. TXI-PY-CEPH CO~i~O
(SY-1510-A)
11~7'7'~
me present invention relates to novel acids
havin~ the structure
A~-CH ~ ~C~2
f ~o N-Al
COOH
.
sometimes hereinafter also written as
~=N
~N~f~ CH2S-J~ J--A1
COOH
herein A is
R - IH - C - or~ C ~ C -
~1
. . ~ op,2
wherein Rl is hydrogen or formyl,
R is
. ~ . or
Y
and Y ~s hydrogen, chlorine, bromine, fluorine, trifluoromethyl,
amino, nitro, hydroxy, (lower)alkyl of 1-4 carbon ato~s or
(lower)alkoxy of 1-4 carbon atoms,
R2 is (lower)alkyl of 1-4 ca.rbon atoms; and
~herein Al is methyl or -(CH2)nCOOII and n is one or tuo;
_
11~77'~4
the easily hydrolyzed esters and.the non~toxic, p~armaceu-
tically acceptable salts of those acids.
Said easily hydrolyzed esters Or the acids of
formula I include those having the group of the formula
~J
-C~
wherein when W represents hydrogen, Z represents (lower)-
alkanoyl, benzoyl, naphthoyl, furoyl, thenoyl~
nltrobenzoyl, methylbenzoyl, halobenzoyl~ phenyl-
benzoyl, N-phthallmldo, N-succlnlmldo, N-saccharlno,
N-(lower)alkylcarbamoyl, (lower)alXoxy, (lo~Yer)-
alkylthlo, phenoxy, carbalkoxy, carbobenzoxy,
carbamoyl, benzyloxy, chlorobenzyloxy, carbophenoxy,
carbo-tcrt,-buto~y or (lower)alkyl~ulronyl, and ~hen
W represent~ carbalkoxy, Z representQ carbalkoxy and,
when W represents phenyl, Z represents benzo~l or
cyano or whereln W and Z taken together represent
2-oxocy¢loal~yl contaln~ng 4 to 8 carbon stom8
lncluslve,
As set rorth below ln more detall the present
lnventlon also provlde~ salts Or these acids, The
~tereocheml8try Or the blcycllc nucleu~ 18 that
~ound ln Cephalosporln C.
A preferred embodiment of the present in~ention
consists of the acids having the D configuration in the 7-
æide chain and the formula II
1147724
o ~S
R-lCH-d-NH-CH- CH \ ~CH2 ~ ~ II
OR ~ C--N ~C~CH2 S ~ N, ~ N~(CH~)nC2H
COOH O
wherein n is one or two and Rl is hydrogen or formyl
~nd R is
~ or ~
and Y is hydrogen, chlorlne, bromine, fluorine,
trlfluoromethyl, amino, nitro, hydroxy, lower-
alkyl or 1-4 carbon atoms or lower alkoxy of 1-4
carbon atoms and the nontoxlc, pharmaceutically
acceptable salts of those acids and the easily
hydrolyzed estçrs of thoæe aclds lncludlng
especlally the plvaloyloxymethyl, acetoxymethyl,
acetonyl, phenacyl and methoxymethyl esters and
the 811yl esters such a~ the trlmethylsllyl ester.
. A further pre~erred embodiment of this invention
consists of the compounds of formula II, ~rherein
R is 2-thienyl, 3-thienyl, phenyl, chlorophenyl, bromophenyl,
trifluoromethylphenyl, tolyl or methoxyphenyl.
Particularly preferred embodiments of this
invention comprises the acids havin~ the D confi~uration
in the 7-sidechain and the formul~
CH-C-NH-CH - CH CH2 ~ ~l
o~/ ~ C ~ 2 N ~ N-(CH2~nC02H
COOH O
1~4'~724
wherein n is one or two and tlleir nontoxic, plla~n<aceuticall~
acceptable salts and easily hydrolyzed esters.
Also included in this invention are the compounds
(used as either intermediates or metabolic precursors)
in which the ~-hydroxy group is "blo~ked" by substituents
~uch a6 dichloroacetyl (U.K. 962,021~ and U. K. 1,328,340),
formyl (U.S. 3,6~1,021), trimethyls~lyl or tetrahydro-
pyren~l (U.K. 1,32~,340).
There is also provided ~y the present invention a
compound having the fol~ula
O ~S
R-CH-C-NH-CH - CH ll~2 ~ ~
OR O~C N\ C - CH2 - S - ~N,N~N lCH2) n 2
COOM
whereln n $s one or two, R is hydrogen or formyl and R is
~ or ~
and Y $s hydrogen, chlorine, bromine, fluor$ne, tr$fluoro-
methyl, amino, nitro, hydroxy, lower alkyl of 1-4 carbon
atoms or lower alkoxy of 1-4 carbon atoms and M is
-ICHOC(C~2)nR' -CIHOC(C~2)nCl R8
.
11~7724
o . o
- CHX~OR or - CH-S-C-R6
Rl 11
n ~s O to 4; R is hydrogen, alkyl having 1 to B carbon
atoms, cycloalkyl of 3 to 6 carbon atoms, phenyl, Cl-C4
phenalkyl, pyridyl, thienyl, or pyrrolyl; R2 is hydrogen,
methyl or ethyl; R7 and R8 are each hydrogen, alkyl having
1 to 6 carbon atoms, phenyl, pyridyl, or thienyl; R4 and RS
are each hydrogen or alkyl of 1 to 4 carbon atoms; R6 is
al~yl having 1 to 4 carbon atoms, phenyl, phenalkyl having
1 to 4 carbon atoms, pyridyl, thiadiazolyl, amino or Cl-C4
alkylamino; X is N~i or oxygen; and each phenyl group is
unsubstituted or ~ubstituted with one or two substituents
selected from the group consisting of alkyl having 1 to 6
carbon atoms, alkoxy having 1 to 4 carbon atoms, hydroxy,
amino, NHRl, N(Rl)2, nitro, fluoro, chloro, bromo or carboxy,
or a nontoxic, pharmaceutically acceptable salt thereof.
There is also provided by the present invention a
compound hav~ng the formula
R-CH-C-NH-CH - CH / CH2 ~ S
1R1 "C . N\ ~ 2 N~b~N ( 2) nCO2H
COOM
wherein n iS sne or two, R is hydrogen or for~yl and R 15
~ or ~
i~7724
and Y is hydrogen~ chlorine, bromine, fluorine, trifluoro-
methyl, amino, nitro, hydroxy, lower alkyl of 1-4 carbon
atoms or lower alkoxy of 1-4 carbon atoms and M is selected
from the group consisting of
CH3 O
- CH - O - C - R6
1 2H5
- CH - C - R
R5 0
~ CH _ x2 _ C - oR7
wherein R5 i~ a hgdrogen atom, ~ methyl or an ethyl group;
x2 18 -0-~ -NH-; R6 i8 a basic group.such as alkyl or aralkyl
8ubstituted wlth substituted or unsubstituted NH2~ 5uch
a8 alkyl-NHCH3, aralkyl-NHCH3,
alkyl-NH~),
aralkyl-NH ~ ~ -IH ~ ~ -CH2NH2~ ~IH-CH2
NH2 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 aralkyl group such as benzyl or
naphthylmethyl; a heterocyclic group and wherein the alkyl,
cycloalkyl, aryl, aralkyl and heterocyclic groups may be
substituted with one or more groups selected from the class
consisting of amino groups, substituted amino group5 such
as methylamino f diethylamino or acetamido groups, the halogen
77Z~
groups such as fluorine, chlorine or bromine, nitro groups,
al~oxy groups such as methoxy, ethoxy, propyloxy, isopropyloxy,
butoxy or isobutoxy; or a nontoxic, pharmaceutically acceptable
~alt thereof.
There is also provided by the present invention a compound
having the formula
O ~S
R-CN-C-N~I-CH - CH C 2 ~ 1
IR1 ~ - N \ ~ 2 ~ N~ ~ N (CH2)nC02H
COO~I
wherein n is one or two, R is hydrogen or formyl and R is
~ or ~
and Y is hydrogen, chlorine, bromine, fluorine, tri~luoro-
methyl, amino, nitro, hydroxy, lower alkyl of 1-4 carbon
~tom6 or lower alkoxy of 1-4 carbon atoms and M is
/C--Y
-CB -N
wherein Y is alkyl of one to six carbon atoms, phenyl,
benzyl, alkoxy of one to six carbon atoms, or benzyloxy
Z i~ alkyl of one to six carbon atoms, phenylbenzyl,
alkoxy of one to six carbon atoms, cyclopentyl, cyclo-
hexyl and phenyl, or Y~Z taken together are a 3-benzoxa-
zolidine ring; or a nontoxic, pharmaceutically acceptable
salt thereof.
1147~2~
- Also included ~lithin the present invention are
pharmaceutical compositions comprising a mixture of an
antibacterially effective amount of 2 COmpOUl)d 0~ the
present invention and a semisynthetic penicillin or another
cephalosporin or a cephamycin or a ~-lactamase inhibitor
or an aminoglycoside antibiotic,
In the treatment of bacterial infections in man,
the compolmds of tllis invention Or fol~ula II are administered
parenterally, in accordance with conventional procedures for
antibiotic administration, in an amount of from about 5
to 200 mg,/kg,/day and preferably about 5 to 20 mg./kg./-
day in di~ided dosage, e,g. three to four times a day.
m ey are administered in dosage units containin~, for
example, 125, 250 or 500 mg, of active ingredient with
suitable physiolo~ically acceptable carriers or excipients,
m e dosage units are preferably in the fo~n of liquid
preparations such as solutions or suspensions.
Another preferred embodiment of this invention
comprises the acids havin~ the forrnu]a III
2 ~ ~J
R2 1I N~ c~C-C~2S N~ ~ Al III
COO~
herein Al is methyl or -(CH2)nC0~ and n is one or two
and ~2 is alkyl containing 1-4 carbon atoms)
the easily hydrolyzed esters and the non-toxic pharmaceutically
acceptable salts of those acids as hereinbefore set forth.
The compounds of formula III Or the present
in~ention are syn isomers or else are mixtures of syn and
anti isomers containing at least 75$ of the syn isomer,
Preferably such mixtures of isomers contain at least
90~ of the syn isomer and not more than 10~" of the anti
1~77~4
isomer Most preferably the compounds of formula III are
syn isomers essen~ially free of the corresponding anti
isomer.
~ le preferred embodiments of the present invention
are the syn isomers of the compounds of Formula III
wherein R2 is methyl or ethyl, n is one or two in its
acid or pivaloyloxymethyl, acetoxymethyl, methoxymethyl,
acetonyl, phenacyl, p-nitrobenzyl, ~ trichloroethyl,
3-phthalidyl or 5-indanyl ester for3n.
~ eference to the syn (cis) isomeric form
refer~ to the configuration of the group oR2 ~rith respect
to the carboxamido group.
There is also provided by the present inven-
tion a compound having the formula
~Lc - C - NH-CIII CIH ICH2 ~=NI
;C N~ "C-C~12-S ~N~N~ - (CH2)nCOOH
C - OR~
wherein R~ 18 alkyl contalning 1-4 carbon atoms, n ls
one or twoand R3 ls selected from the group
conslstlng of
CH~ O
- CH - O - C _ R6
1 2 5 11 6
- CH - C - R,
75 1l
- CH - X - C - OR
_ g _
~7724
wherein R is a h.ydrogen atom, a methyl or an ethyl group;
x2 18 -0-, -NH-; R is a basic group such as alkyl or aralkyl
substituted with substituted or unsubstituted NH2, such
as alkyl-NHCH~, aralkyl-NHCH3,
alkyl-NH ~ ,
aralkyl-NH ~ ~ -~H ~ ~ -CH2NH2~ -IH-CH2
NH2 NH2
R7 ls an al~yl group such as a methyl, ethyl, propyl,
lsopropyl, butyl, isobutyl, pentyl or 2-ethyl-hexyl group;
a cycloalkyl group such as cyclopropyl, cyclobutyl, cyclo-
pèntyl, cyclohexyl or cycloheptyl; an aryl group such as
phenyl or naphthyl; an aralkyl group suCh as benzyl or
naphthyl~ethyl; a heterocycllc group and whereln the
alkyl, cycloalkyl, aryl, aralkyl and heterocyclic groups
may be substitUted wlth one or more groups seleCted from
the class cons~stlng of amino groups, sUbstltuted amlno
groups such as methylamino, diethylamino or acetamido
groups, the halogen groups such as fluorine, chlorine or
bromine, nitro groups, alkoxy groups such as methoxy,
ethoxy, propyloxy, isopropyloxy, butoxy or isobutoxy;
or a nontoxlc, pharmaceutically acceptable salt thereof,
said compound being at least 75% by weight in the form of
lt6 ~ isomer and preferably ln the form of its syn
isomer essentially free of the corresponding anti lsomer.
-- 10 --
77Z4
There is also provided by the present inven-
tion a compound having the formula
C - C - NM-C~--CH ICH2 ~ NI
N_o~ C N C,C CH2 s N,N~ - (CH2)nCOOH
l_OM Q
whereln R~ i5 alkyl containing 1-4 carbon atoms, n i~
one or two and M is
/C -Y
CH2 N\
Z
whereln Y is alkyl of one to six carbon atoms, phenyl,
benzyl, alkoxy of one to six carbon atoms, or benzyloxy;
Z 18 alkyl of one to æix carbon atoms, phenylbenzyl,
alkoxy of one to six carbon atoms, cyclopentyl, cyclo-
hexyl and phenyl, or Y~Z taken together are a ~-benzoxa-
zolidine ring; or a nontoxlc, pharmaceutically acceptable
salt thereof, said compound belng at least 75% by welght
ln the form of its syn isomer and preferably in the form
o~ lts ~ isomer essentially free or the corresponding
anti lsomer.
There is also provided by the present inven-
tion a compound having the formula
o
C - C - NH-CII CIH ICH2 ~ N
N_ oR2 ~C N C'5 2 S ~,N ~ - CH
lC-o~ O
.. O
~772~
wherein R2 is alkyl containing 1-~ carbon atoms and ~1 is
~Ho3 ( c~l2) nR ~ ~CI HOC ( CH2 ~ nCI~B 8
NR R5
O O
-CHXCOR or - CH-S-C-R
n ls O to 4; R is hydrogen,.alkyl having 1 to 8
carbon atoms, cycloal~yl of ~ to 6 carbon atoms,
phenyl, Cl-.C4 phenalkyl, pyridyl, thienyl, or pyrrolyl;
Rl ls hydrogen, methyl or ethyl; R7 and R8
are each hydrogen, alkyl having 1 to 6 carbon atoms,
phenyl, pyridyl, or thienyl; R4 and R5 are each
hydrogen or alkyl of 1 to 4 carbon atoms; R ls alkyl
havlng 1 to 4 carbon atoms, phenyl, phenalkyl having
1 to 4 carbon atoms, pyridyl, thiadiazolyl, amino or
Cl-C4 alkylamino; X is NH or oxygen; and each phenyl
group is unsubstituted or subs~ltuted with one or two
sub6tltuents selected from the group consisting of
alkyl having 1 to 6 carbon atoms, alkoxy having 1 to
4 carbon atoms, hydroxy, amino, NHRl, N(Rl)2, nitro,
fluoro, chloro, bromo or carboxy, or a nontoxlc,
pharmaceutlcally acceptable salt thereof, sald com-
pound being at least 75% by weight in the form of lts
syn lsomer and preferably ln the form of lts syn
i~omer essentially free o~ the corresponding anti isomer
~1~77'~
There is also provided by the present inven-
tion a compound having the formula
C - C - NH~ CH CH2 ~ N
N_ OR~ ~C- N C~5 CH2 S N~ ~ ~ ~ CH~
C - oR3 o
herein ~ 1~ alkyl containing 1-4 carbon atoms
and R3 is celected from the group
con61stlng of
CH3 0
- I H - O - 11 R6
C 2H5 11
- CH - C - R ,
R5
- CH _ x2 _ C - oR7
whereln R5 18 a hydrogen atom, a methyl or an ethyl group;
x2 15 -O-, -NH-; R is a basic group such as alkyl or aralkyl
substituted with substituted or unsubstituted NH2, such
as alkyl-NHCH3, aralkyl-NHCH3,
alkyl-NH ~ ,
aralkyl-NH ~ f ~ CH2NH2 -fH-CH2~
NH2 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
1~7724
phenyl or naphthyl; an aralkyl group such as benzyl or
naphthylmethyl; a heterocycllc group and wherein the
alkyl, cycloalkyl, aryl, aralkyl and heterocyellc groups
may be substltUted with one or more groUps selected from
the class conslstlng of amlno groups, subQtltuted amino
groups such as methylamino, diethylamlno or acet~mido
groups, the halogen groups such as fluorlne, chlorlne or
bromlne, nitro groups, alkoxy groups such as methoxy,
ethoxy, propyloxy, isopropyloxy, butoxy or lsobutoxy;
or a nontoxic, pharmaceutlcally acceptable salt thereof,
sald compound belng at least 75~ by welght in the form of
its syn lsomer and preferably in the form of lts syn
lsomer essentially free of the corresponding anti lsomer.
There ls also provided by the present lnvcn-
tlon a compound havlng the formula
o
C - C - NH~ CIH ICH2 ~ Nl
N_ oR2 ~C N_ C~ 2 N~ ~ - CH~
~-OM O
wherein R iS alkyl containing 1-4 carbon atoms and M is
~C -Y
CH2 N\
~ "
1~772~
wherein Y is alkyl of one to six carbon atoms, phenyl,
benzyl, alkoxy of one to six carbon atoms, or benzyloxy;
Z is alkyl of one to six carbon atoms, phenylbenzyl,
alkoxy of one to six carbon atoms, cyclopentyl, cyclo-
hexyl and phenyl, or Y+Z taken together are a 3-benzoxa-
zolidine ring; or a nontoxlc, pharmaceutically acceptable
salt thereof, said compound being at least 75~ by weight
ln the form of lts syn isomer and preferably in the form
of its syn isomer essentially free OI the corresponding
anti lsomer.
-- , . _ . . . . .
In the treatment of bacterial lnfectlons
in man, the compounds of this inventlon are admlnls-
tered parenterally in an amount of from about 10 to
90 mg./kg /day and preferably about 14 to 50 mg./kg /day
in dlvided dosage, e.g. two to four tlmes a day. They
are admlnlstered ln dosage units contalning, for example,
125, 250 or 500 mg. of actlve lngredient wlth sultable
physlologlcally acceptable carrlers or exceplents.
The dosage unlts are ln the form of liquld preparatlons
such as solutions or suspensions and preferably are
aqueous solutlons of a sodium or potasslum 6alt which
are in~ected lntravenously or intramuscularly or by
continuous or lntermlttent lnfuslon in concentratlons
of about 125-500 m~m./ml., and preferably, 250 mgm./ml.
as ls customary in therapy wlth cephalosporln antlbi-
otics.
It was an unexpected finding that the leading
compound of the present invention (BB-S510; see below)
having a 2-methyl substituent on the trlazolopyridazinone
showed _ vitro antibacterial potency considerably
superior to that of the corresponding compound lacking
such methyl group (BB-S515; see below).
~7'7~
A further embodiment of this invention comprises
a compound having tlle formula
HS - ~N~
wherein A~ is methyl or -(CH~)nCOC~
where n is one or t~o~
The present invention also provides the process
for the production o~ the antibacterial a~ents of formula I
which comprises reaeting a compound of the formula
~S~
~l2~-lH C~l ~2 ~ N
C - -N~ ~jC-CH 2 -S ~ N,N ~ Al IV
COO~
where Al is as hereinbe~ore defined or a salt or ea6ily hydroly~ed
e8ter or Schiff base ~B wi~h benz~ldehyde or ~llcyl~ldehyde
thereof (including, but not lLmited ~o, tho~e of U.S.
