Note: Descriptions are shown in the official language in which they were submitted.
~11;~941~8
This invention is concerned with cephalosporin
compounds possessing valuable antibiotic properties,
The cephalosporin compounds in this specifica-
tion are named with reference to "cepham" after
5 J, Amer, Chem, Soc,, 1962, 84, 3400, the term "cephem"
referring to the basic cepham structure with one double
bond,
. Cephalosporin antibiotics are widely used in the
treatment of diseases caused by pathogenic bacteria in
10 human beings and animals, and are especially useful 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
patients, In many instances it is desirable to employ a
15 cephalosporin antibiotic 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 cephalo-
sporin antibiotics,
Thus, for example, in our British Patent Specifica-
tion No, 1,399,086, we describe a novel class of cephalo-
sporin antibiotics containing a 7~-(a-etherified oximino)-
acylamido group, the oximino group having the syn
~1~94~
-- 2
configuration. This class of ant;biotic compounds is
characterised by high antibacterial activity against
a range of gram-positive and gram-negative organisms
coupled with particularly high stability to ~-lactamases
produced by various gram-negative organisms.
The discovery of this class of compounds has
stimulated further research in the same area in attempts
to find compounds which have improved properties, for
example against particular classes of organisms
especially gram-negative organisms.
In our British Patent Specification No. 1,496,757,
we describe cephalosporin antibiotics containing a
7~-acylamido group of the formula
R.C.C0 NH-
A
O.(CH2) C (CH2) COOH (A)
RB
(wherein R is a thienyl or furyl group; R and RB may
vary widely and may,for example,be Cl 4 alkyl groups
or together with the carbon atom to whicll they are
attached form a C3 7 cycloalkylidene group, an(l m and
n are each 0 or 1 such that the sum of m and n is 0
or 1), the compounds being _~ isomers or mixtures of
syn and anti isomers containing at least 90% of the
syn isomer. The 3-position of the cephalosporin
molecule may be unsubstituted or may contain one of
a wide variety of possible substituents. These compounds
have been found to have particularly good activity
against gram-negative organisms.
~lZ94~
- 3
Other compounds of similar structure have been
developed from these compounds in further attempts to
find antibiotics having improved broad spectrum anti-
biotic activity and/or high activity against gram-
5 negative organisms. Such developments have involvedvariations in not only the 7~-acylamido group in the
above formula but also the introduction of particular
groups in the 3-position of the cephalosporin molecule.
Thus, for example, in Belgian Patent Specification
10 No. 852,427, there are described cephalosporin antibiotic
compolmds falling within the general scope of our British
Patent Specification No. 1,399,086, and wherein the group
R in formula (A) above may be replaced by a variety of
different organic groups, including 2-aminothiazol-4-yl,
and the oxygen atom in the oxyimino group is attached to
an aliphatic hydrocarbon group which may itself be
substituted by, for example, carboxy. In such compounds, the
substituent at the 3-position may vary widely and may be inter
alia an optiona]ly substituted heterocyclic-thiomethyl ~roup.
20 Many examples of such groups are given in the specifica-
tion including those in which the heterocyclic moiety
of the group is a 3- to 8- membered heterocyclic ring
containing 1 to 4 nitrogen atoms, e,g, an imidazolyl,
pyrazolyl, pyridyl, pyrimidyl or tetrazolyl group which
may be substituted, e g. a 1-methyl-1~l-tetrazol-5-yl
group.
Furthermore, Belgian Patent Specification No.
836,813 describes cephalosporin compounds wherein the
group R in formula (A) above may be replaced by, for
example, 2-aminothiazol-4-yl, and the oxyimino group
11~94,~
-- 4
is a hydroxyimino or blocked hydroxyimino group, e.g. a
methoxyimino group. In such compounds, the 3-position of
the cephalosporin molecule is substituted by a methyl
group which may itself be optionally substituted by any
of a large number of residues of nucleophilic compounds
therein described. Examples of such residues include
the mercapto group ~hich may be attached to a 5- or
6-membered heterocyclic ring which may contain l to 4
heteroatoms selected from oxygen, sulphur and nitrogen,
e.g. pyridyl, pyrimidyl, pyrazolyl, or imidazolyl, which
rings may, if desired, be substituted for example by
lower alkyl groups, In the above mentioned Specifica-
tion no antibiotic activity is ascribed to such
compounds which are only mentioned as intermediates for
the preparation of antibiotics described in that speci-
fication,
Belgian Patent Specification No,853,545 describes
cephalosporin antibiotics wherein the 7~-acylamido side
chain is primarily a 2-(2-aminothiazol-4-yl)
-2-(~y~)-methoxyimino-acetamido group and the substituent
in the 3-position is broadly clefint~d ;n a si!nilar ~a~l~
to that in the above-mentioned Belgian Patent Specifica-
tion No, 836,813, Compounds specifically exemplified in
the SpeciEication include compounds ln which the
3-position is substituted by various heterocyclic-thio
methyl radicals including methyltetrazolylthiomethyl
radicals
We have now discovered that by an appropriate
selection of a small number of particular groups at the
94~8
7~-position in co~bination with a heterocyclic-substituted thiomethyl group at
the 3-position, cephalosporin compounds having particularly advantageous
activity (described in more detail below) against a wide range of co nly en-
countered pathogenic organisms may be obtained.
The prese~t invention provides cephalosporin antibiotics of the
general form~la
NH2
/~ H H
S N l I
\~ C. CO.NH I ~ S (I)
N Ra o N ~ CH2S~3
O. C. COOH ~od3
Rb
(wherein Ra and R , which may be the same or different, each represent a Cl 4
alkyl group (preferably a straight chain alkyl group, i.e. a methyl, ethyl,
0 n-propyl or n-butyl group and particularly a methyl or ethyl group) or Ra and
together with the carbon atom to which they are attached form a C3 7 cyclo-
alkylidene group, preferably a C3 5 cycloalkylidene group; and ~ represents a
C-linked 5- or 6- membered heterocyclic ring containing at least one Cl 4 aLkyl
(e.g. methyl)-substituted qua~ernary nitrogen atom, which ring may also contain
one or more sulphur atoms) and non-toxic salts and non-toxic metabolically
labile esters thereof.
The oompounds according to the invention are syn isomers. Ihe syn
isomeric form is defined by the configuration of the group
Ra
O. C. COOH
R
94~
with respect to the carboxamido group. In this specification the syn configura-
tion is denoted structurally as
NH2
,~
\ - / C.CO.NH
N R
O.C.OOOH
Rb
It will ke understoad that sin oe the compounds according to the inven-
tion are geometric isomers, some admixture with the corresponding anti is~rer
may occur.
The invention also includes within its scope the solvates (especially
the hydrates) of the co~pounds of formula (I). It also includes within its
scope salts of esters of compounds of formula (I).
The compounds according to the present invention may exist in tauto-
meric forms (for example in respect of the 2-aminothiazolyl group) and it will
be understood that such tautomeric forms, e.g. the 2-iminothiæolinyl
1~ ~ 9 ~ ~
form, are included within the scope of the invention
The compounds according to the present invention may
also exist in various zwitterionic forms In such
zwitterionic forms, for example, either the carboxyl
group in the 4-position or the carboxyl group in the
7-side chain may be deprotonated, These zwitterionic
forms, and mixtures thereof, are included within the
scope of the present invention
It will also be appreciated that when R and R
in the above formula represent different Cl 4 alkyl
groups, the carbon atom to which they are attached
will comprise a centre of asymmetry. Such compounds
are diastereoisomeric and the present invention
embraces individual diastereoisomers of these compounds
as well as mixtures thereof,
In formula (I) above, the heterocyclic ring
of the group Y~ may for example contain 1 to 4 nitrogen
atoms and, if desired, one sulphur atom, particular
examples of these heterocyclic rings i.ncluding imidazolyl,
pyrazolyl, pyridyl, pyrimidyl, pyrazinyl, pyridazinyl,
triazolyl, tetr~zolyl, thiazolyl, thiadiazolyl, and
thiazolidi.nyl,
The compounds according to the invention exhibit
broad spectrum antibiotic activity Against gram-
llZ9~¢~8
negative organisms the activity is unusually high.This high activity extends to many ~-lactamase-
producing gram-negative strains. The compounds
also possess high stability to ~-lactamases produced
by a range of gram-negative organisms.
Compounds according to the invention have
been found to exhibit unusually high activity
against strains of Pseudomonas organisms, e,g~ strains
of Pseudomonas aeruginosa as well as high activity
against various members of the Enterobacteriaceae
(e,g. strains of Escherichia coli, ~lebsiella
pneumoniae, Salmonella typhimurium, Shigella sonnei,
Enterobacter cloacae, Serratia marcescens Providence
species, Proteus mirabilis and especially indole-positive
15 Proteus organisms such as Proteus vul~aris and Proteus
__ .
morganii),and strains of Haemophilus influenzae.
1 1 ~ 9 ~
The antibiotic properties of the compounds
according to the invention compare very favourably with
those of the aminoglycosides snch as amikacin or
gentamicin In particu].ar, this app]ies to their
activity against strains of various Pse.udomoll~s
organisms whi.ch are not susceptable to the majority of exist-
ing commercially available antibiotic compoullds.
Unlike the aminoglycosi.des, cephalosporin antibiotics
normally exh;.bit l.ow toxi.city in man The use of amino-
glycosides in l-amAn therapy tends to be l;.mited or
COlllp] icated by tlle hi.ghtoxi.c;ty Of thcse alltibiol:ics.
The cephalospor;.n anti.b;otics of the prc.sellt invenL:ion
thus posscss potent;.ally gre~t advalltages over the
aminogl.ycosi.des~
~on-toxi.c sa].t deri.vaLivcs whi.ch nlay be rorrned
by reaction of ei.ther or both of the carboxyl groups
present in the compounds o~ gelleral ~orlllula (I) i.llclude
i.norgani.c base .salts such as alka]i metal salts (~.g.
sod;um and potass;uln salts) an(l alkal;.lle eArth metal.
salts (e.g. c~lc;uln snlts); nm;llo ~c;d sllts (e ~. lys;ne
and arg:i.n~ salls); org-lllic 1)aSe Sa1tS (e.r" I~IOC.1;11e~
1129~8
- 10
phenylethyl-benzylamine, dibenzylethylenediamine,
ethanolamine, diethanolamine and N-methylglucosamine
salts). Other non-toxic salt derivatives include
acid addition salts, e.g. formed with hydrochloric,
hydrobromic, sulphuric, nitric, phosphoric, formic and
trifluoroacetic acids. The salts may also be in the
form of resinates formed with, for example, a polystyrene
resin or cross-linked polystyrene divinylbenzene
copolymer resin containing amino or quarternary amino
groups or sulphonic acid groups, or with a resin
containing carboxyl groups, e.g. a polyacrylic acid
resin. Soluble base salts (e.g. alkali metal salts
such as the sodium salt) of compounds of formula (I)
may be used in therapeutic applications because of the
15 rapid distribution of such salts in the body upon
administration. Where, however, insoluble salts of
compounds (I) are desired in a particular application,
e,g. for use in depot preparations, such salts may be
formed in conventional manner, for example with
20 appropriate organic amines.
These and other salt derivatives such as the
salts with toluene-p-sulphonic and methanesulphonic
acids may be employed as intermediates in the prepara-
tion and/or purification of the present compounds of
25 formula (I), for example in the processes described
below.
Non-toxic metabolically labile ester derivatives
which may be formed by esterification of either or
both carboxyL groups in the parent compound of formula (I)
11294~E~
include acyloxyalkyl esters, e.g, lower alkanoyl-
oxy-methyl or -ethyl esters such as acetoxy-methyl or -ethyl
or pivaloyloxy-methyl esters, In addition to the above ester
derivatives, the present invention includes within its
scope compounds of formula (I) in the form of other
physiologically acceptable equivalents, i.e. physio-
logically acceptable compounds which, like the meta-
bolically labile esters, are converted in vivo into
the parent antibiotic compound of formula (I).
