LACTAMS

Abstract: A compound of formula (I) wherein R<1> is hydrogen or an electron withdrawing group; one of X<1> to X<4> is a CR<2>R<3> group wherein at least one of R<2> and R<3> is C<1> to C6 aliphatic group, an aromatic group, a heterocyclic group, a basic group or R<4>CONH wherein R<4> is a C1 to C6 aliphatic group, an aromatic group or a heterocyclic group and any remaining R<2> or R<3> is hydrogen; at least one of X<1> to X<4> is a CHR<5> group wherein R<5> comprises a hydrogen bond donor or acceptor; the remaining X<1> to X<4> groups are CH2, O or NR<6> wherein R<6> is hydrogen, a C1 to C6 aliphatic group, an aromatic group or a heterocyclic group; and y is 0 or 1, or a pharmaceutically acceptable salt thereof.

Description: LACTAMS The present invention relates to lactams, processes for their preparation and their use as inhibitors of enzymes comprising a nucleophilic serine or cysteine residue at their active sites. The invention in particular relates to monocyclic γ-lactams or larger ring lactams and their use, for example, as inhibitors of serine proteases such as elastase, thrombin, trypsin and chymotrypsin or as antibiotics. β-Lactam antibiotics are known to exert their biological activity by acylation of nucleophilic serinyl residues at the active site of penicillin binding proteins (PBPs), or transpeptidase enzymes, thus inhibiting the cross-linking of bacterial cell walls. β-Lactams have also been shown to inhibit a range of other enzymes with nucleophilic residues at their active sites, including serine proteases. The inhibition of elastases by β-lactams has been studied in detail. It is known that an initial reaction occurs to form an acyl-enzyme ester complex which then partitions between a conformational change to form a long lived enzyme- inhibitor complex or hydrolysis to give a β-amino acid as shown in Scheme 1. The proposed mechanism involves nucleophilic attack of the oxygen of the serine residue on the carbonyl carbon of the β-lactam. The release of ca. 25 kcal of ring strain energy during ring opening of a β-lactam by a hydroxyl group renders reformation of the β-lactam energetically unfavourable. The nature of the conformational change which results in a long lived enzyme-inhibitor complex has also been studied. It is proposed that the β-lactam initially reacts to form a hydrolytically labile covalent acyl-enzyme complex in which the ester carbonyl is located within an oxyanion hole. Rotation of the ester carbonyl out of the oxyanion hole and displacement of, for example, hydrolytic water gives rise to a hydrolytically stable complex. Enzyme - Ser - OH + non-covalent enzyme-inhibitor complex covalent acyl-enzyme complex nucleophilic attack conformational change Scheme 1 β-Lactams antibiotics are, however, susceptible to degradation by β-lactamase enzymes which neutralise many clinically useful antibiotics by cleaving the β-lactam ring. Many bacteria contain β-lactamases and are therefore resistant to β-lactam antibiotics. Thus efforts have been made to find alternatives to β-lactam compounds. The present invention provides monocyclic γ-lactams and larger ring lactams which may be more stable than β-lactams as they may be less susceptible to decomposition mediated by resistance mechanisms. In a first aspect the present invention provides a compound of formula (I) wherein R 1 is hydrogen or an electron withdrawing group; one of X 1 to X 4 is a CR 2 R 3 group wherein at least one of R 2 and R 3 is a to C 6 aliphatic group, an aromatic group, a heterocyclic group, a basic group or R 4 CONH wherein R 4 is a C, to C 6 aliphatic group, an aromatic group or a heterocyclic group and any remaining R 2 or R 3 is hydrogen; at least one of X 1 to X 4 is a CHR 5 group wherein R 5 comprises a hydrogen bond donor or acceptor; the remaining X 1 to X 4 groups are CH 2 , O or NR 6 wherein R 6 is hydrogen, a to C 6 aliphatic group, an aromatic group or a heterocyclic group; and y is 0 or 1, preferably 0, or a pharmaceutically acceptable salt thereof. Preferably the compounds of formula (I) are those wherein R 1 is as defined above; y is 0; and X 1 is CR 2 R\ X 2 is CH 2 and X 3 is CHR 5 ; or X 1 is CR 2 R 3 , X 2 is CHR 5 and X 3 is CH 2 ; or X 1 is CH 2 , X 2 is CR 2 R 3 and X 3 is CHR 5 . It will be appreciated that R 1 is selected to stabilise the acyl-enzyme complex formed between the compound of formula (I) and the enzyme which is to be inhibited. Stabilisation may be achieved by, for example, choosing an R 1 group which reduces or eliminates the availability of the nitrogen lone pair for attack onto the carbonyl of the acyl-enzyme complex by mesomeric or resonance effects. Accordingly R 1 is typically hydrogen, SO 2 R 7 , CO 2 R 7 , CONHR 7 , COR 7 , C=CR 8 R 9 wherein R 7 is Ci to C 6 alkyl; CH 2 (CF 2 ) 0 . 4 CF 3 ; aryl or heteroaryl, which aryl or heteroaryl are mono-ring, or have two fused rings one of which may be saturated, and which aryl and heteroaryl groups may be substituted by one or more Cj to C 4 alkyl, halo, NR 10 R n , SO 2 R 10 R 11 , CONR 10 R n , C, to C 6 alkyl ester, CN, CH 2 OH, O- to C 6 alkyl, CF 3 or nitro groups; aryl-Cj to C 4 alkyl; aryl- to C 4 -alkyl-NH; or aryl-C 2 to C 4 alkenyl; or such groups wherein aryl is substituted by one or more to C 4 alkyl or halo groups; R 8 and R 9 , which may be the same or different, are COR 12 ; R 10 and R 11 , which may be the same or different, are hydrogen or C, to C 4 alkyl; and R 12 is hydrogen or an aliphatic, aromatic or heteroaromatic group. R 2 and R 3 are selected to enhance productive binding and formation of the acyl-enzyme complex. When X 1 is CR 2 R 3 the groups R 2 and R 3 may be important in positioning the lactam in the active site of the enzyme. In the case of enzymes such as elastase, thrombin, trypsin and chymotrypsin when X 1 is CR 2 R 3 the groups R 2 and R 3 can bind into the SI pocket of the enzyme and should be selected to do so. Suitable R 2 and R 3 groups include ethyl, propyl, isopropyl, butyl, isobutyl, methoxy, ethoxy, propoxy, methylthio, ethylthio, propylthio, basic groups such as amino, amidino and guanidino, benzyl, phenyl, C 6 H 5 CH 2 CONH and C 6 H 5 OCH 2 CONH. R 5 is selected to enhance hydrogen bonding and stability within the acyl- enzyme complex. In the case of, for example, elastase R 5 may be chosen to stabilise the acyl-enzyme complex by displacement of the histidine of the catalytic triad from its catalytic position or to make favourable hydrogen bonds with the NH group of the valine 216 or the C=O group of the serine 214. The R 5 group may be, for example CO 2 R 12 , CONHR 12 , NHCOR 12 , NHSO 2 R 12 , OCOR 12 or OCONHR 12 wherein R 12 is as defined above. R 5 may also be chosen so as to displace the hydrolytic water (i.e. the water molecule positioned within the active site to effect hydrolysis of the acyl- enzyme complex). The present invention also includes the salts of those compounds of formula (I) that have salt forming groups, such as an acidic or basic group. The ^ salts are physiologically tolerable salts. In the case of compounds comprising a basic amino group, the salts are formed with suitable inorganic or organic acids. Inorganic acids are, for example, hydrochloric or sulphuric acid. Suitable organic acids include mono-, di- and tricarboxylic acids, such as acetic, trifluoroacetic, tartaric and citric acid, or sulphonic acids, for example methanesulphonic, trifluoromethanesulphonic or/?