Heterocyclic compounds as modulators of β-catenin / TCF4 interaction

Abstract

Provided herein are compounds or methods for inhibiting β-catenin or disrupting the interaction between β-catenin and T-cell factor 4, which can be used to treat diseases or health conditions, such as cancer, neurodegenerative diseases, metabolic diseases, cardiovascular diseases, fibrosis, and bone diseases. In one aspect, the compound has Formula 1: wherein R 1 Yes‑(CR 4 2) m XR 5 ; R 2 is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, aralkyl, heteroaralkyl, or heteroaryl; and R 3 Yes‑(CR 6 2) n R 7 .

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Singapore Patent Application No. 10201908175Q filed on September 4, 2019, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present disclosure generally relates to methods for inhibiting the Wnt / β-catenin (β-cat) signaling pathway. More specifically, the present disclosure provides small molecule inhibitors of β-cat / T cell factor (TCF4), the interaction leading to reduced downstream Wnt expression. The compounds and methods provided herein can be used to treat, manage or prevent diseases that are improved by inhibiting the interaction between β-cat and TCF4, such as cancer, neurodegenerative diseases, metabolic diseases, cardiovascular diseases, fibrosis and bone diseases. Background Art

[0004] The canonical Wnt / β-cat signaling pathway is an important signal transduction pathway required for normal embryonic development and adult tissue self-renewal. Activation of the pathway is achieved through binding of Wnt ligand proteins to Frizzled and low-density lipoprotein receptor-related protein 6 (LRP6). In the absence of Wnt activation, the destruction complex composed of Axin, casein kinase 1α (CK1α), glycogen synthase kinase 3β (GSK3β), and adenomatous polyposis coli (APC) phosphorylates β-cat, a key nuclear effector of Wnt signaling, and targets it for ubiquitination and proteasomal degradation. When Wnt ligands activate the pathway, the destruction complex reassembles to the membrane, leading to its inactivation. Inhibition of the destruction complex results in the cytoplasmic accumulation of non-phosphorylated β-cat and its subsequent translocation to the nucleus, where it binds to the lymphoid enhancer factor / T cell factor (LEF / TCF) family of transcription factors and numerous other cofactors including Bcl-9 and Pygopus, activating downstream target gene expression.

[0005] Due to the importance of the Wnt pathway in maintaining tissue homeostasis, aberrant activation of the Wnt pathway, especially in stem cells, is often a driver of disease, such as cancer. In particular, it has been demonstrated that loss of function of the APC tumor suppressor gene is an early driver of tumorigenesis in most colorectal cancers (CRC), leading to the formation of adenomas. In addition, Axin2 loss-of-function mutations and β-cat activating mutations are prevalent in hepatocellular carcinoma (HCC), but they can also be found at a lower frequency in CRC. In addition to cancer, dysregulation of the Wnt pathway has also been associated with the development of diseases, such as neurodegenerative diseases, metabolic diseases, and bone diseases. Therefore, many Wnt pathway-targeting agents, including small molecule inhibitors and antibody-based therapeutics, have been reported over the years. Despite the large number of promising discoveries of Wnt-targeted drug entities, most have failed to advance to human clinical trials. There have been a few isolated examples of small molecule inhibitors that have entered Phase I trials; however, to date, none have been approved for clinical use. One challenge these agents face in their development as therapeutics is that there is significant crosstalk between the Wnt signaling pathway and other pathways, such as Notch and Hedgehog. Crosstalk between pathways may limit the specificity of these agents or affect their efficacy in therapeutic responses through compensatory activation. Additionally, driver mutations in various components of the Wnt pathway linear cascade may limit the pathway inhibition efficacy of these agents.

[0006] Therefore, it can be predicted that efficient and specific downregulation of the Wnt pathway can be achieved by inhibiting the β-cat-TCF4 interaction required for the expression of downstream Wnt target genes. The advantages of this strategy are twofold. First, targeting the β-cat-TCF4 interaction is more effective in diseases where mutations are present in downstream components of the Wnt pathway, thereby reducing the effectiveness of upstream inhibitors. Second, this strategy specifically targets the transcriptional function of β-cat without affecting its interaction with E-cadherin (ECAD) at cell-cell adhesion junctions (AJs). It has been proposed that specificity can be achieved by selectively targeting a β-cat-TCF4 binding pocket that is distinct from the β-cat-TCF4 binding pocket.

[0007] Previously, it was reported that a chemical genetic screen based on cell-based RNA interference (RNAi) identified compounds that inhibited the β-cat-TCF4 interaction. From the screen, three compounds; iCRT3, iCRT5, and iCRT14 were found to disrupt the β-cat-TCF4 interaction without affecting the interaction of β-cat with ECAD. Electronic docking of iCRT3 with β-cat predicted that iCRT3 binds to a site on β-cat that binds to the TCF4 extension region (residues 13-25). This predicted iCRT3 binding site on β-cat is lined by Arg469 and Lys435, which form a salt bridge with Asp16 of TCF4 and are critical for the high affinity of TCF4 with β-cat. Due to the importance of this binding pocket for the β-cat-TCF4 interaction, it has been an attractive target site for electronic docking studies to identify inhibitors of the β-cat-TCF4 interaction for many years.

[0008] Therefore, improved methods are needed to inhibit β-cat-TCF4 interaction and the concomitant attenuation of downstream Wnt signaling that address or overcome at least some of the above challenges.

[0009] There is also a need for therapeutic agents that can treat diseases or health conditions that can be ameliorated by inhibiting β-cat or disrupting the interaction between β-cat and TCF4. Summary of the invention

[0010] Using existing protein x-ray crystal structures and biological activities of known inhibitors of the β-cat-TCF4 interaction, we describe a computational model that allows for better prediction of potential small molecule inhibitors of this protein-protein interaction surface. To demonstrate the predictive power of the computational model system, an in silico screening of a small molecule library was performed and 27 compounds were identified as potential binders to β-cat. Experimental validation of the hit compounds identified three potent inhibitors of Wnt reporter activity, of which compound GB1874 was found to induce a strong Wnt tumor suppressor phenotype in both in vitro and in vivo phenotypic assays. Based on these results and further in silico and biological screening, a family of compounds that strongly inhibit Wnt reporter activity was identified.

[0011] In one aspect, provided herein is the use of a compound in the manufacture of a medicament for treating a disease or health condition that is improved by inhibiting β-catenin (β-cat) or disrupting the interaction between β-cat and T-cell factor 4 (TCF4), wherein the compound has Formula 1:

[0012]

[0013] or a pharmaceutically acceptable salt thereof, wherein

[0014] R 1 Yes-(CR 4 2) m XR 5 ;

[0015] R 2 is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, aralkyl, heteroaralkyl, or heteroaryl;

[0016] R 3 Yes-(CR 6 2) n R 7 ;

[0017] R 4 Each occurrence is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, and heteroaralkyl; or R 4 The two examples of together with the carbon or carbons to which they are attached form a 3- to 7-membered cycloalkyl;

[0018] R 5 is hydrogen, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl or heteroaryl;

[0019] R 6 Each occurrence is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, and heteroaralkyl; or R 6 The two examples of together with the carbon or carbons to which they are attached form a 3- to 7-membered cycloalkyl;

[0020] R 7 Yes-(C=O)R 8 、-(C=O)OR 8 or -(C=O)N(R 8 )2; or R 7 is the part with formula 2:

[0021]

[0022] R 8 Each occurrence is 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, they form a 3- to 7-membered heterocycloalkyl group; or R 6 and R 8Together with the atoms to which they are attached, they form a 4- to 7-membered optionally substituted cycloalkyl or a 4- to 7-membered optionally substituted heterocycloalkyl;

[0023] m is an integer selected from 0 to 4;

[0024] n is an integer selected from 0 to 4; and

[0025] X is -O-, -S- or absent.

[0026] In another aspect, the present invention provides a method for inhibiting β-cat or disrupting the interaction between β-cat and TCF4, the method comprising: reacting β-cat with a compound of Formula 1:

[0027]

[0028] or a pharmaceutically acceptable salt thereof, wherein

[0029] R 1 Yes-(CR 4 2) m XR 5 ;

[0030] R 2 is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, aralkyl, heteroaralkyl, or heteroaryl;

[0031] R 3 Yes-(CR 6 2) n R 7 ;

[0032] R 4 Each occurrence is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, and heteroaralkyl; or R 4 The two examples of together with the carbon or carbons to which they are attached form a 3- to 7-membered cycloalkyl;

[0033] R 5 is hydrogen, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl or heteroaryl;

[0034] R 6 Each occurrence is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, and heteroaralkyl; or R 6 The two examples of together with the carbon or carbons to which they are attached form a 3- to 7-membered cycloalkyl;

[0035] R 7 Yes-(C=O)R8 、-(C=O)OR 8 or -(C=O)N(R 8 )2; or R 7 is the part with formula 2:

[0036]

[0037] R 8 Each occurrence is 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, they form a 3- to 7-membered heterocycloalkyl group; or R 6 and R 8 Together with the atoms to which they are attached, they form a 4- to 7-membered optionally substituted cycloalkyl or a 4- to 7-membered optionally substituted heterocycloalkyl;

[0038] m is an integer selected from 0 to 4;

[0039] n is an integer selected from 0 to 4; and

[0040] X is -O-, -S- or absent.

