Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CEFURO~IME ANALOGS J
S~-151~
The cephalosporins o~ the present invention in
general posses~ the usual attributes of such compounds ~nd
are particularly useful in the treatment o~ bacterial lnfec-
tions .
Prior known cephalosporins_-n ludQ those dis-
closed in Unlted Klngdom Patent No. 1, 393; o86, having the
general formula
RU. ~ .CO.WE~S (B)
o~ ~ CH2Y
OOH
w~ereln RU is phenyl; naphthyl; thienyl; ~uryl, benzothien~l;
benzo~uryl; pyridyl or any o~ these groups substituted by halo
(chloro, bromo, iodo or fluoro), hydroxy, lower alkyl, nitro,
amino~ loweralkylamino, dlloweralkylamino, lower àlkanoyl,
l~wcr alkanoyiamino, lower alkoxy, lower alkylthlo or car-
bamoyl; Rb is lower alkyl; cycloalkyl containing 3-7 carbon
atoms; carbocyclic or heterocyclic aryl lower alkyl or any
o~ these groups substituted by h~droxy, carboxy, exterified
carboxy, amido, c~ano~ alkanoyl~ amino, substituted amino,
halogen or lower alkoxy; and Y i~ selected from acetoxy; a
group of formula
( Rd ~
where R and n are as defined in claim 19, a group o~ for-
~ mula -S~ where W is thiadiazolyl, diazolyl, triazolyl,
~ tetraæolyl, thiazolyl, thiatriazolyl, oxazol~, oxadiazolyl,
.
,, ., : . . ' . , :. '
~$ ~
benzimidazolyl, benzoxazolyl triazolopyridyl, purinyl,
pyridyl or pyrim1dyl; an alkylthio group containing 1-4
carbon atoms; a group of formula -O.CO.R9 where R9 is
an alkyl or alkenyl group containing 2-4 carbon atoms,
the group -O.CO.NH.(CH2)mD wherein m is an integer of
~rom 1-4 and D is chlorine, bromine, Lodine or fluorine;
and azido) and non-toxic salts and esters thereof. Methods
for the preparation of the starting acids used to form the
7-substituent, including their separation into syn and
anti isomers, are also described therein and in U.K.
1,404,221.
Presently issued U.S. patents 3,966,717 and
~,971,778 contain at least part of the disclosure o~ U.K.
1,399,086 as does U.S. 3,974,153. See Also Farmdoc
abstracts 17270X and 19177X.
U.S. 3,974,153 claims compounds of the formula
H H
Rl . C . CONH , I ¦ ~
~ ORa 0 ~ ~ CH20.CO.NH2
wherein R1 is ~uryl, thlenyl, or phenyl; and Ra is C~ 4alkyl,
C3 7 cycloalkyl or phenyl; and a physiologically acceptable
salt thereof.
For examples o~ publications in the scienti~ic
; literature see Ryan et al., Antimicrobial Agents and
Chemo~-herapy, 9, 520-525 ~I976) and O'Callaghan et al.,
ibid7 9~ 511-519 (1976) and Norby et al., ibid, 9, 506-
510 (1976).
U.S. 3,819,623 discloses the conversion of the
2-mercapto-1,3,4-thiadiazole-5-acetic acid to 7-(lH-
tetrazol l-yl-acetamido)-3-(5-carboxymethyl~ $4-thia-
.
- 2 -
diazol~2-vlthiomethyl)-3-cephem-4-carboxylic acid and
see also Farmdoc abstract 12921T.
U.S. 3,883,520 and 3,931,160 and Fa~qndoc abstract
22850W make reference to 3-heterocyclicthiomethyl cepha-
losporins containing a nwnber of substituents (including
carboxyl) on the numerous heterocycles included but these
references are completely general in nature and include
no physical constants, yields, methods of synthesiæ or
the llke and do not even name any such compound ~ontaining
a carboxyl substituent. See also Farmdoc 00145W.
U.S. 3,928,336 provides a review of much OI the
older cephalosporin art.
Farmdoc abstract 18830X discloses compounds of
the ~ormula
S N--N
--c~2s N -
COOH (CH2)n-cooH
~where R1 = ac~rl or H; R3 = H or methoxy, n = 1-9). The
compounds of the present lnvention are not described
therein or in the full text o:~ the corresponding patent.
See also F~rmdoc abstract Ol9ôlX.
:;
v
.
.
One of the problems presently ~acing the medical
pro~ession at this time was describecl by Arnold L. Smlth,
M.D. in an article titled Antibiotics and Invasive Haemophilus
influenzae, N. Engl. J. Med.~ ~_3 1329-1331 (June 10,
1976) in which theopening sentence reads as follows:
"Recently, the information service of the Center ~or
Disease Control, the Medical Letter and the American Academy
of Pediatr~cs have sounded the alert that invasive ~trains
o~ nfluenzae isolated throughout the United
States have been found to be resistant to ~mpicillin, many
of the isolates being associated with treatment ~ailureO"
His concluding paragraph reads: "The current situation
portends a dismal future ~or the antibiotic treatment o~
invasive H influenzae disease. An H. in~luenzae resistant
to the second-line drug, chloramphenicolg has been described,
and, more recently, an untypable H. influenzae resistant to
chloramphenlcol and tetracycline was isolated ~rom the
throat of a four-~ear~old girl. m us, both these currently
e~ficacious agents may not be use~ul in the ~uture."
A solution to this problem is provided by the
present invention.
The present invention thus pro~ides compounds
having the ~ormula
C - NH-~E--C~I ~H2 N~ Ni
~ oRl 0 ~ N~ C'~C-CH2~S-C`~7_N
c_OR2 (CH2)nCOOEI
11
~ten written herein as
- 4 -
.... . ..
- C - NH ~ S~ Nl ~IN
N ~ N ~ CH2S ~ N~N
COOR (cH2)ncooH
whereinR is alkyl contalning 1-4 caxbon atoms~ n is one~
two or three and R is hydrogen or a conventional, phar~
maceutically acceptable, easily hydrolyzed ester ~orming
group such as those set forth below ana including the group
having the formula -C~-W
wherein when W represents hydrogen, Z represents (lower)
alkanoyl, benzoyl, naphthoyl, furoyl 3 thenoyl,
nltrobenzoyl, methylbenzoyl, halobenzoyl, phenyl~
benzoyl, N-phthalimidoJ N-succinlmidoJ N-saccharino,
N-(lower)alkylcarbamoyl, (lower)alXoxy, (lower)-
alk~lthio, phenoxy, carbalkoxy, carbobenzoxy,
carbamo~l, benzyloxyJ chlorobenzyloxy7 carbophenoxy,
oarbo tert.-butoxy or ~lower)alky1sulf'0n~19 ~nd ~en
W repre~ents carbalkoxyJ Z repre~ent~ carbalkox~ and,,
when W repre~entæ phenyl9 2 repr~sents benzoyl or
cgano or wherein W and Z taken together repre~ent
2-oxocycloalkyl contaisllrlg 4 to 8 carbon atom~
inclu ive.
A3 3ek :forth below in more detall the present
nt~on al~o pr~vldes sa}ts of theæe acids~ The
~ter~o~he~ tr~ of' the blcy~lIc s~ucleus 1~ that
ro~nd ir~ Cep~alosporln C.
~he compounds o~ the present invention are
isomers or else are mixtures of syn and anti
_~
isomer~ containing at least 75~ of the s~ isomer.
Pre~erabl~ such mixtures of isomers contain at :Least
90% of the syn isomer ~nd~not more than 10~ o~ the anti
-- 5 --
`7'
isomer. Most preferably the compounds are ~ isomers
essentially free of the corresponding anti isomer.
The pre~erred embodiments of the present
invention are the syn isomerg of the compounds of Formula
I ~rherein Rl is methyl or ethyl,
n is one or two, and
R2 is hydrogen, pivaloyloxymethyl, acetoxy-
methyl, methoxymethyl, acetonyl~ phenacyl, p-nitrobenzyl,
trichloroethyl, 3-phthalidyl or 5-indanyl.
Reference to the syn (cis) isomeric ~orm refers
to the configuration of the group ORl with respect to
the carboxamido group
;., :
. ~ .; _ _
~: .
.
.
.
The present invention also provides the process for
the production of the antibacterial agents having the formula -
C - C~ 2
~o~l N~o~l / \ ~.C ~H2 S ~N~N
~O~H t~H23 ~ ~ .
wherein Rl is alkyl containing 1-4 carbon atoms and n is
one, two or three which comprises reacting a compound of the
formula
~S .: .
-~CH CH2
C CH2 S C ~ N ~ N II
aoo~ (C~2)nCOOH
wherein n is one, two or three or a salt or easily hydrolyzed
ester or Schi~f 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 silyl 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 pivaloyl- ~
oxymethyl, acetoxymethyL, methoxymethyl/ acetonyl, phenacyl, ~ ; .
p-nitrobenzyl, ~ trichloroethyl, 3-phthalidyl and 5-indanyl : .
esters) thereof with an organic monocarboxylic acid chloride
having the formula
O
'; ~C~ X
O N ::
~ 1
OR
in which X is chloride, and R' is as defined above, or a
20 functional equivalent thereof as an acylating agent.
- 7- :-
: .
~ `7'
Such functional equivalents :Lnclude ~he
corr~pon~lng aoid ~nhydrides, including mixed
~nhydride~ ~nd p~rticul~rly the mixed ~nhydride~
prep~red from ~tronger ~cids ~uch ~s the l~wer
~liphatic monoester~ of c~rbonic ~ICid9 or ~lkyl
~nd ~ryl sulfonic acid~ ~nd of more hindered
scids ~uch ~ diphenylace~ic ~cid. In ~ddi~lo~p
~n ~cid ~zide or an ~ctive ester or thioe~ter (e.g.
with p-nitrophenyl, 2,4-dinitrophenol, thiophenol,
thioacetic acid) may be us~d or the free ~cid itself
m~y be coupled with compound IX ~fter first re~c~ng
~aid ~ree ~cid wi~h N9NI-dimethylchloroform~inium
chlorid~ Ecf. Gr~t Britain 1~008~170 ~nd Novsk
a~d Weichet, i:l!3~ ' Yxl, 6~ 360 ~l9653] or
by th2 u~e of enzymes or o~ an N~N'-carbonyl~
diimidazole or an N,N'~-carbonylditri~201e lcf.
South Afric~n p~tent ~pecific~tion 63/2684] or
a carbodiimide reagent [especially N,N'~dicyclohexyl-
c~rbodiimide. N9N'-diisopropylcarbodiimide ~r N~cyclo-
hexyl-N'-~2-~rpholinoethyl)carbodiim:Lde; c~, Sheehan
~nd He8~ ~L ~ ., 779 1967 ~1955~, or
~ alkylyl~mine re~gent ~c~ R. Bui~le ~nd H~Go
Viehe, An~w Chem. Internation~l Edition 3 5B2,
`
-- 8 --
~196~)~ or o~ an i~oxa~ol~um aalt reagent [~. R, B.
Woodward, R. A. Olor~on and H, Mager,
, 8~, 1010 (1961)~ o~ o~ a ke~enimine reagent
[crr C. L. Stevens and M. E. Munk, ~ h~m~ ~5~
~, 4065 (195~ ~ or o~ hexachlorocyclotr~phosphatrlaz~ne
or hexabromocyclotrlphosphatrla~ine ~U.S. ~,651~050~ or
of' dlphenylphosphoryl azide [DPPA; ,~ ~E,~ Chem~,
~, 6203-6205 ( 1972) ] or of dlethylphosphor~l cyanide
~DEPC; Tetrahedron letter~ No. 18, pp. 1595-1598 ~1973)~
or o~ diphenyl pho3phite ~Tetrahedron L~tters No. 49,
pp. 5047-5050 (1972)~. Another equlval~nt o~ the acld
chlorlde 1~ a corresponding azolide, i.e., an amlde o~
the corresponding acid whose amide nitro~en ls a member
Or a quaslaromatic ~ive membered ring contalnlng at
least two nltrogen atoms, l~e., ~mldazole~ pyrazole,
the trlazoles, benzlm~dazole, benzotriazole . and their
substituted derivatlves~ A~ an examp~e Or the general
method ~or the preparatlon o~ an azolide, N,N~-carbonyl-
dl~midazole i8 reacted with a carbox~llc acid ln
e~ulmolar proportlons at room temperature in tetr~-
byd~o~uran, chlorororm, dlmethylrormamide or a ~m~lar
în~rt ~o~rent to rOrm the carboxyllc acld lmidazolide
ln prastl~ally quantltative yleld wlth llberatlon o~
carbon dioxlde and one mole o~ lmidazole. I)~carboxylic
ac$d~ yleld dimidazollde. The by~produ~t, ~midazole;
pre~lpîtate~ and may be ~eparated and the lmld~zolide
~olated, but thls 13 not essential. The methbd~ f`or
carrging out the~e r~a¢tlon~ to produ~e a cephalosporln
~nd l;~e methods used to ~olat~ the cephal~porln ~o
produced are well hnown in l~he art.
_ g _
- . . . . .
.
5 ~7
~ entlon ~8 made above o~ th@ UB~ 0~ enzyme~ to
couple the ~ree acid with
compound II. Included ln the sc~pe o~ ~uch proee~es
are the use o~ an eater, e.g. t~e methyl e~ter, Or
~hat ~ree acld w~th enzymes provlded by varlou~ ml~ro
organl~m~, e.g. tho~e de~crlbed by 'r. Taka~ashl et ~1.,
J. Amer. Chem. Soc~ . 4035 4,~37 (1972) an~ by
T, Nara et al., J, Antibiotics (Japan) ~L~L, 321-~23
~1971) and ln UOS. 3,6~2,777.
