Note: Descriptions are shown in the official language in which they were submitted.
2~
This invention is concerned with cephalosporin
compounds possessing valuable antibiotic propertles.
The cephalosporin compouncLs in this
specification are named with reference to "cepham" after
J. Amer Chem. Soc., 1962, 84~ 3400, the term "cephem"
reerring to the basi~ cepham structure with one doubIe
bond.
Cephalosporin an~ibiotics are widely used in
the treatment of diseases caused by pathogenic bacteria
in 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 coumpounds,
and in the treat~ent o~ penicillin-sensitive patients,`
In many instances it i9 desirable to employ a
cephalosporin antibiotic which exhibits activity against
both gram-positive and gram-negative microorganlsms, and
a signiicant ~mo~mt of research has been directed to the
development o various types of broad spectr~m
cephalosporin antibiotics.
Thus, for example7 in our British Patent
Specification No. 1,399yO869 we describe a novel class
of Gephalosporin antibiotics containlng a 7~-(a-etherified
oximino)-acylamido group, the oximino group having the ~y~
. ~ ~ ; . . , -
, . . . : ' '~ , ~ :
tl~
configuration~ This class o~ antibi.otic compounds
is characterised by high antibacterial. activity against
a range of gram-positive and gram-negative organisms
coupled wi.th particularly high stability to ~-lactamases
produced by various gram-negative organismsO
The disoovery of this class of coumpounds 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
NoOl,496,757, we describe cephalosporin antibiotics
containing a 7~-acylamido group of the formula
R~CoCO"NH~ A
" R
~O,(CH2) C (CH2)nCOOH (A)
R
(wherein R is a thienyl or furyl group; RA and RB may vary
widely and mayjfor example~be Cl_4 alkyl groups or
together with ~he carbon atom to which they are attached
form a C3 7 cycloalkylidene group, and m and n are each
0 or 1 such that the sum of m and n is 0 or 1), the
compounds being syn isomers or mixtures of ~y~ and anti
isomers containing at least 90% of the ~y~ isomer. The
3 position of the cephalosporin molecule may be
unsubstituted or may contain one of a wide variety of
possi~le substikuents. These compounds have been found
to have particularly good activity against gram-negative
1~3~
organisms.
Other compounds of similar structure have
been developed from these compounds in further attempts to
find antibiotics having improved broad spectrum antibiotic
activity a~d/o~ hlgh activity against gram-negative
organismsO Such developments have involved variations in
not only the 7~-acylamido group in the above fomula but
also the introduction of particular groups in the 3-position
of the cephalosporin molecule.
Thus, for example, in Belgian Patent Speci-
fication No.852,427, there are described cephalosporin
antibiotic compounds ~alling within the general scope of
our British Patent Specification No. 19399,086, and
where~n ~he group R in the above formula (A) m~y be
replaced by a variety of diferent organic groups~
including 2-aminothiazol-4-yl~ and the oxygen atom in the
oxyim;no group is attached to an aliphatic hydrocarbon
group which may itself be substituted by, for example7
carboxy. In such compounds~ the substituent at khe
3~position is con~ined to an acyloxymethyl, hydroxymethyl,
formyl or optionally substituted heterocyclic-thiomethyl
group ~,
~ urthermore, Belgian Patent Specification No~
~36,813 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 is a
hydroxyim;no or blocked hydroxyimino group, e~gO a
methoxyimino group. In such compounds, the 3-position of
the cephalosporin molecule is substituted by a methyl
group ~hich may itself be optionally substituted by
residues of a large number of nucleophilic compo~mds
therein described~ In the above-mentioned Specification
no antibiotic activity is ascribed to such compounds which
are only mentioned as intermediates for the preparation
of antibiotics described i~ the SpecificationO
Belgian Patent Specification No,853,545
descri~es cephalosporin antibiotics w~erein the
7~-acylamido side chain is primarily a
2-~2~aminothiazol-4-yl)-2-( ~ -methoxyimino-acetamido
group and the substituent in the 3-position is broadly
defined in a similar manner to that in the
above-mentioned Belgian Patent Specification No.836,813.
We have now discovered that by an appropriate
selection of a small number of particular groups at the
7~-position in combination with a methyl group at the
3-position, cephalosporin compounds having particularly
advantageous activity (described in more detail bel~w)
against a wide range of commonly encountered pathogenic
organisms may be obtainedO
The present invention provides cephalosporin
antibiotics of the general formula
NH2
~ H 11
S N
\ - / C.C~ S
N ~a ~ N ~ ~113 (-~
\ O~C COOH COOH
1b
(wherein Ra and R~, which may be the s~me or difer~nt,
I each represents a Gl_4alkyl group (preferably a straight
~3~
chain alkyl group i,eO a methyl, ethyl, n-propyl or
n-butyl group and particularly a methyl or ethyl group) or
Raand Rbtogether with the carbon atom to ~hich they are
attached form a C3 7 cycloalkylidene group~ pref~rably a
G3 5 cycloalkylidene group) and non-toxic salts and non~
toxic metabolically labile esters ~hereof.
