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WO2008043791A2 - Thiophene derivatives for treating hepatitis c - Google Patents

Thiophene derivatives for treating hepatitis c Download PDF

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Publication number
WO2008043791A2
WO2008043791A2 PCT/EP2007/060779 EP2007060779W WO2008043791A2 WO 2008043791 A2 WO2008043791 A2 WO 2008043791A2 EP 2007060779 W EP2007060779 W EP 2007060779W WO 2008043791 A2 WO2008043791 A2 WO 2008043791A2
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WO
WIPO (PCT)
Prior art keywords
compound
formula
amino
pyrimidin
carbonyl
Prior art date
Application number
PCT/EP2007/060779
Other languages
French (fr)
Other versions
WO2008043791A3 (en
Inventor
Charles David Hartley
Jacqueline Elizabeth Mordaunt
Pritom Shah
Martin John Slater
Gemma Victoria White
Original Assignee
Smithkline Beecham Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GB0620399A external-priority patent/GB0620399D0/en
Priority claimed from GB0709629A external-priority patent/GB0709629D0/en
Application filed by Smithkline Beecham Corporation filed Critical Smithkline Beecham Corporation
Publication of WO2008043791A2 publication Critical patent/WO2008043791A2/en
Publication of WO2008043791A3 publication Critical patent/WO2008043791A3/en

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Classifications

    • 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
    • C07D409/12—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 linked by a chain containing hetero atoms as chain links
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
    • C07D491/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D513/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00
    • C07D513/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00 in which the condensed system contains two hetero rings
    • C07D513/04—Ortho-condensed systems

Definitions

  • the present invention relates to novel 2-carboxy thiophene derivatives useful as anti-viral agents. Specifically, the present invention involves novel inhibitors of Hepatitis C Virus (HCV) replication.
  • 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 post-transfusion non A, non-B hepatitis
  • NANBH non-B hepatitis
  • this virus was assigned as a new genus in the Flaviviridae family.
  • 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' 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 1 b isolates) and inter-typically (-85% aa identity between genotype 1 a 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): 2046-2051 ).
  • inhibition of NS5B RdRp activity is predicted to be useful to treat 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 1 a is most abundant in the United States, the majority of sequences in Europe and Japan are from genotype 1 b. Table 1
  • X is chosen from -N(R 3 )M(R 2 ) or -JN(R 2 )(R 3 );
  • M is chosen from -SO 2 -, -SO-, -S-, -C(O)-, -C(S)-, -CH 2 C(O)N(R 4 )-, -CH 2 C(S)N(R 15 )-, -
  • R 4 is C 1-6 alkyl
  • R 8 is chosen from H, Ci_i 2 alkyl, C 2- i 2 alkenyl, C 2- i 2 alkynyl, C 6 -i 4 aryl, C 3 -i 2 heterocycle, C 3- i 2 heteroaralkyl, C 6- i 6 aralkyl;
  • R 15 is chosen from H or C 1-6 alkyl
  • J is chosen from -C(W)-, -C(R 6 )-, -S-, -S(O)-, or -SO 2 -;
  • W is chosen from O, S or NR 7 ;
  • R 7 is chosen from H, C 1-12 alkyl, C 2-12 alkenyl, C 2-12 alkynyl, C 6 -i 4 aryl, C 3-12 heterocycle, C 3- i 2 heteroaralkyl, C 6- i 6 aralkyl;
  • R 6 is chosen from H, Ci_i 2 alkyl, C 6 -i 4 aryl, or C 6- i 6 aralkyl;
  • Y 1 is chosen from a bond, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl;
  • Y is chosen from COOR 16 , COCOOR 5 , P(0)0ROR b , S(O)OR 5 , S(O) 2 OR 5 , tetrazole,
  • R , R ,R 5 10 and R are each independently chosen from H, C 1-12 alkyl, C 2-12 alkenyl, C 2-
  • R a and R b are each independently chosen from H, C 1-12 alkyl, C 2-12 alkenyl, C 2-12 alkynyl, C 6- i 4 aryl, C 3- i 2 heterocycle, C 3- i 8 heteroaralkyl, C 6 -i 8 aralkyl; or R a and R b are taken together with the oxygens to form a 5 to 10 membered heterocycle;
  • R 16 is chosen from H, C 1-12 alkyl, C 2-12 alkenyl, C 2-12 alkynyl, C 6- i 4 aryl, C 3-12 heterocycle, C 3- -isheteroaralkyl, C ⁇ -isaralkyl; provided that R 16 is other than methyl or ethyl;
  • R 1 is chosen from Ci.-i 2 alkyl, C 2 -i 2 alkenyl, C 2 -i 2 alkynyl, C 6- i 4 aryl, C 3- i 2 heterocycle, C 3- 18 heteroaralkyl, C 6- i 8 aralkyl;
  • R 2 is chosen from Ci.-i 2 alkyl, C 2- i 2 alkynyl, C 6 -i 4 aryl, C 3- i 2 heterocycle, C 3- i 8 heteroaralkyl, C 6- i 8 aralkyl;
  • R 3 is chosen from H, C 1-12 alkyl, C 2- i 2 alkenyl, C 2- i 2 alkynyl, C 6 -i 4 aryl, C 3-12 heterocycle, C 3- i ⁇ heteroaralkyl, C 6- i 8 aralkyl;
  • Z is chosen from H, halogen, or Ci -6 alkyl.
  • PCT publication number WO2003/062215 generically discloses certain compounds, including certain 2-carboxy thiophene compounds, having kinase inhibiting activity.
  • the compounds disclosed have the fo
  • V is, inter alia, a group of formula (i)
  • R 1 represents T, N-(CO)R 6 R 7 , N(R 6 )COR 7 , or NC(O)OR 6 R 7 ;
  • T represents H, (hetero)alkyl, alkenyl, cycloalkyl, (hetero)aryl, arylalkyl or alkylaryl;
  • R 8 represents T, NR 3 R 4 , N-C(O)R 3 R 4 , N(R 3 )COR 4 , OR 3 , COR 3 , CO 2 R 3 or OCOR 3 ;
  • R 2 , R 5 represent T, alkylaryl, carboxyl, halo or CF 3 ;
  • R 3 , R 4 , R 6 , R 7 represent T, alkylaryl, COOR 5 or COR 5 ;
  • X, Y, Z represent CH or N
  • U represents S or NH
  • W represents NH, O or S.
  • PCT publication number WO2004/1 10357 generically discloses a range of compounds, including certain 2-carboxy thiophene compounds, having phosphodiesterase 6 delta (PDE6D) modulating activity.
  • the compounds disclosed have the formula (I)
  • R 1 , R 2 , R 3 , and R 4 are independently selected from the group consisting of hydrogen, halogen, alkyl, cycloalkyl, aryl, heteroaryl, NR 5 C(O)R 7 , C(O)NR 5 R 6 , C(O)R 7 and C(O)OR 7 , wherein R 5 , R 6 and R 7 are independently selected from hydrogen, lower alkyl, cycloalkyl or aryl, and where R 5 , R 6 , and R 7 together can optionally form a 3, 4, 5, 6 or 7 membered ring optionally having one or more degrees of substitution.
  • the present invention involves novel 2-carboxy thiophene compounds represented hereinbelow, pharmaceutical compositions comprising such compounds and use of the compounds in treating viral infection, especially HCV infection.
  • the present invention provides a compound of Formula (I) :
  • A represents hydroxy
  • R 1 represents -R x -R ⁇ ;
  • R x represents phenyl (optionally substituted by halo, methyl, ethyl, methoxy or trifluoromethyl) or 5- or 6-membered heteroaryl bonded through a ring carbon atom to the carbon atom of the thiophene;
  • R ⁇ represents 8-, 9- or 10-membered heteroaryl, bonded such that when R x is phenyl or 6- membered heteroaryl, R ⁇ is in the para-position;
  • R 2 represents phenyl which is at least 2,4 disubstituted, the substituents being independently selected from unsubstituted C 1-2 alkyl, C 1-2 alkyl (substituted with one or more fluoro atoms), chloro, fluoro and bromo;
  • R 3 represents linear or branched -C 1-6 alkyl, or -C 3-6 cycloalkyl (each optionally substituted by one or more substituents selected from 5- or 6-membered heteroaryl, 5- or 6-membered heterocyclyl and -Cs-ecycloalkyl), linear or branched -C 2- 6alkyl (optionally substituted by one or more substituents selected from methoxy, ethoxy and fluoro), pyranyl, -CH 2 -pyranyl, tetrahydrofuranyl or -CH 2 -tetrahydrofuranyl (each of which may be optionally substituted by one or more substituents selected from -Ci -2 alkyl, fluoro and methoxy);
  • acetyl refers to -C(O)CH 3 .
  • alkyl refers to an optionally substituted hydrocarbon group.
  • the alkyl hydrocarbon group may be linear, branched or cyclic, saturated or unsaturated. Where the alkyl group is linear or branched, examples of such groups include methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl or hexyl and the like.
  • alkyl hydrocarbon group is unsaturated, it will be understood that there will be a minimum of 2 carbon atoms in the group, for example an alkenyl or alkynyl group.
  • alkyl hydrocarbon group is cyclic, it will be understood that there will be a minimum of 3 carbon atoms in the group.
  • a cyclic group may be substituted with a linear or branched alkyl group. In one aspect, alkyl moieties are saturated.
  • alkenyl refers to a linear or branched hydrocarbon group containing one or more carbon-carbon double bonds. In one aspect the alkenyl group has from 2 to 6 carbon atoms. Examples of such groups include ethenyl, propenyl, butenyl, pentenyl or hexenyl and the like.
  • alkynyl refers to a linear or branched hydrocarbon group containing one or more carbon-carbon triple bonds. In one aspect the alkynyl group has from 2 to 6 carbon atoms. Examples of such groups include ethynyl, propynyl, butynyl, pentynyl or hexynyl and the like.
  • cycloalkyl refers to an optionally substituted, cyclic hydrocarbon group.
  • the hydrocarbon group may be saturated or unsaturated, monocyclic or bridged bicyclic.
  • examples of such groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl and the like.
  • examples of such groups include cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl or cyclooctenyl and the like.
  • the cycloalkyl group has from 5 to 7 carbon atoms.
  • cycloalkyl moieties are cyclohexenyl, cyclopentenyl and cyclohexyl. Unless otherwise stated, the cycloalkyl group may be substituted by one or more optional substituents including -C h alky!
  • alkoxy refers to an -O-alkyl group wherein alkyl is as defined herein. Examples of such groups include methoxy, ethoxy, propoxy, butoxy, pentoxy or hexoxy and the like.
  • 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.
  • aryl moieties contain 6-10 carbon atoms.
  • aryl moieties are unsubstituted, monosubstituted, disubstituted or trisubstituted phenyl.
  • aryl substituents are selected from the group consisting of -Ci -6 alkyl, halo, -OR E , -SR E , -C(O)NR B R C , -C(O)R 0 , -CO 2 H, -CO 2 R 0 , -NR B R C , -NR A C(0)R°, -NR A C0 2 R°, -NR A SO 2 R°, -NR A C(0)NR F R G , -SO 2 NR F R G , -SO 2 R 0 , nitro, cyano, heterocyclyl, - CF 3 , -OCF 3 and phenyl.
  • carbonyl refers to -C(O)-.
  • cyano refers to -CN.
  • halogen or halo refer to a fluorine, chlorine, bromine or iodine atom. References to "fluoro”, “chloro”, “bromo” or “iodo” should be construed accordingly.
  • heteroaryl refers to an optionally substituted, 5, 6, 8, 9 or 10 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.
  • heteroaryl moieties are unsubstituted, monosubstituted, disubstituted or trisubstituted (where applicable) pyridine, pyrazine, thiazole, thiophene, oxadiazole, oxazole, pyrimidine, pyridazine, benzodioxole, benzofuran, benzodioxin, indole, benzimidazole, benzofuran, indole, indazole, isoindole, benzothiophene, benzothiazole, benzoxazole, benzisoxazole, benzisothiazole, benzotriazole, furopyridine, furopyrimidine, furopyridazine, furopyrazine, furotriazine, pyrrolopyridine, pyrrolopyrimidine, pyrrolopyridazine, pyrrolopyra
  • heteroaryl substituents are selected from the group consisting of -Ci -6 alkyl, halo, -OR E , -SR E , -C(O)NR B R C , -C(O)R 0 , -CO 2 R 0 , -NR B R C , -NR A C(O)R°, -NR A CO 2 R D , -NR A SO 2 R D , -NR A C(O)NR F R G , -SO 2 NR F R G , -SO 2 R 0 , oxo, nitro, cyano, heterocyclyl, -CF 3 and phenyl.
  • heterocyclic and “heterocyclyl” refer to an optionally substituted, 5 or 6 membered, saturated or partially saturated, cyclic group containing 1 or 2 heteroatoms selected from N, optionally substituted by hydrogen, -Ci -6 alkyl, -C(O)R 0 , -C(O)NR B R C , -C(O)OH, -SO 2 R 0 , aryl or heteroaryl; O; and S, optionally substituted by one or two oxygen atoms.
  • Ring carbon atoms may be optionally substituted by -C 1-6 alkyl, -0R A , -C(O)R 0 , or -SO 2 R 0 .
  • heterocyclic moieties are unsubstituted or monosubstituted tetrahydro-2H-pyran-4-yl, piperidinyl and tetrahydrofuran- 3-yl.
  • nitro refers to -NO 2 .
  • Et refers to "ethyl
  • IPr refers to “isopropyl”
  • Me refers to “methyl”
  • OBn refers to "benzyloxy”
  • Ph refers to "phenyl”.
  • R A represents hydrogen or -C 1-6 alkyl.
  • R B and R c independently represent hydrogen, -Ci -6 alkyl, aryl, heterocyclyl or heteroaryl; or R B and R c together with the nitrogen atom to which they are attached form a 5 or 6 membered saturated cyclic group.
  • R° is selected from the group consisting of -C 1-6 alkyl, aryl, heterocyclyl, heteroaryl, arylalkyl, and heteroarylalkyl.
  • R E represents hydrogen, -C 1-6 alkyl, arylalkyl, heteroarylalkyl, aryl, heterocyclyl or heteroaryl.
  • R F and R G are independently selected from the group consisting of hydrogen, -Ci -6 alkyl, aryl, heteroaryl, arylalkyl, and heteroarylalkyl; or R F and R G together with the nitrogen atom to which they are attached form a 5 or 6 membered saturated cyclic group.
  • substituted refers to substitution with the named substituent or substituents, multiple degrees of substitution being allowed unless otherwise stated.
  • R x represents phenyl optionally substituted by halo, methyl, methoxy or trifluoromethyl. In a further aspect, R x represents unsubstituted phenyl.
  • R ⁇ represents 8-, 9- or 10-membered bicyclic heteroaryl group. In a further aspect, R ⁇ represents 8- or 9-membered bicyclic heteroaryl group. In a further aspect, R ⁇ represents furo[3,2-b]pyridin-2-yl, pyrazolo[1 ,5-a]pyrimidin-2-yl, imidazo[1 ,2-a]pyridin-2-yl, imidazo[2,1-b][1 ,3]thiazol-6-yl, 7-amino-5-methylpyrazolo[1 ,5-a]pyrimidin-2-yl, 5- methylpyrazolo-[1 ,5-a]pyrimidin-2-yl, 7-aminopyrazolo[1 ,5-a]pyrimidin-2-yl, [1 ,3]oxazolo[4,5- b]pyridin-2-yl, furo[2,3-b]pyridin-5-yl, fur
  • R ⁇ represents furo[3,2-b]pyridin-2-yl, pyrazolo[1 ,5-a]pyrimidin-2-yl, imidazo[1 ,2-a]pyridin-2-yl, imidazo[2,1-b][1 ,3]thiazol-6-yl or 7-aminopyrazolo[1 ,5-a]pyrimidin- 2-yl.
  • R x represents furanyl
  • the bonds to the R y and to the thiophene group are in a 2,4 relationship.
  • the bond to R y may be in the 2-position and the bond to the thiophene may be in the 4 position.
  • R x represents thienyl
  • the bonds to the R x and to the central thiophene group are in a 2,5 relationship.
  • R 2 represents 2,4-disubstituted phenyl wherein the substituents are independently selected from Ci -2 alkyl, trifluoromethyl, chloro, fluoro and bromo. In a further aspect, the substituents are independently selected from methyl, chloro, fluoro, bromo and trifluoromethyl. In a further aspect, the substituents are independently selected from methyl, chloro, fluoro and bromo.
  • R 2 represents 2,4-dichlorophenyl, 4-chloro-2- fluorophenyl, 4-bromo-2-methylphenyl, 2,4-dimethylphenyl, 2-fluoro-4-trifluoromethylphenyl or 2,4-bis(trifluoromethyl)phenyl.
  • R 2 represents 2,4-dichlorophenyl or 4- chloro-2-fluorophenyl.
  • R 3 represents linear or branched -Ci -6 alkyl (optionally substituted by one or more substituents independently selected from 5- or 6-membered heteroaryl, 5- or 6- membered heterocyclyl and -C 3-6 cycloalkyl), linear or branched -C 2-6 alkyl (optionally substituted by one or more substituents selected from methoxy, ethoxy and fluoro), or pyranyl, -CH 2 -pyranyl, tetrahydrofuranyl or -CH 2 -tetrahydrofuranyl (all of which may be optionally substituted by one or more substituents selected from -C 1-2 alkyl, fluoro and methoxy).
  • R 3 represents unsubstituted Ci -6 alkyl, C 2- 6alkyl (substituted by methoxy), unsubstituted pyranyl or unsubstituted tetrahydrofuranyl.
  • R 3 represents 1-methylethyl, 2-(methyloxy)ethyl, 1-methyl-2-(methyloxy)ethyl, tetrahydro-2H- pyran-4-yl or tetrahydro-3-furanyl.
  • R 3 represents unsubstituted linear or branched Ci -6 alkyl.
  • R 3 represents 1-methylethyl.
  • R x represents unsubstituted phenyl
  • R ⁇ represents furo[3,2-b]pyridin-2-yl, pyrazolo[1 ,5-a]pyrimidin-2-yl, imidazo[1 ,2-a]pyridin-2-yl, imidazo[2,1-b][1 ,3]thiazol-6-yl, 7-amino-5-methylpyrazolo[1 ,5- a]pyrimidin-2-yl, 5-methylpyrazolo-[1 ,5-a]pyrimidin-2-yl, 7-aminopyrazolo[1 ,5-a]pyrimidin-2-yl, [1 ,3]oxazolo[4,5-b]pyridin-2-yl, furo[2,3-b]pyridin-5-yl, 5-amino-1 ,3-benzoxazol-2-yl, [1 ,
  • R x represents unsubstituted phenyl
  • R ⁇ represents furo[3,2-b]pyridin-2-yl, pyrazolo[1 ,5-a]pyrimidin-2-yl, imidazo[1 ,2-a]pyridin-2-yl, imidazo[2,1-b][1 ,3]thiazol-6-yl or 7-aminopyrazolo[1 ,5-a]pyrimidin-
  • R 2 represents 2,4-dichlorophenyl, 4-chloro-2-fluorophenyl, 4-bromo-2-methylphenyl,
  • R 3 represents 1-methylethyl, 2-(methyloxy)ethyl, 1-methyl-2-(methyloxy)ethyl, tetrahydro-2H- pyran-4-yl or tetrahydro-3-furanyl; or salts, solvates and esters thereof.
  • the present invention provides a compound of Formula (Ia) : wherein:
  • A represents hydroxy
  • R 1 represents -R x -R ⁇ ;
  • R x represents phenyl (optionally substituted by halo, methyl, ethyl, methoxy or trifluoromethyl) or 5- or 6-membered heteroaryl bonded through a ring carbon atom to the carbon atom of the thiophene;
  • R ⁇ represents 8-, 9- or 10-membered heteroaryl, bonded such that when R x is phenyl or 6- membered heteroaryl, R ⁇ is in the para-position;
  • R 2 represents phenyl which is at least 2,4 disubstituted, the substituents being independently selected from unsubstituted C 1-2 alkyl, C 1-2 alkyl (substituted with one or more fluoro atoms), methoxy, chloro, fluoro and bromo;
  • R 3 represents linear or branched -C 1-6 alkyl, or -C 3-6 cycloalkyl (each optionally substituted by one or more substituents selected from 5- or 6-membered heteroaryl, 5- or 6-membered heterocyclyl and -Cs-ecycloalkyl), linear or branched -C 2- 6alkyl (optionally substituted by one or more substituents selected from methoxy, ethoxy and fluoro), pyranyl or tetrahydrofuranyl (each of which may be optionally substituted by one or more substituents selected from -Ci- 2 alkyl, fluoro and methoxy);
  • the present invention provides at least one chemical entity chosen from a compound of Formula (Ib) :
  • A represents hydroxy
  • R 1 represents -R x -R ⁇ ;
  • R x represents phenyl (optionally substituted by halo, methyl, ethyl, methoxy or trifluoromethyl) or 5- or 6-membered heteroaryl bonded through a ring carbon atom to the carbon atom of the thiophene;
  • R ⁇ represents a 8-, 9- or 10-membered heteroaryl, bonded such that when R x is phenyl or 6- membered heteroaryl, R ⁇ is in the para-position;
  • R 2 represents phenyl which is at least 2,4 disubstituted, the substituents being selected from -Ci -2 alkyl, -C 1-2 alkyl substituted with one or more fluoro atoms, methoxy, chloro, fluoro and bromo;
  • R 3 represents linear or branched -Ci -6 alkyl, or -C 3-6 cycloalkyl (each optionally substituted by one or more substituents selected from methoxy, ethoxy, or 5- or 6-membered heteroaryl or heterocyclyl), or -C 3-6 cycloalkyl, pyranyl or furanyl (each of which may be optionally substituted by one or more substituents selected from -Ci -2 alkyl, fluoro and methoxy);
  • At least one chemical entity means at least one chemical substance chosen from the group of compounds consisting of compounds of Formula I and pharmaceutically acceptable derivatives thereof.
  • compounds useful in the present invention may be chosen from compounds of Formula (I) selected from the group consisting of:
  • the compounds of the present invention may be in the form of their free base or pharmaceutically acceptable salts, pharmaceutically acceptable solvates or pharmaceutically acceptable esters thereof.
  • pharmaceutically acceptable salt complexes are also included in the present invention.
  • pharmaceutically acceptable salts refers to salts that retain the desired biological activity of the subject compound and exhibit minimal undesired toxicological effects.
  • pharmaceutically acceptable salts includes both pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts. These pharmaceutically acceptable salts may be prepared in situ during the final isolation and purification of the compound, or by separately reacting the purified compound in its free acid or free base form with a suitable base or acid, respectively.
  • the invention provides a pharmaceutically acceptable salt of a compound of Formula (I) and embodiments thereof.
  • compounds of Formula I may contain an acidic functional group and may therefore be capable of forming pharmaceutically acceptable base addition salts by treatment with a suitable base.
  • a pharmaceutically acceptable base addition salt may be formed by reaction of a compound of Formula I with a suitable strong base, optionally in a suitable solvent such as an organic solvent, to give the base addition salt which may be isolated for example by crystallisation and filtration.
  • Pharmaceutically acceptable base salts include ammonium salts (for example ammonium or tetraalkylammonium), metal salts, for example alkali-metal or alkaline-earth-metal salts (such as hydroxides, sodium, potassium, calcium or magnesium), organic amines (such as tris [also known as tromethamine or tris(hydroxymethyl)aminomethane], ethanolamine, triethanolamine, choline, isopropylamine, dicyclohexylamine or N-methyl-D-glucamine), cationic amino acids (such as arginine, lysine or histidine) or bases for insoluble salts (such as procaine or benzathine).
  • ammonium salts for example ammonium or tetraalkylammonium
  • metal salts for example alkali-metal or alkaline-earth-metal salts (such as hydroxides, sodium, potassium, calcium or magnesium), organic amines (such as tris [also
  • compounds according to Formula I may contain a basic functional group and may therefore be capable of forming pharmaceutically acceptable acid addition salts by treatment with a suitable acid.
  • a pharmaceutically acceptable acid addition salt may be formed by reaction of a compound of Formula I with a suitable strong inorganic acid (such as hydrobromic, hydrochloric, sulfuric, nitric, phosphoric or perchloric) or a suitable strong organic acid, for example, sulfonic acids [such as p-toluenesulfonic, benzenesulfonic, methanesulfonic, ethanesulfonic, 2-hydroxyethanesulfonic, naphthalenesulfonic (e.g.
  • 2- naphthalenesulfonic ], carboxylic acids (such as acetic, propionic, fumaric, maleic, benzoic, salicylic or succinic), anionic amino acids (such as glutamaic or aspartic), hydroxyl acids (such as citric, lactic, tartaric or glycolic), fatty acids (such as caproic, caprylic, decanoic, oleic or stearic) or acids for insoluble salts (such as pamoic or resinic [e.g. polystyrene sulfonate]), optionally in a suitable solvent such as an organic solvent, to give the salt which is usually isolated for example by crystallisation and filtration.
  • carboxylic acids such as acetic, propionic, fumaric, maleic, benzoic, salicylic or succinic
  • anionic amino acids such as glutamaic or aspartic
  • hydroxyl acids such as citric, lactic, tartaric or glycolic
  • fatty acids such as
  • a pharmaceutically acceptable acid addition salt of a compound of Formula I is a salt of a strong acid, for example a hydrobromide, hydrochloride, hydroiodide, sulfate, nitrate, perchlorate, phosphate p-toluenesulfonic, benzenesulfonic or methanesulfonic salt.
  • a strong acid for example a hydrobromide, hydrochloride, hydroiodide, sulfate, nitrate, perchlorate, phosphate p-toluenesulfonic, benzenesulfonic or methanesulfonic salt.
  • non-pharmaceutically acceptable salts for example oxalates
  • oxalates may be used, for example in the isolation of compounds of Formula (I), and are included within the scope of this invention.
  • the invention includes within its scope all possible stoichiometric and non-stoichiometric forms of the salts of the compounds of Formula (I).
  • Salts and solvates of compounds of Formula (I) which are suitable for use in medicine are those wherein the counterion or associated solvent is pharmaceutically acceptable.
  • salts and solvates having non-pharmaceutically acceptable counterions or associated solvents are within the scope of the present invention, for example, for use as intermediates in the preparation of other compounds of the invention and their pharmaceutically acceptable salts and solvates.
  • Suitable prodrugs for compounds of the invention include : esters, carbonate esters, hemi-esters, phosphate esters, nitro esters, sulfate esters, sulfoxides, amides, carbamates, azo-compounds, phosphamides, glycosides, ethers, acetals and ketals.
  • 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 1-4 alkoxy or amino); or for example -
  • 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 substitute
  • any alkyl moiety present in such esters suitably contains 1 to 18 carbon atoms, particularly 1 to 4 carbon atoms.
  • Any aryl moiety present in such esters suitably comprises a phenyl group.
  • the invention provides a pharmaceutically acceptable salt, solvate or prodrug of a compound of Formula (I).
  • the invention provides a compound of Formula (I) in the form of parent compound, a salt or a solvate.
  • crystalline forms of the compounds of Formula (I) may exist in one or more polymorphic form, which are included in the present invention.
  • the compounds of Formula (I) 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. Racemic compounds may either be separated using preparative HPLC and a column with a chiral stationary phase or resolved to yield individual enantiomers utilising methods known to those skilled in the art. In addition, chiral intermediate compounds may be resolved and used to prepare chiral compounds of the invention.
  • the compounds of Formula (I) may exist in one or more tautomeric forms. All tautomers and mixtures thereof are included in the scope of the present invention. For example, a claim to 2-hydroxyquinolinyl would also cover its tautomeric form, ⁇ -quinolinonyl.
  • Diastereoisomers of compounds of Formula (I) may be obtained according to methods well known in the literature, for example by preparative HPLC or by chromatographic purifications. Racemic compounds may either be separated using preparative HPLC and a column with a chiral stationary phase or resolved to yield individual enantiomers utilising methods known to those skilled in the art. In addition, chiral intermediate compounds may be resolved and used to prepare chiral compounds of the invention.
  • 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.
  • the compounds of the present invention exhibit improved potency against the replication of HCV (1a and 1 b genotypes), and therefore have the potential to achieve greater efficacy in man. High potency in both genotypes is considered to be advantageous.
  • a compound of Formula (I) or pharmaceutically acceptable salts, solvates or esters thereof for use in human or veterinary medical therapy, particularly in the treatment or prophylaxis of viral infection, particularly flavivirus infection, for example HCV infection.
  • 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 include 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 a compound of Formula (I) or pharmaceutically acceptable salts, solvates or esters thereof.
  • a compound of Formula (I) or pharmaceutically acceptable salts, solvates or esters thereof in the manufacture of a medicament for the treatment and/or prophylaxis of viral infection, particularly HCV infection.
  • the compounds of Formula (I) or salts, solvates or esters thereof may be prepared by the processes described hereinafter, said processes constituting a further aspect of the invention.
  • A is a protected hydroxy group, for example an alkoxy, benzyloxy or silyloxy group and R 1 , R 2 , and R 3 are as defined above for Formula (I).
  • R 1 , R 2 , and R 3 are as defined above for Formula (I)
  • an appropriate base for example aqueous sodium hydroxide or lithium hydroxide, optionally in a solvent such as methanol, ethanol, tetrahydrofuran or combinations thereof.
  • the temperature is in the range 20 to 100 0 C.
  • A is te/t-butoxy
  • R 1 , R 2 and R 3 are as defined above for Formula (I)
  • an appropriate acid for example trifluoroacetic acid.
  • the reaction is carried out in a solvent, for example dichloromethane.
  • the temperature is in the range 0 to 50 0 C, for example 15 to 30 0 C.
  • A is silyloxy
  • R 1 , R 2 and R 3 are as defined above for Formula (I)
  • a suitable fluoride source for example tetrabutylammonium fluoride.
  • the reaction is carried out in a suitable solvent, for example tetrahydrofuran.
  • the temperature is in the range 0 to 50 0 C, for example 15 to 30 0 C.
  • A is hydroxy or an alkoxy, benzyloxy or silyloxy group, and R 2 and R 3 are as defined above for Formula (I) and X is a halogen such as bromide or iodide; with a suitable boronic acid R 1 -B(OH) 2 or boronate ester R 1 -B(OR')(OR"), in which R' and R" are independently alkyl or R' and R" together with the carbon atoms to which they are attached form a ring optionally substituted by alkyl, such as a pinacol ester, in the presence of a palladium catalyst such as tetrakistriphenyl phosphine palladium(O) or bis-[(diphenylphosphino)- ferrocene]-palladium(ll) chloride, in the presence of a suitable base such as sodium carbonate, in a suitable solvent such as DMF, 1 ,4-dioxane or dimethy
  • A is hydroxy or an alkoxy, benzyloxy or silyloxy group, and R 2 and R 3 are as defined above for Formula (I) and X is a suitable boronic acid -B(OH) 2 or boronate ester -B(OR')(OR"), in which R' and R" are independently alkyl or R' and R" together with the carbon atoms to which they are attached form a ring optionally substituted by alkyl, such as a pinacol ester, with R 1 -Hal wherein Hal is a halogen such as bromide or iodide, in the presence of a palladium catalyst such as tetrakistriphenyl phosphine palladium(O) or bis- [(diphenylphosphino)-ferrocene]-palladium(ll) chloride, in the presence of a suitable base such as sodium carbonate, in a suitable solvent such as DMF, 1 ,4-dioxane
  • A is an alkoxy, benzyloxy or silyloxy, and R 2 and R 3 are as defined above for Formula (I)
  • a suitable base such as lithium diisopropylamide and a halogen source such as iodine in a suitable solvent such as tetrahydrofuran, heptane, ethylbenzene or combinations thereof and at a temperature in the range -78°C to -20 0 C.
