IE45548B1 - Cephalosporins - Google Patents

Abstract

Compounds of the formula I are prepared by reaction of a compound of the formula V with the compound of the formula VI. The meaning of the substituents A and A<1> can be taken from Claim 1. The compounds prepared can be used as active compounds in pharmaceutical preparations having antibacterial action. They are used e.g. for the control of Shig. dysenteriae, Sal. enteritidis, Haemophilus and Neisseria infections.

Description

The present invention relates to novel acids having the structure sometimes hereinafter also written as wherein A is or O_CJ_ II. n ^OR2 wherein R^ is hydrogen or formyl, R is p. » and Y is hydrogen, chlorine, bromine, fluorine, triflurormethyl, amino, nitro, hydroxy, (lower)alkyl of 1-4 carbon atoms or (lower)alkoxy of 1-4 carbon atoms, R is (lower)alkyl of 1-4 carbon atoms; and wherein A1 is methyl or -(CH2)nCOOH and n is one or two; the easily hydrolyzed esters and the non-toxic, pharmaceutically acceptable salts of those acids.

Said easily hydrolyzed esters of the acids of formula I include those having the group of the formula wherein when W represents hydrogen, Z represents (lower)alkanoyl, benzoyl, naphthoyl, furoyl, thenoyl, nitrobenzoyl, methylbenzoyl, halobenzoyl, phenylbenzoyl, N-phthalimido, N-suecinimido, N-saccharino, N-(lower)alkylcarbamoyl, (lower)alkoxy, (lower)alkylthio, phenoxy, carbalkoxy, carbobenzoxy, carbamoyl, benzyloxy, chlorobenzyloxy, carbophenoxy, carbo-tert.-butoxy or (lower)alkylsulfonyl, and when W represents carbalkoxy, Z represents carbalkoxy and, when W represents phenyl, Z represents benzoyl or cyano or wherein W and Z taken together represent 2~oxocycloalkyl' containing 4 to 8 carbon atoms inclusive. By (lower)-alkanoyl, ”(lower)alkylcarbamoyl”, (lower)alkoxy”, (lower)alkylthioand (lower)alkylsulfonyl we mean alkanoyl, alkylcarbamoyl, alkoxy, alkylthio and alkylsulfonyl groups having up to 4 carbon atoms in the alkyl groups.

As set forth below in snore detail the present invention also provides salts of these acids. The stereochemistry of the bicyclic nucleus is that found in Cephalosporin C.

A preferred embodiment of the present invention consists of the acids having the D configuration in the 7side chain and the formula XI - o 45548 N-(CH,) C0,H 2 n 2 COOH II wherein n is one or two and is hydrogen or formyl and R is or and Y is hydrogen, chlorine, bromine, fluorine, trifluoromethyl, amino, nitro, hydroxy, loweralkyl of 1-4 carbon atoms or lower alkoxy of 1-4 carbon atoms and the nontoxic, pharmaceutically acceptable salts of those acids and the easily hydrolyzed esters of those acids including especially the pivaloyloxymethyl, acetoxymethyl, acetonyl, phenacyl and methoxymethyl esters and the silyl esters such as the trimethylsilyl ester,. The invention also includes the benzaldehyde and salicylaldehyde Schiff bases or acetaldehyde or acetone derivatives of such ccapounds when they contain an amino group.

A further preferred embodiment of this invention consists of the compounds of formula II, wherein R is 2-thienyl, 3-thienyl, phenyl, chlorophenyl, bromophenyl, trifluoromethylphenyl, tolyl or methoxyphenyl·.

Particularly preferred embodiments of this invention comprises the acids having the D configuration in the γ-sidechain and the formula S II COOH wherein n is one or two and their nontoxic, pharmaceutically acceptable salts and easily hydrolyzed esters.

Also included in this invention are the compounds (used as either intermediates or metabolic precursors) in which the α-hydroxy group is blocked by substituents such as dichloroacetyl (British Patent Specification Nos. 962,024 and 1,328,340), formyl (U.S. 3,641,021), trimethylsilyl or tetrahydropyranyl (British Patent Specification No. 1,328,340) and the term ester as used herein includes compounds having such blocked a-hydroxy groups.

There is also provided by the present invention a compound having the formula S.

II o COOM wherein n is one or two, R^ is hydrogen or formyl and R is or and ¥ is hydrogen, chlorine, bromine, fluorine, trifluoromethyl, amino, nitro, hydroxy, lower alkyl of 1-4 carbon atoms or lower alkoxy of 1-4 carbon atoms and M is ,45548 CH-S-C-R η is Ο to 4; R is hydrogen, alkyl having 1 to 8 carbon atoms, cyeloalkyl of 3 to 6 carbon atoms, phenyl, C^-C^ phenalkyl, pyridyl, thienyl, or pyrrolyl; R1 is hydrogen, methyl or ethyl; and R® are each hydrogen, alkyl having 4 5 to 6 carbon atoms, phenyl, pyridyl, or thienyl; R and R are each hydrogen or alkyl of 1 to 4 carbon atoms; R6 is alkyl having 1 to 4 carbon atoms, phenyl, phenalkyl having 1 to 4 carbon atoms, pyridyl, thiadiazolyl, amino or Cj-C^ alkylamino; X is NH or oxygen? and each-phenyl group is unsubstituted or substituted with one or two substituents selected from alkyl having 1 to 6 carbon atoms, alkoxy having 1 to 4 carbon atoms, hydroxy, amino, NHR^, N(R^)2, nitro, fluoro, chloro, bromo or carboxy, or a nontoxic, pharmaceutically acceptable salt thereof.

There is also provided by the present invention a compound having the formula n s R-CH-C-NH-CH—CH ,S.

OR ,2 COOM wherein n is one or two, R2 is hydrogen or formyl and R is or and Y is hydrogen, chlorine, bromine, fluorine, trifluoromethyl, amino, nitro, hydroxy, lower alkyl of 1-4 carbon atoms or lower alkoxy of 1-4 carbon atoms and M is selected from CH, 0 I 3 «6 - CH - 0 - C - R , R5 0 I 2 II 7 - CH - X - C - OR7 κ wherein R is a hydrogen atom, a methyl or an ethyl group; 6 X is -0-, -NH-; R is a basic group such as alkyl or aralkyl substituted with substituted or unsubstituted NH2, such as alkyl-NHCH-j, aralkyl-NHCHj, alkyl' aralkyl· η R is an alkyl group such as a methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl or 2-ethyl-hexyl group; a cycloalkyl group such as cyclopropyl, cyclobutyl, cyclopentyl, oyclohexyl or cycloheptyl; an aryl group such as phenyl or naphthyl; an aralkyl group such as benzyl or naphthylmethyl; a heterocyclic group and wherein the alkyl, cycloalkyl, aryl, aralkyl and heterocyclic groups may be substituted with one or more groups selected from amino groups, substituted amino groups such as methylamino, diethylamino or acetamido groups, the halogen » 7 : 45548 atoms auch as fluorine, chlorine or bromine, the nitro group, alkoxy groups such as methoxy, ethoxy, propyloxy, isopropyloxy, butoxy or isobutoxy; or a nontoxic, pharmaceutically acceptable salt thereof.

There having the is also provided by the present invention a compound formula R-CH-C-NH-CH — CH 01? NvS CHI 2 c-ch2-s- nC02K I COOM wherein n is one or two R is hydrogen or formyl and R is or Y. and Y is hydrogen, chlorine, bromine,' fluorine, trifluoromethyl, amino, nitro, hydroxy, lower alkyl of 1-4 carbon atoms or lower alkoxy of 1-4 carbon atoms and M is II · -CH,-N \ ?Z wherein'Y is alkyl of one to six carbon atoms, phenyl, benayl, alkoxy of one to six carbon atoms, or benzyloxy; Z.is alkyl of one .to six carbon atoms, phenylbenzyl, alkoxy e>£ one to six carbon atoms, cyclopentyl, cyclohexyl and phenyl, or Y+Z taken together are a 3-benzoxazolidine ring; or a nontoxic, pharmaceutically acceptable salt thereof. - 8 _ Also Included within the present invention are pharmaceutical compositions comprising a mixture of an antibacterially effective amount of a. compound of the present invention and a semisynthetic penicillin or another cephalosporin or a cephamycin or a β-lactamase inhibitor or an aminoglycoside antibiotic.

In the treatment of bacterial infections in man, the compounds of this invention of formula CX are administered parenterally, in accordance with conventional procedures for antibiotic administration, in an amount of from about 5 to 200 mg./kg./day and preferably about 5 to 20 mg./kg./day in divided dosage, e.g. three to four times a day.

They are administered in dosage units containing, for example, 125, 250 or 500 mg, of active ingredient with suitable physiologically acceptable carriers or excipients.

The dosage units are preferably in the form of liquid preparations such as solutions or suspensions.

Another preferred embodiment of this invention comprises the acids having the formula III wherein A1 is methyl or -(CHg)nCOOH and n is one or two 2 and R is alkyl containing 1-e- carbon atoms, the easily hydrolyzed esters and the non-toxic pharmaceutically acceptable salts of those acids as hereinbefore set forth.

The compounds of formula III of the present invention are syn isomers or else are mixtures of syn and anti isomers containing at least 75% of the syn isomer. Preferably such mixtures of isomers contain at least 20% of the syn isomer and not more tlian 10% of the anti 45348 isomer. Most preferably the compounds of formula III are syn isomers essentially free of the corresponding anti isomer.

Hie preferred embodiments of the present invention 5 are the syn isomers of the compounds of Formula III wherein R2 is methyl or ethyl, n is one or· two, in its acid or pivaloyloxymethyl, acetoxymethyl, methoxymethyl, acetonyl, phenacyl, p-nitrohenzyl, β,β,β-trichloroethyl, 3-phthalidyl or 5-indanyl ester form. • lo Reference to the syn (cis) isomeric form refers to the configuration of the group OR3 with respect to the carboxamido group.

There is also provided by the present invention a compound having the formula (CH2)nC00H wherein RA is alkyl containing 1-4 carbon atoms, n is 5 one or two .and R is selected from CH,. 0 X.-U 12°5 Ϊ 6 - CH - C - R , R5 ’ 0 I 2 II 1 - CH - X - C - 0Rx I -10 45548 ς wherein R is a hydrogen atom, a methyl or an ethyl group; 6 X is -0-, -NH-J R is a basic group such as alkyl or aralkyl substituted with substituted or unsubstituted NHg, such as alkyl-NHCH^, aralkyl-NHCH^, alkyl-NH f X°> aralkyl-NH isopropyl, butyl, isobutyl, pentyl or 2-ethyl-hexyl group; a cycloalkyl group such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl; an aryl group such as phenyl or naphthyl; an aralkyl group such as benzyl' or naphthylmethyl; a heterocyclic group and wherein the alkyl, cycloalkyl, aryl, aralkyl and heterocyclic groups may be substituted with one or more groups selected from amino groups, substituted amino groups such as methylamino, diethylamino or acetamido groups, the halogen atoms such as fluorine, chlorine or bromine, the nitro group, alkoxy groups such as methoxy, ethoxy, propyloxy, isopropyloxy, butoxy or isobutoxy; or a nontoxic, pharmaceutically acceptable salt thereof, said compound being at least 75$ by weight in the form of its syn isomer and preferably in the form of its syn isomer essentially free of the corresponding anti Isomer. - 11 45548 There is also provided by the present invention a compound having the formula (CH2)nC00H 3. wherein IC is alkyl containing 1-t carbon atoms, n is one or two and M is Z wherein Y is alkyl of one to six carbon atoms, phenyl, benzyl, alkoxy of one to six carbon atoms, or benzyloxy} Z is alkyl of one to six carbon atoms, phenylbenzyl, alkoxy of one to 'six carbon atoms, cyclopentyl, cyclohexyl and phenyl, or Y+Z taken together are a 3-benzoxazolidine ring; or a nontoxic, pharmaceutically acceptable salt thereof, said compound being at least 75$ by weight in the form of its syn isomer and preferably in the form of its syn isomer essentially free of the corresponding anti isomer.

There is also provided by the present invention a compound having the formula - 12 43348 wherein R^ is alkyl containing 1-4 carbon atoms and M is -CHXuuk or - CH-S-u-m R’ n is 0 to 4j R is hydrogen, alkyl having 1 to 8 carbon atoms, cyeloalkyl of 3 to 6 carbon atoms, phenyl, C^-Cjj phenalkyl, pyridyl, thienyl, or pyrrolyl; are each hydrogen, alkyl having 1 to 6 carbon atoms, phenyl, pyridyl, or thienyl; R and R are each hydrogen or alkyl of 1 to 4 carbon atoms; is alkyl I I having 1 to 4 carbon atoms, phenyl, phenalkyl having 1 to 4 carbon atoms, pyridyl, thiadiazolyl, amino or C^-Cjj alkylamino; X is NH or oxygen; and each phenyl group is unsubstituted or substituted with one or two substituents selected from alkyl having 1 to 6 carbon atoms, alkoxy having 1 to 4 carbon atoms, hydroxy, amino, NHR1, I^R1^, nitro,, fluoro, chloro, bromo or carboxy, or a nontoxic, pharmaceutically acceptable salt thereof, said compound being at least 75?6 by weight in the form of its syn ieomer and preferably in the form of its syn isomer essentially free of the corresponding anti isomer. - 13 „ 4SS48 There is also provided by the present invention a compound having the formula CH, wherein I?” is alkyl containing 1-4 carbon atoms and R·7 is selected from the group consisting of - CH - 0 - C - R I 6 - CH - C - R > 'R5 0 1 2 II -c - a hydrogen atom, [-; R^ is a basic substituted with substituted or unsubstituted NHg, such as alkyl-NHCH-j, aralkyl-NHCHj, alkyl-NH aralkyl -NH , -CH-^Q^ , -CBgNHj, or-CH-CHg NHg NHg R1 is an alkyl group such as a methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl or 2-ethyl-hexyl group; a cycloalkyl group such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl; an·aryl group such as 4554.8 phenyl or naphthyl; an aralkyl group such as benzyl or naphthylmethylj a heterocyclic group and wherein the alkyl, cyeloalkyl, aryl, aralkyl and heterocyclic groups may be substituted with one or more groups selected from amino groups, substituted amino groups such as methylamino, diethylamino or acetamido ' groups; the halogen atoms such as fluorine, chlorine or bromine, the nitro group, alkoxy groups such as methoxy, ethoxy, propyloxy, isopropyloxy, butoxy or isobutoxy; or a nontoxic, pharmaceutically acceptable salt thereof, said compound being at least 75# by weight in the form of . its syn isomer and preferably in the form of its syn isomer essentially free of the corresponding anti isomer.

There is also provided by the present Inven15 tion a compound having the formula 0 CH.

C-OM II o wherein R1 is alkyl containing 1-4 carbon atoms and M is II .C-Y Z _ 15 ^SS48 ι wherein Y is alkyl of one to six carbon atoms, phenyl, benzyl, alkoxy of one to six carbon atoms, or benzyloxy; Z is alkyl of one to six carbon atoms, phenylbenzyl, alkoxy of one to six carbon atoms, cyclopentyl, cyclo5 hexyl and phenyl, or Y+Z taken together are a 5-benzoxazolidine ring; or a nontoxic, pharmaceutically acceptable salt thereof, said compound being at least 75% by weight ih the form of its syn isomer and preferably in the form of its syn isomer essentially free of the corresponding anti isomer.

In the treatment of bacterial infections in man, the compounds of this Invention are administered parenterally in an amount of from about 10 to 90 mg./kg./day and preferably about 14 to 50 mg./kg./day in divided dosage, e.g. two to four times a day. They are administered in dosage units containing, for example, I25, 250 or 500 mg, of active ingredient with suitable physiologically acceptable carriers or excepients.

The dosage units are in the form of liquid preparations such as solutions or suspensions and preferably are aqueous solutions of a sodium or potassium salt which are injected intravenously or intramuscularly or by continuous or Intermittent infusion in concentrations of .about 125-500 mgm./ml., and preferably, 250 mgm./ml. as is customary in therapy with cephalosporin antibiotics.

It was an unexpected finding that the leading compound of the present invention (BB-S510; see below) having a 2-methyl substituent on the triazolopyridazinone showed in vitro antibacterial potency considerably superior to that of the corresponding compound lacking such methyl group (BB-S515; see below). - 15 45548 The present invention also provides the process for the production of the antibacterial agents of formula I which comprises reacting a compound of the formula where A1 is as hereinbefore defined ον a salt or easily hydrolyzed ester or Schiff base as with benzaldehyde or salicylaldehyde thereof (including, but not limited to, those of U.S. 3,284,451 and U.K. 1,229,453 and any of the uilyl eeters described in U.S. patent 3,249,622 for use with 7-aminopeniclllanic acid and used in Great Britain 1,073,530 and particularly the pivaloyloxymethyl, acetoxymethyl, methoxymethyl, acetonyl, phenacyl, p-nitrobenzyl, β,β,β-trichloroethyl, 3”phthalldyl and 5-indanyl esters) * with an organic monocarboxylic acid chloride or a functional equivalent thereof as an acylating agent corresponding to the acid AOH. _ 17 -t <$S48 Such functional equivalents include the corresponding acid anhydrides, including mixed I anhydrides and particularly the mixed anhydrides prepared from stronger acids such as the lower aliphatic monoesters of carbonic acid, or alkyl and aryl sulfonic acids and of more hindered acids Buch as diphenylacetic acid. In addition, an acid azide or an active ester or thioester (e.g, with p-nitrophenyl, 2,4-dinitrophenol, thiophenol, thioacetic acid) may be used or the free acid itself may be coupled with compound iv after first reacting said free acid with N,N’-dimethylchloroforraiminium chloride [cf, Great Britain 1,008,170 and Novak and Weichet, Experientia XXI. 6, 360 (1965)] or by the use of enzymes or of an N,N*-carbonyldiimidazole or an Ν,Ν'-carbonylditriazole [cf.

South African patent specification 63/2684] or a carbodiimide reagent [especially Ν,Ν'-dicyclohexylcarbodiiraide, Ν,Ν'-diisopropylcarbodiimide or N-cyclohexyl-N*-(2-morpholinoethyl)carbodiimide; cf, Sheehan and Hess. J, Amer, Chem, Soc.. 77. 1967 (1955)], or of alkylylamine reagent [cf. R. Buijle and H.G.

