CA1100406A - Synergistic pharmaceutical compositions comprising clavulanic acid derivatives with a penicillin or a cephalosporin - Google Patents

Abstract

ABSTRACT
A pharmaceutical composition which comprises a compound of the form-ula II
II
wherein R1 and R2 can be an alkyl, alkenyl, phenylalkyl or phenylalkenyl group optionally inertly substituted by a halogen, OH, OR3, O.CO2R3, SR3, or a COR3 group wherein R3 is a hydrocarbon group of up to 8 carbon atoms and the group NR1R2 contains a total of not more than 16 carbon atoms and esters of compounds of formula II and acid addition salts thereof, said esters having the following formula IV and V
IV
V

wherein R1 and R2 are as defined above and A is an alkyl group of 1 - 8 carbon atoms optionally substituted by halogen or a group of the formula OA4, OCOA4, SA4, SO2A wherein A4 is a hydrocarbon group of up to 6 carbon atoms; A2 is a hydrogen atom, an alkyl group of up to 4 carbon atoms or a phenyl group op-tionally substituted by halogen or by a group A5 or OA5 where A5 is an alkyl group of up to 6 carbon atoms; and A3 is a phenyl group optionally substituted by halogen or by a group A5 or OA5 where A5 is an alkyl group of up to 6 car-bon atoms or being selected from the group of esters consisting of phthalidyl, acetoxymethyl, pivaloyloxyethyl, ?-acetoxyethyl, ethoxycarbonyloxymethyl, and ?-ethoxycarbonyloxyethyl and a penicillin or a cephalosporin and a pharmaceutical carrier therefor.

Description

The present invention relates to pharmaceutical compositions containing antibacterial agents which also possess ~ -lactamase in-hibitory activity.
This application is a division of copending application no.
263,103, ~iled October 12, 1976.
Relgian Patent 827,926 discloses inter alia clavulanic acid, which is the compound of the formula (1):

~"~ O~ 0 ~ ¦

c/~ I\J ~ , .

and its salts and esters. Clavulanic acid was shown to be a useful antibacterial agent which possesses B-lactamase inhibitory properties which allowed it to enhance the anti bacterial ef~ectiveness of penicillins and cephalosporins against many gram-nega-tlve and gram-positive bacteria. tre have now discovered a further group o~ compounds that exhibit antibacterial and B-lactamase inhibitory activity but which have a different spectrum of activity -to cIavulanlc acid and its sal-ts..

The basic .Avention provides corpounds ol -the formula (II)

-2 ~ ~ C~2- NR 1 ~2 C~a~ ' .
and salts and esters thereof wherein Rl i5 an inert organic group of up to 14 carbon atoms and R2 is an inert organic group of up to 16 carbon atoms, the NR1 R2 group containing up to 22 carbon atoms.
The present invention provides for synergistic mixtures of such compounds in combination with a penicillin or a cephalosporin.

~ hen used herein the term 'inert organic group' means any organic group that is itself stable and which does not contain any functional groupings ~Jhich cause rupture of the ~-lactam ring of the compound of the formula ~II). Such groups do not include highly electron withdrawing groups situated in a manner which prevents the amino OI the fo~nula (III~:

~ H N R1 R2 (III) from being sufficiently basic -to form a salt of the carboxyl group of the compound of the formula (I) nor do ~hey contain substituents that render t~e amine of the formula (III) unstable.
Nor~ally R1 and R2 are such that the conjugate acid o~ the amine of the ~ormula H2 NR1R2 hàs a pKa of from 7.0 to 11.2 and .
pref~rabl, has a pKa of B~t~ 10. ~ -_3_ ~ ~

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

More suitably Rl contains not more -than 8 carbon atoms. More suitably R2 contalns not more than 14 carbon atoms. Most suitably the NRlR2 group contains not more than 16 carbon atoms.
Most suitably Rl and R2 can be an alkyl, alkenyl, phenylalkyl or phenylalkenyl group. optionally inertly sub-stituted by a halogen, OH, OR3, O.CO2R3, SR3 Ol a COR3 grouy wherein R3 is a hydrocarbon group of up to 8 carbon atoms and the group NRlR2 contains a total of not more than 16 carbon lQ atoms.

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

Suitable values ~or R1 include hydrocarbon groups (such as alkyl, alkenyl, phenylalkyl, phenylalkenyl or the like) option-ally inerkly substituted by halogen, O~l, OR3, O.CO2R3, SR3, COR3 or the like wherein R3 is a hydrocarbon group of up to 8 carbon atoms.
One group of particularly suitable values for Rl is that of the sub-formula (a):

_ f R6 R5 (a) wherein R~ is a hydrogen atom or a Cl 4 alkyl group, R5 is a hydrogen ato~ or a Cl ~ alkyl group and R6 is an optionally substituted phenyl group.
More suitably R4 is a hydrogen atom. More suitably R5 is a hydrogen atom~
When used herein the term "optionally substituted phenyl Group" means a phenyl group or a phenyl group substituted by halogen, OH, OR7, OCOR7, CO2R7 ~r COR7 where R7 is a hydrocar-bon group o~ up to 7 carbon atoms and more suitably the term means a phenyl group or phenyl group substituted by halogen, C~ 4 alkyl, Cl 4 alkoxyl or hy~roxyl.
Particulaxly suitable groups of the sub-~ormula (a) i~clude the benzyl, methoxybenzyl and chlorobenzyl groups espècially the benzyl group ~. ' - .-. . ... : - ,.... ,, .. , - . . , .. . . ,, ~ , .
. . . . , ... . - . : . : . - : : .
,: . .. . . ,: , : -; , . . .

- A further group of particularly suitable values for R1 ls that of the sub-formula (b):

_-- C ~ C(R10) = CR11 R12 R

wherein each of R~ to R11 is independently a hydrogen atom or a Cl _ 4 alkyl group and R12 is a Cl 4 alkyl group or an optionally substitu-ted phenyl group or a hydrogen atom.

