WO2006078886A2

WO2006078886A2 - Compounds and compositions as wnt signaling pathway modulators

Info

Publication number: WO2006078886A2
Application number: PCT/US2006/002007
Authority: WO
Inventors: Sheng Ding; Jun Liu; Peter G. Schultz
Original assignee: IRM LLC; Scripps Research Institute
Current assignee: IRM LLC; Scripps Research Institute
Priority date: 2005-01-18
Filing date: 2006-01-18
Publication date: 2006-07-27
Anticipated expiration: 2007-07-18
Also published as: WO2006078886A3

Abstract

The invention provides a novel class of compounds and methods of using such compounds to study the activity of the Wnt signaling pathway.

Description

COMPOUNDS AND COMPOSITIONS AS WNT SIGNALING PATHWAY MODULATORS

BACKGROUND OF THE INVENTION

Cross-Reference to Related Applications

[0001] This application claims the benefit of U.S. provisional application No.

60/645,366, filed January 18, 2005. The aforementioned application is incorporated herein by reference in its entirety and for all purposes. Field of the Invention

[0002] The invention provides a novel class of compounds and methods of using such compounds to study the activity of the Wnt signaling pathway.

Background

[0003] The Wnt signaling pathway plays a role in a variety of biological processes by regulating genes involved in cellular adhesion, proliferation and differentiation. In this pathway, the Wnt ligand activates downstream signal transduction through interaction with receptors of the 'Frizzled' family on the cell surface. This interaction, through several cytoplasmic relay components, leads to inhibition of β-catenin phosphorylation by the Axin, APC and GSK-3β complex, and results in accumulation of β-catenin in the cytoplasm, β- catenin then translocates into the nucleus, forms a complex with T cell factor/lymphoid enhancer factor (TCF/LEF) which drives target gene expression. Compounds of the invention, as modulators of the Wnt pathway, offer temporal control over Wnt signaling and provide useful tools for studying embryogenesis and adult tissue homeostasis and regeneration. Summary of the Invention

[0004] In one aspect, the present invention provides compounds of Formula I:

in which: n is selected from 1 , 2 and 3 ;

Ri is selected from hydrogen and Ci-βalkoxy;

R₂, R₃ and R₄ are independently selected from hydrogen and C^aUcyl;

R₅ is selected from C₆-i₂aryl-Co₄alkyl and

wherein any aryl or heteroaryl of R₅ is optionally substituted with 1 to 3 radicals independently selected from hydroxy, cyano and halo; and the N-oxide derivatives, prodrug derivatives, protected derivatives, individual isomers and mixture of isomers thereof; and the pharmaceutically acceptable salts and solvates (e.g. hydrates) of such compounds. [0005] In another aspect, the present invention provides a pharmaceutical composition which contains a compound of Formula I or a N-oxide derivative, individual isomers and mixture of isomers thereof; or a pharmaceutically acceptable salt thereof, in admixture with one or more suitable excipients.

[0006] In another aspect, the present invention provides a method for modulating the Wnt signaling pathway. Compounds of the invention that modulate the Wnt signaling pathway are useful tools for studying biological processes that involve Wnt signaling such as hematopoietic stem cell renewal, mesenchymal progenitor cell differentiations, embryonic limb development, and the like. By offering a temporal control over the Wnt signaling pathway, compounds of the invention can be useful for studying, for example, embryogenesis and adult tissue homeostasis and regeneration.

[0007] In another aspect, compounds of the invention can be used to reveal new physiological effects of Wnt signaling in model organisms and can be used to develop new therapeutic targets. [0008] In another aspect, the present invention provides a method of treating a disease in an animal in which modulation of the Wnt signaling pathway activity, can prevent, inhibit or ameliorate the pathology and/or symptomology of the diseases, which method comprises administering to the animal a therapeutically effective amount of a compound of Formula I or a N-oxide derivative, individual isomers and mixture of isomers thereof, or a pharmaceutically acceptable salt thereof.

[0009] In another aspect, the present invention provides the use of a compound of

Formula I in the manufacture of a medicament for treating a disease in an animal in which aberrant Wnt signaling pathway activity contributes to the pathology and/or symptomology of the disease.

