CA1150727A - Triazolo-pyridazinone derivatives - Google Patents

Abstract

ABSTRACT

The specification discloses a compound having the formula I

wherein A is I

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)nCOOH 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

_ ' TRI -PY-CEPH CO~.BO
~lL3Lsal~727 ~SY-1510-A) The present invention relates to novel acids havin~ the structure A~-CH - ~ Cll~2 -CH2-S ~ o ~T-A
COO~I

sometimes hereinafter also written as CH25~ 1_ COOH

l,Jherein A is :: -RI or~ ~ C -N
. ~ op~2 wherein Rl is hydrogen or formyl, R is ~ . or y ,.

and Y is hydrogen, chlorineJ bromine, ~luorine, trifluoromethyl, amino, nitro, hydroxy, (lower)alkyl of 1-4 carbon at~ns or (lower)alkoxy of 1-4 carbon atomsJ
R2 is (lower)~lkyl of 1-4 carbon atoms; and herein Al is meth~rl or -(CH2)nCOO~I and n is one or two, 72~

.~he easily hydrolyzed esters and.the non-toxic, pharmaceu-tically acceptable salts of those acids Said easily hydrolyzed esters Or the acids of formula I include those h~vin~ the group of the formula ;

~W
-C~
wherein when W represents hydrogen, Z rep~esents (lower)-alkanoyl, benzoyl, naphthoyl, furoyl, thenoyl, nitrobenzoyl, methylbenzoyl, halobenzoyl~ phenyl-benzoyl, N-phthallmldo, N-succlnimldo, N-saccharino, N-(lower)al~lcarbamoyl, (lower)al'~oxyl (lot~er)-alkylthlo, phenoxy, carbalko.~y, carbobenzoxy, carbamoyl, benzyloxy, chlorobenzyloxy, carbophenoxy, ~arbo~tert,-butoxy or (loWer)alkylsulronyl, ~nd ~hen W represent 8 carbalkoxy, Z represent 9 carbalkoxy and, when W repre5entB phenyl, Z represents benzoyl or cyano or whereln W and Z taken together represent

2-oxoc~cloalkyl containlng 4 to 8 carbon atom~
lncluslve~
As 8et rorth below in more detall the pre9ent . inventlon also prov1de~ salt~ Or these aclds, ~he stereochemistry Or the blcycllc nucleus ~s that ~ound ln Cephalosporln C.

A preferred embodiment or the present invention consists of the acids having the D configuration in the 7-side chain and the formula II

~5~727 R~CH-C~NH-CH- CH i CH2 ~ ~
OR ~C N C ~C CH2 N~ ~ N-(C~2~nC02H
COOH o wherein n is one or two and Rl is hydrogen or formyl and R is ~ or ~

and Y 1~ hydrogen, chlorine, brom~ne, fluorine, trlfluoromethylJ amlno, nltro, hydroxy, lower-alkyl of 1-4 carbon atoms or lower alkoxy of 1-4 carbon atoms and the nontoxicJ pharmaceutically acceptable salts of tho~e aclds and the easily hydrolyzed esters Or those aclds int:luding especlally the plvaloylox~nethylJ acetoxymethyl, acetonyl) phenacyl and methoxymethy:L esters and the Rllyl esters such as the trlmethylsilyl ester.

A further pre~erred embodiment of this invention consists o~ the compounds of formula II, trherein R is 2-thienyl, 3-thienyl, phenyl, chlorophenyl, bromopllenyl, trifluorome~hylphenyl, tolyl or methoxyphenyl.
Particularly preferred embodiments of this invention comprises the acids h3ving the D confi~uration in the 7-sidechain and the rormula ~3CH-II-NH CH--C/ \CH2 ~
OH ~C--N ~ ~C-CH2-S N ~ ~-(CH2)nC02H
COOH

~L15~7Z7 wherein n is one ~r t~o and their nontoxlc, p~la~naceutically acceptable salts and easil~ hydrolyzed esters.
Also included in this inven~ion are the compounds (used as either inte~lediates or metabolic precursors) in ~Jhich the ~-hydroxy group is "blocked" by su~stituents such as dichloroac~tyl (U.K. 962,024 and U. X. 1,328,340), f~rmyl (U.S. 3,641,021), trimet~lyls~lyl or tetrahydro-pyran~Jl (U.K. 1,328,340).
~ lere is ~lso provided b~ the present invention a compound ha~ing the rol~ula O S
R-CH-C-N~-C~ - Ch ~ ~ }~2 ¦ ~ Nl H2-s ~N,N ~ N (CH2)n 2 I O
COO~l .

wherein n is one ~r two, ~ is hydrogen or formyl and R is f~
~ or y and Y is hydrogen, ~hlorinef 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 O O
_cHOC(C}12)nR~ -CHOC(C~12)nC~R8 ~' Rl R~ 4R~

~15¢~727 .
o . o - CHX~OR6 or - ~H-S-C-R
I l n is O to 4; R is hydrogen, alkyl having 1 to 8 carbon atoms, cycloalkyl of 3 to 6 carbon atoms, phenyl, Cl-C~
phenal~yl, pyridyl, thienylr or pyrrolyl; ~ is hydrogen, methyl or ethyl; R7 ~nd R8 are each hydrogen, ~lkyl having 1 ts 6 carbon atoms, phenyl, pyridyl, or thienyl; R4 and R5 are each hy~rogen or alkyl of 1 to 4 carbon atoms; R6 is alkyl having 1 to 4 carbon atoms, phenyl, phenalkyl having 1 to 4 carbon atoms, pyridyl, thiadiazolyl, amino or Cl-C4 alkylamino; X is N~ or oxygen; and. each phenyl group is unsubstituted or substituted 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 ~ontoxic, pharmaceutically acceptable salt thereof.

~ here is also provided by the present invention a compound having the formula .
R 11 / ~ H2 ~ N
IR1 ~ N \ ~ -Cff2 S ~ N~N ~ N (CH2)nCO~H

COOI~l wherein n i5 one or two, R is hydrogen or formyl and ~ ~8 ~ or Y

~15~:)7;~'~

and Y i~ hydrDgen, chlorine, bromine, fluorine, trifluoro-methyl, amino, nitro, hydroxy, lower alkyl of 1-4 carbo~
~toms or lower alkoxy of 1-4 carbon atoms and M is selected from the group co~sisting of - CH - - ~ - R6 - CH - C - R

R~ O
2 ll - CH - X - C - 'oR7 wherein R5 iB a hydrogen atom, a methyl or an ethyl group;
X2 iS -O-, -NH-; R6 ~5 a baslc group.such as alkyl or aralkyl substltuted with substituted or unsubstituted NH~, such as alkyl-NHCH3, sralkyl-NHCH~, alkyl-NH ~ , ; aralkyl-~H ~ , -IH ~ , -CH2NH2~ _1H_CH2 . NH~ NH2 .. ` .

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 aràl~yl group such as benzyl or naphthylmethyl; a heterocyclic group and wherein the alkyl, cycloalkyl, aryl, aralkyl and heterocyclic groups may be substitute~ with one or moxe groups selected from the class consisting of amino groups, substituted amino groups such as methylamino, diethylamino or acetamido groups, the halogen .

~S~7Z7 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 accep able salt thereof.

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

~-CH-C-NH-CH - CH l~2 ~ Nl I C-C~ -S ~ ,N N-(CH ) CO H

COO~

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

and Y is hydrogen, chlorine, bromine, fluorine, trifluoro-methyl, amino, nitro, hydroxy, lower alkyl of 1-4 car~on atoms or lower alkoxy Gf 1-4 carbon atoms and M is .

O

/~_y \Z

where~n Y ~ alkyl o~ one to six carbon ~toms, phenyl, benzyl, alkoxy of one to 6iX carbon atoms, or benzyloxy;
Z is alkyl of one to ~ix car~on atoms, phenylbenzyl, alkoxy o~ one to six carbon stoms, cyclopentyl, cy~lo-hexyl and phenyl, or Y+Z taken together are a 3-benzoxa-zolidine ring; or a nontoxic, pharmaceutically acGeptabla ~alt thereof.

~L~5~27 Also included within the present invention are pharmaceutical compositions comprising a mixture of an antibacterially effective amount of a compound of the present invention and a semisynthetic penicillin or another cephalosporin or a cephamycin or a ~-lactamase inhibitor or an aminoglycoside antibiotic.
In the treatment of bacterial infections in man, the compounds of this invention of formula 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 divided dosage, e.g. three to four times a day. They are administered in dosage units containing, for example, 125, 250 or 500 mg. of active ingredient with suitable physiologically acceptable carriers or excipients.
The dosage units are preferably in the form of liquid preparations such as solutions or suspensions.
Another preferred embodiment of this invention comprises the acids having the formula III

-C-NH-CH ~S ~fH IN

N~OR ll ~ C-CH S ~ N - Al III
COOH

wherein Al is methyl or -(CH2)nCOOH and n is one or two and R2 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 of the present invention are s~n isomers or else are mixtures of syn and anti isomers containing at least 75% of the ~y~ isomer.
Preferably such mixtures of isomers contain at least 90% of the ~y~ isomer and not more than 10% of the anti ,~''''9 _ _ ~ S~7;~7 isomer, Most prefer~bly the cor,~pounds Or ~ormula III are s~ isomers essen-~ially free of the co~responding anti isomer.
~ le preferre~ embodiments of the present invention are the syn isomers of the compounds of Formula III
~Jherein R2 is methyl or ethyl, n is one or t~To in its acid or pivaloyloxymethyl, acetoxymethyl, methoxymetllyl, acetonyl~ p}lenacyl, p-nitrobenzyl, ~ richloroethyl,

3-phthalidyl or 5-indanyl ester form.
Reference to the syn (cis) isomeric ~orm refers to the confi6uration o~ the group oR2 ~ith respect to the carboxamido ~roup.

There is also provided by the present inven-t~ on a compound having the formula - C - NH-CH--Cl H ~H2 ~=NI
N_oRa ~c N c"c CH2 s ~N~ ~ (CH2)n c - oR3 o O
wherein R~ ls alkyl containing 1-4 carbon atoms, n 19 ~` ~
one or two and R' iB Belected from the group conslsting of IH~5 11 6 - CH - O - C - R, - CH - C - R , R5 o - C~ - X C - 0~

whereln R5 is a hydrogen atom, a methyl or an ethyl group;
X2 1R -0-~ -NH-~ R6 is a basic group such as alkyl or aral~yl substit`uted with substituted or unsubstltuted NH2, such as alkyl-NHCH~, aralkyl-NHCH3, alkyl-NH ~ , aralkyl-NH ~ ' ~lH ~ , -CH2NH2~ -IH-CH2 R is an alkyl group such as a methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl or 2-ethyl-hexyl group;
a cycloalkyl group such as cyclopropyl, cyclobutyl, cyclo-pentyl, cyclohexyl or cycloheptyl; an aryl group such as phenyl or naphthyl; an aralkyl group such as benzyl or naphthylmethyl; a heterocycllc group and wherein the alkyl, cycloalkyl, aryl, aralkyl and heterocycllc ~roups may be substituted with Dne or more groups selected from the cla6s consistlng of amino ~roups, substltuted amino groups such as methylamlno, diethylamlno or acetamido . groups~ the halogen ~roups such as fluorine, chlorine or bromine, nitro groups, alkoxy groups such as methoxy, ethoxy, propyloxy, isopropyloxy, butoxy or isobutoxy;
or a nontoxic, pharmaceutically acceptable salt thereof, said compound being at least 75% by welght in the form of it syn isomer and preferably in the form of its yn isomer essentially free of the correspond~ng anti ~s~mer %7 There is also provided by the present inven-tion a compound having the formula C - C - NH~ CH CH2 ~ N
C N ,5-C~I2-S N,N ~ - (CH2)n I_OM Q

whereln R~ is alkyl rontaining 1-4 carbon atoms, n 13 one or two and M is ~C Y
-CH2-N~

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-zolidlne ring; or a nontoxlc, pharmaceutically acceptable salt thereof, said compoun~ be~ng at least 75% by welght in the form of its ~ isomer and preferably ln the f~rm o~ its syn isomer essentially free o~ the corresponding anti lsomer.

There is also provided by the present inven-tlon a compound having the ~ormula C ~ C - NH-IH- ~H ICH2 ~ N
~l 2 ~C - N ,5 -CH2-S N ~ I CH

C-OM O

O

whereln R2 is alkyl containing 1-4 carbon atoms and ~ is ~}~oc(cH2)nRJ ~CIHOc(cH2)ncl~R8 NR R

O O
-CHXCOR or - CH-S-C-R
I l 1 1 n ls O to 4; R is hydrogen,.alkyl having 1 to 8 carbon atoms, cycloalk~l of 3 to 6 carbon atoms,`
phenyl, Cl-.C4 phenalkyl, pyridyl, th~enyl, or pyrrolyl;
Rl ls hydrogen, methyl or ethyl; R7 and R8 are each hydrogen, alkyl having 1 to 6 carbon atoms, phçnyl, pyridyl, or thienyl, R4 and R5 are each hydrogen or alkyl of 1 to 4 carbon atoms; R ls alkyl having 1 to 4 carbon atoms, phenyl, phenalkyl having 1 to 4 carbon atoms, pyridyl, thiadiazolyl, amino or Cl-C4 alkylam~no; X is NH or oxygen; and each phenyl group is unsubstituted or subs~ituted with one or two substltuents selected ~rom the group consisting of alkyl ~aving 1 to 6 carbon atoms, alkoxy having 1 to

4 carbon atoms, hydroxy, amino, NHRl, N(Rl)2, nitro, fluoro3 chloro, bromo or carboxy, or a nontoxic, p~armaceutically acceptable salt thereof, said com-pound being at least 75% by welght in the form of its syn isomer and preferably in the ~orm of lt~
l~omer essentially ~ree o~ the corresponding anti isomer, - f2 1150~Z7 I

, There is also provided by the present inven-tion ~ compound having the formula O
C - NH-ICIH- CH ~H2 ~ NI

. - OR~ O ~ N C~5 ~C}l2~s N ~ - CH3 I_OR~ O

wherein ~ ls alkyl contalnin~ 1-4 carbon atoms and R is selected ~r~m the group conslsting of Cl 2H5 11 - CH - C - R , - CH - X - C - OR

wherein R5 is a hydrogen atom, a methyl or an ethyl sroup;
x2 is -O-~ -NH-; R is a basic group such ~s alkyl or aralkyl substituted with substituted or unsubstituted NH2, such as alkyl-NHCH3, aralkyl-NHCH~, alkyl-NH ~ , aralkyl-NH ~ , -IH ~ , CH2NH2~ -IH-CH2 R7 is an alkyl group such as a methyl9 ethyl, propyl, lsopropyl, butyl, isobutyl, pentyl or 2-ethyl-hexyl group;
a cycloalkyl group such as cyclopropyl, cyclobutyl, cyclo-pentyl, cyclohexyl or cycloheptyl; an aryl gr~up sucIl as ' `''` ll~j7~7 phenyl or naphthyl; an ~ralkyl group 6uch as benzyl or naphthylmethyl; a heterocyclic group and whereln the alkyl, cycloalkyl, aryl, aralkyl and heterocycllc groups ~ay be substituted with one or more groups selected from the class conslsting Or amino groups, 6ubstituted amlno groups such as methylamino, diethylamino or acetsmido groups, the halogen groups such as ~luorlne, chlorine or bromine, nitro groups, alkoxy groups such as methoxy, ethoxy, propyloxy, isopropyloxy~ butoxy or lsobutoxy;
or a nontoxlc, pharmaceutlcally acceptable salt thereof, 6aid compound being at least 75~ by weight ln the form of lts syn isomer and preferably in the ~orm of lts syn isomer essentially free of the corresponding anti lsomer.

