Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D499/00—Heterocyclic compounds containing 4-thia-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula:, e.g. penicillins, penems; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulfur-containing hetero ring
  • C07D499/88—Compounds with a double bond between positions 2 and 3 and a carbon atom having three bonds to hetero atoms with at the most one bond to halogen, e.g. an ester or nitrile radical, directly attached in position 2
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
  • A61K31/425—Thiazoles
  • A61K31/429—Thiazoles condensed with heterocyclic ring systems
  • A61K31/43—Compounds containing 4-thia-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula, e.g. penicillins, penems
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• 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:
• GB2042520 discloses certain 2-(ethylthio)penem-3-carboxamide derivatives. Although racemic and other mixtures of 5R and 5S enantiomers of penems are known, there has been little attempt to isolate pure 5S isomers of penems because of the general belief that such isomers are antibacterially inactive.
• the invention therefore provides the use of a 5S penem compound of general formula (I):
• n 0 or 1
• R 2 is hydrogen or a substituent group
• R 3 is an ester- or amide-forming group
• R 1 is hydrogen or an organic group linked via carbon and wherein the group -COR 3 is substantially stable toward in-vivo hydrolysis, in the form of its 5S 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).
• 'aryl' includes phenyl and naphthyl, each optionally substituted with up to five, preferably up to three, groups selected from halogen, mercapto, (C 1-6 ) alkyl, phenyl, (C 1-6 ) alkoxy, hydroxy(C 1-6 )alkyl, mercapto(C 1-6 )alkyl, halo(C 1-6 ) alkyl, hydroxy, amino, quaternary ammonium, nitro, carboxy, (C 1-6 ) alkylcarbonyloxy, (C 1-6 ) alkoxycarbonyl, carboxylic acid, formyl, (C 1-6 ) alkylcarbonyl and phosphoric acid groups.
• 'Aralkyl' includes phenyl C 1-6 alkyl and napthyl C 1-6 alkyl each aryl group optionally substituted as aforesaid.
• 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
• 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 invention.
• '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.
• 'halogen' refers to fluorine, chlorine, bromine and iodine.
• R 2 may suitably be hydrogen.
• R 2 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.
• R 2 may be a group R 4 , CO 2 R 4 , OR 4 , SR 4 or SOR 4 . where each R 4 may be the same or different and may denote hydrogen or an unsubstituted or, preferably, substituted aIkyl group or an aryl or heterocyclyl group.
• R 4 represents a substituted alkyl group
• suitable substituents on the alkyl group include hydroxy, (C 1-5 ) alkoxy, (C 1-4 ) alkoxy (C 1-4 ) alkoxy, aminocarbonyloxy, halogen, mercapto, (C 1-6 ) alkylthio, aryl, arylthio, aryloxy, heterocyclyl, heterocyclylthio, amino, (mono- or di-)-(C 1-6 ) alkylamino, acylamino or acyloxy where the acyl group may be aromatic or aliphatic, for example (C 1-6 ) alkanoyl, (C 1-6 ) alkoxycarbonyl, optionally substituted benzoyl or optionally substituted phenyloxycarbonyl, carboxy, (C 1-6 ) alkoxycarbonyl, azido and alkyl- or aryl-substituted silyloxy.
• 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 3 is preferably resistant to in-vivo hydrolysis.
• R 3 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
• R 3 may be an aryloxy group.
• R 3 may be an optionally substituted (C 1- 10 ) alkoxy group where the optional substituent(s) may be halogen or an organic group, such as (C 1-6 ) alkylcarbonyl, (C 1-6 ) alkoxycarbonyl, (C 1-10 ) alkoxy-substituted-(C 1-10 )-alkoxy, aryl or heterocycyl.
• R 3 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 R 3 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 3 substituted alkyl chain include optionally substituted pyrimidyl, benzopyranyl and furanyl.
• R 3 groups examples 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, t-butyloxy, pentyloxy, t-amyloxy, allyloxy, diphenylmethyloxy, triphenylmethyloxy,
• R 3 is preferably benzyloxy, p-nitrobenzyloxy, p-methoxybenzyloxy, 2,2,2- trichloroethyloxy, methoxy, p-(methoxycarbonyl)benzyloxy or allyloxy.