More suitably each of R8 to Rll is lndependently a hydrogen atom at or a methyl group. Mor~ suitably each of Rg to R11 is independently a hydrogen atom. More suitably R12 is a hydrogen atom or a Cl 4 alkyl groupO

10Yet another particularly sultable ~roup of values for R1~ is that o~ the sub-formula (c) : 18 ,.
C - CH (R10) CHR11 R12 . Rg (c) wherein R8 to R12 are as defined in rela-tion to sub-formula (b.~.
Particularly suitable ~roups R2 include those of the sub~formula (a), (b) and ~c) previously described as suitable ~or R1. The group R2 need not have the same value as the group R1 but it is sometimes convenient for it to be so.
, . . ~

, '' .

.

.
. , . . j . .~ , .
.....
. - :
, Q~
~ .

.
Other particularly suitable groups R2 include those of the sub-formula (d~:

13 14 (d) in R13 is a hydrogen a-tom or a Cl 4 alkyl ~roup and R14 is an alkyl group ofl to 14 carbon atoms optionally subs-tituted by O~, OR15 OCOR15 or COR15 where R15 is a h~Jdrocarbon group of u~
to 8 carbon atoms.

~en R1~ is a hydrogen atom it is preferred that R14 is not a methyl group.
Particularly suitable groups of the sub-formulae (d) include the isopropyl, 2-hydroxyethyl, 2~hydroxypropyl 9 ethyl, 2-acetoxyethyl t 2-~ethoxyethy1 and the like ~roup.
Suitàble esters of the formula include those of the formulae (lV~ and (V): .

. .

~ R

CEI2 \ ' / R
O ~ 3 ~ C~2 ~ N

2 l 2 :

(IV) (V) , . ~ .
and acid addition salts thereof wherein R1 and R2 are as defined in relation to formula (II) and Al is an alkyl group of 1 - 8 carbon ato~s op-tionally substltuted by halogen or a group of the formula oA4, OCOA , SA ,So2A4 wherein A4 is a hydrocarbon group of up to 6 carbon atoms;
A is a hydrogen atom, an alkyl group of up to 4 carbon atoms or a phenyl group optionally substituted by halogen ar ~y a group A5 or oA5 where A5 is an alkyl group of up to 6 carbon atoms; and A3 is a phenyl group optionally subs-ti-tuted by halogen or by a group A5 or oA5 where A5 is an alkyl group of up to 6 carbon atoms.

Suitable acid addition sal-ts include those formed with pharmaceutically acceptable acids which are known to be suitable for forming sal-ts with esters of penicillins or cephalosporins which contain a basic group.

A~further suitable group of esters are those which are readilly in-~ivo hydrolysable. Belgian Patent No. 827926 describes - such in-vivo hydrolysable esters. Suitable in-~ivo hydrolysable esters include phthalidyl, acetoxymethyl, pivaloyloxyme~hyl, ~acetoxyethyl, etho~ycarbonyloxymethyl, C~ethoxycarbonyloxyethyl and -the like esters.

' . _ .
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-When non-esterifled~ the compounds of this inven-tion are normally salted. Such salts are preferably zwitterionic salts of the formula (VI).

~ ,~R1 ~2 O ` ~2 Other salts of the compounds of formula (II) include those with pharmaceutically acceptable salting ions such as those ~escribed in 8elgian Patent No. 827920 and also : those with pharmaceutically acceptable acids. These other salts are not a preferred feature of the invention~

.. , ' ' :

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From the preceeding statements it will be realised that certain particularly suitable compounds of this inven-tion are of the formulae (VII)-~IX) H CE~2 R~ , / \ / CH2 - N /
N ~ 15 (VII) CH2 - Cl~=CE~-CE~R
~H O CH. - N
~ / 2 .. N
o C02H
(VIII) ..

CH2 - CH2 ~ CH2-R12 . . H O CH2 - N

. 15 N "

O ~2 H (IX~

.

. . . . . .

_ g _ .

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

and in-vivo hydrolysable esters thereof whereln R6 and R12 are as described in rela-tion to sub-formulae (a), (b) and (c) and R15 is a CH2 R~, CH2 CH: CHR12, CH2- CH2- CH2-R12 or CHR13 R14 group ~here R13 and R14 are`defined as in rela-tion to sub-formula (d).

Compounds within formulae (VII) can be particularly effective in inhibiting ~-lactamases produced by gram-positive bacteria. Compounds within for~ulae (VII) and (IX) are envisaged as useful fcr -t~.eir broad spectrum of ~-lactamase inhibition.

_ 10 ~~

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.

The present invention provid~s a process for the preparation - of the compounds of the o~nula (II) and salts and esters thereof which process comprises the reaction of an amine of the formula (III) H ~ ,R2 (III) wherein R1 and R~ are as defined in relation to formula (II), and a compound of the formula (X~.

~I ~Y
//r~~~ N ~
O ' ~ ( X ) where C02A is an ester group alld -thereafter if desired converting the thus formed compound into the corresponding carboxylic acid or a salt.

The dlene~of the formula (X) may be formed before the reaction with the amine of thç formula (III~ or it may be prepared in situ. Suitable methods of preparing the diene in situ include *he displacement of sulphate or carbo~ylate moiety from a compound of the formulae ~XI) or (XII) . O ~ (~) (+) -'' 2 CH2 - 0 - S03 HN ~ Q

/~ N -~

s ~XI) .. , ~
,' ' . . .
. . -- . . .. . . . . .

..

H

~ N

(XII) ' wherein A ls as ~efined in relation to 40rmula (x), Q1, Q2 and Q3 are groups such that NQ Q2 Q3 is a ~ertiary amine and Q4 is an organic group.

Suitab1~ -the reaction of Ihe amine of the formula (III) ~itn the col!lpound of the formula (,'), (;~I) or (XII) take place in an aprotic solvent such as acevonitrilei dimethylformami~e or other similar solven-t ~t a non-extreme -temperature, for example - 10 to 50C, more usually - 5 to 25C and conveniently within the range 0 to 20.

