Note: Descriptions are shown in the official language in which they were submitted.
1067Q6r3
Tis application is a divisional of our copending
Patent Application Serial NoO 216,571 filed December 20J 19740
This invention is concerned with improvements in
or relating to cephalosporin compounds, and is more
particularly concerned with a cephalosporin compound possess-
ing valuable antibiotic propertiesO
The cephalosporin compounds in this specification
are named with reference to "cepham" after J.AmerOChemO
SocO, 1962, 84, 3400, the term "cephem" referring to the
basic cepham structure with one double bondO
Cephalosporin antibiotics are widely used in the
treatment of diseases caused by pathogenic bacteria in
; human beings and animals, for example in the treatment of
diseases caused by bacteria which are resistant to other
antibiotics such as penicillin compounds, and in the
: treatment of penicillin-sensitive patientsO In many in-
: stances it is desirable to employ a cephalosporin anti-
biotic which exhibits activity against both gram positive
and gram negative microorganisms, and a significant amount
of research has been directed to the development of various
types of broad spectrum cephalosporin antibioticsD
Considerable interest is currently being direct-
ed to the development of broad spectrum cephalosporin anti-
biotics which possess high activity against gram negative
i
- 2 -
.
1067068
organisms, Existing commercially available ~-lactam
antibiotics tend to exhibit comparatively low activity
against certain gram negative organisms such as Proteus
organisms, which are an increasingly common source of
infection in humans, and are also generally substantially
inactive against Pseudomonas organisms, Several Pseudomonas
organisms are resistant to the majority of existing
commercially available antibiotic compounds, and the
practical therapeutic applications of aminoglycoside
antibiotics such as gentamicin which do exhibit Pseudomonas
activity tend to be lim~ed or complicated by the high
. toxicity of these antibiotics, It is well known that
cephalosporin antibiotics normally exhibit low toxicity
I . in man, sothat the development of broad spectrum
cephalosporin antibiotics possessing high activity
against gram negative organisms such as strains of
Proteus and Pseudomonas fulfils a significant need
in chemotherapy,
The present invention provides a cephalosporin
antibiotic of the formula
.,'' ~
.
.
~067068
1~
H H
C.CO.NH
Il , ,o~ ~d~ CH20cONH2
~ .C.COOH COOH
:~>
. (I)
and non-toxic derivatives thereof, the compounds.
- being ~yn isome~ or existing as mixtures of syn
and anti isomers containing at least 9~/O of the syn
isomer,
5 The compoundsexhibit broad spectrum antibiotic
activity characterised by particularly high activity
against gram negative microorganisms., including those
which produce ~-lactamases, and also possess very
high stability to ~-lactamases produced by a range
. . 10 of gram negative organisms, A characteristic feature
of the compoundsistheir high in vitro activity against
gram-negative organisms such as Enterobacter cloacae,
Serratia marcesce~s and 1~5~ oUS_ es~
. .
. The compounds.have particularly high activity
.
_ ~
~.t i,
0 67 0 6 8
against strains of Escherichia coli,Haemophilus influenzae
and Prote;us organisms, e,g, strains of Proteus morganii
. ~
, . . .
and Proteus mirabilis, The compoundsh ve.also shown
`~ ~, unusually high.activity against Pseudomonas organisms, for
S~: example straing of Pseudomonas aeruginosa.
~.,, , ~ .
The compoundbof the~invention are defined as having
the syn lsomeric form as-regards the configuration of
: the group ~ . -
. ::
O.C,COOH ~ ~
with respect.to the carboxamido group,. In this specification
~,., ~. ~ . . : .
~the syn configuration ls denoted structurally as
. O .CONH- -
N
. .C C ON
. this configuration being as:signed on the basis of the
.
work of Ahmad and Spenser reported in Can,~ Ll
1961, 39, 1340, As indicated above, the compoundsmay
exist as mixtures of syn and anti isomers provided
. 15 that such mixtures contain at least 9~/0 of the syn
: - 5 -
.
.