[0041] In another aspect, provided herein is a method for treating a disease or health condition in a subject in need thereof, wherein the disease or health condition is improved by inhibiting β-cat or disrupting the interaction between β-ca and TCF4, the method comprising administering a therapeutically effective amount of a compound of Formula 1:

[0042]

[0043] or a pharmaceutically acceptable salt thereof, wherein

[0044] R 1 Yes-(CR 4 2) m XR 5 ;

[0045] R 2 is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, aralkyl, heteroaralkyl, or heteroaryl;

[0046] R 3 Yes-(CR 6 2) n R 7 ;

[0047] R 4Each occurrence is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, and heteroaralkyl; or R 4 The two examples of together with the carbon or carbons to which they are attached form a 3- to 7-membered cycloalkyl;

[0048] R 5 is hydrogen, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl or heteroaryl;

[0049] R 6 Each occurrence is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, and heteroaralkyl; or R 6 The two examples of together with the carbon or carbons to which they are attached form a 3- to 7-membered cycloalkyl;

[0050] R 7 Yes-(C=O)R 8 、-(C=O)OR 8 or -(C=O)N(R 8 )2; or R 7 is the part with formula 2:

[0051]

[0052] R 8 Each occurrence is 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, they form a 3- to 7-membered heterocycloalkyl group; or R 6 and R 8 Together with the atoms to which they are attached, they form a 4- to 7-membered optionally substituted cycloalkyl or a 4- to 7-membered optionally substituted heterocycloalkyl;

[0053] m is an integer selected from 0 to 4;

[0054] n is an integer selected from 0 to 4; and

[0055] X is -O-, -S- or absent.

[0056] In another aspect, provided herein is a compound for treating a disease or health condition that is improved by inhibiting β-cat or disrupting the interaction between β-cat and TCF4, wherein the compound has Formula 1:

[0057]

[0058] or a pharmaceutically acceptable salt thereof, wherein

[0059] R 1 Yes-(CR 4 2) m XR 5 ;

[0060] R 2 is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, aralkyl, heteroaralkyl, or heteroaryl;

[0061] R 3 Yes-(CR 6 2) n R 7 ;

[0062] R 4 Each occurrence is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, and heteroaralkyl; or R 4 The two examples of together with the carbon or carbons to which they are attached form a 3- to 7-membered cycloalkyl;

[0063] R 5 is hydrogen, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl or heteroaryl;

[0064] R 6 Each occurrence is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, and heteroaralkyl; or R 6 The two examples of together with the carbon or carbons to which they are attached form a 3- to 7-membered cycloalkyl;

[0065] R 7 Yes-(C=O)R 8 、-(C=O)OR 8 or -(C=O)N(R 8 )2; or R 7 is the part with formula 2:

[0066]

[0067] R 8 Each occurrence is 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, they form a 3- to 7-membered heterocycloalkyl group; or R 6 and R 8Together with the atoms to which they are attached, they form a 4- to 7-membered cycloalkyl or a 4- to 7-membered heterocycloalkyl;

[0068] m is an integer selected from 0 to 4;

[0069] n is an integer selected from 0 to 4; and

[0070] X is -O-, -S- or absent.

[0071] In another aspect, provided herein is a compound for use in therapy, wherein the compound has Formula 1:

[0072]

[0073] or a pharmaceutically acceptable salt thereof, wherein

[0074] R 1 Yes-(CR 4 2) m XR 5 ;

[0075] R 2 is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, aralkyl, heteroaralkyl, or heteroaryl;

[0076] R 3 Yes-(CR 6 2) n R 7 ;

[0077] R 4 Each occurrence is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, and heteroaralkyl; or R 4 The two examples of together with the carbon or carbons to which they are attached form a 3- to 7-membered cycloalkyl;

[0078] R 5 is hydrogen, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl or heteroaryl;

[0079] R 6 Each occurrence is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, and heteroaralkyl; or R 6 The two examples of together with the carbon or carbons to which they are attached form a 3- to 7-membered cycloalkyl;

[0080] R 7 Yes-(C=O)R 8 、-(C=O)OR 8 or -(C=O)N(R8 )2; or R 7 is the part with formula 2:

[0081]

[0082] R 8 Each occurrence is 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, they form a 3- to 7-membered heterocycloalkyl group; or R 6 and R 8 Together with the atoms to which they are attached, they form a 4- to 7-membered optionally substituted cycloalkyl or a 4- to 7-membered optionally substituted heterocycloalkyl;

[0083] m is an integer selected from 0 to 4;

[0084] n is an integer selected from 0 to 4; and

[0085] X is -O-, -S- or absent. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] The above and other objects and features of the present disclosure will become apparent from the following description of the present disclosure when taken in conjunction with the accompanying drawings.

[0087] Figure 1 A modeling and docking platform for β-catenin structure generation for ligand discovery is described. Three crystal structures are used as starting models (N=3). The small molecule training library includes three known β-catenin inhibitors (M=3). In one iteration, each target structure will be further optimized in the presence of each of the three known docking ligands.

[0088] Figure 2An in silico study of predicted compounds for inhibition of Wnt signaling is described. (a) TOPFlash reporter activity of predicted compounds in HEK293T STF cells. Cells were treated with 10 μM of compound and stimulated with 500 ng / mL of Wnt3A for 24 h before measuring luciferase activity. TOPFlash reporter activity of compound-treated cells was normalized to cell viability and presented as fold difference of DMSO-treated cells. Error bars represent standard deviation of 4 replicates. (b) Quadratic dose-response inhibition of TOPFlash reporter activity by iCRT3 and hit compounds. STF cells were treated with different concentrations of compound and stimulated with 500 ng / mL of Wnt3A for 24 h before measuring luciferase activity. Luciferase activity of cells treated with different concentrations of compound was first normalized to cell viability of the same concentration and presented as fold difference of DMSO-treated cells. IC was calculated using four-parameter nonlinear regression 50 Values. Error bars represent the standard deviation of three replicates. (c) Chemical structures of iCRT3 and the top three hit compounds. (d) Predicted binding modes of iCRT3 (i), and three new hit compounds discovered in this study: GB8679 (ii), GB6853 (iii) and GB1874 (iv) in the optimized, most abundant β-catenin structure (partially transparent surface). The docked ligands are highlighted (solid sticks), and the key β-catenin binding site residues (partially transparent sticks). The TCF4 peptide fragment (solid line) in the β-catenin / TCF4 crystal complex structure (PDBID 1JPW) is also shown in the figure to indicate the PPI interface.

[0089] Figure 3Effects of hit compounds on HCT116 cells via the Wnt signaling pathway are depicted. (a) Co-immunoprecipitation of β-catenin and its endogenous binding partner. HCT116 cells were treated with the indicated concentrations of hit compounds for 18 h. β-catenin was immunoprecipitated from protein lysates of treated cells, and the amount of TCF4 and E-cadherin bound to β-catenin was analyzed by Western blot (top panel). Quantification of TCF4 (bottom right panel) or E-cadherin (bottom left panel) binding to β-catenin under different treatments was performed and normalized to DMSO-treated cells. Error bars represent standard deviations of n=4 independent experiments. Two-tailed paired Student's t-test was performed between DMSO control and compound treatment. *P<0.05, ns P>0.05. (b) HCT116 cells were treated with DMSO or 50 μM of compound for 18 h. Expression of Wnt target proteins c-Met, cyclin D1, and survivin was determined by Western blotting. (c) HCT116 cells were treated with DMSO or 50 μM of the compound for 18 h. The expression of Wnt pathway-related proteins E-cadherin, β-catenin, and TCF4 was determined by Western blotting. (d) Representative binding curve of compound GB1874 to β-catenin studied by surface plasmon resonance (SPR). Steady-state response values ​​were plotted according to each concentration of compound GB1874, and the dissociation constant K was calculated using the specific binding and Hill slope curve fitting equation D (n=3). (e) SPR dose response inhibition of β-catenin and GST-TCF4 binding by compound GB1874. IC50 values ​​were calculated using four-parameter nonlinear regression (n=2).

[0090] Figure 4The inhibitory effect of the lead compound on growth and its effect on the stemness of Wnt-driven cells are described. (a) Growth curves of HCT116 cells treated with 10 μM of compound daily. One-way repeated ANOVA with Dunnett's multiple comparison test was performed to determine the significance between compound treatment and DMSO control. *P<0.05, ***P<0.001. (b) Effect of the lead compound on colony formation of HCT116 cells. HCT116 cells were grown in 6-well plates and treated with DMSO or 30 μM of compound for 7 days, and then the cells were fixed and stained with crystal violet. The number of colonies obtained was counted. A two-tailed Student's t-test was performed between compound treatment and DMSO control. ***P<0.001. (c) Effect of the lead compound on spheroid formation of HCT116 cells. HCT116 cells were grown in ultra-low attachment 96-well plates and treated with DMSO or 30 μM of compound for 14 days, and then the number of spheroids with a size of ≥200 μm was determined. 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) by intraperitoneal injection every other day for 2 weeks. Tumor volume was monitored over time. On day 17, two-tailed Student's t-test was performed between control and treatment. *P<0.05.

[0091] Figure 5 Depicted are the in silico predicted docking scores and in vitro cell-based IC50 values ​​for iCRT analogs. The lower the DOCK score, the better the binding prediction. The lower the RANK score, the better the predicted binding. Favorable (1), neutral (0), unfavorable (-1). The more (*), the higher the ranking. nd = not determined.

[0092] Fig. 6A Depicted are the DOCK scores of the top 27 compounds identified from the enamine pharmacological diversity compound library.

[0093] Figure 6B The structures of the top 27 compounds identified from the enamine pharmacological diversity compound library are described.

[0094] Figure 7 Depicted are (a) cell viability of STF cells after 24 h of compound treatment. Viability measurements of compound-treated cells were normalized to DMSO-treated cells. Error bars represent standard deviations of 4 replicates. (b) dose-response effects of iCRT3 and lead compounds on STF cell viability after 24 h of treatment. Viability measurements of compound-treated cells were normalized to DMSO-treated cells. IC was calculated using four-parameter nonlinear regression 50Error bars represent standard deviation of 3 replicates.

[0095] Figure 8 Depicted are (a) sensorgrams of binding of analyte compound GB1874 at different concentrations to β-catenin ligand. (b) sensorgrams of binding of 50 nM β-catenin analyte to GST-TCF4 ligand. β-catenin was pre-incubated with different concentrations of compound GB1874 for 15 minutes before binding to GST-TCF4.