[0010] In another aspect, the present invention provides a process for preparing compounds of Formula I and the N-oxide derivatives, prodrug derivatives, protected derivatives, individual isomers and mixture of isomers thereof, and the pharmaceutically acceptable salts thereof.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0011] "Alkyl" as a group and as a structural element of other groups, for example halo-substituted-alkyl and alkoxy, can be either straight-chained or branched. Ci_₄-alkoxy includes, methoxy, ethoxy, and the like. Halo-substituted alkyl includes trifluoromethyl, pentafluoroethyl, and the like.

[0012] An "Agonist", as used herein, refers to a compound of the invention that mimics or upregulates (e. g. potentiates or supplements) a Wnt-signaling bioactivity. An agonist therapeutic can be a compound that upregulates expression of a member of the Wnt-signaling pathway such as the beta-catenin gene or which increases at least one bioactivity of the beta- catenin protein. An agonist can also be a compound which increases the interaction of a beta-catenin polypeptide with another molecule, e. g, a member of the TCF- 4/LEF family. [0013] "Aryl" means a monocyclic or fused bicyclic aromatic ring assembly containing six to ten ring carbon atoms. For example, aryl may be phenyl or naphthyl, preferably phenyl. "Arylene" means a divalent radical derived from an aryl group. [0014] "Heteroaryl" is as defined for aryl above where one or more of the ring members is a heteroatom. For example heteroaryl includes pyridyl, indolyl, indazolyl, quinoxalinyl, quinolinyl, benzofuranyl, benzopyranyl, benzothiopyranyl, benzo[l,3]dioxole, imidazolyl, benzo-imidazolyl, pyrimidinyl, furanyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, thienyl, etc.

[0015] "Cycloalkyl" means a saturated or partially unsaturated, monocyclic, fused bicyclic or bridged polycyclic ring assembly containing the number of ring atoms indicated. For example, C_3-iocycloalkyl includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc. [0016] "Heterocycloalkyl" means cycloalkyl, as defined in this application, provided that one or more of the ring carbons indicated, are replaced by a moiety selected from -O-, -N=, -NR-, -C(O)-, -S-, -S(O) - or -S(O)₂-, wherein R is hydrogen, C_1-4alkyl or a nitrogen protecting group. For example, C_3-8heterocycloalkyl as used in this application to describe compounds of the invention includes morpholino, pyrrolidinyl, pyrrolidinyl-2-one, piperazinyl, piperidinyl, piperidinylone, l,4-dioxa-8-aza-spiro[4.5]dec-8-yl, etc. [0017] "Halogen" (or halo) preferably represents chloro or fluoro, but may also be bromo or iodo.

[0018] "Treat", "treating" and "treatment" refer to a method of alleviating or abating a disease and/or its attendant symptoms.

[0019] "Wnt pathway" and "Wnt signaling pathway" refer to the pathway by which binding of the Wnt protein to its extracellular receptor is translated into the nucleus and results in transcriptional activation of a variety of genes. The Wnt signaling pathway involves a variety of proteins including Frizzled, Disheveled, Axin, APC, GSK-3 beta, beta- catenin, LEF/TCF transcription factors, etc. Cells from many different species express homologs of the proteins involved in the Wnt signaling pathway and accordingly have functionally equivalent Wnt signaling pathways.

Description of the Preferred Embodiments

[0020] In one embodiment, in relation to a compound of Formula I:

Ri is selected from hydrogen and methoxy;

R₂, R₃ and R₄ are hydrogen; and

R₅ is selected from benzo[l,3]dioxol-5-yl and benzyl optionally substituted with hydroxy.

[0021] Preferred compounds include: N⁴-benzo[l,3]dioxol-5-ylmethyl-6-(3- methoxy-phenyl)-pyrimidine-2,4-diamine; 4-[2-(2-amino-6-phenyl-pyrimidin-4-ylamino)- ethyl]-phenol; N⁴-benzo[l,3]dioxol-5-ylmethyl-6-phenyl-pyrimidine-2,4-diamine; and 4-{2- [2-amino-6-(3-methoxy-phenyl)-pyrimidin-4-ylamino]-ethyl}-phenol. Compounds of the invention are further detailed in the Examples and Table I, infra. [0022] In another embodiment, the invention provides for a method for modulating Wnt signal transduction comprising contacting a cell with an amount of a compound of claim 1, effective to change Wnt signal transduction. The method involves modulating β-catenin and TCF mediated transcriptional control and may or may not include stimulating the activity of GSK-3β. Preferred methods are exemplified in the Assays, infra. [0023] In another embodiment, modulating the Wnt signal transduction results in mimicking Wnt signaling bioactivity.

