WO2004009543A2 - 1-carbonyl-4-cyano-pyrrolidine-2-carboxylic acid derivatives as hepatitis c virus inhibitors - Google Patents

1-carbonyl-4-cyano-pyrrolidine-2-carboxylic acid derivatives as hepatitis c virus inhibitors Download PDF

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WO2004009543A2
WO2004009543A2 PCT/EP2003/008138 EP0308138W WO2004009543A2 WO 2004009543 A2 WO2004009543 A2 WO 2004009543A2 EP 0308138 W EP0308138 W EP 0308138W WO 2004009543 A2 WO2004009543 A2 WO 2004009543A2
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compound
pyrrolidine
cyano
carboxylic acid
ferf
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PCT/EP2003/008138
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French (fr)
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WO2004009543A8 (en
WO2004009543A3 (en
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George Burton
Helen Susanne Goodland
David Haigh
Terence John Kiesow
Thomas W. Ku
Martin John Slater
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Glaxo Group Limited
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Priority to AU2003266243A priority Critical patent/AU2003266243A1/en
Publication of WO2004009543A2 publication Critical patent/WO2004009543A2/en
Publication of WO2004009543A3 publication Critical patent/WO2004009543A3/en
Publication of WO2004009543A8 publication Critical patent/WO2004009543A8/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/427—Thiazoles not condensed and containing further heterocyclic rings
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4402—Non condensed pyridines; Hydrogenated derivatives thereof only substituted in position 2, e.g. pheniramine, bisacodyl
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
    • C07D409/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond

Definitions

  • the present invention relates to novel 4-cyano acyl pyrrolidine derivatives useful as anti- viral agents. Specifically, the present invention involves novel HCV inhibitors.
  • HCV infection is responsible for 40-60% of all chronic liver disease and 30% of all liver transplants.
  • Chronic HCV infection accounts for 30% of all cirrhosis, end-stage liver disease, and liver cancer in the U.S. The CDC estimates that the number of deaths due to HCV will minimally increase to 38,000/year by the year 2010.
  • hepatitis C virus HCV
  • NNBH non-B hepatitis
  • flaviviruses e.g. yellow fever virus and Dengue virus types 1-4
  • pestiviruses e.g.
  • HCV bovine viral diarrhea virus, border disease virus, and classic swine fever virus
  • the HCV genome is approximately 9.6 kilobases (kb) with a long, highly conserved, noncapped 5' nontranslated region (NTR) of approximately 340 bases which functions as an internal ribosome entry site (IRES) (Wang CY et al 'An RNA pseudoknot is an essential structural element of the internal ribosome entry site located within the hepatitis C virus 5' noncoding region' [Article] Rna-A Publication of the Rna Society. 1 (5):526-537, 1995 Jul.). This element is followed by a region which encodes a single long open reading frame (ORF) encoding a polypeptide of -3000 amino acids comprising both the structural and nonstructural viral proteins.
  • ORF long open reading frame
  • this RNA Upon entry into the cytoplasm of the cell, this RNA is directly translated into a polypeptide of -3000 amino acids comprising both the structural and nonstructural viral proteins.
  • This large polypeptide is subsequently processed into the individual structural and nonstructural proteins by a combination of host and virally-encoded proteinases (Rice, CM. (1996) in B.N. Fields, D.M.Knipe and P.M. Howley (eds) Virology 2 nd Edition, p931- 960; Raven Press, N.Y.).
  • 3' NTR which roughly consists of three regions: an - 40 base region which is poorly conserved among various genotypes, a variable length poly(U)/polypyrimidine tract, and a highly conserved 98 base element also called the "3' X-tail" (Kolykhalov, A. et al (1996) J. Virology 70:3363-3371 ; Tanaka, T. et al (1995) Biochem Biophys. Res. Commun. 215:744-749; Tanaka, T. et al (1996) J. Virology 70:3307-3312; Yamada, N. et al (1996) Virology 223:255-261).
  • the 3' NTR is predicted to form a stable secondary structure which is essential for HCV growth in chimps and is believed to function in the initiation and regulation of viral RNA replication.
  • the NS5B protein (591 amino acids, 65 kDa) of HCV (Behrens, S.E. et al (1996) EMBO J. 15:12-22), encodes an RNA-dependent RNA polymerase (RdRp) activity and contains canonical motifs present in other RNA viral polymerases.
  • the NS5B protein is fairly well conserved both intra-typically (-95-98% amino acid (aa) identity across 1b isolates) and inter-typically (-85% aa identity between genotype 1a and 1b isolates).
  • the essentiality of the HCV NS5B RdRp activity for the generation of infectious progeny virions has been formally proven in chimpanzees (A. A. Kolykhalov et al.. (2000) Journal of Virology, 74(4), p.2046-2051).
  • inhibition of NS5B RdRp activity is predicted to cure HCV infection.
  • the present invention involves compounds represented hereinbelow, pharmaceutical compositions comprising such compounds and use of the present compounds in treating viral infection, especially HCV infection.
  • the present invention provides compounds of Formula (I) :
  • A represents OH
  • B represents C(O)R 1 wherein R 1 is selected from the group consisting of C 1-6 alkyl, aryl, heteroaryl, arylalkyl, and heteroarylalkyl;
  • C represents C 1-6 alkyl, aryl, heteroaryl or heterocyclyl
  • D represents hydrogen or C 1-6 alkyl
  • E represents hydrogen, C 1-6 alkyl, aryl or heteroaryl
  • F represents hydrogen, C 1-6 alkyl, heterocyclylalkyl, arylalkyl or heteroarylalkyl; and salts, solvates and esters thereof, provided that when A is esterified to form -OR where R is selected from straight or branched chain alkyl, aralkyl, aryloxyalkyl, or aryl, then R is other than fetf-butyl.
  • the compounds of the present invention may contain one or more asymmetric carbon atoms and may exist in racemic, diastereoisomeric, and optically active forms. All of these racemic compounds, enantiomers and diastereoisomers are contemplated to be within the scope of the present invention.
  • R 1 is aryl or heteroaryl;. more preferably R 1 is phenyl, optionally substituted by halo, C 1-6 alkyl or C 1-3 alkoxy, still more preferably R 1 is phenyl substituted in the para- position by -etf-butyl and additionally optionally further substituted.
  • R 1 is 4-te/f-butylphenyl, 4-terf-butyl-3- chlorophenyl, 4-terf-butyl-3-methylphenyl, or 4-ferf-butyl-3-methoxyphenyl;
  • C is C 1-6 alkyl, aryl or heteroaryl; more preferably C is heteroaryl, especially pyridin-2-yl, 1,3-thiazol-2-yl, 1 ,3-thiazol-4-yl or thien-2-yl; most preferably C is 1,3-thiazol- 2-yl;
  • D is hydrogen or methyl; more preferably D is hydrogen;
  • E is hydrogen
  • F is C 1-6 alkyl, arylalkyl or heteroarylalkyl, more preferably F is C 1-6 alkyl, especially 2-methylpropyl (isobutyl).
  • alkyl refers to an optionally substituted hydrocarbon group.
  • the alkyl hydrocarbon group may be linear, branched or cyclic, saturated or unsaturated. Where the alkyl hydrocarbon group is cyclic, it will be understood that there will be a minimum of 3 carbon atoms in the group. Preferably, the group is saturated. Preferably, the group is linear or branched. Preferred alkyl moieties are C 1-4 alkyl.
  • Optional subsituents include C ⁇ _ 6 alkyl, halo, C(O)NR 4 R 5 , OR 6 , CO 2 R 1 , NR 4 R 5 , NHC(O)R 1 , NHCO 2 R 1 , NHC(O)NR 2 R 3 , SO 2 NR 2 R 3 , SO 2 R 1 , nitro, oxo, and heterocyclyl
  • R 2 and R 3 are independently selected from the group consisting of hydrogen, C 1-6 alkyl, aryl, heteroaryl, arylalkyl, and heteroarylalkyl; or R 2 and R 3 together with the nitrogen atom to which they are attached form a 5 or 6 membered saturated cyclic group
  • R 4 and R 5 are independently selected from hydrogen, C 1-6 alkyl, aryl and heteroaryl
  • R 6 represents hydrogen, C- ⁇ - 6 alkyl, arylalkyl, or heteroarylalkyl.
  • the group is unsubstituted.
  • aryl refers to an optionally substituted aromatic group with at least one ring having a conjugated pi-electron system, containing up to two conjugated or fused ring systems.
  • Aryl includes carbocyclic aryl and biaryl groups, all of which may be optionally substituted. Preferred "aryl” moieties are unsubstituted, monosubstituted, disubstituted or trisubstituted phenyl.
  • Preferred "aryl" substituents are selected from the group consisting of C 1-6 alkyl, halo, OR 6 , C(O)NR 4 R 5 , C(O)R 1 , CO 2 H, CO 2 R 1 , NR 4 R 5 , NHC(O)R ⁇ NHCO 2 R 1 , NHC(O)NR 2 R 3 , SO 2 NR 2 R 3 , SO 2 R 1 , nitro, cyano, oxo, heterocyclyl, CF 3 , pyridine, phenyl, and NO 2 .
  • aryl substituents are selected from the group consisting of C ⁇ alkyl, halo, OR 6 , C(O)NR R 5 , CO 2 R 1 , NR 4 R 5 , NHC(O)R 1 , NHCO 2 R 1 , NHC(O)NR 2 R 3 , SO 2 NR 2 R 3 , SO 2 R 1 , nitro, oxo, heterocyclyl, OC ⁇ alkyl, CF 3 , pyridine, phenyl, and NO 2 .
  • heteroaryl refers to an optionally substituted, 5 or 6 membered, aromatic group comprising one to four heteroatoms selected from N, O and S, with at least one ring having a conjugated pi-electron system, containing up to two conjugated or fused ring systems.
  • Preferred "heteroaryl” moieties include unsubstituted, monosubstituted, disubstituted or trisubstituted thienyl, thiazolyl, and pyridinyl.
  • Preferred "heteroaryl" substituents are selected from the group consisting of C 1-6 alkyl, halo, OR 6 , C(O)NR 4 R 5 , C(O)R ⁇ CO 2 H, CO 2 R ⁇ NR 4 R 5 , NHC(O)R 1 , NHCO 2 R 1 , NHC(O)NR 2 R 3 , SO 2 NR 2 R 3 , SO 2 R 1 , nitro, cyano, oxo, heterocyclyl, CF 3 , pyridine, phenyl, and NO 2 .
  • heteroaryl substituents are selected from the group consisting of C 1-6 alkyl, halo, OR 6 , C(O)NR 4 R 5 , CO 2 R 1 , NR 4 R 5 , NHC(O)R 1 , NHCO 2 R 1 , NHC(O)NR 2 R 3 , SO 2 NR 2 R 3 , SO 2 R 1 , nitro, oxo, heterocyclyl, OC 1-4 alkyl, CF 3 , pyridine, phenyl, and NO 2 .
  • heterocyclic and heterocyclyl refer to an optionally substituted, 5 or 6 membered, saturated cyclic hydrocarbon group containing one to four heteroatoms selected from N, optionally substituted by hydrogen, C 1-6 alkyl, C(O)R 1 , SO 2 R 1 , aryl or heteroaryl; O; and S, optionally substituted by one or two oxygen atoms.
  • Preferred compounds useful in the present invention are selected from the group consisting of: re/-(2S,4R,5R)-2-lsobutyl-1 -(3-methoxy-4-te/ ⁇ -butylbenzoyl)-4-cyano-5-(1 ,3-thiazol-2- yl)pyrrolidine-2-carboxylic acid; re/-(2S,4S,5R)-2-lsobutyl-1-(3-methoxy-4--er--butylbenzoyl)-4-cyano-5-(1 ,3-thiazol-2- yl)pyrrolidine-2-carboxylic acid; fe/-(2S,4R,5R)-2-lsobutyl-1-(3-chloro-4-tert-butylbenzoyl)-4-cyano-5-(1 ,3-thiazol-2- yl)pyrrolidine-2-carboxylic acid; re/-(2S
  • physiologically acceptable salts of the compounds of formula (I) include acid salts, for example sodium, potassium, calcium, magnesium and tetraalkylammonium and the like, or mono- or di- basic salts with the appropriate acid for example organic carboxylic acids such as acetic, lactic, tartaric, malic, isethionic, lactobionic and succinic acids; organic sulfonic acids such as methanesulfonic, ethanesulfonic, benzenesulfonic and p-toluenesulfonic acids and inorganic acids such as hydrochloric, sulfuric, phosphoric and sulfamic acids and the like.
  • organic carboxylic acids such as acetic, lactic, tartaric, malic, isethionic, lactobionic and succinic acids
  • organic sulfonic acids such as methanesulfonic, ethanesulfonic, benzenesulfonic and p-toluenes
  • the present invention also relates to pharmaceutically acceptable solvates of the compounds of Formula (I), for example hydrates.
  • the present invention also relates to pharmaceutically acceptable esters of the compounds of Formula (I), for example carboxylic acid esters -COOR, in which R is selected from straight or branched chain alkyl, for example n-propyl, n-butyl, alkoxyalkyl (e.g. methoxymethyl), aralkyl (e.g. benzyl), aryloxyalkyl (e.g. phenoxymethyl), aryl (e.g. phenyl optionally substituted by halogen, C 1-4 alkyl or C ⁇ alkoxy or amino).
  • any alkyl moiety present in such esters preferably contains 1 to 18 carbon atoms, particularly 1 to 4 carbon atoms. Any aryl moiety present in such esters preferably comprises a phenyl group.
  • A is hydroxy or a protected form thereof, and C, D, E and F are as defined above for Formula (I); with a suitable acylating agent, for example R 1 C(O)-hal, wherein hal is a halo atom, preferably chloro or bromo, C is a carbon atom, and R 1 is as defined for Formual (I).
  • a suitable acylating agent for example R 1 C(O)-hal, wherein hal is a halo atom, preferably chloro or bromo, C is a carbon atom, and R 1 is as defined for Formual (I).
  • the reaction is carried out in a suitable solvent, for example dichloromethane, in the presence of a suitable base, for example triethylamine, and thereafter removing any protecting group.
  • Suitable hydroxy protecting groups can be found, but are not restricted to, those found in T W Greene and P G M Wuts 'Protective Groups in Organic Synthesis', 3 rd Ed (1999), J Wiley and Sons. Protection and deprotection of the hydroxy may be carried out by standard techniques known in the art.
  • Compounds of Formula (II) may be prepared by reaction of a compound of Formula (III) wherein A, C and F are as defined for Formula (I) above; with a compound of Formula (IV)
  • reaction is carried out in a suitable solvent, for example THF, in the presence of a Lewis acid catalyst, such as lithium bromide, and a base, such as triethylamine.
  • a Lewis acid catalyst such as lithium bromide
  • a base such as triethylamine
  • racemic compounds of Formula (I) or (II) may be optionally resolved into their individual enantiomers. Such resolutions may conveniently be accomplished by standard methods known in the art. For example, a racemic compound of Formula (I) or (II) may be resolved by chiral preparative HPLC.
  • the title compound a solid, was prepared from Intermediate 8 and acrylonitrile in a manner analogous to that described for the preparation of Intermediate 2, affording a mixture of epimers at the pyrrolidine C(4)-position.
  • the mixture was separated by chromatography on silica gel, using a gradient elution from cyclohexane-ethyl acetate (10:1 v/v) to cyclohexane-ethyl acetate (7:1 v/v) as eluent to afford the title compound in the earlier eluting fractions.
