Note: Descriptions are shown in the official language in which they were submitted.
This invention relates to cextain novel a-amino- or a-
for~y~ (p-acyloxyphenyl)acetamidocephalosporanic acids
which are useful as antibacterial agents.
The novel cephalosporin derivatives of this invention
comprise the D-(-) compounds of the formula
~3NH2 F--N~H2Y
COOH
wherein Y is hydrogen or S-He~, in which Het represents a 5
or 6 membered heterocyclic ring containing 1 to 4 atoms selected
from N, O or S, said heterocyclic ring being optionally 5ub-
stituted by Cl-C4 alkyl which may be optionally substituted
by a carboxylic acid group or hydroxy, or alkoxyalkyl of up
to 4 carbon atoms; R is hydrogen, Cl-Clo alkyl optionally sub-
stituted by carboxylic acid, or phenyl optionally substituted
by Cl-C4 alkyl, halogen, nitro, amino or trifluoromethyl; R'
is hydrogen, hydroxy, Cl-C4 alkyl, Cl-C~ alkoxy or halogen, and
pharmaceutically acceptable salts thereof, when substantially
free of the L-(~) isomer, and the D-(-) compounds of the
formula I(a)
R'
R-~ H-CO-NH ~ S ~
CHO ~ N ~ CH2Y
COOH
I(a)
wherein Y is S Het, in which Het is 1,2,3-triazol-5-yl, l-N-
methyl-tetrazol-5-yl or 2-methyl-1,3,4-thiadiazol-5-yl; R is
hydrogen, Cl-Clo alkyl optionally substituted by carboxylic
acid, or phenyl optionally substituted by Cl-C4 alkyl, halo-
gen, nitro, amino or trifluoromethyl; R' is hydrogen, hydrox~,
Cl-C4 alkyl; Cl-C~ alko~y or halogen, and pharmaceutically
acceptable salts thexeof, when substantially free of the
5 L-(~) isomer.
In the above compounds, the substituent halogen is
preferably selected from fluorine, chlorine or bromine.
The pharmaceutically acceptable salts r~ferred to above
include the nontoxic carboxylic acid salts, e.g., nontoxic
metallic salts such as sodium, potassium, calcium and alumi-
nium, ~he ammonium salt and salts with nontoxic amines, e.g.,
_ trialkylamines, procaine, dibenzylamine, N-benzyl-~-phene-
thylamine, l-ephenamine, N,N'-dibenzylethylenediamine, N
alkylpiperidine and other c~nines which are used to form
salts of penicillins and cephalosporins. Also included within
the definition of pharmaceutically acceptable salts are the
nontoxic acid addition salts (amine salts), e.gO, salts with
mineral acids such as hydrochloric, hydrobromuc~ hydroiodic,
phosphoric, sulfuric and salts with organic acids such as
maleic, acetic,citric, oxalic, succinic, ben~oi~, tartaric,
fumaric, mandelic, asoorbic and malic.
Examples o~ suitable heterocyclic groups included within
the definition of "Het" in formula I are such heterocyclic
radicals as thienyl, pyrazolyl, imidazolyl, isoimidazolyl,
triazolyl, tetrazolyl, ~hiazolyl, thiadiazolyl, thiatriazolyl,
oxazolyl, oxadiazolyl, isothiazolyl, isoxa~o~yl, pyridylt
pyridazinyl, pyrazinyl, pyrimidinyl and triazinyl. The hetero-
cyclic ring may be unsubstituted or substituted wi~h one or
more of the substituents as mentioned above~
3Q Pre~erred D-(-) compounds of formula I are those wherein
~5~ 3
Y is hydrogen or S-Het, in which Het represents 1, 2, 3-triazolyl,
2~methyl-1,3,4-thiadiazol~5-yl, 2-methyl 1,3,4-oxadiazol-5-yl,
l-N-methyl-tetrazol~5-yl and 1,2,3,4-tetrazolyl; R is hydrogen,
Cl-C4 alkyl or phenyl vptionally substituted by Cl-C4 alkyl,
fluorine, chlorine, nitro, amino or trifluoromethyl; and R' is
hydrogen, or the above D (-) compounds of formula I(a) wherein
Y is S-Het, in which Het is 1,2,3-triazolyl, 2-methyl-1,3,4-
thiaaiazol-5-yl, or l-N-methyl-tetrazol-5-yl; R is hydrogen
or Cl-C4 alkyl; and R' is hydrogen.
More preferred D-(-) com~unds of formula I and I(a) are
those in which Y is hydrogen or S-Het, in which Het is 1,2,3-
triazolyl, l-N--methyl-tetrazol-5-yl or 2-methyl-1,3,4-thiadiazol-
5yl; R is hydrogen or methyl; and R' is hydrogen.
The present invention also incllld~s a process fox the
preparation of a 7-D-(-)~-amino~d(p-ac~loxyphenylacetamido)
cephalosporanic acid of the formula
R-~-O ~ fH-~-NH
~2 ~ ~ H2Y
I ~ :
wherein Y is hydrogen or S-Het, in which Het is a 5 membered
heterocyclic ring containing l to 4 N atoms; and R is Cl-ClO
alkyl, or phenyl, when substantially free of the L-(~) isomer,
which :
.:
:
-- 3 --
.- " .
. .
,., . , . : .
process comprises ~ea~tln~ ~ compound of the formula
H2 N T~S
,~N~7~C~2Y :
COOH II
or a silyl ester or salt thereof in which Y is as defined above
or acetoxy with a corresponding D-(-) acylating agent of an
acid of the formula ~ .
: '
lQ R-~-O ~ fH-COO~
NHB ~ .