There is also provlded b~ the present inYen-tion a compound having the formula O
C - C - NH-CH--CH ~H2 ~ N
N_ oR2 0 / ~ C N ~ - CH~
C-o~ O

O
wherein R2 is alkyl containing 1-4 carbon atoms and ~5 is Il /C -Y
CH2 N\

~i50~7~2'7 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-zolldine ring; or a nontoxic, pharmaceutically acceptable salt thereof, said compoun~ being at least 75% by weight ln the form of its syn isomer and preferably in the form of its syn isomer essentially free OI' the corresponding anti isomer.
.. . . _ . . ...
In the treatment of bacterlal infeCtlons in man, the compounds of this lnventlon are admlni~-tered parenterally ln ~n amount of from about 10 to 90 m4./kg /day and preferAbly about 14 to 50 mg./kg./day ln div~ded dosa~e, e.~. two to four times a day. They are admlnlstered in dosage units contalning, for example, 125, 250 or 500 mg, Or actlve lngredient wlth suitable physiologically acceptable carriers or excepients.
e dosage units are in the form of llquid preparations such a~ solutions or suspensions and preferably are ~queous solutions Or a sodlum or potassiUm 6alt which are in~ected intravenously or lntramuscularly or by continuous or intermittent in~usion in concentrations of about 125-500 mgm /ml., and preferably, 250 mgm./ml as ls customary in therapy with cephalosporln antibi-oti~s.
It was an unexpected finding that the leading compound of the present invention (BB-S510; see below) having a 2-methyl substituent on the tr~azolopyridazinone showed in vitro antibacterial potency considerably super~or to that of the corresponding compound lacking such methyl group (BB-S515; see below).

.

A further embQdiment Qf this inVention comprises a`com-pound ha~ing the formula ~ N
HS ~

wherein Al is methyl or -(CH2)nCOOH
where n is one or two. These compounds are made by a process com-prising reacting a compound of the formula Cl ~ A2 o wherein A2 is methyl or -(CH2)nCOOH or an easily hydrolyzable ester thereof and n is one or two, with NaSH.

The present invention also provides the process for the production of the antibacterial agents of formula I which comprises reacting a compound of the formula ~S~

~2N - fH fH Cl 2 ~ N
~iC-CH2-S-l~N ,N ~ N-A IV

COOH
wherein Al is as hereinbefore defined or a salt or easily hydro-lyzed ester or Schiff base as with benzaldehyde or salicylaldehyde thereof (including, but not limited to, those of U.S. 3,284,451 and U.K. 1,229,453 and any of the silyI esters described in U.S.
patent 3,249,622 for use with 7-aminopenicillanic acid and used in Great Britain 1,073,530 and particularly the pivaloyloxymethyl, acetoxymethyl, methoxymethyl, acetonylt phenacyl, p-nitrobenzyl, ~ -trichloroethyl, 3-phthalidyl and 5-indanyl esters~ thereof with an organic monocarboxylic acid chloride or a functional equivalent thereof as an acylating agent.

l~LSql7;;:7 Such functlonal equivalent~ include the corresponding ~cid anhydrlde~, lncluding m~xed nhydrlde~ and p~r$icularly the mixed anhydrltes prep~red from ~tronger ~cids ~uch a~ the lower aliphstic monoe6t2r~ of carbonlc ~cld, or 21kyl snd aryl sulfonic ~cids ~nd of more hindered ncids such ~ diphenyl~cetic scid. In ~ddition, ~n ~cid azide or ~n actlve e8ter or thloe~ter (e.g.
with p-nitrophenyl, 2,4-dinitrophenol, thiophenol~
thioacetic acid) m~y be used or the free ~cid i~6elf may ~e coupled with compound ~T ~fter irst reacting ~sid free ~cid with N,N'-dimetllylchloroformlminium chloride [cf, Great Brit~in 1,008,170 ~nd Nov~k and Weichet, Experientia XXI, 6, 360 (19S5)] or by the use of en?ymes or of an N,N'~c~rbonyl-dlim~d~zole or nn N,N'-carbonylditri~zole lcf South Afric~n p~tent ~pec~fication 63/26841 or c~rbod~mide reagent [especially N,N'-dicyclohexyl~
c~rbodiimide. NaN' dii60propylc~rbodiimide or N~cyclo-. hexyl-N'-(2-morpholinoe$hyl)c~rbodiimide; c~. Sheeh~n Dnd Hess, J. Amer. Chem. Soc., 77, 1967 (1955)J~ or of alkylyl~mine resgent ~cf. R. Bui~le ~nd H.G.
Vleh~, AnREw~ Chem. Intern~t~onal ~dltlon 3, 582, - 17 ~

~ 7 (1964)~ or v~ an ~soxasol~um ~alt r~ag~nt lcr. R. ~.
Woodward, R. A. Olofson and ~. Mayer, ~ ~r~ Che,~, , 1010 tl961)], or o~ a ketenlmlne rea~ent [c~. C. L. Stevens and M, E. Munk, ~ 5h~m_ ~5~
~Q, 4065 (1958)~ or o~ hexachlorocyc,otrlphosphatriaz~ne or hexabromocyclotrlphosPhatrla2lne (U,S, ~,651,050) or of dlphenylph~sphoryl azlde [DPPA; ~_ A~er Chem~ Soc., q4 J 6203-6205 ( 1972) ~ or of dlethylphosphoryl ~yanlde lD~PC; Tetrahedron Letters No, 18, pp. 1595-1598 (1973) or Or dlphenyl phosphlte ~Tetrahedron Letters No. 49, pp, 5047-5050 tl97~)]. Another equlvalent o~ the acld o~lorlde 19 a correspondlng azollde, i.e., an amlde Or the corresp~ndln6 acld whose amlde nltro6en 1~ a member a ~uasiaromatlc ~lYe membered rlng contalnln~ at least two nltro~en atoms, l,e., i~lidazole, pyrazole, the trla~oles, benzlmidazole, benzotrlazole and the~r ~ubatituted der~lat~ves. A~ an example o~ the genersl method for the preparatlon Or an azolide, N,N'-carbonyl-dlim~dazole Is rea c ted wlth a ca rboxy llc a c ld ln equ~molar pr~port~ons at room temperature in tetr~-bydr~uran, chlorororm, dimethylrormamide or a ~lmllar ~nert 801vent to ~orm the carbo~ylic ac~d lmidazolide In practlcally quantltat~ve yleld with llberstIon o~
carbon dloxlde and one mole o~ ~mida201e. Dicarboxyl~c acid3 yleld dlmldazollde. The by~product, imtda~ole9 preclpltate3 and may be separated and the Imldazolide 1301ated, but thi~ ls not essentlal. The methods ~or carr~in~ out these reactlons to produee a cephalo~por~n and the meth~ds u~ed to l~olate the ~ephalooporln ~o produc~d are well known ln the ar~.

"` ~15~77~'7 Ment~n ~ de aboYe Or the u~e o~ ersz~ne~ to couple ~he rree ac~d wlth oompound IV. Included in the sc~pe o~ Buch pro~es~e~
sre the u~e Or an ester, e.g. the met~yl e~ter, o~
that free ~c~d wlth enzymes pro~ded by variou~ mlero~
organlcm3, e.g. tho~e descrlbed by T. Takahashl et sl "
J, Amer, Chem. Soc., 4(111, 4035-4,0~7 (1972) and by T, Nara et al., J. Antlblotlcs (Japan) ~ J 321-~23 ~1971~ ~nd ln U.S. 3,682,777.
Por the coupling of the organic c~rboxyl~o acid as descrlbed above with compound IV (or a 6~1t or preferably ~n easily hydrolyzed ester of Schiff b~se, as with benz~ldehyde, thereof) it is also convenlent and ef~icient to utilize a~ the coupling agent phosphonitrillc chloride trimer (J, Org. Chem " ~ , 29J9-81, 1968) o~ N-ethoxy-1,2-dihydroquinoline (EEDQ) as described in J. Amer. Chem, : 50c., gO, 823-824 ~nd 1652-1653 (1968) snd U,S. P~tent . 3,455,929. The reaction is prefer~bly c~rried out at 30-35C. iR benzene, ethsnol or tetrahydrofur~n uslng ~bout ; .. equlmolar qusntities of ~11 th~ee re~gent~ followed by ~onventional isolation ~nd removal by conventlonal methods of Any blocking groups present.
. .
. One process of the present invention stated more specifically is the process for the preparation of a product having the D-configuration in the sidechain and.the formula CH-CO~l ~ ~
OH k ~CH2s~N,~N (~H2)~t,0211 COOH

~ llSC~727 wherein n is one or two or a s~lt t~ereof whi~h comprises-the consecutive steps of a. preparing an acylating derivative Or D-mandelic acid having the formula ~ 7 .' o~
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~ C) b. mixing therewith, preferably slo~lly, a solution at about the same temperature in a solvent, preferably aqueous tetrahydrofuran, containing substantially the same number of moles of a tertiary amine, preferably a tertiary alkylamine such as triethylamine and substan-tially the same number of moles of 7-amino-3-(2-carboxy-methyl or 2-carboxyethyl-2,3-dihydro-s-triazolo[4,3-bl-. pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-car~oxylic acid or a ~alt or an easily hydrolyzed Schiff base, as with benzaldehyde, thereof to produce the intermediate acid having the formula ~CH CONH Tf ~ cH ~
1R ~N~ 2S ~N~ ~N-(~H2)nCO2H
COOH O

w~erein n is one or t-~ or a salt thereof wher~in R has the meaning set out above; and c. removing said hydroxyl blocking group R by con-ventional chemical methods to produce said product or salt thereof.
, _ 20 -~L~L5~72~7 In preferred embodiTnents of the present inven-tion R represents formyl which is removed in step C by treatment llith aqueous allcali such as aqueous sodium bicarbonate or R represents dechloroacetyl which is removed in step C by alkaline hydrolysis, prererably at about pH 9-10, or ~ represents trime~hylsilyl ~Jhich 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 ~roup from a cephalosporanic acid of the formula A~ H2-O-C-CH~ V

0/ 0~1 ~here A is a.s defined hereinbefore above, prepared by substituting 7-amino cephalosporanic acid for the 3-thiolated-7-amino cephalosporanic acids in the acylation procedures described herein and elsewhere reported with the apprOpriate thiol HSR3 having the formula, or ~I-S--~ N~N ~ N ~ CH2)nCO~H H-S--~ N~N ~ N-CH~

where n is one or two and then removing the protecting group if any is present as on the carboxyl groupl S¢~727 .

The dlspl~cement of 6uch a 3-~cetoxy group w~th - such ~ th~ol ~y be accomplished ln ~olution ~8 in water or aqueou~ ~etone ~t a temper~ture of ~t le~st soom tEmpersture ~nd prefer~bly w~thln the range of absut 50~ to 100C. in the presence o~ ~ mild b~se ~uch fl8 ~odlum b~c~rbon~te, e.~. prefer~bly neer neutr~lity such aB ~t about pH 6. An excess of the thiol ~8 preferebly e~ployed. The react~on product ia isolated by csr~ful acidifica~ion of the reaction mixture followed by extrac-tion wlth a water-immiscible orgsnic solvant. A8 noted above, the p~ep~rstion of many other 7-acyl~idocephnlospor~n~c ~cids is described in the patent end ~cientiflc llter~ture e.g. ln U.S. Clas8 260-243C.

The ~lts of tha compound~ of thi~ lnvent~o~ ~nclud~
the ~ontoxlc c~rboxyl~c oc~d 8alt~ thereof, incl~ting non-toxic ~etallic 3~1t~ ~uch aa oodium, potessium, c~le~um and alu~inum, tha a~m~nium s~lt and subctltuted ammon~um s~
e.g. ~alts o~ ~uch nontoxic am~nes a~ trlalkyl-amlnes in~ludlng trlethylamlne, procaine, dibenzyl-amine, ~-benzyl-beta-phenethylamlne, l-ephenamlne, N,N~-dlbenzylethylenediamlne, dehydroabietylamine, ~,N'~bls-dehydroabietylethylenediamlne, and other ~mlnes whlch have been used to rorm salts wlth benzylpenlclllin, L-lysine, arginine and histidine.
The pre~erred esters o~ the cephalosporlns Or the pre8ent ~nvention are the pivaloyloxymethyl9 - acetoxymethyl9 methoxymethyl, acet~nyl and phenacyl ~sters. All are use~ul lntermedlates ~n the ~, , productlon of the cephalosporln havlng a ~ree carboxyl group.
_ 22 -S~727 A8 Indicated aboYe, t~ese rlve e~ter~ Or 7-amino-cephalosporanlc acld are each prepared by known method~. One excellent procedure i~ that Or U.S.
patent 3,284,451 ln whi~h sodlum cephalothln 1~
ester~led by reactlon with the corre~pondlng actlve chloro or bromo compound (e,g. phenacyl bromlde, chloroacetone, chloromethyl ether, plvaloyloxy-methyl chlorlde [also called chloromethyl plvalatel,acetoxymethyl chlorlde) and then the thlen~
acet~c acid sldechaln i9 removed enzymatlcally as in the ~ame patent or chemlcally a~ ln U.S, patent 3,575,g~0 and ln Journal o~ Antlblot1c~, xxrv (~ 767-773 (1971)~ In another good method the trlethylamlne salt of` 7-amlnocephalo-8poranle acld i8 reacted dlrectly wlth the aotlve halogen compound, as in Unlted Klnedom 1,229,453, mese esters Or 7-aml~ocephalosporanic acld are then reacted wlth the nucleophlle HSR3 ln the ~ame manner as i9 lllustrated hereln ~or 7-amlnooephalO-sporanlc acld ltselr~ me ~-thlolated es~er o~
7-amlnocephalo~poran~c aoid ~s then coupled ~ît~
the organlc carboxylic acld as be~ore.

The ester of the cephalosporin so obtained is, if not used per se, con~erted to ~ts free acid and, if deslred9 sny salt by ~emov~l of the esteri-fying group, 88 by aqueou~ or enzym~tlc hydroly~ 8 wlth hum~n or ~nim~l serum) or ~cid~c or alkaline hydrolys~s or by treatment with ~odium thlophenoxide a8 tDUght ln U.S. 39~84,451 and, ln the penlc~llin serle3, by Sheehan et al,~ 3, Org~ Chem.
2006-2008 (1964).