1067068
isomer, We prefer, however, the compoundsto be svn
isomers essentially free from the corresponding anti
isomer,
By "non-toxic derivatives" is meant those
derivatives which are physiologically acceptable in the
dosage at which they are administered, Such derivatives
may include, for example, salts, biologically acceptable
esters, l-oxides and solvates (especially hydrates),
It will be appreciated that derivatives such as salts and
esters may be formed by reaction of either or both of the
carboxyl groups present in the compound of formula(I2.
Non-toxic salt derivatives which may be formed
from the compound of general formula(I)include inorganic
base salts such as alkali metal salts (e,g, sodium and
potassium salts) and alkaline earth metal salts (e.g.-
calcium salts), and organic base salts (e.g. procaine,
phenylethylbenzylamine, dibenzylethylenediamine,
ethanolamine, diethanolamine, triethanolamine and
N-methylglucosamine salts).
- 6-
~067068
The salts may also be in the form of resinates formed
with, for example, a polystyrene resin or cross-
linked polystyrene divinylbenzene copolymer resincontaining amino or quaternary amino groups. Use of highly
soluble base salts (e,g, alkali metal salts such as
the sodium salt) of the compound of formula(I)is
generally advantageous in therapeutic applications
because of the rapid distribution of such salts
in the body upon administrationO Where, however,
insoluble salts of the compound (I) are desired in a
~articular application, e g for use in depot preparations,
such salts may be formed in conventional manner, for
example with appropriate organic amines,
Biologically acceptable, metabolically labile
ester derivatives which may be formed fromthe compounds
of formula O include, for example, acyloxymethyl esters,
e g lower alkanoyloxymethyl esters such as acetoxymethyl
or pivaloyloxymethyl esters,
.
_ 7 _ .
1067~68
tnpounds
The e~Ywu*~ according to the invention may
be prepared by any convenient method, for example
by techniques analogous to those described in Canadian
Patent No, 19 027~554
Thus according to one embodiment of the invention
we provide a process for the preparation of the
antibiotic compound of formula (~ (as defined above~
or a non-toxic derivative thereof, said compound being
a ~y~ isomer or existing as a mixture of syn and anti
0 isomers containing at least 9~/0 of the syn isomer,
wherein (A) a compound of the formula
B
H2N ~/ ~
0 ~ \ ~ 1CH20CNH2
COOR
(II)
. - 8 -
.
1067068
Iwherein B is ~S or ~S-~ 0 (~- or ~ ); Rl represents
hydrogen or a carboxyl blocking group, e,g, the residue
of an ester-forming aliphatic or araliphatic alcohol
or an ester-forming phenol, silanol or stannanol (the
said alcohol, phenol, silanol or stannanol preferably
containing 1-20 carbon atoms) or a symmetrical or mixed
anhydride group derived from an appropriate acid; and
the dotted line bridging the 2-, 3- and 4-positions
indicates that the compound may be a ceph-2-em or
ceph-3-em compound~ or an acid addition salt such as
a hydrochloride, hydrobromide, sulphate, nitrate,
phosphate, methane sulphonate or p-toluenesulphonate or
an N-silylated derivative thereof, is condensed with an
acylating agent.corresponding to an acid of formula
~; ~L C . COOH
N
O. C. COOR2 ( I:EI)
' <>
~ '
:
10 670 6 8
(wherein R2 is a carboxyl blocking group, e,g, a
group as hereinbefore defined in connection with Rl );
or ~B) a compound of the formula
H H
. . ~ ~ C.CO.NH ~ B ~
O N . ~ ~ CH20H
.C.COORl COOR
<>
(IV)
(wherein B and the dotted line are as hereinbefore
defined; and each Rl may independently represent hydrogen
or a carboxyl blocking group) is reacted wlth a
carbamoylating agent serving to form a carbamoyloxymethyl
.
or.N-protected carbamoyloxymethy,l group at the 3-position;
whereafter, if necessary and/or desired in each instance,
l~ any of the following reactions (C) in any appropriate
sequence, are carried out:- '
i) conversion of a Q2 isomer into the desired ~3 isomer,
ii) reduction of a compound wherein B is ~S-~O to form
- a compound wherein B is ~S, and
.