Examples of preferred compounds according to
the present invention include those compounds of
formula (I) wherein Y represents an aromatic hetero-
cyclic ring containing 1 to 4 nitrogen heteroatoms
(without any sulphur heteroatoms), particularly a
ring containing 1 or 2 nitrogen heteroatoms, rings
containing only one nitrogen heteroatom being especially
preferred, ~xamples of such Y groups include l-methyl-
pyridinium, l-methylpyrimidinium and l,2-dimethylpyrazolium.
Thus a preerred group of compouncls accorc3ing
to the invention by virtue of their h;gh antibiotic
activity are tho~se o~ the general formula:
N1~2 (ia)
~ 11 1
S
\ / C.CO,NE~ ~)J, ~ -CEl2 S
O,C,COOH (~ 3 N
Rb
CH3
11294~3
_ 12
(wherein R and R have the above-defined meanings) and
their non-toxic salts and non-toxic metabolically labile
esters.
In formula (Ia) R and R preferably each represent
5 a methyl group or together with the carbon atom to which
they are attached form a cyclobutylidene group.
An outstanding compound of formula (Ia)
is (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1-carboxy-
cyclobut-l-oxyimino) acetamido~-3-[(l-methylpyridinium-
10 4-yl)-thiomethyl~ ceph-3-em-4-carboxylate which has the
formula
NH2
11 (Ib)
. ! c CO.NH - o~ N~-CH2 ~ +'
~) ~ COOH COO
to~ether wlth its non-toxic salts (e,g. sodium sal~)
and non-toxic metabolically labile esters. The compound
of formula (Ib) possesses to an outstanding extent the
lS general antibiotic properties set out above for the
compounds of general ormula (I). I-~owever one may
emphasise its excellent activity against strains o~ Pseudomona9.
The compound also possesses useful activity against strains o
Staphy-]-o-coccus aureus. The compound has excellent
_ _
2~ antibacterial properties wh;ch are not impaired by
human serum, and, moreover, the effect of increased
inocula against the compound is low. The compound is
rapidly bactericidal at concentrations close to the
~z9~
min~n~n inhibitory concentration. It is well distributed in the bodies of small
rodents giving useful therapeutic levels after subcutaneous injection. In pri-
mates the co~pound gives high and long lasting serum levels after intramuscular
injection. The serun half-life in primates points to the prob~bilitv of a comr
paratively long half-life in man, with the possibility of less frequent dosages
being required for less serious infections. Experimental infections in mice
with gramrnegative bacteria were successfully treated using the co~pound and, in
particular, excallent protection was obtained against strains of Pseudomonas
aeruginosa, an organism normally not susceptible to treatment with oe phalosporin
antibiotics. The protection was comparable with the treatment with an amino-
glycoside such as amikacin. A~ute toxicity tests with the compound in mioe gave
ID50 values in exoe ss of 1.0 g/kg. No nephrotoxicity was observed in rats at
dosages of 2.0 g/kg.
Another compound possessing not dissimilar properties to the compound
of formula (Ib) is (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(2-carboxyprop-2-
oxyimino)aoetarnido]-3-[(1-methylpyridinium~4-yl)-thiamethyl] oeph-3-em~4-
carboxylate together with its non-toxic salts ~e.g. the sodium salt) and non-
toxic metabolically labile esters.
other examples of preferred compounds according to the present inven-
tion include the following compounds of formula (I) and their non-toxic salts
and non-toxic metabolically labile esters, namely:-
(6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(2-carboxyprop-2-oxyimino) acetamido]
-3-[(1-methylpyridinium-2-yl)-thio~n~thyl~ oe ph-3-em-4-carboxylate;
- 13 -
- 14 -
(6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1-carboxycyclo-
but-l-oxyimino) acetamido~-3-[(1-methylpyridinium-2-yl)
-thiomethyl~ ceph-3-em-4-carboxylate;
(6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(2-carboxyprop-2-
oxyimino) acetamido~-3-[(1-methylpyridinium-3-yl)
thiomethyl~ceph-3-em-4-carboxylate;
(6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(2-carboxyprop-2-
oxyimino) acetamido~-3-[(1,2-dimethylpyrazolium-3-yl)
thiomethyl~ceph-3-em-4-carboxylate;
(6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(2-carboxyprop-2-
oxyimino) acetamido~-3-[(1,3-dimethylimidazolium-2-yl)-
thiomethyl~ ceph-3-em-4-carboxylate; and
(6R97R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(2-carboxyprop-2-
oxyimino) acetamidoJ-3-[(1-methylpyrimidinium-2-yl)thio-
methyl~ceph-3-em-4-carboxylate.
Other compounds according to the present invention include
for example those wherein the groups R , R and Y in
formula (I) are as follows:-
99~
R ~ Rb
a) Alkyl groups
- CH3 C2 5 1-methylpyridinium-2-yl
- C2H5 "
- CH3 ~ C2H5 1-methylpyridinium-3-yl
C 2H5
- CH3 ~ C2H5 1-methylpyridinium-4-yl
C 2 5 "
- CH3 ~ C2H5 1-methyl-pyrimidini.um-2-yl !
C 2H5
- CH3 - CH3 1-methyl-pyrimidinium-4-yl
- CH3 C2 5
C2 5
CH3 ~ C2H5 1,2-dimethyl-pyr~zolium-3-y~
- C2~15 " "
- CH3 - C~H5 1,3-d;methyl-imidazolium-
~ C21-15 " ll
._ . . . . _ . . . . I
- 16 -
_ .............................................. _
Ra--C--Rb Y
_ .
b) Cycloalkylidene groups
_
cyclopropylidene l-methylpyridinium-2-yl
cyclopentylidene .. .
cyclohexylidene .,
cyclopropylidene l-methylpyridinium-3 yl
; cyclobutylidene ..
¦ cyclopentylidene ..
¦ cyclohexylidene ..
cyclopropylidene l-methylpyridinium-4-yl
cyc].opentylidene ..
cyclohexylidene ..
cyclopropylidene l-methyl-pyrimidinium-2-yl
cyclobutylidene ..
cyclopentylidene .-
cyclopropylidene l,2-dimethyl-pyrazolium-3-y
cyclobutylidene ..
cyclopentylidene ..
cyclopropy].idene l,3-dimethyl-imidazolium-2-
cyclobutylidene .-
cyclopenty]idene ..
.. ~ ... .. .. ... .. . . .. .. ... .. .. .... ~ ... . .
94
- 17
The compounds of formula (I) may be used for
treating a variety of diseases caused by pathogenic
bacteria in human beings and animals, such as respiratory
tract infections and urinary tract infections.
According to another embodiment of the invention
we provide a process for the preparation of an antibiotic
compound of general formula (I) as hereinbefore defined
or a non-toxic salt (including internal salt) or non-toxic
.metabolically labile ester thereof which comprises (A)
acylating a compound of the formula
11 '!
H2N , ., B
J - N. ~ -CH2SY~A~
~ooRl (II)
~wherein Y~is as defined above; 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-form;.ng phenol, silanol
or stannanol. (the said al.coho]., phenol., s:i.lanol or
stannanol preferably containing 1-20 carbon atoms);
the dotted line bridging the 2-, 3-, and 4-positions
indi.cates that the compound is a ceph-2-em or ceph-3-em
compound. and A is an anion such as a halide, e g chloride
or bromide, or trifluoroacetate anion~ or a salt, e g an acid
addition salt (formed with, for example, a mineral acid
such as hydrochloricj hydrobromic, sulphuric, nitric or
pkosphoric acid or an organic acid such as methanesulphonic
or toluene-~-sulphonic acid) or an N-silyl derivative
thereof, or alternatively a corresponding compound having a
1129~8
- 18 -
group of formula -COO at the 4-position, with an acid of
formula
R3
.~
S N
C.COOH
N R
\ O.C.COOR (III~
Rb
(~her.ein R and Rb arc as hereinbcfore defined; R2
represents a carboxyl blocking group, c.g. as described
for R ; and R is an amino or protected amino group) or
with an acylating agent corresponding thereto; (B) reacting
a compound of formula
R
~ 11 1~
S ~ ;
\ / C ~o.~ ~ B
~1 Ra J ~ 'L(`l, x
\ O,C COOR ~ooR4
Rb (IV)
(whcrcill Ra, Rb, R3, ~ and thc dottcd linc are as deEincd
above; R4 and R4 may indcpendcntly rcprescnt hydrogcn or
a carboxyl blocking group; and X is a replaceable residue
of a nucleophile, e.g. an acetoxy or dichloroacetoxy group
or a halogen atom such as chlorine, bromine or iodine) or
a salt thereof with a sulphur nucleophile serving to form.
a group of formula -CH2SY4 (wherein Y~ is as defined
above) at the 3-position; or (C) reacting a compound
of formula
1129~
19
C.CO.NE~ , j~ B ~
N Ra o,~ N~ CH2 SY
. ( (,)OR4a CoOR4
Ib (V)
(wherein R , R , R , B and the dotted line are as defined
above; R4 and R4 in this instance are both carboxyl
blocking groups; and Y represents a C-linked 5- or 6-
membered heterocyclic ring containing a tertiary
nitrogen atom) with a Cl 4 alkylating agent serving to
introduce a Cl 4 alkyl group as a substituent on the
said tertiary nitrogen atom in the heterocyclic ring of
the group Y ; whereafter, if necessary and/or desired in
each instance, any of the following reactions, in any
appropriate sequence, are carried out:-
i) conversion of a A -isomer into the desired ~ -
isomer,
ii) reduction of a compound wherein B is ~S > O to
form a compound wherein B is >S,
iii) conversion of a carboxyl group into a non-toxic
salt or non-toxic metabolically labile ester
function, and
iv) removal of any carboxylblocking and/or
N-protecting groups.
In the above-described process (A~, the starting
material of formula (II) is preferably a compound wherein
B is ~S and the dotted line represents a ceph-3-em
llZ~4G~8
- 20 -
compound
Acylating agents which may be employed in the
preparation of compounds of formula (I) include acid
halides, particularly acid chlorides or bromides. Such
acylating agents may be prepared by reacting an acid (III~
or a salt thereof with a halogenating agent e.g.
phosphorus pentachloride, thionyl chloride or oxalyl
chloride.
Acy]ations employing acid halides may be effected
in aqueous and non-aqueous reaction media, conveniently
at temperatures of from -50 to +50C, preferably -20 to
+30C, if desired in the presence of an acid binding
agent. Suitable reaction media include aqueous ketones
such as aqueous acetone, esters such as ethyl acetate,
ha]ogenated hydrocarbons such as methylene chloride,
amides such as dimethylacetamide, nitriles such as
acetonitrile, or mixtures of two or more such soivents.
Suitable acid binding agents 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 compounds of
li294~8
21
formula (I). Acylations employing acids (III) are
desirably conducted in the presence of a condensing agent,
for example a carbodiimide such as N,N'-dicyclohexyl-
carbodiimide or N-ethyl-N'-y-dimethylaminopropylcarbodiimide;
a carbonyl compound such as carbonyldiimidazole; or an
isoxazolium salt such as N-ethyl-5-phenylisoxazolium
perchlorate.
Acylation may also be effected with other amide-
forming derivatives of acids of formula (III) such as, for
example, an activated ester, a symmetrical anhydride or a
mixed anhydride (e.g. formed with pivalic acid or with a
haloformate, such as a lower alkylhaloformate). 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
toluene-p-sulphonic acid). An activated ester may
convenient~y be formed in situ using, for example,
l-hydroxybenzotriazole in the presence of a condensing
agent as set out above. Alternatively, the activated
ester may be preformed.
