Note: Descriptions are shown in the official language in which they were submitted.
This invention relates to a process for preparing
the cephalosporin class o~ antibiotics. In particular it
relates to a process for preparing a novel group of orally
O e~fective cephalosporin antibiotics.
Among the presently used cephat osporin anti~iotics,
cephalexin 17-tD-a-phenylglycylamido)~3-methyl-3-cephem-~-
carboxylic acid] is a particularly valuable antibiotic because
of its oral effectiveness.
In the search for more effective and orally active
an~ibiotics a wide variety o~ cephalosporin antibiotics of
varying ~tructure~ have been synthesized. Notably, the
de~acetoxycephalo~poranic acids (3-methyl-3-cephems~ of
which cephalexin i8 a me~ber have been ex~ensively investi-
~ gated.
-3959 2
.
.
,
' ~
6~Z
Recently, 7~acylamido-3-methoxymethyl-3-cephem-4-
carboxylic acid antibiotics were describe~. The de~cribed
compounds have the structural fea~ure of an alkoxy 3ubs~ituted
methyl group in the 3~po~i-tion of the dihydro~hiazine ring
of the cephalosporin~
The cephalosporin anti~.iotlcs prepa-ed by the
process of the present invention repres~nt a novel class
of cephalosporin ethers having the oxygen atom of an ther
substituent attached directly to the C3 carbon of the
dihydrothiazin~ ring~
The cephalo~porin compounds prepared by the process
of this invention are represented by the following Formula I,
H O H
S ~,
R-C-C-N ~
NH2 ~ N ~ o-Rt
C\O
I O-R2
wherein R is phenyl, hydroxyphenyl, halophenyl, methylphenyl,
methoxyphenyl, 2-thienyl, 3-thienyl or 2-furyl;
ao Rl is hydrogen, methyl, ethyl or 3-methyl-2-but~nyl;
R~ is hydro~en or a ~arboxylic acid protecting ester
orming group;
and when R2 is hydrogen the pharmaceutically acceptable non-
toxic salts thereof; wi h the limitation that when Rl is
hydrogen, R2 i~ a carboxylic acid protecting ester foxming
group.
In the foregoing description of the compounds
prepared by the proce~s of the in~ention, ~he t~rm "hydroxy-
phenyl," ref~rs to 4~hydroxyphenyl, 3-hydroxyphenyl, and
7~-hydroxyphenyl. "~alophenyl~" refers to ~he i~omeric
~-3g5~ 3
'`''' ' ' ' ~ '
~7~
fluoro, chloro and bromo phenyl groups such as 4-fluoro-
phenyl, 4-chlorophenyl, 3-chlorophenyl, 2-chlorophenyl,
3-bromophenyl, and 4-bromophenyl. "Me~hylphenyl," refers
to tho isomeric 2-, 3-, and 4-methylphenyl groups and the
texm "me~hoxyphenyl" xefers to 4-methoxyphenyl, 3-methoxy-
phenyl and 2-methoxyphenyl.
As used hereirl, the term "a carboxylic acid protect-
ing ester forming group," has reference to the ester groups
commonly employed for the protection of the C4 carboxyl group
of the cephalospo.rins and which are characterized by their
ease of removal under basic or acidic hydrolysis conditions
or by catalytic hydrogen~lysis to regenerate the free carboxy-
lic acid function. Illustrative of such carboxylic acid
protectiny groups are the 2,2,2-trichloroethyl, diphenylmethyl
(benzhydryl), p-nitrobenzyl, p-methoxybenzyl, t-butyi,
and trimethylsilyl. The methods used for the preparation
and subsequent removal of the cephalosporin esters described
herein are all known methods which have been previously
described.
~20 The compounds represented by the Formula I wherein
Rl is methyl, ethyl or 3-methyl-2-butenyl and R2 is hydrogen
are valuable antibiotics which are effective when administered
by the oral route.
The antibiotic compounds are prepared by a variety
of ~ynthetic methods. According to one method of prepar~tion
a 7-amino-3-hydroxy 3-cephem-4-carboxylic acid es~ex and
preferably a salt thereo such as the hydrochloride salt is
al~ylated, pre~erably with a diazo compound for example
diaæcmethane, to yield the corresponding 3-ether derivative,
for example the 3-methoxy or 3-ethoxy derivative. The
X-3959
7-amino~3-ether cephem ester is then acylated by known methods
with a derivative of a substituted ~lycine represented by the
formula
H O
..
R-C-C-OH
NH2
wherein R is as defined for ~ormula I. The carboxylic acid pro-
tectin~ ester ~roupr R2, ls removed to provide the antibiotic
compound.
Th.e startin~ materials employed in th.e ahove described
preparation, 7-amino~3-hydroxy-3-cephem-4-carboxylic acid esters,
are prepared, as described in our co-pendin~ Canadian application
No. 186,688, filed Nov. 26, 1973, by reacting a 7-acylamido-
cephalosporanic acid such as 7-phenoxyacetamidocephalosporanic
acid with. a sulfur containing nucleophile accordin~ to known
procedures to effect the nucleophilic displacement of the
acetox~ group of the cephalosporanic acid and provi.de a 7-
acylamido-3-thi:o-substituted methyl-3-cephem-4-carboxylic acid.