- - 10 -
~067~)68
iii) removal of any carboxyl blocking and/or N
protecting groups; and finally (D) recovering
the desired compound of formula (I) or a non-toxic
derivative thereof, if necessary after separation
of isomers.
Non-toxic derivatives of the compounds of formula (I)
may be formed in any convenient way, for example according
to methods well known in the art. Thus, for example, sodiu~
and potassium salts may be formed by reaction of the
cephalosporin acid with sodium 2-ethylhexanoate or
potassium 2-ethylhexanoate, Biologically acceptable
ester derivatives may be formed using conventional
esterifying agents, l-Oxides may be formed by treatment
of the corresponding cephalosporin sulphide with an
appropriate oxidising agent, for example with a peracid
such as metaperiodic acid, peracetic acid, monoperphthalic
acid or m-chloroperbenzoic acid, or with t-butyl
hypochlorite, this last reagent conveniently being
employed in the presence of a weak base such as pyridine,
- 11 -
~ 67 0 ~ 8
Acylating agents which may be employed in the
preparation of the compound of formula(~ include acid
halides, particularly acid chlorides or bromides, Such
acylating agents may be prepared by reacting an acid
. (III3 or a salt thereof with a halogenating agent e,g,
phosphorus pentachloride, thionyl chloride or oxalyl
chloride, Treatment of the sodium, potassium or
triethylammonium salt of the acid (III) with oxalyl
chloride is advantageous in that under these conditions
isomerisation is minimal,
Acylations employing acid halides may be effected
in aqueous and non-aqueous reaction media, conveniently
at temperatures of from -50 to +50C, preferably -20
to +300C, if desired in the presence of an acid binding
agent, Suitable reaction media include aqueous ketones
- 12 -
.
:
1067068
such as aqueous acetone, esters such as ethyl acetate,
halogenated hydrocarbons such as methylene chloride,
amides such as dimethylacetamide, nitriles such as
. acetonitrile, or mixtures of two or more such solvents,
Suitable acid binding age~ include tertiary amines
(e,g, triethylamine or dimethylaniline), inorganic bases
(e,g, calcium carbonate or sodium bicarbonate), and
oxiranes such as lower 1,2-alkylene oxides (e,g, ethylene
oxide or propylene oxide) which bind hydrogen halide
liberated in the acylation reaction,
Acids of formula (III) may themselves be used as
acylating agents in the preparation of the compound of
formula.~4, Acylations employing acids (III) are
desirably conducted in the presence of a condensation
agent, for example a carbodiimide such as N,N'-diethyl-,
- dipropyl- or diisopropylcarbodiimide, N,N'-dicyclohexyl-
carbodiimide or N-ethyl-N'-y-dimethylaminopropylcarbo~
diimide; a carbonyl compound such as carbony~;imidazole;
or an isoxazolinium salt such as N-ethyl-5-phenylisoxaz-
olinium perchlorate, Acylation reactions of this type
are desirably effected in an anhydrous reaction medium,
. e,g, methylene chloride, dimethylformamide or acetontrile,
Acylation may also be effected with other amide-
- 13 -
.
67068
forming derivatives of acids of formula(lII)such as, for
example, a symmetrical anhydride or a mixed anhydride
- (e.g. with pivalic acid or formed with a haloformate
such as a lower alkylhaloformate). The mixed or
symmetrical anhydride may be generated in situ; thus~
for example, a mixed anhydride may be genera~ed using
N-ethoxycarbonyl-2-ethoxy-l, 2-dihydroquinoline~ Mixed
anhydrides may also be formed with phosphorus acids (for
example phosphoric or phosphorous acids), sulphuric acid
or aliphatic or aromatic sulphonic acids ~for example
e~e ~_Sc~ o~C
tolucne~ onlc acid).
