CA1178278A - .beta.-LACTAM ANTIBIOTICS, THEIR PREPARATION AND USE - Google Patents

Abstract

A B S R A C T

The present invention provides the compounds or the formula (I):

Description

,~-Lactam Antibiotics, their preparation and use While the followin~ is a complete disclosure of the inv~ntion, this application, being a division oE Canadian Patent Application Serial No. 357,846 filed August 8, 1980, has claims directed only to the compounds of formula I below.

This invention relates to novel antibacterial carbapenem derivatives, to processes for their preparation and to compositions containing them.

European Patent ~pplication Publication Number 0001628 discloses a group of synthetic antibacterial agents containing a 7-oxo-1-azabicyclo[3.2Ø~hept-2-ene ring system. However all compounds described in that specification were racemic at C-5 and could only be prepared by a very long synthetic sequence. A new process has been discovered that enables new antibacterial agents to be prepared via the intermediacy of a thiol. These new antibacterLal agents can be prepared by a relatively short reaction sequence from natural products and are produced as a desirable sin~le optical isomer at C-5.

2~

The present invention provides the compounds of the formula (I):
Rl H
l`H ~ S-H (I) and salts and esters thereof wherein Rl is'a hydrogen atom or a group selected from OH, OS03H or a salt or Cl ~ alkyl ester thereof, OR ~ SR , OCOR , oG02R3 or OCONHR , where R is a Cl 6 alkyl group or an optionally substituted benzyl group and R is a Cl 6 alkyl group or an optionally substituted benzyl or an optionally substituted phenyl group: with the proviso that when R
is OS03H or a salt or Cl 4 alkyl ester thereof the-C-6 and C-5 hydrogen atoms are cis.

A group of compounds of interest are those of the formula (I) wherein R is a hydrogen atom or a group selected from OH, OS03H or a pharmaceutically accepta'ble salt or a methyl or ethyl ester thereof, OR , SR , OCOR2, oC02R3 or oCONHR3 where R is a Cl 4 alkyl group or a benzyl group and R is a Cl 4 alkyl group or a benzyl, phenyl or p-nitrobenzyl group.

When used herein the term "optionally substituted benzyl" means that the phenyl ring of the benzyl group can be substituted by hydrogen, Cl 3 alkoxy, fluorine, 327~

bromine~ chlorine or nitro. The term "optionally substituted phenyl" means that the phenyl group can be substituted by hydrogen, Cl 3 alkyl9 Cl 3 alkoxy, fluorine, bromine, chlorine or nitro~

S Suitable values for Rl include hydrogen, hydroxy, OS03H or a salt or methyl or ethyl ester thereof, methyl-thio, ethylthio9 phenylthio9 ~en~ylthio, p-nitrobenzylthio, acetoxy 9 pxopionoxy9 benzyloxy and phenylacetyloxy.

Preferred values for Rl include hydrogen, hydroxy, acet oxy and OS03H or salt or methyl or ethyl ester thereofO

Although compounds of the formula (I 3 and salts and esters thereof have antibacterial activity, they are primarily envisaged as starting-mater}als in the processes hereinafter defined.

Suitably for use as an intermediate in such processes any sulphate moiety in the group R in the compounds of the formula (I) is in the form of a quaternary ammonium salt for example the benzyldimethyl-n-hexadecylammonium salt, or is in the form of a methyl or ethyl sulphate ester.

Suitably for use as an intermediate the compound of the formula (I) is in t~le form of a cleavable ester at the ~7~

;~

C-2 carboxyl. Apt cleavable esters include those cleavable by chemical methods such as hydrogenolysis or hydrolysis or by biological methods.

Suitably the carboxylic acid is esterified by a group of the sub-formula (a)~ (b) 9 (C) or ~d):

- CH ~ R (a) ~ R5 - CH ~ R (b) R

CH - R (c) ~ ocoR9 - - CH (d) OR

wherein R is a hydrogen atom or an alXyl, alkenyl or alkynyl group of up to 3 carbon atoms; R is a hydrogen atom or a methyl group; R is a phenyl group or a phenyl group substituted by a fluorine, chlorine or bromine atom or a nitro, methyl or methoxy group; R is a hydrogen atom or a phenyl group or a phenyl group substituted by 7~7~

a fluorine, chlorine or bromine atom or a nitro, methyl or methoxy group; R is a hydrogen atom or a methyl group; R is a Cl 4 alkyl, phenyl or Cl 4 alkoxy group or R is joined to R9 to form a ph*halidyl, dimethyl-5 phthalidyl or dimethoxyphthalidyl group; and R10 is aCl ~ alkyl, phenyl, chlorophenyl or nitrophenyl group;
or CHR4R5 is a phenacyl or bromophenacyl group.

~ avourably R4 is a hydrogen atom or a methyl ethyl, vinyl or ethenyl group. ~avourably RS is a hydrogen atom. Favourably R is a phenyl, p-bromophenyl, p-methoxyphenyl or p nitrophenyl group. ~avourably R
is a hydrogen atom. Favouxably R9 is a methyl, t-bu*yl or ethoxy group or is joined to R . ~avourably R10 is a methyl group~

Preferred groups of the sub~formula a~ include the methyl and ethyl groups.

Preferred groups of the sub-formula b) include the benzyl and p-nitrobenzyl groups~

Preferred groups of the sub-formula c) include the acetoxymethyl, pivaloyloxymethyl, a-ethoxycarbonyloxy-methyl and phthalidyl groups.

A preferred group of the sub-formula d) is the methoxymethyl group.

8~7~

Particularly preferred esterifying groups are the p-nitrobenzyl and phthalidyl groups.

When the compounds of th~ formula (I) and salts and esters thereof are intended for use as antibacterial agents, then suitably the compound is in the form of an in-vivo hydrolysable ester or pharmaceutically acceptable salt. Suitable in-vivo hydrolysable esters include those of sub-formula (c) as hereinbefore definedc Suitable pharmaceutically acceptable salts include those of the alkali and alkaline earth metals, of these the sodium and potassium salts are preferred. These pharmaceutically acceptable salts may be formed at the C-2 carboxyl, and/or at a C-8 sulphate moiety (if present). Thus compounds of the formula (I) wherein Rl is a OS03H group or pharmaceutically acceptable salt thereof may be in the form of a di-salt such as the di-sodium salt or di-potassium salt, or may be in the form of a mono-salt of an in-vivo hydrolysable ester, or may be in the form of a mono-salt o an acid or may be in the form of a di-acid.

The compounds of the formula (I) may have the cis- or trans- geometry about the ~-lactam, that is to say they have the (5R, 6R) or (SR, 6S) configuration.
Alternatively the compounds of the formula tI) may be presented in the form o an cis/trans mixture.

The compounds of the formula (I) may have R or S stereochemistry at C-8 (except of course when Rl is hydrogen) or may be in the form of mixtures thereofO

7~

The compounds of the formula (I) wherein R is OSO3H or a salt or Cl 4 ester thereof are in the 8S~configuration as the necessary intermediates are more readily a~ailable.

~78~7~

The present invention also prov:ides a process for the preparation of a compound of the formula (I~ or salt or ester thereof which process comprises the reaction of a cleavable ester of a oompound of the formula Rl ¦ H
CH~ ~ ~ S-cH=cH-NH-co-Rll (II) N ~ll wherein Rl is as defined in relation to formula (I) and Rll is a methyl or ethyl group; with a source of hypohalous acid~ and optionally thereafter:

i) converting a cleavable ester to a free acid or salt, ii~ converting a cleavable ester to a different cleavable ester?

with the proviso that when Rll is an ethyl group Rl must be OSO3H or a salt or Cl 4 alkyl ester thereof~

Preferably R 1 is a methyl group as the inter-lS mediates are rnore readily available.

Suitably the reaction is perfoxmed at a non-extreme temperature such as -15C to +25C, preferably a~bient. Solvents suitable in this reaction are inert organic solvents optionally in the presence of moisture, for example moist acetone or dioxan.

Prefexred esters for use in this process are those described hereinbefore as prefer~ed esters for compounds of the formula (I). A parti~ularly preferred ester for use in this process is the p-nitrobenzyl ester.

Suitably the hypohalous acid is hypobromous acid or hypochlorous acid~ of these hypobromous acid is preferred. Suitable sources o hypohalous aci~s include N-bromoacetamide, N-c~lloroa~etamide and N-bromopropionamide~

Compounds of t~e formula (II) may be prepared by the methods of European Patent Application Publication Numbers 0005348, 00053~9 0007152, 0008497, Belgian Patent Number 864570, an~. U.K, Patent Number 1489235.

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- 1 o--In a fuxther aspect this invention provides a group of a novel compounds which may be formed from the compounds of the formula (I). Thus the present invention provides the compounds of the formula (III):
Rl H

C~3 - CH ~ ~ -Rl (III) ~ N ~
C02~{
and pharmaceutically acceptable salts and in-vivo hydrolysable esters thereof wherein R is as defined in relation to formula (I) except that any sulphate salt must be pharmaceutically acceptable and Rl is Cl 6 alkyl, C2 6 alkenyl, C3 6 alkynyl wherein the triple bond is not present onthe carbon adjacent to the sulphur atom, aralkyl, Cl 6 alkanoyl, aralkanoyl, aryloxyalkanoyl or arylcar-bonyl, any of such R groups being optionally substituted;
with the proviso that when R12 is 2-aminoethyl R must be SR3 or OS03H or a pharmaceutically acceptable salt or Cl 4 alkyl ester thereof; and with the further proviso that when Rl is OS03H or a pharmaceutically acceptable salt or Cl 4 alkyl ester thereof the C-S and C-6 hydrogen atoms are cis.

When R is a Cl 6 alkyl group suitable substituents include amino, hydroxy, Cl 6 alkanoyloxy, Cl 6 alkoxy, benzoyl, Cl 6 alkanoyl or carboxy or an ester or pharmaceutically acceptable salt thereof~

:~1 78%78 When R is a C2 6 alkenyl group wherein the double bond is not present on the carbon adjacent to the s~lphur atoml suitable substituents include carboxy or an ester or pharmaceutically acceptable salt thereof, or hydroxy or Cl 6 alkoxy.

When R is a C2 6 alkenyl group wherein the double bond is present on the carbon adjacent to the sulphur a$om, suitable substituents are carhoxy or an ester or pharmaceutically acceptable salt thereof.

When used herein the term "aralkyl" means a Cl 6 alkyl group substituted by an aryl group~ examples of such aryl groups being naphthyl, pyrrolyl, furyl, thienyl, indolyl, thionaphthyl, benzofuryl, imidazolyl, thiazolyl, or any of such groups substituted by one or more groups selected from Cl 3 alkyl, phenyl, nitro and amino; or a phenyl group optionally substitued by a halogen atom or a Cl_3 alkoxy, nitxo or acetamido group.

A group of compounds of interest is that of the formula (IV):

CH / ~ S-R13 (IV~

32~

and pharmaceutically acceptable salts and in-vivo hydrolysable esters thereof wherein R14 is a hydrogen atom or a group selected from OH, OSO3H or a pharmaceutically acceptable salt or a methyl or ethyl ester thereof~ OR , SR , OCOR , OC02R or oCONHR3 where R is a Cl 4.alkyl group or a benzyl group and R3 is a Cl 4 alkyl, group or a benzyl, phenyl or p-nitrobenzyl group; and R is Cl 4 alkyl, Cl 4 alkyl suhstituted by a phenyl group optionally substituted by a 1uorine, chlorine or bromine atom or a methoxy, n.itro, amino, acetamido or p-nitroben~yloxycarbonylamino group; a C2_4 alkyl group substituted on o*her than the a-carbon atom by an antino, p-nitrobenzyloxycarbonylantino, hydroxy, p-nitrobenzyloxy-carbonyloxy,.methoxy, acetoxy or Cl 4 alkyloxycarbonyl group;
a Cl_4 acyl group or a benzoyl, phenylacetyl or phenoxy-acetyl group; or a CH=CHCO2H group or a pharmaceutically acceptable salt or ester thereof. In one aspect of the compounds of the formula (IV), the group CH-C~CO2H is esteriied to form a Cl 4 alkyl ester or ~nino Cl 4 alkyl ZQ ester.

A similar group of compounds of interest is that of the formula (V):

CH --C H ~ ~ S- R 1 6 ( V ) ~--N . . _ ( C02~1 ~'7~

and pharmaceutically acceptable salts and in-v.ivo hydrolysable esters thereof wherein either:

R is hydrogen or hydroxy,and R is a Cl_6 alkyl group substituted by amino, benzoyl, Cl 6 alkanoyl or carboxy or ester or pharmaceutically acceptable salt thereof,or R16 is a group selected from C2 6 alkenyl, C3 6 alkynyl wherein the triple bond is not present on the carbon atom adjacent to the sulphur atom, aralkyl, C1 6 alkanoyl, aralkanoyl, aryloxyalkanoyl or arylcarbonyl, any of such groups being optionally substituted;

or R15 is a group selected from OS03H or a salt or Cl 4 alkyl ester thereof, oR2~ SR3, OCOR2, oC02R3 or oCONHR3 wherein R and R3 are as defined in relat.iorl to compounds of the formula (I); and Rl is a group R12 wherein R is as defined in relation to compounds of the formula (III);

with the proviso that when R is 2-aminoethyl R S
must be SR or OS03H or a pharmaceutically acceptable salt or Cl 4 alkyl ester thereof; and with the further proviso that when R is OS03H or a pharmaceutically acceptable salt or Cl 4 alkyl ester thereof the C-5 and C-6 protons are cis.

Suitably R in the compounds of the formula (III) is a hydrogen atom or a group selected from OH, OS03H or a pharmaceutically acceptable salt or Cl 4 ester thereof~
oR2 9 SR ~ OCOR , OC02R or OCONHR , wherein R and R3 are as defined in relation to formula (I).

More suitably Rl is hydrogen, hydroxy, OS03H or a pharmaceutically acceptable salt thereof, or acetoxyO
Of these preferred values are hydroxy and 0503H or a pharmaceutically acceptable salt thereof such as the sodium or potassium salt.

When ~12 is an alkyl group or substituted alkyl group suitably such alkyl groups contain up to 4 carbon atoms, for example R12 aptly may be methyl, ethyl, propyl, butyl, aminomethyl~ aminoethyl, aminopropyl, aminobutyl1 hydroxyethyl, hyd~oxypropyl, hydrobutyl, methoxymethyl, methoxyethyl, ethoxymethyl, ethoxyethyl, propoxypxopyl, me-thoxypropyl, ~ethoxybutyl, acetoxymethyl~
acetoxyethyl, propionoxymethyl, propionoxyethyl, ph~nacyl, acetylmethyl, acetylethyl~ propionylmethyl, propionylethyl, carboxymethyl or pharmaceutically acceptable salt thereof, methoxycarbonylmethyl, ethoxycarbonylmethyl, methoxy-carbonylethyl1 methoxycarbonylpropyl, methoxycarbonylbutyl,ethoxycarbonylethyl, carboxyethyl or pharmaceutically acceptable sal.t thereof, carboxypropyl or pharmaceutically acceptable sal.t thereof, carboxybutyl or pharmaceutically accept,able sa].t thereof, and structural isomers thereof, of these preferred values include methyl, ethyl, propyl, butyl and 2-aminoethyl.

Further suitable values for R include propenyl, butenyl and CH=CH-C02H or a pharmaceutically accept,able salt or ester thereoAf~ for example the sodium or potassium salt or the methyl, ethyll propyl~ aminoethyl, aminopropyl, benzyl or p-nitrobenzyl ester.

When ~ is an aralkyl group more suitably S the alkyl moiety is a methylene or ethylene divalent radical. Suitable examples o~ the aryl moiety are phenyl optionally substituted by one or more substil:uents selected from a halogen atom or a Cl 3 alkoxy, nitrc) or acetamido group; pyrrolyl optionally substituted by a phenyl or Cl 3 alkyl group; thienyl optionally substi*uted by a phenyl or C1 3 alkyl group; furyl optionally substituted by a phenyl or Cl 3 alkyl group; imidazolyl optionally substituted by one or more groups selected from phenyl, nitro, amino, Cl 3 alkyl; and thiazolyl optionally substituted by one or more groups selected from phenyl, nitro9 amino and Cl 3 alkyl.

More suitably R is a benzyl, bromobenzyl, chlorobenzyl, fluorobenzyl1 methoxybenzyl, nitrobenzyl, acetamidobenzyl, thiazolylmethyl, aminothiazolylmethyl, nitrothiazolylmethyl or phenylthiazolylmethyl group.

Suitably also R 2 is a phenethyl, pyrrolylethyl or optionally substituted imidazolylethyl. In a suitable aspect the imidazolyl ring may be substituted at the C-2 position (that is -the carbon atom a to the two nitrogen atoms) by a Cl 3 alkyl or phenyl group.
In another aspect the imidazolyl ring may be further substituted at the C-4 position or the C-5 position by a Cl 3 alkyl, 7l3 phenyl, nitro or amino group; preferably such substituents are on the C-4 position ancl the C-5 position is unsubstituted; alternatively such substituents are on the C~5 position and the C-4 position is unsubstituted.

In a further aspect R12 is a Cl 6 alkanoyl, aralkanoyl, aroxyalkanoyl or aroyl group, for example acetyl, phenylacetyl, phenoxyacetyl or benzoyl. Of these acetyl is preferred.

~he groups speci~ied above for the C-3 substituent R12 are also, where applicable, suitable groups for R
and R

The groups specified above for the C-6 substituent R are also, where applicable 9 suitable groups for and R

Thus it is to be realised that preferred compounds of this invention include those of the formula (VI):

CH ~ S-R18 (VI)

3 ~ ~

or pharmaceutically acceptable salts or in-vivo hydroly-sable esters thereof wherein R is OH or OS03H or a phar~aceutically acceptable salt or Cl ~ alkyl ester thereof, and R is an optionally substitu~ed Cl 6 alkyl group.

A further preferred group of compounds is that o the formula (VII):

~ ~7~7~

J~ ~ S-CH=CII-CO~R

N

or pharmaceutically acceptable salt or cleavable est:er thereof wherein Rl7 is as defined in relatio~ to formula (VI) and Rl9 represents a hydrogen atom, a pharmaceutically acceptable salting ion or a Cl ~ alkyl or amino Cl 4 ~lkyl group.

Suitable and preferred in-vivo hydrolysable ester groups for esterifying the C-2 carboxyl of the compounds of the formulae tIII)-(VII) are those described in relation to compounds o the formula (I). Suitable pharmaceutically acceptable salts of the compounds of the formulae (III)-tVII) include thvse of the alkali and alkaline earth metals, of these the sodium and potassium salts are preferred. These pharmaceutically acceptable salts may be formed at the C-2 carboxyl, and/or at a C-8 sulphate moiety (if present). Thus compounds of the formulae (III)-(VII) wherein the C-6 substituent contains a OS03H group or pharmaceutically acceptable salt thereof may be in the form of a di-salt such as the di-sodium or di potassium salt, or may be in the form of a mono-salt of a in-vivo hydrolysable ester, or may be in the form of a mono-salt of an acid or may be in the form of a di-acid.

J~'7~

I ~

When the thio side-chain at the C-3 position contains an amino group it is preferred that the compounds of the formulae (III)-(VII) are zwitterionic.

The compounds of the formulae (III)-(VII) may have the cis- or trans- geometry about the ~,lactam;
that is to say they have the (5R,6R) or (5R, 6S~
configuration. Alternatively the compounds of the formulae (IIX) (VII) may be presented in the form of a cis/trans mixture.