Por the coupling o~ ~he org~ic c~rboxylic ~cid a3
described above w~th compound II tor ~ ~lt or pre~er~bly
~n e88ily hydrolyzed ~ter of Schif~ b~se, a~ with
benzaldehyde, thereof) it is also convenien~ ~nd eficient
to utilize as the coupl~ng agent phosphonitrilic chloride
~rimer (J. Org. Ch~m.9 ~ 2979Y81~ 1968) or Nethoxy-
1,2--dlhyd~oquinoline ~EE~Q) ~ de~cribed in J. Amer. Chem.
Soc., 90, 823-824 and 1652-1653 (196~) and U.S. P~tent
3,455,92g. The re~ction i~ pre~rably c~rried out ~t 30
35~C. in benzene, eth~nol or tetr~hydrofur~n u~lng ~bout
equimol~r quantitl~s of ~11 th~ee resgent~ foll~w~d by
conventional i~ol~tion ~nd remov~l by conYentional method~
of any blocking groups present.
:An additio~l proGess of the pre~ent invention compri~
~hQ prep~r~tlon of th~ c~mpounds of th~ present ~nventlo~
by the di~pl~c~ent of the 3~sceto~y group of a 7 ~cyl~mino-
; cephalo~por~ic ~cid (prep~r~d by subst~tuting 7-~ino~
.
~ - 10 -
cephalospor~nle ~cid for the 3~thiolat~d-7-~mino~
ceph~lo~por~nic acid~ in the ~cyl~tion procedure~
descrlbed herein and elsewhere r0ported) with ~ th~ol
HSR3 hav~ng the formula N--~
}~8 ~ ~N -
(CH2)nCO~)H
wherein n is one, two or three ~nd then removing the
protecting group if ~ny i8 pre8en~, 39 on iche
~arboxyl group.
The displacement o~ such ~ 3-acetoxy group with
such a thio~ m~y be ~ccomplished in solut~on as ~n w~ter
or flqueous ~cetone at ~ temperature of ~t le~t room
emperature ~nd prefer~bly within l;he r~nge of ~bout
50~ to lOO~C. in the presence of ~ mlld b~se euch 198
sod~um bic~rbonate, e.g. prefer~bly ne~r neutr~l~ty ~uch
98 at abou~ pH 6. An exce~ of the thiol i.8 prefer~bly
employed. The reaction product i8 isol~ted by c~reful
~cidification of the reaction mixture :Eollowed by extr~c-
tion with ~ water-immi3cible org~nic ~olvent. A~ noted
~bove, the prep~r~tion o ~aflny other 7 acyl~mitoceph~Jospor~n~c
~cld~ ~8 de~cribed in the p~tent ~nd scientific li er~tur~,
e.g,, ~n U,.S. Cla3s 26û-243C.
.
5~
~ ne salts of the compounds o~ this invention include
the nontoxic carboxylic acid salts thereof, i.ncluding non-
toxic metallic salts such as sodium, potassium~ calcium and
a-~minum, the ammonium salt and substituted ammonium salts,
e,g, salts o~ such nontoxi.c amines as trialkyl-amines
including triethylamine, procaine, dibenzylamine, N-benzyl-
beta-phenethylamine, l-ephenamine, N,N'-dibenzylethylenedi-
amine, dehydro~bie~ylamine, N,N'-bis-dehydroabietylethylene-
diamineJ and otner amines which have been used to form
salts with benzylpenicillin, L-lysine, arginine and histidine.
m e present invention thus also provides the ~rocess for
the production o~ the antibacterial agents having the ~ormula
O / S ~ N N
C - C -NH-CH - ICH CH2
N~ 1 // ~ C ~ N
OR ~ COOH (CH2)ncOoH
. ''' .
wherein Rl is alkyl containing 1-4 ~arbor, a~oms and n is
one, two or three which comprises reacting a compound of
the formula
fl c~s j . , .
5-C-NH-fH I CH2 l
O ~N ~ C-CH O-C-CH3
COOH
herein Rl is æs described above, with a compound having
the formula
N - N
HS
N
' (CH~)nCQ~
wherein n is one, two or three.
- 12 -
The preferred esters o~ the cephalosporins
o~ the present inventlon are the pivaloyloxyTethyl,
acetoxymethyl, methoxyrneth~l, acet~nyl an~ phenacyl
esters. All are usef'ul lntermediates in the
productlon o~ th~ cepha losporin having a free
~arboxyl group.
As lndicated above., these five e~ter~ o~ 7-amino-
cephalosp~ranlc acld are each prepared by known
method~ One excellent procedure ls that o~ UOS.
p~tent 31284,451 ln which sodium cephalothin is
e~terl~ied by reaction with the corresponding active
chloro or bromo compound (e.g. phenacyl bromide,
chloroacetone, chloromethyl ether, plvalo~loxy-
~ethyl chl~ride ~al30 cal}ed chloromethyl pisralate~,~cetoxymethyl chlorlde) and then the thien~l-
acetlc acid sidechaln 1~ removed enzymatically
a~ ln the ~ame patent or chemlcally a~ ln U.S.
p~tent 3.,575J970 and in Journal Or Antibiotic~,
;2~'-7
X~YIV (11), 767-77~ (1971). Irs ~noth~r good
method the trlethylamine ~al~ o~ 7-aminocephalo-
~poranlc ac~d ls reacted directly wlth the active
halogen compound, as in ITnited Kingl~om 1,229,453.
Ihese e~ters o~ 7-amlnocephalo~3poranlc acld
are then reacted wlth th~ nucleophi:le HSR3 in the ~ame
manner as i~ illustrated hereln ~or 7-a~inocepha:Lo-
sporanlc acld ltself, ~he 3-thiolated ester o~
7-aminocephalosporanic acid ~ then coupled ~ith
the organic carboxylic a~id as be~ore.
The o~te2~ o:~ the
cep~losporin 80 obt~lnzd i8" i not u~ed per 8e9
eo~verted to it~ re~ ~cLd
~nd, i~ de~ired,, any ~lt by removal o~ th~3 1!8t'ri-
ying group" a~ by ~queous or enz~ic hydro~ysi~ (a~
with ht~msn or ~r~ enml) or ~cidlc or ~ line
hydrolysis or by ~reatment wi~ch ~odium '~hiophertcxide
as t3ught irl U.S. 3,284t451 ~nd9 in th2 pel~icill~Ln
~eries, by Sheehan et al., J. Org. ChemO ~),
200~-2~0~ ~1964)"
In ano~her alternatlve ynthesis, the 3-
thiolated 7-aminocephalosporanic acid 1~ prepared
as de~ribed herein and then act~7ated at the 7~
~mlnQ group and flnally e~terlf'ied, a~ by reacti.on
Or the appropriate alcohol with the acid chloride
prepared, ~or example, by reactlon Or the rinal
eepha losporîn wlth thionyl chloride or by vther
es~ent~ally acidic esterl~ca~lvn procedl~.re~,
_ 14 ~
.
~ 8~7
In the treatment of bacterial infections
in man, the compounds of this invention are adminis-
tered parenterally in an amount of ~rom about 10 to
90 mg./kg./day and preferably about 14 to 50 mg./kg./day
in divided dosage, e.g. two to four times a day. They
are administered in dosage units containing, for example,
125, 250 or 500 mg. of active ingredient with suitable
physiologically acceptable carriers or excepients.
The dosage units are in the form of liquid preparations
such as solutions or suspensions and preferably are
aqueous solutions of a sodium or potassium salt which
are in~ected intrarenously or intramuscularly or by
continuous or intermittent in~usion in concentrations
o~ about 125-500 mgm./ml., and preferably, 250 mgm./ml.
as is customary in therapy with cephalosporin antibi-
otics.
`:
,
'
~52~
.:
$~ARI~Ç MATERIALS
l-~a~box~rne-~-hv~ mQ~totetr~
N- N
: ~ HS - ~ ~N
CH2COOH
~) R~~1iization o~ et
~ ~ .` ;,' .
:. .
1~ One hundred and ten gram~ o~ l-methyl-5- :-
mèrcaptotetrazole 19 slurried in 350 ml. o~ boi:Ling
chloro~orm. A near ~olution i9 obta lned .
2. lhe hot ~olution (50-60 ) 1~ rapldly . :
~lltered by vacuum through a hea~ed :Buchner runnel : -
(11 cm. SS No. 604 paper contalning 1~4 to 1/~ lnch
*
Or packed f`llter aid ("Supercel"). me rilter pad
~' i8 washed with 50 ml,. Or 50-60' C. chlorof'orm whlch
i8 added to the flltrate.
3. Ihe rlltrate 18 cooled to appro~cimately
o-60 C. and kept at o-6~ c. rOr 2 hours. The
cr~rstal~ whlcll have rormed are collected by
rlltratlon at o-6 c. and wa~hed wlth 60 ml. o~
~: 20 ~ o-6 c. chloro~orm which l~added to the ~iltra~e.
': ~ me cry~tals (cut A~ are alr dried ak 37-45 ~;
: .~ rOr 1~ hour~
',: ~
. ~ : *Trade ~ark
, .
~ (~ ''.',
`' ~ ' .. - ' ' I; ' ' ' .' '
4. me riltrate 1~ concentrated on the
rotary vacuum evapor~tor (60 C. ~ath) to
approx~mately one-half volume. Thi~ ~lurry
1~ ~ooled to o-60 C. and kept at o-6 c. ~or
2 hours. The cr~stals are collected by
~iltration at o-60 C,, washed wlth 40 ml. o~
o-60 C, chloro~orm whlch ls added to the ~
trate. The crysta 1B (cut Bj are air drled at
37-45 C. ~or 18 hou~R. Crystal cut~ A and
are composlted to give an approxlmate 65
welght yleld.
5, m e riltrate o~ cut B, Step 4 may ~
reworked twice a~ descrlbed in Ste~ ~ to obtain
an additlon~l 15~ recovery.
b) _~ ~L~t Or l-
~~~
1. Five hundred ml. of su~stantially dry andpure tetrahydrofuran in a 2-liter ~ neck ~lask wi~h
stlrrer i9 cooled in a salt-acetone-lce bath to
approxlmately -10 C. Dry nltrogen gas i~ blown
: on the liquld ~urrace.
2. Flve hundred ml. o~ 15.06~ (1.6 N) butyl
h~um ln hexane (Foote Mineral CoO) i~ a~ded
over a ten mlnute period undeF dry rlîtrogen and
~tlrrln~3; to the tetrahydro~uran. The: near
~olut~on 1~ ¢ooled to -5 to -lQ~ C,
. ,
,
17 _
. . . . . . . . . . . . .
iZ~7
30 Fortg~ 81x ~nd four tenth~ gram (46,4 g.)
o~ l methyl-5-mercaptotetrazole (recr7sltalllzed
as ~bove) i8 dissolved ln 200 mlO Or ~ub3tanti~11y
pure and dry tetr?hydrof'uran. me solu'ciLon 1~
~lltered i~ cloudy and then cooled to 5 to. 10 C.
4. me cooled solution o~ ~tep 3 i~ added
over 10 mlnube~ with stlrrâng and under dry nitro-
eE~ 'co th~ butg 1 llthium solution . Ihe temperaturle
should be maintained at -5 C. to ~a~m~m~.
Pr~cipitates ma;y ~orm.
` 5, me mixture 1~ stlrred under dry nitrogen
and 0~ C. to ~10 C. ~or one hal~ hour,
6, Anhydrous carbon dioxlde ga~ i8 bubbled
through at a rapld rat~ and with rapid stlrring
~or 15-30 minutes at approximately amblent
temperature (0 to 10 C.) to no hlgher than
~20 ~
7, The wh~te precipitate which ~orms is
sultab ~ collected by ~iltratlon ln an area o~
low humldlty. The preclpltate 1~ wa~hed with about
75 mlO of tetrahydrofuran.
8. ~h~ preclpitate i~ dl~olved in 250 ~
o~ water (p~ 8.5-9.5). A second la~er of tetra-
hydro~uran may be present. Thi~ mag be remove~
in ~he vacuum rotary e~aporator ~50~ C. bath30
.
.. 18 ..
. .. , .. . . . ~
~ 7
9. The aqueous ~olution 1~ adJu~ted to
p~ 1.6-2.0 with con~ntrated hydrochloric acldu
10. The acld aqueous ~olutlon 1~ extr~cted
twice with 250 mlO pQrtlons of ethyl acetate,
Each 250 ml. ethyl acetate extract i~ bac~
extracted with 100 ml. portlon~ of water. qhe
~a~er extracts are discarded. The ethyl acetate
extracts t~r~ o~ any water l~er3 are filtered
and composlted.
11. m e comblned eth~l acetate ex~ract~ are
concentrated to dryness on the vacuum rotary
evaporator t60 C. bath).
12. The ~ry5tal9 in the ~lask are boll~d wlth
: ~00 ml. o~ chlorQrorm ~or about 2 mlnute~. The
hot ~lurry (50-60 C.) i~.vacuum ~lltered through
ff h~ated ~uchner ~unnel (11 cm-SS-604 paper). The
crgstal~ are wa~hed wlth about 75 ml. of 50 C.
chloro~orm. m e crystal3 are air dried at room
temperature.~or about 3 hours.and then made about
100-~00 me~h.
1~, The 100-200 mesh crystals are trea~ed
: wi~h boiling c~loroform exactl~ a~ de~crlbed in
~tep 12 (the ho~ chloro~orm removes most o~ the
unreacted l~methyl-5-mercaptotetrazole). Yield:
approxlmately 4~ to 50 gram~ o~ crystalline ~-
car~oxym~th~l-5-meraaptotetrazole~ These cry~tal3
... .