. The 3-thi.o-substituted cephem acid is then reduced with zinc/
formic acid in the presence of dimethylformami.de or wi.th. Raney
nick.el in the presence of hydrogen to ~ield a 7~acylamido-3-
exomethylenecepham-4-carboxylic acid. The acid i5 esteri~i.edr
for example ~ith. p-nitrobenzyl bromide and the p-nl.trob.enzyl
ester is reacted with phosphorus pentachlor;de in the presence
of pyridine to effect the clea~a~e o~ th~e phenoxyacetyl group
and provide the 7-amino-3-exomethylenecepham-4-carboxylic
acid ester.
The 7~amino-3-exomethylenecepham-4-carboxylic acid
ester is then reacted with ozone in an inert solvent at a
temperature bet~een about ~80 and 0C. and preferably
; -5-
' '
.
$~
between 80 and -50C. to form an intermediate ozonide.
The ozonide i~ decomposed, in situ, in the cold to provide
a 7-amino 3-hydroxy-3-cephem-4-carboxylic acid ester as
illustrated in the following reaction scheme.
H~N~ O:~ (oZonl(le) r~ OH
C~-O
`0-Rz C\0
0-R~
wherein R2 is a carboxylic acid protecting ester group.
Illustrative o the 7-amino-3-hydroxy~3-cephem-~s-
carboxylic acid esters are p-nitrobenzyl 7-amino-3-hydroxy-
3-cephem-4-carboxylate, p-methoxybenzyl 7-amino-3-hydroxy-
3-cephem-4-carboxylate, and 2,2,2-trichloroe~hyl 7 amino-3-
hydroxy~3-cephem-4-carboxylate.
According to the process of the present invention,
the 7-amino-3-hydroxy-3-cephem-4 carboxylic acid esters are
alkylated at the 3-hydroxy group to provide the 3-alkoxy-
3-cephem esters. The alkoxylation is carried out by
reactin~ the 3~hydroxy ester with a diazo compound, namely
; diazomethane, diazoethane or l-diazo-3-methyl-2~butene.
Al50 ~he 3-hydroxy~3~cephem esters can be alkylated with
methyl iodide in the presence of a base, with an alkyl ester
of sulfuric acid in the presence of a base, Eor example
with dimethyl or diethylsulEate, or with trimethyl oxonium
1uoroborate. Likewise, activated halo compounds in the
pre~ence o~ a base can al~o be used to alkylate the 3-hydroxy-
3-cephem esters. For example, the a-haloethers such as
chloromethyl methyl ether, and bromomethyl ethyl ether;
-haloacid e~ters such as ethyl bromoacetate, ma~hyl chloro-
X 3959 6
acetate and ~thyl ~-bromopropionate and ~he allylic halides
such a~ allyl bromide, allyl chloride and l-bromo-3-methyl-
2-butene can react with the 3-hydroxy-3-cephem esters to
provide respectively the 3-alkoxymethoxy, the 3-carbalkoxy-
methoxy and the 3-allyloxy ether derivatives thereof. Th~
preferred alkylatin~ rea~ents are the ~iazo compounds,
diazornethane, diazoethane and l-dia20-3-methyl-2-butene.
These diazo compounds react with the 3-hydroxy-3-cephem
esters to provide the 3-ethers without side-reaction product
~10 contamination. Other alkylating agents~ for example the
activated halo compounds described above, react to provide
mixtures of the desired 3-ether with 4~alkylated-3-hydroxy-
2-cephem-~-carboxylates and 4-alkylated-3-alkoxy-2-cephem-
4-carboxylates. Such mixtures can be separated chromato-
graphically to provide the desired 3-ether estar.
The etheriication xeaction is carried out by adding
an ~thereal solution of one of the preferred diazo compound~
to a solution of the 3~hydroxy ester in an inert solvent.
The eth~rification proceeds at a sati~factory rate at a temper-
ature between about 20 and 25C. An exce~ of the diazo com-
pound is desixably employedO
Representative of the 7 amino-3-alkoxy nucleus e~ers
which can be prepared and used in this invention are p-nitro-
benzyl 7-amino-3-methoxy-3-cephem-4-carboxylate, p-me~hoxy-
benzyl 7-ami~o-3-methoxy--3-cephem-4-carboxylate, p-nitrobenzyl
7-amino-3-ethoxy-3~cephem-~-carboxylate, 2,2,2-trichloroethyl
7-amino-3-methoxy-3-cephem-4-carboxylate, p-methoxybenzyl
7-amino-3-~3-methyl-2-but0nyl~1-oxy)-3-cephem~4-carboxyla~e
and p-ms~hoxybenzyl 7-amino~3-ethoxy-3-cephem-4-carboxylate.
