HEPATITIS C NS3 PROTEASE INHIBITORS

Abstract: Disclosed is a specific pharmacophoric profile which represents the structure for inhibitors of Hepatitis C NS3 protease. The results from mapping studies of the enzyme with depsipeptide substrates allow the generation of a particular pharmacophoric binding profile. Compounds possessing this motif were shown to be inhibitors of Hepatitis NS3 protease. The inhibitors have use in the treatment of hepatitis C.

Description: Hepatitis C NS3 Protease Inhibitors The present invention relates to inhibitors of hepatitis C NS3 protease, compounds which fit the pharmacophoric profiles of these inhibitors and use of these inhibitors for the treatment of hepatitis C. Field of the invention An analysis of the epidemiologic evidence suggests that there may be three types of non- A non-B hepatitis (NANBH); the water borne epidemic type, the blood or needle associated type, and the sporadically occurring (community acquired) type. It is thought that the recently discovered hepatitis C virus (HCV) infects over 300 million people worldwide and is the major etiological agent of both parenterally transmitted and sporadic NANBH. 1,2 Upon first exposure to HCV approximately 2-% of infected individuals develop acute clinical hepatitis while others appear to resolve spontaneously. However, in most instances the virus establishes a chrome infection which persists for decades. 4 This situation usually results in recurrent and progressively worsening liver inflammation, often leading to more severe disease states such as cirrhosis and hepatocellular carcinoma. 3 Current treatment with mterferon-α appears only to cause remission in 25% of patients, although it is also associated with a decrease in liver cell dysfunction and worsening of the disease. Of those patients who do respond they subsequently relapse when the drug is withdrawn We believe that there is a clear need for orally active compounds which lower viral load and prevent viral replication of HCV. The HCV viπon has a positive strand RNA genome that contains a single large open reading frame encoding a polyprotem of 3010-3033 ammo acid residues. The nonstructural proteins involved in replication of the HCV genome are released by the action of two protemases; NS2-3 and NS3.The action of NS3 protease yields the nonstructural proteins NS4A, NS4B, NS5A and NS5B. The N-termmal domain of NS3 contains a trypsin-hke seπne protease and the catalytic triad of Hιs-57, Asp-81 and Ser- 139 (numbeπng from the start of NS3) are strictly conserved among all HCV deπved sequences. It is thought that, because the NS3 protease is essential for viral replication, then inhibitors of this enzyme will be useful m the treatment of NANBH disease. Description of the related art. As far as we are aware only one, weakly active (2.5 μgml ') small molecule inhibitor of Hep C NS3 has been previously reported in the literature. 5 Depsipeptides have previously been synthesised as substrates for hepatitis C NS3 m order to establish a high throughput assay, 6 7 but have not been previously used to generate a pharmacophoπc profile that will be useful m designing inhibitors of Hepatitis C NS3 protease. Summary of the invention Mapping of HCV NS3 protease with a seπes of depsipeptide substrates of the type 6 (Scheme 1) provided sufficient data (Table 1) for the computer generation of a pharmacophoπc profile of the pockets on the non-pπme side of the catalytic centre. It is known m the art 10 that compounds of the type \2 (Scheme 2) which bind tightly to the non-pπme sites of other seπne proteases, and that do not possess a warhead which binds directly to the catalytic centre, can possess potent inhibitory activity In view of this pharmacophoπc profiles have been identified which descπbe inhibitors of hepatitis C NS3 protease. These pharmacophoric profiles are shown in figures 1.3 and 6. Therefore, compounds possessing a pharmacophoπc profile of the type shown m Figures 1,3 and 6 will be inhibitors of hepatitis C NS3 protease and will be useful m the treatment of NANBH. Moreover, judicious connection of all or some of the important residues required for binding to the non-pπme P*-P 5 sites of the NS3 protease 89 via a non peptide scaffold will allow the preparation of orally active drugs. Although some information of the preferred motifs can be gained from a knowledge of the natural cleavage data, somewhat surpπsmgly we have discovered that an aromatic containing residue, such as homophenylalanyl, l,2,3,4.