  • A is an alkoxy, benzyloxy or silyloxy, and R 2 and R 3 are as defined above for Formula (I), by treatment with a suitable base such as lithium diisopropylamide and a boronate such as B(OR) 3 wherein R is an alkyl group, for example methyl, in a suitable solvent such as tetrahydrofuran, heptane, ethylbenzene or combinations thereof, and at a temperature in the range -78°C to -20 0 C.
  • a suitable base such as lithium diisopropylamide and a boronate such as B(OR) 3 wherein R is an alkyl group, for example methyl
  • a suitable solvent such as tetrahydrofuran, heptane, ethylbenzene or combinations thereof, and at a temperature in the range -78°C to -20 0 C.
  • Compounds of Formula (III) in which A is hydroxy may be prepared from compounds of Formula (III) in which A is an alkoxy, benyloxy or silyloxy group, for example by treatment with an appropriate base, acid or fluoride source as described in relation to the preparation of compounds of Formula (I) from compounds of Formula (II).
  • a an alkoxy, benzyloxy or silyloxy group, and R 3 is as defined above for Formula (I); with a suitable acylating agent, for example R 2 -C(0)-Y, wherein Y is a halo atom, for example chloro or bromo, and R 2 is as defined above for Formula (I).
  • the reaction may be carried out in a suitable solvent, for example dichloromethane or dichloroethane, and optionally in the presence of a suitable base, for example pyridine or triethylamine.
  • the reaction may be carried out at a suitable temperature, for example in the range 20 0 C to 80 0 C and optionally under an inert atmosphere.
  • a phosphine such as triphenylphosphine may optionally be used in place of the base.
  • a an alkoxy, benzyloxy or silyloxy group by treatment with a suitable vinyl ether, or a suitable aldehyde or a suitable ketone in the presence of a suitable acid, such as acetic acid or a Lewis acid such as titanium tetrachloride, and a suitable reducing agent such as sodium triacetoxyborohydride, in a suitable solvent such as dichloromethane.
  • a suitable acid such as acetic acid or a Lewis acid such as titanium tetrachloride
  • a suitable reducing agent such as sodium triacetoxyborohydride
  • compounds of Formula (V) in which A is an alkoxy, benzyloxy or silyloxy group may be prepared from compounds of Formula (Vl) in which A is an alkoxy, benzyloxy or silyloxy are as defined above for Formula (I), by treatment with a suitable alkylating agent R 3 -X' where X' is a halo group such as chloride, bromide or iodide, or X' is a sulphonate ester such as methanesulfonate and R 3 is as defined above for Formula (I), in suitable solvent such as dimethylformamide in the presence of a suitable base such as triethylamine.
  • a suitable alkylating agent R 3 -X' where X' is a halo group such as chloride, bromide or iodide, or X' is a sulphonate ester such as methanesulfonate and R 3 is as defined above for Formula (I)
  • suitable solvent such as
  • A is an alkoxy, benzyloxy or silyloxy group and X is a halo group such as bromo
  • X is a halo group such as bromo
  • R 3 is as defined above for Formula (I)
  • a palladium catalyst such as tris(dibenzylidenacetone)dipalladium in the presence of a reagent such as 2,2'-bis(diphenylphosphino)-1 ,1 '-binaphthyl (BINAP) and a base such as cesium carbonate
  • BINAP 2,2'-bis(diphenylphosphino)-1 ,1 '-binaphthyl
  • BINAP 2,2'-bis(diphenylphosphino)-1 ,1 '-binaphthyl
  • a base such as cesium carbonate
  • Compounds of Formula (IV) in which A is an alkoxy, benzyloxy or silyloxy group and R 2 and RR 33 aarree aass ddeeffiirned above for Formula (I), may also be prepared by reaction of a compound of Formula (VIII) in which A an alkoxy, benzyloxy or silyloxy group, and R 2 is as defined above for Formula (I); with a suitable alkylating agent R 3 -X' in which X' is a halo atom such as chloro, bromo or iodo, or X' is a sulphonate ester such as methanesulfonate and R 3 is as defined above for Formula (I), in a suitable solvent such as dimethylformamide, in the presence of a suitable base such as sodium hydride optionally in the presence of triethylamine.
  • a suitable solvent such as dimethylformamide
  • a suitable base such as sodium hydride optionally in the
  • Compounds of Formula (VIII) in which A is an alkoxy, benzyloxy or silyloxy group may be prepared by reaction of a compound of Formula (Vl) in which A an alkoxy, benzyloxy or silyloxy group, with a suitable acylating agent, for example R 2 -C(O)-Y, wherein Y is a halo atom, for example chloro or bromo, and R 2 is as defined above for Formula (I).
  • the reaction may be carried out in a suitable solvent, for example dichloromethane, optionally in the presence of a suitable base, for example pyridine or triethylamine.
  • a phosphine such as triphenylphosphine may optionally be used in place of the base.
  • Compounds of Formula (II) in which A is an alkoxy, benzyloxy or silyloxy group may also be prepared by reaction of a compound of Formula (IX)
  • a an alkoxy, benzyloxy or silyloxy group, and R 1 and R 3 are as defined above for Formula (I) with a suitable acylating agent, for example R 2 -C(O)-Y, wherein Y is a halo atom, for example chloro or bromo, and R 2 is as defined above for Formula (I).
  • the reaction may be carried out in a suitable solvent, for example dichloromethane or dichloroethane, optionally in the presence of a suitable base, for example pyridine or triethylamine.
  • the reaction may be carried out at a suitable temperature, for example in the range 20 0 C to 100 0 C.
  • a phosphine such as triphenylphosphine may optionally be used in place of the base.
  • Compounds of Formula (IX) in which A is an alkoxy, benzyloxy or silyloxy group and R 1 and R 3 are as defined above for Formula (I), may also be prepared by reaction of a compound of Formula (X) in which X is a halogen such as bromide or iodide, A is an alkoxy, benzyloxy or silyloxy group and R 3 is as defined above for Formula (I), with a suitable boronic acid R 1 -B(OH) 2 or boronate ester R 1 -B(OR')(OR"), in which R 1 is as defined above for Formula (I) and R' and R" are independently alkyl or R' and R" together with the carbon atoms to which they are attached form a ring optionally substituted by alkyl, such as a pinacol ester, in the presence of a palladium catalyst such as tetrakistriphenyl phosphine palladium(O) or bis-
  • A is an alkoxy, benzyloxy or silyloxy group and X is a halogen such as bromide or iodide
  • X is a halogen such as bromide or iodide
  • a suitable vinyl ether or a suitable aldehyde or a suitable ketone in the presence of a suitable acid, such as acetic acid, and a suitable reducing agent such as sodium triacetoxyborohydride, in a suitable solvent such as dichloromethane.
  • a suitable acid such as acetic acid
  • a suitable reducing agent such as sodium triacetoxyborohydride
  • compounds of Formula (X) in which A is an alkoxy, benzyloxy or silyloxy group may be prepared from compounds of Formula (Xl) in which A is an alkoxy, benzyloxy or silyloxy, and X is as defined above for Formula (I), by treatment with a suitable alkylating agent R 3 -X' where X' is a halo group such as chloride, bromide or iodide, or X' is a sulphonate ester such as methanesulfonate and R 3 is as defined above for Formula (I), in suitable solvent such as dimethylformamide in the presence of a suitable base such as triethylamine.
  • Compounds of Formula (X) in which A is an alkoxy, benzyloxy or silyloxy group, R 3 is as defined above and X is a suitable halogen such as bromide or iodide may also be prepared by reaction of a compound of Formula (V) in which A an alkoxy, benzyloxy or silyloxy group, and R 3 is as defined above for Formula (I), by treatment with a suitable base such as lithium diisopropylamide and a halogen source such as iodine in a suitable solvent such as tetrahydrofuran, heptane, ethylbenzene or mixtures thereof and at a temperature in the range -78°C to -20 0 C. Therefore, the invention also provides a process for the preparation of a compound of Formula (X) comprising treating a compound of Formula (V)
  • A an alkoxy, benzyloxy or silyloxy group, and R 3 is as defined above for Formula (I), with a base and a halogen source.
  • P is -COCF 3 or -C0 2 tBu and R 3 is as defined above for Formula (I), by treatment with a halogen source, for example iodine, in a suitable solvent such as THF, heptane, ethylbenzene, or combinations thereof, in the presence of a suitable base such as LDA, at a suitable temperature for example -78 to -20 0 C, optionally in an inert atmosphere.
  • a halogen source for example iodine
  • a suitable solvent such as THF, heptane, ethylbenzene, or combinations thereof
  • a suitable base such as LDA
  • Deprotection of the P group can be carried out for example, when P is -COCF 3 , by treatment with a suitable base such as aqueous potassium carbonate optionally in the presence of an alcohol such as methanol, or when P is -CO 2 1 Bu and by treatment with a suitable acid such as hydrochloric acid or trifluoroacetic acid in a suitable solvent such as 1 ,4-dioxane or dichloromethane.
  • a suitable base such as aqueous potassium carbonate optionally in the presence of an alcohol such as methanol
  • a suitable acid such as hydrochloric acid or trifluoroacetic acid in a suitable solvent such as 1 ,4-dioxane or dichloromethane.
  • P is a suitable protecting group such as -COCF 3 or -C(VBu and P' is hydrogen or a suitable protecting group such as -CO 2 1 Bu.
  • a suitable base such as aqueous potassium carbonate optionally in the presence of an alcohol such as methanol
  • P is -CO 2 1 Bu and P' is hydrogen or -CO 2 1 Bu
  • a suitable acid such as hydrochloric acid or trifluoroacetic acid in a suitable solvent such as 1 ,4-dioxane or dichloromethane.
  • a suitable base such as lithium diisopropylamide and a halogen source such as iodine
  • a suitable solvent such as tetrahydrofuran
  • Compounds of Formula (XIII) in which A is an alkoxy, benzyloxy or silyloxy group as defined above for Formula (I) and P and P' are as described above for Formula (XII), may be prepared by treating compounds of Formula (Vl) with trifluoroacetic anhydride or di-tert-butyl dicarbonate in a suitable solvent such as ether, acetonitrile or acetone, optionally in the presence of a catalyst such as DMAP and a base such as triethylamine.
  • a suitable solvent such as ether, acetonitrile or acetone
  • Compounds of Formula (III) in which A is an alkoxy, benzyloxy or silyloxy group may also be prepared from compounds of Formula (XIV) in which A is an alkoxy, benzyloxy or silyloxy, and R 2 is as defined above for Formula (I), by treatment with a suitable alkylating agent R 3 -X' where X' is a halo group such as chloride, bromide or iodide, or X' is a sulphonate ester such as methanesulfonate and R 3 is as defined above for Formula (I), in suitable solvent such as dimethylformamide in the presence of a suitable base such as triethylamine.
  • Compounds of Formula (XIV) in which A is an alkoxy, benzyloxy or silyloxy group may also be prepared from compounds of Formula (Xl) in which A is an alkoxy, benzyloxy or silyloxy group and X is a halogen such as bromide or iodide, with a suitable acylating agent, for example R 2 -C(O)-Y, wherein Y is a halo atom, for example chloro or bromo, and R 2 is as defined above for Formula (I).
  • the reaction may be carried out in a suitable solvent, for example dichloromethane, in the presence of a suitable base, for example pyridine or triethylamine.
  • a phosphine such as triphenylphosphine may optionally be used in place of the base.
  • the reaction may be carried out at a suitable temperature, for example in the range 20 0 C to 80 0 C.
  • a an alkoxy, benzyloxy or silyloxy group, and R 3 is as defined above for Formula (I) and X is a halogen such as bromide or iodide, with a suitable acylating agent, for example R 2 -C(O)-Y, wherein Y is a halo atom, for example chloro or bromo, and R 2 is as defined above for Formula (I).
  • a suitable solvent for example dichloromethane
  • a suitable base for example pyridine or triethylamine.
  • a phosphine such as triphenylphosphine may optionally be used in place of the base.
  • R' and R" are independently alkyl or R' and R" together with the carbon atoms to which they are attached form a ring optionally substituted by alkyl, such as a pinacol ester, with a suitable acylating agent, for example R 2 -C(0)-Y, wherein Y is a halo atom, for example chloro or bromo, and R 2 is as defined above for Formula (I).
  • a suitable solvent for example dichloromethane
  • a suitable base for example pyridine or triethylamine.
  • a phosphine such as triphenylphosphine may optionally be used in place of the base.
  • R x , R 2 , R 3 , and A are as defined for Formula (II) by reaction with a suitable heteroaryl boronic acid R ⁇ -boronic acid, in the presence of a palladium catalyst such as palladium (II) acetate, a reagent such as 2- dicyclohexylphosphino-2'(N,N-dimethylamino)-biphenyl, and an additional reagent such as caesium fluoride, in a suitable solvent such as 1 ,4-dioxane.
  • a palladium catalyst such as palladium (II) acetate
  • a reagent such as 2- dicyclohexylphosphino-2'(N,N-dimethylamino)-biphenyl
  • an additional reagent such as caesium fluoride
  • Z represents B(OH) 2 , and R x , R 2 , R 3 and A are as defined for Formula (II), by reaction with a suitable heteroaryl halide R ⁇ -hal, in which suitably the halide is bromo or iodo, in the presence of a palladium catalyst such as palladium (II) acetate, a reagent such as 2- dicyclohexylphosphino-2'(N,N-dimethylamino)-biphenyl, and an additional reagent such as caesium fluoride, in a suitable solvent such as 1 ,4-dioxane.
  • a suitable heteroaryl halide R ⁇ -hal in which suitably the halide is bromo or iodo
  • a palladium catalyst such as palladium (II) acetate
  • a reagent such as 2- dicyclohexylphosphino-2'(N,N-dimethyl
  • Compounds of Formula (II)' in which Z is halo may be prepared by reaction of a compound of Formula (III) wherein X is halo, with a boronic acid of Formula Z-R x -boronic acid wherein Z is halo and R x is as defined above for Formula (I) under the conditions described above for the preparation of compounds of Formula (I) and (II) from (IX) and R ⁇ -R x -boronic acid.
  • Compounds of Formula (II)' in which Z is B(OH) 2 may be prepared by reaction of a compound of Formula (III) with a compound of Formula Z-R X -B(OH) 2 wherein R x is as described above for Formula (I), under the conditions described above for the preparation of compounds of Formula (I) and (II) from (III) and R 1 -boronic acid.
  • R 1 represents a 4-ethynylphenyl derivative
  • R 2 , R 3 and A are as defined above for Formula (II)
  • a suitable pyridine the pyridine being substituted with adjacent hydroxy and iodo groups
  • a suitable catalyst such as bis(triphenylphosphine)palladium (II) chloride and copper (I) iodide
  • a suitable solvent such as triethylamine or DMF.
  • the temperature is in the range 50-80 0 C.
  • Compounds of Formula (I) or (II) in which R 1 represents a 4-(pyrrolopyridine)phenyl and R 2 , R 3 and A are as defined above for Formula (II), may be prepared by treatment of a compound of Formula (II)" in which R 1 represents 4-ethynylphenyl and R 2 , R 3 and A are as defined above for Formula (ll)with an appropriate pyridine (the pyridine being substituted by adjacent amino and iodo groups), in the presence of a suitable catalyst such as bis(triphenylphosphine)palladium (II) chloride and copper (I) iodide, in a suitable solvent such as triethylamine.
  • a suitable catalyst such as bis(triphenylphosphine)palladium (II) chloride and copper (I) iodide
  • a suitable solvent such as triethylamine.
  • the temperature is in the range 50-80 0 C.
  • Compounds of Formula (I) or (II) in which R 1 represents a 4-(pyrazolopyrimidine)phenyl and R 2 , R 3 and A are as defined above for Formula (II), may be prepared by treating a compound of Formula (II)" in which R 1 represents 4-(phenyl)-1 H-pyrazole-5-amine and R 2 , R 3 and A are as defined above for Formula (II) with 1 ,1 ,3,3-tetramethoxypropane in a suitable solvent such as acetic acid; suitably the temperature is in the range 90-1 10 0 C.
  • a 2-(4-bromophenyl)imidazo[1 ,2-a]pyridine derivative may be prepared by analogy to methods described in Tetrahedron Letters (2001 ) 42, 3077.
  • a 4-(furopyridine)phenyl bromide may be prepared by treatment of a 4-ethynylphenyl bromide with a suitable pyridine (the pyridine being substituted with adjacent hydroxy and iodo groups), with a suitable catalyst such as bis(triphenylphosphine)palladium (II) chloride and copper (I) iodide, in a suitable solvent such as triethylamine or DMF.
  • a suitable catalyst such as bis(triphenylphosphine)palladium (II) chloride and copper (I) iodide
  • a suitable solvent such as triethylamine or DMF.
  • the temperature is in the range 50-80 0 C.
  • a 4-(pyrazolopyrimidine)phenyl bromide may be prepared by treating a 3-(4-bromophenyl)- 1 H-pyrazole-5-amine with 1 ,1 ,3,3-tetramethoxypropane in a suitable solvent such as acetic acid.
  • a suitable solvent such as acetic acid.
  • the temperature is in the range 90-110 0 C.
  • Esters of compounds of Formula (I), in which A is -OR where R is selected from straight or branched chain alkyl, aralkyl, aryloxyalkyl, or aryl, may also be prepared by esterification of a compound of Formula (I) in which A is hydroxy by standard literature procedures for esterification.
  • compounds of Formula (I), (II), (III), (IV), (VIII), (IX) and (X) which exist as diastereoisomers may optionally be separated by techniques well known in the art, for example by column chromatography or recrystallisation. For example, the formation of an ester using a chiral alcohol, separation of the resulting diastereoisomers, and subsequent hydrolysis of the ester to yield the individual enantiomeric acid of Formula (I) (II), (III), (IV), (VIII), (IX) and (X).
  • racemic compounds of Formula (I), (II), (III), (IV), (VIII), (IX) and (X) 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), (II), (III), (IV), (VIII), (IX) and (X) may be resolved by chiral preparative HPLC.
  • racemic compounds of Formula (I), (II), (III), (IV), (VIII), (IX) and (X) 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 may be resolved by treatment with a chiral acid such as (R)-(-)-1 ,1 '-binaphthyl-2,2'-diyl-hydrogen phosphate or (-)-di-O,O'-p-tolyl-L-tartaric acid, in a suitable solvent, for example isopropanol.
  • a suitable solvent for example isopropanol.
  • the free enantiomer may then be obtained by treating the salt with a suitable base, for example triethylamine, in a suitable solvent, for example methyl te/f-butyl ether.
  • racemic acid compounds may be resolved using a chiral base, for example (S)-alpha methylbenzylamine, (S)-alpha phenylethylamine, (1S, 2S)-(+)-2-amino-1-phenyl-1 ,3-propane-diol, (-) ephidrine, quinine, brucine.
  • a chiral base for example (S)-alpha methylbenzylamine, (S)-alpha phenylethylamine, (1S, 2S)-(+)-2-amino-1-phenyl-1 ,3-propane-diol, (-) ephidrine, quinine, brucine.
  • Individual enantiomers of Formula (II), (III), (IV), (VIII), (IX) and/or (X) may then be progressed to an enantiomeric compound of Formula (I) by the chemistry described above in respect of racemic compounds.
  • Ref 1 Burkholder, Conrad; Dolbier, William R.; Medebielle, Maurice; Ait-Mohand, Samia, Tetrahedron Lett, 42, 17, 2001 , 3077 - 3080.
  • 6-(4-lodophenyl)imidazo[2,1-b][1 ,3]thiazole (2 g) was dissolved in dry 1 ,4-dioxane (40 ml_). To this stirred solution was added potassium acetate (1.8 g), bis(pinacolato)diboron (2.34 g) and 1 ,1 '-bis (diphenylphosphino)ferrocene dichloro palladium (II) (350 mg). The reaction mixture was then heated to 100 0 C, and stirred under nitrogen for 18 h.
  • the reaction was then cooled and recharged with potassium acetate (1.42 g), bis(pinacolato)diboron (1.71 g) and 1 ,1 '-bis (diphenylphosphino)ferrocene dichloro palladium (II) (254 mg).
  • the reaction mixture was then reheated to 100 0 C, and stirred under nitrogen for another 18 h.
  • the reaction was allowed to cool and the solvent removed by evaporation and the residue was partitioned between water and DCM.
  • the organic layer was separated, washed with water, and concentrated by evaporation.
  • the residue was then purified by ISCO companion silica chromatography eluting with a gradient of EtOAc / cyclohexane (0% to 100%), to give the title compound.
  • reaction mixture was evaporated to dryness and the residue partitioned between water and DCM.
  • the aqueous phase was separated and extracted with more DCM (x2).
  • the combined organic phases were dried using a hydrophobic frit and evaporated to dryness. This was purified by ISCO companion silica chromatography eluting with a gradient of ethyl acetate in cyclohexane to give the title compound.
  • n-Butyl lithium (2.94 ml_, 4.7 mmol, 1.6M solution in hexanes) was added dropwise to a solution of Intermediate 28 (2.0 g) in THF (25 ml.) at -78 0 C under nitrogen. A fine yellow suspension was formed and stirred at -78 0 C under nitrogen for 45 mins. Trimethoxyborate (669 ⁇ l_, 5.99 mmol) in THF (3 ml.) was added dropwise, maintaining the internal temp at - 78 0 C. This was stirred at -78 0 C for 30 mins then allowed to warm to RT and stirred for a further 45 mins.
  • Methyl 3-amino-2-thiophenecarboxylate (5 g) was dissolved in acetone (30 ml_), then DMAP (195 mg) and triethylamine (10 ml.) were added followed by dropwise addition of di-te/f-butyl dicarbonate (64 ml_, 1 M solution in THF). The reaction mixture was stirred at room temperature overnight. Di-te/f-butyl dicarbonate (32 ml_, 1 M solution in THF) was added dropwise and the reaction was left to stir at room temperature for 18 h.
  • the compounds of Formula (I) 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 pharmaceutically acceptable salt, solvate or ester thereof in conjunction with at least one pharmaceutically acceptable diluent or carrier.
  • the compounds of Formula (I) can be administered by different routes including oral, parenteral (e.g. intravenous, intraperitoneal, subcutaneous, intramuscular), rectal, topical, transdermal, transmucosal, buccal, sublingual, intranasal or by inhalation administration.
  • parenteral e.g. intravenous, intraperitoneal, subcutaneous, intramuscular
  • rectal topical, transdermal, transmucosal, buccal, sublingual, intranasal or by inhalation administration.
  • oral administration is convenient.
  • the compounds of Formula (I) can be formulated into conventional oral dosage forms such as capsules, tablets and liquid preparations such as syrups, elixirs, concentrated drops, ovules, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed-, modified-, sustained-, pulsed-or controlled-release applications.
  • conventional oral dosage forms such as capsules, tablets and liquid preparations such as syrups, elixirs, concentrated drops, ovules, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed-, modified-, sustained-, pulsed-or controlled-release applications.
  • the tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia.
  • excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine
  • disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates
  • granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC),
  • lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.
  • Solid compositions of a similar type may also be employed as fillers in gelatin capsules.
  • suitable excipients in this regard include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene glycols.
  • the agent may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.
  • injection parenteral administration
  • parenteral administration may be used, e.g., intravenously, intraarterially, intraperitoneal ⁇ , intrathecal ⁇ , intraventricular ⁇ , intraurethrally, intrasternally, intracranially, intramuscularly or subcutaneously administering the agent; and/or by using infusion techniques.
  • the compound may be used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood.
  • Typical parenteral compositions consist of a solution or suspension of a compound of Formula (I) or a pharmaceutically acceptable salt, solvate or ester 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.
  • aqueous solutions should be suitably buffered (suitably to a pH of from 3 to 9), if necessary.
  • the preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art.
  • the compounds of Formula (I) may be formulated in liquid solutions, for example, in pharmaceutically compatible buffers or solutions, such as saline solution, Hank's solution, or Ringer's solution.
  • the compounds of Formula (I) may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms can also be produced.
  • the compounds of Formula (I) may be administered in the form of a suppository or pessary, or may be applied topically in the form of a gel, hydrogel, lotion, salve, solution, cream, ointment or dusting powder.
  • the compounds of Formula (I) may also be dermally or transdermal ⁇ administered, for example, by the use of a skin patch. They may also be administered by the pulmonary or rectal routes. They may also be administered by the ocular route.
  • the compounds may be formulated as micronised suspensions in isotonic, pH adjusted, sterile saline, or, suitably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzylalkonium chloride.
  • a preservative such as a benzylalkonium chloride.
  • they may be formulated in an ointment such as petrolatum.
  • the compounds of Formula (I) may be formulated as a suitable ointment containing the active compound suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water.
  • a suitable lotion or cream suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2- octyldodecanol, benzyl alcohol and water.
  • compositions for rectal administration are conveniently in the form of suppositories containing a conventional suppository base such as cocoa butter.
  • 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.
  • 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.
  • Typical dermal and transdermal formulations comprise a conventional aqueous or nonaqueous vehicle, for example a cream, ointment, lotion or paste or are in the form of a medicated plaster, patch or membrane
  • compositions suitable for buccal or sublingual administration include tablets, lozenges and pastilles, wherein the compound of Formula (I) is formulated with a carrier such as sugar and acacia, tragacanth, or gelatin and glycerin.
  • a carrier such as sugar and acacia, tragacanth, or gelatin and glycerin.
  • the compounds of Formula (I) can also be administered intranasally or by inhalation and is conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurised container, pump, spray or nebuliser with the use of a suitable propellant, e. g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, a hydrofluoroalkane such as 1 ,1 ,1 ,2-tetrafluoroethane (HFA 134AT"") or 1 ,1 ,1 ,2,3,3,3- heptafluoropropane (HFA 227EA), carbon dioxide or other suitable gas.
  • a suitable propellant e. g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, a hydrofluoroalkane such as 1 ,1 ,1
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • the pressurised container, pump, spray or nebuliser may contain a solution or suspension of the active compound, e. g. using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e. g. sorbitan trioleate.
  • Capsules and cartridges for use in an inhaler or insufflator may be formulated to contain a powder mix of the compound and a suitable powder base such as lactose or starch.
  • the amounts of various compounds of Formula (I) 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 of Formula (I)), 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 of the compounds of Formula (I) administered also depend on the routes of administration and the degree of oral bioavailability. For example, for compounds of Formula (I) with low oral bioavailability, relatively higher doses will have to be administered. Oral administration is a convenient method of administration of the present compounds of Formula (I).
  • 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, for example 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, for example once, sufficient to exhibit the desired activity, as is readily apparent to one skilled in the art.
  • 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 flavouring or colouring agent.
  • a liquid carrier for example, ethanol, peanut oil, olive oil, glycerine or water with a flavouring or colouring 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.
  • Genotype 1 b 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 1 M 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), Tote/ 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 1 mg/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 2 ⁇ ⁇ 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 1 h 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.
  • genotype 1 a and genotype 1 b may be demonstrated, for example, using the following cell based assay:
  • test compound 100 ⁇ L of medium containing 10% FCS were added to each well of clear, flat-bottomed 96 well microplates, excepting wells in the top row.
  • Test compound was diluted in assay medium to twice the final required starting concentration from a 40 mM stock solution in DMSO.
  • 200 ⁇ l_ of the starting dilution were introduced into two wells each in the top row and doubling dilutions made down the plate by the sequential transfer of 100 ⁇ l_ aliquots with thorough mixing in the wells; the final 100 ⁇ l_ were discarded.
  • the two bottom rows were not used for compound dilutions.
  • 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 1 b; Lohmann, V., Korner, F., Koch, J-O., Herian, U., Thielmann, L. and Bartenschlager, R., Science, 1999, 285, 110- 113) or at 3 x 10 5 cells/mL (genotype 1 a; Gu, B., Gates, AT., Isken, O., Behrens, S. E. and Sarisky, R.T., J.
  • 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 5 o) for the compound.
  • a 4OmM stock solution in DMSO of each test compound was further diluted into 50 ⁇ l_ of DMSO in the first row of a 96 well, V-bottom microplate, to give 100 times the top concentration of the required dilution series. Aliquots of 25 ⁇ l_ of DMSO were added to each well of the remaining rows, and doubling dilutions of compound were prepared by the serial transfer of 25 ⁇ l_ volumes from the first row onwards.
  • a Plate-mate robot was used to transfer 1 ⁇ l_ volumes from each dilution well into duplicate wells of a clear bottom, black walled, 96 well assay plate (COSTAR #3603). Control wells received 1 ⁇ l_ of DMSO alone.
  • Suspensions were prepared from cultures of Huh-7 cells stably transfected with sub-genomic HCV NS3-NS5B replicons of either genotype 1 b (the ET subline described by
  • genotype 1a (subline 1.19 constructed in-house) linked to a firefly luciferase reporter gene.
  • Monolayers nearing confluency were stripped from growth flasks with versene-trypsin solution and the cells re- suspended in assay medium comprising DMEM (Invitrogen #41965-039) supplemented with
  • Resazurin (Fisher #R/0040/79) was dissolved in 5OmL of phosphate buffered saline and 100 ⁇ l_ of solution added to all wells. The plate was re-incubated at 37°C for a further 2- 4 hours, wrapped in aluminium foil, before reading in a FluoStar Optima at 595nm. All growth medium and Resazurin was removed by aspiration, and an opaque mask applied to the bottom of the plate. A solution of SteadyLite cytolytic buffer/luciferase substrate (Perkin-Elmer #6016987) was prepared according to the manufacturer's instructions, and 25 ⁇ l_ added to each well. The plate was then read for luminescence on a TopCount NXT.
  • Toxicity The Resazurin absorbance values from duplicate wells were averaged and expressed as a percentage of the mean absorbance of compound free control wells to determine comparative cell viability. Compound cytotoxicity was expressed either as the lowest concentration at which a significant reduction in viability was observed or a 50% toxic concentration (CCID 50 ) was determined by plotting percentage cytotoxicity against compound concentration using Grafit software (Erithacus Software Ltd.). Potency : The luminescence values from all compound-free wells containing cells were averaged to obtain a positive control value. The mean luminescence value from the compound-free wells that had received no cells 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 luciferase signal in the presence of a drug is a direct measure of replicon inhibition.
  • GraFit software was used to plot the curve of percentage inhibition against compound concentration and derive the 50% inhibitory concentration (IC 50 ) for the compound.
  • Genotvoe 1a Genotvoe 1 b
  • Compound A corresponds to the compound disclosed as Example 307 in WO2002/100851,
  • Compound B corresponds to the compound disclosed as Example 430 in WO2002/100851,
  • the compounds of Formula (I) which have been tested demonstrate a surprisingly superior potency as HCV polymerase inhibitors, as shown by the IC 50 values in the cell-based assays across both of the 1 a and 1 b genotypes of HCV, compared to Compounds A - E. Accordingly, the compounds of Formula (I) 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.
