Classifications

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  • 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
  • C07D311/78—Ring systems having three or more relevant rings
  • C07D311/92—Naphthopyrans; Hydrogenated naphthopyrans
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
• • 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
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • 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
• • 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
  • C07D311/96—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings spiro-condensed with carbocyclic rings or ring systems
• • 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
  • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
  • C07D413/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings directly linked by a ring-member-to-ring-member bond
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
  • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
  • C07D491/04—Ortho-condensed systems
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
  • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
  • C07D491/04—Ortho-condensed systems
  • C07D491/044—Ortho-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring
  • C07D491/052—Ortho-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring the oxygen-containing ring being six-membered
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
  • C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
  • C07D493/04—Ortho-condensed systems

Definitions

• the present invention relates to compounds having the general formula (I) with the definitions of X 1 -X 4 , A, R 6 and R 7 given below and/or solvates, hydrates and pharmaceutically acceptable salts thereof. Furthermore, the invention relates to the use of said compounds for modulating of the Wnt signalling pathway activity and their use as a medicament, preferably for the treatment of cancer.
• the Wnt signalling pathway plays an important role in the regulation of cell proliferation and differentiation. Aberrant activation of the Wnt signalling pathway is known to promote uncontrolled cell growth and survival and can therefore be a major driving force in a broad spectrum of human cancers and diseases. For example, the inhibition of aberrant Wnt signalling pathway activity in cancer cell lines effectively blocks their growth (N. Barker and H. Clevers “Mining the Wnt pathway for cancer therapeutics”, Nature Reviews, vol. 5, 2007, pages 997-1014; R. Nusse, “Mint signalling in disease and in development”, Cell Research, Vol. 15, 2005, pages 23-32).
• the Wnt signalling pathway involves a large number of proteins regulating the production of Wnt signalling molecules, their interaction with receptors on target cells and the physiological response of target cells resulting from the exposure of cells to the extra-cellular Wnt ligands.
• Frizzled (Fz) receptor complexes on the surface of target cells and activate distinct intracellular pathways that are broadly classified as canonical or non-canonical Wnt signalling pathways.
• the canonical pathway regulates the stability of the protein beta-catenin in a cell and its ability to enter the nucleus of the cell, where it interacts with members of the Tcf/Lef protein family.
• Beta-catenin and Tcf form active transcription factor complexes in the nucleus and activate the Wnt target genes.
• the presence of the beta-catenin in the nucleus is a hallmark in the Wnt signalling pathway indicating its activation.
• An overview of the Wnt signalling pathway can be found in N. Barker and H. Clevers “Mining the Wnt pathway for cancer therapeutics”, Nature Reviews, vol. 5, 2007, pages 997-1014.
• Tcf-beta-catenin complexes in the nuclei of cells leads to activation of the genetic program considered to promote cancer formation by stimulating cell growth, blocking apoptosis and altering cell movement and stem cell properties.
• the artificial disruption of Tcf-beta-catenin complex formation in colon cancer cells effectively blocks target gene activation and inhibits the growth in vitro.
• Drugs designed to inhibit the Wnt signalling pathway and consequently the formation of the Tcf-beta-catenin complex in the nucleus of a cell are therefore expected to hold great potential for the treatment of a range of cancers and other diseases associated with the Wnt signalling pathway.
• An object of the present invention is to provide such compounds. This object is achieved by a compound having the general formula (I)
• Alkyl means a linear or branched saturated aliphatic hydrocarbon group, e.g. methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, n-pentyl, i-pentyl, n-hexyl, i-hexyl and 3-methyl-pentyl and the like.
• Alkenyl means a linear or branched unsaturated aliphatic hydrocarbon group with at least one double bond, e.g. ethenyl, propenyl (allyl), 1-butenyl, 2-butenyl, 1-pentenyl, 2-pentenyl, 1-hexenyl, 2-hexenyl and 3-hexenyl and the like.
• Alkinyl means a linear or branched unsaturated aliphatic hydrocarbon group with at least one triple bond, e.g. ethinyl, propinyl, 1-butinyl, 2-butinyl and the like.
• Cycloalkyl means a saturated 3 to 8-membered hydrocarbon ring e.g. cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.
• Alicyclic means a saturated or unsaturated but non-aromtic 3- to 8-membered hydrocarbon ring like cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclopentadiene, cyclohexane, cyclohexene and cycloheptane.
• Heterocyclyl and “heterocyclic” mean a saturated or unsaturated 3- to 8-membered hydrocarbon ring containing 1 to 4 heteroatoms and/or heteratom containing groups selected from the group N, O, S and C(O) in the ring.
• the one or more heteroatoms and hetero atoms containing groups present in the ring replace a —CH 2 — group or a —CH ⁇ in the ring.
• Preferred C 3 -C 8 heterocyclyl groups containing 1 to 4 heteroatoms and heteroatom containing groups selected from the group N, O, S and C(O) are derived from the following heterocyclic compounds: tetrahydrofurane, pyrrolidine, tetrahydrothiophene, oxazolidine, piperidine, tetrahydropyrane, piperazine, dioxane, morpholine, cyclopentanone and trioxane.
• Aryl and “aromatic” mean an aromatic 3- to 7-numbered hydrocarbon ring e.g. phenyl and the like.
• Heteroaryl and “heteroaromatic” mean an aromatic 3- to 7-numbered hydrocarbon ring containing 1 to 4 heteroatoms selected from the group N, O, and S in the ring. The one or more heteroatoms present in the ring replace a —CH ⁇ group in the ring.
• Preferred C 3 -C 7 heteroaryl groups containing 1 to 4 heteroatoms selected from the group N, O and S are derived from the following heteroaromatic compounds: Pyrrole, pyrazole, imidazole, triazole, tetrazole, furane, thiophene, oxazole, isoxazole, oxadiazole, thiazole, isothiazole, thiadiazole, pyridine, pyridazine, pyrimidine, pyrazine and triazine.
• “Aralkyl” means a C 3 -C 7 aryl group substituted with at least one C 1 -C 8 alkyl group, wherein aryl and alkyl are defined as above. Examples for aralkyl are tolyl, benzyl and xylyl. The aralkyl group may be bound via the C 1 -C 8 alkyl group or via the C 3 -C 7 aryl group.
• Heteroarylalkyl means a C 3 -C 7 heteroaryl group substituted with at least one C 1 -C 8 alkyl group, wherein heteroaryl and alkyl are defined as above.
• the heteroarylalkyl group may be bound via the C 1 -C 8 alkyl group or via the C 3 -C 7 heteroaryl group.
• Halogen comprises F, Cl, Br and I.
• Substituent A is preferably selected from the group consisting of substituted or unsubstituted monocyclic or polycyclic moieties derived from benzene, pyridine, imidazole, furane, benzofurane, indole, pyrazole, triazole, tetrazole, oxadiazole, thiadiazole, thiazole, indazole, benzooxazole, pyrimidine, pyrrolopyridine and oxazole.
• A is phenyl which is optionally substituted by 1 to 5 substituents R which may be same or different.
• R substituents
• R 1 ; R 2 ; R 3 ; R 4 and R 5 independently from each other are selected from the group consisting of H and the substituents as defined for R above. Within this embodiment it is even more preferred if at least 3 of the group of R 1 ; R 2 ; R 3 ; R 4 and R 5 are not H, particularly preferred R 2 ; R 3 and R 4 are not H and most preferred R 1 and R 5 are H and R 2 ; R 3 and R 4 are not H.
• a preferred embodiment of the invention relates to compounds of formula (I), wherein two adjacent R 8 form together an optionally substituted 5- or 6-membered saturated or unsaturated hydrocarbon ring containing up to 3 N-atoms in the ring. Especially preferred, the two adjacent R 8 form together an optionally substituted 5- or 6-membered aromatic ring containing up to 3 N-atoms in the ring. Within this embodiment it is particularly preferred, if the two adjacent R 8 form an optionally substituted 6-membered aromatic ring containing up to 3 N-atoms.