The compounds according to the in~ention are ~X~
is~mer~ The ~y~ isomeric form is defined by the configuxa-
tion of the group
Ra
- O.C~COOH
Ib
with respect to the carboxamido group. In this
specification the sYn configuration is denoted
structurally as
,~12
S N
~ , . CO~NH -
Ra
O,~,,(,OOH
Rb
It will be appreciated that since the compounds
according to the invention are geometric isomers, some ad~
mixture wi~h the corresponding antl isomer may occurO
The invention also includes within its scope
the solva~es (especially the hydrates) of the compounds of
formula (I)o It also includes within its scope salts
of es~ers of compo~mds o~ formula (I)o
The compounds ac~ording to the present
ll~D2'7~
- 6 -
invention may exist in tautomeric forms (in respect of
the 2~aminothiazolyl group) and it will be understood
that such tautomeric forms, e,g. the 2-iminothiazolinyl
form, are included within the scope of the înventionO
It ~ill also be appreciated that when Ra and
R in the above formula represent different Cl 4 alkyl
groups, the carbon ~tom to which they are attached will
comprise a centre of asymmetry, Such compounds are
diastereoisomeric and the present invention embraces
individual diastereo;somers of these compounds as well as
mixtures thereof~
The compounds according to the invention
exhibit broad spectrum antibiotic activity. Against
gram-negative organisms the activity is unusually high.
~S This high activity extends to many ~~lactamase~producing
gram-negative strainsV The compounds also possess high
stability to ~-lactamases produced by a range of
gram-negative oxganisms,
Compounds according to the invention have
been found to exhibit high activity against various
members of the Enterobacteriaceae (eOgO strains of
F.scherichia_coli9 Klebsiella pneumoniae, Salmonella
~E~ &~ sonnei, F,nteroba~ter cloacae,
Serratia marcescens Providence species Proteus mirabilis ~nd
especi~lly indole-positive Proteus organisms such as Proteus
and Proteus mo~ ), as well as against strains
of ~ influenzae and good activity against
strains of Pseudomonas organisms eOg~ strains of
~35Ys~5YL~_ ~3~ ~L o This activity against strains
of indole-positive Proteus and Pseudomonas organisms is
unusual7 bearing in mind the antibacterial activity of
known 3-methyl cephalosporin antibioticsO
~L~3~'~
-- 7 --
In addition to their high antibiotic activity,
the 3-methy~ compounds a~cording to the present invention
possess the further advantage that in comparison with
analogous compounds possesslng more elaborate
3-substituents, they can be manufactured relatively easily
and economically on an industrial scale from
readily available s~arting materials~ iOe. penicillin G
or penicillin V.
Non toxic salt derivatives which may be formed
by reaction~ofeither or hoth of the carboxyl groups
present in the compounds 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); ~mino acid salts (e~g. lysine and
arginine salts); organic base salts (e.gO procaine,
phenylethylbénz~amlne, diben~yle~hylenediamine,
ethanolamlne, diethanolamine and N-methylglucosamine salts30
Other non-toxic salt derivatives include acid addition
salts, e.g. ~ormed with hydrochloric, hydrobromic, sulphuric,
~o nitric, phosphoricg formic and trifluoroacetic acids The
salts ~ay also be in the form of resinates formed with, for
example, a polyst~rene resin or cross-linked polystyrene
dlvinylbenzene copolymer resin containing amino or
quaternary amino groups, sulphonic acid groups~ or with a
resin containing carboxyl groups9 e~g~ a polyacrylic acid
resin~ Soluble base salts ~eOg~ alkali metal salts such
as the sodium salt) of compounds of formula ~I) may be used
in therapeutir applications because of the rapid
distribution of such salts in the body upon administrationO
Where, ho~ver, insoluble salts of compounds ~I) are desired
~L3~
in a particular application, e.g. for use in depot preparations, such salts may
be formed in conventional m~nner, for example with appropriate or~anic amines.
mese and other salt derivatives such as the salts with toluene-p-
sulphonic and methanesulphonic acids may be employed as intermediates un the pre-
paration and/or purification of -the present ccmpoullds of formula (I)l for
example in the processes described below.
Non-toxic me-tabolically labile ester derivatives which may be formed
by esterification of either or both car~oxyl groups in the parent co~pound of
formLla (I) include acyloxyalkyl esters, e.g. lcwer alkanoyloxy-methyl or -ethyl
esters such as aoetoxy-~ethyl or -ethyl or pivaloyloxymethyl esters. In addi-
tion to the above ester derivatives~ the present invention includes within its
sccpe oompounds of formula (I) in the form of other physiologically acceptable
equlvalents, i.e. physiol~gically acceptable compounds which like the metabolic-
ally labile esters, are converted in vivo into the parent antibiotic compound of
formula (I).