[0096] Fig. 9 Depicted are the effects of lead compounds on spheroid formation of (a) DLD-1 and (b) SW480 cells. Cells were grown in ultra-low attachment 96-well plates and treated with DMSO or 30 μM compound for 7 days, and the number of spheroids with a size of ≥200 μm 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. Scale bar represents 1 mm. (c) NSG mice xenografted with HCT116 cells were treated with vehicle control (n=6) or 50 mg / kg GB1874 (n=6) by intraperitoneal injection every other day for 2 weeks. Mouse weight was monitored over time. On day 17, a two-tailed Student's t-test was performed between control and treatment. ns P>0.05.

[0097] Fig.10 The effects of GB1874 and its analogs on the expression of β-catenin (CTNNB1) and Wnt target genes are described.

[0098] Fig.11 GB1874 is described to inhibit the growth of Wnt-driven cells in vivo by inhibiting Wnt signaling. (a) NSG mice xenografted with HCT116 cells were treated with vehicle control (n=6) or 50 mg / kg GB1874 (n=6) by intraperitoneal injection every other day for 2 weeks. Tumor volume was monitored over time (left panel). Two-way ANOVA was performed between vehicle control and treatment tumor volumes. *P<0.05. Error bars represent mean ± SEM. The right panel shows tumor images at the end of treatment. The scale bar represents 1 cm. (b) Representative IHC staining of Ki67 and cyclin D1 expression in tumors (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). A one-tailed Student's t-test was performed for each marker between vehicle control and treatment. **P<0.01, *P<0.05, ns P>0.05. Error bars represent mean ± SD of 3 tumors. DETAILED DESCRIPTION

[0099] The following are some definitions that are helpful in understanding the description of the present invention. These terms are intended as general definitions and should not in any way limit the scope of the present invention to only these terms, but are provided for a better understanding of the following description.

[0100] Unless the context requires otherwise or clearly stated to the contrary, an integer, step or element of the invention described herein as a singular integer, step or element expressly encompasses both the singular and the plural of the integer, step or element.

[0101] In this specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising" will be understood to include one stated step or element or integer or multiple steps or groups of elements or integers, but not to exclude any other steps, elements, integers or multiple elements or groups of integers. Therefore, in the context of this specification, the term "comprising" means "mainly including but not necessarily limited to".

[0102] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It should be understood that the invention includes all such variations and modifications. The invention also includes all steps, features, compositions and compounds mentioned or indicated in this specification, either individually or collectively, and any and all combinations or any two or more of said steps or features.

[0103] 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, wherein the acid group can be a carboxylic acid group, a sulfonic acid group or a phosphonic acid group, or a mixture thereof. Relative to one or more acid groups, the amino group can be "α", "β", "γ" ... to "ω". The main chain of an "amino acid" can be substituted by one or more groups selected from the group consisting of halogen, hydroxyl, guanidine, and heterocyclic groups. Therefore, the term "amino acid" also 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, etc. Amino acid (L) and (D) forms are included within the scope of the present invention.

[0104] As used herein, the term "alkyl group" includes within its meaning monovalent ("alkyl") and divalent ("alkylene") straight or branched chain saturated aliphatic groups having from 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, but is not limited to, methyl, ethyl, 1-propyl, isopropyl, 1-butyl, 2-butyl, isobutyl, tert-butyl, pentyl, 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 propyl, 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.

[0105] The term "alkenyl group" includes within its meaning monovalent ("alkenyl") and divalent ("alkenylene") straight or branched chain unsaturated aliphatic hydrocarbon groups having from 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 of E, Z, cis or trans stereochemistry, as applicable, at any position in the alkyl chain. Examples of alkenyl groups include, but are not limited to, ethenyl, vinyl, allyl, 1-methylethenyl, 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.

[0106] As used herein, the term "alkynyl group" includes within its meaning monovalent ("alkynyl") and divalent ("alkynylene") straight or branched unsaturated aliphatic hydrocarbon radicals having from 2 to 10 carbon atoms and having at least one triple bond at any position in 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-nonynyl, 1-decynyl, and the like.

[0107] As used herein, "aryl" refers to an aromatic monocyclic hydrocarbon ring system or a polycyclic ring system, wherein two or more aromatic hydrocarbon rings are fused (i.e., have a common bond) together, or at least one aromatic monocyclic hydrocarbon ring is fused to one or more cycloalkyl and / or cycloheteroalkyl rings. The aryl group may have 6 to 24 carbon atoms (e.g., C6-C24 aryl groups) in its ring system, which may include multiple condensed rings. In certain embodiments, the polycyclic aryl group may have 8 to 24 carbon atoms. Any suitable ring position of the aryl group may be covalently linked to the defined chemical structure. Examples of aryl groups having only one or more aromatic carbocyclic rings include phenyl, 1-naphthyl (bicyclic), 2-naphthyl (bicyclic), anthracene (tricyclic), phenanthrenyl (tricyclic), pentaphenyl (pentacyclic) and similar groups. 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 benzo derivatives of cyclopentane (i.e., indenyl groups, which are 5,6-bicyclic cycloalkyl / aromatic ring systems), cyclohexane (i.e., tetrahydronaphthyl groups, which are 6,6-bicyclic cycloalkyl / aromatic ring systems), imidazoline (i.e., benzimidazolinyl groups, which are 5,6-bicyclic heterocycloalkyl / aromatic ring systems), and pyran (i.e., benzopyranyl groups, which are 6,6-bicyclic heterocycloalkyl / aromatic ring systems). Other examples of aryl groups include benzodioxanyl, benzodioxolyl, chromanyl, indolinyl groups, and the like. In certain embodiments, aryl groups may be optionally substituted. In certain embodiments, aryl groups are substituted by another aryl group, which may be referred to as biaryl groups. Each aryl group in the biaryl group may be optionally substituted.

[0108] 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 present in the ring system is aromatic and contains at least one ring heteroatom. Polycyclic heteroaryl groups include groups with two or more heteroaromatic rings fused together, and groups with at least one monocyclic heteroaryl ring fused to one or more aromatic carbocyclic rings, non-aromatic carbocyclic rings and / or non-aromatic heteroalkyl rings. The heteroaryl group as a whole may have, for example, 5 to 24 ring atoms and include 1 to 5 ring heteroatoms (i.e., 5 to 20 heteroaryl groups). The heteroaryl group may be connected to a defined chemical structure at any heteroatom or carbon atom to form a stable structure. Typically, the heteroaryl ring does not contain OO, SS or SO bonds. However, one or more N or S atoms in the heteroaryl group may be oxidized (e.g., pyridine N-oxide thiophene S-oxide, thiophene S, S-dioxide). Examples of heteroaryl groups include, for example, 5- or 6-membered monocyclic and 5- to 6-ring bicyclic systems such as those shown below: wherein T is O, S, NH, N-alkyl, N-aryl, N-(arylalkyl) (e.g., N-benzyl), SiH2, SiH(alkyl), Si(alkyl), SiH(arylalkyl), Si(arylalkyl), or Si(alkyl)(arylalkyl). Examples of such heteroaryl rings include pyrrolyl, furanyl, thienyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, isothiazolyl, thiazolyl, thiadiazolyl, isoxazolyl, oxazolyl, oxadiazolyl, indolyl, isoindolyl, phenylpropenyl, benzothiophenyl, quinolyl, 2-methylquinolyl, isoquinolyl, quinoxalinyl, quinazolinyl, benzotriazolyl, benzimidazolyl, benzothiazolyl, benzothiazolyl, benzophenone ... substituted 4,5,6,7-tetrahydroindolyl, tetrahydroquinolinyl, benzothiophene pyridinyl, benzofuranyl, benzothiazolyl, benzoxadiazolyl, benzoxazolyl, cinnolinyl, 1H-indazolyl, 2H-indazolyl, indolizinyl, isobenzofuranyl, naphthyridinyl, 2,3-naphthyridinyl, pteridinyl, purinyl, oxazolidinyl, thiazolyl pyridinyl, imidazolyl pyridinyl, furanyl, thienyl pyridinyl, pyridopyrimidinyl, pyridopyrazinyl, pyridopyridazinyl, thienothiazolyl, thienoxazolyl, thienoimidazolyl, etc. Other examples of heteroaryl groups include 4,5,6,7-tetrahydroindolyl, tetrahydroquinolinyl, benzothiophene pyridinyl, benzofuranyl, etc. In certain embodiments, the heteroaryl group can be optionally substituted.

[0109] As used herein, the term "cycloalkyl" 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, for example, 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.

[0110] The term "cycloalkenyl" as used herein refers to a cyclic unsaturated aliphatic group, including within its meaning monovalent ("cycloalkenyl") and divalent ("cycloalkenylene"), monocyclic, bicyclic, polycyclic or fused polycyclic hydrocarbon radicals having 3 to 10 carbon atoms and having at least one E, Z, cis or trans stereochemical double bond (as applicable) at any position in the alkyl chain. Examples of cycloalkenyl groups include, but are not limited to, cyclopropenyl, cyclopentenyl, cyclohexenyl, and the like.

[0111] 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 are heteroatoms selected from O, N, NH or S. Examples include pyrrolidinyl, piperidinyl, quinuclidinyl, azetidinyl, morpholinyl, tetrahydrophenylthio, tetrahydrofuranyl, tetrahydropyranyl, and the like.

[0112] 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 and at least 1 double bond, wherein 1 to 5 of the ring atoms are heteroatoms selected from O, N, NH or S.

[0113] The term "heteroaromatic group" and variants such as "heteroaryl" or "heteroarylene" as used herein include within their meaning monovalent ("heteroaryl") and divalent ("heteroarylene"), mononuclear, polynuclear, conjugated and fused aromatic radicals having from 6 to 20 atoms, of which 1 to 6 atoms are heteroatoms selected from O, N, NH and S. Examples of such groups include pyridyl, 2,2'-bipyridyl, phenanthrolinyl, quinolinyl, phenylthio and the like.