  • the invention thus provides, in a further aspect, a combination comprising a compound of Formula (I) or a salt, solvate or ester thereof together with at least one other therapeutic agent.
  • a compound of Formula (I) or a salt, solvate or ester thereof When a compound of Formula (I) or a salt, solvate or ester thereof is used in combination with a second therapeutic agent active against the same disease state, the dose of each compound may differ from that when the compound is used alone. Appropriate doses will be readily appreciated by those skilled in the art. It will be appreciated that the amount of a compound of Formula (I) or a salt, solvate or ester thereof required for use in treatment will vary with the nature of the condition being treated and the age and the condition of the patient and will be ultimately at the discretion of the attendant physician or veterinarian.
  • the compounds of Formula (I) or a salt, solvate or ester thereof may be used in combination with other therapeutic agents, for example immune therapies [e.g.
  • Interferon such as Interferon alfa-2a (Roferon-A; Hoffmann-La Roche), interferon alpha-2b (Intron-A; Schering-Plough), interferon alfacon-1 (Infergen; Intermune), peginterferon alpha-2b (Peg-lntron; Schering- Plough) or peginterferon alpha-2a (Pegasys; Hoffmann-La Roche)]
  • therapeutic vaccines such as Interferon alfa-2a (Roferon-A; Hoffmann-La Roche), interferon alpha-2b (Intron-A; Schering-Plough), interferon alfacon-1 (Infergen; Intermune), peginterferon alpha-2b (Peg-lntron; Schering- Plough) or peginterferon alpha-2a (Pegasys; Hoffmann-La Roche)]
  • antifibrotic agents such as corticosteroids or NSAIDs
  • bronchodilators such as
  • HCV NS3 protease inhibitors for example HCV NS3 protease inhibitors, 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 compound of Formula (I) or a salt, solvate or ester thereof together with at least one other therapeutically active agent, for example Interferon, ribavirin and/or an additional anti-HCV agent.
  • at least one compound of Formula (I) or a salt, solvate or ester thereof together with at least one other therapeutically active agent, for example Interferon, ribavirin and/or an additional anti-HCV agent.
  • either the compound of Formula (I) or the second therapeutic agent may be administered first.
  • the combination may be administered either in the same or different pharmaceutical composition.
  • the two compounds When combined in the same formulation it will be appreciated that the two compounds must be stable and compatible with each other and the other components of the formulation. When formulated separately they may be provided in any convenient formulation, conveniently in such manner as are known for such compounds in the art.

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Abstract

Anti-viral agents of compounds of Formula (I) : wherein A, R1, R2 and R3 are as defined in the specification, processes for their preparation and their use in HCV treatment are provided.

Description

COMPOUNDS
FIELD OF THE INVENTION
The present invention relates to novel 2-carboxy thiophene derivatives useful as anti-viral agents. Specifically, the present invention involves novel inhibitors of Hepatitis C Virus (HCV) replication.
BACKGROUND OF THE INVENTION
Infection with HCV is a major cause of human liver disease throughout the world. 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 combination 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 ): 71 S-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 responders, 50-70% relapse within 6 months of cessation of treatment. Recently, with the introduction of pegylated interferon (Peg-IFN), 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 associated 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), 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 244:359-362; 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' 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 1 b isolates) and inter-typically (-85% aa identity between genotype 1 a 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): 2046-2051 ). Thus, inhibition of NS5B RdRp activity (inhibition of RNA replication) is predicted to be useful to treat HCV infection.
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 1 a is most abundant in the United States, the majority of sequences in Europe and Japan are from genotype 1 b. Table 1
Figure imgf000004_0002
Based on the foregoing, there exists a significant need to identify synthetic or biological compounds for their ability to inhibit replication of both genotype 1 a and genotype 1 b of HCV.
PCT publication number WO2002/100851 generically discloses certain compounds, including certain 2-carboxy thiophene compounds, having HCV inhibitory activity. The data provided relates to an HCV polymerase assay utilising the 1 b genotype. The compounds disclosed have the formula (I)
Figure imgf000004_0001
wherein
X is chosen from -N(R3)M(R2) or -JN(R2)(R3);
M is chosen from -SO2-, -SO-, -S-, -C(O)-, -C(S)-, -CH2C(O)N(R4)-, -CH2C(S)N(R15)-, -
CH(R15)-, -C(=N(R8))-, or a bond;
R4 is C1-6alkyl;
R8 is chosen from H, Ci_i2alkyl, C2-i2alkenyl, C2-i2alkynyl, C6-i4aryl, C3-i2heterocycle, C3- i2heteroaralkyl, C6-i6aralkyl;
R15 is chosen from H or C1-6alkyl;
J is chosen from -C(W)-, -C(R6)-, -S-, -S(O)-, or -SO2-;
W is chosen from O, S or NR7;
R7 is chosen from H, C1-12alkyl, C2-12alkenyl, C2-12alkynyl, C6-i4aryl, C3-12heterocycle, C3- i2heteroaralkyl, C6-i6aralkyl;
R6 is chosen from H, Ci_i2alkyl, C6-i4aryl, or C6-i6aralkyl;
Y1 is chosen from a bond, C1-6alkyl, C2-6alkenyl, C2-6alkynyl;
Y is chosen from COOR16, COCOOR5, P(0)0RORb, S(O)OR5, S(O)2OR5, tetrazole,
CON(R9)CH(R5)COOR5, CONR10R11, CON(R9)-SO2-R5, CONR9OH, or halogen;
R , R ,R 510 and R are each independently chosen from H, C1-12alkyl, C2-12alkenyl, C2-
12alkynyl, C3-i2heterocycle, C3-i8heteroaralkyl, C6-i8aralkyl; or R10 and R11 are taken together with the nitrogen to form a 3 to 10 membered heterocycle;
Ra and Rb are each independently chosen from H, C1-12alkyl, C2-12alkenyl, C2-12alkynyl, C6- i4aryl, C3-i2heterocycle, C3-i8heteroaralkyl, C6-i8aralkyl; or Ra and Rb are taken together with the oxygens to form a 5 to 10 membered heterocycle;
R16 is chosen from H, C1-12alkyl, C2-12alkenyl, C2-12alkynyl, C6-i4aryl, C3-12heterocycle, C3- -isheteroaralkyl, Cβ-isaralkyl; provided that R16 is other than methyl or ethyl; R1 is chosen from Ci.-i2alkyl, C2-i2alkenyl, C2-i2alkynyl, C6-i4aryl, C3-i2heterocycle, C3- 18heteroaralkyl, C6-i8aralkyl;
R2 is chosen from Ci.-i2alkyl, C2-i2alkynyl, C6-i4aryl, C3-i2heterocycle, C3-i8heteroaralkyl, C6- i8aralkyl;
R3 is chosen from H, C1-12alkyl, C2-i2alkenyl, C2-i2alkynyl, C6-i4aryl, C3-12heterocycle, C3- iβheteroaralkyl, C6-i8aralkyl;
Z is chosen from H, halogen, or Ci-6alkyl.
PCT publication number WO2003/062215 generically discloses certain compounds, including certain 2-carboxy thiophene compounds, having kinase inhibiting activity. The compounds disclosed have the fo
Figure imgf000005_0001
wherein V is, inter alia, a group of formula (i)
Figure imgf000005_0002
R1 represents T, N-(CO)R6R7, N(R6)COR7, or NC(O)OR6R7;
T represents H, (hetero)alkyl, alkenyl, cycloalkyl, (hetero)aryl, arylalkyl or alkylaryl;
R8 represents T, NR3R4, N-C(O)R3R4, N(R3)COR4, OR3, COR3, CO2R3 or OCOR3;
R2, R5 represent T, alkylaryl, carboxyl, halo or CF3; R3, R4, R6, R7 represent T, alkylaryl, COOR5 or COR5;
X, Y, Z represent CH or N;
U represents S or NH; and W represents NH, O or S.
PCT publication number WO2004/1 10357 generically discloses a range of compounds, including certain 2-carboxy thiophene compounds, having phosphodiesterase 6 delta (PDE6D) modulating activity. The compounds disclosed have the formula (I)
Figure imgf000005_0003
wherein
R1, R2, R3, and R4 are independently selected from the group consisting of hydrogen, halogen, alkyl, cycloalkyl, aryl, heteroaryl, NR5C(O)R7, C(O)NR5R6, C(O)R7 and C(O)OR7, wherein R5, R6 and R7 are independently selected from hydrogen, lower alkyl, cycloalkyl or aryl, and where R5, R6, and R7 together can optionally form a 3, 4, 5, 6 or 7 membered ring optionally having one or more degrees of substitution.
Surprisingly, it has now been found that compounds according to the present invention, generically disclosed in WO2002/100851 , WO2003/062215 or WO2004/110357, and having a specific substitution pattern, exhibit improved properties over those compounds specifically disclosed in WO2002/100851 , WO2003/062215 or WO2004/110357.
SUMMARY OF THE INVENTION The present invention involves novel 2-carboxy thiophene compounds represented hereinbelow, pharmaceutical compositions comprising such compounds and use of the compounds in treating viral infection, especially HCV infection.
DETAILED DESCRIPTION OF THE INVENTION In a first aspect, the present invention provides a compound of Formula (I) :
Figure imgf000006_0001
wherein:
A represents hydroxy;
R1 represents -Rx-Rγ;
Rx represents phenyl (optionally substituted by halo, methyl, ethyl, methoxy or trifluoromethyl) or 5- or 6-membered heteroaryl bonded through a ring carbon atom to the carbon atom of the thiophene;
Rγ represents 8-, 9- or 10-membered heteroaryl, bonded such that when Rx is phenyl or 6- membered heteroaryl, Rγ is in the para-position;
R2 represents phenyl which is at least 2,4 disubstituted, the substituents being independently selected from unsubstituted C1-2alkyl, C1-2alkyl (substituted with one or more fluoro atoms), chloro, fluoro and bromo;
R3 represents linear or branched -C1-6alkyl, or -C3-6cycloalkyl (each optionally substituted by one or more substituents selected from 5- or 6-membered heteroaryl, 5- or 6-membered heterocyclyl and -Cs-ecycloalkyl), linear or branched -C2-6alkyl (optionally substituted by one or more substituents selected from methoxy, ethoxy and fluoro), pyranyl, -CH2-pyranyl, tetrahydrofuranyl or -CH2-tetrahydrofuranyl (each of which may be optionally substituted by one or more substituents selected from -Ci-2alkyl, fluoro and methoxy);
or salts, solvates or esters thereof.
It is to be understood that the present invention covers all combinations of aspects, suitable, convenient and preferred groups described herein.
As used herein, "acetyl" refers to -C(O)CH3.
As used herein unless otherwise specified, "alkyl" refers to an optionally substituted hydrocarbon group. The alkyl hydrocarbon group may be linear, branched or cyclic, saturated or unsaturated. Where the alkyl group is linear or branched, examples of such groups include methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl or hexyl and the like. Where the alkyl hydrocarbon group is unsaturated, it will be understood that there will be a minimum of 2 carbon atoms in the group, for example an alkenyl or alkynyl group. Where the alkyl hydrocarbon group is cyclic, it will be understood that there will be a minimum of 3 carbon atoms in the group. A cyclic group may be substituted with a linear or branched alkyl group. In one aspect, alkyl moieties are saturated. In one aspect, alkyl moieties are
Figure imgf000007_0001
Unless otherwise stated, optional substituents include -C1-6alkyl (unsubstituted), =CH(CH2)tH, fluoro, -CF3, -ORE, -SRE, -C(O)NRBRC, -C(O)R0, -CO2H, -CO2R0, -NRBRC, -NRAC(0)R°,
-NRAC02RD, -NRASO2RD, -NRAC(O)NRFRG, -SO2NRFRG, -SO2R0, nitro, cyano, oxo, aryl, heteroaryl and heterocyclyl.
As used herein, the term "alkenyl" refers to a linear or branched hydrocarbon group containing one or more carbon-carbon double bonds. In one aspect the alkenyl group has from 2 to 6 carbon atoms. Examples of such groups include ethenyl, propenyl, butenyl, pentenyl or hexenyl and the like.
As used herein, the term "alkynyl" refers to a linear or branched hydrocarbon group containing one or more carbon-carbon triple bonds. In one aspect the alkynyl group has from 2 to 6 carbon atoms. Examples of such groups include ethynyl, propynyl, butynyl, pentynyl or hexynyl and the like.
As used herein unless otherwise specified, "cycloalkyl" refers to an optionally substituted, cyclic hydrocarbon group. The hydrocarbon group may be saturated or unsaturated, monocyclic or bridged bicyclic. Where the cycloalkyl group is saturated, examples of such groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl and the like. Where the cycloalkyl group is unsaturated, examples of such groups include cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl or cyclooctenyl and the like. In one aspect, the cycloalkyl group has from 5 to 7 carbon atoms. In one aspect, cycloalkyl moieties are cyclohexenyl, cyclopentenyl and cyclohexyl. Unless otherwise stated, the cycloalkyl group may be substituted by one or more optional substituents including -Chalky! (unsubstituted), =CH(CH2)tH, fluoro, -CF3, -ORE, -SRE, -C(O)NRBRC, -C(O)R0, -CO2H, -CO2R0, -NRBRC, -NRAC(O)R°, -NRACO2RD, -NRASO2RD, -NRAC(O)NRFRG, -SO2NRFRG, -SO2R0, nitro, cyano, oxo, phenyl and heterocyclyl.
As used herein, the term " alkoxy" refers to an -O-alkyl group wherein alkyl is as defined herein. Examples of such groups include methoxy, ethoxy, propoxy, butoxy, pentoxy or hexoxy and the like.
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. In one aspect, "aryl" moieties contain 6-10 carbon atoms. In one aspect, "aryl" moieties are unsubstituted, monosubstituted, disubstituted or trisubstituted phenyl. In one aspect, unless otherwise stated, "aryl" substituents are selected from the group consisting of -Ci-6alkyl, halo, -ORE, -SRE, -C(O)NRBRC, -C(O)R0, -CO2H, -CO2R0, -NRBRC, -NRAC(0)R°, -NRAC02R°, -NRASO2R°, -NRAC(0)NRFRG, -SO2NRFRG, -SO2R0, nitro, cyano, heterocyclyl, - CF3, -OCF3 and phenyl.
As used herein, "carbonyl" refers to -C(O)-.
As used herein, "cyano" refers to -CN.
As used herein, "halogen" or "halo" refer to a fluorine, chlorine, bromine or iodine atom. References to "fluoro", "chloro", "bromo" or "iodo" should be construed accordingly.
As used herein, unless otherwise specified, "heteroaryl" refers to an optionally substituted, 5, 6, 8, 9 or 10 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. In one aspect, "heteroaryl" moieties are unsubstituted, monosubstituted, disubstituted or trisubstituted (where applicable) pyridine, pyrazine, thiazole, thiophene, oxadiazole, oxazole, pyrimidine, pyridazine, benzodioxole, benzofuran, benzodioxin, indole, benzimidazole, benzofuran, indole, indazole, isoindole, benzothiophene, benzothiazole, benzoxazole, benzisoxazole, benzisothiazole, benzotriazole, furopyridine, furopyrimidine, furopyridazine, furopyrazine, furotriazine, pyrrolopyridine, pyrrolopyrimidine, pyrrolopyridazine, pyrrolopyrazine, pyrrolotriazine, thienopyridine, thienopyrimidine, thienopyridazine, thienopyrazine, thienotriazine, thiazolopyridine, thiazolopyrimidine, thiazolopyridazine, thiazolopyrazine, thiazolotriazine, oxazolopyridine, oxazolopyrimidine, oxazolopyridazine, oxazolopyrazine, oxazolotriazine, imidazopyridine, imidazopyrimidine, imidazopyridazine, imidazopyrazine, imidazotriazine, pyrazolopyridine, pyrazolopyrimidine, pyrazolopyridazine, pyrazolopyrazine, pyrazolotriazine, triazolopyridine, triazolopyrimidine, triazolopyridazine, triazolopyrazine, quinoline, naphthyridine, quinoxaline, quinazoline, isoquinoline, cinnoline, pyridopyridazine, pyridopyrimidine, pyridopyrazine, pyrazinopyrazine, pteridine, pyrazinopyridazine, pyrimidopyridazine, pyrimidopyrimidine, imidazothiazole, thiazolooxazole. All isomers of the above heteroaryls are within the scope of this invention. Each heteroaryl group may be attached at any ring carbon or may be attached through nitrogen when the nitrogen is part of a 5-membered ring. In one aspect, unless otherwise stated, "heteroaryl" substituents are selected from the group consisting of -Ci-6alkyl, halo, -ORE, -SRE, -C(O)NRBRC, -C(O)R0, -CO2R0, -NRBRC, -NRAC(O)R°, -NRACO2RD, -NRASO2RD, -NRAC(O)NRFRG, -SO2NRFRG, -SO2R0, oxo, nitro, cyano, heterocyclyl, -CF3 and phenyl.
As used herein, "heterocyclic" and "heterocyclyl" refer to an optionally substituted, 5 or 6 membered, saturated or partially saturated, cyclic group containing 1 or 2 heteroatoms selected from N, optionally substituted by hydrogen, -Ci-6alkyl, -C(O)R0, -C(O)NRBRC, -C(O)OH, -SO2R0, aryl or heteroaryl; O; and S, optionally substituted by one or two oxygen atoms. Ring carbon atoms may be optionally substituted by -C1-6alkyl, -0RA, -C(O)R0, or -SO2R0. In one aspect, unless otherwise stated, "heterocyclic" moieties are unsubstituted or monosubstituted tetrahydro-2H-pyran-4-yl, piperidinyl and tetrahydrofuran- 3-yl.
As used herein, "nitro" refers to -NO2.
As used herein, "oxo" refers to =0.
As used herein, "Et" refers to "ethyl", "iPr" refers to "isopropyl", "Me" refers to "methyl", "OBn" refers to "benzyloxy", and "Ph" refers to "phenyl".
RA represents hydrogen or -C1-6alkyl.
RB and Rc independently represent hydrogen, -Ci-6alkyl, aryl, heterocyclyl or heteroaryl; or RB and Rc together with the nitrogen atom to which they are attached form a 5 or 6 membered saturated cyclic group.
R° is selected from the group consisting of -C1-6alkyl, aryl, heterocyclyl, heteroaryl, arylalkyl, and heteroarylalkyl.
RE represents hydrogen, -C1-6alkyl, arylalkyl, heteroarylalkyl, aryl, heterocyclyl or heteroaryl.
RF and RG are independently selected from the group consisting of hydrogen, -Ci-6alkyl, aryl, heteroaryl, arylalkyl, and heteroarylalkyl; or RF and RG together with the nitrogen atom to which they are attached form a 5 or 6 membered saturated cyclic group.
As used herein, the term "substituted" refers to substitution with the named substituent or substituents, multiple degrees of substitution being allowed unless otherwise stated.
Throughout the specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers but not to the exclusion of any other integer or step or group of integers or steps.
In one aspect, Rx represents phenyl optionally substituted by halo, methyl, methoxy or trifluoromethyl. In a further aspect, Rx represents unsubstituted phenyl.
In one aspect, Rγ represents 8-, 9- or 10-membered bicyclic heteroaryl group. In a further aspect, Rγ represents 8- or 9-membered bicyclic heteroaryl group. In a further aspect, Rγ represents furo[3,2-b]pyridin-2-yl, pyrazolo[1 ,5-a]pyrimidin-2-yl, imidazo[1 ,2-a]pyridin-2-yl, imidazo[2,1-b][1 ,3]thiazol-6-yl, 7-amino-5-methylpyrazolo[1 ,5-a]pyrimidin-2-yl, 5- methylpyrazolo-[1 ,5-a]pyrimidin-2-yl, 7-aminopyrazolo[1 ,5-a]pyrimidin-2-yl, [1 ,3]oxazolo[4,5- b]pyridin-2-yl, furo[2,3-b]pyridin-5-yl, 5-amino-1 ,3-benzoxazol-2-yl, [1 ,3]oxazolo[5,4-b]pyridin- 2-yl, or furo[3,2-c]pyridin-2-yl.
In a further aspect, Rγ represents furo[3,2-b]pyridin-2-yl, pyrazolo[1 ,5-a]pyrimidin-2-yl, imidazo[1 ,2-a]pyridin-2-yl, imidazo[2,1-b][1 ,3]thiazol-6-yl or 7-aminopyrazolo[1 ,5-a]pyrimidin- 2-yl.
In a further aspect, when Rx represents furanyl, the bonds to the Ry and to the thiophene group are in a 2,4 relationship. For example, the bond to Ry may be in the 2-position and the bond to the thiophene may be in the 4 position. In a further aspect, when Rx represents thienyl, the bonds to the Rx and to the central thiophene group are in a 2,5 relationship.
In one aspect, R2 represents 2,4-disubstituted phenyl wherein the substituents are independently selected from Ci-2alkyl, trifluoromethyl, chloro, fluoro and bromo. In a further aspect, the substituents are independently selected from methyl, chloro, fluoro, bromo and trifluoromethyl. In a further aspect, the substituents are independently selected from methyl, chloro, fluoro and bromo. In a further aspect, R2 represents 2,4-dichlorophenyl, 4-chloro-2- fluorophenyl, 4-bromo-2-methylphenyl, 2,4-dimethylphenyl, 2-fluoro-4-trifluoromethylphenyl or 2,4-bis(trifluoromethyl)phenyl. In a further aspect, R2 represents 2,4-dichlorophenyl or 4- chloro-2-fluorophenyl.
In one aspect, R3 represents linear or branched -Ci-6alkyl (optionally substituted by one or more substituents independently selected from 5- or 6-membered heteroaryl, 5- or 6- membered heterocyclyl and -C3-6cycloalkyl), linear or branched -C2-6alkyl (optionally substituted by one or more substituents selected from methoxy, ethoxy and fluoro), or pyranyl, -CH2-pyranyl, tetrahydrofuranyl or -CH2-tetrahydrofuranyl (all of which may be optionally substituted by one or more substituents selected from -C1-2alkyl, fluoro and methoxy). In a further aspect, R3 represents unsubstituted Ci-6alkyl, C2-6alkyl (substituted by methoxy), unsubstituted pyranyl or unsubstituted tetrahydrofuranyl. In a further aspect, R3 represents 1-methylethyl, 2-(methyloxy)ethyl, 1-methyl-2-(methyloxy)ethyl, tetrahydro-2H- pyran-4-yl or tetrahydro-3-furanyl. In a further aspect, R3 represents unsubstituted linear or branched Ci-6alkyl. In a further aspect, R3 represents 1-methylethyl.
In one aspect, there is provided a compound of Formula (I) wherein Rx represents unsubstituted phenyl; Rγ represents 8- or 9-membered bicyclic heteroaryl group; R2 represents 2,4-disubstituted phenyl wherein the substituents are independently selected from Ci-2alkyl, trifluoromethyl, chloro, fluoro and bromo; R3 represents linear or branched -Ci- 6alkyl (optionally substituted by one or more substituents independently selected from 5- or 6-membered heteroaryl, 5- or 6-membered heterocyclyl and -C3-6cycloalkyl), linear or branched -C2-6alkyl (optionally substituted by one or more substituents selected from methoxy, ethoxy and fluoro), or pyranyl, -CH2-pyranyl, tetrahydrofuranyl or -CH2- tetrahydrofuranyl (all of which may be optionally substituted by one or more substituents selected from -Ci-2alkyl, fluoro and methoxy); or salts, solvates and esters thereof.
In a further aspect, there is provided a compound of Formula (I) wherein Rx represents unsubstituted phenyl; Rγ represents furo[3,2-b]pyridin-2-yl, pyrazolo[1 ,5-a]pyrimidin-2-yl, imidazo[1 ,2-a]pyridin-2-yl, imidazo[2,1-b][1 ,3]thiazol-6-yl, 7-amino-5-methylpyrazolo[1 ,5- a]pyrimidin-2-yl, 5-methylpyrazolo-[1 ,5-a]pyrimidin-2-yl, 7-aminopyrazolo[1 ,5-a]pyrimidin-2-yl, [1 ,3]oxazolo[4,5-b]pyridin-2-yl, furo[2,3-b]pyridin-5-yl, 5-amino-1 ,3-benzoxazol-2-yl, [1 ,3]oxazolo[5,4-b]pyridin-2-yl, or furo[3,2-c]pyridin-2-yl; R2 represents 2,4-dichlorophenyl, 4- chloro-2-fluorophenyl, 4-bromo-2-methylphenyl, 2,4-dimethylphenyl, 2-fluoro-4- trifluoromethylphenyl or 2,4-bis(trifluoromethyl)phenyl; R3 represents unsubstituted Chalky!, C2-6alkyl (substituted by methoxy), unsubstituted pyranyl or unsubstituted tetrahydrofuranyl; or salts, solvates and esters thereof.
In a further aspect, there is provided a compound of Formula (I) wherein Rx represents unsubstituted phenyl; Rγ represents furo[3,2-b]pyridin-2-yl, pyrazolo[1 ,5-a]pyrimidin-2-yl, imidazo[1 ,2-a]pyridin-2-yl, imidazo[2,1-b][1 ,3]thiazol-6-yl or 7-aminopyrazolo[1 ,5-a]pyrimidin-
2-yl; R2 represents 2,4-dichlorophenyl, 4-chloro-2-fluorophenyl, 4-bromo-2-methylphenyl,
2,4-dimethylphenyl, 2-fluoro-4-trifluoromethylphenyl or 2,4-bis(trifluoromethyl)phenyl; R3 represents 1-methylethyl, 2-(methyloxy)ethyl, 1-methyl-2-(methyloxy)ethyl, tetrahydro-2H- pyran-4-yl or tetrahydro-3-furanyl; or salts, solvates and esters thereof.
In a further aspect, the present invention provides a compound of Formula (Ia) :
Figure imgf000012_0001
wherein:
A represents hydroxy;
R1 represents -Rx-Rγ;
Rx represents phenyl (optionally substituted by halo, methyl, ethyl, methoxy or trifluoromethyl) or 5- or 6-membered heteroaryl bonded through a ring carbon atom to the carbon atom of the thiophene;
Rγ represents 8-, 9- or 10-membered heteroaryl, bonded such that when Rx is phenyl or 6- membered heteroaryl, Rγ is in the para-position;
R2 represents phenyl which is at least 2,4 disubstituted, the substituents being independently selected from unsubstituted C1-2alkyl, C1-2alkyl (substituted with one or more fluoro atoms), methoxy, chloro, fluoro and bromo;
R3 represents linear or branched -C1-6alkyl, or -C3-6cycloalkyl (each optionally substituted by one or more substituents selected from 5- or 6-membered heteroaryl, 5- or 6-membered heterocyclyl and -Cs-ecycloalkyl), linear or branched -C2-6alkyl (optionally substituted by one or more substituents selected from methoxy, ethoxy and fluoro), pyranyl or tetrahydrofuranyl (each of which may be optionally substituted by one or more substituents selected from -Ci- 2alkyl, fluoro and methoxy);
or salts, solvates or esters thereof.
In a further aspect, the present invention provides at least one chemical entity chosen from a compound of Formula (Ib) :
Figure imgf000012_0002
wherein:
A represents hydroxy; R1 represents -Rx-Rγ;
Rx represents phenyl (optionally substituted by halo, methyl, ethyl, methoxy or trifluoromethyl) or 5- or 6-membered heteroaryl bonded through a ring carbon atom to the carbon atom of the thiophene;
Rγ represents a 8-, 9- or 10-membered heteroaryl, bonded such that when Rx is phenyl or 6- membered heteroaryl, Rγ is in the para-position;
R2 represents phenyl which is at least 2,4 disubstituted, the substituents being selected from -Ci-2alkyl, -C1-2alkyl substituted with one or more fluoro atoms, methoxy, chloro, fluoro and bromo;
R3 represents linear or branched -Ci-6alkyl, or -C3-6cycloalkyl (each optionally substituted by one or more substituents selected from methoxy, ethoxy, or 5- or 6-membered heteroaryl or heterocyclyl), or -C3-6cycloalkyl, pyranyl or furanyl (each of which may be optionally substituted by one or more substituents selected from -Ci-2alkyl, fluoro and methoxy);
and salts, solvates and esters thereof.
As used herein the term "at least one chemical entity" means at least one chemical substance chosen from the group of compounds consisting of compounds of Formula I and pharmaceutically acceptable derivatives thereof.
In one aspect, compounds useful in the present invention may be chosen from compounds of Formula (I) selected from the group consisting of:
3-[[(2,4-Dichlorophenyl)carbonyl](1-methylethyl)amino]-5-(4-imidazo[1 ,2-a]pyridin-2- ylphenyl)-2-thiophenecarboxylic acid;
3-[[(4-Chloro-2-fluorophenyl)carbonyl](1-methylethyl)amino]-5-(4-imidazo[1 ,2-a]pyridin-2- ylphenyl)-2-thiophenecarboxylic acid;
3-[[(4-Bromo-2-methylphenyl)carbonyl](1-methylethyl)amino]-5-(4-imidazo[1 ,2-a]pyridin-
2-ylphenyl)-2-thiophenecarboxylic acid;
3-[[(2,4-Dichlorophenyl)carbonyl](1-methylethyl)amino]-5-(4-pyrazolo[1 ,5-a]pyrimidin-2- ylphenyl)-2-thiophenecarboxylic acid; 3-[[(4-Chloro-2-fluorophenyl)carbonyl](1-methylethyl)amino]-5-(4-pyrazolo[1 ,5- a]pyrimidin-2-ylphenyl)-2-thiophenecarboxylic acid;
3-[[(4-Bromo-2-methylphenyl)carbonyl](1-methylethyl)amino]-5-(4-pyrazolo[1 ,5- a]pyrimidin-2-ylphenyl)-2-thiophenecarboxylic acid;
3-[[(2,4-Dimethylphenyl)carbonyl](1-methylethyl)amino]-5-(4-pyrazolo[1 ,5-a]pyrimidin-2- ylphenyl)-2-thiophenecarboxylic acid;
S-^^-DichlorophenyOcarbony^i-methylethyOaminol-δ^-imidazoP.I-^ti .Slthiazol-θ- ylphenyl)-2-thiophenecarboxylic acid; 3-[[(2,4-Dichlorophenyl)carbonyl](1-methylethyl)amino]-5-(4-furo[3,2-b]pyridin-2- ylphenyl)-2-thiophenecarboxylic acid;
5-[4-(7-Aminopyrazolo[1 ,5-a]pyrimidin-2-yl)phenyl]-3-[[(2,4-dichlorophenyl)carbonyl](1- methylethyl)amino]-2-thiophenecarboxylic acid; 3-[[(4-Chloro-2-fluorophenyl)carbonyl](tetrahydro-3-furanyl)amino]-5-(4-pyrazolo[1 ,5- a]pyrimidin-2-ylphenyl)-2-thiophenecarboxylic acid;
3-[[(2,4-Dichlorophenyl)carbonyl](tetrahydro-3-furanyl)amino]-5-(4-pyrazolo[1 ,5- a]pyrimidin-2-ylphenyl)-2-thiophenecarboxylic acid;
3-[{[2-Fluoro-4-(trifluoromethyl)phenyl]carbonyl}(1-methylethyl)amino]-5-(4-pyrazolo[1 ,5- a]pyrimidin-2-ylphenyl)-2-thiophenecarboxylic acid;
3-[{[2,4-Bis(trifluoromethyl)phenyl]carbonyl}(1-methylethyl)amino]-5-(4-pyrazolo[1 ,5- a]pyrimidin-2-ylphenyl)-2-thiophenecarboxylic acid;
3-{[(4-Chloro-2-fluorophenyl)carbonyl][2-(methyloxy)ethyl]amino}-5-(4-pyrazolo[1 ,5- a]pyrimidin-2-ylphenyl)-2-thiophenecarboxylic acid; 3-{[(2,4-Dichlorophenyl)carbonyl][2-(methyloxy)ethyl]amino}-5-(4-pyrazolo[1 ,5- a]pyrimidin-2-ylphenyl)-2-thiophenecarboxylic acid;
3-[[(4-Chloro-2-fluorophenyl)carbonyl](tetrahydro-2H-pyran-4-yl)amino]-5-(4- pyrazolo[1 ,5-a]pyrimidin-2-ylphenyl)-2-thiophenecarboxylic acid;
3-[[(2,4-Dichlorophenyl)carbonyl](tetrahydro-2/-/-pyran-4-yl)amino]-5-(4-pyrazolo[1 ,5- a]pyrimidin-2-ylphenyl)-2-thiophenecarboxylic acid;
3-{[(4-Chloro-2-fluorophenyl)carbonyl][1-methyl-2-(methyloxy)ethyl]amino}-5-(4- pyrazolo[1 ,5-a]pyrimidin-2-ylphenyl)-2-thiophenecarboxylic acid;
and salts, solvates and esters, and individual enantiomers thereof where appropriate.
