NO761052L - - Google Patents

Description

The present invention relates to antibacterial agents and in particular to a class of cephalosporin derivatives with broad-spectrum antibacterial action.

In particular, the compounds according to the invention consist of a number of 7-(oc-aminophenylacetamido- or α-hydroxyphenylacetamido)-A-cephem compounds or derivatives thereof with an oxadiazolyl-, thiadiazolyl- or triazolyl-thiomethyl nucleus in the position which carries one of a specified series of substituents.

Thus, according to the invention, cephalosporins with the general formula are obtained:

wherein R is H or OH; 4 4 Y is Nf^, OH or OR wherein R is a lower alkanoyl, lower alkoxycarbonyl or benzoyl group; X is O, S, NH or NMe; Z is hydrogen, lower alkyl, benzhydrylphenyl, indan-5-yl or a group of the formula -CH20C0(lower alkyl), -CH(CH3)0C00(lower alkyl) or

R<1> is -CH2OCH2COOH, -CH2OCH2COO(lower alkyl),

-CH OCH_CONR2R3 wherein R<2>and R<3> are each independently hydrogen or lower alkyl or -CONR<2>R<3>wherein 2 3

R and R are as above indicated;

and the pharmaceutically acceptable salts thereof.

By "lower alkyl", "lower alkoxy" and "lower alkanoyl" is meant an alkyl, alkoxy or alkanoyl group containing up to 4 carbon atoms. It should also be understood that the term "lower alkanoyl" includes formyl.

Lower alkyl or alkoxy groups containing 3 or 4 carbon atoms and lower alkanoyl groups containing 4 carbon atoms can be straight or branched chains. Preferred alkyl groups have 1 or 2 carbon atoms except for the group with the formula -CH2OCO(lower alkyl) in which said lower alkyl group is preferably methyl or t-butyl.

The pharmaceutically acceptable salts of the compounds according to the invention include the non-toxic metal salts, particularly of lithium, sodium, potassium, calcium, aluminium; and ammonium and substituted ammonium salts, e.g. salts of trialkylamines, N-ethyl-piperidine, procaine, dibenzylamine, N-benzyl-P-phenylethylamine, 1-ephenamine, N,N<1->dibenzyl-ethylenediamine, dehydroabiethylamine, N,N<1->bis- dehydro-abiethylamine and other amines previously used to form salts with benzylpenicillin. Compounds according to the invention which are sufficiently basic, e.g. those in which Y is a -NH2 group can also form acid addition salts. Thus, the invention also includes salts formed from acids which form non-toxic addition salts containing pharmaceutically tolerable anions, such as the hydrochloride, hydrobromide, sulphate or bisulphate, phosphate or acid phosphate, acetate, maleate, fumarate, lactate, tartrate, the citrate, gluconate, succinate, trifluoroacetate and p-toluenesulfonate salts. It should also be understood that compounds in which Y is -NH2 can exist in a zwitterionic form.

In one aspect according to the invention, R 1 is preferably -CH 0 CH 2 C 0 NH . In another aspect, R is preferably -CONH2, -CH2OCH2COOH or -CH2OCH2COO(lower alkyl). Y is preferably -NH 2 or -OH.

When R is -OH, it is preferably in the para position in the phenyl ring. Y is preferably -NH,2 where R is para-hydroxy. X is preferably NH or S. Z is preferably hydrogen.

Preferred individual compounds are those wherein R, r\ X, Y

and Z is as follows:

These preferred compounds are preferably in the D form.

It should be understood that in the compounds according to the invention in which X is NH, tautomerism between the structure in which the hydrogen atom is in the 4-position and the structure in which the hydrogen atom is in the 1- or 2-position is possible.

The cephalosporin derivatives according to the present invention are capable of existing in epimeric "D" and "L" forms, and the invention encompasses the separate D and L epimers as well as mixtures thereof. However, the D compounds are preferred.

The compounds according to the invention can be prepared in several ways, which include the following: (1) The compounds can be prepared by acylating a 7-amino-3-heterocyclic thiomethyl-A3-cephem derivative with the formula:

with an acylation reagent of the formula:

wherein Y"*" is a protected amino or protected hydroxyl group or a group of the formula ;-OR 4 , or with its functional equivalent as an acylating reagent, e.g. an acid chloride or bromide, an "activated" ester or a mixed anhydride; followed by removal of the protecting group in Y^. The acylation reagent (III) is preferably in the D form. - The acid chloride or bromide can be obtained by conventional methods. E.g. the acid chloride can be obtained by reacting the acid (III) in a suitable solvent with oxalyl or thionyl chloride or with phosgene. ;The preferred "activated" ester has the formula:; ; and can be prepared by reacting the acid (III) with N-hydroxysuccinimide in the presence of a dehydrating reagent, e.g. dicyclohexylcarbodiimide. ;Suitable mixed anhydrides have the formula:; ; 1 ; in which R 5 is a lower alkyl group, particularly preferably an iso-butyl group. The mixed anhydride is typically produced by reacting a solution of the acid (III) in a suitable solvent, e.g. dry tetrahydrofuran containing about 1 equivalent of a suitable base, e.g. triethylamine, with a lower alkyl chloroformate, e.g. iso-butyl chloroformate. The reaction should be carried out at a low temperature, e.g. -lo° to o°C and is generally finished in a few minutes. ;It should be understood that the term "functional equivalent as an acylating reagent" applied to compound (III) also includes the "internally protected" dione of the formula: ; The dione can be prepared by the reaction of phosgene with a compound of the formula: ; Suitable protection groups in Y are those that are usually used in the area. The preferred protecting group for an amino group is a t-butoxycarbonyl group and, for a hydroxyl group, a formyl or dichloroacetyl group. During the preparation of the acid chlorides or bromides, HC1 or HBr is evolved which will protonate any free -NH2 groups represented by Y. The proton may serve as a suitable "protecting group" and no introduction of a t-butoxycarbonyl group may be necessary when the solution contains the protonated acid chloride or bromide which connects directly to compound (II). When the acylation is carried out by reacting the free acid (III) with the compound (II), it is generally necessary to protect any free carboxyl group in (II) for the reaction. The reaction should be carried out in the presence of a dehydrating reagent, e.g. dicyclohexylcarbodiimide (DCC) or 1-ethyl-3-(3-dimethylamino-prop-1-yl)carbodiimide hydrochloride. ;Protection of any free carboxyl group (eg by trimethylsilyl groups) is generally necessary when DCC is used, but may not be necessary when using 1-ethyl-3-(3-dimethylaminoprop-1-yl)carbodiimide hydrochloride. In a typical reaction, compound (II) and a base, e.g. triethylamine, in a suitable solvent, e.g. dry methylene chloride, followed by the addition of trimethylsilyl chloride when some free carboxyl groups are present in (II). After stirring for approximately 1 hour, a solution of compound (III) and a dehydrating reagent, e.g. dicyclohexylcarbodiimid., in a suitable solvent, e.g. methylene chloride and this resulting solution is stirred for several hours. After filtration, the organic filtrate can be shaken with aqueous acid, e.g. hydrochloric acid, to remove all trimethylsilyl protecting groups and after separation, the organic phase is dried over anhydrous magnesium sulfate. Although the reaction can be carried out at room temperature, it is preferred to carry it out at a low temperature, e.g. -5° to o°C. The product obtained after evaporating the reaction mixture to dryness will of course be a cephalosporin with a protected amino or protected hydroxyl group. Such protecting groups can be removed by conventional methods. E.g. a t-butoxycarbonyl group can be removed by acid hydrolysis using e.g. formic acid or trifluoroacetic acid at about o°C, the desired product of formula (I) being obtained in the case of trifluoroacetic acid in the form of its trifluoroacetate addition salt after evaporation of the reaction mixture in vacuo and trituration of the resulting oil with dry ether. The trifluoroacetate salt can be converted to its zwitterionic form or to a pharmaceutically acceptable addition salt thereof by conventional methods in the field. E.g. when Z is a hydrogen atom, the zwitterionic form of the compound can be obtained by treating a suspension of the trifluoroacetate salt in water with caustic soda solution to adjust the pH to about 7.5 followed by filtration and then adding hydrochloric acid to reduce the pH to about 3.5 to precipitate the zwitterionic product. Formyl and dichloroacetyl protecting groups can be removed by aqueous base, e.g. aqueous sodium bicarbonate solution which, when Z is a hydrogen atom, gives the sodium salt of the cephalosporin. The free acid can be obtained by acidification. In a typical method, the hydroxyl-protected cephalosporin is dispersed in aqueous sodium bicarbonate solution, and the solution is overlayered with a suitable water-immiscible organic solvent, e.g. ethyl acetate. After acidifying the aqueous phase to about pH 2, the organic phase is separated, dried and evaporated in vacuo to leave an oil which can be triturated with dry ether to give the free acid form of the desired α-hydroxy-cephalosporin. In a typical process which comprises the reaction of a mixed anhydride (V) with the compound (II), the compound (II) is necessarily dissolved by means of a base, such as triethylamine, in a suitable solvent, e.g. aqueous tetrahydrofuran and mixed with a solution of the anhydride in e.g. tetrahydrofuran. After mixing for about 1 hour, preferably at a temperature of -lo° to o°C, the dressing agent is removed and the solution is allowed to stand for several hours, followed by dilution with water and extraction with a suitable water-immiscible organic solvent, f .ex. ethyl acetate, to remove impurities in the organic phase. After separation, the aqueous phase can be overlaid with a suitable water-immiscible organic solvent, e.g. ethyl acetate, and then acidified to e.g. pH 2 by addition of hydrochloric acid to effect extraction of the desired product into the organic phase. After separation, the organic phase can be dried over anhydrous magnesium sulfate, evaporated in vacuo and the resulting oil crystallized by trituration with dry ether. The product is obviously the protected cephalosporin and the protecting groups can be removed as described above. The reaction of the activated ester (IV) with compound (II) can be carried out in a similar manner. In a typical reaction involving the acylation of the compound (II) with an acid chloride, the acid chloride is dispersed in a suitable solvent, e.g. dry acetone, and added to a solution of the compound (II) in e.g. aqueous acetone containing sodium bicarbonate. After mixing for about 1 hour at a low temperature, e.g. o°C-, the dressing agent can be removed and stirring continued for several hours. Water and a water-immiscible solvent such as ethyl acetate are then added and the pH in the aqueous phase is adjusted to around 2.0 with e.g. hydrochloric acid. After filtration, the organic phase can be separated and the aqueous phase extracted with fresh ethyl acetate. The organic phases can be combined, evaporated in a vacuum and triturated with dry ether. The product is again a cephalosporin with a protected amino or a protected hydroxyl group, and the protecting groups can be removed as described above. The acid bromide can be reacted similarly. The acylation with the "internally protected" dione (VI) can be carried out similarly to the acid chloride. The product is obviously a cephalosporin in which Y is -OH. The compounds with the formula (II) where Z is hydrogen can be obtained by analogous methods to those previously known, e.g. as follows: or alternatively In some cases the second of these pathways gives the purer starting material. The compounds of formula (VIII) are new compounds and are claimed in British Patent Application No. 40447/75. Their manufacture is also described in this pending sbknade. ;The compounds of formula (II) in which Z is other than hydrogen, i.e. the compounds in which Z completes an ester group ring can also be prepared by methods which are analogous to previously known ones, e.g. by esterifying the corresponding amino-protected compound (II) in which Z is hydrogen or an alkali metal atom (e.g. potassium), preferably as follows: The corresponding bromine compounds can be used instead of the chlorine compounds indicated in (iii) and (iv) above. It may also be necessary to protect all free carboxyl groups in R prior to reactions (iii) and (iv). After the reaction, the protecting group or groups can be removed by conventional methods. As is known, in the reactions of the type described under (iv) some isomerism of the double bond can occur in the cephem nucleus of the 2-position. This can be reversed by forming the S-oxide of the product and then reducing in a known manner. (2) The compounds according to the invention in which Y is OH or NH2 and Z is hydrogen can also be prepared by reacting a cephalosporin derivative with the formula: ; wherein Y 7 is OH, NH 2 or a protected hydroxyl or protected amino group and R 6 is a good leaving group, e.g. a chlorine, bromine, iodine or, particularly preferred, an acetoxy group with a heterocyclic thiol of the formula: ; or with a metal or ammonium salt thereof, followed by the necessary removal of any protecting group from Y<2>. ;Compound (VII) is preferably in the D form.;The metal salt is preferably an alkali metal salt, particularly preferably a sodium or potassium salt, e.g. with the formula: ; In the case where R"*" contains a -COOH group, the thiol can be reacted in the form of its di-metal salt, e.g.