Carbamoylation of 3-hydroxymethyl compounds (IV) may
be effected by conventional methods~ Thus, for example,
a 3-hydroxymethyl cephalosporin may be reacted with an
isocyanate of formula R .NC0 (wherein R represents a
labile substituent group) to give a compo~nd containing
a 3-position substituent having the formula -CH20.CONHRa
(wherein Ra has the above defined meaning). The labile N-
protecting substituent is subsequently cleaved~ e.g. by
hydrolysis, to form a 3-carbamoyloxymethyl group. Labile
groups R` which are readily cleavable upon subsequent
treatment include chlorosulphonyl and bromosulphonyl;
':
- 14 -
~067068
halogenated lower aIkanoyl groups such as dichloroacetyl
and trichloroacetyl; and halogenated lower alkoxycarbonyl
groups such as 2,2,2-trichloroethoxycarbonyl. These labile
Ra groups may generally be cleaved by a~id or base
catalysed hydrolysis (e.g. by base catalysed hydrolysis
using sodium bicarbonate).
Another carbamoylating agent of use in the
carbamoylation of 3-hydroxymethyl cephalosporins is cyanic
acid, which is conveniently generated in situ from, for
example~ an alkali metal cyanate such as sodium cyanate,
the reaction being facilitated by the presence of an acid~
e~g. a strong organic acid such as trifluoroacetic acid.
Cyanic acid effectively corresponds to a compound of
formula Ra~NCO wherein Ra is hydrogen, and therefore
converts 3-hydroxymethyl cephalosporin compounds directIy
to their 3-carbamoyloxymethyl analogues.
3-Hydroxymethyl cephalosporins (I~) for use in the
above carbamoylation reactions may~ for example, be
prepared by the methods described in British Patent No.
1,121,308 and Canadian Patents Nos~ 1,027,554 and 1,021,280.
- 15 -
G7068
~2-Cephalosporin ester derivatives obtained in
accordance with the process of the invention may be
converted into the corresponding ~3 derivative by, for
example, treatment of the ~2 ester with a base~
S Ceph-2-em reaction products may also ~e oxidised to
yield the corresponding ceph-3-em l-oxide~ for example by
reaction with a peracid as mentioned previously; the
resulting sulphoxide may, if desired, subsequently be
reduced as described hereinafter to yield the corresponding
ceph-3-em sulphide.
- Where a compound is obtained in which B is >S~ 0
this may be converted to the corresponding sulphide by~
for example, reduction of the corresponding acyloxy-
sulphonium or alkyloxysulphonium salt prepared in situ
by reaction with e.g~ acetyl chloride in the case of an
acetoxysulphonium salt, reduction being eff-ected by~ for
example, sodium dithionite or by iodide ion as in a
solution of potassium iodLde in a water miscible solvent
e.g. acetic acid, tetrahydrofuran, dioxan, dimethyl-
formamide ar dimethylacetamide. The reaction may be
. ' , ' .
~16 -
~.o6~068
effected at a temperature of -20 to +50C.
Where the compound ~ formula(I)is obtained as a
mixture of isomers, the syn isomer may be obtained by, for
example, conventional methods such as crystallisation or
chromatography. Syn and anti isomers may be distinguished
by appropriate techniques, e.g. by their ultraviolet
spectra, by thin layer or~ paper chromatography or by their
proton magnetic resonance spectra. Thus, for example5
the p m.r. spectra of DMS0-d6 solutions of syn compounds
of formula(I)exhibit the doublet for the amide NH at a
lower field than do similar solutions of the corresponding
anti-isomers. These factors may be employed in monitoring
reactions.
Starting materials of formula (III) may be prepared
- 15 in an analogous manner to that described in our copending
Application Serial No. 216571~ for example~ as described
in the Preparation belowO
Carboxyl blocking groups R and, where appropriate~
R used in the preparation of the compound of ~rmula
(I)or in the preparation of necessary starting materials
are desirably groups which may readily be split off at a
suitable stage in the reaction sequence, conveniently as
the last stage. It may, however, be convenient in some
~ 17 -
1067068
instances to employ biologically acceptable, metabolically
labile carboxyl blocking groups such as acyloxymethyl
groups (e.g. pivaloyloxymethyl) and retain these in the
final product to give a biologically acceptable ester
derivative of the compound of formula~I).