Viehe, Angew, Chem, International Edition 3. 582, - 18 45548 (1964)] or of an isoxasolium salt reagent [cf. R, B. Woodward, R. A. Olofson and H. Mayer, J. Amer. Chem, Soc., 83. 1010 (1961)], or·of a ketenimine reagent [cf.'c. L.. Stevens and Μ. E. Munk, J... Amer Chem', Soc., 80, 4065 (1958)] or of hexachlorocyclotrlphosphatrlazlne or hexabromocyclotrlphosphatrlazine (U.S, 3,651,050) or of dlphenylphosphoryl azide [DPPA; J. Amer. Chem, Soc., 94, 6203-6205 (1972)] or of diethylphosphoryl cyanide [DEFC; Tetrahedron Letters No. 18, pp. 1595-1598 (1973)] or of diphenyl phosphite [Tetrahedron Letters No. 49, pp. 5047-5050 (1972)]. Another equivalent of the acid chloride ls a corresponding azolide, i.e., an amide of the corresponding acid whose amide nitrogen ls a member of a quasiaromatic five membered ring containing at least two nitrogen atoms, i.e., imidazole, pyrazole, the triazoles, benzimidazole, benzotrlazole and their substituted derivatives. As an example of the general method for the preparation of an azolide, N,N'-carbonyldllmldazole is reacted with a carboxylic acid in '20 equimolar proportions at room temperature In tetrahydrofuran, chloroform, dimethylformamide or a elmllar Inert solvent to form the carboxylic acid Imldazollde In practically quantitative yield with liberation of carbon dioxide and one mole of Imidazole. Dicarboxylic acldB yield dlmldazolide. The by-product, imidazole, precipitates and may be separated and the imldazollde Isolated, but this Is not essential, The methods for carrying out theBe reactions to produce a cephalosporin find the methods used to isolate the cephalosporin so produced are well known in the art. Η W{ Λ Mention was made above of the use of enzymes to couple the free acid with compound IV. Included in the scope of such processes are the use of an ester, e.g. the methyl ester, of that free acid with enzymes provided by varioue microorganisms, e.g. those described by T. Takahashi et al,, j; Amer. Chem.'Soc,, 94(11). 4035-4.037 (1972) and by T. Nara et al., J. Antibiotics (japan) g4(5). 321-323 (1971) and in U.S. 3,682,777. lo For the coupling of the organic carboxylic acid as described above with compound IV (or a salt or preferably an easily hydrolyzed ester of Schiff base, as with benzaldehyde, thereof) it is also convenient and efficient to utilize as the coupling egent phosphonitrilic chloride trimer (J. Org. Chem., 33(7). 2979-81, 1968) or N-ethoxy1,2-dihydroquinoline (EEDQ) as described in J. Amer. Chem. Soc., 90, 823-824 and 1652-1653 (1968) and U.S. Patent 3,455,929. The reaction is preferably carried out at 3035®C. in benzene, ethanol or tetrahydrofuran using about ..20 equimolar quantities of all three reagents followed by conventional isolation and removal by conventional methods of any blocking groups present.

One process of the present invention stated more specifically is the process'for the preparation of a product having the D-configuration in the sideohain and the formula COOH 0 - 2045548 wherein n is one or two or a salt thereof which comprises the consecutive steps of a, preparing an acylating derivative of D-mandelic wherein the hydroxyl blocking group R represents d-ichloroacetyl, silyl and preferably trimethylsilyl, tetrahydropyranyl or, preferably, formyl in an anhydrous organic solvent such as benzene, ethanol or preferably tetrahydrofuran, at room temperature or belov; and preferably at about 5 Cj b. mixing therewith, preferably slowly, a solution at about the same temperature in a solvent, preferably aqueous tetrahydrofuran, containing substantially the same number of moles of a tertiary amine, preferably a tertiary alkylamine such as triethylamine and substantially the same number of moles of 7-amino-3-(2-carboxymethyl or 2-carboxyethyl-2,3-dihydro-s-triasolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid or a salt or an easily hydrolyzed Schiff base, as with benzaldehyde, thereof to produce having the formula the intermediate acid ^Ν'ύγΝ(αΗ2)ηε02Η 0 : thereof wherein R has wherein n is one or two or a s< the meaning set out above; and c. removing said hydroxyl blocking group R by conventional chemical methods to produce said product or salt thereof. - 21 45548 In preferred embodiments of the present invention R represents formyl which is removed in step C by treatment with aqueous alkali such as aqueous sodium bicarbonate or R represents dechloroacetyl which is removed in step C by alkaline hydrolysis, preferably at about pH 9-10, or R represents tr'imethylsilyl which is removed in step C by exposure to aqueous acid.

Other compounds of formula II are made in like manner.

An additional process of the present invention comprises the preparation of the compounds of the present invention by the displacement of the 3-acetoxy group from a cephalosporanic acid of the formula Where A is as defined hereinbefore above, prepared by substituting 7-amino cephalosporanic acid for the 3-thiolated-7-amino cephalosporanic acids in the acylation procedures described herein and elsewhere reported with the appropriate thiol HSR3 having the·formula H-S—bs ,N—S )nCOOH or H-S—As JIs. /N-CH, w Sr where n is one or two and then removing the protecting group if any is present as on the carboxyl group. _ 22 _ The displacement of such a 3-acetoxy group with such a thiol may be accomplished in solution as in water or aqueous acetone at a temperature of at least room temperature and preferably within the range of about 50* to 100*C, in the presence of a mild base such as sodium bicarbonate, e.g. preferably near neutrality such as at about pH 6, An excess of the thiol is preferably employed. The reaction product is isolated by careful acidification of the reaction mixture followed by extraction with a water-inmiscible organic solvent. As noted above, the preparation of many other 7-acylamidocephalosporanic acids is described in the patent and scientific literature, e.g. in U.S. Class 260-243C.

The salts of the compounds of this invention include the nontoxic carboxylic acid salts thereof, including nontoxic metallic salts such as sodium, potassium, calcium and aluminum, the ammonium salt and substituted ammonium salts, e.g. salts of such nontoxic amines as trialkylamines including triethylamine, procaine, dibenzylamlne, N-benzyl-beta-phenethylamine, 1-ephenamine, Ν, N’-d ibenzylethylenedlamine, dehydroabietylamine, Ν,Ν'-bis-dehydroabietylethylenedIamine, and other amines which have been ueed to form salts with benzylpenicillin, L-Iysine, arginine and histidine.

The preferred esters of the cephalosporins of the present invention are the pivaioyloxymethyl, acetoxymethyl, methoxymethyl, acetpnyl and phenacyl eeters. All are useful intermediates in the production of the cephalosporin having a free carboxyl group. _ 23 45548 As indicated above, these five esters of 7-aminocephalosporanic acid are each prepared by known methods. One excellent procedure is that of U.S. patent 3,284,451 in which sodium cephalothin ls esterified by reaction with the corresponding active chloro or bromo compound (e.g. phenacyl bromide, chloroacetone, chloromethyl ether, pivaloyloxymethyl chloride (also called chloromethyl pivalate], acetoxymethyl chloride) and then the thienyl1° acetic acid sidechaln is removed enzymatically as in the same patent or .chemically as in U.S. patent 3,575,970 and in Journal of Antibiotics, XXXV (11), 767-773(1971). In another good method the triethylamine salt of 7-aminocephalo15 sporanic acid is reacted directly with the active I halogen compound, as in United Kingdom 1,229,453. .

These esters of 7-aminocephalosporanic acid are then reacted with the nucleophile HSr3 in the same manner as is illustrated herein for 7-aminocephalo20 sporanic acid itself. The 3“thiolated ester of 7-aminocephalosporanic acid is then coupled with the organic carboxylic acid as before.

The ester of the cephalosporin so obtained , is, if not used per se,. converted to its free acid and, if desired, any salt by removal of the esterifying group, as by aqueous or enzymatic hydrolysis (aa with human or animal serum) or acidic or alkaline hydrolysis or by treatment with sodium thiophenoxide as taught in U.S. 3,284,451 and, in the penicillin series, by Sheehan et al., J, Org, Chem, 29(7). 2006-2008 (1964).

The cephalosporins obtained by the above method, when they contain an amino group, can be converted to the corresponding benzaldehyde or salicylaldehyde Schiff base, or, with acetaldehyde or acetone to the corresponding derivatives. - 24 45548 In another alternative synthesis, the ?thlolated 7-aminocephalosporanic acid is prepared as described herein and then acylated at the 7 anilno group and finally estehified, as by reaction of the appropriate alcohol with the acid chloride prepared, for example, by reaction of the final cephalosporin with thionyl chloride or by other essentially acidic esterification procedures.

There is further provided by the present invention a 10 pharmaceutical composition comprising an antibacterially effective amount of a compound having the formula R-CH-C-NH-CH—CH OR Ns ΪΗ2 c-ch2-s- I COOM ^x/-tCH2>nCO2H 0 wherein n is one or two, R is hydrogen or formyl and R is or and ϊ is hydrogen, chlorine, bromine, fluorine, trifluoromethyl, amino, nitro, hydroxy, lower alkyl of 1-4 carbon atoms or lower alkoxy of 1-4 carbon atoms and M is hydrogen, pivaloyloxymethyl, acetoxymethyl, methoxymethyl, acetonyl, phenacyl, p-nitrogenzyl, Β,β,β-trichloroethyl, 3-phthalidyl or 5-indanyl and preferably is hydrogen or a nontoxio, pharmaceutically acceptable salt thereof. „ 25 „ 455 48 There is further provided by the present invention a method of treating bacterial infections comprising administering by injection to an infected warm-blooded non-human animal, an effective but nontoxic dose, for example of 250-1000 mgm of a compound having the formula R-CH-C-NH-CH — CH I i 1 OR I COOM III 2 C-CH2-S- .N-(CH2)nCO2H wherein n is one or two, R is hydrogen or formyl and R is or and ϊ is hydrogen, chlorine, bromine, fluorine, trifluoro10 methyl, amino, nitro, hydroxy, lower alkyl of 1-4 carbon atoms or lower alkoxy of 1-4 carbon atoms and M is hydrogen, pivaioyloxymethyl, acetoxymethyl, methoxymethyl, acetonyl, phenacyl, p-nitrobenzyl, β,β,β-trichloroethyl, 3-phthalidyl or 5-indanyl or a nontoxic, pharmaceutically acceptable salt thereof.

There is also provided by the present invention a method for combatting Shig. dysenteriae infections which comprises administering to a warm-blooded non-human mammal infected with an Shig. dysenteriae infection an amount effective for treating said Shig. dysenteriae infection of a composition comprising a oompound having the formula .N~(CH2)nCO2H COOM Rwherein n is one or two, R*^ is hydrogen or formyl and R is or and Y is hydrogen, chlorine, bromine, fluorine, trifluoro5 methyl, amino, nitro, hydroxy, lower alkyl of 1-4 carbon atoms or lower alkoxy of 1-4 carbon atoms and M is hydrogen, pivaloyloxymethyl, acetoxymethyl, methoxymethyl, acetonyl, phenacyl, p-nitrobensyl, 3,6,0-trichloroethyl, 3-phthalidyl or 5-indanyl and preferably is hydrogen or a nontoxic, pharmaceutically acceptable salt thereof.

There is also provided by the present invention a method for combatting Sal, enteritidis infections which comprises administering to a warm-blooded non-human mammal infected with a Sal, enteritidis infection an amount effective for treating said Sal, enteritidis infection of a composition comprising a compound having the formula II o o COOM - 27 _ 4SS48 wherein n is one, or two, R·1, is hydrogen or formyl and R is P “ uY ' and Y is hydrogen, chlorine, bromine, fluorine,, trifluoromethyl, amino, nitro, hydroxy, lower alkyl of 1-4 carbon atoms or lower alkoxy of 1-4 carbon atoms and M is hydrogen, pivaloyloxymethyl, acetoxymethyl, methoxymethyl, acetonyl, phenadyl,. p-nitrobenzyl, β,β,β-trichloroethyl, 3-phthalidyl or 5-indanyl and preferably is hydrogen or a nontoxic pharmaceutically acceptable salt thereof. θ There is further provided by the present invention a pharmaceutical composition comprising an antibacterially effective amount of a compound having the formula wherein R3and A1 are as hereinbefore defined and Ra is hydrogen, pivaloyloxymethyl, • acetoxymethyl, methoxymethyl, acetonyl, phenacyl, pnitrobenzyl, β,β,β-triehloroothyl, 3-phthalidyl or 5-indanyl and preferably is hydrogen or a nontoxic, pharmaceutically acceptable salt thereof, said compound being at least - 28 45548 75$ by weight in the form of its syn isomer and preferably in the form of its syn isomer essentially free of the corresponding anti isomer, and a pharmaceutically acceptable carrier therefor, There is further provided by the present invention a pharmaceutical composition comprising an antibacterially effective amount of the syn isomer of a compound having the formula wherein A1 is as hereinbefore defined or a nontoxic, pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier therefore.

There is further provided by the present invention a method of treating bacterial infections · comprising administering by injection to an Infected warm-blooded non-human animal, an effective but nontoxic dose, for example of 250-1000 mgm. of a compound having the formula O-, I •OR - A1 . 29 _ 48 wherein R3 and A1 are as hereinbefore defined and R3 is hydrogen, pivaloyloxymethyl, acetoxymethyl, methoxymethyl, acetonyl, phenacyl, pnitrobenayl, β,β,β-trichloroethyl, 3-phthalidyl or -indanyl or a nontoxic, pharmaceutically acceptable salt thereof, said compound being at least 75# by weight in the form of its syn isomer and preferably in the form of its syn isomer essentially free of the corresponding anti isomer.

There is further provided by the present invention a method of treating bacterial infections comprising administering by injection to an infected warm-blooded non-human animal, an effective but nontoxic dose, for example of 250-1000 mgm. of the syn isomer of a compound having the formula .

NH-CH—CH IjlHg G-II .C-CHp-S (Z f C-OH . A1 wherein Ax is as hereinbefore defined or a nontoxic, pharmaceutically acceptable salt thereof, and wherein n is preferably 1. - 3tJ There is also provided by the present invention a method for combatting Haemophilus infections which comprises administering to a warm-blooded non-human mammal infected with an Haemophilus infection an amount effective for treating said Haemophilus infection of a composition comprising a compound having the formula Η· 2 wherein A and R are as hereinbefore defined and j_s hydrogen, pivaloyloxymethyl, acetoxymethyl, methoxymethyl, acetonyl, phenacyl, pnitrobenzyl, β,β,β-trichloroethyl, 3-phthalidyl or 5-indanyl and preferably is hydrogen or a nontoxic, pharmaceutically acceptable salt thereof, said compound being at least 75# by weight in the form of its syn isomer and preferably in the form of its syn isomer essentially free of the corresponding anti isomer, and a pharmaceutically acceptable carrier therefor.

There is also provided by the present invention a method for combatting Neisseria infections which comprises administering to a warm-blooded non-human mammal infected with a Neisseria infection an amount effective for treating said Neisseria infection of a composition comprising a compound having the formula _ 31 4 5 5/4 8 1 wherein R and A are as hereinbefore defined and R is hydrogen, pivaloyloxymethyl, acetoxymethyl, methoxymethyl, acetonyl, phenacyl, p5 nitrobenzyl, β,β,β-trichloroethyl, 3-phthalidyl or 5-indanyl and preferably is hydrogen or a nontoxic, pharmaceutically acceptable salt thereof, said compound being at least 75% by weight in the form of its syn isomer and preferably in the form of its syn isomer essentially 10 free of the corresponding anti isomer, and a pharmaceutically acceptable carrier therefor. - 32 45548 > ί'ΤΑΗΤΙΝΟ MATERIALS 2-Furoylcyanide To a suspension of 26.1 g. (0,4 mole) of ground potassium cyanide in 300 ml. of acetonitrile at 5° C. was added 26.1 g. (0.2 mole) of a-furoyl chloride while keeping the temperature below 8° C. The mixture was stirred in the cold for 15 minutes then heated at reflux for 30 minutes. The reaction was cooled, filtered and the acetonitrile was removed at 15 mm. (steam-bath) leaving 24.5 S· of a dark oil which was used without further purification.

An infrared spectrum showed a nitrile band at 2265 cm·1·, 2-Furaneglyoxylic Acid The 24.5 g, of crude 2-furoylcyanide was mixed with l60 ml. concentrated hydrochloric acid at 25° c. with intermittent stirring. The reaction was stored for 24 hours at 25° C. and diluted with 80 ml. of water. The reaction was stirred for 5 minutes and filtered. The fil- . trate was saturated with sodium chloride and extracted with 5 x 120 ml. of 1:1 ether-ethyl acetate solution. The extracts were combined, dried over anhydrous magnesium sulfate and evaporated at 30° C. (15 mm.) to give a brownish-orange solid. The solid was dissolved in methanol, treated with charcoal and evaporated under reduced pressure (15 mm.) to dryness to yield 17 g. of the acid.

. The product was recrystallized from toluene to give 11.5 B· (m.p. 760 0.). The ir and nmr spectra were consistent for the structure, g-Methoxylmino-2-furylacetlc Acid To a solution of 4,5 g. (0.032 mole) of 2-furane30 glyoxylic acid in 40 ml. of 50% alcohol and 3.1 g. (0.037 mole) of methoxyamine hydrochloride in 6 ml. water at 20° C. was added dilute sodium hydroxide solution to pH 4-5.

The solution was stirred at pH 4-5 at 25° C. for 24 hours. The alcohol was removed under reduced pressure (15 mm.) and the solution was adjusted to pH 7-8 with 50% sodium hydroxide solution. The reaction was extracted with 3 x 50 ml. of ether and the aqueous layer was adjusted to pH 1.9 using concentrated hydrochloric acid. The mixture was extracted with 5 x 50 ml. of ethyl acetate. The organic fractions were combined, washed with brine, dried over anhydrous magnesium sulfate and evaporated under reduced pressure (15 mm.) to an oil which-was cooled for one hour in an ice· bath. ,The product was slurried with Skellysolve B and collected to yield 3·1 g. of yellow crystals, m.p. 780 c. An analytical sample was recrystallized from toluene, dried for 16 hours in vacuo over FgOj. at 25° C.

The ir and nmr spectra were consistent for the structure.

Anal. Calc'd for C^WO: C, 49.65; H, 4.17; N, 8.28. Found: C, 49.30} H, 4.21; N, 8.37. 2-Ethoxylmlnofurylacetic Acid . The 7.85 g. (0.056 mole) of furyl-2-glyoxylie acid was dissolved in 100 ml. of water and adjusted to pH 7 with 50% sodium hydroxide. The 6.83 g. (0.070 mole) of ethoxyamine hydrochloride in 10 ml. of water was added, while keeping the pH at 4-5. The reaction was diluted with 25 ml. of alcohol,· s-feirred 3 hours at room temperature and then filtered. The alcohol was removed at 35° C. (15 mm.) and the aqueous portion was adjusted with dilute sodium hydroxide solution to pH 7-8 an4 then - 34 45548 Λ was washed with ether and the washes were discarded. The aqueous fraction was adjusted with 6n hydrochloric acid to pH 1.5 and extracted into 3 x 80 ml. of ethyl acetate.

The acetate fractions were combined, washed with brine 5 and reduced in volume at 35° C. (15 mm.) to an oil. The oil was cooled in an ice bath, triturated with Shellysolve B, collected and dried over PgO^ in vacuo at 25° C.

Yield: 4.8 g,, m.p. 83-850 C. The ir and nmr were consistent for the structure.

Anal. Calc'd for CgHgNO^: C, 52.46; H, 4.95; N, 7.65. Found: C, 52.22; H, 4.94; N, ?.6o.

Sodium a-Ethoxyimino-a-(2-furyl)acetate To 50 ml. of methanol was added 250 mg. (0.0109 mole) of metallic sodium and stirred until all the sodium 15 had dissolved. This sodium methoxide solution was treated with 2.0 g. (O.OI09 mole) of a-ethoxyimino-a-(2-furyl)acetic acid dissolved in 10 ml. of methanol and stirred at room temperature for one hour. The methanol was removed at 40° C. (15 mm.) and the product was dried in vacuo over PgOg at 25° C. to yield 2.22 g. white solid, m.p. decomp. . >240° C, The ir and nmr were consistent for the structure.

Skellysolve B is a petroleum ether fraction of b.p. 6o°-68° C. consisting essentially of n-hexane.

Mil □L.

. VcH, Q-nNa CH^OH (COCI), Q-X, J XCH, g-Furoylcyanide 1.

To a suspension of 78.3 g. of powdered potas- : sium cyanide in 900 mi. acetonitrile at 5° 0. was added 59.25 ml. (68;5 g.) of α-furoyl chloride with vigorous stirring while keeping the temperature at 4-8° C. The .mixture was stirred at 4-8° C. for 15 minutes and then heated at reflux for 30 minutes. The mixture was cooled to 23-25° C,, filtered, washed with 50 ml. of acetonitrile which was added to the filtrate, and the acetonitrile was * removed at 60° C. (15 mm.) leaving 51 g. of as a dark - 36 45548 oil. An IR spectrum showed a nitrile band at 2265 cm-1 and an NMR spectrum showed a ratio of approximately 70/30 of product ^/furoic acid. The crude product ^1 was used without further purification (49# yield of product).