  • Stage B The ferf-butyl ester from Stage A (0.157 g, 0.29 mmol) was dissolved in trifluoroacetic acid (2 mL) and stirred at room temperature for 17 hours. The mixture was evaporated and the residue triturated with diethyl ether to afford the title compound, a solid.
  • the title compound a solid, was prepared in a two-stage process from Intermediate 11 by acylation with 3-methoxy-4-ferf-butylbenzoyl chloride (Stage A) and then trifluoroacetic acid induced ester deprotection (Stage B) in a manner analogous to that described in Example 1.
  • the title compound was spectroscopically identical by 1 H NMR and MS to the compound described in Example 1.
  • compositions for use in therapy comprising a compound of formula (I) or a physiologically acceptable salt or solvate thereof in admixture with one or more physiologically acceptable diluents or carriers.
  • the compounds of the present invention can be administered by different routes including intravenous, intraperitoneal, subcutaneous, intramuscular, oral, topical, transdermal, or transmucosal administration.
  • oral administration is preferred.
  • the compounds can be formulated into conventional oral dosage forms such as capsules, tablets and liquid preparations such as syrups, elixirs and concentrated drops.
  • injection parenteral administration
  • the compounds of the invention are formulated in liquid solutions, preferably, in physiologically compatible buffers or solutions, such as saline solution, Hank's solution, or Ringer's solution.
  • the compounds may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms can also be produced.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, bile salts and fusidic acid derivatives.
  • detergents may be used to facilitate permeation.
  • Transmucosal administration for example, may be through nasal sprays, rectal suppositories, or vaginal suppositories.
  • the compounds of the invention can be formulated into ointments, salves, gels, or creams, as is generally known in the art.
  • the amounts of various compounds to be administered can be determined by standard procedures taking into account factors such as the compound (IC 50 ) potency, (EC 50 ) efficacy, and the biological half-life (of the compound), the age, size and weight of the patient, and the disease or disorder associated with the patient. The importance of these and other factors to be considered are known to those of ordinary skill in the art.
  • Amounts administered also depend on the routes of administration and the degree of oral bioavailability. For example, for compounds with low oral bioavailability, relatively higher doses will have to be administered. Oral administration is a preferred method of administration of the present compounds.
  • the composition is in unit dosage form.
  • a tablet, or capsule may be administered, for nasal application, a metered aerosol dose may be administered, for transdermal application, a topical formulation or patch may be administered and for transmucosal delivery, a buccal patch may be administered.
  • dosing is such that the patient may administer a single dose.
  • Each dosage unit for oral administration contains suitably from 0.01 to 500 mg/Kg, and preferably from 0.1 to 50 mg/Kg, of a compound of Formula (I) or a pharmaceutically acceptable salt thereof, calculated as the free base.
  • the daily dosage for parenteral, nasal, oral inhalation, transmucosal or transdermal routes contains suitably from 0.01 mg to 100 mg/Kg, of a compound of Formula(l).
  • a topical formulation contains suitably 0.01 to 5.0% of a compound of Formula (I).
  • the active ingredient may be administered from 1 to 6 times per day, preferably once, sufficient to exhibit the desired activity, as is readily apparent to one skilled in the art.
  • Composition of Formula (I) and their pharmaceutically acceptable salts which are active when given orally can be formulated as syrups, tablets, capsules and lozenges.
  • a syrup formulation will generally consist of a suspension or solution of the compound or salt in a liquid carrier for example, ethanol, peanut oil. olive oil, glycerine or water with a flavoring or coloring agent.
  • a liquid carrier for example, ethanol, peanut oil. olive oil, glycerine or water with a flavoring or coloring agent.
  • any pharmaceutical carrier routinely used for preparing solid formulations may be used. Examples of such carriers include magnesium stearate, terra alba, talc, gelatin, acacia, stearic acid, starch, lactose and sucrose.
  • composition is in the form of a capsule
  • any routine encapsulation is suitable, for example using the aforementioned carriers in a hard gelatin capsule shell.
  • composition is in the form of a soft gelatin shell capsule
  • any pharmaceutical carrier routinely used for preparing dispersions or suspensions may be considered, for example aqueous gums, celluloses, silicates or oils, and are incorporated in a soft gelatin capsule shell.
  • Typical parenteral compositions consist of a solution or suspension of a compound or salt in a sterile aqueous or non-aqueous carrier optionally containing a parenterally acceptable oil, for example polyethylene glycol, polyvinylpyrrolidone, lecithin, arachis oil or sesame oil.
  • a parenterally acceptable oil for example polyethylene glycol, polyvinylpyrrolidone, lecithin, arachis oil or sesame oil.
  • compositions for inhalation are in the form of a solution, suspension or emulsion that may be administered as a dry powder or in the form of an aerosol using a conventional propellant such as dichlorodifluoromethane or trichlorofluoromethane.
  • a typical suppository formulation comprises a compound of Formula (I) or a pharmaceutically acceptable salt thereof which is active when administered in this way, with a binding and/or lubricating agent, for example polymeric glycols, gelatins, cocoa- butter or other low melting vegetable waxes or fats or their synthetic analogs.
  • a binding and/or lubricating agent for example polymeric glycols, gelatins, cocoa- butter or other low melting vegetable waxes or fats or their synthetic analogs.
  • Typical dermal and transdermal formulations comprise a conventional aqueous or non- aqueous vehicle, for example a cream, ointment, lotion or paste or are in the form of a medicated plaster, patch or membrane.
  • Reaction Conditions were 22 ⁇ M [ 3 H]-UTP (0.75 Ci/mMol), 1 mM-Dithiothreitol, 3.2 mM- MgCI 2 , 20 mM-Tris-HCI, pH7.0, 10 ⁇ g/mL polyA-oligoU, and 90 mM-NaCI. Note that 50mM-NaCI is added with the enzyme.
  • HCV RNA Polymerase Recombinant full-length NS5B (Lohmann et al, J. Virol. 71 (11), 1997, 8416 'Biochemical properties of hepatitis C virus NS5B RNA-dependent RNA polymerase and identification of amino acid sequence motifs essential for enzymatic activity') expressed in baculovirus and purified to homogeneity) was diluted to about 50 ⁇ g protein/mL (dependent on specific activity) in 50mM-Hepes, pH7.0, 0.5M-NaCI, 20%- Glycerol, 0.05%-Triton X-100, 5mM-Dithiothreitol, 0.1mM-EDTA.
  • Substrate Mix was prepared using 5x Concentrated Buffer mix (12 ⁇ L), [ 3 H]-UTP (1 ⁇ Ci/ ⁇ L; 21.7 ⁇ M, 1 ⁇ L), 22 ⁇ M-UTP (100 ⁇ M, 13.2 ⁇ L), 10 ⁇ g/mL polyA-oligoU (100 ⁇ g/mL, 6 ⁇ L), and Water (12.8 ⁇ L), Total 45 ⁇ L
  • the Assay was set up using Substrate Mix (45 ⁇ L), compound (10 ⁇ L), and Diluted Enzyme (added last to start reaction) (5 ⁇ L), Total 60 ⁇ L.
  • the reaction was performed in a U-bottomed, clear, 96-well plate.
  • the reaction was mixed on a plate-shaker, after addition of the Enzyme, and incubated for 2h at 22°C. After this time, the reaction was stopped by addition of 25 ⁇ L of 10OmM-EDTA.
  • a DEAE Filtermat (Part No. 1205-405 from Pharmacia) was pre-washed in water and alcohol and dried. 2 x 20 ⁇ L of the Stopped Assay Mix was spotted onto a square of the DEAE Filtermat. The DEAE Filtermat was washed for 2x 15min in SSC buffer (0.3M- NaCI, 30mM-Na Citrate) followed by 2x 2min in water and 1x 1min in alcohol. The Filtermat was dried and sealed in a bag together with 10mL of OptiScint HiSafe scintillation fluid. The radioactivity present on the filtermat was detected by scintillation counting on a Wallac 1205 Betaplate counter.
  • Reaction Conditions were 0.5 ⁇ M [ 33 P]-GTP (0.2 Ci/mMol), 1 mM Dithiothreitol, 20 mM MgCI 2 , 5mM MnCI 2 , 20 mM Tris-HCI, pH7.5, 1.6 ⁇ g/mL polyC/0.256 ⁇ M biotinylated oligoG13, 10% glycerol, 0.01% NP-40, 0.2 u/ ⁇ L RNasin and 50 mM NaCI.
  • HCV RNA Polymerase Recombinant full-length NS5B (Lohmann et al, J. Virol. 71 (11), 1997, 8416 'Biochemical properties of hepatitis C virus NS5B RNA-dependent RNA polymerase and identification of amino acid sequence motifs essential for enzymatic activity') expressed in baculovirus and purified to homogeneity) was added to 10 nM final concentration.
  • 5x concentrated assay buffer mix was prepared using 1M MnCI 2 (0.25 mL), glycerol (4mL), 10% NP-40 (0.025 mL) and Water (7.225 mL), Total 10 mL.
  • 2x concentrated enzyme buffer contained 1M-Tris-HCI, pH7.5 (0.4 mL), 5M NaCI (0.2 mL), 1 M-MgCI 2 (0.4 mL), glycerol (1 mL), 10% NP-40 (10 ⁇ L), 1 M DTT (20 ⁇ L) and water (7.97 mL), Total 10 mL
  • Substrate Mix was prepared using 5x Concentrated assay Buffer mix (4 ⁇ L), [ 33 P]-GTP (10 ⁇ Ci/ ⁇ L, 0.02 ⁇ L), 25 ⁇ M GTP (0.4 ⁇ L), 0.4 u/ ⁇ L RNasin (0.04 ⁇ L), 20 ⁇ g/mL polyrC/biotinylated-oligorG (1.6 ⁇ L), and Water (3.94 ⁇ L), Total 10 ⁇ L.
  • Enzyme Mix was prepared by adding 1 mg/ml full-length NS5B polymerase (1.5 ⁇ L) to 2.811 mL 2x-concentrated enzyme buffer.
  • the Assay was set up using compound (1 ⁇ L), Substrate Mix (10 ⁇ L), and Enzyme Mix (added last to start reaction) (10 ⁇ L), Total 21 ⁇ L.
  • the reaction was performed in a U-bottomed, white, 96-well plate.
  • the reaction was mixed on a plate-shaker, after addition of the Enzyme, and incubated for 1 h at 22°C. After this time, the reaction was stopped by addition of 40 ⁇ L 1.875 mg/ml streptavidin SPA beads in 0.1 M EDTA.
  • the beads were incubated with the reaction mixture for 1h at 22°C after which 120 ⁇ L 0.1 M EDTA in PBS was added.
  • the plate was sealed, mixed centrifuged and incorporated radioactivity determined by counting in a Trilux (Wallac) or Topcount (Packard) Scintillation Counter.
  • the exemplified compounds all had an IC 50 of ⁇ 10 ⁇ M in one of the above described assays. Accordingly, the compounds of the invention are of potential therapeutic benefit in the treatment and prophylaxis of HCV. Preferred compounds had an IC 50 of ⁇ 1 ⁇ M.
  • a compound of formula (I) or a physiologically acceptable salt or solvate thereof for use in human or veterinary medicine, particularly in the treatment or prophylaxis of viral infection, particularly HCV infection.
  • references herein to treatment includes, but is not limited to prevention, retardation, prophylaxis, therapy and cure of the disease. It will further be appreciated that references herein to treatment or prophylaxis of HCV infection includes treatment or prophylaxis of HCV-associated disease such as liver fibrosis, cirrhosis and hepatocellular carcinoma.
  • a compound of formula (I) or a physiologically acceptable salt or solvate thereof in the manufacture of a medicament for the treatment and/or prophylaxis of viral infection, particularly HCV infection.
  • a method for the treatment of a human or animal subject with viral infection, particularly HCV infection comprises administering to said human or animal subject an effective amount of a compound of formula (I) or a physiologically acceptable salt or solvate thereof.
  • compositions according to the invention may also be used in combination with other therapeutic agents, for example immune therapies (eg. interferon), therapeutic vaccines, antifibrotic agents, anti-inflammatory agents such as corticosteroids or NSAIDs, bronchodilators such as beta-2 adrenergic agonists and xanthines (e.g. theophylline), mucolytic agents, anti-muscarinics, anti-leukotrienes, inhibitors of cell adhesion (e.g.
  • compositions according to the invention may also be used in combination with gene replacement therapy.
  • the invention thus provides, in a further aspect, a combination comprising a compound of formula (I) or a physiologically acceptable salt or solvate thereof together with another therapeutically active agent.
  • compositions comprising a combination as defined above together with a pharmaceutically acceptable carrier thereof represent a further aspect of the invention.

