The present invention relates to compounds which function as Wnt pathway inhibitors and which are capable of reducing the proliferation of tumor cells and/or preventing metastasis. The invention further relates to certain novel compounds, to pharmaceutical formulations containing such compounds and to methods for their preparation.

The Wnt/p-catenin signaling pathway is a key regulator in numerous cellular processes including stem cell maintenance, fate decision and cell cycle control. The main denominator of canonical Wnt signaling, β-catenin, has several cellular functions. At the cell membrane, it is associated with E-cadherin and participates in the formation of the adherens junctions. In the cytoplasm, β-catenin can form complexes with a multitude of proteins, including the β-catenin destruction complex consisting of APC, ΑΧΓΝ2, GSK3 and CKla. Tankyrase 1 and 2 (TNKSl/2) regulate the stability of the β-catenin destruction complex through AXIN2 by poly(ADP-ribosyl)ation. While TNKSl/2 modulates the destruction complex, this complex interferes with TNKS.

Evidence suggests that TNKS is phosphorylated in a cell cycle-dependent manner by GSK3 . TNKS is furthermore controlled by the ubiquitin E3 ligase RNF146 that destabilises TNKSl/2, AXIN2 and itself by ubiqitinylation. The destruction complex regulates the sequential phosphorylation of β-catenin by the kinases CKla at S45 and by GSK3 at S33, S37 and T41. This leads to polyubiquitination by β-TrCP and subsequent degradation of β-catenin by the proteasome.

Active Wnt signaling inhibits the function of the destruction complex, and β-catenin is not phosphorylated at the N-terminus and degraded but enters the nucleus. Nuclear uptake of β-catenin may be further enhanced by the context-dependent C-terminal phosphorylation of β-catenin at S675, leading to increased nuclear translocation. In addition to canonical Wnt signaling, a variety of alternative cellular mechanisms may trigger altered nuclear accumulation of β-catenin. These mechanisms include the Hif-la signaling pathway, down-regulation of E-cadherin or C-terminal phosphorylation of β-catenin through HGF-activated cMet.

Furthermore, PKA-involving mechanisms can lead to C-terminal phosphorylation of β-catenin and enhance its nuclear presence. Recently it was discovered that the primary cilium diverts Jouberin from the nucleus and limits β-catenin nuclear entry. In the nucleus, β-catenin exerts a number of functions, including binding to the transcription factor TCF/LEF by replacing Groucho and activation of downstream transcription of genes, including c-Myc, Cyclin Dl and AXIN2. Nuclear β-catenin levels are not only determined by the rate of β-catenin import to the nucleus, but also by "nuclear trapping" of β-catenin through factors such as TCF/LEF. In general, mutations in genes encoding central components of the Wnt/ -catenin pathway, including the β-catenin destruction complex, lead to the accumulation of nuclear β-catenin and contribute to tumor initiation and progression (Barker et al, Nat. Rev. Drug. Discov. 5 : 997- 1014, 2006; and MacDonald et al, Dev. Cell 17: 9-26, 2009). Wnt activating mutations are found in a broad range of solid tumors including colon cancer, gastric cancer, hepatocellular carcinoma, breast cancer, medulloblastoma, melanoma, non-small cell lung cancer, pancreas adenocarcinoma and prostate cancer (Najdi et al, J. Carcinog. 10: 5, 2011). It has been well established that the majority of intestinal neoplasia with deregulated Wnt signaling harbor truncating mutations in both alleles of the adenomatous polyposis coli (APG) tumor suppressor gene (Nieuwenhuis et al., Crit. Rev. Oncol. Hematol. 61_: 153-61, 2007).

Considerable efforts have been made to identify drugs that inhibit Wnt^-catenin signaling (see Barker et al., supra; Chen et al., Nat. Chem. Biol. 5: 100-7, 2009; Curtin et al., Oncotarget1: 563-77, 2010; Thorne et al, Nat. Chem. Biol. 6: 829-836, 2010; and Verkaar et al, Drug Discovery Today 16: 35-41, 2011). Known inhibitory drugs that interact with biotargets directly associated with canonical Wnt signaling include WAY-316606 for SFRP (Bodine et al., Bone 44: 1063-8, 2009), Niclosamide for frizzled (Chen et al, Biochemistry 48: 10267-74, 2009), NCS668036 for Dsh (Shan et al, Biochemistry 44: 15495-503, 2005), Pyrvinium for CK1 (Thorne et al, supra), XAV939 and IWR-1 for TNKSl/2 (Huang et al, Nature 461 : 614-20, 2009; and Chen et al, Nat. Chem, Biol. 5: 100-7, 2009), 2,4-diamino-quinazoline, Quercetin, PKF115-584 and ICG-001 for β-catenin and its binding to TCF or CREB (Chen et al, Bioorg. Med. Chem. Lett. 19: 4980-3, 2009; Emami et al, Proc. Natl. Acad. Sci. USA 101 : 12682-7, 2004; Lepourcelet et al, Cancer Cell 5: 91-102, 2004; and Park et al, Biochem. Biophys. Res. Commun. 328: 227-34, 2005). TNKSl/2, in particular, are promising biotargets for pharmaceutical agents. TNKSl/2 is not only involved in controlling canonical Wnt signaling, but has been associated with further cellular processes through either protein poly(ADP-ribosyl)ation, or by protein complex formation. These are: (i) telomere maintenance by interacting with TRFl; (ii) spindle formation and stabilization by binding to NuMA; and (iii) glucose metabolism by regulating GLUT4 transport through IRAP.

We have now identified a class of compounds which function as TNKS inhibitors. Although not wishing to be bound by theory, it is believed that these inhibit the PARP domain of TNKS 1 and 2, leading to the stabilisation of AXIN2 followed by increased degradation of β-catenin. Such compounds are suitable for inhibiting the proliferation of tumor cells in general and, in particular, those associated with breast cancer, non-small cell lung cancer, pancreatic and colorectal cancers. They are especially suitable for inhibiting the growth of colon carcinoma cells. The compounds also find use in preventing metastasis.

In one aspect the invention provides compounds of general formula I for use in the treatment and/or prevention of any condition or disease in which it is desirable to inhibit signaling in the Wnt athway:

(I)

(wherein

ring A represents an optionally substituted aryl or heteroaryl group;

ring B represents

an optionally substituted cycloalkyl group, or

an optionally substituted, saturated heterocyclic ring;

Z ¹ represents

an optionally substituted aryl or heteroaryl group,

an optionally substituted, saturated heterocyclic ring, or

a Ci _-6 alkyl group;

Z ² represents an optionally substituted aryl group;

L ¹ and L ² are linkers independently selected from the group consisting of:

-CO-NR-(CR ₂ ) _m - -(CR ₂ ) _m -NR-CO- -CO-0-(CR ₂ ) _m - -(CR ₂ ) _m -0-CO-

-(CR ₂ ) _m -CO- in which each m is an integer from 0 to 2, preferably 0 or 1, and

each R independently represents hydrogen or Ci _-6 alkyl (e.g. C _1-4 alkyl), preferably hydrogen or methyl, e.g. hydrogen)

the stereoisomers, pharmaceutically acceptable salts, and prodrugs thereof. In formula (I), ring A may be linked to the remainder of the molecule via any of the available ring positions. Where this is a 6-membered ring, this will generally be linked to groups L ¹ and L ² in the 1,3- or 1,4-positions (i.e. meta- or para-). Particularly preferably, this will be linked in the para-position. Where ring A is 5-membered, linkage to groups L ¹ and L ² will generally be via the 1,3-position.

Ring A may be an aryl group optionally carrying one or more ring substituents (e.g. 1, 2, 3 or 4 substituents). In a preferred embodiment, the aryl group may be monocyclic, for example an optionally substituted phenyl group. In a particularly preferred embodiment, ring A is a substituted or unsubstituted 1,4-phenylene group.

Where ring A is a heteroaryl group, this may contain one or more heteroatoms selected from oxygen, nitrogen and sulphur and may carry one or more ring substituents (e.g. 1, 2, 3 or 4 substituents). Preferred heteroaryl groups A are those containing at least one nitrogen atom, e.g. a single nitrogen atom. Preferably, the heteroaryl group is monocyclic, e.g. a 6-membered monocycle. A preferred example of a heteroaryl ring is an optionally substituted pyridinyl group.

In the case where ring A is subsituted, the ring substituents may be the same or different. Such substituents are as herein defined and include, for example, halogen atoms (such as CI and F) and Ci _-6 alkyl groups (e.g. methyl). Where the ring is substituted, this may be mono- or polysubstituted. Up to 4 ring substituents may be present, although it is preferred that only one or two are present, e.g. one. In one embodiment, the aryl or heteroaryl ring A is unsubstituted. Where ring B in formula (I) is a cycloalkyl group, this may or may not be substituted.

Preferably, this will be unsubstituted. Preferred B rings are those which are 5- or 6-membered, i.e. cyclopentyl or cyclohexyl. In another embodiment, ring B may represent an optionally substituted, saturated heterocyclic ring. The ring may be substituted by any of the substituents herein defined or may be unsubstituted. Unsubstituted heterocyclic rings are preferred. Suitable heterocyclic rings are those containing one or two heteroatoms selected from oxygen, nitrogen and sulphur (e.g. from oxygen and nitrogen). Those containing a single oxygen or a single nitrogen atom are particularly preferred. Where the heteroatom is nitrogen, this may be substituted by a hydrogen atom or by a C _1-4 alkyl group (e.g. methyl). Preferred heterocyclic rings are those which are 5- or 6-membered (preferably 6-membered), in particular those containing a single oxygen atom, e.g. in the 3- or 4-position. Tetrahydropyranyl is a preferred example of ring B. The use of compounds in which ring A is 1,4-phenylene and ring B is tetrahydropyranyl forms one aspect of the invention. According to this aspect, the invention provides compounds for use as herein described having the general formula II:

(wherein

Z ¹ , Z ² , L ¹ and L ² are as hereinbefore defined;

each X is independently selected from halogen (i.e. F, CI, Br, I) and Ci _-6 alkyl (preferably Ci-3 alkyl); and

n is an integer from 0 to 4, preferably 0 or 1)

the stereoisomers, pharmaceutically acceptable salts, and prodrugs thereof.

In formula II, n denotes the number of ring substituents which may be present. As will be appreciated, when n is zero the ring substituents are hydrogen. Where X is present, n will generally be either 1 or 2, preferably 1. Preferred ring substituents are halogen atoms, e.g. F or CI.

In the compounds herein described, the linkers L ¹ and L ² may be the same or different.

Examples of suitable linkers include -CO- R-, - R-CO-, -CO- R-CR ₂ -, -CR ₂ - R-CO-,

-CO-0-CR ₂ , -CR ₂ -0-CO- and -CO- (in which R is as hereinbefore defined, preferably hydrogen or methyl, e.g. hydrogen). In one embodiment, the linkers L ¹ and L ² are different.

Preferred compounds for use according to the invention are those of general formula III:

(wherein

Z ¹ , Z ² , X and n are as hereinbefore defined; and

R ¹ , R ² , R ³ and R ⁴ are each independently selected from hydrogen and Ci _-6 alkyl (e.g. Ci _-3 alkyl)) the stereoisomers, pharmaceutically acceptable salts, and prodrugs thereof.

In formula III, it is preferred that at least one of R ³ and R ⁴ is hydrogen. Preferably, both R ³ and R ⁴ are hydrogen.

In a preferred embodiment, R ¹ and R ² in formula III each independently represent hydrogen or Ci _-3 alkyl, e.g. hydrogen or methyl. Preferably, both R ¹ and R ² are hydrogen.

In any of the embodiments herein described, Z ¹ may be an optionally substituted aryl or heteroaryl group. Whilst such groups may be bicyclic, generally these will be monocyclic. A preferred aryl ring is optionally substituted phenyl. 5- or 6-membered heteroaryl groups, which may or may not be substituted, are preferred. Those containing 5 ring members are particularly preferred. The heteroaryl groups may contain one or more heteroatoms selected from oxygen and nitrogen. Preferably, these will contain at least one oxygen atom. In the case where Z ¹ is substituted, the ring substituents may be the same or different. Such substituents are as herein defined and include, for example, Ci _-6 alkyl (e.g. Ci _-3 alkyl) and Ci _-6 alkoxy (e.g. Ci _-3 alkoxy). Where any ring substituents are present, it is preferred that only one or two are present, e.g. one. In one embodiment, the aryl or heteroaryl ring is unsubstituted.

In another embodiment, Z ¹ may be a saturated heterocyclic ring, preferably a saturated 5- or 6- membered heterocycle. Such rings containing one or more oxygen and/or nitrogen atoms are preferred, for example those containing one oxygen and one nitrogen atom, or those containing either a single oxygen or single nitrogen atom. In one embodiment, such rings may be substituted. Where substituted, the ring substituents may be the same or different and may be selected from any of those herein defined. In another embodiment, the saturated heterocycle may be unsubstituted.

In a yet further embodiment, Z ¹ may represent a Ci _-6 alkyl group. Such groups may be straight- chained or branched.

Preferred examples of group Z ¹ include the following:

Of these structures, the following are particularly preferred for Z ¹ :

In preferred embodiments, Z ² represents a monocyclic aryl group, e.g. a phenyl group optionally substituted by one or more substituents which may be the same or different. Preferably, only one substituent will be present. Optional substituents include those herein defined, e.g. halogen (preferably CI or F), hydroxy and Ci _-6 alkoxy (preferably C _1-3 alkoxy, e.g. methoxy).

Examples of group Z ² include the following:

Of these structures, the following are particularly preferred for Z ² :

The following are examples of particularly preferred compounds for use in accordance with the invention:

Compound No. Structure

- 10 -

- 12 -

Particularly preferred compounds for use in accordance with the invention are Compound Nos. (1), (2), (4)-(6), (11)-(20) and (23)-(26) and their isomers, pharmaceutically acceptable salts thereof and prodrugs. More particularly preferred compounds for use in accordance with the invention are Compound Nos. (1), (14)-(16), (18), (19), (24) and (26), e.g. Compound Nos. (1),

(12), (14), (15) and (26).

Unless otherwise stated, all substituents are independent of one another. In the case where an asterisk (*) is present in any of the structural formulae of any of the substituents provided herein, this is to be understood as indicating the point of attachment of that substituent to the remainder of the molecule.