Furyl-2-glyoxyllc Acid £ The 51 g. °f crude 2-furoyl cyanide was mixed with 500 ml. concentrated hydrochloric acid at 25° C. The reaction was stirred for 24 hours at 25° C. and then diluted with 240 ml. of water. The mixture was stirred for 5 minutes and filtered. The black filtrate was saturated with sodium chloride and extracted with 6 x 500 ml. of 1:1 ether-ethyl acetate solution, (Note; Initially the extractions were difficult due to the Inability to see the separation of two black phases. As additional ether-ethyl acetate extractions were run the . task was simplified.) The extracts were combined and evaporated to dryness at 60° C. (15 mm.). The resultant solid was dissolved in 600 ml. ether, (Note: Use of alcohol should be avoided at this point as esters may form), treated with 10 g. of charcoal (Darko-KB), filtered after stirring for 0.5 hour and evaporated to dryness at 50° C. (15 mm.) to yield 46.6 g, of^ as a light tan colored acid. This product 2 was SV found to contain a ratio of approximately 56/44 of product^2/furoic acid. This represented a 63$ yield of product 2.

Purification was accomplished by dissolving the above crude product ,2 in HgO (50 mg./ml,), titrating to pH 2.8 with HC1 and extracting with 2 x 200 ml. of 4SS48 S ethyl acetate. Evaporation of the ethyl acetate extracts gave 35$ furoic acid and 15$ product^. The pH 2-.8 aqueous phase was adjusted to pH 0.8 (HC1) and extracted with 2 x 200 ml. ethyl acetate. The organic extracts were combined and washed with 50 ml, HgO. The organic phase was evaporated at 50° C. (15 mm.) yielding a solid with a ratio of approximately 86/14 of product ,2/furoic acid. This solid was then recrystallized by dissolving the product J2 in toluene at 50 mg./ml. at 80° C.j decanting, and leaving to crystallize at room temperature for 18 hours, yielding 13.3 g. of pure acid J2 by NMR. This represented a 51$ yield in the purification and recrystallization step and an overall yield from the 2-furoyl chloride to the pure furyl-2-glyoxylic acid ^2 of 16#. , Syn-a-methoxyiminofurylac etlc Acid £ A solution of 4.5 g. of furyl-2-glyoxylic acid £ in 40 ml. of 50$ ethanol was titrated to pH 6 with IN sodium hydroxide and then 3.1 g. of methoxyamine·Η01 in 6 ml. of HgO at 20° G. was added, The solution was titrated to a constant pH 4.9 and stirred at pH 4.9 for 24 hours at 20-23“ C· The ethanol was then removed at 50“ C. (15 mm.) and the residual aqueous solution was titrated to pH 8 with 50$ sodium hydroxide and washed with 3 x 50 ml. ether (pH adjusted to 8 after each wash). The aqueous layer was titrated to pH 1.9 with •concentrated HC1 and extracted with 5 x 50 ml. ethyl acetate with the pH readjusted to 1.9 after each extraction, The ethyl acetate extracts were combined and 45S48 evaporated to a solid 3 at 50° C. (15 mm.).. This solid was then slurried with 75 ml. of Skellysolve B. The suspension was filtered and the solids were redissolved in 16 ml. of toluene at 80° C. The hot solution was decanted and left to crystallize at 20-23° C. for 18 hours to yield 1.17 g.% (22# yield of product). The NMR was clean and consistent for the structure 3 with a trace of anti isomer present.

Sodium Syn-a-methoxylmlnofurylacetate To 40 ml, of methanol was added 0,l6 g. of sodium. The mixture was stirred until all of the sodium dissolved and then decanted. The resulting sodium methoxide solution was cooled to 3° C. and 1.12 g. of syn-a-methoxyimlnofurylacetic acid^ in 7.8 ml. of methanol was added. The solution was stirred for 10 minutes at' room temperature. The solvent was evaporated at 1)0° C. (15 mm,), The residue was dried by azeotropic distillation with 3 x 20 ml. of benzene at 40° C. (15 mm.). The product^ was dried for 18 hours at 23° C. under high vacuum (0.7 mm.) over PgO^ yielding 1.25 6· (99# yield of product). The NMR showed this product^ to be clean and consistent for the structure with 0.15 mole methanol and a trace of anti isomer.

To 0.6j g. of sodium syn-a-methoxyimlnofurylacetate 4, suspended in 25 ml, of benzene was added four drops of dry dimethylformamide and Ο.31 ml, (1,1 eq.) of oxalyl chloride. This mixture was stirred for 40 minutes at 20-23° C. The benzene was removed at 35° C. (15 mm.) to provide the acid chloride 5 as the gummy - 3S . .-48548 λ 6-0ΜοΓθ-2)3-άΐηγάΓο-2-6ΐηοχγΰα^οπγ1πΐ5ίΗ7Ί-5·-ΪΓΐ&ζοΐοr4,3-b]pyrldazln-3-one ' To a solution of 6-chloro-2,3-dihydro-striazolo[4,3-b]pyridazin-3-one [P. Francavilla and F.

Lauria, J. Het. Chem.,.8, 415 (1971)] (1, 1.00 g., 5.9 m.mole) in dry DMF (30 ml.) was added sodium hydride (50% in paraffin, 0.3 g., 6.3 m.mole) under stirring with formation of yellow crystals. To the mixture was added ethyl ohloroacetate (1.4 ml., 13 m.mole) and the mixture was heated at 90° C. for 8 hours with stirring. After booling, the reaction mixture was poured into water (50 ml.) and extracted with toluene (5 x 40 ml.). The organic extracts were combined, dried over anhydrous sodium sulfate and evaporated at reduced pressure. The residue Was crystallized with benzene-n-hexane to give yellow needles (I.16 g., 77%), m.p. 114-115“ C. (lit. 110? C.). ir: v^Br 1735, 1710 cm-1, uv: λ^°Η 231 nm (e, 260.00) nmr: 6^^3 7-58 (IH, d, J=10 Hz, pyridazine-H), 6.98 (IH, d, J=10 Hz, pyridazine-H), 4.8θ (2H, s, -CHgCO), 4.27 (2H, q, J=7.5 Hz, CHgCHj), 1.29 (5H, t, J=7-5 Hz, CHgCHj).

Anal. Calc'd. for CgH^OjCl: C, 42.12; H, 3.55} H, 21.83; Cl, 13.81, Found: C, 41.54, 41.46; H, 3.22, 3.49; H, 21.51, 21.55; Cl, 15.88, 13.99. 2-Carboxymethyl“2,3-dlhydro-6-mercapto-s-triazolo[4,3-b1pyrldazin-3-one To a solution of 6-chloro-2,3-dihydro-2-ethoxycarbonylmethyl-s-triazolo[4,3-b]pyridazin-3-one (30 g., 0.12 mole) in ethanol (900 ml.) was added NaSH-2H20 (70$ pure, 45.9 g., Ο.36 mole) and the mixture was refluxed for 0.5 hour. The reaction mixture was evaporated at reduced pressure. The residue was dissolved in water (200 ml.) and concentrated KOI was added to the solution to adjust to pH 2. The precipitate of 2-carboxymethyl-2,3dihydro-6-mercapto~s-triazolo[4,3-b]pyridazin-3-one was collected by filtration and washed with water. Yield 18.3 g. (69#). ir: 2900, 2450, 1750, 1660 cm1, τηο,χ uv: xl$NaHCO^aq. 2gQ nm (ε}ι95οο), 3I3 nm (e, 7000) ΙΠ 9.X nmr: ^30-¾ 7.88 (ih, d, J=10 Hz, pyridazine-H), 7.45 (1H, d, J=10 Hz, pyridazine-H), 4.72 (2H, s, CHgCO).

Anal. Calc'd. for C^gN^S: C, 37,17; H, 2.675 N, 24.77; S, 14.17. Found: 0, 37-35, 37.23; H, 2.26, 2.285 N, 23.58, 23.695 S, 14.32. 7-Amlno-3-(2-carboxymethyl-2,3-dihydro-s-trlazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic Acid To a suspension of 7-aminocephalosporanic acid (8.79 g., 32.2 m.mole) in 0.-1 M phosphate buffer (pH 7, -41 » \ . • 149 ml·) were added NaHCOj (8,14 g., 97.0 m.mole) and the thiol 2-carboxymethyl-2,3-dihydro-6-mercapto-s-triazolo[4j3-b]pyridazin-3~on.e (7.30 g., 32,2 m.mole) with stirring. , The mixture was heated at 80° C. for 0.5 hour under Ng stream. The mixture was treated with active carbon and adjusted to pH 3 with concentrated HC1. The resulting precipitate was collected by filtration and washed with water to give 7.59 g (54$) of 7-amino-3-(2-carboxymethyl-2,3-dihydro-s-tri10 azolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4carboxylic acid. - 42 _ •45548 ir: vmx 18003 172°3 l6o°3 154θ» 1470 cm'’:L« UV! xr£xfer (PH 252 11111 (εΛ95ΟΟ), 298 nm (e, 8400). nror: δρ.^Κ2Οθ3 7.56 (IH, d, J=9 Hz, pyridazine-H), 7.05 (IH, d, J=9 Hz, pyridazine-H), 5.45 (IH, d, J=5 Hz, 6-H), 5.05 (IH, d, 5 Hz, 7-H), 4.43 (IH, d, J=l4 Hz, 3-CHg), 4.04 (IH, d, J=14 Hz, 3-CHg), 3-88 (IH, d, J=l8 Hz, 2-H), 3.45 (IH, d, J=l8 Hz, 2-H). 6-Chloro-2-(2-cyanoethyl)-g,5-dihydro-s-triazolo[4,5-b’3pyridasin-3-on.

To a solution of 6-chloro-2,3-dihydro-s-tri~ azolo[4,3-b]pyridazin-3-on [P. Francabilla and F. Lauria, J. Het. Chem. 8, 415 (1971)] (17 g., 0.1 mole) in dry DMF (5OO ml.) was added.potassium tert.-butoxide (0.5 g., 4.5 m.moles) with stirring. Acrylonitrile (6.6 g,, 0.12 mole) in dry DMF (10 ml.) was added to the mixture. The mixture was stirred at 100-110° C. for 24 hours, then poured into water (700 ml.) and extracted with ethyl acetate (5 x 400 ml.). The organic extracts were combined, dried over NagS0^ and evaporated. The residue was crystal20 lized from ethyl acetate to give light yellow needles of 6-chloro-2-(2-cyanoethyl)-2,3-dihydro-s-triazolo[ 4,3-b]pyrid'azin-3-oh (2.5 g., 11$). M.p. 166-168° c. ir: 2230, 1720, 1550, 1500 cm1, uv: ^max^ 575 ™ 2000). . nmr: 6D^°_d6 3.03 (2H, t, J=6.0 Hz, CHg), 4.21 (2H, t, J=6,0 Hz, CHg), 7.23 (IH, d, J=10.0 Hz, pyridazine-H), - 43 _ 7.93 (IH, d, J=10.0 Hz, pyridazine-H).

Anal. Calc'd. for CgHgNgOCl: C, 42.97; H, 2.70; N, 31.32; Cl, 15.86. Found: C, 42.73, 42.56; H, 2.57, 2.50; K, 31.36, 31-68; Cl, 15-96, 15.81. 2-(2-Carboxyettyl)-6-chloro-2,5-dihydro-s-triazolo[4,3-b]pyrldazln-3-on.

A solution of 6-chloro-2-(2-cyanoethyl)-2,3dihydro-s-triazolo[4,3-b)pyridazin-3_on (724 mg.) in 6n-HC1 (15 ml.) was refluxed for 6 hours. The reaction mixture was extracted with ethyl acetate (10 x 20 ml.).

The combined extracts were washed with saturated aqueous sodium chloride (50 ml,), dried over HagSO^ and evaporated to give light yellowjsolid 2-(2-carboxyethyl)-6-chloro-2,3dihydro-s-triazolo[4,3-b]pyridazin-3-on (567 mg., 72%).

M.p. >170° C. (sublimation). ir: v^Br 3400-2400, 1730, 1710, 1540 cm1. Π1&Χ •uv: 377 nm (e 1500). nmr: 6D2°pp^HC°3 2,70 (2H, t, J=7.0 Hz, CHg), 4.24 (2H, t, J=7,0 Hz, CH2), 7.17 (IH, d, 1=10.0 Hz, pyridazine-H), '20 7.7Ο (111, d, J=10.0 Hz, pyridazine-H).

Anal. Calc'd. for CgH^O^Cl: C, 39.-60; H, 2.91; N, 23.095 Cl, 14.61, Found: C, 39-62, 39-48; H, 2.97, 2.67; N, 23.05, 22.70; Cl. 13-93, 14.12. _ 45348 2-(2-Carboxyethyl)-2,3-dihydro-6-niercapto-s-triazQlop4,5-b]pyrldazin-3-on.

A mixture of 2-(2-carboxyethyl)-6-chloro-2,3dihydro-s-triazolo[4,3-b]pyridazin-3-on (567 mg., 2.34 m.moles) and 70% sodium hydrosulfide dihydrate (924 mg., 7.02 m.mole) in water (10 ml.) was stirred at room temperature for two hours. The reaction mixture was adjusted successively to pH 1 with c. HCl, to pH 10 with NaOH and then to pH 1 with c. HCl. The resulting precipitate of 2-(10 carboxyethyl)-2,3-dihydro-6-mercapto-s-triazolo[4,3-b]pyridazin-3-on was collected by filtration and washed with water. Yield: 4l8 mg. (74%). M.p. 174-176° C. ir: 3600-2600, 2440, 1730, 1720 (sh) cm'1, uv: λρίί 7m^ffer S6S nm (s/7<»0), 318 nm (e 6600). nmr: 6DMp°^d6 2.73 (2H, t, J=7.0 Hz, CHg), 4.07 (2H, t, J=7.0 Hz, CHg), 7.30 (IH, d, J=10.0 Hz, pyridazine-H), 7.74 (IH, d, J=10.0 Hz, pyridazine-H).

Anal. Calc'd. for CgHgNjjO^.S: C, 40.00; H, 3-36; N, 23.32; S, 13.35. Found: C, 39-08, 39-06; H, 3-12, 3.20; N, 22.65, 22.70; S, 14.23, 14.29. 7-Amino-5-F2-(2-carboxyethyl)-2,5-dihydro-s-triazolo[4,3-'b]pyridazin-3-on-6-ylthiomethyl]-3-cephem~4-carboxylic Acid.

A mixture of 7-ACA (405 J»g., 1.49 m.moles), the thiol 2-(2-carboxyethyl)-2,3-dihydro-6-mercapto-s25 triazolo[4,3-b]pyridazin-3-on (357 mg., 1.49 m.moles) and NaHCOj (375 mg., 4.47 m.moles) in 0.1 M phosphate buffer (pH 7,· 8 ml.) was stirred at 80° C. for 30 minutes. The reaction mixture was cooled and filtered to remove insolubles.

The filtrate was adjusted to pH 1-2 with c. HCl. The resulting precipitate, 7-amino-3-[2-(2-carboxyethyl)-2,3-dihydros-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl]-3-cephem4-carboxylic acid, was collected by filtration - and washed with water. Yield: 519 mg. (77%). ir: 3600-2200, 1800, 1725, 1620, 1550, 1480 cm1, uv: χΡΐί maxUffer 255 11111 (ε ^000°b 298 nm (e/i'coo). nmr: 0D2°*ggC03 2.20 (2H, t, J=7.0 Hz, CHg), 3-40 (IH, d, J=17-5 Hz, 2-H), 3.85 (IH, d, J=17.5 Hz, 2-H), 4.0010 4.5Ο (4H, m, 3-CH2 and N-CHg-), 5-01 (IH, d, J=4.0 Ηζ,'6-Η), .40 (IH, d, J=4.0 Hz, 7-H), 6.94 (IH, d, J=10.0 Hz, pyridazine-H), 7.44 (IH, d, j=10.0 Hz, pyridazine-H)..

Anal. Calc'd. for C^H^NgOgSg^^HgO: C, 40.09; H, 3.99; N, 17.52; S, 13.57. Found; C, 40.06, 40.12; H, 3.35, 3-34; N, 16.96, 16.98; S, 13.87, 13-98. 7-ACA refers to 7-aminocephalosporanic acid and DMF to dimethylformamide.

I - 46 45548 Scheme 1. Preparation of 7-Amino-3-(2-carboxymethyl2,5-dlhydro-s-triazolo[4,3-b]pyridazin-5-on-6-ylthiomethyl)-3-cephem-4-carboxylic Acid.

NaH/DMF . cich2cooc2h5 > Cl· N-CHgCOOCgHg _ 47 Lt· 6-Chloro-g,3-dihydro-g-ethoxycarbonylmethyl-s-triazoloi-4,3-b]pyridazln-5-on6 (2) To a solution of 6-chloro-2,3-dihydro-striazolo[4-,3-b]pyridazin-3-one [P. Francavilla and F.

Laurie, J. Het. Chem., 8, 415 (1971)] (1, 1,00 g., 5,9 m.mole) in dry DMF (30 ml.) was added sodium hydride (50$ in paraffin, 0.3 g,, 6.3 m.mole) under stirring with formation of yellow crystals. To the mixture was added ethyl chloroacetate (1,4 ml., 13 m.mole) and the mixture v;as heated at 90° C. for 8 hours with stirring. After cooling, the reaction mixture was poured into vrater (50 ml.) and extracted with toluene (5 x 40 ml.). The organic extracts were combined, dried over anhydrous sodium sulfate and evaporated at reduced pressure. The residue was crystallized with benzene-n-hexfinu to give yellowneedles of 2 (I.16 g., 77$), m.p. 114-115“ C. (lit. 110’ C.). ir: υ jjg 1735, 1710 cm-1, uv: λ^ΟΗ 231 nm (g, 26000). nmr; $ £^3 7··5θ (IH, d, J=10 Hz, pyridazine-H), 6.98 (IH, d, J=10 Hz, pyridazine-H), 4.80 (2H, s, -CHgCO), 4.27 (2H, q, J=7.5 Hz, CHgCHj), 1.29 (3H, t, J=7-5 Hz, ch2ch3), Anal. Calc'd. for 0^11^0-01: C, 42.12; Η, 3-53 N, 21.83; Cl, 13.81. Found: 0, 41.54, 41.46; H, 3.22, 3.49; H, 21.51, 21.535 Cl, 13.88, 13.99. _ 48 _ 2-Carboxymethyl-2,3-dihydro-6-mercapto-s-triazolo [4,3-b,7~ pyridazin-3-one (2) To a solution of 6-chloro-2,3-dihydro-2-ethoxycarbonylmethyl-s-triazolo/4,3-£/pyridazin-3-one (2, 30 g., 0.12 mole) in ethanol (900 ml.) was added NaSH«2H2O (70% pure, 45.9 g., 0.36 mole) and the mixture was refluxed for 0.5 hour. The reaction mixture was evaporated at reduced pressure. The residue was dissolved in water (200 ml.) and concentrated HCl was added to the solution to adjust to pH 2. The precipitate (2) was collected by filtration and washed with water. Yield 18.3 g. (69%). ir: vX?* 2900, 2450, 1750, 1660 cm1. ΧΠ3Χ uv: xl%NaHc°3aq. 26O nm (ε, 19500), 313 nm (e, 7000).

HiciX nmr; fip^°d6 7.88 (lH, d, J=10 Hz, pyridazine-H), 7.45 (IH, d, J=10 Hz, pyridazine-H), 4.72 (2H, s, CHjCO).

