Note: Descriptions are shown in the official language in which they were submitted.
~C~ ~Y ec~
This invention relates to certain novel a-amino- or a-
formy~ (p-acyloxyphenyl)acetamidocephalosporanic acids
which are useful as antibacterial agents.
The novel cephalosporin derivatives of this invention
comprise the D-(-) compounds of the formula
R-C-O~ 1' 5~
2 --N~ ~H 2Y
COOH
wherein Y is hydrogen or S-Het, in which ~et represents a 5
or 6 membered heterocyclic ring containing 1 to 4 atoms selected
from M, O or S, said heterocyclic ring being optionally sub-
stituted by Cl-C~ 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 C1-C4 alkyl, halogen, nitro, amino or trifluoromethyl: R'
is hydrogen, hydroxy, Cl-C4 alkyl, Cl-C4 alkoxy or halogen, and
pharmaceutically acceptable salts thereof, when substantially
free of the L (~) isomer, and the D-~-) compounds of the
formula I(a)
l~ -CO-NH ~ ~ ~
OCHO CH2Y
COOH
I(a)
wherein Y is S-Het, in which Het i5 1, 2,3-triazol-5-yl, l-N-
methyl-tetrazol-5-yl or 2-methyl-l,3,4-thiadiazol-5-yl; R is
hydrogen, Cl-Clo alkyl optionally substituted by carboxylic
~ s
~3~ 4
acid, or phenyl optionally substituted by Cl-C4 alkyl, halo-
ge~, nitro, amino or trifluoromethyl; R' is hydrogen, hydroxy,
Cl-C~ alkyl, Cl-C~ alkoxy or halogen, and pharmaceutically
acceptable salts thereof, when substantially free of the
L~ isomer.
In the above compounds, the substituent halogen is
preferably selected from fluorine, chlorine or bromine.
The pharmaceutically acceptable salts referred to above
include the nontoxic carboxylic acid salts, e.g., nontoxic
metallic salts such as sodium, potassium, calcium and alumi
nium, the ammonium salt and salts with nontoxic amines, e.g.,
trialkylamines, procaine~ dibenzylamine, N-benæyl-~-phene-
thylamine, l-ephenamine, N,N'-dibenzylethyleneaiamine, N-
alkylpiperidine and other amines which are used to form
salts of penicillins and cephalosporins. Also încluded within
the definition of pharmaceutically acceptable salts are the
nontoxic acid addition salts (amine salts), e.g., salts with
mineral acids such as hydrodhloric, hydrobromic, hydroiodic,
phosphoric, sulfuric and salts with organic acids such as
20 maleic, acetic,citric, oxalic, succinic, benzoic, tartaric, ~-
fumaric, mandelic, asoorbic and malic.
Examples of suit~ble heterocyclic groups included within
the definition of "Het" in formula I are such heterocyclic -~
radicals as thienyl, pyrazolyl, imidazolyl, isoimidazolyl,
25~ triazolyl, tetrazolyl, thiazolyl, thiadiazolyl, thiatriazolyl,
oxazolyl, oxadiazolyl, isothiazolyl, isoxa201yl, pyridyl,
pyridazinyl, pyrazinyl, pyrimidinyl and triazinyl. The hetero-
cyclic ring may be unsubstituted or substituted with one or
more of the substituents as mentioned above.
3Q Pxeferred D-(~) oompounds of formula I are tho~e wherein
- 2 -
- , , ~
Y is hydrogen or S-Het, in which Het represents 1,2,3-triaæolyl,
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 optionally substituted by Cl-C4 alkyl,
fluorine, chlorine, nitro, amino or txifluoromethyl; 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-
thiadiazol-5-yl, or 1-N-methyl-tetrazol-5-yl; R is hydrogen
or Cl-C4 alkyl; and R' is hydrogen.
More preferred D-~-) compounds 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 includes a process for the
~5 preparation of a compound of the formula
R_~_o~
2 N ~ H2Y
COOH
wherein Y i hydrogen or S-Het, in which Het represents a 5
or 6 membered heterocyclic ring contai.ning 1 to 4 atoms
selected from N, O or S, said heterocyclic ring being op-
tionally substituted by Cl-C4 alkyl which may be further op-
~5 tionally substituted by carboxylic acid, or alk.oxyalkyl of
up to 4 carbon atoms; R is hydrogen Cl-Clo alkyl optionally
substituted by carboxylic acid, or phenyl optionally substi-
tuted by Cl-C4 alkyl, halogen, nitro, amino or trifluoromethyl;
R' is hydrogen, hydroxy, C1-C4 alkyl, Cl-C4 alkoxy or halosen,
and pharmaceutically acceptable salts thereof when substantially
~s~
free of the L- (+) isomer, which process comprises reacting
a compound of the formula
S
H2N T--~ ~
~ N ~ CH2Y
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 o~ the formula
R'
NHB
III
in which R and R' are as defined above and B is an amino-pro-
tecting group, and xemoving the amino-protecting group to pro-
duce the compound of formula I or a pharmac~utically acceptable
salt thereof, and converting by methods known per se the
compound of formula I, wherein Y is acetoxy, to the corre~pond-
in~ pxoduct wherein Y is S-Het and, if desired, either beforP
or after removal of B, converting by methods known 2er se the
product in the form of the free acid or silyl ester or salt
th~reof to the corresponding free acid or pharmaceutically
acceptable salt thereof.