-toluenesulphonic acid. The present invention includes all possible isomers and mixtures thereof, including diastereomeric mixtures and racemic mixtures, resulting from the possible combinations of (R) and (S) stereochemistry. More preferably the compounds of formula (I) are those which possess one or more of the following features: R 1 is hydrogen, ? r -nitrobenzenesulphonyl, αr -toluenesulphonyl, para- trifluoromethylbenzenesulphonyl or methanesulphonyl; y is 0; and X 1 is CH 2 , X 2 is CHCH 2 CH 3 and X 3 is CHCO 2 R 13 wherein R 13 is hydrogen, ethyl or benzyl; or X 1 is CHCH 2 CH 3 or C(CH 2 CH 3 ) 2 , X 2 is CH 2 and X 3 is CHCO 2 R 13 wherein R 13 is as defined above; or X 1 is CHCH 2 CH 3 , X 2 is CHCO 2 R 14 wherein R 14 is hydrogen, methyl or ethyl and X 3 is CH 2 . Most preferably the compounds of formula (I) are those which possess one of more of the following features: R 1 is rø-nitrobenzenesulphonyl, r -toluenesulphonyl, para- trifluoromethylbenzenesulphonyl or methanesulphonyl; y is 0; and X 1 is CH 2 , X 2 is CHCH 2 CH 3 and X 3 is CHCO 2 H; or X 1 is CHCH 2 CH 3 , X 2 is CH 2 and X 3 is CHCO 2 H; or X 1 is CHCH 2 CH 3 , X 2 is CHCO 2 R 14 wherein R 14 is as defined above and X 3 is CH 2 . A number of the compounds of formula (I) may be prepared by alkylation of a pyroglutamic acid. For example the γ-lactam of formula (II) may be prepared as shown in Scheme 2. (2S)-Pyroglutamic acid (1) can be converted to benzyl (2S)-pyroglutamate (2) by treatment with benzyl alcohol and concentrated sulphuric acid in benzene (73%). Tosylation of the benzyl ester (2) to give (3) may be accomplished using lithium b s(trimethylsilyl) amide and/?-toluenesulphonyl chloride in tetrahydrofuran (THF) at -78 °C (94%). Alkylation of the pyroglutamate ester (3) to yield the benzyl ester (4) may then be carried out using ethyl triflate as the alkylating agent (73%). Deprotection of (4) to give the γ-lactam of formula (II) may be performed by hydrogenation (96%). (1) (2) (4) (3) (II) (i) BnOH, c. H 2 SO 4 , Hg, A; (ii) LiN(TMS) 2 , THF, -78 °C ; j p-TsCl, THF, -78°C to RT; (iii) LDA (1.2 eq), THF, -78°C; EtOTf (5.0 eq), -78°C; (iv) H 2 , Pd/c, THF, RT. Scheme 2 Other compounds of formula (I) may be prepared by reduction and cyclisation of 1,2-cyano esters. For example, the γ-lactam of formula (III) may be prepared as shown in Scheme 3. Methyl-2-bromobutyrate may be added to cyanoacetate (5) using sodium methoxide as the base to give the cyano diester (6) (60%). The diastereomeric mixture of the lactams (7) and (8) may then be produced by reduction of (6) using hydrogen and methanolic chloroform followed by refluxing in toluene (86%). Mesylation of the lactam (8) may be accomplished by deprotonation with lithium b/Λ-(trimethylsilyl)amide in THF at -78 °C and treatment of the anion methanesulphonyl chloride (77%). CO 2 Me (i) CO> 2 2M v e < CN 60% MeO 2 C CN (5) (6) (ii),(iii) 86% (III) (8) (7) (i) Na/MeOH, MeOH, RT; EtCHBrCO 2 Me, RT; (ii) MeOH, CHC1 3 , H 2 , 80 p.s.i., RT; (iii) toluene, Δ; (iv) LiN(TMS) 2 , THF, -78 °C; MsCl, -78 °C to RT. Scheme 3 Other compounds of formula (I) may be prepared by Michael addition of an αβ unsaturated ester with a dialkyl acetamidomalonate. For example, the γ-lactam of formula (IN) may be prepared as shown in Scheme 4. The first step may be achieved by refluxing the acid (9) in ethanol saturated with hydrochloric acid (61%), followed by addition of DBU in THF at room temperature (89%). The pyrrolidinone (11) may be obtained by Michael addition of the αβ unsaturated ester (10) with diethyl acetamidomalonate and sodium ethoxide in refluxing ethanol (64%). Saponification followed by decarboxylation of the half- ester may be employed to give the diastereomers (12) and (13) (70%). The benzyl esters (14) and (15) may be formed by heating the diastereomeric mixture of (12 ) and (13) at 130°C in benzyl alcohol in the presence of titanium isopropoxide (80%). The resulting benzyl esters (14) and (15) may be separated by fractional crystallisation from cold (-20 °C) diisopropylether. The N-sulphonyl derivative (16) may be obtained in good yield by deprotonation with lithium bt ' s(trimethylsilyl)amide in THF at -78 °C and treatment of the anion vvithp- toluenesulphonyl chloride (94%). Hydrogenolysis of (16) may then yield the γ- lactam of formula (IV) (95%). (nr (iii) 70% (12) (13) (i)(a) EtOH, HCl, A, (b) DBU, TFIF, RT; (ii) Na/EtOH, C 2 H 5 O 2 CCH(NHCOCH 3 )CO 2 C 2 H 5 , Δ; (iii) NaOH, HCl, Δ (160 °C); (iv) titanium isopropoxide, BnOH, Δ; (v) LiN(TMS) 2 , THF, -78 °C; 7-TsCl, THF, -78°C to RT; (vi) H 2 , Pd/c, THF, RT. Scheme 4 In another aspect the present invention provides non-therapeutic use, for example in an assay, of the compounds of formula (I) as inhibitors of enzymes such as elastases, thrombin, factor Xa, trypsin, chymotrypsin, transpeptidase enzymes or β-lactamases. In a further aspect the present invention provides a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, as active ingredient, in association with a pharmaceutically acceptable carrier, excipient or other additive, if necessary. Also provided is a compound of formula (I), as defined above, or a pharmaceutically acceptable salt thereof, for use in the treatment of the human or animal body. Further, the present invention provides the use of a compound of formula (I), or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for use in the treatment of acute respiratory distress syndrome, cystic fibrosis, emphysema, chronic bronchitis, coagulation disorders or bacterial infections. The pharmaceutical composition containing a compound of formula (I) or salts thereof may be prepared in a conventional way by employing conventional non- toxic pharmaceutical carriers or diluents in a variety of dosage forms and ways of administration. In particular, the compounds of formula (I) can be administered: A) orally, for example, as tablets, troches, lozenges, aqueous or oily suspension, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to any method known in the art for the manufacture of pharmaceutical compositions and such composition may contain one or more agents selected from the group consisting of sweetening agents, flavouring agents, colouring and preserving agents in order to provide elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example maize starch or alginic acid; binding agents, for example maize starch, gelatin or acacia, and lubrificating agents, for example magnesium stearate or stearic acid or talc. The tablets may be uncoated or they may be coated )y known techniques to delay disintegration and absoφtion in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such glyceryl monostearate or glyceryl distearate may be employed. Formulations for oral use may also be presented as Jjard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example calcium carbonate, calcium phosphate or kaolin, or soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil. Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl cellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be naturally-occurring phosphatides, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxyacetamol, or condensation products of ethylene oxide with partial esters derived from fatty acids and an hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol anhydrides, for example polyoxysorbitan monooleate. The said aqueous suspension may also contain one or more preservatives, for example ethyl or n-propyl -hydroxybenzoate, one or more colouring agents, one or more flavouring agents, one or more sweetening agents such as sucrose or saccharin. An oily suspension may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil, coconut oil or in a mineral oil such as liquid paraffin. The oily suspension may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents, such as those set forth above, and flavouring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an antioxidant such as ascorbic acid. Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, a suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavouring and colouring agents may also be present. The pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavouring agents. Syrups and elixirs may be formulated with sweetening agents, for example glycerol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, colouring and flavouring agents. B) parenterally, either subcutaneously or intravenously or intramuscularly, or intrasternally, or by infusion techniques. The pharmaceutical compositions may be in the form of a sterile injectable aqueous or olagenous suspensions. These suspensions may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oils may be conventionally employed including synthetic mono or diglycerides. In addition fatty acids such as oleic acid find use in the preparation of injectables. The daily dose varies according to the activity of the specific compound, the age, weight, and conditions of the subject to be treated, the type and the severity of the disease, and the frequency and route of administration. Typically the daily dose is from 0J to 50 mg per kg of body weight. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage forn\-will vary depending upon the host treated and the particular mode of administration. For example, a formulation intended for oral administration may contain from 5 to 95% of the total composition. Dosage unit forms will generally contain between from 5 to 500 mg of the active compound. The Examples which follow further illustrate the present invention. Examples General Procedures All compounds prepared were racemic unless explicitly indicated. Melting points (m.p.) were obtained using a Kofler heated-stage microscope. Values are quoted to the nearest 1 °C and are uncorrected for systematic errors. Flash column chromatography was accomplished using silica gel 60 GB 254 , using a modification of the procedure of Still (W. C Still et al, J. Org. Chem., 1978, 43, 2923). Thin layer chromatography was performed on Merck silica gel 60 F 254 0.2 mm precoated aluminium backed plates, and visualized by the quenching of ultraviolet fluorescence (254 nm), by staining with iodine, or by treatment with ammonium molybdate solution (10% in IM H 2 SO 4 aq.), potassium permanganate (3g KMnO 4 , 20g K 2 CO 3 , 5 ml 5% NaOH, 300 ml H 2 O), or phosphomolybdic acid (PMA) (230g PMA in 1 gal. 95% EtOH), followed by heating. Retention factors are quoted to the nearest 0.05. Optical rotations were obtained using a Perkin-Elmer 241 polarimeter at 589 nm (Na D-line) at the temperature specified for each compound, with a path length of 10 cm. Concentrations (c) are quoted in g/100 ml. Fourier Transform Infrared (FT-IR) spectra were recorded as thin films or KBr discs, using a Perkin-Elmer Paragon 1000 (FT-IR) spectrometer. Only selected absoφtions are reported. Absorptions are reported in wavenumbers (cm "1 ), using the following abbreviations; w, weak; m, medium; s, strong; br, broad. Proton nuclear magnetic resonance ('H NMR) spectra were recorded at 200 and 500 MHz on Varian Gemini 200 or Brucker AC200, and Brucker AM-500 or AMX500 instruments respectively. Chemical shifts (δ H ) are quoted in parts per million (ppm), relative to tetramethylsilane, and are referenced to the residual protonated solvent peak. Coupling constants (J) are quoted to the nearest 0.5 Hz or 0.1 Hz, on the 200 or 500 MHz machines respectively. The following abbreviations are used to report the spectra: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; dd, doublet of doublets; dt, doublet of triplets; br, broad. Carbon nuclear magnetic resonance ( 13 C NMR) spectra were recorded at 50.31 MHz on Varian Gemini 200 spectrometers using Distortionless Enhancement by Polarisation Transfer (DEPT) editing to aid assignment. Chemical shifts (δ c ) are quoted in ppm and referenced to the appropriate residual deuterated solvent peak. Low resolution mass spectra irn/z) were recorded using a Micromass Platform machine atmospheric pressure chemical ionisation (APCI) or electrospray. Only molecular ions, and major peaks are reported. Intensities are recorded as percentages relative to the largest peak. All solvents were distilled before use. Anhydrous THF was obtained by distillation from sodium/benzophenone ketyl under nitrogen. Anhydrous methanol was prepared by the method of Lund and Bjerrum as described in Nogel (A.L Nogel, Textbook of Practical Organic Chemistry, 4 th Edition, Longman, London, 1984). The fraction of light petroleum ether boiling in the range 40-60°C is referred to as PE 40-60. Solvents were evaporated on a Bϋchi Rl 10 Rotavapor, high boiling sovlents being removed on a Bϋchi Rl 10 Rotavapor equipped with dry-ice condenser at <2 mmHg. All other reagents were purchased from commercial sources and purified in accordance with the recommendations of Perrin and