WO2009026657A1 - Flavonoid ppar agonists - Google Patents

Flavonoid ppar agonists Download PDF

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Publication number
WO2009026657A1
WO2009026657A1 PCT/AU2008/001291 AU2008001291W WO2009026657A1 WO 2009026657 A1 WO2009026657 A1 WO 2009026657A1 AU 2008001291 W AU2008001291 W AU 2008001291W WO 2009026657 A1 WO2009026657 A1 WO 2009026657A1
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Prior art keywords
alkyl
hydrogen
aryl
hydroxyl
ppar
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PCT/AU2008/001291
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French (fr)
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WO2009026657A8 (en
Inventor
David Edward Hibbs
Noeris Kris Salam
Tom Hsun-Wei Huang
Rebecca Roubin
Azadeh Matin
Navnath S. Gavande
Srinivas Nammi
Moon Sun Kim
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The University Of Sydney
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Priority claimed from AU2007904674A external-priority patent/AU2007904674A0/en
Application filed by The University Of Sydney filed Critical The University Of Sydney
Publication of WO2009026657A1 publication Critical patent/WO2009026657A1/en
Publication of WO2009026657A8 publication Critical patent/WO2009026657A8/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
    • C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
    • C07D311/34—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 3 only
    • C07D311/36—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 3 only not hydrogenated in the hetero ring, e.g. isoflavones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
    • C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
    • C07D311/34—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 3 only
    • C07D311/38—2,3-Dihydro derivatives, e.g. isoflavanones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/04—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D407/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00
    • C07D407/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings
    • C07D407/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing three or more hetero rings

Definitions

  • the present invention relates to PPAR agonists, and their use in therapy including the treatment of disease.
  • PPARs peroxisome proliferator-activated receptors
  • PPAR- ⁇ The peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that belong to the nuclear hormone superfamily.
  • PPAR- ⁇ three isoforms of PPAR have been identified: PP AR- ⁇ , - ⁇ and - ⁇ .
  • PPAR- ⁇ is the most abundant receptor expressed in adipocytes and macrophages, where, apart from its involvement in adipocyte differentiation and lipid storage, it serves as the primary receptor modulating insulin sensitization and maintaining lipid and glucose homeostasis.
  • PPAR- ⁇ is the target of numerous drug discovery efforts because of its role in numerous disease states, including Type II diabetes.
  • the thiazolidinediones (TZDs; or glitazones) and the L-tyrosine analogues are anti-diabetic synthetic agonists that selectively target PPAR- ⁇ .
  • Their mode of action begins with sensitizing tissue to insulin, lowering glucose levels and reducing serum lipids in diabetic patients by potently binding, and subsequently activating, PPAR- ⁇ .
  • Rosiglitazone (Avandia®), shown below, is a prototypical TZD and serves as a reference compound for this class, which also includes pioglitazone (Actos®) and troglitazone. Rosiglitazone is active in vivo as an anti-diabetic agent in the ob/ob mouse model and is presently being used as an oral hypoglycaemic agent for the treatment of Type II diabetes.
  • the L-tyrosine analogue class of compounds such as Farglitazar (GI262570) shown below, represent the most potent and selective class of synthetic PPAR- ⁇ agonists currently in existence.
  • PPAR- ⁇ agonists reduce body weight gain which led to a hypothesis that activation of PPAR- ⁇ may mitigate the weight gain induced by PPAR- ⁇ activation in humans.
  • PPAR- ⁇ and PPAR- ⁇ agonists have been implicated in the pathology of various disorders including atherosclerosis, coronary heart disease, obesity and inflammation.
  • Compounds that are dual PPAR- ⁇ and PPAR- ⁇ agonists can have fewer therapeutic side- effects than those that act solely at the PPAR- ⁇ receptor or those that act solely at the PPAR- ⁇ receptor.
  • development of safer and efficacious dual PPAR- ⁇ and PPAR- ⁇ agonists are of considerable therapeutic value.
  • the present invention relates to compounds having PPAR agonist activity, and the therapeutic use thereof.
  • the PPAR agonist is a PPAR- ⁇ agonist.
  • the PPAR agonist is a PPAR- ⁇ agonist.
  • the PPAR agonist is a dual PPAR ⁇ / ⁇ agonist.
  • the invention provides a compound of general formula (Ib):
  • R 1 is selected from hydrogen, hydroxyl, halogen, C 1-4 alkyl, haloC 1-4 alkyl, hydroxyC 1-4 alkyl, O-C 1-4 alkyl, OC(O)-C 1-4 alkyl, C(O)-C 1-4 alkyl, and O-sugar;
  • R 3 , R 4 , R 6 are each independently selected from hydrogen, hydroxyl, halogen, Ci- 4 alkyl, C 3-6 cycloalkyl, haloCi -4 alkyl, hydroxyC 1-4 alkyl, O-Ci -4 alkyl, O-Ci.
  • R 7 -R u are each independently selected from hydrogen, halogen, hydroxyl, C 1-4 alkyl, O-C 1-4 alkyl, O-C 3-6 Cycloalkyl, O-C 1-4 haloalkyl, haloC 1-4 alkyl, hydroxyCi.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising
  • a third aspect of the invention provides for a method of treating or preventing a disease in a vertebrate, the method comprising administering to a vertebrate in need thereof an effective amount of a compound of formula (Ib) according to the first aspect of
  • Y is O or S
  • R 1 and R 2 are each independently selected from hydrogen, hydroxyl, halogen, C 1 . 4 alkyl, haloC 1-4 alkyl, hydroxyC 1-4 alkyl, O-C 1-4 alkyl, OC(O)-C 1-4 alkyl, C(O)-C 1-4 alkyl, CO 2 H, and CO(O)-C 1-4 alkyl;
  • R 3 -R 6 are each independently selected from hydrogen, hydroxyl, halogen, C 3-6 cycloalkyl, haloC 1-4 alkyl, hydroxyC 1-4 alkyl, O-C 1-4 alkyl, O-C 1-4 alkyl-CO 2 R, O-C 3-6 cycloalkyl, O-C 3-6 heterocycloalkyl, O-C 3-6 heteroaryl, N(R) 2 C i -4 alkyl, N(R) 3 Ci- 4 alkyl, O-C 1-4 alkyl-N(R) 2 , O-Ci -4 alkyl-N(R) 3 , C 6-10 aryl, O-C 6- i 0 aryl, O-Ci.
  • R and R , R and R , and R and R together form or and each R is independently selected from hydrogen and Ci -4 alkyl; is 2-pyridyl, 3-pyridyl or 4-pyridyl optionally substituted with one or more of hydrogen, halogen, hydroxyl, Ci -4 alkyl, O-C 1-4 alkyl, O-C 3-6 cycloalkyl, haloC 1-4 alkyl, hydroxyCi -4 alkyl, O-Ci -4 alkyl-CO 2 R, 0-C 3-6 CyClOaIlCyI, O-C ⁇ heterocycloalkyl, 0-C 3 .
  • R 7 -R n are each independently selected from hydrogen, halogen, hydroxyl, C 1-4 alkyl, O-C 1-4 alkyl, O-C ⁇ cycloalkyl, haloCi -4 alkyl, hydroxyCi -4 alkyl, O-Ci -4 alkyl- CO 2 R, 0-C 3-6 CyClOaIlCyI, O-C 3-6 heterocycloalkyl, O-C 3-6 heteroaryl, N(R) 2 C 1-4 alkyl, N(R) 3 C 1-4 alkyl, O-Ci -4 alkyl-N(R) 2 , O-C 1-4 alkyl-N(R) 3) C 6 .
  • l group optionally substituted with one or more of halogen, hydroxyl, C 1-4 alkyl, O-C 1-4 alkyl, O-C 3-6 cycloalkyl, hydroxyCi -4 alkyl, O-Ci -4 alkyl- CO 2 R, O-C 3-6 cycloalkyl, O-C 3-6 heterocycloalkyl, O-C 3-6 heteroaryl, N(R) 2 C i -4 alkyl, N(R) 3 C 1-4 alkyl, O-C 1-4 alky 1-N(R) 2 , O-C 1-4 alkyl-N(R) 3, C 6- i 0 aryl, 0-C 6-10 aryl, O-Ci -4 alkyl- C 6-10 aryl, O-C 1-4 alkyl-C 6-10 heterocycloalkyl, O-C 1-4 alkyl-C 6-10 heteroaryl, 0-CON(R) 2 , CON(R) 2 , CO 2 R, C ⁇ al
  • Y is O or S
  • R 1 and R 2 are each independently selected from hydrogen, hydroxyl, halogen, Ci- 4 alkyl, haloC 1-4 alkyl, hydroxyC 1-4 alkyl, O-Ci -4 alkyl, OC(O)-C i -4 alkyl, C(O)-C M alkyl, CO 2 H, and CO(O)-C i.
  • R 3 -R 6 are each independently selected from hydrogen, hydroxyl, halogen, Ci -4 alkyl, C 3-6 cycloalkyl, haloC ⁇ -4 alkyl, hydroxyC 1-4 alkyl, O-Ci -4 alkyl, O-C 1-4 alkyl-CO 2 R, 0-C 3-6 CyClOaIlCyI, O-C 3-6 heterocycloalkyl, O-C 3-6 heteroaryl, N(R) 2 C i -4 alkyl, N(R) 3 Ci- 4 alkyl, O-Ci -4 alkyl-N(R) 2 , O-Ci -4 alkyl-N(R) 3 , C 6-10 aryl, O-C 6 .i 0 aryl, O-C 1-4 alkyl-C 6-10 aryl, 5 O-C 1-4 alkyl-C 6- i 0 heterocycloalkyl, O-C M alkyl-Ce-io
  • each R is independently selected from hydrogen and Ci -4 alkyl; is 2-pyridyl, 3-pyridyl or 4-pyridyl optionally substituted with one or more of hydrogen, halogen, hydroxyl, Ci -4 alkyl, O-C 1-4 alkyl, O-C 3-6 cycloalkyl, haloC ⁇ -4 alkyl, hydroxyC 1-4 alkyl, O-C 1-4 alkyl-CO 2 R, O-C 3-6 cycloalkyl, O-C 3-6 heterocycloalkyl, 0-C 3- 6 heteroaryl, N(R) 2 C 1-4 alkyl, N(R) 3 C 1-4 alkyl, O-C 1-4 alky 1-N(R) 2 , O-C M alkyl-N(R) 3, C 6- i 5 l oaryl, O-C 6-
  • R 7 -R n are each independently selected from hydrogen, halogen, hydroxyl,
  • Ci -4 alkyl O-Ci -4 alkyl-N(R) 2 , O-Ci -4 alkyl-N(R) 3 , C 6- i 0 aryl, O-C 6- i 0 aryl, O-C 1-4 alkyl-
  • a method for identifying a PPAR agonist comprising: determining ligand-receptor interactions of a candidate compound with at least two structurally distinct docking templates; comparing the ligand-receptor interactions of the candidate compound with the interactions of a known PPAR agonist; and thereby determining whether a candidate compound is a PPAR agonist.
  • the PPAR agonist is a PPAR- ⁇ agonist. In another embodiment the PPAR agonist is a PPAR- ⁇ agonist. In a further embodiment the PPAR agonist is a dual PPAR ⁇ / ⁇ agonist.
  • the docking template may be one or more PPAR crystal structures.
  • a first template, receptor (I) may be derived from of the farglitazar-bound PPAR- ⁇ X-ray complex (PDB: 1FM9); and a second, receptor (II), may be derived from the rosiglitazone-bound PPAR- ⁇ X-ray complex (PDB :1FM6).
  • the method may further comprise testing a compound identified as a PPAR agonist in vitro for PPAR activation efficacy using either a transcriptional factor or a reporter gene luciferase assay.
  • the method may further comprise determining the activity of a compound identified as a PPAR agonist in inducing PPAR mRNA and protein expression, then optionally determining if the activity is abolished in the presence of a known selective PPAR antagonist, such as for example, GW9662.
  • a known selective PPAR antagonist such as for example, GW9662.
  • THP-I human acute monocytic leukaemia cell line
  • C 1-4 alkyl group includes within its meaning monovalent (“alkyl”) and divalent (“alkylene”) straight chain or branched chain saturated aliphatic groups having from 1 to 4 carbon atoms.
  • the alkyl group may be C 1-3 alkyl or Cj -2 alkyl.
  • C 1-4 alkyl includes, but is not limited to, methyl, ethyl, 1- propyl, isopropyl, 1 -butyl, 2-butyl, isobutyl, tert-butyl, and the like.
  • C 2-4 alkenyl group includes within its meaning monovalent (“alkenyl”) and divalent (“alkenylene”) straight or branched chain unsaturated aliphatic hydrocarbon groups having from 2 to 4 carbon atoms and at least one double bond anywhere in the chain.
  • the alkenyl group may be C 2-3 alkenyl. Unless indicated otherwise, the stereochemistry about each double bond may independently be cis, trans, E or Z as appropriate.
  • C 2-4 alkenyl groups include but are not limited to ethenyl, vinyl, allyl, 1-methylvinyl, 1-propenyl, 2-propenyl, 2-methyl-l-propenyl, 2-methyl-l-propenyl, 1-butenyl, 2-butenyl, 3-butentyl, 1,3-butadienyl, and the like.
  • amino refers to groups of the form -NR a R b wherein R a and R b are individually selected from hydrogen, optionally substituted (Ci -4 )alkyl, optionally substituted (C 2-4 )alkenyl, optionally substituted (C 2-4 )alkynyl, optionally substituted (C 6-10 )aryl and optionally substituted aralkyl groups, such as benzyl.
  • the amino group may be a primary, secondary or tertiary amino group.
  • amino acid as used herein includes naturally and non-naturally occurring amino acids, as well as substituted variants thereof. Thus, (L) and (D) forms of amino acids are included in the scope of the term “amino acid”.
  • amino acid includes within its scope glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartate, lysine, arginine, and histidine.
  • the backbone of the amino acid residue may be substituted with one or more groups independently selected from (Ci -6 )alkyl, halogen, hydroxy, hydroxy(C 1-6 )alkyl, aryl (e.g, phenyl), aryl(Ci -3 )alkyl (e.g, benzyl), and (C 3- 6 )cycloalkyl.
  • arylalkyl or variants such as “arylalkyl” as used herein, includes within its meaning monovalent (“aryl”) and divalent (“arylene”), single, polynuclear, conjugated and fused aromatic hydrocarbon radicals attached to divalent, saturated, straight or branched chain alkylene radicals.
  • C 6- I 0 aromatic group refers to monovalent (“aryl”) and divalent (“arylene”) single, polynuclear, conjugated and fused residues of aromatic hydrocarbons having from 6 to 10 carbon atoms.
  • aromatic groups include phenyl, naphthyl, phenanthrenyl, and the like.
  • C 3-6 cycloalkyl refers to cyclic saturated aliphatic groups and includes within its meaning monovalent (“cycloalkyl”), and divalent (“cycloalkylene”), saturated, monocyclic, bicyclic, polycyclic or fused polycyclic hydrocarbon radicals having from 3 to 6 carbon atoms.
  • the cycloalkyl group may be C 3-5 cycloalkyl. Examples of cycloalkyl groups include but are not limited to cyclopropyl, 2- methylcyclopropyl, cyclobutyl, cyclopentyl, 2-methylcyclopentyl, 3-methylcyclopentyl, cyclohexyl, and the like.
  • C 3-6 cycloalkenyl refers to cyclic unsaturated aliphatic groups and includes within its meaning monovalent (“cycloalkenyl”) and divalent (“cycloalkenylene”), monocyclic, bicyclic, polycyclic or fused polycyclic hydrocarbon radicals having from 3 to 6 carbon atoms and having at least one double bond anywhere in the alkyl chain.
  • the cycloalkenyl group may be C 3-5 cycloalkenyl. Unless indicated otherwise, the stereochemistry about each double bond may be independently cis, trans, E or Z as appropriate.
  • Examples of cycloalkenyl groups include but are not limited to cyclopropenyl, cyclopentenyl, cyclohexenyl, and the like.
  • C 3-6 heterocycloalkyl includes within its meaning monovalent (“heterocycloalkyl”) and divalent (“heterocycloalkylene”), saturated, monocyclic, bicyclic, polycyclic or fused hydrocarbon radicals having from 3 to 6 ring atoms, wherein from 1 to 3, ring atoms are heteroatoms independently selected from O, N, NH, or S.
  • the heterocycloalkyl group may be C 3-5 heterocycloalkyl.
  • heterocycloalkyl groups include aziridinyl, pyrrolidinyl, piperidinyl, piperazinyl, quinuclidinyl, azetidinyl, morpholinyl, tetrahydrothiophenyl, tetrahydrofuranyl, tetrahydropyranyl, and the like.
  • C 5-10 heteroaromatic group and variants such as “heteroaryl” or “heteroarylene” as used herein, includes within its meaning monovalent (“heteroaryl”) and divalent (“heteroarylene”), single, polynuclear, conjugated and fused aromatic radicals having from 5 to 10 atoms, wherein 1 to 4, or 1 to 2 ring atoms are heteroatoms independently selected from O, N, NH and S.
  • the heteroaromatic group may be C 5-8 heteroaromatic.
  • heteroaromatic groups include pyridyl (including 2-pyridyl, 3-pyridyl and 4-pyridyl), pyrimidinyl, pyridazinyl, pyrazinyl, 2,2'-bipyridyl, phenanthrolinyl, quinolinyl, isoquinolinyl, imidazolinyl, thiazolinyl, pyrrolyl, furanyl, thiophenyl, oxazolyl, isoxazolyl, isothiazolyl, triazolyl, and the like.
  • halogen or variants such as “halide” or “halo” as used herein refers to fluorine, chlorine, bromine and iodine.
  • heteroatom or variants such as “hetero-” as used herein refers to O, N, and S or the group NH.
  • optionally substituted means the group to which this term refers may be unsubstituted, or may be substituted with one or more groups independently selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, halo, haloalkyl, haloalkynyl, hydroxyl, hydroxyalkyl, alkoxy, thioalkoxy, alkenyloxy, haloalkoxy, haloalkenyloxy, NO 2 , NR a R b , nitroalkyl, nitroalkenyl, nitroalkynyl, nitroheterocyclyl, alkylamino, dialkylamino, alkenylamine, alkynylamino, acyl, alkenoyl, alkynoyl, acylamino, diacylamino, acyloxy, alkylsulfonyl
  • Preferred substituents include C 1-3 alkyl, C 1-3 alkoxy, -CH 2 -(C 1-3 )alkoxy, C 6- Io aryl, -CH 2 -phenyl, halo, hydroxyl, hydroxyl-(C 1-3 )alkyl, and halo-(C 1-3 )alkyl, e.g, CF 3 , CH 2 CF 3 .
  • administering includes contacting, applying, delivering or providing a compound or composition of the invention to an organism, or a surface by any appropriate means.
  • vertebrate includes humans and individuals of any species of social, economic or research importance including but not limited to members of the genus ovine, bovine, equine, porcine, feline, canine, primates (including human and non-human primates), rodents, murine, caprine, leporine, and avian.
  • the vertebrate may be a human.
  • treatment refers to any and all uses which remedy a disease state or symptoms, prevent the establishment of disease, or otherwise prevent, hinder, retard, or reverse the progression of disease or other undesirable symptoms in any way whatsoever.
  • therapeutically effective amount and “diagnostically effective amount”, include within their meaning a sufficient but non-toxic amount of a compound or composition of the invention to provide the desired therapeutic or diagnostic effect. The exact amount required will vary from subject to subject depending on factors such as the species being treated, the age and general condition of the subject, the severity of the condition being treated, the particular agent being administered, the mode of administration, and so forth. Thus, it is not possible to specify an exact “effective amount”. However, for any given case, an appropriate “effective amount” may be determined by one of ordinary skill in the art using only routine experimentation.
  • FIG. 1 Docking poses of farglitazar (magenta), a) and rosiglitazone (blue), b) in the LBD of receptor (I) and (II), respectively. Overlaid are their respective published crystallographic pose (brown) from 1FM9 and 1FM6.
  • Helices AF-2 and 10 are shown in cartoon ribbon style, both coloured blue and yellow in receptors (I) and (II), respectively.
  • Comparisons of a) and b) highlight the similar hydrogen bond interactions with residues in the vicinity of the AF-2 helix: Ser289, His323 and Ty473. Hydrophobic ⁇ - ⁇ interactions with Phe363 are prominent with farglitazar although visibly absent with rosiglitazone.
  • FIG. 1 Predicted complexes of flavonoids in the LBD of PPAR- ⁇ .
  • Flavonoids are predicted to occupy the hydrophobic environment formed by residues Phe282, Phe360 and Phe360, ⁇ -stacking with the latter. Hydrogen bond interactions are made to the receptor from the 7-OH by each flavonoid.
  • FIG. 3 Significant flavonoids to activate PPAR- ⁇ in a cell-based transcriptional factor assay.
  • the PPAR- ⁇ Transcription Factor Assay is a sensitive ELISA method for detecting PPAR- ⁇ transcription factor DNA binding activity in nuclear extracts of THP-I derived macrophage cell line.
  • the ELISA assay was conducted according to the manufacturer's manual (Cayman Chemical, Australia).
  • the cell lines were treated with various concentrations (0.01 - 50 ⁇ M) of rosiglitazone, psi-baptigenin (40c), hesperidin (34), apigenin (8), chrysin (12) and biochanin-A (55).
  • FIG. 4 Cytotoxic profiles of rosiglitazone, psi-baptigenin (40c), hesperidin (34), apigenin (8), chrysin (12) and biochanin-A (55), genistein (56), GW-9662 in THP-I derived macrophage cell line.
  • the HEK 293 cells were transiently transfected with tK-PPREx3-Luc, pSG5- hPPAR- ⁇ and pSV- ⁇ -galactosidase control plasmid.
  • Cells were treated with test compounds (5 ⁇ M and 50 ⁇ M). Rosiglitazone (5 ⁇ M) and GW 1929 (1 ⁇ M) were used as positive controls and DMSO (0.1%) as a negative control.
  • DMSO 0.1%) as a negative control.
  • the cells were lysed and assayed for luciferase and ⁇ -galactosidase activities. The results are expressed as relative luciferase activity (fold difference compared to negative control).
  • Figure 9 PPAR- ⁇ reporter gene activity of compounds 40a, 40c, 4Oe and 4Oi in HEK 293 cell line. The HEK 293 cells were transiently transfected with tK-PPREx3-Luc, pBI- G-hPPAR- ⁇ and pSV- ⁇ -galactosidase control plasmid. Cells were treated with test compounds (5, 50 and 100 ⁇ M).
  • WY-14643 25 ⁇ M
  • Fenofibrate 100 ⁇ M
  • DMSO 0.15%
  • the cells were lysed and assayed for luciferase and ⁇ -galactosidase activities. The results are expressed as relative luciferase activity (fold difference compared to negative control).
  • Figure 10 Cytotoxic profiles of compounds 40a, 40c, 4Oe, 4Oi and fenofibrate in HEK- 293 cell line.
  • the present invention is directed to compounds which are agonists of the PPAR receptors.
  • the invention relates to compounds which are agonists of the PPAR- ⁇ receptor.
  • the present invention also relates to compounds that are agonists of the PPAR- ⁇ receptor.
  • the present invention is further directed to compounds which are dual agonists of the PPAR- ⁇ and PPAR- ⁇ receptors.
  • Compounds according to the present invention may be useful in therapy, including for example, the treatment of Type II diabetes, obesity, hyperlipidemia, cardiovascular disease (e.g, coronary and ischemic heart disease, atherosclerosis, peripheral vascular disease), anti-neoplastic diseases and tumours (e.g, control of cell growth, cell differentiation, motility and apoptosis, neuroblastoma, breast cancer), inflammatory conditions (e.g, inflammatory bowel diseases, psoriasis, chronic inflammatory airway disease, asthma, rheumatoid arthritis), and neurodegenerative diseases (e.g, Parkinson's disease, Alzheimer's disease).
  • cardiovascular disease e.g, coronary and ischemic heart disease, atherosclerosis, peripheral vascular disease
  • anti-neoplastic diseases and tumours e.g, control of cell growth, cell differentiation, motility and apoptosis, neuroblastoma, breast cancer
  • inflammatory conditions e.g, inflammatory bowel diseases, psoriasis,
  • the compounds of the present invention are flavonoids. Included in the flavonoid class of compounds are flavones, flavanones, isoflavones and isoflavanones.
  • the present invention relates to compounds of general formulae (1) and (2):
  • R 1 and R 2 are each independently selected from hydrogen, hydroxyl, halogen, Ci- 4 alkyl, haloC 1-4 alkyl, hydroxyC 1-4 alkyl, O-Ci -4 alkyl, OC(O)-Ci -4 alkyl, C(O)-C M alkyl, CO 2 H, and CO(O)-C i -4 alkyl;
  • R 3 -R 6 are each independently selected from hydrogen, hydroxyl, halogen, Ci -4 alkyl, C 3-6 cycloalkyl, haloC ⁇ -4 alkyl, hydroxyd ⁇ alkyl, O-Ci -4 alkyl, O-Ci_ 4 alkyl-CO 2 R, 0-C 3-6 CyClOaIlCyI, O-C ⁇ heterocycloalkyl, O-C 3-6 heteroaryl, N(R) 2 C M alkyl, N(R) 3 Ci- 4 alkyl, O-C 1-4 alkyl-N(R) 2 , O-C 1-4 alkyl-N(R) 3 , C 6-10 -aryl, O-C 6 .i 0 aryl, O-Ci -4 alkyl-C 6- i O aryl, , O-Ci ⁇ alkyl-C ⁇ -ioheterocycloalkyl, O-Ci -4 alkyl-C 6-
  • R 3 and R 4 , R 4 and R 5 , and R 5 and R 6 together form 0 , or C 5 ; each R is independently selected from hydrogen and C 1-4 alkyl;
  • R 7 -R n are each independently selected from hydrogen, halogen, hydroxyl,
  • Y is O. In other embodiments of formula (1) and formula (2) Y is S.
  • ⁇ is a double bond.
  • formula (1) and formula (2) ' is a single bond.
  • R 5 is selected from hydroxyl, O-Ci -4 alkyl, THP, 0-Ci-
  • R 5 is selected from hydroxyl, O-Ci -4 alkyl, THP, 0-Ci-
  • R 1 and R 2 are independently selected from hydrogen, halogen, Ci -4 alkyl, O- Ci -4 alkyl, CO 2 H, and CO 2 -C 1-4 alkyl.
  • R 1 and R 2 are independently selected from hydrogen, halogen, methyl, ethyl, O-methyl, O-ethyl, t- butoxy, CF 3 , CO 2 H, CO 2 methyl, C0 2 ethyl and CO 2 Bu'.
  • R 1 and R 2 are independently selected from hydrogen, halogen, methyl, CF 3 and CO 2 H.
  • R 7 -R n are each independently selected from hydrogen, halogen, hydroxyl, C 1-4 alkyl, haloC 1-4 alkyl, ;
  • R 7 and R 8 , R 8 and R 9 , R 9 a nd R 10 , and R 10 and R 1 ' together form 0 ⁇ ,
  • R 7 , R 9 and R 11 are each hydrogen, R 8 is hydroxyl or O- methyl and R 10 is hydroxyl or O-methyl.
  • R 7 , R 8 , R 10 and R 11 are hydrogen, and R 9 is hydroxyl, halogen methyl, CF 3 , O-methyl, or CO 2 H.
  • R 8 , R 10 and R 11 are hydrogen and R 7 and R 9 are independently selected from hydroxyl, halogen and O-methyl.
  • R 7 , R 10 and R 11 are each hydrogen and R 8 and R 9 are independently selected from hydroxyl and O-methyl or
  • R 9 and R 10 together form 0 ⁇ .
  • R 3 -R 6 are each independently selected from hydrogen, hydroxyl, halogen, Ci -4 alkyl, haloC ⁇ -4 alkyl, hydroxyCi -4 alkyl, O-Ci -4 alkyl, 0-C 3 . 6 heterocycloalkyl, N(R) 2 C 1-4 alkyl, O-benzyl, 0-C(O)-C i -4 alkyl, CO 2 H, CON(R) 2 and O-sugar.
  • R 3 -R 6 are each independently selected from hydrogen, hydroxyl, methyl, ethyl, CH 2 OH, O-methyl, O-ethyl, t-butoxy, -THP, CF 3 , CO 2 H, - CH 2 NMe 2 , -CH 2 NEt 2 , CH 2 NMeEt, CH 2 NHMe, and CH 2 NHEt.
  • R 3 , R 4 and R 6 are selected from hydrogen, F, methyl and O-methyl; and R 5 is hydroxyl, - THP, O-methyl, O-Ci- 4 alkanoyloxymethyl,-P(O)(OH)(O-methyl),-P(O)(O-methyl) 2 , - P(O)(OH)(O-ethyl), or -P(O)(O-ethyl) 2 .
  • R 3 -R 6 are each independently selected from hydrogen, CF 3 , methyl and O-methyl, and R 5 is hydroxyl, THP, O-methyl, O-C ⁇ alkanoyloxymethyl, -P(O)(OH)(O-methyl), -P(O)(O-methyl) 2 , - P(O)(OH)(O-ethyl), or -P(O)(O-ethyl) 2 .
  • the sugar may be a monosaccharide or a disaccharide. Examples of suitable sugar moieties include but are not limited to glucose, rhamnose, arabinglucose, neohesperidose, apioglucose and rutinose.
  • An embodiment of the invention relates to compounds of formula (1). In another embodiment the invention relates to compounds of formula (2).
  • R 9 is not OH or OCH 3 .
  • R 5 is OH and R 1 , R 3 , R 4 , R 6 , R 7 , R 10 and R 11 are
  • R 8 and R 9 do not together form 0 ⁇ .
  • formula (2) when Y is O, then R 2 -R ! l are not each hydrogen; when Y is O, and R 3 is OCH 3 , then R 2 and R 4 -R n are not each hydrogen; when Y is O, R 5 and R 6 are each OH, then R 2 -R 4 and R 7 -R n are not each hydrogen; when Y is O, R 3 and R 5 are each OH, then R 2 , R 4 and R 6 -R n are not each hydrogen; when Y is O, R 3 , R 5 and R 9 are each OH, then R 2 , R 4 , R 6 -R 8 , R 10 and R 11 are not each hydrogen; when Y is O, R 3 , R 5 , R 8 and R 9 are each OH, then R 2 , R 4 , R 6 , R 7 , R 10 and R 11 are not each hydrogen; when Y is O, R 3 , R 5
  • the invention relates to compounds of general formulae (Ia), (Ib) and (2a) as defined herein. It will be apparent to those skilled in the art that formulae (Ia) and (Ib) are subsets of formula (1) and formula (2a) is a subset of formula
  • R 1 is selected from hydrogen, hydroxyl, methyl, ethyl, O-methyl, halo-C 1-2 alkyl, and CO 2 R';
  • R 3 , R 4 , R 6 , R 7 , R 10 and R 11 are each independently selected from hydrogen, hydroxyl, halogen, C 1-4 alkyl, haloC 1-4 alkyl, hydroxyC 1-4 alkyl, O-Q ⁇ alkyl, N(R') 2 and O- phenyl, wherein the phenyl ring may be substituted with one or more substituents selected from hydroxyl, halogen, and Ci -2 alkyl;
  • R 5 is selected from hydrogen, hydroxyl, C 1-4 alkyl, O-Ci -4 alkyl, O-Ci- 4 alkanoyloxymethyl, CO 2 R', O-C M alkyl-CO 2 R ⁇ OP(O)(OH)(OC,.
  • Y is O.
  • Y is S.
  • formula (Ia) 'I is a double bond. In another embodiment is a single bond.
  • n 1
  • R 1 is selected from hydrogen, methyl, ethyl, CF 3 , and CO 2 R', wherein R' is hydrogen, methyl or ethyl.
  • R 5 is selected from hydroxyl, O-Ci -4 alkyl, O-C 1-4 alkyl-CO 2 R', and CO 2 R', wherein R' is hydrogen, methyl or ethyl.
  • R 5 is selected from O-C 1-4 alkyl, O-Ci -4 alkyl-CO 2 R', and CO 2 R', wherein R 1 is hydrogen, methyl or ethyl.
  • n is 2 and R 5 is OH at least one of R 1 , R 3 , R 4 , R 6 , R 7 , R 10 and R 11 is not hydrogen.
  • a further aspect of the invention relates to compounds of general formula (Ib):
  • R 1 is selected from hydrogen, hydroxyl, halogen, C ⁇ . 4 alkyl, haloC 1-4 alkyl, hydroxyC M alkyl, O-Ci -4 alkyl, OC(O)-C 1-4 alkyl, C(O)-C 1-4 alkyl, and O-sugar;
  • R 3 , R 4 , R 6 are each independently selected from hydrogen, hydroxyl, halogen, Ci- i 5 4 alkyl, C 3-6 cycloalkyl, haloCi ⁇ alkyl, hydroxyCi -4 alkyl, O-Ci -4 alkyl, O-Ci -4 alkyl-CO 2 R,
  • R 5 is selected from hydrogen, hydroxyl, halogen, Ci -4 alkyl, C 3 . 6 cycloalkyl, haloCi.
  • R 3 and R 4 , R 4 and R 5 , and R 5 and R 6 together form ° " 1 , or O" 1 ; each R is independently selected from hydrogen and C 1-4 alkyl; is 2-pyridyl, 3-pyridyl or 4-pyridyl optionally substituted with one or more of halogen, hydroxyl, Ci -4 alkyl, O-C 1-4 alkyl, O-C 3-6 cycloalkyl, haloC 1-4 alkyl, hydroxyCi- 4 alkyl, O-C 1-4 alkyl-CO 2 R, O-C 3-6 cycloalkyl, O-C 3-6 heterocycloalkyl, O-C 3-6 heteroaryl, N(R) 2 C 1-4 alkyl, N(R) 3 C 1-4 alkyl, O-C 1-4 alkyl-N(R) 2 , O-C 1-4 alkyl-N(R) 3 , C 6-10 aryl, 0-C 6-10 aryl,
  • R 7 -R n are each independently selected from hydrogen, halogen, hydroxyl, Ci -4 alkyl, O-C 1-4 alkyl, O-C 3-6 cycloalkyl, O-C) -4 haloalkyl, haloCi -4 alkyl, hydroxyCi.
  • N i iss a C ⁇ -ioaryl group optionally substituted with one or more of halogen, hydroxyl, Ci -4 alkyl, O-C ⁇ -4 alkyl, 0-C 3-6 CyClOaIlCyI, haloC 1-4 alkyl, hydroxyC 1-4 alkyl, O-C ]-4 alkyl- CO 2 R, 0-C 3-6 CyClOaIlCyI, O-C 3-6 heterocycloalkyl, O-C 3-6 heteroaryl, N(R) 2 C 1-4 alkyl, N(R) 3 C 1-4 alkyl, O-C 1-4 alky 1-N(R) 2 , O-C 1-4 alkyl-N(R) 3 , C 6-10 aryl, O-C 6- i 0 aryl, O-C 1-4 alkyl- C 6- ioaryl, O-Ci -4 alkyl-C 6-10 heterocycloalkyl, O-Ci -4
  • Y is O. In another embodiment, Y is S.
  • ⁇ is a double bond. In another embodiment is a single bond.
  • Y is O
  • R 1 is selected from hydrogen, Ci -4 alkyl, haloC 1-4 alkyl, and COOH;
  • R 3 is selected from hydrogen, hydroxyl, O-benzyl and optionally substituted O- benzyl;
  • R 4 and R 6 are each independently selected from hydrogen, and Ci -4 alkyl,
  • R 5 is selected from hydroxyl, THP, and O-C 1-4 alkyl-CO 2 R; is 2-pyridyl, optionally substituted with one or more O-C 1-4 alkyl, or
  • R 7 -R u are each independently selected from hydrogen, halogen, hydroxyl, C 1-4 alkyl, O-C M alkyl, O-C 1-4 haloalkyl, haloC 1-4 alkyl, O-benzyl, CHO, CO 2 H, and OC(O)-C i -4 alkyl, or one or more of R 7 and R 8 , R 8 and R 9 , R 9 and R 10 , and R 10 and R 11 together form each R is independently selected from hydrogen and C 1-4 alkyl; or a pharmaceutically acceptable salt thereof.
  • Y is O
  • R 1 is selected from hydrogen, halogen, d ⁇ alkyl and CF 3
  • R 3 , R 4 and R 6 are selected from hydrogen, methyl, ethyl and hydroxyl
  • R 5 is selected from hydrogen, hydroxyl, C 1-4 alkyl, THP or O-benzyl;
  • R 7 -R u are each independently selected from hydrogen, halogen, hydroxyl, Ci- 4 alkyl, O-C M alkyl, O-C 1-4 haloalkyl, haloC 1-4 alkyl, O-benzyl, CHO, CO 2 H, and OC(O)- C].4alkyl, or one or more of R 7 and R 8 , R 8 and R 9 , R 9 and R 10 , and R 10 and R 11 together form , or ; or a pharmaceutically acceptable salt thereof.
  • Y is O;
  • R 1 is selected from hydrogen, methyl, CF 3 , and COOH;
  • R 3 is selected from hydrogen, hydroxyl, O-benzyl and optionally substituted O- benzyl;
  • R 4 and R 6 are each independently selected from hydrogen, methyl and ethyl, R 5 is selected from hydroxyl and THP;
  • R 7 -R n are each independently selected from hydrogen, fluorine, chlorine, CHO, and CO 2 H or or a pharmaceutically acceptable salt thereof.
  • R 1 is selected from hydrogen, C 1-4 alkyl, haloC 1-4 alkyl, and COOH.
  • R 1 is selected from C 2- 4 alkyl, haloC 2-4 alkyl, hydroxyC 2-4 alkyl, O-C 2-4 alkyl, OC(O)-C 2-4 alkyl and C(O)-C 2-4 alkyl
  • R 1 is selected from C 3 . 4 alkyl, haloC 3-4 alkyl, hydroxyC 3-4 alkyl, O-C 3-4 alkyl, OC(O)-C 3-4 alkyl and C(O)-C 3-4 alkyl.
  • R 1 is selected from hydrogen, methyl, CF 3 , and COOH.
  • R 1 is selected from hydrogen, C 2-4 alkyl, haloC 2-4 alkyl, and
  • R 1 is selected from methyl, ethyl or CF 3 .
  • R 3 is selected from C 2-4 alkyl, haloC 2-4 alkyl, hydroxyC 2-4 alkyl, O-C 2-4 alkyl, OC(O)-C 2 . 4 alkyl and C(O)-C 2-4 alkyl.
  • R 3 is selected from C 3-4 alkyl, haloC 3-4 alkyl, hydroxyC 3- 4 alkyl, O-C 3-4 alkyl, OC(O)-C 3-4 alkyl and C(O)-C 3-4 alkyl
  • R 3 is selected from hydrogen, hydroxyl, O-benzyl and optionally substituted O-benzyl.
  • R 3 is selected from hydroxyl and O- benzyl.
  • R 4 is selected from C ⁇ ⁇ alkyl, haloC 2 . 4 alkyl, hydroxyC 2-4 alkyl, O-C 2-4 alkyl, OC(O)-C 2-4 alkyl and C(O)-C 2-4 alkyl, In another embodiment of formula (Ib), R 4 is selected from hydroxyl, halogen, hydrogen and C 1-4 alkyl. In another embodiment of formula (Ib), R 4 is selected from hydrogen, methyl and ethyl. In another embodiment of formula (Ib), R 4 is selected from halogen, ethyl and OC(O)-C 1-4 alkyl.
  • R 5 is selected from hydroxyl, O-methyl, THP, O-d ⁇ alkanoyloxymethyl, CO 2 R, -P(O)(OH)(O-methyl), -P(O)(O-methyl) 2 , - P(O)(OH)(O-ethyl), and -P(O)(O-ethyl) 2 , wherein R is hydrogen or d. 4 alkyl.
  • R 5 is selected from hydroxyl, O-C 1-4 alkyl, THP, 0-C 1 - 4 alkanoyloxymethyl, and CO 2 R, wherein R is hydrogen or methyl or ethyl.
  • R 5 is selected from hydrogen, hydroxyl, Ci. 4 alkyl, THP or O-benzyl. In another embodiment of formula (Ib), R 5 is selected from hydroxyl, THP and O-C 1-4 alkyl-CO 2 R wherein R is selected from hydrogen and C 1-4 alkyl. In another embodiment of formula (Ib), R 5 is selected from hydroxyl and THP.
  • R 6 is selected from C 2-4 alkyl, haloC 2-4 alkyl, hydroxyC 2-4 alkyl, O-C 2-4 alkyl, OC(O)-C 2-4 alkyl and C(O)-C 2-4 alkyl, In another embodiment of formula (Ib), R 6 is selected from hydroxyl, halogen, hydrogen and Ci -4 alkyl. In another embodiment of formula (Ib), R 6 is selected from hydrogen, methyl and ethyl. In another embodiment of formula (Ib), R 6 is selected from halogen, ethyl and OC(O)-C 1-4 alkyl. In another embodiment R 6 is selected from hydroxyl and O-methyl.
  • formula (Ib) is 2-pyridyl, 3-pyridyl or 4-pyridyl optionally substituted with one or more of halogen, hydroxyl, Ci -4 alkyl, O-d- 4 alkyl, O- C 3-5 cycloalkyl, haloC 1-4 alkyl, hydroxyC 1-4 alkyl, O-C 1-4 alkyl-CO 2 R, O-C 3-6 cycloalkyl, O- C 3-6 heterocycloalkyl, O-C 3-6 heteroaryl, N(R) 2 C 1-4 alkyl, N(R) 3 C M alkyl, O-C )-4 alkyl- N(R) 2 , O-C M alkyl-N(R) 3 , C 6-10 aryl, 0-C 6-10 aryl, O-C 1-4 alkyl-C 6 .i aryl, O-C 1-4 alkyl-C 6- loheterocycloalkyl, O-C
  • formula (Ib) is 2- pyridyl optionally substituted with one or more of halogen, hydroxyl, Ci -4 alkyl, 0-Ci- 4 alkyl, O-C 3-6 cycloalkyl, haloC 1-4 alkyl, hydroxyC 1-4 alkyl, O-Ci -4 alkyl-CO 2 R, 0-C 3- 6 cycloalkyl, O-C 3-6 heterocycloalkyl, O-C 3-6 heteroaryl, N(R) 2 Ci -4 alkyl, N(R) 3 Ci -4 alkyl, O- Ci -4 alkyl-N(R) 2 , O-Ci -4 alkyl-N(R) 3 , C 6 -i O aryl, O-C 6- i 0 aryl, O-Ci -4 alkyl-C 6- i 0 aryl, 0-C 1 .
  • formula (Ib) is 2- pyridyl optionally substituted with one or more O-Ci- 4 alkyl.
  • R 7 -R n are each independently selected from hydrogen, halogen, hydroxyl, C 1-4 alkyl, O-C 1-4 alkyl, O-C 3-6 cycloalkyl, O-C 1-4 haloalkyl, haloC ⁇ 4 alkyl, hydroxyCi.
  • R 7 -R n are each independently selected from C 2-4 alkyl, O-C 2-4 alkyl, 0-C 3- 6 cycloalkyl, O-C 2-4 haloalkyl, haloC 2-4 alkyl and hydroxyC 2-4 alkyl.
  • R 7 , R 9 and R 11 are each hydrogen, R 8 is hydroxyl or O-methyl and R 10 is hydroxyl or O-methyl.
  • R 7 , R 8 , R 10 and R 11 are hydrogen, and R 9 is hydroxyl, halogen methyl, CF 3 , O-methyl, or CO 2 H.
  • R 8 , R 10 and R 11 are hydrogen and R 7 and R 9 are independently selected from hydroxyl, halogen and O-methyl.
  • R 7 , R 10 and R 11 are each hydrogen and R 8 and R 9 are independently
  • R 8 and R 9 together form ⁇ ° °1 ⁇ .
  • R 7 and R 8 together form ° ⁇ . In another embodiment,
  • R 5 is hydroxyl
  • R , R , R and R are
  • R 7 and R 1 ' are hydrogen
  • R 9 is O-methyl
  • R 8 and R 10 and each O-methyl or methyl.
  • At least one of R 1 or R 3 -R u is not hydrogen.
  • R 5 is OH and R 8 and R 10 are O- methyl, at least one of R 1 or R 3 -R 7 is not hydrogen.
  • R 9 when Y is O and R 5 is hydroxyl, R 9 is not hydroxyl or O-methyl; when Y is O, R 5 is hydroxyl and R 9 is halo or methyl, at least one of R 1 , R 3 , R 4 , R 6 , R 7 , R 10 and R 11 is not hydrogen;
  • the sugar may be a monosaccharide or a disaccharide.
  • suitable sugar moieties include but are not limited to glucose, rhamnose, arabinglucose, neohesperidose, apioglucose and rutinose.
  • the present invention also relates to compounds of general formula (2a):
  • Y is O or S
  • R 2 is selected from hydrogen, hydroxyl, methyl, ethyl, O- methyl, haloCi -2 alkyl, and CO 2 R';
  • R 3 , R 4 , R 6 , R 7 , R 8 and R 11 are each independently selected from hydrogen, hydroxyl, halogen, C 1-4 alkyl, haloC 1-4 alkyl, hydroxyC 1-4 alkyl, O-Ci -4 alkyl, N(R') 2 and O- phenyl, wherein the phenyl ring may be substituted with one or more substituents selected from hydroxyl, halogen, and C 1-2 alkyl;
  • R 5 is selected from hydrogen, hydroxyl, C 1-4 alkyl, O-Ci -4 alkyl, 0-C 1 -
  • n 1 or 2; each R' is independently selected from hydrogen and Ci -4 alkyl; and pharmaceutically acceptable salts thereof; with the proviso that when Y is O, R 5 is hydroxyl and n is 1, at least one of R 2 , R 3 , R 4 , R 6 , R 7 , R 8 and R 11 is not hydrogen.
  • Y is O. In another embodiment of formula (2a), Y is S. In embodiments of formula (2a), • represents a double bond. In another embodiment, ' represents a single bond.
  • n 1
  • R 5 is selected from hydroxyl, O-Ci -4 alkyl, 0-Ci- 4 alkyl-CO 2 R', and CO 2 R', wherein R' is hydrogen, methyl or ethyl.
  • R 5 is selected from O-Ci -4 alkyl, O-Ci -4 alkyl-CO 2 R', and CO 2 R', wherein R' is hydrogen, methyl or ethyl.
  • R 2 is selected from hydrogen, methyl, ethyl, CF 3 , and CO 2 R', wherein R' is hydrogen, methyl or ethyl.
  • Compounds of formulae (Ia) and (Ib) are subsets of formula (1) and compounds of formula (2a) and subsets of formula (2).
  • Neoeriocitrin (31) H OH H OR H H OH OH H H
  • the compound is selected from compounds 39a, 39d, 4Od, 39e, 39g, 39h, 4Oh, 39i, 4Oi, 39j, 4Oj, 39k, 40k, 391, 401, 39m, 40m, 39n, 4On, 39o, 40o, 39p, 4Op, 39q, 4Oq, 39r, 4Or, 39s, 40s, 41, 46, 48, 49, 53, 57, 58, 59, 60, 61, 62, 63, 53, 65, 66, 67, 70, 74, 78 and 79 above.
  • the compound is selected from compounds 39a, 39d, 4Od, 39e, 39g, 39h, 4Oh, 39i, 4Oi, 39j, 4Oj, 39k, 40k, 391, 401, 39m, 40m, 39n, 4On, 39o, 40o, 39p, 4Op, 39q, 40q, 39r, 40r, 39s, 40s, 41, 46, 48, 49 and 53 above.
  • the compound is selected from compounds 40a, 40b, 39c, 40c, 4Od, 4Oe, 4Of, 39g, 4Og, 4Oh, 4Oi, 4Oj, 40k, 401, 40m, 4On, 40o, 4Op, 4Oq, 40r, 39s, 40s, 41, 42, 43, 44, 46, 48, 49, 51, 53, 54, and 56 above.
  • the compound is selected from compounds 2, 3, 5, 6, 8, 9, 11, 40a, 40b, 39c, 40c, 4Od, 4Oe, 4Of, 39g, 4Og, 4Oh, 4Oi, 4Oj, 41, 42, 43, 44, 46, 51, 54, and 56 above.
  • the compound is selected from compounds 40a, 5 40c, 4Oe and 4Oi above. In an embodiment of the invention the compound is 40a. In an embodiment of the invention the compound is 40c. In an embodiment of the invention the compound is 40e. In an embodiment of the invention the compound is 4Oi.
  • Reagents and Conditions (a) i. BrCH 2 CO 2 Et, K 2 CO 3 , DMF; ii. KOH, MeOH, H 2 O; (b) P 2 S 5 , Pyridine, 115 0 C for 4 hrs; (c) i. Tf 2 O, Pyridine, DCM; ii. CO, Me 3 SiCH 2 CH 2 OH, Pd(OAc) 2 , 1 ,3-DPPP, Et 3 N, DMSO; (d) CI 3 C(CH 3 ) 2 OH, Cone. NaOH, acetone.
  • Reagents and Conditions (a)DHP, PPTS, DCM; (b) 4-Bromophenol, K 2 CO 3 , DMF; (c) Br 2 /CHC1 3 , 0-25 0 C; (d) Benzamide, 125 0 C; (e) LiBH 4 , THF, 50-60 0 C; (f) DEAD, PPh 3 , THF; (g) P-TsOH, MeOH, THF, 60 0 C.
  • Compounds for use in accordance with the present invention may be PPAR agonists.
  • the compound may be a PPAR- ⁇ agonist.
  • the compound may be a PPAR- ⁇ agonist.
  • compounds in accordance with the present invention may exhibit dual PPAR ⁇ / ⁇ agonist activity.
  • the compound is Psi-baptigenin.
  • the compound is Hesperidin.
  • 'pro-drugs' of the compounds of the invention are so-called 'pro-drugs' of the compounds of the invention.
  • certain derivatives of compounds of formulae (l)-(2) [and (la)-(2a) and (Ib)] which may have little or no pharmacological activity themselves can, when administered into or onto the body, be converted into compounds of the invention having the desired activity, for example, by hydrolytic cleavage.
  • Such derivatives are referred to as 'prodrugs'.
  • Further information on the use of prodrugs may be found in Pro-drugs as Novel Delivery Systems, Vol. 14, ACS Symposium Series (T. Higuchi and W. Stella) and Bioreversible Carriers in Drug Design, Pergamon Press, 1987 (ed. E. B. Roche, American Pharmaceutical Association).
  • Prodrugs in accordance with the invention can, for example, be produced by replacing appropriate functionalities present in the compounds of the invention with certain moieties known to those skilled in the art as 'pro-moieties' as described, for example, in Design of Prodrugs by H. Bundgaard (Elsevier, 1985).
  • Some examples of prodrugs in accordance with the invention include:
  • the compound contains a carboxylic acid functionality (COOH), an ester thereof, for example, a compound wherein the hydrogen of the carboxylic acid functionality of a compound of formulae (l)-(2) [and (la)-(2a) and (Ib)] is replaced by (C-Oalkyl;
  • the compound contains an hydroxyl functionality, an ether thereof, for example, a compound wherein the hydrogen of the alcohol functionality of a compound of formulae (l)-(2) [and (la)-(2a) and (Ib)] is replaced by (Ci-C 4 )alkanoyloxymethyl, or a phosphonate ester thereof; and
  • the compound contains a primary or secondary amino functionality (-NH 2 or -NHR where R ⁇ H), an amide thereof, for example, a compound wherein, as the case may be, one or both hydrogens of the amino functionality of the compound is/are replaced by (Ci-C 6 )alkanoyl.
  • Stereoisomers include all stereoisomers, geometric isomers and tautomeric forms of the compounds of formulae (l)-(2) [and (la)-(2a) and (Ib)], including compounds exhibiting more than one type of isomerism, and mixtures of one or more thereof.
  • the present disclosure encompasses all such compounds, including cis-isomers, trans-isomers, (E)-isomers, (Z)-isomers, ( ⁇ )-enantiomers, (_S)-enantiomers and mixtures thereof including racemic mixtures.
  • acid addition or base salts wherein the counterion is optically active for example, an amino acid, e.g, (/-lactate or /-lysine, etc, or racemic, for example, (//-tartrate or (//-arginine, and the like.
  • Cisl trans isomers may be separated by conventional techniques well known to those skilled in the art, for example, chromatography and fractional crystallisation.
  • the racemate (or a racemic precursor) may be reacted with a suitable optically active compound, for example, an alcohol, or, in the case where the compound of formula (1) or (2) contains an acidic or basic moiety, a base or acid such as 1- phenylethylamine or tartaric acid.
  • a suitable optically active compound for example, an alcohol, or, in the case where the compound of formula (1) or (2) contains an acidic or basic moiety, a base or acid such as 1- phenylethylamine or tartaric acid.
  • the resulting diastereomeric mixture may be separated by chromatography and/or fractional crystallization and one or both of the diastereoisomers converted to the corresponding pure enantiomer(s) by means well known to a skilled person.
  • Chiral compounds of the invention may be obtained in enantiomerically-enriched form using chromatography, typically HPLC, on an asymmetric resin with a mobile phase consisting of a hydrocarbon, typically heptane or hexane, containing from 0 to 50% by volume of isopropanol, typically from 2% to 20%, and from 0 to 5% by volume of an alkylamine, typically 0.1% diethylamine. Concentration of the eluate affords the enriched mixture.
  • Stereoisomeric conglomerates may be separated by conventional techniques known to those skilled in the art - see, for example, Stereochemistry of Organic Compounds by ⁇ . L. ⁇ liel and S. H. Wilen (Wiley, New York, 1994). Therapeutic applications
  • a further aspect of the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising one or more compounds of formula (Ib) or a prodrug thereof, together with a pharmaceutically acceptable adjuvant, diluent or carrier.
  • pharmaceutical compositions comprising one or more compounds of formula (1) or (2) or a prodrug thereof, together with a pharmaceutically acceptable adjuvant, diluent or carrier.
  • compositions comprising one or more compounds of formula (1), (Ib) or (2) may be used in combination with drugs beneficial for treating a targeted condition or disease.
  • the pharmaceutical compositions of the present invention may contain one or more other active ingredients, in addition to a compound of formula (1), (Ib) or (2).
  • the optimal dosage of the drug/s to be administered in combination with the compound/s of the present invention can be readily determined by one of ordinary skill in the art.
  • the additional drug/s may be administered simultaneously or sequentially with the compounds of the present invention. When administered simultaneously, it is preferable to use a pharmaceutical composition in unit dosage form containing the compound/s of the present invention and other drug/s. When administered in combination, either simultaneously or sequentially, the compound/s of the present invention and additional other drug/s may be used in lower doses than when each is used alone.
  • Agents which improve a patient's lipid profile including PPAR alpha agonists such as fenofibric acid derivatives (gemfibrozil, clofibrate, fenofibrate and bezafibrate), PPAR alpha/gamma dual agonists such as KRP-297, muraglitazar, tesaglitazar, farglitazar, and JT-501, PPAR delta, nicotinyl alcohol, nicotinic acid or a salt thereof, bile acid sequestrants (cholestyramine, colestipol, and dialkylaminoalkyl derivatives of a cross-linked dextran), HMG-CoA reductase inhibitors (lovastatin, simvastatin, rosuvastatin, pra), PPAR alpha/gamma dual agonists such as KRP-297, muraglitazar, tesaglitazar, farglitazar, and JT
  • Further active ingredients that may be administered in combination with the compounds of the present invention include ileal bile acid transporter inhibitors, antiobesity compounds such as fenfluramine, dexfenfluramine, phentiramine, subitramine, orlistat, neuropeptide Y5 inhibitors, Mc4r agonists, cannabinoid receptor 1 (CB-I) antagonists/inverse agonists, and beta 3 adrenergic receptor agonists, biguanides including metformin and phenformin, protein tyrosine phosphatase-lB (PTP-IB) inhibitors, dipeptidyl peptidase IV (DP-IV) inhibitors, insulin or insulin mimetics, sulfonylureas including tolbutamide and glipizide or related materials, PPAR gamma agonists and partial agonists such as glitazones and non-glitazones (e.g.
  • alpha-glucosidase inhibitors including acarbose, agonists disclosed in WO097/28149, agents for the treatment of inflammatory conditions such as non-steroidal anti-inflammatory drugs, aspirin, glucocorticoids, azulfidine, and cyclo- oxygenase 2 selective inhibitors, glucagon receptor antagonists, and GLP-I, GIP-I and GLP-I analogs such as exendins (for example exenitide).
  • the compounds of the present invention may also be administered in combination with multiple active compounds, for example, biguanides PPAR agonists, PTP-IB inhibitors, anti-obesity compounds, sulfonylureas, HMG-CoA reductase inhibitors, and DP-IV inhibitors.
  • active compounds for example, biguanides PPAR agonists, PTP-IB inhibitors, anti-obesity compounds, sulfonylureas, HMG-CoA reductase inhibitors, and DP-IV inhibitors.
  • Compounds for use in accordance with the present invention may be PPAR agonists.
  • the compound may be a PPAR- ⁇ agonist.
  • the compound may be a PPAR- ⁇ agonist.
  • compounds in accordance with the present invention may exhibit dual PPAR ⁇ / ⁇ agonist activity.
  • One aspect of the invention is the treatment in vertebrates of diseases that are amenable to amelioration through the activation of PPAR including for example type II diabetes, obesity, hyperlipidemia, cardiovascular disease, anti-neoplastic diseases and tumors, inflammatory conditions and neurogenerative diseases.
  • the present invention relates to a method of treating or preventing a disease in a vertebrate, the method comprising administering to a vertebrate in need thereof an effective amount of a compound of formula (1), (2), (Ia), (Ib) or (2a) as defined herein, or a prodrug thereof, wherein the disease is selected from Type II diabetes, obesity, hyperlipidemia, cardiovascular disease (e.g, coronary and ischemic heart disease, atherosclerosis, peripheral vascular disease), anti-neoplastic diseases and tumours (e.g, control of cell growth, cell differentiation, motility and apoptosis, neuroblastoma, breast cancer), inflammatory conditions (e.g, inflammatory bowel diseases, psoriasis, chronic inflammatory airway disease, asthma, rheumatoid arthritis), and neurodegenerative diseases (e.g, Parkinson's disease, Alzheimer's disease).
  • the disease is selected from Type II diabetes, obesity, hyperlipidemia, cardiovascular disease (e.g, coronary and ischemic heart
  • the disease or condition to be treated is selected from Type II diabetes, obesity, hyperlipidemia, and cardiovascular disease (e.g, coronary and ischemic heart disease, atherosclerosis, peripheral vascular disease).
  • cardiovascular disease e.g, coronary and ischemic heart disease, atherosclerosis, peripheral vascular disease.
  • the disease or condition to be treated is selected from Type II diabetes, obesity and hyperlipidemia.
  • the disease or condition to be treated is Type II diabetes.
  • the method comprises administering an effective amount of a compound of formula (1). In another embodiment the method comprises administering an effective amount of a compound of formula (Ib). In another embodiment the method comprises administering an effective amount of a compound of formula (2).
  • the invention provides for use of a compound of formula (1), (2), (Ia), (Ib) or (2a) as defined herein, or a prodrug thereof, in the manufacture of a medicament for treating a disease selected from Type II diabetes, obesity, hyperlipidemia, cardiovascular disease (e.g, coronary and ischemic heart disease, atherosclerosis, peripheral vascular disease), anti-neoplastic diseases and tumours (e.g, control of cell growth, cell differentiation, motility and apoptosis, neuroblastoma, breast cancer), inflammatory conditions (e.g, inflammatory bowel diseases, psoriasis, chronic inflammatory airway disease, asthma, rheumatoid arthritis), and neurodegenerative diseases (e.g, Parkinson's disease, Alzheimer's disease).
  • a disease selected from Type II diabetes, obesity, hyperlipidemia, cardiovascular disease (e.g, coronary and ischemic heart disease, atherosclerosis, peripheral vascular disease), anti-neoplastic diseases and tumours (e.g, control of cell
  • the disease or condition to be treated is selected from Type II diabetes, obesity, hyperlipidemia, and cardiovascular disease (e.g, coronary and ischemic heart disease, atherosclerosis, peripheral vascular disease).
  • cardiovascular disease e.g, coronary and ischemic heart disease, atherosclerosis, peripheral vascular disease.
  • the disease or condition to be treated is Type II diabetes, obesity, or hyperlipidemia.
  • the disease or condition to be treated is Type II diabetes.
  • salts of the compounds of formulae (l)-(2) [and (Ia)- (2a) and (Ib)] will be pharmaceutically acceptable salts; although other salts may be used in the preparation of the inventive compounds or of the pharmaceutically acceptable salt thereof.
  • pharmaceutically acceptable salt it is meant those salts which, within the scope of sound medical judgement, are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.
  • Pharmaceutically acceptable salts are well known in the art.
  • compositions of formulae (l)-(2) [and (la)-(2a) and (Ib)] may be prepared by methods known to those skilled in the art, including for example, (i) by reacting a compound of formula (1) or (2) with the desired acid or base; (ii) by removing an acid- or base-labile protecting group from a suitable precursor of the compound or by ring-opening a suitable cyclic precursor, for example, a lactone or lactam, using the desired acid or base; or (iii) by converting one salt of the compound to another by reaction with an appropriate acid or base or by means of a suitable ion exchange column.
  • the resulting salt may precipitate out and be collected by filtration or may be recovered by evaporation of the solvent.
  • the degree of ionisation in the resulting salt may vary from completely ionised to almost non-ionised.
  • suitable pharmaceutically acceptable salts of compounds according to the present invention may be prepared by mixing a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, methanesulfonic acid, succinic acid, fumaric acid, maleic acid, benzoic acid, phosphoric acid, acetic acid, oxalic acid, carbonic acid, tartaric acid, or citric acid with the compounds of the invention.
  • a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, methanesulfonic acid, succinic acid, fumaric acid, maleic acid, benzoic acid, phosphoric acid, acetic acid, oxalic acid, carbonic acid, tartaric acid, or citric acid.
  • Suitable pharmaceutically acceptable salts of the compounds of the present invention therefore include acid addition salts.
  • S. M. Berge et al. describe pharmaceutically acceptable salts in detail in J.
  • the salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or separately by reacting the free base function with a suitable organic acid.
  • Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, digluconate, cyclopentanepropionate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanes
  • alkali or alkaline earth metal salts include sodium, lithium potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, triethanolamine and the like.
  • Convenient modes of administration include injection (subcutaneous, intravenous, etc.), oral administration, inhalation, transdermal application, topical creams or gels or powders, or rectal administration.
  • the mode of administration is parenteral.
  • the mode of administration is oral.
  • the formulation and/or compound may be coated with a material to protect the compound from the action of enzymes, acids and other natural conditions which may inactivate the therapeutic activity of the compound.
  • the compound also may be administered parenterally or intraperitoneally.
  • Dispersions of compounds according to the invention may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, pharmaceutical preparations may contain a preservative to prevent the growth of microorganisms.
  • Pharmaceutical compositions suitable for injection include sterile aqueous solutions
  • the composition is stable under the conditions of manufacture and storage and may include a preservative to stabilise the composition against the contaminating action of microorganisms such as bacteria and fungi.
  • Compounds of formulae (l)-(2) [and (la)-(2a) and (Ib)] according to the present invention may be administered orally, for example, with an inert diluent or an assimilable edible carrier.
  • the compound(s) and other ingredients may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into an individual's diet.
  • the compound(s) may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • such compositions and preparations may contain at least 1% by weight of active compound.
  • the percentage of the compound(s) in pharmaceutical compositions and preparations may, of course, be varied and, for example, may conveniently range from about 2% to about 90%, about 5% to about 80%, about 10% to about 75%, about 15% to about 65%; about 20% to about 60%, about 25% to about 50%, about 30% to about 45%, or about 35% to about 45%, of the weight of the dosage unit.
  • the amount of compound in therapeutically useful compositions is such that a suitable dosage will be obtained.
  • pharmaceutically acceptable carrier is intended to include solvents, dispersion media, coatings, anti-bacterial and anti-fungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art.
  • compositions according to the present invention may also be incorporated into the compositions according to the present invention. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the individual to be treated; each unit containing a predetermined quantity of compound(s) is calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the compound(s) may be formulated for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in an acceptable dosage unit. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.
  • the carrier is an orally administrable carrier.
  • Another form of a pharmaceutical composition is a dosage form formulated as enterically coated granules, tablets or capsules suitable for oral administration.
  • compositions of formulae (l)-(2) [and (la)-(2a) and (Ib)] according to the invention also may be administered in the form of a "prodrug".
  • a prodrug is an inactive form of a compound which is transformed in vivo to the active form.
  • Suitable prodrugs include esters, phosphonate esters etc, of the active form of the compound.
  • the compound of formulae (l)-(2) [and (la)-(2a) and (Ib)] may be administered by injection.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by including various anti-bacterial and/or antifungal agents.
  • Suitable agents are well known to those skilled in the art and include, for example, parabens, chlorobutanol, phenol, benzyl alcohol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminium monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the analogue in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilisation.
  • dispersions are prepared by incorporating the analogue into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • Tablets, troches, pills, capsules and the like can also contain the following: a binder such as gum gragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring.
  • a binder such as gum gragacanth, acacia, corn starch or gelatin
  • excipients such as dicalcium phosphate
  • a disintegrating agent such as corn starch, potato starch, alginic acid and the like
  • a lubricant such as magnesium stearate
  • a sweetening agent such as sucrose, lactose or saccharin or a flavouring agent such as peppermint, oil of wintergreen, or
  • tablets, pills, or capsules can be coated with shellac, sugar or both.
  • a syrup or elixir can contain the analogue, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour.
  • any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
  • the analogue can be incorporated into sustained-release preparations and formulations.
  • the pharmaceutical composition may further include a suitable buffer to minimise acid hydrolysis. Suitable buffer agent agents are well known to those skilled in the art and include, but are not limited to, phosphates, citrates, carbonates and mixtures thereof.
  • Single or multiple administrations of the compounds and/or pharmaceutical compositions according to the invention may be carried out.
  • One skilled in the art would be able, by routine experimentation, to determine effective, non-toxic dosage levels of the compound and/or composition of the invention and an administration pattern which would be suitable for treating the diseases and/or infections to which the compounds and compositions are applicable.
  • the optimal course of treatment such as the number of doses of the compound or composition of the invention given per day for a defined number of days, can be ascertained using convention course of treatment determination tests.
  • an effective dosage per 24 hours may be in the range of about 0.0001 mg to about 1000 mg per kg body weight; for example, about 0.001 mg to about 750 mg per kg body weight; about 0.01 mg to about 500 mg per kg body weight; about 0.1 mg to about 500 mg per kg body weight; about 0.1 mg to about 250 mg per kg body weight; or about 1.0 mg to about 250 mg per kg body weight.
  • an effective dosage per 24 hours may be in the range of about 1.0 mg to about 200 mg per kg body weight; about 1.0 mg to about 100 mg per kg body weight; about 1.0 mg to about 50 mg per kg body weight; about 1.0 mg to about 25 mg per kg body weight; about 5.0 mg to about 50 mg per kg body weight; about 5.0 mg to about 20 mg per kg body weight; or about 5.0 mg to about 15 mg per kg body weight.
  • an effective dosage may be up to about 500mg/m2.
  • an effective dosage is expected to be in the range of about 25 to about 500mg/m2, about 25 to about 350mg/m2, about 25 to about 300mg/m2, about 25 to about 250mg/m2, about 50 to about 250mg/m2, and about 75 to about 150mg/m2.
  • a compound of Formula (1) or (2) may be administered in an amount in the range from about 100 to about 1000 mg per day, for example, about 200 mg to about 750 mg per day, about 250 to about 500 mg per day, about 250 to about 300 mg per day, or about 270 mg to about 280 mg per day.
  • compounds in accordance with the present invention may be administered as part of a therapeutic regimen with other drugs. It may desirable to administer a combination of active compounds, for example, for the purpose of treating a particular disease or condition.
  • compound(s) according to the present invention may be administered in combination therapy with known antidiabetic or antilipidemic agents.
  • two or more pharmaceutical compositions may be combined in the form of a kit suitable for co-administration of the compositions.
  • the respective agents may be administered simultaneously, or sequentially in any order.
  • the invention relates to an assay for identifying PPAR agonists.
  • the agonist is a PPAR- ⁇ agonist
  • the invention relates to a method for identifying a PPAR agonist, comprising: determining ligand-receptor interactions of a candidate compound with at least two structurally distinct docking templates; comparing the ligand-receptor interactions of the candidate compound with the interactions of a known PPAR agonist; and thereby determining whether a candidate compound is a PPAR agonist.
  • the present invention has used structure-based virtual screening and in vitro bioassays to identify psi-baptigenin ( ⁇ -baptigenin; or pseudo-baptigenin) and hesperidin as PPAR agonists.
  • the method of the invention may be used to identify PPAR agonist that share little similarity with known ligands.
  • the present invention utilises the approach of "multiple rigid-receptor docking" whereby two structurally distinct PPAR- ⁇ crystal structures are employed as docking templates.
  • the first template, receptor (I), is derived from of the farglitazar-bound PPAR- ⁇ X-ray complex (PDB: 1FM9); and the second, receptor (II), is derived from the rosiglitazone-bound PPAR- ⁇ X-ray complex (PDB: 1FM6) (Gampe et al., 2000). Both show significant structural differences in their relative ligand binding domain (LBD) due to ligand-induced side-chain rearrangements.
  • LBD relative ligand binding domain
  • IFD induced-fit docking
  • PPAR- ⁇ crystal structures as rigid docking templates (PDB: 1FM9 and 1FM6); these show small structural differences in their relative LBD due to ligand-induced side-chain rearrangements.
  • Using two receptors of PPAR- ⁇ in the virtual screening allows a wider range of ligands to be screened.
  • Ligands showing high affinity towards PPAR- ⁇ in silico are subsequently tested in vitro using a PPAR- ⁇ transcriptional factor assay, a PPAR- ⁇ reporter gene luciferase assay and a PPAR- ⁇ reporter gene luciferase assay.
  • the activity of compounds identified by this process in inducing PPAR- ⁇ mRNA and protein expression in vitro may be also examined to see if the effect can be abolished in the presence of GW9662, a potent synthetic PPAR- ⁇ antagonist (Bendixen et al., 2001).
  • Biological data from the transactivation assays may be used to characterize compounds as agonists of human PPAR- ⁇ .
  • TLC Thin layer chromatography
  • silica 0.2 mm, 60F 254
  • UV fluorescence 254 nm
  • Flash vacuum chromatography was performed on silica gel (Merck silica gel 6OH, particle size 5—40 ⁇ m). Chemicals were purchased from Aldrich, Boron Molecular and Indofine Chemical Co. at the highest available grade.
  • the catalyst was washed with H 2 O (3 mL) and CH 2 Cl 2 (5 mL).
  • the aqueous phase was extracted twice with CH 2 Cl 2 .
  • the collected organic extracts were dried (Na 2 SO 4 ), filtered,
  • the catalyst was washed with H 2 O (3 mL) and CH 2 Cl 2 (5 mL). The aqueous phase was extracted twice with CH 2 Cl 2 . The collected organic extracts were dried (Na 2 SO 4 ), filtered, and concentrated under reduced pressure. The crude was purified by flash chromatography (60% EtOAc/hexane) to give 39m (444 mg, 87% yield) as a colourless solid.
  • Example 21 3-(3,5-Dimethoxyphenyl)-7-hydroxy-4H-chromen-4-one (40a) To a solution of 3-(3,5-dimethoxyphenyl)-7-(tetrahydro-2H-pyran-2-yloxy)-4H- chromen-4-one (39a) (800 mg) in MeOH (30 mL) and THF (30 mL) was added /?-TsOH (70 mg) at rt. The resulting mixture was stirred at 60 0 C for 1 h, then Et 3 N (0.6 mL) was added, and volatiles were removed under reduced pressure. Purification by flash chromatography (40% EtOAc/hexane then 5% MeOH/EtOAc) provided 40a (530 mg, 85% yield) as a colorless solid.
  • Example 38 3-(3-(Benzyloxy)phenyl)-7-hydroxy-4/y-chromen-4-one (40r) To a solution of 3-(3-(benzyloxy)phenyl)-7-(tetrahydro-2H-pyran-2-yloxy)-4//- chromen-4-one (39r) (450 mg) in MeOH (30 mL) and THF (30 mL) was added /7-TsOH (40 mg) at rt. The resulting mixture was stirred at 60 °C for 1 h, then Et 3 N (0.3 mL) was added, and volatiles were removed under reduced pressure. Purification by flash chromatography (40% EtOAc/hexane then 5% MeOH/EtOAc) provided 4Or (289 mg, 80% yield) as a colorless solid.
  • Example 44 2-(Benzo[d][l,3]dioxol-5-yl)-l-(5-ethyl-2,4-dihydroxyphenyl)ethanone (45)s With stirring, a rapid current of dry hydrogen chloride is passed for 10 min into a solution of 8-cyanomethyl-l, 6-benzodioxecane (500 mg, 1 mmol) in dry toluene (10 mL) cooled to O 0 C. Then a solution of 4-ethylresorcinol (471 mg, 1.1 mmol) and fused zinc chloride (211 mg, 0.5 mmol) in dry ether (5 mL) is added.
  • a solution of trifluoroacetic anhydride (0.22 mL) is added to a solution of 52 (400 30 mg) in 2 mL of dry pyridine at O 0 C.
  • the reaction mixture was shaken, with ice cooling, for 10-15 min and is left overnight. On the following day, it is heated to 40-50 0 C for 10- 15 min and again left at room temperature for 12 h. Then it is poured into 20-30 mL cold water, and the precipitate is filtered off and crystallized from ethanol to give 53 as a cream colour solid.
  • Anti-actin primary antibody bovine serum albumin (BSA), dimethyl sulfoxide (DMSO), GW9662 and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma-Aldrich (Sydney, Australia). Natural products, totaling 200 compounds, were sourced from the Herbal Medicines Research and Education Center (Faculty of Pharmacy, University of Sydney). Cell Culture The THP-I monocytes and macrophages were grown in RPMI 1640 in the presence of 50 ⁇ M ⁇ -mercaptoethanol.
  • BSA bovine serum albumin
  • DMSO dimethyl sulfoxide
  • PMA phorbol 12-myristate 13-acetate
  • All media contained L-glutamine supplemented with penicillin (100 U/ml)/ streptomycin (100 (g/ml), and 10% (v/v) heat- inactivated fetal bovine serum (FBS) in a humidified atmosphere of 5% CO 2 and 95% O 2 at 37°C.
  • FBS heat- inactivated fetal bovine serum
  • the PPAR- ⁇ antagonist, GW9662 (5 (M) was added 1 h prior to addition of positive control or test samples.
  • Cell-Based Transcriptional Factor Assay The PPAR- ⁇ Transcription Factor Assay is a sensitive ELISA method for detecting
  • PPAR- ⁇ transcription factor DNA binding activity in nuclear extracts of THP- 1 derived macrophage cell line was conducted according to the manufacturer's instructions (Cayman Chemical, Sydney, Australia). The purification of cellular nuclear extract from the cultured cells was prepared with CelLyticTM NuCLEARTM Extraction Kit (Sigma, Sydney, Australia). Cell Proliferation Assay
  • THP-I macrophage cells were seeded for overnight, then treated with various concentrations of rosiglitazone, GW9662 and test compounds (0.01 - 100 ⁇ M) before further incubation for 3 days at 37°C in a humidified atmosphere with 5% CO 2 .
  • MTS (tetrazolium salt) reagents (the CellTiter96® Aqueous One Solution Cell Proliferation Assay, Promega, Sydney, Australia) was added, incubated for 4 h and finally analyzed using a microtiter plate reader (model 3550, Bio-Rad) ( ⁇ : 490 nm).
  • Total mRNA was prepared separately from the THP-I macrophage cells using TRIzol (Invitrogen, Sydney, Australia). The relative levels of specific mRNAs were assessed by RT-PCR as described previously (Abe et al., 2002). Single-stranded cDNA was synthesized from 1 ⁇ g of total RNA using Superscript II RNAse H Reverse Transcriptase, as per instructions of the manufacturer (Invitrogen, Sydney, Australia). PCR was performed on a thermocycler, PTC-200 DNA engine (MJ Research Inc, USA). The required cDNA was synthesized with the Platinum ® Pfx DNA Polymerase method (Invitrogen, Sydney, Australia).
  • the genes examined were PPAR- ⁇ (L40904; 382bp; sense: 5'-GAGCCCAAGTTTGAGTTTGC-S '; 5'-TGGAAGAAGGGAAATGTTGG-S') and ⁇ -actin (NMOOI lOl; 629bp; sense: 5'-GGAGTAACCAGGTCGTCCAA-S'; 5'- GAAGGTGCCCAGAAT ACC AA-3').
  • the PCR samples were electrophoresed on 5-12% acrylamide gel (29:1, acrylamide:N,N'-methylene-bis-acrylamide) in TBE buffer [89 mM Tris-base pH 7.6, 89 mM boric acid, 2 mM EDTA].
  • THP-I cells were seeded and treated with PMA (400 ng/ml) for 72 h to obtain THP- 1 macrophages.
  • the macrophages were treated with 0.1% DMSO, psi-baptigenin (40c) (30 ⁇ M), hesperidin (34) (30 ⁇ M) and rosiglitazone (30 ⁇ M) for 48 h.
  • the cells were washed with PBS and lysed with RIPA lysis buffer for protein extraction.
  • the protein contents in the samples were measured using BCA protein estimation kit and 20 ⁇ g of sample was loaded onto 4-12% NuP AGE® Bis-Tris Gel (Invitrogen, Sydney, Australia).
  • the protein was transferred to PVDF membrane and blocked overnight in skim milk (5% skim milk in tris buffered saline).
  • skim milk 5% skim milk in tris buffered saline.
  • the PVDF membranes were treated with antihuman PPAR- ⁇ mouse monoclonal primary antibody (1:500 dilution; Santa Cruz Biotechnology, USA) followed with horseradish peroxidase-conjugated anti-mouse secondary antibody (1 : 10,000 dilution; Promega, USA).
  • the antibody treatment was performed for Ih followed by 30 min wash with the washing buffer (Tris buffered saline with 0.1% Tween-20). Protein expression was detected by chemiluminescence method (Roche).
  • the PVDF membranes were exposed to X-ray film (Kodak, USA) and developed using the SRX-IOlA X-ray developer (Konica, Taiwan). Quantitation of the results was performed by using the NIH Image J software. After stripping with stripping buffer (Glycine (15 g), SDS (1 g), Tween-20 (10 mL), pH 2.2) the membranes were re-probed with anti-actin primary antibody (1: 10,000 dilution; Sigma, Australia) and re- incubated with the secondary horseradish peroxidase antibody, and protein bands were detected as described above.
  • stripping buffer Glycine (15 g), SDS (1 g), Tween-20 (10 mL), pH 2.2
  • Ligand Preparation Ligands were built, manipulated and adjusted for chemical correctness using
  • Maestro 7.5 (Maestro, version 7.5, Schrodinger, LLC, New York, NY) graphical user interface employing MacroModel 9.1 (MacroModel, version 9.1, Schrodinger, LLC, New York, NY). Geometry minimizations were performed on all ligands using the OPLS_2005 (MacroModel, version 9.1, Schrodinger, LLC, New York, NY) force field and the Truncated Newton Conjugate Gradient (TNCG). Optimizations were converged to a gradient rmsd below 0.05 kj A '1 mol or continued to a maximum of 500 iterations, at which point there were negligible changes in rmsd gradients. The library was seeded with farglitazar and rosiglitazone as reference compounds for the virtual screening. Ligand Docking and Target Preparation Ligands were independently docked into the LBD of two PPAR- ⁇ receptors.
  • Protein preparation and refinement protocols directed by the protein preparation facility (Schrodinger User Manuals, Glide v4.0 Schrodinger, LLC, New York, NY), were performed on both targets to achieve chemical correctness. Briefly, this included deleting crystallographic waters; adding hydrogens; adjusting bond orders and formal charges; neutralizing side-chains distant from the binding site and alleviating potential steric clashes via protein minimization with the OPLS_2005 force field. The tautomeric states of His323 (positively charged) and His449 (N ⁇ protonated) were manually selected to maximize hydrogen bonding.
  • the shape and properties of the binding site were characterized and setup for docking using the receptor grid generation panel (Schr ⁇ dinger User Manuals, Glide v4.0 Schr ⁇ dinger, LLC, New York, NY).
  • a Coulomb-van der Waals (vdW) scaling of 1.0/0.8 was set for receptor/ligand vdW radii, respectively.
  • the top 20 of best-scoring ligands from each docking study were pooled together and selected for the PPAR- ⁇ agonist functional assay. Induced-Fit Docking
  • the IFD protocol was run from the graphical user interface accessible within Maestro 7.5. It was carried out on receptor (II) with psi-baptigenin (40c), apigenin (8), chrysin (12), biochanin-A (55) and genistein (56) as test ligands.
  • the overall procedure has four stages: Briefly, during Stage 1 initial softened-potential Glide docking is performed on a vdW scaled-down rigid-receptor (II); a scaling of 0.7/0.5 was set for receptor/ligand vdW radii, respectively (Sherman et al., 2006).
  • Residues Cys285 and Phe363 were temporarily mutated to alanine for having deviated more than 2.5 A compared to receptor (I). The top 20 poses for each test ligand was retained. In Stage 2, Cys285 and Phe363 were restored to their original residue type, followed by Prime side- chain prediction and minimization for each of the 20 ligand/protein complexes. Backbone residues and ligands were minimized. Residues within 5.0 A of ligand poses were similarly refined, but additionally underwent sampling. A total of 20 induced-fit receptor conformations were generated for each of the 5 test ligands.
  • Stage 3 involved redocking the test ligands into their respective 20 structures that are within 30.0 kcal mol "1 of their lowest energy structure. Finally, the ligand poses were scored in Stage 4 using a combination of Prime and GlideScore scoring functions. The XP scoring function was used in all docking stages.
  • the site targeted in the docking calculations was defined by the position of the molecules: farglitazar and rosiglitazone, observed in complex with PPAR- ⁇ as part of the PPAR- ⁇ /RXR ⁇ heterodimer crystal structure (PDB: 1FM9, receptor (I); and 1FM6 receptor (II), respectively) (Gampe et al., 2000).
  • Ligands were ranked based on docking scores whereby the more negative values correspond to greater predicted binding affinities.
  • the 20 best-scoring compounds from each docking study were combined to create an initial hit-list totaling 40 compounds. Duplicate compounds (ie, compounds scoring in the top 20 against both receptors) were removed.
  • Table 1 List of docked compounds, along with rankings and docking scores determined against receptors (I) and (II).
  • the results for the flavonoids represented the only set to have revealed a common binding pose that produced comparable interaction fingerprints to those presented by farglitazar and rosiglitazone. Therefore, the flavonoids were selected for further study. Of particular interest were the compounds psi-baptigenin, hesperidin, apigenin, chrysin, biochanin-A and genistein. Specifically, the results against receptor (I), where they ranked no worse than 14 th position (Table 1), show each occupying a large hydrophobic pocket enclosed by His449, Phe282, Phe360 and Phe363 ( Figure 2a - e).
  • Noticeable hydrophobic contacts include complimentary ⁇ ... ⁇ ( ⁇ 3.5A) stacking between each of the flavonoid's B-Ring phenyl with the side-chain phenyl of Phe363, analogous to the benzophenone...Phe363 interaction of farglitazar.
  • the A-ring phenyl is directed towards the AF-2 helix where hydrogen-bond contacts are made to Ser289 and His323 via the 7-OH.
  • Such interactions are similarly made by farglitazar and rosiglitazone whereby hydrogen bond contacts are established to the above residues, although via a carboxylic acid and thiazolidinedione group, respectively.
  • Apigenin (8) and genistein (56) were also seen to hydrogen bond to the backbone carbonyl of Phe363. Docking results for these flavonoids against receptor (II) are shown in Figure 6.
  • Hesperidin (34) was predicted to bind favorably into both PPAR- ⁇ receptors, ranking third in both instances.
  • Figure 2f clearly illustrates the two binding poses of hesperidin (34) for both receptors, each appearing almost identical when the LBD are superposed.
  • the overall structure twists around Helix 3 (not displayed) in an orientation resembling the U-shape conformation of both farglitazar and rosiglitazone.
  • the highly polar disaccharide moiety is directed towards the polar region of the AF-2 helix. Hydrogen bond contacts are made to Ser289, His323 and Tyr473, again comparable with farglitazar's carboxylate and rosiglitazone 's thiazolidinedione.
  • Hesperidin's A-ring also roughly corresponds to the methylphenyloxazole and pyridine of farglitazar and rosiglitazone, respectively, with each representing a bulky aromatic group in a partially solvent-exposed region. Unlike the previously described flavonoids, hesperidin (34) did not appear to interact with Phe363.
  • the 29 hits chosen on the basis s of docking potential towards PPAR- ⁇ , were selected for PPAR- ⁇ functional assay. This included compounds that were assigned good docking scores, despite not engaging key residues known involved in activation e.g. the gingerones and ginkolides.
  • the Cayman Chemical PPAR- ⁇ Transcription Factor Assay was employed as a sensitive method for detecting specific transcription factor DNA binding activity in nuclear
  • This assay involves the use of a 96 well enzyme-linked immunosorbent assay (ELISA) whereby specific double stranded DNA (dsDNA) sequences containing the peroxisome proliferator response element (PPRE) is immobilized onto the bottom of wells in a microtitre plate.
  • dsDNA double stranded DNA
  • PPRE peroxisome proliferator response element
  • Any PPAR- ⁇ contained in a nuclear extract bind specifically to the PPRE and the degree of binding is detected by the addition of specific primary is antibody directed against PPAR- ⁇ .
  • a secondary antibody conjugated to horse radish peroxidase was added to provide colorometric readout at 450 nm.
  • flavonoids can be classified into 3 groups: flavone [apigenin (8) and chrysin (12)]; isoflavone [psi-baptigenin (40c), biochanin A (55) and genistein (56)] and flavanone glycoside [hesperidin (34)].
  • flavones and isoflavones the two groups differ with the location of the B-ring phenyl on the 1,4- benzopyrone skeleton, be it at the 2 or 3 position, respectively.
  • Hesperidin (34), a flavanone glycoside has pronounced differences including the bound disaccharide rutinose at the 7 position and the reduction of the 2(3) carbon-carbon double bond.
  • Liang et al. suggested apigenin (8) and two similar flavones induced conformational change upon direct binding with the PPAR- ⁇ LBD, and did so differently to that of rosiglitazone.
  • the results from our docking predictions corresponds well with their experimental outcomes — that is, the flavonoids induce conformational change in PPAR- ⁇ and binds differently to rosiglitazone — however, our docking results provide strong indications they do not necessarily have to be allosteric effectors as they remain capable of achieving the above observations in the native binding domain.
  • receptors (I) and (II) were specifically chosen as each structure presented a distinct conformational difference in PPAR- ⁇ ' s LBD, clearly illustrating the ability of this receptor to accommodate a wide range of potential ligands.
  • the IFD successfully rectified critical side-chain rearrangements in the LBD of receptor (II) to closely resemble the LBD of receptor (I), as well as reproducing ligand poses seen in the initial rigid-receptor docking with receptor (I). For that reason, it is conceivable IFD would have been successful in identifying hits excluded by rigid-receptor docking on receptor (II) had the procedure been used in the initial virtual screen.
  • EXPERIMENTAL PROCEDURE Cell Culture Human embryonic kidney (HEK) 293 cell line was obtained from American Type
  • HEK 293 cells were grown in Dulbecco's modified Eagle's medium/F-12 (DMEM/F-12), containing L-glutamine supplemented with penicillin (100 U/mL), streptomycin (100 mg/mL) and 10% (v/v) heat- inactivated foetal bovine serum in a humidified atmosphere of 5% CO 2 and 95% O 2 at 37 °C. (Bramlett et al, 2003; Frederiksen et al, 2004). Transfection and Luciferase Assay (PPAR- ⁇ )
  • the transfection and luciferase procedures were performed as described previously (Bramlett et al., 2003) with slight modification.
  • the HEK 293 cell line was transfected with tK-PPREx3-Luc plasmid, pSG5-hPPAR- ⁇ plasmid and pSV- ⁇ -galactosidase (Promega, Australia) control plasmid.
  • Cells were transfected with FuGENE 6 transfection reagent (Roche, Australia) in accordance with the manufacturer's instructions. After 24 h at 37°C, cells were harvested and plated into 96-well plates at 5 x 10 4 cells per well in complete transfection media and allowed to attach over night at 37 °C.
  • the cells were then treated with rosiglitazone and GWl 929 as positive controls, DMSO (0.1%) as a negative control and the test samples. After 48 hours, the cells were lysed and assayed for luciferase and ⁇ -galactosidase activities using the Bright-Glo Luciferase Assay system and Beta-Glo Assay system (Promega, Australia), respectively. The results were expressed as relative luciferase activity normalized to the ⁇ -galactosidase signal (fold difference compared to negative control).
  • PPAR- ⁇ Cell Proliferation Assay
  • HEK 293 Human embryonic kidney cell line (HEK 293) was seeded overnight then treated with various concentrations of rosiglitazone, GWl 929 and test compounds (0 - 100 ⁇ M) and incubated for 48 hours at 37°C in a humidified atmosphere with 5% CO 2 .
  • MTS (tetrazolium salt) reagent CellTiter96® Aqueous One Solution Cell Proliferation Assay, Promega, Sydney, Australia
  • the transfection and luciferase procedures were performed as described previously (Bramlett et al., 2003) with slight modification.
  • the HEK 293 cell line was transfected with tK-PPREx3-Luc plasmid, pBI-G-hPPAR- ⁇ plasmid and pSV- ⁇ -galactosidase (Promega, Australia) control plasmid.
  • Cells were transfected with FuGENE 6 transfection reagent (Roche, Australia) in accordance with the manufacturer's instructions. After 24 h at 37°C, cells were harvested and plated into 96-well plates at 5 x 10 4 cells per well in complete transfection media and allowed to attach over night at 37°C.
  • the cells were then treated with WY-14643, Fenofibrate as positive controls, DMSO (0.1%) as a negative control and the test samples. After 48 hours, the cells were lysed and assayed for luciferase and ⁇ -galactosidase activities using the Bright-Glo Luciferase Assay system and Beta-Glo Assay system (Promega, Australia), respectively. The results were expressed as relative luciferase activity normalized to the ⁇ -galactosidase signal (fold difference compared to negative control).
  • PPAR-c ⁇ Cell Proliferation Assay
  • HEK 293 cells were seeded overnight then treated with various concentrations of WY-14643, Fenofibrate and test compounds (0 - 100 ⁇ M) and incubated for 48 hours at 37 0 C in a humidified atmosphere with 5% CO 2 .
  • MTS (tetrazolium salt) reagent CellTiter96® Aqueous One Solution Cell Proliferation Assay, Promega, Sydney, Australia was added and samples were incubated for a further 1-4 hours before finally being analyzed using a BMG POLARstar Galaxy Microplate Reader ( ⁇ : 490 nm).
  • Psi-baptigenin (40c) is currently undergoing pre-clinical evaluation, and while it has shown promising activity against PPAR- ⁇ , there is a need for more selective and potent derivatives. This requires an understanding of the key structure features of psi-baptigenin (40c) and its congeners that must be retained to maintain PPAR- ⁇ activity.
  • a series of compounds (Table 3) have been designed, synthesized and evaluated for PPAR- ⁇ activation activity in the Human Embryonic Kidney cell line (HEK 293) at various concentrations (5, 25 and 50 ⁇ M).

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Abstract

The present invention relates to PPAR agonists, and their use in therapy including the treatment of disease. In particular, the invention relates to flavonoid compounds which are PPAR-gamma agonists and/or PPAR alpha/gamma dual agonists.

Description

FLAVONOID PPAR AGONISTS Technical Field
The present invention relates to PPAR agonists, and their use in therapy including the treatment of disease.
Background
The peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that belong to the nuclear hormone superfamily. To date, three isoforms of PPAR have been identified: PP AR-α, -δ and -γ. PPAR-γ is the most abundant receptor expressed in adipocytes and macrophages, where, apart from its involvement in adipocyte differentiation and lipid storage, it serves as the primary receptor modulating insulin sensitization and maintaining lipid and glucose homeostasis.
PPAR-γ is the target of numerous drug discovery efforts because of its role in numerous disease states, including Type II diabetes. The thiazolidinediones (TZDs; or glitazones) and the L-tyrosine analogues are anti-diabetic synthetic agonists that selectively target PPAR-γ. Their mode of action begins with sensitizing tissue to insulin, lowering glucose levels and reducing serum lipids in diabetic patients by potently binding, and subsequently activating, PPAR-γ.
Rosiglitazone (Avandia®), shown below, is a prototypical TZD and serves as a reference compound for this class, which also includes pioglitazone (Actos®) and troglitazone. Rosiglitazone is active in vivo as an anti-diabetic agent in the ob/ob mouse model and is presently being used as an oral hypoglycaemic agent for the treatment of Type II diabetes. The L-tyrosine analogue class of compounds, such as Farglitazar (GI262570) shown below, represent the most potent and selective class of synthetic PPAR-γ agonists currently in existence.
Figure imgf000002_0001
Rosiglitazone Farglitazar
Unfortunately, despite their excellent potencies, several of the known PPAR-γ agonists have been found to present unwanted adverse therapeutic profiles, such as fluid retention, weight gain and cardiac hypertrophy. Troglitazone, for example, was withdrawn from therapeutic use due to liver toxicity, and Farglitazar failed to pass phase III clinical trials due to the emergence of peripheral oedema. The PPAR-α receptor, mainly expressed in the liver, play an important role in fatty acid oxidation and lipoprotein metabolism. Fibrates (Fenofibrate, Clofibrate) and WY- 14643, shown below, show effects such as lowering triglycerides and elevating HDL levels through activation of PPAR-α. The majority of type II diabetes patients suffer from atherogenic lipid abnormalities in addition to insulin resistance, termed as metabolic syndrome. The importance of controlling both glucose and lipid levels in metabolic syndrome, gave rise to the concept of identifying dual agonists, which can activate both PPAR-α and PPAR-γ. In addition to their hypolipidemic effects, PPAR-α agonists reduce body weight gain which led to a hypothesis that activation of PPAR-α may mitigate the weight gain induced by PPAR-γ activation in humans.
Figure imgf000003_0001
Fenofibrate Wy-14643
PPAR-γ and PPAR-α agonists have been implicated in the pathology of various disorders including atherosclerosis, coronary heart disease, obesity and inflammation. Compounds that are dual PPAR-γ and PPAR-α agonists can have fewer therapeutic side- effects than those that act solely at the PPAR-γ receptor or those that act solely at the PPAR-α receptor. Thus development of safer and efficacious dual PPAR-γ and PPAR-α agonists are of considerable therapeutic value.
There is a need to discover new PPAR agonists. More particularly, there is a need for PPAR-γ and PPAR-α agonists and/or dual PPAR-α/γ agonists that are suitable for use in therapy. A further need exists for PPAR-γ and PPAR-α agonists and/or dual PPAR-α/γ agonists that have a therapeutically acceptable side-effect profile.
Summary The present invention relates to compounds having PPAR agonist activity, and the therapeutic use thereof. In one embodiment the PPAR agonist is a PPAR-γ agonist. In another embodiment the PPAR agonist is a PPAR-α agonist. In a further embodiment the PPAR agonist is a dual PPAR α/γ agonist. In a first aspect the invention provides a compound of general formula (Ib):
Figure imgf000004_0001
(Ib) wherein Y is O or S; s ' represents a single bond or a double bond;
R1 is selected from hydrogen, hydroxyl, halogen, C1-4alkyl, haloC1-4alkyl, hydroxyC1-4alkyl, O-C1-4alkyl, OC(O)-C 1-4alkyl, C(O)-C1-4alkyl, and O-sugar;
R3, R4, R6 are each independently selected from hydrogen, hydroxyl, halogen, Ci- 4alkyl, C3-6cycloalkyl, haloCi-4alkyl, hydroxyC1-4alkyl, O-Ci-4alkyl, O-Ci.4alkyl-CO2R,o O-C3-6cycloalkyl, O-C3-6heterocycloalkyl, O-C3-6heteroaryl, N(R)2Ci-4alkyl, N(R)3Ci- 4alkyl, O-C1-4alkyl-N(R)2, O-C1-4alky 1-N(R)3, C6-10aryl, O-C6-]0 aryl, O-Ci.4alkyl-C6.10aryl, O-Ci-4alkyl-C6-10heterocycloalkyl, O-Ci-4alkyl-C6-i0heteroaryl, O-C(O)-Ci-4alkyl, O- CON(R)2, CON(R)2, CO2R, d-4alkanoyloxymethyl, -P(O)(OH)(OCi-4alkyl), -P(O)(OCi- 4alkyl)2, and O-sugar; s R5 is selected from hydrogen, hydroxyl, halogen, Ci-4alkyl, C3-6cycloalkyl, haloCi-
4alkyl, hydroxyC1-4alkyl, O-d.4alkyl, O-Cialkyl-CO2R, O-C3-6cycloalkyl, THP, N(R)2Ci- 4alkyl, N(R)3Ci-4alkyl, O-Ci-4alkyl-N(R)2, C6-i0aryl, O-C6-i0aryl, O-Ci-4alkyl-C6-i0aryl, O- C(O)-C2-4alkyl, 0-CON(R)2, CON(R)2, CO2R, Ci-4alkanoyloxymethyl, -P(O)(OH)(OCi- 4alkyl), and -P(O)(OC]-4alkyl)2; or 0 one or more of R3 and R4, R4 and R5, and R5 and R6 together form
Figure imgf000004_0002
0^ , or O each R is independently selected from hydrogen and Ci-4alkyl;
Figure imgf000004_0003
is 2-pyridyl, 3-pyridyl or 4-pyridyl optionally substituted with one or more of halogen, hydroxyl, Ci-4alkyl, O-C1-4alkyl, O-C3-6cycloalkyl, haloCi-4alkyl, hydroxyCi.5 4alkyl, O-Ci-4alkyl-CO2R, O-C3.6cycloalkyl, O-C3-6heterocycloalkyl, O-C3-6heteroaryl, N(R)2C1-4alkyl, N(R)3C1-4alkyl, O-C1-4alkyl-N(R)2, O-C1-4alkyl-N(R)3> C6.10aryl, 0-C6-10 aryl, 0-C1-4alkyl-C6.ioaryl, O-Ci-4alkyl-C6-10heterocycloalkyl, O-Ci-4alkyl-C6.10heteroaryl, 0-CON(R)2, CON(R)2, CO2R, Ci-4alkanoyloxymethyl, -P(O)(OH)(OCi-4alkyl), - P(O)(OC1-4alkyl)2, O-sugar, O-benzyl, CHO, CO2H, and OC(O)-C Malkyl, or
Figure imgf000005_0001
wherein R7-Ru are each independently selected from hydrogen, halogen, hydroxyl, C1-4alkyl, O-C1-4alkyl, O-C3-6Cycloalkyl, O-C1-4haloalkyl, haloC1-4alkyl, hydroxyCi. 4alkyl, 0-CMaIlCyI-CO2R, O-C3-6cycloalkyl, O-C3-6heterocycloalkyl, O-C3-6heteroaryl, s N(R)2C1-4alkyl, N(R)3C1-4alkyl, O-C1-4alkyl-N(R)2, O-C1-4alkyl-N(R)3> C6.10aryl, O-C6-i0 aryl, O-Ci-4alkyl-C6-10aryl, O-Ci-4alkyl-C6-i0heterocycloalkyl, O-C1-4alkyl-C6-i0heteroaryl, 0-CON(R)2, CON(R)2, CO2R, Cr4alkanoyloxymethyl, -P(O)(OH)(OC1.4alkyl), - P(O)(OC1-4alkyl)2, O-sugar, O-benzyl, CHO, CO2H, and OC(O)-C 1-4alkyl, or one or more of R7 and R8, R8 and R9, R9 and R10, and R10 and R11 together form
Figure imgf000005_0002
group optionally substituted with one or more of halogen, hydroxyl, Ci-4alkyl, O-C1-4alkyl, O-C3-6cycloalkyl,
Figure imgf000005_0003
CO2R, O-C3-6cycloalkyl, O-C3-6heterocycloalkyl, O-C3-6heteroaryl, N(R)2C 1-4alkyl, N(R)3Ci-4alkyl, O-C1-4alkyl-N(R)2, O-C1-4alkyl-N(R)3, C6-10aryl, 0-C6-10 aryl, O-CMalkyl- i5 C6-10aryl, O-C1-4alkyl-C6-10heterocycloalkyl, 0-C1-4alkyl-C6-1oheteroaryl, 0-CON(R)2, CON(R)2, CO2R, Ci-4alkanoyloxymethyl, -P(O)(OH)(OCj-4alkyl), -P(O)(OCi-4alkyl)2, O- sugar, O-benzyl, CHO, CO2H, and OC(O)-C 1-4alkyl; or a pharmaceutically acceptable salt thereof.
In a second aspect the invention provides a pharmaceutical composition comprising
20 one or more compounds of formula (Ib) according to the first aspect of the invention, or a prodrug thereof, together with a pharmaceutically acceptable adjuvant, diluent or carrier.
A third aspect of the invention provides for a method of treating or preventing a disease in a vertebrate, the method comprising administering to a vertebrate in need thereof an effective amount of a compound of formula (Ib) according to the first aspect of
25 the invention or a prodrug thereof, or a composition according to the second aspect of the invention, or a compound of formula (1) or (2):
30
Figure imgf000006_0001
(1) (2) wherein
Y is O or S;
R1 and R2 are each independently selected from hydrogen, hydroxyl, halogen, C1. 4alkyl, haloC1-4alkyl, hydroxyC1-4alkyl, O-C1-4alkyl, OC(O)-C1-4alkyl, C(O)-C 1-4alkyl, CO2H, and CO(O)-C1-4alkyl;
R3-R6 are each independently selected from hydrogen, hydroxyl, halogen,
Figure imgf000006_0002
C3-6cycloalkyl, haloC1-4alkyl, hydroxyC1-4alkyl, O-C1-4alkyl, O-C1-4alkyl-CO2R, O-C3-6cycloalkyl, O-C3-6heterocycloalkyl, O-C3-6heteroaryl, N(R)2C i-4alkyl, N(R)3Ci- 4alkyl, O-C1-4alkyl-N(R)2, O-Ci-4alkyl-N(R)3, C6-10aryl, O-C6-i0 aryl, O-Ci.4alkyl-C6-10aryl, O-C1-4alkyl-C6-10heterocycloalkyl, O-C1-4alkyl-C6-10heteroaryl, O-C(O)-Ci-4alkyl, O- CON(R)2, CON(R)2, CO2R, Ci-4alkanoyloxymethyl, -P(O)(OH)(OCMalkyl), -P(O)(OC1- 4alkyl)2, and O-sugar; or
one or more of R and R , R and R , and R and R together form
Figure imgf000006_0003
or and each R is independently selected from hydrogen and Ci-4alkyl;
Figure imgf000006_0004
is 2-pyridyl, 3-pyridyl or 4-pyridyl optionally substituted with one or more of hydrogen, halogen, hydroxyl, Ci-4alkyl, O-C1-4alkyl, O-C3-6cycloalkyl, haloC1-4alkyl, hydroxyCi-4alkyl, O-Ci-4alkyl-CO2R, 0-C3-6CyClOaIlCyI, O-C^heterocycloalkyl, 0-C3. eheteroaryl, N(R)2C i-4alkyl, N(R)3C i-4alkyl, O-C1-4alkyl-N(R)2, O-C1-4alkyl-N(R)3) C6- 10aryl, 0-C6-io aryl, 0-Ci.4alkyl-C6-ioaryl, O-d^alkyl-C^ioheterocycloalkyl, 0-Ci-4alkyl- C6-10heteroaryl, 0-CON(R)2, CON(R)2, CO2R, C^alkanoyloxymethyl, -P(O)(OH)(OCi- ar, O-benzyl, CHO, CO2H, and OC(O)-C)-4alkyl, or
Figure imgf000006_0005
R7-Rn are each independently selected from hydrogen, halogen, hydroxyl, C1-4alkyl, O-C1-4alkyl, O-C^cycloalkyl, haloCi-4alkyl, hydroxyCi-4alkyl, O-Ci-4alkyl- CO2R, 0-C3-6CyClOaIlCyI, O-C3-6heterocycloalkyl, O-C3-6heteroaryl, N(R)2C1-4alkyl, N(R)3C1-4alkyl, O-Ci-4alkyl-N(R)2, O-C1-4alkyl-N(R)3) C6.10aryl, 0-C6-10 aryl, O-CMalkyl- C6-10aryl, O-C1-4alkyl-C6-10heterocycloalkyl, O-C1-4alkyl-C6-10heteroaryl, 0-CON(R)2, CON(R)2, CO2R, Cr4alkanoyloxymethyl, -P(O)(OH)(OC1-4alkyl), -P(O)(OC 1-4alky I)2, 0- sugar, O-benzyl, CHO, CO2H, and OC(O)-C1-4alkyl, or one or more of R7 and R8, R8 and R9, R9 and R10, and R10 and R11 together form
Figure imgf000007_0001
l group optionally substituted with one or more of halogen, hydroxyl, C1-4alkyl, O-C1-4alkyl, O-C3-6cycloalkyl,
Figure imgf000007_0002
hydroxyCi-4alkyl, O-Ci-4alkyl- CO2R, O-C3-6cycloalkyl, O-C3-6heterocycloalkyl, O-C3-6heteroaryl, N(R)2C i-4alkyl, N(R)3C 1-4alkyl, O-C1-4alky 1-N(R)2, O-C1-4alkyl-N(R)3, C6-i0aryl, 0-C6-10 aryl, O-Ci-4alkyl- C6-10aryl, O-C1-4alkyl-C6-10heterocycloalkyl, O-C1-4alkyl-C6-10heteroaryl, 0-CON(R)2, CON(R)2, CO2R, C^alkanoyloxymethyl, -P(O)(OH)(OC 1-4alkyl), -P(O)(OCMalkyl)2, O- sugar, O-benzyl, CHO, CO2H, and OC(O)-C 1-4alkyl; or a prodrug thereof, or a pharmaceutically acceptable salt thereof, wherein the disease is selected from Type II diabetes, obesity, hyperlipidemia, cardiovascular disease, anti-neoplastic diseases and tumours, inflammatory conditions, and neurodegenerative diseases.
In a fourth aspect of the invention there is provided for use of a compound of formula (Ib) according to the first aspect of the invention or a prodrug thereof, or a compound of formula (1) or (2):
Figure imgf000007_0003
(1) (2) wherein
Y is O or S;
R1 and R2 are each independently selected from hydrogen, hydroxyl, halogen, Ci- 4alkyl, haloC1-4alkyl, hydroxyC1-4alkyl, O-Ci-4alkyl, OC(O)-C i-4alkyl, C(O)-CMalkyl, CO2H, and CO(O)-C i.4alkyl; R3-R6 are each independently selected from hydrogen, hydroxyl, halogen, Ci-4alkyl, C3-6cycloalkyl, haloCι-4alkyl, hydroxyC1-4alkyl, O-Ci-4alkyl, O-C1-4alkyl-CO2R, 0-C3-6CyClOaIlCyI, O-C3-6heterocycloalkyl, O-C3-6heteroaryl, N(R)2C i-4alkyl, N(R)3Ci- 4alkyl, O-Ci-4alkyl-N(R)2, O-Ci-4alkyl-N(R)3, C6-10aryl, O-C6.i0 aryl, O-C1-4alkyl-C6-10aryl, 5 O-C1-4alkyl-C6-i0heterocycloalkyl, O-CMalkyl-Ce-ioheteroaryl, 0-C(O)-C 1-4alkyl, O- CON(R)2, CON(R)2, CO2R, C^alkanoyloxymethyl, -P(O)(OH)(OCi-4alkyl), -P(O)(OC1- 4alkyl)2, and O-sugar; or
one or more of R3 and R4, R4 and R5, and R5 and R6 together form ° * , or
Figure imgf000008_0001
; and I0 each R is independently selected from hydrogen and Ci-4alkyl;
Figure imgf000008_0002
is 2-pyridyl, 3-pyridyl or 4-pyridyl optionally substituted with one or more of hydrogen, halogen, hydroxyl, Ci-4alkyl, O-C1-4alkyl, O-C3-6cycloalkyl, haloCι-4alkyl, hydroxyC1-4alkyl, O-C1-4alkyl-CO2R, O-C3-6cycloalkyl, O-C3-6heterocycloalkyl, 0-C3- 6heteroaryl, N(R)2C1-4alkyl, N(R)3C 1-4alkyl, O-C1-4alky 1-N(R)2, O-CMalkyl-N(R)3, C6- i5 loaryl, O-C6-i0 aryl, O-Cι-4alkyl-C6-i0aryl, O-Ci-4alkyl-C6-i0heterocycloalkyl, O-Ci-4alkyl- C6-10heteroaryl, 0-CON(R)2, CON(R)2, CO2R, C^alkanoyloxymethyl, -P(O)(OH)(OCi- O-benzyl, CHO, CO2H, and OC(O)-C i-4alkyl, or
Figure imgf000008_0003
R7-Rn are each independently selected from hydrogen, halogen, hydroxyl,
20 Ci-4alkyl, O-Ci-4alkyl, O-C3-6cycloalkyl, haloC1-4alkyl, hydroxyCi-4alkyl, O-Ci-4alkyl-
CO2R, O-C3-6cycloalkyl, O-C3-6heterocycloalkyl, O-C3-6heteroaryl, N(R)2C i-4alkyl,
N(R)3Ci-4alkyl, O-Ci-4alkyl-N(R)2, O-Ci-4alkyl-N(R)3, C6-i0aryl, O-C6-i0 aryl, O-C1-4alkyl-
C6-i0aryl, O-C1-4alkyl-C6-10heterocycloalkyl, O-Ci-4alkyl-C6-10heteroaryl, 0-CON(R)2,
CON(R)2, CO2R, C]-4alkanoyloxymethyl, -P(O)(OH)(OCi-4alkyl), -P(O)(OCi-4alkyl)2, O-
2s sugar, O-benzyl, CHO, CO2H, and OC(O)-C 1-4alkyl, or one or more of R7 and R8, R8 and R9, R9 and R10, and R10 and R11 together form
Figure imgf000008_0004
is a C6-10aryl group optionally substituted with one or more of halogen, hydroxyl,
C1-4alkyl, O-Ci-4alkyl, O-C^cycloalkyl, haloCi-4alkyl, hydroxyCi-4alkyl, O-Ci-4alkyl- CO2R, 0-C3-6CyClOaIlCyI, O-C3-6heterocycloalkyl, O-C3-6heteroaryl, N(R)2C1-4alkyl, N(R)3C1-4alkyl, O-C1-4alkyl-N(R)2, O-C1-4alkyl-N(R)3( C6-10aryl, 0-C6-10 aryl, O-C1-4alkyl- C6-10aryl, 0-C1-4alkyl-C6-1oheterocycloalkyl, O-C1-4alkyl-C6-10heteroaryl, 0-CON(R)2, CON(R)2, CO2R, Ci-4alkanoyloxymethyl, -P(O)(OH)(OC1-4alkyl), -P(O)(OC1-4alkyl)2, 0- sugar, 0-benzyl, CHO, CO2H, and 0C(0)-C1-4alkyl; or a prodrug thereof, in the manufacture of a medicament for treating a disease selected from Type II diabetes, obesity, hyperlipidemia, cardiovascular disease, anti-neoplastic diseases and tumours, inflammatory conditions, and neurodegenerative diseases.
In a fifth aspect of the invention there is provided a method for identifying a PPAR agonist, the method comprising: determining ligand-receptor interactions of a candidate compound with at least two structurally distinct docking templates; comparing the ligand-receptor interactions of the candidate compound with the interactions of a known PPAR agonist; and thereby determining whether a candidate compound is a PPAR agonist.
In one embodiment the PPAR agonist is a PPAR-γ agonist. In another embodiment the PPAR agonist is a PPAR-α agonist. In a further embodiment the PPAR agonist is a dual PPAR α/γ agonist.
In accordance with the fifth aspect of the invention the docking template may be one or more PPAR crystal structures. For example, a first template, receptor (I), may be derived from of the farglitazar-bound PPAR-γ X-ray complex (PDB: 1FM9); and a second, receptor (II), may be derived from the rosiglitazone-bound PPAR-γ X-ray complex (PDB :1FM6).
The method may further comprise testing a compound identified as a PPAR agonist in vitro for PPAR activation efficacy using either a transcriptional factor or a reporter gene luciferase assay.
The method may further comprise determining the activity of a compound identified as a PPAR agonist in inducing PPAR mRNA and protein expression, then optionally determining if the activity is abolished in the presence of a known selective PPAR antagonist, such as for example, GW9662.
Abbreviations
PPAR, peroxisome proliferator-activated receptor LBD, ligand binding domain TZD, thiazolidinediones THP Ether, Tetrahydropyranyl Ether
DHP, 3,4-Dihydro-2H-Pyran
PPTS, Pyridinium-p-toluenesulfonate
AF-2, transcriptional activation function 2 domain EC50, the half maximal effective concentration
THP-I, human acute monocytic leukaemia cell line
RA W264.7, mouse leukaemic monocyte macrophage cell line
ΗEK293, human embryonic kidney cell line
DMSO, N,N-Dimethylsulfoxide PPRE, PPAR response element
XP, extra precision
IFD, induced-fit docking
DMF, N,N-Dimethylformamide
DMF-DMA, N,N-Dimethylformamide-dimethyl acetal THF, tetrahydrofuran
TEA, triethylamine
DME, dimethoxyethane
Definitions The following are some definitions that may be helpful in understanding the description of the present invention. These are intended as general definitions and should in no way limit the scope of the present invention to those terms alone, but are put forth for a better understanding of the following description.
Unless the context requires otherwise or specifically stated to the contrary, integers, steps, or elements of the invention recited herein as singular integers, steps or elements clearly encompass both singular and plural forms of the recited integers, steps or elements.
Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated step or element or integer or group of steps or elements or integers, but not the exclusion of any other step or element or integer or group of elements or integers. Thus, in the context of this specification, the term "comprising" means "including principally, but not necessarily solely".
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. For the avoidance of any doubt, the invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features, in any appropriate order.
As used herein, the term "C1-4 alkyl group" includes within its meaning monovalent ("alkyl") and divalent ("alkylene") straight chain or branched chain saturated aliphatic groups having from 1 to 4 carbon atoms. The alkyl group may be C1-3 alkyl or Cj-2 alkyl. Thus, for example, the term C1-4 alkyl includes, but is not limited to, methyl, ethyl, 1- propyl, isopropyl, 1 -butyl, 2-butyl, isobutyl, tert-butyl, and the like.
The term "C2-4 alkenyl group" includes within its meaning monovalent ("alkenyl") and divalent ("alkenylene") straight or branched chain unsaturated aliphatic hydrocarbon groups having from 2 to 4 carbon atoms and at least one double bond anywhere in the chain. The alkenyl group may be C2-3 alkenyl. Unless indicated otherwise, the stereochemistry about each double bond may independently be cis, trans, E or Z as appropriate. Examples of C2-4 alkenyl groups include but are not limited to ethenyl, vinyl, allyl, 1-methylvinyl, 1-propenyl, 2-propenyl, 2-methyl-l-propenyl, 2-methyl-l-propenyl, 1-butenyl, 2-butenyl, 3-butentyl, 1,3-butadienyl, and the like.
The term "amino" as used herein refers to groups of the form -NRaRb wherein Ra and Rb are individually selected from hydrogen, optionally substituted (Ci-4)alkyl, optionally substituted (C2-4)alkenyl, optionally substituted (C2-4)alkynyl, optionally substituted (C6-10)aryl and optionally substituted aralkyl groups, such as benzyl. The amino group may be a primary, secondary or tertiary amino group.
The term "amino acid" as used herein includes naturally and non-naturally occurring amino acids, as well as substituted variants thereof. Thus, (L) and (D) forms of amino acids are included in the scope of the term "amino acid". The term "amino acid" includes within its scope glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartate, lysine, arginine, and histidine. The backbone of the amino acid residue may be substituted with one or more groups independently selected from (Ci-6)alkyl, halogen, hydroxy, hydroxy(C1-6)alkyl, aryl (e.g, phenyl), aryl(Ci-3)alkyl (e.g, benzyl), and (C3- 6)cycloalkyl.
The term "aralkyl" or variants such as "arylalkyl" as used herein, includes within its meaning monovalent ("aryl") and divalent ("arylene"), single, polynuclear, conjugated and fused aromatic hydrocarbon radicals attached to divalent, saturated, straight or branched chain alkylene radicals.
The term "C6-I0 aromatic group", or variants such as "C6-10 aryl" or "C6-I0 arylene" as used herein refers to monovalent ("aryl") and divalent ("arylene") single, polynuclear, conjugated and fused residues of aromatic hydrocarbons having from 6 to 10 carbon atoms. Examples of aromatic groups include phenyl, naphthyl, phenanthrenyl, and the like.
The term "C3-6 cycloalkyl" as used herein refers to cyclic saturated aliphatic groups and includes within its meaning monovalent ("cycloalkyl"), and divalent ("cycloalkylene"), saturated, monocyclic, bicyclic, polycyclic or fused polycyclic hydrocarbon radicals having from 3 to 6 carbon atoms. The cycloalkyl group may be C3-5 cycloalkyl. Examples of cycloalkyl groups include but are not limited to cyclopropyl, 2- methylcyclopropyl, cyclobutyl, cyclopentyl, 2-methylcyclopentyl, 3-methylcyclopentyl, cyclohexyl, and the like. The term "C3-6 cycloalkenyl" as used herein, refers to cyclic unsaturated aliphatic groups and includes within its meaning monovalent ("cycloalkenyl") and divalent ("cycloalkenylene"), monocyclic, bicyclic, polycyclic or fused polycyclic hydrocarbon radicals having from 3 to 6 carbon atoms and having at least one double bond anywhere in the alkyl chain. The cycloalkenyl group may be C3-5 cycloalkenyl. Unless indicated otherwise, the stereochemistry about each double bond may be independently cis, trans, E or Z as appropriate. Examples of cycloalkenyl groups include but are not limited to cyclopropenyl, cyclopentenyl, cyclohexenyl, and the like.
The term "C3-6 heterocycloalkyl" as used herein, includes within its meaning monovalent ("heterocycloalkyl") and divalent ("heterocycloalkylene"), saturated, monocyclic, bicyclic, polycyclic or fused hydrocarbon radicals having from 3 to 6 ring atoms, wherein from 1 to 3, ring atoms are heteroatoms independently selected from O, N, NH, or S. The heterocycloalkyl group may be C3-5 heterocycloalkyl. Examples of heterocycloalkyl groups include aziridinyl, pyrrolidinyl, piperidinyl, piperazinyl, quinuclidinyl, azetidinyl, morpholinyl, tetrahydrothiophenyl, tetrahydrofuranyl, tetrahydropyranyl, and the like.
The term "C5-10 heteroaromatic group" and variants such as "heteroaryl" or "heteroarylene" as used herein, includes within its meaning monovalent ("heteroaryl") and divalent ("heteroarylene"), single, polynuclear, conjugated and fused aromatic radicals having from 5 to 10 atoms, wherein 1 to 4, or 1 to 2 ring atoms are heteroatoms independently selected from O, N, NH and S. The heteroaromatic group may be C5-8 heteroaromatic. Examples of heteroaromatic groups include pyridyl (including 2-pyridyl, 3-pyridyl and 4-pyridyl), pyrimidinyl, pyridazinyl, pyrazinyl, 2,2'-bipyridyl, phenanthrolinyl, quinolinyl, isoquinolinyl, imidazolinyl, thiazolinyl, pyrrolyl, furanyl, thiophenyl, oxazolyl, isoxazolyl, isothiazolyl, triazolyl, and the like. The term "halogen" or variants such as "halide" or "halo" as used herein refers to fluorine, chlorine, bromine and iodine.
The term "heteroatom" or variants such as "hetero-" as used herein refers to O, N, and S or the group NH.
The term "optionally substituted" as used herein means the group to which this term refers may be unsubstituted, or may be substituted with one or more groups independently selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, halo, haloalkyl, haloalkynyl, hydroxyl, hydroxyalkyl, alkoxy, thioalkoxy, alkenyloxy, haloalkoxy, haloalkenyloxy, NO2, NRaRb, nitroalkyl, nitroalkenyl, nitroalkynyl, nitroheterocyclyl, alkylamino, dialkylamino, alkenylamine, alkynylamino, acyl, alkenoyl, alkynoyl, acylamino, diacylamino, acyloxy, alkylsulfonyloxy, heterocycloxy, heterocycloamino, haloheterocycloalkyl, alkylsulfenyl, alkylcarbonyloxy, alkylthio, acylthio, phosphorus-containing groups such as phosphono and phosphinyl, aryl, heteroaryl, alkylaryl, aralkyl, alkylheteroaryl, cyano, cyanate, isocyanate, CO2H, CO2(alkyl), C(O)NH2, -C(O)NH(alkyl), and -C(O)N(alkyl)2. Preferred substituents include C1-3 alkyl, C1-3 alkoxy, -CH2-(C 1-3)alkoxy, C6-Io aryl, -CH2-phenyl, halo, hydroxyl, hydroxyl-(C1-3)alkyl, and halo-(C1-3)alkyl, e.g, CF3, CH2CF3.
In the context of this specification the term "administering" and variations of that term including "administer" and "administration", includes contacting, applying, delivering or providing a compound or composition of the invention to an organism, or a surface by any appropriate means.
In the context of this specification, the term "vertebrate" includes humans and individuals of any species of social, economic or research importance including but not limited to members of the genus ovine, bovine, equine, porcine, feline, canine, primates (including human and non-human primates), rodents, murine, caprine, leporine, and avian. The vertebrate may be a human.
In the context of this specification, the term "treatment", refers to any and all uses which remedy a disease state or symptoms, prevent the establishment of disease, or otherwise prevent, hinder, retard, or reverse the progression of disease or other undesirable symptoms in any way whatsoever. In the context of this specification the terms "therapeutically effective amount" and "diagnostically effective amount", include within their meaning a sufficient but non-toxic amount of a compound or composition of the invention to provide the desired therapeutic or diagnostic effect. The exact amount required will vary from subject to subject depending on factors such as the species being treated, the age and general condition of the subject, the severity of the condition being treated, the particular agent being administered, the mode of administration, and so forth. Thus, it is not possible to specify an exact "effective amount". However, for any given case, an appropriate "effective amount" may be determined by one of ordinary skill in the art using only routine experimentation.
Brief Description of the Figures
Figure 1. Docking poses of farglitazar (magenta), a) and rosiglitazone (blue), b) in the LBD of receptor (I) and (II), respectively. Overlaid are their respective published crystallographic pose (brown) from 1FM9 and 1FM6. Helices AF-2 and 10 are shown in cartoon ribbon style, both coloured blue and yellow in receptors (I) and (II), respectively. Comparisons of a) and b) highlight the similar hydrogen bond interactions with residues in the vicinity of the AF-2 helix: Ser289, His323 and Ty473. Hydrophobic π-π interactions with Phe363 are prominent with farglitazar although visibly absent with rosiglitazone. These two ligands were assigned as controls in the docking study. Here, the simulated binding poses of both farglitazar into (I), and rosiglitazone into (II) underwent the same procedure as outlined above, and successfully recreated their published crystallographic poses (RMSD = 0.43 A and 0.50 A for farglitazar and rosiglitazone, respectively) (Gampe et al., 2000). Furthermore, they both scored in the top 5% of total docked compounds for their respective receptor forms. Helix 3 (H3) was removed to enhance viewing. Images were created with Maestro v7.5.
Figure 2. Predicted complexes of flavonoids in the LBD of PPAR-γ. a)-e) docking poses of psi-baptigenin (40c), genistein (56), chrysin (12), biochanin A (55) and apigenin (8) in the LBD of receptor (I) (blue ribbons). Flavonoids are predicted to occupy the hydrophobic environment formed by residues Phe282, Phe360 and Phe360, π-stacking with the latter. Hydrogen bond interactions are made to the receptor from the 7-OH by each flavonoid. f) Overlay of the docking pose of hesperidin into receptors (I) and (II) (blue and yellow ribbons, respectively). Poses are virtually identical. Figure 3. Significant flavonoids to activate PPAR-γ in a cell-based transcriptional factor assay. The PPAR-γ Transcription Factor Assay is a sensitive ELISA method for detecting PPAR-γ transcription factor DNA binding activity in nuclear extracts of THP-I derived macrophage cell line. The ELISA assay was conducted according to the manufacturer's manual (Cayman Chemical, Australia). The cell lines were treated with various concentrations (0.01 - 50 μM) of rosiglitazone, psi-baptigenin (40c), hesperidin (34), apigenin (8), chrysin (12) and biochanin-A (55). The purification of cellular nuclear extract from the cultured cells was prepared with CelLytic™ NuCLEAR™ Extraction Kit. All values are means ± SEM (n=3) vs control; *p < 0.05.
Figure 4. Cytotoxic profiles of rosiglitazone, psi-baptigenin (40c), hesperidin (34), apigenin (8), chrysin (12) and biochanin-A (55), genistein (56), GW-9662 in THP-I derived macrophage cell line. Cell viability was determined using the CellTiter96® Aqueous One Solution Cell Proliferation Assay. The results are expressed as % relative cell viability. All values are means ± SEM (n=3) vs control.
Figure 5. In vitro effects of psi-baptigenin (40c) and hesperidin (34). GW9962 attenuated the effects of psi-baptigenin (40c) and hesperidin (34) in a PPAR-γ cell-based transcriptional factor assay (a) and (b) and PPAR-γ mRNA (c). Western blot results showed an increase in protein by the compounds in THP-I derived macrophage cell line (d). Vehicle (0.1% DMSO) or compounds were incubated for 48 h. The PPAR-γ antagonist, GW9662 (5 μM) was added 1 h before the positive control or test samples. Total mRNAs and protein were prepared from the cell pellets using TRIzol and cells lysis, respectively. The relative levels of PPAR-γ mRNAs were assessed by RT-PCR. Results were normalized to β-actin. Protein extracts from cell pellets were subjected to immunoblotting using an anti-PPAR-γ antibody. The results were normalized to actin. Control levels were arbitrarily assigned 1.0. All values are means + SEM (n=3) vs control; *ρ < 0.05, #p < 0.001. Figure 6. Predicted induced-fit complexes of psi-baptigenin (40c), apigenin (8), biochanin-A (55) and genistein (56) in receptor (II). a) Ligand poses resulting from rigid- receptor docking against receptor (II) (red side-chains), b) Ligand poses resulting from IFD against receptor (II). Also shown is each of the ligand 's corresponding induced-fit receptor (blue side-chain), c) Side on view of the ligand poses resulting from IFD against receptor (II) overlaid with the original receptor (II). The side-chain phenyl of Phe363 clearly sterically prohibits favorable docking of the ligands in a rigid-receptor. Figure 7: PPAR-γ reporter gene activity of compounds 40a, 40c, 4Oe and 4Oi in HEK 293 cell line. The HEK 293 cells were transiently transfected with tK-PPREx3-Luc, pSG5- hPPAR-γ and pSV-β-galactosidase control plasmid. Cells were treated with test compounds (5 μM and 50 μM). Rosiglitazone (5 μM) and GW 1929 (1 μM) were used as positive controls and DMSO (0.1%) as a negative control. At the end of the incubation period the cells were lysed and assayed for luciferase and β-galactosidase activities. The results are expressed as relative luciferase activity (fold difference compared to negative control). Figure 8: Cytotoxic profiles of compounds 40a, 40c, 4Oe, 4Oi, rosiglitazone and GW 1929 in HEK-293 cell line. Cell viability was determined using the CellTiter96® Aqueous One Solution Cell Proliferation Assay. The results are expressed as % relative cell viability (n=8). Figure 9: PPAR-α reporter gene activity of compounds 40a, 40c, 4Oe and 4Oi in HEK 293 cell line. The HEK 293 cells were transiently transfected with tK-PPREx3-Luc, pBI- G-hPPAR-α and pSV-β-galactosidase control plasmid. Cells were treated with test compounds (5, 50 and 100 μM). WY-14643 (25 μM) and Fenofibrate (100 μM) were used as positive controls and DMSO (0.1%) as a negative control. At the end of the incubation period the cells were lysed and assayed for luciferase and β-galactosidase activities. The results are expressed as relative luciferase activity (fold difference compared to negative control).
Figure 10: Cytotoxic profiles of compounds 40a, 40c, 4Oe, 4Oi and fenofibrate in HEK- 293 cell line. Cell viability was determined using the CellTiter96® Aqueous One Solution Cell Proliferation Assay. The results are expressed as % relative cell viability (n=8).
Detailed Description
The present invention is directed to compounds which are agonists of the PPAR receptors. In particular the invention relates to compounds which are agonists of the PPAR-γ receptor. The present invention also relates to compounds that are agonists of the PPAR-α receptor. The present invention is further directed to compounds which are dual agonists of the PPAR-γ and PPAR-α receptors.
Compounds according to the present invention may be useful in therapy, including for example, the treatment of Type II diabetes, obesity, hyperlipidemia, cardiovascular disease (e.g, coronary and ischemic heart disease, atherosclerosis, peripheral vascular disease), anti-neoplastic diseases and tumours (e.g, control of cell growth, cell differentiation, motility and apoptosis, neuroblastoma, breast cancer), inflammatory conditions (e.g, inflammatory bowel diseases, psoriasis, chronic inflammatory airway disease, asthma, rheumatoid arthritis), and neurodegenerative diseases (e.g, Parkinson's disease, Alzheimer's disease). In an embodiment, compounds in accordance with the invention may be useful for treating Type II diabetes.
The compounds of the present invention are flavonoids. Included in the flavonoid class of compounds are flavones, flavanones, isoflavones and isoflavanones.
The present invention relates to compounds of general formulae (1) and (2):
Figure imgf000017_0001
(1) (2) wherein Y is O or S; represents a single bond or a double bond;
R1 and R2 are each independently selected from hydrogen, hydroxyl, halogen, Ci- 4alkyl, haloC1-4alkyl, hydroxyC1-4alkyl, O-Ci-4alkyl, OC(O)-Ci-4alkyl, C(O)-CMalkyl, CO2H, and CO(O)-C i-4alkyl;
R3-R6 are each independently selected from hydrogen, hydroxyl, halogen, Ci-4alkyl, C3-6cycloalkyl, haloCι-4alkyl, hydroxyd^alkyl, O-Ci-4alkyl, O-Ci_4alkyl-CO2R, 0-C3-6CyClOaIlCyI, O-C^heterocycloalkyl, O-C3-6heteroaryl, N(R)2C Malkyl, N(R)3Ci- 4alkyl, O-C1-4alkyl-N(R)2, O-C1-4alkyl-N(R)3, C6-10-aryl, O-C6.i0aryl, O-Ci-4alkyl-C6- iOaryl, , O-Ci^alkyl-Cό-ioheterocycloalkyl, O-Ci-4alkyl-C6-i0heteroaryl, O-C(O)-Ci-4alkyl, 0-CON(R)2, CON(R)2, CO2R, C^alkanoyloxymethyl, -P(O)(OH)(OCi-4alkyl), - P(O)(OC i-4alkyl)2, and O-sugar; or
one or more of R3 and R4, R4 and R5, and R5 and R6 together form 0
Figure imgf000017_0002
, or C5 ; each R is independently selected from hydrogen and C1-4alkyl;
Figure imgf000017_0003
is 2-pyridyl, 3-pyridyl or 4-pyridyl optionally substituted with one or more of halogen, hydroxyl, C Malkyl,
Figure imgf000017_0004
hydroxyCi. 4alkyl, O-Ci-4alkyl-CO2R, O-Cj-όCycloalkyl, O-Q-eheterocycloalkyl, O-C3-6heteroaryl, N(R)2Ci-4alkyl, N(R)3C1-4alkyl, O-C1-4alkyl-N(R)2, O-C1.4alkyl-N(R)3, C6-10aryl, 0-C6-10 aryl, O-C1-4alkyl-C6-10aryl, O-CMalkyl-Q-ioheterocycloalkyl, O-Ci^alkyl-Cό-ioheteroaryl, 0-CON(R)2, CON(R)2, CO2R, C1-4alkanoyloxymethyl, -P(O)(OH)(OC1-4alkyl),
Figure imgf000018_0001
wherein R7-Rn are each independently selected from hydrogen, halogen, hydroxyl,
5 Ci-4alkyl, O-C1-4alkyl, O-C3-6cycloalkyl, haloC1-4alkyl, hydroxyCi-4alkyl, O-Ci_4alkyl- CO2R, 0-C3-6CyClOaIlCyI, O-C3-6heterocycloalkyl, O-C3-6heteroaryl, N(R)2C1-4alkyl, N(R)3Ci-4alkyl, O-C1-4alkyl-N(R)2, O-C1-4alkyl-N(R)3> C6-10aryl, O-C6-]0 aryl, O-C1-4alkyl- C6-i0aryl, O-C1-4alkyl-C6-10heterocycloalkyl, 0-d.4alkyl-C6-ioheteroaryl, 0-CON(R)2, CON(R)2, CO2R, Ci-4alkanoyloxymethyl, -P(O)(OH)(OC1-4alkyl), -P(O)(OCMalkyl)2, O- io sugar, O-benzyl, CHO, CO2H, and OC(O)-C1-4alkyl, or one or more of R7 and R8, R8 and R9, R9 and R10, and R10 and R11 together form
Figure imgf000018_0002
is a C6-1oaryl group optionally substituted with one or more of halogen, hydroxyl,
C1-4alkyl, O-C1-4alkyl, 0-C3-6CyClOaIlCyI, haloC1-4alkyl, hydroxyC1-4alkyl, O-Ci-4alkyl- i5 CO2R, O-C3-6cycloalkyl, O-C3-6heterocycloalkyl, O-C3-6heteroaryl, N(R)2C 1-4alkyl,
N(R)3C1-4alkyl, O-C1-4alkyl-N(R)2, 0-C i-4alky 1-N(R)3, C6- loaryl, O-C6-i0 aryl, O-CMalkyl-
C6-10aryl, O-Ci^alkyl-Ce-Kjheterocycloalkyl, 0-C1-4alkyl-C6-ioheteroaryl, 0-CON(R)2,
CON(R)2, CO2R, Ci-4alkanoyloxymethyl, -P(O)(OH)(OC1.4alkyl), -P(O)(OC Malkyl)2, O- sugar, O-benzyl, CHO, CO2H, and OC(O)-C1-4alkyl; 20 and pharmaceutically acceptable salts thereof.
In embodiments of formula (1) and formula (2) Y is O. In other embodiments of formula (1) and formula (2) Y is S.
In an embodiment of formula (1) and formula (2) ■ is a double bond. In another embodiment of formula (1) and formula (2) ' is a single bond. 25 In one embodiment R5 is selected from hydroxyl, O-Ci-4alkyl, THP, 0-Ci-
4alkanoyloxymethyl, CO2R, -P(O)(OH)(O-methyl), -P(O)(O-methyl)2, -P(O)(OH)(O- ethyl), and -P(O)(O-ethyl)2, wherein R is hydrogen or C1-4alkyl.
In another embodiment R5 is selected from hydroxyl, O-Ci-4alkyl, THP, 0-Ci-
4alkanoyloxymethyl, and CO2R, wherein R is hydrogen or C1-4 alkyl. In another 3o embodiment, R1 and R2 are independently selected from hydrogen, halogen, Ci-4alkyl, O- Ci-4alkyl,
Figure imgf000019_0001
CO2H, and CO2-C1-4alkyl. In another embodiment, R1 and R2 are independently selected from hydrogen, halogen, methyl, ethyl, O-methyl, O-ethyl, t- butoxy, CF3, CO2H, CO2methyl, C02ethyl and CO2Bu'. In another embodiment, R1 and R2 are independently selected from hydrogen, halogen, methyl, CF3 and CO2H. In one embodiment, R7-Rn are each independently selected from hydrogen, halogen, hydroxyl, C1-4alkyl, haloC1-4alkyl, ;
or one or more of R7 and R8, R8 and R9, R9 a
Figure imgf000019_0002
nd R10, and R10 and R1 ' together form 0^ ,
or
Figure imgf000019_0003
. In another embodiment, R7, R9 and R11 are each hydrogen, R8 is hydroxyl or O- methyl and R10 is hydroxyl or O-methyl. In another embodiment, R7, R8, R10 and R11 are hydrogen, and R9 is hydroxyl, halogen methyl, CF3, O-methyl, or CO2H. In another embodiment, R8, R10 and R11 are hydrogen and R7 and R9 are independently selected from hydroxyl, halogen and O-methyl. In another embodiment R7, R10 and R11 are each hydrogen and R8 and R9 are independently selected from hydroxyl and O-methyl or
Figure imgf000019_0004
embodiment R9 and R10 together form 0 ^ .
In one embodiment, R3 -R6 are each independently selected from hydrogen, hydroxyl, halogen, Ci-4alkyl, haloCι-4alkyl, hydroxyCi-4alkyl, O-Ci-4alkyl, 0-C3. 6heterocycloalkyl, N(R)2C1-4alkyl, O-benzyl, 0-C(O)-C i-4alkyl, CO2H, CON(R)2 and O-sugar. In another embodiment R3-R6 are each independently selected from hydrogen, hydroxyl, methyl, ethyl, CH2OH, O-methyl, O-ethyl, t-butoxy, -THP, CF3, CO2H, - CH2NMe2, -CH2NEt2, CH2NMeEt, CH2NHMe, and CH2NHEt. In another embodiment R3, R4 and R6 are selected from hydrogen, F, methyl and O-methyl; and R5 is hydroxyl, - THP, O-methyl, O-Ci-4alkanoyloxymethyl,-P(O)(OH)(O-methyl),-P(O)(O-methyl)2, - P(O)(OH)(O-ethyl), or -P(O)(O-ethyl)2. In another embodiment R3-R6 are each independently selected from hydrogen, CF3, methyl and O-methyl, and R5 is hydroxyl, THP, O-methyl, O-C^alkanoyloxymethyl, -P(O)(OH)(O-methyl), -P(O)(O-methyl)2, - P(O)(OH)(O-ethyl), or -P(O)(O-ethyl)2. The sugar may be a monosaccharide or a disaccharide. Examples of suitable sugar moieties include but are not limited to glucose, rhamnose, arabinglucose, neohesperidose, apioglucose and rutinose.
An embodiment of the invention relates to compounds of formula (1). In another embodiment the invention relates to compounds of formula (2).
In an embodiment of formula (1), when Y is O, R3 and R5 are each OH, and R1, R4, R6, R7, R8, R10 and R11 are each hydrogen, then R9 is not OH or OCH3. In another embodiment of formula (1), when Y is O, R5 is OH and R1, R3, R4, R6, R7, R10 and R11 are
<°1 each hydrogen, then R8 and R9 do not together form 0 ^ . In various embodiments of formula (2): when Y is O, then R2-R! l are not each hydrogen; when Y is O, and R3 is OCH3, then R2 and R4-Rn are not each hydrogen; when Y is O, R5 and R6 are each OH, then R2-R4 and R7-Rn are not each hydrogen; when Y is O, R3 and R5 are each OH, then R2, R4 and R6-Rn are not each hydrogen; when Y is O, R3, R5 and R9 are each OH, then R2, R4, R6-R8, R10 and R11 are not each hydrogen; when Y is O, R3, R5, R8 and R9 are each OH, then R2, R4, R6, R7, R10 and R11 are not each hydrogen; when Y is O, R2, R3, R5, R8 and R9 are each OH, then R4, R6, R7, R10 and R11 are not each hydrogen; when Y is O, R3, R4, R5, R6 and R9 are each OCH3, then R7, R8 and R10 are not each hydrogen; when Y is O and R2 is OH, then R3-R* ! are not each hydrogen; when Y is O, R2, R3 and R5 are each OH, then R4 and R^R1 ' are not each hydrogen; when Y is O, R2, R3, R5 and R9 are each OH, then R4, R6-R8, R10 and R11 are not each hydrogen; when Y is O, R2, R5, R8 and R9 are each OH, then R3, R4, R6, R7, R10 and R1 1 are not each hydrogen; when Y is O, R2, R3, R5, R7 and R9 are each OH, then R4, R6, R8, R10 and R1 1 are not each hydrogen; when Y is O, R2, R3, R5, R8 and R9 are each OH, then R4, R6, R7, R10 and R11 are not each hydrogen; when Y is O, R2, R3, R5, R8, R9 and R10 are each OH, then R4, R6, R7 and R11 are not each hydrogen. In various embodiments the invention relates to compounds of general formulae (Ia), (Ib) and (2a) as defined herein. It will be apparent to those skilled in the art that formulae (Ia) and (Ib) are subsets of formula (1) and formula (2a) is a subset of formula
(2).
In a further embodiment the invention relates to compounds of general formula (Ia):
Figure imgf000021_0001
(Ia) wherein Y is O or S; represents a single bond or a double bond;
R1 is selected from hydrogen, hydroxyl, methyl, ethyl, O-methyl, halo-C1-2alkyl, and CO2R';
R3, R4, R6, R7, R10 and R11 are each independently selected from hydrogen, hydroxyl, halogen, C1-4alkyl, haloC1-4alkyl, hydroxyC1-4alkyl, O-Q^alkyl, N(R')2 and O- phenyl, wherein the phenyl ring may be substituted with one or more substituents selected from hydroxyl, halogen, and Ci-2 alkyl;
R5 is selected from hydrogen, hydroxyl, C1-4alkyl, O-Ci-4alkyl, O-Ci- 4alkanoyloxymethyl, CO2R', O-CMalkyl-CO2R\ OP(O)(OH)(OC,.4alkyl), OP(O)(OCi- 4alkyl)2, O-C3-6heterocyclyl, and OC(O)Ci-4alkyl; n is 1 or 2; each R' is independently selected from hydrogen and Ci-4alkyl; and pharmaceutically acceptable salts thereof, with the proviso that when Y is O, R5 is hydroxyl, THP or O-methyl, and n is 1 at least one of R1, R3, R4, R6, R7, R10 and R11 is not hydrogen. In embodiments of formula (Ia), Y is O. In another embodiment, Y is S.
In one embodiment of formula (Ia) 'I is a double bond. In another embodiment is a single bond.
In an embodiment of formula (Ia) n is 1.
In one embodiment R1 is selected from hydrogen, methyl, ethyl, CF3, and CO2R', wherein R' is hydrogen, methyl or ethyl. In one embodiment R5 is selected from hydroxyl, O-Ci-4alkyl, O-C1-4alkyl-CO2R', and CO2R', wherein R' is hydrogen, methyl or ethyl. In another embodiment R5 is selected from O-C1-4alkyl, O-Ci-4alkyl-CO2R', and CO2R', wherein R1 is hydrogen, methyl or ethyl.
In one embodiment, when Y is O, n is 2 and R5 is OH at least one of R1, R3, R4, R6, R7, R10 and R11 is not hydrogen.
A further aspect of the invention relates to compounds of general formula (Ib):
Figure imgf000022_0001
(Ib) wherein I0 Y is O or S;
1 represents a single bond or a double bond;
R1 is selected from hydrogen, hydroxyl, halogen, Cι.4alkyl, haloC1-4alkyl, hydroxyCMalkyl, O-Ci-4alkyl, OC(O)-C1-4alkyl, C(O)-C1-4alkyl, and O-sugar;
R3, R4, R6 are each independently selected from hydrogen, hydroxyl, halogen, Ci- i5 4alkyl, C3-6cycloalkyl, haloCi^alkyl, hydroxyCi-4alkyl, O-Ci-4alkyl, O-Ci-4alkyl-CO2R,
O-C3-6cycloalkyl, O-C3-6heterocycloalkyl, O-C3-6heteroaryl, N(R)2C i-4alkyl, N(R)3Ci- 4alkyl, O-Ci-4alkyl-N(R)2, O-C1-4alkyl-N(R)3> C6-10aryl, O-C6-i0 aryl, O-CMalkyl-C6-10aryl,
O-Ci^alkyl-Ce-ioheterocycloalkyl, O-Ci-4alkyl-C6-10heteroaryl, 0-C(O)-C ]-4alkyl, O-
CON(R)2, CON(R)2, CO2R, d-4alkanoyloxymethyl, -P(O)(OH)(OCMalkyl), -P(O)(OC1-
20 4alkyl)2, and O-sugar;
R5 is selected from hydrogen, hydroxyl, halogen, Ci-4alkyl, C3.6cycloalkyl, haloCi.
4alkyl, hydroxyCi-4alkyl, O-C]-4alkyl, O-Ci alky 1-CO2R, 0-C3-6cycloalkyl, THP, N(R)2Ci- 4alkyl, N(R)3C 1-4alkyl, O-CMalkyl-N(R)2, C6-i0aryl, O-C6-i0aryl, O-Ci-4alkyl-C6-i0aryl, O-
C(O)-C2-4alkyl, 0-CON(R)2, CON(R)2, CO2R, Ci-4alkanoyloxymethyl, -P(O)(OH)(OCi-
2s 4alkyl), and -P(O)(OCMalkyl)2; or
one or more of R3 and R4, R4 and R5, and R5 and R6 together form °"1 , or
Figure imgf000022_0002
O"1 ; each R is independently selected from hydrogen and C1-4alkyl;
Figure imgf000023_0001
is 2-pyridyl, 3-pyridyl or 4-pyridyl optionally substituted with one or more of halogen, hydroxyl, Ci-4alkyl, O-C1-4alkyl, O-C3-6cycloalkyl, haloC1-4alkyl, hydroxyCi- 4alkyl, O-C1-4alkyl-CO2R, O-C3-6cycloalkyl, O-C3-6heterocycloalkyl, O-C3-6heteroaryl, N(R)2C1-4alkyl, N(R)3C1-4alkyl, O-C1-4alkyl-N(R)2, O-C1-4alkyl-N(R)3, C6-10aryl, 0-C6-10 aryl, O-C1-4alkyl-C6-i0aryl, O-C1-4alkyl-C6-10heterocycloalkyl, O-C1-4alkyl-C6-i0heteroaryl, 0-CON(R)2, CON(R)2, CO2R, C1-4alkanoyloxymethyl, -P(O)(OH)(OCi-4alkyl), - P(O)(OCi-4alkyl)2, O-sugar, O-benzyl, CHO, CO2H, and OC(O)-C Malkyl, or
Figure imgf000023_0002
wherein R7-Rn are each independently selected from hydrogen, halogen, hydroxyl, Ci-4alkyl, O-C1-4alkyl, O-C3-6cycloalkyl, O-C)-4haloalkyl, haloCi-4alkyl, hydroxyCi.
4alkyl, O-Cι-4alkyl-CO2R, O-C3-6cycloalkyl, O-C3-6heterocycloalkyl, O-C3.6heteroaryl,
N(R)2C1-4alkyl, N(R)3C1-4alkyl, O-d-4alkyl-N(R)2, O-C1-4alkyl-N(R)3, C6-,0aryl, 0-C6-io aryl, 0-Ci-4alkyl-C6-1oaryl, O-Ci-4alkyl-C6-i0heterocycloalkyl,
Figure imgf000023_0003
0-CON(R)2, CON(R)2, CO2R, Cr4alkanoyloxymethyl, -P(O)(OH)(OC1-4alkyl), - P(O)(OC1-4alkyl)2, O-sugar, O-benzyl, CHO, CO2H, and OC(O)-C 1-4alkyl, or one or more of R7 and R8, R8 and R9, R9 and R10, and R10 and R11 together form
Figure imgf000023_0004
N i iss a Cό-ioaryl group optionally substituted with one or more of halogen, hydroxyl, Ci-4alkyl, O-Cι-4alkyl, 0-C3-6CyClOaIlCyI, haloC1-4alkyl, hydroxyC1-4alkyl, O-C]-4alkyl- CO2R, 0-C3-6CyClOaIlCyI, O-C3-6heterocycloalkyl, O-C3-6heteroaryl, N(R)2C 1-4alkyl, N(R)3C1-4alkyl, O-C1-4alky 1-N(R)2, O-C1-4alkyl-N(R)3, C6-10aryl, O-C6-i0 aryl, O-C1-4alkyl- C6-ioaryl, O-Ci-4alkyl-C6-10heterocycloalkyl, O-Ci-4alkyl-C6-10heteroaryl, 0-CON(R)2, CON(R)2, CO2R, d^alkanoyloxymethyl, -P(O)(OH)(OC1-4alkyl), -P(O)(OC 1-4alkyl)2, O- sugar, O-benzyl, CHO, CO2H, and OC(O)-CMalkyl; or a pharmaceutically acceptable salt thereof.
In one embodiment of formula (Ib) Y is O. In another embodiment, Y is S.
In one embodiment of formula (Ib) ■ is a double bond. In another embodiment is a single bond.
In an embodiment of compounds of general formula (Ib): Y is O;
R1 is selected from hydrogen, Ci-4alkyl, haloC1-4alkyl, and COOH;
R3 is selected from hydrogen, hydroxyl, O-benzyl and optionally substituted O- benzyl;
R4 and R6 are each independently selected from hydrogen, and Ci-4alkyl,
R5 is selected from hydroxyl, THP, and O-C1-4alkyl-CO2R;
Figure imgf000024_0001
is 2-pyridyl, optionally substituted with one or more O-C1-4alkyl, or
Figure imgf000024_0002
wherein R7-Ru are each independently selected from hydrogen, halogen, hydroxyl, C1-4alkyl, O-CMalkyl, O-C1-4haloalkyl, haloC1-4alkyl, O-benzyl, CHO, CO2H, and OC(O)-C i-4alkyl, or one or more of R7 and R8, R8 and R9, R9 and R10, and R10 and R11 together form
Figure imgf000024_0003
each R is independently selected from hydrogen and C1-4alkyl; or a pharmaceutically acceptable salt thereof.
In another embodiment of formula (Ib), Y is O, R1 is selected from hydrogen, halogen, d^alkyl and CF3; R3, R4 and R6 are selected from hydrogen, methyl, ethyl and hydroxyl;
R5 is selected from hydrogen, hydroxyl, C1-4alkyl, THP or O-benzyl; and
Figure imgf000024_0004
wherein R7-Ru are each independently selected from hydrogen, halogen, hydroxyl, Ci- 4alkyl, O-CMalkyl, O-C1-4haloalkyl, haloC1-4alkyl, O-benzyl, CHO, CO2H, and OC(O)- C].4alkyl, or one or more of R7 and R8, R8 and R9, R9 and R10, and R10 and R11 together form
Figure imgf000024_0005
, or ; or a pharmaceutically acceptable salt thereof.
In another embodiment of formula (Ib): Y is O; R1 is selected from hydrogen, methyl, CF3, and COOH;
R3 is selected from hydrogen, hydroxyl, O-benzyl and optionally substituted O- benzyl;
R4 and R6 are each independently selected from hydrogen, methyl and ethyl, R5 is selected from hydroxyl and THP;
Figure imgf000025_0001
wherein R7-Rn are each independently selected from hydrogen, fluorine, chlorine, CHO, and CO2H or
Figure imgf000025_0002
or a pharmaceutically acceptable salt thereof.
In a further embodiment of formula (Ib), R1 is selected from hydrogen, C1-4alkyl, haloC1-4alkyl, and COOH. In another embodiment of formula (Ib), R1 is selected from C2- 4alkyl, haloC2-4alkyl, hydroxyC2-4alkyl, O-C2-4alkyl, OC(O)-C2-4alkyl and C(O)-C2-4alkyl, In another embodiment of formula (Ib), R1 is selected from C3.4alkyl, haloC3-4alkyl, hydroxyC3-4alkyl, O-C3-4alkyl, OC(O)-C3-4alkyl and C(O)-C3-4alkyl. In an embodiment of formula (Ib), R1 is selected from hydrogen, methyl, CF3, and COOH. In another embodiment of formula (Ib), R1 is selected from hydrogen, C2-4alkyl, haloC2-4alkyl, and
COOH. In another embodiment of formula (Ib), R1 is selected from methyl, ethyl or CF3.
In an embodiment of formula (Ib), R3 is selected from C2-4alkyl, haloC2-4alkyl, hydroxyC2-4alkyl, O-C2-4alkyl, OC(O)-C2.4alkyl and C(O)-C2-4alkyl. In another embodiment of formula (Ib), R3 is selected from C3-4alkyl, haloC3-4alkyl, hydroxyC3- 4alkyl, O-C3-4alkyl, OC(O)-C3-4alkyl and C(O)-C3-4alkyl, In another embodiment of formula (Ib), R3 is selected from hydrogen, hydroxyl, O-benzyl and optionally substituted O-benzyl. In another embodiment of formula (Ib), R3 is selected from hydroxyl and O- benzyl.
In an embodiment of formula (Ib), R4 is selected from C∑^alkyl, haloC2.4alkyl, hydroxyC2-4alkyl, O-C2-4alkyl, OC(O)-C2-4alkyl and C(O)-C2-4alkyl, In another embodiment of formula (Ib), R4 is selected from hydroxyl, halogen, hydrogen and C1-4 alkyl. In another embodiment of formula (Ib), R4 is selected from hydrogen, methyl and ethyl. In another embodiment of formula (Ib), R4 is selected from halogen, ethyl and OC(O)-C1-4alkyl. In an embodiment of formula (Ib), R5 is selected from hydroxyl, O-methyl, THP, O-d^alkanoyloxymethyl, CO2R, -P(O)(OH)(O-methyl), -P(O)(O-methyl)2, - P(O)(OH)(O-ethyl), and -P(O)(O-ethyl)2, wherein R is hydrogen or d.4alkyl. In another embodiment of formula (Ib), R5 is selected from hydroxyl, O-C1-4alkyl, THP, 0-C1- 4alkanoyloxymethyl, and CO2R, wherein R is hydrogen or methyl or ethyl. In another embodiment of formula (Ib), R5 is selected from hydrogen, hydroxyl, Ci.4alkyl, THP or O-benzyl. In another embodiment of formula (Ib), R5 is selected from hydroxyl, THP and O-C1-4alkyl-CO2R wherein R is selected from hydrogen and C1-4 alkyl. In another embodiment of formula (Ib), R5 is selected from hydroxyl and THP. In an embodiment of formula (Ib), R6 is selected from C2-4alkyl, haloC2-4alkyl, hydroxyC2-4alkyl, O-C2-4alkyl, OC(O)-C2-4alkyl and C(O)-C2-4alkyl, In another embodiment of formula (Ib), R6 is selected from hydroxyl, halogen, hydrogen and Ci-4 alkyl. In another embodiment of formula (Ib), R6 is selected from hydrogen, methyl and ethyl. In another embodiment of formula (Ib), R6 is selected from halogen, ethyl and OC(O)-C1-4alkyl. In another embodiment R6 is selected from hydroxyl and O-methyl.
In an embodiment of formula (Ib),
Figure imgf000026_0001
is 2-pyridyl, 3-pyridyl or 4-pyridyl optionally substituted with one or more of halogen, hydroxyl, Ci-4alkyl, O-d-4alkyl, O- C3-5cycloalkyl, haloC1-4alkyl, hydroxyC1-4alkyl, O-C1-4alkyl-CO2R, O-C3-6cycloalkyl, O- C3-6heterocycloalkyl, O-C3-6heteroaryl, N(R)2C1-4alkyl, N(R)3CMalkyl, O-C)-4alkyl- N(R)2, O-CMalkyl-N(R)3, C6-10aryl, 0-C6-10 aryl, O-C1-4alkyl-C6.i0aryl, O-C1-4alkyl-C6- loheterocycloalkyl, O-C1-4alkyl-C6.10heteroaryl, 0-CON(R)2, CON(R)2, CO2R, Ci- 4alkanoyloxymethyl, -P(O)(OH)(OCi-4alkyl), -P(O)(OC1-4alkyl)2, O-sugar, O-benzyl,
CHO, CO2H, and OC(O)-C 1-4alkyl. In another embodiment of formula (Ib),
Figure imgf000026_0002
is 2- pyridyl optionally substituted with one or more of halogen, hydroxyl, Ci-4alkyl, 0-Ci- 4alkyl, O-C3-6cycloalkyl, haloC1-4alkyl, hydroxyC1-4alkyl, O-Ci-4alkyl-CO2R, 0-C3- 6cycloalkyl, O-C3-6heterocycloalkyl, O-C3-6heteroaryl, N(R)2Ci-4alkyl, N(R)3Ci-4alkyl, O- Ci-4alkyl-N(R)2, O-Ci-4alkyl-N(R)3, C6-iOaryl, O-C6-i0 aryl, O-Ci-4alkyl-C6-i0aryl, 0-C1. 4alkyl-C6-10heterocycloalkyl, O-C1-4alkyl-C6-10heteroaryl, 0-CON(R)2, CON(R)2, CO2R, Ci-4alkanoyloxymethyl, -P(O)(OH)(OCi-4alkyl), -P(O)(OC 1-4alkyl)2, O-sugar, O-benzyl,
CHO, CO2H, and OC(O)-C1-4alkyl. In another embodiment of formula (Ib),
Figure imgf000026_0003
is 2- pyridyl optionally substituted with one or more O-Ci-4alkyl. In another embodiment of formula (Ib)
Figure imgf000027_0001
is wherein R7-Rn are each independently selected from hydrogen, halogen, hydroxyl, C1-4alkyl, O-C1-4alkyl, O-C3-6cycloalkyl, O-C1-4haloalkyl, haloCμ4alkyl, hydroxyCi. 4alkyl, O-C1-4alkyl-CO2R, O-C3-6cycloalkyl, O-C3-6heterocycloalkyl, O-C3-6heteroaryl, N(R)2C1-4alkyl, N(R)3C1-4alkyl, O-C1-4alky 1-N(R)2, O-Ci-4alkyl-N(R)3, C6-iOaryl, O-C6-i0 aryl, O-C^alkyl-Cό-^aryl, O-Cι-4alkyl-C6-10heterocycloalkyl, O-Ci-4alkyl-C6-10heteroaryl, 0-CON(R)2, CON(R)2, CO2R, Ci-4alkanoyloxymethyl, -P(O)(OH)(OCi-4alkyl), - P(O)(OC1-4alkyl)2, O-sugar, O-benzyl, CHO, CO2H, and OC(O)-C1-4alkyl, or one or more of R7 and R8, R8 and R9, R9 and R10, and R10 and R11 together form
° < , or
Figure imgf000027_0003
. In another embodiment of formula (Ib), is
Figure imgf000027_0002
wherein R7-Rn are each independently selected from C2-4alkyl, O-C2-4alkyl, 0-C3- 6cycloalkyl, O-C2-4haloalkyl, haloC2-4alkyl and hydroxyC2-4alkyl. In another embodiment
of formula (
Figure imgf000027_0004
independently selected from hydrogen, fluorine, chlorine, hydroxyl, methyl, O-methyl, 0-CF3, CF3, O-benzyl,
Figure imgf000027_0005
In another embodiment, R7, R9 and R11 are each hydrogen, R8 is hydroxyl or O-methyl and R10 is hydroxyl or O-methyl. In another embodiment, R7, R8, R10 and R11 are hydrogen, and R9 is hydroxyl, halogen methyl, CF3, O-methyl, or CO2H. In another embodiment, R8, R10 and R11 are hydrogen and R7 and R9 are independently selected from hydroxyl, halogen and O-methyl. In another embodiment R7, R10 and R11 are each hydrogen and R8 and R9 are independently
selected from hydroxyl and O-methyl or R8 and R9 together form <° °1< . In another embodiment, one of R7 and R8, R8 and R9, R9 and R10, and R10 and R1 1 together
form ° < . In one embodiment, R7 and R8 together form ° < . In another embodiment,
Figure imgf000028_0001
In another embodiment of formula (Ib), R5 is hydroxyl, R , R , R and R are
hydrogen,
Figure imgf000028_0002
R7 and R1 ' are hydrogen, R9 is O-methyl and R8 and R10 and each O-methyl or methyl.
In an embodiment of formula (Ib) at least one of R1 or R3-Ru is not hydrogen. In another embodiment of formula (Ib) when R5 is OH and R8 and R10 are O- methyl, at least one of R1 or R3-R7 is not hydrogen.
In embodiments of formula (Ib), when Y is O and R5 is hydroxyl, R9 is not hydroxyl or O-methyl; when Y is O, R5 is hydroxyl and R9 is halo or methyl, at least one of R1, R3, R4, R6, R7, R10 and R11 is not hydrogen;
when Y is O, R5 is hydroxyl or THP and R8 and R9 together form ° * or
Figure imgf000028_0003
, at least one of R1, R3, R4, R6, R7, R10 and R11 is not hydrogen. The sugar may be a monosaccharide or a disaccharide. Examples of suitable sugar moieties include but are not limited to glucose, rhamnose, arabinglucose, neohesperidose, apioglucose and rutinose.
The present invention also relates to compounds of general formula (2a):
Figure imgf000028_0004
(2a) wherein
Y is O or S;
1 represents a single bond or a double bond;
R2 is selected from hydrogen, hydroxyl, methyl, ethyl, O- methyl, haloCi-2alkyl, and CO2R'; R3, R4, R6, R7, R8 and R11 are each independently selected from hydrogen, hydroxyl, halogen, C1-4alkyl, haloC1-4alkyl, hydroxyC1-4alkyl, O-Ci-4alkyl, N(R')2 and O- phenyl, wherein the phenyl ring may be substituted with one or more substituents selected from hydroxyl, halogen, and C1-2alkyl; R5 is selected from hydrogen, hydroxyl, C1-4alkyl, O-Ci-4alkyl, 0-C1-
4alkanoyloxymethyl, CO2R', O-C1-4alkyl-CO2R', OP(O)(OH)(OC1-4alkyl), OP(O)(OC1- 4alkyl)2, O-C3-6heterocyclyl, and OC(O)d-4alkyl; n is 1 or 2; each R' is independently selected from hydrogen and Ci-4alkyl; and pharmaceutically acceptable salts thereof; with the proviso that when Y is O, R5 is hydroxyl and n is 1, at least one of R2, R3, R4, R6, R7, R8 and R11 is not hydrogen.
In embodiments of formula (2a), Y is O. In another embodiment of formula (2a), Y is S. In embodiments of formula (2a), • represents a double bond. In another embodiment, ' represents a single bond.
In embodiments of formula (2a), n is 1.
In an embodiment of formula (2a), R5 is selected from hydroxyl, O-Ci-4alkyl, 0-Ci- 4alkyl-CO2R', and CO2R', wherein R' is hydrogen, methyl or ethyl. In another embodiment of formula (2a) R5 is selected from O-Ci-4alkyl, O-Ci-4alkyl-CO2R', and CO2R', wherein R' is hydrogen, methyl or ethyl.
In an embodiment of formula (2a), R2 is selected from hydrogen, methyl, ethyl, CF3, and CO2R', wherein R' is hydrogen, methyl or ethyl.
Compounds of formulae (Ia) and (Ib) are subsets of formula (1) and compounds of formula (2a) and subsets of formula (2).
Further examples of particular compounds in accordance with embodiments of formula (1) of the present invention include:
Figure imgf000030_0001
Figure imgf000031_0001
Figure imgf000032_0001
Figure imgf000033_0001
Further examples of compounds in accordance with embodiments of Formula (2) include:
Figure imgf000033_0002
Name Substitution
Rz R3 R4 Ra R6 R7 R8 R9 R 10 R 11
Flavone (1) H H H H H H H H H H
7, 2'-Dihydroxyflavone (2) H H H OH H OH H H H H
7, 3 '-Dihydroxy flavone (3) H H H OH H H OH H H H
7, 4'-Dihydroxyflavone (4) H H H OH H H H OH H H
6, 4 '-Dihydroxy flavone (5) H H OH H H H H OH H H
5, 4 '-Dihydroxy flavone (6) H OH H H H H H OH H H
3', 4'-Dihydroxyflavone (7) H OH H H H H H OH H H
Apigenin (8) H OH H OH H H H OH H H
Acacetin (9) H OH H OH H H H OMe H H
Chrysoeriol (10) H OH H OH H H OMe OH H H
Diosmetin (11) H OH H OH H H OH OMe H H
Chrysin (12) H OH H OH H H H H H H
Myricitrin (13) OR OH H OH H H H OH OH O
(R3: OR; R : Rhamnoside)
Linarin (14) H OH H OR H H H OMe H H
Diosmin (15) H OH H OR H H H OMe OH H
(R7: OR; R : Rutinoside) Neodiosmin (16) H OH H OR H H H OMe OH H
Rhoifolin (17) H OH H OR H H H OH H H
(R7: OR; R: Neohesperidoside)
Apiin (18) H OH H OR H H H OH H H
(R7: OR; R: Apioglucoside)
Gossypetin (19) OH OH H OH OH H OH OH H H
Kaempferol (20) OH OH H OH H H H OH H H
Quercetin (21) OH OH H OH H H OH OH H H
Fisetin 3'4'-dimethyl ether OH H H OH H H OMe OMe H H
(22)
Robinetin trimethyl ether (23)OH H H OH H H OMe OMe OMe H
Myricetin trimethyl ether (24)OH OH H OH H H OMe OMe OMe H
Syringetin (25) OH OH H OH H H OMe OH OMe H
Gossypin (26) OH OH H OH OR H OH OH H H
(R8: OR; R: Glucoside)
Figure imgf000034_0001
Name Substitution
R2 R3 R4 R5 R6 R7' R8 R9 R10 R11
Naringenin (27) H OH H OH H H H OH H H
Hesperetin (28) H OH H OH H H OH OMe H H
Naringin (29) H OH H OR H H H OH H H
Neohesperidin (30) H OH H OR H H OH OMe H H
Neoeriocitrin (31) H OH H OR H H OH OH H H
Poncirin (32) H OH H OR H H O OMe H H
(R7: OR; R: Neohesperidoside)
Narirutin (33) H OH H OR H H H OH H H
(R7: OR; R: Rutinoside)
Hesperidin (34) H OH H OR H H OH OCH3H H
(R7: OR; R: Rutinoside) Naringeinin-7-O-glucoside H OH H OR H H H OH H H (35) (R7: OR; R: Glucoside)
Other compounds in accordance with embodiments of the present invention include:
Figure imgf000035_0001
Figure imgf000036_0001
In embodiments of the invention the compound is selected from compounds 39a, 39d, 4Od, 39e, 39g, 39h, 4Oh, 39i, 4Oi, 39j, 4Oj, 39k, 40k, 391, 401, 39m, 40m, 39n, 4On, 39o, 40o, 39p, 4Op, 39q, 4Oq, 39r, 4Or, 39s, 40s, 41, 46, 48, 49, 53, 57, 58, 59, 60, 61, 62, 63, 53, 65, 66, 67, 70, 74, 78 and 79 above.
In embodiments of the invention the compound is selected from compounds 39a, 39d, 4Od, 39e, 39g, 39h, 4Oh, 39i, 4Oi, 39j, 4Oj, 39k, 40k, 391, 401, 39m, 40m, 39n, 4On, 39o, 40o, 39p, 4Op, 39q, 40q, 39r, 40r, 39s, 40s, 41, 46, 48, 49 and 53 above.
In embodiments of the invention the compound is selected from compounds 40a, 40b, 39c, 40c, 4Od, 4Oe, 4Of, 39g, 4Og, 4Oh, 4Oi, 4Oj, 40k, 401, 40m, 4On, 40o, 4Op, 4Oq, 40r, 39s, 40s, 41, 42, 43, 44, 46, 48, 49, 51, 53, 54, and 56 above. In embodiments of the invention the compound is selected from compounds 2, 3, 5, 6, 8, 9, 11, 40a, 40b, 39c, 40c, 4Od, 4Oe, 4Of, 39g, 4Og, 4Oh, 4Oi, 4Oj, 41, 42, 43, 44, 46, 51, 54, and 56 above.
In embodiments of the invention the compound is selected from compounds 40a, 5 40c, 4Oe and 4Oi above. In an embodiment of the invention the compound is 40a. In an embodiment of the invention the compound is 40c. In an embodiment of the invention the compound is 40e. In an embodiment of the invention the compound is 4Oi.
Representative Synthetic Schemes io Compounds in accordance with the present invention can be prepared using methods known to those skilled in the art. Representative synthetic schemes are shown below.
Scheme 1:
I5
Figure imgf000037_0001
40a-s
Reagents and Conditions: (a) DHP, PPTS, DCM, rt for 4 hrs; (b) DMF-DMA, 950C for 3 hrs; (c) I2,
Pyridine, CHCI3, rt for 1.2 hrs; (d) ArB(OH)2, 10% Pd/C, Na2CO3, DME/H2O, 450C for 1-3 hrs; (e) p-TsOH, MeOH, THF, 600C for 1-2 hrs. Compd. R7 R8 R9 R10 X
39a, 40a H OMe H OMe C
Figure imgf000038_0001
39d, 4Od H H CF3 H C
39e, 4Oe H H F H C
39f, 4Of H OMe OMe H C
39g, 40gb H V H C
39h, 4Oh F H F H C
39i, 4Oi H OMe H H C
39j, 40j OMe F H F C 39k, 40k H F F F C
391, 401 OMe H OMe H N
39m, 40m H Me OMe Me C
39n, 4On H H Cl H C
39o, 4Oo H F H H C 39p, 4Op OMe H H H C
39q, 4Oq H OCF3 H H C
39r, 4Or H OCH2Ph H H C
39s, 40s H OCH3 OCH3 OCH3 C aR8 and R9 together = -0-CH2-O-. bR8 and R9 together = -0-CH2-CH2-O-. Scheme 2:
Figure imgf000039_0001
Reagents and Conditions: (a) BBr3i DCM, O0C to rt, 2-8 hrs.
Scheme 3:
Figure imgf000039_0002
Reagents and Conditions: (a) MeI, K2CO3, DMF, rt for 3 hrs
Scheme 4:
Figure imgf000040_0001
Figure imgf000040_0002
Reagents and Conditions: (a) Anhydrous HCI, ZnCI2-Et2O, then aq. HCI, heat; (b) BF3-Et2O1DMF, PCI5, 60-700C for 1-2 hrs.
Scheme 5:
Figure imgf000040_0003
Figure imgf000040_0004
Figure imgf000040_0005
Reagents and Conditions: (a) Ac2O, TEA, 120-1300C for 8 hrs; (b) NaOH, 1 hr;
(c) (CF3CO)2O, pyridine, O0C to rt for 12 hrs. Scheme 6:
Figure imgf000041_0001
Reagents and Conditions: (a) Br-CH2COOEt, K2CO3, DMF1 900C for 8 hrs; (b) NaOH, 2 hr.
Scheme 7:
Figure imgf000041_0002
Figure imgf000041_0003
Reagents and Conditions: (a) Anhydrous HCI, ZnCI2-Et2O, then aq. HCI, heat;
(b) (CF3CO)2O, pyridine, O0C to rt for 12 hrs.
Compounds in accordance with the present invention may also be prepared according to the following representative synthetic schemes. Scheme 8:
Figure imgf000042_0001
Reagents and Conditions: (a) i. BrCH2CO2Et, K2CO3, DMF; ii. KOH, MeOH, H2O; (b) P2S5, Pyridine, 1150C for 4 hrs; (c) i. Tf2O, Pyridine, DCM; ii. CO, Me3SiCH2CH2OH, Pd(OAc)2, 1 ,3-DPPP, Et3N, DMSO; (d) CI3C(CH3)2OH, Cone. NaOH, acetone.
Scheme 9:
Figure imgf000042_0002
Reagents and Conditions: (a) DHP, PPTS, DCM, rt for 4 hrs; (b) Piperonal, KOH, MeOH; (c) I2, DMSO, 10O0C; (d) P-TsOH, MeOH, THF. Scheme 10:
Figure imgf000043_0001
Reagents and Conditions: (a) CH2(NMe)2, Dioxane; (b) 10% Pd/C, MeOH, it
Scheme 11:
Figure imgf000043_0002
Reagents and Conditions: (a)Anhy. HCI, ZnCI2, Et2O; (b) BF3OEt2 MeSO2CI, DMF; (c)
MMee22NNCCHHIO, MeSO2CI1 1000C; (d) i. CICOCOOEt, Pyridine, ii. NaOH; (e) (CF3CO)2O,
Pyridine. Scheme 12:
Figure imgf000044_0001
Reagents and Conditions: (a)DHP, PPTS, DCM; (b) 4-Bromophenol, K2CO3, DMF; (c) Br2/CHC13, 0-250C; (d) Benzamide, 1250C; (e) LiBH4, THF, 50-600C; (f) DEAD, PPh3, THF; (g) P-TsOH, MeOH, THF, 600C.
Compounds for use in accordance with the present invention may be PPAR agonists. In an embodiment of the invention the compound may be a PPAR-γ agonist. In another embodiment of the invention the compound may be a PPAR-α agonist. In other embodiments compounds in accordance with the present invention may exhibit dual PPAR α/γ agonist activity. In one embodiment the compound is Psi-baptigenin. In another embodiment the compound is Hesperidin.
Figure imgf000045_0001
Psi-baptigenin (40c) Hesperidin (34)
Also within the scope of the present invention are so-called 'pro-drugs' of the compounds of the invention. Thus, certain derivatives of compounds of formulae (l)-(2) [and (la)-(2a) and (Ib)] which may have little or no pharmacological activity themselves can, when administered into or onto the body, be converted into compounds of the invention having the desired activity, for example, by hydrolytic cleavage. Such derivatives are referred to as 'prodrugs'. Further information on the use of prodrugs may be found in Pro-drugs as Novel Delivery Systems, Vol. 14, ACS Symposium Series (T. Higuchi and W. Stella) and Bioreversible Carriers in Drug Design, Pergamon Press, 1987 (ed. E. B. Roche, American Pharmaceutical Association).
Prodrugs in accordance with the invention can, for example, be produced by replacing appropriate functionalities present in the compounds of the invention with certain moieties known to those skilled in the art as 'pro-moieties' as described, for example, in Design of Prodrugs by H. Bundgaard (Elsevier, 1985). Some examples of prodrugs in accordance with the invention include:
(i) where the compound contains a carboxylic acid functionality (COOH), an ester thereof, for example, a compound wherein the hydrogen of the carboxylic acid functionality of a compound of formulae (l)-(2) [and (la)-(2a) and (Ib)] is replaced by (C-Oalkyl; (ii) where the compound contains an hydroxyl functionality, an ether thereof, for example, a compound wherein the hydrogen of the alcohol functionality of a compound of formulae (l)-(2) [and (la)-(2a) and (Ib)] is replaced by (Ci-C4)alkanoyloxymethyl, or a phosphonate ester thereof; and (iii) where the compound contains a primary or secondary amino functionality (-NH2 or -NHR where R ≠ H), an amide thereof, for example, a compound wherein, as the case may be, one or both hydrogens of the amino functionality of the compound is/are replaced by (Ci-C6)alkanoyl.
Included within the scope of the present invention are all stereoisomers, geometric isomers and tautomeric forms of the compounds of formulae (l)-(2) [and (la)-(2a) and (Ib)], including compounds exhibiting more than one type of isomerism, and mixtures of one or more thereof. The present disclosure encompasses all such compounds, including cis-isomers, trans-isomers, (E)-isomers, (Z)-isomers, (Λ)-enantiomers, (_S)-enantiomers and mixtures thereof including racemic mixtures. Also included are acid addition or base salts wherein the counterion is optically active, for example, an amino acid, e.g, (/-lactate or /-lysine, etc, or racemic, for example, (//-tartrate or (//-arginine, and the like.
Cisl trans isomers may be separated by conventional techniques well known to those skilled in the art, for example, chromatography and fractional crystallisation.
Conventional techniques for the preparation/isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or the racemate of a salt or derivative) using, for example, chiral high pressure liquid chromatography (HPLC).
Alternatively, the racemate (or a racemic precursor) may be reacted with a suitable optically active compound, for example, an alcohol, or, in the case where the compound of formula (1) or (2) contains an acidic or basic moiety, a base or acid such as 1- phenylethylamine or tartaric acid. The resulting diastereomeric mixture may be separated by chromatography and/or fractional crystallization and one or both of the diastereoisomers converted to the corresponding pure enantiomer(s) by means well known to a skilled person.
Chiral compounds of the invention (and chiral precursors thereof) may be obtained in enantiomerically-enriched form using chromatography, typically HPLC, on an asymmetric resin with a mobile phase consisting of a hydrocarbon, typically heptane or hexane, containing from 0 to 50% by volume of isopropanol, typically from 2% to 20%, and from 0 to 5% by volume of an alkylamine, typically 0.1% diethylamine. Concentration of the eluate affords the enriched mixture. Stereoisomeric conglomerates may be separated by conventional techniques known to those skilled in the art - see, for example, Stereochemistry of Organic Compounds by Ε. L. Εliel and S. H. Wilen (Wiley, New York, 1994). Therapeutic applications
A further aspect of the present invention relates to a pharmaceutical composition comprising one or more compounds of formula (Ib) or a prodrug thereof, together with a pharmaceutically acceptable adjuvant, diluent or carrier. Also disclosed herein are pharmaceutical compositions comprising one or more compounds of formula (1) or (2) or a prodrug thereof, together with a pharmaceutically acceptable adjuvant, diluent or carrier.
Pharmaceutical compositions comprising one or more compounds of formula (1), (Ib) or (2) may be used in combination with drugs beneficial for treating a targeted condition or disease. Accordingly, the pharmaceutical compositions of the present invention may contain one or more other active ingredients, in addition to a compound of formula (1), (Ib) or (2). The optimal dosage of the drug/s to be administered in combination with the compound/s of the present invention can be readily determined by one of ordinary skill in the art. The additional drug/s may be administered simultaneously or sequentially with the compounds of the present invention. When administered simultaneously, it is preferable to use a pharmaceutical composition in unit dosage form containing the compound/s of the present invention and other drug/s. When administered in combination, either simultaneously or sequentially, the compound/s of the present invention and additional other drug/s may be used in lower doses than when each is used alone.
Examples of other active ingredients that may be administered in combination with a compound of formula (1), (Ib) or (2) either separately or in the same pharmaceutical composition, include, but are not limited to the following examples. Agents which improve a patient's lipid profile, including PPAR alpha agonists such as fenofibric acid derivatives (gemfibrozil, clofibrate, fenofibrate and bezafibrate), PPAR alpha/gamma dual agonists such as KRP-297, muraglitazar, tesaglitazar, farglitazar, and JT-501, PPAR delta, nicotinyl alcohol, nicotinic acid or a salt thereof, bile acid sequestrants (cholestyramine, colestipol, and dialkylaminoalkyl derivatives of a cross-linked dextran), HMG-CoA reductase inhibitors (lovastatin, simvastatin, rosuvastatin, pravastatin, fluvastatin, atorvastatin, rivastatin, itavastatin, ZD-4522 and other statins), cholesterol absorption inhibitors such as ezetinibe, acyl CoA: cholesterol acyl transferase (ACAT) inhibitors, such as avasimibe, CETP inhibitors, and phenolic anti-oxidants, such as probucol.
Further active ingredients that may be administered in combination with the compounds of the present invention include ileal bile acid transporter inhibitors, antiobesity compounds such as fenfluramine, dexfenfluramine, phentiramine, subitramine, orlistat, neuropeptide Y5 inhibitors, Mc4r agonists, cannabinoid receptor 1 (CB-I) antagonists/inverse agonists, and beta 3 adrenergic receptor agonists, biguanides including metformin and phenformin, protein tyrosine phosphatase-lB (PTP-IB) inhibitors, dipeptidyl peptidase IV (DP-IV) inhibitors, insulin or insulin mimetics, sulfonylureas including tolbutamide and glipizide or related materials, PPAR gamma agonists and partial agonists such as glitazones and non-glitazones (e.g. rosiglitazone, LY-818, englitazone, balaglitazone, netoglitazone, troglitazone, T-131, LY-300512, MCC-555, and pioglitazone), alpha-glucosidase inhibitors including acarbose, agonists disclosed in WO097/28149, agents for the treatment of inflammatory conditions such as non-steroidal anti-inflammatory drugs, aspirin, glucocorticoids, azulfidine, and cyclo- oxygenase 2 selective inhibitors, glucagon receptor antagonists, and GLP-I, GIP-I and GLP-I analogs such as exendins (for example exenitide).
The compounds of the present invention may also be administered in combination with multiple active compounds, for example, biguanides PPAR agonists, PTP-IB inhibitors, anti-obesity compounds, sulfonylureas, HMG-CoA reductase inhibitors, and DP-IV inhibitors.
Compounds for use in accordance with the present invention may be PPAR agonists. In an embodiment of the invention the compound may be a PPAR-γ agonist. In another embodiment of the invention the compound may be a PPAR-α agonist. In other embodiments compounds in accordance with the present invention may exhibit dual PPAR α/γ agonist activity. One aspect of the invention is the treatment in vertebrates of diseases that are amenable to amelioration through the activation of PPAR including for example type II diabetes, obesity, hyperlipidemia, cardiovascular disease, anti-neoplastic diseases and tumors, inflammatory conditions and neurogenerative diseases.
In another aspect the present invention relates to a method of treating or preventing a disease in a vertebrate, the method comprising administering to a vertebrate in need thereof an effective amount of a compound of formula (1), (2), (Ia), (Ib) or (2a) as defined herein, or a prodrug thereof, wherein the disease is selected from Type II diabetes, obesity, hyperlipidemia, cardiovascular disease (e.g, coronary and ischemic heart disease, atherosclerosis, peripheral vascular disease), anti-neoplastic diseases and tumours (e.g, control of cell growth, cell differentiation, motility and apoptosis, neuroblastoma, breast cancer), inflammatory conditions (e.g, inflammatory bowel diseases, psoriasis, chronic inflammatory airway disease, asthma, rheumatoid arthritis), and neurodegenerative diseases (e.g, Parkinson's disease, Alzheimer's disease). In an embodiment the disease or condition to be treated is selected from Type II diabetes, obesity, hyperlipidemia, and cardiovascular disease (e.g, coronary and ischemic heart disease, atherosclerosis, peripheral vascular disease). In another embodiment the disease or condition to be treated is selected from Type II diabetes, obesity and hyperlipidemia. In another embodiment the disease or condition to be treated is Type II diabetes.
In one embodiment the method comprises administering an effective amount of a compound of formula (1). In another embodiment the method comprises administering an effective amount of a compound of formula (Ib). In another embodiment the method comprises administering an effective amount of a compound of formula (2).
In another aspect the invention provides for use of a compound of formula (1), (2), (Ia), (Ib) or (2a) as defined herein, or a prodrug thereof, in the manufacture of a medicament for treating a disease selected from Type II diabetes, obesity, hyperlipidemia, cardiovascular disease (e.g, coronary and ischemic heart disease, atherosclerosis, peripheral vascular disease), anti-neoplastic diseases and tumours (e.g, control of cell growth, cell differentiation, motility and apoptosis, neuroblastoma, breast cancer), inflammatory conditions (e.g, inflammatory bowel diseases, psoriasis, chronic inflammatory airway disease, asthma, rheumatoid arthritis), and neurodegenerative diseases (e.g, Parkinson's disease, Alzheimer's disease). In an embodiment the disease or condition to be treated is selected from Type II diabetes, obesity, hyperlipidemia, and cardiovascular disease (e.g, coronary and ischemic heart disease, atherosclerosis, peripheral vascular disease). In another embodiment the disease or condition to be treated is Type II diabetes, obesity, or hyperlipidemia. In a further embodiment the disease or condition to be treated is Type II diabetes.
Pharmaceutical and/or Therapeutic Formulations
Typically, for medical use, salts of the compounds of formulae (l)-(2) [and (Ia)- (2a) and (Ib)] will be pharmaceutically acceptable salts; although other salts may be used in the preparation of the inventive compounds or of the pharmaceutically acceptable salt thereof. By pharmaceutically acceptable salt it is meant those salts which, within the scope of sound medical judgement, are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. Pharmaceutically acceptable salts of compounds of formulae (l)-(2) [and (la)-(2a) and (Ib)] may be prepared by methods known to those skilled in the art, including for example, (i) by reacting a compound of formula (1) or (2) with the desired acid or base; (ii) by removing an acid- or base-labile protecting group from a suitable precursor of the compound or by ring-opening a suitable cyclic precursor, for example, a lactone or lactam, using the desired acid or base; or (iii) by converting one salt of the compound to another by reaction with an appropriate acid or base or by means of a suitable ion exchange column.
All three reactions are typically carried out in solution. The resulting salt may precipitate out and be collected by filtration or may be recovered by evaporation of the solvent. The degree of ionisation in the resulting salt may vary from completely ionised to almost non-ionised.
Thus, for instance, suitable pharmaceutically acceptable salts of compounds according to the present invention may be prepared by mixing a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, methanesulfonic acid, succinic acid, fumaric acid, maleic acid, benzoic acid, phosphoric acid, acetic acid, oxalic acid, carbonic acid, tartaric acid, or citric acid with the compounds of the invention. Suitable pharmaceutically acceptable salts of the compounds of the present invention therefore include acid addition salts. S. M. Berge et al. describe pharmaceutically acceptable salts in detail in J.
Pharmaceutical Sciences, 1977, 66:1-19. The salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or separately by reacting the free base function with a suitable organic acid. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, digluconate, cyclopentanepropionate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, triethanolamine and the like.
Convenient modes of administration include injection (subcutaneous, intravenous, etc.), oral administration, inhalation, transdermal application, topical creams or gels or powders, or rectal administration. In one embodiment, the mode of administration is parenteral. In another embodiment, the mode of administration is oral. Depending on the route of administration, the formulation and/or compound may be coated with a material to protect the compound from the action of enzymes, acids and other natural conditions which may inactivate the therapeutic activity of the compound. The compound also may be administered parenterally or intraperitoneally.
Dispersions of compounds according to the invention may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, pharmaceutical preparations may contain a preservative to prevent the growth of microorganisms. Pharmaceutical compositions suitable for injection include sterile aqueous solutions
(where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Ideally, the composition is stable under the conditions of manufacture and storage and may include a preservative to stabilise the composition against the contaminating action of microorganisms such as bacteria and fungi.
Compounds of formulae (l)-(2) [and (la)-(2a) and (Ib)] according to the present invention may be administered orally, for example, with an inert diluent or an assimilable edible carrier. The compound(s) and other ingredients may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into an individual's diet. For oral therapeutic administration, the compound(s) may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Suitably, such compositions and preparations may contain at least 1% by weight of active compound. The percentage of the compound(s) in pharmaceutical compositions and preparations may, of course, be varied and, for example, may conveniently range from about 2% to about 90%, about 5% to about 80%, about 10% to about 75%, about 15% to about 65%; about 20% to about 60%, about 25% to about 50%, about 30% to about 45%, or about 35% to about 45%, of the weight of the dosage unit. The amount of compound in therapeutically useful compositions is such that a suitable dosage will be obtained. The language "pharmaceutically acceptable carrier" is intended to include solvents, dispersion media, coatings, anti-bacterial and anti-fungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the compound, use thereof in the therapeutic compositions and methods of treatment and prophylaxis is contemplated. Supplementary active compounds may also be incorporated into the compositions according to the present invention. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. "Dosage unit form" as used herein refers to physically discrete units suited as unitary dosages for the individual to be treated; each unit containing a predetermined quantity of compound(s) is calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The compound(s) may be formulated for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in an acceptable dosage unit. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.
In one embodiment, the carrier is an orally administrable carrier.
Another form of a pharmaceutical composition is a dosage form formulated as enterically coated granules, tablets or capsules suitable for oral administration.
Also included in the scope of this invention are delayed release formulations. Compounds of formulae (l)-(2) [and (la)-(2a) and (Ib)] according to the invention also may be administered in the form of a "prodrug". A prodrug is an inactive form of a compound which is transformed in vivo to the active form. Suitable prodrugs include esters, phosphonate esters etc, of the active form of the compound.
In one embodiment, the compound of formulae (l)-(2) [and (la)-(2a) and (Ib)] may be administered by injection. In the case of injectable solutions, the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by including various anti-bacterial and/or antifungal agents. Suitable agents are well known to those skilled in the art and include, for example, parabens, chlorobutanol, phenol, benzyl alcohol, ascorbic acid, thimerosal, and the like. In many cases, it may be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminium monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the analogue in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilisation. Generally, dispersions are prepared by incorporating the analogue into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
Tablets, troches, pills, capsules and the like can also contain the following: a binder such as gum gragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring. When the dosage unit form is a capsule, it can contain, in addition to materials of the above type, a liquid carrier. Various other materials can be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules can be coated with shellac, sugar or both. A syrup or elixir can contain the analogue, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the analogue can be incorporated into sustained-release preparations and formulations. Preferably, the pharmaceutical composition may further include a suitable buffer to minimise acid hydrolysis. Suitable buffer agent agents are well known to those skilled in the art and include, but are not limited to, phosphates, citrates, carbonates and mixtures thereof.
Single or multiple administrations of the compounds and/or pharmaceutical compositions according to the invention may be carried out. One skilled in the art would be able, by routine experimentation, to determine effective, non-toxic dosage levels of the compound and/or composition of the invention and an administration pattern which would be suitable for treating the diseases and/or infections to which the compounds and compositions are applicable. Further, it will be apparent to one of ordinary skill in the art that the optimal course of treatment, such as the number of doses of the compound or composition of the invention given per day for a defined number of days, can be ascertained using convention course of treatment determination tests. Generally, an effective dosage per 24 hours may be in the range of about 0.0001 mg to about 1000 mg per kg body weight; for example, about 0.001 mg to about 750 mg per kg body weight; about 0.01 mg to about 500 mg per kg body weight; about 0.1 mg to about 500 mg per kg body weight; about 0.1 mg to about 250 mg per kg body weight; or about 1.0 mg to about 250 mg per kg body weight. More suitably, an effective dosage per 24 hours may be in the range of about 1.0 mg to about 200 mg per kg body weight; about 1.0 mg to about 100 mg per kg body weight; about 1.0 mg to about 50 mg per kg body weight; about 1.0 mg to about 25 mg per kg body weight; about 5.0 mg to about 50 mg per kg body weight; about 5.0 mg to about 20 mg per kg body weight; or about 5.0 mg to about 15 mg per kg body weight. Alternatively, an effective dosage may be up to about 500mg/m2. For example, generally, an effective dosage is expected to be in the range of about 25 to about 500mg/m2, about 25 to about 350mg/m2, about 25 to about 300mg/m2, about 25 to about 250mg/m2, about 50 to about 250mg/m2, and about 75 to about 150mg/m2.
In another embodiment, a compound of Formula (1) or (2) may be administered in an amount in the range from about 100 to about 1000 mg per day, for example, about 200 mg to about 750 mg per day, about 250 to about 500 mg per day, about 250 to about 300 mg per day, or about 270 mg to about 280 mg per day.
Therapeutic advantages may be realised through combination regimens. Thus, compounds in accordance with the present invention may be administered as part of a therapeutic regimen with other drugs. It may desirable to administer a combination of active compounds, for example, for the purpose of treating a particular disease or condition. For example, compound(s) according to the present invention may be administered in combination therapy with known antidiabetic or antilipidemic agents.
Accordingly, it is within the scope of the present invention that two or more pharmaceutical compositions, at least one of which contains a compound according to the present invention, may be combined in the form of a kit suitable for co-administration of the compositions. In combination therapy the respective agents may be administered simultaneously, or sequentially in any order. Screening assay to identify PPAR agonists
Another aspect of the present invention relates to an assay for identifying PPAR agonists. In an embodiment of the invention the agonist is a PPAR- γ agonist Thus, the invention relates to a method for identifying a PPAR agonist, comprising: determining ligand-receptor interactions of a candidate compound with at least two structurally distinct docking templates; comparing the ligand-receptor interactions of the candidate compound with the interactions of a known PPAR agonist; and thereby determining whether a candidate compound is a PPAR agonist. The present invention has used structure-based virtual screening and in vitro bioassays to identify psi-baptigenin (ψ-baptigenin; or pseudo-baptigenin) and hesperidin as PPAR agonists. Thus, the method of the invention may be used to identify PPAR agonist that share little similarity with known ligands.
The present invention utilises the approach of "multiple rigid-receptor docking" whereby two structurally distinct PPAR-γ crystal structures are employed as docking templates. The first template, receptor (I), is derived from of the farglitazar-bound PPAR- γ X-ray complex (PDB: 1FM9); and the second, receptor (II), is derived from the rosiglitazone-bound PPAR-γ X-ray complex (PDB: 1FM6) (Gampe et al., 2000). Both show significant structural differences in their relative ligand binding domain (LBD) due to ligand-induced side-chain rearrangements. Emphasis is placed on hits that exhibit ligand-receptor interactions comparatively achieved by rosiglitazone and/or farglitazar, and particularly if they bare little structural and chemical similarity to them. Promising hits are then tested in vitro for their PPAR-γ activation efficacy using a transcriptional factor assay. Additionally, their activities in inducing PPAR-γ mRNA and protein expression are examined, and gauged if the effects are abolished in the presence of GW9662 (Bendixen et al., 2001) - a selective PPAR-γ antagonist. Finally, resulting lead candidates are further scrutinized using induced-fit docking (IFD) protocols (Sherman et al., 2006), which incorporates receptor flexibility, to comprehensively elucidate their optimum binding pose. One strategy comprises a series of high level virtual screens that utilized two
PPAR-γ crystal structures as rigid docking templates (PDB: 1FM9 and 1FM6); these show small structural differences in their relative LBD due to ligand-induced side-chain rearrangements. Using two receptors of PPAR-γ in the virtual screening allows a wider range of ligands to be screened. Ligands showing high affinity towards PPAR-γ in silico are subsequently tested in vitro using a PPAR-γ transcriptional factor assay, a PPAR-γ reporter gene luciferase assay and a PPAR-α reporter gene luciferase assay. The activity of compounds identified by this process in inducing PPAR-γ mRNA and protein expression in vitro may be also examined to see if the effect can be abolished in the presence of GW9662, a potent synthetic PPAR-γ antagonist (Bendixen et al., 2001). Biological data from the transactivation assays may be used to characterize compounds as agonists of human PPAR-γ.
The invention will now be described in more detail, by way of illustration only, with respect to the following examples. The examples are intended to serve to illustrate this invention and should not be construed as limiting the generality of the disclosure of the description throughout this specification.
EXAMPLES General
1H NMR spectra were recorded at 300 MHz using a Varian Gemini 300 spectrometer. Chemical shifts (6H) are quoted in parts per million (ppm), referenced internally to tetramethylsilane (TMS) at 0 ppm in CDCl3 and Coupling constants (J) are reported in Hertz. Low resolution mass spectra were recorded on a Finnigan/MAT TSQ 7000 LCMS/MS spectrometer and high resolution mass spectra were recorded on a Micromass Qtof II spectrometer; only molecular ions (M+ 1). Thin layer chromatography (TLC) was performed on Merck aluminium backed plates pre-coated with silica (0.2 mm, 60F254) which were developed using UV fluorescence (254 nm). Flash vacuum chromatography was performed on silica gel (Merck silica gel 6OH, particle size 5—40 μm). Chemicals were purchased from Aldrich, Boron Molecular and Indofine Chemical Co. at the highest available grade.
Example 1
3-Iodo-7-(tetrahydro-2i/-pyran-2-yloxy)-4//-chromen-4-one (38)
A solution of DHP (3,4-dihydro-2H-pyran) (9 mL, 98.7 mmol) in CH2Cl2 (54 mL) was added dropwise to a solution of acetophenone (5 g, 32.8 mmol) and PPTS (Pyridinium-p-toluenesulfonate) (296 mg) at room temperature (rt). The resulting mixture was stirred for 4 h at rt, then washed with saturated aqueous NaHCO3 solution, and extracted with CH2Cl2. The collected organic extracts were dried (Na2SO4), filtered, and concentrated under reduced pressure. The crude was diluted with DMF/DMA (6.54 mL, 49.3 mmol) and the resulting mixture was stirred at 95 °C for 3 h. After evaporation of volatiles, the obtained solid was dissolved in CHCl3 (53 mL) and successively treated with pyridine (2.66 mL, 33 mmol) and I2 (16.7 g, 66 mmol). The resulting mixture was stirred at it for 12 h. The reaction was hydrolyzed with saturated aqueous Na2S2O3 solution and stirred for 30 min at rt. The aqueous phase was extracted with CH2Cl2. The collected organic extracts were dried (Na2SO4), filtered, and concentrated under reduced pressure. Purification by flash chromatography (20% EtOAc/hexane then 40% EtOAc/hexane) gave 38 (11 g, 90% yield) as a colorless solid.
1H NMR (300 MHz, CDCl3): δ 8.27 (s, IH), 8.18 (d, IH, J= 9.6 Hz), 7.16-7.10 (m, 2H),
5.57 (m, IH), 3.91-3.80 (m, IH), 3.70-3.62 (m, IH), 2.10-1.90 (m, 3H), 1.82-1.58 (m,
3H).
Mass (+ve ion mode m/z): 373 (M++ 1).
Example 2 3-(3,5-Dimethoxyphenyl)-7-(tetrahydro-2/T-pyran-2-yloxy)-4H-chromen-4-one (39a)
To a solution of 3-iodo-7-(tetrahydro-2H-pyran-2-yloxy)-4//-chromen-4-one (38) (1 g, 1 mmol) in DME (8 mL) and H2O (8 mL) were added Na2CO3 (854 mg, 3 mmol), 3,5- dimethoxyphenylboronic acid (733 mg, 1.2 mmol), and Pd/C (104 mg, 5 mol %). The resulting mixture was stirred for 2 h at 45 °C and then filtered. The catalyst was washed with H2O (3 mL) and CH2Cl2 (5 mL). The aqueous phase was extracted twice with CH2Cl2. The collected organic extracts were dried (Na2SO4), filtered, and concentrated under reduced pressure. The crude was purified by flash chromatography (50% EtOAc/hexane) to give 39a (893 mg, 87% yield) as a colourless solid.
1H NMR (300 MHz, CDCl3): δ 7.37 (d, IH, J= 9.2 Hz), 7.30 (s, IH), 6.90 (d, IH, J= 8.3 Hz), 6.75 (t, IH, J= 8.7 Hz), 6.63 (t, IH, J= 7.9 Hz), 3.85 (s, 3H), 3.93 (s, 3H), 5.52 (m, IH), 3.90-3.81 (m, IH), 3.72-3.62 (m, IH), 2.11- 1.90 (m, 3H), 1.82-1.59 (m, 3H). Mass (+ve ion mode m/z): 383 (M++l).
Example 3 3-(4-Methoxyphenyl)-7-(tetrahydro-2H-pyran-2-yloxy)-4H-chromen-4-one (39b)
To a solution of 3-iodo-7-(tetrahydro-2H-pyran-2-yloxy)-4H-chromen-4-one (38) (500 mg, 1 mmol) in DME (4 mL) and H2O (4 mL) were added Na2CO3 (427 mg, 3 mmol), 4-methoxyphenylboronic acid (245 mg, 1.2 mmol), and Pd/C (71 mg, 5 mol %). The resulting mixture was stirred for 1 h at 45 0C and then filtered. The catalyst was washed with H2O (3 mL) and CH2Cl2 (5 mL). The aqueous phase was extracted twice with CH2Cl2. The collected organic extracts were dried (Na2SO4), filtered, and concentrated under reduced pressure. The crude was purified by flash chromatography (50% EtOAc/hexane) to give 39b (368 mg, 78% yield) as a colourless solid. 1H NMR (300 MHz, CDCl3): δ 8.20 (d, IH, J = 9.2 Hz), 7.91 (s, IH), 7.49 (dd, 2H, J = 2.2 Hz, 6.8 Hz), 7.08 (d, 2H, J= 6.7 Hz), 6.96 (d, 2H, J= 8.8 Hz), 5.55 (m, IH), 3.83 (s, IH), 3.91-3.82 (m,lH), 3.71-3.62 (m, IH), 2.06-1.89 (m, 3H), 1.80-1.59 (m, 3H). Mass (+ve ion mode m/z): 353 (M++l). s
Example 4
S-CBenzoIrflll-Sldioxol-S-yO-T-^etrahydro-ljy-pyran-Z-yloxyJ^H-chromen^-one (39c)
To a solution of 3-iodo-7-(tetrahydro-2H-pyran-2-yloxy)-4H-chromen-4-one (38)
I0 (500 mg, 1 mmol) in DME (4 mL) and H2O (4 mL) were added Na2CO3 (427 mg, 3 mmol), 3,4-methylenedioxyphenylboronic acid (267 mg, 1.2 mmol), and Pd/C (71 mg, 5 mol %). The resulting mixture was stirred for 1 h at 45 0C and then filtered. The catalyst was washed with H2O (3 mL) and CH2Cl2 (5 mL). The aqueous phase was extracted twice with CH2Cl2. The collected organic extracts were dried (Na2SO4), filtered, and is concentrated under reduced pressure. The crude was purified by flash chromatography (50% EtOAc/hexane) to give 39c (472 mg, 96% yield) as a colourless solid. 1H NMR (300 MHz, CDCl3): δ 8.24 (d, IH, J= 9.3 Hz), 7.95 (s, IH), 7.15-7.11 (m, 3H), 7.0 (dd, IH5 J= 1.5 Hz, 8.1 Hz), 6.90 (d, IH, J= 8.1Hz), 6.03 (s, 2H), 5.59 (m, IH), 3.94- 3.84 (m, IH), 3.73-3.65 (m, IH), 2.10-1.89 (m, 3H), 1.80-1.60 (m, 3H).
20 Mass (+ve ion mode m/z): 366 (M++l).
Example 5
7-(Tetrahydro-2H-pyran-2-yloxy)-3-(4-(trifluoromethyl)phenyl)-4H-chromen-4-one (39d)
2s To a solution of 3-iodo-7-(tetrahydro-2H-pyran-2-yloxy)-4H-chromen-4-one (38)
(500 mg, 1 mmol) in DME (4 mL) and H2O (4 mL) were added Na2CO3 (427 mg, 3 mmol), 4-trifluoromethylphenylboronic acid (383 mg, 1.5 mmol), and Pd/C (71 mg, 5 mol %). The resulting mixture was stirred for 1.5 h at 45 °C and then filtered. The catalyst was washed with H2O (3 mL) and CH2Cl2 (5 mL). The aqueous phase was
30 extracted twice with CH2Cl2. The collected organic extracts were dried (Na2SO4), filtered, and concentrated under reduced pressure. The crude was purified by flash chromatography (60% EtOAc/hexane) to give 39d (393 mg, 75% yield) as a colourless solid. 1H NMR (300 MHz, CDCl3): δ 8.27-8.23 (m, 2H), 8.03 (s, IH), 7.73 (s, IH), 7.18-7.14 (m, 4H), 5.61 (m, IH), 3.94-3.85 (m, IH), 3.74-3.65 (m, IH), 2.09-1.90 (m, 3H), 1.83- 1.60 (m, 3H). Mass (+ve ion mode m/z): 391 (M++ 1).
5
Example 6 3-(4-Fluorophenyl)-7-(tetrahydro-2iy-pyran-2-yloxy)-4//-chromen-4-one (39e)
To a solution of 3-iodo-7-(tetrahydro-2H-pyran-2-yloxy)-4H-chromen-4-one (38) (500 mg, 1 mmol) in DME (4 mL) and H2O (4 mL) were added Na2CO3 (427 mg,
I0 3 mmol), 4-fluorophenylboronic acid (282 mg, 1.5 mmol), and Pd/C (71 mg, 5 mol %). The resulting mixture was stirred for 1.5 h at 45 °C and then filtered. The catalyst was washed with H2O (3 mL) and CH2Cl2 (5 mL). The aqueous phase was extracted twice with CH2Cl2. The collected organic extracts were dried (Na2SO4), filtered, and concentrated under reduced pressure. The crude was purified by flash chromatography is (60% EtOAc/hexane) to give 39e (356 mg, 78% yield) as a colourless solid.
1H NMR (300 MHz, CDCl3): δ 8.42 (d, IH, J= 8.7 Hz), 8.01 (s, IH), 7.65 (m, 2H), 7.28 ( t, 2H, J = 8.7 Hz), 6.99 (dd, IH, J = 2.1 Hz, 8.4 Hz), 6.90 (d, IH, J = 2.4 Hz), 5.57 (m, IH), 3.94-3.70 (m, IH), 3.71-3.60 (m, IH), 2.11-1.90 (m, 3H), 1.81-1.63 (m, 3H). Mass (+ve ion mode m/z): 341 (M++l).
20
Example 7 3-(3,4-Dimethoxyphenyl)-7-(tetrahydro-2H-pyran-2-yloxy)-4H-chromen-4-one (39f)
To a solution of 3-iodo-7-(tetrahydro-2H-pyran-2-yloxy)-4//-chromen-4-one (38) (1 gm, 1 mmol) in DME (8 mL) and H2O (8 mL) were added Na2CO3 (854 mg, 3 mmol),
25 3,4-dimethoxyphenylboronic acid (587 mg, 1.2 mmol), and Pd/C (104 mg, 5 mol %). The resulting mixture was stirred for 2 h at 45 0C and then filtered. The catalyst was washed with H2O (3 mL) and CH2Cl2 (5 mL). The aqueous phase was extracted twice with CH2Cl2. The collected organic extracts were dried (Na2SO4), filtered, and concentrated under reduced pressure. The crude was purified by flash chromatography (50%
30 EtOAc/hexane) to give 39f (903 mg, 88% yield) as a colourless solid.
1H NMR (300 MHz, CDCl3): δ 8.22 (d, IH, J= 9.5 Hz), 7.94 (s, IH), 7.22 (m, IH), 7.11- 7.03 (m, 3H), 6.92 (d, 2H, J= 8.4 Hz), 5.57 (m, IH), 3.95 (s, 3H), 3.93 (s, 3H), 3.94-3.70 (m, IH), 3.71-3.60 (m, IH), 2.11-1.90 (m, 3H), 1.81-1.63 (m, 3H). Mass (+ve ion mode m/z): 383 (M++l).
35 Example 8 a-CZ^-DihydrobenzofAHl^ldioxin-ό-ylJ-T-^etrahydro-ZH-pyran-Z-yloxy)^^- chromen-4-one (39g)
To a solution of 3-iodo-7-(tetrahydro-2H-pyran-2-yloxy)-4H-chromen-4-one (38) (500 mg, 1 mmol) in DME (4 mL) and H2O (4 niL) were added Na2CO3 (427 mg,
3 mmol), l,4-benzodioxane-6-boronic acid (290 mg, 1.2 mmol), and Pd/C (71 mg,
5 mol %). The resulting mixture was stirred for 1 h at 45 °C and then filtered. The catalyst was washed with H2O (3 mL) and CH2Cl2 (5 mL). The aqueous phase was extracted twice with CH2Cl2. The collected organic extracts were dried (Na2SO4), filtered, and concentrated under reduced pressure. The crude was purified by flash chromatography
(40% EtOAc/hexane then 5% MeOH/EtOAc) to give 39g (475 mg, 93% yield) as a colourless solid.
1H NMR (300 MHz, CDCl3): δ 8.26 (d, IH, J = 8.1Hz), 7.94 (s, IH), 7.14-6.93 (m, 5H), 5.59 (m, IH), 4.33 (s, 4H), 3.92-3.84 (m, IH), 3.70-3.65 (m, IH), 1.97-1.92 (m, 3H), 1.75-1.66 (m, 3H).
Mass (+ve ion mode m/z): 381 (M++ 1).
Example 9 3-(2,4-Difluorophenyl)-7-(tetrahydro-2H-pyran-2-yloxy)-4H-chromen-4-one (39h) To a solution of 3-iodo-7-(tetrahydro-2H-pyran-2-yloxy)-4H-chromen-4-one (38)
(500 mg, 1 mmol) in DME (4 mL) and H2O (4 mL) were added Na2CO3 (427 mg, 3 mmol), 2,4-difluorophenylboronic acid (318 mg, 1.5 mmol), and Pd/C (71 mg, 5 mol %). The resulting mixture was stirred for 1.5 h at 45 0C and then filtered. The catalyst was washed with H2O (3 mL) and CH2Cl2 (5 mL). The aqueous phase was extracted twice with CH2Cl2. The collected organic extracts were dried (Na2SO4), filtered, and concentrated under reduced pressure. The crude was purified by flash chromatography (60% EtOAc/hexane) to give 39h (370 mg, 77% yield) as a colourless solid. 1H NMR (300 MHz, CDCl3): δ 8.39 (d, IH, J= 9.1 Hz), 8.18 (s, IH, J= 9 Hz), 6.90-7.98 (m, 5H), 5.57 (m, IH), 3.94-3.70 (m, IH), 3.71-3.60 (m, IH), 2.11-1.90 (m, 3H), 1.81- 1.63 (m, 3H). Mass (+ve ion mode m/z): 359 (M++l). Example 10 3-(3-Methoxyphenyl)-7-(tetrahydro-2β-pyran-2-yloxy)-4/ϊ-chromen-4-one (39i)
To a solution of 3-iodo-7-(tetrahydro-2H-pyran-2-yloxy)-4H-chromen-4-one (38) (500 mg, 1 mmol) in DME (4 mL) and H2O (4 mL) were added Na2CO3 (427 mg,
5 3 mmol), 3-methoxyphenylboronic acid (245 mg, 1.2 mmol), and Pd/C (71 mg, 5 mol %). The resulting mixture was stirred for 1 h at 45 °C and then filtered. The catalyst was washed with H2O (3 mL) and CH2Cl2 (5 mL). The aqueous phase was extracted twice with CH2Cl2. The collected organic extracts were dried (Na2SO4), filtered, and concentrated under reduced pressure. The crude was purified by flash chromatography
I0 (50% EtOAc/hexane) to give 39i (383 mg, 81% yield) as a colourless solid.
1H NMR (300 MHz, CDCl3): δ 8.25 (d, IH, J= 9.3 Hz), 7.98 (s, IH), 7.40-6.92 (m, 6H),
5.58 (m, IH), 3.86 (s, 3H), 3.93-3.80 (m,lH), 3.74-3.60 (m, IH), 2.08-1.85 (m, 3H), 1.80-
1.59 (m, 3H).
Mass (+ve ion mode m/z): 353 (M++l).
I5
Example 11
3-(3,5-Difluoro-2-methoxyphenyl)-7-(tetrahydro-2/7-pyran-2-yloxy)-4H-chromen-4- one (39j)
To a solution of 3-iodo-7-(tetrahydro-2H-pyran-2-yloxy)-4//-chromen-4-one (38)
2Q (500 mg, 1 mmol) in DME (4 mL) and H2O (4 mL) were added Na2CO3 (427 mg,
3 mmol), 3,5-difluoro-2-methoxyphenylboronic acid (303 mg, 1.2 mmol), and Pd/C
(71 mg, 5 mol %). The resulting mixture was stirred for 3 h at 45 0C and then filtered.
The catalyst was washed with H2O (3 mL) and CH2Cl2 (5 mL). The aqueous phase was extracted twice with CH2Cl2. The collected organic extracts were dried (Na2SO4), filtered,
25 and concentrated under reduced pressure. The crude was purified by flash chromatography (60% EtOAc/hexane) to give 39j (442 mg, 85% yield) as a colourless solid.
1H NMR (300 MHz, CDCl3): δ 8.24 (d, IH, J = 9.0 Hz), 8.0 (s, IH), 7.17-7.16 (m, 2H), 6.98-6.89 (m, 2H), 5.60 (m, IH), 3.92-3.85 (m, IH), 3.83 (s, 3H), 3.71-3.66 (m, IH), so 2.08-1.92 (m, 3H), 1.80-1.64 (m, 3H). Mass (+ve ion mode m/z): 389 (M++l).
35 Example 12 3-(3,4,5-Trifluorophenyl)-7-(tetrahydro-2H-pyran-2-yloxy)-4^-chromeπ-4-one (39k)
To a solution of 3-iodo-7-(tetrahydro-2H-pyran-2-yloxy)-4H-chromen-4-one (38) (500 mg, 1 mmol) in DME (4 mL) and H2O (4 niL) were added Na2CO3 (427 mg, 3 mmol), 3,4,5-trifluorophenylboronic acid (283 mg, 1.2 mmol), and Pd/C (71 mg, 5 mol %). The resulting mixture was stirred for 3 h at 45 °C and then filtered. The catalyst was washed with H2O (3 mL) and CH2Cl2 (5 mL). The aqueous phase was extracted twice with CH2Cl2. The collected organic extracts were dried (Na2SO4), filtered, and concentrated under reduced pressure. The crude was purified by flash chromatography (60% EtOAc/hexane) to give 39k (394 mg, 78% yield) as a colourless solid.
1H NMR (300 MHz, CDCl3): δ 8.14 (d, IH, J = 9.3 Hz), 7.89 (s, IH), 7.20-7-03 (m, 2H),
5.58 (m, IH), 3.91-3.83 (m, IH), 3.71-3.67 (m, IH), 2.08-1.94 (m, 3H), 1.78-1.64 (m,
3H).
Mass (+ve ion mode m/z): 2>11 (M++ 1).
Example 13
3-(2,6-Dimethoxypyridin-3-yl)-7-(tetrahydro-2//-pyran-2-yloxy)-4H-chromen-4-one (391)
To a solution of 3-iodo-7-(tetrahydro-2H-pyran-2-yloxy)-4//-chromen-4-one (38) (500 mg, 1 mmol) in DME (4 mL) and H2O (4 mL) were added Na2CO3 (427 mg, 3 mmol), 2,6-dimethoxypyridin-3-yl-3-boronic acid (295 mg, 1.2 mmol), and Pd/C (71 mg, 5 mol %). The resulting mixture was stirred for 5 h at 45 °C and then filtered. The catalyst was washed with H2O (3 mL) and CH2Cl2 (5 mL). The aqueous phase was extracted twice with CH2Cl2. The collected organic extracts were dried (Na2SO4), filtered, and concentrated under reduced pressure. The crude was purified by flash chromatography (60% EtOAc/hexane) to give 391 (386 mg, 75% yield) as a colourless solid.
1H NMR (300 MHz, CDCl3): δ 8.23 (d, IH, J = 8.7 Hz), 8.04 (s, IH), 7.72 (d, IH, J = 8.1 Hz), 7.14-7.09 (m, 2H), 6.43 (d, IH, J= 8.1 Hz), 5.60 (m, IH), 3.97 (s, 3H), 3.95 (s, 3H), 3.97-3.84 (m, IH), 3.70-3.66 (m, IH), 1.97-1.91 (m, 3H), 1.79-1.63 (m, 3H). Mass (+ve ion mode m/z): 384 (M++l). Example 14
3-(4-Methoxy-3,5-dimethylphenyl)-7-(tetrahydro-2flr-pyran-2-yloxy)-4H-chromen-4- one (39m)
To a solution of 3-iodo-7-(tetrahydro-2H-pyran-2-yloxy)-4H-chromen-4-one (38) (500 mg, 1 mmol) in DME (4 mL) and H2O (4 mL) were added Na2CO3 (427 mg,
3 mmol), 4-methoxy-3,5-dimethylphenylboronic acid (290 mg, 1.2 mmol), and Pd/C
(71 mg, 5 mol %). The resulting mixture was stirred for 5 h at 45 °C and then filtered.
The catalyst was washed with H2O (3 mL) and CH2Cl2 (5 mL). The aqueous phase was extracted twice with CH2Cl2. The collected organic extracts were dried (Na2SO4), filtered, and concentrated under reduced pressure. The crude was purified by flash chromatography (60% EtOAc/hexane) to give 39m (444 mg, 87% yield) as a colourless solid.
1H NMR (300 MHz, CDCl3): ): 6 8.24 (d, IH, J= 8.8 Hz), 8.02 (s, IH), 7.22 (s, 2H), 6.91 (d, IH, J = 8.1 Hz), 6.88 (s, IH), 5.60 (m, IH), 3.97-3.84 (m, IH), 3.70-3.66 (m, IH), 3.69 (s, 3H), 2.26 (s, 3H), 1.97-1.91 (m, 3H), 1.79-1.63 (m, 3H). Mass (+ve ion mode m/z): 381 (M++l).
Example 15 3-(4-Chlorophenyl)-7-(tetrahydro-2H-pyran-2-yloxy)-4f-r-chromen-4-one (39n) To a solution of 3-iodo-7-(tetrahydro-2//-pyran-2-yloxy)-4H-chromen-4-one (38)
(500 mg, 1 mmol) in DME (4 mL) and H2O (4 mL) were added Na2CO3 (427 mg, 3 mmol), 4-chlorophenylboronic acid (251 mg, 1.2 mmol), and Pd/C (71 mg, 5 mol %). The resulting mixture was stirred for 1 h at 45 °C and then filtered. The catalyst was washed with H2O (3 mL) and CH2Cl2 (5 mL). The aqueous phase was extracted twice with CH2Cl2. The collected organic extracts were dried (Na2SO4), filtered, and concentrated under reduced pressure. The crude was purified by flash chromatography (50% EtOAc/hexane) to give 39n (430 mg, 90% yield) as a colourless solid. 1H NMR (300 MHz, CDCl3): δ 8.15 (d, IH, J = 7.8 Hz), 7.88 (s, IH), 7.45 (d, 2H, J = 9 Hz), 7.34 (d, 2H, J = 8.4 Hz), 7.05 (m, 2H), 5.50 (m, IH), 3.79-3.74 (m, IH), 3.61-3.57 (m, IH), 1.87-1.83 (m, 3H), 1.68-1.58 (m, 3H). Mass (+ve ion mode m/z): 357 (M++l). Example 16 3-(3-Fluorophenyl)-7-(tetrahydro-2//-pyran-2-yloxy)-4/J-chromen-4-one (39o)
To a solution of 3-iodo-7-(tetrahydro-2H-pyran-2-yloxy)-4H-chromen-4-one (38) (500 mg, 1 mmol) in DME (4 mL) and H2O (4 mL) were added Na2CO3 (427 mg,
5 3 mmol), 3-fluorophenylboronic acid (225 mg, 1.2 mmol), and Pd/C (71 mg, 5 mol %). The resulting mixture was stirred for 1 h at 45 °C and then filtered. The catalyst was washed with H2O (3 mL) and CH2Cl2 (5 mL). The aqueous phase was extracted twice with CH2Cl2. The collected organic extracts were dried (Na2SO4), filtered, and concentrated under reduced pressure. The crude was purified by flash chromatography io (50% EtOAc/hexane) to give 39o (402 mg, 88% yield) as a colourless solid.
1H NMR (300 MHz, CDCl3): δ 8.26 (d, IH, J= 9.6 Hz), 8.01 (s, IH), 7.44-7.34 (m, 4H), 7.16-7.08 (m, 2H), 5.60 (m, IH), 3.89-3.85 (m, IH), 3.71-3.68 (m, IH), 1.97-1.92 (m, 3H), 1.80-1.64 (m, 3H). Mass (+ve ion mode m/z): 341 (M++l).
I5
Example 17 3-(2-Methoxyphenyl)-7-(tetrahydro-2/7-pyran-2-yloxy)-4//-chroinen-4-one (39p)
To a solution of 3-iodo-7-(tetrahydro-2H-pyran-2-yloxy)-4H-chromen-4-one (38) (500 mg, 1 mmol) in DME (4 mL) and H2O (4 mL) were added Na2CO3 (427 mg,
20 3 mmol), 2-methoxyphenylboronic acid (245 mg, 1.2 mmol), and Pd/C (71 mg, 5 mol %). The resulting mixture was stirred for 1 h at 45 °C and then filtered. The catalyst was washed with H2O (3 mL) and CH2Cl2 (5 mL). The aqueous phase was extracted twice with CH2Cl2. The collected organic extracts were dried (Na2SO4), filtered, and concentrated under reduced pressure. The crude was purified by flash chromatography
25 (50% EtOAc/hexane) to give 39p (392 mg, 83% yield) as a colourless solid.
1K NMR (300 MHz, CDCl3): δ 8.25 (d, IH, J= 8.7 Hz), 7.95 (s, IH), 7.42-7.33 (m, 3H), 7.15-7.07 (m, 3H), 5.59 (m, IH), 3.93-3.80 (m, IH), 3.82 (s, 3H), 3.74-3.65 (m, IH), 1.97-1.92 (m, 3H), 1.78-1.64 (m, 3H). Mass (+ve ion mode m/z): 353 (M++ 1).
30
Example 18
3-(3-(Trifluoromethoxy)phenyl)-7-(tetrahydro-2/T-pyran-2-yloxy)-4Hr-chromen-4- one (39q)
To a solution of 3-iodo-7-(tetrahydro-2H-pyran-2-yloxy)-4H-chromen-4-one (38) 35 (500 mg, 1 mmol) in DME (4 mL) and H2O (4 mL) were added Na2CO3 (427 mg, 3 mmol), 3-(trifluoromethoxy)phenylboronic acid (332 mg, 1.2 mmol), and Pd/C (71 mg, 5 mol %). The resulting mixture was stirred for 2 h at 45 °C and then filtered. The catalyst was washed with H2O (3 mL) and CH2Cl2 (5 mL). The aqueous phase was extracted twice with CH2Cl2. The collected organic extracts were dried (Na2SO4), filtered, and concentrated under reduced pressure. The crude was purified by flash chromatography (50% EtOAc/hexane) to give 39q (436 mg, 80% yield) as a colourless solid. 1H NMR (300 MHz, CDCl3): δ 8.26 (d, IH, J- 8.1 Hz), 8.02 (s, IH), 7.55-7.45 (m, 3H), 7.28 (d, IH J = 8.1 Hz), 7.16 (d, IH, J = 8.7 Hz), 7.14 (s, IH), 5.60 (m, IH), 3.89-3.85 (m, IH), 3.72-3.65 (m, IH), 1.98-1.93 (m, 3H), 1.79-1.66 (m, 3H). Mass (+ve ion mode m/z): 407 (M++l).
Example 19 3-(3-(Benzyloxy)phenyl)-7-(tetrahydro-2iy-pyran-2-yloxy)-4//-chromen-4-one (39r)
To a solution of 3-iodo-7-(tetrahydro-2//-pyran-2-yloxy)-4H-chromen-4-one (38) (500 mg, 1 mmol) in DME (4 mL) and H2O (4 mL) were added Na2CO3 (427 mg,
3 mmol), 3-(benzyloxy)phenylboronic acid (367 mg, 1.2 mmol), and Pd/C (71 mg,
5 mol %). The resulting mixture was stirred for 2 h at 45 °C and then filtered. The catalyst was washed with H2O (3 mL) and CH2Cl2 (5 mL). The aqueous phase was extracted twice with CH2Cl2. The collected organic extracts were dried (Na2SO4), filtered, and concentrated under reduced pressure. The crude was purified by flash chromatography
(60% EtOAc/hexane) to give 39r (517 mg, 90% yield) as a colourless solid.
1R NMR (300 MHz, CDCl3): δ 8.27 (d, IH, J= 9.3 Hz), 7.99 (s, IH), 7.49-7.35 (m, 7H),
7.18-7.13 (m, IH), 7.05-7.01 (m, IH), 6.96 (d, IH, J= 9 Hz), 6.89 (s, IH), 5.60 (m, IH),
5.13 (s, 2H), 3.88-3.83 (m, IH), 3.74-3.65 (m, IH), 1.97-1.94 (m, 3H), 1.81-1.63 (m, 3H). Mass (+ve ion mode m/z): 429 (M++l).
Example 20
3-(3,4,5-Trimethoxyphenyl)-7-(tetrahydro-2/-r-pyran-2-yloxy)-4H-chromen-4-one (39s) To a solution of 3-iodo-7-(tetrahydro-2H-pyran-2-yloxy)-4H-chromen-4-one (38)
(500 mg, 1 mmol) in DME (4 mL) and H2O (4 mL) were added Na2CO3 (427 mg, 3 mmol), 3,4,5-trimethoxyphenylboronic acid (341 mg, 1.2 mmol), and Pd/C (71 mg, 5 mol %). The resulting mixture was stirred for 5 h at 45 °C and then filtered. The catalyst was washed with H2O (3 mL) and CH2Cl2 (5 mL). The aqueous phase was extracted twice with CH2Cl2. The collected organic extracts were dried (Na2SO4), filtered, and concentrated under reduced pressure. The crude was purified by flash chromatography (60% EtOAc/hexane) to give 39s (498 mg, 93% yield) as a colourless solid. 1H NMR (300 MHz, CDCl3): ): δ 8.26 (d, IH, J= 7.8 Hz), 8.00 (s, IH), 7.16 (s, 2H), 7.13 (d, IH J = 8.7 Hz), 6.82 (s, IH), 5.60 (m, IH), 3.93 (s, 6H), 3.91 (s, 3H), 3.93-3.85 (m, IH), 3.72-3.65 (m, IH), 1.97-1.93 (m, 3H), 1.79-1.69 (m, 3H). Mass (+ve ion mode m/z): 413 (M++l).
Example 21 3-(3,5-Dimethoxyphenyl)-7-hydroxy-4H-chromen-4-one (40a) To a solution of 3-(3,5-dimethoxyphenyl)-7-(tetrahydro-2H-pyran-2-yloxy)-4H- chromen-4-one (39a) (800 mg) in MeOH (30 mL) and THF (30 mL) was added /?-TsOH (70 mg) at rt. The resulting mixture was stirred at 60 0C for 1 h, then Et3N (0.6 mL) was added, and volatiles were removed under reduced pressure. Purification by flash chromatography (40% EtOAc/hexane then 5% MeOH/EtOAc) provided 40a (530 mg, 85% yield) as a colorless solid.
1H NMR (300 MHz, DMSO): δ 8.43 (s, IH), 7.99 (d, IH, J = 9.1 Hz), 6.98 (d, IH), 6.95 (d, IH, J= 8.2 Hz), 6.90 (d, IH, J= 7.8 Hz), 6.78 (d, IH, J= 8.8 Hz), 6.55 (t, IH, J= 7.6 Hz), 3.79 (s, 6H). Mass (+ve ion mode m/z): 299 (M++ 1).
Example 22 7-Hydroxy-3-(4-methoxyphenyl)-4//-chromen-4-one (40b)
To a solution of 3-(4-methoxyphenyl)-7-(tetrahydro-2H-pyran-2-yloxy)-4//- chromen-4-one (39b) (350 mg) in MeOH (30 mL) and THF (30 mL) was added p-TsOU (32 mg) at rt. The resulting mixture was stirred at 60 °C for 1 h, then Et3N (0.3 mL) was added, and volatiles were removed under reduced pressure. Purification by flash chromatography (40% EtOAc/hexane then 5% MeOH/EtOAc) provided 40b (218 mg, 82% yield) as a colorless solid. 1H NMR (300 MHz, DMSO): δ 10.84 (br s, IH), 8.34 (s, IH), 8.0 (d, IH, J = 8.7 Hz), 7.53 (dd, IH, J= 2.5 Hz, 8.3 Hz), 7.01-6.87 (m, 5H), 3.79 (s, 3H). Mass (+ve ion mode m/z): 269 (M++ 1). Example 23 3-(Benzo[</] [l,3]dioxol-5-yl)-7-hydroxy-4//-chromen-4-one (40c)
To a solution of 3-(Benzo[d][l,3]dioxol-5-yl)-7-(tetrahydro-2H-pyran-2-yloxy)-4H- chromen-4-one (39c) (450 mg) in MeOH (30 mL) and THF (30 mL) was added p-TsOH
5 (35 mg) at rt. The resulting mixture was stirred at 60 0C for 1 h, then Et3N (0.3 mL) was added, and volatiles were removed under reduced pressure. Purification by flash chromatography (40% EtOAc/hexane then 5% MeOH/EtOAc) provided 40c (277 mg, 80% yield) as a colorless solid. 1H NMR (300 MHz, DMSO): δ 10.82 (br s, IH), 8.36 (s, IH), 7.99 (d, IH, J = 9 Hz),
I0 7.17-6.88 (m, 5H), 6.06 (s, 2H).
Mass (+ve ion mode m/z): 283 (M++ 1).
Example 24 3-(4-(Trifluoromethyl)phenyl)-7-hydroxy-4//-chromen-4-one (4Od) is To a solution of 3-(4-(trifluoromethyl)phenyl)-7-(tetrahydro-2H-pyran-2-yloxy)-
4H-chromen-4-one (39d) (350 mg) in MeOH (30 mL) and TΗF (30 mL) was added p- TsOH (30 mg) at rt. The resulting mixture was stirred at 60 0C for 1 h, then Et3N (0.3 mL) was added, and volatiles were removed under reduced pressure. Purification by flash chromatography (40% EtOAc/hexane then 5% MeOΗ/EtOAc) provided 4Od (219 mg,
20 80% yield) as a colorless solid.
1H NMR (300 MHz, DMSO): δ 10.85 (s, IH), 8.54 (s, IH), 8.02 (d, IH, J= 8.7 Hz), 7.82 (dd, 2H, J= 1.8 Hz, 9.3 Hz), 7.01-6.92 (m, 4H). Mass (+ve ion mode m/z): 307 (M++l).
25 Example 25
3-(4-Fluorophenyl)-7-hydroxy-4//-chromen-4-oiie (4Oe)
To a solution of 3-(4-fluorophenyl)-7-(tetrahydro-2//-pyran-2-yloxy)-4//-chromen- 4-one (39e) (300 mg) in MeOH (30 mL) and THF (30 mL) was added p-TsOH (30 mg) at it. The resulting mixture was stirred at 60 0C for 1 h, then Et3N (0.3 mL) was added, and
30 volatiles were removed under reduced pressure. Purification by flash chromatography (40% EtOAc/hexane then 5% MeOH/EtOAc) provided 4Oe (187 mg, 83% yield) as a colorless solid.
1H NMR (300 MHz, DMSO): δ 10.84 (br s, IH), 8.42 (s, IH), 8.01 (d, IH, J = 8.7 Hz), 7.65 (m, 2H), 7.28 ( t, 2H, J= 8.7 Hz), 6.99 (dd, IH, J= 2.1 Hz, 8.4 Hz), 6.90 (d, IH, J =
3s 2.4 Hz). Mass (+ve ion mode m/z): 257 (M++l).
Example 26 3-(3,4-Dimethoxyphenyl)-7-hydroxy-4//-chromen-4-one (4Of) To a solution of 3-(3,4-dimethoxyphenyl)-7-(tetrahydro-2H-pyran-2-yloxy)-4H- chromen-4-one (39f) (800 mg) in MeOH (30 mL) and TΗF (30 mL) was added p-TsOΗ (70 mg) at it. The resulting mixture was stirred at 60 °C for 1 h, then Et3N (0.6 mL) was added, and volatiles were removed under reduced pressure. Purification by flash chromatography (40% EtOAc/hexane then 5% MeOΗ/EtOAc) provided 4Of (542 mg, 87% yield) as a colorless solid.
1H NMR (300 MHz, DMSO): δ 10.86 (br s, IH), 8.38 (s, IH), 7.98 (d, IH, J = 8.87 Hz), 7.24-7.03 (m, 6.92 (d, 2H, J= 8.4 Hz), 5.57 (m, IH), 3.95 (s, 3H), 3.93 (s, 3H). Mass (+ve ion mode m/z): 299 (M++ 1).
Example 27
3-(2,3-Dihydrobenzo [b] [1,4] dioxin-6-yl)-7-hydroxy-4H-chromen-4-one (4Og)
To a solution of 3-(2,3-dihydrobenzo[ό][l,4]dioxin-6-yl)-7-(tetrahydro-2H-pyran-2- yloxy)-4H-chromen-4-one (39g) (400 mg) in MeOH (30 mL) and TΗF (30 mL) was added /?-TsOΗ (35 mg) at it. The resulting mixture was stirred at 60 °C for 1 h, then Et3N (0.3 mL) was added, and volatiles were removed under reduced pressure. Purification by flash chromatography (40% EtOAc/hexane then 5% MeOH/EtOAc) provided 4Og (255 mg, 82% yield) as a colorless solid.
1H NMR (300 MHz, DMSO): δ 10.84 (br s, IH), 8.35 (s, IH), 7.99 (d, IH, J = 8.7 Hz), 7.13-6.87 (m, 5H), 4.26 (s, 4H). Mass (+ve ion mode m/z): 297 (M++l).
Example 28 3-(2,4-Difluorophenyl)-7-hydroxy-4//-chromen-4-one (40h)
To a solution of 3-(2,4-difluorophenyl)-7-(tetrahydro-2H-pyran-2-yloxy)-4H- chromen-4-one (39h) (300 mg) in MeOH (30 mL) and TΗF (30 mL) was added p-TsOΗ (30 mg) at it. The resulting mixture was stirred at 60 °C for 1 h, then Et3N (0.3 mL) was added, and volatiles were removed under reduced pressure. Purification by flash chromatography (40% EtOAc/hexane then 5% MeOΗ/EtOAc) provided 4Oh (195 mg, 85% yield) as a colorless solid. 1H NMR (300 MHz, DMSO): δ 10.83 (br s, IH), 8.39 (s, IH), 8.18 (d, IH, J = 9 Hz),
6.90-7.98 (m, 5H).
Mass (+ve ion mode m/z): 275 (M++l).
5 Example 29
3-(3-Methoxyphenyl)-7-Hydroxy-4//-chromen-4-one (40i)
To a solution of 3-(3-methoxyphenyl)-7-(tetrahydro-2H-pyran-2-yloxy)-4//- chromen-4-one (39i) (350 mg) in MeOH (30 mL) and TΗF (30 mL) was added p-ΥsOR (32 mg) at rt. The resulting mixture was stirred at 60 °C for 1 h, then Et3N (0.3 mL) was i0 added, and volatiles were removed under reduced pressure. Purification by flash chromatography (40% EtOAc/hexane then 5% MeOH/EtOAc) provided 4Oi (213 mg, 80% yield) as a colorless solid.
1H NMR (300 MHz, DMSO): δ 10.86 (br s, IH), 8.41 (s, IH), 8.01 (d, IH, J = 8.7 Hz), 7.34-6.90 (m, 5H), 6.90 (dd, IH, J = 2.4 Hz, 8.3 Hz), 3.80 (s, IH). is Mass (+ve ion mode m/z): 269 (M++l).
Example 30 3-(3,5-Difluoro-2-methoxyphenyl)-7-hydroxy-4//-chromen-4-one (40j)
To a solution of 3-(3,5-difluoro-2-methoxyphenyl)-7-(tetrahydro-2H-pyran-2- 20 yloxy)-4H-chromen-4-one (39j) (400 mg) in MeOH (3O mL) and TΗF (3O mL) was added p-TsOΗ (36 mg) at rt. The resulting mixture was stirred at 60 0C for 1 h, then Et3N
(0.3 mL) was added, and volatiles were removed under reduced pressure. Purification by flash chromatography (40% EtOAc/hexane then 5% MeOΗ/EtOAc) provided 4Oj
(272 mg, 87% yield) as a colorless solid. 25 1H NMR (300 MHz, DMSO): δ 10.84 (br s, IH), 8.33 (s, IH), 7.98 (d, IH, J - 8.7 Hz),
7.41 (t, IH, J = 8.7 Hz), 7.10 (d, IH, J = 9 Hz), 6.99 (d, IH, J = 8.7 Hz), 6.92 (s, IH),
3.73 (s, 3H).
Mass (+ve ion mode m/z): 305 (M++ 1).
30 Example 31
3-(3,4,5-Trifluorophenyl)-7-hydroxy-4//-chromen-4-one (40k)
To a solution of 3-(3,4,5-trifluorophenyl)-7-(tetrahydro-2H-pyran-2-yloxy)-4H- chromen-4-one (39k) (350 mg) in MeOH (30 mL) and TΗF (30 mL) was added jo-TsOΗ
(32 mg) at rt. The resulting mixture was stirred at 60 °C for 1 h, then Et3N (0.3 mL) was
35 added, and volatiles were removed under reduced pressure. Purification by flash chromatography (40% EtOAc/hexane then 5% MeOH/EtOAc) provided 40k (244 mg, 90% yield) as a colorless solid.
1H NMR (300 MHz, DMSO): δ 10.83 (br s, IH), 8.55 (s, IH), 8.0 (d, IH, J = 8.7 Hz), 7.63 (t, IH, J= 9.6), 6.97 (d, IH, J= 9 Hz), 6.91 (s, 2H). Mass (+ve ion mode m/z): 293 (M++l).
Example 32 3-(2,6-Dimethoxypyridin-3-yl)-7-hydroxy-4//-chromen-4-one (401)
To a solution of 3-(2,6-dimethoxypyridin-3-yl)-7-(tetrahydro-2H-pyran-2-yloxy)- 4H-chromen-4-one (391) (350 mg) in MeOH (30 mL) and THF (30 mL) was added p-
TsOH (32 mg) at it. The resulting mixture was stirred at 60 °C for 1 h, then Et3N
(0.3 mL) was added, and volatiles were removed under reduced pressure. Purification by flash chromatography (40% EtOAc/hexane then 5% MeOH/EtOAc) provided 401
(226 mg, 83% yield) as a colorless solid. 1H NMR (300 MHz, DMSO): δ 10.85 (br s, IH), 8.26 (s, IH), 7.95 (d, IH, J = 8.7 Hz),
7.63 (d, IH, J = 8.1 Hz), 6.97 (d, IH, J = 9 Hz), 6.89 (s, IH), 6.47 (d, IH, J = 8.1 Hz),
3.91 (s, 3H), 3.84 (s, 3H).
Mass (+ve ion mode m/z): 300 (M++ 1).
Example 33
3-(4-Methoxy-3,5-dimethylphenyl)-7-hydroxy-4/f-chromen-4-one (40m)
To a solution of 3-(4-methoxy-3,5-dimethylphenyl)-7-(tetrahydro-2//-pyran-2- yloxy)-4H-chromen-4-one (39m) (400 mg) in MeOH (30 mL) and TΗF (30 mL) was added /?-TsOΗ (36 mg) at rt. The resulting mixture was stirred at 60 °C for 1 h, then Et3N (0.3 mL) was added, and volatiles were removed under reduced pressure. Purification by flash chromatography (40% EtOAc/hexane then 5% MeOH/EtOAc) provided 40m (264 mg, 85% yield) as a colorless solid.
1H NMR (300 MHz, DMSO): δ 10.83 (br s, IH), 8.32 (s, IH), 7.99 (d, IH, J = 8.7 Hz), 7.22 (s, 2H), 6.96 (d, IH, J= 8.7 Hz), 6.88 (s, IH), 3.68 (s, 3H), 3.26 (s, 3H). Mass (+ve ion mode m/z): 297 (M++ 1).
Example 34 3-(4-Chlorophenyl)-7-hydroxy-4//-chromen-4-one (40n)
To a solution of 3-(4-chlorophenyl)-7-(tetrahydro-2H-pyran-2-yloxy)-4//-chromen- 4-one (39n) (400 mg) in MeOH (30 mL) and THF (30 mL) was added p-TsOH (36 mg) at rt. The resulting mixture was stirred at 60 °C for 1 h, then Et3N (0.3 mL) was added, and volatiles were removed under reduced pressure. Purification by flash chromatography (40% EtOAc/hexane then 5% MeOH/EtOAc) provided 4On (259 mg, 85% yield) as a colorless solid. s 1H NMR (300 MHz, DMSO): δ 10.85 (br s, IH), 8.46 (s, IH), 8.01 (d, IH, J = 8.7 Hz), 6.64 (d, 2H, J= 8.7 Hz), 6.52 (d, 2H, J= 8.4 Hz), 6.99-6.90 (m, 2H). Mass (+ve ion mode m/z): 273 (M++l).
Example 35 o 3-(3-fluorophenyl)-7-hydroxy-4//-chromen-4-one (40o)
To a solution of 3-(3-fluorophenyl)-7-(tetrahydro-2H-pyran-2-yloxy)-4//-chromen- 4-one (39o) (350 mg) in MeOH (30 mL) and THF (30 mL) was added p-TsOH (32 mg) at rt. The resulting mixture was stirred at 60 °C for 1 h, then Et3N (0.3 mL) was added, and volatiles were removed under reduced pressure. Purification by flash chromatographys (40% EtOAc/hexane then 5% MeOH/EtOAc) provided 4Oo (231 mg, 88% yield) as a colorless solid.
1H NMR (300 MHz, DMSO): δ 10.86 (br s, IH), 8.49 (s, IH), 8.01 (d, IH, J = 8.7 Hz), 7.50-7.45 (m, 2H), 7.21-7.23 (m, 2H), 6.99-6.90 (m, 2H). Mass (+ve ion mode m/z): 257 (M++ 1). 0
Example 36 3-(2-Methoxyphenyl)-7-hydroxy-4//-chromen-4-one (40p)
To a solution of 3-(2-methoxyphenyl)-7-(tetrahydro-2H-pyran-2-yloxy)-4H- chromen-4-one (39p) (350 mg) in MeOH (30 mL) and TΗF (30 mL) was added p-TsOΗ5 (32 mg) at rt. The resulting mixture was stirred at 60 °C for 1 h, then Et3N (0.3 mL) was added, and volatiles were removed under reduced pressure. Purification by flash chromatography (40% EtOAc/hexane then 5% MeOΗ/EtOAc) provided 4Op (195 mg, 83% yield) as a colorless solid. 1H NMR (300 MHz, DMSO): δ 10.81 (br s, IH), 8.19 (s, IH), 7.95 (d, IH, J = 8.7 Hz),0 7.39 (t, IH, J= 8.4 Hz), 7.25 (d, IH, J= 7.5 Hz), 7.10-6.88 (m, 4H), 3.72 (s, 3H). Mass (+ve ion mode m/z): 269 (M++l).
5 Example 37 3-(3-(Trifluoromethoxy)phenyl)-7-hydroxy-4/y-chromen-4-one (40q)
To a solution of 3-(3-(trifluoromethoxy)phenyl)-7-(tetrahydro-2H-pyran-2-yloxy)- 4H-chromen-4-one (39q) (400 mg) in MeOH (30 mL) and TΗF (30 mL) was added p- TsOΗ (36 mg) at rt. The resulting mixture was stirred at 60 0C for 1 h, then Et3N (0.3 mL) was added, and volatiles were removed under reduced pressure. Purification by flash chromatography (40% EtOAc/hexane then 5% MeOΗ/EtOAc) provided 4Oq (269 mg, 85% yield) as a colorless solid. 1H NMR (300 MHz, DMSO): δ 10.85 (br s, IH), 8.53 (s, IH), 8.02 (d, IH, J = 8.7 Hz), 7.65-7.58 (m, 3H), 7.40 (d, IH, J= 8.4 Hz), 6.99 (d, IH, J= 8.7 Hz), 6.92 (s, IH). Mass (+ve ion mode m/z): 323 (M++l).
Example 38 3-(3-(Benzyloxy)phenyl)-7-hydroxy-4/y-chromen-4-one (40r) To a solution of 3-(3-(benzyloxy)phenyl)-7-(tetrahydro-2H-pyran-2-yloxy)-4//- chromen-4-one (39r) (450 mg) in MeOH (30 mL) and THF (30 mL) was added /7-TsOH (40 mg) at rt. The resulting mixture was stirred at 60 °C for 1 h, then Et3N (0.3 mL) was added, and volatiles were removed under reduced pressure. Purification by flash chromatography (40% EtOAc/hexane then 5% MeOH/EtOAc) provided 4Or (289 mg, 80% yield) as a colorless solid.
1H NMR (300 MHz, DMSO): δ 10.85 (br s, IH), 8.42 (s, IH), 8.01 (d, IH, J = 9 Hz), 7.50-7.33 (m, 7H), 7.18 (d, IH, J = 7.5 Hz), 7.05 (d, IH, J= 8.7 Hz), 6.98 (d, IH, J = 9 Hz), 6.90 (s, IH), 5.14 (s, 2H) Mass (+ve ion mode m/z): 345 (M++l).
Example 39 3-(3,4,5-Trimethoxyphenyl)-7-hydroxy-4//-chromen-4-one (40s)
To a solution of 3-(3,4,5-trimethoxyphenyl)-7-(tetrahydro-2H-pyran-2-yloxy)-4//- chromen-4-one (39s) (450 mg) in MeOH (30 mL) and THF (30 mL) was added /?-TsOH (40 mg) at rt. The resulting mixture was stirred at 60 °C for 1 h, then Et3N (0.3 mL) was added, and volatiles were removed under reduced pressure. Purification by flash chromatography (40% EtOAc/hexane then 5% MeOH/EtOAc) provided 40s (279 mg, 78% yield) as a colorless solid. 1H NMR (300 MHz, DMSO): δ 10.86 (br s, IH), 8.44 (s, IH), 8.01 (d, IH, J= 8.7 Hz), 6.98 (d, IH, J= 8.7 Hz), 6.90 (s, 2H), 6.89 (s, IH), 3.81 (s, 6H), 3.70 (s, 3H). Mass (+ve ion mode m/z): 329 (M++l).
Example 40 3-(3,5-Dihydroxyphenyl)-7-hydroxy-4//-chromen-4-one (41) To a portion (500 mg) of 3-(3,5-dimethoxyphenyl)-7-hydroxy-4H-chromen-4-one
(40a), dissolved in anhydrous dichloromethane (15 mL) and cooled to 0 C, a 1 M solution of BBr3 in dichloromethane (6 mL) is dropped slowly; the solution is stirred at room temperature for 6 h and then is diluted with iced water. The pH is arranged to 6 with 5% Na2HPO4, the mixture is extracted with ethyl acetate, and the organic layer is separated and washed with brine, dried, and concentrated to yield, after crystallization from dichloromethane/methanol/ethyl acetate, 41 as off white colour solid (258 mg, 57% yield).
1H NMR (300 MHz, DMSO): δ 8.30 (s, IH), 7.99 (d, IH, J= 8.3 Hz), 6.92 (dd, IH, J = 2.2 Hz, 8 Hz), 6.83 (d, IH, J= 2.5 Hz), 6.72 (d, IH, J= 2.2 Hz), 6.42 (d, IH, J= 2.4 Hz), 6.23 (s, IH).
Mass (+ve ion mode m/z): 271 (M++l).
Example 41 3-(4-Hydroxyphenyl)-7-hydroxy-4//-chromen-4-one (42) To a portion (100 mg) of 3-(4-methoxyphenyl)-7-hydroxy-4H-chromen-4-one
(40b), dissolved in anhydrous dichloromethane (5 mL) and cooled to O0C, a 1 M solution of BBr3 in dichloromethane (1.5 mL) is dropped slowly; the solution is stirred at room temperature for 4 h and then is diluted with iced water. The pH is arranged to 6 with 5% Na2HPO4, the mixture is extracted with ethyl acetate, and the organic layer is separated and washed with brine, dried, and concentrated to yield, after crystallization from dichloromethane/methanol/ethyl acetate, 42 as off white colour solid (61 mg, 65% yield). 1H NMR (300 MHz, DMSO): δ 8.26 (s, IH), 7.95 (d, IH, J= 8.7 Hz), 7.37 (dt, 2H, J= 2, 8.6 Hz), 6.93 (dd, IH, J = 2.2, 8.7 Hz), 6.85 (d, IH, J= 2 Hz), 6.80 (dt, 2H, J = 2.2, 8.6 Hz). Mass (+ve ion mode m/z): 255 (M++ 1).
Example 42 3-(3,4-Dihydroxyphenyl)-7-hydroxy-4//-chromen-4-one (43)
To a portion (500 mg) of 3-(3,4-dimethoxyphenyl)-7-hydroxy-4H-chromen-4-one (4Of), dissolved in anhydrous dichloromethane (15 mL) and cooled to O0C, a 1 M solution of BBr3 in dichloromethane (6 mL) is dropped slowly; the solution is stirred at room temperature for 6 h and then is diluted with iced water. The Ph is arranged to 6 with 5% Na2HPO4, the mixture is extracted with ethyl acetate, and the organic layer is separated and washed with brine, dried, and concentrated to yield, after crystallization from 5 dichloromethane/methanol/ethyl acetate, 43 as colourless solid (235 mg, 52% yield).
1H NMR (300 MHz, DMSO): δ 8.37 (s, IH), 7.98 (d, IH, J= 8.5 Hz), 7.63 (dd, IH, J = 2.4 Hz, 7.9 Hz), 7.60 (d, IH, J = 2.3 Hz), 7.52 (d, IH, J= 8.1 Hz), 6.90 (dd, IH, J = 2.5 Hz, 8.2 Hz), 6.83 (d, IH, J= 2.3 Hz). Mass (+ve ion mode m/z): 271 (M++ 1). 0
Example 43 3-(Benzo[d] [l,3]dioxol-5-yI)-7-methoxy-4/y-chromen-4-one (44)
To a solution of (150 mg) of 3-(benzo[c?][l,3]dioxol-5-yl)-7-hydroxy-4H-chromen- 4-one (40c) in dry DMF (5 mL) were added K2CO3 (110 mg, 1.5 mmol) and MeI (0.05s mL, 1.5 mmol). The solution is stirred at room temperature for 3 h and then is extracted with ethyl acetate, and the organic layer is separated and washed with brine, dried, and concentrated to get crude product. The crude was purified by flash chromatography (50% EtOAc/hexane) to give 44 (140 mg, 90% yield) as a colourless solid. 1H NMR (300 MHz, DMSO): δ 8.46 (s, IH), 8.06 (d, IH, J = 9 Hz), 7.20-6.98 (m, 5H),0 6.07 (s, 2H), 3.93 (s, 3H).
Mass (+ve ion mode m/z): 297 (M++l).
Example 44 2-(Benzo[d][l,3]dioxol-5-yl)-l-(5-ethyl-2,4-dihydroxyphenyl)ethanone (45)s With stirring, a rapid current of dry hydrogen chloride is passed for 10 min into a solution of 8-cyanomethyl-l, 6-benzodioxecane (500 mg, 1 mmol) in dry toluene (10 mL) cooled to O0C. Then a solution of 4-ethylresorcinol (471 mg, 1.1 mmol) and fused zinc chloride (211 mg, 0.5 mmol) in dry ether (5 mL) is added. Saturation with hydrogen chloride is continued for 4 h and then kept overnight at room temperature. The solvent0 was decanted and triturated twice with dry toluene. The hot water (50 mL) added and the mixture is kept at 900C and pH 1 for 30 min. The product is separated from the hot solution and washed several times with warm water. The crude was purified by flash chromatography to give 45 (447 mg, 51% yield) as an oily product. 1H NMR (300 MHz, CDCl3): δ 7.34 (s, IH), 6.90-6.85 (m, 3H), 6.28 (s, IH), 5.98 (s, 2H),5 4.22 (s, 2H), 2.53 (q, 2H), 1.18 (t, 3H). Mass (+ve ion mode m/z): 301 (M++l).
Example 45 3-(Benzo[d][l,3]dioxol-5-yl)-6-ethyl-7-hydroxy-4/-r-chromen-4-one (46)
5 With stirring, boron trifluoride etherate (1.30 mL, 6 mmol) is added dropwise to a solution of 45 (600 mg, 1 mmol) in DMF (2.7 mL). Then phosphorus pentachloride (384 mg, 1.1 mmol) is added at such a rate that the temperature of the reaction mixture did not rise above 60-700C. After the completion of the reaction, the reaction mixture was poured into water (100 mL) and the resulting mixture is kept at 700C for 1 h. The precipitate is io filtered and crude product crystallized from isopropanol to give 46 as a white solid.
1H NMR (300 MHz, DMSO): δ 10.87 (br s, IH), 8.33 (s, IH), 7.81 (s, IH), 7.13 (d, IH, J = 1.5 Hz), 7.04 (dd, IH, J= 1.5 Hz, 8.1 Hz), 6.96 (d, IH, J= 8.1 Hz), 6.88 (s, IH), 6.04 (s, 2H), 2.64 (q, 2H), 1.17 (t, 3H). Mass (+ve ion mode m/z): 311 (M++ 1).
I5
Example 46
3-(Benzo[d] [l,3]dioxol-5-yl)-6-ethyl-2-methyl-4-oxo-4//-chromen-7-yl acetate (47) A mixture of 45 (500 mg, 1 mmol), acetic anhydride (0.67 mL 5 mmol), and triethylamine (0.81 mL, 4 mmol) is heated at 120-1300C for 6 h. Then the reaction 20 mixture is added to cold water containing 0.2 ml of hydrochloric acid. The precipitate that deposited was filtered off, washed with water until free from smell, dried, and crystallized from ethyl acetate.
1H NMR (300 MHz, CDCl3): δ 8.88 (s, IH), 7.94 (s, IH), 7.23 (s, IH), 7.09 (d, IH, J= 2
Hz), 6.98 (dd, 2H, J = 7.8 Hz), 6.85 (d, IH, J = 8.1 Hz), 5.99 (s, 2H), 2.66 (q, 2H), 2.38 2s (s, 3H), 2.26 (s, 3H), 1.26 (t, 3H).
Mass (+ve ion mode m/z): 367 (M++ 1).
Example 47 3-(Benzo[d][l,3]dioxol-5-yl)-6-ethyl-7-hydroxy-2-methyl-4H-chromen-4-one (48)
30 A hot solution of 47 (400 mg, 1 mmol) in 5 mL of ethanol is treated with a 5 %
NaOH (1 mL) and the mixture is boiled for 10 min. Then 5 mL water is added and boiling is continued for another 20 min, after which the mixture is neutralized with dilute hydrochloric acid to pH 7. The precipitate that deposited was filtered off and crystallized from ethanol to give 48 as a white solid. 1H NMR (300 MHz, DMSO): δ 10.78 (s, IH), 7.70 (s, IH), 6.95 (d, IH, J= 7.8 Hz), 6.84 (s, IH), 6.81 (d, IH, J= 1.2 Hz), 6.70 (dd, IH5 J= 1.8, 8.1 Hz), 6.05 (s, 2H), 2.62 (q, 2H), 2.22 (s, 3H), 1.15 (t, 3H). Mass (+ve ion mode m/z): 325 (M++l).
Example 48
S-CBenzoldlll-Sldioxol-S-yO-ό-ethyl-Z-^rifluoromethyO-T-hydroxy^H-chromen^- one (49)
A solution of trifluoroacetic anhydride (0.37 niL) is added to a solution of 45 (400 mg) in 2 mL of dry pyridine at O0C. The reaction mixture was shaken, with ice cooling, for 10-15 min and is left overnight. On the following day, it is heated to 40-500C for 10-
15 min and again left at room temperature for 12 h. Then it is poured into 20-30 mL cold water, and the precipitate is filtered off and crystallized from ethanol to give 49 as a white solid. 1H NMR (300 MHz, DMSO): δ 11.22 (s, IH), 7.76 (s, IH), 6.97 (d, IH5 J= 8.1 Hz), 6.92
(s, IH), 6.84 (d, IH5 J= 1.5 Hz), 6.71 (dd, IH, J= 1.8, 8.1 Hz), 6.08 (s, 2H), 2.65 (q, 2H),
1.16 (t, 3H).
Mass (+ve ion mode m/z): 379 (M++l).
Example 49
Ethyl 2-(3-(benzo[d][l,3]dioxol-5-yl)-6-ethyl-2-methyl-4-oxo-4H-chromen-7-yloxy)- acetate (50)
To a solution of 48 (400 mg, 1 mmol)) added ethyl bromoacetate (0.14 mL, 1 mmol) and K2CO3 (511 mg, 3 mmol) in 10 mL DMF, and the mixture is stirred for 5 h at 900C. The reaction mixture extracted twice with CH2Cl2. The collected organic extracts were dried (Na2SO4), filtered, and concentrated under reduced pressure. The crude is crystallized from aqueous ethanol.
1H NMR (300 MHz, DMSO): δ 8.06 (s, IH), 6.75 (s, IH), 6.74 (m, 3H), 6.03 (s, 2H), 4.78 (s, 2H), 3.91 (q, 2H), 2.78 (q, 2H), 2.26 (s, 3H), 1.30 (t, 3H), 1.25 (t, 3H). Mass (+ve ion mode m/z): 411 (M++l). Example 50
2-(3-(Benzo[d][l,3]dioxol-5-yl)-6-ethyl-2-methyl-4-oxo-4//-chromen-7-yloxy)acetic acid (51)
To a solution of 50 (200 mg, 1 mmol)) added 2 mL 5 % NaOH in 5 mL ethanol and 5 the mixture refluxed for 2 h. The reaction mixture was neutralized with IN hydrochloric acid. It is then diluted with water and the precipitate that deposited is filtered off and crystallized from ethanol to give 51 as a white solid.
1H NMR (300 MHz, DMSO): δ 8.08 (s, IH), 6.79 (s, IH), 6.78 (m, 3H), 6.05 (s, 2H),
4.82 (s, 2H), 2.65 (q, 2H), 2.23 (s, 3H), 1.20 (t, 3H). I0 Mass (+ve ion mode m/z): 383 (M++l).
Example 51 2-(4-Fluorophenyl)-l-(2,4-dihydroxyphenyl)ethanone (52)
With stirring, a rapid current of dry hydrogen chloride is passed for 10 min into a is solution of 4-fluorobenzyl cyanide (500 mg, 1 mmol) in dry toluene (10 mL) cooled to
O0C. Then a solution of resorcinol (448 mg, 1.1 mmol) and fused zinc chloride (251 mg,
0.5 mmol) in dry ether (5 mL) is added. Saturation with hydrogen chloride is continued for 4 h and then kept overnight at room temperature. The solvent was decanted and triturated twice with dry toluene. The hot water (50 mL) added and the mixture is kept at
20 900C and pH 1 for 30 min. The product is separated from the hot solution and washed several times with water. The crude was purified by flash chromatography to give 52 (455 mg, 50% yield) as an oily product.
1H NMR (300 MHz, CDCl3): δ 12.11 (br s, IH), 7.55 (d, IH, J = 8.9 Hz), 7.05-6.95 (m, 4H), 6.20-6.10 (m, 2H), 3.95 (s, 2H). 25 Mass (+ve ion mode m/z): 247 (M++l).
Example 52 2-(Trifluoromethyl)-3-(4-fluorophenyl)-7-hydroxy-4H-chromen-4-one (53)
A solution of trifluoroacetic anhydride (0.22 mL) is added to a solution of 52 (400 30 mg) in 2 mL of dry pyridine at O0C. The reaction mixture was shaken, with ice cooling, for 10-15 min and is left overnight. On the following day, it is heated to 40-500C for 10- 15 min and again left at room temperature for 12 h. Then it is poured into 20-30 mL cold water, and the precipitate is filtered off and crystallized from ethanol to give 53 as a cream colour solid. 1R NMR (300 MHz, DMSO): δ 11.18 (s, IH), 7.93 (d, IH, J = 8.7 Hz), 7.36-7.25 (m, 4H), 7.03 (dd, IH, J= 2.4, 9 Hz), 6.95 (d, IH, J= 2.4 Hz). Mass (+ve ion mode m/z): 325 (M++l).
Example 53
Screening assay to identify PPAR-γ agonists (THP-I cell lines)
EXPERIMENTAL PROCEDURE General
Anti-actin primary antibody, bovine serum albumin (BSA), dimethyl sulfoxide (DMSO), GW9662 and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma-Aldrich (Sydney, Australia). Natural products, totaling 200 compounds, were sourced from the Herbal Medicines Research and Education Center (Faculty of Pharmacy, University of Sydney). Cell Culture The THP-I monocytes and macrophages were grown in RPMI 1640 in the presence of 50 μM β-mercaptoethanol. All media contained L-glutamine supplemented with penicillin (100 U/ml)/ streptomycin (100 (g/ml), and 10% (v/v) heat- inactivated fetal bovine serum (FBS) in a humidified atmosphere of 5% CO2 and 95% O2 at 37°C. To induce monocyte differentiation into macrophages the THP-I monocytes were treated with PMA (400 ng/ml) for 72 h, before selective PPAR-γ agonist rosiglitazone, test samples or vehicle (0.1% DMSO) were added and incubated for a further 48 h in culture medium for the macrophage treatment experiments. The PPAR-γ antagonist, GW9662 (5 (M) was added 1 h prior to addition of positive control or test samples. Cell-Based Transcriptional Factor Assay The PPAR-γ Transcription Factor Assay is a sensitive ELISA method for detecting
PPAR-γ transcription factor DNA binding activity in nuclear extracts of THP- 1 derived macrophage cell line. The ELISA assay was conducted according to the manufacturer's instructions (Cayman Chemical, Sydney, Australia). The purification of cellular nuclear extract from the cultured cells was prepared with CelLytic™ NuCLEAR™ Extraction Kit (Sigma, Sydney, Australia). Cell Proliferation Assay
THP-I macrophage cells were seeded for overnight, then treated with various concentrations of rosiglitazone, GW9662 and test compounds (0.01 - 100 μM) before further incubation for 3 days at 37°C in a humidified atmosphere with 5% CO2. MTS (tetrazolium salt) reagents (the CellTiter96® Aqueous One Solution Cell Proliferation Assay, Promega, Sydney, Australia) was added, incubated for 4 h and finally analyzed using a microtiter plate reader (model 3550, Bio-Rad) (λ: 490 nm). mRNA Analysis
Total mRNA was prepared separately from the THP-I macrophage cells using TRIzol (Invitrogen, Sydney, Australia). The relative levels of specific mRNAs were assessed by RT-PCR as described previously (Abe et al., 2002). Single-stranded cDNA was synthesized from 1 μg of total RNA using Superscript II RNAse H Reverse Transcriptase, as per instructions of the manufacturer (Invitrogen, Sydney, Australia). PCR was performed on a thermocycler, PTC-200 DNA engine (MJ Research Inc, USA). The required cDNA was synthesized with the Platinum® Pfx DNA Polymerase method (Invitrogen, Sydney, Australia). The genes examined were PPAR-γ (L40904; 382bp; sense: 5'-GAGCCCAAGTTTGAGTTTGC-S '; 5'-TGGAAGAAGGGAAATGTTGG-S') and β-actin (NMOOI lOl; 629bp; sense: 5'-GGAGTAACCAGGTCGTCCAA-S'; 5'- GAAGGTGCCCAGAAT ACC AA-3'). The PCR samples were electrophoresed on 5-12% acrylamide gel (29:1, acrylamide:N,N'-methylene-bis-acrylamide) in TBE buffer [89 mM Tris-base pH 7.6, 89 mM boric acid, 2 mM EDTA]. The gels were stained with ethidium bromide (10 μg/ml) and photographed on top of a 280 nm UV light box (Biorad® Gel Doc 1000, Australia). The gel images were digitally captured with a CCD camera and analyzed with ImageJ 1.29x (NIH, USA). RT-PCR values are presented as a ratio of the specified gene signal in the selected linear amplification cycle divided by the β-actin signal. Western Blot
THP-I cells were seeded and treated with PMA (400 ng/ml) for 72 h to obtain THP- 1 macrophages. The macrophages were treated with 0.1% DMSO, psi-baptigenin (40c) (30 μM), hesperidin (34) (30 μM) and rosiglitazone (30 μM) for 48 h. Then the cells were washed with PBS and lysed with RIPA lysis buffer for protein extraction. The protein contents in the samples were measured using BCA protein estimation kit and 20 μg of sample was loaded onto 4-12% NuP AGE® Bis-Tris Gel (Invitrogen, Sydney, Australia). After electrophoresis at 200 V for 1 h, the protein was transferred to PVDF membrane and blocked overnight in skim milk (5% skim milk in tris buffered saline). On the subsequent day the PVDF membranes were treated with antihuman PPAR-γ mouse monoclonal primary antibody (1:500 dilution; Santa Cruz Biotechnology, USA) followed with horseradish peroxidase-conjugated anti-mouse secondary antibody (1 : 10,000 dilution; Promega, USA). The antibody treatment was performed for Ih followed by 30 min wash with the washing buffer (Tris buffered saline with 0.1% Tween-20). Protein expression was detected by chemiluminescence method (Roche). The PVDF membranes were exposed to X-ray film (Kodak, USA) and developed using the SRX-IOlA X-ray developer (Konica, Taiwan). Quantitation of the results was performed by using the NIH Image J software. After stripping with stripping buffer (Glycine (15 g), SDS (1 g), Tween-20 (10 mL), pH 2.2) the membranes were re-probed with anti-actin primary antibody (1: 10,000 dilution; Sigma, Australia) and re- incubated with the secondary horseradish peroxidase antibody, and protein bands were detected as described above. Statistical Analysis
All results are expressed as means ± standard error of the mean (SEM). Data was analyzed by 1 -factor analysis of variance (ANOVA). If a statistically significant effect was found, the Student Newman-Keuls test was performed to isolate the difference between the groups. P values less than 0.05 (p<0.05) were considered as indicative of significance. Ligand Preparation Ligands were built, manipulated and adjusted for chemical correctness using
Maestro 7.5 (Maestro, version 7.5, Schrodinger, LLC, New York, NY) graphical user interface employing MacroModel 9.1 (MacroModel, version 9.1, Schrodinger, LLC, New York, NY). Geometry minimizations were performed on all ligands using the OPLS_2005 (MacroModel, version 9.1, Schrodinger, LLC, New York, NY) force field and the Truncated Newton Conjugate Gradient (TNCG). Optimizations were converged to a gradient rmsd below 0.05 kj A'1 mol or continued to a maximum of 500 iterations, at which point there were negligible changes in rmsd gradients. The library was seeded with farglitazar and rosiglitazone as reference compounds for the virtual screening. Ligand Docking and Target Preparation Ligands were independently docked into the LBD of two PPAR-γ receptors.
Docking was performed with Glide 4.0 (Glide, version 4.0, Schrodinger, LLC, New York, NY) utilizing the extra precision (XP) scoring function (Friesner et al., 2006) to estimate protein-ligand binding affinities. The site targeted in the docking calculations was defined by the position of the molecules: farglitazar and rosiglitazone, observed in complex with PPAR-γ as part of the PPAR-γ/RXRα heterodimer crystal structure (PDB: 1FM9, receptor (I); and 1FM6 receptor (II), respectively) (Gampe et al., 2000). Protein preparation and refinement protocols, directed by the protein preparation facility (Schrodinger User Manuals, Glide v4.0 Schrodinger, LLC, New York, NY), were performed on both targets to achieve chemical correctness. Briefly, this included deleting crystallographic waters; adding hydrogens; adjusting bond orders and formal charges; neutralizing side-chains distant from the binding site and alleviating potential steric clashes via protein minimization with the OPLS_2005 force field. The tautomeric states of His323 (positively charged) and His449 (Nε protonated) were manually selected to maximize hydrogen bonding. The shape and properties of the binding site were characterized and setup for docking using the receptor grid generation panel (Schrόdinger User Manuals, Glide v4.0 Schrόdinger, LLC, New York, NY). A Coulomb-van der Waals (vdW) scaling of 1.0/0.8 was set for receptor/ligand vdW radii, respectively. The top 20 of best-scoring ligands from each docking study were pooled together and selected for the PPAR-γ agonist functional assay. Induced-Fit Docking
The IFD protocol was run from the graphical user interface accessible within Maestro 7.5. It was carried out on receptor (II) with psi-baptigenin (40c), apigenin (8), chrysin (12), biochanin-A (55) and genistein (56) as test ligands. The overall procedure has four stages: Briefly, during Stage 1 initial softened-potential Glide docking is performed on a vdW scaled-down rigid-receptor (II); a scaling of 0.7/0.5 was set for receptor/ligand vdW radii, respectively (Sherman et al., 2006). Residues Cys285 and Phe363 were temporarily mutated to alanine for having deviated more than 2.5 A compared to receptor (I). The top 20 poses for each test ligand was retained. In Stage 2, Cys285 and Phe363 were restored to their original residue type, followed by Prime side- chain prediction and minimization for each of the 20 ligand/protein complexes. Backbone residues and ligands were minimized. Residues within 5.0 A of ligand poses were similarly refined, but additionally underwent sampling. A total of 20 induced-fit receptor conformations were generated for each of the 5 test ligands. Stage 3 involved redocking the test ligands into their respective 20 structures that are within 30.0 kcal mol"1 of their lowest energy structure. Finally, the ligand poses were scored in Stage 4 using a combination of Prime and GlideScore scoring functions. The XP scoring function was used in all docking stages.
RESULTS: Multi-Conformational Virtual Screening of a Natural Product Library against PPAR-γ
The structures of PPAR-γ bound with farglitazar and rosiglitazone are well-resolved and have been studied in great detail (Gampe et al., 2000; Nolte et al., 1998). Complexed with either of the two, the shape of the PPAR-γ LBD is strongly influenced by the size and chemical makeup of the ligand. Specifically, in case of the larger farglitazar, side- chain rearrangements in the LBD are induced allowing it to bind residues unattainable by rosiglitazone. When taken as a whole, the differences in binding poses of farglitazar and rosiglitazone yield two structurally distinct receptor forms, each of which can be used as templates in structure-based drug discovery efforts. In the present study, a total of 200 compounds comprising a "natural product library" were docked into the LBD of two PPAR-γ templates. Ligands were docked with Glide 4.0 (Glide, version 4.0, Schrodinger, LLC, New York, NY) utilizing the extra precision (XP) scoring function (Friesner et al., 2006) to estimate protein-ligand binding affinities. The site targeted in the docking calculations was defined by the position of the molecules: farglitazar and rosiglitazone, observed in complex with PPAR-γ as part of the PPAR-γ/RXRα heterodimer crystal structure (PDB: 1FM9, receptor (I); and 1FM6 receptor (II), respectively) (Gampe et al., 2000). Ligands were ranked based on docking scores whereby the more negative values correspond to greater predicted binding affinities. The 20 best-scoring compounds from each docking study were combined to create an initial hit-list totaling 40 compounds. Duplicate compounds (ie, compounds scoring in the top 20 against both receptors) were removed. A final list of 29 candidates was considered as potential PPAR-γ ligands having achieved relatively favorable docking scores (Table 1). Among them were 3 compound families: the flavonoids, the predominant class with 17 compounds; the gingeroids with 10 compounds; and the ginkolides with 2 compounds.
Table 1 List of docked compounds, along with rankings and docking scores determined against receptors (I) and (II).
Figure imgf000083_0001
a Docking scores determined by the extra precision (XP) GlideScore function (Friesner et al., 2006). b Interactions with residues known to contribute to PPAR-γ activation (Gampe et al., 2000; Nolte et al., 1998). Post Docking Evaluation
The 29 ligand:PPAR-γ complexes were carefully scrutinized. Previous studies established that hydrogen bond interactions to Ser289, His323, His449 and Tyr473, and hydrophobic interactions, such as π...π stacking, with residues Phe363 and Phe360, are formed by a number of PPAR-γ agonists (Gampe et al., 2000; Nolte et al., 1998). Accordingly, compounds were examined on their potential to engage these residues, located close to the transcriptional activation function 2 domain (AF-2 helix), and whether they established similar ligand:receptor interactions such as those observed by farglitazar and rosiglitazone in their relative X-ray crystal structure complexes (Figure 1). The results for the flavonoids represented the only set to have revealed a common binding pose that produced comparable interaction fingerprints to those presented by farglitazar and rosiglitazone. Therefore, the flavonoids were selected for further study. Of particular interest were the compounds psi-baptigenin, hesperidin, apigenin, chrysin, biochanin-A and genistein. Specifically, the results against receptor (I), where they ranked no worse than 14th position (Table 1), show each occupying a large hydrophobic pocket enclosed by His449, Phe282, Phe360 and Phe363 (Figure 2a - e). Noticeable hydrophobic contacts include complimentary π...π (<3.5A) stacking between each of the flavonoid's B-Ring phenyl with the side-chain phenyl of Phe363, analogous to the benzophenone...Phe363 interaction of farglitazar. The A-ring phenyl is directed towards the AF-2 helix where hydrogen-bond contacts are made to Ser289 and His323 via the 7-OH. Such interactions are similarly made by farglitazar and rosiglitazone whereby hydrogen bond contacts are established to the above residues, although via a carboxylic acid and thiazolidinedione group, respectively. Apigenin (8) and genistein (56) were also seen to hydrogen bond to the backbone carbonyl of Phe363. Docking results for these flavonoids against receptor (II) are shown in Figure 6.
Hesperidin (34) was predicted to bind favorably into both PPAR-γ receptors, ranking third in both instances. Figure 2f clearly illustrates the two binding poses of hesperidin (34) for both receptors, each appearing almost identical when the LBD are superposed. The overall structure twists around Helix 3 (not displayed) in an orientation resembling the U-shape conformation of both farglitazar and rosiglitazone. The highly polar disaccharide moiety is directed towards the polar region of the AF-2 helix. Hydrogen bond contacts are made to Ser289, His323 and Tyr473, again comparable with farglitazar's carboxylate and rosiglitazone 's thiazolidinedione. Hesperidin's A-ring also roughly corresponds to the methylphenyloxazole and pyridine of farglitazar and rosiglitazone, respectively, with each representing a bulky aromatic group in a partially solvent-exposed region. Unlike the previously described flavonoids, hesperidin (34) did not appear to interact with Phe363. Cell-Based Transcriptional Factor Assay
From the initial 200 natural occurring compounds, the 29 hits, chosen on the basis s of docking potential towards PPAR-γ, were selected for PPAR-γ functional assay. This included compounds that were assigned good docking scores, despite not engaging key residues known involved in activation e.g. the gingerones and ginkolides. Here, the Cayman Chemical PPAR-γ Transcription Factor Assay was employed as a sensitive method for detecting specific transcription factor DNA binding activity in nuclear
I0 extracts. This assay involves the use of a 96 well enzyme-linked immunosorbent assay (ELISA) whereby specific double stranded DNA (dsDNA) sequences containing the peroxisome proliferator response element (PPRE) is immobilized onto the bottom of wells in a microtitre plate. Any PPAR-γ contained in a nuclear extract, bind specifically to the PPRE and the degree of binding is detected by the addition of specific primary is antibody directed against PPAR-γ. A secondary antibody conjugated to horse radish peroxidase was added to provide colorometric readout at 450 nm.
In this study, all 29 compounds were screened for PPAR-γ activity with a concentration range of 0.01 μM to 50 μM. Rosiglitazone was used as a positive control. The results of the PPAR-γ transcriptional factor assay show that 6 out of the 29 hits tested
20 possess biological activity (Figure 3). Psi-baptigenin, hesperidin, apigenin, chrysin and biochanin-A all exhibited EC50 values less than 10 μM, whilst genistein was still below the therapeutic limits at 16.7 μM (Table 2). Rosiglitazone, psi-baptigenin (40c) and hesperidin (34) dose-dependently increased PPAR-γ transcriptional activity and at 50 μM each of the compounds yield a 7.3±0.8, 6.2+0.7, 5.1±0.8, fold increase respectively
25 (Figure 3). Apigenin (8), chrysin (12), biochanin-A (55) and genistein (56) also dose- dependently activated PPAR-γ transcriptional activity albeit less effectively compared to psi-baptigenin (40c) and hesperidin (34) at 50 μM (4.7±1.2; 4.3+1.1; 4.0±0.8; and 3.1+0.4, respectively) (Figure 3).
30 Table 2 Structures and EC50 values for transcriptional factor activity of test compounds in
Figure imgf000086_0001
Each value is the mean ± SEM Cell-Proliferation Assay
To ensure the compounds were non-toxic to THP-I macrophage cell lines, cytotoxicity profiles were investigated (Figure 4). The concentrations were 0.0 lμM to lOOμM. All the compounds except GW9662 showed little or no effect on cell viability (>
5 90% viability remained). GW9962 at 10, 50 and 100 μM decreased the cell viability to 86.6%±3.7, 70.5%±4.6 and 24.1%±5.1, respectively (Figure 4). Co-treatment with both 50 μM rosiglitazone and 5 μM GW9662, 50 μM psi-baptigenin (40c) and 5 μM GW- 9662, 50 μM hesperidin (34) and 5 μM GW9662, yield no effect on cell viability (95.9%±3.2, 97.9%±3.2 and 97.0%±4.6, respectively) (Figure 5a). Whereas co-treatment
I0 with both 50 μM rosiglitazone and 10 (M GW9662, 50 μM psi-baptigenin and 10 μM GW9662, 50 μM hesperidin and 10 μM GW9662, decrease the cell viability to 82.1%±3.0, 81.1%±7.5 and 78.8%±6.1, respectively (Figure 5a). Therefore, for all the in vitro experiments 5 μM GW9662 was used. Gene Expression of PPAR-γ is To further investigate the specificity of psi-baptigenin (40c) and hesperidin (34) toward PPAR-γ, we applied GW9662, a potent non-competitive antagonist of PPAR-γ. The results showed that 5 μM of GW9662 abolished the PPAR-γ transcriptional activation by rosiglitazone (50 μM), psi-baptigenin (40c) (50 μM) and hesperidin (34) (50 μM) (Figure 5b).
20 In the quantitative RT-PCR and immunoblotting experiments rosiglitazone, psi- baptigenin (40c) and hesperidin (34) at 50 μM enhanced PPAR-γ mRNA expression (5.4±0.2 fold, 4.1±0.2 fold and 3.8±0.3 fold, respectively) in the THP-I macrophage cell line (Figure 5c). Additionally, psi-baptigenin (40c) (30 μM) and hesperidin (34) (30 μM) appeared to up-regulate PPAR-γ protein expression (2.9±0.2 fold and 2.5±0.8,
25 respectively) (Figure 5d). Rosiglitazone (30 μM) showed no increase in protein expression. The induction of PPAR-γ mRNA expression by all three compounds was attenuated in the presence of GW9662 (5 μM) (Figure 5c). Induced Fit Docking (IFD)
After examining the biological data, it became evident that all of the active
30 flavonoids, with the exception of hesperidin (34), were those to have favored receptor (I) during the preceding docking screen. Presumably, had receptor (II) been the sole template used in this study, psi-baptigenin (40c), apigenin (8), biochanin-A (55) and genistein (56) would not have been selected for biological testing; they did not rank in the top 20 against receptor (II) (Table 1). In recognition of this fact — which is accredited to the structural
35 differences presented by each receptor's LBD — an attempt was made to re-dock the above into receptor (II), this time incorporating receptor flexibility. Here, psi-baptigenin (40c), apigenin (8), chrysin (12), biochanin-A (55) and genistein (56) underwent an induced fit docking (IFD) procedure that unites rigid-receptor Glide 4.0 XP docking ( Friesner et al., 2006) with the protein structure prediction and refinement package, Prime 1.5 (Prime, version 1.5 Schrόdinger, LLC, New York, NY). Detailed in Sherman et al the IFD procedure has already demonstrated success at predicting the binding poses of farglitazar and rosiglitazone towards each other's respective PPAR-γ-bound co-crystal structure (Sherman et al., 2006). For that reason, a measure of assurance is felt when implementing the IFD protocol in this study. The ensuing IFD results successfully produced docking poses and improved scores that are comparable to the results from the rigid-receptor docking screen on receptor (I). All five ligands now engage the AF-2 helix via A-ring hydroxyl groups and interact with the same residues to those found when docked into receptor (I) (Figure 6b). A minor change is seen with apigenin (8) which now directs the carbonyl towards the top of binding cavity, resembling that of the other flavonoids. The most significant outcome resulting from the IFD on receptor (II) is undoubtedly the re-positioning of Phe363 (Figure 6c). It is evident that in the case of all 5 poses, Phe363 no longer protrudes into the center of the LBD but swings towards Helix 10, allowing favorable π...π stacking interaction with each flavonoid B-ring. Docking pose investigations The results of the transcriptional factor assay identified psi-baptigenin and hesperidin amongst a total of 6 flavonoids with the ability to activate PPAR-γ in the THP- 1 macrophage cell line. These flavonoids can be classified into 3 groups: flavone [apigenin (8) and chrysin (12)]; isoflavone [psi-baptigenin (40c), biochanin A (55) and genistein (56)] and flavanone glycoside [hesperidin (34)]. In the case of flavones and isoflavones, the two groups differ with the location of the B-ring phenyl on the 1,4- benzopyrone skeleton, be it at the 2 or 3 position, respectively. Hesperidin (34), a flavanone glycoside, has pronounced differences including the bound disaccharide rutinose at the 7 position and the reduction of the 2(3) carbon-carbon double bond. Interestingly, none of the these flavonoids share any of the typical bioisosteric features seen on other PPAR-γ agonists, that being a flexible, lipophilic backbone attached to a carboxyl, thiazolidinedione, sulfone or sulfonamide. Instead, with the exception of hesperidin, they are exclusively structurally rigid molecules with one rotatable bond and various sites of hydroxylation. However, despite these differences, the initial docking run and subsequent IFD demonstrated that psi-baptigenin, apigenin, chrysin, biochanin-A and genistein bind in the PPAR-γ LBD in a favourable and consistently predictable manner. Collectively, the predicted results suggest the interactions are comparable to those achieved by farglitazar and rosiglitazone as revealed in their relative X-ray crystal complexes. In the case of hesperidin, the docked pose differs significantly to those predicted for the other flavonoids, presumably due to the added sugar moiety. Nevertheless, its conformation is in a similar framework to the U-shape structure demonstrated by farglitazar and rosiglitazone. Overall, the active flavonoids identified in this study, although visibly different in structure and chemistry to the known PPAR-γ agonists such as farglitazar and rosiglitazone, achieved the necessary ligand-receptor interactions required for PPAR-γ activation. PPAR-γ activation studies
The transcriptional factor assay results in the present study were in agreement with Liang et al that apigenin (8), chrysin (12), biochanin A (55) and genistein (56) activate PPAR-γ in RAW264.7 macrophages with the same rank-order affinity as seen in our study, with apigenin (8) found to be the most potent (Liang et al., 2001). Our results showed both psi-baptigenin (40c) and hesperidin (34) activated PPAR-γ at lower EC50 than apigenin (8), classifying them as two of the most potent naturally-derived PPAR-γ agonists currently known. Based on the results of a limited protease digestion, Liang et al. suggested apigenin (8) and two similar flavones induced conformational change upon direct binding with the PPAR-γ LBD, and did so differently to that of rosiglitazone. In light of this difference, they hypothesized that the flavonoids bind to an allosteric site. Indeed, the results from our docking predictions corresponds well with their experimental outcomes — that is, the flavonoids induce conformational change in PPAR-γ and binds differently to rosiglitazone — however, our docking results provide strong indications they do not necessarily have to be allosteric effectors as they remain capable of achieving the above observations in the native binding domain. The abolishment of their activities on PPAR-γ by selective PPAR-γ antagonist, GW9662, provides further that evidence psi- baptigenin and hesperidin exert their effects directly in the PPAR-γ LBD, duly supporting the docking results.
The up-regulation of PPAR-γ mRNA and protein expression by psi-baptigenin (40c) and hesperidin (8) signify their ability to stimulate PPAR-γ gene expression, compounding their status as PPAR-γ agonists. The lack of protein induction by rosiglitazone is distinctly intriguing, particularly on account of its higher mRNA induction (5.4±0.2 fold) versus that of psi-baptigenin (4.1±0.2 fold). However, this observation does conform with Davies et al. whereby rosiglitazone, amongst other TZDs, failed to induce PPAR-γ protein in HepG2 cells (Davies et al., 2002). Induced-fit docking
In this study, two different 3D structures of PPAR-γ were employed as docking templates: receptors (I) and (II). They were specifically chosen as each structure presented a distinct conformational difference in PPAR-γ' s LBD, clearly illustrating the ability of this receptor to accommodate a wide range of potential ligands. Thus, an attempt was made to avoid any bias based on molecular structure and chemistry in the screening procedure by docking the 200 compounds into each receptor. This procedure may increase the likelihood of finding suitable hits of structural and chemical diversity.
The advantage of using more that one docking template is evident from the observation that the majority of compounds, including the most potent, psi-baptigenin, would not have been identified had receptor (II) been the sole template implemented in the study. Since this was attributed to the different receptor conformations between receptors (I) and (II), an attempt was made to introduce IFD — a docking protocol that incorporates protein flexibility — as a means of preventing such false negative results in future studies, and to further elucidate the ligand's probable binding pose. Receptor (II) presented substantial binding pose variability for psi-baptigenin (40c), apigenin (8), chrysin (12), biochanin-A (55) and genistein (56) (Figure 6a). Resultantly, the IFD successfully rectified critical side-chain rearrangements in the LBD of receptor (II) to closely resemble the LBD of receptor (I), as well as reproducing ligand poses seen in the initial rigid-receptor docking with receptor (I). For that reason, it is conceivable IFD would have been successful in identifying hits excluded by rigid-receptor docking on receptor (II) had the procedure been used in the initial virtual screen. However, although this technology may seemingly reduce the reliance in multiple rigid-receptor dockings, it must be stated that, in this study, docking the 200 compounds into two separate receptors would be more computationally efficient than IFD on the same number of compounds - a consequence presumably caused by the added degrees of freedom in the IFD side-chain prediction and geometry minimization algorithms. Nevertheless, when used in combination in a drug discovery environment, multiple rigid-receptor dockings followed by IFD may confirm the identification of novel hit compounds and propose a detailed ligand:receptor binding complex, with greater levels of certainty than rigid-receptor docking alone. Example 54
Screening assay to identify PPAR-γ and PPAR-α agonists (HEK cell lines) EXPERIMENTAL PROCEDURE Cell Culture Human embryonic kidney (HEK) 293 cell line was obtained from American Type
Culture Collection (USA). All materials used for tissue culture were purchased from Invitrogen, Australia unless specified. HEK 293 cells were grown in Dulbecco's modified Eagle's medium/F-12 (DMEM/F-12), containing L-glutamine supplemented with penicillin (100 U/mL), streptomycin (100 mg/mL) and 10% (v/v) heat- inactivated foetal bovine serum in a humidified atmosphere of 5% CO2 and 95% O2 at 37 °C. (Bramlett et al, 2003; Frederiksen et al, 2004). Transfection and Luciferase Assay (PPAR-γ~)
The transfection and luciferase procedures were performed as described previously (Bramlett et al., 2003) with slight modification. The HEK 293 cell line was transfected with tK-PPREx3-Luc plasmid, pSG5-hPPAR-γ plasmid and pSV-β-galactosidase (Promega, Australia) control plasmid. Cells were transfected with FuGENE 6 transfection reagent (Roche, Australia) in accordance with the manufacturer's instructions. After 24 h at 37°C, cells were harvested and plated into 96-well plates at 5 x 104 cells per well in complete transfection media and allowed to attach over night at 37 °C. The cells were then treated with rosiglitazone and GWl 929 as positive controls, DMSO (0.1%) as a negative control and the test samples. After 48 hours, the cells were lysed and assayed for luciferase and β-galactosidase activities using the Bright-Glo Luciferase Assay system and Beta-Glo Assay system (Promega, Australia), respectively. The results were expressed as relative luciferase activity normalized to the β-galactosidase signal (fold difference compared to negative control). Cell Proliferation Assay (PPAR-γ)
Human embryonic kidney cell line (HEK 293) was seeded overnight then treated with various concentrations of rosiglitazone, GWl 929 and test compounds (0 - 100 μM) and incubated for 48 hours at 37°C in a humidified atmosphere with 5% CO2. MTS (tetrazolium salt) reagent (CellTiter96® Aqueous One Solution Cell Proliferation Assay, Promega, Sydney, Australia) was added and samples were incubated for a further 1-4 hours before finally being analyzed using a BMG POLARstar Galaxy Microplate Reader (λ: 490 nm). Transfection and Luciferase Assay (PPAR-oύ
The transfection and luciferase procedures were performed as described previously (Bramlett et al., 2003) with slight modification. The HEK 293 cell line was transfected with tK-PPREx3-Luc plasmid, pBI-G-hPPAR-α plasmid and pSV-β-galactosidase (Promega, Australia) control plasmid. Cells were transfected with FuGENE 6 transfection reagent (Roche, Australia) in accordance with the manufacturer's instructions. After 24 h at 37°C, cells were harvested and plated into 96-well plates at 5 x 104 cells per well in complete transfection media and allowed to attach over night at 37°C. The cells were then treated with WY-14643, Fenofibrate as positive controls, DMSO (0.1%) as a negative control and the test samples. After 48 hours, the cells were lysed and assayed for luciferase and β-galactosidase activities using the Bright-Glo Luciferase Assay system and Beta-Glo Assay system (Promega, Australia), respectively. The results were expressed as relative luciferase activity normalized to the β-galactosidase signal (fold difference compared to negative control). Cell Proliferation Assay (PPAR-cύ
HEK 293 cells were seeded overnight then treated with various concentrations of WY-14643, Fenofibrate and test compounds (0 - 100 μM) and incubated for 48 hours at 37 0C in a humidified atmosphere with 5% CO2. MTS (tetrazolium salt) reagent (CellTiter96® Aqueous One Solution Cell Proliferation Assay, Promega, Sydney, Australia) was added and samples were incubated for a further 1-4 hours before finally being analyzed using a BMG POLARstar Galaxy Microplate Reader (λ: 490 nm).
Results:
Reporter Gene Luciferase Assay (PPAR-γ) Psi-baptigenin (40c) is currently undergoing pre-clinical evaluation, and while it has shown promising activity against PPAR-γ, there is a need for more selective and potent derivatives. This requires an understanding of the key structure features of psi-baptigenin (40c) and its congeners that must be retained to maintain PPAR-γ activity. A series of compounds (Table 3) have been designed, synthesized and evaluated for PPAR-γ activation activity in the Human Embryonic Kidney cell line (HEK 293) at various concentrations (5, 25 and 50 μM). As illustrated in Figure 7, compounds 40a, 40c, 4Oe and 4Oi exhibited a significant PPAR-γ fold activation activity compared to the known PPAR-γ agonist rosiglitazone (positive control). These results emphasize the importance of the 7-OH, and hydrophobic substituents such as F or OMe moieties on the B-ring. Table 3 Structures and PPAR-γ fold activation using HEK-293 cells (Luciferase assay).
Figure imgf000093_0001
Figure imgf000094_0001
Figure imgf000095_0001
Figure imgf000096_0001
Figure imgf000097_0001
Figure imgf000098_0001
Figure imgf000099_0001
Figure imgf000100_0001
ND- not determined
Cell Proliferation Assay (PPAR-γ)
To ensure the compounds were non-toxic to HEK 293 cell line, cytotoxicity profiles were investigated (Figure 8) at various concentrations (0 - 100 μM). As illustrated in Figure 8, molecules 40c and 4Oi exhibited a cell viability of greater than 90% at lOμM concentrations (comparable to GWl 929), while 40a showed a cell viability of greater than 80% at 10 μM (correlates with Rosiglitazone). Compounds 40c and 4Oi maintained a cell viability of greater than 60% even at 100 μM concentrations, that is significantly much higher than Rosiglitazone and GWl 929 (46% and 33%, respectively). Reporter Gene Luciferase Assay (PPAR-α)
A series of compounds (Table 4) have been designed, synthesized and evaluated for PPAR-α activation activity in the Human Embryonic Kidney cell line (HEK 293) at various concentrations (5, 25, 40, 50 and 100 μM). As illustrated in Figure 9, compounds 40a, 40c, 4Oe and 4Oi exhibited a significant PPAR-α fold activation activity compared to the known PPAR-α agonists WY- 14643 and Fenofibrate (positive controls).
Table 4 Structures and PPAR-α fold activation using HEK-293 cells (Luciferase assay).
Figure imgf000100_0002
Figure imgf000101_0001
Figure imgf000102_0001
Figure imgf000103_0001
Figure imgf000104_0001
Cell Proliferation Assay (PPAR-cύ
To ensure the compounds were non-toxic to HEK 293 cell line, cytotoxicity profiles were investigated (Figure 10) at various concentrations (0 - 100 μM). As illustrated in Figure 10, molecules 40c and 4Oi exhibited a cell viability of greater than 90% at lOμM concentration, while Fenofibrate demonstrated a cell viability of greater than 90% only at 0.1 μM concentration. Molecules 40a, 40c, 4Oe and 4Oi all illustrated a cell viability of more than 67% at 50 μM concentration, however, Fenofibrate showed a decrease in cell viability to 38% at the respective concentration. REFERENCES
Abe A, Kiriyama Y, Hirano M, Miura T, Kamiya H, Harashima H and Tokumitsu Y (2002) Troglitazone suppresses cell growth of KU812 cells independently of PPARgamma. Eur J Pharmacol 436(l-2):7-13.
Bendixen AC, Shevde NK, Dienger KM, Willson TM, Funk CD and Pike JW (2001) IL-4 inhibits osteoclast formation through a direct action on osteoclast precursors via peroxisome proliferator-activated receptor gamma 1. Proc Natl Acad Sci U S A 98(5):2443-2448.
Bramlett KS, Houck KA, Borchert KM, Dowless MS, Kulanthaivel P, Zhang Y, Beyer TP, Schmidt R, Thomas JS, Michael LF, Barr R, Montrose C, Eacho PI, Cao G and Burris TP (2003) A natural product ligand of the oxysterol receptor, liver X receptor. J Pharmacol Exp Ther 307(l):291-296.
Davies GF, McFie PJ, Khandelwal RL and Roesler WJ (2002) Unique ability of troglitazone to up-regulate peroxisome proliferator-activated receptor-gamma expression in hepatocytes. J Pharmacol Exp Ther 300(l):72-77.
Frederiksen KS, Wulff EM, Sauerberg P, Mogensen JP, Jeppesen L and Fleckner J (2004) Prediction of PPAR-alpha ligand-mediated physiological changes using gene expression profiles. J Lipid Res 45(3):592-601.
Friesner RA, Murphy RB, Repasky MP, Frye LL, Greenwood JR, Halgren TA, Sanschagrin PC and Mainz DT (2006) Extra precision glide: docking and scoring incorporating a model of hydrophobic enclosure for protein-ligand complexes. J Med Chem 49(21):6177-6196.
Gampe RT, Jr., Montana VG, Lambert MH, Miller AB, Bledsoe RK, Milburn MV, Kliewer SA, Willson TM and Xu HE (2000) Asymmetry in the PPARgamma/RXRalpha crystal structure reveals the molecular basis of heterodimerization among nuclear receptors. MoI Cell 5(3):545-555. Liang YC, Tsai SH, Tsai DC, Lin-Shiau SY and Lin JK (2001) Suppression of inducible cyclooxygenase and nitric oxide synthase through activation of peroxisome proliferator- activated receptor-gamma by flavonoids in mouse macrophages. FEBS Lett 496(1): 12- 18.
Nestel P (2003) Isoflavones: their effects on cardiovascular risk and functions. Curr Opin Lipidol 14(l):3-8.
Nolte RT, Wisely GB, Westin S, Cobb JE, Lambert MH, Kurokawa R, Rosenfeld MG, Willson TM, Glass CK and Milburn MV (1998) Ligand binding and co-activator assembly of the peroxisome proliferator-activated receptor-gamma. Nature 395(6698): 137-143.
Sherman W, Day T, Jacobson MP, Friesner RA and Farid R (2006) Novel procedure for modeling ligand/receptor induced fit effects. J Med Chem 49(2):534-553.
Aitmambetov A, Khilya VP (1994) Synthetic and modified isoflavonoids. VII. Synthesis of benzodioxocane analogs of pseudobaptigenin. Chem Natural Comp 30(2):204-206 and the references therein.
Felpin FX, Lory C, Sow H, Acherar S (2007) Practical and efficient entry to isoflavones by Pd(0)/C-mediated Suzuki-Miyaura reaction. Total synthesis of geranylated isoflavones. Tetrahedron 63(14):3010-3016.

Claims

CLAIMS:
1. A compound of general formula ( 1 b):
Figure imgf000107_0001
(Ib) wherein 5 Y is O or S;
1 represents a single bond or a double bond;
R1 is selected from hydrogen, hydroxyl, halogen, C1.4a.kyl, haloCi.4alkyl, hydroxyC1-4alkyl, O-CMalkyl, OC(O)-CMalkyl, C(O)-C 1-4alkyl, and O-sugar;
R3, R4, R6 are each independently selected from hydrogen, hydroxyl, halogen, Ci- io 4alkyl, C3-6cycloalkyl, haloCi-4alkyl, hydroxyCi-4alkyl, O-Ci-4alkyl, O-Ci-4alkyl-CO2R,
O-Cs-όCycloalkyl, O-C3.6heterocycloalkyl, O-C3-6heteroaryl, N(R)2C Malkyl, N(R)3Ci- 4alkyl, O-Ci-4alkyl-N(R)2, O-Ci-4alkyl-N(R)3, C6-i0aryl, 0-C6-10 aryl, O-CMalkyl-C6.,0aryl,
O-Ci-4alkyl-C6-10heterocycloalkyl, O-Ci-4alkyl-C6-10heteroaryl, O-C(O)-Ci-4alkyl, O-
CON(R)2, CON(R)2, CO2R, Ci-4alkanoyloxymethyl, -P(O)(OH)(OCi-4alkyl), -P(O)(OCi-
I5 4alkyl)2, and O-sugar;
R5 is selected from hydrogen, hydroxyl, halogen, Ci.4alkyl, C3-6cycloalkyl, haloCi.
4alkyl, hydroxyCi-4alkyl, O-Ci-4alkyl, O-Cialkyl-CO2R, 0-C3-6Cy cloalkyl, THP, N(R)2Ci- 4alkyl, N(R)3Ci-4alkyl, O-C1-4alkyl-N(R)2, C6-i0aryl, O-C6-i0aryl, O-Ci.4alkyl-C6.i0aryl, O-
C(O)-C2-4alkyl, 0-CON(R)2, CON(R)2, CO2R, Ci-4alkanoyloxymethyl, -P(O)(OH)(OCi-
20 4alkyl), and -P(O)(OC1-4alkyl)2; or
one or more of R3 and R4, R4 and R5, and R5 and R6 together form ° * , or
Figure imgf000107_0002
or* . each R is independently selected from hydrogen and Ci-4alkyl;
Figure imgf000107_0003
is 2-pyridyl, 3-pyridyl or 4-pyridyl optionally substituted with one or more of
2s halogen, hydroxyl, Ci-4alkyl, O-Ci^alkyl, O-C3-6cycloalkyl, haloCi-4alkyl, hydroxyCi.
4alkyl, O-Ci-4alkyl-CO2R, 0-C3-6CyClOaIlCyI, O-C^heterocycloalkyl, O-C3-6heteroaryl,
N(R)2C1-4alkyl, N(R)3C1-4alkyl, O-C,-4alkyl-N(R)2, O-C1-4alkyl-N(R)3, C6-i0aryl, 0-C6-10 aryl, O-C1-4alkyl-C6-i0aryl, O-Ci-4alkyl-C6-i0heterocycloalkyl, O-Ci-4alkyl-C6-i0heteroaryl, 0-CON(R)2, CON(R)2, CO2R, C1-4alkanoyloxymethyl, -P(O)(OH)(OCi-4alkyl), O-benzyl, CHO, CO2H, and OC(O)-C i-4alkyl, or
Figure imgf000108_0001
wherein R -R are each independently selected from hydrogen, halogen, hydroxyl, Ci-4alkyl, O-Ci-4alkyl, O-C3-6cycloalkyl, O-C1-4haloalkyl, haloC1-4alkyl, hydroxyCi. 4alkyl, O-benzyl, CHO, CO2H, and OC(O)-C1-4alkyl, or one or more of R7 and R8, R8 and R9, R9 and R10, and R10 and R11 together form
Figure imgf000108_0002
group optionally substituted with one or more of halogen, hydroxyl, Cι-4alkyl, O-C1-4alkyl, O-C3-6cycloalkyl, haloCi-4alkyl, hydroxyCi-4alkyl, O-Ci.4alkyl- CO2R, O-C3-6cycloalkyl, O-C3-6heterocycloalkyl, O-C3-6heteroaryl, N(R)2CMalkyl, N(R)3C1-4alkyl, O-Ci-4alkyl-N(R)2, O-Ci-4alkyl-N(R)3, C6-i0aryl, 0-C6-10 aryl, O-CMalkyl- C6-10aryl, O-C1-4alkyl-C6-10heterocycloalkyl,
Figure imgf000108_0003
0-CON(R)2, CON(R)2, CO2R, Ci-4alkanoyloxymethyl, -P(O)(OH)(OC1-4alkyl), -P(O)(OC1-4alkyl)2, O- sugar, O-benzyl, CHO, CO2H, and OC(O)-C1-4alkyl; or a pharmaceutically acceptable salt thereof.
2. A compound of formula (Ib) according to claim 1 , wherein Y is O.
3. A compound of formula (Ib) according to any one of claims 1 to 2, wherein R1 is selected from hydrogen, halogen, C1-4alkyl, haloCi-4alkyl, O-Ci-4alkyl, and OC(O)- C1-4alkyl.
4. A compound of formula (Ib) according to any one of claims 1 to 3, wherein R1 is selected from hydrogen, methyl, CF3 and COOH.
5. A compound of formula (Ib) according to any one of claims 1 to 4, wherein
Figure imgf000108_0004
is 2-pyridyl, optionally substituted with one or more halogen, hydroxyl, Ci-4alkyl, or O-C1-4alkyl. of formula (Ib) according to any one of claims 1 to 4, wherein
Figure imgf000108_0005
wherein R7- R11 are each independently selected from hydrogen, halogen, hydroxyl, Q- 4alkyl, haloC1-4alkyl, O-Ci-4alkyl, O-C1-4haloalkyl, O-Ci-4alkyl-CO2R, O-C3-6cycloalkyl, O-C3-6heterocycloalkyl, O-C3.6heteroaryl, N(R)2d.4alkyl, N(R)3CMalkyl, O-CMalkyl- N(R)2, O-C1-4alkyl-N(R)3) C6-10aryl, O-C6-i0 aryl, O-Ci-4alkyl-C6-i0aryl, O-C,-4alkyl-C6- loheterocycloalkyl, O-C1-4alkyl-C6-i0heteroaryl, 0-CON(R)2, CON(R)2, CO2R, Ci- 4alkanoyloxymethyl, -P(O)(OH)(OCi-4alkyl), -P(O)(OC1-4alkyl)2, O-sugar, O-benzyl, CO2H, CO2-Ci-4a more of R7 and R8, R8 and R9, R9 and R10, and R10 and
R1 ' together form
Figure imgf000109_0001
7. A compound of formula (Ib) according to any one of claims 1 to 4, wherein
Figure imgf000109_0002
wherein R7- R11 are each independently selected from hydrogen, fluorine, chlorine, methyl, O-methyl, 0-CF3, CF3, O-benzyl, CHO and CO2H or R8 and R9 together form
8. A compound of formula (Ib) according to any one of claims 1 to 7, wherein R5 is selected from hydrogen, hydroxyl, THP, O-C1-4alkyl, 0-CH2CO2R and CO2R wherein R is hydrogen or Ci-4alkyl.
9. A compound of formula (Ib) according to any one of claims 1 to 8, wherein R5 is selected from hydrogen, hydroxyl and THP.
10. A compound of formula (Ib) according to any one of claims 1 to 9, wherein R4 and R6 are each independently selected from hydrogen, Ci-4alkyl, hydroxyl, halogen and O-Ci-4alkyl.
11. A compound of formula (Ib) according to any one of claims 1 to 10, wherein R4 and R6 are each independently selected from hydrogen, methyl and ethyl.
12. A compound of formula (Ib) according to any one of claims 1 to 11, wherein R3 is selected from hydrogen, hydroxyl, halogen, O-benzyl, OCi-4alkyl and CF3.
13. A compound of formula (Ib) according to any one of claims 1 to 12, wherein R3 is selected from hydrogen, hydroxyl, and O-benzyl.
14. A compound of formula (Ib) according to any one of claims
when Y is O, R5 is hydroxyl or THP and R8 and R9 together form
Figure imgf000109_0003
one of R1, R3, R4, R6, R7, R10 and R1 ' is not hydrogen.
15. A compound according to any one of claims 1 to 14 selected from the group consisting of:
Figure imgf000110_0001
Figure imgf000111_0001
Figure imgf000112_0001
16. A pharmaceutical composition comprising one or more compounds of formula (Ib) according to any one of claims 1 to 15, or a prodrug thereof, together with a pharmaceutically acceptable adjuvant, diluent or carrier.
17. A method of treating or preventing a disease in a vertebrate, the method comprising administering to a vertebrate in need thereof an effective amount of a compound of formula (Ib) according to any one of claims 1 to 15, or a prodrug thereof, or a composition according to claim 16, or a compound of formula (1) or (2):
Figure imgf000112_0002
(1) (2) wherein
Y is O or S;
R1 and R2 are each independently selected from hydrogen, hydroxyl, halogen, Ci- 4alkyl, haloCMalkyl, hydroxyCMalkyl, O-Ci-4alkyl, OC(O)-C Malkyl, C(O)-C Malkyl, CO2H, and CO(O)-CMalkyl; R3-R6 are each independently selected from hydrogen, hydroxyl, halogen, Ci-4alkyl,
C3-6cycloalkyl, haloCi-4alkyl, hydroxyCi-4alkyl, O-Ci-4alkyl, O-Ci-4alkyl-CO2R, O-C3-6cycloalkyl, O-C3-6heterocycloalkyl, O-C3-6heteroaryl, N(R)2C Malkyl, N(R)3Ci- 4alkyl, O-C1-4alkyl-N(R)2, O-CMalkyl-N(R)3i C6-i0aryl, Q-C6-I0 aryl, O-Ci-4alkyl-C6-,0aryl, O-CMalkyl-C6-10heterocycloalkyl, O-Ci-4alkyl-C6-10heteroaryl, 0-C(O)-C 1-4alkyl, O- CON(R)2, CON(R)2, CO2R, C^alkanoyloxymethyl, -P(O)(OH)(OCMalkyl), -P(O)(OC1-
Figure imgf000113_0001
and
Figure imgf000113_0002
d with one or more of hydrogen, halogen, hydroxyl, C1-4alkyl, O-C1-4alkyl, O-C3.6cycloalkyl, haloCi-4alkyl, hydroxyCi-4alkyl, O-C1-4alkyl-CO2R, O-C^cycloalkyl, O-C^heterocycloalkyl, 0-C3- eheteroaryl, N(R)2Ci-4alkyl, N(R)3C1-4alkyl, O-C1-4alky 1-N(R)2, O-C1-4alkyl-N(R)3, C6. loaryl, 0-C6-I0 aryl, 0-C1-4alkyl-C6-1oaryl, O-C1-4alkyl-C6-10heterocycloalkyl, O-Ci-4alkyl- C6-10heteroaryl, 0-CON(R)2, CON(R)2, CO2R, C1-4alkanoyloxymethyl, -P(O)(OH)(OCi- 4alkyl), gar, O-benzyl, CHO, CO2H, and OC(O)-CMalkyl, or
Figure imgf000113_0003
R7-Rπ are each independently selected from hydrogen, halogen, hydroxyl,
Ci-4alkyl, O-Ci-4alkyl, 0-C3-6CyClOaIlCyI, haloC1-4alkyl, hydroxyCi.4alkyl, O-Ci-4alkyl- CO2R, O-C3-6cycloalkyl, O-C3-6heterocycloalkyl, O-C3-6heteroaryl, N(R)2C 1-4alkyl, N(R)3C1-4alkyl, O-Ci-4alkyl-N(R)2, O-C,.4alkyl-N(R)3, C6-10aryl, O-C6-i0 aryl, O-Ci-4alkyl- C6-i0aryl, O-d^alkyl-C^ioheterocycloalkyl, O-Ci-4alkyl-C6-10heteroaryl, 0-CON(R)2, CON(R)2, CO2R, d^alkanoyloxymethyl, -P(O)(OH)(OC1-4alkyl), -P(O)(OCi-4alkyl)2, O- sugar, O-benzyl, CHO, CO2H, and OC(O)-C1-4alkyl, or one or more of R7 and R8, R8 and R9, R9 and R10, and R10 and R11 together form
Figure imgf000113_0004
^—^ is a C6-iOaryl group optionally substituted with one or more of halogen, hydroxyl, Ci-4alkyl, O-Ci-4alkyl, 0-C3-6CyClOaIlCyI, haloCi-4alkyl, hydroxyCi-4alkyl, O-Ci-4alkyl-
CO2R, O-C3-6cycloalkyl, O-C3-6heterocycloalkyl, O-C3-6heteroaryl, N(R)2C 1-4alkyl,
N(R)3C1-4alkyl, O-C1-4alkyl-N(R)2, O-Ci-4alkyl-N(R)3, C6-10aryl, O-C6.i0 aryl, O-CMalkyl-
C6-ioaryl, O-d^alkyl-Cό-ioheterocycloalkyl, O-Ci-4alkyl-C6.10heteroaryl, Q-CON(R)2, CON(R)2, CO2R, C]-4alkanoyloxymethyl, -P(O)(OH)(OCMalkyl), -P(O)(OCi-4alkyl)2, O- sugar, O-benzyl, CHO, CO2H, and OC(O)-C1-4alkyl; or a prodrug thereof, or a pharmaceutically acceptable salt thereof, wherein the disease is selected from Type II diabetes, obesity, hyperlipidemia, cardiovascular disease, anti-neoplastic diseases and tumours, inflammatory conditions and neurodegenerative diseases.
18. The method according to claim 17, wherein the disease is selected from Type II diabetes, obesity, hyperlipidemia, and cardiovascular disease.
19. The method according to claim 17 or 18, wherein the cardiovascular disease is selected from the group consisting of coronary and ischemic heart disease, atherosclerosis and peripheral vascular disease.
20. The method according to claim 17, wherein the anti-neoplastic diseases and tumours are selected from the group consisting of control of cell growth, cell differentiation, motility and apoptosis, neuroblastoma and breast cancer.
21. The method according to claim 17, wherein the inflammatory conditions are selected from the group consisting of inflammatory bowel diseases, psoriasis, chronic inflammatory airway disease, asthma and rheumatoid arthritis.
22. The method according to claim 17, wherein the neurodegenerative diseases are selected from Parkinson's disease and Alzheimer's disease.
23. A method for identifying a PPAR agonist, the method comprising: determining ligand-receptor interactions of a candidate compound with at least two structurally distinct docking templates; comparing the ligand-receptor interactions of the candidate compound with the interactions of a known PPAR agonist; and thereby determining whether a candidate compound is a PPAR agonist.
24. The method according to claim 23, wherein the PPAR agonist is a PPAR-γ agonist.
25. The method according to claim 23, wherein the PPAR agonist is a PPAR-α agonist.
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