3,284~451 and U~K. 1~229~45~ and any o~ the
811yl e~ter~ de3crlbed in U.S, patent 3~249,622
ror u~e with 7-amlnopenlcillarllc acld and u~ed
ln &reat Brltaln 1JO73~530 and par~cularly the
pivaloyl~xymethyl, acetoxymethyl, methoxymethyl,
a¢etonyl, phenacyl, p-nltrobenzyl, ~ trl¢hloroethyl,
3-phthalldyl and 5-lndanyl esters) thereof with an
org~nic monocarboxyllc acld chlorlde or a functional
e~ulvalent thereor aa an acylating ~gent. Compound of ~ormula IV
comprising reacting 7-amino-cephalosporanic acid or a salt or
easily hydrolyzable ester thereof with a com-
pound of the formula
N
~-S - N,N ~ N-A
~` 1
where A is as defined above.
- 16 -
772~
Such functlonsl equivalents lnclude the
corresponding ecid anhydrides, including mixed
anhydrldes end partlculsrly the mixed flnhydrites
preperet from ~tronger acids such es the lower
allphatlc monoesters of csrbonic ecid, or alkyl
and sryl ~ulfonic acids snd of more hindered
acids such as diphenyl~cetic ecid. In eddition,
sn scid azidé or an activè éster or thioester (e.g.
with p-nitrophenyl, 2,4-dinitrophenol, thiophenol,
thioecetic acid) m~y be used or the free acid itself
mey be coupled with compound IV sfter flrst re~cting
~eid free scid with N,N'-dimethylchloroformlminium
chloride lcf. Great Britsin 1,008,170 and Novsk
and Weichet, Experlentls XXI, 6, 360 (1965)l or
by the use of enzymes or of an N,N'-csrbonyl-
dllmldazole or sn N,N'-c~rbonylditriazole [cf.
South Afsicen petent speciflcetion 63/2684l or
8 csrbodiimide reegent lespecislly N,N'-dicyclohexyl-
cerbodlimide. N,N'-dii~opropylcsrbodiimide or N-cyclo-
hexyl-N'-(2-morpholinoethyl)carbodlimlde; cf, Sheehen
end Hess, J, Amer, Chem, Soc " 77, 1967 (1955)~, or
of alkylylamine reagent lcf. R, Bui~le and H,G.
Ylehe, An~ew, Chem International Edltion 3, S82,
~7724
(1964)] or Or an lso%asollum salt reagent ~cr. R. ~,
Woodward, R. A. Olorson and H. Mayer, ~ Chem~
Soc~ , 1010 (1961)1, or Or a ketenlmlne resgent
[cr. c. L. Stevens and M, E. Munk, J. Amer~ Che~ ~oc,.
~, 4065 (1958)~ or Or hexachlorocyclotrlphosphatrlaz~ne
or hexabromocyclotrlphosphatriazlne (U,S. 3,651,050) or
o~ diphenylphosphoryl azlde [DPPA; ~, Amer. Chem. ~55~J
44, 6203-6205 (1972)] or of dlethylphosphoryl cyanlde
~EPC; Tetrahedron Letters No. 18, pp. 1595-1598 (1g73)]
or o~ dlphenyl phosphlte ~Tetrahedron Letters No. 49,
pp. 5047-5050 (1972)]. Another equlvalent o~ the acld
ohlorlde 18 a correspondln~ azollde, l.e., an amlde Or
the correspondlng acld whose amlde nltroeen 19 a member
Or a quaslaromatlc flYe mem~ered rln~ contalnlng at
least two nitrogen atoms, l.e., l~ldazole, pyrazole,
the trlazoles, benzlmidazole, benzotrlazole and thelr
substltuted derlvatlves. As an example Or the general
method ror the preparatlon Or an azollde, N,N'-carbonyl-
dl~mldazole i9 reacted wlth a carboxyllc acld ln
equ~molar proportlons at room temperatre in tetrs-
hydroruran, chlorororm, dlmethylrormamlde or a ~imllar
lnert solvent to ~orm the carbo~ylic acld lmldazollde
ln practlcally quantltatlve yleld wlth llberatlon o~
carbon dloxlde and one mole o~ ~mldazole. Dicarboxyllc
aclds yleld dlmldazollde. qhe by-product, lmldazole,
preclpltstes and may be separated and the lmldszollde
~olated, but thls ls not essentlal. The ~ethods for
carrying out these reactlons to produce a cephalo~porln
and the methods used to lsolate the cephalo~porln ~o
produced are well known ln the art.
~14772~
Ment~on ~ac made above Or the u~o o~ enzymes to
couple the ~ree acld wlth
compound IV. Inoluded in the scope Or 8uch processe~
are the u~e Or an e~ter, e.g. the methyl ester, Or
that rree acld w~th enzymes provlded by varlou~ mlcro-
organl~m~? e.g. those descrlbed by T. Takahashl et al.,
J, Amer~ Chem. Soc., ~4(11~, 40~5-4037 (1972) and by
T, Nar~ et 81 " J. Antlblotlcs (Japan) 24~), 321-~23
(1971) and ln U.S. ~,682,777.
~ or the coupllng of the orgsnlc csrboxylic ~c~d ~
de6cribed above with compound IV (or a salt or preferably
~n e~sily hydrolyzed ester of Schiff bsse, 88 with
benzaldehyte, thereof) lt iB 81BO convenlent and effic~ent
to utilize a8 the coupling ~gent phosphonitrilic chloride
trimer (J. Org. Chem., 33(7), 2979-81, 1968) or N-ethoxy-
1,2-dihydroquinoline (EEDQ) ss deccribed in J. Amer. Chem.
Soc., 90, 823-824 and 1652-1653 (1968~ snd U.S. P~tent
3,455,929. The resction i8 preferably c~rriet out ~t 30-
35C. in benzene, eth~nol or tetrahydrofursn w ing ~bout
equlmolar qusntitles of all three reagents followed by
conventionsl isolation and re~ov~l by conventlon~l methot~
of any blocking groups present.
One process of the present in~ention stated
more specifically ~s the process for the preparation of
a product having the D-configuration in the sidechain
and.the formula
CH-CONH ~ ~ ~r
1H ~ N ~ CH2S N~ ~ N (CH2)c2
COOH O
7724
wherein n is one or two or a salt thereof which comprises
the consecutive steps of
a. preparing an acylating derivative of D-mandelic
acid having the formula
CH-COOi-l
01
wherein the hydroxyl blocking group R represents di-
chloroacetyl, silyl and preferably trimethylsilyl, tetra-
hydropyranyl or, preferably, formyl in an anhydrous or-
ganic solvent such as benzene, ethanol or preferably
tetrahydrofuran, at room temperature or below and prefer-
ably at about 5~ CJ
b mixing therewith, preferably slo~ly, a solution
at about the same temperature in a solvent, preferably
aqueous.tet~ahydrofuran, containing substantially the
same number of moles of a tertiary amine, preferably a
tertiary alkylamine such as triethylamine and substan-
tially the same number of moles of 7-amino-3-(2-carboxy-
methyl or 2-carboxyethyl-2,3-dihydro-s-triazolol4,3-b]-
pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid
or a salt or an easily hydrolyzed Schiff base, as with
benzaldehyde, thereof to produce the intermediate acid
having the formula
CH-CONH ~ ~ ~r
1R ~N~ 2S N~ ~N-(CH2)nCO2H
COOH O
wherein n is one or t~ or a salt ther~of wher~in ~ has
the meaning set out above and
c. removing said hydroxyl blocking group R by con-
ventional c~emical methods to produce said product or
salt thereof.
~1477Z~
In preferred embodirnents of the present inven-
tion R represents formyl whic~l is removed in step C by
treatment ~rith aqueous alkali such as aqueous sodium
bicarbonate or R represents dechloroacet-Jl ~?hich is removed
in step C by alkaline hydrolysis, preferably at about
pH 9-10, or ~ represents trimethylsilyl ~Ih:l.ch is removed
in step C by exposure to aqueous acid,
Other compounds of formula II are made in like
manner,
An additional process of the present invention
comprises the preparation of the compounds of the
present invention by the displacement of the 3-acetoxy
group from a cephalosporanic acid of the formula
A~rrl ~ LH2 -O-C-CH, V
0/ OC~I
here A is as defined hereinbefore above,
prepared by substituting 7-amino cephalosporanic acld for
the 3-thiolated-7-amino cephalosporanic acids in the acylation
procedures described herein and else~Jhere reported ~lith the
appropriate thiol HSR3 having the formula
Il-S - ~ ~ N ~ CH2)nCOoH H-S- ~ N-~l~
where n is one or t~Jo and then removing the protecting
group if any is present as on the carboxyl group,
~7724
The d~splacement of such ~ 3-acetoxy group wlth
such a thiol m~y be accompli~hed in solution a8 ln wster
or aqueous acetone at ~ temperature of at least room
temper~ture and preferably within the rsnge of about
50- to lOO-C. ln the presence of a mlld b~se such ~8
sodl~m bic~rbonate, e.g. prefer~bly near neutral~ty such
J8 ~t about pH 6. An excess of the th~ol is preferably
emplo~ed. The re~ction product is lsolated by careful
acldlflcation of the reaction m~xture followed by extrac-
tion with ~ water-immiscible orgsnic solvent As notet
above, the prep~ration of many other 7-acylemldocephalospor~nlc
ac~t~ 1~ te~orlbed in the patent and scient~fic liter~turo,
e.g. in U.S. Cl~ss 260-243C.
The salts of the compounds of this inventlon ~nclut~
the nontoxlc carboxyllc acld sslts thereof, lncludlng non-
toxlc metslllc salts Juch a~ ~odlum, pot~slum, calclum ~nd
alumlnum, the ammonlum sJlt and substltutet ~mmon~um a~lt~,
e.~. salt~ Or Buch nontoxl¢ amlnes as trlalkyl-
amlne~ lncludlng trlethylamlne, procalne, dlbenzyl-
amlne, N-benzyl-beta-phenethylsmlne, l-ephenamlne,
N,N~-dibenzylethylenedlamine, dehydroabietylamlne,
N,N'-bls-dehydroabletylethylenedlamlne, and other
amlnes wh~ch have been used to rorm salts wlth
benzylpenlclllin, L-lysine, arginine and histldine
The preferred esters Or the cephalosporlns
o~ the present inventlon are the pivaloyloxymethyl,
acetoxymethyl, methoxymethyl, acet~nyl and phenacyl
e~ters. All are useful lntermedlates ln the
production Or the cephalosporln having a free
carboxyl group.
- 22 -
~1477Z4
As lndlcated above, these rlve ester~ Or 7-amino-
cephalosporanlc acld are each prepared by known
method~. One excellent procedure ~s that of U S.
patent 3,284,451 ln whlch sodlum cephalothln 1~
ester~r~ed by reactlon wlth the correspondlng actlve
ohloro or bromo compound (e.g. phenacyl bromlde,
chloroacetone, chloromethyl ether, plvaloyloxy-
methyl chlorlae ~also called chloromethyl plvalate],J¢etoxymethyl chlorlde) and then the thlen~l-
acetlc acld sidechaln 19 removed enzymatlcally
as in the same patent or chemlcally a~ ln U.S.
patent 3,575,g70 and ln Journal Or Antlblotlcs,
XXIV (11), 767-773 (1971). In another good
method the trlethylamlne salt o~ 7-am~nocephalo-
~poranlc acld 18 reacted directly wlth the actlve
halogen compound, as ln Unlted Kingdom 1,22g,45~.
- m ese esters o~ 7-amlnocephalosporanlc acld
are then reacted wlth the nucleophlle HSR3 ln the ~ame
manner a8 ~8 lllustrated here~n ror 7-amlnocephalO-
~poranlc acld ltselr. ffl e ~-thlolated ester o~
7-amlnocephalo~poran~c aold ~o then couple~ ~lth
the organlc carboxyllc acld as be~ore
The ester of the cephalosporin so obtained
is, if not used per se, converted to its free acid
and, if desired, sny sslt by removsl of the esterl-
fylng group, ~s by aqueow or enzym~tlc hydroly~
with human or animal serum) or acidic or alkaline
hydrolysis or by trefltment with sodium thlophenoxide
as taught ln U.S. 3,284,451 and, in the penicillin
~erle~, by Sheehan et ~1., J. Org. Chem. 29~7~,
2006-2008 (1964).
~77;Z~
In another alternatlve synthe81s, the 3-
thlolated 7-amlnocephalo~poranic acid 1~ prepared
~B descrlbed hereln and then act~l~ted at the 7-
amlno group and rlnally e~te~l~led, as by reactlon
o~ the appropr~ate alcohol wlth the acld chlorlde
prepared, ~or example, by reactlon Or the rlnal
cephalosporln with thlonyl chlorlde or by ~ther
e~entlally acid~c e8terlflcatlon proc~d~lres.
There ~ further provided by the present invention a
pharmaceutical composition comprising an antibacterially
effective amount of a compound having the formula
O ' S
~-CH-C-NH-CH--CH I 2 ~ I
1R 0"~ ~\ ~ 2 ~N'Nb~N- (CH2) nCO2H
COOM
wherein n i~ one or two, Rl is hydxogen or formyl and R ~s
~ or ~
and Y is hydrogen, chlorine, bromine, fluorine, tr~flùoro-
methyl, amino, nitro, hydroxy, lower alkyl of 1-4 carbon
atoms or lower alkoxy of 1-4 carbon atoms and M is hydrogen,
pivaloyloxymethyl, acetoxymethyl, methoxymethyl, acetonyl,
phenacyl, p-nitrogenzyl, B,B,B-trichloroethyl, 3-phthalidyl
or ~-indanyl and preferably is hydrogen or a nontoxlc,
pharmaceutically acceptable salt thereof.
~477Z4
~ here is further provided by the present invention a
method of treating bacterial infections comprising adminis-
tering by $njection to an infected warm-blooded animal,
including man, an effective but nontoxic dose of 250-1000 mgm.
of a compound having the formula
O ~S
~-C~I-C-NH-CH - CH ~ H2 ~ N~
I ~ ~ C--C~2--5~ N~N ~ N (CH2) n 2
COOM
wherein n is one or two, Rl is hydrogen or formyl and R is
or ~
~nd Y is hydrogen, chlorine, bromine, fluorine, trifluoro-
methyl, amino, nitro, hydroxy, lower alkyl of 1-4 carbon
~tom~ or lower alkoxy of 1-4 carbon Atoms and M i5 hydrogen,
p~valoyloxymethyl, acetoxymethyl, methoxymethyl, acetonyl,
phenacyl, p-nitrobenzyl, B,B,B-trichloroethyl, 3-phthalidyl
or 5-indanyl or a nontoxic, pharmaceutically acceptable salt
thereof.
$here is also provided by the present invention a
method for combatting Shig. dvsenteriac infections which
comprises adminiqtering to a warm-blooded mammal infected
with an ~ . dysenteriae infection an amount effect~ve for
treating said Shig. dysenteriae infection of a composition
comprising a compound having the formula
- 25 -
~477Z~
ol /S
R-C~I--C--NH--CH--CH ~:H2 ~N
* ~,C ~ ~ 2 ~:N'N~N (CH2) nC02H
COOM
~herein n i5 one or two, R is hydrogen or formyl and ~ ~s
or ~
~nd Y is hydrogen, chlorine, bromine, fluorine, trifluoro-
methyl, amino, nitro, hydroxy, lower alkyl of 1-4 carbon
atom~ or lower alkoxy of 1-4 carbon atoms and M is hydrogen,
pivaloyloxymethyl, acetoxymethyl, methoxymethyl, acetonyl,
phenacyl, p-nitrobenzyl, B,B,B-trichloroethyl, 3-phthalidyl
or S-indanyl and preferably is hydrogen or a nontoxic,
pharmaceutically acceptable salt thereof.
There i8 also provided by the pre~ent invention a
method for combatting Sal. enteritidis infections which
compri5e~ admini~tering to a warm-blooded mammal infected
with a Sal. _nteritidis infection an amount effective for
treating said Sal. enteritidis infection of a composition
compri~ing a compound having the formula
O
~-CH-C-NH-CH--CH ~ 2 ~=
ORl ~1 ~ N\ j/ ~H2 5 ~N,N~N lC 2)nC02H
OOM
- 26 --
~77'~4
wherein n is one or two, R~ is hydrogen or formyl and R is
~ or ~
and Y ls hydrogen, chlorine, bromine, fluorine, trifluoro-
methyl, amino, nitro, hydroxy, lower alkyl of 1-4 carbon
atoms or lower alkoxy of 1-4 carbon atoms and M is hydrogen,
pivaloyloxymethyl, acetoxymethyl, methoxymethyl, acetonyl,
phenacyl, p-nitrobenzyl, B,B,B-trichloroethyl, 3-phthalidyl
or 5-indanyl and preferably is hydrogen or a nontoxic
pharmaceutically acceptable salt thereof.
There is further provided by the present
invention a pharmaceutlcal composltion comprlsing an
antlbacterially effective amount of a compound having
the formula
O '.
- C - NH~ H ~H2 ~ r1
OR~ ~C N c"C CH2 S
1_0~ 0
wherein R2and Al are as hereinbefore defined
and R3 ~s hydrogen, pivaloyloxymethyl,
acetoxymethyl, methoxymethyl, acetonyl, phenacyl, p-
nitrobenzyl, ~ -tricllloroetllyl, 3-phthalidyl or 5-indanyl
and preferably is hydrogen or a nontoxic, phar~aceutically
acceptable salt thereof, said compound being at least
_ ~7 _
7724
75% by weight in the form of its syn isomer and
preferably in the form of its syn isomer essentially
free of the corresponding anti isomer, and a pharma-
ceutically acceptable carrier therefor.
There is further provided by the present
invention a pharmaceutical composition comprising an
ant~bacterially effective amount of the syn isomer of
a compound having the formula
C - g - NH-C~I CH IC~z ~ N
L~_oc~ ~ r~ c~5 2 S ~ N,N ~ - A1
IC_OH O
wherein Al is as hereinbefore defined or a nontoxic, pha~a-
ceutically acceptable salt thereof and a pharmaceutically
acceptable carrier therefore.
There ls further provided by the present
lnventlon a method of treating bacterial infections
comprising administering by in~ection to an infected
warm-blooded animal, including man, an effective but
nontoxic dose of 250-1000 mgm. of a compound having the
formula
C - C - NH-C~--CH CH2 ~ Nl
OR o ~ ~ C 2 N~ ~ A
- 28 -
119~ f 72~
herein R2 and Al are as hereinberore defined
and R3 is hydrogen, pivaloyloxymethyl,
acetoxymethyl, methoxymethyl, acetonyl, phenacyl, p-
nltrobenzyl, ~ -trichloroethyl, 3-phthalidyl or
5-indanyl or a nontoxic, pharmaceutically
acceptable salt thereof, said compound being at least
75% by weight in the form of its syn isomer and
preferably in the form of its syn isomer essentially
free of the correspond~ng anti isomer.
... . _ .
There ls further provlded by the present
lnvent~on a method of treatlng bacterlal infectlons
comprislng admlnlstering by lnJection to an lnfected
warm-blooded animal, lncludlng man, an effectlve but
nontoxlc dose of 250-1000 mgm. of the syn lsomer of a
compound havlng the formula
~ I - C - NH-CIH- CH ~H2 ~ N
~ o 7 ~
_OH 0
~Jherein ~1 is as hereinbefore defined or a nontoxic,
pharmaceutically acceptable salt thereof, and
wherein n is preferably 1
- 29 -
~ ~ ~ 7 7 Z ~
m ere is also provided by the present
invention a method for combatting Hae~ophllus infec-
tions which comprises administering to a warm-bl-ooded
mammal lnfected with an Haemophilus infectlon an
amoun~ effective for treating said HaemoPhilus lnfec-
tlon of a composltion comprislng a compound having
the rormNla
C - C - NH-CI Cl~ C~1
1~ 2 ~ C N "C-CH2-S N ~ l A
o~3 o
herein Al and R2 are as hereinbefore defined
and R3 is hydrogen, pivaloyloxymethyl,
acetoxymethyl, methoxymethyl, acetonyl, phenacyl, p-
nitrobenzyl, ~,B,~-trichloroethyl, 3-phthalidyl or 5-lnd~nyl
and preferably is hydrogen or a nontoxic, pharmaceutically
acceptable salt thereof, said compound being at least
75% by weight in the form of its syn isomer and
preferably in the form of its syn isomer essentially
free of the corresponding anti isomer, and a pharma-
ceutically acceptable carrier therefor.
