Heterocyclic compounds as modulators of the β-catenin / TCF4 interaction

Abstract

Provided herein are compounds useful for inhibiting β-catenin or disrupting the interaction of β-catenin with T cell factor 4, or methods for inhibiting β-catenin or disrupting the interaction of β-catenin with T cell factor 4, which are useful for treating diseases or conditions such as cancer, neurodegenerative diseases, metabolic diseases, cardiovascular diseases, fibrosis, and bone diseases. In one aspect, the compounds have formula 1, wherein R 1 Ha-(CR 4 2) m XR 5 and R 2 is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, aralkyl, heteroaralkyl, or heteroaryl; R 3 Ha-(CR 6 2) n R 7 is. TIFF2022547426000045.tif27170

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority from Singapore Patent Application No. 10201908175Q, filed September 4, 2019, the contents of which are incorporated herein by reference in their entirety.

[0002] Technical Field The present disclosure generally relates to methods for inhibiting the Wnt / catenin (β-cat) signaling pathway. More specifically, the present disclosure provides small molecule inhibitors of β-cat / T-cell factor 4 (TCF4) interaction that result in attenuation of downstream Wnt expression. The compounds and methods provided herein are useful for treating, managing, or preventing diseases ameliorated by inhibiting the interaction of β-cat and TCF4, such as cancer, neurodegenerative diseases, metabolic diseases, cardiovascular diseases, fibrosis, and bone diseases. [Background technology]

[0003] The canonical Wnt / β-cat signaling pathway is essential for normal embryonic development and self-renewal of adult tissues. Activation of this pathway occurs through the binding of Wnt ligand proteins to Frizzled and low-density lipoprotein receptor-related protein 6 (LRP6). In the inactive state of Wnt, a degradation complex containing axin (Axin), casein kinase 1α (CK1α), glycogen synthase kinase 3β (GSK3β), and adenomatous polyposis coli (APC) phosphorylates β-cat, the major nuclear effector of Wnt signaling, and targets it for ubiquitination and proteasomal degradation. Activation of this pathway by Wnt ligands results in membrane binding (recruitment) of the degradation complex, resulting in its inactivation. Inhibition of the degradation complex leads to the accumulation of unphosphorylated β-cat in the cytoplasm and subsequent translocation into the nucleus, where it binds to transcription factors of the lymphoid enhancer factor / T-cell factor (LEF / TCF) family and many other cofactors, including Bcl-9 and Pygopus, to activate the expression of downstream target genes.

[0004] Due to the importance of the Wnt pathway for maintaining tissue homeostasis, aberrant activation of the Wnt pathway, particularly in stem cells, is often a contributing factor to diseases such as cancer. In particular, it has been shown that in the majority of colorectal cancers (CRCs), loss of function of the APC tumor suppressor gene is an early factor in tumorigenesis, leading to adenoma formation. In addition, loss-of-function mutations in Axin 2 and activating mutations in β-cat are commonly found in hepatocellular carcinoma (HCC) and, at lower frequencies, in CRC. In addition to cancer, misregulation of the Wnt pathway has also been implicated in the development of neurodegenerative, metabolic, and bone disorders. Therefore, numerous therapeutic approaches targeting the Wnt pathway, including small-molecule inhibitors and antibody-based therapies, have been reported over the years. Despite numerous promising discoveries of Wnt-targeting drugs, most have not yet progressed to human clinical trials. While there are individual examples of small-molecule inhibitors that have progressed to phase 1 trials, none have been approved for clinical use to date. One of the challenges these drugs face in developing as therapeutics is the significant crosstalk between the Wnt signaling pathway and other pathways, such as Notch and Hedgehog. Crosstalk between these pathways may limit the specificity of these drugs or affect their efficacy in therapeutic responses through compensatory activation. In addition, driver mutations in various components within the linear cascade of the Wnt pathway may also limit the pathway-inhibitory efficacy of these drugs.

[0005] Consequently, it is predicted that effective and specific downregulation of the Wnt pathway can be achieved by inhibiting the β-cat-TCF4 interaction, which is required for the expression of downstream Wnt target genes. The advantages of this strategy are twofold. First, targeting the β-cat-TCF4 interaction may be more effective in diseases in which mutations in downstream components of the Wnt pathway render upstream inhibitors less effective. Second, this strategy specifically targets the transcriptional function of β-cat and does not appear to affect its interaction with E-cadherin (ECAD) at cell-cell adherens junctions (AJs). It is proposed that specificity is achieved by selectively targeting the β-cat-TCF4 binding pocket, which is distinct from the β-cat-ECAD binding pocket.

[0006] A cell-based RNA interference (RNAi)-based chemical genetic screen has been reported to identify compounds that inhibit the β-cat-TCF4 interaction. This screen uncovered three compounds, iCRT3, iCRT5, and iCRT14, that disrupt the β-cat-TCF4 interaction without affecting the interaction between β-cat and ECAD. In silico docking of iCRT3 to β-cat predicted that iCRT3 binds to a site on β-cat that binds to the extended region of TCF4 (residues 13–25). This predicted iCRT3 binding site on β-cat is surrounded by Arg469 and Lys435, which form a salt bridge with Asp16 of TCF4 and is essential for TCF4's high affinity for β-cat. Because of its importance for the β-cat-TCF4 interaction, this binding pocket has long served as an attractive target site for in silico docking studies to identify inhibitors of the β-cat-TCF4 interaction.

[0007] Therefore, there is a need for improved methods for inhibiting β-cat-TCF4 interaction and concomitantly attenuating downstream Wnt signaling that address or overcome at least some of the above-mentioned challenges.

[0008] There is also a need for therapeutic agents that can treat diseases or conditions ameliorated by inhibiting β-cat or by interfering with the interaction of β-cat with TCF4. Summary of the Invention [Means for solving the problem]

[0009] Described herein is a computational model that uses existing protein X-ray crystal structures and the biological activity of known β-cat-TCF4 interaction inhibitors to better predict potential small molecule inhibitors of this protein-protein interaction surface. To demonstrate the predictive potential of this computational model system, an in silico screen of a small molecule library was performed, identifying 27 compounds as potential β-cat binders. Experimental validation of the hit compounds identified three potent inhibitors of Wnt reporter activity. Among them, compound GB1874 was found to induce a robust Wnt tumor-inhibitory phenotype in both in vitro and in vivo phenotypic assays. Based on these results and further in silico and biological screens, a group of compounds that potently inhibit Wnt reporter activity was identified.

[0010] In one aspect, provided herein is the use of a compound in the manufacture of a medicament for the treatment of a disease or condition ameliorated by inhibiting β-catenin (β-cat) or by disrupting the interaction of β-cat with T-cell factor 4 (TCF4), wherein the compound has formula 1, or a pharmaceutically acceptable salt thereof: [ka] In the above formula 1, R 1 Ha-(CR 4 2) m XR 5 and R 2is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, aralkyl, heteroaralkyl, or heteroaryl; R 3 Ha-(CR 6 2) n R 7 and R 4 is, at each occurrence, independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, and heteroaralkyl, or R 4 Two instances of (the two R 4 ) taken together with one or more carbons to which they are attached form a 3- to 7-membered cycloalkyl; R 5 is hydrogen, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, or heteroaryl; R 6 is, at each occurrence, independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, and heteroaralkyl, or R 6 Two instances of (the two R 6 ) taken together with one or more carbons to which they are attached form a 3- to 7-membered cycloalkyl; R 7 is -(C=O)R 8 , -(C=O)OR 8 , or -(C=O)N(R 8 )2 or R 7 is a moiety having formula 2, [ka] R 8is, at each occurrence, independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, and heteroaralkyl, or two R 8 together with the nitrogen to which they are attached form a 3- to 7-membered heterocycloalkyl, or R 6 and R 8 together with the atom(s) to which they are attached form an optionally substituted 4- to 7-membered cycloalkyl or an optionally substituted 4- to 7-membered heterocycloalkyl; m is an integer selected from 0 to 4; n is an integer selected from 0 to 4, X is -O-, -S-, or absent It is used.

[0011] In another aspect, provided herein is a method of inhibiting β-cat or disrupting the interaction of β-cat with TCF4, comprising contacting β-cat with a compound of formula 1 or a pharmaceutically acceptable salt thereof: [ka] In the above formula 1, R 1 Ha-(CR 4 2) m XR 5 and R 2 is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, aralkyl, heteroaralkyl, or heteroaryl; R 3 Ha-(CR 6 2) n R 7 and R 4is, at each occurrence, independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, and heteroaralkyl, or R 4 two instances of, taken together with one or more carbons to which they are attached, form a 3- to 7-membered cycloalkyl; R 5 is hydrogen, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, or heteroaryl; R 6 is, at each occurrence, independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, and heteroaralkyl, or R 6 two instances of, taken together with one or more carbons to which they are attached, form a 3- to 7-membered cycloalkyl; R 7 is -(C=O)R 8 , -(C=O)OR 8 , or -(C=O)N(R 8 )2 or R 7 is a moiety having formula 2, [ka] R 8 is, at each occurrence, independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, and heteroaralkyl, or two R 8 together with the nitrogen to which they are attached form a 3- to 7-membered heterocycloalkyl, or R 6 and R 8 together with the atom to which they are attached form an optionally substituted 4- to 7-membered cycloalkyl or an optionally substituted 4- to 7-membered heterocycloalkyl; m is an integer selected from 0 to 4; n is an integer selected from 0 to 4, X is -O-, -S-, or absent It is a method.

[0012] In another aspect, provided herein is a method for treating a disease or condition ameliorated by inhibiting β-cat or by disrupting the interaction of β-cat with TCF4 in a subject in need thereof, comprising administering a therapeutically effective amount of a compound of formula 1 or a pharmaceutically acceptable salt thereof; [ka] In the above formula 1, R 1 Ha-(CR 4 2) m XR 5 and R 2 is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, aralkyl, heteroaralkyl, or heteroaryl; R 3 Ha-(CR 6 2) n R 7 and R 4 is, at each occurrence, independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, and heteroaralkyl, or R 4 two instances of, taken together with one or more carbons to which they are attached, form a 3- to 7-membered cycloalkyl; R 5 is hydrogen, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, or heteroaryl; R 6is, at each occurrence, independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, and heteroaralkyl, or R 6 two instances of, taken together with one or more carbons to which they are attached, form a 3- to 7-membered cycloalkyl; R 7 is -(C=O)R 8 , -(C=O)OR 8 , or -(C=O)N(R 8 )2 or R 7 is a moiety having formula 2, [ka] R 8 is, at each occurrence, independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, and heteroaralkyl, or two R 8 together with the nitrogen to which they are attached form a 3- to 7-membered heterocycloalkyl, or R 6 and R 8 together with the atom to which they are attached form an optionally substituted 4- to 7-membered cycloalkyl or an optionally substituted 4- to 7-membered heterocycloalkyl; m is an integer selected from 0 to 4; n is an integer selected from 0 to 4, X is -O-, -S-, or absent It is a method.

