resistant to gram-positive and gram-negative bacteria and also more resistant to penicillin Use staphylococci. The antibacterial activity of these sulfonate esters is illustrated by the following in vitro determined comparative values ​​obtained using typical compounds. Table I below shows the minimum inhibitory concentration (MIC) for typical compounds against Gram-negative bacteria. The corresponding data are obtained according to the so-called standard gradient plate method.

Table I

<td colspan="7">Antibiotic activity of cephalosporin sulfonates</td>

<td></td><td colspan="6">against gram-negative bacteria</td>

<td>Test organism</td><td></td><td colspan="4">investigated compound¹Minimum inhibitor concentration (mg / ml)</td><td></td>

<td></td><td>1</td><td>2</td><td>3</td><td>4</td><td>5</td><td>6</td>

<td>Shigella sp.</td><td>10.7</td><td>7.0</td><td>6.5</td><td>.19,5</td><td>16.5</td><td>13.6</td>

<td>Escherichia coli</td><td>11.0</td><td>13.1</td><td>8.8</td><td>16.3</td><td>24</td><td>19.5</td>

<td>Klebsiella pneumoniae</td><td>6.5</td><td>7.0</td><td>5.5</td><td>9.3</td><td>16.3</td><td>5.5</td>

<td>Aerobacter aerogenes</td><td>3.5</td><td>3.5</td><td>6.0</td><td>16.5</td><td>12.0</td><td>8.8</td>

<td>Salmonella heather</td><td>2.0</td><td>4.0</td><td>6.0</td><td>125</td><td>13.5</td><td>7.0</td>

<td>Pseudomonas aeruginosa</td><td>&gt; 200</td><td>&gt; 200</td><td>&gt; 200</td><td>&gt; 200</td><td>&gt; 200</td><td>&gt; 200</td>

<td>Serratia marcescens</td><td>110</td><td>120</td><td>'180</td><td>&gt; 200</td><td>&gt; 200</td><td>100</td>

No. 329182

Table I (continued) ¹ The compounds investigated are the following:

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

7- [2- (2-Thienyl) acetamido] -3-ethylsulfonyloxy-3-cephem-4-earic acid

7- [2- (2-Thienyl) acetamido] -3-n-butylsulfonyloxy-3-cephem-4-carboxylic acid

7- [2- (2-Thienyl) acetamido] -3- (p-toluenesulfonyloxy) -3-cephem-4-carboxylic acid

7- [2- (2-Thienyl) acetamido] -3- (4-fluorobenzenesulfonyloxy) -3-cephem-4-carboxylic acid

7- (D-PhenyIglycylamido) -3-methylsulfonyloxy-3-cephem-4-carboxylic acid

In the same study, cephalosporin sulfonate esters also inhibit the growth of clinical isolates of penicillin-resistant staphylococci.

The 7-acylamido-3-cephem-3-sulfonate ester of the formula (I) in which represents hydrogen form 5 pharmaceutically acceptable salts with inorganic and organic bases. Thus, for example, the sodium, potassium or calcium salts can be prepared by reacting the free acids with sodium carbonate, sodium bicarbonate, sodium hydroxide, potassium carbonate or calcium hydroxide. With amines, such as benzylamine, 2-aminoethanol, diethanol or diisopropylamine, amine salts can be produced. Compounds of formula (I) wherein RC (O) - represents the phenylglycyl side chain form acid addition salts with suitable mineral and organic acids. By reaction with hydrochloric acid, hydrobromic acid or sulfuric acid, for example, the corresponding hydrochlorides, hydrobromides or sulfates can be produced. By reacting with p-toluenesulfonic acid can be obtained in a similar manner to the p-toluenesulfonate of the amine. Such salts of the C ^ -Carboxylgruppe and an amino group of the C_?Side chain are useful for purifying the free acid of the antibiotic and for preparing pharmaceutically acceptable formulations suitable for administration.

Those compounds of formula (I) in which R 1 represents an ester group are important intermediates for the preparation of the particular free acid of the antibiotic.