.
There is also prov~ded by the present
inventlon a method for combatting Neisseria lnfec-
tions which comprises administering to a warm-blooded
mammal infected with a Neisseria infection an amount
effect~ve for treating sald Neisserla infection of a
composltion comprising a compound having the formula
11~7'724
CI - C - NH-C~ CIII CH2 ~ N
N_ oRl ~C N f~ CH2 S N~N~
_0~ 0
~herein R2 and ~1 are as hereinbefore defined
and R3 is hydrogen, pivaloyloxymethyl,
acetox~methyl, methoxymethyl, acetonyl, phena.cyl, p-
nitrobenzyl, ~ trichloroethyl, 3-phthalidyl or 5-lndanyl
~nd preferably iæ hydrogen or a r.ontoxic, pharmaceutically
acceptable salt thereof, said compound being at least
75% by weight in the form of its syn isomer and
preferably in the form of its syn isomer essentially
free of the corresponding anti isomer, and a pharma-
ceutically acceptable carrier therefor.
m e present invention also provides the novel
compounds having the formula
~ S--~J ~N_~l
wherein Al is methyl or -(CH~)nCO~I, ~rhere n is one or t~ro,
me invention further provides the novel compounds
having the formula
1~477Z4
H~N-~I --CH ~ C~I~
--N ~ ~C-C~2-S ,~ ~ N_
c-o~3
o
wherein Al is methyl or ~(CHa)nC0~3,
where n is one or two and R3 is hydrogen or a conventional,
pharmaceutica.lly acceptable, easily hydrolyzed ester
forming group; or a nontoxic, pharm~ceutically acceptable
salt thereof,
- 32. -
~ 77Z4
STARTI~G ~TERIALS
2-Furoylcyanide
To a suspension of 26.1 g. (0.4 mole) of ground
potasslum cyanide in 300 ml. of acetonitrile at 5 C. was
added 26.1 g. (0.2 mole) of a-furoyl chloride while keeping
the temperature below 8 C. The mixture was ætlrred ln
the cold for 15 mlnutes then heated at reflux ~or 30
minutes. The reaction was cooled, filtered and the aceto-
nitrile was removed at 15 mm. (steam-bath) leaving 24.5 g.
of a dark oil whlch was used without further purification.
An lnfrared spectrum showed a nitriie band at 226~ cm 1
2-Furaneglyoxylic Acid
The 24.5 g. of crude 2-furoylcyanide was mixed
with 160 ml. concentrated hydrochloric acid at 25 C. with
intermittent stirring. The reactlon was stored for 24
hours at 25 C. and diluted with 80 ml. of water. The
reactlon was stirred for 5 minutes and filtered. The fil-
trate was saturated with sodium chlorlde and extracted wlth
5 x 120 ml. of 1:1 ether-ethyl acetate solution. The extracts
were combined, dried over anhydrous magnesium sulfate and
evaporatéd at 30 C. (15 mm.) to give a brownish-orange
~olid. The solid was dissolved in methanol, treated with
charcoal and evaporated under reduced pressure (15 mm.) to
dryness to yield 17 g of the acid.
The product was recrystallized from toluene to give
11.5 g. (m.p. 76 C.). The ir and nmr spectra were consis-
tent for the structure.
2-Methoxyimino-2-furylacetic Acid
To a solution of 4.5 g. (0.032 mole) of 2-furane-
glyoxyllc acid in 40 ml. of 50% alcohol and 3.1 g. (O.037
- 33 -
7'72 ~ .
mole) of methoxyamine hydrochloride in 6 ml. water at 20
C. was added dllute sodium hydroxlde solutlon to pH 4-5.
The solution was stlrred at pH 4-5 at 25 C. for 24 hours.
The alcohol was removed under reduced pressure (15 mm.) and
the solutlon was ad~usted to pH 7-8 with 50% sodlum
hydroxide solution. The reactlon was extracted with ~ x
50 ml. of ether and the aqueous layer was ad~usted to pH
1.9 using concentrated hydrochloric acid. The mixture wa8
extracted wlth 5 x 50 ml. of ethyl acetate. The organlc
fractlons were combined, washed with brine, drled over
~nhydrous magneslum sulfate and evaporated under reduced
pressure (15 mm.) to an oil ~hich was cooled for one hour
in an ice bath. The product was slurried with Skellysolve
B and collected to yield 3.1 g of yellow crystals, m.p.
78 C. An analytical sample was recrystallized from
toluene, dried for 16 hours in vacuo over P205 at 25 C.
The ir and nmr spectra were consistent for the structure.
Anal. Calc'd for C7 ~ N0: C, 49.65; H, 4.17;
N, 8.28. Found: C, 49.30; H, 4.21; N, 8.37.
~-Ethoxyiminofurylacetic Acid
The 7.85 g. (o.056 mole) of furyl-2-glyoxylic
acid was dissolved ln 100 ml. of water and ad~usted to
pH 7 with 50% sodium hydroxide. The 6.83 g. (0.070 mole)
of ethoxyamine hydrochloride in 10 ml. of water was added,
while keeping the pH at 4-5. The reaction was diluted
with 25 ml. of alcohol, stirred 3 hours at room tempera-
ture and then filtered. The alcohol was remo~ed at 35
C. (15 mm.) and the aqueous portion was ad~usted with
dilute sodium hydroxlde solut~on to pH 7-8 and then
- 34 -
1~77Z4
was washed with ether and the washes were discarded. The
aqueous fraction was ad~usted with 6N hydrochIorlc acid
to pH 1. 5 and extracted into ~ x 80 ml. of ethyl acet~te.
The acetate fractions were combined, washed with brine
and reduced in volume at 35D C, (15 mm.) to an oil, The
oll was cooled in an ice bath, triturated with Skellysolve
B, collected and dried over P205 in vacuo at 25 C.
Yield: 4.8 g " m.p. 83-85 C. The ir and nmr were con-
sistent for the structure,
Anal, Calc'd for C8HgN04: C, 52.46; H, 4.95;
N, 7,65, Found: C, 52,22; H, 4.94; N, 7,60,
Sodium a-Ethoxyimino-a-(2-furyl)acetate
To 50 ml. of methanol was added 250 mg, (0.0109
mole) of metallic sodium and stlrred until all the sodium
had dissolved. This sodlum methoxide solutlon was treated
with 2.0 g, (0,0109 mole) of ~-ethoxyimino-a-(2-furyl)acetlc
acid dissolved in 10 ml, of methanol and stirred at room
temperature for one hour, The methanol was removed at
40 C, (15 mm,) and the product was dried in vacuo over
P205 at 25 C,to yield 2.22 g, white solid, m,p. decomp,
>240 C, The ~r and nmr were consistent for the structure,
"Skellysolve ~" is a petroleum ether fraction
of b,p, 60-68 C, consisting es6entially of n-hexane,
~ 35 -
~1~7724
~-CI ~ ~ ~-tN
H 1 1HC1
_ CH3oNH2-Hcl
~CH3 ~ -OH
Na 3
~ ~ (CCl)2
~a3 5 ~CH3
2-Furoylcyanlde 1
To a suspen610n of 78.~ g. of powdered potas- .
~ium cyanide ln 900 ml. acetonltrlle at 5 C. was added
59.25 ml. (68.5 g.) of a-furoyl chloride wlth vigorous
stirrlng whlle keeping the temperature at 4-8 C. The
mixture was stirred at 4-8 C, for 15 minutes and then
heated at reflux for 30 minutes. The mixture was cooled
to 23-25 C., filtered, washed with 50 ml. of acetonitrile
whlch was added to the filtrate, and the acetonitrile was
removed at 60 C. (15 mm.) leavlng 51 g. of ~ as a dark
- 36 -
1~477'Z4
oil. An IR spectrum showed a nitrile band at 2265 cm 1
and an NMR spectrum showed a ratio of approximately
70/30 of product ~ furoic acid. The crude product ~ was
used without further purification (49% yield of product).
Furyl-2-glyoxylic Acid 2
The 51 g. of crude 2-furoyl cyanlde 1 was
mlxed with 500 ml. concentrated hydrochloric acid at
25 C. The reaction was stirred for 24 hours at 25 C.
and then diluted with 240 ml. of water. The mixture was
stirred for 5 minutes and filtered. The black filtrate
was saturated with sodium chloride and extracted with
6 x 500 ml. of 1:1 ether-ethyl acetate solution. (~ote:
Initlally the extractions were difficult due to the
inability to see the separation of two black phases. As
additional ether-ethyl acetate extractions were run the
task was simplified.) The extracts were combined and
evaporated to dryness at 60 C. (15 mm.). The resultant
solid was dissolved in 600 ml. etherJ (Note: Use of
alcohol should be avoided at thls point as esters may
~orm), treated with 10 g. of charcoal ("Darko-KB"),
flltered after stirring for 0.~ hoùr and evaporated
to drynesS at 50~ C. (15 mm.) to yield 46.6 g. of 2
as a light tan colored acid Thi~ product 2 was
found to contain a ratio of approxlmately 56/44 of
product 2/furoic acid. This represented a 63% yield
of product 2.
Purification was accomplished by dissolving
the above crude product 2 in H20 (50 mg./ml.), titrating
to pH 2.8 with HCl and extr~cting with 2 x 200 ml. of
*Trade Mark
~47724
ethyl acetate Evaporation of the ethyl acetate extracts
gave 35~ furoic acid and 15% product 2. The pH 2. 8
aqueous phase was adjusted to pH o.8 (HCl) and extracted
w1th 2 x 200 ml. ethyl acetate. The organic extracts
were combined and washed with 50 ml. H20. The organic
phase was evaporated at 50 C. (15 mm ) yielding a
solid with a ratio of approximately 86/14 of product
2/furoic acid. This solid was then recrystallized by
dissolving the product 2 in toluene at 50 mg./ml. at
80 C., decanting, and leavlng to crystallize at room
temperature for 18 hours, yielding 13.3 g. of pure acid
2 by NMR. This represented a 51% yield ~n the purifica-
tion and recrystallization step and an overall yield from
the 2-furoyl chlorlde to the pure furyl-2-glyoxylic
acid 2 of 16%.
S~n-~-methoxyiminofurylacetic Acid ~
A solution of 4.5 g. of furyl-2-glyoxylic acid
2 in 40 ml. of 50% ethanol was titrated to pH 6 with lN
sodium hydroxide and then 3.1 g. of methoxyamine HCl in
6 ml. of H20 at 20 C. was added. The solution was
tltrated to a constant pH 4.9 and stirred at pH 4.9 for
2~ hours at 20-23~ C. The ethanol was then removed at
50 C. (15 mm.) and the residual a~ueous solutlon was
tltrated to pH 8 with 50~ sodium hydroxide and wa~hed
with 3 x 50 ml. ether (pH ad~usted to 8 after each
wash). The aqueous layer was t1trated to pH 1.9 with
concentrated HCl and extracted wlth 5 x 50 ml. ethyl
acetate with the pH read~usted to 1.9 after each extrac-
tion. The ethyl acetate extracts were combined and
- 38 -
~14'77'~
evaporated to a solid ~ at 50 C. (15 mm.). This solid
was then slurried with 75 ml. of "Skellysolve B". The
suspension was filtered and the solids were redissolved
in 16 ml. of toluene at 80 C. The hot solution was
decanted and left to crystallize at 20-23 C. for 18
hours to yield 1.17 g. ~ (22% yield of product). The
NMR was clean and consistent for the structure ~ with a
trace of anti isomer present.
Sodium Syn-~-methoxyiminofurylacetate 4
To 40 ml. of methanol was added 0.16 g. of
sodium. The mixture was stirred until all of the sodium
dissolved and then decanted. The resulting sodium
methoxide solution was cooled to 3 C. and 1.12 g. of
syn-a-methoxyiminofurylacetic acid ~ in 7.8 ml. of
methanol was added. The solution was stirred for 10
minutes at room temperature. The solvent was evaporated
at 40 C. (15 mm.). The residue 4 was dried by azeo-
tropic distillation with 3 x 20 ml. of benzene at 40 C.
(15 mm.). The product 4 was dried for 18 hours at 23
C. under high vacuum (0.7 mm,) over P205 yielding 1.25 g,
(99% yield of product). The NMR showed this product ~
to be clean and consistent for the Btructure with 0,15
mole methanol and a trace of anti lsomer,
To 0.~3 g. of sodium syn-~-metnoxyiminofuryl-
acetate 4 suspended in ~5 ml. of benzene was added four
drops of dry dimethylformamide and 0.31 ml. (1.1 eq.
of oxalyl chloride. This mixture was stirred for 40
minutes at 20-23 C. The benzene was removed at 35~ C.
(15 mm.) to provide the acid chloride 5 as the gummy
residue.
- 39 -
7'~4
6-Chloro-2~3-dihydro-2-ethoxycarbonylmethyl-s-tria
-r 4,3-blpyridazin-3-one
To a solut~on of 6-chloro-2,3-dihydro-s-
triazolo[4,3-b]pyridazin-3-one ~ P. Franca-rilla and F.
Lauria, J. Het. Chem., 8, 415 (1971)] (1, 1.00 g., 5 9
m.mole) ln dry DMF (30 ml.) was added sod~um hydride
~50% in paraffin, 0.3 g., 6t3 m.mole) under stirring wlth
formation of yellow crystals. To the mixture was added
ethyl chloroacetate (1.4 ml., 13 m.mole) and the mixture
was heat-ed at 90 C. for 8 hours with stirring. After
cooling, the reaction mixture was poured into water (50
ml.) and extracted with toluene (5 x 40 ml.). The or-
ganic extracts were combined, dried over anhydrous sodium
sulfate and evaporated at reduced pressure. The residue
was crystallized with benzene-n-hexane to give yellow
needles (1.16 g., 71%), m.p. 114-115~ C. (llt.
110 C.).
ir: v KBax 1735, 1710 cm 1
uv: ~maxoH 231 nm (~, 26000)
.nmr: ~ppC13 7.58 (lH, d, J=10 Hz, pyridazine-H), 6 98
(lH, d, J=10 Hz, pyridazine-H), 4.80 (2H, s, -CH2C0),
4.27 (2H, q, J=7.5 Hz, CH2CH3), 1.29 (3H, t, J=7.5 Hz,
CH2CH3 ) .
Anal. Calc'd. for CgHgN403Cl C, 42.12; H, 3.53;
N, 21.83; Cl, 13.81. Found: C, 41.54, 41.46; H, 3.22,
3.49; N, 21.51, 21.53; Cl, 13~88~ 13.99.
- l~o -
i~772~
2-Carboxymethyl-2,3-dihydro-6-mercapto-s-triazolor4,3-bl-
p~ridazin-3-one
To a solution of 6-chloro-2,3-dihydro-2-ethoxy-
carbonylmethyl-s-triazolo[4,3-b]pyridazin-3-one ~30 g ,
0.12 mole) in ethanol (900 ml.)-was added NaSH 2H20 (70%
pure, 45.9 g , o.36 mole) and the mixture was refluxed
for 0 5 hour. The reaction mixture was evaporated at re-
duced pressure. The residue was dissolved in water (200
ml.) and concentrated HCl was added to the solution to
ad~ust to pH 2. The precipitate of 2-carboxymethyl-2,3-
dihydro-6-mercapto-s-triazolo[4,3-b]pyridazin-3-one
was collected by fil-
tration and washed with water. Yield 18.3 g (69~).
lr: vKBax 2900, 2450, 1750, 1660 cm 1.
~l%NaHco3a~ 260 nm (,1950)~ 3~ nm (~ 7
nmr: ~ppMm-d6 7.88 (lH, d, J=10 Hz, pyridazine-H),
7.45 (lH, d, J=10 Hz, pyridazine-H), 4.72 (2H, s, CH2C0).
Anal. Calc'd. for C7H6N403S: C, 37-17; H, 2-~7;
N, 24.77; S, 14.17. Found: C, 37.35, 37.23; H, 2.26,
2.28; N, 23.58, 23.69; S, 14.32.
. .
7-Amino-3-(2-carboxymethyl-2,3-dihydro-s-triazolo~4,3-bl-
pyridazin-3-on-6-ylthIomethyl)-3-cephem-4-carboxylic Acid
To a suspension of 7-aminocephalosporanic acid
(8.79 g., 32.2 m;mole) in 0.1 M phosphate buffer (pH 7,
- 41 -
E~_ r T ~ ~ r ~ < ~
. ~
~:1477Z~
149 ml.) were added NaHC03 (8.14 g , 97.0 m.mole) and the
thiol 2-carboxymethyl-2,3-dihydro-6-mercapto-s-triazolo-
[4,3-b]pyridazin-3-one (7.30 g., 32.2 m.mole) with stir-
ring. The mlxture
was heated at 80 C. for 0.5 hour under N2 stream. The
mixture was treated with active carbon and ad~usted to
pH 3 with concentrated HCl. The resulting precipitate was
collected by filtration and washed with water to give 7.59 g.
(54~0) of 7-amino-3-(2-carboxymethyl-2,3-dihydro-s-tri-
azolo~4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-
carboxylic acid.
- 42 -
.~ .~A.,,
~77~
ir: vmBax 1800, 1720, 1600, 1540, 1470 cm 1.
uv: ~BUffer (pH 7) 252 nm (~,19500)~ 298 nm (~, 8400)
nmr: ~pD~m+K2C03 7.56 (lH, d, J=9 Hz, pyridazine-H),
7.05 (lH, d, J=9 Hz, pyridazine-H), 5.45 (lH, d, J=5 Hz,
6-H), 5.05 (lH, d, 5 Hz, 7-H), 4.43 (lH, d, J=14 Hz,
~-CH2), 4.04 (lH, d, J=14 Hz, 3-CH2~, 3.88 (lH, d, J=18 Hz,
2-H), 3.45 (lH, d, J=18 Hz, 2-H).
6-Chloro-2-(2-cyanoethyl)-2,3-dihydro-s-triazolo[4,3-b]-
pyridazin-3-on.
To a solution of 6-chloro-2,3-dihydro-s-tri-
azolo[4,3-b]pyridazin-3-on ~P. Francabilla and F. Lauria,
J. Het. Chem. 8, 415 (1971)] (17 g., 0.1 mole) in dry
DMF (300 ml.) was added potassium tert.-butoxide (0.5 g.,
4.5 m.moles) with stirring. Acrylonitrile (6.6 g., 0.12
mole) in dry DMF (10 ml.) was added to the mixture. The
mixture was stirred at 100-110 C. for 24 hours, then
poured into water (700 ml.) and extracted wi~h ethyl
acetate (5 x 400 ml.). The organic extracts were combined,
drled over Na2S04 and evaporated. The residue was crystal-
llzed from ethyl acetate to give light yellow needles of
6-chloro-2-(2-cyanoethyl)-2,3-dihydro-s-triazolo~4,3-b~-
pyridazin-3-on (2.5 g., 11%). M.p. 166-168 C.
ir: vmBaxr 2230, 1720, 1550, 1500 cm 1
uv: ~dimxane 373 nm (~ 2000)
nmr: ~Dppm d6 3.03 (2H, t, J=6 0 Hz, CH2), 4.21 (2H,
t, J=6.o Hz, CH2), 7.23 (lH, d, J=10.0 Hz, pyridazine-H),
- 43 -
_ _
~ 77Z~
7 93 (lH, d, J=10.0 Hz, pyridazine-H).