Unless otherwise stated, the term "halo" or "halogen atom" may be fluoro, chloro, bromo, or iodo. Preferably, this is fluoro or chloro.

As used herein, the term "alkyl" refers to a saturated hydrocarbon group and is intended to cover both straight-chained and branched alkyl groups. Examples of such groups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, n-pentyl, iso-pentyl, neo- pentyl, n-hexyl, etc. An alkyl group preferably contains from 1-6 carbon atoms, e.g. 1-4 carbon atoms. Unless otherwise stated, any alkyl group mentioned herein may optionally be substituted by one or more groups, which may be identical or different, for example hydroxy, alkoxy, acyloxy, amino or halogen atoms (e.g. F, CI or Br). Alternatively, the alkyl group may be unsubstituted. As used herein, the term "cycloalkyl" is intended to cover any cyclic alkyl group. Such groups can include mono- or polycyclic ring systems (e.g. having 2 fused rings). These may have from 3-20 carbon atoms, preferably 3-14 carbons, more preferably 3-10 carbons, e.g. 3-7 carbons. Monocyclic rings are preferred, for example those having 5 or 6 ring members. Examples of such groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, etc.

Cyclopentyl and cyclohexyl are particularly preferred.

As used herein, the term "saturated heterocyclic ring" is intended to cover any 3-, 4-, 5-, 6- or 7- membered heterocyclic ring which contains at least one heteroatom selected from nitrogen, oxygen and sulphur. Preferred saturated heterocyclic rings are those containing 5 or 6 ring members. The heterocyclic ring structure may be linked to the remainder of the molecule through a carbon atom or, if present, through a nitrogen atom. Preferably it will be linked to the remainder of the molecule through a carbon atom. Examples of such groups include

pyrrolidinyl, tetrahydrofuranyl, tetrahydrothiophene, pyrazolidinyl, imidazolidinyl, 1,3- dioxolane, thiazolidinyl, isoxazolidinyl, piperidinyl, piperazinyl, morpholinyl, 1,4-dioxane, thiomorpholinyl, 1,4-oxathiane and 1,4-dithiane. Unless otherwise stated, any heterocyclic ring mentioned herein may optionally be substituted by one or more groups, which may be identical or different, for example hydroxy, Ci _-6 alkyl, Ci _-6 alkoxy or halogen atoms (e.g. F, CI or Br).

As used herein, the term "aryl" is intended to cover aromatic ring systems. Such ring systems may be monocyclic or polycyclic (e.g. bicyclic) and contain at least one unsaturated aromatic ring. Where these contain polycyclic rings, these may be fused. Preferably such systems contain from 6-20 carbon atoms, e.g. either 6 or 10 carbon atoms. Examples of such groups include phenyl, 1-napthyl and 2-napthyl. Monocyclic ring systems are preferred and include phenyl.

Unless stated otherwise, any "aryl" group may be substituted by one or more substituents, which may be identical or different, for example Ci _-4 alkyl groups, hydroxy, methoxy and halo groups (e.g. CI or F). As used herein, the term "heteroaryl" is intended to cover heterocyclic aromatic groups. Such groups may be monocyclic or bicyclic and contain at least one unsaturated heteroaromatic ring system. Where these are monocyclic, these comprise 5- or 6-membered rings which contain at least one heteroatom selected from nitrogen, oxygen and sulphur and contain sufficient conjugated bonds to form an aromatic system. Where these are bicyclic, these may contain from 9-11 ring atoms. Examples of heteroaryl groups include thiophene, thienyl, pyridyl, thiazolyl, furyl, pyrrolyl, triazolyl, imidazolyl, oxadiazolyl, oxazolyl, pyrazolyl, imidazolonyl, oxazolonyl, thiazolonyl, tetrazolyl, thiadiazolyl, benzimidazolyl, benzooxazolyl, benzofuryl, indolyl, isoindolyl, pyridonyl, pyridazinyl, pyrimidinyl, imidazopyridyl, oxazopyridyl, thiazolopyridyl, imidazopyridazinyl, oxazolopyridazinyl, thiazolopyridazinyl and purinyl. Preferred heteroaryl groups are monocyclic and include, for example, those containing a single heteroatom (e.g. nitrogen). Unless stated otherwise, any "heteroaryl" may be substituted by one or more substituents, which may be identical or different, for example C _1-4 alkyl groups, hydroxy, methoxy and halo groups.

The term "prodrug" is intended to encompass any compound which under physiological conditions is converted into any of the compounds herein described. Suitable prodrugs include compounds which are hydrolysed under physiological conditions to the desired molecule. The compounds for use according to the invention may in some cases be commercially available, for example from companies such as Chembionet

(h ttp :// ww .chembi onet. i nfo/inde .php?id ^:::: 28) or Ambinter (http://w\¾^.ambinter.cotn/coniact). Alternatively, these may be prepared from readily available starting materials using synthetic methods known in the art. The compounds may, for example, be obtained in accordance with the following methods:

(a) (in order to prepare a compound of formula I in which L ¹ represents a group -CO- H-) reacting a compound of general formula IV:

(IV)

with a compound of general formula V:

Z -CO-O-CH ₃ (V) The reaction is conveniently carried out in a solvent or mixture of solvents, such as for example toluene, expediently at temperatures up to 150°C, preferably at temperatures between -20 and 80°C.

(b) (in order to prepare a compound of formula I in which L ² represents a group -CO- H-CH ₂ -) reacting a compound of general formula VI:

(VI)

with a compound of general formula VII:

(VII)

The reaction is conveniently carried out in a solvent or mixture of solvents, such as for example toluene, expediently at temperatures up to 150°C, preferably at temperatures between -20 and 80°C.

(c) resolving a compound of general formula I obtained into the stereoisomers thereof; and/or

(d) converting a compound of general formula I into a salt thereof, particularly a

pharmaceutically acceptable salt thereof.

The compounds used as starting materials are either known from the literature or may be commercially available. Alternatively, these may be obtained by methods known from the literature.

The invention includes all optical isomers and stereoisomers of the compounds herein disclosed. The compounds herein described may be resolved into their enantiomers and/or diastereomers. For example, where these contain only one chiral centre, these may be provided in the form of a racemate or may be provided as pure enantiomers, i.e. in the R- or S-form. Any of the compounds which occur as racemates may be separated into their enantiomers by methods known in the art, such as column separation on chiral phases or by recrystallisation from an optically active solvent. Those compounds with at least two asymmetric carbon atoms may be resolved into their diastereomers on the basis of their physical -chemical differences using methods known per se, e.g. by chromatography and/or fractional crystallisation, and where these compounds are obtained in racemic form, they may subsequently be resolved into the enantiomers.

The compounds herein described may be converted into a salt thereof, particularly into a pharmaceutically acceptable salt thereof with an inorganic or organic acid or base. Acids which may be used for this purpose include hydrochloric acid, hydrobromic acid, sulphuric acid, sulphonic acid, methanesulphonic acid, phosphoric acid, fumaric acid, succinic acid, lactic acid, citric acid, tartaric acid, maleic acid, acetic acid, trifluoroacetic acid and ascorbic acid. Bases which may be suitable for this purpose include alkali and alkaline earth metal hydroxides, e.g. sodium hydroxide, potassium hydroxide or cesium hydroxide, ammonia and organic amines such as diethylamine, triethylamine, ethanolamine, diethanolamine, cyclohexylamine and

dicyclohexylamine. Procedures for salt formation are conventional in the art.

Whilst certain compounds for use in accordance with the invention are known per se, these are not described for any pharmaceutical purpose. Accordingly, in a further aspect the invention provides a pharmaceutical formulation comprising any compound as herein described, or a pharmaceutically acceptable salt thereof, together with one or more pharmaceutically acceptable carriers or excipients.

Certain compounds herein described are in themselves novel and these form a further aspect of the invention. Viewed from this aspect, the invention provides a compound according to any of the general formulae herein described with the proviso that the compound is other than a compound selected from the group consisting of the compound Nos. (1)-(13), (21) and (22).

In one embodiment the invention provides compounds of general formula la:

(la)

(wherein

ring A represents a substituted aryl or optionally substituted heteroaryl group;

ring B represents

an optionally substituted cycloalkyl group, or

an optionally substituted, saturated heterocyclic ring;

Z ¹ represents

an optionally substituted aryl or heteroaryl group,

an optionally substituted, saturated heterocyclic ring, or

a Ci-6 alkyl group;

Z ² represents an optionally substituted aryl group;

L ¹ and L ² are linkers independently selected from the group consisting of:

-CO- R-(CR ₂ ) _m - -(CR ₂ ) _m - R-CO- -CO-0-(CR ₂ ) _m - -(CR ₂ ) _m -0-CO-

-(CR ₂ ) _m -CO- in which each m is an integer from 0 to 2, preferably 0 or 1, and

each R independently represents hydrogen or Ci _-6 alkyl (e.g. C _1-4 alkyl), preferably hydrogen or methyl, e.g. hydrogen)

the stereoisomers, pharmaceutically acceptable salts, and prodrugs thereof.

Ring A in formula la is preferably a phenyl ring substituted by one or more halogen atoms, for example one halogen atom (e.g. CI). Alternatively, ring A may be an unsubstituted pyridyl group.

In an alternative embodiment, the invention provides compounds of general formula lb

(lb)

(wherein

ring A represents an optionally substituted aryl or heteroaryl group;

ring B represents

an optionally substituted cycloalkyl group, or

an optionally substituted, saturated heterocyclic ring;

Z ¹ represents

a substituted heteroaryl group, or

an optionally substituted heteroaryl group which contains two or more heteroatoms; Z ² represents an optionally substituted aryl group;

L ¹ and L ² are linkers independently selected from the group consisting of:

-CO- R-(CR ₂ ) _m - -(CR ₂ ) _m - R-CO- -CO-0-(CR ₂ ) _m - -(CR ₂ ) _m -0-CO-

-(CR ₂ ) _m -CO- in which each m is an integer from 0 to 2, preferably 0 or 1, and

each R independently represents hydrogen or Ci _-6 alkyl (e.g. C _1-4 alkyl), preferably hydrogen or methyl, e.g. hydrogen)

the stereoisomers, pharmaceutically acceptable salts, and prodrugs thereof.

Z ¹ in formula lb is preferably an optionally substituted, 5-membered heteroaryl group containing a single oxygen atom.

In a further embodiment, the invention provides compounds of general formula Ic:

(Ic)

(wherein

ring A represents an optionally substituted aryl or heteroaryl group;

ring B represents

an optionally substituted cycloalkyl group, or

an optionally substituted, saturated heterocyclic ring;

Z ¹ represents

an optionally substituted aryl or heteroaryl group,

an optionally substituted, saturated heterocyclic ring, or

a Ci-6 alkyl group;

Z ² represents an aryl group substituted by one or more halogen atoms;

L ¹ and L ² are linkers independently selected from the group consisting of:

-CO- R-(CR ₂ ) _m - -(CR ₂ ) _m - R-CO- -CO-0-(CR ₂ ) _m - -(CR ₂ ) _m -0-CO-

-(CR ₂ ) _m -CO- in which each m is an integer from 0 to 2, preferably 0 or 1, and

each R independently represents hydrogen or Ci _-6 alkyl (e.g. C _1-4 alkyl), preferably hydrogen or methyl, e.g. hydrogen)

the stereoisomers, pharmaceutically acceptable salts, and prodrugs thereof. Z ² in formula Ic is preferably a phenyl ring substituted by one or more halogen atoms (e.g. F or CI), e.g. by a single halogen atom.

In a yet further embodiment, the invention provides compounds of general formula Id:

(Id)

(wherein

ring A represents an optionally substituted aryl or heteroaryl group;

ring B represents

an optionally substituted cycloalkyl group, or

an optionally substituted, saturated heterocyclic ring;

Z ¹ represents

an optionally substituted aryl or heteroaryl group,

an optionally substituted, saturated heterocyclic ring, or

a Ci-6 alkyl group;

Z ² represents an optionally substituted aryl group;

L ¹ and L ² are linkers independently selected from the group consisting of:

-CO- R-(CR ₂ ) _m - -(CR ₂ ) _m - R-CO- -CO-0-(CR ₂ ) _m - -(CR ₂ ) _m -0-CO-

-(CR ₂ ) _m -CO- in which each m is an integer from 0 to 2, preferably 0 or 1, and

each R independently represents hydrogen or a Ci _-6 alkyl (e.g. C _1-4 alkyl) group, provided that at least one R group is present and is other than hydrogen)

the stereoisomers, pharmaceutically acceptable salts, and prodrugs thereof. In formula Id, at least one R group is preferably methyl.

In respect of any of the general formulae la, lb, Ic and Id, ring A, ring B, Z ¹ , Z ² , L ¹ and L ² may further be defined according to any of the embodiments herein described in relation to the use of the compounds. Preferred compounds according to the invention are those having the structures (14)-(20) and (23)-(26) as herein described.

The compounds herein described and their pharmaceutically acceptable salts have valuable pharmacological properties, particularly an inhibitory effect on β-catenin. In view of their ability to inhibit signaling in the Wnt pathway, and in particular to reduce the levels of nuclear B- catenin, the compounds herein described and their pharmaceutically acceptable salts are suitable for the treatment and/or prevention of any condition or disease which may be affected by over- activation of signaling in the Wnt pathway, in particular those conditions or diseases which involve activation of B-catenin.

The term "Wnt signaling pathway" is used to refer to the chain of events normally mediated by Wnt, LRP (LDL-receptor related protein), Frizzled and B-catenin, among others, and resulting in changes in gene expression and other phenotypic changes typical of Wnt activity.