[0013] In another aspect, provided herein is a compound for use in the treatment of a disease or condition ameliorated by inhibiting β-cat or by disrupting the interaction of β-cat with TCF4, the compound having formula 1, or a pharmaceutically acceptable salt thereof: [ka] In the above formula 1, R 1 Ha-(CR 4 2) m XR 5 and R 2 is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, aralkyl, heteroaralkyl, or heteroaryl; R 3 (CR 6 2) n R 7 and R 4 is, at each occurrence, independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, and heteroaralkyl, or R 4 two instances of, taken together with one or more carbons to which they are attached, form a 3- to 7-membered cycloalkyl; R 5 is hydrogen, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, or heteroaryl; R 6 is, at each occurrence, independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, and heteroaralkyl, or R 6 two instances of, taken together with one or more carbons to which they are attached, form a 3- to 7-membered cycloalkyl; R 7 is -(C=O)R 8 , -(C=O)OR 8 , or -(C=O)N(R 8 )2 or R 7 is a moiety having formula 2, [ka] R 8 is, at each occurrence, independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, and heteroaralkyl, or two R 8 together with the nitrogen to which they are attached form a 3- to 7-membered heterocycloalkyl, or R 6 and R 8 together with the atom to which they are attached form a 4- to 7-membered cycloalkyl or a 4- to 7-membered heterocycloalkyl; m is an integer selected from 0 to 4; n is an integer selected from 0 to 4, X is -O-, -S-, or absent It is a compound.

[0014] In another aspect, provided herein are compounds for use in therapy, the compounds having Formula 1, or a pharmaceutically acceptable salt thereof: [ka] In the above formula 1 R 1 Ha-(CR 4 2) m XR 5 and R 2 is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, aralkyl, heteroaralkyl, or heteroaryl; R 3 Ha-(CR 6 2) n R 7 and R 4is, at each occurrence, independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, and heteroaralkyl, or R 4 two instances of, taken together with one or more carbons to which they are attached, form a 3- to 7-membered cycloalkyl; R 5 is hydrogen, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, or heteroaryl; R 6 is, at each occurrence, independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, and heteroaralkyl, or R 6 two instances of, taken together with one or more carbons to which they are attached, form a 3- to 7-membered cycloalkyl; R 7 is -(C=O)R 8 , -(C=O)OR 8 , or -(C=O)N(R 8 )2 or R 7 is a moiety having formula 2, [ka] R 8 is, at each occurrence, independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, and heteroaralkyl, or two R 8 together with the nitrogen to which they are attached form a 3- to 7-membered heterocycloalkyl, or R 6 and R 8 together with the atom to which they are attached form an optionally substituted 4- to 7-membered cycloalkyl or an optionally substituted 4- to 7-membered heterocycloalkyl; m is an integer selected from 0 to 4; n is an integer selected from 0 to 4, X is -O-, -S-, or absent It is a compound. [Brief explanation of the drawings]

[0015] The above and other objects and features of the present disclosure will become apparent from the following description of the disclosure when considered in conjunction with the accompanying drawings.

[0016] [Figure 1] Figure 1 depicts the modeling and docking platform used to generate β-catenin structures for ligand discovery. Three crystal structures were used as starting models (N=3). A training library of small molecules contains three known inhibitors of β-catenin (M=3). Within one iteration, all target structures are further optimized in the presence of each of the three known docked ligands. [Figure 2(1)] Figure 2 depicts in silico studies of compounds predicted for Wnt signaling inhibition. (a) TOPFlash reporter activity of predicted compounds in HEK293T STF cells. Cells were treated with 10 μM of compound and simultaneously stimulated with 500 ng / mL of Wnt3A for 24 hours, after which luciferase activity was measured. TOPFlash reporter activity of compound-treated cells was normalized to cell viability and presented as fold change relative to DMSO-treated cells. Error bars represent the standard deviation of four replicates. (b) Second-order dose-response inhibition of TOPFlash reporter activity by iCRT3 and hit compounds. STF cells were treated with different concentrations of compound and simultaneously stimulated with 500 ng / mL of Wnt3A for 24 hours, after which luciferase activity was measured. Luciferase activity of cells treated with various concentrations of compound was first normalized to cell viability at the same concentration and presented as fold change relative to DMSO-treated cells. IC50 values ​​were calculated using four-parameter nonlinear regression. Error bars represent the standard deviation of three replicates. [Figure 2(2)]Figure 2 depicts the in silico analysis of compounds predicted for Wnt signaling inhibition. (c) Chemical structures of iCRT3 and the top three hit compounds. (d) Predicted binding modes of iCRT3 (i) and three novel hit compounds discovered in this study, GB8679 (ii), GB6853 (iii), and GB1874 (iv), in the optimized, most enriched β-catenin structure (partially transparent surface). The docked ligands are highlighted (solid bars), and residues in the key β-catenin binding site are also shown (partially transparent bars). To demonstrate the PPI interface, a TCF4 peptide fragment (solid line) from the β-catenin / TCF4 crystal complex structure (PDBID 1JPW) is also shown. [Figure 3(1)] Figure 3 depicts the effects of hit compounds on HCT116 cells via the Wnt signaling pathway. (a) Co-immunoprecipitation of β-catenin and its endogenous binding partners. HCT116 cells were treated with the indicated concentrations of hit compounds for 18 hours. β-catenin was immunoprecipitated from protein lysates of treated cells, and the amounts of TCF4 and E-cadherin bound to β-catenin were analyzed by Western blot (top panel). The amounts of TCF4 (lower right panel) or E-cadherin (lower left panel) bound to β-catenin under different treatments were quantified and normalized to those of DMSO-treated cells. Error bars represent the standard deviation of four independent experiments. A paired, two-tailed Student's t-test was performed between the DMSO control and compound treatments. * P<0.05, ns P>0.05. [Figure 3(2)] Figure 3 depicts the effects of hit compounds on HCT116 cells via the Wnt signaling pathway. (b) HCT116 cells were treated with either DMSO or 50 μM of compound for 18 hours. The expression of Wnt target proteins c-Met, cyclin D1, and survivin was measured by Western blotting. (c) HCT116 cells were treated with either DMSO or 50 μM of compound for 18 hours. The expression of Wnt pathway-related proteins E-cadherin, β-catenin, and TCF4 was measured by Western blotting. [Figure 3(3)] Figure 3 depicts the effects of hit compounds on HCT116 cells via the Wnt signaling pathway. (d) Representative binding curve of compound GB1874 to β-catenin by surface plasmon resonance (SPR). Steady-state response values ​​were plotted against each concentration of compound GB1874, and the dissociation constant KD (n = 3) was calculated using the specific binding with a Hill slope curve fitting equation. (e) SPR dose-response inhibition of the binding of β-catenin and GST-TCF4 by compound GB1874. IC50 values ​​(n = 2) were calculated using a four-parameter nonlinear regression. [Figure 4(1)] Figure 4 depicts growth inhibition by hit compounds and their effects on stemness of Wnt-driven cells. (a) Growth curves of HCT116 cells treated daily with 10 μM compounds. One-way repeated-measures ANOVA with Dunnett's multiple comparison test was performed to determine significance between compound treatment and DMSO control. * p<0.05, *** p<0.001. (b) Effect of hit compounds on colony formation of HCT116 cells. HCT116 cells were cultured in 6-well plates and treated with DMSO or 30 μM compounds for 7 days, after which the cells were fixed and stained with crystal violet. The number of resulting colonies was counted. A two-tailed Student's t-test was performed between compound treatment and DMSO control. *** p<0.001. [Figure 4(2)]Figure 4 depicts growth inhibition by hit compounds and their effect on stemness of Wnt-driven cells. (c) Effect of hit compounds on spheroid formation of HCT116 cells. HCT116 cells were cultured in ultra-low-attachment 96-well plates and treated with either DMSO or 30 μM of the above compounds for 14 days, after which the number of spheroids ≥200 μm in size was determined. A two-tailed Student's t-test was performed between compound treatment and DMSO control. *** p<0.001, ** p<0.01. Scale bar represents 1 mm. (d) NSG mice xenografted with HCT116 cells were treated with vehicle control (n=6) or 50 mg / kg GB1874 (n=6) via i.p. every other day for 2 weeks. Tumor volume was tracked over time. On day 17, a two-tailed Student's t-test was performed between control and treatment. * p<0.05. [Figure 5(1)] Figure 5 depicts in silico predicted docking scores and in vitro cell-based IC50 values ​​for iCRT analogs. Lower DOCK scores predict better binding. Lower RANK scores predict better binding. Favorable (1), neutral (0), unfavorable (-1). More * indicates higher ranking. nd = not determined. [Figure 5(2)] This is a continuation of Figure 5(1). [Figure 5(3)] This is a continuation of Figure 5(2). [Figure 6A] FIG. 6A shows the DOCK scores of the top 27 compounds identified from the Enamine pharmacological diversity compound library. [Figure 6B(1)] Figure 6B depicts the structures of the top 27 compounds identified from the Enamine pharmacological diversity compound library. [Figure 6B(2)] This is a continuation of Figure 6B(1). [Figure 7]Figure 7 (a) depicts cell viability of STF cells after 24 hours of compound treatment. Viability measurements of compound-treated cells were normalized to DMSO-treated cells. Error bars represent the standard deviation of four replicates. (b) Dose-response effect of iCRT3 and hit compounds on viability of STF cells after 24 hours of treatment. Viability measurements of compound-treated cells were normalized to DMSO-treated cells. IC50 values ​​were calculated using four-parameter nonlinear regression. Error bars represent the standard deviation of three replicates. [Figure 8(1)] FIG. 8 depicts (a) the binding sensorgrams of different concentrations of the analyte GB1874 to β-catenin ligands. [Figure 8(2)] (b) Binding sensorgram of 50 nM β-catenin analyte to GST-TCF4 ligand. β-catenin was pre-incubated with different concentrations of compound GB1874 for 15 min before binding to GST-TCF4. [Figure 9(1)] Figure 9 depicts the effect of hit compounds on spheroid formation in (a) DLD-1 and (b) SW480 cells. The cells were cultured in ultra-low attachment 96-well plates and treated with DMSO or 30 μM compound for 7 days, after which the number of spheroids ≥ 200 μm in size was determined. A two-tailed Student's t-test was performed between compound treatment and DMSO control. *** p<0.001, ** p<0.01, * p<0.05. The scale bar represents 1 mm. [Figure 9(2)] (c) NSG mice xenografted with HCT116 cells were treated with vehicle control (n=6) or 50 mg / kg GB1874 (n=6) via the i.p. route every other day for 2 weeks. Mouse weights were monitored over time. On day 17, a two-tailed Student's t-test was performed between control and treatment. ns P>0.05. [Figure 10] FIG. 10 depicts the effect of GB1874 and its analogs on the expression of β-catenin (CTNNB1) and Wnt target genes. [Figure 11]Figure 11 depicts that GB1874 inhibited Wnt-driven cell growth in vivo through inhibition of Wnt signaling. (a) NSG mice xenografted with HCT116 cells were treated with vehicle control (n=6) or 50 mg / kg GB1874 (n=6) via i.p. every other day for 2 weeks. Tumor volume (left panel) was tracked over time. Two-way ANOVA was performed between vehicle control and treated tumor volume. *P<0.05. Error bars represent mean ± SEM. Images of tumors at the end of treatment are shown in the right panel. The scale bar represents 1 cm. (b) Representative IHC staining of tumors for Ki67 and cyclin D1 expression (left panel). The scale bar represents 100 μm. Quantification of the number of cells expressing cyclin D1 and Ki67 per unit tumor area (right panel). For each marker, a one-tailed Student's t-test was performed between vehicle control and treatment. **P<0.01, *P<0.05, ns P>0.05. Error bars represent the mean±SD of triplicate tumors. DETAILED DESCRIPTION OF THE INVENTION

[0017] Below are some definitions that may be helpful in understanding the description of the present invention. These are intended as general definitions and in no way limit the scope of the present invention to only those terms, but are provided for a better understanding of the following description.

[0018] Unless contradictory to the context or clearly stated to the contrary, integers, steps, or elements of the invention described herein as singular integers, steps, or elements expressly encompass both the singular and plural forms of the described integer, step, or element.

