FUROPYRROLIDINE DERIVATIVES AND THEIR USE AS SERINE PROTEASE INHIBITORS

Abstract: There is provided according to the invention an inhibitor of a serine protease enzyme which is a substituted derivative of trans-hexahydrofuro[3,2-b]pyrrol-2-one.

Description: FUROPYRROLIDINE DERIVATIVES AND THEIR USE AS SERINE PROTEASE INHIBITORS This invention relates to a new class of chemical compounds and their use as inhibitors of serine protease enzymes. These compounds are useful as pharmaceuticals and we provide processes for preparing them and formulations containing them. Serine proteases are a class of proteolytic enzymes characterised by having at the active site a serine residue which interacts with the carbonyl carbon of a peptide bond to cleave the peptide bond via an acyl enzyme intermediate. Under the conventional residue numbering based on homology with the serine protease enzyme chymotrypsin, the active site serine is generally numbered Ser-195. Most members of the family of serine proteases have a histidine and an aspartic acid residue in the active site (numbered His-57 and Asp-102 based on chymotrypsin) which activate the serine hydroxyl group to attack the scissile peptide carbonyl. In a small number of enzymes (notably Herpes virus proteases) the role of Asp-102 is taken by a further histidine residue (which is His-157 in cytomegalovirus protease). Residue mutation studies have shown these three residues to be essential for activity and they are conventionally referred to as the "catalytic triad". Although the mechanism of hydrolysis of peptide bonds by serine proteases is believed to be similar for all enzymes in the family, it is well known that their substrate specificities differ dramatically. In general, specificity is shown for peptide bonds which have a particular moiety α to the scissile peptide carbonyl which in conventional nomenclature is said to be in the P 1 position and to occupy the S-, specificity subsite (see Schlecter and Berger (1967) Biochem Biophys Res Common 27 157). For example, the preferred substrate for thrombin is a peptide containing a basic residue (e.g. arginine i.e. the moiety (CH 2 ) 3 NHC(=NH)NH 2 ) is in the P^ position) whereas the preferred substrate for elastase is a peptide containing a valine residue (i.e. the moiety CH(CH 3 ) 2 is in the P-, position). The X-ray crystal structures of a substantial number of serine protease enzymes have become available in recent years. It can be concluded in explanation of the above observations, that the "catalytic triad" is generally highly conserved in terms of its spacial orientation at the active site and that a major factor in the difference in substrate specificity comes from the shape and character of the S Ϊ specificity subsite. Serine proteases are widespread in the human body and abnormal or excessive activity of serine proteases is implicated in a diverse range of diseases and conditions (see "Proteinase Inhibitors", Barrett and Salveson (1986), Elsevier, p56; Drugs Future (1996), 21 (8), 811-816; Exp. Opin. Ther. Patents (1997) 7(1) 17-28). The following enzymes and associated conditions are exemplary: Neutrophil elastase is found in neutrophil azurophilic granules associated with tissue inflammation and is associated with a number of inflammatory diseases including emphysema, chronic bronchitis and adult respiratory distress syndrome (ARDS). Members of the blood coagulation cascade (e.g. thrombin, Factor Vila, Factor Xa, Factor Xla, Factor Xlla) and members of the fib nolytic cascade (e.g. tissue plasminogen activator and plasmin) are potential targets for treatment of diseases of the vascular system. For example, thrombin is a potential target for the treatment of thrombosis. Tissue plasminogen activator and plasmin may also be implicated in tumour metastasis. Tryptase is present in mast cells and inhibitors of tryptase have shown efficacy in models of asthma. Pancreatic elastase, trypsin and chymotrypsin are associated with digestive disorders such as pancreatitis. Cathepsin G is associated with emphysema. Serine proteases are also widespread in human pathogens especially viruses and these provide an attractive target for the treatment of pathogenic diseases and conditions. For example Herpes viruses encode a serine protease which is crucial for viral replication and is therefore a target for the treatment of conditions caused by these viruses. The Herpes family of viruses is responsible for a wide range of human infectious diseases including chicken pox and shingles (varicella and Herpes zoster viruses, respectively), cold sores and genital herpes (herpes simplex virus), retinitis, pneumonitis and keratitis (human cytomegalovirus, hCMV), as well as diseases caused by Epstein Barr Virus (EBV), human herpes virus 6 (HHV 6), HHV 7 and HHV 8. Hepatitis C virus also encodes a serine protease (known as the NS3 serine protease) which is a target for treatment of Hepatitis C virus infection and associated hepatic damage. It will be appreciated that aside from the enzymes and conditions mentioned above many other serine protease enzymes are known to be suitable targets for pharmaceutical therapy and indeed it can be expected that many more will be identified in the future. We have now invented a novel chemical class of molecules which are capable of inhibiting a wide range of serine protease enzymes. As such they are of potential value in the treatment of diseases as discussed above. More particularly, according to the invention, we provide inhibitors of serine protease enzymes which are substituted derivatives of trans-hexahydrofuro[3,2- b]pyrrol-2-one. Most particularly, this invention relates to inhibitors of serine protease enzymes which are compounds of formula I: (relative stereochemistry indicated) wherein R 1 is a moiety adapted to fit in the S 1 specificity subsite of the enzyme; R 2 is a moiety adapted to optimise the potency, pharmacokinetics, pharmacodynamics, selectivity and physicochemical properties of the inhibitor; and physiologically acceptable salts and solvates thereof. Without being limited by theory, we believe that the translactone template of formula I is highly complementary to the active site of serine proteases and the lactone carbonyl mimics the peptide carbonyl of the enzyme's natural substrate. Time-dependent (acylating) inhibition is believed to occur when attack of the enzyme active site serine on the translactone carbonyl causes opening of the strained lactone ring generating an enzyme acylated at the serine sidechain. The advantages of our invention reside inter alia in that (a) the trans- hexahydrofuro[3,2-b]pyrrol-2-one template is completely new and therefore highly desirable in a medicament especially for the treatment of pathogenic conditions which are prone to drug resistance, (b) the trans-hexahydrofuro[3,2- b]pyrrol-2-one template may be highly functionalised and is therefore ideal for the specific and selective inhibition of a wide range of different enzymes, (c) the trans-hexahydrofuro[3,2-b]pyrrol-2-one template may potentially be functionalised to give high or low metabolic stability. The determination of the optimum substitution of the derivatives