The Wnt pathway plays a central role in the pathology of a variety of cancers. The compounds of the invention are thus particularly suitable for preventing and/or retarding proliferation of tumor cells, in particular carcinomas such as adenocarcinomas. More specifically, the compounds are effective in treatment and/or prevention of the following cancers: colon cancers (such as colorectal cancer), pancreatic cancer (e.g. pancreas adenocarcinoma), gastric cancer, liver cancers (e.g. hepatocellular and hepatoblastoma carcinomas), Wilms tumor of the kidney, medulloblastoma, skin cancers (e.g. melanoma), non-small cell lung cancer, cervical cancer, ovarian cancers (e.g. ovarian endometrial cancer), bladder cancer, thyroid cancers (e.g.

anaplastic thyroid cancer), head and neck cancer, breast cancer and prostate cancer. Particularly preferably, the compounds herein described may be used in the treatment and/or prevention of breast cancer, non-small cell lung cancer, ovarian, thyroid, colorectal and pancreatic cancers. Treatment or prevention of breast, non-small cell lung, pancreatic and colorectal cancers represents a particularly preferred aspect of the invention. As used herein, the term "proliferation" refers to cells undergoing mitosis. The term "retarding proliferation" indicates that the compounds inhibit proliferation of a cancer cell. In preferred embodiments, "retarding proliferation" indicates that DNA replication is at least 10% less than that observed in untreated cells, more preferably at least 25% less, yet more preferably at least 50% less, e.g. 75%, 90% or 95% less than that observed in untreated cancer cells. The term "carcinoma" refers to any malignant growth which arises from epithelial cells.

Exemplary carcinomas include basal cell carcinoma, squamous cell carcinoma and

adenocarcinoma. Adenocarcinomas are malignant tumors originating in the glandular epithelium and include colorectal, pancreatic, breast and prostate cancers.

Viewed from a further aspect the invention thus provides a compound as herein described, or a pharmaceutically acceptable salt thereof, for use in therapy. Unless otherwise specified, the term "therapy" as used herein is intended to include both treatment and prevention.

In a still further aspect the invention provides a compound as herein described, or a

pharmaceutically acceptable salt thereof, for use in the treatment or prevention of colon cancers (such as colorectal cancer), pancreatic cancer, gastric cancer, liver cancers (e.g. hepatocellular and hepatoblastoma carcinomas), Wilms tumor of the kidney, medulloblastoma, skin cancers (e.g. melanoma), non-small cell lung cancer, cervical cancer, ovarian endometrial cancer, bladder cancer, anaplastic thyroid cancer, head and neck cancer, breast cancer or prostate cancer. In another aspect the invention provides the use of a compound as herein described, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for use in a method of treatment or prevention of colon cancers (such as colorectal cancer), pancreatic cancer, gastric cancer, liver cancers (e.g. hepatocellular and hepatoblastoma carcinomas), Wilms tumor of the kidney, medulloblastoma, skin cancers (e.g. melanoma), non-small cell lung cancer, cervical cancer, ovarian endometrial cancer, bladder cancer, anaplastic thyroid cancer, head and neck cancer, breast cancer or prostate cancer.

Also provided is a method of treatment of a human or non-human animal body to combat or prevent colon cancers (such as colorectal cancer), pancreatic cancer, gastric cancer, liver cancers (e.g. hepatocellular and hepatoblastoma carcinomas), Wilms tumor of the kidney,

medulloblastoma, skin cancers (e.g. melanoma), non-small cell lung cancer, cervical cancer, ovarian endometrial cancer, bladder cancer, anaplastic thyroid cancer, head and neck cancer, breast cancer, or prostate cancer, said method comprising the step of administering to said body an effective amount of a compound as herein described or a pharmaceutically acceptable salt thereof.

Small molecules that selectively target the developmental pathways which control pattern formation during embryogenesis, including Wnt signalling pathways, are considered to be valuable for directing differentiation of pluripotent stem cells toward many desired tissue types (see Wang et al, ACS Chemical Biology, 16 November 2010). As modulators of Wnt signalling, the compounds herein described also have effects on the development of cellular differentiation. The compounds described herein therefore have valuable properties for use in regenerative medicine, for example in protocols for lineage specific in vitro differentiation of progenitor cells. By "progenitor cell" is meant a cell with the capacity to differentiate into another cell type, e.g. a stem cell.

According to this aspect, the present invention provides a method (e.g. an in vitro method) of promoting and/or directing cellular differentiation comprising contacting a progenitor cell with an effective amount of a compound as herein described or a pharmaceutically acceptable salt thereof. In particular, the progenitor cell is contacted with said at least one compound under suitable conditions and for a sufficient time for the progenitor cell to differentiate into a new cell type. In a related aspect, the present invention provides the use of at least one compound as herein described for promoting and/or directing cellular differentiation of a progenitor cell, especially in vitro.

Preferably, the progenitor cell is a totipotent or a pluripotent cell, especially a stem cell such as an embryonic stem cell. Preferred are mammalian progenitor cells such as mouse, rat and human cells, especially human cells. Such stem cells may be obtained from established cell cultures or may be derived directly from mammalian tissue by methods known in the art, including non tissue-destructive methods.

In a preferred embodiment, the progenitor cell is promoted and/or directed to differentiate into a new cell type which is a myocyte (e.g. a cardiomyocyte), a neuronal cell (e.g. a dopaminergic neuronal cell), an endocrine pancreatic cell or a hepatocyte or a cell type which may further differentiate into a myocyte, a neuronal cell, an endocrine pancreatic cell or a hepatocyte. Especially preferably, the progenitor cell is an embryonic stem cell and the new cell type is a cardiomyocyte, a dopaminergic neuronal cell, an endocrine pancreatic cell or a hepatocyte, especially a cardiomyocyte. The dosage required to achieve the desired activity will depend on the compound which is to be administered, the patient, the nature and severity of the condition, the method and frequency of administration and may be varied or adjusted according to choice. Typically, the dosage may be expected to be in the range from 1 to 100 mg, preferably 1 to 30 mg (when administered intravenously) and from 1 to 1000 mg, preferably from 1 to 200 mg (when administered orally).

The compounds herein described may be formulated with one or more conventional carriers and/or excipients according to techniques well known in the art. Typically, the compositions will be adapted for oral or parenteral administration, for example by intradermal, subcutaneous, intraperitoneal or intravenous injection. Suitable pharmaceutical forms thus include plain or coated tablets, capsules, suspensions and solutions containing the active component optionally together with one or more conventional inert carriers and/or diluents, such as corn starch, lactose, sucrose, microcrystalline cellulose, magnesium stearate, polyvinylpyrrolidone, citric acid, tartaric acid, water, water/ethanol, water/glycerol, water/sorbitol, water/polyethyleneglycol, propylene glycol, stearyl alcohol, carboxymethylcellulose or fatty substances such as hard fat or suitable mixtures of any of the above.

The pharmacological properties of the compounds herein described can be analysed using standard assays for functional activity. Detailed protocols for testing of the compounds are provided in the Examples.

The invention will now be described in more detail in the following non-limiting Examples and with reference to the accompanying figures in which:

Figure 1 - compound (1) specifically reduces canonical Wnt signaling in reporter cells and in Xenopus embryos (0.1% DMSO is used as a control). Figure 1 A shows a luciferase reporter activity in FIEK293 cells, transiently transfected with ST-Luc and Renilla plasmids, and treated with compound (1) at 0.1-10 μιηοΙ/L. The "+" and "-" denote assays with and without 30% Wnt3a-CM, respectively. The lines show the IC-value levels. Figure IB shows the effect of 1 and 10 μιηοΙ/L compound (1) in NIH/3T3 Shh Light II cells. The "+" and "-" denote assays with and without stimulation with 50% Shh-CM. Cyclopamine is used as the positive control. Figure 1C shows the effect of 1 and 10 μιηοΙ/L compound (1) in HEK293 cells transiently transfected with a F-KB reporter ( F-KB-LUC) and Renilla. The "+" and "-" denote assays with and without 10 ng/ml TNFa. The results in all reporter assays (Figs. A-C) show the mean values of at least three independent experiments and error bars present standard deviations. Figure ID (left) shows the quantification of axis duplication in Xenopus embryos co-injected with 10 pg XWnt8 and 0.5% DMSO (grey), or 10 pg XWnt8 and 2 pmol JW55 (black). The axis duplication rate was significantly (*) reduced after co-injection of compound (1) (P = 0.001). Collected data from three independent assays are shown. Figure ID (right) shows representative images of embryos obtained 36 hours after the injection.

Figure 2 - compound (1) inhibits canonical Wnt signaling in colorectal cancer cells (0.1% DMSO is used as a control. Control bars are shown in grey and compound (l)-treated samples are shown in black). Figure 2 A (left) shows that the inhibition of GSK3 (25 mM LiCl) in HEK293 cells, transiently transfected with ST-Luc and Renilla, is counteracted by 0.1-10 μπιοΙ/L compound (1). The "+" and "-" denote assays with and without 25 mM LiCl. The mean values of three independent assays and standard deviations are shown. Figure 2A (right) shows HEK293 cells transiently transfected with ST-Luc and Renilla plus full-length β-catenin or da- Cat. The "+" and "-" denote assays with and without β-catenin plasmids (wt. β-catenin and da- Cat). 10 μιηοΙ/L compound (1) inhibited luciferase activity induced by ectopic wild-type β-cat (* P < 0.05) while da-Cat induction was unaffected, n = total number of measurements from multiple independent assays. The standard deviations are shown as error bars. Figure 2B shows the compound (l)-mediated reduction of luciferase activity in different colorectal cancer cell lines stably transfected with ST-Luc and Renilla. The left panel shows a dose-dependent reduction of ST-Luc activation detected in HCT-15 and SW480 cells harboring mutant APC. The right panel shows a compound (l)-mediated reduction in HCT116 cells containing a single allele mutation in S45 of β-catenin. The mean values and the standard error of several independent assays are shown. Figure 2C shows that 25 or 10 μιηοΙ/L compound 1 reduced the relative expression of AXIN2, SP5 and NKDI mRNA in the CRC cells SW480 and DLD-1 as shown by real-time RT-PCR analysis. The means of three independent experiments are shown along with error bars depicting standard deviations. Figure 3 - compound (1) mediates increased AXIN2 stability and induces β-catenin degradation (0.1% DMSO is used as a control). Figure 3 A shows a Western blot analysis of lysates from SW480 cells after incubations with compound (1) for 24 hours. Antibodies against AXIN2, ABC (active β-catenin) and ρβ-catenin (N-terminal phosphorylated β-catenin) were used.

ACTIN (cytoplasmic) or LAMEST Bl (nuclear extracts) documented equal protein loading (full- length and uncropped blots are shown in Fig. 9). The blots show representative data derived from multiple experiments. Figure 3B shows the cellular redistribution of β-catenin and stabilization of ΑΧΓΝ2 in SW480 cells incubated with 5 μιηοΙ/L compound (1) for 48 hours. The arrows show clustered AXIN2 and β-catenin. The confocal microscopy images are representative examples from one of several independent experiments.

Figure 4 - compound (1) specifically inhibits T KS1, TNKS2 but not PARP1 in biochemical assays. Figure 4 A shows the results obtained with compound (1). Figure 4B shows the results obtained with XAV939 (logarithmic scale). Figure 4C shows the calculated IC ₅₀ -values in μΜ. Mean values represent two independent experiments and the error bars show standard deviations.

Figure 5 - Scheme showing the proposed effect of compound (1) on protein stability in the β- catenin destruction complex. The dark grey vertical arrow indicates nuclear translocation of β- catenin and expression Wnt target genes in a Wnt "on" state. The light grey horiziontal arrows indicate TNKS, AXIN2 and RNF146 degradation in untreated cells. The black arrows depict the inhibition of the PARP domain of TNKS by compound (1), leading to stabilization of the destruction complex, increased N-terminal phosphorylation of β-catenin and degradation in the proteasome. Figure 6 - Inhibition of canonical Wnt signaling by compound (1) promotes cell cycle arrest and specifically reduces proliferation in SW480 CRC cells. Figure 6A (left) shows FACS scatter plots showing SW480 cells labeled with BrdU and PI after incubation in 10 μιηοΙ/L or 0.1% DMSO. The different cell cycle phases, Gl, S and G2/M, are gated in three different

compartments. Figure 6A (right) is a table showing a representative percent cell cycle phase distribution (mean values) of cells after incubation with compound (1) or DMSO. Figure 6B shows cell growth curve as measured by IncuCyte showing a concentration-dependent decrease of proliferation in SW480 cells compared to the Wnt-independent colorectal cancer control cell line RKO. SW480 and RKO cells were incubated with 0.1% DMSO or exposed to compound (1). The plot shows the mean value of two independent experiments and all relative standard deviations are below 20%. Figure 6C shows a relative cell count (%) of SW480 and Wnt- independent HeLa cells under serial passages in media containing compound (1). The cell count of control cells cultured in 0.05% DMSO was defined as 100%. The graphs depict the mean values of several experiments and the error bars show the standard errors. Figure 6D shows a colony assay that demonstrates a specific decrease in colony formation of SW480 cells cultured in various doses of compound (1) and 1% DMSO. The bar chart to the left shows a dose- dependent reduction of SW480 colonies and unaffected growth of the control cell lines HeLa and RKO. 0.05% DMSO was used as a control. The graphs show the mean values of several measurements from multiple experiments along with error bars depicting standard deviations. The images to the right are from a representative assay show that the general SW480 colony sizes are smaller in increased doses of compound (1).

Figure 7 - Determination of IC ₅₀ -values using XLfit. The IC ₁₀₀ value is defined as the level of the positive control (30% Wnt3a-CM (Fig. 7 A) or 25 mM LiCl (Fig. 7B)) subtracted by the negative control (no stimulation). The ICo-value is defined as the level of the negative control. The grey rectangles depict the ICso-value (Y-axis, relative activities in percentage %) and the concentration in μιηοΙ/L (X-axis). The results in the reporter assays (A and B) show the mean values of at least three independent experiments.

Figure 8 - Full-length and uncropped Western blots of analyzed lysates from SW480 cells exposed to different concentrations of compound (1) for 24 hours (see Fig. 3A). All controls were incubated with 0.1% DMSO. The molecular weights of the proteins are indicated by a line and the sizes in kDa are shown. The AXIN2 steady-state level (Fig. 8A upper panel) was dose- dependency elevated by the presence of compound (1), while the cytoplasmic and nuclear β-catenin levels were decreased (Fig. 8A middle panel and Figure 8B top panel). The anti ABC antibody (active β-catenin) recognizes β-catenin that is not phosphorylated at the N-terminus. Figure 8C shows active degradation of β-catenin, mediated by exposure to compound (1) that was detected by using an antibody that recognizes β-catenin phosphorylated at the N-terminus (ρβ-catenin). Antibodies against ACTIN (cytoplasmic) or LAMEST B l (nuclear) documented equal protein loading (Figure 8B middle and bottom panels). The Figures show representative data taken from multiple experiments. Figure 9 - The distribution and steady-state level of β-catenin as visualized by

immunofluorescence. SW480 CRC cells were exposed to 5 or 1 μιηοΙ/L compound (1) for 48 hours and analyzed with an antibody against total β-catenin and secondary antibody coupled to Alexa488. Control cells were cultured in 0.05% DMSO. The images were captured with identical shutter speed and 40 times magnification.