Examples of compounds of the formula (I) obtainable according to the invention are: 7- [2- (2-thienyl) acetamido] -3-methylsulfonyloxy-3-cephem-4-carboxylic acid,

7- [2- (2-Furyl) -acetamido] -3-ethylsulfonyloxy-3-cephem-4-carboxylic acid, 7-phenylacetamido-3- (p-toluenesulfonyloxy) -3-cephem-4-carboxylic acid, 7- (4 -Chlorophenylmercaptoacetamido) -3-methylsulfonyloxy-3-cephem-4-carboxylic acid, 7-phenoxyacetamido-3-n-propylsulfonyloxy-3-cephem-4-carboxylic acid, 7-cyanoacetamido-3-methylsulfonyloxy-3-cephem-4-carboxylic acid,

7- (D-Mandelamido) -3-methylsulfonyloxy-3-eephem-4-carboxylic acid,

7- (D-phenylglycylamido) -3-methylsulfonyloxy-3-cephem-4-carboxylic acid,

7- (D-4-hydroxyphenylglycylamido) -3-äthylsulfonyloxy-3-cephem-4-carboxylic acid,

7- (O-formyl-D-mandelamido) -3- -3-cephem-4-carboxylic acid (p-toluolsuIfonyIoxy)

7- (tetrazol-l-acetamido) -3-methylsulfonyloxy-3-cephem-4-carboxylic acid,

7- (5-amino-5-carboxyvaleramido) -3- (p-toluenesulfonyloxy) -3-cephem-4-earbonic acid, 7-acetamido-3-benzenesulfonyloxy-3-cephem-4-carboxylic acid,

7- ("-Sulfophenylacetamido) -3-ethylsulfonyloxy-3 -cephem-4-carboxylic acid, 7- (Q! -Carboxyl-4-ehlorophenylacetamido) -3-n-butylsulfonyloxy-3-cephem-4-carboxylic acid and its pharmaceutical harmless non-toxic salts.

The invention will be explained in more detail below with reference to the examples.

A) Preparation of the starting materials.

Protocol 1: p-NitrobenzyI-7-amino-3-methylencepham-4-carboxylate hydrochloride.

To a solution of 965 mg (mmol) of p-nitrobenzyl-7-phenoxyacetamido-3-methylencepham-4-carboxylate in ml of methylene chloride is added 175 mg of dry pyridine and 460 mg of phosphorus pentachloride, and the resulting mixture is stirred for 6 hours at room temperature. Then the mixture is mixed with 1 ml of isobutanol and then allowed to stand at 0 ° C overnight. The resulting crystalline precipitate, namely the p-nitrobenzyl-7-amino-3-methylencepham-4-carboxylate hydrochloride, is filtered off to give 430 mg (58% yield) of the desired product.

Elemental analysis calculated for CH NgO SCI: C, 46.69; H4,18; N10,89;

found: C 46.40; H4,20; N10,62.

IR (Nujol Mull)

Carbonyl absorption at 5.65 (/ 3-lactam) and 5.75 (ester) micron.

No. 3291 82

NMR (DMSO-d_G): Signal fog 6,34 (2d, 2H, C-H), 4,98 (d, 1H, CH),

4.7-4.4 (m, 6H, C-H, ester CH₂, C₄-CH₂ and C-H) and 2.4-1.6 (m, 4H, aromatic H) t.

Protocol 2: p-Nitrobenzyl-7-amino-3-hydroxy-3-cephem-4-carboxylate hydrochloride.

A solution of 4 g of p-nitrobenzyl-7-amino-3-methyleneepham-4-carboxylate hydrochloride in 620 ml

Methanol is cooled in a dry ice-acetone bath, and ozone is bubbled through the cold solution for about 20 minutes. The resulting reaction mixture is then purged with nitrogen to remove residual ozone followed by the addition of 10 g of sodium bisulfite. The reaction mixture is then stirred for 1 h at Eisbadtempe10 ratur, whereupon the potassium iodide-starch test of the mixture is negative.

After evaporation of the mixture in vacuo, the desired reaction product is obtained in the form of an amorphous yellow residue. The residue is recrystallized from acetone to give 3.4 g of p-nitrobenzyl-7-amino-3-hydroxy-3-cephem-4-carboxylate hydrochloride as a crystalline acetone solvate.

IR (Nujol Mull):

Carbonyl absorption band at 5.60 (β-lactam) and 6.04 (ester carbonyl hydrogen, attached to

3-hydroxy) micron.

NMR (DMSO-d); Signals at 7.92 (s, 3H, 1/2 mol acetone),

6.22 (2d, 2H, C₂-H₂)

5.07 (d, 1H, C₆H),

4, 8 - 4.5 (m, 3H, ester CH₂ and C_?H),

2.4 - 1.6 (m, 4H, aromatic Η) t.

Protocol 3: 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

Acetone containing 364 mg (0.5 ml, 3.6 mmol) of triethylamine is added with 962 mg of urea. With stirring at room temperature, the mixture thus obtained is then added dropwise to 730 mg (4.4 mmol).

