Note: Descriptions are shown in the official language in which they were submitted.
_ ' TRI -PY-CEPH CO~.BO
~lL3Lsal~727 ~SY-1510-A)
The present invention relates to novel acids
havin~ the structure
A~-CH - ~ Cll~2
-CH2-S ~ o ~T-A
COO~I
sometimes hereinafter also written as
CH25~ 1_
COOH
l,Jherein A is
:: -
RI or~ ~ C -
N
. ~ op~2
wherein Rl is hydrogen or formyl,
R is
~ . or
y ,.
and Y is hydrogen, chlorineJ bromine, ~luorine, trifluoromethyl,
amino, nitro, hydroxy, (lower)alkyl of 1-4 carbon at~ns or
(lower)alkoxy of 1-4 carbon atomsJ
R2 is (lower)~lkyl of 1-4 carbon atoms; and
herein Al is meth~rl or -(CH2)nCOO~I and n is one or two,
72~
.~he easily hydrolyzed esters and.the non-toxic, pharmaceu-
tically acceptable salts of those acids
Said easily hydrolyzed esters Or the acids of
formula I include those h~vin~ the group of the formula
;
~W
-C~
wherein when W represents hydrogen, Z rep~esents (lower)-
alkanoyl, benzoyl, naphthoyl, furoyl, thenoyl,
nitrobenzoyl, methylbenzoyl, halobenzoyl~ phenyl-
benzoyl, N-phthallmldo, N-succlnimldo, N-saccharino,
N-(lower)al~lcarbamoyl, (lower)al'~oxyl (lot~er)-
alkylthlo, phenoxy, carbalko.~y, carbobenzoxy,
carbamoyl, benzyloxy, chlorobenzyloxy, carbophenoxy,
~arbo~tert,-butoxy or (loWer)alkylsulronyl, ~nd ~hen
W represent 8 carbalkoxy, Z represent 9 carbalkoxy and,
when W repre5entB phenyl, Z represents benzoyl or
cyano or whereln W and Z taken together represent
2-oxoc~cloalkyl containlng 4 to 8 carbon atom~
lncluslve~
As 8et rorth below in more detall the pre9ent
. inventlon also prov1de~ salt~ Or these aclds, ~he
stereochemistry Or the blcycllc nucleus ~s that
~ound ln Cephalosporln C.
A preferred embodiment or the present invention
consists of the acids having the D configuration in the 7-
side chain and the formula II
-- 2 --
~5~727
R~CH-C~NH-CH- CH i CH2 ~ ~
OR ~C N C ~C CH2 N~ ~ N-(C~2~nC02H
COOH o
wherein n is one or two and Rl is hydrogen or formyl
and R is
~ or ~
and Y 1~ hydrogen, chlorine, brom~ne, fluorine,
trlfluoromethylJ amlno, nltro, hydroxy, lower-
alkyl of 1-4 carbon atoms or lower alkoxy of 1-4
carbon atoms and the nontoxicJ pharmaceutically
acceptable salts of tho~e aclds and the easily
hydrolyzed esters Or those aclds int:luding
especlally the plvaloylox~nethylJ acetoxymethyl,
acetonyl) phenacyl and methoxymethy:L esters and
the Rllyl esters such as the trlmethylsilyl ester.
A further pre~erred embodiment of this invention
consists o~ the compounds of formula II, trherein
R is 2-thienyl, 3-thienyl, phenyl, chlorophenyl, bromopllenyl,
trifluorome~hylphenyl, tolyl or methoxyphenyl.
Particularly preferred embodiments of this
invention comprises the acids h3ving the D confi~uration
in the 7-sidechain and the rormula
~3CH-II-NH CH--C/ \CH2 ~
OH ~C--N ~ ~C-CH2-S N ~ ~-(CH2)nC02H
COOH
~L15~7Z7
wherein n is one ~r t~o and their nontoxlc, p~la~naceutically
acceptable salts and easil~ hydrolyzed esters.
Also included in this inven~ion are the compounds
(used as either inte~lediates or metabolic precursors)
in ~Jhich the ~-hydroxy group is "blocked" by su~stituents
such as dichloroac~tyl (U.K. 962,024 and U. X. 1,328,340),
f~rmyl (U.S. 3,641,021), trimet~lyls~lyl or tetrahydro-
pyran~Jl (U.K. 1,328,340).
~ lere is ~lso provided b~ the present invention a
compound ha~ing the rol~ula
O S
R-CH-C-N~-C~ - Ch ~ ~ }~2 ¦ ~ Nl
H2-s ~N,N ~ N (CH2)n 2
I O
COO~l .
wherein n is one ~r two, ~ is hydrogen or formyl and R is
f~
~ or
y
and Y is hydrogen, ~hlorinef bromine, fluorine, trifluoro-
methyl, amino, nitro, hydroxy, lower alkyl of 1-4 carbon
atoms or lower alkoxy of 1-4 carbon atoms and M is
O O
_cHOC(C}12)nR~ -CHOC(C~12)nC~R8 ~'
Rl R~ 4R~
~15¢~727
.
o . o
- CHX~OR6 or - ~H-S-C-R
I l
n is O to 4; R is hydrogen, alkyl having 1 to 8 carbon
atoms, cycloalkyl of 3 to 6 carbon atoms, phenyl, Cl-C~
phenal~yl, pyridyl, thienylr or pyrrolyl; ~ is hydrogen,
methyl or ethyl; R7 ~nd R8 are each hydrogen, ~lkyl having
1 ts 6 carbon atoms, phenyl, pyridyl, or thienyl; R4 and R5
are each hy~rogen or alkyl of 1 to 4 carbon atoms; R6 is
alkyl having 1 to 4 carbon atoms, phenyl, phenalkyl having
1 to 4 carbon atoms, pyridyl, thiadiazolyl, amino or Cl-C4
alkylamino; X is N~ or oxygen; and. each phenyl group is
unsubstituted or substituted with one or two substituents
selected from the group consisting of alkyl having 1 to 6
carbon atoms, alkoxy having 1 to 4 carbon atoms, hydroxy,
amino, NHRl, N(Rl)2, nitro, fluoro, chloro, bromo or carboxy,
or a ~ontoxic, pharmaceutically acceptable salt thereof.
~ here is also provided by the present invention a
compound having the formula
.
R 11 / ~ H2 ~ N
IR1 ~ N \ ~ -Cff2 S ~ N~N ~ N (CH2)nCO~H
COOI~l
wherein n i5 one or two, R is hydrogen or formyl and ~ ~8
~ or
Y
~15~:)7;~'~
and Y i~ hydrDgen, chlorine, bromine, fluorine, trifluoro-
methyl, amino, nitro, hydroxy, lower alkyl of 1-4 carbo~
~toms or lower alkoxy of 1-4 carbon atoms and M is selected
from the group co~sisting of
CH3 O
- CH - - ~ - R6
1 2H5
- CH - C - R
R~ O
2 ll
- CH - X - C - 'oR7
wherein R5 iB a hydrogen atom, a methyl or an ethyl group;
X2 iS -O-, -NH-; R6 ~5 a baslc group.such as alkyl or aralkyl
substltuted with substituted or unsubstituted NH~, such
as alkyl-NHCH3, sralkyl-NHCH~,
alkyl-NH ~ ,
; aralkyl-~H ~ , -IH ~ , -CH2NH2~ _1H_CH2
. NH~ NH2
.. ` .
R7 is an alkyl group such as a methyl, ethyl, propyl,
isopropyl, butyl, isobutyl, pentyl or 2-ethyl-hexyl group;
a cycloalkyl group such as cyclopropyl, cyclobutyl, cyclo-
pentyl, cyclohexyl or cycloheptyl: an aryl group such as
phenyl or naphthyl; an aràl~yl group such as benzyl or
naphthylmethyl; a heterocyclic group and wherein the alkyl,
cycloalkyl, aryl, aralkyl and heterocyclic groups may be
substitute~ with one or moxe groups selected from the class
consisting of amino groups, substituted amino groups such
as methylamino, diethylamino or acetamido groups, the halogen
.
~S~7Z7
groups such as fluorine, chlorine or bromine, nitro groups,
al~oxy groups such as methoxy, ethoxy, propyloxy, isopropyloxy,
butoxy or isobutoxy; or a nontoxic, pharmaceutically accep able
salt thereof.
There is also provided by the present invention a compound
having the formula
O ~S
~-CH-C-NH-CH - CH l~2 ~ Nl
I C-C~ -S ~ ,N N-(CH ) CO H
COO~
wherein n is one or two, R is hydrogen or formyl and ~ is
~ or ~
and Y is hydrogen, chlorine, bromine, fluorine, trifluoro-
methyl, amino, nitro, hydroxy, lower alkyl of 1-4 car~on
atoms or lower alkoxy Gf 1-4 carbon atoms and M is
.
O
11
/~_y
-CH2-N
\Z
where~n Y ~ alkyl o~ one to six carbon ~toms, phenyl,
benzyl, alkoxy of one to 6iX carbon atoms, or benzyloxy;
Z is alkyl of one to ~ix car~on atoms, phenylbenzyl,
alkoxy o~ one to six carbon stoms, cyclopentyl, cy~lo-
hexyl and phenyl, or Y+Z taken together are a 3-benzoxa-
zolidine ring; or a nontoxic, pharmaceutically acGeptabla
~alt thereof.
~L~5~27
Also included within the present invention are
pharmaceutical compositions comprising a mixture of an
antibacterially effective amount of a compound of the
present invention and a semisynthetic penicillin or another
cephalosporin or a cephamycin or a ~-lactamase inhibitor
or an aminoglycoside antibiotic.
In the treatment of bacterial infections in man,
the compounds of this invention of formula II are administered
parenterally~ in accordance with conventional procedures for
antibiotic administration, in an amount of from about 5
to 200 mg./kg./day and preferably about 5 to 20 mg./kg./day
in divided dosage, e.g. three to four times a day. They
are administered in dosage units containing, for example,
125, 250 or 500 mg. of active ingredient with suitable
physiologically acceptable carriers or excipients.
The dosage units are preferably in the form of liquid
preparations such as solutions or suspensions.
Another preferred embodiment of this invention
comprises the acids having the formula III
-C-NH-CH ~S ~fH IN
N~OR ll ~ C-CH S ~ N - Al III
COOH
wherein Al is methyl or -(CH2)nCOOH and n is one or two
and R2 is alkyl containing 1-4 carbon atoms, the easily
hydrolyzed esters and the non-toxic pharmaceutically
acceptable salts of those acids as hereinbefore set forth.
The compounds of formula III of the present
invention are s~n isomers or else are mixtures of syn and
anti isomers containing at least 75% of the ~y~ isomer.
Preferably such mixtures of isomers contain at least
90% of the ~y~ isomer and not more than 10% of the anti
,~''''9 _ _
~ S~7;~7
isomer, Most prefer~bly the cor,~pounds Or ~ormula III are
s~ isomers essen-~ially free of the co~responding anti
isomer.
~ le preferre~ embodiments of the present invention
are the syn isomers of the compounds of Formula III
~Jherein R2 is methyl or ethyl, n is one or t~To in its
acid or pivaloyloxymethyl, acetoxymethyl, methoxymetllyl,
acetonyl~ p}lenacyl, p-nitrobenzyl, ~ richloroethyl,
3-phthalidyl or 5-indanyl ester form.
Reference to the syn (cis) isomeric ~orm
refers to the confi6uration o~ the group oR2 ~ith respect
to the carboxamido ~roup.
There is also provided by the present inven-
t~ on a compound having the formula
- C - NH-CH--Cl H ~H2 ~=NI
N_oRa ~c N c"c CH2 s ~N~ ~ (CH2)n
c - oR3 o
O
wherein R~ ls alkyl containing 1-4 carbon atoms, n 19
~` ~
one or two and R' iB Belected from the group
conslsting of
IH~5 11 6
- CH - O - C - R,
1 2H5 11
- CH - C - R ,
R5 o
2 11 7
- C~ - X C - 0~
whereln R5 is a hydrogen atom, a methyl or an ethyl group;
X2 1R -0-~ -NH-~ R6 is a basic group such as alkyl or aral~yl
substit`uted with substituted or unsubstltuted NH2, such
as alkyl-NHCH~, aralkyl-NHCH3,
alkyl-NH ~ ,
aralkyl-NH ~ ' ~lH ~ , -CH2NH2~ -IH-CH2
NH2 NH2
R is an alkyl group such as a methyl, ethyl, propyl,
isopropyl, butyl, isobutyl, pentyl or 2-ethyl-hexyl group;
a cycloalkyl group such as cyclopropyl, cyclobutyl, cyclo-
pentyl, cyclohexyl or cycloheptyl; an aryl group such as
phenyl or naphthyl; an aralkyl group such as benzyl or
naphthylmethyl; a heterocycllc group and wherein the
alkyl, cycloalkyl, aryl, aralkyl and heterocycllc ~roups
may be substituted with Dne or more groups selected from
the cla6s consistlng of amino ~roups, substltuted amino
groups such as methylamlno, diethylamlno or acetamido
. groups~ the halogen ~roups such as fluorine, chlorine or
bromine, nitro groups, alkoxy groups such as methoxy,
ethoxy, propyloxy, isopropyloxy, butoxy or isobutoxy;
or a nontoxic, pharmaceutically acceptable salt thereof,
said compound being at least 75% by welght in the form of
it syn isomer and preferably in the form of its yn
isomer essentially free of the correspond~ng anti ~s~mer
- 10
%7
There is also provided by the present inven-
tion a compound having the formula
C - C - NH~ CH CH2 ~ N
C N ,5-C~I2-S N,N ~ - (CH2)n
I_OM Q
whereln R~ is alkyl rontaining 1-4 carbon atoms, n 13
one or two and M is
~C Y
-CH2-N~
wherein Y is alkyl of one to six carbon atoms, phenyl,
benzyl, alkoxy of one to six carbon atoms, or benzyloxy;
Z is alkyl of one to six carbon atoms, phenylbenzyl,
alkoxy of one to six carbon atoms, cyclopentyl, cyclo-
hexyl and phenyl, or Y+Z taken together are a 3-benzoxa-
zolidlne ring; or a nontoxlc, pharmaceutically acceptable
salt thereof, said compoun~ be~ng at least 75% by welght
in the form of its ~ isomer and preferably ln the f~rm
o~ its syn isomer essentially free o~ the corresponding
anti lsomer.
There is also provided by the present inven-
tlon a compound having the ~ormula
C ~ C - NH-IH- ~H ICH2 ~ N
~l 2 ~C - N ,5 -CH2-S N ~ I CH
C-OM O
O
-- 11 --
27
whereln R2 is alkyl containing 1-4 carbon atoms and ~ is
~}~oc(cH2)nRJ ~CIHOc(cH2)ncl~R8
NR R
O O
-CHXCOR or - CH-S-C-R
I l 1 1
n ls O to 4; R is hydrogen,.alkyl having 1 to 8
carbon atoms, cycloalk~l of 3 to 6 carbon atoms,`
phenyl, Cl-.C4 phenalkyl, pyridyl, th~enyl, or pyrrolyl;
Rl ls hydrogen, methyl or ethyl; R7 and R8
are each hydrogen, alkyl having 1 to 6 carbon atoms,
phçnyl, pyridyl, or thienyl, R4 and R5 are each
hydrogen or alkyl of 1 to 4 carbon atoms; R ls alkyl
having 1 to 4 carbon atoms, phenyl, phenalkyl having
1 to 4 carbon atoms, pyridyl, thiadiazolyl, amino or
Cl-C4 alkylam~no; X is NH or oxygen; and each phenyl
group is unsubstituted or subs~ituted with one or two
substltuents selected ~rom the group consisting of
alkyl ~aving 1 to 6 carbon atoms, alkoxy having 1 to
4 carbon atoms, hydroxy, amino, NHRl, N(Rl)2, nitro,
fluoro3 chloro, bromo or carboxy, or a nontoxic,
p~armaceutically acceptable salt thereof, said com-
pound being at least 75% by welght in the form of its
syn isomer and preferably in the ~orm of lt~
l~omer essentially ~ree o~ the corresponding anti isomer,
- f2
1150~Z7
I
,
There is also provided by the present inven-
tion ~ compound having the formula
O
C - NH-ICIH- CH ~H2 ~ NI
. - OR~ O ~ N C~5 ~C}l2~s N ~ - CH3
I_OR~ O
wherein ~ ls alkyl contalnin~ 1-4 carbon atoms
and R is selected ~r~m the group
conslsting of
CH3 O
- CH - O - C - R6
Cl 2H5 11
- CH - C - R ,
R5 O
2 11 7
- CH - X - C - OR
wherein R5 is a hydrogen atom, a methyl or an ethyl sroup;
x2 is -O-~ -NH-; R is a basic group such ~s alkyl or aralkyl
substituted with substituted or unsubstituted NH2, such
as alkyl-NHCH3, aralkyl-NHCH~,
alkyl-NH ~ ,
aralkyl-NH ~ , -IH ~ , CH2NH2~ -IH-CH2
NH2 NH2
R7 is an alkyl group such as a methyl9 ethyl, propyl,
lsopropyl, butyl, isobutyl, pentyl or 2-ethyl-hexyl group;
a cycloalkyl group such as cyclopropyl, cyclobutyl, cyclo-
pentyl, cyclohexyl or cycloheptyl; an aryl gr~up sucIl as
'
`''` ll~j7~7
phenyl or naphthyl; an ~ralkyl group 6uch as benzyl or
naphthylmethyl; a heterocyclic group and whereln the
alkyl, cycloalkyl, aryl, aralkyl and heterocycllc groups
~ay be substituted with one or more groups selected from
the class conslsting Or amino groups, 6ubstituted amlno
groups such as methylamino, diethylamino or acetsmido
groups, the halogen groups such as ~luorlne, chlorine or
bromine, nitro groups, alkoxy groups such as methoxy,
ethoxy, propyloxy, isopropyloxy~ butoxy or lsobutoxy;
or a nontoxlc, pharmaceutlcally acceptable salt thereof,
6aid compound being at least 75~ by weight ln the form of
lts syn isomer and preferably in the ~orm of lts syn
isomer essentially free of the corresponding anti lsomer.
There is also provlded b~ the present inYen-
tion a compound having the formula
O
C - C - NH-CH--CH ~H2 ~ N
N_ oR2 0 / ~ C N ~ - CH~
C-o~ O
11
O
wherein R2 is alkyl containing 1-4 carbon atoms and ~5 is
Il
/C -Y
CH2 N\
~i50~7~2'7
wherein Y is alkyl of one to six carbon atoms, phenyl,
benzyl, alkoxy of one to six carbon atoms, or benzyloxy;
Z is alkyl of one to six carbon atoms, phenylbenzyl,
alkoxy of one to six carbon atoms, cyclopentyl, cyclo-
hexyl and phenyl, or Y+Z taken together are a 3-benzoxa-
zolldine ring; or a nontoxic, pharmaceutically acceptable
salt thereof, said compoun~ being at least 75% by weight
ln the form of its syn isomer and preferably in the form
of its syn isomer essentially free OI' the corresponding
anti isomer.
.. . . _ . . ...
In the treatment of bacterlal infeCtlons
in man, the compounds of this lnventlon are admlni~-
tered parenterally ln ~n amount of from about 10 to
90 m4./kg /day and preferAbly about 14 to 50 mg./kg./day
ln div~ded dosa~e, e.~. two to four times a day. They
are admlnlstered in dosage units contalning, for example,
125, 250 or 500 mg, Or actlve lngredient wlth suitable
physiologically acceptable carriers or excepients.
e dosage units are in the form of llquid preparations
such a~ solutions or suspensions and preferably are
~queous solutions Or a sodlum or potassiUm 6alt which
are in~ected intravenously or lntramuscularly or by
continuous or intermittent in~usion in concentrations
of about 125-500 mgm /ml., and preferably, 250 mgm./ml
as ls customary in therapy with cephalosporln antibi-
oti~s.
It was an unexpected finding that the leading
compound of the present invention (BB-S510; see below)
having a 2-methyl substituent on the tr~azolopyridazinone
showed in vitro antibacterial potency considerably
super~or to that of the corresponding compound lacking
such methyl group (BB-S515; see below).
- 15 -
27
.
A further embQdiment Qf this inVention comprises a`com-
pound ha~ing the formula
~ N
HS ~
wherein Al is methyl or -(CH2)nCOOH
where n is one or two. These compounds are made by a process com-
prising reacting a compound of the formula
Cl ~ A2
o
wherein A2 is methyl or -(CH2)nCOOH or an easily hydrolyzable ester
thereof and n is one or two, with NaSH.
