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WO1999012936A1 - Derives de furopyrrolidine et leur utilisation en tant qu'inhibiteurs de serine protease - Google Patents

Derives de furopyrrolidine et leur utilisation en tant qu'inhibiteurs de serine protease Download PDF

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Publication number
WO1999012936A1
WO1999012936A1 PCT/EP1998/005606 EP9805606W WO9912936A1 WO 1999012936 A1 WO1999012936 A1 WO 1999012936A1 EP 9805606 W EP9805606 W EP 9805606W WO 9912936 A1 WO9912936 A1 WO 9912936A1
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Prior art keywords
inhibitor
library
enzyme
compounds
compound
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Inventor
Steven John Coote
Michael Dennis Dowle
Harry Finch
Michael Menteith Hann
Henry Anderson Kelly
Simon John Fawcett Macdonald
Neil Anthony Pegg
Nigel Grahame Ramsden
Nigel Stephen Watson
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Glaxo Group Ltd
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Glaxo Group Ltd
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Priority to AU95361/98A priority Critical patent/AU9536198A/en
Priority to EP98948906A priority patent/EP1015458A1/fr
Priority to JP2000510743A priority patent/JP2001515907A/ja
Publication of WO1999012936A1 publication Critical patent/WO1999012936A1/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
    • C07D491/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/407—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with other heterocyclic ring systems, e.g. ketorolac, physostigmine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • 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.
  • 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.
  • 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.
  • specificity is shown for peptide bonds which have a particular moiety ⁇ 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).
  • the preferred substrate for thrombin is a peptide containing a basic residue (e.g. arginine i.e.
  • 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).
  • 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).
  • ARDS adult respiratory distress syndrome
  • thrombin 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.
  • 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.
  • 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.
  • 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.
  • inhibitors of serine protease enzymes which are substituted derivatives of trans-hexahydrofuro[3,2- b]pyrrol-2-one.
  • this invention relates to inhibitors of serine protease enzymes which are compounds of formula I:
  • 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.
  • 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.
  • 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 ⁇ 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).
  • 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.
  • 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.
  • 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).
  • 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.
  • 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
  • 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.
  • a heteroatom e.g. nitrogen or oxygen
  • Amine groups include primary, secondary and tertiary amine groups including cyclic amine.
  • 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.
  • a library comprising a plurality of substituted derivatives of trans-hexahydrofuro[3,2-b]pyrrolo-2-one will be prepared.
  • the library will comprise a plurality of compounds of formula I
  • 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 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.
  • 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.
  • 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
  • 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 p and R q are respectively amino and hydroxyl protecting groups.
  • Step (i) comprises treating ⁇ -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).
  • a strong base such as sodium hydride
  • 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).
  • 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.
  • 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
  • a Mitsunobu inversion using benzoic acid in the presence of triphenylphospine and diethyl azodicarboxylate and in
  • 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.
  • 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).
  • a base such as an organic base (e.g. triethylamine)
  • 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.
  • a palladium catalyst e.g. palladium-on-carbon
  • R 1 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.
  • 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.
  • Enantiomerically pure compounds may be prepared by chiral separation or by synthesis based on chiral starting materials.
  • 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.
  • 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.
  • binding agents for example syrup, acacia, gelatin, sorbitol, tragacanth, mucilage of starch or polyvinyl pyrrolidone
  • fillers
  • 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.
  • 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.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g. sterile, pyrogen-free water, before use.
  • a suitable vehicle e.g. sterile, pyrogen-free water
  • 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.
  • topical administration as used herein, we include administration by insufflation and inhalation.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • Compounds of the invention may be tested for their thrombin inhibitory activity as determined in vitro by their ability to inhibit human ⁇ -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 100 ⁇ M) 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.
  • IC 50 values the data were analysed using Kineticalc ® with a 4-parameter curve fitting procedure to obtain the IC 50 value.
  • the compounds were preincubated with thrombin for these times prior to adding the chromogenic substrate.
  • 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 ⁇ Ala protease).
  • IC 50 data for test compounds are determined after preincubation of the enzyme with test inhibitor compound for
  • Test compounds are dissolved in DMSO, serially diluted and added at a range of concentrations (from 100 ⁇ M - 0.195 ⁇ M) to a reaction containing 0.5 ⁇ M CMV ⁇ Ala 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 Lys-Ala-pNA
  • 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.
  • 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 400 ⁇ M) was added to tryptase (final cone, of 0.11 ⁇ g.m.
  • 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.
  • the substrate final cone, of 160 ⁇ M
  • trypsin final cone, of 25ng.ml "1
  • 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.
  • IC 50 values the data were analyzed using Kinetiealc ® with a 4- parameter curve-fitting procedure.
  • 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- ⁇ -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.
  • the substrate final cone, of 200 ⁇ M
  • Factor Xa final cone, of 0.02 U.ml "1
  • 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.
  • IC 50 values the data were analyzed using Kinetiealc ® with a 4-parameter curve fitting procedure.
  • 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.
  • the substrate final cone, of 400 ⁇ M
  • Factor Xla final cone, of 0.25 ⁇ g.ml "1
  • 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.
  • IC 50 values the data were analyzed using Kinetiealc ® with a 4-parameter curve fitting procedure.
  • 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.
  • the substrate final cone, of 200 ⁇ M
  • Factor Xlla final cone, of 1.25 ⁇ g.ml "1
  • 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.
  • 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.
  • the substrate final cone, of 750 ⁇ M
  • tissue plasminogen activator final cone, of 1.O ⁇ g.ml "1
  • 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.
  • IC 50 values the data were analyzed using Kinetiealc ® with a 4-parameter curve-fitting procedure.
  • 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.
  • the substrate final cone, of 363 ⁇ M
  • plasmin final cone, of 0.02 U.ml "1
  • 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.
  • IC 50 values the data were analyzed using Kinetiealc ® with a 4-parameter curve fitting procedure.
  • 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.
  • the substrate final cone, of 400 ⁇ M
  • Factor Vila final cone, of 10nM in the presence of recombinant soluble tissue factor at optimal concentration
  • 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.
  • IC 50 values the data were analyzed using. Kinetiealc ® with a 4-parameter curve fitting procedure.
  • 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.
  • the substrate final cone, of 178 ⁇ M
  • chymotrypsin final cone, of 0.2 ⁇ g/mL
  • the reaction monitored for 10 minutes at 405nm using a BioTek EL340 plate reader: the assay was performed at 30°C.
  • IC 50 values the data were analysed using Kinetiealc ® with a 4-parameter curve fitting procedure.
  • the compounds were preincubated with chymotrypsin for these times prior to addition of the chromogenic substrate.
  • 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.
  • a suspension of sodium bicarbonate (218g) in water (600ml) was added to a stirred suspension of ⁇ -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.
  • 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).
  • 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 ethe ⁇ hexane (1 :1) to 100% ether (grade elution) to give the title compound (157.5g) as a colourless oil.
  • Example 2 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;