-D-tetrahydroιsoqumolιne, 3-pyπdyl, tyrosme etc, m the P 3 position of the inhibitor is extremely beneficial for inhibitory activity In a first aspect the invention provides novel inhibitors of hepatitis C NS3 protease. In a further aspect the invention provides compounds which fit a pharmacophoπc profile of a hepatitis C NS3 protease inhibitor. In a further aspect the invention provides use of inhibitors of the invention for the treatment of hepatitis C. In a further aspect the invention provides use of the inhibitors of the invention m the manufacture of a medicament for the treatment of hepatitis C. In a further aspect of the invention there is provided a method of treatment of hepatitis C which compπses admimsteπng a pharmaceutically active amount of an inhibitor of the invention. In a further aspect the present invention provides a composition which compπses a pharmaceutically acceptable amount of an inhibitor of the invention together with a pharmaceutically acceptable earner or diluent. The invention will now be described with reference to the accompanying drawings in which: Figure 1 shows a first pharmacophoric profile of a substrate of hepatitis C NS3 protease. Figure 2 shows the distance and angle constraints of the pharmacophoπc profile of figure 1. Figure 3 shows a second pharmacophoric profile of a substrate of hepatitis C NS3 protease. Figures 4 and 5 show the distance and angle constraints of the pharmacophoπc profile of figure 3. Figure 6 shows a third pharmacophoπc profile of a substrate of hepatitis C NS3 protease. Figure 7 shows the distance and angle constraints of the pharmacophoπc profile of figure 6. Figure 8 shows an example of a compound which fits the pharmacophoπc profile of figure 1. 5 Figure 9 shows an example of a compound which fits the pharmacophoric profile of figure 3. Figure 10 shows an example of a compound which fits the pharmacophoπc profile of figure 6. Experimental 0 NS3 folding and protease activity Chemicals and biochemicals were of the highest available grade and unless otherwise stated purchased from Sigma Chemical Co., Poole, U.K. Puπfied insoluble NS3 protease (15.6 mg/m m 8 M urea) was obtained from the Hepatitis C J8 strain. Refolding of the NS3 protease was earned out by dilution of the 5 protein to 100 nM into 50 mM Tns-HCl; pH 7.4 containing 10 mM CHAPS (Calbiochem, Nottingham, U.K.); 2 mM 2-mercaptoethanol; 10 mM magnesium sulphate and 50% glycerol. The first phase of the folding reaction was carried out at 4°C for 1 h and the solution was kept well stirred. After incubation, the sample was treated with NS4A cofactor (1 equivalent, with respect to the protease, of H- o KKGSISIIGRLHLNDRVWAPKK-OH) to form a 100 nM solution which was kept well stirred further incubated at room temperature (20 °C) for a further 15 mm. The protease solution was adjusted to 10 mM 2-mercaptoethanol and this solution was used for NS3 protease assays. Fmoc-NH Linker-Crown Standard Fftioc Chemistry Scheme 1 Synthesis of Depsipeptide Compounds Depsipeptide substrates were synthesised according to the route shown m Scheme 1. Preparation of Crown Assembly The depsipeptide compounds were synthesised in parallel fashion using Fmoc-Rmk- DA/MDA denvatised macrocrowns (ex Chiron Mimotopes, Australia) loaded at approximately 7 μM per crown. Prior to synthesis each crown was connected to its respective stems and slotted into the 8 x 12 stem holder. Coupling of the ammo acids employed standard Fmoc ammo acid chemistry as descπbed in 'Solid Phase Peptide Synthesis', E. Atherton and R.C. Sheppard, IRL Press Ltd, Oxford, UK, 1989. Removal of N« -Fmoc Protection A 250 ml solvent resistant bath is charged with 200 ml of a 20% pipendme/DMF solution. The multipm assembly is added and deprotection allowed to proceed for 30 minutes. The assembly is then removed and excess solvent removed by bnef shaking The assembly is then washed consecutively with (200 ml each), DMF (5 minutes) and MeOH (5 minutes, 2 minutes, 2 minutes) and left to air dry for 15 minutes. Quantitative UV Measurement of Fmoc Chromophore Release A 1 cm path length UV cell is charged with 1.2 ml of a 20% pipendme/DMF solution and used to zero the absorbance of the UV spectrometer at a wavelength of 290nm. A UV standard is then prepared consisting of 5.0 mg Fmoc-Asp(OBut)-Pepsyn KA (0.08 mmol/g) m 3.2 ml of a 20% pipendme DMF solution. This standard gives Abs 290 = 0.55- 0.65 (at room temperature). An aliquot of the multipm deprotection solution is then diluted as appropπate to give a theoretical Abs 290 = 0.6, and this value compared with the actual expenmentally measured absorbance showing the efficiency of previous coupling reaction. Coupling of Standard Ammo Acid Residues Coupling reactions are performed by charging the appropπate wells of a polypropylene 96 well plate