Note: Descriptions are shown in the official language in which they were submitted.
11183`19
This application is a division of Canadian Patent Application No.
301,66Z, filed April 21, 1978.
The present invention relates to synergistic pharmaceutical
compositions containing ~-lactam antibacterial agents.
Canadian Patent Application No. 263,103 (see also Belgian Patent No.
847,044 and West German Offenlegungsschrift No. P2646003.7) discloses inter
alia the compounds of the formula (I):
CH2XI
2-N (I)
CO2H
and esters thereof wherein Xl is hydrogen atom, an alkyl group of up to 5
carbon atoms, an alkenyl group of up to 5 carbon atoms, a hydroxy alkyl group
of up to 5 carbon atoms or an optionally substituted phenyl group and X2 is
an optionally substituted phenyl group, such compounds were described as
antibacterial agents and ~-lactamase inhibitors.
It has now been discovered that certain secondary amines can be
prepared that are ~-lactamase inhibitors that enhance the effectiveness of
penicillins or cephalosporins and which also have antibacterial properties in
their own right.
The parent application noted above describes a compound of the
formula (II):
-- 2 --
;~
~ 11183 19
Fl_ ~H -N H--CH - R ( I I )
O C02H
or an ester thereof wherein Rl is a hydrogen atom, an
alkyl group of up to 5 carbon atoms, a cycloalkyl group
of 5 or 6 carbon atoms, a hydroxyalkyl group of up to
5 carbon atoms or a ~oiety of the sub-formula (a):
R2
3 (a)
~ Rg
wherein R2 is a hydrogen, fluorine, chlorine or bromine
atom or an alkyl group of 1-3 carbon atoms, an alkoxyl
group of 1-3 carbon atoms, an acyloxyl group of 1-3 carbon
ato~s,a hydroxyl group, an alkoxycarbonyl group containing
1-3 carbon atoms in the alkoxy part, or a group -N(R5)CO.R6,
~ -N~R~5)502R6 or -CO-WR5R6 where R5 is a hydrogen atom or an
: : alkyl group of 1-3 carbon atoms or a phenyl or benzyl group
: and R6 is an alkyl group of 1-3 carbon atoms or a phenyl or
; benzyl group; R3 is a hydrogen, fluorine or chlorine atom
or an alkyl group of 1-3 carbon atoms, an alkoxyl group of
: 15 1-3 carbon atoms or an acyloxyl group of 1-3 carbon atoms;
and R4 is a hydrogen fluorine or chlorine atom or an alkyl
group of 1-3 carbon atoms or an alkoxyl group of 1-3 carbon
atoms.
: - 3 -
. ~- . :
:.
~ . . ,: . :; ,. ,
:. - :. . ~
:: .: - : :
, : : ~
~ 11183~9
The compounds of the formula (II) per se exist in
t;he form of ~witterions, that is they may be represented
as shown in formula (IIa):
CH2-NH2-CH2 R1 (IIa)
~ N
0/ C02~
lf desired wherein Rl is as defined in relation to formula
(II). These zwitterionic compounds form a favoured aspect
of this invention in view of their generally crystalline form
and their greater stability than previously reported
~-lactamase inhibitory amines such as those of the formula
(I).
The esters of the compounds of the formula (II) may
be presented in the form of the free base or in the form
of an acid addition salt.
Suitably Rl is a hydrogen atom. Suitably Rl is
an alkyl group of up to 5 carbon atoms. Suitably Rl is
a hydroxyalkyl group of up to 5 carbon atoms. Suitably
R1 is a phenyl group optionally substituted by a fluorine,
chlorine or bromine atom or an alXyl or alkoxyl group of
up to 3 carbon atoms.
Apt groups R1 include the methyl, ethyl, propyl,
butyl, hydroxymethyl, hydroxyethyl, hydroxypropyl, phenyl,
p-methoxyphenyl, p-methylphenyl and the like groups.
Certain particularly apt groups Rl include the methyl, ethyl
..... . .. .... . . . .
!
~ lil83 ~9
, ,
hydroxymethyl, 2-hydroxyethyl, isopropyl and phenyl groups.
A group of suitable compounds of ~his invention are
those of the formula (II~ or an ester thereof wherein Rl
is a hydrogen atom an alkyl group of up to 5 carbon atoms,
a hydroxyalkyl group of up to 5 carbon atoms or a moiety of
the sub-formula (b):
R2
~ R4 (b)
wherein R2 is a hydrogen, fluorine, chlorine or bromine
atom or an alkyl group of 1-3 carbon atoms, an alkoxyl
group of 1-3 carbon atoms, an acyloxy group of 1-3 carbon
atoms, a hydroxyl group or an alkoxycarbonyl group containing
1-3 carbon atoms in the alkoxy part; R3 is a hydrogen,
fluorine or chlorine atom or an alkyl group of 1-3 carbon
atoms, an alkoxyl group of 1-3 carbon atoms or an acyloxyl
group of 1-3 carbon atoms; and R4 is a hydrogen, fluorine
or chlorine atom or an alkyl group of 1-3 carbon atoms or
an alkoxyl group of 1-3 carbon atoms. As described above
these compounds may be in the form of the zwitterion or an
ester or an acid addition salt of said ester. Suitable
and apt values for Rl include those set forth hereinbefore
in relation to formula (II).
One favoured sub-group of compounds within formula
~II) include those of the formula (III):
-- 5 --
.. , , . . ~
.
" -
: :
~' .
11183~9
~;
~H -NH-CH ~ R4 (III)
N
O t~O2H
and esters thereof wherein R2, R3 and R4 are as defined
hereinbefore.
These compounds of the formula are favourably in the
form of the zwitterion for reasons hereinbefore indicated.
The compounds of the formula (III) may be presented in the
form of the ester and suitably that ester is in the form of
its acid addition salt.
More suitably R2 is a hydrogen, fluorine or chlorine
atom or a methoxyl, ethoxyl, hydroxyl, acetoxyl, propionyloxy,
methyl, ethyll methoxycarbonyl or ethoxy-carbonyl group.
More suitably R3 is a hydrogen, fluorine or chlorine
atom or a methoxyl, ethoxyl, acetoxyl, propionoxyl, methyl
or ethyl group.
More suitably R4 is a hydrogen, fluorine or chlorine
atom or a methoxyl, ethoxyl, acetoxyl, propionoxyl, methyl
or ethyl group.
Most suitably R2 is a hydrogen, fluorine or chlorine
atom or a methoxyl, hydroxyI or methyl group.
Most suitably R3 is a hydrogen, fluorine or chlorine
atom or a methoxyl or methyl group.
- 6
! ,
.' ` ` '
'
' . . .
~ 11183 ~
Most suitably R4 is a hydrogen atom or a methyl
or methoxyl group
Preferably R2 is a hydrogen, fluorine or chlorine
atom or a methyl or methoxyl group.
Preferably R3 is a hydrogen atom or methoxyl group.
Preferably R4 is a hydrogen atom.
The compounds of this invention and particularly
those of the formula (III) show a broad spectrum of
~ lactamase inhibitory activity.
Certain particularly favoured compounds of the formula
(III) include those of the formula (IV):
H Q
~ CH -NH-CH ~ (IV)
,~N
o C02H
wherein Q is a hydrogen, fluorine or chlorine atom or a
methyl, methoxyl, ethyl or ethoxyl group.
Suitably Q is a hydrogen, ~-fluorine, m-fluorine,
p-chlorine or _-chlorine atom or a ~-methyl, m-methyl,
~-methoxyl or m-methoxyl group.
Most suitably Q is a hydrogen, ~-fluorine or
p-chlorine atom or a ~-methyl or ~-methoxyl group.
A compound of the formula (IV) which has shown
particularly good synergistic activity in-vlvo is that
,.. ,". ... . ..... . . ..
., - - .
- : ,' ''' .' ~ ` :
~., ; , .
' -` ';
: '.' '- '
~ 11183`1~
wherein Q is a hydrogen atom. This compound is able to
enhance the effectiveness of penicillins, such as ampicillin
or amoxycillin, and cephalosporins against various
~-lactamase producing strains of gram~negative bacteria
including strains of Klebsiella aerogenes, Escherichia coli,
Proteus mirabilis and the like and especially against
~-lactamase producing strains of gram-positive bacteria
such as Staphylococcus aureus when administered orally and
especially when administered by injection. The compounds
of the formula (IV) also have an advantageously low acute
toxicity r for example no deaths in test animals were observed
when administering therapeutic amounts of the synergist.
In addition such compounds are effective when used alone
in treatment of infections due to ~-lactamase producing
as well as non-~-lactamase producing strains of Staphylo-
coccus aureus. Thus, for example, the compound of the
formula (IV) wherein Q is a hydrogen atom has proved more
effective than ampicillin, cloxacillin or cefazolin in
::
treating certain infections due to Staphylococcus aureus
Russell.
; A further favoured sub-group of compounds within
formula (II) having similar properties to that of the
sub-group of the formula (IV) is that of the formula (V):
H
~CH2-NH-CH
~ N
0 ~02H
- -- 8
: ~ : : , . -- ,
.
. :;
'
r~
,~ 1118349
. .
wherein Ql is a hydrogen r fluorine or chlorine atom or
a methyl, ethyl, methoxyl, ethoxyl or hydroxyl group.
Suitably the OH substituent shown in formula ~VIa)
is para- to the carbon to which the -NH-CH2- moiety is
attached.
Suitably the OH substituent shown in formula (VIa)
is meta- to the carbon atom to which the -NH-CH2- moiety
is attached.
Most suitably Ql is a hydrogen atom or a methyl
or methoxyl group.
The zwitterionic compounds of the formula (IV) and
(V) are normally and preferably in crystalline form.
A further sub-group of favoured compounds of this
invention are those of the formula (II) wherein R2 is a
group N(R5)CO.R6, N(R5)S02R6 or CONR5R6 wherein R5 and R6
are as defined in relation to formula (II) and esters
thereof. Suitably R5 i5 a hydrogen atom. Suitably R5
is an alkyl group of 1-3 carbon atoms such as the methyl
group. Suitably R6 is an alkyl group of 1-3 carbon atoms.
More suitably R6 is a methyl group. Suitable values for
R3 and R4 in such compounds are those specified in relatlon
to the compounds of the formula (II). Such compounds may
be in the form of zwitterions of the parent acid. Esters
of such compounds may be in the form of the free base or
may be in the form of an acid addition salt.
A further particularly favoured sub-group of the
compounds of the formula (II) is that of the formula (VI):
:~' '',' ' ~; ,' ' `
: . .
.- : ~ ~ . ' .
~ ~1183 ~9
H f~N ~R5 ) CO . R6
H2-NH-CH2 ~ R4 tVI)
H
C02H
wherein R3, R4, R5 and R6 are as defined in relation
to formula (II).
The compounds of the formula (VI) are generally
produced in crystalline form and in better than average
yield and have similar activity to those of the formula
(IV) and (V).
In relation to the compounds of formula (VI) it is
more suitable that R5 is a hydrogen atom or an alkyl group
of 1-3 carbon atoms such as a methyl group and most suitably
R5 is a hydrogen atom. In relation to the compounds of
the formula (VI) it is more suitable that R6 is an alkyl
group of 1-3 carbon atoms such as the methyl group.
Favoured values for R3 and R4 for the compounds of
the formula (VI) are as defined in relation to formulae
(II) and (III). Preferably R3 and R4 are both hydrogen atoms.
Suitably in the compounds of the formula (VI) the
-N(R5)COR6 moiety is attached para to the -NH-CH2- moiety.
Suitably in the compounds of the formula (VI) the
-N(R5)COR6 moiety is attached meta to the -NH-CH2- moiety.
A favoured sub-group of compounds of the formula
(VI) are those of the formula (VII):
-- lo --
~, ~ . '-
:
~ 83~9
CH -NH-CH ~ N(R5)COR6
~ N
O C02H
wherein R5 and R6 are as deflned in relation to formula (II).
Particularly suitably R5 is a hydrogen atom or an alkyl group
of 1-3 carbon atoms such as a methyl group. Preferably R5
is a hydrogen atom. Particularly suitably R5 is an alkyl
group of 1-3 carbon atoms and preferably a methyl group.
As has been previously indicated we prefer to prepare
and use the crystalline zwitterionic compounds within formula
(II) such as those of the formulae (III), (IV), (V), (VI)
and (VII) and the like. However, esters of the compounds
of the formulae (II)-(VII) also form part of this invention,
for example as the free base or as the acid addition salt,
since such compounds may also be used to enhance the
effectiveness of penicillins or cephalosporins.
Certain suitable esters of the compounds of the
formula (II)-(VII) include those of the formula (VIII) and
(IX):
H H~
~CH2-NH-CH2 Rl r r~ ~ CH2-NH-CH2 R
N ~ N
C2A1 C02CHA2A3
(VIII) ~IX)
' ' ' '` ~
~ 11183~9
~i. ,,
wherein Rl is as defined in relation to formula (II) or
:Ls a substituted phenyl group as present in a compound of
the formula (III)-~VII) wherein Al is an alkyl group of
1-6 carbon atoms optionally substituted by an alkoxyl or
acyloxyl group of 1-7 carbon atoms; A2 is an alkenyl or
alkynyl group of up to 5 carbon atoms or is a phenyl group
optionally substituted by a fluorine, chlorine, bromine,
nitro or alkyl or alkoxyl of up to 4 carbon atoms; and
A3 is a hydrogen atom, an alkyl group of up to 4 carbon a
atoms or a phenyl group optionally substituted by a fluorine,
chlorine, bromine, nitro or alkyl or alkoxyl of up to
4 carbon atoms.
Suitable esters of the compounds of the formula (II)
include the methyl, ethyl, n-propyl, n-butyl, allyl,
CH2-C--CH, methoxymethyl, acetoxymethyl, propionoxymethyl,
pivaloyloxymethyl, ethoxycarbonyloxymethyl, methoxycarbonyl-
oxyethyl, ethoxycarbonyloxyethyl, dimethoxyphthalidyl, benzyl,
methoxybenzyl, ethoxybenzyl, nitrobenzyl, chlorobenzyl or
the like ester.
Certain favoured groups Al include the methyl, methoxy-
methyl, acetoxymethyl, acetoxyethyl, phthalidyl, ethoxy-
carbonyloxymethyl, ~-ethoxycarbonyloxyethyl and the like
groups.
Certain favoured groups A2 include the phenyl and
4-methoxyphenyl groups. A particularly favoured moiety A3
is the hydrogen atom.
- 12 -
11183~9
Certain other favoured values for ~ include t.hose of
the sub-formulae (c), (d) and (e):
5 6 (c)
-CHA5-COA6 (d)
S -CHA5-C02A6 (e)
wherein A5 is a hydrogen atom or a methyl group and A6 is an
alkyl group of up to 4 carbon atoms or a phenyl or benzyl group
either of ~hich may be substituted by one or two alkyl or
alkoxyl group o~ up to 3 carbon atoms or by a fluorine, chlorine
or bromine atom or a nitro group; or A5 is joined to A6 to
form the residue of an unsubstituted saturated 5- or 6-
membered heteroalicyclic ring or an ortho-phenylene group which
may be substituted by one or two alkyl or alkoxyl groups of up
to 3 carbon atoms or by a fluorine, chlorine or bromine atom
or nitro group.
An apt acylic value for the sub-group of the formula
(C) iS -CH2-OA6.
An apt acylic value for the sub-group of the formula
(d) is -CH2-c-A6
An apt acylic value for the sub-group of the formula
(e) is -CH2-C02A6.
A further apt acvlic value for the sub-group of the
: formula (e~ is -CH(CH3)-C02A6-
Favoured values or A6 in the preceding acylic moieties
include the methyl, ethyl, propyl, butyl, phenyl and benzyl
groups.
- 13 -
.-: :. ~
.
-: :. .: ~
,, :
~ ~ .
f~
~ 11:1~349
Apt cyclic values for the sub-group of the formula
(c) include the tetrahydropyranyl and tetrahydrofuranyl groups.