5~7;~7 In ~ ther altes~natlve synthes~ 8, the 3-th~olated 7-amlnocephalo~poran~ a~ld 13 prepared ~ de9crlbed here~n and then ac~lated at the 7-am~no gr~up ~nd rlnally este~l~led, as by reaction o~ the appropriate alcohol wlth the acid chloride prepared, ~or example, by react~on Or the rinal eephalosporln wlth thlonyl chlorlde or by ~ther eB~entlally acldlc e~terl~lcatlon proc~d~.re~, There iQ further provided by the present invention a pharmaceutical composition comprising an antibacterially effective amount of a compound having the formula O ` S
R-C~l-C NH-CH--CH ~ 2 k ~ C-CH2-S_ ~N~ N (CH2)n 2 Joo~

where~n ~ is one or two, Rl i~ hy~rogen or formyl and R is .
~3 Dr ~

and Y is hydrogen, ~hlorine, bromine, fluorine, trifluoro-methyl, amino, nitro, hydroxy, lower ~lkyl of 1 4 carbon atom~ or lower alkoxy of 1-4 ~arbon atoms and M is hydrogen, pivaloyloxymethyl, acetoxymethyl~ methoxymethyl, acetonyl, phenacyl, p-nitroqenzyl, B,~ trichloroethyl. 3-phthalidyl or 5-~ndanyl and pre~erably ~s hydrogen or a nontoxic, pharmaceutically acceptable salt thereof.

115~ 7 There i~ further provided by the present invention a method of tseating bacterial infections comprising ~dminis-tering by injection to an infected warm-blooded animal, ~ncluding m~n, ~n e~fective but nontoxic dose of 250-1000 mgm.
o~ ~ compound having the form~la R-C~-C-NH-CH--C~ ~}12 ~NI
1R1 ~--N\ ~ 2 N~N (CH2)nCO2H

COOM

wherein n is one or two, Rl is hyclrogen or formyl and R is !
y~ ~ ~

and Y ~s hydroyen, chlorine, bromine, fluorine, trifluoro-. methyl, amino, nitro, hydsoxy, lower alkyl ~f 1-4 carbon atoms or lower alkoxy of 1-4 rarbon atoms and M is hydrogen, pivaloyloxymethyl, acetoxymethyl, methoxymethyl, acetonyl, phenacyl, p-nitrobenzyl, B~ trichloroethyl~ 3-phthalidyl or 5-indanyl or a nontoxic, pharmaceutically acceptable salt ~hereof~ .

There is also provided by the psesent inven~ion a method for combatting ~ . dysenteriae infections w~i~h comprise~ administering to a warm blooded mammal infected w~th ~n ~ . dysenteriae infection an ~mount effective ~or treat~ng ~aid ~ . dysenteriae infe~tion of a composition comprising a compound havlng the formula ~ 25 -7~'~

o R-C~-C-NH-C~ C~ 2 ~ Ni C-CH2-S - ~ ,N N-tCH ) ~ O

COOM

wherein n i9 one or two, R $s hyd~ogen or formyl and ~ is ~ ~r ~

and Y $s hydrogen, chlorine, bromine, fluorine, trifluoro-methyl, amino, nitro, hydroxy, lower alkyl of 1-4 carbon atoms or lower alkoxy of 1-4 car~Dn atoms and M is hydrogen, pivaloyloxymethyl, acetoxymethyl, methoxymethyl, acetonyl, phenacyl, p-nitrobenzyl, ~,B,~-trichloroethyl, 3-phthalidyl or 5-indanyl and preferably is hydrogen or a nontoxic, pharmaceutically acceptable salt thereof.

There is also provided by the present invention a method for com~atting Sal. enteritidis infections which comprises administering to a warm-blooded mammal ~nfected with a Sal. enteritidis infection an amount effective for treating said Sal. enteritidis inection of a composition comprising a compound having the formula o /~
~-CH-C-NH-CH - CH l~2 ~ 1 ' 1R1 ~ _ N\ j/ ~H2 S ~N~ N ~CH2)n~02H
- COOM

1150~727 .

wherein n is one or two, R~ is hydrogen ~r ~ormyl and R is Y~ ~
and Y is hydrogen, chlorine, bromine, fluorineO trifluoro-methyl, amino, nitro, hydroxy, lower alkyl of 1-4 ~arbon atoms or lower alkoxy nf 1~4 carbon atoms and M is hydrogen, pivaloyloxymethyl, a~etoxymethyl, methoxymethyl, acetonyl, pbenacyl, p-nitrobenzyl, ~,B,B-trichl~roethyl, 3-phthalidyl or 5-indanyl and pxeferably is hydrogen or a nonkoxic pharmaceutically acceptable salt thereof.

There is further provi.ded by the present invention a pharmaceutical composition comprising an antibacterially ef~ective amount of a compound ha~ing : the formula O
C - NH~ CI~ ~H2 ~ N
N_ OR~ ~C N 1~ 2 S N~N ~ _A

; I_OR O
O

wherein X2and Al are as hereinbefore defined and R3 is h~Jdrogen, pivaloyloxymethyl~
acetoxymethyl, methoxymethyl, acetonyl, phenacyl, p-nitroben~yl, ~ B-tricllloroetllyl~ 3-phtllalidyl or 5-indanyl and preferably is hydrogen or a nontoxic, phar~aceutically acceptable salt thereof, said compound being at least ~15a3~727 75% by weight ln the form of its syn isomer and preferably in the ~orm 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 antibacter~ally effective amount of the syn isomer of a compound having the formula C - g ~ NH-CllI CH ICH2 ~ N
N--OCH3 o~ ~ C~ U2 S
C-OH O

wherein A~ is as hereinbefore define~ or a nontoxic, pharma-ceutically acceptable salt thereof and a pharmaceutically acceptable carrier there~ore.

There is further provlded by the present lnventlon a method of treating bacterial infectlons comprising administering by in~ection to an infected warm-blooded animal, includlng man, an effective but ~- nontoxic dose of 250-1000 mgm. of a compound having the formula :'~ O
C - C - NH-C~--CH C~2 ~ Nl N--R~ ~// C~C CH2-5~~ ~ - A

Il 7~7 herein R2 and A' are as hereinbe~ore defined and R3 is hydrogen, pivaloylox~methyl, acetoxymethyl, methGxymethyl, acetonyl, phenacyl, p-nitrobenzyl, ~ -trichloroethyl, 3-phthalidyl or 5-indanyl or a nontox~c, pharmaceutically acceptable salt thereof, said compound being at least 75% by weight in the form of lts syn isomer and preferably in the form of its syn isomer essent~ally free of the corresponding anti isomer.
.. _ _ _ . , .

There i8 further provided by the pre~ent lnventlon a method of treatlng bacterial infectlons comprlsing admlnlstering by ln~ectlon to ~n lnfected warm-blooded anlmal, includlng man, an effectlve but nontoxic dose of 250-1000 mgm. of the ~ lsomer of a compound having the formula O ' C - C - NH-CH - CH ~H2 ~ N
~-OCH3 o ~ ~ C~ CH2 S N,N ~ I A~

C-OH O
il herein Al is as hereinbefore defined or a nontoxic, pharmaceutically acceptable salt thereofJ and . wherein n is preferably 1.

1~5072r7 There is al~o provided by the present inventlon Q ~ethod ~or cDmbatting Hae~ophilus ~n~ec-tl~ns which comprl~es administering to a warm-bl~oded ¦ mammal in~ccted with an Haemophilus infectlon an I . amount ef~èctlYe for treating said Haemophilus infec-. t~on of a composition comprising a compound having ¦~ the ~ormNla C - g - NH~ -cl~ C~12 ~ Nl _ o}~2 o ~ ~ c~,c CH2-S ,N ~ -A
o~3 o ' O

herei1l Al ~nd R2 are as hereinberore defined and R3 is hydrogen, pivaloyloxymei;hyl, acetoxymethylJ methoxymethyl, acetonyl, phenacylJ p-`. nltrobenzyl, ~ -trichloroethyl, 3-phthalidyl or 5-lndanyl and preferably is hydro~en or a nontoxic, pharmaceutically acceptable salt thereof, said compound being at least 75~ by weight in the ~orm of its ~ isomer and -: preferably in the form of its s~n isomer essen~ially ~ ~ree of the corresponding anti isomer, and a pharma-: ceutically acceptable carrier there~or.
. .' : There is also provided by the present . invention a method for combatting Nelsserl~ ln~ec-tl~ns which comprises adminl~tering to a warm-bl~oded mammal in~ected wlth a Neisseria infection an amount ! e~fec~ive for treating sald Neisseria ln~ection ~f a r composition cDmpr~sing a compound having the ~ormula r - 3c~ _ 1 ~5u7z7 C - C - ~H-C~--CI~ ~H2 ~ N
N_ OR~ ~ ~ C~ 2 N~N ~ -' C-O~ O
., 11 : O

~Jherein R2 and Al are as hereinbefore defined and R3 is hydrogen, pivaloyloxymethyl, - acetox.~lethyl, methoxymethyl, acetonyl) phenacylJ p-nitrobenzyl, ~ -trichloroet}~yl, 3-phthalidyl or 5-indanyl snd pre~erably is hydrogen or a nontoxic, pharmaceutically jj acceptable salt thereof, said compound bein~ at least 75% by weight in the f`orm of its syn isomer and preferably ln the form of its syn isomer essentially free of the corresponding anti isomer, and a pharma-~ ceutically acceptable carrier therefor.

p m e present invention also provides the novel compounds having the formula S~~ N-Al wherein Al is methyl or ~ 2 )nCO~I, ~7here n is one or tl~o.
. . .
me invention further provides the novel compounds havin6 the formu1a ;. ~

r L5~7~27 ,s~
H~N-~?I ~ C~
N ~ ~C-~12-S ~ ,~I ~ N_ C- oP~3 wherein Al is methyl or ~(CHa)nC0~1, ~ here n is one or two and R3 is hydro~en or a conventional, r pharmaceutically acceptable, easily ~lydrolyzed ester , forming group, or a nontoxic, pharmaceutically accepta~le salt thereof, i" .

: ~ ~
. .
. :
. ~
` ~ ' .
. ~
~ ' :
; `:
, ~

. . - 32. - .

5~7~7`

STARTING MATERIALS
2-Furoylc~anide To a suspenslon of 26.1 g. (0.4 mole) of ground potassium cyanlde ln 300 ml. of acetonitrlle at 5 C. was ~dded 26.1 g. (0.2 mole) of n-furoyl chloride while keeping the temperature below 8 C. The mixture was stirred ln the cold for 15 minutes then heated at reflux for 30 mlnutes. The reaction was cooled, filtered and the aceto-nltrile was removed at 15 mm. (steam-bath) leavlng 24.5 g.
of a dark oil which was used without further purlflcation.
An infrared spectrum showed a nltriie band at 2æ5 cm 1.

2-Furane~lyoxYlic Acid .
The 24.5 g. of crude 2-furoylcyanide was mixed with 160 ml. concentrated hydrochloric acid at 25 C. with intermlttent stirring. The reactLon was stored for 24 hours at 25 C. and diluted with 130 ml. of water. The reaction was stirred for 5 minutes and filtered. The fil-trate was ~aturated w~th sodium chloride and extracted with 5 x 120 ml. of 1:1 ether-ethyl acetate solution. The extracts were combined, dried over anhydrous magnesium sulfate and evaporatèd at ~0 C. (1~ mm.) to give a brownish-orange solid. The solid was disqolved ln 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. 76D C.). The ir and nmr spectra were consis-tent for the structure.

2-Methoxyimino-2-furylacetic Acid To a solutlon of 4.5 g. (0.0~2 mole) of 2-furane-glyoxylic acid in 40 ml. of 50~ alcohol and ~.1 g. (0.0~7 ~5~72~

~ole) ~ methoxyamine hydrochlorlde ln 6 ml. water ~t 20 C. ~as added dilute sodium hydroxlde ~olution to pH 4-5.
The solution was stirred at pH 4-5 at 25 C. ~or 24 hours.
The alcohol was removed under reduced pres6ure (15 mm.) and the solution was ad~usted to pH 7-8 with 50% sodium hydroxide solution. The reaction was extracted wlth 3 x ~0 ml. of ether and the aqueous layer was ad~usted to pH
1.9 using concentrated hydrochlorlc acld. The mixture was extracted with 5 x 50 ml. of ethyl acetate. The organlc fractlons were combined, washed wlth brlne, dried over anhydrous magnesium sulfate and evaporated under reduced pressure (15 mm.) to an oil ~hlch was cooled ~or one hour in an ice bath. The product was slurried with Skellysolve B and collected to yield 3.1 g. o~ yellow crystals, m.p.
78 C. An analytlcal sample wa~; recrystallized from toluene, dried for 16 hours in ~acuo 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.
.~ .

2-Ethoxyiminofurylacetic Acid , .
The 7.85 g. (o.o56 mole) o~ furyl-2-glyoxyllc ~ acid was dissolved in 100 ml. of water and ad~usted to ; pH 7 with 50% sodium hydroxide. The 6.83 g. (0.070 mole) ; of ethoxyamine hydrochloride ln 10 ml. o~ 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 ~iltered. The alcohol was removed at 35 C. (15 mm.) and the aqueous portlon was ad~usted wlth ` dllute sodium hydroxide solution to pH 7-8 and then ~151~q2~

was washed with ether ~nd the washes were discarded. The aqueous ~raction was ad~usted wlth 6N hydrochIoric acid to pH 1.5 and extracted into ~ x 80 ml. of ethyl acetate.
me acetate fractions were comblned, washed with brine and reduced ln volume at 35 C. (15 mm.~ to an oil. The oil 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-slstent 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.

, Sodlum a-Ethoxyimino-~-(2-furyl)acetate To 50 ml. of methanol was added 250 mg. (0.0109 mole) of metallic sodium and stirred untll all the sodium had dissolved. This sodium methoxide solution was treated with 2.0 g. (0.0109 mole) of ~-ethoxyimino-a-(2-furyl)acetic acld dissolved in 10 ml. of methanol and stirred at room temperature for one hour. The methanol was remo~ed at 40 C. (1~ mm.) and the product was dried ln ~acuo over P205 at 25~ C.to yield 2.22 g. white solid, m.p. decomp.
~240~ C. The ir and nmr were consistent for the structure.
"Skellysolve B" is a petroleum ether fraction of b.p. 60-68 C. consisting essentially of n-hexane.
, .

.

5¢~727 .

CI -et-nltril~

H HCl CH3ONH2~HCl 3 ~c ; ~ ~ OH

Na C~ OH
~ ~ .

