Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
TRI-PY-CEPH I
SY-15C,-A
r~r'-~
The cephalospori~s of t~.e present invent~on
in general possess the usual attr~butes of such compounds
and are particularly useful in the treatment of bacterial
infections.
The cephalosporins are a well-known group of
semisynthetic antibacterial agents made originally, for
example, by acylation of the 7-amino group of the nucieus
7-aminocephalosporanic acid (7-ACA) and later by similar
acylation of nuclei derived therefrom, as by modifi-
cation of its substituent at the 3-position. Various
reviews have appeared in the scien~ific literature
(e.g. Cephalosporins and Penicillins - Chemistry ænd
3iology, edited by Edwin H. Flynn, Academic Press,
New York, 1972, and particularly pages 554-569) and
in the patent literature, e.g. as in U. S. Paten~s Nos.
3,687,948; 3,741,965; 3,743,644; 3,759,904;
3,759,905; 3,766,175; 3,766,9o~; 3,769,281;
3,769,801; 3,799,923; 3,812,116; 3,813,3~8;
3,814,754 and 3,814,755 (all United States Class 260-243C).
Issued patents on 3-thiolated cepnalospor ns
in which the 7-subs~tuent is
a) ~-Amino-~-phenylace~a~ido include U. S.
3,641JG21; U. S. 3,734,907; U. S. 3,687,948; U. S.
~'
3,741,965, U.S, 3,757,015, U.S. 2,74~,644, Japan
71/24400 (Farmdoc 46374S), Belg~um 776,222 (Farmdoc
38983T; U.K. 1,328,340 whlch lncludes various
~ub~tltuent~ on the benzene rlng), Belglum 772,592
(Farmdoc 19696T; U.S. 3,687,948, 3,734,907 and
3" 57,012), West Germany 2,202,274 (Farmdoc 50428T)
correspondlng to U.S. ~,759,904, Netherlands 72Q5644
(Farmdoc 76309T; U.S. ~,757,014); and
b) o-, m- or p-aminoethoxyphenylacetamldo a~
Netherlands 72/13968 (Farmdoc 2474oU) corre~pondlng
to U.S. 3,759,905 and
c) o-aminomethylphenylacetamido as U.S. 3,766,176 and
3,766,175 (which also review the older patent llterature
concerning su~stituted 7-phen~lacetamidocephalosporanic
acids) and
d~ N-(phenylacetlmldoyl)amlnoacetamldo as
U.S. 3,692,779; and
e) a-amlno-~-(1,4-cyclohexad~enyl)acetamldo
as ln Belgium 776,222 (Farmdoc 38g~3T; U,K. 1,328,340).
Addltlonal slmilar dlsclosure~ are found ln
U,S. 3,692,779 (Bel~lum 771,189; Farmdoc 12819T),
Ja ~n 72/05550 (Farmdoc 12921T), Japan 72/05551
~Farmdoc 12~22T) J U.S. 3,719,67~ (Belglum 759,570; .
Farmdoc 39819S~, Bel~lum 79~,311 (Fzrmdoc 39702U)
and ~elglum 793,191 (Farmdoc ~9684U).
Issued disclosures of 3-thiolated cephalosporins
in which the 7-substituent is 7-mandelamido (7-~-hydroxy-
phenylacetamido) are found, for example, in U.S. 3,641,021,
France 73.10112, U.S. 3,796,801, Great Britain 1,328,340
(Farmdoc 38983T), U.S. 3,701,775 Japan 48-44293 (Farmdoc
55334U) and in Hoover et al., J. Med. Chem. 17(1),34-41 (1974)
and Wick et al., Antimicrobial Ag. Chemo., 1(3), 221-234
(1972).
U.S. 3,819,623 ~and, for example, also U.K. 1,295,841
and West Germany 1,953,861) discloses specifically and with
working details the preparation of 2-mercapto-1,3,4-thiadiazole-
5-acetic acid and its conversion to 7-(lH-tetrazol-l-yl-acet-
amido)-3-(5-carboxymethyl-1,3,4-thiadiazol-2-ylthiomethyl)-3-
cephem-4-carboxylic acid which is also disclosed in West Germany
Offenlegungsschrift 2,262,262.
U.S. 3,766,175 and 3,898,217 disclose a compound of
the formula
CH2NH2
6~CH2-C--NH-CH--fH CH2
~ C~ 2
I
COOH
wherein R is
N~ 6~N I ~ CH2 or ~ N~N
or a nontoxic pharmaceutically acceptable salt thereof,
and
A compound of the formula
R ~ C-C-N~ ~ S
~CH2H
CH2NH2
C02H
wherein
R is --H or lower alkyl;
R' is --H, ~lower)alkanoyloxy,
N ~ N -
-N ~ , -S ~ S ~ 3 S ~ N ~
n is an integer from 4-7, inclusive; and the pharmaceutically
acceptable addition salts thereof, respectively.
U.S. 3,883,520 and 3,931,160 and Farmdoc Abstract
22850W make reference to 3-heterccyclicthio-methyl cephalos-
porins containing a number 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 synthesis or the like and do not even name
any such compound containing a carboxyl substituent.
U.S. 3,928,336 provides a review of much of the
older cephalosporin art.
U.S. 3,907,786 and 3,946,000 disclose cephalosporins
containing various fused ring bicyclic thiols.
,~
-- 4 --
Farmdoc abs ' ract 1~30X discl3se;, ccm~cunds
of the formula
R ~sI ~~ CX2-S l~ ~,N
COOH ( CH2 )n-cooH
(whQre Rl = acyl or H; ~3 - H or methoY.y; n = 1-9).
me present invention pro~ides compounds having
the structure:
/S\
Rl-~-CH _ IH ~2 ~T
~ C--CH~ N ~T~ ~N-(CHa)ncocH
0// ~ O
1!-~
O
often written herein as
Rl-NH f S ~ ~ N
~r ~ CH2S M,N ~ N-(CH2)~COOH
O COC~ O
wherein n is one or two and Rl is acyl or hydrogen and
esters and non-toxic pharmaceutically acceptable salts thereof.
m e stereochemistry of the bicyclic nucleus is that found in
cephalosporin C.
The esters of the compounds of the for~ula I
include, but are not limited to, those having the group
/W
-CH
wherein, when W represents hydrogen, Z represen~s
(lower)alkanoyl, benzoyl, naphthoyl, furoyl, thenoyl,
nitrobenzoyl, methylbenzoyl, halobenzoyl, phenyl-
benzoyl, N-phthalimido, N-succinimido, ~-saccharino,
N-(lower)alkylcarbamoyl, (lower)alkoxy, (lower)-
alkylthio, phenoxy, carbalkoxy, carbobenzoxy,
^arbamoyl, ben7yloxy~ chlorobenzyloxy, carbophenoxy~
_ ~ _
carbo-tert.-butoxy or (lower)alkylsulfonyl, and when
W represents carbalkoxy, Z represents carbalkoxy-and,
when W represents phenyl, Z represents benzoyl or
cyano or wherein W and Z taken together represent
2-oxocycloalkyl containing 4 to 8 carbon atoms inclusive.
Preferred embodiments of this invention include the
pivaloyloxymethyl, acetoxymethyl, methoxymethyl, acetonyl,
phenacyl, p-nitrobenzyl, ~ 3~-trichloroethyl, 3-phthalidyl-
or 5-indanyl- esters.
Acyl (Rl) comprises the groups having the
structures:
2 ~^`~ 2 H2
R ~ CH2CO- , ~ CH2C- ~nd ~ CH2C-
wherein R is hydrogen, hydroxy or methoxy and R' is hydrogen
or methyl.
A preferred embodiment of the present
invention consists of the compounds of Formula
I wherein Rl has the structure
~3~
2MH2 ~ ~2NH2
2C HO ~ 2
CH2NH2
2C and ~ CH~2NH2
Another preferred embodlment Or the present
lnventlon conslsts Or the compounds of Formula
I whereln Rl h~s the structure
NHCH3 ~ CH2NH~3
~C~I2co- XO~~C}~ ~0-
~2MHC~3
- C~2CO- and ~ ~ 2CNoHCH3
A preferred embod~ment of the present invention
consists of the compounds having the formula
C~ C1~25~ (C~2)ncooH
l-OM
wherein Rl represents
~C~CH2 ~ CH2~R ' C~2N~
R ~ CH2CO- OR ~ CH2CO- OR ~ CH2C-
wherein R is hydrogen, hydroxy or methoxy; R' is hydrogen
or methyl; n is one or two and M is
Ol O
-cHoc(c~ 2 ~nR~-CHOC(CH2)nl \ R3
Rl o Rl o NRg Rs
O O
-CHXCOR6 or -CH-S-C-R~
Rl Rl
n is O to 4; R is hydrogen, alkyl having 1 to 8 carbon
atoms, cycloalkyl of 3 to 6 carbon atoms, phenyl, Cl-C4
phenylakyl, pyridyl, thienyl, or pyrrolyl; Rl is hydrogen,
methyl or ethyl; R2 and R3 are each hydrogen, alkyl having
1 to 6 carbon atoms, phenyl, pyridyl, or thienyl; R4 and
R5 are each hydrogen or alkyl of 1 to 4 carbon atoms; R6
is alkyl ha~ing 1 to 4 carbon atoms, phenyl, phenalkyl
having 1 to 4 carbon atoms, pyridyl, thiadiazolyl, amino
_ g _
or Cl-C4 alkylamino; X is NH or oxygen; ~nd each phenyl
group is unsubstituted or substituted with one or two
su~stituents selected from tne group co~sisting of al~yl
ha~ing 1 to 6 carbon atoms, alkoxy having 1 to 4 carbon
atoms, hydroxy, amino, NHRl, N(Rl)2, nitro, ~luoro, c~loro,
bromo or carboxy, or a nontoxic~ pharmaceutically acceptable
salt tnereo~.
Another preferred embodiment of the present
in~ention consists o~ the compounds having the formula
~ S ~
t I fH 2 ~ ~
~C- N ~ C - C~2S - ~ N,N ~ N-(CH2)nCH
- C-OM
O
wherein Rl represents
CH a NHR ~ CH 2 NHR~ ,CH 2NHR
CH2CO- OR ~ CH~CO- ~ ~ ~ CH~-CO-
wherein R is hydrogen, hydroxy or ~ethoxy; R' is hydrogen
or mathyl; n is one or two and M is selected from the group
consisting of
-CH - O - C - R6 , -CH - C - R6
R5
- CH - x2 - IC - oR7
-- 10 -
$i.~
~hereln R5 i8 a hydrogen atom, a methyl or an ethyl group;
X2 ls -O-, -MH-; R6 is a basic group.such as alkyl or aral~yl
substituted with substituted or unsub~tltuted NH2, such
as aIkyl-NXCH3, aralkyl-N~CH
A
al~rl-NH~
aralkyl-NH ~ , -IH ~ ~ CH2NH2, -IH-CH
MH2 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 gro~F such as
phenyl or naphthyl; an aralkyl group such as benzyl or
naphthylmethyl; a heterocyclic group and wherein the
alkyl, cycloalkyl, aryl~ aralkyl and heterocyclic groups
may be substituted with one or more groups selected from
the class conslsting of amino groups, sub~tituted amino
group~ such as methylamino, diethyl~no or acetamido
groups, the halogen groups such as ~luorine, chlorine or
bromlne, nitro groups, alkoxy groups such as methoxy,
ethoxy, propyloxy, isopropyloxy, butoxy or isobutoxy;
or ~ nontoxic, pharmaceutically acceptable salt thereof,
There is further provided by the present invention
a pharmaceutical composition comprising an antibacterially
effective amount of a compound having the formula
Rl_NH_fH~H CH2C f ~ ~
// ~ C~ 2 ~ ~ (CH2)ncooH
C-OM O
wherein Rl represents
CH NHR'
~ 2 2 HR CH2NHR'
R ~ CH2C- OR ~ CH2CO- \ ~ CH CO-
wherein R is hydrogen, hydroxy or methoxy; R' is
hydrogen or methyl; n is one or two and M is hydrogen, piva-
loyloxymethyl, acetoxymethyl, mPthoxymethyl, acetonyl, phenacyl,
p-nitrobenzyl,~ ,-trichloroethyl, 3-phthalidyl or 5-indanyl
and preferably is hydrogen or a nontoxic, pharmaceutically
acceptable salt thereof.
There is further provided by the present invention
a method of treating bacterial infections comprising administer-
ing by injection to an infected warm-blooded animal, including
man, an effective but nontoxic dose of 250-1000 mgm. of a
compound having the formula
R -NH-fH-C~ CH2 = N
O \ ~N ~ ~ N-(CH2) COOH
l_OM
-12-
wherein Rl represents
CH NHR'
~ 2 ~^~CH2NHR' CH2NHR'
R ~--CH2Co- OR ~--CH2CO- ~ CH2C-
wherein R is hydrogen, hydroxy or methoxy; R' is hydrogen
or methyl; n is one or two and M is hydrogen, pivaloyloxy-
methyl, acetoxymethyl, methoxymethyl, acetonyl, phenacyl,
p-nitrobenzyl, ~ , -trichloroethyl, 3-phthalidyl or 5-
indanyl or a nontoxic, pharmaceutically acceptable salt
thereof.
There is also provided by the present invention
a method for combatting Shig. dysenteriae infections which
comprises administering to a warm-blooded mammal infected
with a ~ . dysenteriae infection an amount effective for
treating said Shig. dysenteriae infection of a composition
comprising a compound having the formula
Rl-NH-fH-CH CH2 ~=NI
o ~ N~ ~ (CH2 ) nCOOH
C-OM
11
wherein R represents
CH NHR'
,~,~cH2NHR~ CH2NHR'
R-~ / ~ CH2Co- OR
~ ~ CH2Co- OR 11 ~ C 2
wherein R is hydrogen, hydroxy or methoxy; R' is hydrogen
or methyl; n is one or two and M is hydrogen, pivaloyloxy-
methyl, acetoxymethyl, methoxymethyl, acetonyl, phenacyl,
~ -13-
p-nitrobenzyl, ~ trichloroethyl, 3-phthalidyl or 5-
indanyl and preferably is hydrogen or a nontoxic, pharma-
ceutically acceptable salt thereof.
There is also provided by the present invention a
method for combatting B. anthracis infections which comprises
administering to a warm-blooded mammal infected with a
B. anthracis infection an amount effective for treating said
B. anthracis infection of a composition comprising a compound
having the formula
R -NH-CH-CH CH2 ~ N
// ~C ~C CH2S~N~N ~N-~CH2)ncOoH
C-OM
o
wherein R represents
2 R
~ ,'^~ ~CH NHR' / 2NHR
R ~ CH2C- OR ~ 2 OR I ~
2 ~ S CH2C-
wherein R is hydrogen, hydroxy or methoxy; R' is hydrogen
or methyl; n is one or two and M is hydrogen, pivaloyloxy-
methyl, acetoxymethyl, methoxymethyl, acetonyl, phenacyl,
p-nitrobenzyl, ~ , -trichloroethyl, 3-phthalidyl or 5-
indanyl and preferably is hydrogen or a nontoxic, pharma-
ceutically acceptable salt thereof.
The present invention also provides the process
for the production of the antibacterial agents having the
structure.
Rl~NH-CH -CH CH2 ~ ~
O ~ ~ // 2 ~ ~ N-(CH2)nCOOH
COOH
wherein n is one or two and Rl is acyl as defined aDove
which comprises reacting a compound of the formula
H2~-CH _ C~ CH2 ~ = N
O ~ \ ~ 2 N~ ~ l~ ~(CH2)ncooH
O
COOH II
wherein n is one or two or a salt or easily hydrolyzed 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 silyl esters
described in U.S. patent 3,249,622 for use with 6-aminopenicil-
lanic acid and used in Great Britain 1,073,530 and particularly
the pivaloyloxymethyl, acetoxymethyl, methoxymethyl, acetonyl,
phenacyl, p-nitrobenzyl,~ trichloroethyl,3-phthalidyl
and 5-indanyl esters) thereof with an ~rganic monocarboxylic
acid chloride or a functional equivalent thereof as an acylat-
ing agent.
-15-
Such functional equivalents include the cor-
responding acid anhydrides, includlng mixed anhydrides
and particularly the mixed anhydrides prepared from
stronger acids such as the lower aliphatic monoesters of
carbonic acid, or alkyl and aryl sulfonic acids and of more
hindered acids such as diphenylacetic acid. In addition, an
acid azide or an active ester or thioester (e.g. with p-nitro-
phenyl, 2,4-dinitrophenol, thiophenol, thioacetic acid) may
be used or the free acid itself may be coupled with compound
II after first reacting said free acid with N,N'-dimethyl-
chloroforminium chloride [cf. Great Britain 1,008,170 and
Novak and Weichet, Experientia XXI, 6, 360 (1965)] or by
the use of enzymes or of an N,N'-carbonyldiimidazole or an
N,N'-carbonylditriazole [cf. South African patent specifica-
tion 63/2684] or a carbodiimide reagent [especially N,N'-
dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide or
N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide; cf. Sheehan
and Hess, _. Amer. Chem. Soc., 77, 1967 (1955)], or of_
alkylylamine reagent [cf. R. Buijle and H. G. Viehe, Angew.