• the two adjacent R 8 may be selected from R 8 bound to X 3 and X 4 (formula (Ia)); R 8 bound to X 2 and X 3 (formula (Ib)) or R 8 bound to X 1 and X 2 (formula (Ic)):
• A is phenyl which is optionally substituted by 1 to 5 substituents R which are same or different.
• Compounds of this embodiment have formula
• the two adjacent R 8 form together a cyclic 5-membered substituted or unsubstituted diether resulting in compounds of formulae
• R 1 ; R 2 ; R 3 ; R 4 and R 5 are not H, particularly preferred R 2 ; R 3 and R 4 are not H and most preferred R 1 and R 5 are H and R 2 ; R 3 and R 4 are not H.
• Another preferred embodiment of the invention concerns the compounds of formula (II) and (IIa) to (IIf), wherein at least 3 of the group of R 1 ; R 2 ; R 3 ; R 4 and R 5 are not H, particularly preferred R 2 ; R 3 and R 4 are not H, and the further substituents are defined as above, and/or solvates; hydrates; and pharmaceutically acceptable salts thereof.
• the solvates; hydrates; and/or pharmaceutically acceptable salts of all compounds of formulae (I), (Ia) to (If), (II) and (IIa) to (IIf) described above are included herein.
• Compounds according to the invention which contain one or more acidic groups can be used according to the invention, e.g. as their alkali metal salts, alkaline earth metal salts or ammonium salts. More precise examples of such salts include sodium salts, potassium salts, calcium salts, magnesium salts or salts with ammonia or organic amines such as, e.g. ethylamine, ethanolamine, triethanolamine or amino acids.
• acids examples include hydrogen chloride, hydrogen bromide, phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid, p-toluenesulfonic acid, napthalenedisulfonic acid, oxalic acid, acetic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, formic acid, propionic acid, pivalic acid, diethylacetic acid, malonic acid, succinic acid, pimelic acid, fumaric acid, maleic acid, malic acid, sulfamic acid, phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid, citric acid, adipic acid and other acids known to a person skilled in the art.
• the invention also includes, in addition to the salt forms mentioned, inner salts or betaines.
• the respective salts of the compounds according to the invention can be obtained by customary methods which are known to the person skilled in the art, for example by contacting these with an organic or inorganic acid or base in a solvent or dispersant, or by anion exchange or cation exchange with other salts.
• pharmaceutically acceptable means approved by a regulatory agency such as the EMA (Europe) and/or the FDA (US) and/or any other national regulatory agency for use in animals, preferably in humans.
• the invention includes all salts of the compounds according to the invention which, owing to low physiological compatibility, are not directly suitable for use in pharmaceuticals but which can be used, for example, as intermediates for chemical reactions or for the preparation of pharmaceutically acceptable salts or which might be suitable for studying Wnt signalling pathway modulating activity of a compound according of the invention in any suitable manner, such as any suitable in vitro assay.
• the present invention furthermore includes all solvates of the compounds according to the invention.
• the present invention furthermore includes derivatives/prodrugs (including the salts thereof) of the compounds according to the invention which contain physiologically tolerable and cleavable groups and which are metabolized in animals, preferably mammals, most preferably humans into a compound according to the invention.
• the present invention furthermore includes the metabolites of the compounds according to the invention.
• metabolites refers to all molecules derived from any of the compounds according to the invention in a cell or organism, preferably mammal.
• metabolites relates to molecules which differ from any molecule which is present in any such cell or organism under physiological conditions.
• the compounds of the invention may be prepared by the three component reaction of a substituted or unsubstituted aromatic or heteroaromatic monocyclic or polycyclic aldehyde (e.g. benzaldehyde, pyridinecarbaldehydes, imidazolecarbaldehyde, furancarbaldehydes, benzofurancarbaldehydes, indolecarbaldehydes, pyrazolecarbaaldehydes, oxazolecarbaldehydes and their derivatives), an OH-substituted aromate or heteroaromate, which may be further substituted (e.g.
• a substituted or unsubstituted aromatic or heteroaromatic monocyclic or polycyclic aldehyde e.g. benzaldehyde, pyridinecarbaldehydes, imidazolecarbaldehyde, furancarbaldehydes, benzofurancarbaldehydes, indole
• the compounds of the formulae (I), (Ia) to (If), (II) and (IIa) to (IIf) can be purified by customary purification procedures, for example by recrystallization or chromatography.
• the starting materials for the preparation of the compounds of the formulae (I), (Ia) to (If), (II) and (IIa) to (IIf) are commercially available or can be prepared according to or analogously to literature procedures.
• the compounds of the invention may serve as a basis for the preparation of the other compounds according to the invention by several methods well known by the person skilled in the art.
• the present invention relates to the discovery that signal transduction pathways regulated by Wnt can be inhibited, at least in part, by compounds of formulae (I), (Ia) to (If), (II), (IIa) to (IIf). As set out in more detail below, these compounds can inhibit proliferation of tumor cells having Wnt modulated activity. Therefore, the compounds according to the invention are suited for modulating the Wnt signalling pathway.
• the term “modulating the Wnt signalling pathway” refers to an effect on the series of events that occur when Wnt proteins bind to cell-surface receptors of the frizzled family resulting in an accumulation of beta-catenin in the cell cytoplasm that reaches the nucleus of a cell, and consequently, the Wnt target genes are expressed.
• the Wnt signalling pathway may be modulated by direct or indirect modulation.
• Direct modulation means an interaction of the inventive compounds with proteins directly involved in the Wnt signalling pathway leading to an increase or decrease of the expression of the Wnt target genes.
• “Indirect modulation” means an increase or decrease of the expression of the Wnt target genes without a direct interaction of the inventive compound with the components involved in the Wnt signalling pathway.
• Examples for the indirect modulation of the Wnt signalling pathway are tankyrase-inhibitors and calcium regulators like siperone, thapsigargine and iononycine.
• Inhibition of tankyrases stabilizes one protein of the Wnt signalling pathway (axin), which inhibits the Wnt signalling pathway
• Inhibition of the decomposition of axin leads to an increase of axin and in turn to the inhibition of the Wnt signalling pathway (Huang et al., Nature 461, pp. 614 to 620 (2009))
• the activation of a receptor may be the mechanism by which these compounds act as described in US 2007/0219257 A1.
• the compounds could affect the activity of a Wnt/Frizzled receptor complex.
• the compounds could affect the activity of the serine/threonine kinase GSK3 ⁇ , which is involved in the down regulation of ⁇ -catenin.
• the compounds could also affect the activity of the APC gene. In the absence of Wnt signal, the APC protein functions to foster degradation of ⁇ -catenin and prevent its nuclear entry.
• Frizzled (Fz) and LRP5/6 The pathway can also be activated by mutations of ⁇ -catenin that render it resistant to degradation.
• the compounds could alter the activity of Dishevelled, which is a positive mediator of Wnt signaling.
• the ability of these compounds to inhibit proliferation of cells may be due to the ability of such molecules to interact with Wnt, or at least to interfere with the ability of those proteins to activate a Wnt-mediated signal transduction pathway.
• Signal transduction antagonists of different structures even ones that bind to the same protein in the signaling pathways, may act in slightly different ways. Accordingly, even if a particular condition caused or contributed to by aberrant or unwanted activation of the Wnt pathway shows little response to treatment by one of the antagonists disclosed herein, another of the antagonists disclosed herein may nonetheless be efficacious.
• One embodiment of the present invention includes the use of compounds of formulae (I), (Ia) to (If), (II) and (IIa) to (IIf) that agonize inhibition of Wnt signaling, such as by inhibiting activation of Wnt downstream components of the signaling pathway, in the regulation of repair and/or functional performance of a wide range of cells, tissues and organs, including normal cells, tissues, and organs.