. Suitably one or more of Q1, Q2 and Q3 is a C 1 6 alkyl group such as a methyl or ethyl group.

: Suitably Q4 is a C1 6 alkyl, benzyl, dichloromethyl or like group. ~ . .
It is frequently advantageous to use the preformed diene ester of the formula (X) rather than -to generate it in situ.

~en a compound of the formula (XI) or (XII) is used ..
: in the process o4 this invention a certain degree of dlrect displacement o~ the leaving group by the amine may take place .~ but i-t is believed that in general most and possibly effectively :all of -the desired compou~d is produced via the cliene.

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

. .
' " ' ' "' " ' ' ' `' ~ . ~ ' The present inventlon also provides a process for the preparation of acids of the formula (II) and -the carboxylate salts thereof which process comprises the de-esterification of a corresponding ester of -the compound of the fo~lula (II) and if desired simul~aneously or sub-sequently sal-ting the carboxyl group.
De-esterification may be brought about by conventional - mild methods such as hydrogenation or mild basic hydrolysis.

Suitable hydrogenolysable esters of the co~pound o~
the formula (11) include benzyl and like esters. Such esters may be clea~ed by hydrogenation using a low or medium pressure of hydrogen, for example about l atmosphere, at an approximately ambient temperature, for example about 12 - 20 C, in a con-ven-tional inert solvent, :for example eth~lol, If a base is pres~t'a basic salt maybe formed.' Normally no base is present and the ~witterionic salt results.

Suitable base hydrolysable esters include acetoxymethyl?
phthalidyl and the lilce ester which undergo hydrolysis when maintained in an aqueous medium at a pH of 7 to 8.5. Such reactions can occur rapidly, for example in 10-60 minutes.
Most suitably such reactions take place in a solvent which is water or water together with an ~rganic solvent such as tetra-hydrofuran. The reaction usually occurs sufficiently rapidly, at 5 - 20C. The pH may be maintained at the correct level ~ 13 -,, . , . - ~ ~ -: ~ : -' ' ' - .' . . .
. . .

:- .

~L~01[~40G

`~
by ~he careful addition of base. This is generally a less suitable me-thod of clea~ing the ester than hydrogerloly5is.

Compounds of the formula (II) t~here both or one of R1 and R2 is a group of the sub-formula (c) as herein before described may be prepared by the reduction of a corresponding compo~md containing a corresponding group of the sub-formula (b). Such reactions are normally effected by hydrogenation in the presence of a transition metal catalyst in conventional manne~

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_ 1 4 -.. .. . . .
' ~ .'. ', " ' ' ' The present invention also provides pharmaceutical compo~
sitions 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 use and may be used for the treatment of infection in mammals including humans.
Suitable forms of t~e compositions of this i.nvention include tablets, capsules, creams, syrups, suspellsions, solu-tions, reconstitutable powders and sterile forms suitable for injection or infusion. Such compositions may contain conven-tional pharmaceutically acceptable materials such as diluents, binders, colours, flavours, preservatives, disintegrant and the like in accorda.nce with conventional pharmaceutical practice in the manner well understood by those skilled in the art of formu-latincJ antibiotics.
Injectable or infusable compositions of salts of a com- :
pound ofc the formula (II) are particularly suitable as high tissue levels of a compound of the formula (II) can occur after administration by injection or infusion. Thus, one preferred composition aspect of this invention comprises a salt of a eompound of the formula (II) in sterile form.
Unit dose eompositions comprisincJ a compound of the formu-la (IIl or a salt or ester t.hereof adapted for oral administra-~ tion form a further preferred eomposition aspect of this inven-tion.

~!: ' ' ' . ~ ................ . . . .
-- .

-The compound of the formula (II) or its salt or estermay be present in the composition as sole therapeutic agent or it may be present together with other therapeutic agen-ts such as a ~-lactam antibiotic. Suitable ~-lactam antibiotics for inclusion in the compositions of this invention include benzyl-penicillin, phenoxymethylpenicillin, carbenicillin, azidocillin, propicillin, ampicillin, amoxycillin, epicillin, ticarcillin, cyclacillin, cefatriazine, pirbenicillin, ~-sulphonyloxybenzyl- ¦
penicillin, cephaloridine, cephalothin, cefazolin, cephalexin, cefoxitin, cephacetrile, cephamandole nafatel cephapirin, ceph- j`
radi~ne, ~-hydroxycephalexin, cefaparole, cephaloglycine, and other well known pencillins and cephalosporins or pro-drugs ...
therefore such as hetacillin, metampicillin, ~-ace-toxyampi~
cillin, the acetoxymethyl, ethoxycarbonyloxymethyl, pivaloyl-oxymethyl or phthalidyl esters of ben~ylpenicillin, ampicillin, amoxycillin, or cephaloglycine or the phenyl, tolyl or indanyl C~es-ters of carbenicillin or ticarc.illin or the like. Such compounds are frequently used in the form of a salt or hydrate. .
Naturally if the penicillin or cephalosporin present in the composition is not suitable for oral administration then the composltion will be adapted for parenteral administration.

~ . :

.