Esters of the compounds of the formula (II) such
as those of the compounds of the formulae (IV) or (V) may
be presented in the form of their acid addition salts if
desired. The acid used to form the salt will most suitably
be pharmaceutically acceptable, but non-pharmaceutically
acceptable acid addition salts are also envisaged, for example
as intermediates in the preparation of the pharmaceutically
acceptable salts by ion exchange. Suitable pharmaceutically
acceptable acid addition salts include those of inorganic
and organic acids, such as hydrochloric, phosphoric, sulphuric,
methanesulphonic, toluenesulphonic, citric, malic, acetic,
lactic, tartaric, propionic, succinic or the like acid.
Most suitably the acid addition salt is provided as a solid
and preferably as a crystalline solid.
Compounds of this invention wherein crystalline form
may be solvated, for example hydrated.
-- 1'1 --
~, 11183~9
The present invention provides a pharmaceutical
composition which comprise a compound of this invention and
a pharmaceutically acceptable carrier.
The compositions of the invention include those in a
form adapted for oral, topical or parenteral u$e and may be
used for the treatment of the infection in mammals including
humans.
Suitable forms of the compositions of this invention
include tablets, capsules, creams, syrups, suspensions,
solutions, reconstitutable powders and sterile forms suitable
for injection or infusion. Such compositions may contain
conventional pharmaceutically acceptable materials such as
diluents, binders, colours, flavours, preservatives,
disintegrant and the like in accordance with conventional
pharmaceutical practice in the manner well understood by
those skilled in the art of formulating antibiotics.
Injectable or infusable compositions of a compound of
the invention are particularly suitable as high blood levels
of the compound can occur after administration by injection
or infusion. Thus, one preferred composition aspect of
this invention comprises a compound of the invention in
sterile form and most suitably in sterile crystalline form.
The zwitterionic compounds of this invention are particularly
suitable for-use in such compositions.
The injectable solution of the compound of this
invention may be made up in a sterile pyro~en-free liquid
:
~ 11~8349
s~ch as water, aqueous ethanol or the like.
Compounds of this invention when in highly pure
crystalline form tend to have relatively low aqueous
solubilities so that if it is desired to administer sub-
stantial quantities of the medicament this can requirefairly large quantities of water for reconstitution. In
these circumstances it is often convenient to administer
the solution intravenously.
An alternative approach to administering the compounds
1~ of this invention and especially those zwitterionic compounds
of the formula (III)~(VII) is to utilise an injectable
suspension. Such suspensions may be made up in sterile
water; sterile saline or the like and may also contain
suspendlng agents such as polyvinylpyrrolidone, lecithin
or the like (for example in the manner described for
amoxycillin trihydrate in Belgian Patent ~o. 839109).
Alternatively such compositions may be prepared in an
acceptable oily suspending agent such as arachis oil or
its equivalent. The use of suspensions can give rise to
20 advantageously prolonged blood levels of the medicament.
Belgian Patent No. 839109 may be consulted for suitable
methods and materials for producing injectable aqueous
suspensions. For use in such suspensions the zwitterionic
compound of this invention should be in the form of fin0
particles as described in said Belgian Patent.
Unit dose compositions comprising a compound of this
~ 16 -
: . . ~: .
. .
. - ; ~.
~ ll~B349
invention adapted for oral administration form a further
suitable composition aspect of this invention. However,
orally administrable forms are generally less favoured than
injectable forms owing to the relatively poor absorption of
the compounds from the gastro-intestinal tract. Despite
this orally administrable compositions are of use as a
synergistically effective blood level can be expected at
high doses and at lower doses such compositions may be used
to treat infections localised in the gastro-intestinal tract.
Unit dose compositions comprising a compound of this
invention adapted for topical administration are also
presented by this invention. In this instance 'topical
administration' also includes local administration to
internal surfaces of mammary glands of cattle, for example
during the treatment of mastitis by intra-mammary administration.
The compound of the formula may be present in the
composition as sole therapeutic agent or it may be present
together with other therapeutic agents such as a penicillin
or cephalosporin. Considerable advantages accrue from the
inclusion of a penicillin or cephalosporin since the resulting
composition shows enhanced effectiveness (synergy).
Suitable penicillins for inclusion in the compositions
of this invention include benzylpenicillin, phenoxymethyl-
penicillin, carbenicillin, azidocillin, propicillin, ampicillin,
amoxycillin, epicillin, ticarcillin, cyclacillin, pirbenicillin,
azlocillin, mezlocillin, celbenicillin, and other known
- 17 -
_ .... . .
' ~: ' . ;.,
~- . .
~ 11183~9
penicillins including pro-dru~s therefore such as their
in-vivo hydrolysable esters such as the acetoxymethyl,
pivaloyloxymethyl, ~-ethoxycarbonyloxyethyl or phthalidyl
esters of ampicillin, benzylpenicillin or amoxycillin, and
aldehyde or ketone adducts of penicillins containing an
6-a-aminoacetamide side chain (such as hetacillin, metampicillin
and analogous derivatives of amoxycillin) or ~-esters of
carbenicillin or ticarcillin such as their phenyl or indanyl
a-esters.
Suitable cephalosporins for inclusion in the compositions
of this invention include cefatrizine, cephaloridine, cephalothin,
cefazolin, cephalexin, cephacetrile, cephamandole nafate,
cephapirin, cephradine, 4-hydroxycephalexin, cefaparole,
cephaloglycin, and other known cephalosporins or pro-drugs
thereof.
Such compounds are frequently used in the form of a
salt or hydrate of the like.
Naturally if the penicillin or cephalosporin present
` in the composition is not suitable for oral administration
then the composition will be adapted for parenteral
administration. As previously indicated such injectable
or infusable compositions are preferred.
Highly favoured penicillins for use in the compositions
of this invention include ampicillin, amoxycillin, carbenicillin
and ticarcillin. Such penicillins may be used as a pharma-
ceutically acceptable salt such as the sodium salt.
'
11183 -~9
Alternatively the ampicillin or amoxycillin may be used
in the form of fine particles of the zwitterionic form
(generally as ampicillin trihydrate or amoxycillin trihydrate)
for use in an injectable suspension, for example, in the
manner hereinbefore described for a compound of this invention.
The preferred penicillin for use in the synergistic
composition is amoxycillin, for example as its sodium salt
or trihydrate.
Particularly suitable cephalosporins for use in the
compositions of this invention include cephaloridine and
cefazolin. Such cephalosporins may be used as a pharma-
ceutically acceptable salt, for example the sodium salt.
When present together with a cephalosporin or penicillin,
the ratio of a compound of the invention to the penicillin or
cephalosporin agent may vary over a wide range of ratios,
such as from 10:1 to 1:10 for example about 3:1, 2:1, 1:1,
1:2, 1:3, 1:4, 1:5 or 1:6, (wt/wt, based on pure free
antibiotic equivalent). Orally administrable compositions
containing a compound of the invention will normally contain
relativel~ more synergist than corresponding inje~table
compositions, for example the ratio in an oral composition
may be from about 3:1 to about 1:1 whereas a corresponding
injectable composition may contain a ratio of about 1:1 to
about 1:3 (compound of the invention: penicillin or cephalo-
sporin).
The total quantity of a compound of the invention
-- 19 --
~ ~1183 :~9
in any unit dosage form will normally be between 25 and 1000 mgan~ will usually be between 50 and 500 mg, for example about
62.5, 100, 125, 150, 200 or 250 mg.
Compositions of this invention may be used for the
treatment of infections of inter alia, the respiratory tract,
the urinary tract and soft tissues in humans and mastitis
in cattle.
Normally between 50 and 1000 mg of the compounds of
the invention will be administered each day of treatment but
more usually between 100 and 750 mg of the compounds of the
invention will be administered per day, for example as 1-6
doses, more usually as 2, 3 or 4 doses.
The penicillin or cephalosporin in the synergistic
composition of this invention will normally be present at
approximately the amount at which it is conveniently used
which will usually be expected to be from about 62.5 to
1000 mg per dose, more usually about 125, 250 or 500 mg per
dose.
One particularly favoured composition of this invention
will contain from 150 to 1000 mg of amoxycillin as the
trihydrate or sodium salt and from 25 to 500 mg of a compound
of this invention.
Most suitably this form of composition will contain
a compound of the formula(III) - ~VIII).
A further particularly favoured composition of this
invention will contain from 150 to 1000 mg of ampicillin or
_ 20 -
..,. : :
: . ,:
~ffl 1118349
a pro-drug therefor and from 25 to 500 m~ of a compound of
this invention.
Most suitably this form of composition will contain
ampicillin trihydrate, ampicillin anhydrate, sodium ampicillin,
hetacillin, pivampicillinhydrochloride, bacampicillin
hydrochloride, or talampicillin hydrochloride. Most suitably
this form of the composition will contain a compound of the
formula (III)-(VIII).
Most suitably the preceding compositions will contain
from 200 to 700 mg of the penicillin component. Most suitably
the preceding composition will comprise from 50 to 250 mg of
a compound of the formula (III)-(VIII) preferably in
crystalline form.
Such compositions may be adapted for oral or parenteral
use except when containing an in-vivo hydrolysable ester of
ampicillin or amoxycillin in which case the compositions will
not be adapted for parenteral administration.
Another particularly favoured composition of this
invention will contain from 200 to 2000 mg of carbenicillin,
ticarcillin or a pro-drug therefor and from 50 to 500 mg
of a compound of the invention.
Suitably this form of composition will contain di-sodium
carbenicillin. Suitably this form of the composition will
contain di-sodium ticarcillin.
More suitably this form of the composition will contain
from 75 to 250 mg of a compound of the formula (III)-(VIII)
preferably in crystalline form. Such compositions containing
- 21 -
_ ..... , . , .. . ~ , . . . . . ~ .
.83~9
di-salts of caxbenicillin and tica~cillin will be adapted
for parenteral administration.
The present invention also provides a method of treating
bacterial infections in humans or domestic mammals which
S comprises the administration of a composition of this invention.
Commonly the infection treated will be due to a strain
of Staphylococcus aureus, Klebsiella aerogenes, Escherichia
coli, Proteus sp. or the like. The organisms believed to
be most readily treated by an antibacterially effective amount
of a compound of this invention is Staphylococcus aureus.
The other organisms named are more readily treated by using
a synergistically effective amount of the compaund of the
invention and a penicillin or cephalosporin. The administration
of the two components may take place separately but in general
it we prefer to use a composition containing both the synergist
and the penicillin or cephalosporin.
The indications for treatment include respiratory tract
and urinary tract infections in humans and mastitis in cattle.
~ ', ~ ' ' .
~ ~1183~9
The present invention also provides a process for
the preparation of a compound of the formula (II) as herein-
before defined or an ester thereof which process comprises
the hydrogenation of a compound of the formula (X):
CH -R
CH2~~1 (X)
O C02H
or an ester thereof wherein Rl is as defined in relation to
formula (II) and R7 is a group of the sub-formula (a~ as
defined in relation to Rl of formula (II); and thereafter if
desired esterifying the zwitterion of the formula (IIa) as
hereinbefore defined which was produced by the hydrogenation
of the compound of the formula (X) or a hydrogenolysable
ester thereof.
When used herein the term "hydrogenolysable ester"
means an ester which on hydrogenation is cleaved to yield
the parent carboxylic acid~
The hydrogenation is normally carried out in the
presence of a transition metal catalyst.
The catalyst we have preferred to use is palladium, -
for example in the form of palladium on carbon (charcoal),
palladium on barium sulphate, palladium on calcium carbonate
or the like.
A favoured catalyst is palladium on carbon (sometimes
referred to as palladium on charcoal); for example 5~, 10~,
20~ or 30% palladium on carbon.
_ 23 -
'
': ~ '
, ~ :
.. . .
~ 8349
The higher palladium content catalyst are particularly
apt as smaller total weights of catalyst can be employed
thereby avoiding possible problems associated with absorption
of product onto the carbon.
A low, medium or high pressure of hydrogen may be
used in this reaction, for example from 1 to 6 atmospheres.
In general if the catalyst used contains a lower percentage
of palladium (for example 5% or 10% palladium) then ~etter
yields of the desired product are obtained using a pressure
of about 3 to 5 atmospheres of hydrogen, for example about
4 atmospheres of hydrogen. In general if the catalyst used
contains a higher percentage of palladium (for example 20
or 30% palladium) then acceptable yields of the desired
product may also be obtained at low and medium pressures of
hydrogen, for example about 1 to 2 atmospheres of hydrogen.
We have found it convenient to use an atmospheric or slightly
superatmospheric pressure of hydrogen in con~unction with
higher palladium content catalysts.
The reaction is normally carried out at a non-extreme
temperature, for example from 0C to 30C and more usually
from 12C to 25C, It is generally convenient to carry out
the reaction at ambient temperature.
Suitable solvents for carrying out the hydrogenation
include ethanol, n-propanol, isopropanol, tetrahydrofuran,
dioxane, ethyl acetate or mixtures of such solvents or such
solvents in the presence of water. A favoured solvent is
aqueous tetrahydrofuran. A further favoured solvent is a
_ 24 -
::
~ 11t33~9
mixture of isopropanol, tetrahydrofuran and water.
Suitably Rl is as group of the sub-formula (b) as
hereinbefore defined.
When Rl is a substituted phenyl group then R7 is more
suitably a si~ilarly substituted phenyl group or is a phenyl
group.
Most suitably R7 is a phenyl group.
Most suitably Rl is a phenyl or substituted phenyl
group as shown in and defined as in relation to any of
formulae (III)-(VII).
We have preferred to carry out the hydrogenation
reaction on a hydrogenolysable ester of a compound o~ the
formula (X) so that a compound of the formula (II) per se
is formed by the hydrogenation. Such hydrogenation reactions
proceeds at least in part via the formation of a compound of
the formula (X). Favoured hydrogenolysable esters include
benzyl and substituted benzyl esters such as methoxybenzyl,
nitrobenzyl (for example the p-nitrobenzyl ester), chlorobenzyl,
brGmobenzyl and like esters. A particularly suitable
hydrogenolysable ester is the benzyl esters. A f`urther
particularly suitable hydrogenolysable ester is the
p-methoxybenzyl ester.
The product may generally be isolated from the reaction
mixture by filtering off the solids (the catalyst, which
should be well washed to remove the product) and then
evaporating the solvent, preferably under low pressure, to
_ 25 -
~ 83~9
yield the initial product. Further purification may be
effected by such conventional methods as chromatography over
cellulose or other mild stationary phase eluting with a
Cl 4 alkanol optionally in the presence of water and optionally
in the presence of tetrahydrofuran. Evaporation of the
combined active fraction (identified by aqueous potassium
permanganate spray on tlc) then yields the desired compound
in pure form. The desired product is normally obtained in
crystalline form (unless it is an unsalted ester). Trituration
under ethanol, isopropanol or the like Cl 4 alkanol or other
conventional solvent such as a ketone, ether or ester solvent
or other conventional solvent (for example of up to 6 carbon
atoms and more suitably of up to 4 carbon atoms) may also be
used to aid crystallisation. Recrystallisation from ethanol
or the like may also be employed. The solvent used in such
processes may advantageously be moist.
Zwitterionic compounds, such as those of the formulae
(III)-(IV) may be obtained from higher yielding reactions by
the addition of an Cl 4 alkanol such as cold ethanol or the
iike to the initial product.
The initial product of the lower yielding reactions
may contain considerably impurities so that it may be
advantageous to wash the initial product by dissolving in a
water immiscible organic solvent and extracting into water.
Evaporation of the aqueous phase, preferably under a good
vacuum, then yields a purer product which may be further
purified if desired as previously described.
,
~ 1~183~9
Unsalted esters of the compounds of the formula (II)
te~ld to be oils so that it is often more convenient for
handling to convert them into solid acid addition salts,
for example by reaction with one equivalent of an acid.
Alternatively the non-hydrogenolysable ester of the compound
of the formula (X) may be hydrogenated in the presence of
one equivalent of an acid, that is they may be hydrogenated
in the form of their acid addition salt.
The compounds of the formula (II) may be hydrogenated
in the form of their acid addition salts with a strong acid
but this is not a preferred form of the~process of this
invention.
The present invention also provides a process for
the preparation of an ester of a compound of the formula (II)
which process comprises the reaction of the compound of the
formula (II) with an esterifying agent.
The zwitterionic compound of the formula (II) may be
dissolved or suspended in a solvent such as dimethylformamide,
hexamethylphosphoramide, dichloromethane, ethyl acetate or
other non-esterifiable solvents and therein esterified.