The 7-amino-3-cephem ~ther acids or esters, prepar~d
X-3959 7
' '
~7~
as de~cribed above, are then acylated with an active deriva-
tive of a phenyl or het~rocyclic substituted glycine repre-
sented by the formula
H O
l 11
R-C-C-OH
N~12
wherein R is as def.in~d for Formula I. The acylation can be
caxried out by following the known methods for the acylation
of 7-aminocephalosporanic acid and 7-aminodesacetoxycephalo-
I sporanic acid. For example, the acid chloride hydrochloride
can be reacted with the 7-amino ether nucleus in the presence
of a hydrogen halide acceptor~ Hydrogen halide acceptors
such as the tertiary amines,pyridine and triethylamine can
be used, however the alkylene oxides such as ethylene oxide
and propylene oxide are preferred hydrogen halide acceptors
in that racemization o the active D-configuration of the
D-phenyl, D-thienyl or D-furyl glycyl halide does not occur
with these acceptors. Alternatively, the free acid of an
amino protected phenyl, thienyl or furyl substituted glycine
can be condensed with the amino ether nucleus in the presence
of a condensing agent such as a carbodiimide, for example,
dicyclohexylcarbodiimide. Also a mixed anhydride ormed with
the acylatLng acid and an acid such as formic or acetic acid
can be used a~ the acylation reagent in the presence of a con-
~ den~ing agent such as N-ethQxycarbonyl-2-ethoxy l,2-dihydro-
quinoline (EEDQ).
By other known acylation methods an activated deriva-
tive of the phenyl1 thienyl or furylglycines such as an active
e~ter, for exampl~, a pentachlorophenyl ester, or an a2ide
can he used. In general, any of the known amide coupling
.~959 8
~,~
.~rr~ '
~ ~ 7 ~ ~ ~
methods can be used in the preparation of the compounds of
the invention.
Illustrative o~ the phenyl and h~terocyclic substi-
tuted glycines which can be used to acylate ~he 7-amino ether
nucleus are the following: D-phenylglycyl chloride hydro-
chloride, D-4-hydroxyphenvlglycyl chloride hydrochloride,
2-thienylglycyl chloride hydrochloride, 3-thieny].glycyl chlo-
ride hydrochloride, 2-furylglycyl chloride hydrochloride,
N-~t-butyloxycarbonyl)-D-phenylglycine, N-(l-carbomethoxy-
2-propenyI)-D-phenyl~lycine, and ~- (t-butyloxycarbonyl3 -2-
thienylglycin~.
Other amino protecting groups such as the p-methoxy-
benæyloxycarbonyl, benzyloxycarbonyl and 2, 2, 2-trichloroethoxy-
carbonyl groups can be used to protect the amino group of the
: acylating agent. Any of the generally used amino protectin~
groups can be employed, the unction of such groups being
merely to protect the reactive amino functio~ during the
acylation reaction.
In a spacific emhodiment of ~his invention p-nitro-
~0 benzyl 7-amino-3-hydroxy-3-cephem-4-carboxylate hydro-
chloride i~ reac ed in methylene chloride with an ethereal
solution of diazome~hane to provide p-nitrobenzyl 7-amino-3-
methoxy-3-cephem-4-carboxylate~
The 7-amino-3-methoxy-3-cephem estex is then acylated
wi~h the mixed anhydride formed with N-(1 carbomethoxy-2-
propenyl)-D-phen~lglycine and methyl chloroormate ~n the
pxesence of dime~hy1benæylamine a~ about 25C. to provide
7-lN-(l-carbomethoxy-2-propenyl)-D-phenylglycylamido]-3-
~ me~hoxy-3-cephem-4-carboxylate. The ~mino protecting group
is then removed by acid hydroly~is and the p-nitrobenzyl ester
K-3959 g
group is removed by catalytic hydrogenolysis with hydrogen
in the presence of 5% palladium on carbon catalyst at p~ 2.5
to yield 7-(D-phenylglycylamido)-3-methoxy-3-cephem-4-
carboxylic acid ~Formula I, R = phen~ 1 = methyl and
R2 - 11 ) .
The antibiotics can be prepared by an alternative
process of the invention hy first acylating ~ 7-amino-3-
hydroxy-3-cephem ~ter and then reacting the 7-~phenyl,
thienyl or furylglycylamido)-3-hydroxy-3-cephem-4-carboxylic
acid product with one of the aforementioned diazo compounds.
The acylation of a 3-hydroxy nucleus ester can be carri~d
out by the methods previously described as useful for
the acylation of a 7 amino ether nucleus. }lowever, it is
preferred to acylate the 3-hydroxy nucleus ester under non-
anhydrous acylation methods. When an anhydrous method of
acylation is employed N,O-diacylation occurs to some extent
to provide a mixture of the desired N acylated product and
the N,O-diacylated product~ for example the diacylated product
represented by the following generalized formula
'~0 0
Il ~ S
R-CH-C-N ~ \ O
NHz ~ L ~ -O~C-CH~R
COOR2 NH2
wher~in R is as defined above and R2 is an ester group. Under
non-anhydrous conditions, for example, when the acylation is
carried out in w~t acetone, we~ ace~oni~rile or with mixtures
of water and wa~er immiscible organic solvPnts, N-acyla~ion
occur~ exclusively.