2-thiopheneacetyl chloride in 20 ml of acetone. After 2.5 h, the reaction mixture is filtered and evaporated. The residue is dissolved in ethyl acetate and the resulting solution is washed successively with water, 5% sodium bicarbonate solution, 5% hydrochloric acid and saturated sodium chloride solution. The washed solution is dried and then evaporated in vacuo to give 1.2 g of reaction product in the form of a crystalline residue. Recrystallization from ethyl acetate gives pure p-nitrobenzyl-7- [2- (2-thienyl) acetamido] -3-hydroxy-3-cephem-4-carboxylate, which has the following spectral properties:

IR (Nujol Mull):

Absorption peaks at 3.0 (amide NH), 5.68 (β-laetamcarbonyl), and 6.1 (amide and ester water 35 bound to 3 OH) micron.

NMR (CDClg / DMSO-dg): signals at

6.54 (2d, 2H, C₂H₂)

6.16 (s, 2H, side chains CHp,

4.90 (d, 1H, C_GH),

4.60 (d, 2H, ester CH),

4.43 (q, 1H, C 1 H),

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

B) Preparation of the sulfonate esters.

Example 1:

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

A solution of 4.75 g (10 mmol) of p-nitrobenzyl-7- [2- (2-thienyl) acetamido] -3-hydroxy-3-cephem-4-carboxylate in 50 ml of dry dimethylacetamide is added with 2 ml of propylene oxide added. 1 equivalent of methanesulfonyl chloride is added to the solution thus obtained, and the mixture is then stirred for a further 3 hours. The reaction mixture is taken up in ethyl acetate and the resulting solution is washed with saturated sodium chloride solution. The washed organic phase is evaporated to dryness in vacuo to yield, as residue, a mixture of the desired reaction product. The reaction product is purified by preparative silica gel thin-layer chromatography using 65% ethyl acetate / hexane.

The purified product is microanalytically analyzed, which is the following elemental composition analysis for ^C21H₁₉^N3 °₉S₃¹ calculated: C 45.56; H, 3.46; N7,59;

found: C45.74; H3,56; N7.30;

S 17,38; S 17.06.

No. 329182

The nuclear resonance spectrum and the infrared absorption spectrum are consistent with the structure of the product formed.

NMR (DMSO-dp: delta values ​​3.47 (s, 3H, methyl),

3.80 (broad s, 2H, side chains CH),

3.91 (q, 2H, CH), ²

5.29 (d, 1H, CH),

5.46 (broad s, 2H, ester CH),

5.84 (q, 1H, CH),

6.86-7.44 (m, 3H, thiophene) and

7.98 (q, 4H, phenyl).

IR (Mull): 1785, 1350 and 1158 cm^-1,

UV (ethanol): 264 mju.

The above product (2 g) is dissolved in a mixed solvent of 15 ml of methanol and 20 ml of tetrahydrofuran, and the solution is mixed with 3 g of pre-reduced catalyst of 5% palladium-on-carbon.

(The catalyst is prereduced in 15 ml of methanol for 1 h before use.) The resulting mixture is hydrogenated for 1.5 h, during which time the theoretically possible hydrogen uptake takes place.

The catalyst is filtered off and the filtrate is evaporated on a rotary evaporator in vacuo to dryness. The residue obtained is dissolved in 20 ml of ethyl acetate and the solution is mixed with 20 ml of cold water. Then set the pg value of the solution with a sodium bicarbonate solution to pH 7 and then separates the organic phase. The aqueous phase is washed with ethyl acetate, then the pjj is adjusted to 2.0 with 1N hydrochloric acid. The organic phase is separated and combined with an ethyl acetate extract of the acidified aqueous layer. The mixture of extract and organic layer is dried over magnesium sulfate and then evaporated to dryness to give the deesterified product, namely the 7- [2- (2-thienyl) acetamido] -3-methylsulfonyloxy-3-cephem 4-carboxylic acid.

NMR (acetone dp: delta values ​​3.33 (s, 3H, methyl),

3.50 - 4.00 (m, 4H, two CHp,

5.10 (d, 1H, CH),

5.88 (d, 1H, C₇H),

6.80 - 7.40 (m, 3H, thiophene).

IR (KBr): 1795, 1175 cm¹,

UV (ethanol): Ä_Max 265 (shoulder).

Electrometric titration (80% aqueous methylcellosolve): pK_a 3, 9.