Figure 10 - Shows a human liver microsome (FILM) test of compound (1), indicating a half-life (t½) of 10.1 minutes. This test was carried out according to standard protocols of Cyprotex, United Kingdom, and indicates rapid liver metabolism.

Example 1 - Preparation of 2-Methyl-oxazole-5-carboxylic acid (3-chloro-4-{[4-(4-methoxy- phenyl)-tetrahydro-pyran-4-ylmethyl]-carbamoyl}-phenyl)-amid e - Compound (26)

(a) Preparation of 4-Amino-2-chloro-N-[4-(4-methoxy-phenyl)-tetrahydro-pyran-4- ylmethyl]- benzamide 3:

To a mixture of [4-(4-Methoxy-phenyl)-tetrahydro-pyran-4-yl]-methylamine 1 (0.1 g, 0.45 mmol) and 4-Amino-2-chloro-benzoic acid 2 (0.078 g, 0.45 mmol) in 10 mL of dichloromethane was added EDCI (0.104 g, 0.54 mmol) at ambient temperature. The reaction mixture was stirred at ambient temperature for 16 h, concentrated and purified by flash chromatography

(hexane/AcOEt: 4/1) to give methyl 4-Amino-2-chloro-N-[4-(4-methoxy-phenyl)-tetrahydro- pyran-4-ylmethyl]-benzamide 3 as a white solid. Yield: 0.07 g, 41%. 1H- MR (300 Hz, CDC1 ₃ ) δ (ppm): 7.60 (d, 1 H), 7.28 (d, 2 H), 6.95 (d, 2 H), 6.52-6.50 (m, 2 H), 6.10 (t, 1 H), 3.95 (s, 2 H), 3.90-3.84 (m, 2 H), 3.82 (s, 3 H), 3.67 (d, 2H), 2.65 - 2.58 (m, 2H), 2.20 - 2.10 (m, 2H), 2.00-1.92 (m, 2H). (b) Preparation of 2-Methyl-oxazole-5-carboxylic acid (3-chloro-4-{[4-(4-methoxy-phenyl)- tetrahydro-pyran-4-ylmethyl]-carbamoyl}-phenyl)-amide - Compound (26)

To a mixture of methyl 4-Amino-2-chloro-N-[4-(4-methoxy-phenyl)-tetrahydro-pyran-4- ylmethylj-benzamide 3 (0.06 g, 0.16 mmol) and 2-Methyl-oxazole-5-carboxylic acid methyl ester 4 (0.025 g, 0.16 mmol) in toluene (10 mL) was added AlMe ₃ (0.12 mL, 2.0 M in toluene, 0.24 mmol) dropwise. The reaction mixture was stirred under reflux for 2 h, treated with water (15 mL) and ethyl acetate (3x20 mL). The organic phase was dried and concentrated. The residue was purified by column chromatography eluting with DCM/MeOH (2: 1) to afford Compound (26) as a white solid. Yield: 0.06 g, 77%.

1H- MR (300 Hz, CDC1 ₃ ) δ (ppm): 7.98 (br s, 1 H), 7.82(d, 1 H), 7.70 (s, 1 H), 7.65 (d, 1 H),

7.46 (d, 1 H), 7.28 (d, 2 H), 6.95 (d, 2 H), 5.99 (t, 1 H), 3.95-3.86 (m, 2 H), 3.81 (s, 3 H), 3.67 (d, 2H), 2.65 - 2.58 (m, 2H), 2.60 (s, 3H), 2.20 - 2.10 (m, 2H), 2.02-1.92 (m, 2H).

MS (M+l): 496.4.

HPLC (Waters 625 LC System): 96%.

Example 2 - Preparation of N-(4-((tetrahydro-4-(4-methoxyphenyl)-2H-pyran-4- yl)methylcarbamoyl)phenyl)-5-methylfuran-2-carboxamide - Compound (14)

(a) Preparation of methyl 4-(5-methylfuran-2-carboxamido)benzoate 3: To a mixture of methyl 4-aminobenzoate 2 (0.65 g, 4.30 mmol) and potassium carbonate (1.82 g, 13.26 mmol) in 10 mL of dichloromethane was added 5-methylfuran-2-carbonyl chloride 1 (0.58 g, 4.42 mmol) dropwise at ambient temperature. The reaction mixture was stirred at ambient temperature overnight. The solid was filtered off. The filtrate was concentrated. The residue was treated with methanol (10 mL) and stirred for 30 min. The solid was filtered to yield methyl 4-(5-methylfuran-2-carboxamido)benzoate 3. Yield: 0.40 g, 36.75% .

1H- MR (300 Hz, CDC1 ₃ ) δ (ppm): 8.13 (s, 1 H), 8.06 (d, 2 H), 7.76 (d, 2 H), 7.18 (d, 1 H), 6.18 (d, 1 H), 3.91 (s, 3 H), 2.41 (s, 3 H).

MS (M+l) 247.10. (b) Preparation of N-(4-((tetrahydro-4-(4-methoxyphenyl)-2H-pyran-4- yl)methylcarbamoyl)phenyl)-5-methylfuran-2-carboxamide - Compound (14)

To a mixture of methyl 4-(5-methylfuran-2-carboxamido)benzoate 3 (0.20 g, 0.77 mmol) and 4- (4-methoxyphenyl)tetrahydropyran-4-yl]methanamine 4 (0.20 g, 0.9 mmol) in toluene (20 mL) was added AlMe ₃ (2.0 M 0.45 mL, 0.9 mmol) dropwise. The reaction mixture was stirred under reflux overnight, treated with IN HC1 (5 mL) and ethyl acetate (50 mL). The organic phase was dried and concentrated. The residue was purified by column chromatography eluting with hexane: ethyl acetate (1 :3) to afford Compound (14) as a white solid. Yield: 0.2 g, 57%.

1H-NMR (300 Hz, CDC1 ₃ ) δ (ppm): 8.13 (s, 1 H), 7.69 (d, 2 H), 7.60 (dd, 4 H), 7.27 (d, 2 H), 7.16 (d, 1 H), 6.98 (d, 2 H), 6.16 (d, 1 H), 5.68 (t, 1 H), 3.85 (s, 3 H), 3.90-3.82 (m, 2 H), 3.67- 3.58 (m, 4 H), 2.40 (s, 3 H), 2.10-2.07 (m, 2 H), 1.98-1.90 (m, 2 H).

MS (M+l) 449.2.

HPLC (Waters 625 LC System): 95%. Example 3 - Preparation of N-(4-((tetrahydro-4-(4-methoxyphenyl)-2H-pyran-4- yl)methylcarbamoyl)-3-chlorophenyl)furan-2-carboxamide - Compound (15)

(15)

(a) Preparation of methyl 2-chloro-4-(furan-2-carboxamido)benzoate 7:

To a mixture of methyl 4-amino-2-chlorobenzoate 6 (0.5 g, 2.69 mmol) and potassium carbonate (1.82 g, 13.26 mmol) in 10 mL of dichlorom ethane was added furan-2-carbonyl chloride 5 (0.37 g, 2.83 mmol) dropwise at ambient temperature. The reaction mixture was stirred at ambient temperature overnight. The solid was filtered off. The filtrate was concentrated. The residue was treated with methanol (10 mL) and stirred for 30 min. The solid was filtered to yield methyl 2-chloro-4-(furan-2-carboxamido)benzoate 7 as a solid. Yield: 0.50 g, 67%.

1H- MR (300 Hz, CDC1 ₃ ) δ (ppm): 8.20 (s, 1 H), 7.96 (dd, 1 H), 7.50 (s, 1 H), 7.35 (d, 1 H), 7.13 (d, 1 H), 6.46 (d, 1 H), 3.92 (s, 3 H).

MS (Turbo Ion Spray TOF MS): 282.0567.

(b) Preparation of N-(4-((tetrahydro-4-(4-methoxyphenyl)-2H-pyran-4-yl)methylca rbamoyl)-3- chlorophenyl)furan-2-carboxamide - Compound (15)

To a mixture of methyl 2-chloro-4-(furan-2-carboxamido)benzoate 7 (0.20 g, 0.715 mmol) and 4-(4-methoxyphenyl)tetrahydropyran-4-yl]methanamine 4 (0.20 g, 0.9 mmol) in toluene (20 mL) was added AlMe ₃ (2.0 M 0.45 mL, 0.9 mmol) dropwise. The reaction mixture was stirred under reflux overnight, treated with IN HC1 (5 mL) and ethyl acetate (50 mL). The organic phase was dried and concentrated. The residue was purified by column chromatography eluting with hexane: ethyl acetate (1 :3) to afford Compound (14) as a white solid. Yield: 0.2 g, 60%.

1H-NMR (300 Hz, CDC1 ₃ ) δ (ppm): 8.13 (s, 1 H), 7.86 (d, 1 H), 7.66 (d, 1 H), 7.52 (dd, 1 H), 7.43 (dd, 1 H), 7.29-7.25 (m, 2 H), 6.92 (dd, 3 H), 6.58 (dd, 1 H), 5.99 (t, 1 H), 3.85 (s, 3 H), 3.90-3.82 (m, 2 H), 3.67-3.58 (m, 4 H), 2.10-2.07 (m, 2 H), 1.98-1.90 (m, 2 H). MS (M+1) 469.4. HPLC (Waters 625 LC System): 96%.

Example 4 - Preparation of Furan-2-carboxylic acid (4-{[4-(4-methoxy-phenyl)-tetrahydro- pyran-4-ylmethyl]-carbamoyl}-phenyl)-methyl amide - Compound (17)

(a ) Preparation of methyl 4-(furan-2-carboxamido)benzoate 8:

To a mixture of methyl 4-aminbenzoate 2 (5 g, 33 mmol) and potassium carbonate (18.2 g, 132.6 mmol) in 100 mL of dichloromethane was added furan-2-carbonyl chloride 5 (4.54 g, 34.8 mmol) dropwise at ambient temperature. The reaction mixture was stirred at ambient temperature overnight. The solid was filtered off. The filtrate was concentrated. The residue was treated with methanol (20 mL) and stirred for 30 min. The solid was filtered to yield methyl

4-(furan-2-carboxamido)benzoate 8 as a solid. Yield: 0.49 g, 60%.

1HNMR (300 Hz, CDC1 ₃ ) δ (ppm): 8.22 (s, 1 H), 8.06 (d, 2 H), 7.72 (d, 2 H), 7.53 (s, 1 H), 7.28

(d, 2 H), 6.58 (d, 1 H), 3.91 (s, 3 H).

MS (Turbo Ion Spray TOF MS): 246.1037.

(b) Preparation of methyl 4-(N-methylfuran-2-carboxamido)benzoate 9:

To a solution of methyl 4-(furan-2-carboxamido)benzoate 8 (0.5 g, 2 mmol) in 10 mL of DMF was added NaH (0.074 g, 3 mmol) portionwise at ambient temperature. The reaction mixture was stirred at ambient temperature for 30 min and methyl iodide (1 mL) was added. The resulting mixture was stirred at ambient temperature for 16 h, treated with 1 N HCl and EtOAc.

The organic layer was washed with H ₂ 0, dried and concentrated to yield methyl 4-(N- methylfuran-2-carboxamido)benzoate 9 as a solid. Yield: 0.5 g, 94%.

1H- MR (300 Hz, CDC1 ₃ ) δ (ppm): 8.06 (d, 2 H), 7.27-7.23 (m, 3 H), 6.26 (s, 2 H), 3.93 (s, 3

H), 3.46 (s, 3 H). MS (Turbo Ion Spray TOF MS): 206.1248.

(c) Preparation of N-(4-((tetrahydro-4-(4-methoxyphenyl)-2H-pyran-4-yl)methylca rbamoyl)-3- chlorophenyl)-N-methylfuran-2-carboxamide - Compound (17)

To a mixture of methyl 4-(N-methylfuran-2-carboxamido)benzoate 9 (0.20 g, 0.77 mmol) and 4- (4-methoxyphenyl)tetrahydropyran-4-yl]methanamine 4 (0.20 g, 0.9 mmol) in toluene (20 mL) was added AlMe ₃ (2.0 M 0.45 mL, 0.9 mmol) dropwise. The reaction mixture was stirred under reflux overnight, treated with IN HC1 (5 mL) and ethyl acetate (50 mL). The organic phase was dried and concentrated. The residue was purified by column chromatography eluting with hexane: ethyl acetate (1 :3) to afford Compound (17) as a white solid. Yield: 0.19 g, 54%.

1H-NMR (300 Hz, CDC1 ₃ ) δ (ppm): 7.62 (dd, 2 H), 7.32-7.20 (m, 6 H), 6.99 (dd, 2 H), 6.25 (dd, 2 H), 5.68 (t, 1 H), 3.85 (s, 3 H), 3.90-3.82 (m, 2 H), 3.67-3.58 (m, 4 H), 3.40 (s, 3 H), 2.10-2.07 (m, 2 H), 1.98-1.90 (m, 2 H).

MS (M+l) 449.3.

HPLC (Waters 625 LC System): 93%.

Example 5 - Preparation of N-[4-[[4-(4-methoxyphenyl)tetrahydropyran-4- yl]methylcarbamoyl]phenyl]oxazole-5-carboxamide - Compound (16)

(a) Preparation of methyl 4-(oxazole-5-carbonylamino)benzoate 11:

To a solution of oxazole-5-carbonyl chloride 10 (0.58 g, 4.42 mmol) in 10 mL anhydrous dichlorom ethane was added methyl 4-aminobenzoate 2 (0.65 g, 4.30 mmol) and potassium carbonate (1.82 g, 13.26 mmol). The reaction mixture was stirred at ambient temperature overnight, then added to 20 mL of water and extracted with ethyl acetate (80 mL). The organic solvent was removed and methanol (10 mL) added and stirred for 30 min. Then the solid was filtered to yield methyl 4-(oxazole-5-carbonylamino)benzoate 11 as a light yellow solid. Yield: 0.40 g, 36.75%.