Anal. Calc'd. for C7H6N4O3S: C, 37.17; H, 2.67; N, 24.77; S, 14.17. Found; C, 37.35, 37.23; H, 2.26, 2.28; N, 23.58, 23.69; S, 14.32. 7-Amino-3-(2-carboxymethyl-2,3-dlhydro-s-triazolo [4,3-bJpyridazin-3-on-6-ylthlomethyl)-3-cephem-4-carboxylic Acid (£) To a suspension of 7-aminocephalosporanic acid (8.79g., 32.2 m.mole) in 0.1 M phosphate buffer (pH 7, 149 ml.) were added NaHCO^ (8,14 g., 97.0 m.mole) and the thiol 2 (7.30 g., 32.2 m.mole) with stirring. The mixture was heated at 80° C. for 0.5 hour under N2 stream. The mixture was treated with active carbon and adjusted to pH 3 with concentrated HCl. The resulting precipitate was collected by filtration and washed with water to give 7.59 g. (54%) of 4. ’45548 ir: ^max 1δ0θ<1720> 1600, 1540, 1470 cm1. w: * SSfer (pH ?) 252 nm (g, 19500), 298 nm (£,8400). nmr: J’ p^’’^2C03 7.56 (lH,'d, J=9 Hz, pyridazine-H), 7.05 (lit, d, J«9 its, pyridazine-H), 5·45 (IH, d, J»5 Hs, 5 6-H), 5.05 (IH, d, 5 Hz, 7-H), 4.43 (IH, d, J=14 Hz, 3-CHg), 4.04 (IH, a, J=14 Hz, 3-CHg), 3-88 (IH, d, J=l8 Hz, 2-H), 3.45 (IH, d, J«l8 Hz, 2-H).

Pivaloyloxymethyl-7-amino-5-(2-carboxymethyl-2,3-dlhydro-striazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem10 4-carboxylate.

Method A. - The title compound is produced by substituting for the 7-amino.cephalosporanic acid used immediately above an equimolar weight of plvaloyloxymethyl 7-amlnocephalosporanate hydrochloride prepared according to Example 2 of U.K. 1,229,453 from 7aminocephalosporanic acid. German 1,904,585 (Parmdoc ' 39,445) is equivalent to U.K. 1,229,453.

Method B. - The title compound is produced by substituting for the 0.025 mole (6.8 g.) 7-amino20 cephalosporanic acid used in the procedure of Example of U.K, 1,229,453 an equimolar weight of 7-amino-3. (2-carboxymethyl~2,3-dihydro-s-triazolo[4.,3-b]pyridazin3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid (_4).

The respective acetoxymethyl, methoxymethyl, acetonyl and phenacyl esters of 7-amino-3-(2-carboxymethyl-2,3dihydrb-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3cephem-4-carboxylic acid are prepared by substituting in Method B above- chloromethyl pivalate used therein an equimolar weight of chloromethyl acetate, chloromethyl methyl ether, 30 chloroacetone and phenacyl bromide, respectively. - 50 Λ3548 Preparation of 7-Amino-3-(2-inethyl-2,3-dihydro-s-triazplo[4,3-¾]pyrldazin-3-on-6-ylthiome thyl-3-c ephem-4carboxylic Acid. 6-Obloro-2,3~dihydro-2-methyl-s-trlazolof 4,3-b]pyrldazin3-one (2) To a solution of 6-chloro-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on.e [P. Francavilla and F. Lauria, J. Het. Chem. 8, 415 (1971)] (1, θ·5 g., 50 m.mol.) in dry DMF (12 ml.) was added NaH (50$ dispersion in paraffin, - 51 4 5848 2.64 g., 55 uumol) find the mixture wnn ut.lvved for 1 hour at room temperature» After methyl Iodide (.'1.Ί g,, 450 m,multi) w«a added» the mixture was «tirred for 40 hours at room temperature, diluted with water (200 ml.) · and extracted with CHCl^ (4 x 100 ml.). The combined extracts were washed with water (3 x 50 ml.), treated With a small amount of carbon and dried with anhydrous NagSOjj. Evaporation of the solvent under reduced pressure afforded pale yellow residue which was crystallized from chloroform-n-hexane. Yield: 7.23 g, (79%).

M. p. 18Ο-18Ϊ” C. ir: 1720 cm1. uv:_ λ™ 233 nm..(e 25200), 363 run (e 1600). nmr: 6^^3 3-72 (3H, s, N-CHj), 6.88 (IH, d, J=10 Hz, ..pyridazine-H), 7.48 (IH, d, J=10 Hz, pyridazine-H).

Anal. Calc'd. for CgHgClN^O: C, 39.04; H, 2.73; N, 30.35; Cl, 19.21. Found: C, 39-24, 39-28; H, 2.54, 2.61; N, 30.63, 30.80; Cl, 19-59, 19-26. 6-Mercapto-2,3-dihyaro-2-methyl-s-triazolo[4,3-b]pyridazin•20 3.-°ηθ (X) A mixture of 2 (6.50 g., 35.7 m.mol.) and NaSH’2Hg0 (70% pure, 9.4 g.) in water (100 ml.) was heated under reflux for 15 minutes. The, mixture was cooled and acidified to pH 1 with concentrated HC1 to precipitate 25 the thiol 3 which was collected by filtration and dissolved in aqueous saturated NaHCO^ (100 ml.), pie solution was treated with a small amount of carbon and acidified with dilute HC1 to precipitate 3 as pale yellow prisms.

Yield: 5.72 g. (89%). M.p. >280° C. ir: 2450 (-SH), 1710 (C=0) cm-1. uv: λ1ΐαχ°°5 261 111,1 (ε l63°°b 515 nm (ε 5800). nmr: δ°2Ορρ°Η 3·6θ (3Η, s, N-CfLj), 7.08 (2H, s, pyridazlne-H).

Anal, calc'd. for Cgl^OS: C, 39·55ί H, 3-32; N, 30.75; S, 17.6ο. Found: C, 39-57, 39-66; H, 3-14, 3,22; N, 30.32, 30.61; S, 17.80, 17.89. 7-Amlno-3-(2-methyl-2,3-dlhydro-s-triazolof 4,3-b]pyridazln3-on-6-ylthioinetftyl)-3-cephem-4-carboxylic Acid (4_) A mixture of 7-aminocephalosporanic acid (7-ACA, .44 g., 20 m.mol.), 3, (3.64 g., 20 m.mol.) and NaHC0? (3.56 g., 40 m.mol.) in 0.1 M phosphate buffer (pH 7, 100 ml.) was heated with stirring at 80° C. for 30 minutes.

The hot mixture was treated with a small amount of carbon and the filtrate was acidified to pH 4 with dilute HC1 to precipitate 4. which was collected by filtration, washed with water (50 ml.) and dried. Yield: 5-73 g. (73#).

M.p. 240-245° C. (dec.). ir: v^Br ^qq (p_iactam c=0), 1725 (C=0), l6l0 and 1410 (COO) cm1. uv: λ^™°3 253 nm (ε20000), 305 (ε 8400). nmr: δΟ2Ο+™°3 3-69 (3H, s, N-CH3), 5-08 (IH, d, J=4.5 Hz, 6-H), 5.48 (IH, d, J=4,5Hz, 7-H), 7.00 (IH, d, J=10 Hz, pyridazlne-H), 7.52 (IH, d, J=10 Hs, pyridazine-H).

Anal. Calc’d. for σ-^Η^ΝβΟ^Ή^: C, 40,76; H, 3.91; N, 20.38; S, 15.55. Found: C, 40.84, 40.63; h, 3.44, 3.31; H, 20.50, 20.36; s, 15.19, 15.57.

Preparation of BB-S515 / BB-S515 BB-S515; 7-K2Z)-2-Methoxyimino(fur-2-yl)acetamido]-3(2,3-dihydro-s-triazolo.r 4,3-b]pyridazin-3-on-6-ylthiomethyl)-5-cephem-4-carboxylic Acid sodium Salt.

To a solution of (2Z)-2-methoxyimino(fur-2-yl)acetic acid (169 mg., 1 m.mole) and triethylamine (0.14 ml.j 1 m.mole) in dichloromethane (2 ml.) was added oxalyl chloride (0.09 ml., 1 m.mole) at 0-5° C. and the ί mixture was stirred for 30 minutes. The solvent was evaporated under reduced pressure to afford an oily residue. A solution of that oily residue in dry acetone (5 ml.), after filtration, was added to a mixture of 7-'amino-3-(2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on6-ylthiomethyl)-3-cephem-4-carboxylic acid (380 mg., m.mole) (U.S. 3,907,786) and sodium bicarbonate (336 mg., 4 m.mol.) ip water (10 ml.) at 0-5° C. The reaction mixture was stirred at 0-5° C. for 2 hours.

Most of the acetone was evaporated at reduced pressure, the aqueous concentrate being washed with ether (2 x 30 ml.) and adjusted to pH 1-2 with concentrated HCl.

The resulting precipitate (338 mg.) was collected by . filtration and dried in vacuo. A suspension of the free acid (303 mg.) in water (10 ml.) was adjusted to pH 6.5 with aqueous NaOH ,(1 N, 0.6 ml.) and filtered to make a clear solution which' was lyophilized to give 7-[(2Z) -2methoxyimino(fur-2-yl)acetamido]-3-(2,3-dihydro-s- 54 triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3~cephem4-carboxylic acid sodium salt as a light brown powder (222 mg,, 46$). M.p. >230° C. (dec.). ir: 3410, l?6o, 1720, l6oo cm-1.

Ill 3.X uv: ^pH7®^fer 256 nm (ε 20600), 274 (E 18800). - 55 _ 45848 Ί Preparation of D-mandelio add oarboxvanhydride CgHg-CH-COOH OH COC1„ °65Π“° D-Mandellc acid carboxyanhydride (2) Phosgene was bubbled through a solution of 2,0 g, (0.013 mole) of D(-)-mandelic acid (1.) in dry tetrahydrofuran for 30 minutes. The solution was allowed to stand overnight after which time it was heated under reflux for 10 minutes. Evaporation of the solvent under reduced pressure afforded an oily residue which was solidified by trituration with n-hexane (20 ml.). The product was collected by filtration and dried in vacuo on KOH, Yield 2.3 g. of D-mandellc acid carboxyanhydride.

IR: ' 1895 ’ 1875 > 1780 cm1.

The preferred and m03t active compounds of the present invention are those having the D configuration at the α-carbon atom in the 7-sidechain, that is, those made from D-mandelic acid or a monosubstituted D-mandelio acid as illustrated here in. In addition, the configuration at the two optically active, asymmetric centers in the β-lactam nucleus is that found in cephalosporin C produced by fermentation and in the 7-aminocephalosporanie acid derived therefrom.

J ~ 56 ,4 5 3 4 8 EXAMPLE 1 3, BB-S488 BB-S488; 7-(D-Mandelamido)-5-(2-carboxymethyl-2,5-dihydros-triazolo[4,3-b]pyrldazin-3-on-6-ylthiomethyl)-5-cephem4-carboxyli,c Acid (3) D-(-)-Mandelic acid O-carboxyanhydride (U.S. Patents 3,167,549, 3,840,531 and 3,910,900), (1, 400 mg., 2.3 m.mole) was added portionwise to a solution of 7-amino-3-(3~carboxymethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-5-on-0-ylthiomethyl)-3-cephem-4-carboxylic acid (2, C57 rag., 1.5 m. mole) and sodium bicarbonate (445 mg., 5.3 m.mole) in 50% aqueous acetone (30 ml.) at ca 0° C. with vigorous stirring. The mixture was stirred for 1 hour at room temperature and evaporated under reduced pressure below 40° C. to remove acetone. The resulting aqueous solution was washed with ether and acidified to pH 1 with dilute HCl to afford a gummy precipitate, which was collected by filtration, washed with water and dissolved in totrahydrofuran (THF) (100 ml.). The THE solution was treated with a small amount 48 s of active carbon, dried over anhydrous sodium sulfate and filtered. The filtrate was evaporated under reduced pressure and the residue was triturated with ether. The pale yellow precipitate was collected by filtration and chro5 · matographed on a silica gel column (Wako-gel C-200, 10 g.) eluted with a solution of chloroform-methanpl (20:1). The fractions containing the desired product were combined and concentrated under reduced pressure. The concentrate was diluted with ether (100 ml.) to precipitate the product (3), which was collected by filtration, washed with ether (30 ml.) and dried. Yield 279 (34$).

M.p. 173-176° C. (dec.). ir: V 3600-2400, 1770, 1720, 1520, 1495, 1365, 1245 cm-1. uv: ''max* 254 nm 1θθθ°Λ 297 nm (g 9000, sh). nmr: ^0-¾ 3.68 (2H, m, 2-H), 4.03 (IH, d, J=l3 Hz,.3-H), 4.34 (IH, d, J=13 Hz, 3-H), 4.64 (2H, s, NCHgCO), 5.00 (IH, d, J=4 Hz, 6-H), 5.02 (IH, s, PhCH), 5.63 (IH, d-d, J=4 & 9 Hz, a-doublet with addition of DgO, J=4 Hz, 7-H), 6.97 (IH, d, J=10 Hz, pyridazine-H), 7.1-7.4 (5H, m, phenyl-H), 7-60 (IH, d, J=10 Hz, pyri- . dazine-H), 8.60 (IH, d, J=9 Hz, disappeared with addition Of DgO, CONH).

Anal. Calc'd..for Cg^g^gOgSg-l/^HgO: C, 47.50 H, 3-64; N, 14.451 8, 11,03. Pound; C, 47.3^1 H,'3.48,« N, 13.90; S, 11.01.

In vitro .activity (Table 1) The MIC's were determined by tne Steers' agar dilution method using Mueller-Hinton agar against 4 grampositive and 28 gram-negative bacteria and the results are shown in Table 1.

In vitro activity (Tables 2 and 3) MIC determinations were performed by serial twofold agar dilution method using Steers' apparatus on Mueller-Hinton agar plate against 51-gram-positive and 95 gram-negative bacteria. The results are shown in Tables 2 and 3.

Media effect on MIC The MIC's were determined by using three kinds of agar media [Nutrient (NA), Mueller-Hinton (MHA) and Heart-Infusion (HIA)]. The results obtained with BB-S488 and cefamandole are shown in Table 4, which indicates little media effect in these cephalosporins.

Blood levels in mice Groups of mice were administered subcutaneously graded doses of 40, 20 and 10 mg./kg.. The blood samples collected from orbital sinuses were assayed by the paper disc-agar diffusion method on Sarclna lutea PCI 1001 plates. The results are shown in Table 5· In vivo activity Comparative in vivo evaluation was made by the standard experimental infection in mice against the - 59 •5 58 48 following pathogenic bacteria: £. aureus Smith E, coli Juhl K. pneumoniae A9977 The results are shown in Table 6. 48 MIC (meg,/ml.)_ Test Organism_ BB-S488 Cefamandole Table 1. The In vitro Antibacterial Activity of IW-S4B8 By Agar Dilution Method (Mueller-Hinton Agar).

S. aureus Smith A9537 0.2 0.05 S. aureus A9497 0.1 0.05 S. aureus BX-1633 A9606 0.4 0.1 St. faecalis A9536 100 50 E. coli NIHJ 0.025 0.025 E. coli ATCC 8739 0.1 0.05 E. coli Juhl A15119 0.2 0.4 E. coli BX-1373 0.2 0.8 E. coli Λ1581Ο 0.1 0.4 E. coli A966O 0.05 0.1 E. coli Λ15147 3.1 0.4 Kl. pneumoniae A9678 3.1 3.1 Kl. pneumoniae A9977 0.05 0.2 Kl. pneumoniae A1513O 0.2 0.8 Kl. pneumoniae A9867 0.2 0.8 Pr. vulgaris A9436 0.1 0.4 Pr..vulgaris A9699 0.2 6.3 Pr. mirabilis A9554 0.05 0.4 Pr, mirabilis A99OO 0.1 0.8 Pr. morganii A9553 >100' >100 Pr. morganii A20031 0.1 0.8 Pr. rettgeri Λ15167 0.05 0.2 Ps. aeruginosa A993O >100 >100 Ps. aeruginosa Λ9843 >100 >100 Shig. dysenteriae 0.025 0.1 Shig. flexneri A9o84 12.5 6.3 Shig. sonnei A9516 0.025 0.0'5 Serr. marcescens A20019 100 100 Enterob, cloacae A9656 3.x 3.1 Sal. enteritidis A9531 0.05 0.1 Sal. typhosa A9!I98 0.05 0,1 B.· anthracis A95O4 0.1 0.1 dS 5 43 '20 >5 In vitro Antibacterial Activity in MuellerTable 2.