Anal. Calc'd. for C8H6N50Cl: C, 42.97; H, 2.70;
N, 31.32; Cl, 15.86. Found: C, 42.73, 42.56; H, 2.57,
2.50; N, 31.36, 31.68; Cl, 15.96, 15.81.
2-(?-Carboxyeth~1~-6-chloro-2,3-dihydro-s-triazolo~4,3-b]-
p~ridazin-3-on.
A solution of 6-chloro-2-(2-cyanoethyl)-2,3-
dihydro-s-triazolo[4,3-b]pyridazin-3-on (724 mg.) in
6N-HCl (15 ml.) was refluxed for 6 hours. The reaction
mixture was extracted with ethyl acetate (10 x 20 ml.).
The combin~d extracts were washed ~ith saturated aqueous
~odium chlorid~ (50 ml.), dried over Na2S04 and evaporated
to gi~e light yello~tsolid 2-(2-carboxyethyl)-6-chloro-2,3-
dihydro-s-triazolo~4,3-b]pyridazin-3-on (567 mg., 72~).
M.p. >170 C. (sublimation).
ir: vKBX 3400-2400, 1730, 1710, 1540 cm 1
uv: ~dimXaxne 377 nm (~ 1500).
nmr: ~ 2 ppm~lc3 2.70 (2H, t, J=7.0 Hz, CH2), 4.24
(2H, t, J=7.0 Hz, CH2), 7.17 (lH, d, J=10.0 Hz, pyridazine-H),
7.70 (lH, d, J=10.0 Hæ, pyridazine-H).
Anal. Calc'd. for C8H7N403Cl: C~ 39.60; H, 2.91;
N, 23.09; Cl, 14.61. Found: C, 39 62, 39.48; ~, 2 97,
2.67; N, 23.05, 22.70; Cl. 13.93, 14.12.
_ 1~4 _
. . ` 7 . ~;
~ ~ ~';'7~
2-(2-Carboxyethyl~-2,3-dihydro-6-mercapto-s-triazolor4~3-b]-
pyridazin-3-on.
A mixture of 2-(2-carboxyethyl)-6-chloro-2,3-
dihydro-s-triazolo[4,3-b]pyridazin-3-on (567 mg., 2.34
m.moles) and 70% sodium hydrosulfide dihydrate (924 mg.,
7.02 m.mole) in water (10 ml.) ~las stirred at room tempera-
ture for two hours. The reaction mixture was adjusted suc-
cessively to pH 1 with c. HCl, to pH 10 with NaOH and then
to pH 1 with c. HCl. The resulting precipitate of 2-(-
carboxyethyl)-2,3-dihydro-6-mercapto-s-triazolo~4,3-b]-
pyridazin-3-on was collected by filtration and washed with
water. Yield: 418 mg. (74%). M.p. 174-176 C.
ir: vmBax 3600-2600, 2440, 1730, 1720 (sh) cm ~.
uv: ~pH 7 buffer 262 nm (~11oOO), 318 nm (~ 6600).
nmr: ~DMppmd6 2.73 (2H, t, J=7.0 Hz, CH2), 4.07 (2H,
t, ~=7.0 Hz, CH2), 7.30 (lH, d, J=10.0 Hz, pyridazine-H),
7.74 (lH, d, J=10 0 Hz, pyridazine-H).
Anal. Calc'd. for C8H~N403S: C, 40.00; H, 3.36;
N, 23.32, S, 13.35. Found: C, 39.o8, 39.06; H, 3.12,
~.20; N, 22.65, 22.70; S, 14.23, 14.29.
. .
7-Amino-3-[2-~ -carboxyethyl)-2,3-dihydro-s-triazolol4,3-bl-
pyridazin-3-on-6-ylthiomethyll-3-cephem-4-carboxylic Acid.
A mixture of 7-ACA (405 mg., 1.49 m.moles), the
thiol 2-(2-carboxye~hyl)-2,3-dihydro-6-mercapto-s-
triazolo~4,3-b~pyridazin-3-on (357 mg., 1.49 m.moles) and
NaHC03 (375 mg., 4.47 m.moles) in 0.1 M phosphate buffer
(pH 7, 8 ml.) was stirred at 80 C for 30 minutes. m e
reaction mixture was cooled and filtered to remove insolubles.
_ 1~5 _
l~g7724
The filtrate was adjusted to pH 1-2 with c. HCl. The result-
ing precipitate, 7-amino-3-~2-(2-carboxyethyl)-2,3-dihydro-
s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl]-3-cephem-
4-carboxylic acid, was collected by filtration and washed
with water. Yield: 519 mg. (77%).
ir: vmBax 3600-2200, 1800, 1725, 1620, 1550, 1480 cm 1.
uv ~pH 7 ~uffer 253 nm (E ~0000), 298 nm (~)-
nmr: ~D2ppK~C3 2-20 (2H, t, J=7.0 Hz, CH2), 3.40 (lH,
d, J=17.5 Hz, 2-H), 3.85 (lH, d, J=17.5 Hz, 2-H), 4.00-
4 50 (4H, m, 3-CH2 and N-CH2-), 5.01 (lH, d, J=4.0 Hz, 6-H),
5.40 (lH, d, J=4.0 Hz, 7-H), 6.94 (lH, d, J=10.0 Hz,
pyrldazine-H), 7.44 (lH, d, J=10.0 Hz, pyridazine-H).
Anal. Calc'd for C16H16I~606S2 3/2 2
H, 3.99; N, 17.52; S, 13.37. Found: C, 40.06, 40.12;
H, 3.33, 3.34; N, 16.96, 16.98, S, 13.87, 13.98.
7-ACA refers to 7-aminocephalosporanic acid and
DMF to dimethylformamide.
. . ,
- 46 -
7724
Scheme 1. Preparation of 7-Amino-3-(2-carboxymethyl-
?,3-dihydro-s-triazolo~4,3-b]pyridazin-3-on-6.-ylthio-
methyl)-~-cephem-4-carboxylic Acid.
N NaH/D.iF ~ ~ T
2 2 5) Cl ~ N,N ~ N-CH2COOC2H~
O O
~.SH ~7 7-ACA
) llS ~,N ~ N-CII~COOH - >
3 o
H2'J I 1~ ~ ~= 1,.
O ~ ~ 2 ~ I~,N ~ I!-CH2COOH
CO,2H
- 47 -
L._ .. .. . . _ .
~77Z4
6-Chloro-2,3-dihydro-2-ethoxycarbonylmethyl-s-triazolo-
~4,3-blpyridazin-3-one (2?
To a solution of 6-chloro-2,3-dihydro-s-
triazolo[L,3-b]pyrid~zi!l-3-ol~e [~. ~r2ncavilla ~!~d F.
Lauria, J. Het. Chem., 8, 415 (1971)~ (1, 1.00 g., 5.9
m.mole) in dry D!~F (30 ml.) w~s added sodium hydrid~
~50~ in p~r~ffin, 0.3 g., 6 3 m.mole) under s~irring with
formation of yello~ crystals. To the mixture W2S ~dded
ethyl chloroacetate (1.~ ml., 13 m.mole) and th~ mixture
~ras heated at 90 C. for 8 hours with stirrin~ fter
cooling, the reaction mixture t:a~ poured ~r.to w~ter-(50
ml.) and extracted l~ith toluene (5 x ~0 ml.). Tile or-
ganic extracts ~lere combin~d, dried ovcr atllly~rouc. sodium
sulfate and evaporated ~t reduced pressurc. Ttle residue
~J~s cry~tallized ~!it l be.lzclle--~-h~x-~n~ tu ~ivc y~llo;;
needles of ~ (1.16 ~., 77~0), m. p. 111~-115 C. (lit
110 C
ir: v KBX 1735, 1710 cm~l.
uv ~ EaH 231 nm (&, 26000).
nmr: ~ ppC 3 7.58 (lH, d, J=10 Hz, pyridazine-H), 6.,o8
(lH, d, J=10 Hz, pyridazine-H), 4.80 (2H, s, -CH2C0),
4 27 (2H, c, J=7.5 Hz, CH2CH3), 1.29 (3H, t, J=7 5 l~z,
CH2C~33 ) .
Anal. Calc'd. for C9~91~03Cl: C, 42.12, Hj 3 53;
N, 21.83; Cl, 13.81. Found: C, ~1.5~, 41.4~; K, 3 22,
3.~9; I~i, 21.51, 21 53; Cl, 13.88, 13.~9.
- 48 -
1~7724
2-Carboxymet~yl-2,3-dihydro~-mercapto-s-triazolo[4,3-b]-
pyridazin-3-one (~)
To a solution of 6-chloro-2,3-dihydro-2-ethoxy-
car~on~lmetllyl-s-triazolo[~ b]oyridazin-;-olle (~, ~0 g.,
0.1~ mole) in ethanol (900 ml.) was added t~ .'}~ 0 (70
purD,. ~5.9 ~., o.~6 mole) and the mixture was rerluxed
for 0.5 hour. The reacticn micture was evaporated at re-
duc~ pressure. The residue was dissolved in ater (200
ml.) and concentrated HCl was added to tllc ~olution to
adjust to p~ 2 The precipitate (3) ~ras collected by fil-
tration ancillashed with water. Yicl~ 18.- g. !~9,')
ir: ~ KBx ~900~ 50, 1150, 1~0 cm 1.
uv ~ l~r!l~C0;~ 60 nm (~,19500), -~lS llm (,7000).
nmr ~ D~lS-d6 7.8~ , d, J--10 117, ~)yrir~ !le~
7.~5 (lH, d~ J=10 ~Iz, p~rid.~zine-~ , CH2CC)).
Anal. Calc'd. for C.~H6N'I~O~ C, 37.17; ~I, 2.67;
N, 2~.77; S, 14.17. ~ou~d: C, -S7-~, 37.2~ 2.2~,
2.28; N, 23.58, 23.69; S, 14.32.
7-Amino-3-(2-carboxymethyl-2,3-dihydro-s-triazolo~ -bt-
-on-~-ylthiomethyl)-3-cephem-4-carbo~lic Acid (4)
To 2 suspension of 7-aminocephalospor~ic acid
(8.79 g., 32.2 m.mole) in 0.1 l~ phosph~tc ~uffer (p~ 7,
149 ml.~ were added NalC03 (8.14 g., 97.0 m.mole) and t~e
thiol ~ ~7.~0 g., 32.2 m.mole) ~ith stirring. '~e m~xture
was heated at 80D C. for 0.5 hollr under N2 str~P~. The
mixture was treated with active carbon and adjusted to
pH 3 with conccntrated :~Cl. The resulting prec-pit~te was
collected by filtration and washed with water to giYe 7.59 g.
(5~) of ~
_ 1~9 _
. . .
1~77~
.ir: 1~ Ka~ 1800, 1720, 1600, 151~0, 1470 cm 1.
u~ \ L~ff~r (Pi~ 7) ^52 n;r. (~, 19500), 29~ llm (E~8400)
nmr: ~) pk3~ co3 7.5G (1~1, d, J=9 Hz, pyrida7.ine-H),
7.05 (1~, d, J=9 Hz, ~yridazine-~{), 5.45 (l~t, d, J=5 Hz,
6-~[), 5.05 (lH, d, 5 Hz, 7-H), 4.43 (lH, d, J=14 ~Iz,
3-C~I2), 4.04 (lH, d, J=14 Hz, 3-CH2), 3.88 (lH, d, J=18 Hz,
2-H), 3.45 (1~, d, J=18 Hz, 2-H).
Pivaloyloxymethyl-7-amino-3- ~ carboxymethyl-2,3-dihydro-s-
triazolo~4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-
4-carboxylate.
Method A. - The title compound i8 produced by
substituting for the 7-amlnocephalosporanlc acld used
lmmedlately above an equimolar welght Or pivaloyloxy-
methyl 7-amlnocephalosporanate hydrochlorlde prepared
accordlng to Example 2 Or U.K. 1,229,45~ from 7-
amlnocephalosporanlc acld. German 1,904,585 (Farmdoc
~9,445) i8 equlvalent to U.K, 1,229,453.
Method B. - The tltle compound ls produced by
8ubstltutlng for the 0.025 mole (6.8 g.) 7-amlno-
cephalosporanlc acld used ln the procedure of Example
2 of U,K. 1,229,453 an equlmol~r welght of 7-amlno-~-
(2-carboxymethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-
3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid (4).
The respective acetoxymethyl, methoxymethyl, acetonyl
and phenacyl esters of 7-amino-3-(2-carboxymethyl-2,3-
dihydro-s-triazolo~4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-
cephem-4-carboxylic acid are prepared by substituting in
Method B above chloromethyl pivalate used therein an equimolar
weight of chloromethyl acetate, chloromethyl methyl ether,
chloroacetone and phenacyl bromide, respectively~
; ` ` ` il~77'~4
Preparation of 7-Amino-3-(2-methyl-2,3-dihydro-s-tria-
zolo~4,3-b~pyridazin-7J-on-6-ylthiomethyl-3-cephem-4
carboxylic Acid.
Cl ~ NH Cl ~ N-CH3
1 ~ 2
,
HS ~ N- CH3
H2~J~ ~ ~=NI
O ~ ~ CH2-S ~ 1~ ~ N-CH3
C2H
.. ~
6-Chloro-2,3-dihydro-2-methyl-s-triazolo~4,~-b]pyridazin-
3-one (2)
To a solution of 6-chloro-2,3-dihydro-s-triazolo-
[4,3-b~pyridazin-~-one {P. Francavilla and F. Lauria, J.
Het. Chem. 8, 415 (1971)] (1, 8.5 g., 50 m mol.) in dry
DMF ~12 ml.) was added NaH (50~ dispersion in paraffin,
- 51 -
~77Z~
2.64 g., 55 m.mol) and the mixture was stirred for 1
hour at room temperature. After methyl iodide (21.3 g.,
150 m.mole) was added, the mixture was stirred for 40
hours at room temperature, diluted with water (200 ml.)
and extracted with CHC13 (4 x 100 ml.). The combined
extracts were washed with water (3 x 50 ml.), treated
with a small amount of carbon and dried with anhydrous
Na2S04. Evaporation of the solvent under reduced pres-
sure afforded pale yellow residue which was crystallized
from chloroform-n-hexane. Yield: 7.23 g. (79~).
M.p. 180-181 C.
ir: vKB0 1720 cm 1
uv: ~Emax 233 nm (~ 25200), 363 nm ( E 1600).
nmr: ~,Cppl3 3.72 (3H, s, N-CH3), 6.88 (lH, d, J=10 Hz,
pyridazine-H), 7.48 (lH, d, J=10 Hz, pyridazine-H).
Anal. Calc'd. for C6H5ClN40: C, 39.04; H, 2.73;
N, 30.35; Cl, 19.21. Found: C, 39.24, 39.28; H, 2.54,
2.61; N, 30.63, 30.80; Cl, 19.59, 19.26.
6-Mercapto-2~3-dihydro-2-methyl-s-triazolo[4,3-b~pyridazin-
3-one (3)
-
A mixture of 2 (6.50 g., 35.7 m.mol.) and
NaSH 2H20 (70% pure, 9.4 g.) in water (100 ml.) was
heated under reflux for 15 minutes. m e mixture was cooled
and acidified to pH 1 with concentrated HCl to precipitate
the thiol 3 which was collected by filtration and dissolved
in aqueous saturated NaHC03 (100 ml.). ~le solution was
treated with a small amount of carbon and acidified with
dilute HC1 to precipitate 3 as pale yellow prisms.
Yield: 5.72 g. (~9~). M.p. >280 C.
- 52 -
i~7 72~
..
ir: vKBxr 2450 (-SH), 1710 (C=0) cm
uv ~1%maxHC03 261 nm (E 16300), 315 nm (E 5800).
nmr ~D2+KH 3-60 ~H, s, N-CH3), 7.o8 (2H, s,
pyrida~ine-H).
Anal. calc'd. for C6H6N40S: C, 39-55; H, 3-32;
N, 30.75; S, 17.60. Found: C, 39.57, 39.66; H, 3.14,
3.22; N, 30.32, 30 61; S, 17.80, 17.89.
7-Amino-3-(2-methyl-2,3-dihydro-s-triazolo~4,3-b]~yridazin-
3-on-6-ylthlomethyl-)--3-cephem-4-c-arboxylic Acid (4)
A mixture of 7-aminocephalosporanic acid (7-ACA,
5.44 g., 20 m.mol.), 3, (3.64 g., 20 m.mol ) and NaHC03
(3 36 g , 40 m.mol.) in 0.1 M phosphate buffer (pH 7, 100
ml.) was heated with stirring at 80 C. for 30 minutes.
The hot mixture was treated with a small amount of carbon
and the filtrate was acidified to pH 4 with dilute HCl to
precipitate 4 which was collected by filtration, washed
with water (50 ml.) and dried. Yield: 5.73 g. (73%).
M.p. 240-245~ C. (dec.).
ir: vmBr 1800 (B-lactam C=0), 17?5 (C=0~, 1610 and
1410 (C00 ) cm~l
uv: ~1%MaHC3 253 nm (~20000), 305 (~ 8400).
nmr: ~D20 ppaHC03 3-69 (3H, s, N-C_~), 5.o8 (lH, d, J=4.5
Hz, 6-H), 5.48 (lH, d, J=4.5Hz, 7-H), 7.00 (lH, d, J=10 Hz,
pyridazine-H), 7.52 (lH, d, ~=10 Hz, pyridazine-H).
Anal- Calc'd. for C14Hl4r~6o4s2 H2
H, 3.91; N, 20.38; S, 15.55. Found: C, 40.84, 40.63;
H, 3.44, 3.31; N, 20.50, 20.36; S, 15.19, 15.57.
t7~4
Preparation of BB-S515
~co / ~
/ BB-S515
BB-S515; 7-~(2Z ~ Methoxyimino(fur-2-yl)acetamido]-3-
(2,~-dihydro-s-triazolo~4,3-b~pyridazin-3-on-6-ylthio-
methyl~-3-cephem-4-carboxylic ACid sodium Salt.
To a solution of (2Z)-2-methoxyimino(fur-2-yl)-
acetic acid (169 mg., 1 m.mole) and triethylamine (0.14
ml., 1 m.mole) in dichloromethane (2 ml.) was added
oxalyl chloride (0.09 ml., 1 m.mole) at 0-5 C. and the
mixture was stirred for 30 minutes. The solvent was
evaporated under reduced pressure to afford an oily resi-
due. A solution of that oily residue in dry acetone
(5 ml ), after filtration, was added to a mixture of
, 7-amino-~-(2,3-dihydro-s-triazolo~4,3-b3pyridazin-3-on-
6-ylthiomethyl)-~-cephem-4-carboxylic acid (~80 mg.,
1 m.mole) (U.S. ~,907,786) and~sodium bicarbonate (~36
mg., 4 m.mol.) in water (10 ml~) at 0-5 C. The
reaction mlxture was stirred at 0-5 C. for 2 hours.
Most of the acetone was evaporated at reduced pressure,
the aqueous concentrate being washed with ether (2 x
30 ml.) and ad~usted to pH 1-2 with concentrated HC1
The resulting precipitate (~38 mg.) was collected by
filtration and dried in vacuo. A suspension of the free
acid (303 mg.) in water (10 ml.) was adjusted to pH 6 5
I with aqueous NaOH ~1 N, o,6 ml.) and filtered to make a
clear solution which was lyophilized to give 7-~(2Z)-2-
methoxyimino(fur-2-yl)acetamido~-3-(2,3-dihydro-s-
- 54 -
1~77~4
triazolo[4,3-b]pyridazin-3-on-6-ylthiometllyl)-3-cephem-
4-carboxylic acid sodium salt as a light brown powder
(222 mg., 46~ .p. >230 C. (dec. ) .
ir: vmaxr 3410, 1760, 1720, 1600 cm 1
uv: ~.PH7muaffer 256 rIm (E 20600), 274 (~ 18800).