of trans- hexahydrofuro[3,2-b]pyrrol-2-one, especially regarding selection of groups R 1 , and R 2 for a particular serine protease enzyme can be made in a conventional manner, namely: (a) by preparation of a number of compounds having sufficient diversity especially in groups R 1 and R 2 , (b) treatment of a sample of the enzyme in question with a sample of each of the compounds so prepared and (c) determining the extent to which inhibition of the enzyme has occurred. Assays for enzyme inhibition will generally be well known and in any event will be capable of being performed by a person skilled in the art. More particularly: Suitable R 1 groups will fit appropriately in the S, specificity subsite of the target enzyme. Choice of group R 1 may be made having regard to the known substrate specificity preferences of the target enzyme, crystallographic information concerning the geometry of the S Ϊ specificity subsite of the target enzyme and/or empirical determination based on screening data (see for example "Proteinase Inhibitors" Barrett and Salveson (1986), Elsevier, p9 and p59). When classified by their primary substrate specificity, there are three major types of serine proteinases: elastase-like, trypsin-like and chymotrypsin-like. The differences between these types can be understood in structural terms - see Kraut J (1977) Am. Rev.Biochem. 46 331-358). For inhibition of neutrophil elastase and elastase-like enzymes, the group R 1 is preferably small and hydrophobic, e.g. C 2- alkyl or C 2-4 alkenyl, especially propyl or isopropyl, particularly isopropyl. For inhibition of chymotrypsin-like enzymes (including chymotrypsin and ccaatthheeppssiinn GG)) tthhee ggrroouupp RR 1 is preferably large and hydrophobic, e.g. (CH 2 ) 1 _ 2 Ph, (CH 2 )o- 2 cyclohexyl, t-butyl Ph represents phenyl or substituted phenyl (e.g. phenyl substituted by C 1-6 alkyl, halogen). Planar aromatic sidechains (e.g. benzyl) are especially preferred. For inhibition of trypsin-like enzymes (including trypsin, thrombin, tryptase, Factor Vila, Factor Xa, Factor Xla, Factor Xlla) the group R 1 is preferably basic e.g.(CH 2 ) 2-4 NHC(=NH)NH 2 , (CH 2 ) 1 . 2 PhC(=NH)NH 2 , (CH 2 ) 3-5 C(=NH)NH 2 , CH 2 (cyclohexyl)NH 2 , (CH 2 ) 1-3 (NH) 0-1 Het ( wherein Het represents a 5 or 6 membered aromatic ring containing 1 or more nitrogen atoms and optionally substituted by amine) or (CH 2 ) 3-5 NH 2 especially (CH 2 ) C(=NH)NH 2 or (CH 2 ) 3 NHC(=NH)NH 2 , particularly (CH 2 ) 4 C(=NH)NH 2 . R 2 will be a moiety adapted to optimise the potency, pharmacokinetics, pharmacodynamics, selectivity and physicochemical properties of the serine protease inhibitor. It may also be adapted to optimise other pharmacological properties such as water solubility and oral activity (if desired). In general, R 2 can vary quite widely and a person skilled in the art would be able to determine from suitable testing if a given R 2 is suitable for the aforementioned purposes or not. Frequently, an increase in potency is achieved when the R 2 moiety binds at a remote specificity subsite such as S 3 , S 4 , or S 5 (see "Proteinase Inhibitors", Barrett and Salveson (1986) Elsevier, p6, 69). It is often preferred that R 2 comprises a CO, SO 2 or COO (especially a CO or SO 2 ) moiety attached directly to the pyrrolidine nitrogen and is, for example, a group of formula R 20 CO, R 20 SO 2 or R 20 OCO (especially R 20 CO or R 20 SO 2 ). R 20 will also be a moiety adapted to optimise the potency, pharmacokinetics, pharmacodynamics, selectivity and physicochemical properties of the serine protease inhibitor and may represent, for example, alkyl (e.g. C 1-8 alkyl), alkenyl (e.g. C 1-8 alkenyl), aryl, alkylaryl (e.g. C 1-8 alkyiaryl), or alkenylaryl (e.g. C^ 8 alkenylaryl). Where used herein, alkyl includes branched and cyclic alkyl. Alkenyl includes branched and cyclic alkenyl. Aryl includes mono and bicyclic aromatic rings optionally containing heteroatoms, e.g. O, N and S atoms (for example 1 to 4 heteroatoms). Alkyl, alkenyl, aryl, alkylaryl and alkenylaryl groups may be optionally substituted, e.g. by amine and halogen and optionally interrupted by a heteroatom (e.g. nitrogen or oxygen) or otherwise functionalised. Amine groups include primary, secondary and tertiary amine groups including cyclic amine. Thus according to the invention we also provide a method of inhibiting a serine protease enzyme which comprises treating it with a compound of the invention. We also provide a method of screening for inhibitors of serine proteases which comprises treating a serine protease enzyme with a compound of the invention and determining the extent to which inhibition has occurred. We also provide a method of identifying an inhibitor of a serine protease enzyme which comprises: (a) preparation of a number of substituted derivatives of trans- hexahydrofuro[3,2-b]pyrrol-2-one; (b) treatment of a sample of the enzyme in question with a sample of each of the derivatives so prepared; and (c) determining the extent to which inhibition of the enzyme has occurred. The extent to which inhibition has occurred may be determined by conventional assay techniques including (but not limited to) chromogenic assays, fluorogenic assays, HPLC and scintillation proximity assays. In one particularly advantageous method of drug discovery, a library comprising a plurality of substituted derivatives of trans-hexahydrofuro[3,2-b]pyrrolo-2-one will be prepared. Preferably the library will comprise a plurality of compounds of formula I wherein R 1 is a moiety adapted to fit in the ST specificity subsite of the enzyme; R 2 is a moiety adapted to optimise the potency, pharmacokinetics, pharmacodynamics, selectivity and physicochemical properties of the inhibitor; and physiologically acceptable salts and solvates thereof. The library will, ideally comprise at least 10 (e.g. 10, 100, 1000 or more) different compounds. A library of compounds of formula I wherein R 1 represents a small and hydrophobic group e.g. C 2- alkyl or C 2-4 alkenyl, especially propyl or isopropyl, particularly isopropyl may be particularly useful for screening for an inhibitor of elastase-like enzymes e.g. neutrophil elastase. A library of compounds of formula I wherein R 1 represents a basic group e.g. (CH 2 ) 2-4 NHC(=NH)NH 2 , (CH 2 ) 1-2 PhC(=NH)NH 2 , (CH 2 ) 3-5 C(=NH)NH 2 , CH 2 (cyclohexyl)NH 2 , (CH 2 ) 1-3 (NH) 0-1 Het ( wherein Het represents a 5 or 6 membered aromatic ring containing 1 or more nitrogen atoms and optionally substituted by amine) or (CH 2 ) 3 . 