• the compounds of formulae (I), (IIa) to (IIf), (II) and (IIa) to (IIf) have therapeutic and cosmetic applications ranging from regulation of neural tissues, bone and cartilage formation and repair, regulation of spermatogenesis, regulation of smooth muscle, regulation of lung, liver and other organs arising from the primitive gut, regulation of hematopoietic function, regulation of skin and hair growth, etc.
• the compounds of formula (I) can be applied to cells that are provided in culture (in vitro), or on cells in a whole animal (in vivo).
• Another embodiment of present invention includes the use of compounds of formulae (I), (Ia) to (If), (II) and (IIa) to (IIf) which antagonize activity of the Wnt pathway resulting in the regulation of repair and/or functional performance of a wide range of cells, tissues, and organs.
• the inventive compounds have therapeutic and cosmetic applications ranging from regulation of neural tissues, bone and cartilage formation and repair, regulation of spermatogenesis, regulation of smooth muscle, regulation of lung, liver and other organs arising from the primitive gut, regulation of hematopoietic function, regulation of skin and hair growth, etc.
• the compounds of the invention can be applied on cells which are provided in culture (in vitro), or on cells in a whole animal (in vivo).
• agonist refers to an agent or analog that binds productively to a receptor or other mediators of the signaling pathway and mimics the biological activity.
• antagonist refers to an agent that binds to receptors or other mediators of the signaling pathway and inhibits the biological activity.
• an antagonist potentiates or recapitulates, for example, the bioactivity of Axin, such as to repress transcription of target genes.
• Wnt antagonist refers not only to any agent that may act by directly inhibiting the normal function of the Wnt proteins, but also to any agent that inhibits the Wnt signaling pathway, and thus antgonizes the function of Wnt.
• Wnt agonist likewise refers to an agent which activates or stabilizes the bioactivity of Wnt, such as to increase transcription of target genes.
• the compounds of the invention are used as Wnt antagonists and used to regulate e.g. proliferation or other biological consequences of mis-expression of Wnt.
• an elevated beta-catenin level in the nucleus of a cell is a hallmark of an aberrant activation of the Wnt signalling pathway and plays a major role in the development of several kinds of cancer.
• the measurement of the beta-catenin level in the nucleus of the cell may be carried out according procedures known to the person skilled in the art.
• the measurement of the Tcf-beta-catenin complex level by means of 6 ⁇ Tcf-luciferase is described below in the experiments.
• the use of a compound for modulating the Wnt signalling pathway resulting in a decrease of the relative amount of Tcf-beta-catenin complex in the nucleus of a cell is preferred.
• Modulating the Wnt signalling pathway can be carried out by contacting a cell with a compound according to the invention.
• said modulation is performed in vitro or in cell culture.
• in vitro and cell culture assays are available.
• the modulation can be performed in animals such as mammals.
• Animal subjects to which the invention is applicable extend to both domestic animals and livestock, raised either as pets or for commercial purposes.
• Exemplary mammals are mice, rats, guinea pigs, monkeys, dogs and cats.
• the modulation can also be carried out in humans.
• the invention also relates to the compounds (I), (Ia) to (If), (II) and (IIa) to (IIf) of the invention for use as a medicament.
• the compounds are as defined above; furthermore the embodiments as described below with respect to the use as medicament, e.g. formulation, application and combination, also apply to this aspect of the invention.
• the pharmaceutical preparation or medicaments comprising the compounds of formulae (I), (Ia) to (If), (II) and (IIa) to (IIf) can be effective for both human and animal subjects.
• Animal subjects to which the invention is applicable extend to both domestic animals and livestock, raised either as pets or for commercial purposes. Examples are dogs, cats, cattle, horses, sheep, hogs, and goats.
• the invention further relates to the use of the compounds of formulae (I), (Ia) to (If), (II) and (IIa) to (IIf) for modulating the Wnt signalling pathway.
• the compounds according to the invention are suited for the use for the preparation of a medicament for modulating the Wnt signalling pathway.
• the present invention provides pharmaceutical preparations or medicaments comprising a compound such as described herein, formulated in an amount sufficient to regulate, in vivo, Wnt pathway, e.g., proliferation or other biological consequences of mis-expression of Wnt.
• the invention further relates to the compounds according to the invention for the preparation of a medicament for the treatment of a disorder or disease associated with an aberrant activation of Wnt signalling in a mammal and to compounds of the invention for the treatment of a disorder or disease associated with an aberrant activation of Wnt signalling in a mammal.
• the disorders or diseases associated with the Wnt signalling pathway are for example cell-proliferative disorders, rheumatoid arthritis, diseases connected with aberrant bone density and Dupuytren disease (superficial fibromatosis).
• a certain cell line e.g. a cancer cell line shows an aberrant activity of the Wnt signalling pathway
• Many investigations on the role of the Wnt signalling pathway in different human cancers are summerized in N. Barker and H. Clevers “Mining the Wnt pathway for cancer therapeutics”, Nature Reviews, vol. 5, 2007, pages 997-1014.
• An aberrant activity of the Wnt signalling pathway in a certain cell, e.g. a cancer cell may be detected by an elevated level of beta-catenin in the nucleus of said cell, by the expression of Wnt target genes or by means of other components of the Wnt signalling pathway.
• the level of beta-catenin in the nucleus of a cell is a hall mark of the Wnt signalling pathway and measuring said level is known to the person skilled in the art.
• the inventive compounds specifically influence the Wnt signalling pathway as demonstrated in the examples by the effect of the inventive compounds on cancer cells which are not associated with an aberrant activity of the Wnt signalling pathway (Hek293T and HepG2) showing no cytotoxic activity and on cancer cells associated with an aberrant activity of the Wnt signalling pathway (human colon carcinoma cells D1d1, HCT116 and SW480) showing low IC 50 values.
• This specific activity of the inventive compounds on the Wnt signaling pathway has the great advantage that the mammals suffering from a particular disease, e.g. a certain kind of cancer, may be selected in advance depending on whether they would benefit from the treatment or not, i.e.
• this feature allows to predict whether the therapy of a cancer patient with compounds of formulae (I), (Ia) to (If), (II) and (IIa) to (IIf) will be successful.
• the prediction allows the selection of those cancer patients who have a high probability of benefiting from therapy with the inventive compounds.
• the therapy can be targeted to those patients who are likely to benefit. Since every therapy may cause undesired side-effects, it is desirable to treat only those patients, where the possibility of side-effects is offset by the potential benefits of therapy. Patients who will most likely not benefit from treatment can be spared the hassles and the risk of side-effects.
• the selection of suitable patients allows a more economical allocation of resources.
• the present invention further comprises a method for predicting whether therapy of a cancer patient with compounds of formulae (I), (Ia) to (If), (II) and (IIa) to (IIf) will be successful comprising the steps of
• sample refers to a sample of a body fluid, to a sample of separated cells or to a sample from a tissue or an organ.
• Samples of body fluids can be obtained by well known techniques and include, preferably, samples of blood, plasma, serum, or urine, more preferably, samples of blood, plasma or serum.
• Tissue or organ samples may be obtained from any tissue or organ by, e.g., biopsy.
• Separated cells may be obtained from the body fluids or the tissues or organs by separating techniques such as centrifugation or cell sorting.
• the sample contains cancer cells. More preferably, the sample is obtained by a biopsy of at least one tumor of the cancer patient.
• cancer patient preferably refers to a mammal, more preferably to a primate and most preferably to a human.
• the patient suffers from cancer, more preferably a cancer selected from the group described below.
• an “effective treatment” of a cancer as referred to in the present application is any treatment which decreases the size of at least one tumor in the patient, which prevents or delays the recurrence of a tumor, which prevents or delays the formation of metastases, which relieves the symptoms caused by the cancer or which prevents or delays the further growth of at least one tumor in the patient. More preferably, the “effective treatment” cures the cancer of the patient.
• Criteria for the decision whether a cancer is cured are known to the person skilled in the art. Preferably, no tumors or cancerous cells are detectable in a cured patient and he/she does not exhibit any symptoms associated with the cancer in question. Preferably, the aforementioned condition lasts for at least 5 years.