In a further 3peciic embodiment of this invention,
p--methoxybenzyl 7-a~ino-3-hydxoxy-3-cephem-4-~arboxylate is
X-3959 10
acylated with N-(t-butyloxycarbonyl)-D-phenylglycine in the
presence of EEDQ to provide p-methoxybenzyl 7-[N-~t-butyloxy-
carbonyl~-D-ph~nylglycylamido]-3-hydroxy-3-cephem-4-carboxy-
late. The acylated product is xeacted with diazomethane in
methylene chloride to provide the ether, p-methoxybenzyl
7-[N-tt-butyloxycarbonyl) D-phenylglycylamido]-3-methoxy-3-
cephem-4-carboxylate. The p-metho~ybenzyl group and the
t-butyloxycarbonyl amino protectin~ group are removed with
trifluoroacetic acid in anisole or p-toluenesulfonic acid in
acetonitrile to provide, 7-(D-phenylglycylamido)-3-me~hoxy-
3-cephem-4-carboxylic acid (Formula I, R = phenyl, R1 = methyl
and R2 = H).
Alternatively, the antibiotics can be prepared
by employing a 7-~phenyl, thienyl or furylglycylamido)-3-
exomethylenecepham-4-carboxylic acid ester as the starting
material. According to this method the ~-amino group
in the side chain i~ protected with a readily removable amino
blocking group for example, the t-butyloxycarbonyl group, the
l-carbomethoxy-2-propenyl group, and the 2,2,2-trichloroethoxy-
~0 carbonyl group, and the protscted compound is reacted with
ozone. The intermedi~te ozonide is decomposed to provide the
correspondingLy ~ubstituted 3-hydroxy-3-cephem ester. Alkyl-
ation of ~he 3-hydroxy 3-cephem ester and preferably alkyla-
tion with a diazo compound, or example diazomethane, yields
the 3-methoxy-~ethoxy or 3-methyl-2~butenyl-1 oxy)-3-cephem-
4-carboxylic acid ester. The carboxylic acid protectinq ester
group and the amino protectin~ group are removed ~y known
procedures to provide the antibiotic compound of the invention.
Fox example, p-nitrobenzyl 7-[N-(t-butyloxycarbonyl~-D~phenyl-
glycylamido]-3-exomethyl~necepham-4-carboxylate is reacted
K-3959 11
with oæone, the intermediate ozonide is decomposad with
sulfur dioxide to provide p-nitr~benzyl 7-~N-~t-butyloxy-
carbonyl)-D-phenylylycylamido~-3-hydroxy-3-ceph~m-4-car~oxy-
late. The 3-hydroxy e~ter is reacted with diazomethane to
provid~ the 3-methoxy derivative. The p-nitrobenzyl ester
group is removed from the 3~methoxy derivat$ve by react$ng
the ester with hydroyer~ in the presence of 5% palladium on
carbon in an acidic medium, ~nd thereafter the t-butyloxy-
carbon~l ~roup is removed by acid hydrolysis to provide the
anti~iotic 7-[D-phenylglycylamido)-3-methoxy-3-cephem-4-
carboxylic acid.
Following the same procedures the corresponding
thienyl and furyl substituted glycyl amides are prepared.
The following compounds are illustrative of the
antibiotic compounds of the invention.
7-(D-Phenyl~lycylamido)-3-methoxy-3-cephem~4-carboxylic acid,
7-(D-3-hydroxyphenylglycylamido)-3-methoxy-3-cephem-4-
carboxylic acid,
7-(D-4-hydroxyphenylgylcylamido)-3-methoxy-3-ceph2m-4-
carboxylic acid,
7-(D-2-hydroxyphenylglycylamido)-3-methoxy-3-cephem-4-
carboxylic acid,
7~D-4-m~hylph2nylglycylamido)-3-msthoxy-3-cephem-4-
carboxylic acid,
7-(D-4-chlorophenylglycylamido)-3-methoxy-3-cephem-4
carboxylic acid,
7-(D-3~m2thoxyphenylglycylamido~-3-methoxy-3-cephem-4-
carboxylic acid,
7-(D-phenylglycylamido)-3-e~hoxy-3-c~phem-4-car~oxylic
acid,
X-3~59 12
~7~
7-~D-phenylglycylamido)~3-(3-me~hyl-2-butenyl-1-oxy)-3-
cephem-4-carhoxylic acid,
7-(D-2-th.ienyl~lycylamido)-3-methoxy-3-cephem-4-carboxylic
acid,
7~(D--2-furyl~lycylamido)-3-methoxy-3-cephem-4--carboxylic
acid,
7-(D-3 thienylglycylamido~-3-methoxy-3-cephem-4-carboxylic
acid, and
7-~D-2~thienylglycylamido)-3~ethoxy-3-cephem-4-carboxylic
~ acid.
~ preferred group of an~ibiotics prepared by the
process Qf this invention are the 3-methoxy-3-cephem acids,
Formula I, Rl = methyl and R~ = H .
~mong the preferred 3-methoxy-3-cephem acids the
7-~D-hydroxyphenylglycylamido~-3-methoxy-3-cephe~-4-~arboxylic
acid~ ara a further pref~rred group cf antibiotics.
The antibiotics pro~ided by the ~roces~ of this
invention lnhibit the growth of microorganis~s pathogenic to
man and animals. These antibiotics are a particularly
u~2ful cla~s of antibiotics in that they are effective in
combating infections caused by gram-positive and gram-negativ~
microorgan~sms whe~ administered parenterally or orally.