Preferred compounds according to the present invention include the
following co~pounds 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-carbo~yprop-2-oxyimino)
acetamido]-3-methyloeph-3-emr4-carboxylic acid;
(6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-(1-carboxycyclobut-1-oxyimlno)
acetamido]-3-methylceph-3-em~4-carboxylic acid; and
: '~;`!
;~
.:
(6R,7R)-7-[(Z)-2-(2~aminothiazol-4-yl)-2-(1-carboxycyclopent-1- :~
yloxyimino) acetamido]-3-methylceph-3-em-4-carbcxylic acid. ~;
Other examples of oompounds of form~la ~I) accoxding to the present
invention include those wherein the groups Ra and ~ have the meanings given in
the following Table
TABLE ~:~
Ra Rb
..
a) Alkyl groups
CH3 C2H5
C2H5 C2H5
: b) Cycloalkylidene groups
cyelopropylidene
cyelohexylidene
The eompounds of formula (I) may be used for treating a variety of
- diseases caused by pathogenie bateria in human beings and anim31s, sueh as
respiratory tract infections and urinary tract infeetions.
Aeeording to another embodiment of the invention we provide a proaess
for the prep#ration of an
~1
;
:
~- - g _
,.~ : . .
?:~ ,
' : ~
- 10 _
antibiotic compound of ge~eral foxmula (I) as hereinbefore
defined or a non-toxic salt or non-toxic meta~olically
labile ester thereof wh;ch cornprises acylating a
compound of the formula
H H
H2N ~ B ~ (II)
~N ~CH3
1~ 1
COOR
~whereln ~ is ~S or ~S~O ~c~- or ~-); Rl represent:s
h~drogen or a carboxyl blocking group, e~g, the r~sLdue
of an ester-fo~ning aliphatic or aralipha~ic alcohol or
an ester-forming phenol, 5ilanol or stannanol (the said
alcohol, phenol~ silanol or stannanol preferably containing
1-~0 carbon atoms) a~d the dotted line bridging the 2~ 9
3-, and 4-pos~tions indicates that the compound is a ceph-
2-em or ceph-3-em cornpound~ or a salt, e.gO an acid
addition salt (formed with~ for example, a mlneral acid
such as hydrochloric~ hydrobromic~ sulphuric~ nitric or
phosphoric acid or an organic acid such as methanesul-
phonic or toluene-p-sulphonic acid) or an N-silyl
derivative thereo~, with an acid of formula
~3~
S N
.COOH
N Ra . (III)
OOR
R
.
(wherein Ra and Rb are as hereinbeore de~ined; R2
represents a carbox~l blocking group~ e.g9 as described
for R ; and R3 i9 an amino or protected amino group) or
with an acylating agen~ corresponding thereto
whereafter, if necessary andjor desired in each instance?
any of the followi~g reactionsD in any appropriate
sequence, are carried out:-
- i) con~ersîon ofa Q2-isomer into the desire~.~3-isomer,
- ii) reduction of a compound wherein B is ~S~ O to form a
compound wherein B is ~S,
iii)conversiorl of a carboxyl group into a non-toxic salt
or mekabolically labile ester unction, and
iv) removal of any carboxyl blocking and/or N-protecting
: . groups~
In the above~described process~ the starting
material of formula ~II) is preferably a compound wherein
B is ~S and the dotted line represents a ceph-3~em
compound.
Acylating agents which may be employed in the
preparation of compounds of fo~mllla ~I) include acid
.
~3~
halides, particularly acid chloride~ or bromides. Such
acylating agents may be prepared by reacting an acid (III)
or a salt thereof with a halogenating age.nt e~g. phosphorus
pentachloride~ thionyl chloride or oxalyl chloride
Acylations employing acid halides may be
effected in aqueous an~ non-aqueous reaction media,
convenie~tly at temperatures of from ~50 to ~50C9
preferably -20 to ~30C, if desired in the presence of
an acid binding agent. Suitable reaction media include
aqueous kPtones such as aqueous acetone, esters such as
ethyl acetate, halogenated hydrocarbons such as methylene
chloride9 amides such as dimethylacetamide, nitriles such
as acetonitrlle, or mixtures of two or more such solvents.
Suitable acid b.inding agents incl~lde tertiary amines
(eOg~ triethylamine or dimethylaniline)~ i.norganic bases
( eOg~ calci~m carbonate or sodi~n bicarhonat:e) 7 and
oxiranes such as lower 1~2~-allcylene oxides (e~gO ethylene
oxide or prop~lene oxide3 which bind hydrogen halide
liberated in the acylation reaction.