• predicting refers to the process of establishing a prognosis whether a treatment will be effective as described above. As will be understood by those skilled in the art, such a decision is usually not intended to be correct for 100% of the subjects to be diagnosed. The term, however, requires that a statistically significant portion of those subjects who can be treated successfully can be correctly determined. Whether a portion is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools, e.g., determination of confidence intervals, p-value determination, Student's t-test, Mann-Whitney test etc. Details are found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983.
• Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99%.
• the p-values are, preferably, 0.1, 0.05, 0.01, 0.005, or 0.0001.
• the probability envisaged by the present invention allows that the diagnosis will be correct for at least 60%, at least 70%, at least 80%, or at least 90% of the subjects of a given cohort or population.
• beta-catenin encompasses also variants of the aforementioned specific beta-catenin. Such variants have at least the same essential biological and immunological properties as the specific beta-catenin. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays, e.g., by ELISA Assays using polyclonal or monoclonal antibodies specifically recognizing the said beta-catenin.
• a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs due to at least one amino acid substitution, deletion and/or addition wherein the amino acid sequence of the variant is still, preferably, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 92%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% identical with the amino sequence of the specific beta-catenin, preferably over the entire length of the specific beta-catenin.
• Variants may be allelic variants or any other species specific homologs, paralogs, or orthologs.
• the variants referred to herein include fragments of the specific beta-Catenin or the aforementioned types of variants as long as these fragments have the essential immunological and biological properties as referred to above.
• the beta-catenin variants have immunological properties (i.e. epitope composition) comparable to those of human beta-catenin.
• the variants shall be recognizable by the aforementioned means or ligands used for determination of the amount of the beta-catenin.
• Such fragments may be, e.g., degradation products of beta-catenin.
• variants which differ due to posttranslational modifications such as phosphorylation or myristylation.
• a cell proliferation disorder is a disorder which is connected with some degree of abnormal cell proliferation. Especially, cell-proliferation disorders are important for the development of cancer.
• a list of cancers associated with an aberrant activation of Wnt signalling pathway can be found in Table 1 of N. Barker and H. Clevers “Mining the Wnt pathway for cancer therapeutics”, Nature Reviews, vol. 5, 2007, pages 997-1014 which is incorporated by reference.
• a further cell-proliferation disorder is proliferative skin disorders which are marked by unwanted or aberrant proliferation of cutaneous tissue, for example X-linked ichtyosis, psoriasis, atopic dermatitis, allergic contact dermatitis, epidermolytic hyperkeratosis and seborrheic dermatitis.
• the invention relates to the use of the compounds according to the invention for the preparation of a medicament for the treatment of cancer or proliferative skin disorder.
• the cancer is selected from cancers showing an increased level of beta-catenin or beta-catenin target genes in comparison to a normal tissue, preferably in the nuclei.
• the normal tissue which is used for the comparison is the tissue the cancer originated from.
• an increased level refers to a level which is increased by a factor of at least 5, at least 10, at least 50, at least 100 or at least 1000.
• beta-catenin is well known to person skilled in the art.
• immunofluoroescence may be used for this purpose.
• a description of this method may be found in Scholer-Dahirel, A., Schlabach, et al. Proceedings of the National Academy of Sciences (2011) www.pnas.org/cgi/doi/10.1073/pnas.1104182108.
• the Wnt-signalling pathway is also believed to be involved in the maintenance of stem or progenitor cells in a growing list of adult tissues that includes e.g. skin, blood, gut, prostate, muscle and the nervous system. Stem and progenitor cells are important for cell regeneration and consequently for aging and aging related processes. Therefore, the compounds of the invention are useable for the preparation of a medicament for the treatment of aging and age-related disorders and/or diseases.
• the compounds of the invention are especially suitable for the use for the preparation of a medicament for the treatment of cancer wherein the cancer is a member of the group multiple myeloma, colon cancer, breast cancer, gastritic cancer, colorectal cancer, lung cancer, prostate cancer, ovarian cancer, bladder cancer, liver cancer, uterine cancer, kidney cancer, leukaemia, gliomas, basal cell carcinoma, rhabdomyosarcoma, mesothelioma, osteosarcoma, medulloblastomas and other primary CNS malignant neuroectodermal tumors.
• the cancer is a member of the group multiple myeloma, colon cancer, breast cancer, gastritic cancer, colorectal cancer, lung cancer, prostate cancer, ovarian cancer, bladder cancer, liver cancer, uterine cancer, kidney cancer, leukaemia, gliomas, basal cell carcinoma, rhabdomyosarcoma, mesothelioma, osteosarcoma, medullob
• Possible disorders or diseases which may be treated by administering a medicament prepared from the compounds for formulae (I), (Ia) to (If), (II) and (IIa) to (IIf) are described in detail in US 2007/0219257 A1. Accordingly the compounds of the present invention are applicable to cell culture techniques wherein, whether for genetic or biochemical reasons, the cells have a Wnt receptor.
• a compound of formulae (I), (Ia) to (If), (II) and (IIa) to (IIf) may be employed in a related method directed towards cells which have a Wnt receptor.
• neurotrophic factors such as nerve growth factor (NGF), ciliary trophic factors (CNTF), and brain derived neurotrophic factor (BDNF).
• NGF nerve growth factor
• CNTF ciliary trophic factors
• BDNF brain derived neurotrophic factor
• One use of the compounds of formulae (I), (Ia) to (If), (II) and (IIa) to (IIf) may be in cultures of neuronal stem cells, such as in the use of such cultures for the generation of new neurons and glia.
• the cultured cells can be contacted with an aromatic compound of the present invention in order to alter the rate of proliferation of neuronal stem cells in the culture and/or alter the rate of differentiation, or to maintain the integrity of a culture of certain terminally differentiated neuronal cells.
• the subject method can be used to culture, for example, sensory neurons or, alternatively, motorneurons: Such neuronal cultures can be used as convenient assay systems as well as sources of implantable cells for therapeutic treatments.
• the compounds of the invention can be used in the treatment of neoplastic or hyperplastic transformations such as may occur in the central nervous system.
• the compounds can be utilized to cause such transformed cells to become either post-mitotic or apoptotic.
• the compounds may, therefore, be used as part of a treatment for, e.g., malignant gliomas, meningiomas, medulloblastomas, neuroectodermal tumors, and ependymomas.
• the compounds of the invention can be used as part of a treatment regimen for malignant medulloblastoma and other primary CNS malignant neuroectodermal tumors.
• the compounds of the invention are used as part of treatment program for medulloblastoma.
• Medulloblastoma a primary brain tumor, is the most common brain tumor in children.
• a medulloblastoma is a primitive neuroectodermal tumor (PNET) arising in the posterior fossa. Histologically, they are small round cell tumors commonly arranged in true rosettes, but may display some differentiation to astrocytes, ependymal cells or neurons (Rorke; Kleihues). PNET's may arise in other areas of the brain including the pineal gland (pineoblastoma) and cerebrum.
• PNET neuroectodermal tumor
• Medulloblastoma/PNET's are known to recur anywhere in the CNS after resection, and can even metastasize to bone. Pretreatment evaluation should therefore include an examination of the spinal cord to exclude the possibility of “dropped metastases”.
• Gadolinium-enhanced MRI has largely replaced myelography for this purpose, and CSF cytology is obtained postoperatively as a routine procedure.
• the compounds of formulae (I), (Ia) to (If), (II) and (IIa) to (IIf) are used as part of a treatment program for hepatocellular carcinoma.
• Hepatocellular carcinoma is a form of cancer that arises from hepatocytes, the major cell type of the live, and is one of the most common tumors involving mutations in the Wnt pathway.
• the compounds of formulae (I), (Ia) to (If), (II) and (IIa) to (IIf) are used as part of treatment program for ependymomas.
