IE43541B1 - Cephem compounds - Google Patents

Abstract

Cephalosporins of formula (I) and their salts are new (where R is H or OH; Y is NH2, OH or OR4, where R4 is lower alkanoyl, lower alkoxycarbonyl or benzoyl; X is O, S. NH or NMe; Z is H, lower alkyl, benzhydrylphenyl, indan-5-yl, R'OCOCH2 (R' = lower alkyl), R'OOCOCH(Me) or phthalidyl; R1 is CH2oCH2COOH, CH2OCH2COOR', CH2OCH2CONR2R3 or CONR2R3; R2 and r3 are H or lower alkyl). Cpds. (I) are antibacterials active against Gram-positive and -negative bacteria.

Description

This invention relates to antibacterial agents, and is particularly concerned with a class of cephalosporin derivatives with broad spectrum antibacterial activity.

Xn particular, the compounds of the invention constitute a serie? of 7-(a-aminophenylacetamido- or α-hydroxyphenylacetamido)-Δ -cephem compounds, or derivatives thereof, having in the 3-position an oxadiazolyl-, thiadiazolyl- or triazolyl-thiomethyl nucleus bearing one of a specified series of substituents.

Thus, according to the invention, there are provided cephalosporins of the general formula:R CH -CONH cooz --(1) 3 5 4 χ wherein R is H or OH; 4 Y is NH2, OH or OR wherein R is a lower alkanoyl, lower alkoxycarbonyl or benzoyl group; X is 0, S, NH or NMe; Z is hydrogen, lower alkyl, benzhydrylphenyl, indan-5-yl or a group of the formula -CHjOCOlower alkyl), -CH(CH2)OCOO(lower alkyl) and is -CH2OCH2COOH, -CH2OCH2COO(lower alkyl), CH2OCH2CONR2R3 wherein R2 and R3 are each independently hydrogen or lower alkyl, or 7 3 a 3 -CONR R wherein R and R are as defined above; 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, may be straight or branched chain.

Preferred alkyl groups have 1 or 2 carbon atoms, except in the group of the formula -CHjOCO(lower alkyl) wherein said lower alkyl group is preferably methyl or t-butyl.

The pharmaceutically acceptable salts of the compounds of the invention include the non-toxic metallic salts, particularly of lithium, sodium, potassium, calcium, aluminium; and ammonium and substituted amnonium salts, e.g. salts of trialkylamines, N-ethyl-piperirline, procaine, dibenzylamine, N-benzyl-B-phenylethylamine, l-ephenomine, Ν,Ν'-dibenzyl-ethylenediamine, dehydrobi3541 - 4 ethylamine, Ν,Ν'-bis-dehydroabiethylamine and other amines previously used to form salts with benzylpenicillin. Compounds of the invention which are sufficiently basic, e.g. those in which Y is an -NH2 group, may also form acid addition salts. Thus, the invention also includes salts formed from acids which form non-toxic acid addition salts containing pharmaceutically acceptable anions, such as the hydrochloride, hydrobromide, sulphate or bisulphate, phosphate or acid phosphate, acetate, maleate, fumarate, lactate, tartrate, citrate, gluconate, succinate, trifluoroacetate, and jo-toluenesulphonate salts. It should also be appreciated that compounds in which Y is -NH2 may exist in a zwitterionic form.

In one aspect of the invention R^ is preferably -CH2OCH2CONH2. In another aspect R1 is preferably -CONH2, -CH2OCH2COOH, or -CH2OCH2COO (lower alkyl). Y is preferably -NH2 or -OH.

When R is -OH, it is preferably in the paraposition of the phenyl ring. Y is preferably -NH2 when R is para-hydroxy. X is preferably NH or S. Z is preferably hydrogen.

Preferred individual compounds are those in which R, R^, X, Y and Z are as follows:- R Y R1 X Z — —— H OH -CONH2 NH H p-OH nh2 -CH2OCH2CO2H NH H p-OH nh2 -CH2OCH2CONH2 NH H p-OH nh2 -conh2 NH H H OH -CH2OCH2COOH NH H H OH -CH2OCH2COOEt S H H OH -CH2OCH2COOH S H H OH -CH2OCH2COOH 0 H These preferred compounds are preferably in the D- form. - 5 It should be appreciated that in the compounds of the invention in which X is NH, tautomerism between the structure in which the hydrogen atom resides in the 4position and the structures in which the hydrogen atom resides in the 1- or 2- position is possible.

The cephalosporin derivatives of the present invention are capable of existing in epimeric D and L forms, and the invention includes the separated D- and Lepimers as well as mixtures thereof. The D- compounds are, however, preferred.

The compounds of the invention may be prepared in a number of ways, including the following:(1) the compounds may be prepared by acylating a 73 amino-3-heterocyclicthiomethyl-A -cephem derivative 15 of the formula:- ---(II) with an acylating agent of the formula:CH COOH ---(Ill) - 6 wherein Y^ is a protected amino or protected hydroxyl group or a group of the formula -C®\ or with its functional equivalent as an acylating agent, e.g. an acid chloride or bromide, an activated ester or a mixed anhydride; followed by the removal of the protecting group in Y1. Acylating agent (III) is preferably in the D- form.

The acid chloride or bromide may be obtained by conventional methods.. For example, the acid chloride may 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:0 and may be prepared by reacting the acid (III) with Nhydroxy-succinimide in the presence of a dehydrating agent, e.g. dicyclohexyl-carbodiimide.

Suitable mixed anhydrides have the formula :CO-O-COOR ---(V) CH wherein R^ is a lower alkyl group, most preferably an isobutyl group. The mixed anhydride is typically prepared by the reaction of a solution of the acid (III) in a suitable solvent, e.g. dry tetrahydrofuran, containing about 1 equivalent of a suitable base, e.g. triethyiamine, with a lower alkyl chloroformate, e.g. iso-butyl chloroformate. The reaction should be carried out at low temperature, e.g. -10° to 0°C, and is generally completed in a few minutes.

It should be understood that the term functional equivalent as an acylating agent when applied to compound (III) also includes the internally protected dione of the formula:- The dione may be prepared by the reaction of phosgene with a compound of the formula:J-CH.COOH.

OH Suitable protecting groups in Y^ are those commonly employed in the art. The preferred protecting group for an amino group is a t-butoxycarbonyl group, and, for a hydroxyl group, a formyl or dichloroacetyl group. )3541 - 8 In the preparation of the acid chlorides or bromides, HCl or HBr is evolved which will protonate any free -NH2 groups represented by Y. The proton may act as a suitable protecting group, and no introduction of a t-butoxycarbonyl group may be necessary, the solution containing the protonated acid chloride or bromide being coupled directly with the 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 groups in (II) prior to reaction. The reaction should be carried out in the presence of a dehydrating agent, e.g. dicyclohexylcarbodiimide (DCC) or l-ethyl-3-(3-dimethylaminoprop1-yl)carbodiimide hydrochloride. Protection of any free carboxyl groups (e.g. by trimethylsilyl groups) is· generally essential when using DCC, but may not be necessary when using l-ethyl-3-(3-dimethylaminoprop-lyl) carbodiimide hydrochloride. In a typical reaction, compound (II) and a base, e.g. triethylamine, are dispersed in a suitable solvent, e.g. dry methylene chloride, followed by the addition of trimethylsilyl chloride when any free carboxyl groups are present in (II). After stirring for about an hour, a solution of compound (III) and a dehydrating agent, e.g. dicyclohexylcarbodiimide, in a suitable solvent, e.g. methylene chloride, is added and the resulting solution stirred for several hours. After filtration, the organic filtrate may be shaken with aqueous acid, e.g. hydrochloric acid, to remove any trimethylsilyl protecting groups, and, after separation, the organic phase dried over anhydrous magnesium sulphate. Although the reaction may be carried out at room temperature, it is preferably carried out at low temperature, e.g. -5° to 0°C. The product obtained after evaporation of the reaction mixture to dryness will of course be a cephalosporin having a protected amino or protected hydroxyl group. Such protecting groups may be - 9 removed by conventional procedures. For example, a tbutoxycarbonyl group may be removed by acid hydrolysis, using e.g. formic acid or anhydrous trifluoroacetic acid at about 0°C, the desired product of the 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 may be converted to its zwitterionic form, or to a pharmaceutically acceptable acid addition salt thereof, by methods conventional in the art. For example, when Z is a hydrogen atom the zwitterionic form of the compound may 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 filtering and then adding hydrochloric acid to reduce the pH to about 3.5 to precipitate the zwitterionic product. Formyl and dichloroacetyl protecting groups may be removed by aqueous base, e.g. aqueous sodium bicarbonate solution, which, when Z is a hydrogen atom, produces the sodium salt of the cephalosporin. The free acid may be obtained by acidification. In a typical procedure, the hydroxyl-protected cephalosporin is dispersed in aqueous sodium bicarbonate solution, and the solution overlayered with a suitable water-immiscible organic solvent, e.g. ethyl acetate. After acidification of the aqueous phase to about pH2, the organic phase is separated, dried, and evaporated in vacuo to leave an oil which may be triurated with dry ether to yield the free acid form of the desired α-hydroxy cephalosporin.

In a typical procedure involving the reaction of a mixed anhydride (V) with the compound (II), the compound (II) is dissolved, if necessary with the aid of a base such as triethyiamine, in a suitable solvent, e.g. aqueous tetrahydrofuran, and mixed with a solution of the anhydride in e.g. tetrahydrofuran. After mixing for about 5541 -loan hour, preferably at a temperature of -10° to 0°C, the coolant is removed and the solution left to stand for several hours, followed by dilution with water and extraction with a suitable water-immiscible organic solvent, e.g. ethyl acetate, to remove impurities into the organic phase. After separation, the aqueous phase may be overlayered with a suitable water-immiscible organic solvent, e.g. ethyl acetate, and then acidified to e.g. pH2 by the addition of hydrochloric acid to induce extraction of the desired product into the organic phase. After separation, the organic phase may be dried over anhydrous magnesium sulphate, evaporated in vacuo, and the resulting oil crystallised by trituration with dry ether. The product is of course the protected cephalosporin, and the protecting groups may be removed as described above. The reaction of the activated ester (IV) with the compound (II), may 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 containing sodium bicarbonate. After mixing for about an hour at low temperature, e.g. 0°C, the coolant may be removed and stirring continued for several hours. Water and a water-immiscible solvent such as ethyl acetate are then]added and the pH of the aqueous phase adjusted to about 2.0 with e.g. hydrochloric acid. After filtration, the organic phase may be separated and the aqueous phase extracted with fresh ethyl acetate. The organic phases may be combined, evaporated in vacuo, and triturated with dry ether. The product is again a cephalosporin having a protected amino or protected hydroxyl group, and the protecting groups may be removed as described above. The acid bromide may be reacted similarly. - 11 Acylation with the internally protected” dione (VI) may be carried out similarly to the acid chloride. The product is, of course, a cephalosporin in which Y is -OH.