Example 2:

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

According to the procedure described in Example 4.7 g (10 mmol) of p-nitrobenzyl-7- [2- (2-thienyl) -acetamido] -3-hydroxy-3-cephem-4-carboxylate is added in the 50th hour for about 3 hours ml of dimethylacetamide containing about 2 ml of propylene oxide reacted with a stoichiometric amount of ethanesulfonyl chloride. The Reaktionsgemiseh is dissolved in ethyl acetate, whereupon the solution is washed with saturated sodium chloride solution. The organic phase is dried and evaporated to dryness to give the reaction product.

The product obtained is purified by thin layer chromatography on silica gel using 65%.

Ethyl acetate / hexane to develop. The purified product, namely p-nitrobenzyl-7- [2- (2-thienyl) acetamido] -3-ethylsulfonyloxy-3-cephem-4-carboxylate, has the following elemental analysis:

Gross formula CHN "OS":

21 3 θ3 calculated: C46.55; H3,73; N, 7.40; S16,95;

found: C 46.32; H, 3.48; N7,5; S16,67.

IR (chloroform): 1800, 1358, 1163 cm^-1,

UV (ethanol): λ 265 ταμ.

NMR (CDCl_G1 Delta values: 1.47 (Τ, 3H, methyl),

3.40 (q, 2H, CH),

3.73 (q, 2H, C 1-4 CH),

3.88 (s, 2H, side chains CHp,

5.13 (d, 1H, C_eH),

5.20 (s, 2H, ester CH ,,),

5.91 (q, IH, C_TH),

6.85 (d, 2H, NH),

7.00-7.44 (m, 3H, thiophene),

7.96 (q, 4H, phenyl).

The above product is hydrogenated to remove the p-nitrobenzyl ester group by the hydrogenation process described in Example 1 to give the 7- [2- (2-thienyl) acetamido] -3-ethyl-60-sulfonyloxy-3-cephem-4 carboxylic acid.

No. 32918

NMR (CDC1₃ + Acetone-dJ: delta values

1.45 (Τ, 3H, methyl),

3.41 (q, 2H, CHp,

3.69 (q, 2H, ring CH),

3.84 (s, 2H, side chains CH),

5.12 (d, 1H, C₆-H),

5.83 (q, 1H, C_?-H),

6.88-7.32 (m, 3H, thiophene).

IR (Mull): 1787, 1350 and 1170 cm^-1,

Electrometric titration in 80% aqueous methyl cellosolve: pK_a 4.15.

Example 3:

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

According to the esterification process described in Example 1, 4.7 g of p-nitrobenzyl-7- [2- (2-thienyl) acetamido] -3-hydroxy-3-cephem-4-carboxylate with the stoichiometric amount of n-Butylsulfonylehlorid in Dimethylacetamide containing propylene oxide reacted to give 2.1 g of the 3-n-Buiylsulfonyloxyderivats.

Elemental Analysis for CH NOS ":

25 ο 9 o calculated: C48.39; H4,23; N7,05;

found: C47.94; H4,27; N 6,31.

NMR (DMSO-d ^: delta values ​​0.87 (t, 3H, methyl),

1.10-1.90 (m, 4H, two CHp,

2.45-2.87 (m, 2H, CH),

3.71 (q, 2H, ring CH),

3.79 (s, 2H, side-chain CHp,

5.28 (d, 1H, C₆H),

5.42 (broad s, 2H, ester CH ^),

5.82 (q, 1H, C_?H),

6.50-6.97 (m, 3H, thiophene),

8.07 (q, 4H, phenyl).

IR (CHCy: 1801, 1355, 1170 cm)¹,

In addition to the n-butylsulfonyl derivative thus obtained, the p-nitrobenzyl ester group is then cleaved off by the procedure described in Example 1 to give the 7- [2- (2-thienyl) acetamido] -3-n-butylsulfonyloxy-3- cephem-4-carboxylic acid.

NMR (CDClj): delta values ​​0.98 (t, 3H, methyl),

1.10-2.00 (m, 4H, two CHp,

2.65-3.00 (m, 2H, CH),

3.60 - 4.10 (m, 4H, C₂ and side chains CH₂)

5.09 (d, 1H, C₆H),

5.78 (q, 1H, C_?H),

6.85-7.38 (m, 3H, thiophene).