Hinton Agar (Gram-positive) Code No, ' Test Organism MIC (meg,/ml.) BB-S488 Cefaman- dole Sa-2 S. aureus Smith A9537 0.4 0.2 Sa-3 S. aureus No. 193 0.8 0,2 Sa-8 S. aureus 0.4 0.2 Sa-Q S. aureus No. I93 0.8·. 0.2 5s-I0 5. aureus A20239 1.6 0.4 Sa-11 S. aureus BX-I033 A9606 0.4 0.2 Sa-12 S. aureus A94-97 0.2 0.1 Sa-29 S. aureus No. 193 1.6 0.8 •3a-33 S. aureus Terajima 0.0125 0.0125 3a-34 S. aureus AI5092 0.8 0.2 Sa-35 S. aureus A15094 0.8 0.4 Sa-36 S. aureus Russell 0.8 0.4 Sa-37 S. aureus A9524 1.6 0.8 Sa-38 S. aureus A9534 0.4 0.2 Sa-39 M aureus A9578 0.8 0.4 Sa-40 3. aureus A9§01 0.8 0.4 3s-4l S. aureus A9S02 0.8 0,2 Sa-44 S. aureus AI5097 25 25 Sa-56 S. aureus A9630 5-1 0.8 ' Sa-57 S. aureus A9748 25 3.1 Sa-58 3, aureus A15O33 12.5 1,6 Sa-§9 S. aureus AI5096 100 6,3 Sa-60 S. aureus Α2θβθ4 50 So -6l S. aureus Α2θδθ5 100 j. j. 6.3 Sa-62 S. aureus Α2θ6θ6 3.1 0.8 Sa-63 S. aureus A20607 >100. 12.5 05,-04 S. aureus Α2θβθ8 100 6.3 5. aureus A20609 100 63 Sa-66 S. aureus A20SI0 100 6.3 Sa-67 S. aureus Λ20611 100 r ί Sa-SB S. aureus Λ20612 1.6 0,4 Sa-69 S. aureus A20613 100 6.3 Sp-1 3. pyogenes S-23 0.4 0.1 Sp-2 - ,B. pyogenes .Dick 0.4 0.1 · Sp-3 3. pyogenes Λ9βθ4 0.4 0.1 Sp-4 S. pyogenes A20065 - 0.2 0.1 sp“§ 5. pyogenes,AI5O4O 0.4 0.1 Sp-b S. pyoge es A20066 0.4 0.1 Sp-7 S. pyogenes Dig 7 0.4 0,1 Sp-8 3. pyogenes Λ15Ο41 0.4 0,1 sp-9 3. pyogenes Λ20201 0.4 '0,1 Sp-10 S. pyogenes A20202 0.4 0.1 Dp-1 D. pneumoniae Type Ii 0.2 0.2 Dp-2 D. pneumoniae Type I Neufeld 0,2 0.2 Dp-3 D. pneumoniae Type III 0.2 0,2 Dp-4 D. pneumoniae A9585 0.2 0.2 Dp-g D. pneumoniae AI5069 0.2 0.2 Dp »6 D. pneumoniae A20167 0.2 0.2 Dp-7 D. pneumoniae Λ20759 0.2 0,2 Dp-8 . D. pneumoniae Λ20769 0,2 0.2 Dp-9 D. pneumoniae A2077Q 0.2 0.2 S2 55 Table 3. In vitro Antibacterial Activity in Mueller- Hinton Agar (Gram-negative) Code No. Test Organism MIC (meg./ml.) Cefaman· BB-S488 dole Ec-1 E. coli NIHJ 0.2 0.1 Ec-3 E. coli Juhl A15H9 0.2 0.8 Ec-4 E. coli A15169 12.5 6.3 Ec-5 E. coli K-12, ML-1630 A2O363 coli A2O3&6 0.2 0.8 Ec-11 E. 50 25 Ec-15 E. coli ATCC 8739 0.2 0.1 Ec-3 4 E. coli A966O 0.1 0.1 Ec-35 E. coli A9435 0.4 0.8 Ec-36 E. coli A15147 3.1 1.6 Ec-40 E. coli Α203βΐ 0.2 0.8 Ec-44 E. coli A9535 0.1 0.1 Ec-45 E. coli A15148 3.1 1.6 Ec-46 E. coli A15164 25 12.5 Ec-47 E. coli A15170 100 50 Ec-49 E. coli A20107 0.4 0.2 Ec-50 E. coli Λ20109 0.2 0.8 Ec-51 E. coli A20343 50 12.5 Ec-56 E. eoli Λ20365 25 12.5 Ec-58 E. coli A9675 0.4 1.6 Ec-59 E. coli A20766 0.2 0.8 Ec-62 E. coli A20895 0.4 0.8 El-1 E. cloacae A9656 3.1 3.1 El-2 E. cloacae A20364 3.1 3.1 El-4 E. cloacae A20650 1.6 1.6 El-6 E. cloacae A9657 0.8 0.8 El-7 E. cloacae A9659 1.6 0.8 El-8 E. cloacae A9655 1,6 1.6 El-9 E. cloacae A20021 >100 100 El-11 E, cloacae A2O344 cloacae A21006 >100 >100 El-12 E. 1.6 3.1 El-14 E. cloacae A2O953 0.8 3.1 Pm-1 P. mirabilis.Λ9554 0.1 0.8 Pm-2 P. mirabilis A99OO 0.2 1.6 Fm-3 P. mirabilis Λ20119 0.4 3.1 Pm-4 P. mirabilis A2O454 0.2 1.6 Pm-5 P. mirabilis A9702 0.1 0.8 Pm-S P. mirabilis A21222 1.6 1.6 Pg-1 P. morganii A9553 >100 >100 Pg-2 P. morganii A20031 0.2 . 1.6 Pg-3 P. morganii A9636 0.8 1.6 Pg-5 P. morganii Λ15166 0.1 0.2 Pg-6 P. morganii Λ20455 0.4 1.6 Pg-7 P. morganii A20457 0.2 0.8 Pg-8 P. morganii Λ15153 0.1 0.8 Pg-9 P. morganii Λ15149 0.8 3.1 Pv-1 P. vulgaris A9436 0.2 0.8 Pv-2 P. vulgaris A9526 6.^5 1.6 Pv-3 P. vulgaris A9699 6.3 50 Pv-4 P. vulgaris ATCC 9920 0.1 0.2 Pv-5 P. vulgar!s Λ9539 25 >100 Pv-6 P. vulgaris Λ9716 0.1 0.8 Pv-7 P. vulgaris A21240 25 >100 45543 Table 3 (Continued) Code No. Test Organism BB-S488 Cefaman· dole Pr-1 ' P. rettgeri A15167 0,1 fk ? Pr-2 P. rettgeri A9637 0.1 0.1 Fr~4 P. rettgeri A20645 0.1 0.3 Pr-ij P. rettgeri A20915 0.2 0.8 Pr-6 P. rettgeri A20920 0.1 0.2 Pn-1 P. inconstans A20615 0.1 0.8 Ps-1 P. stuartii A20745 0,4 0,8 Ps-2 P. stuartii A20894 0.2 0.8 Ps-3 P. stuartii A20911 0.8 0.8 Ps-4 P. stuartii A21051 50 25 Ps-5 P. stuartii A21057 0.2 0.8 Kp-1 K. pneumoniae Dll 0,1 0.3 Kp-2 K. pneumoniae A9678 3.1 1,6 Kp-3 K. pneumoniae A9977 0.1 0.8 Kp-4 K. pneumoniae AI5130 0.2 0.8 Kp-7 K. pneumoniae A9857 0,4 0.8 lip »3 K. pneumoniae A20680 25 12.5 Kp-9 K. pneumoniae A20636 12.5 12.5 hp-10 K. pneumoniae A20328 6.3 Kp-11 K. pneumoniae A20330 1.6 12,5 Kp-12 K, pneumoniae A21228 6 »3 6.¾ IGc-2 Klebsiella sp. A9652 0.4 1.6 Kx-3 Klebsiella sp. A20546 0.2- 0.3 Sm-1 S. marcescens A20019 25 25 Sm-2 S. marcescens A20335 3.1 12.5 Sm-3 S. marcescens A20336 • 6.3 12,5 Sm-4 S. marcescens A20442 6-3 12.5 Sm-5 S. marcescens A20222 3.1 - 12.5 Snt-6 3. marcescens A2O46O 6.3 12.5 Sm-9 S. marcescens A20333 6.3 50 Sm-10 S. marcescens A20334 6-3 50 Sm-11 5.-marcescens A20459 6.3 25 Sin-12 S. marcescens A20461 6.3 50 Se-1 S, enteritidis A9531 0.1 0.2 St-1 S. typhosa 0,1 0.2 3h-l 5. paratyphi 0,1 0,2 St-101 S, typhimurium 0,1 0.2 Sd-1 S. dysenteriae 0.1 0.2 Sr-1 S. flexneri A9684 12.5 3.1 Ss-1 S. sonnei Yale 0.1 0.1 ’ Cx-1 Citrobacter sp, A20673 1.6 1,6 Cx-2 Citrobacter sp, A20694 .1.6 1»6 Cx~3 Citrobacter sp. A20695 1.6 1,0 in 04· h h in oomooooomCMinmoooooooooooHOHOmoooo ο in h oj — _ CO CO-4-^- COCO COVO CUkO OJ rl ino in o ooo H ri Η Η H A A A OJ-=t~ r-ί H4f Η H in H 0] ο o no ο o ino mcMtnmooooooooooo novo oko ο ο ο o o cm hcm oo ο o tnino Η Η Η Η Η. H Α Λ A cM^m hcm hco in mm co cm m cm -=r cm m^f- m -4OOkOOOOOOrl inCMkOkD OOOO OkO OOOOrl OkO OkD OOOO o in cm η ο· ο in tno Η Η Η H A AAA Tabie U. Media Effect on MIC of BB-S488 and Reference Compounds d . §s Ο coco in O CM cm m Η H CM^t· CM HCO H CM in H4O in cm in CO CM σ ο ο ο ο ο o o\o oommoooooooooooowowoooo O O O o o o O rfc HO O Η H H rfc r-l rl H rl rl A A A A A A A COkO in O^f CM Ο Η Ο Ο Ο Ο O OkO o OkOkO ooooooooooooooinoooo Ο Ο ~ -- “ - ~ .---- -.Η H A A oi m mm cmco cm cm hcm cmcoh o O O O O o in cm ο ο o rl r| j—1 rl ι—1 rl rl rl rl A AA A A AAA in in ^?-co cm cmco η h cm co· CO H O mo OOOO OkO OOCMCMOOOOOOOOOOOOOOOOOOO ~ ~ ---- · * - - in ino ο o Η Η H A H CM h m OO o ιηιηπ η o Ο Η H rl rl H A A A A A * . MO O C-kD mo\ in< o> < m • m Μ ίϊϊ tfl ffl 3 3 1) ϋ fc fc . «5 b-IA cntn ο cn ^“0 ra h a 3 d fc d rfc 3 J)O d bt u * KO * ' __ ο -v cnvo cn b- tn tn * * η tn o H * 40* h cn -n-ra· cnncjco σκηο tntntnKo-=s-cnH ο o eno in tn koko a-okh «; tncnincninor^-h b-co cncM cm enn h* b-* * * intntnaici .=Mocncninooinooo«!«;a:cn KO- rfc-KO -d- OSCM CM IU Ο (¾¾ < < < dj a! «! << M te 3 HtnOHHtn<|o)«!ddw ω ra ci d w ra .cooinino riri w ra ·η ·η τΐ η ·η ·η η η η α) ο oh CM Η Η rfc CM d ffl d p 0 d Η Η Η Η τ| Η Η fc Η Η Η Ο Ο Ρ U Η EbBBHEEfcrawraBEdd (U OJMWMHWMMWWWWMWWWWMfcCUPUPH&iCL.Cfcft.tfc Pr 03 cn Cl. to u> d •P O d H I CQ. •fc ·=! rfc inco KO rfc rfc cm Η—j· tnH ipfc η tn-e- inn cm h tn-i- tnr-ι tnH cm h oko η h cm tnH tntntn ι ι ι ι ι ι ι ι ι ι ι ι ι ι t ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι ι d d d ίμ ο ο ο ο ο ο ο η H r-ι ρ. Pi ι-l > b 0 0 ω ho M fc ra n ra G 0 d d U) CO CO CO Pl w ei III Μ Ν M ftl W frl Μ M hl Pr Pi Λι Pi Pi Pr Pi Pi Pi Pr Pr CO CO Pi β} Ί 5Λ8. , Table 5. Subcutaneous Mice Blood Levels Meg./Ml. BB-S488 Cefamandole ::/ Dos e Time 40 mg./kg. 15' 19 18 5 30' 17 11 60' 12 3.9 120' 1.6 .0-3 20 mg./kg. 15’ 7.4 9.5 30' •6 4.1 10 60' 4.3 1 120' 0.7 <0.1 .10 mg./kg, 15' 5 3.8 30' 3 1.5 60' 0.8 . 0.3 15 120' · 0.1 Table 6. In vivo Activity Test Organism Dose BB-S488 Cefamandole S. aureus Smith 25 mg./kg. 5/5* 5/5 6.3 5/5 5/5 5 1.6 5/5 5/5 0.4 0.1 PD50 5/5 2/5 0.12 mg./kg. 1/5 0.6 mg./kg. E. coli Juhl 25 mg./kg 5/5 5/5 10 t 6.3 1.6 0.4 0.1 PD50 5/5 5/5 5/5 2/5 0.12 mg./kg. 5/5 2/5 0/5 1.8 mg./kg. 15 K. pneumoniae 25 mg,/kg. 6.3 1.6 0.4 5/5 5/5 5/5 4/5 5/5 1/5 0/5 0/5 20 0.1 0/5PD50 0.26 mg./kg. 6.25 mg./kg.

*No. of survivors/No. tested . 4SS-48 Test Organism Run of No. W50 Dose (mg./kg.) BB-S 488 Cefam- andole E. cloacae C-811 1 x 10 100 5/5 5/5 Α2δ4'64 (El-19) 25 5/5 5/5 1 6.3 5/5 5/5 1.6 4/5 4/5 0,4 2/5 1/5 PD5O (mg./kg,) 0.54 0.8 Urinary Recovery in Rats # Recovery (0-24 Hrs.) Dose (sc) BB-S488 Cefamandole 10 mg./kg. 38.8 58.3 .

Nephrotoxicity Test in Rabbits No nephrotoxic sign was seen in the rabbits treated with 100 mg./kg. (iv) of BB-S488 while cephaloridine showed severe nephrotoxicity in the comparative test.

Additional PP^q Data (Single sc Treatment) Run No. of LD50 Dose BB-S Cefam- Test Organism No. (mg./kg.) 488 andole K. pneumoniae A9977 (Kp-3) C-805 3x10·'’ 25 5/5 5/5 6.3 5/5 1/5 1.6 5/5 0/5 0.4 4/5 0/5 0.1 0/5 PDp-n (mg./kg.) 0.26 9-4 P. vulgaris A9436 (Pv-l) C-808 1x10 50 . 25 5/5 3/5 12.5 —- 1/5 6.3 5/5 ___ 3-1. — 0/5 15 1.6 3/5 — 0.8 --- 0/5 0.4 1/5 — Ρϋ5θ (mg./kg.) 1.1 36 P. mirabilis A9900 (Pm-2) C-810 lxlO5 50 2/5 20 25 5/5 — 12.5 ___ 1/5 6.3 5/5 — 3.1 —- 0/5 1.6 4/5 — 25 0.8 — 0/5 0.4 0/5 0.2 — 0/5 0.1 0/5 — PD50 (mg./kg.) 1.1 50 . 69 455-48 Stability of BB-S488 Stability of BB-S438 was determined in both a 10% and an 0.02% .solution. The stability is indicated as the relative activity remaining in the test solution at given periods to t.he initial solution. The activity was assayed using paper discs on B. subtilis PCI 219 plate (pH 6). , ίο (1) Stability in a 10% Aqueous Solution .at Room Temperature Remaining Activity (%) Compound pH' ' 0 2 5 7 Days BB-S488 6.1 100 128 90 116 )Unadjusted pH of the 10% solution.

- Compound BB-S488 Remaining Activity (%) 100 T . 2 93 3 78 7 Days 102 100 87 64 56 TOO 20 14 13 0 (2)pH 4: 0.1 M AcOII - NaOAc buffer, pH 7: 0.1 M phosphate buffer. pH 9' 04« HHjjOH-NHjjCl buffer.

W5--- ·· ι'·-.

I - 70 “ 45348 EXAMPLE 2 D-H ClgCHCOCl OCOCHClg O~ CHCOC1 I OCOCHCl H-ACA-S-CMTP(3) --> CHCO-ACA-S-CMTP OH BB-S488 pichloroacetylmandeloyl Chloride (2) A mixture of D(-)-mandelic acid (1, 1.52 g., 5 10 m.mole) and dichloroacetyl chloride (4.41 g., 30 m.mole) was heated at 80-85° C. for 1.5 hrs. and the excess dichloroacetyl chloride was removed under diminished pressure. Tb the residue was added thionyl chloride (2.5 ml.) and the mixture was heated under reflux for 1.5 hrs.

Excess thionyl chloride was removed by distillation and dry benzene was added. Evaporation was repeated. The residual oil was kept over KOH at 1 mm Hg overnight at room temperature to remove dichloroacetyl chloride.

Yield, 2.8 g. (100$). This product was used in the next 48 step without further.purification, ir: 1780, II60 cm1. nmr: S ^4 5.91 (IH, s, PhCH or COCHClg), 6.00 (IH, S, PhCH or COCHClg), 7-32 (5H, s, phenyl-H)'.

BB-S488; 7-(D-Mandelamido)-3-(2-carboxymethyl-2,3-dihydros-triazolort(,5-b]pyridazin-5-on-6-ylthiomethyl)-3-cephem4-carboxylic Acid. (4.) A solution of the above-obtained dichloroacetylmandeloyl chloride (2, 2.8 g., 10 m.mole) in dry acetone (30 ml.) was added dropwise to a stirred solution of 7amino-3-(2-carboxymethyl-2,3-'dihydro-s-triazolo[4j3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid (H-ACA-S-CMTPj(5, 3-94 g., 9 m.mole) and triethylamine (3.54 g., 35 m.mole) in 5θ% aqueous acetone (120 ml.) at 0°-5° C. The.mixture was allowed to rise to room temperature during 1 hour with stirring and was adjusted to . pH 11 with 5% aqueous sodium carbonate (ca .12 ml. was required). The. mixture was allowed to stand at room temperature for 30 minutes, acidified to pH 1 with dilute HCl and evaporated under reduced pressure to remove acetone below 40° C. 'The precipitate was collected by filtration, washed with water (20 ml,) and air-dried.

The dried material was dissolved in THP (150 ml.), stirred for 5 minutes at 40-50° C. and filtered to remove insoluble unreacted 5 (0,54 g., 14% recovery). The filtrate was. chromatographed on a silica gel column (Wakogel, C-200, 30 g.) and eluted with chloroform-methanol (100:5). The eluates were collected in 50 »1. fractions monitoring by tic (silica gel, solvent, CH^CN-water = 4:1, - 72 45S48 detected with lg). The fractions containing the desired product were combined, treated with a small amount of carbon and evaporated under reduced pressure. The residue was triturated with chloroform (50 ml.) to yield, 2.36 g. (46$) of 7-(D-mandelamido)-3-(2-carboxymethyl2,3-dihydro-s-triazolo[4,3-b]pyridazin-3“on~6-ylthiomethyl)-3-cephem-4-carboxylic acid (4_). M.P., 165170° C. (dec.). ir: z?JJr 3600-2500, 1780, 1720, 1500, 1410, 1355, 1220, 1195 cm-1. uv: λ^°!Ι 254 nm (g, 18300),-297 nm (sh, &, 93ΟΟ). nmr: S 3-84 (2H, m, 2-H), 4.17 (2H, d, 13 Hz, 3-H), 4.50 (IH,· d, 13 Hz, 3-H), 4.82 (2H, s, MCHgCOO), .20 (IH, d, 4.5 Hz, 6-H), 5.25 (IH, s, PhCH), 5.87 (IH, a-d, 4,5 & 9 Hz, 7-H, a doublet (J=4.5 Hz) by addition of DgO), 7.25 (IH, d, 11 Hz, pyridazine-H), 7-4-7.7 (5H, m, phenyl-H), 7.90 (HI, d, 11 Hz, pyridazine-H), 9-0 (IH, d, Hz, 7-CONH, disappear by addition of DgO).

Anal. Calc'd for Cg^Ig^OgS^ACHClj! C, 43.08; Η, 3.I65 N, 12.69; S, 9.69. Pound: C, 43.11, 43.22; H, . 2.97, 3.06; n, 12.80, 12.77; s, 9-64.

I . 455 48 EXAMPLE 3 CHCOOH 99% HCOOH CHCOOH SOC1, OGHO CHCOC1 H-ACA-S-CMTP (3) OGHO CHCO-ACA-S-CMTP OGHO 7, BB-S494 O-Foymyl-Pf-)-mancLelic Acid (5) A mixture of D(-)-mandelic acid (1, 5.0 g., 33 m.mole) and 99% formic acid (80 ml.) was heated at 8090° C. for 12 hours, The mixture was evaporated and toluene (100 ml.) was added to the residue and evaporated under reduced pressure to remove formic acid azeotropically. The residue was dissolved in benzene (200 ml.) and the iO solution was washed with water (2 x 50 ml.), The organic layer was separated, dried with anhydrous sodium sulfate and evaporated under reduced pressure. The residual oil. was triturated with cyclohexane (50 ml.) to crystallize. Yield, 3,70 g. (63#) of 0-formyl-D(-)-mandelic acid (5) as colorless prisms. M.P., 56-59° C. (lit. M.P., 55-58° C,). n H), S.05 (IH, s, OCHO), 10.05 (IH, s, COOH, disappeared by addition of DgO). rr - 74 4 S 5 4 8 Q-Formyl-D(-)-mandeloyl Chloride (6) A mixture of 5 (2.0 g., 11 m.mole) and thionyl chloride (10 ml.) was heated under reflux for 2 hours, Evaporation of the excess thionyl chloride and distillation of the residue under reduced pressure afforded the acid chloride 0-formyl-D(-)-mandeloyl chloride (6).

Yield, 1.53 g. (70#). B.P., 120-122° C./15 mmHg. ir: z,11^ 1805, 1740, ll60, 1140 cm-1.