Chem. International Edition 3, 582, (1964)] or of an isoza-
zolium salt reagent [cf. R. B. Woodward, R. A. Olofson and
H. Mayer, J. Amer. Chem. ~., 83, 1010 (1961)], or of a
ketenimine reagent [cf. C. L. Stevens and M. E. Munk,
J. Amer. Chem. Soc., 80, 4065 (1958)] or of hexachloro-
cyclotriphosphatriazine or hexabromocyclotriphosphatriazine
(U.S. 3,651,050) or of diphenylphosphoryl azide [DPPA;
J. Amer. Chem. Soc., 94, 6203-6205 (1972)] or of diethyl-
-
phosphoryl cyanide [DEPC; Tetrahedron Letters No. 18, pp.
1595-1598 (1973)] or of diphenyl phosphite [Tetrahedron
Letters No. 49, pp. 5047-5050 (1972)]. Another equi~alent
of -the acid chloride is a corresponding azolide, i.e., an
amide of the correspondin~ acid whose amide nitrogen is a
-16-
member of a quasiaromatic five membered ring containing at
least two nitrogen atoms, i.e., imidazole, pyrazole, the
triazoles, benzimidazole, benzotriazole and their substituted
derivatives. As an example of the general method for the
preparation of an azolide, N,N'-carbonyldiimidazole is
reacted with a carboxylic acid in equimolar proportions at
room temperature in tetrahydrofuran, chloroform, dimethyl-
formamide or a similar inert solvent to form the carboxylic
acid imidazolide in practically quantitative yield with
liberation of carbon dioxide and one mole of imidazole.
Dicarboxylic acids y eld diimidazolide. The by-product,
imidazole, precipitates and may be separated and the imidazolide
isolated, but this is not essential. The methods for carry-
ing out these reactions to produce a cephàlosporin and the
methods used to isolate the cephalosporin so produced are
well known in the art.
-17-
h~;~
Mention was made above of` ~he use Or enzymes to
couple the free a cld wlth
compound II. Included ln the ~cope Or such proceqses
are the u~e o~ an eqter, e.g. the methyl ester, of
that free acld wlth enzymes provlded by varlous m~cro- -
organlqms, e.g. those de~crlbed by T. Takahashi et al.,
J. Amer, Che~. Soc., o4L11), 4035-4037 tl972) and by
T. Nara et al., J. Antib~otlcs (Japan) 24~L, }21-323
(1971) and in U.S. 3,682,777.
~ or the coupling of the organic carboxylic 2cid as
descrlbed above with compound II (or a salt or preferably
an easily hydrolyzed ester of Schiff base, as with
benzaldehyde, thereof) it is also convenient and efficient
to utilize as the coupling agent phosphonitrilic chloride
trimer (J. Org. Chem., 33(7), 2979-81, 1968) or N-ethoxy-
1,2-dihydroquinoline (EEDQ) as described in 3. Amer. Chem.
Soc., 90, 823-824 and 1652-1653 (1968) and U.S. Patent
3,455,929. The reaction i8 preferably carried out at 30-
35-C. in benzene, ethanol or tetrahydrofuran using about
e~u~molar quantities of all three reagents followed by
conventional isolation a~d removal by conventional methods
of any blocking groups present.
An addi~ional process of the present in~ention comprise~
the preparation of the compounds of the present invention
by the displacement of the 3-acetoxy group of a 7-acyla~ino-
cephalosporanic acid tprepare~ by substituting 7-amino-
- 18 -
. . .
1~3~
cephalosporanic acid for the 3-thiolated-7-amino-
cephalosporanic acids in the acylation procedures
described herein and elsewhere reported) with a thiol
HSR3 having the formula
N
~N'N~N- (CH2)nCOOH
wherein n ls one or two a~d then removing the
protecting group if any is present, as on the aminomethyl
or meth~lamino.nethyl group or on the carboxyl group or
both, The displacement of such a 3-acetoxy group with
such a thiol may be accomplished in solution as in water
or aqueous acetone at a temperature of at least room
temperature and preferably within the range of about
50 to 100C. in the presence of a mild base such as
sodium bicarbonate, e.g. preferably near neu~rality such
as at about pH 6. An excess of the thio7 is preferably
employed. The reaction product is iso7ated by carefu~
acidification of the reaction mixture followed by extrac-
tion with a water-~mmiscible organic solvent. As noted
above, the preparation of many other 7-acylamidocephalosporanic
~cids is described in thc patent and scientific l~terature,
e.g. in U.S. Class 260-243C.
When the organic carboxylic acid contains a
functional group sueh as amino or methylamino it ~s often
- 19 -
desirable to first block (or protect) said group, then
carry out the coupling reaction and finally subject the
resulting compound to chemical removal of the protecting
group, that is, subjecting the resulting compound to
elimination reaction of the protecting group.
The present invention thus also provides the
process for the production of the antibacterial agents
having the formula
Rl-NH-cH 9H CH2 ~ - ~
C --N ~ ~ C~CH2S ~\N ' N ~ N (CH2)nCH
COOH
wherein n is one or two and Rl is acyl as defined above which
comprises reacting a compound of the formula
Rl-NH-C~ CtH CH2 1
~ - N ~ ~C-CH-O-C-CH3
I
COOH
wherein R is acyl (including cephalosporin C itself) with a
compound having the formula
~ ~ ~ N
HS ~ ~ ~ N-(CH2)nCOOH
O
wherein n is one or two.
-20-
! ~ ~J
The salts of the compounds of this invention include
the nontoxic carboxylic acid salts thereof J including non-
toxic metallic ~alts such as sodium, potassium, calcium and
~luminum, the ammonium salt and substituted ammonium salt~,
e.g. salts o~ such nontoxlc amlnes as trlalkyl-
amines lncludlng trlethylamlne, procaine, dibenzyl-
amlne, N-benzyl-beta-phenethylamine, l ephenamlne,
N,N'-dlbenzyleth~lenediamlne, dehydroabietylamine,
N,N'-bls-dehydroabletyleth~lenediamine, N-(lower)-
alkylplperidlne, e.g. N-ethylpiperidlne, and other
amlnes which have been used to form salts ~lith
~enzylpenicillln; and the nontoxic acid addition
salts thereor (i.e., the amine salts) including
the mineral acld addltlon salts such 25 the hydro-
chlorlde, hydrobromide, hydroiodide, sulfate,
sul~amate and phosphate and the organic acid
addltion salts such as the maleate, acetate~
citrate, oxalate, succlnate, benzoate, tartrate,
~umarate, malate, mandelate, ascorbate and the
llke.
Also lncluded ln this lnvention are the
~ompound3 ~uaed as either intermediates or
metabol1c precur~or~) in which the amino group
- 21 -
.. . . . ... _ .... _ ... . . ... . . . . .. . .. . . . .
~$~
ls "blocked" by substltuents such as
2-lodoethoxycarbonyl (U.K. 1,349,673), t-butoxy-
carbonyl, carbobenzyloxy, formyl, o-nltrophenyl-
sulrenyl, ~ trlchloroethoxycarbonyl, 4-oxo-2-
pentenyl-2, 1-carbomethoxy-1-prope~1-2- and the
llke. Partlcularly lncluded ln such blocklng
groups are the ketones (especlally acetone) and
aldehydes (especlally rormaldehyde and acetalde-
hyde) dlsclosed, for example, ln U.S. patents
3,198,804 and 3,347,851 and the ~-ketoesters
and B-dlketones dlsclosed, ~or example, ln U.S.
patent 3 "25,479 and the ~-ketoamldes dlsclo~ed
in Japan 71/24714 (Farmdoc 47,321S).
The preferred esters of the cephalosporins
o~ the presen~ inventlon are the pivaloyloxymethyl,
acetoxymethyl, methoxymethyl, acetonyl and phenacyl
esters. All are useful lnter~edlates ln the
productlon of the cephalosporin havlng a ~ree
carboxyl grou~
As lndlcated above, these flve esters Or 7-amino-
cephalospora~ic acid are each prepared by known
methods. One excellent procedure is that Or U.S.
patent 3,284,451 ln whlch sodlum cephalothin is
e~terlf~ed by reactlon wlth the correspondlng actl~e
chloro or bromo compound (e.g. phenacyl bromlde,
chloroacetone, chloromethyl ether, plvaloyloxy-
methyl chlorlde ~also called chloromethyl plvalate3,acetoxymethyl chloride) and then the thie~yl-
acetlc acid sldechain ls remove~ enzymatlcally
as ln the same patent or chemlcally as in U.S.
patent 3,575,g70 and ~n Journal of Antlbiotics
- 22 -
.
XXI~ (11), 767-77~ (1971). In another good
method the trlethyla~ine ~alt or 7-aminoce~alo-
sporanlc acld ls reacted direc~ly with the actlve
halo~en compound, as ln United ~in~o~ 1,229,453.
These esters of 7-amlnocephalosporanlc acld
are then reacted ~ith the nucleophlle HS~3 in the same
manner as is illustrated herein ~or 7-aminocephalo-
sporanlc acid itselr. The 3-thlolated ester or
7-amlnocephalosporanlc acld 1~ then coupled ~lth
the organlc carbox~llc acld R~-OH as before. Before
or after removal of any blocking group, e.g. on sn
amino group in the 7-sidechain, the ester of the
cephalosporin so obtained is, if not uséd per se,
converted to its free acid, including its zwi~terion
~and, if desired, any s~lt) by removal of the esterl-
fy~ng group, as by aqueous or enzymatic hydrolysis (as
with hum~n or snimal serum) or acidic or alkaline
hydrolysis or by treatment with sodium thiophenoxide
as taught in U.S, 3,284,451 and, in the penicillin
series, by Sheehan et al., J. Org. Chem. 29(7),
2Q06-2008 (1964).
~n another alternative synthesis, the 3-
thiolated 7-aminocephalosporanlc acid ls prepared
as described herein and then ac~?.ated at the 7-
amino group and ~inally esterified, 2S by reaction
Or the appropriate alcohol ~ith the acid chloride
prepared, for example, by reaction Or the inal
cephalosporln ~rith thionyl chlorl~e or by other
essentlally acidlc esteril`icat~on pro~e~ es.
- 23 -
. _ ..... .... . .. . . .
In the treatment of bacterial infections in
man, the compounds of this invention are administered
parenterally in an amount of from about 5 to 200 mg.l
kg./day and preferably about 5 to 20 ~g./k8./day in
divide* 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 excepients.
The dosage units are in the form of liquid prepara-
tions such as solutions or suspensions.
- 24 _
.. . _ ..... .. _ . _ . . . . . .. . . . . . . . .
The other reagents used to prepare the compounds
of the present invention are synthes~zed either as described
in the art (e.g. as in the patents and publications noted
above) or by strictly analogous procedures. For convenience
and purposes of illustration, however, there are given
below so~e specific examples of such syntheses,e.g. to
prepare carboxylic acids containing a free amino group
which is '~locked" with tert.-butoxycarbonyl.
2-(tert~utox~carbonvlamlnometh~ 4-
c~clohexad~n~lacetlc aclal
A ~olution of 16.5 g. (0.1 mole) o~ o-a~lno-
methylphenylacetic acid in 1.5 1 of llquld a~monla
(which had been treated wlth 50 mg. of Li to remove a
trace of molsture) was slo~ly dlluted with 500 ml. of c.ry
t-~uOH~ To the solution ~a~ added ln ~mall portlons
4 g. (0 5 atom) of Li over a period o~ 4 hours and
the m~xture ~as ~tlrred for 16 hours at room tempera ture
removlng the llquld ammonla ln a hood and flnall~
evaporated ~o drgnes~ below 4C C. The resldue ~,~as
d~solved ln 500 ml. Or water and the ~olut'on was
chromatographed on a column of IR-120 (H , 7~0 ml.) resln
and eluted with 1,~ NH40H solution. Nlnhydrln posit~ve
~ractions Or the eluate were combined and evaporated ~o
dr~ne~. The resldue w25 washed with ~our ~0 ml.
por~ion~ of hot ~cetone and recr~stallized from 500 ml.
Or ethanol-~a~er (1:1) to glve 11.2 g. (67~) of colorle~s
nAeedle o-(2-amlnomethyl-1,4-cyclohexadlenyl)acetlc acid.
M.p. 18~ C.
- 25 -
, . , . . . _ . . .. .. . .
~$ ~3c~ ~
IR:~'maJ 16~0, 1520, 1~80, 1356 c~ 1.
NMR ~ D20 ~ ~ C03 2-72 (4H, s, ~ C~ ~, 3.01
(2X, s, CH~CO), 3.20 (2H, s, CH2-N), ~-78 (2H, s,
~C=) .
Anal. Calcd. for CgH13N02: C, 6~ .65; HJ 7 . 84;
N, 8.38.
~ ound: C, 64.77; X, 8.o6; N, 8.44.
Im~roved Procedure ~or the Preoaratior ~
~ = ~ a c ld
CH2NH2
CH2Co2H Ll, ~ert. ~uO~ >
NH3 - TEA-HCl
CH2co2H ~ LiCl ~ (C~5)3N
2NH2
Ihe procedure u3ed b~ elch, Dolf~nl and
Glarrusso ln U.S. patent ~,720,665 ~Example 1) to
make D-2-amlno-2-(1,4-cyclonexadlenyl)acetic acld
was adapted. A solutlon o~ 830 ml. of distllled
liquld ammonia was drled with 40 ~,. Or llthlum
under a~ argon atmosphere. To thls stlrred solutlon
was added 11.0 g. (Q.07 mole) Or 2-amino~eth~lpherlyl-
acetlc acld and 3~0 ml. Or tert. but~rl alcohol. A
total- of 1.6 g. (0.225 mole~ of lithium ~Ja~ add~ to
~ 26 -
the vigorously stirred solution over a period of 2 hours.
The grey mixture was then treated with 35 g. (0.215 mole)
of triethylamine (TEA) hydrochloride and stirred overnight
at room temperature for 18 hours. The tert. butyl alcohol
was removed at 40 (15 mm.) to yield a white residue which
was dried ln vacuo over P2O5 overnight. The solid was
dissolved in 30 ml. of 1:1 methanol-water and added with
stirring to 3.5 1. of 1:1 chloroform-acetone at 5. The
mixture was stirred for 20 min. and the amino acid,
~-(2-aminomethyl-1,4-cyclohexadienyl)acetic acid, was
collected and dried for 16 hours in vacuo over P2O5 to
yield 6.3 g. (58~) of white crystals, m.p. 190 decomp.
The IR and NMR spectra were consistent for the structure.
A solution of 19.31 g. (0.135 m) of tertbutoxy-
carbonylazide in 152 ml. of tetrahydrofuran (THF) was
added to a stirred solution of 14.89 g. (0.09 m~ of
2-aminomethyl-1,4-cyclohexadienylacetic acid and 7.20 g.
(0.18 m) of sodium hydroxide in 281 ml. of water. The
solution was stirred for 18 hr. at 25 and then filtered
thru diatomaceous earth (Super-cel). The THF was removed
at 40 (15 mm) and the residual solution was washed with
ether (2 x 175 ml.) and acidified with 6 N hydrochloric acid
(HCl). The mixture was stirred in an ice-bath and the
precipitate was collected and dried for 18 hr. in vacuo
over P2O5 at 25 to yield 17.3 g. (72.6%) of 2-(tert-
butoxycarbonylaminomethyl)-1,4-cyclohexadienylacetic acid
as a white powder. The IR and NMR spectra were consistent
for the structure.
*Trade Mark
-27-
Preparation of 3-Aminomethyl-2-thiophene Acetic Acid
CH -NH
CH -CO H
A~ Thiophene-3-carboxaldehyde Dimethyl Acetal (2a)
A mixture of thiophene-3-carboxaldehydel)
(322 g., 2.9 moles), trimethoxymethane (636 g., 6
moles) and IR-120 resin (H , 6 g.) in methanol (200
ml.) was refluxed over a period of 4 hours. The resin
was removed and the filtrate was evaporated under re-
duced pressure to give a colorless oil which was dis-
tilled under reduced pressure. Yield 423 g. (94%),
b.p. 90-95 C. 13 mmHg.
ir:v maiqX 3150, 1045, 1025 cm 1
nmr ~ ppm 3-21 (6H, s, OCH3), 5.43 (lH, s,
CH ~ ), 7.0-7.4 (3H, m, thiophene-H )
O--
1) S. Gronowitz, Arkev, kemi., 8, 411 (1955).
B. 2-E'ormylthiophene-3-carboxaldehyde Dimethylacetal (3a)
To a stirred solution of 2 a(423 g., 2.68 moles)
in anhydrous ether (1 L) was added dropwise in 1 hour a
freshly prepared solution of n-butyllithium (27 moles) in
ether keeping a gentle reflux under dry N2. Reflux being
continued for 0.5 hour, a solution of DMF (dimethylformamide)
(432 g., 6 moles) in anhydrous ether (0.8 L) was added
dropwise to the mixture over a period of 0.75 hour with
vigorous stirring. After the complete addition the mixture
was stirred overnight, poured onto crushed ice (1 Kg.)
with stirring and allowed to rise to room temperature.