[0114] As used herein, the term "halogen" or variations such as "halide" or "halo" refers to fluorine, chlorine, bromine, and iodine.

[0115] As used herein, the term "heteroatom" or variations such as "heteroatom-" refers to O, N, NH, and S.

[0116] As used herein, the term "alkoxy" refers to a straight or branched chain alkoxy group. Examples include methoxy, ethoxy, n-propoxy, isopropoxy, tert-butoxy, and the like.

[0117] As used herein, the term "amino" refers to a a R b A group in which R a and R b Individually selected from the group including, but not limited to, hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, and optionally substituted aryl groups.

[0118] As used herein, the term "aromatic group" or variants 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.

[0119] 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 groups.

[0120] 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 groups.

[0121] As used herein, the term "optionally substituted" means that the group to which the term refers may be unsubstituted or may be substituted with one or more groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, sulfanyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, halo, carboxyl, haloalkyl, haloalkynyl, hydroxy, alkoxy, sulfanyloxy, alkenyloxy, haloalkoxy, haloalkenyloxy, nitro, amino, nitroalkyl, nitroalkenyl, nitroalkynyl, nitroheterocyclyl, alkylamino, dialkylamino, alkenylamine, alkynylamino, acyl, alkenoyl, alkynoyl, amido, diamido, acyloxy, alkylsulfonyloxy, heterocyclooxy, heterocycloamino, haloheterocycloalkyl, alkylthio, alkylcarbonyloxy, alkylthio, acylthio, phosphorus-containing groups such as phosphono and phosphinyl, aryl, heteroaryl, alkylaryl, alkylheteroaryl, cyano, cyanate, isocyanate, -C(O)NH(alkyl) and -C(O)N(alkyl).

[0122] The present invention includes within its scope all isomeric forms of the compounds disclosed herein, including all diastereomers, racemates and enantiomers. Thus, formula (I) and (II) should be understood to include, for example, E, Z, cis, trans, (R), (S), (L), (D), (+) and / or (-) forms of the compounds, as the case may be.

[0123] In the context of the present invention, the term "administering" and variations of the term including "administer" and "administration" include contacting, applying, delivering or providing the compound or composition of the present invention to an organism or surface by any appropriate means.

[0124] The term "subject" as used herein refers to an animal, typically a mammal or human, that will become or has become a subject of treatment, observation and / or experiment. When the term is used in conjunction with the administration of a compound or drug, the subject has become the subject of treatment, observation and / or administration of the compound or drug. In this specification, the term "subject" includes humans and individuals of any species with social, economic or research importance, including but not limited to members of sheep, cattle, horses, pigs, cats, dogs, primates (including humans and non-human primates), rodents, mice, goats, hares and birds. In certain embodiments, the subject is a human. In certain embodiments, the term "subject" may refer to a cell sample, a tissue sample or an organ sample derived from a subject, including, for example, a cultured cell line, a biopsy, a blood sample or a liquid sample containing cells.

[0125] The term "substituted" is intended to mean that one or more (e.g., 1, 2, 3, 4, or 5; in some embodiments, 1, 2, or 3; in other embodiments, 1 or 2) hydrogen atoms on the group indicated in the "substituted" expression are replaced with one or more organic or inorganic groups selected from the indicated one or more organic or inorganic groups, or with suitable organic or inorganic groups known to those skilled in the art, provided that the normal valence of the indicated atom is not exceeded and the substitution results in a stable compound. Suitable indicated organic or inorganic groups include, for example, alkyl, alkenyl, alkynyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocyclic, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, alkylamino, dialkylamino, trifluoromethylthio, difluoromethyl, amido, nitro, trifluoromethyl, trifluoromethoxy, carboxyl, carboxylalkyl, keto, thioketo, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylsilanyl, and cyano. In addition, suitable groups may 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)(OR)2, -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)NR2, -C(=S)NR2, -C(=NR)NR2, wherein each X is independently a halogen (or "halo" group): F, Cl, Br or I; each R is independently H, an alkyl, an aryl, a heterocycle, a protecting group or a prodrug moiety. As will be readily appreciated by those skilled in the art, when the substituent is a keto group (i.e., =O) or a thioketo group (i.e., =S), etc., then two hydrogen atoms on the substituted atom are replaced by the substituent.

[0126] In the context of this specification, the term "treatment" refers to any and all uses of treating a disease state or symptom, preventing the development of a disease, or otherwise preventing, hindering, slowing or reversing the progression of a disease or other undesirable symptoms in any way.

[0127] In the context of this specification, the term "therapeutically effective amount" includes within its meaning a sufficient amount of the compound or composition of the invention to provide the desired therapeutic effect. The exact dosage required varies from subject to subject, depending on factors such as the type being treated, the age and general condition of the subject, the severity of the condition being treated, the specific agent to be administered, the mode of administration, etc. Therefore, it is not possible to specify an exact "effective amount". However, for any given situation, an appropriate "effective amount" can be determined by a person of ordinary skill in the art using only routine experimentation.

[0128] The present disclosure provides a compound that can be used to inhibit β-cat or disrupt the interaction between β-cat and TCF4, wherein the compound has Formula 1:

[0129]

[0130] or a pharmaceutically acceptable salt thereof, wherein

[0131] R 1 Yes-(CR 4 2) m XR 5 ;

[0132] R 2 is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, aralkyl, heteroaralkyl, or heteroaryl;

[0133] R 3 Yes-(CR 6 2) n R 7 ;

[0134] R 4 Each occurrence is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, and heteroaralkyl; or R 4 The two examples of together with the carbon or carbons to which they are attached form a 3- to 7-membered cycloalkyl;

[0135] R 5 is hydrogen, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl or heteroaryl;

[0136] R 6 Each occurrence is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, and heteroaralkyl; or R 6 The two examples of together with the carbon or carbons to which they are attached form a 3- to 7-membered cycloalkyl;

[0137] R 7Yes-(C=O)R 8 、-(C=O)OR 8 or -(C=O)N(R 8 )2; or R 7 is the part with formula 2:

[0138]

[0139] R 8 Each occurrence is 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, they form a 3- to 7-membered heterocycloalkyl group; or R 6 and R 8 Together with the atoms to which they are attached, they form a 4- to 7-membered optionally substituted cycloalkyl or a 4- to 7-membered optionally substituted heterocycloalkyl;

[0140] m is an integer selected from 0 to 4;

[0141] n is an integer selected from 0 to 4; and

[0142] X is -O-, -S- or absent.

[0143] In certain embodiments, the compound of Formula I does not include the following compounds:

[0144]

[0145] In certain embodiments, X is -O-, -S-, or absent. In the absence of X, R 1 Can have the structure: -(CR 4 2) m R 5 , where m, R 4 and R 5 As defined in any one or more of the embodiments described herein.

[0146] 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; m is 1. In certain embodiments, X is absent, and m is 1 or 2.

[0147] In certain embodiments, m is 0 to 3, 0 to 2, 0 to 1, or 1-2.

[0148] In certain embodiments, n is 0-3, 0-2, 0-1, or 1-2.

[0149] In certain embodiments, R 2 is alkyl, aralkyl, heteroaralkyl; or -(CR9 2) p R 10 , wherein p is an integer selected from 0 to 4; R 9 Each occurrence of is independently hydrogen or alkyl; and R10 is alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, aralkyl, or heteroaralkyl. In certain embodiments, R 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.

[0150] In R 2 Yes-(CR 9 2) p R 10 In the case of , p can be 0 to 3, 0 to 2, 0 to 1, or 1 to 2. In certain embodiments, R 9 Each occurrence is independently hydrogen or alkyl. 2 Yes -(CH2)R 10 or -(CHMe)R 10 ; and R 10 As defined herein.

[0151] In certain embodiments, R 4 Each occurrence is hydrogen or alkyl; or R 4 The two examples of together with the carbon or carbons to which they are attached form a 3-7-membered or 3-6-membered cycloalkyl. 4 It's hydrogen.

[0152] In certain embodiments, R 5 is cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl or heteroaryl. 5 is aryl or heteroaryl. In certain embodiments, R 5 is optionally substituted phenyl.

[0153] In certain embodiments, R 7 Yes-(C=O)R 8 、-(C=O)OR 8 or -(C=O)N(R 8 )2, where R 8 is alkyl, cycloalkyl, aryl; or two R 8 Together with the nitrogen to which they are attached, they form a 3- to 7-membered heterocycloalkyl group; or R 6 and R 8Together with the atoms to which they are attached they form a 4- to 7-membered optionally substituted cycloalkyl or a 4- to 7-membered optionally substituted heterocycloalkyl.

[0154] In two R 8 The 3- to 7-membered heterocycloalkyl groups may contain 1 or 2 heteroatoms selected from oxygen, sulfur and nitrogen in their ring system when taken together with the nitrogen to which they are attached to form a 3- to 7-membered heterocycloalkyl group.

[0155] In R 6 and R 8 When taken together with the atoms to which they are attached to form a 4 to 7 membered optionally substituted heterocycloalkyl, the compound may have Formula 5:

[0156]

[0157] or a pharmaceutically acceptable salt thereof, wherein R 1 , R 2 , R 6 and R 8 wherein each of the above is 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 1 or 2 heteroatoms selected from oxygen, sulfur and nitrogen in its ring system); Y is -O-, -N(R 8 )- or not present; or the compound may have formula 6:

[0158]

[0159] or a pharmaceutically acceptable salt thereof, wherein R 1 , R 2 , R 6 and R 8 wherein each of the above is 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 1 or 2 heteroatoms selected from oxygen, sulfur and nitrogen in its ring system); Y is -O-, -N(R 8 )—or does not exist.

