Ubiquitin ligase inhibitors for cancer treatment

Abstract

The present invention relates to a new class of compounds and compositions comprising the same and their use as drugs for treating cancer.

Description

Technical Field

[0001] The present invention relates to a new class of compounds and compositions comprising the same and their use as drugs for treating cancer. Background Art

[0002] Carcinoma is the most common type of cancer and originates from epithelial cells. The transition from adenoma to carcinoma is associated with the loss of E-cadherin and, therefore, with the disruption of cell-cell contacts. E-cadherin is a tumor suppressor that is downregulated during the epithelial-to-mesenchymal transition (EMT); in fact, the loss of E-cadherin is a predictor of poor prognosis. Hakai is an E3 ubiquitin ligase protein that mediates E-cadherin ubiquitination, endocytosis and eventual degradation, leading to alterations in cell-cell contacts. Although E-cadherin is the most established substrate of Hakai activity, other regulated molecular targets of Hakai may also be involved in cancer cell plasticity during tumor progression. In other work, the authors of the present invention have employed the iTRAQ approach to explore new molecular pathways involved in Hakai-driven EMT during tumor progression. Their results show that Hakai can regulate cytoskeleton-related proteins, extracellular matrix metalloproteinases, and mitochondria. It has an important impact on secretory proteins, RNA-related proteins, and proteins involved in metabolism. In addition, it is emphasized that during the EMT driven by Hakai, the expression of several proteasome subunits decreased significantly. Since proteasome inhibitors are increasingly used in cancer treatment, these findings suggest that E3 ubiquitin ligases (such as Hakai) may be better than proteasomes for using new specific inhibitors for tumor subtypes undergoing EMT (such as cancer, tumors with mesenchymal phenotypes, or tumors in which Hakai expression is detected relative to normal tissue enhancement). However, to date, compounds that can effectively inhibit Hakai-mediated ubiquitination and are particularly suitable as therapeutic tools for treating cancer have not been disclosed.

[0003] The present invention provides such compounds, including enantiomers and pharmaceutically acceptable salts thereof, which selectively and effectively inhibit Hakai-mediated ubiquitination, preferably without affecting Hakai protein levels, while representing excellent anticancer drugs useful for treating various cancers (such as carcinoma). BRIEF DESCRIPTION OF THE DRAWINGS

[0004] Figure 1Computer simulation and in vitro screening of inhibitors of the E3 ubiquitin ligase Hakai. (A) Chemical structures of Hakin-1 and Hakin-5. (B) Predicted binding poses of Hakin-1 (left panel) and Hakin-5 (right panel) molecules docked within the Hakai dimer, as determined by the CRDOCK docking program. (C) In vivo Hakai-dependent ubiquitination assay in 293T cells transfected with Flag-Hakai, v-Src, and HA-ubiquitin in the presence of DMSO or compound Hakin-1. (D) In ​​vivo Hakai-dependent ubiquitination assay in 293T cells transfected with Flag-Hakai, v-Src, and HA-ubiquitin in the presence of DMSO or compound Hakin-5. (E) In vivo ubiquitination assay in 293T cells transfected with v-Src and HA-ubiquitin in the presence of DMSO or compound Hakin-1. (F) Effect of Hakin-1 on Hakai-dependent ubiquitination of the E-cadherin complex. pcDNA-Flag-Hakai, pcDNA-myc-E-cadherin, pSG-v-Src, and pBSSR-HA-ubiquitin were transiently transfected into 293T cells. Immunoprecipitation was performed using anti-E-cadherin antibody before western blotting with the indicated antibodies.

[0005] Figure 2 .Hakin-1 induces cytotoxicity and epithelial phenotype of epithelial tumor cell lines. (A) HT29 and LoVo cells were treated with increasing concentrations of Hakin-1 or Hakin-5, and cell viability was measured by MTT assay. The assay was performed with 6 replicates and expressed as mean ± SD of three independent experiments. (B) Cell viability of MDCK, Hakai-MDCK cell lines (clone 4 and clone 11) was measured using Hakin-1 (upper panel) or Hakin-5 (lower panel) as shown in (A). (CD) Phase contrast images of HT29 and LoVo cell lines (C) and MDCK, Hakai-MDCK cell lines (clone 4 and clone 11) (D) under treatment with Hakin-1 or Hakin-5. Images were acquired using a 20x objective.

[0006] Figure 3.Hakin-1 induces mesenchymal-epithelial transition in epithelial tumor cell lines. (A) Western blot analysis of EMT markers after treatment with Hakin-1 in HT-29 cell line (left figure), and quantification by densitometry (right figure) is shown. (B) Western blot analysis of EMT markers after treatment with Hakin-1 in LoVo cells (left figure), and quantification by densitometry (right figure) is shown. GAPDH is used as a loading control. Quantification is performed as shown in Materials and Methods, and Western blot data represent three experiments. Data show the average of three independent experiments and are expressed as mean ± standard deviation (*p < 0.05; **p < 0.01; ***p < 0.001). (C) Immunofluorescence of E-cadherin in HT-29 and LoVo cell lines after 48h of DMSO presence or Hakin-1 treatment. Images were obtained with a 40x objective. Quantification was performed using Image J program, and the results are presented as mean ± SD of three independent experiments (**p < 0.01; ***p < 0.001). Scale bar represents 250 μM for HT29 cells and 175 μM for LoVo cells.

[0007] Figure 4 .Antiproliferative and anticancer effects of Hakin-1 in tumor epithelial cells. (A) HT29 cells and LoVo cells were treated with Hakin-1 for 48 h, and proliferation was measured by BrdU assay as shown in Materials and Methods. The results are expressed as the mean ± standard deviation of eight replicates, and the experiment was repeated three times (*p < 0.05; **p < 0.01; ***p < 0.001). (B) HT29 cells and LoVo cells were treated with Hakin-5 for 48 h, and proliferation was measured as shown in A. (C) MDCK cells and Hakai-MDCK cells were treated with increasing concentrations of Hakin-1 for 48 h, and proliferation was measured as shown in A. (D) Soft agar assay of HT29 (left) cell line and Hakai-MDCK (right) cell line. Colonies were grown for 28 days (HT29) or 21 days (Hakai-MDCK) and counted as shown in Materials and Methods. Quantification of colonies was performed in triplicate and presented as the mean ± SD of three independent experiments (**p < 0.01; ***p < 0.001).

[0008] Figure 5.Hakin-1 reduces cell invasion and cell migration of epithelial tumor cells. (A) Invasion assays in the LoVo cell line were performed as described in Materials and Methods. Cells were treated for 48 h in the presence of DMSO or Hakin-1 and then seeded into invasion chambers. Representative images were taken using a 20x objective (upper panel), and quantification of the invasive cells photographed was shown (lower panel). (B) Invasion assays were performed as shown in A using MDCK cells and Hakai-MDCK cells. (C) Migration assays in HT29 cells were analyzed within 48 h after treatment with DMS or Hakin-1. Cells were seeded in migration chambers as described in Materials and Methods. Representative images are shown (upper panel), and quantification of migrating cells is shown (lower panel). The results are expressed as the mean ± standard deviation of three independent experimental replicates (***p < 0.001).

[0009] Figure 6 .Hakin-1 inhibits tumor growth in xenograft mice. (A) Effect of Hakin-1 on tumor growth in nude mice inoculated with Hakai-MDCK cells in the flank (n=6 tumors). Tumor growth curves are shown in the upper figure. Error bars represent mean ± SEM (*p<0.05). A schematic diagram of the experimental design is shown in the figure below. (B) At the endpoint, H&E staining was performed on Hakai-MDCK tumors treated with DMSO (left figure) or Hakin-1 (right figure). Images were obtained with a 20x objective. The scale bar is 300μM. (C) H&E staining shows tumor cell infiltration of blood vessels. Images were obtained with a 20x objective. The scale bar is 500μM. (D) Immunohistochemistry of the Ki67 marker in Hakai-MDCK tumors treated with DMSO (left figure) or Hakin-1 (right figure). Representative images were obtained with a 40x objective. The scale bar is 500μM. Quantification of the percentage of positive cells is shown in the figure below. (E) shows representative images of Hakai-MDCK tumors in nude mice stained with H&E. The picture (upper figure) was obtained with a 40x objective. The quantification of the number of mitoses in the high-power field of view is shown (lower figure). The scale bar is 500 μM. (F) Immunohistochemistry of CD31 markers in Hakai-MDCK tumors of nude mice injected with DMSO (left figure) or Hakin-1 (right figure). The image was obtained with a 20x objective. The scale bar is 500 μM. The quantification of the number in the field of view is shown in the figure below and is expressed as mean ± SEM (***p < 0.001).