Suitable temperatures for such a reaction range from about 0
to about 25C. Suitable esterifying reagents include
reactive halides and their equivalents, alkyl oxonium salts
and the like.
When a reagent such as a reactive iodide, chloride,
bromide, tosylate, mesylate or the equivalent is used, the
resulting salt is generally suitable for use in a composition
- 27 -
` ~
~ 1`~183 ~9
of this invention. Alternatively, the salt may be converted
to a free base or alternative salt. When an alkyl oxonium
salt is used, it ls preferred to convert the resulting
tetrafluoroborate to the free base or alternative salt. The
various aforementioned salts may be converted to the free base
by neutralisation, for example by contacting a solution of the
salt in water with an organic phase, neutralising the salt by
adding a base and extracting the liberated amine into the
organic phase. This amine may thereafter be re-salted by
reacting with an appropriate acid, for example in a dry
organic solvent. It is generally preferred to use not more
than one equivalent of acid for this process. Alternatively,
the originally formed salt may be converted into the
alternative salt using an ion exchange material, for example,
by passing an aqueous solution of one salt through a bed
of an anion exchange resin in the form of the desired salt
such as the chloride form.
The salts may normally be obtained in solid form by
dissolving in a fairly polar organic solvent (such as ethanol,
tetrahydrofuran or the like) and then precipitating using a
non-polar solvent such as diethyl ether, cyclohexane or the
like.
The salts of the esters of the compounds of the formula
(II) may normally be obtained in crystalline form by conventional
methods such as trituration under (or crystallisation or
recrystallisation from) a suitable organic solvent such as
. .
~ :- L
: ., ' .
:' , . :
.
11~83 ~9
ether, acetone, acetonitrile, tetrahydrofuran or the like.
The present invention also provides a process ~or
the preparation of an ester of the compound of the formula (II)
which process comprises the reaction of an acid addition salt
of the compound of the formula (II) with an alcohol in the
presence of a condensation promoting agent.
Suitable condensation promoting agents for use in this
process include carbodiimides such as dicyclohexylcarb~iimide
and the chemical equivalents thereof.
The acid addition salt may be formed in situ or may be
preformed. The acid employed will normally be a strong acid
such as a methane sulphonic acid, p-toluene sulphonlc or the
like or trifluoroacetic acid or the like.
The reaction is normally carried out in an inert
organic solvent. When the ester being formed is that of a
liquid alcohol it is convenient to use that alcohol as the
solvent or as part of the solvent system. The esterification
is generally performed at a non-extreme temperature such as
0 to 35C, for example from about 10 to 25C. Conveniently
the reaction mixture may be performed at ambient temperature.
The present invention also provides a process or
the preparation of an ester of a compound of the formula (II)
which process comprises the hydrogenation of a corresponding
ester of a compound of the formula (XI):
- 29 -
~ 11183~9
H C0-0-~H2R7
N ~ CH2-Rl (XI)
O C02H
wherein Rl is as defined in relation to formula (II) and
R7 is as defined in relation to formula (X).
Most suitably R7 is a phenyl group.
The ester of the compound of the formula (XI) is
suitably an ester as defined in relation to formula (VIII).
.. .
Particularly suitable esters of the compound of the
formula (XI) include Cl 4 alkyl esters especially the methyl
and ethyl esters.
The hydrogenation may be performed under the same
general conditions as hereinbefore described in relation to
the hydrogenation of a compound of the formula (X).
A favoured solvent is tetrahydrofuran optior.ally in
admixture with water or a Cl 4 alcohol such as ethanol.
lS Conveniently the reaction uses an atmospheric pressure of
hydrogen at an am~ient temperature.
The present invention also provides a process for
:: .
the perparation of an ester of a compound of the formula
(XI) which process comprises the esterification of the
compound of the formula (XI) of a salt thereof.
Most-suitably this esterification is effected by the
-
_ 30 -
. : . .
.. . .
-:: . ,. ... : . :
. , ~ :
- , . ~ .: .
33 ~9
reaction of a salt of a compound of the formula (XI) with
a reactive halide or the chemical equivalent thereof.
~uitable salts of the compound of the formula (XI)
include the lithium, sodium, potassium and like salts.
Suitable esterifying agent include reactive chlGrides, bromides,
iodides, acid anhydrides, tosylates, mesylates and the like.
The esterification may be effected in a conventional
organic solvent such as acetone, dimethylformamide or the
like. The reaction is normally effected at a non-extreme
temperature such as 0 to 35C, for example 10 to 25C.
Convenlently the reaction is performed at ambient temperature.
The acid of the formula (XI) may also be esterified
by reacting with an alcohol in the presence of a condensation
promoting reagent such as a carbodiimide, for example
dicyclohexylcarbodiimide, or the chemical equivalent thereof.
Such reactions may be carried out under conditions similar
to those hereinbefore described for the same type of reaction
carried out on an acid addition salt of a compound of the
formula (II).
20~ The present inventlon also provides a process for
the preparation of a compound of the formula (XI) or a salt
thereof which process comprises the reaction of a compound
of the formula (II), or a salt thereof, as hereinbefore defined
with a compound cf the formula (XII):
,
Y-C0-0-R7 tXII~
wherein R7 is as defined in relation to formula (X) and Y is
- a readily displaceable group.
.__.... ;....... ~ ......... , -
; 1 -
11:183~
Favoured groups Y include the chlorine atom and
chemically equivalent atoms or groups such as the bromine
atom or a OR7 group or the like.
A preferred compound of the formula (XII) is
S benzylchloroformate.
The reaction may be performed under conventional
acylation conditions, for example in non-acylatable organic
solvent such as acetone in the presence of an acid acceptor
such as lithium bicarbonate, sodium carbonate, potassium
.. . . . . .................. . .. . . . .. .. . ..
carbonate or the like at a non-extreme temperature such as
10 to 30C, for example at about 0 to 10C.
We have found it convenient to carry out the acylation
on a salt of the compound of the formula (II) such as an
alkali metal salt and in partlcular the lithium salt.
~rom the preceding descriptions it will be realised
that from a broad process aspect the present invention provides
a process for the preparation of a compound of the formula (II)
as hereinbefore defined or an ester thereof which process
comprises the hydrogenation of a compound of the formula tX)
as hereinbefore defined or an ester thereof; and thereafter
if desired esterifying the zwitterion of the formula tIIa)
as hereinbefore defined which was produced by the hydrogenation
of the compound of the formula (X) or a hydrogenolysable
ester thereof; or thereafter if desired acylating the
zwitterion of the formula (IIa) as hereinbefore defined
which was produced by the hydrogenation of the compound of
- 32 -
,. .. .. . . .
:
~ .
'
33~9
the formula (X) or a hydrogenolysable ester thereof with a com-
pound of the formula (XII) as hereinbefore defined to form ~
compound of the formula (XI) as hereinbefore defined and there-
after esterifying the resulting compound of the formula tXI) or a
salt thereof and subjecting the thus formed ester to hy,droqenat-
ion to yield the desired ester of a compound of the formula (II).
Since the compounds of the formula (XI) and the salts
and ester thereof are as use as intermediates they form part
of this invention. Suitably the compounds of the formula (XI)
are in the form of-an ester of a type hereinbefore described.
Suitably the compounds of the formula ~XI) are in the form
of a salt such as in alkali metal salt, for example the
lithlum salt.
The intermediates of the formula (X) and their esters
may be prepared by the methods of',Canadian Application No.
263,103 or its equivalent.
We have chosen,to name the novel compounds of this
invention as notional derivatives,of deoxyclavulanic acid which
is of the formu,la (XIII):
H 9
O ~ (XIII)
C2H
Thus on this system the amino-substituent is defined as
being attached to the 9-carbon atom.
_ 33 -
,, , :
~ 1~183~9
b'
The processes of this invention may be adapted to
the preparation of the following compounds:
9-N-Benzylaminodeoxyclavulanic acid
9-N-(2-Methoxybenzyl)aminodeoxyclavulanic acid
9-N-(3-Methoxybenzyl)aminodeoxyclavulanic acid
9-N-(4-Methoxybenzyl)aminodeoxyclavulanic acid
9-N-(4-Hydroxybenzyl)aminodeoxyclavulanic acid
9-N-(3-Hydroxybenzyl)aminodeoxyclavulanic acid
9-~-(4-Hydroxy-3-methoxybenzyl)aminodeoxyclavulanic acid
9-N-(3-Hydroxy-4-methoxybenzyl)aminodeoxyclavulanic acid
9-N-(2-Fluorobenzyl)aminodeoxyclavulanic acid
9-N-(3-Fluorobenzyl)aminodeoxyclavulanic acid
9-N-(4-Fluorobenzyl)aminodeoxyclavulanic acid
9-N-(2-Methylbenzyl)aminodeoxyclavulanic acid
9-N-(3-Methylbenzyl)aminodeoxyclavulanic acid
9-N-(4-Methylbenzyl)aminodeoxyclavulanic acid
9-N-(2-Acetamidobenzyl)aminodeoxyclavulanic acid
9-N-(3-Acetamidobenzyl)aminodeoxyclavulanic acid
:~ ~ 9-N-(4-Acetamidobenzyl)aminodeoxyclavulanic acid
: 9-N-(4-Propionamidobenzyl)aminodeoxyclavulanic acid
9-N-(4-Chlorobenzyl)aminodeoxyclavulanic acid
: 9-N-(2-Hydroxyethyl)aminodeoxyclavulanic acid
9-N-Ethylaminodeoxyclavulanic acid
9-N-(2-Hydroxypropyl)aminodeoxyclavulanic acid
9-N-Propylaminodeoxyclavulanic acid
The present invention also provides esters of the
preceding compounds which esters may be in the form o~ acid
- 34 -
,
;. ' ~
: -:
~ 11~83~9
addition salts. Suitable esters include:
methyl
ethyl
methoxymethyl
benzoylmethyl
acetylmethyl
benzyl
4-methoxybenzyl
2-hydroxyethyl
carboxymethyl
Suitable acid addition salts are normally and preferably
those with pharmaceutically acceptable acids.
The following Descriptions illustrate the processes
ùsèd to prepare intermediates. The secondary amines used
to displace acyloxyl sroups from the acylated clavulanic
acid derivatives ma~ be prepared by the hydrogenation of a
Schiffs base prepared by the reaction of an aldehyde and primary
amine in conventlonal manner. The following Demonstrations
illustrate the activities of the compounds of this invention.
The following Examples illustrate the invent1on.
,
.
, - .
. .
'
~ 11183~9
Des ~
Benzyl 9-(N-benzyl-N-2-hydroxyeth~ minodeoxYclavulanate
Clavudiene benzyl ester (0.5g) in acetonitrile (10ml)
was cooled to 0C, N-benzyl-2-hydroxyethylamine (0.36g) was
added and the reaction mixture stirred for 2~ hours. Ethyl
acetate (100ml) was added and the mixture evaporated to low
volume. The residue was subjected to column chromatography
using ethyl acetate as eluent. The product, isolated in low
yield, had an ir spectrum (liquid film) as follows:
3400 (Broad, -OH), 1800 (~-lactam C=O), 1740 (ester C=O),
1700 (C=C),1695cm 1 (aromatic protons).
. - 36 -
,, ~,,.,,, .~ . ... .. .
~ 11183~9
_scription 2
p-Methoxybenzyl 9-(N-benzyl-N-2-hydroxyethyl)amino-deoxyclavulanat
p-Methoxybenzyl trichloroacetyblavulanate (5.37 mM) in
dry dimethylformamide (75cm3) at -10C was treated with
N-benzyl-N-2-hydroxyethylamine (1.55 cm3) and stirred at this
temperature for 5 hours. The mixture was poured into ethyl
acetate (150cm3) and washed with water (3 x 100cm3), dried and
evaporsted to an oil (0.38g) Rf (SiO2/ethyl acetate:cyclohexane;
0.13,~(film) 3400, 1810, 1750, 1620cm 1.
- 37 -
, . . ~ , - :
^
11183`~g
D ~5L~
BenzYl 9-(N-benzYl-N-ethyl)aminodeoxyclavulanate
Benzyl trichloroacetylclavulanate (9.2mM) in dry
dimethylformamide (30cm3) at -60C was treated with
N-benzylethylamine (2.74cm3) and stirred for 3~ hours at
this temperature. The mixture was poured into ethyl acetate
(150cm3) and washed with water (3 x 100cm3), dried and
evaporated in vacuo to yield an oil ~4.43g). Rf (SiO2/ethyl
acetate:cyclohexane; 1:1) 0.3, ~(film) 1804, 1750cm 1.
. - 38 -
~ 1118349
Description 4
Benzyl 9-(N-benzyl-N-isopropyl)aminodeoxyclavulanate
Clavudiene benzyl ester (0.5g) in acetonitrile (10ml)
was cooled in ice-water. N-isopropylbenzylamine (0.39 g,
1,3 moles) was added with stirring. The reaction mixture
was allowed to warm to room temperature and stirred for
3 hours. Ethyl acetate (100ml) was added, and the solution
evaporated to low bulk in vacuo. The residue was subjected to
colu~n chromato graphy on silica gel using cyclohexane and
ethyl acetate as eluents. The product was eluted after the
unreacted diene.
_ ~9 _
~ 1118349
D cription 5
zyl 9-(N,N-dibenzyl)aminodeoxyclavulanate
Benzyl dichloroacetyl_clavulanate (0.8g) was dissolved
in dry dimethylformamide and cooled to 0C, treated
with dibenzylamine (768~1; 0.004 mol) in dry dimethylformamide
(4ml) over 15 minutes, the temperature being maintained at 0C.
The resulting yellow solution was stirred at 0 for 2~ hours and
at room temperature for 4 hours. Ethylacetate was added (100ml)
and the solution washed with water (3 x 25ml), dried and
evaporated. The oil was purified by fast gradient elution
on silica gel using ethyl acetate/cyclohexane as the eluting
solvent (Yield 0.33g). Rf (SiO2/ethylacetate:cyclohexane; 1:1)
= 0.76 ~(film) 1810, 1755, 1700; ~(CDC13) 2.80 (1H, d, J 16Hz,
6~-CH) 3.05 (2H, d, J 7Hz, 9-C~ ), 3.17-3.37 (1H, m, 6a-CH) 3.32
(4H, s, 2 x NCH2C6H5), 4.59 (1H, t, J 7Hz, 8-C_), 4.90 (1H, s,
3-CH), 5.03 (2H, s, OCH2C6H5) 5.57 (1H, d, J 3Hz, 5a-CH), 7.20
(15H, s, 3 x C ~5).
- 40 -
.
: .
~ 1118349
Description 6
enzyl 9-(N,N-dibenzyl)aminodeoxyclavulanate
Clavudiene benzyl ester (271 mg) in dry acetonitrile
(4 ml) at 0C was treated with dibenzylamine (197 mg) in dry
acetonitrile (2ml) over 5 minutes. The reaction mixture was
stirred at 0C for 2 hours and at room temperature for 2 hours.
The solvent was removed by evaporation and the residue dissolved
in ethyl acetate, washed with water, dried,evaporated and
fractionated on silica-gel to yield the desired product which
was purified by chromatography.
- 41 -
.. _ .. .. . ... .. ... . . . . ~
111~3349
D cription 7
B zyl 9-[N-benzyl-N-(dl-2-hydroxypropyl)laminodeoxyclavulanate
Benzyl dichloroacetylclavulanate (0.8 g) in dry dimethyl-
formamide (20 ml) was cooled to 0C and a solution of d~-1-
benzylamino-propan-2-ol (0.65 g) in dry dimethylformamide was
added slowly. Stirring was continued for 4 hours at 0C. A
more polar component was formed (thin layer chromatograph) and
worked up as described in description 5 and chromatographed
to give the deslred product (0.16 g).