BB-S494; 7-(D-0-Formylmandelamido)-3r(2-carboxvmethvl-2.3dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)3-cephem-4-carboxylic Acid (7) A solution of 0-formyl-D(~)-mandeloyl chloride (6) (1.0 g., 5*1 m.mole) in dry acetone (10 ml.) was added dropwise to a cold (0 to 5° C.) solution of 7-amino-3(2-carboxymethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid (3., 1.75 g., 4.m.mole) in 50# aqueous acetone (70 ml.) containing sodium bicarbonate (1.34 g,, 16 m.mole). The mixture was stirred for 30 minutes at room temperature and washed with ether. The aqueous layer was acidified to pH 1 with dilute HC1. The separated oily gum was Collected and dissolved · in THF (100 ml.). The solution was treated with a small amount of carbon and dried with anhydrous sodium sulfate. Evaporation of the solvent under reduced pressure to 10 ml. and dilution with ether afforded the title compound (7) as a pale yellow amorphous powder, 0.91 g. (38#).

M.P., 172-176° C.(dec.). ir: J 56QO_24oo, 1775, 1720, 1550, 1355, 1230, 1Πα.Λ. 1160 cm'1. uv: EtOH 25i, m β, 20800), 207 nm (5h, £, 10500). 45348 nrnr; § ^O-dg 3.4-4.5 (4H, m, 2-H and 3-H), 4.67 (2H, s, NCHgCOO), 4.97 (1H, d, 4 Hz, 6-H), 5-66 (IH, d-d, 4 & 8 Hz, 7-H), 7.2-7.5 (5H, m, phenyl-H), 7.54 • - (1H, d, 10 Hz, pyridazine-H), 8.29 (IH, s, CHO), 9.29 (IH, d, 8 Hz, 7-CONH, disappeared by addition of DgO).

Anal. Calc'd. for C2^H20N609S2-lHg0: C, 46.60; H, 3.58; N, 13.59; S, 10.37. Found: C, 46.70, 47.20; H, 3.25, 3.34; N, 13.37, 13-78; S, 10.84.

EXAMPLE 4 ' BB-S488; 7.-(D-Mandelamido)-5-(2-carboxymethyl-2>3dihydro-s-triazolo-f 4,3-b]pyridazin-3-on-6-ylthiomethyl)3-cephem-4-carboxylic Acid (4J A mixture of 7-(D-0~formylmandelamido-3-(2-car~ boxymethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on13 6-ylthiomethyl)-3-cephem-4-carboxylic acid (7) (484 mg., 0.81 m.mole) and sodium bicarbonate (748 mg., 8.9 m.mole) in water (4 ml.) was stirred for 4 hours at room temperature, and acidified to pH 1 with dilute HCl. The precipitate (500 mg.) was collected by filtration, washed with water (.2 ml.) and chromatographed on silica gel column (Wako-gel, C-200, 5 g·). The column was eluted with chloroform containing increasing methanol (3-5$) as eluent, and the fractions containing the product were combined, treated with a small amount of carbon and eva25 porated under reduced pressure. The residue was triturated with ether to give. 277 mg. of 7-(D-mandelamido)-3-(2-carboxymethyl-2,3-dihydro~s-triazolo[4,3-bJpyridazin-3-on-6ylthiomethyl)-3-cephem-4-carboxylic acid (BB-S488;, 4).

The nmr-estimation of this product showed 10$ of 7 still remained. 45S48 ir: Ν 3600-2400, 1770, 1720, 1520, 1495, 1365, ΙΙΙα,Λ 1230 cm1. uv: * max1 25it m 20000), 297 η® (sh, s> 9600).

EXAMPLE 5 Sodium Salt of BB-S488 Sodium-2-ethylhexanoate (SEH) (4.0ml., 1M solution in ethyl acetate) was added to a solution of 7-(D-mandelamido)-3-(2-carboxymethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4carboxylic acid (4_) (2.25 g., 3-93 m.mole) in THF (200 ml.). The precipitate was collected by filtration, washed with THF (50 ml.) and dried at βθ° C./l mmHg for 3 hours. Yield of sodium 7-(D-rnandelamido)-3-(2-carboxymethyl-2,3dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)3-cephem-4-carboxylate, 1.96 g. (bio-yield, 97%), M.P., 230-240° C. (dec.). The pH of the 10# aqueous solution was 3.6. ir: 36ΟΟ-3ΟΟΟ, 1765, 1710, 1605, 1390, 1360, 1190, 1080, 1065 cm1. uv: * max61 ““(Elcm) 252 (357)j 510 (sh’ lH0)’ nmr: 6 5jt5 dj lg HZj -5.97 dj ig HZj 2-H), 4.15 (IH, d, 14 Hz, 3-H), 4.53 (Hl, d, 14 Hz,' 3-H), .16 (HI, d, 4.5 HZ, 6-H), 5.36 (Hl, s, PhCH), 5-73 (HI, d, 4.5 Hz, 7-H), 7.13 (Hl, d, 10 Hz, pyridazine-H), 7-57 (5H, s, phenyl-H), 7.69 (IH, d, 10 Hz, pyridazine-H).

Anal. Calc'd. for C25IIigN60gS2Wa-5/4H20: C, 44.77; H, 3.51; N, 13.62; S, 10.39· Found: C, 44.93, 44.79; H, 3.31, 3.15; N, 13.41, 13-33; S, 10.19.

EXAMPLE 6 Aqueous IN. sodium hydroxide solution was added dropwise to a suspension of 7-(D-mandelamido)-3-(2carboxymethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-35 on-6-ylthiomethyl)-3-cephem-4-carboxylic acid (4.)(3-51 g.) in Water (20 ml.) to adjust to pH 6.0. The solution was lyophilized to yield 3,4 g. (bio-yield, 97%) of. disodium 7-(D-mandelamido.) -3-(2-carboxymethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-0 ephem-410 carboxylate. · M.p., >240 C. (dec.). The pH of the % aqueous solution was 5.4. ir: 4 3600-3000, 1760, 1710, 1605, 1390, 1360, II90, 1080, 1060 cm-1. . uv: zlmSer nn <El?m) 252 510 (12U)’ nmr: f 3-43 (IH, d, 19 Hz, 2-H), 3·90 (IH, d, 19 Hz, 2-H), 4.15 (IH, d, 14 Hz, 3-H), 4.53 (IH, d, 14 Hz, 3-H), 4.75 (2H, s, NCHgCO), 5-22 (IK, d, 4.5 Hz, 6-H), 5.42 (IH, s, PhCH), 5-73 (IH, d,. 4.5 Hz, 7-H), 7-22 (IH, d, 10 Hz, pyridazine-H), 7.65 (5H, s, phenyl-H), 7.77 (IH, d, 10 Hz, pyridazine-H).

Anal. Calc'd, for Cg3Hl8Ng08S2Na2’3/2H20: C, . 42.92; H, 3.29; N, 13.06; s, 9-96. Found: C, 42.90, 45.195 H, 3.06, 3.01; N, 13.04, 13.03; S, 9-97.

I Example 7 Substitution of the D-mandelic acid carboxyanhydride in the procedure of Example 1 of an equimolar weight of the carboxyanhydrides prepared 5 in similar fashion from the monosubstituted Dmandelic acids D-2-chloro-mandelic acid, D-3-chloro-mandelic acid, D-4-ehloro-mandellc acid, D-2-bromo-tnandelic acid, D-J-bromo-mandelic acid, D-4-bromo-mandelic acid, D-2-fluoro-mandelic acid, D-J-fluoro-mandelic acid, D-4-fluoro-mandelic acid, D-2-trifluoromethyl-mandelic acid, D-3-tr ifluorome thy1-ma nde11c acid, D-4-trifluoromethyl-mandelic acid, D-2-amino-mandelic acid, D-J-amino-mandelic acid, D-4-amino-mandelic acid, D-2-nitro-mandelic acid, D-J-nitro-mandelic acid, D-4-nitro-mandellc acid, 25 D-2-hydroxy-mandelic acid, D-3-hydroxy-mandelic acid, D-4-hydroxy-mandelic acid, 4SS48 D-2-methyl-mandelic acid,· D-3_methyl-mandelic acid, D-4-methyl-mandelic acid, D-2-methoxy-mandelic acid, .

D-J-methoxy-mandelic acid and D-4-methoxy-mandelic acid respectively produces 7-(D-2-ehloro-mandelamido)-3-(2-carboxymethyl-2,3dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid, '7-(D~3-chloro-mandelamido)-3-(2-carboxymethyl-2,3dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl) -3-cephetn-4-carboxy lie acid, 7-(D-4-chloro-mandelamido)-3-(2-carboxymethyl-2,3dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio15 methyl)-3-cephem-4-carboxylic acid, 7-(D-2-bromo-mandelamidoj-3-(2-carboxymethyl-2.,3dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4‘-carboxylic acid, 7-(D-3-bromo-mandelamido)-3-(2-carboxymethyl-2,3, 20 dihydro-s-triazolo[4,3-'b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid, 7-(D-4-bromo-mandeiamido)-3-(2-carboxymethyl-2,3dihydro-S“triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid, 7-(D“2-fluoro-mandelamido)-3-(2-carboxymethyl-2,3dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthioroethyl)-3-cephem-4-carboxylic acid, - 80 45S48 7-(D-3-fluoro-mandelamido)-5-(2-carboxymethyl-2,3dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid, 7_(D_4_fluoro-mandelamido)-3-(2-carboxymethyl-2,3dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-c ephem-4-carboxylie acid, 7-(D-2-trifluoromethyl-mandelamido)-3-(?-carboxymethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on6- ylthiomethyl)-3-cephem-4-carboxylic acid, 7- (D-3-trifluoromethyl-mandelamido)-3-(2-carboxymethyl-2,3-dihydro-s-triazolo[ lt,-3-b]pyridaziri-3“on6- ylthiomethyl)-3-cephem-4-carboxylic acid, 7- (D-4-trifluoromethyl-mandelamido)-3-(2-carboxymethyl-2,3-dihydro-s-triazolo[ 4,3-b]pyridazin-3-on6- ylthiomethyl)-3-cephem-4-carboxylic acid, 7- (D-2-amino-mandelamido)-3-(2-carboxymethyl-2,3~ dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-5-cephem-4-carboxylic acid, 7-(D-3-amino-mandelnmldo)-3-(2-carboxymethyl-2,3dihydro-fi-triazolo[4,3-b]pyridazin-3-on-b-ylthiomethyl·) -3-cephem-4-carboxylie acid, 7-(D-4-amino-mandelamido)-3- (2-c arboxymethy1-2,5dihydro-s-triazolo[4,3-b]pyridaain-3-on-6-ylthiomethyl )-3-cephem-4-carboxy lie acid, 7-(D-2-nitro-mandelamido)-3-(2-carboxymethyl-2,3- . dihydro-s-tria2olo[4,3-b]pyridazin-3-on-Cj-ylthiomethyl)-3-cephem-J)-carboxylic acid, 7-(D-3-nitro-mandelamido)-3-(2-carboxymethyl-2,3dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethy1)-3-cephem-4-carboxylic acid, , 45548 7_(D-4-nitro-mandelamido )-3-{2-cnrboxymetl\v 1-2,,·>dihydro-s-triazolo[4,3“b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid, 7-(D-2-hydroxy-mandelamido)-3-(2-carboxymethyl-2,35 ,dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid, 7-(D~3-hydroxy-mandelamido)-3-(2-c arboxymethyl-2,3dihydro-s-triazolo[4j3-b]pyridazin-3-on-6-ylthiomethyl) -3-cephem-4-carboxylic acid, 7-(D-4-hydroxy-mandelamido)-3-(2-carboxymethyl-2,3dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl) -3-cephem-4-carboxylic acid, 7-(D-2-methyl-mandelamido)-3-(2-carboxymethyl-2,3dihydro-s-triazolo[ 4,3->ij pyridoxin--S-on-i’-ylthlo15 methyl)-3-cephem-4-carboxylic acid, 7-(D-3-methyl-mandelamido)-3-(2-carboxymethyl~2,3dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio~ methyl)-3-cephem-4-carboXylic acid, 7-(D-4-methyl-mandelamido)-3-(2-carboxymethyl-2,?20 dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylie acid, 7-(D-2-methoxy-mandelamido)-3-(2-c arboxymethyl-2,3. dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid, 7-(D-3-methoxy-mandelamido)-3-(2-carboxyrnethyl-2,3dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid, and 7-(D-4-methoxy-mandelamido)-3-(2-carboxymethyl-2,3dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio30 methyl)-3-cephem-4-carboxylic acid, respectively. e -2 45548 EXAMPLE 8 Substitution for the D-mandelic acid carboxyanhydride in the procedure of Example 1 of an equimolar weight of the carboxyanhydride prepared in similar fashion from D-2-thiopheneglycolic acid and D-J-thiopheneglycolic acid respectively produces 7-(D-a-hydroxy-2-thienylacetamido)-3-(2-carboxymethyl2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylie acid and 7-(D-a-hydroxy10 3-thienylacetamido)-3-(2-carboxymethyl-2,3-dihydro-striazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem4-carboxylic acid, respectively.

EXAMPLE 9 7-(D-a-Hydroxy-a-phenylacetamido)-5-(2-carboxymethyl15 2,3“dihydrO“S-triazolo[4,3-b]p,yrldazin-3-on-6-ylthiomethyl )-3-cephem-4 -carboxylic Acid Prepared From 7-D-Mandelamidocephalosporanic Acid. 0.27 Mole of sodium 7-D-mandelamldocephalosporanate is suspended in 1000 ml. of 0.1 M phosphate buffer of pH 6.4 and there is added 0.31 moles of 2-carboxymethyl-2,3-dihydro-6-mercapto-s-triazolo[4,3-b]pyridazin-3-one. The solution is heated at 55° C. under a nitrogen atmosphere for five hours. After one hour the pH is adjusted to 6.4 by addition of a small amount of 40$ H^POi(. At the end of the five hour heating period the solution is cooled to 23° C. and the pH adjusted to 2 by the addition I 5548 of J N HCl under a layer of ethyl acetate. The product is extracted into ethyl acetate and stirred for 15 min. at 23° G. with 2 g. of (Darco KB) decolorizing charcoal. The mixture is then filtered through a-pad of diatomaceous earth (Celite) and the ethyl acetate removed from the filtrate under ' vacuum. The residue is triturated to a solid with diethyl ether, collected by filtration and dried over under vacuum to yield solid 7-(D-a-hydroxy a-phenylacefcamido)-3-(2-carboxymethyl-2,3-dihydro-striazolo[4,3 -ΐ> ]pyridazin-3-on-6-ylthiomethyl)-3cephem-4-carhoxylic acid.

Example 10 - 84 45548 BB-S 527; 7-[D(-)-Mandelamido]-3-[2-(2-carboxyethyl)-2,3dihydro-s-triazolo-(4,3-blpyridazin-3-on-6-ylthiomethyll3-cephem-4-carboxylic acid To a mixture of 7-amino-3-[2-{2-carboxyethyl)-2,35 dihydro-s-triazolo(4,3-b]pyridazin-3-on-6-ylthiomethyl]3-cephem-4-carboxylic aoid (679 mg, 1.5 m mol) and NaHCO3 (445 mg, 5.3 m mol) in 50% aqueous acetone was added D(-)-mandelic aoid O-carboxyanhydride (400 mg, 2.3 m mol) at O°C. The mixture was stirred at room temperature for 1 hour and evaporated to remove the organic solvent. The aqueous solution was washed with ether (3 x 10 ml), adjusted to pH 1 with dil. HC1 and filtered to collect the crude product, which was dissolved in THF (10 ml), filtered to remove insolubles and evaporated under reduced pressure. The oily residue was triturated with ether. The solid (476 mg) was chromatographed on a column of silica gel (Wako-gle C-200, g) and eluted with MeOH-CHCl-j (MeOH: 0-3%). Fractions which contained the desired product were combined and evaporated to yield 287 mg (33%) of BB-S 527. M.p. >15'5°C (dec.), ir: VKB^ 3600 - 2400, 1780, 1720, 1550, 1520 cm-1. ΓΠαΧ uv: buffer 253 nm (e 20000), 298 nm (ε 9000).’ ΓΠαΧ Anal. Calc'd. for C24H22N6°8S2,3/2n2°! C, 46.98; H, 4.11; N, 13.70; S, 10.45. Found: C, 47.25, 47.39; H, 3.80, 3.76; N, 12,87, 12,77? S, 10.17. 45S48 The sodium salt of BB-S 527 A suspension of the free acid of BB-S 527 {240 mg, 0.4 m mol) in water (5 ml) was adjusted at pH 6.8 with 1 N-NaOH (0.7 ml).to give the clear solution, which was freeze-dried to leave 234 mg (91%, bio-yield) of the sodium salt of BB-S 527 as pale yellow powder. M.p. >210°C (dec.). ir: 3600 — 2800, 1770, 1710, 1600 cm’1, uv: λρΗ 7 Buffer 253 nm (ε 21000) 298 nm (ε 9300). ΠιαΧ Anal, Calc’d. for C^H^NgOgS^a^E^O: C, 43.24; H, 3.63; N, 12.61; S, 9,62. Found: C, 43.39, 43.43; H, 3.20, 3.36; N, 12.63, 12.68; S, 9.42, 9.22.