The present invention also provides the process for the
production of the antibacterial agents of formula I which comprises
reacting a compound of the formula
~S~
~2N - fH fH Cl 2 ~ N
~iC-CH2-S-l~N ,N ~ N-A IV
COOH
wherein Al is as hereinbefore defined or a salt or easily hydro-
lyzed ester or Schiff base as with benzaldehyde or salicylaldehyde
thereof (including, but not limited to, those of U.S. 3,284,451
and U.K. 1,229,453 and any of the silyI esters described in U.S.
patent 3,249,622 for use with 7-aminopenicillanic acid and used in
Great Britain 1,073,530 and particularly the pivaloyloxymethyl,
acetoxymethyl, methoxymethyl, acetonylt phenacyl, p-nitrobenzyl,
~ -trichloroethyl, 3-phthalidyl and 5-indanyl esters~ thereof
with an organic monocarboxylic acid chloride or a functional
equivalent thereof as an acylating agent.
l~LSql7;;:7
Such functlonal equivalent~ include the
corresponding ~cid anhydrlde~, lncluding m~xed
nhydrlde~ and p~r$icularly the mixed anhydrltes
prep~red from ~tronger ~cids ~uch a~ the lower
aliphstic monoe6t2r~ of carbonlc ~cld, or 21kyl
snd aryl sulfonic ~cids ~nd of more hindered
ncids such ~ diphenyl~cetic scid. In ~ddition,
~n ~cid azide or ~n actlve e8ter or thloe~ter (e.g.
with p-nitrophenyl, 2,4-dinitrophenol, thiophenol~
thioacetic acid) m~y be used or the free ~cid i~6elf
may ~e coupled with compound ~T ~fter irst reacting
~sid free ~cid with N,N'-dimetllylchloroformlminium
chloride [cf, Great Brit~in 1,008,170 ~nd Nov~k
and Weichet, Experientia XXI, 6, 360 (19S5)] or
by the use of en?ymes or of an N,N'~c~rbonyl-
dlim~d~zole or nn N,N'-carbonylditri~zole lcf
South Afric~n p~tent ~pec~fication 63/26841 or
c~rbod~mide reagent [especially N,N'-dicyclohexyl~
c~rbodiimide. NaN' dii60propylc~rbodiimide or N~cyclo-
. hexyl-N'-(2-morpholinoe$hyl)c~rbodiimide; c~. Sheeh~n
Dnd Hess, J. Amer. Chem. Soc., 77, 1967 (1955)J~ or
of alkylyl~mine resgent ~cf. R. Bui~le ~nd H.G.
Vleh~, AnREw~ Chem. Intern~t~onal ~dltlon 3, 582,
- 17 ~
~ 7
(1964)~ or v~ an ~soxasol~um ~alt r~ag~nt lcr. R. ~.
Woodward, R. A. Olofson and ~. Mayer, ~ ~r~ Che,~,
, 1010 tl961)], or o~ a ketenlmlne rea~ent
[c~. C. L. Stevens and M, E. Munk, ~ 5h~m_ ~5~
~Q, 4065 (1958)~ or o~ hexachlorocyc,otrlphosphatriaz~ne
or hexabromocyclotrlphosPhatrla2lne (U,S, ~,651,050) or
of dlphenylph~sphoryl azlde [DPPA; ~_ A~er Chem~ Soc.,
q4 J 6203-6205 ( 1972) ~ or of dlethylphosphoryl ~yanlde
lD~PC; Tetrahedron Letters No, 18, pp. 1595-1598 (1973)
or Or dlphenyl phosphlte ~Tetrahedron Letters No. 49,
pp, 5047-5050 tl97~)]. Another equlvalent o~ the acld
o~lorlde 19 a correspondlng azollde, i.e., an amlde Or
the corresp~ndln6 acld whose amlde nltro6en 1~ a member
a ~uasiaromatlc ~lYe membered rlng contalnln~ at
least two nltro~en atoms, l,e., i~lidazole, pyrazole,
the trla~oles, benzlmidazole, benzotrlazole and the~r
~ubatituted der~lat~ves. A~ an example o~ the genersl
method for the preparatlon Or an azolide, N,N'-carbonyl-
dlim~dazole Is rea c ted wlth a ca rboxy llc a c ld ln
equ~molar pr~port~ons at room temperature in tetr~-
bydr~uran, chlorororm, dimethylrormamide or a ~lmllar
~nert 801vent to ~orm the carbo~ylic ac~d lmidazolide
In practlcally quantltat~ve yleld with llberstIon o~
carbon dloxlde and one mole o~ ~mida201e. Dicarboxyl~c
acid3 yleld dlmldazollde. The by~product, imtda~ole9
preclpltate3 and may be separated and the Imldazolide
1301ated, but thi~ ls not essentlal. The methods ~or
carr~in~ out these reactlons to produee a cephalo~por~n
and the meth~ds u~ed to l~olate the ~ephalooporln ~o
produc~d are well known ln the ar~.
"` ~15~77~'7
Ment~n ~ de aboYe Or the u~e o~ ersz~ne~ to
couple ~he rree ac~d wlth
oompound IV. Included in the sc~pe o~ Buch pro~es~e~
sre the u~e Or an ester, e.g. the met~yl e~ter, o~
that free ~c~d wlth enzymes pro~ded by variou~ mlero~
organlcm3, e.g. tho~e descrlbed by T. Takahashl et sl "
J, Amer, Chem. Soc., 4(111, 4035-4,0~7 (1972) and by
T, Nara et al., J. Antlblotlcs (Japan) ~ J 321-~23
~1971~ ~nd ln U.S. 3,682,777.
Por the coupling of the organic c~rboxyl~o acid as
descrlbed above with compound IV (or a 6~1t or preferably
~n easily hydrolyzed ester of Schiff b~se, as with
benz~ldehyde, thereof) it is also convenlent and ef~icient
to utilize a~ the coupling agent phosphonitrillc chloride
trimer (J, Org. Chem " ~ , 29J9-81, 1968) o~ N-ethoxy-
1,2-dihydroquinoline (EEDQ) as described in J. Amer. Chem,
: 50c., gO, 823-824 ~nd 1652-1653 (1968) snd U,S. P~tent
. 3,455,929. The reaction is prefer~bly c~rried out at 30-
35C. iR benzene, ethsnol or tetrahydrofur~n uslng ~bout
; .. equlmolar qusntities of ~11 th~ee re~gent~ followed by
~onventional isolation ~nd removal by conventlonal methods
of Any blocking groups present.
. .
. One process of the present invention stated
more specifically is the process for the preparation of
a product having the D-configuration in the sidechain
and.the formula
CH-CO~l ~ ~
OH k ~CH2s~N,~N (~H2)~t,0211
COOH
- 19 -
~ llSC~727
wherein n is one or two or a s~lt t~ereof whi~h comprises-
the consecutive steps of
a. preparing an acylating derivative Or D-mandelic
acid having the formula
~ 7
.' o~
wherein the hydroxyl blocking group R represents di-
chloroacetyl, silyl and preferably trimethylsilyl, tetra-
hydropyranyl or, preferably, formyl in an anhydrous or-
ganic solvent such as benzene, ethanol or preferably
tetrahydrofuran, at room temperature or below and prefer-
ably at about 5~ C)
b. mixing therewith, preferably slo~lly, a solution
at about the same temperature in a solvent, preferably
aqueous tetrahydrofuran, containing substantially the
same number of moles of a tertiary amine, preferably a
tertiary alkylamine such as triethylamine and substan-
tially the same number of moles of 7-amino-3-(2-carboxy-
methyl or 2-carboxyethyl-2,3-dihydro-s-triazolo[4,3-bl-
. pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-car~oxylic acid
or a ~alt or an easily hydrolyzed Schiff base, as with
benzaldehyde, thereof to produce the intermediate acid
having the formula
~CH CONH Tf ~ cH ~
1R ~N~ 2S ~N~ ~N-(~H2)nCO2H
COOH O
w~erein n is one or t-~ or a salt thereof wher~in R has
the meaning set out above; and
c. removing said hydroxyl blocking group R by con-
ventional chemical methods to produce said product or
salt thereof.
, _ 20 -
~L~L5~72~7
In preferred embodiTnents of the present inven-
tion R represents formyl which is removed in step C by
treatment llith aqueous allcali such as aqueous sodium
bicarbonate or R represents dechloroacetyl which is removed
in step C by alkaline hydrolysis, prererably at about
pH 9-10, or ~ represents trime~hylsilyl ~Jhich is removed
in step C by exposure to aqueous acid.
Other compounds of formula II are made in like
manner,
An additional process of the present invention
comprises the preparation of the compounds of the
present invention by the displacement of the 3-acetoxy
~roup from a cephalosporanic acid of the formula
A~ H2-O-C-CH~ V
0/ 0~1
~here A is a.s defined hereinbefore above,
prepared by substituting 7-amino cephalosporanic acid for
the 3-thiolated-7-amino cephalosporanic acids in the acylation
procedures described herein and elsewhere reported with the
apprOpriate thiol HSR3 having the formula,
or
~I-S--~ N~N ~ N ~ CH2)nCO~H H-S--~ N~N ~ N-CH~
where n is one or two and then removing the protecting
group if any is present as on the carboxyl groupl
S¢~727
.
The dlspl~cement of 6uch a 3-~cetoxy group w~th
- such ~ th~ol ~y be accomplished ln ~olution ~8 in water
or aqueou~ ~etone ~t a temper~ture of ~t le~st soom
tEmpersture ~nd prefer~bly w~thln the range of absut
50~ to 100C. in the presence o~ ~ mild b~se ~uch fl8
~odlum b~c~rbon~te, e.~. prefer~bly neer neutr~lity such
aB ~t about pH 6. An excess of the thiol ~8 preferebly
e~ployed. The react~on product ia isolated by csr~ful
acidifica~ion of the reaction mixture followed by extrac-
tion wlth a water-immiscible orgsnic solvant. A8 noted
above, the p~ep~rstion of many other 7-acyl~idocephnlospor~n~c
~cids is described in the patent end ~cientiflc llter~ture
e.g. ln U.S. Clas8 260-243C.
The ~lts of tha compound~ of thi~ lnvent~o~ ~nclud~
the ~ontoxlc c~rboxyl~c oc~d 8alt~ thereof, incl~ting non-
toxic ~etallic 3~1t~ ~uch aa oodium, potessium, c~le~um and
alu~inum, tha a~m~nium s~lt and subctltuted ammon~um s~
e.g. ~alts o~ ~uch nontoxic am~nes a~ trlalkyl-
amlnes in~ludlng trlethylamlne, procaine, dibenzyl-
amine, ~-benzyl-beta-phenethylamlne, l-ephenamlne,
N,N~-dlbenzylethylenediamlne, dehydroabietylamine,
~,N'~bls-dehydroabietylethylenediamlne, and other
~mlnes whlch have been used to rorm salts wlth
benzylpenlclllin, L-lysine, arginine and histidine.
The pre~erred esters o~ the cephalosporlns
Or the pre8ent ~nvention are the pivaloyloxymethyl9
- acetoxymethyl9 methoxymethyl, acet~nyl and phenacyl
~sters. All are use~ul lntermedlates ~n the
~, ,
productlon of the cephalosporln havlng a ~ree
carboxyl group.
_ 22 -
S~727
A8 Indicated aboYe, t~ese rlve e~ter~ Or 7-amino-
cephalosporanlc acld are each prepared by known
method~. One excellent procedure i~ that Or U.S.
patent 3,284,451 ln whi~h sodlum cephalothln 1~
ester~led by reactlon with the corre~pondlng actlve
chloro or bromo compound (e,g. phenacyl bromlde,
chloroacetone, chloromethyl ether, plvaloyloxy-
methyl chlorlde [also called chloromethyl plvalatel,acetoxymethyl chlorlde) and then the thlen~
acet~c acid sldechaln i9 removed enzymatlcally
as in the ~ame patent or chemlcally a~ ln U.S,
patent 3,575,g~0 and ln Journal o~ Antlblot1c~,
xxrv (~ 767-773 (1971)~ In another good
method the trlethylamlne salt of` 7-amlnocephalo-
8poranle acld i8 reacted dlrectly wlth the aotlve
halogen compound, as in Unlted Klnedom 1,229,453,
mese esters Or 7-aml~ocephalosporanic acld
are then reacted wlth the nucleophlle HSR3 ln the ~ame
manner as i9 lllustrated hereln ~or 7-amlnooephalO-
sporanlc acld ltselr~ me ~-thlolated es~er o~
7-amlnocephalo~poran~c aoid ~s then coupled ~ît~
the organlc carboxylic acld as be~ore.
The ester of the cephalosporin so obtained
is, if not used per se, con~erted to ~ts free acid
and, if deslred9 sny salt by ~emov~l of the esteri-
fying group, 88 by aqueou~ or enzym~tlc hydroly~ 8
wlth hum~n or ~nim~l serum) or ~cid~c or alkaline
hydrolys~s or by treatment with ~odium thlophenoxide
a8 tDUght ln U.S. 39~84,451 and, ln the penlc~llin
serle3, by Sheehan et al,~ 3, Org~ Chem.
2006-2008 (1964).
5~7;~7
In ~ ther altes~natlve synthes~ 8, the 3-
th~olated 7-amlnocephalo~poran~ a~ld 13 prepared
~ de9crlbed here~n and then ac~lated at the 7-
am~no gr~up ~nd rlnally este~l~led, as by reaction
o~ the appropriate alcohol wlth the acid chloride
prepared, ~or example, by react~on Or the rinal
eephalosporln wlth thlonyl chlorlde or by ~ther
eB~entlally acldlc e~terl~lcatlon proc~d~.re~,
There iQ further provided by the present invention a
pharmaceutical composition comprising an antibacterially
effective amount of a compound having the formula
O ` S
R-C~l-C NH-CH--CH ~ 2
k ~ C-CH2-S_ ~N~ N (CH2)n 2
Joo~
where~n ~ is one or two, Rl i~ hy~rogen or formyl and R is
.
~3 Dr ~
and Y is hydrogen, ~hlorine, bromine, fluorine, trifluoro-
methyl, amino, nitro, hydroxy, lower ~lkyl of 1 4 carbon
atom~ or lower alkoxy of 1-4 ~arbon atoms and M is hydrogen,
pivaloyloxymethyl, acetoxymethyl~ methoxymethyl, acetonyl,
phenacyl, p-nitroqenzyl, B,~ trichloroethyl. 3-phthalidyl
or 5-~ndanyl and pre~erably ~s hydrogen or a nontoxic,
pharmaceutically acceptable salt thereof.
- 24 -
115~ 7
There i~ further provided by the present invention a
method of tseating bacterial infections comprising ~dminis-
tering by injection to an infected warm-blooded animal,
~ncluding m~n, ~n e~fective but nontoxic dose of 250-1000 mgm.
o~ ~ compound having the form~la
R-C~-C-NH-CH--C~ ~}12 ~NI
1R1 ~--N\ ~ 2 N~N (CH2)nCO2H
COOM
wherein n is one or two, Rl is hyclrogen or formyl and R is
!
y~ ~ ~
and Y ~s hydroyen, chlorine, bromine, fluorine, trifluoro-
. methyl, amino, nitro, hydsoxy, lower alkyl ~f 1-4 carbon
atoms or lower alkoxy of 1-4 rarbon atoms and M is hydrogen,
pivaloyloxymethyl, acetoxymethyl, methoxymethyl, acetonyl,
phenacyl, p-nitrobenzyl, B~ trichloroethyl~ 3-phthalidyl
or 5-indanyl or a nontoxic, pharmaceutically acceptable salt
~hereof~ .
There is also provided by the psesent inven~ion a
method for combatting ~ . dysenteriae infections w~i~h
comprise~ administering to a warm blooded mammal infected
w~th ~n ~ . dysenteriae infection an ~mount effective ~or
treat~ng ~aid ~ . dysenteriae infe~tion of a composition
comprising a compound havlng the formula
~ 25 -
7~'~
o
R-C~-C-NH-C~ C~ 2 ~ Ni
C-CH2-S - ~ ,N N-tCH ) ~ O
COOM
wherein n i9 one or two, R $s hyd~ogen or formyl and ~ is
~ ~r ~
and Y $s hydrogen, chlorine, bromine, fluorine, trifluoro-
methyl, amino, nitro, hydroxy, lower alkyl of 1-4 carbon
atoms or lower alkoxy of 1-4 car~Dn atoms and M is hydrogen,
pivaloyloxymethyl, acetoxymethyl, methoxymethyl, acetonyl,
phenacyl, p-nitrobenzyl, ~,B,~-trichloroethyl, 3-phthalidyl
or 5-indanyl and preferably is hydrogen or a nontoxic,
pharmaceutically acceptable salt thereof.
There is also provided by the present invention a
method for com~atting Sal. enteritidis infections which
comprises administering to a warm-blooded mammal ~nfected
with a Sal. enteritidis infection an amount effective for
treating said Sal. enteritidis inection of a composition
comprising a compound having the formula
o /~
~-CH-C-NH-CH - CH l~2 ~ 1 '
1R1 ~ _ N\ j/ ~H2 S ~N~ N ~CH2)n~02H
- COOM
- 26 -
1150~727
.
wherein n is one or two, R~ is hydrogen ~r ~ormyl and R is
Y~ ~
and Y is hydrogen, chlorine, bromine, fluorineO trifluoro-
methyl, amino, nitro, hydroxy, lower alkyl of 1-4 ~arbon
atoms or lower alkoxy nf 1~4 carbon atoms and M is hydrogen,
pivaloyloxymethyl, a~etoxymethyl, methoxymethyl, acetonyl,
pbenacyl, p-nitrobenzyl, ~,B,B-trichl~roethyl, 3-phthalidyl
or 5-indanyl and pxeferably is hydrogen or a nonkoxic
pharmaceutically acceptable salt thereof.
There is further provi.ded by the present
invention a pharmaceutical composition comprising an
antibacterially ef~ective amount of a compound ha~ing
: the formula
O
C - NH~ CI~ ~H2 ~ N
N_ OR~ ~C N 1~ 2 S N~N ~ _A
; I_OR O
O
wherein X2and Al are as hereinbefore defined
and R3 is h~Jdrogen, pivaloyloxymethyl~
acetoxymethyl, methoxymethyl, acetonyl, phenacyl, p-
nitroben~yl, ~ B-tricllloroetllyl~ 3-phtllalidyl or 5-indanyl
and preferably is hydrogen or a nontoxic, phar~aceutically
acceptable salt thereof, said compound being at least
- 27 -
~15a3~727
75% by weight ln the form of its syn isomer and
preferably in the ~orm of its syn isomer essentially
free of the corresponding anti isomer, and a pharma-
ceutically acceptable carrier therefor.
There is further provided by the present
invention a pharmaceutical composition comprising an
antibacter~ally effective amount of the syn isomer of
a compound having the formula
C - g ~ NH-CllI CH ICH2 ~ N
N--OCH3 o~ ~ C~ U2 S
C-OH O
wherein A~ is as hereinbefore define~ or a nontoxic, pharma-
ceutically acceptable salt thereof and a pharmaceutically
acceptable carrier there~ore.
There is further provlded by the present
lnventlon a method of treating bacterial infectlons
comprising administering by in~ection to an infected
warm-blooded animal, includlng man, an effective but
~- nontoxic dose of 250-1000 mgm. of a compound having the
formula
:'~ O
C - C - NH-C~--CH C~2 ~ Nl
N--R~ ~// C~C CH2-5~~ ~ - A
Il
- 28 -
7~7
herein R2 and A' are as hereinbe~ore defined
and R3 is hydrogen, pivaloylox~methyl,
acetoxymethyl, methGxymethyl, acetonyl, phenacyl, p-
nitrobenzyl, ~ -trichloroethyl, 3-phthalidyl or
5-indanyl or a nontox~c, pharmaceutically
acceptable salt thereof, said compound being at least
75% by weight in the form of lts syn isomer and
preferably in the form of its syn isomer essent~ally
free of the corresponding anti isomer.
.. _ _ _ . , .
There i8 further provided by the pre~ent
lnventlon a method of treatlng bacterial infectlons
comprlsing admlnlstering by ln~ectlon to ~n lnfected
warm-blooded anlmal, includlng man, an effectlve but
nontoxic dose of 250-1000 mgm. of the ~ lsomer of a
compound having the formula
O '
C - C - NH-CH - CH ~H2 ~ N
~-OCH3 o ~ ~ C~ CH2 S N,N ~ I A~
C-OH O
il
herein Al is as hereinbefore defined or a nontoxic,
pharmaceutically acceptable salt thereofJ and
. wherein n is preferably 1.