5 NH 2 especially (CH 2 ) 4 C(=NH)NH 2 or (CH 2 ) 3 NHC(=NH)NH 2 , particularly (CH 2 ) 4 C(=NH)NH 2 may be especially useful for screening for an inhibitor of a trypsin-like enzyme (e.g. thrombin or tryptase). A library of compounds of formula I wherein R 1 represents a large and hydrophobic group e.g. (CH 2 ) 1-2 Ph, (CH 2 ) 0-2 cyclohexyl or t-butyl may be useful for screening for an inhibitor of a chymotrypsin-like enzyme e.g. chymotrypsin or cathepsin G. Library technology will be known to a person skilled in the art and is reviewed in Drug Discovery Today (1996) 1(4) 134-144 and Annual Reports in Combinatorial Chemistry and Molecular Diversity 1. Ed. Moos Walter H, Pavia Michael R, Kay Brian K, Ellington Andy D. The library may be a solid phase or a solution phase library. It may be a discrete library or a pooled library. We also provide a method of treatment of a disease in which serine protease activity is implicated which comprises administering to a patient an effective amount of compound of the invention; and use of a compound of the invention in the manufacture of a medicament for the treatment of a disease in which serine protease activity is implicated. It will be appreciated that references herein to treatment extend to prophylaxis as well as the treatment of established conditions. A particularly preferred embodiment of the invention relates to the application of compounds of the invention in the inhibition of neutrophil elastase, thrombin, and tryptase. Compounds of the invention may be prepared from compounds of formula II (relative stereochemistry indicated) wherein R 1 is a moiety adapted to fit in the S 1 specificity subsite of the enzyme; or a protected derivative thereof, by sequential reaction to introduce the desired R 2 substituent. Conditions for such sequential reactions will be known to a person skilled in the art. Generally these reactions will consist of alkylations (usually with an alkyl halide), sulphonylations (with a sulphonyl halide) or acylations (reaction with a carboxylic acid, acid halide or anhydride). Compounds of formula II may be prepared following Scheme 1 below (compounds are drawn with relative stereochemistry): Scheme 1 β-Alanine-HCI (IX) (ix) (XII) (wherein R p and R q are respectively amino and hydroxyl protecting groups). Steps (i) to (xi) in Scheme 1 hereinabove are discussed below. Step (i) comprises treating β-alanine hydrochloride to introduce a suitable amino protecting group. Step (ii) comprises treating a compound of formula (III) with diethylfumarate in the presence of a strong base such as sodium hydride in a suitable solvent such as an aromatic hydrocarbon (e.g. toluene) or an ether (e.g. tetrahydrofuran). The reaction may conveniently be effected at an elevated temperature (e.g. reflux). Step (iii) comprises treating a compound of formula (IV) with dimethylsulfoxide in the presence of sodium chloride solution at an elevated temperature (e.g. reflux). Step (iv) comprises a stereospecific reduction of a compound of formula (V) using a borohydride such as sodium borohydride in the presence of a lanthanide salt (e.g. cerium trichloride) and in a suitable solvent such as an alcohol (e.g. ethanol) at about room temperature, followed by a Mitsunobu inversion using benzoic acid in the presence of triphenylphospine and diethyl azodicarboxylate and in a suitable solvent such as an ether (e.g. tetrahydrofuran) at about room temperature. Step (v) comprises removing the benzoyl group from a compound of formula (VI) using a base such as an inorganic base, for example a carbonate (e.g. potassium carbonate) in a solvent such as an alcohol (e.g. methanol) at about room temperature. Step (vi) comprises treating a compound of formula (VII) to introduce a suitable hydroxyl protecting group. Step (vii) comprises treating a compound of formula (VIII) with reagents capable of introducing the group R 1 . R 1 may generally be introduced by treating a compound of formula (VIII) with R 1 Hal (where Hal is a halogen atom such as bromine) in the presence of a strong base, such as lithium hexamethyldisilazide in a solvent such as THF at a temperature from 78°C to +20°C. Step (viii) comprises removing the hydroxyl protecting group R q from a compound of formula (IX). Step (ix) comprises a base-catalysed hydrolysis of a compound of formula (X). The hydrolysis may conveniently be carried out using an alkali metal hydroxide such as lithium hydroxide or potassium hydroxide. Step (x) comprises cyclising a compound of formula (XI) in the presence of a base such as an organic base (e.g. triethylamine) in the presence of an activating agent such as pyridinium salt (e.g. 2-chloro-1-methylpyridinium iodide). The reaction may be performed in a suitable inert solvent (e.g. DCM) at a temperature of 0-100°C. The pyridinium salt and the reaction is completed by adding 4-dimethyl aminopyridine in an aromatic hydrocarbon solvent (e.g. toluene) and heating the mixture (e.g. at reflux). Step (xi) comprises removing the amino protecting group R p . Compounds of formula (II) in which R p represents a benzyloxycarbonyl group may conveniently be prepared by hydrogenation/hydrogenolysis in the presence of a palladium catalyst (e.g. palladium-on-carbon) in a solvent such as ethyl acetate. In Scheme 1 , a diastereomeric separation may be necessary to obtain the compound of desired stereochemistry. Compounds of formula (I) may also be prepared from another compound of formula (I) following one or more conventional chemical transformations. It will be apparent to a person skilled in the art that the above synthetic processes for the preparation of compounds of formula (I) may be modified so as to include or omit protecting groups or so as to use alternative protecting groups (for example those described in T W Greene "Protective Groups Inorganic Synthesis", 2nd Ed (1991) J Wiley & Sons) in the course of routine optimisation of experimental conditions. For example, when R 1 contains an amidine moiety, it may be preferred to introduce substituent R 1 (e.g. as in Scheme 1) as the oxadiazolinone derivative. This may be suitably O or N protected in subsequent chemical processes. Treatment of this derivative with hydrogen over Pd/C yields the free amidine. The invention embraces compounds of the invention in racemic form as well as in a form in which one enantiomer predominates or is present exclusively. The invention embraces compounds of the invention in racemic form as well as in a form in which one enantiomer predominates or is present exclusively. Generally, we prefer to provide a compound of formula (I) in diastereoisomerically and enantiomerically pure form. For inhibition of elastase we prefer the diastereoisomers having relative stereochemistry shown in formula (1a) For inhibition of thrombin and tryptase we also prefer the diastereoisomers having relative stereochemistry shown in formula (la). Enantiomerically pure compounds may be prepared by chiral separation or by synthesis based on chiral starting materials. The present invention also covers the physiologically acceptable salts of the compounds of the invention. Suitable physiologically acceptable salts include inorganic base salts such as alkali metal salts (for example sodium and potassium salts) and ammonium salts and organic base salts. Suitable organic base salts include amine salts such as trialkylamine (e.g. triethylamine), dialkylamine (e.g. dicyclohexylamine), optionally substituted benzylamine (e.g. phenylbenzylamine or p-bromobenzylamine), procaine, ethanolamine, diethanolamine, N-methylglucosamine and tri(hydroxymethyl)methylamine salts and amino acid salts (e.g. lysine and arginine salts). Suitable inorganic and organic acid salts include the hydrochloride, trifluoroacetate and tartrate. The compounds of the invention may be formulated for