The compounds of the formula (II) wherein Z is hydrogen may be obtained by methods analogous to those of the prior art, e.g. as follows:- co2h 43S41 In some cases the second of these routes produces the purer starting material.

The compounds of the formula (VIII) may be prepared by a number of routes, including the following: 41 (a) The thiadiazoles may be prepared by catalysed cyclisation of a compound of the the acidformula :---(IX) formula (I)) or an alkali the compound of the formula ΝΗ-NH L 1 S '“//Xi (wherein R1 is as defined for metal salt thereof.

Xn a typical procedure (IX) is added slowly to concentrated sulphuric acid at low temperature, e.g. 0°C. After a short period, the reaction mixture may be carefully added to an ice/water 10 mixture to precipitate the desired product, which may be filtered off, washed with water and dried.

The compounds of the formula (IX) may be prepared by conventional methods, e.g. by reacting a compound of the formula:15 r'conhnh2 ---(X) with carbon disulfide in the presence of base. (b) The oxadiazoles may be prepared by the base-catalysed cyclisation of a compound of the formula (IX) or alkali metal salt thereof.

In a typical reaction the compound (IX) may be reacted in a suitable solvent with potassium hydroxide in absolute alcohol. 3 S 4 i - 14 (c) The compounds in which X is NMe may be prepared by cyclising a compound of the formula (XI) which may itself be prepared by reacting compound (X) with methy1isothiocyanate, according to the following reaction scheme :ch3ncs (XI) In some cases the compound (XI) may be cyclised to the end product merely by heating, although the presence of a base .such as sodium hydride is sometimes necessary.

In a typical reaction, a solution of methyl isothiocyanate in a suitable solvent, e.g. dry tetrahydrofuran, is added slowly to a solution of the hydrazide (X) in a suitable solvent, e.g. absolute alcohol, and the reaction mixture is then stirred at room temperature for several hours, filtered, and the filtrate evaporated in vacuo and triturated with e.g. dry ether to obtain the intermediate (XI). It is not, however, essential to isolate the intermediate. The intermediate may then be added to a solution of sodium hydride in a suitable, solvent, e.g. dry ethanol, and the reaction mixture heated e.g. under reflux for several hours, cooled, and acifified with e.g. dilute hydrochloric acid to a low pH, e.g. pH2. The organic solvent may then be removed in vacuo, the aqueous residue extracted with a suitable - 15 solvent, e.g. ethyl acetate, and the organic phase dried and evaporated to dryness to obtain the desired product. (d) The compounds in which X is NH may be prepared by procedures similar to those described in (c) above but using isothiocyanic acid (HNCS) in place of methyl isothiocyanate.

Preferably, however, the compounds in which X is NH are prepared by cyclising a compound of the formula:NH NH B wherein B is a benzyl group or p-methoxylbenzyl group, to produce a compound of the formula :- B and then removing the group B.

Generally, the compound (XII) cyclises on heating, although the presence of a base such as sodium hydroxide may sometimes be necessary. The group B, when necessary, may be removed by e.g. reaction with trifluoroacetic acid (or other suitable strong acid), or reduction with hydrogen. 541 The compounds of the formula (XII) may be prepared by reacting a compound of the formula:, (X) RCONHNH2 with benzyl- or £-methoxybenzyl-isothiocyanate. Often under the reaction conditions compound (XII) will automatically cyclise to the end product .

Thus, in a typical reaction the isothiocyanate and the compound (X) are heated e.g. under reflux in a suitable solvent, e.g. absolute alcohol, for several hours. The reaction mixture is then filtered, the filtrate evaporated in vacuo, and the resulting oil triturated with e.g. dry ether to obtain the desired intermediate. The intermediate is then preferably-deprotected by a suitable strong acid, e.g. trifluoroacetic acid. This is typically carried out by heating the intermediate in a mixture of trifluoroacetic acid and anisole for a few hours, cooling, evaporating the reaction mixture in vacuo, and triturating the resulting product with a suitable solvent.

Alternatively, the intermediate (XII) in which R^ is -CONH2 or -CH2OCH2CONH2 may be prepared as follows :Ri'COO(C1-C4 alkyl) + B. NHCSNHNHj + BNHCSNHNHCOid (R1 = CONR2R3 or -CH2OCH2CONR2R3 only). 2 3 (e) The compounds in which R is a -CH,0CH9C0NR R or 2 3 ί ί -CONR R group may be prepared from the corresponding compounds in which R1 is a -CH2OCH2COO (C^-C4 alkyl) or -COO(C^-Cd alkyl) group, respectively, - 17 2 3 by reaction with a compound of the formula R R NH. When ammonia is used it is preferably in the form of ammoniasaturated ethanol.

The compounds in which R1 is a -000(0^-0^ alkyl) group may be prepared by methods (a) to (d) described above but utilizing starting materials in which R1 is a -000(0^-0^ alkyl) group.

The compounds in which R^ is a -GHjOCHjCOOH group may be prepared from their C^-C^ alkyl esters by acid or alkaline hydrolysis.

It should be appreciated that when R^ in routes (a) to (d) described above is a -CHjOCI^COO(0^-0^ alkyl) group, hydrolysis of this group to the corresponding free acid may automatically occur under the reaction conditions, particularly in base-catalysed cyclisations.

The esters may be obtained from the corresponding acids by esterification e.g. with a C^-C^ alkanol.

The reactions in (e) above may be carried out before or after the removal of group B. (5) The salts of the compounds of the formula (VIII) may be prepared by conventional methods, e.g. by reacting the thiol with an aqueous or ethanolic solution of the appropriate alkali metal hydroxide.

The compounds of the formula (II) in which Z is other than hydrogen, i.e. those compounds wherein Z completes an ester grouping, may also be prepared by methods analogous to those of the prior art, e.g. by esterifying the corresponding amino-protected compound (II) in which Z is hydrogen or an alkali metal atom (e.g. potassium), typically as follows:3S4i -18 (iii) 3 5 41 COOCH(CH3)OCO2(lower /M - an alkali metal atom, alkyl) preferably potassium7 - 20 The corresponding bromo compounds may be used in place of the chloro-compounds referred to in (iii) and (iv) above.

It may also be necessary to protect any free carboxyl groups in R1 prior to reactions (iii) and (iv) . Following reaction, the protecting group or groups may be removed by conventional procedures.

As is well known, in the reactions of the type described under (iv), some isomerism of the double bond in the cephem nucleus to the 2- position may occur. This can be reversed by forming the S-oxide of the product and then reducing, in known manner. (2) The compounds of the invention in which Y is OH or NH2 and Z is hydrogen may also be prepared by reacting a cephalosporin derivative of the formula:- wherein Y is OH, NH9 or a protected hydroxyl or * 6 protected amino group and R is a facile leaving group, e.g. a chloro, bromo, iodo, or, most preferably, an acetoxy group, with a heterocyclic thiol of the formula:4 33 41 - 21 Ν -Ν ---(VIII) or with a metal or ammonium salt thereof, followed by, if necessary, the removal of any 2 protecting groups from Y .

Compound (VII) is preferably in the D- form.

The metal salt is preferably an alkali metal salt, most preferably a sodium or potassium salt, e.g. of the formula:---(VIIIA) In the case where R^ contains a -COOH group, the thiol may be reacted in the form of its di-metal salt, e.g.

N-N IA KS CH2OCH2COOK ---(VIIIB) Y is preferably a protected amino group, e.g. a 15 t-butyloxy carbonylamino group, or a free hydroxyl group. Unprotected amino groups tend to react with the β-lactam system of the cephalosporin under the reaction 4334 - 22 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 anion:N_N and at a temperature of from 50° to 75°C. After several hours, the reaction mixture may be cooled and overlayered with a suitable water-immiscible organic solvent, e.g. ethyl acetate, to extract impurities into the organic phase. After separation, the aqueous phase may be overlayered with fresh ethyl acetate and then treated with aqueous hydrochloric acid to reduce the pH to a low value, e.g. pH2, to induce extraction of the product into the organic phase. After separation, the organic phase may be washed with brine, dried over anhydrous magnesium sulphate, and evaporated in vacuo.

The resulting oil may be crystallised by trituration with dry ether. Any amino- or hydroxy- protecting groups in the cephalosporin product may be removed as described in method (1) above.

The starting materials of the formula (VII) are either known compounds or may be prepared by methods analogous to those of the prior art. (3) The compounds of the formula (I) in which Z is other than hydrogen may be prepared by esterification of the corresponding compounds of the formula (I) in which Z is hydrogen or an alkali metal atom (preferably potassium), any free hydroxyl or amino groups represented by Y, and any free carboxyl groups in R.\ being if necessary protected prior to the esterification reaction and de-protected afterwards.

The esterifications may for example be carried out using the reagents described in route (1) subsections (iii) and (iv). (4) Salts of the compounds of the invention may be prepared, if necessary, by standard techniques.

For example, preparation of a sodium or potassium salt may be carried out by dissolving a compound in which Z is H in a suitable solvent and adding a solution of the appropriate alkali metal acetate in the same solvent. After reaction, the salt is typically isolated by concentration of the reaction mixture by partial evaporation in vacuo and adding the concentrate to a large volume of a suitable solvent, thereby precipitating the salt. Acid addition salts of those compounds of the invention in which Y is an amino group may be prepared by dispersing the cephalosporin in water, acidifying to a low pH (e.g. pH2) with the appropriate acid, e.g. hydrochloric acid, and evaporating the product to dryness, preferably by freezedrying.