III -
in which R is as defined above and B is an amino-pro-
tecting group, and removing the amino-prote~ting group, and ;
when Y is acetoxy converting the thus-obtained compound to the
corresponding final compound wherein Y i~3 S-Het and, when the .
compound of formula II is in the form of the silyl ester or .
salt thereof, either before or after removal of B, converting
the thus-obtained compou~d to the corresponding final product.
In a preferred embodiment, the acylating agent is of the
formula
R~C O ~ ~ CH - COCl
NH2JHCl
wherein R ~s ~s dP~ined above.
In the preparation of the novel cephalosporin compounds
of the present invention, a corresponding 7-amino-cephalosporanic
acid compound of formula II or salt thereof is acylated by known
me~hods with the appropriate D-(-) acylating agent of formula
III7
In the case o$ 3-thiolated-7-aminocephalosporanic acid
4--
. ,~ ~ . .
intermediate of formula II, when Y is S-Het, said intermediate
may he prepared by dlsplacement of the 3 acetoxy group of 7-
aminocephalosporanic acid or a salt thereof with th~ appropriate
heterocyclic thiol or a salt thereof. The displacement of an
ester group with a thiol group is a known reaction and i5
preferably accomplished in aqueous solution wit~ heating.
The intermediate II may, if desired, be converted prior
to the acylation reaction to a silyl ester or acid addition
salt thereof. The ~ilyl esters may be prepared by the methods
described in the literature, e.g. U. S. Patent 3,249,622. The
silyl ester group may be removed following the acylation re-
action by hydrolysis.
Prior to the acylation reaction the amino group of the
acylating agent III may be protected by a conventional amino-
blocking group B respectively, which may be readily removedat the conclusion of the reaction by methods known per se.
.
Examples of suitable amino-protecting or blocking groups include
t-butoxycarbonyl, carbobenzyloxy, 2-hydroxy-1 naphthcarbonyl,
trichloroethoxycarbonyl, 2-ethoxycarbonyl-1-methylvinyl and
2~methoxycarbonyl-1-methylvinyl. A particularly valuable
blocking group is a proton, as in the compound of the formula
~'
R~-O ~ ICH - COCl
NH2 . ~Cl
~5
For example, following the acylation coupling reaction, it can
be easily removed by neutralization. Obviously other func-
tionally equivalent blocking groups for an amino group can be
used and such grollps are considered within the scope of this
--5--
invention.
In addition to the above, the present invention also in-
cludes a process for the preparation of a compound of the
formula I(a) wherein Y is S-Het, in which Het is 1,2,3-triazol-
5-yl, 1-N-methyl-tetrazol-5-yl or 2-methyl-1,3,4 thiadiazol-
5-yl; R is hydrogen, Cl-C10 alkyl optionally substituted by
carboxylic acid, or phenyl optionally substituted by Cl-C4
alkyl, halogen, nitro, amino or trifluoromethyl; R' is hydro-
gen, hydroxy, Cl-C~ alkyl, Cl-C~ alkoxy or halogen, and pharma-
ceutically acceptable salts thereof, whsn substantially free
of the L- (~) isomer, which process comprises reacting a com-
pound of the formula II, in which Y is as immediately defined
above or a silyl ester or salt thereof with a corresponding
D~t~) acylating agent of an acid of the formula
R'
R--~--o~H-COOr:~
OCHO
~ a~
in which R and R' are as defined immediately above, to produce
the co~pound of formula Ita) or a pharmaceutically acceptable
salt thereof, and, if desired, converting by methods known
se the product in the form of the free acid or silyl
ester or salt thereof to the corresponding free acid or pharma-
ceutically acceptable salt thereof.
Acylation of a 7-amino group of a cephalosporin is a
well-known reaation and any of the functional equivalents
of ~ormula III or III(a) commonly used as acylating agents for
primary amino groups may be employed. Examples of suitable
acylating derivatives of the free acid include the correspondin~
~5~ L3
acid c~nhydrides, mixed anhydrides, e.g. alko~yformic anhydrides,
acid halides, acid azides, active esters and active thioesters.
The free acid may be coupled with compound II after first re-
acting said free acid with N,N'-dimethylchloroformunium chloride
or by the use of enzymes or of an N,N'-carbonyldiimudazole or an
N,N'-carbonylditriazole or a carbodiimide reagent, e.g.
N,N-diisopropylcarbodiimide. N,N'-dic~clohexylcarbodiimide
or N-cyclohexylcarbodiimide or N-cyclohexyl-N'-~2-morphilino-
ethyl) carbodiimide or of an alkylylamine reagent or of an
isoxasolium salt reagent. Another equivalent of the free
acid is a corresponding azolide, i.e., an amide of the
corresponding acid whose amide nitrogen is a member of a
quasiaromatic five member~d ring containing a~ least two
nitrogen atoms, i.e. imidazole, pyrazole, ~he triazoles,
ben~iimidazole, benzotriazole and their substituted deriva-
tives. A reactive derivative of the phenylglycine
acid of formula III is the N-carboxy c~hydride tLeuch's
anhydride~. In this structure the group which activate the
carboxyl group also serves to protect th~ amino group. A
particularly preferred acylating agent is the acid chloride
hydrochloride of the formula
: R~
R-C-O ~ C5 - COCl
~S NH2~Cl
which also ser~es a dual function of carboxyl activation and
amino protection. Mention was made above of the use of
enzymes to couple the free acid wi~h its blocked amino group
with compound III. Included in the scope of such processes
_ 7 _ :
..
,,. ,.:: ,
.,, ,. , ., ; . .... ; .:, . .. : : . :
- . . . . , ,. , ,, . .. , . : .
~L~5~3
are the use of an ester, e.g. the methyl ester, of that
free acid with enzymes provided by various microorganisms,
~ e.g. those described ~y T. Takahashi et al., J. Amer. Chem. Soc.,
94(11), 4035-4037 ~1~72) and by T. Nara et al., J Antibio$ics
(Japan), 24(5), 321-323 (1971) and in West Germany 2,216,113.