- 29 -
1~5072r7
There is al~o provided by the present
inventlon Q ~ethod ~or cDmbatting Hae~ophilus ~n~ec-
tl~ns which comprl~es administering to a warm-bl~oded
¦ mammal in~ccted with an Haemophilus infectlon an
I . amount ef~èctlYe for treating said Haemophilus infec-
. t~on of a composition comprising a compound having
¦~ the ~ormNla
C - g - NH~ -cl~ C~12 ~ Nl
_ o}~2 o ~ ~ c~,c CH2-S ,N ~ -A
o~3 o
' O
herei1l Al ~nd R2 are as hereinberore defined
and R3 is hydrogen, pivaloyloxymei;hyl,
acetoxymethylJ methoxymethyl, acetonyl, phenacylJ p-
`. nltrobenzyl, ~ -trichloroethyl, 3-phthalidyl or 5-lndanyl
and preferably is hydro~en or a nontoxic, pharmaceutically
acceptable salt thereof, said compound being at least
75~ by weight in the ~orm of its ~ isomer and
-: preferably in the form of its s~n isomer essen~ially
~ ~ree of the corresponding anti isomer, and a pharma-
: ceutically acceptable carrier there~or.
. .'
: There is also provided by the present
. invention a method for combatting Nelsserl~ ln~ec-
tl~ns which comprises adminl~tering to a warm-bl~oded
mammal in~ected wlth a Neisseria infection an amount
! e~fec~ive for treating sald Neisseria ln~ection ~f a
r composition cDmpr~sing a compound having the ~ormula
r - 3c~ _
1 ~5u7z7
C - C - ~H-C~--CI~ ~H2 ~ N
N_ OR~ ~ ~ C~ 2 N~N ~ -
' C-O~ O
., 11
: O
~Jherein R2 and Al are as hereinbefore defined
and R3 is hydrogen, pivaloyloxymethyl,
- acetox.~lethyl, methoxymethyl, acetonyl) phenacylJ p-
nitrobenzyl, ~ -trichloroet}~yl, 3-phthalidyl or 5-indanyl
snd pre~erably is hydrogen or a nontoxic, pharmaceutically
jj acceptable salt thereof, said compound bein~ at least
75% by weight in the f`orm of its syn isomer and
preferably ln the form of its syn isomer essentially
free of the corresponding anti isomer, and a pharma-
~ ceutically acceptable carrier therefor.
p m e present invention also provides the novel
compounds having the formula
S~~ N-Al
wherein Al is methyl or ~ 2 )nCO~I, ~7here n is one or tl~o.
. . .
me invention further provides the novel compounds
havin6 the formu1a
;. ~
- 31 -
r
L5~7~27
,s~
H~N-~?I ~ C~
N ~ ~C-~12-S ~ ,~I ~ N_
C- oP~3
wherein Al is methyl or ~(CHa)nC0~1,
~ here n is one or two and R3 is hydro~en or a conventional,
r pharmaceutically acceptable, easily ~lydrolyzed ester
,
forming group, or a nontoxic, pharmaceutically accepta~le
salt thereof,
i" .
: ~ ~
. .
. :
. ~
` ~ ' .
. ~
~ ' :
; `:
, ~
. . - 32. - .
5~7~7`
STARTING MATERIALS
2-Furoylc~anide
To a suspenslon of 26.1 g. (0.4 mole) of ground
potassium cyanlde ln 300 ml. of acetonitrlle at 5 C. was
~dded 26.1 g. (0.2 mole) of n-furoyl chloride while keeping
the temperature below 8 C. The mixture was stirred ln
the cold for 15 minutes then heated at reflux for 30
mlnutes. The reaction was cooled, filtered and the aceto-
nltrile was removed at 15 mm. (steam-bath) leavlng 24.5 g.
of a dark oil which was used without further purlflcation.
An infrared spectrum showed a nltriie band at 2æ5 cm 1.
2-Furane~lyoxYlic Acid
.
The 24.5 g. of crude 2-furoylcyanide was mixed
with 160 ml. concentrated hydrochloric acid at 25 C. with
intermlttent stirring. The reactLon was stored for 24
hours at 25 C. and diluted with 130 ml. of water. The
reaction was stirred for 5 minutes and filtered. The fil-
trate was ~aturated w~th sodium chloride and extracted with
5 x 120 ml. of 1:1 ether-ethyl acetate solution. The extracts
were combined, dried over anhydrous magnesium sulfate and
evaporatèd at ~0 C. (1~ mm.) to give a brownish-orange
solid. The solid was disqolved ln methanol, treated with
charcoal and evaporated under reduced pressure (15 mm.) to
dryness to yield 17 g. of the acid.
The product was recrystallized from toluene to give
11.5 g. (m.p. 76D C.). The ir and nmr spectra were consis-
tent for the structure.
2-Methoxyimino-2-furylacetic Acid
To a solutlon of 4.5 g. (0.0~2 mole) of 2-furane-
glyoxylic acid in 40 ml. of 50~ alcohol and ~.1 g. (0.0~7
- 33 - -
~5~72~
~ole) ~ methoxyamine hydrochlorlde ln 6 ml. water ~t 20
C. ~as added dilute sodium hydroxlde ~olution to pH 4-5.
The solution was stirred at pH 4-5 at 25 C. ~or 24 hours.
The alcohol was removed under reduced pres6ure (15 mm.) and
the solution was ad~usted to pH 7-8 with 50% sodium
hydroxide solution. The reaction was extracted wlth 3 x
~0 ml. of ether and the aqueous layer was ad~usted to pH
1.9 using concentrated hydrochlorlc acld. The mixture was
extracted with 5 x 50 ml. of ethyl acetate. The organlc
fractlons were combined, washed wlth brlne, dried over
anhydrous magnesium sulfate and evaporated under reduced
pressure (15 mm.) to an oil ~hlch was cooled ~or one hour
in an ice bath. The product was slurried with Skellysolve
B and collected to yield 3.1 g. o~ yellow crystals, m.p.
78 C. An analytlcal sample wa~; recrystallized from
toluene, dried for 16 hours in ~acuo over P205 at 25 C.
The ir and nmr spectra were consistent for the structure.
Anal. Calc~d for C7 ~ N0: C, 49.65, H, 4.17;
~ N, 8.28. Found: C, 49.30; H, 4.21; N, 8.37.
.~ .
2-Ethoxyiminofurylacetic Acid
, .
The 7.85 g. (o.o56 mole) o~ furyl-2-glyoxyllc
~ acid was dissolved in 100 ml. of water and ad~usted to
; pH 7 with 50% sodium hydroxide. The 6.83 g. (0.070 mole)
; of ethoxyamine hydrochloride ln 10 ml. o~ water was added,
while keeping the pH at 4-5. The reaction was diluted
with 25 ml. of alcohol, stirred 3 hours at room tempera-
ture and then ~iltered. The alcohol was removed at 35
C. (15 mm.) and the aqueous portlon was ad~usted wlth
` dllute sodium hydroxide solution to pH 7-8 and then
- 34 -
~151~q2~
was washed with ether ~nd the washes were discarded. The
aqueous ~raction was ad~usted wlth 6N hydrochIoric acid
to pH 1.5 and extracted into ~ x 80 ml. of ethyl acetate.
me acetate fractions were comblned, washed with brine
and reduced ln volume at 35 C. (15 mm.~ to an oil. The
oil was cooled in an ice bath, triturated with Skellysolve
B, collected and dried over P205 in vacuo at 25 C.
Yield: 4.8 g., m.p. 83-85 C. The ir and nmr were con-
slstent for the structure.
Anal. Calc'd for C8HgN04 C, 52.46; H, 4.95;
N, 7.65. Found: C, 52.22, H, 4.94; N, 7.60.
,
Sodlum a-Ethoxyimino-~-(2-furyl)acetate
To 50 ml. of methanol was added 250 mg. (0.0109
mole) of metallic sodium and stirred untll all the sodium
had dissolved. This sodium methoxide solution was treated
with 2.0 g. (0.0109 mole) of ~-ethoxyimino-a-(2-furyl)acetic
acld dissolved in 10 ml. of methanol and stirred at room
temperature for one hour. The methanol was remo~ed at
40 C. (1~ mm.) and the product was dried ln ~acuo over
P205 at 25~ C.to yield 2.22 g. white solid, m.p. decomp.
~240~ C. The ir and nmr were consistent for the structure.
"Skellysolve B" is a petroleum ether fraction
of b.p. 60-68 C. consisting essentially of n-hexane.
,
.
- 35 -
.
5¢~727
.
CI -et-nltril~
H HCl
CH3ONH2~HCl
3 ~c ; ~ ~ OH
Na C~ OH
~ ~ .
1~1 ~ (CCl)2 r~l ~
:i . ~fi- -ONa ~ C I
CH3 ~ IbCH3
,,
. ~
:~.
...
?-Furo~lcyanide 1
To a suspension of 78.~ g. of powdered potas-
~:~ sium cyanide in 900 ml. acetonitrile at 5 C. was added
59.25 ml. (68.5 g.) o~ ~-furoyl chloride with ~igorous
stirring while keeping the temperature at 4-8 C. The
- mixture was stirred at 4-8 C. for 15 minutes and then
heated at reflux for 30 minutes. The mixture was cooled
::~ to 23-25 C., filtered, washed with 50 ml. of acetonitrlle
. whlch was added to the filtrate, and the acetonitrile was
removed at 60 C. (15 mm.) leaving 51 g. of 1 as a dark
- 36 -
. . -- . _
72~
~il. An IR spectrum showed a nitrile band at 2265 cm 1
and an NMR spectrum showed a ratio of approximately
70/30 of product ~ furoic acld. The crude product ~ was
used without further purification (49% yield of product).
Furyl-2-glyoxylic Acid 2
The 51 g. of crude 2-furoyl cyanlde 1 was
mixed with 500 ml. concentrated hydrochloric acld at
25 C. The reaction was stirred for 24 hours at 25~ C.
and then diluted with 240 ml. of water. The mlxture was
stirred for 5 minutes and filtered. The black filtrate
was saturated with sodium chloride and extracted with
6 x 500 ml. of 1:1 ether-ethyl acetate solution. (Note:
Initially the extractions were difficult due to the
inability to see the separation of two black phases. As
additional ether-ethyl acetate extractions were run the
task was slmplified.) The extracts were combined and
evaporated to dryness at 60 C. (15 mm.). The resultant
solld was dissolved in 600 ml. ether, (Note: Use of
alcohol should be a~oided at this point as es~ers may
form)~ treated with 10 g. of charcoal ("Darko-KB"),
filtered after stlrring for 0.5 hour ana evaporated
to dryness at 50 C. ~15 mm.) to yield 46.6 g. of 2
as a light tan colored acid. Thls product 2 was
found to contain a ratio of approxlmately 56/44 o~
product 2/furoic acid. This represented a 63% yield
of product 2.
Puri~ication was accomplished by dissolving
the above crude product 2 in H20 (50 mg./ml.), titrating
to pH 2.8 with HCl and extracting with 2 x 200 ml. of
*Trade Mark
1' 115~727
ethyl acetate. Evaporation o~ the ethyl acetate extracts
gave 35% furoic acid and 15% product 2, The pH 2.8
aqueous phase was ad~usted to pH o.8 ~HCl~ and extracted
w1th 2 x 200 ml. ethyl acetateO The organic extracts
were combined and washed with 50 ml. H20. The organic `
phase was evaporated at 50 C. (15 mm. 3 yielding a
601id with a ratio o~ approximately 86/14 o~ product
2/furoic acid. This solid was then recrystallized by
dissolving the product 2 ln toluene at 50 mg./ml. at
80~ C., decanting, and leaving to crystallize at room
temperature for 18 hours, yielding 13.3 g. of pure acid
2 by NMR. This represented a 51% yield in the purifica-
tion and recrystallization step and an overall yleld from
.. .
the 2-furoyl chloride to the pure furyl-2-glyoxylic
acid 2 o~ 16%.
Syn-~-methoxyiminofurylacetic Acid ~
A solution of 4.5 g. of furyl-2-glyoxylic ac~d
2 in 40 ml. of 50% ethanol was titrated to pH 6 with lN
sodium hydroxide and then ~.1 g. of methoxyamine HCl in
6 ml. nf H20 at 20 C. was added. The solution was
titrated to a constant pH 4.9 ~nd stlrred-at pH 4.9 for
~4 hours at 20-23~ C. The ethanol was then removed at
50 C. (15 mm.~ and the residual aqueous ~olution was
titrated to pH 8 with 50% sodium hydroxide and washed
with 3 x 50 ml. ether (pH adjusted to 8 a~ter each
wash~. me aqueous layer was titrated to pH 1.9 with
concentrated HCl and extracted with 5 x 50 ml. ethyl
acetate with the pH read~usted to 1.9 after each extrac-
tion. The ethyl acetate extracts were combined and
38 -
.,
`
` ~S~7;;~7
evaporated to a solid ~ at 50 C. (15 mm.~. Thls solld `
was then slurried with 75 ml. of "Skellysolve B". me
suspension was filtered and the solids were redissolved
in 16 ml. of toluene at 80 C. The hot solution was
decanted and left to crystallize at 20-23 C. for 18
hours to yield 1.17 g. ~ (22% yield of product). m e
NMR was clean and consistent for the structure ~ with a
trace of anti isomer present.
Sodium S~ methoxyiminofurylacetate 4
~ .
To 40 ml. of methanol was added 0.16 g. of
sodium. The mixture was stirred until all of the sodium
dissolved and then decanted. The resulting sodium
methoxide solution was cooled to 3 C. and 1.12 g. o~
~ -methoxyiminofurylacetic acid ~ in 7.8 ml. of
methanol was added. The solution was stirred for 10
` minutes at room temperature. The solvent was evaporated
at 40 C. (15 mm.). The residue 4 was dried by azeo-
tropic distillation with 3 x 20 ml. of benzene at 40 C.
(15 mm.). The product,4 was dried for 18 hours at 2~
C. under high vacuum (0.7 mm;) over P205 yielding 1.25 g.
9% yleld of product). The NMR showed this product ~
to be clean and consistent for the structure with 0.15
mole methanol and a trace of anti isomer.
To 0.63 ~. of sodium syn-~-methoxylmlnofuryl-
acetate 4 suspended in ~5 ml. of benzene was added four
drops of dry dimethylformamide ~nd 0.31 ml. (1.1 eq.
of oxalyl chloride. This mixture was stirred for 40
minutes at 20-23 C. The benzene was removed at 35 C.
(15 mm~) to provide the acid chloride 5 as the gummy
residue.
39
l~S07Z7
6-Chloro-2,3-dihydro-2-ethoxycarbonylmethyl-s-t-rla
3-blpyridazin-3-one
To a solution of 6-chloro-2,3-dlhydro-s-
triazolo~4,3-b]pyridazin-3-one [P. Francavilla and F.
Lauria, J. Het. Chem., 8, 415 (1971)] (1, 1.00 g., 5.9
m.mole) ln dry DMF (30 ml.) was added sodium hydrlde
(50% in paraffin, 0.3 g., 6!3 m.mole) under stirring with
formation of yellow crystals. To the mixture was added
ethyl chloroacetate (1.4 ml., 13 m.mole) and the mixture
was heated at 90 C. for 8 hours with stirring. After
cooling, the reaction mixture was poured into water (50
ml.) and extracted with toluene (5 x 40 ml.). The or-
ganic extracts were combined, dried over anhydrous sodium
sulfate and e~aporated at reduced pressure. The residue
was crystallized with benzene-n-hexane to give yellow
needles (1.16 g., 77%), m.p 114-115~ C. (lit.
110 C . ) .
; ir: ~KBar 1735, 1710 cm 1
uv; ~EtOH 231 nm (E ~ 26000)
nmr: ~ppC13 7.58 (lH, d, J=10 Hz, pyr~dazine-H), 6.98
(lH, d, J=10 Hz~ pyridazine-H), 4.80 (2H~ s, -CH2CO),
4.27 (2H, q, J=7.5 Hz, CH2CH3), 1.29 (3H, t, J=7.5 Hz,
CH2CH3 ),
Anal. Calc'd. for CgHgN403Cl C, 42.12; H, 3.53;
N, 21.83; Cl, 13.81. Found: C, 41.54, 41.46; H, 3.~2,
3.49; N, 21.51, 21.~3; Cl, 13.88, 13.99.
- 40 -
1~ S~727
?-Carboxymethyl-2,3-dlhydro-6-mercapto-~-trlazolo ~ 3-~ _
p~ridazin-3-one
To a solu~ion of 6-chloro-293-dlhydro-2-ethoxy-
carbonylmethyl-s-triazolo~4,3-b]pyridazin-3-one (30 g.,
0.12 mole) in ethanol (900 ml.) was added NaSH 2H20 (70%
pure, 45.9 g., 0.36 mole) and the mixture was refluxed
for 0.5 hour. The reaction mixture was e~aporated at re-
duced pressure. The residue was dissolved in water (200
ml.) and concentrated HCl was added to the solution to
adjust to pH 2. The precipitate of 2-carboxymethyl-2,3-
dihydro-6-mercapto-s-triazolo~4,3-b~pyridazin-3-one
was collected by fil-
tration and washed with water. Yield 18.3 g. (69~).
lr: vKBax 2900, 2450, 1750, 1660 cm 1.
uv: ~l~xaHCO3aq 260 nm (E~19500), 3i3 nm (E, 7000)
~` nmr: ~pDMS0-d6 7.88 (lH, d, J=10 Hz, pyridazine-H),
7.45 (lH, d, J=10 Hz~ pyridazine-H), 4.72 (2H, s, CH2C0).
Anal. Calc'd. for C7H6N403S: C, 37.17; H, 2-67;
N, 24.77; S, 14.17. Found: C, 37.35, 37.23; H, 2.269
2.28; N, 23.58, 2~.69; S, 14.32.
. : .
7-Amino-3-(2-carboxymethyl-2,3-dihydro-s-triazoio[4,3-bl-
pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic Acid
To a suspenslon of 7-aminocephalosporanic acid
(8.79 g., 32.2 m.mole) ln 0.1 M phosphate buffer (pH 7,
~ 41 -
~L~5C~7Z7 `
149 ml.) were added NaHC03 (8.14 g., 97.0 m.mole) and-the
thiol 2-carboxymethyl-2,3 dihydro-6-mercapto-s-triazolo-
[4,3-b~pyridazin-3-one (7.30 g.~ 32.2 m.mole) with stir-
ring. The mixture
was heated at 80 C. for 0.5 hour under N2 stream. The
mixture was treated with active carbon and ad~usted to
pH 3 with concentrated HCl. m e resulting precipitate was
collected by filtration and washed with water to give 7.59 g.
(54%) of 7-amino 3-(2 carboxymethyl-2,3-dihydro-s-tri-
azolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-
carboxylic acid.
.~ .
- 42 -
;
~L~ Cj~7;~7
ir vKBar 1800, ~720, 1600, 1540, 1470 cm 1.
uY ~Buffer (pH 7) 252 nm (~,19500), 298 nm (~, 8400).
nmr: ~p~mO+K2C3 7.56 (lH, d, J=9 Hz, pyridazine-H),
7.05 (lH, d~ J=9 Hz, pyridazine-H), 5.45 (lH, d, J=5 Hz,
6-H), ~.05 (lH, d, 5 Hz, 7-H), 4;43 (lH, d, J=14 Hz,
~-CH2), 4.04 (lH, d, J=14 Hz, 3-CH2), 3.88 (lH, d, J=18 Hz~
2-H), 3.45 (lH, d, J=18 Hz, 2-H).
6-Chlo hyl)-2,3-dihydro-s-triazolo~4,3-b]-
pyridazin-3-on.
To a solution of 6-chloro-2,3-dihydro-s-tri-
azolo[4,3-b]pyridazin-3-on [P. Francabilla and F~ Lauria,
J. Het. Chem. 8, 415 (1971)] (17 g., 0.1 mole) in dry
DMF (300 ml.) was added potassium tert.-butoxide (0;5 g.,
4.5 m.moles) with stirring. Acrylonitrile (6.6 g., 0.12
mole) in dry DMF (10 ml.) was added to the mixture. The
mixture ~as stirred at 100-110 C. for 24 hours, then
poured into water (700 ml.) and extracted with ethyl
acetate (5 x 400 ml.). The organic extracts were combined,
dried over Na2S04 and evaporated. The residue was crystal-
lized from ethyl acetate to give light yellow needles o~
. 6-chloro-2-(2-cyanoethyl)-2,3-dihydro-s-triaz~lo[4,3-b]-
pyridazin-3-on (2.5 g., 11%). M.p. 165-168 C.
ir: vmBx 2230, 1720, 1550, 15nO cm 1.
uv: ~dimXaxne 373 nm ( E 2000 ) .
nmr: ~Dppm d6 3.03 (2H, t, J-6.o Hz, CH2), 4.21 (2H,
t, J-6.o Hz, CH2), 7.23 (lH3 d, J=10.0 Hz, pyridazine-H),
_ 43 _
1~ 5137Z7
.