The compounds of the formulae (III)-(VII) may have R or S stereochemistry at C-8 (except of cGurse when the C~6 substituent is ethyl) or may be in the form of mixtures thereof. The compounds of the formulae (III)-(VII) wherein the C-6 substituent contains a 0503H group or pharmaceutically acceptable salt thereof or Cl 4 alkyl ester thereof are in the 8S~configuration as the necessary intermediates are more readily available.

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_ 19 _ In a further aspect of this invention there is provided a process for the preparation of a compound of the formul~ (VIII):
Rl ¦ H

r~ N - ~ (VIII) . and pharmaceutically acceptable salts and in-vivo hydrolysable esters thereof wherein R is as defined in relation to formula (I) except that any sulphate salt must be pharmaceutically acceptable, and R20 is Cl 6 alkyl, C3 6 alkenyl wherein the double bond is not present on the carbon atom adjacent to the sulphur atom, C3 6 alkynyl wherein the triple bond is not present on the carbon adjacent to the sulphux atom, aralkyl, Cl 6 alkanoyl, aralkanoyl, aryloxyalkanoyl or arylcarbonyl, any of such R groups being optionally substituted; with the proviso that when R is OSO3H or a pharmaceutically lS acceptable salt or Cl 4 alkyl ester thereof the C-5 and C-6 protons are cis; which process comprises the reaction of a cleavable ester of a compound of the formula (I) with a compound of the formula (IX):

X R (IX) wherein X is a leaving group, in the presence of an acid ~O acceptor; and subsequently:

t;;~

i) converting any cleavable ester group which is not in-vivo hydrolysable into a free -acid, a pharmaceutically acceptable salt or in-vivo hydrolysable ester group;
ii) optionally converting any cleavable ester group which is in~vivo hydrolysable into a free acid, a pharmaceutically acceptable salt or a different in-vl o hydrolysable ester group.

Suitable acid acceptors are carbonates and bicarbonates such as anhydrous potassium carbonate. The reactlon is generally carrie~ ou-t in a dry polar solvent such as dimethyl-formamide. Suitably the reaction is performed at a non-extreme temperature, ~or example -30C to -~ 60C, more sultably -10 C
to -~ ~0C and preferably at ambient temperature.

Suitably X is a chlorine, bromine or iodine atom or is a sulphonate ester moiety such as a tosylate or mesylate, of these values iodine and chlorine are pre~erred. In an alternative aspect when R20 is a methyl or ethyl group, the leaving group X may be dimethyl ether or diethyl ester respectively. In other words X is derived from a trimethyl-oxonium salt or a triethyloxonium salt. Such salts are conveniently presented as their tetrafluoroborates. Such alkylations involving a trimethyloxonium or triethyloxonium salt are preferably performed in a a halogenated hydrocarbon solvent for example dichloromethane or chloroform, at a depressed temperature for example -80C to 0C, more suitably -70~ to -30 C.

",, - 20 . . .

27~

In another aspect of this invention there is provided a pxocess for the preparati.on of a compound of the formula (X):

Rl I H
CH ~ ~ -S-CH=CH C02R (X) and pharmaceutically acceptable salts and in-vivo ., hydrolysable esters thereof wherein R' is defined in relation to formula (I) and R21 i5 a hydrogen atom~ a pharmaceutically acceptable salting ion or group such that C02R 1 is an ester group; which process comprises the reaction of a cleavable ester of a compound of the formula (I) with a compound of the formula (XI):

H~C--C-C02R21 (XI) in the presence of an acid acceptor; and subsequently:

i) converting any cleavable ester group which is not in-vivo hydrolysable into a free 1~ acid, a pharmaceutically acceptable salt or in-vivo hydrolysable ester group;

ii) optionally converting any cleavable ester group which is in-vivo hydrolysable into a free acid, a pharmaceutically acceptable salt or a different in-vivo hydrolysable ester group.

~ ~L7~$

Suitable acid acceptors are carbonates and bicarbonates such as anhydrous potassium carbonate. The reaction is generally carried out in a dry polar solvent such as dimethylformamide. Suitably the reaction is performed at a non-extreme temperature for example ~30C
to + 60C, more suitably -10C to ~ 40C and preferably at ambient temperature.

The group Rl may be varied after the modification of the 2-position side chain. Such methods of modi~,'ication IQ can be those which are used in modifying the C-6 position substituents of natural products of the carbapenem type. Thus included herein by cross~reference are European Patent Application Publication Numbers 0004132 0005348, 0005349 and 0007152.

In the processes hereinbefore described for the preparation of compounds of the formulae (VIII) and (X) any amino grollp present can be conveniently protected in conventional manner, or example as a p-nitrobenzyloxy-carbonylamino groupO Similarly any hydroxy group present can be conveniently protected in conventional manner, for example as a p-nitrobenzyloxycarbonyloxy group.

Methods of removing protecting groups, cleaving any ester moiety, and converting a free acid or salt to a pharmaceutically acceptable salt or ester~ are as detailed in the hereinbefore mentione~ European Patent Application Publications~ In addition 6-ethylidene compounds of the formula (XII):

~7~

_ ~3 -CH3-CH ~ ~ ~ S-R (XII) r N ~

and salts and cleavable esters thereof are envisaged as intermediates in the processes of this invention. I'he compounds o the formula ~XII) may be readily converted to 6~ethyl compounds by known processes such as reduction9 In a particularly preferred aspect the processes of this invention are performed on hydrogenolysable esters, ~or ex~mple the p-nitrobenzyl ester, which may be cleaved using an approximately atmospheric pressure of h,vdrogen at arnbie~t temperature with a transition metal catalyst for exarnple palladium, preferably 5% or 10% palladium on carbon~ The compounds of the sormulae (I~ and (III) and their pharmaceutically acceptable salts and in-vivo hydrolysable esters may be employed in the treatment of bacterial infections such as those due to Staphylococcus ~ Escherichia coli and ~lebsiella aeroqenes. Thus the present invention provides a pharmaceutical composition which cornprises a compound of the formulae (I) or (III) in the form of its pharmaceutically acceptable salt or in-vivo hydrolysable ester and a pharmaceutically acceptable carrier.

~78~

_ 24 -.
.

The compositions of this invention may be prepared by conventional methods of preparing antibiotic compositions and in conventional manner may be adapted for oral, topical or parenteral administration.

Aptly, the compositions of this invention are in the form of a unit-dose composition adapted or oral administration.

Alternatively the compositions of this invention are in the form of a unit dose composition adapted for administration by injection.

Unit-dose forms according to this invention will normally contain from 50 to 500 mgs of a compound of this invention, for example about 62.59 100, 125, 150, 200, 250 or 300 mgs. Such compositions may be administered from 1 to 6 times a day or more conveniently 2, 3 or 4 times a day so that the total daily dose for a 70 kg adult is about 200 to 2000 mg, for example about 400, 600, 750, 1000 or 1500 mg.

The compositions of this invention may be used to treat infections of the respiratory tract, urinary tract or soft tissues in humans,or mastitis in cattle.

The carriers used in the compositions of this invention may include diluents, binders, disintegrants, 7L~

lubricants, colours, flavouring agents or preservatives in conv~ntional manner. Thus suitable agents include lactose, starch, sucrose, calcium phosphate9 sorbitol, polyvinylpyrxolidone, acacia, gelatin, tragacanth, potato s*arch or polyvinylpolypyrrolidone, magnesium stearate or sodium lauryl sulphate.

Orally administrable forms of the compositions of this invention are most suitably in the form of unit~
dose units such as tablets or capsules.

The present invention also provides synergistic pharmaceutical compositions which comprise a pharmaceutical composition as hereinbefore described which als~ contains a penicillin or a cephalosporin.

Suitable penicillins for inclusion in the com-]5 positions of this invention include benzyl penicillin,phenoxymethylpenicillin, ampicillin or a pro~drug therefor, amoxycillin or a pro-drug therefor, caxbenicillin or a pro-drug therefor, ticarcillin or a pro-drug therefor, suncillin, sulbenicillin, azlocillin or mezlocillin.

Particularly suitable penicillins for inclusion in orally administrable compositions of this invention include ampicillin and its orally administrable pro-drugs, amoxycillin and its orally administrable pro-dru~s and orally administrable pro-drugs of carbenicillin.

Thus particularly suitable penicillins include ampicillin anhydrate, ampicillin trihydrate, sodium ampicillin, talampicillin hydrochloride, pivampicillin hydrochlorlde and bacampicillin hydrochloride; amoxycillin trihydrate, sodium amoxycillin; and the sodium salts of the phenyl and 5-indanyl a-esters of carbenicillin.

A preferred penicillin for inclusion in the orally administrable compositions o this invention is amoxycillin trihydrate. A further preferred penicillin for inclusion in the orally administrable coropositions of this invention is ampicillin trihydrate.

Particularly suitable penicillins for inclusion in injectably administrabl~ compositions of this invention include injectable salts such as the sodium salt of ampicillin, amoxycillin, carbenicillin and ticarcillin.

A preferred penicillin for inclusion in the injectably administrable compositions of this invention is sodium amoxycillin. A further preferred penicillin for inclusion in the injectably administrable compositions of this invention is sodium ampicillin.

Particularly suitable cephalosporins for inclusion in the compositions of this invention include cephaloridine, cephalexin, cephradine, cefazolin and cephalothin.

1~7~3%7~

A particularly suitable cephalosporin for inclusion in the orally administrable compositions of this invention is cephalexin.

Particularly suitable cephalosporins for inclusions in the injectably administrable compositions of this invention include cephaloridine, cefa201in and cephradine, generally as their pharmaceutically acceptable salt.

The weight ratio between compound of tlis invention and penicillin or cephalosporin is generally from 10:1 to 1:10, more usually from 5:1 to 1:5 and normally rom 3:1 to 1:3.

The penicillin or c~phalosporin is generally utilised in its conventionally administered amount.

Suitable methods of formulation include those described in the aforementioned European Patent Applications~

The following Examples serve to illustrate the invention.

27i!3 2qg -Example 1 - -Nitrobenzyl (5R,6S)-3-methylthio-6-~(S)-1-hydroxyethyl]-7-oxo-1-azabicyclo[3.2 0]hept 2-ene-2-carboxylate CH~ H CH,H
HO r~ ~CQCH3~

~ N ~ fi~---N
6 co PNB d C02P~B oo2 el) 2 (e2) CH~ ~1 HO ~SCH3 Step A (e3) C02PNB
5- A solution of the ester (el) (160mg) in acetone (3ml) conta~ning water (10 drops) was cooled to -20. A
solution of N-bromoacetamide (55mg) in aceto~e (0~5ml) was added with stirring, and stirring was continued for 20 min at -20. Chlorofonn (30ml) was added and the solution was washed with water (30ml). The dried (MgS04) organic layer was concentrated in vacuo to afford a foam (119mg) which contained the thiol(e2); vmax (CHC13) 1775, 1705 cm 1. -Step B
The product from Step A was dissolved in DMF ~lml) and to the solution was added anhydrous potassi~n carbonate (25mg) and meth~l iodide (0.2ml). After stirring the mixture vigorously for 20 min, ethyl acetate (30ml) was added, and the organic layer was washed with water (2x30ml) and brine (30ml). The solution was dried (MgSO~) and the solvent evaporated in vacuo to leave a residue which was chromatographed on silica using 2~ EtOH in CHC13 to elute.
The title meth~lthio-derivative (e3) was obtained as a white solid (29mg); vmax (KBr) 3450 (br) 1765 and 1695 cm 1;
~ma~ (EtOEI) 320 (11,900) and 266nm (11,300); ~DMF-d7) 1.29 (3H,d,J 6.5 Hz, CH3CH), 2.45 (3H,s,CH3S), 3.22 and 3.45 (each lH,dd,J 9 and 18 Hz, 4-CH2), 3.50 (lH,dd,J 3 and 4 Hz, 6-CH), ca. 4.1 (lH,m,CH3CH), 4.25 (lH,dt,J 3 and 9 Hz, 5-CH), 5.17 (lH,d,J 4.5 Hz, OH), 5.32 and 5.57 ~each lH,d, 7~
_ 29 -J 14 Hz, Cl~2C02), 7.82 and fi.27 (each 2}1,d,J 9 llz, CGil~-N02). L ~I~, 378.0884. C17l~l8N206S requires M, 378.OB82].

Example 2 - Benzyl ~5~,6S)-3-methylthio-6-~(S)-l-hydroxy ethyl)-7-ox_-1-aza~icyclo~3.2.0]hept-~-ene-~-carboxylate C~ CH~
N

h d~ C02C~I2Ph ~0 ~ SC~3 Ste~ ~ (e6) C2 2 A solution of the trimethylsilyl ether (e4) (1~3mg) in acetone (3ml) containing water (3 drops~ was treated with ~-bromoacetamide (39mg~ at -20 with stirring. After 20 min ethyl acetate (30ml) was added and the solution was washed with water (30ml) followed by brine (30ml), before drying (MgS04) and co~centrating in vacuo. To the residue was added chloroform followed by ether and the solid which precipitated was removed by filtration. The mother liquors were concentrated in vacuo to leave a foam (86mg) which contained the thiol (e5); vmax (CHC13) 1775 and 1700cm 1.
Step B
The product from Step A was dissolved in DMF (lml), and anhydrous potassium carbonate (20mg) followed by methyl iodide (0.5ml) were added to the solution. After stirring the mixture for 1.5h, ethyl acetate (30ml) was added and the organic solution was washed with water (2x30ml) and brine (30ml). The dried (MgS04) solution was concentrated in vacuo and the residue chromatographed on silica gel using 20% petroleum ether (60-80) in ethyl acetate to elute. The title methylthio-derivative (e6) was obtained as a gum (3mg); vmax (CHC13) 3400 (br), 1780 and 1700cm 1;

~7~
_ 30 -AmaX (EtOH) 319nmi ~(CHC13) 1.36 (311,d,J 6.5 ~Iz, CH3CEI), 2.37 (3H,s,SCH3), ca. 3.2 (3H,m,6-CH and 4-CH2), ca. 4.15 (2H,m,5-CH and CH.CH3), 5.30 (~H,AA'X,CH2Ph) and 7~35 ( ,m,CH2Ph). [M / 333.1034. C17HLgN04S requires M, 333.1033].

Example ~ Nitrobenzyl (5R,6S)-3-ethy~hi~ 6 hydrox~ethyll-7-oxo-l-azabicy~10[3.2.0]
carboxylate CH H CH H
Ho 'I s ~ NHCOCH3 H~SH
N ~ ~ N

(el) (le2) CH
HO~ SCH2cH3 Method 1 (e7) C02PNB
10 Step A
A solution of the ester (el) (500mg) in acetone (lOml) containing water tlml) was treated with N-bromoacetamide (154mg) with stirring at -20 for 20 min. Work-up as described in Example 1, Step A afforded a foam (519mg) which contained the thiol (e2).
Step B
The product from Step A was treated with ethyl iodide (0.75ml) and anhydrous K2C03 (250mg) in D~F (5ml) in a way analogous to that described in Example 1~ Step B. Work-up as also described therein gave a product which was chromatographed on silica using a gradient elution with ethyl acetate/petroleum ether mixtures (from 80% to 100%
ethyl acetate). The title ethylthio-derivative was obtained as a white crystalline solid (120 mgs);
25 m.p- 180-183 ; vmax (KBr) 3490, 1760 and 1700 cm AmaX (EtOH) 320 (12,600) and 266nm (11,300). ~DMF-d7) 1.26 (3H,t,J 7.5 Hz, CH3CH2), 1.27 (3H,d,J 6.5, CH3CH), 271~

2.94 (lH,q,J 7.5 Hz, SCH2CH3), 3.33 (lH,d,J 9 Hz, 4-CH2), 3. 49 (lH,dd,J 3 and 4 Hz, 6-CH), 4.09 (lH,m,CH3CH),

4~24 (lH,dt,J 3 and 9 Hz, 5-CH), 5.13 (lH,d,J 5 Hz, OH),

5.~9 and 5.54 (each lH,d,J 14 Hz, CH2C02), 7.82 and 8~26 (each 2H,d,J 8.5 Hz, C~H4-N02). ~M , 392.1045, C18H20N206S requires M, 392.1040~.
Method 2 _ Step A
The ester (el) (200mg). was converted into the thiol derivative (e2) by ~he method described in Example 1, Step A.
Step B
A ~olution o~ the product from Step A in dichlo.romethane (lOml) was stirred vigorously with anhydrous potassium carbonate (120mg) and triethyloxonium tetrafluoroborate (84mg) at -10 for 20 mln. The mixture was allowed to warm to room tempexature ~or 10 min, and was then diluted with more methylene chloride ~20ml). The solution was washed with water and dilute brine, then dried ~MgS04) and -concentrated in vacuo. The residue was chromatographed on a column of silica gel using 2Q~petroleum ether in ethyl acetate followed by ethyl acetate to elute.
The ethyl~hio-derivative (e7), identical in all respects to the product isolated by method 1, was obtained as a white solid (19mg).

ExampLe 4 - ~-Nitrobenzyl (5R,6S)--3-p-bromobenzylthio-6-[(S~ hydroxyethyl]-7-oxo-l-azabicyclo E 3 . 2 . o] hept-2-ene-2-caxboxylate S~I HO~SCH2~3Br C02PNB ~e8) C02PNB

~ ~ '7~
_ ~2 -Step A
The ester (el) (200mg) was converted into the thiol derivative te2) by the method describecl in Example l, Step A.
Step B
The product from S~ep A was dis~olved in D~F (2ml) and to the solution were added anhydrous potassium carbonate (1~3mg) and p-bromobenzyl bromi~e (223mg)~ The mixture was stirred for O.Sh at r.~. and was then diluted with ethyl 10 acetate (SOml). The organic solution was washed with water (2x30ml) and bri~e (30ml), then dried (MgSO~ and concentrated in vacuo. Chxomatography o the residue on a silica-gel column using 30% petroleum ether in ethyl.
aceta~e to elute at firs~,gradually increasing the polarity o t~e eluant to 100% ethyl acetate, afvrded the title p-bromobenzylthio-derivative (e8) as a solid which was further puri~ied by trituration with ethyl acetate-e~her (1:2) and ~iltration~ The final product was obtained as a crystalline solid (35mg); ~max ~KBr) 1770 and 1695 ~m l;
~max (EtO~) 321 (l4,300) and 266nm (12,350); ~(DMF-d7) 1.2? (3H,d,J 6.5 Hz, CH3CH), ca. 3.3-3.6 (3H,m,4-CH2 and

6-CH), ca. 4.l ~2H,m,5-CH and CH3C ), 4.28 (2H,s,SCH2), 5~19 (lH,d,J S Hz, OH), 5.30 and 5.57 (each lH,d,J 14 EIz, CH2C02), 7.~0 and 7.58 (each 2H,d,J 8.5, C6H4-Br), 7~8l and 2S 8.27 (each 2H,d, 9 Hz, C6H4-N02~.