¹ HNMR (300 Hz, CD ₃ OD) δ (ppm): 8.39 (s, 1H), 8.03 (d, 2H), 7.90 (s, 1H), 7.84 (d, 2H), 3.89 (s, 3H). MS (M+l) 247.10.

(b) Preparation of N-[4-[[4-(4-methoxyphenyl)tetrahydropyran-4

yl]methylcarbamoyl]phenyl]oxazole-5-carboxamide - Compound (16)

To a mixture of methyl 4-(oxazole-5-carbonylamino)benzoate 11 (0.20 g, 0.812 mmol) and 4-(4- methoxyphenyl)tetrahydropyran-4-yl]methanamine 4 (0.20 g, 0.89 mmol) in anhydrous toluene (20 mL) was added AlMe ₃ (2.0 M 0.41 mL, 0.812 mmol) dropwise. The reaction mixture was stirred under reflux overnight, cooled to ambient temperature and IN HCl solution (5 mL) added. The mixture was extracted with ethyl acetate (2x50 mL). The organic phase was concentrated. The residue was purified by column chromatography eluting with hexane: ethyl acetate (1 :3) to afford Compound (16) as a white solid. Yield: 0.095 g, 26.8%.

1H-NMR (300 Hz, CDC1 ₃ ) δ (ppm): 8.11 (s, 1 H), 7.98 (s, 1 H), 7.86 (s, 1 H), 7.67 (d, 2 H), 7.60 (d, 2 H), 7.27 (d, 2 H), 6.98 (d, 2 H), 5.67 (t, 1 H), 3.85 (s, 3 H), 3.90-3.82 (m, 2 H), 3.67-3.58 (m, 4 H), 2.10-2.07 (m, 2 H), 1.98-1.90 (m, 2 H).

MS (M+l) 436.3.

HPLC (Waters 625 LC System): 96%.

Example 6 - N-(4-((tetrahydro-4-(4-methoxyphenyl)-2H-pyran-4-yl)methylca rbamoyl)-2- chlorophenyl)furan-2-carboxamide - Compound (19)

(19) (a) Preparation of methyl 3-chloro-4-(furan-2-carboxamido)benzoate 3:

To a mixture of methyl 4-amino-3-chlorobenzoate 2 (1.0 g, 5.38 mmol) and potassium carbonate (1.50 g, 11.0 mmol) in 10 mL of dichlorom ethane was added furan-2-carbonyl chloride 1 (0.70 g, 5.38 mmol) dropwise at ambient temperature. The reaction mixture was stirred at ambient temperature overnight. The solid was filtered off. The filtrate was concentrated. The residue was treated with methanol (10 mL) and stirred for 30 min. The solid was filtered to yield methyl 3-chloro-4-(furan-2-carboxamido)benzoate 3. Yield: 0.69 g, 45.8%.

1H- MR (300 Hz, CDC1 ₃ ) δ (ppm): 8.88 (brs, 1H), 8.68 (d, 1H), 8.11 (d, 1H), 8.00 (dd, 1H), 7.59 (m, 1H), 7.30 (dd, 1H), 6.60 (dd, 1H), 3.92 (s, 3H).

(b) Preparation of N-(4-((tetrahydro-4-(4-methoxyphenyl)-2H-pyran-4-yl)methylca rbamoyl)-2- chlorophenyl)furan-2-carboxamide - Compound (19)

To a mixture of methyl 3-chloro-4-(furan-2-carboxamido)benzoate 3 (0.18 g, 0.68 mmol) and 4- (4-methoxyphenyl)tetrahydropyran-4-yl]methanamine 4 (0.16 g, 0.78 mmol) in toluene (20 mL) was added AlMe ₃ (2.0 M 0.41 mL, 0.82 mmol) dropwise. The reaction mixture was stirred under reflux overnight, cooled to ambient temperature and then treated with IN HC1 (5 mL) and ethyl acetate (50 mL). The organic phase was dried and concentrated. The residue was purified by column chromatography eluting with hexane: ethyl acetate (1 :3) to afford Compound (19) as a white solid. Yield: 0.118 g, 37%. 1H-NMR (300 Hz, CDC1 ₃ ) δ (ppm): 8.78 (brs, 1H), 8.55 (d, 1H), 7.78 (s, 1H), 7.56 (s, 1H), 7.36 (d, 1H), 7.25 (d, 2H), 6.95 (d, 2H), 6.58 (t, 1H), 5.75 (t, 1H), 3.83 (s, 3H), 3.86-3.83 (m, 2H), 3.63-3.57 (m, 4H), 2.12-2.03 (m, 2H), 1.96-1.88 (m, 2H).

MS (M+l) 469.10.

HPLC (Waters 625 LC System): 95%.

Example 7 - Preparation of 6-(furan-2-carboxamido)-N-((tetrahydro-4-(4-methoxyphenyl)-2 H- pyran-4-yl)methyl)pyridine-3-carboxamide - Compound (20)

(a) Preparation of methyl 6-(furan-2-carboxamido)pyridine-3-carboxylate 6:

To a solution of furan-2-carbonyl chloride 1 (0.43 g, 3.28 mmol) in 10 mL anhydrous dichlorom ethane was added methyl 6-aminopyridine-3-carboxylate 5 (0.50 g, 3.28 mmol) and potassium carbonate (0.90 g, 6.60 mmol). The reaction mixture was stirred at ambient temperature overnight, 100 ml water was added and extracted with ethyl acetate (200 mL). The organic solvent was removed and the residue was purified by column chromatography eluting with hexane:ethyl acetate (2: 1) to get methyl 6-(furan-2-carbonylamino)pyridine-3-carboxylate 6 as a white solid. Yield: 0.34 g, 42.0%.

1H- MR (300 Hz, CDC1 ₃ ) δ (ppm): 8.95 (d, IH), 8.36 (dd, IH), 7.45 (d, IH), 7.43 (d, IH), 7.22 (d, IH), 6.49 (dd, IH), 3.94 (s, 3H).

MS (M+l) 247.10.

(b) Preparation of 6-(furan-2-carboxamido)-N-((tetrahydro-4-(4-methoxyphenyl)-2 H-pyran-4- yl)methyl)pyridine-3-carboxamide - Compound (20)

To a 100 ml flask with magnetic stirring bar and reflux condenser with methyl 6-(furan-2- carbonylamino)pyridine-3-carboxylate 6 (0.17 g, 0.69 mmol) and 4-(4- methoxyphenyl)tetrahydropyran-4-yl]methanamine 4 (0.17 g, 0.80 mmol) in anhydrous toluene (20 ml) was added AlMe ₃ (2.0 M 0.41 mL, 0.812 mmol). The reaction was carried out at reflux overnight. The reaction mixture, at ambient temperature, was added to IN HC1 solution (5 ml) and extracted with ethyl acetate (2 ^χ 100 mL). The organic phase was concentrated and purified by column chromatography eluting with hexane: ethyl acetate (1 :3) to afford Compound (20) as a white solid. Yield: 80 mg, 26.6%. 1H-NMR (300 Hz, CDC1 ₃ ) δ (ppm): 8.84 (s, 1H), 8.55 (d, 1H), 8.35 (dd, 1H), 7.90 (dd, 1H), 7.56 (t, 1H), 7.31-7.26 (m, 3H), 6.98 (d, 2H), 6.59 (t, 1H), 5.60 (t, 1H), 3.85 (s, 3H), 3.91-3.83 (m, 2H), 3.69-3.59 (m, 4H), 2.17-2.07 (m, 2H), 2.00-1.90 (m, 2H).

MS (M+l) 436.30.

HPLC (Waters 625 LC System): 98%.

Example 8 - N-(4-((tetrahydro-4-(4-methoxyphenyl)-2H-pyran-4-yl)methylca rbamoyl)-3- chlorophenyl)-N-methylfuran-2-carboxamide - Compound (18)

(a) Preparation of methyl 4-(furan-2-carboxamido)benzoate 8:

To a mixture of methyl 4-aminobenzoate 7 (5 g, 33 mmol) and potassium carbonate (18.2 g, 132.6 mmol) in 100 mL of dichloromethane was added furan-2-carbonyl chloride 1 (4.54 g, 34.8 mmol) dropwise at ambient temperature. The reaction mixture was stirred at ambient temperature overnight. The solid was filtered off. The filtrate was concentrated. The residue was treated with methanol (20 mL) and stirred for 30 min. The solid was filtered to yield methyl 4-(furan-2-carboxamido)benzoate 8 as a solid. Yield: 4.9 g, 60%.

1H-NMR (300 Hz, CDCI ₃ ) δ (ppm): 8.22 (s, 1 H), 8.06 (d, 2 H), 7.72 (d, 2 H), 7.53 (s, 1 H), 7.28 (d, 2 H), 6.58 (d, 1 H), 3.91 (s, 3 H). MS (Turbo Ion Spray TOF MS): 246.1037.

(b) Preparation of N-(4-(N-((tetrahydro-4-(4-methoxyphenyl)-2H-pyran-4-yl)methy l)-N- methylcarbamoyl)-3-chlorophenyl)furan-2-carboxamide - Compound (18)

To a mixture of methyl 4-(furan-2-carboxamido)benzoate 8 (0.20 g, 0.77 mmol) and (tetrahydro- 4-(4-methoxyphenyl)-2H-pyran-4-yl)-N-methylmethanamine 9 (0.20 g, 0.9 mmol) in toluene (20 mL) was added AlMe ₃ (2.0 M 0.45 mL, 0.9 mmol) dropwise. The reaction mixture was stirred under reflux overnight, cooled to ambient temperature and then treated with IN HCl (5 mL) and ethyl acetate (50 mL). The organic phase was dried and concentrated. The residue was purified by column chromatography eluting with hexane: ethyl acetate (1 :3) to afford Compound (18) as a white solid. Yield: 0.19 g, 54%.

1H- MR (300 Hz, CDC1 ₃ ) δ (ppm): 7.62 (dd, 2 H), 7.32-7.20 (m, 6 H), 6.99 (dd, 2 H), 6.25 (dd, 2 H), 5.68 (t, 1 H), 3.85 (s, 3 H), 3.90-3.82 (m, 2 H), 3.67-3.58 (m, 4 H), 3.02 (s, 3 H), 2.10-2.07 (m, 2 H), 1.98-1.90 (m, 2 H).

MS (M+l) 449.2.

HPLC (Waters 625 LC System): 91%. Example 9 - Preparation of 5-Methyl-[l,3,4]oxadiazole-2-carboxylic acid (3-chloro-4-{[4-(4- methoxy-phenyl)-tetrahydro-pyran-4-ylmethyl]-carbamoyl}-phen yl)-amide - Compound (25)

(a) Preparation of acetic acid hydrazide 2:

A mixture of hydrazine monohydrate (20 mL, 400 mmol) and ethyl acetate 1 (30 mL, 300 mmol) in ethanol (80 mL) was stirred under reflux overnight to provide acetic acid hydrazide 2 as a white solid. Yield: 23.8 g, 100%. This was used for the next step without further purification. 1H- MR (300 Hz, CDCI ₃ ) δ (ppm): 6.90 (br s, 1 H), 3.90 (br s, 2 H), 1.98 (s, 3 H). (b) Preparation of (Ν'- Acetyl -hydrazino)-oxo-acetic acid ethyl ester 3:

To a solution of acetic acid hydrazide 2 (10 g, 140 mmol) and TEA (58.6 mL, 420 mmol) in DCM (400 ml) was added Chloro-oxo-acetic acid ethyl ester (18 mL, 160 mmol) dropwise at 0°C. The reaction mixture was stirred at ambient temperature for 16 h and concentrated. The residue was subjected to column chromatography (50-100% ethyl acetate/hexanes) to give (N'-Acetyl-hydrazino)-oxo-acetic acid ethyl ester 3 as a yellow solid. Yield: 22 g, 91%. This was used for the next step without further purification.

1H- MR (300 Hz, CDC1 ₃ ) δ (ppm): 3.98 (q, 2 H), 2.10 (s, 3 H), 1.38 (t, 3 H). (c) Preparation of (N'-Acetyl-hydrazino)-oxo-acetic acid ethyl ester 4:

To a solution of (N'-Acetyl-hydrazino)-oxo-acetic acid ethyl ester 3 (10 g, 57.8 mmol) and TEA (10.5 mL, 75.1 mmol) in DCM (100 ml) was added p-TsCl (13.2 g, 69.3 mmol) portionwise at ambient temperature. The reaction mixture was stirred at ambient temperature for 16 h, washed with H ₂ 0, dried and concentrated. The residue was subjected to column chromatography (12.5% to 25%) ethyl acetate/hexanes) to give (N'-Acetyl-hydrazino)-oxo-acetic acid ethyl ester 4 as a white solid. Yield: 4.2 g, 47%.

1H- MR (300 Hz, CDCI ₃ ) δ (ppm): 4.53 (q, 2 H), 2.65 (s, 3 H), 1.48 (t, 3 H).

(d) Preparation of 4-Amino-2-chloro-N-[4-(4-methoxy-phenyl)-tetrahydro-pyran-4- ylmethyl]- benzamide 7:

To a mixture of C-[4-(4-Methoxy-phenyl)-tetrahydro-pyran-4-yl]-methylamine 5 (0.1 g, 0.45 mmol) and 4-Amino-2-chloro-benzoic acid (0.078 g, 0.45 mmol) in 10 mL of dichloromethane was added EDCI (0.104 g, 0.54 mmol) at ambient temperature. The reaction mixture was stirred at ambient temperature for 16 h, concentrated and purified by column chromatography

(hexane/AcOEt: 4/1) to give methyl 4-Amino-2-chloro-N-[4-(4-methoxy-phenyl)-tetrahydro- pyran-4-ylmethyl]-benzamide 7 as a white solid. Yield: 0.07 g, 41%.