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Abstract

L'invention concerne un inhibiteur d'une enzyme sérine protéase, ledit inhibiteur consistant en un dérivé substitué de trans-hexahydrofuro[3,2-b]pyrrol-2-one.
PCT/EP1998/005606 1997-09-09 1998-09-07 Derives de furopyrrolidine et leur utilisation en tant qu'inhibiteurs de serine protease Ceased WO1999012936A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AU95361/98A AU9536198A (en) 1997-09-09 1998-09-07 Furopyrrolidine derivatives and their use as serine protease inhibitors
EP98948906A EP1015458A1 (fr) 1997-09-09 1998-09-07 Derives de furopyrrolidine et leur utilisation en tant qu'inhibiteurs de serine protease
JP2000510743A JP2001515907A (ja) 1997-09-09 1998-09-07 フロピロリジン誘導体およびそのセリンプロテアーゼとしての使用

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GBGB9719176.1A GB9719176D0 (en) 1997-09-09 1997-09-09 New therapeutic method
GB9719176.1 1997-09-09

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WO1999012936A1 true WO1999012936A1 (fr) 1999-03-18

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006044650A3 (fr) * 2004-10-15 2006-12-14 Genencor Int Criblage differentiel competitif

Citations (1)

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Publication number Priority date Publication date Assignee Title
US5618825A (en) * 1994-03-11 1997-04-08 Pharmacopeia, Inc. Combinatorial sulfonamide library

Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
US5618825A (en) * 1994-03-11 1997-04-08 Pharmacopeia, Inc. Combinatorial sulfonamide library

Non-Patent Citations (4)

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Title
ANTONIO R. DE FARIA ET AL.: "[2+2] Cycloaddition Reaction of cyclic Enecarbamates and Enamides with Ketenes...", TETRAHEDRON LETTERS, vol. 34, no. 1, 1993, Great Britain, pages 27 - 30, XP002093078 *
DAVID W. KNIGHT ET AL.: "Homoproline homologation by enolare Claisen rearrangement.....", JOURNAL OF CHEMICAL SOCIETY PERKIN TRANSACTIONS I, 1997, Great Britain, pages 2089 - 2097, XP002093081 *
JEREMY COOPER ET AL.: "Baker's yeast Reductions of beta-Oxopyrrolidinecarboxylates:...", JOURNAL OF CHEMICAL SOCIETY PERKIN TRANSACTIONS I, 1993, Great Britain, pages 1313 - 1317, XP002093080 *
MIKKEL THANING ET AL.: "A short Enantiodivergent Synthesis of the Geissman-Waiss Lactone", JOURNAL OF ORGANIC CHEMISTRY, vol. 55, no. 4, 1990, Cleveland Ohio, pages 1406 - 1408, XP002093079 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006044650A3 (fr) * 2004-10-15 2006-12-14 Genencor Int Criblage differentiel competitif
JP2008517275A (ja) * 2004-10-15 2008-05-22 ジェネンコー・インターナショナル・インク 競合示差スクリーニング

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AU9536198A (en) 1999-03-29
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JP2001515907A (ja) 2001-09-25

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