IR (CHCl 3): 1787, 1360 cm "¹,

Electrometric titration in 80% aqueous methyl cellosolve: pKa 4.75,

Example 4:

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

A solution of 9.5 g (20 mmol) of p-nitrobenzyl-7- [2- (2-thienyl) acetamido] -3-hydroxy-3-cephem-4-carboxylate in 30 ml of dimethylacetamide and 30 ml of propylene oxide is added with ice bath temperature with

4.2 g (22 mmol, 1.1 equiv.) Of p-toluenesulfonyl chloride. The reaction mixture is stirred at ice bath temperature for about 15 hours and then at room temperature for about 3 hours. The mixture is then evaporated to remove excess propylene oxide and the concentrate is dissolved in ethyl acetate. The resulting solution is washed with saturated sodium chloride solution and dried. Evaporation of the dried solution under reduced pressure gives the crude tosylate ester in the form of a dry residue. The residue is dissolved in ethyl acetate and the resulting solution is chromatographed on water-deactivated silica gel (Woelm silica gel, 10% water) packed in a glass column. The column is eluted with 45% by volume of hexane in ethyl acetate. There are collected 4 fractions with about 100 ml volume. Fractions 2 and 3 are combined and evaporated under reduced pressure to give 4.75 g of p-nitrobenzyI-7- [2- (2-thienyl) -aeetamido] -3- (p-toluenesulfonyloxy) -3-cephem-4 carboxylate which is mixed with the corresponding 2-cephem isomer.

A solution of 1.26 g of the isomer mixture in 20 ml of methylene chloride is added under ice-bath temperature with a solution of 0.4 g of m-chloroperbenzoic acid in 20 ml of methylene chloride. The mixture

The mixture is stirred for 40 minutes, after which it is evaporated to dryness. The residue is treated with isopropanol, then the whole is filtered, washed with ether and dried to give 1.1 g of product, namely p-nitrobenzyl-7- [2- (2-thienyl) -acetamido] -3- (p- toluenesulfonyloxy) -3-cephem-4-carboxylate sulfoxide. The resulting sulfoxide has the following elemental composition:

Gross formula CH "N" OS ":

23 3 10 3 calculated: C 50.23; H3,59; N6,51;

Found: C49.98; H3,30; N6,53.

The delta²cephemsulfoxide is reduced to the delta as follows ³-cephemsulfid:

A solution of 1.0 g of the sulfoxide in 25 ml of acetonitrile containing 5 ml of dimethylformamide is added under ice-bath temperature while stirring with 0.157 g of phosphorus tribromide. The mixture is then stirred for 1 h in the cold. The mixture is then treated with ethyl acetate and saturated aqueous sodium chloride solution. The product is extracted with ethyl acetate, and the organic phase is washed three times with saturated sodium chloride solution. The organic phase is dried and evaporated to give 1.1 g of product, namely p-nitrobenzyl-7- | 2- (2-thienyl) -acetamido] -3- (p-toluenesulfonyloxy) 15 -3-cephem-4 carboxylate.

The product obtained above is hydrogenated over a catalyst before reduction from 5% palladium on carbon in methanol / tetrahydrofuran according to the procedure described in Example 1, whereby the product obtained is the free carboxylic acid, namely the 7- [2- (2-thienyl ) acetamido] -3- -3-cephem-4-carboxylic acid (p-toluenesulfonyloxy).

NMR (CDC1 / delta values ​​2.47 (s, 3H, methyl),

3.40 - 4.10 (m, 4H, C₂ and side chains CH /

5.05 (d, 2H, C_eH),

5.80 (q, 1H, C_T-H),

6.85-7.38 (m, 3H, thiophene),

7.61 (q, 4H, phenyl).

IR (CHC1 / 1790, 1380, 1170 cm)¹,

Electrometric titration (80% aqueous methylcellosolve): pK_a 4.4.

UV (ethanol): Ä_Max 265 m ^ (shoulder).

Example 5: 7- [2- (2-Thienyl) acetamido] -3- (4-fluorobenzenesulfonyloxy) -3-cephem-4-carboxylic acid.

Following the sulfonation and deesterification process described in Example 1, p-nitrobenzyl-7- [2- (2-thienyl) acetamido] -3-hydroxy-3-cephem-4-carboxylate is reacted with 4-fluorobenzenesulfonyl chloride in dimethylacetamide in the presence of Propylene oxide and the resulting sulfonation product, namely p-nitrobenzyl-7- [2- (2-thienyl) acetamido] -3- (4-fluorobenzenesulfonyloxy) -3-cephem-4-carboxylate, is hydrogenated in methanol-tetrahydrofuran over prereduced 5% plagium-on-charcoal catalyst, whereupon the product 7-, 2- (2-thienyl) -acetamido] -3- (4-fluorobenzenesulfonyloxy) -3- cephem-4-carboxylic acid.