~5 -
1~7724
Pre~aratlon o~ D-mandellc acid carbo~YanhYdrlde
COCl
C6H5-CH-cOOH ~ 6 5
1 2
D-Mandellc acld carboxvanhvdrlde (2)
Phosgene was bubbled through a solutlon of 2 0 g.
(0,013 mole) of D(-)-mandelic acld (1) ln dry tetra-
hydroruran rOr 30 minutes. The solutlon wa~ allowed
to 3tand overnlght after which tlme it was heated
under rerlux ~or 10 mlnutes. Evaporatlon Or the
solvent under reduced pre~sure afrorded an oily
resldue whlch was ~olldlfled by trlturatlon wlth
n-hexane (20 ml.). The product was collected by
rlltratlon and drled ln yacuo on KOH, Yield 2 ~ B-
o~ ~-mandellc acld carboxyanhydrlde.
IR: ~ naUJ 1895, 1875, 1780 cm 1
l'he pre~erred and most actlve compounds Or
the pre~ent lnventlon are those ha~lng the D
conriguration at the a-carbon atom in the 7-~ide-
chain, that i~, those made f~om D-mandellc acld or
a monosubstituted D-mandellc acid as lllustrated here-
ln In addltlon, the configuration at the two
optically actlve, as~mmetrlc centers in the ~-lactam
nucleus 19 that round ln cephalosporln C produced by
~ermentatlon and ln the 7-amlnocephalosporanlc acld
derlved therefrom.
~47724
EXAMPLE 1
6 5 1 \0 H2N~ ~ ~7
O--C/ N~L CH2 S N~N~ N-CH2C02H
o C02H O
C6H5-cH-coNH~s~
o ~LCH2-S l~N~N~N-CH2C2H
C02H
~, BB-S488
BB-S488; 7-(D-Mandelamido~-~-(2-carboxymethyl-2,3-dihydro-
s-triazolo[4,~-b~pyridazin-3-on-6-ylthiomethyl)-3-cephem-
4-carboxylic Acid (~)
D-(-)-Mandelic acid 0-carboxyanhydride (U S Patents
~,167,549, ~,840,531 and ~,910,900~, (1, 400 mg, 2.~ m.mole)
was added portionwise to a solution of 7-amino-~-(2-carboxy-
methyl-2,~-dihydro-s-triazolo[4,3-b]pyridazin-~-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid (2, 657 mg., 1 5 m mole)
and sodium bicarbonate (445 mg, 5 3 m mole) in 50~ aqueous
acetone (~0 ml ) at ca 0 C. with vigorous stirring. The
mixture was stirred for 1 hour at room temperature and
evaporated under reduced pressure below 40 C to remove
acetone The resulting aqueous solution l"as washed with
ether and acidified to pH 1 with dilute HCl to afford a
gummy precipitate, which was collected by filtration,
washed with water and dissolved in tetrahydrofuran (THF)
(100 ml.). The THF solution was treated with a small amount
- 57 -
, , _
~1477Z4
of active carbon, dried over anhydrous sodium sulfate and
filtered. The filtrate was evaporated under reduced pres-
sure and the residue was triturated with ether. The pale
yellow precipitate was collected by filtration and chro-
matographed on a silica gel column (Wako-gel C-200, 10
g.) eluted with a solution of chloroform-methanol (20:1).
The fractions containing the desired product were combined
and concentrated under reduced pressure. The concentrate
was diluted with ether (100 ml.) to precipitate the
product (3), which was collected by filtration, washed
with ether (30 ml.) and dried. Yield 279 mg (34~).
M.p. 173-176 C. (dec ).
ir: ~ KBax 3600-2400, 1770, 1720, 1520, 1495, 1~65,
1245 cm~l.
uv: ~ EtxH 254 nm (~ 18000), 297 nm (~ 9000, sh).
nmr ~ DMS-d6 3 68 (2H, m, 2-H), 4.03 (lH~ d~ J=13
Hz, 3-H), 4.34 (lH, d, J=13 Hz, 3-H~, 4.64 (2H, s,
NCH2CO), 5.00 (lH, d, J=4 Hz, 6-H), 5.02 (lH, s, PhCH),
5.63 (lH, d-d, J=4 & 9 Hz, a doublet with addition of
D20, J=4 Hz, 7-H), 6.97 (lH, d, J=10 Hz, pyridazine-H~,
7.1-7.4 (5H, m, phenyl-H), 7.60 (lH, d, J=10 Hz, pyri-
dazine-H), 8.60 (lH, d, J=9 Hz, disappeared with addi-
tion of D20, CONH).
Anal. Calc'd. for C23H20N608S2 / 2
H, 3.64; N, 14.45; S, 11.03. Found: C, 47.34; H, 3.48;
N, 13.90; S, 11 01.
*Trade Mark
- 5~ -
11 1477;~4
In vitro activity (Table 1)
The MIC's were determined by tne Steers' agar
dilution method using Mueller-Hinton agar against 4 gram-
positive and 28 gram-negative bacteria and the results are
shown in Table 1.
In vitro activity (Tables 2 and ~)
MIC determinations were performed by serial two-
fold agar dilution method using Steers' apparatus on
Mueller-Hinton agar plate against 51-gram-positive and 95
gram-negative bacteria. The results are shown in Tables
2 and ~.
Media effect on ~IC
The MIC's were determined by using three kinds
of agar media [Nutrient (NA), Mueller-Hinton (MHA) and
Heart-Infusion (HIA)], The results obtained with BB-S488
and cefamandole are shown in Table 4, which indicates
little media effect in these cephalosporins.
lood levels in mice
Groups of mice weré administered subcutaneously
graded doses of 40, 20 and 10 mg.~kg.. The blood sam-
ples collected from orbital sinuses were assayed by the
paper disc-agar diffusion method on Sarcina lutea PCI 1001
plates. The results are shown in Table 5.
In vivo activity
Comparative in vivo evaluation was made by the
standard experimental infection in mice against the
_ ~9 _
7f~
following pathogenic bacteria:
S. aureus Smith
-
E. coli Juhl
K. pneumoniae A9977
The results are shown in Table 6,
~77;~4
Table 1. The in vitrG Antibacterial Activity of BB-S~88
_
By Agar Dilution Method (Mueller-Hinton Agar).
_ MIC (mcg./ml.)
Test Organism BB-S488 Cefamandole
_ . . _ . _ .
S. aureus Smith A9537 0.2 .5
S. aureus A9497 0.1 0.05
S. aureus BX-1633 A9606 0,4 0.1
St. faecalis A9536 100 50
E. coli NIHJ 0.025 0.025
E. coli ATCC 8739 0.1 0.05
E. coli Juhl A15119 0.2 0.4
E. coli BX-1373 0.2 o.8
E. coli A15810 0.1 0.4
E. coli A9660 .5 0.1
E. coli A15147 3.1 0.4
Kl. pneumoniae A9678 3.1 3 1
K1. pneumoniae A9977 0.05 0.2
Kl. pneumoniae A15130 0.2 o.8
Kl. pneumoniae A9867 0.2 o.8
Pr. vulgaris A9436 0.1 0.4
Pr. vulgaris A9699 0.~ 6.3
Pr. mirabilis A9554 0.05 0,4
Pr. mirabilis A9900 0.1 o.8
Pr. morganii A9553 >100 >100
Pr. morganii A20031 0.1 o.8
Pr. rettgeri A15167 0,05 0.2
Ps. aeruginosa A9930 >100 >100
Ps. aeruginosa A9843 >100 >100
Shig. dysenteriae 0.025 0.1
Shig. flexneri A9o84 12.5 6.3
Shig. sonnei A9516 0.025 ~5
Serr. marcescens A20019 100 100
Enterob. cloacae A9656 ~.1 3.1
Sal. enteritidis A9531 0.05 0.1
Sal. typhosa A9498 0.05 0.1
B. anthracis A9504 0.1 0.1
~77;;~'~
Table ?. In vitro Antibacterial Activity in MueIler-
Hinton Agar (Gram-positive)
MIC (mcg./ml.) _
Code Cefaman-
No. I Test Organism BB-S488dole
_
Sa-2 S. aureus Smith A9537 0.4 0.2
Sa-3 S. aureus No. 193 o.8 0.2
Sa-8 S. aureus 0.4 0.2
Sa-9 S. aureus l~o. 193 o.8 0.2
Sa-10 S. aùreus A20239 1.6 0,4
Sa-ll S. aureus BX-1633 A9606 0,4 0.2
Sa-12 S. aureus A9497 0.2 0.1
Sa-29 S. aureus No. 193 1.6 o.8
Sa-33 S. aureus Terajima 0.0125 0.0125
Sa-34 S. aureus A15092 o.8 0.2
Sa-3~ S. aureus A15094 o.8 0.4
Sa-3~ S, aureus Russell o.8 0.4
Sa-37 S. aureus A9524 1.6 o.8
Sa-38 S. aureus A9534 o.4 0.2
Sa-39 S. aureus A9578 o.8 0.4
Sa-40 S. aureus A9601 o.8 0.4
Sa-41 S. aureus A9602 o.8 0.2
Sa-44 S. aureus A1~097 25 25
Sa-56 S. aureus A9f~30 3.1 o.8
Sa-57 S. aureus A9748 25 3.1
Sa-58 S. aureus A15033 12.5 1.6
Sa-59 S. aureus A15096 100 6.3
Sa-60 S. aureus A20604 50 3 1
Sa-61 S. aureus A20605 100 6 ~
Sa-62 S. aureus A20606 -~.1 o.8
Sa-6~ ~S. aureus A20607 >100 12.5
Sa-64 S. aureus A20608 100 6.3
Sa-65 S. aureus A20609 100 6.3
Sa-66 S. aureus A20610 100 6.3
Sa-67 S. aureus ~20611 100 6.3
Sa-68 ,S. aureus A20612 1.6 0.4
Sa-69 S. aureus A20613 100 6.3
Sp-1 S. pyo~enes S-23 0.4 0.1
Sp~2 S. ~yogenes Dick o.l~0.1
Sp-3 S. pyogenes A9604 0,4 0.1
Sp-4 S. pyogenes A20065 0.2 0.1
Sp-5 S. pyogenes A15040 0,4 0.1
Sp-6 S. pyogenes A20066 0.4 0.1
Sp-7 S. pyogenes Dig 7 0,l~0.1
Sp-8 S. pyogenes A15041 o.l~0.1
Sp-9 S. pyogenes A20201 0.4 0.1
Sp-10 S. pyogenes A20202 0.4 0.1
Dp-l D. pneumoniae Type II 0.2 0.2
Dp-2 D. pneumoniae Type I Neufeld 0.2 0.2
Dp-3 D. pneumoniae Type III 0.2 0,2
Dp-4 D. pneumoniae A9585 0.2 0.2
Dp-5 D. pneumoniae A15069 0.2 0.2
Dp-6 D. pneumoniae A20167 0.2 0.2
Dp-7 D. pneumoniae A20759 0.2 - 0.2
Dp-8 D. pneumoniae A207~9 0.2 0.2
Dp-9 D. pneumoniae A20770 0.2 0.2
- 62 -
'~ 77Z4
Table 3. In vitro Antibacterial Activity in Mueller-`
Hinton Agar (Gram-negative)
MIC (mcg./ml.)_
Code , Cefaman-
No. Test Organism BB-S488 dole
.
Ec-l E. coli NIHJ 0.2 0.1
Ec-3 E. coli Juhl A15119 0.2 o.8
Ec-4' E. coli A15169 12.5 6,3
Ec-5 E. coli K-12 ML-1630 A20363 0.2 o.8
Ec-ll E. coli A203~6 5 25
Ec-15 E. coli ATCC 8739 0.2 0.1
Ec-34 E. coli A9660 0.1 0.1
Ec-~5 E. coli A9435 0,4 o.8
Ec-3~ E. coli A15147 3,1 1.6
Ec-40 E. coli A20361 0.2 o.8
Ec-44 E. coli A9535 0.1 0.1
Ec-45 E. coli A15148 3.1 1.6
Ec-46 E. coli A15164 25 12.5
Ec-47 E. coli A15170 100 50
Ec-49 E. coli A20107 0.4 0.2
Ec-50 E. coli A20109 0.2 o.8
Ec-51 E. coli A20343 5 ~_2.5
Ec-56 E. coli A20365 25 12.5
Ec-58 E. coli A957~ 0,4 1.6
Ec-59 E. coli A207~6 0.2 o.8
Ec-62 E. coli A20895 0.4 o.8
El-l E. cloacae A9656 3.1 3.1
El-2 E. cloacae A20364 3.1 3.1
El-4 E. cloacae A20650 1.6 1.6
El-6 E. cloacae A9657 o.8 o.8
El-7 E. cloacae A9659 1.6 o.8
El-8 E. cloacae A9655 1.6 1.6
El-9 E. cloacae A20021 >100 100
El-ll E. cloacae A20344 >100 >100
El-12 E. cloacae A21006 1 .G 3.1
El-14 E. cloacae A20953 , o.8 3.1
Pm-l P. mirabilis ~9554 0.1 o.8
Pm-2 P. mirabilis A9900 0.2 1.6
Pm-3 P. mirabilis A20119 4 3.1
Pm-4 P. mirabilis A20454 0.2 1.6
Pm-5 P. mirabilis A9702 0.1 o,8
Pm-6 P. mirabilis A21222 1.6 1.6
Pg-l P. morganii A95~3 >100 >100
Pg-2 P. morganii A20031 0.2 .1.6
Pg-3 P. morganii A9636 o,8 1,6
Pg-5 P. morganii A15166 0.1 0.2
Pg-6 P. morganii A20455 0.4 1.6
Pg-7 P. morganii A20~57 0.2 o.8
Pg-8 P. morganii A15153 0.1 o.8
Pg-9 P. morganii A15149 o.8 3.1
Pv-l P, vulgaris A9436 0.2 o.8
Pv-2 P. vulgaris A9526 6.3 , 1,6
Pv-3 P. vulgaris A9699 6.3 5
Pv-~ P. vulgaris ATCC 9920 , 0.1 0.2
Pv-5 P, vulgaris A9539 25 >100
Pv-6 P. vulgaris A971~ 0.1 o.8
Pv-7 P. vulgaris A21240 25 >loo
- 63 -
~477Z4
Table 3 (Continued)
Code Cefaman-
No. Test OrganismBB-S488 dole
Pr-l P. rettgeri A15167 0.1 0.2
Pr-2 P. rettgeri A9637 0.1 0.1
Pr-4 P. rettgeri A20645 0.1 0.1
Pr-5 P. rettgeri A20915 0.2 o.8
Pr-6 P. rettgeri A20920 0.1 0.2
Pn-l P. inconstans A20615 0.1 o.8
Ps-l P. stuartii A20745 0.4 o.8
Ps-2 P. stuartii A20~94 0.2 o.8
Ps-3 P. stuartii A20911 o.8 o.8
Ps-4 P. stuartii A21051 5 25
Ps-5 P. stuartii A21057 0.2 o.8
Kp-l K. pneumoniae Dll 0.1 o.8
Kp-2 K. pneumoniae A96783.1 1.6
Kp-3 K. pneumoniae A99770.1 o.8
Kp-4 K. pneumoniae A15130 0.2 0 8
Kp-7 K. pneumoniae A98670.4 o:8
Kp-8 K. pneumoniae A20680 25 12.5
Kp-9 K. pneumoniae A206~6 12.5 12.5
Kp-10 K. pneumoniae A20328 6.3 3.1
Kp-ll K. pneumoniae A20330 1.6 12.5
Kp-12 K. pneumoniae A21228 6.3 6.~
Kx-2 Klebsiella sp. A9662 0.4 1.6
Kx-3 Klebsiella sp. A20346 0.2 o.8
Sm-l S. marcescens A20019 25 25
Sm-2 S. marcescens A20335 3.1 12.5
Sm-3 S. marcescens A203~6 6.3 12.5
Sm-4 S. marcescens A20442 6.3 12.5
Sm-5 S. marcescens A20222 3.1 12.5
Sm-~ S. marcescens A20460 6.3 12.5
Sm-9 S. marcescens A20333 G.3 5
Sm-10 S. marcescens A20334 6.3 5
Sm-ll S. marcescens A20459 6.3 25
Sm-12 S. marcescens A20461 6.3 5
Se-l S. enteritidis A9531 0.1 0.2
St-1 S. typhosa 0.1 0.2
Sh-1 S. paratyphi 0.1 0.2
St-101 S. typhimurium 0.1 0.2
Sd-l S. dysenteriae 0.1 0.2
Sr-l S. flexneri A9684 12.5 3.1
Ss-l S. sonnei Yale 0.1 0.1
Cx-l Citrobacter sp. A20673 1.6 1.6
Cx-2 Citrobacter sp. A20694 1.6 1.6
Cx-3 Citrobacter sp. A20695 1,6 1.6
~1~7724
U~
. . C~ I o~ ~ 0O~ o~o~ C~CU~C~I ~
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o U~ o U~ o o o o
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O C~J ~I N O O O O lr~L~ O
. ~ ~ A
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A ~ ~
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''C1 0 0 0 N N ~ ~ ~I N J N H oCI H N --1~ O Ir~ N 1~ 0 N
~ ~ 8 g g ~ 8 g ~ ~ o o g o o o N O N g O O O
O A ~1~1 ~I H r-l ~1
C~ ~\ A A /\ A A
a) L~ ,
0 0~C) O ~ ~ ~ ~ Na:~ N ~'J ~1 C~l ~J CO ~ ~ N
CO ¢
~A;1 J ¦--¦ ~I O O ~0 O O ~0 ~D O O O O O O O O O O O O O O L~ O O O O
U:) ~ O O O O O O O O 1~ N O O O
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0 H H N r~ ~t 0 N N 0 H H N ::~; ~ 0 ~
¢ O~OOOOOO~OOOC~i0000000000000000000
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00~ ~ A A /~ A A'l ~
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I~ ~ O ~
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H ~3 ~ r-l * * ~~ H O ~ ~ O * H L~ ~1 0 ~ t~:~
a~ u~ ¢ ~ ~ J a~ O ~ t~ D J C~ l O O O~ O
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c C ~* ~ * * * ~ ~ ¢ ¢ ~ ~) o~ O o Is~ o o o ¢ ¢ d
o ~d ~ H o a~ d ~.o 0 ~ J ~o a~ a~ ¢ ¢ cs~ N N H N N C'J ¢
tl~ ~ I ~, ¢ ~ .J ~ ~ a~ N N o a~ ~ ¢ ¢ ¢ ¢ ¢ ¢ ¢ ¢ ¢
S~ ~ X ~ 1 ~ ¢ ¢ ¢ ~
~ o ~ m ¢ tn ~ ~ o ~ o ~ O r~
a) . ~1 H ~ H r l C~,1 rl H H r( ,1 rl h rl rl rl U Cl ~ O
q~ ~u~ ¢ ¢ ¢ ¢ ¢ ~ d O O H h h ~ s~ O ~ ~ ~ Ul cq ,1 ~
rn ~ J c~ ~J fi fi H ~ d bD h. h h ~ a) W h
E-) h h h O ~ H H H H H ~ O O O ~ rl H ~1 5-~ h h ~ h ~ ~ h h
n~ ~ O o o o o o o ~~ rl O O O a
................ ,~
~ O ~ ~
rl ~) N H~t ~\~l r~,l H ~ II~H N r-l r~ r~l ~H N r~ N~ l H CU r~ ~ H
,D ~ O I ~ ~ ~ I I I I I ! I I I I I
t~ O ._ n~ ~ ~ ~ c) C) C) C) C) C) C) H H H p Pl ~ ~ '' F ~ 1 bl\ b0 ~ tl~ tn tl~ ~ r~
E~ ~" . c~ ~ r~ r~ r l r~ r~ r~ ~ l sl ~A p~ p~ ~; p; r~l p~ p~ p~ ~ *
- 65 -
1147724
Table 5. Subcutaneous Mice Blood Levels
Mcg./Ml.