Suitable carboxyl blocking groups are well known in
the art, a list of representative blocked carboxyl groups
being included in Canadian Patent No~ 1,027,554. Preferred
blocked carboxyl groups include aryl lower alkoxycarbonyl
groups such as ~-methoxybenzyloxycarbonyl~ ~-nitro-
benzyloxycarbonyl and diphenylmethoxycarbonyl; lower
alkoxycarbonyl groups such as t-butoxycarbonyl; and lower
haloalkoxycarbonyl groups such as 2,2,2-trichloroethoxy-
carbonyl.~ The carboxyl blocking group may subsequently
be removed by any of the appropriate methods disclosed
in the literature; thus~ for example, acid or base
catalysed hydrolysis is applicable in many cases, as are
enzymically-catalysed hydrolyses.
The antibiotic compounds of the invention~ i.e. the
compound of form~la(I)and non-toxic derivatives thereof,
may be formulated for administration in any convenient
way, by analogy wLth other antibiotics and the invention
therefore includes within its scope pharmaceutical
- 18 -
.
~,o67~6~
compositions comprising an antibiotic compound in
accordance with the invention adapted for use in human or
veterinary medicine. Such compositions may be presented
for use in conventional manner with the aid of any
necessary pharmaceutical carriers or excipients~
The antibiotic compounds according to the invention
may be formulated for injection and may be presented in
unit dose form in ampoules, or in multi-dose containers with
an added preservative. The compositions may take such
forms as suspensions, solutions~ or emulsions in oily or
aqueous vehicles, and may contain formulatory agents
such as suspending, stabilising and/or dispersing agents.
Alternatively the active ingredient may be in powder form
for reconstitution with a suitable vehicle, e.g. sterile,
pyrogen-free water, before use.
The antibiotic compounds may also be presented in a
form suitable for absorption by the gastro-inte~tinal tract.
Tablets and capsules for oral administration may be in unit
dose presentation form, and may contain conventional
excipients such as binding agents~ for example syrup, acacia,
gelatin, sorbitol, tragacanth or polyvinylpyrrolidone; fillers
for example lactose~ sugar,
- 19 ~
~067068
maize-starch, calcium phosphate, sorbitol or glycine;
lubricants, for example magnesium stearate, talc,
polyethylene glycol or silica; disintegrants, for
example potato starch; or acceptable wetting agents such
as sodium lauryl sulphate~ The tables may be coated
according to methods well known in the art. O~al liquid
preparatio~ may be in the form of~ for example, aqueous
or oily suspensions, solutions~ emulsions, syrups or
elixirs, or may be presented as a dry product for reconstitutior
with water or other suitable ve~icle before use. Such
liquid preparatio~ may contain conventional additives
such as suspending agents, for example sorbitol syrup,
methyl cellulose, glucose/sugar syrup, gelatin, hydroxy-
ethylcellulose, carboxymethyl cellulose, aluminium
sterate gel or hydrogenated edible fats; emulsifying
agents, for example lecithin~ sorbitan mono-oleate or
acacia9 non-aqueous vehicles (which may include edible
oils), for example almond oil, fractionated coconut oil,
oily esters~ propylene glycol or ethyl alcohol; and
preservatives~ for example methyl or propyl p-hydroxy-
benzoates or sorbic acid. The antibiotic compounds may
also be formulated as suppositories,e.g. containing
conventional suppository bases such as cocoa butter
- 20 -
.
106'7068
or other glyceride~
~7 e~,c,~e
Compositions for veterinary mcdici may, for
example~ be formulated as intrammary preparations in
either long acting or quick-release bases.
The compositions may contain from 0.1% upwards~ e.g~
0.1-99%, preferably from 10-60% of the active material,
depending on the method of administration. When the
compositions comprise dosage units, each unit will
preferably contain 50-1500mg of the active ingredient.
The dosage as employed for adult human treatment will
preferably range from 500 to 5000 mg per day, depending
on the route and frequency of administration, although
in treating Pseudomonas infections higher daily doses
may ~e required.
The antibiotic compounds according to the invention
may be administered in combination with other therapeutic
agents such as antibiotics, for example penicillins,
tetracyclines or other cephalosporins.