-28-
s~
The organic layer was separated and the water layer was
saturated with NaCl and extracted thoroughIy with ether
(2 x 200 ml.). The ether extracts were combined, dried
over MgS04 and concentrated. The residue was distilled
under reduced pressure and the pale yeIlow oil was
collected at 100-125 C., 0.7 mmHg. Yield, 277 g.
(56%).
ir: v maxq 3110, 1660, 1100 cm 1.
nmr: ~npat 3.40 (6H, s, OCH ) 5 86 (lH s
CH ), 7.27 (lH, d, J=6Hz, thiophene-H ~), 7.81 (1 H,
d-d, J-1.5 and 6Hz, thiophene-H~), 10.34 (lH, d, J-1.5 Hz,
-CHO).
C) l-Methylsulfinyl-l-methylthio-2-(3-carboxaldehyde
ethYleneacetal-2-thienYl)ethylene (4b)
Preparation of 4b was carried out according to
the procedure similar to that reported by K. Ogura et al.4).
Triton B (40% in methanol, 2 ml. in THF ~tetrahydrofuran)
(5 ml.) was added to a solution of methyl methylthiomethyl
sulfoxide ) (2.5 g., 20 m. moles) and 2-formyl-3-thio-
phenecarboxaldehyde ethylene acetal3) (3b). The mixture
was refluxed for about one hour and concentrated under
reduced pressure.
-29-
.3~
The residue was dissolved in benzene (I50 ml) and extracted
with water (3 x 20 ml). The organic layer was dried over
MgSO4 and eYaporated to dryness under reduced pressure.
The residue was column-chromatographed on silica gel ~80 g)
eluting with benzene (500 ml) and chloroform (500 ml)
successively. From the chloroform eIuate 4.9 g (85~) of
the product 4b was isolated as a pale yellow oil.
ir: v laiq 3110, 1600 cm 1.
nmr: ~ ppC13 2.42 (3H, s, S^CH3), 2.78 (3H, s, ~
, ~,,
SO-CH3), 4.15 (4H, m, CH2CH2-), 6.12 (lH, s, -
CH ~ ), 7.34 (lH, d, J 5 4.5Hz, thiophene-H~), 7.40
(lH, d, J=4.5Hz, thiophene-Hq), 8.28 (lH, s, -CH=).
The semioarbazone of 4 was prepared by a usual
manner and crystallized from ethanol-DMF. M.p. 212-
213C .
Anal- Calcd- for CloH13N3O2S2 C, 39-58; H~ 4-32;
N, 13.85; S, 31.70.
Found: C, 39.46; H, 4.24; N, 14.05; S, 31.63.
2) K. Ogura, et al. Bull Chem. Soc. (Japan), 45, 2203 ('72)
3) D.W-. McDowell et al., J. Org. Chem. 31, 3592 (i66)
4) K. Ogura, et al., Tetrahedron Letters, 1383 (1972).
-30-
D) l-Methylsulfinyl-l-methylthio-2-(3-carboxaldehyde
dimethylacetal-2-thienyl)ethylene (4a).
The compound 4a was prepared by the procedure similar
to that for 4b. Triton B (40% in methanol, 50 ml) was
added to a solution of methyl methylthiomethylsulfoxide -~
(72 g., 0.58 mole) and 3a (108 g, 0.58 mole) in THF
(300 ml) and the mixture was refluxed for 4 hours.
Separation by column chromatography with silica gel
(400 g) eluting with chloroform (5 L) gave 130.5 g (78%)
of 4a as a pale yellow oil.
ir: v maxq 3100, 1580, 1100, 1050 1
nmr: ~ pcpm4 2.42 (3H, s, S-CH3), 2.70 (3H, s, SO-CH3),
3.34 (6H, s, OCH3), 5.56 (lH, s, CH ~ ), 7.20 (lH, d,
o
J=6Hz, thiophene-H~), 7.40 (lH, d, J=6Hz, thiophene-H~),
8.12 (lH, s, -CH=).
E) Ethyl 3-formyl-2-thienylacetate ) (5)
. .
Dry hydrogen chloride (33 g) was absorbed in anhydrous
ethanol (500 ml). To this solution 4a (130 g, 0.45 mole)
was added and the mixture heated under reflux for 5 mins.
The reaction mixture was diluted with water and evaporated
under reduced pressure. The residue was extracted with
benzene (2 x 100 ml) and the benzene extracts were combined,
washed with water (50 ml), dried over MgSO4 and evaporated
to dr~ness. The oily residue was column-chromotographed
on silica gel (400 g) eluting with chloroform (5 L).
,'~3
Fractions containing the desired product were combined
and concentrated. The residual oil (60 g) was distilled
under reduced pressure to afford 23 g (23%) of 5, boiling
at 120-126 C/l mm Hg.
max 3110, 1730, 1670 cm 1
CDCl
nmr: ~ ppm3 1.30 (3H, t, J=6Hz, -CH2CH3), 4.25
(2H, q, J=6Hz, -CH2CH3), 4.26 (2H, s, -CH2CO), 7.25
(lH, d, J=SHz, thiophene-H~), 7.48 (lH, d, J=5Hz,
thiophene-H~), 10.15 (lH, s, CHO).
The analytical sample of 5 was submitted as the 2,4-
dinitrophenylhydrazone which was crystallized from
chloroform. M.p. 178-179 C.
i v nujol 1720, 1610, 1570 cm 1
max
Anal- Calcd. for C15H14N4O6S: C, 47.62; H, 3.73; N,
14.81; S, 8.47.
Found: C, 47.33; H, 3.47; N, 14.77; S, 8.68.
According to the similar procedure 2.2 g (7.6 m moles)
of the ethylene acetal 4b was treated with 1.1 g of dry
hydrogen chloride in 800 ml of anhydrous ethanol to afford
S which was purified by column chromatography on silica
gel (30 g). Elution with chloroform gave 663 mg (44%) of
5 as a pale yellow oil.
- 32 -
134~i~
F) Ethyl 3-formyl-2-thienylacetate oxime (6)
Sodium carbonate (1.7 g, 16 m mole) was added to a -
solution of the aldehyde 5, (3.14 g, 16 m mole) and
hydroxylamine hydrochloride (2.2 g, 32 m mole) in 50%
aq. ethanol (40 ml) at 5 C with stirring. The reaction
mixture was warmed up to room temperature. After 2.5 hrs.,
the reaction mixture was concentrated under reduced pressure.
The residue was extracted with benzene (3 x 50 ml). The
benzene extracts were washed with water (10 ml), dried
over MgSO4, and evaporated under reduced pressure.
Separation by column chromatography on silica gel (60 g)
gave 2.7 g (80%) of colorless oil 6.
ir- v maq 3400~ 1730, 1620 cm 1.
nmr: ~ ppm on d6 1.23 (3H, t, J-7.5Hz, -CH2CH3), 4.01
(2H, s, -CH2CO), 4.14 (2H, q, Js7.5Hz, -C_2CH3), 7.31
(2H, s, thiophene-H), 8.26 (lH, s, -CH~), 10.15 (lH,
s, NOH, disappeared by addition of D2O).
G) The ~-lactam of 3-aminomethyl-2-thienylacetic
acid (7).
.
Method A: Catalytic reduction
A mixture of the oxime 6 (2.65 g. 12.4 m moles),
10% palladium on charcoal, dry hydrogen chloride
(1.4 g, 37.2 m moles) in anhydrous ethanol (68 ml) was
hydrogenated overnight under atmospheric pressure at
room temperature. The catalyst was exchanged twice
and the reaction was carried out over a period of 3 days.
The catalyst was removed and the filtrate was concentrated
under reduced pressure. To the residue was added water
(10 ml) and the mixture washed with ethyl acetate (2 x 10 ml).
The aqueous layer was adjusted to pH 9 with sodium carbonate,
saturated with sodium chloride, and extracted with ethyl
acetate (3 x 20 ml). The ethyl acetate extracts were dried
over MgSO4, treated with charcoal, and evaporated under
reduced pressure. Recrystallization from ethyl acetate
gave 417 mg (22%) of colorless needles 7 melting at
194-l95oc .
ir: v max 3200, 1650, 1480 cm
nmr ~ DMSO-d6 3.53 (2H, t, J=3Hz, -CH2CO-), 4.36
(2H, d-t, J=3, 1.5Hz, changed to a triplet by addition
of D2O, J=3Hz, CH2N), 6.95 (lH, d, J=4.5Hz, thiophene-
H O, 7.45 (lH, d, J=4.5Hz, thiophene-Ha), 8.0 (lH, m,
disappeared by addition of D2O, NH).
Anal. Calcd. for C7H7NOS: C, 54.88; H, 4.61; N,
9.14; S, 20.93.
Found: C, 55.04; H, 4.45; N, 9.13; S, 20.50.
Method B: Zn-dust reduction
To a solution of the oxime 6 (18.3 q, 86 m moles)
in acetic acid (200 ml), zinc dust (17 g, 258 m moles)
was added portionwise over a period of 1 hr. at 40-50C
with vigorous stirring. The reaction mixture was stirred
-34-
overnight at room temperature and heated at 60 C
for 4 hours. The contents were filtered and the filtrate
was concentrated under reduced pressure. To the residual
oil was added water (100 ml) and the mixture washed with
ether (2 x 50 ml). The aqueous solution was layered with
ethyl acetate (100 ml) and adjusted to pH 10 with sodium
carbonate. The precipitate was filtered off. The filtrate
was extracted with ethyl acetate. The ethyl acetate
extracts were washed with water (10 ml), dried over MgSO4,
and evaporated under reduced pressure. The residual solid
was triturated with benzene.
Crystallization from ethyl acetate gave 2.7 g (21%) of the
lactam 7 which was identical to Method A in the IR and the
NMR spectra.
H. 3-Aminomethyl-2-thienylacetic acid (8)
A mixture of the lactam 7 (2.88 g, i8.8 m moles) and
6N hydrochloric acid (50 ml) was heated under reflux for
3 hrs. The reaction mixture was concentrated under reduced
pressure. To the residue was added water (20 ml) and the
mixture treated with charcoal and evaporated under reduced
pressure. The trituration of the residue with THF gave the
amino acid 8 hydrochloride (3.72 g, 95%; m.p. 171-172 C;
ir (KBr) cm ; 3450, 3000, 1700, 1200; nmr (D2O)ppm;
4.80 (2H, s, -CH2CO), 4.27 (2H, s, CH2-N), 7.26
(lH, d, J=6Hz, thiophene-_~), 7.53 (lH, d, J_6Hz,
thiophene-H~). The hydrochloride (3.71 g, 17.9 m moles)
was dissolved in water (10 ml) chromatographed on a
-35-
column of IR-120 (H, 30 ml) and developed successively
with water (100 ml) and 5N-NH40H (2 L). The ammonia
eluate was evaporated to dryness. The residue was
crystallized from aqueous acetone to give 3.0 g (98%)
of 8 m.p. 223-225 C.
ir: v ma 3000, 1620, 1520 cm 1
nmr:~ 2 2 3 3.20 (2H, s, -CH2CO), 4.13
ppm
~2H, s, CH2N), 7.04 (lH, d, J-6Hz, thiophene-H~).
7.30 (lH, d, J=6Hz, thiophene-H~).
Anal. Calcd. for C7HgNO2S: C, 49.10; H, 5.30;
N, 8.18; S, 18.73.
Found: C, 48.53; h, 5.22; N, 7.98; S, 18.97.
I. 3-t-Butoxycarbonylaminomethyl-2-thienylacetic
acid (_)
A mixture of 3-aminomethyl-2-thienylacetic acid 8
(3.1 g, 18 m. moles) and triethylamine (8 g. 80 m moles)
in 50~ aqueous acetone (80 ml) was added dropwise
t-butoxycar~onyl azide (5.7 g. 40 m moles) over a period
of 20 mins. at 0C with vigorous stirring. The reaction
mixture was stirred overnight at room temperature and
concentrated under reduced pressure. The concentrate was
washed with ether (2 x 20 ml), adjusted to pH 3 with conc.
HCl and extracted with ethyl acetate (2 x 50 ml). The
ethyl acetate extracts were washed with saturated
-36-
aqueous sodium chloride, dried over MgS04, treated
with charcoal and evaporated under reduced pressure.
The residue was triturated with n-hexane and c~ystallized
from n-hexane and benzene to give 4.5 g (92~) of colorless
5 needles 9, melting at 62 - 63C.
ir: v NUJOL* 3350, 1700 cm 1
max
nmr ~ CDC13 1.43 (9H, s, BOC-_), 3.27 t2H, s,
CH2CO), 4.16 (2H, d, J=6Hz, CH2-N, a singlet when D20
was added), 5.00 (lH , br, -NH- , disappeared by addition
of D20), 6.30 (lH, broad s, -COOH, disappeared by addition
of D2O), 6.86 (lH, d, J=6Hz, thiophene-_~), 7.06
( lH, d, J= 6Hz, thiophene-_~).
Anal. Calcd. for C12H17NO4S: C, 52.89; H, 6.29;
N, 5.14; S, 11.77.
Found: C, 53.30; H, 6.39; N, 5.13; S, 11.72.
1~13~;
PreParation of 2-N-Methylaminomethyl-4-methoxy-(and 4-
hydroxy-~phenylacetic Ac_ds
HO ~ CH2NH2 Ts-Cl H ~ H2NH-TS CH I
CH2C02H > ~ H2CO2H 3
CH
H~CO ~ CH2N-Ts Na/liq.NH3 H3C0 ~ CH2NH-cH3
2 2H CH2C02K
4a
IHBr
H0 ~ CH2CO2H
4b
2-N-Tosylaminomethyl-_-hydroxyphenylacetic Acid (2)
To a solution of 14.56 g. (0.08 mol.) of 2-amino-
methyl-4-hydroxyphenylacetic acid (1) (U.S. 3,823,141) and
13 g. (0.32 mol.) of sodium hydroxide in 200 ml. of water
was added dropwise with stirring at 65-70 C. a solution
of 18.5 g. (0.097) mol.) of p-toluenesulfonyl chloride in
50 ml. of dry ether and the mixture was kept at the same
temperature for one hour. The mixture being cooled, the
aqueous layer was separated, washed with ether (2 x S0 ml.)
acidified with 6N HCl and extracted with 400 ml. of ethyl
acetate. The extract was washed with
-38-
i7~
S;~
water and a saturated aqueous NaCl solution, dried with
Na2SO4 and treated with active carbon (1 g.). The fil-
trate was concentrated to dryness and the residue was
crystallized from ethyl acetate to give 11.0 g. (40.5%)
of 2 melting at 212-215 C.
ir: max 3240, 1700, 1380, 1330, 1150 cm 1
uv: ~m%aKX2cO3 230 nm (~: 7,750)
nmr:~ ppm d6 2.47 (3H, s, Ar-CH3), 3.60 (2H, s,
CH2CO), 3.93 (2H, d, J=6.0 Hz, CH2N), 6.6-8.2 (7H, m,
phenyl-H).
2-(N-Methyl-N-tosylamino)methyl-4-methoxyphenylacetic
Acid (3)
A mixture of 11 g. ~0.033 mol.) of 2, 10.3 ml.
(0.17 mol.) of methyl iodide and 9.2 g. (0.24 mol.) of
sodium hydroxide in 100 ml. of water was heated at
80-90 C. for 45 minutes in a sealed tube with occasional
shaking. The mixture was washed with ethyl acetate (30
ml.) and the water layer was acidified with 6N HCl and
extracted with ethyl acetate (3 x 30 ml). The combined
extracts were washed with water (30 ml.) and a saturated
aqueous NaCl solution (30 ml.) treated with active carbon
(1 g.) and dried over Na2SO4. The filtrate was evaporated
to dryness and the residue was crystallized from benzene
to give 8 g. (66.5%) of the N,O-dimethyl derivative 3
melting at 146-150C.
- ir: v mar 1690, 1500, 1340, 1280, 1150 cm
uv: ~Etax 229 nm (~: 20500), 278 nm (ç: 2400).
-39-
'a~l
., ..~
L~
ppm 2.52 (3H, s, N-CH3), 2.47 (3H, s,
Ar-CH3), 3.67 (2H, s, CH2CO), 3.74 (3H, s, OCH3), 4.10
(2H, s, CH2N), 6.7-7.8 (7H, m, Ar-H), 11.5 (lH, ~r-s,
COOH).
Anal. Calc'd. for C18H21NO5S: C, 59.49; H,
5.82; N, 3.84; S, 8.82. Found: C, 59.48; H, 5.68;
N, 3.37; S, 9.22.
2-N-Methylaminomethyl-4-methoxyphenylacetic Acid (4a)
To a solution of liquid ammonia (300 ml.) was
added 9.4 g. (0.026 mol.) of 3 at -50 C. and the
mixture was stirred until a clear solution was obtained
at the same temperature. To the solution was added 3.3 g
(0.14 g. atom) of Na in small pieces at -40 C. and the
mixture was stirred for 2 hours. Ammonia was evaporated
and the _esidue was dissolved in 100 ml. of water carefully.