~n especially preferred antibiotic compound
pr~pared by the process o this invention is 7-(D-phenyl-
glycylamido)~3~methoxy-3--cephem-4-carboxylic acid.
In Tabl2 I, which f~llows, the minimum inhibitory
concentration (MIC~ for this ~ompound against clinical i~olate3
of penicillin resi~tant S~ lococcus, both in the presence
and ab~ence of ~erum, are listed. The MIC values were deter-
mined by the Gradient Pl~te technique ~Bryson and Sz~balski,
_cience, 116, 45 (1952)._
E-395~ 13
~7~
able I _ _ -
7-~D-Phenylglycylamido)-3-methoxy-3-cephem-4-carboxylic acid
Antibiotic Activity vs. Penicillin Resistant Staphylococcus
Clinical I~olate Minimum Inhibitory Concentration
(mcg/ml)
~bsent Serum Serum
V- ~1 11.4 >20
V-- 32 1~.4 >20
X_~o01 >20 >20
V 84 4.4 11.2
~- 1.1 0.6 0.5
lethicillin res.istant ~E~h~oco cus.
In Table II below, the MIC values for 7-(D-phenyl-
glycylamido)~3-methoxy-3-cephem-4--carboxylic acid against
representative gram-ne~ative organisms is presented. The
data were obtained by the Gradient Plate technique.
Table II
Antibiotic Activity vs. Gram-Negativ~ Organisms
OrganismMinimum Inhibitory Concentration
2a (mcg/ml)
~ ~e~ 7.2
E~cher~ahia coli 6.6
KlebLiel1a pneumoniae 5.1
Aero~act~r a~ 3.6
.
Salmonella ~ 3.8
P~udom~nas ~ >200
Serratia marce~cen~ 124
___ __
The antimicrobial activity exhibited by 7-(D phenyl-
glycyl~mido)-3 methoxy-3-cephem-4-carboxylic acid is illus-
~30 tr~tive of the activity of the compounds prepared by th~
~-3~59 14
~37~
process of the inv~ntion.
A~ previously mentioned the highly preferred csm-
pound prepared by the process of this invention, 7-(D-phenyl-
glycylamido)-3-methoxy-3-cephem-4-carboxylic acid, is a highly
effective anti~iotic when administered orally. For example
the ED50 values listed bel~w were obtained with the above
named compound in mice infected with the listed micro-
organisms.
Microorganism ED50 (mg/kg. oral)
_taph~lococcus EX~Lenes 1.0
_ococcus E~e_ o iae 24.1
~ s a reus 5.2
Escherichia coli 12 2
The ED50 values wexe determined as de~cribed by
~ W. E. Wick et al., Journal of Bacteriology, 81 ~No. 2] 233-
; ~35 (1961).
The antibiotic compounds prepared by tha process
of this invention can be administered in the free acid form
or in ~he form of a pharmaceutically acceptable non toxic
salt such as the sodium or pota~sium salt. Such salts are
prepared by reacting the antibiotic acid with a sui~abl~
ba~e such a~ sodium carbonate, sodium bicarbonate, sodium
hydroxide, or potassium carbonate.
For example, the highl~ preferred antibiotic is
eff~ctive $rl combating infections when admini~tered orally
at a do~e between about 100 and 500 mg qOi.d. The antibiotic
ca~ be administered in a ~ui~able oral pharmaceu ical form
for example in gelatin capsule~.
The following examples ar~ provided to further illus-
trate the invention.
~-395~ 15
7~
Example 1
p-Nitrobenzyl 7-amino-3-hydroxy-3-cephem 4-carboxylate hydro-
chloride.
A solution of 3.85 ~. of p-nitrobenzyl 7-amino-3-
methylenecepham-4-car~oxylate hydrochloride in 600 ml. of
methanol was cooled i.n an acetone~dry ice bath. Ozone was
bubbled through the reaction mixture for approximately 20
minutes at which time the reaction mixture developed a
faint blue coloration. Nitrogen was then passed through
the reaction mixture to expel excess ozone. Next, the
intermediate ozonide was decomposed by passing sulfur dioxide
~as through the reaction mixture until the mixture gave a
negative potassium iodide-starch test.
The reaction mixture was evaporated in vacuo and
the residue was ~issolved in 200 ml. of 0.lN hydrogen chloride
in methylene chloride. The so~ution was evaporated to dryness
and the residual reaction product was dissolved in ace-tone.
On cooling, 3.15 ~. o p-nitrobenzyl 7-amino 3-hydroxy-3-
cephem-4-carboxylate hydrochloride precipitated as a crystal~
line solid.
I.R. ("Nujol"* Mull): -
Carbonyl absorption at
5.55 (~-lactam carbonyl) and
5.02 (ester carbonyl hydrogen bonded to 3
hydroxy~ microns.
Electrometric titr tion (66% DMF) pKa 4.0 and 6,3.