[0024] In another embodiment, the Wnt signal transduction results in upregulating the Wnt signaling bioactivity.

[0025] In another embodiment, compounds of the invention can be used to modulate the Wnt signal pathway and thereby can be useful for studying biological processes that involve Wnt signaling, such as stem cell renewal, mesenchymal progenitor cell differentiation, embryonic limb development and the like.

Pharmacology and Utility

[0026] The Wnt gene family encodes secreted ligand proteins that serve key roles in differentiation and development. This family comprises at least 15 vertebrate and invertebrate genes including the Drosophila segment polarity gene wingless and one of its vertebrate homologues, integrated from which the Wnt name derives. The Wnt proteins, via their signal transduction components and links to the hedgehog development pathway, appear to facilitate a number of developmental and homeostatic processes. [0027] β-catenin protein transduces the Wnt signal from the cytoplasm to the nucleus. In the absence of Wnt signaling, β-catenin is constitutively degraded by the proteasome and can be found in a multimeric complex with conductin (or axin), APC (Adenomatous Polyposis CoIi) and GSK-3β. GSK-3β, a serine/threonine kinase, phosphorylates β-catenin thus stimulating its degradation by the proteasome. Upon Wnt signaling, the GSK-3β kinase is inactivated leading to stabilization of the β-catenin protein, β-catenin is then released from the multimeric complex and translocates into the nucleus. [0028] Once in the nucleus, β-catenin interacts with the LEF/TCF (Lymphoid

Enhancer Factor/T-Cell Factor) family of HMG (High Mobility Group) box transcription factors. The LEF/TCF factors are stimulated through interaction with β-catenin to become potent transactivators of a number of genes including c-myc, cyclin Dl, c-jun and hedgehog

(Wi).

[0029] The invention provides for an agonist therapeutic, which can mimic or potentiate the activity of the Wnt-signaling pathway by, for example, promoting, mimicking, or potentiating the interaction between β-catenin and a member of the TCF/LEF family. An agonist therapeutic of the invention has utility for disorders of cell fate and can be administered: (1) in diseases or disorders involving an absence or decreased (relative to normal, or desired) levels of Wnt function, for example, in patients where any member of the Wnt-signaling pathway is lacking, genetically defective, biologically inactive or underactive, or under expressed; and (2) in diseases or disorders wherein in vitro (or in vivo) assays indicate the utility of Wnt agonist administration.

[0030] An agonist therapeutic is administered (1) in diseases or disorders involving an absence or decreased levels of Wnt function, for example, inpatients where β- catenin is lacking, genetically defective, biologically inactive or underactive, or under expressed; and (2) in diseases or disorders wherein in vitro (or in vivo) assays indicate the utility of Wnt agonist administration. [0031] Absence of Wnt pathway function (or decreased levels of function) can be readily detected, e. g., by obtaining a patient tissue sample (e. g., from biopsy tissue) and assaying it in vitro for protein levels, structure and/or activity of the expressed β-catenin protein. Many methods standard in the art can be employed to detect Wnt signal modulation and/or decreased or increased activity of the Wnt signal pathway component proteins. In vitro assays which can be used to determine whether administration of a specific agonist therapeutic is indicated, include in vitro cell culture assays in which a patient tissue sample is grown in culture, and exposed to or otherwise administered a therapeutic, and the effect of such a therapeutic upon the tissue sample is observed.

[0032] Compounds of the invention can be used as therapeutics for the promotion of cell growth, upon a patient cell sample from tissue having or suspected of having a hypo- proliferative disorder, such as disorders including diseases or conditions associated with insufficient cell proliferation, such as stimulation of tissue repair, tissue regeneration, wound healing, neovascularization, and the like.

[0033] In accordance with the foregoing, the present invention further provides useful tools for the study of the Wnt signal pathway and/or for preventing or treating any of the diseases or disorders described above in a subject in need of such treatment, which method comprises administering to said subject a therapeutically effective amount (See, "Administration and Pharmaceutical Compositioiis", infra) of a compound of Formula I or a pharmaceutically acceptable salt thereof. For any of the above uses, the required dosage will vary depending on the mode of administration, the particular condition to be treated and the effect desired.