7.93 (lH, d3 J=10 0 Hz, pyridazine-H).
Anal. Calc'd. for C8H6N50Cl: C, 42.97; H, 2.70;
N, 31.32; Cl3 15.86. Found: C, 42.73, 42.56; H, 2.57,
2.50; N, 31.36, 31.68; Cl, 15.96, 15081.
2-(2-Carboxyethyl)-6-chloro-2~3-d ~ azolo~4,3-b
pyridazin-3-on.
A solution of 6-Chloro-2-(2-cyanoethyl)-2~3
dihydro-s-triazolo[4,3-b]pyridazin-3-on (724 mg.) in
6N-HCl (15 ml.) was refluxed for 6 hours. The reaction
mixture was extracted with ethyl acetate (10 x 20 ml.).
The combined extracvs were washed with saturated aqueous
sodium chloride (50 ml.), dried over Na2SQ4 and evaporated
to give light yello~J~solid 2-(2-carboxyethyl)-6-chloro-2,3-
dihydro-s-triazolo[4,3-b]pyridazin-3-~n (567 m~., 72~).
M.p. >170~ C. (sublimation).
ir: ~KBx 3400-2400, 1730, 1710, 1540 cm 1.
- uv: ~dioxane 377 nm (E 1500)
nmr ~D2~panHC3 2.70 ~2H, t, J=7.0 Hz, CH2), 4-24
(2H, t, J=7.0 Hz, CH2), 7.17 (lH, d, J=10.0 Hz, pyrid~zine-H)3
7.70 (lH, d, J=lO.Q Hz, pyridazine-H).
Anal. Calc'd. ~or C8H7N4Q3Cl: C, 39.60; H, 2.91;
N, 23.09; Cl, 14.61. Found: C, 39.62, 39.48; H, 2.97,
2.67, N, 23.05, 22.70, Cl. 13.93, 14.12.
' ~5~7~7
2-~2-Carboxyethyl~-2,3-dihydro-6-mercapto-s-tr-iazolo~4~3-b
pyridazin-3-on.
A mixture of 2-(2-carboxyethyl)-6-chloro-2,3-
dihydro-s-triazolo[4,3-b]pyridazin-3-on (567 mg., 2.34
m.moles) and 70% sodium hydrosulfide dihydrate (924 mg.,
7.02 m.mole) in water (10 ml.) ~as stirred at room tempera-
ture for two hours. The reaction mixture was adjusted suc-
cessively to pH 1 with c. HCl, to pH 10 with NaOH and then
to pH 1 with c. HCl. The resulting preci~itate of 2-(-
carboxyethyl)-2,3-dihydro-6-mercapto-s-triazolo[4,3-b]-
pyridazin-3-on was collected by filtration and washed with
water. Yield: 418 mg. (74%). M.p. 174-176 C.
ir: vmBax 3600-2600, 2440, ;1730, 1720 (sh) cm 1.
uv: ~pH 7 buffer 262 nm (E/7o~0), 318 nm (E 6600).
nmr: ~D~ppmd6 2.73 (2H, t, J=7.0 Hz, CH2), 4.07 (2H,
t, J=7.0 Hz, CH2), 7.30 (lH, d, J=10.0 Hz, pyridazine-H),
7.74 (lH, d, J=10.0 Hz, pyridazine-H).
!
Anal. Calc'd. for C8H8N403S: C, 40.00; H, 3.36;
N, 23.32; S~ 13.35. Found: C, 39.o8, 39.o6; H, 3.12,
3.20; Ny 22.65, 22.70; S, 14.23, 14.29.
7-Amino-3-~2-(2-carboxyethyl)-2,3-dihydro-s-triazolo~4,3-b~-
pyridazin-3-on-6-ylthiomethyl]-3-cephem-4-carboxylic Acid.
- A mixture of 7-ACA (405 mg., 1.49 m.moles), the
thiol 2-(2-carboxye~hyl3-2,3-dihydro-6-mercapto-s-
triazolo~4,3-b]pyridazin-3-on (357 mg., 1.49 m.moles) and
NaHC03 (375 mg., 4.47 m.moles) in 0.1 M phosphate buffer
(pH 7, 8 ml.) was stirred at 8Q C. for 30 minutes. nle
reaction mixture was cooled and filtered to remove insolubles.
- 1~5 -
- 1~5~q27
The filtrate was adjusted to pH 1-2 with c. HCl. The result-
ing precipitate, 7-amlno-3-[2-(2-carboxyethyl)-2,3-dihydro-
s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl]-3-cephem-
4-carboxylic acid, was collected by filtration and washed
with water. Yield: 519 mg. (77%).
ir: vKBX 3600-2200, 1800, 1725, 1620, 1550, 1480 cm 1.
~pH 7 buffer 253 nm (E ~0000), 298 nm (~/ 0 )
` nmr: ~D2ppK~C3 2-20 (2H, t, J=7.0 Hz, CH2), 3.40 (lH,
d, J=17.5 Hz, 2-H), 3.85 (lH, d, J=17.5 Hz, 2-H), 4.00-
4.50 (4H, m, 3-CH2 and N-CH2-), 5.01 (lH, d, J=4.0 Hz, 6-H),
5.40 (lH, d, J=4.0 Hz, 7-H), 6.94 (lH, d, J=10.0 Hz,
pyridazine-H), 7.44 (lH, d, J=10.0 Hz, pyridazine-H).
Anal- Ca1c~d- for C16H16~606S2 3/ 2
H, 3.99; N, 17.52; S, 13.37. Found: C, 40.06, 40.12;
H, 3.33, 3.34; N, 16.96, 16.98; S, 13.87, 13.98.
7-ACA refers to 7-aminocephalosporanic acid and
DMF to dimethylformamide.
.
- 46 -
...
- ~.15~7Z7
Scheme 1. Preparation of 7-Amino-3-(2-carboxymethyl-
2,3-dihydro-s-triazolo[4,3-b]pyrida-zin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic Acid.
_~=N~ ClCH2COOC H
C N"N~n~NH 2 5 ) Cl ~ N ~ N-CH2COOC2H5
~N~SH ~ ~ 7-~CA
HS N,N ~ N-Ctl~COOH
O
H2N ~ ~ M
N ~ CH2S ~ I~,N ~ N-CH2COOH
C02H
47 -
7;~7
6-Chloro-2,3-dihydro-2-ethoxycarbonylmethyl-s-triazolo-
[4,3-b]pyridazin-3-one (2)
To a solution of 6-chloro-2,3-dihydro-s-
triazolo[4,3-b]pyridazin-3-one [P. Francavilla and F.
Lauria, J. Het. Chem., 8, 415 (1971)] (1, 1.00 g., 5.9
m.mole) in dry DMF (30 ml.) was added sodium hydride
(50% in paraffin, 0.3 g., 6.3 m.mole) under stirring with
formation of yellow crystals. To the mixture was added
ethyl chloroacetate (1.4 ml., 13 m.mole) and the mixture
was heated at 90 C. for 8 hours with stirring. After
cooling, the reaction mixture was poured into water (50
ml.) and extracted with toluene (5 x 40 ml.). The or-
ganic extracts were combined, dried over anhydrous sodium
sulfate and evaporated at reduced pressure. The residue
was crystallized with benzene-n-hexane to give yellow
needles of 2 (1.16 g., 77~, m.p. 114-115 C. (lit.
110 C.).
ir: ~ max 1735, 1710 cm 1.
uv: ~ Emtax~I 231 nm (, 26000).
nmr: ~ ppC13 7.58 (lH, d, J=10 Hz, pyridazine-H), Ç.98
(lH, d, J=10 Hz, pyridazine-H), 4.80 (2H, s, -CH2CO),
4.27 (2Hl q, J=7.5 Hz, CH2CH3), 1.29 (3H, t, J=7.5 Hz,
CH2CH3 ) .
Anal. Calc'd. for CgHgH4O3Cl C, 42.12; H, 3.53;
N, 21.83; C1, 13.81. Found: C, 41.54, 41.46; H, 3.22,
3.49; N, 21.51, 21.53; Cl, 13.88, 13.99.
l~LS~
2-Carboxymethyl-2,3-dihydro-6-mercapto-s-triazolo[4,3-b]-
pyridazin-3-one (3)
-
To a solution of 6-chloro-2,3-dihydro-2-ethoxy-
carbonylmethyl-s-triazolo[4,3-b]pyridazin-3-one (2, 30 g.,
0.12 mole) in ethanol (900 ml.) was added NaSH 2H2O (70%
pure, 45.9 g., 0.36 mole) and the mixture was refluxed
for 0.5 hour. The reaction mixture was evaporated at re-
duced pressure. The residue was dissolved in water (200
ml.3 and concentrated HCl was added to the solution to
adjust to pH 2. The precipitate (3) was collected by fil-
tration and washed with water. Yield 18.3 g. (69%).
ir ~ max 2900, 2450, 1750, 1660 cm
uv: ~ 1%NaHCO3aq 260 nm (, 19500), 313 nm (~, 7000).
nmr: ~ ppm d6 7.88 (lH, d, J=10 Hz, pyridazine-H),
7.45 (lH, d, J=10 Hz, pyridazine-H), 4.72 (2H, s, CH2CO).
Anal. Calc'd. for C7H6N4O3S: C, 37.17; H, 2.67;
N, 24.77; S, 14.17. Found: C, 37.35, 37.23; H, 2.26,
2.28; N, 23.58, 23.69; S, 14.32.
7-Amino-3-(2-carboxymethyl-2,3-dih~dro-s-triazolo[4,3-b3-
pyridazin-3-on-6-ylthiomethyl)-3 cephem-4-carboxylic Acid (4)
To a suspension of 7-aminocephalosporanic acid
(8.79 g., 32.2 m.mole) in 0.1 M phosphate buffer (pH 7,
149 ml.) were added NaHCO3 (8.14 g., 97.0 m.mole) and the
thiol 3 (7.30 g., 32.2 m.mole) with stirring. The mixture
was heated at 80 C. for 0.5 hour under N2 stream. The
mixture was treated with active carbon and adjusted to
pH 3 with conc~ntrated HCl. The resulting precipitate was
collected by filtration and washed with water to give 7.59 g.
(54~) of 4.
- 49 -
'~
if ~:
5~P7~7
ir: ~ K~x 1800, 1720, 1600, 1540, 1470 cm 1.
uffer (P~ 7~ ~5~ nm (~,19500), 298 ~n (E,8400)-
n!nr~ m~lK2 3 7.5G (1~1, d, J=9 Hz, pyrida~ine-H),
7.05 (ln, d, J=9 Hz, pyridazine-~), 5.45 (1~, d, J=5 Hz,
:`~ 6-~), 5.05 (lH, d, 5 Hz, 7-~l), 4.43 (lH, d, J=14 Hz~
3-C~I2), 4.04 (lH, ~, J=14 Hz, 3~CH2), 3.88 (lH, d~ J=18 Hz,
~- 2-H), 3.45 (1~l, d~ J=18 Hz, 2-H).
Pivaloylox~nethyl-~-amino-3-(2-carboxymethyl-2,3-dihydro-s-
triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl~-3-cephem-
4-carboxylate.
Method A - The tltle compound i9 produced by
~ubstitutlng for the 7-amlnocephalosporanlc acid used
immedlately above an equlmolar weight Or pivaloyloxy-
i . methyl 7~aminocephalosporanate hydrochloride prepared
according to Example 2 of U.K. 1,229~453 from 7-
amlnocephalosporanic acld. German 1,904,585 (Farmdoc
39,445) ls equlvalent to U~K, 1~229~453,
. Method B. - The title compound is produced by
Qubstltutlng for the 0 025 mole (6.8 g.) 7-amlno-
: cephalosporanlc ac~d used ln the procedure o~ Example
~` 2 Or U.K. 1,229,453 an equlmolar welght o~ 7-amlno-3-
.~ (2-carboxymetnyl-2,3-dihydro-s-triazolo[4,~-b]pyridazin-
3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid (4).
The respective acetoxymethyl, methoxymethyl, acetonyl
and phenacyl esters of 7-amino-3-(2-carboxymethyl-2~3-
dihydro-s-triazolo[4,3-b]pyridazin-3-o~-6-ylthiomethyl)-3-
cephem-4-carboxylic acid are prepared by substituting in
Method B above chloromethyl pivalate used therein an equimolar
weight of chloromethyl acetate, chloromethyl methyl ether,
chloroacetone and phenacyl bromide, respectively.
.
~ ,. . .
27
Preparation of 7-Amino-3-(2-methYl-2~3-dihydro-s-tria-
zo ~ 4,3-b]pyridazin-3-on-6-ylthiomethyl-3-cephem-4-
carboxylic Acid.
Cl ~ NH C1 ` ~,N ~ N-CH~
1 / 2
~/ ,.
~I ' '
HS N'N~ ~ N- CH3
O
'. ,
1 ~
H2N T~
N ~ 2 ~ l~f ~ N CH3
C2H
! 4
6-Chloro-_,3-dihydro-2-methyl-s-triazolo~4,3-b]pyridazi_-
3-one (2)
To a solution of 6-chloro-2,3-dihydro-s-triazolo-
[4,3-b]pyridazin-3-one [P. Francavilla and F. Lauria, J.
Het. Chem. 8, 415 (1971)] (1, 8.5 g., 50 m.mol.) in dry
DM~ (12 ml.) was added NaH (50~ dispersion in paraffin,
. ' .
~ ~L15~27
.
2.64 g., 55 m.mol) and the mixture was stirred for 1
hour at room temperature. After methyl iodide (21.3 g.,
150 m.mole) was addedg the mixture was stirred for 40
hours at room temperature, diluted with water (200 ml.)
and extracted with CHC13 (4 x 100 ml.). The combined
extracts were washed with water (3 x 50 ml.), treated
with a small amount of carbon and dried with anhydrous
Na2S04. Evaporation of the solvent under reduced pres-
sure af~orded pale yellow residue which was crystallized
from chloroform-n-hexane. Yield: 7.23 g. (79~).
M.p. 180-181 C.
ir: vKB0 1720 cm 1
uv: ~Eta~I 233 nm (~ 25200), 363 nm ( E 1600).
nmr: ~Cppl3 3.72 (3H, s, N-CH3), 6.88 (lH, d, J=10 Hz,
pyridazine-H), 7.48 (lH, d, J=10 Hz, pyridazine-H).
Anal. Calc'd. fo~ C6H~ClN40: C, 3~.04; H, 2.73;
N, 30.35; C1, 19.21. Found: C, 39.24, 39.28; H, 2.54,
2.61; N, 30.63, 30.80; Cl, 19.59, 19.26.
~,
6-Mercapto-2,3-dihydro-2-methyl-s-triazolo~4,3-b]pyridazin-
3-one (3)
A mixture of 2 (6.50 g., 35.7 m.mol.) and
NaSH ~H20 (70~ pure, 9.4 g.) in water (100 ml.) was
heated under reflux for 15 minutes. The mixture was cooled
and acidified to pH 1 with concentrated HC1 to precipitate
the thiol 3 which was collected by filtration and dissolved
in aqueous saturated NaHC03 (100 ml.). The solution was
treated with a small amount of carbon and acidified with
dilute HCl to precipitate 3 as pale yellow prisms.
Yield: 5.72 ~ 95'). M.p. >280 C.
.
( ~
" ~ 27
:
ir: vKax 2450 (-SH), 1710 (C=0) cm 1
uv: ~1%maHC3 261 nm ( E 16300), 315 nm (~ 5800).
nmr: ~ 2 ppm 3.60 (3H, s, N-CH3) 3 7.o8 (2H s
pyridazine-H).
Anal. calc'd. for C6H6N40S: C, 39.55; H, 3.32;
N, 30.75; S, 17.60. Found: C, 39.57, 39.66; H, 3.14,
3.22; N, 30.32, 30.61; S, 17.80, 17.89.
7-Amino-3-(2-methyl-2,3-dihydro-s-triazolo~4~3-b]pyridazin-
3-on-6-ylthlomethyl~-3-cephem-4-carboxyllc Acid (4)
A mixture of 7-aminocephalosporanic acid (7-ACA,
5.44 g., 20 m.mol.), 3, (3.64 g., 20 m.mol.) and NaHC03
(3.36 g., 40 m.mol.) in 0.1 M phospllate buffer (pH 7, 100
ml.) was heated with stirrlng at 80~ C. for 30 minutes.
The hot mixture was treated with a small amount of carbon
and the filtrate was acidi~ied to pH 4 lJith dilute HCl to
prec~pitate 4 which was collected by filtration, washed
with water (50 ml.) and dried. Yield: 5.73 g. (73~).
M.p. 240-245 C. (dec ~.
ir: vKBX 1800 (~-lactam C=0), 1725 (C=0), 1610 and
1410 (C00 ) cm~l
uv: ~ ~NaHCo3 253 nm (E20000), 305 (E 8400).
D O
nmr: ~ 2 ppm 3 3.69 (3H, s, N-CH3), 5-08 (lH~ d~ J=4.5
Hz, 6-H), 5.48 (lH, d, J=4.5Hz, 7-H), 7.00 (lH, d, J=10 Hz,
p~ridazine-H), 7.52 (lH, d, ~=10 Hz, pyridazine-H).
Anal. CalC~d~ for C14H14~604S2 H2
- H, 3.91; N, 20.38; S, 15.55. Found: C, 40.84, 40.63;
H, 3.44, 3.31; N~ 20.50, 20.36; S~ 15.19, 15.57.
- 53 -
`~ 56C~27 - -
I`''' .
Preparation of BB-S515
CO-N ~ S ~ N
No~N~ CH2S ~N ,N ~ NH
OCH3 C02Na
BB-S515
r BB-S515; 7-~(2Z)-2-Methoxyimino(fur-?-yl)acetamido~-3-
(?~3-dihydro-s-triazolo~4,3-b~pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic ACid Sodium Salt.
To a solution of (2Z)-2-methoxyimino(fur-2-yl)-
acetic acid (169 mg., 1 m.mole) and triethylamine (0.14
ml., 1 m.mole) in dichloromethane (2 ml.) was added
oxalyl chloride (0.09 ml., 1 m.mole) at 0-5 C. and the
mixture was stirred for 30 minutes. The solvent was
evaporated under reduced pressure to a~ford an oily resi-
~ due. A solution of that oily residue in dry acetone
`~ (5 ml.), after filtration, was added to a mixture of
. , 7-amino-~-(2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-
~` ~ 6-ylthiomethyl)-3-cephem-4-carboxylic acid (380 mg.,
1 m.mole) (U.S. ~,907,786) and sodium bicarbonate (336
mg., 4 m.~ol.) in water (10 ml.) at 0-5~ C. The
¦ ~ reaction mixture was stirred at 0-5 C~ for 2 hours.
¦ Most of the acetone was evaporated at reduced pressure,
the aqueous concentrate being washed with ether (2 x
30 ml.) and adjusted to pH 1-2 with concentrated HCl.
The resulting precipltate (338 mg.) was collected by
filtration and dried in vacuo. A suspension of the free
acid (303 mg.) in water tlO ml.) was adjusted to pH 6.5
with aqueous NaOH ~1 N, o.6 ml.) and filtered to make a
clear solution which was lyophilized to ~ive 7-~(2Z)-2-
methoxyimino(fur-2-yl)acetamido]-3-(2,3-dihydro-s-
. .
-
- 54 -
1~ 5~1~Z~
triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-
4-carboxylic acid sodium salt as a light brown powder
(222 mg., 46%). M.p. >230 C. (dec,),
. ' ir: vKBaxr 3410, 1760, 1720, 1600 cm 1,
uv ~,pH7Buffer 256 ~m (E 20600), 274 (~ 18800)-
i:
.
1: '
.
'
.
:~ :
L5û~27
.
.
Pre~aratlon of D-man.delic acid carb~o~yanhYdride
COC 1 . - '
6 5 ~ 2 ~ C H ~o
OH d~ ~
o
.
-Mandel~c acld carboxvanhYdrlde (2)
Pho~gene was bubbled through a-solution of 2.0 g.
(0.01~ mole) of D(-)-~andelic acld (1) ln dry tetra-
hydroruran ror 30 mlnutes. The solutlon was allowed
to stand overnlght arter which tlme lt wa~ heated
under reflux rOr 10 mlnutes. Evaporatlon of the
solvent under reduced pressure afrorded an oily
resldue whlch was solldl~led by trlturatlon wlth
n-hexane (20 ml,). The product was collected by
flltratlon and drled ln ~Q~ on KOH. Yield 2.3 g.
of D-mandellc acld carboxyanhydrlde.
I .
