Note: Descriptions are shown in the official language in which they were submitted.
1t~9~:;373
The present invention relates to novel ureido
substituted cephalosporin compounds which are highly active
broad spectrum antibiotics especially useful in the treatment
of infections attributable to the gram-negative microorganisms.
Cephalosporin compounds having a ureido or a
substituted ureido substituent in the ~-position of the
7-acylamido side chain have been described. In U.S. Patent
3,673,183 issued June 27, 1972, a-ureidocephalosporanic
acids are disclosed. Acyloxymethyl esters of a-ureidocyclo-
hexadienylacetamidocephalosporins are described in U.S.
Patent 3,708,479 and of a-aminobenzylpenicillin in U.S.
Patent 3,697,507. Penicillins and cephalosporins having an
~-(3-imidoylureido)arylacetamido side chain are described in
U.S. Patents 3,634,405 and 3,646,024, respectively. a-3-
Acylureidobenzylpenicillins also are known wherein a wide
variety of acyl groups are attached to the terminal nitrogen
of the a-ureido group of the 6-arylacetamido side chain.
The compounds provided by this invention differ
structurally from the compounds of the prior art in that the
cephalosporin dihydrothiazine ring is substituted in the
3-position with a heterocyclicthiomethyl group. In addition,
the cephalosporin antibiotics described herein can be
characterized as expanded spectrum cephalosporin antibiotics
in that they not only possess the usual high level of
activity against gram-positive microorganisms but they also
possess a high level of activity against a broad spectrum
of gram-negative microorganisms which the prior art compounds
did not possess.
X-4087A 2
~L~96373
This invention relates to new ureido substituted
cephalosporin antibiotic compounds represented by the following
general formula I -
O H O H
R--C--N--C~C-N--t--~ ~
Rl o~ CH2--R2 I
COOR3
wherein R is a 3-substituted ureido group represented by the
ormula
H O .
l 11
R' '--N--C--N--
R '
wherein R'' is hydrogen, Cl-C3 alkyl, allyl,
propargyl, C3-C6 cycloalkyl, phenyl, benzyl, or
furfuryl; R' is hydrogen or methyl;
or R is a cyclic ureido group of the formula
O
Y- N~ ~N .
(CH2)n
wherein Y is hydrogen, acetyl or methanesulfonyl,
and _ is 2 or 3;
l~ i
. --3
~09~i37~
R1 is phenyl, hydroxyphenyl, halophenyl, hydroxy substituted
halophenyl,
~ or
R2 is acetoxy,
-S-~ ~ \5 ~ or -S- ~ N N
Z Z
wherein Z is Cl-C lower alkyl;
R3 is hydrogen, indanyl, phthalidyl, an acyloxymethyl group
of the formula
o
-CHz{~_y~
wherein Y' is C1-C4 alkyl or phenyl, and when R3 is
hydrogen, the pharmaceutically acceptable non-toxic salts
thereof.
~ -4-
~gt~73
In the foregoing definition, the cyclic ureido groups
represented are the five-membered imidazolidine-2-one-1-yl
group (n = 2) and the six-membered hexahydropyrimidine-2-one-1-yl
group represented by the formulae
R
y ~ ~ and Y ~ ~
\~/
wherein Y is H-, CH3S02- or (CH3)2C(O)-.
The term "halogen" as used herein refers to fluoro,
chloro, and bromo and preferably chloro.
Representative of the substituted phenyl groups, (Rl)
hydroxyphenyl, halophenyl, and hydroxy substituted
~'~ ```
~L~9G37~
halophenyl are 4-chlorophenyl, 4-bromophenyl, 3-fluoro-
phenyl, 4-hydroxyphenyl, 3-hydroxyphenyl, 3-chloro-4-
hydroxyphenyl, 2-chlorophenyl, 3,4-dichlorophenyl, 3,5-
dichlorophenyl, 3,5-dihydroxyphenyl, 2-hydroxyphenyl, 4-
hydroxy-3,5-dichlorophenyl, and 4-hydroxy-3,5-dibromophenyl.
The term "Cl-C4 lower alkyl", represented by Z in
the above formula, refers to methyl, ethyl n-propyl, iso-
propyl, n-butyl, t-butyl, and like straight and branched
chain Cl-C4 hydrocarbon radicals.
The term C3-C6 cycloalkyl refers to cyclopropyl,
cyclobutyl, cyclopentyl and cyclohexyl.
The cephalosporin antibiotic compounds represented
by the formula I, when R3 is hydrogen, are prepared by
acylating a 7-(D-a-amino-a-arylacetamido)-3-cephem-4-
carboxylic acid represented by the formula II,
O H
H2N-CH-C-N-I - t ~ 'I
Rl o~ ~ ~ -CH2-R2 II
COOH
wherein Rl and R2 have the previously defined meaningsJ
1~963''~3
O H
H2N-CH-C-N--
R1 o~t ~ ~ ~-CH2-R2 II
COOH
wherein Rl and R2 are as defined above, with a compound of
the formula
O
R-C-A
wherein R is as defined above and
A is -Cl or {)~ NO2
~ /
and if desired converting the acid so obtained
wherein R3 is hydrogen to the corresponding ester
wherein R3 is other than hydrogen ~
and where desired, when R is hydrogen, forming a
pharmaceutically acceptable, non-toxic salt of said compound
of formula I.
The compounds of formula I wherein R is the 1,3-
disubstituted ureido group
-6a-
1~963~;'3
H~O
l ll
R''-N-C-N- (Formula I, R'=CH3)
CH3
and R" is other than hydrogen are prepared by acylating the
cephalosporin compound II with the corresponding carbamoyl
chloride,
.
H O O
11 11
R''-N-C-N-C-CI
CH3
- The compounds of the formula I wherein R is a cyclic ureido
group are prepared by the acylation of II with a cyclic
ureido carbamoyl chloride of the formula
Il O
Y~
~ CHa)n
wherein Y is hydrogen, acetyl or methanesulfonyl, and
n is 2 or 3.
The compounds of the formula I wherein R is a
3-substituted ureido group
~L~i9G,~73
~1 0 .
I 11
R''-N-C-N- (Formula I, R'=H~
H
are prepared by the acylation of II with a p-nitrophenyl
carbamate
H O H O
R''-N-C-N-C-O--~ ~ --NOz
=--
The starting materials represented by the formula
II wherein Rl is a phenyl, substituted phenyl, or thienyl
group and R2 is a l-lower alkyl-lH-tetrazole-5-yl group or a
5-lower alkyl-1,3,4-thiadiazole-2-yl group are described by
Ryan in U.S. Patent 3,641,021. Compounds of the formula II
wherein Rl is a furyl group are prepared by the acylation of
a 7-amino nucleus compound of the formula
H~N-~
t
COOH
with the anhydride formed with a-(t-butyloxycarbamido)-
furanacetic acid and isobutylchloroformate. Following the
X-4087A 8
~9~i~73
acylation the t-butyloxycarbonyl protecting group is removed
by known methods, for example, with trifluoroacetic acid in
the cold, or alternatively with p-toluenesulfonic acid in
acetonitrile as described by Chauvette in U.S. Patent
3,769,281.
The starting materials represented by formula II
wherein R2 is the acetoxy group are prepared by the acylation
of 7-aminocephalosporanic acid with a phenyl, thienyl, or
furyl glycine. The compound of the formula II wherein Rl is
phenyl and R2 is acetoxy is the well known antibiotic,
cephaloglycin.
The acylation of a compound of the formula II with
the 1,3-disubstituted-ureidocarbamoyl chloride
H O O
l 11 11
R ' ' -N-C-N-C-C I
CH3
or the cyclic ureidocarbamoyl chloride involves the acylation
of the free a-amino group in the 7-position side chain. The
acylation is carried out in an inert solvent in the presence
of a hydrogen halide acceptor at a temperature between
about -5C. and 20C. and preferably at about 0-5C. Sol-
vents such as acetonitrile, tetrahydrofuran, dimethylform-
amide and dimethylacetamide can be used in the acylation. A
preferred solvent is acetonitrile. Should the starting
material be insoluble or partly insoluble in the solvent, it
can be solubilized by the addition of a silylating agent
such as bis-(trimethylsilyl~acetamide tBsA) before the
X-4087A 9
~96373
; the addition of the carbamoyl chloride.
Hydrogen halide acceptors which can be used include
the tertiary amines such as triethylamine and pyridine and
the alkylene oxides such as propylene oxide or butylene
oxide.