Acylation reactions involving the free acids or
their above-mentioned amide-forming derivatives are
desirably efected in an anhydrous reaction medium, e.g.
methylene chloride, tetrahydrofuran, dimethylformamide or
acetonitrile.
If desired, the above acylation reactions may be
carried out in the presence of a catalyst such as 4-
dimethylaminopyridine.
The acids of formula (III) and acylating agents
corresponding thereto may, if desired, be prepared and
employed in the form of their acid addition salts. Thus,
for example, acid chlorides may conveniently be employed
~1294~3
- 22
as their hydrochloride salts, and acid bromides as their
hydrobromide salts.
In process (B) above, the sulphur nucleophile
may be used to displace a wide variety of substituents X
from the cephalosporin of formula (IV). To some extent
the facility of the displacement is related to the pKa of
the acid HX from which the substituent is derived. Thus,
atoms or groups X derived from strong acids tend, in
general, to be more easily displaced than atoms or groups
derived from weaker acids. The facility of the
displacement is also related, to some extent, to the
precise character of the sulphur nucleophile. The latter
nucleophile may be employed for example in the form of
an appropriate thiol or thione.
The displacement of X by the sulphur nucleophile
may conveniently be effected by maintaining the reactants
in solution or suspension. The reaction is advantageously
effected using from 1 to 10 moles of the nucleophile.
Nucleophilic displacement reactions may
conveniently be carried out on those compounds of
formula (IV) wherein the substituent X is a halogen
atom or an acyloxy group, for example as discussed below.
Acyloxy ~roups
Compounds of formula (IV) wherein X is an acetoxy
group are convenient starting materials for use in the
nucleophilic displacement reaction with the sulphur
nucleophile. Alternative starting materials in this class
include compounds of formula (IV) in which X is the residue
of a substituted acetic acid e.g. chloroacetic acid,
dich]oroacetic acid and trifluoroacetic acid.
Displacement reactions on compounds (IV) possessing
X substituents of this class, particularly in the case where
~lZg4~8
- 23
X is an acetoxy group, may be facilitated by the
presence in the reaction medium of iodide or thiocyanate
ions.
The substituent X may also be derived from formic
acid, a haloformic acid such as chloroformic acid,or a
carbamic acid.
When using a compound of formula (IV) in which
X represents an acetoxy or substituted acetoxy group, it
is generally desirable that the group R4 in formula (IV)
should be a hydrogen atom and that B should represent ~S.
In this case, the reaction is advantageously effected in
an aqueous medium.
Under aqueous conditions, the pH value of the
reaction solution is advantageously maintained in the
range 6-8, if necessary by the addition of a base. The `
base is conveniently an alkali metal or alkaline earth
metal hydroxide or bicarbonate such as sodium hydroxide
or sodium bicarbonate.
When using compounds of formula (IV) in which X
is an acetoxy group, the reaction is conveniently effected
at a temperature oE 30C to llOC, preerably 50 to 80C.
Halo~ens
Compounds oE formula (IV) in which X is a chlorine,
bromine or iodine atom can also be convenlently used as
starting materials in the nucleophilic displacement
reaction with the sulphur nuce]ophile. When ~Ising
compounds of formula (IV) in this class, B may represent
~ S and R4may represent a carboxyl blocking group. The
reaction is conveniently effected in a non-aqueous
medium which preferably comprises one or more organic
solvents, advantageously of a polar nature such as
ethers, e.g. dioxan or tetrahydrofuran, esters, e.g.
~:~294~l~
- 24
ethyl acetate, amides, e.g. formamide and N,N-dimethyl-
formamide, and ketones e.g. acetone. Other suitable
organic solvents are described in more detail in British
Patent Specification No. 1,326,531. The reaction medium
should be neither extremely acidic nor extremely basic.
In the case of reactions carried out on compounds
of formula (IV) in which R4 and R4 are carboxyl blocking
groups and the resulting Y group contains a quaternary
nitrogen atom, the product will be formed as the
corresponding halide salt which may, if desired, be
subjected to one or more ion exchange reactions to
obtain a salt having the desired anion.
When using compounds of formula (IV) in which X
is a halogen atom as described above, the reaction is
conveniently effected at a temperature of -20 to +60,
preferably 0 to +30C.
~ len the incoming nucleophile does not yield a com-
pound containing a quaternised nitrogen atom, the reaction
is generally effected in the presence of an acid
scavenging agent for example a base such as triethylamine
or calcium carbonate.
In process (C) above, the compound of formula (V)
is advantageously reacted with an alkylating agent of the
formula R~Z wherein R5 is a Cl 4 alkyl gro~lp and Z is a
leaving group such as a halogen atom (e.g. iodine, chlorine
or bromine~ or a hydrocarbylsulphonate (e.g. mesylate or
tosylate) group. Alternatively, a di-Cl 4-alkyl sulphate,
e.g. dimethyl sulphate, may be employed as the alkylating
agent. Iodomethane is preferred as the alkylating agent.
The alkylation reaction is preferably carried out at a
temperature in the range of O to 60C, advantageously 20
~ ..
il294~8
to 30-C. Where the alkylating agent is liquid under the
reaction conditions, as in the case of iodomethane, this
agent can itself serve as a solvent. Alternatively, the
reaction may be conveniently effected in an inert solvent
such as an ether e.g. tetrahydrofuran, an amide, e.g.
dimethylformamide, a lower alkanol, e.g. ethanol, a lower
dialkylketone, e.g. acetone,a halogenated hydrocarbon
e.g. dichloromethane or an ester e.g. ethyl acetate.
The compound of formula (V) used as starting
material in process (C) may be prepared for example by
reaction of a compound of formula (IV) (as defined above)
with an appropriate sulphur nucleophile in an analogous
manner to the nucleophilic displacement reaction
described with respect to process (B). If desired the
above nucleophile may be used in the form of a metal
thiolate salt.
The reaction product may be separated from the
reaction mixture, which may contain, for example,
unchanged cephalosporin starting material and other
substances, by a variety of processes including
recrystallisation, ionophoresis, column chromatography
and use of ion-exchangers (for example by chromatography
on ion-exchange resins) or macroreticular resins.
~ -Cephalosporin ester derivatives obtained in
accordance with the process of the invention may be
converted into the corresponding Q -derivative by, for
example, treatment of the ~ -ester with a base, such as
pyridine or triethylamine.
A ceph-2-em reaction product may also be oxidised
to yield the corresponding ceph-3-em l-oxide, for example
by reaction with a peracid, e.g. peracetic or m-
chloroperbenzoic acid; the resulting sulphoxide may, if
~lZ94
~6
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
acyloxysulphonium or alkoxysulphonium salt prepared
in situ by reaction with e.g. acetyl chloride in the
case of an acetoxysulphonium salt, reduction being
effected by, for example, sodium dithionite or by iodide
ion as in a solution of potassium iodide in a water-
miscible solvent e.g. acetic acid, acetone,
tetrahydrofuran, dioxan, dimethylformamide or
dimethylacetamide. The reaction may be effected at a
temperature of from -20 to +50C.
Metabolically labile ester derivatives of the
compounds of formula (I) may be prepared by reacting a
compound of formula (I) or a salt or protected
derivative thereof with the appropriate esterifying agent
such as an acyloxyalkyl halide (e.g. iodide)
conveniently in an inert organic solvent such as
dimethylformamicle or acetone, fol1Owed,~here necessary9 by
removal of any protecting ~roups.
Base salts of the compound of formula (I) may be
formed by reactin~ an acid of formula (I) with an
appropriate base. Thus, for example, sodium or
potassium salts may be prepared using the respective
2-ethylhexanoate or hydrogen carbonate salt. Acid
addition salts may be prepared by reacting a compound
of formula (I) or a metabolically labile ester
derivative thereof with the appropriate acid.
Where a compound of formula (I) is obtained as
a mixture of isomers, the ~y_ isomer may be obtained by,
1~294~3
- 27
for example, conventional methods such as crystallisation
or chromatography.
For use as starting materials for the preparation
of compounds of general formula (I) according to the
invention, compounds of general formula (III) and acid
halides and anhydrides corresponding thereto in their syn
isomeric form or in the form of mixtures of the syn
isomers and the corresponding anti isomers containing at
least 90% of the ~_ isorner are preferably used.
Acids of formula (III) (provided that R and R
together with the carbon atom to which they are attached
do not form a cyclopropylidene group) may be prepared by
etherification of a compound of formula
R3
,J~
(VI)
\O~l
(wherein R is as hereinbefore defined and R represents a
carboxyl blocking group), by reaction with a compound of
general formu.la
Ra
T.~.COOR2
Rb (VII)
(wherein R and R and R are as hereinbefore defined
and T is halogen such as chloro, bromo or iodo;
sulphate; or sulphonate such as tosylate), followed by
removal of the carboxy]. blocking group R . Separation
of isomers may be effected either before or after such
2 9
28
etherification. The etherification reaction is generally
carried out in the presence of a base, e.g. potassium
carbonate or sodium hydride, and is preferably
conducted in an organic solvent, for example
dimethylsulphoxide, a cyclic ether such as
tetrahydrofuran or dioxan, or an N,N-disubstituted amide
such as dimethylformamide. Under these conditions the
configuration of the oxyimino group is substantially
unchanged by the etherification reaction. The reaction
should be effected in the presence of a base if an acid
addition salt of a compound of formula (VI) is used.
The base should be used in sufficient quantity to
neutralise rapidly the acid in question.
Acids of general formula (III) may also be
prepared by reaction of a compound of formula
R3
S~
\ - l CO.COOR (VIII)
(wherein R and R are as hereinbefore defined) with a
compound of formula
lRa 2
l~2N.O.C.COOR
Ib ~IX)
(wherein Ra, Rb and R2 are as defined above), followed by
removal of the carboxyl blocking group R , and where
necessary by the separation of s~n and anti isomers.
The last mentioned reaction is particularly
applicable to the preparation of acids of formula (III)
wherein Ra and Rb together with the carbon atom to which
1129~8
- 29
they are attached form a cyclopropylidene group. In this
case, the relevant compounds of formula (IX) may be
prepared in conventional manner, e.g. by means of the
synthesis described in Belgian Patent Specification No.
866,422 for the preparation of t-butyl l-amino-oxycyclo-
propane carboxylate.
The acids of formula (III) may be converted to the
corresponding acid halides and anhydrides and acid
addition salts by conventional methods, for example as
described hereinabove.
Where X is a halogen (i.e. chlorine, bromine or
iodine) atom in formula (IV), ceph-3-em starting
compounds may be prepared in conventional manner, e.g. by
halogenation of a 7~-protected amino-3-methylceph-3-em-4-
carboxylic acid ester l~-oxide, removal of the 7~-
protecting group, acylation of the resulting 7~-amino
compound to form the desired 7~-acylamido group, e.g.
in an analogous manner to process (A) above, followed
by reduction o the l~-oxide group later in the sequence.
This is described in British Patent No. 1,326,531. The
corresponding ceph-2-em compounds may be prepared by the
method of Dutch published Patent Application No.
G,902,013 by reaction of a 3-methylceph-2-em compound
with N-bromosuccinimide to yield the corresponding 3-
bromomethylceph-2-em-compound.
Where X in formula (IV) is an acetoxy group, such
starting materials may be prepared for example by
acylation of 7-aminocephalosporanic acid, e.g. in an
analogous manner to process (A) above. Compounds of
formula (IV) in which X represents other acyloxy groups
can be prepared by acylation of the corresponding 3-
llZ~8
30 -
hydroxymethyl compounds which may be prepared for example
by hydrolysis of the appropriate 3-acetoxymethyl compounds,
e.g. as described for example in British Patent Specifications
Nos. 1,474,519 and 1,531,212.