IR ~ maUJ 1895, 1875, 1780 cm 1,
. - '
The preferred and most actlve compounds of
the pre~ent invention are those havlng the D
configuratlon at the a-carbon atom in the 7-~ide-
chain, that is, those mad~ from D-mandellc acid or
a mono~ub~tituted D-mandelic acid as illustrated here-
in, In additlon, the configùration at the two
optically active, asymmetric centers in the ~-lactam
nucleu~ 19 that ~ound ln cephalosporln C produced by
fermentatlon and in the 7-aminocephalosporanic acld
derlved there~rom.
-
- 56 -
)727
EXAMPLE 1
/0
C6H5-CH-C \ H N ~ ~ ~ N
O ~C / o ~ ~ CH2 S N,N ~ N-CH2C2H
O C2H. O
-- ~ C6H5-cH-coNH~s~
1H ~ N ~ CH2-S N~N ~ N-CH2C2H
C02H O
3, BB-S488
.
BB-S488, 7-(D-Mandelami ~ -3-(2-carboxymethyl-2,3-dihydro-
s-triazolo~4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-
4-carboxylic Acid (3)
. . . ~
D-(-)-Mandelic acid 0-carboxyanhydride (U.S. Patents
3,167,549, 3,840,531 and 3,910,900), (1, 400 mg., 2.3 m.mole)
was adde~ portlonwise to a solution of 7-amino-~-(2-carboxy-
methyl-?,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid (2, 657 mg., 1.5 m. mole)
and sod;um blcarbonate (445 mg., 5.3 m.mole) in 50~ aqueous
acetone (30 ml.) at ca 0 C. with vigorous stlrring. The
~mixture was stirred for 1 hour at room temperature and`
evaporated under reduced pressure below 40 C. to remove
acetone. The resulting aqueous solution was washed ~lith
ether and acidified to pH l with dilute HCl to afford a
gummy precipitate~ which was collected by tiltration,
washed with water and dissolved in tetrahydrofuran (TliF)
(100 ml.). The THF solution was treated with a small amount
~ 57 -
_ .~ . . . ; . . . _ .~ . . ............................... .
. . . .. ~ ,
,
7 ~`
of active carbon, dried over anhydrous sodium sulfate and
filtered. The filtrate was evaporated under reduced pres-
sure and the residue was triturated with ether. The pale
yellow precipitate was collected by filtration and chro-
matographed on a silica gel column (Wako-gel C-200, 10
g.) eluted with a solution of chloroform-methanol (20:1).
The fractions containing the desired product were combined
and concentrated under reduced pressure. Thè concentrate
was diluted with ether (100 ml.) to precipitate the
product (3), which was collected by filtration, washed
with ether (30 ml.) and dried. Yield 279 mg (34~).
M.p. 173-176 C. (dec.).
ir: ~ Kax 3600-2400, 1770, 1720, 1520, 1495, 1365,
1245 cm~l.
uv: ~ EtxH 254 nm ( 18000), 297 nm ( 9000, sh).
nmr ~ DMS-d6 3.68 (2H, m, 2-~T), 4.03 (lH~ d~ J=13
Hz, 3-H), 4.34 (lH, d, J=13 Hz, 3-H), 4.64 (2H, s,
NCH2CO), 5.00 (lH, d, J=4 Hz, 6-H), 5.02 (lH, s, PhCH),
5 .63 (lH, d-d, J=4 & 9 Hz, a doublet with addition of
D20, J=4 Hz, 7-H), 6.97 (lH, d, J=10 Hz, pyridazine-H),
7,1-7.4 (5H, m, phenyl-H), 7.60 (lH, d, J=10 Hz, pyri-
dazine-_), 8.60 (lH, d, J=9 Hz, disappeared with addi-
tion of D20, CONH).
Anal. Calc'd. for C23H20N60gS2 / 2
H, 3.64; N, 14.45; S, 11.03. Found: C, 47.34; H, 3.48;
N, 13.90; S, 11.01.
*Trade ~Iark
- 5~ -
'7
In vitro activit~ (Table 1)
The MIC's were determined by tne Steers' agar
dilution method using Mueller-Hinton agar against 4 gram-
positive and 28 gram-negative bacteria and the results are
shown in Table 1.
.' ' .
In vitro_activity (Tables 2 and ~)
MIC determinations were performed by serial two-
fold agar dilution method using Steers' apparatus on
Mueller-Hinton agar plate against 51-gram-positive and 95
gram-negative bacteria. The results are shown in Tables
2 and 3.
.
Media effect on MIC
~ . . .
The MIC's were determined by using three kinds
of agar media [Nutrient (NA), Mueller-Hinton (MHA) and
Heart-Infusion (HIA)]. The results obtained with BB-S488
and ceramandole are shown in Table 4, which indicates
little media effect in these cephalosporins.
Blood levels in mice
Groups o~ mice were administered subcutaneously
graded doses of 40, 20 and 10 mg./kg.. The ~lood sam-
ples collected from orbital sinuses were assayed by the
paper disc-agar diffusion method on Sarcina lutea PCI lO01
plates. The results are shown in Table 5.
In vivo activity
Comparative in vivo evaluation was made by the
standard experimental infection in mice against the
. - 59 -
,
~ ~LSg:~727 --~
following pathogenic bacteria
S. aureus Smith
E. coli Juhl
K, pneumoniae A9977
The results are shown in Table 6.
, - \
\
. . \ .
.: \
\
. . 60 _
: . .
~ 11 S ~ ~7 2 7
Table 1. The in vitro Antibacterial Activity o~ BB-S~88
.
By Agar Dilution ~lethod (Mueller-Hinton Agar).
~IC (mcg,/ml.)
Test Organism _ BB-S488 Cefamandole
S. aureus S~ith A9537 0.2 0.05
S. aureus A9497 0.1 0.05
S. aureus BX-1633 A9606 0,4 0.1
St. ~aecalis A9536 IOO 50
E. coli NIHJ 0.025 0.025
E. coli ATCC 8739 0.1 0.05
E. coli Juhl A15119 0.2 0.4
E. coli BX-1373 0.2 o.8
E. coli A15810 0.1 0.4
E. coli A9660 0.05 0.1
E. coli A15147 3.1 0.4
Kl. pneumoniae Q9678 3.1 3.1
ISl. pneumoniae A9977 0.05 0.2
Kl. pneumoniae A15130 0.2 0.8
Kl. pneumoniae A9867 0.2 o.8
Pr. vulgaris A9436 0.1 0.4
Pr. vulgaris A9699 0.2 6.3
Pr. mirabilis A9554 0-05 0.4
Pr. mirabilis ~g900 0.1 o.8
Pr. morganii A9553 >100 >100
Pr. morganii A20031 0.1 o.8
Pr. rettgeri A15167 0,05 o,~
Ps. aeruginosa A9930 >100 >100
Ps. aeruginosa A98l~3 >100 >100
- Shig. dysenteriae 0.025 0.1
Shig. flexneri A9684 12.5 6.3
Shig. sonnei A9516 0.025 0-05
Serr. marcescens A20019 100 100
Enterob. cloacae A9656 3.1 3.1
Sal. enteritidis A9531 0.05 0.1
Sal. typhosa A9498 0.05 0.1
B. anthracis A9504 0.1 0.1
- 61 -
- . .. ...
~7~
Table ?. In vitro Antibac~erial Activity in Mueller-
.... .. . .
Hinton Agar (Gram-positive~
.
MIC (mcg./ml )
Code Cefaman-
No. ' Test Organism BB-S488 dole
.
Sa-2 S. aureus Smith A9537 0.4 0.2
Sa-3 S. aureus No. 193 o.8 0 2
Sa-8 S. aureus 0.4 0 2
Sa-9 S. aureus No. 193 o.8 0.2
Sa-10 S. aureus A20239 1.6 0 4
Sa-ll S. aureus BX-1633 A9606 0.4 0 2
Sa-12 S. aureus A9497 0.2 0.1
Sa-29 S. aureus No. 193 1.6 0 8
Sa-33 S. aureus Terajima 0.0125 0 0125
Sa-34 S. aureus A15092 o.8 0 2
Sa-35 S. aureus A15094 o.8 0 4
Sa-36 S. aureus Russell o.8 O 4
Sa-37 S. aureus A9524 1.6 0 8
Sa-38 S. aureus A9534 0.4 0 2
Sa-~9 S. aureus A9578 o.8 0 4
Sa-40 S. aureus A9601 o.8 o. L~
Sa-41 S. aureus A9602 o.8 0 2
Sa-44 S. aureus A15097 25 25.
Sa-56 S. aureus A9~30 3.1 o.8
Sa-57 S. aureus A9748 25 ~.1
Sa-58 S. aureus A15033 12.5 1 6
Sa-59 S. aureus A15096 100 6 3
Sa-60 S. aureus A2060ll 50 3 1
Sa-61 S. aureus A20605 100 6 3
Sa-62 S. aureus A20606 3.1 o.8
Sa-6~ S. aureus A20607 >100 12.5
Sa-64 S. aureus A20608 100 6 ~
Sa-65 S. aureus A20609 100 6 3
Sa-66 S. aureus A20610 100 6
Sa-67 S. aureus A20611 100 6 3
Sa-68 S. aureus A20612 1.6 0 4
Sa-69 S. aureus A20613 100 6 3
Sp-l S. pyogenes S-23 0.4 0 1
Sp-2 S. pyogenes Dick 0.4 0 1
Sp-3 S. pyogenes A9604 - 0.4 0 1
Sp-4 S. pyogenes A20065 0.2 0 1
Sp-5 S. pyogenes A15040 o,4 0.1
Sp-6 S. pyogenes A20066 0.4 0 1
Sp-7 S. pyogenes Dig 7 0.ll 0 1
Sp-8 S. pyogenes A15041 0.4 0.1
Sp-9 S. pyogenes A?0201 0.4 0.1
Sp-10 S. pyogenes A?0202 0.4 0.1
Dp-l D. pneumoniae Type II 0.2 0.2
Dp-2 D. pneumoniae Type I Neufeld 0.2 0.2
Dp-3 D. pneumoniae Type III 0.2 0.2
Dp-4 D. pneumoniae A9585 0.2 0.2
Dp-5 D. pneumoniae A15069 0.2 0.2
Dp-6 D. pneumoniae ~201G7 0.2 0.2
Dp-7 D. pneùmoniae A20759 0.2 O ?
Dp-8 D. pneumo!liae A20769 0.2 0 2
Dp-9 D. pne~loniae A20770 0.2 0.?
- 62 -
~ , . . . .
~L5~ 7
Table 3. In vitro Antibacterial Activity in Mueller-
.
Hinton Agar ~ram-negative~
MIC (mcg./ml.)
Code , Cefaman-
No. Test Organism BB-S488 dole
... _ . ...
Ec-l E. coli NIHJ 0.2 0.1
Ec-3 E.-coli Juhl A15119 0.2 o.8
Ec-4 E. coli A15169 12.5 6.3
Ec-5 E. coli K-12~ ML-1630 A203630.2 o.8
Ec-ll E. coli A20366 5 25
Ec-15 E. coli ATCC 8739 0.2 0.1
Ec-34 E. coli A9660 0.1 0.1
Ec-35 E. coli A9435 0.4 o.8
Ec-3~ E. coli A15147 3.1 1.6
Ec-40 E. coli A20361 0.2 o.8
Ec-44 E. coli A9535 0-.1 0.1
Ec-45 E. coli A15148 3.1 1.6
Ec-46 E. coli A15164 25 12.5
Ec-47 E. coli A15170 100 50
Ec-49 E. coli A20107 0.4 0.2
Ec-50 E. coli A20109 0.2 o.8
Ec-51 E. coli A203ll3 50 12.5
Ec-56 E. coli A20365 25 12.5
Ec-58 E. coli A9575 0.4 1.6
Ec-59 E. coli A20766 0.2 o.8
Ec-62 E. coli A20895 0.4 o.8
El-l E. cloacae A9656 3.1 3.1
El-2 E. cloacae A20361l 3.1 3.1
El-4 E. cloacae A20650 1.6 1.6
El-6 E. cloacae A9557 o.8 o.8
El-7 E. cloacae A9659 1.6 o.8
El-8 E. cloacae A9655 1.6 1.6
El-9 E. cloacae A20021 >100 100
El-ll E. cloacae A20344 >100 ~100
El-12 E. cloac~e A21006 1.~
El-14 E. cloacae A20953 o.8 3.1
Pm-l P. mirabilis A9554 0.1 o.8
Pm-2 P. mirabilis A9900 0.2 1.6
Pm-3 P. mirabilis A20119 , 0~4 3.1
Pm-4 P. mirabilis A20~54 0.2 1.6
Pm-5 P. mirabilis A9702 0.1 o.8
Pm-6 P. mirabilis A21222 1.6 1.6
Pg-l P. morganii A9553 >100 >100
Pg-2 P. morganii A20031 0.2 .1.6
Pg-3 P. morganii A9636 o.8 1.6
Pg~5 P. morganii A15166 0.1 0.2
Pg-6 P. morganii A20455 0.4 1.6
Pg-7 P. morg~nii A20~57 0.2 o.8
Pg-8 P. morganii A15153 0.1 o.8
Pg-9 P. morganii A15149 0.8 3.1
Pv-l P. vulgaris A9436 0.2 0.8
Pv-2 P. vulgaris A9526 ~,3 1.6
Pv-3 P. vulgaris A9699 6.3 5
Pv-4 P. vulg~ris ~TC~ 9920 0.1 0.2
Pv-5 P. vulgaris A9539 25 >100
Pv-6 P. vulgaris A9716 0.1 0.8
Pv-7 P. vulgaris A21240 25 >100
- 63 -
. ~ 7
.
Table 3 (Continued)
Code Cefaman-
No, Test Organism BB-S488 dole
. " .. ..
Pr-l P. rettgeri A15167 0.1 0.2
Pr-2 P. rettgeri A9637 0.1 0.1
Pr-4 P. rettgeri A20645 0.1 0.1
Pr-5 P. rettgeri A20915 0.2 o.8
Pr-~ PO rettgeri A20920 0.1 0.2
Pn-l P. inconstans A206150.1 o.8
Ps-l P. stuartii A20745 0.4 0 8
Ps-2 P. stuartii A20894 0.2 0 8
Ps-3 P. stuartii A20911 o.8 o.8
Ps-4 P. stuartii A21051 5 25
Ps-5 P. stuartii A21057 0.2 o.8
Kp-l K. pneumoniae Dll 0.1 o.8
Kp-2 K. pneumoniae A9678 3.1 1.6
Kp-3 K. pneumoniae A9977 0.1 o.8
Kp-4 K. pneumoniae A151300.2 o.8
Kp-7 K. pneumoniae A9867 0.4 o.8
Kp-8 K. pneumoniae A2068025 12.5
Kp-9 K. pneumoniae A2063612.5 12.5
Kp-10 K. pneumoniae A203286,3 3.1
Kp-ll K. pneumoniae A203301.6 12.5
Kp-12 K. pneumoniae A212286.3 6.3
Kx-2 Klebsiella sp. A96620.4 1.6
Kx-3 Klebsiella sp. A20346 0.2 o.8
Sm-l S. marcescens A2001925 25
Sm-2 S. marcescens A203353.1 12.5
Sm-3 S. marcescens A203366.3 12.5
Sm-4 S. m~rcescens A204426.3 12.5
Sm-5 S. marcescens A202223.1 12.5
Sm-6 S. marcescens A204606.3 12.5
Sm-g S~ marcescens A20333G.3 5
Sm-10 S. marcescens A203346.3 5
Sm-ll S. marcescens A204596.~ 25
Sm-12 S. marcescens A204616.3 5
Se-l S. enteritidis A95~10.1 0.2
St-l S. typ}los~ 0.1 0.2
Sh-l S. paraty~hi 0.1 0.2
St-101 S. typhimurium 0.1 0.2
Sd-l S dysenteriae 0.1 0.2
Sr-l S flexneri A9684 12.5 3.1
Ss-l S. sonnei Yale 0.1 0.1
Cx-l Citrobacter sp. A20673 1.6 1.6
Cx-2 Citrobacter sp. A20694 1.6 1.6
Cx-3 Citrobacter sp. A20695 1.6 1.6
- 64 -
U~
. . N~ ~ OJ ~ ~ ~ COCO~ C0~ C0~ N~ N ~ J
. O O ~ O O O O O ~ N ~ ~ O O O O O O O O O O O ~ O ~ O ~ O O O O
O ~ ~ N ~O ~ O O O O
~1 A ~ ~
O . N ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ N
OO~OOO~O~N~OOOOOOOOOOO~O~oO~OOOO
S O N ~ N O O O O ~ ~ O
~ . ~
V N ~ ~ ~ N ~ ~ ~ ~ ~ ~ ~ ~ N ~ N ~ ~ ~ N ~ ~ ~
C O O~ O O O O O ~ ~N~ O O O O O~ O O O O ~ O~O O~ O O O O
O ~ N~ O O ~O
~1 ~1 . ,1 ~1
~\
~ . U~
COC0 O N N ~ ~ ~ N~ N ~ ~ N ~ O ~N ~ CO N
Cl~ . . . . . . . ~ . . . . . . . . . . . . . . .
O ~ O O O O O O O O~O O O ~O O O O O O O O O O O O N O N O O O O
~, ~ 00 0 00 0 0 ~1 ~10 0
~1 ~
O
O~
0 0~ O~ N ~ ~ N0 N N ~ N N0 H ~ N
C 0 H O ~ O O O O O O~ O O ~O ~ O C; O O O O O O O O O O O O ~ O O O O
OO O OO O ~ NOOO
~1 ~1 ~1
~ m ~ ~ ~ ~ ~ ~\~
0~ ,~N ,1~ ~ _~0NN0~1~1 N~J 0
~a
¢ O ~O O O O O O~ O O N N O O O O O O O O O O O O O O O O O O O
~d ~ O O OU~ 1 0 0 ~ U~ O O
~,~ ~ ~ ~ ~1
0
:` 0
u~
m
*
~o *
, O ~D O * * ~ ~ O
. ~ 'O~ ~1 * * C--~ O ~ ~ O * ~ O ~
X U~ ¢ ~ ~ ~ ~N~ O~ O ~Lr~ ~ ~,~ O O ~O
.~ .~ ~ O~d~O~0~N~
r' ~ ~ ~ ~ ~ ~ * * * LS~ ~ ¢ ~: ~ ~ ~ ~ Lr~ O O U~ O O O ¢ ¢ ¢
O ~ ~ ~ O ~ ~i.o0 ~ ~ O O ~ ~¢ ¢ ~N N ~ N N N
~ .~ I ~¢ ~o ~ ~N N ~ ~ ~¢ ¢ ¢ ¢ ¢ ¢ ¢ ¢ ¢
.~ ~ ~ X ~ ~ rl 1~) ~I r~ ~\¢ ~ ¢ t~
~ o ~ m d ~ ~ ~ O ~ ~ ~ O ~ O ~
H ~ N ~ ~ ~ N ~ ~ ~ ~ h ~ ~ ~ U
. u~ ~ ¢ ¢ ¢ ¢ ¢ ~ o o ~ C ~
u~ ~ F ~ P R ~ bD h. h h ~1 ~ bD h
~1 o ~ nd ~ :3 C) bD hO ~ O bD bD
E-l h h h O rl-l ~1 ` I ~1 ~I r-l H 0 0 0 Q~ h h h ~ h h h ~
t~ ~ d O O O O O O O r~ I C C~ rl O O O O ~ ~ ~ t~ c
Q
~ O
'~ ~ , ~ ~ ~ ~ ~ ~ ~ N ~ ~ ~ ~ N ~ ~ ~ ~ N
~ o V ~ ~ ~ ~ ~ v ~) v v v v ~ ~ ~ O- P~ ~ bO bO ~
v~ ~q ~ q ri~ ~ ril ~ r~ r~ r l rL~ r~ r~ p~ ? ~ ~ *
- 65 ~
.
l`` ': ,. ',, ;
j Table 5. Subcutaneous Mice Blood Levels
! , Mcg./Ml.
. . Dose _ Time BB-S488 Cefamandole
40 mg./kg. 15' 19 18
30' 17 11
60' 12 3.9
120' 1.6 0,3
.
20 mg./kg. 15' 7.4 9.5
0~ 6 4.1
60l 4.3
120' 0.7 ~0.1
.;~ ' .
~ 10 mg./k~. 15' 5 3.8
'~ 3' 3 1.5
I 60' o,.8 0.3
I 120' -- 0.1
D
~1
r .
. ~ . .
~ .
-i ,
~ . ~
.
- 66.-
- : . .
; ~ ~
llSV7Z7
Table 6. In vivo Activity
.. .