[0019] Throughout this specification, unless contradictory to the context, the word "comprise" or variations such as "comprises" or "comprising" will be understood to mean the inclusion of the stated steps or elements or integers or group of steps or elements or integers, but not to the exclusion of any other steps or elements or integers or group of elements or integers. Thus, in the context of this specification, the term "comprising" means "including principally, but not necessarily solely."

[0020] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. The invention is to be understood as including all such variations and modifications. The invention also includes all steps, features, compositions, and compounds referred to or shown in this specification, individually or collectively, as well as any and all combinations of any two or more of such steps or features.

[0021] In the context of this specification, the term "amino acid" is defined as having at least one primary, secondary, tertiary, or quaternary amino group and at least one acid group, which may be carboxylic, sulfonic, or phosphonic, or a mixture thereof. The amino group may be "α," "β," "γ,"..., through "ω" with respect to the acid group(s). The backbone of the "amino acid" may be substituted with one or more groups selected from halogen, hydroxy, guanido, and heterocyclic groups. Thus, "amino acid" includes within its scope glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, serine, threonine, cysteine, tyrosine, asparagine, glutamic acid, aspartic acid, glutamine, lysine, arginine, and histidine, taurine, betaine, N-methylalanine, and the like. Both (L) and (D) amino acids are within the scope of the present invention.

[0022] As used herein, the term "alkyl group" includes within its meaning monovalent ("alkyl") and divalent ("alkylene") straight- or branched-chain saturated aliphatic groups having 1 to 10 carbon atoms, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. For example, the term alkyl includes methyl, ethyl, 1-propyl, isopropyl, 1-butyl, 2-butyl, isobutyl, tert-butyl, amyl, 1,2-dimethylpropyl, 1,1-dimethylpropyl, pentyl, isopentyl, hexyl, 4-methylpentyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 1,2,2-trimethylpropyl, 1,1,2-trimethylpropyl ... Examples of alkyl aryl include, but are not limited to, 1,2,3-trimethylpropyl, 2-ethylpentyl, 3-ethylpentyl, heptyl, 1-methylhexyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 4,4-dimethylpentyl, 1,2-dimethylpentyl, 1,3-dimethylpentyl, 1,4-dimethylpentyl, 1,2,3-trimethylbutyl, 1,1,2-trimethylbutyl, 1,1,3-trimethylbutyl, 5-methylheptyl, 1-methylheptyl, octyl, nonyl, decyl, and the like.

[0023] The term "alkenyl group" includes within its meaning monovalent ("alkenyl") and divalent ("alkenylene") straight or branched chain unsaturated aliphatic hydrocarbon groups having 2 to 10 carbon atoms, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, and having at least one double bond, where applicable, anywhere in the alkyl chain, of either E, Z, cis, or trans stereochemistry. Examples of alkenyl groups include, but are not limited to, ethenyl, vinyl, allyl, 1-methylvinyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1,3-butadienyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1,3-pentadienyl, 2,4-pentadienyl, 1,4-pentadienyl, 3-methyl-2-butenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,3-hexadienyl, 1,4-hexadienyl, 2-methylpentenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 1-octenyl, 1-nonenyl, 1-decenyl, and the like.

[0024] As used herein, the term "alkynyl group" includes within its meaning monovalent ("alkynyl") and divalent ("alkynylene") straight- or branched-chain unsaturated aliphatic hydrocarbon groups having 2 to 10 carbon atoms and having at least one triple bond anywhere along the carbon chain. Examples of alkynyl groups include, but are not limited to, ethynyl, 1-propynyl, 1-butynyl, 2-butynyl, 1-methyl-2-butynyl, 3-methyl-1-butynyl, 1-pentynyl, 1-hexynyl, methylpentynyl, 1-heptynyl, 2-heptynyl, 1-octynyl, 2-octynyl, 1-nonyl, 1-decynyl, and the like.

[0025] As used herein, "aryl" refers to an aromatic monocyclic hydrocarbon ring system or a polycyclic ring system in which two or more aromatic hydrocarbon rings are fused together (i.e., have a common bond) or at least one aromatic monocyclic hydrocarbon ring is fused to one or more cycloalkyl and / or cycloheteroalkyl rings. An aryl group can have 6 to 24 carbon atoms in its ring system (e.g., C6-C8). 24 Polycyclic aryl groups can contain multiple fused rings. In certain embodiments, polycyclic aryl groups can have from 8 to 24 carbon atoms. Any suitable ring position of the aryl group can be covalently linked to the defined chemical structure. Examples of aryl groups having only aromatic carbocyclic ring(s) include groups such as phenyl, 1-naphthyl (bicyclic), 2-naphthyl (bicyclic), anthracenyl (tricyclic), phenanthrenyl (tricyclic), pentacenyl (pentacyclic), and the like. Examples of polycyclic ring systems in which at least one aromatic carbocyclic ring is fused to one or more cycloalkyl and / or cycloheteroalkyl rings include, among others, benzo derivatives of cyclopentane (i.e., an indanyl group, which is a 5,6-bicyclic cycloalkyl / aromatic ring system), benzo derivatives of cyclohexane (i.e., a tetrahydronaphthyl group, which is a 6,6-bicyclic cycloalkyl / aromatic ring system), benzo derivatives of imidazoline (i.e., a benzimidazolinyl group, which is a 5,6-bicyclic cycloheteroalkyl / aromatic ring system), and benzo derivatives of pyran (i.e., a chromenyl group, which is a 6,6-bicyclic cycloheteroalkyl / aromatic ring system). Other examples of aryl groups include benzodioxanyl, benzodioxolyl, chromanyl, indolinyl, and the like. In certain embodiments, aryl groups can be optionally substituted. In certain embodiments, an aryl group is substituted with another aryl group, which can be referred to as a biaryl group. Each of the aryl groups in a biaryl group can be optionally substituted.

[0026] As used herein, "heteroaryl" refers to an aromatic monocyclic ring system containing at least one ring heteroatom selected from oxygen (O), nitrogen (N), sulfur (S), silicon (Si), and selenium (Se), or a polycyclic ring system in which at least one ring in the ring system is aromatic and contains at least one ring heteroatom. Polycyclic heteroaryl groups include those containing two or more fused heteroaryl rings as well as those containing at least one monocyclic heteroaryl ring fused to one or more aromatic carbocyclic rings, non-aromatic carbocyclic rings, and / or non-aromatic cycloheteroalkyl rings. Heteroaryl groups as a whole can have, for example, 5 to 24 ring atoms and can contain 1 to 5 ring heteroatoms (i.e., 5- to 20-membered heteroaryl groups). A heteroaryl group can be attached to the defined chemical structure at any heteroatom or carbon atom that results in a stable structure. Generally, heteroaryl rings do not contain O-O, S-S, or S-O bonds. However, one or more N or S atoms in a heteroaryl group may be oxidized (e.g., pyridine N-oxide, thiophene S-oxide, thiophene S,S-dioxide). Examples of heteroaryl groups include, for example, the 5- or 6-membered monocyclic and 5- to 6-membered bicyclic ring systems shown below, where T is O, S, NH, N-alkyl, N-aryl, N-(arylalkyl) (e.g., N-benzyl), SiH, SiH(alkyl), Si(alkyl), SiH(arylalkyl), Si(arylalkyl), or Si(alkyl)(arylalkyl).Examples of such heteroaryl rings include pyrrolyl, furyl, thienyl, pyridyl, pyrimidyl, pyridazinyl, pyrazinyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, isothiazolyl, thiazolyl, thiadiazolyl, isoxazolyl, oxazolyl, oxadiazolyl, indolyl, isoindolyl, benzofuryl, benzothienyl, quinolyl, 2-methylquinolyl, isoquinolyl, quinoxalyl, quinazolyl, benzotriazolyl, benzimidazolyl, and benzothiazolyl. Examples of heteroaryl groups include benzoisothiazolyl, benzisoxazolyl, benzoxdiazolyl, benzoxazolyl, cinnolinyl, 1H-indazolyl, 2H-indazolyl, indolizinyl, isobenzofuryl, naphthyridinyl, phthalazinyl, pteridinyl, purinyl, oxazolopyridinyl, thiazolopyridinyl, imidazopyridinyl, furopyridinyl, thienopyridinyl, pyridopyrimidinyl, pyridopyrazinyl, pyridopyridazinyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, and the like. Further examples of heteroaryl groups include 4,5,6,7-tetrahydroindolyl, tetrahydroquinolinyl, benzothienopyridinyl, benzofuropyridinyl, and the like. In certain embodiments, heteroaryl groups can be optionally substituted.

[0027] The term "cycloalkyl," as used herein, refers to a cyclic saturated aliphatic group and includes within its meaning monovalent ("cycloalkyl") and divalent ("cycloalkylene") saturated, monocyclic, bicyclic, polycyclic, or fused polycyclic hydrocarbon radicals having 3 to 10 carbon atoms, e.g., 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, 2-methylcyclopropyl, cyclobutyl, cyclopentyl, 2-methylcyclopentyl, 3-methylcyclopentyl, cyclohexyl, and the like.

[0028] The term "cycloalkenyl," as used herein, refers to a cyclic, unsaturated aliphatic group and includes within its meaning monovalent ("cycloalkenyl") and divalent ("cycloalkenylene") monocyclic, bicyclic, polycyclic, or fused polycyclic hydrocarbon radicals having 3 to 10 carbon atoms and at least one double bond, if applicable, anywhere in the alkyl chain, of either E, Z, cis, or trans stereochemistry. Examples of cycloalkenyl groups include, but are not limited to, cyclopropenyl, cyclopentenyl, cyclohexenyl, and the like.

[0029] As used herein, the term "heterocycloalkyl" includes within its meaning monovalent ("heterocycloalkyl") and divalent ("heterocycloalkylene") saturated, monocyclic, bicyclic, polycyclic, or fused hydrocarbon radicals having 3 to 10 ring atoms, of which 1 to 5 ring atoms are heteroatoms selected from O, N, NH, or S. Examples include pyrrolidinyl, piperidinyl, quinuclidinyl, azetidinyl, morpholinyl, tetrahydrothiophenyl, tetrahydrofuranyl, tetrahydropyranyl, and the like.

[0030] As used herein, the term "heterocycloalkenyl" includes within its meaning monovalent ("heterocycloalkenyl") and divalent ("heterocycloalkenylene") saturated, monocyclic, bicyclic, polycyclic, or fused polycyclic hydrocarbon radicals having 3 to 10 ring atoms, having at least one double bond, and in which 1 to 5 ring atoms are heteroatoms selected from O, N, NH, or S.

[0031] As used herein, the terms "heteroaromatic group" and variations such as "heteroaryl" or "heteroarylene" include within their meaning monovalent ("heteroaryl") and divalent ("heteroarylene") mononuclear, polynuclear, conjugated, and fused aromatic radicals having from 6 to 20 atoms, of which from 1 to 6 are heteroatoms selected from O, N, NH, and S. Examples of such groups include pyridyl, 2,2'-bipyridyl, phenanthrolinyl, quinolinyl, thiophenyl, and the like.

[0032] As used herein, the term "halogen" or variations such as "halide" or "halo" refers to fluorine, chlorine, bromine and iodine.

[0033] As used herein, the term "heteroatom" or variations thereof such as "hetero" refers to O, N, NH, and S.

[0034] The term "alkoxy" as used herein refers to a straight or branched chain alkyloxy group, examples of which include methoxy, ethoxy, n-propoxy, isopropoxy, tert-butoxy, and the like.

[0035] As used herein, the term "amino" refers to -NR a R b where R a and R b are individually selected from the group including, but not limited to, hydrogen, optionally substituted alkyl groups, optionally substituted alkenyl groups, optionally substituted alkynyl groups, and optionally substituted aryl groups.