Administration and Pharmaceutical Compositions

[0034] In general, compounds of the invention will be administered in therapeutically effective amounts via any of the usual and acceptable modes known in the art, either singly or in combination with one or more therapeutic agents. A therapeutically effective amount may vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the compound used and other factors. In general, satisfactory results are indicated to be obtained systemically at daily dosages of from about 0.03 to 2.5mg/kg per body weight. An indicated daily dosage in the larger mammal, e.g. humans, is in the range from about 0.5mg to about lOOmg, conveniently administered, e.g. in divided doses up to four times a day or in retard form. Suitable unit dosage forms for oral administration comprise from ca. 1 to 50mg active ingredient. [0035] Compounds of the invention can be administered as pharmaceutical compositions by any conventional route, in particular enterally, e.g., orally, e.g., in the form of tablets or capsules, or parenterally, e.g., in the form of injectable solutions or suspensions, topically, e.g., in the form of lotions, gels, ointments or creams, or in a nasal or suppository form. Pharmaceutical compositions comprising a compound of the present invention in free form or in a pharmaceutically acceptable salt form in association with at least one pharmaceutically acceptable carrier or diluent can be manufactured in a conventional manner by mixing, granulating or coating methods. For example, oral compositions can be tablets or gelatin capsules comprising the active ingredient together with a) diluents, e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine; b) lubricants, e.g., silica, talcum, stearic acid, its magnesium or calcium salt and/or polyethyleneglycol; for tablets also c) binders, e.g., magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and or polyvinylpyrrolidone; if desired d) disintegrants, e.g., starches, agar, alginic acid or its sodium salt, or effervescent mixtures; and/or e) absorbents, colorants, flavors and sweeteners. Injectable compositions can be aqueous isotonic solutions or suspensions, and suppositories can be prepared from fatty emulsions or suspensions. The compositions may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers. In addition, they may also contain other therapeutically valuable substances. Suitable formulations for transdermal applications include an effective amount of a compound of the present invention with a carrier. A carrier can include absorbable pharmacologically acceptable solvents to assist passage through the skin of the host. For example, transdermal devices are in the form of a bandage comprising a backing member, a reservoir containing the compound optionally with carriers, optionally a rate controlling barrier to deliver the compound to the skin of the host at a controlled and predetermined rate over a prolonged period of time, and means to secure the device to the skin. Matrix transdermal formulations may also be used. Suitable formulations for topical application, e.g., to the skin and eyes, are preferably aqueous solutions, ointments, creams or gels well-known in the art. Such may contain solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives.

[0036] Compounds of the invention can be administered in therapeutically effective amounts in combination with one or more therapeutic agents (pharmaceutical combinations). Where the compounds of the invention are administered in conjunction with other therapies, dosages of the co-administered compounds will of course vary depending on the type of co-drug employed, on the specific drug employed, on the condition being treated and so forth.

[0037] The invention also provides for a pharmaceutical combinations, e.g. a kit, comprising a) a first agent which is a compound of the invention as disclosed herein, in free form or in pharmaceutically acceptable salt form, and b) at least one co-agent. The kit can comprise instructions for its administration.

[0038] The terms "co-administration" or "combined administration" or the like as utilized herein are meant to encompass administration of the selected therapeutic agents to a single patient, and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time. [0039] The term "pharmaceutical combination" as used herein means a product that results from the mixing or combining of more than one active ingredient and includes both fixed and non-fixed combinations of the active ingredients. The term "fixed combination" means that the active ingredients, e.g. a compound of Formula I and a co- agent, are both administered to a patient simultaneously in the form of a single entity or dosage. The term "non-fixed combination" means that the active ingredients, e.g. a compound of Formula I and a co-agent, are both administered to a patient as separate entities either simultaneously, concurrently or sequentially with no specific time limits, wherein such administration provides therapeutically effective levels of the 2 compounds in the body of the patient. The latter also applies to cocktail therapy, e.g. the administration of 3 or more active ingredients. Processes for Making Compounds of the Invention

[0040] The present invention also includes processes for the preparation of compounds of the invention. In the reactions described, it can be necessary to protect reactive functional groups, for example hydroxy, amino, imino, thio or carboxy groups, where these are desired in the final product, to avoid their unwanted' participation in the reactions. Conventional protecting groups can be used in accordance with standard practice, for example, see T. W. Greene and P. G. M. Wuts in "Protective Groups in Organic

Chemistry", John Wiley and Sons, 1991.

