CA1147724A - Tri-py-ceph combo - Google Patents

Abstract

ABSTRACT
The specification discloses a compound having the formula 1 wherein A is or wherein R1 is hydrogen or formyl;
R is or ;
Y is hydrogen, chlorine, bromine, fluorine, trifluoro-methyl, amino, nitro, hydroxy, (lower) alkyl of 1-4 carbon atoms or (lower)alkoxy of 1-4 carbon atoms;
R2 is (lower) alkyl of 1-4 carbon atoms and wherein A1 is methyl or -(CH2)n COOH and n is one or two; or an easily hydrolyzed ester or nontoxic pharmaceutically acceptable salt thereof. The specification further discloses a compound of formula I wherein A is H. There is also disclosed a process for the preparation of the compounds, an easily hydrolyzed ester or nontoxic pharmaceutically acceptable salt thereof. The compound of the formula I according to the present invention is effective in the treatment of bacterial infections in man.

Description

. TXI-PY-CEPH CO~i~O
(SY-1510-A) 11~7'7'~

me present invention relates to novel acids havin~ the structure A~-CH ~ ~C~2 f ~o N-Al COOH
.

sometimes hereinafter also written as ~=N
~N~f~ CH2S-J~ J--A1 COOH
herein A is R - IH - C - or~ C ~ C -~1 . . ~ op,2 wherein Rl is hydrogen or formyl, R is . ~ . or Y

and Y ~s hydrogen, chlorine, bromine, fluorine, trifluoromethyl, amino, nitro, hydroxy, (lower)alkyl of 1-4 carbon ato~s or (lower)alkoxy of 1-4 carbon atoms, R2 is (lower)alkyl of 1-4 ca.rbon atoms; and ~herein Al is methyl or -(CH2)nCOOII and n is one or tuo;
_ 11~77'~4 the easily hydrolyzed esters and.the non~toxic, p~armaceu-tically acceptable salts of those acids.
Said easily hydrolyzed esters Or the acids of formula I include those having the group of the formula ~J
-C~
wherein when W represents hydrogen, Z represents (lower)-alkanoyl, benzoyl, naphthoyl, furoyl, thenoyl~
nltrobenzoyl, methylbenzoyl, halobenzoyl~ phenyl-benzoyl, N-phthallmldo, N-succlnlmldo, N-saccharlno, N-(lower)alkylcarbamoyl, (lower)alXoxy, (lo~Yer)-alkylthlo, phenoxy, carbalkoxy, carbobenzoxy, carbamoyl, benzyloxy, chlorobenzyloxy, carbophenoxy, carbo-tcrt,-buto~y or (lower)alkyl~ulronyl, and ~hen W represent~ carbalkoxy, Z representQ carbalkoxy and, when W represents phenyl, Z represents benzo~l or cyano or whereln W and Z taken together represent

2-oxocy¢loal~yl contaln~ng 4 to 8 carbon stom8 lncluslve, As set rorth below ln more detall the present lnventlon also provlde~ salts Or these acids, The ~tereocheml8try Or the blcycllc nucleu~ 18 that ~ound ln Cephalosporln C.

A preferred embodiment of the present in~ention consists of the acids having the D configuration in the 7-æide chain and the formula II

o ~S
R-lCH-d-NH-CH- CH \ ~CH2 ~ ~ II
OR ~ C--N ~C~CH2 S ~ N, ~ N~(CH~)nC2H
COOH O
wherein n is one or two and Rl is hydrogen or formyl ~nd R is ~ or ~

and Y is hydrogen, chlorlne, bromine, fluorine, trlfluoromethyl, amino, nitro, hydroxy, lower-alkyl or 1-4 carbon atoms or lower alkoxy of 1-4 carbon atoms and the nontoxlc, pharmaceutically acceptable salts of those acids and the easily hydrolyzed estçrs of thoæe aclds lncludlng especlally the plvaloyloxymethyl, acetoxymethyl, acetonyl, phenacyl and methoxymethyl esters and the 811yl esters such a~ the trlmethylsllyl ester.

. A further pre~erred embodiment of this invention consists of the compounds of formula II, ~rherein R is 2-thienyl, 3-thienyl, phenyl, chlorophenyl, bromophenyl, trifluoromethylphenyl, tolyl or methoxyphenyl.
Particularly preferred embodiments of this invention comprises the acids havin~ the D confi~uration in the 7-sidechain and the formul~

CH-C-NH-CH - CH CH2 ~ ~l o~/ ~ C ~ 2 N ~ N-(CH2~nC02H
COOH O

1~4'~724 wherein n is one or two and tlleir nontoxic, plla~n<aceuticall~
acceptable salts and easily hydrolyzed esters.
Also included in this invention are the compounds (used as either intermediates or metabolic precursors) in which the ~-hydroxy group is "blo~ked" by substituents ~uch a6 dichloroacetyl (U.K. 962,021~ and U. K. 1,328,340), formyl (U.S. 3,6~1,021), trimethyls~lyl or tetrahydro-pyren~l (U.K. 1,32~,340).
There is also provided ~y the present invention a compound having the fol~ula O ~S
R-CH-C-NH-CH - CH ll~2 ~ ~
OR O~C N\ C - CH2 - S - ~N,N~N lCH2) n 2 COOM

whereln n $s one or two, R is hydrogen or formyl and R is ~ or ~

and Y $s hydrogen, chlorine, bromine, fluor$ne, tr$fluoro-methyl, amino, nitro, hydroxy, lower alkyl of 1-4 carbon atoms or lower alkoxy of 1-4 carbon atoms and M is -ICHOC(C~2)nR' -CIHOC(C~2)nCl R8 .
11~7724 o . o - CHX~OR or - CH-S-C-R6 Rl 11 n ~s O to 4; R is hydrogen, alkyl having 1 to B carbon atoms, cycloalkyl of 3 to 6 carbon atoms, phenyl, Cl-C4 phenalkyl, pyridyl, thienyl, or pyrrolyl; R2 is hydrogen, methyl or ethyl; R7 and R8 are each hydrogen, alkyl having 1 to 6 carbon atoms, phenyl, pyridyl, or thienyl; R4 and RS
are each hydrogen or alkyl of 1 to 4 carbon atoms; R6 is al~yl having 1 to 4 carbon atoms, phenyl, phenalkyl having 1 to 4 carbon atoms, pyridyl, thiadiazolyl, amino or Cl-C4 alkylamino; X is N~i or oxygen; and each phenyl group is unsubstituted or ~ubstituted with one or two substituents selected from the group consisting of alkyl having 1 to 6 carbon atoms, alkoxy having 1 to 4 carbon atoms, hydroxy, amino, NHRl, N(Rl)2, nitro, fluoro, chloro, bromo or carboxy, or a nontoxic, pharmaceutically acceptable salt thereof.

There is also provided by the present invention a compound hav~ng the formula R-CH-C-NH-CH - CH / CH2 ~ S
1R1 "C . N\ ~ 2 N~b~N ( 2) nCO2H

COOM

wherein n iS sne or two, R is hydrogen or for~yl and R 15 ~ or ~

i~7724 and Y is hydrogen~ chlorine, bromine, fluorine, trifluoro-methyl, amino, nitro, hydroxy, lower alkyl of 1-4 carbon atoms or lower alkoxy of 1-4 carbon atoms and M is selected from the group consisting of - CH - C - R

~ CH _ x2 _ C - oR7 wherein R5 i~ a hgdrogen atom, ~ methyl or an ethyl group;
x2 18 -0-~ -NH-; R6 i8 a basic group.such as alkyl or aralkyl 8ubstituted wlth substituted or unsubstituted NH2~ 5uch a8 alkyl-NHCH3, aralkyl-NHCH3, alkyl-NH~), aralkyl-NH ~ ~ -IH ~ ~ -CH2NH2~ ~IH-CH2 . . .

R7 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, cyclo-pentyl, 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 the class consisting of amino groups, substituted amino group5 such as methylamino f diethylamino or acetamido groups, the halogen 77Z~

groups such as fluorine, chlorine or bromine, nitro groups, al~oxy groups such as methoxy, ethoxy, propyloxy, isopropyloxy, butoxy or isobutoxy; or a nontoxic, pharmaceutically acceptable ~alt thereof.

There is also provided by the present invention a compound having the formula O ~S
R-CN-C-N~I-CH - CH C 2 ~ 1 IR1 ~ - N \ ~ 2 ~ N~ ~ N (CH2)nC02H
COO~I

wherein n is one or two, R is hydrogen or formyl and R is ~ or ~

and Y is hydrogen, chlorine, bromine, fluorine, tri~luoro-methyl, amino, nitro, hydroxy, lower alkyl of 1-4 carbon ~tom6 or lower alkoxy of 1-4 carbon atoms and M is /C--Y
-CB -N

wherein Y is alkyl of one to six carbon atoms, phenyl, benzyl, alkoxy of one to six carbon atoms, or benzyloxy Z i~ alkyl of one to six carbon atoms, phenylbenzyl, alkoxy of one to six carbon atoms, cyclopentyl, cyclo-hexyl and phenyl, or Y~Z taken together are a 3-benzoxa-zolidine ring; or a nontoxic, pharmaceutically acceptable salt thereof.

1147~2~

- Also included ~lithin the present invention are pharmaceutical compositions comprising a mixture of an antibacterially effective amount of 2 COmpOUl)d 0~ the present invention and a semisynthetic penicillin or another cephalosporin or a cephamycin or a ~-lactamase inhibitor or an aminoglycoside antibiotic, In the treatment of bacterial infections in man, the compolmds of tllis invention Or fol~ula II 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 di~ided dosage, e,g. three to four times a day.
m ey are administered in dosage units containin~, for example, 125, 250 or 500 mg, of active ingredient with suitable physiolo~ically acceptable carriers or excipients, m e dosage units are preferably in the fo~n of liquid preparations such as solutions or suspensions.
Another preferred embodiment of this invention comprises the acids havin~ the forrnu]a III

2 ~ ~J
R2 1I N~ c~C-C~2S N~ ~ Al III
COO~

herein Al is methyl or -(CH2)nC0~ and n is one or two and ~2 is alkyl containing 1-4 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 Or the present in~ention 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 90~ of the syn isomer and not more than 10~" of the anti 1~77~4 isomer Most preferably the compounds of formula III are syn isomers essen~ially free of the corresponding anti isomer.
~ le preferred embodiments of the present invention 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-nitrobenzyl, ~ trichloroethyl,

3-phthalidyl or 5-indanyl ester for3n.
~ eference to the syn (cis) isomeric form refer~ to the configuration of the group oR2 ~rith respect to the carboxamido group.

There is also provided by the present inven-tion a compound having the formula ~Lc - C - NH-CIII CIH ICH2 ~=NI
;C N~ "C-C~12-S ~N~N~ - (CH2)nCOOH
C - OR~

wherein R~ 18 alkyl contalning 1-4 carbon atoms, n ls one or twoand R3 ls selected from the group conslstlng of CH~ O
- CH - O - C _ R6 - CH - C - R, 75 1l - CH - X - C - OR

_ g _ ~7724 wherein R is a h.ydrogen atom, a methyl or an ethyl group;
x2 18 -0-, -NH-; R is a basic group such as alkyl or aralkyl substituted with substituted or unsubstituted NH2, such as alkyl-NHCH~, aralkyl-NHCH3, alkyl-NH ~ , aralkyl-NH ~ ~ -~H ~ ~ -CH2NH2~ -IH-CH2 R7 ls an al~yl group such as a methyl, ethyl, propyl, lsopropyl, butyl, isobutyl, pentyl or 2-ethyl-hexyl group;
a cycloalkyl group such as cyclopropyl, cyclobutyl, cyclo-pèntyl, cyclohexyl or cycloheptyl; an aryl group such as phenyl or naphthyl; an aralkyl group suCh as benzyl or naphthyl~ethyl; a heterocycllc group and whereln the alkyl, cycloalkyl, aryl, aralkyl and heterocyclic groups may be substitUted wlth one or more groups seleCted from the class cons~stlng of amino groups, sUbstltuted amlno groups such as methylamino, diethylamino or acetamido groups, the halogen groups such as fluorine, chlorine or bromine, nitro groups, alkoxy groups such as methoxy, ethoxy, propyloxy, isopropyloxy, butoxy or isobutoxy;
or a nontoxlc, pharmaceutically acceptable salt thereof, said compound being at least 75% by weight in the form of lt6 ~ isomer and preferably ln the form of its syn isomer essentially free of the corresponding anti lsomer.

There is also provided by the present inven-tion a compound having the formula C - C - NM-C~--CH ICH2 ~ NI
N_o~ C N C,C CH2 s N,N~ - (CH2)nCOOH
l_OM Q

whereln R~ i5 alkyl containing 1-4 carbon atoms, n i~
one or two and M is /C -Y
CH2 N\
Z
whereln Y is alkyl of one to six carbon atoms, phenyl, benzyl, alkoxy of one to six carbon atoms, or benzyloxy;
Z 18 alkyl of one to æix carbon atoms, phenylbenzyl, alkoxy of one to six carbon atoms, cyclopentyl, cyclo-hexyl and phenyl, or Y~Z taken together are a ~-benzoxa-zolidine ring; or a nontoxlc, pharmaceutically acceptable salt thereof, said compound belng at least 75% by welght ln the form of its syn isomer and preferably in the form o~ lts ~ isomer essentially free or the corresponding anti lsomer.

There is also provided by the present inven-tion a compound having the formula o C - C - NH-CII CIH ICH2 ~ N
N_ oR2 ~C N C'5 2 S ~,N ~ - CH

lC-o~ O

.. O

~772~

wherein R2 is alkyl containing 1-~ carbon atoms and ~1 is ~Ho3 ( c~l2) nR ~ ~CI HOC ( CH2 ~ nCI~B 8 O O
-CHXCOR or - CH-S-C-R

n ls O to 4; R is hydrogen,.alkyl having 1 to 8 carbon atoms, cycloal~yl of ~ to 6 carbon atoms, phenyl, Cl-.C4 phenalkyl, pyridyl, thienyl, or pyrrolyl;
Rl ls hydrogen, methyl or ethyl; R7 and R8 are each hydrogen, alkyl having 1 to 6 carbon atoms, phenyl, pyridyl, or thienyl; R4 and R5 are each hydrogen or alkyl of 1 to 4 carbon atoms; R ls alkyl havlng 1 to 4 carbon atoms, phenyl, phenalkyl having 1 to 4 carbon atoms, pyridyl, thiadiazolyl, amino or Cl-C4 alkylamino; X is NH or oxygen; and each phenyl group is unsubstituted or subs~ltuted with one or two sub6tltuents selected from the group consisting of alkyl having 1 to 6 carbon atoms, alkoxy having 1 to

4 carbon atoms, hydroxy, amino, NHRl, N(Rl)2, nitro, fluoro, chloro, bromo or carboxy, or a nontoxlc, pharmaceutlcally acceptable salt thereof, sald com-pound being at least 75% by weight in the form of lts syn lsomer and preferably ln the form of lts syn i~omer essentially free o~ the corresponding anti isomer ~1~77'~

There is also provided by the present inven-tion a compound having the formula C - C - NH~ CH CH2 ~ N
N_ OR~ ~C- N C~5 CH2 S N~ ~ ~ ~ CH~
C - oR3 o herein ~ 1~ alkyl containing 1-4 carbon atoms and R3 is celected from the group con61stlng of - CH - C - R , - CH _ x2 _ C - oR7 whereln R5 18 a hydrogen atom, a methyl or an ethyl group;
x2 15 -O-, -NH-; R is a basic group such as alkyl or aralkyl substituted with substituted or unsubstituted NH2, such as alkyl-NHCH3, aralkyl-NHCH3, alkyl-NH ~ , aralkyl-NH ~ f ~ CH2NH2 -fH-CH2~

R7 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, cyclo-pentyl, cyclohexyl or cycloheptyl; an aryl group such as 1~7724 phenyl or naphthyl; an aralkyl group such as benzyl or naphthylmethyl; a heterocycllc group and wherein the alkyl, cycloalkyl, aryl, aralkyl and heterocyellc groups may be substltUted with one or more groUps selected from the class conslstlng of amlno groups, subQtltuted amino groups such as methylamino, diethylamlno or acet~mido groups, the halogen groups such as fluorlne, chlorlne or bromlne, nitro groups, alkoxy groups such as methoxy, ethoxy, propyloxy, isopropyloxy, butoxy or lsobutoxy;
or a nontoxic, pharmaceutlcally acceptable salt thereof, sald compound belng at least 75~ by welght in the form of its syn lsomer and preferably in the form of lts syn lsomer essentially free of the corresponding anti lsomer.