Y 2 is preferably a protected amino group, e.g. a t-butyl-oxycarbonylamino group or a free hydroxyl group. Unprotected amino groups tend to react with the (3-lactam system of the cephalosporin under the reaction conditions. In most cases, however, there is no need to protect an α-hydroxyl group.

The reaction is typically carried out in a phosphate buffer solution at a pH of from 6.5 to 8.0 to ensure the existence of the cation:

and at a temperature of from 5o° to 75°. After several hours, the reaction mixture can be cooled and overlaid with a suitable water-immiscible organic solvent, e.g. ethyl acetate, to extract contaminants into the organic phase. After separation, the aqueous phase can be overlaid with fresh ethyl acetate and then treated with aqueous hydrochloric acid to

reduce the pH to a low value, e.g. pH 2, to effect the extraction of the product into the organic phase. After separation, the organic phase can be washed with brine, dried over anhydrous magnesium sulfate and evaporated in a vacuum. The resulting oil can be crystallized by trituration with dry ether. Any amino or hydroxy protecting group in the cephalosporin product can be removed as described in method (1) above.

The starting materials with the formula (VII) are either known compounds or can be prepared by a method analogous to those previously known. (3) The compounds of the formula (I) in which Z is different from hydrogen can be prepared by esterification of Qe corresponding compounds of the formula (I) in which Z is hydrogen or an alkali metal atom (preferably potassium), any free hydroxyl or amine group being prepared by Y and any free carboxyl group in R<1> if necessary are protected prior to the esterification reaction and deprotected afterwards.

The esterifications can e.g. is carried out using the reagents described in route (1) subsections (iii) and (iv).

(4) Salts of the compounds according to the invention can be prepared if necessary by standardized techniques. E.g. the preparation of a sodium or potassium salt can be carried out by dissolving a compound in which Z is H in a suitable solvent and adding a solution of the suitable alkali metal acetate in the same solvent. After the reaction, the salt is preferably isolated by concentrating the reaction mixture by partial evaporation in a vacuum and adding the concentrate to a large volume of a suitable solvent and thereby precipitating the salt. Acid addition salts of these compounds according to the invention in which Y is an amino group can be prepared by dispersing the cephalosporin in water, acidified to a low pH (e.g. pH 2)

with the suitable acid, e.g. hydrochloric acid, and evaporate the product to dryness, preferably by freeze drying.

The in vitro evaluation of the compounds according to the invention

as antibacterial agents was carried out by determining the minimum inhibitory concentration (M.I.C.) of the test compound in a suitable medium at which growth of the particular microorganism was prevented from occurring. In practice, agar (brain/heart infusion agar): plates, each incorporating the test compound at a particular concentration, were inoculated with a standardized number of cells of the test organism and each plate was then incubated for 24 hours at 37° C The plates were then examined for the presence or absence of bacterial growth and the corresponding M.I.C. number noted. Microorganisms which were used in such experiments and against which the compounds were active included strains of Escher. ichia coli, Klebsiella pneumoniae, Aerobacter aerogenes, Serratia marcescens, Proteus mirabilis, Proteus vulq aris, Staphylococcus aureus and Streptococcus pyogenes.

M. I.C. values in [J-g/ml for the compounds according to the invention

is given in the following table:

The compounds according to the invention can be administered alone, but will generally be administered in admixture with one pharmaceutical carrier selected taking into account the intended route of administration and usual pharmaceutical practice. E.g. they can be administered orally in the form of tablets containing such excipients as starch or lactose, or in capsules either alone or mixed with excipients, or in the form of elixirs

or suspensions containing flavoring or coloring matter. They can be injected parenterally, e.g. intravenously, intramuscularly or subcutaneously. For parenteral administration, they are preferably used in the form of a sterile aqueous solution which may contain other dissolved substances, e.g. sufficient salts or glucose to make the solution isotonic.

It is expected that a suitable total daily dosage (oral or parenteral) for an average adult person. (7o kg) will be 1 range from 125 mg to 1 g of the active compound taken

2 to 4 times a day. The doctor will in each individual case

determine the most appropriate dose which will depend on the age, weight and response of the patient.

Thus, the invention also provides a pharmaceutical composition containing a compound of the formula (I) as stated herein or a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable diluent or carrier. The invention also includes within its scope a method for treating animals, including human beings, to cure them of diseases caused by gram-positive or gram-negative bacteria, which consists in administering to the animal an antibacterially effective amount of a compound with the formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutical composition as indicated above.

The following examples illustrate the invention and the production of the starting materials. The compounds were tested for purity by thin-layer chromatography (t.l.c.) on silica gel and/or by high-pressure liquid chromatography. Infrared spectra (IR) were recorded as solutions in a suitable solvent, e.g. chloroform, as KBr discs or as Nujol slurries.

Nuclear magnetic resonance spectra were taken at 60MHz

(except example 2 - loo MHz) for solutions of the compounds in e.g. perdeutero dimethylsulfoxide (DMSOd^) or deuterium oxide (D2CO using a suitable internal reference standard, e.g. tetramethylsilane. The protons giving rise to the signals are underlined.

EXAMPLE 1

A. 7-( D - tert -butoxycarbonylamino- p -hydroxyphenylacetamido)-3-(3-carboxymethoxymethyl-1,2,4-triazol-5-yl)thiomethylcef-3-em-4-carboxylic acid.