To the solution was added 100 ml. of Amberlite I~-C 50
(ammonium type) and the mixture was stirred for 30 minutes
at room temperature. The resin was remo~ed and the filtrate
was treated with barium acetate until no more precipitate
was observed. The precipitate was filtered off and the
filtrate was chromatographed with a column of IR-120 ion-
exchange resin (H , 100 ml.) by eluting with 5-10% ammonia.
The eluate (2 L) containing the desired product was
evaporated to dryness below 50 C and the residue was
triturated with acetone to give 4.4 g. (81~) of 4a, m.p.
225-227 C.
max 1590, 1380, 1260, 1035 cm
-40-
-
~$1~
nmr: ~ 2 2.77 ~3H, s, N-CH3), 3.6 (2H, s, CH2C~),
ppm
3.87 (3H, s, OCH3~, 4.18 (2H, s, CH2N), 6.8-7.4 (3H, m,
phenyl-H).
2-N-Methylaminomethyl-4~hydroxyphenylacetic Acid (_)
A mixture of 2.9 g. (0.014 mol.) of 4a in 30
ml. of 48% hydrobromic acid was refluxed for 5 hours and
the solution was evaporated to dryness. The residue was
dissolved in 50 ml. of water. The solution was chroma-
tographed on a column of Amberlite IR-120 (H , 50 ml.)
eluting with 5-10% ammonia. The eluate was collected
in 250 ml. fractions. Fractions containing the product
were combined and evaporated to dryness below 50 C.
The residue was triturated with acetone to give 1.3 g.
(48.5%) of 4b, which was crystallized from 80~ ethanol.
M.p. 218-221 C.
ir: KBr 2000-3400, 1610, 1540, 1460, 1380, 1270 cm
uv:~ 1%K2CO3243 nm (:4700)~ 297 nm (~: 1350).
max
nmr: ~ ~20~NaOH2.64 (3H, s, N-CH3), 3.47 (2H, s,
ppm
CH2CO), 3.94 (2H, s, N-CH2), 6.5-7.2 (3H, m, phenyl~
Anal. Calc'd. for CloH13NO3: C, 61.53; H, 6.71;
N, 7.17. Found: C, 61.44; H, 6.81; N, 7.20.
2-N-t-Butoxycarbonyl-N-methylaminomethyl-4-methoxyphenyl-
acetic Acid (_, R = CH3)
A mixture of 1.05 g. (5 m.mol.) of 4a, 1.43 g.
(6 m.mol.) of t-butyl 4,6-dimethylpyrimidin-2-ylthiol-
carbonate and 1.4 ml. of triethylamine in 40 ml. of 50%
THF was stirred at room temperature for 20 hours. Most
*Trade Mark
-41-
of the THF was evaporated and the resulting aqueous solu-
tion (ca. 20 ml.) was washed with ether. The water layer
was acidified with 6N HCl and extracted with ether (3 x
10 ml.). The ethereal extracts were washed wi~h wat~r
(10 ml.) and a saturated aqueous NaCl solution (10 ml.),
treated with a small amount of active carbon and dried
over Na2SO4. The filtrate was evaporated to dryness to
give 1.0 g. (77.5%) of 5 (R = CH3) as an oil.
nmr ~ CDcl3 1.47 (9H, s, BOC-_), 2.77 (3H, s, N-CH3),
ppm
3.60 (2H, s, CH2CO), 3.79 (3H, s, O-CH3), 4.49 (2H, s,
CH2N), 6.1-7.3 (3H, m, phenyl-_).
2-N-t-Butoxycarbonyl-~-metnylaminomethyl-4-hydroxyphenyl-
acetic Acid (5, R = H)
A mixture of 1 g. (4.78 m.mol.) of 4b, 1.5 g.
(6.3 m.mol.) of t-butyl 4,6-dimethylpyrimidin-2-ylthiol-
carbonate and 2.1 ml. of triethylamine in 50 ml. of 50~
aqueous THF solution was stirred at room temperature for
20 hours. The mixture was concentrated to 20 ml. under
reduced pressure. The concentrate was washed with ether
(10 ml.), acidified with 6N HCl and extracted with ethyl
acetate (2 x 100 ml.). The combined extracts were washed
with water (30 ml.) and a saturated aqueous NaCl solution
(2 x 30 ml.), treated with a small amount of active car-
bon and dried over anhydrous Na2SO4. The filtrate was
evaporated ~o dryness to give 1.3 g. (92%) of 5 (R = H)
as an oil.
ir: v max 3000-3600, 1570, 1260, 1150 cm 1.
nmr: ~ 3 1.44 (9H, s, C(CH3)3), 2.73 (3H, s, N-CH3),
-42-
3.54 (2H, s, CH2CO), 4.38 (2H, s, CH2N), 6.5-7.3 (3H, m,
phenyl-H).
Preparation o~ Ortho-N-methylaminomethyl-phenylacetic Acid.
O2H CH ~ SO2C
H2CO2H CH3
CH2NS~CH3 HBr
CH2C02H - -
Na/liq~NH3
H2C02H CH3
B0C CH3-c-0c0-
CH3
- 43 -
3~
o-(p-Toluenesulfonylaminomethyl)ph-nylacetic Acid (2)
To a stirred solution of o-aminomethylphenyl-
acetic acid hydrochloride (7.50 g., 37 m.mol.) and sodium
hydroxide (4.74 g., 118 m.mol.) in water (100 ml.) was
added p-toluenesulfonyl chloride (7.64 g., 40 m.mol.)in
portions at 60 C. The mixture was stirred for 1 hour
at the same temperature and acidified with hydrochloric
acid. The mixture was extracted with ethyl acetate (4 x
50 ml.). The combined extracts were washed with water,
treated with a small amount of carbon and dried. The
solvent was evaporated under reduced pressure and the
residue crystallized from ethyl acetate to afford 2 as
colorless prisms. Yield, 9.84 g. (84%). M.p. 155-
156C
ir:v mUa~ 3300~ 1705, 1355, 1170 cm 1
nmr: ~ ppm 6 2.38 (3H, s, CH3), 3.65 (2H, s, CH2CO),
3.97 (2H, d, J=5 Hz, CH2N), 7.1-8.2 (9H, m, phenyl-H &
NH).
Anal. Calc'd. for C16H17NO4S: C, 60.17; H,
5.37; N, 4.39; S, 10.10. Found: C, 60.11, 60.15; H,
5,43, 5.40; N, 4.28, 4.30: S, 9.72, 9.80.
o-(N-p-Toluenesulfonyl-N-methylaminomethyl)phenylacetic
Acid (3)
A mixture of 2 (9.0 g.,28 m.mol.) sodium hy-
droxide (6.0 g.) and methyl iodide (6 ml.) in water
(60 ml.) was heated in a sealed tube for 30 minutes at
70 C. After cooling, the reaction mixture was acidified
with hydrochloric acid to separate pale yellow precipitate
which was crystallized from ethyl acetate-n-hexane to
give colorless prisms, 3. Yield, 8.5 g. (91%). M.p.
162-163 C.
v max 2700-2300, 1700, 1600, 1345, 1200, 925 cm 1
D20+KOH
nmr: ~ppm 2.37 (3H, s, CH3), 2.49 (3H, s, CH3),
3.80 (2H, s, CH2CO), 4.18 (2H, s, CH2N), 7.0-8.0 (8H,
m, phenyl-H).
Anal. Calc'd. for C17HlgNO2: C, 61.24; H,
5.74; N, 4.20; S, 9.61. Found: C, 61.31, 61.36; H,
5.73, 5.71; N, 4.51, 4.29; S, 9.63, 9.55.
N-Methylaminomethylphenylacetic Acid (4)
Method A (using hydrobromic acid) - A mixture of
28.6 g. (0.086 mol.) of 3 and 20 g. (0.213 mol.) of phenol
in 260 ml. of 48% hydrobromic acid was refluxed for 30
minutes. The mixture was cooled, diluted with the same
volume of water and washed with ethyl acetate (2 x 50 ml.).
The aqueous layer was evaporated to dryness in diminished
pressure to give an oil which was chromatographed on a
column of Amberlite IR-120 (H form, 200 ml.) eluting with
5~ ammonium hydroxide solution. The eluate (2.5 1) was
collected and evaporated to dryness under reduced pressure.
The residue was triturated with acetone and crystallized
from ethanol to afford 6.7 g. (43.5%~ of 4 as colorless
needles, melting at 168-170 C. (dec.).
Method B (using metallic sodium in liquid
ammonia) - To a mixture of 3 (35 g., 0.105 mol.) in
liquid ammonia (1000 ml.) was added 13.3 g. (0.578 atom)
-45-
of sodium in small pieces under vigorous stirring over
a period of 2 hours. The ammonia was evaporated with
stirring on a water-bath in a well-ventilated hood and
finally under reduced pressure to remove it completely.
The residue was dissolved in ice water (~00 ml.) and
the solution was stirred with ion-exchange resin IRC-50
(H form, 400 ml.) for 0.5 hour at room temperature.
The resin was filtered off and to the filtrate was added
an aqueous 1 M solution of barium acetate until no
more precipitate was formed (ca 50 ml. of the barium
acetate solution was required). The mixture was fil-
tered and the filtrate was chromatographed on a column
of IR-120 (H form, 400 ml.) as in Method A to give
13.6 g. (72%) of 4.
o-(N-~ethyl-N-t-butoxycarbonylaminomethyl)phenylacetic
Acid ~
t-Butyl 4,6-dimethylpyrimidin-2 ylthiolcarbonate
(11 g., 0.048 mol.~ was added in one portion to a mixture
of 4 (7.2 g., 0.04 mol.) and 1,1,3,3-tetramethylguanidine
(6.9 g., 0.06 mol.) in 50% aqueous THF and the mixture
was stirred overnight at room temperature. The THF being
evaporated under reduced pressure, the aqueous solution
was acidified to pH 2 with dil. hydrochloric acid and
extracted with ethyl acetate ~2 x 20 ml.). The combined
extracts were washed with water, treated with a small
amount of active carbon and evaporated under diminished
pressure. The residue was triturated with hexane and
crystallized from n-hexane-ether to afford 9.2 g. (8~%)
of 5 as colorless prisms. M.p. 96-98 C.
3Q
*Trade Mark
-46-
ir: `~ Kax 1730, 1630, 1430, 1830, 1250 cm 1,
nmr: ~ CDC13 1.49 (9H, s, t-butyl), 2.78 ~3H, s,
N-CH3), 3.72 (2H, s, CH2CO), 4.25 (2H, s, CH2N), 7.28
(4H, s, phenyl), 9.83 (lH, s, -COOH).
Anal. Calc'd. for C15H21NO4: C, 64.50; H,
7.58; N, 5.01. Found: C, 64.69; H, 7.66; N, 4.89.
-
- 47 -
~13~
Preparation of 3-N-methylaminomethyl-2-thienylacetic
Acid.
CH~NOH ~ NaH, C
S CH2C02C2H5 ~ ~ ~ ~ benzene
3 ) ~ CH2~~CH3
3 / 4
H3
2N-BOC
~ S ~CH2cooH
3-Aminomethyl-2-thienylacetic Acid ~--lactam (2)
Glacial acetic acid (140 ml.) was added dropwise
with stirring to a mixture of 2-ethoxycarbonylmethylthiophene-
3-carboxaldehyde oxime (1) (41 g., 0.19 mole) and zinc dust
(65.4 g., 1 mole) in methanol, and the mixture was stirred
under reflux for 4 hours. The mixture was cooled and insolubles
were removed by filtration and washed with methanol (3 x 50
ml.). The filtrate was combined with the washings and evap-
orated _ vacuo to dryness, the residue being extracted with
methanol (5 x 100 ml.). The methanol extracts were
combined and evaporated under reduced pressure. To the
-48-
~$~
residue was added water (50 ml.) and the mixture was
adjusted to pH 10 with Na2CO3 and extracted with chloro-
form (3 x 100 ml.). The combined chloroform extracts
were washed with water (10 ml.), dried over MgSO4, and
evaporated under reduced pressure. The residual oil
(30 g.) was trituxated with hot benzene (150 ml.). The
colorless needles were collected by filtration and re-
crystallized from ethyl acetate to give the lactam 2
(7.7 g., 26%), melting at 195~196C.
W max 232 nm (~, 6500)
Anal. Calc'd. for C7H7NOS : C, 54.88; H, 4.61;
N, 9.14; S, 20.93. Found: C, 55.04; H, 4.45; N, 9.13;
S, 20.50.
3-N-Methylaminomethyl-2-thienylacetic Acid ~-lactam (3)
To a suspension of sodium hydride (50% in paraf-
fin, 1.82 g., 38 m. moles) in absolute benzene (500 ml.)
was added the lactam 2 (4.85 g., 32 m.moles) with stir-
ring under nitrogen atmosphere and the mixture was re-
fluxed for 2 hours. Methyl iodide (22.7 g., 160 m.moles)
was added in one portion at room temperature and the mix-
ture was again refluxed for 2 hours. Ice-water (50 g.)
was added to the mixture and organic layer was separated.
The aqueous layer was extracted successively with ben-
zene (2 x 50 ml.) and chloroform (50 ml.) The extracts
were combined and dried on MgSO4. The solvent was
e~aporated under reduced pressure. To the residue was
added a hot mixture of benzene-n-hexane (1:1, 100 ml.)
to recover 2 as needles ~2.02 g., 42%). The filtrate
-49-
~'
~3~
was evaporated and the residue was crystallized from
benzene-n-hexane to afford colorless plates 3. Yield:
2.7 g. (51~). M.p. 98-100 C.
ir v nUjol 1620 cm 1
max
~ CHCl
nmr: max 3.15 (3H, s, N-CH3), 3.72 (2H, t, J=3Hz,
CH2CO), 4.53 (2H, t, J=3Hz, -CH2-N), 6.87 (lH, d, J=4.5Hz,
thiophene-He), 7.30 (lH, d, J=4.5 Hz, thiophene-H~).
uv: ~ MeOH 232 nm (E, 6700)
max
Anal. Calc'd. for C8Hg~OS C, 57.46; H, 5.42;
N, 8.38; S, 19_17. Found: C, 57.56; H, 5.26; N, 8.31;
S, 19.13.
3-(N-Methylaminomethyl)-2-thienylacetic Acid (4)
A mixture of the lactam 3 (3.5 g., 21 m. moles)
and 6N HCl (100 ml.) was heated under reflux for 12 hours.
The mixture was treated with carbon and concentrated to
dryness under reduced pressure. The residual oil was dis-
solved in water (10 ml.) and chromatographed on a column
of IR=120 (H+, 50 ml.). The column was eluted with water
(200 ml.) and 5N NH40H ~3 L.~. The amino acid 4 (3.0 g.,
77%) was isolated by evaporation of the ammonia eluates
followed by crystallization from aqueous acetone.
M.p. 181-182 C.
ir KBr 1570, 1360 cm
nmr: ~ 2 2.21 (3H, s, N-CH3), 3.80 (2H, s, CH2CO),
ppm
4.20 (2H, s, CH2-N), 7.19 (lH, d, J=6Hz, thiophene-H~),
7.46 (lH, d, J=6Hz, thiophene-HN).
uv: ~ ~2
max 237 nm (~, 7600)
-5~-
~ 3c ~ ~
Anal. Calc~d. for C8HllNO2S C, 51.87; H,
5.99; N, 7.56; S, 17.31. Found: C, 51.67; H, 6.50;
N, 7.28; S, 16.69.
3-(N-t-butoxycarbonyl-N-methylaminomethyl)-2-thienyl-
AcetiC Acid (_)
To a mixture of 3-N-methylaminomethyl-2-
thienylacetic acid 4 (2.7 g., 14.6 m.moles) and tri-
ethylamine (6 g., 60 m. moles) in 50% a~ueous acetone
(60 ml.) was added dropwise t-butoxycarbonyl azide ~4.2
g., 29.2 m.moles) over a period of 20 minutes at 0 C.
with vigorous stirring. The reaction mixture was
stirred overnight at room temperature and concentrated
under reduced pressure. The concentrate was washed with
ether (2 x 20 ml.), adjusted to pH 2 with concentrated
HCl and extracted with ethyl acetate (2 x 50 ml.). The
ethyl acetate extracts were washed with a saturated
aqueous NaCl solution, dried on MgSO4~ treated with
charcoal and evaporated under reduced pressure. The
residue was triturated with n-hexane and crystallized
from n-hexane-benzene to give 3.68 g. (88%) of colorless
needles 5 melting at 82-83 C.
ir: v max 1730, 1640 cm 1
nmr: ~ CDC13 1.47 (9H, s, BOC-H), 2.78 (3H, s, N-CH3),
3.87 (2H, s, CH2-CO), 4.48 (2H, s, CH2-N), 6.91 (lH, d,
J=6Hz, thiophene-H~), 7.20 (lH, d, J=6Hz, thiophene-H~),
10.63 (lH, s, CO2H, disappeared by addition of D2O).
Anal. Calc'd. for C13HlgNO4S: C, 54.72; H,
6.71; N, 4.91; S, 11.24 Found: C, 54.91; H, 6.85;
N, 4.92; S, 11.19.