[0036] As used herein, the term "aromatic group," or variations such as "aryl" or "arylene," refers to monovalent ("aryl") and divalent ("arylene") mononuclear, polynuclear, conjugated, and fused residues of aromatic hydrocarbons having 6 to 10 carbon atoms. Examples of such groups include phenyl, biphenyl, naphthyl, phenanthrenyl, and the like.

[0037] As used herein, the term "aralkyl" includes within its meaning monovalent ("aryl") and divalent ("arylene") mononuclear, polynuclear, conjugated, and fused aromatic hydrocarbon radicals attached to divalent saturated, straight- and branched-chain alkylene radicals.

[0038] As used herein, the term "heteroaralkyl" includes within its meaning monovalent ("heteroaryl") and divalent ("heteroarylene") mononuclear, polynuclear, conjugated, and fused aromatic hydrocarbon radicals attached to divalent saturated, straight- and branched-chain alkylene radicals.

[0039] As used herein, the term "optionally substituted" means that the group to which this term refers may be unsubstituted or, independently, may be substituted with alkyl, alkenyl, alkynyl, thioalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, halo, carboxyl, haloalkyl, haloalkynyl, hydroxyl, alkoxy, thioalkoxy, alkenyloxy, haloalkoxy, haloalkenyloxy, nitro, amino, nitroalkyl, nitroalkenyl, nitroalkynyl, nitroheterocyclyl, alkylamino, dialkylamino, alkenylamine, alkynylamine. This means that the alkyl group may be optionally substituted with one or more groups selected from phosphorus-containing groups such as aryl, heteroaryl, alkylaryl, alkylheteroaryl, cyano, cyanato, isocyanato, —C(O)NH(alkyl), and —C(O)N(alkyl).

[0040] The present invention includes within its scope all isomers, including all diastereomeric isomers, racemates and enantiomers, of the compounds disclosed herein. Thus, Formulas (I) and (II) should be understood to include, for example, the E-, Z-, cis-, trans-, (R), (S), (L), (D), (+), and / or (-) forms of the compounds, as appropriate.

[0041] In the context of the present invention, the term "administering" and variations thereof, including "administer" and "administration", includes contacting, applying, delivering, or providing a compound or composition of the present invention to an organism or surface by any suitable means.

[0042] As used herein, the term "subject" refers to an animal, typically a mammal or human, that is or has been the object of treatment, observation, and / or experimentation. When the term is used in connection with the administration of a compound or agent, the subject is the subject that has been the object of treatment, observation, and / or administration of the compound or agent. In this context, the term "subject" includes humans and individuals of any species of social, economic, or research importance, including, but not limited to, members of the genera Ovis, Bovis, Evis, Sus, Fevis, Canis, Primates (including humans and non-human primates), Rodentia, Murine, Capricorn, Leporidae, and Avian. In certain embodiments, the subject is a human. In certain embodiments, the term "subject" can refer to a cell, tissue, or organ sample derived from a subject, including, for example, a cultured cell line, a biopsy, a blood sample, or a fluid sample containing cells.

[0043] The term "substituted" is intended to indicate that one or more (e.g., 1, 2, 3, 4, or 5, in some embodiments 1, 2, or 3, and in other embodiments 1 or 2) hydrogen atoms on the group designated with "substituted" have been replaced with a selection from the designated organic or inorganic groups, or with suitable organic or inorganic groups known to those of ordinary skill in the art, provided that the normal valence of the designated atom is not exceeded and the substitution results in a stable compound. Suitable designated organic or inorganic groups include, for example, alkyl, alkenyl, alkynyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, alkanoyl, alkoxylcarbonyl, amino, alkylamino, dialkylamino, trifluoromethylthio, difluoromethyl, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylsilyl, and cyano. Additionally, suitable designated groups include, for example, -X, -R, -O-, -OR, -SR, -S-, -NR2, -NR3, =NR, -CX3, -CN, -OCN, -SCN, -N=C=O, -NCS, -NO, -NO2, =N2, -N3, -NRC(=O)R, -C(=O)R, -C(=O)NR2, -S(=O)2O-, -S(=O)2R, -OS(=O)2OR, -S(=O)2NR2, -OP(=O)(OR)2, -P(=O) Examples of suitable substituents include -OR, -C(=O)R, -C(=O)X, -C(S)R, -C(=O)OR, -C(=O)O-, -C(=S)OR, -C(=O)SR, -C(=S)SR, -C(=O)NR, -C(=S)NR, and -C(=NR)NR, where each X is independently a halogen (or "halo" group), F, Cl, Br, or I, and each R is independently H, an alkyl, an aryl, a heterocycle, a protecting group, or a prodrug moiety. As will be readily understood by those skilled in the art, when a substituent is keto (i.e., =O) or thioxo (i.e., =S), or the like, two hydrogen atoms on the substituted atom are replaced by the substituent.

[0044] In the context of this specification, the term "treatment" refers to any and all uses that ameliorate the condition or symptoms of a disease, prevent the establishment of a disease, or in any way prevent, hinder, slow or reverse the progression of a disease or other undesirable condition.

[0045] In the context of this specification, the term "therapeutically effective amount" includes within its meaning a sufficient amount of a compound or composition of the present invention to produce the desired therapeutic effect. The exact amount required will vary from subject to subject, depending on factors such as the species being treated, the age and general condition of the subject, the severity of the condition being treated, the particular agent being administered, the method of administration, etc. Therefore, it is not possible to specify an exact "effective amount." However, for any given case, an appropriate "effective amount" may be determined by one of ordinary skill in the art using only routine experimentation.

[0046] The present disclosure provides compounds useful for inhibiting β-cat or interfering with the interaction of β-cat with TCF4, the compounds having formula 1, or a pharmaceutically acceptable salt thereof: [ka] In the above formula 1, R 1 Ha-(CR 4 2) m XR 5 and R 2 is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, aralkyl, heteroaralkyl, or heteroaryl; R 3 is -(CR 6 2) n R 7 and R 4is, at each occurrence, independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, and heteroaralkyl, or R 4 two instances of, taken together with one or more carbons to which they are attached, form a 3- to 7-membered cycloalkyl; R 5 is hydrogen, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, or heteroaryl; R 6 is, at each occurrence, independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, and heteroaralkyl, or R 6 two instances of, taken together with one or more carbons to which they are attached, form a 3- to 7-membered cycloalkyl; R 7 is -(C=O)R 8 , -(C=O)OR 8 , or -(C=O)N(R 8 )2 or R 7 is a moiety having formula 2, [ka] R 8 is, at each occurrence, independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, and heteroaralkyl, or two R 8 together with the nitrogen to which they are attached form a 3- to 7-membered heterocycloalkyl, or R 6 and R 8 together with the atom to which they are attached form an optionally substituted 4- to 7-membered cycloalkyl or an optionally substituted 4- to 7-membered heterocycloalkyl; m is an integer selected from 0 to 4; n is an integer selected from 0 to 4, X is -O-, -S-, or absent A compound is provided.

[0047] In certain embodiments, the compounds of formula I do not include the following compounds: [ka]

[0048] In certain embodiments, X is -O-, -S-, or absent. In instances where X is absent, R 1 is the structure - (CR 4 2) m R 5 wherein m, R 4 , and R 5 is as defined in any embodiment(s) described herein.

[0049] In certain embodiments, m is 0 to 3, 0 to 2, 0 to 1, or 1 to 2. In certain embodiments, X is O or S and m is 1. In certain embodiments, X is absent and m is 1 or 2.

[0050] In certain embodiments, m is 0-3, 0-2, 0-1, or 1-2.

[0051] In certain embodiments, n is 0-3, 0-2, 0-1, or 1-2.

[0052] In certain embodiments, R 2 is alkyl, aralkyl, heteroaralkyl, or -(CR 9 2) p R 10 In this formula, p is an integer selected from 0 to 4, and R 9 is independently at each occurrence hydrogen or alkyl; R 10is alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl. 10 is optionally substituted phenyl, optionally substituted furan, optionally substituted thiophene, optionally substituted pyrrole, optionally substituted imidazole, optionally substituted oxazole, optionally substituted isoxazole, optionally substituted thiazole, optionally substituted pyridine, optionally substituted pyrazine, or optionally substituted triazine.

[0053] R 2 -(CR 9 2) p R 10 In examples where R is , P can be 0 to 3, 0 to 2, 0 to 1, or 1 to 2. In certain embodiments, R 9 is, at each occurrence, independently hydrogen or alkyl. In certain embodiments, R 2 Ha-(CH2)R 10 or -(CHMe)R 10 and R 10 is as defined herein.

[0054] In certain embodiments, R 4 is, at each occurrence, hydrogen or alkyl, or R 4 Two instances of, taken together with one or more carbons to which they are attached, form a 3- to 7-membered or 3- to 6-membered cycloalkyl. In certain embodiments, R 4 is hydrogen.

[0055] In certain embodiments, R 5 is cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, or heteroaryl. In certain embodiments, R 5 is aryl or heteroaryl. In certain embodiments, R 5 is an optionally substituted phenyl.

[0056] In certain embodiments, R 7 is -(C=O)R 8 , -(C=O)OR 8 , or -(C=O)N(R 8 )2 and R 8 is alkyl, cycloalkyl, aryl, or two R 8 together with the nitrogen to which they are attached form a 3- to 7-membered heterocycloalkyl, or R 6 and R 8 together with the atom to which they are attached form an optionally substituted 4- to 7-membered cycloalkyl or an optionally substituted 4- to 7-membered heterocycloalkyl.

[0057] The Two R's 8 In the instances where, together with the nitrogen to which they are attached, form a 3- to 7-membered heterocycloalkyl, the 3- to 7-membered heterocycloalkyl can contain one or two heteroatoms selected from oxygen, sulfur, and nitrogen in its ring system.

[0058] R 6 and R 8 In the example where, together with the atom to which they are attached, form an optionally substituted 4- to 7-membered heterocycloalkyl, the compound can have formula 5, or a pharmaceutically acceptable salt thereof: [ka] In the above formula 5, R 1 , R 2 , R 6 and R 8 are each independently as defined herein, m is 0 to 4, A represents a 4- to 7-membered optionally substituted cycloalkyl or a 4- to 7-membered optionally substituted heterocycloalkyl containing one or two heteroatoms selected from oxygen, sulfur, and nitrogen in the ring system, and Y is -O-, -N(R 8)- or absent, or the compound can have formula 6, or a pharmaceutically acceptable salt thereof: [ka] In the above formula 6, R 1 , R 2 , R 6 , and R 8 are each independently as defined herein, m is 0 to 3, A represents a 4- to 7-membered optionally substituted cycloalkyl or a 4- to 7-membered optionally substituted heterocycloalkyl containing one or two heteroatoms selected from oxygen, sulfur, and nitrogen in the ring system, and Y is -O-, -N(R 8 )- or does not exist.

[0059] In certain embodiments, R 8 is C1~C 10 alkyl, C3-C7 cycloalkyl, biaryl, or polycyclic hydrocarbon, or two R 8 together with the nitrogen to which they are attached form a 3- to 7-membered, 4- to 7-membered, 5- to 7-membered, or 5- to 6-membered optionally substituted heterocycloalkyl, or the compound has formula 6 where A is a 5- to 6-membered optionally substituted cycloalkyl.

[0060] R 7 In examples where is a moiety having formula 2, the compound can be represented by formula 7, or a pharmaceutically acceptable salt thereof: [ka] In the above formula, each R 1 , R 2 , R 6 , and m are each independently as defined herein.