In the preparation of the novel cephalosporin compounds
o~ the present invention, a corresponding 7-amino-cephalosporanic
acid compound of formula II or salt thereof is acylated by kno~n
methods with the appropr:iate D-(-) acylating agent of formula
III.
In the case of 3 thiolated-7-aminocephalosporanic acid
--4--
~56~
intermediate of formula II, when Y is S-Het, said intermediate
may be prepared by displacement of the 3~acetoxy group of 7-
aminoc~phalosporanic acid or a salt thereof with the appropriate
heterocyclic thiol or a salt thereof. The displacement of an
ester group with a thiol group is a known reaction and is
preferably accomplished in aqueous solution with heating.
The intermediate II may, if de~ired, be converted prior
to the acylation reaction to a silyl ester or acid addition
salt hereof. The silyl 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-
lS blocklng group B respectively, which may be re~dily rPmovedat the conclusion of the reaction by methods known ~ se. -
Examples of suitable amino-protecting or blocking groups include
t~butoxycarbonyl, carbobenzyloxy, 2-hydroxy-1-naphthGarbonyl,
trichloroethoxycaxbonyl, 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'
R-C-O ~f ~ coc 1
NH2 . HCl
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 groups are considered within the scope of this
invention.
In addi~ion to the abo~e, the pr~sent invention also in-
cludes a proce~s 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, l~N-methyl-tetraæol-5-yl or 2-m~thyl-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, halogen, ~itro, amino or trifluoromethyl; R' is hydro-
gen, hydroxy, Cl-C4 alkyl, Cl-C4 alkoxy or halogen, and pharma-
ceutically acceptable salts thereof, when 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-(-) acylating agent of an acid of the formula
R'
R-~- ~ H-COOH
OCHO
III(a) .
20 in which R and R' are as defined immediately above, to produce
the compound of formula I(a) or a pharmaceutically acceptable
sal~ thereof, and, if desired, converting by methods known
per se the product in the form of the fxee acid or silyl
ester or salt thereof to the corresponding free acid or pharma-
ceutically acc~ptable salt th~reof.
Ln one aspect of t~is i`n~ention there is- provided a
process for the preparation o~ a 7-D-(-)d-formyloxy-~(p-acyloxy-
phenylacétamido)cephalosporanic acid of the formula
fH~CO-NN ~ ~ S ~
OCHO ~ CH2Y
~0()~
I(a)
- 6 -
: . ,
.,
~ \
wherein Y is S-Het, in which Het is l-N-methyl-tetrazol-5-yl;
R is hydrogen or Cl C10 alkyl; when substantially free of
the L (~) isomer. The process comprises reacting a ompound
of the formula
S .
~2N~ ~ :
~N~C~2Y
COOR
or a silyl ester or salt thereof~ in which Y is as deined
above, with a corresponding D-(-~ acylating a~ent of an acid
of the formula
~ ~ ~ ~ H-COOH ,~
OCHO
III5a)
in which R is as defin~d ab3ve.- ~ha~l the compoun* ~f
formula II is in the form of the silyl ester or salt thereof,
~0 the thus-obtained compound ~e converted to the corresponding ~:
final product.
Acylation of ~ 7-~ino grt~up of ~ c:ephalo~porin 1$ a : .
well-known reae~ion and any of the functional equivalents :
of formula III or III (a) commonly used as acylating agents for
25 primary amino group~ may be employed. Examples of suitable
~cylating d rivat~ves c~f the free acid include th~ c:orr~ponaing ~ .
' .
- 6(a) - :
~ ,.
~56~
acid anhydrides, mixed anhydrides, e.g. alko~yformic c~nhydrid~s,
acid halides, acid azides, acti~e esters and active thioesters.
The free acid may be coupled with compound II after fi~st re-
acting said free acid with N,N'-dimethylchloroforminium chloride
or by the use of enzymes or of an N,N'-carbonyldiimidazole or an
N,N'-carbonylditriazole or a carbodiimide reagent, e.g.
N,N-diisopropyl carbodiimide. N,NI-di Gy clohexylcarbodiimide
or N-cyclohexylcarbodiimide or N-cyclohexyl-N'-(2-morphilino-
ethyl) carbodiimide or of an alkylyl~mine reagent or o~ an
isoxasolium salt reagent. ~nother 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 memkered ring containing at least t~o
nitrogen atoms, i.e. imidazole, pyrazole, the triazoles,
benziimidazole, benzotriazole c~nd their substituted deriva-
tives. A reactive derivative of the phenylglycine
acid of formula III is the N-oarboxy c~hydride ~Leuch's
anhydride). In ~his structure the group which activate the
carboxyl group al~o serves to protect the ~mino group. A
particularly preferred acylating agent is the acid Ghloride
hydrochloride of the ~ormula
: R~
R-C-O ~ CH - COCl
~5 N~2~HCl
which also serves a dual function of carboxyl activation and
amino protection. Mention was made above of the use of
enzymes to couple the free acid with its blocked c~mino group
with compound III. Included in the scope of such processes
. . . ' ' ' , ' '' . .. ' ... ' . , " ~ .