[0041] Compounds of Formula I can be prepared by proceeding as in the following Reaction Scheme I:

Reactions Scheme I

(2) (D

[0042] wherein n, Ri, R₂, R₃, R₄ and R₅ are as described in the Summary of the

Invention. A compound of Formula I is formed by reacting a compound of formula 2 and formula 3 in the presence of a suitable solvent (for example, water, acetonitrile, or the like) and in the presence of a suitable catalyst (for example, Pd(PPh₃)₄, or the like). The reaction proceeds at a temperature range of about 100⁰C to about 18O⁰C and can take up to about 10 hours to complete.

[0043] A detailed example of the synthesis of a compound of Formula I can be found in the Examples, infra. Additional Processes for Making Compounds of the Invention

[0044] A compound of the invention can be prepared as a pharmaceutically acceptable acid addition salt by reacting the free base form of the compound with a pharmaceutically acceptable inorganic or organic acid. Alternatively, a pharmaceutically acceptable base addition salt of a compound of the invention can be prepared by reacting the free acid form of the compound with a pharmaceutically acceptable inorganic or organic base.

[0045] Alternatively, the salt forms of the compounds of the invention can be prepared using salts of the starting materials or intermediates.

[0046] The free acid or free base forms of the compounds of the invention can be prepared from the corresponding base addition salt or acid addition salt from, respectively.

For example a compound of the invention in an acid addition salt form can be converted to the corresponding free base by treating with a suitable base (e.g., ammonium hydroxide solution, sodium hydroxide, and the like). A compound of the invention in a base addition salt form can be converted to the corresponding free acid by treating with a suitable acid

(e.g., hydrochloric acid, etc.).

[0047] Compounds of the invention in unoxidized form can be prepared from N- oxides of compounds of the invention by treating with a reducing agent (e.g., sulfur, sulfur dioxide, triphenyl phosphine, lithium borohydride, sodium borohydride, phosphorus trichloride, tribromide, or the like) in a suitable inert organic solvent (e.g. acetonitrile, ethanol, aqueous dioxane, or the like) at 0 to 80⁰C.

[0048] Prodrug derivatives of the compounds of the invention can be prepared by methods known to those of ordinary skill in the art (e.g., for further details see Saulnier et al., (1994), Bioorganic and Medicinal Chemistry Letters, Vol. 4, p. 1985). For example, appropriate prodrugs can be prepared by reacting a non-derivatized compound of the invention with a suitable carbamylating agent (e.g., 1,1-acyloxyalkylcarbanochloridate, para- nitrophenyl carbonate, or the like).

[0049] Protected derivatives of the compounds of the invention can be made by means known to those of ordinary skill in the art. A detailed description of techniques applicable to the creation of protecting groups and their removal can be found in T. W. Greene, "Protecting Groups in Organic Chemistry", 3^rd edition, John Wiley and Sons, Inc., 1999.

[0050] Compounds of the present invention can be conveniently prepared, or formed during the process of the invention, as solvates (e.g., hydrates). Hydrates of compounds of the present invention can be conveniently prepared by recrystallization from an aqueous/organic solvent mixture, using organic solvents such as dioxin, tetrahydrofuran or methanol.

[0051] Compounds of the invention can be prepared as their individual stereoisomers by reacting a racemic mixture of the compound with an optically active resolving agent to form a pair of diastereoisomeric compounds, separating the diastereomers and recovering the optically pure enantiomers. While resolution of enantiomers can be carried out using covalent diastereomeric derivatives of the compounds of the invention, dissociable complexes are preferred (e.g., crystalline diastereomeric salts). Diastereomers have distinct physical properties (e.g., melting points, boiling points, solubilities, reactivity, etc.) and can be readily separated by taking advantage of these dissimilarities. The diastereomers can be separated by chromatography, or preferably, by separation/resolution techniques based upon differences in solubility. The optically pure enantiomer is then recovered, along with the resolving agent, by any practical means that would not result in racemization. A more detailed description of the techniques applicable to the resolution of stereoisomers of compounds from their racemic mixture can be found in Jean Jacques, Andre Collet, Samuel H. Wilen, "Enantiomers, Racemates and Resolutions", John Wiley And Sons, Inc., 1981.