A solution of 3-acetoxymethyl-7-(D-tert-butoxycarbonyl-amino-p-hydroxyphenylacetamido)-cef-3-em-4-carboxylic acid (4.25 g) and 3-carboxymethoxymethyl-1,2,4-triazole -5-thiol (1.53 g)

in pH 7.0 phosphate buffer (100 ml) was brought to pH 7.2 by addition of aqueous 2N sodium hydroxide solution, and the resulting solution was then heated at 70°C for 3 hours. The reaction mixture was cooled to room temperature, extracted once with ethyl acetate and the organic layer discarded. The aqueous layer was overlaid with fresh ethyl acetate and the pH was adjusted to 2.5 by addition of aqueous 2N hydrochloric acid. The organic phase was separated, washed with brine, separated again and finally dried over sodium sulfate. Evaporation of this dried extract and trituration of the resulting residue with dry ether afforded the product, 7-(D-a-tert-butoxycarbonylamino-p-hydroxyphenylacetamido)-3-(3-carboxy-methoxymethyl)-1,2,4-triazole-5 -yl)thiomethylcef-3-.em-4-carboxylic acid as a white solid, yield 1.6 g.

B. 7-(D-g-amino-p-hydroxyphenylacetamido)-3-(3-carboxymethoxy-methyl-1,2,4-triazol-5-yl)thiomethylcef-3-em-4-carboxylic acid;

trifluoroacetate salt.

The product from Example 1 (1.5 g) was stirred in ice-cold trifluoroacetic acid (12 ml) for 10 minutes. This solution was then added to dry ether (2oo ml) and the off-white precipitate thus obtained was filtered off and dried in vacuo. This product was purified by trituration with 50 ml of 5:1 v.v. ethyl acetate/ethanol for 1 hour. The yield of the dried product, the trifluoroacetate salt of 7-(D-a-amino-jD-hydroxyphenylacetamido)-3-(3-carboxy-methoxymethyl-1,2,4-triazol-5-yl)thiomethylcef-3-em-4-carboxylic acid , was 1.1 g.

21

I.R. (KBr) V max. = 1.765 cm<-1>(p-lactam carbonyl)

N.M.R. (DMSOd6) <f =3.42 (broad singlet; 2-CH_2)

4,o Ringlett) (_CH OCH .CO H)

4.4 (singlet) —2 ~22

4.82 (multiplet; 6-H and α-H)

5.55 (unresolved multiplet; 7-H)

6.64 (doublet, J = 7 c/s; 2 aromatic

protons)

7.15 (doublet, J = 7 c/s; 2 aromatic

protons)

9.3 (doublet, J = 8 c/s; CO-NH) p.p.m.

EXAMPLES 2 AND 3

The following trifluoroacetate salts were prepared by procedures similar to those described in Example 1, starting from the same cephalosporin as Example 1 and the appropriate thiol:

EXAMPLE 4

7-(D-g-hydroxyphenylacetamido)-3-(3-carbamoyl-1,2,4-triazol-5-yl)thiomethylcef-3-em-4-carboxylic acid.

Aqueous 2N sodium hydroxide was added to a solution of 3-acetoxy-methyl-7-(D-α-hydroxyphenylacetamido)cef-3-em-4-carboxylic acid (27.6 g) and the ammonium salt of 3-carbamoyl-1,2,4- triazole-5-thiol (10 g) in pH 7.0 phosphate buffer to raise the pH to 7.0, and this solution was then heated at 70°C for 3 hours. The reaction mixture was then cooled to room temperature and acidified to pH 2.0 by addition of aqueous 2N hydrochloric acid. Extraction of the reaction mixture with ethyl acetate and evaporation gave a yellow solid (3 g), which by high pressure liquid chromatography (hplc) was found to be about 50% pure and this was discarded. The remaining aqueous portion contained a gummy residue which was washed thoroughly with water and finally triturated with a solution of isopropanol/ethyl acetate. The residue from this demolition, yield 15 g, was found to be around 70% pure by high pressure liquid chromatography analysis. Evaporation of the isopropanol/ethyl acetate filtrate gave a solid (5.7 g), which was found to be about 80% pure. This latter material was chromatographed over silica gel, and a 95% (hplc) sample was obtained by elution with a 6% solution of methanol in chloroform, yielding 1.56 g of 7-(D-α-hydroxyphenylacetamido)-3-(3-carbamoyl- 1,2,4-triazol-5-yl)thiomethylcef-3-em-4-carboxylic acid.

I.R. (KBr) V max. = 1.765 cm"<1>((3-lactam carbonyl)

N.M.R. (DMSOd6) = 3.6 (broad singlet; 2^CH2)

4,o (doublet; J = 13c/s) \

4.35 (doublet; J = 13c/s)J -2

5,o (multiplet; 6-H and a-H)

5.55 (doublet; J = 5c/s)|

5.7o (doublet; J = 5c/s)J1-

7.25 (multiplet; aromatic protons) 8.55 (doublet, J = 8c/s; CO-NH) p.p.m.

EXAMPLE 5-12

The following compounds were prepared by similar procedures to those described in Example 4, starting from the same cephalosporin as Example 4 and the appropriate thiol or salt thereof:

EXAMPLE 13

A. 7-aminc— 3-( 3- carbamoyl-1, 2, 4- triazol-5- yl) thiomethylcef- 3-em- 4- carboxylic acid

7-Formamidocephalosporanic acid (100 g) was dissolved in pH 7.0 phosphate buffer solution (1600 ml) and the pH was adjusted to 7.0 with 2N sodium hydroxide solution. 3-Carbamoyl-5-mercapto-1,2,4-triazole (48 g) was then added and the pH was again adjusted to 7.0 with 2N sodium hydroxide solution. The resulting mixture was heated at 70°C for 5 hours and then cooled to room temperature and its pH adjusted to 0.5 with concentrated hydrochloric acid. The mixture was diluted with methanol (1500 ml) and stirred at room temperature for 3 hours before it was cooled to 50°C. The pH was then adjusted to 3.9 with ammonium hydroxide solution and after stirring for 2 hours at 5°C the precipitation of 7-amino-3-(3-carbamoyl-1,2,4-triazol-5-yl)thiomethylcef-3- em-4-carboxylic acid (47.9 g) filtered off, washed with water, then with acetone and ether and finally dried in vacuo at room temperature.