The in vitro evaluation of the compounds of the invention as anti-bacterial agents was performed by determining the minimum inhibitory concentration (M.I.C.) of the test compound in a suitable medium at which growth of the particular micro-organism failed to occur.

In practice, agar (brain/heart infusion agar) plates, each having incorporated therein the test compound at a particular concentration were inoculated with a standard number of cells of the test micro-organism and each plate was then incubated for 24 hours at 37°C. The - 24 plates were then observed for the presence or absence of the growth of bacteria and the appropriate M.I.C. value noted. Micro-organisms used in such tests and against which the compounds were active included strains of Escherichia coli, Klebsiella pneumoniae, Aerobacter aerogenes, Serratia marcescens, Proteus mirabilis, Proteus vulgaris, Staphylococcus aureus and Streptococcus pyogenes.

M.I.C. values in micrograms/ml. for the compounds of the invention are given in the following Table:TABLE iple of •fi H a) (ϋ •fi fi 1 w Φ fi Φ Oi 0 Ul fi Φ 0 ω CQ •fi r—j CQ •fi aureus CQ Φ fi Φ tn 0 >< 0« >ound 0 0 0 fi fi Φ φ 0 •fi Λ fi Π3 CQ fi CQ A «ΰ fi (0 Oi 0 fi (ΰ vo σ> ’tf ro H fi in Η O o in o cn •fi VO Hi O fi o S O •fi rfi □ VO o o 0 O 43 CN h o Φ o o 6 o > o 0 o 0 CN q rf 44 rf (0 < u u 0 < 0 a •fi rfi Φ cn 0 in •η ω CO Γ- CQ Γ- 0 CM fi tn •fi in rd m +) to 3 m 3 in -P O 0 ω 43 (0 Φ Φ XJ 0, 43 43 0 fi •P +1 ft Φ 0 0) fi fi 0 0 rd fi ω r-j fl) Φ fi fi •P‘ +> w « cn ft PM cn cn 1.56 3.12 3.12 >100 3.12 3.12 12.5 1.56 6.25 6.25 12.5 >100 12.5 12.5 6.25 0.78 12.5 3.12 3.12 >100 3.12 6.25 25 3.12 Lso id 1.56 0.78 1.56 100 1.56 1.56 0.39 0.39 29) 1.56 0.39 0.39 100 0.39 0.39 1.56 0.39 1.56 0.78 1.56 100 12.5 6.25 0.39 0.19 1.56 0.78 1.56 100 1.56 1.56 0.39 0.09 0.78 0.78 0.78 100 1.56 3.12 3.12 0.78 1.56 0.78 0.78 100 0.78 0.78 3.12 0.39 3 5 41 - 25 TABLE (continued) Example No. of 0) nJ •rl β | to Φ c Φ Cn cens ω •rl ω ω tf Φ P 3 1 tf ! 05 Φ α φ tn 0 Compound r4 0 to Φ •H •rl μ ω ! Gj ϋ β Φ o Λ tf 3 1 tn Gi nJ Jh tf tn ο ϊ 3 nJ k Η ο ί ϋ •H (0 0) ε r-l •rl ε ui go 0 ο rn U 0 ω r-f O -P o tf o r~i to 0 ο ϋ Ο 0 ό υ ό •Η O ω o tn o rd U 0 IN Ή ri! nJ rtj 2 u 2 o •Ρ ο to tn XI tn tf m GJ > Φ r· pCJ Μ ftM XJ in O in 4J m +j in ft ο φ Ο Φ μ 0 0 tf Ρ 0 Φ p P -Ρ •Ρ w « 05 Λ C4 ω to 10 50 25 12.5 >100 100 25 12.5 0.39 11 3.1 6.2 6.2 100 12.5 12.5 1.56 0.098 12 0.39 0.19 0.39 100 0.78 0.78 0.78 0.012 14 6.2 6.2 6.2 >100 12.5 12.5 6.2 0.098 15 6.2 12.5 6.2 - 50 50 6.2 0.19 16 50 50 25 >100 100 100 50 0.78 17 50 50 50 50 50 50 25 0.78 18 100 100 100 >100 100 100 50 0.78 19 50 50 50 >100 100 100 100 0.78 20 100 100 100 >100 100 100 50 0.78 21 100 >100 >100 >100 >100 100 50 0.39 22 25 25 25 >100 25 25 25 0.39 23 100 100 100 100 - 100 6.2 0.19 24 12.5 6.2 12.5 >100 25 25 6.2 0.19 25 6.2 6.2 6.2 >100 25 25 6.2 0.098 26 25 6.2 6.2 >100 12.5 12.5 25 0.39 TABLE (continued) unple . of ipound •H (1) ίβ •rl c 0 Φ a φ O' 0 U) a φ o ω •H rM ai •r| aureus pyogenes 0 0) φ •rl . M ω 0 fi 0) o Λ ίβ cs ω & ίβ n ίβ Cn ϋ 3 (0 te Μ rH ϋ ϋ •H ίβ M E •rl 3 0 ϋ Λ io H G\ 0) r—1 E Ml > o □ in 0 η o <0 Η O u o nJ O r—1 ID 0 o 0 Ο •H CN Φ o 0 o •Η O in O 03 O H Q Ο CN H < nJ fSj +» < 3 O CS □ +) υ 0) H ω co Λ m ίβ CO Φ > Φ r- X! r-1 βιΜ Λ m Λ in 0 in U M3 +i in +i m fhO Φ Ο □ Φ k 0 O (β μ Ifl r—I Φ Φ P •μ w M W CM CM ω CQ 6.2 1.6 3.1 >100 3.12 3.12 50 1.6 L2.5 3.12 3.12 >100 6.2 3.12 12.5 1.56 The compounds of the invention can be administered alone but will generally be administered in admixture with a pharmaceutical carrier selected with regard to the intended route of administration and standard pharmaceutical practice. For example, they may be administered orally in the form of tablets containing such excipients as starch or lactose, or in capsules either alone or in admixture with excipients, or in the form of elixirs or suspensions containing flavouring or colouring agents.

They may be injected parenterally, for example, intravenously intramuscularly or subcutaneously. For parenteral administration, they are best used in the form of a sterile aqueous solution which may contain other solutes, for example, enough salts or glucose to make the solution isotonic.

It is expected that a suitable total daily dosage (oral or parenteral) for an average adult human being (70 kg.) will be in the range of from 125 mg. to 1 gm of the active compound taken 2 to 4 times a day. The physician will in any case determine the most suitable dose which will depend on the age, weight, and response of the patient.

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

The following Examples illustrate the invention and the preparation of the starting materials. The compounds were tested for purity by thin layer chromatograph (t.l.c.) on silica gel and/or by high pressure liquid chromatography. Infra-red (IR) spectra were recorded as solutions in a suitable solvent, e.g. chloroform, as KBr discs or as Nujol (Registered Trade Mark) mulls. Nuclear magnetic resonance spectra were recorded at 60MHz (except Example 2- 100 MHz) for solutions of the compounds in e.g. perdeutero dimethyl sulfoxide (DMSOdg) or deuterium oxide (D20) using a suitable internal referance standard, e.g. tetramethylsilane. The protons responsible for the signals are underlined. - 28 EXAMPLE 1 A. 7-(D-g-tert-butoxycarbonylamino-p-hydroxyphenylacetamido-)-3-(3-carboxymethoxymethyl-l,2,4-triazol5-yl)thiomethylceph-3-em-4-carboxylic acid.

A solution of 3-acetoxymethyl-7- (D-g-tert-butOxycarbonylamino-£-hydroxyphenylacetamido)-oeph-3-em-4carboxylic acid (4.25 g) and 3-carboxymethoxymethyl-l,2,4triazole-5-thiol (1.53 g) in pH 7.0 phosphate buffer (100 ml) was brought to pH 7.2 by the addition of aqueous 2N sodium hydroxide solution, and the resulting solution was then heated at 70° C for three hours. The reaction mixture was cooled to room temperature, extracted once with ethyl acetate and the organic layer discarded. The aqueous layer was overlayered with fresh ethyl acetate and the pH was adjusted to 2.5 by the addition of aqueous 2N hydrochloric acid. The organic phase was separated, washed with brine, separated again and finally dried over sodium sulphate. Evaporation of this dried extract and trituration of the resulting residue with dry ether gave the product, 7-(D-g-tert-butoxycarbonylamino-p-hydroxyphenylacetamido)-3-(3-carboxymethoxymethyl)-1,2,4triazol-5-yl)thiomethylceph-3-em-4-carboxylic acid, as a white solid, yield 1.6 g.

B. 7-(D-g-amino-£-hydroxyphenylacetamido)-3-(3-carboxymethoxymethyl-1,2,4-triazol-5-yl)thiomethylceph-3em-4-carboxylic acid; trifluoroacetate salt.

The product of Example 1 (1.5 g) was stirred in ice-cold trifluoroacetic acid (12 ml) for ten minutes. This solution was then added to dry ether (200 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 one hour. Yield of the dried product, the trifluoroacetate salt of 7-(D-a-amino-£~hydroxyphenylacetamido)-3-(3-carboxymethoxymethyl-l,2,4-triazol~5-yl) thio5 methylceph-3-em-4-carboxylic acid, was 1.1 g.

(KBr) max. V = 1,765 cni (2 -lactam carbonyl) (DMSOdg) = 3.42 (broad singlet; 2-CH2) 4.0 (singlet) (-CH.OCH,.CO,H) 4.4 (singlet) _ 4.82 (multiplet? 6-H and a-H) 5.55 (unresolved multiplet; 7-H) 6.64 (doublet, J = 7c/s; 2 aromatic protons) 7.15 (doublet, J = 7c/s; 2 aromatic protons) 9.3 (doublet, J = 8c/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 No. R1 Characterising data 2 -CONH2 I.R. (KBr) v „ = 1760 cm-1 max. (β -lactam carbonyl) 1660 οη1 (amide carbonyls) N.M.R. (DMSOdg) fi = 7.35, doublet (J = 8c/s) (Aromatics) 6.85, doublet (J = 8c/s) (Aromatics) 5.68, broad singlet (7-H) 4.96, multiplet (6-H and a-H) 3.54, broad singlet (2-CH2 ) p.p.m. 3 5 41 Example No. R1 Characterising data 3 -ch2och2conh2 I.R. (KBr) v _ = 1760 cm-1 (β -lactam carbonyl) = 1660 cm”·'· (amide carbonyls) N.M.R. (DMSOdg) δ = 7.26, doublet (Aromatics) 6.77, doublet (Aromatics) 5.62, broad singlet (7-H) 4.97, doublet (J = 5c/s), (6-H) 4.80, singlet (a-H) 4.46, singlet (onaCH2) 4.32, singlet (one CH2) 4.08, singlet (one CH2 ) 3.48, broad singlet (2-CH2)p.p.m.