1H- MR (300 Hz, CDCI ₃ ) δ (ppm): 7.60 (d, 1 H), 7.28 (d, 2 H), 6.95 (d, 2 H), 6.52-6.50 (m, 2 H), 6.10 (t, 1 H), 3.95 (s, 2 H), 3.90-3.84 (m, 2 H), 3.82 (s, 3 H), 3.67 (d, 2H), 2.65 - 2.58 (m, 2H), 2.20 - 2.10 (m, 2H), 2.00-1.92 (m, 2H).

(e) Preparation of 5-Methyl-[l,3,4]oxadiazole-2-carboxylic acid (3-chloro-4-{[4-(4-methoxy- phenyl)-tetrahydro-pyran-4-ylmethyl]-carbamoyl}-phenyl)-amid e - Compound (25)

To a mixture of methyl 4-Amino-2-chloro-N-[4-(4-methoxy-phenyl)-tetrahydro-pyran-4- ylmethylj-benzamide 7 (0.06 g, 0.16 mmol) and (N'-Acetyl-hydrazino)-oxo-acetic acid ethyl ester 4 (0.025 g, 0.16 mmol) in toluene (10 mL) was added AlMe ₃ (0.12 mL, 2.0 M in toluene, 0.24 mmol) dropwise at ambient temperature. The reaction mixture was stirred under reflux for 2 h, cooled to ambient temperature, and then treated with water (15 mL) and ethyl acetate (3x20 mL). The organic phase was dried and concentrated. The residue was purified by column chromatography eluting with DCM/MeOH (2: 1) to afford Compound (25) as a white solid. Yield: 0.06 g, 77%.

1H- MR (300 Hz, CDC1 ₃ ) δ 8.82 (s, 1 H), 7.85 (s, 1 H), 7.70 (d, 1 H), 7.52 (d, 1 H), 7.28 (d, 2 H), 6.88 (d, 2 H), 5.98 (t, 1 H), 3.92-3.82 (m, 2 H), 3.80 (s, 3 H), 3.68 (d, 2H), 3.65 - 3.58 (m, 2 H), 2.68 (s, 3 H), 2.20 - 2.10 (m, 2H), 2.00-1.92 (m, 2 H).

MS (M+l) 483.2.

HPLC (Waters 625 LC System): 99%.

Example 10 - Preparation of N-(4-((4-(3-methoxyphenyl)-l-methylpiperidin-4- yl)methylcarbamoyl)-3-chlorophenyl)-5-methylfuran-2-carboxam ide- Compound (23)

(23)

(a) Preparation of 4-(4-methoxyphenyl)-l-methylpiperidine-4-carbonitrile 3:

To a mixture of 2-(4-methoxyphenyl)acetonitrile 1 (5 g, 34 mmol) and 2-chloro-N-(2- chloroethyl)-N-methylethanamine 2 (6.5 g, 34 mmol) in DMF (20 mL) was added NaH (5.4 g, 60%, 136 mmol) portionwise. The reaction mixture was stirred at ambient temperature overnight, treated with water (15 mL) and ethyl acetate (3x20 mL). The organic phase was dried and concentrated. The residue was purified by column chromatography eluting with AcOEt to provide 4-(4-methoxyphenyl)-l-methylpiperidine-4-carbonitrile 3 as a yellow oil. Yield: 5 g, 64%.

1H- MR (300 Hz, CDC1 ₃ ) δ (ppm): 7.42 (d, 2 H), 6.91 (d, 2 H), 3.79 (s, 3 H), 2.89 - 2.98 (m, 2 H), 2.40 - 2.51 (m, 2 H), 2.39 (s, 3 H), 2.10 - 2.19 (m, 4 H).

(b) Preparation of (4-(4-methoxyphenyl)-l-methylpiperidin-4-yl)methanamine 4:

To a mixture of LAH (0.81 g, 21.5 mmol) in anhydrous THF (15 ml) cooled to 0°C a solution of 4-(4-methoxyphenyl)-l-methylpiperidine-4-carbonitrile 3 (1 g, 4.3 mmol) in anhydrous THF (10 mL) was added slowly. The reaction mixture was stirred at 0°C for 3 h. A solution of NaOH (2N, 9 mL) was added dropwise, filtered and the filtrate washed with AcOEt. The organic layers were combined and dried over Na ₂ S0 ₄ , and concentrated to give (4-(4-methoxyphenyl)-l- methylpiperidin-4-yl)methanamine 4 as a yellow oil. Yield: 1 g, 100%. The product was used for the next step reaction without further purification.

(c) Preparation of methyl 4-(5-methylfuran-2-carboxamido)-2-chlorobenzoate 7:

To a mixture of methyl 4-amino-2-chlorobenzoate 6 (0.51 g, 2.77 mmol) and triethylamine (1.2 mL, 8.31 mmol) in 10 mL of dichl or om ethane was added 5-methylfuran-2-carbonyl chloride 5 (0.40 g, 2.77 mmol) dropwise at ambient temperature. The reaction mixture was stirred at ambient temperature for 4h. HC1 (1M, 20 mL) was added to the reaction and the mixture extracted with AcOEt (2x20mL). The organic layers were combined and washed with K ₂ C0 ₃ solution (2M), dried over Na ₂ S0 ₄ , filtered and concentrated to give a crude product, which was purified by chromatography on silica gel (hexane/ AcOEt: 4/1) to give methyl 4-(5-methylfuran- 2-carboxamido)-2-chlorobenzoate 7 as a white solid. Yield: 0.45 g, 55.3%.

1H- MR (300 Hz, CDC1 ₃ ) δ (ppm): 8.13 (s, 1 H), 7.60 - 7.90 (m, 3 H), 7.18 - 7.20 (m, 1 H), 6.19 - 6.21 (m, 1 H), 3.91 (s, 3 H), 2.42 (s, 3 H). (d) Preparation of N-(4-((4-(3-methoxyphenyl)-l-methylpiperidin-4-yl)methylcarb amoyl)-3- chlorophenyl)-5-methylfuran-2-carboxamide - Compound (23)

To a mixture of methyl 4-(5-methylfuran-2-carboxamido)-2-chlorobenzoate 7 (0.22 g, 0.75 mmol) and (4-(3-methoxyphenyl)-l-methylpiperidin-4-yl)methanamine 4 (0.35 g, 1.5 mmol) in toluene (20 mL) was added AlMe ₃ (0.75 mL, 2.0 M in toluene, 1.5 mmol) dropwise. The reaction mixture was stirred under reflux for 2 h, cooled to ambient temperature and treated with water (15 mL) and ethyl acetate (3x20 mL). The organic phase was dried and concentrated. The residue was purified by column chromatography eluting with DCM/MeOH (2: 1) to afford Compound (23) as a white solid. Yield: 0.20 g, 54%.

1H- MR (300 Hz, CDC1 ₃ ) δ (ppm): 8.03 (br s, 1 H), 7.84(d, 1 H), 7.65 (d, 1 H), 7.45 (dd, 1 H), 7.26 - 7.31 (m, 2 H), 7.16 (d, 1 H), 6.91 (d, 2 H), 6.16 - 6.19 (m, 1 H), 5.99 (t, 1 H), 3.81 (s, 3 H), 3.67 (d, 2H), 2.56 - 2.65 (m, 2H), 2.40 (s, 3H), 2.33 - 2.42 (m, 2H), 2.27 (s, 3H), 1.94 - 2.22 (m, 4H).

MS (M+l) 496.4.

HPLC (Waters 625 LC System): 96%.

Example 11 - Preparation of N-(4-((4-(3-chlorophenyl)-tetrahydro-2H-pyran-4- yl)methylcarbamoyl)-3-chlorophenyl)-5-methylfuran-2-carboxam ide - Compound (24)

(a) Preparation of 4-(4-chlorophenyl)-tetrahydro-2H-pyran-4-carbonitrile 3:

To a mixture of 2-(4-chlorophenyl)acetonitrile 1 (10 g, 65.9 mmol) and l-(2-chloroethoxy)-2- chloroethane 2 (7.73 g, 65.9 mmol) in DMF (50 mL) cooled to 0°C was added NaH (7.9 g, 60%, 197.7 mmol) slowly. The reaction mixture was stirred at ambient temperature overnight, then treated with water (15 mL) and acidified with HC1 (35%) to pH 3, extracted with ethyl acetate (3x20 mL) and the organic layers combined and washed with water (3x3 OmL). The organic phase was dried and concentrated. The residue was purified by column chromatography eluting with hexane/AcOEt: 4/1 to 4-(4-chlorophenyl)-tetrahydro-2H-pyran-4-carbonitrile 3 as a yellow solid. Yield: 10 g, 67%.

1H- MR (300 Hz, CDC1 ₃ ) δ (ppm): 7.40 - 7.49 (m, 4 H), 4.03 - 4.18 (m, 2 H), 3.81 - 3.90 (m, 2 H), 1.97 - 2.19 (m, 4 H).

(b) Preparation of (4-(4-chlorophenyl)-tetrahydro-2H-pyran-4-yl)methanamine 4:

To a mixture of LAH (0.85 g, 22.5 mmol) in anhydrous THF (15 ml) cooled to 0°C a solution of 4-(4-chlorophenyl)-tetrahydro-2H-pyran-4-carbonitrile 3 in anhydrous THF (10 mL) (1 g, 4.5 mmol) was added slowly. The reaction mixture was stirred at ambient temperature for 4 hrs. A solution of NaOH (2N, 10 mL) was added dropwise, then filtered and washed with CH ₂ C1 ₂ . The organic layer was dried over Na ₂ S0 ₄ , filtered and concentrated to give (4-(4-chlorophenyl)- tetrahydro-2H-pyran-4-yl)methanamine 4 as a yellow oil. Yield: 1 g, 99%. The crude product was used for the next step without further purification.

(c) Preparation of methyl 4-(5-methylfuran-2-carboxamido)-2-chlorobenzoate 7:

To a mixture of methyl 4-amino-2-chlorobenzoate 6 (0.51 g, 2.77 mmol) and triethylamine (1.2 mL, 8.31 mmol) in 10 mL of dichl or om ethane was added 5-methylfuran-2-carbonyl chloride 5 (0.40 g, 2.77 mmol) dropwise at ambient temperature. The reaction mixture was stirred at ambient temperature for 4h. HC1 (1M, 20 mL) was added to the reaction mixture and extracted with AcOEt (2x20mL). The organic layers were combined and washed with K ₂ C0 ₃ solution (2M), dried over Na ₂ S0 ₄ , filtered and concentrated to give the crude product, which was purified by chromatography on silica gel (hexane/ AcOEt: 4/1) to give methyl 4-(5-methylfuran-2- carboxamido)-2-chlorobenzoate 7 as a white solid. Yield: 0.45 g, 55.3%.

1H- MR (300 Hz, CDC1 ₃ ) δ (ppm): 8.13 (s, 1 H), 7.60 - 7.90 (m, 3 H), 7.18 - 7.20 (m, 1 H), 6.19 - 6.21 (m, 1 H), 3.91 (s, 3 H), 2.42 (s, 3 H).

(d) Preparation of N-(4-((4-(3-chlorophenyl)-tetrahydro-2H-pyran-4-yl)methylcar bamoyl)-3- chlorophenyl)-5-methylfuran-2-carboxamide - Compound (24)

To a mixture of methyl 4-(5-methylfuran-2-carboxamido)-2-chlorobenzoate 7 (0.20 g, 0.68 mmol) and (4-(3-chlorophenyl)-tetrahydro-2H-pyran-4-yl)methanamine 4 (0.30 g, 1.36 mmol) in toluene (20 mL) was added AlMe ₃ (0.68 mL, 2.0 M in toluene, 1.36 mmol) dropwise. The reaction mixture was stirred under reflux for 3 hrs, cooled to ambient temperature, and treated with IN HC1 (15 mL) and ethyl acetate (50 mL). The organic phase was dried and concentrated. The residue was purified by column chromatography eluting with hexane: ethyl acetate (1 :2) to afford Compound (24) as a white solid. Yield: 0.11 g, 33%.

1H-NMR (300 Hz, CDC1 ₃ ) δ (ppm): 8.04 (br s, 1 H), 7.84 - 7.86 (m, 1 H), 7.60 (d, 1 H), 7.28 - 7.49 (m, 5 H), 7.14 - 7.18 (m, 1 H), 6.17 - 6.20 (m, 1 H), 6.01 (br s, 1 H), 3.84 - 3.94 (m, 2 H), 3.75 (t, 2 H), 3.59 - 3.70 (m, 2 H), 2.41 (s, 3 H), 1.96 - 2.22 (m, 4 H). MS (M+l) 487.3.

HPLC (Waters 625 LC System): 98%.

Example 12 - Synthetic scheme for the preparation of Compound (1)

+

Examples 13 to 16 are examples of formulations in which reference to the "active substance" denotes one or more compounds as herein described, including the salts thereof. Example 13 - Tablets containing 100 mg of active substance

Each tablet contains:

active substance 100.0 mg

lactose 80.0 mg

corn starch 34.0 mg

polyvinylpyrrolidone 4.0 mg

magnesium stearate 2.0 mg The active substance, lactose and corn starch are mixed together and uniformly moistened with an aqueous solution of the polyvinylpyrrolidone. The moist composition is screened (2.0 mm mesh size) and dried at 50°C. The lubricant is added and the final mixture is compressed to form tablets. Final weight of each tablet is 220 mg Example 14 - Tablets containing 150 mg of active substance

Each tablet contains:

active sub stance 150.0 mg

powdered lactose 89.0 mg

corn starch 40.0 mg

colloidal silica 10.0 mg

polyvinylpyrrolidone 10.0 mg

magnesium stearate 1.0 mg The active substance is mixed with lactose, corn starch and silica and moistened with an aqueous polyvinylpyrrolidone solution. The moist composition is passed through a screen with a mesh size of 1.5 mm. The resulting granules are dried at 45°C, then mixed with the magnesium stearate. Tablets are pressed from the mixture. Each tablet weighs 300 mg. Example 15 - Ampoules containing 10 mg active substance

Each ampoule contains:

active substance 10.0 mg

0.01 N hydrochloric acid q.s.

double-distilled water ad 2.0 ml

The active substance is dissolved in the necessary amount of 0.01 N HCl, made isotonic with common salt, sterile filtered and transferred into 2 ml ampoules.