- 42 -
.
e~
11183 19
Description 8
B~n
Benzyl trichloroacetylclavulanate (2.5g; 5.8mm) in dry
dimethyl-formamide (50cm3) at -10 was treated with N-benzyl-N-
(dl-2-hydroxypropyl)amine (1.9 equivalents) in dimethylformarnide
(20cm3) dropwise. The reaction mixture was stirred at -10 for
4~ hours, poured onto cold (0) ethylacetate and the organic
layer washed with water (5 x 75cm3). After drying, the ethyl
acetate solution was passed through a ~cm x 2~cm dia~column
of silica gel eluting with ethyl acetate (75cm3) until the
eluate was no longer coloured. The ethyl acetate solution
was extracted with dilute acetic acid (3 x 50cm3), the aqueous
extracts were combined and treated with sodium bicarbonate
in the presence of fresh ethyl acetate (80cm3) with vigourous
stirring until the pH of the aqueous phase was 8. The ethyl
acetate phase was dried and evaporated in vacuo to yield product
as a yellow oil (700mg) ; R f (SiO2/ethylacetate:cyclohexane;
1:1)= 0.28, detection by aqueous potassium permanganate spray;
~ max (KBr) 3410 (broad), 1795, 1740, 1700 cm 1; ~[(CD3)2CO]
0.98 (3H, d, J 6Hz, CHCH3), 2.28j2.30 (2H, 2 x d, J 6Hz, NCH2-
CH(OH)), 2.91,2.94 (1H, 2 x d, J 17Hz, 6~-CH), 3.15 (2H, d, J
7Hz, 9-C_2), 3.2-3-9 (5H, m, NC_2C6H5, -CH2CH(OH)CH3, 6a-CH,
CHO~ , 4.72 (1H, t, J 7Hz, 8-CH), 5.06 (1H, s, 3-CH).
- 43 -
~ 1118349
Description 9
Benzyl 9-N-benzY1-N-(4-methoxybenzYl)aminodeoxyclavulanate
Benzyl dichloroacetylclavulanate (4.8 g; 12 mM) in
dimethylformamide (50 ml) at 0 was treated with N-benzyl-N-
4-methoxybenzylamine (5.2 g; 1.9 equivalents) dropwise. The
reaction mixture was stirred at 0 for 1 hour and then at 20
for 5 hours. The reaction mixture was then poured into cold
(0) ethyl acetate and the organic layer washed with water
(3 x 75 ml) and saturated brine (5 x 75 ml). The ethyl acetate
phase was dried and evaporated in vacuo to yield 6.6 g of a
coloured oil. This oil was chromatographed on silica gel,
eluting with ethyl acetate - cyclohexane; 1:1. Fractions containin
the title compound were collected and evaporated in vacuo to
yield benzyl 9-[N-benzyl-N-(4-methoxybenzyl)amino ]deoxyclavulanate
(2.24 g) as a colourless oil. Rf (SiO2/ethyl acetate:
cyclohexane; 1:1) = 0.70
~)~film) 1805, 1750, 1690, 1610, 1510, 1~00, 1250, 1175, 740,
700 -1
~; (CI)C13) 2.87 (1H, d, J 17Hz, 6~CH), 3.10 (2H, d, J 7Hz, 9C_2),
3.~3 (1H, dd, J 17 Hz and 3Hz, 6aCH), 3.38, 3.43 (4H, 2 x s,
NCH2C6H5 and NCH2C6H40CH3), 3.70 (3H, s, OC_3), 4.71 (1H, t, J
207Hz, 8CH), 5.00 (1H, s, 3C_), 5.10 (2H, s, OCH2C6H5), 5.51 (1H, d,
J 3Hz, 5aCH), 6.75 (2H, d, J 8Hz, aromatic, ortho to methoxyl)~
7 09-7.20 (12H, m, aromatic meta to methoxyl and 2 x C6_5).
-- 44 --
;
.. ..
~.'. .
- ,
,
.
~ 11183 ~9
Description 10
Benzyl 9-[N-benzyl-N-(4-fluorobenz~l~]aminodeoxyclavulanate
Benzyl dichloroacetylclavulanate (8.5 g; 21 mM) in
dimethylformamide (75 cm3) at 0 was treated dropwise with
N-benzyl-N-4-fluorobenzylamine (8.1 g; 1.9 equivalents).
The reaction mixture was stirred at 0 for 1 hour and 5 hours
at 20. The reaction mixture was poured into cold (0) ethyl
acetate (150 cm3) and the organic layer was then washed with
water (3 x 75 cm3) and saturated brine (4 x 75 cm3). The
ethyl acetate phase was dried and evaporated in vacuo to yield
10 g of a coloured oil. 3 g of this crude product was
chromatographed on silica gel, eluting with ethyl acetate -
cyclohexane; 1:1. Fractions containing the title compound
were collected and evaporated in vacuo to yield benzyl 9-[N-
benzyl-N-(4-fluorobenzyl)]aminodeoxyclavulanate (1 g )
as a colourless oil. Rf (SiO2/ethyl acetate: cyclohexane;
1;1)= 0~70, detection by potassium permanganate spray.
(film) 1805, 1750, 1690, 1508, 1305, 1220, 820, 740, 700 cm 1
~ (CDC13) 2.90 (1H, d, J 17Hz, 6,BCH), 3.09 (2H, d, J 7 Hz, 9CH2)
3.38 (1H, dd, J 17Hz and 3Hz, 6aC_), 3.39, 3.42 (4H, 2 x s, NCH2-
C6H5 and NC_2C6H4F), 4-70 (1H, t, J 7Hz, 8CH), 5.02 (1H, s,
3-CH), 5.12 (2H, s, OCH2C6H5), 5.54 (1H, d, J 3Hz, 5aCH), 6.80 -
7.25 (14H, m, 2 x CH2C6H5, and CH2C6_4F).
-- 45 ~
. : . . .~ . . . .
33~9
Description 11
Benzyl 9-[N-benzyl-N-(4-methylbenzyl)]aminodeoxyclavulanate
Benzyl dichloroacetylclavulanate (8.5 g; 21 m~) in
dimethylformamide (75 cm3) at 0 was treated with N-benzyl-
N-(4-methylbenzyl)amine (8.40 g; 1.9 equivalents), dropwise
and then stirred for 1 hour at 0 and 3 hours at 20. The
reaction mixture was poured into cold (0) ethyl acetate
(150 cm3j and the organic phase washed with water (3 x 75 cm3)
and saturated brine (4 x 75 cm3). The ethyl acetate phase
was dried and evaporated in vacuo to yield 11 g o:E a coloured
oil. 1 g of this crude product was chromatographed on silica
gel, eluting with ethyl acetate ; cyclohexane; 1:1. Fractions
containing the title compound were collected and were
evaporated in vacuo to yield 250 mg (25%) of a colourless oil;
Rf (SiO2 /ethyl acetate:cyclohexane; 1:1) = 0.70; detection
by aqueous potassium permanganate spray.
Further fractions containing the title compound in approximately
80% purity were collected, yield = 0.6 g.
~1 (film) OI purest product: 1805, 1750, 1690, 1510, 1495, 1450,
1300, 1230, 1175, 1120, 1080, 1040, 1020, 965, 890, 800, 740, 700
-1 .
~ (CDC13) 2.28 (3H, s, -C6H4CH3), 2.88 (1H, d, J 17 Hz, 6~CH),
3.10 (2H, d, J 7 Hz, 9C_ 2)~ 3.35 (1H, dd, J 17 Hz and 3Hz, 6aCH),
3.42 (4H, s, NCH2C6H5 and NCH2C6H4CH3), 4.71 (1H, t, J 7 Hz, 8CH),
5.00 (1H, s, 3CH), 5.10 (2H, s, OCH2C6H5), 5.52 (1H, d, J 3Hz,
5~CH ), 6.95 - 7.27 (14H, m, 2 x CH2C6~5 and CH2C6~4CH3).
- ~6 -
,
,
: ,
~ 11183~9
Description 12
.
Ben;~yl 9-~N-benzyl-N(4-benzoxy-3-methoxybenzyl)benzyl]amino-
x~clavulanate.
Benzyl dichloroacetyl-clavulanate (1.9 g; 4.7m~) in dry
dimethylformamide (50cm3) at 0C was treated with N~benzyl-N-
(4-benzoxy-3-methoxybenzy ~mine (3 g; 1.9 equival~nts) dropwise.
The reaction mixture was stirred at 0C for 1 hour then at
20C for 5 hours. The reaction mixture was then poured into
cold (0) ethyl acetate and the organic layer washed with
water (3 x 75 cm3) and saturated brine (5 x 75 cm3). The ethyl
acetate phase was dried and evaporated in vacuo to yield a coloured
oil. This oil was chromatographed on silica gel, eluting with
ethyl acetate - cyclohexane; 1:1. Fractions were collected
containing the title compound;these were evaporated in vacuo
to yield benzyl 9-[N-benzyl-N-(4-benzoxy-3-methoxybenzyl)]amino-
deoxyclavulanate (0.1 g) as acolourless oil.
Rf (SiO2/ethyl acetate:cyclohexane; 1:1) = 0.70.
~ (film) 1800, 1750, 1675, 1590, 1510, 1450, 1380, 1300, 1260,
1225S 1160, 1140, 1080, 1015, 805, 740, 700 cm~1.
~ (CDC13) 2.90 (1H, d, J 17Hz, 6~CH), 3.03 (2H, d, J 7 Hz, 9CH2),
3.37 (1H, dd, J 17 and 3 Hz, 6aCH), 3.37, 3.42 (6H, 2 x s,
NCH2C6H5 and NC_2C6H3 OCH3, OCH2C6H5), 3 ( 3
(1H, t, J 7 Hz, 8CH), 5.01 (1H, s, 3CH), 5.54 (1H, d, J 3 Hz, 5aCH),
6.70 - 7.34 (18H, m, 3 x CH2C6H5 and 2 protons ortho to the CH2
of CH2(C6H3)(0CH3)0cH2c6H5-
- 47 -
3 ~9
Description 13
_
Benzyl 9-~N-(4-benz~loxYbenz~l)-N-benzyl]aminodeoxyclavulanate
Benzyl dichloroacetylclavulanate (7.2 g; 18 mm), in dry
dimethylformamide (75 cm3) at 0 was treated with N-(4-benzoxy-
benzyl)-N-benzylamine (1.9 equivalents) and stirred at 0 for
3 hours, then poured into ethyl acetate (150 cm3) and washed with
water (5 x 50 cm3) and brine (3 x 50 cm3), dried (anhydrous
magnesium sulphate) and evaporated in vacuo to yield a coloured
oil,` This oil was chromatographed on silica eluting with
ethylacetate/cyclohexane; 1:1. Fractions were collected
containing the title compound (detection by aqueous potassium
permanganate spray), RF (SiO2 ethylacetate/cyclohexane; 1:1) = 0.81.
Combined fractions were evaporated to an oil, yield of approx-
imately 70% pure material = 2.7 g, pure fractions were collected
for spectroscopy. ~(film) 1800, 1745, 1690, 1600, 1595, 1510,
1450, 1380, 1300, 1230, 1170, 1129, 1080, 1045, 1020, 830, 740,
700 cm 1; ~(CDC13) 2.87 (1H, d, J 17Hz, 6~CH), 3.09 (2H, d, J
7Hz 9CH2), 3.33 (2H, dd, J 17Hz and 3Hz, 6~CH), 3.37, 3.42 (4H,
' -2C6H5 and NCH2C6H40CH2C6H5), 4.70 (1H t J 7Hz 8CH)
4.97 (3H, broad S, C6H40CH2C6H5 and 3CH3, 5.10 (2H, S, C02CH2C6H5),
5.51 (1H, d, J 3 Hz, 5~- CH), 6.80 (2H, d, J 9Hz aromati~ protons
ortho to benzyloxy), 7.0 - 7.30 (17H, m, N-CH2C6H5, OCH2C6_5,
C02CH2C6~5, aromatic protons meta to benzyloxy).
- 48 -
,, ,
,~
Q 11183`~9
Description 14
Benzyl 9-~(N-3.4-dimethoxvbenzyl)~J-benzylamino]deoxyclavulanate
Benzyl dichloroacetylcla~ulanate (6.56 g; 16 mm) in dry
dimethylformamide (50 cm3) at 0 was treated with N-(3,4-
dimethoxybenzyl)-N-benzylamine (8 g; 1.9 equivalents) in 30 cm3
dimethylformamide and stirred for 2 hours at 0 then 2 hours at
10. Then poured into cold ethyl acetate (150 cm3) and washed with
water (5 x 50 cm3) and saturated brine (5 x 50 cm3), dried
(anhydrous magnesium sulphate) and evaporated in vacuo to yield
an oil; 10.1 g. 1.2 g of this crude product was chromatographed
on silica gel eluting with ethylacetate/cyclohexane; 1:1. Fractions
were collected containing the title compound.
Rf = 0.74 (SiO2/ethylacetate:cyclohexane; 1:1), detection by
aqueous potassium permanganate spray. Combined fractions were
evaporated in vacuo to yield an oil; 0.35 g. ~(film) 1804, 1750,
1690, 1510, 1480, 1305, 1260, 1235, 1175, 1155, 1145, 1120, 1030,
151015, 807, 740, 700 cm 1.
~ (CDC13) 2.91 (1H, d, J 17Hz, 6~CH3, 3.12 (2H, d, J 7Hz, 9CH2),
3.38 (1H, dd, J 17 Hz and 3Hz, 6cl-CH), 3.39, 3.43 (4H, 2 x s
2 x NGH2), 3.83 (6H, S, 2 x OCH~), 4.73 (1H, dt, J 7Hz andc 1Hz,
8CH), 5.01 (1H, d, J~1Hz, 3CH), 5.12 (2H, S, OCH2C6H5), 5.55
(1H, d, J 3Hz, 5~-CH), 6.73 - 6.83 (3H, m, aromatic protons ortho
to CH2), 7.23 (10H, m, 2 x C6H5).
-- 49 --
~ 83`~9
. .
D ription 15
Benzyl 9-~N-(4-acetYlaminobenzyl~-N-benzyl]aminodeoxyclavulanate
Benzyl dichloroacetylclavulanate (4.4 g; 11 mm) in dry
dimethylformamide (50 cm3) at 0 was treated with N-(4-acetamido-
benzyl)benzylamine (5.3 g; 1.9 equivalents) dropwise in 20 cm3
dimethylformamide with stirring. Stirring was continued for 4
hours at 0 then poured into ethyl acetate (200cm3) and washed
with water (5 x 50 cm3) and brine (3 x 50 cm3), dried (anhydrous
magnesium sulphate) and evaporated to a foam. This crude product
was chromatographed on silica eluting with ethylacetate-cyclohexane;
1:1 graduating to neat ethylacetate. Fractions were collected
containing the title compound (detection by aqueous potassium
permanganate spray). Rf (SiO2/ethylacetate-cyclohexane; 1:1) =
0.30. Combined fractions were evaporated in vacuo to give a crisp
foam, 2.51 g ~(Nujol Mull) 3300, 1800, 1745, 1690, 1665,
1600, 1530, 1510, 1450, 1410, 1370, 1310, 1260, 1170, 1120, 1040,
1015, 740, 700 cm . ~(CDC13) 2.10 (1H? S, CH3CONH), 2.91 t1H, d,
J 17Hz, 6~CH), 3.10 (2H, d, J 7Hz, 9CH2)~ 3.37 (1H, dd, J 17 and
3 Hz, 6aCH), 3.40, 3.43 (4H, 2 x s, 2 x NCH2), 4.69 (1H, t, J 7Hz,
8CH), 4.98 (1H, s, 3CH), 5.11 (2H, s, C02CH2C6H5), 5.33 (1H, d, J
3Hz~ 5~CH)~ 7-12 - ?-41 (15H, m, 2 x C6Hs, CH2C6H4 - p-NHCOCH
CONH).
- 50 -
..;
~1~83 ~9
Description 16
Benzyl 9-[N-(2-fluorobenzYl)-N-benzYllaminodeoxYclavulanate
Benzyl dichloroacetylclavulanate (8g; 0 02M) in dry
dimethylformamide (100 cm3) at 0 was treated with N-(2-fluoro-
benyl)benzylamine (1.9 equivalents) dropwise in dimethylformamide
(30cm3) and stirred for 4 hours at 0 then 2 hours at 20. The
mixture was poured into ethylacetate (200cm3) and washed with
water (4 x 50cm3) and saturated brine (5 x 50cm3) dried (anhydrous
magnesium sulphate) and evaporated to an oil, yield = 10g. 2g of
this crude product was chromatographed on silica eluting with
ethylacetate/cyclohexane (1:2). Fractions were collected
containing the title compound, Rf (SiO2/ethylacetate-cyclohexane;
1:1) = 0.78 (detection by aqueous potassium permanganate spray).