,,, :, . . .
,, . . . .. . : .
,, ,:, , , ~ . :
' ' . '', ,': :. , , . : .
are the use of an ester, e.g. the methyl ester, of that
free acid with en~ymes provided by various microorganisms,
~ e.g. those described ~y T. Takahashi et al., J. Amer. Chem. Soc.,
94(11), 4035-4037 Clq72) and by T. Nara et al., J. Antibiotics
(Japan), 24(5), 321-323 ~1971) and in West Germany 2,216,113.
The particular process conditions, e.g. temperature,
solvent, reaction time, etc. selected for the coupling re-
action are determined by the nature of the acylation method
used and are known to ~hos~ skilled in the art. Generally
it is useful to add an organic tertiary amine~ e.g. triethyl-
amine, N,N-dime~hylaniline, ethylpiperidine~ 2,6~1utidine or
quinoline, to serve as a proton acceptor or salt-forming agent.
The compounds of ~he presen~ invention may be isolated
in any o the ways customaxily employed for the isolation of
similar cephalosporins. Thus, the product ma~ be obtained
as the neutral molecule and, in the case of compounds of
formula I, this i5 probably more accurately represented as the
zwitterion, or it may be isolated as a salt. Formation of the
desired pharmaceutically accepta~le carboxylic acid or acid
addit~ion salt is carxied out by known methods, e.g. xeaction
of the acid wi~h an appropriate base o~ acid~
At the conclusion o the acylation reaction the product
obtained may be converted (before or after removal of the amino-
protecting group) by methods known per se to another desired
product of formula I. For example, th~ product of formula I
or I(a) in the form of a silyl ester ox salt thereof may be
converted to the free acid product or pharmaceutically acceptable
salt thereof by removal of the silyl ester group, e.g. by
hydrolysis.
The pharmaceutically active compounds of the present inven-
~ion are potent antibacterial agents useful in the txeatment of
.. . . .
.. .. . . . . ..
~s~
infectious diseases in poultry and animals, including man, caused
by many Gram-positive and Gram-negative bacteria. The active
compounds are also of value as nutrition~l supplements in animal
feeds and as agents for ~he treabment of mastitis in cattle.
The preferred compounds have also been unexpectedly round to
be e~ficiently absorbed upon oral administration.
The novel medicaments provided by the present invention
~ay ~e formulated as pharmaceutical compositions comprising,
in addition to the ~ctive ingredientO a ph?rmaceutically
ac~eptable carrier or diluent. The compounds may be administered
~o h orally and parenterally. The pharmaceutical preparations
may be in solid form such as ~apsules, tablets or emulsions.
In the tr~atment of bacterial infections in.man, the compoun~s
of this invention may ~e administered parenterally in an amount of
~rom about 5 to 200 mg./kg.~day in divided dosage, e.g~ 3 to ~
times a day. They ar~ administered in dosage units co~tainins
eOg. 125, 2$0 or 500 mg. of active ingredient with suitable
physiologically accepta~le carriers or excipients.
The following illustrates the preparation of starting
2~ materials used Ln the production of the novel compounds of
the invention.
/ ,~' /
,:
~;tartin~ ~aterials
Synthesis of po*assium_l,2,3-~riazole-~-thiolate
O
Il N~
~bc-~a=c=s~ CH2N~ OH
`S
163~19 42.04 ~05.24
~ SH ~ SK
10 N / ~ N
N N
H H
101.13 13g.23 .
The synthesis of the thiol was accomplished by a pro-
cedure essentially identical to that described in the litera-
ture [J. GoPrdler and G. Gnad, Chem. Ber. ~9, 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
2~ with vigorous stirri~g, 0.685 N ethereal diazomethane (453
ml., 310 mmoles~. When the addition was completed, the mix-
t~re 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
- ~he pure material. A small second crop ~21,1 g~) was obtained .
by evaporation of the mother liquor in vacuo. The total : -
yield w~s therefore 4Q%.
- 10 - ' ';'
' ,.' ' - . ' ' ', ; ,~'''' "', . ' :' ' ' ' . ' ; ' , ' '
.,~ , .
-
1,2,3-Triazole-5-thiol
A solution of the above benzamido compound (8.2 ~., 40
mmoles~ in 2N sodium hydroxide (80 ml.~ 160 mmoles) w~s heated
under reflux temperature in a nitrogen atmosphere for 24 hours.