~ -51-
-
3~
The use of an "en-amine" blocking group with a
prospecti~e 7-side chain containing a free amino group
prior to acylation of a nucleus such as II herein is
well known as from U.S. 3,223,141, U.S. 3,813,390, -
U.S. 3,813,391, U.S. 3,823,141 and Belgium 773,773.
Sodium 2-[N-(l-carbethoxypropen-2-yl)aminomethyl]-1,4-
cyclOnexadienYl acetate (4)
To a stirred solution of 460 mg. (0.02 g. atom) of
metallic sodium in 100 ml. of absolute EtOH was added 3.34 g.
(0.02 mole) of 2-aminomethyl-1,4-cyclohexadienylacetic acid
and 3.1 g. (0.024 mole of ethyl acetoacetate and the mixture
was heated to reflux for 4 hours with stirring. The hot
reaction mixture was filtered and the filtrate was allowed
to keep cold overnight to give 2.0 g. of colorless
needles 4 melting at 264 C. The additional product
(3.3 g.) was obtained by concentration of the mother
liquid. The total yield was 5.3 g. (88%) .
IR v maU~ 3300, 1635, 1600, 1570, 1300, 1275, 1170,
1090 cm 1.
NMR: ~ D2O 1.23 (3H, t, 7Hz, CH2CH3), 1.96 & 2.25
ppm
(3H, s, C=C-CH3-, cis & trans), 2.70 (4H, s, _2C ~=),
3.04 (2H, s, CH2CO), 3.66 & 3.95 (2H, s, C_2-N, cis &
trans), 4.07 (2H, q, 7Hz, CH2CH3), 4.45 & 4.56 (lH,s,
=/ , cis & trans), 5.76 (2H, s, H ~ )
Anal- Calcd. for C15H20NO4Na: C, 59.79; H, 6.69;
N, 4.64.
Found: C, 59.69; H, 6.76; N, 4.75.
-52-
2-t-Butoxycarbon~laminomethyl-4-hydroxyphenylacetic
acid is prepared, for example, accordin~ to U.S. 3,82~,141.
o-(N-Methylaminometh~l)phenylacetic acid ~-lactam
~ -CH3
Sodium hydride (57Z in paraffin, 4.3 g.; 0.11 mol.) was
washed with ~ry n-hexane and suspended in dry benzene (100
ml.). To the suspension was added a solution of o-am~no-
methylphenylacetic acLd ~-lactam (U.S. 3,796,716) (14.7 g.,
0.1 mol.) in dry benzene or xylene (200 ml.) with stirrlng
under a nitrogen atmosphere. The mixture was refluxed for
one hour and cooled to room temperature. To the mixture was
added methyl iodide (18 ml.) in one portion and the mixture
was refluxed again for 1.5 hours. The reaction mixture was
cooled to room temperature and poured into ice-water (100 ml.).
The aqueous layer was separated from the organic layer and
extracted with CHC13 (2 x 50 ml.). The extracts were combined
with the organic layer and dried on ~gS04. The solvent was
removed and the o~ly residue was distilled in vacuo to afford
14~9 g. (92%) of o-(N-methylaminomethyl)phenylacetic acid
~-lactam, boiling at 130-135C/2 mmHg., m.p. 35-37C.
lr: vmax 3300, 1620, 1490 cm 1,
nmr: ~ppml3 3.12 (3H, s), 3.59 (2H, t, J_ 1.5 Hz), 4.48
(2HI t, J- 1.5 Hz), 7.21 (4H, br-s).
- 53 -
Anal. Calc'd. for CloHllN0-1/4H20: C, 72.49; H, 6.84;
N, 8.45. Found: C, 72.78, 72.70; H, 6.76, 6.81; N, 8.49,
8.51.
o-N-MethYlaminomethYlphen21acetic acid
2~ NHCH
CH2cooH
A mixture of the above-produced o-(N-methylaminomethyl)-
phenylacetic acid ~-lactam (5.0 g., 0.031 mol) and conc
hydrochloric acid (500 ml,) was refluxed for 40 hours. The
mixture was evaporated ~Inder reduced pressure, and the re-
sidual oil was dissolved in water (20 ml.) and treated with
a small amount of active carbon. The flltrate was washed
with benzene (50 ml.) and evaporated to drynes~. The
residual oil was crystallized by trituration with THF (or
scetone) to give colorless needles of o-N-methylamino-
methylphenylacetic acid hydrochloride (4.5 g., 67%). Anal.
Calc'd- for CloHl3No2.Hcl C, 55.69; H, 6.54; N, 6.49;
Cl, 16.44. ~ound: C, 55.65, 55.74; H, 6.62, 6.60; N,
6.53, 6.53; Cl, 16.36.
Some unreacted starting material was recovered fr.~m
~he benzene layer and the THF washings (1.2 g., 2470, b.p.
140-143C/2mmHg~,
- 54 -
An aqueous solution of o-N-methylaminomethylphenylacetlc
acid hydrochloride (5 g.) was column chromatographed with
IR-120 ion-exchange resin (H , 70 ml,) and eluted with 3N
NH40H (2 1) to afford 3.9 g. (937O) of o-N-methylaminomethyl-
phenylacetic acid as needles.
ir:~KBX 1650, 1470 cm 1,
The following examples are given in illustration
of, but not in limitation of, the present invention.
All temperatures are in degrees Centigrade. 7-Amino-
cephalosporanic acid is abbreviated as 7-ACA; -ACA
represents the moiety having the structure.
-NH ~H _C ~ \ H
C
COOH
and thus 7-ACA can be represented as H-ACA-O-C-CH3.
Methyl isobutyl ketone is represented as MIBK.
"Skellysolve B" is a petroleum ether fraction of
B.P. 60-68 C. consisting essentially of n-hexane.
LA-l resin is a mixture of secondary amines
wherein each secondary amine has the formula
IR
3( 2)10 2 f
R3
wherein each of R , R and R is a monovalent aliphatic
hydrocarbon radical and wherein Rl, R2 and R3 contain
in the aggregate from eleven to fourteen carbon atoms.
This particular mixture of secondary amines, which is
sometimes referred to in these examples as "Liquid
Amine Mixture No. II," is a clear amber liquid having
the following phy~ical characteristics: viscosity at 25 C.
of 70 cpd., specific gravity at 20 C. of 0.826;
refractive index at 25 C. of 1.4554; distillation range
at 10 mm., up to 170 C. - 0.5%, 170-220 C. - 3%,
220-230 C. - 90% and above 230 C - 6.5%.
-56-
IR-120 is also called Amberlite IR-120 and
is a strong cation exchange resin containing sulfonic
acid radicals. Amberlite IR-120 is a commercially
available cation exchange resin of the polystyrene
sulfonic acid type; it is thus a nuclear sulfonated
polystyrene resin cross-lined with divinyl benzene
obtained by the procedure given by Kunin, Ion
Exchange Resins, 2nd. Edition (1958), ~ohn Wiley and
Sons, Inc. Therein see pages 84 and 87 for example.
Amberlite IRC-50 is a commercially available
cation exchange resin of the carboxylic type; it is
a copolymer of methacrylic acid and divinylbenzene.
Dicyclohexylcarbodiimide is abbreviated as
DCC, tetrahydrofuran as THF, thin layer chromatography
as TLC, p-toluenesulfonyl as Ts and methanol as MeOH.
When the following instrumental readings are
given, for infrared nu if used is written v, for
ultraviolet lambda is written as ~, with molar absorp-
tivity as epsilon (~) and for nuclear magnetic resonance
(nmr) delta is written as~ and tau as r ( ~ = 10- J ) .
-57-
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Synthesis (Schemes 1, 2 and 3)
The 3-side chain thiol, 2-carboxymethyl-2,3-
dihydro-s-triazolo~4,3-b~pyridazin-3-Qn-6-ylthiol (3),
was prepared by N-ethoxycarbonylmeth~lation of 6-chloro
2,3-dihydro-s-triazolo[4~3-b]pyridazin-3-One (1) with
sodium hydride and ethyl chloroacetate in D~IF (dimethyl-
formamide) and subsequent thiolation with sodium hydro-
sulfide (Scheme 1). Condensation of 7-ACA (7-amino-
cephalosporanic acid) with 3 carried out by refluxing in
phosphate buffer (pH 7) to give the 3-substituted-thio
7-ACA (4), which was coupled with an appropriate N-BOC-
protected amino acid by the active ester method using
2,4-dinitrophenol (DNP). The resulting N-BOC-protected
cephalosporins 7 and 11 were deblocked with TFA (tri-
fluoroacetic acid) and converted to the monosodium salt
with N sodium hydroxide (Schemes 2 and 3).
- 58 -
1~3'~
Scheme 1. Pre~aration of 7-Amino-3-(2-c~rbo~ethyl-
2?3-dihydro-s-triazolo~4,3-b]pyridazin-3-on-6-ylthio-
methyl~-3-cephem-4-carboxylic Acid.
N NaH/DMF - ~ N
H2COOC2H
Cl ~ N,N ~ NH -- 5~ Cl- ~ N,N ~ N-cH2cooc2H5
NaSH ~ Nl 7-ACA
> HS-~N~N ~ N-CH2COOH
o
H2N ~ ~ N
0
CG2H
. . ~
- 59 ~
liî~f~
Scheme 2. Preparation of ~-r (o-Aminomethylphenyl~-
acetamidol-3-(2-carboxymethyl-2,~-dihydro-s-triazolo-
[4,~-blpyridazin-3-on-6-ylthiomethyl)-3-cephem-4-
carboxylic Acids.
R2 ~ N-BOC 2, 4-DNP/DCC
COOH
Rl
R2 ~ COO ~ + 4
N02
Rl
R2 ~N-BOC
CONH ~ ~ ~ N
N ~
C 02H O
7 TFA
Rl ~
R2 ~ `CONH ~ ~ IN
N~f~LCH2S ~r~ N-CH2cOOH
CO2~
a: R1 = H, R2 = H 8a: BB-S469
b R = CH3, R = H 80: BB-S479
c~ R = CH3, R - OH 8c: BB-S478
- 6C -
Scheme 3. Preparation of 7-(3-Aminomethyl-2-thienyl-
acetamido)-3-(2-carboxymethyl-2,~-dihydro-s-triazolo-
~4,3-bl~yridazin-3-on-6-ylthiomethyl)-3-cephem-4-
carboxylic Acids.
~' .
N-BOC 2,4-DNP/DCC
" COOH
~oo ~3No2 . _ >
N2
R
-BOC
NH T~ N
,~N~CH2s N'Nb~ N-cH2cooH
C02H O
11 CF3COOH
. IR ,
~S ~ ONH ~ ~
CH2S N'N`~ N-cH2cooH
C2H o
a: R = H 12a: BB-S483
b R = CH3 12b: BB-S472
- 61 -
6-chloro-2~3-dlhydro-2-ethoxycarbonylmethyl-s-tria
r 4~blp.yridazin-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
~ormation 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 co~bined, dried over anhydrous ~odium
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: v KBx 1735, 1710 cm 1
uv: 1 EaxH 231 nm (8, 26000)
nmr: ~ pCpC13 7.58 (lH, d, J=10 Hz, pyridazine-H), 6.98
(lH, d, J=10 Hz, pyridazine-H), 4.80 (2H, s, -CH2C0),
4.27 (2H, q, J=7.5 Hz, CH2CH3), 1.29 (3H, t, J=7.5 Hz,
CH2CH3 ) .
Anal. Calc'd. for CgHgN403Cl C, 42.12; H, 3.53;
N, 21.83; Cl, 1~.81. Found: C, 41.54, 41.46; H, 3.22,
3.49; N, 21.51, 21.53; Cl, 13.88, 13.99.
- 62 -
3 ~i3~_v~
2-Carboxymeth~1-2~3 ~ ydro-6-mercapto-s-triazolor4,3-bl-
~ridazin-3-one (3)
To a solution of 6-chloro-2,3-dihydro-2-ethoxy-
carbonyimethyl-s-triazolo~4,3-b~pyridazin-3-one (2, 30 g.,
0.12 mole) in ethanol (900 ml.) was added NaSH 2H20 (70
pure, 45.9 g., o.36 mole) and the mixture was refluxed
for 0.5 hour. The reaction mixture was evaporated at re-
duced pressure. The residue was dissolved in water (200
ml.) and concentrated HCl was added to the solution to
ad~ust to pH 2. The precipitate (3) was collected by fil-
tration and washed with water. Yield 18.3 g. (69~).
ir: ~ Ka~xr 2900, 2450, 1750, 1660 cm 1
uv ~ 1%NaHC03aq- 260 nm (~,19500), 313 nm (~ 700)
nmr: ~ D~S-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, 2.26,
2.28; N, 23.58, 23.69; S, 14.32.
7-Amino-3-(2-carboxymethyl-2,3-dihydro-s-triazolor4,3-bl-
2yridazin-3-on-6-ylthiomethyl)-3-ce~hem-4-carboxylic Acid (4)
.
To a suspension of 7-aminocephalosporanic acid
(8.79 g., 32.2 m.mole) ln 0.1 M phosphate buffer (pH 7,
149 ml.) were added NaHC03 (8.14 g., 97.0 m.mole) and the
thiol 3 (7.30 g., 32.2 m.mole) with stirrlng. The mix~ure
was heated at 80 C. for 0.5 hour under N2 stream. ~1e
mixture was treated with active carbon and adjusted to
pH 3 with concentrated HCl. The resulting precipitate was
collected by filtration and washed with water t~ give 7.59 g.
(54~) of 4.
- 63 -
ir~ xr 1800, 1720, 1600, 1540, 1470 cm~l
uv: ~ Buffer (P~ 7~ 252 nm (~,19500), 29& nm (~, 8400).
nmr:C~ 3~fK2C3 7.56 (lH, d, J=9 Hz, pyridazlne-H),
7.o5 (lH, d, J=9 Hz, pyridazine-H), 5.45 (lH, d, J=~ Hz,
6-H), 5.05 (lH, d, 5 Hz, 7-H), 4.43 (lH, d, J=14 H ,
3-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).
EX.4MPLE 1
Pre~aration of BB-S469
~C CH2NH2 ~f
N ~ H2 S N'N ~ N-C~2COOH
C2H
8a, BB-S469
2,4-Dlnitro~henyl o-(N-butoxycarbonylaminomethyl) ~ -
acetate (6a)
To a mixture of o-(N-butoxycarbonylaminomethyl)-
phenylacetic acid (5a, 13g., 49 m.mole) and 2,4-dinitro-
phenol (9.02 g., 49 m.mole) in dry ethyl acetate (123 ml.)
was added dicyclohexylcarbodiimide (DCC) (10.1 g., 49 m.
mole) under water cooling (5-15 C.), and the mixture was
stirred for 30 minutes at the same temperature and then
~or 40 mlnutes at room temperature. The resulting preci-
pitate was filtered off and the filtrate was evaporated to
give 23.9 g. of the active ester 6a, which was used in the
next acylation reaction without further purification.
ir: ~ maBx 1775, 1700, 1600, 1530 cm 1,
7-r o- (N-Butoxycarbonylaminomethyl)phenylacetamido]-3-(2-
. . ~ _ ~ . . ,
carboxymethyl~2 3-dihydro-s-triazolo~4,3-blpyridazin-3
on-6-ylthiomethyl)-3-cephem-4-carboxylic Acidj(7a)
To a cold (0 C.) mixture of 4 (4.38 g., 10
m.mole), Et3N (4.5 ml., 30 m.mole), CH~CN (20 ml.) a~d
water (20 ml.) was added a solution of 2,4-dinitrophenyi
- 65 -
L~
o-(N-butoxycarbonylaminomethyl)phenylacetate (6a, 4.79 g )
in THF (tetrahydrofuran) (20 ml.). After stirring at roo~
temperature overnight, THF and CH3CN in the reaction mix-
ture were removed at reduced pressure and the resulting
aqueous solution was adjusted to-pH 2 with dilute HCl and
extracted with ethyl acetate (10 x 30 ml.). The organic
extracts were dried over sodium sulfate and evaporated.
The residue was chromatographed on a column of silica gel
(60 g.~ and eluted with CHC13 and 3% MeOH-CHC13 successively
to give 2.40 g. (37~) o~ 7a, mp. >161 C. (dec.).
ir: ~ mKBaX 1780, 1720 cm~l.
u~: ~ pH 7 Bu~fer 252 nm (~, 19800), 298 nm ~, 8900).
nmr: ~ ppmo+D2 7.67 (lH, d, J=9.0 Hz, pyridazine-H),
7.10 (4H, s, phenyl-H), 7.05 (lH, d, J=9.0 Hz, pyridazine-
H), 5 66 (lH, d, J=4.5 Hz, 7-H), 5.07 (lH, d, J=4.5 Hz,
6-H), ~.71 (2H, s, N-CH2-C0), 4.4-4.0 (~H, m, 3-CH2 &
CH2-N), 3.8-3.5 (4H, m, 2-H & CH2C0), 1.42 (9H, s, t-Butyl-H)
~nal. Calc'd- for C28H31N707S2 2H2
H, 5.20; N, 14.46; S, 9.46. Found- C, 49.97, 49.95; H,
- 4 79, 4.62; N, 14.00, 13.84; S, 9.37, 9.32.