Example 5 - ~Nitrobenzyl (5R,6S)-3-P-nitrobenzylthio-6-~(S)-l-hydroxyethyl]-7-oxo-l-azabicYclo[3.2.0]hept-2-ene-2-carboxylate H ~"~ ~ S~l ~ SC112 ~ No2 C02PNB (e9) C02PN~

~7~

The thiol (e2) was prepared from the ester (el) (50mg) by the method described in Example 1, Step A.
Step B
The product from Step A was treated with p-nitrobenzyl bromide (24mg) and K2C03 (30mg) in a manner analogous to that described in Example 4, Step B. Work-up and chromatography as also described therein gave the title p-nitrobenzylthio-derivative (e9) (3mg); vmax (partial solution in CHC13) 1780 and 1710 tbr) cm 1, Example 6 - Sodium (5~,6S) 3-methylthio-6 ~(S)-l-hydroxy-ethyl3=7-oxo-1-a~abicyclo[3.2.0~hept-2-ene-2-carbox~

~ ~ SCH3 (e3) C02PN~ O (elO) 2 a 5~ Pd on C catalyst (60mg) was shaken with hydrogen in 30% aqueous dioxan (lOml) at ambient pressure and temperature for 0.5h. A solution of the ester (e3) in 30% aqueous dioxan (2ml) was added to the vessel and hydroyenation was continued for a further 3.25h. Sodium bicarbonate (lOmg) was added and the mixture was filtered through Celite*washing the pad well with water (20ml), The filtrate was concentrated in vacuo to ca. 20ml and the aqueous solution was washed with ethyl acetate (3x30ml), before concentrating in vacuo to a volume of ca. 5ml. The resulting solution was loaded onto a column (lSx2.5cm) of Biogei P2 which was eluted with water. Fractions containing the title sodium salt (elO) were identified by the chromophore at ~max (H20) 302nm in the W spectrum These were collected and combined to afford an aqueous * TRADE MARK

'7~
. 34 -solution of the salt (elO~ (ca~ llmg estimated by UV).
The salt could be obtained as ahygroscopic solid by removal of the solvent in vacuo(aided by the addition of ethanol and toluene, respectively).

Example 7 - Sodium (5R,6S)-3-ethylthio-6-[(S)-l-hydroxy-ethyl]-?-oxo-l-azabicyclo~3.2.0~hept-2-ene-2-carboxylate CH ll C~
~lO~ ~ SC~I2CH3 HO ' ~ SCH2CH3 N ~ -N
(e7) C02PNB (ell) C02~Na Hydrogenolysis of the ethyl~hio-derivative (e7) l120mg) was per~ormed in the manner described in Ex~-~ple 6.
(150mg 5% Pd-C, 26mg NaHC03j. After Biogel P2 chromatography (column 25x2.5cm) the title salt (ell) (32mg) was obtained in aqueous solution; AmaX 302nm. Part of the solution was concentrated in vacuo, the final traces of water being removed by azeotroping from ethanol and then toluene, to afford the salt (ell) as a hygroscopic solid; vmax (KBr~
1750 and 1590 cm 1; ~(D20) 1.21 (3H,t,J 7.5 Hz, CH3CH2), 1.26 (3H,d,J 6.5 Hz, CH3CH), 2.78 (2H,q,J 7.5 Hz,SCH2CH3), 3.14 (2H,m,4-CH2), 3.39 (lH,dd,J 3 and 5.5 Hz, 6-CH), 4.02 (lH,m,5-CH) and 4.15 (l~l,m,CH3CH), (reference HOD at ~4.60).

7~7~

Example 8 - Benzyldimethyl-n-hexade~a_m nium salt of p-nitrobenzyl (5R,6R)-3-methylthio-6-[(S)~l-hydroxy-sulphonyloxyethyl]~7-oxo-1-a~abicyclo~3 ~ t-2-ene-2-carboxylate CH H H
M B N+-O SO ~ S ~ NY,COC~3 16 33 ~ N ~
0 ~0 PNB
(el2) 2 ~_ ~ SH ~e2BZ~ 3S ~ ~ SCH3 H33 16 N C16H33 ~N
(~13) C02PNB (el4) C02PNB

S Step A
A solution of the quaternary ammonium salt (el2) (409mg) in 7% aqueous acetone ~7.5ml) was cooled to -20, and a solution of N-bromoacetamide ~70mg) in acetone (lml) was added w.ith stirring. After 20 min at -20 chloroform (50ml) was added, and the solution was washed with dilute brine (At this stage it was necessary to filter the mixture through Celite in order to brçak up the emulsion which had formed~. The organic layer was dried (Mg-S04) and.
the solvent removed in vacuo to afford a foam (380mg) which contained the thiol (el3); ~max (CHC13~ 1775 and 1700 cm 1.
Step B
A solution of the product from Step A in DMF (3ml) was stirred with anhydrous potassium carbonate (75mg) and methyl iodide (0.3ml) at room temperature for 20 min.
Chloroform (SOml) was added and the organic solution washed with brine (2x50ml), water (2x50ml) and brine again (50ml). The dried (MgS04) solution was evaporated in vacuo and the residue chromatographed on silica gel using a - 3~ -gradient elution ~f chloroform to 30~ ethanol in chloroform.
Fractions containing the product (t.l.c.) were combined and concentrated in vacuo to afford the title methylthio~
derivative (el4) as a foam (118mg); vmax ~CHC13) 1775 and 5 1700 cm ; ~max (EtOH) 318 and 268nm.

Example 9 - p-Nitrobenzyl (5R,6R)-_-methylthio-6-[(S)-l-ethoxysulphonyloxyeth~l]-7-oxQ-l-azabicyc10[3-2-]hePt~
2~ene-2-carboxylate C H ~ ~ SC~13 EtO3SO ~ ,SC~I3 (~14) C02PNB ~15) C02PNB

A solution of the quaternary ammonium salt (el4) ~115mg) in dichloromethane (lOml) was stirred with triethyloxonium tetrafluoroborate (30mg) for 15 min at room temperature. Chloroform (20ml) was then added and the solution was washed with water (20ml), dried (MgS04~ and concentrated in vacuo. The product was rapidly chromatogxaphed on silica gel (230-400 mesh ASTM) using 20~ petroleum ether in ethyl acetate to elute. The title diester (el5) was obtained as a gum (29mg); v x (CEIC13) 1780 and 1700 cm 1; ~max (EtOH) 317 and 267nm; ~(CDC13) 1.42 (3H,t,J 7 Hz, CH3CH2), 1.66 (3H,d,J 6 Hz, CH3CH), 2 41 (3H,s,SCH3), 3.10 (lH,dd,J 18 and 10 Hz, 4-CH Hb), 3.36 (lH,dd,J 18 and 9 Hz, 4-CHaHb), 3.84 (lH,dd,J 6 and 10 Hz, 6-CH), 4.35 (2H,q,J 7 Hz, OCH2CH3), 4.37 (lH,m,5-CH), 5.03 (lH,m,CH3CH), 5.20 and 5.48 (each lH,d,J 13.5,CH~C02), 7.60 and 8.19 (each 2H,d,J 8.5 Hz, C6H4N02).

~L~ 7~7~3 _ 37 -Example 10 - ~Nitrobenzyl (5R,6R)-3-ethylthio-6-~(S)-l-etho~y ulphonylox~ethyl]-7- xo-1-azabicyclo[3.2.0]he~ 2-ene-2-carboxylate Cl~
+_ ~ S ~ NHCCCI13 O~PNB
¦ (el2) ~33C16 ~ SH EtO3SO ~ Et Step A (el3) C02PNB (el6) C02PNB
The ~ua~ernary ammonium salt (el2) (660mg) was converted into the thiol derivative (el3) by the method described in Example 8, Step A.
Step B
The product from Step A was dissolved in dry dichloromethane ~15ml) and to the solution was added anhydrous potassium carbonate (205mg) and Meerwein's reagent ( 282mg) . The mixture was stirred for 15 min at room temperature and was then diluted with chloroform (30ml). The organic solution was washed with very dilute brine, dried (MgS04) and concentrated in vacuo. Rapid chromatography of the residue on silica gel (230-400 ASTM) using 20% petroleum ether /ethyl acetate to elute afforded the title diester as a foam (189mg); vmax (CHC13) 1780 and 1705 cm ; ~(CDC13) 1.33 (3H,t,J 7 Hz, CH3CH2S), 1.42 (3H,t,J 7 Hz, CH3CH20), 1.66 (3H,d,J 6 Hz, CH3CH), 2.90 (2H,q,J 7 Hz, CH3CH2S), ca. 3.25 (2H,m,4-CH2), 3.84 (lH,dd, J 6 and 10 Hz, 6-CH), ca. 4.30 (lH,m,5-CH), 4.35 (2H,q,J 7 Hz, CH3CH2S), ca. 5.05 (lH,m,CHCH3), 5.21 and 5.48 (each lH,d,J 14 Hz, CH2C02), 7.61 and 8.20 (each 2H,d,J 8.5 Hz, C6H4-N02). (The n.m.r. spectrum revealed that some impurity was present in the product).

- 38 _ ~ 2~

Exam~le 11 - p-Nitrobenzyl (5R)-3-ethylthio-6-[(E)-ethylidene]-7-oxo-l~azabicyclo[3.2.0]hept-2-ene-2-carboxvlate C H lI H
EtO3S0 ~ ~ Et ~ Et N ~ N ~
(el6) C02PNB(el7) CO~PNB

The diester (el6) ~60mg) was stirred with anhydrous S potassium carbonate (41mg) in DMF (0.5ml) at room temperatur0 for 30 min. Ethyl acetate (30ml) was added and the solution was washed with water (2x30ml) and ~rine (20ml) ~efore drylng (MgS04) and evaporating in vacuo.
The product was chromatographed on silica gel using 30 petroleum ether in ethyl acetate to elute. The title e~hyli~ene derivative (el7) was isolated as a gum;
vmax (CHC13) 1770 and 1705 cm ; ~(CDC13) 1.32 (3H,t, 7 Hæ, CH3CH2), 1.83 (3H,d,J 7 Hz, CH3CH), 2.84 (2~,~,J 7 Hz, SCH2CH3), 3.04 and 3.27 (each lH,dd,J 18 lS and 9 Hz, 4-CH2), 4.77~1EI, brt,J 9 Hz, 5-CH), 5.23 and 5.53 (each lH,d,J 13.5 Hz, C~2C02), 6.43 (l~,qd,J 7 and 1.5 Hz, CH3CH), 7.17 and 8.19 (each 2H,d,J 8.5 Hz, C6H4-N02).

8~

Example 12 - Benzyldimethyl-n-hexadecylammonium salt of p-nitrobenzyl (5R,6R)-3-ethylthio-6-[(S)-l-hydroxysulpho-r oxyethyl]-7-oxo-l-azabic ~10[3.2~0]hept-2-ene-2-carboxyla-te c~

1 6 3 3 ~ Nl ICCX~I i3 0;;~ PN E~
~ (el2~

2 1 3 ~ Me2BzN o3so ~ 5~t Step A (~Il) C02FNB (el 02PNU -8) The quaterna~ ammonium salt (el'2) (660mg) was converted into the thiol(el3) by the method described in Exàmple 8, Step A.
S Step B
The thiol derivative ~el3) was treated with ethyl iodide and potassium carbonate in DMF in a manner analogous to that described in Example 8, Step B. The title ethylthio-derivative (elR) was obtained as a foam (240mg); ~ ax (CHC13) 1780 and 1700 cm ; AmaX (EtOH) 317 and 267nm.

Æxample_13 - Sodium salt of p-nitrobenzyl (5R,6R)-3-ethylthio-6-[(S)-l-hydr xysulphonyloxyethyl]-7-oxo-l-aza bicyclo[3.2.0]hept-2-ene-2-carboxylate Method 1 ~. T r ~1 ~ Et Na 03S~ ~ s~t.

(eL6) 2 ( C~2PN~

~. 11.~7~7~
~o A solution of the diester (el6) (150mg) in DMF (2ml) was stirred at room temperature in the presence of sodium iodide (172mg) for 4.5h. The solution was evaporated to dryness and the residue chromatographed on silica gel using a gradient elution from chloroform to 25~ ethanol in chloroform. Fractions containing the required product (t.l.c. and u.v.) were combined and evaporated in vacuo to afford the title compound as a solid (23mg); ~max (EtOH) 318 and 267nm, (H20) 317 and 275nm; ~max (KBr) 1765 and 1695 cm 1 Method 2 S~t 3 H ll te~8) C02PNB ~19) CO~rNL

~ column (lOx2.5 cm) of Amberlyst A2~ resin was washed successively with lOOml quantities of methanol, water, ethanol and 30% CHC13 in EtOH. A solution of the quaternary ammonium salt (el8) (19Omg) in 30% CHC13 in EtOH (5ml) was loaded on to the column which was then eluted successively with lOOml portions of 30~ CHC13 in EtOH, ethanol and water. Finally elution with a mixture (1:1) of 5% NaCl in H20 and methanol afforded a solution of the mono sodium salt (el9). This may be further purified by silica gel chromatography as described in method 1.

* TRADE MARK

Example 14 - Disodium (5R,6R?-3-ethylthio-6-~(S)-l-Sulphonato-oxyethyl~-7-oxo-1-azabicyclot3.2.0}hept~ uc==~=5~5~yl~e Na~-O SO \) ~ \~SEt N +~O SO ~ ~ / ~ Et C02PNB ( 20) C2 Na 5% Pd on C catalyst (15mg) was shaken with hydrogen in 30% aqueous dioxan (4ml~ for 30 min. A solution of the ester (el9) (7mg) in 30% aqueous dioxan (lml) was introduced lnto the vessel and hydrogenation was continued for 3h.
NaHC03 (2mg) was added to the mixture which was filtered over Celite, washing the pad with water (lOml), and the solution was concentrated in vacuo to _. lOml. The aqueous solution was washed with ethyl acetate (3xl5ml) and then concentrated in vacuo -to ca. 3ml. The solu-tion was loaded onto a column of Biogel P2 (2.5xlO cm) which was then eluted with water.
Fractions con-taining the -title disodium salt (e20) were identified by the absorption in -the UV spectrum at ~max 298nm.

Example 15 = p-Nitrobenzyl(5R,6S?-3-acet~lthio-6-[(S)-l-acetoxyethyl]-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carbox~late CH ~ CH 3 cooJ~ ,/\ S H

,J~T
N~ C02PNB
O `CO PNB (e22) C~-~
CH3C00~ 3 ~ (e23) 2 The acetoxy derivative (e21) (9Omg) was dissolved in acetone (2ml) containing water (3 drops) The solution was cooled to ~20 and a solution o N bromoacet~nide in acetone (O.Sml) was added with stixring. After 25 min at -20 the solution was diluted with chloroform (25ml) and washed with water (25ml). The organic layer was dried (MgS04) and concentrated in vacuo to afford a gum, which contained the thiol (e22), vmax (CHC13) 1780, 1730 and 1700 sh cm 1.

The product was dissolved in pyridine (lml) and to the stirred solution was added acetyl chloride (3 drops). After 15 min the mixture was evaporated to dryness and the product was partitioned between chloroform (25ml) and water (25ml). The organic layer was washed with pH 3 phosphate buffer (20ml), dilute aqueous sodium bicarbonate (20ml) and water (20ml), and then dried (MgS04) and concentrated in vacuo. The residue was chromatographed on silica gel using 50% petroleum ether-ethyl acetate to elute. The first-eluted component was the title diacetyl derivative (e23) (Smg); vmax (CHC13) 1790r 1740 and ca. 1700 sh cm v~nax (EtOH) ca. 300 sh and 267 nm~ ~(CDC13)inter alia 1.43 (3H,d,J 6.5 Hz, CH3CH), 2.11 (3H,s,CH3CO), 2.40 (3H,s,COCH3) and ca. 5.15 (lH,m,CH3CH) ~ ~ 's~7~.

_ ~3 _ Exan)ple 16 - p ~ R GS)-3-(2-ethoxycarbonyl-ethen lthio)-6-[(S)-l-acetoxvethvl]-7-oxo-1 azabicyclo Y ~
[3.2.0]hept-2-ene-2-carboxylate ~ / CE13COO ~ i~/S~C~CO~Et C~L C021:'N13 ( 24 ) 2 CH~ H

-t Cll ~COO J~ =J 2 (~25 ) C2~ B
A solution of the acetoxy~clerivative (e21) (330mg) in a mixture of acetone (7ml) and water (0.75ml) was cooled to -20. A solution of N-bromoacetamide (93mg) in acetone (lml) was added with stirring, and stirring at -20 was continued for 20 min. Chloroform (50ml) was added and the organic solution was wash.ed with water 150ml), dried (MgS04) and evaporated in vacuo to afford a foam, which contained the thiol/thione (e22).
Step B
The product from Step A was dissolved in DMF (3ml) and 1~ to the solution was added ethyl propiolate (66mg) followed by anhydrous potassium carbonate (93mg) with stirring.
After stirring for 15 min at ambient temperature, ethyl acetate (40ml) was added and the solution was washed with water (3x30ml) and brine (30ml). Evaporation of the dried (MgS04) solution gave a residue which was chromatographed on sllica gel using 20% petroleum ether in ethyl acetate to elute.
The first-eluted component (8mg) was the (E)-isomer of the title compound (e24); vmax (CHC13) 1790, 1735 and 1710 cm ; ~max (EtOH) 333 and 267 nm.
The next-eluted product (51mg) was the (Z)-isomer of the title compound (e25); vmax (CHC13) 1790, 1740 and 1710 cm ; ~max (EtOH) 335 and 267 nm; ~(CDC13) 1.30 ~.7~
_ 44 -(3H,t,J 7 Hz, CH3CH2), 1.42 (3H,~,J 6.5 Hz, CH3CH), 2.-08 (3H,s,CH3C0), 3.18 and 3.44 (each lH,d,J 9 and 18 Hz, 4-CH2), 3.49 (lH,dd,J 3 and 4 H~, 6-CH), 4.12 llH,dt,J 3 and 9 Hz, 5-CH) overlapping with 4.22 (2H,q,J 7 Hz, C~3CH2), 5.24 (lH,m,CH3CH), 5.27 and 5.52 (each lH,d,J 14 Hz, CH2C02), 5.97 and 7.22 (each lH,d,J 10 Hz, CH=CH), 7.68 and 8.21 (each 2H,d,J 9 Hz, C6H4-N02).

~ ~ 78~

Example 17 ~-Nitrobe~zyl (5R,6S)-3-[2-(2-~-nitrobenzyloxycarbonylamino-ethoxycarbonyl)ethenylthio]-6-L(S)-l-hydroxyethyl]-7-oxo-1-azabicycloL3.2.0]hept-2-ene-2-carboxylate H ~ ~ ~\ S COCH3 (el) CO2PNB

' ` ~C2 (e26) 2 Step A
A.solution of the ester (el) (250 mg) in dioxan (5 ml) containing water (0.75 ml) was stirred with N-bromoacetamide (75 ml) for 5 minutes at ambient temperature. Chloroform (30 ml) was added and the solution was washed with 0.05M phosphate buffer (pH7) solution (20 ml) and brine (20 ml). The solution was dried (MgSO4) and evaporated in vacuo -to afford a foamy product which contained the thiol/thione (e2)~
Step B
The product from step A was dissolved in DMF (3 ml) and to the solution was added with stirring, anhydrous potassium carbonate (75 ml) followed by 2-(_-ni-trobenzyloxycarbonylamino-ethyl)propiolate (200 mg). After stirring for 15 minutes the reaction mixture was diluted with ethyl acetate (30 ml) and the organic solution was washed with water (3 x 30 ml) and brine ~ ~7~715 (30 ml). Evapora-tion of the dried (MgSO~) organic layer gave a product which was chromatographed on a column of sil:ica yel using ethyl acetate fol.lowed by 10~ ethanol ethyl acetate. The - 45a --le~ ~

major product obtained (81 mg) consisted of p-nitr~benz~l (5R,6S)-3~[2-(2-p-nitrobenzyloxycarbonylaminoethoxycarbonyl) ethenyl~hio~-6-[(S) l~hydroxyethyl~-7-oxo-1-azabicyclo [3.2Ø~hept--2-ene-2-carboxylate, as a mixture of the Z-isomer (e26) a-nd *he corresponding E-isomer (ca. 85:15);
A (E OH) 337 and 265 nm. ~max (CHC13) 1780 and 1720 c~ ; ~ ~M~-d7) 1 29 (3H, d~ J 6.5 Hz, CH3CH), 3.30-3.70 (SH9 m, 4-CH2, CH2N and 6-CH), 4.0-4,5 (4H, m, OCH2, 5-CH and CH3CH) 9 ca. 5.2 (lH, OH), 5.23 (2H9 s, CH2Ar), 5.37 and 5.60 (each lH, d, J 14 Hz, CH2Ar), 6.06 ~lH, d, J SCH for Z-isomer) ca~ 7.40 - 8.05 (6H, m, 4 x aromatic protons and _ CHCO~) and 8.25 (4H? d, J 9 Hz~
aromatic protons). The E-isomer shows inter alia ~ 6.23 (lH, d, J 16 Hz, SCH =).