The compounds of the present invention may be in the form of their free base or pharmaceutically acceptable salts, pharmaceutically acceptable solvates or pharmaceutically acceptable esters thereof.
Also included in the present invention are pharmaceutically acceptable salt complexes. As used herein, the term "pharmaceutically acceptable salts" refers to salts that retain the desired biological activity of the subject compound and exhibit minimal undesired toxicological effects. For a review on suitable salts see Berge et al, J. Pharm. ScL, 1977, 66, 1-19. The term "pharmaceutically acceptable salts" includes both pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts. These pharmaceutically acceptable salts may be prepared in situ during the final isolation and purification of the compound, or by separately reacting the purified compound in its free acid or free base form with a suitable base or acid, respectively. The salt may precipitate from solution and be collected by filtration or may be recovered by evaporation of the solvent. Therefore, according to a further aspect, the invention provides a pharmaceutically acceptable salt of a compound of Formula (I) and embodiments thereof. In certain embodiments, compounds of Formula I may contain an acidic functional group and may therefore be capable of forming pharmaceutically acceptable base addition salts by treatment with a suitable base. A pharmaceutically acceptable base addition salt may be formed by reaction of a compound of Formula I with a suitable strong base, optionally in a suitable solvent such as an organic solvent, to give the base addition salt which may be isolated for example by crystallisation and filtration. Pharmaceutically acceptable base salts include ammonium salts (for example ammonium or tetraalkylammonium), metal salts, for example alkali-metal or alkaline-earth-metal salts (such as hydroxides, sodium, potassium, calcium or magnesium), organic amines (such as tris [also known as tromethamine or tris(hydroxymethyl)aminomethane], ethanolamine, triethanolamine, choline, isopropylamine, dicyclohexylamine or N-methyl-D-glucamine), cationic amino acids (such as arginine, lysine or histidine) or bases for insoluble salts (such as procaine or benzathine).
In certain embodiments, compounds according to Formula I may contain a basic functional group and may therefore be capable of forming pharmaceutically acceptable acid addition salts by treatment with a suitable acid. A pharmaceutically acceptable acid addition salt may be formed by reaction of a compound of Formula I with a suitable strong inorganic acid (such as hydrobromic, hydrochloric, sulfuric, nitric, phosphoric or perchloric) or a suitable strong organic acid, for example, sulfonic acids [such as p-toluenesulfonic, benzenesulfonic, methanesulfonic, ethanesulfonic, 2-hydroxyethanesulfonic, naphthalenesulfonic (e.g. 2- naphthalenesulfonic)], carboxylic acids (such as acetic, propionic, fumaric, maleic, benzoic, salicylic or succinic), anionic amino acids (such as glutamaic or aspartic), hydroxyl acids (such as citric, lactic, tartaric or glycolic), fatty acids (such as caproic, caprylic, decanoic, oleic or stearic) or acids for insoluble salts (such as pamoic or resinic [e.g. polystyrene sulfonate]), optionally in a suitable solvent such as an organic solvent, to give the salt which is usually isolated for example by crystallisation and filtration. In one embodiment, a pharmaceutically acceptable acid addition salt of a compound of Formula I is a salt of a strong acid, for example a hydrobromide, hydrochloride, hydroiodide, sulfate, nitrate, perchlorate, phosphate p-toluenesulfonic, benzenesulfonic or methanesulfonic salt.
Other non-pharmaceutically acceptable salts, for example oxalates, may be used, for example in the isolation of compounds of Formula (I), and are included within the scope of this invention.
The invention includes within its scope all possible stoichiometric and non-stoichiometric forms of the salts of the compounds of Formula (I).
Those skilled in the art of organic chemistry will appreciate that many organic compounds can form complexes with solvents in which they are reacted or from which they are precipitated or crystallized. These complexes are known as "solvates". For example, a complex with water is known as a "hydrate". Solvates of the compound of Formula (I) are within the scope of the invention. Therefore, the present invention also relates to solvates of the compounds of Formula (I), for example hydrates.
Salts and solvates of compounds of Formula (I) which are suitable for use in medicine are those wherein the counterion or associated solvent is pharmaceutically acceptable. However, salts and solvates having non-pharmaceutically acceptable counterions or associated solvents are within the scope of the present invention, for example, for use as intermediates in the preparation of other compounds of the invention and their pharmaceutically acceptable salts and solvates.
It will be appreciated by those skilled in the art that certain protected derivatives of compounds of Formula (I), which may be made prior to a final deprotection stage, may not possess pharmacological activity as such, but may, in certain instances, be administered orally or parenterally and thereafter metabolised in the body to form compounds defined in the first aspect which are pharmacologically active. Such derivatives may therefore be described as "prodrugs". All protected derivatives and prodrugs of compounds defined in the first aspect are included within the scope of the invention. Examples of suitable pro-drugs for the compounds of the present invention are described in Drugs of Today, Volume 19, Number 9, 1983, pp 499 - 538 and in Topics in Chemistry, Chapter 31 , pp 306 - 316 and in "Design of Prodrugs" by H. Bundgaard, Elsevier, 1985, Chapter 1 (the disclosures in which documents are incorporated herein by reference). It will further be appreciated by those skilled in the art, that certain moieties, known to those skilled in the art as "pro-moieties", for example as described by H. Bundgaard in "Design of Prodrugs" (the disclosure in which document is incorporated herein by reference) may be placed on appropriate functionalities when such functionalities are present within the compounds of Formula (I). Suitable prodrugs for compounds of the invention include : esters, carbonate esters, hemi-esters, phosphate esters, nitro esters, sulfate esters, sulfoxides, amides, carbamates, azo-compounds, phosphamides, glycosides, ethers, acetals and ketals.
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 -C1-4alkoxy or amino); or for example -
CH2OC(O)R' or -CH2OCO2R' in which R' is alkyl (e.g. R' is f-butyl). Unless otherwise specified, any alkyl moiety present in such esters suitably contains 1 to 18 carbon atoms, particularly 1 to 4 carbon atoms. Any aryl moiety present in such esters suitably comprises a phenyl group.
In one aspect, the invention provides a pharmaceutically acceptable salt, solvate or prodrug of a compound of Formula (I).
In one aspect, the invention provides a compound of Formula (I) in the form of parent compound, a salt or a solvate.
Furthermore, some of the crystalline forms of the compounds of Formula (I) may exist in one or more polymorphic form, which are included in the present invention.
It will be appreciated that the compounds of Formula (I) 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. Racemic compounds may either be separated using preparative HPLC and a column with a chiral stationary phase or resolved to yield individual enantiomers utilising methods known to those skilled in the art. In addition, chiral intermediate compounds may be resolved and used to prepare chiral compounds of the invention.
The compounds of Formula (I) may exist in one or more tautomeric forms. All tautomers and mixtures thereof are included in the scope of the present invention. For example, a claim to 2-hydroxyquinolinyl would also cover its tautomeric form, α-quinolinonyl.
Diastereoisomers of compounds of Formula (I) may be obtained according to methods well known in the literature, for example by preparative HPLC or by chromatographic purifications. Racemic compounds may either be separated using preparative HPLC and a column with a chiral stationary phase or resolved to yield individual enantiomers utilising methods known to those skilled in the art. In addition, chiral intermediate compounds may be resolved and used to prepare chiral compounds of the invention.
As used herein, 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.
The compounds of the present invention exhibit improved potency against the replication of HCV (1a and 1 b genotypes), and therefore have the potential to achieve greater efficacy in man. High potency in both genotypes is considered to be advantageous.
There is provided as a further aspect of the present invention a compound of Formula (I) or pharmaceutically acceptable salts, solvates or esters thereof for use in human or veterinary medical therapy, particularly in the treatment or prophylaxis of viral infection, particularly flavivirus infection, for example HCV infection.
It will be appreciated that reference 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 include treatment or prophylaxis of HCV-associated disease such as liver fibrosis, cirrhosis and hepatocellular carcinoma.
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 pharmaceutically acceptable salts, solvates or esters thereof.
According to another aspect of the invention, there is provided the use of a compound of Formula (I) or pharmaceutically acceptable salts, solvates or esters thereof in the manufacture of a medicament for the treatment and/or prophylaxis of viral infection, particularly HCV infection.
PROCESSES
The compounds of Formula (I) or salts, solvates or esters thereof may be prepared by the processes described hereinafter, said processes constituting a further aspect of the invention.
Compounds of Formula (I) in which A is hydroxy may be prepared from a compound of Formula (II)
Figure imgf000018_0001
in which A is a protected hydroxy group, for example an alkoxy, benzyloxy or silyloxy group and R1, R2, and R3 are as defined above for Formula (I). For example when A is methoxy or ethoxy, and R1, R2, R3 and R4 are as defined above for Formula (I), by treatment with an appropriate base, for example aqueous sodium hydroxide or lithium hydroxide, optionally in a solvent such as methanol, ethanol, tetrahydrofuran or combinations thereof. Suitably, the temperature is in the range 20 to 1000C. Alternatively, when A is methoxy or ethoxy and R1, R2 and R3 are as defined above for Formula (I), by treatment with lithium iodide in a suitable solvent such as pyridine, lutidine or collidine, suitably in the temperature range 100-1700C.
For example when A is te/t-butoxy, and R1, R2 and R3 are as defined above for Formula (I), by treatment with an appropriate acid, for example trifluoroacetic acid. Suitably, the reaction is carried out in a solvent, for example dichloromethane. Suitably, the temperature is in the range 0 to 500C, for example 15 to 300C.
For example when A is silyloxy, and R1, R2 and R3 are as defined above for Formula (I), by treatment with a suitable fluoride source for example tetrabutylammonium fluoride. The reaction is carried out in a suitable solvent, for example tetrahydrofuran. Suitably, the temperature is in the range 0 to 500C, for example 15 to 300C.
Compounds of Formula (I) in which A is hydroxy, or (II) in which A is an alkoxy, benzyloxy or silyloxy group and R1 and R3 are as defined above for Formula (I), may be prepared by reaction of a compound of Form
Figure imgf000019_0001
in which A is hydroxy or an alkoxy, benzyloxy or silyloxy group, and R2 and R3 are as defined above for Formula (I) and X is a halogen such as bromide or iodide; with a suitable boronic acid R1-B(OH)2 or boronate ester R1-B(OR')(OR"), in which R' and R" are independently alkyl or R' and R" together with the carbon atoms to which they are attached form a ring optionally substituted by alkyl, such as a pinacol ester, in the presence of a palladium catalyst such as tetrakistriphenyl phosphine palladium(O) or bis-[(diphenylphosphino)- ferrocene]-palladium(ll) chloride, in the presence of a suitable base such as sodium carbonate, in a suitable solvent such as DMF, 1 ,4-dioxane or dimethyoxyethane, or combinations thereof, optionally in the presence of water, at a temperature in the range 50- 1000C, optionally under an inert atmosphere.
Compounds of Formula (I) in which A is hydroxy, or (II) in which A is an alkoxy, benzyloxy or silyloxy group and R1 and R3 are as defined above for Formula (I), may be prepared by reaction of a compound of Formula (III)'
Figure imgf000019_0002
in which A is hydroxy or an alkoxy, benzyloxy or silyloxy group, and R2 and R3 are as defined above for Formula (I) and X is a suitable boronic acid -B(OH)2 or boronate ester -B(OR')(OR"), in which R' and R" are independently alkyl or R' and R" together with the carbon atoms to which they are attached form a ring optionally substituted by alkyl, such as a pinacol ester, with R1-Hal wherein Hal is a halogen such as bromide or iodide, in the presence of a palladium catalyst such as tetrakistriphenyl phosphine palladium(O) or bis- [(diphenylphosphino)-ferrocene]-palladium(ll) chloride, in the presence of a suitable base such as sodium carbonate, in a suitable solvent such as DMF, 1 ,4-dioxane or dimethyoxyethane, or combinations thereof, optionally in the presence of water, at a temperature in the range 50-1000C, optionally under an inert atmosphere. Compounds of Formula (III) in which A is an alkoxy, benzyloxy or silyloxy group may be prepared from compounds of For
Figure imgf000020_0001
in which A is an alkoxy, benzyloxy or silyloxy, and R2 and R3 are as defined above for Formula (I), by treatment with a suitable base such as lithium diisopropylamide and a halogen source such as iodine in a suitable solvent such as tetrahydrofuran, heptane, ethylbenzene or combinations thereof and at a temperature in the range -78°C to -200C.
Compounds of Formula (III)' in which A is an alkoxy, benzyloxy or silyloxy group may be prepared from compounds of For
Figure imgf000020_0002
in which A is an alkoxy, benzyloxy or silyloxy, and R2 and R3 are as defined above for Formula (I), by treatment with a suitable base such as lithium diisopropylamide and a boronate such as B(OR)3 wherein R is an alkyl group, for example methyl, in a suitable solvent such as tetrahydrofuran, heptane, ethylbenzene or combinations thereof, and at a temperature in the range -78°C to -200C.
Compounds of Formula (III) in which A is hydroxy may be prepared from compounds of Formula (III) in which A is an alkoxy, benyloxy or silyloxy group, for example by treatment with an appropriate base, acid or fluoride source as described in relation to the preparation of compounds of Formula (I) from compounds of Formula (II).
Compounds of Formula (IV) in which A is an alkoxy, benzyloxy or silyloxy group and R3 is as defined above may be prepared by reaction of a compound of Formula (V)
Figure imgf000020_0003
in which A an alkoxy, benzyloxy or silyloxy group, and R3 is as defined above for Formula (I); with a suitable acylating agent, for example R2-C(0)-Y, wherein Y is a halo atom, for example chloro or bromo, and R2 is as defined above for Formula (I). The reaction may be carried out in a suitable solvent, for example dichloromethane or dichloroethane, and optionally in the presence of a suitable base, for example pyridine or triethylamine. The reaction may be carried out at a suitable temperature, for example in the range 200C to 800C and optionally under an inert atmosphere. A phosphine such as triphenylphosphine may optionally be used in place of the base.
Compounds of Formula (V) in which A is an alkoxy, benzyloxy or silyloxy group may be prepared by reaction of a compound of Formula (Vl)
Figure imgf000021_0001
in which A an alkoxy, benzyloxy or silyloxy group, by treatment with a suitable vinyl ether, or a suitable aldehyde or a suitable ketone in the presence of a suitable acid, such as acetic acid or a Lewis acid such as titanium tetrachloride, and a suitable reducing agent such as sodium triacetoxyborohydride, in a suitable solvent such as dichloromethane. Alternatively, compounds of Formula (V) in which A is an alkoxy, benzyloxy or silyloxy group may be prepared from compounds of Formula (Vl) in which A is an alkoxy, benzyloxy or silyloxy are as defined above for Formula (I), by treatment with a suitable alkylating agent R3-X' where X' is a halo group such as chloride, bromide or iodide, or X' is a sulphonate ester such as methanesulfonate and R3 is as defined above for Formula (I), in suitable solvent such as dimethylformamide in the presence of a suitable base such as triethylamine.
Compounds of Formula (V) may also be prepared by reacting a compound of Formula (VII)
Figure imgf000021_0002
in which A is an alkoxy, benzyloxy or silyloxy group and X is a halo group such as bromo, with an amine R3NH2 wherein R3 is as defined above for Formula (I), in the presence of a palladium catalyst such as tris(dibenzylidenacetone)dipalladium in the presence of a reagent such as 2,2'-bis(diphenylphosphino)-1 ,1 '-binaphthyl (BINAP) and a base such as cesium carbonate, in a suitable solvent such as toluene and in a suitable temperature range such as 80-120°C.
Compounds of Formula (IV) in which A is an alkoxy, benzyloxy or silyloxy group and R2 and RR33 aarree aass ddeeffiirned above for Formula (I), may also be prepared by reaction of a compound of Formula (VIII)
Figure imgf000022_0001
in which A an alkoxy, benzyloxy or silyloxy group, and R2 is as defined above for Formula (I); with a suitable alkylating agent R3-X' in which X' is a halo atom such as chloro, bromo or iodo, or X' is a sulphonate ester such as methanesulfonate and R3 is as defined above for Formula (I), in a suitable solvent such as dimethylformamide, in the presence of a suitable base such as sodium hydride optionally in the presence of triethylamine.
Compounds of Formula (VIII) in which A is an alkoxy, benzyloxy or silyloxy group may be prepared by reaction of a compound of Formula (Vl) in which A an alkoxy, benzyloxy or silyloxy group, with a suitable acylating agent, for example R2-C(O)-Y, wherein Y is a halo atom, for example chloro or bromo, and R2 is as defined above for Formula (I). The reaction may be carried out in a suitable solvent, for example dichloromethane, optionally in the presence of a suitable base, for example pyridine or triethylamine. A phosphine such as triphenylphosphine may optionally be used in place of the base. Compounds of Formula (II) in which A is an alkoxy, benzyloxy or silyloxy group may also be prepared by reaction of a compound of Formula (IX)
Figure imgf000022_0002
in which A an alkoxy, benzyloxy or silyloxy group, and R1 and R3 are as defined above for Formula (I) with a suitable acylating agent, for example R2-C(O)-Y, wherein Y is a halo atom, for example chloro or bromo, and R2 is as defined above for Formula (I). The reaction may be carried out in a suitable solvent, for example dichloromethane or dichloroethane, optionally in the presence of a suitable base, for example pyridine or triethylamine. The reaction may be carried out at a suitable temperature, for example in the range 200C to 1000C. A phosphine such as triphenylphosphine may optionally be used in place of the base.
Compounds of Formula (IX) in which A is an alkoxy, benzyloxy or silyloxy group and R1 and R3 are as defined above for Formula (I), may also be prepared by reaction of a compound of Formula (X)
Figure imgf000023_0001
in which X is a halogen such as bromide or iodide, A is an alkoxy, benzyloxy or silyloxy group and R3 is as defined above for Formula (I), with a suitable boronic acid R1-B(OH)2 or boronate ester R1-B(OR')(OR"), in which R1 is as defined above for Formula (I) and R' and R" are independently alkyl or R' and R" together with the carbon atoms to which they are attached form a ring optionally substituted by alkyl, such as a pinacol ester, in the presence of a palladium catalyst such as tetrakistriphenyl phosphine palladium(O) or bis-
[(diphenylphosphino)-ferrocene]-palladium(ll) chloride, in the presence of a suitable base such as sodium carbonate, in a suitable solvent such as DMF, 1 ,4-dioxane or dimethyoxyethane, or combinations thereof, optionally in the presence of water, at a temperature in the range 50-1000C, optionally under an inert atmosphere.
Compounds of Formula (X) in which A is an alkoxy, benzyloxy or silyloxy group, R3 is as defined above for Formula (I) and X is a halogen such as bromide or iodide, may be prepared by reaction of a compound of Formula (Xl)
Figure imgf000023_0002
in which A is an alkoxy, benzyloxy or silyloxy group and X is a halogen such as bromide or iodide, by treatment with a suitable vinyl ether, or a suitable aldehyde or a suitable ketone in the presence of a suitable acid, such as acetic acid, and a suitable reducing agent such as sodium triacetoxyborohydride, in a suitable solvent such as dichloromethane. Alternatively, compounds of Formula (X) in which A is an alkoxy, benzyloxy or silyloxy group may be prepared from compounds of Formula (Xl) in which A is an alkoxy, benzyloxy or silyloxy, and X is as defined above for Formula (I), by treatment with a suitable alkylating agent R3-X' where X' is a halo group such as chloride, bromide or iodide, or X' is a sulphonate ester such as methanesulfonate and R3 is as defined above for Formula (I), in suitable solvent such as dimethylformamide in the presence of a suitable base such as triethylamine.
Compounds of Formula (X) in which A is an alkoxy, benzyloxy or silyloxy group, R3 is as defined above and X is a suitable halogen such as bromide or iodide, may also be prepared by reaction of a compound of Formula (V) in which A an alkoxy, benzyloxy or silyloxy group, and R3 is as defined above for Formula (I), by treatment with a suitable base such as lithium diisopropylamide and a halogen source such as iodine in a suitable solvent such as tetrahydrofuran, heptane, ethylbenzene or mixtures thereof and at a temperature in the range -78°C to -200C. Therefore, the invention also provides a process for the preparation of a compound of Formula (X) comprising treating a compound of Formula (V)
Figure imgf000024_0001
in which A an alkoxy, benzyloxy or silyloxy group, and R3 is as defined above for Formula (I), with a base and a halogen source.
Compounds of Formula (X) in which A is an alkoxy, benzyloxy or silyloxy group, R3 is as defined above for Formula (I) and X is a halogen such as bromide or iodide, may also be prepared by reaction of a compound of Formula (Xl)'
Figure imgf000024_0002
wherein P is -COCF3 or -C02tBu and R3 is as defined above for Formula (I), by treatment with a halogen source, for example iodine, in a suitable solvent such as THF, heptane, ethylbenzene, or combinations thereof, in the presence of a suitable base such as LDA, at a suitable temperature for example -78 to -200C, optionally in an inert atmosphere. Deprotection of the P group can be carried out for example, when P is -COCF3, by treatment with a suitable base such as aqueous potassium carbonate optionally in the presence of an alcohol such as methanol, or when P is -CO2 1Bu and by treatment with a suitable acid such as hydrochloric acid or trifluoroacetic acid in a suitable solvent such as 1 ,4-dioxane or dichloromethane.
Compounds of Formula (Xl)' in which P is -COCF3 and R3 is as defined above for Formula (I), may be prepared from a compound of Formula (V)
Figure imgf000024_0003
in which A an alkoxy, benzyloxy or silyloxy group, and R3 is as defined above for Formula (I), in treatment with trifluoroacetic anhydride in a suitable solvent such as diethylether. Compounds of Formula (Xl)' in which P is C02tBu and R3 is as defined above for Formula (I), may be prepared from a compound of Formula (V), by treatment with trifluoroacetic anhydride or di-tert-butyl dicarbonate in a suitable solvent such as ether, acetonitrile or acetone, optionally in the presence of a catalyst such as DMAP and a base such as triethylamine.
Compounds of Formula (Xl) in which A is an alkoxy, benzyloxy or silyloxy group as defined above for Formula (I) and X is a halogen such as iodide may be prepared from a compound of Formula (XII)
Figure imgf000025_0001
in which P is a suitable protecting group such as -COCF3 or -C(VBu and P' is hydrogen or a suitable protecting group such as -CO2 1Bu. For example, when P is -COCF3 and P' is hydrogen, by treatment with a suitable base such as aqueous potassium carbonate optionally in the presence of an alcohol such as methanol, or when P is -CO2 1Bu and P' is hydrogen or -CO2 1Bu, by treatment with a suitable acid such as hydrochloric acid or trifluoroacetic acid in a suitable solvent such as 1 ,4-dioxane or dichloromethane.
Compounds of Formula (XII) in which A is an alkoxy, benzyloxy or silyloxy group as defined above for Formula (I), X is a halo atom such as iodide and P is a suitable protecting group such as -COCF3 and P' is hydrogen, or P is -CO2 1Bu and P' is hydrogen or -CO2 1Bu, may be prepared by reaction of a compound of Formula (XIII)
Figure imgf000025_0002
with a suitable base such as lithium diisopropylamide and a halogen source such as iodine, in a suitable solvent such as tetrahydrofuran, and at a temperature in the range -78°C to - 200C.
Compounds of Formula (XIII) in which A is an alkoxy, benzyloxy or silyloxy group as defined above for Formula (I) and P and P' are as described above for Formula (XII), may be prepared by treating compounds of Formula (Vl) with trifluoroacetic anhydride or di-tert-butyl dicarbonate in a suitable solvent such as ether, acetonitrile or acetone, optionally in the presence of a catalyst such as DMAP and a base such as triethylamine.
Compounds of Formula (III) in which A is an alkoxy, benzyloxy or silyloxy group may also be prepared from compounds of Formula (XIV)
Figure imgf000026_0001
in which A is an alkoxy, benzyloxy or silyloxy, and R2 is as defined above for Formula (I), by treatment with a suitable alkylating agent R3-X' where X' is a halo group such as chloride, bromide or iodide, or X' is a sulphonate ester such as methanesulfonate and R3 is as defined above for Formula (I), in suitable solvent such as dimethylformamide in the presence of a suitable base such as triethylamine.
Compounds of Formula (XIV) in which A is an alkoxy, benzyloxy or silyloxy group may also be prepared from compounds of Formula (Xl) in which A is an alkoxy, benzyloxy or silyloxy group and X is a halogen such as bromide or iodide, with a suitable acylating agent, for example R2-C(O)-Y, wherein Y is a halo atom, for example chloro or bromo, and R2 is as defined above for Formula (I). The reaction may be carried out in a suitable solvent, for example dichloromethane, in the presence of a suitable base, for example pyridine or triethylamine. A phosphine such as triphenylphosphine may optionally be used in place of the base. The reaction may be carried out at a suitable temperature, for example in the range 200C to 800C.
Compounds of Formula (III) in which A is an alkoxy, benzyloxy or silyloxy group and R2 and R3 are as defined above may also be prepared by reaction of a compound of Formula (X)
Figure imgf000026_0002
in which A an alkoxy, benzyloxy or silyloxy group, and R3 is as defined above for Formula (I) and X is a halogen such as bromide or iodide, with a suitable acylating agent, for example R2-C(O)-Y, wherein Y is a halo atom, for example chloro or bromo, and R2 is as defined above for Formula (I). The reaction may be carried out in a suitable solvent, for example dichloromethane, in the presence of a suitable base, for example pyridine or triethylamine. A phosphine such as triphenylphosphine may optionally be used in place of the base.
Compounds of Formula (III)' in which A is an alkoxy, benzyloxy or silyloxy group and R3 is as defined above may also be prepared by reaction of a compound of Formula (X)'
Figure imgf000027_0001
in which A an alkoxy, benzyloxy or silyloxy group, and R3 is as defined above for Formula (I) and X is a suitable boronic acid -B(OH)2 or boronate ester
-B(OR')(OR"), in which R' and R" are independently alkyl or R' and R" together with the carbon atoms to which they are attached form a ring optionally substituted by alkyl, such as a pinacol ester, with a suitable acylating agent, for example R2-C(0)-Y, wherein Y is a halo atom, for example chloro or bromo, and R2 is as defined above for Formula (I). The reaction may be carried out in a suitable solvent, for example dichloromethane, in the presence of a suitable base, for example pyridine or triethylamine. A phosphine such as triphenylphosphine may optionally be used in place of the base.
Compounds of Formula (X)' in which A is an alkoxy, benzyloxy or silyloxy group and R3 is as defined above and X is a suitable boronic acid -B(OH)2 or boronate ester -B(OR')(OR"), in which R' and R" are independently alkyl or R' and R" together with the carbon atoms to which they are attached form a ring optionally substituted by alkyl, such as a pinacol ester, may also be prepared by reaction of a compound of Formula (V) in which A an alkoxy, benzyloxy or silyloxy group, and R3 is as defined above for Formula (I), by treatment with a suitable base such as lithium diisopropylamide and a boronate source such as B(OR)3 wherein R is an alkyl group, for example methyl, in a suitable solvent such as tetrahydrofuran, and at a temperature in the range -78°C to -200C.
Compounds of Formula (Vl) and (VII) are commercially available or well known in the art.
Compounds of Formula (I) in which A is hydroxy, or (II) in which A is an alkoxy, benzyloxy or silyloxy group, may be Formula (II)'
Figure imgf000027_0002
in which Z represents a halo substituent, and Rx, R2, R3, and A are as defined for Formula (II) by reaction with a suitable heteroaryl boronic acid Rγ-boronic acid, in the presence of a palladium catalyst such as palladium (II) acetate, a reagent such as 2- dicyclohexylphosphino-2'(N,N-dimethylamino)-biphenyl, and an additional reagent such as caesium fluoride, in a suitable solvent such as 1 ,4-dioxane. Compounds of Formula (I) in which A is hydroxy, or (II) in which A is an alkoxy, benzyloxy or silyloxy group, may also be nd of Formula (II)'
Figure imgf000028_0001
in which Z represents B(OH)2, and Rx, R2, R3 and A are as defined for Formula (II), by reaction with a suitable heteroaryl halide Rγ-hal, in which suitably the halide is bromo or iodo, in the presence of a palladium catalyst such as palladium (II) acetate, a reagent such as 2- dicyclohexylphosphino-2'(N,N-dimethylamino)-biphenyl, and an additional reagent such as caesium fluoride, in a suitable solvent such as 1 ,4-dioxane.
Compounds of Formula (II)' in which Z is halo may be prepared by reaction of a compound of Formula (III) wherein X is halo, with a boronic acid of Formula Z-Rx-boronic acid wherein Z is halo and Rx is as defined above for Formula (I) under the conditions described above for the preparation of compounds of Formula (I) and (II) from (IX) and Rγ-Rx-boronic acid.
Compounds of Formula (II)' in which Z is B(OH)2 may be prepared by reaction of a compound of Formula (III) with a compound of Formula Z-RX-B(OH)2 wherein Rx is as described above for Formula (I), under the conditions described above for the preparation of compounds of Formula (I) and (II) from (III) and R1-boronic acid.
Boronic acids Z-Rx-boronic acid, Rγ-Rx-boronic acid and Rγ-boronic acid are commercially available or may be prepared by analogy to methods provided in Organometallics (1983) 2, 1316, Chem Revs. (1995) 95, 2457, Journal of Org Chem (2004) 69, 1999, SynLett (2004) (5), 892, Bioorg Med Chem (2005) 13, 2305, Tetrahedron Letters (2004) 44, 9359 and Tetrahedron Letters (2005) 45, 6657.
Compounds of Formula (I) or (II) in which R1 represents a 4-(furopyridine)phenyl and R2, R3 and A are as defined above for Formula (II), may be prepared by treatment of a compound of Formula (II)"
Figure imgf000028_0002
in which R1 represents a 4-ethynylphenyl derivative, and R2, R3 and A are as defined above for Formula (II), with a suitable pyridine (the pyridine being substituted with adjacent hydroxy and iodo groups), with a suitable catalyst such as bis(triphenylphosphine)palladium (II) chloride and copper (I) iodide, in a suitable solvent such as triethylamine or DMF. Suitably the temperature is in the range 50-800C. For examples of furopyridine synthesis see Bioorganic and Medicinal Chemistry Letters (2002) 12, 1399, Synthesis (1986) 749.