19 _
,. .. .
may contain 0.02 to 0,05 moles o~ 1-methyl-5-
mercaptotetrazole.
14. The cry~tal~ o~ step 13 are ~lu~ried
with 250 ml. o~ eth~l ether at room temperature
ror 3-5 mlnute~. m e mixture ls ~lltered. .The
inqoluble~ ~O.5-5%) may be a contaminatlng
symmetrlcal mercaptotetrazole ketone o~ the
~ollowlng tentatlve structure:
.
~ - N O M _ N
N~C ~N CH2 C 2 ~ N ~ C~N
SH . SH
GAU~ION: This compound ~ a~3~ at approximatel~
205-2~0 C.
159 The ether rlltrate o~ ~tep 14 ls
é~aporated to dryness on t~e vacuu~ rotar~
evaporator (~0 C. bath). Approximately 42 to
~8 grams of crg~talllne 1-carboxymethyl-5-mercapto-
tetrazole containing approximately 0~01-0.05 mole
o~ l-meth~1-5-mercaptotetra~ole is recovered.
16. Ihe crystals are dissolved in 420 mlO
o~ absolute ethanol (approxlmately 100 m~./ml.).
The solutio~ ls warmed to 50-60~ C~
170 To the hot solution Q~ step 16, 310 mlO
o~ a 41% ~odlum ?-ethylhexanoate (SEH~ solution
in lsopropanol 1~ added with verg rapid stirring
over ~ 10 minute period. A crystalline precip~tate
~orm~ m e m~ture i~ slurried at 50-60D C. ~or
nute3~ ~
_ 20 _
: . . . . . . . .
`7'
18~ me m~xture 15 ~lltered hot (50~60 C.)
through a heated ~uchner ~unnel (1~ cm-SS-No. 60
paper~. me crystals are wa~hed wlth 75 ml. Or
50 C. ethanol,
l9o qhe ethanol damp cry~tal~ of ~tep 18
are slurried ln 200-~00 ml. o~ ethanolO q~e
B~Urry 1~ passed through a 200 mesh ~creen. Th~
slurry i~ heated to 50-60 C. rOr 5 minute~ with
rapld ~tirring tunreacted mono-sodium l-methyl-5-
mer~aptotetrazole i8 very ~olubïe in hot ethanol),
20. ~he cry8tals are collected at 50-60 C,
~n a 11 cm-SS No. 604 paper ln a heated Buch~er
rl~lne~ ! ory3tal9 are wa~hed wlth 75-100 ml.
of ethanol ~nd vacuum drieà at 50-60~ C~ ~or
24-48 hours. Yleld: 40-48 gram~ o~ dl-~odlum
l-carboxymethyl-5-merc~ptotetrazole (rree o~ 1-
methyl-5-mercaptotetrazole a9 observed b~ NMR).
.
.. . .
~ 21 ,_
~ id.
~I~N-C~2C2Na O~_CN2-O-C-C~
C~12N
.
H2N ~ S ~ N,N~N
CH2 S ~ 2 2
C02H
1~ Int~ a 3 necked ~la~k set up with an
a~lta~o~, a temperature regulator~thermometer
and a nitrogen lnlet tube, place 18 grams
(oOo66 mole) of 7-amlnocephalosporanlc acid,
.
' :.
.
: . ' . '
_ 2~
.. ..
(wh~ch hs~ pre~erabl~ been recry~tallized by
the koluen~sul~onlc acld procedure) and ~00
ml. o~ 0.1 M pH 6.4 phosphate bu~er ~20.7
gram~ Or sodium pho~phate, monoba~ic .IH20
8.5 gram~ o~ ~odium phosphate, dlba3i~,
anhydrous~ q.~. to 2 llter~).
2~ With agltatlon of the mlxture descrlbed
ln ~t~p 1, add 1~5 grams of ~odium blsul~l~e and
16 gram~ (0.078 moles) o~ 1-o~rboxymethgl-5-
mer~aptotetraæole di~odlum.
3. With agl~atlon contlnuing, bubble
nitrogen through the m~xture for 10 minutes.
4. Ma~ntainlng agltation and nitrogen
ln~low, heat the slurry over a 20 mlnute perlod
to 56 C, During thi~ tlme inter~rai3 6.5 gram3
o~ ~odlum blcarbonate is added ln 8mall lncrement~.
: 5. With ~ontinued agltation and nitro~en
inPlow, mainta~ the temperature ~ the ~olutlon
at 56 C. ~or ~ hours. m e pH should remaln at
between 6,2 - 6,6.
6. Cool the reaction mixture ln an ice bath
to ~ C,
7~ Add 50 ml~ ~ a 1:1 phosphorlc acid/water
:~ ~olutlon ~o the mlxture or concentrated HCl to
a pH Or 2.0 ~ 3.0,
8~ Collect the product by ~iltration. Wa.~h
~he ril~er oake with 20 mlO o~ cold water ~ollowed
: b~ 200 ml. Or c~ld methanol.
. .
- 23 -
,~ ~
J/ ;~ '
.
,
521~7
9. A~r dr~ the ~olld to constant weight.
~A typical run produced 14.5 gram~ o~' ~roduct,~
~i~ produGt may var~ ~n color rrom yellow to
dark brown.
10" Pa~ the product through a 200 mesh
~talnle~ st~el ~reen.
11. Suspend 10 ,grams o~ the 200 mesh powder
in 2û0 ml. o~ propanol wlth rapid ~tirrlng.
12" Add 2.0 ml. o~ ~ancenkrated hydrochlor~
10 acid and ~tir vlgorously for 0.5 hour at room
. t~mperature .
l~io Fllter the slurry. Wash the brown ~olid~
~ith 20 ml. Or n-prop~nol and add the wash to the.
~iltrate (save the ~llt~r cake ~or posslble recover~
o~ additlonal product).
dd 1.5 gransor charcoal ("Darco G-601'3
to the n-propanol ~lltrate o~ step 13~ Slurry.
~or 0.5 hQur. Remove the carborl b~ ~iltratlon.
Wa~h the carbon with 20 ml. o~ n-propanol and addl the
20 wash to the riltrate.
15. Wlth rapid stlrring, ad~ trlethylamine to
th~ n-propanol f`lltra~e to an apparer~'c p~I o~ 3fO~
~ Cr~tal~ ~orm. Slurry ~or 10 mlnute~
ï6. Coll2e~t the whlte cry~tals by ~lltratlon
and w~sh with 30 ml. o~ n-propanol~ 50 ml. Or
m~thanol, and vacuum drr ~t ~0~ C. ~or 24 hour~O
~leld: ~ to 8 ~rams o~ 7-am~no-3~ carbox~methyl-
'cetr~zol-5-ylthlomethyl)-~c~p~em 4-~arboxyllc acidO -
*Trade Mark
i 24
- . . .
~ 7
170 An alternate procedure ror the purl~i-
cat1~n o~ 7-amino-3~ car~ox~lme~h;ylte~razol-5-
~- ylthlomethyl)-3-cephem-~-carboxylic acid ~ollow~:
a~ Slurr~ lO grams of the 200 mesh product
(~rom ~tep 10) ln 75 ml. Or 1 N hydrochloric acld .
ror lO-l5 min~tes at room temperature. Filter
to remove dark brown solids.
b) Add 2,5 gram~ o~ charcoal t"D~r~o ~-60")
and 3lurry ~or 0.5 hour.
c) Remo~e the carbon by ~lltratlon, Wa~h
the ¢arbon wlth 15 ml. of water and add the wa~h
to the ~îltrate,
d) Wi~h rapid stlrrlng, add concentrated
annnonium hydroxide to ~he filtrate to pH 2,5-
3 . O, Crysiea ls f`orm . . .
e) Slurry the cry~tal mass ~or 25 mlnute~.
Remo~re the crystals by ~lltration. Wash the
, ~ . .
~ry~tals with 30 ml. of water9 50 ml. o~ methanol3
and ~acuum dry at room temperature. Yleld: 4-7
gram~ ~r near whlte crystal~,
~' " , .
.
.
. ~ 25 -
'
: , :. ...,, ~ , . , - ..
.. . .
~`7
Preparation of l-Carbox~eth~lte-trazol-5-thiol
__
NS ~N ~ N
~H2)2 C02H
A) ~ ~ate
. ~-alanine ethyl ester hydrochloride (9~.6 g,),
triethylamine ~I23.$ g.) and methylene chloride (400 ml.)
were mixed together and cooled to -10 C. Carbon disul-
~ide (46.5 g. ) dissolved in 150 ml. of chloroform w~s
added to the above solution during a two-hour period
while keeping the temperature at about -10 C. A~ter
the addition was co~plete, the temperature was allowed
to warm to 10 C. ~or about 10 minutes. The solution
wa8 ag~ir cooled to -10 C. ~nd 66.3 g. of e~hyl chl~ro-
formate in 60 ml. of chloro~orm was added dropwise over
a 40-minute peri.od with stirring. Thë temperature was
allowed to rise to room temperature ~or 30 mi~utes and
again cooled to 0 ~ .; an additional 61 . 6 g . o~ tri-
ethylamine was added at 0 C'. an~ then the solution was
stirred at room temperature for 3 hours.
The mixture was treated with water and the
organlc phase collected, washed with 2 x 250 ml. o~
2N HCl, then 2 x 250 ml. o~ NaHC03, the~ 2 x 250 ml. o~
water~ The organic phase was dried over Na2S04 and ~he ..
301~ent removed in vacuo to produce 93.7 g. of an oil ..
fou~d ~ be ~he desired product. The IR and NMR spec-
, ~ .
tra ~ere consis~ent with the structureO
Sodlum azide (2~.7 g.~ was dlssol~ed ln 400 mlO
~ 26 -
.
.. . . , . ., . ~ . . , . ~ . ..
~ . ..
.. . . .
~ 7
of water and heated to 60 C. in a nitrogen ~tmosphere,
2-CarboethoxyethyllsoCyanate (4609 g.) dissolved ln
50 ml. o~ Skellysol~e B (essentially n-hexane) was
added to the heated sodium azide solution. The 80-
lution was stirred for about 150 minutes at about
70-72 C., then cooled to 3Q C. in an ice bath. Fi~ty
percent sodium h~rdroxide solution was added until the
pH was 12. me mlxture was heated for 40 minutes at
70 C. and cooled to 15 C. in an ice bath. The pH
was ad~usted to 2 using con~entrated ~Cl End then ex-
tracted with ethyl acetate (4 x 150 ml. ) . The ethyl
acetate extracts were washed with water, then dried
over sodium sulfate~ The solvent was evaporated in
vacuo and the product was coliected as crystals from
methylene chloride to yield 19. 5 g . of title product .
'.
I Alternate Synthesis o~ l-Car~o~methy~-5-m
tetfazole
..
f~ 2C02C;!~15 ~ C52 ~ ~JaN3
H2 a~ UaOH
.
~ ~ N
~N - C~2~02~ + Na2S ~ c2H5o~
:' ' ' '.
SH ... ..
. , ' ,
'' ' ~
*Trade Mark
' ~"
27- ;
.
,, . -, . . . .
Z~7
To a stirred mixture of 13. 95 g tO.10 m) o~
glycine ethyl ester hydroc:hloride~ 8.o gO (0.20 m.)
of sodium hydroxi~le and 8 ~ 37 g lO . ~Ll mJ of car~on
disulfide w~s added a solut~on o~ 7~47 g tO.115 ~1
o~ sodium azida in 125 ml of water. . The solut~o
was heated at reflu~E ~or 6 1~2 hrs~ d stored
'~6 hrs. at 25. ~he darX bro~m mix~ure wa~
filte~ed and the filtrata ac:d~ o p~ lo~;
. with con~O hydrochloric acid. The ~olution wa~
earbon treated and the yellow ~iltra~e was
extracted 4 x lOQ Jnl with ethyl acetate. ~h~
ethyl acetate was washed wit}~ water, dried ove~
magnesium sul~ate and evaporated at 40 tl5 mrr~]
to an oil. ~he oil was tritura~ea ~ith methylene
e:hloride asld the product was collected~ The
.~ ,..
gample was dried in Yai::UO ov~r phosphorus pantoxid~
~or 16 hr9. at 25~. The ix and s~mr sp~c~ra ~r~re
con~ist~nt for tha ~tructure.
Referenc:e: G~rman Patent 106645.
,
`
i' :
,
:: :
<. .
, ~ ' . .
- 28 ~
.
,, . . ~ ' ' .
. . .
;2
?-Carboetho~ymeth~l Isothio~ te
_ ~
IH2_NH2 ~ (C2H5)3N fH2-NH-c-sH .
C-OC2H5 ~HCl , - ~ O~ I OC2Hs (C2H5)3 N
f2~5
: O-C-Cl
~ , 1l .
CH2~NS f H2 MH I; s 11 O~'~H;
11-C?H5~ (C2H5)3N11 W2H5 S O
O . I O .
NaN3
- N CH2C02C2H5 OH ~ ~ ~ C ~ C0
~r ~ rT
~N;~--~n N`N~ SH
Carboethoxymethyl I othioc~ 2
Carbon disul~ide (22.8 g.~ 0.3 molO) in ~hloro-
~or~ (40 ml.) was added over a perlod o~ one hour to a
~tlrred suspens~on of glycine ethyl es~er hydrochlor~de
(41.7 g. 0.~ mol.) and trlethylamlne ~60.7 g., o~6 mol~)
i~ methylene chloride (300 ml.) at -10 C. The reaction
mixtur~ was allowed to warm up to 10 C~ and was stlrred
~or 10 minutes at this temper~ture. The mixture ~a8
tre~ted dropwiæe with ethyl chloro~ormate (32.6 g,
0.3 mol.) ln ch~oroform (50 ml.) at 0-5 C. over a
~' ' .