~:~78;~78 Example 18 (5R,6S)-3-[2-(Z)-(2-Aminoethoxycarbonyl)ethenyl~thio]-6-[(S)-l -hydroxyethyl~-7-oxo-1-azabicyclo~3.2.0]hept-2-ene-2-carboxylic acid J
CO2PNB H~ ¦ 3 (e26) ) H ~ y S ICO

(e27) A mixkure of the ester (e26) (80 mg), dioxan (10 ml), ethanol (0.8 ml), water (3 ml), 0.05M pH7 phospha-te buffer (4 ml) and 10% palladium on charcoal (110 mg) was shaken in an atmosphere of hydrogen for 2 hours. The mixture was filtered through Celite washing with water (15 ml) and the solution was then eoncentrated to a volume of ca. 20 ml. The aqueous solution was washed with ethyl acetate-ether (1:1;2 x 50 ml) and then concentrated to ca. 5 ml before loading onto a column of XAD-2 (2.5 x 10 em). Elution with water gave fractions containing (5R,6S)-3-L2-(Z)-(2-aminoethoxycarbonyl)ethenylthio]--6-L(S)-1-[3.2.0]hydroxyethyl~-7-oxo-1-azabicyclo[3.2.0]hept-2--ene-2-carboxylie acid (e27); ~max (H2O) 325 nm. (contains ca. 15% of eorresponding E-isomer).

~ ~ 78~

Example 19 p-Nitrobenzyl (5R,6R)-3-[2-(Z)-(2~ itrobenzyloxycarbonyl~
aminoethoxycarbonyl)ethenylthio~-6-~(S)-L-hydroxyethyl~-7-oxo-1-azabicyclo[3.2 0]hept-2-ene-2-carboxylate __ _ _ C 2PNB ~ H CH3 (e28) HO /~, S ~~,NE~C02PNB

(e29) Step A
The ester (e28) (300 mg) was trea-ted with N-bromo-acetamide (92 mg) as described in Example 17.
Step B
The product from step A was treated with 2-~-nitro-benzyloxycarbonylaminoethylpropiolate (300 mg) as described in Example 17. p-Nitrobenzyl (5R,6R)-3-r2-(2-p-nitrobenzyl-oxycarbonylaminoethoxycarbonyl)ethenylthio]-6-L(S)-l-hydroxy-ethyl~-7-oxo-1-azabicyclo[3.2.0~hept-2-ene-2-carboxylate (e29) was obtained ini-tially as a foam (166 mg) which was crystallised from acetone-ether to afford a solid (120 mg). The ratio of Z-and E- isomers was ca. 95 5;1~max (E-tOH) 334 and 264 nm., max (KBr) 1785 and 1710 cm , ~ (DMF-d7) 1.32 (3H, d, J 6.5 Hz, CH3CH), ca. 3.3-3.8 (5H, m, 4-CH2, NCH2 and 6-CH), ca. 4.05-4.55 (4H, ml OCH2, 5-CH and CH3CH), 5.15 (lH, br, OH), 5.24 (2H, s, CH2Ar), 5.37 and 5.59 (each lH, d, J 14 Hz, ArC 2) 6.10 (lH, d, J 10 Hz, SCH= for Z-isomer) and ca. 7.4-7v95 and 8.2-8.35 (lOH, m, 8 x aromatic protons, =CHCO2 and NH).

Example 20 (5R,6R)-3-~2-(Z)-(2-Aminoethoxycarbonyl)ethenylthio~6-[(S)~ hydroxyethyl]-7-oxo-1-azabi _clo[3 2.0]hept-2-ene-2-carboxylic acid -H" ¦ ~ H
/` ~ ~ S =7~ 'N~IC02PNB

CO PNB
2 CH~
(e29) _ ~ H ~ ¦ H H

/ \ ~ ~ ,S ~ f 2'" ~ 2 (e30) The ester (e29) (150 mg) was disso]ved in a mixture of dioxan (10 ml) and 0.05M pEi7 phosphate buffer (4.5 ml), and the solution was shaken with 5% palladium on charcoal (200 mg) in an atmosphere of hydrogen for 2 hours. The mixture was filtered over Celite, washed with water (20 ml), and the solution was then concentrated in vacuo to a volume of ca. 20 ml. The solution was washed with ethyl acetate (4 x 25 ml), and was then freeze-dried to afford (5R,6R)-3-~2-(Z)-(2-aminoethoxycarbonyl)ethenylthio~-6-C(S)-l-hydroxyethyl~-7-oxo-1-azabicyclo~3.2.0~ hept-2-ene-2-carboxylic acid (e30) as a solid; ~ max (H2O) 325 nm., vmax (KBr) 1750, 1690 and 1565-1610 (br) cm . (The product contained ca. 5% of the corresponding E-isomer).

~7~7~

_ 50 -The Sodium salt of ~-nitxobenzyl (5R,6R~-3- L ~Z)-2-~-nitrobenzyl-oxycarbonylethenylth _ J-6-~(S)-l-hydroxysulphonyloxyethyl~-7-oxo-1-azab ~

0350 ~ ~ aiO350 ~ ¦ S ~ 2 ~NBzMe2 ~ ~ NHCOCH3 C16H33 ~02PNB C02PNB
(el2~ (e31) The mono quaternaryammonium derivative (el2) tl.448 9) was dissolved in dioxan (20 ml) containing water (1.5.ml) and N-bromoacetamide (220 mg) was added with stirring.
After 4.5 minutes, the solution was diluted with CHC13 (40 ml) and was then washed with water (50 ml) and brine (30 ml). Evaporation of the dried (MgS04) organic layer gave a foam which contained the thiol (el3)~

Step B A solution of the product from step A in DMF (10 ml) was stirred at room temperature with ~-nitrobenzyl propiolate (500 mg) and potassium carbonate (220 mg). After 20 minutes ethyl aceta~e (70 ml) was added and the organic solution was washed with water (3 x 50 ml) and brine (50 ml). The solution was dried (MgS04) and evaporated in vacuo to give a crude product which was chromatographed on silica gel using chloroform followed by 5%, 10%, 15% and 20% ethanol in ctlloroform respectively, to elute. ~ractions containing the major single component were combined and evaporated to give a foamy product (0.54 9). [vmax (CHC13) 1785 and 1710 cm 1.~

7~8 Step C To a solution of the product from step B in ~cetone (5 ml) was added a solution o sodi~lm iodide (80 mg) in acetone (1 ml). Scratching of the vessel with a glass rod induced precipitation of a white solid. More acetone was added and the solid filtered off and washed with acetone followed b.V ether. The solid (193 mg) consisted o the sodium salt of ~-nitrobenzyl (SR, 6R)-3-[ (7~-2-~-nitrobenzyloxycarbonyl-ethenylthio~-6-[(S)-l-hydroxysulphonyloxyethyl~-7-oxo-l-azabicyclo [3.2.0Jhept_2-ene_2-carboxylate (e31).
A max (H20) 335 (20,410) and 272 (20l150), ~max (KBr) 1775 and 1710 cm 1, c, (DM~-d7) 1.48 (3H, d, J 6.5 Hz, CH3CH), ca. 3.2-3.6 (lH, m, 4-CHa), 3.7-4.1 (2H, m, 6-CH
and 4-CHb), 4.25-4.80 (2H~ m, 5-CH ancl CH3CH), ~.41 (2H, s, CH2Ar), 5.38 and 6.60 (each lH, d, J 14 Hz, CH~Ar), 6.24 tlH, d, J 10 Hz, SCH=), ca. 7.70~7.95 (m) and 8.29 (d, J g Hz) ~Total 9H, 2 x C6H4-N02 and =CHC02).

Exa~ple 22 The Trisodium salt of (5R,6R)-3- C(Z)-2-carboxyethenylthiol-6-~(S)-1-hydroxysulphonyloxyethyl~-7-oxo-1-azabicyclor3.2. b~
hept-2-ene-2-carboxylic acid _ H~
Na O3SO
>~/ S~ ~02PNB
~ N y (e31) ~ H~CH3 `1 H ~l Na 2 a (e32) A solution of the ester (e31) (150 rng) in 25% aqueous dioxan (20 ml) was hydrogenated in the presence of 5% palladium on earbon (200 mg) for 3~5 hours after previously prehydrogenat-ing the ca-talyst for O.S hours. Sodium bicarbonate (38 mg) was added to the mixture which was then filtered over Hiflo washing with water (20 ml~. The filtrate was concentrated to ea. 20 ml and then washed with ethyl acetate (3 x 20 ml). The aqueous sol.ution was further concentrated to ca. 5 ml and then chromatographed on a column of Biogel P2 (2.5 x 25 cm).
Fractions containing the produet were eombined and evaporated in vaeuo to afford the trisodium salt of (SR,6R)-3-[(Z)-2-earboxyethenylthio]-6- r(s~ -l-hydroxysulphonyloxyethyl~-7-oxo-l-azabicycloC3.2.0]hept-2-ene-2-carboxylic acid (e32) as a white solid (60 mg); ~ max (H2O) 325 nm., ~ max (KBr) 1755, 1630 sh and 1580 cm 1, 7~
_ 53 Example 23 r- Nitroben~L-l-5Rr~R ! - ~- r 2~-nitrober ~ _ thio1-6 r ( s, l-hvdroxyethyl 1-7-oxo- 1 azabic~. 2 t ' ~
hept-2~ene-2-ca~ æ,_(E~ and (Z~_iso_ers H ~ ~ H3 COzPNB --~2PNB

(e28) ~ (e33) H~
H0~ ~ ~ S ~f 2PNB

(e3~) Step A A solution of the ester (e28) (300 mg) in a mixture of dioxan (6 ml) and water (0.9 ml) was stirred with N-bromoacetamide (90 mg) at room temperature for 4.5 minutes.
Chloroform (30 ml) was added and the organic solution was washed with 0.05M pH7 phosphate bu~fer (20 ml) followed by dilute brine (Z0 ml). Evaporation of the dried (MgS0~) organic layer afforded a foam.

~ ~'7~;27~
- 5~l -Step B The product ~rom step A was dlssolved in DMF (3 ml) and potassium carbonate (45 mg) and ~nitrobenzyl propiolate ~275 mg) were added. The mixture was stirred for 25 minutes and was then diluted with ethyl acetate (30 ~1). The organic solution was washed with water (3 x 30 ml) and brine (20 ml)~ then dried (MgS04) and evaporated in vacuo. The residue was chromatographed on silica-~el using a gradient elution of 50~0 ethyl acetate-petrol to 100% ethyl acetate.
Two main products were obtained The first (28 mg) corresponded to p-nitroben~yl (5R,6R)-3-[(E)-2-~-nitro-benzyloxycarbonylethenylthio]-6-C(S)~l-hydroxyethyl~-7-oxo-l-azabicyclo ~3.2.0~hept-2-ene-2-carboxylate (e33);
A ax (EtOH) 335 and 265 nm., vmax (CHC13) 1785 and 1715 cm , ~ ~Me2CO) 1.35 (3H, d, J 6 Hz, CH3CH), ca.
lS 3.25~3.90 (3H, m, 4-CH2 and 6-CH), ca. 4,45 (3H, m~ 5 CH, CH3CH and OH), 5.35 (2H, s, CH2Ar), 5.30 and 5.55 (each lH, d, J 14 Hz, ArCH2~, 6.30 (lH, d, J 15 Hz, SCH=), 7.62-8.30 (9H, m, 2 x C6H4-N02 and =CHC02).

The second pxoduct (100 mg) was the corresponding Z-isomer (e34) AmaX (EtOH) 335 (21,998) and 263 (Zl,785), vmax (KBr) 1780 and 1710 cm , ~ (DMF-d7) 1.31 (3H, d, J 6.5 Hz, CH3CH), ca. 3.40-3.85 (3H, m, 4-CH2 and 6-CH), 4~05-4.55 (2H, m, 5-CH and CHCH3), 5.11 (lH, d, J 4.5 Hz, OH), 5.42 (2H, s, CH2Ar), 5.39 and 5.61 (each lH, ~1 J 13 Hz, C_2Ar), 6.26 ~lH, d, J 10 Hz, SCH=), 7.07-8.05 (5H, m, =CH.C02 and 4 x aromatic protons) and 8.28 (4H, d, J 9 Hz~
aromatic protons) [M, 525.0838 corresponds to M - CH3C=OH].

:~7~

Example 24 -carboxyethenylthio~-6-r (s )-l-hydroxvethyll-7-oxo-1-azabicycloL3.2.0]he~t-2-ene-2-carboxylic acid HJ ~72P~B = ~ a Na~

O

(e3~) (e35) The ester (e34) (95 mg) was hydrogenolysed in the manner described in Example 22. After chromatography on Biogel P2* ( 10 x 2.S cm) and evaporation of the solvent, the disodium salt of (5R,6R)-3-~(Z)-2-carboxyethenylthio]-6-~S)-l-hydroxyethyl]-7-oxo-1-azabicyclo [3.2.0~hept-2-ene-2-carboxylic acid te35~ was obtained as a white solid (28 I9); ~ max (H20) 325 nm-~ v max (~Br) 1750 and 1590 cm * TRADE MARK

7~3 Delllonstration a. Antibacterial effectiveness of compounds of Examples 6 and 7 in agar.

M.I.C. (~g/ml) Organism . ~ _ _ Ex.6 Ex.7 . ........... , - . . _ Citrobacter freundii E8 12.5 2.5 Enterobacter cloacae Nl 28 5.0 Escherichia coli 0111 12.5 5.0 Escherichia coli JT39 12.5 2.5 Klebsiella aerogenes A 12.5 2.5 Proteus mirabilis C977 50 12.5 Proteus morganii 1580 2S 2.5 Proteus rettgeri WM16 25 5.0 Proteus vulgaris WO91 25 5.0 Pseudomonas aeruginosa ~ >100 >50 Salmonella typhimurium CT10 12.5 2.5 Serratia marcescens US20 12.5 5.0 Shigella sonnei MB 11967 12.5 5.0 ~acillus subtilis A 25 5.0 Staphylococcus aureus Oxford 3.1 1.2 Staphylococcus aureus Russell 6.2 1.2 Staphylococcus aureus 1517 100 NG
Streptococcus faecalis I >100 >50 Streptococcus pneumoniae CN33 _ NG
Streptococcus pyogenes CN10 _ ~ _ 1.2 _ 57 ~ 'B

. Antibacterial effectiveness of the compound of Ex~nple 14 and ~ 13902 in agar.

~ ,. . _ ~
Organism Ex. 14 13902 -- . _, . .. , .. __ Escherichia coli ~1 -0.1 '0.1 NCTC 15418'0.1'0.1 E8 '0.1'0.1 '0.1'0.1 Ba78R~ 0.20.8 JT4R-~ 0.41.6 ~T20R~ 0.81.6 E96R~ ~0.10.2 JT425C~ ~0.10.8 JT414C-~ 0.20.4 ~ , . --Klebsiella aerogenes T219 ~0.1-0.1 R112 '0.10.2 I281 '0.10.2 Va~R+ 0.86.2 ~ . _ _ .. , ._ Proteus mirabilis T3180.4 '0.1 8~9 0.~0.2 Proteus morganii I 0.4 0.2 T361 0.40.2 Proteus rettgeri I 0.4 0.2 RllO 0.20.2 Proteus vulgaris X 0.4 0.4 NCTC 4395 0 2 .1 - 58 - ~ ~ 7 b. contd~

_ . ~ , ~ _ Organism Ex~ 14 13902 __ _ _ _ ____ Enterobacter cloacae T749 6.2 12.5 NCTC 10005 6.2 12.5 Enterobacter aerogenes T660 0.2 0.4 NCTC 10006 0.4 0.4 Citrobacter freundii W18 1.6 6.2 T221 1.6 6.2 Serratia marcescens SM27 0~4 1.6 Wol46 1.6 3.1 . _ ___ _= _ ~
Pseudomonas aeru~inosa W975 100 50 Dalgleish 50 50 , _. _ _. . _ Staphylococcus aureus Smith 0.4 1.6 RTCC 25923 0.4 1.6 ~9~ 1.6 3.1 T67~ 0.8 0.8 T150+ 0.4 1.6 Streptococcus faecalis C90 12.5 6.2 TllOl 12.5 6.2 _ Bacteroides fragilis2118 0.2 0.2 B3 0.4 0.4 WS12o.2 0.2 BC161.6 1.6 VPl 8243 0.4 0.2 WS41 0.2 0.4 WSl 6.2 6.2 BC4 0.8 1.6 7~
_ 59 -DEMONSTRATION OF EFFECTIVENESS

ORGANISM MIC (~.g/ml) in agar of compound of Example , . _ . _ . _ _ Citrobacter freundii E83.1 0.2 25 1.2 Enterobactex cloacae N13.1 002 0.5 O.S
E.scherichia coli 0111 3.1 ~ 0.1 l.Z 0.2 Escherichia coli JT 39 3.1 0.2 1.2 5.0 Klebsiella aerogenes A 3.1 ~0.1 1.2 0.2 Proteus mirabilis C97712.5 0.4 1.2 0.5 Proteus morganii 1580 3.1 _ 0.5 0.5 Proteus rettgeri WM16 6.2 0.2 1.2 0.5 Proteus vulgaris WO91 6.2 0.4 1.2 0.5 Pseudomonas aeruginosa A 25 25 ~50 ~50 Salmonella typhimurium CT10 3.1 ~ 0.1 1.2 0.5 Serratia marcescens US20 1.2 ~ 0.1 5.0 2.5 Shigella sonnei M8 11967 3.1 0.2 1.2 0.5 Bacillus subtilis A _ c 0.1 0.5 ~0.1 Staphylococcus aureus Oxford ~ 0.1 ~ 0.1 2.5 0.5 Staphylococcus aureus Russell 0.4 ~ 2.5 0.5 Staphylococcus aureus 1517 6.2 0.2 25 5.0 Streptococcus faecalis I '2.5 ~ 0.1 12.5 2.5 Strep~ococcus pneumoniae CN33 _ ~ 0.1 _ ~0.1 Streptococcus pyogenes CN10 ~0,1 ~ 0.1 _ ~0.1 E.coli ESS _ ~ 0.1 l.Z 50.1 827~
~.~

Example_25 Sodium (5R,6S)-6-[(S?-l~acetoxyeth ~
ethox~_rbonyl=ethenylthio]-7-oxo-l-azabicyclo[3.2.o]
.
hept-2-ene-2-carboxylate.