V~
i When present in a phrmaceutical composition together with a ~-lactam antibiotic, the ratio of a compound oE the formula (II) or its salt or ester present to ~-lactam antibiotic present may vary over a wide range of ratios, for example 10:1 to 1:3 and advantageously may be ~rom 5:1 to 1:2, for example, 3:1 to 1:1.
The total quantit~ of antibacterial agents present in any unit dosage form will normally be between 50 and 1500 mg and will usually be between 100 and 1000 mg.
Compositions of this invention may be used for tl~e treat-ment o~ infections on inter alia, the respiratory tract, the urinary tract and soft tissues and mastitis in cattle.
Normally between 50 and 3000 mg of the compounds of the invention will be administered each day of treatment but more usually between 100 and 1000 mg of the compounds of the inven-tion will be administered per day, for e~ample as 1 - 6 doses, more usually 2 - 4 doses.
The penicillin or cephalosporin in synergistic composi-tions of this invention will normally be present by up to or at approximately the amount at which it is conventionally used.
Particularly ~avoured compositions of this invention will contain from 150 - 1000 mg of amoxycillin, ampicillin or a pro-drug therefore and from 50 - 500 mg of a compound of the formula (II) or a salt or in-vivo hydrolysa~le ester thereof and more suitably from 200 - 500 mg of amoxycillin, ampicillin or a pro-drug therefore and from 50 - 250 mg of a compound of the formula (II) or a salt or in-vivo hydrolysable ester thereof.

. .

..

.

_ The ma-terials present 1~ s~ch compositions may be hydrated if required, for e~ample ampicillin trihydrate or z~oxycillin trihydrate may be employed. The wei~h-ts of the a~-tlbiotics i~ such composi-tions are expressed on -the basis OL antlbiotlc -theoreticall~ available from t~e composition and not on the basis o~ the weight OL pro-drug.

. .
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E~MPLE 1 Benzyl 3-(2-dibenzylaminoethylidene)-7-oxo-1-azabicylo . .
L3,2,0~ heptane-2_carboxylate (e2) li ~--) (-~) Il CH2 - O - S - O HN (CH3) 3 / 2 6 5 _ N ? I CH - N
~0 CH2C6H5 C2C~2C6H5 C02CH2C6Hs (e 1) (e 2) Crude benzyl clavulanyl sulphate trimethylammonium salt (el) (500 mg) in Ereshly distilled dimethylformam:ide (5ml) was treated with a solution of dibenzylamine (450 mg) in dimethyl-formamide (2ml) dropwise over lS minutes. The reaction was stirred at room temperature for 2 hours and diluted with ethyl acetate/petroleum ether (b.p. 60-80) l/l (50ml). The solution was chromatographed directly on silica gel to yield as the first eluted product benzyl dibenzylaminodeoxy clavulanate (e2) as a light yellow oil (84 mg.). ~echromatography afforded pure (e2) as a colourless oil (38 mg.) The preparation of (el) is described in United States Patent ; 4,061,~49.

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

~0~36 ~ N,m.r. (CDCl~ .00 (lH, d, J= 17Hz, 6~-CH); 3.20 - (2H~ d,J= ~Hz), CH2N); 3.S2 (1H, dd, J= 17Hz, 6a--CH);

3.57 [4H, s, N(CH2Ph)2~; 4,84 (lH, br.t., J=8Hz, olefinic CH);
5.13 (iH, br.s., 3-~H); 5.25 (2H, s, CO~CH2Ph); 5.6~ (lH, d, J=2.5 Hz, 5-CH); 7,l~1~(15H, s, aromatic protons).

When the compound prepared by -the method of F,xample l was tested against certain enzymes by the ~-lactamase inhibition : assay described in Belgian Patent No. ~27926 the following 50 values were obtained:

Appro,~imate ~n~yme IS~ (in m~/ml) ~ coli JT4 0.02 E. _ JT10 0.24 Klebsiella aero~enes AE70 0.40 StaPhYlococcus aureus (Russell) 0.06 Psèudom~ aeru~inosa 0.14 Citrobacter man-tio G.15 .

_ ~0 -. . - .
' . .
., - , : : . : . ~ ....
.

- . - : : .
:: .

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

)4~
.

7-oxo-4-1-azabicyclo [3t2,0~ 2 - carboxyla-te ,Ç~ >~ "r~
Co~ C~ 5 O ~ ~ c~3 (e 2) (e3) The comnound (e2j 100 m~) in tetrah~Jdrofur~n (5 ml) ~as hydrog~na-ted at ambient temperature and pressur~ o~er 10j' , Pd'/C (50 m~) for onë hour. The ~olution ~as filtered through keirselguhr, the sol~ent evaporated, the residue dissolved in eth~Jlaceta-te (10 mlj and ex-tracted with wa-ter ' (4 * 10 ml); The water,was evaporated to yield a gum which was dissolved in methanol (5 ml) and,treated with excess/
diazomethane at 0C, Evaporation cf the solven-t and chromatography yield the desired compound (methyl dibenzylamino-deoxyclavulanate (e 3) as a colourless gum \ i.r. (CHCl3) 1800, 1755, 1700 cm 1 n.m.r. (CDCl3) 2.94 (1H,d,J=17H7, 6~ -CH); 3.17 (2H, d, J=7Hz, -CH-CH2); 3.41 (1H, dd, J= 1i Hz, J1 = 2.5Hz, 6c~ -CH);' 3.53 (4H,s, NCCH C6H5]2); 3,74 (~H, sl C0 C,~ ; 4.78 (1H,brit, J=7Hz9 = CH - CH2) 5,01 (1H, br. s, 3-CH); 5.60 (1H, d, J=2,5Hz~
5-CH~ 7~30 ~(10H,s N[CH~ C6H5]~ ~ ] 5 =,0.4 (c~0.81; ~OH) .
. `.~

- 2~ -~ '~-'' : ' , - . . . . . . .. .. . . . . . .
, . . .
, :,,: . . . .
, , , -X~lPL,~ ~ ~e~.vl. ~I-benzyl~ hydrox~ hylamino-deo~vclavulanate ", ~ / j O ~l 2 ~C~ C ~i`
//~
o cO2CH2C6H5 ~ `co2cy~2c6H5 . ' . O

(e 4) (e 5) Clavudiene benzyl ester (0,5~) ln ~oetor~itrile (10 ml) ~las cooled to C , N-benzyl-2-hydrox~Jethylamine (0.~6g)~ras added ancl ~he reac-tion mi,Y*ure s~irred for 2~ hours. Ethy~
' acetate (100ml) wa.s added and the mixtllre evaporated -to lo volume, The resid~le ~:~as subjected -to colwnn chomatography using ethyl acetate as eluen~. The pro~lct, ~as isola-ted in lo~ ield had an ir spect,rum (liquid film) as follows:
3400 (broad, -()t~ 00 (~-lactam ~-0), l7l~0 (ester C=O), 1700 (~=C), 695 cm 1 (aromatic protons). The n.m.r. spectrum was consistent ~lith the desired product (e5).
' . . .