The following examples iIlustrate the invention. All
temperatures are in C. The structure of the products
U)~S
-wer~ verified by p.m.r. spectroscopy and i.r. spectroscopy.
_ 21 -
11~67068
PREPARATION
2-(1-t-ButoxYcarbonYlcyclobut-l-oxyimino~-2-(fur-2-yl~
acetic acid (sYn-isomer)
A solution of 2-(fur-2-yl)-2-hydroxyimino acetic acid
S (syn-isomer) (7.05g) in dimethylsulphoxide (lOOml) was
added dropwise, over 30 minutes, to a stirred solution of
potassium t-butoxide (11.2g) in dimethylsulphoxide (200ml)
under an atmosphere of dry nitrogen. Stirring was con-
tinued for a further 30 minutes and then a solution of
t-butyl l-bromocyclobutanecarboxylate (12.1g~ in dimethyl-
sulphoxide (25ml) was added over 30 minutes to the reaction
mixture at room temperature. After addition was complete
the resulting solution was stirred for a further hour at
650. The reaction mixture was poured on to ice and
acidified to pH 1.5 under ether. The two layers were
separated and the aqueous layer was q~racted with more
ether. The combined ether extracts were washed once with
water, then extracted with aqueous sodium bicarbonate
~ solution. The combined alkaline extracts were acidified
under methylene chloride to pH 1.6 with concentrated
hydrochloric acid and the acid solution was extracted
further with methylene chloride. The combined organic
extracts were washed (water and saturated brine), dried
- and concentrated to give a solid which was crystallised
from carbon tetrachloride to ~ive the title compound
m.p. 113-114C and ~ max (pH6 buffer) 278nm (~17~200).
.
- 22
106'706B
Example
(6R.7R)-3-carbamoyloxymethYl-7-c2-(l-carboxycyclobut
l-oxyimino)-2-(fur-2-Yl)-acetamido]ceE~h-3-em-4-
carbox~lic acid (svn-isomer)
A solution of 2-(1-t-butoxycarbonylcyclobut-1-oxyimino)-
2-(fur-2-yl) acetic acid (syn isomer) (0.93g) in
methylene chloride containing a few drops of N,N-
dimethylformamide and triethylamine (0.75ml) was
treated with oxalyl chloride (0.30ml) at 0-5 for ca.
1 hour. The mixture was then evaporated to dryness.
The residue was suspended or dissolved in 25ml acetone
and added to a stirred, ice-cold solution of (6R,7R)-
7-amino-3-carbamoyloxymethyl-ceph-3-em-4-carboxylic acid
(0.90g) in a mixture-of acetone and water containing
sodium hydrogen carbonate (0.83g). The reaction mixture
was stirred for 45 minutes, allowing the temperature to
rise to room temperature, whereafter the acetone was
- removed under reduced pressure.
.
- 23 -
~C~67068
The pH was adjusted to 1.5 - 2~0 and the product
extracted into ethyl acetate (alternatively ether or
methylene chloride may b~ used). The organic layer was
washed with water and/or saturated brine) dried and evaporated
to give (6R~7R)-3-carbamoyloxymethyl-7-~2-(~buto~ycarbonyl-
cyclobut-l-oxyimino)~-(fur-~2-yl)-acetamido~ceph-3-em-4-
carboxylic acid (sYn-isomer~ which was characterised by
optical rotation and/or by spectroscopy.
The t-butyl ester ~1.13g) was deprotected by treating with
trifluoracetic acid containing anisole at room temperature
for at least 5 minutes. The reaction mixture was evaporated
in vacuo and the product isolated by tri~uration or by
.
distributing between ethyl acetate (or ether) and an
aqueous solution of sodium hydrogen carbonate~ separating
the aqueous extracts, acidifying these extracts under
ethyl acetate and isolating the title dicarboxylic acid,(0.99g)
Ca~26(DMSO); ~ max (pH 6 phosphate buffer) 277.5 nm
(~15~100)~ ~ max (N-yol- Trade Mark) 1788 cm 1; ~ (d6-DMSO)
values include 0.34 (NH)j 4.12 (7-H~ and 7.56 ànd 8.10
(cyclobutyl protons).
.
- 24 -