The solution ~as cooled to 0 in ice, ~nd concentrated hydro-
chloric acid (26 ml. ) was added, while a continuous stream of
nitrogen was passed through the solution. The benæoic 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 fil-trate was --
immediately extracted with ethyl acetate, the extract was washed
with saturated salt solution, dried over magnesium sulfate and
then evaporated 'in vacuo. The viscous oil which remained was
immediately evaporatively distilled in vacuo (70-75/0.001 mm. ?
to give an oil (2.84 g., 70~ which solidified (m.p. 52-59;
Goerdler reported mOp. 60) spontaneously. -'
Potassi'um 1','2~3-Triaz'o'l'e`-`5`-'th'io'la-te
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 thendiluted with anhydrous ether until crystallization of the salt
was completed. The solid was collected by ~iltration, 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 ~'
via 5-amino-1,2,3-thiadiazole [G. Goerdler and G. Gnad, Chem.
Ber. ~9, 1618 (1966)~.
~5~
É~
NEICO,0 L ~ ~H2 ~
S-
S
OH J~ ..
El~ ~ N 8 7 %
~N ~ 93% M~ S ~NH N
EI 2
5-Amino-1, 2, 3-thiadiazole can be prepared by an alternative route~ . .
not involvin~ diazomethane [D. L. Pain and R. Slack, J. Chem.
Soc. 5166 (1965)].
O . :
~\N--CEIzCE10 + ElzNNE~CO2Et ~ '
O '. '. ~' ' '. ' '.
2C3 Q !!~ ... ~ .
~,~N-CH2CEI--N--NHC02Et .....
8 93% ~;
O
r\ N
o ~ S ~ '' '' "' ' '
8g% " ;"~
N~ . .
3 0
7 7 %
- 12 -
Synthesis of 7-Amino-3-(lr2!3-triazol-5-ylthiomethyl)-3-
cephem 4 r carboxylic_acid
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 for 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-txiazole 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 10.5, was adju~ted to pH 8.5 with
42% phosphoric acid. 7-Aminocephalosporanic acld (21.8 g.,
0.08 mole) was added and the mixture heated at 50 for 4 hours.
The clear solution was cooled in an ice bath and adjusted to
pH 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.) was brought into solution
with 600 ml. of methanol and 40 ml. of conc. HCl. After
carbon treatment the solution was diluted with 1.5 1. of ice
water and extracted once with ethyl acetate. The aqueous
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 ~iltration, washed
with water and methanol and dried in vacuo over phosphorus
pentoxide; 11~4 g. The IR and NMR spectra were ~ully
consistent for the de~ired product.
13-
~5~
Anal. Calcd. for CloHllN53S2 C, 38.42; H~ ; ~
Found: C, 38.27, 38.25; H, 3.76,
3.40; N, 21.02, 21.00; H2O, 1.70.
Purification of 7-Amino-3-( ~ hiomethyl)-3-
_ _ .
cephem-4-carbox~lic acid (II~
Crude 7-amino~3-(1,2,3-triazol-5-ylthiomethyl)-3-
cephem 4-carboxylic acid (16.1 g.) containing approximately
20 mole % of 7-aminocephalosporanic acid as an impurity, was
1~ brought into solution with 600 ml. of methanol and 40 ml.
of conc. HCl~ After carbon treatment, the solution was
diluted with 1.5 1. of ice water and extracted once with
ethyl acetate. m e aqueous phase was concentrated at reduced
pressure to ramove methanol. The cold aqueous concentrate was
then adjusted slowly to pH 4.0 with 20% sodium hydroxide
causing the product to crystallize. The product was collected
by filtrati~n, washed with water ancl methanol and dried in
vacuo over phosphorus pentoxide; 11.4 g. The NMR spectrum
indicated that this product contained about 7 mole % of 7-
aminocephalosporanic acid as an impurity.
The 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 C10~15N503S2 C, 38-42; H~
Found: C, 39.06, 38.53; H, 3.56,
3.Sl; N, 22.05, 21,60; H2O, 1.78,
-14-
~L~5~
7-Amino-3-tl,2 ~ t ~ omethyl)-3-cephem-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 phospha~e 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% H3PO~. The resulting precipitate was filtered
off, washed with cold water (50 ml.) and air dried. 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.
Anal. Calcd. for CloHllN503S2: C, 38~39; H,
Found: C, 38.36; ~, 3.78.
acid (II)
Two hundxed seventy-two g~ (1.0 mole) of 7-amino-
cephalosporanic-acid was su~pended 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 (Note: The sodium
bicarbonate was added in portions). Then 143 g. (1.0 mole) of
5 mercapto-1~ 1,2,3-triazole potassium salt was added and
the mixture stirred at 55 C. + 1~ C. under a nitrogen
atmosphere for 4 hou~s. After 1 hr. the pH was readjusted to
6.4 ~y addition of a small amoun~ of 40% H3PO4. At the end
*
of the 4 hr. heating period, 50 g. of "Darco KB" decolorizing
*~rade Mark
-15-
room tem~era~ure the yield ~as lQ~ g.; dec. pt. 230C.
The IR and NMR were consistent for the desired structure.