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~ 22 ~

.:
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o~

,~,:f, .
E.XAMPLE 4 Benzvl N-benzyl~N~ ~Ylamino deoxvclavulanate ~3)2 2 ~ ~ ~ / CH2C~5 O CO~,~,C~ 5 2, c?
. (e4) ` (e6) Theester(e4)(o.5g) in acetoni-trile (10 ml) was cooled in ice-water. N-is~opy~zyl~une (0.39gt 1.~ moles) was added with stirring. The reaction mixture ~as allowed to warm to room temperature and s-tirred ~or 3 hours. Ethyl acetate (100 ml) was added, and the solution e~aporated to low bulk in vacuo. The residue was subjected to~column chromatography on silica gel using cycloheY~ane and ethyl acetate as eluents. The product (e6) was eluted after the unreacted diene It had Ir spectrum (liquid film) as follo~s: 'l803, (~-lactam C=0) 1845 (ester C=0) 1700 (C=C) 695 cm 1 (aromatlc protons)0 The n.m.r. spectr~n was consistent with the desired p-oduct .~

_ 23 -. .

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

EXAMPLE 5 Benzvl clibenzYl;~minodeo~cyclavulana-te ~ C~OC~CHCQ2 11 0 C~-~(C~
c4 c~,2c~e~ 'c~c~

(e7) (e2) . Benzyl dichloroacetylclavulanate (e7) (~.8g) w~s dissol~ed in dry di~athyforrnamide and cooled to 0C, treated ~rith dibenzylamine (768~1; 0.004 ~lol) in dry dimethylforma~ide (4ml) over 15 minu-tes, the tempPrature bein~ ~.aintained 2t 0 C.
: 5 The resul-ting yello~r solution was stirred at 0 for 2~ hours and a~
room temperature for 4 hours. Ethylacetate ~as added (100ml) and the solution washed with water (3 x 25rnl), dried and evaporated. The product was purified by fast gradient elution on silica gel using ethyl acetate/cyclohe~ane as the eluting solven~ ( yield 0.33g) i.r. (film) 1810, 1755, 1700 cm n.m.r.
(CDCl3)2.92 (d, IH,J 17Hz, 6~H) 3.12 (2H, d, J 8Hz, = CH CH2N) 3.38 (IH,dd,J =17Hzj J1~2~5 Hz, 6O~ --H) 3.46 (l~H,s,N(CH2 Ph)~

4.72 (IH, dt, J 8Hz, = CH CH2 )5.01 (IH,bs, CH C02Bz) 5-12 (2H, s, C02CH2Ph) 5.53 ~IH,d, J 2.5 Hz, 5~CH) 7.22 (15H,S, aromatic-H) , .

' . ' 1, ~ .
. .

.
. -~ , . .
~5 EXAMPI,E 6 Be~l dibenzylaminodeoxyclavulanate ~ O ~ / ~2~ 7 ,~l-N(C~?

C~c~

. (e 4) (e 2) .

The diene (e4)t27l r,~) ~ dry ace-toni~rile (4 ml) at 0 was treated ~ith dibenzylamine (197 mg) in dry acetonitrite : (2ml) over 5 minutes. The reaction mixture was stirred at 0C f`or 2 hours and at room temperature for 2 hours. The solvent was removed by evaporation and the residue dissolved in ethyl acetate, washed wi-th water, dried evaporated and fractionated on silica-gel to yield the desired product (e 2) which was purified by:chromatography.

,~

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.. . .
. .

,, ~10~

EXA~P_E 7 Benzyl ~
amino = 9 - deoxyclavulanate ,~3 cH -~ o~
~--C Ha ~ o ~/ 1 `c~r (é7) `~ ~ C~æ ~l+5 ` co~

Benzyl dichloroacetylclavulana~e (e7) (0.8g) in dry dimethylformamide(20ml~las cooled to 0 C and a solution of dl -1 - ben~ylamino ~ 2 - proDanol (o.65g) in dry dimethylformamide was added~1o~lly. S~irring was continued for 4 hours at 0 C.

A more pola r component was formed (thin lay2r chromatograph) and worked up as described in example 5 and chromatographed to gi~e the desired product (e 8) (o.l6 g) i.r~ (film)3450, 1808, 1750, 1700 cm 1 n.m.r. (CDCl3) 0.9 (3H, d, J 6~z , C~. CH3) 2-32 (2H~ ddt J 7~k, J 2Hz~ CH2 CoHH. CH3) 2.97 (1H, dd, J 17.5 Hz, J 1.5Hz 6B -H) 3.17 (2H, d, J 7HZ, = CH.CH2) 3.56 (1H, dd, J 17.5HZ, J1 2.5 HZ, 6~ H) 3.48 (2H, s~ N CH2Ph) 4.75 (1H, bt, J 7H , = CH
CH2) 5.1 (1H, s, C-3) 5.18 (2H,s, C02 CHph) 5.63 (1H, d,J 2.5 Hz

5~ ) 7.21 ? 7.3 (~10H, s, aromatic - H) . .
~, ''`, ~ - 2 6 -:

.~

Benzvl dialIylaminodeoxvclavulanate C~(~=C~, 2 t~)~C ~ C ~ -" ~>
O C~C~ 5 `~,~.2c~

(~ 7~ (e9 .