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Abstract

Novel anti-viral agents of Formula (I) wherein: A represents OH; B represents C(O)R1 wherein R1 is selected from the group consisting of C1-6alkyl, aryl, heteroaryl, arylalkyl, and heteroarylalkyl; C represents C1-6alkyl, aryl, heteroaryl or heterocyclyl; D represents hydrogen or C1-6alkyl; E represents hydrogen, C1-6alkyl, aryl or heteroaryl; and F represents hydrogen, C1-6alkyl, heterocyclylalkyl, arylalkyl or heteroarylalkyl; and salts, solvates and esters thereof, processes for their preparation and methods of using them in HCV treatment are provided.

Description

COMPOUNDS
FIELD OF THE INVENTION
The present invention relates to novel 4-cyano acyl pyrrolidine derivatives useful as anti- viral agents. Specifically, the present invention involves novel HCV inhibitors.
BACKGROUND OF THE INVENTION
In the US, an estimated 4.5 million Americans are chronically infected with HCV. Although only 30% of acute infections are symptomatic, greater than 85% of infected individuals develop chronic, persistent infection. Treatment costs for HCV infection have been estimated at $5.46 billion for the US in 1997. Worldwide over 200 million people are estimated to be infected chronically. HCV infection is responsible for 40-60% of all chronic liver disease and 30% of all liver transplants. Chronic HCV infection accounts for 30% of all cirrhosis, end-stage liver disease, and liver cancer in the U.S. The CDC estimates that the number of deaths due to HCV will minimally increase to 38,000/year by the year 2010.
Due to the high degree of variability in the viral surface antigens, existence of multiple viral genotypes, and demonstrated specificity of immunity, the development of a successful vaccine in the near future is unlikely. Alpha-interferon (alone or in combiation with ribavirin) has been widely used since its approval for treatment of chronic HCV infection. However, adverse side effects are commonly associated with this treatment: flu-like symptoms, leukopenia, thrombocytopenia, depression from interferon, as well as anemia induced by ribavirin (Lindsay, K.L. (1997) Hepatology 26 (suppl 1):71S-77S). This therapy remains less effective against infections caused by HCV genotype 1 (which constitutes -75% of all HCV infections in the developed markets) compared to infections caused by the other 5 major HCV genotypes. Unfortunately, only ~50-80% of the patients respond to this treatment (measured by a reduction in serum HCV RNA levels and normalization of liver enzymes) and, of those treated, 50-70% relapse within 6 months of cessation of treatment. Recently, with the introduction of pegylated interferon, both initial and sustained response rates have improved substantially, and combination treatment of Peg- IFN with ribavirin constitutes the gold standard for therapy. However, the side effects asociated with combination therapy and the impaired response in patients with genotype 1 present opportunities for improvement in the management of this disease.
First identified by molecular cloning in 1989 (Choo, Q-L et al (1989) Science 244:359- 362), hepatitis C virus (HCV) is now widely accepted as the most common causative agent of post-transfusion non A, non-B hepatitis (NANBH) (Kuo, G et al (1989) Science 244:362-364). Due to its genome structure and sequence homology, this virus was assigned as a new genus in the Flaviviridae family. Like the other members of the Flaviviridae, such as flaviviruses (e.g. yellow fever virus and Dengue virus types 1-4) and pestiviruses (e.g. bovine viral diarrhea virus, border disease virus, and classic swine fever virus) (Choo, Q-L et al (1989) Science 244:359-3; Miller, R.H. and R.H. Purcell (1990) Proc. Natl. Acad. Sci. USA 87:2057-2061), HCV is an enveloped virus containing a single strand RNA molecule of positive polarity. The HCV genome is approximately 9.6 kilobases (kb) with a long, highly conserved, noncapped 5' nontranslated region (NTR) of approximately 340 bases which functions as an internal ribosome entry site (IRES) (Wang CY et al 'An RNA pseudoknot is an essential structural element of the internal ribosome entry site located within the hepatitis C virus 5' noncoding region' [Article] Rna-A Publication of the Rna Society. 1 (5):526-537, 1995 Jul.). This element is followed by a region which encodes a single long open reading frame (ORF) encoding a polypeptide of -3000 amino acids comprising both the structural and nonstructural viral proteins.
Upon entry into the cytoplasm of the cell, this RNA is directly translated into a polypeptide of -3000 amino acids comprising both the structural and nonstructural viral proteins. This large polypeptide is subsequently processed into the individual structural and nonstructural proteins by a combination of host and virally-encoded proteinases (Rice, CM. (1996) in B.N. Fields, D.M.Knipe and P.M. Howley (eds) Virology 2nd Edition, p931- 960; Raven Press, N.Y.). Following the termination codon at the end of the long ORF, there is a 3' NTR which roughly consists of three regions: an - 40 base region which is poorly conserved among various genotypes, a variable length poly(U)/polypyrimidine tract, and a highly conserved 98 base element also called the "3' X-tail" (Kolykhalov, A. et al (1996) J. Virology 70:3363-3371 ; Tanaka, T. et al (1995) Biochem Biophys. Res. Commun. 215:744-749; Tanaka, T. et al (1996) J. Virology 70:3307-3312; Yamada, N. et al (1996) Virology 223:255-261). The 3' NTR is predicted to form a stable secondary structure which is essential for HCV growth in chimps and is believed to function in the initiation and regulation of viral RNA replication.
The NS5B protein (591 amino acids, 65 kDa) of HCV (Behrens, S.E. et al (1996) EMBO J. 15:12-22), encodes an RNA-dependent RNA polymerase (RdRp) activity and contains canonical motifs present in other RNA viral polymerases. The NS5B protein is fairly well conserved both intra-typically (-95-98% amino acid (aa) identity across 1b isolates) and inter-typically (-85% aa identity between genotype 1a and 1b isolates). The essentiality of the HCV NS5B RdRp activity for the generation of infectious progeny virions has been formally proven in chimpanzees (A. A. Kolykhalov et al.. (2000) Journal of Virology, 74(4), p.2046-2051). Thus, inhibition of NS5B RdRp activity (inhibition of RNA replication) is predicted to cure HCV infection.
Based on the foregoing, there exists a significant need to identify synthetic or biological compounds for their ability to inhibit HCV. SUMMARY OF THE INVENTION
The present invention involves compounds represented hereinbelow, pharmaceutical compositions comprising such compounds and use of the present compounds in treating viral infection, especially HCV infection.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides compounds of Formula (I) :
Figure imgf000004_0001
wherein:
A represents OH;
B represents C(O)R1 wherein R1 is selected from the group consisting of C1-6alkyl, aryl, heteroaryl, arylalkyl, and heteroarylalkyl;
C represents C1-6alkyl, aryl, heteroaryl or heterocyclyl;
D represents hydrogen or C1-6alkyl;
E represents hydrogen, C1-6alkyl, aryl or heteroaryl; and
F represents hydrogen, C1-6alkyl, heterocyclylalkyl, arylalkyl or heteroarylalkyl; and salts, solvates and esters thereof, provided that when A is esterified to form -OR where R is selected from straight or branched chain alkyl, aralkyl, aryloxyalkyl, or aryl, then R is other than fetf-butyl.
It will be appreciated that the compounds of the present invention may contain one or more asymmetric carbon atoms and may exist in racemic, diastereoisomeric, and optically active forms. All of these racemic compounds, enantiomers and diastereoisomers are contemplated to be within the scope of the present invention.
It will further be appreciated that certain compounds of the present invention may exist in different tautomeric forms. All tautomers are contemplated to be within the scope of the present invention.
Preferably, R1 is aryl or heteroaryl;. more preferably R1 is phenyl, optionally substituted by halo, C1-6alkyl or C1-3alkoxy, still more preferably R1 is phenyl substituted in the para- position by -etf-butyl and additionally optionally further substituted. Especially preferred is 4-terf-butylphenyl, optionally 3-substituted by halo, C1-3alkyl or C1-3alkoxy, especially bromo, chloro, methyl or methoxy; most preferably R1 is 4-te/f-butylphenyl, 4-terf-butyl-3- chlorophenyl, 4-terf-butyl-3-methylphenyl, or 4-ferf-butyl-3-methoxyphenyl;
Preferably, C is C1-6alkyl, aryl or heteroaryl; more preferably C is heteroaryl, especially pyridin-2-yl, 1,3-thiazol-2-yl, 1 ,3-thiazol-4-yl or thien-2-yl; most preferably C is 1,3-thiazol- 2-yl;
Preferably, D is hydrogen or methyl; more preferably D is hydrogen;
Preferably, E is hydrogen;
Preferably, F is C1-6alkyl, arylalkyl or heteroarylalkyl, more preferably F is C1-6alkyl, especially 2-methylpropyl (isobutyl).
As used herein, "alkyl" refers to an optionally substituted hydrocarbon group. The alkyl hydrocarbon group may be linear, branched or cyclic, saturated or unsaturated. Where the alkyl hydrocarbon group is cyclic, it will be understood that there will be a minimum of 3 carbon atoms in the group. Preferably, the group is saturated. Preferably, the group is linear or branched. Preferred alkyl moieties are C1-4alkyl. Optional subsituents include Cι_ 6alkyl, halo, C(O)NR4R5, OR6, CO2R1, NR4R5, NHC(O)R1, NHCO2R1, NHC(O)NR2R3, SO2NR2R3, SO2R1, nitro, oxo, and heterocyclyl wherein R2 and R3 are independently selected from the group consisting of hydrogen, C1-6alkyl, aryl, heteroaryl, arylalkyl, and heteroarylalkyl; or R2 and R3 together with the nitrogen atom to which they are attached form a 5 or 6 membered saturated cyclic group; R4 and R5 are independently selected from hydrogen, C1-6alkyl, aryl and heteroaryl; and R6 represents hydrogen, C-ι-6alkyl, arylalkyl, or heteroarylalkyl. Preferably, the group is unsubstituted.
As used herein, "aryl" refers to an optionally substituted aromatic group with at least one ring having a conjugated pi-electron system, containing up to two conjugated or fused ring systems. "Aryl" includes carbocyclic aryl and biaryl groups, all of which may be optionally substituted. Preferred "aryl" moieties are unsubstituted, monosubstituted, disubstituted or trisubstituted phenyl. Preferred "aryl" substituents are selected from the group consisting of C1-6alkyl, halo, OR6, C(O)NR4R5, C(O)R1, CO2H, CO2R1, NR4R5, NHC(O)R\ NHCO2R1, NHC(O)NR2R3, SO2NR2R3, SO2R1, nitro, cyano, oxo, heterocyclyl, CF3, pyridine, phenyl, and NO2. Another preferred group of "aryl" substituents are selected from the group consisting of C^alkyl, halo, OR6, C(O)NR R5, CO2R1, NR4R5, NHC(O)R1, NHCO2R1, NHC(O)NR2R3, SO2NR2R3, SO2R1, nitro, oxo, heterocyclyl, OC^alkyl, CF3, pyridine, phenyl, and NO2. As used herein, "heteroaryl" refers to an optionally substituted, 5 or 6 membered, aromatic group comprising one to four heteroatoms selected from N, O and S, with at least one ring having a conjugated pi-electron system, containing up to two conjugated or fused ring systems. Preferred "heteroaryl" moieties include unsubstituted, monosubstituted, disubstituted or trisubstituted thienyl, thiazolyl, and pyridinyl. Preferred "heteroaryl" substituents are selected from the group consisting of C1-6alkyl, halo, OR6, C(O)NR4R5, C(O)R\ CO2H, CO2R\ NR4R5, NHC(O)R1, NHCO2R1, NHC(O)NR2R3, SO2NR2R3, SO2R1, nitro, cyano, oxo, heterocyclyl, CF3, pyridine, phenyl, and NO2. Another preferred group of "heteroaryl" substituents are selected from the group consisting of C1-6alkyl, halo, OR6, C(O)NR4R5, CO2R1, NR4R5, NHC(O)R1, NHCO2R1, NHC(O)NR2R3, SO2NR2R3, SO2R1, nitro, oxo, heterocyclyl, OC1-4alkyl, CF3, pyridine, phenyl, and NO2.
As used herein, "heterocyclic" and "heterocyclyl" refer to an optionally substituted, 5 or 6 membered, saturated cyclic hydrocarbon group containing one to four heteroatoms selected from N, optionally substituted by hydrogen, C1-6alkyl, C(O)R1, SO2R1, aryl or heteroaryl; O; and S, optionally substituted by one or two oxygen atoms.