Compounds of formula(II) may also be prepared in con-
ventional manner, e.g. by nucleophilic displacement of a
corresponding 3-acyloxymethyl or 3-halomethyl compound with
the appropriate nucleophile, e.g. as described in British Patents
Nos.1,012,943 and 1,241,657.
A further method for the preparation of the starting
materials of formula (II) comprises deprotecting a corresponding
protected 7~-amino compound in conventional manner, e.g.
using PC15 A new compound of formula (II) is 7-amino-3-
C(l-methylpyridinium-4-yl)thiomethyl~ceph-3-em-4-carboxylate
It should be appreciated that in some of the above
transformati~ns it may be necessary to protect any sensitive
groups in the molecule of the compound in question to avoid
undesirable side reactions. ~or example, during any of the
reaction sequences referred to above it may be necessary to
protect the NH2 group of the aminothiazolyl moiety, for example
by tritylation, acylation (e.g. ch]oroacetylation), protonation
or other conventional method. The protecting group may there-
after be removed in any convenient way which does not cause
breakdown of the desired compound, e.g. in the case of a
trityl group by using an optionally halogenated carboxylic
acid, e.g. acetic acid, Eormic acid, chloroacetic acid or
trifluoroacetic acid or using a mineral acid, e.g. hydrochloric
acid or mixtures of such acids, preferably in the presence of
a protic solvent such as water, or, in the case of a chloro-
acetyl group, by treatment with thiourea.
Carboxyl blocking groups used in the preparation ofcompounds of formula (I) or in the preparation of
necessary starting materials are desirably groups which
11 ~ 9 ~`8
may readily be split off at a suitable stage in the
reaction sequence, conveniently at the last stage. It
may, however, be convenient in some instances to einploy
non-toxic metabolically labile carboxyl blocking groups
such as acyloxy-methyl or -ethyl groups (e.g.
acetoxy methyl or-ethyl or pivaloyloxymethyl) and retain
these in the final product to give an appropriate ester
derivative of a compound of formula (I).
Suitable carboxyl blocking groups are well known
in the art, a list of representative blocked carboxyl
groups being included in British Patent No. 1,399,086.
Preferred blocked carboxyl groups include aryl lower
alkoxycarbonyl groups such as p-methoxybenzyloxycarbonyl,
p-nitrobenzyloxycarbonyl and diphenylmethoxycarbonyl;
lower alkoxycarbonyl groups such as t-butoxycarbonyl; and
lower haloalkoxycarbonyl groups such as 2,2,2-
trich]oroethoxycarbonyl. Carboxyl blocking group(s) 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 may be
formulated ~or administration in any convenient way, by
analogy with other antibiotics and the invention therefore
includes within its scope pharmaceutical 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
llZ94@8
- 32
may be formulated for injection and may be presented in
~mit dose form in ampoules, or in multi-dose containers,
if necessary with an added preservative. The
compositions may also 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.
If desired7 such powder formulations may contain
an appropriate non-toxic base in order to improve the
water-solubility of the active ingredient and/or to
ensure that when the powder is reconstituted with water,
the pH of the resulting aqueous formulation is
physiologically acceptable. Alternatively the base may
be present in the water with which the powder is
reconstituted. The base may be, for example, an
inorganic base such as sodium carbonate, sodium
bicarbonate or sodium acetate, or an organic base such as
lysine or lysine acetate.
The antibiot;c compounds may also be formulatecl as
suppositorles, e.g. containing conventional suppository
bases s~lch as cocoa butter or other g]ycerides.
Compositions for veterinary medicine may, for
example, be formulated as intramammary preparations in
either long acting or quick-release bases.
The compositions may contain from 0.1% upwards, e.g.
0.1-99% of the active material, depending on the method
of administration. When the compositions comprise
dosage units, each unit will preferably contain
llZ94~P8
50-1500 mg of the active ingredient. The dosage as
employed for adult human treatment will preferably
range from 500 to 6000 mg per day, depending on the
route and frequency of administration. For example,
in adult human treatment 1000 to 3000 mg per day
administered intravenously or intramuscularly will
normally suffice. In treating Pseudomonas infections
higher daily doses may be required.
The antibiotic compounds according to the
inventlon may be administered in combination with other
therapeutic agents such as antibiotics, for example
penicillins or other cephalosporins.
The following Examples illustrate the invention.
All temperatures are in C. ~Petrol~ means petroleum
ether (b.p. 40-60).
Proton magnetic resonance (p.m.r.) spectra were
determined at 100 ~z. The integrals are in agreement
with the assignments, coupling constants, J, are in Hz,
the signs not being determined; s = singlet, d = doublet,
dd = double doublet, m = multiplet, q = quartet and
ABq = AB ~uartet.
llZ94
- 34-
Preparation 1
Ethyl (Z~-2-(2-aminothiazol-4-yl)-2-(hydroxyimino)acetate
To a stirred and ice-cooled solution of ethyl
aceto~cetate (292 g) in glacial acetic acid (296 ml) was
added a solution of sodium nitrite (180 g) in water
(400 ml) at such a rate that the reaction temperature was
maintained below 10C. Stirring and cooling were continued
for about 30 min., when a solution of potassium chloride
(160 g) in water (800 ml) was added. The resulting
mixture was stirred for one hour. The lower oily phase
was separated and the aqueous phasewas extracted with diethyl
ether. The extract was combined with the oil, washed
successively with water and saturated brine, dried, and
evaporated. The residual oil, which solidified on
standing, was washed with petrol and dried in vacuo over
potassium hydroxide, giving ethyl (Z)-2-(hydroxyimino)-
3-oxobutyrate (309 g).
A stirred and ice-cooled solution of ethyl
(Z)-2-(hydroxyimino)-3-oxobutyrate (150 g) in
dichloromethane (400 ml) was treated dropwise with
sulphuryl chloride (140 g). The resulting solution was
kept at room temperature for 3 days, then evaporated.
The residue was dissolved in diethyl ether, washed with
~ater until thewashings were almost neutral, dried, and
evaporated. The residual oil (177 g) was dissolved
in ethanol (500 ml) and dimethylaniline (77 ml) and
thiourea (42 g) were added with stirring. After two hours,
the ~roduct was collected by filtration, washed with
ethanol and dried to give the title compound (73 g);
m.p.188 (decomp.).
1~2~8
Preparation 2
Ethyl ~Z~-2-hydroxyimino-2-(2-tritylaminothiazol-4-yl)-
acetate, h~drochloride,
Trityl chloride (16.75 g) was added portionwise
over 2 hours to a stirred and cooled (-30) solution of the
product of Preparation 1 (12.91 g) in dimethylformamide
(28 ml) containing triethylamine (8.4 ml). The mixture
was allowed to warm to 15 over one hour, stirred for a
further 2 hours and then partitioned between water (500 ml)
and ethyl acetate (S00 ml). The organic phase was
separated, washed with water (2 x 500 ml) and then shaken
with lN HCl (500 ml). The precipitate was collected,
washed successively with water (100 ml), ethyl acetate
(200 ml) and ether (200 ml) and dried in vacuo to provide
the title compound as a white solid (16.4 g); m.p. 184 to
186 (decomp).
Preparation 3
Ethyl (~-2-~2-t-butoxycarbonylprop-2-oxyimino)-2-(2-trityl-
aminothiazol-4-yl)acetate
Potassium carbonate (34.6 g) and t-butyl
2-bromo-2-methylpropionate (24.5 g) in dimethylsulphoxide
(25 ml) were added to a stirred solution under nitrogen
of the product of Preparation 2 (49.4 g) in
dimethylsulphoxide (200 ml) and the mixture was stirred at
room temperature or 6 hours. The mixture was poured into
water (2 1), stirred for 10 mins., and filtered. The solid
was washed with water and dissolved in ethyl acetate
(600 ml). The solution was washed successively with
water, 2N hydrochloric acid, water, and saturated brine,
dried, and evaporated. The residue was recrystallised
from petroleum ether (b.p 60-80) to give the title
llZ9
- 36
compound (34 g), m.p. 123.5 to 125
Preparation 4
(Z)-2-(2-t-Butoxycarbonylprop-2-oxyimino)-2-(2-tritylamino-
thiazol-4-~l)acetic acid
The product of Preparation 3 (2 g) was
dissolved in methanol (20 ml) and 2N sodium hydroxide
(3.3 ml) was added. The mixture was refluxed for 1.5
hours and then concentrated, The residue was taken up
in a mixture of water ~50 ml), 2N hydrochloric acid
(7 ml), and ethyl acetate (50 ml). The organic phase
was separated, and the aqueous phase extracted with
ethyl acetate, The organic solutions were combined,
washed successively with water and saturated brine,
dried, and evaporated, The residue was recrystallised
from a mixture of carbon tetrachloride and petrol to
give the title compound (1 g), m.p. 152 to 156 (decomp).
Preparation 5
Ethyl (Z)-2-(2-tritylaminothiazol-4-yl)-2-(1-t-butoxy-
carbonylcyclobut-l-oxyimino) acetate.
The product of Preparation 2 (55.8 g) was
stirred under nitrogen in dimethylsulphoxide (400 ml)
with potassium carbonate (finely ground, 31.2 g) at room
temperature. After 30 minutes, t-butyl l-bromocyclobutane
carboxylate (29.2 g) was added. After 8 hours urther
potassium carbonate (31.2 g) was added. More potassium
carbonate (6 x 16 g portions) was added during the next
three days and further t-butyl l-bromocyclobutane
carboxylate (3.45 g) was added after 3 days. After 4
days in all, the mixture was poured into ice-water (ca.
3 litres) and the solid was collected by filtration and
112g4
- 37 -
washed well with water and petrol. The solid was
dissolved in ethyl acetate and the solution washed with
brine (twice), dried with magnesium sulphate and
evaporated to a foam. This foam was dissolved in ethyl
acetate-petrol(1:2) and filtered through silica gel (500 g).
Evaporation gave the title compound (60 g) as a foam,
V (CHBr3) 3400 (NH) and 1730 cm 1 (ester),
Preparation 6
(Z)-2-(1-t-Butoxycarbonvlcyclobut-l-ox~,imino)-2-(2-trityl-
aminothiazol-4-yl) acetic acidO
A mixture of the product of Preparation 5
(3,2g) and potassium carbonate (1,65 g) was refluxed in
methanol (180 ml) and water (20 ml) for 9 hours and the
mixture was cooled to room temperatureO The mixture was
concentrated and the residue partitioned between ethyl
acetate and water, to which was added 2N HCl (12.2 ml)~
The organic phase was separated and the a~ueous phase
extracted with ethyl acetate, The combined organic
extracts were washed with ~aturated brine, dried and
evaporated to give the title compound (2,3 g); ~max
(ethanol) 265 nm (ElCm 243),
Preparation 7
Diphenylm ~ ~-
butoxycarbonylprop-2-oxyimino)-2-(2-tritvlarninothiazol-
4-yl)acetamido~ceph-3-em-4-carboxy].ate, l-Oxide
Diphenylmethyl (lS.6R,7R)-7-amino-3-bromomethyl-
ceph-3-em-4-carboxylate, l-oxide hydrobromide (1.40 g) was
shaken with dichloromethane (100 ml) and half-saturated
aqueous sod;um bicarbonate solution (lOO ml). The
~lZ94~8
- 38 -
filtered organic layer ~ras dried over anhyclrous sodium sulphate
and evaporated to give the free 7-amine as a white solid (l.05 g).
