WO2007039142A1 - Composes de heteroarylmethyl-c (4) -methoxymethyl acyl pyrrolidine et leur utilisation dans le traitement d'infections virales, en particulier du virus de l'hepatite c - Google Patents

Composes de heteroarylmethyl-c (4) -methoxymethyl acyl pyrrolidine et leur utilisation dans le traitement d'infections virales, en particulier du virus de l'hepatite c Download PDF

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WO2007039142A1
WO2007039142A1 PCT/EP2006/009234 EP2006009234W WO2007039142A1 WO 2007039142 A1 WO2007039142 A1 WO 2007039142A1 EP 2006009234 W EP2006009234 W EP 2006009234W WO 2007039142 A1 WO2007039142 A1 WO 2007039142A1
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formula
butyl
thiazol
compound
compounds
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PCT/EP2006/009234
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English (en)
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David Haigh
Charles David Hartley
Peter David Howes
Richard Lewis Jarvest
Linos Lazarides
Fabrizio Nerozzi
Stephen Allan Smith
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Glaxo Group Limited
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Publication of WO2007039142A1 publication Critical patent/WO2007039142A1/fr

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    • 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
    • C07D417/14—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 three or more hetero rings
    • 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
    • A61P31/14—Antivirals for RNA viruses
    • 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 C(4)-methoxymethyl acyl pyrrolidine derivatives useful as anti-viral agents. Specifically, the present invention involves novel Hepatitis C Virus (HCV) inhibitors.
  • HCV Hepatitis C Virus
  • 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.
  • Alpha-interferon (alone or in combination with ribavirin) has been widely used since its approval for treatment of chronic HCV infection.
  • 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 ): 71 S-77S).
  • HCV hepatitis C virus
  • NANBH non-B hepatitis
  • 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 1 noncoding region' RNA- A Publication of the RNA Society. 1 (5): 526-537, 1995 JuI.). 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 1 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 1 b isolates) and inter-typically (-85% aa identity between genotype 1a and 1 b 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.
  • genotype 1 Although the predominant HCV genotype worldwide is genotype 1 , this itself has two main subtypes, denoted 1a and 1 b. As seen from entries into the Los Alamos HCV database (www.hcv.lanl.gov) (Table 1 ) there are regional differences in the distribution of these subtypes: while genotype 1a is most abundant in the United States, the majority of sequences in Europe and Japan are from genotype 1b. Table 1
  • genotype 1a makes it highly desirable to identify an anti-viral agent that is able to inhibit both genotype 1a and genotype 1 b. This means a wider patient pool would be able to benefit from treatment with the same agent.
  • PCT publication number WO2004/037818 generically discloses certain compounds, including certain acyl pyrrolidine compounds, having HCV inhibitory activity.
  • the assay is directed to the 1 b genotype.
  • the compounds disclosed have the formula (I)
  • A represents hydroxy
  • D represents aryl or heteroaryl
  • E represents hydrogen, Ci. 6 alkyl, aryl, heteroaryl or heterocyclyl
  • G represents hydrogen or C 1-6 alkyl optionally substituted by one or more substituents selected from halo, OR 1 , SR 1 , C(O)NR 2 R 3 , CO 2 H, C(O)R 4 , CO 2 R 4 , NR 2 R 3 , NHC(O)R 4 ,
  • R 1 represents hydrogen, C 1-6 alkyl, arylalkyl, or heteroarylalkyl
  • R 2 and R 3 are independently selected from hydrogen, C 1-e alkyl, aryl and heteroaryl; 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 is selected from the group consisting of C 1-6 alkyl, aryl, heteroaryl, arylalkyl, and heteroarylalkyl;
  • R 5 and R 6 are independently selected from the group consisting of hydrogen, C 1-6 alkyl, aryl, heteroaryl, arylalkyl, and heteroarylalkyl; or R 5 and R 6 together with the nitrogen atom to which they are attached form a 5 or 6 membered saturated cyclic group; and J represents 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 tert-butyl.
  • the present invention involves C(2)-heteroarylmethyl-C(4)-methoxymethyl acyl pyrrolidine compounds represented hereinbelow, pharmaceutical compositions comprising such compounds and use of the compounds in treating viral infection, especially HCV infection.
  • the present invention provides at least one chemical entity chosen from compounds of Formula (Ia) :
  • A represents hydroxy
  • D represents 4-fert-butyl-3-ethenylphenyl or 4-terf-butyl-5-ethenyl-2-fluorophenyl;
  • E represents 1 ,3-thiazol-2-yl
  • G represents methoxymethyl
  • J represents 1 ,3-thiazol-4-ylmethyl or 1H-pyrazol-1-ylmethyl
  • the relative stereochemistry of racemic compounds of Formula (Ia) is represented by Formulae (Ip) or (Iq): ve stereochemistry
  • A represents hydroxy (that is, not esterified).
  • the compounds of Formula (Ia) are represented by compounds of Formula (Ip).
  • the compounds of Formula (Ia) are represented by compounds of Formula (Ia).
  • J represents 1 ,3-thiazol-4-ylmethyl. In another aspect J represents 1 H- pyrazol-1-ylmethyl.
  • D represents 4-te/t-butyl-3-ethenylphenyl. In another aspect D represents 4-terf-butyl-5-ethenyl-2-fluorophenyl.