Acids of formula SIII~ may themselves be used
as acylating agents in the preparation of compounds of
formula (I). Acylations employing acids (III) are
desirably conducted .in ~he presence of a condensing agent,
for example a carbodiimide such as N,N'-dicyclohexyl-
carbodiim1de or N~ethyl~N~-y-dimethylaminopropyl-
carbodiimide; a carbonyl compound such ascarbonyldiimidazole; or 2n ~soxazoliu~ salt such as
N-ethyl-5-phenylisoxazolium pe-rchlorateO
Acylation may also be effected with other
amide-forming derivatives of acids of formula (III~ such
as~ for example~ an activated ester9 a symmetrical
`s
~3~
anhydride or a mixed anhydride (e.g. forr~3d with pivalic acid or with a halo-
formate, such as a lcwer alkylhaloforrnate). Mixed anhydrides may also be forr~d
with phosphorus acids (for example phosphoric or phosphorous acids), sulphuric
acid or aliphatic or arcmatic sulphonic acids (for example toluene-p-sulphonic
acid). An activated ester may conveniently be formed in situ using, for example,
l-hydroxybenzotriazole in the presence of a condensing agent as set out above.
- Alte m atively, the activated estex may be preformed.
Acylation reactions involving the free acids or their above-nEntioned
amide-forming derivatives are desirably effected in an anhydrous reaction medium,
e.g. methylene chloride, tetrahydrofuran, dimethylformamide or aoetonitrile.
If desired, the acylation reactions mc~y 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 orm of their acid c~ddition
salts. Thus, or example, acid chlorides mc~y conveniently be employed as their
hydrochloride salts, and acid bromides as their hydrobromide salts.
The reaction product may be separated from the rec~ction mixture, which
may contain, for example, unchanged caphalosporin starting material and other
substances, by a variet~ of prccesses incl~lding recrystallisation, ionophoresis,
column chrcmatography and use of ion-exchangers (for example by chromatography
on ion-exchange resins) or macroreticular resins.
~ - 13 -
~9 3C~79
~ -Cephalosporin ester derivatives obtained in accordance ~ith the pro-
oess of the invention may be converted into the corresponding Q3-derivatives by,
for example, treatment of the A -ester with a base, such as pyridine or triethyl-
amine.
A ceph-2-em reaction product may also be oxidised to yield the corres-
ponding ceph-3-em l-oxide, for example by reaction with a peracid, e~g. per-
acetic or m-chloroper~enzoic acid; the resulting sulphoxide may, if desired, sub-
sequently be reduoed as described hereinafter -to yield the corresponding ceph-3-
em sulphide.
Where a ccmpovnd is obtained in which B is ~O -this may be converted
to the corresponding sulphide by, for example, reduction of the corresponding
acyloxysulphonium or alkoxysulphonium salt prepared in situ by reactioll with e.g.
aoetyl chloride in the case of an acetoxysulphonium salt, reduction being
effected by, Eor example, sodium dithionite or by iodide ion c~s in a solution of
potassium iodide m a water-miscible solven-t e.g. acetic acid, acetone, tetra-
hydrofuran, dioxan, dimethylformamide or dirnethylacetamide. me reaction may ke
effected at a temperature of from -20 to +50 &.
Metabolically labile ester derivatives of the compounds of formwla (I~
rnay be prepared ky reacting a compound of form~la (I) or a salt or protected
derivative thereof with the appropriate esterifying agent such as an acyloxy-
alkyl halide (e.g. iodide) conveniently in an inert organic solvent such as
dimethylformamide or acetone, followed, where necessary, by removal of any pro-
tecting groups.
Base salts of the compounds of formula (I) rnay be formed by reacting
- 14 -
~. .,
, .
;, .
~L3~ 79
an acid of ormula (I) with the appropriate base. Thus, for exc~mple, 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 camr
pound of formula (I) or a metabolically labile ester derivative thereof with the
appropriate acid.
: Where a cQmpound of formula (I) is obtained as a mixture of isomers,
the syn isomer may be obtained by, for example, corlventional methods such as
: crystallisation or chrQmatography.
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 isomers
form or in the form of mixtures of the syn isomers and the cor:resp~nding anti
isQmers containing at least 90% of the _y~ isomer are preferably used.
Acids oE forrnula (III) (provided that Ra and ~ together with the
carbon ato.m to which they are attached do not form a cyclopropylidene group) may
be prepared by etherification of a compound of formula
(IV)
S N
.COOR
N\ OH
. ' - ' ~ .:
.
~L~3~'~9
(wherein R3 is as herein~efore defined and R represents a carboxyl blocking
group), by reaction with a compound of general formula
Ra
T.C.COOR (V)
R
(wherein Ra and Rb are as hereinbefore defined and T is halogen such as chlor~,
bromo or iodo; sulphate; or sulphonate such as tosylate), follcwed by removal of
the carboxyl blocking group R . Separation of isomers may be effected either
; before or after such etherification. The etherification reaction is generally
carried out m the presence of a base, e.g. potassium carbonate or sodium hydride,
and is preferably oonducted 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
oxyinuno groups 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 (IV~ 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
ocmpound of formula
- 16 -
':' ' , : -
,
R
S N
~ ~ CO.COOR
(wherein R3 and R4 are as hereinbefore defined) wi~h a ccmpound of formula
Ra
H2N.O.C.COOR (VII)
R
(wherein Ra, Rb and R2 are as defined above), followed by removal of ~he carboxyl
blocking group R4, and where necessary by the separation of ~y~ and anti isomers.