• Ependymomas account for approximately 10% of the pediatric brain tumors in children. Grossly, they are tumors that arise from the ependymal lining of the ventricles and microscopically form rosettes, canals, and perivascular rosettes.
• compositions comprising one or more of the inventive compounds can also be utilized for both cell culture and therapeutic uses involving generation and maintenance of non-neuronal tissue.
• the present invention makes use of the discovery that Wnt is apparently involved in controlling the development of stem cells responsible for formation of the digestive tract, liver, lungs, and other organs which derive from the primitive gut. Therefore, for example, compounds of formulae (I), (Ia) to (If), (II) and (IIa) to (IIf) can be employed for regulating the development and maintenance of an artificial liver which can have multiple metabolic functions of a normal liver.
• the compounds of formulae (I), (Ia) to (If), (II) and (IIa) to (IIf) can be used to regulate the proliferation and differentiation of digestive tube stem cells to form hepatocyte cultures which can be used to populate extracellular matrices, or which can be encapsulated in biocompatible polymers, to form both implantable and extracorporeal artificial livers.
• compositions of inventive compounds can be utilized in conjunction with transplantation of such artificial livers, as well as embryonic liver structures, to regulate uptake of intraperitoneal implantation, vascularization, and in vivo differentiation and maintenance of the engrafted liver tissue.
• the compounds of formulae (I), (Ia) to (If), (II) and (IIa) to (IIf) can be employed therapeutically to regulate such organs after physical, chemical or pathological insult.
• therapeutic compositions comprising the compounds of formulae (I), (Ia) to (If), (II) and (IIa) to (IIf) can be utilized in liver repair subsequent to a partial hepatectomy.
• inventive compounds can be used to control or regulate the proliferation and/or differentiation of pancreatic tissue both in vivo and in vitro.
• the compounds of the present invention may provide therapeutic benefits, with the general strategy being, for example, the correction of aberrant insulin expression, or modulation of differentiation. More generally, however, the present invention relates to the use of the compounds of formulae (I), (Ia) to (If), (II) and (IIa) to (IIf) for inducing and/or maintaining a differentiated state, enhancing survival and/or affecting proliferation of pancreatic cells, by contacting the cells with the subject inhibitors.
• the compounds of the invention could be used as part of a technique to generate and/or maintain such tissue both in vitro and in vivo.
• modulation of the function of Wnt can be employed in both cell culture and therapeutic uses involving generation and maintenance ⁇ -cells and possibly also for non-pancreatic tissue, such as in controlling the development and maintenance of tissue from the digestive tract, spleen, lungs, colon, and other organs which derive from the primitive gut.
• the compounds of the invention can be used in the treatment of hyperplastic and neoplastic disorders effecting pancreatic tissue, particularly those characterized by aberrant proliferation of pancreatic cells.
• pancreatic cancers are marked by abnormal proliferation of pancreatic cells which can result in alterations of insulin secretory capacity of the pancreas.
• certain pancreatic hyperplasias such as pancreatic carcinomas, can result in hypoinsulinemia due to dysfunction of ⁇ -cells or decreased islet cell mass.
• the compounds of the invention can be used to enhance regeneration of the tissue after anti-tumor therapy.
• the present invention makes use of the apparent involvement of Wnt in regulating the development of pancreatic tissue.
• the compounds of the invention can be employed therapeutically to regulate the pancreas after physical, chemical or pathological insult.
• the subject method can be applied to cell culture techniques, and in particular, may be employed to enhance the initial generation of prosthetic pancreatic tissue devices
• Manipulation of proliferation and differentiation of pancreatic tissue for example, by altering Wnt, can provide a means for more carefully controlling the characteristics of a cultured tissue.
• pancreatic islets Early progenitor cells to the pancreatic islets are multipotential, and apparently coactivate all the islet-specific genes from the time they first appear. As development proceeds, expression of islet-specific hormones, such as insulin, becomes restricted to the pattern of expression characteristic of mature islet cells. The phenotype of mature islet cells, however, is not stable in culture, as reappearance of embryonal traits in mature B-cells can be observed. By utilizing the compounds of the invention, the differentiation path or proliferative index of the cells can be regulated.
• manipulation of the differentiative state of pancreatic tissue can be utilized in conjunction with transplantation of artificial pancreas so as to promote implantation, vascularization, and in vivo differentiation and maintenance of the engrafted tissue.
• manipulation of Wnt function to affect tissue differentiation can be utilized as a means of maintaining graft viability.
• Many other tumors may, based on evidence such as involvement of the Wnt pathway in these tumors, or detected expression of Wnt or its receptors in these tissues during development, be affected by treatment with the compounds of the invention.
• compositions comprising one or more of the compounds of the invention can be used in the in vitro generation of skeletal tissue, such as from skeletogenic stem cells, as well as the in vivo treatment of skeletal tissue deficiencies.
• the present invention particularly contemplates the use of compounds of the invention to regulate the rate of chondrogenesis and/or osteogenesis.
• skeletal tissue deficiency it is meant a deficiency in bone or other skeletal connective tissue at any site where it is desired to restore the bone or connective tissue, no matter how the deficiency originated, e.g. whether as a result of surgical intervention, removal of tumor, ulceration, implant, fracture, or other traumatic or degenerative conditions.
• the compounds of the invention can be used as part of a regimen for restoring cartilage function to a connective tissue. They are useful in, for example, the repair of defects or lesions in cartilage tissue which is the result of degenerative wear such as that which results in arthritis, as well as other mechanical derangements which may be caused by trauma to the tissue, such as a displacement of torn meniscus tissue, meniscectomy, a laxation of a joint by a torn ligament, malignment of joints, bone fracture, or by hereditary disease.
• the compounds of the invention may also be useful for remodeling cartilage matrix, such as in plastic or reconstructive surgery, as well as periodontal surgery. They may also be applied to improving a previous reparative procedure, for example, following surgical repair of a meniscus, ligament, or cartilage. Furthermore, it may prevent the onset or exacerbation of degenerative disease if applied early enough after trauma.
• One embodiment of the present invention relates to the treating of the afflicted connective tissue with a therapeutically effective amount of compounds of the invention to regulate a cartilage repair response in the connective tissue by managing the rate of differentiation and/or proliferation of chondrocytes embedded in the tissue.
• connective tissues as articular cartilage, interarticular cartilage (menisci), costal cartilage (connecting the true ribs and the sternum), ligaments, and tendons are particularly amenable to treatment in reconstructive and/or regenerative therapies using the subject method.
• regenerative therapies include treatment of degenerative states which have progressed to the point of which impairment of the tissue is obviously manifest, as well as preventive treatments of tissue where degeneration is in its earliest stages or imminent.
• the compounds of the invention can be used as part of a therapeutic intervention in the treatment of cartilage of a diarthroidal joint, such as a knee, an ankle, an elbow, a hip, a wrist, a knuckle of either a finger or toe, or a tempomandibular joint.
• the treatment can be directed to the meniscus of the joint, to the articular cartilage of the joint, or both.
• the compounds of the invention can be used to treat a degenerative disorder of a knee, such as which might be the result of traumatic injury (e.g., a sports injury or excessive wear) or osteoarthritis.
• the compounds of the invention may be administered as an injection into the joint with, for instance, an arthroscopic needle.
• the injected agent can be in the form of a hydrogel or other slow release vehicle described above in order to permit a more extended and regular contact of the agent with the treated tissue.
• the present invention further contemplates the use of the compounds of the invention in the field of cartilage transplantation and prosthetic device therapies.
• problems arise, for instance, because the characteristics of cartilage and fibrocartilage vary between different tissue: such as between articular, meniscal cartilage, ligaments, and tendons, between the two ends of the same ligament or tendon, and between the superficial and deep parts of the tissue.
• the zonal arrangement of these tissues may reflect a gradual change in mechanical properties, and failure occurs when implanted tissue, which has not differentiated under those conditions, lacks the ability to appropriately respond.