H.. ~ ~ S ~ H ~ ~ . C2Et CH3COO ¦ CH C~O ~
O ~ N 'O2PN~ 3 C2 Na (e25) (e36) 5~ Pd on C catalyst (75 mg) was prehydrogenatecl ~or 0.5 h in a mixture of dioxan (6 ml) and water (3 ml).
solution of the ester (e 25) (50 m~) in dioxan (1 ml) was introduced into the vessel and hydrogenation was continued for 4 h. Sodium bicarbonate (9 mg) was then added and the mixture was filtered over Celite washing the pad well with water. The solution was concentrated to ca. 10 ml and was then washed with ethyl acetate ~30 x 3 ml) before again concentrating in vacuo to ca. 5 ml . The aqueous solution was chromatographed on a column of Biogel P2 (10 x 2.5 cm) and fractions containing the desired salt (e 36) were identified by u.v. These were combined and the solvent evaporated in vacuo, azeotroping out water withethanol 9 and then ethanol with toluene, to afford the title compound (e 36) as an off-white solid (15 mg);
~max.(H2O) 323 nm; ~max.(KBr) 1770, 1735, 1695 and 1575 cm .7~3~7~

Example 26 p-Nitrobenzyl (5R,65)-6-[(S)-l-acetox~ethvl]-3-ethylthio-7-oxo-1-azabicyclo~3.2.0]hep~-2-ene-2-carbo~ylate.
___ _~

CH

(e21) ~ (e22) ~ CH~SEt (e3~) Step A
A solution of the derivative ~e 21~ (50 m~) in a mixture of dioxan (1 ml) and water (0~2 ml) was stirred with N-bromoacetamide (14 mg) for 15 min. at room t~mpexature. CHC13 ~20 ml) was added and the organic solution was washed with water, then dried (MgSO~) and c~ncentrated in vacuo to afford the thiol (e 22).

St~e_~
The product from step A was dissolved in DMF (0. 5ml).
and the solution was treated with ethyl iodide (0.2 ml) and K2CO3 (15 mg) in the manner described in Example 1, step B. Work-up as therein described followed by chromatography on silica using 30% petrol in ethyl aoetate to elute afforded the title-derivative (e 37) as an oil (17 rll~); vmax. (CHC13) 1785, 1735 and 1700 cm 1; ~max.
(EtOH) 320 and 267 nm.; ~(CDC13) 1.32 (3H, t, J 7.5 Hz, CH3CH2), 1.41 (3H, d, J 6.5 Hz, CH3CH), 2.08 (3H, s, CH3CO), 2.84 (2H, q, J 7.5 Hz, CH2S), 3.15 (2H, centre of ABX, 4-CH2), 3.42 (lH, m, 6-CH), 4.09 (lH, m, 5-CH), ca.
5.25 (lH, m, CH3CH), 5.23 and 5.52 (each lH, d, J 14 Hz, CH2Ar), 7.66 and 8.22 (each 2H, d, J 9 Hz, C6H~-NO2).
~M , 434.1136. C20H22N2O7S requires 434.1145].

7~7~3 Example 27 p-Nitrobenzyl_(5R,6S)~3-(2-ethoxycarbonyletheny~hio)~
6=[(S)-l-hydroxyethyl]=7-oxo-1-azabicyclo[3.2.0}hept-2-ene-2-carboxylate. 3 H H

(el) ~ (e2) ~ HO ~ ~ ~ O2Et (e38) C~ C02E' N

(e39) 2 Step A
The ~ster (e 1) (250 mg) was converted into the thiol (e 2) by the method described in Example 17, step A.

Step B
The thiol (e 2) was treated with ethyl propiolate ~110 mg) and anhydrous potassium carbonate (75 mg) in the manner described in Example 16, step B. Work-up and chromatography as described therein af~orded two products.

The first product (20 mg) consisted mainly of the E-isomer of the title compound ~e 38); ~max. (CHC13) 1780 and 1700 cm 1; ~max. (EtO~) 335 and 267 nm.;
[M+~ 462-1132i C21H22N28S requires 462-1097]-The second, more polar component (48 mg) was the Z-isomer of the title compound (e 39), and was obtained as a crystalline solid; vmax (KBr) 1775 and 1700 cm 1;
~max. (EtOH) 336 (21,600) and 2.64 (13,100) nm;
~ (DMF-d7) 1.26 (3H, t, J 7 Hz, CH3CH2) overlapping with - 63 - ~7~7~

1.29 (3H, d, J 7 Hz, CH3CH), ea. 3.25~3.80 (3H, m, 4-CH2 and 6-CH~, 4-18 (2H, q, J 7 Hzr OCH2CH3), ca- 4.05-4.50 t2H, m, 5-CH and CHCH3), 5.24 (lH, d, J 4.5 Hz, OH), 5.38 and 5.62 (each lH, d, J 14 Hz, CH2Ar), 6.07 and 7.84 (each 1~, d, J 10 Hz, CH-C~), 7.87 and 8.28 ~each 2H~ d~
J 9 Hz, C6H4-NO2); [M~, 462-1089, C21H22N2O8S requires 462.1093].

Example 28 p-Nitrobenz~1_(5R,6R)-3-ethyl~hio-6 [(S)~ droxyet`hyl]
-7-oxo-1-azahic~clo [3q2~0] hept-2-en- 2 _~r~g~ylgb~.

(e28)~1 ~ ~ ~ SEt (e40) (e~l) Step A
The ester (e 28) (500 m~) was treated with N~bromoacet~
amide (154 mg) in a mixture of dioxan (10 ml) and water (1.5 ml) uslng the methodology of Example 17, step A.
The product contained the thiol (e 40).

Step B
A solution of the thiol (e 40) in DMF (7 ml) was stirred with ethyl iodide (0.5 ml) and anhydrous potassium carbonate (154 mg) for 25 min. a~ room tempexature.
Ethyl acetate ~50 ml) was added and the solution was washed with water (50 ml) and brine (3 x 30 ml). The organic layer was dried (M~So4) and evaporated in ~acuo to yield a crude product which was chromatographed on silica using 20% petrol in ethyl acetate ~o elute. The major product was the title compound (e 41), isolated as a foam (185 mg) which afforded crystals from ethyl acetate -petrol-ether m.p.l49-151; ~max (EtO}I) 319 (12,400) and 265 nm (10,800); ~max. (CHC13) 1780 and 1700 cm 1;
~ (CDC13) 1.32 (3H, t, J Hz, CH3CH2), 1.39 (3H, d, J 6 Hz, CH3CH), ca. 2.0 (lH, br, OH), 2.88 (2H, q, J 7 Hz, CH2S), 3.08 (lH, dd~ J 18 and 9.5 Hz, 4-CHa), 3.53 (lH, dd, J 18 and 9 Hz, 4-CHb), 3.56 (lH, dd, J 5.5 and 8.5 Hz, 6-CH), 4.0-4.35 (2H, m, 5-CH and CH3C_), 5.20 and 5.49 (each lH, d, J 14 Hz, C_2Ar), 7.63 and 8.19 (each 2H, d, J 8.5 Hz, C6H4-N02 ) .

~ :~7~7~
_ 6s -Ex mple 29 Sodium ( SR, 6R)-3-ethylthio-6-[(S)-l-h~ydrox~ethyl]-7-oxo-l-azabicv~Q [3.2.0]hept- 2 - ene - ? -carboxylate.

(e41) ______~ C ~ ~ ~ SEt O/ N CO -N +
(e42) Using the method of Example 6, the ester (e 41) (95 mg) was hydro~enolysed to afford the sodium salt (e 42) as a white solid after chromatography on Biogel P2; ~max.
tH20) 300 nm; vmax.(KBr) 174S and 1590 nm.

- 66 - ~ ~ 7~ r~

Example 30 Sodium salt of p-n _ obenæyl (5R,6 -3-[(Z)-ethoxycarbonylethenylthio]-6-[(S)-l- h~dr~x ~ x -7-oxo-1-azabicyclo~3.2.0]hept-2-ene-2-carboxylate.

(el2) ~ (el3) --~ H-_~5 Na O3S
~---- N

(e43!

~
The monoquaternaryammonium salt tl.5~9) was converted into the thiol (e 13) by the method desc ribed in Example 21, step A.

Stey B
The thiol (e 13) was treated with ethyl propiolate 1350 mg) and potassium carbonate (247 mg) in an analogous manner to that described in Example 21, step B.
Chromatography of the product as therein described afforded a oam ~790 mg); vmax. (CE~C13) 1780 and 1705 cm Step C
The product from step B was dissolved in acetone (2.5 ml) and a solution of sodium iodide (131 mg) in acetone (1 ml) was added. The resulting white precipitate was filtered and washed with acetone and ether to afford the title compound (e 43) (400 mg); ~max.(H2O) 333 (20,300) and 270 (12,800 nm.) vmax.(XBr) 1775 and 1700 cm ; ~(DMF-d7) 1.25 t3H, t, J 7 Hz, CH3CH~, 1.57 (3H, d, J 6.5 Hz, CH3CH), ca. 3.10-4.0 (2H, m, 4-CH2), 3.79 (lH, dd, J 5.5 and 11.5 Hz, 6-CH), 4.18 (2H, q, J 7 Hz, CH3CH2O), ca. 4.2-4.7 (2H, m, 5-CH and CHCH3), 5.38 and 5.59 (each lH, d, J 14 Hz, CH2CO), 6.11 (lH, d, J 10 ~7 ~ 67 --Hz, CH=CH S), 7.73 (lH, d, J 10 Hz, CH=CII.C02), 7.86 and 8 ~ 29 (each 2H, d, J 9 H~, C6H4NO2 ) . The product contained ca. 10% of the corresponding E-isomer as seen from the signal at ~ 6. 26 (lH, d, J 15 Hz, SCH=CH) .

xample 31 Disodium salt of (5Rt6R)-3~t (Z)-? ethoxycarbonyl-ethenylthio]-6-[(S)-l-hydroxysulphonylGxyethyl]-7 oxo-l-azabicyclo[3.2.0Jhept-2-ene-2-carboxylic acid.
CH H H

Na~ O3 ~ ~ ~
CO -N +
(e4~) The ester (e 43) (348 mg) was hydrogenolysed as described in Example 22. After chromato~raphy on Biogel P2 (20 x 3.5 cm) the aqu~ous solution containing the product was freeze~dried ~o afford the ti~le disodium salt (e 44) (130 mg); vmax. (KBr) 1750, 1685, 1600 and 10 1570 cm 1; ~max. (H2O) 324 nm.; ~(D2O) 1.29 (3H, t, J 7 Hz, CH3CH2), 1.52 (3H, d, J 6 Hz, CH3CH), 3.22 ~lH~ dd, J
10 and 17 Hz, 4-CHa), 3.53 (lH, dd, J 9 and 17 ~z, 4 CHb), 3.89 (lH, dd, J 5.5 and 9 HZr 6-C~I), 4.20 (2H, q, J 7 Hz, OCH2CH3), ca. 4.40 (lH, m, 5-CH), ca. 4.8 (lH, m, CH3CH), 15 5.99 (lH, d, J 10 Hz, CH=CH.S). and 7.59 (lH, d, J 10 Hz, CH=CH.CO2) ~7~
_ 69 -Example 32 Sodium salt of p-nitrobenzyl ~5R,6R)~3-ethylthio-6-~(S)-l hydroxysulphonyloxyethyl~-7-oxo--1-azabicyclo [3.2.0]he~t-2-ene-2-carboxylate.

H ~
(el2)__~ (el3)~ el8)- ~ - ~ _ ~ ~ Et Na O35O ~_ N ~

(el9) S ~
-The derivative (e 12) (3.5 g) ~as converted into the thiol (e 13~ by the method o Example 21, step A.

Ste~ ~
The thiol (e 13) was trea~ed with ethyl iodide in the manner as described in Example 8, step B to afford the ethylthio-derivative (e 18) (1.48 g).

Ste~ C
A solution of the quaternaryammonium compound (e 18) in acetone ~15 ml) was mixed with a solution of sodium iodide (268 m~ in acetone ~5 ml). A white solid crystallised out of the solution and this was filtered and washed with acetone and ether. The produc~ (500 mg) consisted of the title compound (e 19); ~max. (H2O) 317 (12,470) and 273 nm (10,750); ~max. (KBr) 1770 and 1695 cm 1; ~(DMF-d7) 1.27 (3H, t, J 7.5 Hz, CH3CH2), 1.46 (3H, d, J 6 Hz, CH3CH), 2.94 (2H, q, J 7.5 Hz, CH2 CH3), 3.17 (lH, dd, J 19 and 9.5 Hz, 4-CHa), 3.71 (lH, dd, J
5.5 and 11 Hz, 6-CH), 3.95 (lH, dd, J 19 and 8.5 Hz, 4-CHb), ca. 4.3 (lH, m, 5-CH), ca. 4.55 (lH, m, CH3CH), 5.31 and 5.55 (each lH, d, J 13.5, CH2Ar) and 7.80 and 8~27 (each 2H, d, J 8~5 Hz, C6H4-NO2).

~ ~ ~7~
_ 70 -Example 33 Disodium salt o~ (5R!6R)-3-ethylthio-6-t(S)~
hydroxysulphonyl-oxy~thyl]-7-oxo-1 aza icyclo[3.2.0]hept-2-ene-2-car oxylic acid.
_ (e 19)~ e 20 The ester (el9~ (1.12 g) was hydrogen.olysed with 5 Pd-C (l.S g~ in 30% aqueous dioxan (100 ml~ using the procedure of Example 14. After work-up (190 mg NaHCO3), as therein described, and chromatography on a column of Diaion HP20*(3.5 x lS cm), eluting with water, fractions containillg the product were combined and reeze-dri~d.
The resulting solid (460 mg) consisted o~ the ~itle disodium salt (e 20); ~max.(H2O) 300 nm (7900); vmax.
(XBr) 1750 and lS95 cm 1; [a]D (c.l, H2O) ~32; ~(D2O) 1.20 (3H, t, J 7 Hz, CH3CH2), 1.45 ~3H, d, J 6.5 Hz, CH3CH), 2.80 (2H, q, J 7 Hz,, CH2CH3), 3.04 (lH, dd, J 10 and 18 Hz, 4-CHa), 3.35 (lH, dd, J 9 and 18 Hz, 4 CHb), 3.78 (lH, dd, J 9 and 5 Hzl 6-CH), ca~ 4~2 (lE~, m, 5-CH), and ca. 4.75 (lH, m, CH3CH) .

* TRADE MARK

7~ 7~ 713 Example 34 p-Nltrobenzyl (5R,6S)-3-(2-p-nitrobenzyloxycarbonyl~
aminoethylthio)-6-~!S)-l-hydroxyethyl] 7-oxo l-azabicyclo ~3.2.0]hept-2-ene-2-carboxylate.

~el) ~ H ~ ~v~NHco2pNB
N

~e45) S ~.
Following the procedure of Example 17, step A, the ester (e 1) (200 mg) was converted to the thiol (e 2).

St~p ~
The thiol (e 2) was treated with 2-p-nitrobenzyloxy-carbonylamin oethyl bromide (135 mg) and potassium carbonate (62 mg) in DMF (3 ml) in a similar way to that described in Example 1, step B. Work-up and ~uxmatography on silica using ethyl acetate to elute afford~d the title-derivative (e 4S) (31 mg); ~max. (EtOH) 319 and 267 nm;
vmax~ (~Br) 1775 and 1700 cm 1; ~(DMF-d7) 1.28 (3H, d, J 6 Hz, CH3CH), ca. 3.0-3.6 (7H, m, SC1~2C~2N, 4-CH2 and 6-CH), ca. 4.15 (2H, m, 5-CH and CH3CH), 5.15 (lH, d, OH), _.
5.24 (2H, s, CH2Ar), 5.32 and 5.57 (each lH, d, J 14 Hz, CH2Ar), ca. 7.6-7.9 (5H, aromatic protons and NH) and 8.24 (4H, d, J 9 Hz, aromatic protons).

- 7~ 7~

Example 35 (5R,6S?-3-(2-Aminoethylthio)-6-[(S)-l hydroxyethyl]
_-oxo-l-azabicyclo~3.2.0]hept-2-ene-2-~arboxylic acid.
C~l (e45) ~ H.. ~ '~ ~ ~ S ~'~*H

~ --C02~
(e46) A mixture of the ester (e 45) (30 mg), 10% Pd on C
5 (50 mg), dioxan (4 ml), ethanol (0~3 ml), water (1 ml) and 0.05 M pH 7 phospha~e buf~er (1.3 ml) was shaken under a hydrogen atmosphere for 2 hours. The mixture was then filtered over Celite, washing with water (15 ml), and concentrated in vacuo to a volume of ca. 10 ml. The aqueous solution was washed with ethyl a~etate ~x 3), and then concentrated in vacuo and chromatographed on a column of XAD-2 (8 x 1.2 cm), eluting with water.
Fractions containing the title zwitterion (e 46) were identified by the u.v. absorption at 297 nm.

~:~7~7~

Example 36 p-Nit obenzyl (5R,6R)-3-(2-~ obenzyloxycarbonyl-aminoethylthio)-6-~(S)-l-hydroxyet~yl~w7-oxo-l-azabicyclo [3.2.0]hept-2-ene-2-carboxylate.

H ~ ~ ~ S ~ ~ CO PNB
(e283~~~~-~ J N ~ 2 o C02P~B
(e~7, Using the procedure of Example 34 the ester (e Z8) (500 mg) was conver~ed into the title derivati~e (e 47), which could be cr~stallised from ethyl acetate-ether;
vmax. (KBr) 1775 and 1740~1700 (br) cm 1; ~max. (EtOH) 315 (11,830) and 264 (18,740) nm.; ~(DMF-d7) 1.31 (3H, d, J 6.5 Hz, CH3CH), ca. 3.5-3.75 (7H, m, 4-CH2, SCH~CH2N
and 6-CH), 3.95-4.45 ~H, m, 5-CH and CHC~3), 5.08 (lH, d, J 5 Hz, OH), 5.27 (2H, s, ArCH2), S.35 and 5.58 ~each lH, d, J 14 Hz, CH2Ar), ca. 7.6-7.9 (5~1, m, aroma~ic protons and NH) and 8.28 (4H t d, J 9 Hz, aromatic protonsj.

Example 37 (5R,6R)-3-(2-Aminoethylthio)-6-~(S)-l-h~d~.~v~ ~ -7-oxo-l-azabicyc-10[3..2.0]hept-2-e~
c~3 (e47) ~ H~ ~ I ~ ~~'\ NH

O ~C02H
(e48~
Using the process described in Example 35, the diester (e 47) (135 mg) was converted into the title compound (e 48) ~11 mg); Amax. (H20) :296 nm.

_ 75 _ ~7~2~

Example 38 Sodium salt of p-nitrobenzyl (5R,6R)-3-_(2-~-n_tro-benzyloxycarbonylaminoethylthio?-6-[(S)-l-hydrox~sulphonyl~
ox~ethyl3-7-oxo-1-azabicyclo[3.2.03hept -ene-2~ar~1ate.

Na 03S ~ H ~_,~ NHC02PNB

(e49) Steps A and B
Using processes analogous to those of Example 32 the ester ~e 12) (1.25 g) was converted inko the thiol~/ 13) which was then alkylated with 2-p-nitrobenzyloxycarbonyl-aminoethyl bromide (300 mg).

Ste~ C
The product was treated with sodium iodide (50 mg) in acetone ~10 ml) and the resulting colloidal solution was chromatographed on silica gel using a gradient elution of CHC13 to 35% ethanol in chloroform. Fractions containing the product (e 49) were ~ombined and concentrated in vacuo to afford the title compound as a solid ~64 mg~;
~max. (H~O) 310 sh and 274 nm; vmax. (KBr) 1775 and 1700 cm _ 76 - ~7B~7~

F.xample 39 _ .
Mono-sodium salt of (5R,6R)-3-(2-aminoethylthio)-6-[(S)-l-hydroxysulphonyloxyethyl3--7-oxo--l-ar~abicyclo [3.2 03hept-2-ene-2-carboxylate.