with the pattern of activated solutions required duπng a particular round of coupling. Macrocrown (approx 7 μ mole) standard couplings are performed in DMF (500 μl). Coupling of an Amino-acid Residue To Appropπate Well Whilst the multipm assembly is drying, the appropnate N α -Fmoc ammo acid pfp esters (10 equivalents calculated from the loading of each crown) and HOBt (10 equivalents) required for the particular round of coupling are accurately weighed into suitable containers. Alternatively, the appropπate N α -Fmoc ammo acids (10 equivalents calculated from the loading of each crown), desired coupling agent e.g. HBTU (9.9 equivalents calculated from the loading of each crown) and activation e.g. HOBt (9.9 equivalents calculated from the loading of each crown), NMM (19.9 equivalents calculated from the loading of each crown) are accurately weighed into suitable containers. The protected and activated Fmoc ammo acid denvatives are then dissolved in DMF (500 μl for each macrocrown e.g. for 20 macrocrowns, 20 x 10 eq. x 7 μ moles of denvative would be dissolved m 10 ml DMF). The appropπate derivatives are then dispensed to the appropnate wells ready for commencement of the 'coupling cycle'. As a standard, coupling reactions are allowed to proceed for 6 hours. The coupled assembly was then washed as detailed below. Coupling of L-lactic Acid L-lactic acid (lOeq per macrocrown) and HOBt (lOeq per macrocrown) were dissolved m dichloromethane (450 μL per macrocrown) and cooled with ιce-stιrnng to 0°C. Diisopropylcarbodiamide (lOeq per macrocrown) m dichloromethane (50μL per macrocrown) was added and the mixture stirred at 0°C for lhr. The resultant symmetric anhydride solution was apportioned to the appropriate wells (500μL per well), the pm assembly for coupling, added and reaction left for 5hrs. The coupled assembly was then washed as detailed below. Esterification Using Fmoc-Abu-F / 4-dimethylaminopyridine Fmoc-Abu-F (lOeq per macrocrown) was dissolved in dichloromethane (450μL per well) and the pin assembly for esterification soaked in the acid fluoride solution for 5mins. 4- dimethylaminopyridine (2eq per macrocrown) in dichloromethane (50μL per macrocrown) was added to each well and the reaction left at RT for lhr followed by washing as detailed below. Washing Following Coupling If a 20%) piperidine/DMF deprotection is to immediately follow the coupling cycle, then the multipin assembly is briefly shaken to remove excess solvent washed consecutively with (200 ml each), MeOH (5 minutes) and DMF (5 minutes) and de-protected. If the multipin assembly is to be stored or reacted further, then a full washing cycle consisting of brief shaking then consecutive washes with (200 ml each), DMF (5 minutes) and MeOH (5 minutes, 2 minutes, 2 minutes) is performed. Following these general methods, the single depsipeptides shown in Table 1 were sequentially assembled by applying the appropriate coupling procedure at the correct cycle during synthesis. Acidolvtic Mediated Cleavage of Peptide-Pin Assembly Acid mediated cleavage protocols are strictly performed in a fume hood. A polystyrene 96 well plate (1 ml/well) is labelled, then the tare weight measured to the nearest mg. Appropriate wells are then charged with a trifluoroacetic acid/triisopropylsilane (95:5, v/v, 600 μl) cleavage solution, in a pattern corresponding to that of the multipin assembly to be cleaved. The multipin assembly is added, the entire construct covered in tin foil and left for 2 hours. The multipin assembly is then added to another polystyrene 96 well plate (1 ml/well) containing trifluoroacetic acid/triisopropylsilane (95:5, v/v, 600 μl) (as above) for 5 minutes. Work up of Cleaved Peptides The primary polystyrene cleavage plate (2 hour cleavage) and the secondary polystyrene plate (5 minute wash) are then placed in the SpeedVac and the solvents removed (minimum drying rate) for 90 minutes. The contents of the secondary polystyrene plate are transferred to their corresponding wells on the primary plate using an acetonitrile/water/acetic acid (50:45:5, v/v/v) solution (3 x 150 μl) and the spent secondary plate discarded. Analysis of Products A 5μL aliquot from each well is diluted to 100 μl with 0.1% aq. TFA, then a 10 μL aliquot from this plate diluted with a further 100 μl 0.1% aq. TFA. The double diluted plate is analysed by HPLC-MS. Final Lvophilisation of Peptides The plate is covered with tin foil, held to the plate with an elastic band. A pin prick is placed in the foil directly above each well and the plate placed at -80°C for 30 minutes. The plate is then lyophilised on the 'Heto freeze drier' overnight. Finally, the dried plate