administration in any convenient way, and the invention therefore also includes within its scope pharmaceutical compositions for use in therapy, comprising a compound of the invention or a physiologically acceptable salt or solvate thereof in admixture with one or more physiologically acceptable diluents or carriers. There is also provided according to the invention a process for preparation of such a pharmaceutical composition which comprises mixing the ingredients as considered appropriate for the indication. The compounds of the invention may, for example, be formulated for oral, buccal, parenteral, topical or rectal administration. Tablets and capsules for oral administration may contain conventional excipients such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, mucilage of starch or polyvinyl pyrrolidone; fillers, for example, lactose, microcrystalline cellulose, sugar, maize- starch, calcium phosphate or sorbitol; lubricants, for example, magnesium stearate, stearic acid, talc, polyethylene glycol or silica; disintegrants, for example, potato starch, croscarmellose sodium or sodium starch glycollate; or wetting agents such as sodium lauryl sulphate. The tablets may be coated according to methods well known in the art. 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 constitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, for example, sorbitol syrup, methyl cellulose, glucose/sugar syrup, gelatin, hydroxymethyl cellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats; emulsifying agents, for example, lecithin, sorbitan mono-oleate or acacia; non-aqueous vehicles (which may include edible oils), for example almond oil, fractionated coconut oil, oily esters, propylene glycol or ethyl alcohol; or preservatives, for example, methyl or propyl f hydroxybenzoates or sorbic acid. The preparations may also contain buffer salts, flavouring, colouring and/or sweetening agents (e.g. mannitol) as appropriate. For buccal administration the compositions may take the form of tablets or lozenges formulated in conventional manner. The compounds may also be formulated as suppositories, e.g. containing conventional suppository bases such as cocoa butter or other glycerides. The compounds of the invention may also be formulated for parenteral administration by bolus injection or continuous infusion and may be presented in unit dose form, for instance as ampoules, vials, small volume infusions or pre- filled syringes, or in multi-dose containers with an added preservative. The compositions may take such forms as solutions, suspensions, or emulsions in aqueous or non-aqueous vehicles, and may contain formulatory agents such as anti-oxidants, buffers, antimicrobial agents and/or toxicity adjusting agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g. sterile, pyrogen-free water, before use. The dry solid presentation may be prepared by filling a sterile powder aseptically into individual sterile containers or by filling a sterile solution aseptically into each container and freeze-drying. By topical administration as used herein, we include administration by insufflation and inhalation. Examples of various types of preparation for topical administration include ointments, creams, lotions, powders, pessaries, sprays, aerosols, capsules or cartridges for use in an inhaler or insufflator or drops (e.g. eye or nose drops). Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents and/or solvents. Such bases may thus, for example, include water and/or an oil such as liquid paraffin or a vegetable oil such as arachis oil or castor oil or a solvent such as a polyethylene glycol. Thickening agents which may be used include soft paraffin, aluminium stearate, cetostearyl alcohol, polyethylene glycols, microcrystalline wax and beeswax. Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilising agents, dispersing agents, suspending agents or thickening agents. Powders for external application may be formed with the aid of any suitable powder base, for example, talc, lactose or starch. Drops may be formulated with an aqueous or non-aqueous base also comprising one or more dispersing agents, solubilising agents or suspending agents. Spray compositions may be formulated, for example, as aqueous solutions or suspensions or as aerosols delivered from pressurised packs, with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, 1 ,1 ,1 ,2,3,3,3-heptafluoropropane, 1 ,1 ,1 ,2- tetrafluorethane, carbon dioxide or other suitable gas. Capsules and cartridges for use in an inhaler or insufflator, of for example gelatin, may be formulated containing a powder mix of a compound of the invention and a suitable powder base such as lactose or starch. Compounds of the invention may also be used in purification and diagnostic applications involving serine protease enzymes. For example, an immobilised compound of the invention may allow a serine protease capable of binding that compound to be isolated. A tagged compound of the invention may enable a serine protease capable of binding that compound to be identified. ABBREVIATIONS BOC t-butyloxycarbonyl CBZ Benzyloxycarbonyl (BOC) 2 0 Di-tert-butyldicarbonate THF Tetrahydrofuran LHMDS Lithium bis (trimethylsilyl)amide DMPU 1 ,3-dimethyl-3,4,5,6-tetrahydro 2 (1 H) pyrimidinone DMAP 4-dimethylaminopyridine DMF Dimethylformamide EDC 1-(3-N,N-dimethylaminopropyl)-3- ethylcarbodiimide DEAD diethylazodicarboxylate DCM dichloromethane TMEDA tetramethylethylenediamine DMSO dimethylsulphoxide The invention will be illustrated by reference to the following examples: Assay Examples In the foregoing, enzyme activity is generally determined at a 15 minute timepoint. Enzyme kinetics may be investigated by determining enzyme activity at other timepoints (e.g. 0, 30 minutes). Assay Example 1 In vitro assay for inhibition of human neutrophil elastase Assay contents: 50mM Tris/HCI (pH 8.6) 150mM NaCI 11.8nM purified human neutrophil elastase Suitable concentrations of compound under test diluted with water from a 10mM stock solution in dimethylsulphoxide. Values above are final concentrations after the addition of substrate solution (see below). The mixture above is incubated for 15 minutes at 30°C at which time the remaining elastase activity is measured for 10 minutes in a BioTek 340i plate- reader, after the addition of 0.6mM MeO-succinyl-Ala-Ala-Pro-Val-p-nitroanilide. The rate of increase in absorbance at 405nm is proportional to elastase activity. Enzyme activity is plotted against concentration of inhibitor and an IC50 determined using curve fitting software. Assay Example 2 In vitro assay for inhibition of human thrombin Compounds of the invention may be tested for their thrombin inhibitory activity as determined in vitro by their ability to inhibit human α-thrombin in a chromogenic assay, using N-p-tosyl-Gly-Pro-Lys p-nitroanilide as the chromogenic substrate. All dilutions were made in a buffer consisting of: 50mM HEPES, 150 mM NaCI, 5mM CaCI 2 , 0.1 % PEG and at pH7.4. Briefly, the substrate (final cone, of 100μM) was added to thrombin (final cone, of 1 nM) and the reaction monitored for 10mins at 405nm using a Biotek EL340 plate reader; the assay was performed