  • the chemical entities of the present invention exhibit an improved genotype-1a/1b profile against HCV polymerase, and therefore have the potential to achieve efficacy in man over a broad patient population.
  • the term 'genotype-1a/1 b profile' means potency as an inhibitor of HCV polymerase enzyme in wildtype HCV of the 1a genotype and of the 1 b genotype. High potency in both genotypes is considered to be advantageous.
  • references herein to therapy and/or 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 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 at least one chemical entity chosen from compounds of Formula (Ia) and pharmaceutically acceptable salts, solvates or esters thereof.
  • the compounds of the present invention may contain one or more asymmetric carbon atoms and may exist in racemic, diastereoisomeric, and optically active forms.
  • chemical entities useful in the present invention may be chosen from compounds of Formula (Ia) selected from the group consisting of: re/-(2R,4S,5R)-1-(4-fert-butyl-3-ethenylbenzoyl)-4-(methoxymethyl)-5-(1,3-thiazol-2-yl)-2-
  • compositions are also included in the present invention.
  • Suitable pharmaceutically acceptable salts of the compounds of formula (Ia) 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-tolu
  • the present invention also relates to solvates of the compounds of Formula (Ia), for example hydrates.
  • the present invention also relates to pharmaceutically acceptable esters of the compounds of Formula (Ia), 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), alkoxycarbonylalkyl (e.g. methoxycarbonylmethyl), acyloxyalkyl (e.g. pivaloyloxymethyl), aralkyl (e.g. benzyl), aryloxyalkyl (e.g. phenoxymethyl), aryl (e.g. phenyl optionally substituted by halogen, or C 1-4 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.
  • the compound of Formula (Ia) is in the form of parent compound, a salt or a solvate.
  • the term "pharmaceutically acceptable” used in relation to an ingredient (active ingredient such as an active ingredient, a salt thereof or an excipient) which may be included in a pharmaceutical formulation for administration to a patient refers to that ingredient being acceptable in the sense of being compatible with any other ingredients present in the pharmaceutical formulation and not being deleterious to the recipient thereof.
  • A' is a protected hydroxy group, for example an alkoxy, benzyloxy or silyloxy, for example tri-(C 1-4 alkyl)-silyloxy group
  • D, E, G and J are as defined above for Formula (Ia), by deprotection.
  • 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', 3 rd Ed (1999), J Wiley and Sons.
  • the reaction is carried out in a solvent, for example dichloromethane.
  • the temperature is in the range 0 to 50°C, suitably 20 to 30 0 C.
  • a suitable catalyst for example palladium-on- carbon.
  • the reaction is carried out in a solvent, for example ethanol.
  • the temperature is in the range 0 to 50 0 C.
  • A' is allyloxy
  • D, E, G and J are as defined above for Formula (Ia)
  • a suitable catalyst for example tetrakis(triphenylphosphine)palladium(0)
  • a suitable proton source for example phenylsilane.
  • the reaction is carried out in a suitable solvent, for example dichloromethane.
  • A' is tri(methyl)silyloxy
  • D, E, G and J are as defined above for Formula (Ia)
  • a suitable fluoride source for example tetrabutylammonium fluoride.
  • the reaction is carried out in a suitable solvent, for example tetrahydrofuran.
  • A" is hydroxy or an alkoxy, benzyloxy or a silyloxy, for example a tri-(C 1-4 alkyl)- silyloxy, group, and E, G, and J are as defined above for Formula (Ia); with a suitable acylating agent, for example D-C(O)-hal, wherein hal is a halo atom, preferably chloro or bromo, and D is as defined above for Formula (Ia).
  • the reaction is carried out in a suitable solvent, for example dichloromethane, in the presence of a suitable base, for example triethylamine.
  • the temperature is in the range 0 to 50 0 C, more preferably 20 to 30 0 C.
  • the reaction may be carried out at the reflux temperature of the solvent.
  • Compounds of Formula (Ia) or (II) may also be prepared by methylation of a compound of formula (IV) in which A" is as defined above for Formula (III), and G' represents hydroxymethyl using a suitable base for example sodium hydride or sodium tert-butoxide and a suitable methylating agent such as methyl iodide.
  • a suitable base for example sodium hydride or sodium tert-butoxide
  • a suitable methylating agent such as methyl iodide.
  • the reaction is carried out in a suitable solvent, for example dimethylformamide, dichloromethane, acetonitrile or a mixture thereof.
  • the reaction is carried out at a temperature in the range 0 to 50 0 C, suitably 20 to 30 0 C.
  • L represents CHO or CO 2 Y wherein Y represents hydrogen or alkyl.
  • a suitable reducing agent for example lithium borohydride, lithium triethylborohydride, sodium borohydride, sodium triacetoxyborohydride, borane/dimethyl sulfide complex or lithium aluminium hydride, or suitable combinations thereof, in a suitable solvent or mixture thereof for example tetrahydrofuran or methanol.
  • the reaction is carried out at a temperature in the range -78 to -20 0 C.
  • a compound of Formula (V) in which L represents CO 2 Y wherein Y represents hydrogen may be prepared from a compound of Formula (V) in which L represents CO 2 Y wherein Y represents alkyl.
  • a compound of Formula (V) in which L represents CO 2 Me may be converted into a compound of Formula (V) in which L represents CO 2 H by hydrolysis, for example base catalysed hydrolysis using a suitable base such as sodium methoxide in a suitable solvent such as methanol.