B. 7-(D-α-Hydroxyphenylacetamido)-3-(3-carbamoyl-1,2,4-triazol-5-y1)thiomethylcef-3-em-4-carboxylic acid

D-O-(dichloroacetyl)mandelic acid chloride (72 g) in acetone (120 ml)

was added over 45 minutes to a solution of 7-amino-3-(3t-carbamoyl-1,2,4-triazol-5-yl)thiomethylcef-3-em-4-carboxylic acid (45 g) prepared in Part A and sodium bicarbonate (27 g) in water (72o ml) and acetone (6oo ml) at o°C. The pH was maintained at 7.5 during the addition by adjusting with 2N sodium hydroxide solution. The mixture was allowed to warm to room temperature and then stirred for 1 hour. The acetone was removed in vacuo, the pH raised to 9.5 by the addition of sodium carbonate solution and after 30 minutes the pH was again adjusted to 2 with concentrated hydrochloric acid. The reaction mixture was then extracted with a mixture of tetrahydrofuran (310 ml) and ethyl acetate (310 ml).

The organic phase was washed with water and brine and evaporated to dryness. The resulting oil was triturated with ether to give 7-(D-α-hydroxyphenylacetamido)-3-(3-carbamoyl-1,2,4-triazol-5-yl)thiomethylcef-3-em-4-carboxylic acid as a near- white solid, yield 23.3 g, determined by hplc to be identical to the product of Example 4.

EXAMPLE 14

By a similar method as in Example 13, part B, 7-(D-α-hydroxyphenylacetamido)-3-(2-carbamoyl-1,3,4-oxadiazol-5-yl)thiomethylcef-3-em-4-carboxylic acid prepared from 7-amino-3-(2-carbamoyl-1,3,4-oxadiazol-5-yl)thiomethylcef-3-em-4-carboxylic acid and D-O-formylmandelic acid chloride.

I.R. (Nujol slurry) i) max. = 1.7 7o ({3-lactam carbonyl) cm<-1>N.M.R. (DMSOdg) = 7.25, singlet (aromatics)

5.58, multiplet (7-H)

5,oo, multiplet (6-H and a-H)

4.3o, broad singlet (3-CH2-S)

3.55, multiplet (2-CH2) p.p.m.

EXAMPLE 15

A. 7-amino-3-(2-carbamoyl-1,3,4-oxadiazol-5-yl)thiomethylcef-3-em-4-carboxylic acid

Solutions of 7-aminocephalosporanic acid (5.44 g) and 2-carbamoyl-1,3,4-oxadiazole-5-thiol (3.19 g) in dilute aqueous sodium hydroxide solution at pH approx. 7.0 was mixed (total volume about 75 ml) and the resulting mixture stirred and heated in a water bath at 70°C for ■! hour. During the reaction, the pH of the solution was kept at 6.5 - 7.o by the addition of aqueous 2N sodium hydroxide solution. After this time, the reaction mixture was rapidly cooled to room temperature, 2N hydrochloric acid was added to adjust the pH to 3.5, and the resulting brown precipitate was collected by filtration. The product, 7-amino-3-(2-carbamoyl-1,3,4-oxadiazol-5-yl)thiomethylcef-3-em-4-carboxylic acid, was washed thoroughly with acetone to give a brown powder (3.1 g).

B. 7-(D-α-tert-butoxycarbonylamino)phenylacetamido-3-(2-carbamoyl-1,3,4-oxadiazol-5-yl)thiomethylcef-3-em-4-carboxylic acid D-N-tert-butoxycarbonylphenylglycine (1,9 g) was dissolved in dry tetrahydrofuran (THF) (25 ml) and the solution cooled to 5°C. Triethylamine (0.76 g) was added with stirring, the reaction cooled to -10°C and treated for 2 minutes with isobutyl chloroformate (1.0 g). The resulting solution of the mixed anhydride was stirred at -10° for 15 minutes.

The cephalosporin product from Part A. (1.8 g) was added to an ice-cold solution of THF (13 ml) and water (13 ml) containing triethylamine (0.5 g) and stirred to effect dissolution. The resulting solution was added over 7 minutes with stirring to the solution of said mixed anhydride at -5°C. After a further 30 minutes at this temperature, the reaction mixture was allowed to stand for 2 hours during which time the cooling was removed. Water (25 mL) was added and the reaction extracted once with ethyl acetate. The ethyl acetate extract was dried over anhydrous magnesium sulfate, evaporated in vacuo and the product triturated with dry ether to give an off-white solid (CROP I-53o rag). The aqueous phase was overlaid with ethyl acetate, the pH adjusted to 2.0 by the addition of aqueous 2N hydrochloric acid and the organic layer separated and dried over magnesium sulfate. The solvent was evaporated in vacuo and the product triturated with dry ether to give an off-white solid (CROP 2-800 mg). By thin layer chromatographic analysis, CROP I and 2 were found to be identical as 7-(D-α-tert-butoxycarbonylamino)phenylacetamido-3-(2-carbamoyl-1,3,4-oxadiazol-5-yl)thiomethylcef-3-em -4-carboxylic acid and were merged.

C. 7-(D-g-aminophenylacetamido)-3-(2-carbamoyl-1,3,4-oxadiazol-5-yl)thiomethylcef-3-em-4-carboxylic acid trifluoroacetate salt

The product from part B. (500 mg) was added to ice-cold trifluoroacetic acid (5 ml) with stirring. After 6 minutes, the brown solution was evaporated in vacuo and the oily residue triturated with dry ether. The product, 7-(D-α-aminophenylacetamido)-3-(2-carbamoyl-1,3,4-oxadiazol-5-yl)thiomethylcef-3-em-4-carboxylic acid trifluoroacetate salt, was filtered, washed thoroughly with dry ether and dried in vacuum. The yield of off-white solid was 5oo mg.

D. 7-(D-α-aminophenylacetamido)-3-(2-carbamoyl-1,3,4-oxadiazol-5-yl)thiomethylcef-. 3 - em- 4- carboxylic acid

To obtain the zwitterionic form of the desired product, the trifluoroacetate salt prepared in C. above (400 mg) was suspended in water (5 ml) and the pH of the mixture was adjusted to 7.5 by the addition of 2N sodium hydroxide solution.

The solution was filtered to remove insoluble material and

pH in the filtrate adjusted to 3.5 by adding 2N hydrochloric acid. After 2 days at 5°C, the product, 7-(D-α-aminophenylacetamido)-3-(2-carbamoyl-1,3,4-oxadiazol-5-yl)thiomethylcef-3-em-4-carboxylic acid in zwitterionic form, collected by filtration, washed with little water and dried in vacuo. The yield of off-white solid was llo mg. The product was characterized using thin-layer chromatography and infrared and nuclear magnetic resonance spectroscopy.