EXAMPLE 4 7- (D-ct-hydroxyphenylacetamido)-3-(3-carbamoy1)-1,2,4triazol-5-yl) thiomethylceph-3-em-4-earboxylic acid.

Aqueous 2N sodium hydroxide was added to a solution of 3-acetoxymethyl-7-(D-a-hydroxyphenylacetamido)ceph-3- 32 em-4-carboxylic acid (27.6 g) and the ammonium salt of 3carbamoyl-l,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 three hours. The reaction mixture was then cooled to room temperature and acidified to pH 2.0 by the addition of aqueous 2N hydrochloric acid. Extraction of the reaction mixture with ethyl acetate and evaporation gave a yellow solid (3 g), found by high pressure liquid chromatography (hplc) to be about 50% pure and this was discarded. The aqueous portion remaining contained a gummy residue which was washed well with water and finally triturated with a solution of isopropanol/ethyl acetate. The residue from this trituration, yield 15 g, was found to be about 70% pure by high pressure liquid chromatographic analysis. Evaporation of the isopropanol/ethyl acetate filtrate gave a solid (5.7 g), found to be about 80% pure. This latter material was chromatographed on silica, a 95% pure (hplc) sample being obtained by elution with a 6% solution of methanol in chloroform, yield 1.56 g of 7-(D-a-hydroxyphenylacetamido) -3-(3-carbamoyl-l,2,4-triazol-5-yl) thiomethylceph3-em-4-carboxylic acid.

I.R. (KBr) Vmax= 1,765 cm 1 (8 -lactam carbonyl) N.M.R. (DMSOdg) 6=3.6 (broad singlet; 2-CH2) 3-CH2-S5.0 (multiplet; 6-H and a-H) .55 (doublet, J = 5c/sf\ $ 7-H .70 (doublet, J = 5c/s)J — 7.25 (multiplet; aromatic protons) 8.55 (doublet, J= 8c/s; CO-NH) p.p.m. 4.0 (doublet; J = 13c/s) 4.35 (doublet;J = 13c/sj 43S41 - 33 EXAMPLE 5 to 12 The following compounds were prepared by procedures similar to those described in Example 4, starting from the same cephalosporin as Example 4 and the appropriate thiol or salt thereof:- Example No. X R1 Characterising data 5 NH -CH2OCH2COOH I.R. (KBr) = 1,760 ΙΙίαΛ « (β -lactam carbonyl) cm-·'· N.M.R. (DMSOdg) 6 = 3.68 (broad (singlet; 2- CH2 4.2 (singlet tunresolved quartet; 3- CH2~s + one ch2) 4.65 (singlet; one CH2) 5.06 (multiplet; 6-H and a-H) 3541 mpie 0. X R1 Characterising data ntin- 3) NH -CH2OCH2COOH 5.65 (doublet, J = 5c/s) 7-H 5.80 (doublet, J = 5c/s) 7-H 7.42 (multip- let, aromatics) 8.73 (doublet, J = 9c/s; -CONH)p.p.m. nple 5. X R1 Characterising data ) s -CH20CH2C0NH2 I.R. (Nujol) = 1,775 cm-1 max · (β-lactam carbonyl) 1,670 cm-1 (amide carbonyl) N.M.R.(DMSOdg) δ = 7.3, broad singlet (Aromatics) 5.65, broad singlet (7-H) 5.04, centre of doublet (J = 4.5c/s)(6-H) 4.90, singlet (a-H) 3.95, broad singlet (2 methylenes) 3.64, broad singlet (2-CH2)p.p.m. 3 5 41 Example No. X R1 Characterising data 7 _ S -CH2OCH2CO2Et I.R. (KBr) vmaXi = 1,775 (β -lactam -1 carbonyl) cm N.M.R. (DMSOdg) δ = 1.15 (centre of triplet, J = 6.5c/s; -co2ch2ch3) 3.6 (broad singlet; 2-¾) 4.18 (complex pattern; -co2ch2ch3 and 3-CH2 and one Cg2) 4.90 (singlet; one CH2) 4.96 (multiplet; 6-H and a-H) 5.6 (poorly resolved doublet of doublets; 7-H) 7.25 (multiplet; aromatics) 8.52 (doublet; J = 9c/s CONH; exchanges with D20)p.p.m. nple ), X R1 Characterising data NMe -CH2OCH2CONH2 I.R. (KBr) vm=„ = 1,770 cm-1 max · (β -lactam carbonyl) 1,670 cm-1 (amide carbonyl) N.M.R. (DMSOdg) ό = 7.45, multiplet(aromatics) 4.75, singlet (methylene) 4.15, single (methylene) 3.70, singlet (N-CH^) 3.55, broad singlet (2-CH2) pie X R1 Characterising data NMe -CH2OCH2COOH I.R. (Nujol Mull) V__„ = 1,775 max · (6 -lactam carbonyl) cm’'1 N.M.R.(DMSOdg) 6 = 3.6 (multiplet; 2-CH2 and N-CH3) 4.2 (multiplet; -CH2~S and one ch2) 4.7 (broad singlet; - ch2> 5.1 (multiplet; 6-H and a-H) *3541 Example No. X 1- R1 Characterising data 9 (contin- ued) 5.7 (poorly resolved doublet of doublets; 7-H) 7.38 (broad singlet; aromatics) 8.74 (doublet J = 8c/s; CONH)p.p.m.

Example NO. X R1 Characterising data 10 0 -CH2OCH2COOH I.R. (KBr) vma]t| = 1,775 (8 -lactam carbonyl) cm 1 N.M.R.(DMSOdg) δ = 9.62, doublet (J = 7.5C/S) (CONH) = 7.30, singlet (aromatics) 5.65, broad singlet(7-H) 5.05, multiplet (6-H and a-H) 4.76, singlet (one C£2) 4.15, singlet (one CH2) 541 nple D. X rl Characterising data 3 itin- I) 3.55, multiplet (2-CH2)p.p.m. iple }. X R1 Characterising data NMe -CONH2 I.R. (Nujol Mull) \> = 1,770 IuclX · (β-lactam carbonyl) 1,680 (amide carbonyl) cm * N.M.R. (DMSOdg) δ = 8.55, doublet (J = 9c/s)(Amide NH) = 7.80, doublet (J = 18c/s)(Amide NH) 7.28, broad singlet (Aromatics) 5.00, multiplet (6-H and a-H) 4.15, broad singlet (3-CH2-S) 3.70, centre of multiplet (N-CH^ and 2-CH2) p.p.m. 43341 Example No. X R1 Characterising data 12 i s -CH2OCH2COOH I.R. (KBr) vmax> = 1,755 (β -lactam carbonyl) = 1,665 (amide carbonyl) I i i 1 N.M.R. (D20) 6 = 7.42, singlet (Aromatics) 5.58, doublet (J = 4c/s) (7-H) 5.25, singlet (Ph.CH(OH)-) 5.00, multiplet (6-H and one CH2) 4.05, singlet (one CH2) 3.66, doublet (J = 18c/s) (2-CH2) 3.25, doublet (J = 18c/s) (2-CH2)p.p.m. 541 - 40 EXAMPLE 13 A. 7-Amino-3-(3-carbamoyl-l,2,4—triazol-5-yl) thiomethylceph-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-l,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 being cooled to 5°C. The pH was then adjusted to 3.9 with ammonium hydroxide solution and after stirring for 2 hours at 5°C the precipitate of 7-Amino-3(3-carbamoyl-l,2,4-triazol-5-yl)thiomethylceph-3-em-4carboxylic acid (47.9 g) was filtered off, washed with water, then with acetone and ether, and finally dried in vacuo at room temperature.

B. 7-(D-a-hydroxyphenylacetamido)-3-(3-carbamoyl1,2,4-triazol-5-yl)thiomethylceph-3-em-4carboxylic acid.

D-0-(DichloroacetylJmandeloyl chloride (72 g) in acetone (120 ml) was added over 45 minutes to a solution of 7-Amino-3-(3-carbamoyl-l,2,4-triazol-5-yl)thiomethylceph-3-em-4-carboxylic acid (45 g) prepared in Part A and sodium bicarbonate (27 g) in water (720 ml) and acetone (600 ml) at 0°C. The pH was maintained at 7.5 throughout the addition by adjustment with 2N sodium hydroxide solution. The mixture was allowed to warm to room temperature and then stirred for 1 hour. The acetone was removed under vacuum, the pH raised to 9.5 by the addition of sodium carbonate solution and, after 30 43341 - 41 minutes, the pH was re-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-ahydroxyphenylacetamido)-3-(3-carbamoyl-l,2,4-triazol-5yl) thiomethylceph-3-em-4-carboxylic acid as an off white solid, yield 23.3 g, confirmed by h.p.l.c. to be identical to the product of Example 4.

EXAMPLE 14 By a method similar to that of Example 13 Part B, 7-(D-cx-hydroxyphenylacetamido)-3-(2-carbamoyl-l,3,4oxadiazol-5-yl)thiomethylceph-3-em-4-carboxylic acid was prepared from 7-amino-3-(2-carbamoyl-l,3,4-oxadiazol-5yl)thiomethylceph-3-em-4-carboxylic acid and D- 0formylmandeloyl chloride.

I.R. (Nujol Mull) v 1,770 (β -lactam carbonyl) cm ' TU3X» N.M.R. (DMSOdg) = 7.25, singlet (Aromatics) .58, multiplet (7—H) .00, multiplet (6-H and a-H) 4.30, broad singlet (3-CH2~S) 3.55, multiplet (2-CH2)p.p.m.

EXAMPLE 15 (A) 7-Amino-3-(2-carbamoyl-l,3,4-oxadlazol-5-yl)thiomethylceph-3-em-4-carboxylic acid.