There ls also provided by the present lnvcn-tlon a compound havlng the formula o C - C - NH~ CIH ICH2 ~ Nl N_ oR2 ~C N_ C~ 2 N~ ~ - CH~

~-OM O

wherein R iS alkyl containing 1-4 carbon atoms and M is ~C -Y
CH2 N\

~ "

1~772~

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, cyclo-hexyl and phenyl, or Y+Z taken together are a 3-benzoxa-zolidine ring; or a nontoxlc, pharmaceutically acceptable salt thereof, said compound being at least 75~ by weight ln the form of lts syn isomer and preferably in the form of its syn isomer essentially free OI the corresponding anti lsomer.
-- , . _ . . . . .
In the treatment of bacterial lnfectlons in man, the compounds of this inventlon are admlnls-tered parenterally in an amount of from about 10 to 90 mg./kg /day and preferably about 14 to 50 mg./kg /day in dlvided dosage, e.g. two to four tlmes a day. They are admlnlstered ln dosage units contalning, for example, 125, 250 or 500 mg. of actlve lngredient wlth sultable physlologlcally acceptable carrlers or exceplents.
The dosage unlts are ln the form of liquld preparatlons such as solutions or suspensions and preferably are aqueous solutlons of a sodium or potasslum 6alt which are in~ected lntravenously or intramuscularly or by continuous or lntermlttent lnfuslon in concentratlons of about 125-500 m~m./ml., and preferably, 250 mgm./ml.
as ls customary in therapy wlth cephalosporln antlbi-otics.
It was an unexpected finding that the leading compound of the present invention (BB-S510; see below) having a 2-methyl substituent on the trlazolopyridazinone showed _ vitro antibacterial potency considerably superior to that of the corresponding compound lacking such methyl group (BB-S515; see below).

~7'7~

A further embodiment of this invention comprises a compound having tlle formula HS - ~N~

wherein A~ is methyl or -(CH~)nCOC~
where n is one or t~o~

The present invention also provides the process for the production o~ the antibacterial a~ents of formula I
which comprises reaeting a compound of the formula ~S~
~l2~-lH C~l ~2 ~ N
C - -N~ ~jC-CH 2 -S ~ N,N ~ Al IV

COO~
where Al is as hereinbe~ore defined or a salt or ea6ily hydroly~ed e8ter or Schiff base ~B wi~h benz~ldehyde or ~llcyl~ldehyde thereof (including, but not lLmited ~o, tho~e of U.S.
3,284~451 and U~K. 1~229~45~ and any o~ the 811yl e~ter~ de3crlbed in U.S, patent 3~249,622 ror u~e with 7-amlnopenlcillarllc acld and u~ed ln &reat Brltaln 1JO73~530 and par~cularly the pivaloyl~xymethyl, acetoxymethyl, methoxymethyl, a¢etonyl, phenacyl, p-nltrobenzyl, ~ trl¢hloroethyl, 3-phthalldyl and 5-lndanyl esters) thereof with an org~nic monocarboxyllc acld chlorlde or a functional e~ulvalent thereor aa an acylating ~gent. Compound of ~ormula IV
comprising reacting 7-amino-cephalosporanic acid or a salt or easily hydrolyzable ester thereof with a com-pound of the formula N
~-S - N,N ~ N-A

~` 1 where A is as defined above.

772~

Such functlonsl equivalents lnclude the corresponding ecid anhydrides, including mixed anhydrldes end partlculsrly the mixed flnhydrites preperet from ~tronger acids such es the lower allphatlc monoesters of csrbonic ecid, or alkyl and sryl ~ulfonic acids snd of more hindered acids such as diphenyl~cetic ecid. In eddition, sn scid azidé or an activè éster or thioester (e.g.
with p-nitrophenyl, 2,4-dinitrophenol, thiophenol, thioecetic acid) m~y be used or the free acid itself mey be coupled with compound IV sfter flrst re~cting ~eid free scid with N,N'-dimethylchloroformlminium chloride lcf. Great Britsin 1,008,170 and Novsk and Weichet, Experlentls XXI, 6, 360 (1965)l or by the use of enzymes or of an N,N'-csrbonyl-dllmldazole or sn N,N'-c~rbonylditriazole [cf.
South Afsicen petent speciflcetion 63/2684l or 8 csrbodiimide reegent lespecislly N,N'-dicyclohexyl-cerbodlimide. N,N'-dii~opropylcsrbodiimide or N-cyclo-hexyl-N'-(2-morpholinoethyl)carbodlimlde; cf, Sheehen end Hess, J, Amer, Chem, Soc " 77, 1967 (1955)~, or of alkylylamine reagent lcf. R, Bui~le and H,G.
Ylehe, An~ew, Chem International Edltion 3, S82, ~7724 (1964)] or Or an lso%asollum salt reagent ~cr. R. ~, Woodward, R. A. Olorson and H. Mayer, ~ Chem~
Soc~ , 1010 (1961)1, or Or a ketenlmlne resgent [cr. c. L. Stevens and M, E. Munk, J. Amer~ Che~ ~oc,.
~, 4065 (1958)~ or Or hexachlorocyclotrlphosphatrlaz~ne or hexabromocyclotrlphosphatriazlne (U,S. 3,651,050) or o~ diphenylphosphoryl azlde [DPPA; ~, Amer. Chem. ~55~J
44, 6203-6205 (1972)] or of dlethylphosphoryl cyanlde ~EPC; Tetrahedron Letters No. 18, pp. 1595-1598 (1g73)]
or o~ dlphenyl phosphlte ~Tetrahedron Letters No. 49, pp. 5047-5050 (1972)]. Another equlvalent o~ the acld ohlorlde 18 a correspondln~ azollde, l.e., an amlde Or the correspondlng acld whose amlde nltroeen 19 a member Or a quaslaromatlc flYe mem~ered rln~ contalnlng at least two nitrogen atoms, l.e., l~ldazole, pyrazole, the trlazoles, benzlmidazole, benzotrlazole and thelr substltuted derlvatlves. As an example Or the general method ror the preparatlon Or an azollde, N,N'-carbonyl-dl~mldazole i9 reacted wlth a carboxyllc acld ln equ~molar proportlons at room temperatre in tetrs-hydroruran, chlorororm, dlmethylrormamlde or a ~imllar lnert solvent to ~orm the carbo~ylic acld lmldazollde ln practlcally quantltatlve yleld wlth llberatlon o~
carbon dloxlde and one mole o~ ~mldazole. Dicarboxyllc aclds yleld dlmldazollde. qhe by-product, lmldazole, preclpltstes and may be separated and the lmldszollde ~olated, but thls ls not essentlal. The ~ethods for carrying out these reactlons to produce a cephalo~porln and the methods used to lsolate the cephalo~porln ~o produced are well known ln the art.

~14772~

Ment~on ~ac made above Or the u~o o~ enzymes to couple the ~ree acld wlth compound IV. Inoluded in the scope Or 8uch processe~
are the u~e Or an e~ter, e.g. the methyl ester, Or that rree acld w~th enzymes provlded by varlou~ mlcro-organl~m~? e.g. those descrlbed by T. Takahashl et al., J, Amer~ Chem. Soc., ~4(11~, 40~5-4037 (1972) and by T, Nar~ et 81 " J. Antlblotlcs (Japan) 24~), 321-~23 (1971) and ln U.S. ~,682,777.

~ or the coupllng of the orgsnlc csrboxylic ~c~d ~
de6cribed above with compound IV (or a salt or preferably ~n e~sily hydrolyzed ester of Schiff bsse, 88 with benzaldehyte, thereof) lt iB 81BO convenlent and effic~ent to utilize a8 the coupling ~gent phosphonitrilic chloride trimer (J. Org. Chem., 33(7), 2979-81, 1968) or N-ethoxy-1,2-dihydroquinoline (EEDQ) ss deccribed in J. Amer. Chem.
Soc., 90, 823-824 and 1652-1653 (1968~ snd U.S. P~tent 3,455,929. The resction i8 preferably c~rriet out ~t 30-35C. in benzene, eth~nol or tetrahydrofursn w ing ~bout equlmolar qusntitles of all three reagents followed by conventionsl isolation and re~ov~l by conventlon~l methot~
of any blocking groups present.

One process of the present in~ention stated more specifically ~s the process for the preparation of a product having the D-configuration in the sidechain and.the formula CH-CONH ~ ~ ~r 1H ~ N ~ CH2S N~ ~ N (CH2)c2 COOH O

wherein n is one or two or a salt thereof which comprises the consecutive steps of a. preparing an acylating derivative of D-mandelic acid having the formula CH-COOi-l wherein the hydroxyl blocking group R represents di-chloroacetyl, silyl and preferably trimethylsilyl, tetra-hydropyranyl or, preferably, formyl in an anhydrous or-ganic solvent such as benzene, ethanol or preferably tetrahydrofuran, at room temperature or below and prefer-ably at about 5~ CJ

b mixing therewith, preferably slo~ly, a solution at about the same temperature in a solvent, preferably aqueous.tet~ahydrofuran, containing substantially the same number of moles of a tertiary amine, preferably a tertiary alkylamine such as triethylamine and substan-tially the same number of moles of 7-amino-3-(2-carboxy-methyl or 2-carboxyethyl-2,3-dihydro-s-triazolol4,3-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 the intermediate acid having the formula CH-CONH ~ ~ ~r 1R ~N~ 2S N~ ~N-(CH2)nCO2H
COOH O

wherein n is one or t~ or a salt ther~of wher~in ~ has the meaning set out above and c. removing said hydroxyl blocking group R by con-ventional c~emical methods to produce said product or salt thereof.

~1477Z~

In preferred embodirnents of the present inven-tion R represents formyl whic~l is removed in step C by treatment ~rith aqueous alkali such as aqueous sodium bicarbonate or R represents dechloroacet-Jl ~?hich is removed in step C by alkaline hydrolysis, preferably at about pH 9-10, or ~ represents trimethylsilyl ~Ih:l.ch 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 A~rrl ~ LH2 -O-C-CH, V

0/ OC~I

here A is as defined hereinbefore above, prepared by substituting 7-amino cephalosporanic acld for the 3-thiolated-7-amino cephalosporanic acids in the acylation procedures described herein and else~Jhere reported ~lith the appropriate thiol HSR3 having the formula Il-S - ~ ~ N ~ CH2)nCOoH H-S- ~ N-~l~

where n is one or t~Jo and then removing the protecting group if any is present as on the carboxyl group, ~7724 The d~splacement of such ~ 3-acetoxy group wlth such a thiol m~y be accompli~hed in solution a8 ln wster or aqueous acetone at ~ temperature of at least room temper~ture and preferably within the rsnge of about 50- to lOO-C. ln the presence of a mlld b~se such ~8 sodl~m bic~rbonate, e.g. prefer~bly near neutral~ty such J8 ~t about pH 6. An excess of the th~ol is preferably emplo~ed. The re~ction product is lsolated by careful acldlflcation of the reaction m~xture followed by extrac-tion with ~ water-immiscible orgsnic solvent As notet above, the prep~ration of many other 7-acylemldocephalospor~nlc ac~t~ 1~ te~orlbed in the patent and scient~fic liter~turo, e.g. in U.S. Cl~ss 260-243C.

The salts of the compounds of this inventlon ~nclut~
the nontoxlc carboxyllc acld sslts thereof, lncludlng non-toxlc metslllc salts Juch a~ ~odlum, pot~slum, calclum ~nd alumlnum, the ammonlum sJlt and substltutet ~mmon~um a~lt~, e.~. salt~ Or Buch nontoxl¢ amlnes as trlalkyl-amlne~ lncludlng trlethylamlne, procalne, dlbenzyl-amlne, N-benzyl-beta-phenethylsmlne, l-ephenamlne, N,N~-dibenzylethylenedlamine, dehydroabietylamlne, N,N'-bls-dehydroabletylethylenedlamlne, and other amlnes wh~ch have been used to rorm salts wlth benzylpenlclllin, L-lysine, arginine and histldine The preferred esters Or the cephalosporlns o~ the present inventlon are the pivaloyloxymethyl, acetoxymethyl, methoxymethyl, acet~nyl and phenacyl e~ters. All are useful lntermedlates ln the production Or the cephalosporln having a free carboxyl group.

~1477Z4 As lndlcated above, these rlve ester~ Or 7-amino-cephalosporanlc acld are each prepared by known method~. One excellent procedure ~s that of U S.
patent 3,284,451 ln whlch sodlum cephalothln 1~
ester~r~ed by reactlon wlth the correspondlng actlve ohloro or bromo compound (e.g. phenacyl bromlde, chloroacetone, chloromethyl ether, plvaloyloxy-methyl chlorlae ~also called chloromethyl plvalate],J¢etoxymethyl chlorlde) and then the thlen~l-acetlc acld sidechaln 19 removed enzymatlcally as in the same patent or chemlcally a~ ln U.S.
patent 3,575,g70 and ln Journal Or Antlblotlcs, XXIV (11), 767-773 (1971). In another good method the trlethylamlne salt o~ 7-am~nocephalo-~poranlc acld 18 reacted directly wlth the actlve halogen compound, as ln Unlted Kingdom 1,22g,45~.
- m ese esters o~ 7-amlnocephalosporanlc acld are then reacted wlth the nucleophlle HSR3 ln the ~ame manner a8 ~8 lllustrated here~n ror 7-amlnocephalO-~poranlc acld ltselr. ffl e ~-thlolated ester o~
7-amlnocephalo~poran~c aold ~o then couple~ ~lth the organlc carboxyllc acld as be~ore The ester of the cephalosporin so obtained is, if not used per se, converted to its free acid and, if desired, sny sslt by removsl of the esterl-fylng group, ~s by aqueow or enzym~tlc hydroly~
with human or animal serum) or acidic or alkaline hydrolysis or by trefltment with sodium thlophenoxide as taught ln U.S. 3,284,451 and, in the penicillin ~erle~, by Sheehan et ~1., J. Org. Chem. 29~7~, 2006-2008 (1964).

~77;Z~

In another alternatlve synthe81s, the 3-thlolated 7-amlnocephalo~poranic acid 1~ prepared ~B descrlbed hereln and then act~l~ted at the 7-amlno group and rlnally e~te~l~led, as by reactlon o~ the appropr~ate alcohol wlth the acld chlorlde prepared, ~or example, by reactlon Or the rlnal cephalosporln with thlonyl chlorlde or by ~ther e~entlally acid~c e8terlflcatlon proc~d~lres.

There ~ further provided by the present invention a pharmaceutical composition comprising an antibacterially effective amount of a compound having the formula O ' S
~-CH-C-NH-CH--CH I 2 ~ I
1R 0"~ ~\ ~ 2 ~N'Nb~N- (CH2) nCO2H
COOM

wherein n i~ one or two, Rl is hydxogen or formyl and R ~s ~ or ~

and Y is hydrogen, chlorine, bromine, fluorine, tr~flùoro-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-nitrogenzyl, B,B,B-trichloroethyl, 3-phthalidyl or ~-indanyl and preferably is hydrogen or a nontoxlc, pharmaceutically acceptable salt thereof.

~477Z4 ~ here is further provided by the present invention a method of treating bacterial infections comprising adminis-tering by $njection to an infected warm-blooded animal, including man, an effective but nontoxic dose of 250-1000 mgm.
of a compound having the formula O ~S
~-C~I-C-NH-CH - CH ~ H2 ~ N~
I ~ ~ C--C~2--5~ N~N ~ N (CH2) n 2 COOM

wherein n is one or two, Rl is hydrogen or formyl and R is or ~

~nd Y is hydrogen, chlorine, bromine, fluorine, trifluoro-methyl, amino, nitro, hydroxy, lower alkyl of 1-4 carbon ~tom~ or lower alkoxy of 1-4 carbon Atoms and M i5 hydrogen, p~valoyloxymethyl, acetoxymethyl, methoxymethyl, acetonyl, phenacyl, p-nitrobenzyl, B,B,B-trichloroethyl, 3-phthalidyl or 5-indanyl or a nontoxic, pharmaceutically acceptable salt thereof.

$here is also provided by the present invention a method for combatting Shig. dvsenteriac infections which comprises adminiqtering to a warm-blooded mammal infected with an ~ . dysenteriae infection an amount effect~ve for treating said Shig. dysenteriae infection of a composition comprising a compound having the formula ~477Z~

ol /S
R-C~I--C--NH--CH--CH ~:H2 ~N
* ~,C ~ ~ 2 ~:N'N~N (CH2) nC02H

COOM

~herein n i5 one or two, R is hydrogen or formyl and ~ ~s or ~

~nd Y is hydrogen, chlorine, bromine, fluorine, trifluoro-methyl, amino, nitro, hydroxy, lower alkyl of 1-4 carbon atom~ or lower alkoxy of 1-4 carbon atoms and M is hydrogen, pivaloyloxymethyl, acetoxymethyl, methoxymethyl, acetonyl, phenacyl, p-nitrobenzyl, B,B,B-trichloroethyl, 3-phthalidyl or S-indanyl and preferably is hydrogen or a nontoxic, pharmaceutically acceptable salt thereof.