[0061] In certain embodiments, the compound has formula 3, or a pharmaceutically acceptable salt thereof: [ka] In the above formula 3, R 4 is, at each occurrence, independently selected from the group consisting of hydrogen and alkyl; R 5 is aryl or heteroaryl, R 6 is, at each occurrence, independently selected from the group consisting of hydrogen and alkyl; R 7 is -(C=O)R 8 , -(C=O)OR 8 , or -(C=O)N(R 8 )2 or R 7 is a moiety having formula 4, [ka] R 8 is, at each occurrence, independently selected from the group consisting of alkyl, cycloalkyl, and aryl, or R 8 Two instances of, taken together with the nitrogen to which they are attached, form a 5- to 6-membered heterocycloalkyl, or R 6 and R 8 together with the atoms to which they are attached form a 5- to 6-membered cycloalkyl; R 9 is, at each occurrence, independently selected from the group consisting of hydrogen and alkyl; R 10 is cycloalkyl, aryl or heteroaryl; X is —O— or absent.

[0062] In certain embodiments, the compound is R 4 is hydrogen and R 5 is aryl and R 9 is, at each occurrence, independently selected from the group consisting of hydrogen and alkyl; R 10 is aryl and R 6 is, at each occurrence, independently selected from the group consisting of hydrogen and alkyl; R 7 -(C=O)OR 8or -(C=O)N(R 8 )2 and R 8 is, at each occurrence, independently selected from the group consisting of alkyl, cycloalkyl, and aryl, or R 8 Two instances of have formula 3, taken together with the nitrogen to which they are attached, form a 5-6 membered heterocycloalkyl.

[0063] In certain embodiments, the compound has formula 8, or a pharmaceutically acceptable salt thereof: [ka] In the above formula 8, q is an integer selected from 0 to 2, t is 1 or 2; R 5 is optionally substituted phenyl, R 9 is hydrogen or alkyl, R 10 is optionally substituted phenyl, R 11 is independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, heteroaralkyl, -SR, -OR, -O(C=O)R, -O(C=O)OR, -O(C=O)N(R), -NR, or -N(R)(C=O)N(R), wherein R ... hydrogen, alkyl, aryl, or heteroaryl, or two R's together with the nitrogen or nitrogens to which they are attached form a 3- to 7-membered heterocycloalkyl.

[0064] In certain embodiments, the compound has q=0 or 1, t=1, and R 9 is hydrogen or C1-C3 alkyl, and R 11 is alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl.

[0065] In certain embodiments, the compound is [ka] or a pharmaceutically acceptable salt thereof.

[0066] Pharmaceutically acceptable salts of the compounds described herein can include, for example, conventional non-toxic salts or quaternary ammonium salts of the compounds from non-toxic organic or inorganic acids. For example, such conventional non-toxic salts include salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, and nitric acid, as well as salts prepared from organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, palmitic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, and isothioic acid.

[0067] In other cases, the compounds described herein may contain one or more acidic functional groups and, therefore, can form pharmaceutically acceptable salts with pharmaceutically acceptable bases. In these instances, the term "pharmaceutically acceptable salts" can refer to relatively non-toxic inorganic and organic base addition salts of the compounds described herein. These salts can likewise be prepared in situ during the administration vehicle or dosage form manufacturing process, or can be prepared separately by reacting the purified compound in its free acid form with a suitable base, such as a hydroxide, carbonate, or bicarbonate salt of a pharmaceutically acceptable metal cation, ammonia, or a pharmaceutically acceptable organic primary, secondary, or tertiary amine. Representative alkali or alkaline earth salts include lithium, sodium, potassium, calcium, magnesium, and aluminum salts. Representative organic amines useful for forming base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, and the like.

[0068] Inhibition of β-cat or disruption of the interaction of β-cat with TCF4 may be carried out in vitro, in vivo, or ex vivo.

[0069] Inhibition of β-cat or disruption of the interaction between β-cat and TCF4 may be carried out by contacting one or more of the compounds described herein with at least one of β-cat and the β-cat-TCF4 complex.

[0070] Contacting β-cat or the β-cat-TCF4 complex with a compound of formula 1 can result in one or more of inhibition of β-cat function, inhibition of the interaction between β-cat and TCF4, and disruption of the β-cat / TCF4 complex, and can also result in attenuation of Wnt signaling.

[0071] Thus, methods of inhibiting β-cat or disrupting the interaction between β-cat and TCF4 can be used to treat diseases in which inhibition of β-cat, disruption of the β-cat / TCF4 complex, and / or attenuation of the Wnt signaling pathway results in an improvement in the disease or health condition.

[0072] Disrupting the interaction between β-cat and TCF4 can refer to either or both of inhibiting the association of β-cat and TCF4, or inducing dissociation of the β-cat-TCF4 complex to form β-cat and TCF4.

[0073] The amount or concentration of a compound of Formula 1 required for administration can be readily determined by one of ordinary skill in the art using well-known methods and the disclosure provided herein.

[0074] Provided herein are methods for treating a disease or condition ameliorated by inhibiting the interaction of β-cat and TCF4 in a subject in need thereof, comprising administering a therapeutically effective amount of a compound described herein. In certain embodiments, the subject is a human.

[0075] The present disclosure also provides the compounds described herein for use in treating a disease or condition ameliorated by inhibiting β-cat or by disrupting the interaction of β-cat with TCF4.

[0076] The present disclosure further provides the use of the compounds described herein in the manufacture of a medicament for the treatment of a disease or condition ameliorated by inhibiting β-cat or by disrupting the interaction of β-cat with TCF4.

[0077] The disease or condition can be cancer, a neurodegenerative disease, a metabolic disease, a cardiovascular disease, fibrosis, or a bone disease.

[0078] In certain embodiments, the disease or condition is cancer characterized by aberrant activation of one or more of the Wnt signaling pathway, the Myc signaling pathway, and the Hippo signaling pathway.

[0079] In certain embodiments, the disease or condition is cancer characterized by aberrant activation of the Wnt / β-cat signaling pathway.

[0080] Aberrant Wnt / β-cat pathway signaling has been implicated in many different cancers, and multiple genetic abnormalities in this pathway may contribute to tumor promotion and progression. 1 Activation of the Wnt / β-cat pathway is associated with colorectal cancer (colon cancer). 2 ,melanoma 3 , hepatoblastoma 4 , medulloblastoma 5 , prostate cancer 6 , and endometrioid adenocarcinoma of the uterus and ovary 7~10 It is one of the most frequently observed signal transduction abnormalities in several human cancers, including those involving the Wnt / β-cat pathway. Activation of the Wnt / β-cat pathway is also commonly observed in metaplastic carcinomas of the breast. 11 .

[0081] Thus, the provided methods can be used to treat cancers selected from the group consisting of colorectal cancer (colon cancer), hepatocellular carcinoma, melanoma, liver cancer, breast cancer, prostate cancer, leukemia, thyroid cancer, brain cancer, medulloblastoma, hepatoblastoma, desmoid tumor, osteoma, head and neck cancer, and endometrioid adenocarcinoma of the uterus and ovary.

[0082] In certain embodiments, the cancer is colorectal cancer, which is characterized by aberrant activation of the Wnt / β-cat signaling pathway. [Example]

[0083] Generation of virtual chemical libraries The training libraries are iCRT3, iCRT5, and iCRT14. 12 Three known inhibitors of the β-cat-TCF4 complex, including 14 By ZINC Database13 The validation library consists of 63 iCRT3 analogs synthesized in the DasGupta lab, and 150 property-matched computational decoys selected from the library. The virtual screening library consists of 10,240 compounds purchased from Enamine (https: / / enamine.net / ).

[0084] Molecular docking screen Before docking, spheres and grids were generated. By augmenting the ligand-derived spheres with receptor-derived spheres, 45 matching spheres were generated to help orient the database compounds within the site. The ligand-derived spheres were initially represented by the positions of non-hydrogen atoms in the crystal structure of the TCF4 peptide fragment, and in subsequent rounds, these were replaced by the docking pose of the iCRT ligand. The receptor-derived spheres were generated using SPHGEN. 15 The docking screen was generated using the program DOCK version 3.6. 16 The docked compounds were ranked by their docking energy, which is the sum of the van der Waals term, the Poisson-Boltzmann electrostatic term, and the ligand desolvation penalty term.

[0085] Docking performance evaluation The accuracy of the structural model in ligand prediction is evaluated by the degree of improvement (enrichment) of the top-scoring compounds compared to known ligands. This improvement is similar to the initial improvement factor EF1 and the area under the curve (AUC) of the receiver operating characteristic (ROC), but with more emphasis on the initial improvement. 16、17 The overall improvement was measured by logAUC.

[0086] cell line Wnt STF reporter cells (HEK 293 cells stably transfected with the TOPFlash reporter) and STF3A reporter cells (STF cells constitutively secreting Wnt3A) were kind gifts from David Virshup (Duke-NUS Graduate Medical School, Singapore). HCT116, DLD-1, and SW480 cell lines were obtained from ATCC. Hippo pathway TEAD reporter (catalog no. 60618) and Myc signaling pathway reporter (catalog no. 60520) cell lines were purchased from BPS Bioscience, Inc.

[0087] Culture medium STF, STF3A, and DLD-1 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco catalog number 11965084) supplemented with 10% fetal bovine serum (FBS, Hyclone, catalog number SV30160.03) and 100 U / mL penicillin-streptomycin (Gibco, catalog number 15140122). HCT116, SW480, and Myc signaling pathway reporter cells were cultured in McCoy's 5A medium (Gibco, catalog number 16600108) supplemented with 10% FBS and 100 U / mL penicillin-streptomycin. The Myc signaling pathway reporter cell medium was further supplemented with 400 μg / mL G-418 sulfate (Gold Biotechnology, catalog number G-418-5). Hippo pathway TEAD reporter cells were cultured in minimum essential medium (MEM) containing Earle's balanced salts (EBSS) (Hyclone, catalog number SH30024.01) supplemented with 10% FBS, 100 U / mL penicillin-streptomycin, 1% MEM non-essential amino acids (NEAA, Gibco, catalog number 11140050), 1 mM sodium pyruvate (Gibco, catalog number 11360070), 400 μg / mL G-418 sulfate, and 10 μg / mL insulin (Sigma-Aldrich, catalog number I1882-100MG). The medium for spheroid formation was DMEM / F-12 (Gibco, catalog number 11320082) supplemented with B27 (Gibco, catalog number 12587010), 100 U / mL penicillin-streptomycin, 20 ng / mL EGF (Gibco, catalog number PHG0313), 20 ng / mL bFGF (Gibco, catalog number PHG0023), and 3% Matrigel (Corning, catalog number 354234).

[0088] TOPFlash (Wnt signaling) reporter screen STF cells were seeded at 20,000 cells per well in 100 μL of culture medium in 96-well plates (Corning, catalog nos. 3903 and 3904). The next day, candidate compounds in DMSO were added to the cells at a final concentration of 10 μM with 1% DMSO. Cells were also stimulated with Wnt3A-conditioned medium. After 24 h of incubation, cell viability was measured using PrestoBlue cell viability reagent (Invitrogen, catalog no. A13262) according to the manufacturer's protocol, while TOPFlash reporter activity was measured using Steady-Glo luciferase reagent (Promega, catalog no. E2550).

[0089] Dose response of reporter strains Wnt signaling reporter Wnt STF reporter cells were seeded at 20,000 cells per well in 100 μL of culture medium in a 96-well plate (Corning, catalog no. 3903; Falcon, catalog no. 353072). The following day, compounds diluted 5-fold in DMSO were added to the cells to a final concentration of 1% DMSO. Cells were also stimulated with 500 ng / mL recombinant human Wnt3A (RnD systems, catalog no. 5036-WN-010). After 24 h of incubation, cell viability was measured using the Cell Counting Kit-8 (CCK-8, Dojindo, catalog no. CK04) cell viability reagent according to the manufacturer's protocol, while TOPFlash reporter activity was measured using the Steady-Glo luciferase reagent.