Compounds of Formula (I) or (II) in which R1 represents a 4-(pyrrolopyridine)phenyl and R2, R3 and A are as defined above for Formula (II), may be prepared by treatment of a compound of Formula (II)" in which R1 represents 4-ethynylphenyl and R2, R3 and A are as defined above for Formula (ll)with an appropriate pyridine (the pyridine being substituted by adjacent amino and iodo groups), in the presence of a suitable catalyst such as bis(triphenylphosphine)palladium (II) chloride and copper (I) iodide, in a suitable solvent such as triethylamine. Suitably the temperature is in the range 50-800C. For examples of pyrrolopyridine synthesis see Heterocycles (1986) 24, 31 , Tetrahedron (2003) 59, 1571 , Synlett (1992) 515.
Compounds of Formula (I) or (II) in which R1 represents phenyl substituted by a 4- imidazo[1 ,2-a]pyridine-2-yl and R2, R3 and A are as defined above for Formula (II), may be prepared by analogy to methods described in Tetrahedron Letters (2001 ) 42, 3077.
Compounds of Formula (I) or (II) in which R1 represents a 4-(pyrazolopyrimidine)phenyl and R2, R3 and A are as defined above for Formula (II), may be prepared by treating a compound of Formula (II)" in which R1 represents 4-(phenyl)-1 H-pyrazole-5-amine and R2, R3 and A are as defined above for Formula (II) with 1 ,1 ,3,3-tetramethoxypropane in a suitable solvent such as acetic acid; suitably the temperature is in the range 90-1 100C.
Suitable methods for the preparation of compounds incorporating the above discussed Rγ derivatives may be found in the chemical literature, for example those described in Comprehensive Heterocyclic Chemistry, Edited by A.R. Katritzky and CW. Rees, Pergamon 1984, and Heterocyclic Chemistry, Edited by J.A. Joules and K. Mills, 4th Ed, Blackwell Science.
Compounds of Formula RY-RX-B(OR')2 or RY-B(OR')2 for use in the preparation of compounds of Formula (II) are available commercially or may be prepared from compounds of Rγ-Rx-hal or Rγ-hal by methods well known in the art.
Compounds of Formula Rγ-Rx-hal or Rγ-hal for use in the preparation of compounds of Formula (II) are available commercially or may be prepared by methods well known in the art. Some examples of heteroaryl halide preparation are given below (but are not limited to these examples).
A 2-(4-bromophenyl)imidazo[1 ,2-a]pyridine derivative may be prepared by analogy to methods described in Tetrahedron Letters (2001 ) 42, 3077.
A 4-(furopyridine)phenyl bromide, may be prepared by treatment of a 4-ethynylphenyl bromide with a suitable pyridine (the pyridine being substituted with adjacent hydroxy and iodo groups), with a suitable catalyst such as bis(triphenylphosphine)palladium (II) chloride and copper (I) iodide, in a suitable solvent such as triethylamine or DMF. Suitably the temperature is in the range 50-800C. For examples of furopyridine synthesis see Bioorganic and Medicinal Chemistry Letters (2002) 12, 1399, Synthesis (1986) 749.
A 4-(pyrazolopyrimidine)phenyl bromide may be prepared by treating a 3-(4-bromophenyl)- 1 H-pyrazole-5-amine with 1 ,1 ,3,3-tetramethoxypropane in a suitable solvent such as acetic acid. Suitably the temperature is in the range 90-1100C.
Esters of compounds of Formula (I), in which A is -OR where R is selected from straight or branched chain alkyl, aralkyl, aryloxyalkyl, or aryl, may also be prepared by esterification of a compound of Formula (I) in which A is hydroxy by standard literature procedures for esterification.
It will be appreciated that compounds of Formula (I), (II), (III), (IV), (VIII), (IX) and (X) which exist as diastereoisomers may optionally be separated by techniques well known in the art, for example by column chromatography or recrystallisation. For example, the formation of an ester using a chiral alcohol, separation of the resulting diastereoisomers, and subsequent hydrolysis of the ester to yield the individual enantiomeric acid of Formula (I) (II), (III), (IV), (VIII), (IX) and (X).
It will be appreciated that racemic compounds of Formula (I), (II), (III), (IV), (VIII), (IX) and (X) 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), (II), (III), (IV), (VIII), (IX) and (X) may be resolved by chiral preparative HPLC. Alternatively, racemic compounds of Formula (I), (II), (III), (IV), (VIII), (IX) and (X) 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. For example, a racemic compound may be resolved by treatment with a chiral acid such as (R)-(-)-1 ,1 '-binaphthyl-2,2'-diyl-hydrogen phosphate or (-)-di-O,O'-p-tolyl-L-tartaric acid, in a suitable solvent, for example isopropanol. The free enantiomer may then be obtained by treating the salt with a suitable base, for example triethylamine, in a suitable solvent, for example methyl te/f-butyl ether. Alternatively, racemic acid compounds may be resolved using a chiral base, for example (S)-alpha methylbenzylamine, (S)-alpha phenylethylamine, (1S, 2S)-(+)-2-amino-1-phenyl-1 ,3-propane-diol, (-) ephidrine, quinine, brucine. Individual enantiomers of Formula (II), (III), (IV), (VIII), (IX) and/or (X) may then be progressed to an enantiomeric compound of Formula (I) by the chemistry described above in respect of racemic compounds.
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.
Various of the synthetic procedures described above in general terms (and below in specific terms) may involve heating the reactants. It will be appreciated that heating may be carried out by various conventional methods but also with the use of a microwave reactor.
EXAMPLES ABBREVIATIONS
AcOH acetic acid
BINAP 2,2'-bis(diphenylphosphino)-1 ,1 '-binaphthyl
DCE 1 ,2-dichloroethane
DCM dichloromethane
DMAP 4-(dimethylamino)pyridine
DME 1 ,2-dimethoxyethane
DMF N,N-dimethylformamide
EtOAc ethyl acetate
EtOH ethanol
Et2O diethyl ether h hours
HCI hydrochloric acid
HPLC high pressure liquid chromatography
ISCO Companion automated flash chromatography equipment with fraction analysis by UV absorption available from Presearch.
LDA lithium Diisopropylamide (2M in tetrahydrofuran/heptane/ethyl benzene)
MDAP HPLC reverse phase HPLC on a Cis column using a two-solvent gradient elution with (A) water containing formic acid (0.1 %) and (B) acetonitrile-water (95:5 v/v) containing formic acid
(0.05%) as the eluents, and analysis of the fractions by electrospray mass spectroscopy.
MeCN acetonitrile MeOH methanol mins minutes NH2 SPE aminopropyl capped silica ion-exchange solid phase extraction cartridge
OASIS HLB cartridge Sample extraction cartridge available from Waters
PdCI2(dppf) [1 ,1 '-bis(diphenylphosphino)ferrocene]dichloro-palladium(ll)
Pd(PPh3)4 tetrakis(triphenylphosphine) palladium RT room temperature
SPE silica packed solid phase extraction column
TFA trifluoroacetic acid
THF tetrahydrofuran
All mass spectroscopy was performed using electrospray as the method of ionisation.
Intermediate 1 2-(4-Bromophenyl)furo[3,2-b]pyridine
Figure imgf000032_0001
To 4-bromophenylactetylene (4.3 g) was added 2-iodo-3-hydroxypyridine (5.25 g), bis(triphenylphosphine)palladium dichloride (1.5 g), copper (I) iodide (0.58 g) and triethylamine (100 ml_). The reaction was stirred at 9O0C, under nitrogen, for 4 h. The reaction was cooled, diluted with EtOAc (400 ml.) and washed twice with saturated ammonium chloride solution, brine and then concentrated. The crude product was dissolved in DCM, filtered and purified by ISCO companion silica chromatography eluting with a gradient of ethyl acetate in cyclohexane (30-100%) to give the title compound. MS calcd for (C13H8BrNO + H)+: 274/276 MS found (electrospray): (M+H)+ = 274/276
Intermediate 2
2-[4-(4,4,5,5-Tetramethyl-1 ,3,2-dioxaborolan-2-yl)phenyl]furo[3,2-fe]pyridine
Figure imgf000032_0002
A mixture of Intermediate 1 (250 mg), bis(pinacolato)diboron (345 mg), potassium acetate (265 mg) and 1 ,1 '-bis (diphenylphosphino)ferrocene dichloro palladium (II) (48 mg) in dry 1 ,4-dioxane (5 ml.) was heated to 100°C under nitrogen for 7 h. The solvent was evaporated and the residue was partitioned between water (10 ml.) and DCM (20 ml_). The aqueous phase was extracted further with DCM (10 ml.) and the combined organics evaporated. The residue was purified by SPE chromatography eluting with cyclohexane/EtOAc (2:1 then 1 :1 ). Further purification by SPE chromatography, eluting with cyclohexane then cyclohexane/EtOAc (3:1 ) to (1 :1 ) to (1 :2) then EtOAc, EtOAc/MeCN (1 :1 ) and finally MeCN gave the title compound. MS calcd for (Ci9H20BNO3+ H)+: 322 MS found (electrospray): (M+H)+ = 322 Intermediate 3 2-(4-Bromophenyl)pyrazolo[1,5-a]pyrimidine
Figure imgf000033_0001
A solution of 3-(4-bromophenyl)-1 H-pyrazol-5-amine (5.00 g) in AcOH (80 ml.) was treated with 1 ,1 ,3,3-tetramethoxypropane (4.13 g) and the mixture heated at 11O0C for 1.5 h. On cooling to room temperature, the precipitated solid was isolated by filtration, washed with water (3 x 10 ml.) and dried in vacuo at 400C. This was recrystallised from acetic acid and dried in vacuo at 400C to give the title compound. MS calcd for (C12H8BrN3+ H)+: 274/276
MS found (electrospray): (M+H)+ = 274/276
Intermediate 4 2-[4-(4,4,5,5-Tetramethyl-1 ,3,2-dioxaborolan-2-yl)phenyl]pyrazolo[1,5-a]pyrimidine
Figure imgf000033_0002
A mixture of Intermediate 3 (100 mg), bis(pinacolato)diboron (134 mg), potassium acetate
(108 mg) and 1 ,1 '-bis (diphenylphosphino)ferrocene dichloropalladium (II) (20 mg) in dry 1 ,4- dioxane (2 ml.) was heated at 100°C under nitrogen for 15 h. The solvent was evaporated and the residue partitioned between water (10 ml.) and DCM (20 ml_). The aqueous phase was extracted further with DCM (10 ml.) and the combined organics evaporated. This was purified by SPE chromatography, eluting with cyclohexane/EtOAc (3:1 ) to give the title compound.
MS calcd for (Ci8H20BN3O2+ H)+: 322
MS found (electrospray): (M+H)+ = 322
Intermediate 5
2-[4-(4,4,5,5-Tetramethyl-1 ,3,2-dioxaborolan-2-yl)phenyl]imidazo[1 ,2-a]pyridine
Figure imgf000033_0003
A mixture of 2-(4-bromophenyl)imidazo[1 ,2-a]pyridine1 (300 mg), bis(pinacolato)diboron (508 mg), potassium acetate (393 mg) and 1 ,1 '-bis (diphenylphosphino)ferrocene dichloro palladium (II) (58 mg) in dry 1 ,4-dioxane (6 ml_) was heated at 100°C under nitrogen for 24 h.
The solvent was evaporated and the residue was partitioned between water (10 ml.) and
DCM (20 ml_). The aqueous phase was extracted further with DCM (10 ml.) and the combined organics evaporated. The residue was purified by SPE chromatography eluting with cyclohexane/EtOAc (3:1 ). Further purification by SPE (silica) eluting with cyclohexane then cyclohexane/EtOAc [(3:1 ) followed by (1 :1 )1 gave the title compound. MS calcd for (Ci9H21BN2O2+ H)+: 321 MS found (electrospray): (M+H)+ = 321
Ref 1 : Burkholder, Conrad; Dolbier, William R.; Medebielle, Maurice; Ait-Mohand, Samia, Tetrahedron Lett, 42, 17, 2001 , 3077 - 3080.
Intermediate 6 6-[4-(4,4,5,5-Tetramethyl-1 ,3,2-dioxaborolan-2-yl)phenyl]imidazo[2,1-b][1,3]thiazole
Figure imgf000034_0001
6-(4-lodophenyl)imidazo[2,1-b][1 ,3]thiazole (2 g) was dissolved in dry 1 ,4-dioxane (40 ml_). To this stirred solution was added potassium acetate (1.8 g), bis(pinacolato)diboron (2.34 g) and 1 ,1 '-bis (diphenylphosphino)ferrocene dichloro palladium (II) (350 mg). The reaction mixture was then heated to 100 0C, and stirred under nitrogen for 18 h. Further, potassium acetate (1.8 g), bis(pinacolato) diboron (2.3 g) and 1 ,1 '-bis (diphenylphosphino)ferrocene dichloropalladium (II) ( 350 mg,) was added. The reaction mixture was then left to stir at 1000C for a further 24 h. The solvent was then removed by evaporation and the residue was partitioned between water and DCM. The layers were separated using a hydrophobic frit, and the organic phase was concentrated by evaporation to give a solid. This was purified by ISCO companion silica chromatography eluting with a gradient of EtOAc / cyclohexane (10% to 50%), to give the title compound MS calcd for (C17H19BN2O2S+ H)+: 327 MS found (electrospray): (M+H)+ = 327
Intermediate 7 2-(4-Bromophenyl)pyrazolo[1,5-a]pyrimidin-7 -amine
Figure imgf000034_0002
5-Amino-3-(4-bromophenyl)pyrazole (5 g) was dissolved in acetic acid (80 ml.) and trans-3-
(dimethylamino)-acrylonitrile (2.3 ml.) was added. The reaction heated to 11 O0C for 3 h, and was then allowed to cool to room temperature before being evaporated in vacuo. The residue was treated with saturated sodium bicarbonate solution and the resulting precipitate was filtered off, washed with water and dried in a vacuum oven for 18 h to give the title compound.
MS calcd for (C12H9N4Br+ H) + : 289/291
MS found (electrospray): (M+H)+= 289/291 Intermediate 8
2-[4-(4,4,5,5-Tetramethyl-1 ,3,2-dioxaborolan-2-yl)phenyl]pyrazolo[1,5-a]pyrimidin-7- amine
Figure imgf000035_0001
Intermediate 7 (1.5 g) was dissolved in dry 1 ,4-dioxane (30 ml_). To this stirred solution was added potassium acetate (1.42 g), bis(pinacolato)diboron (1.71 g) and 1 ,1 '-bis (diphenylphosphino)ferrocene dichloro palladium (II) (254 mg). The reaction mixture was heated to 100 0C, and stirred under nitrogen for 18 h. The reaction was then cooled and recharged with potassium acetate (1.42 g), bis(pinacolato)diboron (1.71 g) and 1 ,1 '-bis (diphenylphosphino)ferrocene dichloro palladium (II) (254 mg). The reaction mixture was then reheated to 100 0C, and stirred under nitrogen for another 18 h. The reaction was allowed to cool and the solvent removed by evaporation and the residue was partitioned between water and DCM. The organic layer was separated, washed with water, and concentrated by evaporation. The residue was then purified by ISCO companion silica chromatography eluting with a gradient of EtOAc / cyclohexane (0% to 100%), to give the title compound.
MS calcd for (C18H21BN4O2+ H)+: 337 MS found (electrospray): (M+H)+ = 337
Intermediate 9
Methyl 3-[(1 -methylethyl)amino]-2-thiophenecarboxylate
Figure imgf000035_0002
To methyl-3-amino-2-thiophene carboxylate (10.0 g) in DCM (200 ml.) under nitrogen was added 2-methoxypropene (18.4 ml_), glacial acetic acid (1 1.2 ml.) and sodium triacetoxyborohydride (20.2 g) portionwise. The reaction mixture was stirred at room temperature overnight. 2-Methoxypropene (9.1 ml.) and sodium triacetoxyborohydride (10.1 g) were added and the reaction mixture was stirred at room temperature for 3 h. Water (200 ml.) was added to the reaction mixture which was neutralised by addition of solid sodium bicarbonate. The DCM was separated and washed with sodium bicarbonate solution. The organic extract was dried by passing through a hydrophobic frit and evaporated to dryness to give the title compound. MS calcd for (C9H13NO2S + H)+ : 200 MS found (electrospray) : (M+H)+ = 200 Intermediate 10
Methyl 3-[(1-methylethyl)(trifluoroacetyl)amino]-2-thiophenecarboxylate
Figure imgf000036_0001
A solution of Intermediate 9 (9.1 g) in diethylether (180 mL) was cooled to O0C. Trifluoroacetic anhydride (9.5 mL) was added and the reaction mixture was stirred for 1 h then warmed to room temperature and evaporated to dryness. The residue was azeotroped with toluene (x3) to give the title compound. MS calcd for (CnH12F3NO3S + H)+ : 296 MS found (electrospray) : (M+H)+ = 296
Intermediate 11
Methyl 5-iodo-3-[(1 -methylethyl)amino]-2-thiophenecarboxylate
Figure imgf000036_0002
LDA (15.2 mL) was cooled to -780C under nitrogen. A solution of Intermediate 10 (3.0 g) in anhydrous THF (30 mL) was added dropwise keeping the internal temperature between - 730C and -650C. The reaction mixture was stirred at -740C for 1 h. A solution of iodine (5.1 g) in anhydrous THF (30 mL) was added dropwise over -30 min maintaining the internal temperature <-65°C. Saturated NH4CI (aq) (15 mL) was added dropwise after 15 mins and the reaction mixture was warmed to room temperature before washing with 5% sodium thiosulphate solution. The organic was separated and the aqueous extracted with EtOAc (x2). The combined organics were dried with sodium sulphate and evaporated to dryness. The residue was purified by ISCO companion silica chromatography eluting with a gradient of ethyl acetate in cyclohexane to give the title compound. MS calcd for (C9H12INO2S + H)+ : 326 MS found (electrospray) : (M+H)+ = 326
Intermediate 12
Methyl 5-(4-imidazo[1 ,2-a]pyridin-2-ylphenyl)-3-[(1 -methylethyl)amino]-2- thiophenecarboxylate
Figure imgf000036_0003
Intermediate 11 (2.0 g), 2-[4-(4,4!5!5-tetramethyl-1 !3,2-dioxaborolan-2-yl)phenyl]imidazo[1 !2- a]pyridine (a synthesis of which is described in Intermediate 5) (2.8 g), Pd(PPh3)4 (955 mg) and Na2CO3 (2.6g in H2O (-15 ml.)) were dissolved in DMF (180 ml.) and were heated at 1000C for 3 h. The reaction mixture was evaporated to dryness and the residue partitioned between water and DCM. The aqueous phase was separated and extracted with more DCM (x2). The combined organic phases were dried using a hydrophobic frit and evaporated to dryness. This was purified by ISCO companion silica chromatography eluting with a gradient of ethyl acetate in cyclohexane to give the title compound. MS calcd for (C22H21N3O2S + H)+ : 392 MS found (electrospray) : (M+H)+ = 392
Intermediate 13
Methyl 3-[[(2,4-dichlorophenyl)carbonyl](1 -methylethyl)amino]-5-(4-imidazo[1 ,2- a]pyridin-2-ylphenyl)-2-thiophenecarboxylate
Figure imgf000037_0001
Intermediate 12 (200 mg), 2,4-dichlorobenzoyl chloride (243 mg), and triphenylphosphine (334 mg) were dissolved in anhydrous DCM (6 ml.) and were heated at reflux for 2 days. The reaction mixture was cooled and sodium bicarbonate solution was added to the reaction mixture which was stirred at room temperature for 1 h. The organic was separated and dried by passing through a hydrophobic frit and was evaporated to dryness. This was purified by ISCO companion silica chromatography eluting with a gradient of ethyl acetate in cyclohexane to give the title compound. MS calcd for (C29H23CI2N3O3S + H)+ : 564/565/566 MS found (electrospray) : (M+H)+ = 564/565/566
Intermediate 14
Methyl 3-[[(4-chloro-2-fluorophenyl)carbonyl](1 -methylethyl)amino]-5-(4-imidazo[1 ,2- a]pyridin-2-ylphenyl)-2-thiophenecarboxylate
Figure imgf000037_0002
Similarly prepared to the process described for Intermediate 13, from Intermediate 12 (165 mg), 2-fluoro-4-chlorobenzoyl chloride (203 mg) and PPh3 (276 mg, 2.5 eq) in DCM (6 ml.) . MS calcd for (C29H23CIFN3O3S + H)+ : 548/550 MS found (electrospray) : (M+H)+ = 548/550
Intermediate 15
Methyl 3-[[(4-bromo-2-methylphenyl)carbonyl](1 -methylethyl)amino]-5-(4-imidazo[1 ,2- a]pyridin-2-ylphenyl)-2-thiophenecarboxylate
Figure imgf000038_0001
Similarly prepared to the process described for Intermediate 13, from Intermediate 12 (200 mg), 2-methyl-4-bromobenzolyl chloride (217 mg) and PPh3 (334 mg, 2.5 eq) in DCM (6 ml_). MS calcd for (C30H26BrN3O3S + H)+ : 588/590 MS found (electrospray) : (M+H)+ = 588/590
Intermediate 16
Methyl 3-[(1 -methylethyl)amino]-5-(4-pyrazolo[1 ,5-a]pyrimidin-2-ylphenyl)-2- thiophenecarboxylate
Figure imgf000038_0002
Methyl 5-iodo-3-[(1-methylethyl)amino]-2-thiophenecarboxylate (a synthesis of which is described in Intermediate 1 1 ) (2.1 g), 2-[4-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2- yl)phenyl]pyrazolo[1 ,5-a]pyrimidine (a synthesis of which is described in Intermediate 4) (2.9 g), Pd(PPh3)4 (1.0 g) and Na2CO3 (2.8g in H2O (-15 mL)) were dissolved in DMF (190 mL) and were heated at 1000C for 3 h. The reaction mixture was evaporated to dryness and the residue partitioned between water and DCM. The aqueous phase was separated and extracted with more DCM (x2). The combined organic phases were dried using a hydrophobic frit and evaporated to dryness. This was purified by ISCO companion silica chromatography eluting with a gradient of ethyl acetate in cyclohexane to give the title compound.
MS calcd for (C21H20N4O2S + H)+ : 393 MS found (electrospray) : (M+H)+ = 393
Intermediate 17
Methyl 3-[[(2,4-dichlorophenyl)carbonyl](1 -methylethyl)amino]-5-(4-pyrazolo[1 ,5- a]pyrimidin-2-ylphenyl)-2 -thiophenecarboxylate
Figure imgf000039_0001
Intermediate 16 (150 mg), 2,4-dichlorobenzoyl chloride (250 mg), and triphenylphosphine (250 mg) were dissolved in anhydrous DCM (6 ml.) and were heated at reflux overnight. The reaction mixture was cooled and sodium bicarbonate solution was added to the reaction mixture which was stirred at room temperature for 1 h. The organic was separated and dried by passing through a hydrophobic frit and was evaporated to dryness. This was purified by ISCO companion silica chromatography eluting with a gradient of ethyl acetate in cyclohexane to give the title compound. MS calcd for (C28H22CI2N4O3S + H)+ : 565/567/568 MS found (electrospray) : (M+H)+ = 565/567/568
Intermediate 18
Methyl 3-[[(4-chloro-2-fluorophenyl)carbonyl](1 -methylethyl)amino]-5-(4-pyrazolo[1 ,5- a]pyrimidin-2-ylphenyl)-2-thiophenecarboxylate
Figure imgf000039_0002
Similarly prepared to Intermediate 17 from Intermediate 16. MS calcd for (C28H22CIN4O3SF + H)+ : 549/551 MS found (electrospray) : (M+H)+ = 549/551
Intermediate 19
Methyl 3-[[(4-bromo-2-methylphenyl)carbonyl](1 -methylethyl)amino]-5-(4-pyrazolo[1 ,5- a]pyrimidin-2-ylphenyl)-2-thiophenecarboxylate
Figure imgf000039_0003
Similarly prepared to Intermediate 17 from Intermediate 16. MS calcd for (C29H25BrN4O3S + H)+ : 589/591 MS found (electrospray) : (M+H)+ = 589/591 Intermediate 20
Methyl 3-[[(2,4-dimethylphenyl)carbonyl](1 -methylethyl)amino]-5-(4-pyrazolo[1 ,5- a]pyrimidin-2-ylphenyl)-2-thiophenecarboxylate
Figure imgf000040_0001
Similarly prepared to Intermediate 17 from Intermediate 16. MS calcd for (C30H28N4O3S + H)+ : 525 MS found (electrospray) : (M+H)+ = 525
Intermediate 21
Methyl 3-[[(2,4-dichlorophenyl)carb mino]-2-thiophenecarboxylate
Figure imgf000040_0002
To a solution of methyl 3-[(1-methylethyl)amino]-2-thiophenecarboxylate (a synthesis of which is described in Intermediate 9) (5 g) in pyridine (45 ml.) was added 2,4- dichlorobenzoyl chloride (5.3 ml_). The reaction mixture was heated at 7O0C under nitrogen overnight. The reaction mixture was partitioned between EtOAc and sodium bicarbonate solution. The organics were washed with brine, dried with sodium sulphate and evaporated to dryness. This was purified by ISCO companion silica chromatography eluting with a gradient of ethyl acetate in cyclohexane to give the title compound. MS calcd for (C16H15NO3SCI2 + H)+ : 372/373/374 MS found (electrospray) : (M+H)+ = 372/373/374
Intermediate 22
Methyl 3-[[(2,4-dichlorophenyl)carbonyl](1 -methylethyl)amino]-5-iodo-2- thiophenecarboxylate
Figure imgf000040_0003
LDA (9.5 ml.) was cooled to -780C under nitrogen. A solution of Intermediate 21 (3.0 g) in anhydrous THF (30 ml.) was added dropwise keeping the internal temperature between - 730C and -650C. The reaction mixture was stirred at -740C for 15 min. A solution of iodine (6.1 g) in anhydrous THF (30 ml.) was added dropwise over -20 min maintaining the internal temperature <-65°C. Sat. NH4CI (aq) (10 ml.) was added dropwise after 15 min and the reaction mixture was warmed to room temperature before washing with 5% sodium thiosulphate solution. The organic was separated and the aqueous extracted with EtOAc (x2). The combined organics were dried with sodium sulphate and evaporated to dryness. The residue was purified by ISCO companion silica chromatography eluting with a gradient of ethyl acetate in cyclohexane to give the title compound. MS calcd for (Ci6H14NO3SCI2I + H)+ : 498/500/502 MS found (electrospray) : (M+H)+ = 498/500/502
Intermediate 23 Methyl 3-[[(2,4<lichlorophenyl)carbonyl](1 -methylethyl)amino]-5-(4-imidazo[2,1- fe][1,3]thiazol-6-ylphenyl)-2-thiophenecarboxylate
Figure imgf000041_0001
Intermediate 22 (334 mg), 6-[4-(4,4!5!5-tetramethyl-1 !3,2-dioxaborolan-2- yl)phenyl]imidazo[2,1-b][1 ,3]thiazole (a synthesis of which is described in Intermediate 6) (307 mg), Pd(PPh3)4 (104 mg) and Na2CO3 (284 mg in H2O (~3 ml.)) were dissolved in DMF (8 ml.) and were heated at 1000C for 2 h. The reaction mixture was evaporated to dryness and the residue partitioned between water and DCM. The aqueous phase was separated and extracted with more DCM (x2). The combined organic phases were dried using a hydrophobic frit and evaporated to dryness. This was purified by ISCO companion silica chromatography eluting with a gradient of ethyl acetate in cyclohexane to give the title compound.
MS calcd for (C27H21CI2N3O3S2+ H)+ : 570/572/573 MS found (electrospray) : (M+H)+ = 570/572/573
Intermediate 24
Methyl S-I^^-dichlorophenylJcarbonyllti -methylethylJaminol-S^-furoIS^-felpyridin- 2-ylphenyl)-2-thiophenecarboxylate
Figure imgf000042_0001
Similarly prepared to Intermediate 23 from Intermediate 22 (334 mg, 0.67 mmol) and Intermediate 2 (302 mg, 0.94 mmol, a synthesis of which is described as Intermediate 2). MS calcd for (C29H22CI2N2O4S + H)+ : 565/567/568 MS found (electrospray) : (M+H)+ = 565/567/568
Intermediate 25
Methyl 5-[4-(7-aminopyrazolo[1 ,5-a]pyrimidin-2-yl)phenyl]-3-[[(2,4- dichlorophenyl)carbonyl](1 -methylethyl)amino]-2-thiophenecarboxylate
Figure imgf000042_0002
Similarly prepared to Intermediate 23 from Intermediate 22 (334 mg, 0.67 mmol) and Intermediate 8 (316 mg, 0.94 mmol, a synthesis of which is described as Intermediate 8). MS calcd for (C28H23CI2N5O3S + H)+ : 580/582/583 MS found (electrospray) : (M+H)+ = 580/582/583
Intermediate 26
Methyl 3-[[(4-chloro-2-fluorophenyl)carbonyl](1-methylethyl)amino]-5-phenyl-2- thiophenecarboxylate
Figure imgf000042_0003
Methyl 3-[(1-methylethyl)amino]-5-phenyl-2-thiophenecarboxylate (a synthesis of which is described in Intermediate 27) (400 mg), 4-chloro-2-fluorobenzoyl chloride (700 mg), and triphenylphosphine (951 mg) were dissolved in anhydrous DCM (8 ml.) and were heated at reflux overnight. The reaction mixture was cooled and sodium bicarbonate solution was added to the reaction mixture which was stirred at room temperature for 30 min. The organic was separated and dried by passing through a hydrophobic frit and was evaporated to dryness. This was purified by ISCO companion silica chromatography eluting with a gradient of ethyl acetate in cyclohexane to give the title compound. MS calcd for (C22Hi9CIFNO3S + H)+ : 432/434 MS found (electrospray) : (M+H)+ = 432/434
Intermediate 27
Methyl 3-[(1-methylethyl)amino]-5-phenyl-2-thiophenecarboxylate
Figure imgf000043_0001
To a solution of S-amino-δ-phenyl-thiophene^-carboxylic acid methyl ester (4.92 g, 21.1 mmol) in DCM (1 10 ml.) was added 2-methoxypropene (8.23 ml_, 84.4 mmol), acetic acid (4.83 ml_, 84.4 mmol) and sodium triacetoxyborohydride (8.94 g, 42.2 mmol) and the mixture stirred overnight. Ethyl acetate and water were added, the aqueous phase adjusted to pH7 using sodium bicarbonate and then extracted with ethyl acetate. The combined organic extracts were washed with brine and dried over sodium sulphate. The crude product was purified by silica chromatography using a Flashmaster Personal apparatus (Argonaut) eluting with DCM / cyclohexane (1 :4) to give the title compound. MS calcd for (Ci5H17NO2S+ H)+: 276 MS found (electrospray): (M+H)+ =276
Intermediate 28 5-(4-Bromophenyl)-2-(triphenylmethyl)-2H-tetrazole
Figure imgf000043_0002
A mixture of 5-(4-bromophenyl)-1H-tetrazole (2 g, 8.89 mmol), trityl chloride (2.47 g, 8.89 mmol), tetrabutyl ammonium bromide (130 mg) and 2N sodium hydroxide solution (4.79 ml_, 9.33 mmol) in DCM (15 ml.) was stirred vigorously at RT for 3 h. The mixture was diluted with DCM (50 ml.) and water (30 ml_). The layers were separated and the aqueous extracted further with DCM (3 x 20 ml_). The combined organic fractions were dried (hydrophobic frit) and evaporated to give a solid. This was applied in DCM to a 20 g SPE (silica) and eluted with DCM gave the title compound. 1H NMR (400MHz, CDCI3) δ 8.05 (2H, d), 7.6 (2H, d), 7.4-7.1 (15H, m).