The last mentioned reaction is particularly applicable to the prepara-
tion of acids of formula (III) wherein Ra and Rb together with the carbon atom
to which they are attached Eorm a cyclopropylidene group. In this case, the
relevant compounds of formula (VII) 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-oxycyclopropane c æboxylate.
The acids of formula (III) may be converted to the correspo~ding acid
, ~
` ~L3 ~'~
halides and anhydrides and acid addition salts by conventional methods.
It should be appreciated that in same of the above transformatians it
may be ne oe ssars~ to protect any sensiti~e groups in the molecule of the com-
pound in question to avoid undesirable side reactians For example, during any
of the reaction sequen oes referred to above it may be ne oessary to protect the
NH2 group of the ~ninothiazolyl moietyr for example by tritylation/ acylation
(e.g. chloroaoetylation), protonation or other conventional method. The protect-
ing group may thereafter be removed in any convenient wa~ which does not cause
breakdown of the desired comQound, e.g. in the case of a trityl group by using
an optionally halogenated carboxylic acid, e.g. acetic acid, formic acid,
chloroacetic acid or trifluoroacetic acid or using a mineral acid, e.g~ hydro-
chloric acid or mixtures of such acids, preferably in the presence of a protic
solvent such as water, or in the case of a chloroacetyl group, by treatment with
thiourea.
Carbo~yl blocking groups used in the preparation of compounds of
formula ~I) or in the pr~paration of necessary starting materials are desirably
groups which may readily be split off at a suitable stage in the reaction
sequence, conveniently at the last stage~ It may, hcwever, be convenient in
some instances to e~,ploy non-toxic metabolically labile carboxyl blocking groups
such as acyloxy-methyl or -ethyl (e.g. acetoxy-methyl or -ethyl and pivaloyloxy-
methyl) and retain these in the final prDduct to give an appropriate ester
derivative of a compound of formula (I).
- 18 -
~3~;27~
- lg -
Suitable carboxyl blocking groups a~e well
known in the art, a list of representative blocked
carboxyl groups being included in British Patent NoO
1,399,086~ Preferred blocked carboxyl groups include
5 aryl lower alkoxycarbonyl groups such as p methoxybenzyl-
oxycarbonyl, p-nitrobenzyloxycarbonyl and diphenyl-
methoxycarbonyl; lower alkoxycarbonyl groups such as
t-butoxycarbonyl; and lower haloalkoxycarbonyl groups
such as 2,2,2-trichloroethoxycarbonyl. Carboxyl
blocking group(s) may su~sequently be removed by any of
the appropriate methods disclosed in the literature; thus,
for example, acid or base catalysed hydrolysis is
applicable in manycases, as are enæymically-catalysed
hydrolyses,
The antibiotic compounds of the invention
may be formulated for administration in any convenient
way9 by analogy with other antibiotics and the invention
therefore includes within its scope pharmaceutical
composikions comprising an antibiotic com~ound in
accordance with the invention adapted for use in human or
veterinary medicineO Such compositions may be presented
for use in conventional rnanner with the aid of any
necessary pharmaceutical carriers or excipients.
The antibiotic compounds according to the
invention ma~ be ormulated for injection and may be
presented in unitdoseform in ampoules, or in multi dose
containers~ if necessary with an added preservative.
The compositions may also take such forms as suspensions9
solutions, or emulsions in oily or aqueous vehicles9 and
may contain formulatory agents such as suspending,
. .
stabilising and/or dispersing agen-ts. Alternatively the active ingredient may
be in powder form for reconstitution with a sui-table vehicle, e.g. sterile,
pyrogen-free water, before use.
If desired, such p~wder fonmulations may contain an appropriate non-
toxic base in order to improve the water-solubility o~ -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 pcwder is reconstituted. The
base may be, for example, an inorganic base such as sodium carbonate, sodium
bic æbonate or sodium acetate, or an organic base such as lysine or lysine
acetate.
The antibiotic compounds may also be formulated as suppositories, e.g.
containing oonventional suppository bases such as cocoa butter or other
glycerides.
Compositions for veterinary medicine may, for example, be form~lated
as intramammary preparations in either long acting or quick-release bases.
The ccmpositions may oontain from 0.1% upwards, e.g. 0.1-99% of the
active material, depending on the method of administration. When the composi-
tions co~lprise dosage unitsr each unit will preferably contain 50-1500 nxJ of the
active ingredient. The dosage as employed for adult human treatment will pre-
ferably range from 500 to 6000 mg per day, depending on the route and frequency
of administration. For example, in adult human treatment 1000-3000 mg per day
administered intravenously or
- 20 -
:
~ : .
-21~
intramuscularly will normally suf~iceO In treating
infections higher daily doses may be
required.
The antibiotic compou~ds according to the
invention may be administered in comb:ination 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).