Combined fractions were evaporated in vacuo to yield an oil,
yield - 0.4g~ ~(film) 1800, 1745, 1690, 1490, 1450, 1305, 1230.
1180, 1120, 1045, 1020, 760, 700 cm 1, ~ (CDC13) 2.94 (1H, d, J
17HZ, 6~CH), 3.13 (2H, d, J 7HZ, 9CH2), 3.41 (1H, dd, J 17 and 3HZ,
6~CH), 3.50, 3.55 (4H, 2 x s, 2 x NCH2), 4.75 (1H, t, J 7HZ, 8CH),
5.02 (1H, s, 3C_), 5.14 (2H, s, C02C_2C6H5), 5~57 (1H, d, J 3HZ,
5_~-CH), 6.94 - 7.47 (4H, m, CH2C ~ F), 7.24, 7.26 (10H, 2 x s,
2 x CH2C ~5)
~ ~183 ~9
Description 17
Benz~l 9-[N-(2-methoxYbenzyl)-N-benzyllaminodeoxyclavulanate.
Benzyl dichloroacetylclavulanate (21 mm) in dry
dimethylformamide (70cm3) at 0 was treated with N-2-methoxybenzyl-
N-benzylamine (1.9 equivalents) in dimethylformamide (30cm3) and
stirred for 3~ hours at 0 then 1~ hours at 20. The mixture
was poured into ethylacetate (200cm3) and washed with water
(5 x 50cm3) and saturated brine (5 x 50cm3), dried (anhydrous
magnesium sulphate) ~nd evaporated in vacuo to yield an oil, 13g.
5g of this crude product was chromatographed on silica eluting
with ethylacetate:cyclohexane (1:1). Fractions were collected
containing the title compound, Rf (SiO2: ethyl acetate:cyclohexane;
1:1) = 0.74. Combined fractions were evaporated in vacuo to yield
an oil, 1.2g ~ (film) 1805, 1745, 750, 700 cm 1. ~(CDC13)
2.93 (1H, s, J 17Hz~6~CH), 3.17 (2H, d, J 7Hz, 9CH2), 3.40 (1H, dd,
J 17 and 3Hz, 6 aCH), 3.54. 3.57 (4H, 2 x s, 2 x NCH2), 3.75 (3H,
s, OCH~), 4.86 (1H, t, J 7Hz, 8CH), 5.02 (1H, s, 3CH), 5.14 (2H, s,
C02CH2C6H5), 5.57 (1H, d, J 3Hz, 5 aCH), 6.75 - 7.45 (14H, m, 2 x CH
C6H~,, CH2C6H40CH3).
- 52 -
,.
. .
. ~ ;
11~83~9
Description 18
Benz~l trichloroacetylclavulanate
Benzyl clavulanate (5.78g, 20mmol) in dry methylene
chloride (100ml) was cooled to -30C and treated with pyridine
(1.61 ml). Trichloroacetyl chloride (2.23 ml, 20mmol)
in dry methylene chloride (10ml) was then added dropwise over
a period of 10 mins. After a further 10 mins. at -30C the
reaction mixture was poured into dilute hydrochloric acid
(100ml, 2M). The organic phase was washed wi-th water, sodium
bicarbonate solution, brine, dried and evaporated to afford
the product as an oil, 7.81g (90%). ~max (film) 1800, 1750
and 1680cm 1.
- 53 -
~ ~8349
Description 19
Benz l dichloroacet lclavulanate
Y _ Y
Benzyl clavulanate (20.2g; 70mm) in dichloromethane
(100cm3) was treated with dry pyridine (1 equivalent) and cooled
to -20. Dichloroacetyl chloride (10.3g; 1 equivalent) was
added in dichloromethane (20cm3), dropwise, and the reaction
stirred for 20 minutes. The mixture was washed wi-th water
(5 x 100cm3) and saturated brine (5 x 100cm3), dried (anhydrous
magnesium sulphate) and evaporated in vacuo to an oil, yield
= 27.5g (98%). ~(film) 1800, 1745, 1690, 1295, 1170, 1120, 1085,
io42, 1020, 1000, 955, 890, 815, 742, 700cm 1. ~(CDC13) 3.05
(1H, d, J 17Hz, 6~CH), 3.50 (1H, dd, J17 and 3Hz, 6a CH), 4.82
(3H, s, 8C_ and 9CH2), 5.10 (1H, s, 3CH), 5.17 (2H, s, CH2C6H5),
,
5.70 (1H, d, J 3Hz, 5 C_), 5.90 (1H, s, C_C12), 7.32 (5H, s,
CH2C6H5 ) -
~ ~183~9
D _ ription 20
B_ yl monochloroacetYlclavulanate
Benzyl clavulanate (2.51g, 8.7 mmol) was dissolved in
methylene chloride (30ml) and treated with pyridine (0.775ml,
9.60 mmol) at room temperature. The reaction mixture was
cooled to -30C and chloroacetyl chloride (0.69ml, 8.7 mmol)
in methylene chloride (10ml) was added dropwise over ten minutes.
After stirring at -30C for a further ten minutes the reaction
mixture was poured into dilute hydrochloric acid and extracted with
methylene chloride. The organic phase was washed successively
with dilute hydrochloric acid, sodium bicarbonate solution, brine,
and dried (MgS04). Evaporation in vacuo afforded a pale yellow
oil homogeneous by t.l.c., 3.10g. ~max (CHC13) 1803, 1755
(br) and 1700cm 1-
: :
~1183 ~9
D ription 21
Met~l dichloroacetvlclavulanate
Methyl clavulanate (1.03g; 4.8 mm) in dichlormethane
(30cm3) was treated with pyridine (1 equivalent) and cooled
to -20, then treated with dichloroacetyl chloride (1 equivalent)
and stirred for 10 minutes. The solution was washed with water
(2 x 50cm3) and saturated brlne (5 x 50cm3),dried (anhydrous
magnesium sulphate) and evaporated in vacuo to an oil, 1.31g.
~ (film) 1805, 1750, 1690, 1300, 1240, 1165, 1045, 1010,
960, 890, 820cm 1. ~ (CDC13) 3.08 (1H, d, J 17Hz, 6~CH), 3.51
(1H, dd, J 17 and 3HZ, 6aCH), 3.77 (3H, s, C02CH3), 4.85 (3H,
s, 8CH and 9CH2), 5.08 (1H, s, 3CH), 5.72 (1H, d, J 3HZ, 5 a~
CH), 5.91 (1H, s, CHC12).
- 56 -
83~g
.~ ..
Description 22
Meth~l 9-~N-(4-acetamidobenzyl)-N-benz~l]amin_deox~clavulanate
Methyl dichloroacetylclavulanate (1.25g; 3.86mm) in
dimethylfomamide (30cm3) was treated at 0 with N-(4-acetamido-
benzyl) benzylamine (1.9 equivalents) and stirred for 4hours;
poured into ethylacetate (200cm3) and washed with water (5 x 50cm3)
and saturated brine (5 x 50cm3), dried (anhydrous magnesium
sulphate) and evaporated in vacuo to an oil. This oil was
chromatographed on silica eluting with ethylacetate-cyclohexane
1:1 grading to neat ethylacetate; fractions were collected
containing the title compound, Rf (SiO2/ethylacetate) = o.60.
Combined fractions were evaporated in vacuo to yield an oil,
yield = 0.61g. ~J(film) 3300 (broad), 1800, 1750, 1690, 1670,
750, 700cm 1. ~(KBr) (3650-3150), 1800, 1750, 1690, 1665, 1600,
1530, 1515, 1412, 1370, 1312, 1265, 1240, 1200, 1180,1120, 1010,
745, 700cm 1.
~ (CDC13) 2.13 (3H, s, COCH3), 2.~5 (1H, d, J 17Hz, 6~CH), 3.14
(2H, d, J 7Hz, 9CH2), 3.41 (1H, dd, J = 17 and 3Hz, 6~CH), 3.48,
3.52 (4H, 2 x s, 2 x NCH2), 3.73 (3H, s, C02CH3), 4-74 (1H~ t~ J
7Hz, 8CH), 4.97 (1H, s, 3C_), 5.57 (1H, d, J 3Hz, 5aCH), 7.17 -
7.45 (9H, m, CH2C6H5 and CH2C6H4NHCOCH~).
- 57 ~
.
''
~ 83`~9
Description 23
Benzyl 9-N-benzyl-N-(4-h~droxy-~-methoxy-benzyl)
ami nodeoxYclavulanate
Benzyl dichloroacetylclavulanate (4.07 g 10.2 mm)
in dimethylformamide (75 cm3) at 0 was treated with
N-benzyl-N-(4-hydroxy-3-methoxybenzyl) amine (4.7 g;
1.9 equivalents) in dimethylformamide (20 cm3), dropwise
and stirred at 0 for % hr then at 20 for 1~ hrs. The
reaction mixture was poured into ethylacetate (200 cm3)
and the organic phase washed with water (3 x 75 cm3) and
saturated brine (4 X 75 cm3). The ethyl acetate phase
was dried and evaporated in vacuo to yield a coloured foam.
This foam was chromatographed on silica gel, eluting with
ethylacetate-cyclohexane; 1:1. Fractions were collected
containing the title compound and were evaporated in vacuo
to yield 2.32 g (44%) of a colourless oil; Rf (SiO2/
ethylacetate: cyclohexane; 1:1) = 0.66, detection by
aqueous potassium permanganate spray. v(film) 3500 broad,
1800, 1750, 1695, 1610, 1602, 1512, 1450, 1430, 1380, 1302,
1270, 1230, 1180, 1155, 1120, 1080, 1032, 1015, 820, 800,
750, 700 cm . ~(CDC13) 2.85 (lH, d, J 17Hz, 6~CH),
3.10 (2H, d, J7Hz, 9CH2), 3.31 (lH, dd, J17 and 31Hz,
6~CH), 3.35, 3.41 (4H, 2 x s, NCH2C6H5 and NCH2C6H3(0H,OCH3)),
- 58 -
;' '"
11183~9
3..74 (3H, s, OCH3), 4.80 (lH, t, J 7Hz, 8C~),
5.00 (lH, s, 3-CH), 5.09 (2H, s, OCH2C6H5),
5.25 (lH, broad s, exchanges with D20~C6H3(0H, OCH3),
5.51 (lH, d, J 3Hz, 5~CH~, 6.71 - 6.78 t3H, m,
protons in trisubstituted phenyl group), 7.20 (10H, broad s,
2 x CH2C6H5).
- 59 -
:
''
~ 1~183`~9
Example 1
(2-Hydroxyethyl)aminodeox~clavulanic acid
p-Methoxybenzyl 9-[N-benzyl-N-(2-hydroxyethyl)]amino-
deoxyclavulanate (0.38g) in ethanol and tetra1lydrofuran (1:1
(100cm3) was hydrogenated with 10% palladium on carbon as
catalyst (0.15g) for 23 hours. The mixture-was filtered
through celite and the clear filtrate evaporated in vacuo
to yield a coloured oil. This oil was dissolved in ethyl
acetate (100cm3) and extracted with water (50cm3). The
aqueous extract was evaporated in vacuo to yield a pale
yellow oil. This oil was chromatographed on a cellulose
column, eluting with butanol/propan-2-ol/water 7:7:5.
Fractions were collected containing only (1), Rf (SiO2; butanol:
propan-2-ol:water, 7:7:6)= 0.20, detection by aqueous potassium
permanganate spray. The combined fractions containing only
9-N-(2-hydroxyethyl)aminodeoxyclavulanic acid were evaporated
1~5 in vacuo to yield a white solid, (50mg); ~(D20) 3.1. (1H, d,
J 17Hz, 6~-CH), 3.05-3.17 (2H, m, NCH2CH20H), 3.57 (1H, dd, J
17Hz and 3Hz, 6~-CH), 3.70-3.84 (4H, m, NCH2CH20H, 9-CH2),
4.80 (1H, t, J 8Hz, 8C_), 4.99 (1H, s, 3-C_), 5.76 (1H, d, J 3Hz,
5a-CX). ~CH3CN was used as an internal standard, ~ CH3CN = 2.00];
~ (B r) 3000-3600, 1785, 1620cm 1.
- 60 -
, ., . . , - . .
~ ,
~-\
83~9
~.
Example 2
Benzylaminodeoxyclavulanic acid
Benzyl 9-(N,N-dibenzylamino)deoxyclavulanate (0.43g)
in ethanol and tetrahydrofuran, 1:1 (75cm3) with 1cm3 water
was hydrogenated with 5% palladium on carbon (0.43g) as catalyst.
The hydrogenation was carried out at 55 psi for 5 hours. The
mixture was filtered through celite and the clear filtrate
evaporated in vacuo to yield a coloured oil. This oil was
dissolved in ethyl acetate (80cm3) and washed with water
(~ x 30cm3). The combined a~ueous extracts were evaporated
in vacuo to yield a pale yellow oil. This oil was chromatographed
on a cellulose column, eluting with butanol/propan-2-ol/water;
4:4:1. Fractions were collected containing 9-N-benzylaminodeoxy-
clavulanic acid,detection by aqueous potassium permanganate
spray; R~ (SiO2; butanol/propan-2-ol/water, 7:7:6) = 0.45.
The combined fractions were evaporated in vacuo to yield a solid.
This solid was washed with ethanol and then dried to yield
9-N-benzylaminodeoxyclavulanic acid (36mg). ~(KBr) (3680-3150'
(3100-2900)~ (2900-2300), 1800, 1694, 1610, 1460, 1400, 1305,
1190, 1020, 895, 755, 700 cm 1; ~(D20 + 5% DMS0 D-6), 3.13 (1H,
d, J 17H~, 6~-CH), 3.62 (1H, bd, J 17Hz, 6a-C_), 3.77 (2H, d, J
8Hz, 9-CH2), 4.22 (2H, s, CH2C6H5), 4.84 (1H, t, J 8Hz, 8-CH),
5.01 (1H, s, 3-CH), 5.77 (1H, bs, 5a-CH), 7.48 (5H, s, C6_5).
The compound of this invention was produced as fine needles,
(ie. in crystalline form). Crystalllne 9-N-benzylaminodeoxy-
clavulanic acid is normally colourless. Chemical Analysis of the
product indicated that the crystals contained water.
_ 61 _
..... . . .. . . .
,
~` ~il83 ~9
Example 3
9-N-Ethylaminodeoxyclavulanic Acid
Benzyl 9-(N-benzyl-N-ethyl)aminodeoxyclavulanate [3.44 g;
obtained by the reaction of benzyl trichloroacetylclavulanate
(3 g~ with 2 e~uivalents of N-benzylethylamine] in ethanol
and tetrahydrofuran, 1:1 (100cm3) with 1cm3 water was hydrogenated
with 10~ palladium on carbon (1g) as catalyst. The hydrogenation
was carried out for 16 hours at atmospheric pressure. The
mixture was filtered through celite and the clear filtrate
evaporated in vacuo to yield an oil. This oil was dissolved
in ethyl acetate (100cm3) and extracted with water (3 x 40cm3).
The combined aqueous extracts were evaporated in vacuo to yield
a pale yellow oil. This oil was chromatographed on a cellulose
column, eluting with butanol/propan-2-ol/water; 4:4:1. Fractions
were collected containing 9-ethylaminodeoxyclavulanic acid; Rf
(SiO2; butanol/propan-2-ol/water; 7:7:6)= 0.17, detection by
aqueous potassium permanganate spray. The combined fractions
were evaporated in vacuo to yield 9-N-ethylaminodeoxyclavulanic ac
as a colour}ess crystalline solid (13% overall yield from benzyl
trichloroacotylclavulanate); ~(KBr) (3700-3250), (3200-2900),
(2900-2600), (2600-2400), 1790, 1695, 1625, 1460, 1400, 1305,
1190, 1120, 1045, 1020, 900, 800, 745 cm 1; ~(D20) 1.22 (3H,
t, J 7Hz, -CH2CH3), 2.89 - 3.20 (3H, m, -CH2CH3 and 6~-CH), 3.57
(1H, broad d, J 17Hz, 6a-CH), 3.68 (2H, d, J 8Hz, 9-CH2), 4.78
(1H, t, J 8Hz, 8-CH), 5.00 (1H, s, 3-CH), 5.73 (1H,broad s, 5-CH)
- 62 -
. . ~ :.