1~1 ~ (CCl)2 r~l ~
:i . ~fi- -ONa ~ C I

CH3 ~ IbCH3 ,, . ~
:~.
...
?-Furo~lcyanide 1 To a suspension of 78.~ g. of powdered potas-~:~ sium cyanide in 900 ml. acetonitrile at 5 C. was added 59.25 ml. (68.5 g.) o~ ~-furoyl chloride with ~igorous stirring while keeping the temperature at 4-8 C. The - mixture was stirred at 4-8 C. for 15 minutes and then heated at reflux for 30 minutes. The mixture was cooled ::~ to 23-25 C., filtered, washed with 50 ml. of acetonitrlle . whlch was added to the filtrate, and the acetonitrile was removed at 60 C. (15 mm.) leaving 51 g. of 1 as a dark . . -- . _ 72~

~il. 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 acld. 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 mixed with 500 ml. concentrated hydrochloric acld at 25 C. The reaction was stirred for 24 hours at 25~ C.
and then diluted with 240 ml. of water. The mlxture was stirred for 5 minutes and filtered. The black filtrate was saturated with sodium chloride and extracted with

6 x 500 ml. of 1:1 ether-ethyl acetate solution. (Note:
Initially the extractions were difficult due to the inability to see the separation of two black phases. As additional ether-ethyl acetate extractions were run the task was slmplified.) The extracts were combined and evaporated to dryness at 60 C. (15 mm.). The resultant solld was dissolved in 600 ml. ether, (Note: Use of alcohol should be a~oided at this point as es~ers may form)~ treated with 10 g. of charcoal ("Darko-KB"), filtered after stlrring for 0.5 hour ana evaporated to dryness at 50 C. ~15 mm.) to yield 46.6 g. of 2 as a light tan colored acid. Thls product 2 was found to contain a ratio of approxlmately 56/44 o~
product 2/furoic acid. This represented a 63% yield of product 2.
Puri~ication was accomplished by dissolving the above crude product 2 in H20 (50 mg./ml.), titrating to pH 2.8 with HCl and extracting with 2 x 200 ml. of *Trade Mark 1' 115~727 ethyl acetate. Evaporation o~ the ethyl acetate extracts gave 35% furoic acid and 15% product 2, The pH 2.8 aqueous phase was ad~usted to pH o.8 ~HCl~ and extracted w1th 2 x 200 ml. ethyl acetateO The organic extracts were combined and washed with 50 ml. H20. The organic `
phase was evaporated at 50 C. (15 mm. 3 yielding a 601id with a ratio o~ approximately 86/14 o~ product 2/furoic acid. This solid was then recrystallized by dissolving the product 2 ln toluene at 50 mg./ml. at 80~ C., decanting, and leaving to crystallize at room temperature for 18 hours, yielding 13.3 g. of pure acid 2 by NMR. This represented a 51% yield in the purifica-tion and recrystallization step and an overall yleld from .. .
the 2-furoyl chloride to the pure furyl-2-glyoxylic acid 2 o~ 16%.

Syn-~-methoxyiminofurylacetic Acid ~
A solution of 4.5 g. of furyl-2-glyoxylic ac~d 2 in 40 ml. of 50% ethanol was titrated to pH 6 with lN
sodium hydroxide and then ~.1 g. of methoxyamine HCl in 6 ml. nf H20 at 20 C. was added. The solution was titrated to a constant pH 4.9 ~nd stlrred-at pH 4.9 for ~4 hours at 20-23~ C. The ethanol was then removed at 50 C. (15 mm.~ and the residual aqueous ~olution was titrated to pH 8 with 50% sodium hydroxide and washed with 3 x 50 ml. ether (pH adjusted to 8 a~ter each wash~. me aqueous layer was titrated to pH 1.9 with concentrated HCl and extracted with 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 ., `
` ~S~7;;~7 evaporated to a solid ~ at 50 C. (15 mm.~. Thls solld `
was then slurried with 75 ml. of "Skellysolve B". me 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). m e NMR was clean and consistent for the structure ~ with a trace of anti isomer present.

Sodium S~ 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. o~
~ -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 2~
C. under high vacuum (0.7 mm;) over P205 yielding 1.25 g.
9% yleld of product). The NMR showed this product ~
to be clean and consistent for the structure with 0.15 mole methanol and a trace of anti isomer.
To 0.63 ~. of sodium syn-~-methoxylmlnofuryl-acetate 4 suspended in ~5 ml. of benzene was added four drops of dry dimethylformamide ~nd 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.

l~S07Z7 6-Chloro-2,3-dihydro-2-ethoxycarbonylmethyl-s-t-rla 3-blpyridazin-3-one To a solution of 6-chloro-2,3-dlhydro-s-triazolo~4,3-b]pyridazin-3-one [P. Francavilla and F.
Lauria, J. Het. Chem., 8, 415 (1971)] (1, 1.00 g., 5.9 m.mole) ln dry DMF (30 ml.) was added sodium hydrlde (50% in paraffin, 0.3 g., 6!3 m.mole) under stirring with formation of yellow crystals. To the mixture was added ethyl chloroacetate (1.4 ml., 13 m.mole) and the mixture was heated 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 e~aporated at reduced pressure. The residue was crystallized with benzene-n-hexane to give yellow needles (1.16 g., 77%), m.p 114-115~ C. (lit.
110 C . ) .
; ir: ~KBar 1735, 1710 cm 1 uv; ~EtOH 231 nm (E ~ 26000) nmr: ~ppC13 7.58 (lH, d, J=10 Hz, pyr~dazine-H), 6.98 (lH, d, J=10 Hz~ pyridazine-H), 4.80 (2H~ s, -CH2CO), 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.~2, 3.49; N, 21.51, 21.~3; Cl, 13.88, 13.99.

1~ S~727 ?-Carboxymethyl-2,3-dlhydro-6-mercapto-~-trlazolo ~ 3-~ _ p~ridazin-3-one To a solu~ion of 6-chloro-293-dlhydro-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., 0.36 mole) and the mixture was refluxed for 0.5 hour. The reaction mixture was e~aporated at re-duced pressure. The residue was dissolved in water (200 ml.) and concentrated HCl was added to the solution to adjust 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.
uv: ~l~xaHCO3aq 260 nm (E~19500), 3i3 nm (E, 7000) ~` nmr: ~pDMS0-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, 2-67;
N, 24.77; S, 14.17. Found: C, 37.35, 37.23; H, 2.269 2.28; N, 23.58, 2~.69; S, 14.32.
. : .
7-Amino-3-(2-carboxymethyl-2,3-dihydro-s-triazoio[4,3-bl-pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic Acid To a suspenslon of 7-aminocephalosporanic acid (8.79 g., 32.2 m.mole) ln 0.1 M phosphate buffer (pH 7, ~ 41 -~L~5C~7Z7 `

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 mixture 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. m e resulting precipitate was collected by filtration and washed with water to give 7.59 g.
(54%) of 7-amino 3-(2 carboxymethyl-2,3-dihydro-s-tri-azolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid.

.~ .

;

~L~ Cj~7;~7 ir vKBar 1800, ~720, 1600, 1540, 1470 cm 1.
uY ~Buffer (pH 7) 252 nm (~,19500), 298 nm (~, 8400).
nmr: ~p~mO+K2C3 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), ~.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-Chlo hyl)-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 ~as stirred at 100-110 C. for 24 hours, then poured into water (700 ml.) and extracted with ethyl acetate (5 x 400 ml.). The organic extracts were combined, dried over Na2S04 and evaporated. The residue was crystal-lized from ethyl acetate to give light yellow needles o~
. 6-chloro-2-(2-cyanoethyl)-2,3-dihydro-s-triaz~lo[4,3-b]-pyridazin-3-on (2.5 g., 11%). M.p. 165-168 C.

ir: vmBx 2230, 1720, 1550, 15nO cm 1.
uv: ~dimXaxne 373 nm ( E 2000 ) .
nmr: ~Dppm d6 3.03 (2H, t, J-6.o Hz, CH2), 4.21 (2H, t, J-6.o Hz, CH2), 7.23 (lH3 d, J=10.0 Hz, pyridazine-H), _ 43 _ 1~ 5137Z7 .

7.93 (lH, d3 J=10 0 Hz, pyridazine-H).
Anal. Calc'd. for C8H6N50Cl: C, 42.97; H, 2.70;
N, 31.32; Cl3 15.86. Found: C, 42.73, 42.56; H, 2.57, 2.50; N, 31.36, 31.68; Cl, 15.96, 15081.

2-(2-Carboxyethyl)-6-chloro-2~3-d ~ azolo~4,3-b pyridazin-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 combined extracvs were washed with saturated aqueous sodium chloride (50 ml.), dried over Na2SQ4 and evaporated to give light yello~J~solid 2-(2-carboxyethyl)-6-chloro-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-~n (567 m~., 72~).
M.p. >170~ C. (sublimation).
ir: ~KBx 3400-2400, 1730, 1710, 1540 cm 1.
- uv: ~dioxane 377 nm (E 1500) nmr ~D2~panHC3 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, pyrid~zine-H)3 7.70 (lH, d, J=lO.Q Hz, pyridazine-H).
Anal. Calc'd. ~or C8H7N4Q3Cl: C, 39.60; H, 2.91;
N, 23.09; Cl, 14.61. Found: C, 39.62, 39.48; H, 2.97, 2.67, N, 23.05, 22.70, Cl. 13.93, 14.12.

' ~5~7~7 2-~2-Carboxyethyl~-2,3-dihydro-6-mercapto-s-tr-iazolo~4~3-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.) ~as 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 preci~itate 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 1.
uv: ~pH 7 buffer 262 nm (E/7o~0), 318 nm (E 6600).

nmr: ~D~ppmd6 2.73 (2H, t, J=7.0 Hz, CH2), 4.07 (2H, t, J=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 C8H8N403S: C, 40.00; H, 3.36;
N, 23.32; S~ 13.35. Found: C, 39.o8, 39.o6; H, 3.12, 3.20; Ny 22.65, 22.70; S, 14.23, 14.29.

7-Amino-3-~2-(2-carboxyethyl)-2,3-dihydro-s-triazolo~4,3-b~-pyridazin-3-on-6-ylthiomethyl]-3-cephem-4-carboxylic Acid.
- A mixture of 7-ACA (405 mg., 1.49 m.moles), the thiol 2-(2-carboxye~hyl3-2,3-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 8Q C. for 30 minutes. nle reaction mixture was cooled and filtered to remove insolubles.

- 1~5 -- 1~5~q27 The filtrate was adjusted to pH 1-2 with c. HCl. The result-ing precipitate, 7-amlno-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: vKBX 3600-2200, 1800, 1725, 1620, 1550, 1480 cm 1.
~pH 7 buffer 253 nm (E ~0000), 298 nm (~/ 0 ) ` 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, pyridazine-H), 7.44 (lH, d, J=10.0 Hz, pyridazine-H).
Anal- Ca1c~d- for C16H16~606S2 3/ 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.

.

...

- ~.15~7Z7 Scheme 1. Preparation of 7-Amino-3-(2-carboxymethyl-2,3-dihydro-s-triazolo[4,3-b]pyrida-zin-3-on-6-ylthio-methyl)-3-cephem-4-carboxylic Acid.

_~=N~ ClCH2COOC H
C N"N~n~NH 2 5 ) Cl ~ N ~ N-CH2COOC2H5 ~N~SH ~ ~ 7-~CA
HS N,N ~ N-Ctl~COOH

O

H2N ~ ~ M
N ~ CH2S ~ I~,N ~ N-CH2COOH

7;~7 6-Chloro-2,3-dihydro-2-ethoxycarbonylmethyl-s-triazolo-[4,3-b]pyridazin-3-one (2) To a solution of 6-chloro-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-one [P. Francavilla and F.
Lauria, J. Het. Chem., 8, 415 (1971)] (1, 1.00 g., 5.9 m.mole) in dry DMF (30 ml.) was added sodium hydride (50% in paraffin, 0.3 g., 6.3 m.mole) under stirring with formation of yellow crystals. To the mixture was added ethyl chloroacetate (1.4 ml., 13 m.mole) and the mixture was heated 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 of 2 (1.16 g., 77~, m.p. 114-115 C. (lit.
110 C.).
ir: ~ max 1735, 1710 cm 1.
uv: ~ Emtax~I 231 nm (, 26000).
nmr: ~ ppC13 7.58 (lH, d, J=10 Hz, pyridazine-H), Ç.98 (lH, d, J=10 Hz, pyridazine-H), 4.80 (2H, s, -CH2CO), 4.27 (2Hl q, J=7.5 Hz, CH2CH3), 1.29 (3H, t, J=7.5 Hz, CH2CH3 ) .
Anal. Calc'd. for CgHgH4O3Cl C, 42.12; H, 3.53;
N, 21.83; C1, 13.81. Found: C, 41.54, 41.46; H, 3.22, 3.49; N, 21.51, 21.53; Cl, 13.88, 13.99.

l~LS~

2-Carboxymethyl-2,3-dihydro-6-mercapto-s-triazolo[4,3-b]-pyridazin-3-one (3) -To a solution of 6-chloro-2,3-dihydro-2-ethoxy-carbonylmethyl-s-triazolo[4,3-b]pyridazin-3-one (2, 30 g., 0.12 mole) in ethanol (900 ml.) was added NaSH 2H2O (70%
pure, 45.9 g., 0.36 mole) and the mixture was refluxed for 0.5 hour. The reaction mixture was evaporated at re-duced pressure. The residue was dissolved in water (200 ml.3 and concentrated HCl was added to the solution to adjust to pH 2. The precipitate (3) was collected by fil-tration and washed with water. Yield 18.3 g. (69%).
ir ~ max 2900, 2450, 1750, 1660 cm uv: ~ 1%NaHCO3aq 260 nm (, 19500), 313 nm (~, 7000).

nmr: ~ ppm d6 7.88 (lH, d, J=10 Hz, pyridazine-H), 7.45 (lH, d, J=10 Hz, pyridazine-H), 4.72 (2H, s, CH2CO).
Anal. Calc'd. for C7H6N4O3S: C, 37.17; H, 2.67;
N, 24.77; S, 14.17. Found: C, 37.35, 37.23; H, 2.26, 2.28; N, 23.58, 23.69; S, 14.32.

7-Amino-3-(2-carboxymethyl-2,3-dih~dro-s-triazolo[4,3-b3-pyridazin-3-on-6-ylthiomethyl)-3 cephem-4-carboxylic Acid (4) To a suspension of 7-aminocephalosporanic acid (8.79 g., 32.2 m.mole) in 0.1 M phosphate buffer (pH 7, 149 ml.) were added NaHCO3 (8.14 g., 97.0 m.mole) and the thiol 3 (7.30 g., 32.2 m.mole) with stirring. The mixture was heated at 80 C. for 0.5 hour under N2 stream. The mixture was treated with active carbon and adjusted to pH 3 with conc~ntrated HCl. The resulting precipitate was collected by filtration and washed with water to give 7.59 g.
(54~) of 4.

'~

if ~:
5~P7~7 ir: ~ K~x 1800, 1720, 1600, 1540, 1470 cm 1.
uffer (P~ 7~ ~5~ nm (~,19500), 298 ~n (E,8400)-n!nr~ m~lK2 3 7.5G (1~1, d, J=9 Hz, pyrida~ine-H), 7.05 (ln, d, J=9 Hz, pyridazine-~), 5.45 (1~, d, J=5 Hz, :`~ 6-~), 5.05 (lH, d, 5 Hz, 7-~l), 4.43 (lH, d, J=14 Hz~
3-C~I2), 4.04 (lH, ~, J=14 Hz, 3~CH2), 3.88 (lH, d~ J=18 Hz, ~- 2-H), 3.45 (1~l, d~ J=18 Hz, 2-H).
Pivaloylox~nethyl-~-amino-3-(2-carboxymethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl~-3-cephem-4-carboxylate.
Method A - The tltle compound i9 produced by ~ubstitutlng for the 7-amlnocephalosporanlc acid used immedlately above an equlmolar weight Or pivaloyloxy-i . methyl 7~aminocephalosporanate hydrochloride prepared according to Example 2 of U.K. 1,229~453 from 7-amlnocephalosporanic acld. German 1,904,585 (Farmdoc 39,445) ls equlvalent to U~K, 1~229~453, . Method B. - The title compound is produced by Qubstltutlng for the 0 025 mole (6.8 g.) 7-amlno-: cephalosporanlc ac~d used ln the procedure o~ Example ~` 2 Or U.K. 1,229,453 an equlmolar welght o~ 7-amlno-3-.~ (2-carboxymetnyl-2,3-dihydro-s-triazolo[4,~-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-o~-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.