Example 16 - Ampoules containing 50 mg of active substance Each ampoule contains:

active substance 50.0 mg

0.01 N hydrochloric acid q.s.

double-distilled water ad 10.0 ml

The active substance is dissolved in the necessary amount of 0.01 N HCl, made isotonic with common salt, sterile filtered and transferred into 10 ml ampoules.

Example 17 - In vivo efficacy and specificity of Wnt pathway inhibitors

To evaluate the efficacy and specificity of Compound (1) it was tested in three different luciferase reporter assays:

1) a Luciferase assay in HEK293 cells transiently transfected with SuperTOPFlash plasmid (ST-Luc) (Veeman MT et al., Curr. Biol. 13 : 680-685, 2003);

2) a stable reporter line for sonic hedgehog pathway, Shh Light II cells (Glil-Luc - purchased from ATCC and maintained according to supplier's recommendations); and

3) HEK293 cells transiently transfected with a NF-KB-Luciferase reporter (NF-KB-LUC - purchased from Promega).

Experimental

1) Transfection was performed using 80000 HEK293 cells (American Type Culture Collection - cells maintained according to supplier's recommendations) seeded in 48-well plates coated with poly-L lysine. After 24 hours, 0.25 μg total plasmid DNA (0.23 μg SuperTOPFlash + 0.02 μg pRL-TK (Renilla) - Promega) and 0.75 μΐ FuGE E6 (Roche) was combined in a total volume in 25 μΐ Opti-MEM ^® (Invitrogen) as described by the manufacturer (Roche). The transfection mixture was added to the plated cells and media were changed after 24 hours. All luciferase assays contained a minimum of three replicates for each differently treated sample unless indicated otherwise.

The Luciferase assay was performed by incubating transfected cells for an additional 24 hours with various concentrations of compound (1) in 30% Wnt3a-CM (Wnt3a containing conditioned medium from L Wnt3a cells was harvested as described by ATCC) or 25 mM LiCl. All treated reporter cells were finally lysed and the firefly luciferase and Renilla activities were measured on a 20/20n Luminometer (Turner BioSystems) as described in the Dual-Glo™ Luciferase Assay System Technical Manual (Promega).

2) The Shh Light II assay was performed by seeding 100000 Light II cells in 48-well plates and incubating them for 48 hours with 10 or 1 μΜ of compound (1) in 50% Shh-CM (Shh conditioned medium was harvested from PANC-1 cells that carried lentivirus (produced using ViraPower Lentiviral Systems (Invitrogen)) with Shh cDNA (mouse Shh cDNA was cloned into a pLenti6.2-GW/EmGFP Expression Control Vector)).

3) Transfection was performed as described in part (1) above, but using 0.23 μg F-KB- Luciferase (Promega) and 0.02 μg pRL-TK (Renilla) (Promega). Transfected cells were incubated for 24 hours with 1 or 10 μΜ of compound (1) in 10 ng/ml rTNF-a (R&D Systems).

XLfit (idbs) was used to determine the ICso-values of inhibition experiments. A Langmuir Binding Isotherm was used to fit the data points:

fit = ((A+(B*x))+(((C-B)*(l-exp((-l *D)*x)))/D))

res = (y-fit)

SigmaPlot ^® 11 (Systat Software Inc.) was used to perform all statistical analyses. For comparisons of two groups, normal distributions of the datasets were first analyzed with the Shapiro-Wilk tests. When the Shapiro-Wilk test passed (P = > 0.05), a Student's t-test was performed. If the Shapiro-Wilk test failed (P = < 0.05), a Mann-Whitney rank sum test was applied. When performing Students t-test and Mann-Whitney rank sum tests, P < 0.05 was regarded as a statistically significant difference. In determining the IC ₅₀ -values using XLfit, the IC ₁₀₀ value was defined as the level of the positive control (30% Wnt3a-CM) subtracted by the negative control (no stimulation). The IC ₀ - value is defined as the level of the negative control. The grey line depicts the ICso-value (Y-axis, relative activities in percentage %) and the concentration in μιηοΙ/L (X-axis). Results in the reporter assays (Figure 7A) shows the mean values of at least three independent experiments.

Results

Wnt3a-induced HEK293 cells containing a transiently transfected ST-Luc (SuperTop-luciferase) reporter showed inhibition by compound (1) with an IC ₅₀ of 470 nM (Fig. 1A, left panel and Fig. 7A). In contrast, Shh Light II cells (Glil-Luc reporter) that were activated with 50% SHH-CM were not inhibited by 10 or 1 μιηοΙ/L compound (1). Also, T Fa-activated HEK293 cells containing a transiently transfected F-KB-Luciferase ( F-KB-LUC) plasmid showed no pathway inhibition in the presence of 10 or 1 μιηοΙ/L of compound (1) (Fig. IB and C).

The results of these in vivo experiments suggest that compounds according to the invention are specific inhibitors of the Wnt signaling pathway.

Example 18 - In vivo selectivity and specificity of Wnt pathway inhibitors

The selectivity and specificity of compound (1) was further tested in an in vivo Xenopus axis duplication assay, a highly stringent test for examining inhibitors of canonical Wnt signaling. When injected into the ventral blastomeres of four-cell stage X. laevis embryos, XWnt8 mRNA induces Wnt signaling resulting in the formation of a second body axis. By using selective Wnt inhibitors, the axis duplication can be inhibited and a normal phenotype restored (Waaler et al, Cancer Res (2011) TV.197-205). This assay provides a reliable method to test biological effects of compounds with potential effects on Wnt signaling.

Experimental

Capped XWnt8 mRNA was synthesized from a linearized plasmid template using the

mMESSAGE mMACHINE kit (Ambion). 4 nl XWnt8 mRNA (10 pg), with 2 pmol Compound (1) or a corresponding volume of DMSO, was injected into the equatorial regions of the two prospective ventral blastomeres of four-cell stage Xenopus embryos. The embryos were incubated at 19 °C and axis duplication was scored after 36 hours.

Results

Co-injection of 10 pg XWnt8 and 2 pmol of compound (1) resulted in a significant 51% reduction of the axis duplication incidence in developing frog embryos when compared to the DMSO vehicle control (z-test, P = 0.001) (Fig. ID, left). This result provides evidence that compounds of the invention act as specific and effective inhibitors of canonical Wnt signaling. Example 19 - Point of action in the Wnt pathway

To identify the point at which compound (1) acts on the canonical Wnt signaling pathway, the effect of compound (1) was monitored after LiCl-induced activation of the pathway. The activity of GSK3 , a serine/threonine protein kinase which is part of the β-catenin destruction complex, is reduced in the presence of LiCl (Phiel et al, Annu Rev Pharmacol Toxicol (2001) 4J_:789-813), rendering the N-terminal phosphorylation of β-catenin by GSK3 ineffective. This causes nuclear accumulation of β-catenin and activation of Wnt signaling. Therefore HEK293 cells were transiently transfected with a SuperTOPFlash reporter, and subsequently induced with LiCl and exposed to various doses of compound (1).

Experimental

Transient transfection of FEK293 cells was performed as described in Example 6, part (3). Following transfection, cells were incubated for 24 hours with different doses of compound (1) in 25mM LiCl. All treated reporter cells were finally lysed and the firefly luciferase and Renilla activities were measured on a 20/20n Luminometer (Turner BioSystems) as described in the Dual-Glo(TM) Luciferase Assay System Technical Manual (Promega).

Results

The LiCl-activating effect was counteracted by compound (1) in a dose-dependent manner with an IC ₅ o-value of 360 nM, indicating that compound (1) acts at the level of, or downstream of, the destruction complex (Fig. 2A, left and Fig. 7B). Example 20 - Direct action on β -catenin

To investigate a potential direct effect of compound (1) on β-catenin, HEK293 cells were transiently transfected with the ST-Luc reporter and with a constructs encoding various components of the Wnt pathway. These components were (i) wild-type β-catenin; and (ii) β- catenin with point mutations in the N-terminal phosphorylation sites (S33, S37, T41, S45) that is resistant to degradation and functions as dominant active (da-Cat).

Experimental

Transfection was performed as described in Example 6, part (3), but using β-catenin variants co- transfected in the following plasmid combinations:

(i) 0.215 μg ST-Luc + 0.02 ^g Renilla + O.O^g β-catenin; and

(ii) 0.23 μg ST-Luc + 0.02 ^g Renilla + 0.2 ng da-Cat.

β-catenin plasmid version co-transfected HEK293 cells were exposed to 10 μΜ compound (1) for 24 hours from time of transfection. A minimum of six replicates were performed for each treatment.

Results

Expression of wild-type β-catenin led to an increased ST-Luc reporter activity that could be reduced by 55% when the transfected cells were exposed to 10 μιηοΙ/L of compound (1)

(normality test failed, rank sum test: P = 0.001) (Fig. 2A, right). In contrast, the activation of the pathway by da-Cat could not be inhibited by 10 μιηοΙ/L compound (1), further indicating that compound (1) acts at the level of the destruction complex (normality test failed, rank sum test: P = 0.457) (Fig. 2A, right).

Example 21 - Inhibition of canonical Wnt signaling in colorectal cancer cell lines in vitro

Mutations in the APC gene, which occur in nearly all colorectal cancers (Barker et al., Nat Rev Drug Discov (2006) 5 :997-1014), lead to ineffective degradation of β-catenin and aberrant up- regulation of Wnt signaling. The effect of compound (1) on cell lines containing mutant APC genes was therefore investigated. Cell lines SW480 and HCT-15 are mutated in codon 1338 and 1417 of the APC gene, respectively. Cell line HCT116 carries a point mutation in the CK la- dependent phosphorylation site S45 of one β-catenin allele. However, S45-mutated β-catenin may still be phosphorylated in the remaining GSK3 phosphorylation sites (e.g. S33, S37, T41), with resulting semi-regulated β-catenin turn-over (Wang et al, Cancer Res (2003) 63 :5234-5).

Experimental

Cell lines SW480, HCT-15 and HCT116 were purchased from ATCC (American Type Culture Collection) and maintained according to the supplier's recommendations.

Stable transfection constructs were prepared for ST-Luc by subcloning the ST-Luc cassette into a pCIneo plasmid (Promega), giving rise to the construct "ST-Luc-neo" - selection on 500-1000 μg/mL Genetecin (G418, Sigma), and for Renilla by subcloning the pRL-TK (Renilla) cassette into pPUR (Promega), giving rise to the construct "pRL-TK-puro" - selection on 1-5 μg/ml Puromycin (Sigma).

50,000 HCT-15 or SW480 cells stably transfected with ST-Luc-neo and pRL-TK-puro were seeded in 48-well plates and cultured overnight. The medium was changed to 0.05% DMSO or different concentrations of compound (1). The cells were then incubated for another 48 hours. At incubation end, all reporter cells were lysed and the firefly luciferase and Renilla activities were measured in a 20/20n Luminometer (Turner BioSystems) as described in the Dual-Glo (TM) Luciferase Assay System Technical Manual (Promega).

Results

Stably transfected SW480 or HCT-15 cells were incubated with various doses of compound (1). A dose-dependent reduction of luciferase activity was detected in all cell lines. Compound (1) was effective in the range of 1-5 μπιοΙ/L in SW480 cells and 0.01-5 μπιοΙ/L in HCT-15 cells (Fig. 2B, left). In HCT116 cells, compound (1) was effective in the range of 0.01-5 μπιοΙ/L (Fig. 2B, right). A basal luciferase expression level at ~ 50% was reached after exposure to 5 μπιοΙ/L in the colorectal cancer cells.

Example 22 - Specific inhibition of Wnt target gene expression

A real-time RT-PCR analysis was employed to confirm specific inhibition of expressed Wnt target genes. The effect of compound (1) on the expression of endogenous Wnt target genes was examined by real-time PCR in the cell lines SW480 and DLD-1. Compared to SW480 cells, the APC mutation in DLD-1 contains a more extensive C-terminal deletion (Yang et al., J Biol Chem (2006) 281 : 17751-7).

Experimental

100000 DLD-1 (American Type Culture Collection) or SW480 cells were seeded in 12-well plates. After 24 hours, compound (1) was added to a final concentration of 10 or 25 μιηοΙ/L. The medium containing compound (1) was changed daily for three days. mRNA was harvested using GenElute™ Mammalian Total RNA Miniprep Kit (Sigma). cDNA was synthesized from the purified mRNA with an AffinityScript™ QPCR cDNA Synthesis Kit (Stratagene). Real-time RT-PCR (SYBR Green PCR Master mix, Stratagene) was performed in Mx3000P ^® QPCR System real-time thermal cycler (Stratagene). owing primers used were (obtained from Eurofins MWG Operon):

AXIN2 forward: 5' -CCC AAGCCCC AT AGTGCCC AAAG-3 ' (SEQ ID NO: 1)

AXIN2 reverse: 5' -CAGGGGAGGCATCGCAGGGTC- 3' (SEQ ID NO: 2)

SP5 forward: 5' -GCGGCGAGGGGC AAGGGC-3 ' (SEQ ID NO: 3)

SP5 reverse: 5' - CGCCGAGGCATGGACACCCG-3' (SEQ ID NO: 4)

NKD1 forward: 5' -TCACTCCAAGCCGGCCGCC-3' (SEQ ID NO: 5)

NKD1 reverse: 5' -TCCCGGGTGCTTCGGCCTATG-3 ' (SEQ ID NO: 6)

GAPDH forward: 5' - GCCCCCTCTGCTGATGCCCCCA-3' (SEQ ID NO: 7)

GAPDH reverse: 5' - TGGGTGGC AGTGGC ATGG-3 ' (SEQ ID NO: 8)

Results were calculated as the ratio of expression of target gene to a control gene {GAPDH). Results

In SW480 cells, a dose-dependent decline in the expression of the three target genes was observed (Fig. 2C, left):

AXIN2 (25 μιηοΙ/L: 55% and 10 μιηοΙ/L: 45%);

SP5 (25 μιηοΙ/L: 38% and 10 μιηοΙ/L: 15%); and

NKD1 (25 μιηοΙ/L: 31% and 10 μιηοΙ/L: 7%).

A similar reduction was observed in DLD-1 cells after exposure to 10 μιηοΙ/L of Compound (1), (Fig. 2C, right):

AXIN2: 72%; SP5: 38%; and

NKDI: 66%.