Example 2
p-~i~robenzyl 7-amino-3-hydroxy-3~cephem-4-carboxylate~
Four ~illimole o~ p-nitrobenzyl 7-amino-3-hydroxy-
3-cepheM-4-carboxylata hydrochloride (prepare~ as described
- 16 -
-3~9
* Trademar~ fsr a highly-refinea mineral oil. It is a colorl ss,
~odorless transparent oily liquid comprising a mixture of
~hydrocarbons~
in Example 1) was dissol~ed in water and ethyl acetate was
adde~ to the solution. The p~I of the slurry was adjusted
from pH 2.2 to pE~ 5 with lN sodium hydroxide. The ethyl
acetate layer was separated and was washed with water and
dried over ma~nesium sulfate. The dried ethyl acetate layer
was evaporated to dryness to yield 1.2 g. of p-nitroben2yl
7-amino-3-hydroxy-3-cephem-4 carboxylate as a crystalline
rasidue.
Elemental analysis for: C14EI13N306S:
Theory: C, 47.86; II, 3.73; N, 11.96
Found: C, 47.87; ~I, 4.00; N, 12.11
I.R. (Nujol Mull):
Carbonyl absorption at 5.6S (broad, ~-lactam and
ester) and 6.0 (amide) microns.
N.M.R. ~DMS0 d6):
signals at 6.63 (2d, 2H, C2~
5.31 (d, lH, C6H),
4.89 (d, lH, C7~
4.62 (s, 2H, ester CH2),
4.30 (broad s, 2H, 7 N-EI),
2.5-1.8 (m, 4~, aromatic ~i) and
1.2 (d, lH, C30~1) tau.
Example 3
-
p-Nitrobenzyl 7-amino-3-methoxy-3-cephem-4-carboxylate.
To a stirred suspension of 445 mg. of p-nitro~enzyl
7~amino-3 hydroxy-3-cephem 4-carboxylate hydrochloride (pre-
pared as described by ~xample 1) in 35 ml. of dry tetrahydro-
furan was added one equivalent of triethylamine followed ~y
10 ml. of an ethereal solution of diazomethane in excess.
A~ter 30 min. the solvent and excess diazomethane were evapo-
~-3959 17
.~ . . . . . . . .
~7~ 2
rated and the residue was dissolved in a mixture of water and
ethyl acetate. The organic layer was separated and was washed
with water and dried. The dried ethyl acetate solution was
evaporated to dryness to yield 310 mg. o~ p-nitrobenzyl
7-amino-3-methoxy-3-cephem-4-carboxylate. The product was
obtained crystalline by trituration wi~h diethyl ether.
Elemental analysis ~or C15lll$N3O6S:
Theory: C, 49.31; E~, 4.14; N, 11.~0
Found: C, 49.51; H, 4.40; ~1, 11.25
I.R. ("Nujol" Mull): absorption peaks at 2.99 (amide),
5.75 (broad, ~-lactam and ester carbonyl), and
5~98 (amide carbonyl) microns.
U.V. (ethanol~ a~sorption maximum 268 m~,
~-14,~00.
N.M.R. (DMSO d6): signals at 7.10 (broad s, 2H, C7NH2),
6.22 ~s, 2~1, C2H2)~ 6.20 (5, 3H, C3 methoxyl~, S.27
(d, 1~, C6H), 4.93 (d, 1~, C7H), 4.60 (s, 2H, ester
CH2), and 2.35-1.6 (q, 4H, aromatic H) tau.
Ex~ ~le 4
. ,
O p-Nitrobenzyl 7-amino-3-methoxy-3-cephem-4-carboxylate
hydrochloride.
To a stirred suspension of 445 mg. of p-nitrobenzyl
7-amino-3-hydroxy-3-cephem-4-carboxylate hydrochloride in 30
ml. of methylene chloride was added 131 mg. of mono-trimethyl-
'
silyi acetamide and the mixture was stirred at room tempera-
ture for 30 mina An e hereal solution of excess diazomethane
-
was added and after 20 min. the mixture was evaporated to
xemove solvent and excess diazomethane. The residue was
trQated with 1 ml. of methanol and then dissolved in an ethyl
8 a~e~ate-water mix~ure. The ethyl ace~ate layer wa~ separated,
Sg 18
Ç~
,
- ., . '' ~
~7~
washed with water and dried. Hydrogen chloride was passed
through the dried ethyl acetate l~yer to precipitate the
reaction product, p-nitrobenzy]. 7-amino-3-methoxy-3-cephem-
4-carboxylate hydrochloride.
~M.R. (DMSO d6): signals at 6.97 (broad s, 3H, NEI3 ),
' 2 H2)~ 6.23 ¦s, 3E-I, C3 methoxyl), 5 39
(d, l~1, C611), 5~05 (~, lH, C71I) and 2.5-1.92 (q, 4H,
aromatic ~I) tau.
Example 5
p-Ni~robenzyl 7-[N-(t-butyloxycarbonyl)-D-phenylglycylamido3-
3-methoxy~3-cephem 4-carboxylate.
A mixture o 365 mg. o~ p-nitrobenz~l 7-amino-3-
methoxy--3-cephem-4-carboxylate, 256 mg. of N-(t-butyloxycarb-
onyl)~-D-~-phenylglycine, and 273 mg. of N-ethoxycarbonyl-2-
ethoxy-l,2-dihydroquinoline, in 20 ml. of THF and lO ml. of
acetone was stirred at room temperature for 16 hours. The
reaction mixture was evaporated to remove volatiles and the
residue was dissolv~d in a mixture of water and eth~l ac.etate.