.

~ ,. . .

Preparation of 7-Amino-3-(2-methYl-2~3-dihydro-s-tria-zo ~ 4,3-b]pyridazin-3-on-6-ylthiomethyl-3-cephem-4-carboxylic Acid.

Cl ~ NH C1 ` ~,N ~ N-CH~

~/ ,.

~I ' ' HS N'N~ ~ N- CH3 O
'. , 1 ~
H2N T~
N ~ 2 ~ l~f ~ N CH3 ! 4 6-Chloro-_,3-dihydro-2-methyl-s-triazolo~4,3-b]pyridazi_-3-one (2) To a solution of 6-chloro-2,3-dihydro-s-triazolo-[4,3-b]pyridazin-3-one [P. Francavilla and F. Lauria, J.
Het. Chem. 8, 415 (1971)] (1, 8.5 g., 50 m.mol.) in dry DM~ (12 ml.) was added NaH (50~ dispersion in paraffin, . ' .

~ ~L15~27 .
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 addedg 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 af~orded 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: ~Eta~I 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. fo~ C6H~ClN40: C, 3~.04; H, 2.73;
N, 30.35; C1, 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 ~H20 (70~ pure, 9.4 g.) in water (100 ml.) was heated under reflux for 15 minutes. The mixture was cooled and acidified to pH 1 with concentrated HC1 to precipitate the thiol 3 which was collected by filtration and dissolved in aqueous saturated NaHC03 (100 ml.). The solution was treated with a small amount of carbon and acidified with dilute HCl to precipitate 3 as pale yellow prisms.
Yield: 5.72 ~ 95'). M.p. >280 C.

.

( ~
" ~ 27 :
ir: vKax 2450 (-SH), 1710 (C=0) cm 1 uv: ~1%maHC3 261 nm ( E 16300), 315 nm (~ 5800).
nmr: ~ 2 ppm 3.60 (3H, s, N-CH3) 3 7.o8 (2H s pyridazine-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]pyridazin-3-on-6-ylthlomethyl~-3-cephem-4-carboxyllc 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 phospllate buffer (pH 7, 100 ml.) was heated with stirrlng at 80~ C. for 30 minutes.
The hot mixture was treated with a small amount of carbon and the filtrate was acidi~ied to pH 4 lJith dilute HCl to prec~pitate 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: vKBX 1800 (~-lactam C=0), 1725 (C=0), 1610 and 1410 (C00 ) cm~l uv: ~ ~NaHCo3 253 nm (E20000), 305 (E 8400).
D O
nmr: ~ 2 ppm 3 3.69 (3H, s, N-CH3), 5-08 (lH~ d~ J=4.5 Hz, 6-H), 5.48 (lH, d, J=4.5Hz, 7-H), 7.00 (lH, d, J=10 Hz, p~ridazine-H), 7.52 (lH, d, ~=10 Hz, pyridazine-H).
Anal. CalC~d~ for C14H14~604S2 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.

`~ 56C~27 - -I`''' .
Preparation of BB-S515 CO-N ~ S ~ N
No~N~ CH2S ~N ,N ~ NH
OCH3 C02Na r BB-S515; 7-~(2Z)-2-Methoxyimino(fur-?-yl)acetamido~-3-(?~3-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 a~ford 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-b]pyridazin-3-on-~` ~ 6-ylthiomethyl)-3-cephem-4-carboxylic acid (380 mg., 1 m.mole) (U.S. ~,907,786) and sodium bicarbonate (336 mg., 4 m.~ol.) in water (10 ml.) at 0-5~ C. The ¦ ~ reaction mixture was stirred at 0-5 C~ for 2 hours.
¦ Most of the acetone was evaporated at reduced pressure, the aqueous concentrate being washed with ether (2 x 30 ml.) and adjusted to pH 1-2 with concentrated HCl.
The resulting precipltate (338 mg.) was collected by filtration and dried in vacuo. A suspension of the free acid (303 mg.) in water tlO ml.) was adjusted to pH 6.5 with aqueous NaOH ~1 N, o.6 ml.) and filtered to make a clear solution which was lyophilized to ~ive 7-~(2Z)-2-methoxyimino(fur-2-yl)acetamido]-3-(2,3-dihydro-s-. .

-1~ 5~1~Z~

triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid sodium salt as a light brown powder (222 mg., 46%). M.p. >230 C. (dec,), . ' ir: vKBaxr 3410, 1760, 1720, 1600 cm 1, uv ~,pH7Buffer 256 ~m (E 20600), 274 (~ 18800)-i:

.

1: ' .

' .

:~ :
L5û~27 .
.

Pre~aratlon of D-man.delic acid carb~o~yanhYdride COC 1 . - ' 6 5 ~ 2 ~ C H ~o OH d~ ~
o .
-Mandel~c acld carboxvanhYdrlde (2) Pho~gene was bubbled through a-solution of 2.0 g.
(0.01~ mole) of D(-)-~andelic acld (1) ln dry tetra-hydroruran ror 30 mlnutes. The solutlon was allowed to stand overnlght arter which tlme lt wa~ heated under reflux rOr 10 mlnutes. Evaporatlon of the solvent under reduced pressure afrorded an oily resldue whlch was solldl~led by trlturatlon wlth n-hexane (20 ml,). The product was collected by flltratlon and drled ln ~Q~ on KOH. Yield 2.3 g.
of D-mandellc acld carboxyanhydrlde.
I .
IR ~ maUJ 1895, 1875, 1780 cm 1, . - ' The preferred and most actlve compounds of the pre~ent invention are those havlng the D
configuratlon at the a-carbon atom in the 7-~ide-chain, that is, those mad~ from D-mandellc acid or a mono~ub~tituted D-mandelic acid as illustrated here-in, In additlon, the configùration at the two optically active, asymmetric centers in the ~-lactam nucleu~ 19 that ~ound ln cephalosporln C produced by fermentatlon and in the 7-aminocephalosporanic acld derlved there~rom.

-)727 C6H5-CH-C \ H N ~ ~ ~ N
O ~C / o ~ ~ CH2 S N,N ~ N-CH2C2H
O C2H. O

-- ~ C6H5-cH-coNH~s~
1H ~ N ~ CH2-S N~N ~ N-CH2C2H

3, BB-S488 .
BB-S488, 7-(D-Mandelami ~ -3-(2-carboxymethyl-2,3-dihydro-s-triazolo~4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic Acid (3) . . . ~
D-(-)-Mandelic acid 0-carboxyanhydride (U.S. Patents 3,167,549, 3,840,531 and 3,910,900), (1, 400 mg., 2.3 m.mole) was adde~ portlonwise to a solution of 7-amino-~-(2-carboxy-methyl-?,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-methyl)-3-cephem-4-carboxylic acid (2, 657 mg., 1.5 m. mole) and sod;um blcarbonate (445 mg., 5.3 m.mole) in 50~ aqueous acetone (30 ml.) at ca 0 C. with vigorous stlrring. The ~mixture was stirred for 1 hour at room temperature and`
evaporated under reduced pressure below 40 C. to remove acetone. The resulting aqueous solution was washed ~lith ether and acidified to pH l with dilute HCl to afford a gummy precipitate~ which was collected by tiltration, washed with water and dissolved in tetrahydrofuran (TliF) (100 ml.). The THF solution was treated with a small amount ~ 57 -_ .~ . . . ; . . . _ .~ . . ............................... .
. . . .. ~ , , 7 ~`

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. Thè 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: ~ Kax 3600-2400, 1770, 1720, 1520, 1495, 1365, 1245 cm~l.
uv: ~ EtxH 254 nm ( 18000), 297 nm ( 9000, sh).
nmr ~ DMS-d6 3.68 (2H, m, 2-~T), 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-_), 8.60 (lH, d, J=9 Hz, disappeared with addi-tion of D20, CONH).
Anal. Calc'd. for C23H20N60gS2 / 2 H, 3.64; N, 14.45; S, 11.03. Found: C, 47.34; H, 3.48;
N, 13.90; S, 11.01.

*Trade ~Iark - 5~ -'7 In vitro activit~ (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 3.

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

Blood levels in mice Groups o~ mice were administered subcutaneously graded doses of 40, 20 and 10 mg./kg.. The ~lood sam-ples collected from orbital sinuses were assayed by the paper disc-agar diffusion method on Sarcina lutea PCI lO01 plates. The results are shown in Table 5.

In vivo activity Comparative in vivo evaluation was made by the standard experimental infection in mice against the . - 59 -, ~ ~LSg:~727 --~

following pathogenic bacteria S. aureus Smith E. coli Juhl K, pneumoniae A9977 The results are shown in Table 6.

, - \
\
. . \ .

.: \

\

. . 60 _ : . .

~ 11 S ~ ~7 2 7 Table 1. The in vitro Antibacterial Activity o~ BB-S~88 .
By Agar Dilution ~lethod (Mueller-Hinton Agar).
~IC (mcg,/ml.) Test Organism _ BB-S488 Cefamandole S. aureus S~ith A9537 0.2 0.05 S. aureus A9497 0.1 0.05 S. aureus BX-1633 A9606 0,4 0.1 St. ~aecalis A9536 IOO 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 0.05 0.1 E. coli A15147 3.1 0.4 Kl. pneumoniae Q9678 3.1 3.1 ISl. pneumoniae A9977 0.05 0.2 Kl. pneumoniae A15130 0.2 0.8 Kl. pneumoniae A9867 0.2 o.8 Pr. vulgaris A9436 0.1 0.4 Pr. vulgaris A9699 0.2 6.3 Pr. mirabilis A9554 0-05 0.4 Pr. mirabilis ~g900 0.1 o.8 Pr. morganii A9553 >100 >100 Pr. morganii A20031 0.1 o.8 Pr. rettgeri A15167 0,05 o,~
Ps. aeruginosa A9930 >100 >100 Ps. aeruginosa A98l~3 >100 >100 - Shig. dysenteriae 0.025 0.1 Shig. flexneri A9684 12.5 6.3 Shig. sonnei A9516 0.025 0-05 Serr. marcescens A20019 100 100 Enterob. cloacae A9656 3.1 3.1 Sal. enteritidis A9531 0.05 0.1 Sal. typhosa A9498 0.05 0.1 B. anthracis A9504 0.1 0.1 - . .. ...

~7~

Table ?. In vitro Antibac~erial Activity in Mueller-.... .. . .
Hinton Agar (Gram-positive~
.
MIC (mcg./ml ) Code Cefaman-No. ' Test Organism BB-S488 dole .
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 No. 193 o.8 0.2 Sa-10 S. aureus 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 0 8 Sa-33 S. aureus Terajima 0.0125 0 0125 Sa-34 S. aureus A15092 o.8 0 2 Sa-35 S. aureus A15094 o.8 0 4 Sa-36 S. aureus Russell o.8 O 4 Sa-37 S. aureus A9524 1.6 0 8 Sa-38 S. aureus A9534 0.4 0 2 Sa-~9 S. aureus A9578 o.8 0 4 Sa-40 S. aureus A9601 o.8 o. L~
Sa-41 S. aureus A9602 o.8 0 2 Sa-44 S. aureus A15097 25 25.
Sa-56 S. aureus A9~30 3.1 o.8 Sa-57 S. aureus A9748 25 ~.1 Sa-58 S. aureus A15033 12.5 1 6 Sa-59 S. aureus A15096 100 6 3 Sa-60 S. aureus A2060ll 50 3 1 Sa-61 S. aureus A20605 100 6 3 Sa-62 S. aureus A20606 3.1 o.8 Sa-6~ S. aureus A20607 >100 12.5 Sa-64 S. aureus A20608 100 6 ~
Sa-65 S. aureus A20609 100 6 3 Sa-66 S. aureus A20610 100 6 Sa-67 S. aureus A20611 100 6 3 Sa-68 S. aureus A20612 1.6 0 4 Sa-69 S. aureus A20613 100 6 3 Sp-l S. pyogenes S-23 0.4 0 1 Sp-2 S. pyogenes Dick 0.4 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 o,4 0.1 Sp-6 S. pyogenes A20066 0.4 0 1 Sp-7 S. pyogenes Dig 7 0.ll 0 1 Sp-8 S. pyogenes A15041 0.4 0.1 Sp-9 S. pyogenes A?0201 0.4 0.1 Sp-10 S. pyogenes A?0202 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 ~201G7 0.2 0.2 Dp-7 D. pneùmoniae A20759 0.2 O ?
Dp-8 D. pneumo!liae A20769 0.2 0 2 Dp-9 D. pne~loniae A20770 0.2 0.?

~ , . . . .

~L5~ 7 Table 3. In vitro Antibacterial Activity in Mueller-.
Hinton Agar ~ram-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 A203630.2 o.8 Ec-ll E. coli A20366 5 25 Ec-15 E. coli ATCC 8739 0.2 0.1 Ec-34 E. coli A9660 0.1 0.1 Ec-35 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 A203ll3 50 12.5 Ec-56 E. coli A20365 25 12.5 Ec-58 E. coli A9575 0.4 1.6 Ec-59 E. coli A20766 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 A20361l 3.1 3.1 El-4 E. cloacae A20650 1.6 1.6 El-6 E. cloacae A9557 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. cloac~e A21006 1.~
El-14 E. cloacae A20953 o.8 3.1 Pm-l P. mirabilis A9554 0.1 o.8 Pm-2 P. mirabilis A9900 0.2 1.6 Pm-3 P. mirabilis A20119 , 0~4 3.1 Pm-4 P. mirabilis A20~54 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 A9553 >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. morg~nii A20~57 0.2 o.8 Pg-8 P. morganii A15153 0.1 o.8 Pg-9 P. morganii A15149 0.8 3.1 Pv-l P. vulgaris A9436 0.2 0.8 Pv-2 P. vulgaris A9526 ~,3 1.6 Pv-3 P. vulgaris A9699 6.3 5 Pv-4 P. vulg~ris ~TC~ 9920 0.1 0.2 Pv-5 P. vulgaris A9539 25 >100 Pv-6 P. vulgaris A9716 0.1 0.8 Pv-7 P. vulgaris A21240 25 >100 . ~ 7 .
Table 3 (Continued) Code Cefaman-No, Test Organism BB-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-~ PO rettgeri A20920 0.1 0.2 Pn-l P. inconstans A206150.1 o.8 Ps-l P. stuartii A20745 0.4 0 8 Ps-2 P. stuartii A20894 0.2 0 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 A9678 3.1 1.6 Kp-3 K. pneumoniae A9977 0.1 o.8 Kp-4 K. pneumoniae A151300.2 o.8 Kp-7 K. pneumoniae A9867 0.4 o.8 Kp-8 K. pneumoniae A2068025 12.5 Kp-9 K. pneumoniae A2063612.5 12.5 Kp-10 K. pneumoniae A203286,3 3.1 Kp-ll K. pneumoniae A203301.6 12.5 Kp-12 K. pneumoniae A212286.3 6.3 Kx-2 Klebsiella sp. A96620.4 1.6 Kx-3 Klebsiella sp. A20346 0.2 o.8 Sm-l S. marcescens A2001925 25 Sm-2 S. marcescens A203353.1 12.5 Sm-3 S. marcescens A203366.3 12.5 Sm-4 S. m~rcescens A204426.3 12.5 Sm-5 S. marcescens A202223.1 12.5 Sm-6 S. marcescens A204606.3 12.5 Sm-g S~ marcescens A20333G.3 5 Sm-10 S. marcescens A203346.3 5 Sm-ll S. marcescens A204596.~ 25 Sm-12 S. marcescens A204616.3 5 Se-l S. enteritidis A95~10.1 0.2 St-l S. typ}los~ 0.1 0.2 Sh-l S. paraty~hi 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 U~
. . N~ ~ OJ ~ ~ ~ COCO~ C0~ C0~ N~ N ~ J
. O O ~ O O O O O ~ N ~ ~ O O O O O O O O O O O ~ O ~ O ~ O O O O
O ~ ~ N ~O ~ O O O O
~1 A ~ ~
O . N ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ N
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O ~ O O O O O O O O~O O O ~O O O O O O O O O O O O N O N O O O O
~, ~ 00 0 00 0 0 ~1 ~10 0 ~1 ~
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0 0~ O~ N ~ ~ N0 N N ~ N N0 H ~ N
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OO O OO O ~ NOOO
~1 ~1 ~1 ~ m ~ ~ ~ ~ ~ ~\~
0~ ,~N ,1~ ~ _~0NN0~1~1 N~J 0 ~a ¢ O ~O O O O O O~ O O N N O O O O O O O O O O O O O O O O O O O
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. u~ ~ ¢ ¢ ¢ ¢ ¢ ~ o o ~ C ~
u~ ~ F ~ P R ~ bD h. h h ~1 ~ bD h ~1 o ~ nd ~ :3 C) bD hO ~ O bD bD
E-l h h h O rl-l ~1 ` I ~1 ~I r-l H 0 0 0 Q~ h h h ~ h h h ~
t~ ~ d O O O O O O O r~ I C C~ rl O O O O ~ ~ ~ t~ c Q