• meniscal cartilage is used to repair anterior cruciate ligaments, the tissue undergoes a metaplasia to pure fibrous tissue.
• the compounds of the invention can be used to particularly address this problem, by helping to adaptively control the implanted cells in the new environment and effectively resemble hypertrophic chondrocytes of an earlier developmental stage of the tissue.
• the compounds of the invention can be applied to enhancing both the generation of prosthetic cartilage devices and to their implantation.
• the need for improved treatment has motivated research aimed at creating new cartilage that is based on collagen-glycosaminogly-can templates (Stone et al., Clin. Orthop. Relat. Red 252:129 (1990)), isolated chondrocytes (Grande et al., J. Orthop. Res. 7:208 (1989); and Takigawa et al., Bone Miner 2:449 (1987)), and chondrocytes attached to natural or synthetic polymers (Walitani et al., J. Bone Jt. Surg.
• chondrocytes can be grown in culture on biodegradable, biocompatible highly porous scaffolds formed from polymers such as polyglycolic acid, polylactic acid, agarose gel, or other polymers which degrade over time as function of hydrolysis of the polymer backbone into innocuous monomers.
• the matrices are designed to allow adequate nutrient and gas exchange to the cells until engraftment occurs.
• the cells can be cultured in vitro until adequate cell volume and density has developed for the cells to be implanted.
• One advantage of the matrices is that they can be cast or molded into a desired shape on an individual basis, so that the final product closely resembles the patient's own ear or nose (by way of example), or flexible matrices can be used which allow for manipulation at the time of implantation, as in a joint.
• the implants are contacted with a subject aromatic compound during certain stages of the culturing process in order to manage the rate of differentiation of chondrocytes and the formation of hypertrophic chrondrocytes in the culture.
• the implanted device is treated with a compound of formulae (I), (Ia) to (If), (II) and (IIa) to (IIf) in order to actively remodel the implanted matrix and to make it more suitable for its intended function.
• the artificial transplants suffer from the same deficiency of not being derived in a setting which is comparable to the actual mechanical environment in which the matrix is implanted.
• the ability to regulate the chondrocytes in the matrix by the subject method can allow the implant to acquire characteristics similar to the tissue for which it is intended to replace.
• the compounds of the invention are used to enhance attachment of prosthetic devices.
• the compounds of the invention can be used in the implantation of a periodontal prosthesis, wherein the treatment of the surrounding connective tissue stimulates formation of periodontal ligament about the prosthesis.
• the compounds of the invention can be employed as part of a regimen for the generation of bone (osteogenesis) at a site in the animal where such skeletal tissue is deficient.
• administration of a compound of the present invention can be employed as part of a method for regulating the rate of bone loss in a subject.
• preparations comprising subject compounds can be employed, for example, to control endoch-ondral ossification in the formation of a “model” for ossification.
• the compounds of the invention also have wide applicability to the treatment or prophylaxis of disorders afflicting epithelial tissue, as well as in cosmetic uses. In general, this includes a step of administering to an animal an amount of a subject aromatic compound effective to alter the growth state of a treated epithelial tissue.
• the mode of administration and dosage regimens will vary depending on the epithelial tissue(s) which is to be treated. For example, topical formulations will be preferred where the treated tissue is epidermal tissue, such as dermal or mucosal tissues.
• scarring can be an important obstacle in regaining normal function and appearance of healed skin. This is particularly true when pathologic scarring such as keloids or hypertrophic scars of the hands or face causes functional disability or physical deformity. In the severest circumstances, such scarring may precipitate psychosocial distress and a life of economic deprivation.
• Wound repair includes the stages of hemostasis, inflammation, proliferation, and remodeling. The proliferative stage involves multiplication of fibroblasts and endothelial and epithelial cells. Through the use of the compounds of the invention, the rate of proliferation of epithelial cells in and proximal to the wound can be controlled in order to accelerate closure of the wound and/or minimize the formation of scar tissue.
• the present treatment can also be effective as part of a therapeutic regimen for treating oral and paraoral ulcers, e.g., resulting from radiation and/or chemotherapy.
• Such ulcers commonly develop within days after chemotherapy or radiation therapy.
• These ulcers usually begin as small, painful irregularly shaped lesions usually covered by a delicate gray necrotic membrane and surrounded by inflammatory tissue.
• a lack of treatment results in proliferation of tissue around the periphery of the lesion on an inflammatory basis.
• the epithelium bordering the ulcer usually demonstrates proliferative activity, resulting in loss of continuity of surface epithelium.
• a treatment for such ulcers which includes application of a compound of formulae (I), (Ia) to (If), (II) and (IIa) to (IIf) can reduce the abnormal proliferation and differentiation of the affected epithelium, helping to reduce the severity of subsequent inflammatory events.
• the compounds of the invention and compositions thereof can also be used to treat wounds resulting from dermatological diseases, such as lesions resulting from autoimmune disorders such as psoriasis.
• Atopic dermititis refers to skin trauma resulting from allergies associated with an immune response caused by allergens such as pollens, foods, dander, insect venoms and plant toxins.
• antiproliferative preparations of subject compounds can be used to inhibit lens epithelial cell proliferation to prevent post-operative complications of extracapsular cataract extraction.
• Cataract is an intractable eye disease and various studies on a treatment of cataract have been made. But at present, the treatment of cataract is attained by surgical operations. Cataract surgery has been applied for a long time and various operative methods have been examined. Extracapsular lens extraction has become the method of choice for removing cataracts. The major medical advantages of this technique over intra-capsular extraction are lower incidence of aphakic cystoid macular edema and retinal detachment. Extracapsular extraction is also required for implantation of posterior chamber type intraocular lenses which are now considered to be the lenses of choice in most cases.
• a disadvantage of extracapsular cataract extraction is the high incidence of posterior lens capsule opacification, often called after-cataract, which can occur in up to 50% of cases within three years after surgery.
• After-cataract is caused by proliferation of equatorial and anterior capsule lens epithelial cells which remain after extracapsular lens extraction. These cells proliferate to cause Sommerling rings, and along with fibroblasts which also deposit and occur on the posterior capsule, cause opacification of the posterior capsule, which interferes with vision. Prevention of after-cataract would be preferable to treatment.
• the present invention provides a means for inhibiting proliferation of the remaining lens epithelial cells.
• such cells can be induced to remain quiescent by instilling a solution containing a preparation of a compound of formulae (I), (Ia) to (If), (II) and (IIa) to (IIf) into the anterior chamber of the eye after lens removal.
• the solution can be osmotically balanced to provide minimal effective dosage when instilled into the anterior chamber of the eye, thereby inhibiting subcapsular epithelial growth with some specificity.
• the compounds of the invention can also be used in the treatment of corneopathies marked by corneal epithelial cell proliferation, as for example in ocular epithelial disorders such as epithelial downgrowth or squamous cell carcinomas of the ocular surface.
• Hair is basically composed of keratin, a tough and insoluble protein; its chief strength lies in its disulphide bond of cystine.
• Each individual hair comprises a cylindrical shaft and a root, and is contained in a follicle, a flask-like depression in the skin.
• the bottom of the follicle contains a finger-like projection termed the papilla, which consists of connective tissue from which hair grows, and through which blood vessels supply the cells with nourishment.
• the shaft is the part that extends outwards from the skin surface, whilst the root has been described as the buried part of the hair.
• the base of the root expands into the hair bulb, which rests upon the papilla.
• Cells from which the hair is produced grow in the bulb of the follicle; they are extruded in the form of fibers as the cells proliferate in the follicle.
• Hair “growth” refers to the formation and elongation of the hair fiber by the dividing cells.
• the common hair cycle is divided into three stages: anagen, catagen and telogen.
• anagen the epidermal stem cells of the dermal papilla divide rapidly.
• Daughter cells move upward and differentiate to form the concentric layers of the hair itself.
• the transitional stage, catagen is marked by the cessation of mitosis of the stem cells in the follicle.