  • a compound of Formula (V) in which L represents CHO or CO 2 Y wherein Y represents hydrogen or alkyl may be prepared from a compound of Formula (Vl) in which L represents CHO or CO 2 Y wherein Y represents hydrogen or alkyl, and A", E, and J are as defined above for Formula (III); with a suitable acylating agent, for example D-C(O)-hal, wherein hal is a halo atom, preferably chloro or bromo, and D is as defined above for Formula (Ia).
  • a suitable solvent for example dichioromethane
  • a suitable base for example triethylamine.
  • the reaction is carried out at a temperature in the range 0 to 50 0 C, suitably 20 to 30 0 C.
  • the reaction may be carried out under reflux.
  • a compound of Formula (V) in which A" is hydroxy may be converted to a compound of Formula (V) in which A" is an alkoxy, benzyloxy or silyloxy group by standard hydroxy protecting techniques.
  • a compound of Formula (V) in which A" is an alkoxy, benzyloxy or silyloxy group may be converted to a compound of Formula (V) in which A” is hydroxy by standard deprotecting techniques.
  • 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', 3 rd Ed (1999), J Wiley and Sons.
  • a compound of Formula (Vl) may be prepared by reaction of a compound of Formula (VII)
  • L represents CHO or CO 2 Y wherein Y represents hydrogen or alkyl.
  • the reaction is carried out in a suitable solvent, for example THF or acetonitrile, optionally in the presence of a Lewis acid catalyst, such as lithium bromide or silver acetate, and a base, such as triethylamine, 1 ,8-diazabicyclo[5,4,0]undec-7-ene (DBU) or tetramethyl guanidine.
  • a suitable solvent for example THF or acetonitrile
  • a Lewis acid catalyst such as lithium bromide or silver acetate
  • a base such as triethylamine, 1 ,8-diazabicyclo[5,4,0]undec-7-ene (DBU) or tetramethyl guanidine.
  • reaction is carried out in a suitable solvent, for example THF or acetonitrile, in the presence of an acid, such as acetic acid, or the reaction may be carried out by heating compounds of Formula (VII) and Formula (VIII) in a suitable solvent, for example toluene, xylene or acetonitrile in the absence of a catalyst.
  • a suitable solvent for example THF or acetonitrile
  • an acid such as acetic acid
  • a compound of Formula (Vl) may also be prepared in a one pot synthesis by reaction of a compound of Formula (X) (see below for structure) with a compound of Formula (VIII) and a compound of Formula E-CHO.
  • the reaction is carried out in a suitable solvent, for example THF or acetonitrile, optionally in the presence of a Lewis acid catalyst, such as lithium bromide or silver acetate, and a base, such as triethylamine, 1 ,8- diazabicyclo[5,4,0]undec-7-ene (DBU) or tetramethyl guanidine.
  • a suitable solvent for example THF or acetonitrile
  • a Lewis acid catalyst such as lithium bromide or silver acetate
  • a base such as triethylamine, 1 ,8- diazabicyclo[5,4,0]undec-7-ene (DBU) or tetramethyl guanidine.
  • DBU triethylamine, 1
  • a compound of Formula (III) may be prepared by appropriate manipulation of a compound of Formula (IX)
  • N-protected compound of Formula (IX) may be converted into a compound of Formula (III), in which G represents methoxymethyl and the N-atom is protected, by methylation. Deprotection of the N-atom by standard procedures results in the compound of Formula (III).
  • Compounds of Formula (IX) may be prepared by reduction of an optionally N-protected compound of Formula (Vl) in which L represents CO 2 Y and Y represents alkyl, using a suitable reducing agent, for example lithium borohydride or sodium borohydride, in a suitable solvent for example tetrahydrofuran.
  • a suitable reducing agent for example lithium borohydride or sodium borohydride
  • Deprotection of the N-atom by standard procedures results in the compound of Formula (IX).
  • the N-protecting group is CBZ
  • deprotection may be achieved by catalytic hydrogenolysis.
  • the N-protecting group is f-butoxycarbonyl
  • deprotection may be achieved by treatment with a suitable acid, for example trifluoroacetic acid.
  • J is 1 H-pyrazol-1-ylmethyl
  • M is a metal cation, for example potassium
  • a suitable acid for example 10% aqueous hydrochloric acid, in the presence of Amberlyst 120 (H + ).
  • Formula (X) in which J is 1 H-pyrazol-1-ylmethyl, and A" is hydroxy by conventional esterification or protecting group procedures.
  • a compound of Formula (X) in which J is 1 H-pyrazol-1-ylmethyl, and A" is ferf-butoxy may be prepared by treatment of a compound of Formula (X) in which J is 1 H-pyrazol-1-ylmethyl, and A" is hydroxy, with an appropriate terf-butyl transfer agent, such as tert-butylacetate in the presence of a suitable acid catalyst, such as 70% perchloric acid.
  • reaction is carried out at a temperature in the range 50-70 0 C, suitably 60 0 C.
  • J is 1 ,3-thiazol-4-ylmethyl and A" is an alkoxy, benzyloxy or tri-(C 1-4 alkyl)-silyIoxy group with an acid, for example 15% aqueous citric acid.