. . Dose Time BB-S488 Cefamandole
40 mg./kg. 15~ 19 18
30' 17 11
60' 12 3.9
120' 1.6 0.3
20 mg./kg. 15' 7.4 9.5
30~ 6 4.1
60' 4.~ 1
120' 0.7 <0.1
10 mg./kg. 15' 5 3.8
3' 3 1.5
60' o.8 0.3
120' -- 0.1
. . .
- 66.-
~1~77Z~
Table 6. In vivo Activity
.
Test Organism Dose BB-S488 Cef~na~dole
S. aureus Smith 25 mg./kg. 5/5* 5/5
6.3 5/5 5/5
1.6 5/5 5/5
0.4 5/5 1/5
0.1 2/5
PD~o 0.12 mg./kg. o.6 mg./kg.
E. coli Juhl25 mg./kg 5/5 5/5
6.3 5/5 5/5
1.6 5/5 2/5
4 5/5 0/5
0.1 2/5
PD50 0.12 mg./kg. 1.8 mg./kg.
K. ~neumoniae25 mg./kg. 5/5 5/5
A9977
6.3 5/5 1/5
1.6 5/5 /5
0.4 4/5 0/5
0.1 0/5
PD50 0.26 mg./kg.6.25 mg./kg.
*No. of survivors/No. tested
- 67 -
_ _
~14772~
Run No. of Dose BB-S Cefc~n-
Test Organism No. LD50 (mg./kg.) 488 andole
E. cloacae C-811 1 x 10100 5/5 5/5
A204~4 ~El-l9)
5/5 5/5
6.3 5/5 5/5
1.6 4/5 4/5
0,4 2/5 1/5
PDso (mg./kg,~ 0.54 o.8
_ _ _
Urinary Recovery in Rats
- ~ Recovery (0-24 Hrs.
Dose tsc) BB-S488 Ce~amandole
10 mg./kg. 38.8 58.3
Nephroto ~ Test in Rabbits
No nephrotoxic sign was seen in the rabbits
treated with 100 mg./kg. (iv) of BB-S488 while cephalori-
dine showed severe nephrotoxicity in the comparative test,
- 68 -
. _ -
~77Z4
Additional ~ O Data (~in~le sc l`re~t;n~e~
Run D Dose BB-S Cefam-
Test Organism No. L 5o (mg./kg.) 488 andole
K pneumoniae c-805 3xlO 25 5/5 5/5
6 ~ 3 5/5 1/5
1.6 5/5 0/5
0.4 4/5 0/5
o,1 o/5 _ __
PD50 (mg./kg.) 0.26 9.4
P. vulgaris C-808 lxlo 50 --- 3/5
A94-3~ (Pv-l)
5/5 ___
12.5 --- 1/5
6 - 3 5/5 ___
3.1 --- o/5
1.6 3/5 -- -
o .8 - -- 0/5
0.4 1/5 - - -
PD50 (mg./kg.) 1.1 36
P. mirabilis C-810 lx103 50 --- 2/5
A9900 (~m-2)
5/5 ___
12.5 --- 1/5
6.~ 5/5 ---
3.1 - _ o/5
1.6 4/5 ---
o.8 --- 0~5
o .4 0/5 - - -
0.2 --- /5
o .1 0/5 - - -
PD50 (mg~/kg.) 1.1 50
. . .
77Z4
Stability of BB-S488
Stability of BB-S488 was determined in both a
10~ and an 0.02% solution. The stability is indicated as
the relative activity remaining in the test solution at
given periods to the initial solution. Tlle activity was
a~ayed using paper discs on B. subtilis PCI 219 plate
_
(pH 6),
(1) Stability in a 10~ Aqueous Solution at Room Temperature
1 Remaining Activity (~
Compound ~ 0 2 3 7 Days
BB-S488 6,1 100 128 90 116
(l)Unadjusted pH of the 10~ solution.
2~ Remaining ActivitY (%)
Compoundp~( 0 1 2 3 7 Days
r4 100 93 78 102
BB-S488 ~ 7 100 87 64 56
~ 10~ 20 14 1~ 0
(2)
pH 4: O. 1 M AcOH - NaOAc buffer.
pH 7: 0.1 M phosphate buffer.
pH 9: 0,1 M NH40H-NH4Cl buffer.
-- 70 -
~1~7729~
EXA~lPLE 2
~CHCOOH C12CIICOCl ~CHCOOH 1 SOC12
OH OCOCHC12J
D~
CHcocl H-AC~-S-CMTP(3~ [~ CHCO-ACA-S-CMTP~
OCOCHC12 OCOCHC12
2C3
HCO-ACA-S-CMTP
OH
BB-S48
Dichloroacetylmandeloyl Chloride (2~
A mixture of D(-)-mandelic acid (1, 1.52 g,,
10 m.mole) and dichloroacetyl chloride (4.41 g.~ ~0
m.mole) was heated at 80-85~ C. for 1.5 hrs. and the
excess dichloroacetyl chloride was removed under diminished
pressure. To the residue was added thionyl chloride (2.5
ml.) and the mixture was heated under reflux for 1.5 hrs,
Excess thionyl chloride was removed by distillation and
dry benzene was added. Evaporation was repeated. The
residual oil was ~ept over KOH at 1 mm llg overnight at
room temperature to remove dichloroacetyl chloride.
Yield, 2.8 g. (100~). This product was used in the next
- 71 -
. ` 11~7;~4
step without further purification.
ir: ~ laq. 1780, 1160 cm 1.
nmr: ~ pC14 5.91 (lH, s, PhCH or COCHC12), 6.oo (lH,
s, PhCH or COCHC12), 7.32 (5H, s, phenyl-H~.
BB-S488,_7-(D-Mandelamido)-3-(2-carboxymethyl-2,3-dihydro-
s-triazolo~4,~-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-
4-carboxylic Acid (4)
A solution of the above-obtained dichloroacetyl-
mandeloyl chloride (2, 2.8 g., 10 m.mole~ in dry acetone
(30 ml.) was added dropwise to a stirred solution of 7-
amino-3-(2-carboxymethyl-2,3-dihydro-s-triazolo[4,3-b]-
pyridazin-3-on-6-ylthiomethyl)-~-cephem-~-carbo~.ylic
acid (H-ACA-S-CMTP)(~, 3.94 g., 9 m.mole) and triethyl-
amine (3.54 g., 35 m.mole) in 50% aqueous acetone (120 ml.
at 0-5 C. The mixture was allowed to rise to room tem-
perature during 1 hour with stirring and was adjusted to
pH 11 with 5~ aqueous sodium carbonate (ca 12 ml. was
required). The mixture was allowed to stand at room tem-
perature for 30 minutes, acidified to pH 1 with dilute
HCl and evaporated under reduced pressure to remove
acetone below 40 C. The precipitate was collected by
filtration, washed with water (20 ml.~ and air-dried.
The dried material was dissolved in THF (150 ml.~,
stirred for 5 minutes at 40-50 C. and filtered to remove
insoluble unreacted ~ (0.54 g., 14~ recovery~. The fil-
trate was chromatographed on a silica gel column (T~Jako-
gel, C-200, ~0 g.) and eluted with chloroform-methanol
(100:5). The eluates were collected in 50 ml. fractions
monitoring by tlc (silica gel, solvent, CH3CM-water = 4:1,
1~77Z4
detected with I2). The fractions containing the desired
product were combined, treated with a small amount of
carbon and evaporated under reduced pressure. The resi-
due was triturated with chloroform (50 ml.) to yield,
2.~6 g. (46~) of 7-(D-mandelamido)-3-(2-~arboxymethyl-
2,3-dihydro-s-triazolo[4,3-b~pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid (4). M.P., 165-
170 C. (dec.).
ir: ~ KBax 3600-2500, 1780, 1720, 1500, 1410, I355,
1220, 1195 cm 1.
uv: ~ maH 254 nm (e, 18300), 297 nm (sh, &, 9300).
nmr: ~ ppSO d6 3.84 (2H, m, 2-H), 4.17 (211, d, 13 Hz,
3-H), 4.50 (lH, d, 13 Hz, 3-H), 4.~2 (2H, s, NCH2COO),
5.20 (lH, d, 4.5 Hz, 6-H), 5.25 (lH, s, PhCH), 5.87 (lH,
d-d, 4.5 & 9 Hzj 7-H, a doublet (J=4.5 Hz) by addition of
D20), 7.25 (lH, d, 11 Hz, pyrid~zine-H), 7.4-7.7 (5H, m,
phenyl-H), 7.90 (lH, d, 11 Hz, pyridazine-l~), 9.0 (lH, d,
9 Hz, 7-CONH, disappear by addit~on f ~2)
Anal. Ca1C'd for C23H20N608S23/ C 3
H, 3.16; N, 12.69; S, 9.69. Found: C, 43 11, 43.22; H,
2.97, 3.06, N, 12.80, 12.77; S, 9.64.
~47~7Z4
EXAMPLE 3
/~ 99~ HCOOH G\ SOCl
_CHCOOH -- ---) ~ CHCOOH 2
OH OCHO
H-ACA-S-CMTP (3) /~
CHCOCl~ ~ ~ CHCO-ACA-S-CMTP
OCHO OCHO
6 7, BB-S494
0-Formyl-D(-)-mandelic ~cid t5~
A mixture of D(-)-mandelic acid (1, 5.0 ~., 33
m.mole) and 99~ formic acid (80 ml.) was heated at 80-
90 C. for 12 hours. The mixture was evaporated and
toluene (100 ml ) was added to the residue and evaporated
under reduced pressure to remove formic acid azeotropically.
The residue was dissolved in benzene (200 ml.~ and the
solution was washed with water (2 x 50 ml.). The organic
layer was separated, dried with anhydrous sodium sulfate
and evaporated under reduced pressure. The residual oil
was triturated with cyclohexane (50 ml.) to crystallize.
Yield, 3.70 g. (63$~ of 0-formyl-D(-)-mandelic acid (5)
as colorless prisms. M.P., 56-59 C. (lit. M.P., 55-58 C. ) .
ir: ~ ~ar 3400-2800, 1755, 1720, 1160, 990 cm I,
nmr: ~; ppml~ 5.98 (lH, s, PhCH), 7.31 (5~1, m, phenyl-
H), 8~.o5 (lH, s, OCHO), 10.05 (lH, s, COOH, disappeared
by addition of D20).
- 74 -
1~47'7;~
0-Formyl-D(-~-mandeloyl Chloride (6~
A mixture of 5 (2.0 g., 11 m.mole) and thionyl
chloride (10 ml.) was heated under reflux for 2 hours.
Evaporation of the excess thionyl chloride and distilla-
tion of the residue under reduced pressure afforded the
acid chloride 0-formyl-D(-)-mandeloyl chloride ~6)
Yleld, 1.53 g. (70~). B.P., 120-122 C./15 mmHg.
ir: ~ miq 1805, 1740, 1160, 1140 cm~l.
BB ~ -(D-0-Formylmandelamido)-3-(2-carboxymethyl-2,3-
dihydro-s-triazolor4,3-b]pyridazin-3-on-6-ylthiomethyl)-
3-cephem-4-carboxylic Acid (7)
A solution of 0-formyl-D(-)-mandeloyl chloride
(6) (1 0 g., 5.1 m.mole) in dry acetone (10 ml.) was added
dropwise to a cold (0 to 5 C.) solution of 7-amino-3-
(2-carboxymethyl-2,3-dihydro-s-triazolo[4,3-b~pyridazin-
3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid (3, 1.75
g., 4 m.mole) in 50~ aqueous acetone (70 ml.) containing
sodium bicarbonate (1.34 g.~ 16 m.mole). The mixture was
stirred for 30 minutes at room temperature and washed with
ether. The aqueous layer was acidified to pH 1 with dilute
HCl. The separated oily gum was collected and dissolved
in THF (100 ml.). The solution l~as treated with a small
amount of carbon and dried with anhydrous sodium sulfate.
Evaporation of the solvent under reduced pressure to 10
ml. and dilution with ether afforded the title compound
(7) as a pale yellow amorphous powder, 0.91 g (~8%)
M.P., 172~176 C.(dec.).
ir: ~ mBax ~600-2400, 1775, 1720, 1550, 1355, 12~0,
1160 cm~l
uv: ~ mt~I 254 nm (, 20800), 297 nm (sh, ~, 10500)
- 75 -
1. .
~477'~
nmr: ~ ppMmO d6 3.4-4.5 (4H, m, 2-H and 3-H), 4.67 (2H,
s, NCH2COO), 4.97 (lH, d, 4 Hz, 6-H), 5.66 (lH, d-d, 4
& 8 Hz, 7-H), 7.2-7.5 (5E~, m, phenyl-H), 7,64
(lH, d, 10 Hz, pyridazine-H), 8.29 (lH, s, CHO), 9.29
(lH, d, 8 Hz, 7-CONH, disappeared by addition of D20).
Anal. Calc'd. for C24H20N609S2 lI2
H, 3.58; N, 13.59; S, 10.37. Found: C, 46.70, 47.20;
H, 3.25, 3.34; N, 13.37, 13.78; S, 10.84.
EXAMPLE 4
BB-S488; 7-(D~ andelamido)-3-(2-carboxymethyl-2,3-
dihydro-s-triazolo-~4,3-b~pyridazin-3-on-6-ylthiomethyl)-
~-cephem-4-carboxylic Acid (4~
A mixture of 7-(D-O- formylmandelamido-3-(2-car-
boxymethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-
6-ylthiomethyl)-~-cephem-4-carboxylic acid (7) (484 mg.,
0 81 m.mole~ and sodium bicarbonate (748 mg., 8.9 m.mole)
in water (4 ml.) was stirred for 4 hours at room tempera-
ture, and acidified to pH 1 with dilute HCl. 'rhe preci-
pitate (500 m~.) was collected by filtration, washed with
water (2 ml.) and chromatographed on silica gel column
(Wako-gel, C-200, 5 g.). The column was eluted with
chloroform containing increasing methanol (3-5~) as
eluent, and the fractions containinG the product ~ere
combined, treated with a small amount of carbon and eva-
porated under reduced pressure. The residue ~las triturated
with ether to give 277 mg. of 7-(D-mandelamido)-3-(2-car-
boxymethyl-2,3-dihydro-s-triazolo~4,3-b]pyridazin-3-on-6-
ylthiomethyl)-3-cephem-4-carboxylic acid tBB-S488; 4).
The nmr-estimation of this product sho~Jed 10~ of 7 still
remained.
- 7~ -
~77Z4
ir: ~ KBar 3600-2400, 1770, 1720, 1520, 1495, 1365,
1230 cm~l.
uv: ~ EaH 254 nm (~, 20000), 297 nm (sh, ~, 9600).
EXAMPLE 5
Sodium Salt of BB-S488
Sodium-2~ethylhexanoate (SEH) (4.0 ml., 1 M
solution in ethyl acetate) was added to a solution of
7-(D-mandelamido)-3-(2-carboxymethyl-2,3-dihydro-s-tri-
azolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-
carboxylic acid (4) (2.25 g., 3.93 m.mole) in THF (200
ml.). The precipitate was collected by filtration, washed
with THF (50 ml.~ and dried at 60 C.~l mmHg for 3 hours.
Yield of sodium 7-(D-mandelamido)-3-(2-carboxymethyl-2,3-
dihydro-s-triazolo~4,3-b~pyridazin-3-on-6-ylthiomethyl)-
3-cephem-4-carboxylate,1.96 ~- (bio-yield, 97%),
M.P., 230-240 C. (dec.). The pH o the 10~ aqueous
solution was 3.6.
ir: J m~x 3600-3000, 1765, 1710, 1605, 1390, 1360, ll9P,
1080, 1065 cm~l. -
uv: ~ maater nm(El'cm) 252 (357), 310 (sh, 140).
nmr: ~ pD~m 3.43 (lH, d, 19 Hz, 2-H), 3.87 (lH, d, 19 llz,
2- ), 4.15 (lH, d, 14 Hz, 3-H), 4.53 (lH, d, 14 Hz, 3-_),
5.16 (lH, d, 4.5 Hz, 6-H), 5.36 (lH, s, PhCH), 5.73 (lH,
d, 4.5 Hz, 7.H), 7.13 (lH, d, 10 Hz, pyridazine-H), 7.57
(5H, s, phenyl-H), 7.69 (lH, d, 10 Hz, pyridazine-H).
Anal. Calc'd. for C23HlgN60~S2N 5/ 2
H, 3.51; N, 13.62; S, 10.39. Found: C, 44.93, 44.79; H,
3.31, 3.15; N, 13.~1, 13.33; S, 10.19.
- 77 -
24
EXAMPLE 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 ~qater (20 ml.) to adjust to pH 6.o. The solution was
lyophilized to yield 3.4 g. (bio-yield, 97%) of disodium
7-(D-mandelamido)-3-(2-carboxymethyl-2,3-dihydro-s-tri-
azolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-
carboxylylate. M.p., >240 C. (dec.). The pH of the
5% aqueous solution was 5.4.
ir: J mBar 3600-3000, 1760, 1710, 1605, 1390, 1360,
1190, 1080, 1060 cm~l.
uv: /1 mater nm (El'Cm) 252 (320), 310 (124).
nmr: ~ pp2m 3.43 (lH, d, 19 Hz, 2-H), 3.90 (lH, d, 19
Hz, 2-H), 4.15 (lH, d, 14 Hz, 3-H), 4.53 (lH, d, 14 Hz,
3-H), 4.75 (2H, s, NCH2C0), 5.22 (lH, d, 4.5 Hz, 6-H),
5.42 (lH, 8, PhCH), 5.73 (lH, d, 4.5 Hz, 7-H), 7.22 (lH,
d, 10 Hz, pyridazine-H), 7.65 (5H, s, phenyl-H), 7.77
(lH, d, 10 Hz, pyridazine-H).
Anal. Calc~d. for C23H18N68S2Na2 3/ 2
42.92; H, 3.29; N, 13.06; S, 9.96. Found: C, 42.90,
43.19; H, 3.o6, 3.01; N, 13.04, 13.03; S, 9.97.
- - 78 -
~ .
