5 S PENEM DERIVATIVES, THEIR PREPARATION AND USE

This invention relates to novel penem compounds, the use of such substances in the treatment of bacterial infections, and to novel formulations which incorporate such substances.

Penems are a known class of compound derived from the acid nucleus:

in which the numbering system used herein is also indicated. This nucleus can exist in two isomeric forms, and the 5R isomers, ie having the configuration below at the 5-position:

are known to have antibacterial activity resulting from their interaction with penicillin binding proteins (PBP\'s). This activity is generally confined to the free acid or anion, so such penems are normally used in antibacterial therapy in the form of salts of the carboxylate anion, or as in-vivo hydrolysable esters. The 5S isomers, having the configuration below at the 5-position:

are generally antibacterially inactive, because of non-interaction with PBP\'s. The preparation of 55 penems, for example the parent acid and the acetonyl ester, are known from H.R. Pfaendler et al., J.Am. Chem. Soc. 1979, 101(21) p6306-6310.

GB2036015, GB2037277, GB2013674, EP0399228, US4282236, GB1604751, ZA8504947 and J. Chem. Soc. Chem. Comm. 1988, 544 describe certain penem-3-carboxy ester derivatives but with no antibacterial activity.

GB2042520 discloses certain 2-(ethylthio)penem-3-carboxamide derivatives. Although racemic and other mixtures of 5R and 55 enantiomers of penems are known, there has been little attempt to isolate pure 55 isomers of penems because of the general belief that such isomers are antibacterially inactive. It has now been discovered that certain 55 isomers of penem esters inhibit the

bacterial enzyme leader peptidase 1, and are of potential use in the treatment of bacterial infections. Such compounds may be prepared in an isomerically enriched state relative to racemic mixtures.

The invention therefore provides the use of a 55 penem compound of general formula (I):

(I) wherein n is 0 or 1, R^ is hydrogen or a substituent group, R^ is an ester- or amide- forming group, and R ¹ is hydrogen or an organic group linked via carbon and wherein the group -COR^ is substantially stable toward in-vivo hydrolysis, in the form of its 55 isomer, as an inhibitor of the bacterial enzyme leader peptidase 1.

The invention also provides a pharmaceutical composition, in particular for use in the treatment of bacterial infections especially by the mechanism of inhibition of the bacterial enzyme leader peptidase 1, which comprises a compound of formula (I) and a pharmaceutically acceptable carrier.

The invention also provides a compound of formula (I) for use as an active therapeutic substance, particularly for use in treating bacterial infections, especially by the mechanism of inhibition of bacterial leader peptidase 1 enzyme.

The invention also provides the use of a compound of formula (I) in the manufacture of a medicament for use in the treatment of bacterial infections, especially by the mechanism of inhibition of the bacterial enzyme leader peptidase 1.

The invention also provides a method of treatment of bacterial infections in mammals, especially by the mechanism of inhibition of the bacterial enzyme leader peptidase 1, which comprises the administration to a mammal in need of such treatment, an effective amount of a compound of formula (I).

When used herein the term \'aryl\' includes phenyl and naphthyl, each optionally substituted with up to five, preferably up to three, groups selected from halogen, mercapto, (C g) alkyl, phenyl, (Cι_6) alkoxy, hydroxy(Cι_6)alkyl, mercapto(Cι_6)alkyl, halo(Cι_6) alkyl, hydroxy, amino, quaternary ammonium, nitro, carboxy, (Cι_6) alkylcarbonyloxy, (C\. ) alkoxycarbonyl, carboxylic acid, formyl, (C\. ) alkylcarbonyl and phosphoric acid groups. \'Aralkyi\' includes phenyl C\.β alkyl and napthyl Ci .5 alkyl each aryl group optionally substituted as aforesaid. The terms \'heterocyclyl\' and \'heterocyclic\' as used herein include aromatic and non-aromatic, single and fused, rings suitably containing up to four hetero-atoms in

each ring selected from oxygen, nitrogen and sulphur, which rings may be unsubstituted or substituted by, for example, up to three groups selected from halogen, (Cι_6) alkyl, (C\. ) alkoxy, halo (Cι_6) alkyl, hydroxy, carboxy, carboxy salts, carboxy esters such as (C\. ) alkoxycarbonyl or C\. ) alkoxycarbonyl (C\. ) alkyl, aryl and oxo groups. Each heterocyclic ring suitably has from 4 to 7, preferably 5 or 6, ring atoms. The term \'heteroaryl\' refers to heteroaromatic heterocyclic ring or ring system, suitably having 5 or 6 ring atoms in each ring. A fused heterocyclic ring system may include carbocyclic rings and need include only one heterocyclic ring. Compounds within the invention containing a heterocyclyl group may occur in two or more tautometric forms depending on the nature of the heterocyclyl group; all such tautomeric forms are included within the scope of the -Λvention.

When used herein the terms \'alkyl\', \'alkenyl\', \'alkynyl\' and \'alkoxy\' include straight and branched chain groups containing from 1 to 6 carbon atoms, such as methyl, ethyl, propyl and butyl. A particular alkyl group is methyl.

When used herein the term \'halogen\' refers to fluorine, chlorine, bromine and iodine.

In the compound of formula (I) R ² may suitably be hydrogen. When R ² is other than hydrogen it may suitably be an organic group linked to the penem ring 2-position by a carbon, oxygen or sulphur atom. For example R ² may be a group R ⁴ , CO2R ⁴ , OR ⁴ , Sk or SOR ⁴ - where each R ⁴ may be the same or different and may denote hydrogen or an unsubstituted or, preferably, substituted Ikyl group or an aryl or heterocyclyl group.

For example when R ⁴ represents a substituted alkyl group suitable substituents on the alkyl group include hydroxy, (C1.5) alkoxy, (C1.4) alkoxy (Cj^) alkoxy, aminocarbonyloxy, halogen, mercapto, (Cχ.6) alkylthio, aryl, arylthio, aryloxy, heterocyclyl, heterocyclylthio, amino, (mono- or di-)-(Cj.6) alkylamino, acylamino or acyloxy where the acyl group may be aromatic or aliphatic, for example (Cι_6> alkanoyl, (Cι_6) alkoxycarbonyl, optionally substituted benzoyl or optionally substituted phenyloxycarbonyl, carboxy, (Cj.g) alkoxycarbonyl, azido and alkyl- or aryl-substituted silyloxy.

Suitable examples of R ² substituents other than hydrogen include methyl, hydroxymethyl, hydroxyethylthio, phenyl, 4-chlorophenyl, 4-methoxyphenyl, 4- methoxycarbonylphenyl, aminocarbonyloxymethyl, p- nitrobenzyloxycarbonylaminomethyl, ethyhhio, methylthio, t- butyldiphenylsiloxymethyl and benzyl.

R ² is preferably hydrogen, hydroxymethyl, hydroxyethylthio, phenyl or aminocarbonyloxymethyl.

The group COR^ is preferably resistant to in-vivo hydrolysis.

When R3 is an ester-forming group it may suitably be selected from any such group which is known to be substantially stable to in-vivo metabolic degradation

R3 may be an aryloxy group. Alternatively R^ may be an optionally substituted (CJ.JO) alkoxy group where the optional substituent(s) may be halogen or an organic group, such as (Cμβ) alkylcarbonyl, (C\. ) alkoxycarbonyl, (Cj.io) aU-Oxy-substituted-(Ci_io)-alkoxy, aryl or heterocycyl. Typically such an alkoxy group R3 may be substituted by one or two such substituents at the terminal positions on the alkyl chain if the chain contains more than one carbon atom.

Examples of optional aryl substituents on an R3 substituted alkoxy chain include phenyl and substituted phenyl where the substituent(s) may be selected from halogen, carboxylic acid, carboxy ester, amino, quaternary ammonium, alkoxy, hydroxy or phosphorous acids. Examples of heterocyclyl substituents on an R^ substituted alkyl chain include optionally substituted pyrimidyl, benzopyranyl and furanyl. Examples of such R^ groups include methoxy, benzyloxy, p- methoxybenzyloxy, 2,2,2-trichloroethoxy, p-nitrophenyloxy, acetonyloxybenzyloxy, m- and p-(methoxycarbonyl)benzyloxy, benzoylmethyloxy, p-nitrobenzyloxy, 4-pyridylmethyloxy, 2,2,2-trichloroethyloxy, 2,2,2-tribromoethyloxy, l-butyloxy, pentyloxy, i-amyloxy, allyloxy, diphenylmethyloxy, triphenylmethyloxy, adamantyloxy, 2-benzyloxyphenyloxy, 4-methylthiophenyloxy, tetrahydrofuran-2- yloxy, tetrahydropyran-2-yloxy, pentachlorophenyloxy, acetonyloxy, β-toluenesulphonylethyloxy, methoxymethyloxy, 4-(4-nitrobenzyloxycarbonyl)- benzyloxy, 4-carboxybenzyloxy and salts thereof, 4-phenylbutyloxy, 2-(2- methoxyethoxy)ethoxy, 4-bromobenzyloxy and (7-methoxy-2-oxo-2H-l-benzopyran- 4-yl)methoxy

R3 is preferably benzyloxy, p-nitrobenzyloxy, p-methoxybenzyloxy, 2,2,2- trichloroethyloxy, methoxy, p-(methoxycarbonyl)benzyloxy or allyloxy.

When R3 is an amide-forming group it may be a group NR^2 where each R^ may be the same or different and may be independently selected from hydrogen, aryl such as optionally substituted phenyl or optionally substituted (Cj_6) alkyl, where the optional substituents on the (C1.5) alkyl group may be selected from the same list of substituents from which substituents on an alkyl group R ⁴ may be selected. Alternatively the two R^ moieties may represent the residue of a nitrogen containing heterocyclic ring as defined above. Alternatively NR^ may represent the residue of an amino acid, for example a natually occurring α-amino acid such as glycine, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, cystine, methionine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, phenylalanine, tyrosine, proline, hydroxyproline, histidine or tryptophan, in which the amino group of the amino acid forms an amide link to the carbonyl group shown at the 3-position

- A -

on the penem ring system.

Suitably one of R^ may be hydrogen and the other may be an optionally substituted alkyl group, such as an aryl substituted alkyl group such as benzyl. Examples of such airiide-forming groups R^ include benzylarnino, N- methylbenzylamino, pyrrolidino, anilino, diemylaπiino, p-methoxyrjenzylamino, morpholino, 2-(meΛoxycarbonyl)ethylamino, t-butylamino and (3-pyridylmemyl)arnino.

R3 is preferably pyrrolidino

R* may suitably be hydrogen. Alternatively R* may suitably be a substituent group selected from optionally substituted (Ci-6) alkyl, optionally substituted (C\. ) alkenyl and C\.(- alkanoyl where the optional substituents on the alkyl group of these substituents may be selected from the same list of substituents from which substituents on an alkyl group R ⁴ may be selected. R* is preferably Cj_4 alkyl substituted in the 1 -position by a substituent linked via oxygen, such as hydroxy, (C\- 5)alkoxy such as methoxy, arninocarbonyloxy, benzoyloxy or (C\. ) alkanoyloxy optionally substituted by a group such as benzyloxycarbonylamino. When the carbon

Alternatively R* alkyl may suitably be substituted by a group R^-COO- or

R^CO-NH- where R^ is the residue of an arnino acid, for example a naturally occurring α-amino acid. Alternatively the substituent on R* may be an azido group, N ₃ or a peptide residue, e.g. of formula R9-NH-Crø ¹⁰ -CO[-ϊrøCHR ¹⁰ -C ]n-X- where R^ is an amino-substituting group, for example an optionally substituted Ci -g alkanoyl or Cj-g alkoxycarbonyl group, R ¹ ^ i _s a residue derived from a naturally occurring arnino acid, such as the above-mentioned α-amino acids, X is O or NH and n is an integer 1 to 4 more particularly 1 to 3 such as 1 to 2. An example of such a peptide residue is (CH3)3CO.CONH.CH(CH3).CONH.CH(CH ₂ .CH(CH3)2).CONH. Preferably R ¹ is hydrogen, 1-acetoxyethyl or 1 -hydroxyethyl. Suitably the compound of formula (I) may include 50% or more preferably

60% or more, more preferably 70% or more, even more preferably 80% or more, desirably 90% or more of the 5S isomer. It is especially preferred that the compound of formula (I) is substantially the pure 55 isomer.

Some of the compounds of this invention may be crystallised or recrystallised from solvents such as organic solvents. In such cases solvates may be formed. This

invention includes within its scope stoichiometric solvates including hydrates as well as compounds containing variable amounts of water that may be produced by processes such as lyophilisation.

Since the antibiotic compounds of the invention are intended for use in pharmaceutical compositions it will readily be understood that they are each provided in substantially pure form, for example at least 60% pure, more suitably at least 75% pure and preferably at least 85%, especially at least 95% pure (% are on a weight for weight basis). Impure preparations of the compounds may be used for preparing the more pure forms used in the pharmaceutical compositions; these less pure preparations of the compounds should contain at least 1 %, more suitably at least 5% and preferably from 10 to 49% of a compound of the formula (I).

Some compounds of formula (I) are known, alternatively they may be prepared by methods analogous to those described in J.A.C.S 1979, 101(21), p6306- 6310, for example by cyclisation of compounds of formula (II):

(π) where R ² * is R ² or a substituent which can be converted into or replaced by R ² , R31 is a group R^ or a group which can be converted into or replaced by R^, R- 1 is a group R- or a group which can be converted into R*, where R ² , R^ and R* are as defined in formula (I), and R^ is an organic radical such as phenyl.

Examples of groups R ² * convertible into R ² include -CH2OS-R3 which is convertible to -CK OH and other derivatives, or an ester attached to a phenyl group which is convertible to an acid.

When R ² in formula (I) is hydrogen, compounds of formula (II) in which R ² * is hydrogen may be obtained from the corresponding compound where the substituent on sulphur is a β-(alkoxycarbonyl)vinyl group such as β-(ethoxycarbonyl)vinyl or

CH,

R31 ay for example be a group OR^l where R^l is a carboxylic acid protecting group which may be removed by a conventional procedure to form an acid

of formula (HI):

OH) Further compounds of formula (I) may be prepared using known ester- or amide-forming reactions from the parent acid (III) or an acylating derivative thereof, where R21, R! 1 and n are as defined in formulae (I) and (II) above, and in particular wherein the acid is isomerically enriched in the 55 isomer. The preparation of such acids as the pure 55 isomers is for example described in Pfaendler J.A.C.S. op cit.

Suitable acylating derivatives include symmetrical or mixed anhydrides. The acylation may be effected in the presence of an acid binding agent for example, tertiary amine (such as pyridine or dimethylaniline), molecular sieves, an inorganic base (such as calcium carbonate or sodium bicarbonate) or an oxirane, which binds hydrogen halide liberated in the acylation reaction. The oxirane is preferably a (C\. 6)-l,2-alkylene oxide - such as ethylene oxide or propylene oxide. The ac ation reaction may be carried out at a temperature in the range -50°C to +50°C, preferably -20°C to +20°C, in aqueous or non-aqueous media such as water, acetone, tetrahydrofuran, ethyl acetate, dimethylacetamide, dimethylformamide, acetonitrile, dichloromethane, 1,2-dichloroethane, or mixtures thereof. Alternatively, the reaction - may be carried out in an unstable emulsion of water-immiscible solvent, especially an aliphatic ester or ketone, such as methyl isobutyl ketone or butyl acetate. The acylation with acid anhydride is suitably carried out in the presence of a basic catalyst such as pyridine or 2,6-lutidine.

Suitable mixed anhydrides are anhydrides with, for example, carbonic acid monoesters, trimethyl acetic acid, thioacetic acid, diphenylacetic acid, benzoic acid, phosphorus acids (such as phosphoric, phosphorous, and phosphinic acids) or aromatic or aliphatic sulphonic acids (such as p.-toluenesulphonic acid or methanesulphonic acid).

Alternative acylating derivatives of acid (HI) are the acid azide, or activated esters such as esters with 2-mercaptopyridine, cyanomethanol, p.-nitrophenol, 2,4-dinitrophenol, thiophenol, halophenols, including pentachlorophenol, monomethoxyphenol, ϋ-hydroxy succinimide, ϋ-hydroxybenzotriazole, or 8-hydroxj. uinoline; or amides such as H-acylsaccharins, ϋ-acylthiazolidin-2-thione or ϋ-acylphthalimides; or an alkylidene iminoester prepared by reaction of the acid (HI) with an oxime.

Other reactive acylating derivatives of the acid (III) include the reactive intermediates formed by reaction in situ with a condensing agent such as a carbodiimide, for example, NJ_f -diethyl-, dipropyl- or diisopropylcarbodiim.de, ϋ^-di-cyclohexyl-carbodiimide, or H-ethyl-M\'-[3-(dimethylamino)propyl]- carbodiimide; a suitable carbonyl compound, for example, H-J-f -carbonyldiimidazole or H^-T-carbonyldi- triazole; an isoxazolinium salt, for example, ϋ-e-hyl-5-pheny-isoxazolinium-3-sulphonate or H-t-butyl-5- methylisoxazolinium perchlorate; or an H-alkoxycarbonyl 2-alkoxy-l,2-dihydroquinoline, such as ϋ-ethoxycarbonyl 2-ethoxy-l,2-dihydroquinoline. Other condensing agents include Lewis acids (for example BBr - C5EL5); or a phosphoric acid condensing agent such as diethylphosphorylcyanide. The condensation reaction is preferably carried out in an organic reaction medium, for example, methylene chloride, dimethylformamide, acetonitrile, alcohol, benzene, dioxan or tetrahydrofuran.

A further method of forming the acylating derivative of the acid of formula (LTJ) is to treat the acid of formula (HI) with a solution or suspension preformed by addition of a carbonyl halide, preferably oxalyl chloride, or a phosphoryl halide such as phosphorus oxychloride, to a halogenated hydrocarbon solvent, preferably dichloromethane, containing a lower acyl tertiary amide, preferably N^-dimethylformamide. The acid (HI) or its acylating derivative may be caused to react with an alcohol of formula HO-R^ or an amine of formula H-NR^, where R3 and R^ are as defined above. Methods of formation of esters and amides from such alcohols and amines are well known in the art of organic chemistry.

Suitable examples of such alcohols include benzyl alcohol, p-methoxybenzyl alcohol, 2,2,2-trichloroethanol, methanol, p-nitrophenol, and hydroxyacetone.

Suitable examples of amines include benzylamine, N-methylbenzylamine, pyrrolidine, aniline, methylamine, p-methoxybenzylamine, morpholine, 2-(methoxycarbonyl)ethylamine, t-butylamine and (3-pyridylmethyl)amine.

Suitably an ester may be formed by reaction of an acylating derivative of the acid (IH) with the alcohol HO-R^ in a suitable solvent Alternatively esters of formula (I) may for example be prepared by reaction of a metal salt of the acid of formula (HI), for example the potassium salt, which may itself be prepared by reaction of the acid (HI) with a basic compound of the metal such as the carbonate, with for example a halide of formula X-R^ where X is a halide radical such as iodide. Esters may also be formed by the reaction of the free acid with a diazoalkane or diphenyldiazoalkane in an inert solvent at ambient temperature.

Suitably an amide may be formed by reaction of the acid (UI) or an N- acylating derivative with the amine H-NR^ in a suitable solvent, if desired in the

presence of an acid binding agent such as triethylamine or pyridine. A suitable N- acylating derivative of the acid (LU) is a mixed anhydride for example as formed by reaction of the acid (HI) with ethylchloroformate in the presence of an acid binding agent such as triethylamine in an organic solvent such as tetrahydrofuran for example at a temperature of 0 to -20°C. Reaction of the anhydride with the amine may be carried out with the anhydride so formed in situ.

Certain compounds of formula (I) as defined above are believed to be novel, and as such constitute a further aspect of this invention.

In particular amides of formula (I) as defined above wherein R ² is other than an S-linked organic group when R-3 is an amino or di-lower alkyl amino group are believed to be novel, and as such constitute a further aspect of this invention.

The compositions of the invention include those in a form adapted for oral, topical or parenteral use and may be used for the treatment of bacterial infection in mammals including humans. The compounds according to the invention may be formulated for administration in any convenient way for use in human or veterinary medicine, by analogy with other antibiotics.

The composition may be formulated for administration by any route, such as oral, topical or parenteral, especially oral. The compositions may be in the form of tablets, capsules, powders, granules, lozenges, creams or liquid preparations, such as oral or sterile parenteral solutions or suspensions.

The topical formulations of the present invention may be presented as, for instance, ointments, creams or lotions, eye ointments and eye or ear drops, impregnated dressings and aerosols, and may contain appropriate conventional additives such as preservatives, solvents to assist drug penetration and emollients in ointments and creams.

The formulations may also contain compatible conventional carriers, such as cream or ointment bases and ethanol or oleyl alcohol for lotions. Such carriers may be present as from about 1% up to about 98% of the formulation. More usually they will form up to about 80% of the formulation.

Tablets and capsules for oral administration may be in unit dose presentation form, and may contain conventional excipients such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, orpolyvinylpyrollidone; fillers, for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tabletting lubricants, for example magnesium stearate, talc, polyethylene glycol or silica; ^\' disintegrants, for example potato starch; or acceptable wetting agents such as sodium lauryl sulphate. The tablets may be coated according to methods well known in normal pharmaceutical practice. Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or

may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives, such as suspending agents, for example sorbitol, methyl cellulose, glucose syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats, emulsifying agents, for example lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which may include edible oils), for example almond oil, oily esters such as glycerine, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid, and, if desired, conventional flavouring or colouring agents. Suppositories will contain conventional suppository bases, e.g. cocoa-butter or other glyceride.

For parenteral administration, fluid unit dosage forms are prepared utilizing the compound and a sterile vehicle, water being preferred. The compound, depending on the vehicle and concentration used, can be either suspended or dissolved in the vehicle. In preparing solutions the compound can be dissolved in water for injection and filter sterilised before filling into a suitable vial or ampoule and sealing.

Advantageously, agents such as a local anaesthetic, preservative and buffering agents can be dissolved in the vehicle. To enhance the stability, the composition can be frozen after filling into the vial and the water removed under vacuum. The dry lyophilized powder is then sealed in the vial and an accompanying vial of water for injection may be supplied to reconstitute the liquid prior to use. Parenteral suspensions are prepared in substantially the same manner except that the compound is suspended in the vehicle instead of being dissolved and sterilization cannot be accomplished by filtration. The compound can be sterilised by exposure to ethylene oxide before suspending in the sterile vehicle. Advantageously, a surfactant or wetting agent is included in the composition to facilitate uniform distribution of the compound.

The compositions may contain from 0.1% by weight, preferably from 10-60% by weight, of the active material, depending on the method of administration. Where the compositions comprise dosage units, each unit will preferably contain from 50-500mg of the active ingredient. The dosage as employed for adult human treatment will preferably range from lOOmg to 5g per day, depending on the route and frequency of administration. Such a dosage corresponds to 1.5 to 70mg/kg per day. Suitably the dosage is from 5 to 20 mg/kg per day. No unacceptable toxicological effects are expected when a compound of formula (I) or a pharmaceutically acceptable salt or in vivo hydrolysable ester thereof is administered in the above-mentioned dosage range.

The compound of formula (I) may be the sole therapeutic agent in the compositions of the invention or a combination with other antibiotics may be

employed.

The compounds of the present invention are of potential use as antibacterial agents against a wide range of organisms including both Gram-negative organisms such as Exoli and Gram-positive organisms such as S.aureus.

The following Examples illustrate the preparation of compounds of the invention and intermediates thereto.

Example 1 - Benzyl (55)-penem-3-carboxylate

la (45)-(cis-β-Carbomenthyloxyvinylmercapto)azetidin-2-one

fractional crystallization

(i) To a solution of (-)-cis-β-carbomenthyloxyvinylisothiuronium chloride (32. lg, O.lmol), prepared by the method of Pfaendler H.R. et al., (J. Amer. Chem. Soc, 101, 1979, 6306) in ethanol (400ml) at -10°C was added a pre-cooled aqueous sodium hydroxide solution (200ml, 1.0N, 0.2mol). The resulting suspension containing the sodium salt of (-)-cis-β-carbomenthyloxyvinylmercaptan was used immediately for the subsequent reaction.

(ii) A pre-cooled solution of 4-acetoxyazetidin-2-one (12.9g, 0. lmol) in ethanol (200ml) was added to the above suspension of sodium (-)-cis-β- carbomenthyloxyvinyl mercaptide at -10°C. The internal temperature was maintained at -10°C during the addition. The reaction solution was then stirred at -10°C for 30 min. and 5°C for 15 min. Methylene chloride and saturated sodium chloride solution was added and the organic solution was separated, dried (MgSO4) and evaporated. The residue was chromatographed over silica gel, eluting with ethyl acetate-hexane mixtures to yield the epimeric mixture of azetidinones as a white semi-solid (26. lg). This semi-solid was recrystallized from dichloromethane/n-pentane to yield a white solid (0.902g) which was recrystallised an additional three times from dichloromethane/n-pentane until the crystalline solid obtained had a constant melting point The material thus obtained was the pure (45)-(cis-β- carbomenthyloxyvinylmercapto)- azetidin-2-one (0.66g), m.p. 130-131°C; [ α ]D ² 3 - 79° (c 1.0, CHCI3), δ H (CDCI3) 0.77 (3H, d, J 7Hz), 0.90 (6H, 2 x d, / 6.5 and 7Hz), 0.9-2.12 (m, 9H), 3.08 (IH, ddd, J 15.5, 2, 1Hz), 3.52 (IH, ddd, J 15.5, 5, 2Hz), 4.76 (IH, dt, J 11 and 4.5Hz), 4.92 (IH, dd, J 5 and 2.5Hz), 6.01 (IH, d, J

10Hz), 6.28 (IH, broad resonance), 7.11 (IH, d, J 10Hz).

The original mother liquors were evaporated and recrystallized again from dichloromethane n-pentane. Repeated recrystallisation of the solid thus obtained (from dichloromethane/n-pentane) gave an additional quantity of the pure (45)-(cis-β- carbomenthyloxyvinylmercapto)azetidin-2-one (1.66g), m.p. 130°C; [ α ]τj ² 3 -80° (c 1.0, CHC1 ₃ ).

lb. Benzyl [(45)-(cis-β-Carbomenthyloxyvinylmercapto)-2-oxoazetidin-l- yI]- triphenylphosphoranylideneacetate

Benzyl glyoxylate. monohydrate (0.439g; 2.415mM) was dissolved in benzene (100ml) and the solution was heated to reflux with the pr ision for the removal of water (Dean and Stark apparatus) for lh. The solution was then allowed to cool to room temperature and the solution .... the (45)-(cis-β- carbomenthyloxyvinylmercapto)azetidin-2-one (0.500g; 1.61mM) in dry benzene (10ml) was added. Triethylamine (4 drops) was added and the solution was stirred at room temperature for 4h. The solution was then evaporated at reduced pressure and azeotroped twice from toluene to yield the diastereoisomeric hydroxyacetates as a foam. This foam was dissolved in dry tetrahydrofuran (50ml) and cooled to - 10°C under an atmosphere of argon. 2,6-Lutidine (0.282ml) and thionyl chloride (0.177ml) were added and stirring was continued at -10°C for 15 min. The solution was then filtered and the filtrate was evaporated to yield the diastereoisomeric chloroacetates as a foam. The chloroacetates were dissolved in 1,4-dioxan (50ml) and triphenylphosphine (1.68gm) was adde ^*■ the stirred solution. When all the triphenylphosphine had dissolved, the s. .on was evaporated to small volume (approx 10ml). 2,6-Lutidine (0.228ml) was added and the solution was stirred at room temperature for 16h. The solution was then partitioned between ethyl acetate and water. The organic solution was washed with 5% citric acid solution, saturated sodium hydrogen carbonate solution, brine, dried (MgSO4) and evaporated. The residue was chromatographed over silica gel, eluting with ethyl acetate-hexane

mixtures, to yield the pure title phosphorane as a white foam (0.740g), v _m ax (CH C-2) 1763, 1700, 1638, 1582cm- ¹ .

Ic Benzyl (55)-Penem-3-carboxylate

Benzyl [(45)-(cis-β-carbomenthyloxyvinylmercapto)-2-oxoazetidin-l- yl]- triphenylphosphoranylideneacetate (0.740g) was dissolved in ethyl acetate and cooled to 0°C under a stream of argon. Trifluoroacetic acid (15ml) was added and the solution was stirred at 0°C for 15 min. and then cooled to -78°C. Ozone was bubbled through the solution for 2h, when tl.c. indicated complete ozonolysis. The excess ozone was blown off with argon and triphenylphosphine (0.315g) was added.

Saturated sodium hydrogen carbonate solution was added and the solution allowed to warm up to room temperature. The organic solution was washed with sat sodium hydrogen carbonate solution, brine, dried (MgSO4) and evaporated/The residue was dissolved in dichloromethane (20ml) and stirred at room temperature for 2h, when tl.c. indicated that cychzation to the bicyclic penem was complete. The solution was evaporated at reduced pressure and the residue chromatographed over silica gel. Elution with ethyl acetate-hexane mixtures afforded the title compound contaminated with menthyl glyoxylate (0.183g). The impure product was therefore rechromatographed over silica gel, eluting with 0%, 1% and 2% ethanol in dichloromethane, to provide the pure title compound as a white crystalline solid (0.051g), m.p. (EtOAc-hexane) 136-140°C; [ α ] _D ²³ -206° (c 1.0, CH ₂ α ₂ ); v _max (CH ₂ C-2) 1800, 1720, 1562cm" ¹ ; δ _H (CDCI3) 3.59 (IH, dt J 16.5, 1.5Hz), 3.86 (IH, ddd, J 16.5, 4, 1Hz), 5.22 (IH, d, / 12.5Hz) and 5.28 (IH, d, J 12.5Hz) (all ABq), 5.80 (IH, dd, J4, 2Hz), 7.2-7.45 (6H, m) (Found: C, 59.65; H, 4.15; N, 5.4; S, 12.15%; M+, 261.0464. C^ 2NO3S requires: C, 59.75; H, 4.25; N, 5.35; S, 12.25%; M, 261.0460).

Example 2 - Methyl (5R5)-penem-3-carboxylate

A solution of 4-nitrobenzyl (5R5)-penem-3-carboxylate [U.K. Pat. Appl.

2,036,015 (Beecham Group)] (30mg) in dioxane (5ml) and water (1.25ml) was hydrogenated at atmospheric pressure for 2h. in the presence of 10% palladium on

carbon catalyst (30mg). The product was filtered through Celite and a solution of potassium carbonate (8mg) in water (5ml) added to the filtrate. After a period of 0.25h the solution was evaporated under reduced pressure until just turbid and then washed twice with ethyl acetate. The remaining aqueous solution was freeze-dried to give the crude potassium salt This salt was stirred in dry dimethylformamide (1ml) and iodomethane (28mg, 12 μl) was added. The mixture was stirred at room temperature under argon for 3h. and then evaporated under reduced pressure. The residue was partitioned between ethyl acetate and brine; the organic phase was separated, washed with brine, dried and evaporated. The residue was purified by chromatography on silica gel 60 using ethyl acetate/hexane 1:1 as solvent to give a gum which was crystallised from ethyl acetate/hexane to give the methyl ester (7mg); m.p. 91-93°C; v _max (CHC1 ₃ ) 2970, 1800, 1720 and 1565cm\' ¹ ; δ _H (CDCI3) 3.58 (IH, d, J 17.0Hz with fine splitting, 6-H), 3.82 (3H, s, OCH3), 3.86 (IH, dd, J 17.0 & 3.9Hz, 6-H), 5.80 (IH, dd, J 3.9 & 1.9Hz, 5-H), and 7.27 (IH, s, 2-H); + (El), mlz 185.

Example 3 • Benzyl (5R)-penem-3-carboxylate

p-Methoxybenzyl (5R)-penem-3-carboxylate was converted to the sodium salt of (5R penem-3-carboxylic acid by the procedure described by M. Ohtani et al (J. Org. Chem., 1984, 49, 5271). This was then dissolved in dry N,N- dimethylformamide and stirred at room temperature for 2hr. with benzyl bromide (2 equiv.). The solution was diluted with ethyl acetate and washed with water, brine, dried (MgSO4) and evaporated. The residue was chromatographed over silica gel, eluting with a gradient of 10-50% ethyl acetate and hexane to yield the title compound as a white solid, [α] _D ²³ + 201° (C 1.0, CH2CI2) (ZA 8504947, 1986, Merck).

Example 4 - Benzyl (5R5)-penem-3-carboxylate

This was prepared according to the method of GB 2,036,015 (1980) to give the title compound, m.p. 118-119°C λm∞ (EtOH) 260 and 319 nm; Vmax (CHCI3) 1795, 1715 cm- ¹ δ (CDCI3) 3.49 (IH, ddd, / 16, 2 and 2Hz), 3.83 (IH, dd, / 16 and 4Hz), 5.23 (2H, s), 5.74 (IH, dd, / 4 and 2Hz), 7.24 (IH, d, J 2Hz), 7.35 (5H, s) (Found: C, 59.6; H, 4.1; N, 5.2; S, 12.3%. C13H1 !NO ₃ S requires C, 59.7; H, 4.2; N, 5.4; S, 12.3%).

Example 5 - p-Nitrobenzyl (5R5)-penem-3-carboxylate

This was prepared according to the method of GB 2,036,015 (1980) to give the title compound p 163-165°, λ^^ (EtOH) 319nm; v _max (CHCI3) 1800, 1715 cm" ¹ ; δ _H (CDCI3) 3.57 (IH, ddd, J = 17, 2, lHz), 3.95 (IH, ddd, J = 17, 4, 2Hz), 5.22 and 5.45 (2H, ABq, J = 14Hz), 5.81 (IH, dd, J 4, 2Hz), 7.35 (IH, d, J 1Hz), 7.57 (2H, d, J = 9Hz) and 8.23 (2H, ά, J = 9Hz) (Found: C, 51.0; H, 3.3; N, 9.1; S, 10.3%. Ci3H ₁₀ N ₂ O ₅ S requires C, 50.98; H, 3.27; N, 9.15; S, 10.45%).

Example 6 - p-Methoxybenzyl (5R5)-penem-3-carboxyIate

This was prepared according to method of example 2 using p-methoxybenzyl bromide to give the title compound, v _max (CHCI3) 1798, 1715 cm" ¹ ; δjj (CDCI3) 3.55 (IH, dt), 3.75-3.95 (4H, s + m), 5.18 (2H, ABq), 5.78 (IH, dd, J 1.85, 4.0 Hz), 6.90 (2H, d, J 8.7Hz), 7.25 (IH, s), 7.33 (2H, d, J 8.7Hz) (Found: M ⁺ 291.0571; C14H13NO4S requires 391.0565).

Example 7 - p-Methoxybenzyl (5R)-penem-3-carboxylate

p-Methoxybenzyl (5R, 65)-6-bromopenem-3-carboxylate (J.C.S., Chem. Commun., 1989, p.371) (2.0g) was dissolved in acetonitrile (80ml) and stirred at room temperature for 30min. wim zinc powder (1.4g) and \M ammonium acetate solution. The resulting suspension was filtered through Celite, diluted with ethyl acetate and washed with sodium hydrogen carbonate solution, brine, dried (MgSO4) and evaporated. The residue was chromatographed over silica gel, eluting with a gradient of 20— >50% EtOAc/hexane to yield the title compound as a pale yellow solid (0.50g), [α] _D ² 3 + 169° (C1.0, CHCI3).

Example 8 - Trichloroethyl (5R5)-penem-3-carboxylate

This was prepared in an analogous manner to example 4 to give the title compound, mp 119-121°, X^^ (EtOH) 322; v _max (CHCI3) 1800, 1730 cm\' ¹ ; δfj (CDCI3) 3.53 (IH, ddd, / 16, 2 and 1 Hz), 3.89 (IH, dd, J 16 and 4Hz), 4.74 and 4.89 (2H, ABq, / 12Hz), 5.31 (IH, dd, J 4 and 2Hz), 7.42 (IH, d, J 1Hz). (Found: C, 32.3; H, 2.0; N, 4.9; S, 10.6%. C8H6NO3SCI3 requires C, 31.7; H, 2.0; N, 4.6; S, 10.6%).

Example 9 - p-Nitrobenzyl (5R5)-2-(hydroxymethyl)penem-3-carboxy.ate

This was prepared according to the method described in GB 2,037,277B to give the title compound, m.p. 137-9°, ^ax (EtOH) 265 and 322 nm; v _max (0103)

3600-3010, 1795, 1705 cm\' ¹ ; δ _H (CDC1 ₃ ) 3.11-3.40 (IH, bs, exch D ₂ O), 3.51 (IH, dd, J = 2, 16Hz), 3.84 (IH, dd, J = 4, 16Hz) 4.67 (2H, bs), 5.22 and 5.47 (2H, ABq, / = 14Hz), 5.67 (IH, dd, J = 2, 4Hz), 7.20 (2H, d, J = 8Hz), 8.20 (2H, d, / = 8Hz). (Found: C, 50.21, H, 3.43; N, 8.26; S, 9.52%; C14H12N2O6S requires C, 50.0; H, 3.60; N, 8.33; S, 9.53%).

Example 10 - Allyl (5R5)-penem-3-carboxylate

Allyl [(4R5) - tritylthio-2-oxoazetidin-l-yl] triphenyl phosphoranylidene acetate (0.500g; 0.711 mM was dissolved in dry acetonitrile (25ml). To the stirred solution was added 4-dimethylaminopyridine (0.104g) and a solution of silver nitrate (0.185g) in acetonitrile. The solution was stirred at room temperature for 1 hour. The solution was then cooled to 5°C and 4-dimethylaminopyridine (0.087g), sodium iodide (1.066g) followed by acetic-formic anhydride (0.57ml) were added in rapid succession. The cooling bath was removed and the solution was stirred at ambient temperature for 30 min. The reaction solution was then partitioned between ethyl acetate and water. The organic solution was washed with IN HC1, water, saturated sodium hydrogen carbonate solution, brine, dried (MgSθ4) and evaporated at reduced pressure. The crude product was chromatographed over silica gel. Elution with a gradient of 10 to 25% ethyl acetate/hexane provided the title compound as a pale yellow oil (0.090g), Vmax (CH ₂ Cl2) 1800, 1720cm\' ¹ ; δ _H (CDCI3) 3.58 (IH, dt J 16.6, 1.5 Hz), 3.88 (ddd. 16.5, 4.0, 0.7 Hz), 4.72

(2H, m), 5.2-5.5 (2H, m), 5.70 (IH, dd, J 2, 4 Hz), 5.85 - 6.10 (lH,m), 7.30 (IH, d, 1 Hz), m e 211.0300 (C9H9NO3S requires M, 211.0303).

Example 11 - -Nitrobenzyl (S5)-penem-3-carboxylate

11a p-Nitrobenzyl [(45) - (cis-β-Carbomenthyloxy •vinylmercapto) -2- oxoazetidin -l-yl] • triphenylphosphoranylideneacetate p-Nitrobenzyl glyoxylate-monohydrate was reacted with (45) - (cύ- V carbomenthyloxyvinyl mercapto) azetidmone by the procedure of example lb. to yield the title phosphorane as a foam, \ _max (CH2 CI2) 1762, 1698, 1638, 1618, 1581, cm" ¹ .

lib p-Nitrobenzyl (55)-Penem-3-carboxylate Ozonolysis of the phosphorane from example 1 la. by the procedure of example lc. provided the title compound as a white crystalline solid from ethyl acetate/hexane, [α]D ² 3-151° (C 1.0, CH ₂ C-2); Vπ^ (CH2CI2) 1800, 1722, 1610

cm" ¹ ; δH (CDCI3) 3.62 (IH, dt J 16.5 and lHz) and 3.90 (IH, dd, J 16.5 and 4Hz). 5.27 (1HΛ J 13.5 Hz) and 5.41 (IH, d, J 13.5Hz), 5.83 (IH, dd, J 2 and 4 Hz), 7.38 (IH, s), 7.60 (2H, d, J 8.7 Hz), 8.23 (2H, d, J 8.7 Hz); 1^^ (EtOH) 316 (ε 7,300), 263nm (εl0,021); m e 306.0310 (Ci3H ₁₀ N2θ ₅ S requires M, 306.0310).

Example 12 - Methyl (55)-penem-3-carboxylate

12a (55)-Penem-3-carboxylic acid p-Nitrobenzyl (55) - penem - 3 - carboxylate (0.250g; 0.817mM) was dissolved in 30% aqueous 1, 4-dioxan (50ml) and shaken for 1 hour at room • temperature and pressure with hydrogen in the presence of 10% palladium on carbon catalyst (0.375g). The reaction suspension, after the addition of a solution of sodium bicarbonate (0.076g) in water (2ml), was filtered over Celite, washing well with water. The filtate was evaporated to small volume and partitioned between ethyl acetate and water. The aqueous layer was separated, evaporated to small volume and applied to a column of Diaion HP20-SS, which was eluted with water. Those fractions which contained the sodium salt of the title acid were combined and freeze- dried to yield a pale yellow solid.

12b. Methyl (55) • Penem - 3 - carboxylate

The sodium salt from example 12a. was suspended in dry dimethylformamide (5ml) and stirred at room temperature for 14 hours with methyl iodide (0.5ml). The solvent was then evaporated at reduced pressure and the residue was partitioned between ethyl acetate and water. The organic solution was washed with brine, dried (MgS04) and evaporated. The residue was chromatographed over silica gel, eluting with a gradient of 10 to 50% ethyl acetate-hexane, to yield the title ester as a white solid, λmax (EtOH) 315 (ε 7081), 255nm (ε 2651);v _max (CH ₂ Cl ₂ ) 1800, 1728, 1568 cm" ¹ ; ^δ H (CDC13) 3.59 (IH, d, J 16.5Hz), 3.8 - 3.95 (4H,overlapping s + dd), 5.80 (IH, dd, / 1.7 and 3.8Hz), 7.28 (IH, s), m e M ⁺ 185.0147 (C7H7NO3S requires: M, 185.0147).

Example 13 - 4-(Methoxycarbonyl)benzyI (5R5)-pen-2-em-3-carboxylate

The method of Example 2 was used with methyl 4-(bromethyl) benzoate to prepare this ester in 40% yield; m.p. 135-7°C (ethyl acetate hexane) _; v _max (CHCI3) 2960, 1800, 1725 sh, 1715 and 1615 cm\' ¹ ; δ _H (CDCI3) 3.60 (IH, dt J 16.6 & 1.6 Hz, 6-H), 3.87 (IH, d, J 16.6 & 4.0 Hz, 6-H), 3.92 (3H, s, OCH3), 5.25 and 5.35 (2H, ABq, J 13.1 Hz, OCH ₂ Ar), 5.81 (IH, dd, J 4.0 and 1.6Hz, 5-H), 7.32 (IH, s, 2-H), 7.47 (2H, d, J 8.2 Hz, Ar-H) and 8.04 (2H, d, J 8.2 Hz, Ar-H); (Found: C,

56.3; H, 4.0; N, 4.5%. requires C, 56.4; H, 4.1; N, 4.4%).

Example 14 - 4-[(4-Nitrobenzyl)oxycarbonyl]benzyl (5R5)-pen-2-em-3- carboxylate

The method of Example 2 was used to prepare the penem ester from 4-nitrobenzyl 4-(bromethyl) benzoate in 53% yield; m.p. 132-5°C (ethyl acetate hexane); v _max (CHCI3) 1800, 1720, 1610, 1560 and 1525 cm\' ¹ ; δκ(CDCl3) 3.60 (IH, dt, / 16.6 & 1.6 Hz, 6-H) 3.87 (IH, ddd, J 16.6, 3.9 & 0.7 Hz, 6-H), 5.26 and 5.37 (2H, ABq, J 13.2 Hz, OCI^Ar), 5.46 (2 s, OO^Ar), 5.81 (IH, dd, J 3.9 & 1.6 Hz, 5-H), 7.33 (IH, s, 2-H), 7.50 (2H, d, J 8.3Hz, Ar-H), 7.60 (2H, d, / 8.7 Hz, Ar-H) 8.09 (2H, d, J 8.3 Hz Ar-H) and 8.26 (2H, d, J 8.7 Hz, Ar-H); (Found: C, 57.6; H, 3.8, N, 6.2%. C2iH ₁₆ N ₂ 7S requires C, 57.3; H, 3.7; N, 6.4%).

Example 15 - Sodium 4-[[(5R5)-pen-2-em-3-carbonyl]oxymethyl]benzoate

A solution of 4-[(4-nitrobenzyl) oxycarbonyl] benzyl (5R5) -pen-2-em-3- carboxylate (33mg) in dioxane (10ml) and water (2.5ml) was hydrogenated at atmospheric pressure for 3 h in the presence of 10% palladium on carbon catalyst (33mg). The reaction mixture was filtered through Celite and a solution of sodium carbonate (4.0mg) in water (5ml) added to the filtrate. After 0.5h the solution was evaporated until it just became turbid and then washed with ethyl acetate. The aqueous solution was freeze-dried to give the title sodium salt as a pale yellow solid (16mg; 65%); x^^. (KBr) 3400, 1786, 1709 and 1597 cm" ^1\' ^ (D2O) 3.62 (IH, brd, / 17.1 Hz, 6-H), 3.87 (IH, dd,/ 17.1 & 3.8 Hz, 6-H), 5.27 (2H, s, OCH ₂ Ar), 5.82 (IH, dd, J 3.8 & 1.7Hz, 5-H), 7.45 (2H, d, / 8.1 Hz, Ar-H), 7.62 (IH, s, 2-H) and 7.83 (2H, d, J 8.1 Hz, Ar-H).

Example 16 - n-Pentyl (5R5)-pen-2-em-3-carboxylate

A solution of 4-nitrobenzyl (5R5)-penem-3-carboxylate (50mg) in dioxane (10ml) and water (2.5ml) was hydrogenated at atmospheric pressure for 2h in the presence of 10% palladium on carbon catalyst (50mg). The product was filtered through Celite and a solution of potassium carbonate (14mg) in water (5ml) added to the filtrate. After a period of 0.25h the solution was evaporated under reduced pressure until just turbid and then washed twice with ethyl acetate. The aqueous solution of the potassium salt was saturated with sodium chloride and then vigorously stirred wim ethyl acetate/tetrahydro-furan 1 : 1 (20ml). The mixture was acidified to

pH 2.0 with 0.5N hydrochloric acid and the organic phase was separated, dried and evaporated. The crude acid was stirred in dry dimethylformamide (1ml) at room temperature under argon and treated with 1-iodopentane (48mg) and caesium fluoride (37mg). After a period of 18h the mixture was evaporated and the residue partitioned between ethyl acetate and dilute aqueous sodium hydrogencarbonate. The organic phase was separated; it was washed with brine and then dried over sodium sulphate and evaporated. The residue was chromatographed on silica gel 60 using ethyl acetate/hexane (4 : 6) as eluent to give the pentyl ester as a gum (lOmg); v _max (CHCI3) 2980, 2950, 2880, 1800, 1715 and 1560cm" ¹ ; ^ (CDCI3), 0.91 (3H, t, J 7.0Hz, CH3), 1.29-1.43 (4H, m, CH ₂ \'s), 1.69 (2H, pent, J 7.OHz, OCH2CH2,), 3-57 (IH, dd J 16.5 & 1.9 Hz, 6-H) 3-85 (IH, dd, J 16.5 & 3.9 Hz, 6-H), 4.12-4.29 (2H, m, OCH ₂ ), 5.79 (IH, dd, / 3.9 & 1.9 Hz, 5-H) and 7.24 (IH, s, 2-H); [Found : M+> 241 (El). CπHι ₅ NO ₃ S requires M, 241].

Example 17 - 4-Phenylbutyl (5R5)-pen-2-em-3-carboxy.ate

The method of Example 16 was used to prepare the title penem ester from (4- iodobutyl) benzene in 24% yield; v _max (CHCI3) 2930, 2860, 1800, 1710 and 1560 cm-1; δH; (CDCI3) 1.67-1.80 (4H, m, OCH ₂ CH ₂ CH2). ² -60-2.70 (2Η, m, CH ₂ Ar), 3.57 (IH, dt 16-6 & 1.7 Hz, 6-H). 3-85 (IH, dd, J 16.6 & 3-9 Hz, 6-H), 4.14-4.30 (2H, m, OCH ₂ ) 5.78 (IH, dd, / 3-9 & 1.7Hz, 5-H), and 7.15-7.34 (6H, m, 2-H and Ar-H\'S): [Found : M+» 303 (El). Ci6Hi ₇ NO3S requires M, 303].

Example 18 - 3-(Methoxycarbonyl)benzyl (5RS)-pen-2-em-3-carboxylate

A solution of 4-nitrobenzyl (5R5)-peneπ_-3-carboxylate (50mg) in dioxane

(10ml) and water (2.5ml) was hydrogenated at atmospheric pressure for 2h in the presence of 10% palladium on carbon catalyst (50mg). The product was filtered through Celite and a solution of potassium carbonate (14mg) in water (5ml) added to the filtrate. After a period of 0.25h the solution was evaporated under reduced pressure until just turbid and then washed twice with ethyl acetate. The aqueous solution of the potassium salt was saturated with sodium chloride and then vigorously stirred with ethyl acetate/tetrahydro-furan 1:1 (20ml). The mixture was acidified to pH. 2.0 with 0.5N hydrochloric acid and the organic phase was separated, dried and evaporated. A solution of this crude acid in dry DMF (1ml) was treated with methyl 3-(bromomethyl) benzoate (45mg) and powdered potassium carbonate (14mg). The reaction mixture was stirred at room temperature under argon for 18h and was then evaporated. The residue was partitioned between ethyl acetate and brine; the organic phase was separated and then washed with brine and dried over sodium sulphate. The

solution was evaporated and the residue was chromatographed on silica gel 60 using ethyl acetate/hexane (4:6) as eluant to give the penem ester (25mg); m.p. 100-2° C(ethyl acetate hexane); v _max . (CHCI3) 2960, 1800, 1725 and 1560 cm" ¹ ; δjj (CDCI3) 3.59 (IH, dt / 16.6 & 1.9 Hz, 6-H), 3.86 (IH, dd, / 16.6 & 3.9 Hz, 6-H), 3.93 (3H, s, OCH3), 5.25 and 5.33 (2H, ABq, J 12.6 Hz, OCH ₂ ), 5.80 (IH, dd, / 3.9 & 1.9 Hz, 5-H), 7.30 (IH, s, 2ϋ), 7.46 (IH, t, / 7.7Hz, Ar5\'-H, 7.62 (IH, d, / 7.7 Hz, AT 6\'-H), 8.01 (IH, d, /7.7Hz, Ar4\'-H) and 8.07 (IH, s, Aτ2\'-H): (Found: C, 56.9; H, 4.0; N, 4.6%. requires C, 56.4; H. 4.1; N, 4.4%).

Example 19 - 2-(2-Methoxyethoxy)ethyl (5R5)-pen-2-em-3-carboxylate

The method of Example 18 was used to prepare the penem ester from 1 -iodo-

2-(2-methoxyethoxy) ethane as a gum in 16% yield; δH(CDCl3) 3.39 (3H, s, OCH3), 3.53-3.69 (5H, m, 6-H and OCH2CH2OMe), 3.76 (2Η, t, / 4.9 Hz, CO ₂ CH ₂ CH2θ), 3.85 (1Η, dd, / 16.5 & 3.9 Ηz, 6-Η), 4.35 and 4.41 (2H, 2dt, /12.0 & 4.9 Hz, CO ₂ CH ₂ ), 5.79 (1Η, dd, J3.9 & 1.9 Ηz, 5-Η) and 7.30 (IH, s, 2-H); [Found: M+, 273 (EI). C11H15NO5S requires M, 273].

Example 20 - 4-Bromobenzyl (5R5)-pen-2-em-3-carboxylate

The method of example 18 was used to prepare the penem ester from 4- bromo-l-(bromomethyl) benzene in 54% yield; m.p. 108°C (ethyl acetate/hexane); v _max . (CHCI3) 1800, 1720 and 1565 cm\' ¹ ; *H (CDCI3) 3.58 (IH, dt / 16.6 & 1.8 Hz, 6-H), 3.86 (IH, ddd, / 16.6, 4.0, 0.6 Hz, 6-H), 5.15 and 5.24 (2H, ABq, / 12.6 Hz, CH ₂ Ar), 5.79 (IH, dd, / 4.0 & 1.8 Hz, 5-H), 7.28 (2H, d, / 8.2 Hz, Ar-H), 7.29 (IH s, 2-H) and 7.50 (2H, d,/ 8.2 Hz, Ar-H); (Found: C.45.8; H, 2.8; N, 4.2%. Ci3Hκ)BrN03S requires C, 45.9; H, 3.0; N, 4.1%).

Example 21 - (7-Methoxy-2-oxo-2H-l-benzopyran-4-yl)methy. (5RS)-pen-2-em- 3-carboxylate

The method of Example 18 was used to prepare the penem ester from 4-

(bromomethyl)-7-methoxycoumarin in 51% yield; m.p. 170-2°C; v _max (KBr) 1786, 1723, 1710, 1614, 1560 and 1510 cm" ¹ ; δ _H [(CD3) ₂ S0/C ₅ D ₅ N 1:1] 3.75 (IH, brd, / 16.8 Hz, 6-H), 3.91 (3H, s, OCH3), 4.08 (IH, dd/ 16.8 & 4.0Hz, 6-H), 5.58 and 5.74 (2H, 2dd, J 16.0 & 1.3 Hz, O^Ar), 5.99 (IH, dd, / 4.0 & 1.8, 5-H), 6.64 (IH, s, 2- H), 7.01-7-12 (2H, m, 6\'-H & 8\'-H), 7.77 (IH, d, / 8.8 Hz, 5\'Η) and 8.08 (IH, s, 3 ^* - H); [Found: M H+, 360 (NH3DCI). Cι ₇ H ₁₃ NO ₆ S requires M, 359].

Example 22 - Methoxymethyl (5R5)-pen-2-em-3-carboxylate

The method of Example 18 was used to prepare the penem methoxymethyl ester from chloromethyl methyl ether in 36% yield; m.p. 109-110°C (ethyl acetate/hexane); v _max (CHCI3) 2970, 1800, 1720 and 1560 cm" ¹ ; ^~ ~ (CDCI3) 3.52 (3H, s, OCH3) 3.62 (IH, dt / 16.6 & 1.9Hz, 6-H), 3.87 (IH, dd, / 16.6 & 3.9Hz, 6- H), 5.30 and 5.44 (2H, ABq, / 5.9 Hz, OCH ₂ O), 5.81 (IH, dd, / 3.9 & 1.9 Hz, 5-H) and 7. 35 (lH, s, 2-H); (Found: C, 44.8; H, 4.0; N, 6.5%; M+ 215. C ₈ H ₉ NO4S requires C, 44.7; H.4.2; N, 6.5; Λ , 215).

Example 23 - Methoxymethyl (55)-pen-2-em-3-carboxylate

The racemic methoxymethyl ester described in example 22 was subjected to preparative h.p.l.c. on a chiral stationary phase according to the following conditions:

Sample Presentation: Smgml" ¹ Ethanol 1ml injection

The second of the enantiomers to be eluted from the column was the required (55)-penem; [α] ²⁵ £> - 258.

Example 24 - Allyl (5R5)-6-(l-acetoxyethyl)penem-3-carboxylate

24a. Allyl [3 - d-hydroxyethyl)-4-trityIthio-2-oxoazetidin-l-yI] triphenylphosphoranyiidene-acetate

Allyl [(4R5) - tritylthio-2-oxoazetidin-l-yl] triphenyl phosphoranylidene acetate (3.0g; 4.27mM) was dissolved in dry tetrahydrofuran (75ml) and cooled to - 78°C under an atmosphere of argon. A solution of lithuim diiosopropylamide (5.34ml of a 2.0M solution in heptane/tetrahydrofuran/ethylbenzene) was added and the solution was stirred at -78°C for 30 minutes. A solution of acetaldehyde (1.2ml)

in tetrahydrofuran was men added and stirring was continued at -78°C for an additional 30 minutes before quenching the reaction by the addition of glacial acetic acid (2ml). The reaction solution was partitioned between ethyl acetate and water and allowed to reach room temperature. The organic solution was washed with IN hydrochloric acid solution, water, saturated sodium hydrogen carbonate solution, water, brine, dried (MgSθ4) and evaporated at reduced pressure. The residue was chromatographed over silica gel, eluting with a gradient of 10 to 75% ethyl acetate - hexane, to yield the diastereoisomeric mixture of title phosphoranes as a pale yellow foam (2.44g), v _max (CH2CI2) 1755, 1622cm" ¹ ; m/e (ammonia DO) 748 (MH ⁺ ).

24b. Allyl [3-(l-acetoxyethyl)-4-tritylthio-2-oxoazetidin- ^* 1-yl] triphenyiphosphoranylidene acetate

The diastereoisomeric mixture of alcohols from example 24a. (2.44g) was dissolved in dry dichloromethane (200ml) and stirred at room temperature for 16 hours witii triethylamine (1.32ml), acetic anhydride (0.96ml) and 4-dimethylamino¬ pyridine (0.25g). The organic solution was then washed with IN hydrochloric acid solution, water, saturated sodium hydrogen carbonate solution, brine, dried (MgSθ4) and evaporated. The residue was chromatographed over silica gel, eluting with ethyl acetate - hexane mixtures to yield the diastereoisomeric title phosphoranes as a white foam (1.48g) v _max (CH ₂ α ₂ ) 1755, 1740, 1620 cm" ¹ .

24c AllyI(5R5)-6-(l-acetoxyethyl)penem-3-carboxylate

The diastereoisomeric phosphoranes from example 24b. (0.750g; 0.95mM) were

dissolved in dry acetonitrile (25ml) and stirred at room temperature for 2 hours with 4-dimemylaminopyridine (0.139g) and a solution of silver nitrate (0.363g) in acetonitrile. Acetic-formic anhydride (0.8ml), 4-dime-hylaminopyridine (0.118g) and sodium iodide (1.42g) were then added and stirring was continued at room temperature for 1 hour. The reaction solution was partitioned between ethyl acetate and water. The organic layer was washed with saturated sodium hydrogen carbonate solution, water, brine and dried (MgSθ4). The solution was allowed to stand at room temperature for 30 minutes before evaporating at reduced pressure. The residue was chromatographed over silica gel (25g) eluting with a gradient of 10 to 50% ethyl acetate-hexane.

The first eluted component was a single pure isomer (isomer A) of the title compound, obtained as a colourless gum (O.OlOg), v _max (CH2CI2) !800, 1748, 1725 cm" ¹ ; δ _H (CDCI3) 1.53 (3H, d / 6 Hz), 2.07 (3H, s), 4.07 (IH, ddd, / 0.7, 3.8, 10.3 Hz), 4.6 - 4.8 (2H, m), 5.2-5.45 (3H, m), 5.84 (IH, d, / 4.0Hz), 5.85-6.05 (IH, m), 7.31 (IH, d. 0.8Hz).

The second eluted component was also a pure isomer (isomer B) of the title penem (0.041g), v _max (CH ₂ C1 ₂ ) 1801, 1748, 1728cm" ¹ ; δ _H (CDCI3) 1.45 (3H, d, J 6.5 Hz), 2.12 (3H,s), 4.03 (IH, overlapping ddd, / 1,2, 4.5 Hz), 4.6-4.85 (2H,m), 5.2- 5.45 (2H,m), 5.62 (IH, d, / 2Hz), 5.88-6.06 (lH,m),7.24 (IH, d, / 1Hz). Continued elution gave a mixture of isomers (0.039g), followed by a third pure isomer (isomer C) of the title penem (0.056g), v _max (CH2CI2) 1802, 1748, 1722 cm" ¹ ; δ _H (CDCI3) 1.37 (3H, d J 6.3 Hz), 2.09 (3H,s), 4.08 (IH, dd / 4.4, 7.8 Hz), 4.6-4.82 (2H,m), 5.25-5.58 (3H,m), 5.82 (IH, d, /4.4Hz), 5.88-6.05 (lH,m), 7.28 (lH, d, /0.7 Hz).

Example 25 - p-Nitrobenzyl (5RS, 6RS)-6-[(SΛ)-l-acetoxyethyl]-2-(2- hydroxyethylthio)penem-3-carboxylate

A solution of p-nitrobenzyl [3- (15R-acetoxyethyl)-4- (2-hydroxyethyl trithiocarbonyl)-2-oxoazetidin-l-yl] triphenylphosphoranylidene acetate (4.47g) [Prepared by methods described in EP 0, 041, 768 (16, 12.81)] in xylene (1 1) was heated at reflux under argon for 5.5h. The reliction mixture was evaporated to dryness and chromatographed twice on silica gel eluting with ethyl acetate/methylene chloride (1:9) and ether/ethyl acetate (9 : 1) to give the title compound (297mg), mp 151-152°, λmax (EtOH) 260 nm, 333 nm; v _max (CHCI3) 1795, 1730, 1690 cm\' ¹ ; δ H(CDC1 ₃ ) 1.55 (3H, d, J 6Hz). 2.08 (3H, s), 2.14 (IH, br s), 3.11-3.32 (2H,m), 3.85- 3.95 (2H,m), 4.03 (IH, dd, J 4.0 and 9.8 Hz), 5.22 and 5.47 (2H, ABq, J 13 Hz) 5.19- 5.31 (IH, m), 5.76 (IH, d, J 4 Hz), 7.61(2H, d, J 9Hz), 8.23 (2H, d J 9 Hz) (Found: M ⁺ 468.0647, C19H20N2O8S2 requires M, 468.0657).

Example 26 - Methyl (5R5)-2-pheny.penem-3-carboxy.ate

26a (4R5)-Benzoylthioazetidin-2-one

Thiobenzoic acid (1.52g, llmM) was dissolved in dichloromethane (30ml) and the solution cooled to -10°C. Sodium hydroxide (0.44g, llmM) was dissolved in water (30ml) and added to the cooled solution above. After 5 min. 4- acetoxyazetidin-2-one (1.29g, lOmM) as a suspension in dichloromethane (20ml) was added to the cooled reaction mixture which was then allowed to warm to room temperaure and stirred for 16 hrs. The reaction mixture was then acidified to pH3 with 5M hydrochloric acid and the dichloromethane layer separated. The aqueous layer was extracted wim dichloromethane (3 x 50ml) and the organic layers were combined and washed with saturated aqueous sodium bicarbonate solution, followed by drying over anhydrous magnesium sulphate. The dichloromethane solution was filtered and evaporated to give a pale yellow solid which was purified by column chromatography on silica-gel 60, eluting with ethyl acetate hexane mixture (1 : 1). The title compound was isolated (1.54g) as a white solid. v _max (CH2CI2) ^85,

1670, cm\' ¹ ; 5H(CDC1 ₃ ) 7.93 (2H,m,Ar), 7.63 (lH,m,Ar), 7.50(2H,m,Ar), 6.55 (IH, br s, N-H), 5.41 (IH, dd, J 5.3, 2.5 Hz, 4-H), 3.55 (IH, ddd, J 15.2, 5.2, 2 Hz, 3-H) and 3.11 (IH, ddd, J 15.3, 3, 2.5 Hz, 3-H); MS (El) m/z 207.

26b (4R5)-Benzoylthio-l-(l-hydroxy-l-methoxycarbonylmethyl)-azet idin-2-one Methyl glyoxylate (1.76g, 16mM) was dissolved in dry benzene (80ml) and refluxed with provision for azeotropic removal of water for 1 hr. The solution was allowed to cool to room temperature and 4-benzoylthio-azetidin-2-one (l.Og, 4.8mM) was added followed by 3 drops of triethylamine. The reaction mixture was stirred for 1 hr. and then evaporated, taken up in toluene (15ml), filtered to remove triethylamine hydrochloride and re-evaporated to give the title compound (1 : 1 mixture of epimers) which was used in the next step without purification.

26c (4R5)-Benzoylthio-l-(l-chloro-l-methoxycarbonylmethyl)-azeti din-2-one

4-Benzoylthio-l-(l-hydroxy-l-methoxycarbonylmethyl)azetid in-2-one (4.8mM), as a crude (1 : 1) mixture of epimers, was dissolved in dry tetrahydrofuran (50ml) and the solution cooled to -10°C. 2,6-Lutidine (0.84ml) followed by thionyl chloride (0.52ml) were added. There was an immediate white precipitate and stirring was contined at -10°C for 1 hr. The reaction mixture was filtered to remove lutidine hydrochloride, and the solvent evaporated to leave a yellow oil which was re- evaparated from toluene (2 x 10ml) to remove thionyl chloride residues and used in the next step without further purification.

26d (4R5)-Benzoylthio-l-(l-methoxycarbonyl-l-

triphenylphosphoranylidenemethyl)-azetidin-2-one

4-Benzoylthio- 1 - ( 1 -chloro- 1 -methoxycarbonylmethyl)-azetidin-2-αιιe (4.8mM) was dissolved in dry dioxan (30ml) and triphenylphosphine (2-52g, 9.6mM) was added. The solution was evaporated to give a syrup and this was stirred at loom temperature and 2,6-lutidine (0.67ml, 5.7mM) was added. Stirring was continued for 16 hrs. and then the reaction mixture was diluted with ethyl acetate (30ml) and filtered to remove 2,6-lutidine hydrochloride. The organic layer was washed with IN hydrochloric acid (1 x 10ml), brine (1 x 10ml) and dried over anhydrous magnesium sulphate. The solution was filtered and evaporated to give a brown solid which was purified by column chromatography on silica gel 60 eluting with ethyl acetate-nexane mixture (1 : 1) giving the title compound as a white foam (1.59g).

26e Methyl (5RS)-2-phenylpenem-3-carboxylate

(4R5)-Benzoylthio- 1 -( 1 -methoxycarbonyl- 1 -triphenylphosphoryanylidene methyl)-azetidin-2-one (1.59g) was dissolved in dry toluene (100ml) and the solution refluxed for 4 hrs. The reaction mixture was allowed to cool and evaporated to give a cream solid which was purified by column chromatography on silica-gel 60 eluting with ethyl acetate-hexane mixtures (1: 1) to give the title compound (0.515g) as a white solid. v _max (CHCI2) 1800, 1720 cm" ¹ ; ^δ H(CDCl3) ca, 7.45 (5H, m, Ar), 5.77 (IH, dd, J 3.9, 1.8 Hz, 5-H) 3.85 (IH, dd, J 16.4, 3.8 Hz, 6-H), 3.72 (3H, s, CO2CH3) and 3.59 (IH, dd, J 16.5 1.8 Hz, 6-H); MS m/z 261, (Found: M+261.0464 C13H11NO3S requires M+ 261.0460).

Example 27 • Methyl (5R5)-2-(4-methoxyphenyl)penem-3-carboxylate

Prepared by a method analogous to example 26. v _max (CH2C-2) 1 8, 1718, cm" ¹ , δ _H (CDCI3) 7.51 (2H, d, J 8.8 Hz, Ar), 6.99 (2H, d, J 8.9 Hz, Ar), 5.70 (IH, dd, J 3.8, 1.8 Hz, 5-H), 3.84 (3H, s, CO2CH3), 3.83 (IH, dd, J 16.1, 3.8Hz, 6-H), 3.54 (3H, s, OCH3) and 3.56 (IH, dd, J 16.2, 1.6Hz, 6-H); MS m z 291, (Found: M+291. 0568, C14H13NO4S requires M+291.0565).

Example 28 - Methyl (5R5)-2-(4-chlorophenyl)penem-3-carboxylate

Prepared by a method analogous to example 26. v _max (CH2CI2) 1798, 1720 cm-1; δH(CDCl ₃ ) 7.44 (2H, d, J 8.6 Hz, Ar), 7.37 (2H, d, J 8.6 Hz, Ar), 5.77 (IH, dd, J 3.8, 1.6 Hz, 5-H), 3.87 (IH, dd, J 16.4, 3.8 Hz, 6-H), 3.73 (3H, s, CO2CH3) and 3.60 (IH, dd, J 16.4, 1.6 Hz, 6-H); MS m/z 295, (Found: M+295.0067, C13H10NO3CIS requires M+ 295.0070).

Example 29 - Methyl (5R5)-2-(4-methoxycarbonylphenyl)penem-3-carboxylate

Prepared by a method analogous to example 26. v _max (CH2CI2) 1805, ^{1 2} cm\' ¹ ; 5H(CDC1 ₃ ) 8.05 (2H, d, J 6.8 Hz, Ar), 7.52 (2H, d, J 6.8 Hz, Ar), 5.80 (IH, dd, J 3.8, 1.7 Hz 5-H), 3.93 (3H, s, Ar CO ₂ CH ₃ ), 3.89 (IH, dd, J 16.5, 3.9 Hz, 6-H), 3.70 (3H, s, CO2CH3) and 3.62 (IH, dd, J 16.48, 1.8 Hz, 6-H); MS m/z 319, (Found: M+319.0517 requires M ⁺ 319. 0514).

Example 30 - Methyl (5R5)-2-benzylpenem-3-carboxylate

Prepared by a method analogous to example 26. v _max (CH2CI2) 1800, 1718 cm" ¹ ; 5H(CDC1 ₃ ) ca 7.28 (5H, m, Ph), 5.58 (IH, dd, J 3.8, 1.5 Hz, 5-H), 4.23 (IH, d, J 15.3 Hz, CH2-Ph) 4.13 (IH, d, J 15.2 Hz, CH ₂ Ph), 3.87 (3H, s, CO2CH3) 3.74 (IH, dd, J 16.3, 3.8 Hz, 6-H) and 3.45 (IH, dd, J 16.3, 1.5 Hz 6-H); MS m/z 275, (Found: M ⁺ 275.0618 C ₁₄ H ₁₃ NO3S requires M+ 275.0616.

Example 31 - Benzyl (5R5) -2-(hydroxymethyl)penem-3-carboxylate

The title compound was prepared by a method analogous to that described in UK Patent 2,037, 277B. v _max (CH ₂ α ₂ ) 1798, 1718 cm" ¹ ; δH(CDCl3) ca 7.38 (5H, m, Ph), 5.65 (IH, dd, J 3.9 1.7 Hz 5-H), 5.30 (IH, d, J 12.7 Hz, CH2 ^~ Ph), 5.24 (IH, d, J 12.7 Hz, CH ₂ Ph), 4.68 (lH,d, J 15.7 Hz, CH2OH), 4.60 (IH, d, J 16 Hz, CH ₂ OH), 3.80 (IH, dd, J 16.5, 3.9 Hz, 6-H) and 3.54 (IH, dd, J 16.4, 1.7 Hz, 6-H); MS m/z 291 (Found: M+291-0563 C14H13NO4S requires 291.0565).

Example 32 - Benzyl (5R5)-2-(carbamoyloxymethyl)penem-3-carboxylate

Benzyl (5R5)-2-hydroxymethyl-penem-3-carboxylate (57mg) was dissolved in dry dichloromethane (1ml) and trichloroacetyl-isocyanate (25μl) was added. The reaction mixture was stirred at room temperature for 30 min. under argon and then diluted with ethyl acetate (10ml) and washed wim saturated aqueous sodium bicarbonate (5ml), water (5ml) and brine (5ml). The organic layer was dried over anhydrous magnesium sulphate and evaporated to give a brown oil. This oil was taken up in methanol (1ml) and stirred for 5 hrs. at room temperature after the addition of 2,6-lutidine (46μl). The reaction mixture was then diluted with ethyl acetate (10ml) and washed with 5% citric acid (5ml), saturated aqueous sodium bicarbonate (5ml), water (5ml) and brine (5ml). The organic layer was dried over anhydrous magnesium sulphate and evaporated to give an oil which was purified by column chromatography on silica-gel 60 eluting with ethyl acetate-hexane mixtures

(1 : 1) yielding the title compound as a gummy solid (23mg). v _max (CH2CI2 1805, 1748, 1715 cm" ¹ ; ^δ H(CDCl3) ca 7.5-7.25 (5H, m, Ph), 5.67 (IH, dd, J 3.9, 1.8 Hz, 5- H), 5-47 (IH, d, J 15.5 Hz, CH2OCONH2), 5.30 (IH, d, J 12.5 Hz, CH Ph), 5.14 (IH, d, J 12.5 Hz, CH ₂ Ph), 5.11 (IH, d, J 15.5 Hz, CH ₂ OCONH ₂ ), 4.70(2H, br s, NH ₂ ) 3.80(1H, dd, J 16.5, 3.9 Hz, 6-H) and 3.53 (IH, dd, J 16.4, 1.7 Hz, 6-H); MS (NH3 C.I.) MNH4+ 352.

Example 33 - Methyl (5R5)-2-(carbarnoyloxymethyl)penern-3-carboxyIate

The title compound was prepared by a method analogous to that described in example 32. v _max (CH ₂ C1 ₂ ) 1805, 1745, 1718, cm" ¹ ; ^δ H (CDCI3) 5.68 (IH, dd, J 3.9, 1.6 Hz, 5-H), 5.47 (IH, d, J 15.4 Hz, CH ₂ OCONH ₂ ), 5.11 (IH, d, J 15.4 Hz, CH2OCONH2). ⁴ -8 ( ² H, br.s. NH ₂ ) 3.84 (3H, s, CO2CH3), 3.77 (IH, dd, J 16.5, 3.9 Hz, 6-H), and 3.54 (IH, dd, J 16.5, 1.9Hz, 6-H); MS (NH3 C.I.) MNH4+ 276.

Example 34 - Benzyl (5R5)-2-phenylpenem-3-carboxylate

The title compound was prepared by a method analogous to that described in example 26. v _max (CH ₂ Cl2) 1800, 1718 cm" ¹ ; ^δ H(CDCl3) 7.50 - 7.15 (10H, m, Phx2), 5.77 (IH, dd, J 3.8, 1.7 Hz, 5-H), 5.17 (IH, d, J 12.6 Hz, CH ₂ Ph), 5.10 (IH, d, J12.6 Hz, CH ₂ Ph), 3.84 (IH, dd, J 16.4, 3.9 Hz, 6-H) and 3.59 (IH, dd, J 16.4, 1.8 Hz, 6-H); MS m/z M ⁺ 337, (Found: M+337.0776, Ci9H ₁₅ NO ₃ S requires M ⁺ 337.0773).

Example 35 - Benzyl (5R5)-2-(4-chlorophenyl)penem-3-carboxylate

The title compound was prepared by a method analogous to that described in example 26. v _max (CH2CI2) ¹⁸ 00, 1718 cm" ¹ ; H(CDCl ₃ ) 7.45-7.15 (10H, m, 2xPh), 5.77 (IH, dd, J 3.8, 1.7 Hz, 5-H), 5.15 (IH, d, J12.6 Hz), CH ₂ Ph), 5.09 (IH, d, J 12.6 Hz, CH ₂ Ph), 3.84 (IH, dd, J 16.4, 3.8 Hz, 6- H) and 3.60 (IH, dd, J 16.4, 1.8 Hz, 6-H); MS m/z H+ 371, (Found: M+ 371.0383, CιoH ₁₄ αNθ3S required M ⁺ 371.0383).

Example 36 - Benzyl (5R5)-2-methyl penem-3-carboxylate

The title compound was prepared by a method analogous to that described in example 26. v _max (CH2CI2) 1798, 1720 cm" ¹ ; ^δ H(CDα ₃ ) 7.5 - 7.3 (5H, m, Ph), 5.62 (IH, dd, J 3.9, 1.8 Hz, 5-H), 5.30 (IH, d, J 12.4 Hz, CH ₂ Ph), 5.22 (IH, d, J 12.5 Hz, CH ₂ Ph), 3.76 (IH, dd, J 16.2, 3.9 Hz, 6-

H), 3.46 (IH, dd, J 16.4, 1.8 Hz, 6-H) and 2.35 (3H, s CH3); MS m z M+ 275, (Found: M+275.0620, C ₁₄ H ₁₃ NO3S requires M+ 275.0616).

Example 37 - Benzyl (5R5)-2-methy.penem-sulphoxide-3-carboxylate

Benzyl (5R5) -2-methyl penem-3-carboxylate (27.5mg) was dissolved in dichloromethane (5ml) and cooled to 0°C. m-Chloro-perbenzoic acid (19mg) was added and the reaction mixture stirred at 0°C for 2hr. Dichloromethane (60ml) was added and the organic phase washed with 10% aqueous sodium sulphate solution (15ml), 10% aqueous sodium carbonate solution (15ml) and brine (20ml) before being dried over anhydrous magnesium sulphate. The solution was filtered and evaporated to give the title compound as a clear oil (12mg) (5 : 1 ratio of isomers) ^δ H (CDCI3), 7.5-7.3 (5H, m, Ph), 5.35 (2H, s, CH ₂ Ph), 4.97 (IH, dd J 5.5, 3.0 Hz, 5-H, minor isomer), 4.82 (IH, dd, J 5.7, 3.3 Hz, 5-H, major isomer), 3.64 (IH, dd, J 16.9, 3.4 Hz, 6-H major isomer) 3.46 (IH, dd, J 16.8, 5.7 Hz, 6-H major isomer), 2.57 (3H, s, CH3 major isomer) and 2.42 (3H, s, CH3 minor isomer); MS m z M+291 (Found: M+ 291.0566, C1 H13NO4S requires 291.0565).

Example 38 - p-Nitrobenzyl (5R5)-2-(methoxymethoxymethyl)penem-3- carboxylate

A solution of the alcohol (Example 9) (lOOmg, 0.3 mmol), 2,6-lutidine (320 mg, 3 mmol) and bromomethyl methyl ether (375 mg, 3 mmol) in dichloromethane (20ml) was refluxed under argon for an hour. The reaction mixture was washed with sodium bicarbonate solution followed by citric acid solution and was then dried and evaporated. Chromatography on silica gel, eluting with ethyl acetate/petroleum mixtures afforded the title compound (65 mg), as a light yellow amorphous solid. m.p. 94-5° (light yellow needles from ethyl acetate- /petroleum), max (EtOH) 261 (12,400), 318 (10,000) nm. v _{max <} (CHCI3) 1795, 1705, 1580 cm" ¹ . δ _H (CDCI3) 3.36 (3H, s), 3.49 (IH, dd, / = 2, 16Hz), 3.83 (IH, dd, / = 4, 16Hz), 4.62 (2H, s),

4.64 and 4.88 (2H, centres of ABq, / = 16Hz); 5.20 and 5.44 (2H, centres of ABq, / = 13Hz), 5.65 (IH, dd, / = 2, 4Hz), 7.59 (2H, d, / = 8Hz), 8.20 (2H, d, / = 8Hz). (Found: C, 50.26; H, 4.14; N, 7.14; S, 8.60%. Cι ₆ H ₁₆ N2θ ₇ S requires: C, 50.52; H, 4.24; N, 7.37; S, 8.43%).

Example 39 - Acetonyl (5R5)-2-(4-nitrobenzy.oxycarbony_aminomethyl)per Λ 3-carboxylate

The title compound was prepared by a procedure analogous to that decribed

for example 26; m.p. (dioxane petroleum) 180-2°; v _max (nujol) 1810, 1735, 1700cm- ¹ ; λm∞. (EtOH) 317nm (ε 8740); δ _H (d6-DMSO) 2.10 (3H, s), 3.48 (IH, dd, / 2, 16Hz), 3.87 (IH, dd, / 4, 16Hz), 4.38 (2H, d, / 6Hz), 4.83 (2H, s), 5.21 (2H, s), 5.70 (IH, dd, / 2, 4Hz), 7.60 (2H, d, / 8Hz), 8.04 (IH, t, / 6Hz), 8.23 (2H, d, / 8Hz); mlz (Found M+ 435.0736: Cι ₈ H ₁₇ N ₃ O ₈ S requires M+ 435.0736); (Found: C, 49.6; H, 4.0; N, 9.5%. C ₁₈ H ₁₇ N3θ ₈ S requires: C, 49.7; H, 3.9; N, 9.7%).

Example 40 - /»-Nitrobenzyl (5R5)-2-(4- nitrobenzyloxycarbonylaminomethyl)penem-3-carboxylate

The title compound was prepared by a procedure analogous to that described for example 26; v _max> (CHC1 ₃ ) 1795, 1720 cm" ¹ ; mlz M+ 514 (Found M+ 514.0776. C32H18N4O9S requires 514.0794).

Example 41 - Allyl (55, 65)-6-[l-(R)-hydroxyethyl]penem-3-carboxylate

41a Allyl [(35, 4R)-3-[l-R-(t-butyIdimethylsilyIoxy)ethyI)]-2-oxo-4- (trityIthio)azetidin-l-yl]triphenyIphosphoranylidene acetate

A solution of (35, 4R)-3-[l-R-(t-butyldimethylsilyloxy)ethyH4-(trityl- thio)azetidin-2-one (lg) and allyl glyoxylate (342 mg) in benzene (10 ml) was refluxed under a Dean-Stark trap for 1 hour. The mixture was cooled to room temperature, treated with triethylamine (0.1 ml), left for 11/2 hours and evaporated. The residue was taken up in THF (18 ml), cooled to -20°C and treated with 2,6- lutidine (0.47 ml) and thionyl chloride (0.234 ml). After warming to room temperamre, the mixture was filtered through celite and the filtrate evaporated. The residue was taken up in benzene (50ml) and evaporated, then treated with dioxan (1.5 ml), 2,6-lutidine (0.47 ml) and triphenylphosphine (4g) and stirred overnight at 40°C. The mixture was taken up in ethyl acetate (100 ml), washed with 0.5N hydro- chloric acid (100 ml) and water, dried and evaporated. The residue was chromatographed on silica, eluting with ethylacetate/hexane 1 :3, to provide the title compound as a pale yellow foam (1.2g). v _maX4 (CHCI3) 1750, 1610 cm"l.

41b Allyl (5R, 65)-6-[l-i?-(t-butyldimethylsilyloxy)ethyl]penem-3-carboxyla te

The phosphorane (from 41a) above (1.2 g) was dissolved in dichloromethane (1-5 ml)/methanol (10 ml) and treated with pyridine (0.146 ml) and a 0.15 M solution of silver nitrate in methanol (12.1 ml). After stirring 2 hour at room temperature, the solution was evaporated and the residue taken up in chloroform (100ml) and evaporated. The residue was redissolved in dichloromethane (17.5 ml), ice-cooled, and treated sequentially with acetic formic anhydride (1.1 ml) 4-dimethylamino-

pyridine (0.17 g) and triethylamine hydrochloride (1.93 g). After vigorous stirring for I/2 hour, the mixture was filtered through celite and the filtrate diluted with ethyl acetate (100 ml), washed with 0.5N hydrochloric acid (100ml), water (50ml) and sodium bicarbonate solution (50 ml), dried and evaporated. The residue was taken up in small volume of toluene, allowed to stand for 11/2 hours at room temperature and applied to a silica column. Elution with 5% then 10% ethyl acetate/hexane provided the title compound (437 mg). Crystallisation from hexane/pentane gave colourless needles, m.p. 91-3°C. (Found: C 55.3, H, 7.6, N 3.8, S 8.7%. C ₁₇ H2 NO4SSi requires C 55.3, H, 7.4, N 3.8, S 8.7%) v _max . (KBr) 1771, 1705, 1650, 1557cm" ¹ .

41c Allyl (55, 65)-6-[l-R-(t-butyldimethyIsiIyloxy)ethyl]penem-3- carboxylate

Allyl (5R, 65)-6-[l-R-(t-butyldimethylsilyloxy)ethyl]penem-3-carboxylat e (247 mg) was dissolved in 100 ml of ethyl acetate and placed in the vessel of a Hanovia photochemical reactor. Argon was bubbled through the stirred solution for 2 hours, and then the solution was irradiated with a medium pressure UN lamp through a pyrex filter for 2 hour. The solution was evaporated and the residue chromatographed on silica, eluting with 5% then 8% ethyl acetate hexane. The first penem to be eluted was the title compound, obtained as a solid (112 mg). v _max> (KBr) 1796, 1712, 1650, 1557cm" ¹ . FAB MS (matrix 3-ΝOBA/Νa) mlz 392 (100%, MNa+)._ [α] _D ²¹ - 214° (c. .68; CHCI3).

41d Allyl (55, 65)-6-[l-(Jf)-hydroxyethyl]penem-3-carboxylate

Allyl (55, 65)-6-[l-R-(t-butyldimethylsilyloxy)ethyl]penem-3-carboxylat e (11 mg) was treated with acetic acid (0.036 ml) and then a 1M solution of tetrabutylammonium fluoride in tetrahydrofuran (0.2 ml). After stirring at room temperature for 4 hours, the mixture was diluted with ethyl acetate (5 ml), washed with water (5 ml), sodium bicarbonate solution (5 ml) and water (5 ml), dried and evaporated. The residue was chromatographed on silica, eluting with ethyl acetate hexane 1:2 to provide the title compound as a colourless gum (6.4 mg). v _max (CHCI3) 3600, 3500, 1795, 1710, 1650, 1555 cm" ¹ , δπ (CDCI3) 1.48 (3H, d, / 6Hz), 1.83 (IH, broad s), 3.89 (IH, ddd,/ 10, 4 and .7 Hz), 4.2-4.5 (IH, broad m), 4.6-4.8 (2H, m), 5.2-5.5 (2H, m), 5.84 (IH, d, /4Hz), 5.85-6.0 (IH, m), 7.31 (IH, d, / .7 Hz). MS (E.I.) mlz 255 (10%, M+), 170 (100%), 112 (80%).

Example 42 • Allyl (55R, 6R5)-6-ethylpenem-3-carboxylate

42a (3R5, 45R)-3-Ethyl-4-tritylthio-N-f.butyldimethylsilyl-azetidin- 2-one

A solution of 4-tritylthio-N-t-butyldimethylsilyl-azetidin-2-one (3g) in dry tetrahydrofuran (20 ml) was added dropwise to a solution of LDA (diisopropylamine 1 ml + 0.83 M n-butyl lithium 8.6 ml) in tetrahydro- furan (15 ml) cooled to -76° (bath temperature) under argon. After 15 min. ethyl iodide (3 ml) was added. The reaction was diluted with ethyl acetate after 0.5 hr, washed sequentially with saturated ammonium chloride solution and brine before being dried (MgSO4) and evaporated. Chromatography on S1O2 eluting with n-hexane/ethyl acetate (20:1) gave the title compound (3.02 g), δ _H (CDCI3) 0.22 (6H, s), 0.68 (3H, t / 7.2 H3), 0.90-1.12 (1 IH, m), 3.04-3.12 (IH, m), 3.9 (IH, d, / 1.63 Hz), 7.18-7.5 (15H, m).

42b Allyl (55R, 6RS)-ό-ethy.-penem-3-carboxy.ate (3R5, 45R)-3-Emyl-4-tritylthic-N-^utyldimemylsilyl-azetidin-2-one was converted to the phosphorane by standard methods (JACS 1979, 101 (21), p 6306- 6310 and example 26 b,c,d). The final cyclisation was analogous to that in example 24c, with the addition that the worked up reaction was warmed to 40°C in ethyl acetate for 0.5 hr before chromatography. Elution with ethyl acetate/n-hexane (1:4) gave the title compound as an oil in 48% yield, v _max (KBr) 1778, 1710, 1648 cm" ¹ δ _H (CDCI3) 1.08 (3H, t /7.4 Hz), 1.8-2.06 (2H, m), 3.77 (IH, m), 4.61-4.82 (2H, m), 524-5.42 (2H, m), 5.50 (IH, d, / 1.6Hz), 5.81-6.04 (IH, m), 7.24 (IH, d, / 1.2 Hz) (Found: M+ 239.0618, Ci ^KΝC^S requires M, 239.0616).

Example 43 - Allyl (55R, 6SR)-6-ethylpenem-3-carboxylate

Allyl [55R, 5R5]-6-ethyl-penem-3-carboxylate (0.024 g) in toluene was heated to 100°C for 4.5 hours when Hplc indicated a near 1:1 mixture of cis\'.trans isomer. Evaporation of the reaction and preparative Hplc with acetonitrile/water (0.47:1) as eluent gave the title compound as a waxy solid (2.6 mg), δπ (CDCI3) 1.03 (3H, t Z7.37 Hz), 1.8-2.14 (2H, m), 3.86-3.94 (IH, m,), 4.66-4.82 (2H, m), 5.22- 5.45 (2H, m), 5.83 (IH, d, / 4.13), 5.83-6.04 (IH, m), 7.25 (IH, d, /0.82 Hz).

Example 44 - Allyl (5R5, 65R)-6-[(R5)-l-acetoxyethyI]penem-3-carboxylate

44a N-t-butyldimethylsilyl-3-(l-hydroxyethyI)-4-(triphenylmethyl - thio)azetidin-2-one

N-^utyldimethyisUyl-4-(triphenylmethylthio)azetidin-2-one (3.0 g; 6.536 mM) was dissolved in dry THF (75 ml) and cooled to -78°C under an atmosphere of argon. A solution of lithium disopropylamide (1.5 M solution in heptane THF/ethylbenzene; 5.23 ml; 7.84 mM) was added and the solution was stirred at -78°C for 30 min. A solution of acetaldehyde (0.438 ml) in THF (5 ml) was added and stirring was continued at -78°C for 30 minutes. The reaction was quenched by the addition of glacial acetic acid (2 ml) and the reaction solution was partitioned between ethyl acetate and water. The organic solution was washed with IN HC1, water, sat ΝaHCθ3 solution, brine, dried (MgSO4) and evaporated. The residue was chromatographed over silica gel (60 g), eluting with a gradient of 10- 25% ethyl acetate-hexane to yield a diastereoisomeric mixture of the title compound (1.15 g), as a white foam.

44b 3-(l-Hydroxyethyl)-4-(triphenylmethylthio)azetidin-2-one

The diastereoisomeric mixture of silylated azetidmone from preparation 44a (3.68 g) was dissolved in methanol (100 ml) and stirred at -10°C for 2 hrs with a solution of potassium fluoride (0.467 g) in methanol, under an atmosphere of argon. The reaction solution was partitioned between ethyl acetate and water and the organic solution was wa with brine, dried (MgSO4) and evaporated.

Silica ge. jdumn chromatography gave a diasteroisomeric mixture of the title azetidinones as a white foam (2.2 lg), v _maX- (CH2CI2) 3600, 3400, 1770 cm" ¹ .

44c 3-(l-Acetoxyethyl)-4-(triphenylmethylthio)azetidin-2-one

The azetidinones from preparation 44b (2.21 gm) were dissolved in dry dichloromethane and cooled in an ice-bath, with stirring. Triethylamine (0.786 ml), 4-dimethylaminopyridine (0.069 g) and acetic anhydride (0.642 ml) were added and the reaction solution was stirred at 5°C for 1.5 hr. The solution was then washed with IN HC1, water, sat N-1HCO3 solution, brine, dried (MgSO4) and evaporated. Silica gel column chromatography gave the title azetidinones as white foam (1.89 gm), ^v max. (CH ₂ C-2) 3400, 1780, 1740cm ^"1 .

44d Allyl [3-(l-acetoxyethyI)-4-triphenylmethylthio-2-oxoaze.idiπ-l-y .]- triphenylphosphoranylidene acetate

Allyl glyoxylate monohydrate was reacted with the diastereoisomeric mixture of azetidinones from preparation 44c (0.092 g) according to the procedure of example 1 to yield the title phosphoranes (0.870 g) as a white foam, v _max . (CH2CI2) ¹⁷ 55, 1622 cm" ¹ .

44e Allyl (5R5, 6SR)-6-[(RS)-l-acetoxyethyl]penern-3-carboxylate

The diastereoisomeric phosphoranes from preparation 44d (0.800 gm) were reacted according to the procedure described in Example 24c to yield the title penem which was purified by silica gel chromatography. Elution with a gradient of lO→ 25% ethyl acetate-hexane gave the pure (5R5, 65R, 8R5)-isomer of the title

compound as the first eluted component (0.036 g); λmax (EtOH) 314 nm ( e 5754), 258 nm (ε 3137); _ra3 ^ (CH ₂ C1 ₂ ) 1800, 1742, 1722 cm\' ¹ ; δ _H (CDCI3), 1.42 (3H, d, / 6.44 Hz), 2.09 (3H, s), 3.94 (IH, dt, / 1.0, 1.8, 7.7 Hz), 4.6-4.85 (2H, m), 5 -5.45 (3H, m), 5.71 (IH, d, / 1.8Hz), 5.85-6.05 (IH, m), 7.26 (IH, d, / 1.0Hz); (Found: mle 297.0675. Cι ₂ H ₁₅ NO ₅ S requires: M+ 297.0671).

Continued elution provided a mixture of diastereoisomers (0.125 gm), followed by the pure (5R5, 65R, 85R)-isomer (0.052 gm) which corresponded with isomer B from example 24c.

Example 45 • Methyl (55)-2-phenyipenem-3-carboxyIate

The racemic methyl ester described in example 26 was subjected to preparative hplc on a chiral stationary phase as for example 23 to give the required (5)-enantiomer [α]o ²⁵ - 141°.

Example 46 - p-Nitrobenzyl (5R5)-2-(ethylthio)penem-3-carboxylate

The compound was prepared as described in GB patent 2,036,015A to give the title compound, m.p. 131°, 7^^ (EtOH) 263 and 340nm; Vm^ (CHCI3) 1790, 1690cm ¹ ; δ _H (CDCI3) 1.36 (3H, t / 8Hz), 2.96 (2H, q, / 8Hz), 3.46 (IH, dd, / 16, 2Hz), 3.83 (IH, dd, / 16, 4Hz), 5.18 and 5.45 (2H, ABq, / 14Hz), 5.68 (IH, dd, / 4, 2Hz), 7.59 (2H, d, / 9Hz), 8.17 (2H, d, / 9Hz) (Found M+ 366.0354, C15H14N2O5S2 requires 366.0343).

Example 47 - Benzyl (5RS)-2-(methylthio)penem-3-carboxylate

The compound has prepared as described in GB patent 2,036,015A to give the title compound m.p. 136-137°; λmax 257 and 339nm v^^^ (CHCI3) 1795, 1685cm" ¹ δ _H (CDCI3) 2.50 (3H, s), 3.43 (IH, dd, / 16, 2Hz), 3.79 (IH, dd,/4, 2Hz), 7.20- 7.50 (5H, s) (Found: C, 54.6; H, 4.4; N, 4.6; S, 20.8%). 4H13NO3S2 requires C, 54.7; H, 4.2; N, 4.6; S, 20.9%).

Example 48 • Benzyl (5R5)-2-(ΛbutyldiphenylsHoxymethyl)penern-3-carboxylate

The compound was prepared as described in GB 2,037,277B to give the title compound, m.p. 89-89°; ^^ (EtOH) 265 and 323nm; Vmax (CHCI3) 1785, 1700, 1580cm- ¹ ; δ _H (CDCI3) 1.04 (9H, s), 3.44 (IH, dd, / 2, 16Hz), 5.56 (IH, dd, /, 2, 4Hz), 7.20-7.78 (15H, m) (Found: C, 67.8; H, 5.90; N, 2.87; S, 6.10%). C3oH3iNO4SSi requires C, 68.02; H, 5.90; N, 2.64; S, 6.05%).

Example 49 - />-Nitrobenzyl (5S, 65, 8R)-2-hydroxymethyI-6-(l- hydroxyethyl)penem-3-carboxylate

49a p-Nitrobenzyl (55, 65, 8R)-2-hydroxymethyl-6-[l-(/- butyldimethylsiIyloxy)ethyl]penem-3-carboxylate p-Nitrobenzyl (5R, 65, 8R)-2-hydroxymethyl-6-[l-(t-butyldimethyl- silyloxy)ethyl]penem-3-carboxylate (/. Antibiotics, 1983, 36, 938) (lg) was photoisomerised by a procedure analogous to that described in example 41c giving the title compound (313mg) as a pale yellow solid, Vm^ (CH2CI2) 1795cm-l; δπ (CDCI3) 0.13 (6H, s), 0.88 (9H, s), 1.45 (3H, d, / 6Hz), 3.37 (IH, t / 7Hz), 3.91 (IH, dd, / 10, 4Hz), 4.41 (IH, m), 4.66 (2H, d, / 7Hz), 5.23 (IH, d, / 13.7Hz), 5.49 (IH, d, / 13.7Hz), 5.71 (IH, d, / 4Hz), 7.62 (2H, d, / 8.7Hz) and 8.25 (2H, d / 8.7Hz); m/z M+ 494.

49b p-Nitrobenzyl (55, 6S, 8R)-2-hydroxymethyI-6-(l-hydroxyethyI)penem-3- carboxylate p-Nitrobenzyl (55, 65, 8R)-2-hydroxymethyl-6-[l-t-(butyldimethyl- silyloxy)ethyl]penem-3-carboxylate (98.8mg) was dissolved in dry tetrahydrofuran (1ml) and acetic acid (136μl) and a IM solution of tetrabutylammonium fluoride in tetrahydrofuran (780μl) were added. The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was diluted with ethyl acetate (20ml) and the solution washed with saturated aqueous sodium bicarbonate solution (1 x 20ml) and brine (1 x 20ml). The organic layer was dried over anhydrous magnesium sulphate and after filtration was evaporated in vacuo to give a yellow oil. The crude product was chromatographed on silica gel 60 eluting with ethyl acetate/hexane (4:1) to give me title compound as a pale yellow solid (48mg). yf _mSiX (CH2CI2) 1792cm" ¹ ; δ _H (CDCI3) 1.5 (3H, d, / 6.2Hz) 2.54 (2H, brs), 3.89 (IH, dd / 10.3, 4.1Hz), 4.39 (IH, m), 4.64 (IH, d, / 15.8Hz), 4.72 (IH, d, / 15.8Hz), 5.23 (IH, d, / 13.8Hz), 5.46 (IH, d, / 13.7Hz), 5.73 (IH, d, / 4.0Hz), 7.62 (2H, d, / 8.7Hz) and 8.22 (2H, d, / 8.7Hz); m/z M+ 380.

Example 50 - Allyl (55, 65)-6-[(R)-l-acetoxyethyl]penem-3-carboxylate

The racemic ester (isomer A) of example 24 was subjected to preparative chiral hplc as described in Example 23 to give the title compound [α]τ _j ² 4 - 204°.

Example 51 - Methyl (5R5)-2-(hydroxymethyl)penem-3-carboxylate

The tide compound was prepared by a method analogous to mat described in U.K Patent 2,037,277B. v _max (CH ₂ C1 ₂ ) 1978,1715 cm\' ¹ , δ (CDCI3) 3.55 (IH, dd, I 16.4, 1.6Hz, 6-H), 3.80 (IH, d, 13.9Hz, 6-H), 3.88 (3H, s, CO2CH3), 4.64 (2H, br s, CH2-OH), 5.69 (IH, dd, 13.9, 1.6Hz, 5-H). Found: M ⁺ , 215.0255. C ₈ H ₉ NO4S requires M, 215.0252.

Example 52 - 4-Nitrobenzyl (5R5, 6RS) and (5R5, 65R)-2-ethylthio-6- ethylpenem-3-carboxyiate

a) 1-Acetoxybut-l-ene

A mixture of 1-butanal (74ml), acetic anhydride (119ml) and potassium acetate (lO.lg) was stirred at 120°C for 17 hours. The mixture was then cooled, neutralised with aqueous sodium carbonate solution and the organic phase separated and dried wim anhydrous magnesium sulphate to give a pale yellow oil which was distilled to give the title compound (7.5g), b.p. 122-4°C, v _max (film) 1755, 1678cm" ¹ .

b) (3R5, 4RS) and (3R5, 4SR)-4-Acetoxy-3-ethylazetidin-2-one

1-Acetoxybut-l-ene (2.28g) and chlorosulphonyl isocyanate (2.83g) were mixed and stirred at room temperature for 2.5 hours. The reaction mixture was cooled in an icebath and a mixture of ice and sodium bicarbonate/sodium sulphite (35:1) was added in water/dichloromethane solution. The organic phase was separated and dried (anhydrous magnesium sulphate), filtered and die solvent removed in vacuo. The crude product was chromatographed on kieselgel-60, eluting with ethylacetate-petrol mixtures (2:3 to 1:1) to give the title compound as a mixture of isomers (Ug); v _max (CHCI3) 3420, 1785, 1725 cm\' ¹ .

c) (3RS, 4RS) and (3R5, 4SR)-3-ethyl-4-(ethylxanthyl)azetidin-2-one

The mixture of (3R5, 4R5) and (3R5, 45R 4-acetoxy-3-ethylazetidin-2-one (l.Og) in dichloromethane (25ml) at 5°C, was treated with potassium ethylxandiate (1.15g) in water (25ml) also at 5°C and the mixture stirred vigorously, After 15 minutes, the mixture was allowed to warm to room temperature and stirring continued for 1 hour. The organic phase was separated and the aqueous phase extracted with dichloromethane (3 x 25ml). The organic layers were combined, dried with anhydrous magnesium sulphate, filtered and evaporated to give the tide compound as a yellow oil (1.3g); (εm) 301nm (14,400), 237 (3,500); v _max (CHCI3) 3410, 1770cm" ¹ ; δ _H (CDCI3) 1.10 (3H, t, 18Hz), 1.40 (3H, t J 8Hz) 1.65-2.12 (2H, m), 3.07-3.36 (IH, m), 3.40 (2H, q, 18Hz), 5.36 (IH, d, J 2Hz), 6.60 (IH, br s). Found: M ⁺ , 235.0153. C ₈ Hi3NOS ₃ requires M, 235.0160]

d) 4-Nitrobenzyl (5R5, 6RS) and (5R5, 65R)-2-ethylthio-6-ethylpenem-3- carboxylate

The mixture of (3R5, 4R5) and (3R5, 45R)-3-ethyl-4-(ethylxanthyl)azetidin-2- one prepared above was converted to the title compound by a me od analogous to that described in example 26; v _max (CHCI3) 1790, 1695, 1605cm" ¹ ; λ ax (C ₂ H ₅ OH) 338nm (11,300), 261 (17,200); Found: M+ 394.0672. Cι ₇ H ₁₈ N O ₅ S2 requires M 394.0654).

Example 53 - 4-Nitrobenzyl (5R5, 65R)-6-[(5R)-l-acetoxyethyl]-2- (ethylthio)penem-3-carboxylate

A solution of p-nitrobenzyl [3-[(R5)-l-acetoxyethyl]-4-(ethyltrithiocarbonyl)- 2-oxoazetidin-l-yl](triphenylphosphoranylidene)acetate (l.Og) (prepared by methods described in EP 0041768) in xylene 01) was heated to reflux under argon for 10 h. The reaction mixture was evaporated to dryness and chromatographed on silica gel eluting with ethyl acetate/petroleum edier gradient to give the tide compound (120mg), mp 131-3°C; ^ (C ₂ H ₅ OH) 334nm (ε 11,000) and 260nm (ε 16,200); Vmax (CHCI3) 1795, 1730, 1695cm" ¹ , δ _H (CDCI3) 1.39 (3H, t, H Mz), 1.54 (3H, d, 16.0Hz), 2.06 (3H, s), 2.96-3.13 (2H, m), 4.01 (IH, dd, 14.0 and 9.9 Hz), 5.21 and 5.47 (2H, ABq, 1 13.7Hz overlaying a q Q 7.4Hz) at 5.26, together 3H), 5.75 (IH, d, 14Hz), 7.61 (2H, d, 18.7Hz), 8.22 (2H, d, 18.7Hz). Found: M ⁺ 452.0729; C, 50.5; H, 4.2; N, 6.1; S, 13.8. requires M 452.0712; C, 50.5; H, 4.4; N, 6.2; S, 14.2.

Example 54 - N-Benzyl (5R5)-penem-3-carboxamide

A solution of 4-nitrobenzyl (5R5)-penem-3-carboxylate [U.K. Pat. Appl.

2,036,015 (Beecham Group)] (30mg) in dioxane (5ml) and water (1.25ml) was hydrogenated at atmospheric pressure for 2h in the presence o 10% palladium on carbon catalyst (30mg). The product was filtered through Celite and a solution of potassium carbonate (8mg) in water (5ml) added to the filtrate). After a period of 0.25h the solution was evaporated under reduced pressure until just turbid and then

washed twice with ethyl acetate. The aqueous solution of the potassium salt was saturated with sodium chloride and then vigorously stirred with ethyl acetate/tetrahydro- furan 1:1 (10ml). The mixture was acidified to pH 2.0 with 0.5N hydro- chloric acid and the organic phase was separated, dried and evaporated. The residue of the crude free acid was stirred in dry tetrahydrofuran (2ml) at - 10 to - 15°C under argon and treated with triethylamine (18mg, 24 μl followed after 5 mins. by ethyl chloroformate (19mg, 17 μl). The mixed anhydride was allowed 0-25h to form and then benzylamine (1 lmg, 12 μl) was added. The reaction mixture was stirred at - 10 to -15°C for 1.5h and then evaporated. The residue was partitioned between brine arid ethyl acetate; the organic phase was separated, washed with brine, dried and evaporated. Chromatography of the residual gum on silica gel 60 using chloroform as eluant, followed by crystallisation from ethyl acetate hexane gave the benzylamide (llmg); m.p. 85-86°C; v _max (CHCI3) 3420, 2990, 1785, 1660, 1580 and 1515cm\' ¹ ; δ H (CDCI3) 3.64 (IH, ddd, / 16.6, 2.0 and 1.1Hz, 6-H), 3.85 (IH, dd, / 16.6 and 3.8Hz, 6-H), 4.49 (IH, dd, / 15.0 and 5.7Hz, PhCH), 4.60 (1Η, dd, / 15.0 and 6.1Hz, PhCH), 5.82 (1Η, dd, / 3.8 and 2.0Hz, 5-H), 6.78 (IH, br, NH), 7.16 (IH, s, 2-H), and 7.20-7.40 (5H, m, Aryl-H); MR+ (NH3 DCI), mlz 261.

Example 55 - l-[(5R5)-Pen-2-em-3-carbonyi]pyrπ>Iidine

The method of example 54 was used to prepare die penem amide from pyrrolidine as a colourless gum; Vm^ (CHCI3) 2970, 2880, 1790, 1610 and 1545cm" ¹ ; δπ (CDCI3) 1.80-2.07 (4H, m, pyrrolidine-H\'s), 3.51-3.75 (4H, m, pyπohdine-H ^* s). 3.58 (IH, dt, J 16.5 & 1.8Hz, 6-H), 3.81 (IH, ddd, J 16.5, 3.8 & 0.6Hz, 6-H), 5.83 (IH, dd, J 3.8 & 1.8Hz, 5-H) and 7.10 (IH, s, 2-H); [Found: M+, 224 (El). C10H12N2O2S requires M, 224].

Example 56 - (5R5)-N-Phenylpen-2-em-3-carboxamide

A solution of 4-nitrobenzyl (5R5)-penem-3-carboxylate (50mg) in die s

(10ml) and water (2.5ml) was hydrogenated at atmospheric pressure for 2h in , presence of 10% palladium on carbon catalyst (50mg). The product was filtered through Celite and a solution of potassium carbonate (14mg) in water (5ml) added to the filtrate. After a period of 0.25h the solution was evaporated under reduced

pressure until just turbid and then washed twice with ethyl acetate. The aqueous solution of the potassium salt was saturated with sodium chloride and then vigorously stirred with ethyl acetate/tetrahydro- furan 1:1 (20ml). The mixture was acidified to pH 2.0 with 0.5N hydrochloric acid and the organic phase was separated, dried and evaporated. A solution of the crude acid in dry THF (2ml) was stirred at -15°C under argon and to this was added triethylamine (27μl) followed after 5 mins by diphenylphosphoryl chloride (40 μl). A period of 15 mins was allowed for mixed anhydride formation and then aniline (18μl) and triethylamine (27 μl) were added. The reaction mixture was allowed to warm to room temperature over 2h and it was then evaporated to dryness. The residue was partitioned between ethyl acetate and brine; the organic phase was separated and washed with further brine prior to being dried over sodium sulphate and evaporation to a gum. The gum was chromatographed on silica gel 60 using ethylacetate hexane (3:7) as eluant to give the penem amide (9mg, 23%); m.p. 139-141°C (ethyl acetate hexane); Vmax (CHCI3) 3420, 1790, 1675, 1605, 1575, 1530 and 1500cm- ¹ ; δ _H (CDCI3) 3.73 (IH, ddd, J 16.4, 2.0 & 1.1Hz, 6-H), 3.93 (IH, dd, J 16.4 & 3.8Hz, 6-H), 5.91 (IH, dd, J 3.8 & 2.0Hz, 5-H), 7.13 (IH, t, J 7.8Hz, Ar-H), 7.24 (IH, s, 2-H), 7.34 (2H, t J 7.8Hz, Ar- H) and 7.57 (2H, d, J 7.8Hz, Ar-H). [Found: C, 57.8; H, 4.1; N, 11.1%; M+ 246.0462 (El). Ci2H ₁₀ N ₂ O2S requires C, 58.5; H, 4.1; N, 11.4%; M 246.0463].

Example 57 - (5R5)-N-Benzyl-N-methylpen-2-em-3-carboxamide

The method of example 56 was used to prepare the penem amide from N-methylbenzylamine in 22% yield as a colourless gum; Vmax (CHCI3) 3000, 1795, 1625 and 1550cm- 1 ; δ _H (CDCI3) 3.02 (3H, s, NCH3), 3.58 (IH, d, J 16.4Hz, 6-H), 3.81 (IH, dd, J 16.4 & 3.7Hz, 6-H), 4.70 (2H, s, PhCH2), 5.80 (IH, br s, 5-H) and 7.20-7.45 (6H, m, 2-H and Ar-Hs). [Found: M+, 274 (NH3 DCI). Ci4H ₁ N ₂ O2S requires M, 274].

Example 58 - (5R5)-N,N-Diethylpen-2-em-3-carboxamide

The mediod of example 56 was used with diethylamine to prepare the penem diethylamide (24%) as a colourless gum; v _max (CHCI3) 3000, 2950, 1800, 1620 and 1550cm" ¹ ; δπ (CDCI3) 1.19 (6H, t, J 7.1Hz, NCH ₂ CH ₃ ), 3.46 (4H, d, J 7.1Hz, NCH2CH3), 3.58 (IH, ddd, J 16.6, 2.0 & 1.2Hz, 6-H), 3.82 (IH, ddd, J 16.6, 3.9 & 0.6Hz, 6-H), 5.82 (IH, dd, J, 3.9 & 2.0Hz, 5-H) and 6.82 (IH, s, 2-H). [Found: M+, 226 (El). C10H14N2O2S requires M, 226].

Example 59 - (5RS)-N-(4-Methoxyphenyl)pen-2-em-3-carboxamide

The method of example 56 was applied to p-amsidine to produce the penem amide in 49% yield; m.p. 129-132°C (ethyl acetate/hexane); v _max (CHCI3) 3420, 3010, 2960, 2940, 2850, 1785, 1670, 1600, 1575 and 1515cm- ¹ ; δ _H (CDCI3) 3.71 (IH, ddd, J 16.9, 1.9 & 1.1Hz, 6-H), 3.80 (3H, s, OCH3), 3.91 (IH, ddd, J 16.9, 3.9 & 0.6Hz, 6-H), 5.90 (IH, dd, J 3.9 & 1.9Hz, 5-H), 6.87 (2H, d, J 9.0Hz, Ar-H), 7.21 (IH, s, 2-H), 7.48 (IH, d, J 9.0Hz, Ar-H) and 8.21 (IH, br s, NH). [Found: C, 56.7; H, 4.4; N, 10.6%; M+, 276 (El). Ci ₃ H ₁₂ N ₂ O3S requires C, 56.5; H, 4.4; N, 10.2%; M, 276].

Example 60 - N-[(5R5)-Pen-2-em-3-carbonyl]morpholine

The method of example 56 was used to prepare the penem amide from morpholine as a colourless gum; v _max (CHCI3) 3000, 2980, 2940, 2870, 1795, 1620 and 1550cm\' ¹ ; δ _H (CDCI3) 3.61 (IH, dt, J 15.9 & 2.0Hz, 6-H), 3.60-3.85 (8H, m, morpholine-H\'s), 3.84 (IH, dd, J 15.9 & 3.9Hz, 6-H), 5.83 (IH, dd, J 3.9 & 2.0Hz, 5- H) and 6.97 (IH, s, 2-H). [Found: M+, 240 (El). CioH ₁₂ N ₂ O3S requires M, 240].

Example 61 - (5R5)-N-(Pyrid-3-ylmethyl)pen-2-em-3-carboxamide

The method of example 56 was used to prepare the penem amide from 3-pyridinemethanamine as a colourless solid (30%); m.p. 146°C (with decomposition); v^^x (CHCI3) 3430, 3000, 1790, 1665, 1580 and 1520cm" ¹ ; δπ (CDCI3) 3.66 (IH, ddd, J 16.8, 2.0 & 0.8Hz, 6-H), 3.86 (IH, dd, J 16.8 & 3.8Hz, 6- H), 4.52 and 4.61 (2H, 2dd, J 17.0 & 5.9Hz, NCH ₂ ), 5.84 (IH, dd, J 3.8 & 2.0Hz, 5- H), 6.88 (IH, v. br, NH), 7.18 (IH, s, 2-H), 7.29 (IH, dd, J 8.0 & 4.6Hz, pyr 5-H), 7.70 (IH, br d, J 8.0Hz, pyr 4-H), 8.53 (IH, d, J 4.6Hz, pyr 6-H) and 8.58 (IH, d, J i.OHz, pyr 2-H). [Found: C, 55.4; H, 4.0; N, 15.7%; M+, 261.0571]. C12H11N3O2S requires C, 55.2; H, 4.2; N, 16.1%; M, 261.0572].

Example 62 - Methyl 3-[(5R5)-pen-2-em-3-carbonyIamino]propionate

The method of example 56 was applied to β alanine methyl ester (prepared from its hydrochloride by stirring with triethylamine in tetrahydrofuran for lh) to prepare the tide amide as a colourless gum in 37% yield; Vmax (CHCI3) 3440, 3000, 2960, 1795, 1740, 1665, 1580 and 1520cm" ¹ ; δ _H (CDCI3) 2.60 (2H, t, J 6.1Hz, CH ₂ CO), 3.62 (2H, q, 16.1Hz, CH ₂ N), 3.63 (IH, d, 1 16.6Hz with fine coupling, 6- H), 3.72 (3H, s, OCH3), 3.86 (IH, dd, 1 16.6 & 3.9Hz, 6-H), 5.82 (IH, dd, 13.9 & 2.0Hz, 5-H), 6.90 (IH, br, NH) and 7.11 (IH, s, 2-H). Found: M ⁺ _. 256.0517.

C10H12N2O4S requires 256.0518.

Example 63 - (5R5)-N-t-Butylpen-2-em-3-carboxamide

The mediod of example 56 was used to prepare the penem t-butylamide from t-butylamine in 19% yield; m.p 116-119°C; v _max (CHCI3) 3420, 2980, 1780, 1660, 1580 and 1520cm" ¹ ; δ _n (CDCI3) 1.40 (9H, s, t-butyl), 3.63 (IH, d, 1 16.6Hz with fine coupling, 6-H), 3.83 (IH, dd, 1 16.6 & 3.8Hz, 6-H), 5.82 (IH, dd, 13.8 & 2.0Hz, 5-H), 6.37 (IH, brs, NH) and 7.02 (IH, s, 2-H). Found: C, 52.5; H, 6.3, N, 12.2%. C10H14N2O2S requires C 53.1; H, 6.3; N, 12.4%.

Example 64 - Benzyl (5R5)-2-benzylpenem-3-carboxylate

A method analogous to that used in example 26 was used to prepare the tide compound. δ _H (CDCI3) 3.43 (IH, dd, 1 16.4 & 1.6Hz, 6-H), 3.73 (IH, dd, 1 16.4 & 3.9Hz, 6-H), 4.11 & 4.22 (2H, ABq, 1 15.3Hz, 2-CH ₂ ), 5.25 & 5.35 (2H, ABq, I 12.5Hz, OCH2), 5.57 (IH, dd, 13.9 & 1.6Hz, 5-H), 7.20-7.47 (10H, m, Ar-Hs). Found: M ⁺ , 351.0921. C ₂ oH ₁₇ NO ₃ S requires M, 351.0929.

Example 65 - Allyl (55, 65)-6-[(R)-l-(carbamoyloxy)ethyl]ρenem-3-carboxylate

Allyl (55, 65)-6-[(R)-l-hydroxyethyl]penem-3-carboxylate (lOOmg) was dissolved in ethyl acetate (0.3ml) and trichloroacetyl isocyanate (0.3ml) and left for 5h. The mixture was diluted with ethyl acetate (20ml), washed with sodium bicarbonate solution, dried and evaporated. A chloroform solution of die residue was applied to a silica column and eluted slowly with ethyl acetate/hexane 1:1 to give the tide compound as a solid (95mg). Crystallisation from ethyl acetate/hexane gave colourless needles, m.p. 161-3°C; [α] _D ²¹ - 290° (c 0.7; CHCI3); Vmax (KBr) 3439, 3357, 3291, 3211, 1776, 1717, 1661, 1620, 1553cm" ¹ ; δ _H (CDCI3) 1.54 (3H, m, I 6.3Hz), 4.06 (IH, dd, 19.8 & 3.6Hz), 4.60 (2H, broad s), 4.6-4.8 (2H, m), 5.1-5.4 (3H, m), 5.83 (IH, d, 14Hz), 5.8-6.1 (IH, m), 7.32 (IH, d, 10.8Hz); λmax (C ₂ H ₅ OH) 254nm (ε 2856), 312nm (ε 7788); m/z (FAB, matrix 3-NOBA/Na) 321 (MNa+, 65%).

Example 66 - Allyl (55, 65)-6-[(R)-l-benzoyloxyethyl]penem-3-carboxylate

Allyl (55, 65)-6-[(R l-hydroxyethyl]penem-3-carboxylate (lOOmg) was dissolved in dichloromethane (5ml), stirred and ice-cooled. The solution was treated with triethylamine (0.42ml), 4-dimethylaminopyridine (catalytic quantity) and

benzoyl chloride (0.33ml) and stirred for 2h at room temperature. It was diluted with ethyl acetate (50ml), washed with 0.5N HCl (50ml), water and sodium bicarbonate solution, dried and evaporated. The residue was chromatographed on silica, eluting with ethyl acetate/hexane mixtures, to give the tide compound (83mg). Crystallisation from ethyl acetate/hexane gave colourless needles m.p. 146-8°C; [a]-* - 283° (c 0.5; CHCI3); Vmax (KBr) 1791, 1702, 1649, 1554cm" ¹ ; δ _H (CDCI3) 1.67 (3H, d, 16.3Hz), 4.23 (IH, dd, 19.8 & 4Hz), 4.6-4.8 (2H, m), 5.2-5.5 (2H, m), 5.5-5.7 (IH, m), 5.91 (IH, d, 14Hz), 5.9-6.1 (IH, m), 7.33 (IH, s), 7.4-7.7 (3H, m) 7.9-8.1 (2H, m); ^-- (C2H5OH) 229nm (ε 14975), 313nm (ε 7150). Found: C, 60.2; H, 4.7; N, 4.0; S 8.7% Ci ₈ Hi ₇ NO5S requires C, 60.2; H, 4.8; N, 3.9; S, 8.9%.

Example 67 • Allyl (55, 65)-6-[(R)-l-methoxyethyl]penem-3-carboxylate

Allyl (55, 65)-6-[(R)- 1 -hydroxyethyl]penem-3-carboxylate (50mg) was dissolved in nitromethane (2ml), ice-cooled, stirred vigorously and treated with anhydrous, powdered sodium carbonate (375mg) and trimethyloxonium fluoroborate (250mg). After 5h, the mixture was filtered through celite, diluted with dichloromethane (20ml), washed with sodium bicarbonate solution, dried and evaporated. The residue was chromatographed on silica, eluting with etivyl acetate/hexane mixtures, to give the tide compound (9mg) as a colourless gum. [α] _D ²ⁱ - 210° (c 0.9; CHCI3); v,^ (CHCI3) 1790, 1710, 1650, 1555cm- ¹ ; δ _H (CDCI3) 1.40 (3H, d, 15.6Hz), 3.32 (3H, s), 3.7-4.0 (2H, m), 4.6-4.9 (2H, m), 5.2- 5.5 (2H, m), 5.81 (IH, d, 13.7Hz), 5.85-6.05 (IH, m), 7.32 (IH, s); λmax (C2H5OH) 253nm (ε 2693), 314nm (ε 6520). m/z (E.I.) 269 (M+, 10%); m/z (NH3CL) 270 (MH+, 80%), 287 (MNH4+ 100%).

Example 68 - Allyl (55, 6S)-6-[(R)-l-[(N-(benzyloxycarbonyl)-(5)- alanyl)oxy]ethyl]penem-3-carboxylate

Allyl (55, 65)-6-[(R)-l-hydroxyethyl]penem-3-carboxylate (50mg), N- (benzyloxycarbonyl)-L-alanine (180mg) and 4-dimethylaminopyridine (24mg) were dissolved in dichloromethane (1ml), cooled to -15°C and treated wim dicyclohexylcarbodiimide (165mg). The mixture was s -ed for 0.5h, dilutedwith ethyl acetate (20ml), washed with 0.5N HCl, water and sodium bicarbonate solution, dried and evaporate. The residue was chromatographed on silica, eluting with ethyl acetate hexane 1:3. Fractions containing the product were evaporated, the residue taken up in ethyl acetate/hexane 1 :3 (3ml) and filtered to remove a solid byproduct The filtrate was evaporated to give die tide compound (54mg) as a colourless gum.

Vmax (CHCI3) 3420, 1790, 1715, 1650, 1560, 1510cm- ¹ ; δ _H (CDCI3) 1.40 (3H, d, 17.2Hz), 1.50 (3H, d, 16.2Hz), 4.06 (IH, dd, 1 10.1 & 3.8Hz), 4.28 (IH, quintet, 1 7.2Hz), 4.6-4.8 (2H, m), 5.11 (2H, s), 5.15-5.45 (4H, m), 5.78 (IH, d, 13.8Hz), 5.8- 6.1 (IH, m), 7.29 (IH, s), 7.35 (5H, s); m/z (FAB, matrix 3-NOBA/Na) 483 (MNa+, 55%).

Compounds of the examples are described in the literature as follows: GB2037277 - examples 9, 31, 48 J. Chem. Soc. Chem. Commun. 1988, 544 - example 2 ZA8504947 - example 36

GB2036015 - examples 4, 5, 46, 47

US4282236 - example 31

There is no disclosure of antibacterial activity for the compounds in these references.

Biological Activity

Inhibition of bacterial leader peptidase 1

Introduction: Leader Peptidase 1 (LP1) cleaves the pre-Maltose Binding Protein (pre-MBP) based nonapeptide (H-Phe-Ser-Ala-Ser-

Ala-Leu-Ala-Lys-lle-NH2) to the heptapeptide (H-Phe-Ser- Ala-Ser-Ala-Leu-Ala-O") and a dipeptide residue (1). The nonapeptide substrate and the heptapeptide cleavage product are detected by reverse-phase gradient HPLC. Inhibitors of LP1 will stop or slow the cleavage of the pre-MBP based peptide.

Assay: All solutions are prepared with HPLC grade H2O. The following are mixed in order: -

• When using a solvent other than H2O to dissolve die compound, a volume of solvent is added to die control peptide and control assay equal to that in the test samples. • Duplicate samples of the compounds to be tested are assayed to determine a mean reduction in activity.

• Bacterial Leader Peptidase is prepared from strains bearing the plasmid pRD8 (2) and isolated according to the method of Wolf «fl/ 1982 (3). Samples are pre-warmed at 37°C for 5 min.

5 μl ImM substrate peptide in 50mM triethanolamine pH8.0, ImM EDTA, 1% triton X-100, is added to each sample (final vol = 20 μl).

Samples incubated in a 37°C water bath for 2h.

Samples centrifuged for 3 sec. then 20 μl 0.1% TFA is added to each to stop the reaction.

Samples centrifuged for 5 min. Preferably me samples are now analysed on me HPLC, though they can be stored at -20°C prior to analysis.

HPLC Analysis:

The HPLC set-up is as follows:

Results: The activity of a compound is expressed as the mean % reduction in

LPl activity. This is calculated as the reduction in product peak area compared to the control, when the reduction in product corresponds to an increase in substrate peak area.

Using this methodology, the two enantiomers and the racemate of benzyl penem-3-carboxylate (examples 1, 3 and 4) gave the following results:

It was merefore concluded that the LPl inhibition properties observed from the racemate arise from the 5S isomer.

(1) Dev, I.K. P.H. Ray & P. Novak (/. Biological Chemistry 1990, 265, (33), p 20069).

(2) Dalbey, R.E. & W. Wickner (/. Biolgical Chemistry 1985, 260 p 15925) (3) Wolfe, P.B., P. Silver & W. Wickner (/. Biological Chemistryl 1982, 257 p 7898).

The plasmid pRD8 was a gift from Prof. W. Wickner - UCLA.

The compounds of the Examples were tested for inhibitory effect against leader peptidase (I). The results are listed in the table below:

I

*> I

I I

I

(_π

I

I

UI tv) I pNB = p-nitrobenzyl Bz = benzyl Ph = phenyl 1 comparative example showing inactivity of the R isomer.

5 2 @10μM

3 As sulphoxide

4 cis/trans monoethyl mixture