• R 3 When R 3 is an amide-forming group it may be a group NR 5 2 where each R 5 may be the same or different and may be independently selected from hydrogen, aryl such as optionally substituted phenyl or optionally substituted (C 1-6 ) alkyl, where the optional substituents on the (C 1-6 ) alkyl group may be selected from the same list of substituents from which substituents on an alkyl group R 4 may be selected.
• the two R 5 moieties may represent the residue of a nitrogen containing heterocyclic ring as defined above.
• NR 5 2 may represent the residue of an amino acid, for example a natually occurring ⁇ -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 on the penem ring system.
• a natually occurring ⁇ -amino acid such as glycine, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, cystine, me
• R 5 may be hydrogen and the other may be an optionally substituted alkyl group, such as an aryl substituted alkyl group such as benzyl.
• airiide-forming groups R 3 include benzylarnino, N-methylbenzylamino, pyrrolidino, anilino, diethylamino, p-methoxybenzylamino, morpholino, 2-(methoxycarbonyl)ethylamino, t-butylamino and
• R 3 is preferably pyrrolidino
• R 1 may suitably be hydrogen.
• R 1 may suitably be a substituent group selected from optionally substituted (C 1-6 ) alkyl, optionally substituted (C 1-6 ) alkenyl and C 1-6 alkanoyl where the optional substituents on the alkyl group of these substituents may be selected from the same list of substituents from which
• R 1 is preferably C 1-4 alkyl substituted in the 1-position by a substituent linked via oxygen, such as hydroxy, (C 1 - 5 )alkoxy such as methoxy, arninocarbonyloxy, benzoyloxy or (C 1-6 ) alkanoyloxy optionally substituted by a group such as benzyloxycarbonylamino.
• a substituent linked via oxygen such as hydroxy, (C 1 - 5 )alkoxy such as methoxy, arninocarbonyloxy, benzoyloxy or (C 1-6 ) alkanoyloxy optionally substituted by a group such as benzyloxycarbonylamino.
• R 1 alkyl may suitably be substituted by a group R 8 -COO- or
• the substituent on R 1 may be an azido group, N 3 or a peptide residue, e.g.
• R 9 is an amino-substituting group, for example an optionally substituted C 1-6 alkanoyl or C 1-6 alkoxycarbonyl group
• R 10 is a residue derived from a naturally occurring amino acid, such as the above-mentioned ⁇ -amino acids
• X is O or NH
• 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 (CH 3 ) 3 CO.CONH.CH(CH 3 ).CONH.CH(CH 2 .CH(CH 3 ) 2 ).CONH.
• R 1 is hydrogen, 1-acetoxyethyl or 1 -hydroxyethyl.
• the compound of formula (I) may include 50% or more preferably
• the compound of formula (I) is substantially the pure 5S isomer.
• 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.
• 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).
• R 21 is R 2 or a substituent which can be converted into or replaced by R 2
• R 31 is a group R 3 or a group which can be converted into or replaced by R 3
• R 11 is a group R 1 or a group which can be converted into R 1
• R 2 , R 3 and R 1 are as defined in formula (I)
• R 81 is an organic radical such as phenyl.
• R 21 convertible into R 2 examples include -CH 2 OS-R 3 which is convertible to -CH 2 OH and other derivatives, or an ester attached to a phenyl group which is convertible to an acid.
• compounds of formula (II) in which R 21 is hydrogen may be obtained from the corresponding compound where the substituent on sulphur is a ⁇ -(alkoxycarbonyl)vinyl group such as ⁇ -(ethoxycarbonyl)vinyl or
• R 31 may for example be a group OR 91 where R 91 is a carboxylic acid protecting group which may be removed by a conventional procedure to form an acid of formula (III):
• 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 1 - 6 )-1,2-alkylene oxide - such as ethylene oxide or propylene oxide.
• the acylation 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.
• 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
• Alternative acylating derivatives of acid (III) are the acid azide, or activated esters such as esters with 2-mercaptopyridine, cyanomethanol, p.-nitrophenol, 2,4-dinitrophenol, thiophenol, halophenols, including pentachlorophenol, monomethoxyphenol, N-hydroxy succinimide, N-hydroxybenzotriazole, or
• amides such as N-acylsaccharins, N-acylthiazolidin-2-thione or N-acylphthalimides; or an alkylidene iminoester prepared by reaction of the acid (III) 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, N,N'-diethyl-, dipropyl- or diisopropylcarbodiimide,
• N,N'-di-cyclohexyl-carbodiimide or N-ethyl-N'-[3-(dimethylamino)propyl]-carbodiimide
• a suitable carbonyl compound for example, N,N'-carbonyldiimidazole or N,N-carbonyldi- triazole
• an isoxazolinium salt for example,
• N-ethoxycarbonyl 2-ethoxy-1,2-dihydroquinoline Other condensing agents include Lewis acids (for example BBr 3 - C 6 H 6 ); 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 (III) is to treat the acid of formula (III) 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
• the acid (III) or its acylating derivative may be caused to react with an alcohol of formula HO-R 3 or an amine of formula H-NR 5 2, where R 3 and R 5 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,
• esters of formula (I) may for example be prepared by reaction of a metal salt of the acid of formula (III), for example the potassium salt, which may itself be prepared by reaction of the acid (III) with a basic compound of the metal such as the carbonate, with for example a halide of formula X-R 3 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.
• an amide may be formed by reaction of the acid (III) or an N- acylating derivative with the amine H-NR 5 2 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 (III) is a mixed anhydride for example as formed by reaction of the acid (III) 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.
• 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
• compositions 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.
• 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.
• suitable 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;
• 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
• 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.
• 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
• Suppositories will contain conventional suppository bases, e.g. cocoa-butter or other glyceride.
• 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.
• the compound can be dissolved in water for injection and filter sterilised before filling into a suitable vial or ampoule and sealing.
• agents such as a local anaesthetic, preservative and buffering agents can be dissolved in the vehicle.
• 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.
• a surfactant or wetting agent is included in the composition to facilitate uniform distribution of the compound.
• 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 100mg 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.
• 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 E.coli and Gram-positive organisms such as S.aureus.
• Benzyl glyoxylate. monohydrate (0.439g; 2.415mM) was dissolved in benzene (100ml) and the solution was heated to reflux with the provision for the removal of water (Dean and Stark apparatus) for 1h. The solution was then allowed to cool to room temperature and the solution of the (45)-(cis- ⁇ -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.
• triphenylphosphine (1.68gm) was added to the stirred solution. When all the triphenylphosphine had dissolved, the solution 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 (MgSO 4 ) 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 max
• Benzyl [(4S)-(cis- ⁇ -carbomenthyloxyvinylmercapto)-2-oxoazetidin-1-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 t.l.c. indicated complete ozonolysis. The excess ozone was blown off with argon and triphenylphosphine (0.315g) was added.
• 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 (MgSO 4 ) and evaporated.
• Example 6 - p-Methoxybenzyl (5RS)-penem-3-carboxyIate This was prepared according to method of example 2 using p-methoxybenzyl bromide to give the title compound, v max (CHCl 3 ) 1798, 1715 cm -1 ; ⁇ H (CDCl 3 ) 3.55 (1H, dt), 3.75-3.95 (4H, s + m), 5.18 (2H, ABq), 5.78 (1H, dd, J 1.85, 4.0 Hz), 6.90 (2H, d, J 8.7Hz), 7.25 (1H, s), 7.33 (2H, d, J 8.7Hz) (Found: M + 291.0571; C 14 H 13 NO 4 S requires 391.0565).
• Example 2 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 (CHCl 3 ) 2960, 1800, 1725 sh, 1715 and 1615 cm -1 ; ⁇ H (CDCl 3 ) 3.60 (1H, dt J 16.6 & 1.6 Hz, 6-H), 3.87 (1H, d, J 16.6 & 4.0 Hz, 6-H), 3.92 (3H, s, OCH 3 ), 5.25 and 5.35 (2H, ABq, J 13.1 Hz, OCH 2 Ar), 5.81 (1H, dd, J 4.0 and 1.6Hz, 5-H), 7.32 (1H, 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.
• Example 2 The method of Example 2 was used to prepare the penem ester from
• 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.
• Example 16 The method of Example 16 was used to prepare the title penem ester from (4-iodobutyl) benzene in 24% yield; v max (CHCl 3 ) 2930, 2860, 1800, 1710 and 1560 cm-1; ⁇ H; (CDCl 3 ) 1.67-1.80 (4H, m, OCH 2 CH 2 CH 2 ), 2.60-2.70 (2 ⁇ , m, CH 2 Ar), 3.57 (1H, dt J 16-6 & 1.7 Hz, 6-H).
• Example 19 2-(2-Methoxyethoxy)ethyl (5RS)-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; ⁇ H(CDCl 3 ) 3.39 (3H, s,
• Example 21 (7-Methoxy-2-oxo-2H-1-benzopyran-4-yl)methyl (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.
• Example 22 Methoxymethyl (5RS)-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 (CHCl 3 ) 2970, 1800, 1720 and 1560 cm -1 ; ⁇ H (CDCl 3 ) 3.52 (3H, s, OCH 3 ) 3.62 (1H, dt J 16.6 & 1.9Hz, 6-H), 3.87 (1H, dd, J 16.6 & 3.9Hz, 6-H), 5.30 and 5.44 (2H, ABq, J 5.9 Hz, OCH 2 O), 5.81 (1H, dd, J 3.9 & 1.9 Hz, 5-H) and 7.
• 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:
• 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-dimethylaminopyridine (0.139g) and a solution of silver nitrate (0.363g) in acetonitrile.
• Acetic-formic anhydride (0.8ml), 4-dimethylaminopyridine (0.118g) and sodium iodide (1.42g) were then added and stirring was continued at room
• the first eluted component was a single pure isomer (isomer A) of the title compound, obtained as a colourless gum (0.010g), v max (CH 2 Cl 2 ) 1800, 1748, 1725 cm -1 ; ⁇ H (CDCl 3 ) 1.53 (3H, d J 6 Hz), 2.07 (3H, s), 4.07 (1H, ddd, J 0.7, 3.8, 10.3 Hz), 4.6 - 4.8 (2H, m), 5.2-5.45 (3H, m), 5.84 (1H, d, J 4.0Hz), 5.85-6.05 (1H, m), 7.31 (1H, d. 0.8Hz).
• the second eluted component was also a pure isomer (isomer B) of the title penem (0.041g), v max (CH 2 Cl 2 ) 1801, 1748, 1728cm -1 ; ⁇ H (CDCl 3 ) 1.45 (3H, d, J 6.5 Hz), 2.12 (3H,s), 4.03 (1H, overlapping ddd, J 1,2, 4.5 Hz), 4.6-4.85 (2H,m), 5.2- 5.45 (2H,m), 5.62 (1H, d, J 2Hz), 5.88-6.06 (1H,m),7.24 (1H, d, J 1Hz).
• reaction mixture was filtered to remove lutidine hydrochloride, and the solvent evaporated to leave a yellow oil which was reevaparated from toluene (2 ⁇ 10ml) to remove thionyl chloride residues and used in the next step without further purification.
• Example 28 Methyl (5RS)-2-(4-chlorophenyl)penem-3-carboxylate Prepared by a method analogous to example 26.
• Example 29 Methyl (5RS)-2-(4-methoxycarbonylphenyl)penem-3-carboxylate Prepared by a method analogous to example 26.
• Benzyl (5RS)-2-hydroxymethyl-penem-3-carboxylate (57mg) was dissolved in dry dichloromethane (1ml) and trichloroacetyl-isocyanate (25 ⁇ l) was added. The reaction mixture was stirred at room temperature for 30 min. under argon and then diluted with ethyl acetate (10ml) and washed with 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 (46 ⁇ l).
• 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).
• Example 33 Methyl (5RS)-2-(carbamoyloxymethyl)penern-3-carboxyIate
• the title compound was prepared by a method analogous to that described in example 32.
• 4.8 (2H, br.s.
• Benzyl (5RS) -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 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 1 / 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).
• N-t-butyldimethyisilyl-4-(triphenylmethylthio)azetidin-2-one (3.0 g;
• 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 washed with brine, dried (MgSO 4 ) and evaporated.
• 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 [ ⁇ ] D 25 - 141°.
• Example 48 Benzyl (5RS)-2-(t-butyldiphenylsiloxymethyl)penem-3-carboxylate
• the compound was prepared as described in GB 2,037,277B to give the title compound, m.p. 89-89°; ⁇ max (EtOH) 265 and 323nm; v max (CHCl 3 ) 1785, 1700, 1580cm -1 ; ⁇ H (CDCl 3 ) 1.04 (9H, s), 3.44 (1H, dd, J 2, 16Hz), 5.56 (1H, dd, J, 2, 4Hz), 7.20-7.78 (15H, m) (Found: C, 67.8; H, 5.90; N, 2.87; S, 6.10%).
• Example 50 Allyl (5S, 6S)-6-[(R)-1-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 [ ⁇ ] D 24 - 204°.
• Example 51 Methyl (5RS)-2-(hydroxymethyl)penem-3-carboxylate
• the title compound was prepared by a method analogous to mat described in U.K Patent 2,037,277B.
• C 8 H 9 NO 4 S requires M, 215.0252.
• the method of example 54 was used to prepare die penem amide from pyrrolidine as a colourless gum; v max (CHCl 3 ) 2970, 2880, 1790, 1610 and 1545cm -1 ; ⁇ H (CDCl 3 ) 1.80-2.07 (4H, m, pyrrolidine-H's), 3.51-3.75 (4H, m, pyrrolidine-H's).
• diphenylphosphoryl chloride 40 ⁇ l. A period of 15 mins was allowed for mixed anhydride formation and then aniline (18 ⁇ l) and triethylamine (27 ⁇ l) 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
• Example 59 (5RS)-N-(4-Methoxyphenyl)pen-2-em-3-carboxamide
• the method of example 56 was applied to p-anisidine to produce the penem amide in 49% yield; m.p. 129-132°C (ethyl acetate/hexane); v max (CHCl 3 ) 3420, 3010, 2960, 2940, 2850, 1785, 1670, 1600, 1575 and 1515cm - 1 ; ⁇ H (CDCl 3 ) 3.71 (1H, dddd, J 16.9, 1.9 & 1.1Hz, 6-H), 3.80 (3H, s, OCH 3 ), 3.91 (1H, ddd, J 16.9, 3.9 & 0.6Hz, 6-H), 5.90 (1H, dd, J 3.9 & 1.9Hz, 5-H), 6.87 (2H, d, J 9.0Hz, Ar-H), 7.21 (1H, s, 2-H), 7.48 (1H,
• Example 63 - (5RS)-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 (CHCl 3 ) 3420, 2980, 1780, 1660, 1580 and 1520cm -1 ; ⁇ n (CDCl 3 ) 1.40 (9H, s, t-butyl), 3.63 (1H, d, 1 16.6Hz with fine coupling, 6-H), 3.83 (1H, dd, 1 16.6 & 3.8Hz, 6-H), 5.82 (1H, dd, 13.8 & 2.0Hz, 5-H), 6.37 (1H, brs, NH) and 7.02 (1H, s, 2-H). Found: C, 52.5; H, 6.3, N, 12.2%. C 10 H 14 N 2 O 2 S requires C 53.1; H, 6.3; N, 12.4%.
• pre-MBP Protein (pre-MBP) based nonapeptide (H-Phe-Ser-Ala-Ser- Ala-Leu-Ala-Lys-Ile-NH 2 ) 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.
• HPLC set-up is as follows:
• 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.
• the plasmid pRD8 was a gift from Prof. W. Wickner - UCLA.

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