Armarego (D.D. Perrin and Armarego, Purification of Laboratory Chemicals, Pergamon Press, Oxford, 1988) or used as obtained. Preparation of the γ-Lactam of Formula (II) Benzyl (2S)-pyroglutamate (2) A suspension of (2S)-pyroglutamic acid (1) (10.013g, 77.5 mmol), benzyl alcohol (8.4 ml, 81.2 mmol), and c. sulphuric acid (2 drops of 98%) in benzene (25 ml) was heated at reflux using Dean-Stark apparatus for 51 h until a clear yellow solution had formed. The reaction was cooled and concentrated in vacuo to yield a pale yellow oil which was redissolved in DCM (100 ml), washed with K 2 CO 3 (IN, 20 ml), and brine (2 x 20 ml). The organic layer was dried (MgSO 4 ), filtered, and concentrated in vacuo to give a pale yellow oil, which was purified by flash column chromatography (silica, EtOAc) to give the title compound as a colourless oil which solidified on extended standing. The resulting white solid was recrystallised from Et 2 O for analytical purposes (12.411 g, 56.6 mmol, 73%); mp 44-46°C (from Et 2 O) (lit., 43-44.5 °C); (Found: C, 65.46; H, 5.98; N, 6.44. C 12 H 13 NO 3 requires C, 65.74; H, 5.98; N, 6.39%); v max (KBr discycm "1 3298 br m (N-H), 2963w (C-H), 2886w (C- H), 1745s (C=O), 1697s (C=O), 1664s, 1456m, 1245m, 1218m, and 1194m; δ H (200 MHz; CDC1 3 ) 2.14-2.56 (4H, m, CHjCH,), 4.26-4.32 (1H, m, CHN), 5.20 (2H, s, CH 2 C 6 H 5 ), 6.64 (1H, br s, NH), 7.37 (5H, s, C___); δ c (50.31 MHz; CDC1 3 ) 24.65 (CH 2 CH), 29.19 (CH 2 CO), 55.55 (CHN), 67.23 (CH 2 C 6 H 5 ), 128.52, 128.75, 128.85 (aromatic CH, C 6 H 5 ), 135.40 (ipso-C, CCH 2 ), 172.35, 178.68 (2 x CO); m/z (APCI+, NH 3 ) 220 (MH + , 100%). Benzyl (2S)-l-(p-toluenesuIphonvPpyroglutamate (3) Lithium b/ ' -?(trimethylsilyl)amide (1.0M in THF, 11.4 ml, 11 A mmol) was added dropwise to a stirred solution of benzyl (2S)-pyroglutamate (2) (2.500 g, 11.4 mmol) in dry THF (170 ml) at -78 °C under argon. The mixture was stirred for 15 min and then a solution of/?-toluenesulphonyl chloride (2.440 g, 12.5 mmol) in dry THF (20 ml) was added rapidly. The mixture was stirred at -78 °C for a further 15 min and at room temperature for a further 90 min. The reaction was quenched with saturated NH 4 C1 solution (200 ml), the mixture partitioned, and the aqueous layer washed with EtOAc (3 x 20 ml). The combined organic layers were dried (MgSO 4 ), filtered, and concentrated in vacuo to yield a white solid which was purified by flash column chromatography (silica, Et 2 O:PE 40-60, 3: 1) and recrystallisation from Et 2 O to give the title compound as white crystals (3.977 g, 10.7 mmol, 94%); mp 103- 104°C (from Et 2 O); [α] 23 D -40.6 (c 1.00 in CHC1 3 ); (Found: C, 61.03; H, 5.01; N, 3.78. C, 9 H, 9 NSO 5 requires C, 61.1 1 ; H, 5.13; N, 3.75%); v max (KBr discycm "1 3059w, 2973w, 1757s (C=O), 1740s (C=O), 1460m, 1364s, 1355s, 1169s, 1146s, and 1088m; δ H (200MHz; CDC1 3 ) 2.00-2.67 (4H, m, CH^CHJ, 2.44 (3H, s, Ts CH 3 ), 4.93-4.98 (IH, m, CHN), 5.19, 5.28 (2H, AB quartet, J 12.5, CH Hj), 7.30 (2H, d, J 8.5, Ts CH), 7.39 (5H, s, C ,), 7.94 (2H, d, 8.5, Ts CH); δ c (50.31MHz; CDC1 3 ) 21.60 (Ar CH 3 ), 23.12 (CH 2 CH), 30.36 (CH 2 CO), 59.44 (CHN), 67.69 (OH 2 C 6 H 5 ), 128.49, 128.89, 129.26, 129.51 (aromatic CH, C__ 5 , Ts), 134.97, 135.16 (ipso- C,CCH 2 , CCH 3 ), 145.63 (ipso-C, CSO 2 ), 170.97, 173.00 (2 x CO); m/z (APCI+ NH 3 ) 374 (MH + , 100%). Benzyl (2S, 4R)-l-(p-toluenesulphonylV4-ethylpyroglutamate (41 Lithium όw(trimethylsilyl)amide (1.0M in THF, 917 μl, 917 μmol) was added dropwise to a stirred solution of benzyl (2S)-l-(p-toluenesulphonyl)- pyroglutamate (3) (285 mg, 763 μmol) in dry THF (5 ml) at -78 °C under argon and stirred for 35 min. Ethyl triflate (500 μl, 3.86 mmol) was added dropwise, the mixture stirred at -78 °C for a further 6 h, and the reaction quenched with saturated NH 4 C1 solution (60 ml). The mixture was allowed to warm to room temperature and extracted with DCM (6 x 25 ml). The combined organic extracts were washed with saturated NaHCO 3 solution (3 x 50 ml), dried (MgSO 4 ), filtered, and concentrated in vacuo to yield a colourless oil which was purified by flash column chromatography (silica, j Pr 2 O: PE 40-60, 3:2) to give (4) as a diastereomeric mixture together with the ring opened product (224 mg, 557 μl, 73%) characterised as follows: benzyl (2S,4R)-l-(/?-toluenesuIphonyI)-4-ethylpyrogIutamate (4) δ H (200MHz; CDC1 3 ) 0.91 (3H, t, 77.5, CH 3 CH 2 ), 1.30-1.43 (IH, m, one of O CH,), 1.77-1.83 (IH, m, one of CH 3 CHJ, 2.00-2.61 (3H, m, CH^CH), 2.43 (3H, s, Ts CH 3 ), 4.92 (IH, dd, J9.5, 1.5, CHN), 5.19, 5.28 (2H, AB quartet, J 12.5, CH_Ph), 7.30 (2H, d, J 8.5, Ts CH), 7.39 (5H, s, C 6 Hs), 7.93 (2H, d, J8.5, Ts CH); δ c (50.31MHz; CDC1 3 ) 10.92 (CH 3 CH 2 ), 21.63 (Ts CH 3 ), 22.88 (CH 3 CH 2 ), 29.61 (CH 2 (CH) 2 ), 42.30 (CH 3 CH 2 CH), 57.59 (CHN), 67.72 (CH 2 O), 128.45, 128.92, 129.43 (aromatic CH, C*H 5 , Ts), 134.86, 135.11 (ipso-C, CCH 2 , CCH 3 ), 145.59 (ipso-C, CSO 2 ), 170.96, 174.75 (2 x CO); m/z (APCI+, NH 3 ) 402 (MH + , 100%). benzyl (2S,4S)-l-(p-toluenesulphonyl)-4-ethylpyroglutamate δ„(200MHz; CDC1 3 ) 0.86 (3H, t, .77.5, CHjCH,), 1.21-1.43 (IH, m, one of CH.CH,), 1.66-1.87 (IH, m, one of CH.CHJ, 2.04-2.69 (3H, m, CHjCH), 2.44 (3H, s, Ts CH,), 4.82 (IH, dd, ./ 6.5, 8.5, CHN), 5.28 (2H, s, CH_.Ph), 7.31 (2H, d, 78.5, Ts CH), 7.40 (5H, s, CβHs), 7.98 (2H, d, J 8.5, Ts CH); m/z (APCI+,NH 3 ) 402 (MH + , 100%). (S)-l-benzyI-5-ethyl-2-N- ;-toluenesulphonylaminogIutanιic diester δ H (200MHz; CDC1 3 ) 1.26 (3H, t, 77.0, CI^CH,), 1.79 (IH, m, one of CH^H,), 2.04-2.21 (IH, m, one of CH^H^), 2.35-2.54 (2H, m, CH^CH), 2.41 (3H, s, Ts CH , 3.95-4.06 (IH, m, CHΝ), 4J2, 4J6 (2H, AB quartet, J 7.0, CH^Ph), 7.20 (2H, d, J 8.5, Ts CH), 7J6 (5H, s, C 6 Hs), 7.69 (2H, d, 78.5, Ts CH); δ c (50.31MHz; CDC1 3 ) 14.02 (CH 3 CH 2 ), 21.48 (Ts CH 3 ), 28.19 (CH 2 CH), 29.57 (CH 2 CO), 54.89 (CHΝ), 60.73, 67.56 (CH 3 CH 2 O and CH 2 Ph), 127.44, 128.42, 128.82, 129.90 (aromatic CH, C 6 H 5 , Ts), 134.81, 136.49 (ipso-C, CCH 2 , CCH 3 ), 143.89 (ipso-C, CSO 2 ), 171.44, 172.84 (2 x CO); m/z (APCI+, ΝH 3 ) 420 (MH + , 100%). (IS, 4R)-l-(p-Toluenesulphonyl)-4-ethylpyroglutamic acid (γ-Lactam (II)) A solution of benzyl (2S, 4R)-l-(p-toluenesulphonyl)-4-ethylproglutamate (4) (155 mg, 0J86 mmol) in dry THF (10 ml) and Pd on carbon catalyst was stirred under an atmosphere of hydrogen for 15 minutes. The reaction mixture was filtered through celite® and the celite ® was washed with DCM (3 x 25 ml). The combined organic washes were concentrated in vacuo to give the γ-lactam (II) as a colourless oil which solidified on standing (115 mg, 0J69 mmol, 96%). The solid was recrystallised from EtOAc/PE 40-bO to give a white crystalline solid for analytical purposes; m.p. 70-71 °C; [α] 2 5 D -61J (c 1.0 in CHC1 3 ); v max (KBr discycm "1 2968m, 293 lw, 2877w, 1739br s (CONSO 2 R), 1715 (CO 2 H), 1596m, 1366s (SO 2 ), 1278s, 1234s, 1191s (SO 2 ), 1088m and 816m (/?