The solid (1.015 g) and the product of Preparation 4 (1.23 g)
were dissolved in N,N-dimethylformamide (21 ml) and to the
5 solution was added l-hydroxybenzotriazole monohydrate (394 mg)
and N,N'-dicyclohexylcarbodiimide (530 mg). The solution,
which soon developed a precipitate,was stirred at 20 for 18
hours. and was then filtered, and the residue was washed with
ethyl acetate (2 x 50 ml). The combined filtrate and washings
lO were washed with 2N-hydrochloric acid (2 x lOO ml) and water
(2 x 100 ml). The organic layer was dried over anhydrous
sodium sulphate and evaporated to a brown foam (2.645 g) which
was triturated with ether:petrol - l:l (50 ml) to give a solid
(2.3!~;). The solid was partly purified on a column of silica
gel (70 to 230 mesh, 50 g) eluted with chloroform in 75 ml
fractions. Eractions 3 and 4 yieLded a foam (1.085 g) which
was purified by preparative thin-layer chromatography in ethyl
acetate:to]uene = 1:3 to give a product (Rf ca. 0.5) as a foarn
(494 mg). A solution of the product in a lit:tle ethyl acetate
20 was added to stirred petrol and the precipitate was filtered
ofi~ and dried in vacuo to give the title compouncl (424 mg)
s a solid, [c~ + 6~ (ClIC1.3, c 0.53 ); AmaX ( O
(El/ 188)~ Aj ~ 237.5 nm (Elcm 249), Ainf lcm
inf ( ~cm
l~LZ9~8
- 39 -
a) t-ButYl~(6~R~7R)-3-Acetoxymethyl-7-~(z)-2-(2-t-butoxy-
carbonylprop-2-oxyimino)-2-(2-tritylaminothiazol-4-yl)
acetamidoJceph-3-em-4-carboxYlate
A stirred solution of the product of Preparation 4
(572 mg) and t-butyl (6R,7R)-3-acetoxymethyl-7-aminoceph-
3-em-4-carboxylate (328 mg) in dimethylformamide (10 ml)
was cooled to 0', and l-hydroxybenzotriazole (150 mg~ was
added, followed by dicyclohexylcarbodiimide (225 mg). The
mixture was warrned to room temperature, stirred for 5 hours,
and allowed to stand overnight. The mixture was filtered,
and the white solid washed with a ]ittle ether. The filtrate
and washings were diluted with water (50 ml) and extracted
with ethyl acetate. The organic extracts were combined,
washed successively with water, 2N hydrochloric acid, water,
sodium bicarbonate solution, and saturated brine, dried and
evaporated. The residue was eluted through a silica column
with ether. The product-containing eluate was collected
and concentrated to give the title compound (533 mg). A
portion was recrystallised ~rom di-isopropy] ether and had
m.p. 103 to 113- (decomp.);[alD + 8.5- (c 1.0, DMSO),
b) (6R~7R)-3-Acetoxymethyl-7-r(Z)-2-(2-aminothiazol-4-yl~-
2-(2-carboxyprop-2-oxyimino)acetamido~ceph-3-em-4-
carboxylic acid
Tri~luoroacetic acid (18 ml) was added to a solution
of the product of Stage a) (2.4 g) in anisole (18 ml) at 0'.
The mixture was stirred at room temperature for 2 hours
and concentrated. The residue was dissolved in ethyl
acetate and extracted with saturated sodium bicarbonate
solution. The pH of the aqueous extracts was adjusted to 6,
and the solution washed w;th ethyl acetate. The aqueous
2 g ~
- 40 -
phase was acidified to pH 1.5 under ethyl acetate,
saturated with sodium chloride, and extracted with ethyl
acetate. The combined organic extracts were washed with
saturated brine, dried, and evaporated. The residue was
dissolved in warm 50V/o formic acid (20 ml) and allowed to
stand for 2 hours. The mixture was diluted with water
(50 ml), and iltered. The filtrate was concentrated.
The residue was taken up in water (50 ml), refiltered, and
lyophilized to give the title compound (920 mg), ~
(pH 6 buffer) 236 nm (ElCm50), ~inf 255 nm (ElCm 235),
296 nm (ElCmO3); ~D + 20.0- (c 1.0, DM~O).
c) (6R.7R~-7-r(z)-2~-Aminothiazol-4-yl)-2-~2-~arb~o~y~r
2-oxyimino)acetamido~-3-(1-methylpyrid_nium-2-ylthio-
methyl) ceph-3-em-4-carboxylate, mono-sodium salt
lS The product of stage b) (1.58g) and sodium hydrogen
carbonate (0.50g.) were warmed with water (2ml), and the
mixture was then treated successively with N-methylpyrid-2-
thione (564mg.), sodium iodide (2.7g) and sodium bicarbonate
(ca, 400mg., to give a mixture of pH7). Water (0.4ml) was
added and the rnixture was heated ~mder nitrogen at 65- for
5 hours. The cooled mixture was then applied to a column
of Amberlite X~D-2 resin (lOOg.) and eluted with water in
lOOml. fractions (ractions 1 to 10) and then water : ethanol
3:1 in lOOml. fractions (11 to 14)~ Fractions 10 to 12 were
combined and evaporated to 250ml. and washed with ether
(4 x 125ml.) and lyophilised to a pale brown residue
(594mg.) The residue (500mg.) was dissolved in water
(150ml.) and the solution was washed with ethyl acetate
~3 x 150ml.) and lyophilised in the presence of formic acid
to a foam which was triturated with ether and dried
in vacuo to give the title compound as a formate salt
-
11;~94~8
(464mg), AmaX (pH6 buffer) 241 nm (ElCm88), Ainf 261nm
(E 261) A. 304nm (E L74);v (Nujol) 1538 1670
lcm ' 1nf lcm max
(CONH), 1600 (broad; CO2 ), 1767cm (~-lactam).
Example 2
a) t-Buty1 (6R~7R)-3-Acetoxymethyl-7-[(z)--2--(-l---t---but
carbonylcyclo-bu-t--~ --o-xyimino)-2-(2-tritylaminothia
4-yl)_acetamido~ceph-3-em-4-carb-xylate
A stirred solution of the product of Preparation 6
(24.2 g) and t-butyl (6R,7R)-3-acetoxymethyl-7-aminoceph-
3-em-4-carboxylate (13.6 g) in dimethylformamide (300 ml)
was cooled to 0-, treated with l-hydroxybenzotriazole mono-
hydrate (4.5 g), followed by dicyclohexylcarbodiimide
(6.4 g) and the product isolated substantially
as described in Example 1 (a) to give the title compound
(12,8 g), m.p. 113,5 to 116.5-(de~omp), [a~D + 15,0-
(c 1.0, DMSO),
b) (6R~7R ~3-Acetoxymethyl-7-r(Z)-2-(2-aminothiazol-4-yl)
-2-(1-carboxy,cyclobut-1-o~xyimin,o~cetamiclolc,eph-~-em-4-
carboxylic a_id
Trifluoroacetic acid (100 ml) was a<lded to a mixture
of the product of Stage a) (12.5 g) and anisole
(5ml) at 0-. The mixture was treated substantially as
described in Example 1 b) to give the tit~e compound
(4 g), AmaX (pH 6 buffer) 246 nm (E1%264" Ai, f 295 nm
(ElCml8)~ [a~D ~ 27.3- (_ 1.0, DMSO).
c) ~6R~7R)-7-r(z)-2-c2~Aminothiazol-4-yl)-2-(l-c~rb
but-1-~yimino)acetamido~-3-(1-methylpyridlnium-2- -
ylthiomethyl)_ceph-3-em-4-carboxylate, mono-sodium salt.
The product from Stage a) (1.60 g) and sodium
3L~Z9~8
- 42 -
bicarbonate (0.50 g) were warmed with water (2 ml) and to
the suspension was added l-methylpyrid-2-thione (564 mg)
and then more sodium bicarbonate (ca 350 mg) to give a
mixture of pH ca 6.5. Sodium iodide (2,7 g) was added and
the slight]y cloudy solution was heated at 65~ for 5 hours
and then cooled and applied to a column of Amberlite
XAD-2 (100 g). The column was eluted with water in ca
150 ml. fractions (1 to 5) and then with water:ethanol =
3:1 in 150 ml. fractions (6 to 10). Fractions 4 to 7 were
combined and the ethanol was removed by evaporation.
The solution was washed with ethyl acetate (3x300 ml) and
ether (200 ml) and evaporated until the organic solvents
had been removed. It was then lyophilised to give a brown
foam which was washed with ether and filtered and dried
in vacuo to give the title~ compound (8l6 mg) as a foam,
[~D + 211 (H20:EtOH 1~ 0084);% ~max (pE-16 buffer)
lcm ' max nm (Elcm~91)-
~1294~8
Example 3
a) (6R 7R)-3-Acetoxymethyl-7r(Z)-2-(2-aminothiazol-4-yl)
-2-(2-carboxyprop-2-oxyimino)acetamido~ceph-3-em-4-
carboxylic acid Trifluoroacetate salt
The product from Ex~mple 1 a) (31g) w~s dissolved in
trifluoroacetic acid (90 ml) with ice-water cooling
After five minutes the mixture was warmed to 20-.
After one hour the solution was poured into water (900 ml).
Five minutes later, ether (450 ml) was added and the
mixture was stirred for ten minutes. The ether layer,
after separation, was extracted with further water (5 ml).
The combined aqueous layers were washed with ether three
times, on each occasion the organic layer being back
extracted with water. The combined aqueous layers were
evaporated to dryness and the residue was triturated with
ether and clried to give the title comJ~ (15.4g) vmax
(Nujol) 3700 - 2200 (01~ and NH3), 1784 (~-lactam), 1730
(ester), 1670 and 1545 (amide), 1680 (acids) and 1650 cm
(CF3C00 ),
llZ94~8
- 44 -
b) (6R~ R)-7,[(Z)-2-(2-Aminothiazol-4-yl)-2-(2-carboxyprop-
2-oxyimino)acetamido~ methy~p~ridinium-4-yl)-
thiomethyl~ceph-3-em-4-carboxylate mono-sodium salt
A suspension of the product from Stage a) (2.11 g )
in water (3 ml) was treated portionwise with sodium bicarbon-
ate (1.06 g ) and warmed until effervescence ceased. The
solution was treated successively under nitrogen with
sodium iodide (4 g) and N-methylpyrid-4-thione (0.75 g),
and kept at 70- for 4 hours.
The mixture was cooled and diluted with water and the
resulting solution was poured into acetone (800 ml) with
vigorous stirring The precipitated material was isolated
by filtration and washed with a little acetone and some
ether and dried in vacuo to give a powder (2.97 g).
The product was purified by dissolving it in water
(10 ml) and applying the solut on to a column (ca. 330 mm
B i x 25 mm) containing "Amberlite' XAD-2 resin (100 g).
The column was eluted with water (9 x 50 ml fractions
collected) followed by 25% ethanol in water (7 x S0 ml
fractions collected). Appropriate fractions were combined;
ethanol re~oved in vacuo and the res;due freeze-dried to
give the titLe compound in two crops:-
(i) 0.253 g (isolated from the aqueous fractions)
had A ax (pl-16 buEfer) 231.5 nm (El/ 369) and 303nm(El/ 336)
with an inElection at 255 nm (ElCm268) and
(ii) 0.900 g (isolated from the aqueous ethanol
fractions) had ~maX(pH6 buffer) 231.5 nm (ElCm378) and
303 nm (El 379) with an inflection at 255 nm (El/ 275)
and ~(D20) 1.68 (d, J 6Hz) and
2.23 (d, J 6Hz) (pyridyl protons), 3.15 (s, thiazol-5-yl
proton), 4.30 (d, J 5Hz, 7-H), 4.84 (d, J 5Hz, 6-H), 5.72
~f ~de /~/ark
- 45 -
(obscured ABq, 3-CH2), 5.88 (s, NCH3), 6.44 (centre of
ABq, J ]5Hz, 2-H2) and 8.55 (s, CMe2).