1~477~
ExamDle 7
Substitution of the D-mandellc acid carboxy-
anhydrlde in the procedure of Example 1 Or an
equlmolar welght Or the carboxyanhydride~ prepared
ln ~lmllar fa~hlon rrom the monosub~tltuted D-
mandelic acids
D-2-chloro-mandellc acld,
D-3-chloro-mandellc acld,
D-4-chloro-mandellc acld,
D-2-bromo-mandellc acld,
D-~-bromo-mandellc acld,
D-4-bromo-mandelic acld,
D-2-rluoro-mandellc acld,
D-3-fluoro-mandellc acld,
D-4-fluoro-mandelic acld,
D-2-trlfluoromethyl-mandellc acld,
D-~-trlfluoromethyl-mandellc acid,
D-4-trlrluoromethyl-mandellc acld,
D-2-amino-mandellc acld,
D-3-amlno-mandellc acld,
D-4-amlno-mandelic acld,
D-2-nitro-mandellc acid,
D-3-nltro-mandellc acid,
D-~-nitro-mandellc acld,
D-2-hydroxy-mandellc acld,
D-3-hydroxy-mandellc acld,
D-~-hydroxy-mandellc acid,
- 79 -
~47724
D-2-methyl-mandellc acld,-
D-~-methyl-mandellc acld,
D-4-methyl-mandellc acld,
D-~-methoxy-mandellc acld,
D-~-methoxy-mandellc acld and
D-4-methoxy-mandellc acld respectlvely produces
7-(D-2-chloro-mandelamido)-3-(2-carboxymethyl-2,3-
dlhydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid,
7-(D-3-chloro-mandelamido)-3-(2-carboxymethyl-2,3-
dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid,
7-(D-4-chloro-mandelamido)-3-(2-carboxymethyl-2,3-
dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid,
7-(D-2-bromo-mandelamido)-3-(2-carboxymethyl-2,3-
dihydro-s-triazolo[4,3-b]pyridazin-3-on-~-ylthio-
methyl)-3-cephem-4-carboxylic acid,
7-(D-3-bromo-mandelamido)-3-(2-carboxymethyl-2,3-
dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-
methyl)-3-cephem-1~-carboxylic acid,
7-(D-4-bromo-mandelamido)-3-(2-carboxyrnethyl-2,3-
dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid,
7-(D-2-fluoro-mandelamido)-3-(2-carbox~nethyl-2,3-
dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid,
- 80 -
1~47'724
7-(D-3-fluoro-mandelamido)-3-(2-carboxymethyl-2,3-
dihydro-s-triazolo[4,3-b~pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid,
7-(D-4-fluoro-mandelamido)-3-(2-carboxymethyl-2,3-
dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid,
7-(D-2-trifluoromethyl-mandelamido)-3-(2-carboxy-
methyl-2,3-dihydro-s-triazolo[4,3-b~pyridazin-3-on-
6-ylthiomethyl)-3-cephem-4-carboxylic acid,
7-(D-3-trifluoromethyl-mandelamido)-3-(c-carboxy-
methyl-2,3-dihydro-s-triazolo[4,3-b~pyridazin-3-on-
6-ylthiomethyl)-3-cephem-4-carboxylic acid,
7-(D-4-trifluoromethyl-mandelamido)-3-(2-carboxy-
methyl-2,3-dihydro-s-triazolo[4,3-b~pyridazin-3-on-
6-ylthiomethyl)-3-cephem-4-carboxylic acid,
7-(D-2-amino-mandelamido)-3-(2-carboxymethyl-2,3-
dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid,
7-(D-3-amino-mandelamido)-3-(2-carboxymethyl-2,3-
dlhydro-s-triazolo[lJ,3-b]pyridazin-3-on-~-ylthio-
methyl)-3-cephem-4-carboxylic acid,
7-(D-4-amino-mandelamido)-3-(2-carboxymethyl-2,3-
dihydro-s-triazolo[4,3-b]pyridaain-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid,
7-(D-2-nitro-mandelamido)-3-(2-carboxymethyl-2,3-
dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid,
.7-(D-3-nitro-mandelamido)-3-(2-carboxymethyl-2,3-
dihydro-s-triazolo[4,3-b~pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid,
- 81 -
1~77Z~
7-(D-4-nitro-mandelamido)-3-(2-carboxymethyl-2,3-
dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid,
7-(D-2-hydroxy-mandelamido)-3-(2-carboxymethyl-2,3-
dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid,
7-(D-3-hydroxy-mandelamido)-3-(2-carbox~nethyl-2,3-
dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid,
7-(D-4-hydroxy-mandelamido)-3-(2-carboxymethyl-2,3-
dihydro-s-triazolo[4,3-b]pyridazin-~-on-6-ylthio-
methyl)-3-cephem-4-car~oxylic acid,
7-(D-2-methyl-mandelamido)-3-(2-carboxymethyl-2,3-
dihydro-s-triazolo[4,3-b]pyrid~zin-3-oll-5-yltllio-
methyl)-3-cephem-4-carboxylic acid, -
7-(D-3-methyl-mandelamido)-3-(2-carboxymethyl-2,3-
dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid,
7-(D-4-methyl-mandelamido)-3-(2-carboxymethyl-2,3-
dlhydro-s-triazolo[4,3-b~pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid,
7-(D-2-methoxy-mandelamido)-3-(2-carboY~ymethyl-2,3-
dihydro-s-triazolo[4,3-b~pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid,
7-(D-3-methoxy-mandelamido)-3-(2-carboxymethyl-2,3-
dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid, and
7-(D-4-methoxy-mandelamido)-3-(2-carboxymethyl-2,3-
dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-
methyl)-3-cephem-~-carboxylic acid, respectively.
- 82 -
77~4
'EXAMPLE 8
Sub~titutlon for the D-mandelic acid
carboxyanhydrlde in the procedure of Example 1 of an
equimolar weight of the carboxyanhydride prepared
in ~lmilar fashion from D-2-thlopheneglycollc acld
and D-~-thiopheneglycollc acid respectively produces
7-(D-a-hydroxy-2-thienylacetamido)-3-(2-carboxymethyl-
2,3-dihydro-s-triazols[4,3-b]pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid and 7-(D-a-hydroxy-
3-thienylacetamido)-3-(2-carboxymethyl-2,3-dihydro-s-
~riazolo~,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-
4-carboxylic acid, 'respectively.
EXAIIPI~ 9
7-(D-~-Hydroxy-~-phenylacetamido)-3-(2-carboxymethyl-
2,3-dihydro-s-triazolo~4,3-blpyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic Acid ~re~red From
7-D-Mandelc~midocephalospor.~nic Acid.
0,27 Mole o~ sodium 7-D-mandelamidocephalo-
eporanate ls suspended ln 1000 ml. of 0 1 M phosphate
burrer Or pH 6.4 and there is added 0.31 moles of
2-carboxymethyl-2,3-dihydro-6-mercapto-s-triazolo-
~4,3-b]pyrid~zin-3-one. The solution is
heated at 55 C. under a nitrogen atmosphere ~or five
hours. After one hour the pH is ad3usted to 6,4 by
addltion of a small amount of 40~o H~P04. At the end o~
the five hour heating period the solution is cooled
to 23 C. and the pH ad~usted to 2 by the addition
- 83 -
11477~4
of 3 N HCl under a layer of ethyl acetate. The
product 1~ extracted lnto ethyl acetate and stlrred
ror 15 mln. at 23 C. wlth 2 g, of ("Darco KB")
decolorlzlng charcoal. The mixture is then flltered
through a pad of dlatomaceous earth ("Cellte") and
the ethyl acetate removed ~rom the filtrate under
vacuum. The re~ldue i8 trlturated to a 3011d with
dlethyl ether, collected by flltratlon and drled
over P205 under vacuum to yleld solld 7-(D-a-hydroxy-
~-phenylacetamldo)-3-(2-carboxymethyl-2,3-dihydro-s-
triazolo~4,3-b~pyridazin-3-on-6-y]thiomet}lyl)-3-
cephem-4-carboxylic acid.
Example 10
H2N~S~ ~
0~ ~CH2 - SI~N~~N--Cli2C1~2COOH
C21i o
C61i5~
6~
\ / O
C \ H s _ N
OH ~ ~ c~2_ ~ ~ ~ 3-C112C 2
CO2~ o
77~4
BB-S 527; 7-[D(-)-Mandelamido~-3-12-(2-carboxyethvl)-2,3-
dihydro-s-triazolo-[4,3-b]pyridazin-3-on-6-ylthiomethyl]-
3-cephem-4-carboxylic acid
To a mixture of 7-amino-3-[2-(2-carboxyethyl)-2,3-
dihydro-s-triazolo~4,3-b]pyridazin-3-on-6-ylthiomethyl]-
3-cephem-4-carboxyllc acid (679 mg, 1.5 m mol) and NaHCO3
(445 mg, 5.3 m mol) in 50% aqueous acetone was added D-
(-)-mandelic acid O-carboxyanhydride (400 mg, 2.3 m mol) at
OC. The mixture was stirred at room temperature for 1 hour
and evaporated to remove the organic solvent. The aqueous
solution was washed with ether (3 x 10 ml), adjusted to pH
1 with dil. HCl and fiLtered to collect the crude product,
which was di~solved in TI~F (10 ml), filtered to remove
insolubles and evaporatea under reduced pressure. The oily
residue was triturated with ether. The solid ~476 mg) was
chromatographed on a column of silica gel (Wako-gle C-200,
10 g) and eluted with MeOII-CHC13 (MeOII: 0-3%). Fractions
which contained the desired product were combined and evapor-
ated to yield 287 mg ~33~) of BB-S 527. ~I.p. >155C (dec.).
irs ~Kar 3600 - 2400, 1780, 1720, 1550, 1520 cm 1.
u~: ~PI~ 7 buffer 253 nm (E 20000), 298 nm (E 9000) -
Anal. Calc'd- for C24H22N6852 / 2
4.11; N, 13.70; S, 10.45. Found: C, 47.25, 47.39; ~, 3.80,
3.76; N, 12.87, 12.77; S, 10.17.
- 85 -
1147'724
The sodium salt of BB-S 527
A suspension of the free acid of BB-S 527 (240 mg, 0.4
m mol) in water (5 ml) was adjusted at pH 6.8 with 1 N-NaOH
(0.7 ml) to give the clear solution, which was freeze-dried
to leave 234 mg (91%, bio-yield) of the sodium salt of BB-S
527 as pale yellow powder, M.p. ~210C (dec.).
ir: vmaBr 3600 - 2800, 1770, 1710, 1600 cm~l.
ApH 7 Buffer 253 nm ( 21000) 298 nm ( 9300)
Anal. Calc'd. for C24H20N6O8S2Na2~2H2O: C, 43.24: H,
3.63; N, 12.61; S, 9.62. Found: C, 43.39, 43.43; }1, 3.20,
3.36; N, 12.63, 12.68; S, 9.42, 9.22.
In vitro antibacterial activity of BB-S 527 compared
with BB-S 488 and cefamandole (determined by Steers' agar
dilution method on Mueller-Hinton agar plate)
MIC (mcq/ml)
. .
Organism B-S 527 BB-S 488 cefamandole
S. aureus Smith 1.6 0.8 0.2
S. aureus 0.4 0.4 0.1
S. aureus BX-1633 3.1 3.1 0.4
St. faecalis >100 >100 >100
E. coli NIHJ 0.4 0.2 0.05
E. coli A~CC 8?3912.5 6.3 3.1
E. coli Juhl 0.4 0.2 0.8
E. coli BX-1373 6.3 3.1 3.1
E. coli 0.1 0.1 0.1
E. coli 0.1 0.05 0.1
E. coli 6.3 3.1 1.6
- 86 -
~ . , ; _ , , . , . ____ .
~14772~
Xl. pneumoniae 6.3 3.1 3.1
Kl. pneumoniae 0.2 0.1 0.8
Kl. pneumoniae 0.8 0.4 0.8
Kl. pneumoniae 0.4 0.2 0.8
Pr. vulgaris 0.1 0.1 0.2
Pr. vulgaris 12.5 0.8 50
Pr. mirabilis 0.2 0.05 0.8
Pr. mirabilis 0.1 O.G5 0.2
Pr. morganii >100 >100 >100
Pr. morganii 0.4 0.2 0.8
Pr. rettgeri 0.2 0.2 0.4
Ps. aeruginosa >100 >100 >100
Ps. aeruginosa >100 >100 >100
Shig. dysenteriae 0.025 0.025 0.1
Shig. flexneri 50 25 6.3
Shig. sonnei 0.1 0.05 0.2
Serr. marcescens >100 >100 100
Enterob. cloacae 6.3 3.1 3.1
Sal. enteritidis 0.05 0.025 0.05
Sal. typhosa 0.1 0.05 0.1
. anthracis 0.4 0.2 0.4
- 87 -
1~4'~'7Z4
ExamDle ~
Sub~titution for the D-mandellc acid carboxy-
anhydride in the procedure Or ExamplelO of an
equlmolar weight o~ the carbox~anhydrldes prepared
ln ~imllar ~ashlon from the monosubstltuted D-
mandelic aclds
D-2-chloro-mandelic acid,
D-3-chloro-mandelic acld,
D-4-chloro-mandelic acid,
D-2-bromo-mandellc acid,
D-3-bromo-mandelic acid,
D-4-bromo-mandellc acid,
D-2-rluoro-mandellc acld,
D-~-rluoro-mandelic acld,
D-4-~luoro-mandelic acid,
D-2-trlrluoromethyl-mandelic acid,
D-~-trl~luoromethyl-mandelic acld,
D-4-trl~luoromethyl-mandellc acid,
D-2-amlno-mandellc acld,
D-3-amlno-mandellc acid,
D-4-amlno-mandellc acld,
D-2-nitro-mandellc acid,
D-3-nitro-mandellc acld,
D-4-nltro-mandelic acld,
D-2-hydroxy-mandellc acid,
D-3-hydroxy-mandellc acld,
D-~-hydrox~-mandellc acid,
- 88 -
~, _ ,. , ___ . , , . , .. ,, _
~4~772~
D-2-methyl-mandellc acid,
D-3-methyl-mandellc acid,
D-4-methyl-mandelic acld,
D-2-methox~-mandellc acld,
D-~-methoxy-mandelic acid and
D-4 methoxy-mandelic acld respectlvely produces
7-(D-2-chloro-mandelamido)-3-(2-carboxyethyl-2,3-
dihydro-s-triazolo[4,3-b]pyridazln-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid,
7-(D-3-chloro-mandelamido)-3-(2-carboxyethyl-2,3-
dihydro-s-triazolo[~ -b]pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acld,
7-(D-4-chloro-mandelamido)-~-(2-carboxyethyl-2,3-
dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid,
7-(D-2-bromo-mandelamido)-3-(2-carboxyethyl-2~3-
dihydro-e-triazolo[4,3-b]pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid,
7-(D-3-bromo-mandelamido)-3-(2-carboxyethyl-2,3-
dihydro-s-triaz~10[4,3-b~pyridazin-3-on-6 ylthio-
methyl)-3-cephem-4-carboxylic acid,
7-(D-4-bromo-mandel~mido)-3-(2-carboxyethyl-2,3-
dihydro-s-triazolo[4,3-b~pyridazin-3-on-6-ylthio-
metnyl)-3-cephem-4-carboxylic acid,
7-(D-2-fluoro-mandelamido)-3-(2-carboxyethyl-2,3-
dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-
methyl3-3-cephem-4-carboxylic acid,
- 89 -
1~77;~4
7-(D-3-fluoro-mandelamido)-3-(2-carboxyethyl-2~3-
. dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid,
7-(D-4-fluoro-mandelamido)-3-(2-carboxyethyl-2,3-
dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid,
7-(D-2-trifluoromethyl-mandelamido)-3-(2-carboxy-
ethyl-2,3-dihydro-s-triazolo[4,3-b~pyridazin-3-on-
6-ylthiomethyl)-3-cephem-4-carboxylic acid,
7-(D-3-trifluoromethyl-mandelamido)-3-(2-carboxy-
ethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-
6-ylthiomethyl)-3-cephem-4-carboxylic acid,
7-(D-4-trifluoromethyl-mandelamido)-3-(2-carboxy-
ethYl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-
6-ylthiomethyl)-3-cephem-4-carboxylic acid,
7-(D-2-amino-mandelamido)-3-(2-carboxyethyl-2,3-
dihydro-s-triazolo[~,3-b]pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid,
7-(D-3-amino-mandelamido)-3-(2-carboxyethyl-2,3-
dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid,
7-(D-4-amino-mandelamido)-3-(2-carboxyethyl-2,3-
.. dihydro-s-triazolo[4,3-b~pyridaain-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid,
7-(D-2-nitro-mandelamldoJ-3-(2-carboxyethyl-2,3- .
dihydro-s-triazolo~4,3-b~pyridazin-3-on-6-ylthio-
methyl)-~-cephem-4-carboxylic acid,
7-(D-3-nitro-mandelamido)-3-(2-carboxyethyl-2,3-
dihydro-s-triazolo[4,3-b~pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid,
- 9 -
i~7724
7-(D-4-nitro-mandelamido)-3-(2-carboxyethyl-2,3-
dihydro-s-triazolo~4,3-b]pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid,
7-(D-2-hydroxy-mandelamido)-3-(2-carboxyethyl-2,3-
dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid,
7-(D-3-hydroxy-mandelamido)-3-(2-carboxyethyl-2,3-
dlhydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid,
7-(D-4-hydroxy-mandelamido)-3-(2-carboxy~thyl-2,3-
dihydro-s-triazolo[4,3-b]pyrldazin-3-on-6-ylthlo-
methyl)-3-cephem-4-carboxylic acid,
7-(D-2-methyl-mandelamido)-3-(2-carboxyethyl-2,3-
dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-
methyl)-3-cephem-~-carboxylic acid,
7-(D-3-methyl-mandelamido)-3-(2-carboxyethyl-2,3-
dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid,
7-(D-4-methyl-mandelamido)-3-(2-carboxyethyl-2,3-
dihydro-s-triazolo~4,3-b]pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid,
7-(D-2-methoxy-mandelamido)-3-(2-carboxyet~yl-2~3
. dihydro-s-triazolo[4,~-b]pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic ac~d,
7-(D-3-methoxy-mandelamido)-3-(2-carboxyethyl-2,3-
dihydro-s-triazolo~4,3-b]pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid, and
7-(D-4-methoxy-mandelamido)-3-(2-carboxyethyl-2,3-
dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid, respectively.
- 91 -
1~ ~'77~4
EX~MPLE 12
Substitution for the D-mandelic acid carboxyanhydride
in the procedure of Example 10 of an equimolar weight or
the carboxyanhydride prepared in similar fashion from D-2-
thiopheneglycolic acid and D-3-thiopheneglycolic acid
respect~vely produces 7-(D-~-hydroxy-2-thienylacetamido)-
3-~2-carboxyethyl-2,3-dihydro-s-triazolo~4,3-b]pyridazin-
3-on-6-ylthiomethyl]-3-cephem-4-carboxylic acid and 7-
~D-a-hydroxy-3-thienylacetamido)-3-(2-carboxyethyl-2,3-
dihydro-s-triazolo~4,3-blpyridazin-3-on-6-ylthiomethyl)-
3-cephem-4-carboxylic acid, respectively.
1~47'7;~4
.
EXAMPLE 13
C0-N p C~2-S ~ -CH2COONa
C2Na
7-~(2Z)-2-Methoxyimino(fur-2-yl)acetamido]-3-(2-carboxy-
methyl-2,3-dihydro-s-triazolo~4,3-b]pyridazin-~-on-6-
ylthiomethyl)-~-cephem-4-carboxylic Acid Disodium
-
Salt; BB-S511,
To a solution of (2Z)-2-methoxyimino(fur-2-yl)-
acetic acid (507 mg., 3 m,moles) and triethylamine (0,42
ml,, 3 m,moles) in dichloromethane (6 ml,)was added oxalyl
chloride (0,26 ml., ~ m.moles) at 0-5 C. and the mixture
was stirred for 30 minutes, The mixture was evaporated at
reduced pressure to give an oily residue of the acid
chloride which was dissolved in dry acetone (10 ml,),
A~ter filtration the acetone solution was added to a mix-
ture of 7~amino-3-(2-carboxymethyl-2,3-dihydro-s-triazolo-
[4,~-b~pyridazin-3-on-6-ylthiomethyl)-3-cephem-~-
carboxylic acid (1,31 g " 3 m,moles) and NaHC0~ (504 mg "
6 m,moles) in water (20 ml,) at 0-5 C, The mixture was
stirred at 0-5 C, for 2 hours with the acetone being
removed under reduced pressure, The aqueous solution was
washed with ether (2 x 50 ml,) and adjusted to pH 1-2 with
conc, HCl to afford a precipitate which was collected by
filtration, washed with water and dried in vacuo, The
solid was dissolved in THF (tetrahydrofuran) (~0 ml,) and
- 93 -
1~77Z~
filtered. To the filtrate was added 1 M-SEH (sodium
ethylhexanoate) in ethyl acetate (3 ml.) and the result-
ing precipitate was collected by filtration and dried
in vacuo. Yield of 7-[(2Z)-2-methoxyimino(fur-2-yl)acet-
amido~-3-(2-carboxymethyl-2,3-dihydro-s-triazolo[4,~-b3-
pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid
di~odium salt was 1 0 g. (54%) mp >210 C. (dec.).
ir: vmBax 1770, 1710, 1680, 1610, 1550 cm 1.
uv ~pH mabUffer 257 nm (E 25000), 277 nm (~ 24000).
nmr: ~DMppmd6 7.7o (lH, br, furan-Ha), 7.52 (lH, d,
J=9.5 Hz, pyridazine-H), 6.87 (lH, d, J=9.5 Hz, pyridazine-H),
6 5-6.6 (2H, m, furan-H~), 5.58 (lH, m, 7-H), 5.00 (lH, d,
J=4.5 Hz, 6-H), 3.84 (3H, s, OCH3).