  • the reaction is carried out in a suitable solvent or mixture thereof, for example THF or water.
  • reaction is carried out in the presence of a suitable base such as potassium £-butoxide.
  • a suitable solvent for example THF.
  • reaction is carried out in the presence of a suitable catalyst, for example lithium iodide.
  • the reaction is carried out at a temperature in the range -10 0 C to room temperature, suitably at O 0 C.
  • a suitable acid halide forming reagent for example oxalyl chloride or thionyl chloride.
  • the reaction is carried out in the presence of a suitable catalyst, for example dimethylformamide or diethylformamide.
  • a suitable solvent for example dichloromethane, at a temperature in the range 0 to 50 0 C, for example 20 to 3O 0 C.
  • the reaction is carried out using thionyl chloride under reflux.
  • the compound of Formula (XV) may be prepared by reaction of a compound of Formula
  • the temperature is in the range 0 to 50 0 C, suitably 20 to 3O 0 C.
  • Compounds of Formula (XVI) may be prepared by reaction of a compound of Formula (XVII) in which P' is a suitable protecting group, for example tert-butyl, with a suitable olefinating reagent, for example methyltriphenylphosphonium bromide.
  • a suitable base for example potassium tert-butoxide.
  • the reaction is carried out in a suitable solvent, for example THF.
  • the temperature is in the range 0 to 50 0 C, suitably 20 to 30 0 C.
  • P' is a suitable protecting group, for example tert-butyl, with a suitable formylating reagent, for example DMF.
  • the reaction is carried out in the presence of a suitable base, for example n-butyl lithium in hexane.
  • a suitable solvent for example THF.
  • the temperature is in the range 0 to -78°C, suitably -78°C.
  • the compound of Formula (XVIII) may be prepared by protection of the compound of Formula (XIX)
  • the reaction is carried out in the presence of a suitable alcohol, for example tert-butyl alcohol.
  • a suitable base for example DBU.
  • an activating agent for example 1 ,1 '-carbonyldiimidazole.
  • the reaction is carried out in a suitable solvent, for example DMF.
  • the temperature is in the range 0 to 50°C, suitably 40 0 C.
  • the compound of Formula (XV) in may also be prepared by reaction of a compound of Formula (XIX) with an ethenyl transfer reagent, for example potassium ethenyltrifluoroborate.
  • an ethenyl transfer reagent for example potassium ethenyltrifluoroborate.
  • the reaction is carried out in the presence of a suitable catalyst, for example [1 ,1 '-bis(diphenylphosphino)ferrocene]dichloropalladium(ll), complex with CH 2 CI 2 .
  • a suitable base for example caesium carbonate.
  • the reaction is carried in a suitable solvent, for example a mixture of THF and water.
  • the reaction is carried out under reflux.
  • a suitable acid halide forming reagent for example oxalyl chloride or thionyl chloride.
  • the reaction is carried out in the presence of a suitable catalyst, for example dimethylformamide or diethylformamide.
  • a suitable solvent for example dichloromethane, at a temperature in the range 0 to 50 0 C, for example 20 to 30 0 C.
  • the reaction is carried out using thionyl chloride under reflux.
  • the compound of Formula (XX) may be prepared by reaction of the compound of Formula (XX)
  • the reaction is carried out in the presence of a suitable catalyst, for example [1 ,1 '- bis(diphenylphosphino)ferrocene]dichloropalladium(ll), complex with CH 2 CI 2 .
  • a suitable catalyst for example [1 ,1 '- bis(diphenylphosphino)ferrocene]dichloropalladium(ll), complex with CH 2 CI 2 .
  • the reaction is carried out in the presence of a suitable base for example caesium carbonate.
  • the reaction is carried in a suitable solvent, for example a mixture of THF and water.
  • the reaction is carried out under reflux.
  • the compound of Formula (XXI) may be prepared by reaction of the compound of Formula (XXII)
  • the compound of Formula (XXII) may be prepared by treatment of the compound of
  • reaction is carried out in a suitable solvent, for example pyridine and water. In one aspect, the reaction is carried out under reflux.
  • a suitable oxidising agent for example potassium permanganate.
  • the reaction is carried out in a suitable solvent, for example pyridine and water. In one aspect, the reaction is carried out under reflux.
  • the compound of Formula (XXIII) may be prepared by reaction of the compound of Formula (XXIV)
  • reaction is carried out in a suitable solvent, for example dimethylformamide. In one aspect, the reaction is carried out at a temperature in the range 50-80 c C.
  • the compound of Formula (XXIV) may be prepared by reaction of the compound of Formula (XXV)
  • reaction is carried out in a suitable solvent, for example dimethylformamide.
  • the compound of Formula (XXV) may be prepared by reaction of the compound of Formula (XXVI)
  • the reation is carried out at a temperature in the range 50-80 0 C.
  • Compounds of Formula (Ia) in which A is an ester may be prepared by esterification of a compound of Formula (Ia) in which A is hydroxy by standard literature procedures for esterification.
  • the present invention provides a method for the interconversion of C(4)-epimers of a compound of formula (V) or (Vl) in which L represents CHO or CO 2 Y wherein Y represents hydrogen or alkyl, A" is as defined above for formula (III) and E, and J are as defined above for formula (Ia).
  • L represents CHO or CO 2 Y wherein Y represents hydrogen or alkyl
  • A" is as defined above for formula (III) and E
  • J are as defined above for formula (Ia).