~ Pre~aration o~ D (~-2-amino-2-t4-acetoxyphenyl)acetic acid
Method A ~in-acetic acid as solvent~
203.5 g (1 Mole) of D(-)p-hydroxyphenylglycine chloride,
800 ml of acetic acid and 314 g( 4 Moles~ of acetyl chloride
are stirred 48 hours at room temperature. The solid is
collected, washed three times with acetone ~3 x 250 ml) and
twice with ethanol (2 x 250 ml) and dried at 40. Yield
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 4.5 with 20% 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 glycine3.
~D (1~ HCl N/10) ~ -104.5
Method B cin methylene chloride~
4.07 g ~0.02 Mole~ of D(-)p-hydroxyphenylglycine hydrochloride,
30 ml of methylene chloride and 6.28 g t p.08 Mole~ of acetyl ;~
chloride -are stirred 48 hours at room temperature. The solid
is collected, washed ~wice with acetone and twice with ethanol. ;~
Yield 4.17 g (84.5%3. 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 ml of tri*luoroace~ic acid at room
temperature. After dissolution, 1.57 g( 0.02 Mole3 of acetyl
chloride is added. After a slightly exothermic reaction, a
- 17 -
,, : . .,. . , , ~ ., , , .
,,:, ,
~igii8~
charcoal was added and, after stirred for 15 min. at 55 C.,
the slurry was filtered hot through a diatomaceous ear~h
["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 ~Cl. 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. ~f
methanol and air dried.
The crude product was suspended in 3000 ml. of 50%
methanol-water and 300 gO (1.5 mole) of ~-toluenesulfonic
acid was added. The mixture was stirred for 15 min. and then
50 g. of "Darco XB" decolorizing charcoal was added. A~ter
stirring for 15 min. at 22 C., the slurry was filtered
through a 'ICelite" pad and the pad washed with 2 x 100 ml.
of 50% methanol;water. The pH of the combined filtrates was
adjusted to 4.0 by addition of approximately 210 ml. of tri-
ethylamine. After cooling at O 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 a~d air dried.
This material was suspended in 2000 ml. of water and 84
g. (1 mole) of sodium bicarbonate 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
tered through a "Celite" pad. The product was washed with
2 x 100 ml. of water and the pH of the combined filtrates
was adjusted to 3O5 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 by filtration was washed
with 2 x 200 ml. of cold water and 2 x 1000 ml. of acetone.
After drying over P2Os in a vacuum desiccator for 14 hr. at
*Trade Mark
-16-
solid appears. The suspension is stirred 1 lf2 hours at roo~
temperature and t~e trifluoroacetic acid is removed in
~ vacuum. The remaining solid is collec~ed, washed with
methylene chloride and with ethanol. The DC-]2-amino-2-
(4-acetoxyphenyl~acetic acid is identical to that
prepared by method A or B.
Yield~ g(75%)
Preparation of D (-)2-amino-2-~4-pivalyloxyphenyl2acetic
~. .
acid hydro~hloride
1.67 g (0.01 Mole) of D(-~p-hydroxyphenylglycine is added
- to 10 ml of txiEluoroacetic 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 from isopropanol.
Anal. Cl = 11.8% (calculated 12.3%)
W ~max. 205 nm and 220 nmO
Preparation of D ~-),2~amino-2~(4-benzoyloxyphenyl)acetic_
acid hyd_och oride
This compound is prepared according to the same procedure
as used for the pivalyloxy derivative. Yield: 2.7 g (87%).
25~ An analytical sample is recrystallized from ethanol.
Anal. cl. = 11.3% (calculated 11.5%)
W ~max. 205 nm and 234 nm
- 18 -
~: :
,
hydrochloride
83~6 g. (0.40 mole) of D(-)2-amino-2-(4-acetoxyphenyl)-
acetic acid and 1.25 1. of anhydrous me~hylene chloride are
cooled to -5 C. with stirring. Then 152 g. of phosphorous
pentachloride are slowly added followed by 4 ml. of dimethyl
formamide. The mixture is stirred 4 hours at 0 C. The
solid is collected, wa~hed with anhydrous methylene chloride
and vacuum dried at room temperature. Yield: 61 g. (57.5~).
Anal. Total chlorine = 27.2% (Theory 26.9%)
Preparation of D-t-)2-formyloxy-2-~4-formyloxyphenyl)acetic
a _
A solution of 3.6 g. (0.02 mol~) of D-5-)-2-hydroxy-2-(4-
hydroxyphenyl)acetic acid in 50 ml. of 97% aqueous formic acidwas 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 sulfate, fil-
~ tered and evaporated to aford the desired product.
Preparation of D~(-)2-formvloxy-2-(4~acetoxY~henvl)acetic
acid
The D-~-)2-formyloxy-2-(4-formyloxyphenyl)acetic acid
obtained above is dissolved in 10 ml. of acetyl chloride and
the xesulting mixture wa6 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 ac~ording to NMR.