~11 3~ 79
Preparation 1
Ethyl (Z)-2-(2-aminothiazol-4-yl)-2-(hydroxyimino)acetate
To a stirred and ice-cooled solution of ethyl acetoacetate (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 abo~lt 30 min., when a solution of
potassium chloride (160 g) in water (800 ml) was a~ded. The resulting mLxture
was stirred for one hour~ The lower oily phase was separated and the a~ueous
phase was extracted with diethyl ether. The extract was ocmbined with the oil,
washed successively with water and saturated brine, dried, and evaporated. me
residual oil, which solidified on standing, was washed with petrol and dried Ln
va over potassium hydro~ide, giving ethyl (Z)-2-(hydroxyimino)-3-oxobutyrate
(309 g)-
A stirred and ice-oooled solution of ethyl (Z)-2-(hydroxyimino)-3-
oxobutyrate (150 g) in dichlorcmethane (400 ml) wa~s ~reated dropwise with
sulphuryl chloride (140 g). m e resulting solution was kept at room temperature
for 3 days, then evaporated. The residue w~s dissolved in diethyl ether, washed
with water until the washings 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 pro-
duct was collected by filtration, washed with ethanol and dried to give the
title compound (73 g); m.p. 188 (deoomp.).
- 22 -
-
~ .
~L3~
Preparation 2
Ethyl (Z)-2-hydroxyimino-2-(2--tritylaminothiæ ol-4-yl)-acetate, hydrochloride
l~ityl 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 triethyL1mine (8.4 ml). me mixture was
allcwed to warm to 15 over one hour, stirred for a further 2 hours and then
partitioned between water ~500 ml) and ethyl aoetate (500 ml). me 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 (Z)-2-(2-t-butoxycarbonylprop-2-oxylmino)-2-(2-tritylaminothiazol-4-yl)
acetate
Potassium carbonate (34.6 g) and t-butyl 2-brcmo-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 for 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 frcm petroleum ether
(b.p~ 60-80) to give the title compound (34 g), m.p. 123.5 to 125.
- 23 -
. ~,
., ~ ~ ,
.
Preparation 4
(Z)-2-(2-t-Butox~carbonylprop-2-oxyimino)-2-(2-tritylaminothiazol-4-yl)acetic
acid
The product of Preparation 3 (2 g) was dissolved in methanol (20 ml)
and 2N sodium hydroxide (3.3 ml) was added. me mixture was refluxed for 1.5
hours and then concen-trated. The residue was taken up in a mlxture of water
(50 ml), 2N hydrochloric acid (7 ml), and ethyl aoe tate (50 ml). m e organic
phase was separated, and the aqueous phase extracted with ethyl acetate. The
organic solutions were cambined, washed suc oessively with wa-ter and saturated
brine, dried, and evaporated. m e residue was recrystallised frcm a mixture of
caxbon tetrachloride and petrol to give the title ccm~ound (1 g), m.p. 152 to
156 (decamp).
Prep æ ation 5
Ethyl (Z)-2-(2-tritylam mothiazol-4-yl)-2-tl-t-butoxycarbonylcyclobut-1-oxyimino)
aoetate
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
roam temperature. After 30 minutes, t-butyl l-bramocyclobutane carboxylate
(29.2 g) was added. After 8 hours further 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-bramocyclobutane 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 washed well with
- 24 -
~3~9~
water and petrol. The solid was dissolved in ethyl acetate and the solution
washed with brine (twice), dried with magnesium sulphc~te and evaporated to a
foam. mis 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, ~max (CHBr3) 3400 (NH) and 1730 cm 1 (ester).
Preparation 6
(Z)-2-(1-t-Butoxycar~onylcyclobut-l-ox_1mino)-2-(2-tritylaminothiazol-4-yl)
acetic acid
:
A mi~ture of the product of Preparation 5 (3.2 g) and potassium
carbonate (1.65 g) was refluxed in methanol (180 ml) and water (20 ml) for 0
hours and the mixture was cooled to roam temperature. The mixture was concen-
trated 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 aqueous phase
extracted with ethyl acetate. The combined organic extracts wer~ washed with
saturated brine, dried and evaporated to give the title compound (2.3 g); ~max
(ethanol) 265 nm OElcm 243).
Preparation 7
Ethyl(Z)-2-(1-t-butoxycarbonylcyclopent-1-yloxyimino)-2-(2-tritylam~nothiazol-4-
yl)a oe tate
me product of Preparation 2 (10 g) was stirred with t-b~Ityl 2-bromo-
cylcpentanecar~oxylate (7 g) in dimethylsulphoxide (40 ml) containing potassium
carbonate (10 g) under nikrogen ak 21 for 21 hours. The mixture was poured
into ice-water (500 ml) and -the grey solid was collected by filtration, washed
wikh water and air dried.
~36~27~
2~ -
Recrystallisation of this solid from methanol ~500ml~ gave
the title ~ (11~7g)~ m.pO 179-~180, vmax ~CHBr3)
3410 (NH), 1735 (ester)~ 1275 (ester) and 755 cm ~phenyl)~
~.