Example 23 - Gene expression analysis following Wnt pathway inhibition

A microarray analysis using an Illumina ^® array was performed in triplicate to investigate differential gene expression in cells treated with a Wnt pathway inhibitor.

Experimental

mRNA from three independent experiments, SW480 exposed to 25 μιηοΙ/L compound (1) as described in Example 22, was amplified for hybridization on Illumina ^® BeadChips using the Illumina ^® TotalPrep RNA amplification Kit (Ambion) # IL1791, using 400 ng of the total RNA. The m vitro transcription reaction was incubated overnight (14 hr). Labeled cRNA was hybridized to the Illumina Human-6 v3 BeadChips (Illumina) at 58°C overnight, according to the Illumina Whole-Genome Gene Expression Protocol for BeadStation (Illumina). The hybridized BeadChip was stained with streptavidin-Cy3 (Amersham) for visualization and scanned with an Illumina® BeadArray Reader. The scanned images were imported into BeadStudio 3.1.3.0 (Illumina) for extraction and quality control. Results

Several Wnt target genes (Wnt homepage http://wv^v.stanford.edu/group/nusse¾ab/cgi-biri/\v¾t were differently expressed. A list of differentially expressed genes of interest is given below in Tables 1 and 2. Table 1 shows examples of genes that were down-regulated on addition compound (1); and Table 2 shows examples of genes that were up-regulated. The degree of modulation is an indication of the degree of change between the non-treated and compound (1) treated samples. Tables 1 and 2 are sorted to show the most down-regulated (or up-regulated) genes first. Up-regulated genes of interest included (Log2 > 0.5): WISP3, TCF7, PLAUR, EFNB2 and NOTUM. Down-regulated genes of interest included (Log2 < -0.5): AXIN2, NKDI, DKK1, MMP7, ID2, GAST, FZD2, EDN1, CYR61, SOX18 and members of the SPANX gene family (SPANXA1, SPANXB2, SPANXC and SPANXE). The Illumina analysis also revealed other interesting genes with modified expression, including SFRP5, CSNK1G2, ALPL, PPARG, KLF4, KLF5 and KLF6. Table 1

" " denotes very strong down-regulation (log ² ratio of between -2 and -2.5); "- -" denotes strong down-regulation (log ² ratio of between -1.5 and -2); and "-" denotes moderate down- regulation (log ² ratio of between -1 and -1.5). Table 2

"+ + + " denotes very strong up-regulation (log ² ratio of between 1.5 and 1. 75) ; "++ " denotes strong up-regulation (log ² ratio of between 1.25 and 1.5); and "+ " denotes moderate up- regulation (log ² ratio of between 1 and 1.25). Example 24 - Destabilisation of β-catenin by increasing cytoplasmic AXIN2 levels

Previous studies have shown that an increase in AXIN2 steady-state levels induced by TNKSl/2 or CKla inhibitors is accompanied by a decrease in β-catenin concentrations (e.g. Huang et al, Nature (2009) 461 : 614-20). Increased levels of AXIN2 protein promote degradation of β- catenin even in cells with truncated APC (e.g. Chen et al, Nat Chem Biol (2009) 5 : 100-7). The effect of compound (1) on intracellular levels of AXIN2 and β-catenin were thefore investigated by Western blotting and immunocytochemistry. Experimental

For immunoblotting, 100000 SW480 cells per well were seeded in 12-well plates and treated with compound (1) at various concentrations for 24 hours. Lysates were immunoblotted using the following primary antibodies:

monoclonal active-P-catenin (#05-665ABC) (Millipore);

β-catenin (610153, BD Transduction Laboratories™);

phospho-P-catenin (Ser33/37/Thr41, Cell Signaling Technology);

AXIN2 (76G6, Cell Signaling Technology);

ACTIN (A2066, Sigma); and

LAMIN B1 (abl6048-100, Abeam).

Primary antibodies were visualized with secondary HRP-conjugated antibodies (sc-2313 or sc- 2314, Santa Cruz Biotechnology) and enhanced chemiluminescent substrate (Pierce ^® ECL Western Blotting Substrate, Thermo Scientific).

For immunocytochemical studies, 50000 SW480 cells were seeded in 24-well plates on glass slides and exposed to 1 or 5 μιηοΙ/L compound 1 for 48 hours. After the incubation, the cells were fixed in 4% PFA in PBS for 10 minutes. Immunostaining was performed as described in standard protocols. Primary antibodies used were β-catenin (610153, BD Transduction

Laboratories™) or AXIN2 (76G6, Cell Signaling Technology). Secondary antibodies used were DyLight549 (555) donkey-anti-mouse and Cy2-donkey-anti -rabbit (both Jackson

ImmunoResearch, 1 : 1000). The samples were imaged using a Zeiss Axiovert 200M

Fluorescence/Live cell Imaging Microscope at 40 times magnification. A Zeiss LSM780 at 63 times magnification was used for confocal microscopy. Results

Figures 3 A and 8 indicate that Western Blot analysis of SW480 cells lysates revealed a dose- dependent increase of cytoplasmic AXIN2 after treatment with compound (1) (range: 10 μιηοΙ/L -100 nM). Furthermore, an antibody against the active and non-phosphorylated form of β- catenin (active β-catenin, ABC) identified reduced levels of β-catenin in the cytoplasm of compound (l)-treated cells. In addition, a modest reduction of total β-catenin and a substantial decrease of nuclear ABC were observed, and an increase in phosphorylated β-catenin (ρβ- catenin) levels was seen, indicating ongoing β-catenin degradation. To gain further insight into the changes in cellular distribution of ΑΧΓΝ2 and β-catenin, compound (l)-treated SW480 cells were analyzed by immunofluorescence. A general reduction of total β-catenin, both in the cytoplasmic and nuclear compartments, was detected at the doses of 5 and 1 μπιοΙ/L (equal shutter speed) (Fig. 9). Confocal microscopy (equal shutter speeds) revealed, in accordance with the Western blot analysis, that the levels of cytoplasmic AXIN2 were significantly increased (Fig. 3B) and large protein foci, probably representing accumulated destruction complexes, were observed (Fig. 3B, arrows). Clusters of co-localized cytoplasmic β- catenin and AXIN2 have previously been detected in SW480 cells after treatment with Wnt antagonists. Enhanced phosphorylation and resulting degradation of β-catenin after compound (1) exposure appeared to be orchestrated by stabilization of AXIN2 in the destruction complex (Figs. 3 and 5).

Example 25 - Inhibition of the PARsylation activity of T KS1 and TNKS2

By inhibiting the PARP domain of TNKSl/2, compounds XAV939 and IWR-1 prevent auto- PARsylation of TNKSl/2 and PARsylation of AXIN2. This leads to the stabilization of AXF 2 followed by an increased activity of the β-catenin destruction complex. To test whether compound (1) decreased canonical Wnt signaling by inhibiting the PARP domain of TNKSl/2, biochemical assays for monitoring the activity of TNKSl/2 and PARP were performed. Experimental

The inhibitory activity of compound (1) at various doses (in duplicate) was tested using

Chemiluminescent Assay Kits (BPS Bioscience, Nordic Biosite) against TNKS1 (Cat No. 80564), TNSK2 (Cat No. 80566) and PARPl (Cat No. 80551). The procedures were performed according to the manufacturer's protocols.

A fluorescence polarization competition assay was performed as follows. 2 mg of XAV939 including a primary amino group (TC scientific) was dissolved in 250 μΙ_, DMSO and mixed with 4 mg NHS-fluorescein (Thermo Scientific) in 100 μΙ_, DMSO and 100 μΙ_, sodium phosphate (20 mM). The solution was incubated at room temperature overnight. The labeled product

(XAV939-fluorescein) was isolated from the excess reactants by semi -preparative reverse phase liquid chromatography, using a 10 mm i.d. x250 mm 5 AQ C ₁₈ column (ACE). The mobile phase was ACN/0.1% FA (aq) (43/57, v/v). The eluent was dried with a SpeedVac concentrator overnight. The product was examined by LC-MS (Bruker) and the purity was assessed to be >99%. The product was weighed and a stock solution of 1 mM was prepared with DMSO. A working solution of 1 μΜ was prepared by dilution with water. The inactive analog (TC scientific) was tested in a ST-Luc assay with Wnt3a activation as described in Example 6, part (1). A F200 PRO fluorescence polarizer (Tecan) with appropriate filters (EX 485/EM 535) was employed for the competition assay.

Results

Compound (1) decreased auto-PARsylation of TNKS1/2 in vitro with ICso-values of 1.9 μπιοΙ/L and 830 nmol/L, respectively (Fig. 4A). However, in contrast to XAV939, but similar to IWR-1, compound (1) exhibited no inhibition of PARPl at doses up to 20 μπιοΙ/L (Fig. 4A).

These results indicate that compounds of the invention block canonical Wnt signaling by specifically inhibiting auto-PARsylation of TNKS1/2 (Fig. 5) while leaving PARPl activity unaffected.

Example 26 - Reduction in growth of SW480 colon cancer

Colorectal cancer cells can enter cell cycle arrest as a result of antagonized canonical Wnt signaling (e.g. Suzuki et al., Nat Genet (2004) 36: 417-22). Various proliferation assays were therefore performed to see whether compound (l)-mediated inhibition of canonical Wnt signaling would affect colorectal cancer cell growth. Experimental

Cell cycle analysis was performed by seeding 100000 SW480 cells per well in 12-well plates and exposing the cells to 10 μιηοΙ/L compound (1) or 0.1% DMSO for three days. The cell culture medium was changed daily. After 30 minutes of incubation with 10 μιηοΙ/L BrdU, the cells were trypsinized, fixed in 4% PFA and stained with mouse anti-BrdU (Roche, 1 : 100) followed by binding of anti-mouse Alexa Fluor ^® 488 (Invitrogen). Counterstaining with 10 μg/mL propidium iodide (PI) was followed by treatment with RNase I (both from Sigma). The samples were analyzed in a PAS-PPCS flow cytometer (Partec). Cell proliferation kinetics were monitored using IncuCyte™. SW480 cells were exposed to 10, 5 or 1 μιηοΙ/L of compound (1) for nine days and the confluency was measured every two hours. In parallel, the colorectal cancer cell line RKO (ATCC), which contains wild-type APC and β- catenin and exhibits Wnt-independent cell growth, was used as a control. Long-term culture of cells was performed by seeing 40000 SW480 cells per well in 24-well plates (as duplicates). HeLa cells (ATCC), which are Wnt growth-independent cervical cancer cells, were used as a control. A day after seeding, the cell medium was changed to 0.05% DMSO (control) or 5, 2.5 or 1 μιηοΙ/L solutions of compound (1). The cells were split 1 :5 twice a week while cultured in the compound (l)-containing or control solutions. At each passage, a proportion of the discarded cells were counted by a PAS-PPCS flow cytometer to determine the proliferation rate (Partec).

In addition, SW480 cells were grown under low serum conditions (1% FBS) along with various concentrations of compound (1) (5, 1 and 0.1 μιηοΙ/L) and the formation of colonies was measured. HeLa and RKO cells were similarly grown in control experiments.

Results

Flow cytometry cell cycle analysis of SW480 cells incubated with compound (1) and labeled with BrdU and propidium iodide revealed that treatment with compound (1):

(a) lowered the proportion of cells in the S phase from 28.4% in DMSO-treated

controls to 22.2%;

(b) raised moderately the cell fraction in the Gl phase from 37.3% to 38.8%; and (c) increased the number of cells in G2/M phase from 34.3% to 39%. Results are shown in Fig. 6A.

Cell proliferation kinetic measurements indicated that all doses of compound (1) reduced SW480 cell growth and the most robust effect was detected at a dose of 10 μιηοΙ/L compound (1). The confluency was reduced to 55% relative to the DMSO control at the end of experiment (Fig. 6B). In contrast, the control cells (RKO) reached 100% confluency within seven days and were not affected by treatment with 10 μιηοΙ/L compound (1) (Fig. 6B).

When proliferation of SW480 cells was examined by consecutive passages in the presence of 5, 2.5 or 1 μιηοΙ/L compound (1), a dose-dependent decrease of cell numbers over three passages was noted in SW480 cells in the presence of compound (1). In contrast, HeLa cells remained unaffected (Fig. 6C).

When the formation of clonies was quantified, a concentration-dependent reduction of colony numbers in SW480 cells was observed. No reduction in colony numbers in the control cell lines HeLa and RKO was observed when cultured in 5 μιηοΙ/L compound (1) (Fig. 6D, left). All SW480 colonies, which formed in the presence of compound (1), were substantially smaller (Fig. 6D, right). Taken together, these data show that compound (l)-mediated inhibition of canonical Wnt signaling resulted in reduced cell cycle progression, proliferation and colony formation in the colorectal cancer cell line SW480 in vitro.

Example 27 - IC ₅₀ values of compounds

IC ₅₀ values were calculated using STF/REN HEK293 cells (stably transfected with ST-Luc (7 X TCF binding sites) and Renilla (Promega) plasmids). HEK293 cells (80,000 cells per well) were seeded in 48-well plates coated with poly-L lysine. 24 hours after seeding, the cells were incubated for an additional 24 hours with various compound concentrations in 50% Wnt3a containing conditioned media (from L Wnt3a-expressing cells (ATCC)). After compound exposure, the cells were lysed and the firefly luciferase and Renilla activities were measured on a 20/20n Luminometer (Turner BioSystems) as described in the Dual-Glo™ Luciferase Assay System Technical Manual (Promega). XLfit (idbs) was used to determine the IC ₅₀ values in inhibition experiments. The data were fit to the following formula:

IC ₅₀ : Langmuir Binding Isotherm:

fit = ((A+(B*x))+(((C-B)*(l-exp((-l *D)*x)))/D))

res = (y-fit)

Table 3 shows the IC ₅₀ values of certain compounds. All values shown are average values from multiple experiments.

Table 3

Compound No. IC ₅₀ μΜ ± Standard

Deviation

(1) 1.23 0.6

(12) 0.79 0.05

(13) >10 -

(14) 0.38 0.2

(15) 0.36 0.1

(16) 4.1 1.1

(17) >10 -

(18) 4.9 1.1

(19) 5.1 1.5

(20) >10 -

(21) >10 -

(22) >10 -

(23) >10 -

(24) 4.26 1.4

(25) >10 -

(26) 0.69 0.040