Preferred compounds useful in the present invention are selected from the group consisting of: re/-(2S,4R,5R)-2-lsobutyl-1 -(3-methoxy-4-te/τ-butylbenzoyl)-4-cyano-5-(1 ,3-thiazol-2- yl)pyrrolidine-2-carboxylic acid; re/-(2S,4S,5R)-2-lsobutyl-1-(3-methoxy-4--er--butylbenzoyl)-4-cyano-5-(1 ,3-thiazol-2- yl)pyrrolidine-2-carboxylic acid; fe/-(2S,4R,5R)-2-lsobutyl-1-(3-chloro-4-tert-butylbenzoyl)-4-cyano-5-(1 ,3-thiazol-2- yl)pyrrolidine-2-carboxylic acid; re/-(2S,4R,5R)-2-lsobutyl-1-(3-methyl-4-tett-butylbenzoyl)-4-cyano-5-(1 ,3-thiazol-2- yl)pyrrolidine-2-carboxylic acid; re/-(2S,4R,5R)-2-lsobutyl-1-(3-methoxy-4-te τ-butylbenzoyl)-4-cyano-5-(thien-2- yl)pyrrolidine-2-carboxylic acid; re/-(2S,4R,5R)-2-lsobutyl-1-(3-methoxy-4-ter/-butylbenzoyl)-4-cyano-5-(pyhdin-2- yl)pyrrolidine-2-carboxylic acid; re/-(2S,4R,5R)-2-lsobutyl-1-(3-methoxy-4-tert-butylbenzoyl)-4-cyano-5-(1 ,3-thiazol-4- yl)pyrrolidine-2-carboxylic acid; re/-(2S,4S,5R)-2-lsobutyl-1-(3-methoxy-4-ter--butylbenzoyl)-4-cyano-5-(1 ,3-thiazol-4- yl)pyrrolidine-2-carboxylic acid; re/-(2S,4R,5R)-2-lsobutyl-1-(3-methoxy-4-terτ-butylbenzoyl)-4-cyano-4-methyl-5-(1,3- thiazol-2-yl)pyrrolidine-2-carboxylic acid;
/-e/-(2S,4S,5R)-2-lsobutyl-1-(4-terf-butylbenzoyl)-4-cyano-5-(1 ,3-thiazol-2-yl)pyrrolidine-2- carboxylic acid;
and salts, solvates, esters and individual enantiomers thereof. Also included in the present invention are pharmaceutically acceptable salt complexes. The present invention also covers the physiologically acceptable salts of the compounds of formula (I). Suitable physiologically acceptable salts of the compounds of formula (I) include acid salts, for example sodium, potassium, calcium, magnesium and tetraalkylammonium and the like, or mono- or di- basic salts with the appropriate acid for example organic carboxylic acids such as acetic, lactic, tartaric, malic, isethionic, lactobionic and succinic acids; organic sulfonic acids such as methanesulfonic, ethanesulfonic, benzenesulfonic and p-toluenesulfonic acids and inorganic acids such as hydrochloric, sulfuric, phosphoric and sulfamic acids and the like.
The present invention also relates to pharmaceutically acceptable solvates of the compounds of Formula (I), for example hydrates.
The present invention also relates to pharmaceutically acceptable esters of the compounds of Formula (I), for example carboxylic acid esters -COOR, in which R is selected from straight or branched chain alkyl, for example n-propyl, n-butyl, alkoxyalkyl (e.g. methoxymethyl), aralkyl (e.g. benzyl), aryloxyalkyl (e.g. phenoxymethyl), aryl (e.g. phenyl optionally substituted by halogen, C1-4alkyl or C^alkoxy or amino). Unless otherwise specified, any alkyl moiety present in such esters preferably contains 1 to 18 carbon atoms, particularly 1 to 4 carbon atoms. Any aryl moiety present in such esters preferably comprises a phenyl group.
Compounds of Formula (I) may be prepared by reaction of a compound of Formula (II)
Figure imgf000007_0001
in which A is hydroxy or a protected form thereof, and C, D, E and F are as defined above for Formula (I); with a suitable acylating agent, for example R1C(O)-hal, wherein hal is a halo atom, preferably chloro or bromo, C is a carbon atom, and R1 is as defined for Formual (I). Preferably the reaction is carried out in a suitable solvent, for example dichloromethane, in the presence of a suitable base, for example triethylamine, and thereafter removing any protecting group. Suitable hydroxy protecting groups can be found, but are not restricted to, those found in T W Greene and P G M Wuts 'Protective Groups in Organic Synthesis', 3rd Ed (1999), J Wiley and Sons. Protection and deprotection of the hydroxy may be carried out by standard techniques known in the art.
Compounds of Formula (II) may be prepared by reaction of a compound of Formula (III)
Figure imgf000008_0001
wherein A, C and F are as defined for Formula (I) above; with a compound of Formula (IV)
Figure imgf000008_0002
wherein D and E are as defined for Formula (I). Preferably, the reaction is carried out in a suitable solvent, for example THF, in the presence of a Lewis acid catalyst, such as lithium bromide, and a base, such as triethylamine.
Compounds of Formula (III), (IV) and R1C(O)-hal are known in the art or may be prepared by standard literature procedures.
With appropriate manipulation and protection of any chemical functionality, synthesis of compounds of Formula (I) is accomplished by methods analogous to those above and to those described in the Experimental section. Suitable protecting groups can be found, but are not restricted to, those found in T W Greene and P G M Wuts 'Protective Groups in Organic Synthesis', 3rd Ed (1999), J Wiley and Sons.
It will be appreciated that compounds of Formula I and/or II which exist as diastereoisomers may optionally be separated by techniques well known in the art, for example by column chromatography.
It will be appreciated that racemic compounds of Formula (I) or (II) may be optionally resolved into their individual enantiomers. Such resolutions may conveniently be accomplished by standard methods known in the art. For example, a racemic compound of Formula (I) or (II) may be resolved by chiral preparative HPLC.
EXAMPLES Intermediate 1 2-[N-(1 ,3-Thiazol-2-ylmethylene) tanoic acid, ferf-butyl ester
Figure imgf000008_0003
A stirred mixture of 2-amino-4-methyl-pentanoic acid terf-butyl ester, hydrochloride salt (5.00 g, 22.34 mmol), 1 ,3-thiazole-2-carboxaldehyde (2.53 g, 22.34 mmol) and triethylamine (3.10 mL, 22.3 mmol) in dichloromethane (60 mL) were heated under reflux under nitrogen for 19 hours. The reaction mixture was allowed to cool to room temperature, washed twice with water, dried over Na2SO4 and evaporated to give the title compound as an oil. 1H NMR (CDCI3): δ 8.46 (s, 1H), 7.94 (d, 1H), 7.44 (d, 1H), 4.07 (dd, 1H), 1.89-1.74 (m, 2H), 1.64-1.52 (m, 1 H), 1.48 (s, 9H), 0.96 (d, 3H) and 0.90 (d, 3H).
Intermediate 2 re/-(2S,4R,5R)-4-Cyano-2-isobutyl-5-(1,3-thiazol-2-yl)pyrrolidine-2-carboxylic acid, terf-butyl ester
Racemic;
Relative stereochemistry shown
Figure imgf000009_0001
Acrylonitrile (1.17 mL, 17.7 mmol) was added to a solution of 2-[N-(1 ,3-thiazol-2~ ylmethylene)amino]-4-methylpentanoic acid, terf-butyl ester (Intermediate 1 ; 4.00 g, 14.2 mmol) in anhydrous THF (20 L) at 0°C. Lithium bromide (2.47 g, 28.4 mmol) was added and the mixture stirred at 0°C for 5 minutes prior to the addition of triethylamine (2.47 mL, 17.7 mmol). The resulting mixture was stirred at room temperature for 18 hours then diluted with ethyl acetate and washed with saturated aqueous ammonium chloride solution and brine, dried (Na2SO4) and evaporated. The crude product mixture was purified by chromatography on silica gel using cyclohexane-ethyl acetate (10:1 v/v) as eluent to afford the title compound, a solid.
MS calcd for (C17H25N3O2S + H)+: 336. Found: (M+H)+ = 336.
1H NMR (CD3OD): δ 7.65 (1 H, d), 7.40 (1 H, d), 4.70 (1 H, d), 3.15 (1 H, m), 2.80 (1 H, dd), 2.00 (1H, dd), 1.80 (1H, m), 1.60 (2H, m), 1.30 (9H, s) and 0.85 (6H, m). The pyrrolidine NH proton exchanges with the solvent. Continued elution of the chromatography column afforded the rel-(2S,4S,5R)- diastereoisomer, described as Intermediate 3 below.
Intermediate 3 re/-(2S,4S,5R)-4-Cyano-2-isobutyl-5-(1,3-thiazol-2-yl)pyrrolidine-2-carboxylic acid, terf-butyl ester
Racemic; Relative stereochemistry shown
Figure imgf000009_0002
Continued elution of the chromatography column described in Intermediate 2, using cyclohexane-ethyl acetate (7:3 v/v) as eluent afforded the title compound, the rel- (2S,4S,5R)-diastereoisomer. MS calcd for (d^NsOzS + H)+: 336. Found: (M+H)+ = 336. 1H NMR (CD3OD): δ 7.80 (1 H, d), 7.55 (1 H, d), 4.90 (1 H, d), 3.70 (1 H, m), 2.80 (1H, dd), 2.30 (1 H, d), 1.75 (3H, m), 1.50 (9H, s), 0.95 (6H, m). The pyrrolidine NH proton exchanges with the solvent.
Intermediate 4
2-[N-(Thien-2-ylmethylene)amino]-4-methylpentanoic acid, ferf-butyl ester
Figure imgf000010_0001
The title compound was prepared according to the method described for Intermediate 1 , substituting thiophene-2-carboxaldehyde in place of thiazole-2-carboxaldehyde. 1H NMR (CDCI3): δ 8.38 (s, 1 H), 7.43 (dt, 1 H), 7.36 (dd, 1H), 7.08 (dd, 1 H), 3.94 (dd, 1 H), 1.87-1.71 (m, 2H), 1.59 (m, 1 H), 1.46 (s, 9H), 0.95 (d, 3H) and 0.89 (d, 3H).
Intermediate 5 re/-(2S,4R,5R)-4-Cyano-2-isobutyI-5-(thien-2-yI)pyrrolidine-2-carboxylic acid, tert- butyl ester
Racemic;
Relative stereochemistry shown
Figure imgf000010_0002
The title compound, a solid, was prepared from Intermediate 4 and acrylonitrile in a manner analogous to that described for the preparation of Intermediate 2, affording a mixture of epimers at the pyrrolidine C(4)-position. The mixture was separated by chromatography on silica gel, using cyclohexane-ethyl acetate (11 :1 v/v) as eluent to afford the title compound in the earlier eluting fractions. MS calcd for (C18H26N2O2S + H)+: 335. Found: (M+H)+ = 335.
1H NMR (CD3OD): δ 7.35 (1 H, d), 7.15 (1 H, d), 7.00 (1 H, t), 4.65 (1 H, d), 3.00-2.90 (1 H, m), 2.85 (1H, dd), 2.10 (1 H, dd), 1.80-1.60 (3H, m), 1.45 (9H, s) and 0.95 (6H, m). Pyrrolidine NH proton exchanges with solvent.
Intermediate 6
2-[N-(Pyridin-2-ylmethylene)amino]-4-methylpentanoic acid, ferf-butyl ester
Figure imgf000010_0003
The title compound was prepared according to the method described for Intermediate 1 , substituting pyridine-2-carboxaldehyde in place of thiazole-2-carboxaldehyde. 1H NMR (CDCI3): δ 8.65 (1H, d), 8.40 (1H, s), 8.10 (1H, d), 7.75 (1 H, t), 7.35 (1 H, t), 4.05 (1H, dd), 1.90-1.75 (2H, m), 1.60-1.50 (1H, m), 1.45 (9H, s), 0.95 (3H, d) and 0.90 (3H, d).
Intermediate 7 re/-(2S,4R,5R)-4-Cyano-2-isobutyl-5-(pyridin-2-yl)pyrro!idine-2-carboxylic acid, ferf- butyl ester
Racemic;
Relative stereochemistry shown
Figure imgf000011_0001
The title compound, a solid, was prepared from Intermediate 6 and acrylonitrile in a manner analogous to that described for the preparation of Intermediate 2, affording a mixture of epimers at the pyrrolidine C(4)-position. The mixture was separated by chromatography on silica gel, using a gradient elution from cyclohexane-ethyl acetate (9:1 v/v) to cyclohexane-ethyl acetate (1 :1 v/v) as eluent to afford the title compound in the earlier eluting fractions.
MS calcd for (Cι9H27N3O2 + H)+: 330. Found: (M+H)+ = 330.
1H NMR (CD3OD): δ 8.60 (1 H, d), 7.85 (1 H, m), 7.55 (1 H, d), 7.40 (1 H, m), 4.45 (1 H, d), 3.10-3.00 (1 H, dd), 2.90-2.80 (1 H, dd), 2.20-2.10 (1 H, dd), 1.85-1.70 (3H, m), 1.50 (9H, s), 1.00 (3H, d) and 0.95 (3H, d). Pyrrolidine NH proton exchanges with solvent.
Intermediate 8
2-[N-(1,3-Thiazol-4-ylmethylene) tanoic acid, ferf-butyl ester
Figure imgf000011_0002
The title compound was prepared according to the method described for Intermediate 1 , substituting thiazole-4-carboxaldehyde in place of thiazole-2-carboxaldehyde.
1H NMR (CDCI3): δ 8.85 (1 H, d), 8.50 (1 H, s), 8.00 (1 H, d), 4.00 (1 H, dd), 1.90-1.75 (2H, m), 1.60-1.50 (1 H, m), 1.50 (9H, s), 0.95 (3H, d) and 0.90 (3H, d).
Intermediate 9 re/-(2S,4R,5R)-4-Cyano-2-isobutyl-5-(1 ,3-thiazol-4-yl)pyrrolidine-2-carboxylic acid, ferf-butyl ester Racemic;
Relative stereochemistry shown
The title compound, a solid, was prepared from Intermediate 8 and acrylonitrile in a manner analogous to that described for the preparation of Intermediate 2, affording a mixture of epimers at the pyrrolidine C(4)-position. The mixture was separated by chromatography on silica gel, using a gradient elution from cyclohexane-ethyl acetate (10:1 v/v) to cyclohexane-ethyl acetate (7:1 v/v) as eluent to afford the title compound in the earlier eluting fractions.
MS calcd for (C17H25N3O2S + H)+: 336. Found: (M+H)+ = 336.
1H NMR (CD3OD): δ 8.90 (1H, d), 7.55 (1 H, d), 4.45 (1 H, d), 3.10-3.00 (1 H, dd), 2.70 (1 H, dd), 2.10-2.00 (1 H, dd), 1.70-1.60 (3H, m), 1.40 (9H, s), 0.85 (3H, d) and 0.80 (3H, d). Pyrrolidine NH proton exchanges with solvent.
Continued elution of the chromatography column afforded the rel-(2S,4S,5R)- diastereoisomer, described as Intermediate 10 below.
Intermediate 10 re/-(2S,4S,5R)-4-Cyano-2-isobutyl-5-(1,3-thiazol-4-yl)pyrrolidine-2-carboxylic acid, ferf-butyl ester
Racemic;
Relative stereochemistry shown
Figure imgf000012_0002
Continued elution of the chromatography column described in Intermediate 9 afforded the title compound, the re/-(2S,4S,5R)-diastereoisomer.
MS calcd for (C17H25N3O2S + H)+: 336. Found: (M+H)+ = 336.
1H NMR (CD3OD): δ 9.00 (1 H, d), 7.70 (1H, d), 4.70 (1 H, d), 3.60 (1 H, m), 2.80-2.75 (1 H, dd), 2.30 (1 H, dd), 1.80-1.70 (2H, m), 1.65 (1 H, dd), 1.55 (9H, s), 1.00 (3H, d) and 0.95 (3H, d). Pyrrolidine NH proton exchanges with solvent.