S
~.
The Product rom Preparation 7 (625mg) was refluxed with
2N sodium hydroxide solution (O.5ml~ and water ~lml) in
methanol (12ml) for seven hours. The mixture was left
to cool overnight. After dilution with water,
orthophosphoric acid was added to adjust the solution to '
pH 2O The precipitate was extracted with ether and the
combîned extracts were washed with brine~ Ater drying
with magnesium sulpha~e, the solven~ was evaporated to g;.ve
a gum (493mg), Recrystallisation from di-isopropyl
ether gave the title ~ (356mg) mOpO 171-173,
~m~x (CHBr3) 2500-3500 (OH and NH), 1755 (ester), 1692
(acid) and 755 and 770 cm (phenyl)O
:,
- 27
Example 1
r~ony~prop-
2 ox~imino~-2- ~-trit~laminothiazol 4-Yl? acetamido~-3
m ~
A stirred solution of the product of Preparation 4
(2.~6 g) and diphenylmethyl (6R, 7R~-7 amino-3-methyl-
ceph-3-em-4-carboxylate (2~09 g) in dimethylformamide
(50 ml) was cooled to 0' and treated with hydroxybenzo-
triazole ~745 mg) and dicyclohexylcarbodiimide (1.14 g)O
The mixture was warmed to room temperature and stirred
overnight. The mixture was filtered, and the white solid
washed with a little ethyl acetate. The filtrate and
washings were dilutecl with water (100 ml) and extracted
with ethyl acetate, The organic extracts were combined,
washed successively with 2N hydrochloric acid, water,
sodium bicarbonate solution, and saturated brine, dried,
and evaporated. The residue was eluted through a silica
column with ethyl acetateO The product containing eluate
was collected and concentrated to give the_title
(3.6 g)~Amax(ethanol) 238 nm (El 283~, A inf
263.5 nm (E1%186); ~ D0 ~ 6.5- (c 1.0, DMSO).
b ~ )-7-~ Z)-2- ~ 4 ~ 2~ 2-carboxyprop-
2-oxyimino) acetamido ~ -4-carbox~lic
acid
_
Trifluoroacetic acid (20 ml) was added to a solution
of the product of stage a) (2.8 g) in anisole (20 ml) at 0-.
The mixture was stirred at room temperature for 2 hours and
concentrated. The residue was dissolved in ether and
re-evaporated, The residue was dissolved in ethyl acetate
and exLracted with saturated sodium bicarbonate solution.
:
- 2~ ~
The pH of the aqueous ex~racts wa~s adjuted to 6, and the
solut;on washed with ethyl acetate The aqueous phase
was acidified to pH 1 .S under ethyl acetate 9 and extracted
with ethyl acetate The combined org~nic extracts were
washed with saturated briney dried 9 and evaporated
The residue was dissolved in warm 75% aqueous formic acid
(40 ml) and allowed to stand for 1 hour~ The mixture was
diluted with water and filtered, The filtrate was
concentrated, The residue was taken up in water,
refiltered, and lyophilized to give ~ ~md
; (900 mg), ~. f(pH 6 buffer) 236 nm ~El~ 287)~ 261 nm
n ~O/ c
(El' 254), 296 nm (ElCmll5); vmax (Nujol~ 1530, 1665
(CONH), 1720 ~C02H), 1765 cm (~-lactam~O
Exc~mple 2
~L~L~
cyclob t-l-ox~imino)-2-~2-tritylAminothiAzol~4 ~:L~-
_
acetamido1-3~ L ~ ~w ~ç~b~x~
A stirred solution of the product of Preparation 6
(2.2 g) and diphenylmethyl ~6R~7R~-7-amino-3-methylceph~
3-em-4~carboxylate (2.2 g) in dimethylformamide (45 ml)
was cooled to 0-~ and l-hydroxybenzotriazole (655 mg)was
added, followed by dicyclohexylcarbodiimide (982 mg)O
The mixture was warmed to room temperature and sti.rred
overnight. The mixture was filtered. The filtrate was
diluted with water (300 ml) and extracted with et.hyl acetate.
The organic extracts were combined, washed successively with
2N hydrochloric acid, sodium bicarbonate solution, and
saturated brine, dried, and evaporated. The residue was
eluted through a silica column with ether-petrol (3-1).
The app~opriate ractions were concen~rated to give
~3~27~
- 29-
the title compound ~12vjl g~, Ainf (~ElCm238), 302 nm (ElCm 67); v max (CHBr3) 1520, 1682
(CONH), 1722 (C02R), 1788 cm ~ lactam).
b~ ~6R~7~)-7-r~2)-2-(2-Aminothiazol-4-yl?-2~ carboxy
~ lceph-3-em-4-
carbox~lic acid
Trifluoroacetic acid (8 ml) was added to a solution
~-~ of the product of Stage a)(l.9 g) in anisole (2 rnl) at 0-.