.. .
. 29 - .
period o~ .5 hour~ The mixture wa~ Rtirred ~or another
40 minutes at r~om temperature and triethylamine (30,4
g., 0.3 mol.) was added dropwise over a period of 15
mlnutes. The mixture was ~tirred ~or 1~4 hour at
0 C. and an additional 1/2 hour at room temperature
and finally stored over night at 25 C. The mlxture
was washed with water (250 ml.), 2N hydrochloric acid
(2 x 300 ml.)~ 5% ~odium bicarbonate solution.~ x 300
ml.) and ~ried o~er anhydrous sodium sul~ate, The
~olvent was evaporated at ~0 C. (15 mm.) to yield
38.1 g. o~ crude isothiocy~late. Upon storage over-
night, a ~ed dye wa8 produced. Thi~ may be eliminated
by distillation of the isothlocyanate.*
5-M2rcaptotetrazole-l-acetic acid ~
To a solution o~ sodium azide (4.9 g., 0.075
mol.) in water (~00 ml.) heated to 6,o c. under nitro-
gen~ was added the isothiocyanate (7.3 g.g 0.5 mol.)
bver a period o~ 1/4 hour. The mixture wa~ heated at
70-75 C. ~or 2 hours, cooled to 5 C. and 50% sodium
hydroxide was added to pH 12. The solution was heated
to 75 C. for 1 hour, cooled to 5 C. and ad~usted to
~H 2 with concentrated hydrochloric acid and filtered
~hrough di~tomaceous e~r~h ("Superce1"). The ~oluti~n
w~ extr2~ted ~ th e~hyl 3cet~te (4 x 120 ml,), c~on
tr~t~d ~nd evaporated eo an oil ~ 30~ 15 mmO3~ The
8~dUe W~3 ~lurried with chloro~orm to y~eld 1 g~ of
scid. The nmr and ir spectra were identical with
authenkic 5~mercaptot;etrazole-1-acetic aeid.
*The i~othiocyanate may be distilled at 104-106 Ct
(7 mm.3 T~B. Johnson ar~d A.G. Ren~rew, JACS 47
240-~45 (1~25)
- 30-
,,
-
, , .. , . , . - . . .
~ o a ~olut~on of 4û.B g. (0.63 ~1~3 of ~odiu7n
~zid~ in 400 ml. of w~ter at 60-C. WB8 ~dded 66.8 13~ ~0~,42
mole) of methoxyc~rbonyl propyl i~othioc:y~n~te [D.l.~
Garmsise, et al., J. Ame~. Chem. Sc~c-9 809 3332 (1958)1
d~op~ise. The mixture was he~ted ~t 78~C. ~or 2 hours,
cooled to room temper~ture ~nd sd3usted to pH 12 w~Lth Sû%
~odiu~ hydroxid~ solution. Tho solution w~ then he~ted
~t reflux ~or 1-1/2 hour~, cooled 'co 27~C. 3nd ad~u~ted to
0 pH 2 with 6N hydrochloric flcid. The mix~ure w~ filtered
through di~tom~ceous earth ('q)ic~lite") ~nd the c~lke was
wa3hed with 100 ml. of ethyl ~cet~t~. The filtr~te W~8
~xtracted with 4 x 100 ml. of ethyl ~cet~teO The extr~clts
were combi~ed~ wash~d with w~te~ ~nd dried by ~eotropic ~:~
dis~illation to precipitate ~n oil which cryst~llized on
cool~ng in ~n ice-b~th to yield 22 g~ l-c~rboxypropyl 2~
m~rc~ptotetrazole~ The nmr ~pectru~ was consl~tent for the ~;
cture.
~ ~ ~~ ,
~
To ~ Bu3p~nsion of 22 g. (0~0~1 mole~ of 7
~mlnoceph~lo~poran~c ~cid in 350 ml. of pH 6.4 O~lM
phosph~te buffer wa~ ~dded 16.9 g. ~0.089 mole~ of 1-
c~rboxypropyl-5-~erc~ptotetr~zole ~nd lr5 gL of ~odium
b~sulfi~e. The mix~ure w~ he~ed under ni~rogen ~o 55~C.
*Trade Mark
~ ",
: ~
,
~nd solld Yod~um bic~rbon~te Wfll3 added u~t~ he mixture
bec~me cle~r (pH 7~5). ~he ~olut~on ~8 he~ted for 3.5
hour~ cooled to lOaC. ~nd ad~u~ted to pH 2 with 6N
hyds~ochloric ~cid. Th~ precipit~te Wa~l collec~d,, w~h~d
with cold w~ter snd ~in~lly w~th m~th~nol ~nd ~r-dri~d
to yield 17.5 g. of 7 ~mino-3 (l~c~rboxypropyltetr~zol~5~r
ylthiomethyl)-3-cephem-4-c~rboacylil: ~cid. Th~ ~mple w3s
recryst~llized from 100 ml. of m~thanol ~'ch con~entr~'ced
hydrochloric ~cld ~dded dropwi~e until the mlxture w~s
cle~, The 301u~ion W~8 ~d~usted to pH 5 with co~centr~ted
amm`onitml hydroxlde and the precipit~te w~ coll~cted to
yield 607 g. The nmr ~nd ir spectr~ ~ro consiston~ for
~he ~tr~ctureO
Preparation o~ 7-Amlno-3-(1-carboxypropyltetrazolyl-5-
thiomethyl)-3-cephem-4-carboxylic Acid.
O
H2NCH2CH2CH2C-OC ~ HCl ~ (C2H5)3N CH2~12
¦--ICH2CH23 - OC.~3 1 C2~50-C-Cl
L G~ J
S (C2Hs)~NH
O
CH2CH?C OCH C 11
1 3 (C ~ ) N I ~ NaN
CH2NH-CI-S-~-OC2H5 _ ~ CH2-N=C=S - ~
S
-32-
L5~
~ NaOH
Nl - ~ ~ HCl N~ N
NaS -C~N ~ ~ XS - C~N~N
(cH~)3co2cH~ (~H2)3C02H
SH
H2NT~S ~ (CH2)3C02H
N ~ CH2-0-C-C ~ `N
CO H
S
~LCH S-ll IN
~OZH ( CH2 ) ~;CO2H
N-~utyrylmethoxy Isothiocyanate.
To a mixture of 153.6 g. (1.0 mole) of 4-amino-
butyric acid methyl ester hydrochloride 202~4 g. (2.0 mole)
trieth~lamine and 600 ml. of methyIene chloride cooled to
-15 C. was added 7~.1 g. (1.0 mole) of carbon disulfide in
;~ 200 ml. chloroform during a 60 minute period. The mixture
was warmed to 10 C., stirred 10 minutes, cooled to 0 C.
and 108.5 g. (1~0 mole) of ethyl chloroformate in 80 ml.
of chloroform was added during a 20~minute period (0-5 C.).
m e mixture was stirred without ice bath for 7Q minutes
and warmed to 18 C. The reaction was again cooled to 0
C. and 101.2 g. (1.0 mole) o~ triethylamine was added
during a 20-mlnute period. The reaction was stlrred for 15
minutes at 0 C. then for 1.5 hours withou-t the ice bath.
The mixturewas washed with 250 ml. of water,
2 x ~00 ml. of 2N hydrochloric acid, 2 x 300 ml. of 5~
sodium bicarbonate and 2 x 300 ml. of water. The organic
phase was dried over anhydrous magneæium sulfate and
~,
-33-
.~
reduced in volume 15 mm. to a ~ellow oil. Yleld: 126 g,
oil. The ir was consistent for the structure.
1 Carboxypro~yl 5-mercaptotetrazole
To 40.8 g. ~oO63 mole) of sodium azide in 400
ml. of water at 60 C. under a brisk nitrogen flow was
added dropwise 66.8 g. (O.~2 mole) of N-butyr~lmethoxy
isothiocyanate~ The reaction was warmed at 78 C. ~or
2 hours under nitrogen. The reaction was caoled to 25
C., 50% sodium hydroxide was added to pH 12 and the
mixture was refluxed for 1.5 hours and then cooled. The
mixture was ad~usted to pH 2 using 6N h~drochl~ric acid
(caution: hydrogen azide). The mixture was filtered
through Super cel and the cake was washed with ethyl
acetate. The washings were added to the ~iltrate and
the phases were separated. The aqueous fraction was ex-
tracted inta 4 x 100 ml. of ethyl acetate and the org~nic
~ractions were combined. The organic phase was washed
with water, dried over anh~drous magnesium sulfate and
reduced ~n volume at 35 C. (15 mm.) to an oil which was
allowed to stand in an ice bath. The product was collected
nd dried in vacuo over P20 at 25 C. Yield: 22 g,
o~f-white soIid. The nmr was consistent ~or structure.
7-~mi~ (1-carboxypropyltetrazol~l-5-thiometh~ll -3-
cephem-4-c_rbo~lic Acid.
To a nitrogen purged solution o* 1.5 g. (1.4 x
10 2 mole) of sodium bisulfite in 350 mlO of pH 6.4 phos-
phate bu~fer was added 22 g. (8.1 x 10 2 mole) of 7-amino-
cephalosporanic acid, 16085 g, (9.0 x 10 mole) o~
l-carbox~propyl-5-mercaptotetrazole and enough sodium
~34-
;. . .. ... , ..... . : ~- . -
- . . . . .
~15Z~ 7
bicarbonate to form a clear solution~ The reaction was
warmed for ~.5 hours at 56 C0 under a brlsk nitrogen
flow, then cooled to 10 C~ The pH was ad~usted to 2.5
using 6N hydrochloric acid and the mixture was stirred
in an ice bath to aid precipitation. The product was.
collected and then washed with cold water, methanol and
acetone.
The product was recrystallized from ~ethanol-
hgdrochloric acid and dried in acuo over P~05 at 25 C.
Yield: 6.7 g. The ir and nmr were consistent ~or the
structure.
~ 7
2-Furoylcy--a-nide
To a suspension of 26.1 g. (0.4 mole) of ground
potassium cyanide in ~00 ml. of acetonitrile at 5 C0 was
added 26.1 g. (O.Z mole) of a-furoyl chlorlde while keeping
the temperature below 8 C. The mix-ture was stirred in
the cold for 15 minutes then heated at re~lux ~or 30
minutes~ The reaction was cooled, ~i:Ltered and the aceto~
nitrile was removed at 15 mm. (steam-bath) leaving 24.5 g.
of a dark oll which was used without further purification.
An infrared spectrum showed a nitrile band at 2265 cm 1,
2-Furaneglyoxylic Acid
The 24.5 g. o~ crude 2-furoylcyanide was mixed
with 160 ml, concentrated hydrochloric acid at 25 C. with
intermittent Rtirring. The reaction was stor~d ~or 24
hour~ at 25 C. and diluted with 80 ml. o~ water. The
reaction was stlrred for 5 minutes and filtered. m e *il-
trate was saturated with sodium chloride and extracted with
5 x 120 ml. o~ 1:1 ether-ethyl acetate solution. The extracts
were combined, dried over anhydrous magnesium sulfate and
evaporated at 30 C. (15 mm.) to gi~e a brownish-orange
solid. The solid was dissolved in methanol, treated with
charcoal and evaporatea under reduced pressure (15 mm.) t9
dryness to ~ield 17 g. of the acid.
The product was recrystallized from toluene to give
11.5 g. (m,p. 76 C.). The lr and nmr spectra were consis-
~ent ~or the structure.
2-Methoxyimino-2-~urylacetic Acid
To a solutlon o~ 4.5 g. (0~032 mole) o~ 2-furane-
gl~oxylic acid in 40 ml, of 50~ alcohol and ~.1 g. (0.037
-36-
: , ~ . . . . .. .
mo~e) of metho~yamine h~drochloride in 6 ml. water at 20
C. was added dilute sodium hydroxide solution to pH 4-5.
The solution was stirred at pH 4-5 at 25 C. for 24 hours.
m e alcohol was rem~ved under reduced pressure (15 mm.) and
the solution was ad~usted to pH 7-8 with 50% sodium
hydroxide solution. The reaction was extracted with 3 x
50 ml. o~ ether and the aqueous layer was ad~usted to pH
1.9 using concentrated hydrochloric acid. The mixture was
extracted with 5 x 50 ml. of ethyl acetate. The organic
fractions were combined, washed with brine, dried over
anhydrous magnesium sul~ate and evaporated under reduced
pressure (15 mm.) to an oil ~hich was cooled ~or one hour
in an ice bath. The product was slurried with Skellysolve
B and collected to yield 3.1 g. of yellow crystals, m.p.
78 C. An analytical sample was recrystallized from
toluene, dried ~or 16 hours in vacuo over P205 at 25 C.
me 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; N3 8.~7.
7-Amino~ carbox~ethyltetrazol-5-ylthio ethyl)-3
cephem-4-carboxylic Acid
To a mixture of 5 g. (0.0286 mole) of 5-mercapto~
tetrazol~l-propionic acld and 7.8 g. (0.0286 mole) o~
7-aminocephalosporanic acid in 150 ml. of .lM phosphate
buf~er (pH 6.4) was added with stirring solid sodium
bicarbonate until the solution became clear (pH 7.5).
The solution was heated at 55 C. under nitrogen ~or
4 hours and acidified with 1:1 ~ P04. The precipitate
was collected, washed with water and final~y with a small
~olume o~ methanol. The solid was then slurried with 150
-37-
.
.
6~
ml. o~ methanol and concentrated hydrochloric acid added
dropwise untll the-mixture became clear. The solution
was treated with carbon, filtered and neutralized with
concentrated ammoni~un hydroxide to pH 4.5. The solid
was collected, washed with methanol to weigh 5.2 g. m.p.