Solutions of 7-aminocephalosporanic acid (5.44 g) and 2-carbamoyl-l,3,4-oxadiazole-5-thiol (3.19 g) in dilute aqueous sodium hydroxide solution at pH ca. 7.0 were mixed (total volume about 75 ml) and the resulting mixture stirred and heated in a water-bath at 70°C for one hour. During the reaction the pH of the solution - 42 was maintained at 6.5 - 7.0 by the addition of aqueous 2N sodium hydroxide solution. After this time the reaction mixture was cooled rapidly to room temperature, 2N hydrochloric acid was added to adjust the pH to 3.5, and the resulting brown precipitate collected by filtration. The product, 7-amino-3-(2-carbamoyl-l,3,4-oxadiazol5-yl)thiomethylceph-3-em-4-carboxylic acid, was washed well with acetone to yield a tan powder (3.1 g).

(Bl 7-(D-a-tert-butoxycarbonylamino)phenylacetamido3-(2-carbamoyl-l,3,4-oxadiazol-5-yl)thiomethylceph-3-em-4-carboxylic acid D- N-tert-butoxycarbonylpheny! glycine (1.9 g) was dissolved in dry tetrahydrofuran (THF)(25 ml) and the solution cooled to 5°C. Triethyiamine (0.76 g) was added with stirring, the reaction mixture cooled to -10°C and treated over two minutes with isobutyl chloroformate (1.0 g). The resulting solution of the mixed anhydride was stirred at -10° C for fifteen minutes.

The cephalosporin product of part (A)(1.8 g) was added to an ice-cold solution of THF (13 ml) and water (13 ml) containing triethyiamine (0.5 g) and stirred to effect solution. The resulting solution was added over seven minutes with, stirring to the solution of the said anhydride at -5°C. After a further thirty minutes at this temperature the reaction mixture was left for two hours during which time the coolant was removed. Water (25 ml) was added and the reaction extracted once with ethyl acetate. The ethyl acetate extract was dried over anhydrous magnesium sulphate, evaporated in vacuo, and the product triturated with dry ether to yield an offwhite solid (CROP 1-530 mg). The aqueous phase was overlayered with ethyl acetate, the pH adjusted to 2.0 by the addition of aqueous 2N hydrochloric aoid, and the organic layer separated and dried over magnesium sulphate. The solvent was evaporated in vacuo and the product 33 41 - 43 triturated with dry ether to yield an off-white solid (CROP 2-800 mg). By thin layer chromatographic analysis Crops I and 2 were found to be identical, viz. 7-(D-atertbutoxycarbonylamino)phenylacetamido-3-(2-carbamoyl1,3,4-oxadiazol-5-yl)thiomethylceph-3-em-4-carboxylic acid, and were combined.

(C) 7-(D-a-aminophenylacetamido)-3-(2-carbamoyl-l,3,4oxadiazol-5-yl)thiomethylceph-3-em-4-carboxylic acid trifluoroacetate salt.

The product from part (B) (500 mg) was added with stirring to ice-cold trifluoroacetic acid (5 ml). After six minutes the brown solution was evaporated in vacuo and the oily residue triturated with dry ether. The product, 7-(D-a-aminophenyl acetamido)-3-(2-carbamoyl-l,3, -4-oxadiazol-5-yl)thiomethylceph-3-em-4-carboxylic acid trifluoroacetate salt, was filtered, washed well with dry ether, and dried in vacuo. The yield of off-white solid was 500 mg.

(D) 7-(D-a-aminophenylacetamido)-3-(2-carbamoyl-l,3,4oxadiazol-5-yl)thiomethylceph-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 the pH of the filtrate adjusted to 3.5 by addition of 2N hydrochloric acid. After two days at 5°G the product, 7-(D-a-aminophenyl acetamido)3-(2-carbamoyl-l,3,4-oxadiazol-5-yl)thiomethylceph-3em-4-carboxylic acid in zwitterionic form, was collected by filtration, washed with a little water and dried in vacuo. The yield of off-white solid was 110 mg. The product was characterised by means of thin layer chroma541 tography and infra-red and nuclear magnetic resonance spectroscopy.

I.R. (Nujol) v = 1760 cm (β-lactam carbonyl), 1690 cm-3 (4 - carboxyl), EXAMPLES 16 to 28 The following cephalosporanic acid derivatives were prepared by similar procedures to that described in Example 15 starting from 7-aminocephalosporanic acid and the appropriate heterocyclic thiol. The products were recovered in the form or forms indicated (in most cases the trifluoroacetate (TFA) salt was not converted to the zwitterion as in Example 15), and were characteri sed by means of thin layer or high pressure liquid chromatography, and by infra-red and nuclear magnetic resonance spectroscopy. - 45 13341 Example No. R X R1 Form Isolated 16 HO- 0 -CONH2 zwitterion and TFA salt 17 H 0 -CONHCH3 TFA salt 18 H 0 -CON(Me)2 TFA salt 19 HO- 0 -CON(Me)2 TFA salt 20 H 0 -CONHEt fl 21 HO- 0 -CONHEt II 22 HO- 0 -CH2OCH2CO2Et II 23 H s -conhch3 II 24 H N.CH3 -conh2 II 25 HO- n.ch3 -conh2 It 26 HO- n.ch3 -CH2OCH2CO2Et II 27 HO- n.ch3 -ch2och2co2h II 28 HO- s -ch2och2co2h II If desired, the trifluoroacetate salts may be converted to the zwitterionic form of the cephalosporin by the method of part (D) of Example 15.

Characterising Data for the compounds of Examples to 28 is set out below:EXAMPLE 16 I.R. (Mull) v = 1,760 (8 -lactam carbonyl) 1,670 (amide carbonyl) cm di EXAMPLE 17 I.R. υ = 1,775 (β -lactam carbonyl) JiioX« 1,675 (amide carbonyl) cm-·4 N.M.R. (DMSOdg) δ = 9.6, doublet (J = 8c/s)(Amide NH) 9.12, doublet (J = 5c/s)(CONHCH3) 7.5, singlet (Aromatics) .76, unresolved multiplet (7-H) .05, multiplet (6-H and a-H) 4.58, doublet (J = 13c/sA (3-CH2“S) 4.25, doublet (J = 13c/s)J 3.57, multiplet (2-CH2) 2.82, doublet (J = 5c/s)(CONHCH3)p.p.m.

EXAMPLE 18 I.R. (KBr) = 1,775 (8 -lactam carbonyl) 1,660 (Amide carbonyl) cm- N.M.R. (DMSOdg) δ = 7.5, singlet (Aromatics) , 5.8, broad singlet (7-H) .1, multiplet (6-H and a-H) 4.35, broad singlet (3-CH2“S) 3.6, multiplet (2-CH2) 3.3 singlet (N-CHj) 3.05, singlet (N-CH3) p.p.m.

EXAMPLE 19 I.R. (KBr) = 1,770 (β -lactam carbonyl) v IuaX» η 1,660 (amide carbonyl) cm N.M.R. (DMSOdg) δ = 7.32, doublet (J = 7c/s)(Amide -NH) 7.25, doublet (J = 8c/s)(Aromatics) 6.72, doublet (J = 8c/s)(Aromatics) 5.70, unresolved multiplet (7-H) 4.95, unresolved multiplet (6-H and -H) 4.30, broad singlet (3-GH2-S~) 3.58, broad singlet (2-CH2) - 47 - I.R. (KBr) 1 v max. 3.36, singlet (N-CHj) 3.00, singlet (Ν-CHj) p.p.m. EXAMPLE 20 = 1,780 (β -lactam carbonyl) 1,670 (amide carbonyl) cm-''' N.M.R. (DMSOdg) δ = 7.4, singlet (Aromatics) 5.7, broad singlet (7-H) 5.0, multiplet (6-H and a-H) 4.3, broad singlet (3-CH2~S) 3-4, complex multiplet (2-CH2 and conhch2ch3) 1.1, centre of triplet (J = 7c/s) (CONHCH2CH3)p.p.m. I.R. (KBr) ' v max. EXAMPLE 21 = 1,775 (β -lactam carbonyl) 1,670 (amide carbonyl) cm I.R. (KBr) υ max. EXAMPLE 22 = 1,780 (β -lactam carbonyl) 1,760 (ester carbonyl) 1,675 (amide carbonyl) cm~“ N.M.R. (DMSOdg) δ = = 3.3, doublet (J = 8c/s)(Aromatics) 4.75, doublet (J = 8c/s)(Aromatics) 5.70, broad multiplet (7-H) 3.60, broad singlet (2-CH2) 1.20, centre of triplet (J = 7c/s)p.p.m. I.R. (KBr) ' v max. EXAMPLE 23 = 1,775 (β -lactam carbonyl) 1,665 (Amide carbonyl) cm”·1· N.M.R. (DMSOdg) δ =; 9.18, doublet (J = 5c/s)(Amide NH) 7.47, singlet (Aromatics) 5.77, broad multiplet (7-H) I - 48 - 5.00, multiplet 4.30, multiplet 3.55, multiplet 2.7 6, centre of (C0NHCH3)p (6-H and a-H) (3-CH2-S) (2-¾) doublet (J=5c/s) .p.m.

EXAMPLE 24 I.R. (Nujol Mull) = 1,775 (β -lactam carbonyl) ν ΙΠ3.Λ · 1,680 (amide carbonyl) cm N.M.R. (DMSOdg) δ == 7.4, singlet (Aromatics) .65, unresolved multiplet (7-H) 4.95, unresolved multiplet (6-H and a-H) 4.15, broad singlet (3-CH2-S) 3.75, singlet (N-CH^) 3.6, multiplet (2-(¾) p.p.m.

EXAMPLE 25 I.R (KBr) v max.

N.M.R. (DMSOdg) δ 1,775 (β -lactam carbonyl) 1,675 (Amide carbonyl) cm 7.25, doublet (J = 8c/s)(Aromatics) 6.75, doublet (J = 8c/s)(Aromatics) 5.65, multiplet (7-H) 4.90, multiplet (6-H and a-H) 4.10, broad singlet (3-CH2~S) 3.70, singlet (¢1-¾) 3.55, multiplet (2-CH,,) p.p.m.

EXAMPLE 26 I.R.

(KBr) v max.