":~
11183~9
.~
Example 4
~-N ~ -2-Hydroxypropyl)aminodeox~clavulanic acid
Benzyl 9-[N-benzyl-N(dl-2-hydroxypropyl)]aminodeoxy-
clavulanate (3.5 mM) in ethanol and tetrahydrofuran, 1:1
(75cm3) with 1cm3 water was hydrogenated with 10% Palladium
on carbon (0.9 g) as cataylst. The hydrogenation was
carried out at atmospheric pressure for 21 hours. Thin layer
chromatography showed one major spot at Rf (SiO2/butanol:propan-
2-ol:water, 7:7:6) = 0.24. Detection by potassium permanganate
- spray. The mixture was filtered through celite and the clear
filtrate evaporated in vacuo to yield an oil. This oil was
dissolved in ethyl acetate (100cm3) and extracted with water
(3 x 50cm3). The combined aqueous extracts were evaporated in
vacuo to yield a pale yellow oil. This oil was chromatographed
on a cellulose column, eluting with butanol/propan-2-ol/water,
4:4:1. Fractions were collected containing 9- N-(dl-2-hydroxy-
propyl)]aminodeoxyclavulanic acid, Rf (SiO2/butanol:propan-2-ol:
water, 7:7:6) = 0.24. The combined fractions were evaporated
in vacuo to yield 9-N-(dl-2-hydroxypropyl)aminodeoxyclavulanic
acid as a colourless oil in a 12% yield; S(D20) 1.21 (3H, d, J
6Hz, -CH2CH(OH)C_3), 2.7 - 3.2 (3H, m, 6~-CH, CH2CH(OH)CH3),
3.57 (lH, broad d, J 17Hz, 6~-CH), 3.76 (2H, d,J8Hz, 9-CH2),
3~.83 - 4.21 (1H, m, -CH2CH(OH)CH3), 4.80 (1H, t, J 8Hz, 8-CH),
5.01 (1H, s, 3-CH), 5 75 (1H, broad s, 5~-CH).
_ 63 -
,
~ ~83~9
Example 5
9-(4-Fluorobenzyl~aminodeoxyclavulanic acid
Benzyl 9-N-benzyl-N-(4-fluorobenzyl)aminodeoxy-
clavulanate (7 g of approximately 50~ pure material)
in 100 cm3 tetrahydrofuran - ethanol (50~) plus 4 cm3 water,
was hydrogenated at 55 p.s.i. for 21 hours in the presence
of 4 g palladium on carbon ~10%). The mixture was
flltered through celite and the clear filtrate evaporated
to a coloured oil. This oil was dissolved in ethyl
acetate (80 cm3) and extracted with water (3 x 30 cm3).
The combined aqueous extracts were evaporated i~ vacuo
to yield a coloured oil. This oil was chromatographed
on a cellulose column, eluting with butanol/propan-2-ol/
water; 4 5 4:1. Fractions containing the title compound
were colIected,(detection by aqueous potassium permanganate
spray; Rf (SiO2/butanol/propan-2-ol/water, 7:7:6) = 0.63).
- 64 -
- ' - ' ':
~1183~9
~`
The combined fractions were evaporated ln vacuo
to yield a colourless material which crystallised ~n
the addition of ethanol and cooling. The
crystals were washed with cold (0C)ethanol and dried to
yield 9-(4-fluorobenzyl)aminodeoxyclavulanic acid (367 mg).
(KBr) (3700 - 3120), (3120 - 2900), (2900 - 2650),
(2650 - 2500), (2500 - 2300), 1805, 1695, 1580, 1515,
1470, 1410, 1340, 1303, 1283, 1230, 1185, 1165, 1045,
1008, 992, 895, 858, 835, 773 cm 1,
- 65 -
'~ ' .
183~
xample 6
9-N-(4-Methylbenzyl)aminodeox~clavulanic acid
Benzyl 9-N-benyzl-~-(4-methylbenzyl)aminodeoxy-
clavulanate (10.6 g of approximately 50% pure material)
in 100 cm3 tetrahydrofuran - ethanol (50%) plus 4 cm3
water was hydrogenated at 55 p.s.i. for 21 hours
in the presence of 5 g palladium on carbon (10%). The
mixture was filtered through celite and the clear
filtrate evaporated in vacuo to a coloured oil. This
oil was chromatographed on cellulose, eluting with
butanol/propan-2-ol/water: 4:4:1. Fractions containing
the title compound were collected, detection by aqueous
;~ ~ potasslum permanganate spray; Rf (Si02; butanol/propan-2-ol/
water, 7:7:63 = 0.60. The combined fractions were
lS e~v~ap~rated in vacuo to yield an oil to whiah was added
ethanol. On cooling a qolourless crystalline solid
formed. Thia was filtered off, washed with cold ethanol
and dried to yield 85 mg of zwitterionic 9-~-(4-methyl-
benzyl3aminodeoxyclavulanic acid as fine needles.
- 66 -
. .... . ,.. ... .... ... . .. . ... ... . .. . . , . , . . ~ .
..
.,
- . :,
'. : ,~ . ~ . :
~ .
-- 11183~9
(KBr) (3670 - 3150), (3150 - 2870), (2870 - 2500),
(2500 ~ 2250), 1790, 1690, 1590, 1460, 1395, 1300, 1195,
1110, 1035, 1015, 1000, 990, 940, 892, 805, 748, 555,
485, 435 cm 1
,
:
~:
- 67 -
. .
-
.. ~: . . . -
~: . . , :
.
~ 11183 ~
Example 7
9-N-(4-Methoxybenzyl~aminodeox~clavulanic acid
Benzyl 9-N-benzyl-N-(4-methoxybenzyl)aminodeoxy-
clavulanate (1.4 g) in lOO cm3 tetrahydrofuran -
S ethanol (50~) plus 2 cm3 water was hydrogenated at 55 p.s.i. for21 hours in the presence of l g palladium on carbon (10~).
The reaction mixture was filtered through celite and the
filtraté evaporated in vacuo to yield a coloured foam.
This foam was chromatographed on cellulose eluting with
butanol - isopropanol - water; 4:4:1. Fractions containing
the title compound only were collected. Rf (SiO2/butanol -
isopropanol - water; 7:7:6) - 0.60, detection by aqueous
potassium permanganate spray. These fractions wereevaporated
in vacuo to yield a colourless residue. Trituration of
the resldue undercooled ethanol yielded solid zwitterionic
: 9-N-(4-methoxybenzyl)aminodeoxyclavulanic acid as fine needles,
~ : (33 mg).
:
' ' .
- 68 -
~' :
~1~8349
(KBr) (3600), (3500 - 3150), (3150 - 2880), (2880 - 2780),
(2780 - 2680), (2680 - ~520), (2520 - 2250), 1790,
1695, 1600, 1517, 1470, 1400, 1305, 1255, 1200, 1184,
1125, 1030, 995, 950, 900, 840, 820, 767, 760, 575,
545, 445 cm 1.
. - .69 ~
11183~9
_xamPle 8
s-N- ( 4-Hydroxy-3-methoxybenzyl)aminodeoxYclavulanic acid
Benzyl 9-N-(4-benzoxy-3-methoxybenzyl)benzylamino-
deoxyclavulanate (0.7 g of approximately 50% pure material)
in tetrahydrofuran - ethanol (50~) 50 cm3 plus 2 cm3 water
was hydrogenated at 55 p.s,i.for 15 hours in the presence
of 0.5 g palladium on carbon (10%). The reaction mixture
was filtered through celite and the filtrate evaporated in
vacuo to yield a coloured oil. This oil was dissolved in ethyl
acetate (50 cm3) and extracted with water (2 x 25 cm3). The
combined aqueous extracts were evaporated in vacuo to yield
a coloured foam. This foam was chromato~raphed on a cellulose
column, eluting with butanol/propan-2-ol/water 4:4:1. Fractions
containing the title compound were collected and evaporated
in vacuo to yield 9-N-(4-hydroxy-3-methoxybenzyl)aminodeoxy-
clavulanic acid as a white solid (30mg). Rf (SiO~/butanol-propan- -
1~5 2-ol-water; 7:7:6) = 0.56. The p.m.r. spectrum was consistent
with the desired product.
- 70
:,
. .
~.
~ 11183 ~9
_ample 9
9-N-(4-Hydrox~-3-methoxybenzyl)aminodeoxyclavulanic acid
Benzyl 9-N-(4-hydroxy-3-methoxybenzyl)benzyl
aminodeoxyclavulanate (2.27 g) in 80 cm3 tetrahydrofuran -
ethanol (50%) plus 2 cm3 water, was hydrogentated at 55 psi
for 6 hours in the presence of 2 g palladium on charcoal
(10%). The mixture was filtered through celite and the
clear filtrate evaporated in vacuo to yield a pale yellow
foam. To this foam was added methanol (5 cm3), then to this
solution was added dry acetone (70 cm3) and the resultant
precipitated white solid filtered off and dried, yield
0.88 g. 0.83 g of this crude material was chromatographed
on cellulose with butanol/propan-2-ol/water; 4:4:1. Fractions
were collected containing the title compound and were
evaporated in vacuo to yield a white solid. Propan-2-ol was
added (20 cm3) followed by methanol (10 cm3) and the solution
cooled (0C). The crystalline product was filtered off and
washed with cold (0) methanol and dried to yield 9-N-(4-hydroxy-
3-methoxybenzyl)aminodeoxyclavulanic acid as fine needle~ (102mg).
Rf (SiO2/butanol-propan-2-ol-water; 7:7:6) = 0.60.
- 71 -
.,
.. ,~.,. ,,.. ~....... . ~
11183-~9
Example 10
Benzylaminodeoxyclavulanic acid
Benzyl 9-N,N-di-benzylaminodeoxyclavulanate (13g) was
dissolved in 60 cm3 tetrahydrofuran plus 50 cm3 of aqueous
propan-2-ol (H20: IPA = 2:5). To this was added 6 g of
palladium on charcoal (10%) which had been previously
washed with water (to neutral filtrate). The reaction
mixture was hydrogenated at 55 psi for 6~ hours, filtered
through celite (pH of the filtrate was 5.37) and washed with
aqueous tetrahydrofuran (50%, 150 cm3) followed by aqueous
ethanol (50%, 200 cm3). The filtrate from the final washing
was collected separately and evaporated to yield a white
solid. Ethanol was added (20cm3) and cooled (0), then
filtered and washed with ice-cold ethanol, and dried in vacuo
to yield 9-~-benzylaminodeoxyclavulanic acid as a finely
~ crystalline solid (0.~2 g). The main filtrate was evaporated
and ethanol added (50 cm3) resulting in rapid crystallisation.
The solid was filtered off and washed with ice-cold ethanol
and dried in vacuo to yield 9-N-benzylaminodeoxyclavulanic acid
as a very slightly off-white crystalline solid (1.57 g). The
filtrate was again evaporated but this time ethanol was added
(50 cm3). Cooling yielded fine crystals, which were filtered
off and washed with ice-cold methanol, and dried in vacuo to
yield the 9-N-benzylaminodeoxyclavulanic acid as a finely
crystalline solid (0.25 g). The total yield was 2.14 g.
(assuming the dibenzylamino compound was 90% pure this represents
a yield of 30%).
- 72-
., ' , ~ .
: '
~ 111~3 ~9
The coloured residue was evaporated and redissolved in ethanol.
This solution was applied to a cellulose column 180cm x 4~ cm
and partially eluted with ethanol (50 to 70 cm3) and then with
butanol/isopropanol/water; 4:4:1. Fractions were collected
containing virtually only 9-N-benzylaminodeoxycla~ulanic acid were
evaporated and methanol added; the resulting fine crystals
of the desired compound were filtered off and washed with cold
methanol to yield further 67 mg.
~ max (nujol) 3620, 3540, 3400 broad, 3200 broad, 2800-2500,
2500-2300, 1810, 1690, 1610, 1575, 1395, 1185l 1145, 1115, 1080,
1060, 1040 ? 1030, 1015, 1005, 990, 945, 895, 865, 850, 815, 790,
755, 700 cm~1,
Cu Ka radiation, 36kV, 26mA, scan speed ~ 20/min,
scanned 33 -~ 20 20: Reflections at the following approx.
angles 20: 11.5, 13.5, 15.2, 15.8, 16.4~ 17.25, 18.2, (broad),
19 5, 21.0, 21.8, 22.5, 23.1, 24.1, 24.3, 25.2, 25.5, 26.0, 27.5,
28.4, 28.9, 29.6, 32.6, (major reflections underlined).
.
~ 3~9
Example 11
9-N-(4-Hydroxybenzyl)-aminodeoxyclavulanic acid
Benzyl 9-N(4-benzoxybenzyl) benzylamino deoxyclavulanate (4 g of
approximately 50% pure material) in tetrahydro~uran (60 cm ) and aqueous
propan-2-ol (50~; 50 cm ) was hydrogenated at 55 psi i'or 6~ hours in
the presenee oi' 2 g palladium on earbon (10%). The mixture wa~ filtered
through celite and the catalyst~/q5hecl . with aqueous ethanol (200 cm ).
the ~iltrate was evaporated in vaeuo to yield a coloured ~oam. The i'oam
was dissolved in ethanol (lO cm ) and dry ether added (100 em ), the
resultant precipitate was ~iltered oii' and dried, dissolved in a ~mall volume
ol ethanol and chromatographed on eellulose, eluting with ethanol (60 cm ) then
butanol/ propan-2-ol/water; ~:4:1). Fraetlons were collected eontaining the
title compound (detection by aqueous potassium permanganate sprsy), Ri
(S;02/butanol/propan-2-ol/water; 7:7:6) = 0.67. Combined ~ractions were
evaporated in vacuo to yield an oil, trlturation with cold methanol yielded
9-N(4-hydroxybenzyl) aminodeoxyelavulanio aeld a~ i'ine cry~tals; (4.7 mg).
~)~ujol :mu ~)3575, 3350, 3175, 1780, 1695, 1620, 1580, lS20, 1310,
1200, 1125, 1105, 1075, lOS0, 1020, 1005, 985, 895, 840, 750, 720 cm 1,
- 7 4 ~
. ~ . . . .
~:: . . : , :: :
: ~ ~ ,, -
'
',
1118349
Example 12
~,-N-(3,4-Dimethoxybenzyl)aminodeoxyclavulanate
Using the procedures of the foregoing Examples, hydrogenation
of benzyl 9-N-(3,4-dimethoxybenzyl)aminodeoxyclavulanate yields
zwitterionic 9-N-(3,4-dimethoxybenzyl)aminodeoxyclavulanate.
- 75 -
,, , ; :
.: : .
.: ' ,., ' .: ~
~ 11183~9
Example 13
9-N-(4-Acetamidobenzyl)aminodeoxYclavulanic acid
-
Benzyl 9-N-(4-acetamidobenzyl)benzylaminodeoxyclavulanate
(0.9g) in tetrahydrofuran (50 cm3) and water (10 cm3) was
hydrogenated at atmospheric pressure in the presence of
0.3g palladium on carbon (10%) for 40 minutes. The mixture
5 was filtered through celite and the catalyst washed with
aqueous tetrahydrofuran (1:1, 100cm3) and aqueous ethanol
(1:1,150cm3). The clear filtrate was evaporated in vacuo,
ethanol was added (20cm3) and after cooling,colourless crystals
were filtered off and dried to give 9-N-(4-acetamidobenzyl)-
aminodeoxyclavulanic acid (309 mg). The filtrate was evaporated
in vacuo after which the addition of cold methanol yielded
a further amount of the title compound (37 mg). Rf (SiO2/
butanol-propan-2-ol-water; 7:7:6) = 0.45; detection by aqueous
potassium permanganate spray. ~(Nujol Mull) (3600-3440), 3380,
(3350-3200), 3180, 3120, 2720, (2670-2520), 2450, (1805-1785),
1690, 1670, 1600, 1530, 1310, 1200, 1110, 1070, 1050, 1035,
1020, 1000, 990, 940, 895, 840, 750 cm 1
S (D20/DMSO) 2.01 (3H, s, CH3CONH), 2.88 (1H, d, J 17Hz,
6~CH), 3.38 - 3.53 (3H~, m, 6aCH, 9CH2), 3.95 (2H, s, NCH2),
4.61-4.75 (2H, m, 3CH, 8CH), 5.62 (1H, broad s, 5aCH), 7.30-
7.56 (4H, ABq, J 9Hz, CH2C6H4-p-NHCOCH3~.