3B-S46 ~ (A~inomethylphenyl)acetamidol-3-(2-carboxy
methyl-2?3-dihydro-s-triazolo~4~3-b ~ yridazln-3-on-6-
ylthiomethyl~-3-cephem-4-carboxylic Acid (8a~
. _
Tri~luoroacetic acid (3.4 ml.) was ~dds~ to 7~
(2.33 g.) at 0 C. and the mixture stirred for 15 minutes
at room temperature. To the mixture was added dry ether
(100 ml.), and the precipitate was collected by filtration
and washed with dry ether (2 x 50 ml.) TA~e solid was
- 66 -
- ~
dissolved in a mixture of CH3CN (100 ml.) and water
(14 ml.) and the solution was ad~usted to pH 4-5 with
concentrated NH40H to afford a precipitate which was
collected by filtration and washed with CH3CN (2 x 50
ml.) to give 1.759. (82%) of 8a as the ammonium salt.
M.p. >160 C. (dec.).
ir: V KBar 1765, 1710, 1640, 1590 cm 1
uv: ~ H 7 Buffer 252 nm (~, 23900), 298 nm (~, 10900).
Anal. Calc'd. for 524H22N707S2 NH4 H2 C, 46-51;
H, 3.96; N, 18.08; S, 10.35. Found: C, 46.52, 46.26;
H, 4.15, 4.19; N, 17.60, 17.48; S, 10.89, 10.41.
Pre~aration of BB-S469 Monosodium Salt
To a suspension of 8a (1.58 g.) in 50% acetone-
water (30 ml.) was added a 10~ solution to ad~ust the pH
to 7.7. An additional amount of acetone was added and
the precipitate was collected by filtration and washed with
acetone to give 1.38 g. (87%) of monosodium salt of 8a,
m.p. >170 C. (dec.).
ir: ~ maxr 1765, 1710, 1640, 1600, 1540, 1485 cm 1.
uv ~ pH 7 Buffer 252 nm (~, 20600), 298 nm (~, 9200)-
nmr: ~ ~ +K2C3 7.52 (lH, d, J=9 Hz, pyridazine-H), 7.27
(4H, s, phenyl-H), 7.03 11H, d, J=9 Hz, pyridazine-H~, 5.62
(lH, d, J=4.5 Hz, 7-H), 5.07 (lH, d, J=4.5 Hz, 6-H).
Anal. Calc'd. for C24H22N707S2 2
H, 4.07; N, 15.23; S, 9.96 Found: C, 44-.44, 44.95;
h, 3.68, 3.90; N, 16.50, 16.67; S, 10.38, 10.45.
- ~7 -
EXAMPLE 2
Preparation of BB-S472
l~ CH2CONH ~ ~ ~=j
N ~
C2H
12b, BB-S472
7-(3-N-t-Butoxycarbonyl-N-methylaminomethyl-2-thienyl-
acetam do)-3-(2-carboxymethyl-2,3-dihydro-s-triazolo-
~4,3-b~pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-
carboxylic Acid. (llb)
A mixture of the BOC-protected a~ino acid (lOb,
513 mg., 1.8 m.mole), 2,4-dinitrophenol (400 mg., 2.16
m.mole) and DCC (445 mg., 2.16 m.mole) in THF (5 ml.) was
stirred at room temperature for 12 hours. The precipitated
urea was removed and the ~iltrate was added to a mixture of
7-amino-3-(2-carboxymethyl-2,3-dihydro-s-triazolo[4,3-b]-
pyridazln-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid
(4, 800 mg., 1.8 m.mole) and triethylamine (0.76 ml., 5.4
m.mole) in water (5 ml.) at 0 C. with stirring. Stirring
was continued until active ester had disappeared on tlc
(slli~a gel plate; Rf 0.95; solvent system, CHC13:MeOH=
3~ The reaction mixture was diluted with water (20
ml.), layered with AcOEt (50 ml.) and adjusted to pH 2
with concentrated HCl at 5 C. The organic layer was
separated and aqueous layer was extracted with AcOEt (3 x
50 ml.). The AcOEt extracts were combined, washed with
sat. aq. NaCl, dried over MgS04 and concentrated under
- 68 -
~ f~5 ~
reduced pressure. The residual oil (1.9 g.) was chroma-
tographed on silica gel (40 g.). The column was eluted
successively with CHC13 (400 ml.), 3% MeOH-CHC13 (100 ml.)
and 10~ MeOH-CHC13 while monitoring with tlc ~silica gel
plate, solvent system MeOH:CHC13-1:2, detected with I2).
From the CHC13 eluate was recovered a mixture of 2,4-
DNP and the BOC-protected amino acid 9 and from 3~
MeOH-CHC13 eluate 50 mg. of 9. The des~red product (llb)
(Rf 0.4, solvent system CHC13:MeOH=3:1) was obtained by
evaporation of the eluate with 10% MeOH-CHC13. Yield
490 mg. (39~). M.p. 215-220 C.
ir ~ KaBX 3400, 1780, 1720, 1680, 1550 cm~l.
u~: ~ pH 7 Buffer 245 nm (~, 23000), 260 nm (~, 18000),
300 nm (~, 7900).
nmr: ~ ppSO d6 1.42 (9H, s, BOC-H), 2.75 (3H, s, N-CH3),
H H S ~ N
~ 3-C~ ), 4.72 (2H, s, BOC-N-CH2), 5.10 (lH, d, J=4.5 Hz,
6-H), 5.70 (lH, d-d, J=4.5 & 10.5 Xz, changed to a doublet
J=4.5 by addition of D20,7-H), 6.85 (lH, d, J=4.5 Hz,
thiophene H~), 7.19 (lH, d, J=9 Hz, pyridazine H), 7.34
(lH, d, J=4.5 thiophene Ha), 7.72 (lH, d, J=9 Hz, pyri-
dazine-H), 9.11 (lH, d, J=10.5 Hz, disappeared by addition
of D20, NH)-
Anal CalC~d- for C28H31~7gS~ H20
H, 4060; N, 13.55; S, 13.29. ~ound: C, 46.67; H, 4.71;
N, 12.79; S, 12.81.
- 69 -
BB-S472; 7-(3-Methylaminomethyl-2-thienylacetamido)-3_
(2-car ~ methyl-2,3-dihydro-s-triazolor4,3-blP~ridazin
~-on-6-ylthiomethyl1-3-cephem-4-carboxylic Acid (12b)
Trifluoroacetic acid (0.4 ml.) was added to llb
(~00 mg., O.57 m.mole) at O C. a~d the mixture was stirred
at room temperature for 15 minutes. To the reaction mlx-
ture was added anhydrous ether (10 ml.) to separate a
precipitate which was collected by filtration, washed
with anhydrous ether (2 x 10 ml.~ and suspended in aceto-
nitrile (10 ml.). The suspension was adjusted to pH 4 with
concentrated NH40H and stirred for 10 minutes. The solid
was collected by filtration, washed with acetonitrile (2 x
5 ml.) and dried at 60 C./lmmHg for 7 hours to afford
310 mg. (90~) of 12b melting at 188-191 C. (dec.).
ir: L~maBx 3400~ 1770, 1720, 1680, 1550 cm 1
uv: ~ max 245 nm (~, 22400), 260 nm (, 18700),
300 n.~ (, 8600).
Anal. Calc'd. for C23H23N707S3 ~H20: C, 41-87;
H, 4.43; N, 14.86; S, 14.58. ~ound: C, 42.0~; H, 3.59;
N, 14.79; S, 14.35.
Pre aration of BB-S472 Monosodium Salt
.P
To a s-~spension of 12b (230 mg., o.38 m.mole) in
0. 5 ml. of deioni7ed water was added N NaOH to adjust to
pH 8.9. Acetone (15 ml.) was added to the solution. The
precipitate was collected by filtration, ~ashed with ace-
tone (2 x 5 ml), and dried at 60 C./lmmHg for 7 hours to
afford 170 mg. (71~) of monosodium salt of BB-S472, m.p.
>210 C. (dec.).
- 70 -
ir: ~mK~Y 3400~ 1765, 1710, 1680, 1600 cm~l.
~ ~H 7 3uffer 2'5 nm (~, 21800), 260 ~ 5)~
300 nm (~, 7800).
nmr: ~ D~ 2.72 (3H, s, N-CH3), 3.45 (lH, d, J= ~ Hz,
2-H), 3.75 (lH, d, J=18 Xz, 2-H), 3.95 (2r:, s, S ~ CH2C0),
4.18 (4H, m, ~ CH2N & 3-CH2), 4.57 (2H, s, N-CH2), 5.00
(lH, d, J=4.5 Hz, 6-X), 5.53 (lH, d, J=4.5 Hz, 7-H), 6.97
(lH, d, J=9 Hz, pyridazine-H), 7.03 (lH, d, J=4.5 ~z,
thiophene-H~), 7.34 (lH, d, J=4.5 Hz, thiophene-Ha),
7.48 (lH, d, J=9 Hz, pyridazine-H).
Anal. Calc'd. for C23H22N707S3Na l/2H20: C, 43-40,
H, 3.64; M, 15.40; S, 15.11. Found: C, U3,c5; H, .o8;
N, 14.18; S, 13.91.
~3h~;~
EXAMPLE ~
Preparati n of BB-S478
H0 ~ CH2NH-cH3 S
CH2CONH ~ ~ ~ J
2 S~N~N~ N-cH2cooH
C2H
8c, BB-S478
~ N-t-Butoxycarbonyl-N-methylaminomethyl)-4-hydroxy-
phenylacetamidol-3-(2-carboxymethyl-2,3-dihydro-s-triazolo-
~ ~yridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic
Acid (7c)
A mixture of 2-N-t-butoxycarbon~l-N-methylamino-
methyl-4-hydroxyphenylacetic acid (5c), ~708 mg., 2.4
m.mole), 2,4-dinitrophenol (478 mg., 2.6 m.mole) and DCC
(536 mg., 2.6 m.mole) in dry THF (20 ml.) was stirred at
room temperature for 2 hours. The precipitated urea was
removed by filtration. The filtrate was added to a solu-
tion of 7-amino-3-(2-carboxymethyl-2,~-dihydro-s-triazolo-
[4,5-b]pyridazin-~-on-6-ylthiomethyl)-3-cephem-4-carboxylic
acid (4) (876 mg., 2 m.mole) in 20 ml. of water ^ontaining
triethylamine (o.84 ml., 6 m.mole) and he mixture was
stirred at room temperature for 18 hours. After concen-
trating to 20 ml. the aqueous solution was washed with
ether, acidified with 6N HCl and extracted with 200 ml. of
ethyl acetate The extract was filtered to remove insol-
ubles, washed with water and a saturated aqueous NaCl solu-
tion and dried. The solution was evaporated to dryness and
the oily residue was chromatographed on a silica gel (Wako-
gel C-200, 25 g.) eluting with chloroform and ~ of
- 72 -
chloroform-methanol. The fractions containing the desired
product (monitored by tlc; Rf 0.3; solvent system,
CHC13:MeOH=2:1) were collected and evaporated to dryness.
The oily residue was triturated with ether-r.-hexane to
give 630 mg. (44~) of the product 7c melting at 200-210 C.
(slow dec.).
ir: ~ KBaxr 1780, 1720, 1660, 1400, 1240, 1150 cm 1
uv: ~ max 252 nm (~, 13000), 300 nm (E, 5400).
nmr ~ ppm 6 1 39 (9H, s, C-CH3), 2.73 (3H, s, N-CH3),
.3-3.9 (4H, m, CH2CO & 2-H), 4.35 (4H, m, CH2N & 3-H),
4.48 (2H, s, NCH2CO), 5.03 (lH, d, 4.5 Hz, 6-H), 5.61 (lH,
d-d, 8 & 4.5 Hz, 7-H), 6.4-7.2 (3H, m, phenyl-H), 5.98
(lH, d, 10 Hz, pyridazine-H), 7.61 (lH, d, 10 Hz, pyridazine-
H), 8.87 (lH, d, 8 Hz, NH).
Anal. CalC'd- for C30H33N710S2
4.65; N, 13.70; S, ~.96. Found: C, 50.98; H, 5.36; N,
11.88; S, 7.60.
BB-S478; 7-(2-N-Meth~aminomethyl-4-hydroxypheny--lacetamido)-
3-~2-N~carboxymethyl-2,3-dihydro-s-triazolo~4,5-blpyridazin-
3-on-6-ylthiomethyl)-3-ceDhem-4-carboxyl-ic Acid ( _ )
A mixture of 7c (570 mg., o.8 m.mole) and tri-
fluoroacetic acid (1.5 ml.) was stirred at 10 C. for 30
minutes and the mixture was diluted with ether (50 ml.)
to separate the trifluoroacetate of 8c which was collected
by filtration and then dissolved in a mixture of 10 ml. of
acetonitrile and 5 ml. of water and then filtered. The
filtrate was adjusted to pH 6 with concentrated ammoni
hydroxide and the mixture was diluted wilh acetonitrile
- 73 ~
(100 ml.). The resulting precipitate was collected by
filtration, washed with acetonitrile and dried in vacuo
over P205 to give 370 mg. (75%) of 8c, melting at 215 -
220 C. (dec.).
ir: ~ mKBar 1770, 1710, 1600, 1380, 1350 cm 1
uv: ~ pH 7 BUffer 252 nm (~, 19000), 300 nm (P, 9100).
nmr: ~ ~~K2C3 2.75 (3H, s, N-CH~), 2.9-3.3 (4H, m,
CH2C0 & 2-H), 4.0-4.3 (4H, m, CH2N & 3-H), 4.57 (2H, s,
NCH2C0), 4.81 ( lH, d, 4. 5 Hz, 6 -H), 5. 53 (lH, d, 4.5 Hz,
7-H), 6.6-7.5 (5H, m, phenyl-H & pyridazin-H).
Anal. Calc'd. for C25H25N7ogs2 5/2H20 C~ 45 -45;
H, 4.58; N, 14.84; S, 9.71. Found: C, 45.69; H, 4.21;
N, 15.03; S, 9.46.
Preparation of Monosodium Salt of BB-S478
To a suspension of 8c (308 mg., 0.5 m.molej iIl
water (2 ml. ) was added 0.3-0.4 ml. of N NaOH and the
mlxture was stirred at room temperature; the pH of the
resulting solution was 9.2. Acetone (20 ml.) was slowly
added to the solution. The resulting precipitate was
collected by filtration, washed with acetone (10 ml.) and
dried in vacuo over P205 to give 290 mg. (91~) of the
monosodium salt of BB-S478, melting at 230-235 C. (dec.).
ir: ~ KaBxr 1770, 1700, 1600~ 1390, 1350 cm 1
uv: ~ pH 7 Bu fer 250 nm (~, 18000), ~00 nm (~, 8400).
Anal. CalC'd. for C25H24N708S2N 5~ 2
43.98; H, 4.28; N, 14.36; S, 9.39. ~ound: 5, 43.96;
H, 4.14; N, 13.51; S, 9.34.
- 74 -
EXAMPLE 4
Preparation of BB-S479
CH2NH-CH~; S
~!LCH2CONH r ~ ~ ~ N
2-S~N,N~f N-cH2cooH
C2H
8b, BB-S479
7-~o-(N-Butoxycarbonyl-N-methy-laminomethyl)phenylacetamido]
3-~2-carboxymethyl-2~3-dihydro-s-triazolo~4~3-b~pyridazin
3-on-6-ylthiomethyl)-3-cephem-4-carboxylic Acid (7b)
To a cold (0 C.) mixture of ~ (5.4 g., 12
m-mole)~ Et3N (5.5 ml.), CH3C~ (25 ml.) and water (25 ml.)
was added a solution of 2,4-dinitrophenYl o-(N-butoxy-
carbonyl-N-methylaminomethyl)phenylacetate ~6b) in THF
~prepared from o-(N-butoxycarbonyl-N-methylaminomethyl)-
phenylacetic acid (5b) (3.48 g., 13.5 m.mole), 2,4-dinitro-
phenol (2.49 g., 13.5 m.mole) and DCC (2.79 g., 13.5 m.mole)
in dry THF (37 ml.)~. The mixture was stirred at room tem-
perature overnight. THF and CH3CN were removed from the
reaction mixture by evaporation under reduced pressure
and the resulting aqueous solution was washed with ether
(3 x 30 ml.), adjusted to pH 2-3 with dllute HC1 and ex-
tracted with ethyl acetate (4 x 30 ml.). The orgarlc
extracts were combined, dried over sodium sulfate and
evaporated. The residue was chrom.ato&ra?'led on a col~mn
of SiO2 (100 g.). After washing with CHC13, the column
was eluted with 3~ MeOH in CHC13 to afford ~ desired
fraction containing 7b. Yield 3.8 g. (45-,~). m.p. >200
C. (dec.).
- 75 -
ir: ~ KBx 1780, 1720, 1680 cm 1.
uv: ~ max 250 nm (, 18800) ? 297 nm (~, 8400)
nmr: ~ pDpSO+320 7.62 (lH, d, J=10.5 Hz, pyridazlne-H),
7.14 (4H, s, phenyl-H), 6.98 (lH, d, J=10.5 Hz, pyridazine_
H), 5.61 (lH, d, J=4.5 Hz, 7-H), 5.03 (lH, d, J=4.5 Hz,
6-H), 4.67 (2H, s, N-CH2), 4.42 (2H, s, CH2-N), 4.4-4.0
(2H, m, 3-CH2), 3.8-3.4 (4H, m, 2-H & CH2-CO), 2.72 (3H,
s, N-CH3), 1.38 (9H, s, BOC-H)-
Anal- Calcld. for C~oH33N7o9s2 5/ 2
H, 5.14; N, 13.16; S, 8.61. Found: C, 48.25, 48.23; H,
4.52, 4.46; N, 12.93, 12.86; S, 8.68.