Intermediate 29
Methyl 3-[[(2,4-dichlorophenyl)carbonyl](1 -methylethyl)amino]-5-[4-(1H-tetrazol-5- yl)phenyl]-2-thiophenecarboxylate
Figure imgf000044_0001
n-Butyl lithium (2.94 ml_, 4.7 mmol, 1.6M solution in hexanes) was added dropwise to a solution of Intermediate 28 (2.0 g) in THF (25 ml.) at -780C under nitrogen. A fine yellow suspension was formed and stirred at -780C under nitrogen for 45 mins. Trimethoxyborate (669 μl_, 5.99 mmol) in THF (3 ml.) was added dropwise, maintaining the internal temp at - 780C. This was stirred at -780C for 30 mins then allowed to warm to RT and stirred for a further 45 mins. Water (30 ml.) was added slowly and the mixture extracted with EtOAc ( 2 x 40 ml_), dried (Na2SO4) and evaporated to give an oil. This was triturated with diethyl ether (40 ml.) and the resulting solid (A) was filtered off. Tetrakis(triphenylphosphine)palladium (0) (23 mg, 0.02 mmol) was added to a mixture of Intermediate 22 (200mg, 0.40 mmol) and the solid (A) (185mg, 0.40 mmol) in 1 ,4-dioxane (3 ml.)/ 2N sodium carbonate solution (1 mL) and heated to 1000C under nitrogen for 4 h. The reaction was allowed to cool and the solvent evaporated, acidified with 2N HCI and the phases separated using an aqueous extraction cartridge. This was evaporated to give a solid, which was applied in the minimum volume of DCM to a 2Og Si SPE cartridge. This was eluted with cyclohexane then cyclohexane/EtOAc [(9:1 ) gradient to (7:3)]. Further elution with EtOAc to MeCN to acetone to MeOH. These latter fractions were combined and evaporated, dissolved in MeOH and applied to a 5g NH2 SPE cartridge. Elution was with MeOH (5 x column volumes) then 10% acetic acid/MeOH (5 x column volumes). The acetic acid/MeOH fractions were combined and evaporated to give the title compound.
MS calcd for (C23H19CI2N5O3S + H)+: 516/518/520 MS found (electrospray): (M+H)+ = 516/518/520
Intermediate 30
Methyl 3-[[(2,4-dichlorophenyl)carbonyl](1 -methylethyl)amino]-5-phenyl-2- thiophenecarboxylate
Figure imgf000044_0002
To a solution of S-amino-δ-phenyl-thiophene^-carboxylic acid methyl ester (a synthesis of which is described in Intermediate 27 (0.10 g; 0.36 mmol) and triphenylphosphine (0.14 g; 0.55 mmol) in dichloromethane (40 ml.) was added 2,4-dichlorobenzoyl chloride (76 μl_; 0.55 mmol) and the reaction mixture stirred at reflux under nitrogen for 2 days. After cooling to room temperature DCM was added to take the volume to ~ 50 ml_, then the solution was neutralised with 2M HCI, washed with water, dried using a hydrophobic frit and evaporated to dryness. The crude product was purified by 40 g silica ISCO chromatography eluted with 0 - 100% EtOAc in cyclohexane to give the title compound. MS calcd for (C22Hi9CI2NO3S + H)+: 448/450/452 MS found (electrospray): (M+H)+ = 448/450/452
Intermediate 31
Methyl 3-{[1-methyl-2-(methyloxy)ethyl]amino}-5-phenyl-2-thiophenecarboxylate
Figure imgf000045_0001
To a solution of methyl S-amino-δ-phenyl^-thiophenecarboxylate (2.0 g; 8.58 mmol) and acetic acid (5 ml.) in dry dichloromethane (100 ml.) was added methoxyacetone (0.91 g; 10.3 mmol) and the reaction mixture stirred at room temperature under nitrogen for 10 minutes. Sodium triacetoxyborohydride (3.6 g; 17.2 mmol) was added in 3 portions and the reaction mixture warmed to reflux under nitrogen for 3 days. A further equivalent sodium triacetoxyborohydride (1.8 g; 8.6 mmol) was added and the reaction mixture stirred to reflux under nitrogen for 2 days. Sodium bicarbonate solution (100 ml.) was added and the mixture stirred for 30 minutes, passed through a hydrophobic frit and evaporated to dryness. The crude product was purified by 120 g silica ISCO chromatography eluted with 0 - 100%
EtOAc in cyclohexane to give the title compound.
MS calcd for (Ci6H19NO3S + H)+: 306
MS found (electrospray): (M+H)+ = 306
Intermediate 32
Methyl 3-{[(2,4-dichlorophenyl)carbonyl][1 -methyl-2-(methyloxy)ethyl]amino}-5-phenyl-
2-thiophenecarboxylate
Figure imgf000045_0002
To a solution of Intermediate 31 (0.17 g; 0.56 mmol) and triphenylphosphine (0.22g; 0.84 mmol) in dry dichloromethane (25 ml.) was added 2,4-dichlorobenzoyl chloride (1 16 μl_, 0.84 mmol) and the reaction mixture heated at reflux under nitrogen for 4 days then cooled to room temperature. The reaction mixture was neutralised with 2M HCI, washed with water, using a hydrophobic frit and evaporated to dryness to give the crude title compound. MS calcd for (C23H2ICI2NO4S + H)+: 478/480/482 MS found (electrospray): (M+H)+ = 478/480/482
Intermediate 33
Methyl 3-(tetrahydro-3-furanylamino)-2-thiophenecarboxylate
Figure imgf000046_0001
Methyl 3-amino-2-thiophenecarboxylate (3.8 g), dihydro-3(2H)-furanone (2.5 g) and glacial acetic acid (4.4 g) were dissolved in DCM (75 ml_). Sodium triacetoxyborohydride (12.6 g) was added portion-wise and the reaction was stirred at room temperature over a weekend.
Water (70 ml.) was added and the reaction mixture was neutralised by the addition of solid sodium bicarbonate. The organics were separated, dried by passing through a hydrophobic frit and evaporated in vacuo. The crude material was purified by ISCO Companion silica chromatography, eluting with a gradient 5-100% EtOAc in cyclohexane to give the title compound.
MS calcd for (Ci0H13NO3S + H)+: 228
MS found (electrospray): (M+H)+ = 228
Intermediate 34
Methyl 5-iodo-3-(tetrahydro-3-furanylamino)-2-thiophenecarboxylate
Figure imgf000046_0002
A solution of LDA (2.0M solution in THF/heptane/ethyl benzene, 7.9 ml.) was cooled to -780C under nitrogen. Intermediate 33 (0.9 g) was dissolved in dry THF (9 ml.) and was added dropwise to the cooled LDA solution, maintaining an internal temperature between -780C and -640C. After stirring at -780C for 1 h, a solution of iodine (2 g) in dry THF (9 mL) was added dropwise, maintaining an internal temperature between -780C and -640C. The reaction was stirred at -780C for 10 mins, then the reaction was quenched with saturated NH4CI solution (15 mL). The reaction was allowed to warm to room temperature before being washed with 5% sodium thiosulphate solution. The organics were separated and the aqueous was washed with EtOAc (x 2). The combined organics were dried by passing through a hydrophobic frit and were evaporated in vacuo. The crude material was purified by reverse phase ISCO Companion chromatography, using a C18 cartridge, eluting with a gradient 30- 90% MeCN in water to give the title compound. MS calcd for (C10H12INO3S + H)+: 354 MS found (electrospray): (M+H)+ = 354 Intermediate 35
Methyl 3-[[(4-chloro-2-fluorophenyl)carbonyl](tetrahydro-3-furanyl)amino]-5-iodo-2- thiophenecarboxylate
Figure imgf000047_0001
Intermediate 34 (380 mg) was dissolved in DCM (10 ml_), then 4-chloro-2-fluorobenzoyl chloride (415 mg) and triphenylphosphine (704 mg) were added. The reaction was stirred at 4O0C overnight. The reaction mixture was cooled, sodium bicarbonate solution was added and the reaction was stirred for 30 mins. The mixture was separated, the organics were dried by passing through a hydrophobic frit and were evaporated in vacuo. The crude material was purified by ISCO Companion silica chromatography, eluting with a gradient 5- 100% EtOAc in cyclohexane to give the title compound. MS calcd for (C17H14CIFI NO4S + H)+: 510/512 MS found (electrospray): (M+H)+ = 510/512
Intermediate 36
Methyl 3-[[(4-chloro-2-fluorophenyl)carbonyl](tetrahydro-3-furanyl)amino]-5-(4- pyrazolo[1 ,5-a]pyri
Figure imgf000047_0002
A mixture of Intermediate 35 (140 mg), 2-[4-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2- yl)phenyl]pyrazolo[1 ,5-a]pyrimidine (106 mg, a synthesis of which is described as Intermediate 4), sodium carbonate (117 mg) in water (2 ml.) and tetrakis(triphenylphosphine)palladium (0) (32 mg) was dissolved in DMF (6 ml.) and was heated under nitrogen at 9O0C for 2 h. The reaction was cooled to room temperature and was evaporated in vacuo. The residue was partitioned between DCM and water and the organics were dried by passing through a hydrophobic frit. The organics were evaporated in vacuo and the crude material was purified by ISCO Companion silica chromatography, eluting with a gradient 5-100% EtOAc in cyclohexane to give the title compound. MS calcd for (C29H22CIFN4O4S + H)+: 577/579 MS found (electrospray): (M+H)+ = 577/579
Intermediate 37 Methyl 3-[[(2,4-dichlorophenyl)carbonyl](tetrahydro-3-furanyl)amino]-5-iodo-2- thiophenecarboxylate
Figure imgf000048_0001
Intermediate 34 (452 mg) and 2,4-dichlorobenzoyl chloride (0.139 ml.) were dissolved in DCE (5 ml.) and were heated at 9O0C overnight. The reaction mixture was cooled to room temperature and saturated sodium bicarbonate was added. The organics were separated and the aqueous was washed with DCM (x 2). The combined organics were dried by passing through a hydrophobic frit and were evaporated in vacuo to give the title compound. MS calcd for (Ci7H14CI2INO4S + H)+: 526/528/530 MS found (electrospray): (M+H)+ = 526/528530
Intermediate 38
Methyl S-K^^-dichlorophenylJcarbonyl^tetrahydro-S-furanylJaminol-S-^-pyrazoloII.S- a]pyrimidin-2-ylphenyl)-2-thiophenecarboxylate
Figure imgf000048_0002
A mixture of Intermediate 37 (420 mg), 2-[4-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2- yl)phenyl]pyrazolo[1 ,5-a]pyrimidine (307 mg, a synthesis of which is described as Intermediate 4), sodium carbonate (338 mg) in water (4 ml.) and tetrakis(triphenylphosphine)palladium(0) (93 mg) in DMF (12 ml.) and was heated under nitrogen at 9O0C for 2 h. The reaction was evaporated in vacuo and the residue was partitioned between DCM and water. The organics were dried by passing through a hydrophobic frit and were evaporated in vacuo. The crude material was purified by ISCO Companion silica chromatography, eluting with a gradient 10-100% EtOAc in cyclohexane to give the title compound.
MS calcd for (C29H22CI2N4O4S + H)+: 593/595/597 MS found (electrospray): (M+H)+ = 593/595/597
Intermediate 39
Methyl 3-[{[2-fluoro-4-(trifluoromethyl)phenyl]carbonyl}(1-methylethyl)amino]-5-(4- pyrazolo[1 ,5-a]pyrimidin-2-ylphenyl)-2-thiophenecarboxylate
Figure imgf000049_0001
A mixture of methyl 3-[(1-methylethyl)amino]-5-(4-pyrazolo[1 ,5-a]pyrimidin-2-ylphenyl)-2- thiophenecarboxylate (188 mg, a synthesis of which is described as Intermediate 16), 2- fluoro-4-(trifluoromethyl)benzoyl chloride (217 mg) and triphenylphosphine (314 mg) was dissolved in DCM (6 ml.) and the reaction was stirred at 450C overnight. Sodium bicarbonate solution was added to the reaction and this was stirred at room temperature for 1 h. The organics were separated, dried by passing through a hydrophobic frit and evaporated in vacuo. The crude material was purified by ISCO Companion silica chromatography, eluting with a gradient 5-100% EtOAc in cyclohexane to give the title compound.
MS calcd for (C29H22F4N4O3S + H)+: 583 MS found (electrospray): (M+H)+ = 583
Intermediate 40
Methyl 3-[{[2,4-bis(trifluoromethyl)phenyl]carbonyl}(1-methylethyl)amino]-5-(4- pyrazolo[1 ,5-a]pyrimidin-2-ylphenyl)-2-thiophenecarboxylate
Figure imgf000049_0002
A mixture of methyl 3-[(1-methylethyl)amino]-5-(4-pyrazolo[1 ,5-a]pyrimidin-2-ylphenyl)-2- thiophenecarboxylate (188 mg, a synthesis of which is described as Intermediate 16), 2,4- bis(trifluoromethyl)benzoyl chloride (265 mg) and triphenylphosphine (314 mg) was dissolved in DCM (6 ml.) and the reaction was stirred at 450C overnight. Sodium bicarbonate solution was added to the reaction and this was stirred at room temperature for 1 h. The organics were separated, dried by passing through a hydrophobic frit and evaporated in vacuo. The crude material was purified by ISCO Companion silica chromatography, eluting with a gradient 5-100% EtOAc in cyclohexane to give the title compound. MS calcd for (C30H22F6N4O3S + H)+: 633 MS found (electrospray): (M+H)+ = 633
Intermediate 41 Methyl 3-(bis{[(1,1 -dimethylethyl)oxy]carbonyl}amino)-2-thiophenecarboxylate
Figure imgf000050_0001
Methyl 3-amino-2-thiophenecarboxylate (5 g) was dissolved in acetone (30 ml_), then DMAP (195 mg) and triethylamine (10 ml.) were added followed by dropwise addition of di-te/f-butyl dicarbonate (64 ml_, 1 M solution in THF). The reaction mixture was stirred at room temperature overnight. Di-te/f-butyl dicarbonate (32 ml_, 1 M solution in THF) was added dropwise and the reaction was left to stir at room temperature for 18 h. The reaction was quenched by dropwise addition of 2-amino-5-diethylaminopentane (10.12 g) and was stirred for 1.5 h then evaporated in vacuo. The residue was partitioned between DCM and 1 N HCI and was washed with water. The organics were dried using a hydrophobic frit and were evaporated in vacuo. The crude material was purified by ISCO Companion silica chromatography, eluting with a gradient 5-10% EtOAc in cyclohexane, followed by 5-50% EtOAc in cyclohexane to give the title compound. 1H NMR (CDCI3) δ 7.45 (1 H, d), 6.91 (1 H, d), 3.86 (3H, s), 1.42 (18H, s).
Intermediate 42
Methyl 3-(bis{[(1 J ^imethylethylJoxylcarbony^aminoJ-δ-iodo^-thiophenecarboxylate
Figure imgf000050_0002
A solution of LDA (1.8M solution in THF/heptane/ethyl benzene, 34 ml.) was cooled to -780C. Intermediate 41 (7.35 g) was dissolved in dry THF (70 ml.) and was added dropwise maintaining an internal temperature between -780C and -7O0C. A solution of iodine (10.4 g) in dry THF (70 ml.) was added dropwise, maintaining an internal temperature between -780C and -7O0C. The reaction was quenched immediately with saturated NH4CI solution (100 ml.) and was allowed to warm to room temperature before being washed with 5% sodium thiosulphate solution. The organics were separated and the aqueous was washed with EtOAc (x 2). The combined organics were dried over sodium sulphate and evaporated in vacuo. The crude material was purified by ISCO Companion silica chromatography, eluting with a gradient 5-50% EtOAc in cyclohexane to give the title compound. 1H NMR (CDCI3) δ 7.08 (1 H, s), 3.84 (3H, s), 1.44 (18H, s).
Intermediate 43 Methyl S-amino-δ-iodo^-thiophenecarboxylate
Figure imgf000051_0001
Intermediate 42 (3 g) was dissolved in DCM (30 mL) and TFA (7.5 mL) was added. The reaction mixture was stirred at room temperature for 4 h and was evaporated in vacuo. The residue was partitioned between DCM and saturated sodium bicarbonate solution. The organics were dried by passing through a hydrophobic frit and were evaporated in vacuo to give the title compound.
MS calcd for (C6H6INO2S + H)+: 284
MS found (electrospray): (M+H)+ = 284
Intermediate 44
Methyl 3-{[(4-chloro-2-fluoropheny -5-iodo-2-thiophenecarboxylate
Figure imgf000051_0002
Intermediate 43 (1 g) was dissolved in DCM (40 mL) and triethylamine (1.3 mL) was added, followed by 4-chloro-2-fluorobenzoyl chloride (1.4 g). The reaction was stirred at room temperature for 2 h. A precipitate was observed. The reaction was acidified with 2N HCI and was stirred at room temperature for 1.5 h. The solid was filtered off to give the title compound.
MS calcd for (C13H8CIFINO3S + H)+: 439/441
MS found (electrospray): (M+H)+ = 439/441
Intermediate 45
Methyl 3-{[(4-chloro-2-fluorophenyl)carbonyl][2-(methyloxy)ethyl]amino}-5-iodo-2- thiophenecarboxylate
Figure imgf000051_0003
Intermediate 44 (200 mg) was dissolved in dry DMF (5 mL) and sodium hydride (20 mg, 60% in mineral oil) was added. The reaction mixture was stirred at room temperature under nitrogen for 30 mins. 2-Bromoethyl-methyl ether (0.185 mL) was added and the reaction was stirred at room temperature for 6 h, at 5O0C for 18 h then room temperature for 3 days. MeOH (5 ml.) was added and the reaction mixture was stirred at room temperature for 30 mins before evaporating in vacuo to give the title compound. MS calcd for (Ci6H14CIFINO4S + H)+: 497/499 MS found (electrospray): (M+H)+ = 497/499
Intermediate 46
Methyl 3-{[(4-chloro-2-fluorophenyl)carbonyl][2-(methyloxy)ethyl]amino}-5-(4- pyrazolo[1 ,5-a]pyrimidi
Figure imgf000052_0001
Intermediate 45 (140 mg), 2-[4-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2- yl)phenyl]pyrazolo[1 ,5-a]pyrimidine (105 mg, a synthesis of which is described as Intermediate 4), sodium carbonate (115 mg) in water (2 ml.) and tetrakis(triphenylphosphine)palladium(0) (31 mg) were dissolved in DMF (6 ml.) and were stirred at 1000C for 2 h. The reaction was evaporated in vacuo and the residue was partitioned between DCM and water. The organics were dried by passing through a hydrophobic frit and were evaporated in vacuo. The crude material was purified by ISCO Companion silica chromatography, eluting with a gradient 5-100% EtOAc in cyclohexane to give the title compound. MS calcd for (C28H22CIFN4O4S + H)+: 565/567 MS found (electrospray): (M+H)+ = 565/567
Intermediate 47
Methyl 3-{[(2,4-dichlorophenyl)car iophenecarboxylate
Figure imgf000052_0002
Methyl 3-amino-2-thiophenecarboxylate (1 g), 2,4-dichlorobenzoyl chloride (2.7 g) and triphenylphosphine (3.3 g) were dissolved in DCM (20 ml.) and were heated at 450C overnight. Sodium bicarbonate was added and the mixture was stirred at room temperature for 30 mins. The organics were separated and the aqueous was extracted with DCM. The combined organics were dried by passing through a hydrophobic frit and evaporated in vacuo. The crude material was purified by ISCO Companion silica chromatography, eluting with a gradient 5-100% EtOAc in cyclohexane to give the title compound. MS calcd for (Ci3H9CI2NO3S + H)+: 330/332/334 MS found (electrospray): (M+H)+ = 330/332/334
Intermediate 48
Methyl 3-{[(2,4-dichlorophenyl)carbonyl][2-(methyloxy)ethyl]amino}-2- thiophenecarboxylate
Figure imgf000053_0001
Intermediate 47 (1.45 g) was dissolved in DMF (55 ml.) and the solution was cooled in an ice bath before sodium hydride (191 mg, 60% in mineral oil) was added portion-wise. The reaction mixture was allowed to warm to room temperature over 1 h. 2-Bromoethyl methylether (0.359 ml.) in DMF (3 ml.) was added dropwise and the reaction was stirred at room temperature for 3 days. The reaction was then heated at 4O0C for 24 h and was then treated with MeOH and stirred at room temperature for 30 mins. The reaction was evaporated in vacuo and was partitioned between EtOAc and water. The aqueous was extracted with EtOAc (x 2) and the combined organics were washed with water and brine, and dried using a hydrophobic frit. The organics were evaporated in vacuo and the crude material was purified by ISCO Companion silica chromatography, eluting with a gradient 5- 100% EtOAc in cyclohexane to give the title compound. MS calcd for (C16H15CI2NO4S + H)+: 388/390/392 MS found (electrospray): (M+H)+ = 388/390/392
Intermediate 49
Methyl 3-{[(2,4-dichlorophenyl)carbonyl][2-(methyloxy)ethyl]amino}-5-iodo-2- thiophenecarboxylate
Figure imgf000053_0002
A solution of LDA (1.8M solution in THF/heptane/ethyl benzene, 3.2 ml.) was cooled to - 780C. Intermediate 48 (750 mg) was dissolved in dry THF (7.5 ml.) and was added dropwise maintaining an internal temperature between -780C and -7O0C. A solution of iodine (980 mg) in dry THF (7.5 ml.) was added dropwise, maintaining an internal temperature between - 780C and -7O0C. The reaction was quenched immediately with saturated NH4CI solution (15 ml.) and was allowed to warm to room temperature before being washed with 5% sodium thiosulphate solution. The organics were separated and the aqueous was washed with EtOAc (x 2). The combined organics were dried over sodium sulphate and evaporated in vacuo. The crude material was purified by ISCO Companion silica chromatography, eluting with a gradient 5-50% EtOAc in cyclohexane. The material was purified further using a Supelco ABZ + plus 150 x 30mm 12 μm column with a flow rate 40 mL/min, 12 injections of 1 ml. each, eluting with a gradient of 45-99% MeCN/formic acid to give the title compound. MS calcd for (Ci6H14CI2INO4S + H)+: 514/516/518 MS found (electrospray): (M+H)+ = 514/516/518
Intermediate 50
Methyl 3-{[(2,4-dichlorophenyl)carbonyl][2-(methyloxy)ethyl]amino}-5-(4-pyrazolo[1,5- a]pyrimidin-2-ylphenyl)
Figure imgf000054_0001
Intermediate 49 (204 mg), 2-[4-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2- yl)phenyl]pyrazolo[1 ,5-a]pyrimidine (152 mg, a synthesis of which is described as Intermediate 4), sodium carbonate (168 mg) in water (1 ml.) and tetrakis(triphenylphosphine)palladium(0) (46 mg) were dissolved in DMF (6 ml.) and were stirred at 1000C for 2 h. The reaction was cooled and evaporated in vacuo. The residue was partitioned between DCM and water. The organics were dried by passing through a hydrophobic frit and were evaporated in vacuo. The crude material was purified by ISCO Companion silica chromatography, eluting with a gradient 5-100% EtOAc in cyclohexane to give the title compound. MS calcd for (C28H22CI2N4O4S + H)+: 581/583/585 MS found (electrospray): (M+H)+ = 581/583/585
Intermediate 51
Methyl 3-(tetrahydro-2H-pyran-4-ylamino)-2-thiophenecarboxylate
Figure imgf000054_0002
To a solution of methyl 3-amino-2-thiophenecarboxylate (8 g) in DCM (160 ml.) was added tetrahydro-4H-pyran-4-one (9.2 ml.) and glacial acetic acid (8.6 ml.) followed by portion-wise addition of sodium triactoxyborohydride (21 g). The reaction was stirred at room temperature overnight. Water (160 ml.) was added and the reaction was stirred at room temperature for 30 mins and was then neutralised by addition of solid sodium bicarbonate. The organics were separated, dried by passing through a hydrophobic frit and evaporated in vacuo. The crude material was purified by ISCO Companion silica chromatography, eluting with a gradient 5-50% EtOAc in cyclohexane to give the title compound. MS calcd for (CnH15NO3S + H)+: 242 MS found (electrospray): (M+H)+ = 242
Intermediate 52
Methyl 5-iodo-3-(tetrahydro-2H-pyran-4-ylamino)-2-thiophenecarboxylate
Figure imgf000055_0001
A solution of LDA (2M solution in THF/heptane/ethyl benzene, 16.6 ml.) was cooled to -780C.
Intermediate 51 (2 g) was dissolved in dry THF (20 ml.) and was added dropwise maintaining an internal temperature between -780C and -7O0C. The reaction mixture was stirred at -780C for 1 h. A solution of iodine (4.2 g) in dry THF (20 ml.) was added dropwise, maintaining an internal temperature between -780C and -7O0C. The reaction mixture was stirred at -780C for 1 h 10 mins. The reaction was quenched with saturated NH4CI solution and was allowed to warm to room temperature before being washed with 5% sodium thiosulphate solution. The organics were separated and the aqueous was washed with EtOAc (x 2). The combined organics were dried using a hydrophobic frit and evaporated in vacuo. The crude material was purified by reverse phase ISCO Companion chromatography, using a C18 cartridge, eluting with a gradient 30-95% MeCN in water to give the title compound. MS calcd for (CH H14I NO3S + H)+: 368 MS found (electrospray): (M+H)+ = 368
Intermediate 53
Methyl 3-[[(4<:hloro-2-fluorophenyl)carbonyl](tetrahydro-2Hφyran-4-yl)amino]-5-iodo-
2-thiophenecarboxylate
Figure imgf000055_0002
To a solution of Intermediate 52 (500 mg) in DCM (8 ml.) was added 4-chloro-2- fluorobenzoyl chloride (524 mg) and triphenylphosphine (890 mg). The reaction was stirred at 4O0C overnight. Sodium bicarbonate solution (5 ml.) was added and the reaction was stirred for 30 mins. The organics were separated, dried by passing through a hydrophobic frit and evaporated in vacuo. The crude material was purified by ISCO Companion silica chromatography, eluting with a gradient 5-100% EtOAc in cyclohexane to give the title compound.
MS calcd for (Ci8H16CIFINO4S + H)+: 524/526
MS found (electrospray): (M+H)+ = 524/526
Intermediate 54
Methyl 3-[[(4-chloro-2-fluorophenyl)carbonyl](tetrahydro-2H-pyran-4-yl)amino]-5-(4- pyrazolo[1 ,5-a]pyrimidin
Figure imgf000056_0001
Intermediate 53 (310 mg), 2-[4-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2- yl)phenyl]pyrazolo[1 ,5-a]pyrimidine (228 mg, a synthesis of which is described as Intermediate 4), sodium carbonate (250 mg) in water (2 ml.) and tetrakis(triphenylphosphine)palladium(0) (68 mg) were dissolved in DMF (6 ml.) and were stirred at 1000C for 1 h. The reaction was cooled and evaporated in vacuo. The residue was partitioned between DCM and water. The organics were dried by passing through a hydrophobic frit and were evaporated in vacuo. The crude material was purified by ISCO Companion silica chromatography, eluting with a gradient 5-100% EtOAc in cyclohexane to give the title compound. MS calcd for (C30H24CIFN4O4S + H)+: 591/593 MS found (electrospray): (M+H)+ = 591/593
Intermediate 55
Methyl 3-[[(2,4-dichlorophenyl)carbonyl](tetrahydro-2H-pyran-4-yl)amino]-5-iodo-2- thiophenecarboxylate
Figure imgf000056_0002
To a solution of Intermediate 52 (500 mg) in DCM (8 ml.) was added 2,4-dichlorobenzoyl chloride (0.381 ml.) and triphenylphosphine (890 mg). The reaction was stirred at 4O0C overnight. Sodium bicarbonate solution (5 ml.) was added and the reaction was stirred for 30 mins. The organics were separated, dried by passing through a hydrophobic frit and evaporated in vacuo. The crude material was purified by ISCO Companion silica chromatography, eluting with a gradient 5-100% EtOAc in cyclohexane to give the title compound.
MS calcd for (C18H16CI2INO4S + H)+: 540/542/544
MS found (electrospray): (M+H)+ = 540/542/544
Intermediate 56
Methyl 3-[[(2,4-dichlorophenyl)carbonyl](tetrahydro-2H-pyran-4-yl)amino]-5-(4- pyrazolo[1 ,5-a]pyrimidin
Figure imgf000057_0001
Intermediate 55 (223 mg), 2-[4-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2- yl)phenyl]pyrazolo[1 ,5-a]pyrimidine (159 mg, a synthesis of which is described as Intermediate 4), sodium carbonate (178 mg) in water (2 ml.) and tetrakis(triphenylphosphine)palladium (0) (47 mg) were dissolved in DMF (6 ml.) and were stirred at 1000C for 1 h. The reaction was cooled and evaporated in vacuo. The residue was partitioned between DCM and water. The organics were dried by passing through a hydrophobic frit and were evaporated in vacuo. The crude material was purified by ISCO Companion silica chromatography, eluting with a gradient 5-100% EtOAc in cyclohexane to give the title compound. MS calcd for (C30H24CI2N4O4S + H)+: 607/609/61 1 MS found (electrospray): (M+H)+ = 607/609/611
Intermediate 57
Methyl 5-iodo-3-{[1 -methyl-2-(methyloxy)ethyl]amino}-2-thiophenecarboxylate
Figure imgf000057_0002
A solution of methyl S-amino-δ-iodo^-thiophenecarboxylate (0.60 g, a synthesis of which is described as Intermediate 43), methoxyacetone (0.39 ml.) and titanium (IV) isopropoxide (0.74 ml.) in dry DCM (30 ml.) was warmed to reflux under nitrogen for 18 h. Sodium triacetoxyborohydride (1.35 g) was added and the reaction was heated at reflux under nitrogen for 4 days. The reaction was evaporated in vacuo and the residue was taken into water (30 ml.) and was extracted with DCM (3 x 30 ml_). The organics were dried by passing through a hydrophobic frit and evaporated in vacuo. The crude material was purified by ISCO Companion silica chromatography, eluting with a gradient 0-100% EtOAc in cyclohexane to give the title compound.
1H NMR (DMSO-d6) δ 7.18 (1 H, s), 3.85-3.77 (1 H, m), 3.69 (3H, s), 3.36-3.33 (2H, m), 3.28 (3H, s), 1.12 (3H, d), amine proton not seen.