N.M.R. (DMSOdg) δ 1,780 (β -lactam carbonyl) 1,765 (Ester carbonyl) 1,680 (Amide carbonyl) cm-1 7.45, doublet (J = 8c/s)(Aromatics) 6.76, doublet (J= 8o/s)(Aromatics) 5.75, broad singlet (7-H) 4.96, multiplet (6-H and a-H) 13541 - 49 4.70, singlet (one CH,,) 4.10, multiplet (two CH2) 3.58, broad singlet (N-CH3 and 2-CH2) 1.00, centre of triplet, J = 6c/s (CO2CH2CH3) p.p.m.

EXAMPLE 27 I.R. (KBr) ,, = 1,775 (β -lactam carbonyl) 1,675 (Amide carbonyl) cm N.M.R. (DMSOdg) δ -· 7.28, doublet (J = 8c/s)(Aromatics) 6.74, doublet (J ' = 8c/s)(Aromatics) .68, broad singlet (7-H) 4.95, multiplet (6-H and a-H) 4.68, singlet (one CH2) 4.08, singlet (one CH,,) 3.55, singlet (N-CHj) 3.35, multiplet (2-CH2) p.p.m.

EXAMPLE 28 I.R. (KBr) ,, = 1,765 (β -lactam carbonyl) V ιίΐαΧ · η 1,670 (Amide carbonyl) om N.M.R. (DMSOd,) δ = 9.55 (doublet, J = 8c/s)(Amide N-H) D — 7.35 (centre of doublet, J = 8c/s) (Aromatics) 6.83 (centre of doublet, J = 8c/s) (Aromatics) .80 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. 4i - 50 EXAMPLE 29 A. (D)-5-phenyl-l,3-dioxolan-2,4-dione A solution of phosgene (56.9 g) in toluene (500 ml) was added over 1 hour to (D)-a-hydroxyphenylaeetic acid (45.6 g) in tetrahydrofuran (450 ml) at room temperature and then the mixture was heated at 45°C for 6 hours. The organic solvent was removed under vacuum and the residue was crystallised from carbon tetrachloride to yield (D)-5-phenyl-l,3-dioxolan-2,4-dione as a white solid (49.4 g) , m.p. 76-77°C.

Analysis;Found: C, 60.37; H, 3.68% Calculated for CgHgO^. C, 60.67; H, 3.39% ; B. 7-(D-a-hydroxyphenylacetamido)-3-(3-carbamoyl)1,2,4-triazol-5-yl) thiomethyl-ceph-3-em-4carboxylic acid (D)-5-phenyl-l,3-dioxolan-2,4-dione (50.4 g) was added over 15 minutes to a solution of 7-amino-3- ζ(3carbamoyl-1,2,4-triazol-5-yl)thiomethylj ceph-3-em-4carboxylic 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 on silica using up to 10% methanol in chloroform as eluant gave the pure product, 7-(D-ahydroxyphenylacetamido) -3-(3-carbamoyl-l,2,4-triazol-5yl)thiomethyl which was chromatographically and - 51 spectroscopically identical to the material prepared in Example 4.

EXAMPLE 30 Diphenylmethyl 7-(D-ci-hydroxyphenylacetamido)-3(3-carbamoyl-l,2,4-triazol-5-yl)thiomethylceph-3em-4-carboxylate The product of Example 4 (100 mg) was added to an ice-cooled stirred solution of diphenyl diazomethane (PhjCHNj)(45.1 mg) in ethyl acetate (15 ml) and the reaction mixture was kept under refrigeration 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 sulphate. The organic layer was evaporated in vaeuo and the residue triturated with dry ether to give the diphenylmethyl ester of 7-(D-ct-hydroxyphenylacetamido)-3(3-carbamoyl-l,2,4-triazol-5-yl)thiomethylceph-3-em-4carboxylic acid as a white solid (99 mg), m.p. 133-135°C. A sample recrystallised from isopropanol analysed as follows :Found: C, 58.56; H = 4.32% Calculated for ¢32^28^6^6^2: C' H = 4.30% I.R. , = 1778 (β -lactam carbonyl) cm \ V JnSX» EXAMPLE 31 3-(3-Carbamoyl-l,2,4-triazol-5-yl)thiomethyl-7(D-ct-formyloxyphenylacetamido)ceph-3-em-4carboxylic acid Ν,Ο-Bis(trimethylsilyl) acetamide (800 mg) was added to a suspension of the product of Example 13 Part A (400 mg) in dry THF (9 ml), and the mixture was warmed at 50°C for 20 minutes after which time a clear 4i solution was obtained. This solution was cooled to room temperature and a solution of D-O-formylmandeloyl chloride (0.23 g)(prepared as described in German Offenlegungschrift No. 2,506,622 {(Lilly)) in dry THF (1 ml) added. After stirring for two 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^) and evaporated in vacuo to yield 0.5g of crude material. Trituration of this crude material with dry acetone gave the required product, 3-(3-carbamoy1-1,2,4-triazol-5-yl)thiomethyl7-(D-a-formyloxyphenylacetamido)ceph-3-em-4-carboxylic acid, as an off-white solid (140 mg).

I.R. (KBr) = 1,770 cm 1 (β-lactam carbonyl) U.V. (EtOH) E^ = 169 (@270nm) N.M.R.(DMSOdg) δ = 3.5 (broad singlet, 2-CH2) 3.95 (centre of doublet, J=13c/s) 4.30 (centre of doublet, J=13c/s) 4.95 (doublet, J=5c/s, H-6) .60 (very broad multiplet, H-7) 6.08 (singlet, α-Βί) 7.35 (broad singlet, aromatics) 7.76 (broad singlet, - CONH-) 8.26 (sharp singlet, - O.CHO) 9.20 (doublet, Jf7c/s, -CONH)p.p.m.

EXAMPLE 32 Pivaloyloxymethyl 3-(3-carbamoyl-l,2,4-triazol5-yl)thiomethyl-7-(D-a-hydroxyphenylacetamido)ceph-3-em-4-carboxylate Sodium bromide (28mg) was added to a stirred solution of the chloromethyl ester of pivalic acid (40mg) 3 S 41 - 53 in dry dimethylformamide (1.5ml). After 15 minutes the product of Example 4 (123mg) was added and, after lo minutes, a solution of dicyclohexylamine (50mg) in dry DMF (0.rml) was added dropwise over approximately 15 minutes,. The resulting reaction mixture was stirred for 17 hours at room temperature after which time 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 sulphate. 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-ct-hydroxyphenylacetamido)-ceph-3-em-4-carboxylate, as an off-white solid (34mg).

T-R· (KBr) V max = 1,751 i(broad)(β-lactam and ester carbonyls) cm N.M.R. (DMSOdg) δ = 1.18 (sharp singlet, t-butyl), 3.95 (centre of a doublet, J=13c/s) )ch2s 4.40 (centre of a doublet, J=13c/s) 5.10 (multiplet, 6-H and a-H) 5.80 (multiplet, 7-H and methylene of ester group) 7.30 (broad singlet, aromatic protons) 7.86 (multiplet, -C0NH2) 8.68 (doublet, J=7c/s, -C0NH-)p.p.m.

The following Preparations illustrate the preparation of certain of the starting materials:41 - 54 Preparation 1 Part A Potassium 3-(ethoxycarbonylmethoxyacetyl)dithiocarbazate Ethoxycarbonylmethoxyacetohydrazide (EtO.CO.CH2OCH2 CONHNH2) (15 g) was suspended in ethanol at 0°C (200 ml) and carbon disulphide (26.7 ml) was added dropwise. A solution of potassium hydroxide (5.6 g) in ethanol (60 ml) was then added over thirty minutes and the reaction mixture stirred at 0°C for a further thirty minutes. Finally the coolant was removed and the reaction mixture stirred overnight. The produot, potassium 3-(ethoXycarbonylmethoxyacetyl)dithiocarbazate (Et0.C0.CH2QCH2 CONH NHCS2K), was filtered off from the reaction mixture, washed successively with ethanol and ether and finally dried in vacuo, (Yield = 43.5 g). This product was used in the next stage without further purification.

Part E 2-(EthoxycarbonyImethoxymethyl)-1,3,4-thiadiazole-5-thiol The product of part A (4 g) was added portionwise, with stirring, over fifteen minutes to ice-cooled concentrated sulphuric acid (20 ml). After the final addition the reaction mixture was stirred for a further ten minutes and then added carefully to an ice/water mixture (200 g). The insoluble product, 2-(ethoxycarbonylmethoxymethyl )-1, 3, 4-thiadiazole-5- thiol, was filtered off, washed with water and finally dried in vacuo , (Yield 2.4 g). A small analytical sample recrystallised from 20% ethanol/water as white needles, m.p. 8O°-82°.

Analysis Found: C, 35.83; H, 4.25; N, 11.85% C7H1ON2°3S2 rec3uires: C, 35.90; H, 4.30; N, 11.96% - 55 Preparation 2 Di-Potassium salt of 2-(Carboxymethoxymethyl)-1,3,4thiadiazole-5-thiol The product of Preparation 1, Part B (6.0 g) was added to a stirred solution of potassium hydroxide (4.5 g) in absolute alcohol (150 ml), and, after one hour, the insoluble product was filtered off, washed successively with alcohol and ether, and finally dried in vacuo. The yield of pale yellow solid was 7.0 g, and this analysed as the monohydrate of the di-potassium salt of 2-(carboxymethoxymethyl)-l,3,4-thiadiazole-5-thiol, m.p. 269-70° (d).

Analysis: Found: C, 20.5; H, 1.92; N, 9.19% C5H4N2O3S2K2.H2O requires: C, 20.00;H, 2.00; N, 9.33% Preparation 3 2-(Carbamoylmethoxymethyl)-1,3,4-thiadiazole-5-thiol m.p. 216-217°C, was prepared from the product of Preparation 1 by treating it with ammonia-saturated ethanol in a similar procedure to that described in Preparation 15, Part C hereinafter.

Analysis:Found: C, 29.13; H, 3.48; N, 19.51% Cj-HyNjOjSj requires: C, 29.23; H, 3.44; N, 20.46% Preparation 4 2-(N-methylcarbamoyl)-1,3,4-thiadiazole-5-thiol was prepared from the corresponding 2-ethoxycarbonyl compound by reaction with methylamine. The starting 2-ethoxycarbonyl compound was prepared in a similar manner to Preparation 1, starting from, the ethyl ester >41 of oxalic acid monohydrazide (EtOOC.CONHNB^) and carbon disulphide.