Intermediate 11
Enantiomer A of re/-(2S,4R,5R)-4-Cyano-2-isobutyl-5-(1 ,3-thiazol-2-yl)pyrrolidine-2- carboxylic acid, ferf-butyl ester
Chiral;
Relative stereochemistry shown
Figure imgf000012_0003
The enantiomers of Intermediate 2 were separated on chiral HPLC using Chiralcel OD as the stationary phase and heptane-ethanol (95:5 v/v) as eluent. The title compound was the first eluting enantiomer (Enantiomer A). This material was spectroscopically identical by 1H NMR and MS to that described as Intermediate 2.
Intermediate 12 re/-(2S,4R,5R)-4-Cyano-2-isobutyl-4-methyl-5-(1,3-t iazol-2-yl)pyrrolidine-2- carboxylic acid, ferf-butyl ester
Racemic;
Relative stereochemistry shown
Figure imgf000013_0001
The title compound, a solid, was prepared from Intermediate 1 and 1-cyano-1- methylethene in a manner analogous to that described for the preparation of Intermediate 2, affording a mixture of epimers at the pyrrolidine C(4)-position. The mixture was separated by chromatography on silica gel, using cyclohexane-ethyl acetate (10:1 v/v) as eluent to afford the title compound in the earlier eluting fractions.
MS calcd for (Cι8H27N3O2S + H)+: 350. Found: (M+H)+ = 350.
1H NMR (CD3OD): δ 7.85 (1 H, d), 7.60 (1 H, d), 4.95 (1 H, s), 2.75 (1 H, d), 2.45 (1 H, d), 1.90-1.70 (3H, m), 1.50 (9H, s) and 1.00-0.95 (9H, m). Pyrrolidine NH proton exchanges with solvent.
Example 1 re/-(2S,4R,5R)-2-lsobutyl-1 -(3-methoxy-4-ferf-butylbenzoyl)-4-cyano-5-(1 ,3-thiazol-2- yl)pyrrolidine-2-carboxylic acid
Figure imgf000013_0002
Sfage A: 3-Methoxy-4-ferf-butylbenzoyl chloride (0.203 g, 0.89 mmol) was added to a solution of re/-(2S,4R,5R)-4-cyano-2-isobutyl-5-(1 ,3-thiazol-2-yl)pyrrolidine-2-carboxylic acid, ferf-butyl ester (Intermediate 2; 0.250 g, 0.74 mmol) and triethylamine (0.13 mL, 0.93 mmol) in dry dichloromethane (5 mL). The mixture was stirred at room temperature overnight then diluted with additional dichloromethane and washed with saturated aqueous sodium bicarbonate solution and brine, dried (Na2SO4) and evaporated. The residual gum was chromatographed on silica gel using a gradient elution from cyclohexane to cyclohexane-ethyl acetate (4:1 v/v) to afford the ferf-butyl ester of the title compound.
Stage B: The ferf-butyl ester from Stage A (0.157 g, 0.29 mmol) was dissolved in trifluoroacetic acid (2 mL) and stirred at room temperature for 17 hours. The mixture was evaporated and the residue triturated with diethyl ether to afford the title compound, a solid.
MS calcd for (C25H31N3θ4S + H)+: 470. Found (M+H)+= 470.
1H NMR (CDCI3): δ 7.70 (1 H, d), 7.30 (1 H, d), 7.20 (1 H, d), 7.00 (1H, d), 6.60 (1 H, s), 5.65 (1 H, d), 3.75 (3H, s), 3.60 (1 H, dd), 3.30 (1H, dd), 2.60 (1H, dd), 2.50 (1 H, dd), 2.00 (1H, dd), 1.85 (1H, br), 1.30 (9H, s) and 1.10 (6H, m).
Example 2 re/-(2S,4S,5R)-2-lsobutyl-1-(3-methoxy-4-ferf-butylbenzoyl)-4-cyano-5-(1,3-thiazol-2- yl)pyrrolidine-2-carboxylic acid
Figure imgf000014_0001
The title compound, a solid, was prepared from re/-(2S,4S,5R)-4-cyano-2-isobutyl-5-(1 ,3- thiazol-2-yl)pyrrolidine-2-carboxylic acid, ferf-butyl ester (Intermediate 3) in a manner directly analogous to that described for the corresponding re/-(2S,4R,5R)-diastereoisomer (Example 1).
MS calcd for (C25H3ιN3O4S + H)+: 470. Found (M+H)+ = 470.
1H NMR (CDCI3): δ 7.90 (1 H, d), 7.40 (1 H, d), 7.20 (1 H, d), 6.65 (1 H, d), 6.30 (1 H, s), 5.65 (1 H, d), 3.80 (1 H, m), 3.60 (3H, s), 2.85 (1H, t), 2.65 (1 H, dd), 2.40-2.30 (2H, m), 1.85 (1 H, br), 1.30 (9H, s) and 1.10 (6H, m). Carboxylic acid proton not seen.
Example 3 re/-(2S,4R,5R)-2-lsobutyl-1-(3-chloro-4-ferf-butylbenzoyl)-4-cyano-5-(1,3-thiazoI-2- yl)pyrrolidine-2-carboxylic acid
Figure imgf000014_0002
The title compound, a solid, was prepared in a two-stage process from Intermediate 2 by acylation with 3-chloro-4-ferf-butylbenzoyl chloride (Stage A) and then trifluoroacetic acid induced ester deprotection (Stage B) in a manner analogous to that described in Example 1. MS calcd for (C24H28CIN3O3S + H)+: 474/476. Found (M+H)+ = 474/476. 1H NMR (CD3OD): δ 7.50 (1 H, d), 7.40-7.35 (2H, m), 7.25 (1H, d), 7.10 (1 H, s), 5.80 (1 H, d), 3.80 (1H, dd), 3.35 (2H, m), 2.50 (1H, br), 2.10 (1H, dd), 1.95 (1H, br), 1.45 (9H, s), 1.15 (3H, d) and 1.00 (3H, d). Carboxylic acid proton exchanges with solvent.
Example 4 re/-(2S,4R,5R)-2-lsobutyl-1-(3-methyl-4-ferf-butylbenzoyI)-4-cyano-5-(1,3-thiazol-2- yl)pyrrolidine-2-carboxylic acid
Figure imgf000015_0001
The title compound, a solid, was prepared in a two-stage process from Intermediate 2 by acylation with 3-methyl-4-ferf-butylbenzoyl chloride (Stage A) and then trifluoroacetic acid induced ester deprotection (Stage B) in a manner analogous to that described in Example
1.
MS calcd for (C25H3ιN3θ3S + H)+: 454. Found (M+H)+ = 454.
1H NMR (CD3OD): δ. 7.45 (1H, d), 7.30 (1 H, br), 7.25 (1H, d), 7.10 (1 H, d), 6.90 (1 H, br), 5.80 (1 H, d), 3.80 (1H, dd), 2.70 (2H, m), 2.45 (3H, s), 2.10-2.05 (2H, m), 1.95 (1 H, br),
1.40 (9H, s), 1.15 (3H, d) and 1.00 (3H, d). Carboxylic acid proton exchanges with solvent.
Example 5 re/-(2S,4R,5R)-2-lsobutyl-1-(3-methoxy-4-ferf-butylbenzoyl)-4-cyano-5-(thien-2- yl)pyrrolidine-2-carboxylic acid
shown
Figure imgf000015_0002
The title compound, a solid, was prepared in a two-stage process from Intermediate 5 by acylation with 3-methoxy-4-ferf-butylbenzoyl chloride (Stage A) and then trifluoroacetic acid induced ester deprotection (Stage B) in a manner analogous to that described in
Example 1.
MS calcd for (C26H32N2O4S + H)+: 469. Found (M+H)+ = 469.
1H NMR (CD3OD): δ. 7.20 (1 H, d), 7.15 (1 H, d), 6.90 (1H, d), 6.65 (1 H, br d), 6.60 (1 H, t),
6.55 (1 H, br), 5.60 (1 H, d), 3.70 (4H, m), 2.70-2.65 (2H, m), 2.50 (1 H, br d), 2.05-1.95 (2H, m), 1.35 (9H, s), 1.20 (3H, d) and 1.05 (3H, d). Carboxylic acid proton exchanges with solvent. Example 6 re/-(2S,4R,5R)-2-lsobutyl-1-(3-methoxy-4-ferf-butylbenzoyl)-4-cyano-5-(pyridin-2- yl)pyrrolidine-2-carboxylic acid
Figure imgf000016_0001
The title compound, a solid, was prepared in a two-stage process from Intermediate 7 by acylation with 3-methoxy-4-.erf-butylbenzoyl chloride (Stage A) and then trifluoroacetic acid induced ester deprotection (Stage B) in a manner analogous to that described in Example 1. MS calcd for (C27H33N3O4 + H)+: 464. Found (M+H)+ = 464.
1H NMR (CD3OD): δ 8.35 (1 H, d), 7.65 (1H, t), 7.40 (1 H, d), 7.25 (1 H, t), 7.15 (1 H, d), 6.90 (1H, d), 6.50 (1H, s), 5.50 (1H, d), 3.70 (3H, s), 3.65 (1H, m), 2.80-2.70 (2H, m), 2.50 (1H, br), 2.20 (1H, dd), 2.00 (1 H, m), 1.30 (9H, s), 1.20 (3H, d) and 1.05 (3H, d). Carboxylic acid proton exchanges with solvent.
Example 7 re/-(2S,4R,5R)-2-lsobutyl-1-(3-methoxy-4-ferf-butylbenzoyl)-4-cyano-5-(1,3-thiazol-4- yl)pyrrolidine-2-carboxylic acid
Figure imgf000016_0002
The title compound, a solid, was prepared in a two-stage process from Intermediate 9 by acylation with 3-methoxy-4-ferf-butylbenzoyl chloride (Stage A) and then trifluoroacetic acid induced ester deprotection (Stage B) in a manner analogous to that described in Example 1. MS calcd for (C25H3ιN3O4S + H)+: 470. Found (M+H)+ = 470. 1H NMR (CD3OD): δ 8.70 (1 H, s), 7.45 (1 H, s), 7.15 (1 H, d), 6.90 (1 H, d), 6.60 (1 H, s), 5.65 (1 H, d), 3.75 (3H, s), 3.70 (1 H, m), 2.80 (1 H, m), 2.65 (1H, t), 2.45 (1H, dd), 2.15 (1 H, dd), 1.95 (1H, m), 1.35 (9H, s), 1.15 (3H, d) and 1.05 (3H, d). Carboxylic acid proton exchanges with solvent.
Example 8 re/-(2S,4S,5R)-2-lsobutyl-1-(3-methoxy-4-ferf-butylbenzoyl)-4-cyano-5-(1,3-thiazol-4- yl)pyrrolidine-2-carboxylic acid
Figure imgf000017_0001
The title compound, a solid, was prepared in a two-stage process from Intermediate 10 by acylation with 3-methoxy-4-ferf-butylbenzoyl chloride (Stage A) and then trifluoroacetic acid induced ester deprotection (Stage B) in a manner analogous to that described in Example 1.
MS calcd for (C25H3iN3O4S + H)+: 470. Found (M+H)+ = 470.
1H NMR (CD3OD): δ 9.00 (1H, s), 7.75 (1H, s), 7.20 (1 H, d), 6.70 (1 H, d), 6.50 (1 H, s), 5.80 (1 H, d), 4.10 (1 H, dd), 3.70 (3H, s), 2.70 (2H, d), 2.25 (2H, br), 2.00 (1 H, br), 1.30 (9H, s) and 1.10 (6H, d). Carboxylic acid proton exchanges with solvent.
Example 9
Enantiomer A of re/-(2S,4R,5R)-2-lsobutyl-1 -(3-methoxy-4-ferf-butylbenzoyl)-4- cyano-5-(1,3-thiazol-2-yl)pyrrolidine-2-carboxylic acid
stereochemistry shown
Figure imgf000017_0002
The title compound, a solid, was prepared in a two-stage process from Intermediate 11 by acylation with 3-methoxy-4-ferf-butylbenzoyl chloride (Stage A) and then trifluoroacetic acid induced ester deprotection (Stage B) in a manner analogous to that described in Example 1. The title compound was spectroscopically identical by 1H NMR and MS to the compound described in Example 1.
Example 10 re/-(2S,4R,5R)-2-lsobutyl-1-(3-methoxy-4-ferf-butylbenzoyl)-4-cyano-4-methyl-5-(1,3- thiazol-2-yl)pyrrolidine-2-carboxylic acid
Figure imgf000018_0001
The title compound, a solid, was prepared in a two-stage process from Intermediate 12 by acylation with 3-methoxy-4-fe/f-butylbenzoyl chloride (Stage A) and then trifluoroacetic acid induced ester deprotection (Stage B) in a manner analogous to that described in
Example 1.
MS calcd for (C26H33N3O4S + H)+: 484. Found (M+H)+ = 484.
1H NMR (CD3OD): δ 7.85 (1 H, d), 7.65 (1 H, d), 7.30 (1 H, d), 6.80 (1H, d), 6.50 (1 H, br),
6.00 (1 H, s), 3.70 (3H, s), 2.90 (1 H, d), 2.75-2.70 (1 H, br), 2.60 (1 H, d), 2.20-2.10 (2H, m),
1.35 (9H, s) and 1.15 (9H, m). Carboxylic acid proton exchanges with solvent.
Example 11 re/-(2S,4S,5R)-2-lsobutyl-1-(4-ferf-butylbenzoyl)-4-cyano-5-(1,3-thiazol-2- yl)pyrrolidine-2-carboxylic acid
Figure imgf000018_0002
The title compound, a solid, was prepared in a two-stage process from Intermediate 3 by acylation with 4-ferf-butylbenzoyl chloride (Stage A) and then trifluoroacetic acid induced ester deprotection (Stage B) in a manner analogous to that described in Example 1. MS calcd for (C2 H29N3O3S + H)+: 440. Found (M+H)+ = 440. 1H NMR (CDCI3): δ 7.87 (1 H, d), 7.38 (1 H, d), 7.24 (2H, d), 6.94 (2H, d), 6.53 (1 H, d), 3.80 (1 H, m), 2.85 (1 H, t), 2.65 (1 H, dd), 2.39 (1 H, dd), 2.31 (1 H, dd), 1.86 (1H, m), 1.25 (9H, s), 1.12 (6H, t).
The compounds according to 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 formula (I) or a physiologically acceptable salt or solvate thereof in admixture with one or more physiologically acceptable diluents or carriers.
The compounds of the present invention can be administered by different routes including intravenous, intraperitoneal, subcutaneous, intramuscular, oral, topical, transdermal, or transmucosal administration. For systemic administration, oral administration is preferred. For oral administration, for example, the compounds can be formulated into conventional oral dosage forms such as capsules, tablets and liquid preparations such as syrups, elixirs and concentrated drops.
Alternatively, injection (parenteral administration) may be used, e.g., intramuscular, intravenous, intraperitoneal, and subcutaneous. For injection, the compounds of the invention are formulated in liquid solutions, preferably, in physiologically compatible buffers or solutions, such as saline solution, Hank's solution, or Ringer's solution. In addition, the compounds may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms can also be produced.
Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, bile salts and fusidic acid derivatives. In addition, detergents may be used to facilitate permeation. Transmucosal administration, for example, may be through nasal sprays, rectal suppositories, or vaginal suppositories.
For topical administration, the compounds of the invention can be formulated into ointments, salves, gels, or creams, as is generally known in the art.
The amounts of various compounds to be administered can be determined by standard procedures taking into account factors such as the compound (IC50) potency, (EC50) efficacy, and the biological half-life (of the compound), the age, size and weight of the patient, and the disease or disorder associated with the patient. The importance of these and other factors to be considered are known to those of ordinary skill in the art.
Amounts administered also depend on the routes of administration and the degree of oral bioavailability. For example, for compounds with low oral bioavailability, relatively higher doses will have to be administered. Oral administration is a preferred method of administration of the present compounds.