Example 4
a) t-Butyl_(6R 7R)-3-Acetoxymethyl-7-r(Z)-2-(1-t-butoxy-
5 carbonylcyclobut-l-oxyimino)-2-(2-trity-la-m-i-n-o-t-h-i-a
4-yl) acetamido~ceph-3-em-4-carboxylate
A stirred solution of the product of Preparation 6
~24.2 g) and t-butyl (6R,7R)-3-acetoxymethyl-7-aminoceph-
3-em-4-carboxylate (13 6 g) in dimethylformamide (300 ml)
was cooled to 0-, and l-hydroxybenzotriazole monohydrate
(4.5 g) added, followed by dicyclohexylcarbodiimide (6.4 g).
The mixture was warmed to room temperature and stirred over-
night. The mixture was filtered, and the white solid
washed with a little ether. The filtrate and washings were
diluted with water (1.51) and extracted with ethyl acetate.
The organic extracts were combined, washed successively with
water and saturated brine, dried, and evaporated. The
residue was taken up in ether, filtered, and re-evaporated.
The required product was isolated after elution through two
silica columns with ether and concentrating the appropriate
fractions. The residues were recrystallised rom di-isopro-
pyl ether to give the title compound (12.8 g), ~.p 113.5
to 116.5-;[a~D + 15,0- (c 1.0, DMS0)
b) (6R,7R)-3-Acetoxymethyl-7-r(Z)-2-(2 Aminothi~zol-4-yl)-2-
(l-carboxycyclobut~l-oxyimino)acetamidolceph-3-em-4-t-but
carboxylic Acid Hydrochloride
t-Butyl(6R,7R)-3-acetoxymethyl-7-~(~)-2-(l-t-butoxy-
carbonylcyclobut-l-oxyimino)-2-(2-tritylaminothiazo1 4-yl)
acetamido~ceph-3-em-4-carboxylate (17.9 g) in formic acid
(72 ml) was cooled to +12- and concentrated hydrochloric
acid (6 ml) added. The mixture was stirred for l~ hours
~1~9~
- 46 -
at 18-20- and then cooled to 6-. The solid was filtered
off, washed with formic acid and the combined filtrate and
wash added over 15 minutes to stirred isopropyl ether
(2 1). The solid was filtered off, washed with isopropyl
ether (25 ml) and dried at 40 in vacuo to give the
title_compound (11.47 g), ~(DMSO d6) 0.29 (d, J 9), 2.98
(s,thiazol-5-yl proton), 4.10 (dd, J4 and 9, 7-H), 4.78
(d, J 4,6-H)9 4.98 and 5.29 (ABq,J 13, 3-CH2-), 6.38
(s,2-H2) 7.2-7.8 and 7.8-8.3(cyclobutyl protons), 7.98
(s, OCOCH3).
c)(6R,7R)-7-r(;Z)-2-(2-Aminothi~zol-4-yl)-2-(1-carboxycyclo-
but-l-oxy
methyl)ceph-3-em-4-carboxylate
A suspension of (6R,7R)-3-acetoxymethyl-7-~(Z)-2-(2-
aminothiazol-4-yl)-2-(1-carboxycyclobut-1-Oxyimino)-
acetamido~ceph-3-em-4-carboxylic acid hydrochloride (2~30 g )
in water (5 ml) was treated portionwise with sodium bicar-
bonate (1006 g ) and warmed until effervescence ceased.
Sodium iodide (4 g ) was added to the solution fol]owed by
N-methylpyrid-4-thione (0 75 g ) The mixture was covered
with nitrogen and incubated at 70- Eor 4 hours.
The resulting solution was cooled to room temperature
and poured into stirred acetone (800 ml) The precipitated
solid was isolated by filtration, washed wlth acetone
(10 ml), diethyl ether (10 ml), and dried in vacuo to give
a powder (2.7 g).
The product was dissolved in water (10 ml) and the pH
of the solution adjusted to 4.5 with acetic acid. The solu-
tion was treated with a mixture of "Amberlite" LA2 resin
(10 ml), ethyl acetate (10 ml) and acetic acid (0.75 ml)
and stirred at ambient temperature for 1 hour. The aqueous
llZ94~
layer was separated and washed with ethyl acetate (5 ml).
Ethyl acetate dissolved in the aqueous extract was removed
by rotary evaporation. The resulting solution was
readjusted to pH 405 with acetic acid.
The solution was run onto a column (ca. 330 mm x 25 mm)
of "Amberlite" XAD-2 resin. The column was eluted with
water (ca. 1.2 1), followed by water/éthanol (2/1).
Appropriate fractions (caO 1.2 1) were combined and
reduced to a small volume (ca. 25 ml) by rotary evaporation.
The residue was freeze-dried to give the title compound
(530 mgO).
This material had ~ (pH 6 buffer), 231 nm
(El% 351) and 303 nm (El~ 428) with an inflection at 255 nm
(ElCm 267). ~ (D20 + NaHC03) 1.64 (d, J 6Hz) and 2.21
(d, J ~Hz) (pyridyl protons), 3.07 (s, thiazol-5-yl proton),
4,21 (d, ~ 4Hz, 7-H), 4.75 (d, J 4Hz, 6-H), 5.5 to 5.8
(obscured ABq, 3-CH2), 5.84 (s, ~CH3), 6.4 (centre of ABq
J 15 Hz, 2-H2), 7.58 and 8.1 (cyclobutyl protons).
94~8
- 48 -
Exam~le 5
a) Diphenylmethyl C6R,7R~-7-r(Z)-2-(2-t-butQxycarbn~ylprop-
2-oxy,imino,~2-(2-tritylaminothiazol-4-yl)-acetamido
bromome~hylceph-3-em-4-carboxylate
Phosphorus pentachloride (8,8 g) was dissolved with
stirring in methylene chloride (400 ml). The solution was
cooled to -20-C and treated with the product of Preparation
4 (22,8 g) and the resultant clear solution stirred for ca
30 min at -15-C, Triethylamine (15,8 ml) was added and the
mixture maintained at -15-C for a further 5 min. before
being added over 5 min,to a solution of diphenylmethyl
(6R,7R)-7-amino-3-bromomethylceph-3-em-4-carboxylate
hydrochloride (19,8 g) and triethylamine (5,6 ml) in
methylene chloride (400 ml) stirred and maintained through-
out at -15 to -20-C, The cooling was removed after 10 min
and the mixture was stirred whilst the temperature rose to
ambient over ca 30 min,
The mixture was poured into water (1 1) and methylene
chloride (400 ml), shaken and the organic layer collected~
The water was backwashed with methylene chloride (l00
The initial methylene chloride layer was washed with
dilute sodium bicarbonate solution (to ca pH 8) and then
~1294~8
- 49 -
this aqueous layer further washed with the backwash.
The organic layers were combined and washed with half
saturated brine solution. After drying the methylene
chloride layer over magnesium sulphate the solvent was
evaporated to afford a dry foam-solid. This was collected
from the evaporation flask and dried in vacuo at 40- to
afford the title compound (40.25 g) as a buff solid
~ (CDC13) 3.03 (s, -CH.Ph2), 3.27 (s, thiazol-5-yl proton),
4.04 (dd, J 4Hz + 9Hz, 7-H), 5.0 (d, J 4Hz, 6-H), 5.72
(s, 3-CH2, 6.3-6.7 (ABq 2-H2), 8.37 + &~41 (2 x s, C-Me2).
b) Diphenylmethy] ~ carbonvlprop-2-
oxyimino)-2-(2-tritylaminothiazol-4-yl)-acetamido~-3
methylpyridin
bromide
The product from Stage a) (40 g), was dissolved in
tetrahydrofuran (500 ml) and whilst being stirred was
treated with N-methylpyrid-4-thione (5.0 g). After stirr-
ing at ambient temperature for 6 hours cliethyl ether
(2.0 1) was added The mixture was stirred ~or 1 hour
then cooled at 0' ~or 16 hours. The solid was removed by
~iltration, washed well with diethyl ether and dried for
16 hours Ln vacuo to give the title c~e~ ,(39.9 g);
`~ (CDC13) 1.1 ~ 1.3 (broad d, pyridinium protons adjacent
to N-Me), 2.2-2.4 (broad d, pyridinium protons adjacent to
C-S), 3.07 (s, CH.Ph2), 3.27 (s, thiazol-S-yl proton),
3.36 (broad s Ph3CNH), 3~9 - 4.2 (dd, J 511z + 9Hz 7-H),
4.90 (d,J SHz, 6-H), 5,64 (broad s C3-CH2 + NMe) 6.1 - 6.6
~broad m, 2-H2), 8.38 + 8.40 (2 x s,`G-Me2): v (Nujol)
1802 (~-lac~am), 1708cm (C02H).
1~946~8
- 50 -
-c) ( .: /_
-2-oxy;mino)acetamidol-3-(l-methylpyridinium-4-ylthio-
methyl)ceph-3-em-4-c rboxylate
The product from Stage b) (40 g) was taken up in tri-
fluoroacetic acid (320 ml) and anisole (80 ml) and stirred
in an ice bath for 1~ hours. The solution was poured into
water (3 1) and diethyl ether (105 1) and shaken vigorouslyO
The aqueous layer was separated and washed further with
diethyl ether (3 x 1.5 1) before being evaporated to dry-
ness at ca 50-C in vacuo The resultant foam was trLturated
_ _ _ _ .
with acetone ca 500 ml and the solid filtered, washed with
diethyl ether and dried in vacuo at 40- to give the title
.
compound (12.2.~ g) whose nmr spectroscopic properties
resembled those of the product of Example 3b). ~ (pH6
( lcm 320), 303nm (ElCm 385) and ~ 258nm
(ElCm 233). Fluorine assay (2~8%) indi.cated that the product
contained some trifluoracetic acid.
E ~
a) Diphenylmethyl (6R~7R)-7-ami.no-3-(l-rnethylp-y dinium-4-
,v_c _ me~,~vl~-ceph-3-em-4-ca_ oxylate bromide,
D;.phenylmethyl (6R,7R)-7-amino-3-bromomethylceph-3-em-
4-carboxylate hydrochloride (500 mg) was suspended in
:. ,
9 ~ ~ 8
- 51 -
tetrahydrofuran (20 ml) and treated with triethylamine
(0.14 ml). After stirring for ca 5 minutes at ca 0' the
solution was clarified and treated with N-methylpyrid-4-
thione (150 mg). The mixture was stirred
for 2 hours at ambient temperature, then stored at 0- for
16 hours. The white solid was filtered off, washed with
tetrahydrofuran and diethyl ether and dried in vacuo at
ambient temperature for 16 hours to yield the title
compound (500 mg)~(DMS0-d6) 1 26 (d, pyridinium protons
adjacent to NMe), 2.09 (d,pyridinium protons), 3.01
(s, CH.Ph2), 4.88 (d, J 5Hz,7-H), 5.08 (d, J 5Hz, 6-H),
5.77 (s, 3-CH2 and NMe), 6.15 and 6.5G (m, 2-H2).
b) Diphenylmethyl (6R,7R)-7 1 Z)-2-(2-t-butoxycarbpn~lprop-2-
oxyimino)-2-(2-tritylaminothiazol-4-yl)-acetamido~-3-
(1-methylpyridinium-4-yl-thiometh~l)cePh-3-em-4-
carboxylate bromide
Phosphonls pentachloride (170 mg) in methylene
chloride (8 ml) was cooled to O-C and the product of
Preparation 4 (0 44 g) was addedO After stirring for 30
minutes at O-C triethylamine (0.25 ml)was added and the
solution stirred at 0- for a further 5 minutes before being
added over 10 minutes to a stirrecl solution o~ the product
from Stage a) (450 mg ) in methylene chloride (5 ml) at
O-C. The mixture was stirred at ambient temperature for
2 hours, stored for 16 hours at ca 0- and then poured into
a mixture of ethyl acetate (50 ml) and water (50 ml).