Preparation 5 2- (Ethoxycarbonylmethoxymethyl) -1,3 , 4-oxadiazole-5-thiol A solution of potassium hydroxide (0.22 g) in alcohol (2.5 ml) was added to a suspension of ethoxycarbonylmethoxyacetohydrazide in alcohol (4 ml), followed by carbon disulphide (0.95 ml) and dimethyl sulphoxide (0.54 ml). The reaction mixture was then refluxed for twelve hours, cooled, filtered and the filtrate evaporated In vacuo. The resulting oil was dissolved in water, filtered, and the filtrate acidified to pH 2,0 with aqueous 2N hydrochloric acid. Extraction of the reaction mixture with ethyl acetate and evaporation of the solvent yielded the product, 2-(ethoxycarbonylmethoxymethyl) -1,3, 4-oxadiazole-5-thiol, as a white solid (0.5 g), m.p, 101-3°.

AnalysisiPound: C, 38.82; H, 4.45; N, 12.86% C7H1ON2°4S re<JuireS: C, 38.53; H, 4.62; N, 12.84% Preparation 6 Di-Potassium salt of 2-(Carboxymethoxymethyl)-l,3,4oxadiazole-5-thiol The product of Preparation 5 (2.0 g) was stirred at room temperature in aqueous 2N potassium hydroxide (20 ml) for four hours. The resulting solution was overlayered with ethyl acetate and aqueous 2N hydrochloric acid was added to reduce the pH to 2.0. The organic layer was separated, dried (MgSO^) and evaporated to yield a solid which was shown by thin layer chromatographic analysis to contain some unreacted starting - 57 naterial. The solid {1.0 g) was then stirred overnight vizh a sclucxcn cf ethanol -,10 mi; containing potassi«!u hydroxide (0.5 g) and the resulting insoluble solid, the di-potassium salt of 2-(carboxymethoxymethyl)-1,3,45 oxadiazole-5-thiol was filtered off, washed with alcohol and dried in vacuo (yield 1.0 g; m.p. 207-10°(d)).

A sample of the salt was converted to the free acid by treatment with dilute hydrochloric acid and had a melting point of 126-9° and the following analysis.

Analysis:Found: C, 31.69,- H, 3.15; N, 14.63% CgHgNjO^S requires: C, 31.58; H, 3.16; N, 14.74% Preparations 7 to 10 The following oxadiazoles were prepared by the reaction of 2-ethoxycarbonyl-l,3,4-oxadiazole-5-thiol with, respectively, ammonia, methylamine, ethylamine and dimethylamine in a manner similar to that described In Preparation 15 Part (C) hereinafter. The starting 2-ethoxycarbonyl compound was prepared in a manner similar to Preparation 5 but utilising as the starting hydrazide EtOOC.CONHNH2. 4 2- 58 Ν-Ν □aration No. R1 m.p. (°C) Analysis % (calculated in brackets) C H N 7 -conh2 237°(d) 24.92 (24.82 2.09 2.08 28.53 29.95) 8 -conhch3 - - - - . 9 -CONHEt 210°(d) 35.00 (34.67 4,14 4.08 24,00 24.26) 3 -con(ch3)2 185-7° 34.81 (34.67 3.99 4.08 23.76 24.26) Preparation 11 Part A A solution of methyl isothiocyanate (3.52 g) in dry tetrahydrofuran (20 ml) was added over thirty minutes to a suspension of ethoxycarbonylmethoxyacetohydrazide (8.50 g) in absolute alcohol (50 ml) and the reaction mixture was stirred for a further two hours.

The solution was filtered and the clear filtrate evaporated in vacuo. Trituration of the residue with dry ether yielded the compound CH3NHCSNHNHCOCH2OCH2CO.OEt, as a white solid (8.0 g).

Analysis: Found: C, 38.77; H, 6.21; N, 17.03% c8h15N3°4S requires: C, 38.55; H, 6.02; N, 16.87% - 59 Part B The product of Part A (2.49 g) was added to a solution of sodium hydride (0.3 g) in dry alcohol (50 ml) and the reaction mixture was refluxed for four hours.

The reaction mixture was then cooled and divided equally into two portions (i) and (ii). Aqueous 2N hydrochloric acid was added to portion (i) to reduce the pH to 2.0 and the organic solvent was then removed in vacuo.

Extraction of the aqueous residue with ethyl acetate and evaporation of this dried (MgSO^) extract gave 3-(ethoxycarbonylmethoxymethyl)-4-methyl-1,2,4-triazole-5-thiol as a white solid, m.p. 86-88°C, (Yield 300 mg).

I.R. (KBr) ,, = 1,740 (Ester carbonyl) cm 1 v ill cl X · N.M.R. (DMSOdg) δ = 1.15 (centre of triplet, J = 7 c/s, ester CH^) 3.5 (singlet, N-CH^) 4.1 (multiplet; CH2 and ester CH2) 4.6 (singlet; Cih,) p.p.m.

Preparation 12 Preparation of 3-Carboxymethoxymethyl-4-methyl-l,2,4triazole-5-thiol Portion (ii) from Preparation 11 was evaporated in vacuo, the residue dissolved in water, acidified with aqueous 2N hydrochloric acid and the solution then extracted with ethyl acetate. The dried (MgSO^) extract was evaporated in vacuo to yield a white solid shown by thin layer chromatographic analysis to be a 50/50 mixture of the ester of Preparation 11 and required acid. This mixture was then stirred in aqueous 2N sodium hydroxide for two hours, acidified with 2N hydrochloric acid and extracted with ethyl acetate. The dried(MgSO^) organic extract was evaporated in vacuo to yield 3-(carboxymethoxymethyl) -4-methyl-l, 2, 4-triazole-5-thiol as a white 13541 - 60 solid (0.25 g) , m.p. 155-156°.

Analysis:Found: C, 35.68; H, 4.48; N, 20.73% C6HgN3O3S requires: C, 35.46; H, 4.43; N, 20.69% Preparations 13 and 14 The following compounds were prepared by the reaction of ethanolic ammonia with the corresponding compounds in which R1 is -CH2OCH2COOEt and -COOEt, respectively, in a manner similar to that described in Preparation IS part (C) hereinafter: N -N IL HS / j R1 CH, paration No. R1 m.p. (°C) Analysis % (Found in Brackets) C H N S 13 -CH2OCH2CONH2 152-153° - - - -* 14 -conh2 273-275°(d) 30.28 3.81 36.20 19.72 (30.28 3.82 35.44 20.20) The starting ester for Preparation 14 was prepared similarly to Preparation 11, starting from methyl isothiocyanate and EtOOC.C0NHNH2. 3 5 41 *N.M.R. (DMSOdg) 6 6 = 3.45 (singlet, n-ch3) 4.10 (singlet,cil2> 4.60 (singlet, ch2).

Preparation 15 A. 3-Ethoxycarbonyl-4-(para-methoxybenzyl)-1,2,4triazole-5-thiol A solution of para-methoxybenzyl isothiocyanate (8.95 g) and EtOOC.CONHNH2 ¢6.8 g) in alcohol (50 ml) was refluxed overnight. The hot solution was filtered, the filtrate evaporated in vacuo and the resulting oil triturated with dry ether. The solid so obtained, 3(ethoxyearbonyl)-4-(p-methoxybenzyl)-1,2,4-triazole-5thiol, (7.2 g) Was recrystallised from alcohol/water to yield 4 g of pure product. An analytical sampl-3 was obtained by a further reerystallisation from ethyl acetate/petrol b.p. 60/80°, and had m.p. 142-144°C.

Analysis Pound: C, 53.29; H, 5.12; N, 14.70% requires: C, 53.25; H, 5.16; N, 14.33% B. 3-Ethoxycarbonyl-l,2,4-triazole-5-thiol A mixture of the product of Part A (4.8 g) in 4:1 v.v. trifluoroacetic acid/anisole (20 ml) was heated at 60°C for four hours. The solution was cooled, evaporated in vacuo and the residue triturated with petrol b.p. 60/80° to give an orange solid. This solid was then triturated with dry ether to give 3-ethoxycarbonyl-l,2,4triazole-5-thiol as a white solid (1.9 g), m.p. 192-193°C (decorap.).

S41 - 62 Analysis:Found: C, 34.60,- H, 4.07; N, 24.07% C5H7N3O2S requires: C, 34.69; H, 4.08; N, 24.28% C. 3-Carbamoyl-l,2,4-triazole-5-thiol The product of Part B (14.5 g) was heated at 100°C overnight in a bomb with saturated ammoniacal ethanol (200 ml). The reaction mixture was evaporated in vacuo to yield 12.0 g of the ammonium salt of 3-carbamoyl-l,2,4· triazole-5-thiol. An analytical sample of the free thiol was obtained by acidification of an aqueous solution of the ammonium salt, the free thiol precipitating as an off-white solid, m.p. 276°(d), Analysis:- (free thiol) Found: c, 25.97; H, 3.00; N, 37.75% C3H^N^OS requires: c, 25.01; H, 2.80; N, 38.89% I.R. (KBr) V max. 1640 (Amide carbonyl) cm’1 Preparation 16 By a procedure similar to that described in Preparation 15 part A, 3-ethoxycarbonylmethoxymethyl4-(£-methoxybenzyl)-l,2,4-triazole-5-thiol was prepared, starting from p-methoxybenzylisothiocyanate and ethoxycarbonylmethoxyacetohydrazide (EtOOC.CH2OCH2 C0NHNH2) The ethoxycarbonyl group was then hydrolysed to the free acid using aqueous 2N caustic soda, and the £methoxybenzyl group was then removed using trifluoroacetic acid in a procedure similar to that described in Preparation 15 part B to give 3-carboxymethoxymethyl1,2,4-triazole-5-thiol, m.p. 180-182°.