In vitro antibacterial activity of BB-S 527 compared With BB-S 488 and cefamandole (determined by Steers’ agar dilution method on Mueller-Uinton agar plate) 3 5 MIC (mcg/ml) Organism BB-S 527 BB-S 488 cefamandole S. aureus Smith 1.6 0.8 0.2 / S. aureus 0.4 0.4 0.1 . S. aureus BX-1633 3.1 3.1 0.4 20 St. faecalis >100 >100 >100 E. coli NIHJ. 0.4 0.2 0.05 E. coli ATCC 8739 12.5 6.3 *3.1 Ε» coli juhl 0.4 0.2 0.8 E. coli BX-1373 6.3 3.1 3.1 25 E, coli 0.1 0.1 0.1 E. coli 0.1 0.05 0.1 E. coli 6.3 3.1 1.6 - 86'·' 4S548 Kl. pneumoniae 6.3 3.1 3.1 Kl. pneumoniae 0.2 0.1 0.8 Kl. pneumoniae 0.8 0.4 0.8 Kl. pneumoniae 0.4 0.2 0.8 5 Pr. vulgaris 0.1 0.1 0.2 Pr. vulgaris 12.5 0.8 50 Pr. mirabilis 0.2 0.05 0.8 Pr. mirabilis 0.1 0.05 •0.2 Pr. morganii >100 >100 >100 10 Pr. morganii 0.4 0.2 0.8 Pr. rettgeri 0.2 0.2 0.4 Ps. aeruginosa >100 >100 >100 Ps. aeruginosa >100 >100 >100 Shig, dysenteriae 0.025 0.025 0.1 15 Shig. flexneri 50 25 6.3 Shig. sonnei 0.1 0.05 0.2 Serr. marcescens >100 >100 100 Enterob. cloacae 6.3 3.1 3.1 Sal. enteritidis 0.05 0.025 0.05 20 Sal. typhosa 0.1 0.05 0.1 B. anthracis 0.4 0.2 0.4 4'SS48 Example 11 Substitution for the D-mandelic acid carboxyanhydride in the procedure of Example 10 of an equimolar weight of the carboxyanhydrides prepared in similar fashion from the monosubstituted Dmandelic acid3 D-2-ehloro-mandelic acid, D-3-ohloro-mandelic acid, .D-4-chloro-mandelic acid, D-2~br0mo-mandeIic acid, D-3-bromo-mandelic acid, D-4-bromo-mandelic acid, D-2-fluoro-mandelie acid, D-3~fluoro-mandelic acid, D-4-fluoro-mandelic acid, D-2-trifluoromethyl-mandelic acid, D~3“trifluoromethyl-mandelic acid, D-4-trifluoromethyl-mandelic acid, D-2-amino-mandelic acid, D-3-amino-mandelic acid, D-4-amino-mandelic acid, D-2-hitro-mandelic acid, D“3“nltro-mandelic acid, D-4-nitro-mdndelic acid, D-2-hydroxy-mandelic acid, D-p-hydroxy-mandelic acid, D-4-hydroxy-mandelic acid, - 88 45548 D-2-methyl-mandelic acid, D-J-methyl-mandelic acid, D-4-methyl-mandelic acid, D-2-methoxy-mandelic aeid, D-J-methoxy-mandelic acid and D-4-methoxy-mandelic acid respectively produces 7-(D-2-chloro-mandelamido)-3-(2-carboxyethyl-2,3dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid, 10 7-(D-3-chloro-mandelamido)-3-(2-carboxyethyl-2,3dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl )-3-cephem-4-carboxylic acid, 7-(D-4-chloro-mandelamido)-3-(2-carboxyethyl-2,3dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio15 methyl)-3-cephem-4-carboxylic acid, 7-(D-2-bromo-mandelamido)-3-(2-carboxyethyl-2,3dihydro-s-triazolo£4,3-b]pyridazin-3-on-6-ylthiomethyl )-3-cephem-4-carboxylic acid, 7-(D-3-bromo-raandelaraido)-3-(2-carboxyethyl-2,320 dihydro-s-triazdlo[4,3-b]pyridazin-3-on-6-ylthiomethyl )-3-cephem-4-carboxy lie acid, 7-(D-4-bromo-mandelamido)-3-(2-carboxyethyl-2,3dihydro-s-triazolo[4,3-b]pyridazin-3-on~6-ylthiomethyl)-3-cephem-4-carboxylic acid, 7- (D-2-f luoro-mandelainido.).-3- (2-carboxyethyl-2,3dihydro-s-triazolo[4,5-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid, ! ; - es 1 ' 45S48 7-(D-3-flu.oro-mandelaraido)-3-(2-carboxyethyl-2,3dihydro-s-triazolo[4,3-'b]pyvidazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid, 7-(D-4-fluoro-mandelamido)-3-(2-carboxyethy 1-2,35 dihydro-s-triazolo[4,3-h]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid, 7- (D-2-trif luorome'thyl-mandelainido) -3- (2-carboxyethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on6-ylthiomethyl)-3-eephem-4-carboxylic acid, 1° . 7-(D-3-trifluoro3nethyl-mandelamido)-3-(2-carboxyethyl-2,3-dihydro-s-triazolo[4J3rb]pyi'idazin-3-on6- ylthiomethyl)-3-cephem-4-carboxylic acid, 7- (D-4-trifTuoromethyl-»andelamido)-3-(2-carboxyethy 1-2,3-diliydro-s-triazolo[4,3-b]pyridazin-3-on15 6-jrlthiomethyi)-3-cephem-4-carboxylic acid, 7-(D-2-amino-mandelainido)-3-(2-carboxyethyl-2,3dihydro-s-tfiazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid, 7-(D-3~amiho-mandelamido)-3-(2-carboxyethyl-2,320 dihydfo-s-triazolof 4,3-b]pyridazin-3-on-6-ylthioBiethyl)-3-cephem-4-carboxylic acid, 7~(D-4-amixio-mandelami.do)-3- (2-carboxyethy 1-2,3dihydro-s-triazolo[4,3-h]pyridaain-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid, 7-(D-2-nitro-mandelamido)-3-(2~carboxyethyl-2,3- . dihydro~s-triazolo[4,3-b]pyridazin-3~on-6-ylthiomethyl)-3~cephem-4-carboxylic acid, 7(D-3-nitro-mandelamido)-3-(2-carboxyethyl-2,3dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio30 methyl)-3-cephem-4-carboxylic acid, 7-(D-4-nitro-mandelamido)-3-(2-carboxyathyl-2,3dihydro-s-triazolo[4,3-b]pyrldazin-3~on-6-ylthiomethyl)-3-oephem-4-carboxylic acid, 7- (D-2-hydroxy-mandelamido)-3- (2-carboxyethyl-2,35 diliydro-s-triazolof 4,3-b]pyrldazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid, 7- (D-3-hydroxy-mandelamido) -3-(2-carboxjethyl-2, 3dihydro-s-triazolo[4,3-bipyridazin-3-on-6-ylthiomethyl)-3-cephem-4~carboxylic acid, 10 7-(D-4-hydroxy-mandelamido )-3-( 2-carboxyathyl-2,3dihydro-s-triazolo[4,3-b]pyridazin-3-on-6~ylthiomethyl)-3-cephem-4-carboxylic acid, - (D-2-methyl-mandelamido) -3 - (2-c arboxyathyl -2,3 dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio15 methyl)-3-cephem-4-carboxyllc acid, 7- (D-3-methyl-mandelamido) -3- (2-carboxyathyl~2,3dibydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl )-3-cephem-4-carboxylic acid, 7-(D-4-methyl-mandelaraido)-3-(2-carboxyethyl-2,320 dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid, 7-(D-2-methoxy-mandelamido)-3-(2-carboxyethyl-2,3dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid, 25 7-(D-3-methoxy-mandelamido)-3-(2-carboxyethyl-2,3dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid, and 7-(D-4-methoxy-mandelamido)-3-(2-carboxyethyl-2,3dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio30 methyl)-3-cephem-4-carboxylic acid, respectively. - 91 4554® EXAMPLE 12 Substitution for the D-mandelic acid carboxyanhydride in the procedure of Example 10 of an equimolar weight of the carboxyanhydride prepared in similar fashion from D-25 thiopheneglyoolio acid and D-3-thiopheneglycolic acid respectively produces 7-(D-a-hydroxy-2-thienylacetamido)3-(2t-carboxyethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin3-on-6-ylthiomethyl]-3-cephem-4-carboxylic acid and 7(D-a-hydroxy-3-thienylacetamido)-3-(2-carboxyethyl-2,310 dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)3-cephem-4-carboxylic acid, respectively. ι - 92 45548 EXAMPLE 13 C02Na CH2C00Na 7-f(2Z)-2-Methoxyimino(fur-2-yl)acetamiao)-3-(2-carboXymethyl-2,3-dihydro-s-triazolo{4,5-b]pyridazin-3-on-6ylthlomethyl)-3-cephem-4-carboxylic Acid Disodium Salt; BB-S5H.

To a solution of (2Z)-2-methoxyimino(fur-2-yl)acetic acid (507 mg., 3 m,moles) and triethylamine (0.42 ml., 3 m.moles) in dichloromethane (6 ml.) was added oxalyl chloride (0.26 ml., 3 m.moles) at 0-5° C. and the mixture was stirred for 30 minutes. The mixture was evaporated at reduced pressure to give an oily residue of the acid chloride which was dissolved in dry acetone (10 ml.).

After filtration the acetone solution was added to a mixture of 7-amino-3-(2-carboxymethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4carboxylic acid (1.31 g., 3 m.moles) and NaHCO^ (504 mg., m.moles) in water (20 ml.) at 0-5° C. The mixture was stirred at 0-5° C. for 2 hours with the acetone being, removed under reduced pressure. The aqueous solution was washed with ether (2 x 50 ml.) and adjusted to pH 1-2 with cone. HC1 to afford a precipitate which was collected by filtration, washed with water and dried in vacuo. The solid was dissolved in THF (tetrahydrofuran) (40 ml.) and 43548 filtered. To the filtrate was added 1 M-SEH (sodium . ethylhexanoate) in ethyl acetate (3 ml.) and the resulting precipitate was collected by filtration and dried in’ vacuo. Yield of 7-[(2Z)-2-methoxyimino(fur-2-yl)acet5 amido]-3-{2-carboxymethyl~2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid disodium salt was 1.0 g. (54#) mp >210° C. (dec.). ir: 1770, 1710, l680,-l6l0, 1550 cm”1. uv: λρΗ ^Uffer 257 nm (ε 25000), 277 nm (ε 24000). nmr: SDMp°”d6 7.70 (IH, br, furan-Ηα), 7..52 (IH, d, . J=9<5 Hz, pyridazine-H), 6.87 (IH, d, J=9-5 Hz, pyridazine-H), 6,5-6.6 (2H, m, furan-Ηβ), 5.58 (IH, m, 7-H), 5-00 (IH, d, J=4.5 Hz, 6-H), 3.84 (3H, s, OCB^).

Anal. Calc'd. for CggH^^OgSgNag’HgO: C, 40.55; . H, 2.94; N, 15.05; S, 9-84. Found: C, 40.81, 41.02; H, 3.08, 3.22; N, 14.69, 14.86; S, 9-70, 9·θ2.

EXAMPLE 14 Η CO-N.

XOCH. <Ί p Ahx\^J-ciu;h COOH 3 θ COgH 7-((2Z)-2-Methoxyimino(fur-2-yl)acetamido]-3-(g-(g-carboxyethyl)-2,3-dihydro-s-triazolo( 4,3-b]pyridazin-3-on5 6-ylthiomethyl]-3-cephem-4-carboxylic Aeid Sodium Salt; BB-S526.

The acid chloride prepared from (2Z)-2~methoxylmino(fur-2-yl)acetic acid (169 rag,, 1 m.mole) was dissolved in dry acetone (5 ml.) and filtered to remove in10 solubles. The filtrate was added to a solution of 7-amino-3-[2-(2-carboxyethyl)-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl]-3-cephem-4-car• boxylic Acid, (452 mg., 1 m.mole) and NallCO^ (336 mg., • 4 m.mole) in water (10 ml.). The reaction mixture was stirred for 2 hours in an ice-water bath. Acetone was removed at reduced pressure. The aqueous solution was Washed with ether (2 x 10 ml.) and adjusted to pH 1-2 with cone. HCl. The resulting precipitate was collected by filtration, washed with water and dried under reduced pressure. A solution of the precipitate in THP (10 ml.) was treated with active carbon. A SEH solution in ethyl acetate (1 M>0.8 ml.) was added to the THF solution to give 7-((2Z)-2-methoxyimino(fur-2-yl)acetamido]-3-(2-(2carboxyethyl)-2,3-dihydro-s-triazolo[ 4,3-b]pyridazin-325 on-6-ylthiomethyl]»3-cephem-4-carboxylic acid sodium salt which was collected by filtration. Yield: 410 mg. (66% as mono Na salt). Mp. >205’ C. (dec.). “ 4SS48 ir: 3600-2800, 1770, 1710, 1600, 1550 cm-1.

IuclX uv: λρΗ Z=5Uffer 257 nm (ε 27000), 276 ( e 26100). max nmr: , -δ0Μρ^δ+Π2° 2.70 (2H, t, J=6 Hz, CHg), 3.3-4.5 (9H, m, 2-H, 3-CHg, CHg and OCHg), 4.96 (IH, d, J=5.5 Hz, 6-H), 5.53 (IH, d, J=5.5 Hs, 7-H), 6.55 (2H, m, furan-H), 6.92 (lH, d, J=10 Hz, pyridazine-H), 7.53 (IH, d, J=10 Hz, pyridazine-H), 7.68 (IH, br, furan-H).

Anal. Calc'd. for CggHgQ^OgSgNa-HgO: C, 42.92; H, 3.45; N, 15.23; S, 9.96. Pound: C, 43.08, 42.77; H, 3.20, 3.03; N, 14.96, 14.76; S, 9.96.

TABLE 7 In vitro Activity Using Mueller-Hinton Agar By the Serial Dilution Method Geometric Mean of MIC BB-S511 (Ex. 13) (Meg./ml.) BB-S526 (EX. 14) Cefuroxime S. aureus E3 strains) 1.97 1.6 1.24 E. Coli (7) 0.58 0.78 1.28 20 Kl. pneumoniae (4) 0.47 0.93 3.1 Proteus (6) 0.021 0.061 0.88 shig. (3), Serr. (1) . Enterab. (1), Sal.(2) B. anthraois (1). 1.11 2.41 4.06 25 S. pyogenes (5) 0.032 0.032 0.025 S. viridans (5) 0.15 0.4 0.1 D. pneumoniae (5) 0.037 0.056 0.0125 M. meningitidis (5) 2.37 3.60 1.6 30 N. gonorrhoeae (5) 1.36 1.6 0.4 H. influenzae (7) . 0.64 0.71 1.16 Cefuroxime is sodium ι SR,7R-3-carbamoyloxymethyl-7-(2Z)-2- methoxyimino (fur-2-yl)acetamidoceph-3-em-4-carboxylate.

TABLE 8 Geometric Means of MIC's Against 3 Strains of S. aureus and 27 Strains of Gram-negative Bacteria (meg./ml., Mueller-Hinton Agar) No. of Strains BB-S511 BL-S786 S. aureus 1 1.6 1.6 S aureus, Penicillin-R 2 0.6 1.6 E. coli 6 0.2 0.2 E. coli, Cephalosporin-R 1 6.3 12.5 K. pneumoniae 4 0.7 0.3 Indole (-) Proteus 2 0.1 0.2 Indole (+) Proteus 3 0.05 0.3 Indole (+) Proteus, Cfephalosporin-R 2 6.3 50.1 S. marcescens 1 25 >100 E. cloacae 1 3.1 1.6 Shigella, Salmonella 5 0.3 0.5 P. aeruginosa 2 >100 >100 BL-S786 is 7-/a-(2-aminomethylphenyl) acetamido J7-3-/(l-car- boxymethyltetrazol-5-ylthio)methyl7-3-cephem-4-carboxylic acid.

TABLE 9 Geometric Means of MIC's Aganist 18 Strains of S. marcescens BB-S511 BL-S786 .4 85.9 EXAMPLE 15 Substitution of an equimolar weight of 2-ethoxyimino-2-(fur-2-y-l)acetic acid for the 2-methoxyiminofuryl acetic acid used in the procedures of Examples 13 and 14 produces 7-(2-ethoxyimino-2-furylacetamido)-3-(2-carboxym'ethyl-2,3-dihydro-s -triazolof 4,3-b]pyridazin-3-on-6ylthiomethyl)-3-cephem-4-carboxylic acid and 7-(2-ethoxyimino-2-furylacetamido)-3-[2-(2-carboxyethyl)-2,3-dihydr0s-triaz olo[ 4,3-b ]pyridazin-3-όη-6-ylthiomethyl]-3-c ephem4-carboxylic acid, respectively.

EXAMPLE 16 Substitution of an equimolar weight of 2-n-propoxyimino-2-(fur-2-yl)acetic acid for the 2-methoxyiminofuryl acetic acid used in the procedures of Examples 13 and 14 produces 7-(2-n-propoxyimino-2-furylacetamido)-3-(2-carboxymethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6ylthiomethyl)-3-cephem-4-carboxylic acid and 7-(2-n-propoxyimino-2-furylacetamido)-3-(2-(2-carboxyethy1)-2,3-dihydros.-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl]-3-cephem4-carboxylic acid, respectively.

EXAMPLE 17 Substitution Of an equimolar weight of 2-n-butoxyimino-2-(fur-2-yl)acetic acid for the 2-methoxyiminofuryl acetic acid used in the procedures of Examples 13 and l4 produces 7-(2-n-butoxyimino-2-furylacetamido)-3-(2-carboxymethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6ylthiomethyl)-3-cephem-4-carboxylic acid and 7-(2-n-butoxy25 imino-2-furylacetamido)-3-[2-(2-carboxyethyl)-2,3-dihydro· s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl]-3-cephemI 4-carboxylic acid, respectively.

EXAMPLE 18 The products of Examples 13-17 nn: prepared os syn isomers essentially free of the corresponding anti isomers by the use in the procedures of those examples of purified syn isomers of the appropriate 2-alkoxyimino-2-(fur-2-yl)acetic acid. Conversion of part Of the syn isomer to anti isomer during preparation of the acid chloride from the acid is substantially avoided by minimizing its exposure to •hydrogen chloride, e.g. by first converting the acid to its anhydrous sodium salt and by treating that salt with oxalyl chloride under anhydrous conditions in the presence of a hydrogen ion acceptor such as dimethylformamide.

Such syn isomers are also named as (2Z)-2alkoxyimino-2-(fur-2-yl)acetic acids. 5848 EXAMPLE 19 An injectable pharmaceutical composition is formed by adding sterile water or sterile saline solution (2 ml.) to 100-500 mgm. of ?-[(2J5)-2-methoxyimino5 (fur-2-yl)acetamidoI-3-(2-carboxymethyl-2,3-dihydro-striazolo[4,2-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem4-carboxylie acid disodium salt.

Pharmaceutical compositions of the sodium and potassium salts of the other compounds of the present invention, preferably in the form of the pure syn isomer, are formulated in a similar manner.

When the compounds are first prepared in the form of the free acid they are converted to the desired, highly vzater soluble potassium salt by treat15 ment with potassium 2-ethylhexanoate using the procedure of Example 13.

It is occasionally advantageous to have admixed with said solid cephalosporin as a stabilizing and/or solubilizing agent a sterile, anhydrous solid such as sodium carbonate, potassium carbonate or lithium carbonate (e.g. in about 5 or 6 percent by weight of the weight of the cephalosporin) ox· such as L-lysine, arginine or histidine (e.g) in about 20-50% by weight of the weight of the cephalosporin) or such as a sodium, potassium or calcium salt of levulinic acid, citric acid, ascorbic acid, tartaric acid or pyruvic acid (e.g. in about 25-200% by weight of the weight of the cephalosporin) or such as sodium bicarbonate, ammonium carbamate alkali metal or ammonium phosphates or N-methyl30 glucamine (per British Specification No, 1,380,741). - 100 EXAMl’bl' BB-S510; 7-[(2Z)-2-Methoxyimlno(fur-2-yl)acetamido]-3(2-methyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on6-ylthiomethyl)-3-cephem-4-carboxylic Acid.Sodium Salt.

To a solution of (2Z)-2-methoxyimino(fur-2-yl)acetic acid (253 mg., 1.5 m.mol.) and triethylamine (0.2 ml., 1.5 m.mol.) in dichloromethane (3 ml.) was added oxalyl chloride (0.13 ml., 1.5 m.mol.) at 0-5° C. and the mixture was stirred for 30 minutes and evaporated at reduced pressure to give the acid chloride as an oil which was dissolved in dry acetone (5 ml.) and filtered to remove insolubles. The acetone solution was added to a mixture of 7-amino-3-(2-methyl-2,3-dihydro-s-triazclo[4,3-b]pyridazin-3-on~6-ylthiomethyl)-3-cephem-4-carboxylic acid (591 mg., 1.5 m.mol.) and NaHCO^ (504 mg., m.mol.) in water (10 ml.) at 0-5° C. The reaction mixture was stirred at 0-5° C. for 3 hours. Acetone was removed at reduced pressure and the residual aqueous solution was washed with ether (2 x 30 ml.) and adjusted to pit 1-2 with concent rated IIC1. 'J'ho precipitate which was collected by filtration, washed with water and dried in vacuo, was dissolved in THF (30 ml.) and filtered to remove insolubles. To the THF solution was added a solution of sodium ?-ethylhexanoate (SEH, 1 M, 1.5 ml.) in “ 101 45SA8 ethyl acetate and the resulting precipitate was collected by filtration and dried in vacuo. Yield: 492 mg. of 7-[(2Z)-2-methoxyimino(fur-2-yl)acetamido]-3-(2-methyl2,3-dihydro-s-triazolo[4,3-b]pyridazin-3“On-6-ylthio5 methyl)-3-cephem-4-carboxylie acid sodium salt (58%), M.p. >l80° C. (dec.). ir: 1770, 1720, 1670/1600, 1550 cm-1, uv: λΡ.Η7^ΓβΓ 257 nm (ε 22600), 277 nm (e 22300). nmr: 7-53 (IH, d, J=1.5 Hz, furan-Ha), 7.35 (IH, a, J=9„5 Hz, pyridazine-H), 6.90 (IH, d,J=9.5'Hz, pyridazine-H), 6.72 (IH, d, J=3.0 Hz, furan-Hp), 6.48 (IH, q, J=1.5 and 3.0 Hz, furan-HP), 5-72 (IH, d, J=4.5 Hz, 7-H), 5.14 (IH, d, J=4<5 Hz, 6-H), 2.94 (3H, s, O-CH^), 3.61 (3H, s, N-CHj).