I.R. (Nujol) V max. = 1.76o cm ((3-lactam carbonyl) ,

1.69o cm<-1>(4-carboxyl).

EXAMPLE 16 - 28

The following cephalosporanic acid derivatives were prepared by similar procedures as described in Example 15 starting from 7-aminocephalosporanic acid and the appropriate heterocyclic thiol. The products were recovered in the form or forms as indicated (in most cases the trifluoroacetate (TFA) salt was not converted to the zwitterion as in Example 15), and were characterized by thin layer or high pressure liquid chromatography and by infrared and nuclear magnetic resonance spectroscopy.

If desired, the trifluoroacetate salts can be converted to the zwitterionic form of the cephalosporin by the method of Part D. of Example 15.

Characterizing data for the compounds in Examples 16-28 are listed below:

EXAMPLE 16

I.R. (slurry) ]) max. = 1.76o ((3-lactam carbonyl)

1.67o (amide carbonyl) cm EXE MPEL 17

I.R. D max. = 1.775 ((3-lactam carbonyl)

1.67 5 (amide carbonyl) cm<->"'"

N.M.R. (DMSOd6) é =9.6, doublet (J = 8c/s)(amide NH)

9,12, doublet (J = 5c/s)(CONHCH3)

7.5, singlet (aromatics)

5.76, unresolved multiplet (7-H)

5.05, multiplet (6-H and a-H)

4.58, doublet (J = 13c/s)| A_rH

4.25, doublet (J = 13c/s)jK-2'

3.57, multiplet (2-CH )

2.82, doublet (J = 5c/s)(CONHCH3) p.p.m.

EXE MPEL 18

I.R. (KBr) ) J max. = 1.775 (p-lactam carbonyl)

1.66o (amide carbonyl) cm<1>

N.M.R. (DMSOd6) é =7.5, singlet (aromatic substances)

5.8, wide singlet (7-H)

5,1, multiplet (6-H and a-H)

4.35, broad singlet (3-CH-S) 3.6, multiplet ('2-CH2)

3,3, singlet (N-CH3)

3.o5, singlet (N-CH3) p.p.m.

EXAMPLE 19

I.R. (KBr) )) max. = 1.77o (p-lactam carbonyl)

I.660(amide carbonyl) cm<->"'"

N.M.R. (DMSOd6) ( f = 7.32, doublet (J = 7c/s)(amide -NH)

7.25, doublet (J = 8c/s) (aromatics)

6.72, doublet (J = 8c/s) (aromatics). 5.7o, unresolved multiplet (7-H) 4.95, unresolved multiplet (6-H and -H) 4.3o, broad singlet (3-CH2-S-)

3.58, wide singlet (2-CH2)

3.36, singlet (N-CH3)

3,oo, singlet (N-CH^) p.p.m.

EXAMPLE 2o

I.R. (KBr) V max. = 1.78o ([3-lactam carbonyl)

1.67o (amide carbonyl) cm 1

N.M.R. (DMSOd ) J 7.4, singlet (aromatics)

5.7, wide singlet (7-H)

5.0, multiplet (6-H and a-H)

4.3, broad singlet (3-CH2-S)

3.4, complex multiplet (2-CH2and CONHCH2CH3) 1.1, center of triplet (J = 7c/s)

(CONHCH2CH3) p.p.m.

EXAMPLE 21

I.R. (KBr) V max. = 1.775 ((3-lactam carbonyl)

,1.67o (amide carbonyl) cm""<1>"

EXAMPLE 22

I.R. (KBr) 1/ max. = 1.78o (p-lactam carbonyl)

1.76o (ester carbonyl)

1.675 (amide carbonyl) cm"

N.M.R. (DMSOd6) cf =3.3, doublet (J = 8c/s) (aromatics)

4.75, doublet (J = 8c/s) (aromatics)

5.7o, wide multiplet (7-H)

3.6o, broad singlet (2-CH2)

1.2o, centrium of triplet (J = 7c/s) p.p.m.

EXAMPLE 23

I.R. (KBr) y max. = 1.775 (p-lactam carbonyl)

1.665 (amide carbonyl) cm<-1>

N.M.R. (DMSOd6) é = 9.18, doublet (J = 5c/s)(amide NH)

7.47, singlet (aroma fabrics)

5.77, broad multiplet (7-H)

5,oo, multiplet (6-H and a-H).

4.3o, multiplet (3-CH2-S)

3.55, multiplet (2-CH2)

2.76, center of doublet (J = 5c/s) (CONHCH.^)

p.p.m.

EXAMPLE 24

I.R. (Nujol slurry) max. = 1.775 ((3-lactam carbonyl)

1.68o (amide carbonyl) cm<-1>N.M.R. (DMSOd6) ( f 7.4, singlet (aromatic substances)

5.65, unresolved multiplet (7-H) 4.95, unresolved multiplet (6-H and a-H) 4.15, broad singlet (3-CH -S)

3.75, singlet (N-CHg)

3.6, multiplet (2-CH2) p.p.m.

EXAMPLE 25

I.R. (KBr) V'max. =1.775((3-lactam carbonyl)

1.67 5 (amide carbonyl) cm<-1>

N.M.R. (DMSOd,) cf = 7.25, doublet (J = 8c/s) (aromatics)

6.75, doublet (J = 8c/s) (aromatics)

5.65, multiplet (7-H)

4.9o, multiplet (6-H and a-H)

/ 4,lo, wide singlet (3-CH2-S)

3.7o, singlet (N-CHg)

3.55, multiplet (2-CH2) p.p.m.

EXAMPLE 26

I.R. (KBr) 1) max. = 1.78o (p-lactam carbonyl).

1.765 (ester carbonyl)

0I.680(amide carbonyl) cm<-1>

N.M.R. (DMSOdb,)</= 7.45, doublet (J = 8c/s)(aromatics)

6.76, doublet (J = 8c/s) (aromatics)

5.75, wide singlet (7-H)

4.96, multiplet (6-H and a-H)

4.7o, singlet (a CH2)

4,lo, multiplet (two CH2)

3.58, broad singlet '(N-CH3and 2-CH2) l,oo, center of triplet, J = 6c/s(C02CH2CH3)

p.p.m.