There i8 also provided by the pre~ent invention a method for combatting Sal. enteritidis infections which compri5e~ admini~tering to a warm-blooded mammal infected with a Sal. _nteritidis infection an amount effective for treating said Sal. enteritidis infection of a composition compri~ing a compound having the formula O
~-CH-C-NH-CH--CH ~ 2 ~=
ORl ~1 ~ N\ j/ ~H2 5 ~N,N~N lC 2)nC02H

OOM

~77'~4 wherein n is one or two, R~ is hydrogen or formyl and R is ~ or ~

and Y ls hydrogen, chlorine, bromine, fluorine, trifluoro-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-nitrobenzyl, B,B,B-trichloroethyl, 3-phthalidyl or 5-indanyl and preferably is hydrogen or a nontoxic pharmaceutically acceptable salt thereof.

There is further provided by the present invention a pharmaceutlcal composltion comprlsing an antlbacterially effective amount of a compound having the formula O '.
- C - NH~ H ~H2 ~ r1 OR~ ~C N c"C CH2 S
1_0~ 0 wherein R2and Al are as hereinbefore defined and R3 ~s hydrogen, pivaloyloxymethyl, acetoxymethyl, methoxymethyl, acetonyl, phenacyl, p-nitrobenzyl, ~ -tricllloroetllyl, 3-phthalidyl or 5-indanyl and preferably is hydrogen or a nontoxic, phar~aceutically acceptable salt thereof, said compound being at least _ ~7 _ 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 pharma-ceutically acceptable carrier therefor.
There is further provided by the present invention a pharmaceutical composition comprising an ant~bacterially effective amount of the syn isomer of a compound having the formula C - g - NH-C~I CH IC~z ~ N
L~_oc~ ~ r~ c~5 2 S ~ N,N ~ - A1 IC_OH O

wherein Al is as hereinbefore defined or a nontoxic, pha~a-ceutically acceptable salt thereof and a pharmaceutically acceptable carrier therefore.

There ls further provided by the present lnventlon a method of treating bacterial infections comprising administering by in~ection to an infected warm-blooded animal, including man, an effective but nontoxic dose of 250-1000 mgm. of a compound having the formula C - C - NH-C~--CH CH2 ~ Nl OR o ~ ~ C 2 N~ ~ A

119~ f 72~

herein R2 and Al are as hereinberore defined and R3 is hydrogen, pivaloyloxymethyl, acetoxymethyl, methoxymethyl, acetonyl, phenacyl, p-nltrobenzyl, ~ -trichloroethyl, 3-phthalidyl or

5-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 correspond~ng anti isomer.
... . _ .

There ls further provlded by the present lnvent~on a method of treatlng bacterlal infectlons comprislng admlnlstering by lnJection to an lnfected warm-blooded animal, lncludlng man, an effectlve but nontoxlc dose of 250-1000 mgm. of the syn lsomer of a compound havlng the formula ~ I - C - NH-CIH- CH ~H2 ~ N
~ o 7 ~
_OH 0 ~Jherein ~1 is as hereinbefore defined or a nontoxic, pharmaceutically acceptable salt thereof, and wherein n is preferably 1 ~ ~ ~ 7 7 Z ~

m ere is also provided by the present invention a method for combatting Hae~ophllus infec-tions which comprises administering to a warm-bl-ooded mammal lnfected with an Haemophilus infectlon an amoun~ effective for treating said HaemoPhilus lnfec-tlon of a composltion comprislng a compound having the rormNla C - C - NH-CI Cl~ C~1 1~ 2 ~ C N "C-CH2-S N ~ l A
o~3 o herein Al and R2 are as hereinbefore defined and R3 is hydrogen, pivaloyloxymethyl, acetoxymethyl, methoxymethyl, acetonyl, phenacyl, p-nitrobenzyl, ~,B,~-trichloroethyl, 3-phthalidyl or 5-lnd~nyl 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 pharma-ceutically acceptable carrier therefor.

.
There is also prov~ded by the present inventlon a method for combatting Neisseria lnfec-tions which comprises administering to a warm-blooded mammal infected with a Neisseria infection an amount effect~ve for treating sald Neisserla infection of a composltion comprising a compound having the formula 11~7'724 CI - C - NH-C~ CIII CH2 ~ N
N_ oRl ~C N f~ CH2 S N~N~

_0~ 0 ~herein R2 and ~1 are as hereinbefore defined and R3 is hydrogen, pivaloyloxymethyl, acetox~methyl, methoxymethyl, acetonyl, phena.cyl, p-nitrobenzyl, ~ trichloroethyl, 3-phthalidyl or 5-lndanyl ~nd preferably iæ hydrogen or a r.ontoxic, 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 pharma-ceutically acceptable carrier therefor.

m e present invention also provides the novel compounds having the formula ~ S--~J ~N_~l wherein Al is methyl or -(CH~)nCO~I, ~rhere n is one or t~ro, me invention further provides the novel compounds having the formula 1~477Z4 H~N-~I --CH ~ C~I~
--N ~ ~C-C~2-S ,~ ~ N_ c-o~3 o wherein Al is methyl or ~(CHa)nC0~3, where n is one or two and R3 is hydrogen or a conventional, pharmaceutica.lly acceptable, easily hydrolyzed ester forming group; or a nontoxic, pharm~ceutically acceptable salt thereof, - 32. -~ 77Z4 STARTI~G ~TERIALS
2-Furoylcyanide To a suspension of 26.1 g. (0.4 mole) of ground potasslum 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 ætlrred ln the cold for 15 mlnutes then heated at reflux ~or 30 minutes. The reaction was cooled, filtered and the aceto-nitrile was removed at 15 mm. (steam-bath) leaving 24.5 g.
of a dark oil whlch was used without further purification.
An lnfrared spectrum showed a nitriie band at 226~ cm 1 2-Furaneglyoxylic Acid The 24.5 g. of crude 2-furoylcyanide was mixed with 160 ml. concentrated hydrochloric acid at 25 C. with intermittent stirring. The reactlon was stored for 24 hours at 25 C. and diluted with 80 ml. of water. The reactlon was stirred for 5 minutes and filtered. The fil-trate was saturated with sodium chlorlde and extracted wlth 5 x 120 ml. of 1:1 ether-ethyl acetate solution. The extracts were combined, dried over anhydrous magnesium sulfate and evaporatéd at 30 C. (15 mm.) to give a brownish-orange ~olid. 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 g. (m.p. 76 C.). The ir and nmr spectra were consis-tent for the structure.

2-Methoxyimino-2-furylacetic Acid To a solution of 4.5 g. (0.032 mole) of 2-furane-glyoxyllc acid in 40 ml. of 50% alcohol and 3.1 g. (O.037 7'72 ~ .

mole) of methoxyamine hydrochloride in 6 ml. water at 20 C. was added dllute sodium hydroxlde solutlon to pH 4-5.
The solution was stlrred at pH 4-5 at 25 C. for 24 hours.
The alcohol was removed under reduced pressure (15 mm.) and the solutlon was ad~usted to pH 7-8 with 50% sodlum hydroxide solution. The reactlon was extracted with ~ x 50 ml. of ether and the aqueous layer was ad~usted to pH
1.9 using concentrated hydrochloric acid. The mixture wa8 extracted wlth 5 x 50 ml. of ethyl acetate. The organlc fractlons were combined, washed with brine, drled over ~nhydrous magneslum sulfate and evaporated under reduced pressure (15 mm.) to an oil ~hich 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.
78 C. An analytical sample was recrystallized from toluene, dried for 16 hours in vacuo over P205 at 25 C.
The ir and nmr spectra were consistent for the structure.
Anal. Calc'd for C7 ~ N0: C, 49.65; H, 4.17;
N, 8.28. Found: C, 49.30; H, 4.21; N, 8.37.

~-Ethoxyiminofurylacetic Acid The 7.85 g. (o.056 mole) of furyl-2-glyoxylic acid was dissolved ln 100 ml. of water and ad~usted 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, stirred 3 hours at room tempera-ture and then filtered. The alcohol was remo~ed at 35 C. (15 mm.) and the aqueous portion was ad~usted with dilute sodium hydroxlde solut~on to pH 7-8 and then 1~77Z4 was washed with ether and the washes were discarded. The aqueous fraction was ad~usted with 6N hydrochIorlc acid to pH 1. 5 and extracted into ~ x 80 ml. of ethyl acet~te.
The acetate fractions were combined, washed with brine and reduced in volume at 35D C, (15 mm.) to an oil, The oll was cooled in an ice bath, triturated with Skellysolve B, collected and dried over P205 in vacuo at 25 C.
Yield: 4.8 g " m.p. 83-85 C. The ir and nmr were con-sistent for the structure, Anal, Calc'd for C8HgN04: C, 52.46; H, 4.95;
N, 7,65, Found: C, 52,22; H, 4.94; N, 7,60, Sodium a-Ethoxyimino-a-(2-furyl)acetate To 50 ml. of methanol was added 250 mg, (0.0109 mole) of metallic sodium and stlrred until all the sodium had dissolved. This sodlum methoxide solutlon was treated with 2.0 g, (0,0109 mole) of ~-ethoxyimino-a-(2-furyl)acetlc 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 P205 at 25 C,to yield 2.22 g, white solid, m,p. decomp, >240 C, The ~r and nmr were consistent for the structure, "Skellysolve ~" is a petroleum ether fraction of b,p, 60-68 C, consisting es6entially of n-hexane, ~ 35 -~1~7724 ~-CI ~ ~ ~-tN

_ CH3oNH2-Hcl ~CH3 ~ -OH

Na 3 ~ ~ (CCl)2 ~a3 5 ~CH3 2-Furoylcyanlde 1 To a suspen610n of 78.~ g. of powdered potas- .
~ium cyanide ln 900 ml. acetonltrlle at 5 C. was added 59.25 ml. (68.5 g.) of a-furoyl chloride wlth vigorous stirrlng whlle 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 whlch was added to the filtrate, and the acetonitrile was removed at 60 C. (15 mm.) leavlng 51 g. of ~ as a dark 1~477'Z4 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 ~ was used without further purification (49% yield of product).

Furyl-2-glyoxylic Acid 2 The 51 g. of crude 2-furoyl cyanlde 1 was mlxed 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. (~ote:
Initlally 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. etherJ (Note: Use of alcohol should be avoided at thls point as esters may ~orm), treated with 10 g. of charcoal ("Darko-KB"), flltered after stirring for 0.~ hoùr and evaporated to drynesS at 50~ C. (15 mm.) to yield 46.6 g. of 2 as a light tan colored acid Thi~ product 2 was found to contain a ratio of approxlmately 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 H20 (50 mg./ml.), titrating to pH 2.8 with HCl and extr~cting with 2 x 200 ml. of *Trade Mark ~47724 ethyl acetate Evaporation of the ethyl acetate extracts gave 35~ furoic acid and 15% product 2. The pH 2. 8 aqueous phase was adjusted to pH o.8 (HCl) and extracted w1th 2 x 200 ml. ethyl acetate. The organic extracts were combined and washed with 50 ml. H20. 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 2 in toluene at 50 mg./ml. at 80 C., decanting, and leavlng to crystallize at room temperature for 18 hours, yielding 13.3 g. of pure acid 2 by NMR. This represented a 51% yield ~n the purifica-tion and recrystallization step and an overall yield from the 2-furoyl chlorlde to the pure furyl-2-glyoxylic acid 2 of 16%.

S~n-~-methoxyiminofurylacetic Acid ~
A solution of 4.5 g. of furyl-2-glyoxylic acid 2 in 40 ml. of 50% ethanol was titrated to pH 6 with lN
sodium hydroxide and then 3.1 g. of methoxyamine HCl in 6 ml. of H20 at 20 C. was added. The solution was tltrated to a constant pH 4.9 and stirred at pH 4.9 for 2~ hours at 20-23~ C. The ethanol was then removed at 50 C. (15 mm.) and the residual a~ueous solutlon was tltrated to pH 8 with 50~ sodium hydroxide and wa~hed with 3 x 50 ml. ether (pH ad~usted to 8 after each wash). The aqueous layer was t1trated to pH 1.9 with concentrated HCl and extracted wlth 5 x 50 ml. ethyl acetate with the pH read~usted to 1.9 after each extrac-tion. The ethyl acetate extracts were combined and ~14'77'~

evaporated to a solid ~ 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 ~ with a trace of anti isomer present.

Sodium Syn-~-methoxyiminofurylacetate 4 To 40 ml. of methanol was added 0.16 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-methoxyiminofurylacetic acid ~ in 7.8 ml. of methanol was added. The solution was stirred for 10 minutes at room temperature. The solvent was evaporated at 40 C. (15 mm.). The residue 4 was dried by azeo-tropic distillation with 3 x 20 ml. of benzene at 40 C.
(15 mm.). The product 4 was dried for 18 hours at 23 C. under high vacuum (0.7 mm,) over P205 yielding 1.25 g, (99% yield of product). The NMR showed this product ~
to be clean and consistent for the Btructure with 0,15 mole methanol and a trace of anti lsomer, To 0.~3 g. of sodium syn-~-metnoxyiminofuryl-acetate 4 suspended in ~5 ml. of benzene was added four drops of dry dimethylformamide and 0.31 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 residue.

7'~4 6-Chloro-2~3-dihydro-2-ethoxycarbonylmethyl-s-tria -r 4,3-blpyridazin-3-one To a solut~on of 6-chloro-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-one ~ P. Franca-rilla and F.
Lauria, J. Het. Chem., 8, 415 (1971)] (1, 1.00 g., 5 9 m.mole) ln dry DMF (30 ml.) was added sod~um hydride ~50% in paraffin, 0.3 g., 6t3 m.mole) under stirring wlth formation of yellow crystals. To the mixture was added ethyl chloroacetate (1.4 ml., 13 m.mole) and the mixture was heat-ed at 90 C. for 8 hours with stirring. After cooling, the reaction mixture was poured into water (50 ml.) and extracted with toluene (5 x 40 ml.). The or-ganic 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 (1.16 g., 71%), m.p. 114-115~ C. (llt.
110 C.).
ir: v KBax 1735, 1710 cm 1 uv: ~maxoH 231 nm (~, 26000) .nmr: ~ppC13 7.58 (lH, d, J=10 Hz, pyridazine-H), 6 98 (lH, d, J=10 Hz, pyridazine-H), 4.80 (2H, s, -CH2C0), 4.27 (2H, q, J=7.5 Hz, CH2CH3), 1.29 (3H, t, J=7.5 Hz, CH2CH3 ) .
Anal. Calc'd. for CgHgN403Cl C, 42.12; H, 3.53;
N, 21.83; Cl, 13.81. Found: C, 41.54, 41.46; H, 3.22, 3.49; N, 21.51, 21.53; Cl, 13~88~ 13.99.

- l~o -i~772~

2-Carboxymethyl-2,3-dihydro-6-mercapto-s-triazolor4,3-bl-p~ridazin-3-one To a solution of 6-chloro-2,3-dihydro-2-ethoxy-carbonylmethyl-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 , o.36 mole) and the mixture was refluxed for 0 5 hour. The reaction mixture was evaporated at re-duced pressure. The residue was dissolved in water (200 ml.) and concentrated HCl was added to the solution to ad~ust to pH 2. The precipitate of 2-carboxymethyl-2,3-dihydro-6-mercapto-s-triazolo[4,3-b]pyridazin-3-one was collected by fil-tration and washed with water. Yield 18.3 g (69~).
lr: vKBax 2900, 2450, 1750, 1660 cm 1.
~l%NaHco3a~ 260 nm (,1950)~ 3~ nm (~ 7 nmr: ~ppMm-d6 7.88 (lH, d, J=10 Hz, pyridazine-H), 7.45 (lH, d, J=10 Hz, pyridazine-H), 4.72 (2H, s, CH2C0).
Anal. Calc'd. for C7H6N403S: C, 37-17; H, 2-~7;
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-dihydro-s-triazolo~4,3-bl-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, E~_ r T ~ ~ r ~ < ~
. ~

~:1477Z~

149 ml.) were added NaHC03 (8.14 g , 97.0 m.mole) and the thiol 2-carboxymethyl-2,3-dihydro-6-mercapto-s-triazolo-[4,3-b]pyridazin-3-one (7.30 g., 32.2 m.mole) with stir-ring. The mlxture was heated at 80 C. for 0.5 hour under N2 stream. The mixture was treated with active carbon and ad~usted to pH 3 with concentrated HCl. The resulting precipitate was collected by filtration and washed with water to give 7.59 g.