The carbamoyl chlorides
H 0 0
1 11 11
R''-N-C-N-C-CI
CH3
are prepared by reacting the 1,3-disubstituted urea with
phosgene in a dry, inert solvent such as dichloroethane,
dichloromethane or tetrahydrofuran. The reaction is prefer-
ably carried out in the cold at about 0-5C.
The symmetrical 1,3-dimethylurea affords but one
carbamoyl chloride, namely, N-methylaminocarbonyl-N-
methylcarbamoyl chloride
H 0 0
1 11 11
CH3-N-C-N-C-CI (R"=methyl)
CH3
since both nitrogen atoms of the urea are equivalent.
The unsymmetrical ureas, wherein R" is a group
other than methyl, can form two carbamoyl chlorides on
reaction with phosgene. The desired N-methylcarbamoyl
chloride
H 0 0
11
R''-N-C-N - C-CI
CH3
is separated from the undesired isomeric chloride by frac-
tional crystallization from mixtures of polar and non-polar
X-4087A 10
1~9~3~
organic solvents such as mixtures of diethyl ether and
petroleum ether, and acetone or ethyl acetate mixed with
hexane or petroleum ether.
The 1,3-disubstituted ureas are prepared by well
known methods or are commercially available.
Illustrative N-substituted-aminocarbonyl-N-methyl-
carbamoyl chlorides are represented by the foregoing formula
wherein R" is ethyl, H2C=CH-CH2-(allyl), HC-C-CH2-(propargyl),
phenyl, benzyl, 2-furfuryl, cyclopropyl and cyclohexyl.
The cyclic ureido carbamoyl chlorides
Il o
Y-N ~~C-CI
(CH~)n
wherein Y and n are as defined above are prepared by react-
ing the substituted (Y=acetyl or methylsulfonyl) or unsubsti-
tuted (Y=H), imidazolidine-2-one(n=2) or hexahydropyrimidine-
2-one(n=3) with phosgene in a dry, inert solvent at about
0-10C.
The cyclic ureido carbamoyl chlorides used to
prepare the cephalosporins of the formula I are imidazol-
idine-2-one-1-ylcarbonyl chloride, 3-(methylsulfonyl)-
imidazolidine-2-one-1-ylcarbonyl chloride, hexahydropyrimidino-
2-one-1-ylcarbonyl chloride, 3-(methylsulfonyl)hexahydropyrimi-
dine-2-one-1-yl-carbonyl chloride, and 3-(acetyl)hexahydro-
pyrimidine-2-one-1-ylcarbonyl chloride.
The preparation of the compounds represented by
X-4087A 11
1~9~i37~
the formula I wherein R3 is hydrogen and R' is CH3 is illus-
trated by the following description of the preparation of
7-[a-(3-methylcarbamoyl-3-methyl-1-ureido)-a-phenylacet-
amido]-3-(1-methyl-lH-tetrazole-5-ylthiomethyl)-3-cephem-4-
carboxylic acid. 7-(D--Amino-a-phenylacetamido)-3-(1-
methyl-lH-tetrazole-5-ylthiomethyl)-3-cephem-4-carboxylic
acid is suspended in acetonitrile containing an excess of
propylene oxide. A small excess of bis-(trimethylsilyl)-
acetamide is added to the suspension with stirring to effect
solution. The solution is cooled to about 5C. and a molar
equivalent of N-methylaminocarbonyl-N-methylcarbamoyl chlor-
ide is added dropwise or portionwise with stirring. After
stirring for about 2-4 hours in the cold, the reaction
mixture is allowed to warm to room temperature. The product
is preferably isolated via extraction with an organic sol-
vent such as ethyl acetate. The reaction mixture is poured
i~,o a mixture of ethyl acetate and water, the aqueous phase
;3 separated, acidified, and the product extracted with
ethyl acetate.
The compounds of the formula I wherein R' is
hydrogen are prepared by acylating the ~-amino cephalosporin
starting material II with a p-nitrophenyl carbamate
H 0 H 0
R' ' -N-C-N-C-O--~ >--NO2
wherein R" has the previously defined meanings. These esters
are prepared by reacting urea or the mono-substituted urea,
~-4087A 12
~'
~96373
R"-NH-C(O)-NH2 in an inert solvent such as THF with p-nitro-
phenyl chloroformate. For example, methylurea is reacted in
dry THF at about 0C. with p-nitrophenyl chloroformate to
form p-nitrophenyl N-(methylcarbamoyl)carbamate.
As is the case in the preparation of the above
described carbamoyl chlorides, the reaction of urea or a
mono-substituted urea with p-nitrophenyl chloroformate
affords two isomeric p-nitrophenyl carbamates.
The desired carbamate is formed by the acylation
of the Nl(unsubstituted)urea nitrogen while the undesired
carbamate ester is fo~med by the acylation of the N3(sub-
stituted), or the R"-N-, urea nitrogen. Usually the two
products are formed in equal amounts.
The desired p-nitrophenyl N-(substituted carbamoyl)
carbamates form isocyanates when treated with silylating
agents such as bis-(trimethylsilyl)acetamide (BSA) or mono-
(trimethylsilyl)acetamide (MSA). The reaction is illus-
trated by the scheme:
H 0 H 0 ._ H 0
1 11 1 11 ~ ~ 1 11
R''-N-C-N-C-0~ -N02 BSA ~ R''-N~C-N=C=0
-- +
H0~ -N02
The undesired p-nitrophenyl carbamate
X-4087A 13
~6373
R' '-N-C-NH2
C=O
o~ -NO2
.
formed along with the above desired carbamate is incapable
of forming an isocyanate with the silylating agent.
In the acylation of an a-aminoarylacetamido
cephalosporin, II, to form the cephalosporin I wherein R'=H,
the mixture of both carbamates obtained as described above
is conveniently used. The acylation reaction is carried out
in an inert dry solvent in the presence of an excess of a
silylating agent such as BSA or MSA. The p-nitrophenyl
N-(substituted carbamoyl)carbamate forms the isocyanate ln
situ which then reacts with the ~-amino group of II to form
the product.
Illustrative p-nitrophenyl carbamates useful in
preparing the compounds of the formula I wherein R'=H are
p-nitrophenyl N-(ethylcarbamoyl)carbamate, p-nitrophenyl
N-tcyclopropylcarbamoyl)carbamate, p-nitrophenyl N-(phenyl-
carbamoyl)carbamate, p-nitrophenyl N-(phenylcarbamoyl)-
carbamate, p-nitrophenyl N-(propargylcarbamoyl)carbamate,
p-nitrophenyl N-(allylcarbamoyl)carbamate, p-nitrophenyl
N-(benzylcarbamoyl)carbamate and p-nitrophenyl N-(carbamoyl)-
carbamate (R"=H).
The acylation of an a-aminoarylacetamido cephalo-
sporin, II, with the above p-nitrophenyl carbamates is
conveniently carried out in dry acetonitrile at about room
temperature (20-25C.). Ir. order to insure anhydrous condi-
tions the acylation is preferably carried out in an atmos-
X-~087A 14
~9~3q3~
phere of a dry inert gas such as nitrogen or argon. A sily-
lating agent such as BSA or MSA is added in excess and
serves two functions. It first serves to solubilize the
-aminoarylacetamidocephalosporin II via formation of solu-
ble silyl derlvatives (for example a silyl ester of II) and
secondly the excess reacts with the p-nitrophenyl carbamate
to generate, in situ, the isocyanate as described above.
. .
The acylation reaction is carried out as follows.
A suspension of the a-aminoarylacetamido-cephalosporin II,
in dry acetonitrile is treated with excess BSA. After a
homogeneous solution is obtained the mixture of p-nitro-
phenyl carbamates is added in an amount sufficient to provide
at least one molar equivalent of the desired p-nitrophenyl
carbamate isomer per compound II.
The reaction mixture is stirred at room tempera-
ture for between 1 and 3 hours after which the product is
recovered.
The cephalosporin product (formula I, R'=H, R3=H)
is conveniently recovered by extracting the reaction mixture,
after dilution with water, with a water-immiscible organic
solvent such as ethyl acetate, amyl acetate or other suit-
able solvent. The extraction is carried out at acid pH and
' preferably at about pH 2.5. The extract is washed, dried
and evaporated to yield the cephalosporin antibiotic of the
invention.
In an exemplary preparation of the cephalosporins
of the formula I wherein R' is hydrogen, 7-[a-amino-a-(2-
thienyl)acetamido]-3-(5-methyl-1,3,4-thiadiazole-2-ylthio-
methyl)-3-cephem-4-carboxylic acid is reacted in dry aceton-
itrile in the presence of excess bis-(trimethylsilyl)-
X-4087A 15
1~963~73
acetamide with p-nitrobenzyl N-(methylcarbamoyl)carbamate to
provide, after recovery, 7-[a-(3-methylcarbamoyl-1-ureido)-~-
(2-thienyl)acetamido]-3-(5-methyl-1,3,4-thiadiazole-2-yl-
thiomethyl)-3-cephem-4-carboxylic acid.
According to the above described preparative
procedures the compounds of the formula I wherein R is a
1,3-disubstituted ureido group of the formula
H O
1 11
R' '-N-C-N- (R'=CH3)
CH3
are prepared with the above described carbamoyl chlorides.
The compounds I, wherein R' is hydrogen, are prepared as
described above with a p-nitrophenyl N-(substituted carba-
moyl) carbamate,
H O H O
I 11 1 11 ~ ~
R''-N-C-N-C-0--~ / -N02
Illustrative of the cephalosporin antibiotics of
the formula I wherein R3 is hydrogen are the following.
X-4087A 16
1~6.~73
R Rl R2
CH3N-C-N- phenyl -S~
CH3
4-hydroxy- CH3
do phenyl do
4-chloro-
do phenyl do
4-hydroxy-
3-chloro-
do phenyl do
do 2-thienyl do
do 2-furyl do
do 3-thienyl do
do phenyl -S-~ -CH3
4-hydroxy-
do phenyl do
do 2-thienyl do
H~ phenyl -S-~
3-hydroxy- CH3
do phenyl do
do 2-thienyl do
3-chloro-
do phenyl do
do phenyl do
4-hydroxy-
do phenyl do
X-4087A 17
1~39~Y~3
-- --1 R2
H~ phenyl_s_~\ ~-CH3
3-chloro-
4-hydroxy--
do phenyl do
do 2-thienyl do
do 2-furyl do
do phenylacetoxy
4-hydroxy-
do phenyl do
do 2-thienyl do
O ~ .
CH3-C-N \~- phenyl acetoxy
1--
4-hydroxy-
do phenyl
CH3
20CH3-SO2-~ phenyl do
do 2-thienyl do
do 2-furyl do
CH3-SO2-~ 1- phenyl do
\~/
X-4087A 18
~L~ ~3~;3~7 3
R Rl R2
do do acetoxy
4-hydroxy-
do phenyl do
H do do ~ ~~
1 11
CH3-N-C-N- phenyl acetoxy
CH3
4-hydroxy-
do phenyl do
3-chloro-
4-hydroxy-
do phenyl do
do 2-thienyl do
H 0
1 11
R''-N-C-N~
R'
(R") (R') ~ _ ~
phenyl H phenyl --~\/Y CH3
do CH3 do acetoxy
benzyl H do ~
CH3
4-hydroxy-
2-furfuryl H phenyl do
allyl H phenyl do
do CH3 do acetoxy
X-4087A 19
~9~373
R Rl R2
(R") (R')
3-chloro-
4-hydroxy-
propargyl H phenyl do
do H 2-thienyl do
do CH3 do ~
CH3
do H phenyl do
cyclo-
propyl H do do
do H do acetoxy
do CH3 do do
cyclo-
pentyl H do do
4-chloro- N N
do H phenyl \S/
4-hydroxy-
do H phenyl do
do CH3 phenyl
CH3
X-4087A 20
1~9637'3
The cephalosporin antibiotics of the formula I
wherein R3 is hydrogen are converted to the acyloxymethyl
esters, wherein R3 is represented by the group
O
-CH2-O-C-Y'
by reacting an alkali metal salt of the cephalosporin car-
boxylic acid, for example, the lithium, sodium, or potassium
; salt, with an acyloxymethyl halide of the formula
O
X-CH2-O-C-Y'
wherein X is chloro or bromo and Y' has the same meanings as
previously defined. Acyloxymethyl halides which can be
; employed include chloromethyl acetate, bromomethyl acetate,
bromomethyl propionate, chloromethyl pivaloate, and benzo-
yloxymethyl chloride.
The preparation of the acyloxymethyl esters of the
formula I is carried out by reacting the alkali metal salt
form of the parent acid in an inert solvent with a slight
molar excess of the bromo or ch]oromethyl ester, e.g.,
bromomethyl acetate at room temperature or at slightly
elevated temperatures up to about 40-45C. Solvents such as
acetone, tetrahydrofuran, dioxane, dimethylformamide, and
methylene chloride can be used.
The indanyl esters of the formula I wherein R3 is
X-4087A 21
~C~9G373
1 `
are prepared by reacting 5-indanol in an inert solvent such
as dioxane or tetrahydrofuran with the free acid form of a
compound of the formula I wherein R3 is hydrogen, in the
presence of a condensing agent such as a diimide, for example,
dicyclohexyldiimide. T~e reaction is carried out with
stirring at about 20-35C. for about 6 to 8 hours. The
indanyl ester is isolated by first diluting the reaction
mixture with water and filtering the reaction mixtu~ to
remove the insoluble dicyclohexylurea. The ester is then
extracted from the filtrate.
Alternatively, the indanyl esters can be prepared
by reacting a mixed acid anhydride formed with a cephalo-
sporin acid of the formula I and acetic acid with 5-indanol.
The phthalidyl esters of the formula I wherein R3
is the phthalidyl group
X-4087A 22
63~73
are obtained by reacting bromopht:halide of t-he Eormula
with a salt of a cephalosporin acid of the formula I. The
esterification can be carried out in dimethylformamide,
dimethylacetamide, dioxane, tetrahydrofuran, or mixtures
thereof by slowly warming a mixture of equimolar amounts of
the cephalosporin acid salt, for example, the sodium or
potassium salt and bromophthalide.
I~lustrative esters of the formula I are:
acetoxymethyl 7-[a-(3-methylcarbamoyl-3-methyl-1-
ureido)--phenylacetamido]-3-(1-methyl-lH-tetrazole-5-ylthio-
methyl)-3-cephem-4-carboxylate;
pivaloyloxymethyl 7-[a-(3-methylcarbamoyl-3-
methyl-1-ureido]-a-(4-hydroxyphenyl)acetamido]-3-t5-methyl-
1,3,4-thiadiazole-2-ylthiomethyl)-3-cephem-4-carboxylate;
phthalidyl 7-[a-(3-methylaminocarbamoyl-3-methyl-
l-ureido)--phenylacetamido]-3-(1-methyl-lH-tetrazole-5-
ylthiomethyl)-3-cephem-4-carboxylate;
acetoxymethyl 7-[a-(3-methylcarbamoyl-3-methyl-1-
ureido)-a-(3-chloro-4-hydroxyphenyl)acetamido]-3-(1-methyl-
lH-tetrazole-5-ylthiomethyl)-3-cephem-4-carboxylate;
X-4087A 23
1~ 373
acetoxy~ethyl 7-[~-(3-methylcarbamoyl-1-ureido)-a-
(2-thienyl)acetamido]-3-(1-methyl-lH-tetrazole-5-ylthio-
methyl)-3-cephem-4-carboxylate; and
pivaloyloxymethyl 7-[~-(3-methylcarbamoyl-1-ureido)-
~-phenylacetamido]-3-(1-methyl-lH-tetrazole-5-ylthiomethyl)-
3-cephem-4-carboxylate.
The cephalosporin compounds of this invention, in
the free acid form (formula I, R3=H), form pharmaceutically
acceptable salts with inorganic bases such as the alkali
metal carbonates and bicarbonates. For example, the sodium
and potassium salts can be formed with sodium and potassium
carbonate by following conventional procedures.
Salts can also be prepared with basic organic
amines, such as methylamine, diethylamine, cyclohexylamine,
dicyclohexylamine, ethanol amine, diethanol amine, and
tris-~hydroxymethyl)aminomethane. Such salts can be used to
formulate the antibiotics into suitable pharmaceutical forms
for parenteral administraticn.
The cephalosporin antibiotics of this invention
are highly effective in inhibiting the growth of a wide
spectrum of pathogenic microorganisms of both the gram-
positive and gram-negative type.
A number of cephalosporin antibiotics are known
which are effective against gram-positive microorganisms but
are limited in the spectrum of activity against the gram-
negative microorganisms. Other cephalosporin antibiotics
which have been synthesized have demonstrated enhanced
gram-negative activity; however, they possess either a lower
spectrum of activity against the gram-positive organisms
X-4087A 24
1~963~73
or a decreased level of activity against these organisms.
The antibiotics described herein ~have enhanced gram-negative
activity both with respect to spectrum and level of activity
as well as activity against the gram-positive microorganisms.
Thus, the antibiotics described herein can be characterized
as cephalosporins having an expanded spectrum of activity.
These cephalosporin antibiotics exhibit high
levels of activity against -the Pseudomonas, Enterobacter
sp., indole (+) and (-), Pro-teus sp., Aerobacter, Serratia,
and Klebsiella gram-negative microorganisms. They also are
effective in controlling the growth of penicillin resistant
Staphylococcus as well as the Streptococcus D group, e.g.,
S. faecalis.
The antibiotic activity of the cephalosporin
compounds of the formula I is illustrated by the data pre-
sented in Table I for representative compounds. The values
in the table are the minimum inhibitory concentrations (MIC)
for the test compounds against the indicated microorganisms.
The MIC values were obtained in the Gradient Plate ln vitro
method for determining antibiotic activity.
X-4087A 25
1~9~373
Z
H ^ 1:'~ ~ ~ ~ ~ ~1 N ~ 0
O ~i ~ (~
P~ ~ A
V~
O
o ) ~ o ~ ~ ~ ~ n o o
C) ~ N
O
o
a ~ ~ 1-- ~ ~ O O ~D ,~ O O
m ~ ~
H :~ O U~
u~ U a) "'
~ o o ~ ~ o o
H O
E~ ~ ~
~ ~ ~D
O ~ ~ ~ ~ ~ ~ ~ a~ C~ O O O
H
E~
H
H O O Ir) O o o co u~
o o
H O
.u ~ ~ ~ ~ ,fa
O ~ ~ O E O r ~ ~ o ~:r
X--4087A 26
~963 7~
U~
u
O O ~D Ln 0~ Ul U~ Ln O In
~ O O U~ r o o o o ~
~n u~ ~ ~ ~ ~ ~ Q~
æ ~ ~
H ~ U~
p,, o ,~ .. ... .. .
~ ~ o ~ l` o o
O ~) ~1 ~J ~1 0
A U~
~1 A ri
o o u~ a~ o o ~ o o o
~1 ~ O ~1 ~1 H ~ ') t~ O ~ ~ g
~1 U
O ~1 U
C) U ~
H O O ~ '1 ~1 0 0 0 ~1
~r ~ o e~ I o ~I t`') o 11') ~
O u~ ~ ~ A ~d
-- O H U~
H H E~ H
W ~ k ~ u~ o o ~) o ~ 0
-rl
E~ O
o o S~
F~l ~ `D U) In N O O N ~r O ~J
H ~1 ~ ~Ir--l ~D O N
H ~ ~ ,_1
E~ ~ ~ ~ ~
~¢ , R ,, 3 .
~ o a) ,~ ~ ~ u~
H rl ~i tJ a~ _) U E~
E~ ~ ~ O ~ s~In U~
o ,- o a) s~ ~1 a) ~ ,,
Hm ~ U ~ ~ ~ td S~ ~ ~ ~
H rl Q~ ,a U~ 11~ ~ U~ ~
rl 11~ ~ t~ ~15 E~ O-rlc_~
t~l fd U ~1 ~\~1 O 1~1 O
~, ,,,~ o u a~ E~.,, ~ o
O ~s~ ,
~ ~U ~ 0~ ~ ~ S~ ~ ~ o ~ X E~
,C u~ ~ ~ ~ tn a) ~r
1~:1 X ~:C u~ P-, u~ ~ ~> X ~ ~I N
X-4087A 27
~9~373
A. 7-[a-(3-methylcarbamoyl-3-methyl-1-ureido)-a-(4-
hydroxyphenyl)acetamido]-3-(1-methyl-lH-tetrazole-5-ylthio-
methyl) 3-cephem-4-carboxylic acid.
B. 7-[a-(3-methylcarbamoyl-3-methyl-1-ureido)-a-(4-
hydroxyphenyl)acetamido]-3-(5-methyl-1,3,4-thiadiazole-2-
ylthiomethyl)-3-cephem-4-carboxylic acid.
C. 7-[a-(3-methylcarbamoyl-3-methyl-1 ureido)-a-
phenylacetamido]-3-acetoxymethyl-3-cephem-4-carboxylic acid.
D. 7-[a-(imidazolidine-2-one-1-ylcarbonylamino)-a-
phenylacetamido]-3-(1-methyl-lH-tetrazole-5-ylthiomethyl)-3-
cephem-4-carboxylic acid.
E. 7-[a-(imidazclidine-2-one-1-ylcarbonylamino)-a-
phenylacetamido]-3-acetoxymethyl-3-cephem-4-carboxylic acid.
F. 7-[a-(3-methylcarbamoyl-3-methyl-1-ureido)-a-phenyl-
acetamido]-3-(1-methyl-lH-tetrazole-5-ylthiomethyl)-3-cephem-
4-carboxylic acid.
H. 7-[a-(3-carbamoyl-1-ureido-a-phenylacetamido]-3-
(l-methyl-lH-tetrazole-5-ylthiomethyl)-3-cephem-4-carboxylic
acid.
I. 7-[a-(3-methylcarbamoyl-1-ureido)-a-(2-thienyl)-
acetamido]-3-(1-methyl-lH-tetrazole-5-ylthiomethyl)-3-
cephem-4-carboxylic acid.
J. 7-[a-(3-methylcarbamoyl-1-ureido)-a-phenylacetamido)-
3-(1-methyl-lH-tetrazole-5-ylthiomethyl)-3-cephem-4-car-
boxylic acid.
K. 7-[a-(3-methylcarbamoyl-3-methyl-1-ureido)-a-
(2-thienyl)acetamido]-3-(1-methyl-lH-tetrazole-5-ylthio-
methyl)-3-cephem-4-carboxylic acid.
L. Sodium 7-la-(3-methylcarbamoyl-1-ureido)-a-(4-
hydroxyphenyl)acetamido]-3-(5-methyl-1,3,4-thiadiazole-2-yl-
X-4087A 28
'1~9~i373
thiomethyl)3-cephem-4-carboxylat:e.
M. 7-[~-(3-phenylcarbamoyl-1-ureido)-~-phenylace-
tamido]-3-(1-methyl-lH-tetrazole-5-ylthiomethyl)-3-cephem-4-
carboxylic acid.
In the standard agar dilution method, the MIC
values for the above test compounds A through E against
Staphylococcus aureus were as follows: A, 2; B, l; C, 0.5;
. . .
D, 2; and E, 0.5 mcg./ml.
The cephalosporin antibiotics of the formula I,
wherein R3 is hydrogen, and the pharmaceutically acceptable
salts thereof are useful in combating infections in warm
blooded mammals when administered parenterally in non-toxic
doses between about 10 and 500 mg./kg. The indanyl, phthal-
idyl and acyloxymethyl esters of the formula I are useful
antibiotics when administered orally in non-toxic doses of
between about 50 and 750 mg./kg. of body weight.
A preferred group of cephalosporin antibiotics of
this invention are represented by the formula I wherein R is
0
CH~-N-C-N- or H-N \N-
H R ~
R' is H or methyl;
Rl is phenyl, hydroxyphenyl, hydroxy-substituted halo-
phenyl, or 2-thienyl;
R2 iS
X-4087A 29
~96,3~3
-S-~
CH3
R3 is hydrogen and the pharmaceutically acceptable
salts thereof.
A further preferred group of antibiotics are represented
by the formula I when
R is
CH3-N-C-N-
H R'
R' is hydrogen or methyl;
Rl is phenyl, hydroxyphenyl, or hydroxy substituted
halophenyl or 2-thienyl;
R2 iS
\S
2 0 CH3
R3 is hydrogen and the pharmaceutically acceptable
non-toxic salts thereof.
Exemplary of the preferred antibiotics are 7-[a-(3-
methylcarbamoyl-3-methyl-1-ureido)-a-phenylacetamido]-3-(1-
X-4087A 30
1~9~i3'73
methyl-lH-tetrazole-5-ylthiomethyl)--3-cephem-4-carboxylie
acid,
7-[~-(3-methylcarbamoyl-3--methyl-1-ureido)-~-(4-
hydroxyphenyl)aeetamido]-3-(S-methyl-1,3,4-thiadiazole-2-
ylthiomethyl)-3-cephem-4-carboxylic acid;
7-[~-(3-methylcarbamoyl-3--methyl-1-ureido)--
(4-hydroxyphenyl)acetamido]-3-(1-methyl-lH-tetrazole-2-
ylthiomethyl)-3-eephem-4-carboxylic acid,
7-[~-(3-methylcarbamoyl-1-ureido)-~-phenylacet-
amido]-3-(1-methyl-lH-tetrazole-5-ylthiomethyl)-3-cephem-4-
carboxylic acid,
7-[~-(3-methylcarbamoyl-3-methyl-1-ureido)-a-(2-
thienyl)acetamido]-3-(1-methyl-lH-tetrazole-5-ylthiomethyl)-
3-cephem-4-earboxylie acid,
7-[-(3-methylearbamoyl-1-ureido)-a-(2-thienyl)-
aeetamido]-3-(1-methyl-lH-tetrazole-5-ylthiomethyl-3-cephem-
4-earboxylie aeid.
7-[~-(imidazolidine-2-one-1-ylearbonylamino)-~-
phenylaeetamido]-3-(1-methyl-lH-tetrazole-5-ylthiomethyl)-3-
eephem-4-earboxylie aeid,
7-[a-(3-methylearbamoyl-3-methyl-1-ureido)-~-(3-
chloro-4-hydroxyphenyl)acetamido]-3-(1-methyl-lH-tetrazole-
5-ylthiomethyl)-3-eephem-4-earboxylie aeid.
7-1~-(3-methylearbamoyl-3-methyl-1-ureido)-~-(3,5-
diehloro-4-hydroxyphenyl)aeetamido]-3-(1-methyl-lH-tetrazole-
5-ylthiomethyl)-3-eephem-4-earboxylie aeid, and the pharma-
eeutieally aeeeptable, non-toxie salts thereof.
The in vivo effeetive dose (ED50) in mg./kg. for
representative antibioties of the formula I has been deter-
mined in experimental infeetions in miee. Table II lists
X-4087A 31
63'73
the ED50 dose vs Streptococcus ~ogenes and Escherichia coli
infections. In the table, the antibiotics are identified
with the R", R', and Rl-R3 terms of Formula I.
-4087A 32
1~963~73
TABLE II
Test Compound ED50(mg/kg x 2) i.p.
R" R' R R R S. pyogenes E. coli
~ 2 3 --
CH3 H phenyl tet H 0.7 72
4-hydroxy
CH3 CH3 phenyl tet H 0.7 72
H H phenyl tet H 0.7 <72
4-hydroxy 2
CH3 H phenyl thiad H 0.7 <72
CH3 H 2-thienyl tet H ~7.2 <72
CH3 CH3 phenyl acetoxy H<7.2 ~7.2
l/tet=l-methyl-lH-tetrazole-5-ylthio-
2/thiad=5-methyl-1,3,4-thiadiazole-2-ylthio-,
X-4087A 33
~96373
The following examples are provided to further describe
this invention and are not to be construed as limiting
thereof.
In the examples the following abbreviations have
the meaning indicated below.
BSA - bis-(trimethylsilyl)acetamide
THS - tetrahydrofuran
DMF - dimethylformamide
NMR - nuclear magnetic resonance spectrum
IR - infrared absorption spectrum
UV - ultraviolet absorption spectrum.
Example 1
N-Methylcarbamoyl-N-methylcarbamoyl chloride.
To a cold suspension of 22 g. (0.25 m.) of sym-di-
methylurea in dichloroethane was added dropwise with stirring
a cold solution of 30 g. (0.3 m.) of phosgene in 90 ml. of
dichloroethane. After the addition of the phosgene sclution
was complete, the reaction mixture was allowed to stir at
room temperature for one hour and was then heated to 80C.
and purged with nitrogen for one hour. The reaction mixture
was evaporated under reduced pressure and the residual gum
was extracted twice with 350 ml. portions of ether. The
extracts were combined and evaporated to provide 25 g. of
the carbamoyl chloride.
Example 2
Imidazolidine-2-one-1-ylcarbonyl chloride.
To a stirred suspension of 35 g. of 2-imidazolidine
in 500 ml. of dry tetrahydrofuran cooled in an ice bath was
added a cold solution of 40 g. of phosgene in 100 ml. of dry
tetrahydrofuran. The reaction mixture was stirred at room
X-4087A 34
~963~3
temperature for about 16 hours and was filtered to remove
insolubles. The filtrate was concentrated under reduced
pressure and the reaction product precipitated from the
concentrate on the addition of acetone and petroleum ether.
The product was collected by filtration and dried on the
filter.
Example 3
Preparation of 7-[a-(3-methylcarbamoyl-3-methyl-1-ureido)-a-
(4-hydroxyphenyl)acetamido]-3-(1-methyl-lH-tetrazole-5-
ylthiomethyl)-3-cephem-4-carboxylic acid.
To a suspension of 483 mg. (1 mmole) of 7-[D-a-
amino--(4-hydroxyphenyl)acetamido]-3-(1-methyl-lH-tetrazole-
5-ylthiomethyl)-3-cephem-4-carboxylic acid in 8 ml of dry
acetonitrile containing 2 ml. of propylene oxide maintained
under an atmosphere of dry argon was added 1 ml. of bis-ttri-
methylsilyl)acetamide. When solution was obtained, the
reaction vessel and solution were cooled to 0-5C. A solu-
tion of 150 mg. (1 mmole) of N-methylaminocarbonyl-N~methyl-
carbamoyl chloride in 2 ml. of dry acetonitrile was added
dropwise to the cold solution with stirring. The reaction
mixture was stirred for 2 hours and was allowed to warm to
room temperature. The reaction mixture was poured into a
mixture of water and ethyl acetate and the pH of the mixture
was adjusted to pH 9. The aqueous layer was separated and
relayered with fresh ethyl acetate. The pH of the aqueous
layer was adjusted to pH 2.5 and the ethyl acetate layer was
separated, was washed with brine and dried over sodium
sulfate. The dried ethyl acetate solution containing the
reaction product was evaporated to dryness in vacuo to
X-4087A 35
73
obtain the product as a faintly yellow powder. The product
was dissolved in ethyl acetate and in part precipitated by
adding petroleum ether to the solution. The precipitate of
product was filtered and dried to yield 138 mg. Additional
product was recovered by evaporation of the filtrate.
NMR (60 MHz., DMSO d6): 9.8 (d, J=7, lH), 9.3 (d,
J=8, lH), 7.4-6.5 (m, 5H), 5.85-5.50 (g, lH), 5.50-5.30 (d,
J=7, lH), 4.9 (d, J=5, lH), 4.3 (broad, 2H), 3.5 (s, 3H),
3.6 (broad, 2H), 3.1 (s, 3H) and 2.65 (d, J=3, 3H) delta.
Elemental analysis (percent) for C22H25NgO7S2
Theory: C, 44.66; H, 4.26; N, 21.31
Found: C, 44.32; H, 4.34; N, 19.32.
Example 4
Preparation of 7-[a-(3-methylcarbamoyl-3-methyl-1-ureido)-a-
(4-hydroxyphenyl)acetamido]-3-(5-methyl-1,3,4-thiadiazole-2-
ylthiomethyl)-3-cephem-4-carboxylic acid.
To a suspension of 2.12 g. (4 mmole) of 7-[D-a-
amino-a-(4-hydroxyphenyl)acetamido]-3-(5-methyl-1,3,4-thia-
diazole-2-ylthiomethyl)-3-cephem-4-carboxylic acid in 32 ml.
of dry acetonitrile containing 8 ml. of propylene oxide
maintained under an atmosphere of dry argon was added 4 ml.
of bis-(trimethylsilyl)acetamide with stirring. When solu-
tion had occurred, the solution was cooled to O~C. and 600
mg. (4 mmole) of N-methylaminocarbonyl-N-methylcarbamoyl
chloride in 8 ml. of dry acetonitrile were added dropwise
with stirring. The reaction mixture was stirred for 2 hours
during which time the temperature of the mixture was allowed
to rise to room temperature. The reaction mixture was
poured into a water-ethyl acetate mixture and the pH of the
X-4087A 36
g~,373
a~ueous layer was adjusted to pH 8.5. The aqueous layer was
separated and layered with fresh ethyl acetate. The pH of
the aqueous layer was then adjusted to about pH 2.5 and the
ethyl acetate layer was separated, washed with brine and
dried over sodium sulfate. The dried solution was evaporated
to dryness under reduced pressure to yield about 1.6 g. of
the product as a light yellow solid. The product ~as trit-
urated with ethyl acetate and the insoluhle product (645
mg.) was filtered. The filtrate was concentrated by evapora-
tion to yield 423 mg. of precipitated product. The latter
filtrate was evaporated to dryness to yield 560 mg. The
above three product fractions were shown to be the same when
chromatographed on silica gel thin layer chromatograms using
chloroform:methanol (7:3, v:v) for development and either
iodine vapors or ultraviolet light for visualization of the
developed plates.