Preferably the composition is in unit dosage form. For oral application, for example, a tablet, or capsule may be administered, for nasal application, a metered aerosol dose may be administered, for transdermal application, a topical formulation or patch may be administered and for transmucosal delivery, a buccal patch may be administered. In each case, dosing is such that the patient may administer a single dose.
Each dosage unit for oral administration contains suitably from 0.01 to 500 mg/Kg, and preferably from 0.1 to 50 mg/Kg, of a compound of Formula (I) or a pharmaceutically acceptable salt thereof, calculated as the free base. The daily dosage for parenteral, nasal, oral inhalation, transmucosal or transdermal routes contains suitably from 0.01 mg to 100 mg/Kg, of a compound of Formula(l). A topical formulation contains suitably 0.01 to 5.0% of a compound of Formula (I). The active ingredient may be administered from 1 to 6 times per day, preferably once, sufficient to exhibit the desired activity, as is readily apparent to one skilled in the art.
Composition of Formula (I) and their pharmaceutically acceptable salts which are active when given orally can be formulated as syrups, tablets, capsules and lozenges. A syrup formulation will generally consist of a suspension or solution of the compound or salt in a liquid carrier for example, ethanol, peanut oil. olive oil, glycerine or water with a flavoring or coloring agent. Where the composition is in the form of a tablet, any pharmaceutical carrier routinely used for preparing solid formulations may be used. Examples of such carriers include magnesium stearate, terra alba, talc, gelatin, acacia, stearic acid, starch, lactose and sucrose. Where the composition is in the form of a capsule, any routine encapsulation is suitable, for example using the aforementioned carriers in a hard gelatin capsule shell. Where the composition is in the form of a soft gelatin shell capsule any pharmaceutical carrier routinely used for preparing dispersions or suspensions may be considered, for example aqueous gums, celluloses, silicates or oils, and are incorporated in a soft gelatin capsule shell.
Typical parenteral compositions consist of a solution or suspension of a compound or salt in a sterile aqueous or non-aqueous carrier optionally containing a parenterally acceptable oil, for example polyethylene glycol, polyvinylpyrrolidone, lecithin, arachis oil or sesame oil.
Typical compositions for inhalation are in the form of a solution, suspension or emulsion that may be administered as a dry powder or in the form of an aerosol using a conventional propellant such as dichlorodifluoromethane or trichlorofluoromethane.
A typical suppository formulation comprises a compound of Formula (I) or a pharmaceutically acceptable salt thereof which is active when administered in this way, with a binding and/or lubricating agent, for example polymeric glycols, gelatins, cocoa- butter or other low melting vegetable waxes or fats or their synthetic analogs.
Typical dermal and transdermal formulations comprise a conventional aqueous or non- aqueous vehicle, for example a cream, ointment, lotion or paste or are in the form of a medicated plaster, patch or membrane.
No unacceptable toxological effects are expected when compounds of the present invention are administered in accordance with the present invention. ASSAY
The potential for compounds of the invention to inhibit NS5B wildtype HCV polymerase activity may be demonstrated, for example, using one of the following in vitro assays:
In Vitro Detection of inhibitors of HCV RNA-dependent RNA Polymerase Activity (A)
Incorporation of [3H]-UMP into RNA was followed by absorption of the RNA polymer onto a DEAE glass fibre filter. A synthetic template consisting of 16mer oligoU hybridised to polyrA (10:1 w/w) was used as a homopolymer substrate.
Reaction Conditions were 22 μM [3H]-UTP (0.75 Ci/mMol), 1 mM-Dithiothreitol, 3.2 mM- MgCI2, 20 mM-Tris-HCI, pH7.0, 10 μg/mL polyA-oligoU, and 90 mM-NaCI. Note that 50mM-NaCI is added with the enzyme.
HCV RNA Polymerase (Recombinant full-length NS5B (Lohmann et al, J. Virol. 71 (11), 1997, 8416 'Biochemical properties of hepatitis C virus NS5B RNA-dependent RNA polymerase and identification of amino acid sequence motifs essential for enzymatic activity') expressed in baculovirus and purified to homogeneity) was diluted to about 50 μg protein/mL (dependent on specific activity) in 50mM-Hepes, pH7.0, 0.5M-NaCI, 20%- Glycerol, 0.05%-Triton X-100, 5mM-Dithiothreitol, 0.1mM-EDTA.
5x Concentrated Buffer mix was prepared using 1M-Tris-HCI (pH7.0, 1 mL), 1 M-MgCI2 (0.16mL), 1 M-Dithiothreitol (0.05mL), 5M-NaCI (0.4mL), and Water (8.4mL), Total 10mL
Substrate Mix was prepared using 5x Concentrated Buffer mix (12μL), [3H]-UTP (1 μCi/μL; 21.7μM, 1μL), 22 μM-UTP (100 μM, 13.2 μL), 10 μg/mL polyA-oligoU (100 μg/mL, 6μL), and Water (12.8 μL), Total 45μL
The Assay was set up using Substrate Mix (45μL), compound (10μL), and Diluted Enzyme (added last to start reaction) (5μL), Total 60μL.
The reaction was performed in a U-bottomed, clear, 96-well plate. The reaction was mixed on a plate-shaker, after addition of the Enzyme, and incubated for 2h at 22°C. After this time, the reaction was stopped by addition of 25μL of 10OmM-EDTA.
A DEAE Filtermat (Part No. 1205-405 from Pharmacia) was pre-washed in water and alcohol and dried. 2 x 20μL of the Stopped Assay Mix was spotted onto a square of the DEAE Filtermat. The DEAE Filtermat was washed for 2x 15min in SSC buffer (0.3M- NaCI, 30mM-Na Citrate) followed by 2x 2min in water and 1x 1min in alcohol. The Filtermat was dried and sealed in a bag together with 10mL of OptiScint HiSafe scintillation fluid. The radioactivity present on the filtermat was detected by scintillation counting on a Wallac 1205 Betaplate counter. After subtraction of background levels without enzyme, any reduction in the amount of radioactivity incorporated in the presence of a compound, compared to that in the absence, was taken as a measure of the level of inhibition. Ten concentrations of compounds were tested in two- or threefold dilutions. From the counts, percentage of inhibition at highest concentration tested or IC50s for the compounds were calculated using Grafit3 or Grafit4 software packages.
In Vitro Detection of inhibitors of HCV RNA-dependent RNA Polymerase Activity (B)
Incorporation of [33P]-GMP into RNA was followed by absorption of the biotin labelled RNA polymer by streptavidin containing SPA beads. A synthetic template consisting of biotinylated 13mer-oligoG hybridised to polyrC was used as a homopolymer substrate.
Reaction Conditions were 0.5 μM [33P]-GTP (0.2 Ci/mMol), 1 mM Dithiothreitol, 20 mM MgCI2, 5mM MnCI2, 20 mM Tris-HCI, pH7.5, 1.6 μg/mL polyC/0.256 μM biotinylated oligoG13, 10% glycerol, 0.01% NP-40, 0.2 u/μL RNasin and 50 mM NaCI.
HCV RNA Polymerase (Recombinant full-length NS5B (Lohmann et al, J. Virol. 71 (11), 1997, 8416 'Biochemical properties of hepatitis C virus NS5B RNA-dependent RNA polymerase and identification of amino acid sequence motifs essential for enzymatic activity') expressed in baculovirus and purified to homogeneity) was added to 10 nM final concentration.
5x concentrated assay buffer mix was prepared using 1M MnCI2 (0.25 mL), glycerol (4mL), 10% NP-40 (0.025 mL) and Water (7.225 mL), Total 10 mL.
2x concentrated enzyme buffer contained 1M-Tris-HCI, pH7.5 (0.4 mL), 5M NaCI (0.2 mL), 1 M-MgCI2 (0.4 mL), glycerol (1 mL), 10% NP-40 (10 μL), 1 M DTT (20 μL) and water (7.97 mL), Total 10 mL
Substrate Mix was prepared using 5x Concentrated assay Buffer mix (4μL), [33P]-GTP (10 μCi/μL, 0.02μL), 25 μM GTP (0.4 μL), 0.4 u/μL RNasin (0.04 μL), 20 μg/mL polyrC/biotinylated-oligorG (1.6 μL), and Water (3.94 μL), Total 10 μL.
Enzyme Mix was prepared by adding 1 mg/ml full-length NS5B polymerase (1.5 μL) to 2.811 mL 2x-concentrated enzyme buffer.
The Assay was set up using compound (1μL), Substrate Mix (10 μL), and Enzyme Mix (added last to start reaction) (10 μL), Total 21 μL.
The reaction was performed in a U-bottomed, white, 96-well plate. The reaction was mixed on a plate-shaker, after addition of the Enzyme, and incubated for 1 h at 22°C. After this time, the reaction was stopped by addition of 40 μL 1.875 mg/ml streptavidin SPA beads in 0.1 M EDTA. The beads were incubated with the reaction mixture for 1h at 22°C after which 120 μL 0.1 M EDTA in PBS was added. The plate was sealed, mixed centrifuged and incorporated radioactivity determined by counting in a Trilux (Wallac) or Topcount (Packard) Scintillation Counter.
After subtraction of background levels without enzyme, any reduction in the amount of radioactivity incorporated in the presence of a compound, compared to that in the absence, was taken as a measure of the level of inhibition. Ten concentrations of compounds were tested in three- or fivefold dilutions. From the counts, percentage of inhibition at highest concentration tested or IC50s for the compounds were calculated using Grafit3 or Grafit4 software packages.
The exemplified compounds all had an IC50 of <10μM in one of the above described assays. Accordingly, the compounds of the invention are of potential therapeutic benefit in the treatment and prophylaxis of HCV. Preferred compounds had an IC50 of <1μM.
Thus, there is provided as a further aspect of the present invention a compound of formula (I) or a physiologically acceptable salt or solvate thereof for use in human or veterinary medicine, particularly in the treatment or prophylaxis of viral infection, particularly HCV infection.
It will be appreciated that reference herein to treatment includes, but is not limited to prevention, retardation, prophylaxis, therapy and cure of the disease. It will further be appreciated that references herein to treatment or prophylaxis of HCV infection includes treatment or prophylaxis of HCV-associated disease such as liver fibrosis, cirrhosis and hepatocellular carcinoma.
According to another aspect of the invention, there is provided the use of a compound of formula (I) or a physiologically acceptable salt or solvate thereof in the manufacture of a medicament for the treatment and/or prophylaxis of viral infection, particularly HCV infection.
In a further or alternative aspect there is provided a method for the treatment of a human or animal subject with viral infection, particularly HCV infection, which method comprises administering to said human or animal subject an effective amount of a compound of formula (I) or a physiologically acceptable salt or solvate thereof.
The pharmaceutical compositions according to the invention may also be used in combination with other therapeutic agents, for example immune therapies (eg. interferon), therapeutic vaccines, antifibrotic agents, anti-inflammatory agents such as corticosteroids or NSAIDs, bronchodilators such as beta-2 adrenergic agonists and xanthines (e.g. theophylline), mucolytic agents, anti-muscarinics, anti-leukotrienes, inhibitors of cell adhesion (e.g. ICAM antagonists), anti-oxidants (eg N-acetylcysteine), cytokine agonists, cytokine antagonists, lung surfactants and/or antimicrobial and anti-viral agents (eg ribavirin and amantidine). The compositions according to the invention may also be used in combination with gene replacement therapy.
The invention thus provides, in a further aspect, a combination comprising a compound of formula (I) or a physiologically acceptable salt or solvate thereof together with another therapeutically active agent.
The combination referred to above may conveniently be presented for use in the form of a pharmaceutical formulation and thus pharmaceutical formulations comprising a combination as defined above together with a pharmaceutically acceptable carrier thereof represent a further aspect of the invention.
The individual components of such combinations may be administered either sequentially or simultaneously in separate or combined pharmaceutical formulations. Appropriate doses of known therapeutic agents will be readily appreciated by those skilled in the art.
All publications, including but not limited to patents and patent applications cited in this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference as though fully set forth.