• the resting stage is known as telogen, where the hair is retained within the scalp for several weeks before an emerging new hair developing below it dislodges the telogen-phase shaft from its follicle. From this model it has become clear that the larger the pool of dividing stem cells that differentiate into hair cells, the more hair growth occurs. Accordingly, methods for increasing or reducing hair growth can be carried out by potentiating or inhibiting, respectively, the proliferation of these stem cells.
• the compounds of the invention can be employed as a way of reducing the growth of human hair as opposed to its conventional removal by cutting, shaving, or depilation.
• the present method can be used in the treatment of trichosis characterized by abnormally rapid or dense growth of hair, e.g. hypertrichosis.
• subject compounds can be used to manage hirsutism, a disorder marked by abnormal hairiness.
• the compounds of the invention can also provide a process for extending the duration of depilation.
• a subject compound will often be cytostatic to epithelial; cells, rather than cytotoxic, such agents can be used to protect hair follicle cells from cytotoxic agents which require progression into S-phase of the cell-cycle for efficacy, e.g. radiation-induced death.
• Treatment by the compounds of the invention can provide protection by causing the hair follicle cells to become quiescent, e.g., by inhibiting the cells from entering S phase, and thereby preventing the follicle cells from undergoing mitotic catastrophe or programmed cell death.
• compounds of the invention can be used for patients undergoing chemo- or radiation-therapies which ordinarily result in hair loss.
• the subject treatment can protect hair follicle cells from death which might otherwise result from activation of cell death programs.
• the administration of compounds of the invention can be stopped with concommitant relief of the inhibition of follicle cell proliferation.
• the compounds of the invention can also be used in the treatment of folliculitis, such as folliculitis decalvans, folliculitis ulerythematosa reticulata or keloid folliculitis.
• folliculitis such as folliculitis decalvans, folliculitis ulerythematosa reticulata or keloid folliculitis.
• a cosmetic preparation of a compound of formulae (I), (Ia) to (If), (II) and (IIa) to (IIf) can be applied topically in the treatment of pseudofolliculitis, a chronic disorder occurring most often in the submandibular region of the neck and associated with shaving, the characteristic lesions of which are erythematous papules and pustules containing buried hairs.
• the compounds of the invention can be used to induce differentiation and/or inhibit proliferation of epithelially derived tissue.
• Such forms of these molecules can provide a basis for differentiation therapy for the treatment of hyperplastic and/or neoplastic conditions involving epithelial tissue.
• preparations can be used for the treatment of cutaneous diseases in which there is abnormal proliferation or growth of cells of the skin.
• the pharmaceutical preparations of the invention are intended for the treatment of hyperplastic epidermal conditions, such as keratosis, as well as for the treatment of neoplastic epidermal conditions such as those characterized by a high proliferation rate for various skin cancers, as for example basal cell carcinoma or squamous cell carcinoma.
• the compounds of the invention can also be used in the treatment of autoimmune diseases affecting the skin, in particular, of dermatological diseases involving morbid proliferation and/or keratinization of the epidermis, as for example, caused by psoriasis or atopic dermatosis.
• psoriasis squamous cell carcinoma
• keratoacanthoma actinic keratosis
• psoriasis which is characterized by scaly, red, elevated plaques on the skin
• the keratinocytes are known to proliferate much more rapidly than normal and to differentiate less completely.
• the preparations of the present invention are suitable for the treatment of dermatological ailments linked to keratinization disorders causing abnormal proliferation of skin cells, which disorders may be marked by either inflammatory or non-inflammatory components.
• therapeutic preparations of an inventive compound e.g., which promotes quiescense or differentiation, can be used to treat varying forms of psoriasis, be they cutaneous, mucosal orungual.
• Psoriasis as described above, is typically characterized by epidermal keratinocytes which display marked proliferative activation and differentiation along a “regenerative” pathway.
• Treatment with an antipro-liferative compound of the invention can be used to reverse the pathological epidermal activiation and can provide a basis for sustained remission of the disease.
• Actinic keratoses are superficial inflammatory premalig-nant tumors arising on sun-exposed and irradiated skin. The lesions are erythematous to brown with variable scaling.
• Current therapies include excisional and cryosurgery. These treatments are painful, however, and often produce cosmetically unacceptable scarring.
• treatment of keratosis can include application, preferably topical, of a composition containing at least one compound of formulae (I), (Ia) to (If), (II) and (IIa) to (IIf) in amounts sufficient to inhibit hyperproliferation of epidermal/epidermoid cells of the lesion.
• Acne represents yet another dermatologic ailment which may be treated by the compounds of the invention.
• Acne vulgaris for instance, is a multifactorial disease most commonly occurring in teenagers and young adults, and is characterized by the appearance of inflammatory and noninflammatory lesions on the face and upper trunk.
• the basic defect which gives rise to acne vulgaris is hypercornification of the duct of a hyperactive sebaceous gland. Hypercornification blocks the normal mobility of skin and follicle microorganisms, and in so doing, stimulates the release of lipases by Propinobac-terium acnes and Staphylococcus epidermidis bacteria and Pitrosporum ovale, a yeast.
• Treatment with an antiproliferative compound of formulae (I), (Ia) to (If), (II) and (IIa) to (IIf), particularly topical preparations, may be useful for preventing the transitional features of the ducts, e.g. hypercornification, which lead to lesion formation.
• the subject treatment may further include, for example, antibiotics, retinoids and antiandrogens.
• the present invention also provides a method for treating various forms of dermatitis.
• Dermatitis is a descriptive term referring to poorly demarcated lesions which are either pruritic, erythematous, scaly, blistered, weeping, fissured or crusted. These lesions arise from any of a wide variety of causes.
• the most common types of dermatitis are atopic, contact and diaper dermatitis.
• seborrheic dermatitis is a chronic, usually pruritic, dermatitis with erythema, dry, moist, or greasy scaling, and yellow crusted patches on various areas, especially the scalp, with exfoliation of an excessive amount of dry scales.
• the compounds of the invention can also be used in the treatment of stasis dermatitis, an often chronic, usually eczematous dermatitis.
• Actinic dermatitis is dermatitis that due to exposure to actinic radiation such as that from the sun, ultraviolet waves or x- or gamma-radiation.
• the compounds of the invention can be used in the treatment and/or prevention of certain symptoms of dermatitis caused by unwanted proliferation of epithelial cells.
• Such therapies for these various forms of dermatitis can also include topical and systemic corticosteroids, antipuritics, and antibiotics.
• Ailments which may be treated by the compounds of the invention are disorders specific to non-humans, such as mange.
• the compounds of the invention can be used in the treatment of human cancers, particularly basal cell carcinomas and other tumors of epithelial tissues such as the skin.
• compounds of the invention can be employed, in the subject method, as part of a treatment for basal cell nevus syndrome (BCNS), and other other human carcinomas, adenocarcinomas, sarcomas and the like.
• BCNS basal cell nevus syndrome
• the compounds of the invention are used as part of a treatment of prophylaxis regimen for treating (or preventing) basal cell carcinoma.
• the deregulation of the Wnt signaling pathway may be a general feature of basal cell carcinomas caused by ptc mutations. Consistent overexpres-sion of human ptc mRNA has been described in tumors of familial and sporadic BCCs, determined by in situ hybridization. Mutations that inactivate ptc may be expected to result in overexpression of mutant Ptc, because ptc displays negative autoregulation. Likewise, mutations that inactivate Wnt may be expected to result in overexpression of mutant Wnt, because Wnt displays negative autoregulation.
• the compounds of the invention can also be used to treat patients with BCNS, e.g., to prevent BCC or other effects of the disease which may be the result of Wnt-mediated disorders.
• Basal cell nevus syndrome is a rare autosomal dominant disorder characterized by multiple BCCs that appear at a young age.
• BCNS patients are very susceptible to the development of these tumors; in the second decade of life, large numbers appear, mainly on sun-exposed areas of the skin. This disease also causes a number of developmental abnormalities, including rib, head and face alterations, and sometimes polydactyl), syndactyl), and spina bifida.