[0010] Figure 7.Hakin-1 treatment reduces mesenchymal markers and micrometastasis formation in tumor xenografts in the lungs of nude mice. (AC) Immunohistochemical staining of Hakai (A), E-cadherin (B) and N-cadherin (C). Representative images were obtained using a 20x objective (upper figure). The lower figure shows the quantification of significant protein expression intensity. (D) Immunohistochemical staining and protein expression quantification of cortical actin (Cortactin) antibody are shown (upper and lower figures, respectively). Images were obtained with a 40x objective. (E) H&E staining of mouse lungs. Representative images were obtained with a 10x objective. (F) Real-time quantitative PCR was performed using primers for the HA epitope and Hakai to detect the presence of DNA of Hakai-MDCK cells entering the mouse lungs. Results are expressed as mean ± SEM (***p < 0.001). Scale bar is 500 μM.

[0011] Figure 8 .Hakin-5 does not affect EMT marker expression. (A) Western blot of E-cadherin, cortical actin, and Hakai in HT29 cells after 48 h of Hakin-5 treatment. (B) Immunofluorescence of E-cadherin in HT29 cells treated with Hakin-5 for 48 h. Images were taken with a 40x objective. Scale bar is 250 μM.

[0012] Fig. 9 .The role of Hakin-1 in human cancer cells. Breast cancer MCF7 cells, prostate cancer PC-3 cells, bladder cancer 5637 cells, kidney cancer ACHN cells and liver cancer HepG2 cells were treated with Hakin-1 for 48 h, and proliferation was measured by BrdU assay as indicated in Materials and Methods. The results are expressed as the mean ± standard deviation of eight replicates, and the experiment was repeated three times (*p < 0.05; **p < 0.01; ***p < 0.001).

[0013] Fig.10 .Hakin-1 does not affect apoptosis in vivo in a xenograft mouse model. Tunel assays were performed as described in Materials and Methods. Representative images are shown (left), and quantification of the number of positive cells (right) is also presented as mean ± SEM. Images were taken with a 20x objective. Scale bar is 125 μM.

[0014] Fig.11 .Intact cellular morphology and tissue architecture of liver and kidney in vivo in a xenograft mouse model treated with Hakin-1. H&E staining of liver (upper panel) and kidney (lower panel) of nude mice treated with 5 mg / kg of DMSO or Hakin-1. Images were taken with a 10x objective. Scale bar 500 μM.

[0015] Fig.12Effect of selected analogs on the cytotoxicity of the HT29 epithelial tumor cell line. Cells were treated with an increasing concentration range (50 μM, 100 μM, 250 μM and 500 μM) of the following substances: (A) ketophenyls: A-1, A-7, A-8 and A-9; (B) ketoheteroaryls: A-23 and A-25; (C) cyclic amides: A-10 and A-16; and (D) benzamides: A-6.1. Cell viability was measured by MTT assay. The assay was performed in 6 replicates and is presented as the mean ± SD of two independent experiments.

[0016] Fig.13 Effect of analog inhibitors on Hakai-dependent ubiquitination. In vivo Hakai-dependent ubiquitination assay in 293T cells transfected with Flag-Hakai, v-Src, and HA-ubiquitin in the presence of DMSO or selected analogs. (A) Ketophenyls: A-7 and A-9; (B) Ketophenyls: A-9, and cyclic amides: A-10 and A-16; and (C) Ketoheteroaryls: A-23.

[0017] Fig.14 .Compound 1 (Hakin-1), chemical structure, physicochemical parameters and binding mode (docking).

[0018] Fig.15 .Total XIC of all metabolites found in mouse samples at 0 min.

[0019] Fig.16 .Total XIC of all metabolites found in the 10-min mouse samples.

[0020] Fig.17 .Total XIC of all metabolites found in the 20-min mouse samples.

[0021] Fig.18 . Analytical data for metabolite analysis of the parent compound Hakin-1.

[0022] Fig.19 . Analytical data of metabolite analysis of the reduced metabolite MKH1.

[0023] Fig. 20 .Synthesis route of MKH1. The synthesis was carried out according to the given scheme.

[0024] Fig.21 .Structural summary of K1, K2, K8 and K33.

[0025] Fig. 22. Immunofluorescence images and quantification of E-cadherin expression at cell-cell contacts in human colon cancer cells treated with K1 and K2 compounds. The colorectal adenocarcinoma cell line HT-29 was exposed to 100 μM or 20 μM of the indicated compounds or DMSO vehicle control for 48 h. (A) Representative images of E-cadherin expression at cell-cell junctions were taken. Scale bar: 100 μm. (B) Quantification of E-cadherin expression at cell-cell contacts. Each mean is the mean of 12, 6, or 3 wells, ns, not significant; *P<0.05; **P<0.01; ***P<0.001; P<0.0001 indicates a difference from control, which was determined by analysis of variance followed by Dunnett's procedure or unpaired t-test.

[0026] Fig.23 . Immunofluorescence images and quantification of E-cadherin expression at cell-to-cell contacts in human colon cancer cells treated with nocodazol. (A) Immunofluorescence images of E-cadherin in HT-29 cell lines treated with nocodazole and DMSO vehicle control at the indicated concentrations and times. Scale bar: 100 μm. (B) Quantification of E-cadherin expression at cell-to-cell contacts in HT-29 cell lines treated with nocodazole and DMSO vehicle control at the indicated concentrations and times. Each mean is the mean of 12, 6, or 3 wells, ns, not significant; *P<0.05; **P<0.01; ***P<0.001; P<0.0001 indicates a difference from the control, which was determined by analysis of variance followed by Dunnett's procedure or unpaired t-test.

[0027] Fig.24 .Cell cycle analysis of human colon cancer cells exposed to indicated compounds or DMSO. CyteSeer software can measure relative DNA content by quantifying the total integrated intensity (TII) of the nuclear image (Ni) on the nuclear mask (Nm). HT-29 cells were treated with K1, K2 compounds or 0.1% DMSO vehicle control for 48 hours. Note: Ploidy histograms for 48 hours or DMSO vehicle control contain cell numbers from 12 wells, respectively.

[0028] Fig.25.Cell cycle analysis of human colon cancer cells exposed to nocodazole or DMSO. CyteSeer software can measure relative DNA content by quantifying the total integrated intensity (TII) of the nuclear image (Ni) on the nuclear mask (Nm). HT-29 cells were treated with nocodazole or DMSO as vehicle control for the indicated time and concentration for 24 hours. Note: Ploidy histograms for the 48 hour or 24 hour DMSO vehicle control contain cell numbers from 12 or 6 wells, respectively, and therefore have different y-axes compared to the ploidy histograms for the test compounds, which contain data from only 3 replicate wells.

[0029] Fig.26 .Immunofluorescence images of E-cadherin expression at cell-cell contacts in human colon cancer cells treated with MKH1. The colorectal adenocarcinoma cell line HT-29 was exposed to 5 μM, 20 μM, and 100 μM MKH1 or DMSO vehicle control for 48 hours, and representative images of E-cadherin expression at cell-cell junctions were taken (upper panels). Quantification was performed using the Image J program (lower panels), and the results are presented as mean ± SD of three independent experiments (**p < 0.01; ***p < 0.001).

[0030] Fig. 27 .Immunofluorescence images of E-cadherin expression at cell-to-cell contacts in human colon cancer cells treated with K8. The colorectal adenocarcinoma cell line HT-29 was exposed to 5 μM, 20 μM, 50 μM and 100 μM K8 or DMSO vehicle control for 48 hours, and representative images of E-cadherin expression at cell-to-cell junctions were taken (upper panel). Quantification was performed using the Image J program (lower panel), and the results are presented as mean ± SD of three independent experiments (**p < 0.01; ***p < 0.001), and K1 was used as a positive control.