- 76 -
.. , . ,~ . .. . .. .. . .
~ ~ ,
- '
~ 1118349
Example 14
9-N-(2-Methoxybenzyl)amin~deoxyclavulanic acid
Benzyl 9-N-(2-methoxybenzyl)benzylaminodeoxyclavulanate
(8g of crude product) in tetrahydrofuran (100 cm3) and water
(10cm3) was hydrogenated in the presence of 10% palladium
on carbon (1g) at atmospheric pressure for 1~ hours. The
mixture was filtered and the catalyst washed with aqueous
tetrahydrofuran (100cm3), the filtrate was evaporated in
vacuo, to the residue was added ethyl acetate (150 cm3) and then
washed with water (2 x 75cm3). The combined aqueous extracts
were evaporated in vacuo to a foam (3g). This foam was
chromatographed on cellulose eluting with butanol/propan-
2-ol/water: 8:8:1. Fractions were collected containing the
title compound, Rf (SiO2/ butanol-propan-2-ol-water; 7:7:6) o.60
(detection by aqueous potassium permanganate spray). Combined
fractions were evaporated in vacuo. On addition of cold
ethanol colourless crystals formed; the crystals were filtered
off and washed with cold ethanol and dried to yield 9-N-(2-
; methoxybenzyl)aminodeoxyclavulanic acid (140mg) ~(Nujol mull)
~3320-3220), (2750-2100), 1800, 1685, 1610, 1300, 1260, 1185,
1120, 1085, 1070, 1055, 1030, 1020, 1005, 935, 900, 775, 755,
745 cm 1
- 77 -
-
.
~ lllS349
Example 15
9-N-(2-Fluorobenzyl)aninodeox~clavulanic acid
Benzyl 9-N-(2-fluorobenzyl)benzylaminodeoxyclavulanate
(0.35g) in tetrahydrofuran (30cm3) and water (3cm3) was
hydrogenated in the presence o~ 30% palladium on carbon
(40mg) at atmospheric pressure for 4 hours. A further
quantity (40mg) of the same catalyst was added and hydrogenolysis
continued for 15 hours. The mixture was filtered and the catalys-
washed with aqueous ethanol (50cm3) and evaporated in vacuo.
Addition of cold ethanol yielded 9-N-(2-fluorobenzyl)amino-
deoxyclavulanic acid as acolourless crystalline solid (47mg)
Rf (SiO2/butanol-propan-2-ol-water; 7:7:6) = 0.51 (detection
by aqueous potassium permanganate spray. ~(KBr), (3680-3140),
(3140-2880), (2880-2500), (2500-2200), 1785, 1694, 1615, 1496,
1457, 1380, 1308, 1240, 1193, 1110, 1043, 1020, 900, 865 cm~1.
~(D20) 3.05 (1H, d, J 17Hz, 6~CH), 3.54 (1H, dd, J 17 and 3Hz
6~CH), 3.72 (2H, d, J 7Hz, 9CH2), 4.23 (2H, s, NC-2C6H4F), 4.77
(1H, t, J 7Hz, 8CH), 4.95 (lH, s, 3CH), 5.70 (1H, d, J 3Hz,
5aCH), 7.05-7.48 (4H, m, CH2C ~4F).
- 78 -
. .
~ .
':
i/
~1183~9
'~
Example 16
_arboxymethyl 9-N-benzylaminodeoxyclavulanate.
Benzyloxycarbonylmethyl 9-N-benzyloxycarbonyl-N-benzyl-
aminodeoxyclavulanate (279mg) was dissolved in tetrahydrofuran
(20ml) and water (2ml) and hydrogenated at normal temperature
and pressure over 10% palladium on carbon. After the reaction
was complete (as judged by tlc) the ca-talyst was filtered off,
washed well with aqueous tetrahydrofuran and the combined
filtrated evaporated in vacuo to yield carboxymethyl 9-N-
benzylaminodeoxyclavulanate. The residue was triturated with
acetone/ether to give carboxymethyl 9-N-benzylamino-
deoxyclavulanate as a white solid which was filtered off
. .
and dried in a desiccator. ~ max (Nujol) 1795, 1745, 1690,
1610 cm~l. i ~
,
79
~'
~ 11183`~9
_xample 17
Methyl 9-N-(4-acetamidobenzyl)aminodeoxyclavulanate hydr~en
~L)-malate
Methy~ 9-N-(4-acetamidobenzyl)benzylaminodeoxyclavulanate
(0.5g) in tetrahydrofuran (15 cm3) and water (5cm3) was
hydrogenated at atmospheric pressure for 2~ hours in the
presence of 0.1g palladium on carbon catalyst (30%) and malic
acid (0.155g; 1 equivalent). The mixture was filtered and the
catalyst washed with aqueous tetrahydrofuran (10cm3). The
filtrate was evaporated in vacuo to an oil. This oil was
dissolved in ethanol (10cm3) and triturated with ether to
yield a solid. This solid was washed with ether and dried
in vacuo to yield methyl 9-N-(4-acetamidobenzyl)aminodeoxy-
clavulanate hydrogen L-malate as a solid (343 mg).. ~(Nujol)
1800, 1745, 1690, 1670, 1600 cm 1.
- 80 -
'
: ,
~ 1~183 ~9
Example 18
Methoxymethyl 9-N-benzylaminodeox~
9-N-Benzylaminodeoxyclaw lanic acid (162mg) in
dimethylformamide (lOmls) at 20 was treated with chlorodimethyl
ether (42 ml, 1 equivalent) and stirred for 4-5 minutes (when
tlc showed no further change). At this point the solution
contains the hydrochloride salt of methoxymethyl 9-N-benzyl-
aminodeoxyclavulanic acid;(R~ HCl salt 0.8 on SiO2 using
butanol:propanol: water 7:7 6). The mixture was poured
into ethyl acetate (100ml) and water (50mll and stirred
vigourously while adding sodium bicarbonate (solid) until the
pH was 9.5. The organic phase was washed with water (6 x 50 ml)
and saturated brine (3 x 50 ml?, dried (anhydrous magnesium
sulphate) and evaporated to yield methoxymethyl 9--N-benzylamino-
deoxyclaw lanate as an oil (154mg). (Rf, SiO2/ethyl acetate
0.17).
~ max (film) = 3325 (broad), 1800, 1750, 1700, 745, 705 cm 1.
- 81 -
- ~, ~ ,, '' .
~118349
Example 19
M~thoxymethyl 9-benzylaminodeoxyclavulanate hydrochloride
9-Benzylaminodeoxyclavulanic acid (0.4g) in dimethyl-
formamide (15ml) was treated at room temperature with
chlorodimethyl ether (1 equivalent) and stirred for 5
minutes, the evaporated in vacuo to yield an oil. This oil
was dissolved in ethanol (5ml) and added-dropwise to diethyl
ether (250ml) with vigorous stirring. The resultant precipitate
was filtered off, washed with dry diethyl ether, and dried
in vacuo to yield methoxymethyl 9-benzylaminodeoxyclavulanate
hydrochloride as a white solid (320mg).
~ (Nujol) 2800-2460, 2460-2300, 1800, 1750, 1695, 750, 700 cm 1.
~ (CD30D) values include: 3.10 (1H, d, J 17Hz, 6~-CH), 3.43 (3H,
s, OCH3), 3.74 (2H, d, J 7Hz, 9-CH2), 4.15 (2H, s, CH2C6H5),
5.27 (2H, s, CH20CH3), 5.77 (1H, d, J 3Hz, 5 -CH), 7.40 ~5H, s,
CH2C ~5).
- 82 -
....... ,., _
,. .
,
I
11~8349
xa~ple 20
Ethyl 9-N-benzylaminodeoxyclavulanate
A suspension of 9-N-benzylaminodeoxyclavulanic acid
(576 mg) in methylene chloride (20ml) was treated with a
solution of triethyloxonium tetrafluoroborate (380mg ) in
methylene chloride (20ml) and stirred at room temperature
for 3 hours until no further change was seen by tlc. The
solvent was removed by evaporation to yield ethyl 9-N-benzyl-
aminodeoxyclavulanate tetra~luoroborate. This residuewas
dissolved in aqueous sodium bicarbonate (roughly one equivalent)
and extracted with methylene chloride (2 x 20 ml). The
organic phase was washed with brine (20ml), dried (anhydrous
magnesium sulphate) and evaporated to yield ethyl 9-N-benzyl-
aminodeoxyclavulanate as an oil (250mg). Rf =`0.2 on SiO2
using ethyl acetate.
max (film) = 3300 (broad), 1800, 1745, 1695 cm 1.
- 83
.... , , . , .. , , . .. . . . .............. . -
,
-
~ ~1183~9
Example 21
Benzyloxymethyl 9-N-benzylaminodeoxyclavulanate hydrochloride
9-N Benzylaminodeoxyclavulanate (288mg) was dissolved
in dry dimethyl~ormamide (15ml) at room temperature and to this
solution was added benzyloxymethyl chloride (157mg) in
dimethylformamide (1ml). The reaction mixture was stirred
at room temperature for a further four hours (tlc showed
reaction was complet~ and the solvent was removed in vacuo.
The residue was dissolved in acetone, filtered through Celite
and dry ether added. On cooling and scratching,benzyloxymethyl
9-N-benzylaminodeoxyclavulanate hydrochloride crystallised
out and was collected by filtration (220mg).[a]2 = + 15.6
(1.0; MeOH); ~max (Nujol) 2725 (b), 1795, 1766, 1700cm 1; ~ max
(KBr) 2940 (b), 2700~2840 (b), 1795, 1767, 1698 cm~1; ~(CD30D~
3.08 (1H, d, J 17Hz, 6~-CH), 3.57 (1H, dd, J 3.5 and 17Hz,
6~-CH), 3.68 (2H, broad d, 9CH2), 4.11 (2H, s, NH2CH2C6H5),
4.69 (2H, s, obscur2d by water peak, CH20CH2C6H5), 4.85 (1H,
t, J 7Hz, 8CH), 5.23 (1H, s, 3-CH), 5.38 (2H, s, C02CH20),
5.71 (1H, d, J 3Hz, 5-C_), 7.23 (5H, s, aromatic -H), 7.37
(5H, s, aromatic -H).
- 84 -
' ' '-
. ` - ~ . ~
~ 11:183 ~g
Example 22
4--Nitrobenz~loxymethy1 9-N-benzylaminod~eoxYclavulanate
hydrochloride
-
9-N-Benzylaminodeoxyclavulanate(0.4g) in dry dimethyl-
formamide (15 cm3) at room temperature was treated with
4-nitrobenzyloxymethylchloride (1 equivalent) and stirred far
seven minutes. The solvent was evaporated in vacuo and
5 acetone added (20 cm3) followed by petroleum spirit (8~-100)
and diethyl ether (1:1, 200cm3), the mix~ure was cooled
overnight and the resultant solid filtered off and washed
with ether. Drying in vacuo afforded 4-nitrobenzyloxymethyl
9-N-benzylaminodeoxyclavulanate hydrochloride as an off-white
solid (0.41g). Rf (SiO2/ethanol: chloroform, 1:4) = 0.58.
V max (film) 1805, 1755, 1700 cm 1. ~(DMS0) 3.15 (1H, d, J
17Hz, 6~CH) 3.62 (1H, dd, J 17 and 3 Hz, 6-a-CH) 3.5 (2H,
broad m, 9-CH2), 4.03 (2H, broad s, sharps on shaking with
D20, NC_2C6H5), 4-86 (2H, s, CH20CH2C6H5N02), 4.96 (1H, t, J
7Hz, 8C_), 5.37 (1H, s, 3CH), 5.45 (2H, s, CH20CH2C6H4N02),
5.70 (1H, d, J 3Hz, 5~-CH), 7.27 - 7-60 (7H, m, CH2C6 5 and
protons meta to nitro group of nitrobenzyl), 8.12 (2H, d, J
9Hz, protons ortho to nitro group of nitrobenzyl), 9.72 (2H,
broad s, exchanges with D20, NH~2)
11183~
Example 2~
9-N-Benzylaminodeoxyclavulanic acid p-toluene sul ~ .
9-N-Benzylaminodeoxyclavulanic acid (288 mg)
was suspended in benzyl alcohol (15 ml) and ~-toluene sulphonic
acid (190 mg) added to form the title compound.
- 86 - -
.
E ple 24
BenzYl 9-N-benzylaminodeoxyclavulanate p-toluene sulphonate
Dicyclohexylcarbodiimide (206 mg) was added to the
solution obtained in Example 23 and the mixture stirred at
room temperature overnight. The solution was loaded onto a
column of silica gel and the product obtained by gradient elution
with chloroform/ethanol, finally eluting with 10:1. The title
compound was obtained as a white solid on trituration with
acetone/ether. ~ max (Nujol) 1810, 1750, 1700 cm 1,
. - 87 -
.
~, . .
.
~ 83~9
Example 25
Ethyl 9-N-benzylaminodeoxyclavulanate p-toluene sul~honate
9-N-Benzylaminodeoxyclavulanic acid (288 mg) was
suspended in ethanol (15 ml) and p-toluene sulphonic acid
(190 mg) added to form the acid addition salt. Dicyclohexyl-
carbodiimide (206 mg)was added to the solution and the reaction
mixture stirred for several hours at room temperature. The
solvent was removed and the product purified by column chromato-
graphy on silica gel, elu-ting with butanol/propan-2-ol/water
4:4:1. Fractions containing the title compound were combined
and evaporated; addition of ethanol and ether to the residue
gave the product as a white solid which was filtered off and
drled. ~(Nujol mull) 1810, 1750, 1700 cm 1,
- 88 -
_ . ... _ , .. _ _ _ _ _. . .. , . ... _. _ . ~ _.. __ , _ .. _ . . ... .. ~ . . . ... .. . . _ _ . .. .
~ .
3~9
Example 26
Phenacyl 9-N-benzYlaminodeoxyclavu-lanate hydrobromide
9-N-Benzylaminodeoxyclavulanic acid (288 mg) in
dimethylformamide,(15 ml) was treated with phenacyl bromide
(199 mg) and the resulting solution stirred at room temper-
ature for 1~ hours. The dimethylformamide was evaporated in
vacuo, the residue chromatographed on silica gel, and the title`
compound was eluted with chloroformtethanol, 10:1. Evaporation
of the fractions followed by trituration with acetone/ether gave
the required salt as a pale yellow solid. ~max (KBr) 1798, 1755,
1698 cm~1
.
- 89 -
: . . . ~, . ,
. . .. .
. .: . . ~ :
- ~
L .: . :
Example 27
Phenacyl 9-N-benzYlaminodeoxyclavula-ate hydroiodide
Phenacyl bromide (199 mg) was dissolved in acetone (2 ml)
and a solution of sodium iodide (1.1 equivalent) in acetone
(2 ml) added. An immediate precipitate of sodium bromide was
obtained. After stirring the mixture for 10 minutes, the
precipitate was filtered off and the filtrate added to a suspension
of 9-N-benzylaminodeoxyclavulanic acid (288 mg) in dimethyl-
formamide (15 ml). The solution was stirred at room temperature
for 2 hours and the solvent removed. Fractionation of the crude
product on silica gel, eluting with chloroform: ethanol, lO:l,
gave the title compound. Work-up and trituration with acetone/
ether gave the product as a pale yellow solid. ~max (Nujol) 1800,
1750, 1695 cm 1,
-- 90 --
, . .
,~34~
Example 28
Lithium 9-N-carbobenzoxy-N-benzylaminodeoxyclavulanate
9-N-Benzylaminodeoxyclavulanic acid (1.15g) in dimethyl-
formamide (20ml) containing one equivalent of lithium bicarbonate
(544mg; 10.8ml 5% solution) was treated dropwise with a solution
of benzylchloroformate (684mg) in acetone (lOml) at 0. The
reaction mixture was stirred at this temperature for 1 hour.