Benzyl dichloroacety'calvulanate (e7) (o Lg) ~ s dissolved in dry dimethylformamide (7 mls) and cooled to 0C~ A solution of diallylamine (246 ul ; 0.002 mol) in dry dimethyl~ormamide (2 ml) was added the temperature was maintained a-t 0C. The solution was stirred for 2 hours at 0 and at 15 for 15 minutes. Ethyl acetate (75 ml) was added and the solution washed with`water (2 x 35ml)dried and evaporated The more polar product was isolated by column chromatography on silica gel and was obtained as a yellow oil (0.18g); i.r.(film) 1804, 1755? 1695, l640 cm 1-n.m~r. (CDC13), 2.96 (IH,d, J l7Hz, 6~-H) 2.97 (4H, d, J 6Hz, N(C~2 CHacH2) 3.13 (2H, d, J 7Hz, = C'H CH2N ' ), 3h1. (IH,dd,J 2.5Hz

6 ~-H) 4.68 (.~,dt, J 7H~, = CH CH2)4.97 - 5 13 ~5H, m,3-CH and N ~ ~H2CH = CH2]2) 5.l3 ~2H, s super~sed on broad m, Co2CH2Ph), 5.62 (lH, d, J 2.5Hz, 5-Hj, 5.75 (2~i, m, N ~ 2~-~ ), 7.3 (5H, s, cH2Ph).

.1 . ., ~ ' ' . .

EXAM LE 9 _p-Methoxybenzyl d.iallylaminodeoxycla~rulanate C tt - ~ C l~3) ~ 3 ~o~ JC~l t~

`C~-C~ ~C~3 ~ C~ C~3 (e 10) (e 11) The sulphate (e10) (0.46g) was dissolved in dry dimethyl-formamide (7 ml) and cooled :to 0C,diallylamine (0.175g) in dry dimethylformamide (2 ml~)~ was added slowly dropwise.
The reaction mixture was stirred at 0 for 2 hours and wor~ed up as in Example 5. Column chromatograph gave the product (e11 ) as a yellow oil.

.

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

EXAMP1E 10 5~ diben7~ ~ -deox~clavulanic_acid ~=~C` ~ d. ~1 5) ~ c ~ I2 ~ (( '!2 C~ ~l 5 ):2.

C ~ ,c~ d~c~ 5 (e~) (es~
A solution of the benzyl ester (e~) (330 mg) ln ethanol (20 ml) was hydrogenolysed at ambiant temperature and pressure using 10~/o ~d/C (110 mg). ~le reac-tion ~as complete after 15 mi~utes.

~he catal~Jst was filtered off and the filtrate evaporated, the crude product was dissolvad in ethyl acetate (20 ml) and extracted with water (5 x 5 ml). The water was removed in vacuo to give the desired product ~e 1~ as a pale yellow gum (yield 41,b).

i,r. 3400 (O)j 1800, 1'00 (W), 1620 (b) cm 1 .

.:

29 -~
`

' , ~ .

~ ' ' . . ' ~

~ 6 ~ yl_ M-benzyl_- N - norbornyla~inodeo~,~lavulanat2 , .,. ' ~

H ~ _,o ~

(~13) Clavudiene benzyl ester (0.5g~ in acetonitile (10 ml) was trsated at -15 ~ri~h 2 (M~benzyl) ~o~ornylamine (1.3 moles, 0.48g).
Allowed to ~rarm to room temperatu~e during about 2 hours, stood l hour~
added ethyl acetate (100 ml), evaporated to small volume. Residue subjected to colu~n chromatography on silica gel usin~ gradient elution with cyclohexane and ethyl acetate. ~he solvents were removed under reduced pressure to yield the desired product (e13) as a pale yellow oil. I.r. spectrum as follows:1802 cm 1 ~ -lactam C--D.
.m.r. 0.9 - 3.7 (unassignablo multiple peaks) , 1~ 4.65( 1H,t,.CH~ ), 4t97 (2H, C6 H5 CH2)~ 5 . - 3 3 .--1 .'' , .

. .

-, `~ 4~
:
~ PLE 1? 9-~M,M-~ipro~lamino~-9-deox~clav~anio ~cid .: .

C~$~ c~l (e q) (e 1~) ~en~-vrl 9 - (M, N-diallylamlno) -9-deo~yclavulanate (e 9) was dissolved in ethanol (10 ml) and hydrogenolysed at room tem~erature and pressu~e using 10Q' Pd/C catalyst (70 mg)~ ~fter ilt~ation thrOU~I
kiesel~uhr the solvent was evaporated and the residue dissolved in ethyl acetate (25 ml) and -the product extracted with water (3 ~ 10 ml). Evaporati~n in vacuo g~ve the zwitterion (e 14) as a yellow gum in ~5,h yield.
.

(i.r. (film) 3400 (b), 2500 (b), 1790, 1695, 1625 cm n.m.r. (D20) 0.~3 (6E t J 7~z N (CH2 CH2 CH3)2) 1,6 (4H, ~, N(CH2 C ~'2) 6~ - H~ N(CH2CH2CH3)2) 3.25 (I~, dd, J 18Hz, J 3Hz, 6 -H) ~.75 (2H, d, J 8~z, = CH.CH2) 4.66 (IH, t,obscured by HOD peak, = CH CH2) 4.95 I~, g,-H) 5.72 (IH, d, J 3Hz, 5 - X) , ,~ . . . '' ' ' ; ` - 3~ -: . . , ' :
:

, ~C~ 6 -deo~c'avulanio acid IV~ J , ~ lo 2 ~Id~/S ~=fi~a~

~'C (~ O `~
- ~ CO, ~

(a 1~) ( 7 ~-- ) The zwitterion ~ras prepared by hydrogonolysis of 'oenzyl 9 -[DL - N - benzyl -N - (2-hydroxypropyl) amino~ - 9 - deoxyclavulanzte (e 8) (90 mg) using 1~b pd/C catalyst (33 mg) in ethanol (10 ml) at ambient temperature and pressure. The reaction ~/as complete in 30 minutes~ ~le catalyst was removed by filtra-tion through kieselguhr and the ~iltrate was evaporated. ~he residual gum was taken up in ethyl acetate (20 ml) and extracted with water (4 x 10 ml). ~he aqueous extracts we~e combined and e~aporated in ~acuo to give the desi~ed pro~uct (e 15) a~
a pale yellow gum in 52~/o ~ield.
l.r. (film) 3400 (b) 1790~ 1695~ 1620 cm 1~

The Field Desorption mass spectrum showed a peak at m/e 347 corresponding to (M+l) and a peak a-t m/e 165 (ph~2N~ 2cHo~IcH3).