The particular process conditions, P.g. temperature,
solvent, reaction time, etc. selected for the coupling re
action are determined by the nature of the acylation method
u~ed and are known to those skilled in the art. Generally
it is useful to add an organic tertiary amine, e.g. triethyl-
amine, N,N-dimethylaniline, ethylpiperidine, 2,6-lutidine or
quinoline, to s~rve as a proton acceptor or salt-forming agent.
The compounds of the present invention may be isolated
in any of the ways customarily employed for the isolation of
similar cephalosporins. Thus, the product may be obtained
as the neutral molecule and, in the case of compounds of
formula I, this is probably more accurately represented as the
zwitterion, or it may be isolated as a salt. Formation of the
desired pharmacautically acceptable carbox~lic acid or acid
addit~on salt is carried out by known methods, e.g. reaction
of the acid with an appropriate base or acid.
At the conclusion of the acylation reaction the product
obtained may be converted tbefore or after removal of the amino-
protecting group1 by methods known ~ se to another desired
product of formula I. For example; the product of formula I
or I(a~ in the form of a silyl ester or salt thereof may be
converted to the free acid product or pharmaceutically acceptable
sal$ thereof by removal of thé silyl ester group, e.g. by
hydroly~is. :
The pharmaceutically active compounds of the present inven-
tion are potent antibacteri~l agents useful in the treatment o~
. ... . .... : ' ' ' ', ~, ',' ',
,
3~5~ 3
inf~ctious diseases in poultry and animals, including man,
caused by many Gram positive and Gram-negative bacterial The
active compounds are also of value as nutritional supplements
in animal feeds and as agents for the treatment of mastitis in
cattle. The preferred compounds have also been unexpectedly
found to be efficiently absorbed upon oral administration.
The novel medicaments provided by the present invention
may be formulated as pharmaceutical compositions comprising,
in addition to the active ingredient, a pharmaceutically
acceptable carrier or diluent. The compounds may be administered
both orally and parenterally. The pharmaceutical preparations
may be in solid form such as capsules, tablets or emulsions.
In the treatment of bacterial infections in man, the compounds
of this invention may be administered parenterally in an amount
of from about 5 to 200 mg./kg./day in divided dosage, e.g. 3 to 4
times a day. They are administered in dosage units containing
e.g. 125, 250 or 500 mg~ of active ingxedient with suitable
physiologically acceptable carriers or excipients.
The following illustrates the preparation of staxting
materials used in the production of the novel compounds of
the invention.
,,. ~
.. . .
_ g - " ' ~
: .'
:
~: . . ,, .: ....... , :. . . . . . . ~
L3 ~:
Star*in~ Materials
.
Synthesis of potassium 1,2,3-triazole-~-thiolate
. o
Il N
~C-N=C=S ~ CH2N2 ~ OH-~
N~ ~ -~HCO
163.1g ~.0~ 205.24
.... ....
n
N ~N ~ ~
N N
H H
-
101~13 139.23
The synthesis of the thiol was accomplished by a pro-
cedure essentially identical to that described in the litera-
IS ture [~. Gverdler and G. Gnad, Chem. Ber. 99, 1618 (1966)].
: 5 Benzamide~1,2,3-thiadiazole
To a stirred solution of benzoylisothiocyanate (50.6 g.,
310 mmoles) in commercial anhydrous ether (400 ml.)~ main-
tained at 0 and in a nitrogen atmosphere, was added dropwise
wi~h vigorous stirring, 0.685 N e~hereal diazomethane (453 :~
~1., 310 mmoles). When the addition was completed, the mix-
t~r~ was stirred for 1 hour at 0, the solid was collected
by filtration and dried in vacuo. The melting point of the
~ crude material (23.3 g.) thus obtained was observed somewhere
in the region 232 to 257C. Goerdler reported m.p. 267 for
the pure material. A small second crop (2.1 g.~ was nbtained
by evaporation of the mothex liquor in vacuo. The total . : :
yield was therefore 40%.
' . .
10
,.
~i6~3L3
1,2,3-Triazole-5-thiol
A solution of the above benzamido compound (8.2 ~O~ 40
mmoles) in 2N sodium hydroxide (80 ml'., 160 mmoles) was heated
under reflux temperature in a nit~ogen atmosphere for 24 hours.
The solution was cooled to 0 in ice, and concentrated hydro-
chloric acid (26 mla) was added, while a continuous stream of
nitrogen was passed through the solution. The benzoic acid
which precipitated was collected by filtration; the filtrate
was saturated with sodium chloride and the additional benzoic
acid which separated was removed by filtration. The filtrate
was immediately extracted with ethyl acetate~ the extract was
washed with saturated salt solution, dried over magnesium ~' -
sul~ate and then evaporated'ln vacuo. The viscous oil which
remained ~as immediately evaporatively distilled ln vacuo ~70- '
75/0.001 mm.) to give an oil (2.84 g., 70%) which solidified ~'
(m.p. 52-59; Goerdler reported m.p. 60) spontaneously.
Potassium '1',2,3-Tr'ia'zole~5-thi'o'late ~ '
To a solution of the above thiol (2.84 g., 28.1 mmoles)
in absolute ethanol (28 ml.) was added 1.93 N alcoholic potas- ;'
sium hydroxide solution (14.5 ml.). The solution was then
diluted with anhydrous ether until crystallization of the salt
was completed. The solid was collected by filtration, washed
with ether~ and dried' in vacuo. The salt obtained in this
manner (3.65 g., 93%) had m.p. 225 with decomposition.
It is important to note that the conversion of the
benzamido thiadiazole to the triazole thiol is known to proceed
v'ia 5-amino-1~2,3-thiadiazole [G. Goerdler and G. Gnad, Chem.