>130 C. deco~p. The ir and nmr were consistent for the
structure.
2-Ethoxyiminofurylacetic Acid
,, , , ~ . . .. . .. .
The 7.85 g. (o.o56 mole) of furyl-2-glyoxylic
acid was dissolved in 100 ml. of water and adjusted to
pH 7 with 50% sodium h~droxide. The 6.83 g. (0.070 mole)
o~ ethoxyamine hydrochloride in 10 ml. o~ water was added,
while keeping the pH at 4-5. The reaction was diluted
with 25 ml. of alcohol, ~tirred 3 hours at room tempera~
ture and then filtered. The alcohol was removed at ~5
C. (15 mm ) and the a~ueous portion was adjusted with
dilute sodium hydroxide solution to pH 7-8 and then
was washed with ether and the washes were discarded. The
aqueous ~raction was ad~usted with 6N hydrochloric acid
to pH 1.5 and extracted into ~ x 80 ml. of ethyl acetate.
m e acetate fractions were combined, washed with brine
and reduced in volume at ~5 C. (15:mm.) to an oil. m e
oil was cooled in an ice bathg tr~turated wlth Skell~solve
B, collected ~nd dried o~er P2~5 in vacuo at 25 C-
Yield: 4.8 g., m.p. 83-85 C. The ir and nmr were con-
sistent for the structure.
Anal Calc'd for C8HgN04: C, 52.46; H, 4.95;
N, 7.65. Founa: C~ 52.22; H, 4.94; ~ 7.60.
-38-
,
5~7
Sodium_a-Ethoxyimino-a-(2-furyl)acetate
To 50 ml. o~ methanol wa~ added 250 mg. (0.0109
mole) o~ metallic sodium and stirred until all the sodium
had dissolved. This sodium methoxide solution was treated
with 2.0 g. (0.0109 mole) of a-ethoxyimino-a-(2-furyl)acetic
acid dissolved in 10 ml. of methanol and stirred at room
temperature for one hour. The methanol was removed at
40 C. (15 mm.) and the product was dried in vacuo over-
P205 at 25 C.to yield 2,22 g. white solid, m.p. decomp.
~240 C. m e ir and nmr were consistent for the ~tructure.
"Skellysolve B" is a petroleum ether fraction
o~ b.p. 60-68 C. consisting essentially of n-hexane.
-39
,,
,' . : ' ' .
5~
Descri~tion of the Preferred Embodiments
E2~AMPLE_ 1
7- ~2-Methoxyimino-2-furylacetamido)-3 (1 C~rOo~ et _ -
tetrazol~l-5-thiomethyl)-3-cephem-4-carbox~lic Acid
. ~
~L-S1095)
A suspension o~ 750 mg. (0 0039 mole) o~ sodium
2-methoxyimino~uryl-acetate in 25 ml. of benzene and 2
drops of d~ethylformamide was stirred vlgorously while
0.35 ml. ~0.0039 mole) of 02alyl chloride was adde-d dropwise~
The suspension was stirred ~or 45 minutes and the salt~ which
formed were recovered b~ filtration. The benzene was removed at
40 (15 mm.) and the light yellow oil was disqolved in 20 ml.
o~ acetone and added to a solution of 1.6 g. (0,0039 mole)
of 7-amino-3-(1-carboxyethyltetra~ol~5-ylthiomethy1~-3-
cephem-4-carboxyllc acid in 25 ml of water and 500 mg. of
sodium bicarbonate at 5 C. The solution was stirred ~or 1/2 hour
at 5 C. and the acetone was evaporated under reduced
pressure at 40 C. (15 mmO) diluted with 25 ml. of water
and acidified with 1:1 phosphoric acid. The mixture was
extracted with 3 x 50 ml. o~ ethyl ace~ate and the organic
layer was separated, washed with water and evaporated to a
gummy residuer After the solid was slurried with ether,
the free acid was dissolved in acetone and treated with
750 mg. o~ potassium~2-ethylhe~anoate. The pota~sium salt
was collec~ed, washed with acetone and dried over ~25 to
glve 800 mg. m p. ~1~0 C. slow decomp.
Anal Calc'd ~or ClgH17K2N707S2 1-1/2 H20 C~ 37-21;
E, 3.52; N, 15 90. Found: C, 37.17, H~ 3.31; N~ 13.89.
nmr (D20 ppm S) 7 75, d, 1~ -CH-00; 6.~5~ d; 1, =C~I-CH=;
606-6.8 m., l=CH-CH=; 5.8~, d, 1~ N-C~-; 5.25 ZL~ CE-S;
~40-
Z~7
4 .6, T, 2, N C~-CH2-C02H~ 4.0-4 .6 M2~C-CH2-SO3 4.0, S~ 3,
N-OC ~ ; 3.3-4.0 m 2 S-CH2-C=, 2.85, T, 2, CH2~C02H.
ir ( B r cm 1) 3100-3600, OH, NH, 1765 ~-lact~m carbonyl;
1675 NHC ~ 1600 C02-.
EXAMPLE 2
7-(2-Methoxyimino-?-furylacetamido)-3-(1-carboxymeth~l-
tetrazolyl-5-thiomethyl)-3-ce~hem-4-carbox li cid (BL-S1080)
To 25 ml. o~ methanol was added 100 mg. (0.00425
mole) of metallic sodium and the mixture was stirred unti7
all of the sodium had dissolved. The sodium methoxide
solution was cooled to 3 C. and 0.179 g. (0.00425 mole~
of methoxyiminofurylacetic acid in 5 ml. methanol was added.
The solutlon was ætirred for 10 minutes at room temperature
and the solvent was evaporated at 30 C. (15 ~m.) and dried
by azeotropic distillation with 3 x 20 ml. of benzene (15
The sodlum 2-methoxyimlno-2-furyl-acetate was
suspended in 25 ml. of benzene and treated with 4 drop~ of
dry dimethyl~ormamide. A total of 1.1 g. (O. oo8s mole)
o~ oxalyl chlorlde was added and solution was stirred for
40 minutes at 25 C, r~he benzene was remo~ed at 35 r~ C .
(15 mm.) and the acid chloride was dissolved in 10 ml.
acetone. A solution o~ 1.2 g. (0.0032 mole) of 7-amino-
3~ carboxyDIethgltetrazolyl-s-thiomethyl)-3-cephem-4~
carboxylic acid in 20 ml. of water and o.68 g. ~0.0081
mole) of sodium bicarbonate was cooled to 3 C. The
acetone solution o~ the above acid chloride was added ~nd
the reactlon was stirred ~or 40 minutes without the ic~
bath. The acetone was removed at 30 C. (15 mm.) and
the aqueous solution was ad~usted to pH 1.8 using 6N
.
6t~
hydrochloric acid. The product was extracted with 3 x
60 ml. of ethyl acetate. The organic fractions were com-
bined, washed with brine and aæeotroped at 30 C. (15 mm.)
to an amorphous solid. The residue was treated with 50 ml.
of ether and the product was collect~d and dried in vacuo
o~er P205 to yield 750 mg. of a light tan solid, m.p. >120
C decomP. Anal. CalC'd for C18~17N78S2 / ( 2 5)2
H20: C, 42.17; H, 4.o6; N~ 17.20. Found: C, 42.16; H,
.96; N, 16.66. The nmr spectra showed the compound to be
a mixture of 75% syn and 25~ a_ti isomers with diethyl ether
as an impurity or solvate. The syn pattern is listed below:
R
nmr (DMSO ppm S) 9.75 d, 1 C-N~-; 7.75, S~ 1, =CH-O-; q
6.5-6.8 m, 2, =CH-CH-; 5.6-509, m, 1, N-CH- 5.3, 5, 2, N-CH2-C-;
5.15, d, 1, -CH-S; 3.95_4.65 m 2, CH2-S, 3.9, S 3 OCH3;
3.4-4.0 m, 2, -S-CH2-C=.
Separate anti patterns: 9.55 d, 1, -CO-NH-; 7.25
d, 1, =CH-CH=j 4.0, S, ~, OC ~ .
~AMPLE, ~
'~ ?~-(2-Methoxyiminofurylace-tamido)-3-(1-carboxypropyltetra-
zolyl-5-thiomethyl)-3-cephem_4-carbox~lic Acid. (BL-S1081
O
C - C - N~l ~ S 71 1,
OCH~ ~ ~ CH2-S -C~N~N
C02H (CH2)3
C02H
To 25 ml. of methanol was added 96 mg. (4.16 x
10 3 mole) o~ metal~ic sodium which was stirred until all
of the sodium had dissolved. This sodium methoxide solu-
tion was cooled to 7 C. and 70~ mg. (4.16 x 10 3 mole) of
-~2-
: .
:L$~ `7
2-methoxyiminofurylacetic acid in 5 ml of methanol was
added. The reaction was stirred for 10 minutes at room
temperature, the methanol removed at 35 C. (15 mm.) and
the sample was azeotroped with 3 x 30 ml. benzene at 35
C. (15 mm.).
To a stirred suspension of the above sodium
2-methoxyiminofurylacetate in 25 ml. o~ benzene with 3
drops of dimethyl~ormamide was added 1.06 g. (8.3 x
10 3 mole) oxalyl chloride and stlrred 1 hour at room
temperature. The benzene was removed at 35 C. (15 mm.)
and the acid chloride was dissolved in 10 ml. o~ acetone.
The acid chloride solution was added to a solu-
tion of 1.28 g. (3.2 x 10 3 mole) of 7-amino-3~ carboxy-
propyl-tetrazolyl-5-thiomethyl)-3-cephem-4-carboxylic acid
and 0.672 g. (8 x 10 3 mole) sodium bicarbonate in 25 ml.
o~ water at 3 C. The solution was stirred 40 minutes
with the ice bath removed. The reaction was filtered,
the acetone removed at 30 C. (15 mm.) and the aqueous
~ra~tion was ad~usted to pH 1.8 with 6N ~Cl. The sample
was layered with ethyl acetate and then filtered through
Super~cel. The phases were separated ancl the aqueous
phase wa~ ~urther extracted with 3 x ~0 ml. of ethyl
acetateO The organic ~ractions were combined, treated
with charcQal, filtered through Super-cel~ then azeotroped
at 30 C , (15 mm.) to an amorphous solid. This residue
was triturated with excess ether and the product collected,
dried over P205 at 25 C~ yielding 370 mg. of an off-
white solid. M.p. dec. ~83 C. The ir and nmr were con-
sistent ~or the structure.
-43-
.
~ `7
EXAMPLE 4
7-L2-Ethoxyimino-2-(fur-2-yl~acetamido]-3~(1~carboxymethy
tetrazolyl-5-thiomethyl)-3~cephem-4-carboxylic Acid Syn-
isomer. (BL-S1105)
o
C - C - NH ~ ~ Nl IN
N-OC2H5 ~ N ~ CH2-S -C~N~N
G02H IH2
C02H
To a stirred suspension of l.l g. (5.~ x lO 3
mole) of sodium a-ethoxyimino-a-(2-furyl)acetate in 40 ml~
o~ benzene with 6 drops of dimethylformamide was added
706 mg. (5.57 x 10 3 mole) o~ oxalyl chloride and stirred
~ ~or one hour at room temperature. The benzene was removed
i, at 40 C. (15 mm.) and the acid chloride was dissolved in
5 ml. o~ acetone.
The acid chloride solution was added to a solu-
tion of 1.89 g. (5.o8 x 10-3 mole) of 7-amino-3-(1~
carboxymethyltetrazolyl-5-thiomethyl)-3-cephem-4-carboxylic
acid and 1.28 g. (1.52 x lO 2 mole) of sodium carbonate in
40 ml. of water at ~ C. The reaction was stirred ~or 40
mlnutes~ filtered and the acetone removed at reduced pressure
at ~5 C. (15 mm.). The aqueous solution was layered
with ethyl acetate~ ad~usted to pH 1.9 with 40% phosphoric
acid and ~iltered. The phases were separated and the
aqueous portion was extracted with 2 x 60 ml. o~ ethyl
acetate. The ethyl acetate ~ractions were combined5 washed
with brlne, dried over anhydrous magnesium sul~ate and
reduced in volume to an oil at 35 C. (15 mm.)~ The oil
was layered with excess ether and allowed to stand at
~44-
room tem~erature for 16 hours. The product was collected
and dried in vacuo over P205 at 25 C. to ~ield 800 mg. tan
solid. M.p. dec. >75 C.
Anal. Calc'd for ClgHlgM708S2 1/2(C2H5)2
43.90; H, 4.29; N, 17.06. Found: C, 4~.90; H, 4.24;
N, 16.21.
The ir and nmr were consistent ~or the structure
of a hemi diethyl etherate.
EXAMPLE 5
Substitution o~ an equimolar weight of sodium
2-ethoxy~mino-2-(fur-2-yl)acetate for the sodium
2-methoxyimino~uryl acetate used in the procedures o~
Examples 1 and 2 produces 7-(2 ethoxyimino-2-furylacet-
amido)-3-(1-carboxyethyltetrazol-5-ylthiomethyl)-3-
cephem-4-carboxylic acid and 7-(2-ethoxyimino-2-furyl-
acetamido)-3-(1-carboxymethyltetrazol-5-ylthiomethyl)-3-
cephem-4-carboxylic acid, respectively.
EXAMPLE 6
Substitution of an equimolar weight o~ sodium
2-n-propoxyimino-2-(fur-2-yl)acetate for the sodium
2-methoxyiminofuryl acetate used in the procedures of
Examples 1 and 2 produces 7-(2-n-propoxyimino-2-furylacet-
amldo)-3~ carboxyethyltetraæol-5-ylthiomethyl)-~-
cephem-4-carboxylic acid and 7-(2-n-propoxyimino-2-fur~l-
acPtamido)-3~ carboxymethyltetrazol-5-ylthiomethyl)-3-
cephem-4-carboxylic acid, respectively.