Test Organism Dose BB-S488 Cefam3nd~1e
S. aureus Smith 25 mg./kg. 5/5* 5/5
6-3 5/5 5/5
; 1-6 5/5 5/5
0.4 . 5/5 1/5
0,1 2/5
PD~o 0.12 mg./kg. o.6 mg./kg.
E. coli Juhl25 mg./kg 5/5 5/5
.~ 6-3 5/5 5/5
1.6 5/5 2/5
0.4 5/5 0/5
0.1 c/5
PD50 12 mg./kg. 1.8 mg./kg.
K. pneumoniae25 mg./kg.5/5 5/5
A3977
i 6.3 5/5 1/5
: 1.6 5/5 /5
.4 4/5 0/5
: 0.1 0/5
~P ~
PD50 0.26 mg./kg.6.25 mg./kg.
f
, : *No. of survivors/No.:tested
. ~ . .
. . .
.
.
,
.
.~
~15i~727
Run No. of Dose. BB-S Cefam-
Test Organism No~ LD50 (mg./kg.) 488 andole
E. cloacae C-811 1 x 10 100 5/5 5/5
A2~4~4 (El-l9)
` 25 5/5 5/5
: 6.3 5/5 5/5
1.6 4/5 4/5
0.4 2/5 1/5
50 (mg./kg.) O. 54 o.8
Urinary Recovery in Rats
% Recovery (0-24 Hrs.
Dose (sc) BB-S488 Ce~amandole
. .
10 mg./kg. 38.8 58.3
Ne ~rotoxicity Test in Rabbits
No nephrotoxic si~n was seen in the rabbits
treated with 100 mg./kg. tiv) o~ BB-S488 while cephalori-
dine showed severe nephrotoxicity in the comparative test.
.
.
.
_................................... , ~ `
Addltion~il P' ~O I)eta (Stn~le sc TreaCmcllt) 1150'727
Run No. o~ Dose BB-S Cefam-
Test Organism No. LD50 (mg./kg ~ 488 andolè
:
r K pneumoniae C-805 3x103 25 5/5 5/5
6.3 5/5 1/5
1.6 5/5 0/5
0.4 4/5 0/5
o . 1 0!5 ---
PDso (mg./kg,) 0.26 9.4
_ . .
A94U3~1-g ~ 1) C-808 lxlO 50 --- 3/5
5/5 ___
12-5 --~ 1/5
6.3 5/5 ~~~
3.1 --- 0/5
1.6 3/5 __~
.8 --- 0/5
0.4 1/5 ---
PD50 tmg-/kg.) 1.1 36
P. mirabilis C-810 lx103 50 ~~~ 2/5
A9900 ~Ym-2)
5/5 -_-
12.5 --- 1/5
6.3 5/5 ---
3.1 --- 0/5
1 6 4/5 ---
o.8 --- 0~5
0.4 0/5 ___
; 0.2 --- 0/5
0.1 0/5 ~~~
PD50 (mg./~g.~ 1.1 50
- _ ~
69
` ~ 727
!
Stability of BB-S488
! Stability of BB-S488 was determlned in both a
10% and an 0.02% solution. The stability is indicated as
the relative activity remaining in the test solution at
given periods to the initial solution. Tile activity was
assayed using paper discs on B. subtilis PCI 219 plate
(pH 6),
.
(1) Stability in a 10% A~ueous Solution at Room Temperature
~l\ Remaining ActivitY (~
Compound pH~ ~ O 2 ~ 3 7 Da~Js
BB-S488 6,1 100 128 90 116
(l)Unadjusted pH of the 10~ solution.
~2 Remaining Activity (%)
CompoundpH ) O1 ~~~~ 2 3 7 Dayis~
4 100 93 78 102
BB-S488 ~ 7 100 87 64
10020 14 13 0
i~ (2)pH 4: 0.1 M AcOH - NaOAc buffer.
pH 7: 0.1 M phosphate buffer.
pH 9: 0.1 M NH~OH-hH4Cl buffer.
~ .
.
- 70 -
~ .
~ ~ "
CHCOOH C12CHCOCl ~ CHCOOH SOC12
....... _~ ~ l
OH OCOCHC12
D-(-)
CHCoCl H-Ac~-s-cMTp(~) [ ~ CHCO-ACA-S-CMTP~
2 OCOCHC12 OCOCHC12
.. /
, ~/. Na2C03
CHCO-ACA-S-C~ITP
4 OH
. BB-S488
Dichloroacetylmandeloyl Chloride (2)
_
A mixture of D(-)-mandelic acid (1, 1.52 g.,
10 m.mole) and dichloroacetyl chloride (4.41 g., 30
m.mole) was heated at 80-85 C. for 1.5 hrs. and the
excess dichloroacetyl chloride was removed under diminished
pressure. To the residue was added thionyl chloride (2.5
ml.) and the mixture was heated under reflux for 1.5 hrs.
Excess thionyl chloride was removed by distillation and
dry benzene was added. Evaporation was repeated. The
residual oil was kept over KOH at 1 mm Hg overnight at
room temperature to remove dichloroacetyl chloride.
Yield, 2.8 g. (100~). This product was used in the next
- 71 -
~I~L5~7~
.
step without further purification.
ir: ~ miaq 1780, 1160 cm 1
nmr: ~ ppl4 5091 (lH, s, PhCH or COCHC12), 6.oo (lH,
S7 PhCH or COCHCl2)3 7.32 (5H, s, phenyl~
.
BB-S488; 7-~D-Mandelamido)-3-(2-carbo~ymethyl-2~,-dihydro-
s-triazolo~4 "-blpyridazin-3-on-6-ylthiomethyl)_3-cephem-
4-carboxylic Acid. (4)
A solution of the above-obtained dichloroacetyl-
mandeloyl chloride (2, 2.8 g., 10 m.mole) in dry acetone
(30 ml.) was added dropwise to a stirred solution of 7-
amino-3-(2-carboxymethyl-2,3-dihydro-s-triazolo[4,3-b]-
pyridazin-3-on-6-ylthiomethyl)-3-ce~hem- !1 -C arboxylic
acid (H-ACA-S-C~.TP)(3, 3.94 g., 9 m.mole) and trietilyl-
amine (3.54 g., 35 m.mole) in 50% a~ueous acetone (120 ml.)
at OD-5 C. The mixture was allo~red to rise to room tem-
i perature during 1 hour with stirring and was adjusted to
,~ pH 11 with 5~ aqueous sodium carbonate (ca 12 ml. was
required). The mixture was allo~Jed to stand at room te~-
perature for 30 minutes, acidified to pH 1 with dilute
HCl ~nd eva~orated under reduced pressure to remove
acetone below 40 C. The precipitate W25 collected by
filtration~ washed with water (20 ml.) and air-dried.
The dried m~terial was dissolved in THF (150 ml.),
stirred for 5 minutes at 40_50C G. ~nd filtered to remove
insoluble unreacted ~ (0.54 g., 14~ recovery). The fil-
trate was chromatographed on a silica gel column (~ ko-
gel, C-200, 30 g.) and eluted with chloroform-methanol
(100:5). The eluates were collected in 50 ml. fractions
; monitoring by tlc (silica gel, solvent, GH3C~l-water = 4:1,
. - 72 -
~ 1 lSV~
.
detected with I2?. The fractions containing the desired
product were combined, treated with a small amount of
carbon and evaporated under reduced pressure. The res~-
due was triturated with chloroform (50 ml.) to yield,
2.36 g. (46~) o~ 7-(D-mandelamido)-3-(2-carboxymethyl-
2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthlo-
methyl)-3-cephem-4-carboxylic acid (4). M.P., 165-
:~ . 170 C. (dec.).
ir: ~ KBax 3600-2500, 1780, 1720, 1500, 1410, 1355,
1220, 1195 cm 1
`uv: ~ EtaxH 254 nm (e, 18300), 297 nm (sh, ~, g300).
nmr ~ DMS-d6 3.84 (~H, m, 2-H)~ 4-17 (2~l~ d~ 13 Hz,
3-H), 4.50 (lH, d, 13 Hz, 3-H), 4.82 (2H, s, NCH2COO),
5.20 (lH, d, 4.5 Hz, 6-H), 5.25 (lH, s, PhCH), 5.87 (lH,
!d-d, 4.5 ~ 9 Hzj 7-H, a doublet (J=4.5 Hz) by addition of
D20), 7.25 (lH, d, 11 Hz, pyridazine-H), 7.4-7.7 (5H, m,
~!phenyl-H), 7.90 (lH, d, 11 Hz, pyridazine-H), 9.0 (lH, d,
9 Hz, 7-CONH, disappear by addition of D20).
Anal. Calc'd for C23H2oN60gS~}/4CHC13: C, 43- o8;
H, 3~16; ~, 12.69; S, 9.69. Found: C, 43.11, 43.22; H, .
2.97, ~.o6; N, 12.8~, 12.77; s, 9.64.
,
'~.
. . .
- 73 ~
~; . ;
'` ~Li5q~7;2 7
AMPLE 3
=~ 99% HCOOH ~==\ SOCl
7HCOOX) ~ ~CHCOOH
OH OCHO
~==\ H-ACA-S-CI~rP (3) ~
CHCOCl> ~ CHCO-ACA-S-C~ITP
OCHOOCHO
6 7, BB-S494
O-Formyl-D(-)-mandelic Acid (5)
A mixt~re of D(-)-mandelic acid (1, 5.0 ~ 3
m.mole) and 99;J formic acid (80 ml.) ~tas heated at 80-
90 C. for 12 hours. The mixture was evaporated and
toluene (100 ml.) was added to the residue and evaporated
under reduced pressure to remove formic acid azeotropically
The resi~ue ~las dissolved in benzene (200 ml.) and the
solution was washed with water (2 x 50 ml.). The organic
layer was separated, dried with anhydrous sodium sulfate
and evaporated under reduced pressure. The residual oil
was triturated with cyclohexane (50 ml.) to crystallize.
Yield, 3.70 g. (63~) of O-formyl-D(-)-mandelic acid (5)
as colorless prisms. M.P.~ 56-59 C. (lit. M.P.~ 55-58~ C.).
ir: ~ m~ar 3400-2800, 1755, 1720, 1160, 990 cm~l.
nmr: ~ ppDml3 5.98 (lH, s, PhGH), 7.31 (5H, m, phenyl-
H), 8.o5 (lH, s, OCHO), 10.05 (lH, s, COOH, disappeared
by addition of D20).
_ 74
,
5~727
, 0-F rmyl-D(-)-mandeloyl Chloride (6)
A mixture of 5 (2.0 g., 11 m.mole) and thionyl
chloride (10 ml.) was heated under reflux for 2 hours.
Evaporation of the excess thionyl chloride and distilla-
tion of the resîdue under reduced pressure afforded the
acid chloride 0-formyl-D(-)-mandeloyl chloride ~6).
r Yield, 1.53 g. (70%). B.P., 120-122 C./15 mmHg.
ir: ~maq- 1805, 1740, 1160, 1140 cm 1.
t BB-S494; 7-(D-0-Formylmandelamido)-3-(2-carboxymethvl-2,3-
dihydro-s-triazolo[4~3-blpyridazin-3-on-6-ylthiomethyl)
3-cephem-4-carboxylic Acid (7)
; A solution of 0-fDrmyl-D(-)-mandeloyl chloride
(6) (1.0 g., 5.1 m.mole) in dry acetone (10 ml.) was added
¦ dropwise to a cold (0 to 5 C.) solution of 7-amino-3-
(2-carboxymethyl-2,3-dihydro-s-triazolo[413-b]pyridazin-
3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid (3, 1.75
g., 4.m.mole) in 50~ aqueous acetone (70 ml.) containing
I sodium bicarbonate (1.34 g., 16 m.mole). The mixture was
¦ stirred for 30 minutes at room temperature and washed with
ether. The aqueous layer was a~idified to pH 1 with dilute
HCl. The separated oily gum was collected and dissolved
in THF (100 ml.). The solution was treated with a small
~mount of carbon and dried with anhydrous sodium sulfate.
Evaporation o~ the solvent under reducea pressure to 10
mlO and dilution with ether afforded the title compound
(7) as a pale yellow amorphous po~der, 0.91 g. (38~).
M.P., 172-176 C.(dec~).
ir: ~ rmBax 3600-2400, 1775, 1720, 1550, 1355, 1230,
1160 cm~l.
uv: ~ mtl~ 254 nm (, 20800), 297 nm ~sh~ io500).
nmr: S ppm d6 3.4-4.5 (4H, m, 2-H and 3-H), 4.67 (2H,
s, NCH2COO), 4.97 (lH, d, 4 Hz, 6-H), 5.66 (lH, d-d, 4
& 8 Hz, 7-H), 7.2-7.5 (5H, m, phenyl-H), 7.64
~lH, d, 10 Hz, pyridazine-H), 8.29 (lH, s, CHO), 9.29
(lH? d, 8 Hz, 7-CONH, disappeared by addition of D20).
Anal Calc'd. for ~24H20N609S2 lH2
Hj 3.58; N, 13.59, S, 10.37. Found: C, 46.70, 47.20;
H, 3.25, 3.34; N, 13.37, 13.78; S, 10.84.
- EXAMPLE 4
BB-S488; 7-(D-~andelamido)-3-(2-carbo~ymethyl-2~3-
dihydro-s-triazolo-~4,3-b]pyridazin-3-on-6-ylthiomethyl~-
3-cephem-4-carboxylic Acid (4)
A mixture of 7-(D-O-formylmandelamido-3-t2-car-
boxymethyl-2,3-dihydro-s-triazolo[4,~-b]pyridazin-3-on-
6-ylthiomethyl)-3-cephem-4-carboxylic acid (7) (484 mg.,
0.81 m.mole) and sodium bicarbonate (748 mg., 8.9 m.mole)
in water (4 ml.) was stirred for 4 hours at room tempera-
ture, and acidi~ied to pH 1 with dilute HCl. The preci-
pitate (500 mg.) was collected by filtration, waslled with
water (2 ml.) and chromatographed on silica gel column
(Wako-gel, C-200, 5 g.). The column was eluted with
.~ chloroform containing increasing methanol (~-5~) as
eluent, and the fractions containin~ the product ~ere
combined, treated with a small amount of carbon and eva-
porated under reduced pressure. The residue was triturated
with ether to give 277 mg. of 7-(D-mandelamido)-3-(2-car-
boxymethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-
`ylthiomethyl)-3-cephem-~-carboxylic acid (B~-S488; 4).
The nmr-estimation of this product sho~led 10~ of 7 still
remained.
- 7G -
5~7
ir: ~ KBax 3600-2400, 1770, 1720~ 1520, 1495, 1365,
1230 cm~l. ``
uv: ~ EtaxH 254 nm (~, 20000), 297 nm (sh, ~, 9600)
'
EXAMPLE 5
Sodi~m Salt of BB-S488
.
Sodium-2-ethylhexanoate (~EH) (4.0 ml., 1 M
solution in ethyl acetate) was added to a solution of
j 7-(D-mandelamido)-3-(2-carboxymethyl-2,3-dihydro-s-tri-
azolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-
; carboxylic acid (4) (2.25 g., 3.93 m.mole) in THF (200
i ml.). The precipitate was collected by filtration, washed
l~ ~1ith THF (50 ml.) and dried at 60~ C./l mmHg for 3 hours.
Yield of sodium 7-(D-mandelamido)-3-(2-carboxymethyl-2,3-
! dihydro-s-triazolo~4~3-b]pyridazin-3-on-6-ylthiomethyl)-
3-cephem-4-carboxylate,l.96 ~ bio yield, 97~),
M.P., 230-240 C. (dec.). The pH of the 10~ aqueous
solution was 3.6.
ir: ~ max 3600-3000, 1765, 1710, 1~05, 1390, 1360, 1190,
1080, 1065 cm~l.
uv: ~ ~mater nm(~l'c~m)~252 (357), 310 (sh, 140).
nmr: ~ ~ m 3.43 (lH, d, 19 Hz, 2-H), 3.87 (lH, d, 19 I~z,
2-H), 4.15 (lH, d, 14 Hz, 3-H), 4.53 (lH, d, 14 Hz, 3-H),
r 5.16 (lH, d, 4.5 Hz, 6-H), 5.36 (lH, s~ PhCH), 5.73 (lH,
d, 4.5 Hz, 7-H), 7.13 (lH, d, 10 Hz, pyridazine-H), 7.57
(5H, s, phenyl-H), 7.69 (lII, d, 10 Hz, pyridazine-H).
Anal- C~lc'd. for C23HlgN60~S2Na 5/4H20: C, 44-77;
H, 3.51; N, 13.62~ S, 10.39. Found: C9 44.93, 44.79; H,
3.31, 3.15; N~ 13.~1, 13.33; S, 10.19.
. ;' ' ~ .
- 77 ~
'
, , . :
~:~LSq~27
EXAMPIE_6
Aqueous lN sodium hydroxide solution was added
dropwise to a suspension of 7-(D-mandelamido)-3-(2-
carboxymethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-
on-6-ylthiomethyl)-3-cephem-4-carboxylic acid (4)(3.51 g.)
in water (20 ml.) to adjust to pH 6.o. The solution was
lyophilized to yield 3.4 g. (bio-yield, 97~) of disodium
7-(D-mandelamido)-3-(2-carboxymethyl-2,3-dihydro-s-tri-
azolo[4,7-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-
carboxylylate. M.p., >240~ C. (dec.). The pH of the
5% aqueous solution was 5.4.
ir: ~ KBax 3600-3000, 1760, 1710, 1605, 1390, 1360,
1190, 1080, 1060 cm~l.
uv: ~1 mater nm (El~m) 252 (320), 310 (124).
nmr: ~ p~ 3.43 (lH, d, 19 Hx, 2-H), 3.90 (lH, d, 19
Hz, 2-~), 4.15 (lH, d, 14 Hz, 3-~_), 4.53 (lH, d, 14 Hz,
~~H)g 4.75 (2H, s, NCH2C0), 5.22 (lH, d, 4.5 Hz, 6-H),
5.42 (lH~ s, PhCH), 5.73 (lH, d7 4.5 Hz, 7-H)~ 7.22 (lH,
d, 10 Hz, pyridazine-H), 7.65 (5H, s, phenyl-H), 7 77
(lH, d, 10 Hz, pyridazine-H).
A 1 Calctd- for C23Hl8N6o8s2Na2 3/ 2
42.92; H, 3.29; N, 13.06; S, 9.96. Found: C, 42.90,
43.19, H, 3.06, 3.01; N, 13.04, 13.03; S, 9.97.
- - 78 -
~L~ 51~7'27
ExamDle 7
Sub~titution of the D-mande'llc acid carbox~-
anhydrlde in the procedure of Example 1 of an
equlmoiar weight Or the carbox~anhydrldes prepared'
In similar fashion from t'he monosub~tituted D-
mandelic acid~
D-2-chloro-mandellc acld,
: D-3-chloro-mandellc acid,
D-4-chloro-mandellc acld,
D-2-bromo-mandellc acld,
: D-3-bromo-mandelic acld,
D-4-bromo-mandellc acld,
D-2-~luoro-mandellc acld,
D-3-rluoro-mandellc acld,
D-4-fluoro-mandelic acid,
D-2-trifluoromethyl-mandellc acid,
.' D-3-trifluoromethyl-mandelic acid,
D-4-trl~luoromethyl-mandelic acid,
~ D-2-amino-mandellc acld,
,: ~D-3-amlno-mandelic acld,
: D-~-amlno-mandelic acld,
D-2-nltro-mandellc acid,
D-3-nltro-mandelic acld,
D-4-nitro-mandelic acld,
D-2-hydroxy-mandelic acid,
D-3-hydroxy-mandelic acid,
-~D-~-hydroxy-mandelic acld,
::~
,
:: - 79 -
, . . . . .
111 50727
,
D-2-methyl-mandellc acid,
D-3-methyl-mandelic acld,
D-4-methyl-mandellc acid,
D-2-methoxy-mandelic acid,
D-3-methoxy-mandelic acld and
D-4-methoxy-mandellc acld respectlvely produces
7-(D-2-chloro-mandel~nido)-3-(2-carboxyrnetllyl-2,3-
dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-yltllio-
methyl)-3-cephem-4-carboxylic acid,
7-(D-3-chloro-mandelamido)-3-(2-carboxymethyl-2,3-
dihydro-s-triazolo[4,3-b]pyridazin-3-on-5-ylthio-
methyl)-3-cephem-4-carboxylic acid,
7-(D-4-chloro-mandelamido)-3-(2-carboxymethyl-2,3-
dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid,
7-(D-2-bromo-mandelamido)-3-(2-carboxymethyl-2,3-
dihydro-s-triazolo[4,3-b~pyridazin-3-on-G-ylthio-
methyl)-3-cephem-4-carboxylic acid,
7-(D-3-bromo-mandelamido)-3-(2-carbox~nethyl-2,3-
dihydro-s-triazolo[493-b]pyridazin-3-on-6-ylthio-
methyl)-3-cephem-1~-carboxylic acid,
7-(D-4-bromo-mandelamido)-3-(2-carboxymethyl-c,3-
dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid,
7-(D-2-~luoro-mandelamido)-3-(2-carbox~nethyl-2,3-
dihydro-s-triazolo~4,3-b]pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid,
- 80 -
` ~SC~7
.