[0090] Wnt STF3A reporter cells were seeded at 20,000 cells per well in 100 μL of culture medium in 96-well plates (Corning, catalog no. 3903; Falcon, catalog no. 353072). The following day, compounds diluted 5-fold in DMSO were added to the cells to a final concentration of 1% DMSO. After 24 h of incubation, cell viability was measured using the Cell Counting Kit-8 (CCK-8, Dojindo catalog no. CK04) cell viability reagent according to the manufacturer's protocol, while TOPFlash reporter activity was measured using the Steady-Glo luciferase reagent.

[0091] Myc signaling reporter Myc reporter (Luc)-HCT116 cells were seeded at 25,000 cells per well in 100 μL of assay medium in 96-well plates (Corning, Cat. No. 3903; Falcon, Cat. No. 353072). Assay medium consisted of growth medium without G-418. The following day, compounds diluted 4-fold in DMSO were added to the cells to a final concentration of 1% DMSO. After 24 hours of incubation, cell viability was measured using PrestoBlue cell viability reagent, while luciferase reporter activity was measured using Steady-Glo luciferase reagent.

[0092] Hippo signaling reporter Hippo TEAD reporter cells were seeded at 100,000 cells per well in 100 μL of assay medium in 96-well plates (Corning, Cat. No. 3903; Falcon, Cat. No. 353072). Assay medium consisted of growth medium without G-418. The following day, compounds diluted 5-fold in DMSO were added to the cells to a final concentration of 1% DMSO. After 24 hours of incubation, cell viability was measured using CCK-8 cell viability reagent, while luciferase reporter activity was measured using Steady-Glo luciferase reagent.

[0093] Dose-response study using CRC cell lines HCT116, DLD-1, and SW480 cells were seeded at 5,000 cells per well in 100 μL of growth medium in a 96-well plate (Corning, catalog no. 3903). The following day, compounds diluted 5-fold in DMSO were added to the cells to a final concentration of 1% DMSO. After 24 hours of incubation, cell viability was measured using CellTiter-Glo Luminescent Cell Viability Reagent (Promega, catalog no. G7573) according to the manufacturer's protocol.

[0094] Data analysis of dose-response studies For each plate, the average signal from DMSO-treated wells was calculated, and the signal from each treated well was normalized to the average DMSO signal from its respective plate. Normalized values ​​were plotted against the concentrations tested using GraphPad Prism 5, and EC values ​​were calculated using three- or four-parameter nonlinear regression. 50 value was determined.

[0095] Proliferation assay HCT116 cells were seeded at 3,000 cells per well in 100 μL of growth medium in a 96-well plate (Corning, Catalog No. 3903) and allowed to attach overnight. Cells were then treated with compounds at a final concentration of 10 μM in 0.1% DMSO for 4 consecutive days, with daily medium changes. Daily cell proliferation was measured using the CellTiter-Glo Luminescent Cell Viability Assay (Promega, Catalog No. G7573) according to the manufacturer's protocol.

[0096] Spheroid formation assay HCT116, DLD-1, and SW480 cells were seeded at 150 cells per well in 200 μL of spheroid medium in a 96-well ultra-low attachment plate (Corning, catalog no. 3474). The compounds were added to a final concentration of 30 μM with 0.5% DMSO on the day of cell seeding. Cells were cultured at 37°C and 5% CO2. Images of the formed spheroids were captured using an Operetta CLS system (Perkin-Elmer) on days 7 and 10 after cell seeding.

[0097] Western blotting HCT116 cells were treated with compounds at a final concentration of 50 μM with 1% DMSO for 18 hours. Cells were lysed on ice in RIPA buffer (Thermo, Cat. No. 89900) containing cOmplete™ protease inhibitor cocktail (Roche, Cat. No. 11697498001) and PhosSTOP phosphatase inhibitor cocktail (Roche, Cat. No. 04906837001). 30 μg of total cellular protein per sample was separated by SDS-PAGE and blotted onto a PVDF membrane (Millipore, Cat. No. IPVH00010). Primary antibodies were incubated overnight at 4°C, and blots were detected with either IRDye 800CW goat anti-rabbit antibody (LI-COR, Catalog No. 926-32211) or IRDye 680RD goat anti-mouse antibody (LI-COR, Catalog No. 926-68070) at a dilution of 1:5000. Signals were visualized using a LI-COR Odyssey CLx imaging system, and bands were quantified using LI-COR Image Studio data analysis software. The primary antibodies used were Met (Cell Signaling Technology, catalog no. 8198, 1:1000), cyclin D1 (Cell Signaling Technology, catalog no. 2978, 1:1000), survivin (Santa Cruz, catalog no. sc-10811, 1:500), ECAD (Cell Signaling Technology, catalog no. 3195, 1:1000), TCF4 / TCF7L2 (Cell Signaling Technology, catalog no. 2569, 1:1000), β-cat (Cell Signaling Technology, catalog no. 8480, 1:1000), and β-actin (Abcam, catalog no. ab8226, 1:3000).

[0098] Co-immunoprecipitation HCT116 cells were treated with the indicated concentrations of compounds for 18 hours. Cells were lysed on ice using a lysis buffer containing 20 mM HEPES, pH 7.5, 137 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 1 mM dithiothreitol (DTT), 1% Triton X-100, 10% glycerol, cOmplete protease inhibitor cocktail (Roche, catalog no. 11697498001), and 1 mM Na3VO4. Each sample of the cleared protein lysate was incubated overnight at 4°C with 4 μg of anti-β-cat antibody (Sigma, catalog no. C7207), followed by incubation with 50 μL of Dynabeads Protein G slurry (Invitrogen, catalog no. 10004D) for 1 hour at 4°C. The beads were then washed three times with lysis buffer at 4°C, and bound proteins were eluted by heating the beads with 30 μL (per sample) of SDS sample buffer at 95°C for 7 minutes. Proteins were separated by SDS-PAGE and blotted onto PVDF membranes (Millipore, catalog no. IPVH00010). Primary antibodies were incubated overnight at 4°C, and blots were detected with either IRDye 800CW goat anti-rabbit antibody (LI-COR, catalog no. 926-32211) or IRDye 680RD goat anti-mouse antibody (LI-COR, catalog no. 926-68070) at a dilution of 1:5000. Signals were visualized using a LI-COR Odyssey CLx imaging system, and bands were quantified using LI-COR Image Studio data analysis software. The primary antibodies used were E-cadherin (Abcam, catalog no. ab15148, 1:1000), TCF4 / TCF7L2 (Cell Signalling Technology, catalog no. 2569, 1:1000), and β-cat (Cell Signalling Technology, catalog no. 8480, 1:1000).

[0099] Protein expression and purification BL21(DE3) cells harboring the pET-28β-cat expression vector encoding His-tagged human β-cat(134-668) were cultured at 37°C until OD 600 The cells were cultured until the RI was 0.6–0.8. The cells were then induced with 500 μM isopropyl-β-D-thiogalactopyranoside (IPTG) for 6–8 h at 23°C. The cells were lysed by sonication in lysis buffer containing 20 mM Tris (pH 8.8), 250 mM NaCl, 2 mM DTT, 5% glycerol, 0.1% Triton X-100, and cComplete protease inhibitor cocktail (EDTA-free, Roche, catalog no. 11873580001). The lysate was incubated with Ni-NTA agarose beads (Qiagen, catalog no. 30210) for 1 h at 4°C, after which the beads were washed with lysis buffer containing 20 mM imidazole. Bound proteins were eluted with a buffer containing 20 mM Tris (pH 8.8), 250 mM NaCl, 0.5 mM tris(2-carboxyethyl)phosphine (TCEP), 5% glycerol, and 250 mM imidazole.

[0100] The GST-tagged TCF4 N-terminal domain was similarly expressed in BL21(DE3) cells, induced with IPTG for 2–4 h at 23°C, and then purified from glutathione agarose beads (Pierce, catalog no. 16100) using a buffer containing 20 mM Tris (pH 8.8), 250 mM NaCl, 0.5 mM TCEP, 5% glycerol, and 10 mM reduced glutathione.

[0101] Surface plasmon resonance (SPR) studies Binding to β-cat Surface plasmon resonance experiments were performed on a CM5 Series S sensor chip (GE Healthcare, catalog number BR-1005-30) using a BIACORE T100 (GE Healthcare). His-tagged β-cat (60 kDa, >90% purity by SDS-PAGE) was immobilized using amine coupling chemistry. The surfaces of flow cells 1 and 2 were activated with a 1:1 mixture of 0.1 M NHS (N-hydroxysuccinimide) and 0.1 M EDC (3-(N,N-dimethylamino)propyl-N-ethylcarbodiimide) at a flow rate of 5 μL / min. Flow cell 2 was immobilized with 1 μM of the ligand in PBS, pH 6.0, at a density of 8000 RU. Flow cell 1 was left blank to serve as the reference surface. Both surfaces were blocked with 1 M ethanolamine (pH 8.0). To collect binding data, compound GB1874 in PBS (pH 7.5) containing 5% DMSO was injected over two flow cells at concentrations of 125 μM, 100 μM, 80 μM, 60 μM, 40 μM, and 20 μM at a flow rate of 30 μL / min and a temperature of 25°C. The complexes were allowed to associate and dissociate for 120 and 600 seconds, respectively. The surface was regenerated by injecting 50 mM NaOH for 30 seconds. RU responses were collected and plotted against the concentrations of GB1874 tested using GraphPad Prism 5. The dissociation constant, K D was obtained by non-linear specific binding using the Hill slope curve fitting function.

[0102] Inhibition of β-cat-TCF4 interaction GST-tagged TCF4 N-terminal domain (31 kDa, >90% purity by SDS-PAGE) was immobilized onto a CM5 Series S sensor chip using amine coupling chemistry. A 0.5 μM concentration of ligand in 10 mM citrate buffer, pH 4.0, was immobilized on flow cell 4 at a density of 400 RU, while flow cell 3 was left blank to serve as the reference surface. For inhibition studies, 50 nM His-tagged β-cat was preincubated with different concentrations of compound GB1874 in PBS containing 5% DMSO for 15 min at room temperature. This mixture was injected over the two flow cells at a flow rate of 30 μL / min at 25 °C. The complex was allowed to associate and dissociate for 60 s, respectively, and the surface was regenerated by injecting 50 mM NaOH for 30 s. RU responses were collected and plotted against the concentrations of GB1874 tested using GraphPad Prism 5. The inhibitory IC of compound GB1874 was calculated. 50 Values ​​were obtained by four-parameter nonlinear regression.