-45-
.
.
EXAMPLE 7
Substitution of an equimolar weight of sodium
2-n-butoxyimino-2-(~ur-2-yl)acetate for the sodium
2-methoxyiminofuryl acetate used in the procedures of
Examples 1 and 2 produces 7-(2-n-butoxyimino-2-furylacet-
amido) ~ carboxyethyltetrazol-5-ylthiomethyl)-3-
cephem-4-carboxylic acid and 7-(2-n-butoxyimino-2-furyl-
acetamido)-~ carbox~methyltetrazol-5-ylthiomethyl)-~-
cephem-4-carboxylic acid, respecti~ely.
EXAMPLE 8
The products of Examples 1-7 are prepared as syn
lsomers essentially free of the corresponding anti isomers
by the use in the procedures of those examples o~ purified
syn isomers of the appropriate 2-alkoxyimino-2-(fur-2-yl)-
acetic acid. Conversion of part of the ~n isomer to anti
isomer durlng preparation of the acid chloride from the acid
is substantially avoided by minimlzing its exposure to
hydrogen chloride, e.g. by ~irst convertin~ the acid to its
anhydrous sodium salt and by treating that salt with oxalyl
chloride under anhydrous conditions in the presence of a
hydrogen ion acceptor such as dimethylformamide.
-46-
~ ~52~
EXAMPLE 9
~ n in~ectable pharmaceutical composition is
formed by adding sterile water or sterile saline solu-
tion (2 ml.) to 100-500 mgm. o~ di-potassium 7-(2-
methoxyimino-2-furylacetamido)-3-(1 carboxyethyltetra~
zolyl-5-thiomethyl)-3-cephem-4-carboxylate.
Pharmaceutical compositions of the sodium
and potassium salts of the other compounds o~ the
present invention, preferably in the form o~ the pure
isomer, are formulated in a similar manner.
When the compounds are first prepared in
the ~orm of the ~ree acid they are converted to the
desired, highly water soluble potassi~n salt by treat-
ment with potassium 2-ethylhexanoate using the procedure
o~ Example 1.
It is occasionally advantageous to have ad-
mixed with said solid cephalosporin as a stabllizing
and/or solubilizing agent a sterile, anhydrou~q solld
such as sodium carbonate, potassium car~onate or lithium
carbonate(e.g. in about 5 or 6 percent b~ weight o~ the
weight of the cephalosporin) or such as ~-lysine, argi-
nine or histidine (e.g. in about 20-50~ b~ weight o~
the weight o~ the cephalosporin) or such as a sodium,
pota sium or calcium salt o~ levulinic acid, citric acid,
ascorbic acid, tartaric acid or pyruvic acid (e.g. in
about 25-200% by weight of the weight o~ the cephalos~
porin) or such as sodium bicarbonate~ ammonium car-
bamate alkali metal or ~mmonium phosphates or N-methyl-
glucamine (PeF o-K- 1,300,741).
_1~7_
.
.
. : . : :
.
EXAMPLE 1~
7-(2-Methoxyimino-2-furylacetamido)-3~ carbox~methyl
etrazolyl-5-th~ometh~ 3-ce~em-4-carbo~
(BL-S1080)
To ~.0 g. (0.0156 mole) of' sodium 2-methoxy-
imino-2-furyl acetate suspended in 60 ml. of ben2ene
was added 6 drops of dimethyl~ormamide and 1,98 g,
(0.0156 mole) of oxalyl chloride. The resulting solu-
tion was stirred at 25 C. ~or 45 minutes. The benzene
was removed at 40 C. (15 mm.) and the acid chloride was
dissolved in 4 ml. of acetone. A solution o~ 5.8 g.
(0.0156 mole) of 7-amino-3-(1-carboxymethyltetrazolyl-
5-thiomethyl)-~-cephem-4-carboxylic acid and 2.5 g.
(0.03 mole) of sodium bicarbonate in 80 ml. of water was
cooled to 3 C. and the acid chloride solution was added.
The reaction mixture was stirred ~or 20 minutes at 3 C,
and 20 minutes wlth ice-bath removed. A pH o~ 7 was
maintained while stirring. The mixture was ~iltered and
the acetone removed at 35 C. (15 mm.). The aqueou~ por-
tion was lagered with ethyl acetate, adjusted to pH 1.8
with 6N hydrochloric acid and extracted 2 x 80 ml. with
ethyl acetate. m e organic ~ractions were combined,
washed 2 x 40 ml. with brine, dried over anhydrous mag-
ne~ium sulfate and evaporated at 35 C, (15 mm.) to an
amorphous solld whlch was triturated with ether. The
free acid was collected and air dried to yleld 4.5 g.
~he acid was dissolved in acetone and treated with 1.2 g.
(o.oo65 mole) of potassium 2-eth~lhexanoate dissolved in
acetone. The product was collected and dried in vacuo
over P205 at 25 C. to yield 2.49 g. of the potassium
~alt. M.p. >130 C. slow decomp. The ~mr spec-tra
-~,8-
52~
indicated the compound to be a mixture of 95~ s~n and
5~ anti isomers.
Analysis Calc'd for C18H16KN70gS2: C, ~8.49,
~, 2.87; K~ 6.96; N, 17.45. Found: C, ~8.o6; H, 2.68;
K~ 6.65; N, 17.34.
EXAMPLE 11
Preparation of Sterile, Lyophilized Parenteral-grade
BL-S1080: L-~ysine Salt (Lab~l Claim is 250 Mg. of
BL-S1080 Activity~Ml.)
FORMULA
BL-S1080 free-acid (potency=1000~1050 mcg./mg.~ *1 0.25 Gram
Parenteral-grade L-Lysine *2 0.0875 Gram
Water for Injection, U.S.P. qs to 1 ml.
*1 Label claim is 0.25~gram BL-S1080 activity as the
free-acid. The amount of BL-S1080 free-acid
required is calculated as follows:
0.25 gm. x 1000 _ _ Weight in grams of
Potency of BL-SI~O free~acid ~ln mcg.7~. ~ BL-SlQ80 free-acid.
This wei~ht may also be increased by adding
increments based on the following factors:
;
1) O~erbatch required for shelf life (stability),
2) Overfill required for vial, syringe and
needle holdup,
~ ) Machine fill variability.
*2: Th~s represents ~5~ of the weight of the BL-S1080
free-acid, assumlng a potency of 1000 mcg~/mg.
-49-
.
~ . . . ..
~ 7
Manu~acturing Instructions
1. Slurry lO0 grams o~ pyrogen-free BL-S1080
free-acid (1000 mcg./mg.) in 250 ml. of Water for In~ec
tion, U.S.P. at 20-24 C.
2. Add with rapid stirring over a 5-minute
inter~al 35 grams of pyrogen-free L-Lysine. A p~ 7.4-8.0
solution or near solution is obtained. Add Water for
In~ection, U.S.P. to a final volume of 400 ml. (a lO
gram "Darco KB"-0.5 hour slurry o~ this step is optimal).
3. Pass the solution through suitable ~ilters
to remove particles and bacteria.
4. Using sterile technique, ~ill the requlred
volume of sterile, pyrogen-free solution o~ step 3 into
steril~zed glass vials.
Steps 2 to 4 inclusive should be completed within
3 hours. Freeze immediately.
Under sterile condi-tions lyophlli~e ~or 48
hours. Maintaining vacuum contlnue at 50 C. for 24
hours. Cool to 22-25 C. and then release vacuum to
atmospheric pressure with sterile nitrogen.
6. Aseptically stopper under nitrogen and cap.
`:
:, -
,
-50-
~~PLE 12
Preparation of BL-S10_
C1 acetonitrile ~0. g~CN
_ ....
H HCl
; E;3 CH30NH2 HCl
0 ~ 0
. ~ ~ 3 ~D~H3 "~
Na C~I3OH
.
q (COCl)2 Irll 8
~Na.
~3 Glf3
/
:Ha~S~ /
02H H;~CO2~F/
~N~ C~12-S~p~l
bCH3 ~2~ a2~
P~H~ BL-S 1 o80 ,~
Na~ ~L-~ 1080 ,
-51
ii2~
2-Furoylcy~nide 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.) of a-furoyl chloride with vigorous
stirring while keeping the temperature at 4-8 C. The
mixture was stirred at 4-8 C. for 15 minutes and then
heated at reflux for 30 minutes. The ~ixture was cooled
to 23-25 C., filtered, washed with 50 ml. of acetonitrile
which was added to the filtrate, and the acetonitrile was
removed at 60 C. (15 mm.) leaving 51 g. o~ ~ as ~ dark
oil. An IR spectrum showed a nitrile band at 2265 cm 1
and an NMR spectrum showed a ratio o~ approximately
70/30 o~ product ~ furoic acid. The crude product ~ was
used without ~urther puri~ication (49~ yield of product).
Furyl-2-glyoxy~ic Acid ~
The 51 g. of crude 2-furoyl cyanide 1 was
mixed with 500 ml. concentrated hydrochloric acid at
25 C. The reaction was stirred ~or 2~ hours at 25 C.
and then diluted with 240 ml. of water. The mixture was
stirred for 5 minutes and filtered. The black ~iltrate
was saturated with sodium chloride and extracted with
6 x 500 ml. o~ 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
addit'onal ether-ethyl acetate extractions were run the
task was simplified.) The extracts were combined and
evaporated to dryness at 60 C. (15 mm.). The resultant
solid was dissolved in 600 ml~ ether, (Note: Use of
alcohol should be avoided at this point as esters may
form), treated with 10 g. of charcoal ('iDar~o-g~
.. . ~.
; -52-
, .......... . . . ...
filtered after stirring for 0.5 hour and evaporated
to dryness at 50 C. (15 mm.) to yield 46.6 g. o~ 2
as a light tan colored acid. This product 2 was
found to contain a ratio of approximately 56/44 of
product 2/furoic acid. This represented a 63~ yield
of product 2.
Purification was accomplished by dissolving
the above crude product 2 in ~2 (5 mg./ml.), titrating
to pH 2.8 with HCl and extracting with 2 x 200 ml. of
ethyl acetate. Evaporation of the ethyl acetate extracts
gave 35% furoic acid and 15% product 2. The pH 2.8
aqueous phase was adjusted to pH o.8 (HCl) and extracted
with 2 x 200 ml. ethyl acetate. The organic extracts
were comblned and washed with 50 ml. H20. The organic
phase was e~aporated at 50 C. (15 mm,) yielding a
solid with a ratio of approximately 86/14 o~ product
2/furoic acid. This solid was then recrystallized by
dis~olving the product 2 in 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 puri~ica-
tion and recrystallization step and an overall yleld from
the 2-furoyl chloride to the pure furyl-2-glyoxylic
acid 2 o~ 16~.
~ 3
. ~ ~ .
A solution of ~.5 g. o~ furyl-2-glyoxylic acid
in 40 ml. of 50~ ethanol was titrated to pH 6 with lN
sadium hydroxide and then ~.l g. of methoxyamine~HCl in
6 ml. of H20 at 20 C. was added, me solution was
titrated to a constant pH 4O9 and stirred at pH 409 for
-53~
.
: . . , : . '
. .
.
6~
24 hours at 20-23 C. The ethanol was then removed at
50 C. (15 mm.~ and the residual aqueous solution was
titrated to pH 8 with 50~ sodium hydroxide and washed
with 3 x 50 ml. ether (pH ad~usted to 8 after each
wash). The aqueous layer was titrated to pH 1.9 with
concentrated HCl and extracted with 5 x 50 ml. ethyl
acetate with the pH readjusted to 1.9 after each extrac-
tion. The ethyl acetate extracts were combined and
evaporated to a solid ~ at 50 C. (15 mm.). This solid
was then slurried with 75 ml. of "Skelly601ve B". The
suspension was filtered and the solids were redissolved
in 16 ml. of toluene at 80 C. The hot solution was
decanted and left to crystallize at 20-23 C. for 18
hours to yield 1.17 g. ~ (22~ yield of product). The
NMR was clean and consistent for the structure ~ with a
trace o~ anti 1somer present.
~ .
Sodium Syn-a-methoxyiminofurylacetate
,~,,
To 40 ml. o~ methanol was added 0.16 g. o~
sodium. The mixture was stirred until all of the sodium
dlssolved and then decanted. The resulting sodium
methox~de 801ut~0n was cooled to ~ C. and 1.12 g. o~
s~-a-methoxyiminofurylacetic acid ~ in 7.8 ml. of
methanol was added. The solution was stirred ~or 10
mlnutes at room temperature. The solvent was e~aporated
at 40 C. (15 mm.). The residue ~ was dried by azeo-
tropic distillation with ~ x 20 ml. of benzene at 40 C,
(1~ mm~). m e product 4 was dried for 18 hours at 2~
C~ und r high vacuum (o.T mm.) over P205 y~elding 1.25 g.
(99~ yield of product). The NMR showed this product
-5~-
..
~5267
to be clean and consistent for the structure with 0.15
mole methanol and a trace of anti isomer,
7-(Syn-a methoxyiminofurylacetamido)-3~ carboxymethyl-
tetrazol~5-ylthiomethyl~3-ce~hem-4-carboxylic Acid 5
To o,6~ g, of sodium syn-a-methoxyiminofuryl-
acetate ~ suspended in 25 ml, of benzene was added four
drops o~ dry dimethylformamide and 0,31 ml. (l,l eq.)
of oxalyl chlorlde, This mlxture was stirred for 40
minutes at 20-2~ C, The benzene was removed at 35 C.