:: -
Ber.''99, 1618 (1966)~
'' '
'
. ', . ," , ' .
: ': ' . .
~35~ 3
.. _
N~ ~
NHCO0 ~ ~ l~I2
N OH ~
I/ ~ ~ N N 87%
N N N~ S ~ ~ N
H 93%
5-Amino-1,2,3-thiadiazole can be prepared by an alternative :~ .
route, not involving diazomethane [D. L. Pain and R. Slack, ~ :
J. Chem. Soc. 5166 (1965)]~
O `~
~N-CH2CH + H2NNHC2Et -~
o ,: ~
: ~ N-CH2CH-N-NHCO2Et
O 93%
N
O ~N :
89%
N ~
30 . ~ ~ H ~ ~:
- 12 - 77%
~ . .
:. ; , ,::, ~ .
., . ~ I
Synthesis of 7-Ami~o-3-(1,2,3~triazol-5-ylthiomethyl)-3-
cep~em-4-carboxylic acld
The reactions were conducted under a nitrogen atmosphere
in a reaction vessel protected from light. The water and
phosphate buffer were gassed vigorously with nitrogen prior
to use to displace oxygen.
5-Amino-1,2,3~thiadiazole (10.3 g., 0.102 mole) was added
to a solution of 8.16 g. of sodium hydroxide in 100 ml. of
water. The mixture was heated rapidly to reflux and then re-
fluxed or 10 min. to rearrange 5-amino-1,2,3-thiadiazole to
5-mercapto-1,2,3-triazole. To the reaction mixture containing
5-mercapto-1,2,3-triazole cooled in an ice bath was added
1100 ml. of ice cold O.lM pH 6.4 phosphate buffer. The
solution, which was at pH 10.5t was adjusted to pH 8.5 with
42~ phosphoric acid. 7-Aminocephalosporanic acid (21.8 g.,
0O08 mole) was added and the mixture heated at 50~ for 4 hours~
The clear solution was cooled in an ice bath and adjusted to
p~ 4.5 with conc. HCl. The precipitated product was collected
by filtration, washed with water and air dried; 16.2 g.
The crude product (15.2 g.) wa-~ brought in o solution
with 600 ml. of methanol and 40 ml. of conc. ~C1. After
carbon treatment the solution was diluted with 1.5 1. of ice
water and extxacted once with ethyl acetate. The aqueou~
phase was concentrated at reduced pressure to remove methanol.
The cold aqueous concentrate was adjusted slowly to pH 4.0
with 20% sodium hydroxide causing crystallization of the
product. The product was collected by filtration, washed
with water and methanol and dried in vacuo over phosphorus
pentoxide; 11.4 g. The IR and NMR spectra were fully
consistent for the desired product.
-13- ;
Anal. Calcd- for CloHllN53S2 C, 38-42;
Found: C, 38.27, 38.26; H~ 3.76,
3.40; N, 21.02, 21.00; H2O, 1070.
Purific tion of 7-Amino-3 (1,2,3-t ~ thiom~ 3-
~ .
Crude 7-amino 3-(1,2,3-triazol-5-ylthiomethyl3-3- -
cephem-4-carboxylic acid (16.1 g.) containing approximately
20 mole % of 7-aminocephalosporanic acid as an impurity, was
brought into solution with 600 ml. of methanol and 40 ml.
of conc. ~Cl. Ater carbon treatment, the solution was
diluted with 1.5 lo of ice water and extracted once with
ethyl acetate. The aqueous phase was concentrated at reduced
pressure to remove methanol. rhe cold aqueous concentrate was
then adjusted slowly to pH ~.0 with 20% sodium hydroxide
causing the product to crystallize. The product was collected
by filtration, washed with water and methanol and dried in
vacuo over phosphorus pentoxide; 11.4 g. The NMR spectrum
indicated that this product contained about 7 mole ~ of 7
aminocephalo~poranic acid as an impurity.
~ he above purification procedure was repeated on 11.4 g.
of the product using 425 ml. of methanol, 28 ml. of conc. HCl
and 1 1. of ice water yielding 8.0 g. of product. The NMR
spectrum was fully consistent for the desired product and
indicated no trace of 7-aminocephalosporanic acid as an
impurity.
Anal. Calcd. for CloH15N5O3S2: C, 38.42; X, 3.55; N, 22.40.
Found: C, 39.06, 38.53; H, 3.56,
3.51; N, 22.05, 21,60; H2O, 1.780
3n
:
-14~
~6~3
7-Amino-3-(1,2,3-t_iazole-5- lthlometh ~ hem-4-
carboxylic acid (II)
.
Ten grams (0.075 mole) of 5-mercapto-1,2,3-triazole
potassium salt was added to a stirred slurry of 19 g. (0.07
mole) of purified 7-aminocephalosporanic acid and 5.9 g.
(0.07 mole~ of NaHCO3 in 350 ml. of 0.1 M phosp~ate buffer
(pH 6.4) and the mixture heated and stirred at 55 C~ for
3 1/2 hours under a nitrogen atmosphere. The resulting
solution was cooled to 22 C. and adjusted to pH 5.5
with 40~ H3PO4. The resulting precipitate was filtered
off, washed with cold water (50 ml.) and air dri~d. The
yield of 7-amino-3-(1,2,3-triazole-5-ylthiomethyl)-3-
cephem~4-carboxylic acid was 8 g., dec. pt. 230 C. IR
analysis showed some decomposition of the ~-lactam ring
but it was used "as is" for the next step.
~nal. Calcd. for ClOHllN5O3S2 C, 38-39; ~,
Found: C, 38.36; ~, 3~78.