[0031] Fig.28 .Immunofluorescence images of E-cadherin expression at cell-cell contacts in human colon cancer cells treated with K33. The colorectal adenocarcinoma cell line HT-29 was exposed to 20 μM and 100 μM K8 or DMSO vehicle control for 48 hours, and representative images of E-cadherin expression at cell-cell junctions were taken (upper panel). Quantification was performed using the Image J program (lower panel), and the results are presented as mean ± SEM of three independent experiments (**p<0.01; ***p<0.001), and K1 was used as a positive control.

[0032] Fig.29Effects of MKH1 and K8 on proliferation of human colon cancer cells. Colon cancer HCT116 cells were treated with the indicated compounds for 48 hours, and proliferation was measured by the BrdU assay as described in Materials and Methods. Results are presented as mean ± SD of eight replicates, and the experiment was repeated three times (*p<0.05; **p<0.01; ***p<0.001).

[0033] Fig.30 .Hakin-1, K1, MKH1 and K8 inhibitors reduce cell invasion of human epithelial tumor cells. As described in Materials and Methods, invasion assays were performed in the HT29 cell line. Before cells were seeded into the invasion chamber, a dose / response experiment was performed by treating cells for 48 hours in the presence of inhibitors Hakin-1, MKH1, K1 and K8 or DMSO as a control. The quantification of the invasive cells photographed is shown. The results are expressed as mean ± standard deviation of three independent experiments (*p<0.05; **p<0.01; ***p<0.001).

[0034] Fig.31 .Cytotoxicity analysis of K1, MKH1 and K8 at tested concentrations. (A) HCT116 colon cancer cells and non-cancerous HEK293T cells were treated with increasing concentrations of K1 (A), MKH1 (B) and K8 (C), and cell viability was measured by MTT assay as described in Materials and Methods. The assay was repeated 6 times and is presented as the mean ± SD of three independent experiments.

[0035] Fig.32 Effects of K1, MKH1 and K8 inhibitors on Hakai-induced ubiquitination In vivo Hakai-dependent ubiquitination assay in 293T cells transfected with Flag-Hakai and HA-ubiquitin in the presence of DMSO or compounds K1, MKH1 and K8. DETAILED DESCRIPTION

[0036] The present invention is based on our previous identification of 4-tetrazolylbenzoic acid Hakin-1, Hakin-2 and Hakin-6 (from compounds #1, 2 and 6 below) as Hakai inhibitors that can compete for the HYB binding site that is only present in the Hakai dimer. As discussed in the inventor's previous patent application (PCT / EP2019 / 081522), Hakai has been reported to be involved in tumor progression; therefore, inhibitors of the interaction between Hakai and E-cadherin may be useful for treating cancer. In this sense, as shown in the examples of PCT / EP2019 / 081522 (which are reproduced in full herein), inhibition of tumor progression was demonstrated in vitro and in vivo using compound #1.

[0037]

[0038] Therefore, they solved the technical problem of providing a compound with excellent anticancer effect and low toxicity in PCT / EP2019 / 081522. These compounds can be advantageously used as medicines, in particular for the treatment of a variety of cancers, such as cancer, in particular cancers of the gastrointestinal tract including the mouth (oral cancer), esophagus, stomach and small intestine and large intestine (such as rectal cancer or colon cancer). Also included are skin cancer, breast (breast cancer), pancreatic cancer, lung cancer, head and neck cancer, liver cancer, ovarian cancer, cervical cancer, uterine cancer, gallbladder cancer, penile cancer and urinary bladder cancer (such as kidney cancer, prostate cancer or bladder cancer). In fact, in general, as shown in the examples of PCT / EP2019 / 081522, the data show that Hakin-1 is a specific inhibitor of Hakai-mediated ubiquitination without affecting Hakai protein levels (Example 1). In addition, Hakin-1 can inhibit the proliferation of Hakai-MDCK cells, while no effect was detected on MDCK cells ( Figure 4 c). Hakin-1 also inhibits cell proliferation in other epithelial cell lines (such as breast cancer MCF7 cells, prostate cancer PC-3 cells, bladder cancer 5637 cells, liver cancer HepG2 cells, and kidney cancer ACHN cells). All these findings support that Hakin-1 exerts its anti-tumor effects by acting on cell proliferation, oncogenic potential, cell motility, and invasion. In addition, PCT / EP2019 / 081522 further showed that Hakin-1 treatment significantly inhibited tumor growth in nude mice without systemic toxicity ( Fig.11 ). In addition, Hakin-1 resulted in a significant reduction in micrometastases detected in the lungs of Hakai-MDCK xenograft mice compared with control DMSO-treated mice, whereas no micrometastases were detected in the lungs of non-transformed MDCK-injected mice ( Figure 7 f). This result emphasizes that Hakin-1 suppresses lung metastasis in vivo.

[0039] Compound #1 (also known as Hakin-1, as mentioned above) is a 1,5-disubstituted tetrazole, a chemically and metabolically stable pharmacophore fragment commonly used in drug development (Tetrazole Derivatives as Promising Anticancer Agents, EAPopova et al., Anticancer Agents Med Chem. 2017 Mar 27. doi: 10.2174 / 1871520617666170327143148, Epub ahead of print). The 1-position of the tetrazole ring is substituted with a 4-carboxyphenyl group. The 5-position of the tetrazole ring is connected to another benzene ring via a mercaptomethylcarbonyl linker.

[0040]

[0041] Important physicochemical parameters such as molecular weight, calculated lipophilicity logP, and polar surface area are within the range required for orally absorbed drugs that are unlikely to cross the blood-brain barrier. Based on the docking study snapshot of compound 1 at the Hakai HYB site, the carboxyphenyl moiety creates 3 important hydrogen bonding interactions with the protein and may mimic the binding mode of the phosphotyrosine substrate. Other hydrogen bonds are formed by two of the tetrazole nitrogen atoms, which provides additional stability and maintains the tetrazole-carboxyphenyl unit at both ends in a defined binding mode. Based on the above, in PCT / EP2019 / 081522, the following subunits are described as essential and are considered as the core structure for identifying derivatives of compound #1:

[0042]

[0043] Starting from this common structural core, many modifications can be made to optimize the activity (and other properties) of these compounds and to provide analogs of these compounds. In this sense, it is pointed out in PCT / EP2019 / 081522 that there are several opportunities to introduce modifications in different parts of the molecule, which represents an important goal for optimizing the process and providing analogs of compounds #1, #2 and #6. In this regard, the medicinal chemistry project will start to focus on the chemical space around compound #1, followed by the chemical space around compounds #2 and #6. With this in mind, they provide a set of different analogs of compounds #1, #2 and #6 that can be used for the invention described in PCT / EP2019 / 081522. It is worth noting that they can be used as drugs, in particular for the treatment of various cancers (such as cancers, in particular tumors arising from the epithelial layer of the gastrointestinal tract including the mouth (oral cancer), esophagus, stomach, and small and large intestines (such as rectal cancer or colon cancer)). Also included are cancers of the skin, breast (breast cancer), pancreas, lung, head and neck, liver, ovary, cervix, uterus, gallbladder, penis, and urinary bladder (such as kidney, prostate, or bladder).