The solvent was removed and the residue triturated with acetone/
ether, the resulting white solid lithium 9-N-carbobenzoxy-N-benzyl-
aminodeoxyclavulanate was filtered off and dried in a desiccator
in vacuo (1.6g). Rf (SiO2: n-butanol: isopropanol: water; 7:7:6) =
0-66; [a] D = + 28-3 (C=1.6; water); ~max (nujol) 1778, 1682,
1620cm , ~ ((CD3)2 SO) 2.68 (lH, d, J 17.5Hz, 6~-CH), 3.42 (lH,
dd, obscured by water peak, 6~ -CH), 3.8 (2H, d, J 7.5Hz, 9-CH2),
4-32 (2H, s, NCH2C6H5), 4.52 (2H, m, 3-C_ and 8-CH), 5.07
(2H, s, N.CO.O.CH2), 5.52 (lH, d, J 3Hz, 5-CH), 7.21 (5H, s,
aromatic-H), 7.28 (5H, s, aromatic-_).
-- 91 --
~' . , -
~L"" , .,
., ' '
3~9
~xample 29
Methyl 9-N-carbobenzoxy-N-benzylaminodeox~clavulanate
Lithium 9-N-Carbobenzoxy-N-benzylaminodeoxyclavulanate
(428mg) was dissolved in dimethylformamide (10ml) and methyl
iodide (710mg) added. The reaction mixture was stirred at
room temperature for 4 hours. The solvent was removed and the
residue chromatographed on silica gel eluting with ethyl
acetate: cyclohexane (1:1). The title compound was obtained
after evaporation of the solvent as a colourless oil (244mg).
Rf (Ethylacetate-cyclohexane; 1:1) = 0.75; [a]D= + 13.21
(C=1.12; MeOH); ~max (film) 1805, 1750, 1690 cm 1; ~(CDCl3)
2.86 (1H, d, J 17.5Hz, 6~-CH), 3.42 (1H, dd, J 17.5 and 3Hz, 6a-
CH), 3.72 (3H (3H, s, C02CH~), 3.96 (2H, d, J 7.5Hz, CH.C 2N),
4-44 (2H~ s~ Nca2Ph)~ 4-67 (1H, bt, J 7.5Hz, CHCH2), 4.94 (1H,
d, J 1.5Hz, 3-CH), 5.16 (2H, s, C02CH2Ph), 5.53 (1H, d, J 3Hz,
5-CH), 7.22, 7.3 (10H, 2 x s, Ar-H).
- 92 -
xample 30
Ethyl 9-N-carbobenzoxy-N-benzylaminodeoxvclavulanate
-
Ethyl iodide (780mg) was added to a solutio~ of
lithium 9-N-carbobenzoxy-N-benzylaminodeoxyclavulanate (428mg)
in dimethylformamide (1Oml) containing about 2 drops water.
The solution was allowed to stir at room temperature for
5 seven hours. The solvent was removed by e~aporation and the
residue chromatographed on silica gel. The product was
isolated by elution with ethyl acetate: cyclohexane; 1:1 and
was obtained after evaporation of the solvent as a colourless
oil (307mg). Rf (ethyl acetate: cyclohexane; 1:1) = 0.78;
[~]20= + 9.6 (C=1.38; MeOH); ~max (film) 1B02, 1745, 1695 cm
~ (CDC13) 1.25 (3H, t, J 8Hz, CH2CH3), 2.86 (1H, d, J 17.5Hz,
6~-CH), 3.41 (1H, dd, J 17.5Hz and 3Hz, 6-CH), 3.96 (2H, d, J
8Hz, CHCH2N), 4.17 (2H, q, J 8Hz, C_2CH3), 4.43 (2H, s, NCH2-
C6H5), 4 92 (1H, d, J 1.5Hz, 3-CH), 4.67 (1H, bt, J 8Hz, 8-CH),
5.17 (2H, s, CO.O.C_2C6H5), 5.53 (1H, d, J 3Hz, 5-CH), 7.23,
7.30 (10H, 2 x s, Ar-H).
- 93 -
- ' L - '
, ` .
' ' , ~ ' ~ , ,
~ 3~
Example 31
Methyl 9-N-benzylaminodeoxyclavulanate h~ro~ L-malate
Methyl 9-N-benzyloxycarbonyl-N-benzylaminodeoxyclav~lanate
(218 mg) in tetrahydrofuran (15 ml) and water (1 ml) was
hydrogenated at N.T.P. in the presence of L-malic acid (67mg)
and 10% palladium on carbon (73 mg) for two hours. The catalyst
was removed by filtration and the filtrate evaporated to
dryness. Trituration with propan-2-ol/ether gave the title salt
as an off-white solid. ~max (Nujol) 1800, 1745, 1695, 1~25 cm 1.
_ g ~ _
~34~
Example 32
Ethyl 9-N-benzylaminodeoxyclavulanate hydrogen L-malate
A solution containing ethyl 9-N-benzyloxycarbonyl-~-
benzylaminodeoxyclavulanate (140 mg) and L-malic acid (42mg)
in tetrahydrofuran (15ml) and water (1ml) was hydrogenated
over 10% palladium on carbon (45 mg) at N.T.P. for two hours.
The catalyst was filtered off, washed with water and the
filtrate evaporated. Trituration of the residue with propan-
2-ol/ether gave the product as an off-white solid. ~ max (Nujol)
1800, 1745, 1695, 1630 cm 1.
`~ _ 9 5_
'
~i83~9
.,
Example 33
Benzyloxycarbonylmeth~l 9-N-benzylox~carbonyl-N-benzylamino-
d xyclavulanate
9-N-Benzyloxycarbonyl-N-benzylaminodeoxyclavulanic acid
lithium salt (428 mg) was suspended in dimethylformamide (15 ml)
and three drops of water added; benzyl bromoacetate(343 mg) was
added to the resulting solution and the reaction mixture stirred
at room temperature for five hours. Thin layer chromatography
showed the reaction to be complete, the solvent was removed and
the residue chromatographed on silica gel, eluting with ethyl
acetate/cyclohexane (1:1). The combined ~ractions were evaporated
in vacuo to yield a colourless oil (384 mg).Rf (SiO2: ethyl acetate:
cyclohexane, 1:1) = 0.71; ~a]D = + 12.2 (C = 1.~; MeOH); ~max
(film) 1805, 1750 (b), 1705 (sh), 1690 cm~1; ~(CDC13) 2.86 (1H, d,
J 17.5Hz, 6~-CH), 3.38 (1H, dd, J 17.5 and 3Hz, 6-CH), 3.94 (2H,
d, J 8Hz, 9-CH2). 4.44 (2H, s, NCH2Ph), 4.65 (2H, s, C02CH2C02),
4.74 (1H,bt,partially obscured by signal at 4.65), 5.04 (1H, d, J,
1.5Hz, 3-C_), 5.15 (4H, s, NC02CH2Ph and CH2C02C_2Ph), 5.52 (1H, d,
J 3Hz, 5-CH), 7.22, 7.29 (15H, 2 x s, aromatic -H).
- 96 -
.. , .. ~ . . . ~ ....... .................. . . .. , ..... . .. ... . . . . ~
~ 8349
Example 34
2-Hydroxyethyl 9-N-benzyloxycarbonyl-N-benzylaminodeoxy~ _ te
2-Iodoethanol (860 mg) was added to a solution of lithium
9-N-benzyloxycarbonyl-N-benzylaminodeoxyclavulanate (428mg) in
dimethylformamide (15 ml) and water (3 drops). The solution
was stirred for 14 hours at room temperature and the solvent
removed; the residue was purified by column chroma-tography on
silica gel, eluting with ethyl acetate/cyclohexane, 1:1. The
product was obtained as a colourless oil. Rf (SiO2: ethyl
acetate:cyclohexane, 1:1) = 0.29; ~max (film) 1800, 1745, 1700 cm~1.
_ g q _
~- `
11183~9
Example ~5
Methyl ~-N-benzylaminodeoxYclavulanate hYdro~en succinate
Methyl 9-N-benzyloxycarbonyl-N-benzylaminodeoxyclavulanate
(109mg) was dissolved in tetrahydrofuran/methanol (10ml) and
hydrogenated at N.T.P. over 10~ palladium on carbon (38mg).
The catalyst was filtered off and the solvent evaporated to
yield the product as a gum. ~max (film) 1800, 1740, 1695, 1615 cm 1
:
:
: '
- 98 -
~ . . ,: .
-
.
' '
83~9
ExamPle 36
Methyl 9-N-benzylaminodeox~clavulanate hydro~en L-tartrate
Methyl 9-N-benzyloxycarbonyl-N-benzylaminodeoxyclavulanate
(87mg) in tetrahydrofuran (10ml) and water (1ml) was hydrogenated
at atmospheric pressure in the presence of (+)-tartaric acid
(30mg) and 10% palladium on carbon (30mg) for ~ hour. The
mixture was filtered through celite, the catalyst washed with
water, and the filtrate was evaporated to dryness. Trituration
of the residue with acetone/ether gave the product as a white
solid. ~max (Nujol) 1797, 1743, 1700, 1620 cm 1.
, ... . ... . .. . . .
:1.:118~4g .
Example 37
Composi-tions
a. 100mg of sterile 9-N-benzylaminodeoxyclavulanic acid
may be dissolved in 5ml of sterile water for injection to yield
an injectable solution.
b. 100mg of sterile 9-N-benzylaminodeoxyclavulanic acid and
sterile sodium amoxycillin equivalent to 250mg pure free acid
may be dissolved in 8ml of sterile water for injection to yield
an injectable solution.
c. 50mg of sterile 9-N-benzylaminodeoxyclavulanic acid and
sterile sodium amoxycillln equivalent to 250mg pure free acid
may be dissolved in 5ml of sterile water for injection to yield
an injectable solution.
Similar compositions may be prepared which contain 9-N-(4-acetam-
idobenæyl)aminodeoxyclavulanic acid, 9-N-(4-hydroxybenzylamino)-
deoxyclavulanic acid or 9-N-(4-methoxybenzyl)aminodeoxyclavulanic
acid in place of the 9-N-benzylaminodeoxyclavulanic acid.
.
-- 1 00 _
83L~g
Demonstration 1
In-vit~ ActivitY
The MIC values for ampicillin alone and in 1 ~g/ml or
5 ~g/ml of the compounds of the Examples were determined by
the microtitre method. The results were as follows:
-- 101 --
__ _ __ ... .,_ __ _ ., . .. _ .. .... .... . .. .. . . . . . ., .. .... . . . . . . . . . .. ... . . . ~
.
,
~ 3 ~
. "
~ 0 o ;~ ~ J
O I + 0~0~D ~ ~00
~ ~ o O O O O O O O O
tH O O O O O O O O
O + L~ O ~ O O Ll~
~ l C~l
~ ,
0 b~J ~ L~ J O ~- N J 0
P~ ~, ~ O~ ~ Ir~ ~I ~ ~ ,
~ ~ h 0 + ~ D 1~ ~> (~ t~l ~
~o P~o o 8 8 g o g g g
~ + U~ o U~ o o ~
h ~ . /~ l .
Il~ I~ 0
. ~ ~ ~ + ~ O ~ ~ O
a) a) u~
CH ~1 ~ ~ ~- ~D N , ~- N
O ~ s~! ~ + ~ ~
~ ~ o 8 og g g o 8 8 g
~ ^ + U~ o U~ o o ~ ~ U~
t ~ ~_ I'~
~ .
~O ~ ~ L~ O O i ~ O O O
~) t~q + 0 VO
O 1~ ~ ~ O N ~D ~D 1~ 0 0
. . ~ ~ + O O O O O O O O
. ~d o Ll~ ~
. ~) ~ O N 8 ~ 8 8 ~ N c\i
. + ~ Lf~ ~D
a~
4~z;
~ ~ ~ ~ ~ U~ ~ ~
~ ~ i '
~ U~C~
_ . _ ._
- 102 --
~118~4~
In-vivo Activity
The synergistic effect of the compounds of this
invention in animals is demonstrated by the co-administration
of the compound of Example 2 and amoxycillin (as the sodium
salt) to test animals infected by E. coli JT 39. This micro-
organism produces considerable quantities of a ~-lactamase
which degrades amoxycillin so reducing its antibaceterial
effectiveness in-vivo. However when the compound of Example
2 is administered to the test animal at the same time as
amoxycillin the inhibition of the ~-lactamase allows the
amoxycillin to exhibit its antibacterial activity. The
following results were obtained when amoxycillin alone or
together with the compound of Example 2 or sodium clavulanate
was administered sub-cutaneously to mice infected by E. coli
JT 39:
Test Compounds CD50 (mglk~ x 2)
Sodium amoxycillin alone 200
Sodium amoxycillin + 2 mg/kg
oP compound of Example 2 3. 3
Sodium amoxyclllin + 1 mg/kg
o~ compound of Example 2 8.7
Sodiu~ amoxycillin ~ 2mg/kg
of sodium clavulanate 14.5
Sodium amoxycillin + 1mg/kg
of sodium clavulanate 58
(Sodium amoxycillin was prepared by dissolving amoxycillin
trihydrate in a sodium carbonate/sodium bicarbonate buffer).
- 103 -
~llB349
,~' .
The compound of Example 2 was also effective in
protecting amoxycillin from the ~-lactamase of E. coli JT 39
in-vivo when administered orally but was less effective
per given weight than when administered sub-cutaneously.
The compound of Example 2 was not observed to produce
toxic effects during these -tests.
- 104 -
_._ ., ,.. , .. , .~.. .... .. ....... .
~ ;
, -. ' ,'
~ 83~
~, .
Demonstration 2
Demonstration of Effectiveness as Synergist
The following approximate CD50 values were obtained for
amoxycillin in the presence of the compounds of certain
Examples when administered ~ub-cutaneously 1 and 5 hours post
infection against a peritoneal infection due to E.coli JT39.
Test 1 CD50
Amoxycillin alone 1000 mg/kg x 2
Amoxycillin + Comp Ex 2 at 2mg/kg 4.5 mg/kg x 2
Amoxycillin + Comp Ex 2 at 1mg/kg 6.3 mg/kg x 2
Amoxycillin + Comp Ex 7 at 2mg/kg 6.4 mg/kg x 2
Amoxycillin + Comp Ex 7 at 1mg/kg 7.5 mg/kg x 2
Amoxycillin + Comp Ex 9 at 2Tng/kg 4.5-8 mg/kg x 2
Amoxycillin + Comp Ex 9 at 1mg/kg 12 mg/kg x 2
Cefazolin alone 10.5 mg/kg x 2
Test 2 CD50
Amoxycillin alone 1000 mg/kg x 2
Amoxycillin + Comp Ex 6 at 2 mg~kg 3.1 mg/kg x 2
Amoxycillin + Comp Ex 6 at 1 mg/kg 8 mg/kg x 2
Amoxycillin + Comp Ex 2 at 2 mg/kg 4.5 mg/kg x 2
Amoxycillin + Comp Ex 2 at 1 mg/kg 8.5 m~/kg x 2
Test 3 CD50
Amoxycillin alone 1000 mg/kg x 2
Amoxycillin + Comp Ex 13 at 2 mg/kg 4.4 mg/kg x 2
Amoxycillin + Comp Ex 2 at 2 mg/kg 5.8mg/kg x 2
- l05 -
'
'
` ~ ~83~
Demonstration 3
Demonstration of Effectiveness as ~nti-Bacterial
Mice were infected with staphylococcus aureus Russell (thigh
lesion with 0,2 ml im) and thereafter were dosed sub-
cutaneously at 1, 3 and 5 hours post infection with a
solution of the test compound. The following results were
obtained:
Com~ound Dosa~e (m ~kg) Protection (,
9-N-Benzylaminodeoxyclavulanic 5 69.8
acid 10 89.7
99.3
9-N-p-Methoxybenzylaminodeoxy- 5 28.0
clavulanic acid 10 80.8
87.6
Cloxacillin 20 61.6
91.7
Cefazolin 20 39.0
71.2
The LD50 f 9-N-benzylaminodeoxyclavulanic acid in mice is
greater than 1000 mg/kg on intra peritoneal i~'ection and
greater tk~n 500 mg/kg on sub-cutaneous administration.
- 106 _
___ _ . . _ ~ _. .. _.. . . .... .. . ... _ _.. , . _ .... _._ _ _ . . _ . .. ~