? ~ hiomethyl)_3-cephem-4-carbo~_ic
acld (II)
Two hundred seventy-two g. (1.0 mole) of 7-amino~
caphalosporanic acid was suspended in 3000 ml. of O.lM phosphate
buffer, pH 6.4, and 150 ml. of methyl isobutyl ketone followed
by 84 g. (1.0 mole) of sodium bicarbonate tNote: The sodium
bicarbonate was added in portions). Then 143 g. (1.0 mole) of
5-mercapto~ 1,2~3-triazole potassiwm salt was added and
the mixture stirred at 55 C. 1~ C. under a nitrogen
atmosphere for 4 hours. After 1 hr. the pH was readjusted to
6.4 by addition of a small amount of 40% H3PO4. At the end
of the 4 hr. heating period, 50 g. of "Darco KB" decolorizing
*Trade Mark
15-
~5~3
charcoal was added and, after stirred for 15 min. at 55 C.,
the slurry was filtered hot through a diatomaceous earth
("Celite") pad. The pad was washed with 3 x 100 ml. water.
The pH of the combined filtrates was adjusted while hot to
4.5 by slow addition of 6 N HCl. After cooling 30 min. at
0 C., the crude product was collected by filtration, washed
with 2 x 200 ml. of cold water followed by 2 x 1000 ml. of
methanol and air dried.
The crude product was suspended in 3000 ml. of 50~
methanol-water and 300 g. (1.5 mole) of ~-toluenesulfonic
acid was added. The mixture was stirred for 15 min. and then
50 g. of "Darco KB" decolorizing charcoal was added. A~ter
stirring for 15 min. at 22 C., the slurry was ~iltered
through a "Celite~" pad and the pad washed with 2 x 100 ml.
o 50% methanol-water. The pH of the combined filtrates was
adjusted to 4.0 by addition of apprc)ximately 210 ml. of tri-
ethylamine. After cooling at 0 C. for 1 hour the product was
collected by filtration, washed with 2 x 400 ml. of 50%
methanol~water and then 2-1000 ml. of methanol and air dried.
This material was suspended in 2000 ml. of water and 84
g. (1 mole) of sodium ~icarbonate was added. After stirring
for 10 min. at 22 C., 50 g. of "Darco KB" charcoal was added
and, after stirring for 15 min. at 22 C. the slurry was fil-
ter~d thxough a "Celite" pad. The product was washed with
~5 2 x 100 ml. of water and the pH of the combined filtrates
was adjusted to 3~5 by slow addition of 6 N HCl. After stir-
ring for 10 min. at 22 C., the mixture was cooled to 0 C.
for 1 hr. The product was collected b~ filtration was washed
with 2 x 200 ml. of cold water and 2 x 1000 ml. of acetone.
After drying over P2O5 in a vacuum desiccator for 14 hr. at
*Trade Mark
-16-
~5~ 3
room temperature the yield wa~ lQ~,g.; dec. pt~ 230Co
Th~ IR and NMR were consistent for the desired structure.
. . .
~ Preparation of D (-)-2-amino-2-~4-acetoxyphenyl3acetic acid
Method A ~in acetic acid as solvent~
203.5 g ~1 Mole) of D~-)p-hydrox~phenylglycine chloride,
800 ml of acetic acid and 314 g( 4 Moles) of acetyl chloride
are stirred 48 hours at room temperature. The solid i5
collected, washed three times with acetone (3 x 250 ml) and
twice wi~h ethanol (2 x 250 ml) and dried at 40O. Yield
10 210 g (85.4%). This hydrochloride is dissolved in 3.0 1
of water; the solution is cooled to ~5 to 10C and the pH
adjusted to 4O5 with 2Q% NH40H. The suspension is stirred
1 hour at 5C and the solid collected, washed twice with
water and twice with acetone, and dried at 40C. Yield
133 g~ ~4~ from D(-)p-hydroxy phenyl glycine).
~D (1~ HCl N/10) ~ -104.5 :' '''-'
Me thod B (in methylene chloride~ r. .... .
4.07 g (0.02 Mole) of D(,-)p-hydroxyphenylglycine hydrochloride,
30 ml of methylene chloride and 6.28 g ( p.0$ Mole) of acetyl
2Q chloride are stirred 48 hours at room temperature. ~he solid
is collected~ washed twice with acetone and twice with ethanol.
Yield 4.17 g (84.5~). Anal. cl = 14.8% (calculated 14.4%~
Method C (in trifluoroacetic acid)
1.67 g ~0.01 Mole) of D(-)p-hydroxyphenylglycine is added
with stirring, to 10 1~ of tri~luoroacetic acid at room
temperature. After dissolution, 1~57 gC~.02 Mole) of acetyl
chloride is added. A~ter a slightly exothermic reaction, a
. . ' ' . ,'
~ .
- 17 -
~!3
solid appPars. The suspension is stirred 1 1~2 hours at room
temperature and th~ trifluoroacetic aci~ is remov d in
~ vacuum. The remaining solid is collected, washed with
methylene chloride and with ethanol. The D(-~2-anuno-2-
(4-acetoxyphenyl~acetic acid is identical to that
prepared by method A or B.
Yield: 1.9 g(75~)
Preparation of D (-) 2-amino-2-(4-pivalyloxyphenyl~ acetic
acid hydrochloride
1.67 g ~0.01 Mole) of D(-~p-hydroxyphenylglycine is added
- to 10 ml of trifluoroacetic acid, followed by 2.4 g (0.02
Mole~ of pivalyl chloride. The resulting solution is
stirred 24 hours at room temperature and vacuum concentrated
to dryness. The solid is collected and washed with ether.
Yield: 2.56 g (89%)~
This hydrochloride is recrystallized Erom isopropanol.
Anal. Cl = 11.8% (calculated 12.3~)
W ,~,maxO 205 nm and 220 nm.