[0052] In summary, the compounds of Formula I can be made by a process, which involves:

(a) that of reaction scheme I; and

(b) optionally converting a compound of the invention into a pharmaceutically acceptable salt;

(c) optionally converting a salt form of a compound of the invention to a non-salt form;

(d) optionally converting an unoxidized form of a compound of the invention into a pharmaceutically acceptable N-oxide; (e) optionally converting anN-oxide form of a compound of the invention to its unoxidized form;

(f) optionally resolving an individual isomer of a compound of the invention from a mixture of isomers;

(g) optionally converting a non-derivatized compound of the invention into a pharmaceutically acceptable prodrug derivative; and

(h) optionally converting a prodrug derivative of a compound of the invention to its non-derivatized form.

[0053] Insofar as the production of the starting materials is not particularly described, the compounds are known or can be prepared analogously to methods known in the art or as disclosed in the Examples hereinafter.

[0054] One of skill in the art will appreciate that the above transformations are only representative of methods for preparation of the compounds of the present invention, and that other well known methods can similarly be used.

Examples

[0055] The present invention is further exemplified, but not limited, by the following examples that illustrate the preparation of compounds of Formula I according to the invention.

Example 1

^-BenzorLSIdioxol-S-ylmethyl-β-rS-methoxy-υhenvD-pyrimidine-Z^-diamine

[0056] 2-Amino-4,6-dichloropyrimidine (100mg, 0.61 mmol) is dissolved 1- butanol (5mL). Diisopropylethylamine (DIEA; 117μL, 0.67mmol) and piperonylamine (92.2mg, 0.61mmol) are then added to the mixture. The reaction mixture is heated at 7O⁰C for 12 hours and the desired product, N^-benzofl^JdioxoI-S-ylmethyl-^β-chloro-pyrimidine- 2,4-diamine, is purified by column chromatography.

[0057] N⁴-benzo[ 1 ,3]dioxol-5-ylmethyl-6-chloro-pyrimidine-2,4-diamine

(0.036mmol), Pd(PPh₃)₄ (2.1mg, 0.0018mmol), Na₂CO₃ (15.2mg, 0.144mmol), acetonitrile/water (1:1; 2mL) and 3-methoxy-phenylboronic acid (l lmg, 0.072mmol) are mixed together under Argon. The reaction mixture is heated at 150oC for 20 minutes in a microwave reactor to give N⁴-benzo[l,3]dioxol-5-ylmethyl-6-(3-methoxy-phenylV pyrimidine-2.4-diamine which is further purified by preparative RP-HPLC and lyophilized to a white powder: ¹H NMR (40OmHz, DMSOd₆): δ (ppm) 3.84 (s, 3H), 4.54 (d, 2H₅ J=5.8), 6.0 (s, 2H), 6.39 (s, IH), 6.85 (m, 2H), 6.96 (s, IH), 7.19 (m, IH), 7.28 (m, 2H), 7.52 (m, IH), 9.13 (s, IH); MS (m/z): 351.1455.

[0058] By repeating the procedures described in the above examples, using appropriate starting materials, the following compounds of Formula I, as identified in Table 1, are obtained.

Table 1

Assays

[0059] β-catenin/TCF transcriptional activity is dependent on Wnt signaling and can be used to monitor activity of the Wnt signaling pathway. A reporter based cellular assay is used to measure the transcriptional activity of β-catenin/TCF in 384-well cell culture plates. The reporter construct encodes the luciferase gene driven by β-catenin/TCF responsive elements. 293T cells are transiently transfected with the reporter construct in T75 cell culture flasks and replated into 384-well cell culture plates after overnight culture. After 24 hours, cells are treated with lOμM of a compound of the invention and incubated for a further 24 hours. Luciferase activity is measured and ECs₀S are measured when the signal is inhibited by 50%. [0060] Compounds of Formula I preferably have an EC50 of less than or equal to lμM. For example. N⁴-benzo[l ,31dioxol-5-ylmemyl-6-f3-methoxy-phenyiypyrimidine-2,4- diamine (Example 1) induces β-catenin and TCF dependent transcriptional activity in a dose dependent manner with an EC₅₀ of 0.7μM. Compounds 2, 3 and 4 of table 1 have an EC₅₀ of l.OμM, 0.9μM and 0.8μM, respectively.