.
;7-(D-3-fluoro-mandelamido)-3-(2-carboxymethyl-2,3-
dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid,
7-(D-4-fluoro-mandelamido)-3-(2-carboxymethyl-2,3-
dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid~
7-(D-2-trifluoromethyl-mandelamido)-3-(2-carboxy-
methyl-2,3-dihydro-s-triazolo[4,3-b3pyridazin-3-on-
6-ylthiomethyl)-3-cepllem-4-carboxylic acid,
7-(D-3-trifluoromethyl-mandelamido)-3-(2-carboxy-
methyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-
6-ylthiomethyl)-3-cephem-4-carboxylic acid,
7-(D-4-trifluoromethyl-mandelamido)-3-(2-carboxy-
meth~l-2,, dihydro-s-triazolo[4,3-b]p~rri~a.zin-3-on-
6-ylthiomethyl)-3-cephem-4-carboxylic acid,
7-(D-2-amino-mandelamido)-3-(2-carboxymethyl-2,3-
dihydro-s-triazolo[~,3-b]p~yrida.zin-3-on-G-ylthio-
. methyl)-3-cephem-4-carboxylic acid,
. 7-(D-3-amino-mandel~mido)-3-(2-carboxymethyl-2,3-
dihydro-s-triazolo[1~,3-b~pyrida~in-3-on-~-yltllio-
methyl)-3-cephem-4-carboxylic acid,
. 7-(D-4-amino-mandelamido)-3-(2-carboxymethyl-2,3-
¦i . dihydro-s-triazolo[4,3-b]pyridaain-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid,
7-(D-2-nitro-mandelamido)-3-(2-carboxymethyl-2,3-
. dihydro-s-triazolo[4,3-b~pyridazin-3-oll-6-ylthio-
methyl)-3-cephem-4-carboxylic acid,
7-(D-3-nitro-mandelamido)-3-(2-carboxymethyl-2,3-
. dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-yltllio-
methyl)-3-cepllem-4-carboxylic acicl,
.
- 81 -
- ~5i~7
7-(D-4-nitro-mandelamido)-3-(2-carboxymethyl-2,3-
dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid,
7-(D-2-hydroxy-mandelamido)-3-(2-carboxymethyl-2,3-
.dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid,
7-(D-3-hydroxy-mandelamido)-3-(2-carboxymethyl-2,~-
dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid,
7-(D-4-hydroxy-mandelamido)-3-(2-carboxymethyl-2,3-
dihydro-s-triazolo~4,3-b]pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid,
7-(D-2-methyl-m~ndelamido)-3-(2-carboxymetllyl-2,~-
dihydro-s-triazolo[4,3-b~pyrida.zi!l-3-oll-6-yltllio-
methyl)-3-cephem-4-carboxylic a.cid,
7-(D-3-methyl-mandelamido)-3-('-carboxymethyl-2,3-
dihydro-s-triazolo[4,3-b]pyrida2in-3-on-6-ylthio-
i methyl)-3-cephem-4-carboxylic acid,
, 7-(D-4-methyl-mandelamido)-3-(2-cl.rboxymet~yl-2,3-
: '.
: : dihyd~o-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-
methyl)-~i-cephem-4-carboxylic aci(l,,
7-(D-2-methoxy-mandelamido)-3-(2-carbox~rrnethyl-2,3-
i dihydro-s-triazolo~,3-b]pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid,
. 7-(D-3-methoxy-mandelamido)-3-(2-carboxymethyl-2,3-
dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid, and
7-(D-4-methoxy-mandelamido)-3-(2-carboxymethyl-2,3-
dihydro-s-triazolo[4,3-b]pyrida~in-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid, respectively.
- 82 _
SC~'727
I`` '
EXAM~LE 8
Sub8tltutlon for the D-mandellc acid
carbo~yanhydride ln the procedure of Example 1 of an
equlmolar weight o~ the carboxyanhydride prepared
in ~imllar ~ashlon from D-2-thiopheneglycollc àcid
and D-3 thlopheneglycollc ac~d respectively produces
7-(D-~-hydroxy-2-thienylacetamido)-3-(2-carboxymethyl-
2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid and 7-(D-a-hydroxy-
3-thienylacetamido)-3-(2-carboxymethyl-2,3-dihydro-s-
~riazolo~4,3-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-
4-carboxyllc acid, respectively.
~X~IIPLr 9
7-(D-~- ydroxy-~-p!lenylacetamido)-3-(2-carbo~J~etIl~l-
2,3-dilydro-s-tria7olo~4,3-bl~yridazin-3-on-6-ylthio-
methyl~-3-cepIlem-4-carboxylic Acid ~'rer~red ~rom
7-D-MaIldel~midocenIlalospor~nic Acid.
I . .. _ _ .,.
- 0.27 Mole o~ sodium 7-D-mandelamldocephalo-
I sporanate ~s suspended ln 1000 ml. of 0~1 M phosphate
bur~er Or pH 6.4 and there ls added 0.31 moles of
2-carboxymethyl-2,~-dihydrO-6-mercapto-s-triazolo-
. ~4.3-b]pyrida~in-3-one. The solution is
heated at 55 C. under a nltrogen atmosphere for five
hour3. After one hour the pH ls ad~usted to 6 4 by
addltlon of a small amount of 40~ H3P04. At the end of
- the five hour heating perlod the solution is cooled
to 23 C. and the pH adjusted to 2 by the additlon
.. .
` - 83 -
. , .
~.Si~27
.
Or 3 N HCl under a layer of ethyl acetate, The
product i9 extracted lnto ethyl acetate and stlrred
~or 15 mln. at 2~ C. with 2 g. o~ ("Darco KB")
decolorlzlng charcoal. The mixture ls then filtered
through a pad o~ diatomaeeous earth ("Celite") and
the ethyl acetate removed rrom the flltrate under
vacuum. The re idue is triturated to a solid with
diethyl ether, collected by ~iltration and drled
oYer P205 under vacuum to yield solid 7-(D-a-hydroxy-
~-phenylacetamido)-3-(2-carboxymethyl-2,3-dihydro-s-
tri~zolo[ 4 ,3-b~ pyridazin-3-on-6- ylthiometl~yl ) -3-
cephem-4-carbo.Yylic acid.
~x ampl e 1 o
H2N~L 2 5~ H_CH2CH2CODH
C2H o
C 6H5 0
~ ~/
o
N ~S N
OH . ~LCH2~ CH2C~32COOII
C02H
_
`~ 11507Z7
BB-S 527; 7-lD~ Mandelamido]-3-[2-~2-carboxyeth~1)-2~3-
.
dihydro-s-triazolo-[4,3-b]pxrldazin-3-on-6~1thiomethyl]-
3-cePhem-4-carboxvlic acid
To a miY~Ure of 7-amino-3-[2-(2-carboxyethyl)-2,3-
dihydro-s-triazolo~4,3 b]pyridazin-3-on-6-ylthiomethyl]-
3-cephem-4-carboxyli~ acid (679 mg, 1.5 m mol) and NaHCO3
(445 mg, 5.3 m mol) in 50~ aqueous acetone was added D-
mandelic acid O-carboxyanhydride (400 mg, 2.3 m mol) at
OC. The mixture was stirred at room temperature for 1 hour
and evaporated to remove the organic solvent. ~he aqueous
sclution was washed with ether (3 x 10 ml), adjusted to p}3
1 with dil. HCl and filtered to collect the crude product,
which was dissolved in THF ~10 ml), filtered to remove
insolubles and evaporatea under reduced pressure. The oily
residue was triturated with ether. The solid ~476 mg) was
chromatographed on a column of silica gel (Wako-glP C-200,
10 g) and eluted with MeOII-CIIC13 ~MeOII: 0-3%). Fractions
w~)ich contained the desired product were combined and evapor
ated to yield 287 mg t33~) of BB-S 527. ~.p. >155C (dec.).
ir: ~KaDx 3600 - 2400, 1780, 1720, 1550, 1520 cm 1.
~pl~ 7 buffer 253 nm ( E 20000), 298 nm t
max
Anal. ~alc'd. for C24H22N6ogs2-3/2H2o C~ 46-98; ~
4.11; N, 13.70; S, 10.45. Found: C, 47.25, 47.39; H, 3.80,
3.76; N, 12.87, 12.77; S, 10.17.
. . .
, .
- - 85 -
.
~S~27
The sodium salt of BB-S 527
. . _ . . _
A suspension of the free acid of BB-S 527 (240 mg, 0.4
m mol) in water (5 ml) was adjusted at pH 6.8 with 1 N-NaOH
t0~7 ml) to gi~e the clear solgtion, which was freeze-dried
to leave 234 mg (91%, bio-yield) of the sodium salt of BB-S
527 as pale yellow powder. M.p. >210C (dec.).
ir: ~max 3600 - 2800, 1770, 1710, 1600 cm 1.
pH 7 Buffer 253 nm (~ 21000~ 298 nm ( E 9
Anal. calc'd. for C24H20N6O8S2Na22~2O
3.63; N, 12.61; S, 9.62. Found: C, 43.39, 43.43: ~, 3.20,
3.36; N, 12.63, 12.68; S, 9.42, 9.22.
In vitro antibacterial activity of BB-S 527 compared
with BB-S 4B8 and cefamandole (determined by Steers' agar
dilution method on Mueller-Hinton agar plate)
MIC tmcq/ml) _ __
Or~anism BB-S 527 BB-S 488 cefamandole
S. aureus Smith 1.6 0.8 0.2
S. aureus 0.4 0.4 0.1
. S. aureus BX-1633 3.1 3,1 0.4
~ . .
St. faecalis `~100 >100 >100 .
E. coli NIHJ 0.4 0.2 0.05
E. coli ATCC 8739 12.5 6.3 3.1
E. coli Juhl 0.4 0.2 0~8
E. coli BX-1373 6.3 3.1 3.1
E. coli 0.1 0.1 0.1
E. coli 0.1 0.05 0.1
E. coli 6.3 3.1 1.6
- 86 -
.
`'` 1150~27
Xl, pneumoniae 6.3 3.1 3O1
Kl. pneumoniae 0.2 0.1 0.8
Kl. pn~umoniae 0.8 0.4 0.8
Kl. pneumoniae 0.4 0.2 0,8
~r. vulgaris 0.1 0.1 0.2
Pr. vulgaris 12.5 0.8 50
Pr. mirabilis 0.2 O.OS 0.8
PrO mirabilis 0.1 0.05. 0.2
PrO morganii >100 ~lD0 >100
Pr. morganii 0.4 0.2 0.8
Pr. rettgeri 0.2 0.2 0.4
Ps. aeruginosa >100 >100 >100
Ps. aeruginosa >100 >100 >100
Shig. dysenteriae 0.025 0.025 0.1
Shig. flexneri 50 25 . 6.3
: Shig. sonnei 0.1 0.05 0.2
Serr. marcescens >100 >100 100
Enterob. cloacae 6.3 3.1 3.1
SalO enteritidis 0.05 0.025 0.05
~! Sal. typhosa 0.1 0.05 0.1
B. anthracis 0.4 0.2 0.4
. ~ .
. .
- 87 -
51~727
Substitution f~r the D-mandellc acld carboxy-
anhydrlde in the procedure o~ Examplelo o an
equlmolar welght Or the carbox~anhydrides prepared
ln slmllar ~a~hlon rrom the monosubstltuted D-
mandelic acid~
D-2-chloro-mandelic acid,
D-3-chloro-mandelic acld,
D-4-chloro-mandellc acid~
D-2-bromo-mandellc ac ld,
D-3-bromo-mandelic acid,
D-4-bromo-mandellc acid,
D-2-rluoro-mandelic acld,
D-~-rluoro-mandelic acid,
D-4-rluoro-mandellc acld,
D-2-trirluoromethyl-mandellc acid,
D-3-trifluoromethyl-mandellc acid,
D-4-trlrluoromethyl-mandellc acld,
D-Z-amlno-mandellc acid,
D-3-amlno-mandelic acld,
D-4-amlno-mandellc acid,
D-2-nitro-mandelic acid,
D-3-nltro-mandelic acld,
D-4-nitro-mandelic acid,
D-2-hydroxy-mandellc acld~
D-3-hydroxy-mandelic acid,
D-~-hydroxy-mandellc acid~
.
- 8~ -
.
50727
.
D-2-methyl-mandeli~ acid,
D-3-methyl-mandellc acld9
D-4-meth~l-mandellc acld,
D-2-methoxy-mandelic acld,
D-3-m~thoxy-mandelic acid and
D-4-methox~-~andel~c acid re~pectively produces
7-(D-2-chloro-mandelamido)-3-(2-carboxyethyl-2~3-
dihydro-s-triazolo[4,3-b~pyridazin-3-on-6-ylthio-
methyl) 3-cephem-4-carboxyl~c acid,
7-(D-3-chloro-mandelamldo)-3-(2-carboxyethyl-2,3-
dihydro~s-triazolo~4,3-b]pyrldazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acld,
7~(D-~-chloro-mandelamido)-3-(2-carboxyethyl-2,3-
dlhydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid,
7-(D-2-bromo-mandelam~do)-3-(2-carboxyethyl-2~3-
dihydro-s-triazolo~4,3-b]pyridazin-3-on-6-ylthlo-
; methyl)-3-cephem-4-ca~boxylic acid,
:~ 7-(D-3-bromo-mandel~ldo)-3-(2-carboxy~thyl-2,3-
dihydro-s-triaz~10l4,3-b]pyridazin-3-on-6-ylthio-
methylj-3-cephem-4-carboxylic acid,
. 7-(D-4-bromo-mandelamido)-3-(2-carboxyethyl-2,3-
dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-
methyl)-~-cephem-4-carboxylic acid~
7-(D-2-fluoro~mandelamido)-3-(2-carboxyetllyl-2,3-
dihydro-s-triazoloL4S3-b]pyridazin-3-on-6-y-thio-
methyl 3 -~-cephem 4-carboxylic acid,
- 89 -
15i~7Z7
.
7-(D-3~1uoro-mandelamido)-3-(2-carboxyethyl-2,3-
. dihydro-s-triazolot4,~-b]pyridazin-3-on-6-ylthio_
~ethyl)-3-cephem-4~carboxylic acid,
7-(D-4-rluoro-mandelamido)-3-(2-carboxyethyl-2~3-
dihydro-s-triazolo~4,3-b]pyridazin-3-on-6-ylthio-
methyl)-~-cephem-4-carboxylic acid,
7-(D-2-tri~luoromethyl-mandelamido)-3-(2-carboxy-
ethyl-2~3-dihydro-s-tr~azolo[4,3-b]pyridazin-3-on-
6-ylthiomethyl)-3-cephem-4-carboxylic acid,
7-(D-3-trifluoromethyl-mandelamido)-3-(2-carboxy-
ethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-
6-ylthiomethyl)-3-cephem-4-carboxylic acid,
7-(D-4-trifluoromethyl-mandelamido)-3-(2-carboxy-
ethyl_2~3_dihydro-s-triazolo[4,3-b]pyridazin-3-on-
t 6-ylthiomethy1)-3-cephem-4-carboxylic acid,
7-(D-2-amino-mandelamido)-3-(2-carboxyethyl-2,3-
dlhydro-s-triazolo~4,3-b]pyridazin-3-on-6-ylthio-
I methyl)-3-cephem-4-carboxylic acid,
:I 7-(D-3-amino-mandelamido)-3-(2-carboxyethyl-2,3-
~ dihydro-s-triazolo[4,3-b3pyridazin-3-on-6-ylthio-
; methyl)-3-cephem-4-carboxylic acid,
. 7-(D-4-amino-mandelamido)-3-(2-carboxyethyl-2,3-
.. dihydro-s-triazolo[4,3-b]pyridaain-3-on-6-ylthio-
: methyl)-3-cephem-4-carboxylic acid,
7-(D-2-nitro-mandelamido~-3-(2-carboxyethyl-2,3-
dihydro-s-triazolo[493-b3pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-car~oxylic acid,
7-(D-3-nitro-mandelamido)-3-(2-carboxyethyl-2,3-
dihydro-~-triazolo[4,3-b3pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid~ -
- 90 -
.
5(~7;~7
,
7 (D-4-nitro-mandelamido)-3-(2-carboxye~hyl-2,3-
dihydro-s-triazolo~4~3-b]pyridaz1n-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid,
7-(D-2-hydroxy-mandelamido)-3-(2-carboxyethyl-2,3-
dlhydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid,
7-(D-3-hydroxy-mandelam~do)-3-t2-carboxyethyl-2,3-
dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-
methyl)-3-ceph2m-4-carboxylic acid,
7-(D-~-hydroxy-mandelamido)-~-(2-carboxy~thyl-2,3-
dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid,
7-(D-2-methyl-mandelamido)-3-(2-carboxyethyl-2,3-
dihydro-s-triazolol4,3-b]pyridazin-3-on-6-ylthio-
. methyl)-3-cephem-4-carboxylic acidl
7-(D-3-methyl-mandelamido)-3-(2-carboxyethyl-2,3-
dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid,
1~
; 7-(D-4-methyl-mandelamido)-3-(2-carboxy2thyl-2,3-
dlhydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid~
. .!
~ 7-(D-2-methoxy-mandelamido)-3-(2-carboxyetllyl-2,3-
¦:: dihydro-s-triazolo[4,3-b]pyrida~.in-3 on-6-yltliio-
methyl~ cephem-4-carboxylic acid,
~: ~7-(~-3-methoxy-mandelamido)-3-(2-carboxyethyl-2,3-
dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthio-
;~ methyl)-3-cephem-4-carboxylic acid, and
7-(D-4-methoxy-mandelamido)-3-(2-carboxyethyl-2~3
~ d~hydro-s-triazolo[4,~-b]pyridazin-3-on-6-ylthio-
:~ methyl)-3-cephem-4-carboxylic acid, respecti~ely.
.
115~ 7Z~
EX~MPLE 12
Substitution for the D-mandelic acid carboxyanhydride
in the procedure ~f Example 10 of an equimolar weight of
the c~rboxyanhydride prepared in similar ~ashion from D-2-
thiopheneglycolic acid and D-3-thiopheneglycolic acid
respectively produces 7-(D ~-hydroxy-2-thienylacetamido)-
3-(2-carboxyethyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-
3-on-6-ylthiomethyll-3-cephem-4-carboxylic acid and 7~
(D--hy~roxy-3-thienylacetamido)-3-~2-carboxyethyl-2~3-
dihydro-s-triazolo~4,3-b]pyridazin-3-on-6-yl~hiomethyl)-
3-cephem-4-carboxylic acid, respectively.
.
~ /
~. ' /
~'~ ''" /
/
` /
`~ /
.
. . . _ 92
27
.
EXA~IPLE 13
`OCH3 0 ~ 2 S ~ r ~ -CH2COONa
7-~(2Z)-2-Methoxyimino(fur-2-yl)acetamidol-3-(2-carboxy-
methyl-2,3_dihydro-s-triazolo[4,3-b~pyridazin-3-on-6-
ylthiomethyl~-3-cephem-4-carboxylic Acid Disodium
Salt; BB-S511.
To a solution of (2Z)-2-methoxyimino(fur-2-yl)-
acetic acid (507 mg., 3 m.moles~ and triethylamine (0.42
ml., 3 m.moles) in dichloromethane (6 ml.)was added oxalyl
chloride (0.26 ml., 3 m.moles) at 0-5 C. and the mixture
was stirred for 30 minutes. The mixture was evaporated at
reduced pressure to give an oily residue of the acid
chloride which was dissolved in dry acetone (10 ml.).
't ~ A~ter filtration the acetone solution was added to a mix-
~¦ ture of 7-amino-3-(2-carboxymethyl-2,3-dihydro-s-triazolo-
4,3-b]pyridazin-3-on-6-ylthiomethyl)-~-cephem-~-
carboxylic acid (1.~1 g., 3 m.moles) and NaHC03 (504 mg.,
6 m moles) in water (20 ml.) at 0-5 C. The mixture was
stlrred at 0-5 C. for 2 hours with the acetone being
removed under reduced pressure. The aqueous solution was
` ` washed with ether (2 x 50 ml.) and adjusted to pH 1-2 with
conc. HCl to afford a precipitate which was collected by
filtration, washed with water and dried in vacuo. The
solid was dissolved in THF (tetrahydrofuran) (40 ml.) and
- 93 ~
.