Preparation of D ~-),2-amino-2-(4-benzoyloxyphenyl)acetic
_id hydrochloride
This compound is prepared according to the same procedure
as used for the pivalyloxy derivakive. Yield: 2.7 g ~87%).
25' An analytical sample is recrystallized from ethanol. ~ -
Anal. cl. = 11.3% (calculated 11.5%)
UV ~max. 205 nm and 234 nm
- 18 -
... . .. . ... .
~S~3
Preparation of D (-)2-amino-2-(4-aceto~henyl)acetx~ chloride
hydrochloride
83.6 g. (0.40 mole) of D(-)2-amino-2-(4-acetoxyphenyl)-
acetic acid and 1.25 1. of anhydrous methylene chloride are
cooled to -5 C. with stirring. Then 152 gO of phosphorous
pentachloxide are slowly added followed by 4 ml. of dimethyl
formamide. The mixture is stirred 4 hours at 0 C. The
solid is collected, washed with anhydrous methylene chloride
and vacuum dried at room temperature. Yield: 61 g. (57O5~).
Anal. Total chlorine - 27.2% (Theory 26.9%)
Pre aration of D~(-)2-form~__xy~2-(4-f_rmxloxy~h n
P
acid
~ . _
A solution of 3.6 g~ (0.02 mole) of D-~-)-2-hydroxy-2-(4-
hydroxyphenyl)acetic acid in 50 ml. of 97% aqueous formic acid
was allowed to react at 22 C. for approximately 68 hours.
The excess formic acid was removed by distillation at 22 C.
under reduced pressure. The residue is extracted with diethyl
ether; the ethereal layer is dried over sodium sul~ate, fil-
tered and evaporated to afford the desired product.
Prevaration of D-(-)2-formvloxY-2-(4-acetoxY~henvl)acetic
acid
The D-(-)2-formyloxy 2-t4-formyloxyphenyl)acetic acid
obtained above is dissolved in 10 ml. of acetyl chloride and
the resulting mixture was allowed to stand at 22 C. for 20
hours. The excess acetyl chloride was distilled off under re-
duced pressure; the residue was treated with benzene and the
benzene then removed under vacuum to afford the desired prod-
uct which analyzed as 60~ pure according to NMR.
--19--
The following examples are gi~en in illustration of,bii. not in limitation of, the prPsent invention. All temper-
atures are in degrees Centigrage. 7-Aminocephalosporanic
acid is a~breviated as 7-ACA and 7-aminodesacetoxycephalosporanic
aci~ as 7-ADCA.
Exam~le 1
7-D(-)2-amino-2-(4 aceto~yphenylacet_mido)desacetoxy-
cephalosporanic acid - (acetoxy ce~halexine) - RN 1394
15.27 g ~ poO714 Mole~ of 7~ADCA are stirred in 500 ml of anhydrous
methylene chloride; 120 ml of methylene chloride are distilled
off and 11.8 ml o hexamethyldisilazane are added. The
mixture is stirred and refluxed 20 hours (after about 10-15
hours all the 7-ADCA is run in solution~. The above
solution is cooled to 0C and 120 ml of methylene chloride
followed by the addition of 9.5 ml of dimethylaniline and
7 ml of a solution of dimethyl~niline hydrochloride in
methylene chloride (30%). Then 20 g (0.0756 Mole) of
D(-)2-amlno-2~4 -acetoxyphenyl~acetyl chloride hydrochloride
are added in small portions (~1 1/2 hours) a~ 0C. :
The mixtuxe is stirred 30 min. at +10C and 4 hours at ~20OC
and-allowed to stand overnight at ~5C. Then S ml of methanol
followed by 240 ml of water are added~ The pH is adjusted
at 2.5 with triethylamine and ~he mixture is filtered
, through a celite pad; then the pH is checked and the aqueous
phase is separated, washed twice 52 x 150 ml) with methylene
c~loride and treated with charcoal. .~.
The solution i~ adjusted to pH 4~5 and vacuum concentrated to
a volume of ~ 150 ml. m e suspension is allowed to stand
- 20 ~
~ ~s~
ov~rrlight at f5C and th~ solid colleeted and washed with
wa ~cer and ace tone, and dried at 40c C .
Yield: 15,1 g ( - 50~O of 75-80-^,' pure ma~erial)
¦ ~D ( 1% H2O) -- +107
5 14 g of this crude material is suspended in 30 ml of
wat~3r (p~ = 3,2): hydrochloxic acid t~6%7 is added to p~
1, 3 and ~he resulting solutio;~ is charcoal treated and
filt~red through a celite pæ~d and adjusted to pH 4 4, 5
otter stirring~ After 2 hours at û- ~5C the R~ 139~
10 collç~cted, i~ashed with wat~r and acetone and dried at ~'.O~C.
Yield: 7 g,
~D(l,-~ ~l2O) ~ +i33- :
T`ne infrared and nucl~ar magnetic resonance s~ectra are
consistent with the d~sired product.
Biological Data
Table I shows comparative MIC dlata for BL-S 578-4
(~-hydroxy analogue of cephalexin) and (p-acetoxycephalexin
(RN 1394). Minimal inhibitory concentrations were detexmined
by the 2-fold broth dilution method utilizing equimolar con-
centrations o~ each compound.
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ul Q ~ u~ o O .sa ~ m o
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-- 22 --
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... . .
Example
7~ -12-Am~no-2-(4-acetoxyphenylacetamido]-3~ 2~3-tria
- _-yl)-thiomethyl3-3-cephem-4-carbo~ylic acid: ~N 1396
62.6 g (0.2 Mole) of 7-amino-3-~(1,2,3-triazcl-5-yl)thiomathyl]-
3-cephem-4-carboxylic acid (7-TACA), 1.5 1 of methylene chloride
and 6Q.2 g (0.374 le~ of hexamethyl disilazane are stirred
and refluxed 20 hours with a slight pressure o~ nitrogen
(after about 2 hours all 7-TACA is run in solution~.