Elemental analysis (percent) for C23H25N7O7S3:
Theory: C, 45.46; H, 4.15; N, 16.13
Found: C, 45.53; H, 4.47; N, 14.85.
Electrometric tritration in 66 percent DMF showed
the presence of two titratable groups:
PKa 4.8 and 12.2
and an apparent molecular weight as calculated from the
titration data of 587 (calculated MW=607). The infrared
absorption spectrum (mineral oil mull) showing the character- -
istic ~-lactam carbonyl absorption at about 2920 wave numbers
and the NMR spectrum were in agreement with the expected
product.
X-4087A 37
~96~7:~
UV absorption spectrum (methanol)
A max 303 ~=9,246
max 275 ~=9,273
max 229 ~=16,254
Example 5
Preparation of 7-[a-(3-methylcarbamoyl-3-methyl-1-ureido)-a-
phenylacetamido]-3-acetoxymethyl-3-cephem-4-carboxylic acid.
The above-named compound was ~repared by the
reaction of 7-(D-a-amino-a-phenylacetamido)-3-acetoxymethyl-
3-cephem-4-carboxylic acid (cephaloglycine) with N-methyl-
aminocarbonyl-N-methylcarbamoyl chloride by following the
reaction conditions employed in Example 3. The product (158
mg.) was obtained as a white crystalline solid.
Nuclear magnetic resonance spectrum (60 MHz., DMSO
d6): 7.45 (s, 5H), 5.9-5.4 (m, 2H), 5.2-4.4 tm, 3H) 3.5
~broad, 2H), 3.15 (s, 3H), 2.7 (s, 3H) and 2.05 (s, 3H)
delta.
Example 6
Preparation of 7-[D-a-(imidazolidine-2-one-1-ylcarbonyl-
amino)-~-phenylacetamido]-3-acetoxymethyl-3-cephem-4-carboxy-
lic ncid.
A suspension of 17.66 g. of cephaloglycine di-
hydrate in 150 ml. of tetrahydrofuran-water (80 percent THF)
was cooled in an ice bath and the pH of the suspension was
adjusted to 7.8-8.2 by the addition of triethylamine. To
the cold (0C.) suspension was added in small portions 5.94
X-4087A 38
G373
g. of N-chlorocarbonylimidazolidine-2-one. Throughout the
addition of the acid chloride, the pH of the reaction mixture
was maintained between 7.5 and 8n 0 by adding triethylamine
as required. Following the addition of the acid chloride,
the reaction mixture was stirred at 0C. for 30 minutes and
then at room temperature for another 20 minutes. The reaction
mixture (pH 7.5) was diluted with 130 ml. of water and then
evaporated to remove most of the THF. The aqueous phase was
extracted once with ether and was then layered with ethyl
acetate. The pH of the aqueous layer was adjusted to 1.5 to
2.0 with dilute hydrochloric acid. The ethyl acetate layer
was separated and was washed with water and dried over
magnesi~m sulfate. Evaporation of the dried ethyl acetate
solution under reduced pressure provided the product as an
amorphous solid.
Example 7
By following the acylation procedures and condi-
tions described by Example 6, 7-(D--amino-a-phenylacetamido)-
3-(1-methyl-lH-tetrazole-5~ylthiomethyl)-3-cephem-4-carboxy-
lic acid is reacted with N-chlorocarbonylimidazolidine-2-one
to provide 7-[D-(a-(imidazolidine-2-one-1-ylcarbonylamino)-a-
phenylacetamido]-3-(1-methyl-lH-tetrazole-5-ylthiomethyl)-3-
cephem-4-carboxylic acid.
Example 8
By following the procedures and by employing the
solvents and conditions described in the acylation of Example
6, 7-(D-a-amino-a-phenylacetamido)-3-(5-methyl-1,3,4-thia-
diazole-2-ylthiomethyl)-3-cephem-4-carboxylic acid is reacted
with chlorocarbonylimidazolidine-2-one to provide 7-[D-a-
X-4087A 39
37~
(imidazolidine-2-one-1-ylcarbonylamino)-a-phenylaceta~ido]-3-
(5-methyl-1,3,4-thiadiazole-2-ylthiomethyl)-3-cephem-4-car-
boxylic acid.
Example ~
Preparation of Sodium 7-[-(3-methylcarbamoyl-1-ureido)-a-
(4-hydroxyphenyl)acetamido]-3-(5-methyl-1,3,4-thiadiazole-2-
ylthiomethyl)-3-cephem-4-carboxylate.
To a suspension of 2.88 g. (6 mmole) of sodium
7-[D-a-amino-a-(4-hydroxyphenyl)acetamido]-3-(5-methyl-1,3,4-
thiadiazole-2-ylthiomethyl)-3-cephem-4-carboxylate in 48 ml.
of dry acetontrile at room temperature were added 6 ml. of
BSA. When an homogeneous solution was obtained 5.2 g. of
p-nitrophenyl methylcarbamoylcarbamate were added. The
reaction mixture was stirred for one hour and was poured
into a mixture of water and ethyl acetate. The pH was
adjusted to 6 and the ethyl acetate phase was separated and
replaced with fresh ethyl acetate. The pH of the aqueous
phase was adjusted to pH 2.5. The aqueous layer was separ-
ated and discarded. The ethyl acetate phase was washed with
dilute hydrochloric acid (pH 2.0) and fresh water was
added. The pH was finally adjusted to 5.5 and the aqueous
phase lyophilized to yield 1.3 g. of the product as a light
yellow powder.
NMR(DMSO-d6): 2.7 (s, 6H, NHCH3 & thiadiazole CH3),
3.5 (broad, 2H, C2-H2), 5O0 (d, J=5, lH, C6-H),
5.4-5.9 (m, 2H, C7-H & side chain CH), 6.9 (d,
J=9, 2H, aromatic) and 7.4 (d, J=9, 2H, aromatic)
delta.
X-4087A 40
~9~i3,7;~
Example 10
Preparation of Sodium 7-[a-(3-methylcarbamoyl-3-methyl-1-
ureido)-~-phenylacetamido]-3-(5-methyl-1,3,4-thiadiazole-2-
y]thiomethyl)-3-cephem-4-carboxylate.
To a suspension of 2.78 g. of sodium 7-(D-a-amino-
a-phenylacetamido)-3-(5-methyl-1,3,4-thiadiazole-2-ylthio-
methyl)-3-cephem-4-carboxylate in 48 ml of dry acetonitrile
containing 12 ml. of propylene oxide were added 6 ml. of
bis-(trimethylsilyl)acetamide. When a homogeneous solution
was obtained the solution was cooled to about 0C. and a
solution of 6 mmoles of N-methylaminocarbonyl-N-methyl-
carbamoyl chloride in 12 ml. of dry acetonitrile was added.
The reaction mixture was stirred for 2 hours.
The product was recovered by following the work-up
procedures described in Example 9 to yield 1.1 g. of the
sodium salt.
IR (mull): ~-lactam carbonyl absorption at about
2920 cm 1
NMR (DMSO d6): 2.7 (Broad, 6H, NHCH3 & Thiadiazole
CH3), 3.1 (s, 3H, N-CH3), 4.4 (Broad, 2H,
C(3')H2), 4.9 (d, J=4.5, lH, C(6)H), 5.4-5.9 (m,
2H, C(7)H & Side Chain CH), 9.4 (d, J=9, lH,
NH) and 10.0 (d, J=7, lH, NH) delta.
Example 11
Preparation of 7-la-(3-methylcarbamoyl-3-methyl-1-ureido)-a-
(2-thienyl)acetamido]-3-(1-methyl-lH-tetrazole-5-ylthio-
methyl)-3-cephem-4-carboxylic acid.
To a suspension of 234 mg. of 7-[a-amino-a-(2-
thienyl) acetamido]-3-(1-methyl-lH-tetrazole-5-ylthio
X-4087A 41
i373
methyl)-3-cephem-4-carboxylic acid in 4 ml. of dry aceton-
itrile containing 1 ml. of propylene oxide was added under
argon 0.5 ml. of BSA to form a homogeneous solution. The
solution was cooled to 0C. and a solution of 75 mg. of
N-methylaminocarbonyl-N-methylcarbamoyl chloride in 1 ml. of
acetonitrile. The reaction mixture was stirred for 2 hours
and was then poured into a mixture of water-ethyl acetate.
The pH of the aqueous phase was adjusted to pH 6 and the
organic phase separated. Fresh ethyl acetate was added to
the aqueous phase and the pH adjusted to 2.5 with dilute
hydrochloric acid. The organic phase was separated, dried
and evaporated to dryness to yield the product.