Anal. Calc’d. for C^H-j^OySgNa'l/^THF-HgO: C, 44.44; H, 3.89; S, 10.32. Found: C, 44.89; H, 3-92; s, 9.67. 102 “ 4554 8 TABLE 10 In vitro Activity Using Mueller-Hinton Agar By the Serial Dilution Method Geometric Mean of MIC (Meg./ml.) BB-S510 (EX.20) BB-S515 Cefuroxime S. aureus (3 strains) 0.62 2.48 1.24 E. Coli (7) 2.11 2.33 1.28 10 Kl. pneumoniae (4) 6.3 3.1 3.1 Proteus (6) 1.39 1.11 0.88 Shig. (3) ,Serr (1) Enterab.(1),Sal.(2) B. anthracis (1) 6.26 5.26 4.06 15 S. pyogenes (5) 0.0125 0.032 0.025 S. viridans (5) 0.13 0.59 0.1 D. pneumoniae (5) 0.021 0.1 0.0125 N. meningitidis (5) 1.03 5.45 1.6 N. gonorrhoeae (5) 0.26 2.07 0.4 20 H. influenzae (7) 0.35 2.11 1.16 Cefuroxime is sodium 6R,7R-3-carbamoyloxymethyl-7-(2Z)-2methoxyimino (fur-2-yl)acetamidoceph-3-em-4-carboxylate. 103 48S48 TABLE 11 Geometric Means of MIC's Against 3 strains of S. aureus and 27 strains of Gram-negative Bacteria (meg./ml., Mueller-Hinton Agar) No. of Strains BB-S510 BL-S786 S. aureus 1 0.1 1.6 S. aureus, Penicillin-R 2 0.1 1.6 E. coli 6 0.1 0.2 E. coli, Cephalosporin-R 1 3.1 12.5 10 K. pneumoniae 4 2.6 0.3 Indole (-) Proteus 2 1.1 0.2 Indole (+) Proteus 3 0.2 0.3 Indole (+) Proteus Cephalosporin-R 2 6.3 50.1 15 S. marcescens 1 6.3 >100 E. cloacae 1 3.1 1.6 Shigella, Salmonella 5 0.5 0.5 P. aeruginosa 2 >100 >100 BL-S786 is 7-/ά-(2-aminomethylphenyl) acetamido/-3-/(1-car20 boxymethyltetrazol-5-ylthio)methyl/“3Cephem-4-carboxylicacid.

TABLE 12 Geometric Means of MIC's Against 18 Strains of S. roarcescens BB-S510 BL-S786 7.9 85.9 - 104 4554S EXAMPLE 21 Substitution of an equimolar weight of 2-ethoxyimino-2-(fur-2-yl)acetic acid for the 2-methoxyiminofuryl acetic acid used in the procedure of Example 20 produces 7-(2-ethoxyimino-2-furylacetamido)-3-(2-methyl-2,3-dihydros-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem4-carboxylie acid.

EXAMPLE 22 Substitution of an equimolar weight of 2-n-propoxy10 imino-2-(fur-2-yl)acetic acid for the 2-methoxyiminofuryl acetic acid used in the procedure of Example 20 produces 7-(2-n-propoxyimino-2-furylacetamido)-3-(2-methyl-2,3dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)3-cephem-4-carboxylic acid.

EXAMPLE 23 Substitution of an equimolar weight of 2-n-butoxyimino-2-(fur-2-yl)acetlc acid for the 2-methoxyiminofuryl acetic acid used in the procedure of Example 20 produces 7-(2-n-butoxyimino-2-furylacetamido)-3-(2-methyl-2,320 dihydro-s-triazolo[4,3-b]pyridazin-3-on“6-ylthiomethyl)3-eephem-4-carboxylic acid. - 105 4SS48 EXAMPLE The products of Examples 20-23 were prepared as syn Isomers essentially free of the corresponding anti isomers by the use in the procedures of those examples of purified 5 syn Isomers of the appropriate 2-alkoxyimino-2-(fur-2~yl)~ acetic acid. Conversion of part of the syn isomer to anti isomer during preparation of .the acid chloride from the acid is substantially avoided by minimizing its exposure to hydrogen chloride, e.g. -by first converting the acid to its anhydrous sodium salt and by treating that salt with oxalyl chloride under anhydrous conditions in the presence of a hydrogen ion acceptor such as dimethylformamide.

Such syn isomers are also named as (2Z)-2alkoxyimino-2-(fur-2-yl)acetic acids. - 106 EXAMPLE 25 An injectable pharmaceutical composition is formed by adding sterile water or sterile saline solution ( 2 ml.) to 100-500 mgm. of 7-/(2Z)-2-methoxyimino5 (fur-2-yl)acetamidq7-3-(2-methy1-2,3-dihydro-striazolo/4,3-b7pyridazin-3-on-6-ylthiomethyl)-3-cephem4-carboxylic acid and disodium salt.

Pharmaceutical compositions of the sodium and potassium salts of the other compounds of the present invention, preferably in the form of the pure syn isomer, are formulated in a similar manner.

When the compounds are first prepared in the form of the free acid they are converted to the desired, highly water soluble potassium salt by treat15 ment with potassium 2-ethylhexanoate using the procedure of Example 20.

It is occasionally advantageous to have admixed with said solid cephalosporin as a stabilizing and/or solubilizing agent a sterile, anhydrous solid such as sodium carbonate, potassium carbonate or lithium carbonate (e.g. in about 5 or 6 percent by weight of the weight of the cephalosporin) or such as L-lysine, arginine or histidine (e.g. in about 20-50% by weight of the weight of the cephalosporin) or such as a sodium, potassium or calcium salt of levulinic acid, citric acid, ascorbic acid, tartaric acid or pyruvic acid (e.g. in about 25-200% by weight of the weight of the cephalosporin) or such as sodium bicarbonate, ammonium carbamate, alkali metal or ammonium phosphates or N-methyl30 glucamine (per British Patent Specification No. 1,380,741) - 107 I 4SS48

Claims (14)

1. CLAIMS:1. A compound having the formula /S ANH-CH-CH '/ COOH -N C-C.H--S 2 wherein A is R-CH· OR wherein R is hydrogen or formyl; R is (w 0 II X 0R 2 or Y is hydrogen, chlorine, bromine, fluorine, trifluoromethyl, amino, nitro, hydroxy, (lower)alkyl of 1 to 4 10 carbon atoms or (lower)alkoxy of 1 to 4 carbon atoms; R is (lower)alkyl of 1 to 4 carbon atoms and i wherein A is methyl or~(CH 2 ) n C00H and n is one or two; or an easily hydrolyzed ester or nontoxic pharmaceutically acceptable salt thereof, or the benzaldelhyde or salicylaldehyde 15 Schiff base or acetaldehyde or acetone derivative of such a compound when it contains an amino group.

2. A compound according to Claim 1 having the Dconfiguration in the 7-side chain and the formula 108 wherein n is one or two, R is hydrogen or formyl and R is or and Y is hydrogen, chlorine, bromine, fluorine, trifluoromethyl amino, nitro, hydroxy, lower alkyl of 1 to 4 carbon atoms or lower alkoxy of 1 to 4 carbon atoms.

3. A compound according to Claim 2, wherein R is chlorophenyl, bromophenyl, trifluoromethylphenyl, tolyl or methoxyphenyl.

4. A compound according to Claim 2, wherein R is Ij

5. A compound according to Claim 2, wherein R is phenyl.

6. A compound according to Claims 2, 3, 4, or 5 wherein R is hydrogen.

7. A compound according to any of Claims 2, 3, 4 or 5 wherein R is formyl. 0. A compoind according to Claim 1 having the formula III - 109 /43548 ί wherein A is methyl or — (CH^^COOH and n is one or two 2 and R is alkyl containing 1 to 4 carbon atoms, the easily hydrolyzed esters and the non-toxic pharmaceutically acceptable salts of those acids. 5 9. A compound according to Claim 8 having the Formula (crt 2 ) n co°H wherein R is alkyl containing 1 to 4 carbon atoms, n is one or two and R is hydrogen or a conventional, pharmaceutically acceptable, easily hydrolyzed ester forming group; or a non10 toxic, pharmaceutically acceptable salt thereoF, said compound being at least 75?i by weight in the form of its syn isomer. 10. A syn isomer of a compound according to Claim 9 having the formula NH-CH—CH ? H 2 c ,,c-ch 2 - s c-or3 o {CH g ) n COOH •I 15 wherein R is alkyl containing 1 to 4 carbon atoms, π is one or two and R is hydrogen, pivaloyloxymethyl, acetoxymethyl, methoxymethyl, acetonyl, phenacyl, p-nitrobenzyl, ρ,^,β-trichloroethyl, 3-phthalidyl or 5-indanyl or a non-toxic, pharmaceutically acceptable salt thereof. 11. A syn isomer of a compund according to Claim 9 having the formula . 110 c-απ ο (C H 2) n c °0H wherein R is alkyl containing 1 to ί carbon atoms and n is one or two or a non-toxic pharmaceutically accejptable salt thereof. 5 12. A syn isomer of a compound according to Claim 11 having the formula wherein n is one or two or a non-toxic, pharmaceutically acceptable salt thereof. 13. A syn isomer of a compound according to Claim 9 having the formula wherein R 5 is hydrogen or a conventional, pharmaceutically acceptable, easily hydrolyzed ester forming group and n is one 15 . or two or a non-toxic, pharmaceutically acceptable salt thereof. -Ill 4^348 14. A compound of any of the Claims 6 to 13, wherein n is one. 15. A compound of any of the Claims 8 to 13, wherein n is two. 16. A compound according to Claim 8 having the formula 1 3 wherein R is alkyl containing 1 to 4 carbon atoms and R is hydrogen or a conventional, pharmaceutically acceptable, easily hydrolyzed ester forming group; or a non-toxic, pharmaceutically acceptable salt thereof, said compound being at least 75?» by weight in the form of its syn isomer. 17. A byn isomer of a compound according to Claim 16 having the formula 1 3 wherein R is alkyl containing 1 to 4 carbon atomB and R is hydrogen, pivaloyloxymethyl, acetoxymethyl, methoxymethyl, acetonyl, phenacyl, p-nitrobenzyl, β, β, ^-trichloroethyl, 3-phthalidyl or 5-indanyl or e non-toxic pharmaceutically acceptable salt thereof. 18. A syn isomer of a compound according to Claim 16 having the formula 112 Assa ο ο q wherein R is alkyl containing 1 to 4 carbon atoms or a non-toxic pharmaceutically acceptable salt thereof. 19. The syn isomer of a compound according to Claim 16 5 having the formula O-E OCH, or a non-toxic, pharmaceutically acceptable salt thereof. 20. The syn isomer of a compound according to Claim 16 having the formula wherein R^ is hydrogen or a conventional, pharmaceutically acceptable, easily hydrolyzed ester forming group, or a non toxic, pharmaceutically acceptable salt thereof. - 113 1' 21» A compound according to any one of the preceding claims io the form of the sodium or potassium salt. Z2. A compound according to any one of the preceding claims in the form of the pivaloyloxymethyl, acetoxymethyl, acetonyl, 5 phenacyl or methoxymethyl ester. 23. A compound according to Claim 1 having the formula R-CH-C-NH-CH — CH 0» COOM wherein n is one or two, R is hydrogen or formyl and R is or 10 and Y is hydrogen, chlorine, bromine, fluorine, trifluoromethyl, amino, nitro, hydroxy, lower alkyl of 1 to 4 carbon atoms or lower alkoxy of 1 to 4 carbon atoms and M is -CH0C(CH 2 ) n R, S CH0C(CH 2 ) n < R8 Rl HR^rS 11445548 chxEor 6 or - CH-S-C-R n is 0 to 4; R is hydrogen, alkyl having 1 to 8 carbon atoms, cyeloalkyl of 3 to 6 carbon atoms, phenyl, C^-C^ phenalkyl, pyridyl, thienyl, or pyrrolyl; R is hydrogen, methyl or ethyl 7 8 '5 R and R are each hydrogen, alkyl having 1 to 6 carbon atoms, phenyl, pyridyl or thienyl; R^ and R 5 are each hydrogen or alkyl of 1 to 4 carbon atoms; R^ is alkyl having 1 to 4 carbon atoms, phenyl,phenalkyl having 1 to 4 carbon atoms, pyridyl, thiadiazolyl, amino or C^-C^ alkylamino; X is NH or oxygen; 10 and each phenyl group is unsubstituted or substituted with one or two substituents selected from alkyl having 1 to 6 carbon atoms, alkoxy having 1 to 4 carbon atoms, hydroxy, amino, 1 1 • NHR , N(R ) 2 , nitro, fluoro, chloro, bromo or carboxy, or a non toxic, pharmaceutically acceptable salt thereof, 15 24. A compound according to Claim 1 having the formula COOH wherein n is one or two, R z is hydrogen or formyl and R is or o-115 “ 4 5548 and Y is hydrogen, chlorine, bromine, fluorine, trifluoromethyl, amino, nitro, hydroxy, lower alkyl of 1 to 4 carbon atoms or lower alkoxy of ί to 4 carbon atoms and M is selected from

8. 6 CH - 0 - C - R, c 2 h 5 0 CH - C - R CH C - OR wherein R 5 is a hydrogen atom, a methyl or an ethyl group; 2 ¢5 7 X 4 is -0-,. -NH-; R u is a basic group.; R' is an alkyl group, a cycloalkyl group, an aryl group, an aralkyl group or a heterocyclic group, which groups may be substituted with one

9. 10 or more groups selected from amino groups, substituted amino groups, halogen atoms, nitro groups, or alkoxy groups; or a non-toxic pharmaceutically acceptable salt thereof. 25. A compound according to Claim 1 having the formula 0 R-C1I-C-UH-CH — CH (2 ill I,ft, “’W 'C' I COOM 15 wherein n is one or two, R ic hydrogen or formyl and R is 116 4 5 5 4 8 or and Y is hydrogen, chlorine, bromine, fluorine, trifluoromethyl, amino, nitro, hydroxy, lower alkyl of 1 to 4 carbon atoms or lower alkoxy-of 1 to 4 carbon atoms and M is II - CH 2“ N \ λ Ζ wherein Y is alkyl of one to six carbon atoms, phenyl, benzyl, alkoxy of one to six carbon atoms, or benzyloxy; Z is alkyl of . one to six carbon atoms, phenylbenzyl, alkoxy of one to six carbon atoms, cyclopentyl, cyclohexyl and phenyl, or Y+Z taken 10 together are a 3-benzoxazolidine ring; or a non-toxic, pharmaceutically acceptable salt thereof. 26. A procesa for the.preparation of a compound of the formula wherein A is 117 485 48 R-CH-ΟΟΗ 1 or wherein ι R is hydrogen R is or formyl; or 5 Y is hydrogen, chlorine, bromine, fluorine, trifluoromethyl, amino, nitro, hydroxy, (lower)alkyl of 1 to 4 carbon atoms or (lowerJalkoxy of 1 to 4 carbon atoms; R is (lower)alkyl of 1 to 4 carbon atoms, and <1 10 wherein A is methyl or -(CHg^COOH and n is one or two; or an easily hydrolyzed ester or non-toxic pharmaceutically acceptable salt thereof; characterized by reacting a compound of the formula II ch 2 occh 3 15 in Which A is H or A as defined above or a salt or easily - 118 45548 hydrolyzable ester thereof with a compound of the formula H-S 'Ύ H Ν-Α 1

10. 1 2 wherein A is as defined above, and, when A is H, treating the resulting compound with an acylating agent of the formula A- X in which X is halide or a functional equivalent thereof and A is as defined above, which when the A group contains a free amino or hydroxyl, said groups are blocked by conventional protecting groups and subsequently removed to afford a compound of the formula I, and, if desired, converting a resulting free acid of a compound of the formula I to the corresponding ester or noh-toxic pharmaceutically acceptable salt thereof, and, if desired, converting in a resulting salt or easily hydrolyzable ester of a compound of the formula 1 said compound to the corresponding free acid of the formula I. 27. The process according to Claim 26 wherein a resulting free acid of the formula I is converted to an ester selected from the pivaloyloxymethyl-, acetoxymethyl-, methoxymethyl-, acetonyl-, phenacyl-, ρ-nitrobenzyl-, β ,/,^-trichloroethyl-, 3-phthalidyl- or 5-indanyl-oxy. 28. The process according to Claim 26 or 27 wherein A is 119 45 5 48 wherein R^ is hydrogen or formyl and R is chlorophenyl, bromophenyl, trifluoromethyl phenyl, tolyl or methoxyphenyl. 29. The process according to Claim 27 or 28 wherein R is 5 30. The process according to Claim 26 or 27 wherein A is 31. The process according to any of Claims 26 to 30 wherein A is methyl. 32. The process according to any of Claims 26 to 30 wherein •1 10 A is -(CH,)„C00Hj where n is one or two. Ζ n , 33, The process of Claim 32 where n XG one. 34. The process of Claim 32 where n is two. 35. The process of any one of Clsims 26 to 34 where A has the D-configuration.

11. 15 36. 'The process' according to any one of -Claims 26, 27 and 31 to 35 farther ccnprising converting the resulting ccnpound when it has an amirio group to the corresponding benzaldehyde or salicylaldehyde Schiff base. 37. The process according to any one of Claims 26, 27 and 31 to 35 comprising treating the resulting compound when it has an amino grotp with

12. 20 - acetaldehyde at acetone to afford a corresponding derivative thereof. - 120 38. A process for the preparation.of a compound of the formula CH ch 2 -s->^>: N N-A 1 «I wherein A is methyl or ~(CH 2 ) n CD0H and n is one or two; 5 characterized by reacting 7-amirio-caphalosporanic acid or a salt or easily hydrolyzable ester or Schiff base thereof with a compound of the formula where A is as defined above, 10 39. A process for the production Of the antibacterial agents of formula I which comprises reacting a compound of the formula H 2 N-CH-Cil CH, // I XV com 121 where A id as hereinbefore defined or a salt or easily hydrolyzed ester or Schiff base thereof with an organic monocarboxylic acid chloride or a functional equivalent thereof as an acylating agent corresponding to the acid 5 AOH. ' 40. A process according to Claim 39 substantially as described herein and exemplified. 41. A compound according to Claim 1 substantially as described herein. 10 42. A compound according to Claim 1 substantially as described herein with reference to any one of Examples 1 to 24. 43. A process according to Claim. 28 substantially as described herein and exemplified. 15 44. . Compounds when prepared by the process of any one of Claims Z6 to 40 or 43.. . 45. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an antibacterially effective amount of a compound in accordance with any one 20 of Claims 1 to 26, 41, 42 or 44. 46. A pharmaceutical composition according to Claim 45 further comprising a semi-synthetic penicillin or another cephalosporin or a cephamycin or a /^lactamase inhibitor or an aminoglycoside antibiotic.

13. 25 47. A pharmaceutical composition substantially as herein described and exemplified. 48. A method of treating bacterial infections comprising administering by injection to an infected warm-blooded non-human animal, an effective but non-toxic dose of a

14. 30 compound in accordance with any one of Claims 1 to 25. F, R. KELLY & 00., AGENTS FOR THE APPLICANTS,