Claims

Claims
1. A compound of formula (I)
Figure imgf000025_0001
wherein:
A represents OH;
B represents C(O)R1 wherein R1 is selected from the group consisting of C1-6alkyl, aryl, heteroaryl, arylalkyl, and heteroarylalkyl;
C represents C1-6alkyl, aryl, heteroaryl or heterocyclyl;
D represents hydrogen or C1-6alkyl;
E represents hydrogen, C1-6alkyl, aryl or heteroaryl; and
F represents hydrogen, d-6alkyl, heterocyclylalkyl, arylalkyl or heteroarylalkyl; and salts, solvates and esters thereof, provided that when A is esterified to form -OR where R is selected from straight or branched chain alkyl, aralkyl, aryloxyalkyl, or aryl, then R is other than ferf-butyl.
2. A compound as claimed in claim 1 wherein R1 represents 4-ferf-butylphenyl optionally 3-substituted by halo, C1-3alkyl or Cι-3alkoxy.
3. A compound as claimed in claim 1 or 2 wherein C represents heteroaryl.
4. A compound as claimed in any one of claims 1 to 3, wherein D represents hydrogen or methyl.
5. A compound as claimed in any one of claims 1 to 4, wherein F represents C^ 6alkyl.
6. A compound as claimed in claim 1 selected from the group consisting of: re/-(2S,4R,5R)-2-lsobutyl-1-(3-methoxy-4-ferf-butylbenzoyl)-4-cyano-5-(1 ,3-thiazol-2- yl)pyrrolidine-2-carboxylic acid; re/-(2S,4S,5R)-2-lsobutyl-1-(3-methoxy-4-ferf-butylbenzoyl)-4-cyano-5-(1 ,3-thiazol-2- yl)pyrrolidine-2-carboxylic acid; re/-(2S,4R,5R)-2-lsobutyl-1-(3-chloro-4-ferf-butylbenzoyl)-4-cyano-5-(1 ,3-thiazol-2- yl)pyrrolidine-2-carboxylic acid; re/-(2S,4R,5R)-2-lsobutyl-1-(3-methyl-4-ferf-butylbenzoyl)-4-cyano-5-(1,3-thiazol-2- yl)pyrrolidine-2-carboxylic acid; re/-(2S,4R,5R)-2-lsobutyl-1-(3-methoxy-4- erf-butylbenzoyl)-4-cyano-5-(thien-2- yl)pyrrolidine-2-carboxylic acid; re/-(2S,4R,5R)-2-lsobutyl-1-(3-methoxy-4-ferf-butylbenzoyl)-4-cyano-5-(pyridin-2- yl)pyrrolidine-2-carboxylic acid; re/-(2S,4R,5R)-2-lsobutyl-1-(3-methoxy-4-ferf-butylbenzoyl)-4-cyano-5-(1,3-thiazol-4- yl)pyrrolidine-2-carboxylic acid; re/-(2S,4S,5R)-2-lsobutyl-1-(3-methoxy-4-ferf-butylbenzoyl)-4-cyano-5-(1,3-thiazol-4- yl)pyrrolidine-2-carboxylic acid; re/-(2S,4R,5R)-2-lsobutyl-1-(3-methoxy-4-ferf-butylbenzoyl)-4-cyano-4-methyl-5-(1 ,3- thiazol-2-yl)pyrrolidine-2-carboxylic acid; re/-(2S,4S,5R)-2-lsobutyl-1 -(4-ferf-butylbenzoyl)-4-cyano-5-(1 ,3-thiazol-2-yl)pyrrolidine-2- carboxylic acid;
and salts, solvates, esters and individual enantiomers thereof.
7. A method for the treatment of a viral infection which comprises administering to a subject in need thereof, an effective amount of a compound as claimed in claim 1.
8. A method as claimed in claim 7 which involves inhibiting HCV.
9. A method as claimed in claim 7 in which the compound is administered in an oral dosage form.
10. A compound of Formula (I) as claimed in any of claims 1 to 6 for use in medical therapy.
1 1. A compound as claimed in claim 10 wherein the medical therapy is the treatment of viral infection.
12. A compound as claimed in claim 11 wherein the viral infection is HCV.
13. Use of a compound of Formula (I) as claimed in any of claims 1 to 6 in the manufacture of a medicament for the treatment and/or prophylaxis of viral infection.
14. Use as claimed in claim 13, wherein the viral infection is HCV.
15. A pharmaceutical composition comprising a compound of Formula (I) as claimed in any of claims 1 to 6, together with a pharmaceutically acceptable dileunt of carrier therefor.
16. A process for the preparation of a compound of Formula (I) as defined in claim , said process comprising the acylation of a compound of Formula (II)
Figure imgf000027_0001
wherein A is hydroxy or a protected form thereof, and C, D, E, and F are as defined in claim 1 for Formula (I), with an acylating agent of Formula R1C(O)-hal wherein hal is a halo atom and R1 is as defined in claim 1 for Formula (I), thereafter removing any protecting group.
PCT/EP2003/008138 2002-07-22 2003-07-18 1-carbonyl-4-cyano-pyrrolidine-2-carboxylic acid derivatives as hepatitis c virus inhibitors WO2004009543A2 (en)

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WO2006050035A1 (en) 2004-10-29 2006-05-11 Schering Corporation Substituted 5-oxo pyrazoles and [1,2,4]triazoles as antiviral agents
EP2494991A1 (en) 2007-05-04 2012-09-05 Vertex Pharmaceuticals Incorporated Combination therapy for the treatment of HCV infection
US20130209403A1 (en) * 2010-09-08 2013-08-15 Ruprecht-Karls-Universitaet Use of inhibitors of phospholipase a2 for the treatment or prevention of flavivirus infection

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WO2003037894A1 (en) * 2001-11-02 2003-05-08 Glaxo Group Limited 4-(5-membered)-heteroaryl acyl pyrrolidine derivatives as hcv inhibitors
WO2003037893A1 (en) * 2001-11-02 2003-05-08 Glaxo Group Limited Acyl dihydro pyrrole derivatives as hcv inhibitors

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Publication number Priority date Publication date Assignee Title
WO2004076415A1 (en) * 2003-02-27 2004-09-10 Glaxo Group Limited 1- (hetero)aroyl-pyrrolidine-2-carboxylic acid derivatives useful as anti.viral agents
WO2006050035A1 (en) 2004-10-29 2006-05-11 Schering Corporation Substituted 5-oxo pyrazoles and [1,2,4]triazoles as antiviral agents
US7115749B2 (en) 2004-10-29 2006-10-03 Schering Corporation Substituted 5-oxo pyrazoles and [1,2,4]triazoles as antiviral agents
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EP2494991A1 (en) 2007-05-04 2012-09-05 Vertex Pharmaceuticals Incorporated Combination therapy for the treatment of HCV infection
US20130209403A1 (en) * 2010-09-08 2013-08-15 Ruprecht-Karls-Universitaet Use of inhibitors of phospholipase a2 for the treatment or prevention of flavivirus infection

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GB0216985D0 (en) 2002-08-28

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