--19--
The follo~ing examplec are given in illustration o~,but not in limitation of, the present in~ention. All temper-
atures are in degrees Centigrage. 7-Aminocephalosporanic
acid is a~breviated as 7-ACA and 7-aminodesacetoxycephalOspOraniC
acid as 7-~D Q.
Example 1
7~ 2-amino-2-~4-acetoxyphenylacetamudo)desaceto~y-
cephalospor nic acid - (acetoxy cephalexine) - RN 1394
15.27 g C P .0~14 Mole) of 7 ADCA are stirred in 500 ml of anhydrous
methylene chloride; 120 ml o~ methylene chloride are distilled
10 off and 11.8 ml of hexamethyldisilazane are added. The
mixture is stirred and re~luxed 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 ~.5 ml o~ dimethylaniline and
7 ml of a solution of dimethylaniline hydrochloride in
methylene chloride (30%). Then 20 g (0.0756 Mole) of
D~-~2-amino-2~4 -acetoxyphenyl)acetyl chloriae hydrochlorid~
are added in small portions (~1 1/2 hours) a~ O~C.
The mixture is ~tirred 30 min. at +lO~C and 4 hours at ~20OC
and allowed to stand overniyht at ~5CO Then 5 ml of methanol
followed by 240 ml of water are added. ~he pH is adjusted
at 2.S with triethylamine and the mixture is filtered
, through a celite pad; then the pH is checked and the aqueous
phase is separated, washed twice (2 x 15n m~) with methylene
chloride and treated with charcoal.
The solution is adjusted to pH 4.5 and vacuum concentrated to ~:
a volume of ~ lS0 ml. The suspension is allowed to stand
- 20 -
'
ov~r~ight a ~5C: arld the solid c911ected and washed wit~
water and acetone, and dried at 40Co
~ield: 15,1 g (~ so^æ of 75-80-~,' pure material3
(~D (1% H;20) = ~107
5 14 g of this crude material is suspended în 30 ml o
wa'~:er (p~ ~ 3,2) hydrochloric acid (36~o) is added to pH
1, 3 and the resulting solutio;~ is charcoal treated and
filt~red through a celite pad and 2djusted to pH 4-4, 5
over stirring. ~fter 2 hours at 0- 1 5C the RN 139~ i~
10 collected, ~shed wit~ water and acete~ne and dried at ~.'0C.
Yield: 7 g,
~ D t~ R20) 3 t 133.
~e infrared and nuclear magnetic resonance s~ectra are
consistent wi~h the desired product.
siological Data
- Table I sh~ws comparative MIC data for sL-s 578-4
(~-hydroxy analogue of cephalexin) and (~-acetoxycephalexin
(RN 1394). Minimal inhibitory concentrations were determined
by the 2-fold broth dilution method utilizing equimolar con-
centrations of each compound.
.
l ~
~x
O a) ~r co c~
a~ o 1~ ~ ~
....
,~, ~ C~ O ~I N ~ a~ Ln Ln Ln Ln CO n
~'J a. ~) ~ ~D ~1 ~ ~ ~ ~ N ~J
I ~1) Z ~ r~
~ V P:;
~ I 1:
I ~C ~0~
I` o a~ ....
Ln ~ ~ O ~1 ~ l CO Ln Ln Ln Ln ~D Ln
~J ~ ~1 ~ ~7 -1 rf) N N ~3 N ~I N
u~ :>l.C ~1 ,1 ~1 ~1 ,~
~ ,. .
.'
a ~ ~ :
n ~ ~ ~1 m r~, ~ o Ln
E~ CD O ~ ~ O O C~ ~ ~I r~ I~ ~10 ~ er ~S In Ln Ln :~
H ~ In D In U~ D In Ln m ~D ~ Ln ~l Ln I
Q~
E l o _
O ~ ~ Ln ,Iq
~t * ~ ~ ~ O ~ '--
r~ r o o--O
I I I ~1 ~ I o--_
o a c~
~,1--I ~ ~ I I I I I ~~ I I I h ~ :
o o o o ~ o o c~
rl ~ .~i ~ ~ ~ ~ bq ~ '~ ~ O
5) Ul~ bO tOn L~ . ~:
o ~ 3 o X a~ I O ~ O V D O
h . ~rl ~ M Lln ~ ~ a~ c ~l
O ~ u~ 0 0 ~ ~ a o
3 o ~ h O O O ~rl
~ O O
h ~u ~
tr h ~ h
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.. . : ~ ~
Example 2
7-[DC-)2-Amino-2~4-acetoxyphenylacetamido]-3-l(1,2,3-triazol-
5~ thiomethyl]-3-cephem-4-carbo~y~ic acid: RN 139Ç
62.6 g (0.2 Mole~ of 7-amino-3-l(1,2,3-triazol-5-yl)thiomethyl]--
3-cephem-4-carboxylic acid (7-TACA~, 1.5 1 of methylene chloride
and 60~2 g (0O374 mole) o hexamethyl disilazane are stirred
and refluxed 20 hours with a slight pressure of nitrogen
~after about 2 h~urs all 7-TACA is run in solution).