The mixture was shaken thoroughly and the ethyl acetate
layer collected After evaporation of the organic solvent
the solid residue was triturated with a little diethyl
ether, filtered, washed with more diethyl ether and dried
l~Z94C~8
- 52 -
in vacuo at 40- to yield the title compound (680 mg) as a
powder whose nmr spectrum resembled that of the product of
Example 5 b). vmax (Nujol) 1786 (~-lactam), 1680 and 1545
(amicle), 2600 and 1710cm (C02H).
c) (6R,_7R)-7-r,(z~2-Aminothiazol-4-yl)-2-(2-carboxyprop-2
oxyimino)-acetamidol-3-(1-methylpyridinium-4-ylthio-
methyl)ceph-3-em-4-carboxylate.
The product from Stage b) (400 mg) was treated with
anisole (1 ml) and trifluoroacetic acid (4 ml) for ca 1 hour
at 0- and the product isolated substantially as described
in Example 5(c) to yield the title compound whose nmr
spectroscopic properties resembled those of the product of
Example 3 b), Amax (pH6 buffer) 230nm (ElCm 347)~ 302.5nm
(Elcm 392) and Ainf 249.5nm (Elcm 242).
l~Z94~,~
- 53 -
Example 7
a)Diphenylmethyl (6R,7R)-7-[~-2- - ~ o~ar~bon lcyclo-
but-l-oxyimino)-2-(2-tritylaminothiazol-4-yl)acetamido~
-3-b-~omomethylceph-3-em-4-carboxylate.
Phosphorus pentachloride (9.2 g ) in methylene
chloride (400 ml) was cooled to -20C before the addition
of the product of Preparation 6 (23.32 g). The solution
was stirred at -20 for ca 30 minutes when triethylamine
(12.32 mL) was added. After a further 5 minutes at -20
the solution was added over 5 minutes to a solution of
diphenylmethyl (6R,7R)-7-amino-3-bromomethylceph-3-em-4
carboxylate hydrochloride (19.8 g ) and triethylamine
(5.6 ml) in methylene chloride (400 ml) maintained at
-20C. After lO minutes the cooling was removed and the
reaction allowed to warnt to ambient temperature over ca
30 minutes when it was poured into water(L 1) and
methylene chloride (400 ml), The title com~ound (42 g)
was isolated in a similar manner to that described in
Example 5a). ~ (CDCl3) 3.03 (s, C~IPh2), 3.24 (s,
thiazol-5-yl proton), 4.05 (dd, J 5Hz + 9Hx, 7-H), 4 95
(d, J5Hz, 6-H) 5.72 (s, 3-CH2,Br), 6 3-6.7 (broad q,
2-H2), 7.2-8.1 (m, cyclobutyl protons).
l~LZ9~8
b) Diphenylmethyl (6R,7R)-7-~(Z)-2-~-t-butoxycarbonyl-
cyclobut-l-oxyimino)-2-t2-tritylaminothiazol-4-vl)-
acetamido]-3-[1-methylpyridinium-4-ylthiomethyl)ceph-3-em
4-carboxy]ate bromide
The product from Stage a) (42 g) in tetrahydrofuran
(500 ml), was treated with N-methylpyrid-4-thione
(5.2 g) and stirred for 3 hours at ambient temperature
After storage for 16 hours at ca 0 the solution was stirred
and diethyl ether (2.0 1) added. The solid was filtered
off, washed well with diethyl ether and dried at 40 in
vacuo to afford the title comPound (40.5 g) ~(CDC13) 1.18
(broad s, pyridyl protons adjacent to NMe), 2.32 (broad s,
pyri~yl protons adjacent to C - S), 3.05 (s, CH Ph2),
3.22 (s, thiazol-5-yl proton) 4.07, (dd, J 5Hz and 9Hz, 7-H),
4 88 (d, J 5Hz 6-H), 5.62 (broad s, 3-CH2 and NMe), 6 1 -
6.7 (m, 2-H2) 7.2 - 8.2 (m, cyclobutylprotons),
c) (6R,7R)-7- r ~Z)- 2-(2-Aminothiazol-4-y:l)-2-(l-ca~oxy
cyclobut-l-oxyimino)-acetamido~-3-(1-methyl-
pyridinium-4-ylthiomethyl)ceph-3-em-4-carboxylate
The product from Stage a) (40 5 g) was stirred in tri-
fluoroacetic acid (320 ml) and anisole (80 ml) at ca 0-
for 1~ hours The mixture was poured into water (3 1) and
diethyl ether (1.5 1). The title compound was isolated
as described in Example 5 (c) ~nd had spectroscopic
5 ~ e# ~ ffl~ing those o the product of Example 4 c).
~1294Ç~3
Fluorine assay (ca 4.2%) indicated that the product
contained some trifluoroacetic acid.
Example 8
a) Diphenvlmethyl (lS,6R,7R)-7-r~Z)-2-(2-t-Butoxycarb~nyl-
S prop-2-oxyimino)-2-~2-tritylamin-ot-h~ azol~-v )~
3-r ~l-methylpyrimidinium-2-~y~hiome-t-h-y-llceph--3---e-m-4
carboxylate ~ ide
The product of Preparation 7 (1.02 g) in dry tetra-
hydrofuran (10 ml) was treated with 1,2-dihydro-1-methyl-
pyrimidine-2-thione (158 mg) and the mixture was stirred
at 25' for 1~ hours. The resulting solution was added
slowly to stirred ether (150 ml) and the precipitate was
filtered off and dried in vacuo to give the title compound
(1.043 g) as a solid, [~D + 56- (c 0.66, CHC13),
(EtOH) 364 nm (ElCml49) with inflections at 240 nm
(ElCm250) and 275 nm (ElCml40).
b) Diphenylmethy1 (6R~7R)-7l(Z)~(2-t-Butox~carbonylpro~-2
oxyimino~-2-(2-tritylaminothiazol-4-yl)acetamido~-3-
L~l-methy]pyrimidinium-2-yl ~ iomethvlJceph-3-em-4-
carboxyl~te iodide.
The product from Stage a) (912 mg) in acetone (10 ml)
was treated with potassium iodide (525 mg) and the suspens-
ion was stirred at 25- for 5 minutes and was then cooled
to -10-. Acety] chloride (0.115 ml) was added and the
mixture was stirred at 0- to ~2- for 50 minutes.
The mixture was then added to a stirred solution of sodium
metabisulphite (600 mg) in water (60 ml). The precipitate
l~Z94&-~
- 56 -
was filtered off and washed with water and dried in vacuo
over phosphorus pentoxide to give a solid (880 mg).
A solution of the solid in acetone (5 ml) was treated
with potassium iodide (525 mg) and cooled to -10- and then
treated with acetyl chloride (0.115 ml). The mixture was
stirred at 0- to +2- for 40 minutes, and was then added to
a stirred solution of sodium metabisulphite (1 g) in water
(60 ml). The precipitate was filtered off and washed with
water and dried in vacuo over phosphorus pentoxide to give
the title_compound (940 mg) as a solid, m.p. 142 to 156-
(decomp), [a~D -67- (c 0.45, acetone).
c) (6R~7R)-7-r~)-2-(2-Aminothiazol-4-yl)-2-(2-carboxyprop-2
oxyimin~)acetamido~-3-r~l-methylpyrimidinium-2-yl)thio-
methyl~-ceph-3-em-4-carboxylate, trifluoroacetate
The product from Stage b) (800 mg) was wetted with
anisole (0.5 ml) and treated with trifluoroacetic acid
(2 ml), The mixture was swirled at 24- for 2~ minutes, and
was then evaporated to an oil which was triturated with
ether. The solid (560 mg) was filtered of r ancl washed with
ether and dried in vacuo. It was then treated with anisole
(0.14 ml) and trifluoroacetic acicl (l6 m]) at 24- for 15
minutes. The mixture was filtered and the residue was
washed with trifluoroacetic acid (5 ml). The combined
filtrates were evaporated to an oil which was triturated
with ether. The solid was filtered off and washed with
ether and dried in vacuo to give the title comFound (409 mg)
as a solid,[a~D -45- (c 0.53, H20:EtOH = 1~ max
(pH6 buffer) 253 nm (El 297) with an inflection at 291 nm,
(E % 181).
lcm
llZ94~8
- 57 -
Exam~ _ 9
(6R,7R)-7-r (Z)-2-~2-Aminothiazol-4-Yl)-2-(~-prop-2
oxyimino)-acetamido~-3-r_(112-dimethylpyrazolium-3-yl)thio-
meth_llceph-3-em-4-carboxylate, sodium salt
The product of Example 3 a) (264 mg) and sodium
bicarbonate (135 mg) were warmed with water (0.3 ml) until
a solution had formed. 1,2-Dimethylpyrazol-4-ine-3-thione
(97 mg), sodium iodide (450 mg), and water (0.1 ml) were
added, and the mixture was heated at 70- for 4 hours with
occasional swirling. The mixture was then allowed to cool
and was diluted with water (0.5 ml) and was added slowly
to stirred acetone (100 ml). The precipitate was
filtered off and washed with acetone and with ether and
dried in vacuo to give a solid (318 mg). The solid (282 mg)
was dissolved in a little water and passed through a column
of 'Amberlite' XAD-2 (100 g). Elution was with water
(66 ml. fractions: 1 to 6) and then water:ethanol = 3:1
(66 ml. fractions: 7 to 12). Fractions 7 to 10 were
combined and evaporated to ca. 150 ml and freeæ-dried to a
white foam (158 mg) which was triturated with ether to give
the title_compound (133 mg) as a white solicl, [~D -16'
(c 0,21, water), AmaX(pH6 buffer) 234 nm (ElCm302) with an
inflection at 256 nm (Elcm255).
.. .
llZ94,~
- 58 -
Example_l0
a) Diphenylmethyl (lS 6R 7R)-7-~(Z?- 2-(2-t-Butoxycarbonyl-
prop-2-oxyimino)-2-~2-tritylaminothiazol-4-yl)acetamido~=
3-(pyrid-3-ylthiomethyl~ceph-3-em-4-carboxylate
l-oxide
A mixture of the product from Preparation 7 ~1 54 g) and
3-mercaptopyridine (0.200 g) in dry tetrahydrofuran (12 ml)
was treated with triethylamine (0 224 ml). The grey suspens-
ion was stirred for 10 minutes at 22- then partitioned
between water (150 ml) and ethyl acetate (150 ml).
The organic phase was washed with water (2 x 50 ml) and
then dried and evaporated to give a foam (1.546 g).
This foam was purified by chromatography on a column
of silica (Merck Kieselgei~60, 70-230 mesh, 50 g) eluted
with toluene:ethyl acetate (2:1)
Appropriate fractions were collected and evaporated to
give the title compound (1.385 g) as a foam, [~D + 23-
(c 0.81, CHC13), and vmax (C~IBr3) 3390 (NH), 1804
(~-lactam), 1725 cm 1 (C02R).
b) Diphenylme~ lS,6R,7R)-7-r~Z)-2-(2-t-Butoxycarbonyl-
prop-2-oxyit~ino)-2-(2-tritylaminothi-az-o-l-4-yl)acetamiclo~-
3-r(l-methylpyriclinium-3-yl)thiomethyl~ceph-3-em-4-carboxy-
late~_l-oxide, loclide Salt
The product from Stage a) (1.28 g) in iodotnethane (5 ml)
was allowed to stand at 22- for 11/4 hours, then ether
(50 ml) was added The precipitate was triturated and
filtered off and washed with ether and dried in vacuo to
give the title compound (1.30 g) as a solid, [~D ~ 21-
(c 0.29, CHC13), and vmax (Nujol) 3380 (NH), 1800
(~-lactam), 1730cm (C02R).