[0044] To find those analogs that are similar to compounds #1 and #2, the core structure is linked to a ring structure (Cy = any ring) to provide a first class of analogs described as:

[0045]

[0046] These types of compounds in PCT / EP2019 / 081522 are grouped as follows according to the following structural subclasses a) to c):

[0047] a) Ketophenyls (including compound #1):

[0048]

[0049] It is important to note that these structures can contain substituents on the benzene ring or in another part of the molecule, for example, on the carboxyl benzene ring or on the carbon atom between the sulfur and the carbonyl group. An example of the latter is shown below:

[0050]

[0051] Furthermore, the following examples illustrate the introduction of modifications such as a cyclopropyl bridge, or the addition of substituents on a carboxyphenyl ring:

[0052]

[0053] Specific examples of ketophenyl compounds that can be used to implement the present invention are shown below:

[0054]

[0055] 4-(5-((2-(4-nitrophenyl)-2-oxoethyl)thio)-1H-tetrazol-1-yl)benzoic acid

[0056]

[0057] 4-(5-((2-(4-acetylphenyl)-2-oxoethyl)thio)-1H-tetrazol-1-yl)benzoic acid

[0058]

[0059] 4-(5-((2-(4-(methylsulfonyl)phenyl)-2-oxoethyl)thio)-1H-tetrazol-1-yl)benzoic acid

[0060]

[0061] 4-(2-((1-(4-carboxyphenyl)-1H-tetrazol-5-yl)thio)acetyl)benzoic acid

[0062]

[0063] 4-(5-((2-(4-carbamoylphenyl)-2-oxoethyl)thio)-1H-tetrazol-1-yl)benzoic acid

[0064]

[0065] 4-(5-((2-oxo-2-(4-sulfamoylphenyl)ethyl)thio)-1H-tetrazol-1-yl)benzoic acid

[0066]

[0067] 4-(5-((2-(4-(Methanesulfonamido)phenyl)-2-oxoethyl)thio)-1H-tetrazol-1-yl)benzoic acid

[0068]

[0069] 4-(5-((2-(4-(cyclopropanecarboxamido)phenyl)-2-oxoethyl)thio)-1H-tetrazol-1-yl)benzoic acid

[0070]

[0071] 4-(5-((2-(4-bromophenyl)-2-oxoethyl)thio)-1H-tetrazol-1-yl)benzoic acid

[0072]

[0073] 4-(5-((2-(4-cyanophenyl)-2-oxoethyl)thio)-1H-tetrazol-1-yl)benzoic acid

[0074]

[0075] 4-(5-((2-oxo-2-(4-trifluoromethyl)phenyl)ethyl)thio)-1H-tetrazol-1-yl)benzoic acid

[0076]

[0077] 4-(5-((2-(4-cyclopropylphenyl)-2-oxoethyl)thio)-1H-tetrazol-1-yl)benzoic acid

[0078]

[0079] 4-(5-((2-(4-(cyclopropylamino)phenyl)-2-oxoethyl)thio)-1H-tetrazol-1-yl)benzoic acid

[0080]

[0081] 4-(5-((2-(4-morpholinophenyl)-2-oxoethyl)thio)-1H-tetrazol-1-yl)benzoic acid

[0082]

[0083] 4-(5-((2-(4-(4-methylpiperazin-1-yl)phenyl)-2-oxoethyl)thio)-1H-tetrazol-1-yl)benzoic acid

[0084]

[0085] 4-(5-((2-(4-methoxyphenyl)-2-oxoethyl)thio)-1H-tetrazol-1-yl)benzoic acid

[0086]

[0087] 4-(5-((2-(4-(5-fluoropyridin-3-yl)phenyl)-2-oxoethyl)thio)-1H-tetrazol-1-yl)benzoic acid.

[0088] b) Ketoheteroaryls:

[0089] Other substructures provided by PCT / EP2019 / 081522, formed by linking a core structure to a ring structure (Cy = any ring), are classified as ketoheteroaryls. In this sense, a typical exploration in medicinal chemistry is to replace the phenyl group present in compound #1 with a heteroaryl group, which represents a similar aromatic ring with an additional heteroatom. PCT / EP2019 / 081522 provides 6 examples of analogs belonging to this category:

[0090]

[0091] Six-membered heteroaryl groups that are structurally closer to phenyl (such as pyridine, pyrimidine, pyridazine) as shown below also form part of PCT / EP2019 / 081522:

[0092]

[0093] In addition, specific examples of ketone heteroaryl compounds that can be used to implement the invention identified in PCT / EP2019 / 081522 are as follows:

[0094]

[0095] 4-(5-((2-oxo-2-(pyridin-4-yl)ethyl)thio)-1H-tetrazol-1-yl)benzoic acid

[0096]

[0097] 4-(5-((2-(6-aminopyridin-3-yl)-2-oxoethyl)thio)-1H-tetrazol-1-yl)benzoic acid

[0098]

[0099] 4-(5-((2-(6-hydroxypyridin-3-yl)-2-oxoethyl)thio)-1H-tetrazol-1-yl)benzoic acid

[0100]

[0101] 4-(5-((2-(6-morpholinopyridin-3-yl)-2-oxoethyl)thio)-1H-tetrazol-1-yl)benzoic acid

[0102]

[0103] 4-(5-((2-(5-cyanopyridin-2-yl)-2-oxoethyl)thio)-1H-tetrazol-1-yl)benzoic acid

[0104]

[0105] 4-(5-((2-oxo-2-(pyrimidin-4-yl)ethyl)thio)-1H-tetrazol-1-yl)benzoic acid

[0106]

[0107] 4-(5-((2-oxo-2-(pyridazin-4-yl)ethyl)thio)-1H-tetrazol-1-yl)benzoic acid

[0108]

[0109] 4-(5-((2-oxo-2-(pyrazin-2-yl)ethyl)thio)-1H-tetrazol-1-yl)benzoic acid.

[0110] c) Cyclic amides (including compound #2):

[0111] Still further substructures provided by PCT / EP2019 / 081522, formed by connecting a core structure to a ring structure (Cy = any ring), are listed as cyclic amides, as shown below:

[0112]

[0113] In particular, substituted indoline (and indole) analogs are shown below:

[0114]

[0115] More specifically, any compound herein substituted on the benzene ring is as follows:

[0116]

[0117] 4-(5-((2-(5-bromoindolin-1-yl)-2-oxoethyl)thio)-1H-tetrazol-1-yl)benzoic acid

[0118]

[0119] 4-(5-((2-(5-cyanoindolin-1-yl)-2-oxoethyl)thio)-1H-tetrazol-1-yl)benzoic acid

[0120]

[0121] 4-(5-((2-(5-cyclopropylindolin-1-yl)-2-oxoethyl)thio)-1H-tetrazol-1-yl)benzoic acid

[0122]

[0123] 4-(5-((2-(5-morpholinoindolin-1-yl)-2-oxoethyl)thio)-1H-tetrazol-1-yl)benzoic acid

[0124]

[0125] 4-(5-((2-oxo-2-(5-(pyridin-3-yl)indolin-1-yl)ethyl)thio)-1H-tetrazol-1-yl)benzoic acid

[0126]

[0127] 4-(5-((2-(5-fluoroindolin-1-yl)-2-oxoethyl)thio)-1H-tetrazol-1-yl)benzoic acid

[0128]

[0129] 1-(2-((1-(4-carboxyphenyl)-1H-tetrazol-5-yl)thio)acetyl)indoleline-5-carboxylic acid

[0130]

[0131] 4-(5-((2-(5-carbamoylindolin-1-yl)-2-oxoethyl)thio)-1H-tetrazol-1-yl)benzoic acid. Modifications in other parts of the molecule:

[0132]

[0133] 4-(5-((1-(5-cyanoindolin-1-carbonyl)cyclopropyl)thio)-1H-tetrazol-1-yl)benzoic acid

[0134]

[0135] 2-Fluoro-4-(5-((1-(5-morpholinoindolin-1-carbonyl)cyclopropyl)thio)-1H-tetrazol-1-yl)benzoic acid

[0136]

[0137] 4-(5-((1-(5-Carbamoylindoline-1-carbonyl)cyclopropyl)thio)-1H-tetrazol-1-yl)benzoic acid

[0138]

[0139] 3-Cyclopropyl-4-(5-((2-(5-fluoroindolin-1-yl)-2-oxoethyl)thio)-1H-tetrazol-1-yl)benzoic acid

[0140]

[0141] 3-Cyano-4-(5-((2-(5-cyclopropylindolin-1-yl)-2-oxoethyl)thio)-1H-tetrazol-1-yl)benzoic acid.