BB-S479; 7-[o-(Methylaminomethyl)phenylacet&mido]-3-(2-
carboxymethyl-2,3-dihydro-s-triazolo~4,3-blpyridazin-3-
on-6-,ylthiomethyl)-3-cephem-4~carboxylic Acid (8b)
Trifluoroacetic acid (7 ml.) was added to the
t-BOC-derivative 7b (3.8 g., 5.5 m.mole) at 0 C., and
the mixture was stirred for 20 minutes at room temperature.
Dry ether (100 ml.) was added to the mixture. The re~ulting
precipitate was collected by filtration and washed with dry
ether (3 x 100 ml.). The precipitate was dissolved in a
mixture of CH35N (120 ml.) and water (18 ml.) and the
solution was adjusted to pH 5-6 with concentrated NH~OH
to give anoily precipitate which was triturated with
CH3CN to form solid material. The product 8b was collected
by filtration, washed with CH3CN and dried. Yield 2.55 g.
(77%~.
ir: ~ KBr 1770, 1710, 1600, 1550 cm~l.
- 76 -
Pre~aration of ~lonosodium Salt of B~-S479
To a soluti3r. of B3-S479 (&b) (2.54 ~ .3
m.mole) in water (25 ml.), N NaOH (ca. 3 ml.) was added
under cooling (the pH o~ the solution ~Jas 10). A large
amount of acetone was added to the solution and the preci-
pitate was collected b~ filtration and washed with acetone
to give 1.94 g. (84~) of monosodium salt of BB-S479.
M.p. >200 C. (dec.).
ir: ~ maBxr 1770, 1710, 1600, 1550 cm 1,
v ~ pH 7 Buffer 250 nm (~, 19400), 297 nm (~, 8700).
Anal. Calc!d for C2sH241~r707S2rra 1/2~20 C~
47.61; H, 4.00; N, 15.55; S, 10.17. Found: C, 47.~3,
47.43; H, 4.67, 4.68; N, 15.97, 15.70; S, 9.25, 9.8~.
~ 3
EXAMPL~ 5
Pre~aration of ~B-~483
CH2coNH ~ ~ ~ N
N~CH2 S N~N~ N-CH2cooH
C2H
12a, BB-S483
7-f3-N-t-Butox~carbonylaminomethyl-2-thienylacetamido ~ -
~ arboxymethyl-2~3-dihydro-s-triazoloL4~3-blpyridazin-
3-on-~- lthiomethyl)-3-ce hem-4-carboxylic Acid (lla)
_ Y . - . P _ ~ _
A ~ixture of the BOC-protected amino acid (9,
410 mg., 1 56 m.mole), 2,4-dinitrophenol (313 mg., 1.7
m.mole) and DCC (353 mg., 1.7 m.mole) in THF (5 ml.) was
stirred at room temperature for 12 hours. The precipitated
urea was removed and the filtrate was added to a mixture of
7-amino-3-(2-carboxymethyl-2,3-dihydro-s-triazolo[4,3-b]-
pyridazin-3-on-6-ylthiomethyl)-3-cephem-4-carboxylic acid
(4, 683 mg., 1.56 m.mole) and triethylamine (0.62 ml.,
4.68 m.mole) in water (5 ml.) at 0 C. with stirring.
Stirring was continued at room temperature until the active
ester disappeared on tlc (silica gel plate; Rf 0 95; sol-
vent system, CHC13:~eOH=3:1). The reactio~ mixture was
diluted with water (20 ml.), layered with AcOEt (50 ~1.)
~nd ad~usted to pH 2 with concentrated HCl at 5 C. The
organic layer was separated and the aqueous layer was ex-
tr~cted with AcOEt (3 x 50 ml.). The AcOEt (etnyl aceta e)
extracts were combined, washed with satur~ted aqueous NaCl,
dried over MgS04 ar.d concentrated under reduced pressure.
- 78 -
-
The residual oil (1.8 g.) was chromatographed on sllica
gel (40 g.). ~ne column was eluted successively with
CHC13 (400 ml.) and 3~ MeOH-CHC13 (500 ml.). The eluate
was monitored with tlc (silica gel plate, solvent system
CHC13:MeOH=2:1, detected with I2) The desired product
lla (Rf 0.2) was obtained by evaporation of the MeOH-
CHC13 eluate. Yield 450 mg. (42~), melting at 155-160 C.
ir: ~ KaBxr 3300, 1775, 1720, 1680 cm 1.
uv: ~ pH 7 Buffer 245 nm (~, 23900), 260 nm (~, 19200),
300 nm (~, 87~o).
nmr ~ DMS-d6 1.39 (SH, s, BOC-H), 3 76 (4H, br-s,
2-H & S CH2GO), 4.05 (2H, d, J=6 Hz, ch~nged to a singlet
by addition of D20, BOCNH-CH2), 4.20 (2H, m, 3-CH2), 4.69
(2H, s, N-CH2C02), 5.o6 (lH, d, J-4.5 Hz, 6-H), 5.62 (lH,
d-d, J=4.5 ~ 9 Hz, changed to a doublet J=4.5 Hz by addi-
tion of D20, 7-H), 6.83 (lH, d, J=4.5 Hz, thiophene-H~),
7.00 (lH, m, disappeared by addition of D20, NHB05), 7.04
(lH, d, J=9 Hz, pyridazine-H), 7.12 (lH, d, J=4.5 Hz,
thiophene-Ha), 7.65 (lH, d, J=9 Hz, pyridazine-H), 8.97
(lH, d, J=9 Hz, disappeared by addition of D20, 7-NH).
Anal. Calc'd. for C27H29N709S3: C, 46-88; H,
4.23; N, 14.17; S, 13.90. Found: C, 46.42; H, 4.37;
N, 13.49; S, 13.61.
- 79 -
BB-S 48~; 7-(3-Aminomethyl-2-thlenylacetamldo)-3-(2-
carboxymethYl-2,7-dihydro-s-tr~azolo~4,~-b]pyridazin-3-
on-6-ylthiomethyl)-3-cephem-4-carboxylic Acid (12a)
Trifluoroacetic acid (0.4 ml.) was added to lla
(410 mg., 0.59 m.mole) at 0 C. and the mixture was stirred
at room temperature for lS minutes. ~o the reaction mix-
ture was added anhydrous ether (10 ml.) to separate a
precipitate which was collected by filtration, washed with
anhydrous ether (2 x 10 ml.) and suspended in acetonitrile
(10 ml.). The suspension was adJusted to pH 4 with concen-
trated NH40H and stirred for 10 minutes. The precipitate
was collected by filtration, washed with acetonitrile (2 x
5 ml.) and dried at 60 C./lmmHg for 7 hours to afford
310 mg. (88%) of 12a, melting at above 200 C. (slow dec.).
ir: ~ maBr 3400, 3150, 1760, 1700, 1680, 1600 cm 1
uv ~ maHx7 ~uffer 245 nm (~, 17100), 260 nm (~, 14100)~
300 nm (~, 6500).
Anal. Calc'd. for C22H21N707S3 3H20: C, 40-90;
H, 4.21; N, 15.17; S, 14.89. Found: C, 40.39; H, 3.62;
N, 15.87; S, 14.35.
PreParation of Monosodium Salt of BB-S483
___
To a suspension of 12a (280 mg., 0.47 m.mole) in
0.5 ml. of deionized water was added N NaOH to adjust to
pH 9.5 and insoluble material was collected by filtration.
Acetone (15 ml.) was added to the filtrate to separate the
precipitate which was collected by filtratlon, washed with
acetone (2 x 5 ml.) and dried at 70 C./lmmHg for 7 hours
to afford 220 mg. (76%) of monosodium salt of 12a.
M.p. >210 C. (slow dec.).
- 80 -
ir: ~ KaBxr 3400, 3250, 1760, 1710, 1650, 1600, 1550 cm 1.
u~: ~ pHax7 Buffer 245 nm (~, 19900), 260 nm (~, 16400),
300 nm (e, 6900).
nmr: ~ ~ m ~.60 (2H, m, 2-H), 3.91 (2H, s, CH2C0), 4.12
(2H~ s, CH2-NH2), 4-20 (2H, m, 3-CH2), 4.55 (2H, s,
N-CH2C0), 4.95 (lH, d, J=4.5 Hz, 6-H), 5.50 (lH, d, J=4.5
Hz 7-H), 6.94 (lH, d, J=9 Hz, pyridazine-H), 6.99 (lH, d,
J=4.5 Hz, thiophene-H~), 7.28 (lH, d, J=4.5 Hz, thiophene-
Ha), 7.32 (lH, d, J=9 Hz, pyridazine-H).
C22H20N707S3Na 1/2cH3cocH3
C, 43.92; H, 3.61; N, 15.26; S, 14.97. Found: C, 43.48;
H, 4.56; N, 15.28; S, 13.91.
Solubility
All of the monosodium salts in this series showed
more than 10% solubility in water.
NePhrotoxicity
A preliminary nephrotoxicity study was carried
out by administration of the test compound to a group o~
two rabbits at 100 mg./kg. intravenously. The results ob-
tained with BB-S469 and BB-S479 indicated that they might
have little nephrotoxic potential.
In vitro Activity (Table 1)
. .
The MIC's were determined by the serial dilution
method using Mueller-Hinton agar against 51 gram-positive
and 96 gram-negative bacteria. The 147 test organisms were
classified into 16 groups according to the genera and the
- 81 -
types of antibiotic resistance, 5 groups for gram-positive
and 11 for gram-negative bacteria. In Table 1 is shown
the in vitro activity in terms of geometric mean of MIC's.
BB-S472 and BB-S479 showed better overall activity than
their non-N-methylated analogs, BB-S483 and BB-S469,
respectively. BB-S479 was superior to BB-S472 in some
species of gram-negative bacteria. Comparing with cefam-
andole, BB-S479 was more active against most of the test
organisms except against Providencia species and Staphylo-
cocci.
- 82 _
*
0 ~ O C~J0~
,~ ,~ ~ coooo o~ ,i ~oo a~oOo~ o
~3 ~/ ~1 ~t ~ O CU ~ ~ O ~ Lf~ ~D ~t ~ F
~ O O ~'JOOO ~ ~1 ~00 ~\O'C~. O
V _
0 ~ ~ -~O O ~ ~ C~ ~
1~ ~ O 1--I 00 0 0 0 r-i U~ 0 0 C--C~ 0 N
~_ a~
._ ~1
\ _ ~ o~ o O C~ D
~ U X r~ C~ C~ ~~
a ~ Ox
~V CO ^ J O C~l 00 ~D ~t r~ ~CO ~ n
~: C~ . O ~10 0 0 0 ~0 0 Ctl O O C-_ O O O ~1
mm ~ .
~ ~ _ ~ ~ OOC~ coco ~ ~ ~
O 0~ m
~ m-- _ _ _ 5
E~ a~- ~- I O ~ ~0
X O ~ O O O O ~ ~ ' O O ~1 ~ O O O
mm w ~1 ~ :"
¢ - ~ ~ '
.~ ~: ~ ~ 0 ~ ) J U~ O ~ ~
¢ . ~ . -- bD I
h ~ ~1 a) ~ a)~ .~ + .~ ~ . ~DC ~ :~
~I cq ,~ ~ ,C S-~ ^~ O ~ cq ~ , C O _ X
O ~ c ~cq~ ~ ~ c ,~ ~.Q~ O ~-~ ' c'q c) ~ V
cq I I c(l ~ O 1~ O O ~ O ~q h Q~ ~ 3 ~ O cq ~1 ~1 0 V C~
c'~ ~ ~ c~ c) ~ a) O a~ a) ~ ~ ~ ~ ,Q E~ V S~ O U ~
Lq ~rl ~ 14 ~ cq ~ V O O O a) 0 a) cq O ~ ~5 H ~ V
E~ u~lc~ c~l C/~ ~: ~ ~ s~ c) ,~ c~l~ ~ U ~ ~; *
- 83 -
,
~i3
E~AMP r .~ 6
Substitution ln the procedure of Example 3 for
the 2-N-t-butoxycarbonyl-N-methyl~minomethYl-4-hydroxy- -
phenylacetic acid used therein of an equimolar weight of
2-N-t-butoxycarbonylaminomethyl-4-hydroxyphenylacet~c
acid and of 2-N-t-butoxycarbonylaminomethyi-4-methoxy-
phenylacetic acid and of 2-N-t-butoxycarbonyl-N-methyl-
aminomethyl-4-methoxyphenylacetic acid, respectively,
produces the com~ounds having tile structures
HO~CH2NH2 ~S~
CH2CONH-CH-CH SH2 ~F
~C--N~ ~C--CH2S ~ N~N ~ N-CH2COOH
C-OH
o and
3 ~ CH2NH2 ~S~
CH2CoNH-~H-cl~ C~'2 ~F
o// ~C~ CE~2S ~N'N~'N-CH2COoH
C-OH
o and
3 ~ CH2N~CH3
CH2CONH-CH-CH CH2 ~ N
/C--N~C--CH2S ~ N,N ~ N-~H2COoH
O C~ O
C-OH
respectively.
- 84 -
1 ~ 1 3
EYA~IPLE 7
Pre~aration of BB-S493
.
NH-BOC
COOH
NH-BOC
CONH ~ S~ ~ N
N ~ CH2S ~ N~N ~ N-CH2C2H
C02H O
CONH ~ ~ N
N ~ CH2S N~ J-CH2C2H
C2H
3, BB-S493
7-~(2-N-t-Butoxycarbonylaminomethyl-1,4-cyclohexadienyl)-
ac_tamido]-3-(2-N-carboxymeth~l-s-triazolo~4,5-bIpyridazin-
3-on-6-ylthiomethyl~-3-cephem-4-carboxylic Acid (2)
A mixture of 2-N-t-butoxycarbonylæminomethyl-
1,4-cyclohexadienylacetic acid (1, 640 mg,, 2.4 m,mole)~
2,4-dinitrophenol (422 mg., 2.4 m,mole) and DCC (494 mg,,
2.4 m,mole) in 10 ml, of dry THF was stirred for 1.5 hours
at room temperature, The precipitated urea ~as removed by
filtration. The filtrate was added in one portion to a
- 85 -
l~i3'~`~
solution of 7-~nino-3-(2-N-carbox~rmethyl-s-triazol[4,5-b~-
pyridazin-3-on-6-ylthiomethyl)-3-Cephem-4-Carboxylic acid
in 10 ml. of water containing triethylamine (0.56 ml.,
4 m.mole) and the mixture was stirred at room temperature
for 3 hours. The reaction mixture was concentrated to
10 ml. under reduced pressure, washed with ether (3 x 10
ml )~ acidified with 6N hydrochloric acid and extracted
with ethyl acetate (5 x 10 ml.). The combined extracts
were washed with a saturated saline solution and dried
wlth anhydrous Na2S04. The solvent was evaporated and the
residue was chromatographed on silica gel (Wako-gel C-200,
~0 g.) eluting with chloroform-methanol (0-50%). The
fractions containing the desired product wer~ collected.
The solvent was removed and the residue was triturated
with ether-n-hexane to give 410 m~g. (30~) of the product
2. M.p. 110 C. (decomp.).
ir: ~J K13xr 1780, 1730, 1610, 1530, 1250, 1160 cm 1
uv: ~ BUaxf (pH 7) 252 nm (~, 19000), 300 nm (~, 8600, sh).
Anal. Calc'd for C29H33N709S2 2 2
H, 5.15; N, 13.54; S, 8.86. Found: C, 48.07; H, 4.64;
N, 12.70; S, 8.39.
BB-S493, 7- r ( 2-Aminomethyl-1,4-cyclohexadienyl~acetamido]-
_ _ _ _ _ _ _ _ _
3-(2-N-carboxymethyl-s-triazolo~4,5-b~pyridazin-3-on-6-
ylthiometh_1~-3-cephem-4-carboxylic AC ~ d (_)
The N-BOC-protected cephalosporin 2 (350 mg.,
0.51 m.mole) was treated with trlfluoroacetic acid (r~A)
(1 ml.) for 30 minutes at room temperature. To the mlxture
was added ether (50 ml.) to give the TFA salt of 3, which
was collected by filtration and then dissolved in a mixture
- 86 ~
l~i3'~
of acetonitrile (5 ml.) and water (2 ml.). The solution
was treated with a small amount of active carbon, ad~usted
to pH 6 with concentrated ammonium hydroxide. The preci-
pitate was collected, washed with acetonitrile (5 ml.) and
dried in vacuo to afford 235 mg. (78~) of 3. l`I.p. 220-
230 C. (decomp.).
lr: L~ KBax 1770, 1740, 1710, 1650, 1600, 1550 cm 1.
uv: ~ BUaxf (pH 7) 252 nm (~, 20000), 300 nm (~ 9000, sh)-
Anal. Calc'd- for C24H25N707S2 2
H, 4.49; N, 16.19; S, 10.59. Found: C, 47.77; H, 4.o6;
N, 16.49; S, 10.64.