Analysis:Found: C, 32.13; H, 3.73; N, 22.47% *3Sdl - 63 C5H2N3O3S requires: C, 31.75; H, 3.73; N, 22.22% Preparation 17 3-Carbamoylmethoxymethyl-l,2,4-triazole-5-thiol was prepared from 3-ethoxycarbonylmethoxymethyl-4-(£methoxybenzyl)-1,2,4-triazole-5-thiol by reaction with ammoniacal ethanol in a manner similar to Preparation 15 part C and then removal of the £-methoxybenzyl group with trifluoroacetic acid in a manner similar to Preparation 15 part B.

N.M.R. (D20) δ = 4.5, singlet (methylene), = 4.05, singlet (methylene) Preparation 18 3-(N-Ethylenecarbamoylmethoxymethyl)-1,2,4triazole-5-thiol was prepared by a procedure similar to that of Preparation 17 starting from 3-ethoxycarbonylmethoxymethyl-4-(£-methoxybenzyl)-1,2,4-triazole-5thiol as in Preparation 17 but using ethylamine in place of ammoniacal ethanol.

N.M.R. (DMSOdg) = 4.7, singlet (methylene) = 4.0, singlet (methylene) = 3.35, singlet (J = 8 c/s) (conhch2ch3) = 3.15, doublet (J = 8 c/s) (co.nhch2ch3) = 1.05, centre of triplet (J = 8 c/s)(CONHCH2CH3) p.p.m.

Preparation 19 A. l-0xamoyl-4-(p-methoxybenzyl)thiosemicarbazide Sodium methoxide (5.4 g; 0.1M) and 4-(^methoxybenzyl) thiosemicarbazide (21.1 g; 0.1M) were stirred in methanol (200 ml) for 5 minutes. Ethyl oxamate 541 (11.7 g; 0.1M) was added and the mixture refluxed for 3 hours. Most of the solvent was evaporated under reduced pressure, water (200 ml) was added and the pH adjusted to 2 with dilute hydrocholoric acid. The resulting solid was filtered and dried at 50°C In vacuo to give l-oxamoyl-4-(g-methoxybenzyl)thiosemicarbazide (25.7 g; 91% yield), m.p. 185-19O°C.

AnalysisiFound: C, 46.3; H, 4.87; N, 18.83% C11H14N4°3S * * retluires: c' 46·8'' H, 5.00; N, 19.15% B. 3-Carbamoyl-4-(p-methoxybenzyl)-1,2,4-triazole5-thiol Sodium hydroxide (0.4 g; 0.01M) and l-oxamoyl-4(g-methoxybenzyl)thiosemicarbazide (2.82 g; 0.01M) were heated in water (20 ml) on a steam bath for 2 hours. The clear solution was cooled and acidified with dilute hydrochloric acid. The resulting solid was filtered and dried at 50°C in vacuo to give 3-carbamoyl-4-(gmethoxybenzyl)-l,2,4-triazole-5-thiol (2.5 g; 94% yield), m.p. 24O-242°C. A sample was crystallised from a mixture of dimethylformamide and ether; m.p. 243-245°C.

Analysis:Found: C, 49.0; H, 4.51; N, 20.5% C^^H^2N^O2S requires: C, 49.9; H, 4.58; N, 21.2% C. 3-Carbamoyl-l,2,4-triazole-5-thiol 3-Carbamoyl-4-(g-methoxybenzyl)-1,2,4-triazole-5thiol (2.0 g; 0.0076M), trifluoroacetic acid (8 ml) and anisole (2 ml) were heated at 70°C for 3¾ hours. Most of the trifluoroacetic acid was removed by evaporation under reduced pressure and the residue triturated with ether. The solid was filtered, washed with ether and - 65 dried at 50°C in vacuo to give 3-carbamoyl-l,2,4triazole-5-thiol (1.0 g; 91% yield), m.p. 276-278°C.

The infrared spectrum was identical to that of a reference sample prepared as in Preparation 15 Part C.

Claims (33)

1. Compounds of the formula :R COOZ ---(I) wherein R is H or OH; 4 4 Y is NH 2 , OH or OR wherein R is a lower alkanoyl, lower alkoxycarbonyl or benzyl group; X is 0, S, NH or NMe; Z is hydrogen, lower alkyl, benzhydryl, phenyl, indan-5-yl or a group of the formula -CH 2 0C0(lower alkyl) ,-CH(CH 3 )OCOO(lawer alkyl) and R 1 is -CH.OCH.COOH, -CH,0CH.C00(lower alkyl), >7 Z 3 -CHgOCHjCONR R J wherein R and R are each independently hydrogen or lower alkyl, or 2 3 2 3 -CONR R wherein R and R are as defined above; and the pharmaceutically acceptable salts thereof.

2. A compound as claimed in claim 1 wherein Y is NH 2 or OH and Z is hydrogen.

3. A compound as claimed in claim 2 wherein R^ is -ch 2 och 2 conh 2 .

4. 3 5 41 - 67 4. A compound as claimed in claim 2 wherein R 1 is -C0NH 2 , -CH 2 OCH 2 COOH or -CH 2 OCH 2 COO(lower alkyl).

5. A compound as claimed in any one of claims 2 to 4 wherein R is H and Y is OH.

6. A compound as claimed in any one of claims 2 to 4 wherein R is para-OH and Y is NH 2<

7. A compound as claimed in any one of claims 2 to 6 wherein X is NH or S.

8. A compound as claimed in any one of claims 2 to 7 which is in the D- form.

9. A compound as claimed in claim 1 in which R is Η, Y is OH, R 1 is CONH 2 , X is NH and Z is H, and which is in the D- form.

10. A compound as claimed in claim 1 in which R is £-0H, Y is NH 2 , R 1 is -CH 2 OCH 2 COOH, X is NH and Z is H, and which is in the D- form.

11. A compound as claimed in claim 1 in which R is £-0H, Y is NH 2 , R 1 is -CH 2 OCH 2 CONH 2 , X is NH and Z is H, and which is in the D- form.

12. A compound as claimed in claim 1 in which R is £-0H, Y is NH 2 , R 1 is -CONH 2 , X is NH and Z is H, and which is in the D- form.

13. A compound as claimed in claim 1 in which R is Η, Y is OH, R 1 is -CH 2 OCH 2 COOH, X is NH and Z is H, and which is in the D- form.

14. A compound as claimed in claim 1 in which R is -68-. Η, Υ is OH, R 1 is -CH 2 0CH 2 C00Et, X is S and Z is H, and which is in the D- form.

15. A compound as claimed in claim 1 in which R is Η, Y is OH, R 1 is -CH 2 0CH 2 C00H, X is S and Z is H, and which is in the D- form.

16. A compound as claimed in claim 1 in which R is Η, Y is OH, R 1 is -CH 2 0CH 2 C00H, X is 0 and Z is H, and which is in the D- form.

17. A process for preparing a compound of the formula (I) as claimed in claim 1, which comprises reacting a 7-amino-3-heterocyclyl3 thiomethyl- Δ -cephem derivative of the formula:- wherein r\ X and Z are as defined in claim 1, with an acylating agent of the formula:66 COOH CH ---(Ill) wherein R is as defined in claim 1, and Y^ is a protected amino or protected hydroxyl group 4 4 or a group of the formula -OR wherein R - 69 is as defined in claim 1, or with its functional equivalent as an acylating agent, followed by removing the protecting group from Y' L , any free carboxyl groups in (II) being if 5 necessary protected prior to reaction and deprotected after reaction.

18. A process as claimed in claim 17, wherein said functional equivalent as an acylating agent is an acid chloride or bromide, activated ester, 10 or mixed anhydride of the compound of the formula (III).

19. A process as claimed in claim 18, wherein the activated ester has the formula :15 wherein ---(IV) R and Y are as defined in claim 17.

20. A process as claimed in claim 18 wherein said mixed anhydride has the formula :CO-O-COOR wherein R and Y ---(V) are as defined in claim 17 and R Si 70 is a lower alkyl group.

21. A process as claimed in any one of claims 17 to 20 wherein Y 1 is a protected amino or protected hydroxyl group and Z is hydrogen.

22. A process as claimed in claim 17 wherein said functional equivalent as an acylating agent has the formula:.CO CH ---(VI) CO wherein R is as defined in claim 17.

23. A process for preparing a compound of the formula (I) as claimed in claim 1 in which Y is OH or NH 2 and Z is hydrogen, which comprises reacting a cephalosporin derivative of the formula:- wherein R is as defined in claim 1, Y is OH, NH 2 or a protected amino or protected hydroxyl group, and R 2 * * * 6 is a facile leaving group, with a heterocyclic thiol of the formula:- 71 43541 HS Ν Ν Ror a metal or ammonium salt thereof, wherein R 1 and X are as defined in olaim 1, followed by, if necessary, the removal of any protecting group in S.

24. A process as claimed in claim 23, where Y is OH or t-butoxyoarbonylamino.

25. A process as claimed in claim 23 or 24 wherein the compound (VIII) is reacted in the form of its alkali metal salt.

26. A process as claimed in any one of claims 23 to 25 wherein said facile leaving group is a chloro, bromo, iodo or acetoxy group.

27. A process for preparing a compound of the formula (I) as claimed in claim 1 in which Z is as defined in claim 1 other than hydrogen, which comprises esterifying a compound of the formula (I) in which Z is hydrogen, if necessary after conversion to its alkali metal salt form, any free hydroxyl or amino groups represented by Y, and any free carboxyl groups in r\ being if necessary, protected prior to the esterification and de-protected afterwards.

28. A process as claimed in claim 17 substantially as hereinbefore described in any one of Examples 13 to

29. - 72 29. A process as claimed in claim 23 substantially as hereinbefore described in any one of Examples 1 to 12.

30. The process as claimed in claim 27 substantially as hereinbefore described in Example 30.

31. A compound of the formula (I) as claimed in claim 1 which has been prepared by a process as claimed in any one of claims 17 to 30.

32. A pharmaceutical composition comprising a compound of the formula (I) or pharmaceutically acceptable salt thereof as claimed in any one of claims 1 to 16 and 31 together with a pharmaceutically acceptable diluent or carrier.

33. A method of treating non-human animals to cure them of diseases caused by Gram-positive or Gram-negative bacteria, which comprises administering to the animal an antibacterially effective amount of a compound of the formula (I) or pharmaceutically acceptable salt thereof as claimed in any one of claims 1 to 16 and 31 or pharmaceutical composition as claimed in claim 32.