(15 mm,) and the acld chloride (the gummy residue) was
dissolved in 10 ml. acetone (NaCl insolubles were present
but not removed). A solution of o.g8 g. 7-amlno-3-(l-
carboxymethyltetrazol-5-ylthiomethyl)~~-cephem-4-
carboxylic acid in 20 ml, H20 and 0,55 g. of sodium
bicarbonate ~pH 6.4) was cooled to 3 C, The acetone
solution o~ the above acid chloride was added and the
reaction was stirred for 40 minutes at ambient tempera-
ture (pH 3-4 3 -
The acetone was removed at 30 C. (15 mm,)and the remaining aqueous solution was adjusted to pH
1.8 with 6N HCl. The product ~ was extracted with
3 x 60 ml. ethyl acetate. The combined solvent extracts
were backwashed with 50 ml. H20 and then evaporated to
dryne~s (wlth periodic additlons of fresh dry ethyl
ace'cate until the water present had been azeotropicall~
removed) at ~0 C. (15 mm,) to leave an amorphous solid.
m is solid residue was triturated with 50 ml. of ether
and collected by filtration yieldlng 1,02 g, ~ ~64%
yield o~ product), The NMR was consistent for BL-Sl080
with the following con-taminations: about lO~ anti isomer9
~5~2~7
about 10~ furoic acid, trace dimethyl~ormamide and
ether.
Disodium Salt of BL-S1080 2.5 H20 ~
1. To 0.97 g. of 5 (BL-S1080) suspended in
10 ml H20 was added lN NaOH until a constant pH of 7
was obtained and all o~ the BL-S1080 had gone into solu-
tion. This solution was then lyophilized ~or 18 hours
yielding O.93 g. Na2 salt of BL-S1080. The NMR of this
product was consistent for BL-S1080 with about 10% anti-
isomer, about 10% furoic acid and a trace of dimethyl-
~ormamide. A bioautograph showed ~ust one round spot.
2. Alternate Method for Making Na Salt of
BL-S1080 6
Compound ~ (BL-S1080) (0.5 g.) was dissolved
in 5 ml. ethanol, filtered, 5 ml. H20 added and titrated
to pH 7 with lN NaOH. The ethanol was e~aporated at 50
C. (15 mm ) and the remaining solution was l~ophilized
*or 13 hours yielding 0.44 g. Na2 salt o~ BL-S1080. The
IR and NMR were consistent for BL-S1080 with about 10
anti-isomer and about 10~ furoic acid.
Anal. Calc~d ~or C18H17N708S2~a2
~, 3.01; N, 17.28. Found (corrected ~or water content):
C, 38.28; H, 3.29; N, 16.~1
Anal. Calc'd for C18H17N708S2Na2 205 2
(H20), 7.3~. Found: KF (H20), 8.o6
-56-
. :
:;
Laboratory Evaluatio~
Bacteria. The organisms, preponderantly oi
recent clinical or{gin, were obtained ~rom numerous ~ources
o~ broad geographical distribution. Obligate anaerobes
were maintained in Egg Meat Medium (Di~co); cobacterium
was stored on Lowenstein Medium {Jensen Modificatlon;
Di~co). The techniques of storlng all other organismæ
have been described previously (Leitner et al., BL-S6403
A Cephaloæporln with a Broad Spectrum of Antibacterial
Activity: Properties in vitro, Antimlcrob. Agents Chemo-
ther. 7:298-305 (1975~.
Antibiotic spectrum. The growth-inhibitory ac-
.
tivity of . the . compounds was determined
by ths antibiotic dilution technique. Procedures were as
. ~ollows:
. ~ excluding M~cobaeterium~.
Except ~or Haemophilus and Neisserla, the assay was per-
.
~ormed in Mueller-Hlnton Medium (Di~co3, For ~astidlous
organlsms, i.e., Streptococcus~ Listeria, Pasteurella,
Bordetella and Vibrio, the medium was supplemented with
4% defi~rinated sheep blood. The antibiotlc susceptibili~y
o~ _ e ~ and Neieeeria was determlned in GC Medium
Bas~ (BBL) supplemented with 1% Hemoglobin (BBL? and 1% :
Isovitalex (BBL).
O~e mi~ht broth cultures o~ an exponentially
growlng:culture (Neisseria) served as the source of
inoculumO A volume of approximately O.003 ml~ of th~
-57-
undiluted or diluted culture was app:Lied to the sur~ace
o~ the antibiotlc-containing agar plates with the
inoculatar o~ Steers et al., An Inocula Replicating
Apparatus for Routine Testing of Bac1;erial Susceptibi-
lity to Antibiotics, Antibiot. Chemother. 2:~07-311
(1959). Cultures of Neisseria, Streptococcus pneumoni~e,
S. viridans and S. pyo~enes were usecl without dllution;
.
those of all other organisms were diluted 100-~old. m e
~noculum contained about 103 vlable cells ~or Neiiierli,
105 for S. pneumonlae and S. ~ , 106 for S. virldans,
and 104 for all other species. The culture plate~ were ln-
cubated at ~7 C. elther overnight or for 24 hours
(Haemo~hilus) and the minimum inhibitory concentratio~
(MIC), i.e., the lowe~t concentration o~ antibiotic which
prevents visible ~rowth~ was recorded,
~ n in Blood. Male Swiss-
Webster mice~ weighing 19-22 g., were given 0.2 ml. of
ant~biotlc ~olutions at appropriate concentrations by
intramuscular inJection. The vehicle was 0.01% phos-
phate bu~er at pH 7Ø Elght animals were used ~or
each dose level. Blood
~amples (0.03 ml.) were obtained ~'rom the orbital sinuses
by means o~ heparinized capillar~ tubes~(Clay Adams~ at
0.25, 0.5, 1 and 1.5 hours after adminIstration of the
compound. Paper disc~ 6~35 mm. in diameter~ were im-
pregnated with the blood and the antib~otic activity
~ : :
.~ '
-58-
- . ,
: ; -
... . , : .
assa~ed by the di~fusion technique using Seed Agar (BBL)
inoculated with Bacillus subtilis ATCC 66~3. A standard
_
llne relating the dlameter of the i~libition zone tQ drug
concentration was obtained by assayirlg the compounds at
known concentrations in heparinized mouse blood.
~ . The
procedures were identical with those published pre-
viou31y ~Leitner et al., BL-S640, a Cephalo~porin With
a B~oad Spectrum of Antibacterial Activit~: Bioa~ail-
ab$1ity and Therapeutic Properties in Rodents, Anti-
mlcrob. Agents Chemother. 7:306~~}0, (1975)], except that:
~) the hog gastric mucin used ln infections wlth
5~s~ 2e_l9~ aureus No. 2 was purchased from American
Laboratories~ Inc., Omaha~ Neb. (lot ~o. 15416~) Rnd b)
that the medium used to suspend all other organisms con-
tained ~% (rather than 4%) hog gastric mucin (type 1701W,
Wilson Laboratories~ Inc.~ Park Forest South~ Ill.).
-59-
_
o~
o o ,, .,
X o CU ~ CU o oo ~ ,~ C~ o ~ U~
~o CU C~
~ .
a)
V
_ . . . _ ._ __ _
,~ oo r~
oU~ O O CO j~ 00 0 CU O 00 ::~ N CU O i~ 15~
bD 1:l ~ 1~ ~~D C`J CU rl CU
E3 ~ f~
~ _ __ _ _ . _.
O
~. O 0~
~_
~n o ~ c~O 00 ~ ~ C~J o L~
h I
r I A ~I
o . L~ ~ O O ~ ~ C~
L~ ~ ~I LO L~
; L~ ~ > O ~ ~~ ~ O ~O U~ ~O
p.,~ ~ ~ ~ ~ a~ ~ Ln ~ o ~ ~ ~
'U~ h c~ r~ 1 rl rl N h
m
.
~ . . ~
o ~ _ _ _ _ . C~
.
* .
*
IIIIIIIIIII~I~I
ooooooooooooooooooo
l ~ ~ ~ s~ ~
* a~ ; tn O
~Q ~ ~ I ~ ~a
~ c,3 * ~ ~ U~ q~
.' ~ rl ~ ~ ~ ~1 ~ +~ ~ a) a) tq ~q ~ O
h S~ O ~' X a~ rl rl O c) a) a) O +
o o ~
E~ a~ * h ~ V C~ m O
~ ~ o o ,~ ti~ '3 ¢
a) ~ O O O ct;
rl rl ~ ~ h S- h ~ 1
~ o o ~ ~-rl
h h ~i ~ ~ ~h .la .~ ~~ ~1
h ~ 5 S~ *
u~ l * *
. , .
i
~60-
Mouse Blood Levels After I.M. Administration**
__ __ ___~_
Blooa Level ~mcgO/~L~ )
Dose 0.25 0.5 1 1.5
ComPound fm~./k~.) Hr. A~ter Ac~ministration
~ ~ __ __
BL-S1080 40 46.3 3~4 15 <8.9
21.1 16,4 <8.1 <8.1
______ ____ ___________ ____ __ __ ____ __ _ ___ __ __ _____
BL-51095 40 45.8 45~1 27 3 .<_5_9
BL-S1081 40 35 20.7 8.4 ~l~,g
17.4 10.7 <~.7 ~-7
_ _ _ _ _ _ _ _ _ _ _ ~ _ _ _ _ _ _ _ __ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _
Cefuroxime 40 34-3 22,8 7.1 <1.8
16.7 10.4 2.4 <1.8
8.6 5.9 ~1.8 <1.~
__ _____________ ____ _______________ _______ _____
BL-S1105 40 39.3 27.3 ~ , <10.
E~ficacy of Intramuscular Treatment of Mice
Systemically Infected with Various Organisms**
Challenge P~50/treatment (mg/kg)
(No. of BL-S ~~ ~ BL S
1080 oxime ~ 1095 1105
H. influenzae 5 x 10 3.1 13 29
A9729 6 x 106 1.4 ~3 25
_ _ ___ ______ _ _ __ _ ___ __ ___ ___ _ __ ___ __ __ _____ _ ___ _ _ _ __ _ _ _ __ __
E. coli 7 x 105 7.1 3 6.3 7.1
A15119 7 x 105 6.~ 2-3* o,4
P. mlrabilis 3 x 10 3.1 5.4 o.8
A9900 5 x 106 0.6 _ o.7
P. vulgaris 4 x 105 0.2 0.29 1.6
A9436 ___ ________ __ ___ ____. _____
_______________ _____________ ____
S. pyogenes 2 x 10~ 1.6 0.2 1.6
A9604 5 x 102 1.6 _ 1.6
S. pneumoniae 1 x 104 207 Or4 1~6
A9585 4 x 103 2.7 0.9 1.6
S. aureus 8 x 108 19 0~6 ~ 11 8~2
~9606 8 ~ 108 1~3 1.0 _
1 x 109 1~ _ 6 ~ _ _
_ __ _ __
* A~erage result o~ 3 or more previous tests
** N~t all o~ these experiments were conducted simultaneously.
BL~S786 ls 7-(2-aminomethylphenylacetamido)-3-(1-c:a:rboxy-
methyltetrazol-5-ylthiomethyl)-3-cephem-4-carbo~ylic acid.
-61-
- .
Microbiology Comp_un S_recnln~
Com~vund BL-S1081 YS. Haemophilus Influenzae
Secondary in vitro Screening Results - MIC ~mcg./m
Orga ni sm
llumb e r ~i~iai 1;~1
A9729 .5 1 .5
~9832 .5 1 .1
A20173 .5 1 .5
~20177 .5 .5 ~5
A20178 .13 .5 ,1~
~20181 .25 1 ,1~ .
A20188 .25 1 . .13
A20189 .25 1 .13, .
AaQ192 .13 .5 .0~ .
A20193 .25 1 ~5
A21515 .25 .5 .1
~21517 .25 .5 .13
A21519 .25 .5 .13
A21520 .25 .5 .063
A21522 .032 '5 .13
A21523 .25 .5 .13
___ ~
~crobiology Compound Screen~ng
In~luenzae
:
Secondary in vitro Screening Results - MIC (mcg./ml.)
Organism
I:umber _
A9729 ~13 .5 4
A9832 .032 .25 4
A9833 .0~2 .25 4
A20177 .0~3 .25 4
: A20178 .063 .5 4
A20187 . o63 . 25 4
A20~88 . o63 . 25 4
A20189 .o63 .25 4
. ~20191 ,063 .25 8
A20192 .032 .25
A21515 .063 .13 2
A21517 .063 .13 4
A21518 .032 .13 4
` A21~19 .032 .25 8
A21522 ,032 .13 4
A21523 032 _ _........... 4
:.
_62-
.
Microbiology Com~ound Screening
Compound BL-S1081_vs. Neisseria_Gonorrhoeae
Secondary in vitro Screening Results - MIC_(mcg.~ml.
Organism - - ~ -~ _ _
Number BL-S108I Cefuroxlme BL-S1080
__ _ _ ~
A20142 .032 . o63 .13
A20143 .032 . oo8 . 016
A21440 .032 .032 .016
~21442 .13 .13 .13
A21444 .13 .032 ~o~3
A21448 .016 .016 .016
A21449 .016 .016 .016
A21453 .016 .016 ~oo8
A21455 .063 .13 .13
A21456 2 1 2
A21461 ,o63 .063 . o63
A21462 1 .5 2
A21467 .13 .25 .25
A21468 .016 .032 .oo8
~214~9 .063 .25 .25
A21470 13 .13 .25
.
Mlcrobiolog~ Compound Screening
Compound BL-S1080 vs. Neisseria Gonorrhoeae
Secondary in vitro Screening Results - MIC (mcg./ml.)