- 87 -
In vitro AntibaCterial Activity of BB-S493 Compared with
_
BB-S479 and Cefamandole (Determined by Steers' Ag2r Dilu-
tion Method on Mueller-Hinton Agar Plate)
~IC (mcg./ml.)
Organism BB-S493 BB-S479 Cefamandole
S. aureus Smith A9537 0.4 0.4 0.1
S. aureus A9497 0.2 0.1 0.05
S. aureus BX-1633 A9606 0.4 0.4 0.2
St. faecalis A9536 100 100 50
E. coli NIHJ 0.1 0.05 0.025
E. coli ATCC 8739 0.2 0.05 0.05
-E. coli Juhl A15119 0.2 0.1 0.4
E. coli BX-1373 0.4 0.2 0.4
E. coli A15810 0.2 0.1 0.2
E. coli A9660 0.1 0.05 0.1
E. coli A15147 6.3 3.1 3.1
Kl. pneumoniae A9678 0.4 0.4 1.6
Kl. pneumoniae A9977 0.2 0.1 0.4
Kl. pneumoniae A15130 0.2 0.1 0.4
Kl. pneumoriae A9867 0.2 0.1 o.8
Pr. vulgaris A9436 0.4 0.1 0.2
Pr. vulgaris A96gg 6.3 o.8 25
Pr. mirabilis A9554 0.2 0.1 o.8
Pr. mirabilis A9900 0.2 0.1 o.8
Pr. morganii A9553 >100 ~100 >100
Pr. morganii A20031 0.4 0.1 o.8
Pr. rettgeri A15167 0.1 0.1 0.1
Ps. aeruginosa A9930 ~100 ~100 ~100
Ps. aeruginosa A9843 >100 >100 ~100
Shig. dysenteriae 0.05 0.025 0.2
Shig. flexneri A9684 25 12.5 3.1
Shig. sonnei A3516 0.05 0.025 0.05
Serr. mareescens A20019 100 25 59
Enterob. cloacae A9656 6.3 1,6 3.1
Sal. enteritidis A953i 0.1 0-05 0.1
Sal. typhosa A9498 0.1 0.05 0.1
B. anthracis A9504 0.0125 0.025 0.2
- 88 -
~Sl;;~
'~ o r~
cn IS~ lr~ o ~ ~
m o o o o o o o o o
~1
O ~ ~ ~D CO 0 a~ ~ c~ c~ cO
O O O' O O' O` ~ J 0
C~
C~ C~
~ m o o
C~ ~ C~
riq ~3 cn ~ u~
~ ~o m o o
~ o ~
u~ P~ ~ ~O C`J O~ U~ CU
~; _ cn ~ ~ C~
e m o o o o o o
Co ~
~o Co
~ m o o
~; C~J
~ ~,~ C~
~ m o o
mn $ ~ ~D a~
m cn ~ ,~ ~J C`J
m o o o o o o o
¢ ~q ~q
~1 ~D ~ ol
H ¦ C/~ I F~3 ¦
- 89
Ad~itional Starting ~Iate~ials
6-Chloro-2-(2-cyanoethyl~-2~3-d~hydro-~-trlazolo[4,3-b~-
~ridazin-3-on.
To a solution of 6-chloro-2,3-dihydro-s-tri-
azolo[4,3-b]pyridazin-3-on [P. Francabilla and F. Laurla,
J. Het. Chem. 8, 415 (1971)~ (17 g., 0.1 mole) in dry
DMF (300 ml.) was added potassium tert.-butoxide (0.5 g.,
.5 m.moles) with stirring. Acrylonitrile (6.6 g., 0.12
mole) in dry D~ (10 ml.) was added to the mixture. The
mixture was stirred at 100-110 C. for 24 hours, then
poured into water (700 ml.) and extracted 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 of
6-chloro-2-(2-cyanoethyl)-2,3-dihydro-s-triazolo[4,3-b]-
pyridazin-3-on (2.5 g., 11%). M.p. 166-168 C.
ir: vKBax 2230, 1720, 1550, 1500 cm~l.
uv: ~dioxane 373 nm (~ 2000).
nmr: ~Dpp-d6 3.03 (2H, t, J-6.o Hz, CH2), 4.21 (2H,
t, J=6.o Hz, CH2), 7.23 (lH, d, J=10.0 Hz, pyridazine-H),
7.93 (lH, d, J=10.0 Hz, pyridazine-H).
Anal. Caic~d. for C8H6N5CCl: C, 42-97; H~ 2-70;
N, 31.32; Cl, 15.86. Found: C, 42.73, 42.56; H, 2.57,
2.50; N, 31.36, 31.68; Cl, 15.96, 15.81.
- 9o
l~i3'~
2-(2-Carboxyethyl)-6-chloro-2,?3-dihydrO-s-triaZolor4~3-bl-
pyridazin-3-on.
A solution of 6-Chloro-2-(2-cyanoethyl)-2~3-
dihydro-s-triazolo[4,3-b]pyridazin-3-on (724 mg.) ~n
6N-HCl (15 ml.) was refluxed for 6 hours. The reaction
mixture was extracted with ethyl acetate (10 x 20 ml.).
The combined extracts were washed with saturated aqueous
sodium chloride (50 ml.), drie~ over Na2S04 and evaporated
to give light yellow~solid 2-(2-carboxyethyl)-6-chloro-2,3-
dihydro-s-triazolo[4,3-b]pyridazin-3-on (567 mg., 72%).
M.p. ~170 C. (sublimation).
ir: vKBax 3400-2400, 1730, 1710, 1540 cm~l.
~dioxane 377 nm (~ 1500).
nmr: ~ 2 ppm ~ 2.70 (2H~ t, J=7.0 Hz, CH2), 4.24
(2H, t, J=7.0 Hz, CH2), 7.17 (lH, d, J=10.0 Hz, pyridazine-H),
7.70 (lH, d, J=10.0 Hz, pyridazine-H).
Anal. Calc'd. for C8H7N403Cl: C, 39.60; H, 2.91;
N, 23.09; Cl, 14.61. Found: C, 39.62, 39 48; H, 2.97,
2.67; N, 23.05, 22.70; Cl. 13.93, 14.12.
- 91 -
-~ .
2-(2-Carboxyethyl)-2,3-dihydro-6-mercapto-s-triazolor4,3-bl-
~yrldazin-3-on.
A mixture of 2-(2-carboxyethyl)-6-chloro-2,3-
dlhydro-s-triazolo~4,3-b]pyrldazin-3-on (567 mg., 2.3~
m moles) and 70~ sodium hydrosulfide dihydrate (924 mg.,
7.02 m.mole) ln water (10 ml.) was stirred at room tempera-
ture for two hours. The reaction mixture was ad~usted suc-
cesslvely to pH 1 wlth c. HCl, to pH 10 wlth NaOH and then
to pH 1 with c. HCl. The resulting precipltate o~ 2-(-
carboxyethyl)-2,3-dihydro-6-mercapto-s-triazolot4,3-b]-
pyridazin-3-on was collected by filtration and washed wlth
water. Yield: 418 mg. (74%). M.p. 174-176 C.
lr: vKBax 3600-2600, 2440, 1730, 1720 (sh) cm 1.
~pH 7 buffer 262 nm (~ 17000),318 nm (e 660o).
nmr: ~DMppmd6 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, JalO.O 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; N, 22.65, 22.70; S, 14.23, 14.29.
7-Amino-3-r2-(2-carboxyethyl)-2,3-dihydro-s-triazolo~4,3-bl-
ridazin-3-on-6-ylthiomethyll-3-cephem-4-carboxyllc Acid.
P~
A mixture of 7-ACA (405 mg., 1.49 m.moles), the
thiol 2-(2-carboxyethyl)-2,3-dihydro-6-mercapto-s-
triazolo[4,3-b]pyridazin-3-on (35? mg., 1.49 m.moles) and
NaHC03 (375 mg., 4.47 m.moles) in 0.1 M phosphate buffer
(pH 7, 8 ml.) was stirred at 80 C. for 30 minutes. The
reaction mixture was cooled and filtered to remove insolubles.
- 92 -
The filtrate was ad~usted to pH 1-2 wi~h c. HCl. The result-
lng precipitate, 7-amino-3-[2-(2-carboxyethyl)-2,3-dihydro-
s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl~-~-cephem-
4-carbo~vlic acid, was collected by filtration and washed
wlth water. Yield: 519 mg. (77~).
ir: vKaBr 3600-2200, 1800, 1725, 1620, 1550, 1480 cm~l.
u~: ~pH mabuffer 253 nm ( 20000), 298 nm ( 10000).
n~r: ~D20ppK~Co3 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- Calcld~ for C15H16r~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.
- 93 -
Example 8
7-[o-(N-Butoxycarbonyl-N-methylaminomethyl)phenylacetamido]-
3-[2-(2-carboxyethyl)-2,3-dihydro-s-triazolo[4,3-b]pyridazin-
3-on-6-ylthiomethyl]-3-cephem-4-carboxylic acid.
To a mixture of 7-amino-3-[2-(2-carboxyethyl)-2,3-
dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-ylthiomethyl]-3-
cephem-4-carboxylic acid (452 mg., 1 m.mole) and triethyl-
amine (0.46 ml., 3.3 m.mole) in 50~ aqueous acetonitrile
(4 ml.) was added a THF solution (3 ml.) of 2,4-dinitro-
phenyl o-(N-t-butoxycarbonyl-N-methylaminomethyl)phenyl-
acetate prepared from 0-(N-t-butoxycarbonyl-N-methylamino-
methyl)phenylacetic acid (283 mg., 1.1 m.mole), 2,4-dinitro-
phenol (202 mg., 1.1 m.mole) and DCC (227 mg., 1.1 m.mole).
The mixture was stirred at room temperature overnight and
concentrated under reduced pressure to remove the organic
solvents. The aqueous concentrate was washed with ether
(3 x 20 ml.)., acidified with c.HC1 to pH 1 - 2 and extracted
with ethyl acetate (5 x 20 ml.). The combined extracts were
dried with anhydrous Na2SO4 and evaporated to dryness. The
residue was chromatographed on a column of silica gel (Wako
gel, C-200, 10 g.) by eluting with a mixture of MEOH-CHC13
(MeOH: 0 to 3%-). The combined eluates which contained the
desired product were evaporated to give 359 mg. (50~) of
the title compound. M.p. >150 C. (dec.).
ir: v ax 3600-2400, 1780, 1720, 1680, 1550, 1490 cm
uv: ~ max Buffer 253 nm (~19800), 298 nm (~9400).
nmr: ~ ppm 6 1 37 (9H, s, t-Bu-H), 2.70 (3H, s, N-C_3),
2.70 (2H, t, J=7.0 Hz, -CH2-), 3.2 - 4.S (lOH, m), 5.01 (lH,
-94-
~ ~ 3
d, J=5 Hz, 6-H), 5.60 (lH, d-d, J=5 & 8 Hz, the 8 Hz coupling
disappeared by addition of D2O, 7-H), 6.93 (lH, d, J=10 Hz,
pyridazine-H). 7.58 (lH, d, J=10 Hz, pyridazine-H).
Anal- Calcd- for C31H35N79S2 5/2H2
N, 12.92; S, 8.45. Found: C, 49.32; 49.36; H, 4.70, 4.63; N,
12.52, 12.53; S, 8.44, 8.43.
BB-S 525; 7-[o-(N-Methylaminomethyl)phenylacetamido)-3-[2-(2-
carboxyethyl)-2,3-dihydro-s-triazolo[4,3-b]pyridazin-3-on-6-
ylthiomethyl]-3-cephem-4-carbQ-xylic acid.
A mixture of trifluoroacetic acid (1 ml.) and the
BOC-protected cephalosporin prepared above (302 mg., 0.42 m.
mole) was allowed to stand at room temperature for 15 min. and
then diluted with ether (10 ml.). The resulting precipitate
was collected by filtration and washed with dry ether (2 x 10
ml.) to afford 263 mg. of solid which was dissolved in a
mixture of water (6 ml.) and acetonitrile (3 ml.). The stirred
solution was adjusted at pH 4 with 1 N-NaOH (0.36 ml.) and
diluted with acetonitrile (100 ml.) to give the precipitate
(187 mg.), which was suspended in water (4 ml.) and adjusted
at pH 9 with sodium hydroxide (1 _, 0.3 ml.). The solution
was treated with a small amount of active carbon and freeze-
dried to leave the ll~nosodium salt of BB-S 525. Yield 106 mg.
(39~). M.p. >180 C. (dec.).
ir: " Br 3600 - 2400, 1770, 1710, 1600, 1490,1400 cm
max
uv: ~ pH 7 Buffer 253 nm (~19800), 298 nm (~8800).
max
nmr: ~ 2 2.70 (2H, m, -CH2-), 2.75 (3H, s, N-CH3), 4.4-
max
3.4 (lOH, m), 4.92 (lH, d, J=4.0 Hz, 6-H), 5.55 (lH, d, J=4.0
Hz, 7-H), 6.93 (lH, d, J=9.5 Hz, pyridazine-H), 7.28 (4H, s,
Ph-H),
-95-
r
7.40 (lH, d, J=9.5 Hz, p~ridazine~H).
Anal. Calcd. for C2~H26N707S2 . Na . 3H20: C, 45.28;
H, 4.68; N, 14.22; S, 9.30. Found: C, 45.34, 44.84; H, 4.01,
3.85; N, 14.14, 14.08; S, 9.76.
In vitro antibacterial activity of BB-S 525 compared with
BB-S 479 and cefamandole (determined by Steers' agar dilution
m thod on Mueller-Hinton agar plate)
MIC (mcg./ml)
Organism BB-S 525BB-S 479 Cefamandole
S. aureus Smith 0.4 0.4 0.2
S. aureus 0.2 0.2 0.05
S. aureus BX-1633 0.8 0.8 0.2
St. faecalis >100 >100 lG0
E. coli NIH~ 0.2 0.1 C.l
E. coli ~TCC 8739 6.3 3.1 6.3
E. coli Juhl 0.4 0.2 0.4
E. coli BX-1373 0.4 0.2 0.2
E. coli 0.2 0.1 0.1
E. coli 0.2 0.05 0.05
E. coli 6.3 3.1 1.6
Kl. pneumoniae 1.6 0.8 3.1
Kl. pneumoniae 0.2 0.1 0.2
Kl. pneumoniae 0.2 0.2 0.8
Kl. pneumoniae 0.2 0.2 0.8
Pr. vulgaris 0.2 0.2 0.2
Pr. ~ulgaris 3.1 0.8 50
Pr. mirabilis 0.4 0.1 0.4
Pr. mirabilis 0.2 0.1 0.2
Pr. morganii >100 >100 3.1
Pr. morganii 0.2 0.2 0.8
Pr. rettgeri - 0.8 0.8 0.1
Ps. aeruginosa ~100 >100 >100
Ps. aeruginosa >100 >100 >100
Shig. dysenteriae 0.1 0.1 0.1
Shig. ~lexneri 12.5 12.5 3.1
Shig. sonnei 0.1 0.1 0.2
Serr. marcescens 25 12.5 50
Enterob. cloacae 3.1 3.1 1.6
Sal. enteritidIs 0.2 0.1 0.1
Sal. typhosa 0.2 0.1 0.1
R. anthracis 0.2 0.2 0.05
- 96 -
~XAMpr.~ g
~ ubstitution in the procedure ~f Example 8 for
the 2-ll-t-butoxycar~onyl-N-methylamino.methyl-4-hydroxy-
phenyl~cetic acid used therein of an e~uimolar weight of
2-N-t-butoxycarbonylaminomethyl-4-hydroxyphenylacetic
acid and of 2-N-t-butoxycarbonylaminomethyl-4-methoxy-
phenylacetic acid and of 2-N-t-butoxycarbonyl-N-methyl-
aminomethyl-4-methoxyphenylacetic acid, respectively,
produces the compounds having the structures
HO~CH2NH2
~S~
CH2CO.YH-CH-CH SH ~ N
~C N ~ ~C--CR2S ~ N~N ~ N-C~ C~ COOH
C-OH
O and
3 ~ C~2NH2 ~S~
CH2COIYH-CH-CH C~12 ~ N
~C--N~ ~C--C~2S ~ N~N ~ N-C~ CX~COOH
C-OH
B and
3 ` ~ CH2~CH3
~NH-c~-cH CH ~r
~C N ~ ~C - CH2S ~ ~N ~ N-C~ C~ COOH
C-OH
O respectlvely.
'l
1~
- 97 -
` i~