:

' -- . . . ...

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

. . .
- : - . .

~0~4~

Ac-tiv~y a, The compounds of this invention do not have a high level of acute toxicity9 for e~ample the compounds of Examples 8 and 12 have LD50 values o~ greater than 500 mg/kg in mice when administered by the sub-cutaneous or oral routes.

b The effectiveness of t~e com~ounds of this invention as s-~ergis~s can be demonstLated by con~ ntional ~IC tests in which ampicillin alone, ampicillin and synergist and synergist alone are compared. The follo~ing test results were obtained using a strain of Staphyloco~cus aureus Russell uninhibited by 250 J~g/n~ o:~ an~icill~ and unhibited by 5~g/n~ of synergist:

~_~
MIC of ampicillin -Compound of Example (ug/ml) in presence o. of l. ~g/ml ~f synergist ___ _ ~
2 30~f2 1 7. 8 8 . <0.09 10 . - 0.156 ' ~ :

PREPARATION 1. General Proc~dure for Pre~ ulphonate The following process for the preparation of benzyl clavulana-te -O- sulphona~e trimethylamine salt (e1) may be adapted for the preparation o~ analogous esters by substituting the appropriate ester of clavulanic acid for (~ 1).

Benzyl clavulanate (57.8 mg) in dry dimethylformamide (0.8 ml) was treated ~/ith the trim2thylamine - sulphur -t~ioxide comple.c (55.6 m~) and left at ambient tem~erature for 18 hours.
The solvent ~as removed in vacuo and the res~due extr2cted ~rith chlorofor~. The chloro~orm solutlon was evaporated and the residue was extrac~ed several times with diethyl either to remove benzyl clavulanate. The e-ther ln~soluble oil was shown to be the -trimethylamine salt o~ benzyl clavulanate -O- sulphonate (el) which was produced in 600,6 yield. ~In the n.m.r. the -CH20S03 proton appeared at a doublet at about 4. 71~'as compared to abou~ 4.~24 ~in ~he starting mate-ial).

:

.

.

...... . . . . .
.

. .

4~

PRP~ARATION ~ General Procedure for Pre~aration of Diene Esters The following pro~ess ~or the preparation o~ the benzyl ester of cla w ladiene may be adapted to the preparation of other corresponding esters by replacing the benzyl clavulanate star-ting material by the corresponding ester of cla~ulanic acid.

~enzylcla-~ulanate (0.2 ~-) was added t~ dr-~ dimethyl~
sulpho,cide (6 ml) and dry benzene (3 ml) containing dicyclo-he~Jlcarbodiimide (0.43 g.). Anhydrous orthophosphoric acid (0.069 g) in dimethyl sulphoxide (2 ml) was added and the mixture s-tirred at room temperature for 4 hours. Thin layer chromatography showed a faster moving spot whi.ch gave a blue `' fluorescence at 366 n.m. The dicyclohexylurea was filtered off and benzene added to the filtrate, the organic phase was washed with water dried and evaporated,. Fractionation on silica gel gave the product as a colourless oil in 71~o yield.
The diene was stored as a solution in acetone containing hydroquinone (0,01%) as a stabiliser.

Icr. (film): ~ 1810, 1700, 1628, 1,56~ cm 1 .

-- , .

4~

EXAMPLE 15 Meth~ 2-(N-ben _l~ norbonylami_o_eox~cl vulanate ~.
cl~2oll L~-~ ~CI~2~

N `C0 CH
~C02Na 2 3 (e16) (e17) Sodium clavulanate tetrahydrate (el6) (2,9g) in dime-thylormamide (25 ml) was treated with iodomethane (25 ml) at room tempera-ture over l hour.
Acetonitrile (10 ml) was added and removed by evaporation under reduced pressure (to remove excess methyliodide). The residual dime-thylformamide solution was cooled in ice, and phthalic anhydride (1,5g) and trie-thylamine (5 ml) added, ~fter 1 hour at below 5C, acetoni-trile (2 x 25 ml portions was added and evaporated under reduced pressure ea~h time, 2-N-ben%ylnor-bornylamine (4g) was added and the reaction allowed to stir at 5C overnight.
Most of the dimethylformamide was evaporated in vacuo. The residue was 1Q dissolved in 1!1 ethyl acetate-cyclohexane (lO0 ml), treated ~ith silica gel tl5 (chromatography gxade), filtered off, evapoxated to small bulk, and the residue subjected -to gradient chromatography on silica gel using ethyl acetate and cyclohexane as eluents. ~he least polar ~-lactam containing material was isola-ted by evaporation of the solvents from fractions containing it~ to yield ethyl 2-(N-benz~l)norbornylaminodeoxyclavulanate (el7) as a yellow oil (50 me). It had I.r, 1800 (~-lacta~ 1750 (ester C = 0) 1695 cm~1 ~C=C).

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

' - - , . . .

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

X~PLE 16 ~enz~] N-ben~ N-ethylaJ~inodeoxyclavul~nate C;l~ HJ-~

CO CH C

~ .
(e 18) Clavudiene ben~yl ester (0.44g) in acetonitrile (8 ml) was cooled to - 10C
and treated with N-benzylethylamine (0.27g). The reaction mixture was main-tained at - 5 to 0C for 1~ hours. E-thyl acetate (70 ml) was added and the mixture evaporated to small volume. The residue was sub~ected to column chromatography on silica gel using a gradient elution, starting with

7:1 cyclohexane/ethyl acetate increasing q~ickly to 2:1.