[0160] In certain embodiments, R 8 It 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 they form a 3 to 7, 4 to 7, 5 to 7 or 5 to 6 membered optionally substituted heterocycloalkyl; or a compound has Formula 6 wherein A is a 5 to 6 membered optionally substituted cycloalkyl.

[0161] In R 7 In the case of a moiety having Formula 2, the compound can be represented by Formula 7:

[0162]

[0163] or a pharmaceutically acceptable salt thereof, wherein R 1 , R 2 , R 6 Each of and m is each independently as defined herein.

[0164] In certain embodiments, the compound has Formula 3:

[0165]

[0166] or a pharmaceutically acceptable salt thereof, wherein

[0167] R 4 is independently selected at each occurrence from the group consisting of hydrogen and alkyl;

[0168] R 5 is aryl or heteroaryl;

[0169] R 6 is independently selected at each occurrence from the group consisting of hydrogen and alkyl;

[0170] R 7 Yes-(C=O)R 8 、-(C=O)OR 8 or -(C=O)N(R 8 )2; or R 7 is a part having formula 4:

[0171]

[0172] R 8 Each occurrence is independently selected from the group consisting of alkyl, cycloalkyl and aryl; or R 8 Together with the nitrogen to which they are attached, the two examples form a 5- to 6-membered heterocycloalkyl; or R 6 and R 8 Together with the atoms to which they are attached, they form a 5- to 6-membered cycloalkyl group;

[0173] R 9 is independently selected at each occurrence from the group consisting of hydrogen and alkyl;

[0174] R 10 is cycloalkyl, aryl or heteroaryl; and

[0175] X is -O- or absent.

[0176] In certain embodiments, the compound has Formula 3, wherein R 4 is hydrogen; R 5 is an aryl group; R 9Each occurrence is independently selected from the group consisting of hydrogen and alkyl; R 10 is an aryl group; R 6 Each occurrence is independently selected from the group consisting of hydrogen and alkyl; R 7 is -(C=O)OR 8 or -(C=O)N(R 8 )2, where R 8 Each occurrence is independently selected from the group consisting of alkyl, cycloalkyl and aryl; or R 8 The two examples of together with the nitrogen to which they are attached form a 5- to 6-membered heterocycloalkyl.

[0177] In certain embodiments, the compound has Formula 8:

[0178]

[0179] or a pharmaceutically acceptable salt thereof, wherein

[0180] q is an integer selected from 0 to 2;

[0181] t is 1 or 2;

[0182] R 5 is optionally substituted phenyl;

[0183] R 9 is hydrogen or alkyl;

[0184] R 10 is optionally substituted phenyl;

[0185] R 11 Each occurrence 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)2, -NR2 or -N(R)(C=O)N(R)2, wherein each occurrence of R is independently hydrogen, alkyl, aryl or heteroaryl; or two R together with the nitrogen or nitrogens to which they are attached form a 3- to 7-membered heterocycloalkyl.

[0186] In certain embodiments, the compound has Formula 8, q is 0 or 1; t is 1; R 9 is hydrogen or C1-C3 alkyl; R 11 is alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl.

[0187] In certain embodiments, the compound is selected from the group consisting of:

[0188]

[0189] or a pharmaceutically acceptable salt thereof.

[0190] Pharmaceutically acceptable salts of the compounds described herein may include conventional non-toxic salts or quaternary ammonium salts of the compounds, such as 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, aminosulfonic acid, phosphoric acid, nitric acid, and the like; and 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, isothiocarboxylic acid, and the like.

[0191] In other cases, the compounds described herein may contain one or more acidic functional groups and are therefore capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases. In these examples, the term "pharmaceutically acceptable salts" may refer to relatively nontoxic, inorganic and organic base addition salts of the compounds described herein. These salts can also be prepared in situ during the administration of the carrier or the manufacture of the dosage form, or by reacting the purified compound in the form of a free acid with a suitable base (e.g., a pharmaceutically acceptable hydroxide, carbonate or bicarbonate of a metal cation), with ammonia, or with a pharmaceutically acceptable organic primary, secondary or tertiary amine alone. Representative alkali or alkaline earth salts include lithium, sodium, potassium, calcium, magnesium and aluminum salts, etc. Representative organic amines for forming base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, etc.

[0192] Inhibition of β-cat or disruption of the interaction between β-cat and TCF4 can be performed in vitro, in vivo or ex vivo.

[0193] β-cat can be inhibited or the interaction between β-cat and TCF4 can be disrupted by contacting at least one of β-cat and the β-cat-TCF4 complex with one or more compounds described herein.

[0194] Contacting β-cat or the β-cat / TCF4 complex with a compound of Formula 1 may result in one or more of the following: inhibition of one or more β-cat functions, inhibition of the interaction between β-cat and TCF4, and disruption of the β-cat / TCF4 complex, and may further result in attenuation of Wnt signaling.

[0195] Therefore, methods of inhibiting β-cat or disrupting the interaction between β-cat and TCF4 can be used to treat diseases where inhibition of β-cat, disruption of the β-cat / TCF4 complex, and / or attenuation of the Wnt signaling pathway results in disease or improved health status.

[0196] Disrupting the interaction between β-cat and TCF4 may refer to one or both of inhibiting the binding of β-cat to TCF4 or inducing the dissociation of the β-cat-TCF4 complex to form β-cat and TCF4.

[0197] The amount or concentration of the compound of Formula 1 desired for administration can be readily determined by one of ordinary skill in the art using well-known methods and the disclosure provided herein.

[0198] Provided herein is a method of treating a disease or health condition that is improved by inhibiting the interaction between β-cat and TCF4 in a subject in need thereof, the method comprising administering a therapeutically effective amount of a compound described herein. In certain embodiments, the subject is a human.

[0199] The present disclosure also provides a compound as described herein for use in treating a disease or health condition that is improved by inhibiting β-cat or disrupting the interaction between β-cat and TCF4.

[0200] The present disclosure further provides the use of a compound as described herein in the manufacture of a medicament for treating a disease or health condition that is improved by inhibiting β-cat or disrupting the interaction between β-cat and TCF4.

[0201] The disease or health condition may be cancer, neurodegenerative disease, metabolic disease, cardiovascular disease, fibrosis, and bone disease.

[0202] In certain embodiments, the disease or health condition is cancer characterized by aberrant activation of one or more of the Wnt, Myc, and Hippo signaling pathways.

[0203] In certain embodiments, the disease or condition is cancer characterized by aberrant activation of the Wnt / β-cat signaling pathway.

[0204] Abnormalities in the Wnt / β-cat signaling pathway have been implicated in many different cancers, and defects in many genes within this pathway may contribute to tumor promotion and progression. 1. Activation of the Wnt / β-cat pathway is one of the most common signaling abnormalities in many types of human cancers, such as colorectal cancer. 2. Melanoma, 3. Hepatoblastoma, 4. Medulloblastoma, 5. Prostate cancer, 6. Endometrioid adenocarcinoma of the uterus and ovary. 7-10. Wnt / β-V pathway activation is also common in breast metaplastic carcinomas. 11.

[0205] Thus, the provided methods can be used to treat a cancer selected from the group consisting of colorectal 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 uterine and ovarian endometrioid adenocarcinoma.

[0206] In certain embodiments, the cancer is colorectal cancer characterized by aberrant activation of the Wnt / β-cat signaling pathway.

[0207] Example

[0208] Generation of virtual chemical libraries

[0209] The training library consisted of three known β-cat-TCF4 complex inhibitors (including iCRT3, iCRT5, and iCRT14, 12. ) and through the DUD-E method 14. From ZINC database 13. The validation library consisted of 63 iCRT3 analogs synthesized by the DasGupta laboratory. The virtual screening library consisted of 150 property-matched computational decoys selected as negative controls. https: / / enamine.net / ) was purchased from 10,240 compounds.

[0210] Molecular docking screening

[0211] Spheres and grids were generated before docking. 45 matching spheres were generated for positioning database compounds in the site by augmenting the ligand-derived spheres with the receptor-derived spheres. The ligand-derived spheres were represented by the non-hydrogen atom positions of the TCF4 peptide fragment crystal structure at the beginning and replaced by the docking site of the iCRT ligand in the following rounds. The molecular surface of the binding site was generated using SPHGEN 15. The receptor-derived spheres were calculated using the program DOCK version 3.6. 16. The docked compounds are ranked by docking energy, which is the sum of van der Waals, Poisson-Boltzmann electrostatics, and ligand desolvation penalty terms.

[0212] Docking performance evaluation

[0213] The accuracy of structural models in ligand prediction was assessed by the enrichment of known ligands in the top-scoring compounds, measured by the early enrichment factor EF1 and the overall enrichment logAUC, which is similar to the area under the receiver operating characteristic (ROC) curve (AUC) but gives more weight to early enrichment. 16,17.

[0214] Cell lines

[0215] Wnt-STF reporter cells (HEK 293 cells stably transfected with TOPFlash reporter) and STF3A reporter cells (STF cells with intrinsic secretion of Wnt3A) were a gift from David Virshup (Duke-NUS Graduate Medical School, Singapore). HCT116, DLD-1, and SW480 cell lines were obtained from ATCC. Hippo pathway TEAD reporter (Cat. No. 60618) and Myc signaling pathway reporter (Cat. No. 60520) cell lines were purchased from BPS Bioscience, Inc.

[0216] Culture medium

[0217] STF, STF3A and DLD-1 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco catalog number 11965084), which was supplemented with 10% fetal bovine serum (FBS, Hyclone catalog number SV30160.03) and 100U / 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), which was supplemented with 10% FBS and 100U / mL penicillin-streptomycin. The culture medium of the Myc signaling pathway reporter cells was additionally supplemented with 400μg / mL G-418 sulfate (GoldBiotechnology catalog number G-418-5). Hippo pathway TEAD reporter cells were cultured in minimal 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 culture 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).