H \ H H
~- ~ H03S~ ~ ~ ~ / ~N~
N
C2 Na (e50~
S The method of Example 35 was used to hydrogenolyse the derivative (e 49) (60 mg) to afford an ac~ueous solution of the title compound (e 50); ~max. (H20) 296 nm.

~ 77 ~ ~.17~7~

Example 40 p-Nitrobenzyl (5~,6S)-3-(2-hydroxyethylthio~-6-[(S)-l-hydroxyethyl]-7-oxo-1-aæablcyclo~3.2.03hept-2-ene-2-carboxylate.

(el).~ ~ S

(e51) S Employing the processes of Example 28, the ester (e 1) (300 mg) was converted into the derivative (e 51) by the use of 2-iodoethanol as the alkylating agent in step B. The title compound (e 51) was obtained as a white solid (67 mg); ~max. (EtOH) 320 and 268 nm.;
vmax. (KBr) 1765 and 1700 cm 1; ~(DMF-d7) 1.29 (3H, d, J 6.5 Hz, CH3CH), 3.06 (2H, t, J 7 Hz, SCH2), ca. 3.35 (2H, m, centre of ABX, 4-CH2), ca. 3.50 (lH, m, 6-CH), ca. 3.70 (2H, m; OCH2), 3.95-4.40 ~2H, m, 5-CH and CH3CH), ca. 5.10 (2H, br, 2 x OH), 5.31 and 5.58 (each lH, d, J
13.5 Hz, CH2Ar) and 7.83 and 8. 27 (each 2H, d, J 9 Hæ, ArCH2)- [M+, 408.1000. C18H20N2O7S requires 408.0989].

Example 41 Sodium (5R,6S) 3-(2-hydroxyethy~hio)-6 [(S)-l--hydroxyethyl]-7-oxo-1-a~abicxclo[3.2._Jhept-2-ene-2-carboxylate.

H. ~ ~ ~ " S ~ OH

(e51) ~ ~ CO Na+
(e52) The ester (e 51) ~60 mg) was hydrogenolysed in the manner of Example 6 to afford the title salt (e 52) ~18 mg); ~max. (H2O) 302 nm.

~L~7~7~3 Ex~ple 42 ~ -Nitrobenzyl ~5R,6R)-3-(2~hydroxy__hylthio)-6-[ (S) -1-hydroxye~hyl ~-7-oxo-1-a:zabicyclo~;~ 2~0.. Jhept~2=
ene~2-carbo~cylate .

H ~ ~", S~,~ OH
(e28) ~ HO

~eS3) The methodology of Example 28 was used to convert the ester (e 28) (500 mg) into the derivative (e 53), via the thiol (e 40), employin~ 2-iodoethanol a6 the alkylating agent in steo B. The product (e 53) was obtained as a foam (138 mg) which could be crystallised from ethyl acetate-ether; ~max. (EtOH) 317 (11,500) and 266 (10,300) nm; vmax. (KBr) 1780, 1765 and 1690 cm 1;
~(DMF~d7) 1.29 (3H, d, J 6 Hz, CH3CH), 3.07 (2H, t, J 6 Hz, CH2S), 3.2-3.85 (5H, m, 4-CH2, 6-CH and OCH2), ca 3.95-4.45 (2H, m, S-CH and CHCH3), 5.05 (2H, br, 2 x OH), }S 5.28 and 5.54 (each lH, d, J 13 Hz, CH2Ar) and 7.80 and 8.26 (each 2H, d, J 8.5 HZ, ArCH2). [M+, 408.1001, C18H20N2O7S requires 408.0991].

- ~o --7~

Examp1e._43 Sodium (5R,6R)-3-(2-hydroxyethylthio)-6~[(S)-l . ~
hydroxyathyl]-7-oxo-1-azabicyc1o~3.2.0]he~t-2-ene-2 carboxylate.

(e53) > ~ `~/ ~ OH

(e54) Elydrogenolysis of the ester (e 53) (70 mg) in the manner described in EXample 6 af~orded the title sodium salt (e 54) (17 mg); ~max. (H2O) 298 nm; vmax. (KBr) 1750 and 1590 cm 1.

.~ ~7~7~

Example 44 Methyl (5R,6S) 3-p-bromophen~ylthio-6-[(S)-l-hydroxyethyl}-7 oxo-1-azabicyclo[3. 2.0]hept-2-ene-2-carboxylate.
-(e55) (e56) The derivative (e 55) (150 mg) was treated with a) N~bromoacetamide and b) p-bromophenacyl bromide using the method of Example 28 to afford the title compound (e 57) (42 mg); ~max. (EtOH) 315 and 260 nm.;
vmax. (CHC13) 1785, 1735 and 1700 cm 1; ~(CL ~13) 1.37 (3H, d, J 6.5 Hz, CH3CH), 2.05 (3H, s, CH3CO), 3.02 (lH, d, J 9 and 17 Hz, 4-CHa), 3.34 (lH! dd, J 9.5 and 17 Hz, 4-CHb), 3.37 (lH, m, 6-CH), 3O77 (3H, s, OCH3), 4.08 (2H, s, C~2S), ca. 4.05 (lH, m, 5-CH), 5.22 (lH, m, CH3CH), 7~59 and 7.79 (each 2H, d, J 8 Hz, C6_4-Br).
[M+, 481.0203. C20H20NO6SBr requires 481.0197].

A little of the product was recrystallised from ethyl acetate-petrol m.p. 160-162.

~L~'7 Example 45 p-Nitrobenzyl (5R,6S)~6-~(S)~l-acetoxyethyl]-3-propar~ylthio-7-oxo-1-azabicyclo E 3.2,0Jhept-2-carboxylate.

CH3 ~ SCH2-CH

(e21) ~ CH3C~O ¦ l (eS7) Employin~ the methodology of Example 28, the ester (e 21) t500 mg) was converted into the derivative (e 57) by the use fpropargyl bromide in step B. The title compound (e 57) was obtained as an oil (235 mg~;
vmax. (CHC13) 1780, 1730 and 1700 sh; ~(CDC13) 1.41 (3H, d, J 6.5 Hz, CH3CH), 2.G8 t3H, s, CH3CO), ~.33 (lH, t, J 2 Hz, ~ CH), 3.05-3065 (5H, m, CH2C _ , 6-CH
and 4-CH2), 4.12 (lH, dtj J ca. 3 and 9 Hz, 5-CH3, ca.
5.25 (lH, m, CH3CH), 5.22 and 5.50 (each lH, d, J 14 Hz, CH2Ar), 7.63 and 8.21 teach 2H, d, J 9 Hz, CH~Ar) [M+, 444-0989- C21H20N27S requires 444.0979].

7~

Example 46 Sodium salt of p-nitrobenzyl--(5R~6R)-3-pr~parqylthi -6-[(S)-l-hydxoxysulphonyloxyethyl]-7-oxo-1-azabicyclo [3.2.0]hept~2-ene-2-carboxylate.

CH ~ ~ SCH2=CH

(el2) ~-----~ O ~ ~ CO2PNB

(e58~
By an anologous process to that described in Example 32, the mono-quaternaryammonium salt (e 12) (2.8 g) was transformed into the title rnono sodlum salt (e 58) which was obtained a~ a whi~e solid (515 mg); Amax. (H2O) 310 (12,991) and 270 (11,257) nm.; ~max. (KBr) 1770 and 1700 ~m 1. ~(DME~-d7) 1.37 (3H, d, J 6 Hz, CH3CH), ca.
2.95-~.40 (2H, m, 4-CHa and = CH), ca. 3.60-3.95 (4H, m, 4-CHb, 6-CH and CH2C-), 4.15-4.60 (2H, m, 5-CH and CHCH3), 5.27 and 5.46 (each lH, d, J 13 Hz, CH2Ar), 7.69 and 8.23 (each 2H, d, J 9 Hz, ArCH2); ~]D (c.l, H2O) -16 ; [Found: C, 44.7; H, 3.6; N, 5.2~
ClgH17N2OgS2Na requires C, 45.2; H, 3.4; N, 5-5%]-Example 47 Disodium salt of (5R,6R)-3-allylthlo-6-[(S)-l-hydroxysulphonyloxy eth~l_ 7-oxo-1-azab~yc ~ 3.? O ]hçpt-2-ene-2-carboxylic acid and disodium salt of (5R,6R)-3-~ropargylthio-6-~(S)-l-hydroxysulphonyloxyethyl]-7-oxo-1-azabicyclo~3_2.0]hept-2-ene~2-carboxylic acid.
__ ( e 5 8 ,~ ~ ~ SCH2CH=CH2 Na 03S
N ~ C0 -N
(e5g) ~ ~ SC~2C~CH
Na~-03SO I i R
N ~~ C0 -N
(e60) The ester (e 58) (400 mg) was hydrogenated over 5~
Pd-C (500 mg) in the manner described in Example 6. The product, purified by chromatography on Biogel P2, consisted of a mixture of thP title compounds (e 59~ and (e 60) (130 mg) (ca. 4:1 by h.p.l.c. analysis)i ~max. (H20) 302 nm; vmax. (KBr) 1755 and 1595 cm 1.

- 85 ~ 7~7~

E~ple 48 p-Nitrobenzyl (5R,6R)-3-allylthlo-6-[(S)-1 hydroxyethyl]-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate.
CH
H . ~ / SCH2CH=CH2 (e2~3~ - HO
N ~ CO2PNB
(e61) Using the pEocess employed in Example 28 wlth allyl bromide as ~he alkylating agent in step B~ the ester (e 28) (300 mg) was converted ko the title derivative (e 61) (115 mg) which was obtained as a crystalline solid;
m.p. 137-139, [a]D (CØ5, CHC13) +24, Amax. (EtOH) 317 ~12,772) and 266 (11,069) nm.; vmax. (CH2C12) 3590, 1780 and 1705 cm 1; ~(CDC13) 1.39 (3H, d, J 6 Hz, CH3CH), 1.85 (lH, d, J 5.5 Hz, OH), 3.10 (lH, dd, J 9.5 and 19 Hz, 4-CHa), ca. 3.38-3.60 (4H, m, CH2S, 4CHb and 6-CH) 4.0-4.45 (2H, m, 5-CH and CHCH3), 5.10-5.60 (4H, m, ArCH2 and = CH2~, 5.60~6.10 (lH, m, CH=), 7.63 and 8.19 (each 2H, d, J 9 Hz,C6H4 NO2). ~Found: C, 56.30; H, 4.99;
N, 6.75~; M+, 404.1050. ClgH20N2O6S requires C, 56.42;
H, 4.98; N, 6.93~; L!+, 404.1042].

7l~7~

Example 49 Sodium (5R,6R)-3-propylthio-6~ l-hydroxye_hyl]-7-oxo-1-azabic~yclo[3.2.03hept-2-ene--2-carboxylate, and sodium (5R,6R)-3-al}ylthio-6 [(S)-l~hydroxyethyl~-7-oxo-1-azabicyclo{3.2.03hept=2-ene-2-carboxylate.
(e6~ o ~ CH2CH2CH3 H ~--r~ SCH2CH=CH2 N 02~Na~ ~ - N CO~ Na (e62) (e~3) Hydrog~nolysis of the ester (e 61) (80 mg) as described in Example 6 afforded a mixture (ca. ~:1 by h.p.l.c.) of the title derivatives (e 62) and (e 63) ~max. (H20) 300 nm.

Example 50 Sodium salt of~ trobenzyl (5R,6R)~3-allylthio-6-[(S?-l-hydroxysulphollyloxyethyl]-7-oxo-1-azabicyclo ~3.2.0~he~t-2-ene-2-carboxylate.
-H`~ H H
(el~3 ~ >Na~ O3S ~ ~

(e6~) Conversion of compound (e 12) (2.8 g) to the derivative (e 64) was achieved by th~ process of Example 32, using allyl bromide as the alkylating agent in step B. The title compound (e 64) was obtained as a white solid (408 mg~; ~max. (EtOH) 317 (11,083) and 266 (9758) nm. ~max. (KBr) 1775, 1695 and 1260-1220 cm l; ~(DMF-d7) 1.47 (3H, d, J 6.5 Hz, CH3CH), 3.22 (lH, dd, J 18 and 10 Hz, 4-CHa), 3.63 (2H, d, J 5.5 Hz, SCH2), ca. 3.5-4.1 (2H, m, 6-CH and 4-CHb), ca. 4.2-4.8 (2H~ m, 5-CH and CHCH3), 5.10-5.62 (4H, m, C_2Ar and = CH2), 5.75-6.20 (lH, m, HC=), 7.80 and 8.27 (each 2H, d, J 9 Hz, C6H~-NO2) ;:3 L ~ L ~

Example 51 Disodium salt of (5R.6R)-3~E~ropylthio-6-[~S)-l-.. .. _ .. _ hydroxysulphonyloxveth ~ -7-oxo-1-a~abicYclo~3.?.~. ~eP~-2-ene-2-carboxylic_acid _nd disodium salt of (5R,6R) 3-. ~
allylthio-6-E(~)-l-hydroxy~ulphonyloxyethyl3-7-oxo-1-azabicyclo[3.2.03hept-2-ene-2-carboxylic acid.
~ H H
H / ~--"--~SCH2CH2CH3 (e64) ~ (eS9) ~ Na~~O3SO
- N J - CO ~Na~

(e65) Hydrogenolysis o the ester (e 64) ~300 mg~ by the method described in Example 6 afforded a mixture (ca. 3:1) o~ the title-compounds (e 65) and (e 59)~ An aqueous solution of the product was freeze-dried to give a solid (92 mg); ~max. (H2O) 302 nm.

- 89 - ~7~7~

Exam~le 52 p-Nitrobenzyl _(5R,6R)-3-(2-p-nitrobenzyloxycarbonyl-aminoethylthio?~6-[(S)-l-eth ~ 1]-7-oxo-1 azabicyclo _ [3.2.0 ~ t-2-ene.-2-carbxoylat .
~ CH~

EtS ~ -' ~ NHCOCH3 /~ N ~ ~

(e66) ~e67) H H
E ~ ~ ~ NHCO2PNB
/r N
O CO PNB
(e6,3) 2 The ethylthlo-derivative (e 66) (270 mg) was treated with N-bromoacetamide (77 mg) as described in Example 17 step A to afford the thiol (e 67). Alkylation of (e 67) with 2-p-nitrobenzyloxycarbonylaminoethyl bromide (338 mg) in the manner of Example 34, step B, afforded the title derivative (e 68) (27 mg); Amax. (EtOH) 319 nm.; vmax.
(CH2C12) 3450, 1780 and 1705 sh cm 1; ~(CDC13) 1.27 (3H, t, J 7 Hz, CHiCH2), 1.39 (3H, d, J 6.5 Hz, CH3CH), 2.61 (2H, q, J 7 Hz, CH2S), 2.90-3.60 (8~1, m, 4-CH2, SCH2CH2N, 6-CH and CHCH3), 4.16 (lH, m, 5-CH), 5.20 (2H, s, C_2Ar), ca. 5.25 ~lH, br, NH), S.23 and 5.52 (each 1~, d, J 14 Hz, ArCH2), 7.48 and 7.64 (each 2H, d, J 14 Hz, aromatic protons) and 8.20 (4H, d, J 14 Hz, aromatic protons).

-go~ ~7~

Example 53 p-Nitrobenzyl [5R,6R)-3-(2-p~nitrobenzyloxycarbonyl-aminoeth~lthio)-6-[(R)-l-ethylthioethyl]-7-oxo-1-azabic ~3.2,0]hept-2-en_-2-carbox~
SEt SEt 3 ----~ CH
N
o~ ~ C02PNB ~ 02PN~
(e69) (e7~) SEt \ H H ~
CH ~ ~ ~ ~ NHCO2PNB
O N `CO2P~3 te71 ) Compound (e 69) (600 mg) was con~erted, via the thiol (e 70), into the derivative (e 71) by use of the methods outlined in Example 52. The title compound (e 71) was obtained as a crystalline solid (131 mg); m.p.
205-206; ~max. (EtOH) 319 (13,125) and 266 (21,235);
vmax. (KBr) 1775 and 1725-1700 br cm 1; ~(DMF-d7) 1.22 (3H, t, J 7 Hz, CH3CH2), 1.41 ~3H, d, J 6.5 Hz, CH3CH), 2.64 (2H, q, J 7 Hz, CH2S), ca~ 3.0-3.6 ~8H, m, 4-CH2, 6-CH, CH3CH and SCH2CH2N), 4.17 (lH, m, 5~CH), 5.25 (2H, s, CH2Ar), 5.33 and 5.58 (each lH, d, CH2Ar), ca. 7.65 (lH, br, NH), 7.67 and 7.82 (each 2H, d, J 9 Hz, aromatic protons) and 8.27 (4H, d, J 9 Hz, aromatic protons).
[Found: C, j3~4; H, 4.8; N, 8.8~. C28H30N4OgS2 requires C, 53.3; H, 4.8; N, 8.9%].

~7~

Exam~le 54 (5R, 6R) - 3 ~2-Aminoethylthio)-6-~(R)-l-eth~lthioeth -7-oxo~ y~lo 3 2.0]hept-2-ene-2-carboxylic acid.
SEt (e71) - _ > ~ i NH2 C~'~ C02H
(e72) A mixture of the thioether ~e 71) (100 mg), 5~ Pd on S C (150 mg), 0.05M, pH 7 phosphate buf~er (5 ml) and dioxan (12 ml) was shaken in an atmosphere of hydrogen for 2 h. A further quantity (100 mg~ o~ 5~ Pd on C was added and hydrogenation was continued for a further 2.25 h. The mixture was filtered through Celite, washing the pad well with water (30 ml); and the filtrate was evaporated in vacuo to a volume of _ . 20 ml. The aqueous solution was washed with ethyl acetate (3 x 30 ml) and then further concentrated in vacuo to a volume of ca.
15 ml. This solution contained the title zwitterionic derivative (e 72); ~max. ~H2O) 298 nm.

~ ~'7 Example 55 P-Nitrobenzyl (5R,6R)-3-methy ~ o-ethyl)-7-oxo-l-azabicyclo[3.2.0]hept-2--ene~2-car~oxylate~

SEt Et ~S ~ COC ~ , SCH3 `CO2PNB /~ CO2PNB
(e66)~(e69) (e73) A mixture of the esters (e66) and (e 69) (200 mg) S wa~ convert~d into dexivative (e 733 using the methodology of Example 17 r with iodomethane as the alkylating agent in step B. The title compound (e 73) was obtained as a gum (50 mg); vmax. (CH2C12~ 1780 and 1705 cm 1; ~(C~C13) ca. 1.15-1.50 (3H, m, C_3CH2), 2.38 (3H, s, CH3S), 2.59 (2H, q, J 7 Hz, SCH2CH3), ca. 2.85-3.60 (4H, m, 4-CH2, 6-CH and CH3CH), 4.15 (lH, m, 5-CH), 5.22 and 5.52 (each lH, d, J 14 Hz, CH2Ar), 7.66 and 8.22 (each 2H, d, J 9 Hz, ArCH2) [M+, 422~.

_ 93 ~ 7~

Example 56 ~ -Nltrobenzyl (5R,6R)-3_[2-(pyrrol-1-yl)ethylthio]-6-[(S)-l-hydroxyet~_]-7-oxo-1-azabic~clo[3.2.0]hept-2-ene-2-carboxylate.

~ E ~

(e7~) In a reaction sequence similar to that of Example 28, the ester te 28) (500 my) was converted first to the thiol (e 40) which was alkylated with 1-(2-bromoethyl~pyrrole to afford the title compound (e 74). After purification on a silica column using 50% to 20% petrol-ethyl acetate to elute, compound (e 74) was obtained as a ~oam (78 mg);
~max. (EtOH) 317 and 267 nm.; vmaxO (CH2C12) 3580, 1780 and 170S cm l; ~(CDC13) 1.36 (3~, d, J 6~5 Hz, CH3CH), 1.91 ~lH7 br, OH), 2.53 (lH, dd, J 10 and 18 Hz, 4-CHa), 2.99 (lH, dd, J 9 and 18 Hz, 4-CHb), 3.12 (2H, t, J 6.5 E~z, CH2S), 3.48 (lH, dd, J 5.5 and g Hz, 6-CH), 4.10 (4H, m, CHCH3, 5-CH and CH2N), 5.19 and 5.48 (each lH, d, J 14 Hz, CH2Ar), 6.13 (2H, m, pyrrole B-CH), 6.67 (2H, pyrrole ~-CH), 7.63 and 8.20 (each 2H, d, J 9 Hz, ArCH2).

_ 9~} -~.78~'78 Example 57 (5R,6R) 6-[(S)-l-Hydrox~ethyl]-3-[2-(1 pyrrolyl) ethylthio]-7 oxo-l azabicyclo[3.2.0]hept-2 ene-2-carboxylic acid.

(e74) N ~.~ CO

(e75) The procedure of Example 20 was used to convert:
the ester (e 74) (70 mg) into the zwitterion (e 75) (13 mg).
The product was purified by passing through a short column of Biogel P2 (5 x 2 cm) and freeze drying the result:ing solution. The ti~le compound was obtained as a solid;
~max. (H2O) 300 nm.

_ ~5 -~ ~7~3~7~

Sodium salt of p-nitrobenzyl (5R,6R ~ rol-L-ylethyl)-6-[(S)-1 hydroxysulphon~loxyethyl]-7-oxo-1-azabicyclo[3.2.0~hept-2-ene-2 carboxylate.

C~
~3 H H /~
H ~ ~,_S ~N
(el2) - ~ +~
Na O35O
0~ C02PNB
(e~6) Steps A and B
The method of Example 32 was used to convert the quaternaryammonium salt (e 12) (2.8 g) into the thiol (e 13), which was then alkylatefl with 1-(2-br~th~l) pyrrole.

10 ~
To a solution of the product from step B in acetone (10 ml) a solution of sodium iodide (124 mg) in acetone (5 ml) was added. The resulting solution was cooled and the solid formed (quaternary-ammonium iodide) was removed by filtration. The filtrate was concentrated in vacuo and then chromatographed on silica using a gradient elution of CHC13 to 30~ EtOH-CHC13. The requisite fractions were combined and evaporated in vacuo to afford the title compound (e 76) (196 mg); ~max. (H2O) 316 and 273 nm; vmax. (KBr) 1765 and 1700 cm 1; ~(DMF-d7) 1.48 (3H, d, J 6.5 Hz, CH3CH), ca. 3.2 (lH, m, 4-CHa), 3.28 (2H, m, SCH2), ca. 3.7 (2H, m, 6-CH and 4-CHb), 4.22 (2H, m, CH2N), ca. 4.55 (2H, m, 5 CH and CHCH3), 5.31 and 5.55 (each lH, d, J 14 Hz, CH2Ar~ 6.00 (2H, m, pyrrole ~-CH), 6.91 (2H, m, pyrrole ~-CH), 7.80 and 8.27 (each 2H, d, _ 9 Hz, ArCH2).

_ 96 - ~7~

Example 59 Monosodium salt of (SR,6R)-3-(2-Pyrrol-l-ylethylthio)-6-~(S)-l-hydroxysulphonYloxyethyl~-7-oxo-1-azabicyclo [3.2.0]hept-2-ene-2-carboxylic acid.

H ~ S
(e76) ~ HO3SO I ~
N ~ CO2 Na (e77) The procedure of Example 20 was used to hydrogenolyse the ester (e 76) (150 mg). Chromatog~phy on Biogel P2 (10 x 2.5 cm) aorded the title compound ~e 77) (46 mg).
The solution was freeze-dried to give a solid; ~max.
(H2O) 300 nm.; vmax. (KBr) 1750 and 1600 br cm 1.

_ 97 ~ 7~

Example_60 D-Nitrobenzvl ~ ,6S)-3 13-(p-nitrobenz~l-oxycarbonylamino~rop~lthio~l-6-[~ 1-hydroxvethyl~ 7 oxo-l-azabicy_1O~3.2Ø3hept-2-ene-2 carboxylate CH CH

o~ ~N ~ C0zPNB H0 (el) (e23 CH
H- ~ ~ ~ ~ ~ C02PN3 te78) C02PNB
The ester (el, 500 mg, 1.119 mM) was dissolved in 1,4-clioxan (10 ml) containing water (30 drops). A
solution of M~bromoacetamide (155 mg, 1.119 mM) in 1,4-dioxan was then added and the solution stirred at room temperature for 4.5 minutes. Chloroform (50 ml) was added and the organic phase was washed with pH 7.0, 0.05 M phosphate buffer, saturated sodium chloride solution and dried over anhydrous magnesium sulphate.
~iltration and removal of the solvent at reduced pressure yielded the crude thiol (e2) as a gum, ~ max (CHC13) 1780, 1730 and 1700 cm 1.

The crude thiol was dissolved in dry dimethyl-formamide ~10 ml) and stirred at room temperature for 25 minutes with anhydrous potassium carbonate (155 mg, 1.119 mM) and 3-(N-p-nitrobenzyloxycarbonyl) amino-1-bromopropane (355 mg; 1.119 mM). Ethyl acetate was added and the organic solution washed with watert saturated sodium chloride solution and dried over anhydrous magnesium sulphate. After filtration the solvent was removed at reduced pressure to give a - 98 ~ 'J~

p~le yellow oil, which w~s chromatographed over silica gel (50 gm). Elution with 5% ethanol/chloroform afforded the title compound (e ~ ) (155 mg) as a white solid. This solid was digested in die-~hyl ether and collected by filtration (145 mg), m.p. 164-168 C, v m~x tKBr3 3420, 1765, 1700 1608 cm ; A max (EtOH) 320 nm (~m 13,400) and 266 nm ~m 20,050); ~H (d7-DM~) 1.28 (3H, d, J 6.6Hz, CH3CH), 1.86 (2H, m, CH2CH2CH2), 2-99 (2H, t, SCH2), 3.24 (t, CH2NH), 3-33 (d~ 4 C~z), 3-49 (lH, t, J6-8 4.3Hz, 6-CH), 4.05 (lH, m, CH3CH), 4~24 (lH, dt, J 4_5 9.0Hz, Js_6 2,9Hz, 5-CH) 9 5.12 (lH~ cl, JOH 8 4.6Hz, OH), 5.23-5.65 (4H, S~AB, 2 x CH2Ar)7 7.39 (lH, bro~d reson~nce, NH), 7.68 (2H, d, Ar protons) 9 7.82 (2H, d, Ar protons), 8.25 (4H, d, Ar protons~; In/e 286, lS3~ 136, no M ; (~und: C, 53.71; H9 4.60; N, C27H28N4010S requireS: C~ 53.99; H, 4 70; N 9 33%) _ 99 ~ 7~

Example 61 (~6~=3-L3-Aminopropvlthio)-6-~(s)-l-hvdroxyeth -7-oxo-1-azabicyclo[3.2Ø~hept-2-ene-2-carboxylic acid HO~ ~b HO ~ ~,S ~ NH2 ~ N- ~
'' C02PNB 0/ co2~l (e7~) (e79) A solution of the ester (e78)~ 130 mg) in 1,4-dioxan (15 ml), water (4.5 ml), ethanol (1.35 ml) and 0.05 M pH7.0 phosphate buffer (6 ml) was hydrogenated in the presence of 10% palladium on carbon (200 mg) for 2 hours. The suspension was then filtered over Celite, washing with water (20 ml). The filtrate was concentrated to about 20 ml and washed with ethyl acetate (3 x 25 ml). The aqueous solution was futher concentrated to about 10 ml and chromatographed on a column of XAD-2, eluting with water~ ~ractions containing the pr~duct ~ere combined to yield the title compound (e79) (14.5 mg) in aqueous solution, A max (H20) 299nm.

~7~ 7~

Example 62 p-Nitrobenzvl ~R16R~-3~3-(p-nitrob~y~
ox~carbonylan!ino~ropylthio¦-6-L(S~ hydroxyethyl J -7-oxo~l-azabicvclo~3.2Ø~hept-2-ene-2-carbo~ylate CH~ CH3 H- ~ I-r YOiNHCOCH3 ) ~ ~ ~ SH

O~Z~- N C02PNB ~ CO2PNB
(e28) (e~O) CH~

/~- N -O CO~PNB
te8o) The title compound (e80) was prepared as a white solid in 20% yield from the ester (e28), adopting the procedure described in example 60., m.p. 104-106 C (ethyl acetate/diethyl ether), Amax (EtOH) 317 nm (Em 12,360) 266 nm ~Em 18,890); v max ~KBr) 3410, 1770, 1700~ 1608 cm ~ (d7-DM~) 1.31 (3H, d, J 6Hz, CH3CH), 1.9 (2H, m, CH2 CH2CH2), 3-03 (2H, t, SCH2), 3.22 ~t, CH2NH), 3.35 (d, 4-CH2), 3.6 (m, lH, 6-CH), 3~9-~5 (2H, m, 8-CH + 5~CH), 5.06 (lH, d, J 6Hz, OH), 5.23 (2H, St CH2Ar), 5044 (2H, AB, CH2Ar), 7.4 (lH, broad resonance, NH), 7.6-8.4 (8H, 2xAB, 2xAr); m/e 555, 514, 286, 136, no M~

~8~7~

Example 63 ~5R,6R)~3-(3_Am.~nopropvlthio)-6-~S)-l-hyclroxyethvl¦

H / ~ f ~ HC02PNB ~ r ~ ~5~/~ NH2 N ~ ~ 02PNB 0 N -~~-~~
(e80) ~e81) The ester te80) was hydrogenolysed as in exarnple 61 to yielcl (5R,6R)-3-~3-aminopropylthio)-6-[~S)-l hydroxyethyl~-7-oxo 1-azabicycloC3.2~0~hept-2-ene-2-carboxylic acid (e81) in 26% yield as an aqueous solution (based on Em 8,500). A sample was freeze dried to give a pale yellow fluffy solid, A max (H20) 292 nm, v max tKBr) 3420, 1750, 1600 (broad) cm - 10~
7~

Example 64 p-Nitrob_ nzyl_~5R, 6S)-3- (p-n~tr.obenzYloxycarbonvl-methylthiol-6-[(S~ hydroxyethvl~-7-o _ l-azabicyclo ~302Ølhept-2-ene-2-carbo~late -\ S CH CO PNB

) - ~, iL I 1 i `C02PNB
o (e~2) The diester (e82) (150 mg) was p.repared as a pale yellow solid by reaction of the ester (el) (500 rng) with N~bromoacetarllide, ollowed by p-nitrobenzyl bromoacetate, utilising the procedure outlined :in example 60~, ~ max (EtOH) 309 nm, 265 nm (Em 15,355);
max tKBr) 3500, 176S, 1737, 1700, 1608, 1550, 1520 1348 cm 1; ~H (d7-DMF) 8.26 (d) + 7.79 tt~ 8H, aromatic protons), 5.40 ts ~ ~, 4H, CH2Ar protons); 5.20 (lH, d, OH~, 4.0 - 4.5 (s ~ m, 4H, CH2C02PNB ~ 5-CH + 8-CH)~
1.27 (3H, d, CH3CH); m/e 513, 286, 168, 136, 78 no M .

- 103 ~ 7~7~

Example 65 Disodium ~ c~ O~
hvdroxyethyl~-7-oxo-1-azabi ~ .Olhept-2=ene-2=
carbox~ e H ~ ~ 2 2Na (e82~
~ N ~C2Na (e83) 5% Palladium on carbon catalyst (75 mg) was shaken with hydrogen in 30% aqueous 1,4 dioxan (10 ml~
at ambient pressure and ~emperature for 0.5 hours. A
solutîon of the diester (e82) (50 mg) in 1,4-dio.Yan (10 ml) was added and the hydrogenation was continued for a further 3.5 hours. Sodium bicarbonate (15 mg) was added and the mix~ure was filtered through Celite, washing well with water (30 ml). The filtrate was concentrated at reduced pressure to approximately 20 ml and washed with ethyl acetate ~3 x 25 ml). The aqueous solution was further concentrated to about 10 ml and chromatographed on a column of Biogel P-2, eluting with water. Fractions containing the title compound (e83) were identified by the chromophore at A max (H20) 300 nm in the U.V. spectrum. These fractions were combined and freeze dried to afford a pale yellow solid (9 mg).

-- 10~ --7~

Example 66 p-Nitrobenzyl ~5RI6R~- -(p-nitrobenzyloxycarbonyl-methylthio)-6-L~ hydroxyethyl~-7-oxo-1-azabicyclo ~ ~ / S~H2C02PNB
(e28)~
~ N------~` C02PNB
O
(e84) The diester (e84) (179 mg) was prepared as a pale yellow solid by reaction of the thiol (e40), derived from the ester (e28) (SOO mg) with p-nitrobenzyl bromoacetate, by the method described in example 60., A max (EtOH~
5 311 nm (Em 11,120), 265 nm (Em 21,197); ~ max (KBr) 3490, 1760 (broad), 1700, 1608, 1555, 1520, 1348 cm J ~H
(d7-DMF) 8.26 (d) and 7.78 (t, 8H, aromatic protons?, 5.40 (4H, q + s, CH2Ar protons), 5.03 (lH, d, OH), 3.8 -4.5 (s ~ m, 4H, CH2C02PNB + 5-CH -~ 8-CH), 3.2 - 3.8 (m, 4 CH2 ~ 6-CH), 1-28 (3H, d, CH3CH); m/e 513, 471, 286, 2279 168, 136, no M+.

~ 7~3~

Example 67 Disodium t5R.6R~-3-[carboxvmethvlthio)-6-r(S~
hydroxyethyl~-7-oxo-1-azabicyclo[3.2.0 l e~
carboxylate CH
H ~ -~ ~ ~ 2 2 (e84) ~ HO ~ r ~
N `C2Na -(e85) The diester (e84) (75 mg) was hydrogenolysed in the mannex described in example 65. Biogel P-2 column chromatography~ eluting with water afforcled the title compound te85) (15 mg) in aqueous solution, A max (H20 300 nm. A sample was freeze dried to yield a yellow solid.

Z7~

Example p-Nitrobenzyl (5R,6S~-3- L 2- ~ thyl-4-nitroimidazol~
l-yl~ethylthio ~6-~(S)-l-hvdroxyethyl~-7-oxo-1-azabicyclo A ~ ~V~

(el~-- ) H ~ ~ 02PNB

~e86) The title compound (e86) (97 mg) was prepared as a pale yellow solid by reacting the thiol (e2), derived from ester (el) (500 mg) with 1-(~-bromoethyl)-2-methyl-4-nitroimidazole, adopting the procedure described in example 60., A max (EtOH) 308 nm (Em i544g), 270 nm ~Em 13539), ~ max (KBr) 34209 1770, 1700, 1605, 1540~
1520, 1345, 1333, 750, 738 cm 1, ~H (d7-DM~) 8-39 (lH~ S, imidazole-CH), 8.25 (2H, d~ aromatic protons), 7.81 (2H, d, aromatic protons), 5.44 (2H, q, CH2Ar), 5.14 (lH, d, OH), 4.43 (2H, t, CH2N), (dt~ 5-CH)~ 2-43 (3H~ s, imidazole-CH3). 1.27 (3H, d, CH3C~), m/e 330.0846(Cl~Hl4 N206 requires 330.0852) 7 286, 187, 128, 44, no M~.

Example 69 ethylthioJ-6-(S~ hydroxyethyl~-7-oxo-1-azabicyclo ~3.2Ø~hept-2-ene-2-carboxylic acid (e86 ~ ~ ~ `~ ~ ~N

(e87) Hydrogenolysis of the ester (e86) (70 mg) by the procedure described in example 61 gave an aqueous solution containing (5R,6S)-3-C2-(2~methyl-4-amino-imidazol-l-yl) ethylthio~-6-[(S)-l-hydroxyethyl~-7-oxo-1-azabicyclo [3.2Ø~hept-2-ene-2~carboxylic acid (87). A sample was freeze-dried to yield a white fluf~y solid, A max (H~0) 298 nm, ~ max (KBr) 3400, 1750, 1665, 1590, 1400 cm - 108 - 1~7~Z7~

Exa~ple 70 p-Nitrobenzyl (5R,6R)-3-~2-(2-methyl-4-nitroimidazol-l-yl)ethylthi~ -6-~(S~ hyd_~yethyl~-7-oxo-1-azabicvclo ~3 2Ø~hept-2-ene-2-carboxylate H \ ~ : ~ ~ ~ ~ N~

(e28)~ F l `C02PNB
(e8~) The title cornpound (e88) (85 mg) was prepared as a white solid by reacting the thiol (e40), derived from the ester (e28) (500 mg) with 1-(~-bromoethyl)-2-methyl-4-nitroimidazole, utilising the procedure described in example 60., A max (EtOH) 308 nm (Em 11,961), 275 nm, v max (CHBr3) 3400, 1775, 1698, 1605, 1563, 1540, 1515, 1320, 743 cm 1 9 ~H (d7-DMF) 8.39 (lH, S, imidazole CH~, 8.27 (2H, d~ aromatic protons), 7.81 (2H, S, aromati~
protons~, 5.45 (2H, q, CH2Ar protons), 4.45 (2H, t, CH2N), 2.44 (3H, S, imidazole CH3~, 1.30 (3H, d, CH3CH).

~7~

Example 71 (5R,6Rl-3-~?-(2-Methyl-4-amino-imidazol-1~
ethylthio]~6-[(S)-l-hydroxy~thyl~ 7-oxo-1-a~abicyclo [3.2Ø~h~pt-2-ene-2-carboxylic acid (e~8~
N~ C02H
(e89) Hydrogenolysis of the ester (e88) (60 mg) by the procedure described in example 61 gave an aqueous solution containing the title compound (e89), A max (H20) 297 nm, ~ max (KBr) 3400, 1750, 1665, 15959 1400 cm - 110- ~ 27~

xample 72 p-Nitrobenzyl (5R,6S~-3-[2-(2-rnethyl-5-nitroimidazol l-yl)ethylthio~-6-~S~-l-hydroxyethyl~-7-oxo-1-azabicyclo [3 ?-- ~hept-2-ene-2-carboxylate (el) ~ ~ H 9 ~ N02 (e90) p-Nitrobenzyl (5R,6S)-3-[2-methyl-5-nitroimiclazol-l-yl)ethylthio~-6-~(S)-l-hydroxyethyl~-7-oxo-1-azabicyclo [3.2Ø~hept-2-ene-2-carboxylate (e90) (40 mg) was '7 obtained as a white solid by the reaction of the thiol (e2) derived from the ester (el) (250 mg) with 1-(~-brornoeth~ 2-rnethyl-5-nitroimidazole, utilising the procudure described in example 60., A max (EtOH) 312 nm (Em 17925), 268 nm (Em 12885), ~ max (KBr) 3420, 1775, 1700, 1608, 1520, 1332, 1350, 1365, 742 cm ~ ~H (d7-DMF) 8.25 (2H, d, aromatic protons), 8.01 (lH, s, irnidazole CH), 7.80 (2H, d, arornatic protons), 5.44 (2H, q, CH2Ar), 5.19 (lH, d, OH), 4.68 (2H, t, CH2N), 4.2 (2H, dt + m, 5-CH + 8-CH), 2.52 (3H, s, imidazole CH3), 1.28 (3H, d, CH3CH).

8~

Example 73 (5R,6S2-3-[2-(2-Methyl-5-amino-imidazol-1-yl) eth,ylthiol-6-C(S)-l-hvdro~ethyl~-7-oxo 1-azabicyclo L3 . ? . 0. ~hept-2-ene-2-carboxylic_acid ~ H3 c~3 H H S~
H0 ~ NH2 (e90 ~ ~ C02H
~ e9 1 ) The title compound (e91 ) was obtained in aqueous solution by hydrogenolysis o:f the ester (e90) (30 mg).
The procedure outlined in example 61 was followed with the exception that hydrogenolysis time was extended ~o 5 3 hours. A sample was freeze dried to yield a white fluffy solid~ A max (H20) 297 nm, v max (KBr) 3400, 1750, 1600, 1390 cm 7~Z~

Example 74 p-Nitrobenzvl (SR,6R~-3-~2~(2~m~~ L~L ~=LL o ~ ~3 l-vl )et}~ylthio,~-6-L(S'L~ hvdr~_ xo-l-azabicvclo r 3.2Ø ~ept-2-ene-2--carboxylate (e28) _~ (e40) ~ ~ ~ ¢

(e92) The title compound (e92) (77 mg) was prepared by reaction of the thiol (e40) derived from the ester (e28) (227 mg) with 1~ bromoethyl)-2-methyl-5-nitroimidazole, adopting the procedure described in example 60., A max (EtOH) 310 nm (Em 17677), 266 nm (Em 12969), v max (KBr) 3400, 1770, 1700, 1608, 1520, 740 cm 1, ~H (CDC13) 8.18 (2H, d, aromatic protons), 7.93 (lH, S, imidazole CH), 7.63 ~2H, d, aromatic protons), 5.34 (2H, q, CH2Ar), 4.51 (2H, t, CH2N), 4.0-4.4 (m, 2H, 5-C_ + 8-CH), 2.9-3.8 (5H, m, SCH2 + 6-CH * 4-CH2), 2.62 (lH, broad res., OH), 2.49 (3H, S, imidazole CH3), 1.38 (3H, d, CH3C~

Example 75 (5R,6R)-3-[2-t2-Methyl-5-aminoimidazol-1-vl) ethvlthio~-6-[(S~ hydroxyeth~lL~7-oxo-1-aza _cyclo ~3.2.0 7 hept-2-ene-2-carboxylic acid H0 ~ NH2 o C02H
(e93) The title compound (e93) was prepared by hydrogenol~ysis o.~ the ester (e92) (30 mg~, following the procedure outlined in example 61 with the exception that the hydrogenation time was extended to 4 hours. ~reeze drying gave a white solid, A max (H20) 293 nm, ~ max (KBr) 3400, 1750, 1600, 1390 cm 1.

3~

Example 76 p-Nitrobenzyl (5R,6S)-3-~(2-p-nitrobenzyloxv-carbonylaminothiazol-4-vl~methylthio~-6-[(S)-l-hydroxyethyl~-7-oxo-1-azabicyclo[3.2.0~hept-2-~ne 2-carboxylate NHC02PNB
C~13 N
, s "l~

(el)_~(e2~ ~ N C02PNB (e94) ClCH2 ~ C02PNB
N
PNB
(e95) Following the procedure describecl in example 60, react;on of the thiol (e2) ~erived from the ester (el) (250 mg) with the intermediate (e95) gave, after silica gel column chromatography~ p-nitrobenzyl (5R,6S)-3-[(2-p-nitrobenzyloxycarbonylaminothiazol-4-yl)methylthio~-6-L(S)-l-hydroxyethyl~-7-oxo-1 azabicyclo[3.2Ø~hept-2-ene-2-carboxylate (e94) (70 mg) as a white solid, A max (EtOH) 319 nm (Em 12286), 263 nm (Em 25630) 9 ~ max (KBr) 3440, 1770, 1730, 1700, 1610, 1550, 1520, 1350, 1335 cm 1, ~H (d7-DMF) 7.7-8.4 (8H, m, aroma-tic pro~ons), 7.13 (lH, ~, thiazole CH), 5.1-5.7 (5 + q + broad d, 2 x CH2Ar +
OH, 5H), 4.0-4.4 (4H, s + m, SCH2 ~ 5-C_ + 8-CH), 3.2-3-9 (m, 4-C_2 ~ 6-CH), 1.28 (3H, d, CH3CH).

~ 115 -3~

Example 77 p-Nitrobenzvl (SR,6RL=~-L2-p-nitrobenzyloxycarbonyl-aminothiazo1-5-yl)methylthio~-6-~(S)-l-hy~roxyeth oxo-l-azabicyclo[3.2Ø~hept-2-ene-2-carboxylate NHCO PNB
CH3 N ~ 2 .~ S
~e28) ~(e40) ~ H /
o ~ N 2 N
(e~6) The title compound (e96) (95 mg) was prepared as a white solid by reaction of the ester (e28) (250 mg) with N-bromoacetamide, followed by intermediate (e95), utilising the procedure outlined in example 60., ~ max ~
(EtOH) 316 nm (Em 13729), 264 nm (Em 27411), ~ max (KBr) 3400 (broad), 3390 (sharp), 3080, 3110, 1770, 17307 1695, 1608, 1550, 1345, 1330 cm 1, ~H (d7-DM~) 7.7-8.4 (8H, m, aromatic protons), 7.15 (lH, s, thiazole-CH), 5.2-5.6 (4H, s ~ q, C_2Ar protons), 4.0-4.4 (s + m, 4H, SCH2 + 5-CH + 8-CH), 1.32 (3H, d, CH3CH).

~ ~7~7~

Example 78 (5R,6R)~3- r ( 2- minothiazol-4-vl!methvlthiol-6-r(S~-l-hydroxyethvll-7-oxo-1-azabicycloL _.Olhept-2-ene-2-carboxyl_c acid ~ H N ~ NH2 (e96) ~~~ r ~ S ~S

(e97) The ester (e96) (60mg) was hydrogenolysed, as in example 61 to yield (5R,6R)-3-[(2-aminothiazol-~-yl) methylthio]-6-[(S)-l-hydroxyethyl]-7-oxo-1-azabicyclo C3.2.0]hept-2-ene-2-carboxylic acid (e97) (22mg based on Em 8t500 at A max 297 nm in the u~v. spectrum) as an aqueous solution. A white fluffy solid was obtained on freeze-drying, A max (H20) 261nm, 297nm, v max (KBr) 3400, 1750, 1590cm 1.

7~3 Exa~ e 79 ~-Nitrobenzvl (5R,6S)-3-r2-pl-enYlthiazol-4-vl~
methylthio1-6-~(S)-l-hydroxyethyl1-7-oxo-1-azabicyclo ~3.2.01he~t-2-ene-2-carboxylate (el)-~(e2) ~ H ~ H N ~ Ph (e98) p-Nitrobenzyl (5R,6S)-3-[(2-phenylthiazol 4-yl) methylthio]-6-C(S)-l~hydroxyethyl~-7-oxo-l-azabicyclo [3.2.0]hept-2-ene-2-carboxylate (e98) (148mg) was prepared as a pale yellow solid by reaction of the thiol (e2) derived from the ester (el) (500mg) with 2-phenyl-4-iodomethyl thiazole, following the procedure described in example 60, A max (EtOH) 290nm (Em 23059); ~ max (KBr) 3500, 1768, 1698, 1608, 1546, 1519, 1348, 1332cm~l;
SH (~ 7-DM~) 7.4-8.4 (lOH, m, p-nitrobenzyl protons +
phenyl protons + thiazole-CH), 5.44(2H, q, CH2Ar), 5.19(1H, d, OH, disappears on D20)9 4.42(2H,s,SCH2), 4.0-4.4 (2H,m, S-C_+~-CH), 3.5(3H,m,4 C_2+6-CH), 1.29(3H,d,CH3CH); m/e 537 (relative intensity) (1%), 491 (10), 451 (10), 286 (30), 207 (55), 175 (100), 136 (20).

- 118- 1~7~

E~ample ~O
p-Nitroben?vl(SR,6~ 3~ el-~ylti~:iazol-4-yl)rnethyl-thiol-6-~(S~ hvdroxyethyll-7-oxo-1-azabicvclo~3.2.01hept-2-ene-2-carboxylate (e28)-~(e40) t HO ~ N ~ h ~e99) The title compound (e99) (198mg) was obtained as a white solid by reaction of the thio (e40), derived from the ester (e28) (500mg) with 2-phenyl-4-iodomethyl-thiazole, adopting the procedure described in example 60, A max (EtOH) 300nm (Em 21622); V max (KBr) 3430, 3110, 1776, 1695, 1608, 1550, 1520, 1348, 1328cm~l;
-DMF)7.4-8.3(10H,m,p-nitrobenzyl protons + phenyl protons + thiazole-CH), 5.43(2H,q,CH2Ar), 5.10(lH,d,OH, disappears on D20), 4.47(2H,s,SC_2), 3.9-4.4(2H,m,5-C_ +
8-CH), 3.5-3.9(3H,m,4-CH2+6-CH), 1.33(3H,d,C_3CH), m/e (relative intensity) 451 (4%), 418(2), 286(50), 207(100), 174(95), 104(55), 71(72).

3L~7~3~7~

Example ~ 1 (5R,6R)-3-r(2-Phenvlthiazol-4-vl)methvl ~ -r (S)-l-hvdroxyethyll-7-oxo-1- zabicyclo~3.2 Olhept-2-ene-2-carboxylic acid CH3 Ph (e99~ HO ~ ~ S

co2 (elQO) The ester (e99) (90mg) was hydrogenolysed, as in example 611 to yield the title compound (elOO) (19mg based on Em 17,000 at ~ max 297nm in the u.v. spectrum) as an aqueous solution. A white fluffy solid was obtained on S freeze-drying, ~ max (HzO) 297nm, v max (KBr) 3400, 1740, 1600cm 1 - 120 ~ L7 Example 8~
~?-Nitrobenz\Jl (5R)-3-~ ethylthio~-~-Z-ethylidene-7-oxo-1-azabicyclo~3.2Ø1hept-2-el-e-2-H H
H~ SCH2C 3 ~ ~SCH2CH3 O Co2pNB o C02PNB
(e7) (elOl) p-Nitrobenzyl (SR,65)-3-ethylthio-6-C(S)~l-hydroxy-ethyl]-7-oxo-1 azabicyclo~3.2.0~hept-2-ene-2-carboxylate (e7) (237mg; 0.60mM) was dissolved in dry tetrahydrofuran (40ml) and cooled to 0C. Triphenylphosphine (634mg;
5 2.42mlvl) was added with stirring, followed by a solution of diethylazodicarboxylate (421mg; 2.42mM) in dry tetrahydrofuran (20ml). The solution was then allowed to reach room temperature and stirred for a further 20 minutes. The solvent was removed at reduced pressure and 10 the residue partitioned between ethyl acetate and water.
The organic layer was washed with an additional volume of water, saturated sodium chloride solution and dried over anhydrous magnesium sulphate. Filtration and removal of the solvent at reduced pressure gave the crude ethylidene 15 as an orange oil. This oil was dissolved in the minimum volume of ethyl acetate/hexane (1:1) and chromatographed over silica gel (20mg), eluting with a graclient of :,0-75% ethyl acetate/hexane. The title ethylidene derivative (elOl) was obtained as a pale yellow oil (185mg).
20 Tritura-tion with diethyl ether/hexane gave a pale yellow solid (13Qrng), ~ max (Cl{Br3) 17557 1700, 1605cm 1, - 121 - ~ 6~ ~ 7 Exam~]e 83 benZyl(5R 6R)-3-ethvlthiO-~>-eth ~ _ L3. 2.0 ~ t-2=en_-2-car_Ox ~

~ H H
(elOlH3C J r ~ Sc},2c,l3 SC~l~c~l3 (elO2) (elo3) the ethyLidene (elOl) (1 (lml) waS COoled to lo&
stirred solution was added a solution of sodium borohydride (52mg) in O.O5M pH7.0 phosphate buffer (2ml). The e was allowed to reach 5 continued at, this temperature for 1 hour. Ethyl acetate (50ml) was added and the organic solution washed with water, saturated sodium chloride solution, and dried over anhydrous magnesiurn sulphate. Filtration and removal of 10d u c ed p re s s u re g a ve th ilica gel c o 1 u mn c h ro t el~ltin9 With 1:1 hexal1e/ethyl acetate gave a mixture of p-nitrobenzylt5R~6R)-3-ethylthio-6 ~hept-2-ene-2-Carbox nitrobenzyl(5R~6s)-3-ethylthio-6-et~yl-7-oxo-l-azabicyclo [3~2~o~hept-2-ene-2-carboxylate (elO3) (ratio 5:1 by h.p.l.c. ) as a colourless oil (14mg)~ Crystallisation from ether/hexane gave a white solid, m.p~. 10~-115C, max (EtOH) 322nm, 267nm, v max (CHBr3) 1775, 1700, 1605, 1550cm ~ H (CVC13) 1-06(3H, t,CH3CH2CH), 1.32(3~t~CH3 CH2S), 1~85(2H~m~CH3CH2CH)~ 2.85(2H,q,SCH2), 2.7-3.3(3H~
m,4-C_2~6-CH), 3.97(1H,dt,5-CH), 5.37(2H,q,CH2Ar), 7.65(2H, d~aromatic protons), 8.21(2H,d~aromatic protons), m/e (relative intensity) 376.1048(32%; Cl8H20N2oss requires 376.1091~, 347(2), 306(100), 273(20), 255(10), 170(20), 136(35), 126(40).

- 123 - ~7~

Example 84 Sodium(SR.6R~-3-eth~hio-6-ethyl-7-oxo-1-azabicvclo [3.2!0~hept-2-ene-2-carboxylate H H H H
H3C L ~ SC~12CH3 ~ SCH2CH3 C02PNB -N _ C02Na (elO2) (elO4) 5% Palladium on carbon catalyst (lSmg) was shaken with hydrogen in 30~0 aqueous 1,4-dioxan (Sml) at ambient pressure and temperature for 20 minutes. A solution of the ester (elO2) (lOmg) in 1,4-dioxan (5ml) was added and hydrogenation was continued for a fur~her 3.5 hours.
Sodium bicarbonate (3mg) was added and the suspension filtered through Celite, washing well with water (25ml).
The filtrate was concentrated at reduced pressure to approximately 15ml and washed with ethyl acetate (3x25ml).
The aqueous solution was further concentrated to about lOml and column chromatographed over Biogel P-2, eluting with water. Fractions containing sodium (SR,6R)-3-ethylthio-6~ethyl-~-oxo-1-azabicyclo[3.2.0]hept 2-ene-2-carboxylate (elO4) were identified by the chromophore at A max (H20) 1~ 300nm in the u.v. spectrum and combined (1.6mg based on Em 8,000).

Claims (18)

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

1. A process for the preparation of a compound of formula (I):

(I) and salts and esters thereof wherein R1 is a hydrogen atom or a group selected from OH, OSO3H or a salt or a C1-4 alkyl ester thereof, OR2, SR3, OCOR2, OCO2R3 or OCONHR3, where R2 is a C1-6 alkyl group or an optionally substituted benzyl group, R3 is a C1-6 alkyl group or an optionally substituted benzyl or an optionally substituted phenyl group; with the proviso that when R1 is OSO3H or a salt or C1-4 alkyl ester thereof the C-6 and C-5 hydrogen atoms are cis;
which comprises reacting a cleavable ester of a compound of the formula (II):

(II) wherein R1 is as defined in claim 1 and R11 is a methyl or ethyl group; with a source of hypohalous acid;

and, where required, i) converting a cleavable ester to a free acid or salt, ii) converting a cleavable ester to a different cleavable ester;

with the proviso that when R11 is an ethyl group R1 must be OSO3H or a salt or C1-4 alkyl ester thereof.

2. A process as claimed in claim 1 wherein the hypohalous acid is hypobromous acid.

3. A process as claimed in claim 2 wherein the source of hypobromous acid is N-bromoacetamide.

4. A compound of the formula I as defined in claim 1 and salts and esters thereof, whenever prepared by the process of any of claims 1, 2 and 3, or by an obvious chemical equivalent thereof.

5. A process for the preparation of a compound of formula (e2):

wherein -PNB is p-nitrobenzyl, which comprises reacting an ester of the formula (e1):

with N-bromoacetamide.

66. A process for the preparation of a compound of formula (e5):

which comprises reacting an ester of the formula (e4):

with N-bromoacetamide.

7. A process for the preparation of a compound of formula (e13):

wherein -PNB is p-nitrobenzyl, which comprises reacting an ester of the formula (e12):

with N-bromoacetamide.

8. A process for the preparation of a compound of formula (e22):

wherein -PNB is p-nitrobenzyl, which comprises reacting an ester of the formula (e21):

with N-bromoacetamide.

9. A process for the preparation of a compound of formula (e40):

(e40) wherein -PNB is p-nitrobenzyl, which comprises reacting an ester of the formula (e28):

(e28) with N-bromoacetamide.

10. A process for the preparation of a compound of formula (e67):

(e67) wherein -PNB is p-nitrobenzyl, which comprises reacting an ethylthio derivative of the formula (e66):

with N-bromoacetamide.

11. A process for the preparation of a compound of the formula (e70):

wherein -PNB is p-nitrobenzyl, which comprises reacting an ester of the formula (e69):

with N-bromoacetamide.

12. p-Nitrobenzyl (5R,6s)-3-thiol-6-[(s)-1-hydroxyethyl]
-7-oxo-1-azabicyclo[3 2.0]hept-2-ene-2-carboxylate, being the compound (e2), whenever prepared by the process of claim 5 or by an obvious chemical equivalent.

13. Benzyl (5R,6S)-3-thiol-6-[(S)-1-hydroxyethyl]-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate, being the compound (e5), whenever prepared by the process of claim 6 or by an obvious chemical equivalent.

14 The benzyldimethyl-n-hexadecylammonium salt of p-nitrobenzyl (5R,6R)-3-thiol-6-[(S)-1-hydr0xy-sulphonyloxy-ethyl]-7-oxo-1-azabicyclo[3.2.0]hept=2-ene-2-carboxylate, being the compound (e13), whenever prepared by the process of claim 7 or by an obvious chemical equivalent.

15. p-Nitrobenzyl(5R,6S)-3-thiol-6-[(S)-1-acetoxyethuyl]
-7-oxo-1-azabicyelo[3.2.0]hept-2-ene-2-carboxylate, being the compound (e22), whenever prepared by the process of claim 8 or by an obvious chemical equivalent.

16 p-Nitrobenzyl(5R,6R)-3-thiol-6-[(S)-1-hydroxyethyl]
-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate, being the compound (e40), whenever prepared by the process of claim 9 or by an obvious chemical equivalent.

17. p-Nitrobenzyl (SR,6R)-3-thiol-6-((S)-1-ethylthio-ethyl]-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate, being the compound (e67), whenever prepared by the process of claim 10 or by an obvious chemical equivalent.

18. p-Nitrobenzyl (5R,6R)-3-thiol 6-[(R)-1-ethylthio ethyl]-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate, being the compound (e70), whenever prepared by the process of claim 11 or by an obvious chemical equivalent.