Intermediate 58
Methyl 3-{[1 -methyl-2-(methyloxy)ethyl]amino}-5-(4-pyrazolo[1 ,5-a]pyrimidin-2- ylphenyl)-2-thiophenecarboxylate
Figure imgf000058_0001
A mixture of Intermediate 57 (0.45 g), 2-[4-(4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2- yl)phenyl]pyrazolo[1 ,5-a]pyrimidine (0.49 g, a synthesis of which is described as Intermediate 4), sodium carbonate (0.40 g) and tetrakis(triphenylphosphine)palladium(0) (70 mg) in 1 ,4- dioxane (1.5 ml.) and water (0.5 ml.) was heated in a microwave at 1000C for 20 mins, then 12O0C for 30 mins. The reaction was evaporated in vacuo, taken into water and extracted with EtOAc (x 2). The organics were dried using a hydrophobic frit and were evaporated in vacuo. The crude material was purified by ISCO Companion silica chromatography, eluting with a gradient 0-100% EtOAc in cyclohexane, then 0-100% MeOH in DCM. The material was triturated with Et2O and was allowed to stand before filtering to give the title compound. MS calcd for (C22H22N4O3S + H)+: 423 MS found (electrospray): (M+H)+ = 423
Intermediate 59
Methyl S'-acetyl^-H^^-dichlorophenylJcarbonylKI-methylethylJamino]^^1- bithiophene-5-carboxylate
Figure imgf000058_0002
A mixture of methyl 3-[[(2,4-dichlorophenyl)carbonyl](1-methylethyl)amino]-5-iodo-2- thiophenecarboxylate (100 mg, a synthesis of which is described as Intermediate 22), (5- acetyl-2-thienyl)boronic acid (41 mg), sodium carbonate (64 mg) and tetrakis(triphenylphosphine)palladium(0) (23 mg) in 1 ,4-dioxane (1.5 ml.) and water (0.5 ml.) was heated in a microwave at 1000C for 10 mins. The reaction mixture was evaporated in vacuo. The residue was taken into water, extracted with DCM, dried using a hydrophobic frit and was evaporated in vacuo. The crude material was purified by ISCO Companion silica chromatography, eluting with a gradient 0-100% EtOAc in cyclohexane to give the title compound.
MS calcd for (C22Hi9CI2NO4S2 + H)+: 496/498/500
MS found (electrospray): (M+H)+ = 496/498/500
Example 1
S-I^^-DichlorophenylJcarbonyllti -methylethylJaminol-S^-imidazoII^-alpyridin^- ylphenyl)-2-thiophenecarboxylic acid
Figure imgf000059_0001
Intermediate 13 (104 mg) was dissolved in tetrahydrofuran (1.5 ml.) and ethanol (1.5 ml.) and treated with sodium hydroxide solution (2N, 1.0 ml_). The reaction mixture was stirred at room temperature overnight. DCM (12 ml.) and HCI (2N, 5.0 ml.) were added and the reaction mixture was stirred for 10 min. The organic fractions were dried using a hydrophobic frit and evaporated to dryness. The residue was purified by MDAP HPLC to give the title compound.
MS calcd for (C28H21CI2N3O3S + H)+ : 550/552/553
MS found (electrospray) : (M+H)+ = 550/552/553
1H NMR (DMSO-de) δ 8.54 (1 H, br.d), 8.50 (1 H, s), 8.03 (2H, Y2AABB'), 7.80 (2H, 1/2AA'BB'),
7.69 (1 H, s), 7.68-7.50 (2H, m, excess), 7.42 (1 H, dd), 7.35-7.22 (2H, m), 6.92 (1 H, dt), 4.88
(1 H, m), 1.40 (3H, d), 1.06 (3H, d) carboxylic acid proton not seen.
Example 2
3-[[(4-Chloro-2-fluorophenyl)carbonyl](1 -methylethyl)amino]-5-(4-imidazo[1 ,2- a]pyridin-2-ylphenyl)-2-thiophenecarboxylic acid
Figure imgf000059_0002
Similarly prepared to Example 1 from Intermediate 14.
MS calcd for (C28H21CIFN3O3S + H)+ : 534/536
MS found (electrospray) : (M+H)+ = 534/536
1H NMR (CD3OD) δ 8.46 (1 H, br.d) 8.30 (1 H, s), 8.00 (2H, 1/2AA'BB'), 7.76 (2H, Y2AABB'), 7.59 (1 H, br.d), 7.46 (1 H, d), 7.45-7.34 (2H, dd+ddd), 7.09 (1 H, dd), 7.045 (1 H, dd), 6.97 (1 H, dd), 5.00 (1 H, m), 1.45 (3H, d), 1.15 (3H, d) carboxylic acid proton not seen.
Example 3
3-[[(4-Bromo-2-methylphenyl)carbonyl](1 -methylethyl)amino]-5-(4-imidazo[1 ,2- a]pyridin-2-ylphenyl)-2-thiophenecarboxylic acid
Figure imgf000060_0001
Intermediate 15 (44 mg) was dissolved in tetrahydrofuran (1.5 ml.) and ethanol (1.5 ml.) and treated with sodium hydroxide solution (2N, 1.0 ml_). The reaction mixture was stirred at room temperature overnight. DCM (12 ml.) and HCI (2N, 5.0 ml.) were added and the reaction mixture was stirred for 10 min. The organic fractions were dried using a hydrophobic frit and evaporated to dryness. The residue was purified by MDAP HPLC. The compound was further purified by loading onto an aminopropyl cartridge (5 g) in MeOH. The column was washed with MeOH (4 column volumes) and the compound eluted with 10%
AcOH in MeOH. The appropriate fractions were evaporated to dryness to give the title compound.
MS calcd for (C29H24BrN3O3S + H)+ : 574/576
MS found (electrospray) : (M+H)+ = 574/576 1HNMR (DMSO-de) δ 8.52 (1 H, d), 8.42 (1 H, s), 7.96 (2H, d), 7.69-7.53 (3H, m), 7.47 (1 H, d),
7.32-7.18 (3H, m), 7.07 (1 H, m), 6.89 (1 H, m ), 4.82 (1 H, septet), 2.31 (3H, s), 1.31 (3H, d),1.08 (3H, d), carboxylic acid proton not seen.
Example 4
S-I^^-DichlorophenylJcarbonyllti -methylethylJaminol-S^-pyrazoloII .S-alpyrimidin- 2-ylphenyl)-2-thiophenecarboxylic acid
Figure imgf000060_0002
Intermediate 17 (124 mg) was dissolved in tetrahydrofuran (1.5 ml.) and ethanol (1.5 ml.) and treated with sodium hydroxide solution (2N, 1.0 ml_). The reaction mixture was stirred at room temperature overnight. The reaction mixture was evaporated to dryness and was partitioned between 2N HCI and DCM. The organic fractions were dried using a hydrophobic frit and evaporated to dryness. The residue was purified by MDAP HPLC to give the title compound.
MS calcd for (C27H20CI2N4O3S + H)+ : 551/553/554
MS found (electrospray) : (M+H)+ = 551/553/554
1H NMR (DMSO-de) δ 9.15 (1 H, d), 8.56 (1 H, m), 8.12 (2H, d), 7.84 (2H, d), 7.70 (1 H, br),
7.48 (1 H, s), 7.34 (3H, m), 7.07 (1 H, m), 4.86 (1 H, septet), 1.39 (3H, d), 1.05 (3H, d), carboxylic acid proton not seen.
Example 5
S-I^-Chloro^-fluorophenylJcarbonyllti -methylethylJaminol-S^-pyrazoloII.S- a]pyrimidin-2-ylphenyl)-2-thiophenecarboxylic acid
Figure imgf000061_0001
Similarly prepared to Example 1 from Intermediate 18 (170 mg).
MS calcd for (C27H20CIFN4O3S + H)+ : 535/537
MS found (electrospray) : (M+H)+ = 535/537
1H NMR (CDCI3) δ 8.75 (1 H, d), 8.52 (1 H, m), 8.09 (2H, d), 7.74 (2H, d), 7.39 (1 H, t), 7.34
(1 H, m), 7.07 (1 H, s), 7.02 (1 H, m), 6.87 (2H, m), 5.16 (1 H, septet), 1.47 (3H, d), 1.17 (3H, d), carboxylic acid proton not seen.
Example 6
S-I^-Bromo^-methylphenylJcarbonyllti -methylethylJaminol-S^-pyrazoloII.S- a]pyrimidin-2-ylphenyl)-2-thiophenecarboxylic acid
Figure imgf000061_0002
Similarly prepared to Example 1 from Intermediate 19 (81 mg).
MS calcd for (C28H23BrN4O3S + H)+ : 575/577
MS found (electrospray) : (M+H)+ = 575/577
1H NMR (CDCI3) δ 8.77 (1 H, d), 8.53 (1 H, br), 8.06 (2H, d), 7.79 (1 H, br), 7.68 (1 H, br), 7.45
(1 H, br), 7.21 (2H, s), 7.09 (1 H, s), 7.06 (1 H, s), 6.88 (1 H, m), 5.15 (1 H, br), 2.41 (3H, br.s),
1.54-1.05 (6H, br.m, rotomers), carboxylic acid proton not seen. Example 7
S-I^^-DimethylphenylJcarbonyllti -methylethylJaminol-S^-pyrazoloII .S-alpyrimidin- 2-ylphenyl)-2-thiophenecarboxylic acid
Figure imgf000062_0001
Similarly prepared to Example 1 from Intermediate 20 (96 mg).
MS calcd for (C29H26N4O3S + H)+ : 511
MS found (electrospray) : (M+H)+ = 511
1H NMR (393K, DMSOd6) δ 9.03 (1 H, d), 8.53 (1 H, m), 8.09 (2H, d), 7.79 (2H, d), 7.48 (1 H, s), 7.21-7.07 (2H, m), 7.02 (1 H, m), 6.92 (1 H, s), 6.82 (1 H, br.d), 4.66 (1 H, br), 2.33 (3H, s),
2.19 (3H, s), 1.26 (6H, br.d), carboxylic acid proton not seen.
Example 8
S-I^^-DichlorophenylJcarbonylKI -methylethylJaminol-S^-imidazo^.i -felli.Slthiazol- 6-ylphenyl)-2-thiophenecarboxylic acid
Figure imgf000062_0002
Intermediate 23 (241 mg) was dissolved in tetrahydrofuran (1.5 ml.) and ethanol (1.5 ml.) and treated with sodium hydroxide solution (2N, 1.0 ml_). The reaction mixture was stirred at room temperature overnight. DCM (12 ml.) and HCI (2N, 5.0 ml.) were added and the reaction mixture was stirred for 10 min. The organic fractions were dried using a hydrophobic frit and evaporated to dryness. The residue was loaded onto an aminopropyl cartridge (10 g) in MeOH/DMSO. The column was washed with MeOH (4 column volumes) and the compound eluted with 10% AcOH in MeOH. The appropriate fractions were evaporated to dryness to give the title compound. MS calcd for (C26H19CI2N3O3S2 + H)+ : 556/558/559 MS found (electrospray) : (M+H)+ = 556/558/559
1H NMR (CDCI3) δ 7.87 (2H, Y2AABB'), 7.80 (1 H, s), 7.61 (2H, Y2AABB'), 7.48 (1 H, d), 7.39 (1 H, br.d), 7.34 (1 H, s), 7.18 (1 H, s), 7.08 (1 H, br.d), 6.91 (1 H, d), 5.12 (1 H, m), 1.44 (3H, d), 1.13 (3H, d) carboxylic acid proton not seen.
Example 9 3-[[(2,4-Dichlorophenyl)carbonyl](1 -methylethyl)amino]-5-(4-furo[3,2-fe]pyridin-2- ylphenyl)-2-thiophenecarboxylic acid
Figure imgf000063_0001
Similarly prepared to Example 1 from Intermediate 24 (136 mg).
MS calcd for (C28H20CI2N2O4S + H)+ : 551/552/553
MS found (electrospray) : (M+H)+ = 551/552/553
1H NMR (DMSO-de) δ 8.54 (1 H, d), 8.14-8.02 (3H, m), 7.90 (2H, d), 7.83-7.75 (2H, m), 7.48
(1 H, m), 7.42-7.29 (3H, m), 4.86 (1 H, septet), 1.41 (3H, d), 1.06 (3H, d), carboxylic acid proton not seen.
Example 10
5-[4-(7-Aminopyrazolo[1 ,5-a]pyrimidin-2-yl)phenyl]-3-[[(2,4-dichlorophenyl)carbonyl](1- methylethyl)amino]-2-thiophenecarboxylic acid
Figure imgf000063_0002
Intermediate 25 (124 mg) was dissolved in tetrahydrofuran (1.5 ml.) and ethanol (1.5 ml.) and treated with sodium hydroxide solution (2N, 1.0 ml_). The reaction mixture was stirred at room temperature overnight. DCM (12 ml.) and HCI (2N, 5.0 ml.) were added and the reaction mixture was stirred for 10 min. The organic fractions were dried using a hydrophobic frit and evaporated to dryness. The residue was purified by MDAP HPLC. The compound was further purified by HPLC on a ABZ+plus column (100 x 21.2 mm, diameter 5 uM) using 0.1%TFA / Water and 0.05% TFA / MeCN, with a gradient of 30-65% in 25 min at 20mL/min. The eluent was evaporated to dryness to give the title compound. MS calcd for (C27H21CI2N5O3S + H)+ : 566/568/569 MS found (electrospray) : (M+H)+ = 566/568/569
1H NMR (CD3OD) δ 8.23-8.13 (3H, m), 7.79 (2H, d), 7.59 (1 H, s), 7.41 (1 H, d), 7.31 (1 H, m), 7.22 (1 H, m), 7.00 (1 H, s), 6.42 (1 H, d), 4.99 (1 H, septet), 1.47 (3H, d), 1.14 (3H, d), carboxylic acid and amine protons not seen.
Example 11 S-I^-Chloro^-fluorophenylJcarbonyllttetrahydro-S-furanylJaminol-δ^-pyrazoloII.S- a]pyrimidin-2-ylphe
Figure imgf000064_0001
Intermediate 36 (171 mg) was dissolved in MeOH (3 ml.) and THF (3 ml_). 2N Sodium hydroxide solution (2 ml.) was added and the reaction mixture was stirred at room temperature for 2 days. The reaction was diluted with DCM (15 ml.) and 2N HCI (5 ml.) was added. The reaction mixture was stirred at room temperature for 30 mins. The organics were separated, dried using a hydrophobic frit and evaporated in vacuo. The crude material was purified using an NH2 SPE cartridge, eluting with 10% 2N HCI in MeOH, followed by MDAP HPLC to give the title compound. MS calcd for (C28H20CIFN4O4S + H)+: 563/565 MS found (electrospray): (M+H)+ = 563/565
1H NMR (DMSO-de) δ 9.15 (1 H, d), 8.56 (1 H, dd), 8.13 (2H, d), 7.88-7.75 (3H, m), 7.37-7.28 (3H, m), 7.21 (1 H, dd), 7.07 (1 H, dd), 5.16 -5.06 (1 H, m), 4.02 (1 H, d), 3.85-3.56 (2.5H, m), 3.00-2.81 (0.5H, br, m), 2.45-1.76 (2H, m), carboxylic acid proton not seen.
Example 12
S-I^^-DichlorophenylJcarbonyllttetrahydro-S-furanylJaminol-S^-pyrazoloII .S- a]pyrimidin-2-ylphenyl)-2-thiophenecarboxylic acid
Figure imgf000064_0002
Intermediate 38 (405 mg) was dissolved in MeOH (3 ml.) and THF (3 ml_). 2N Sodium hydroxide solution (2 ml.) was added and the reaction mixture was stirred at room temperature overnight. DCM (10 ml.) and 2N HCI (5 ml.) were added and the reaction was stirred at room temperature for 30 mins. The organics were separated using a hydrophobic frit and were evaporated in vacuo. The crude material was purified using an NH2 SPE cartridge, eluting with 10% 2N HCI in MeOH, followed by MDAP HPLC to give the title compound.
MS calcd for (C28H20CI2N4O4S + H)+: 579/581/583 MS found (electrospray): (M+H)+ = 579/581/583 1H NMR (DMSO-d6) δ 9.15 (1 H, dd), 8.56 (1 H, dd), 8.12 (2H, d), 7.85-7.74 (3H, m), 7.53-7.51 (1 H, m), 7.39-7.27 (3H, m), 7.07 (1 H, dd), 5.13-5.05 (1 H, m), 4.04 (1 H, d), 3.83-3.72 (1 H, m), 3.69-3.50 (2H, m, obscured by water peak), 2.45-1.73 (2H, m), carboxylic acid proton not seen.
Example 13
3-[{[2-Fluoro-4-(trifluoromethyl)phenyl]carbonyl}(1 -methylethyl)amino]-5-(4- pyrazolo[1 ,5-a]pyrimidin-2-ylphenyl)-2-thiophenecarboxylic acid
Figure imgf000065_0001
To a solution of Intermediate 39 (105 mg) in MeOH (1.5 mL) and THF (1.5 mL) was added 2N sodium hydroxide solution (1 mL) and the reaction mixture was stirred overnight. DCM (10 mL) and 2N HCI (5 mL) were added and the reaction was stirred for 30 mins. The organics were separated using a hydrophobic frit and were evaporated in vacuo. The crude material was purified by reverse phase ISCO Companion chromatography, using a C18 cartridge, eluting with a gradient 40-95% MeCN in water to give the title compound. MS calcd for (C28H20F4N4O3S + H)+: 569 MS found (electrospray): (M+H)+ = 569 1H NMR (CD3OD) δ 8.92 (1 H, d), 8.49 (1 H, dd), 8.09 (2H, d), 7.77 (2H, d), 7.61 (1 H, m), 7.53 (1 H, d), 7.40 (1 H, d), 7.32 (1 H, d), 7.09 (1 H, s), 6.99 (1 H, dd), 5.02 (1 H, quintet), 1.47 (3H, d), 1.16 (3H, d), carboxylic acid proton not seen.
Example 14 3-[{[2,4-Bis(trifluoromethyl)phenyl]carbonyl}(1 -methylethyl)amino]-5-(4-pyrazolo[1 ,5- a]pyrimidin-2-ylpheny
Figure imgf000065_0002
To a solution of Intermediate 40 (138 mg) in MeOH (1.5 mL) and THF (1.5 mL) was added 2N sodium hydroxide solution (1 mL) and the reaction mixture was stirred overnight. DCM (10 mL) and 2N HCI (5 mL) were added and the reaction was stirred for 30 mins. The organics were separated using a hydrophobic frit and were evaporated in vacuo. The crude material was purified by reverse phase ISCO Companion chromatography, using a C18 cartridge, eluting with a gradient 40-95% MeCN in water. The material was purified further by MDAP HPLC to give the title compound. MS calcd for (C29H20F6N4O3S + H)+: 519 MS found (electrospray): (M+H)+ = 519 1H NMR (CD3OD) δ 8.97-8.90 (1 H, m), 8.54-8.48 (1 H, m), 8.27-8.05 (3H, m), 7.92-7.65 (4H, m), 7.43 (0.35H, s), 7.26 (0.65H, s), 7.14-7.08 (1 H, m), 7.04-6.97 (1 H, m), 5.00-4.91 (1 H, m), 1.42 (2H, d), 1.29 (1 H, d), 1.17 (2H, d), 1.12 (1 H, d), contains rotamers, carboxylic acid proton not seen.
Example 15
3-{[(4-Chloro-2-fluorophenyl)carbonyl][2-(methyloxy)ethyl]amino}-5-(4-pyrazolo[1 ,5- a]pyrimidin-2-ylphenyl)
Figure imgf000066_0001
To a solution of Intermediate 46 (157 mg) in THF (1.5 ml.) and EtOH (1.5 mL) was added 2N sodium hydroxide solution and the reaction mixture was stirred at room temperature overnight before being diluted with 2N HCI. The organics were separated, dried by passing through a hydrophobic frit and evaporated in vacuo. The crude material was purified by reverse phase ISCO Companion chromatography, using a C18 cartridge, eluting with a gradient 40-95% MeCN in water. The material was freeze-dried from 1 ,4-dioxane to give the title compound.
MS calcd for (C27H20CIFN4O4S + H)+: 551/553 MS found (electrospray): (M+H)+ = 551/553 1H NMR (DMSO-d6) δ 9.15 (1 H, d), 8.56 (1 H, dd), 8.13 (2H, d), 7.81 (2H, d), 7.71 (1 H, s), 7.38-7.32 (3H, m), 7.23 (1 H, dd), 7.07 (1 H, dd), 4.12-4.03 (1 H, m), 3.94-3.84 (1 H, m), 3.69- 3.60 (1 H, m), 3.56-3.48 (1 H, m), 3.22 (3H, s), carboxylic acid proton not seen.
Example 16
3-{[(2,4-Dichlorophenyl)carbonyl][2-(methyloxy)ethyl]amino}-5-(4-pyrazolo[1,5- a]pyrimidin-2-ylphenyl)
Figure imgf000066_0002
To a solution of Intermediate 50 (123 mg) in THF (1.5 mL) and EtOH (1.5 mL) was added 2N sodium hydroxide solution (1 mL) and the reaction was stirred at room temperature overnight. The reaction was diluted with DCM (15 mL) and was acidified with 2N HCI. The mixture was stirred at room temperature for 30 mins. The organics were separated, dried by passing through a hydrophobic frit and evaporated in vacuo. The crude material was purified by reverse phase ISCO Companion chromatography, using a C18 cartridge, eluting with a gradient 40-95% MeCN in water to give the title compound.
MS calcd for (C27H20CI2N4O4S + H)+: 567/569/571
MS found (electrospray): (M+H)+ = 567/569/571
1H NMR (DMSO-de, 392K) δ 9.02 (1 H, d), 8.53 (1 H, dd), 8.10 (2H, d), 7.72 (2H, d), 7.52-7.29
(4H, m), 7.17 (1 H, s), 7.02 (1 H, dd), 3.98 (2H, br), 3.62 (2H, br), 3.26 (3H, s), carboxylic acid proton not seen.
Example 17
3-[[(4-Chloro-2-fluorophenyl)carbonyl](tetrahydro-2W-pyran-4-yl)amino]-5-(4- pyrazolo[1 ,5-a]pyrimidin-2-ylphenyl)-2-thiophenecarboxylic acid
Figure imgf000067_0001
To a solution of Intermediate 54 (278 mg) in THF (3 ml.) and EtOH (1.5 ml.) was added 2N sodium hydroxide solution (2 ml_), and the reaction was stirred at room temperature overnight. DCM (12 ml.) and 2N HCI (5 ml.) were added and the mixture was stirred for 30 mins before being passed through a hydrophobic frit and evaporated in vacuo. The crude material was purified by reverse phase ISCO Companion chromatography, using a C18 cartridge, eluting with a gradient 40-95% MeCN in water. The material was purified further using a NH2 SPE cartridge, eluting with 10% 2N HCI in MeOH to give the title compound. MS calcd for (C29H22CIFN4O4S + H)+: 577/579 MS found (electrospray): (M+H)+ = 577/579
1H NMR (CD3OD) δ 9.05 (1 H, d), 8.59 (1 H, dd), 8.13 (2H, d), 7.81 (2H, d), 7.55 (1 H, d), 7.40- 7.34 (1 H, m), 7.17 (1 H, s), 7.13-7.03 (3H, m), 4.89-4.82 (1 H, m, partially obscured by water peak), 4.05 (1 H, dd), 3.95 (1 H, dd), 3.63-3.48 (2H, m), 2.16-2.09 (1 H, m), 2.00-1.84 (2H, m), 1.55-1.42 (1 H, m), carboxylic acid proton not seen.
Example 18 S-I^^-DichlorophenylJcarbonyllttetrahydro^H-pyran^-ylJaminol-S^-pyrazoloII .S- a]pyrimidin-2-ylphenyl)
Figure imgf000067_0002
To a solution of Intermediate 56 (304 mg) in THF (3 ml.) and EtOH (1.5 ml.) was added 2N sodium hydroxide solution (2 mL) and the reaction was stirred at room temperature overnight. DCM (12 mL) and 2N HCI (5 mL) were added and the mixture was stirred for 30 mins before being passed through a hydrophobic frit and evaporated in vacuo. The crude material was purified by reverse phase ISCO Companion chromatography, using a C18 cartridge, eluting with a gradient 40-95% MeCN in water. The material was purified further using a NH2 SPE cartridge, eluting with 10% 2N HCI in MeOH to give the title compound. MS calcd for (C29H22CI2N4O4S + H)+: 593/595/597 MS found (electrospray): (M+H)+ = 593/595/597
1H NMR (CD3OD) δ 9.03 (1 H, d), 8.57 (1 H, dd), 8.11 (2H, d), 7.77 (2H, d), 7.60 (1 H, s), 7.39 (1 H, d), 7.34 (1 H, d), 7.23 (1 H, dd), 7.15 (1 H, d), 7.06 (1 H, dd), 4.88 (1 H, m, obscured by water peak), 4.05 (1 H, dd), 3.95 (1 H, dd), 3.64-3.49 (2H, m), 2.23-2.16 (1 H, m), 1.98-1.85 (2H, m), 1.53-1.40 (1 H, m), carboxylic acid proton not seen.
Example 19 3-{[(4-Chloro-2-fluorophenyl)carbonyl][1 -methyl-2-(methyloxy)ethyl]amino}-5-(4- pyrazolo[1 ,5-a]pyrimidin-2-ylphenyl)-2-thiophenecarboxylic acid
Figure imgf000068_0001
A solution of Intermediate 58 (130 mg), 4-chloro-2-fluorobenzoyl chloride (120 mg) and triphenylphosphine (160 mg) in dry DCM (25 mL) was heated to reflux under nitrogen for 6 days. The reaction mixture was evaporated in vacuo, taken into DCM and washed with sodium bicarbonate solution. The organics were dried using a hydrophobic frit and were evaporated in vacuo. The crude material was purified by ISCO Companion silica chromatography, eluting with a gradient 0-100% EtOAc in cyclohexane. This gave no separation. The material was purified further by ISCO Companion silica chromatography, eluting with a gradient 0-20% MeOH in DCM. This gave no separation. The fractions were evaporated in vacuo.
To the residue was added 2M lithium hydroxide (1 mL) and MeOH (2 mL), and the mixture was stirred at room temperature for 3 days. The reaction was evaporated in vacuo and the residue was acidified with 2M HCI and extracted with DCM (x 3). The combined organics were dried using a hydrophobic frit and were evaporated in vacuo. The crude material was purified by NH2 SPE cartridge, eluting with MeOH (3 x column volumes) followed by 10%AcOH in MeOH (3 x column volumes). The acidic fractions were combined and evaporated in vacuo. The material was purified further by MDAP HPLC, and was freeze- dried from 1 ,4-dioxane to give the title compound. MS calcd for (C28H22CIFN4O4S + H)+: 565/567 MS found (electrospray): (M+H)+ = 565/567
1H NMR (CD3OD) δ 8.94 (1 H, d), 8.52 (1 H, dd), 8.13-8.08 (2H, m), 7.79-7.74 (2H, m), 7.54 (1 H, d), 7.47-7.37 (1 H, m), 7.14-6.98 (4H, m), 5.16 (0.7H, quartet), 4.74 (0.3H, quartet), 3.61 (1 H, d), 3.51 (2H, s), 3.33-3.31 (1 H, m), 3.29 (1 H, s), 1.51 (1 H, d), 1.14 (2H, d), contains rotamers, carboxylic acid proton not seen.
Example A
3-[[(4-Chloro-2-fluorophenyl)carbonyl](1 -methylethyl)amino]-5-phenyl-2- thiophenecarboxylic acid
Figure imgf000069_0001
Intermediate 26 (530 mg) was dissolved in tetrahydrofuran (4.0 mL) and ethanol (4.0 mL) and treated with sodium hydroxide solution (2N, 1.5 mL). The reaction mixture was stirred at room temperature overnight. DCM (12 mL) and HCI (2N, 5.0 mL) were added and the reaction mixture was stirred for 1 h. The organic fractions were dried using a hydrophobic frit and evaporated to dryness. The residue was purified by ISCO companion reverse phase chromatography. The compound was eluted with a gradient of 40-95% MeCN in Water. The appropriate fractions were evaporated to dryness to give the title compound. MS calcd for (C2i H17CIFNO3S + H)+ : 418/420 MS found (electrospray) : (M+H)+ = 418/420
1H NMR (CD3OD) δ 7.65 (2H, d), 7.47-7.33 (5H, m), 7.10-6.99 (2H, m), 4.98 (1 H, septet), 1.43 (3H, d), 1.13 (3H, d), carboxylic acid proton not seen.
Example B
S-I^^-DichlorophenylJcarbonyllti -methylethylJaminol-S-^-tiW-tetrazol-S-ylJphenyl]- 2-thiophenecarboxylic acid
Figure imgf000069_0002
2N NaOH (1 mL) was added to a solution of Intermediate 29 (50 mg, 0.09 mmol) in THF (1 mL)/MeOH (2 mL). The solution was left to stir for 24 h, then evaporated and the residue acidified to pH 1.0 with 2N HCI. The resulting suspension was applied to a 1 g OASIS cartridge and eluted with water (3 x column volumes) then MeOH ( 3 x column volumes). The appropriate MeOH fractions were combined and evaporated to give a solid which was purified further by MDAP to give the title compound. MS calcd for (C22H17CI2N5O3S + H)+: 502/504/506 MS found (electrospray): (M+H)+ = 502/504/506 1H NMR (DMSO-de) δ 8.15-7.96 (dd 4H), 7.8 (s, 1 H), 7.7-7.3 (m, 3H), 4.87 (1 H, m), 1.4-0.9 (dd, 6H): the carboxylic acid and tetrazole protons are assumed to be exchanged with moisture in the solvent.
Example C 3-[[(2,4-Dichlorophenyl)carbonyl](1 -methylethyl)amino]-5-phenyl-2- thiophenecarboxylic acid
Figure imgf000070_0001
To a solution of Intermediate 30 (0.16 g; 0.36 mmol) in methanol (5 ml.) was added 2M sodium hydroxide solution (2 ml.) and the reaction stirred at room temperature for 20 hours. The reaction mixture was evaporated to dryness, dissolved in water, acidified with 2M HCI, extracted with EtOAc, dried using a hydrophobic frit and evaporated to dryness. The crude product was purified using a Zorbax SB Phenyl column, 150 x 21.2 mm ID 7 μM, using 40 -
100 % (MeCN + 0.05 % v/v TFA) in (water + 0.1% v/v TFA) at 20 ml. / min over 30 minutes.
It was then applied to a 1 g aminopropyl ion-exchange cartridge and eluted with 1 ,4-dioxane then 10% AcOH in methanol. Next it was purified by 12 g silica ISCO chromatography eluted with 0 - 20% MeOH in DCM, and finally recrystallised from hot methanol, filtered, washed with ethanol and dried to give the title compound.