at room temperature. To obtain IC 50 values the data were analysed using Kineticalc ® with a 4-parameter curve fitting procedure to obtain the IC 50 value. To determine the IC 50 at 15mins, the compounds were preincubated with thrombin for these times prior to adding the chromogenic substrate. Assay Example 3 In vitro pNA assay of viral serine protease inhibitor activity The hCMV serine protease used is a mutant of the 30K protease lacking the internal cleavage site (Ala142/Ala143) and which has been cloned in E.coli to produce active enzyme (hCMV δAla protease). IC 50 data for test compounds are determined after preincubation of the enzyme with test inhibitor compound for 15 minutes. Test compounds are dissolved in DMSO, serially diluted and added at a range of concentrations (from 100μM - 0.195μM) to a reaction containing 0.5μM CMV δAla protease, 100mM HEPES pH7.5, 0.2mM EDTA, 10mM NaCI, 1 mM DTT, and 30% glycerol. The reaction mixture is pre-incubated at 32°C for 15 minutes prior to addition of 4mM oligopeptide substrate (Arg-Glu-Ser-Tyr-Val- Lys-Ala-pNA) and then analysed at 32°C in a BIO-TEK Bio Kinetics Reader EL340L The plate reader monitors production of pNA and calculates the reaction rates over 30 minutes. The rates are plotted against inhibitor concentration and IC 50 values determined. Assay Example 4 In vitro assay for inhibition of human mast cell tryptase. Compounds of the invention may be tested for their tryptase inhibitory activity as determined in vitro by their ability to inhibit human lung mast cell tryptase in a chromogenic assay, using N-p-Tosyl-Gly-Pro-Lys-p-nitroanilide as the chromogenic substrate. Compounds were diluted from a 10mM stock solution in dimethylsulphoxide. All dilutions were made in a buffer consisting of: 10mM Tris- HCI, 120mM NaCI, pH 7.4. Briefly, the substrate (final cone, of 400μM) was added to tryptase (final cone, of 0.11μg.m. "1 ) and compound at appropriate concentrations and the reaction monitored for 30 minutes at 405nm using a Molecular Devices Thermomax microplate reader; the assay was performed at room temperature. To obtain IC 50 values the data were analyzed using curve fitting software. To determine the IC 50 at 30 mins. the compounds were preincubated with tryptase for this time prior to addition of the chromogenic substrate. Assay Example 5 In vitro assay for inhibition of trypsin Compounds of the invention may be tested for their trypsin inhibitory activity as determined in vitro by their ability to inhibit bovine trypsin in a chromogenic assay, using N-Benzoyl-lle-Glu-Gly-Arg-p-nitroanilide as the chromogenic substrate. Compounds were diluted from a 10mM stock solution in dimethylsulphoxide. All dilutions were made in a buffer consisting of: 50mM Tris- HCI, 15mM CaCI 2 , pH 8.4. Briefly, the substrate (final cone, of 160μM) was added to trypsin (final cone, of 25ng.ml "1 ) and compound at appropriate concentrations incubated for 15 minutes and the reaction monitored for 10 minutes at 405nm using a BioTek EL340 plate reader; the assay was performed at 37°C. To obtain IC 50 values the data were analyzed using Kinetiealc ® with a 4- parameter curve-fitting procedure. Assay Example 6 In vitro assay for inhibition of Factor Xa Compounds of the invention may be tested for their Factor Xa inhibitory activity as determined in vitro by their ability to inhibit human Factor Xa in a chromogenic assay, using N-α-Benzyloxycarbonyl-D-Arg-Gly-Arg-p-nitroanilide as the chromogenic substrate. Compounds were diluted from a 10mM stock solution in dimethylsulphoxide. All dilutions were made in a buffer consisting of: 50mM Tris-HCI, 150mM NaCI, 5mM CaCI 2 , pH 7.4. Briefly, the substrate (final cone, of 200μM) was added to Factor Xa (final cone, of 0.02 U.ml "1 ) and compound at appropriate concentrations incubated for 15 minutes and the reaction monitored for 10 minutes at 405nm using a BioTek EL340 plate reader; the assay was performed at 37°C. To obtain IC 50 values the data were analyzed using Kinetiealc ® with a 4-parameter curve fitting procedure. Assay Example 7 In vitro assay for inhibition of Factor Xla Compounds of the invention may be tested for their Factor Xla inhibitory activity as determined in vitro by their ability to inhibit human Factor Xla in a chromogenic assay, using L-Pyroglutamyl-Pro-Arg-p-nitroanilide as the chromogenic substrate. Compounds were diluted from a 10mM stock solution in dimethylsulphoxide. All dilutions were made in a buffer consisting of: 8.1 mM NaH 2 PO 4 , 147mM KH 2 PO 4 , 2.7mM KCI, 137mM NaCI, pH 7.2. Briefly, the substrate (final cone, of 400μM) was added to Factor Xla (final cone, of 0.25μg.ml "1 ) and compound at appropriate concentrations incubated for 15 minutes and the reaction monitored for 10 minutes at 405nm using a BioTek EL340 plate reader; the assay was performed at 25°C. To obtain IC 50 values the data were analyzed using Kinetiealc ® with a 4-parameter curve fitting procedure. Assay Example 8 In vitro assay for inhibition of Factor Xlla Compounds of the invention may be tested for their Factor Xlla inhibitory activity as determined in vitro by their ability to inhibit human Factor Xlla in a chromogenic assay, using H-D-Pro-Phe-Arg-p-nitroanilide as the chromogenic substrate. Compounds were diluted from a 10mM stock solution in dimethylsulphoxide. All dilutions were made in a buffer consisting of: 28mM NaBarbitone, 125mM NaCI, 1mM EDTA, pH 7.35. Briefly, the substrate (final cone, of 200μM) was added to Factor Xlla (final cone, of 1.25μg.ml "1 ) and compound at appropriate concentrations incubated for 15 minutes and the reaction monitored for 10 minutes at 405nm using a BioTek EL340 plate reader; the assay was performed at 25°C. Assay Example 9 In vitro assay for inhibition of tPA Compounds of the invention may be tested for their tissue plasminogen activator inhibitory activity as determined in vitro by their ability to inhibit human tissue plasminogen activator in a chromogenic assay, using MeSO 2 -D-CHT-Gly-Arg-p- nitroanilide as the chromogenic substrate. Compounds were diluted from a 10mM stock solution in dimethylsulphoxide. All dilutions were made in a buffer consisting of: 50mM Tris-HCI, 150mM NaCI, pH 8.4. Briefly, the substrate (final cone, of 750μM) was added to tissue plasminogen activator (final cone, of 1.Oμg.ml "1 ) and compound at appropriate concentrations incubated for 15 minutes and the reaction monitored for 10 minutes at 405nm using a BioTek EL340 plate reader; the assay was performed at 30°C. To obtain IC 50 values the data were analyzed using Kinetiealc ® with a 4-parameter curve-fitting procedure. Assay Example 10 In vitro assay for inhibition of plasmin Compounds of the invention may be tested for their plasmin inhibitory activity as determined in vitro by their ability to inhibit human plasmin in a chromogenic assay, using H-D-Val-Leu-Lys-p-nitroanilide as the chromogenic substrate. Compounds were diluted from a 10mM stock solution in dimethylsulphoxide. All dilutions were made in a buffer consisting of: 50mM Tris-HCI, 150mM NaCI, 5mM CaCI 2 , pH 7.4. Briefly, the substrate (final cone, of 363μM) was added to plasmin (final cone, of 0.02 U.ml "1 ) and compound