is weighed. The total cleaved peptide is quantified (by weight) and the average content of each peptide calculated. Since all the peptides present have originated from the same peptide-pin assembly, cleaved under identical conditions, it is reasonable to assume that the contents of each well are roughly equimolar. Synthesis of Inhibitors of Hepatitis C NS3 protease Inhibitors of the protease were synthesised according to the route shown in scheme 2. -CONH-Crown 9 0-(CH -CONH-Crown 10 11 12 Scheme 2 Depsipeptide Substrate Kinetics The biological data for compounds of the type 6. (Scheme 1) are shown below in Table 1. Note that 2-aminobenzoyl-Glutamyl-Norleucyl-Glutamyl-Glutamyl-AbuΨ[COO]Ala-3- nitrotyrosinyl-Aspartyl-NH 2 , (Table 1 ) is a shorthand representation of N- aminobenzoyl-L-glutamyl -L-norleucinyl-L-glutamyl-L-2-aminobutanoyl-L-2- hydroxypropanoyl-L-3-nitrotyrosinyl-aspartyl amide 13 (Scheme 3) and is a specific example for the generalised structure 6 (Schemes 1 and 3). Other compounds described below are similarly represented. 13 Scheme 3 Table 1. General Synthesis of Alkyl Amide Singles. Preparation of Multipin Assembly Whilst weanng standard plastic gloves, the Fmoc-Rink-DA/MDA macrocrowns are assembled (simply clipped) onto stems and slotted into the 8 x 12 stem holder m the 5 desired pattern for synthesis. Removal ofN -Fmoc Protection A 250 ml solvent resistant bath is charged with 200 ml of a 20% pipendme/DMF solution. The multipm assembly is added and deprotection allowed to proceed for 30 minutes. The assembly is then removed and excess solvent removed by brief shaking. The 0 assembly is then washed consecutively with (200 ml each), DMF (5 minutes) and MeOH (5 minutes, 2 minutes, 2 minutes) and left to air dry for 15 minutes. Quantitative UV Measurement of Fmoc Chromophore Release A 1 cm path length UV cell is charged with 1.2 ml of a 20% pipendme/DMF solution and used to zero the absorbance of the UV spectrometer at a wavelength of 290nm. A UV 5 standard is then prepared consisting of 5.0 mg Fmoc-Asp(OBut)-Pepsyn KA (0.08 mmol/g) m 3.2 ml of a 20% pipendme/DMF solution. This standard gives Abs 290 = 0.55- 0.65 (at room temperature). An aliquot of the multipm deprotection solution is then diluted as appropπate to give a theoretical Abs 290 = 0.6, and this value compared with the actual experimentally measured absorbance showing the efficiency of previous coupling o reaction. Coupling of5(4-formyl-3-hvdroxyphenoxy)pentanoιc acid to pins 5(4-formyl-3-hydroxyphenoxy)ρentanoιc acid (lOeq), l-hydroxybenzotπazole.H 2 0 (lOeq), BOP (9.95eq) and NMM (19.9eq) were dissolved in DMF (0.5mL per well) and agitated for 30secs. 500μL of solution was dispensed to each well of a 96-well 5 polypropylene plate. 10 x H.-Gly-MA/DMA-Macrocrown loaded onto pins / pin holder was added to the acylation mixture containing wells and the reaction left for 4hrs. The pm assembly was removed from the plate, shaken free of excess liquid then immersed m DMF (200mL) for 5mms. The assembly was again shaken then immersed in MeOH (200mL, 3 x 5mms) and allowed to air dry. Coupling Of Amines to Backbone Aldehyde The amines were dissolved in DMF /!% AcOH( 450μL per well) and dispensed into appropπate wells. The p assembly was then added and left for 5 minutes. After this time Na(AcO) 3 BH (lOeq per well in lOOμL DMF per well) was added and the reaction left 4hrs with occasional agitation to remove any gas bubbles formed. The pm assembly was removed from the plate, shaken free of excess liquid then immersed m DMF / H 2 0 (200mL, 9.1 , v/v) for 5mms. The acetate salt was neutralised by treatment of the pm assembly with 20% pipeπdine / DMF (200mL. v/v) for 30mms. The assembly was shaken then immersed DMF (200mL) for 5mms, then MeOH (200mL, 3 x 5mms) and allowed to air dry. Coupling Of Symmetric Anhydride Amino Acid Residues To Aminated Resin Amino acid ( 20eq ) was dissolved dichloromethane (3.5mL) / DMF ( small quantity as required) and stirred / ice-cooled. Diisopropylcarbodnmide ( 1 Oeq ) was added and the mixture left to stir at 0°C for 30mms, then warmed up to room temperature over 30 minutes. The solutions or gels were then dispensed as well as possible (Phe was a problem) into a 96 well plate as appropπate and the reactions left 20 hours in a saturated atmosphere of DCM The coupled assembly was then washed as detailed below. Coupling Of Standard Amino Acid Residues Coupling reactions are performed by charging the appropπate wells of a polypropylene 96 well plate with the pattern of activated solutions required duπng a particular round of coupling. Macrocrown (approx 7 μmole) standard couplings are performed m