(54~0) of 7-amino-3-(2-carboxymethyl-2,3-dihydro-s-tri-azolo~4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid.

.~ .~A.,, ~77~

ir: vmBax 1800, 1720, 1600, 1540, 1470 cm 1.
uv: ~BUffer (pH 7) 252 nm (~,19500)~ 298 nm (~, 8400) nmr: ~pD~m+K2C03 7.56 (lH, d, J=9 Hz, pyridazine-H), 7.05 (lH, d, J=9 Hz, pyridazine-H), 5.45 (lH, d, J=5 Hz, 6-H), 5.05 (lH, d, 5 Hz, 7-H), 4.43 (lH, d, J=14 Hz, ~-CH2), 4.04 (lH, d, J=14 Hz, 3-CH2~, 3.88 (lH, d, J=18 Hz, 2-H), 3.45 (lH, d, J=18 Hz, 2-H).

6-Chloro-2-(2-cyanoethyl)-2,3-dihydro-s-triazolo[4,3-b]-pyridazin-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 (300 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 wi~h ethyl acetate (5 x 400 ml.). The organic extracts were combined, drled over Na2S04 and evaporated. The residue was crystal-llzed from ethyl acetate to give light yellow needles of 6-chloro-2-(2-cyanoethyl)-2,3-dihydro-s-triazolo~4,3-b~-pyridazin-3-on (2.5 g., 11%). M.p. 166-168 C.

ir: vmBaxr 2230, 1720, 1550, 1500 cm 1 uv: ~dimxane 373 nm (~ 2000) nmr: ~Dppm d6 3.03 (2H, t, J=6 0 Hz, CH2), 4.21 (2H, t, J=6.o Hz, CH2), 7.23 (lH, d, J=10.0 Hz, pyridazine-H), _ _ ~ 77Z~

7 93 (lH, d, J=10.0 Hz, pyridazine-H).
Anal. Calc'd. for C8H6N50Cl: C, 42.97; H, 2.70;
N, 31.32; Cl, 15.86. Found: C, 42.73, 42.56; H, 2.57, 2.50; N, 31.36, 31.68; Cl, 15.96, 15.81.

2-(?-Carboxyeth~1~-6-chloro-2,3-dihydro-s-triazolo~4,3-b]-p~ridazin-3-on.
A solution of 6-chloro-2-(2-cyanoethyl)-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on (724 mg.) in 6N-HCl (15 ml.) was refluxed for 6 hours. The reaction mixture was extracted with ethyl acetate (10 x 20 ml.).
The combin~d extracts were washed ~ith saturated aqueous ~odium chlorid~ (50 ml.), dried over Na2S04 and evaporated to gi~e light yello~tsolid 2-(2-carboxyethyl)-6-chloro-2,3-dihydro-s-triazolo~4,3-b]pyridazin-3-on (567 mg., 72~).
M.p. >170 C. (sublimation).
ir: vKBX 3400-2400, 1730, 1710, 1540 cm 1 uv: ~dimXaxne 377 nm (~ 1500).

nmr: ~ 2 ppm~lc3 2.70 (2H, t, J=7.0 Hz, CH2), 4.24 (2H, t, J=7.0 Hz, CH2), 7.17 (lH, d, J=10.0 Hz, pyridazine-H), 7.70 (lH, d, J=10.0 Hæ, pyridazine-H).
Anal. Calc'd. for C8H7N403Cl: C~ 39.60; H, 2.91;
N, 23.09; Cl, 14.61. Found: C, 39 62, 39.48; ~, 2 97, 2.67; N, 23.05, 22.70; Cl. 13.93, 14.12.

_ 1~4 _ . . ` 7 . ~;

~ ~ ~';'7~

2-(2-Carboxyethyl~-2,3-dihydro-6-mercapto-s-triazolor4~3-b]-pyridazin-3-on.
A mixture of 2-(2-carboxyethyl)-6-chloro-2,3-dihydro-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.) ~las stirred at room tempera-ture for two hours. The reaction mixture was adjusted suc-cessively to pH 1 with c. HCl, to pH 10 with NaOH and then to pH 1 with c. HCl. The resulting precipitate of 2-(-carboxyethyl)-2,3-dihydro-6-mercapto-s-triazolo~4,3-b]-pyridazin-3-on was collected by filtration and washed with water. Yield: 418 mg. (74%). M.p. 174-176 C.
ir: vmBax 3600-2600, 2440, 1730, 1720 (sh) cm ~.
uv: ~pH 7 buffer 262 nm (~11oOO), 318 nm (~ 6600).

nmr: ~DMppmd6 2.73 (2H, t, J=7.0 Hz, CH2), 4.07 (2H, t, ~=7.0 Hz, CH2), 7.30 (lH, d, J=10.0 Hz, pyridazine-H), 7.74 (lH, d, J=10 0 Hz, pyridazine-H).
Anal. Calc'd. for C8H~N403S: C, 40.00; H, 3.36;
N, 23.32, S, 13.35. Found: C, 39.o8, 39.06; H, 3.12, ~.20; N, 22.65, 22.70; S, 14.23, 14.29.

. .
7-Amino-3-[2-~ -carboxyethyl)-2,3-dihydro-s-triazolol4,3-bl-pyridazin-3-on-6-ylthiomethyll-3-cephem-4-carboxylic Acid.
A mixture of 7-ACA (405 mg., 1.49 m.moles), the thiol 2-(2-carboxye~hyl)-2,3-dihydro-6-mercapto-s-triazolo~4,3-b~pyridazin-3-on (357 mg., 1.49 m.moles) and NaHC03 (375 mg., 4.47 m.moles) in 0.1 M phosphate buffer (pH 7, 8 ml.) was stirred at 80 C for 30 minutes. m e reaction mixture was cooled and filtered to remove insolubles.

_ 1~5 _ l~g7724 The filtrate was adjusted to pH 1-2 with c. HCl. The result-ing precipitate, 7-amino-3-~2-(2-carboxyethyl)-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl]-3-cephem-4-carboxylic acid, was collected by filtration and washed with water. Yield: 519 mg. (77%).
ir: vmBax 3600-2200, 1800, 1725, 1620, 1550, 1480 cm 1.
uv ~pH 7 ~uffer 253 nm (E ~0000), 298 nm (~)-nmr: ~D2ppK~C3 2-20 (2H, t, J=7.0 Hz, CH2), 3.40 (lH, d, J=17.5 Hz, 2-H), 3.85 (lH, d, J=17.5 Hz, 2-H), 4.00-4 50 (4H, m, 3-CH2 and N-CH2-), 5.01 (lH, d, J=4.0 Hz, 6-H), 5.40 (lH, d, J=4.0 Hz, 7-H), 6.94 (lH, d, J=10.0 Hz, pyrldazine-H), 7.44 (lH, d, J=10.0 Hz, pyridazine-H).
Anal. Calc'd for C16H16I~606S2 3/2 2 H, 3.99; N, 17.52; S, 13.37. Found: C, 40.06, 40.12;
H, 3.33, 3.34; N, 16.96, 16.98, S, 13.87, 13.98.
7-ACA refers to 7-aminocephalosporanic acid and DMF to dimethylformamide.

. . , Scheme 1. Preparation of 7-Amino-3-(2-carboxymethyl-?,3-dihydro-s-triazolo~4,3-b]pyridazin-3-on-6.-ylthio-methyl)-~-cephem-4-carboxylic Acid.

N NaH/D.iF ~ ~ T
2 2 5) Cl ~ N,N ~ N-CH2COOC2H~

O O

~.SH ~7 7-ACA
) llS ~,N ~ N-CII~COOH - >

3 o H2'J I 1~ ~ ~= 1,.
O ~ ~ 2 ~ I~,N ~ I!-CH2COOH

CO,2H

L._ .. .. . . _ .

~77Z4 6-Chloro-2,3-dihydro-2-ethoxycarbonylmethyl-s-triazolo-~4,3-blpyridazin-3-one (2?
To a solution of 6-chloro-2,3-dihydro-s-triazolo[L,3-b]pyrid~zi!l-3-ol~e [~. ~r2ncavilla ~!~d F.
Lauria, J. Het. Chem., 8, 415 (1971)~ (1, 1.00 g., 5.9 m.mole) in dry D!~F (30 ml.) w~s added sodium hydrid~
~50~ in p~r~ffin, 0.3 g., 6 3 m.mole) under s~irring with formation of yello~ crystals. To the mixture W2S ~dded ethyl chloroacetate (1.~ ml., 13 m.mole) and th~ mixture ~ras heated at 90 C. for 8 hours with stirrin~ fter cooling, the reaction mixture t:a~ poured ~r.to w~ter-(50 ml.) and extracted l~ith toluene (5 x ~0 ml.). Tile or-ganic extracts ~lere combin~d, dried ovcr atllly~rouc. sodium sulfate and evaporated ~t reduced pressurc. Ttle residue ~J~s cry~tallized ~!it l be.lzclle--~-h~x-~n~ tu ~ivc y~llo;;

needles of ~ (1.16 ~., 77~0), m. p. 111~-115 C. (lit ir: v KBX 1735, 1710 cm~l.

uv ~ EaH 231 nm (&, 26000).
nmr: ~ ppC 3 7.58 (lH, d, J=10 Hz, pyridazine-H), 6.,o8 (lH, d, J=10 Hz, pyridazine-H), 4.80 (2H, s, -CH2C0), 4 27 (2H, c, J=7.5 Hz, CH2CH3), 1.29 (3H, t, J=7 5 l~z, CH2C~33 ) .
Anal. Calc'd. for C9~91~03Cl: C, 42.12, Hj 3 53;
N, 21.83; Cl, 13.81. Found: C, ~1.5~, 41.4~; K, 3 22, 3.~9; I~i, 21.51, 21 53; Cl, 13.88, 13.~9.

1~7724 2-Carboxymet~yl-2,3-dihydro~-mercapto-s-triazolo[4,3-b]-pyridazin-3-one (~) To a solution of 6-chloro-2,3-dihydro-2-ethoxy-car~on~lmetllyl-s-triazolo[~ b]oyridazin-;-olle (~, ~0 g., 0.1~ mole) in ethanol (900 ml.) was added t~ .'}~ 0 (70 purD,. ~5.9 ~., o.~6 mole) and the mixture was rerluxed for 0.5 hour. The reacticn micture was evaporated at re-duc~ pressure. The residue was dissolved in ater (200 ml.) and concentrated HCl was added to tllc ~olution to adjust to p~ 2 The precipitate (3) ~ras collected by fil-tration ancillashed with water. Yicl~ 18.- g. !~9,') ir: ~ KBx ~900~ 50, 1150, 1~0 cm 1.
uv ~ l~r!l~C0;~ 60 nm (~,19500), -~lS llm (,7000).
nmr ~ D~lS-d6 7.8~ , d, J--10 117, ~)yrir~ !le~
7.~5 (lH, d~ J=10 ~Iz, p~rid.~zine-~ , CH2CC)).
Anal. Calc'd. for C.~H6N'I~O~ C, 37.17; ~I, 2.67;
N, 2~.77; S, 14.17. ~ou~d: C, -S7-~, 37.2~ 2.2~, 2.28; N, 23.58, 23.69; S, 14.32.

7-Amino-3-(2-carboxymethyl-2,3-dihydro-s-triazolo~ -bt--on-~-ylthiomethyl)-3-cephem-4-carbo~lic Acid (4) To 2 suspension of 7-aminocephalospor~ic acid (8.79 g., 32.2 m.mole) in 0.1 l~ phosph~tc ~uffer (p~ 7, 149 ml.~ were added NalC03 (8.14 g., 97.0 m.mole) and t~e thiol ~ ~7.~0 g., 32.2 m.mole) ~ith stirring. '~e m~xture was heated at 80D C. for 0.5 hollr under N2 str~P~. The mixture was treated with active carbon and adjusted to pH 3 with conccntrated :~Cl. The resulting prec-pit~te was collected by filtration and washed with water to giYe 7.59 g.
(5~) of ~

_ 1~9 _ . . .

1~77~

.ir: 1~ Ka~ 1800, 1720, 1600, 151~0, 1470 cm 1.
u~ \ L~ff~r (Pi~ 7) ^52 n;r. (~, 19500), 29~ llm (E~8400) nmr: ~) pk3~ co3 7.5G (1~1, d, J=9 Hz, pyrida7.ine-H), 7.05 (1~, d, J=9 Hz, ~yridazine-~{), 5.45 (l~t, d, J=5 Hz, 6-~[), 5.05 (lH, d, 5 Hz, 7-H), 4.43 (lH, d, J=14 ~Iz, 3-C~I2), 4.04 (lH, d, J=14 Hz, 3-CH2), 3.88 (lH, d, J=18 Hz, 2-H), 3.45 (1~, d, J=18 Hz, 2-H).

Pivaloyloxymethyl-7-amino-3- ~ carboxymethyl-2,3-dihydro-s-triazolo~4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylate.
Method A. - The title compound i8 produced by substituting for the 7-amlnocephalosporanlc acld used lmmedlately above an equimolar welght Or pivaloyloxy-methyl 7-amlnocephalosporanate hydrochlorlde prepared accordlng to Example 2 Or U.K. 1,229,45~ from 7-amlnocephalosporanlc acld. German 1,904,585 (Farmdoc ~9,445) i8 equlvalent to U.K, 1,229,453.
Method B. - The tltle compound ls produced by 8ubstltutlng for the 0.025 mole (6.8 g.) 7-amlno-cephalosporanlc acld used ln the procedure of Example 2 of U,K. 1,229,453 an equlmol~r welght of 7-amlno-~-(2-carboxymethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid (4).
The respective acetoxymethyl, methoxymethyl, acetonyl and phenacyl esters of 7-amino-3-(2-carboxymethyl-2,3-dihydro-s-triazolo~4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid are prepared by substituting in Method B above chloromethyl pivalate used therein an equimolar weight of chloromethyl acetate, chloromethyl methyl ether, chloroacetone and phenacyl bromide, respectively~

; ` ` ` il~77'~4 Preparation of 7-Amino-3-(2-methyl-2,3-dihydro-s-tria-zolo~4,3-b~pyridazin-7J-on-6-ylthiomethyl-3-cephem-4 carboxylic Acid.

Cl ~ NH Cl ~ N-CH3 1 ~ 2 , HS ~ N- CH3 H2~J~ ~ ~=NI
O ~ ~ CH2-S ~ 1~ ~ N-CH3 .. ~

6-Chloro-2,3-dihydro-2-methyl-s-triazolo~4,~-b]pyridazin-3-one (2) To a solution of 6-chloro-2,3-dihydro-s-triazolo-[4,3-b~pyridazin-~-one {P. Francavilla and F. Lauria, J.
Het. Chem. 8, 415 (1971)] (1, 8.5 g., 50 m mol.) in dry DMF ~12 ml.) was added NaH (50~ dispersion in paraffin, ~77Z~

2.64 g., 55 m.mol) and the mixture was stirred for 1 hour at room temperature. After methyl iodide (21.3 g., 150 m.mole) was added, the mixture was stirred for 40 hours at room temperature, diluted with water (200 ml.) and extracted with CHC13 (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 Na2S04. Evaporation of the solvent under reduced pres-sure afforded pale yellow residue which was crystallized from chloroform-n-hexane. Yield: 7.23 g. (79~).
M.p. 180-181 C.
ir: vKB0 1720 cm 1 uv: ~Emax 233 nm (~ 25200), 363 nm ( E 1600).

nmr: ~,Cppl3 3.72 (3H, s, N-CH3), 6.88 (lH, d, J=10 Hz, pyridazine-H), 7.48 (lH, d, J=10 Hz, pyridazine-H).
Anal. Calc'd. for C6H5ClN40: 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-dihydro-2-methyl-s-triazolo[4,3-b~pyridazin-3-one (3) -A mixture of 2 (6.50 g., 35.7 m.mol.) and NaSH 2H20 (70% pure, 9.4 g.) in water (100 ml.) was heated under reflux for 15 minutes. m e mixture was cooled and acidified to pH 1 with concentrated HCl to precipitate the thiol 3 which was collected by filtration and dissolved in aqueous saturated NaHC03 (100 ml.). ~le 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. (~9~). M.p. >280 C.

i~7 72~
..

ir: vKBxr 2450 (-SH), 1710 (C=0) cm uv ~1%maxHC03 261 nm (E 16300), 315 nm (E 5800).
nmr ~D2+KH 3-60 ~H, s, N-CH3), 7.o8 (2H, s, pyrida~ine-H).
Anal. calc'd. for C6H6N40S: C, 39-55; H, 3-32;
N, 30.75; S, 17.60. Found: C, 39.57, 39.66; H, 3.14, 3.22; N, 30.32, 30 61; S, 17.80, 17.89.