  • the rel- (2R, 4S, 5R)-diastereoisomer of a compound of formula (V) and/or (Vl) may be converted into the rel-(2R, 4R, 5R)-diastereoisomer where appropriate.
  • Such epimerisation of these rel-(4S, 5R)-diastereoisomers into the corresponding rel-(4R, 5R)-diastereoisomers may be accomplished by treatment of a compound of formula (V) and/or (Vl) with a suitable base, in the presence of a suitable solvent.
  • a suitable base such as sodium methoxide
  • a suitable solvent such as methanol.
  • racemic compounds of Formula (Ia), (II), (III), (IV), (V), (Vl) and/or (IX) 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 (Ia), (II), (III), (IV), (V), (Vl) and/or (IX) may be resolved by chiral preparative HPLC.
  • racemic compounds of Formula (Ia), (II), (III), (IV), (V), (Vl) and/or (IX) which contain an appropriate acidic or basic group, such as a carboxylic acid group or amine group may be resolved by standard diastereoisomeric salt formation with a chiral base or acid reagent respectively as appropriate. Such techniques are well established in the art.
  • a racemic compound of Formula (Vl) where L is CO 2 Me may be resolved by treatment with a chiral acid such as (R)-(-)-1 , 1 '- binaphthyl-2,2'-diyl-hydrogen phosphate, in a suitable solvent, for example dichloromethane, isopropanol or acetonitrile.
  • a suitable solvent for example dichloromethane, isopropanol or acetonitrile.
  • the enantiomer of Formula (Vl) may then be obtained by treating the salt with a suitable base, for example triethylamine, in a suitable solvent, for example methyl tert-butyl ether.
  • individual enantiomeric compounds of Formula (III), (Vl) and/or (IX) may be prepared by general methods of asymmetric synthesis using, where appropriate, chiral auxiliaries or chiral catalytic reagents and additionally performing any suitable functional group interconversion step as hereinbefore described, including the addition or removal of any such chiral auxiliary.
  • Such general methods of asymmetric synthesis are well known in the art and include, but are not restricted to, those described in "Asymmetric Synthesis,” Academic Press, 1984 and/or “Chiral Auxiliaries and Ligands in Asymmetric Synthesis", Wiley, 1995.
  • suitable general chiral auxiliaries include chiral alcohols such as menthol or 1-phenylethanol; chiral oxazolidinones such as 4-benzyloxazolidin-2-one or 4-isopropyloxazolidin-2-one; chiral sultams such as camphor sultam; or chiral amines such as 1-phenylethylamine or 2-amino-2-phenylethanol.
  • Suitable general chiral catalytic reagents include chiral basic amines and chiral ligands such as N-methylephedrine, 1-phenyl-2-(1-pyrrolidinyl)-1-propanol, 3-(dimethylamino)- 1 ,7,7-trimethylbicyclo[2.2.1]-heptan-2-ol, 3,4-bis(diphenylphosphanyl)-1-(phenylmethyl)- pyrrolidine, chinchonine, chinchonidine, sparteine, hydroquinine or quinine, BINAP or chiral bis(oxazoline) (BOX) ligands and derivatives, optionally in the presence of a metal salt, for example M m X x where M is silver, cobalt, zinc, titanium, magnesium, or manganese, and X is halide (for example chloride or bromide), acetate, trifluoroacetate, p- toluene
  • L 1 represents CO 2 Y or CO 2 Y 1 wherein Y represents hydrogen or alkyl, Y 1 represents a chiral auxiliary, and A", E, and J are as defined above for Formula (Vl), and * denotes an enantioenriched chiral centre can be prepared by reaction of a compound of Formula (VII), as hereinbefore defined, with a compound of Formula (Villa)
  • L 1 represents a chiral ester group CO 2 Y 1 wherein Y 1 represents a chiral auxiliary and thereafter optionally carrying out any conversion of CO 2 Y 1 into CO 2 Y by standard methods for removal of chiral auxiliaries.
  • chiral ester CO 2 Y 1 may be derived from a chiral alcohol Y 1 OH, for example menthol, by standard esterification techniques.
  • reaction of a compound of Formula (VII) with a compound of Formula (Villa) is carried out in a suitable solvent, for example THF or acetonitrile, optionally in the presence of a Lewis acid catalyst, such as lithium bromide or silver acetate, and a base, such as triethylamine, 1 ,8-diazabicyclo[5,4,0]undec-7-ene (DBU) or tetramethyl guanidine.
  • a suitable solvent for example THF or acetonitrile
  • a Lewis acid catalyst such as lithium bromide or silver acetate
  • a base such as triethylamine, 1 ,8-diazabicyclo[5,4,0]undec-7-ene (DBU) or tetramethyl guanidine.
  • reaction is carried out in a suitable solvent, for example THF or acetonitrile, in the presence of an acid, such as acetic acid, or the reaction may be carried out by heating compounds of Formula (VII) and (Villa) in a suitable solvent, for example toluene, xylene or acetonitrile in the absence of a catalyst.
  • a suitable solvent for example toluene, xylene or acetonitrile
  • the reaction is carried out in the presence of a suitable chiral catalytic reagent, for example (-)-N-methylephedrine, and a suitable metal salt, for example manganese (II) bromide, in a suitable solvent, for example acetonitrile.