EXAMPLE 27

I.R. (KBr) V max. = 1.775 ((3-lactam carbonyl)

1.675 (amide carbonyl) cm<-1>

N.M.R. (DMSOd6) 6 = 7.28, doublet (J = 8c/s) (aromatics)

6.74, doublet (J = 8c/s) (aromatics)

5.68, wide singlet (7-H)

4.95, multiplet (6-H and a-H)

4.68, singlet (a CH2)

4,o8, singlet (a CH2)

3.55, singlet (N-CH3)

3.35, multiplet (2-CH2) p.p.m.

EXAMPLE 28

I.R. (KBr) )) max. = 1.765 (p-lactam carbonyl)

1.67o (amide carbonyl) cm

N.M.R. (DMSOd b) é =9.55 (doublet, J = 8c/s)(amide N-H)

7.35 (center of doublet, J = 8c/s)(aromatics fer )

6.83 (center of doublet, J =. 8c/s)(aromatics)

5.8o unresolved multiplet (7-H)

4.96 multiplet (6-H and a-H)

4.18 singlet (methylene)

3.62 broad singlet (2-CH2) p.p.m.

EXAMPLE 29

A. (D)-5-phenyl-1,3-dioxolane-2,4-dione

A solution of phosgene (56.9 g) in toluene (500 ml) was added over 1 hour to (D)-α-hydroxyphenylacetic acid (45.6 g) in tetrahydrofuran (450 ml) at room temperature and the mixture was then warmed at 45°C for 6 hours. The organic solvent was removed under vacuum and the residue crystallized from carbon tetrachloride to give (D)-5-phenyl-1,3-dioxolane-2,4-dione as

a white solid (49.4 g), m.p. 76 - 77<9>.

Analysis:

Found: C, 60.37; H/3.68%

Calcd for CgH 6 O 4 : C, 60.67; H, 3.39%

B• 7-( D- a- hydroxyphenylacetamido)- 3-( 3- carbamoyl- 1, 2, 4- triazol-5- yl) thiomethyl- cef- 3- em- 4- carboxylic acid

(D)-5-phenyl-1,3-dioxolane-2,4-dione (50.4 g) was added over 15 minutes to a solution of 7-amino-3-[(3-carbamoyl-1,2 ,4-triazol-5-yl)thiomethyl-7-cef-3-em-4-carboxylic acid (84.0 g) in aqueous buffer (1075 ml) at pH 6.8. The solution was stirred for 45 minutes at room temperature, a mixture of ethyl acetate and tetrahydrofuran (1:1; 840 ml) was added and the pH of the aqueous phase reduced to 2.0 with concentrated hydrochloric acid. The organic layer was separated, washed with water (800 ml), dried and evaporated under reduced pressure. The residue was triturated with ether to give the crude product as a pale yellow solid (80.2 g). Chromatography over silica gel using up to 10% methanol in chloroform as eluent gave the pure product, 7-(D-a-hydroxyphenyl-acetamido)-3-(3-carbamoyl-1,2,4-triazol-5-yl) thiomethyl which was

chromatographically and spectroscopically identical to the material prepared in example 4.

EXAMPLE 3o

Diphenylmethyl- 7-( D- a- hydroxyphenylacetamido)- 3-( 3- carbamoyl-1, 2, 4- triazol- 5- yl) thiomethylcef- 3- em- 4- carboxylate

The product of Example 4 (100 mg) was added to an ice-cooled stirred solution of diphenyldiazomethane (Ph 2 CHN 2 ) (45.1 mg) in ethyl acetate (15 ml) and the reaction mixture was kept under cooling for about 20 hours. The reaction mixture was then washed with aqueous sodium bicarbonate solution and then water, the organic layer separated and dried over magnesium sulfate. The organic layer was evaporated in vacuo and the residue triturated with dry ether to give the diphenylmethyl ester of 7-(D-a-hydroxyphenylacetamido)-3-(3-carbamoyl-1,2,4-triazol-5-yl)thiomethylcef-3-em -4-carboxylic acid as a white solid (99 mg), m.p. 133 - 135°C. A sample recrystallized from isopropanol gave the following analysis:

Found: C, 58.56; H = 4.32%

Calculated for C32H28N6°6S2: C'58'52'H = 4/3o%

I.R. V max. = 1.778 (p-lactam carbonyl) cm<-1>.

EXAMPLE 31

3-(3-carbamoyl-1,2,4-triazol-5-yl)thiomethyl-7-(D-a-formyloxy-phenylacetamido)cef-3-em-4-carboxylic acid

N,O-bis(trimethylsilyl)acetamide (800 mg) was added to a suspension of the product from Example 13, Part A (400 mg) in dry THF (9 mL) and the mixture was heated at 50°C for 20 minutes after which a clear solution was obtained. This solution was cooled to room temperature and a solution of D-O-formylmandelic acid chloride (0.23 g) (prepared as described in German Laid-Open No. 2.506.622 (Lilly)) in dry THF (1 ml) was added. After stirring for 2 hours, ethyl acetate (10 ml) and water (5 ml) were added and this mixture was stirred for a further 10 minutes. The organic phase was separated, washed twice with water, separated, dried (MgSO 4 ) and evaporated in vacuo to give 0.5 g of crude material. Trituration of this crude product with dry acetone afforded the desired product, 3-(3-carbamoyl-1,2,4-triazol-5-yl)thiomethyl-7-(D-α-formyloxyphenylacetamido)cef-3-em-4-carboxylic acid as an off-white solid (14o mg).

I.R. (KBr) D max. = 1.77o cm ((3-lactam carbonyl)

UV (EtOH) E<1>= 169 (@27o nm)

N.M.R. (DMSOd5) / = 3.5 (broad singlet, 2-CH2).

3.95 (center of doublet, J = 13c/s(3_CH _s_ 4.3o (center of doublet, J = 13c/sj —2

4.95 (doublet, J = 5c/s, H-6)

5.6o (very wide multiplet, H-7)

6,o8 (singlet, a-H)

7.35 (wide singlet, aroma substances)

7.76 (broad singlet, -CONH2)

8.26 (sharp singlet, -O.CHO)

9.2o (doublet, J = 7c/s, -CONH) p.p.m.