7-Amino-3-(2-methyl-2,3-dihydro-s-triazolo~4,3-b]~yridazin-3-on-6-ylthlomethyl-)--3-cephem-4-c-arboxylic Acid (4) A mixture of 7-aminocephalosporanic acid (7-ACA, 5.44 g., 20 m.mol.), 3, (3.64 g., 20 m.mol ) and NaHC03 (3 36 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 HCl 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: vmBr 1800 (B-lactam C=0), 17?5 (C=0~, 1610 and 1410 (C00 ) cm~l uv: ~1%MaHC3 253 nm (~20000), 305 (~ 8400).

nmr: ~D20 ppaHC03 3-69 (3H, s, N-C_~), 5.o8 (lH, d, J=4.5 Hz, 6-H), 5.48 (lH, d, J=4.5Hz, 7-H), 7.00 (lH, d, J=10 Hz, pyridazine-H), 7.52 (lH, d, ~=10 Hz, pyridazine-H).
Anal- Calc'd. for C14Hl4r~6o4s2 H2 H, 3.91; N, 20.38; S, 15.55. Found: C, 40.84, 40.63;
H, 3.44, 3.31; N, 20.50, 20.36; S, 15.19, 15.57.

t7~4 Preparation of BB-S515 ~co / ~

BB-S515; 7-~(2Z ~ Methoxyimino(fur-2-yl)acetamido]-3-(2,~-dihydro-s-triazolo~4,3-b~pyridazin-3-on-6-ylthio-methyl~-3-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., 1 m.mole) in dichloromethane (2 ml.) was added oxalyl chloride (0.09 ml., 1 m.mole) at 0-5 C. and the mixture was stirred for 30 minutes. The solvent was evaporated under reduced pressure to afford an oily resi-due. A solution of that oily residue in dry acetone (5 ml ), after filtration, was added to a mixture of , 7-amino-~-(2,3-dihydro-s-triazolo~4,3-b3pyridazin-3-on-6-ylthiomethyl)-~-cephem-4-carboxylic acid (~80 mg., 1 m.mole) (U.S. ~,907,786) and~sodium bicarbonate (~36 mg., 4 m.mol.) in water (10 ml~) at 0-5 C. The reaction mlxture 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 ad~usted to pH 1-2 with concentrated HC1 The resulting precipitate (~38 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 I with aqueous NaOH ~1 N, o,6 ml.) and filtered to make a clear solution which was lyophilized to give 7-~(2Z)-2-methoxyimino(fur-2-yl)acetamido~-3-(2,3-dihydro-s-1~77~4 triazolo[4,3-b]pyridazin-3-on-6-ylthiometllyl)-3-cephem-4-carboxylic acid sodium salt as a light brown powder (222 mg., 46~ .p. >230 C. (dec. ) .
ir: vmaxr 3410, 1760, 1720, 1600 cm 1 uv: ~.PH7muaffer 256 rIm (E 20600), 274 (~ 18800).

~5 -1~7724 Pre~aratlon o~ D-mandellc acid carbo~YanhYdrlde COCl C6H5-CH-cOOH ~ 6 5 D-Mandellc acld carboxvanhvdrlde (2) Phosgene was bubbled through a solutlon of 2 0 g.
(0,013 mole) of D(-)-mandelic acld (1) ln dry tetra-hydroruran rOr 30 minutes. The solutlon wa~ allowed to 3tand overnlght after which tlme it was heated under rerlux ~or 10 mlnutes. Evaporatlon Or the solvent under reduced pre~sure afrorded an oily resldue whlch was ~olldlfled by trlturatlon wlth n-hexane (20 ml.). The product was collected by rlltratlon and drled ln yacuo on KOH, Yield 2 ~ B-o~ ~-mandellc acld carboxyanhydrlde.

IR: ~ naUJ 1895, 1875, 1780 cm 1 l'he pre~erred and most actlve compounds Or the pre~ent lnventlon are those ha~lng the D
conriguration at the a-carbon atom in the 7-~ide-chain, that i~, those made f~om D-mandellc acld or a monosubstituted D-mandellc acid as lllustrated here-ln In addltlon, the configuration at the two optically actlve, as~mmetrlc centers in the ~-lactam nucleus 19 that round ln cephalosporln C produced by ~ermentatlon and ln the 7-amlnocephalosporanlc acld derlved therefrom.

~47724 6 5 1 \0 H2N~ ~ ~7 O--C/ N~L CH2 S N~N~ N-CH2C02H
o C02H O

C6H5-cH-coNH~s~
o ~LCH2-S l~N~N~N-CH2C2H

~, BB-S488 BB-S488; 7-(D-Mandelamido~-~-(2-carboxymethyl-2,3-dihydro-s-triazolo[4,~-b~pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic Acid (~) D-(-)-Mandelic acid 0-carboxyanhydride (U S Patents ~,167,549, ~,840,531 and ~,910,900~, (1, 400 mg, 2.~ m.mole) was added portionwise to a solution of 7-amino-~-(2-carboxy-methyl-2,~-dihydro-s-triazolo[4,3-b]pyridazin-~-on-6-ylthio-methyl)-3-cephem-4-carboxylic acid (2, 657 mg., 1 5 m mole) and sodium bicarbonate (445 mg, 5 3 m mole) in 50~ aqueous acetone (~0 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 l"as 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 tetrahydrofuran (THF) (100 ml.). The THF solution was treated with a small amount , , _ ~1477Z4 of active carbon, dried over anhydrous sodium sulfate and filtered. The filtrate was evaporated under reduced pres-sure and the residue was triturated with ether. The pale yellow precipitate was collected by filtration and chro-matographed on a silica gel column (Wako-gel C-200, 10 g.) eluted with a solution of chloroform-methanol (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 mg (34~).
M.p. 173-176 C. (dec ).
ir: ~ KBax 3600-2400, 1770, 1720, 1520, 1495, 1~65, 1245 cm~l.
uv: ~ EtxH 254 nm (~ 18000), 297 nm (~ 9000, sh).
nmr ~ DMS-d6 3 68 (2H, m, 2-H), 4.03 (lH~ d~ J=13 Hz, 3-H), 4.34 (lH, d, J=13 Hz, 3-H~, 4.64 (2H, s, NCH2CO), 5.00 (lH, d, J=4 Hz, 6-H), 5.02 (lH, s, PhCH), 5.63 (lH, d-d, J=4 & 9 Hz, a doublet with addition of D20, J=4 Hz, 7-H), 6.97 (lH, d, J=10 Hz, pyridazine-H~, 7.1-7.4 (5H, m, phenyl-H), 7.60 (lH, d, J=10 Hz, pyri-dazine-H), 8.60 (lH, d, J=9 Hz, disappeared with addi-tion of D20, CONH).
Anal. Calc'd. for C23H20N608S2 / 2 H, 3.64; N, 14.45; S, 11.03. Found: C, 47.34; H, 3.48;
N, 13.90; S, 11 01.

*Trade Mark - 5~ -11 1477;~4 In vitro activity (Table 1) The MIC's were determined by tne Steers' agar dilution method using Mueller-Hinton agar against 4 gram-positive and 28 gram-negative bacteria and the results are shown in Table 1.

In vitro activity (Tables 2 and ~) MIC determinations were performed by serial two-fold 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 ~.

Media effect on ~IC
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.

lood levels in mice Groups of mice weré administered subcutaneously graded doses of 40, 20 and 10 mg.~kg.. The blood sam-ples collected from orbital sinuses were assayed by the paper disc-agar diffusion method on Sarcina 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 _ ~9 _ 7f~

following pathogenic bacteria:
S. aureus Smith -E. coli Juhl K. pneumoniae A9977 The results are shown in Table 6, ~77;~4 Table 1. The in vitrG Antibacterial Activity of BB-S~88 _ By Agar Dilution Method (Mueller-Hinton Agar).

_ MIC (mcg./ml.) Test Organism BB-S488 Cefamandole _ . . _ . _ .
S. aureus Smith A9537 0.2 .5 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 o.8 E. coli A15810 0.1 0.4 E. coli A9660 .5 0.1 E. coli A15147 3.1 0.4 Kl. pneumoniae A9678 3.1 3 1 K1. pneumoniae A9977 0.05 0.2 Kl. pneumoniae A15130 0.2 o.8 Kl. pneumoniae A9867 0.2 o.8 Pr. vulgaris A9436 0.1 0.4 Pr. vulgaris A9699 0.~ 6.3 Pr. mirabilis A9554 0.05 0,4 Pr. mirabilis A9900 0.1 o.8 Pr. morganii A9553 >100 >100 Pr. morganii A20031 0.1 o.8 Pr. rettgeri A15167 0,05 0.2 Ps. aeruginosa A9930 >100 >100 Ps. aeruginosa A9843 >100 >100 Shig. dysenteriae 0.025 0.1 Shig. flexneri A9o84 12.5 6.3 Shig. sonnei A9516 0.025 ~5 Serr. marcescens A20019 100 100 Enterob. cloacae A9656 ~.1 3.1 Sal. enteritidis A9531 0.05 0.1 Sal. typhosa A9498 0.05 0.1 B. anthracis A9504 0.1 0.1 ~77;;~'~

Table ?. In vitro Antibacterial Activity in MueIler-Hinton Agar (Gram-positive) MIC (mcg./ml.) _ Code Cefaman-No. I Test Organism BB-S488dole _ Sa-2 S. aureus Smith A9537 0.4 0.2 Sa-3 S. aureus No. 193 o.8 0.2 Sa-8 S. aureus 0.4 0.2 Sa-9 S. aureus l~o. 193 o.8 0.2 Sa-10 S. aùreus A20239 1.6 0,4 Sa-ll S. aureus BX-1633 A9606 0,4 0.2 Sa-12 S. aureus A9497 0.2 0.1 Sa-29 S. aureus No. 193 1.6 o.8 Sa-33 S. aureus Terajima 0.0125 0.0125 Sa-34 S. aureus A15092 o.8 0.2 Sa-3~ S. aureus A15094 o.8 0.4 Sa-3~ S, aureus Russell o.8 0.4 Sa-37 S. aureus A9524 1.6 o.8 Sa-38 S. aureus A9534 o.4 0.2 Sa-39 S. aureus A9578 o.8 0.4 Sa-40 S. aureus A9601 o.8 0.4 Sa-41 S. aureus A9602 o.8 0.2 Sa-44 S. aureus A1~097 25 25 Sa-56 S. aureus A9f~30 3.1 o.8 Sa-57 S. aureus A9748 25 3.1 Sa-58 S. aureus A15033 12.5 1.6 Sa-59 S. aureus A15096 100 6.3 Sa-60 S. aureus A20604 50 3 1 Sa-61 S. aureus A20605 100 6 ~
Sa-62 S. aureus A20606 -~.1 o.8 Sa-6~ ~S. aureus A20607 >100 12.5 Sa-64 S. aureus A20608 100 6.3 Sa-65 S. aureus A20609 100 6.3 Sa-66 S. aureus A20610 100 6.3 Sa-67 S. aureus ~20611 100 6.3 Sa-68 ,S. aureus A20612 1.6 0.4 Sa-69 S. aureus A20613 100 6.3 Sp-1 S. pyo~enes S-23 0.4 0.1 Sp~2 S. ~yogenes Dick o.l~0.1 Sp-3 S. pyogenes A9604 0,4 0.1 Sp-4 S. pyogenes A20065 0.2 0.1 Sp-5 S. pyogenes A15040 0,4 0.1 Sp-6 S. pyogenes A20066 0.4 0.1 Sp-7 S. pyogenes Dig 7 0,l~0.1 Sp-8 S. pyogenes A15041 o.l~0.1 Sp-9 S. pyogenes A20201 0.4 0.1 Sp-10 S. pyogenes A20202 0.4 0.1 Dp-l 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-5 D. pneumoniae A15069 0.2 0.2 Dp-6 D. pneumoniae A20167 0.2 0.2 Dp-7 D. pneumoniae A20759 0.2 - 0.2 Dp-8 D. pneumoniae A207~9 0.2 0.2 Dp-9 D. pneumoniae A20770 0.2 0.2 '~ 77Z4 Table 3. In vitro Antibacterial Activity in Mueller-`
Hinton Agar (Gram-negative) MIC (mcg./ml.)_ Code , Cefaman-No. Test Organism BB-S488 dole .
Ec-l E. coli NIHJ 0.2 0.1 Ec-3 E. coli Juhl A15119 0.2 o.8 Ec-4' E. coli A15169 12.5 6,3 Ec-5 E. coli K-12 ML-1630 A20363 0.2 o.8 Ec-ll E. coli A203~6 5 25 Ec-15 E. coli ATCC 8739 0.2 0.1 Ec-34 E. coli A9660 0.1 0.1 Ec-~5 E. coli A9435 0,4 o.8 Ec-3~ E. coli A15147 3,1 1.6 Ec-40 E. coli A20361 0.2 o.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 A20109 0.2 o.8 Ec-51 E. coli A20343 5 ~_2.5 Ec-56 E. coli A20365 25 12.5 Ec-58 E. coli A957~ 0,4 1.6 Ec-59 E. coli A207~6 0.2 o.8 Ec-62 E. coli A20895 0.4 o.8 El-l 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 o.8 o.8 El-7 E. cloacae A9659 1.6 o.8 El-8 E. cloacae A9655 1.6 1.6 El-9 E. cloacae A20021 >100 100 El-ll E. cloacae A20344 >100 >100 El-12 E. cloacae A21006 1 .G 3.1 El-14 E. cloacae A20953 , o.8 3.1 Pm-l P. mirabilis ~9554 0.1 o.8 Pm-2 P. mirabilis A9900 0.2 1.6 Pm-3 P. mirabilis A20119 4 3.1 Pm-4 P. mirabilis A20454 0.2 1.6 Pm-5 P. mirabilis A9702 0.1 o,8 Pm-6 P. mirabilis A21222 1.6 1.6 Pg-l P. morganii A95~3 >100 >100 Pg-2 P. morganii A20031 0.2 .1.6 Pg-3 P. morganii A9636 o,8 1,6 Pg-5 P. morganii A15166 0.1 0.2 Pg-6 P. morganii A20455 0.4 1.6 Pg-7 P. morganii A20~57 0.2 o.8 Pg-8 P. morganii A15153 0.1 o.8 Pg-9 P. morganii A15149 o.8 3.1 Pv-l P, vulgaris A9436 0.2 o.8 Pv-2 P. vulgaris A9526 6.3 , 1,6 Pv-3 P. vulgaris A9699 6.3 5 Pv-~ P. vulgaris ATCC 9920 , 0.1 0.2 Pv-5 P, vulgaris A9539 25 >100 Pv-6 P. vulgaris A971~ 0.1 o.8 Pv-7 P. vulgaris A21240 25 >loo ~477Z4 Table 3 (Continued) Code Cefaman-No. Test OrganismBB-S488 dole Pr-l P. rettgeri A15167 0.1 0.2 Pr-2 P. rettgeri A9637 0.1 0.1 Pr-4 P. rettgeri A20645 0.1 0.1 Pr-5 P. rettgeri A20915 0.2 o.8 Pr-6 P. rettgeri A20920 0.1 0.2 Pn-l P. inconstans A20615 0.1 o.8 Ps-l P. stuartii A20745 0.4 o.8 Ps-2 P. stuartii A20~94 0.2 o.8 Ps-3 P. stuartii A20911 o.8 o.8 Ps-4 P. stuartii A21051 5 25 Ps-5 P. stuartii A21057 0.2 o.8 Kp-l K. pneumoniae Dll 0.1 o.8 Kp-2 K. pneumoniae A96783.1 1.6 Kp-3 K. pneumoniae A99770.1 o.8 Kp-4 K. pneumoniae A15130 0.2 0 8 Kp-7 K. pneumoniae A98670.4 o:8 Kp-8 K. pneumoniae A20680 25 12.5 Kp-9 K. pneumoniae A206~6 12.5 12.5 Kp-10 K. pneumoniae A20328 6.3 3.1 Kp-ll K. pneumoniae A20330 1.6 12.5 Kp-12 K. pneumoniae A21228 6.3 6.~
Kx-2 Klebsiella sp. A9662 0.4 1.6 Kx-3 Klebsiella sp. A20346 0.2 o.8 Sm-l S. marcescens A20019 25 25 Sm-2 S. marcescens A20335 3.1 12.5 Sm-3 S. marcescens A203~6 6.3 12.5 Sm-4 S. marcescens A20442 6.3 12.5 Sm-5 S. marcescens A20222 3.1 12.5 Sm-~ S. marcescens A20460 6.3 12.5 Sm-9 S. marcescens A20333 G.3 5 Sm-10 S. marcescens A20334 6.3 5 Sm-ll S. marcescens A20459 6.3 25 Sm-12 S. marcescens A20461 6.3 5 Se-l S. enteritidis A9531 0.1 0.2 St-1 S. typhosa 0.1 0.2 Sh-1 S. paratyphi 0.1 0.2 St-101 S. typhimurium 0.1 0.2 Sd-l S. dysenteriae 0.1 0.2 Sr-l S. flexneri A9684 12.5 3.1 Ss-l S. sonnei Yale 0.1 0.1 Cx-l Citrobacter sp. A20673 1.6 1.6 Cx-2 Citrobacter sp. A20694 1.6 1.6 Cx-3 Citrobacter sp. A20695 1,6 1.6 ~1~7724 U~
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Table 5. Subcutaneous Mice Blood Levels Mcg./Ml.