Elemental analysis for: C20H23NgO6S3
Theory: C, 41.30; H, 3.99; N, 21.68
Found: C, 41.78; H, 4.14; N, 21.73.
UV(methanol): ~ max 238 ~ 17,475
max 270 ~ 9,000
Example 12
Preparation of p-nitrophenyl methylcarbamoylcarbamate.
Methylurea (3.7 g., S0 mmole) and p-nitrophenyl
chloroformate (50 mmole) were allowed to react under nitro-
gen in 10 ml. of dry THF. The reaction mixture became clear
initially and the product began to precipitate from the
clear solution. The mixture was stirred for about 18 hours
and the precipitated product was filtered. The product was
washed with water and diethyl ether to yield 5.9 g. of
approximately a 50:50 mixture of p-nitrophenyl methylcar-
bamoylcarbamate and p-nitrophenyl carbamoyl-N-methylcarbamate
as shown by NMR.
X-4087A 42
~39~3~3
Example 13
Preparation of 7-[~-(3-methylcarbamoyl-1-ureido)-phenyl-
acetamido]-3-(1-methyl-lH-tetrazole-5-ylthiomethyl)-3-
cephem-4-carboxylic acid.
A suspension under argon of 2 mmoles of 7-(a-amino-
a-phenylacetamido]-3-(1-methyl-lH-tetrazole-5-ylthiomethyl)-
3-cephem-4-carboxylic acid in 16 ml. of dry acetonitrile was
solubilized by adding 2 ml of BSA. With stirring, 4 mmoles
of the product mixture obtained as described by Example 12
(mixture of p-nitrophenyl methylcarbamoylcarbamate and
p-nitrophenyl carbamoyl-N-methylcarbamate) were added. The
reaction mixture became clear in a few minutes and was
stirred for about 2 hours.
The reaction mixture was poured into a mixture of
water-ethyl acetate and the pH adjusted to 6. The ethyl
acetate layer was separated and discarded. The aqueous
phase was relayered with fresh ethyl acetate and the pH of
the aqueous phase was the adjusted to pH 2.5. The organic
layer was separated, washed with water, dried and evaporated
in vacuo. The product residue was triturated with diethyl
ether to yield 600 mg. of product as a faintly yellow powder.
Elemental analysis for: C21H23NgO6S2
Theory: C, 44.91; H, 4.13; 22.45; S, 11.42
Found: C, 44.66; H, 4.34; 22.29; S, 11.28.
UV(methanol): ~ max 272 11 , 091
NMR (DMSO d6): 2.6 (d, J5H2, 3H, NHCH3), 3.9 (S, 3H),
-CH3 on tetrazole), 3.55 (Broad, 2H, CH2), 5.0
(d, J=5, lH, C(6)H), 5.4-5.9 (m, 2H, C(7)H & Side
X-4087A 43
~91 i:373
Chain CH), 7.3 (S, 5H, Q), 8.4 (d, J-7, lH, NH),
8.8 (S, lH, NH) and 9.4 (d, J=9, lH, NH) delta.
Example 14
Preparation of 7-~a-(3-methylcarbamoyl-1-ureido)-a-(4-hydro-
xyphenyl)acetamido]-3-(5-methyl-1,3,4-thiadiazole-2-
ylthiomethyl)-3-cephem-4-carboxylic acid.
To a suspension of 988 mg. (2 mmole) of 7-[a-
amino-a-(4-hydroxyphenyl)acetamido]-3-(5-methyl-1,3,4-thia-
diazole-2-ylthiomethyl)-3-cephem-4-carboxylic acid in 16 ml.
of dry acetonitrile were added 2 ml. of BSA under argon. An
homogeneous solution was obtained and 1.43 g. (6 mmole) of
the product mixture containing 50% of p-nitrophenyl methyl-
carbamoylcarbamate (Example 12) were added. After stirring
for one hour the product was recovered from the reaction
mixture by following the work-up procedures described in
Example 13. The product, 524 mg., was obtained as a light
i yellow powder.
UV(methanol): A max 232 ~ 16,072
~ max 275 ~ 14,309
IRtmull): ~-lactam carbonyl absorption at about
2900 cm 1
Example 15
Preparation of 7-[a-(3-methylcarbamoyl-1-ureido)-a-(2-
thienyl) acetamido]-3-(1-methyl-lH-tetrazole-5-ylthiomethyl)-
3-cephem-4-carboxylic acid.
To a suspension of 435 mg. (0.93 mmole) of 7-[D-a-
amino-a-(2-thienyl)acetamido]-3-(1-methyl-lH-tetrazole-5-yl-
X-4087A 44
, ~ ~
~9637~
thiomethyl)-3-cephem-4-carboxylic acid in 8 ml. of dry
acetonitrile was added 1 ml. of b:is-(trimethylsilyl)acetamide.
After an homogeneous solution was obtained 1.4 g. of p-nitro-
phenyl methylcarbamoylcarbamate were added.
The reaction mixture was stirred at room tempera-
ture for 90 minutes. The product was recovered from the
reaction mixture by following the work-up procedures describ-
ed in Examp]e 11. The product was obtained as a white powder
weighing 341 mg.
Elemental analysis for: C29H21NgO6S3:
Theory: C, 40.20; H, 3.73; N, 22.21; S, 16.95
Found: C, 39.86; H, 4.02; N, 22.88; S, 14.66.
I.R.(mull) ~-lactam carbonyl absorption at about 2920 cm 1.
U.V.(methanol): ~ max 235 ~ 15,459
~ max 272 ~ 9,360
NMR(DMSO d6): 2.6(d, J=4.5, 3H, NHCH3), 3.6 (Broad, 2H,
C(2)H2), 3.9 (5, 3H, tetrazole CH3), 4.25 (Broad,
2H, C(3')H2), 5.1 (d, J=5, lH, C(6)H), 5.6-5.9(m,
2H, C(7)H & Side Chain CH), 6.9-7.6(m, 4H,
Thiophene & lNH), 8.4 (d, J=8, lH, NH), 8.9
(S, lH, NH) and 9.5 (d, J=8.5, lH, NH) delta.
Example 16
Preparation of p-nitrophenyl phenylcarbamoylcarbamate.
To a stirred solution of 6.8 g. of phenylurea in
50 ml. of dry THF maintained at 0C. under nitrogen were
X-4087A 45
~96373
added 5.05 g. of p-nitrophenyl chloroformate. The reaction
mixture was allowed to warm to room temperature and was
stirred for about 18 hours. The mixture was evaporated to
dryness and the residue dissolved in ethyl acetate. The
solution was washed twice with water, twice with brine, and
was filtered through sodium sulfate. The filtrate was evapor-
ated to dryness to yield 4.9 g. of the product as a white
product.
Example 17
Preparation of 7-[~-(3-phenylcarbamoyl-1-ureido)-a-phenyl-
acetamido]-3-(1-methyl-lH-tetrazole-5-ylthiomethyl)-3-cephem-
4-carboxylic acid.
A suspension of 950 mg. (2 mmole) of 7-(D-a-amino-
a-phenylacetamido)-3-(1-methyl-lH-tetrazole-5-ylthiomethyl)-
3-cephem-4-carboxylic acid in 16 ml. of dry acetonitrile at
0C. was solubilized under nitrogen with 2 ml. of BSA. With
stirring 1.2 g. of p-nitrophenyl N-phenylcarbamoylcarbamate,
prepared as described in Example 16, were added to the
solution. The reaction mixture was stirred for one hour and
the product recovered by following the work-up procedures
described in Example 11. The product was obtained as a
light yellow powder weighing 839 mg.
NMR(DMSO d6): 3.6 (Broad, 2H C(2)-H2), 3.95 (S, 3H,
Tetrazole-CH3), 4.3 (Broad, 2H, C(3')-H2), 5.0 (d,
J=5, lH, C(6)-H), 5.5-5.9 (m, 2H, C(7)-H & Side
Chain CH), 7.0-7.6 (Broad, lOH, Aromatic), 8.4 (d,
J=7.5, lH, NH), 9.1 (s, lH, NH), 9.6 (d, J=9,
lH, NH), 9.8 (S, lH, NH).
X-4087A 46
1~6373
Example 18
Preparation of 7-[a-(3-benzylcarbamoyl-1-ureido)-a-phenyl-
acetamido]-3-(1-methyl-lH-tetrazole-5-ylthiomethyl)-3-
cephem-4-carboxylic acid.