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'~ ~ , ~ ~ ~ ~ ~ ~ ~ N ~ ~ ~ ~ N ~ ~ ~ ~ N
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v~ ~q ~ q ri~ ~ ril ~ r~ r~ r l rL~ r~ r~ p~ ? ~ ~ *

- 65 ~
.

l`` ': ,. ',, ;
j 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 0~ 6 4.1 60l 4.3 120' 0.7 ~0.1 .;~ ' .
~ 10 mg./k~. 15' 5 3.8 '~ 3' 3 1.5 I 60' o,.8 0.3 I 120' -- 0.1 D
~1 r .
. ~ . .
~ .

-i , ~ . ~

.

- 66.-- : . .
; ~ ~

llSV7Z7 Table 6. In vivo Activity .. .

Test Organism Dose BB-S488 Cefam3nd~1e S. aureus Smith 25 mg./kg. 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 0.4 5/5 0/5 0.1 c/5 PD50 12 mg./kg. 1.8 mg./kg.
K. pneumoniae25 mg./kg.5/5 5/5 i 6.3 5/5 1/5 : 1.6 5/5 /5 .4 4/5 0/5 : 0.1 0/5 ~P ~
PD50 0.26 mg./kg.6.25 mg./kg.
f , : *No. of survivors/No.:tested . ~ . .
. . .

.

.
, .
.~

~15i~727 Run No. of Dose. BB-S Cefam-Test Organism No~ LD50 (mg./kg.) 488 andole E. cloacae C-811 1 x 10 100 5/5 5/5 A2~4~4 (El-l9) ` 25 5/5 5/5 : 6.3 5/5 5/5 1.6 4/5 4/5 0.4 2/5 1/5 50 (mg./kg.) O. 54 o.8 Urinary Recovery in Rats % Recovery (0-24 Hrs.
Dose (sc) BB-S488 Ce~amandole . .
10 mg./kg. 38.8 58.3 Ne ~rotoxicity Test in Rabbits No nephrotoxic si~n was seen in the rabbits treated with 100 mg./kg. tiv) o~ BB-S488 while cephalori-dine showed severe nephrotoxicity in the comparative test.
.

.

.
_................................... , ~ `

Addltion~il P' ~O I)eta (Stn~le sc TreaCmcllt) 1150'727 Run No. o~ Dose BB-S Cefam-Test Organism No. LD50 (mg./kg ~ 488 andolè
:
r K pneumoniae C-805 3x103 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 0!5 ---PDso (mg./kg,) 0.26 9.4 _ . .
A94U3~1-g ~ 1) C-808 lxlO 50 --- 3/5 5/5 ___ 12-5 --~ 1/5 6.3 5/5 ~~~
3.1 --- 0/5 1.6 3/5 __~
.8 --- 0/5 0.4 1/5 ---PD50 tmg-/kg.) 1.1 36 P. mirabilis C-810 lx103 50 ~~~ 2/5 A9900 ~Ym-2) 5/5 -_-12.5 --- 1/5 6.3 5/5 ---3.1 --- 0/5 o.8 --- 0~5 0.4 0/5 ___ ; 0.2 --- 0/5 0.1 0/5 ~~~
PD50 (mg./~g.~ 1.1 50 - _ ~

` ~ 727 !

Stability of BB-S488 ! Stability of BB-S488 was determlned 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. Tile activity was assayed using paper discs on B. subtilis PCI 219 plate (pH 6), .

(1) Stability in a 10% A~ueous Solution at Room Temperature ~l\ Remaining ActivitY (~
Compound pH~ ~ O 2 ~ 3 7 Da~Js BB-S488 6,1 100 128 90 116 (l)Unadjusted pH of the 10~ solution.

~2 Remaining Activity (%) CompoundpH ) O1 ~~~~ 2 3 7 Dayis~

BB-S488 ~ 7 100 87 64 i~ (2)pH 4: 0.1 M AcOH - NaOAc buffer.
pH 7: 0.1 M phosphate buffer.
pH 9: 0.1 M NH~OH-hH4Cl buffer.

~ .

.

~ .

~ ~ "
CHCOOH C12CHCOCl ~ CHCOOH SOC12 ....... _~ ~ l D-(-) CHCoCl H-Ac~-s-cMTp(~) [ ~ CHCO-ACA-S-CMTP~

.. /

, ~/. Na2C03 CHCO-ACA-S-C~ITP

. BB-S488 Dichloroacetylmandeloyl Chloride (2) _ A mixture of D(-)-mandelic acid (1, 1.52 g., 10 m.mole) and dichloroacetyl chloride (4.41 g., 30 m.mole) was heated at 80-85 C. for 1.5 hrs. and the excess dichloroacetyl chloride was removed under diminished pressure. 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 kept over KOH at 1 mm Hg overnight at room temperature to remove dichloroacetyl chloride.
Yield, 2.8 g. (100~). This product was used in the next ~I~L5~7~
.
step without further purification.
ir: ~ miaq 1780, 1160 cm 1 nmr: ~ ppl4 5091 (lH, s, PhCH or COCHC12), 6.oo (lH, S7 PhCH or COCHCl2)3 7.32 (5H, s, phenyl~

.
BB-S488; 7-~D-Mandelamido)-3-(2-carbo~ymethyl-2~,-dihydro-s-triazolo~4 "-blpyridazin-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)-3-ce~hem- !1 -C arboxylic acid (H-ACA-S-C~.TP)(3, 3.94 g., 9 m.mole) and trietilyl-amine (3.54 g., 35 m.mole) in 50% a~ueous acetone (120 ml.) at OD-5 C. The mixture was allo~red to rise to room tem-i 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 allo~Jed to stand at room te~-perature for 30 minutes, acidified to pH 1 with dilute HCl ~nd eva~orated under reduced pressure to remove acetone below 40 C. The precipitate W25 collected by filtration~ washed with water (20 ml.) and air-dried.
The dried m~terial was dissolved in THF (150 ml.), stirred for 5 minutes at 40_50C G. ~nd filtered to remove insoluble unreacted ~ (0.54 g., 14~ recovery). The fil-trate was chromatographed on a silica gel column (~ ko-gel, C-200, 30 g.) and eluted with chloroform-methanol (100:5). The eluates were collected in 50 ml. fractions ; monitoring by tlc (silica gel, solvent, GH3C~l-water = 4:1, . - 72 -~ 1 lSV~
.

detected with I2?. The fractions containing the desired product were combined, treated with a small amount of carbon and evaporated under reduced pressure. The res~-due was triturated with chloroform (50 ml.) to yield, 2.36 g. (46~) o~ 7-(D-mandelamido)-3-(2-carboxymethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthlo-methyl)-3-cephem-4-carboxylic acid (4). M.P., 165-:~ . 170 C. (dec.).
ir: ~ KBax 3600-2500, 1780, 1720, 1500, 1410, 1355, 1220, 1195 cm 1 `uv: ~ EtaxH 254 nm (e, 18300), 297 nm (sh, ~, g300).
nmr ~ DMS-d6 3.84 (~H, m, 2-H)~ 4-17 (2~l~ d~ 13 Hz, 3-H), 4.50 (lH, d, 13 Hz, 3-H), 4.82 (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, pyridazine-H), 7.4-7.7 (5H, m, ~!phenyl-H), 7.90 (lH, d, 11 Hz, pyridazine-H), 9.0 (lH, d, 9 Hz, 7-CONH, disappear by addition of D20).
Anal. Calc'd for C23H2oN60gS~}/4CHC13: C, 43- o8;
H, 3~16; ~, 12.69; S, 9.69. Found: C, 43.11, 43.22; H, .
2.97, ~.o6; N, 12.8~, 12.77; s, 9.64.

, '~.
. . .

- 73 ~

~; . ;
'` ~Li5q~7;2 7 =~ 99% HCOOH ~==\ SOCl 7HCOOX) ~ ~CHCOOH

OH OCHO

~==\ H-ACA-S-CI~rP (3) ~
CHCOCl> ~ CHCO-ACA-S-C~ITP

OCHOOCHO
6 7, BB-S494 O-Formyl-D(-)-mandelic Acid (5) A mixt~re of D(-)-mandelic acid (1, 5.0 ~ 3 m.mole) and 99;J formic acid (80 ml.) ~tas 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 resi~ue ~las 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 O-formyl-D(-)-mandelic acid (5) as colorless prisms. M.P.~ 56-59 C. (lit. M.P.~ 55-58~ C.).

ir: ~ m~ar 3400-2800, 1755, 1720, 1160, 990 cm~l.
nmr: ~ ppDml3 5.98 (lH, s, PhGH), 7.31 (5H, m, phenyl-H), 8.o5 (lH, s, OCHO), 10.05 (lH, s, COOH, disappeared by addition of D20).

_ 74 , 5~727 , 0-F rmyl-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 resîdue under reduced pressure afforded the acid chloride 0-formyl-D(-)-mandeloyl chloride ~6).
r Yield, 1.53 g. (70%). B.P., 120-122 C./15 mmHg.
ir: ~maq- 1805, 1740, 1160, 1140 cm 1.
t BB-S494; 7-(D-0-Formylmandelamido)-3-(2-carboxymethvl-2,3-dihydro-s-triazolo[4~3-blpyridazin-3-on-6-ylthiomethyl) 3-cephem-4-carboxylic Acid (7) ; A solution of 0-fDrmyl-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[413-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 I 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 a~idified to pH 1 with dilute HCl. The separated oily gum was collected and dissolved in THF (100 ml.). The solution was treated with a small ~mount of carbon and dried with anhydrous sodium sulfate.
Evaporation o~ the solvent under reducea pressure to 10 mlO and dilution with ether afforded the title compound (7) as a pale yellow amorphous po~der, 0.91 g. (38~).
M.P., 172-176 C.(dec~).
ir: ~ rmBax 3600-2400, 1775, 1720, 1550, 1355, 1230, 1160 cm~l.
uv: ~ mtl~ 254 nm (, 20800), 297 nm ~sh~ io500).

nmr: S ppm 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 (5H, 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 ~24H20N609S2 lH2 Hj 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-carbo~ymethyl-2~3-dihydro-s-triazolo-~4,3-b]pyridazin-3-on-6-ylthiomethyl~-3-cephem-4-carboxylic Acid (4) A mixture of 7-(D-O-formylmandelamido-3-t2-car-boxymethyl-2,3-dihydro-s-triazolo[4,~-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid (7) (484 mg., 0.81 m.mole) and sodium bicarbonate (748 mg., 8.9 m.mole) in water (4 ml.) was stirred for 4 hours at room tempera-ture, and acidi~ied to pH 1 with dilute HCl. The preci-pitate (500 mg.) was collected by filtration, waslled 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 (~-5~) as eluent, and the fractions containin~ the product ~ere combined, treated with a small amount of carbon and eva-porated under reduced pressure. The residue was 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-~-carboxylic acid (B~-S488; 4).

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

5~7 ir: ~ KBax 3600-2400, 1770, 1720~ 1520, 1495, 1365, 1230 cm~l. ``
uv: ~ EtaxH 254 nm (~, 20000), 297 nm (sh, ~, 9600) ' Sodi~m Salt of BB-S488 .
Sodium-2-ethylhexanoate (~EH) (4.0 ml., 1 M
solution in ethyl acetate) was added to a solution of j 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 i ml.). The precipitate was collected by filtration, washed l~ ~1ith 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,l.96 ~ bio yield, 97~), M.P., 230-240 C. (dec.). The pH of the 10~ aqueous solution was 3.6.

ir: ~ max 3600-3000, 1765, 1710, 1~05, 1390, 1360, 1190, 1080, 1065 cm~l.
uv: ~ ~mater nm(~l'c~m)~252 (357), 310 (sh, 140).
nmr: ~ ~ m 3.43 (lH, d, 19 Hz, 2-H), 3.87 (lH, d, 19 I~z, 2-H), 4.15 (lH, d, 14 Hz, 3-H), 4.53 (lH, d, 14 Hz, 3-H), r 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 (lII, d, 10 Hz, pyridazine-H).
Anal- C~lc'd. for C23HlgN60~S2Na 5/4H20: C, 44-77;
H, 3.51; N, 13.62~ S, 10.39. Found: C9 44.93, 44.79; H, 3.31, 3.15; N~ 13.~1, 13.33; S, 10.19.

. ;' ' ~ .
- 77 ~

' , , . :

~:~LSq~27 EXAMPIE_6 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 water (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[4,7-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: ~ KBax 3600-3000, 1760, 1710, 1605, 1390, 1360, 1190, 1080, 1060 cm~l.
uv: ~1 mater nm (El~m) 252 (320), 310 (124).

nmr: ~ p~ 3.43 (lH, d, 19 Hx, 2-H), 3.90 (lH, d, 19 Hz, 2-~), 4.15 (lH, d, 14 Hz, 3-~_), 4.53 (lH, d, 14 Hz, ~~H)g 4.75 (2H, s, NCH2C0), 5.22 (lH, d, 4.5 Hz, 6-H), 5.42 (lH~ s, PhCH), 5.73 (lH, d7 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).
A 1 Calctd- for C23Hl8N6o8s2Na2 3/ 2 42.92; H, 3.29; N, 13.06; S, 9.96. Found: C, 42.90, 43.19, H, 3.06, 3.01; N, 13.04, 13.03; S, 9.97.