[0142] Furthermore, PCT / EP2019 / 081522 further teaches that the above-mentioned different substructures a) to c) provided by connecting the core structure to a ring structure (Cy = any ring) can be further substituted as follows:

[0143] 1. Examples of structures having substitutions on the mercaptoacetyl linker

[0144]

[0145] 4-(5-((2-methyl-1-(4-nitrophenyl)-1-oxopropan-2-yl)thio)-1H-tetrazol-1-yl)benzoic acid

[0146]

[0147] 4-(5-((1-(4-carbamoylphenyl)-2-methyl-1-oxopropan-2-yl)thio)-1H-tetrazol-1-yl)benzoic acid

[0148]

[0149] 4-(5-((2-methyl-1-(4-(methylsulfonamido)phenyl)-1-oxopropan-2-yl)thio)-1H-tetrazol-1-yl)benzoic acid

[0150]

[0151] 4-(5-((1-(4-cyanophenyl)-2-methyl-1-oxopropan-2-yl)thio)-1H-tetrazol-1-yl)benzoic acid

[0152]

[0153] 4-(5-((1-(4-nitrobenzoyl)cyclopropyl)thio)-1H-tetrazol-1-yl)benzoic acid

[0154]

[0155] 4-(5-((1-(4-carbamoylbenzoyl)cyclopropyl)thio)-1H-tetrazol-1-yl)benzoic acid

[0156]

[0157] 4-(5-((1-(4-methylsulfonamido)benzoyl)cyclopropyl)thio)-1H-tetrazol-1-yl)benzoic acid

[0158]

[0159] 4-(5-((1-(4-cyanobenzoyl)cyclopropyl)thio)-1H-tetrazol-1-yl)benzoic acid.

[0160] 2. Examples of structures having substitution on the carboxyphenyl moiety

[0161]

[0162] 2-Cyano-4-(5-((2-(4-nitrophenyl)-2-oxoethyl)thio)-1H-tetrazol-1-yl)benzoic acid

[0163]

[0164] 4-(5-((2-(4-carbamoylphenyl)-2-oxoethyl)thio)-1H-tetrazol-1-yl)-2-fluorobenzoic acid

[0165]

[0166] 2-Cyclopropyl-4-(5-((2-(4-(methylsulfonamido)phenyl)-2-oxoethyl)thio)-1H-tetrazol-1-yl)benzoic acid

[0167]

[0168] 4-(5-((2-(4-cyanophenyl)-2-oxoethyl)thio)-1H-tetrazol-1-yl)-3-fluorobenzoic acid

[0169]

[0170] 3-Cyano-4-(5-((2-(4-nitrophenyl)-2-oxoethyl)thio)-1H-tetrazol-1-yl)benzoic acid

[0171]

[0172] 3-Cyclopropyl-4-(5-((2-(4-(methylsulfonamido)phenyl)-2-oxoethyl)thio)-1H-tetrazol-1-yl)benzoic acid.

[0173] On the other hand, a completely new group or class of compounds derived from the expansion of the core structure with nitrogen atoms, carbon atoms and cyclic groups to provide compounds similar to compound #6 is provided in PCT / EP2019 / 081522 under the subclass benzylamides:

[0174]

[0175] Compounds belonging to this class are listed below in this article:

[0176]

[0177] Other compounds that belong to this class of compounds are:

[0178]

[0179] 4-(5-((2-((3-cyanobenzyl)amino)-2-oxoethyl)thio)-1H-tetrazol-1-yl)benzoic acid

[0180]

[0181] 4-(5-((2-((3-cyclopropylbenzyl)amino)-2-oxoethyl)thio)-1H-tetrazol-1-yl)benzoic acid

[0182]

[0183] 4-(5-((2-((3-morpholinobenzyl)amino)-2-oxoethyl)thio)-1H-tetrazol-1-yl)benzoic acid

[0184]

[0185] 4-(5-((2-oxo-2-((3-(pyridin-3-yl)benzyl)amino)ethyl)thio)-1H-tetrazol-1-yl)benzoic acid

[0186]

[0187] 4-(5-((2-((3-carbamoylbenzyl)amino)-2-oxoethyl)thio)-1H-tetrazol-1-yl)benzoic acid

[0188]

[0189] 4-(5-((2-((3-fluorobenzyl)amino)-2-oxoethyl)thio)-1H-tetrazol-1-yl)benzoic acid.

[0190] There are modifications in other parts of the molecule:

[0191]

[0192] 4-(5-((1-((3-cyanobenzyl)carbamoyl)cyclopropyl)thio)-1H-tetrazol-1-yl)benzoic acid

[0193]

[0194] 3-Cyano-4-(5-((2-((3-cyclopropylbenzyl)amino)-2-oxoethyl)thio)-1H-tetrazol-1-yl)benzoic acid

[0195]

[0196] 4-(5-((1-((3-morpholinobenzyl)carbamoyl)cyclopropyl)thio)-1H-tetrazol-1-yl)benzoic acid

[0197]

[0198] 2-Fluoro-4-(5-((2-oxo-2-((3-(pyridin-3-yl)benzyl)amino)ethyl)thio)-1H-tetrazol-1-yl)benzoic acid

[0199]

[0200] 4-(5-((2-((3-carbamoylbenzyl)amino)-2-oxoethyl)thio)-1H-tetrazol-1-yl)-3-cyclopropylbenzoic acid

[0201]

[0202] 4-(5-((1-((3-fluorobenzyl)amino)-2-methyl-1-oxopropan-2-yl)thio)-1H-tetrazol-1-yl)benzoic acid.

[0203] Heteroaryl groups are:

[0204]

[0205] 4-(5-((2-oxo-2-((pyridin-4-ylmethyl)amino)ethyl)thio)-1H-tetrazol-1-yl)benzoic acid

[0206]

[0207] 4-(5-((2-oxo-2-((pyrimidin-4-ylmethyl)amino)ethyl)thio)-1H-tetrazol-1-yl)benzoic acid

[0208]

[0209] 4-(5-((2-oxo-2-((pyridazin-4-ylmethyl)amino)ethyl)thio)-1H-tetrazol-1-yl)benzoic acid

[0210]

[0211] 4-(5-((2-(((5-cyanopyridin-2-yl)methyl)amino)-2-oxoethyl)thio)-1H-tetrazol-1-yl)benzoic acid.

[0212] It is pointed out herein that the following general formula covers all compounds shown in PCT / EP2019 / 081522, and any pharmaceutically acceptable salts thereof:

[0213]

[0214] in:

[0215] -A represents a group selected from aryl, heteroaryl and cyclic amide, wherein the aryl, heteroaryl and cyclic amide are optionally substituted by 1 or 2 groups independently selected from the following:

[0216] a) Halogen atoms, NO2, -CN, -N(R a )R b 、-OR a 、-C(=O)R a 、-C(=O)OR a 、-C(=O)N(R a )R b 、-OC(=O)-R a 、-N(R c )C(=O)R b 、-NR c S02R a 、-SO2N(R a )R b 、-SR a 、-S(O)R a 、-S(O)2R a ;

[0217] b) a linear or branched C1-C6 alkyl group optionally substituted by 1, 2 or 3 halogen atoms;

[0218] c) a C3-C6 cycloalkyl group optionally comprising 1 or 2 heteroatoms selected from O, S and N, and the ring is optionally substituted with a C1-C3 alkyl group;

[0219] d) phenyl or C5-C6 heteroaryl, each optionally substituted by a halogen atom, a cyano group, a C1-C3 alkyl group or a cyclopropyl group;

[0220] -R a , R b and R c Each independently represents:

[0221] a) Hydrogen atoms,

[0222] b) a linear or branched C1-C ... 12 Alkyl, C3-C6 cycloalkyl and C4-C6 heterocycloalkyl: carbonyl, halogen atom, hydroxyl, phenyl, C3-C6 cycloalkyl, linear or branched C1-C6 alkoxy, amino, alkylamino, dialkylamino, linear or branched C1-C6 alkylcarbonyl;

[0223] c) phenyl or C5-C6 heteroaryl optionally substituted by 1, 2 or 3 substituents selected from the group consisting of a halogen atom, a cyano group, a linear or branched C1-C6 alkyl group, a linear or branched C1-C6 haloalkyl group, a hydroxyl group, a linear or branched C1-C6 alkoxy group, an amino group, an alkylamino group, a dialkylamino group;

[0224] d) R a and R b Together with the nitrogen atom to which they are attached, they form a 3- to 8-membered ring, the ring optionally containing other heteroatoms selected from O, N and S, and the ring is optionally substituted with 1, 2 or 3 substituents selected from the following: carbonyl, straight chain or branched C1-C6 alkyl, straight chain or branched C1-C6 haloalkyl, straight chain or branched C1-C6 alkylcarbonyl;

[0225] - x and y are integers independently selected from 0 and 1;

[0226] -R 1 represents a group selected from hydrogen, cyclopropyl or linear or branched C1-C6 alkyl, wherein the alkyl is optionally substituted by 1, 2 or 3 halogen atoms;

[0227] -When y is 0, R 1 The two or three carbon atoms of the nitrogen atom may form a five-membered or six-membered ring together with the adjacent nitrogen atom and the two adjacent carbon atoms of the aromatic ring to which the nitrogen atom is attached;

[0228] -R 2 and R 3Each independently represents a group selected from hydrogen, cyclopropyl or linear or branched C1-C6 alkyl; optionally, R 2 and R 3 can form a 3- or 4-membered spiro ring together with the carbon atoms to which they are both attached;

[0229] - z is an integer selected from 0, 1, 2 or 3;

[0230] -R 4 represents a group selected from -CN, cyclopropyl or a linear or branched C1-C6 alkyl group, the alkyl group being optionally substituted by 1, 2 or 3 halogen atoms; wherein the group R 4 If present, replaces the one present in R 4 A hydrogen atom of one of the CH groups in the benzene ring to which it is attached.