[0218] TOPFlash (signal) report filter

[0219] STF cells were seeded in 96-well plates (Corning catalog number 3903 & 3904) at 20,000 cells in 100 μL of culture medium per well. The next day, candidate compounds in DMSO were added to the cells at a final concentration of 10 μM, 1% DMSO. Cells were also stimulated with Wnt3A conditioned medium. After 24 hours of incubation, cell viability was determined using PrestoBlue Viability Reagent (Invitrogen catalog number A13262) according to the manufacturer's protocol, while TOPFlash reporter activity was determined using Steady-Glo luciferase reagent (Promega catalog number E2550).

[0220] Reporter dose response

[0221] Wnt signaling reporter

[0222] Wnt STF reporter cells were seeded in 96-well plates (Corning catalog number 3903, Falcon catalog number 353072) with 20,000 cells in 100 μL of culture medium per well. The next day, 5-fold dilutions of the compound in DMSO were added to the cells at a final concentration of 1% DMSO. The cells were also stimulated with 500 ng / mL recombinant human Wnt3A (RnD system, catalog number 5036-WN-010). After 24 hours of incubation, cell viability was determined using a cell counting kit-8 (CCK-8, Dojindo catalog number CK04) cell viability reagent according to the manufacturer's protocol, while TOPFlash reporter activity was determined using a Steady-Glo luciferase reagent.

[0223] STF3A reporter cells were seeded at 20,000 cells in 100 μL of culture medium per well in 96-well plates (Corning catalog number 3903, Falcon catalog number 353072). The next day, 5-fold dilutions of the compounds in DMSO were added to the cells at a final concentration of 1% DMSO. After 24 hours of incubation, cell viability was determined using a cell counting kit-8 (CCK-8, Dojindo catalog number CK04) cell viability reagent according to the manufacturer's protocol, while TOPFlash reporter activity was determined using a Steady-Glo luciferase reagent.

[0224] Myc signaling reporter

[0225] Myc reporter (Luc)-HCT116 cells were seeded in 96-well plates (Corning catalog number 3903, Falcon catalog number 353072) with 25,000 cells in 100 μL of assay medium per well. The assay medium consisted of growth medium without G-418. The next day, 4 times the dilution of the compound in DMSO was added to the cells at a final concentration of 1% DMSO. After 24 hours of incubation, cell viability was determined using PrestoBlue cell viability reagent, while luciferase reporter activity was determined using Steady-Glo luciferase reagent.

[0226] Hippo Signal Report

[0227] Hippo TEAD reporter cells were seeded in 96-well plates (Corning catalog number 3903, Falcon catalog number 353072) at 100 μL of assay medium per well. The assay medium consisted of growth medium without G-418. The next day, 5-fold dilutions of the compounds in DMSO were added to the cells at a final concentration of 1% DMSO. After 24 hours of incubation, cell viability was determined using CCK-8 cell viability reagent, while luciferase reporter activity was determined using Steady-Glo luciferase reagent.

[0228] Dose-response studies in CRC cell lines

[0229] HCT116, DLD-1 and SW480 cells were seeded in 96-well plates (Corning catalog number 3903) at 5,000 cells per well in 100 μL of growth medium. The next day, 5-fold dilutions of the compounds in DMSO were added to the cells at a final concentration of 1% DMSO. After 24 hours of incubation, cell viability was determined using CellTiter-Glo luminescent cell viability reagent (Promega catalog number G7573) according to the manufacturer's protocol.

[0230] Data analysis for dose-response studies

[0231] The average signal of the DMSO-treated wells of each plate was calculated and the signal of each treated well was normalized to the average DMSO signal of the corresponding plate. Normalized values ​​were plotted versus test concentration using GraphPad Prism 5 and EC was determined by three-parameter or four-parameter nonlinear regression. 50 value.

[0232] Proliferation assay

[0233] HCT116 cells were seeded in 96-well plates (Corning catalog number 3903) at 3,000 cells per well in 100 μL of growth medium and allowed to adsorb overnight. The cells were then treated with compounds at a final concentration of 10%, 0.1 M DMSO for 4 consecutive days, with the medium replaced every day. Cell proliferation was measured daily using the CellTiter Glo luminescent cell viability assay (Promega catalog number G7573) according to the manufacturer's protocol.

[0234] Spheroid formation assay

[0235] HCT116, DLD-1 and SW480 were seeded with 150 cells in 200 μL of spheroid medium per well on 96-well ultra-low attachment plates (Corning catalog number 3474). Compounds were added at a final concentration of 30 μM, 0.5% DMSO on the day of cell seeding. Cells were grown at 37°C, 5% CO2, and images of spheroids formed were taken on days 7 and 10 after cell seeding using the Operetta CLS system (Perkin Elmer).

[0236] Western blotting

[0237] HCT116 cells were treated with compounds at a final concentration of 50 μM, 1% DMSO for 18 hours. Cells were lysed on ice in RIPA buffer (Thermo catalog number 89900), which contained cOmpleteTM protease inhibitor cocktail (Roche catalog number 11697498001) and PhosSTOP phosphatase inhibitor cocktail (Roche catalog number 04906837001). 30 μg of total cell protein per sample was resolved by SDS-PAGE and blotted onto PVDF membrane (Millipore catalog number PVH00010). One pole antibody was incubated overnight at 4°C and blotted with IRDye 800CW goat anti-rabbit antibody (LI-COR catalog number 926-32211) or IRDye 680RD goat anti-mouse anti-rabbit (LI-COR catalog number 926-68070) at a dilution of 1:5000. Signals were visualized using the LI-COR Odyssey CLx imaging system, and bands were quantified using LI-COR ImageStudio data analysis software. The primary antibodies used were Met (Cell Signalling Technology catalog number 8198, 1:1000), cyclin D1 (Cell Signalling Technology catalog number 2978, 1:1000), survivin (Santa Cruz catalog number sc-10811, 1:500), ECAD (Cell Signalling Technology catalog number 3195, 1:1000), TCF4 / TCF7L2 (Cell Signalling Technology catalog number 2569, 1:1000), β-cat (Cell Signalling Technology catalog number 8480, 1:1000), β-actin (Abcam catalog number ab8226, 1:3000).

[0238] Co-immunoprecipitation

[0239] HCT116 cells were treated with compounds at specified concentrations for 18 hours. Cells were lysed on ice in lysis buffer containing 20mM HEPES, pH 7.5, 137mM NaCl, 1.5mM MgCl2, 1mM EGTA, 1mM dithiothreitol (DTT), 1% Triton X-100, 10% glycerol, cOmplete protease inhibitor cocktail (Roche catalog number 11697498001) and 1mM Na3VO4. Each clarified protein lysate sample was incubated overnight at 4°C with 4 μg anti-β-cat antibody (Sigma catalog number C7207), followed by incubation of 50 μL Dynabeads Protein G (Invitrogen catalog number 10004D) slurry at 4°C for 1 hour. The beads were then washed three times with lysis buffer at 4°C, and the bound proteins were eluted by heating the beads at 95°C for 7 minutes with 30 μL (each sample) SDS sample buffer. Proteins were resolved by SDS-PAGE and blotted onto PVDF membranes (Millipore catalog number IPVH00010). Primary antibodies were incubated overnight at 4°C and blotted with IRDye800CW goat anti-rabbit antibody (LI-COR catalog number 926-32211) or IRDye 680RD goat anti-mouse antibody (LI-COR catalog number 926-68070) at a dilution of 1:5000. Signals were visualized using the 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 number ab15148, 1:1000), TCF4 / TCF7L2 (Cell Signalling Technology catalog number 2569, 1:1000), and β-cat (Cell Signalling Technology catalog number 8480, 1:1000).

[0240] Protein expression and purification

[0241] BL21 (DE3) cells carrying the pET-28 β-cat expression vector of His-tagged human β-cat (134 to 668) were cultured at 37°C until OD600 = 0.6 to 0.8. The cells were then induced with 500 μM isopropyl-β-D-thiogalactopyranoside (IPTG) at 23°C for 6 to 8 hours. The cells were lysed by sonication in a lysis buffer containing 20 mM Tris (pH 8.8), 250 mM NaCl, 2 mM DTT, 5% glycerol, 0.1% Triton X-100 and cOmplete protease inhibitor cocktail (EDTA-free, Roche catalog number 11873580001). The lysate was incubated with Ni-NTA agarose beads (Qiagen catalog number 30210) at 4°C for 1 hour, and then the beads were washed with a lysis buffer containing 20 mM imidazole. The bound protein was 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.

[0242] The GST-tagged TCF4 N-terminal domain was similarly expressed in BL21(DE3) cells, induced with IPTG for 2 to 4 hours at 23°C, and purified from glutathione agarose beads (Pierce catalog number 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.

[0243] Surface Plasmon Resonance (SPR) Studies

[0244] Binding to β-cat

[0245] Surface plasmon resonance experiments were performed on CM5 series S sensor chips (GE Healthcare catalog number BR-1005-30) using BIACORE T100 (GE Healthcare). His-tagged β-cat (60 kDa, purity >90% based on SDS-PAGE) was immobilized using amine coupling chemistry. The surfaces of flow cells 1 and 2 were activated using 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. The ligand was immobilized at a density of 8000 RU in PBS at a concentration of 1 μM and pH 6.0 on flow cell 2, and flow cell 1 was left blank as a 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 into two flow cells at concentrations of 125, 100, 80, 60, 40 and 20 μM at a flow rate of 30 μL / min and a temperature of 25°C. The complex was allowed to bind and dissociate for 120 and 600 seconds, respectively. The surface was regenerated by injecting 50 mM NaOH for 30 seconds. The RU response was collected and plotted against the tested GB1874 concentrations using GraphPad Prism 5. The dissociation constant K was obtained by nonlinear specific binding using the Hill slope curve fitting function. D .