The s~lution is cooled to 0C and 3Q . 4 ml of dimethylaniline
followed by 20.4 ml of a solution of dimethylaniline
hydrochloride in methylene chloride (30%3 and 1. 35 g of
.
imidazole are addedO
Then 60.5 g ~0.22~ mole) of 2-amino-2-~4-acetoxyphenyl)acetyl
chloride hydrochloride are added in small portions (~ 1 1~2
hours) at 0C. The mixture is then stirred 3 hours at 20C
and let stand overnight at ~5C. 25 ml of methanol ~ollowed
by 75Q ml of water are th~n added. The pH is adjusted to
2.3 - 2.5 with triethylamine and the l~xture is filtered
through a celite pad. The aqueous phase is separated, washed
twice with methylene ~hloride and charcoal treated.
The solution is adjusted to pH 4.3 and stirred 2 hours at
~5C. The solid is colleoted, washed twice with water and -~
dried at 40C. Yield: 53 g (about 5Q~)O
This crude material is purified twice as follows: ~ ~
The solid is treated with eight volumes of 0.5N hydrochloric ~ ~-
acid and the suspen ion is decolorized with charcoal. An :~ :
equal volume of methanol is added to the solution and the pH
adjusted to 2 - 2.1; after 15 minut~s a small amount of
suspended solid is collected and discarded. The ~iltrate is
adjusted to pH 4. The precipitated solid is collected, washed with
- 23 - -
. .
: . :
.. . .
.
~Si6~
MeOH/H2O (50/50) and pure methanol.
Yield: 25 g. (after two purifications)n
- IR consistent with assigned structure
- Moisture (KF) : 5.1
- Chemical assay
- Iodometric assay 885 mcg/mg
- Potentiometry Amine assay: 97%
Bioloyical Data
Table II shows comparative MIC data for the above prepared
7-lD( )2-amlno 2-(4-acetoxyphenylacetamido)]-3-[1,2,3-triazole-
5-yl)-thiomethyl]-3-cephem-4-carboxylic acid, RN-1396, and its
p hydroxy analogue, BL-S640. Minimal inhibitory concentrations
were determined by the 2-fold broth dilutio~ method utilizing
equimolar concentrations of each compound.
-24-
~0 Q~
~P ~ ~ O O
U u~ h 1-lc~ ~ o ~r OD o N o~ ~1 ~ o r~) m u~ lo ~1
1 1 ~ Z~ N ~ ~ t~
~m~ 1\~ ~
..
,.,
C~
-. ID O O r~
U~ O ~1 0 _I ~ O O er O 1~ ~ In m In O
H ~ ¦ " -
_ r~
c~ ~ ~ O Cl c~ ~ O Ll~ r
. ~ ~.) . ~ ~ o ~ D O .
. V ' '
m
æ
_ dP O -
~ ff~ . o
.. .~C * ~r-l ~ ~ O ~ + . : .
. . _~ I `~ ~ ~ . .: :
. I I Cl r~ O .
. - . O O ~
~1 ~ p ~ I O O O I r-l ~ I I IS~l ~ ....
O ~~ O O Ofl~ O :
.
I "-1 V~ ~ O
~ * ~ u~ w ,s~
o ~ O ~ l O ~ aJ a) o O ..
r~ c U~ ~ ~ m 2, ~ + - .
~, h '--~- U U ~: . .
L: LQ ~ ~ 1 ~
a) . O, h ~1 p h ~ U t.) C) :1 . . ..
h ,_1 . ., ~ ~ J.? ~J~P C3 .
h , ~ - ~ Sl h cq U~ * ~1~ .
æ ~ ~ :
" ':'' '
- 25 - . . ~
,. . ..i ., ; , ;,. . ..
.. , . . , . : :.. :. .: .. . . .. .... . .
56E~
Example 3
7-D-(-)[2-(4-aceto~y~ n~ 2-for~yl$ey3g~t~a~ thyl-
, 2 ~ 3 ! 4-tetrazol-5-yl~thiom th~1 3-ce~hem-4-carboxylic acid
To a solution of 4.8 g. (0.02 mole) of crude D-(-)2-
form~loxy-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 dropwise to a solu-
tion of 6.3 g. (0.02 mole) of 7 amino-3-(1-methyl-1,2,3,4-
tetrazol-5-yl)thiomethyl-3-cephem-4-carboxylic acid, 5.6 g.
of sodium bicarbo~ate, 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 was 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 sulfateO
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 ha~e 85-90% acetyl and 50-60% formyl.
7-D-(-)[2-(4-form~loxyphenyl)-2-form~l~ cetamido]-3~ methyl
1,2,3,4-ketrazol-5~yl)thiomethyl-3-cephem-4-carboxylic acid
The same procedure as in Example 3 was followed to react
2.5 g. (0.01 mole) of D-~-)2-formyloxy-2-(4-formylox~phenyl)-
acetic acid with 3.28 g. (0.01 mole) of 7-amino-3~ methyl-
1,2,3,4-tetrazol-5yl)thiomethyl-3-cephem-4-carboxylic acid to
-26~-
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-pr~duct.