DMF (500 μl). Coupling of an Ammo-acid Residue To Appropriate Well Whilst the multipm assembly is drying, the appropπate N α -Fmoc ammo acid pfp esters (10 equivalents calculated from the loading of each crown) and HOBt (10 equivalents) required for the particular round of coupling are accurately weighed into suitable containers. Alternatively, the appropπate N α -Fmoc ammo acids (10 equivalents calculated from the loading of each crown), desired coupling agent e.g. HBTU (9.9 equivalents calculated from the loading of each crown) and activation e.g. HOBt (9.9 equivalents calculated from the loading of each crown), NMM (19.9 equivalents calculated from the loading of each crown) are accurately weighed into suitable containers. The protected and activated Fmoc ammo acid deπvatives are then dissolved m DMF (500 μl for each macrocrown e.g. for 20 macrocrowns, 20 x 10 eq. x 7 μmoles of deπvative would be dissolved 10 000 μl DMF). The appropπate denvatives are then dispensed to the appropnate wells ready for commencement of the 'coupling cycle'. As a standard, coupling reactions are allowed to proceed for 6 hours. The coupled assembly was then washed as detailed below. Washing Following Coupling If a 20% pipendme/DMF deprotection is to immediately follow the coupling cycle, then the multipm assembly is bnefly shaken to remove excess solvent washed consecutively with (200 ml each), MeOH (5 minutes) and DMF (5 minutes) and de-protected (see 6.2). If the multipm assembly is to be stored or reacted further, then a full washing cycle consisting bnef shaking then consecutive washes with (200 ml each), DMF (5 minutes) and MeOH (5 minutes, 2 minutes, 2 minutes) is performed. Coupling OfBenzoic Anhydride To Last Residue Benzoic Anhydride (20eq) is dissolved in DMF (500 μl for each macrocrown e.g. for 20 macrocrowns, 20 x 10 eq. x 7 μmoles of deπvative would be dissolved m 10 000 μl DMF) to which NMM (40eq) was added. The solution is then dispensed to the appropπate wells ready for commencement of the 'coupling cycle '.The reaction was then left for 2 hours. The coupled assembly was then washed as detailed below and treated with 20% pipeπdine in DMF followed by the standard washing cycle before cleavage. Following these general methods, the single peptide inhibitors shown in Table 2 were sequentially assembled by applying the appropπate coupling procedure at the correct cycle dunng synthesis. Acidolvtic Mediated Cleavage of Peptide-Pin Assembly Acid mediated cleavage protocols are stnctly performed m a fume hood. A polystyrene 96 well plate (1 ml/well) is labelled, then the tare weight measured to the nearest mg. Appropπate wells are then charged with a trifluoroacetic acid/tπisopropylsilane (95:5, v/v, 600 μl) cleavage solution, m a pattern corresponding to that of the multipm assembly to be cleaved. The multipm assembly is added, the entire construct covered in tin foil and left for 2 hours. The multipm assembly in then added to another polystyrene 96 well plate (1 ml/well) containing trifluoroacetic acid/tnisopropylsilane (95:5, v/v, 600 μl) (as above) for 5 minutes. Work up of Cleaved Peptides The pnmary polystyrene cleavage plate (2 hour cleavage) and the secondary polystyrene plate (5 mmute wash) are then placed m the GeneVac and the solvents, no heating required. The contents of the secondary polystyrene plate are transferred to their corresponding wells on the primary plate using an acetonitrile/water/acetic acid (50:45:5, v/v/v) solution (3 x 150 μl) and the spent secondary plate discarded. Analysis of Products A 5μL aliquot from each well is diluted to 100 μl with 0.1% aq. TFA, then a lOμL aliquot from this plate diluted with a further 100 μl 0.1% aq. TFA. The double diluted plate is analysed by HPLC-MS. Final Lyophihsation of Peptides The plate is covered with tm foil, held to the plate with an elastic band. A pm pπck is placed m the foil directly above each well and the plate placed at -80°C for 30 minutes. The plate is then lyophihsed on the Ηeto freeze dner' overnight. Finally, the dned plate is weighed. The total cleaved peptide is quantified (by weight) and 5 the average content of each peptide calculated. Since all the peptides present have ongmated from the same peptide-pm assembly, cleaved under identical conditions, it is reasonable to assume that the contents of each well are roughly eqmmolar. Inhibitor names and lab numbers: 24-14) N-benzoyl-L-glutamyl -L-norleucyl -L-homophenylalanyl -L-leucyl - 3-methyl l o butan - 1 - amide [SEQ ID 1 ] 24-22) N-benzoyl-L-glutamyl -L-norleucyl -L-homophenylalanyl -L-phenyl - 3-methyl butan -1- amide [SEQ ID 2] 24-38) N-benzoyl-L-glutamyl -L-norleucyl -L-homophenylalanyl -L-leucyl - 2-phenyl ethyl -1- amide [SEQ ID 3] 15 24-46) N-benzoyl-L-glutamyl -L-norleucyl -L-homophenylalanyl -L-phenyl - 2-phenyl ethyl -1- amide [SEQ ID 4] 24-15) N-benzoyl-L-glutamyl -L-norleucyl - 1,2,3 ,4-D-tetrahydroιsoqumolme-3- carboxamidyl -L-leucyl - 3-methyl butan -1- amide [SEQ ID 5] 24-19) N-benzoyl-L-glutamyl -L-norleucyl - 1,2,3 ,4-D-tetrahydroιsoqumohne-3- 20 carboxamidyl -L-glutamyl - 3-methyl butan -1- amide [SEQ ED 6] 24-23) N-benzoyl-L-glutamyl -L-norleucyl -I,2,3,4-D-tetrahydroιsoqumolme-3- carboxamidyl -phenyl- 3-methyl butan -1- amide [SEQ ID 7] 24-39) N-benzoyl-L-glutamyl -L-norleucyl -l,2,3,4-D-tetrahydroιsoqumolme-3- carboxamidyl -L-leucyl - 2-phenyl ethyl -1- amide [SEQ ID 8] 24-47) N-benzoyl-L-glutamyl -L-norleucyl l,2,3,4-D-tetrahydroisoquinoline-3- carboxamidyl -L-phenyl - 2-phenyl ethyl -1- amide [SEQ ID 9] 24-95) N-benzoyl-L-glutamyl -L-norleucyl - 1,2,3, 4-D-tetrahydroisoquinoline-3- carboxamidyl -L-phenyl - ethyl -1- amide [SEQ ID 10] Table 3. Pharmacophoπc Profile Definition and Specification Methodology Collections of compounds with biological activity for HCV NS3 J8 are provided as training sets. Each compound m a training set undergoes full conformational analysis 12 . A representative number of conformers are generated over a given energy range above the lowest energy conformation 13 M This information is used to derive a pharmacophore (based on seven chemical feature type rules) 15 that correlates to the observed biological activity. It is assumed that the molecules m a training set all act at the same target m the same manner of action. From the available data, three motifs are identified that correspond to compounds that are recognised by HCV NS3 J8 as substrates. These motifs are as follows: MOTIF 1 Negative Iomzable - Hydrophobe - Hydrophobe - Hydrophobe MOTIF 2 Negative Iomzable - Hydrophobe - Aromatic - Hydrophobe MOTIF 3 Negative Iomzable - Hydrophobe - Aromatic or Hydrophobe - Negative Iomzable These represent the chemical functionality required at positions P5 - P4 - P3 - P2 the HCV NS3 J8 active site. A HYDROPHOBE feature is defined as * a contiguous set of atoms that are not adjacent to any concentrations of charge (charged atoms or electronegative atoms), in a conformation such that the atoms have surface accessibility, including phenyl, cycloalkyl, isopropyl and methyl. * this may also include residues which have a partial hydrophobic character such as Lysyl or Glutammyl ammo acid sidechains. A NEGATIVE IOMZABLE feature is defined as 5 * atoms or groups of atoms that are likely to be deprotonated at physiological pH, such as trifluromethyl sulfonamide hydrogens, sulfonic acids (centroid of the three oxygens), phosphonic acids (centroid of the three oxygens), sulphmic, carboxylic or phosphmic acids (centroid of the two oxygens), tetrazoles and negative charges not adjacent to a positive charge). l o A RING AROMATIC feature is defined as * matching aromatic nngs with five or six member atoms A pharmacophore* consisting of at least the following chemical features can be used to descπbe MOTIF 1 • Three HYDROPHOBE" features and a NEGATIVE IONIZABLE feature. 15 The HYDROPHOBE features are represented by spheres 1 7 Angstroms radius (up to 2.7 Angstroms). The NEGATIVE IONIZABLE feature is similarly represented by a sphere 1.7 Angstroms radius (up to 2.7 Angstroms) see figure 1. The absolute sphere centroid positions of each feature are descnbed as 20 follows: * Negative Iomzable 1 has Cartesian XYZ co-ordmates of -8.207, -3.059, -3.78 * Hydrophobe 2 has co-ordinates of -2.975, 4.725, -0.229 Hydrophobe 3 has co-ordinates of 6.065, 2.205, 3.991 Hydrophobe 4 has co-ordmates of 3.385, -2.935, -1.149 The distance and angle constraints for these are described m figure 2: see figure 2. The tolerances on all distances between these features is +/- 0.5 Angstroms and the geometric angles +/- 20 Degrees. * In this context, the use of the term "pharmacophore" is not meant to imply any pharmacological activity. The term refers to those chemical features and their distnbution m three-dimensional space which constitute and epitomise the preferred requirements for molecular interaction with the receptor. In this case the receptor being the catalytic active site of the protease HCV NS3 J8. Similarly, a pharmacophore consisting of at least the following chemical features can be used to descπbe MOTIF 2: Two HYDROPHOBE features, a NEGATIVE IONIZABLE feature and a RING AROMATIC feature. The HYDROPHOBE features are represented by spheres 1.7 Angstroms radius (up to 2.7 Angstroms). The NEGATIVE IONIZABLE feature is similarly represented by a sphere 1.7 