The solution is cooled to 0C and 30.4 ml of dimethylaniline,
followed by 2n .4 ml of a solution of dimethylaniline
hydrochloride in methylene chloride (30%) and 1.35 g of
imidazole are added.
Then 60.5 g (0.22~ mole~ of 2-amino-2-t4-acetoxyphenyl)acetyl
chloride hydrochloride are added in small portions (~ 1 1~
hour ) at 0C. The mixture is then stirred 3 hours at 20C
and let stand overnight at +5C. 25 ml of methanol followed
by 75~ ml of water are then added. The pH is adjusted to
2.3 - 2.5 with triethylamine and the l~xture is filtered
through a celite pad. The a~ueous phase is separated, washed
twice with n~thyle~e chloridP ~nd charcoal treated.
The solutio~ is adjusted to p~ 4.3 and stirred 2 hours at
~ .
~5C. The solid is collected, washed twice with wat~r and
dried at 40C. Yield: 53 g (about 5û%). ~:
This crude material is purified twice as follows:
The solid is treated with eight volumes of 0.5N hydrochloric
acid and the suspension is decolorized with charcoal. An
equal volume o~ methanol is adde~ to the solution and the pH
adjusted to 2 - 2.1; aftex 15 minutes a small amount of
suspended solid is colle~ted and discarded. The filtrate is
adjusted to pH 4. The precipitated solid is c~llected, washed with
- ~3 -
.
. . .. . , ~ :
MeO~/H20 (50/50) and pure methanol.
Yield: 25 g. (after two purifications).
- IR consistent with assigned structure
- Moisture (KF) : 5.1%
- Chemical ascay
- Iodometric assay 885 mcg/mg
- Potentiometry Amine assay: 97
~ ~: D~t~ .
Table II shows comparative MIC data for the above prepared
7-[~( )2~amino~2-(4-acetoxyphenylacetamido)]-3-[1,2,3-triazole-
S~yl)-thiomethyl]-3~cephem-4-carboxylic acid, RN-1396, and its
p-hydroxy analogue, BLWS640. Minimal inhibitory concentrations
were determined by the 2-fold broth dilution method utilizing
equimolar concentrations of each compoundO
3~ .
-24- :
D
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o ~
O ~ O ~ O ~ ~ ~1~ O ~ ~ , " .
: ::
o ~r
~r o o ~
. ~ . :
,~ o ,l ~ o o ~ o ~1 o ~ In L~ ul o ~
~ u~
r~
î
H ~ . .
a~ . ~,. . .,~ .
_ In ~ r` t~ o Y~
~ c~~ o a ~ ~ ,~ o ln ~ u~ ~
E~ O . U~ n Ln ~D ~1~ CS~ U~ c~ o u~
H C~~1 ~ ~1 1~ N C~
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* ~ O ~1 1
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~ ~ + a~ !
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~1~ o c~V ~
: ~ ~~ O ~I r~l r-~ O ~ O O O a~ n~ ..
O
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h ~ ~ m ~ ~ + .
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rl 5 ~ O O ~ ~a dP r-l
~ s~ ~ a
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'
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. ,
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- 25 - `,
.
,
Example 3
7-D-(-)[2-(4-acetoxyphenyl)-2-ormyloxyacetamido]-3~ methy~
1,2l 3,4-tetrazol-S-yl)thiomethyl-3-cephem-4 carboxylic acid
To a solution of 4.8 g. (0~02 mole) of crude D-(-)2-
formyloxy-2-(4-acetoxyphenyl)acetic acid and 25 ml. of diethyl
ether was added one drop of dimethylformamide and 5 ml. of
oxalyl chloride. After stirring at 22 C. for 1 hour, the
solvent was removed and the residue dissolved in 25 ml. of
acetone, the resulting solution was added dropwis~ to a solu-
tion of 6.3 gO (0.02 mole) of 7 amino-3~ methyl-1,2,3l4-
tetrazol-5-yl~thiomethyl-3 ceph~m-4-carboxylic acid, 5.6 g.
of sodium bicarbonate, 300 ml. of water and 80 ml. of acetone
at approximately 3 C. The reaction mixture was stirred for
1 hour at 3 to 5 C. followed by removal of the acetone. The
pH of the residue wa~ adjusted to 2.0 by the addition of 40%
aqueous phosphoric acid under a layer of ethyl acetate~ The
aqueous layer was extracted with 2 x 100 ml. of ethyl acetate
and the combined organic layers dried over sodium sulfate.
The organic layers were filtered and the filtrate evaporated
under vacuum to an oil~ Trituration of the oil with diethyl
... .
ether afforded 8 g. of solid product which analyzed by NMR
to have 85 90~ acetyl and 50-60% formyl.
Example 4
7-D-_(-)[2-(4-~orm~ x~phenyl) 2-formyloxyacetamidol-3~ methyl-
1,2,3,4--tetrazol 5-yl)thiomethyl-3-c~phem-4-carboxylic acid
The same procedure as in Example 3 was followed to react
2.S g. (0.01 mole) of D-(-)2-formyloxy-2-(4-formyloxyphenyl)-
acetic acid with 3.28 g. ~0.01 mole) of 7 amino-3-(1-me~hyl-
1,2,3,4-tetrazol-5yl~thiomethyl-3-cephem 4-carboxylic acid to
-2~-
~L~5~
afford after trituration with diethyl ether 4.2 g. of desired
product which solids had a decomposition point of 160-5 C.,
an infrared and NMR spectrum consistent with its structure but
containing a by-product.