Microanalysis ~f the desired product gave:
Calc'd. for C20~1gN6O8S2
~ The~y % Found
.
C 44.86 46.47
H 3.38 4.14
N 15~70 .13.50
K.F. (H2O) 1.78
2~ -.
- :
-27
In addition to the above, the compounds of the instant
invention are also valuable as intermediates for the prepara-
tion of other pharmaceutically active compounds. For example,
the instant a-formyloxy or a-amino-a-(p-acyloxyphenyl)ac tamido
cephalosporanic acids may be converted to the corresponding
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 circum~tances.
We have f~und 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-~-(p-hydroxyphenylacetamido~desacetoxy-
cephalosporanic acid.
Accordingly t the present invention also provides for a
novel process for preparing 7-D-~-)a-amino-a-(p-h~dro~y-
phenylacetamido)desacetoxycephalosporanic acid, hydrate or a
pharmaceutically acceptable salt thereof, which process comprises
treating in an aqueous solution 7-D-(~ amino-a-(p-acetoxy-
phenylacetamido)desacetoxycephalosporanic acid with an esterase
at a pH between about 5.0 and about 7.5; isolating the product
by methods known ~ se, and, if desired, converting by methods
known ~ se the product in the form of the free acid or hydrate
to the corresponding pharmaceutically acceptable salt thereof.
A preferred embodiment is the preparation of 7-D~ a-
amino-a-(p-hydroxyphenylacetamido)desacetoxycephalosporanic
acid, hydxate or a pharmaceutically acceptable salt which
pxocess comprises treating in aqueous solution 7-D (-)-a-amino-
-28-
~5~
~-(p-acetoxyphenylacetamido)desacetoxycephalosporanic acid with
an esterase selected from human serum, animal serum, citrus
esterase, wheat bran, wheat germ, and bacillus subtilis at a
pH between about 5.0 and about 7.5 and at a concentration of
5 about 5 to about 10 mg./ml. of esterase per total volume of the
aqueous solution; isolating the product by methods known
se, and/ if desired, converting the product in the form of
free acid or hydrate to the corresponding pharmaceutically
. acc~ptable salt thereof.
A commercially preferred embodiment of the present inven-
tion is the preparation of 7~D-(-)a-amino-~-(p-hydr~xyphenyl-
acetamido)desacetoxycephalosporanic acid, hydrates or pharma-
ceutically acceptable salts thereof, which process comprises:
treating in an aqueous sollltion 7 D-(-)-a-amino ~-
(p-acetoxyphenylacetami~o)c phalosporanic acid with an esterase
selected from citrus esterase, wheat bran, and wheat gexm at
a p~ between about 5.0 and about 7.5 and at a concentration of
abou~ 5 to about 10 mg./mlO of esterase per total volume of
the aqueous solution; and
isolating the product by methods known per se, a~d,
if desir~d, converting the product in the form of the free acid
or hydrate to the corresponding pharmaceutically acceptable salt
thexeof.
. Of special commercial intexest is the process for prepar-
25 ing 7 D- (-)a-amino-a ~p-hydroxyphenylacetamido)desacetoxy-
cephalosporanic acid, hydrat~ or pharmaceutically acceptable
salt thereof comprising:
treating in an agueous solution 7-D~ amino~
(p-acetoxyphenylacetamido)desacetoxycephalosporanic acid with
the commercially available esterase, coarse wheat bran, at a
--2g-
8~
pH be~ween 5.5 and 6.0 or optionally in the presence of ~ buf-
fer at a pH of 7.0 at a concentration of about 10 mg./ml. of
esterase 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 thereof.
The 7-D-(-)a-amino-a-(p-hydroxyphenylacetamido)desacetoxy-
cephalosporanic acid prepared by the instant invention is
known to be a potent antibacterial agent useful in the treat-
ment of infectious diseases in poultry and animals, including
man, caused by ~any Gram-positive and Gram--negative bacteria.
The following example illustrates the preparation of
p-acetoxycephalexin according to the invention.
Ex~Mæ~E A
Solutions o~ 0.5 mg./ml. of 7-D-(-) a-amino-a- ~p-acetoxy-
phsnylacetamido)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- ~-) a-amino-a-(p-hydroxy-
phenylacetamido)desacetoxycephalosporanic acid (p-hydroxy-
cephalexin) were also prepared in both normal saline and
human serum.
Rll the above solutions were incubated at 37~ CO with
shaking and sampled for chxomatography at 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 dxied and developed in a solvent system
containing 30 parts butylacetate; 15 parts n-butanol; 40 parks
acetic acid; and 24 parts water. The strips were then bio-
*~rade Mark
-30
l~S6~4
autographed on plates seeded with bacillus subtilis at a pH
of 6Ø
The biochromatograms indicated that p-acetoxycephalexin
i5 quickly hydrolyzed to the p-hydroxy form in human serum
but appears stable in normal saline.
.
-31-