MS calcd for (C2i H17CI2NO3S + H)+: 434/436/438
MS found (electrospray): (M+H)+ = 434/436/438 1H NMR (DMSO-de): δ 13.20-14.00 (1 H, bs), ([7.80-7.86 (m), 7.71 (d), 7.62 (s), 7.38-7.55
(m), 7.29-7.36 (m)] [BOTH ROTS], total 8H), 4.80-4.90 (1 H, m, [MAJOR ROT]), 3.83-3.90 (1 H, m,
[MiNOR ROT]), 1.37 (d, 3H, [MAJOR ROT]), 1.27 (d, 3H, [MINOR ROT]), 1.14 (3H, d, [MAJOR ROT]),
0.95 (3H, d, [MiNOR ROT])
Example D
3-{[(2,4-Dichlorophenyl)carbonyl][1-methyl-2-(methyloxy)ethyl]amino}-5-phenyl-2- thiophenecarboxylic acid
Figure imgf000071_0001
To a solution of Intermediate 32 (0.56 g) in methanol (5 ml.) was added 2M sodium hydroxide solution (1 ml.) and the reaction stirred at room temperature for 3 days. The reaction mixture was evaporated to dryness, dissolved in water, acidified with 2M HCI, extracted with EtOAc, dried using a hydrophobic frit and evaporated to dryness. The crude product was purified by dry loading onto a 40 g silica cartridge in THF and purifying by ISCO chromatography eluted with 0 - 100% MeOH in DCM, then purified by MDAP HPIC to give the title compound.
MS calcd for (C22Hi9CI2NO4S + H)+: 464/466/468
MS found (electrospray): (M+H)+ = 464/466/468
1H NMR (DMSO-de): δ 13.5-13.9 (1 H, bs), 7.31-7.80 (9H, m, [BOTH ROTS]), 4.95 (1 H, sept,
[MAJOR ROT]), 4.66 (1 H, sept, [MINOR ROT]), 3.53-3.64 (3H, m, [BOTH ROTS]), 3.37 (3H, s,
[MAJOR ROT]), 3.24-3.40 (2H, m, [BOTH ROTS]), (1.39 (d), 1.23 (s), 1.03 (d), total 5H [BOTH
ROTS]
Example E
S'-Acetyl-^I^^-dichlorophenylJcarbonyllti-methylethylJaminol^^'-bithiophene-S- carboxylic acid
Figure imgf000071_0002
A mixture of Intermediate 59 (53 mg) and lithium iodide (71 mg) in pyridine (2 ml.) was heated in a Reacti-vial at 12O0C for 1 day and was then cooled to room temperature. The mixture was evaporated in vacuo and the residue was taken in to water, acidified with 2M HCI and extracted with EtOAc (x 2). The organics were dried using a hydrophobic frit and were evaporated in vacuo. The crude material was purified by ISCO Companion silica chromatography, eluting with a gradient 0-50% MeOH/DCM. The material was purified further by MDAP HPLC and was freeze-dried from 1 ,4-dioxane to give the title compound. MS calcd for (C21H17CI2NO4S2 + H)+: 482/484/486 MS found (electrospray): (M+H)+ = 482/484/486
1H NMR (CD3OD) δ 13.76 (1 H, br), 7.88 (1 H, d), 7.68-7.23 (5H, m), 4.79 (1 H, quintet), 2.52 (3H, s), 1.35 (3H, d), 1.05 (3H, d), rotamers observed. The compounds of Formula (I) 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 pharmaceutically acceptable salt, solvate or ester thereof in conjunction with at least one pharmaceutically acceptable diluent or carrier.
The compounds of Formula (I) can be administered by different routes including oral, parenteral (e.g. intravenous, intraperitoneal, subcutaneous, intramuscular), rectal, topical, transdermal, transmucosal, buccal, sublingual, intranasal or by inhalation administration. For systemic administration, oral administration is convenient.
For oral administration, for example, the compounds of Formula (I) can be formulated into conventional oral dosage forms such as capsules, tablets and liquid preparations such as syrups, elixirs, concentrated drops, ovules, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed-, modified-, sustained-, pulsed-or controlled-release applications.
The tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia.
Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.
Solid compositions of a similar type may also be employed as fillers in gelatin capsules. Suitable excipients in this regard include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the agent may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.
Alternatively, injection (parenteral administration) may be used, e.g., intravenously, intraarterially, intraperitoneal^, intrathecal^, intraventricular^, intraurethrally, intrasternally, intracranially, intramuscularly or subcutaneously administering the agent; and/or by using infusion techniques. For parenteral administration, the compound may be used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. Typical parenteral compositions consist of a solution or suspension of a compound of Formula (I) or a pharmaceutically acceptable salt, solvate or ester 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. The aqueous solutions should be suitably buffered (suitably to a pH of from 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art. For example, the compounds of Formula (I) may be formulated in liquid solutions, for example, in pharmaceutically compatible buffers or solutions, such as saline solution, Hank's solution, or Ringer's solution. In addition, the compounds of Formula (I) may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms can also be produced.
Alternatively, the compounds of Formula (I) may be administered in the form of a suppository or pessary, or may be applied topically in the form of a gel, hydrogel, lotion, salve, solution, cream, ointment or dusting powder. The compounds of Formula (I) may also be dermally or transdermal^ administered, for example, by the use of a skin patch. They may also be administered by the pulmonary or rectal routes. They may also be administered by the ocular route. For ophthalmic use, the compounds may be formulated as micronised suspensions in isotonic, pH adjusted, sterile saline, or, suitably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzylalkonium chloride. Alternatively, they may be formulated in an ointment such as petrolatum.
For application topically to the skin, the compounds of Formula (I) may be formulated as a suitable ointment containing the active compound suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water.
Alternatively, it can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2- octyldodecanol, benzyl alcohol and water.
Compositions for rectal administration are conveniently in the form of suppositories containing a conventional suppository base such as cocoa butter. 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.
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. Typical dermal and transdermal formulations comprise a conventional aqueous or nonaqueous vehicle, for example a cream, ointment, lotion or paste or are in the form of a medicated plaster, patch or membrane
Compositions suitable for buccal or sublingual administration include tablets, lozenges and pastilles, wherein the compound of Formula (I) is formulated with a carrier such as sugar and acacia, tragacanth, or gelatin and glycerin.
The compounds of Formula (I) can also be administered intranasally or by inhalation and is conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurised container, pump, spray or nebuliser with the use of a suitable propellant, e. g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, a hydrofluoroalkane such as 1 ,1 ,1 ,2-tetrafluoroethane (HFA 134AT"") or 1 ,1 ,1 ,2,3,3,3- heptafluoropropane (HFA 227EA), carbon dioxide or other suitable gas. In the case of a pressurised aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The pressurised container, pump, spray or nebuliser may contain a solution or suspension of the active compound, e. g. using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e. g. sorbitan trioleate.
Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of the compound and a suitable powder base such as lactose or starch.
The amounts of various compounds of Formula (I) 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 of Formula (I)), 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 of the compounds of Formula (I) administered also depend on the routes of administration and the degree of oral bioavailability. For example, for compounds of Formula (I) with low oral bioavailability, relatively higher doses will have to be administered. Oral administration is a convenient method of administration of the present compounds of Formula (I).
Suitably 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, for example 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, for example once, sufficient to exhibit the desired activity, as is readily apparent to one skilled in the art.
Compounds of Formula (I) 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 flavouring or colouring 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.
ASSAYS
The potential for compounds of Formula (I) to inhibit NS5B wildtype HCV polymerase activity, genotype 1 b, may be demonstrated, for example, using the following in vitro assay:
In Vitro Detection of inhibitors of HCV RNA-dependent RNA Polymerase Activity 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.
Genotype 1 b Full-Length Enzyme Reaction Conditions were 0.5 μM [33P]-GTP (20 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 4 nM final concentration.
5x concentrated assay buffer mix was prepared using 1 M MnCI2 (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-MgCI2 (0.4 mL), glycerol (1 mL), 10% NP-40 (10 μL), 1 M DTT (20 μL) and water (7.97 mL), Tote/ 1O 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), 40 u/μL RNasin (0.1 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 2.81 mL 2x-concentrated enzyme buffer.
The Assay was set up using compound (1 μL), Substrate Mix (10 μL), and Enzyme Mix (added last to start reaction) (10 μL), Total 2λ 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 1 h 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 compound of Formula (I) 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 GraFit 3, GraFit 4 or GraFit 5 software packages or a data evaluation macro for Excel based on XLFit Software (IDBS).
The potential for compounds of Formula (I) to inhibit HCV replication, genotype 1 a and genotype 1 b, may be demonstrated, for example, using the following cell based assay:
Replicon ELISA cell based assay Method
100 μL of medium containing 10% FCS were added to each well of clear, flat-bottomed 96 well microplates, excepting wells in the top row. Test compound was diluted in assay medium to twice the final required starting concentration from a 40 mM stock solution in DMSO. 200 μl_ of the starting dilution were introduced into two wells each in the top row and doubling dilutions made down the plate by the sequential transfer of 100 μl_ aliquots with thorough mixing in the wells; the final 100 μl_ were discarded. The two bottom rows were not used for compound dilutions. 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 105 cells/mL (sub-line 5-15; genotype 1 b; Lohmann, V., Korner, F., Koch, J-O., Herian, U., Thielmann, L. and Bartenschlager, R., Science, 1999, 285, 110- 113) or at 3 x 105 cells/mL (genotype 1 a; Gu, B., Gates, AT., Isken, O., Behrens, S. E. and Sarisky, R.T., J. Virol., 2003, 77, 5352-5359). 100 μl_ of cell suspension were added to all wells and the plates incubated at 37°C for 72 hours in a 5% CO2 atmosphere. Following incubation, the assay medium was aspirated from the plates. The cell sheets were washed by gentle immersion in phosphate buffered saline (PBS), which was then aspirated off, and fixed with acetone:methanol (1 :1 ) for 5 minutes. Following a further wash with PBS, 100 μL of ELISA diluent (PBS + 0.05% v/v Tween 20 + 2% w/v skimmed milk powder) were added to all wells and the plates incubated at 37°C for 30 minutes on an orbital platform. The diluent was removed and each well then received 50 μL 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 μL 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 μL 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 μL 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 (IC5o) for the compound.
Replicon Luciferase cell based assay Method
A 4OmM stock solution in DMSO of each test compound was further diluted into 50μl_ of DMSO in the first row of a 96 well, V-bottom microplate, to give 100 times the top concentration of the required dilution series. Aliquots of 25μl_ of DMSO were added to each well of the remaining rows, and doubling dilutions of compound were prepared by the serial transfer of 25μl_ volumes from the first row onwards. A Plate-mate robot was used to transfer 1 μl_ volumes from each dilution well into duplicate wells of a clear bottom, black walled, 96 well assay plate (COSTAR #3603). Control wells received 1 μl_ of DMSO alone.
Suspensions were prepared from cultures of Huh-7 cells stably transfected with sub-genomic HCV NS3-NS5B replicons of either genotype 1 b (the ET subline described by
Pietschmann,T., Lohmann, V., Kaul, A., Krieger, N., Rinck, G., Rutter, G., Strand, D. &
Bartenschlager, R., Journal of Virology, 2002, 76, 4008-4021 ) or genotype 1a (subline 1.19 constructed in-house) linked to a firefly luciferase reporter gene. Monolayers nearing confluency were stripped from growth flasks with versene-trypsin solution and the cells re- suspended in assay medium comprising DMEM (Invitrogen #41965-039) supplemented with
5% v/v foetal calf serum, 1% v/v non-essential amino acids solution, 100 units/ml penicillin,
100μg/ml streptomycin and 2mM L-glutamine. 100μl_ of suspension containing either 15,000 cells (genotype 1 b luciferase replicon) or 20,000 cells (genotype 1a luciferase replicon) were added to all wells, except medium controls, of the assay plate and the plate incubated for 48 hours at 37°C in a 5% CO2 atmosphere.
One tablet of Resazurin (Fisher #R/0040/79) was dissolved in 5OmL of phosphate buffered saline and 100μl_ of solution added to all wells. The plate was re-incubated at 37°C for a further 2- 4 hours, wrapped in aluminium foil, before reading in a FluoStar Optima at 595nm. All growth medium and Resazurin was removed by aspiration, and an opaque mask applied to the bottom of the plate. A solution of SteadyLite cytolytic buffer/luciferase substrate (Perkin-Elmer #6016987) was prepared according to the manufacturer's instructions, and 25μl_ added to each well. The plate was then read for luminescence on a TopCount NXT.
Data Analysis
Toxicity : The Resazurin absorbance values from duplicate wells were averaged and expressed as a percentage of the mean absorbance of compound free control wells to determine comparative cell viability. Compound cytotoxicity was expressed either as the lowest concentration at which a significant reduction in viability was observed or a 50% toxic concentration (CCID50) was determined by plotting percentage cytotoxicity against compound concentration using Grafit software (Erithacus Software Ltd.). Potency : The luminescence values from all compound-free wells containing cells were averaged to obtain a positive control value. The mean luminescence value from the compound-free wells that had received no cells 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 luciferase signal in the presence of a drug is a direct measure of replicon inhibition. GraFit software was used to plot the curve of percentage inhibition against compound concentration and derive the 50% inhibitory concentration (IC50) for the compound.
Results
All compounds listed below were assayed at least twice and the mean data are included in the table below.
Figure imgf000079_0001
Figure imgf000080_0001
Activity ranges
Genotvoe 1a Genotvoe 1 b
+ >5.00 i μM + >5.00 μM
++ 1.00- ■ 5.00 μM ++ 1.00- 5.00 μM
+++ 0.50- -1.00μM +++ 0.50- ■LOOμM
++++ 0.10- -0.50μM ++++ 0.10- ■0.50μM
+++++ 0.05- -0.10μM +++++ 0.05- ■O.IOμM
++++++ 0.01 - -0.05μM ++++++ 0.01- ■0.05μM
+++++++ 0.005 1-0.01 μM +++++++ 0.005 -0.01 μM
++++++++ <0.005 μM ++++++++ <0.005 μM
Compound A corresponds to the compound disclosed as Example 307 in WO2002/100851,
3-[[(4-Chloro-2-fluorophenyl)carbonyl](1-methylethyl)amino]-5-phenyl-2-thiophenecarboxylic acid
Compound B corresponds to the compound disclosed as Example 430 in WO2002/100851,
3-[[(2,4-Dichlorophenyl)carbonyl](1-methylethyl)amino]-5-[4-(1/-/-tetrazol-5-yl)phenyl]-2- thiophenecarboxylic acid
Compound C corresponds to the compound disclosed as Example 149 in WO2002/100851,
3-[[(2,4-Dichlorophenyl)carbonyl](1-methylethyl)amino]-5-phenyl-2-thiophenecarboxylic acid
Compound D corresponds to the compound disclosed as Example 561 in WO2002/100851,
3-{[(2,4-Dichlorophenyl)carbonyl][1-methyl-2-(methyloxy)ethyl]amino}-5-phenyl-2- thiophenecarboxylic acid.
Compound E corresponds to the compound disclosed as Example 358 in WO2002/100851,
5'-acetyl-4-[[(2,4-dichlorophenyl)carbonyl](1-methylethyl)amino]-2,2'-bithiophene-5-carboxylic acid.
Compounds A to E may be made according to the processes described in WO2002/100851 or as described hereinabove.
Structures of Compounds A-E are shown below for the avoidance of doubt.
Figure imgf000081_0001
The compounds of Formula (I) which have been tested demonstrate a surprisingly superior potency as HCV polymerase inhibitors, as shown by the IC50 values in the cell-based assays across both of the 1 a and 1 b genotypes of HCV, compared to Compounds A - E. Accordingly, the compounds of Formula (I) are of great potential therapeutic benefit in the treatment and prophylaxis of HCV.
The pharmaceutical compositions according to the invention may also be used in combination with other therapeutic agents. The invention thus provides, in a further aspect, a combination comprising a compound of Formula (I) or a salt, solvate or ester thereof together with at least one other therapeutic agent.
When a compound of Formula (I) or a salt, solvate or ester thereof is used in combination with a second therapeutic agent active against the same disease state, the dose of each compound may differ from that when the compound is used alone. Appropriate doses will be readily appreciated by those skilled in the art. It will be appreciated that the amount of a compound of Formula (I) or a salt, solvate or ester thereof required for use in treatment will vary with the nature of the condition being treated and the age and the condition of the patient and will be ultimately at the discretion of the attendant physician or veterinarian. The compounds of Formula (I) or a salt, solvate or ester thereof may be used in combination with other therapeutic agents, for example immune therapies [e.g. Interferon, such as Interferon alfa-2a (Roferon-A; Hoffmann-La Roche), interferon alpha-2b (Intron-A; Schering-Plough), interferon alfacon-1 (Infergen; Intermune), peginterferon alpha-2b (Peg-lntron; Schering- Plough) or peginterferon alpha-2a (Pegasys; Hoffmann-La Roche)], 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 [e.g. N-acetylcysteine], cytokine agonists, cytokine antagonists, lung surfactants and/or antimicrobial, anti-viral agents [e.g. ribavirin and amantidine], and anti-HCV agents [for example HCV NS3 protease inhibitors, e.g. VX950 (telapravir; Vertex) or SCH503034 (Schering Plough)], or HCV NS5b polymerase inhibitors [for example HCV796 (Wyeth) or R1626 (Roche)], RNAi agents or cyclophilin inhibitors. 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 at least one compound of Formula (I) or a salt, solvate or ester thereof together with at least one other therapeutically active agent, for example Interferon, ribavirin and/or an additional anti-HCV agent.
The combinations 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.
When administration is sequential, either the compound of Formula (I) or the second therapeutic agent may be administered first. When administration is simultaneous, the combination may be administered either in the same or different pharmaceutical composition.
When combined in the same formulation it will be appreciated that the two compounds must be stable and compatible with each other and the other components of the formulation. When formulated separately they may be provided in any convenient formulation, conveniently in such manner as are known for such compounds 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 imgf000083_0001
wherein:
A represents hydroxy;
R1 represents -Rx-Rγ;
Rx represents phenyl (optionally substituted by halo, methyl, ethyl, methoxy or trifluoromethyl) or 5- or 6-membered heteroaryl bonded through a ring carbon atom to the carbon atom of the thiophene;
Rγ represents 8-, 9- or 10-membered heteroaryl, bonded such that when Rx is phenyl or 6- membered heteroaryl, Rγ is in the para-position;
R2 represents phenyl which is at least 2,4 disubstituted, the substituents being independently selected from unsubstituted C1-2alkyl, C1-2alkyl (substituted with one or more fluoro atoms), chloro, fluoro and bromo;
R3 represents linear or branched -C1-6alkyl, or -C3-6cycloalkyl (each optionally substituted by one or more substituents selected from 5- or 6-membered heteroaryl, 5- or 6-membered heterocyclyl and -C3-6cycloalkyl), linear or branched -C2-6alkyl (optionally substituted by one or more substituents selected from methoxy, ethoxy and fluoro), pyranyl, -CH2-pyranyl, tetrahydrofuranyl or -CH2-tetrahydrofuranyl (each of which may be optionally substituted by one or more substituents selected from -Ci-2alkyl, fluoro and methoxy);
or salts, solvates or esters thereof.
2. A compound as claimed in Claim 1 , wherein Rx represents phenyl optionally substituted by halo, methyl, methoxy or trifluoromethyl.
3. A compound as claimed in Claim 1 or Claim 2, wherein Rx represents unsubstituted phenyl.
4. A compound as claimed in Claims 1 to 3 wherein Rγ represents 8- or 9-membered bicyclic heteroaryl group.
5. A compound as claimed in Claims 1 to 4 wherein Rγ represents furo[3,2-b]pyridin-2- yl, pyrazolo[1 ,5-a]pyrimidin-2-yl, imidazo[1 ,2-a]pyridin-2-yl, imidazo[2,1-b][1 ,3]thiazol-6-yl, 7- amino-5-methylpyrazolo[1 ,5-a]pyrimidin-2-yl, 5-methylpyrazolo-[1 ,5-a]pyrimidin-2-yl, 7- aminopyrazolo[1 ,5-a]pyrimidin-2-yl, [1 ,3]oxazolo[4,5-b]pyridin-2-yl, furo[2,3-b]pyridin-5-yl, 5- amino-1 ,3-benzoxazol-2-yl, [1 ,3]oxazolo[5,4-b]pyridin-2-yl, or furo[3,2-c]pyridin-2-yl.
6. A compound as claimed in Claims 1 to 5 wherein Rγ represents furo[3,2-b]pyridin-2- yl, pyrazolo[1 ,5-a]pyrimidin-2-yl, imidazo[1 ,2-a]pyridin-2-yl, imidazo[2,1-fe][1 ,3]thiazol-6-yl or 7-aminopyrazolo[1 ,5-a]pyrimidin-2-yl.
7. A compound as claimed in Claims 1 to 6 wherein R2 represents 2,4-disubstituted phenyl wherein the substituents are independently selected from C1-2alkyl, trifluoromethyl, chloro, fluoro and bromo.
8. A compound as claimed in Claims 1 to 7, wherein R2 represents 2,4-dichlorophenyl, 4-chloro-2-fluorophenyl, 4-bromo-2-methylphenyl, 2,4-dimethylphenyl, 2-fluoro-4- trifluoromethylphenyl or 2,4-bis(trifluoromethyl)phenyl.
9. A compound as claimed in Claim 1 to 8, wherein R3 represents linear or branched - Ci-6alkyl (optionally substituted by one or more substituents independently selected from 5- or 6-membered heteroaryl, 5- or 6-membered heterocyclyl and -C3-6cycloalkyl), linear or branched -C2-6alkyl (optionally substituted by one or more substituents selected from methoxy, ethoxy and fluoro), or pyranyl, -CH2-pyranyl, tetrahydrofuranyl or -CH2- tetrahydrofuranyl (each of which may be optionally substituted by one or more substituents selected from -Ci-2alkyl, fluoro and methoxy).
10. A compound as claimed in Claims 1 to 9, wherein R3 represents unsubstituted C1- 6alkyl, C2-6alkyl (substituted by methoxy), unsubstituted pyranyl or unsubstituted tetrahydrofuranyl.
11. A compound as claimed in Claims 1 to 10, wherein R3 represents 1-methylethyl, 2- (methyloxy)ethyl, 1-methyl-2-(methyloxy)ethyl, tetrahydro-2H-pyran-4-yl or tetrahydro-3- furanyl.
12. A compound as claimed in Claim 1 , wherein Rx represents unsubstituted phenyl; Rγ represents 8- or 9-membered bicyclic heteroaryl group; R2 represents 2,4-disubstituted phenyl wherein the substituents are independently selected from Ci-2alkyl, trifluoromethyl, chloro, fluoro and bromo; R3 represents linear or branched -Ci-6alkyl (optionally substituted by one or more substituents independently selected from 5- or 6-membered heteroaryl, 5- or 6-membered heterocyclyl and -C3-6cycloalkyl), linear or branched -C2-6alkyl (optionally substituted by one or more substituents selected from methoxy, ethoxy and fluoro), or pyranyl, -CH2-pyranyl, tetrahydrofuranyl or -CH2-tetrahydrofuranyl (all of which may be optionally substituted by one or more substituents selected from -d^alkyl, fluoro and methoxy; and salts, solvates and esters thereof.
13. A compound as claimed in Claim 1 or Claim 12 wherein Rx represents unsubstituted phenyl; Rγ represents furo[3,2-b]pyridin-2-yl, pyrazolo[1 ,5-a]pyrimidin-2-yl, imidazo[1 ,2- a]pyridin-2-yl, imidazo[2,1-b][1 ,3]thiazol-6-yl, 7-amino-5-methylpyrazolo[1 ,5-a]pyrimidin-2-yl, 5-methylpyrazolo-[1 ,5-a]pyrimidin-2-yl, 7-aminopyrazolo[1 ,5-a]pyrimidin-2-yl,
[1 ,3]oxazolo[4,5-b]pyridin-2-yl, furo[2,3-b]pyridin-5-yl, 5-amino-1 ,3-benzoxazol-2-yl, [1 ,3]oxazolo[5,4-b]pyridin-2-yl, or furo[3,2-c]pyridin-2-yl; R2 represents 2,4-dichlorophenyl, 4- chloro-2-fluorophenyl, 4-bromo-2-methylphenyl, 2,4-dimethylphenyl, 2-fluoro-4- trifluoromethylphenyl or 2,4-bis(trifluoromethyl)phenyl; R3 represents unsubstituted Ci-6alkyl, C2-6alkyl (substituted by methoxy), unsubstituted pyranyl or unsubstituted tetrahydrofuranyl; and salts, solvates and esters thereof.
14. A compound as claimed in Claims 1 , 12 or 13 wherein Rx represents unsubstituted phenyl; Rγ represents furo[3,2-b]pyridin-2-yl, pyrazolo[1 ,5-a]pyrimidin-2-yl, imidazo[1 ,2- a]pyridin-2-yl, imidazo[2,1-b][1 ,3]thiazol-6-yl or 7-aminopyrazolo[1 ,5-a]pyrimidin-2-yl; R2 represents 2,4-dichlorophenyl, 4-chloro-2-fluorophenyl, 4-bromo-2-methylphenyl, 2,4- dimethylphenyl, 2-fluoro-4-trifluoromethylphenyl or 2,4-bis(trifluoromethyl)phenyl; R3 represents 1-methylethyl, 2-(methyloxy)ethyl, 1-methyl-2-(methyloxy)ethyl, tetrahydro-2H- pyran-4-yl or tetrahydro-3-furanyl; and salts, solvates and esters thereof.
15. A compound of Formula (I) which is:
3-[[(2,4-Dichlorophenyl)carbonyl](1-methylethyl)amino]-5-(4-imidazo[1 ,2-a]pyridin-2- ylphenyl)-2-thiophenecarboxylic acid;
3-[[(4-Chloro-2-fluorophenyl)carbonyl](1-methylethyl)amino]-5-(4-imidazo[1 ,2-a]pyridin-2- ylphenyl)-2-thiophenecarboxylic acid;
3-[[(4-Bromo-2-methylphenyl)carbonyl](1-methylethyl)amino]-5-(4-imidazo[1 ,2-a]pyridin-2- ylphenyl)-2-thiophenecarboxylic acid; 3-[[(2,4-Dichlorophenyl)carbonyl](1-methylethyl)amino]-5-(4-pyrazolo[1 ,5-a]pyrimidin-2- ylphenyl)-2-thiophenecarboxylic acid;
3-[[(4-Chloro-2-fluorophenyl)carbonyl](1-methylethyl)amino]-5-(4-pyrazolo[1 ,5-a]pyrimidin-2- ylphenyl)-2-thiophenecarboxylic acid;
3-[[(4-Bromo-2-methylphenyl)carbonyl](1-methylethyl)amino]-5-(4-pyrazolo[1 ,5-a]pyrimidin-2- ylphenyl)-2-thiophenecarboxylic acid;
3-[[(2,4-Dimethylphenyl)carbonyl](1-methylethyl)amino]-5-(4-pyrazolo[1 ,5-a]pyrimidin-2- ylphenyl)-2-thiophenecarboxylic acid;
S-^^-DichlorophenyOcarbony^i-methylethyOaminol-δ^-imidazoP.I-^ti .Slthiazol-θ- ylphenyl)-2-thiophenecarboxylic acid; 3-[[(2,4-Dichlorophenyl)carbonyl](1-methylethyl)amino]-5-(4-furo[3,2-b]pyridin-2-ylphenyl)-2- thiophenecarboxylic acid;
5-[4-(7-Aminopyrazolo[1 ,5-a]pyrimidin-2-yl)phenyl]-3-[[(2,4-dichlorophenyl)carbonyl](1- methylethyl)amino]-2-thiophenecarboxylic acid;
3-[[(4-Chloro-2-fluorophenyl)carbonyl](tetrahydro-3-furanyl)amino]-5-(4-pyrazolo[1 ,5- a]pyrimidin-2-ylphenyl)-2-thiophenecarboxylic acid;
3-[[(2,4-Dichlorophenyl)carbonyl](tetrahydro-3-furanyl)amino]-5-(4-pyrazolo[1 ,5-a]pyrimidin- 2-ylphenyl)-2-thiophenecarboxylic acid;
3-[{[2-Fluoro-4-(trifluoromethyl)phenyl]carbonyl}(1-methylethyl)amino]-5-(4-pyrazolo[1 ,5- a]pyrimidin-2-ylphenyl)-2-thiophenecarboxylic acid;
3-[{[2,4-Bis(trifluoromethyl)phenyl]carbonyl}(1-methylethyl)amino]-5-(4-pyrazolo[1 ,5- a]pyrimidin-2-ylphenyl)-2-thiophenecarboxylic acid; 3-{[(4-Chloro-2-fluorophenyl)carbonyl][2-(methyloxy)ethyl]amino}-5-(4-pyrazolo[1 ,5- a]pyrimidin-2-ylphenyl)-2-thiophenecarboxylic acid;
3-{[(2,4-Dichlorophenyl)carbonyl][2-(methyloxy)ethyl]amino}-5-(4-pyrazolo[1 ,5-a]pyrimidin-2- ylphenyl)-2-thiophenecarboxylic acid;
3-[[(4-Chloro-2-fluorophenyl)carbonyl](tetrahydro-2H-pyran-4-yl)amino]-5-(4-pyrazolo[1 ,5- a]pyrimidin-2-ylphenyl)-2-thiophenecarboxylic acid;
3-[[(2,4-Dichlorophenyl)carbonyl](tetrahydro-2/-/-pyran-4-yl)amino]-5-(4-pyrazolo[1 ,5- a]pyrimidin-2-ylphenyl)-2-thiophenecarboxylic acid;
3-{[(4-Chloro-2-fluorophenyl)carbonyl][1-methyl-2-(methyloxy)ethyl]amino}-5-(4-pyrazolo[1 ,5- a]pyrimidin-2-ylphenyl)-2-thiophenecarboxylic acid; or salts, solvates or esters, or individual enantiomers thereof where appropriate.
16. A method of treating or preventing viral infection which comprises administering to a subject in need thereof, an effective amount of a compound of Formula (I) as claimed in claim 1 or pharmaceutically acceptable salts, solvates or esters thereof.
17. A method as claimed in claim 16 which involves inhibiting HCV replication.
18. A method as claimed in claim 16 or 17 in which the compound is administered in an oral dosage form.
19. A compound of Formula (I) as claimed in Claim 1 or pharmaceutically acceptable salts, solvates or esters thereof, for use in medical therapy.
20. A compound as claimed in claim 19 wherein the medical therapy is the treatment of viral infection.
21. A compound as claimed in claim 20 wherein the viral infection is HCV.
22. Use of a compound of Formula (I) as defined in claim 1 or pharmaceutically acceptable salts, solvates or esters thereof, in the manufacture of a medicament for the treatment and/or prophylaxis of viral infection.
23. Use as claimed in claim 22 wherein the viral infection is HCV.
24. A pharmaceutical formulation comprising a compound of Formula (I) as claimed in claim 1 or pharmaceutically acceptable salts, solvates or esters thereof in conjunction with at least one pharmaceutically acceptable diluent or carrier.
25. A process for the preparation of a compound of Formula (X)
Figure imgf000087_0001
in which X is a halogen such as bromide or iodide, A is an alkoxy, benzyloxy or silyloxy group and R3 is as defined above for Formula (I), comprising treating a compound of
Formula (V)
Figure imgf000087_0002
in which A an alkoxy, benzyloxy or silyloxy group, and R is as defined above for Formula (I), with a base and a halogen source.
26. A combination comprising a compound of Formula (I) as defined in Claim 1 or a salt, solvate or ester thereof, together with at least one other therapeutically active agent.
27. A combination as claimed in Claim 26, wherein the other therapeutically active agent is selected from Interferon, ribavirin and/or an additional anti-HCV agent.
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