Anal. Calcld- for C22H17lJ7gS2Na2 2
H, 2.94; N, 15.05; S, 9.84. Found: C, 40.81, 41.02;
H, 3.o8, 3.22, N, 14.69, 14.86; S, 9.70, 9.62.
_ 9~ _
~7'7;~4
EXAMPLE 14
J~OCH3 ~CH2-5 (~ I-CH2CH2COOH
7-l(2Z¦-2-Methoxyimino(fur-2-y~ acetamido]-3-~2-(2-car-
boxyethyl)-2~3-dihydro-s-triazolor4,3-b]pyridazin-3-on-
6-ylthiomethyl]-3-cephem-4-carboxylic Acid Sodium Salt;
BB-S526
The acid chloride prepared from (2Z)-2-methoxy-
imino(fur-2-yl)acetic acid (169 mg., 1 m.mole) was dis-
solved in dry acetone (5 ml.) and filtered to remove in-
solubles. The filtrate was added to a solution of
7-amino-3-[2-(2-carboxyethyl)-2,3-dihydro-s-triazolo-
[4,3-b]pyridazin-3-on-6-ylthiomethyl]-3-cephem-4-car-
boxylic acid,(452 mg., 1 m.mole) and NaHC03 (~36 mg.,
4 m.mole) in water (10 ml.). The reaction mixture was
stirred for 2 hours in an ice-water bath. Acetone was
removed at reduced pressure. The aqueous solution was
washed with ether (2 x 10 ml.) and ad~usted to pH 1-2 with
conc. HCl. The resulting precipita~e was collected by
filtration, washed with water and dried under reduced
pressure. A solution of the precipitate in THF (10 ml.)
was treated with active carbon. A SEH solution in ethyl
acetate (1 ~ o.8 ml.) was added to the THF solution to
give 7-[(2Z)-2-methoxyimino(fur-2-yl)acetamido~-3-[2-(2-
carboxyethyl)-2,3-dihydro-s-triazolo~4,3-b]pyridazin-3-
on-6-ylthiomethyl]-3-cephem-4-carboxylic acid sodium salt
which was collected by filtration. Yield: 410 mg. (66
as mono Na salt). ~Ip ~205D C. (dec.).
- 95 -
~477'~4
ir: ~mBr 3600-2800, 1770, 1710, 1600, 1550 cm 1
uv: ~pH mabxUffer 257 nm (e 27000), 276 (~ 26100).
nmr: ~DMppOmd6 D20 2.70 (2H, t, J=6 Hz, CH2), 3.3-4.5
(9H, m, 2-H, 3-CH2, CH2 and OCH3), 4.96 (lH, d, J=5.5 Hz,
6-H), 5.53 (lH, d, J=5.5 Hz, 7-H), 6.55 (2H, m, furan-H),
6.92 (lH, d, J=10 Hz, pyridazine-H), 7.53 (lH, d, J=10
Hz, pyridazine-H), 7 68 (lH, br, furan-H).
Anal. Calc'd. for C2~H20N709S2Na H20: C, 42-92;
H, 3.45; N, 15.23, S, 9.96. Found: C, 43.08, 42.77;
H, 3.20, 3.03; N, 14.96, 14.76; S, 9.96.
TABLE 7
In vitro Activity Usin~ rlueller-Hinton Agar
~~ By~the Serial ~lutioll ;letllod
~ Geometric ~ean- of MIC
(Mcg./ml.)
BB-S511 BB-S526
~Ex. 13? (Ex. 14) Cefuroxime
S. aureus (~ strains) 1.97 1 6 1 24
E. coli (7) o 58 0.78 1.28
Kl. pneumoniae (4) 0.47 0 93 3.1
Proteus (6) 0.021 0.061 o.88
Shig.(3), Serr.(l)
Enterab.(l), Sal.(2) 1.11 2.41 4. o6
B. anthracis (1)
S. pyogenes (5) 0.032 0.032 0.025
S. ~iridans (5) 0.15 0.4 0.1
D. pneumoniae (5) 0 037 0.056 0.0125
N. meningitidis (5) 2.37 3.60 1.6
N. gonorrhoeae (5) 1.36 1.6 0.4
H. influenzae (7~ 0.64 0.71 1.16
Cefuroxime is sodium 6R,7R-3-carbamoyloxymethyl-7-(2Z)-2-
methoxyimino(fur-2-yl)acetamidoceph-3-em-4-carboxylate.
_ 9~ _
'7'~4
TABLE 8
Geometric Means of MIC's Against 3 Strains
of S. aureus and 27 Strains of Gram-negative Bacteria
(mcg./ml., Mueller-Hinton Agar)
No. of
Strains BB-S511 BL-S786
S. aureus 1 1.6 1.6
S. aureus, Penicillin-R 2 o.6 1.6
E. coli 6 0.2 0.2
E. coli, Cephalosporin-R 1 6.312.5
K. pneumoniae 4 o,7 o,3
Indole (-) Proteus 2 0.1 0.2
Indole(~) Proteus 3 o,o5o,3
Indole(~) Proteus, Cephalosporin-R 2 6.~ 50.1
S. marcescens 1 25 >100
E. cloacae 1 ~.1 1.6
Shigella, Salmonella 5 o.3 o,5
P. aeruginosa 2 >100 >100
BL-S786 is 7-[a-(2-aminomethylphenyl)acetamido]-3-[(l-car
boxymethyltetrazol-5-ylthio)methyl]-3-cephem-4-carboxylic
acid,
TABLE 9
Geometric ~leans of IIIC's Against 18 Strains
. _ .
of S. marcescens
BB-S511 BE-S786
.
5,4 85-9
- 97 -
l~f~7'7~4
EXAMP~E 15
. Substitution of an equimolar weight of 2-ethoxy-
imino-2-(fur-2-yl)acetic acid for the 2-methoxyiminofuryl
acetic acid used in the procedures. of Examples 13 and 14
produces 7-(2-ethoxyimino-2-furylacetamido)-3-(2-carboxy-
methyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-~-on-6-
ylthiomethyl)-3-cephem-4-carboxylic acid and 7-(2-ethoxy-
imlno-2-furylacetamido)-3-[.2-(2-carboxyethyl)-2,3-dihydro-
s-triazolo[4,3-b]pyridazin-3-~n-6-ylthiomethyl~-~-cephem-
4-carboxylic acid, respectively.
EXAMPLE 16
Substitution of an equimolar ~leight of 2-n-propoxy-
imino-2-(fur-2-yl)acetic acid for the 2-methoxyiminofuryl
acetic acid used in the procedures of Examples 13 and 14
produces 7-(2-n-propoxyimino-2-furylacetamido)-3-(2-carboxy-
methyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-
ylthiomethyl)-3-cephem-4-carboxylic acid and 7-(2-n-propoxy-
lmino-2-furylacetamido)-3-~2-(2-c~rboxyethyl).-2,3-dihydro-
s-triazolo[4,3-b]pyridazin-3-on-6-Ylthiomethyl]-3-cephem-
4-carboxylic acld, respectively.
. . .
EXAMPLE 17
Substitution of an equimolar weight of 2-n-butoxy-
imino-2-(fur-2-yl)acetic acid for the 2-methoxyiminofuryl
acetic acid used in the procedures of Examples 13 ~.nd 14
produces 7-(2-n-butoxyimino-2-furylacetamido)-3-(2-carboxy-
methyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-
ylthiomethyl)-3-cephem-4-car~oxylic acid and 7-(2-n-butoxy-
: - 98 -
~147724
lmino-2-furylacetamido)-~-[2-(2-carboxyethyl)-2,~-dihydro-
s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl]-3-cephem-
4-carboxylic acid, respectively.
EXAMPLE 18
The products of Examples 1~-17 ~re prepared es s~n
isomers essentially free of the corresponding anti isomers
by the use in the procedures of those examples of purifled
syn isomers of the appropriate 2-alkoxyimino-~-(fur-2-yl)-
acetic acid. Conversion of part of the syn isomer to anti
l~omer durlng preparation of the acid chloride from the acid
ls substantlally avoided by minimizing its exposure to
hydrogen chloride, e.g. by first converting the acid to its
anhydrous sodium salt and by treating that salt with oxalyl
chloride under anhydrous conditions in the presence of a
hydrogen ion acceptor such as dimethylformamide
~ u~r~ syn ~ b ~ U as ~G )
alkoxyimino-2-(fur-2-yl)acetic acids.
_ 99 _
~77~
.
EXAMPLE 19
An in~ectable pharmaceutical composition is
formed by adding sterile water or sterile saline solu-
tion 52 ml.) to 100-500 mgm. of 7-[(2Z~-2-methoxyimino-
(fur-2-yl)acetamido]-3-(2-carboxymethyl-2,~-dihydro-s-
triazolo[4,~-b]pyridazin-~-on-6-ylthiomethyl)-3-cephem-
4-carboxylic acid disodium salt.
Pharmaceutical compositions of the sodium
and potassium salts of the other compounds of the
present invention, preferably in the form of the pure
syn lsomer, are formulated in a similar manner.
When the compounds are first prepared in
the form of the free acid they are converted to the
deslred, highly water soluble potassium salt by treat-
ment wlth potassium 2-ethylhexanoate using the procedure
of Example 13.
It is occasionally advantageous to have ad-
mixed with said solid cephalosporin as a stabillzlng
and/or solubilizlng agent a sterlle, anhydrous solid
such as sodlum carbonate, potassium carbonate or llthium
carbonate(e.g. ln about 5 or 6 percent by weight of the
weight of the cephalospor~n) or such as L-lysine, argi-
nine or hlstidine (e.g. in about 20-50% by weight of
the weight o~ the cephalosporin) or such as a sodium,
potassium or calcium salt of levulinic acid, citric acid,
ascorbic acid, tartaric acid or pyruvic acid (e.g. in
about 25-200% by weight of the weight of the cephalos-
porin) or such as sodium bicarbonate, ammonium car-
bamate alkali metal or ammonium phosphates or N-methyl-
glucamine (per U.~. 1,380,7~
- 100 -
1~47'7'~4'
~Aiil'Il ~O
CO-N S ~ N
OCH3 ~ 02Na ~ N ~ N-CH3
BB-S510
BB-S510; 7-~(2Z)-2-Methoxyimino(fur-2-yl)acetamido~-3-
(2-metilyl-2,~-dihydro-s-triazolor4 ? 3-b~pyridazin-3-on-
6- ~ hyl)-3-cephem-4-carboxylic Acid.Sodium Salt
To a solution of (2Z)-2-methoxyimino(fur-2-yl)-
acetic acid (2~3 mg., 1.5 m mol.) and triethylamine (0 2
ml , 1.5 m.mol.~ in dichloromethane (~ ml.~ was added
oxalyl chloride (0.1~ ml., 1.5 m.mol.) at 0-5 C. and
the mixture was stirred for 30 minutes and evaporated
at reduced pressure to give the acid chloride as an oil
which was dissolved in dry acetone (5 ml.) and filtered
to remove insolubles. The acetone solution was added to
a mixture of 7-amino-~-(2-methyl-2,~-dihydro-s-triazolo-
[4,3-b]pyridazin-~-on-6-ylthiomethylj-3-cephem-4-car-
boxylic acid (591 mg., 1 5 m.mol.) and NaHCO~ (504 mg.,
6 m.mol.) in water (10 ml.) at 0-5 C. The reaction
mixture was stirred at 0-5 C. for 3 hours. Acetone was
removed at reduced pressure and the residual aqueous
solution was washed with ether (2 x ~0 ml.) and adjusted
to pH 1-2 with concentrated HC1. The precipitate which
was collected by filtration, washed with water and dried
in vacuo, was dissolved in THF (~0 ml.) and filtered to
remove insolu~les. To the THF solution was added a solu-
tion o~` sodium 2-ethylllexanoate (SEH, l M, 1.5 ml.) in
-- 101 -
77Z~
ethyl acetate and the resulting precipitate was collected
by filtration and dried in vacuo. Yield: 492 mg. of
7-[(2Z)-2-methoxyimino(fur-2-yl)acetamido]-3-(2-methyl-
2,3-dihydro-s-triazolor4,3-b~pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid sodium salt (58~).
M.p. >180 C. (dec.).
ir: v~Bx 1770, 1720, 1670, 1600, 1550 cm 1
uv ~pH7Buaffer 257 nm (E 22600), 277 nm (E 22300~.
nmr: ~p ~ 7.53 (lH, d, J=1.5 Hz, furan-Ha), 7.35 (lH,
d, J=9 5 Hz, pyridazine-H), 6.90 (lH, d~ J=9.5 Hz, pyri-
dazine-H~, 6.72 (lH, d, J=3.0 Hz, furan-H~), 6.48 (lH,
q, J=1.5 and 3.0 Hz, furan-H~), 5.72 (lH, d, J=4.5 Hz,
7-H), 5.14 (lH, d, J=4.5 Hz, 6-H), 2.94 (3H, s, 0-CH3),
3 61 (3H, 6 ~ N-CH3~
Anal. Calc'd. for C21H18N707S2Na / 2
C, 44.44; H, 3.89; S, 10.32. Found: C, 44.89; H, 3.92;
S, 9.67.
- 102 -
~1~77'~4
TABLE 10
In,,vitro ACttivity.-lr-n$lMutellelrl-H~intdon Ag~,r
By lle Serla Dl u
Geometric Mean of MIC
(Mcg./ml.)
BB-S510
~Ex. 20) BB-S515 Cefuroxime
S. aureus (3 strains) 0.62 2.48 1.24
E. coli (7) 2,11 2.33 1.28
Kl, pneumoniae (4) 6,.3 3~1 3~1
Proteus (6) 1.39 1.11 o.88
Shig.(3), Serr.(l)
Enterab.(l~, Sal.~2~ 6.26 5.26 4.o6
B. anthracls (1)
S. pyogenes (5) 0.0125 0.032 0.025
S. viridans (5) 0.13 0.59 0.1
D. pneumonlae (5) 0.021 0.1 0.0125
N. meningitidis (5) 1.03 5.45 1.6
N. gonorrhoeae (5) 0,25 2,07 0,4
H, influenzae (7) 0,35 2,11 1,16
Cefuroxime is sodium 6R~7R-3-carbamoyloxymethyl-7-(2Z)-2-
methoxyimino(fur-2-yl)acetamidoceph-3-em-4-carboxylate,
- 103 -
~147'~Z4
TABLE 11
Geometric Means of MIC's A~ainst ~ Strains
of S. aureus and 27 Strains of Gram-negative Bacteria
(mcg./ml., Mueller-Hinton Agar)
No. of
Strains BB-S510 BL-S786
S, aureus 1 0.1 1.6
S. aureus, Penicillin-R 2 0,1 1.6
E. coli 6 0,1 0.2 .
E. coli, Cephalosporin-R 1 3,1 12.5
K. pneumoniae 4 2.6 o,3
Indole (-) Proteus 2 1.1 0.2
Indole~+) Proteus ~ 0,2 0,3
Indole(~) Proteus, Cephalosporin-R 2 6,~ 50.1
S. marcescens 1 6.3 >100
E. cloacae 1 3.1 1.6
Shigella, Salmonella 5 0,5 0.5
P. aeruginosa 2 >100 >100
BL-S786 is 7-[a-(2-aminomethylphenyl)acet~mido3-3-[(1-car-
boxymethyltetrazol-5-ylthio)methyl]-3-cephem-4-carboxylic
acid.
TABLE 12
.. Geometric Means of ~IC's Against 18 Strains
of S. marcescens
BB-S510 BL-S786
-
7,9 85-9
- 104 -
7"7;~4
EX~PLE 21
Substitution of an equ~molar weight of 2-ethoxy-
imino-2-(fur-2-yl)acetic acid for the 2-methoxyiminofuryl
acetic acid used in the procedure of Example 20 produces
7-(2-ethoxyimino-2-furylacetamido)-3-(2-methyl-2,3-dihydro-
s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-
4-carboxylic acid.
EXAMPLE 22
Substitution of an equimolar weight of 2-n-propoxy-
imino-2-(fur-2-yl)acetic acid for the 2-methoxyiminofuryl
acetic acid used in the procedure of Example 20 produces
7-(2-n-propoxyimino-2-furylacetamido~-3-(2-methyl-2,3-
dihydro-s-triazolo~4,3-b]pyridazin-3-on-6-ylthiomethyl)-
3-cephem-4-carboxylic acid.
ExAl`~pLE 23
Substitution of an equlmolar weight of 2-n-butoxy-
imino-2-(fur-2-yl)acetic acid for the 2-methoxyiminofuryl
acetic acld used in the procedure of Example 20 produces
7-(2-n-butoxyimino-2-furylacetamido)-3-(2-methyl-2,3-
dihydro-s-triazolo~4,3-b]pyrid~zin-3-on-6-ylthiomethyl~-
3-cephem-4-carboxylic acid.
- 105 -
11477Z4
EXA~IPLE 24
_
The products of Examples 20-23 were prepared as syn
isomers essentially free of the corresponding anti isomers
by the use in the procedures of those examples of purified
syn isomers of the appropriate 2-alkoxyimino-2-(fur-2-yl)-
acetlc acid. Converæion of part of the syn isomer to anti
isomer durlng preparation of the acid chloride from the acid
is substantially avoided by minimizing its exposure to
hydrogen chloride, e,g, by first converting the acid to its
anhydrous sodium salt and by treating that salt with oxalyl
chloride under anhydrous conditions in the presence of a
hydrogen ion acceptor such as dimethylformamide,
Such syn isomers are also named as (2Z)-2-
alkoxyimino-2-(fur-2-yl)acetic acids,
- 106 -
~1477~g
EXAMPLE 25
An ln~ectable pharmaceutical composition i6
formed by addlng sterile water or sterlle saline 801u-
tion (2 ml.) to 100-500 mgm. of 7c[~2Z)-2-methoxyimino-
(fur-2-yl)acetamido]-3-(2-methyl-2,~-dih~ydro-s-
triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-
4-carboxylic acid disodium salt.
Pharmaceutical composltlons of the sodlum
and potassium salts of the other compounds of the
present lnv~ntion, preferably ln the fa~m of the pure
syn isomer, are formulated in a similar manner.
When the compounds are first prepared ln
the form Or the free acid they are converted to the
desired, highly water soluble potassium salt by treat-
ment with potassium 2-ethylhexanoate using the procedure
of Example 20.
It is occaslonally advantageous to have ad-
mixed with said solid cephalosporin as a stabilizing
and/or solubilizing agent a sterile, anhydrous solid
such as sodium carbonate, potassium carbonate or lithium
carbonate(e.g. in about 5 or 6 percent by weight of the
weight of the cephalosporin) or such as L-lysine, argi-
. nine or histidine (e g. in about 20-50~ by weight of
the weight o~ the cephalosporin) or such as a sodium,
potassium or calcium salt of levulinic acid, citric acid,
ascorbic acid, tartaric acid or pyruvic acid (e g. in
about 25-200% by weight of the weight of the cephalos-
porin) or such as sodium bicarbonate, ammonium car-
bamate,alkali metal or ammonium phosphates or N-methyl-
glucamine (per U.K. 1,380,7lJl).
- 107 -