Organism _
Number BL-S1080 Ce~uroxime BL-S786
~ _ _
A20142 o002 ,0005 . oo8
A20143 .001 .0005 . oo8
A20144 .063 .063
A21440 .oo8 .002 .5
A21442 .004 .OQl .5
A21444 . oo8 . oo
A21446 . 016 . oo8
~ A21447 . oo8 . 002 . 5
: ~2~448 .004 .001 ~5
A2~449 , oo8 . ool . 5
A21450 .032 . oo8 2
~21455 .13 .063
A214~6 .25 .13
A214~1 . oo8 . oo8 . 5
A21462 .25 ,25 4
`: A21469 032 ~13 2
__
-6~-
.
,
Microbiology Com~;?ound Screening
Com~ound BL-S1081
Primary in vltro Screening Results - MIC (mcg./ml.)
~L-S BL-S Ce~ur-
Or~anism 108.1 1080 oxime
Diplococcus pneumoniae A9585 .004 -- .004
Streptococcus pyogenes . A9604 .004 -- .004
Staphylococcus aureus A9537 8 8 .25
Staph, aureus'50% serum A9537 16 63
Staph. aureus at 10 3 Dil A96064 __ ,5
Staph. aureus at 10 Dil ~9606 4 __ ,5
Sa~monella enterltidis A9531 .o6 __ . o6
Escherichia coli A15119 4
Escherichia coli A9675 4 - 4
Klebsiella pneumoniae A9977 1 __ 1
Klebslella pneumoniae A1513032 ~~ 32
Prdteus mirabilis A9900.13 ~~ .13
Proteus mo~ganii A1515363 ~~ 63
Pseudomonas ae~uginosa A9843A~125 -- >125
Serratia marcescens A2001932 __ 125
St. aur. Meth~Res.10 ~ DilA15097 32 - ~ 4
Enterobacter cloacae A9656>125 -- >125
Enterobacter cloacae A9657 8 __ 2
Enterobacter closcae A9659 63 __ 125
:
:::
:
~.
'
-6l~_
;2!~ 7
Microbiolo~y Comp~ Screening
~d BI~S1105
Primar~r in vitro Screening Results - MIC (~cg./mlO)
BL-S BL-S BI~S Ce~
Organism 1105 1080 786 o~ime
_
Diplococcus pneumoniae A9585 . 016 . 016 . 016 . 004
Streptococcus pyogenes A960~ i .O~i .03 "004
Staphylococcus aureus A9537 4 ~ 1 5
Staph aureus~50~ serum A9537 63 ~3 4
Staph aureus at 10 ~ Dil A9606 4 8
Staph ~ureus at 10 Dil A9606 4 8 2
Sa~monella enteritidis A9531 .5 .5 oO16 .13
~scherlchia coli A15119 8 2 .5 8
Escherichia coli A9675 4 2 8 63
Klebsiell~ pne~lmoniae A9977 2 1 .016 2
Xlebsiella pneumoniae A151~0 63 3~ 1 ~2
Proteus mirabilis A9900 .25 .016 .03 .03
Proteus morganii A15153 63 63 63 32
Pseudomonas aeruginosa A9843A 125 ~125 ~125 >125
Serratia marcescen~ A20019 63 125 125 >125
St.aur.Meth-Res~10 3 Dil A15097 32 32 4 4
Enterobacter cloacae A9656 >125 125 125 >125
Enterobacter cloacae A9657 4 2 .5 2
Enterobacter cloacae A9659 >125 >125 >125 >125
,
-
. , .
_6 5--
There is also provided by the present in~en
tion a compound having the formula
i ~ C - NH-C~--CH ~CH2 ~ - N
~b_ N ,C-C~I2~-S-C~7~N
l_~M (C~2)nC
O
wherein R is alkyl containing 1-4 carbon atoms, n is
one, two or three and M ls
M is-CI~OC(CH2)nR, ~R~C(cH2)nc~ 3
~ - fHXCOR or - CH-S-~-R6
R ~1
n i8 0 to 4; R is hydrogen, alkyl having 1 to 8
carbon atoms, cycloalkyl of 3 to 6 carbon atoms~
phenyl~ Cl-C4 phenalkyl, pyridyl, thienyl, or
propyl; Rl i~ hydrogen~ methyl or eth~l, R2 and R3
are e~ch hydrogen~ alkyl having 1 to 6 carbon atoms,
phenyl, pyridyl, o.r thienyl; R4 and R5 are each
hydrogen or alkyl of 1 to 4 carbon atoms; R is alkyl
ha~lng 1 to 4 c~rbon atoms, phenyl, phenal~yl having
1 to 4~carbon atoms, pyridyl3 thiadiazolyl, amino or
G1~54 alk~lamino; X is NH or oxygen; and each phenyl
group is unsubstltuted or subs~ituted with one or two
~ubstltuents selected ~rom the group consisting of
alkyl having 1 to ~ carbon atoms, alkoxy having 1 to
4 carbon atoms~ hydroxy~ amino, NHRl, N(Rl)2~ nitro,
fluoro, chloro, bromo or carboxy, or a nontoxic,
-6~-
.
~ ~5 ~ 7
pharmaceutically acceptable salt thereof~ said com-
pound being at least 75~ by weight in the form of its
isomer and preferably in the form of its ~
i.somer essentially free o~' the corresponding anti isomer.
.~ .
:
. I .
` _67-
,
.
5~7
There is also provided by the present inven-
tion a compound having the formula
O
f - C - NH- ~EI--CH ~CH2 11--N
N_ OR1 ~ N--C~ C~I2 S-C~. N~~
C-OR3 tCH2~nCOO~
o
wherein Rl is alkyl containing 1-4 carbon atoms, n i~
one, two or three and R~ is selected ~rom the group
consisting of
- CH3 0
- CH - 0 - ~ R6
: 12~5 11 6
- CH - C - R,
.1 . R5
- CH _ x2 ~ ORl
wherein R5 is a h,ydrogen atom, a methyl or an ethyl group;
x2 ls -O-, ~ ; R is a basic group.such as alkyl or aralkyl
substituted with substituted or unsubstituted NH2, such
as alkyl-NHCH~, aralkyl-NHC ~ ,
. .
alkyl-NH~,
aralkyl-NH ~ , -fH ~ , -C~2NH2, -IH-cH2 ~ '
` NH2 NH2
: R7 ls an alkyl group such as a meth~l, ethyl, propyl,
i90propyl, but~l, lsobut~l, pentyl or 2-ethyl-hexyl group,
a ~cycloalkyl group such as cyclopropyl, cyclobutyl3 cyclo-
pentyl, cyclohexyl or cycloheptyl; an aryl group such as
,
~ 68-
` - 1 ~
.
phenyl or naphthyl; an aralkyl group such as benzyl or
naphthylmethyl; a heterocyclic group and wherein the
alkyl, cycloalkyl, aryl, aralkyl and heterocyclic groups
may be substituted with one or more groups selected ~rom
the class consisting of amino groups, substituted amino
groups such as methylamino, diethylam.ino or acetamido
groups3 the halogen groups such as ~luorine3 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 weight in the form of
its ~ isomer and preferably in the form of its s~
isomer essentially ~ree o~ the corresponding anti isomer.
-69-
. . , , ~ ,
~ 7
There is also provided by the present ~nven-
tion a compound having the formula
O ;
~Lc I NH-C~CH ICH2
ORl o~ N C~5-C~2-s-c~N - N '`
C-OM (CH2)ncooH
wherein Rl is ~lkyl conta~ning 1-4 carbon atoms, n is
one, two or three and M i~
Il .
C Y
- CH2 N\
wherein Y is alkyl of one to six carbon atoms, phenyl,
benzyl, alkoxy of one to ~ix carbon atoms, or benzyloxy;
Z is alkyl of one to six carbon atoms, phen~lbenzyl3
alkoxy of one to six carbon atoms~ cyclopentyl, cyclo-
hexyl and phenyl, or Y+Z taken together are a ~-benzoxa-
zolidlne ring; or a nontoxic, pharmaceutically acceptable
salt thereof, said compoun~ bein~ at least 75~ by weight
in the form of its syn isomer and preferably in the form
of its s~ isomer essentially free o~ the corresponding
anti isomer.
`:
-7-
Also included within the present invention
are pharmaceutical compositions comprising a mlx~ure
o~ an antibacterially effective amount o~ a compound
of the present invention and a semisynthetic penici].-
lin or another cephalosporin or a cephamycin or a
~-lactamase inhibitor or an aminoglycoside antibiotic
There is further provided by the present
invent~on a pharmaceutical composition comprising an
antibacterially e~fective amount of a compound having
the formula
C - NH-III CI~ ~H2 ~l - N
N_oRl ~C N C'~C-CH2-S-C--N'N
C-OR ~C~I2)ncOoH
il
wherein Rl is alkyl containing ~-4 carbon atoms, n is
one~ two or three and R is hydrogen, pivaloyloxymethyl,
acetoxymethyl a methoxymethyl~ acetonyl, phenacyl, p-
nitrobenzyl, ~ -trichloroethyll ~-phthalidyl or
nitrobenzyl, ~ -trichloroethyi3 ~-phthalidyl or 5-indanyl
a~d preferably i5 hydrogen or a nontoxic, pharmaceutically
75% by weight in the ~orm of its syn isomer and
pre~erably in the form o~ its syn isomer essentially
free o~ the corresponding anti isomer, and a pharma-
ceutically acceptable carrier therefor.
There is ~urther pro~ided by the present
invention a pharmaceutical composition comprising an
antibacterially e~fective amount o~ the syn isomer o~
a compound having t~e formula
-71-
.. . .
$~
C - C - NH~ -qH ICH2 3 ~
~C N "C-CH2-S-C~N N
C- OH (CH2)nc~ooH
ti
wherein n is one, two or three or a nontoxic, ~harma-
ceutically acceptable salt thereo~ and whereln n is
preferably one, and a pharmaceuticall~ acceptable carrier
- there~or.
There is further provided by the present
invention a method of treating bacterial infections
comprising administering by in~ection to an in~ected
warm-blooded animal~ including man, an e~ective but
nontoxic dose o~ 250-1000 mgm. of a compound having the
formula
G - C - NH-~H - CIH CH2 ~l - N
N~_o~l ~C--N~ "C-CH~-S-C~N_N
: ~-OR (CH2)ncooH
.,~ , O
wherei~ Rl is al~yl containing 1-4 carbon atoms, n is
one,~two or three and-R2 is hydrogen, pivaloyloxymethyl,
: acetoxymethyl, methoxymethyl~ acetonyl, phenacyl, p-
nitrobenzyl~ B,~ trichloroethyl, 3-phthalidyl or
5-lndanyl or a nontoxic, pharmaceutically
acceptable salt thereo~, said compound being at least
:
~ 75%~by weight in the form of its s isomer and
pr~:~erably in the ~or~ of its syn isomer essentially
~ree of the corresponding anti isomer
~: : -72-
24~
There is further provided by the present
invention a method of treating bacterial in~ections
comprising administering by in~ection to an infected
warm-blooded animal, including man, an effective but
nontoxic dose of 250-1000 mgm~ of the syn isomer of a
compound having the ~ormula
C - NH~ I C~2 7 - N
N--~oc~S 0~ ~ C,C-c~I2-s-c~N - N
~-OH (CH2)
o
wherein n is one~ two or three or a nontoxic, ~harma-
c~utlcally acceptable salt thereof and wherein n is
preferably one.
.
. .
.~
-7~-
2~
There is also provided by the present
invention a method for combatting Haemophilus infec-
tions which comprlses administerlng to a warm-blooded
mammal in~ected with an Haemophilus infection an
amount effective for treating sald HaemoPhilus infec-
tion of a composition comprising a compound having
the formula
C - C ~ NH~ CH C~I2 Nll --N
--ORl ~C l!~ C,,C-CH2-S-C_N~N
~-OR ~cH2)ncooH
O
w~erein Rl is alkyl containing 1-4 carbon atoms, n is
one, two or three and R2 is hydrogen, pivaloyloxymethyl,
acetoxymethyl, methoxymethyl, acetonyl, phenacyl, p-
nitrobenæyl, ~ trichloroethyl, ~-ph~halidyl or
nitrobenzyl~ trichloroethyi, 3-phthalidyl or 5-indanyl
and preferably is hydrogen or a nontoxic, pharmaceutically
75% by weight in the form of its syn lsomer and
preferabiy in the form of its syn isomer essen-tially
free of the corresponding anti isomer, an~ a pharma-
ceutically acceptable carrier therefor.
-74-
- . . .
.
There is also provided by.the present
invention a method for combatting N isseria in~ec-
tions which comprises administering to a warm-blooded
mammal in~ected with a Neisseria in~ection an amount
effective ~or treating said Neisseria infection of a
composition comprising a compound having the for~ula
o
C C NH- ,~C,H SH2 Nll--N
N~ OR1 ~;C--N~ C ,C CH2 S C N~N
c_oR2 ~C~2)nCOO~I
. Il
wherein Rl i8 al~yl containing 1-4 carbon atoms, n is
o~e~ two or three and R is hydrogen, pivaloyloxymethyl,
acetoxymethyl, methoxymethyl, acetonylj. phenacyl, p-
nitrobenzyl, ~ trichloroethyl, 3-phthalidyl or
nitrobenzyl~ trichloroethyi~ 3-phthalidyl or 5-indanyl
and preferably is hydrogen or a nontox~c, pharmaceutically
75% by weight in the ~orm of its syn isomer and
pre~erably in the form of its syn isomer essentially
free oP the corresponding anti isomer, and a pharma-
ce~ticall~ acceptable carrier there~or.
. .
~,
-75-