. . Dose Time BB-S488 Cefamandole 40 mg./kg. 15~ 19 18 30' 17 11 60' 12 3.9 120' 1.6 0.3 20 mg./kg. 15' 7.4 9.5 30~ 6 4.1 60' 4.~ 1 120' 0.7 <0.1 10 mg./kg. 15' 5 3.8 3' 3 1.5 60' o.8 0.3 120' -- 0.1 . . .

- 66.-~1~77Z~

Table 6. In vivo Activity .

Test Organism Dose BB-S488 Cef~na~dole S. aureus Smith 25 mg./kg. 5/5* 5/5 6.3 5/5 5/5 1.6 5/5 5/5 0.4 5/5 1/5 0.1 2/5 PD~o 0.12 mg./kg. o.6 mg./kg.
E. coli Juhl25 mg./kg 5/5 5/5 6.3 5/5 5/5 1.6 5/5 2/5 0.1 2/5 PD50 0.12 mg./kg. 1.8 mg./kg.
K. ~neumoniae25 mg./kg. 5/5 5/5 6.3 5/5 1/5 1.6 5/5 /5 0.4 4/5 0/5 0.1 0/5 PD50 0.26 mg./kg.6.25 mg./kg.

*No. of survivors/No. tested _ _ ~14772~

Run No. of Dose BB-S Cefc~n-Test Organism No. LD50 (mg./kg.) 488 andole E. cloacae C-811 1 x 10100 5/5 5/5 A204~4 ~El-l9) 6.3 5/5 5/5 1.6 4/5 4/5 0,4 2/5 1/5 PDso (mg./kg,~ 0.54 o.8 _ _ _ Urinary Recovery in Rats - ~ Recovery (0-24 Hrs.
Dose tsc) BB-S488 Ce~amandole 10 mg./kg. 38.8 58.3 Nephroto ~ Test in Rabbits No nephrotoxic sign was seen in the rabbits treated with 100 mg./kg. (iv) of BB-S488 while cephalori-dine showed severe nephrotoxicity in the comparative test, . _ -~77Z4 Additional ~ O Data (~in~le sc l`re~t;n~e~

Run D Dose BB-S Cefam-Test Organism No. L 5o (mg./kg.) 488 andole K pneumoniae c-805 3xlO 25 5/5 5/5 6 ~ 3 5/5 1/5 1.6 5/5 0/5 0.4 4/5 0/5 o,1 o/5 _ __ PD50 (mg./kg.) 0.26 9.4 P. vulgaris C-808 lxlo 50 --- 3/5 A94-3~ (Pv-l) 5/5 ___ 12.5 --- 1/5 6 - 3 5/5 ___ 3.1 --- o/5 1.6 3/5 -- -o .8 - -- 0/5 0.4 1/5 - - -PD50 (mg./kg.) 1.1 36 P. mirabilis C-810 lx103 50 --- 2/5 A9900 (~m-2) 5/5 ___ 12.5 --- 1/5 6.~ 5/5 ---3.1 - _ o/5 1.6 4/5 ---o.8 --- 0~5 o .4 0/5 - - -0.2 --- /5 o .1 0/5 - - -PD50 (mg~/kg.) 1.1 50 . . .

Stability of BB-S488 Stability of BB-S488 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 the initial solution. Tlle activity was a~ayed using paper discs on B. subtilis PCI 219 plate _ (pH 6), (1) Stability in a 10~ Aqueous Solution at Room Temperature 1 Remaining Activity (~
Compound ~ 0 2 3 7 Days BB-S488 6,1 100 128 90 116 (l)Unadjusted pH of the 10~ solution.

2~ Remaining ActivitY (%) Compoundp~( 0 1 2 3 7 Days r4 100 93 78 102 BB-S488 ~ 7 100 87 64 56 ~ 10~ 20 14 1~ 0 (2) pH 4: O. 1 M AcOH - NaOAc buffer.
pH 7: 0.1 M phosphate buffer.
pH 9: 0,1 M NH40H-NH4Cl buffer.

~1~7729~

EXA~lPLE 2 ~CHCOOH C12CIICOCl ~CHCOOH 1 SOC12 D~

CHcocl H-AC~-S-CMTP(3~ [~ CHCO-ACA-S-CMTP~

HCO-ACA-S-CMTP
OH

Dichloroacetylmandeloyl Chloride (2~
A mixture of D(-)-mandelic acid (1, 1.52 g,, 10 m.mole) and dichloroacetyl chloride (4.41 g.~ ~0 m.mole) was heated at 80-85~ C. for 1.5 hrs. and the excess dichloroacetyl chloride was removed under diminished pressure. To 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 ~ept over KOH at 1 mm llg overnight at room temperature to remove dichloroacetyl chloride.
Yield, 2.8 g. (100~). This product was used in the next . ` 11~7;~4 step without further purification.
ir: ~ laq. 1780, 1160 cm 1.

nmr: ~ pC14 5.91 (lH, s, PhCH or COCHC12), 6.oo (lH, s, PhCH or COCHC12), 7.32 (5H, s, phenyl-H~.

BB-S488,_7-(D-Mandelamido)-3-(2-carboxymethyl-2,3-dihydro-s-triazolo~4,~-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic Acid (4) A solution of the above-obtained dichloroacetyl-mandeloyl chloride (2, 2.8 g., 10 m.mole~ in dry acetone (30 ml.) was added dropwise to a stirred solution of 7-amino-3-(2-carboxymethyl-2,3-dihydro-s-triazolo[4,3-b]-pyridazin-3-on-6-ylthiomethyl)-~-cephem-~-carbo~.ylic acid (H-ACA-S-CMTP)(~, 3.94 g., 9 m.mole) and triethyl-amine (3.54 g., 35 m.mole) in 50% aqueous acetone (120 ml.
at 0-5 C. The mixture was allowed to rise to room tem-perature 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 tem-perature 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 THF (150 ml.~, stirred for 5 minutes at 40-50 C. and filtered to remove insoluble unreacted ~ (0.54 g., 14~ recovery~. The fil-trate was chromatographed on a silica gel column (T~Jako-gel, C-200, ~0 g.) and eluted with chloroform-methanol (100:5). The eluates were collected in 50 ml. fractions monitoring by tlc (silica gel, solvent, CH3CM-water = 4:1, 1~77Z4 detected with I2). The fractions containing the desired product were combined, treated with a small amount of carbon and evaporated under reduced pressure. The resi-due was triturated with chloroform (50 ml.) to yield, 2.~6 g. (46~) of 7-(D-mandelamido)-3-(2-~arboxymethyl-2,3-dihydro-s-triazolo[4,3-b~pyridazin-3-on-6-ylthio-methyl)-3-cephem-4-carboxylic acid (4). M.P., 165-170 C. (dec.).
ir: ~ KBax 3600-2500, 1780, 1720, 1500, 1410, I355, 1220, 1195 cm 1.
uv: ~ maH 254 nm (e, 18300), 297 nm (sh, &, 9300).

nmr: ~ ppSO d6 3.84 (2H, m, 2-H), 4.17 (211, d, 13 Hz, 3-H), 4.50 (lH, d, 13 Hz, 3-H), 4.~2 (2H, s, NCH2COO), 5.20 (lH, d, 4.5 Hz, 6-H), 5.25 (lH, s, PhCH), 5.87 (lH, d-d, 4.5 & 9 Hzj 7-H, a doublet (J=4.5 Hz) by addition of D20), 7.25 (lH, d, 11 Hz, pyrid~zine-H), 7.4-7.7 (5H, m, phenyl-H), 7.90 (lH, d, 11 Hz, pyridazine-l~), 9.0 (lH, d, 9 Hz, 7-CONH, disappear by addit~on f ~2) Anal. Ca1C'd for C23H20N608S23/ C 3 H, 3.16; N, 12.69; S, 9.69. Found: C, 43 11, 43.22; H, 2.97, 3.06, N, 12.80, 12.77; S, 9.64.

~47~7Z4 /~ 99~ HCOOH G\ SOCl _CHCOOH -- ---) ~ CHCOOH 2 OH OCHO

H-ACA-S-CMTP (3) /~
CHCOCl~ ~ ~ CHCO-ACA-S-CMTP

OCHO OCHO
6 7, BB-S494 0-Formyl-D(-)-mandelic ~cid t5~
A mixture of D(-)-mandelic acid (1, 5.0 ~., 33 m.mole) and 99~ formic acid (80 ml.) was heated at 80-90 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 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. ) .

ir: ~ ~ar 3400-2800, 1755, 1720, 1160, 990 cm I, nmr: ~; ppml~ 5.98 (lH, s, PhCH), 7.31 (5~1, m, phenyl-H), 8~.o5 (lH, s, OCHO), 10.05 (lH, s, COOH, disappeared by addition of D20).

1~47'7;~

0-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 distilla-tion of the residue under reduced pressure afforded the acid chloride 0-formyl-D(-)-mandeloyl chloride ~6) Yleld, 1.53 g. (70~). B.P., 120-122 C./15 mmHg.
ir: ~ miq 1805, 1740, 1160, 1140 cm~l.

BB ~ -(D-0-Formylmandelamido)-3-(2-carboxymethyl-2,3-dihydro-s-triazolor4,3-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~pyridazin-3-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 HCl. The separated oily gum was collected and dissolved in THF (100 ml.). The solution l~as 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 (~8%) M.P., 172~176 C.(dec.).

ir: ~ mBax ~600-2400, 1775, 1720, 1550, 1355, 12~0, 1160 cm~l uv: ~ mt~I 254 nm (, 20800), 297 nm (sh, ~, 10500) 1. .

~477'~

nmr: ~ ppMmO d6 3.4-4.5 (4H, m, 2-H and 3-H), 4.67 (2H, s, NCH2COO), 4.97 (lH, d, 4 Hz, 6-H), 5.66 (lH, d-d, 4 & 8 Hz, 7-H), 7.2-7.5 (5E~, m, phenyl-H), 7,64 (lH, d, 10 Hz, pyridazine-H), 8.29 (lH, s, CHO), 9.29 (lH, d, 8 Hz, 7-CONH, disappeared by addition of D20).
Anal. Calc'd. for C24H20N609S2 lI2 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.

BB-S488; 7-(D~ andelamido)-3-(2-carboxymethyl-2,3-dihydro-s-triazolo-~4,3-b~pyridazin-3-on-6-ylthiomethyl)-~-cephem-4-carboxylic Acid (4~
A mixture of 7-(D-O- formylmandelamido-3-(2-car-boxymethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-~-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 tempera-ture, and acidified to pH 1 with dilute HCl. 'rhe preci-pitate (500 m~.) 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 ~ere combined, treated with a small amount of carbon and eva-porated under reduced pressure. The residue ~las triturated with ether to give 277 mg. of 7-(D-mandelamido)-3-(2-car-boxymethyl-2,3-dihydro-s-triazolo~4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid tBB-S488; 4).

The nmr-estimation of this product sho~Jed 10~ of 7 still remained.

- 7~ -~77Z4 ir: ~ KBar 3600-2400, 1770, 1720, 1520, 1495, 1365, 1230 cm~l.
uv: ~ EaH 254 nm (~, 20000), 297 nm (sh, ~, 9600).

Sodium Salt of BB-S488 Sodium-2~ethylhexanoate (SEH) (4.0 ml., 1 M
solution in ethyl acetate) was added to a solution of 7-(D-mandelamido)-3-(2-carboxymethyl-2,3-dihydro-s-tri-azolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic 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 60 C.~l mmHg for 3 hours.
Yield of sodium 7-(D-mandelamido)-3-(2-carboxymethyl-2,3-dihydro-s-triazolo~4,3-b~pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylate,1.96 ~- (bio-yield, 97%), M.P., 230-240 C. (dec.). The pH o the 10~ aqueous solution was 3.6.

ir: J m~x 3600-3000, 1765, 1710, 1605, 1390, 1360, ll9P, 1080, 1065 cm~l. -uv: ~ maater nm(El'cm) 252 (357), 310 (sh, 140).

nmr: ~ pD~m 3.43 (lH, d, 19 Hz, 2-H), 3.87 (lH, d, 19 llz, 2- ), 4.15 (lH, d, 14 Hz, 3-H), 4.53 (lH, d, 14 Hz, 3-_), 5.16 (lH, d, 4.5 Hz, 6-H), 5.36 (lH, s, PhCH), 5.73 (lH, d, 4.5 Hz, 7.H), 7.13 (lH, d, 10 Hz, pyridazine-H), 7.57 (5H, s, phenyl-H), 7.69 (lH, d, 10 Hz, pyridazine-H).
Anal. Calc'd. for C23HlgN60~S2N 5/ 2 H, 3.51; N, 13.62; S, 10.39. Found: C, 44.93, 44.79; H, 3.31, 3.15; N, 13.~1, 13.33; S, 10.19.

Aqueous lN sodium hydroxide solution was added dropwise to a suspension of 7-(D-mandelamido)-3-(2-carboxymethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid (4)(3.51 g.) in ~qater (20 ml.) to adjust to pH 6.o. The solution was lyophilized to yield 3.4 g. (bio-yield, 97%) of disodium 7-(D-mandelamido)-3-(2-carboxymethyl-2,3-dihydro-s-tri-azolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylylate. M.p., >240 C. (dec.). The pH of the 5% aqueous solution was 5.4.

ir: J mBar 3600-3000, 1760, 1710, 1605, 1390, 1360, 1190, 1080, 1060 cm~l.
uv: /1 mater nm (El'Cm) 252 (320), 310 (124).

nmr: ~ pp2m 3.43 (lH, d, 19 Hz, 2-H), 3.90 (lH, d, 19 Hz, 2-H), 4.15 (lH, d, 14 Hz, 3-H), 4.53 (lH, d, 14 Hz, 3-H), 4.75 (2H, s, NCH2C0), 5.22 (lH, d, 4.5 Hz, 6-H), 5.42 (lH, 8, PhCH), 5.73 (lH, d, 4.5 Hz, 7-H), 7.22 (lH, d, 10 Hz, pyridazine-H), 7.65 (5H, s, phenyl-H), 7.77 (lH, d, 10 Hz, pyridazine-H).
Anal. Calc~d. for C23H18N68S2Na2 3/ 2 42.92; H, 3.29; N, 13.06; S, 9.96. Found: C, 42.90, 43.19; H, 3.o6, 3.01; N, 13.04, 13.03; S, 9.97.

~ .