  • a suitable chiral catalytic reagent for example (-)-N-methylephedrine
  • a suitable metal salt for example manganese (II) bromide
  • a suitable solvent for example acetonitrile.
  • the reaction is carried out at a temperature in the range -30 0 C to room temperature, suitably at -20 0 C.
  • the reaction is carried out in the presence of a suitable chiral catalytic reagent, for example S-BINAP, and a suitable metal salt, for example silver acetate, in the presence of a suitable base, for example diisopropylethylamine, in a suitable solvent, for example acetonitrile optionally co-solvated with toluene.
  • a suitable chiral catalytic reagent for example S-BINAP
  • a suitable metal salt for example silver acetate
  • a suitable base for example diisopropylethylamine
  • a suitable solvent for example acetonitrile optionally co-solvated with toluene.
  • the reaction is carried out at a temperature in the range -15 0 C to room temperature, suitably at -5°C.
  • the major chiral diastereoisomer of a compound of Formula (Via) or Formula (VIb) arising from such an asymmetric reaction may be further enantioenriched by conventional purification techniques well known in the art, for example by chromatography, or by fractional crystallisation.
  • a favourable crystallisation method is the fractional crystallisation of a salt of the major chiral diastereoisomer, for example the hydrochloride salt or the (R)-(-)-1 , 1 '-binaphthyl-2,2'-diyl-hydrogen phosphate salt.
  • the hydrochloride salt of a compound of Formula (Via) or Formula (VIb) may be prepared by treating a compound of Formula (Via) or Formula (VIb) with anhydrous hydrogen chloride in a suitable solvent, for example diethyl ether. Preferably the reaction is carried out at a temperature in the range -10 to 10 0 C.
  • a suitable solvent for example diethyl ether.
  • the reaction is carried out at a temperature in the range -10 to 10 0 C.
  • the (R)-(-)-1 ,1'-binaphthyl-2,2'-diyl-hydrogen phosphate salt of a compound of Formula (Via) or Formula (VIb) may be prepared as herein before described for the resolution of a racemic compound of Formula (Vl).
  • a chiral auxiliary from a group in which L 1 represents CO 2 Y 1 to afford a group in which L 1 represents CO 2 Y is readily accomplished by standard methods, for example treatment with a hydrolytic reagent such as sodium hydroxide or an alkoxide such as sodium methoxide as appropriate, in a suitable solvent such as methanol.
  • a hydrolytic reagent such as sodium hydroxide or an alkoxide such as sodium methoxide as appropriate
  • a chiral compound of Formula (Via) or Formula (VIb) may be converted into a chiral compound of Formula (IX) in which G' represents hydroxyalkyl, and A", E, and J are as defined above for Formula (III) by treatment with suitable reagents for accomplishing the functional group interconversion of the group L or L 1 into group G'.
  • a compound of Formula (Via) in which L 1 represents CO 2 Y 1 and Y 1 is as defined above may be treated with a suitable reducing agent, for example lithium aluminium hydride, in a suitable solvent, for example tetrahydrofuran.
  • a chiral compound of Formula (Via) or Formula (VIb) may be converted into a chiral compound of Formula (IV) in which G' represents hydroxyalkyl, by first acylating the pyrrolidine nitrogen atom as described above for the transformation of a compound of Formula (Vl) into a compound of Formula (V) and then subsequently by treatment with suitable reagents for accomplishing the functional group interconversion of the group L or L 1 into group G' as described above for the transformation of a compound of Formula (Via) or Formula (VIb) into a chiral compound of Formula (IX).
  • chiral compounds of Formula (Ia), (II), (IV) and/or (V) may be prepared from chiral compounds of Formula (III), (Vl) and (IX).
  • the mixture so obtained was purified by chromatography on silica gel using cyclohexane-ethyl acetate as eluent (gradient elution from 8:1 v/v via 4:1 v/v to 1 :1 v/v) to give the title compound as an oil.
  • Methyltriphenylphosphonium bromide (1.35 g) in dry THF (25 ml.) was stirred at room temperature under nitrogen. A 1.0 M solution of potassium terf-butoxide in THF (3.8 ml.) was added and the resulting solution was stirred for 30 minutes at room temperature. To this was added 3-formyl-4-tert-butylbenzoic acid, tert-butyl ester (Intermediate 2; 0.50 g) and the mixture was stirred for a further 2 hours. The reaction mixture was quenched with water and extracted with diethyl ether. The combined extracts were dried (MgSO 4 ), filtered and concentrated in vacuo.
  • Part C The 4-(chloromethyl)-1 ,3-thiazole (formed in Part B) was dissolved in THF (100 mL) and added dropwise (dropping funnel) over 30 minutes to the reaction mixture from Part A, keeping the reaction at ice-bath temperature. Solid anhydrous lithium iodide (1 g, 7.5 mmol) was added directly to the reaction mixture 5 minutes after addition of the alkylating agent had started. The dropping funnel was rinsed with further dry THF (50 mL) which was added to the reaction.
  • the reaction was stirred at ice-bath temperature for 45 minutes, allowed to warm to room temperature over 30 minutes and was stirred at room temperature for an additional 2.5 hours before being partitioned between a mixture of saturated brine (400 ml_), water (200 mL) and ethyl acetate (800 mL). The organic layer was separated and the aqueous layer re-extracted with further ethyl acetate (2 x 300 mL). The combined organic layers were dried over sodium sulphate and evaporated to give the title compound (57.8 g, crude) which was used without further purification.