-subst. ArCH); δ H (500MHz; CDC1 3 ) 0.92 (3H, t, ./7.5, CT CH,), 1.35-1.44 (IH, m, 1 x O^CH,), 1.80-1.88 (IH, m, 1 x CH 3 CH 2 ), 2J2-2J9 (IH, m, 1 x C^)!^), 2.39-2.43 (IH, m, 1 x C^H,), 2.45 (3H, s, ArCH,), 2.54-2.61 (IH, m, C(4)H), 4.90 (IH, d, 79.5, C(2)H) and 7.35, 7.98 (4H, AA'BB', Ar-H); a 2D COSY experiment was consistent with the connectivity as indicated; δ c (50MHz; CDC1 3 ) 11.0 (CH 3 CH 2 ), 21.7 (ArCH 3 ), 22.9 (CH 3 CH 2 ), 29.6 (C(3)), 42.4 (C(4)), 57.4 (C(2)), 129.0, 129J (Ar- , 134.6, 145.6 (2 x ipso- ^ 174.5 and 176.0 (2 x CO); m/z (CI, NH 3 ) 329 [MNH 4 + , 100%], 312 [MH + , 38], 175 [14], 158 [19], 139 [10] and 1 12 [20]; (Found MH + 312.0906. C 14 H 17 NO 5 S requires MH + 312.0905(7)). Preparation of the γ-Lactam of Formula (III) Dimethyl-2-cvano-3-ethyl-l,4-butanedioate (6) Sodium (2.409 g, 105 mmol) was dissolved in anhydrous methanol (60 ml), methyl cyanoacetate (10.345 g, 104 mmol) was added and the mixture stirred at room temperature for 30 min. Methyl 2-bromobutyrate (10.6 ml, 92J mmol}_was added and the mixture stirred at room temperature for 68 h. The reaction was quenched with acetic acid (20 ml 50% aq. v/v), washed with saturated NaHCO 3 solution (150 ml), and extracted with EtOAc (6 x 100 ml). The combined organic extracts were dried (MgSO 4 ), filtered, and concentrated in vacuo to yield a yellow oil. The oil was purified by distillation (92-100°C; 0.5 Torr), and flash column chromatography (silica, 'Pr 2 O:PE 40-60, 7:3) to give an inseparable mixture of diastereomers (ca. 1 : 1 ratio by __ NMR analysis) of the title compound as a colourless oil (10.930 g, 54.9 mmol, 60%); R f 0.35 (Et 2 O: 40-60 PE, 75:25); δπ(200MHz; CDC1 3 ) 0.91-1.09 (6H, m, 2 x CHjCH,), 1.60-2.09 (4H, m, 2 x CH^CH,), 2.99-3.14 (2H, m, 2 x CH 3 CH 2 CH), 3.72 (6H, s, 2 x 3.76 (IH, d, 78.5 Hz, CHCN), 3.80 (6H, s, 2 x CO 2 CH 3 ), 3.93; δ c (50J l MHz; CDC1 3 ) 10.46, 11.47 (2 x CH 3 CH 2 ), 22.68, 22.98 (2 x CH 3 CH 2 ), 38.24 (2 x CH 3 CH 2 CH), 45.25, 46.17 (2 x CHCN), 52.40, 53.66 (4 x COiCH,), 114.83 (2 x CN), 165.72, 165.83, 172.00, 172.59 (4 x CO 2 CH 3 ); m/z (APCI-, NH 3 ) 198 (M-H, 100%). cts-3-Ethyl-4-(methoxycarbonyl)pyι olidin-2-one (7)- and trfl«s-3-Ethyl-4-(methoxycarbonyl)pyrrolidin-2-one (8) To a solution of diethyl-2-cyano-3 -ethyl- 1,4-butanedioate (6) (2.663 g, 13.4 mmol) in anhydrous methanol (20 ml) was added PtO 2 (455 mg, 2.00 mmol) and chloroform (6.6 ml, 80.5 mmol). The mixture was stirred under an atmosphere of hydrogen, at 80 psi for 72 h. The catalyst was removed by filtration through Celite® (the catalyst was observed to glow red, giving out heat), and the solvent removed in vacuo to yield a thick yellow oil which was taken up in IM H 3 PO 4 (10 ml), and extracted with Et 2 O (3 x 50 ml). The combined organic extracts were dried (MgSO 4 ), filtered, and concentrated in vacuo to yield unreached starting material. The aqueous layer was concentrated in vacuo to yield a thick yellow oil, basified with saturated K 2 CO 3 solution (10 ml), and extracted with EtOAc (3 x 50 ml). The combined organic extracts were dried (MgSO 4 ), filtered, and concentrated in vacuo to yield a yellow oil which was dissolved in toluene (50 ml) and refluxed for 24 h. The mixture was concentrated in vacuo and purified by flash column chromatography (silica, EtOAc: EtOH, 49: 1) to yield the partially separated diastereomers cw-3-ethyl- 4-(methoxycarbonyl)pyrrolidin-2-one (7), and trøt?s-3-ethyl-4- (methoxycarbonyl)pyιτolidin-2-one (8), (ca. 1 : 1 ratio by Η NMR analysis), as white crystals which were recrystallised from Et 2 O for analytical puφoses (1.963 g, 11.5 mmol, 86%>), and characterised as follows: c«-3-EthyI-4-(methoxycarbonyl)pyrrolidin-2-one (7) mp 68-70 °C (from Et 2 O, -20 °C); (Found: C, 56.14; H, 7.75: N, 8.10. C 8 H 13 NO 3 requires C, 56.13: H, 7.65: N, 8.18%); v max (KBr discycm "1 3215 br m (N-H), 2972m (C-H), 1724s (C=O), 1700s (C=O), 1366m, 1208s, and 1183s; δ H (200MHz; CDC1 3 ) 1.04 (3H, t, 77.5 CF CHz), 139-1.61 (IH, m, one of CHsCHj), 1.69-1.91 (IH, m, one of CH 3 CH 2 ), 2.58 (IH, pseudc q, CHCO 2 Me), 3.41-3.75 (3H, m, CHjN, CHCON), 3.75 (3H, s, C0 2 Α,, 5.93 (IH, br s, NH); δ c (50.31MHz; CDC1 3 ) 11.47 (CH 3 CH 2 ), 20.09 (CH 3 CH 2 ), 42.59 (CH 2 N), 43.21 (CHCON), 44.85 (CHCO 2 Me), 51.58 (CO 2 CH 3 ), 172.36, 178.71 (2 x CO); m/z (APCI+, NH 3 ), 172 (MH + , 100%). trα«-s-3-ethyl-4-(methoxycarbonyl)pyrroIidin-2-one (8) mp 65-66°C (from Et 2 O, -20°C); (Found: C, 56.21 : H, 7.80; N, 8.21. C 8 H 13 NO 3 requires C, 56.13; N, 7.65; N, 8.18%); v max (KBr discycm "1 3206br m (N-H), 2961m (C-H), 1737s (OO), 1679s (C=O), 1234w, and 1178m (C-O); δ H (200MHz; CDC1 3 ) 1.00 (3H, t, 77.5, CH,CH 2 ), 1.56-1.96 (2H, m, CTLCH , 2.64-2.75 (IH, m, CHCON), 3.09 (IH, pseudo q, CHCO 2 Me), 3.52-3.57 (2H, m, CH^N), 3.77 (3H, s, CO 2 CH 3 ), 5.64 (IH, br s, NH); δ c (50.31MHz; CDC1 3 ) 10.42 (CH 3 CH 2 ), 22.72 (CH 3 CH 2 ), 42.77 (CH 2 N), 44.06 (CHCON), 45.59 (CHCOMe), 52.31 (CO 2 CH 3 ), 173.77, 178.72 (2 x CO); m/z (APCI+ NH 3 ) 172 (MH + , 100%). tmns-3-Ethyl-l-(methanesulphonyl)-4-(methoxycarbonyl)pyrrolidin-2-one (γ- Lactam (III)) Lithium b/ trimethylsilyl)amide (1.0M in THF, 1J8 ml, 1J8 mmol) was added dropwise to a stirred solution of trα«.v-3-ethyl-4-(methoxycarbonyl)pyrrolidin-2-one (8) (202 mg, 1.18 mmol) in dry THF (15 ml) at -78° C under argon. The mixture was stirred for 15 min and then methanesulphonyl chloride (11 1 μl, 1.45 mmol) was added rapidly. The mixture was stirred at -78 °C for a further 15 min and at room temperature for a further 2 h. The reaction was quenched with saturated NH 4 C1 solution (60 ml), the mixture partitioned, and the aqueous layer washed with EtOAc (3 x 20 ml). The combined organic extracts were dried (MgSO 4 ), filtered, and concentrated in vacuo to yield a white solid which was purified by recrystallisation from Et 2 O to give the title compound as white crystals (225 mg, 903 μmol, 77%); mp 68-70°C (from Et 2 O, -20°C); (Found: C, 43.12; H, 5.81; N, 5.43. C 9 H 15 NSO 5 requires C, 43.36; H, 6.06; N, 5.62%); v max (KBr discycm "1 2967w, 1736s (C=O), 1348s, 1244w, 1165s, and 984m; δ H (200MHz; CDC1 3 ) 1.01 (3H, t, 77.5, CHjCHj), 1.60-1.99 (2H, m, CH^ϋ), 2.82-2.93 (IH, m, CHCON), 3.03 (IH, pseudo q, CHCO 2 Me), 3.37 (3H, s, Ms CHj), 3.77 (3H, s, CO.CH,), 3.87 (IH, dd, 77.5, 10.2, one of CH^N), 4.07 (IH, dd, 77.5, 10.2, one of CL N); δ c (50.31MHz; CDC1 3 ) 10.43 (CH 3 CH 2 ), 22.72 (CH 3 CH 2 ), 40.45 (SO 2 CH 3 ), 41.38 (CHCON), 46.34 (CH 2 N), 47.65 (CHCO 2 Me), 52.75 (CO 2 CH 3 ), 172.34, 174.43 (2 x CO); m/z (APCI+, NH 3 ) 250 (MH + , 100%). Preparation of γ-Lactam of Formula (IV) E-Ethyl-pent-2-enoate (10) A solution of tr. ---2-pentenoic acid (9) (25 g, 0.25 mol) in EtOH (70 ml) was saturated with HCl gas and refluxed. After 24 h the excess solvent was removed in vacuo, the residue washed with NaHCO 3 (10% w/w, 30 ml), the mixture partitioned, and the aqueous layer extracted with Et 2 O (3 x 50 ml). The combined organic extracts were dried (K 2 CO 3 ), filtered, and concentrated in vacuo to give a yellow oil which was purified by flash column chromatography (silica, Et 2 O; PE 40-60, 1 :4) to give ethyl-3-chloro-pent-2-enoate as a colourless oil (25 g, 0J5 mol, 61%); δ H (200MHz; CDC1 3 ) 0.99 (3H, t, 77.5, CH 3 CH 2 CH 2 ), 1.21 (3H, t, 77.0, CHjCH.O), 1.62-1.91 (2H, m, CH 3 CH 2 CH), 2.67 (2H, d, 77.0 Hz, CH 2 CO); 4J2 (2H, q, 77.0 Hz, CH 3 CH 2 O), 4J3-4.30 (IH, m, CH 3 CH 2 CH); δ c (50.31MHz; CDC1 3 ) 10.49 (CH 3 CH 2 CH), 13.91 (CH 3 CH 2 O), 30.95, (CH 3 CH 2 CH), 43.16 (CH 2 CO), 59.99 60.71 (CH 2 O), 170.45 (CO); m/z (APCI+, NH 3 ) 165 (MH + , 100%). DBU was added to the 4-ethyl-3-chloro-pent-2-enoate (5 g, 30 mmol) in THF (10 ml) and the mixture stirred at room temperature for 3 h. The solvent was removed in vacuo. The residue was dissolved in EtOAc (20 ml) and washed with water (2 x 10 ml). Organic extracts were combined, dried (MgSO 4 ), and concentrated in vacuo to give a colourless oil. The mixtuie was purified by flash column chromatography (silica, Et 2 O; PE 40-60, 1 :4) to yield the title compound as a colourless oil (3.5 g, 27 mmol, 89%); δ H (200MHz; CDC1 3 ) 0.97 (3H, t, 77.5, CHjCH.CH), 1J8 (3H, t, 77.0, CH 3 CH 2 O), 2.04-231 (2H, m, CH.CHjCH), 4.07 (2H, q, 77.0 Hz Q^CH^O), 5.70 (IH, d, 7 16.0 Hz, CHCO), 6.91 (IH, dt, 76.5, 16.0, CH 3 CH 2 CH); δ c (5031MHz; CDC1 3 ) 11.52 (CH 3 CH 2 CH), 13.61 (2 x CH 3 CH 2 O), 24.70 (CH 3 CH 2 CH), 59.42 (CH 2 CO), 120.25 (CHCO), 150.02 (CH 3 CH 2 CH), 166.17 (CO); m/z (APCI+, NH 3 ) 129 (MH + , 100%). 4-Ethyl-5-di(ethoxycarbonvI)pyrrolidin-2-one (11) Sodium (322 mg, 14.0 mmol) in dry EtOH (15 ml) was added to a stirred solution of diethyl acetamidomalonate (10.980 g, 50.134 mmol) in dry EtOH (200 ml). E-Ethyl- pent-2-enoate (10) (9.642 g, 75.24 mmol) was added dropwise to the solution, and the mixture refluxed. After 64 h the mixture was cooled to room temperature, and neutralised with glacial acetic acid. The EtOH was removed in vacuo to give a yellow/brown oil which was suspended in Et 2 O yielding white crystals which were filtered off and recrystallised from water-acetone to yield the title compound as white crystals (8.206 g, 31.89 mmol, 64%); mp 109-110°C (from water-acetone) (lit, 107- 109°C (from water-acetone)); (Found C, 56.01; H, 7.60; N, 5.46. Cι 2 H 19 NO 5 requires C, 56.02; H, 7.44; N, 5.44%); v max (KBr discycm "1 3210br w (N-H), 2967m (C-H), 2936w (C-H), 1752s (C=O), 1730s (C-O), 1701s (CO), 1264m, 1252w, 1230m, and 1063w; δ H (200MHz; CDC1 3 ) 0.97 (3H, t, 77.0, CHjCH.CH), 1.09-133 (IH, m, one of CH 3 CH 2 CH), 1.30 (6H, t, 77.0, 2 x CHjCH.O), 1.71-1.90 (IH, m, one of CH 3 CH 2 CH), 2J9 (IH, dd, 79.0, 16.5, one of CH^N), 2.56 (IH, dd, 78.5, 16.5, one of CH^N), 2.79-2.95 (IH, m, CHCH 2 ), 4J 5-4.38 (4H, m, 2 x CHjO), 6.44 (IH, br s, NH); δ c (5031MHz; CDC1 3 ) 12.02 (CH 3 CH 2 CH), 13.74, 13.92 (2 x CH 3 CH 2 O), 23.56 (CH 3 CH 2 CH), 35.10 (CH 2 CO), 42.45 (CHCH 2 ), 62.32 (2 x CH 2 O), 71.56 (ipso-C,CN), 168.34, 168.74 (2 x CO 2 Et), 176.59 (CH 2 CO); m/z (APCI+ NH 3 ) 258 (MH + , 100%). trα«-y-4-Ethyl-5-(ethoxycarbonyl)pyrroIidin-2-one (12) c s-4-Ethyl-5-(ethoxycarbony0pyrrolidin-2-one (13) NaOH (IM, 19 ml) was added to a stirred solution of 4-ethyl-5-di(ethoxycarbonyl)- pyrrolidin-2-one (11) (5 g, 19 mmol) in EtOH (50 ml) and the mixture refluxed. After 24 h the mixture was cooled to room temperature, HCl (IN, 19 ml) was added, and the solution evaporated in vacuo. The residual half-ester was heated at 160° C for 1 h, cooled, dissolved in Et 2 O, and filtered and concentrated in vacuo to give (11), and (12) as a white crystalline solid in the ratio (ca. 35:65 by *H NMR analysis), (2.5 g. 14 mmol, 70%), characterised as follows: trα«s-4-Ethyl-5-(ethoxycarbonyI)pyrrolidin-2-one (12) mp 58-60°C (from Tr 2 O) (lit. 59-60 from Et 2 O); δ H (200MHz; CDC1 3 ) 0.98 (3H, t, 7 7.5, CH^CH.CH), 1.30 (3H, t, 77.0, CH 3 CH 2 O), 1.40-1.61 (IH, m, one of CH 3 CH 2 CH), 1.69-1.91 (IH, m, one of CH 3 CHaCH), 2.07 (IH, dd, 75.5, 16.0, one of CH 2 CO), 237-2.53 (IH, m, CHCH 2 ), 2.56 (IH, dd, 78.8, 16.0, one of CH.CO), 3.87 (IH, d, 75.0, CHN), 4.23 (2H, q, 77.0 , CH 3 CH 2 O), 5.84 (IH, br s, NH); δ c (50.31MHz; CDC1 3 ) 1 1.32 (CH 3 CH 2 CH), 13.95 (CH 3 CH 2 O), 27.66 (CH 3 CH 2 CH), 35.42 (CH 2 CO), 40.37 (CHCH 2 ), 60.72 (CHN), 61.53 (CH 3 CH 2 O), 172.24, 177.55 (2 x CO); m/z (APCI+, NH 3 ) 186 (MH + , 100%). c-s-4-Ethyl-5-(benzoxycarbonyl)pyrrolidin-2-one (15), and trfl«-.-4-EthyI-5-(benzoxycarbonyl)pyrrolidin-2-one (14) Titanium isopropoxide (250 μl, 840 μmol) was added to a stirred solution of 4-ethyl- 5-(ethoxycarbonyl)pyrrolidin-2-one (518 mg, 2.80 mmol) in benzyl alcohol (30 ml, 290 mmol), and the mixture heated at 130°C for 24 h. The cooled mixture was quenched with 5 N HCl (aq) (4 ml), washed with saturated K 2 CO 3 (30 ml), and extracted with EtOAc (1 x 60 ml). The organic layer was washed with brine (1 x 10 ml), dried (MgSO 4 ), filtered, and the solvent removed in vacuo using a Kugelrhor to give a pale yellow oil which was purified by flash column chromatography (silica, EtOAc:PE 40-60, 7:3) to give a mixture of both of the title compounds as a white solid (555 mg, 2.24 mmol, 80%). The diastereomers were separated by fractional crystallisation from ; Pr 2 O (-20°C) and characterised as follows: cjs-4-EthyI-5-(benzoxycarbonyl)pyrroIidin-2-one (15) mp 86-88°C (from Ψr0 2 - 20°C); (Found: C, 67.99; H, 6.91; N, 5.64. C 14 H 17 NO 3 requires C, 68.00; H, 6.93; N, 5.66%); v max (KBr discycm "1 3173br m, 3090br m, 2960m, 293 lw, 2877w, 1748s (C=O), 1702s (C=O), 1374m, 1194s, and 740m; δ H (200MHz; CDC1 3 ) 0.89 (3H, t, 77.5, CH^CH,), 1.08-130 (IH, m, one of CH 3 CH_,), 135-1.57 (IH, m, one of ^ K,), 2J5 (IH, dd, 79.5, 16.5, one of CH..CO), 238 (IH, dd, 78.0, 16.5, one of CH^CO), 4.49-2.69 (IH, m, CHCH 2 ), 4.27 (IH, d, 78.0, CHN), 5J8 (2H, s, CH^Ph), 6.78 (IH, br s, NH), 7.37 (5H, s, ϋ); δ c (5031MHz; CDC1 3 ) 12.02 (CH 3 CH 2 ), 23.27 (CH 3 CH 2 ), 34.77 (CH 2 CO), 40.38 (CHCH 2 ), 59.56 (CHN), 67.12 (CH 2 Ph), 128.85 (aromatic CH, C^Hs), 135.22 (ipso-C, CCH 2 ) 171.47, 178.68 (2 x CO); m/z (APCI-, NH 3 ) 248 (MH + , 100%). trans-4-Ethyl-5-(benzoxycarbonyl)pyrrolidin-2-one (14) mp 78°C (from 'PrO^ -20°C); (Found: C, 68.15; H, 6.95; N, 5.59. C 14 H 17 NO 3 requires C, 68.00; H, 6.93; N, 5.66%); v max (KBr discycm "1 