The mixture was stirred for 5 minutes and trifluoroacetic
acid (32 ml) was added. Tne mixture was stirred at room
temperature for 30 minutes and concentrated. The residue
was dissolved in ethyl acetate and re-evaporated. The
: residue was dissolved in ethyl acetate ~nd extracted with
saturated sodiurn bicarbonate solution, The aqueous extracts,
at pH 7 to 7,5, were washed with ethyl acetate, acidified
to pH 1.5 under ethyl acetate, and extrac~ed with ethyl
acetate. The combined organic extracts were dried and
; evaporated. The residue was dissolved in formic acid
(30 ml), water (9 ml) added, and the mixture stirred at
room temperature ~or 2 hours. The mixture was diluted with
water (200 ml) and filtered. The filtrate was concentrated.
The residue was taken up in water, refiltered, and lyo-
philized to give the title ~e~ (730 mg)9 Amax
(pH 6 bufer) 241 nm (E~% 272), Ainf252 nm (ElCm 269),
291 nm (ElCm 129); [a~ D ~ 62.5- (c 1 0, DMSO).
E~e~
,,
a) Diphenylmethyl ~6R~7R)-7-r~Z)-2-(l-t-butoxycarbonylcyc-
lo,Pent-l-yloxyimino~-2-(2~trit~1aminothi.azol 4-yl)acetamido
o
Th~ product of Preparation ~ (lg) was dissolved in tetra-
- hydrofuran ~25ml) and diphenylmethyl (6R,7R)-7 amino-3-
~L~3~;~7~
methylceph-3 em-4~carbo~ylate (76~mg~ and l~hydroxybenzo-
triazole hydrate (380mg) were added with stirring. When a
clear solution had formedg dicyclohexylcarbodiimide
(520mg) was added and the mixture was stirred at 21~ for
24 hours. The solution was filtered and the filtrate was
concentr~ted, This solution was percolated through a short
column of neutral alumina (30g) with elution by ethyl
acetate: 60~-80~ petroleum ether (1~3). Ihe product was
loaded onto a column of silica ~kieselgel 60G of 15~M
particle size; SOg), This column was eluted with ethyl
acetate: 60-80- petroleum ether (1:3) at a pressure of
81b/sq. in. and e~aporation of the appropriate fractions
gave the title compound ~SlOmg) as a oam, ~JD
~ 25-2 ( 0~95, CMC13), vmax (CHBr3) 3405,3275 ~NH),
1790 (~-lactam), 1727 (esters), 1683 and LS27 cm~l (amide).
~ othiazol~y~l) ~ rboxy~yc-
carboxylic acid,_trifluoroace ate salt.
The product from Stage a) (408mg) was mixed with
anisole (0,3ml) and trifluoroacetic acid (2ml~ was added.After 2 hours at 21-, the solution was concentratecl and the
residue was poured ;nto water (20ml). This mixture was
washecl with ether (three times) each time the ether being
back extracted with water, The combined a~ueous layers were
evaporated to dryness and the residue was triturated with
ether to give the ~L~ (88mg) Am~X ~pH6 buffer)
239,5nm (E %304)~ A, 290 (E V/ol3l~ v (NuJol) 3700-
, lcm lnfl J l~m max -1
2100 (NH3, NH and OH), 1770 ~-lactam~ and 7 680 cm
(amide and acid)O
~3~'æ7~
31
PHARMACY ~AMPLES
Formula Per Vial
(6R, 7~)-7-~(Z)-2-(2-Aminothia~ol 4 yl)-2-(2-carboxyprop
5 -2-oxyimino)acetamido]-3-methylceph 3-em-4-carboxylic
acid 500mg
Sodium Carbonate3 anhydrous 113mg
Method
Blend the sterlle cephalosporin antibiotic with sterile
10 sodium carbonate under aseptic conditions. Fill.
aseptical~y into glass vials under a blanket of sterile
nitrogen. Close the vials using rubber discs or plugs~ .
held in position by aluminium overseals, thereby
preventing gaseous exchange or ingress of micro-organi.sms.
Reconstitute the product by dissolving in Water for
Injections or other suitable sterile vehicle shortly
before administrat ion .
EXAMPLE B - Dry ~
Fill sterile (6R, 7R)-7-[(~)-2-(2-aminothiazol-4 yl)
~2-(1-carboxycyclob~t-1-oxyimino)acetamido]~3-
methylceph-3-em-4-carboxylic acid9 disodium salt into
glass vials such that each vial contains an amount
equivalent to 500mg of the antibiotic acidO Carry out
the filling aseptically under a blanket of sterile
nitrogen, Close the vials using rubber discs or plugs,
held in position by aluminium overseals 9 thereby
:L~3~Z7~
- 32 -
.
preventing gaseous exchange or ingress of micro-organisms~
Reconstitute the product by dissolving in Water for
Injections or other suitable sterile vehicle shortly
before administration.