The ethyl acetate layer was separated and was washed with 5
hydrochloric acid and water and was dried. The dried layer
was then evaporated to dryness and the product obtained
crystalline by ~ri~uration of the residue with diethyl ether.
Elemental analysis or C2~H3~N4OgS
Theory: C, 56.I8; H, 5.05; N, 9.36
Found: C, 55.95; H, 5.l6i N, 9.30
I.R. (Nujol Mull): absorption peaks at 3.01 (amide NH~,
5.67, 5.84, 5.90 and 6.06 Icarbonyl~ microns.
~ N.M.R. (CDCl3~: signals at 8.60 (s, 9H, t-butyl),
: 6 75 (~, 2~, C2H~), 6.23 (s, 3H, C3 methQ~yl)/
5.20-3.90 (m, 5H, C6H, sid~-chain CH2, ester CH3
.30 and C7~), 2,80-1.70 (m, 9H, aromatic H) tauO
~-3~59 l9
Example_6
7-~D-Phenylglycylamido)~3-methoxy-3-cep~lem-4-carboxylic acid
A aolution of 600 mg. of p-nitrobenzyl 7-[N- (t-~u~tl-
oxycarbonyl) D-~-phenylglycylamido3~3-methoxy-3-cephem-4-
carboxylate ~prepared as described in Example 5) in 25 ml.
of THF and 6~ ml. o metha~ol containin~ 3 drops of iH hydro-
chloric acid was hydrogenated at room temperature under 50 psi
hydrogen pressure for 3 hr. in the presence of 600 mg. of
pre-reduced 5% palladium-on-carbon.
The catalyst was filtered and was washed with THF.
The filtrate and wash were combined and evaporated to dryness
in vacuo. The residue was dissolved in a mixture of water
and ethyl acetate and the ~olution was cooled in an ice-water
bath~ The pH of the cold solution was adjusted to pH 2.5 and
the ethyl acetate layer ~eparated. The ethyl acetate layer
was washed, dried and evaporated to dryness to yield 7-EN-(t-
butyloxycarbonyl)-D-~-phenylglycylamido]-3-methoxy-3-cephem-
4-carboxylic acid as a pale yellow amorphous solid.
The carboxylic acid product was dissolved in 5 ml.
of acetonitrile and 380 mg. of p-toluenesulfonie acid mono-
hydrate were added to the solution. The mixture was allowed
~o stand at roo~ temperature for 3 hr. Thereafter 1 ml. of
water was added to the mixture and the pH was adjusted to plI
4.5 with triethylamineO
On cooling, 7~(D-~-phenylglycylamido)~3-methoxy-3-
cephem-4-carboxylic acid precipitated as a crystalline solid
ha~ing ~pec ral propertie~ in agreement with those of the
produ~t obtained by Example 8~
~,
X-3959 20
:
~ '
' ' ~"-
~7~
Exam~e 7
p-Nitrobenzyl 7-[N~ carbomethoxy-2-propenyl)-D-phenylylycyl-
amid~-3-methoxy-3-cephem-4-carboxylate~
To 45 ml. of acetonitrile containing 6 drop~ of
dimethylbenzylamine were added 815 mg. of methyl 3-a-carboxy-
benzylaminocrotonate sodium sa].t and the mixture was cooled
in a dxy ice-carbon tetrachloride bath.
To the cold solution were added with stirring 303
mg. of methyl chloroformate and after 20 min. a solution of
1.1 g. of p-nitrobenzyl 7-amino-3-~ethoxy-3-cephem-4-carboxy-
late in 45 ml. of acetone was addedO The reaction mixture
wa~ stirred in the cold for 30 min. and then at room temper-
ature for 2 hr.
The reaction mixture was filtered and evaporated in
; vacuo. The residue was dissolved in ethyl acetate and the
solution was washed with water and dried over magnesium
sulfate. The dried solution was evaporated to dryness and
; the residue recrystallized from ethanol to yield 1.1 g. of
~ cxy~talline reaction product, p-nitrobenzyl 7-~N-(l-carbo-
methoxy-2 propenyl~-D phenylglycylamido]--3~methoxy-3-cephem-
4-caxboxylate, melting at about 135 to 145C. with decomposi-
: tion.
Elemental analysis or C~8H28N409S:
Theory: C, 5G.37; H, 4.73; N~ 9039
Foun~: C, 56.09; H, 4.57; N, 9027.
7~(D-a-Phenylglycylamido)~3-methoxy-3~cephem-4-carboxylic acid
A ~olution of 500 mg. ~f p-nitrobenzyl 7-[N~ carbo-
methoxy-2-prop2nyl)-D-phenylglycylamido~-3-me~hoxy-3-~ephem-
4-carboxylate (prepared as described by Example 7) in 30 ml.