3221br m (N-H), 2957m (C-H), 2934m (C-H), 1743s (C=O), 1684 (C=O), 1253m, and 734m; δ H (200MHz; CDC1 3 ) 0.96 (3H, t, 77.5, CHsCH,), 1.40-1.61 (IH, m, one of CH 3 CHι), 1.68-1.89 (IH, m, one of CH^FQ, 2.06 (IH, dd, 75.5, 16.0, one of CH^CO), 234-2.49 (IH, m, CHCH 2 ), 2.55 (IH, dd, 79.0, 16.0, one of CI . CO), 3.93 (IH, d, 75.0, CHN), 5.20 (2H, s, CHjPh), 6.36 (IH, br s, NH), 7.37 (5H, s, C 6 __); δ c (5031MHz; CDC1 3 ) 11.36 (CH 3 CH 2 ), 27.73 (CH 3 CH 2 ), 35.36 (CH 2 CO), 40.40 (CHCH 2 ), 60.68 (CHN), 67.28 (CH 2 Ph), 128.52, 128.90 (aromatic CgHs), 135.33 (ipso-C, CCH 2 ), 172.10, 177.41 (2 x CO). trflH.v-4-Ethyl-5-(benzoxycarbonyl)-l-(p-toluenesulphonyl)pyrrolidin-2-one (16) Lithium (1.0M in THF, 411 μl, 411 μmol) was added dropwise to a stirred solution of trα/w-ethyl-5-(benzoxycarbonyl)pyrrolidin-2-one (14) (101 mg, 41 1 μmol) in dry THF (15 ml) at -78°C under argon. The mixture was stirred for 15 min and a solution of/ oluenesulphonyl chloride (94 mg, 480 μmol) in dry THF (5 ml) at -78 °C was added rapidly. The mixture was stirred at -78 °C for a further 10 min and at room temperature for a further 3 h. The reaction was quenched with saturated NH 4 C1 solution (60 ml), the mixture partitioned, and the aqueous layer extracted with EtOAc (3 x 20 ml). The combined organic extracts were dried (MgSO 4 ), filtered, and concentrated in vacuo to yield a colourless oil, which was purified by flash column chromatography (silica, Et 2 O:PE 40-60, 1 : 1) to give (16) as a colourless oil (155 mg, 386 μmol, 94%); δ H (200MHz; CDC1 3 ) 0.94 (3H, t, 77.5, CHjCH,), 1.38-1.69 (2H, m, CH 3 CH_.), 2J0-2.29 (2H, m, one of CH 2 CO, CHCH 2 ), 2.62-2.74 (IH, m, one of CH^CO), 4.61 (IH, d, 72.5, CHN), 5.21, 5.29 (2H, ABq, 7 12.5, CH. ), 7.30 (2H, d, 78.0, Ts CH), 7.38 (5H, s, C 6 H 5 ), 7.92 (2H, d, 78.0, Ts CH); δ c (5031MHz; CDC1 3 ) 10.89 (CH 3 CH 2 ), 21.62 (Ts CH 3 ), 27.39 (CH 3 CH 2 ), 36.49 (CH 2 CO), 37.90 (CHCH 2 ), 64.50 (CHN), 67.64 (CH 2 O), 128.45, 128.81, 128.89, 129.15, 129.54 (aromatic CH, Q;H 5 , Ts), 134.98, 135.98, 135.23 (ipso-C, CCH 2 , CCH 3 ), 145.60 (ipso-C, CSO 2 ), 170.78, 172.57 (2 x CO); m/z (APCI+, NH 3 ) 402 (MH + , 100%). trans-4-Ethyl-l-(p-toluenesulphonyl)-pyrrolidin-2-one-5-carboxylic acid (γ-Lactam (IV)) A solution of tr π----4-ethyl-5-(benzoxycarbonyl)-l-(p-toluenesulphonyl)-pyrrolidin- 2-one (81 mg, 200 μmol) in dry THF (5 ml) was stirred under an atmosphere of hydrogen, and the reaction followed by TLC. On completion of reaction (70 min) the mixture was filtered through Celite® washing with EtOAc (3 x 25 ml). The combined organic extracts were filtered through glass wool, and the solvent removed in vacuo to yield the γ-lactam as a white crystalline solid (60 mg. 190 μmol, 95%); δ H (200MHz; CDC1 3 ) 0.96 (3H, t, 77.5, CHjCH,), 135-1.73 (2H, m, CHsCI^), 2J8 (IH, dd, 73.0, 17.5, one of CtLOO), 230-2.45 (IH, m, CHCH 2 ), 2.44 (2H, s, Ts CH 3 ), 2.78 (IH, dd, 78.5, 17.5, one of CH^CO), 4.56 (IH, d, 72.0, CHN), 7.35 (2H, d, 78.5, Ts CH), 7.97 (2H, d, 78.5, Ts CH), 9J3 (IH, br s, CO 2 H), δ c (5031MHz; CDC1 3 ) 10.81 (CH 3 CH 2 ), 21.60 (Ts CH 3 ), 27.49 (CH 3 CH 2 ), 36.45 (CH 2 CO), 37.88 (CHCH 2 ), 64.47 (CHN), 129.04, 129.69 (aromatic CH, Ts), 134.82 (ipso-C, CCH 3 ), 145.86 (ipso-C,CS0 2 ), 172.99, 175.99, 175.92 (2 x CO); m/z (APCI-,NH 3 ) 310 (M- H, 100%). The following compounds were prepared by processes analogous to those described above: Ethyl (2S, 4R)-l-(p-nitrobenzenesulphonyl)-4-ethylpyroglutamate M.p. 60.5-61.5 °C. By "PNBS", as used herein, is meant -nitrobenzenesulphonyl and by "Et", as used herein, is meant ethyl. (2S, 4R)-l-(p-Toluenesulphonyl)-4-ethylpyroglutamic acid M.p. 70-71°C. By "Ts", as used herein, is meant /?-toluenesulphonyl. (2S, 4R)-l-(p-Nitrobenzenesulphonyl)-4-ethylpyroglutamic acid Isolated as a brown oil. (2S, 4R)-l-(p-Toluenesulphonyl)-4-benzylpyroglutamic acid M.p. 91-92°C. By "Ph", as used herein, is meant phenyl. Ethyl (IS, 4R)-4-ethylpyroglutamate M.p. 122-123°C. Ethyl (2S-4S)-4-ethylpyroglutamate Isolated as a colourless oil. Benzhydryl (2S, 4R)-4-ethylpyroglutamate M.p. 156-157°C. Benzyl (2S, 4R)-4-ethylpyroglutamate M.p. 55-56°C. By "Bn" as used herein, is meant benzyl. Benzyl (2S, 4S)-4-ethylpyroglutamate M.p. 71-73°C. (3R, 5S)-3-Ethyl-5-hvdroxymethylpyrrolidin-2-one M.p. 46-47° C. (3S- 5S)-3-Ethyl-5-hvdroxymethylpyrrolidin-2-one Isolated as a colourless oil. (2S, 4R)-4-Benzylpyroglutamic acid Isolated as a colourless oil. Benzyl (2S)-4,4-diethylpyroglutamate Isolated as a colourless oil. Ethyl (2S)-4,4-diethylpyroglutamate Isolated as a colourless oil. Benzyl (2S- 4S)-4-benzylpyroglutamate Isolated as a colourless oil. Benzyl (2S, 4R)-4-benzylpyroglutamate M.p. 98-99.5°C. Ethyl (2S, 4R)-l-(p-toluenesulphonyl)-4-ethylpyroglutamate M.p. 79-80°C. Ethyl (2S. 4S)-l-(p-nitrobenzenesulphonyl)-4-ethylpyroglutamate M.p. 91-92°C. Benzyl (2S, 4R)-l-(p-nitrobenzenesulphonyl)-4-ethylpyroglutamate M.p. 84-85°C. Benzyl (IS, 4R)-l-(p-toluenesulphonyl)-4-ethylpyroglutamate Isolated as a colourless oil. Benzhydryl (2S- 4R)-l-(p-nitrobenzenesulphonyl)-4-ethylpyroglutamate M.p. 108-109°C. Ethyl (2SVl-(p-nitrobenzenesulphonyI)-4-4-diethylpyroglutamate M.p. 99-100°C. Benzyl (2S)-l-(p-toluenesulphonyl)-4-4-diethylpyroglutamate Isolated as a colourless oil. Benzyl (2S, 4R)-l-(p-toluenesulphonyl)-4-benzylpyroglutamate I Ts M.p. 111-112°C. (2S)-l-(p-Toluenesulphonyl)-4,4-diethylpyroglutamic acid M.p. 117-118°C. trans-3-Ethyl-l-(methanesulphonyl)pyrrolidin-2-one-4-carboxylic acid Isolated as a white solid. By "Ms", as used herein, is meant methanesulphonyl. 3-Ethyl-pyrrolidin-2-one-4-carboxylic acid M.p. 158-161°C. tr««-v-3-Ethyl-4-(methoxycarbonyl)-l-(p-nitrobenzenesulphonyl)pyrrolidin-2- one M.p. 144-146°C. By "Me", as used herein, is meant methyl. t «s-3-Ethyl-4-(methoxycarbonyl)-l-(/ oluenesulphonyl)pyrrolidin-2-one M.p. 103-106°C. c -3-Ethyl-4-(methoxycarbonyl)-l-(p-toluenesulphonyl)pyrrolidin-2-one M.p. 104-105°C. tra«-v-4-Ethyl-5-(ethoxycarbonyl)-l-(p-toluenesulphonyl)-pyrrolidin-2-one M.p. 78-81°C. c*s-4-Ethyl-5-(ethoxycarbonyl)-l-(p-toluenesulphonyl)pyrrolidin-2-one M.p. 88-90°C. c.s-4-Ethyl-l-(p-toluenesulphonyI)pyιτolidin-2-one-5-carboxylic acid M.p. 189-192°C. trfl»y-4-Ethyl-5-(benzoxycarbo-ιyl)-l-(methanesulphonyl)pyrrolidin-2-one M.p. 69-70°C. cts-4-Ethyl-5-(benzoxycarbonyl)-l-(p-toluenesulphonyl)pyrrolidin-2-one M.p. 108-109°C. c-s-4-Ethyl-5-(benzoxycarbonyl)-l-(methanesulphonyI)pyι olidin-2-one M.p. 117-119°C. The data obtained for the above compounds were found to be in accordance with predictions. Inhibition Studies Examples of active compounds against elastase. i Assays followed standard procedures according E.G. Del Marr; C. Largman; J.W. Brodrick; M. Fassett; M.C. Geokas; Biochemistry. 1980, 19, 468-472.