Angstroms radius (up to 2.7 Angstroms). The RING AROMATIC is represented as two equal size spheres (1.6 Angstroms radius up to 2.0 Angstoms) whose centroids are 3.1 Angstroms apart. One sphere corresponds to the position of an aromatic ring moiety and the other to the projected point of the electron pi stacking of the aromatic πng system. see figure 3. The absolute sphere centroid positions of each feature are described as follows: * Negative Iomzable 1 has Cartesian XYZ co-ordinates of 4.907, -1.284, 3.039 * Hydrophobe 2 has co-ordmates of 1.496, 3.212, -3.793 * Hydrophobe 3 has co-ordinates of -4.324, -4.228, -3.313 * Ring Aromatic centroid 4 has co-ordmates of -0.798, - 1.230, 2.330 * Ring Aromatic projected point 5 has co-ordmates of -4.324, -4.228, -3.313 The distance and angle constraints for these are descnbed in figures 4 and 5: see figures 4 and 5. The tolerances on all distances between these features is +/- 0.5 Angstroms and the geometπc angles +/- 20 Degrees. A pharmacophore consisting of at least the following chemical features can be used to describe MOTIF 3: Two HYDROPHOBE features and two NEGATIVE IONIZABLE features. The HYDROPHOBE features are represented by spheres 1.7 Angstroms radius (up to 2.7 Angstroms). The NEGATIVE IONIZABLE features are similarly represented by spheres 1.7 Angstroms radius (up to 2.7 Angstroms). see figure 6. The absolute sphere centroid positions of each feature are descnbed as follows: * Negative Iomzable 1 has Cartesian XYZ co-ordmates of -8.551, 0.769, -0.895 * Hydrophobe 2 has co-ordmates of -0.697, 1.087, -5.655 * Negative Iomzable 3 has co-ordinates of 6.098, 1.653, 4.709 * Hydrophobe 4 has co-ordmates of 0.503, -2.453, 2.784 The distance and angle constraints for these are descnbed m figure 7: see figure 7. Compounds that exemplify the pharmacophore Bz-Glu-Nle-hphe-Leu-R where R = 3-methyl butylamme shown mapped to MOTIF 1 and Bz-Glu-Nle-hphe-Phe-R where R = 2-phenylethyl amine is shown mapped to MOTTF 2 and Bz-Glu-Nle-dTic-Glu-R where R = 3-methyl butylamme shown mapped to MOTIF 3. see figures 8, 9 and 10. Those skilled m the art will appreciate that the compounds of the invention may essentailly consist of an ammo acid (aa) sequence (or non peptide mimetic thereof) or may include a sequence corresponding to one of the pharmacophoric motifs described herein. For example the sequence might consist of or include the sequence [aa] n wherein n is any integer from 4 upwards, for example wherein n is 4,5,6,7,8,9,10,11 or 12. References 1. Q.L. Choo et al., Science, 1989, 244, 359-362. 2. G. Kuo et al., Science, 1989, 244, 362. 3. E.E. Mast, Semin. Virol., 1993, 4, 273-283. 4. S. Iwarson, FEMS Microbiol. Rev., 1994, 14, 201-204 5. M. Chu et al., Tetrahedron Letters, 1996, 37, 7229-7232 6. a) C. Steinkuhler, L. Tomei and R. De Francesco, J. Biol. Chem. 1996, 271, 6367- 6373 and references therein, b) M. Taliani et al., Analytical Biochemistry, 1996, 240. 60-67. c) C. Steinkuhler et al., Italian Patent Application (1995) RM95A000573. 7. E. Bianchi, C. Steinkuhler, M. Taliani, A. Urbani, R. De Francesco and A. Pessi, Analytical Biochemistry, 1996, 232, 239-244. 8. R. A. Love et al., Cell, 1996, 87, 331-342 9. J.L. Kim et al., Cell, 1996, 87, 343-355 10.T.J. Tucker et al., J. Med. Chem., 1997, 40, 830-832 11. J. Comp. Chem., 1986, 7, 565-577 12. J.Comp.Chem., 1995, 16, 171-187 13. J.Cheminf.Comp.Sci., 1995, 35, 285-294 14. J.Cheminf.Comp.Sci., 1995, 35, 295-304 15. J.Chem.Inf.Comp.Sci., 1994, 34, 1297-1308

Claims: Claims 1. A compound having biological activity as an inhibitor of Hepatitis C NS3 protease, which compound is recognised by HCV NS3 J8 as a substrate, and having at least four chemical functionalities for interacting with at least the P 5 -P 4 -P 3 -P 2 pockets of the HCV NS3 J8 catalytically active site, which functionalities are each provided by an ammo acid residue (or a wholly or partly non-peptide mimetic thereof), wherein said functionalities provide a pharmacophoric motif selected from the group consisting of: MOTIF 1 Negative Iomzable - Hydrophobe - Hydrophobe - Hydrophobe MOTIF 2 Negative Iomzable - Hydrophobe - Aromatic - Hydrophobe MOTIF 3 Negative Iomzable - Hydrophobe - Aromatic or Hydrophobe - Negative Iomzable wherein; a HYDROPHOBE feature is defined as * a contiguous set of atoms that are not adjacent to any concentrations of charge (charged atoms or electronegative atoms), in a conformation such that the atoms have surface accessibility, including phenyl, cycloalkyl, isopropyl and methyl, (this may also include residues which have a partial hydrophobic character such as Lysyl or Glutammyl ammo acid sidechams) and: a NEGATIVE