[0231] However, the same inventor of PCT / EP2019 / 081522 has now realized that other analogs of compounds #1, #2 and #6 useful in the invention described in PCT / EP2019 / 081522 are not covered by the above chemical formula. It is worth noting that these new compounds should also be used as drugs, in particular for the treatment of various cancers, such as carcinomas, especially tumors arising from the epithelial layer of the gastrointestinal tract including the mouth (oral cancer), esophagus, stomach and small and large intestines (such as rectal cancer or colon cancer). Such medical uses should also include skin cancer, breast (breast cancer), pancreatic cancer, lung cancer, head and neck cancer, liver cancer, ovarian cancer, cervical cancer, uterine cancer, gallbladder cancer, penile cancer and urinary bladder cancer (such as kidney cancer, prostate cancer or bladder cancer).

[0232] In this sense, as already pointed out above, the interactions of the compounds described as useful in PCT / EP2019 / 081522 with proteins such as Fig.14 , where the major interactions originate from the right side of the molecule as the acid interacts with tyrosine and histidine residues and the tetrazole interacts with the main chain NH in the protein sequence. Importantly, however, the A group and linker structure of the general structure of formula (I) occupy a larger pocket on the left side of the molecule and the carbonyl function does not appear to interact with proteins, thus suggesting that it is not essential for activity. Furthermore, while the nitro group in compound 1 can interact with proteins, screening suggests that even if this is the case, this interaction may not be strictly essential. For these reasons, in some cases, having an amide / ketone may be advantageous even if no interaction with proteins occurs, as it provides conformational rigidity and reduces the lipophilicity of the compound of formula (I).

[0233] For these reasons, in the present invention, we provide here other analogs of the following compounds #1, #2 and #6 that are useful in the invention described in PCT / EP2019 / 081522 but are not covered by the general chemical formula (I):

[0234]

[0235] The above-mentioned compound (1) is also referred to herein as compound K1.

[0236] Based on the available data, it is clearly plausible that this group of compounds (hereinafter referred to as "Group 1A") would be active because they retain the key pharmacophores in the right-hand phenyltetrazolyl / carboxylic acid and are of a size that fits into the pocket. In addition, the larger lipophilic groups of each of the above compounds will be prone to interacting with proteins due to the hydrophobic nature of the pocket. On the other hand, in most cases, the smaller groups have polar hydrophilic groups (6, 7) - which can be accommodated without interacting with the protein surface - these polar groups can provide advantages in terms of solubility and lower lipophilicity to reduce interaction / metabolism with key metabolic enzymes such as cytochrome P450.

[0237] In addition, we provide here other analogs of the following compounds #1, #2 and #6 that are useful in the invention described in PCT / EP2019 / 081522 but are not covered by the above chemical formula (I) (hereinafter referred to as "Group 3A"):

[0238]

[0239]

[0240] It can be noted that in the original Markush formula (I), (A) can be selected from aryl, heteroaryl or cycloalkyl, which occupy a large pocket with the potential to accommodate more other groups. Therefore, it is entirely reasonable that non-cyclic amides (for example, those compounds 14, 15, 16, 17, 19, 20, 21, 22 or 23 mentioned above) can be accommodated inside (because they fit in this pocket) and can interact hydrophobically with the protein. On the other hand, it would also be predicted that the protein structure (compound 24) can tolerate substitution on the group (Y).

[0241] In addition, we provide here other analogs of the following compounds #1, #2 and #6 that are useful in the invention described in PCT / EP2019 / 081522 but are not covered by the above chemical formula (I) (hereinafter referred to as "Group 2A"):

[0242]

[0243] Compound (27) is also referred to herein as K2.

[0244] These compounds are not covered by the existing Markush formula (I) because they contain an acid group in the meta position of the benzene ring, whereas only para derivatives were covered in the original application. Fig.14 ) suggests that the acid interacts with the histidine in the protein. In this sense, if there is movement in the protein, or flexibility in the binding mode or protein, it makes perfect sense that small adjustments would allow the meta-acid to interact in the same way as the para-acid without affecting other bond interactions (since these groups are in close proximity).

[0245] In this sense, the acids (a-para) and (b-meta) which show close proximity are compared.

[0246]

[0247] Furthermore, even without flexibility, the meta-acid can still make some interactions - see Figures (d) and (e), which show that one of the oxygens can always occupy the same region of space when comparing the para-isomer and the meta-isomer. It is also worth noting that the acid can make hydrogen bond donors and acceptors interact, because the bond can flip between multiple forms (tautomerism), and either interaction mode is possible in each case (compare (b) and (c), and (d) and (e)). Moreover, in two cases (compounds 29 and 31 above), the chlorine atom is present in the para position next to the meta-acid. In both cases, the complete hydrogen bond network of the acid can be maintained, because the chlorine has also been shown to behave like a hydrogen bond donor and interact with the carbonyl group (J Med Chem. 2010, 53(14):5061–5084).

[0248] In addition, we provide herewith the following other analogs / metabolites (hereinafter referred to as derivatives of Compound #1) that are useful in the invention described in PCT / EP2019 / 081522.

[0249]

[0250] Isomers of the above compounds are also encompassed in the present invention, as shown below.

[0251]

[0252] The above compounds (34) and (35) are also referred to herein as MKH1. Fig. 20 The chemical synthesis of MKH1 is shown in FIG.

[0253] The derivative of compound #1 is a novel metabolite of compound #1. The metabolite was identified after incubation of compound #1 with mouse hepatocytes. Mass spectrometry showed that the molecular weight of the metabolite was two mass units higher than that of the parent, indicating that reduction had occurred. Using a combination of high performance liquid chromatography and mass spectrometry (LCMS), the metabolite was shown to be more polar than the parent, indicating that reduction of the ketone had occurred, which was subsequently confirmed by exploring the fragments present in the mass spectrometry studies. This metabolite is more polar than the parent and therefore has reduced lipophilicity, and in addition, the metabolite contains additional atoms with a three-dimensional geometry (the carbonyl group present in the parent is planar in nature, while the alcohol is not planar). The combination of reduced lipophilicity and reduced planarity is associated with increased aqueous solubility, which is beneficial for oral drug molecules. In addition, reduced lipophilicity is generally associated with other superior developability of drug molecules, including a reduced potential for drug-drug interactions and a reduced potential for toxicity.