Microanalysis of the desired product gave:
Calc'd. for C20H18N6O8S2
% The~ %_Found
C ~4.86 46.47
H 3.38 4.14
N 15.70 13.50
K.F. (H2O) 1.78
lS
~5~
In addition to the above, the compounds of the instant
invention are also valuable as intermediates for ~he prepara-
tion of other pharmaceutically active compounds. For example,
the instant a-formyloxy or a-amino-a-(p-acyloxyphenyl)acetamido
cephalosporanic acids may be converted to the corre~ponding
p~hydroxy compounds which are known to be potent antibacterial
agents useful in the treatment of infectious diseases in poultry
and animals, including man, caused by many Gram-positive and
Gram-negative bacteria. The conversion can be carried out
chemically by simple acid or base hydrolysis in an aqueous
medium in most circumstances.
We have found that 7~D~(-ja-amino-a-(p-acetoxyphenyl-
acetamido)desacetoxycephalosporanic acid, although stable in
normal saline, is hydrolyzed enzymatically to the known and
potent 7-D-(-)a-amino-a-(p-hydroxyphlenylacetamido)desacetoxy-
cephalosporanic acid.
Accordingly, the present invention also provides for a
novel process for preparing 7-D~ amino-a-(p-hydroxy-
phenylacetamido)de~acetoxycephalosporanic acid, hydrate or a
pharmaceukically acceptable salt thereof, which process comprises
treating in an aqueous solution 7-D-(-)a-amino -(p acetoxy-
phenylacetamido~des~cetoxycephalosporanic acid with an esterase
at a pH between about 5.0 and about 7.5; isolating the product
by m~thods known ~ se, and, if desired, converting by methods
kno~m ~ se the product in the form of the free acid or hydrate
to the corresponding phaxmaceutically acceptable salt thereof.
A preferred embodiment is the preparation of 7-D~
- amino-a-(p-hydroxyphenylacetamido)desacetoxycephalosporanic
acid, hydrate or a pharmaceutically acceptable salt which
process comprises treating in aqueous solution 7 D-(-)-a-amino-
-2~-
:~5~ 3
a-(p~acetoxyphenylacetamido)desaceto~ycephalosporanic acid with
an esterase selected from human serum, animal serum, citrus
esterase, wheat bran, wheat germr and bacillus subtilis at a
pH between about 5.0 and about 7.5 and at a concentration of
about 5 to about 10 mg./ml. of esterase per total volume of the
aqueous solution; isolating the product by methods known
s , and, if desired, converting the product in the form of
free acid or hydrate to the corresponding pharmaceutically
; acceptable salt thereof.
A commercially preferred embodimPnt of the present inven-
tion is the preparation of 7~D-(-)a-amino-a-~p-hydroxyphenyl-
acetamido)desacetoxycephalosporanic acid, hydrates or pharma-
ceutically acceptable salts thereof, which process comprises:
treating in an aqueous solution 7 D-(-)-a-amino-a-
(p-acetoxyphenylacetamido)cephalosporanic acid with an esterase
selected rom citrus esterase, wheat bran, and wheat germ at
a pH between about 5.0 and about 7.5 and at a concentration of
about 5 to about 10 mg./ml. of esterase per total volume of
the'aqueous solution; and
isolating the product by methods known per se, and,
if desired, converting the product in the form of the free acid
or hydrate to the corresponding pharmaceutically acceptable salt
~hereof.
. Of special commercial interest is the process for prepar
ing 7~ )a-amino-a-(p-hydroxyphenylacetamido)desacetoxy-
cephalosporanic acid, hydrate or pharmaceutically acceptable
salt the,reof comprising:
treating in an aqueous solution 7-D~( )-a-amino-a-
(p~acetoxyphenylacetamido)desacetoxycephalosporanic acid with
the commercially available esterase, coarse wheat bran, at a
-29-
i6~3
pH between 5.5 and 6.0 or optionally in the presence of a buf-
fer at a pH of 7.0 at a concentration of about 10 mg./ml. of
~sterase per total volume of solution; and
isolating the product by methods known per se, and,
if desired, converting the product in the form of a free acid
or hydrate to the corresponding pharmaceutically acceptable
salt thereofu
The 7-D-(-)a-~mino-~-(p-hydroxyphenylacetamido~desacetoxy-
cephalosporanic acid prepared by the instan~ inven~ion is
known to be a potent antibacterial agent useful in the treat-
ment of infectious diseases in poultry and animals, including
man, caused by many Gram-positive and Gram-negative bacteriaO
~ he following example illustrates the preparation of
p-acetoxycephalexin aceording to the invention.
EXAMPLE A
Solutions of 0.5 mg./ml. of 7-D-(-)a-amino-a-(p-acetoxy-
phenylacetamido)desacetoxycephalosporanic acid (p-acetoxycepha-
lexin) in normal saline and in human serum were prepared. Stan-
dard solutions o~ 0.5 mg./ml. of 7-D~ amino-a-(p-hydroxy-
phenylacetamido)desacetoxycephalosporanic acid (p-hydroxy- ;
cephalexin) were also prepared in both normal saline and
human serum.
A11 the above solutions were incubated at 37 C. with
shaking and sampled for chromatography a~ time intervals of
0, 2, 4, 8 and 24 hours. The solutions, approximately 5 micro-
liters per strip, were spotted on Whatman No. 1 half-inch
strips which were dried and developed in a solvent system
containing 80 parts buty:Lacetate; 15 parts n-butanol; 40 parts
acetic acid; and 24 parts water. The strips were then bio-
*Trade Mark
~30-
~5~ 3
autographed on plates seeded with bacillus subtilis at a pH
o~ 6Ø
The biochromatograms indicated that p-acetoxycephalexin
is quickly hydrolyzed to thP p-hydroxy form in human serum
but appears stable in normal saline.
I5