1~477~

ExamDle 7 Substitution of the D-mandellc acid carboxy-anhydrlde in the procedure of Example 1 Or an equlmolar welght Or the carboxyanhydride~ prepared ln ~lmllar fa~hlon rrom the monosub~tltuted D-mandelic acids D-2-chloro-mandellc acld, D-3-chloro-mandellc acld, D-4-chloro-mandellc acld, D-2-bromo-mandellc acld, D-~-bromo-mandellc acld, D-4-bromo-mandelic acld, D-2-rluoro-mandellc acld, D-3-fluoro-mandellc acld, D-4-fluoro-mandelic acld, D-2-trlfluoromethyl-mandellc acld, D-~-trlfluoromethyl-mandellc acid, D-4-trlrluoromethyl-mandellc acld, D-2-amino-mandellc acld, D-3-amlno-mandellc acld, D-4-amlno-mandelic acld, D-2-nitro-mandellc acid, D-3-nltro-mandellc acid, D-~-nitro-mandellc acld, D-2-hydroxy-mandellc acld, D-3-hydroxy-mandellc acld, D-~-hydroxy-mandellc acid, ~47724 D-2-methyl-mandellc acld,-D-~-methyl-mandellc acld, D-4-methyl-mandellc acld, D-~-methoxy-mandellc acld, D-~-methoxy-mandellc acld and D-4-methoxy-mandellc acld respectlvely produces 7-(D-2-chloro-mandelamido)-3-(2-carboxymethyl-2,3-dlhydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-methyl)-3-cephem-4-carboxylic acid, 7-(D-3-chloro-mandelamido)-3-(2-carboxymethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-methyl)-3-cephem-4-carboxylic acid, 7-(D-4-chloro-mandelamido)-3-(2-carboxymethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-methyl)-3-cephem-4-carboxylic acid, 7-(D-2-bromo-mandelamido)-3-(2-carboxymethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-~-ylthio-methyl)-3-cephem-4-carboxylic acid, 7-(D-3-bromo-mandelamido)-3-(2-carboxymethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-methyl)-3-cephem-1~-carboxylic acid, 7-(D-4-bromo-mandelamido)-3-(2-carboxyrnethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-methyl)-3-cephem-4-carboxylic acid, 7-(D-2-fluoro-mandelamido)-3-(2-carbox~nethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-methyl)-3-cephem-4-carboxylic acid, 1~47'724 7-(D-3-fluoro-mandelamido)-3-(2-carboxymethyl-2,3-dihydro-s-triazolo[4,3-b~pyridazin-3-on-6-ylthio-methyl)-3-cephem-4-carboxylic acid, 7-(D-4-fluoro-mandelamido)-3-(2-carboxymethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-methyl)-3-cephem-4-carboxylic acid, 7-(D-2-trifluoromethyl-mandelamido)-3-(2-carboxy-methyl-2,3-dihydro-s-triazolo[4,3-b~pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid, 7-(D-3-trifluoromethyl-mandelamido)-3-(c-carboxy-methyl-2,3-dihydro-s-triazolo[4,3-b~pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid, 7-(D-4-trifluoromethyl-mandelamido)-3-(2-carboxy-methyl-2,3-dihydro-s-triazolo[4,3-b~pyridazin-3-on-6-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-ylthio-methyl)-3-cephem-4-carboxylic acid, 7-(D-3-amino-mandelamido)-3-(2-carboxymethyl-2,3-dlhydro-s-triazolo[lJ,3-b]pyridazin-3-on-~-ylthio-methyl)-3-cephem-4-carboxylic acid, 7-(D-4-amino-mandelamido)-3-(2-carboxymethyl-2,3-dihydro-s-triazolo[4,3-b]pyridaain-3-on-6-ylthio-methyl)-3-cephem-4-carboxylic acid, 7-(D-2-nitro-mandelamido)-3-(2-carboxymethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-methyl)-3-cephem-4-carboxylic acid, .7-(D-3-nitro-mandelamido)-3-(2-carboxymethyl-2,3-dihydro-s-triazolo[4,3-b~pyridazin-3-on-6-ylthio-methyl)-3-cephem-4-carboxylic acid, 1~77Z~

7-(D-4-nitro-mandelamido)-3-(2-carboxymethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-methyl)-3-cephem-4-carboxylic acid, 7-(D-2-hydroxy-mandelamido)-3-(2-carboxymethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-methyl)-3-cephem-4-carboxylic acid, 7-(D-3-hydroxy-mandelamido)-3-(2-carbox~nethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-methyl)-3-cephem-4-carboxylic acid, 7-(D-4-hydroxy-mandelamido)-3-(2-carboxymethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-~-on-6-ylthio-methyl)-3-cephem-4-car~oxylic acid, 7-(D-2-methyl-mandelamido)-3-(2-carboxymethyl-2,3-dihydro-s-triazolo[4,3-b]pyrid~zin-3-oll-5-yltllio-methyl)-3-cephem-4-carboxylic acid, -7-(D-3-methyl-mandelamido)-3-(2-carboxymethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-methyl)-3-cephem-4-carboxylic acid, 7-(D-4-methyl-mandelamido)-3-(2-carboxymethyl-2,3-dlhydro-s-triazolo[4,3-b~pyridazin-3-on-6-ylthio-methyl)-3-cephem-4-carboxylic acid, 7-(D-2-methoxy-mandelamido)-3-(2-carboY~ymethyl-2,3-dihydro-s-triazolo[4,3-b~pyridazin-3-on-6-ylthio-methyl)-3-cephem-4-carboxylic acid, 7-(D-3-methoxy-mandelamido)-3-(2-carboxymethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-methyl)-3-cephem-4-carboxylic acid, and 7-(D-4-methoxy-mandelamido)-3-(2-carboxymethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-methyl)-3-cephem-~-carboxylic acid, respectively.

77~4 'EXAMPLE 8 Sub~titutlon for the D-mandelic acid carboxyanhydrlde in the procedure of Example 1 of an equimolar weight of the carboxyanhydride prepared in ~lmilar fashion from D-2-thlopheneglycollc acld and D-~-thiopheneglycollc acid respectively produces 7-(D-a-hydroxy-2-thienylacetamido)-3-(2-carboxymethyl-2,3-dihydro-s-triazols[4,3-b]pyridazin-3-on-6-ylthio-methyl)-3-cephem-4-carboxylic acid and 7-(D-a-hydroxy-3-thienylacetamido)-3-(2-carboxymethyl-2,3-dihydro-s-~riazolo~,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid, 'respectively.

EXAIIPI~ 9 7-(D-~-Hydroxy-~-phenylacetamido)-3-(2-carboxymethyl-2,3-dihydro-s-triazolo~4,3-blpyridazin-3-on-6-ylthio-methyl)-3-cephem-4-carboxylic Acid ~re~red From 7-D-Mandelc~midocephalospor.~nic Acid.
0,27 Mole o~ sodium 7-D-mandelamidocephalo-eporanate ls suspended ln 1000 ml. of 0 1 M phosphate burrer Or pH 6.4 and there is added 0.31 moles of 2-carboxymethyl-2,3-dihydro-6-mercapto-s-triazolo-~4,3-b]pyrid~zin-3-one. The solution is heated at 55 C. under a nitrogen atmosphere ~or five hours. After one hour the pH is ad3usted to 6,4 by addltion of a small amount of 40~o H~P04. At the end o~
the five hour heating period the solution is cooled to 23 C. and the pH ad~usted to 2 by the addition 11477~4 of 3 N HCl under a layer of ethyl acetate. The product 1~ extracted lnto ethyl acetate and stlrred ror 15 mln. at 23 C. wlth 2 g, of ("Darco KB") decolorlzlng charcoal. The mixture is then flltered through a pad of dlatomaceous earth ("Cellte") and the ethyl acetate removed ~rom the filtrate under vacuum. The re~ldue i8 trlturated to a 3011d with dlethyl ether, collected by flltratlon and drled over P205 under vacuum to yleld solld 7-(D-a-hydroxy-~-phenylacetamldo)-3-(2-carboxymethyl-2,3-dihydro-s-triazolo~4,3-b~pyridazin-3-on-6-y]thiomet}lyl)-3-cephem-4-carboxylic acid.

Example 10 H2N~S~ ~

0~ ~CH2 - SI~N~~N--Cli2C1~2COOH
C21i o C61i5~

6~
\ / O

C \ H s _ N

OH ~ ~ c~2_ ~ ~ ~ 3-C112C 2 CO2~ o 77~4 BB-S 527; 7-[D(-)-Mandelamido~-3-12-(2-carboxyethvl)-2,3-dihydro-s-triazolo-[4,3-b]pyridazin-3-on-6-ylthiomethyl]-3-cephem-4-carboxylic acid To a mixture of 7-amino-3-[2-(2-carboxyethyl)-2,3-dihydro-s-triazolo~4,3-b]pyridazin-3-on-6-ylthiomethyl]-3-cephem-4-carboxyllc acid (679 mg, 1.5 m mol) and NaHCO3 (445 mg, 5.3 m mol) in 50% aqueous acetone was added D-(-)-mandelic acid O-carboxyanhydride (400 mg, 2.3 m mol) at OC. 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. HCl and fiLtered to collect the crude product, which was di~solved in TI~F (10 ml), filtered to remove insolubles and evaporatea 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, 10 g) and eluted with MeOII-CHC13 (MeOII: 0-3%). Fractions which contained the desired product were combined and evapor-ated to yield 287 mg ~33~) of BB-S 527. ~I.p. >155C (dec.).

irs ~Kar 3600 - 2400, 1780, 1720, 1550, 1520 cm 1.
u~: ~PI~ 7 buffer 253 nm (E 20000), 298 nm (E 9000) -Anal. Calc'd- for C24H22N6852 / 2 4.11; N, 13.70; S, 10.45. Found: C, 47.25, 47.39; ~, 3.80, 3.76; N, 12.87, 12.77; S, 10.17.

1147'724 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. ~210C (dec.).

ir: vmaBr 3600 - 2800, 1770, 1710, 1600 cm~l.

ApH 7 Buffer 253 nm ( 21000) 298 nm ( 9300) Anal. Calc'd. for C24H20N6O8S2Na2~2H2O: C, 43.24: H, 3.63; N, 12.61; S, 9.62. Found: C, 43.39, 43.43; }1, 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-Hinton agar plate) MIC (mcq/ml) . .
Organism B-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 St. faecalis >100 >100 >100 E. coli NIHJ 0.4 0.2 0.05 E. coli A~CC 8?3912.5 6.3 3.1 E. coli Juhl 0.4 0.2 0.8 E. coli BX-1373 6.3 3.1 3.1 E. coli 0.1 0.1 0.1 E. coli 0.1 0.05 0.1 E. coli 6.3 3.1 1.6 ~ . , ; _ , , . , . ____ .

~14772~

Xl. 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 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 O.G5 0.2 Pr. morganii >100 >100 >100 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 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 Sal. typhosa 0.1 0.05 0.1 . anthracis 0.4 0.2 0.4 1~4'~'7Z4 ExamDle ~
Sub~titution for the D-mandellc acid carboxy-anhydride in the procedure Or ExamplelO of an equlmolar weight o~ the carbox~anhydrldes prepared ln ~imllar ~ashlon from the monosubstltuted D-mandelic aclds D-2-chloro-mandelic acid, D-3-chloro-mandelic acld, D-4-chloro-mandelic acid, D-2-bromo-mandellc acid, D-3-bromo-mandelic acid, D-4-bromo-mandellc acid, D-2-rluoro-mandellc acld, D-~-rluoro-mandelic acld, D-4-~luoro-mandelic acid, D-2-trlrluoromethyl-mandelic acid, D-~-trl~luoromethyl-mandelic acld, D-4-trl~luoromethyl-mandellc acid, D-2-amlno-mandellc acld, D-3-amlno-mandellc acid, D-4-amlno-mandellc acld, D-2-nitro-mandellc acid, D-3-nitro-mandellc acld, D-4-nltro-mandelic acld, D-2-hydroxy-mandellc acid, D-3-hydroxy-mandellc acld, D-~-hydrox~-mandellc acid, ~, _ ,. , ___ . , , . , .. ,, _ ~4~772~

D-2-methyl-mandellc acid, D-3-methyl-mandellc acid, D-4-methyl-mandelic acld, D-2-methox~-mandellc acld, D-~-methoxy-mandelic acid and D-4 methoxy-mandelic acld respectlvely produces 7-(D-2-chloro-mandelamido)-3-(2-carboxyethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazln-3-on-6-ylthio-methyl)-3-cephem-4-carboxylic acid, 7-(D-3-chloro-mandelamido)-3-(2-carboxyethyl-2,3-dihydro-s-triazolo[~ -b]pyridazin-3-on-6-ylthio-methyl)-3-cephem-4-carboxylic acld, 7-(D-4-chloro-mandelamido)-~-(2-carboxyethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-methyl)-3-cephem-4-carboxylic acid, 7-(D-2-bromo-mandelamido)-3-(2-carboxyethyl-2~3-dihydro-e-triazolo[4,3-b]pyridazin-3-on-6-ylthio-methyl)-3-cephem-4-carboxylic acid, 7-(D-3-bromo-mandelamido)-3-(2-carboxyethyl-2,3-dihydro-s-triaz~10[4,3-b~pyridazin-3-on-6 ylthio-methyl)-3-cephem-4-carboxylic acid, 7-(D-4-bromo-mandel~mido)-3-(2-carboxyethyl-2,3-dihydro-s-triazolo[4,3-b~pyridazin-3-on-6-ylthio-metnyl)-3-cephem-4-carboxylic acid, 7-(D-2-fluoro-mandelamido)-3-(2-carboxyethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-methyl3-3-cephem-4-carboxylic acid, 1~77;~4 7-(D-3-fluoro-mandelamido)-3-(2-carboxyethyl-2~3-. dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-methyl)-3-cephem-4-carboxylic acid, 7-(D-4-fluoro-mandelamido)-3-(2-carboxyethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-methyl)-3-cephem-4-carboxylic acid, 7-(D-2-trifluoromethyl-mandelamido)-3-(2-carboxy-ethyl-2,3-dihydro-s-triazolo[4,3-b~pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid, 7-(D-3-trifluoromethyl-mandelamido)-3-(2-carboxy-ethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid, 7-(D-4-trifluoromethyl-mandelamido)-3-(2-carboxy-ethYl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid, 7-(D-2-amino-mandelamido)-3-(2-carboxyethyl-2,3-dihydro-s-triazolo[~,3-b]pyridazin-3-on-6-ylthio-methyl)-3-cephem-4-carboxylic acid, 7-(D-3-amino-mandelamido)-3-(2-carboxyethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-methyl)-3-cephem-4-carboxylic acid, 7-(D-4-amino-mandelamido)-3-(2-carboxyethyl-2,3-.. dihydro-s-triazolo[4,3-b~pyridaain-3-on-6-ylthio-methyl)-3-cephem-4-carboxylic acid, 7-(D-2-nitro-mandelamldoJ-3-(2-carboxyethyl-2,3- .
dihydro-s-triazolo~4,3-b~pyridazin-3-on-6-ylthio-methyl)-~-cephem-4-carboxylic acid, 7-(D-3-nitro-mandelamido)-3-(2-carboxyethyl-2,3-dihydro-s-triazolo[4,3-b~pyridazin-3-on-6-ylthio-methyl)-3-cephem-4-carboxylic acid, i~7724 7-(D-4-nitro-mandelamido)-3-(2-carboxyethyl-2,3-dihydro-s-triazolo~4,3-b]pyridazin-3-on-6-ylthio-methyl)-3-cephem-4-carboxylic acid, 7-(D-2-hydroxy-mandelamido)-3-(2-carboxyethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-methyl)-3-cephem-4-carboxylic acid, 7-(D-3-hydroxy-mandelamido)-3-(2-carboxyethyl-2,3-dlhydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-methyl)-3-cephem-4-carboxylic acid, 7-(D-4-hydroxy-mandelamido)-3-(2-carboxy~thyl-2,3-dihydro-s-triazolo[4,3-b]pyrldazin-3-on-6-ylthlo-methyl)-3-cephem-4-carboxylic acid, 7-(D-2-methyl-mandelamido)-3-(2-carboxyethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-methyl)-3-cephem-~-carboxylic acid, 7-(D-3-methyl-mandelamido)-3-(2-carboxyethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-methyl)-3-cephem-4-carboxylic acid, 7-(D-4-methyl-mandelamido)-3-(2-carboxyethyl-2,3-dihydro-s-triazolo~4,3-b]pyridazin-3-on-6-ylthio-methyl)-3-cephem-4-carboxylic acid, 7-(D-2-methoxy-mandelamido)-3-(2-carboxyet~yl-2~3 . dihydro-s-triazolo[4,~-b]pyridazin-3-on-6-ylthio-methyl)-3-cephem-4-carboxylic ac~d, 7-(D-3-methoxy-mandelamido)-3-(2-carboxyethyl-2,3-dihydro-s-triazolo~4,3-b]pyridazin-3-on-6-ylthio-methyl)-3-cephem-4-carboxylic acid, and 7-(D-4-methoxy-mandelamido)-3-(2-carboxyethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-methyl)-3-cephem-4-carboxylic acid, respectively.

1~ ~'77~4 EX~MPLE 12 Substitution for the D-mandelic acid carboxyanhydride in the procedure of Example 10 of an equimolar weight or the carboxyanhydride prepared in similar fashion from D-2-thiopheneglycolic acid and D-3-thiopheneglycolic acid respect~vely produces 7-(D-~-hydroxy-2-thienylacetamido)-3-~2-carboxyethyl-2,3-dihydro-s-triazolo~4,3-b]pyridazin-3-on-6-ylthiomethyl]-3-cephem-4-carboxylic acid and 7-~D-a-hydroxy-3-thienylacetamido)-3-(2-carboxyethyl-2,3-dihydro-s-triazolo~4,3-blpyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid, respectively.