[0246] Inhibition of β-cat-TCF4 interaction

[0247] GST-tagged TCF4 N-terminal domain (31 kDa, >90% purity based on SDS-PAGE) was immobilized on a CM5 series S sensor chip using amine coupling chemistry. The ligand was immobilized on flow cell 4 at a density of 400 RU at a concentration of 0.5 μM in 10 mM citrate buffer (pH 4.0), and flow cell 3 was left blank as a 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 minutes at room temperature. The mixture was injected into two flow cells at a flow rate of 30 μL / min and a temperature of 25°C. The complex was allowed to bind and dissociate for 60 seconds respectively, and the surface was regenerated by injecting 50 mM NaOH for 30 seconds. The RU response was collected and plotted against the tested GB1874 concentrations using GraphPad Prism 5. The inhibition IC of compound GB1874 was obtained by four-parameter nonlinear regression. 50 value.

[0248] Mouse xenografts and compound treatment

[0249] HCT116 cells (1×106 cells per site) were implanted subcutaneously into the right flank of 5- to 7-week-old NSG (NOD scid gamma) mice (Jackson Laboratory, stock number 005557). When the tumor volume reached 60 to 100 mm 3 At , the animals were randomly divided into a treatment group or a control group. The mice in the treatment group were administered compound GB1874 at 50 mg / kg by intraperitoneal injection every other day. At the same time, the animals in the control group were administered with a diluent without the compound. Compound GB1874 was prepared by dissolving it in DMSO to a concentration of 60 mg / mL and diluting it in saline with 5% PEG 300 and 5% Tween-80 to a final concentration of 3 mg / mL, 5% DMSO. The length and width of the tumor were measured with a vernier caliper every 2 days. Tumor volume was estimated using the following modified ellipsoid formula: Tumor volume = 1 / 2 (length × width 2 ). When the tumor in the control group reached 2000mm 3 When , the mice were euthanized.

[0250] result

[0251] Generation of β-cat structural model

[0252] The final structural model of β-cat used for prospective ligand prediction was derived from three β-cat crystal structures, including β-cat-Tcf3, β-cat-TCF4, and β-cat-BCL9-TCF4 complexes (PDB codes: 1G3J, 18. 1JPW 19. and 2GL7 20. ), after 2 phases: 1) optimization (training) and 2) blind testing (validation) based on ligand enrichment measured by EF1 and logAUC. Optimization was done by an in-house automated modeling and docking platform ( Figure 1 ). First, MetaPocket 21. A putative ligand binding site was predicted in the β-cat crystal structure (PDB code: 1JPW), which overlaps with the charged groove generated by the β-cat armadillo repeats 4 to 9, which in turn overlaps with the β-cat-TCF4 interaction interface. Therefore, this site is the focus of the current study. 22.The three crystal structures of β-cat were sampled for side chains and three additional structures were introduced. This set of 6 β-cat structures was used as the starting point for training. Third, to more extensively explore the β-cat conformational space, the training library (153 compounds) was docked with all 6 β-cat structures, and the docking performance in terms of ligand enrichment was evaluated and PLOP (Protein Local Optimization Program) was used in the presence of each docking ligand (iCRT3, iCRT5 and iCRT14). 23. Optimize binding site side chains. Repeat this step until some optimized structures show ligand enrichment performance better than an arbitrary threshold: logAUC>=40 and EF1>=30.

[0253] Prediction of small molecule inhibitors of β-cat-TCF4 interaction

[0254] The best β-cat structure in the training phase showed an overall ligand enrichment logAUC of 44.2 and an early ligand enrichment EF1 of 33.3 in the training library. iCRT3, the most potent known inhibitor, ranked best among the 153 compounds in the training library. The interactions between β-cat and iCRT3 in the complex structure generated by docking were analyzed. The oxazolyl group in iCRT3 formed a hydrogen bond with R469, a cation-π interaction with K508, and a salt bridge with E17 of TCF4 in the β-cat-TCF4 crystal structure ( Figure 2 D). At the same time, the phenyl group in iCRT3 fits into the pocket outlined by R386, N426, and P463, which have been shown to interact with I19 and F21 of TCF4. In addition, the amide group of iCRT3 connecting the oxazolyl and phenyl groups is stabilized by another hydrogen bond with E462 in β-cat, and the ethylphenyl group of iCRT3 fills the pocket outlined by K508, V511, and R515.

[0255] With the help of a validation library of 63 iCRT3 analogs, this most abundant β-cat structure was blindly tested ( Figure 5 ). Taking into account both the docking grade determined by DOCK energy scoring and the docking pose determined by manual intervention, 19 ligand candidates were selected from the virtual screening ( Figure 5 ). Meanwhile, all 63 compounds were tested in a Wnt reporter assay in which HEK 293 cells were stably transfected with a single concentration of the TOPFlash reporter (STF cells), and 12 compounds showed activity (data not shown). These 12 compounds were further tested against STF cells over a range of concentrations, and five compounds had IC 50 The values ​​were comparable or lower than those of the known inhibitor iCRT3 ( Figure 5). In fact, all five of these strong inhibitors were prioritized as top ligand candidates by docking, indicating that the most enriched structures generated by training can predict new ligands.

[0256] After functional validation of novel ligand predictions, a library of 10,240 small molecules (purchased from Enamine) was computationally screened against the most abundant β-cat structures. The 500 highest-scoring hit compounds (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 β-cat residues, such as hydrogen bonds, and (3) chemical novelty of their scaffolds.

[0257] Functional validation of predicted inhibitors using TOPFlash / Wnt-reporter assays

[0258] The 27 compounds identified by the docking study were first tested at 10 μM for their ability to inhibit Wnt signaling in STF reporter cells ( Figure 2 From this screen, three compounds were identified that inhibited Wnt signaling by more than 50% on average, while being less than 25% toxic to cells, compared to DMSO controls ( Figure 7 A) Dose response studies of 3 lead compounds on STF reports showed that they have low micromolar IC 50 Inhibits Wnt signaling pathway ( Figure 2 B) Although they also affect the viability of reporter cells, cell viability EC 50 Values ​​are reporting cell IC 50 At least 4 times the value ( Figure 7 B). Encouragingly, these three hit compounds are approximately 2 to 5 times more potent inhibitors of the Wnt pathway compared to our previously reported compound iCRT3. Interestingly, these three hit compounds have different chemical structures ( Figure 2 C).

[0259] Specificity of candidate inhibitors

[0260] To further understand the mechanism of action of candidate lead compounds, the effects of these small molecules on the activities of other signaling pathways were studied using reporter cell lines. The initial screening and dose-response studies were conducted in the STF reporter cell line, in which Wnt signaling must be activated by adding exogenous Wnt3A. The effects on the STF3A reporter cell line (STF reporter cell line with endogenous expression of Wnt3A) were also studied. Therefore, Wnt signaling is constitutively active in the STF3A reporter cell line. Based on the IC50 values ​​obtained, both iCRT3 and compound GB1874 were identified as effective inhibitors of the Wnt pathway regardless of the initial activation state (Table 1). However, although compounds GB6853 and GB8679 can effectively inhibit the activation of the Wnt pathway (STFIC 50 values ​​were 4.8 μM and 7.0 μM, respectively), but when the Wnt pathway was already activated (STF3A IC 50 This may be due to the different abilities of these compounds to disrupt pre-existing (e.g. STF3A cells) versus newly formed (e.g. STF cells) β-cat-TCF4 complexes.

[0261] We also investigated the effects of the compounds on a Myc reporter cell line, in which the expression of the luciferase gene is controlled by a Myc response element. Since one of the Wnt target genes is Myc, inhibition of Wnt signaling will also result in inhibition of Myc signaling. The potency of these compounds against the Myc reporter was similar to that of the STF3A reporter, in which potent inhibitors of the Wnt pathway were also potent inhibitors of the Myc reporter (Table 1).

[0262] Finally, candidate hit compounds were tested against the Hippo pathway reporter, where the TEAD response element drives the expression of luciferase. Since activation of the Hippo pathway leads to reduced TEAD transcriptional activation, compounds that reduce the reporter signal can activate the Hippo pathway, and vice versa. Interestingly, iCRT3 was found to activate the Hippo pathway, and compound GB8679 was a potent inhibitor of the Hippo pathway (Table 1). Since the Wnt pathway can crosstalk with the Hippo pathway, 24. Therefore, it is suspected that the inhibition of Hippo signaling by GB8679 may indirectly affect its effectiveness in reducing the expression of Wnt target genes.

[0263] In silico prediction of different binding modes for three candidate small molecule binders of β-cat

[0264] The predicted binding modes of two hit compounds GB8679 and GB6853 in β-cat are similar to that of iCRT3. For example, the two compounds occupy the two pockets arranged by R386, N426, P463 and K508, V511, R515 respectively with their large number of hydrophobic groups. At the same time, the cleft between K508 and R469 is also filled by the thiazole groups in the two compounds, forming hydrogen bonds and salt bridges ( Figure 2 D). However, the third hit compound GB1874 shows a different binding mode from iCRT3 and the other two hit compounds. It still fills the cleft between K508 and R469 with its triazole group and the two pockets arranged by R386, N426, P463 and K508, V511, R515 with its bulky hydrophobic groups. In addition, the ethylphenyl group in GB1874 fits in the pocket arranged by H470, R474 and K435. Strikingly, this pocket in the β-cat-TCF4 crystal complex structure is occupied by the TCF4 D16 residue (forming a salt bridge with K435 in β-cat) and represents a key β-cat-TCF4 interaction. 25. Single amino acid substitutions at D16 on TCF4 or K435 on β-cat were sufficient to disrupt their interaction and significantly reduce downstream transcriptional activity. 25,26. Taken together, it can be hypothesized that this unique binding mode of GB1874 allows it to more effectively disrupt the β-cat-TCF4 interaction compared to iCRT3 and the other two hit compounds.