By following the procedure described in Example
17, 2 mmoles of 7-(a-amino-a-phenylace~amido)-3-(1-methyl-lH-
tetrazole-5-ylthiomethyl)-3-cephem-4-carboxylic acid were
dissolved in acetonitrile with BSA and reacted with 1.26 g.
of p-nitrophenyl N-benzylcarbamoylcarbamate to yield the
title compound.
NMR(DMSO d6): 3.6(Broad, 2H, C(3)H2), 3.9(S, 3H,
Tetrazole CH3), 4.3 (Broad, 4H, C(3')-H2 and
benzyl CH2), 5.0 (d, J=5, lH, C(6)H), 5.4-5.9
(m, 2H, C(7)H & Side Chain CH), 7.2-7.6 tm, lOH,
Aromatic), 7.8 (t, J=5.5, lH), 8.4 (d, J=7,
lH, NH), 8.95 (S, lH, NH), 9.5 (d, J=8, lH, NH).
Example 19
Preparation of 7-[a-(3-furfurylcarbamoyl-1-ureido)-a-
phenylacetamido]-3-(1-methyl-lH-tetrazole-5-ylthiomethyl)-3-
cephem-4-carboxylic acid.
By following the procedure described in Example
17, 2 mmoles of 7-(a-amino-~-phenylacetamido)-3-(1-methyl-lH-
tetrazole-5-ylthiomethyl)-3-cephem-4-carboxylic acid were
reacted at 0C. in dry acetonitrii~ with BSA and 1.22 g. of
p-nitrophenyl N-furfurylcarbamoylcarbamate to obtain the
title compound.
X-4087A 47
1~96~3~3
NMR(DMSO d6): 3.55 (Broad, 2H, C(2)-H2), 3.9 (S, 3H,
Tetrazole CH3), 4.3 (Bxoad, 4H, C(3')H2 & Thiophene
CH2), 5.0 (d, J=5, lH, C(6)H), 5.4-5.9 (m, 2H,
C(7)-H & Side Chain CH), 6.3 (m, 2H, thiophene
aromatic), 7.2-7.9 (m, 7H, aromatic & NH), 8.4
(d, J=7, lH, NH), 8.9 (s, lH, NH), 9.4 (d,
J=8, lH, NH).
Example 20
Preparation of p-nitrophenyl carbamoylcarbamate.
To a mixture of 1.20 g. (20 mmole) of urea and
2.02 g. (10 mmole) of p-nitrophenyl chloroformate in a dry
flask under nitrogen were added 10 ml. of dry acetonitrile.
The reaction mixture momentarily became clear and then a
precipitate formed. The mixture was tirred for 18 hours at
room temperature and the precipitate was filtered, washed
with water and dried under vacuum. The dried prcduct was
triturated with ether and redried to yield 1.0 g. of crystal-
line product.
Example 21
Preparation of 7-[a-(3-carbamoyl-1-ureido)-~-phenylacetamido~-
3-(1-methyl-lH-tetrazole-5-ylthiomethyl)-3-cephem-4-carboxy-
lic acid.
To a suspension of 2 mmole of 7-(a-amino-~-phenyl-
acetamido)-3-(1-methyl-lH-tetrazole-5-ylthiomethyl)-3-
cephem-4-carboxylic acid in 20 ml. of dry acetonitrile under
argon were added 3/4 ml. of BSA. When an homogeneous solu-
tion was obtained it was evaporated to dryness. The residual
dry off-white powder was dissolved in DMF and the solution
X-4087A 48
~Gi9~373
added under argon with stirring to a solution of 20 mmole of
p-nitrophenyl carbamoylcarbamate (Example 20) in 10 ml. of
DMF containing 20 mg. of l-hydroxybenzotriazole monohydrate.
The reaction mixture was stirred for 72 hours at room tem-
perature and then was diluted with water and the product
extracted with ethyl acetate at a pH of about 2.5. The
extract was washed, dried and evaporated to yield the product
as an amorphous powder.
Elemental analysis for: C20H2lN9o6s2
Theory: C, 43.87; H, 3.87; N, 23.02; S, 11.71
Found: C, 43.77; , 4.00; N, 22.86; S, 11.41.
UV(methanol): ~ max 250 ~ 8,957
NMR (DMS0 d6): 3.55 (Broad, 2H, C(2)H2), 3.95 (S, 3H,
tetrazole-CH3), 4.45 (Broad, 2H, c(3')H2), 5.0(d,
J=5, lH, C(6)H), 5.4-5.9 (m, 2H, C(7)H & Side
Chain CH), 6.75 (Broad S, 2H, -NH2), 7.4 (S, 5H,
phenyl), 8.51 (d, J=7, lH, NH), 8.8 (S, lH, NH~
and 9.5 (d, J=10, lH, NH) delta.
Example 22
Preparation of 3-(methylsulfonyl)imidazolidine-2-one-1-
ylcarbonyl chloride.
To a suspension of 10.7 g. of imidazolidone-2 in
100 ml. of dry THF were added dropwise with stirring at room
temperature 15.7 g. of methanesulfonyl chloride. The reac-
tion mixture was stirred for one-hour at about 40C. and was
then heated at the reflux temperature for one-hour.
The reaction mixture was evaporated in vacuo and
the thick syrupy residue was dried in vacuo for about 18
X-4087A 49
1~63~;~
hours. The dried residue was crystallized from warm acetone
to yield 7.1 g. of N-methylsulfonyl-imidazolidone-2. The
percent elemental composition of the product was determined
by microanalysis:
Calculated for: C4H8N2O3S:
Theory: C, 29.26; H, 4.91; N, 17.06; S, 19.53
Found: C, 29.47; H, 4.96; N, 17.17; S, 19.50.
A solution of 4.1 g. of the above product in
dioxane was treated with 7 g. of trimethylchlorosilane and 5
g. of triethylamine.
The solution was heated at the reflux temperature
for about 2.5 days and was cooled to room temperature. The
precipitate of triethylamine hydrochloride was filtered and
the filtrate treated with 3 ml. of phosgene. The filtrate
was allowed to stand at room temperature for 2 days and was
evaporated in vacuo to dryness. The residue was crystallized
from warm acetone and the product further dried in vacuo to
yield 2.8 g. of the title compound melting at about 178C.
Example 23
Preparation of 7-[-[3-(methylsulfonyl)imidazolidine-2-one-
l-ylcarbonylamino]a-(2-thienyl)acetamido]-3-(1-methyl-lH-
tetrazole-5-ylthiomethyl)-3-cephem-4-carboxylic acid.
To a solution of 200 mg. of 7-[~-amino-~-(2-
thienyl) acetamido]-3-(1-methyl-lH-tetrazole-5-ylthiomethyl)-
3-cephem-4-carboxylic acid in 25 ml. of dichloromethane were
added 2 molar equivalents of triethylamine and a pinch of
sodium sulfate. The mixture was filtered and 100 mg. of 3-
X-4087A 50
1~6373
methylsulfonylimidazolidine-2-one-1-ylcarbonyl chloride were
added to the filtrate. The reaction mixture was stirred for
3 hours in a water bath after which the dichloromethane was
evaporated. The residue was extracted at pH 2 with ethyl
acetate and the extract was washed with water and dried.
The dried extract was evaporated in vacuo and the residue
recrystallized twice from acetone:diethyl ether:petroleum
ether to yield 109 mg. of the product.
UV(methanolJ: ~ max 270 ~ 9,328
233 ~ 15,995
NMR(DMSO d6): 3.45 (S, 3H, CH3SO2-), 3.65 (AB, 2H,
C(2)-H2), 3.8(Broad, 4H, CH2CH2), 3.95 (S, 3H,
tetrazole CH3), 4.6 (AB, 2H, C(3')-H2), 5.05 (d,
J=5, lH, C(6)-H), 5.75 (dd, J=8, J2=5' lH,
C(7)-H), 5.88 (d, J=7.5, lH, Side Chain CH),
6.94-7.15 (m, 2H, thiophene aromatic), 7.4-7.5
~dd, lH, thiophene aromatic), 8.72 (d, J=7,
lH, NH) and 9.51 (d, J=8.5, lH, NH) delta.
Following the preparative methods described in the
above examples, 7-[a-(3-methylcarbamoyl-3-methyl-1-ureido)-a-
~4-hydroxyphenyl)acetamido]-3-(1-methyl-1,2,3-triazole-5-
ylthiomethyl)-3-cephem-4-carboxylic acid and 7-[D-a-
(imidazolidine-2-one-1-ylcarbonylamino)-~-phenylacetamdio]-3-
(l-methyl-1,2,3-triazole-5-ylthiomethyl)-3-cephem-4-carboxy-
lic acid are prepared.
X-4087A 51