~L~ 51~7'27 ExamDle 7 Sub~titution of the D-mande'llc acid carbox~-anhydrlde in the procedure of Example 1 of an equlmoiar weight Or the carbox~anhydrldes prepared' In similar fashion from t'he monosub~tituted D-mandelic acid~
D-2-chloro-mandellc acld, : D-3-chloro-mandellc acid, D-4-chloro-mandellc acld, D-2-bromo-mandellc acld, : D-3-bromo-mandelic acld, D-4-bromo-mandellc acld, D-2-~luoro-mandellc acld, D-3-rluoro-mandellc acld, D-4-fluoro-mandelic acid, D-2-trifluoromethyl-mandellc acid, .' D-3-trifluoromethyl-mandelic acid, D-4-trl~luoromethyl-mandelic acid, ~ D-2-amino-mandellc acld, ,: ~D-3-amlno-mandelic acld, : D-~-amlno-mandelic acld, D-2-nltro-mandellc acid, D-3-nltro-mandelic acld, D-4-nitro-mandelic acld, D-2-hydroxy-mandelic acid, D-3-hydroxy-mandelic acid, -~D-~-hydroxy-mandelic acld, ::~
, :: - 79 -, . . . . .

, D-2-methyl-mandellc acid, D-3-methyl-mandelic acld, D-4-methyl-mandellc acid, D-2-methoxy-mandelic acid, D-3-methoxy-mandelic acld and D-4-methoxy-mandellc acld respectlvely produces 7-(D-2-chloro-mandel~nido)-3-(2-carboxyrnetllyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-yltllio-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-5-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-G-ylthio-methyl)-3-cephem-4-carboxylic acid, 7-(D-3-bromo-mandelamido)-3-(2-carbox~nethyl-2,3-dihydro-s-triazolo[493-b]pyridazin-3-on-6-ylthio-methyl)-3-cephem-1~-carboxylic acid, 7-(D-4-bromo-mandelamido)-3-(2-carboxymethyl-c,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-methyl)-3-cephem-4-carboxylic acid, 7-(D-2-~luoro-mandelamido)-3-(2-carbox~nethyl-2,3-dihydro-s-triazolo~4,3-b]pyridazin-3-on-6-ylthio-methyl)-3-cephem-4-carboxylic acid, ` ~SC~7 .
.
;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-b3pyridazin-3-on-6-ylthiomethyl)-3-cepllem-4-carboxylic acid, 7-(D-3-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-4-trifluoromethyl-mandelamido)-3-(2-carboxy-meth~l-2,, dihydro-s-triazolo[4,3-b]p~rri~a.zin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid, 7-(D-2-amino-mandelamido)-3-(2-carboxymethyl-2,3-dihydro-s-triazolo[~,3-b]p~yrida.zin-3-on-G-ylthio-. methyl)-3-cephem-4-carboxylic acid, . 7-(D-3-amino-mandel~mido)-3-(2-carboxymethyl-2,3-dihydro-s-triazolo[1~,3-b~pyrida~in-3-on-~-yltllio-methyl)-3-cephem-4-carboxylic acid, . 7-(D-4-amino-mandelamido)-3-(2-carboxymethyl-2,3-¦i . 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-oll-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-yltllio-methyl)-3-cepllem-4-carboxylic acicl, .

- ~5i~7 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-carboxymethyl-2,~-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-3-on-6-ylthio-methyl)-3-cephem-4-carboxylic acid, 7-(D-2-methyl-m~ndelamido)-3-(2-carboxymetllyl-2,~-dihydro-s-triazolo[4,3-b~pyrida.zi!l-3-oll-6-yltllio-methyl)-3-cephem-4-carboxylic a.cid, 7-(D-3-methyl-mandelamido)-3-('-carboxymethyl-2,3-dihydro-s-triazolo[4,3-b]pyrida2in-3-on-6-ylthio-i methyl)-3-cephem-4-carboxylic acid, , 7-(D-4-methyl-mandelamido)-3-(2-cl.rboxymet~yl-2,3-: '.
: : dihyd~o-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-methyl)-~i-cephem-4-carboxylic aci(l,, 7-(D-2-methoxy-mandelamido)-3-(2-carbox~rrnethyl-2,3-i dihydro-s-triazolo~,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]pyrida~in-3-on-6-ylthio-methyl)-3-cephem-4-carboxylic acid, respectively.

- 82 _ SC~'727 I`` ' EXAM~LE 8 Sub8tltutlon for the D-mandellc acid carbo~yanhydride ln the procedure of Example 1 of an equlmolar weight o~ the carboxyanhydride prepared in ~imllar ~ashlon from D-2-thiopheneglycollc àcid and D-3 thlopheneglycollc ac~d respectively produces 7-(D-~-hydroxy-2-thienylacetamido)-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-a-hydroxy-3-thienylacetamido)-3-(2-carboxymethyl-2,3-dihydro-s-~riazolo~4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxyllc acid, respectively.

~X~IIPLr 9 7-(D-~- ydroxy-~-p!lenylacetamido)-3-(2-carbo~J~etIl~l-2,3-dilydro-s-tria7olo~4,3-bl~yridazin-3-on-6-ylthio-methyl~-3-cepIlem-4-carboxylic Acid ~'rer~red ~rom 7-D-MaIldel~midocenIlalospor~nic Acid.
I . .. _ _ .,.
- 0.27 Mole o~ sodium 7-D-mandelamldocephalo-I sporanate ~s suspended ln 1000 ml. of 0~1 M phosphate bur~er Or pH 6.4 and there ls added 0.31 moles of 2-carboxymethyl-2,~-dihydrO-6-mercapto-s-triazolo-. ~4.3-b]pyrida~in-3-one. The solution is heated at 55 C. under a nltrogen atmosphere for five hour3. After one hour the pH ls ad~usted to 6 4 by addltlon of a small amount of 40~ H3P04. At the end of - the five hour heating perlod the solution is cooled to 23 C. and the pH adjusted to 2 by the additlon .. .

` - 83 -. , .

~.Si~27 .
Or 3 N HCl under a layer of ethyl acetate, The product i9 extracted lnto ethyl acetate and stlrred ~or 15 mln. at 2~ C. with 2 g. o~ ("Darco KB") decolorlzlng charcoal. The mixture ls then filtered through a pad o~ diatomaeeous earth ("Celite") and the ethyl acetate removed rrom the flltrate under vacuum. The re idue is triturated to a solid with diethyl ether, collected by ~iltration and drled oYer P205 under vacuum to yield solid 7-(D-a-hydroxy-~-phenylacetamido)-3-(2-carboxymethyl-2,3-dihydro-s-tri~zolo[ 4 ,3-b~ pyridazin-3-on-6- ylthiometl~yl ) -3-cephem-4-carbo.Yylic acid.

~x ampl e 1 o H2N~L 2 5~ H_CH2CH2CODH
C2H o ~ ~/
o N ~S N
OH . ~LCH2~ CH2C~32COOII

_ `~ 11507Z7 BB-S 527; 7-lD~ Mandelamido]-3-[2-~2-carboxyeth~1)-2~3-.
dihydro-s-triazolo-[4,3-b]pxrldazin-3-on-6~1thiomethyl]-3-cePhem-4-carboxvlic acid To a miY~Ure of 7-amino-3-[2-(2-carboxyethyl)-2,3-dihydro-s-triazolo~4,3 b]pyridazin-3-on-6-ylthiomethyl]-3-cephem-4-carboxyli~ 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. ~he aqueous sclution was washed with ether (3 x 10 ml), adjusted to p}3 1 with dil. HCl and filtered to collect the crude product, which was dissolved in THF ~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-glP C-200, 10 g) and eluted with MeOII-CIIC13 ~MeOII: 0-3%). Fractions w~)ich contained the desired product were combined and evapor ated to yield 287 mg t33~) of BB-S 527. ~.p. >155C (dec.).

ir: ~KaDx 3600 - 2400, 1780, 1720, 1550, 1520 cm 1.
~pl~ 7 buffer 253 nm ( E 20000), 298 nm t max Anal. ~alc'd. for C24H22N6ogs2-3/2H2o C~ 46-98; ~
4.11; N, 13.70; S, 10.45. Found: C, 47.25, 47.39; H, 3.80, 3.76; N, 12.87, 12.77; S, 10.17.

. . .

, .

.

~S~27 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
t0~7 ml) to gi~e the clear solgtion, 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: ~max 3600 - 2800, 1770, 1710, 1600 cm 1.
pH 7 Buffer 253 nm (~ 21000~ 298 nm ( E 9 Anal. calc'd. for C24H20N6O8S2Na22~2O
3.63; N, 12.61; S, 9.62. Found: C, 43.39, 43.43: ~, 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 4B8 and cefamandole (determined by Steers' agar dilution method on Mueller-Hinton agar plate) MIC tmcq/ml) _ __ Or~anism BB-S 527 BB-S 488 cefamandole S. aureus Smith 1.6 0.8 0.2 S. aureus 0.4 0.4 0.1 . S. aureus BX-1633 3.1 3,1 0.4 ~ . .
St. faecalis `~100 >100 >100 .

E. coli NIHJ 0.4 0.2 0.05 E. coli ATCC 8739 12.5 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 .

`'` 1150~27 Xl, pneumoniae 6.3 3.1 3O1 Kl. pneumoniae 0.2 0.1 0.8 Kl. pn~umoniae 0.8 0.4 0.8 Kl. pneumoniae 0.4 0.2 0,8 ~r. vulgaris 0.1 0.1 0.2 Pr. vulgaris 12.5 0.8 50 Pr. mirabilis 0.2 O.OS 0.8 PrO mirabilis 0.1 0.05. 0.2 PrO morganii >100 ~lD0 >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 SalO enteritidis 0.05 0.025 0.05 ~! Sal. typhosa 0.1 0.05 0.1 B. anthracis 0.4 0.2 0.4 . ~ .
. .

51~727 Substitution f~r the D-mandellc acld carboxy-anhydrlde in the procedure o~ Examplelo o an equlmolar welght Or the carbox~anhydrides prepared ln slmllar ~a~hlon rrom the monosubstltuted D-mandelic acid~
D-2-chloro-mandelic acid, D-3-chloro-mandelic acld, D-4-chloro-mandellc acid~
D-2-bromo-mandellc ac ld, D-3-bromo-mandelic acid, D-4-bromo-mandellc acid, D-2-rluoro-mandelic acld, D-~-rluoro-mandelic acid, D-4-rluoro-mandellc acld, D-2-trirluoromethyl-mandellc acid, D-3-trifluoromethyl-mandellc acid, D-4-trlrluoromethyl-mandellc acld, D-Z-amlno-mandellc acid, D-3-amlno-mandelic acld, D-4-amlno-mandellc acid, D-2-nitro-mandelic acid, D-3-nltro-mandelic acld, D-4-nitro-mandelic acid, D-2-hydroxy-mandellc acld~
D-3-hydroxy-mandelic acid, D-~-hydroxy-mandellc acid~

.

- 8~ -.

.

D-2-methyl-mandeli~ acid, D-3-methyl-mandellc acld9 D-4-meth~l-mandellc acld, D-2-methoxy-mandelic acld, D-3-m~thoxy-mandelic acid and D-4-methox~-~andel~c acid re~pectively produces 7-(D-2-chloro-mandelamido)-3-(2-carboxyethyl-2~3-dihydro-s-triazolo[4,3-b~pyridazin-3-on-6-ylthio-methyl) 3-cephem-4-carboxyl~c acid, 7-(D-3-chloro-mandelamldo)-3-(2-carboxyethyl-2,3-dihydro~s-triazolo~4,3-b]pyrldazin-3-on-6-ylthio-methyl)-3-cephem-4-carboxylic acld, 7~(D-~-chloro-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-2-bromo-mandelam~do)-3-(2-carboxyethyl-2~3-dihydro-s-triazolo~4,3-b]pyridazin-3-on-6-ylthlo-; methyl)-3-cephem-4-ca~boxylic acid, :~ 7-(D-3-bromo-mandel~ldo)-3-(2-carboxy~thyl-2,3-dihydro-s-triaz~10l4,3-b]pyridazin-3-on-6-ylthio-methylj-3-cephem-4-carboxylic acid, . 7-(D-4-bromo-mandelamido)-3-(2-carboxyethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-methyl)-~-cephem-4-carboxylic acid~
7-(D-2-fluoro~mandelamido)-3-(2-carboxyetllyl-2,3-dihydro-s-triazoloL4S3-b]pyridazin-3-on-6-y-thio-methyl 3 -~-cephem 4-carboxylic acid, 15i~7Z7 .
7-(D-3~1uoro-mandelamido)-3-(2-carboxyethyl-2,3-. dihydro-s-triazolot4,~-b]pyridazin-3-on-6-ylthio_ ~ethyl)-3-cephem-4~carboxylic acid, 7-(D-4-rluoro-mandelamido)-3-(2-carboxyethyl-2~3-dihydro-s-triazolo~4,3-b]pyridazin-3-on-6-ylthio-methyl)-~-cephem-4-carboxylic acid, 7-(D-2-tri~luoromethyl-mandelamido)-3-(2-carboxy-ethyl-2~3-dihydro-s-tr~azolo[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-t 6-ylthiomethy1)-3-cephem-4-carboxylic acid, 7-(D-2-amino-mandelamido)-3-(2-carboxyethyl-2,3-dlhydro-s-triazolo~4,3-b]pyridazin-3-on-6-ylthio-I methyl)-3-cephem-4-carboxylic acid, :I 7-(D-3-amino-mandelamido)-3-(2-carboxyethyl-2,3-~ dihydro-s-triazolo[4,3-b3pyridazin-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-mandelamido~-3-(2-carboxyethyl-2,3-dihydro-s-triazolo[493-b3pyridazin-3-on-6-ylthio-methyl)-3-cephem-4-car~oxylic acid, 7-(D-3-nitro-mandelamido)-3-(2-carboxyethyl-2,3-dihydro-~-triazolo[4,3-b3pyridazin-3-on-6-ylthio-methyl)-3-cephem-4-carboxylic acid~ -.

5(~7;~7 , 7 (D-4-nitro-mandelamido)-3-(2-carboxye~hyl-2,3-dihydro-s-triazolo~4~3-b]pyridaz1n-3-on-6-ylthio-methyl)-3-cephem-4-carboxylic acid, 7-(D-2-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-3-hydroxy-mandelam~do)-3-t2-carboxyethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-methyl)-3-ceph2m-4-carboxylic acid, 7-(D-~-hydroxy-mandelamido)-~-(2-carboxy~thyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-methyl)-3-cephem-4-carboxylic acid, 7-(D-2-methyl-mandelamido)-3-(2-carboxyethyl-2,3-dihydro-s-triazolol4,3-b]pyridazin-3-on-6-ylthio-. methyl)-3-cephem-4-carboxylic acidl 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, 1~
; 7-(D-4-methyl-mandelamido)-3-(2-carboxy2thyl-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-carboxyetllyl-2,3-¦:: dihydro-s-triazolo[4,3-b]pyrida~.in-3 on-6-yltliio-methyl~ cephem-4-carboxylic acid, ~: ~7-(~-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 ~ d~hydro-s-triazolo[4,~-b]pyridazin-3-on-6-ylthio-:~ methyl)-3-cephem-4-carboxylic acid, respecti~ely.

.