~he material was collected in fractions just after the star-ting material.
~he solvents were evaporated to yield 45 mg of the title compound (e 18) as a pale yellow oil. I.r. 1803 (~-lactam) 1750 (ester) 1700 (C=C) cm~l :

- 37 ~

: ' ~

:

, `` . ' ` , `' ;` ' ~' . ' , , ' `' ' , ' '

Claims (19)

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

1. A pharmaceutical composition which comprises a compound of the formula II
II
wherein R1 and R2 can be an alkyl, alkenyl, phenylalkyl or phenylalkenyl group optionally inertly substituted by a halogen, OH, OR3, O.CO2R3, SR3, or a COR3 group wherein R3 is a hydrocarbon group of up to 8 carbon atoms and the group NR1R2 contains a total of not more than 16 carbon atoms and esters of compounds of formula II and acid addition salts thereof, said esters having the following formula IV and V
IV
V

wherein R1 and R2 are as defined above and A1 is an alkyl group of 1 - 3 carbon atoms optionally substituted by halogen or a group of the formula OA4, OCOA4, SA4, SO2A4 wherein A4 is a hydrocarbon group of up to 6 carbon atoms; A2 is a hydrogen atom, an alkyl group of up to 4 carbon atoms or a phenyl group op-tionally substituted by halogen or by a group A5 or OA5 where A5 is an alkyl group of up to 6 carbon atoms; and A3 is a phenyl group optionally substituted by halogen or by a group A5 or OA5 where A5 is an alkyl group of up to 6 car-bon atoms or being selected from the group of esters consisting of phthalidyl, acetoxymethyl, pivaloyloxymethyl, ?-acetoxyethyl, ethoxycarbonyloxymethyl, and ?-ethoxycarbonyloxyethyl and a penicillin or a cephalosporin and a pharmaceutical carrier therefor.

2. A pharmaceutical composition as claimed in Claim 1 which contains from 150-1000 mg. of amoxycillin, ampicillin or a pro-drug therefor and from 50-500 mg. of a compound of the formula II or a salt or in vivo hydrolysable ester thereof.

3. A pharmaceutical composition as claimed in Claim 1 which contains from 200-500 mg. of amoxycillin, ampicillin or a pro drug therefor and from 50-250 mg. of a compound of the formula II or a salt or in vivo hydrolysable ester thereof.

4. A pharmaceutical composition as claimed in Claim 1, 2 or 3 which contains benzyl-3-(2-dibenzyl-aminoethylidene)-7-oxo-1-azabicyclo-[3,2,0]-heptane-2-carboxylate and ampicillin.

5. A pharmaceutical composition as claimed in Claim 1, 2 or 3 which contains methyl-3-(2-dibenzylaminoethylidene)-7-oxo-4,1-azabicyclo-[3,2,0]-2-carboxylate and ampicillin.

6. A pharmaceutical composition as claimed in Claim 1, 2 or 3 which contains benzyl diallylaminodeoxyclavulanate and ampicillin.

7. A pharmaceutical composition as claimed in Claim 1, 2 or 3 which contains 9-(N,N-dibenzylamino)-9-deoxyclavulanic acid and ampicillin.

8. A pharmaceutical composition as claimed in claim 1, 2 or 8 which contains 9-(N,N-dipropylamino)-9-deoxyclavulanic acid and ampicillin.

9. A pharmaceutical composition as claimed in claim 1, 2 or 3 which contains benzyl-3-)2-dibenzyl-aminoethylidene)-7-oxo-1-azabicyclo-[3,2,0]-heptane-2-carboxyate and amoxycillin.

10. A pharmaceutical composition as claimed in claim 1, 2 or 3 which contains methyl-3-(2-dibenzylamino-ethylidene)-7-oxo-4,1-azabicyclo-[3,2,0]-2-carboxylate and amoxycillin.

11. A pharmaceutical composition as claimed in claim 1, 2 or 3 which contains benzyl diallkylaminodeoxyclavulanate and amoxycillin.

12. A pharmaceutical composition as claimed in claim 1, 2 or 3 which contains 9-(N,N-dibenzylamino)-9-deoxyclavulanic acid and amoxycillin.

13. A pharmaceutical composition as claimed in claim 1, 2 or 3 which contains 9-(N,N-dipropylamino)-9-deoxyclavulanic acid and amoxycillin.

14. A pharmaceutical composition as claimed in claim 1 which comprises ticarcillin and a compound of the formula II or a salt or in vivo hydrolysable ester thereof.

15. A pharmaceutical composition as claimed in claim 1 wherein the ratio of the compound of the formula II or a salt or in vivo hydrolysable ester thereof to antibiotic is from 10:1 to 1:3.

16. A pharmaceutical composition as claimed in claim 15 wherein the ratio is from 5:1 to 1:2.

17. A pharmaceutical composition as claimed in claim 15 wherein the ratio is from 3:1 to 1:1.

18. A pharmaceutical composition as claimed in claim 15, 16, or 17 wherein the antibiotic is amoxycillin, ticarcillin, ampicillin or a pro-drug therefor.

19. A pharmaceutical composition as claimed in claim 15, 16, or 17 wherein the antibiotic is amoxycillin ticarcillin, ampicillin or a pro-drug therefor and the compound of formula II is selected from benzyl-3-(2-dibenzyl-aminoethylidene)-7-oxo-1-azabicyclo-[3,2,0]-heptane-2-carboxylate, methyl-3-(2-dibenzylaminoethylidene)-7-oxo-4,1,-azabicyclo-[3,2,0]-2-carboxylate, benzyl diallylaminodeoxyclavulanate, 9-(N,N,-dibenzylamino)-9-deoxyclavulanic acid and 9-(N,N-dipropylamino)-9-deoxyclavulanic acid.