[0103] Mouse xenografts and compound treatment HCT116 cells (1 × 10 per site) 6 Cells) were subcutaneously implanted into the right flank of 5- to 7-week-old NSG (NOD scid gamma) mice (Jackson Laboratory, stock number 005557). Tumor volumes were 60-100 mm. 3 At the time of reaching 1000 mg / kg, the animals were randomly assigned to either a treatment group or a control group. Mice in the treatment group were administered 50 mg / kg of compound GB1874 by intraperitoneal injection every other day. At the same time, mice in the control group were administered a compound-free diluent. Compound GB1874 was dissolved in DMSO to a concentration of 60 mg / mL and diluted with 5% PEG300, 5% Tween-80 in saline to a final concentration of 3 mg / mL, 5% DMSO. The length and width of the tumor were measured using calipers every two days. Tumor volume was estimated using the following modified ellipsoid formula: tumor volume = 1 / 2(length × width) 2 ) The control group had tumors of 2000 mm 3 Mice were euthanized when they reached

[0104] result Generation of β-cat structural model The final structural models of β-cat used for future ligand prediction are the β-cat-Tcf3 complex, the β-cat-TCF4 complex, and the β-cat-BCL9-TCF4 complex (PDB codes: 1G3J, 1G4J, 1G5J, 1G6J, 1G7J, 1G8J, 1G9J, 1G10J, 1G11J, 1G12J, 1G13J, 18 , 1JPW 19 , and 2GL7 20 The three β-cat crystal structures, including the β-cat β-catenin complex, were obtained through two stages: 1) optimization (training) and 2) blind testing (validation) based on the ligand improvement measured by EF1 and logAUC. Optimization was achieved by an in-house automated modeling and docking platform (Figure 1). First, we used MetaPocket 21 We predicted a putative ligand-binding site in the crystal structure of β-cat (PDB code: 1JPW) using SCWRL. This site overlaps with the charged groove formed by armadillo repeats 4-9 of β-cat, and this charged groove overlaps with the β-cat-TCF4 interaction interface. Therefore, we focused on this site in the present study. Second, we compared the three crystal structures of β-cat with SCWRL. 22 Side chain sampling was performed using the β-cat protein synthesis algorithm, and three other structures were introduced. This set of six β-cat structures served as the starting point for training. Third, to explore the conformational space of β-cat more broadly, the training library (153 compounds) was docked to all six β-cat structures, and the docking performance was evaluated by the degree of ligand improvement. The docking performance was evaluated by PLOP (Protein Local Optimization Program) in the presence of each docked ligand (iCRT3, iCRT5, iCRT14). 23 The side chains of the binding site were optimized using the method described above. This process was repeated until several optimized structures showed ligand improvement performance superior to arbitrary thresholds (logAUC≧40 and EF1≧30).

[0105] Prediction of small molecule inhibitors of β-cat-TCF4 interaction The best β-cat structure in the training phase demonstrated an overall ligand refinement of the training library (logAUC = 44.2) and an initial ligand refinement of EF1 = 33.3. Among the 153 compounds in the training library, iCRT3, the most potent known inhibitor, was evaluated as the best. The interactions between β-cat and iCRT3 in the complex structure generated by docking were analyzed. The oxazole group of iCRT3 formed a hydrogen bond with R469 and a cation-π interaction with K508, which formed a salt bridge with E17 of TCF4 in the β-cat-TCF4 crystal structure (Figure 2D). Meanwhile, the phenyl group of iCRT3 was packed into a pocket surrounded by R386, N426, and P463, which were shown to interact with I19 and F21 of TCF4. In addition, the amide group of iCRT3 connecting the oxazole and phenyl groups was stabilized by another hydrogen bond with E462 of β-cat, and the ethyl phenyl group of iCRT3 filled a pocket surrounded by K508, V511, and R515.

[0106] This most improved (enriched) β-cat structure was subjected to a blind test using a validation library containing 63 iCRT3 analogues (Figure 5). Taking into account both the docking rank based on the DOCK energy score and the docking poses determined by human intervention, 19 candidate ligands were selected from the virtual screening (Figure 5). All 63 compounds were tested at a single concentration in a Wnt reporter assay using HEK 293 cells stably transfected with the TOPFlash reporter (STF cells). Twelve compounds demonstrated activity (data not shown). These 12 compounds were further tested at various concentrations against STF cells. Five of the compounds had IC values ​​equal to or lower than those of known iCRT3 inhibitors. 50 The results showed that the docking results were consistent with the previous results (Figure 5). In fact, all five of these potent inhibitors were preferentially ranked as the top ligand candidates by docking, suggesting that the most improved structures generated by training can predict novel ligands.

[0107] After functionally validating the methodology for novel ligand prediction, a library of 10,240 small molecules (purchased from Enamine) was computationally screened against the most improved β-cat structures. Five hundred top-scoring hits, representing 4.0% of the screened library, were manually analyzed. Twenty-seven compounds (Figure 6) were selected for experimental testing based on three criteria: (1) disruption of the β-cat-TCF4 interaction, (2) formation of favorable interactions with the β-cat residue, such as hydrogen bonds, and (3) chemical novelty of the scaffold.

[0108] Functional validation of predicted inhibitors using TOPFlash / Wnt-reporter assay The 27 compounds identified from the docking study were first screened at 10 μM for their ability to inhibit Wnt signaling in STF-reporter cells (Figure 2A). From this screen, three compounds were identified. These compounds inhibited Wnt signaling by an average of more than 50% compared to the DMSO control while causing less than 25% toxicity to the cells (Figure 7A). Dose-response studies of the three hit compounds against the STF reporter demonstrated that these compounds had low micromolar IC 50 These compounds also affected the viability of reporter cells, but the EC value of cell viability was not significantly different from that of the control group (Figure 2B). 50 Values ​​are IC of reporters 50 The ATP values ​​were at least fourfold greater than those of the previous study (Figure 7B). Encouragingly, the three hit compounds were approximately two to five times more potent inhibitors of the Wnt pathway than our previously reported compound, iCRT3. Interestingly, the chemical structures of the three hit compounds were different (Figure 2C).

[0109] Specificity of inhibitor candidates To further understand the mechanism of action of the hit candidate compounds, we investigated the effects of these small molecules on the activity of other signaling pathways using reporter cell lines. Initial screening and dose-response studies were performed in the STF reporter line, which requires the addition of exogenous Wnt3A to activate Wnt signaling. We also examined the effects on the STF3A reporter cell line, an STF reporter cell line that endogenously expresses Wnt3A. As a result, Wnt signaling is constitutively activated in the STF3A reporter cell line. The obtained IC 50 Based on these values, both iCRT3 and compound GB1874 were determined to be potent inhibitors of the Wnt pathway, regardless of the initial activation state (Table 1). However, compounds GB6853 and GB8679 could potently inhibit Wnt pathway activation (STF IC 50 values ​​of 4.8 μM and 7.0 μM, respectively), they are ineffective when the Wnt pathway is already activated (STF3A IC above 100 μM). 50 This is thought to be due to the difference in the ability of these compounds to disrupt pre-existing β-cat-TCF4 complexes (e.g., in STF3A cells) and newly formed β-cat-TCF4 complexes (e.g., in STF cells).

[0110] We also examined the effects of the above compounds on a Myc reporter cell line, in which luciferase gene expression is under the control of a Myc response element. Because Myc is one of the Wnt target genes, inhibiting Wnt signaling would also inhibit Myc signaling. The efficacy of the compounds on the Myc reporter mirrored that of the STF3A reporter; potent inhibitors of the Wnt pathway in the STF3A reporter were also potent inhibitors of the Myc reporter (Table 1).

[0111] Finally, the candidate hit compounds were tested against the Hippo pathway reporter. In the Hippo pathway reporter, the TEAD response element drives luciferase expression. When the Hippo pathway is activated, the transcriptional activation of TEAD is reduced, so compounds that reduce the reporter signal activate the Hippo pathway, and vice versa. Interestingly, we found that the Hippo pathway can be activated by iCRT3, and that the compound GB8679 is a potent inhibitor of the Hippo pathway (Table 1). Because the Wnt pathway may crosstalk with the Hippo pathway, 24 Therefore, it was suspected that inhibition of Hippo signaling by GB8679 might indirectly affect its efficacy in reducing the expression of Wnt target genes.

[0112] Different binding modes predicted in silico for three candidate small molecule binders of β-cat The binding modes of the two hit compounds, GB8679 and GB6853, to β-cat are similar to those of iCRT3. For example, both compounds occupy two pockets surrounded by R386, N426, P463, and K508, V511, and R515, respectively, with their large hydrophobic groups. Meanwhile, the thiazole group of both compounds also fills the cleft between K508 and R469, forming hydrogen bonds and salt bridges (Figure 2D). However, the third hit compound, GB1874, exhibited a binding mode different from that of iCRT3 and the other two hit compounds. GB1874 occupies a triazole group in the cleft between K508 and R469 and a hydrophobic group in the two pockets surrounded by R386, N426, P463, and K508, V511, and R515. In addition, the ethylphenyl group of GB1874 was found to be packed into a pocket surrounded by H470, R474, and K435. Remarkably, in the crystal structure of the β-cat-TCF4 complex, this pocket was occupied by the D16 residue of TCF4, which forms a salt bridge with K435 of β-cat, thus playing a key role in the interaction between β-cat and TCF4. 25A single amino acid substitution at D16 in TCF4 or K435 in β-cat is sufficient to disrupt these interactions and significantly reduce downstream transcriptional activity. 25、26 Taken together, we hypothesized that this unique binding mode of GB1874 would enable it to more potently disrupt the β-cat-TCF4 interaction compared with iCRT3 and the other two hit compounds.

[0113] Hit compounds disrupt β-cat-TCF4 interaction and reduce expression of Wnt target genes Since the above compounds were identified through in silico docking studies with β-cat protein, we investigated the ability of other compounds to disrupt the β-cat-TCF4 interaction in biologically relevant cell lines. Consequently, we performed co-immunoprecipitation (co-IP) of β-cat and its interacting partners in HCT116 CRC cells. Western blot analysis of proteins bound to β-cat upon compound treatment revealed that, of the three hit compounds, only compound GB1874 was able to reduce the β-cat-TCF4 interaction to the same extent as iCRT3 (Figure 3A). Interestingly, compound GB1874, while affecting the β-cat-TCF4 interaction, had little effect on the β-cat-ECAD interaction (Figure 3A).

[0114] We next examined the effects of the hit compounds on the expression of Wnt target genes. HCT116 cells were treated with 50 μM compounds, and the expression of Wnt target genes, including c-Met, cyclin D1, and survivin, was analyzed by Western blotting. Compound GB1874 was most effective in reducing the expression of these proteins (Figure 3B). In fact, the reduction in Wnt target protein expression by compound GB1874 was comparable to that of iCRT3. In addition, while compound GB1874 reduced the expression of Wnt target genes, it had little effect on the expression of β-cat itself or its partner proteins, such as ECAD and TCF4 (Figure 3C). These results suggest that GB1874 can regulate the nuclear transcriptional function of β-cat by specifically disrupting the β-cat-TCF4 interaction, but does not affect its membrane function at E-cad-mediated adherens junctions.

[0115] In contrast, compounds GB6853 and GB8679 had little effect on the expression of the Wnt target genes examined (Figure 3B). These compounds were also less effective than compound GB1874 in disrupting the β-cat-TCF4 interaction (Figure 3A). These results, combined with the lack of inhibitory activity in the STF3A reporter cell line (Table 1), suggest that compounds GB6853 and GB8679 are unable to inhibit the Wnt pathway in cells in which Wnt signaling is already activated.

[0116] The compound GB1874 disrupts the β-cat-TCF4 interaction in vitro Because GB1874 compound was predicted to bind to β-cat by in silico docking studies, we conducted experiments to investigate whether GB1874 compound could interact with purified β-cat in vitro and inhibit the interaction between β-cat and TCF4. Surface plasmon resonance (SPR) was employed using purified β-cat (ARM domain repeat region) and a protein encoding the TCF4 N-terminal domain, which is known to interact with β-cat. First, purified β-cat protein was immobilized on an SPR sensor chip, and different concentrations of GB1874 compound were injected onto the β-cat. From the sensorgram (Figure 8A), dose-dependent binding of GB1874 to β-cat was observed. Plotting the steady-state response versus the concentration of GB1874 compound revealed that this compound had a K of 76 ± 13 μM. D The results showed that GB1874 binds to β-cat at a specific site (n = 3, Figure 3D). However, the Hill slope of the best-fit curve was greater than 1, suggesting that compound GB1874 binds to multiple sites on β-cat with positive cooperativity.