` ~S~ 7
filtered. To the filtrate was added 1 M-SEH (sodium
ethylhexanoate) in ethyl acetate (3 ml.) and the result-
ing precipitate was collected by filtration and dried
in vacuo. Yield of 7-~(2Z)-2-methoxyimino(fur-2-yl)acet
amido~-3-(2-carboxymethyl-2,3-dihydro-s-triazolo[4,3-b]-
pyridazin-3 on-6-ylthiomethyl)-3-cephem-4-carboxylic acid
disodium salt was 1.0 g. (54%) mp ~210 C. (dec.).
ir: vmBr 1770, 1710, 1680, 1610, 1550 cm 1.
uv: ~pH mabXuffer 257 nm (E 25000), 277 nm (E 24000).
nmr: ~DMppmd6 7.7o (lH, br, furan-H~), 7.52 (lH, d,
J=9.5 Hz, pyridazine-H), 6.87 (lH, d, J=9.5 Hz, pyridazine-H),
6.5-6.6 (2H, m, furan-H~), 5.58 (lH, m, 7-H), 5.00 (lH, d,
J=4.5 Hz, 6-H), 3.84 (3H, s, OCH3).
Anal. CalC'd- for C22~17N709S2Na2 2
H, 2.94; N, 15.05; S, 9.84. Fol~d: C, 40.81~ 41.02;
H, 3.0-, 3.22; N, 14.69, 14.86; S, 9.70, 9.62.
,
.
.
.
_ 91~ _
7;~7 ?
EXAMPLE 14
O~Co-N ~ CH2-S ~ CI~2CH2C
7-~(2Z)-2-Methoxyimino(fur-2-yl)acetamido~-3-r2-(2-car-
boxyethyl)-2~3-dihydro-s-triazolo~4 ~ ridazin-3-on-
6-ylthiomethyl]-3-cephem-4~carboxylic Acid Sodium Salt,
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 o~
.
7-amino-3-~2-(2-carboxyethyl)-2~3-dihydro-s-triazolo-
[4,3-b]pyridazin-3-on-6 ylthiomethyl]-3-cephem-4-car-
boxylic acid,(452 m~., 1 m.mole~ and ~aHC03 (336 mg.~
! 4 m.mole) in ~ater (10 ml.). ~he reaction mixture was
stirred for 2 hours in an ice-water bath. Acetone was
removed at reduced pressure. The aqueous solution was
washed with ether (2 x 10 ml.) and adJusted to pH 1-2 with
i conc. HCl. The resulting precipita~e was collected by
filtration, washed ~ith water and dried under reduced
pressure. A solution of the precipitate in THF (10 ml.)
was treated with active carbon. A SEH solution in ethyl
acetate (1 ~o.8 ml.) was added to the THF solution to
give 7-[(2Z)-2-methoxyimino(fur-2-yl)acetamido]-3-[2-(2-
carboxyethyl)-2,3-dihydro-s-triazolo~4,3-b]pyridazin-3-
on~6-ylthiomethyl]-3-cephem-4-carboxylic acid sodium salt
which was collected by filtration. Yield: 410 mg. (66
as mono Na salt). Mp. ~205 C. (dec.).
~ .
- 95 -
ci~27`
ir: vmBxr 3600-2800, 1770, 1710, 1600, 1550 cm 1
uv: ~pH mabXuffer 257 nm (E 27000), 276 (E 26100).
nmr: ~DMpSpmd6 D20 2.70 (2H, t, J=6 Hz, CH2)3 3.3-4.5
- (9H~ m, 2-H, 3-CH2, CH2 and OCH3), 4.96 (lH, d, J=5.5 Hz,
6-H), 5.53 (lH, d, J-5.5 Hz, 7-H), 6.55 (2H, m9 furan-H),
6.92 (lH, d, J=10 Hz, pyridazine-H), 7.53 tlH, d, J=10
Hz, pyridazine-H), 7.68 (lH, br, furan-H).
Anal. Calc'd. for C23H20N709S2Na H20: C, 42-92;
H, 3.45; N, 15.23; S, 9.96. Found: C, 43.08, 42.77;
H, 3.20, 3~03; N, 14.96, 14.76; S, 9.96.
TABLE 7
In vitro Activity ~Tsine ~uQller-Hinton A~r
EY the Serial D~lu~ion Metllod
eometric Mean of MIC
(l~cg./ml.)
BB-S511 BB-S526
(Ex. 13) (EX. 14? Cefuroxime
S. aureus (3 strains) 1.97 1.6 1.24
E. coli (7) o.58 0.78 1.28
Kl. pneumoniae ~4) 0.47 0 93 3.1
Proteus (6) 0.021 0.061 o.88
tg (b)(l)err (1~ ) 1.11 - 2.41 4.o6
. B. anthracis (1)
S. pyogenes (5) 0.032 0.032 0.025
S. viridans (5) 0.15 0.4 0.1
D. pneumoniae (5) o.o37 0.056 0.0125
N. meningitidis (5) 2.37 3.60 1.6
N. gonorrhoeae (5) 1.36 1.6 0.4
H. in~luenzae (7) 0.64 -0.71 1.16
Cefuroxime is sodium 6R,7R-3-carbamoyloxymethyl-7-(2Z)-2-
methoxyimino(fur-2-yl)acetamidoceph-3-em-4-carboxylate.
_ 9~ _
;~ 5~7~7
~` ` . ,.
T~BLE B
j Geometric Means of MIC's Against 3 Strains
of S. aureus and 27 Strains of Gram-negative Bacteria
(mcg./ml., Mueller-Hlnton Agar)
No. of
t - Strains BB-S511 BL-S786
S. aureus 1 1.61.6
S. aureus, Penicillin-R 2 o.61.6
¦ E. coli 6 0.20.2
E. coli, Cephalosporin-R 1 6.312.5
K. pneumoniae 4 o 7o 3
Indole (-) Proteus 2 0.10.2
$ Indole(+) Proteus 3 o o5 o 3
Indole(+) Proteus, Cephalosporin-R 2 6.3 50.1
S. marcescens 1 25>100
E. cloacae 1 3.1 1.6
Shigella, Salmonella 5 Q 3 0 5
P. aeruginosa 2 >100~100
BL S786 is 7-[a-(2-aminomethylphenyl)acetzmldo]-3-[(1-car-
boxymethyltetrazol-5-ylthio)methyl]-3-cephem-4-carboxylic
:-
~ .
TABLE 9
Geometric Means of ~IC's Against 18 ~trains
. ~ - .
- ~ of S. marcescens
BB-S511 B~-S786
5.4 85.9
~j ' ' .
~ :
.
. ~
_ 97 _
., .
~ 5~17~7
EXAI~P~E 15
. Substitution of an equimolar weight of 2-ethox~-
imino-2-(fur-2-yl)acetic acid for the 2-methoxyiminofuryl
acetic acid used in the procedures.of Examples 13 and 14
produces 7-(2-ethoxyimino-2-furylacetamido)-3-(2-carboxy-
methyl-2,3-dihydro-s-triazolo~4,~-b]pyridazin-3-on-6-
ylthiomethyl)-3-cephem-4-carboxylic acid and 7-(2-ethoxy-
lmino-2-furylacetamido)-3-[.2-(2-carboxyethyl)-2,3-dihydro-
s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl]-3-cephem-
4-carboxylic acid, respectively.
EXAMPLE 16
Substitution o~ an eauimolar ~-eight of 2-n-propoxy-
imino-2-(fur-2-yl)acetic acid for the 2-methoxyiminofuryl
acetic acid used in the procedures of Examples 13 and 14
produces 7-(2-n-~ropoxyimino-2-furyl ac etamido)-3-(2-carboxy-
methyl-~,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-
ylthiomethyl) 3-cephem-4-carboxylic acid and 7-(2-n-propoxy-
: . imino-2-furylacetamido)-3-[2-(2-carboxyethyl).-2,3-dihydro-
s-triazolo[4,3-b]pyridazin-~-on-6-Ylthiomethyl]-3-cephem-
4-carboxylic acid, respectively.
.
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 ~xamples13 ~nd 14
produces 7-(2-n-butoxyimino-2-furylacet2mido)-3-(2-carboxy-
methyl-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-
ylthiomethyl)-3-cephem-4-carboxylic acid and 7-(2-n-butoxy-
- 98 -
727
imino-2-furylacetamido)-~-[2-(2-carboxyetl~yl)-2,3-dihydro-
s-triazolo[4,3-b~pyridazin-~-on-6-ylthiomethyl]-3-cephem-
4-carboxylic acid, respectively.
,
EXAMPLE 18
The products o~ Examples 13-17 are prepared 3S Sy~
lsomers essentially ~ree of the corresponding anti isomers
by the use ln the procedures of those examples of purified
syn isomers of the appropriate 2-alkoxyimino-2-(fur-2-yl)-
ncetic acid. Conversion of part of the syn isomer to anti
isomer during preparatlon of the ac~d chloride from the acid
is substantially avoided by minimizing its exposure to
hydrogen chloride, e.g. by first converting the acid to its
anhydrous sodium salt and by treatlng that salt with oxalyl
chloride under anhydrous conditions in the presence of a
hydrogen ion acceptor such as dimethylformamide.
Such syn isomer~ are a~so n~m~d as ~2z)-2-
alkoxyimino-2-(fur-2-yl)acetic acids.
_ ~9 _
.
¦ "
~ 50727
.
EXAMPLE 19
An ln3ectable pharmaceutlcal compo~ltion ls
formed by adding sterile water or sterile saline solu-
tion ~2 ml.) to 100-500 mgm. of 7-~(2Z)-2-methoxyimino-
(fur-2-yl)acetamido]-~-(2-carbox~methyl-2,3-dihydro-s-
- triazolo[4,~-b]pyridazin-3-on-6-ylthiomethyl)-3-cephem-
4-carboxylic acid disodium salt.
Pharmaceutical composltions of the sodium
and potassium salts o~ the other compounds of the
present invention, preferably in the form of the pure
-~ syn isomer, are formulated in a similar m~nner.
When the compounds are first prepared in
the form o~ the free acid they are converted to the
deslred, highly water soluble potassium salt by treat-`
ment with potassium 2-ethylhexanoate using the procedure
- of Example 13.
It is occasionally advantageous to have ad-
mixed with sald solid cephalosporin as a stabilizing
and/or solubilizing agent a sterile, anhydrous solid
`:
6uch as sodium carbonate, potassium carbonate or lithium
carbonate(e.g. in about 5 or 6 percent by weight of the
welght of the cephalosporin) or such as L-lysine, argi-
` nlne or histidine (e.g. in about 20-50% by weight of
the weight ol the cephalosporin) or such as a sodium,
potassium or ealcium salt Or levulinic acid, cltric acid,
ascorbic acid, tartaric acid or pyruvic acid (e.g. in
about 25-200% by weight o~ the welght of the cephalos-
porin) or such as sodium bicarbonate, ammonium car-
bamate alkali metal or ammonium phosphates or N-methyl-
BluCamine (per U.~. 1,380,741).
- 100 -
: i
Z7
,r~ 20
~,' ' ' "~
C0-N ~ S ~ ~ N
Il~ o~N~CH2S~N~N~N_(~H3
BB-S510
BB-S510; 7-~(2Z¦-2-~ethoxyimino(fur-2-yl~acetamido]-3-
L2-methyl-2?3-dillydro-s-triaæolo[4,3-b]pyridazin-3-on-
6-ylthiomethyl)-3-cephem-4-carboxylic Acid.Sodium Salt.
To a solution o~ (2Z) 2-methoxyimino(fur-2-yl)-
acetic acid (253 mg., 1.5 m.mol.) and triethylamine (0.2
ml., 1.5 m.mol.) in dichloromethane (~ ml.) was added
oxalyl~chloride (0.13 ml., 1.5 m.mol~) at 0-5 C. and
the mixture was stirred for 30 minutes and evaporated
at reduced pressure to give the acid chloride as an oil
which was dissolved in dry acetone (5 ml.) and riltered
to remove insolubles. The acetone solutlon was added to
a`mixture o~ 7-amino-3-(2-methyl-2,~-dihydro-s-triazolo-
4,3-b]pyridazin-3-on-6-ylthiomethylj-3-cephem-4-car-
boxylic acid (591 mg., 1.5 m.mol.j and NaHC03 (504 mg.,
6 m.mol.) in water (10 ml.) at 0-5 C. The reaction
mixture was stirred at 0-5 C. for 3 hours. hcetone was
removed at reduced pressure and the residual aqueous
soIution was washed with ether (2 x 30 ml.) and adjusted
.
to ~II 1-2 with concentrated HCl. The precipitate which
was collected by filtration, washed with water and dried
:
in vacuo, was dissolved in THF (30 ml.) and filtered to
remove insolubles To the THF solution was added a solu-
tion of sodium 2-ethylllexanoate (SEH, 1 I~I~ 1.5 ml.) in
-- 101 --
.
5~7;~7
ethyl acetate and the resulting precipitate was collected
by filtration and dried in vacuo. Yield: 492 mg. of
7-[(2Z)-2-methoxyimino(fur-2-yl)acetamido]-3-(2-methyl-
2,3-dihydro-s-t~iazolo[4,3-b]pyridazin-3-on-6-ylthio-
methyl)-3-cephem-4-carboxylic acid sodium salt (58~).
M.p. ~180~ C. (dec.).
ir: ~KBax 1770, 1720, 1670, 1600, 1550 cm 1
uv: ~PH7muaxfer 257 nm (E 22600), 277 nm (E 22300).
nmr: ~ ~ 7.53 (lH, d, J=1.5 Hz, furan-Ha), 7.35 (lH,
d, J-9.5 Hz, pyridazine-H), 6.90 (lH, d~ J 9.5 Hz, pyri-
dazine-H), 6.72 (lH3 d, J=3.0 Hz, furan-H~), 6.48 (lH,
q, J=1.5 and 3.0 Hz, furan-H~), 5.72 (lH, d, J=4.5 Hz,
7-H), 5.14 (lH, d, J=4.5 Hz, 6-H), 2.94 (3H, s, 0-C~ ),
3 61 (3H, s~ N-C_3)-
f r C21H~.gN707S2Na 1/2THF H20
C, 44.44; H, 3.89; S, 10.32. Found: C, 44.89~ H, 3.g2;
S, 9.67.
- 102 -
.. . . .. .. ... . . .. ~
~LS~27
.
TABLE 10
In vitro Activ ty Using Mueller-Hinton Ag~r
By he Seria Dl uti~n Ir~
~eomeF~ of MIC
(MCgO/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
~1. pneumoniae (4) 6.3 3,1 3.1
Proteus (6) 1.39 1.11 o.88
Shig.(3), Serr.(l)
Enterab.(l~ Sal.(2) 6.26 5.~6 4.o6
B. anthracls (1)
S. pyogenes (5) 0.0125 0.032 0.025
S. viridans (5) 0.13 0.59 0.1
D. pneumoniae (5) 0.021 0.1 0.01c5
N. meningitidis (5) 1.03 5.45 1,6
N. gonorrhoeae (5) 0.26 2.07 0.4
H. influenzae (7) 0.35 2.11 1.16
Cefuroxime is sodiwn 6R~7R-3-carbamoyloxymethyl-/-(2Z)-2-
methoxyimino(fur-2-yl)acetamidoceph-3-em-4-carboxylate.
; ::
. . .
.
- 103 -
~l 1 A~ 7 2 7
TABL~ 11
Geometric Means of MIC's Against 3 Strains
of S. aureus and 27 Strains_of Gram-negative Bacteria
(mcg./ml., Mueller-Hinton Agar)
No. of
Strains BB-S510 BL-S785
S. aureus 1 0.1 1.6
S. aureus, Penicillin-R 2 0 1 1.6
E. coli 6 0 1 0.2 .
E. coli, Cephalosporin-R 1 3 1 12.5
K. pneumoniae 4 2.6 o.3
Indole (-) Proteus 2 1.1 0.2
Indole~) Proteus 3 0 2 0.3
Indole(+) Proteus, Cephalosporin-R 2 6 ~ 50.1
S. marcescens 1 6.3 >100
E. cloacae 1 3.1 1.6
Shigella, Salmonella 5 0 5 0 5
P. aeruginosa 2 ~100 >100
9L-~786 is 7-[~-(2-aminometllylphenyl)acetamido~-3-~tl-c2r-
boxymethyltetrazol-5-ylthio)methyl~-3-cephem-4-carboxylic
acid.
TABLE 12
~. ~
. -Geometric Means of ~IC's Against 18_Strains
-
of S. marcescens
BB-S510 BL-S786
7.9 85.9
.
.
. - 104 -
.
i. : i
' . ` !
~ ~5~727
EX~PLE 21
Substitution of an equimolar weight of 2-ethoxy-
imino-2-(fur-2-yl)acetic acid for the 2-methoxyiminofuryl
acetic acid used in the procedure of Example 20 produces
7-(2-ethoxyimino-2-furylacetamido)-3-(2-methyl-2,3-dihydro-
. ` . 6 -triazolo[4,7-b~pyridazin-3-on-6-ylthiomethyl)-~_cephem-
4-carboxylic acid.
.
` EXAMPLE 22
Substitution of an equimolar weight of 2-n-propoxy-
lmino-2-(fur-2-yl)acetic acid for the 2-methoxyiminofuryl
acetic acid used in the procedure of Example 20 produces
7-(2-n-propoxyimino-2-furylacetamido)-3-(2-metllyl-2l3-
dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl)-
F 3-cephem-4-carboxylic acid.
XAI;~PLE 23
~ Substitution of an equimolar weight of 2-n-butoxy-
! imino-2-(fur-2-yl)acetic acid for the 2-methoxyiminofuryl
¦ acetic acid used in the procedure of Example 20 produces
. 7-(2-n-butoxyimino-2-furylacetamido)-~-(2-methyl-2,3-
dihydro-s-triazolo[4,3~b]pyridazin-3-on-6-ylthiomethyl)-
3-cephem-4-carboxylic acid.
- 105 -
.
~ ` llS~27
.
.
EXA~PLE 24
The products of Examples 20-23 were prepared as s~rn
isomers essentially free of the corresponding anti isomers
by the use in the procedures of those examples of purified
s~n lsomers of the appropriate 2-alkoxyimino-2-(fur-2-yl)-
acetic acid. Converslon of part o~ the syn isomer to anti
isomer durlng preparation of the acid chloride from the acid
ls substantially avoided by minimizing its exposure to
hydrogen chloride, e.g. by first converting the acid to its
anhydrous sodium salt and by treatlng that salt with oxalyl
chloride under anhydrous conditions in the presence of a
hydrogen ion acceptor such as dimethylformamide.
Such syn isomers are also named as (2Z)-2-
alkoxyimino-2-(fur-2-yl)acetic acids.
.'~ ,
~, '
.
o
` ' ' ' .
- 106 -
:
~5~ 7
~ 25
An ln~ectable pharmaceutical co~po~ition i6
ormed by addlng sterile water or sterile saline solu-
tlon (2 ml.) to 100-500 mgm. of 7-[(2Z)-~-methoxyimino-
(fur-2-yl)acetamido]-3-(2-methyl-2,~-dihydro-s-
triazolo[4,3-b]pyridazin-~-on-6-ylthiomethyl)-3-cephem-
4-carboxylic acid disodium salt.
Pharmaceutical compositions of the sodlum
and potassium salts of the other compounds of the
present inv~ntion, preferably in the f~Im of the pure
lsomer, are formulated ln a similar manner.
When the compounds are first prepared in
the form of the ~ree acid they are converted to the
desired, highly ~ater soluble potassiwn salt by treat-
ment with potassium 2-ethylhexanoate using the procedure
- of Example 20.
It is occasionally advantageous to have ad-
mixed with said solid cephalosporin as a stabilizing
and~or solubilizing agent a sterile, anhydrous solld
such as sodium carbonate, potassium carbonate or llthium
carbona~e(e.g. in about 5 or 6 percent by weight of the
weight of the cephalosporin) or such as L-lysine, argi-
; nine or histidine (e.g. in about 20-50~o by weight of
the weight ot the cephalosporin) or such as a s~dium,
potassium or calcium salt of levulinic acid, citric acid,
ascorbic acid, tartaric acid or pyruvic acid (e.g. in
about 25-200~ by weight of the weight of the cephalos-
porin) or such as sodium bicarbonate, ammonium car-
bamate~alkali metal or ammonium phosphates or N-methyl-
glucamine (per U.K. 1,380~741).
- 107 -
:
