WO2010012037A1 - 6-substituted isoflavonoid compounds and uses thereof - Google Patents
6-substituted isoflavonoid compounds and uses thereof Download PDFInfo
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- WO2010012037A1 WO2010012037A1 PCT/AU2009/000973 AU2009000973W WO2010012037A1 WO 2010012037 A1 WO2010012037 A1 WO 2010012037A1 AU 2009000973 W AU2009000973 W AU 2009000973W WO 2010012037 A1 WO2010012037 A1 WO 2010012037A1
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- A61K31/00—Medicinal preparations containing organic active ingredients
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- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
- A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
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- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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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
Definitions
- the present invention relates to 6-substituted isoflavonoid compounds and compositions comprising same.
- the invention further relates to the use of 6-substituted isoflavonoid compounds for the treatment of various diseases and conditions.
- NSAIDs non-steroidal anti-inflammatory drugs
- their use has always been limited by their gastrointestinal side effects, such as gastric ulceration, perforation and bleeding, as well as acute renal failure and hypertension.
- These shortcomings were met in part by the development of agents which selectively inhibited the inflammatory process driven by COX-2, but left the homeostatic functions managed by COX-I unaffected - the COXIBs.
- the theory was that the prostaglandin PGE 2 produced in response to COX-I, and which provided gut protection remained, but the PGE 2 synthesised in response to COX-2 produced as part of the inflammatory response was suppressed.
- the cardiovascular risks associated with selective COX-2 inhibition appear to be due to the disruption of the homeostasis between COX-2-induced prostacyclin (PGI 2 ), which is anti-thrombotic and vasodilatory, and COX-I -induced TXA 2 , which is prothrombotic and vasoconstrictory (Caughey et al. 2001).
- PGI 2 COX-2-induced prostacyclin
- TXA 2 promotes and PGI 2 prevents the initiation and progression of atherogenesis through control of platelet activation and leukocyte- endothelial cell interaction (Kobayashi et al. 2004). This homeostasis is disturbed to varying degrees whenever the COX pathway is inhibited, regardless of COX isotype selectivity, as has been well demonstrated with the increased cardiovascular and gastrointestinal side effects associated with all NSAIDs.
- 6-substituted isoflavonoid compounds possess useful anti-inflammatory activity.
- certain 6-substituted isoflavanoid compounds may also provide other therapeutic benefits.
- the present invention provides a compound of the general formula (I):
- R 2 , R 3 and R 4 are independently selected from the group consisting of: hydrogen, hydroxy, ORg, OC(O)Rg, OSi(Ri 0 ) 3 , Ci-Ci 0 alkyl, C 3 -C 7 cycloalkyl, amino, aminoalkyl, aryl, arylalkyl, alkylaryl, thiol, COOH, alkylthio, nitro, cyano, halo, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl and heteroaryl,
- R 7 is selected from the group consisting of: hydrogen, R 9 , C(O)Rg, Si(Ri 0 ) 3 and C 3 -
- R 8 is selected from the group consisting of: hydrogen, Ci-Ci 0 alkyl, C 3 -C 7 cycloalkyl, aryl, arylalkyl, nitro, cyano and halo,
- R 9 is selected from the group consisting of: Ci-Ci 0 alkyl, haloalkyl, aryl, arylalkyl and alkylaryl,
- Ri 0 is independently selected from the group consisting of: Ci-Ci 0 alkyl and aryl,
- Rn and Ri 2 are independently selected from the group consisting of: hydrogen, Ci- Cio alkyl and -Y-CO 2 Ri 3 , or Rn and R] 2 together with the nitrogen to which they are attached form a heterocyclic ring comprising 5, 6 or 7 ring members, the heterocyclic ring being optionally substituted with one or more substituents selected from the group consisting of: Ci-Ci 0 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, COOH, COORi 0 , halo, nitro, cyano and aryl,
- Rn is selected from the group consisting of: hydrogen, C 3 -C 7 cycloalkyl, Ci-Ci 0 alkyl, C 2 -C 6 -alkenyl and C 2 -C 6 alkynyl,
- Y is a hydrocarbon chain having between 1 and 15 carbon atoms which may optionally be interrupted by one or more oxygen, nitrogen or sulfur atoms, n is an integer between 1 and 4, the drawing "—" represents either a single bond or a double bond, and salts thereof.
- the compound of the formula (I) may be selected from the group consisting of:
- the present invention provides a pharmaceutical composition
- a pharmaceutical composition comprising a compound of the formula (I), or a pharmaceutically acceptable salt thereof, as defined in the first aspect, and a pharmaceutically acceptable carrier, diluent and/or excipient.
- the present invention provides a method for the prevention and/or treatment of inflammation and/or an inflammatory disease or disorder in a subject in need thereof, said method comprising administration to the subject of a therapeutically effective amount of a compound of the formula (I), or a pharmaceutically acceptable salt thereof, as defined in the first aspect.
- the present invention provides the use of a compound of the formula (I), or a pharmaceutically acceptable salt thereof, as defined in the first aspect in the manufacture of a medicament for the prevention and/or treatment of inflammation and/or an inflammatory disease or disorder.
- the present invention provides a compound of the formula (I), or a pharmaceutically acceptable salt thereof, as defined in the first aspect, for use in the prevention and/or treatment of inflammation and/or an inflammatory disease or disorder.
- the present invention provides the use of a compound of the formula (I) or a pharmaceutically acceptable salt thereof, as defined in the first aspect, as an antioxidant.
- the present invention provides a method for modulation of the immune system in a subject, said method comprising administration to the subject of a therapeutically effective amount of a compound of the formula (I), or a pharmaceutically acceptable salt thereof, as defined in the first aspect.
- the present invention provides the use of a compound of the formula (I), or a pharmaceutically acceptable salt thereof, as defined in the first aspect in the manufacture of a medicament for modulation of the immune system.
- the present invention provides the use of a compound of the formula (I) or a pharmaceutically acceptable salt thereof, as defined in the first aspect, for modulation of the immune system.
- the modulation of the immune system may comprise inhibition or suppression of an immune response.
- the modulation of the immune system may comprise suppression of activation or production of T-cells and/or B-cells.
- the present invention provides a method for inhibiting the proliferation of cells, said method comprising contacting the cells with a compound of the formula (I), or a pharmaceutically acceptable salt thereof, as defined in the first aspect.
- the present invention provides the use of a compound of the formula (I), or a pharmaceutically acceptable salt thereof, as defined in the first aspect in the manufacture of a medicament for inhibiting the proliferation of cells.
- the present invention provides a compound of the formula (I), or a pharmaceutically acceptable salt thereof, as defined in the first aspect, for use in inhibiting the proliferation of cells.
- the present invention provides a method for the prevention and/or treatment of cancer in a subject in need thereof, said method comprising administration to the subject of a therapeutically effective amount of a compound of the formula (I), or a pharmaceutically acceptable salt thereof, as defined in the first aspect.
- the present invention provides the use of a compound of the formula (I), or a pharmaceutically acceptable salt thereof, as defined in the first aspect in the manufacture of a medicament for the prevention and/or treatment of cancer.
- the present invention provides a compound of the formula (I), or a pharmaceutically acceptable salt thereof, as defined in the first aspect, for use in the prevention and/or treatment of cancer.
- the cancer may be selected from the group consisting of: ovarian cancer, leukaemia, prostate cancer, colorectal cancer, pancreatic cancer, glioma, melanoma and lung cancer.
- the present invention provides a method for the prevention and/or treatment of cardiovascular disease in a subject in need thereof, said method comprising administration to the subject of a therapeutically effective amount of a compound of the formula (I), or a pharmaceutically acceptable salt thereof, as defined in the first aspect.
- the present invention provides the use of a compound of the formula (I), or a pharmaceutically acceptable salt thereof, as defined in the first aspect in the manufacture of a medicament for the prevention and/or treatment of cardiovascular disease.
- the present invention provides a compound of the formula (I), or a pharmaceutically acceptable salt thereof, as defined in the first aspect, for use in the prevention and/or treatment of cardiovascular disease.
- treatment refers to any and all uses which remedy a condition, disease, disorder or symptoms thereof, or otherwise prevent, hinder or reverse the progression of a condition, disease, disorder or symptoms thereof, in any way whatsoever. Treatment may be for a defined period of time, or provided on an ongoing basis depending on the particular circumstances of any given individual.
- prevent and “prevention” refer to any and all uses which prevent the establishment or onset of a condition, disease, disorder or symptoms thereof in any way whatsoever.
- the term "therapeutically effective amount” includes within its meaning a non-toxic amount of a compound of formula (I) sufficient to provide the desired therapeutic effect. The exact amount will vary from subject to subject depending on the age of the subject, their general health, the severity of the disorder being treated and the mode of administration. It is therefore not possible to specify an exact “therapeutically effective amount”, however one skilled in the art would be capable of determining a “ therapeutically effective amount” by routine trial and experimentation.
- the term “salts thereof is understood to include acid addition salts, anionic salts and zwitterionic salts, and in particular pharmaceutically acceptable salts.
- salts include, but are not limited to, those formed from: acetic, ascorbic, aspartic, benzoic, benzenesulfonic, citric, cinnamic, ethanesulfonic, fumaric, glutamic, glutaric, gluconic, hydrochloric, hydrobromic, lactic, maleic, malic, methanesulfonic, naphthoic, hydroxynaphthoic, naphthalenesulfonic, naphthalenedisulfonic, naphthaleneacrylic, oleic, oxalic, oxaloacetic, phosphoric, pyruvic, p-toluenesulfonic, tartaric, trifluoroacetic, triphenylacetic, tricarballylic, salicylic, sulphuric, sufamic, sulfanilic and succinic acid.
- Ci-C] 0 alkyl is taken to include straight chain and branched chain monovalent saturated hydrocarbon groups having 1 to 10 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, secbutyl, tertiary butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl and the like.
- Ci-C 6 alkyl is taken to include straight chain and branched chain monovalent saturated hydrocarbon groups having 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, secbutyl, tertiary butyl, pentyl, hexyl and the like.
- C 2 -C 6 alkenyl is taken to include straight chain and branched chain monovalent hydrocarbon radicals having 2 to 6 carbon atoms and at least one carbon-carbon double bond, such as vinyl, propenyl, 2-methyl-2- propenyl, butenyl, pentenyl and the like.
- the alkenyl group may contain from 2 to 4 carbon atoms.
- C 2 -C 6 alkynyl is taken to include straight chain and branched chain monovalent hydrocarbon radicals having 2 to 6 carbon atoms and at least one carbon-carbon triple bond, such as ethynyl, propynyl, butynyl, pentynyl, hexynyl and the like.
- the alkynyl group may contain from 2 to 4 carbon atoms.
- C 3 -C 7 cycloalkyl is taken to include cyclic alkyl groups having 3 to 7 carbon atoms such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.
- alkyl, alkenyl, alkynyl or cycloalkyl group may optionally be substituted by one or more of: acyloxy, hydroxy, halo, alkoxy, nitro or cyano.
- aryl is taken to include monovalent aromatic radicals having between 6 and 30 carbon atoms.
- the aryl group may be selected from the group consisting of: phenyl, biphenyl, naphthyl, anthracenyl and phenanthrenyl.
- the aryl group may be unsubstituted or optionally substituted by one or more of: Ci-C 6 alkyl, halo, acyloxy, hydroxy, alkoxy, silyloxy, nitro or cyano.
- heteroaryl is taken to include monovalent aromatic radicals having between 1 and 12 atoms, wherein 1 to 6, or 1 to 5, or 1 to 4, or 1 to 3, or 1 or 2 atoms are heteroatoms selected from nitrogen, oxygen and sulfur.
- the heteroaryl group may be selected from the group consisting of: furanyl, quinazolinyl, quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, pyrazolyl, tetrazolyl, oxazolyl, isoxazolyl, isothiazolyl, thiazolyl, thienyl, imidazolyl, pyrazinyl, pyridazinyl, pyrimidinyl, pyridyl, triazolyl, benzothiazolyl, benzisothiazolyl, benzoxazolyl, benzisoxazolyl, benzimidazolyl and triazinyl.
- the heteroaryl group may be unsubstituted or optionally substituted by one or more of: alkyl, halo, acyloxy, hydroxy, halo, alkoxy, silyloxy, nitro or cyano.
- halo is taken to include fluoro, chloro, bromo and iodo.
- aminoalkyl is taken to include “alkyl” as defined above, wherein one or more hydrogen atoms have been replaced by one or more amino groups. One or two hydrogen atoms may be replaced by one or two amino groups.
- the aminoalkyl group may be aminomethyl, aminoethyl, aminopropyl and the like.
- arylalkyl is taken to include an "aryl” group as defined above attached to the molecule via a divalent alkylene group.
- arylalkyl groups include benzyl and phenethyl and the like.
- alkylene is taken to include a divalent group derived from a straight or branched chain saturated hydrocarbon group by the removal of two hydrogen atoms.
- Representative alkylene groups include methylene, ethylene, propylene, isobutylene, and the like.
- alkylaryl is taken to include an “alkyl” group as defined above attached to the molecule via a divalent arylene group.
- alkylaryl groups include tolyl, ethylphenyl, propylphenyl, butylphenyl and the like.
- arylene is taken to include an aromatic ring system derived from an aryl group as defined above by the removal of two hydrogen atoms.
- haloalkyl is taken to include monohalogenated, dihalogenated and up to perhalogenated alkyl groups.
- Preferred perhaloalkyl groups are trifluoromethyl and pentafluoroethyl.
- Figure 1 Effect of compound (1) (denoted as NV- 17) on joint scores in rat adjuvant-induced arthritis.
- Figure 2 Effect of incubation with compound (1) on splenocyte proliferation and cytokine production.
- Figure 3 Effect of incubation with compound (1) on splenocyte proliferation and cytokine production.
- Figure 4 Effect of compound (1) (denoted as NV- 17) on the aortic contractility induced by noradrenaline.
- Figure 5 Effect of compound (3) (denoted as NV- 124) on the aortic contractility induced by noradrenaline.
- the present invention provides a compound of the general formula (I):
- R 2 , R 3 and R 4 are independently selected from the group consisting of: hydrogen, hydroxy, ORg, OC(O)Rg, OSi(Ri 0 ) 3 , CrC 10 alkyl, C 3 -C 7 cycloalkyl, amino, aminoalkyl, aryl, arylalkyl, alkylaryl, thiol, COOH, alkylthio, nitro, cyano, halo, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl and heteroaryl,
- R 6 Is R 11 (R 12 )N(CH 2 V,
- R 7 is selected from the group consisting of: hydrogen, R 9 , C(O)Rg, Si(Ri O ) 3 and C 3 - C 7 cycloalkyl
- R 8 is selected from the group consisting of: hydrogen, Ci-Ci 0 alkyl, C 3 -C 7 cycloalkyl, aryl, arylalkyl, nitro, cyano and halo,
- R 9 is selected from the group consisting of: C]-Ci 0 alkyl, haloalkyl, aryl, arylalkyl and alkylaryl,
- Rio is independently selected from the group consisting of: C 1 -Ci 0 alkyl and aryl,
- Rn and Ri 2 are independently selected from the group consisting of: hydrogen, Ci- Cio alkyl and -Y-CO 2 Ri 3 , or Rn and Ri 2 together with the nitrogen to which they are attached form a heterocyclic ring comprising 5, 6 or 7 ring members, the heterocyclic ring being optionally substituted with one or more substituents selected from the group consisting of: Ci-Ci 0 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, COOH, COOR !0 , halo, nitro, cyano and aryl,
- Ri 3 is selected from the group consisting of: hydrogen, C 3 -C 7 cycloalkyl, Ci-C 1 O alkyl, C 2 -C 6 -alkenyl and C 2 -C 6 alkynyl,
- Y is a hydrocarbon chain having between 1 and 15 carbon atoms which may optionally be interrupted by one or more oxygen, nitrogen or sulfur atoms, n is an integer between 1 and 4, the drawing " ⁇ " represents either a single bond or a double bond, and salts thereof.
- R 2 , R 3 and R 4 may be independently selected from the group consisting of: hydrogen, C 1 -C 10 alkyl, halo, hydroxy, OR 9 , OC(O)Rg and OSi(R 10 ) 3 .
- at least one of R 2 , R 3 and R 4 is hydroxy
- R 2 , R 3 and R 4 are independently selected from the group consisting of: hydrogen and hydroxy, wherein at least two of R 2 , R 3 and R 4 are hydrogen and the remaining substituent is hydroxy.
- the hydroxy substituent (when present) may be located at the para position.
- R 7 may be selected from the group consisting of: hydrogen, C(O)Rg and C 1 -C 1O alkyl.
- R 8 may be selected from the group consisting of: hydrogen, Ci-Ci 0 alkyl, aryl, arylalkyl and halo.
- R 9 may be selected from the group consisting of: C]-Ci 0 alkyl, haloalkyl and aryl.
- Rio may be Ci-Ci 0 alkyl.
- Rn and Ri 2 may be independently selected from the group consisting of: -Y- CO 2 Ri 3 , hydrogen and Ci-Ci 0 alkyl, or Ri 1 and Ri 2 together with the nitrogen to which they are attached form a heterocyclic ring comprising 5 or 6 ring members, the heterocyclic ring being optionally substituted with one or more substituents selected from the group consisting of: Ci-Ci 0 alkyl, COOH, COORi 0 and halo.
- Y may be a hydrocarbon chain having between 1 and 10, 1 and 9, 1 and 8, 1 and 7 or 1 and 6 carbon atoms.
- Ri 3 may be Ci-Ci 0 alkyl.
- n may be 1, 2 or 3.
- R 2 , R 3 and R 4 may be independently selected from the group consisting of: hydrogen, hydroxy and OR 9 .
- R 7 may be selected from the group consisting of: hydrogen and Ci-C 6 alkyl.
- R 8 may be selected from the group consisting of: hydrogen, Ci-Ci 0 alkyl and halo.
- R 9 may be selected from the group consisting of: CpC 6 alkyl and haloalkyl.
- Ri 0 may be Ci-C 6 alkyl.
- Rn and Ri 2 may be independently selected from the group consisting of: -Y- CO 2 Ri 3, hydrogen and Ci-C 6 alkyl, or Rn and Ri 2 together with the nitrogen to which they are attached form a heterocyclic ring comprising 5 or 6 ring members, the heterocyclic ring being, optionally substituted with one or more substituents selected from the group consisting of: C]-Ci 0 alkyl, COOH and halo.
- Y may be a hydrocarbon chain having between 1 and 5 carbons.
- Ri 3 may be Ci-C 6 alkyl.
- n may be 1 or 2.
- R 2 , R 3 and R 4 may be independently selected from the group consisting of: hydrogen, hydroxy and OMe.
- R 7 may be selected from the group consisting of: hydrogen and methyl.
- R 8 may be selected from the group consisting of: hydrogen and Ci-C 6 alkyl.
- R 9 may be Ci-C 6 alkyl.
- Rn and Ri 2 may be independently selected from the group consisting of: -Y- CO 2 Ri 3 , hydrogen and methyl, or Rn and Ri 2 together with the nitrogen to which they are attached form a heterocyclic ring comprising 5 ring members, the heterocyclic ring being optionally substituted with a substituent selected from the group consisting of: methyl, COOH and halo.
- Y may be a hydrocarbon chain having 1 or 2 carbon atoms.
- Ri 3 may be selected from the group consisting of methyl, ethyl or propyl, n is 1.
- R 2 , R 3 and R 4 are independently selected from the group consisting of: hydrogen, hydroxy, OR 9 , OC(O)R 9 and OSi(Ri 0 ) 3
- R 7 is selected from the group consisting of: hydrogen, C(O)R 9 and C 1 -Ci 0 alkyl
- R 8 is selected from the group consisting of: hydrogen, Ci-Ci 0 alkyl, aryl, arylalkyl, and halo
- R 9 is selected from the group consisting of: C]-Ci 0 alkyl, haloalkyl and aryl
- Ri 0 is Ci-C] 0 alkyl
- Rn and Rj 2 are independently selected from the group consisting of: -Y-CO 2 Ri 3 , hydrogen and Ci-C) 0 alkyl, or Rn and Ri 2 together with the nitrogen to which they are attached form a heterocyclic ring comprising 5 or 6 ring members, the heterocyclic ring being optionally substituted with one or
- R 2 , R 3 and R 4 are independently selected from the group consisting of: hydrogen and hydroxy, wherein at least one of R 2 , R 3 and R 4 is hydroxy, R 7 is selected from the group consisting of: hydrogen and Ci-Ci 0 alkyl, R 8 is selected from the group consisting of: hydrogen, Ci-Ci 0 alkyl and halo, Rn and Ri 2 are independently selected from the group consisting of: -Y-CO 2 Ri 3 , hydrogen and Ci-Ci 0 alkyl or Rn and Ri 2 together with the nitrogen to which they are attached form a heterocyclic ring comprising 5 ring members, the heterocyclic ring being optionally substituted with one or more substituents selected from the group consisting of: Ci-Ci 0 alkyl, COOMe and COOH, Y is a hydrocarbon chain having between 1 and 6 carbons, Ri 3 is C]-C 6 alkyl and n is 1 or 2.
- R 2 , R 3 and R 4 are independently selected from the group consisting of: hydrogen and hydroxy, wherein at least one of R 2 , R 3 and R 4 is hydroxy, R 7 is selected from the group consisting of: hydrogen and Ci-C 6 alkyl, R 8 is selected from the group consisting of: hydrogen and Ci-C 6 alkyl, Rn and Ri 2 are independently selected from the group consisting of: -Y-CO 2 Ri 3 , hydrogen and Ci-C 6 alkyl or Rn and R n together with the nitrogen to which they are attached form a heterocyclic ring comprising 5 ring members, the heterocyclic ring being optionally substituted with one or more substituents selected from the group consisting of: C]-C 6 alkyl, COOH and COOMe, Y is a hydrocarbon chain having between 1 and 4 carbons, R 13 is Ci-C 6 alkyl, n is 1 or 2 and the double bond at position 3 is present.
- R 2 , R 3 and R 4 are independently selected from the group consisting of: hydrogen and hydroxy, wherein at least one of R 2 , R 3 and R 4 is hydroxy, R 7 is hydrogen, R 8 is selected from the group consisting of: hydrogen and Ci-C 6 alkyl, Rn and Ri 2 are independently selected from the group consisting of: -Y-CO 2 Ri 3 , hydrogen and Q- C 6 alkyl or Rn and Ri 2 together with the nitrogen to which they are attached form a heterocyclic ring comprising 5 ring members, the heterocyclic ring being optionally substituted with one or two substituents selected from the group consisting of: Ci-C 6 alkyl, COOH and COOMe, Y is a hydrocarbon chain having between 1 and 4 carbons, Ri 3 is C 1 - C 6 alkyl, n is 1 or 2 and the double bond at position 3 is present.
- R 2 , R 3 and R 4 are independently selected from the group consisting of: hydrogen and hydroxy, wherein at least one of R 2 , R 3 and R 4 is hydroxy, R 7 is hydrogen, R 8 is hydrogen, Rn and Ri 2 are independently selected from the group consisting of: -Y-CO 2 Ri 3, hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, t- butyl and s-butyl or Rn and Ri 2 together with the nitrogen to which they are attached form a heterocyclic ring comprising 5 ring members, the heterocyclic ring being optionally substituted with one or two substituents selected from the group consisting of: methyl, ethyl and COOH, Y is a hydrocarbon chain having between 1 and 3 carbons, Ri 3 is methyl, ethyl, isopropyl or propyl, n is 1 or 2 and the double bond at position 3 is present.
- R 2 , R 3 and R 4 are independently selected from the group consisting of: hydrogen and hydroxy, wherein at least two of R 2 , R 3 and R 4 are hydrogen and the remaining substituent is hydroxy, R 7 is hydrogen, R 8 is hydrogen, Ri ⁇ and Ri 2 are independently selected from the group consisting of: -Y-CO 2 Ri 3 , hydrogen, methyl, ethyl, n-propyl and isopropyl or Ri i and Ri 2 together with the nitrogen to which they are attached form a heterocyclic ring comprising 5 ring members, the heterocyclic ring being optionally substituted with COOH, Y is -CH 2 - or -CH 2 CH 2 -, Ri 3 is methyl, ethyl, isopropyl or propyl, n is 1 or 2 and the double bond at position 3 is present.
- R 2 , R 3 and R 4 are independently selected from the group consisting of: hydrogen and hydroxy, wherein at least two of R 2 , R 3 and R 4 are hydrogen and the remaining substituent is hydroxy which is located in the para position, R 7 is hydrogen, R 8 is hydrogen, Rn and R] 2 are independently selected from the group consisting of: -Y-CO 2 R] 3 , hydrogen, methyl, ethyl, n-propyl and isopropyl or Rn and Ri 2 together with the nitrogen to which they are attached form a heterocyclic ring comprising 5 ring members, the heterocyclic ring being optionally substituted with COOH, Y is -CH 2 - or - CH 2 CH 2 -, Ri 3 is methyl, ethyl, isopropyl or propyl, n is 1 or 2 and the double bond at position 3 is present.
- R 2 , R 3 and R 4 are independently selected from the group consisting of: hydrogen and hydroxy, wherein at least two of R 2 , R 3 and R 4 are hydrogen and the remaining substituent is hydroxy which is located in the para position, R 7 is hydrogen, R 8 is hydrogen, R 11 and Ri 2 are independently selected from the group consisting of: -Y-CO 2 Ri 3 , hydrogen, methyl, ethyl, n-propyl and isopropyl, Ri 3 is methyl, ethyl, isopropyl or n-propyl, Y is -CH 2 -, n is 1 and the double bond at position 3 is present.
- the compound of the formula (I) may be a compound wherein the pendant phenyl ring located at the 3-position of the benzopyran ring is less activated than the phenyl ring of the actual benzopyran moiety.
- the compounds of formula (I) may have one or more chiral centres.
- the present invention includes all enantiomers and diastereoisomers, as well as mixtures thereof in any proportions.
- the invention also extends to isolated enantiomers or pairs of enantiomers. Enantiomers and diastereoisomers may be separated according to methods well known to those skilled in the art.
- a compound of the formula (X) or (Xl) may be treated with an appropriately functionalised amino compound in the presence of formaldehyde to yield a compound of formula (I) having an amino-containing substituent at the 6-position.
- an appropriately functionalised amino compound in the presence of formaldehyde to yield a compound of formula (I) having an amino-containing substituent at the 6-position.
- the ring cyclisation reactions of compounds of formula (C) may be conveniently performed with methanesulfonyl chloride to give compounds of formula (D).
- the reduction reaction to isoflavanols of formula (E) may be carried out with Pd-C or Pd- alumina in an alcoholic solvent in the presence of hydrogen. Dehydration may be effected with acid or P 2 O 5 for example to afford compounds of the formula (Xl).
- Hydroxy protecting groups include, carboxylic acid esters, e.g. acetate esters, aryl esters such as benzoate, acetals/ketals such as acetonide and benzylidene, ethers such as ortho-benzyl and methoxy benzyl ethers, tetrahydropyranyl ethers and silyl ethers such as /ert-butyldimethyl silyl ether.
- Protecting groups can be removed by, for example, acid or base catalysed hydrolysis or reduction, for example, hydrogenation.
- Silyl ethers may require hydrogen fluoride or tetrabutylammonium fluoride to be cleaved.
- Compounds of the invention include the following:
- the compounds of formula (I) are useful in the prevention and/or treatment of inflammation and inflammatory diseases or disorders.
- inflammatory diseases and disorders include, but are not limited to: conditions associated with high estrogen levels, psoriasis and other inflammatory diseases of the skin, inflammatory lesions, fibromyalgia, sarcoidosis, systemic sclerosis, Alzheimer's disease, proliferative retinopathy, hepatitis, arthritis (including for example, osteoarthritis), inflammatory bowel disease (including, for example, forms of colitis such as ulcerative colitis and Crohn's disease), diverticulitis, ulcerative proctitis, autoimmune disorders (including, for example, systemic lupus erythematosis, rheumatoid arthritis, glomerulonephritis and Sjogren's syndrome), asthma, diseases and disorders involving pulmonary inflammation and atherosclerosis.
- the compounds of formula (I) may also be useful for the prevention and/or treatment of pain, oedema and
- Compounds of formula (I) are advantageous in the prevention and/or treatment of inflammation, inflammatory diseases and disorders in that at physiologically relevant concentrations, they are not associated with adverse cardiovascular events, as is the case with a number of other anti-inflammatory drugs currently in use.
- the compounds of formula (I) demonstrate cardioprotective effects and hence may be suitable for the prevention and/or treatment of cardiovascular diseases, including but not limited to: myocardial infarction, atherosclerosis, cerebrovascular disease, hypertension, angina pectoris, ischemia, coronary artery disease, congestive heart failure and blood vessel diseases.
- the compounds of formula (I), and pharmaceutical compositions comprising same may be used in combination with, or include one or more other therapeutic agents, for example other anti-inflammatory agents, anticholinergic agents (particularly Ml, M2, M1/M2 or M3 receptor antagonists), ⁇ 2-adrenoreceptor agonists, antiinfective agents (e.g. antibiotics, antivirals), or antihistamines.
- Combinations may comprise a compound or compounds of formula (I) or pharmaceutically acceptable salts, solvates or physiologically functional derivatives thereof, together with a corticosteroid and/or an anticholinergic and/or a PDE-4 inhibitor.
- Suitable anti-inflammatory agents include corticosteroids and NSAIDs.
- Suitable corticosteroids which may be used in combination with the compounds of the formula (I) are those oral and inhaled corticosteroids and their pro-drugs which have anti- inflammatory activity.
- Examples include methyl prednisolone, prednisolone, dexamethasone, fluticasone propionate, 6 ⁇ ,9 ⁇ ,-difluoro-17 ⁇ -[(2-furanylcarbonyl)oxy]-l l ⁇ -hydroxy- 16 ⁇ -methyl-3-oxo-androsta- 1 ,4-diene- 17 ⁇ -carbothioic acid S-fluoromethyl ester, 6 ⁇ ,9 ⁇ -difluoro- 11 ⁇ -hydroxy- 16 ⁇ -methyl-3-oxo- 17 ⁇ -propionyloxy-androsta- 1 ,4- diene- 17 ⁇ -carbothioic acid S-(2-oxo-tetrahydro-furan-3S-yl) ester, beclomethasone esters (e.g.
- the 17-propionate ester or the 17,21-dipropionate ester the 17-propionate ester or the 17,21-dipropionate ester
- budesonide flunisolide
- mometasone esters e.g. the furoate ester
- triamcinolone acetonide e.g. the furoate ester
- rofleponide e.g. the rofleponide
- ciclesonide e.g. the butixocort propionate.
- Preferred corticosteroids include fluticasone propionate, and 6 ⁇ ,9 ⁇ -difluoro-17 ⁇ -[(2-furanylcarbonyl)oxy]-l 1 ⁇ -hydroxy- 16 ⁇ -methyl- 3-oxo-androsta-l,4-diene-17 ⁇ -carbothioic acid S-fluoromethyl ester, more preferably 6 ⁇ ,9 ⁇ -difluoro-17 ⁇ -[(2-furanylcarbonyl)oxy]-l l ⁇ -hydroxy-16 ⁇ -methyl-3-oxo-androsta- l,4-diene-17 ⁇ -carbothioic acid S-fluoromethyl ester.
- Suitable NSAEDs include sodium cromoglycate, nedocromil sodium, phosphodiesterase (PDE) inhibitors (e.g. theophylline, PDE4 inhibitors or mixed PDE3/PDE4 inhibitors), leukotriene antagonists, inhibitors of leukotriene synthesis, iNOS inhibitors, tryptase and elastase inhibitors, b-2 integrin antogonists and adenosine receptor agonists or antagonists (e.g. adenosine 2 ⁇ agonists), cytokine antagonists (e.g. chemokine antagonists) or inhibitors of cytokine synthesis.
- PDE phosphodiesterase
- the co-administration of compounds may be simultaneous or sequential. Simultaneous administration may be effected by the compounds being in the same unit dose, or in individual and discrete unit doses administered at the same or similar time. Sequential administration may be in any order as required, and may require an ongoing physiological effect of the first or initial compound to be current when the second or later compound is administered, especially where a cumulative or synergistic effect is desired.
- the present inventors have also discovered that compounds of formula (I) possess potent oxidation-inhibiting properties. Accordingly, the compounds of formula (I) may be useful in a wide range of applications as antioxidants, and may conveniently be included in food stuffs or drinks and consumed accordingly.
- the compounds of formula (I) may be useful in the modulation of the immune system.
- the compounds of formula (I) may be immunosuppressive and thus find utility in the treatment of conditions associated with inappropriate immune responses, for example inflammatory bowel disease and rheumatoid arthritis.
- the compounds of formula (I) may be useful in inhibiting the proliferation of cells, and hence beneficial in the prevention and/or treatment of diseases and disorders associated with abberant cell proliferation, for example cancer.
- cancers examples include, but are not limited to: gastrointestinal tumours, cancer of the liver and biliary tract, pancreatic cancer, prostate cancer, testicular cancer, lung cancer, skin cancer (for example melanoma), breast cancer, non-melanoma skin cancer (for example basal cell carcinoma and squamous cell carcinoma), ovarian cancer, uterine cancer, cervical cancer, cancer of the head and neck, bladder cancer, sarcomas and osteosarcomas, Kaposi sarcoma, AEDS-related Kaposi sarcoma, renal carcinoma, leukaemia, colorectal cancer and glioma.
- the cancer may be a primary or secondary cancer.
- chemotherapeutic and other anti-cancer agents include: taxol, fluorouracil, cisplatin, oxaliplatin, ⁇ -interferon, vincristine, vinblastine, angioinhibins, doxorubicin, bleomycin, mitomycin C, phenoxodiol, NV- 128, methramycin, TNP -470, pentosan polysulfate, tamoxifen, LM-609, CM-101 and SU-101.
- the co-administration of compounds of the formula (I) and chemotherapeutic or other anti-cancer agents may be simultaneous or sequential.
- Simultaneous administration may be effected by a compound of the formula (I) being in the same unit dose as a chemotherapeutic or other anti-cancer agent, or the compound of the formula (I) and the chemotherapeutic or other anti-cancer agents may be present in individual and discrete unit doses administered at the same, or at a similar time.
- Sequential administration may be in any order as required, and may require an ongoing physiological effect of the first or initial compound to be current when the second or later compound is administered, especially where a cumulative or synergistic effect is desired.
- Pharmaceutical compositions and routes of administration may be in any order as required, and may require an ongoing physiological effect of the first or initial compound to be current when the second or later compound is administered, especially where a cumulative or synergistic effect is desired.
- the compounds of formula (I) are useful as therapeutic agents in the treatment or prevention of various diseases or conditions in a subject.
- the compounds of formula (I) may be administered to a subject in the form of pharmaceutical compositions.
- Pharmaceutical compositions include those suitable for oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous and intraarticular), inhalation (including use of metered dose pressurised aerosols, nebulisers or insufflators), rectal and topical (including dermal, buccal, sublingual and intraocular) administration.
- the most suitable route may depend upon, for example, the condition and disorder of the recipient.
- compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing one or more compounds of the formula (I) into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association one or more compounds of the formula (I) with a liquid carrier or finely divided solid carrier, or both and then, if necessary, shaping the product into the desired composition.
- an effective dosage of a compound of the formula (I) is expected to be in the range of about O.OOOlmg to about lOOOmg per kg body weight per 24 hours; about O.OOlmg to about 750mg per kg body weight per 24 hours; about O.Olmg to about 500mg per kg body weight per 24 hours; about O.lmg to about 500mg per kg body weight per 24 hours; about O.lmg to about 250mg per kg body weight per 24 hours, or about l.Omg to about 250mg per kg body weight per 24 hours.
- an effective dose range is expected to be in the range of about l.Omg to about 200mg per kg body weight per 24 hours; about l.Omg to about lOOmg per kg body weight per 24 hours; about l.Omg to about 50mg per kg body weight per 24 hours; about 1.Omg to about 25mg per kg body weight per 24 hours; about 5. Omg to about 50mg per kg body weight per 24 hours; about 5. Omg to about 20mg per kg body weight per 24 hours, or about 5. Omg to about 15mg per kg body weight per 24 hours.
- an effective dosage may be up to about 500mg/m 2 .
- an effective dosage is expected to be in the range of about 25 to about 500mg/m 2 , about 25 to about 350mg/m 2 , about 25 to about 300mg/m 2 , about 25 to about 250mg/m 2 , about 50 to about 250mg/m 2 , or about 75 to about 150mg/m 2 .
- compositions suitable for buccal (sublingual) administration include lozenges comprising a compound of the formula (I) in a flavoured base, usually sucrose and acacia or tragacanth; and pastilles comprising a compound of the formula (I) in an inert base such as gelatine and glycerin or sucrose and acacia.
- compositions suitable for oral administration may be presented as discrete units such as gelatine or HPMC capsules, cachets or tablets, each containing a predetermined amount of a compound of formula (I) as a powder, granules, as a solution or a suspension in an aqueous liquid or a non-aqueous liquid, or as an oil-in-water liquid emulsion or a water- in-oil liquid emulsion.
- the compound of formula (I) may also be present as a paste.
- the compound When compounds of the formula (I) are formulated as capsules, the compound may be formulated with one or more pharmaceutically acceptable carriers such as starch, lactose, microcrystalline cellulose, silicon dioxide and/or a cyclic oligosaccaride such as cyclodextrin. Additional ingredients may include lubricants such as magnesium stearate and/or calcium stearate.
- pharmaceutically acceptable carriers such as starch, lactose, microcrystalline cellulose, silicon dioxide and/or a cyclic oligosaccaride such as cyclodextrin.
- Additional ingredients may include lubricants such as magnesium stearate and/or calcium stearate.
- Suitable cyclodextrins include ⁇ -cyclodextrin, ⁇ -cyclodextrin, ⁇ - cyclodextrin, 2-hydroxyethyl- ⁇ -cyclodextrin, 2-hydroxypropyl-cyclodextrin, 3- hydroxypropyl- ⁇ -cyclodextrin and tri-methyl- ⁇ -cyclodextrin.
- the cyclodextrin may be hydroxypropyl- ⁇ -cyclodextrin.
- Suitable derivatives of cyclodextrins include Captisol® a sulfobutyl ether derivative of cyclodextrin and analogues thereof as described in US patent No. 5,134,127.
- Tablets may be prepared by compression or moulding, optionally with one or more accessory ingredients.
- Compressed tablets may be prepared by compressing in a suitable machine the compound of formula (I) in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant (for example magnesium stearate or calcium stearate), inert diluent or a surface active/dispersing agent.
- Moulded tablets may be made by moulding a mixture of the powdered compound of formula (I) moistened with an inert liquid diluent, in a suitable machine.
- the tablets may optionally be coated, for example, with an enteric coating and may be formulated so as to provide slow or controlled release of the compound of formula (I) therein.
- compositions for parenteral administration include aqueous and non-aqueous sterile injectable solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient, and which may include suspending agents and thickening agents.
- a parenteral composition may comprise a cyclic oligosaccaride such as hydroxypropyl- ⁇ -cyclodextrin.
- the compositions may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example saline or water- for-injection, immediately prior to use.
- Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
- Dry powder compositions for topical delivery to the lung by inhalation may, for example, be presented in capsules and cartridges of, for example gelatine, or blisters or for example laminated aluminium foil, for use in an inhaler or insufflator.
- Compositions generally contain a powder mix for inhalation of the one or more compounds of the formula (I) and a suitable powder base (carrier substance) such as lactose or starch. Use of lactose is preferred.
- Each capsule or cartridge may generally contain between 20 ⁇ g-10mg of a compound of formula (I), optionally in combination with another therapeutically active ingredient.
- the compound or compounds of the formula (I) may be presented without excipients.
- Packaging of the composition may be for unit dose or multi- dose delivery.
- compositions suitable for transdermal administration may be presented as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged period of time.
- patches suitably comprise the compound of the formula (I) as an optionally buffered aqueous solution of, for example, 0.1 M to 0.2 M concentration with respect to the compound.
- compositions suitable for transdermal administration may also be delivered by iontophoresis, and typically take the form of an optionally buffered aqueous solution of the active compound.
- Suitable compositions comprise citrate or Bis/Tris buffer (pH 6) or ethano I/water and contain from 0.1 M to 0.2 M of a compound of the formula (I).
- Spray compositions for topical delivery to the lung by inhalation may, for example be formulated as aqueous solutions or suspensions or as aerosols, suspensions or solutions delivered from pressurised packs, such as a metered dose inhaler, with the use of a suitable liquefied propellant.
- suitable propellants include a fluorocarbon or a hydrogen-containing chlorofluorocarbon or mixtures thereof, particularly hydro fluoroalkanes, e.g.
- the aerosol composition may be excipient free or may optionally contain additional composition excipients well known in the art, such as surfactants e.g. oleic acid or lecithin and cosolvents e.g. ethanol.
- Pressurised compositions will generally be retained in a canister (e.g. an aluminium canister) closed with a valve (e.g. a metering valve) and fitted into an actuator provided with a mouthpiece.
- Medicaments for administration by inhalation desirably have a controlled particle size.
- the optimum particle size for inhalation into the bronchial system is usually 1-10 ⁇ m, preferably 2-5 ⁇ m. Particles having a size above 20 ⁇ m are generally too large when inhaled to reach the small airways.
- lactose it will typically be present as milled lactose, wherein not more than 85% of lactose particles will have a MMD of 60-90 ⁇ m and not less than 15% will have a MMD of less than 15 ⁇ m.
- compositions for rectal administration may be presented as a suppository with carriers such as cocoa butter or polyethylene glycol, or as an enema wherein the carrier is an isotonic liquid such as saline.
- Additional components of the compositions may include a cyclic oligosaccaride, for example, a cyclodextrin, as described above, such as hydroxypropyl- ⁇ - cyclodextrin, one or more surfactants, buffer salts or acid or alkali to adjust the pH, isotonicity adjusting agents and/or anti-oxidants.
- compositions suitable for topical administration to the skin preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil.
- Carriers which may be used include Vasoline, lanoline, polyethylene glycols, alcohols, and combination of two or more thereof.
- the compound of the formula (I) is generally present at a concentration of from 0.1% to 5% w/w, or from 0.5% to 2% w/w. Examples of such compositions include cosmetic skin creams.
- the compounds of the formula (I) may be provided in the form of food stuffs, such as being added to, admixed into, coated, combined or otherwise added to a food stuff.
- food stuff is used in its widest possible sense and includes liquid compositions such as drinks, including dairy products and other foods, such as health bars, desserts, etc.
- Food compositions comprising compounds of the formula (I) can be readily prepared according to standard practices.
- compositions for the treatment of the therapeutic indications herein described are typically prepared by admixture of the compounds of the formula (I) with one or more pharmaceutically or veterinary acceptable carriers and/or excipients as are well known in the art.
- the carrier must, of course, be acceptable in the sense of being compatible with any other ingredients in the composition and must not be deleterious to the subject.
- the carrier or excipient may be a solid or a liquid, or both, and is preferably formulated with the compound as a unit-dose, for example, a tablet, which may contain up to 100% by weight of the active compound, preferably from 0.5% to 75% by weight of the compound of the formula (I).
- composition may also be administered or delivered to target cells in the form of liposomes.
- Liposomes are generally derived from phospholipids or other lipid substances, and are formed by mono- or multi-lamellar hydrated liquid crystals that are dispersed in an aqueous medium.
- liposomes used in administering or delivering a composition to target cells are synthetic cholesterol (Sigma), 1 ,2-distearoyl- sn-glycero-3-phosphocholine (DSPC; Avanti Polar Lipids), 3-7V-[(-methoxy poly(ethylene glycol)2000)carbamoyl]-l,2-dimyrestyloxy-propylamine (PEG-cDMA), or 1,2-di- ⁇ - octadecenyl-3-(N,yV-dimethyl)aminopropane (DODMA).
- synthetic cholesterol Sigma
- DSPC 1 ,2-distearoyl- sn-glycero-3-phosphocholine
- PEG-cDMA 3-7V-[(-methoxy poly(ethylene glycol)2000)carbamoyl]-l,2-dimyrestyloxy-propylamine
- DODMA 1,2-di- ⁇ - octadecenyl
- compositions may also be administered in the form of microparticles.
- Biodegradable microparticles formed from polylactide (PLA), polylactide-co-glycolide (PLGA), and ⁇ -caprolactone have been extensively used as drug carriers to increase plasma half life and thereby prolong efficacy (R. Kumar, M., 2000, J. Pharm. Pharmaceut. Sci. 3(2) 234-258).
- compositions may incorporate a controlled release matrix that is composed of sucrose acetate isobutyrate (SABB) and organic solvent or organic solvent mixtures.
- SABB sucrose acetate isobutyrate
- Polymer additives may be added to the vehicle as a release modifier to further increase the viscosity and slow down the release rate.
- a compound of the formula (I) may be added to the SAD3 delivery vehicle to form SAIB solution or suspension compositions.
- SAIB solution or suspension compositions When the formulation is injected subcutaneously, the solvent diffuses from the matrix allowing the S AIB-drug or S AIB-drug-polymer mixtures to set up as an in situ forming depot.
- NFKB is a ubiquitous transcription factor central to cellular responses to stimuli such as stress, proinflammatory cytokines (eg IL-I or TNF- ⁇ ), free radicals, ultraviolet irradiation, and bacterial or viral antigens. Its inhibition provides an anti-inflammatory strategy.
- the assay utilizes a genetically modified THP-I cell line and GeneBLAzer® beta- lactamase technology (Invitrogen Corp).
- the human THP-I monocyte/macrophages contain a stably-transfected beta-lactamase reporter gene under control of the NFkB response element. They respond to stimulation with TNF ⁇ , which leads to activation of the NFkB signaling pathway.
- Co-incubation of cells with TNFa and test material allows quantitative determination of the ability of test material to inhibit TNF ⁇ -stimulated beta- lactamase production.
- An inflammatory index was calculated as the ratio of beta- lactamase product to beta-lactamase substrate.
- THP-I cells were seeded into wells of a 96-well plate (50 x 10 3 cells/well) in the presence of RPMI 1640 medium (70 ⁇ l). TNF ⁇ was added to each well (lO ⁇ l) to give a final concentration of 7.5 ng/ml. Dialyzed bovine serum was added (lO ⁇ l). Test compounds were dissolved in DMSO (10 ⁇ L) (5 wells). Each plate contained a no-cell control (4 wells), a no-serum control (4 wells) and two serum controls. Plates were incubated for 5h at 37°C to allow for NFkB-stimulated beta-lactamase production.
- CCF4-AM LiveBLAzerTM FRET B/G Substrate (CCF4-AM) substrate was then added to the assay.
- CCF4-AM is a F ⁇ rster resonance energy transfer (FRET) based substrate for beta-lactamase developed by Invitrogen Corp. Once CCFA-AM enters a cell, it is converted to negatively charged CCF4 by endogenous esterases. Excitation of this substrate at 409nm leads to efficient FRET between the coumarin and fluorescein moieties, resulting in a green fluorescence detectable at 530nm. The presence of beta-lactamase leads to cleavage of CCF4 and results in a loss of FRET, resulting in a robust blue fluorescent signal detectable at 460nm.
- FRET F ⁇ rster resonance energy transfer
- beta-lactamase a marker of NFkB-promoter activity
- a product to substrate ratio blue/green fluorescence ratio: 460nm/530nm.
- the determination of inflammatory index had a within-plate CV of 2.1% and a between-plate CV of 8.9%.
- compound (1) significantly reduced the promoter activity of NFKB at lO ⁇ M and lOO ⁇ M. It did so in the absence of cytotoxicity.
- Compound (2) was active at the highest concentration only, and again in the absence of cytotoxicity.
- Inflammation involves the recruitment of inflammatory cells from the circulation and their transendothelial migration. This process is predominantly mediated by cellular adhesion molecules, which are expressed on the vascular endothelium and on circulating leukocytes in response to several inflammatory stimuli.
- Vascular cell adhesion molecule- 1 (VCAM-I) induces firm adhesion of inflammatory cells at the vascular surface. Consequently, inhibition of VCAM-I is a potential therapeutic target for the control of inflammation in general and arthritis in particular.
- TNF ⁇ -stimulated endothelial cell activation was assessed by measuring surface expression of cell adhesion molecules with an ELISA method.
- Human arterial endothelial cells (HAEC) in growth medium (Cell Applications Inc.) were seeded into 96-well plates at a density of 10,000 cells per well. Plates were incubated overnight at 37°C in a humidified incubator to allow for cells to become confluent.
- TNF ⁇ (10 ⁇ l, 2ng/ml) was added to each well, which contained 100 ⁇ l of medium.
- Test compounds were diluted in DMSO-containing medium (2.5% DMSO) to give a concentration of 100 and 300 ⁇ M. They were added to wells so that final concentrations were 10 and 30 ⁇ M. DMSO-containing medium alone was added to zero concentration control wells. All samples were measured in quadruplicate (4 wells per treatment).
- Adhesion molecule expression was detected by addition of sheep anti-mouse antibody/ horseradish peroxidase conjugate. Plates were allowed to stand for 30 minutes - monolayers were then washed, and sheep anti-mouse antibody/horseradish peroxidase conjugate (1:500 in lOO ⁇ L HBSS with 10% heat-inactivated human serum and 0.05% Tween 20) was added and left for 30 minutes. After further washing, 150 ⁇ L ABTS substrate (Kirkegaard and Perry Laboratories) was added to each well and allowed to develop for 15 minutes. Optical density was measured at 405nm with an ELISA reader (Titertek Multiscan, Flow Laboratories).
- LTs Leukotrienes
- AA arachidonic acid
- LTs are products of the 5 -lipoxygenase (5-LO) pathway.
- LTs play a role in allergic and inflammatory diseases, amplifying inflammation by causing increased vascular permeability, vasodilation and smooth muscle contraction. In addition, they are potent chemotactic agents.
- inhibition of 5-LO indirectly reduces the expression of TNF ⁇ Inhibition of LTs is an anti-inflammatory strategy.
- the pathway for LTB 4 synthesis involves initial release of AA from phospholipids by a Ca-dependent PLA 2 .
- the free AA is then oxygenated at by 5-LO (requiring enzyme activation by FLAP) to generate an epoxide intermediate (LTA 4 ).
- LTA 4 is then converted to LTB 4 by LTA 4 hydrolase.
- LTB 4 is metabolised (and deactivated) by a cytochrome P- (CYP) 450 ⁇ -hydrolase to produce 20-hydroxy and 20-carboxy metabolites. These metabolites are also measured in the HPLC assay.
- Neutrophils were isolated from citrated human venous blood to >90% purity by centrifugation through Ficoll, dextran sedimentation and lysis of erythrocytes (Boyum 1986). Cells were washed in HEPES buffered Hanks solution (HBHS) and then suspended at 4.5 million cells/mL in HBHS containing 0.1% BSA (HBHS+BSA).
- compound (1) was active in inhibiting the synthesis of LTB 4 and its metabolites.
- the IC 50 for the production of LTB 4 was 4.3 ⁇ M.
- compound (3) was active in inhibiting the synthesis Of LTB 4 and the IC 50 for its production was 5.4 ⁇ M. Whilst it inhibited the production of 20-OH-LTB 4 , the production of 20-COOH-LTB 4 was enhanced .
- Nitric oxide a molecular messenger synthesized by nitric oxide synthase (NOS) from L-arginine and molecular oxygen, is involved in a number of physiological and pathological processes.
- NOS nitric oxide synthase
- Three structurally distinct isoforms of NOS have been identified: neuronal (nNOS), endothelial (eNOS) and inducible (/NOS).
- nNOS neuronal
- eNOS endothelial
- /NOS inducible
- Excess NO produced by /NOS has been implicated in inflammation.
- NO causes apoptosis and dedifferentiation of articular chondrocytes by the modulation of extracellular signal-regulated kinase (ERK), p38 kinase, and protein kinase C (PKC).
- ERK extracellular signal-regulated kinase
- PKC protein kinase C
- eNOS-derived NO also regulates endothelial cell adhesion molecule expression, leukocyte adhesion, and extravasation -significant increases in constitutive expression of adhesion molecules ICAM-I and P-selectin, leukocyte rolling, adhesion, and extravasation were seen in the vasculature of tissues from eNOS knock-out mice compared to their wild-type controls. Accordingly, selective inhibition of /NOS and upregulation of eNOS would be an advantageous as an anti-inflammatory strategy, as well as provide a cardioprotective effect.
- the mouse macrophage cell line RAW 264.7 was cultured in DMEM supplemented with foetal calf serum (FCS), 2mM glutamine and 50U/ml penicillin/streptomycin. Cells were treated with either the compounds of the formula (I) (in 0.025% DMSO) or vehicle alone, and added one hour before 50ng/ml LPS, which induces /NOS and the production of NO. After incubation for 16hrs, culture media was collected. Nitrite concentration is a quantitative indicator of NO production and was determined by the Griess Reaction. Briefly, lOO ⁇ L of Griess reagent was added to 50 ⁇ L of each supernatant in duplicate. The absorbance at 550nm was measured (Molecular Devices, SpectraMax 250 microplate spectrophotometer, CA, USA), and nitrite concentrations were determined against a standard curve of sodium nitrite.
- HCAECs were grown as described above. Because cell viability was less than 100% at 30 and lOO ⁇ M, eNOS experiments were conducted at one concentration (lO ⁇ M). After incubation, total RNA was extracted using TRI reagent (Sigma, St Louis, MO, USA), following the manufacturer's protocol. RNA was quantitated and normalized to lOOng/ ⁇ l using the SYBR Green II assay (Molecular Probes, Eugene, OR, USA) before being reverse transcribed using iScript (Bio-Rad, Hercules, CA, USA).
- eNOS sense 5'- CCA TCT ACA GCT TTC CGG CGC-3' and antisense 5'-CTC TGG GGT GGC CTT CAG CA-3'
- 18S sense 5'-CGG CTA CCA CAT CCA AGG AA-3'and antisense 5'- GCT GGA ATT ACC GCG GCT-3'
- mRNA levels were determined by real-time PCR using iQ SYBR Green Supermix (Bio-Rad) in an iCycler iQ RealTime thermocyler detection system (Bio-Rad Laboratories). The cycling parameters were 95 0 C for 30 seconds, 62°C for 30 seconds, and 72 0 C for 30 seconds for 40 cycles, and real-time data was collected at each cycle. There were six replicates.
- Adjuvant-induced arthritis in genetically susceptible rodents is a well accepted animal model of chronic joint inflammation such as that experienced in rheumatoid arthritis. It is responsive to anti-inflammatory and immunosuppressive agents.
- DA.CD45.1 Male Dark Agouti (DA) strain (DA.CD45.1) rats were fed with either compound (l)-treated feed or placebo-treated feed for seven days prior to the injection of Complete Freund's adjuvant (0.1ml) into the base of the tail. The treated feeds were continued throughout the experiment. Arthritis, which became evident at Day 8 was subjectively scored each day by an operator blinded to the identity of treatments and using a scoring system as follows:
- Air pouches were raised on the dorsum of female Dark Agouti rats, approximately seven weeks of age. To promote the formation of a cellular membrane lining the inside of each pouch, the pouches were maintained by re-inflating on days 2 and 5 after the initial injection of air. On re-inflation, the pouch was first deflated to ensure the needle was positioned correctly, before being re-inflated with 2 mL of sterile air. Using this protocol, the pouches remained inflated until use on day 7, when they were injected with 0.5 ml of either test compound or vehicle control. After 15 min, air pouches were injected with serum-treated zymozan (STZ - 500 ⁇ g).
- STZ - 500 ⁇ g serum-treated zymozan
- Table 8 Effect of test compounds on the number of cells in the exudate of the air- pouch cavity and on the number of PMN in the air pouch wall
- Control was DMSO / PBS, the vehicle for the test compounds.
- the ratio of DMSO/PBS was 1: 100. Wan ⁇ SD. c unpaired t-test (2-tailed).
- AA the immediate precursor of the eicosanoids
- AA induces an early (10-15min) increase in both PGE 2 and LTC 4 synthesis which precedes the increase in ear thickness.
- PMA protein kinase C
- PKC protein kinase C
- LTs leukotrienes
- PGs prostaglandins
- mice Groups of 5-6 female BALB/c mice (ARC, WA, Australia), weighing 15 -2 Ig were injected intraperitoneally (i/p) with selected compounds of the formula (1) at 25mg/kg delivered in polyethylene glycol (PEG) 400: phosphate buffered saline (PBS) 1:1 or ethanol: propanediol: PBS 4:9:7 either 30min prior to or immediately before the inflammogen was applied to the ears.
- PEG polyethylene glycol
- PBS phosphate buffered saline
- propanediol PBS 4:9:7 either 30min prior to or immediately before the inflammogen was applied to the ears.
- Mice were anaesthetised using isoflurane and baseline thickness of both ears was measured using a spring micrometer.
- Each mouse received a total of 20 ⁇ L of either AA in ethanol (50mg/ml or 200mg/ml) or PMA in either ethanol or acetone (0.2mg/ml) applied to the inner and outer surfaces of each pinna (i.e. 0.5mg or 2mg AA or 2 ⁇ g PMA per ear). Mice were anaesthetised again to re-measure the ears at lhr post- AA application and 5hr after PMA.
- the antioxidant (free radical trapping) activity of test compounds was assessed using the stable free radical compound 2,2-diphenyl-l-picrylhydrazyl (DPPH).
- DPPH stable free radical compound 2,2-diphenyl-l-picrylhydrazyl
- a stock solution of DPPH was prepared at a concentration of 0.1 mM in ethanol and mixed for 10 minutes prior to use.
- Compound (1) was reacted with DPPH for 20 minutes, after which time the absorbance at 517nm was determined.
- the change in absorbance at 517nm was compared to a reagent blank (DPPH with ethanol alone).
- the IC 50 value was estimated as the concentration of the test compound that caused a 0.6 change in absorbance (with 1.2 absorbance units representing total scavenging of the DPPH radical).
- LDL low density lipoprotein
- Oxidized low density lipoprotein is pro-inflammatory, it can cause endothelial dysfunction and it readily accumulates within the arterial wall (Rosenson 2004). Oxidized lipoproteins are thought to provoke a number of changes in cell functions that promote atherogenesis, via an inflammatory response. Therefore, inhibition of the oxidation of LDL can be anti-atherogenic, anti-inflammatory and cardioprotective.
- LDL LDL was passed through a second PDlO column and diluted with chelex treated PBS (10OmM) to give a standard protein concentration of 0.1 mg/niL, ie final concentration per reaction. Oxidation reactions were initiated by the addition of freshly prepared Cu 2+ solution, such that the final concentration Of CuSO 4 was 5 ⁇ M.
- LDL was pre-treated with either compound (1) or compound (3), at final concentrations of 0.1, 1.0, 10 and lOO ⁇ M, for 2 minutes at room temperature prior to the addition of copper solution and subsequently incubated at 37°C.
- the extent of lipoprotein oxidation was determined by measuring the formation of lipid— peroxides on aliquots removed every 30-minute over a 3-hour period. Peroxides were determined at each time point by the ferrous oxidation-xylenol orange (FOX) assay using standard hydrogen peroxide curve (5 to 200 ⁇ M). Compound (1) was examined in at least two separate experiments performed on separate days.
- AAPH AAPH (1.22 gm) was dissolved in 7.5 ml of PBS to yield a 4 x stock at 600 mM and 50 ⁇ l aliquots (final concentration of 150 mM) were then added to each well to initiate the lysis assay.
- Peroxyl-induced RBC lysis assays were performed in 96-flat bottom well microtitre plates with a total volume of 200 ⁇ l per well. Turbidity of RBC suspensions were monitored using a Tecan microplate reader at 690 nm (37°C) with gentle vortexing. Assays were performed in quadruplicate and readings were taken every 5 minutes over 5 hours. RBC lysis curves were constructed by plotting absorbance (mean of 4 readings) against time. Time to half-lysis was calculated by taking the highest absorbance reading (no lysis) and the lowest absorbance reading (maximum lysis). The sum of these two readings divided by two gave the absorbance at half-lysis. Simple regression analysis was used to calculate the time at which half-lysis absorbance occurred.
- compound (1) demonstrated antioxidant activity by delaying the AAPH-induced time to half-lysis of red blood cells.
- Rheumatoid arthritis is a chronic, inflammatory, multisystem, autoimmune disorder that usually manifests with polyarthritis.
- the pathogenesis involves a T-cell mediated 'attack' on the synovium.
- IBD Inflammatory bowel disease
- Both diseases are often treated with a combination of anti-inflammatory and immunosuppressive therapies.
- Selected compounds of the invention were therefore tested in order to determine whether they have immunosuppressive activity in addition to anti-inflammatory activity.
- mice Male Skh-1:HR1 (hairless) mice, approximately six weeks old were killed by cervical dislocation. Single cell suspensions were made from the spleen and erythrocytes were lysed in buffer (0.14M NH4C1, 17mM Tris, pH 7.2). The remaining splenocytes were cultured in RPMI-1640 (Gibco) supplemented with 10% (v:v) FBS, 20OmM L- glutamine, penicillin/streptomycin and 5OmM 2-mercaptoethanol.
- Splenocytes were added to quadruplicate wells containing either concanavalin A (ConA, Sigma-Aldrich - 0.4 ⁇ g/well), LPS (Sigma-Aldrich - l ⁇ g/well) or no mitogen, as well lO ⁇ M of test compound in DMSO.
- Samples were analysed after a 3 day incubation at 37°C in 5% CO2 in air.
- Methylthiazoletetrazolium (MTT) is bioreduced by viable cells into a coloured formazan product that is soluble in DMSO.
- MTT Methylthiazoletetrazolium
- MTT was added to each well, incubated for a further 4hrs and then colour developed with 0.04N HCl in isopropanol.
- Culture supernatants were stored after collection at -80°C and both T and B cells analysed by ELISA (BD Biosciences) for IFN- ⁇ ( a Th-I cytokine) and for T cells alone, IL-6 (a Th-2 cytokine).
- Compounds (1) and (3) were examined in two individual mice each. Compound (1) was markedly and significantly immunosuppressive to T cells and to a lesser extent, B cells. This effect was further evidenced by a concomitant reduction on the synthesis of INF- ⁇ and IL-6 into the supernatant (see Figures 2 and 3).
- compound (3) had little effect on cell numbers, but did decrease the cytokines produced by T cells in particular.
- PGI 2 is the main COX product of endothelial cells, produced from PGH 2 by the action of the enzyme prostacyclin synthase.
- PGI 2 additionally protects from cardiovascular disease by pleiotropic effects on vascular smooth muscle cells (VSMC). Genetic deletion of the prostacyclin receptor in mice reduced the development of atherosclerosis, intimal hyperplasia and restenosis, possibly via PGI 2 inhibition of VSMC proliferation and migration. Its production is inhibited indirectly by NSAIDs, via inhibition of COX, and it is this effect that contributes to the increase in adverse cardiovascular events associated with all NSAIDs. Therefore, a desirable cardioprotective attribute of an anti-inflammatory agent would be lack of inhibition of endothelial PGI 2 synthesis.
- HUVECs were predominantly epithelioid but included some spindle shaped cells. Cells were mainly healthy and viable, as indicated by their translucent appearance and adherence to the culture dish. Floating cells were presumed non-viable. Confluent growth in the central region of the wells resulted in the classic "cobblestone" appearance. In non-confluent areas, adherent cells had a "stretched" appearance.
- TXA 2 produced in activated platelets by TXS has prothrombotic properties by stimulating platelet aggregation and vasoconstriction. Inhibiting TXS selectively would therefore be anti-thrombotic. This might be evidenced by a shunting of substrate to enable an increase in the synthesis of PGE 2 .
- the effect of test compounds was examined in human monocytes and the murine macrophage cell line, RAW 264.7. COX inhibitory activity would be evidenced by substantial inhibition of both PGE 2 and TXB 2 .
- U937 cells were thawed and resuspended in RPMI and 10% FCS at 2 x 10 5 cells per ml. The cells were incubated in 5% CO 2 at 37°C and expanded in growing culture to at least 6.4 x 10 7 total cells. The cells were then resuspended in fresh medium and cultured with 5 ⁇ M retinoic acid (RA) at 2 x 10 5 cells per ml for a further 3 days (72h). RA treated cells were washed 2x in serum-free RPMI and resuspended in serum-free medium at 5 x 10 6 cells per ml. The cells were aliquotted into Teflon tubes at ImI per tube.
- RA retinoic acid
- the mouse macrophage cell line RAW 264.7 was cultured in DMEM supplemented with foetal calf serum (FCS), 2mM glutamine and 50U/ml penicillin/streptomycin. Cells were treated with either test compounds (in 0.025% DMSO) or vehicle alone, and added one hour before 50ng/ml LPS. After incubation for 16hrs, culture media was collected for PGE 2 or TXB 2 measurement by ELISA (Cayman Chemical), and TNF ⁇ measurement using an ELISA (Becton Dickinson).
- vasodilatory capacity of the compounds of formula (1) was examined ex situ using the rat aortic ring assay.
- the addition of noradrenaline to the test bath causes the rings to contract, and if that vasoconstriction is inhibited by a test agent i.e. it antagonises the effect of noradrenaline, it suggests that that agent may have vasodilatory activity.
- PK profiles of compounds (1) and (3) were examined following oral administration in PEG 400/PBS 1 :1 at a dose of 25mg/kg. For each experiment, three animals were allocated per time point (15 min, 30 min, 60 min, 90 min, 4 hr and 24 hr). Mice were killed using cervical dislocation and serum collected via cardiac puncture. Faeces and urine, where available, were also collected. Samples were stored at - 80 0 C and analysed by LC-MS in-house. The limit of detection was 20ng/ml.
- AUC represents the area under the serum concentration versus time curve, expressed as ⁇ M*hour/L. This number assesses absorption and clearance in a relative way. The higher the number, the more compound has been absorbed and the longer it has remained in circulation.
- AUCf ree refers to the area under the curve for unconjugated or free analogue
- AUC tota i refers to the area under the curve for the free and conjugated analogue combined.
- [max] refers to the maximum concentration observed in serum.
- the ratio of the AUCf ree to the AUC to tai gives a relative measure of how much of the administered compound i.e. 'free' remains either in circulation compared to how much of the compound is conjugated. Therefore, if the ratio is relatively high, it suggests that much of the compound remains unconjugated, whereas if the ratio is relatively low, it suggests that conjugation (and thus perhaps urinary excretion) occurs rapidly.
- t v is the half life, i.e. the time taken for the serum concentration to fall by half.
- the elimination of a drug is usually an exponential (logarithmic) process, so that a constant proportion is eliminated per unit time.
- Table 23 PK profile of compound (1) following a single oral dose in mice
- the human colorectal cell line HT-29 (HTB-38TM), human prostate lines PC-3 (CRL-1435TM) and DU-145 (HTB-81TM), and the human melanoma line SK-Mel-28 (HTB-72TM) were cultured in RPMI 1640 medium (Gibco, Cat#21870-076).
- the human prostate cell line LNCaP Clone FGC (CRL- 1740TM), human leukemic cell line CCRF-CEMTM (CCL-119TM) human colorectal adenocarcinoma cell line HCT- 15(CCL-225TM) and the human lung cancer cell lines NCI-H23 (CRL-5800TM) and NCI- H460 (HTB- 127TM) were cultured in RPMI 1640, supplemented to contain 1OmM HEPES (Sigma, Cat#H0887), 4.5g/L Glucose (Sigma, Cat#G8769) and ImM Sodium Pyruvate (Sigma, Cat#S8636).
- 1OmM HEPES Sigma, Cat#H0887
- 4.5g/L Glucose (Sigma, Cat#G8769)
- ImM Sodium Pyruvate (Sigma, Cat#S8636).
- the human melanoma cell line MM200 was obtained as a gift from Prof. Peter Hersey (University of Newcastle) and cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco, Cat#l 1960-069).
- DMEM Dulbecco's Modified Eagle's Medium
- the human melanoma cell line MM96L were obtained as gift from Professor Peter Parsons (Queensland Institute of Medical Research) and cultured in RPMI 1640.
- the human ovarian cancer cell lines A2780 and CP70 were obtained as gifts from Dr. Gil Mor (Yale University).
- A2780 was cultured in RPMI 1640 medium.
- CP70 was cultured in DMEM/Hams F-12 1:1 (Gibco, Cat#l 1320-082) supplemented with 1OmM HEPES, Ix non essential amino acids (Sigma, Cat#M7145), 5.0g/L sodium bicarbonate (Sigma, Cat#S5761), and ImM sodium pyruvate.
- the breast cancer cell line MDA-MB-468 (HTB- 132TM) was cultured in DMEM/Hams F-12 1:1.
- the human pancreatic cancer cell line, HPAC (CRL-2119TM) was routinely cultured in DMEM/Hams F-12 1:1 and supplemented with 15mM HEPES, 0.002 mg/ml insulin (Sigma, Cat#I9278), 0.005mg/ml transferrin (Sigma, Cat#T8158), 40ng/ml hydrocortisone (Sigma, Cat#H0135) and 10 ng/ml epidermal growth factor (Sigma, Cat#E4269).
- the human Glioma cell line Hs 683 (HTB-138 TM) was cultured in DMEM.
- IC 50 values were determined for each cell line. Cells were seeded in 96-well plates at an appropriate cell density as determined from growth kinetics analysis and cultured for 5 days in the absence and presence of the test compounds. Cell proliferation was assessed after the addition of 20 ⁇ l of 3-4,5 dimethylthiazol-2,5-diphenyl tetrazolium bromide (MTT, 2.5 mg/ml in PBS, Sigma) for 3-4hrs at 37 0 C according to manufacturer's instructions. IC 50 values were calculated from semi-log plots of % of control proliferation on the y-axis against log dose on the x-axis.
- MTT 3-4,5 dimethylthiazol-2,5-diphenyl tetrazolium bromide
- compound (1) demonstrated activity (ie IC 50 ⁇ 20 ⁇ M) in a number of cancer cell lines.
- Compounds (2) and (3) were less active.
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US13/056,608 US20120003270A1 (en) | 2008-07-30 | 2009-07-30 | 6-substituted isoflavonoid compounds and uses thereof |
CN2009801393999A CN102164904A (en) | 2008-07-30 | 2009-07-30 | 6-substituted isoflavonoid compounds and uses thereof |
JP2011520279A JP2011529450A (en) | 2008-07-30 | 2009-07-30 | 6-substituted isoflavonoid compounds and uses thereof |
CA2738510A CA2738510A1 (en) | 2008-07-30 | 2009-07-30 | 6-substituted isoflavonoid compounds and uses thereof |
EP09802278A EP2324003A4 (en) | 2008-07-30 | 2009-07-30 | 6-substituted isoflavonoid compounds and uses thereof |
AU2009276293A AU2009276293A1 (en) | 2008-07-30 | 2009-07-30 | 6-substituted isoflavonoid compounds and uses thereof |
MX2011001225A MX2011001225A (en) | 2008-07-30 | 2009-07-30 | 6-substituted isoflavonoid compounds and uses thereof. |
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US20060153781A1 (en) * | 2002-09-06 | 2006-07-13 | Novogen Research Pty Ltd, A Corporation | Repair of dna mutagenic damage |
US20060167083A1 (en) * | 2002-04-09 | 2006-07-27 | Kelly Graham E | Therapeutic methods and compositions involving isoflav-3-ene and isoflavan structures |
US20060183728A1 (en) * | 2002-10-02 | 2006-08-17 | Kelly Graham E | Combination chemotherapy compositions and methods |
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US20060167083A1 (en) * | 2002-04-09 | 2006-07-27 | Kelly Graham E | Therapeutic methods and compositions involving isoflav-3-ene and isoflavan structures |
US20060153781A1 (en) * | 2002-09-06 | 2006-07-13 | Novogen Research Pty Ltd, A Corporation | Repair of dna mutagenic damage |
US20060183728A1 (en) * | 2002-10-02 | 2006-08-17 | Kelly Graham E | Combination chemotherapy compositions and methods |
US20080069900A1 (en) * | 2002-10-02 | 2008-03-20 | Novogen Research Pty Ltd | Combination chemotherapy compositions and methods |
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WO2011115819A3 (en) * | 2010-03-15 | 2012-01-05 | Genus Oncology, Llc | Small molecule inhibitors of muc1 and methods of identifying the same |
US8952054B2 (en) | 2010-03-15 | 2015-02-10 | Dana-Farber Cancer Institute, Inc. | Small molecule inhibitors of MUC1 and methods of identifying the same |
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