CA1118349A - Anti-bacterial agents - Google Patents

Abstract

ABSTRACT
A synergistic pharmaceutical composition which comprises a penicillin or a cephalosporin and a compound of the formula (II):

(II) or an ester thereof or a pharmaceutically acceptable acid addition salt of such an ester wherein R1 is a hydrogen atom, an alkyl group of up to 5 carbon atoms, a cycloalkyl group of 5 or 6 carbon atoms, a hydroxylalkyl group of up to 5 carbon atoms or a moiety of the sub-formula (a):

Description

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):

2-N (I) 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):

;~

~ 11183 19 Fl_ ~H -N H--CH - R ( I I ) 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):

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):

~ 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):

.. , , . . ~

.
" -: :

~' .
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.

! , .' ` ` ' ' ' . . .

~ 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

: ~ : : , . -- , .
. :;
' 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

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

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

(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.

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.

.-: :. ~
.
-: :. .: ~
,, :
~ ~ .

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 _ .... . .
' ~: ' . ;., ~- . .

~ 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 ~ ~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 _ ..... , . , .. . ~ , . . . . . ~ .

.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) 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 ` ~

~ 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):

~ 11183~9 H C0-0-~H2R7 N ~ CH2-Rl (XI) 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 ,. .. .. . . .
:
~ .
' 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):

O ~ (XIII) 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 , ;. ' ~

: -:

~ 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.

, . . ~ , - :

^
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).

.
: .

~ 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.

.. _ .. .. . ... .. ... . . . . ~

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).

.

e~

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).

~ 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, ~; (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).

;

.. ..
~.'. .

- , , .

~ 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)0cH2c6H5-3 ~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).

,, , ,~

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).

~ 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).

..;

~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).

,.

. .
. ~ ;

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.

~ ~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).

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)), ;' '"

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).

:
'' ~ 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.

, ., . . , - . .

~ , ~-\
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) . . ~ :.

":~
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).

- ' - ' ':

~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, '~ ' .

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.

. .... . ,.. ... .... ... . .. . ... ... . .. . . , . , . . ~ .

..
., - . :, '. : ,~ . ~ . :
~ .

-- 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 , :

~:

. .
-.. ~: . . . -~: . . , :

.

~ 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).
:
' ' .

~' :

~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.

:, . .
~.

~ 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.

., .. ,~.,. ,,.. ~....... . ~

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%).

., ' , ~ .
: ' ~ 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 ~

. ~ . . . .
~:: . . : , :: :
: ~ ~ ,, -' ', 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.

,, , ; :

.: : .
.: ' ,., ' .: ~

~ 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~.

.. , . ,~ . .. . .. .. . .
~ ~ , - ' ~ 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 -.

~ 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).

. .

~ .
':

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 ~
, ~' ~ 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.

' : , ~ 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.

- ~, ~ ,, '' .

~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).

....... ,., _ ,. .
, 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.

.... , , . , .. , , . .. . . . .............. . -, -~ ~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).

' ' '-. ` - ~ . ~

~ 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.

.

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, _ . ... _ , .. _ _ _ _ _. . .. , . ... _. _ . ~ _.. __ , _ .. _ . . ... .. ~ . . . ... .. . . _ _ . .. .
~ .

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 .

: . . . ~, . , . . .. .

. .: . . ~ :
- ~
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, , . .

,~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-_).

~' . , -~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).

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).

- ' 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).

.. , .. ~ . . . ~ ....... .................. . . .. , ..... . .. ... . . . . ~

~ 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 :
:
: ' ~ . . ,: .
-.

' ' 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:

__ _ __ ... .,_ __ _ ., . .. _ .. .... .... . .. .. . . . . . ., .. .... . . . . . . . . . .. ... . . . ~
.
, ~ 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~
_ . _ ._ ~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).

~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.

_._ ., ,.. , .. , .~.. .... .. ....... .

~ ;
, -. ' ,' ~ 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 _ ___ _ . . _ ~ _. .. _.. . . .... .. . ... _ _.. , . _ .... _._ _ _ . . _ . .. ~

Claims (28)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY OR PRIVILEGE
IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A synergistic pharmaceutical composition which comprises a penicillin or a cephalosporin and a compound of the formula (II):

(II) or an ester thereof or a pharmaceutically acceptable acid addition salt of such an ester wherein R1 is a hydrogen atom, an alkyl group of up to 5 carbon atoms, a cycloalkyl group of 5 or 6 carbon atoms, a hydroxylalkyl group of up to 5 carbon atoms or a moiety of the sub-formula (a):

wherein R2 is a hydrogen, fluorine, chlorine or bromine atom or an alkyl group of 1-3 carbon atoms, an alkoxy group of 1-3 carbon atoms, an acyloxyl group of 1-3 carbon atoms, hydroxyl group, an alkoxycarbonyl group containing 1-3 carbon atoms in the alkoxy part, or a group -N(R5)CO.R6, -N(R5)SO2R6, -CO-NR5R6 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 alkoxy group of 1-3 carbon atoms or an acyloxy 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 alkoxy group of 1-3 carbon atoms and when required a pharmaceutically acceptable carrier therefor.

2. A synergistic pharmaceutical composition which comprises a penicillin or a cephalosporin and a compound of the formulae (III) - (VII):

(III) (IV) (V) (VI) (VII) and esters thereof wherein R2 is 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 a 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, Q is a hydrogen, fluorine or chlorine atom or a methyl, methoxyl, ethyl or ethoxyl group, Q1 is a hydrogen, fluorine or chlorine atom or a methyl, ethyl, methoxyl, ethoxyl or hydroxyl group, and R5 and R6 are as defined in relation to formula (II) in claim 1 and when required a pharmaceutically acceptable carrier therefor.