1~47'7;~4 .

C0-N p C~2-S ~ -CH2COONa C2Na 7-~(2Z)-2-Methoxyimino(fur-2-yl)acetamido]-3-(2-carboxy-methyl-2,3-dihydro-s-triazolo~4,3-b]pyridazin-~-on-6-ylthiomethyl)-~-cephem-4-carboxylic Acid Disodium -Salt; BB-S511, 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., ~ 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,), A~ter filtration the acetone solution was added to a mix-ture of 7~amino-3-(2-carboxymethyl-2,3-dihydro-s-triazolo-[4,~-b~pyridazin-3-on-6-ylthiomethyl)-3-cephem-~-carboxylic acid (1,31 g " 3 m,moles) and NaHC0~ (504 mg "
6 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 conc, HCl to afford a precipitate which was collected by filtration, washed with water and dried in vacuo, The solid was dissolved in THF (tetrahydrofuran) (~0 ml,) and 1~77Z~

filtered. To the filtrate was added 1 M-SEH (sodium ethylhexanoate) in ethyl acetate (3 ml.) and the result-ing precipitate was collected by filtration and dried in vacuo. Yield of 7-[(2Z)-2-methoxyimino(fur-2-yl)acet-amido~-3-(2-carboxymethyl-2,3-dihydro-s-triazolo[4,~-b3-pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid di~odium salt was 1 0 g. (54%) mp >210 C. (dec.).
ir: vmBax 1770, 1710, 1680, 1610, 1550 cm 1.
uv ~pH mabUffer 257 nm (E 25000), 277 nm (~ 24000).

nmr: ~DMppmd6 7.7o (lH, br, furan-Ha), 7.52 (lH, d, J=9.5 Hz, pyridazine-H), 6.87 (lH, d, J=9.5 Hz, pyridazine-H), 6 5-6.6 (2H, m, furan-H~), 5.58 (lH, m, 7-H), 5.00 (lH, d, J=4.5 Hz, 6-H), 3.84 (3H, s, OCH3).
Anal. Calcld- for C22H17lJ7gS2Na2 2 H, 2.94; N, 15.05; S, 9.84. Found: C, 40.81, 41.02;
H, 3.o8, 3.22, N, 14.69, 14.86; S, 9.70, 9.62.

_ 9~ _ ~7'7;~4 J~OCH3 ~CH2-5 (~ I-CH2CH2COOH

7-l(2Z¦-2-Methoxyimino(fur-2-y~ acetamido]-3-~2-(2-car-boxyethyl)-2~3-dihydro-s-triazolor4,3-b]pyridazin-3-on-6-ylthiomethyl]-3-cephem-4-carboxylic Acid Sodium Salt;

The acid chloride prepared from (2Z)-2-methoxy-imino(fur-2-yl)acetic acid (169 mg., 1 m.mole) was dis-solved in dry acetone (5 ml.) and filtered to remove in-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 NaHC03 (~36 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 ad~usted to pH 1-2 with conc. HCl. The resulting precipita~e was collected by filtration, washed with water and dried under reduced pressure. A solution of the precipitate in THF (10 ml.) was treated with active carbon. A SEH solution in ethyl acetate (1 ~ o.8 ml.) was added to the THF solution to give 7-[(2Z)-2-methoxyimino(fur-2-yl)acetamido~-3-[2-(2-carboxyethyl)-2,3-dihydro-s-triazolo~4,3-b]pyridazin-3-on-6-ylthiomethyl]-3-cephem-4-carboxylic acid sodium salt which was collected by filtration. Yield: 410 mg. (66 as mono Na salt). ~Ip ~205D C. (dec.).

~477'~4 ir: ~mBr 3600-2800, 1770, 1710, 1600, 1550 cm 1 uv: ~pH mabxUffer 257 nm (e 27000), 276 (~ 26100).
nmr: ~DMppOmd6 D20 2.70 (2H, t, J=6 Hz, CH2), 3.3-4.5 (9H, m, 2-H, 3-CH2, CH2 and OCH3), 4.96 (lH, d, J=5.5 Hz, 6-H), 5.53 (lH, d, J=5.5 Hz, 7-H), 6.55 (2H, m, furan-H), 6.92 (lH, d, J=10 Hz, pyridazine-H), 7.53 (lH, d, J=10 Hz, pyridazine-H), 7 68 (lH, br, furan-H).
Anal. Calc'd. for C2~H20N709S2Na H20: C, 42-92;
H, 3.45; N, 15.23, S, 9.96. Found: C, 43.08, 42.77;
H, 3.20, 3.03; N, 14.96, 14.76; S, 9.96.

In vitro Activity Usin~ rlueller-Hinton Agar ~~ By~the Serial ~lutioll ;letllod ~ Geometric ~ean- of MIC
(Mcg./ml.) ~Ex. 13? (Ex. 14) Cefuroxime S. aureus (~ strains) 1.97 1 6 1 24 E. coli (7) o 58 0.78 1.28 Kl. pneumoniae (4) 0.47 0 93 3.1 Proteus (6) 0.021 0.061 o.88 Shig.(3), Serr.(l) Enterab.(l), Sal.(2) 1.11 2.41 4. o6 B. anthracis (1) S. pyogenes (5) 0.032 0.032 0.025 S. ~iridans (5) 0.15 0.4 0.1 D. pneumoniae (5) 0 037 0.056 0.0125 N. meningitidis (5) 2.37 3.60 1.6 N. gonorrhoeae (5) 1.36 1.6 0.4 H. influenzae (7~ 0.64 0.71 1.16 Cefuroxime is sodium 6R,7R-3-carbamoyloxymethyl-7-(2Z)-2-methoxyimino(fur-2-yl)acetamidoceph-3-em-4-carboxylate.

_ 9~ _ '7'~4 Geometric Means of MIC's Against 3 Strains of S. aureus and 27 Strains of Gram-negative Bacteria (mcg./ml., Mueller-Hinton Agar) No. of Strains BB-S511 BL-S786 S. aureus 1 1.6 1.6 S. aureus, Penicillin-R 2 o.6 1.6 E. coli 6 0.2 0.2 E. coli, Cephalosporin-R 1 6.312.5 K. pneumoniae 4 o,7 o,3 Indole (-) Proteus 2 0.1 0.2 Indole(~) Proteus 3 o,o5o,3 Indole(~) Proteus, Cephalosporin-R 2 6.~ 50.1 S. marcescens 1 25 >100 E. cloacae 1 ~.1 1.6 Shigella, Salmonella 5 o.3 o,5 P. aeruginosa 2 >100 >100 BL-S786 is 7-[a-(2-aminomethylphenyl)acetamido]-3-[(l-car boxymethyltetrazol-5-ylthio)methyl]-3-cephem-4-carboxylic acid, Geometric ~leans of IIIC's Against 18 Strains . _ .
of S. marcescens .
5,4 85-9 l~f~7'7~4 EXAMP~E 15 . Substitution of an equimolar weight of 2-ethoxy-imino-2-(fur-2-yl)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-carboxy-methyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-~-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid and 7-(2-ethoxy-imlno-2-furylacetamido)-3-[.2-(2-carboxyethyl)-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-~n-6-ylthiomethyl~-~-cephem-4-carboxylic acid, respectively.

Substitution of an equimolar ~leight of 2-n-propoxy-imino-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-carboxy-methyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid and 7-(2-n-propoxy-lmino-2-furylacetamido)-3-~2-(2-c~rboxyethyl).-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-Ylthiomethyl]-3-cephem-4-carboxylic acld, respectively.
. . .

Substitution of an equimolar weight of 2-n-butoxy-imino-2-(fur-2-yl)acetic acid for the 2-methoxyiminofuryl acetic acid used in the procedures of Examples 13 ~.nd 14 produces 7-(2-n-butoxyimino-2-furylacetamido)-3-(2-carboxy-methyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-car~oxylic acid and 7-(2-n-butoxy-: - 98 -~147724 lmino-2-furylacetamido)-~-[2-(2-carboxyethyl)-2,~-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl]-3-cephem-4-carboxylic acid, respectively.

The products of Examples 1~-17 ~re prepared es s~n isomers essentially free of the corresponding anti isomers by the use in the procedures of those examples of purifled syn isomers of the appropriate 2-alkoxyimino-~-(fur-2-yl)-acetic acid. Conversion of part of the syn isomer to anti l~omer durlng preparation of the acid chloride from the acid ls substantlally 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 ~ u~r~ syn ~ b ~ U as ~G ) alkoxyimino-2-(fur-2-yl)acetic acids.

_ 99 _ ~77~
.

An in~ectable pharmaceutical composition is formed by adding sterile water or sterile saline solu-tion 52 ml.) to 100-500 mgm. of 7-[(2Z~-2-methoxyimino-(fur-2-yl)acetamido]-3-(2-carboxymethyl-2,~-dihydro-s-triazolo[4,~-b]pyridazin-~-on-6-ylthiomethyl)-3-cephem-4-carboxylic 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 lsomer, are formulated in a similar manner.
When the compounds are first prepared in the form of the free acid they are converted to the deslred, highly water soluble potassium salt by treat-ment wlth potassium 2-ethylhexanoate using the procedure of Example 13.
It is occasionally advantageous to have ad-mixed with said solid cephalosporin as a stabillzlng and/or solubilizlng agent a sterlle, anhydrous solid such as sodlum carbonate, potassium carbonate or llthium carbonate(e.g. ln about 5 or 6 percent by weight of the weight of the cephalospor~n) or such as L-lysine, argi-nine or hlstidine (e.g. in about 20-50% by weight of the weight o~ 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 cephalos-porin) or such as sodium bicarbonate, ammonium car-bamate alkali metal or ammonium phosphates or N-methyl-glucamine (per U.~. 1,380,7~

1~47'7'~4' ~Aiil'Il ~O

CO-N S ~ N
OCH3 ~ 02Na ~ N ~ N-CH3 BB-S510; 7-~(2Z)-2-Methoxyimino(fur-2-yl)acetamido~-3-(2-metilyl-2,~-dihydro-s-triazolor4 ? 3-b~pyridazin-3-on-6- ~ hyl)-3-cephem-4-carboxylic Acid.Sodium Salt To a solution of (2Z)-2-methoxyimino(fur-2-yl)-acetic acid (2~3 mg., 1.5 m mol.) and triethylamine (0 2 ml , 1.5 m.mol.~ in dichloromethane (~ ml.~ was added oxalyl chloride (0.1~ 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-~-(2-methyl-2,~-dihydro-s-triazolo-[4,3-b]pyridazin-~-on-6-ylthiomethylj-3-cephem-4-car-boxylic acid (591 mg., 1 5 m.mol.) and NaHCO~ (504 mg., 6 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 ~0 ml.) and adjusted to pH 1-2 with concentrated HC1. The precipitate which was collected by filtration, washed with water and dried in vacuo, was dissolved in THF (~0 ml.) and filtered to remove insolu~les. To the THF solution was added a solu-tion o~` sodium 2-ethylllexanoate (SEH, l M, 1.5 ml.) in 77Z~

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-methyl-2,3-dihydro-s-triazolor4,3-b~pyridazin-3-on-6-ylthio-methyl)-3-cephem-4-carboxylic acid sodium salt (58~).
M.p. >180 C. (dec.).
ir: v~Bx 1770, 1720, 1670, 1600, 1550 cm 1 uv ~pH7Buaffer 257 nm (E 22600), 277 nm (E 22300~.
nmr: ~p ~ 7.53 (lH, d, J=1.5 Hz, furan-Ha), 7.35 (lH, d, J=9 5 Hz, pyridazine-H), 6.90 (lH, d~ J=9.5 Hz, pyri-dazine-H~, 6.72 (lH, d, J=3.0 Hz, furan-H~), 6.48 (lH, q, J=1.5 and 3.0 Hz, furan-H~), 5.72 (lH, d, J=4.5 Hz, 7-H), 5.14 (lH, d, J=4.5 Hz, 6-H), 2.94 (3H, s, 0-CH3), 3 61 (3H, 6 ~ N-CH3~

Anal. Calc'd. for C21H18N707S2Na / 2 C, 44.44; H, 3.89; S, 10.32. Found: C, 44.89; H, 3.92;
S, 9.67.

~1~77'~4 In,,vitro ACttivity.-lr-n$lMutellelrl-H~intdon Ag~,r By lle Serla Dl u Geometric Mean of MIC
(Mcg./ml.) ~Ex. 20) BB-S515 Cefuroxime S. aureus (3 strains) 0.62 2.48 1.24 E. coli (7) 2,11 2.33 1.28 Kl, pneumoniae (4) 6,.3 3~1 3~1 Proteus (6) 1.39 1.11 o.88 Shig.(3), Serr.(l) Enterab.(l~, Sal.~2~ 6.26 5.26 4.o6 B. anthracls (1) S. pyogenes (5) 0.0125 0.032 0.025 S. viridans (5) 0.13 0.59 0.1 D. pneumonlae (5) 0.021 0.1 0.0125 N. meningitidis (5) 1.03 5.45 1.6 N. gonorrhoeae (5) 0,25 2,07 0,4 H, influenzae (7) 0,35 2,11 1,16 Cefuroxime is sodium 6R~7R-3-carbamoyloxymethyl-7-(2Z)-2-methoxyimino(fur-2-yl)acetamidoceph-3-em-4-carboxylate, ~147'~Z4 Geometric Means of MIC's A~ainst ~ Strains of S. aureus and 27 Strains of Gram-negative Bacteria (mcg./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 K. pneumoniae 4 2.6 o,3 Indole (-) Proteus 2 1.1 0.2 Indole~+) Proteus ~ 0,2 0,3 Indole(~) Proteus, Cephalosporin-R 2 6,~ 50.1 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-[a-(2-aminomethylphenyl)acet~mido3-3-[(1-car-boxymethyltetrazol-5-ylthio)methyl]-3-cephem-4-carboxylic acid.

.. Geometric Means of ~IC's Against 18 Strains of S. marcescens -7,9 85-9 7"7;~4 EX~PLE 21 Substitution of an equ~molar weight of 2-ethoxy-imino-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-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid.

Substitution of an equimolar weight of 2-n-propoxy-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-2,3-dihydro-s-triazolo~4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid.

ExAl`~pLE 23 Substitution of an equlmolar weight of 2-n-butoxy-imino-2-(fur-2-yl)acetic acid for the 2-methoxyiminofuryl acetic acld used in the procedure of Example 20 produces 7-(2-n-butoxyimino-2-furylacetamido)-3-(2-methyl-2,3-dihydro-s-triazolo~4,3-b]pyrid~zin-3-on-6-ylthiomethyl~-3-cephem-4-carboxylic acid.

EXA~IPLE 24 _ 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 syn isomers of the appropriate 2-alkoxyimino-2-(fur-2-yl)-acetlc acid. Converæion of part of the syn isomer to anti isomer durlng 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)-2-alkoxyimino-2-(fur-2-yl)acetic acids, ~1477~g An ln~ectable pharmaceutical composition i6 formed by addlng sterile water or sterlle saline 801u-tion (2 ml.) to 100-500 mgm. of 7c[~2Z)-2-methoxyimino-(fur-2-yl)acetamido]-3-(2-methyl-2,~-dih~ydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid disodium salt.
Pharmaceutical composltlons of the sodlum and potassium salts of the other compounds of the present lnv~ntion, preferably ln the fa~m of the pure syn isomer, are formulated in a similar manner.
When the compounds are first prepared ln the form Or the free acid they are converted to the desired, highly water soluble potassium salt by treat-ment with potassium 2-ethylhexanoate using the procedure of Example 20.
It is occaslonally advantageous to have ad-mixed 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, argi-. nine or histidine (e g. in about 20-50~ by weight of the weight o~ 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 cephalos-porin) or such as sodium bicarbonate, ammonium car-bamate,alkali metal or ammonium phosphates or N-methyl-glucamine (per U.K. 1,380,7lJl).

Claims (2)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A process for the preparation of a compound of the formula IV

wherein Al is methyl or -(CH2)nCOOH and n is one or two;
comprising reacting 7-amino-cephalosporanic acid or a salt or easily hydrolyzable ester thereof with a com-pound of the formula where A' is as defined above.

2. A compound of the formula wherein A' is methyl or -(CH2)n COOH and n is one or two;
whenever prepared or produced by the process of Claim 1 or by an obvious chemical equivalent thereof.