  • saturated brine 400 ml_
  • water 200 mL
  • ethyl acetate 800 mL
  • the organic layer was separated and the aqueous layer re-extracted with further ethyl acetate (2 x 300 mL).
  • the combined organic layers were dried over sodium sulphate and evaporated to give the title compound (57.8 g, crude) which was used without
  • reaction mixture was stirred for 30 minutes and was then cooled to 22°C before (1 R, 2S)-(-)-N-methylephedrine (17.6 g) was gradually added to the stirred reaction mixture.
  • the reaction mixture was stirred for 10 minutes at 22°C and was then cooled to -20°C.
  • Methyl acrylate (9.2 mL) was added and stirring continued for 2 hours at a temperature between -23 0 C and -2O 0 C.
  • the reaction mixture was quenched at -20 0 C by the addition of saturated ammonium chloride solution, and then extracted with ethyl acetate.
  • compositions for use in therapy comprising a compound of formula (Ia) or a pharmaceutically acceptable salt, solvate or ester thereof in admixture with one or more pharmaceutically 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 pharmaceutically 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 (Ia) 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 (Ia).
  • a topical formulation contains suitably 0.01 to 5.0% of a compound of Formula (Ia).
  • 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.
  • compositions comprising a compound of Formula (Ia) and/or a pharmaceutically acceptable salt, solvate or ester thereof 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 non-CFC propellant such as 1 ,1 ,1 ,2-tetrafluoroethane or 1 ,1 ,1 ,2,3,3,3- heptafluoropropane.
  • a conventional non-CFC propellant such as 1 ,1 ,1 ,2-tetrafluoroethane or 1 ,1 ,1 ,2,3,3,3- heptafluoropropane.
  • a typical suppository formulation comprises a compound of Formula (Ia) 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.
  • genotype 1a and genotype 1b may be demonstrated, for example, using the following in vitro assays:
  • HCV RNA Polymerase [Recombinant NS5B with C-terminal 21 amino acid deletion and C- terminal 6His-tag (Ferrari et al. J. Virol. 73(2), 1999, 1649. 'Characterization of soluble hepatitis C virus RNA-dependent RNA polymerase expressed in Escherichia coli. 1 ) expressed in E. coli and purified to homogeneity] was added to 25 nM final concentration. Polymerase of genotype 1a was from strain H77 (Yanagi, M., Purcell, R. H., Emerson, S. U. & Bukh, J. (1997), Proceedings of the National Academy of Sciences, USA 94, 8738- 8743) containing a sequence change from valine to isoleucine at position 180.
  • Reaction Conditions were 25 nM enzyme, 1.5 ⁇ g/ml oligo-rG13/poly-rC and 0.2 ⁇ Ci ⁇ - 33 P- GTP in 0.5 ⁇ M GTP (20 Ci/mMol) , 20 mM Tris pH 7.5, 23 rtiM NaCI, 3 mM DTT, 5 mM MgCI 2 , 1 mM MnCI 2 .
  • Enzyme was diluted to 500 nM concentration in 20 mM Tris-HCI, pH 7.5, 25 r ⁇ M NaCI and 3 mM DTT.
  • 4x concentrated assay buffer mix was prepared using 1M Tris-HCI, pH7.5 (1 mL), 5M NaCI (0.25 mL), 1M DTT (0.12 mL) and Water (8.63 mL), Total 10 mL.
  • 2x concentrated first reagent was prepared using 4x concentrated assay buffer mix (5 ⁇ L), 40 u/ ⁇ L RNasin (0.1 ⁇ L), 20 ⁇ g/mL polyrC/biotinylated-oligorG (1.6 ⁇ L), 500 nM enzyme (1 ⁇ L ) and Water (2.3 ⁇ L), Total 10 ⁇ L/well.
  • 2x concentrated second reagent was prepared using 1 M MgCI 2 (0.1 ⁇ L), 1 M MnCI 2 (0.02 ⁇ L), 25 ⁇ M GTP (0.4 ⁇ L), Q-[ 33 P]- GTP (10 ⁇ Ci/ ⁇ L, 0.02 ⁇ L) and water (9.5 ⁇ L), Total 10 ⁇ L/well.
  • the assay was set up using compound (1 ⁇ L in 100% DMSO), first reagent (10 ⁇ L), and second reagent (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 1h at 22°C. After this time, the reaction was stopped by addition of 60 ⁇ L 1.5 mg/ml streptavidin SPA beads (Amersham) in 0.1 M EDTA in PBS.
  • the beads were incubated with the reaction mixture for 1h at 22°C after which 100 ⁇ 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.
  • Genotype 1b Full-Length Enzyme Reaction Conditions were 0.5 ⁇ M [ 33 P]-GTP (20 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 4 nM final concentration.
  • 5x concentrated assay buffer mix was prepared using 1M MnCI 2 (0.25 ml_), glycerol (2.5ml_), 10% NP-40 (0.025 mL) and Water (7.225 ml_), Total 10 mL
  • 2x concentrated enzyme buffer contained 1 M-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 1O 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), 40 u/ ⁇ L RNasin (0.1 ⁇ L), 20 ⁇ g/mL polyrC/biotinylated-oligorG (1.6 ⁇ L), and Water (3.94 ⁇ L), Total 10 ⁇ L.