- 59 -
c) Di~henylmethy1_(6R,7R), ~ Z)-2-(2-t-Butoxycarbonyl-
pro~-2-~y'~i~A~ t~tylaminothiazol-4-yl)-
acetamidol-3-r(l-methvlpvridinium-3-yl~thiomethyl~ceph-3-
em-4-c~ late, Iodide Salt
A solution of the product from Stage b) (1.18 g)
in acetone (5 ml) was treated with potassium iodide
(0.665 g) and then cooled to - 10- and stirred and
treated with acetyl chloride (0.143 ml). The
mixture was stirred at 0 to +2- for 30 minutes after
which more acetone (5 ml) was added. After a further
30 minutes at 0- to +2- the mixture was diluted
with a solution of sodium metabisulphite (0.6 g) in water
(60 ml) and the precipitate was triturated to a solid
which was filtered off, washed with water and dried
in vacuo to give a solid (1.3 g).
The product was partitioned between chloroform
(containing some ethyl acetate) and dilute aqueous sodium
metabisulphite solution. The organic layer was washed
with water (twice) and dried nnd evapora~ed to a foam
(1.194 g).
A solution of this product (ca 1.19 g) in N,N ~limethyl-
formamide (5 ml) was stirred with potassium iodide (0.665 g)
at 22- for 15 minutes and then the mixture was cooled to
-10- and treated with acetyl chloride (0015 ml). The solut-
ion was stirred at 0- to +2- for 1 hour and was then added
slowly to a stirred solution of sodium metabisulphite (1 g)
in water (100 ml). The precipitate was filtered off and
washed with water. It was then partitioned between chloro-
form and dilute sodium metabisulphite solution. The organic
layer was washed with water (twice) and brine then dried
and evaporated to give the title compound (1.137 g) as a
:~lZ94~8
- 60 -
foam; [~D + 8 (_ 0.5, CHC13), and vmax (CHBr3), 3405
and 3275 (NH), 1793 (~-lactam), 1725 (CO2R), 1684 and 1527
(CONH), 1624 (C=N) and 758 cm (phenyl)O
d) (6R,7R)-7-~ Z~-2-(2-Aminothiazol-4-Yl)-2-(2-carboxv-
pro
thiomethyl~-ceph-3-em-4-carboxylic acid, Trifluoro-
acetate Salt
_ _
The product of Stage c) (0.99 g) was treated with
anisole (1 ml) and trifluoroacetic acid (4 ml) as
described in Example 8 c) to give the title compound
(0.583 g) as a solid, [a~D + 27- (c 0.69, H~O:EtOH = 1:1),
A (pH 6 buffer) 232 nm (E % 286) and 256 nm
maOx 1 cm
(El/m 254).
Example 11
a) Diphenylmethyl ~lS,6R lR)-7-r(%)-2-(2-t-Butoxy-
carbonylprop-2-oxYimino~-2-(2-tritvlamino-
thiazol-4-yl)acetamido~-3-~1l3-dimethylimidazolium-2
yl)thiomethyl~ceph-3-em-4-carboxylate. l-Oxide,
~romide Salt
-- .
The product of Preparation 7 (0.825 g ) in dry tetra-
hydrofuran (4 ml) was treated with 1,3-dimethylimidazol-4-
ine-2-thione (0.108 g) and the mixture was stirred at 24-
for 3 hours. The resulting solution was added to stirred
ether (150 ml) and the precipitate was filtered off and
dried in vacuo to give the title compound (0.834 g) as a
solid, m.p. 150- to 160- (decomp); [~D + 19- (c 0.78,
CHC13).
b) Di~ ~ 1 (6R,7R)-7-r(Z)-2-(_2-t-Butoxy-
carbonylprop-2-oxyimino)-2-(2-tritylamino-
thiazol-4-yl)acetamido~-3-r(~3-dimec~'mkl~
yl~thiomethyllceph-3-em-4-carboxylate, Iodide Salt
l:lZ94~P8
- 61 -
The product obtained in Stage a) (0.725 g) in acetone
(5 ml) was treated substantially as described in Example 8 b)
to give the title_compound (0.759 g) as a solid, m.p~ 135-
to 145 (decomp.); [a~D +10 (C 0.29, CHC13).
c)__(6R,7R)-7-r(Z)-2-(2-Aminothiazo1-4-yl)-?-(?-carboxy-
prop-2-oxyimino)acetamido,1-3-L(1,3-dimethvlimidazolium-
2-yl)thiomethyllceph-3-em-4---c-arboxvl-ic Acid, Trifluoro-
acetate Salt
_
The product obtained from Stage b) (0.65 g) was
10 treated with anisole (0.5 ml) and treated with trifluoro-
acetic acid (2ml) and the product isolated substantially as
described in Example 8 c ) to give the title compound (0.273 g)
as a solid [aJD +110 7(_ 0.52, EtOH:H20 = 1:1); ~max
(pH6 buffer) 235 nm (Elcm3o7)~ Ainf253 nm (ElCn2145),
15 Ainf275 nm (El/194).
1~294~8
-- ~2 --
Example 12
a) (6R,7R)-7-~(Z)-2-(2-Aminothiazol-4-yl)-2-(1-carboxycyclo-
but-l-oxyimino)-acetarnido]-3-(pyrid-4-yl-thiomethyl)ceph-3-
em-4-carboxylate
The product of Example 2(b) (12 g) was added to a solu-
tion of sodium iodide (21 g) in water (20 ml) at ambient
temperature. Sodium bicarbonate (3.72 g) was then added
over 40 minutes. 4-Mercapto-pyridine (3.5 g) was then added
and the reaction mixture heated to 70OC for 4 hours. lhe
mixture was then cooled to ambient temperature arc~ added to
acetone (2 1) to give a solid (14 g) containing the title
compound.
~(DMS0-d6) includes:- 3.24 (s, aminothiazole proton),
1.69 (d, J = 5Hz) + 2.68 (d, J = 5Hz) ~yridinium protons),
4.2-4.5 (m, 7-H), 4.98, 7.2-7.8, + 7.8-8.4 (m, cyclobutane
proton).
b) (6R,7R)-7-~(Z)-2-(2-Aminothiazol-4-yl)-2-(1-carboxycyclo-
but-l-oxyimino)-acetamido~-3-(1-methylpyridinium-4-yl-thio-
20 methyl)ceph-3-em-4-carboxylate
The solid from Stage a) (5 g) was dissolved in methanol
(30 ml) and water (20 ml) at ambient temperature. A solution
of methyl iodide (0.84 g) in methanol (10 ml) was then added.
After 24 hours at ambient temperature, the solvent was removed
25 and the residue triturated with acetone t;o give a solid
(3.98 g) containing the title compound.
~ (DMSO-d6) includes:- 5.84 ( " methyl group protons on
pyridinium ring).
Analysis by high pressure liquid chromatography gave a
30 peak having a retention time i~entical to that o~ the product
~:~Z94~38
- 63
of Example 4 c).
Example 13
a) Diphenylmethyl (6R,7R)-7-~(Z)-2-(1-t-butoxycarbonylcyclo-
but-l-oxyimino)-2-(2-tritylaminothiazol-4-yl)acetamido]-3-
(l-methylpyridinium-4-ylthiomethyl)-ceph-3-em-4-carboxylate
Bromide
Phosphorus pentachloride (0.92 g) in methylene chloride
(50 ml) was cooled to 0C and the product of Preparation 6
(2.33 g), was added. The solution was stirred at ca. -200
for 30 minutes when triethylamine (1.25 ml) was added. After
a further 5 minutes at -20, the solution was added to a
stirred suspension of the product of Example 6 a) in methylene
chloride (40 ml) at -10. The mixture was stirred at ambient
temperature Lor ca. 2 hours and became almost clear before
being poured into water (200 ml) and ethyl acetate (300 ml).
The mixture was shaken and the ethyl acetate layer separated.
After evaporation of the solvent in vacuo, the solid residue
was dissolved in tetrahydrofuran (30 ml) and treated with
diethyl ether (90 rnl). The solid that precipi~ated was Lil-
tered, washed with diethyl ether and dried in vacuo at 40
to yield the title compound (2.3 g) as a powder, whose
spectroscopic properties resembled those of the product oL
Example 7 b).
b) (6R,7R)-7-C(Z)-2-(2-Aminothiazol-4-yl)~2-(1-carboxycy~lo-
but-l-oxyimino)-acetamido]-3-(1-methylpyridinium-4-ylthio- .
~ethyl)ceph-3-em-4-carboxylate
The product from Stage a) (0.9 g) was stirred in 98%
formic acid (7 ml) for 15 minutes at room temperature before
11~94~
_ 64 -
conc. hydrochloric acid (0.2 ml) was added and stirrin~ con-
tinued for a total of l~ hours. The solvent was evaporated
under high vacuum at room temperature and the resultant
solid foam triturated with acetone (30 ml). The solid was
filtered, washed with diethyl ether and dried in vacuo to
afford the title compound (0.5g) whose nmr spectrum resembled
that of the product of Example 4 c) as a powder,
~max (pH 6 buffer) 226 nm (ElCm286), 303.5 nm (El 346)
and ~inf 255 nm (El 215).
~1294~8
PHARMACY EXAMPLES
... .... ~
Example A - Dry Powder for Injection
Formula Per Vial
~6R~7R)-7-C(Z)-2-(2-Arninothiazol-4-yl)-2-~2-carboxyprop-2
oxyimino)acetamido]-3-(l-methylpyridinium-4-yl)thiomethyl-
ceph-3-em-4-carboxylate 500mg
Sodium acetate, anhydrous 69mg
Method
The cephalosporin antibiotic was blended with sodium acetate
and filled into a glass vial. The vial headspace was purged
with nitrogen and a combination seal applied by crimping.
The product was dissolved, as for administration, by the
addition of 2ml Water for Injections.
Example B - Intramammary Injection (Veterinary)
Formula
(6R,7X)~7-L(Z)-2-Aminothiazol-4-yl)-2-(2-carbo~yprop-2-
25 oxyimino)acetamido]-3-(l-methylpyridinium-4-yl)thio-
methylceph-3-em-4-carboxylate,monosodium salt200mg
Polysorbate 60 3.0% w/v )
White beeswax 6.0% w/v ) to 5.0g
Arachis Oil 91.0% w/v
11294~8
- 66 -
Method
Heat the last three ingredients together at 150C for one
hour and then cool to room temperature with stirring. Add
the sterile milled antibiotic aseptically to this vehicle
and refine with a high speed mixer. Fill the product asep-
tically into sterile plastic syringes, using a fill weightof 5.00g per container.
Example C - Dry Powder for Injection
Formula Per Vial
(6R,7R)-7-~(Z)-2-(2-Aminothiazol-4-yl)-2-(1-carboxycyclobut-
l-oxyimino)acetamido]-3-(1- methylpyridinium-4-yl)thiomethyl-
ceph-3-em-4-carboxylate 500mg
Meglumine 161mg
Method
The cephalosporin antibiotic was blended with meglumine and
illed into a glass vial. The vial headspace was purged with
nitrogen and a combination seal applied by crimping. The
product was dissolved, as for administration, by the addition
of 2ml Water for Injections.
Example D - Dry Powder 'or Iniection
Fill the sterile (6R,7R)-7-C(Z)-2-(2-aminothiazol-4-yl)-2-
(l-carboxycyclobut-l-oxyimino)acetamido]-3-(1-methylpyridi-
nium-4-yl)thiomethylceph-3-em-4-carboxylate, monosodium salt
~1294~8
- 67 -
into glass vials such that each vial contains an amount
equivalent to l.OOg of the antibiotic acidO Carry out the
filling aseptically under a blanket of sterile nitrogen,
Close the vials using rubber disks or plugs, held in position
by aluminium overseals, thereby preventing gaseous exchange
or ingress of microorganisms. Reconstitute the product
by dissolving in Water for Injections or other suitable
sterile vehicle shortly before administrationO
Polysorbate 60 is a polyoxyethylene sorbitan monostearate
containing about 20 ethylene oxide units and meglumine is
N-Methylglucamine BP.
: .