115~ 7Z~

EX~MPLE 12 Substitution for the D-mandelic acid carboxyanhydride in the procedure ~f Example 10 of an equimolar weight of the c~rboxyanhydride prepared in similar ~ashion from D-2-thiopheneglycolic acid and D-3-thiopheneglycolic acid respectively produces 7-(D ~-hydroxy-2-thienylacetamido)-3-(2-carboxyethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyll-3-cephem-4-carboxylic acid and 7~
(D--hy~roxy-3-thienylacetamido)-3-~2-carboxyethyl-2~3-dihydro-s-triazolo~4,3-b]pyridazin-3-on-6-yl~hiomethyl)-3-cephem-4-carboxylic acid, respectively.

.

~ /

~. ' /
~'~ ''" /
/

` /
`~ /

.

. . . _ 92 .

EXA~IPLE 13 `OCH3 0 ~ 2 S ~ r ~ -CH2COONa 7-~(2Z)-2-Methoxyimino(fur-2-yl)acetamidol-3-(2-carboxy-methyl-2,3_dihydro-s-triazolo[4,3-b~pyridazin-3-on-6-ylthiomethyl~-3-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., 3 m.moles) at 0-5 C. and the mixture was stirred for 30 minutes. The mixture was evaporated at reduced pressure to give an oily residue of the acid chloride which was dissolved in dry acetone (10 ml.).
't ~ A~ter filtration the acetone solution was added to a mix-~¦ ture of 7-amino-3-(2-carboxymethyl-2,3-dihydro-s-triazolo-4,3-b]pyridazin-3-on-6-ylthiomethyl)-~-cephem-~-carboxylic acid (1.~1 g., 3 m.moles) and NaHC03 (504 mg., 6 m moles) in water (20 ml.) at 0-5 C. The mixture was stlrred 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) (40 ml.) and - 93 ~
.

` ~S~ 7 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[4,3-b]-pyridazin-3 on-6-ylthiomethyl)-3-cephem-4-carboxylic acid disodium salt was 1.0 g. (54%) mp ~210 C. (dec.).
ir: vmBr 1770, 1710, 1680, 1610, 1550 cm 1.
uv: ~pH mabXuffer 257 nm (E 25000), 277 nm (E 24000).

nmr: ~DMppmd6 7.7o (lH, br, furan-H~), 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. CalC'd- for C22~17N709S2Na2 2 H, 2.94; N, 15.05; S, 9.84. Fol~d: C, 40.81~ 41.02;
H, 3.0-, 3.22; N, 14.69, 14.86; S, 9.70, 9.62.

, .

.

.

_ 91~ _ 7;~7 ?

O~Co-N ~ CH2-S ~ CI~2CH2C

7-~(2Z)-2-Methoxyimino(fur-2-yl)acetamido~-3-r2-(2-car-boxyethyl)-2~3-dihydro-s-triazolo~4 ~ ridazin-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 o~

.
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 m~., 1 m.mole~ and ~aHC03 (336 mg.~
! 4 m.mole) in ~ater (10 ml.). ~he reaction mixture was stirred for 2 hours in an ice-water bath. Acetone was removed at reduced pressure. The aqueous solution was washed with ether (2 x 10 ml.) and adJusted to pH 1-2 with i conc. HCl. The resulting precipita~e was collected by filtration, washed ~ith 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). Mp. ~205 C. (dec.).

~ .

ci~27`

ir: vmBxr 3600-2800, 1770, 1710, 1600, 1550 cm 1 uv: ~pH mabXuffer 257 nm (E 27000), 276 (E 26100).
nmr: ~DMpSpmd6 D20 2.70 (2H, t, J=6 Hz, CH2)3 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, m9 furan-H), 6.92 (lH, d, J=10 Hz, pyridazine-H), 7.53 tlH, d, J=10 Hz, pyridazine-H), 7.68 (lH, br, furan-H).
Anal. Calc'd. for C23H20N709S2Na 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 ~Tsine ~uQller-Hinton A~r EY the Serial D~lu~ion Metllod eometric Mean of MIC
(l~cg./ml.) (Ex. 13) (EX. 14? Cefuroxime S. aureus (3 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 tg (b)(l)err (1~ ) 1.11 - 2.41 4.o6 . B. anthracis (1) S. pyogenes (5) 0.032 0.032 0.025 S. viridans (5) 0.15 0.4 0.1 D. pneumoniae (5) o.o37 0.056 0.0125 N. meningitidis (5) 2.37 3.60 1.6 N. gonorrhoeae (5) 1.36 1.6 0.4 H. in~luenzae (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~ _ ;~ 5~7~7 ~` ` . ,.
T~BLE B
j Geometric Means of MIC's Against 3 Strains of S. aureus and 27 Strains of Gram-negative Bacteria (mcg./ml., Mueller-Hlnton Agar) No. of t - Strains BB-S511 BL-S786 S. aureus 1 1.61.6 S. aureus, Penicillin-R 2 o.61.6 ¦ E. coli 6 0.20.2 E. coli, Cephalosporin-R 1 6.312.5 K. pneumoniae 4 o 7o 3 Indole (-) Proteus 2 0.10.2 $ Indole(+) Proteus 3 o o5 o 3 Indole(+) Proteus, Cephalosporin-R 2 6.3 50.1 S. marcescens 1 25>100 E. cloacae 1 3.1 1.6 Shigella, Salmonella 5 Q 3 0 5 P. aeruginosa 2 >100~100 BL S786 is 7-[a-(2-aminomethylphenyl)acetzmldo]-3-[(1-car-boxymethyltetrazol-5-ylthio)methyl]-3-cephem-4-carboxylic :-~ .

Geometric Means of ~IC's Against 18 ~trains . ~ - .
- ~ of S. marcescens BB-S511 B~-S786 5.4 85.9 ~j ' ' .
~ :

.

. ~
_ 97 _ ., .

~ 5~17~7 EXAI~P~E 15 . Substitution of an equimolar weight of 2-ethox~-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,~-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid and 7-(2-ethoxy-lmino-2-furylacetamido)-3-[.2-(2-carboxyethyl)-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl]-3-cephem-4-carboxylic acid, respectively.

Substitution o~ an eauimolar ~-eight 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-~ropoxyimino-2-furyl ac etamido)-3-(2-carboxy-methyl-~,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl) 3-cephem-4-carboxylic acid and 7-(2-n-propoxy-: . imino-2-furylacetamido)-3-[2-(2-carboxyethyl).-2,3-dihydro-s-triazolo[4,3-b]pyridazin-~-on-6-Ylthiomethyl]-3-cephem-4-carboxylic acid, 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 ~xamples13 ~nd 14 produces 7-(2-n-butoxyimino-2-furylacet2mido)-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-butoxy-imino-2-furylacetamido)-~-[2-(2-carboxyetl~yl)-2,3-dihydro-s-triazolo[4,3-b~pyridazin-~-on-6-ylthiomethyl]-3-cephem-4-carboxylic acid, respectively.

, The products o~ Examples 13-17 are prepared 3S Sy~
lsomers essentially ~ree of the corresponding anti isomers by the use ln the procedures of those examples of purified syn isomers of the appropriate 2-alkoxyimino-2-(fur-2-yl)-ncetic acid. Conversion of part of the syn isomer to anti isomer during preparatlon of the ac~d 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 treatlng that salt with oxalyl chloride under anhydrous conditions in the presence of a hydrogen ion acceptor such as dimethylformamide.
Such syn isomer~ are a~so n~m~d as ~2z)-2-alkoxyimino-2-(fur-2-yl)acetic acids.

_ ~9 _ .

¦ "
~ 50727 .

An ln3ectable pharmaceutlcal compo~ltion ls formed by adding sterile water or sterile saline solu-tion ~2 ml.) to 100-500 mgm. of 7-~(2Z)-2-methoxyimino-(fur-2-yl)acetamido]-~-(2-carbox~methyl-2,3-dihydro-s-- triazolo[4,~-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid disodium salt.
Pharmaceutical composltions of the sodium and potassium salts o~ the other compounds of the present invention, preferably in the form of the pure -~ syn isomer, are formulated in a similar m~nner.
When the compounds are first prepared in the form o~ the free acid they are converted to the deslred, highly water soluble potassium salt by treat-`
ment with potassium 2-ethylhexanoate using the procedure - of Example 13.
It is occasionally advantageous to have ad-mixed with sald solid cephalosporin as a stabilizing and/or solubilizing agent a sterile, anhydrous solid `:
6uch as sodium carbonate, potassium carbonate or lithium carbonate(e.g. in about 5 or 6 percent by weight of the welght of the cephalosporin) or such as L-lysine, argi-` nlne or histidine (e.g. in about 20-50% by weight of the weight ol the cephalosporin) or such as a sodium, potassium or ealcium salt Or levulinic acid, cltric acid, ascorbic acid, tartaric acid or pyruvic acid (e.g. in about 25-200% by weight o~ the welght of the cephalos-porin) or such as sodium bicarbonate, ammonium car-bamate alkali metal or ammonium phosphates or N-methyl-BluCamine (per U.~. 1,380,741).

: i ,r~ 20 ~,' ' ' "~
C0-N ~ S ~ ~ N
Il~ o~N~CH2S~N~N~N_(~H3 BB-S510; 7-~(2Z¦-2-~ethoxyimino(fur-2-yl~acetamido]-3-L2-methyl-2?3-dillydro-s-triaæolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic Acid.Sodium Salt.
To a solution o~ (2Z) 2-methoxyimino(fur-2-yl)-acetic acid (253 mg., 1.5 m.mol.) and triethylamine (0.2 ml., 1.5 m.mol.) in dichloromethane (~ ml.) was added oxalyl~chloride (0.13 ml., 1.5 m.mol~) at 0-5 C. and the mixture was stirred for 30 minutes and evaporated at reduced pressure to give the acid chloride as an oil which was dissolved in dry acetone (5 ml.) and riltered to remove insolubles. The acetone solutlon was added to a`mixture o~ 7-amino-3-(2-methyl-2,~-dihydro-s-triazolo-4,3-b]pyridazin-3-on-6-ylthiomethylj-3-cephem-4-car-boxylic acid (591 mg., 1.5 m.mol.j and NaHC03 (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. hcetone was removed at reduced pressure and the residual aqueous soIution was washed with ether (2 x 30 ml.) and adjusted .
to ~II 1-2 with concentrated HCl. The precipitate which was collected by filtration, washed with water and dried :
in vacuo, was dissolved in THF (30 ml.) and filtered to remove insolubles To the THF solution was added a solu-tion of sodium 2-ethylllexanoate (SEH, 1 I~I~ 1.5 ml.) in .

5~7;~7 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-t~iazolo[4,3-b]pyridazin-3-on-6-ylthio-methyl)-3-cephem-4-carboxylic acid sodium salt (58~).
M.p. ~180~ C. (dec.).
ir: ~KBax 1770, 1720, 1670, 1600, 1550 cm 1 uv: ~PH7muaxfer 257 nm (E 22600), 277 nm (E 22300).

nmr: ~ ~ 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 (lH3 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-C~ ), 3 61 (3H, s~ N-C_3)-f r C21H~.gN707S2Na 1/2THF H20 C, 44.44; H, 3.89; S, 10.32. Found: C, 44.89~ H, 3.g2;
S, 9.67.

.. . . .. .. ... . . .. ~

~LS~27 .

In vitro Activ ty Using Mueller-Hinton Ag~r By he Seria Dl uti~n Ir~
~eomeF~ of MIC
(MCgO/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 ~1. 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.~6 4.o6 B. anthracls (1) S. pyogenes (5) 0.0125 0.032 0.025 S. viridans (5) 0.13 0.59 0.1 D. pneumoniae (5) 0.021 0.1 0.01c5 N. meningitidis (5) 1.03 5.45 1,6 N. gonorrhoeae (5) 0.26 2.07 0.4 H. influenzae (7) 0.35 2.11 1.16 Cefuroxime is sodiwn 6R~7R-3-carbamoyloxymethyl-/-(2Z)-2-methoxyimino(fur-2-yl)acetamidoceph-3-em-4-carboxylate.

; ::

. . .

.

~l 1 A~ 7 2 7 TABL~ 11 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-S510 BL-S785 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 3 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 9L-~786 is 7-[~-(2-aminometllylphenyl)acetamido~-3-~tl-c2r-boxymethyltetrazol-5-ylthio)methyl~-3-cephem-4-carboxylic acid.

~. ~
. -Geometric Means of ~IC's Against 18_Strains -of S. marcescens 7.9 85.9 .

.

. - 104 -.

i. : i ' . ` !
~ ~5~727 EX~PLE 21 Substitution of an equimolar 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-. ` . 6 -triazolo[4,7-b~pyridazin-3-on-6-ylthiomethyl)-~_cephem-4-carboxylic acid.

.
` EXAMPLE 22 Substitution of an equimolar weight of 2-n-propoxy-lmino-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-metllyl-2l3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-F 3-cephem-4-carboxylic acid.

XAI;~PLE 23 ~ 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 procedure of Example 20 produces . 7-(2-n-butoxyimino-2-furylacetamido)-~-(2-methyl-2,3-dihydro-s-triazolo[4,3~b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid.

.

~ ` llS~27 .
.

EXA~PLE 24 The products of Examples 20-23 were prepared as s~rn isomers essentially free of the corresponding anti isomers by the use in the procedures of those examples of purified s~n lsomers of the appropriate 2-alkoxyimino-2-(fur-2-yl)-acetic acid. Converslon of part o~ the syn isomer to anti isomer durlng preparation of the acid chloride from the acid ls substantially avoided by minimizing its exposure to hydrogen chloride, e.g. by first converting the acid to its anhydrous sodium salt and by treatlng 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.

.'~ , ~, ' .

o ` ' ' ' .

:

~5~ 7 ~ 25 An ln~ectable pharmaceutical co~po~ition i6 ormed by addlng sterile water or sterile saline solu-tlon (2 ml.) to 100-500 mgm. of 7-[(2Z)-~-methoxyimino-(fur-2-yl)acetamido]-3-(2-methyl-2,~-dihydro-s-triazolo[4,3-b]pyridazin-~-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid disodium salt.
Pharmaceutical compositions of the sodlum and potassium salts of the other compounds of the present inv~ntion, preferably in the f~Im of the pure lsomer, are formulated ln a similar manner.
When the compounds are first prepared in the form of the ~ree acid they are converted to the desired, highly ~ater soluble potassiwn salt by treat-ment with potassium 2-ethylhexanoate using the procedure - of Example 20.
It is occasionally advantageous to have ad-mixed with said solid cephalosporin as a stabilizing and~or solubilizing agent a sterile, anhydrous solld such as sodium carbonate, potassium carbonate or llthium carbona~e(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~o by weight of the weight ot the cephalosporin) or such as a s~dium, 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. 1,380~741).

:

Claims (2)

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

1. A procecs for the preparation of a compound having the formula wherein A1 is methyl or -(CH2)n COOH and n is one or two;

comprising reacting a compound of the formula wherein A2 is methyl or -(CH2)nCOOH or an easily hydrolyzable ester thereof and n is one or two, with NaSH.

2. A compound having the formula wherein A1 is methyl or -(CH2)nCOOH and n is one or two, whenever prepared or produced by the process of Claim 1 or by an obvious chemical equivalent thereof.