at appropriate concentrations incubated for 15 minutes and the reaction monitored for 10 minutes at 405nm using a BioTek EL340 plate reader; the assay was performed at 37°C. To obtain IC 50 values the data were analyzed using Kinetiealc ® with a 4-parameter curve fitting procedure. Assay Example 11 In vitro assay for inhibition of Factor Vila Compounds of the invention may be tested for their Factor Vila inhibitory activity as determined in vitro by their ability to inhibit human Factor Vila in a chromogenic assay, using H-D-lle-Pro-Arg-p-nitroanilide as the chromogenic substrate. Compounds were diluted from a 10mM stock solution in dimethylsulphoxide. All dilutions were made in a buffer consisting of: 20mM Tris- HCI, 150mM NaCI, 5mM CaCI 2 , 0.1% bovine serum albumin, pH 7.5. Briefly, the substrate (final cone, of 400μM) was added to Factor Vila (final cone, of 10nM in the presence of recombinant soluble tissue factor at optimal concentration) and compound at appropriate concentrations incubated for 15 minutes and the reaction monitored for 30 minutes at 405nm using a BioTek EL340 plate reader; the assay was performed at 37°C. To obtain IC 50 values the data were analyzed using. Kinetiealc ® with a 4-parameter curve fitting procedure. Assay Example 12 In vitro assay for inhibition of chymotrypsin Compounds of the invention may be tested for their chymotrypsin inhibitory activity as determined in-vitro by their ability to inhibit human pancreatic chymotrypsin in a chromogenic assay, using MeO-Succ-Arg-Pro-Tyr-pNA hydrochloride as the chromogenic substrate. Compounds were diluted from a 10mM stock solution in dimethylsulphoxide. All dilutions were made in a buffer consisting of 50mM Tris-HCI, 150mM NaCI, 25mM CaCI 2 , pH 8.4. Briefly, the substrate (final cone, of 178μM) was added to chymotrypsin (final cone, of 0.2μg/mL) and compound at appropriate concentrations and the reaction monitored for 10 minutes at 405nm using a BioTek EL340 plate reader: the assay was performed at 30°C. To obtain IC 50 values the data were analysed using Kinetiealc ® with a 4-parameter curve fitting procedure. To determine the IC 50 at 15 mins. the compounds were preincubated with chymotrypsin for these times prior to addition of the chromogenic substrate. Assay Example 13 In vitro assay for inhibition of cathepsin G Compounds of the invention may be tested for their Cathepsin G inhibitory activity as determined in vitro by their ability to inhibit human neutrophil Cathepsin G in a chromogenic assay, using N-succinyl-Ala-Ala-Pro-Phe-p- nitroanilide as the chromogenic substrate. Compounds were diluted from a 10mM stock solution in dimethylsulphoxide. All dilutions were made in a buffer consisting of: 100mM HEPES, 300mM NaCI, pH 7.2. Briefly, the enzyme (1.25ug/mL final ), buffer and compound at appropriate concentrations were incubated for 15 mins at 30°C. Substrate (7.25mM final ) was added and the reaction monitored at 30°C for 30 minutes at 405nm using a BioTek EL340 plate reader. To obtain IC 50 values the data were analyzed using Microsoft Excel® within ActivityBase® with a 4-parameter curve fitting procedure (XLFIT®). Assay Example 14 In vitro assay for plasma stability Stability of the compounds of the invention to exposure to undefined esterolytic (and other) activity was assessed in rat plasma and blood. Briefly, compounds were mixed with fresh rat plasma or rat blood, then incubated at 37°C and at various times after mixing were extracted by precipitation with acetonitrile. Reverse phase high performance liquid chromatography was used to quantify compounds at each time point. Half-lives for the compounds were calculated from the time-course data by log-linear regression. Compounds were considered to be unstable if the half life was less than 10 minutes. Compound Examples Intermediates Intermediate 1 3-(Benzyloxycarbonylamino)propionic acid ethyl ester A suspension of sodium bicarbonate (218g) in water (600ml) was added to a stirred suspension of β-alanine hydrochloride (181g) in 1 ,4-dioxan (200ml) at 20°C. The mixture was cooled to 0-5°C and a solution of benzyl chloroformate (239g) in 1 ,4-dioxan (200ml) was added over 40min. The mixture was stirred at 0-5°C for 5h, filtered and concentrated. It was then diluted with ethyl acetate (800ml), washed with water (2x400ml) and brine (400ml), dried (MgSO 4 ) and concentrated in vacuo to give the title compound as a colourless oil (300g). Analysis Found: C.62.3; H,6.6; N,5.5; C 13 H 17 NO 4 requires: C.62.4; H.6.8; N, 5.7%. Intermediate 2 5-Ethoxycarbonylmethyl-4-oxopyrrolidine-1 ,3-dicarboxylic acid 1 -benzyl ester 3- ethyl ester A solution of Intermediate 1 (169g) in dry toluene (100ml) was added over 1 h to a stirred suspension of sodium hydride (80% suspension in oil, 22g) in dry toluene (900ml) at 20°C. The mixture was stirred for 10min before a solution of diethylfumarate (127g) in dry toluene (100ml) was added over 30min. The reaction was then stirred at reflux for 3.5h. The reaction mixture was cooled to 20°C and acidified to pH5 with hydrochloric acid (2N; ca.300ml). The mixture was extracted with ethyl acetate (3x600ml). The combined extracts were washed with water (1000ml) and brine (1000ml) and dried (MgSO 4 ). The solvent was removed in vacuo to give the title compound (252g) as a brown oil. T.l.c. Silica, ether, Rf 0.41. Intermediate 3 2-Ethoxycarbonylmethyl-3-oxopyrrolidine-1-carboxylic acid benzyl ester An emulsion of Intermediate 2 (252g) in dimethylsulfoxide (2.2 litres) and brine (545ml) was stirred vigorously at reflux for 5h. The reaction mixture was cooled to 20°C and diluted with water (2 litres). The mixture was extracted with ethyl acetate (3x1 litres), and the combined extracts were washed with water (2 litres) and brine (2 litres) and dried (Na 2 SO 4 ). The solvent was evaporated and the residue was purified by flash column chromatography using etheπhexane (1 :1) to 100% ether (gradient elution) to give the title compound (138g) as a colourless oil. T.l.c. Silica, ether, Rf 0.53. Intermediate 4 trans-3-Benzyloxy-2-ethoxycarbonylmethylpyrrolidine-1 -carboxylic acid benzyl ester Sodium borohydride (2.5g) was added over 15min to a stirred suspension of Intermediate 3 (20g) and cerium chloride heptahydrate (24g) in ethanol (200ml) at 20°C and the mixture was stirred for 2.5h. The reaction was quenched by the dropwise addition of saturated aqueous ammonium chloride (250ml) and brine (250ml) and was then extracted with ethyl acetate (3x500ml). The combined extracts were dried (Na 2 SO ) and evaporated, the residual oil was dissolved in dry tetrahydrofuran (600ml) and benzoic acid (12g) and triphenylphosphine (26g) were added. A solution of diethyl azodicarboxylate (17g) in dry tetrahydrofuran (25ml) was then added dropwise over 0.5h at 20°C. The solution was stirred at 20°C for 20h and then concentrated in vacuo. The residue was partitioned between sodium bicarbonate solution (1 litre) and ethyl acetate (3x500ml). The combined extracts were dried (Na 2 SO 4 ), concentrated in vacuo and purified by flash column chromatography on silica (Merck 9385) using etheπhexane (1 :3) as eluent to give the title compound (12.6g) as a yellow oil (eluent increased to etheπhexane (1 :2) once product started to collect). T.l.c. Silica, etheπhexane (1 :1), Rf 0.32. Intermediate 5 trans-3-Hydroxy-2-ethoxycarbonylmethylpyrrolidine-1 -carboxcyclic acid benzyl ester Potassium carbonate (4.0g) was added to a stirred solution of Intermediate 4 (4.0g) in absolute ethanol (50ml) at 20°C and the suspension was stirred at 20°C for 2 days. Hydrochloric acid (2N, 30ml) was then added and the mixture was extracted with ethyl acetate (3x40ml). The combined extracts were dried (MgSO 4 ) and concentrated in vacuo to a yellow oil which was purified by flash chromatography on silica (Merck 9385) using etheπhexane (10:1) as eluent to give the title compound as a yellow oil (1.6g). Analysis Found: C,62.35; H.6.9; N.4.5; C 16 H 21 NO 5 requires: C,62.5; H.6.9; N,4.6%. Intermediate 6 trans-3-(Butyldimethylsiloxy)-2-ethoxycarbonylmethylpyrrolidine-1 -carboxylic acid benzyl ester Imidazole (4.6g) and t-butylchlorodimethylsilane (8.8g) were added to a stirred solution of Intermediate 5 (10.5g) in dry dimethylformamide (120ml) at 20°C. The resultant solution was stirred at 20°C for 16h. Water (300ml) was added and the mixture was extracted with ethyl acetate (3x40ml). The combined extracts were dried (MgSO 4 ) and concentrated in vacuo to a yellow oil. Purification by flash chromatography on silica (Merck 9385) using etheπhexane (1 :5) as eluent yielded the title compound as a colourless oil (13.8g). T.l.c. Silica, etheπhexane (1 :3), Rf 0.30. Intermediate 7 trans-3-(t-Butyldimethylsilyloxy)-2-(1-ethoxycarbonylbut-3-enyl)pyrrolidine-1- carboxylic acid benzyl ester Lithium hexamethyldisilazide (1 M in hexane, 37ml) was added dropwise over 20min to a stirred solution of Intermediate 6 (10.4g) in dry tetrahydrofuran (140ml) at -78°C. The solution was stirred at -78°C for 2h. Allyl bromide (3.8g) was added over 5min and the solution was stirred at 78°C for 1 hr and at 20°C for a further 3h. Saturated aqueous ammonium chloride (100ml) was added and the mixture extracted with ethyl acetate (3x40ml). The combined extracts were dried (MgSO 4 ) and concentrated in vacuo to yield a yellow oil which was purified by flash chromatography on silica (Merck 9385) using etheπhexane (1 :5) as eluent to give the title compound as a pale yellow oil (6.9g). Analysis Found: C,65.05; H,8.6; N,3.3; C 25 H 41 NO 5 Si requires: C.64.8; H,8.9; N,3.0%. Intermediate 8 trans-2-(1-Ethoxycarbonylbut-3-enyl)-3-hydroxypyrrolidine-1 -carboxylic acid benzyl ester Tetrabutylammonium fluoride (1 M in tetrahydrofuran, 750ml) was added over 10min to a solution of Intermediate 7 (280g) in dry tetrahydrofuran. The mixture was stirred at 20°C under nitrogen for 18h. The solvent was removed in vacuo and the residue was purified by flash column chromatography on silica (Merck 9385) using etheπhexane (1 :1) to 100% ether (grade elution) to give the title compound (157.5g) as a colourless oil. T.l.c. Silica, ether Rf, 0.71/0.67 (diastereomers). Intermediate 9 trans-2-(1-Carboxybut-3-enyl)-3-hydroxypyrrolidine-1 -carboxylic acid benzyl ester A suspension of lithium hydroxide (16.0g) in water (50ml) was added to a solution of Intermediate 8 (45. Og) in a mixture of tetrahydrofuran (700ml) and water (80ml). The suspension was stirred at 50°C for 18h. The reaction was allowed to cool. Water (500ml) was added and the mixture was washed with diethyl ether (3x150ml). The aqueous layer was acidified to pH4 with dilute hydrochloric acid (2N, 100ml) and extracted with ethyl acetate (3x400ml). The combined extracts were washed with brine (500ml), dried (MgSO 4 ) and evaporated to give the title compound (34g) as a pale yellow oil. T.l.c. Silica, ether, Rf 0.32. Intermediate 10 rel-(3R,3aR,6aS)-3-Allyl-2-oxohexahydrofuro[3,2-b]pyrrole-4-carboxylic acid benzyl ester 2,4,6-Trichlorobenzoyl chloride (26g) was added to a solution of Intermediate 9 (10g) in dry dichloromethane (600ml) and triethylamine (3.6g) at 20°C. The mixture was stirred at 20°C for 3h. It was then diluted with toluene (1600ml) and added dropwise over 4h to a stirred solution of 4-dimethylaminopyridine (17.22g) in dry toluene (800ml) at reflux. The mixture was stirred at reflux for a further 1 h. The reaction was allowed to cool and the solvent was removed in vacuo. Water (300ml) and dilute hydrochloric acid (2N, 300ml) were added and the mixture was extracted with ethyl acetate (3x500ml). The combined extracts were dried (MgSO 4 ), concentrated and purified twice by flash column chromatography on silica (Merck 9385) using etheπhexane (1 :1) as eluent to give the title compound as an off-white solid (9.08g). Analysis Found: C,68.5; H,6.6; N,4.65; C 17 H 19 NO 4 requires: C,67.8; H.6.4; N,4.7%. Intermediate 11 rel(3R,3aR,6aS)-3-Propylhexahydrofuro[3,2-b]pyrrole-2-one 5% Palladium/carbon catalyst was stirred in ethyl acetate (50ml) under a hydrogen atmosphere for V 2 h. Intermediate 10 (5.0g) in ethyl acetate (150ml) was added to the catalyst suspension and the mixture was stirred under a hydrogen atmosphere for 2h. The catalyst was filtered off and the filtrate was evaporated to give the title compound (2.7g) as a white solid. T.l.c. Silica, ethyl acetate:methanol (19:1), Rf 0.21 Analysis Found: C.63.85; H,8.8; N.7.9; C 9 H 15 NO 2 requires: C,63.9; H,8.9; N,8.3%. Compound Example 1 rel-(3R,3aR,6aS)-4-(2-Oxo-3-propylhexahydrofuro[3,2-b]pyrrole-4- sulfonyQbenzoic acid Intermediate 11 (0.05g), 4-(chlorosulfonyl)benzoic acid (0.076g) and triethylamine (0.076g) were mixed at 0°C in dichloromethane (5ml). The mixture was allowed to warm to room temperature. After 3 days the reaction was diluted with dichloromethane (10ml) and hydrochloric acid (1 M 10ml). The organics were then washed with brine and dried (MgSO ). The solvent was removed in vacuo and the residue purified by flash chromatography on silica (Merck 9385) using ethyl acetate: hexane (1 :1) as eluent to give the title compound (0.006g) as a white solid. Proton NMR (CH 3 OD) 51.01 (3H,t), 1.35 (2H,m), 1.6 (2H,m), 1.75-1.92 (2H,m), 2.07 (1 H,m), 2.3 (1 H,m), 2.92-3.03 (2H,m), 3.65-3.81 (2H,m), 4.09 (1 H, ddd), 7.93 and 8.25 (4H, AA'BB') Analysis Found: C.54.5; H,5.5; N,3.6; C 16 H 19 NO 6 S requires: C,54.4; N.5.4; N,4.0%. Compound Example 2 rel-(3R,3aR,6aS)-[3-(2-Oxo-3-propylhexahydrofuro[3,2-b]pyrrole-4- sulfonylmethyl)benzoic acid benzyl ester A mixture of Intermediate 11 (0.025g), triethylamine (0.017g) and 3- chlorosulfonylmethyl-benzoic acid benzyl ester (0.052g) in dichloromethane (2.5ml) was stirred at room temperature for 2.5h. The mixture was concentrated in vacuo and then examined by flash column chromatography on silica (Merck 9385) using etheπhexane (4:1) as eluent to give the title compound (0.040g) as a white solid. Proton NMR (CDCI 3 ) δθ.87 (3H,t), 1.0-1.8 (4H,m), 2.01 (1 H,m), 2.24-2.45 (2H,m), 3.17 (1 H,dd), 3.52 (1 H,m), 3.7-3.84 (2H,m), 4.3 (2H,AB), 5.37 (2H,s), 7.35-7.47 (5H,m), 7.51 (1 H,t), 7.62 (1 H, ddd), 8.03 (1 H,t), 8.12 (1 H,ddd). Analysis Found: C,62.4; H,5.9; N,2.9; C 24 H 29 NO 6 S requires: C,62.7; H,6.4; N,3.0%. Compound Example 3 rel-(3R,3aR,6aS)-3-(2-Oxo-propylhexahydrofuro[3,2-b]pyrrole-4- sulfonylmethyl)benzoic acid A mixture of Example 2 (0.034g) and 5% palladium on carbon catalyst (0.030g) in ethyl acetate (25ml) was stirred vigorously at room temperature under a hydrogen atmosphere for 2h. The catalyst was filtered off and the filtrate concentrated in vacuo. The residue was redissolved in ethyl acetate and the solution filtered and evaporated to give the title compound (0.032g). Analysis Found: C,56.3; H,6.0; N,3.6; C 17 H 21 NO 6 S requires: C,55.6; H.5.8; N,3.8% Proton NMR (d 6 -DMSO) δθ.82 (3H,t), 1.14 (2H,m), 1.38 (2H,m), 1.53 (2H,m), 1.67 (2H,m), 2.07 (2H,m), 2.34 (2H,m), 2.83 (1 H, m), 3.32 (1 H,dd), 3.6 (1 H,m), 3.94 (1 H,t), 4.08 (1 H,ddd), 4.67 (2H, AB), 7.56 (1 H,t), 7.68 (1 H,dt), 7.75 (1 H,dt), 8.05 (1 H,t). Compound Example 4 rel-(3R,3aR,6aS)-4-(1 H-lndol-2-carbonyl)-3-propyl-hexahydro-furo[3,2-b]pyrrol- 2-one 1 H-lndole-2-carbonyl chloride * (162mg) was added to a stirred solution of Intermediate 11 (100mg) and triethylamine (0.2ml) in dichloromethane (10ml) at room 20°. The suspension was stirred for 1.5h. Water (20ml) was added and the mixture was extracted with dichloromethane (3x20ml). The combined extracts were dried (MgSO 4 ) and concentrated in vacuo to give a foam (304mg). Purification of this material by flash chromatography on (Merck 9385) silica using etheπhexane (3:1) as eluent gave the title compound (118mg) as a pale yellow foam. T.l.c. silica, etheπhexane (3:1); Rf 0.53 N.m.r. (CDCI 3 ) δ 9.23(1 H, brs), 7.69(1 H, brd), 7.41(1 H, brd), 7.32(1 H,dt), 7.16(1 H, dt), 6.91(1 H, d), 4.41- 4.18(2H, m), 4.16-4.0(1 H, m), 3.59(1 H, brt), 2.97(1 H, m), 2.55(1 H, m), 2.35- 2.10(3H, m), 1.75(1 H, m), 1.40(1 H, m), 1.03(3H, t) . * Ref. W H Parsons et al J Med Chem (1989) 32, 1681 Biological Data The results obtained by testing example compounds in the example assays are indicated below: O Data in columns 1-13 is indicated at IC 50 (μM) Data in column 14 is indicated as t 1 2

Claims: Claims: 1. An inhibitor of is a substituted derivative of trans-hexa 2. An inhibitor of is a compound of formula I: (relative stereochemistr wherein R 1 is a moiety a subsite of the enzyme; R 2 is a moiety adapted t okinetics, pharmacodynamics, sel perties of the inhibitor; and physiologically acc f. 3. Use of a substituted derivative of trans-hexahydrofuro[3,2-b]pyrrol-2- 6. An inhibitor according to claim 5 wherein R 1 represents propyl or isopropyl. 7. An inhibitor according to claim 2 wherein R 1 represents (CH 2 ) 2-4 NHC(=NH)NH 2 , (CH 2 ) 1-2 PhC(=NH)NH 2ι (CH 2 ) 3 . 5 C(=NH)NH 2 , CH 2 (cyclohexyl)NH 2 , (CH 2 ) 1-3 (NH) 0 . 1 Het ( wherein Het represents a 5 or 6 membered aromatic ring containing 1 or more nitrogen atoms and optionally substituted by amine) or (CH 2 ) 3-5 NH 2 for use as an inhibitor of a trypsin-like enzyme, e.g. thrombin or tryptase. 8. An inhibitor according to claim 2 wherein R 1 represents benzyl for use as an inhibitor of a chymotrypsin-like enzyme, e.g. Cathepsin G. 9. A compound of formula II (relative stereochemistry indicated) wherein R 1 is a moiety adapted to fit in the ST specificity subsite of the enzyme. 10. A compound according to claim 9 wherein R 1 represents (CH 2 ) 2 _ 4 NHC(=NH)NH 2 , (CH 2 ) 1-2 PhC(=NH)NH 2 , (CH 2 ) 3-5 C(=NH)NH 2 , CH 2 (cyclohexyl)NH 2 , (CH 2 ) 1 . 3 (NH) 0-1 Het ( wherein Het represents a 5 or 6 membered aromatic ring containing 1 or more nitrogen atoms and optionally substituted by amine) or (CH 2 ) 3-5 NH 2 . 11. A compound according to claim 9 wherein R 1 represents C 2-4 alkyl or C 2-4 alkenyl. 12. A method of treatment of chronic bronchitis or ARDS which comprises administering to a patient an effective amount of a neutrophil elastase inhibitor according to claim 5 or 6. 13. A method of treatment of diseases of the vascular system especially thrombosis which comprises administering to a patient an effective amount of a thrombin inhibitor according to claim 7. 14. A method of treatment of asthma which comprises administering to a patient an effective amount of a tryptase inhibitor according to claim 7. 15. A library comprising a plurality of substituted derivatives of trans- hexahydrofuro[3,2-b]pyrrol-2-one. 16. A library comprising a plurality of compounds of formula I (relative stereochemistry indicated) wherein R 1 is a moiety adapted to fit in the S 1 specificity subsite of the enzyme; R 2 is a moiety adapted to optimise the potency, pharmacokinetics, pharmacodynamics, selectivity and physicochemical properties of the inhibitor; and pharmaceutically acceptable salts and solvates thereof. 17. A library according to claim 15 or 16 comprising at least 10 different compounds. 18. A library according to claim 16 or claim 17 wherein R 1 represents C 2-4 alkyl or C 2-4 alkenyl. 19. A library according to claim 16 or claim 17 wherein R 1 represents (CH 2 ) 2-4 NHC(=NH)NH 2 , (CH 2 ) 1-2 PhC(=NH)NH 2ι (CH 2 ) 3-5 C(=NH)NH 2 , CH 2 (cyclohexyl)NH 2 , (CH 2 ) 1-3 (NH) 0 . 1 Het ( wherein Het represents a 5 or 6 membered aromatic ring containing 1 or more nitrogen atoms and optionally substituted by amine) or (CH 2 ) 3-5 NH 2 . 20. A library according to any one of claims 15 to 19 which is a solid phase library. 21. A library according to any one of claims 15 to 19 which is a solution phase library. 22. A library according to claim 20 or 21 which is a discrete library. 23. A library according to claim 20 or 21 which is a pooled library. 24. Use of a library according to any one of claims 15 to 18 for screening for an inhibitor of neutrophil elastase. 25. Use of a library according to any one of claims 15, 16, 17 and 19 for screening for an inhibitor of thrombin or tryptase. 26. A method of screening for an inhibitor of a serine protease enzyme which comprises treating a serine protease enzyme with an inhibitor according to any one of claim 1 or claim 2 and determining the extent to which inhibition has occurred. 27. A method of identifying an inhibitor of a serine protease enzyme which comprises: (a) preparation of a number of substituted derivatives of trans- hexahydrofuro[3,2-b]pyrrol-2-one; (b) treatment of a sample of the enzyme in question with a sample of each of the derivatives so prepared; and (c) determining the extent to which inhibition of the enzyme has occurred.

Inventors: COOTE STEVEN JOHN, DOWLE MICHAEL DENNIS, FINCH HARRY, HANN MICHAEL MENTEITH, KELLY HENRY ANDERSON, MACDONALD SIMON JOHN FAWCETT, PEGG NEIL ANTHONY, RAMSDEN NIGEL GRAHAME, WATSON NIGEL STEPHEN, COOTE, STEVEN, JOHN, DOWLE, MICHAEL, DENNIS, FINCH, HARRY, HANN, MICHAEL, MENTEITH, KELLY, HENRY, ANDERSON, MACDONALD, SIMON, JOHN, FAWCETT, PEGG, NEIL, ANTHONY, RAMSDEN, NIGEL, GRAHAME, WATSON, NIGEL, STEPHEN
Applicants: COOTE STEVEN JOHN, DOWLE MICHAEL DENNIS, FINCH HARRY, GLAXO GROUP LTD, HANN MICHAEL MENTEITH, KELLY HENRY ANDERSON, MACDONALD SIMON JOHN FAWCETT, PEGG NEIL ANTHONY, RAMSDEN NIGEL GRAHAME, WATSON NIGEL STEPHEN, COOTE, STEVEN, JOHN, DOWLE, MICHAEL, DENNIS, FINCH, HARRY, GLAXO GROUP LIMITED, HANN, MICHAEL, MENTEITH, KELLY, HENRY, ANDERSON, MACDONALD, SIMON, JOHN, FAWCETT, PEGG, NEIL, ANTHONY, RAMSDEN, NIGEL, GRAHAME, WATSON, NIGEL, STEPHEN
Application year: 1998
Main classification: C07D491
IPC codes: A61K31, A61P7, A61P11, A61P31, A61P43, C07B61, C07D491, C12Q1