• the compounds of the invention can be used to prevent or treat such tumor types in BCNS and non-BCNS patients.
• Studies of BCNS patients show that they have both genomic and sporadic mutations in the ptc gene, suggesting that these mutations are the ultimate cause of this disease.
• the present invention provides pharmaceutical preparations and methods for controlling the formation of megakaryocyte-derived cells and/or controlling the functional performance of megakaryocyte-derived cells.
• certain of the compositions disclosed herein may be applied to the treatment or prevention of a variety hyperplastic or neoplastic conditions affecting platelets.
• the invention relates to pharmaceutical compositions comprising at least one compound according to the invention.
• the invention relates to pharmaceutical compositions comprising at least one compound according to the invention in a mixture with an inert carrier, where said inert carrier is a pharmaceutical carrier.
• carrier refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered.
• Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, including but not limited to peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered orally.
• Saline and aqueous dextrose are preferred carriers when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions are preferably employed as liquid carriers for injectable solutions.
• Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
• the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations and the like.
• the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
• Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin. Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.
• the invention further relates to a process for the preparation of a medicament comprising the steps of:
• the compounds according to the invention used for the preparation of a medicament for the modulation of the Wnt signalling pathway in a mammal may be administered in any convenient route.
• the compounds are formulated to be compatible with the desired route of administration and may be administered together with other biologically active agents.
• the compounds may be formulated for the intravenous, intradermal, subcutanus, intramuscular, intraperitoneal, epidural, oral, transdermal, transmucosal, rectal or pulmonary administration. Administration can be systemic or local. Pulmonary administration can be employed by use of an inhaler or nebulizer, and formulation with an aerosolizing agent, for example. In another embodiment, the compound can be delivered in a vesicle, in particular a liposome (Langer (1990) Science 249, 1527.
• the compound can be delivered via a controlled release system.
• a pump may be used (Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14, 201; Buchwald et al. (1980) Surgery 88, 507; Saudek et al. (1989) N. Engl. J. Med. 321, 574).
• polymeric materials can be used (Ranger and Peppas (1983) Macromol. Sci. Rev. Macromol. Chem. 23, 61; Levy et al. (1985) Science 228, 190; During et al. (1989) Ann. Neurol. 25, 351; Howard et al. (1989) J. Neurosurg. 71, 858).
• a controlled release system can be placed in proximity of the therapeutic target, i.e., the brain, thus requiring only a fraction of the systemic dose (e.g. Goodson, 1984, In: Medical Applications of Controlled Release, supra, Vol. 2, 115). Other controlled release systems are discussed in the review by Langer (1990, Science 249, 1527).
• Toxicity and therapeutic efficacy of the compounds of the invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals.
• the results obtained from the cell culture assays and animal studies can be used in formulating the range of dosage for use in medicaments in humans.
• the specific dosage for any particular subject is influenced by several factors, e.g. by the activity of the specific compound used, the age, body weight, general health, gender and diet of the subject, the time and the route of administration and the rate of excretion.
• 1,4-Dihydroxy-naphthalene (704 mg, 4.4 mmol), 3-bromo-4,5-dimethoxy-benz-aldehyde (1077 mg, 4.4 mmol) and malononitrile (295 mg, 4.4 mmol) were taken in 40 ml ethanol at room temperature, charged with DABCO (48.4 ⁇ l, 1.46 mmol) and then refluxed with stirring under LC-MS control for 18 h. The reaction mixture was then cooled down to room temperature. The mixture was diluted with water to about 100 ml, stirred at room temperature for 1 h and the precipitates were separated by filtration. It was washed well with 50% aqueous ethanol and dried under vacuum (1.68 g, 3.7 mmol, 84%).
• a reporter gene based assay describing the modulation of the TCF4 transcription factor was used. More specifically 4000 Hek293T cells were seeded into 384 high density plates. 24 h after seeding a Wnt-sensitive reporter (6 ⁇ TCF-luciferase (Firefly) (pTOP-FLASH; “Armadillo coactivates transcription driven by the product of the Drosophila segment polarity gene dTCF”, Cell, 1997, 88(6), pages 789-99)) and constitutively expressed control reporter ( Renilla luciferase pCMV-RL) were transfected into Hek293T.
• pTOP-FLASH “Armadillo coactivates transcription driven by the product of the Drosophila segment polarity gene dTCF”, Cell, 1997, 88(6), pages 789-99
• Wnt signaling was stimulated by cotransfecting mouse Wnt1, mouse Frizzled 8 and human LRP6 according to “Casein kinase 1 gamma couples Wnt receptor activation to cytoplasmic signal transduction”, Nature, 2005, 438(7069), pages 867-872.
• 24 h after pathway stimulation compounds were added at a concentration of 10 microM and allowed to incubate for 24 h.
• the respective compound was applied in increasing concentrations yielding final concentrations per well of 5 nM-100 microM.
• IC 50 -values of the cytotoxic activity of compounds (1), (4), (6), (7), (16) to (20), (29) to (32) and (39) to (43) against Hek293T and HepG2 are shown in table 2.
• HCT116 human colorectal cancer cells
• SW480 denoted (2)
• Dld-1 denoted (3)
• human fibroblasts HCT68, denoted (4)
• Mc Coy's (1)
• Dulbecco's Modified Eagles Medium (2)-(4)
• fetal calf serum 1% penicillin/streptomycin
• Cells were grown in T75 flasks at 37° C., 5% CO2 and trypsinized when 60-80% confluent by adding 2 ml of 0.25% Trypsin-EDTA (ethylenediaminetetraacetic acid) solution. Cells were then re-suspended into culture medium yielding approx. 750 cells, suspended in 45 microl medium, were plated into each well of a black 384-well plate for fluorescence imaging experiments. 24-36 hr later 5 microl compound solution (100 microM compound dissolved in ultra pure water containing 1% DMSO (dimethylsulfoxide)), were added to achieve a final concentration of 10 microM and were incubated for at least 72 hr.
• Trypsin-EDTA ethylenediaminetetraacetic acid
• Compound incubation was terminated by (a) fixation and (b) permeabilisation of cells, followed by (c) fluorescence labeling of cell nuclei for cytometric quantification or by immunocytochemistry for microscopic evaluation of cell morphology.
• the three steps were performed by replacing the solution of the previous step in each well of a 384-well plate by (a) 30 microl PBS (phosphate buffered saline) containing 5% PFA (paraformaldehyde), (b) 30 microl PBS containing 0.2% TritonX-100 and (c) 10 microl PBS containing Hoechst-33342 for cytometry or Hoechst-33342, FITC-(fluorescein isothiocyanate-) labeled alpha-tubulin antibodies and TRITC-(tetramethylrhodamine isothiocyanate-)labeled phalloidin for microscopy.
• PBS phosphate buffered saline
• PFA paraformal
• Cell line-specific cytotoxicity was quantified by counting the number of Hoechst labeled nuclei per well of a 384-well plate using a plate cytometer. Data obtained with the cytometer was analysed using standard data analysis software.
• Identified hits were further evaluated by visual inspection of fluorescence micrographs obtained from imaging using a conventional fluorescence microscope.
• the Wnt pathway inhibitory activity of compounds (57) to (70) was investigated according to the procedure described in example 54.
• the IC 50 -values of the Wnt pathway inhibitory activity of compounds (57) to (70) were below 100 nM.
• the cytotoxic activity of compounds (57) to (70) was investigated according to the procedure described in example 55.
• Sample preparation The racemate was dissolved in a 1:1 mixture of ethanol and acetonitrile with a final concentration of 10 mg/ml. Injection volume: 2 ml per injection.
• the Wnt pathway inhibitory activity was determined as described in example 54.
• the influence of the racemate and the two separated enantiomeres on colon cancer cell lines Dld1 and HCT116 was determined according to the procedure described in example 56. The results are shown in Table 7.

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