[0061] The Wnt signal is transduced through its receptor, resulting in formation of a heterodimeric complex of β-catenin and TCF, which drives down stream target expression. In order to test if compounds of the invention induce reporter gene expression in a TCF- dependent manner, a dominant negative TCF4 was constructed and transfected into cells in the absence/presence of compounds of the invention. The dominant negative TCF4 contains a DNA binding domain but lacks the β-catenin interaction motif, leading to sequestration of TCF factor binding sites and subsequent blocking of β-catenin and TCF dependent transcriptional activity. The gene encoding the dominant negative TCF4 is generated by PCR and cloned into an eukaryotic expression vector pcDNA3. Dominant negative TCF4 is co-transfected with the Wnt-responsive reporter construct into 293T cells, and 16-24 hours later the compounds (agonists) are added to cells at the final concentration of 1-10 μM. After another 24 hours, luciferase activity is measured and EC5₀S are calculated when the signal is activated by 50%.

[0062] Compounds of the invention block β-catenin and TCF dependent reporter gene expression in the presence of the dominant negative TCF4, suggesting that compounds of the invention function in the canonical Wnt pathway through the TCF factors. Compounds of the invention activate the Wnt pathway without inhibiting GSK-3β.

Whole organism model

[0063] Compounds of the invention are tested for their ability to function in a whole organism model. During early Xenopus development, Wnt antagonism plays an essential role in head specification. Expression of Wnt inhibitors results in large heads and forebrains. Conversely, in activation of Wnt inhibitors or overexpression of Wnt activators leads to microcephalic embryos. This development process provides an experimental means to test the compounds of the invention's ability to modulate Wnt signaling in vivo. Embryos are treated with 10μM of a compound of the invention or the vehicle, DMSO, at a stage 10.5 (gastrulation stage) for 24 hours and allowed to develop until stage 40 (tadpole stage). The phenotypes are observed using techniques known in the art.

[0064] Compounds of the invention show substantial head defects ranging from significantly diminished heads and complete loss of eyes to reduced eyes and heads while the posterior structures of tadpoles remained intact. This phenotype is consistent with Wnt overexpression in head specification during early embryonic development suggesting that the compounds of the invention mimic the effects of Wnt at the whole organism level.

[0065] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. AU publications, patents, and patent applications cited herein are hereby incorporated by reference for all purposes.

Claims

WE CLAIM:

1. A compound of Formula I:

in which: n is selected from 1 , 2 and 3;

Ri is selected from hydrogen and Ci-βalkoxy;

R₂, R₃ and R₄ are independently selected from hydrogen and C^aUcyl;

R₅ is selected from Q-^aryl-Co^alkyl and Cs-ioheteroaryl-Co^alkyl; wherein any aryl or heteroaryl of R₅ is optionally substituted with 1 to 3 radicals independently selected from hydroxy, cyano and halo; and the pharmaceutically acceptable salts, hydrates, solvates and isomers thereof.

2. The compound of claim 1 in which:

Ri is selected from hydrogen and methoxy; R₂, R₃ and R₄ are hydrogen; and R₅ is selected from benzo[l,3]dioxol-5-yl and benzyl optionally substituted with hydroxy.

3. The compound of claim 2 selected from: N⁴-benzo[l,3]dioxol-5-ylmethyl-6-(3- methoxy-phenyl)-pyrimidine-2,4-diamine; 4-[2-(2-amino-6-phenyl-pyrimidin-4-ylamino)- ethyl] -phenol; N⁴-benzo[l,3]dioxol-5-ylmethyl-6-phenyl-pyrimidine-2,4-diamine; and 4-{2- [2-amino-6-(3-methoxy-phenyl)-pyrimidin-4-ylamino]-ethyl}-phenol.

4. A method for modulating Wnt signal transduction comprising contacting a cell with an amount of a compound of claim 1, effective to change Wnt signal transduction.

5. The method of claim 4 in which modulating the Wnt signal transduction results in mimicking Wnt signaling bioactivity.

6. The method of claim 4 in which modulating the Wnt signal transduction results in upregulating the Wnt signaling bioactivity.

7. The method of claim 4 in which modulating the Wnt signal pathway is used to study biological processes that involve Wnt signaling.

8. The method of claim 7 in which the biological process is stem cell renewal.

9. The method of claim 7 in which the biological process is mesenchymal progenitor cell differentiation.

10. The method of claim 7 in which the biological process is embryonic limb development.

11. A pharmaceutical composition comprising a therapeutically effective amount of a compound of Claim 1 in combination with a pharmaceutically acceptable excipient.

12. A method for treating a disease or disorder in an animal in which modulation of the Wnt signal pathway can prevent, inhibit or ameliorate the pathology and/or symptomology of the disease, which method comprises administering to the animal a therapeutically effective amount of a compound of Claim 1.