3. A synergistic pharmaceutical composition which comprises a penicillin or a cephalosporin and a compound of the formula (VIII) or (IX):

(VIII) (IX) wherein R1 is as defined in claim 10 or CH2R1 is a group as defined for the 9-N-substituent in formulae (III) - (VII) as defined in claim 10; A1 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 atoms or a phenyl group optionally substituted by a fluorine, chlorine, bromine, nitro or alkyl or alkoxyl of up to 4 carbon atoms and when required a pharmaceutically acceptable carrier therefor.

4. A synergistic pharmaceutical composition as claimed in claim 1 which includes 9-N-(2-hydroxyethyl) aminodeoxyclavulanic acid.

5. A synergistic pharmaceutical composition as claimed in claim 1 which includes 9-N-benzylaminodeoxyclavulanic acid.

6. A synergistic pharmaceutical composition as claimed in claim 1 which includes 9-N-ethylaminodeoxyclavulanic acid.

7. A synergistic pharmaceutical composition as claimed in claim 1 which includes 9-N-(dl-2-hydroxypropyl) aminodeoxyclavulanic acid.

8. A synergistic pharmaceutical composition as claimed in claim 1 which includes 9-(4-fluorobenzyl) aminodeoxyclavulanic acid.

9. A synergistic pharmaceutical composition as claimed in claim 1 which includes 9-N-(4-methylbenzyl) aminodeoxyclavulanic acid.

10. A synergistic pharmaceutical composition as claimed in claim 1 which includes 9-N-(4-methoxybenzyl) aminodeoxyclavulanic acid.

11. A synergistic pharmaceutical composition as claimed in claim 1 which includes 9-N-(4-hydroxy-3-methoxybenzyl) aminodeoxyclavulanic acid.

12. A synergistic pharmaceutical composition as claimed in claim 1 which includes 9-N-(4-hydroxy-3-methoxybenzyl) aminodeoxyclavulanic acid.

13. A synergistic pharmaceutical composition as claimed in claim 1 which includes 9-N-benzylaminodeoxyclavulanic acid.

14. A synergistic pharmaceutical composition as claimed in claim 1 which includes 9-N-(4-hydroxybenzyl) aminodeoxyclavulanic acid.

15. A synergistic pharmaceutical composition as claimed in claim 1 which includes 9-N-(3,4-dimethoxybenzyl) aminodeoxyclavulanate.

16. A synergistic pharmaceutical composition as claimed in claim 1 which includes 9-N-(4-acetamidobenzyl) aminodeoxyclavulanic acid.

17. A synergistic pharmaceutical composition as claimed in claim 1 which includes 9-N-(2-methoxybenzyl) aminodeoxyclavulanic acid.

18. A synergistic pharmaceutical composition as claimed in claim 1 which includes 9-N-(2-fluorobenzyl) aminodeoxyclavulanic acid.

19. A synergistic pharmaceutical composition as claimed in claim 1, 2 or 3 wherein the penicillin is selected from benzylpenicillin, phenoxymethylpenicillin, carbenicillin, azidocillin, propicillin, ampicillin, amoxycillin, epicillin, ticarcillin, cyclacillin, pirbenicillin, azlocillin, mezlocillin, celbenicillin and pro-drugs therefor.

20. A synergistic pharmaceutical composition as claimed in claim 1, 2 or 3 wherein the cephalosporin is selected from cefatrizine, cephaloridine, cephalothin, cefazolin, cephalexin, cephacetrile, cephamandole mafate, cephapirin, cephradine, 4-hydroxycephalexin, cefaparole, cephaloglycin and pro-drugs therefor.

21. A synergistic pharmaceutical composition as claimed in claim 1, 2 or 3 wherein therein is included the sodium salt of amoxycillin, the trihydrate salt of amoxycillin, cephaloridine, cefazolin, the sodium salt of cephaloridine or the sodium salt of cefazolin.

22. A synergistic pharmaceutical composition as claimed in claim 1, 2 or 3 wherein the ratio of the compound to the penicillin or cephalosporin is in the ratio range of from 10:1 to 1:10.

23. A synergistic pharmaceutical composition as claimed in claim 1, 2 or 3 wherein the unit dosage form is from between 25 and 1000 mg.

24. A synergistic pharmaceutical composition as claimed in claim 1, 2 or 3 wherein from between 50 and 1000 mg of the compounds as defined in claims 1, 2 or 3 are administered each day of treatment.

25. A synergistic pharmaceutical composition as claimed in claim 1, 2 or 3 wherein the penicillin or cephalosporin is present in an amount of about 62.5 to 1000 mg per dose.

26. A synergistic pharmaceutical composition as claimed in claim 2 or 3 which includes from 150 to 1000 mg of amoxycillin as the trihydrate or sodium salt and from 25 to 500 mg of a compound of the formulae (III) to (VIII) as defined in claims 2 and 3.

27. A synergistic pharmaceutical composition as claimed in claim 2 or 3 which includes from 150 to lO00 mg of ampicillin or a pro-drug therefor and from 25 to 500 mg of a compound of the formulae (III) to (VIII) as defined in claims 2 or 3.

28. A synergistic pharmaceutical composition as claimed in claim 2 or 3 which includes from 200 to 700 mg of a penicillin selected from ampicillin tirhydrate.