Claims: CLAIMS 1. A compound of formula (I) wherein R 1 is hydrogen or an electron withdrawing group; one of X 1 to X 4 is a CR 2 R 3 group wherein at least one of R 2 and R 3 is a C x to C 6 aliphatic group, an aromatic group, a heterocyclic group, a basic group or R 4 CONH wherein R 4 is a to C 6 aliphatic group, an aromatic group or a heterocyclic group and any remaining R 2 or R 3 is hydrogen; at least one of X 1 to X 4 is a CHR 5 group wherein R 5 comprises a hydrogen bond donor or acceptor; the remaining X 1 to X 4 groups are CH 2 , O or NR 6 wherein R 6 is hydrogen, a Cj to C 6 aliphatic group, an aromatic group or a heterocyclic group; and y is 0 or 1, or a pharmaceutically acceptable salt thereof. 2. A compound according to claim 1, wherein X 1 is CR 2 R 3 , X 2 is CH 2 and X 3 is CHR 5 ; or X 1 is CR 2 R 3 , X 2 is CHR 5 and X 3 is CH 2 ; or X 1 is CH 2 , X 2 is CR 2 R 3 and X 3 is CHR 5 ; and R 1 , R 2 , R 3 and R 5 are as defined in claim 1, or a pharmaceutically acceptable salt thereof. 3. A compound according to claim 1 or 2, wherein R 1 is hydrogen, SO 2 R 7 , CO 2 R 7 , CONHR 7 , COR 7 , C-CR 8 R 9 wherein R 7 is C, to C 6 alkyl; CH 2 (CF 2 ) 0 . 4 CF 3 ; aryl or heteroaryl, which aryl or heteroaryl are mono-ring, or have two fused rings one of which may be saturated, and which aryl and heteroaryl groups may be substituted by one or more C, to C 4 alkyl, halo, NR 10 R π , SO 2 R 10 R", CONR 10 R", C, to C fi alkyl ester, CN, CH 2 OH, O-C, to C ft alkyl, CF 3 or nitro groups, aryl-C, to C„ alkyl; aryl-C, to C 4 -alkyl-NH, or aryl-C 2 to C 4 alkenyl; or such groups wherein aryl is substituted by one or more C, to C 4 alkyl or halo groups; R s and R 9 , which may be the same or different, are COR 12 ; R 10 and R 11 , which may be the same or different, are hydrogen or Cj to C 4 alkyl; and R 12 is hydrogen or an aliphatic, aromatic or heteroaromatic group, or a pharmaceutically acceptable salt thereof 4. A compound according to any one of claims 1 to 3, wherein at least one of R 2 and R 3 is ethyl, propyl, isopropyl, butyl, isobutyl, methoxy, ethoxy, propoxy, methylthio, ethylthio, propylthio, basic groups such as amino, amidino and guanidino, benzyl, phenyl, C 6 H 5 CH 2 CONH or C 6 H 5 OCH 2 CONH, or a pharmaceutically acceptable salt thereof 5. A compound according to any one of the preceding claims, wherein R 5 is CO 2 R 12 , CONHR 12 , NHCOR 12 , NHSO 2 R 12 , OCOR 12 or OCONHR 12 , wherein R 12 is as defined in claim 3, or a pharmaceutically acceptable salt thereof 6. A compound according to any one of the preceding claims, wherein R 1 is hydrogen, joα/' -nitrobenzenesulphonyl, ?αrα-toluenesulphonyl, para- trifluoromethylbenzenesulphonyl or methanesulphonyl; and X 1 is CH 2 , X 2 is CHCH 2 CH 3 and X 3 is CHCO 2 R 13 wherein R 13 is hydrogen, ethyl or benzyl; or X 1 is CHCH 2 CH 3 or C(CH 2 CH 3 ) 2 , X 2 is CH 2 and X 3 is CHCO 2 R 13 wherein R 13 is as defined above; or X 1 is CHCH 2 CH 3 , X 2 is CHCO 2 R 14 wherein R 14 is hydrogen, methyl or ethyl and X 3 is CH 2 , or a pharmaceutically acceptable salt thereof. 7. A compound according to any one of the preceding claims, wherein R 1 is » rα-nitrobenzenesulphonyl, ?αr -toluenesulphonyl, αr7- trifluoromethylbenzenesulphonyl or methanesulphonyl; and X 1 is CH 2 , X 2 is CHCH 2 CH and X 3 is CHCO 2 H, or X 1 is CHCH 2 CH , X 2 is CH 2 and X 3 is CHCO 2 H, or X 1 is CHCH 2 CH 3 , X 2 is CHCO 2 R 14 wherein R 14 is as defined in claim 6 and X 3 is CH 2 , or a pharmaceutically acceptable salt thereof 8. A compound of formula (I) as claimed in claim 1, or a pharmaceutically acceptable salt thereof, specifically identified herein. 9. A pharmaceutical composition comprising a compound of formula (I), as claimed in any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, in association with a pharmaceutically acceptable carrier or excipient. 10. A compound of formula (I), as claimed in any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, for use in the treatment of the human or animal body. 11. Use of a compound of formula (I), as claimed in any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for use in the treatment of acute respiratory distress syndrome, cystic fibrosis, emphysema, chronic bronchitis, coagulation disorders or bacterial infections. 12. A method of treating a human or animal suffering from, or susceptible to, acute respiratory distress syndrome, cystic fibrosis, emphysema, chronic bronchitis, coagulation disorders or bacterial infections, which method comprises administering thereto a non-toxic and effective amount of a compound of formula (I), as claimed in any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof. 13. Non-therapeutic use of a compound of formula (I), as claimed in any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, as an inhibitor of elastases, thrombin, factor Xa, trypsin, chymotrypsin, transpeptidase enzymes or β- lactamases.

Inventors: SCHOFIELD CHRISTOPHER JOSEPH, SCHOFIELD, CHRISTOPHER, JOSEPH
Applicants: ISIS INNOVATION, SCHOFIELD CHRISTOPHER JOSEPH, ISIS INNOVATION LIMITED, SCHOFIELD, CHRISTOPHER, JOSEPH
Application year: 1999
Main classification: C07D207
IPC codes: A61K31, A61P11, A61P31, C07D207, C07D211, C12N9