This compound has the following elementary analysis.

Gross formula for CH ".NOSi:

25 2 7 3 calculated: C 45.78; H3,03; N 5.62; F3,81;

found: C, 46.04; H3,31; N5,33; F3,89.

NMR (CDC1 / Delta values ​​3.48 - 4.10 (broad m, 4H, C₂ and side chains CH /

5.05 (d, 1H, C_G-H),

5.77 (q, 1H, C_?-H),

6.80-7.48 and 7.77-8.16 (broad m, 7H, thiophene and phenyl H).

IR (CHC1 / 1792, 1385 and 1160 cm "¹,

Electrometric titration (80% aqueous methylcellosolve): pK_a 4.25.

Example 6:

7- (D-phenylglycylamido) -3-methylsulfonyloxy-3-cephem-4-carboxylic acid.

A solution of 11.1 g of p-nitrobenzyl-7-amino-3-hydroxy-3-cephem-4-carboxylate hydrochloride in

500 ml of tetrahydrofuran is mixed with 15.1 g of sodium bisulfite. The mixture is stirred at room temperature for 1 h, whereupon 6.4 g of N- (p-butyloxycarbonyl) phenylglycine and 6.25 g of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) are added. The mixture is stirred for 7 h at room temperature, whereupon it narrows to remove tetrahydrofuran. This concentrate is dissolved in ethyl acetate and the resulting solution is washed successively with sodium bicarbonate solution, dilute hydrochloric acid and saturated sodium chloride solution and then dried. The dried solution is evaporated to dryness to give 11.14 g of p-nitrobenzyl-7- (nt-butyloxycarbonyl) -D-phenylglycylamido) -3-hydroxy-3-cephem-4-carboxylate.

A solution of 11.14 g of the above product in 50 ml of dimethylacetamide containing 25 ml of propylene oxide is added at room temperature with 1.47 ml of methanesulfonyl chloride. The resulting mixture is stirred for about 3 h, whereupon 1.47 ml of methanesulfonyl chloride are added and the mixture is stirred for a further 15 h. The

No, 329182

The reaction mixture is diluted with ethyl acetate, and the resulting solution is extracted four times with saturated sodium chloride solution. The washed organic phase is dried and evaporated to a crude product of p-nitrobenzyl-7- (Nt-butyloxycarbonyl-D-phenylglycylamldo) -3-methylsulfonyloxy-3-cephem-4-carboxylate. The product is purified by dissolving in methylene chloride and precipitating the solution

Dilute with hexane. The purified product (8.09 g) is filtered and dried.

To remove the p-nitrobenzyl ester group, this product is hydrogenated over prereduced 5% palladium on carbon by the procedure described in Example 1 to give 4.21 g of the free acid.

The free acid (1.545 g) is dissolved in 3 ml of dry acetonitrile, and the solution is added to 10.7 g of p-toluenesulfonic acid. The reaction mixture is stirred overnight at room temperature. The mixture is then mixed with water and the pg value is adjusted to pH 5.0 with sodium bicarbonate solution. The mixture is then heated to remove acetonitrile, after which the aqueous residue is filtered. The pg value of the filtrate is then adjusted to pg 4.0, after which it is freeze-dried. The freeze-dried mixture is treated with acetone and filtered. The solid is dissolved in 15 ml of water, 15 and the resulting solution is added at ice bath temperature with about 5 ml of acetone. The product thus obtained, namely the 7- (D-phenylglycylamido) -3-methylsulfonyloxy-3-cephem-4-carboxylic acid, crystallized from the cold solution, followed by filtration, washing with cold water and with acetone and drying and 143 mg of the desired product is obtained with the following elemental composition:

Elemental analysis for CH N "OS:

lb 11 i 7 2 calculated: C 44.92; H 4.01; N, 9.83;

found: C44,13; H4,24; N 9,26.

Electrometric titration in 80% aqueous methyl cellosolve: pK_a 3.6 and 6.75.

IR (Mull): 1780, 1360 and 1178 cm¹,

UV (pg 6 buffer): Ä_Max 261 mp (e = 8,400).

NMR (DMSO-d₆Delta values: 3.28 (s, 3H, methyl),

3.55 (q, 2H, 0, -CH ,,),

4.92-5.1 (m, 2H, C_GH and CH of the side chain),

5.68 (q, 1H, C_?-H),

7.48 (m, 5H, phenyl).