EXAMPLE 32

Pivaloyloxymethyl-3-(3-carbamoyl-1,2,4- tria2ol-5-yl)thiomethyl-7-(D-a-hydroxyphenylacetamido)-cef-3-e m-4- carboxylate

Sodium bromide (28 mg) was added to a stirred solution of the chloromethyl ester of pivalic acid (40 mg) in dry dimethylformamide (1.5 ml). After 15 minutes, the product from example 4

(123 mg) was added and after 10 minutes a solution of dicyclohexylamine (50 mg) in dry DMF (0.5 ml) was added dropwise over approximately 15 minutes. The resulting reaction mixture was stirred for 17 hours at room temperature after which water and ethyl acetate were added. The organic layer was separated and washed successively with aqueous sodium bicarbonate, water, 1-N hydrochloric acid, water and finally dried over anhydrous magnesium sulfate. The solvent was removed in vacuo and the residue triturated with dry ether to give the product pivaloyloxymethyl-3-(3-carbamoyl-1,2,4-triazol-5-yl)thiomethyl-7-(D-a-hydroxyphenyl-acetamido)-cef- 3-em-4-carboxylate as an off-white solid (34 mg).

I.R. (KBr) V max. = 1.755 (broad) ((3-lactam and ester carbonyl) cm-1 N.M.R. (DMSOdg) S = 1.18 (sharp singlet, t-butyl)

3.95 (center of a doublet, J = 13c/s)^H ^ 4.4o (center of a doublet, J = 13c/s)~2 5.lo (multiplet, 6-H and a-H)

in

5.8o (multiplet, 7-H and the methylene of ester group)

7.3o (broad singlet, aromatic proton) 7.86 (multiplet, -CONH2)

8.68 (doublet, J = 7c/s, -CONH-) p.p.m.

Claims (14)

1. Analogous method for the production of cephalosporins with the formula: wherein R is H or OH; 4 4 Y is NH 2 , OH or OR in which R is lower alkanoyl, lower alkoxycarbonyl or benzoyl; X is O, S, NH or NMe; Z is hydrogen, lower alkyl, benzhydryl, phenyl or indan-5-yl or a group of the formula -CH 0C0(lower alkyl), -CH(CH)OCOO(lower alkyl) or and R <1> is -CH2 OCH2 COOH, -CH2 OCH2 COO(lower alkyl), 2 3 2 3 -CH OCH-CONR R wherein R and R are independently hydrogen 2 3 2 3 or lower alkyl or -CONR R wherein R and R are as above; and pharmaceutically acceptable salts thereof; characterized by (A) reacting a 7-amino-3-heterocyclic thiomethyl-A 3-cephem derivative with the formula: wherein X, R<1> and Z are as indicated above with an acylating agent of the formula: in which R is as defined above and Y<1> is a protected amino- or protected hydroxyl-4 4 group or a group of the formula -OR in which R i is as defined above, or with its functional equivalent as an acylating agent, followed by the removal of the protecting group from Y<1> , any free carboxyl group in (II) being protected as necessary for the reaction and deprotected after the reaction; or (B) to prepare compounds of formula (I) wherein Y is OH or NH^ and Z is H, by reacting a cephalosporin derivative of the formula: wherein R is as above, Y 2 is OH, NH^ or a protected hydroxyl or protected amino group, and r, 6 R is a good leaving group with a heterocyclic thiol of the formula: in which -R1 and X are as above defined, or with a metal or ammonium salt thereof, followed by the necessary removal of any protecting group from Y 2 ; these procedures (A) and (B) are followed by, where applicable, (i) converting a compound of formula (I) into a pharmaceutically acceptable acid addition or metal salt by reaction with a suitable acid or metal compound; or (ii) conversion of a compound of formula (I) in which Z is hydrogen or an alkali metal salt thereof to a compound of formula (I) in which Z is as defined above different from hydrogen, by esterification, any free hydroxyl or amino group in Y and any free carboxyl group in R1 is, if necessary, protected prior to the reaction and deprotected after the reaction. 2. Analogy method according to claim 1, characterized in that Zi the compound with the formula (II) is hydrogen and Y<1> in the compound with the formula (III) is a protected amino or protected hydroxyl group. 3. Analogy method according to claim 1, characterized in that said functional equivalent as an acylating agent is an acid chloride or bromide, activated ester or mixed anhydride of the compound with the formula (III). 4. Analogy method according to claim 3, characterized in that this activated ester has the formula: wherein R and Y <1> are as defined in claim 1. 5. Analogy method according to claim 3, characterized by this mixed anhydride having the formula: wherein R and Y are as defined in claim 1 and R 5 is a lower alkyl group, preferably an isobutyl group. 6. Analogy method according to claim 1 or 2, characterized in that this functional equivalent as an acylating agent has the formula: wherein R is as defined in claim 1. 7. Analogy method according to claim 1, characterized in that R in the compound (VII) is a chlorine, bromine, iodine or acetoxy group. 8. Analogy method according to claim 1 or 7, characterized in that this metal salt of the compound (VIII) is an alkali metal salt. 9. Analogy method according to claims 1 - 8, characterized in that R1 is -CH2 OCH2 CONH2 . 10 . Analogous method according to claims 1 - 8, characterized in that R1 is -CH2 OCH2 COOH, -CH2 OCH2 CO0 (lower alkyl) or -CONH2 .. 11. Analogy method according to claim 1, characterized in that the compound (III) or (VII) is in the D form. 12. Analogous method according to claim 1, characterized by reacting a compound of the formula (II) in which X is NH, Z is hydrogen and R <1> is -CONH2 with a compound of the formula (III) or functional equivalent thereof in which R is hydrogen and Y <1> is a protected hydroxyl group, this compound (III) being in the D form. 13. Analogy method according to claim 12, characterized in that the functional equivalent of the compound (III) has the formula: 14. Analogous method according to claim 1, characterized by reacting a compound of the formula (VII) in which R is H, Y 2 is a hydroxyl or protected hydroxyl group and R 2 is an acetoxy group with a compound of the formula (VIII) in which X is NH and R<1> is -CON^, or with an alkali metal or ammonium salt thereof, said compound (VII) being in the D-form.