~-39sg 21
of acetonitrile and 15 ml. of water was acidified to pH 1 with
concentrated hydrochloric acid and immediately back titrated
to pH 2.5 with lN sodium hydroxide. The mixture was evapo-
rated to dryness and th~ residue dlssolved in a solvent mix-
ture o~ 40 ml. of TIIF and 80 ml. of methanol.
The ~olution was charged into a Parr low pressure
hydrogenation apparatus and hydrogenated under a hydrogen
pressure of 50 psi at room temperature for 2.5 hr. in the
presence of 500 mg. of 5% palladium-on-carbon. The catalyst
~10 had be~n pre-reduced in ethanol for 30 min. under 50 psi
hydrogen pressure at room temperature.
The catalyst was filtered and washed with THF and
with water. The filtrate and catalyst wash~s were combin~d
and evaporated in vacuo to rem~ve volatile solvents. The
aqueou~ residue wa~ slurried with ethyl acetate and the pH
of the slurry was ad~usted to p~I 4.5 with lN sodium hydroxide.
The aqueous layer was separated, washed with ethyl ac~tate
and then concentrated in vacuo to a volume of 2 ml.
The aqueous concentrate was diluted with 1 mln of
2~ acetonitrile and the solution was cooled ~o precipi~ate 122
mg. of 7-~D-phenyl~lycylamido)-3-methoxy-3-cephem-4-carboxylic
acid as ~he crystallin~ dihydra~e.
EIemen~al analysis for C16H17N305S.2H20:
Theory: C, 48.10; H, 5.30, N, 10.52
Found: C, 47~80; ~, 4.74; N, 10.21.
Nujol Uull): ab~orptio~ peaks a~ 2.95 (amide NH),
5.75, 5.96 t~-lactam ~nd amide carbonyl~), a~d
6.25 ~c~rboxylate) microns.
N.M.R. ~D~0/DCl): signals at 6.58 (2d, 2H~ C~H2~, 6.10
~, 3H, C3 met~oxyl), 4.87 (d, lH, C6H~, 4.70 (s,
X-3959 ~2
lH, ~ -CH), 4.i4 (d, lH, C7H) and 2.41 (s, 5H,
aromatic H) tauO
U.~. (pH 7 buffer): ~ max 265 m~u, ~ = 7,500.
Electrometric titration (80% aqueous DMF):
pKa 6.2 and 7.3.
Example 9
Following the procedures described in Examples 7
and 8, p-nitrobenzyi 7-[N~(l-carbomethoxy-2-propenyl)-D-2-
thienylglycylamido]-3-methoxy-3~cephem-4-carboxylate is
prepared by reacting p-nitrobenzyl 7-amino-3-methoxy-3-cephem-
4-carboxylate with methyl 3-[[~2-thienyl3(carboxy)methyl]amino]-
crotonate sodi~m salt and methyl chloroformate, and the
l-carbomethoxy-2-propenyl amino protecting group is removed
by acid hydrolysis followed by the catalytic hydrogenolysis
of the p-nitrobenzyl ester group to provide 7-(D-2-thienyl-
glycylamido)-3-methoxy-3-cephem-4-carboxylic acid.
Example lO
Following the procedures described in Examples 7
and 8, p-nitrobenzyl 7-~N-(l-carbomethoxy-2-propenyl)-D-4-
hydroxyphenylglycylamido~-3-methoxy-3-cephem~4-carboxylate is
prepared by reaction of p-nitrobenzyl 7-amino-3-methoxy-3-
cephem-4-carboxylate with methyl 3- C~ -carboxy-4-hydroxybenzyl-
aminocrotonate sodium .salt and methyl chlorofor~ate. The
l-car~ome~hox~-2-propenyl amino protecting group and the
.
p-nitrobenzyl ester group are removed to provide 7-(D-4-
phenylglycylamido~-3-me~hoxy-3-cephem-4-carboxylic acid.
Example ll
p-Nitxokenzyl ~ (D-phenylglycylamido)-3-hydroxy-3-cephem-4-
carboxylate.
3~ To a solution of 446 mg. of p-nitrobenzyl 7-amino-
3-hydroxy-3-cephem-4-carboxylate in 20 ml. of acetonitrile
containing 10 ml. of propylene oxide was added 206 mg. of
X-395~ - 23 -
D phenylc31ycyl chloride hydrochloride. The re~ction mixture
was stirred for 16 hours at room temperature and was then
evaporated in vacuo. The residue was tritu~ated with ace-
tonitrile to remove soluble impurities. The residue was then
; ~ ' '
~ ~ I
: i
X~3959 - 23a -
: ~ : : .
, ; ' , i .
~76~2
dried in vacuo ~o yield 315 mg. of p-nitrobenzyl 7-(D-
phenylglycylamido)-3-hydroxy-3-cephem-4-carboxylate.
Elemental analysis for C22H20N~O7S:
Theory: C, 54.54; ~I, 4.16; N, 11.57
Found: C, 54.99; ~1, 4.29; N, 11.02
~ "
t ~ I.R. (Nujol Mull): absorption peaks at 3.01 (amicle),
5.75 ~-lactam carbonyl), and 6.10 (broad, amide,
and hydrogen bonded ester carbonyl) microns.
,. . .
"~
, ~ .
:
X-3g5g 24
, ,,;"
., .