[0117] To confirm whether compound GB1874 can directly inhibit the interaction between β-cat and TCF4 in vitro, an SPR competition experiment was developed. Instead of β-cat, the TCF4 N-terminal domain was immobilized on an SPR sensor chip. 50 nM β-cat was preincubated with different concentrations of compound GB1874 and then injected over the TCF4 N-terminal domain. As evident from the sensorgram (Figure 8B), compound GB1874 had an IC of 25 ± 8 μM (n = 2). 50 It was found that the interaction between β-cat and TCF4 was inhibited at high levels (Figure 3E).

[0118] Candidate hit compounds affect proliferation and stemness of "Wnt-dependent" cancer cells Apart from disrupting the β-cat-TCF4 interaction and reducing the expression of Wnt target genes, we also conducted experiments to determine whether the hit compounds would induce phenotypes reminiscent of reduced Wnt activity in biologically relevant cancer cell lines. First, we examined the effects of candidate compounds GB6853, GB8679, and GB1874 in HCT116 colorectal cancer (CRC) cells. HCT116 cells harbor a heterozygous mutation in the β-cat gene, resulting in a deletion of the Ser45 residue in the protein. Ser45 is the site of phosphorylation by CK1α, a key member of the degradation complex, which subsequently targets β-cat for degradation by the proteasome. Therefore, mutation of Ser45 prevents the degradation of mutant β-cat, resulting in constitutive activation of Wnt signaling. We hypothesized that inhibiting Wnt signaling downstream of β-cat in HCT116 cells would have a significant effect on cell growth.

[0119] HCT116 cells were treated daily with 10 μM of each compound, and growth was monitored using the CellTiter-Glo viability assay. For comparison, compounds such as IWP-2, XAV939, and iCRT3, which target different components along the Wnt signaling cascade, were included. 27As shown in Figure 4A, treatment with any of the three hit compounds (GB1874, GB6853, and GB8679) effectively suppressed HCT116 cell growth. Notably, all of these compounds were more effective than iCRT3 in growth inhibition assays. Compounds IWP-2 and XAV939 (inhibitors of porcupine and tankyrase, respectively) inhibit Wnt signaling upstream of the β-cat-TCF4 interaction, but were less effective in inhibiting HCT116 cell growth. Compounds GB8679, GB6853, and GB1874 at 30 μM significantly reduced the viability of cancer stem-like cells within the HCT116 population, as evidenced by their effects on colony formation efficiency (Figure 4B) and spheroid formation assays (Figure 4C). In addition, the above hit compounds also affected the spheroid formation of APC mutant CRC cell lines DLD-1 and SW480, although to different degrees (Figures 9A and 9B). Next, we examined the effect of the above compounds on the viability of CRC cells. EC 50 From the values ​​(Table 2), compound GB1874 showed the strongest effect on CRC cell lines, followed by compound GB8679.

[0120] GB1874 inhibits the growth of murine tumor xenografts Based on the above studies, compound GB1874 was identified as the most potent compound against Wnt-driven CRC cells. To examine its in vivo efficacy, NSG mice were inoculated with HCT116 cells in the flank. Tumor-bearing mice were then treated with either vehicle control or 50 mg / kg of compound GB1874 by ip injection every other day (qad). Compound GB1874 effectively inhibited the growth of HCT116 xenografts in vivo (Figure 4D), while simultaneously causing minimal systemic toxicity in mice (Figure 9C).

[0121] [Table 1]

[0122] [Table 2]

[0123] Using the GB1874 structure as a scaffold, we searched chemical databases for GB1874 analogs. These compounds were then docked into our in silico model of β-catenin to predict potential potent β-catenin binders. From this prediction, eight structural analogs of GB1874 were identified and tested.

[0124] These compounds were first tested for their ability to inhibit Wnt reporter activity using STF and STF3A reporter cells (Table 3). In general, the inhibitory IC50 for STF cells was 50 The values ​​were lower compared to the corresponding values ​​for STF3A cells, indicating that these compounds were less active against biological systems with constitutive Wnt activation, with the exception of GB1874A, which was more potent against STF3A cells than STF cells.

[0125] GB1874 IC 50 Value (STF IC 50 = 6.8 μM, STF3A IC 50 = 27 μM) with GB1874F (STF IC 50 = 5.4 μM, STF3A IC 50 =10 μM), GB1874G (STF IC 50 = 3.9 μM, STF3A IC 50 =6.7 μM), and GB1874H (STF IC 50 = 5.0 μM, STF3A IC 50= 11 μM), it was found that increasing the size of the C3 substituent of the 1,2,4-triazole did not affect the activity of the compounds against the STF and STF3A reporters. Because the new GB1874 analogs mostly differed in the C3 substituent of the 1,2,4-triazole, it was not possible to elucidate the effects of changing the substituents at other positions. Therefore, to gain a deeper understanding of the structure-activity relationship of GB1874, it was necessary to purchase or synthesize more GB1874 analogs.

[0126] [Table 3(1)] [Table 3(2)]

[0127] We examined the effects of GB1874 analogs on the expression of Wnt target genes in HCT116 CRC cells. The results (Figure 5) showed that the degree of downregulation of Wnt target genes correlated with the compound's potency in inhibiting the STF reporter. The most potent compounds were iCRT3, GB1874, GB1874C, GB1874E, GB1874F, GB1874G, and GB1874H. These compounds potently inhibited Wnt target genes such as AXIN2, BIRC5, BMP4, and CCND1, while having little effect on the expression of the β-catenin (CTNNB1) gene.

[0128] The top hit compound, GB1874, was also tested in primary patient-derived colorectal cancer (CRC) cell lines (Table 4). GB1874 was found to exhibit more potent activity against primary CRC cell lines compared to the tankyrase inhibitor XAV939.

[0129] [Table 4]

[0130] HCT116 xenograft tumors obtained in the in vivo study were analyzed. As shown in Figure 4D and Figure 11A, treatment of HCT116 xenografts with 50 mg / kg GB1874 via i.p. every other day (qad) inhibited their growth. Immunohistochemical staining and quantification indicated that tumor growth inhibition was associated with decreased expression of the Wnt target gene cyclin D1 and the proliferation marker Ki67 (Figure 11B). [Industrial Applicability]

[0131] The compounds disclosed herein can be used to inhibit β-cat or disrupt the interaction of β-cat with TCF4, and therefore can be used to treat a disease or condition selected from the group consisting of cancer, neurodegenerative diseases, metabolic diseases, cardiovascular diseases, fibrosis, and bone diseases.

[0132] Various other modifications and adaptations of the present invention will become apparent to those skilled in the art after reading the above disclosure without departing from the spirit and scope of the present invention. All such modifications and adaptations are intended to fall within the scope of the appended claims.

[0133] References 1. Polakis, 2000, Genes Dev. 14:1837-1851. 2. Morin et al., 1997, Science 275:1787-1790. 3. Rubinfeld et al., 1997, Science 275:1790-1792. 4. Koch et al., 1999, Cancer Res. 59:269-273. 5. Zurawel et al., 1998, Cancer Res. 58:896-899. 6. Voeller et al., 1998, Cancer Res. 58:2520-2523. 7. Schlosshauer et al., 2000, Mod. Pathol. 13:1066-1071. 8. Mirabelli-Primdahl et al., 1999, Cancer Res. 59:3346-3351. 9. Saegusa and Okayasu, 2001, J.Pathol. 194:59-67. 10. Wu et al., 2001, Cancer Res. 61:8247-8255. 11. Hayes et al., 2008, Clin.Cancer Res. 14:4038-4044. 12. Gonsalves FC, Klein K, Carson BB, Katz S, Ekas LA, Evans S et al., An RNAi-based chemical genetic screen identifies three small-molecule inhibitors of the Wnt / wingless signalling pathway. Proc Nat.Acad Sci USA 2011;108, 5954-63. 13. Irwin JJ, Sterling T, Mysinger MM, Bolstad ES, Coleman RG. ZINC: a free tool to discover chemistry for biology. J Chem Inf Model 2012; 52: 1757-68. 14. Mysinger MM, Carchia M, Irwin JJ, Shoichet BK. Directory of useful decoys, enhanced (DUD-E): better ligands and decoys for better benchmarking. J Med Chem 2012; 55: 6582-94. 15. Kuntz ID, Blaney JM, Oatley SJ, Langridge R, Ferrin TE. A geometric approach to macromolecule-ligand interactions. J Mol Biol 1982;161: 269-88. 16. Mysinger MM、Shoichet BK. Rapid context-dependent ligand desolvation in molecular docking. J Chem Inf Model 2010; 50: 1561-73. 17. Fan H、Irwin JJ、Webb BM、Klebe G、Shoichet BK、Sali A. Molecular docking screens using comparative models of proteins. J Chem Inf Model 2009; 49: 2512-27. 18. Graham TA、Weaver C、Mao F、Kimelman D、Xu W. Crystal structure of a beta-catenin / TCF4 complex. Cell 2000;103: 885-96. 19. Poy F、Lepourcelet M、Shivdasani RA、Eck MJ. Structure of a human TCF4-β-catenin complex. Nat Struct Biol 2001;8:1053-7. 20. Sampietro J、Dahlberg CL、Cho US、Hinds TR、Kimelman D、Xu W. Crystal structure of a beta-catenin / BCL9 / TCF4 complex. Mol Cell 2006;24: 293-300. 21. Zhang Z、Li Y、Lin B、Schroeder M、Huang B. Identification of cavities on protein surface using multiple computational approaches for drug binding site prediction. Bioinformatics 2011; 27: 2083-8. 22. Krivov GG、Shapovalov MV、Dunbrack RL Jr. Improved prediction of protein side-chain conformations with SCWRL4. Proteins 2009; 77: 778-95. 23. Sherman W、Day T、Jacobson MP、Friesner RA、Farid R. Novel procedure for modeling ligand / receptor induced fit effects. J Med Chem 2006; 49: 534-53. 24. Piccolo S、Dupont S、Cordenonsi M. The biology of Yap / TAZ: Hippo signalling and beyond. Physiol Rev 2014; 94: 1287-312. 25. Fasolini M、Wu X、Flocco M、Trosset JY、Oppermann U、Knapp S. Hot Spots in TCF4 for the Interaction with β-Catenin. J Biol Chem 2003; 278: 21092-8. 26. Liu J、Wang H、Zuo Y、Farmer SR. Functional Interaction between Peroxisome Proliferator-Activated Receptor γ and β-Catenin. Mol Cell Biol 2006; 26: 5827-37. 27. Kahn M. Can we safely target the WNT pathway? Nat Rev Drug Discovery 2014; 13: 513-32.

Claims

1. 1. Use of a compound in the manufacture of a medicament for the treatment of cancer, said compound comprising: 【Chemical 1】 or a pharmaceutically acceptable salt thereof use.

2. 2. The use of claim 1, wherein the cancer is selected from the group consisting of colorectal cancer, hepatocellular carcinoma, melanoma, breast cancer, prostate cancer, and leukemia.

3. An in vitro method for inhibiting β-cat or disrupting the interaction of β-cat with TCF4, comprising contacting β-cat with a compound; The compound is 【Chemistry 2】 or a pharmaceutically acceptable salt thereof method.

4. 1. A composition for use in the treatment of cancer, comprising a compound, said compound comprising: 【Chemistry 3】 or a pharmaceutically acceptable salt thereof composition.

5. 5. The composition of claim 4, wherein the cancer is selected from the group consisting of colorectal cancer, hepatocellular carcinoma, melanoma, breast cancer, prostate cancer, and leukemia.

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