  • Enzyme Mix was prepared by adding 1mg/ml full-length NS5B polymerase (1.5 ⁇ L) to 2.81 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 1h 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 0 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.
  • genotype 1a and genotype 1b may be demonstrated, for example, using the following cell based assays:
  • Huh-7 HCV replicon cell monolayers nearing confluency were stripped from growth flasks with versene-trypsin solution and the cells were resuspended in assay medium at either 2 x 10 5 cells/mL (sub-line 5-15; genotype 1b; Lohmann, V., Korner, F., Koch, J-O., Herian, U., Thielmann, L. And Bartenschlager, R., 1999, Science, 285, pp 110-113) or at 3 x 10 5 cells/mL (genotype 1a; Gu, B., Gates, AT., Isken, O., Behrens, S.E.and Sarisky, R.T., J.
  • the diluent was removed and each well then received 50 ⁇ L of a 1/200 dilution of anti-HCV specific, murine, monoclonal antibody (either Virostat #1872 or #1877), except for wells in one of the compound-free control rows which received diluent alone to act as negative controls.
  • the plates were incubated at 37°C for 2 hours and washed 3 times with PBS/0.05% Tween 20, then 50 ⁇ L of horseradish peroxidase conjugated, anti-mouse, rabbit polyclonal serum (Dako #P0260), diluted 1/1000, were added to all wells.
  • the plates were incubated for a further hour, the antibody removed and the cell sheets washed 5 times with PBS/Tween and blotted dry.
  • the assay was developed by the addition of 50 ⁇ L of ortho-phenylenediamine/peroxidase substrate in urea/citrate buffer (SigmaFast, Sigma #P-9187) to each well, and colour allowed to develop for up to 15 minutes.
  • the reaction was stopped by the addition of 25 ⁇ L per well of 2 M sulphuric acid and the plates were read at 490 nm on a Fluostar Optima spectrophotometer.
  • the substrate solution was removed and the plates were washed in tap water, blotted dry and the cells stained with 5 % carbol fuchsin in water for 30 minutes. The stain was discarded and the cell sheets washed, dried and examined microscopically to assess cytotoxicity. Data analysis
  • the absorbance values from all compound-free wells that had received both primary and secondary antibodies were averaged to obtain a positive control value.
  • the mean absorbance value from the compound-free wells that had not received the primary antibody was used to provide the negative (background) control value.
  • the readings from the duplicate wells at each compound concentration were averaged and, after the subtraction of the mean background from all values, were expressed as a percentage of the positive control signal.
  • the quantifiable and specific reduction of expressed protein detected by the ELISA in the presence of a drug can be used as a measure of replicon inhibition.
  • GraFit software (Erithacus Software Ltd.) was used to plot the curve of percentage inhibition against compound concentration and derive the 50% inhibitory concentration (IC 50 ) for the compound.
  • Genotype 1a GenotvDe 1 b enzyme * ⁇ 0.75 ⁇ M # ⁇ 0.20 ⁇ M
  • Compound A corresponds to the racemic compound disclosed as Example 11 in
  • Compound D corresponds to the racemic compound disclosed as Example 24 in
  • Compound F corresponds to the racemic compound disclosed as Example 33 in
  • WO2004/037818 re/-(2R,4S,5R)-2-benzyl-1-(3-methoxy-4-tert-butylbenzoyl)-4- methoxymethyl-5-(1 ,3-thiazol-2-yl)-pyrrolidine-2-carboxylic acid.
  • Compound G corresponds to the racemic compound disclosed as Example 40 in
  • the compounds of the present invention which have been tested demonstrate a surprisingly superior genotype- 1 a/1 b profile, as shown by the IC 50 values in the enzyme and cell-based assays across both of the 1a and 1b genotypes of HCV, compared to Compounds A - G. Accordingly, the compounds of the present invention are of great potential therapeutic benefit in the treatment and prophylaxis of HCV.
  • 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 at least one chemical entity chosen from compounds of formula (Ia) and pharmaceutically acceptable salts, solvates or esters thereof, together with at least one other therapeutically active agent.
  • compositions comprising a combination as defined above together with at least one pharmaceutically acceptable diluent or 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.

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Abstract

L'invention porte sur des agents antiviraux de formule (Ia) dans laquelle A représente hydroxyle; D représente 4-tert-butyl-3-éthénylphényle ou 4-tert-butyl-5-ethenyl-2-fluorophényle; E représente 1,3-thiazol-2-yl; G représente méthoxyméthyle; J représente 1,3-thiazol-4-ylméthyle ou 1H-pyrazol-1-ylméthyle; et sur des sels, solvates et esters desdits composés, à condition que, lorsque A est estérifié pour former un groupe OR, R étant choisi dans un groupe alkyle à chaîne ramifiée, alors R est différent de tert-butyl; l'invention concerne également des procédés de préparation et d'utilisation desdits composés dans le traitement du VHC.
PCT/EP2006/009234 2005-09-23 2006-09-21 Composes de heteroarylmethyl-c (4) -methoxymethyl acyl pyrrolidine et leur utilisation dans le traitement d'infections virales, en particulier du virus de l'hepatite c WO2007039142A1 (fr)

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