[0265] Hit compounds disrupt β-cat-TCF4 interaction and reduce expression of Wnt target genes

[0266] Since these compounds were identified through electronic docking studies on β-cat protein, the ability of other compounds to disrupt β-cat-TCF4 interaction in biologically relevant cell lines was investigated. Therefore, co-immunoprecipitation (co-IP) of β-cat and its interacting partners was performed in HCT116 CRC cells. Western blot analysis of proteins bound to β-cat under compound treatment showed that among the 3 hit compounds, only compound GB1874 was able to reduce β-cat-TCF4 interaction to the same extent as iCRT3 ( Figure 3 A). Encouragingly, although compound GB1874 was observed to affect the β-cat-TCF4 interaction, it had minimal effect on the β-cat-ECAD interaction ( Figure 3 A).

[0267] Subsequently, the effects of the hit compounds on the expression of Wnt target genes were investigated. HCT116 cells were treated with 50 μM of the 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 3 B). In fact, the effect of compound GB1874 on reducing the expression of Wnt target proteins is comparable to that of iCRT3. In addition, although compound GB1874 reduces the expression of Wnt target genes, it has little effect on the expression of β-cat itself or its partner proteins, such as ECAD and TCF4 ( Figure 3 C) These results suggest that GB1874 can regulate the nuclear transcriptional function of β-cat by specifically disrupting the β-cat-TCF4 interaction without affecting its membrane function at E-cad-mediated adherens junctions.

[0268] On the other hand, compounds GB6853 and GB8679 had minimal effects on the expression of the studied Wnt target genes ( Figure 3 B). These compounds were also less effective than compound GB1874 in disrupting the β-cat-TCF4 interaction ( Figure 3 A). These results, together with their lack of inhibitory activity in the STF3A reporter cell line (Table 1), suggest that they are unable to inhibit the Wnt pathway in cells where Wnt signaling is already active.

[0269] Compound GB1874 disrupts β-cat-TCF4 interaction in vitro

[0270] Since the GB1874 compound was predicted to bind to β-cat by electronic docking studies, experiments were performed to determine whether it could interact with purified β-cat in vitro and inhibit its interaction with TCF4. Surface plasmon resonance (SPR) studies were performed using purified proteins encoding β-cat (ARM-domain repeat region) and the N-terminal domain of TCF4, which is known to interact with β-cat. The purified β-cat protein was first immobilized on an SPR sensor chip, and different concentrations of the compound GB1874 were injected onto the β-cat. According to the sensor map ( Figure 8 A), a dose-dependent binding of compound GB1874 to β-cat was observed. The plot of steady-state response value versus compound GB1874 concentration indicates that the compound binds to β-cat, K D 76±13μM (n=3, Figure 3 D) However, the Hill slope of the best fit curve is greater than 1, indicating that compound GB1874 binds to multiple sites on β-cat with positive cooperativity.

[0271] To determine whether compound GB1874 can directly inhibit the interaction between β-cat and TCF4 in vitro, an SPR competition experiment was performed. The TCF4 N-terminal domain was immobilized on the SPR sensor chip instead of β-cat. Then 50nM β-cat pre-incubated with different concentrations of compound GB1874 was injected into the TCF4 N-terminal domain. From the sensorgram ( Figure 8 B) It can be clearly seen that compound GB1874 was found to inhibit the interaction between β-cat and TCF4, IC 50 The value was 25±8μM (n=2) ( Figure 3 E).

[0272] Candidate hit compounds affect the proliferation and stemness of "Wnt-addicted" cancer cells

[0273] In addition to disrupting the β-cat-TCF4 interaction and reducing the expression of Wnt target genes, experiments were performed to determine whether the hit compounds would induce phenotypes suggestive of reduced Wnt activity in biologically relevant cancer cell lines. First, the effects of candidate compounds GB6853, GB8679, and GB1874 on HCT116 colorectal cancer (CRC) cells were investigated. HCT116 cells have a heterozygous mutation in the β-cat gene, resulting in the loss of the Ser45 residue in the protein. Ser45 is the site of CK1α-triggered phosphorylation and is a key member of the destruction complex, which subsequently targets β-cat for proteasomal degradation. Therefore, the Ser45 mutation results in constitutive activation of Wnt signaling because the mutated β-cat cannot be degraded. It was hypothesized that inhibiting Wnt signaling downstream of β-cat in HCT116 cells would have a greater effect on their growth.

[0274] HCT116 cells were treated daily with 10 μM of each compound and their growth was monitored using the CellTiter-Glo viability assay. For comparison, compounds targeting different components along the Wnt signaling cascade were included, such as IWP-2, XAV939, and iCRT3. 27. like Figure 4As shown in A, treatment with any of the three hit compounds (GB1874, GB6853, and GB8679) effectively inhibited the growth of HCT116 cells. Notably, they were all more effective than iCRT3 in the growth inhibition assay. Compounds IWP-2 and XAV939 (porcupine and tankyrase inhibitors, respectively) inhibit Wnt signaling upstream of the β-cat-TCF4 interaction and are less effective in inhibiting the growth of HCT116 cells. Compounds GB8679, GB6853, and GB1874 also significantly reduced the survival of cancer stem-like cells in the HCT116 population at 30 μM, which can be seen by their effects on colony formation efficiency ( Figure 4 B) and spheroid formation assay ( Figure 4 In addition, the hit compound also affected the sphere formation of APC mutant CRC cell lines DLD-1 and SW480 ( Fig. 9 A and 9B), although to varying degrees. The effects of these compounds on CRC cell viability were then investigated. ECs against HCT116, DLD-1, and SW480 cell lines 50 The values ​​(Table 2) showed that compound GB1874 was the most effective against CRC cell lines, followed by compound GB8679.

[0275] GB1874 inhibits the growth of mouse xenografts

[0276] Based on the above studies, compound GB1874 was identified as the most effective compound against Wnt-driven CRC cells. To study its in vivo efficacy, HCT116 cells were inoculated into the flanks of NSG mice. Subsequently, tumor-bearing mice were treated with solvent control or compound GB1874 (50 mg / kg) by intraperitoneal injection every other day (qad). Compound GB1874 effectively inhibited the growth of HCT116 xenografts in vivo ( Figure 4 D), while producing minimal systemic toxicity in mice ( Fig. 9 C).

[0277] Table 1. Effects of lead compounds on different signaling pathways

[0278]

[0279]

[0280] Table 2. Effects of hit compounds on Wnt-driven CRC cell viability

[0281]

[0282] Using the structure of GB1874 as a scaffold, chemical databases were searched for analogs of GB1874. These compounds were then linked to our electronic model of β-catenin to predict strong candidate binders of β-catenin. Eight structural analogs of GB1874 were identified and tested.

[0283] We first tested the ability of these compounds to inhibit Wnt reporter activity using STF and STF3A reporter cells (Table 3). In general, the inhibition IC50 values ​​for STF cells were lower compared to the corresponding values ​​for STF3A cells, indicating that these compounds are less active against biological systems with constitutive Wnt activation. The exception was GB1874A, where it was more potent against STF3A cells compared to STF cells.

[0284] Compare GB1874(STF IC 50 =6.8μM, STF3A IC 50 =27μM) and GB1874F (STF IC 50 =5.4μM, STF3A IC 50 =10μM)、GB1874G(STF IC 50 =3.9μM, STF3A IC 50 =6.7μM) and GB1874H (STF IC 50 =5.0μM, STF3A IC 50 =11μM) 50 values, it was observed that increasing the size of the C3 substituent of the 1,2,4-triazole did not affect the activity of the compounds reported for STF or STF3A. Since the new analogs of GB1874 differ mostly at the C3 substituent of the 1,2,4-triazole, the effects of changing the substituents at other positions cannot be inferred. Therefore, in order to better understand the structure-activity relationship of GB1874, more analogs of GB1874 need to be purchased or synthesized.

[0285] Table 3. Structural analogs of GB1874 and their activity against STF and STF3A reporter cell lines

[0286]

[0287]

[0288] The effects of GB1874 analogs on the expression of Wnt target genes in HCT116 CRC cells were examined. Figure 5) As can be seen, the extent of downregulation of Wnt target genes correlates with the potency of the compounds in inhibiting STF reporters. The most potent compounds were iCRT3, GB1874, GB1874C, GB1874E, GB1874F, GB1874G, and GB1874H. These compounds effectively inhibited Wnt target genes such as AXIN2, BIRC5, BMP4, and CCND1, while having less effect on β-catenin (CTNNB1) gene expression.

[0289] We also tested the top hit compound GB1874 on primary patient-derived colorectal cancer (CRC) cell lines (Table 4). GB1874 was found to be more potent against primary CRC cell lines compared to the Tankyrase inhibitor XAV939.

[0290] Table 4. EC50 values ​​of GB1874 and XAV939 against primary patient-derived colorectal cancer cell lines

[0291]

[0292] HCT116 xenograft tumors obtained from in vivo studies were analyzed. Figure 4 D and again Fig.11 As shown in A, treatment of HCT116 xenografts with GB1874 at 50 mg / kg ip every other day (qad) inhibited the growth of HCT116 xenografts. By immunohistochemical staining and quantification, it was found that the inhibition of tumor growth was associated with reduced expression of Wnt target genes, cyclin D1, and proliferation marker Ki67 ( Fig.11 B).

[0293] Industrial Applicability

[0294] The compounds disclosed herein are useful for inhibiting β-cat or disrupting the interaction between β-cat and TCF4 and are therefore useful for treating a disease or health condition selected from the group consisting of cancer, neurodegenerative diseases, metabolic diseases, cardiovascular diseases, fibrosis and bone disease.

[0295] Obviously, various other modifications and adaptations of the present invention which do not depart from the spirit and scope of the present invention will become apparent to those skilled in the art after reading the foregoing disclosure, and all such modifications and adaptations are within the scope of the appended claims.

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Claims

1. Use of a compound in the manufacture of a medicament for treating colorectal cancer, wherein the compound is selected from the group consisting of: and or a pharmaceutically acceptable salt thereof.

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