IONIZABLE feature is defined as * atoms or groups of atoms that are likely to be deprotonated at physiological pH, such as tπfluromethyl sulfonamide hydrogens, sulfonic acids (centroid of the three oxygens), phosphonic acids (centroid of the three oxygens), sulphmic, carboxylic or phosphmic acids (centroid of the two oxygens), tetrazoles and negative charges not adjacent to a positive charge) and; a RING AROMATIC feature is defined as * matching aromatic πngs with five or six member atoms and wherein; (l) a pharmacophore consisting of at least the following chemical features can be used to describe MOTIF 1: Three HYDROPHOBE features and a NEGATIVE IONIZABLE feature, m which the HYDROPHOBE features are represented by spheres 1.7 Angstroms radius (up to 2.7 Angstroms); the NEGATIVE IONIZABLE feature is similarly represented by a sphere 1.7 Angstroms radius (up to 2.7 Angstroms); The absolute sphere centroid positions of each feature are descnbed as follows: * Negative Iomzable 1 has Cartesian XYZ co-ordinates of -8.207, -3.059, -3.78 * Hydrophobe 2 has co-ordmates of -2.975, 4.725, -0.229 * Hydrophobe 3 has co-ordmates of 6.065, 2.205, 3.991 * Hydrophobe 4 has co-ordmates of 3.385, -2.935, -1.149 and the distance and angle constraints for these are descnbed figure 2: and wherein; (n) a pharmacophore consisting of at least the following chemical features can be used to describe MOTIF 2: Two HYDROPHOBE features, a NEGATIVE IONIZABLE feature and a RING AROMATIC feature, in which the HYDROPHOBE features are represented by spheres 1.7 Angstroms radius (up to 2.7 Angstroms); the NEGATIVE IONIZABLE feature is similarly represented by a sphere 1.7 Angstroms radius (up to 2.7 Angstroms); the RING AROMATIC is represented as two equal size spheres (1.6 Angstroms radius up to 2.0 Angstroms) whose centroids are 3.1 Angstroms apart, one sphere corresponds to the position of an aromatic πng moiety and the other to the projected point of the electron pi stacking of the aromatic nng system; The absolute sphere centroid positions of each feature are descnbed as follows: * Negative Iomzable 1 has Cartesian XYZ co-ordinates of 4.907, -1.284, 3.039 * Hydrophobe 2 has co-ordmates of 1.496, 3.212, -3.793 * Hydrophobe 3 has co-ordmates of -4.324, -4.228, -3.313 * Ring Aromatic centroid 4 has co-ordmates of -0.798, -1.230, 2.330 * Ring Aromatic projected point 5 has co-ordmates of -4.324, -4.228, -3.313 and distance and angle constraints for these are described in figures 4 and 5: and wherein; (in) a pharmacophore consisting of at least the following chemical features can be used to descnbe MOTIF 3: Two HYDROPHOBE features and two NEGATIVE IONIZABLE features, m which the HYDROPHOBE features are represented by spheres 1.7 Angstroms radius (up to 2.7 Angstroms); the NEGATIVE IONIZABLE features are similarly represented by spheres 1.7 Angstroms radius (up to 2.7 Angstroms); The absolute sphere centroid positions of each feature are described as follows: * Negative Iomzable 1 has Cartesian XYZ co-ordmates of -8.551, 0.769, -0.895 * Hydrophobe 2 has co-ordmates of -0.697, 1.087, -5.655 * Negative Iomzable 3 has co-ordmates of 6.098, 1.653, 4.709 * Hydrophobe 4 has co-ordinates of 0.503, -2.453, 2.784 and the distance and angle constraints for these are descnbed m figure 7. and wherein; (iv) the tolerances on all distances between features are +/- 0.5 Angstroms and the tolerances on all geometnc angles are +/- 20 degrees for all three Motifs. 2. A compound according to claim 1 selected from the group consisting of SEQ ID Nos. 1-10, or a wholly or partly non peptide mimetic thereof, or N- or C-terminal deπvatives thereof, or analogues thereof by virtue of conservative ammo acid deletion, addition or substitution. 3. A composition which compπses a pharmaceutically acceptable amount of an inhibitor according to claim 1 or 2 together with a pharmaceutically acceptable earner or diluent. 4. Use of an inhibitor according to claim 1 or 2 or a composition according to claim 3 in the manufacture of a medicament for the treatment of hepatitis C. 5. Use of an inhibitor according to claim 1 or 2 or a composition according to claim 3 for the treatment of hepatitis C. 6. A method of treatment of hepatitis C which compnses admmisteπng to a patient a pharmaceutically active amount of an inhibitor according to claim 1 or 2 or a composition according to claim 3

Inventors: HART TERANCE, QUIBELL MARTIN, HART, TERANCE, QUIBELL, MARTIN
Applicants: HART TERANCE, PEPTIDE THERAPEUTICS LTD, QUIBELL MARTIN, HART, TERANCE, PEPTIDE THERAPEUTICS LIMITED, QUIBELL, MARTIN
Application year: 1998
Main classification: C07K5
IPC codes: A61K38, A61P1, A61P31, C07K5