[0254] Therefore, based on the above, it is pointed out herein that the following general formula covers all compounds of Group 1A, and any solvates, isomers or pharmaceutically acceptable salts thereof:

[0255]

[0256] in:

[0257] -A represents a group selected from C1-C4 alkyl (preferably C1-C2 alkyl), aryl (such as phenyl), heteroaryl and cyclic amide; wherein optionally, any one of these groups is substituted by 1, 2 or 3 groups independently selected from the following:

[0258] a) Halogen atoms (such as chlorine or bromine atoms), hydroxyl groups, NO2, -CN, -N(R a )R b 、-OR a 、-C(=O)R a 、-C(=O)OR a 、-C(=O)N(R a )R b 、-OC(=O)-R a 、-N(R c )C(=O)R b 、-NR c S02R a 、-SO2N(R a )R b 、-SR a 、-S(O)R a 、-S(O)2R a ;

[0259] b) a linear or branched C1-C6 alkyl group optionally substituted by 1, 2 or 3 halogen atoms, preferably a methyl group;

[0260] c) a C3-C6 cycloalkyl group optionally comprising 1 or 2 heteroatoms selected from O, S and N, and the ring is optionally substituted with a C1-C3 alkyl group;

[0261] d) phenyl or C5-C6 heteroaryl, each optionally substituted by a halogen atom, a cyano group, a C1-C3 alkyl group or a cyclopropyl group;

[0262] -R a , R b and R c Each independently represents:

[0263] a) Hydrogen atoms,

[0264] b) a linear or branched C1-C ... 12 Alkyl (preferably methyl), C3-C6 cycloalkyl and C4-C6 heterocycloalkyl: carbonyl, halogen atom, hydroxyl, phenyl, C3-C6 cycloalkyl, linear or branched C1-C6 alkoxy, amino, alkylamino, dialkylamino, linear or branched C1-C6 alkylcarbonyl,

[0265] c) phenyl or C5-C6 heteroaryl optionally substituted by 1, 2 or 3 substituents selected from the group consisting of a halogen atom, a cyano group, a linear or branched C1-C6 alkyl group, a linear or branched C1-C6 haloalkyl group, a hydroxyl group, a linear or branched C1-C6 alkoxy group, an amino group, an alkylamino group, a dialkylamino group;

[0266] d) R a and R b Together with the nitrogen atom to which they are attached, they form a 3- to 8-membered ring, the ring optionally containing other heteroatoms selected from O, N and S, and the ring is optionally substituted with 1, 2 or 3 substituents selected from the following: carbonyl, straight chain or branched C1-C6 alkyl, straight chain or branched C1-C6 haloalkyl, straight chain or branched C1-C6 alkylcarbonyl;

[0267] - x and y are integers independently selected from 0 and 1;

[0268] -R 1 and R 2 Each independently represents a group selected from hydrogen, aryl (such as phenyl), cyclopropyl or straight-chain or branched C1-C6 alkyl;

[0269] - z is an integer selected from 0, 1, 2 or 3;

[0270] -R 3represents a halogen atom (such as a chlorine atom), -CN, cyclopropyl, -OR a or a linear or branched C1-C6 alkyl group, the alkyl group may be optionally substituted by 1, 2 or 3 halogen atoms; wherein the group R 3 If present, replaces the one present in R 3 A hydrogen atom of one of the CH groups in the benzene ring to which it is attached.

[0271] Therefore, the first aspect of the present invention relates to compounds of formula II. Preferred compounds are selected from the following list, or solvates, isomers or pharmaceutically acceptable salts thereof:

[0272]

[0273]

[0274] In addition, it is pointed out herein that the following general formula covers all compounds of Group 3A, and any solvates, isomers or pharmaceutically acceptable salts thereof:

[0275]

[0276] in:

[0277] -A represents a group selected from hydrogen and C1-C4 alkyl, the C1-C4 alkyl being optionally substituted by 1, 2 or 3 groups independently selected from:

[0278] a) Halogen atoms, NO2, -CN, -N(R a )R b 、-OR a 、-C(=O)R a 、-C(=O)OR a 、-C(=O)N(R a )R b 、-OC(=O)-R a 、-N(R c )C(=O)R b 、-NR c S02R a 、-SO2N(R a )R b 、-SR a 、-S(O)R a 、-S(O)2R a ;

[0279] b) a linear or branched C1-C6 alkyl group optionally substituted by 1, 2 or 3 halogen atoms, preferably a methyl group;

[0280] c) a C3-C6 cycloalkyl group optionally comprising 1 or 2 heteroatoms selected from O, S and N, and the ring is optionally substituted with a C1-C3 alkyl group;

[0281] d) phenyl or C5-C6 heteroaryl, each optionally substituted by a halogen atom, a cyano group, a C1-C3 alkyl group or a cyclopropyl group;

[0282] -R a , R b and R c Each independently represents:

[0283] a) Hydrogen atoms,

[0284] b) a linear or branched C1-C ... 12 Alkyl (preferably methyl), C3-C6 cycloalkyl and C4-C6 heterocycloalkyl: carbonyl, halogen atom, hydroxyl, phenyl, C3-C6 cycloalkyl, linear or branched C1-C6 alkoxy, amino, alkylamino, dialkylamino, linear or branched C1-C6 alkylcarbonyl,

[0285] c) phenyl or C5-C6 heteroaryl optionally substituted by 1, 2 or 3 substituents selected from the group consisting of a halogen atom, a cyano group, a linear or branched C1-C6 alkyl group, a linear or branched C1-C6 haloalkyl group, a hydroxyl group, a linear or branched C1-C6 alkoxy group, an amino group, an alkylamino group, a dialkylamino group;

[0286] d) R a and R b Together with the nitrogen atom to which they are attached, they form a 3- to 8-membered ring, the ring optionally containing other heteroatoms selected from O, N and S, and the ring is optionally substituted with 1, 2 or 3 substituents selected from the following: carbonyl, straight chain or branched C1-C6 alkyl, straight chain or branched C1-C6 haloalkyl, straight chain or branched C1-C6 alkylcarbonyl;

[0287] -y is 0 or 1;

[0288] -B is a nitrogen atom or an oxygen atom;

[0289] -R 1 is present only when B is a nitrogen atom and is represented by a group selected from hydrogen, cyclopropyl or a linear or branched C1-C6 alkyl group, wherein the alkyl group is optionally substituted by 1, 2 or 3 halogen atoms;

[0290] -When y is 0, R 1 The two or three carbon atoms of the nitrogen atom may form a five-membered or six-membered ring together with the adjacent nitrogen atom and the two adjacent carbon atoms of the aromatic ring to which the nitrogen atom is attached;

[0291] -R2 and R 3 Each independently represents a group selected from hydrogen, cyclopropyl or linear or branched C1-C6 alkyl; optionally, R 2 and R 3 can form a 3- or 4-membered spiro ring together with the carbon atoms to which they are both attached;

[0292] - z is an integer selected from 0, 1, 2 or 3;

[0293] -R 4 represents a group selected from -CN, cyclopropyl or a linear or branched C1-C6 alkyl group, the alkyl group being optionally substituted by 1, 2 or 3 halogen atoms; wherein the group R 4 If present, replaces the one present in R 4 A hydrogen atom of one of the CH groups in the benzene ring to which it is attached.

[0294] Therefore, the second aspect of the present invention relates to compounds of formula III. Preferred compounds are selected from the list consisting of the following, or solvates, isomers or pharmaceutically acceptable salts thereof:

[0295]

[0296]

[0297] It should also be noted that other compounds having structures similar to those falling within Formula III may also be encompassed by this general formula if a ring of 6 or 7 carbon atoms is created or formed between A or R1 and R2 or R3.

[0298]

[0299] The compound with this modification is compound K33, as shown below:

[0300]

[0301] In addition, it is pointed out herein that the following general formula covers all compounds of Group 2A, and any solvates, isomers or pharmaceutically acceptable salts thereof:

[0302]

[0303] in:

[0304] -A represents a group selected from hydrogen, C1-C4 alkyl, aryl, heteroaryl and cyclic amide, wherein C1-C4 alkyl (preferably C1 alkyl), aryl, heteroaryl and cyclic amide are optionally substituted by 1 or 2 groups independently selected from the following:

[0305] a) Halogen atoms, NO2, -CN, -N(R a )Rb 、-OR a 、-C(=O)R a 、-C(=O)OR a 、-C(=O)N(R a )R b 、-OC(=O)-R a 、-N(R c )C(=O)R b 、-NR c S02R a 、-SO2N(R a )R b 、-SR a 、-S(O)R a 、-S(O)2R a ;

[0306] b) a linear or branched C1-C6 alkyl group optionally substituted by 1, 2 or 3 halogen atoms, preferably a methyl group;

[0307] c) a C3-C6 cycloalkyl group optionally comprising 1 or 2 heteroatoms selected from O, S and N, and the ring is optionally substituted with a C1-C3 alkyl group;

[0308] d) phenyl or C5-C6 heteroaryl, each optionally substituted by 1 halogen atom, cyano, C1-C3 alkyl or cyclopropyl;

[0309] -R a , R b and R c Each independently represents:

[0310] a) Hydrogen atoms,

[0311] b) a linear or branched C1-C ... 12 Alkyl, C3-C6 cycloalkyl and C4-C6 heterocycloalkyl: carbonyl, halogen atom, hydroxyl, phenyl, C3-C6 cycloalkyl, straight-chain or branched C1-C6 alkoxy, amino, alkylamino, dialkylamino, straight-chain or branched C1-C6 alkylcarbonyl,

[0312] c) phenyl or C5-C6 heteroaryl optionally substituted by 1, 2 or 3 substituents selected from the group consisting of halogen atoms, cyano, linear or branched C1-C6 alkyl, linear or branched C1-C6 haloalkyl, hydroxy, linear or branched C1-C6 alkoxy, amino, alkylamino, dialkylamino;

[0313] d) R a and R bTogether with the nitrogen atom to which they are attached, they form a 3- to 8-membered ring, the ring optionally containing other heteroatoms selected from O, N and S, and the ring is optionally substituted with 1, 2 or 3 substituents selected from the following: carbonyl, straight chain or branched C1-C6 alkyl, straight chain or branched C1-C6 haloalkyl, straight chain or branched C1-C6 alkylcarbonyl;

[0314] - x and y are integers independently selected from 0 and 1;

[0315] -R 1 represents a group selected from hydrogen, cyclopropyl or linear or branched C1-C6 alkyl, wherein the alkyl is optionally substituted by 1, 2 or 3 halogen atoms;

[0316] -When y is 0, R 1 The two or three carbon atoms of the nitrogen atom may form a five-membered or six-membered ring together with the adjacent nitrogen atom and the two adjacent carbon atoms of the aromatic ring to which the nitrogen atom is attached;

[0317] -R 2 and R 3 Each independently represents a group selected from hydrogen, cyclopropyl or linear or branched C1-C6 alkyl; optionally, R 2 and R 3 can form a 3- or 4-membered spiro ring together with the carbon atoms to which they are both attached;

[0318] - z is an integer selected from 0, 1, 2 or 3;

[0319] -R 4 represents a group selected from -CN, cyclopropyl, halogen (such as chlorine atom) or straight-chain or branched C1-C6 alkyl, wherein the alkyl is optionally substituted by 1, 2 or 3 halogen atoms; wherein the group R 4 If present, replaces the one present in R 4 A hydrogen atom of one of the CH groups in the benzene ring to which it is attached.

[0320] Therefore, the third aspect of the present invention relates to compounds of formula IV. Preferred compounds are selected from the list consisting of the following, and any solvate, isomer or pharmaceutically acceptable salt thereof:

[0321]

[0322]

[0323] Finally, we provide herein other analogs / metabolites useful in the invention described in PCT / EP2019 / 081522. These analogs fall within the scope of the following general structure (V):

[0324]

[0325] wherein n is an integer selected from 0 to 4, and A represents an aryl (preferably phenyl) or heteroaryl group optionally substituted by 1 or 2 groups independently selected from:

[0326] a) Halogen atoms, NO2, -CN, -N(R a )R b 、-OR a 、-C(=O)R a 、-C(=O)OR a 、-C(=O)N(R a )R b 、-OC(=O)-R a 、-N(R c )C(=O)R b 、-NR c S02R a 、-SO2N(R a )R b 、-SR a 、-S(O)R a 、-S(O)2R a ;

[0327] b) a linear or branched C1-C6 alkyl group optionally substituted by 1, 2 or 3 halogen atoms, preferably a methyl group;

[0328] c) a C3-C6 cycloalkyl group optionally comprising 1 or 2 heteroatoms selected from O, S and N, and the ring is optionally substituted with a C1-C3 alkyl group;

[0329] d) phenyl or C5-C6 heteroaryl, each optionally substituted by 1 halogen atom, cyano, C1-C3 alkyl or cyclopropyl;

[0330] -R a , R b and R c Each independently represents:

[0331] a) Hydrogen atoms,

[0332] b) a linear or branched C1-C ... 12 Alkyl, C3-C6 cycloalkyl and C4-C6 heterocycloalkyl: carbonyl, halogen atom, hydroxyl, phenyl, C3-C6 cycloalkyl, straight-chain or branched C1-C6 alkoxy, amino, alkylamino, dialkylamino, straight-chain or branched C1-C6 alkylcarbonyl,

[0333] c) phenyl or C5-C6 heteroaryl optionally substituted by 1, 2 or 3 substituents selected from the group consisting of a halogen atom, a cyano group, a linear or branched C1-C6 alkyl group, a linear or branched C1-C6 haloalkyl group, a hydroxyl group, a linear or branched C1-C6 alkoxy group, an amino group, an alkylamino group, a dialkylamino group;

[0334] d) R a and R b Together with the nitrogen atom to which they are attached, they form a 3- to 8-membered ring, which optionally contains other heteroatoms selected from O, N and S, and which is optionally substituted by 1, 2 or 3 substituents selected from the following: carbonyl, straight chain or branched C1-C6 alkyl, straight chain or branched C1-C6 haloalkyl, straight chain or branched C1-C6 alkylcarbonyl.

[0335] A preferred compound is shown herein as K8:

[0336]

[0337] Compound K8 can be synthesized as shown below.

[0338] -K8 Synthesis

[0339]

[0340] In a preferred embodiment, the "compounds of the present invention" useful in practicing the present invention are selected from those encompassed by any of Formulae II, III, IV or V above, or from the list of compounds explicitly noted above (and their chemical structures) as structures belonging to Group 1A, Group 2A, Group 3A or a derivative of Compound #1 (MKH1) or any other analogues thereof described throughout the present invention.

[0341] Most of the compounds of the present invention can be easily synthesized as shown in Example 1.

[0342] The compounds of the present invention may be in the form of free form or pharmaceutically acceptable salts. Examples of pharmaceutically acceptable salts include: inorganic acid salts, for example, hydrochlorides, sulfates, nitrates, phosphates or hydrobromides, etc.; organic acid salts, for example, acetates, fumarates, oxalates, citrates, methanesulfonates, benzenesulfonates, p-toluenesulfonates or maleates, etc. In addition, when the compound has a substituent such as a carboxyl group, salts formed with bases (for example, alkali metal salts such as sodium salts, potassium salts, etc., or alkaline earth metal salts such as calcium salts, etc.) can be cited.

[0343] The compound of the present invention or its isomer, preferably enantiomer, or pharmaceutically acceptable salt may be any of its intramolecular salt or adduct, or its solvate or hydrate.

[0344] As taught by the present invention, the compounds of the present invention can be used for treatment. In this sense, when the compounds of the present invention or their pharmaceutically acceptable salts are used as active ingredients for medical purposes, they can be used together with pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers are inert carriers suitable for each method of administration, and can be formulated into conventional pharmaceutical preparations (tablets, granules, capsules, powders, solutions, suspensions, emulsions, injections, infusions, etc.). As such carriers, for example, pharmaceutically acceptable adhesives (such as gum arabic, gelatin, sorbitol and polyvinyl pyrrolidone), excipients (such as lactose, sugar, corn starch and sorbitol), lubricants (such as magnesium stearate, talc and polyethylene glycol), disintegrants (such as potato starch) and the like can be cited. When they are used as injection solutions or infusion solutions, they can be prepared by using distilled water for injection, physiological saline, and glucose aqueous solution.

[0345] The administration method of the compound of the present invention and / or the pharmaceutically acceptable salt thereof is not particularly limited, and conventional oral or parenteral administration methods (intravenous, intramuscular, subcutaneous, transdermal, intranasal and others, transmucosal, enteral, etc.) can be used.

[0346] The dosage of the compound of the present invention or its pharmaceutically acceptable salt of the present invention can be optionally set within the range of an effective amount sufficient to show a pharmacological effect according to the efficacy or characteristics of the compound to be used as an active ingredient. The dosage can vary according to the method of administration, the age, weight or condition of the patient.

[0347] Finally, a fourth aspect of the invention relates to a compound of any one of formula (II), (III), (IV) or (V) or any other derivative of compound #1 described herein (such as MKH1) or any pharmaceutically acceptable salt thereof for use in the treatment of cancer. Preferably, for the treatment of cancer. More preferably, wherein the cancer is a cancer selected from the list consisting of: tumors arising from the epithelial layer of the gastrointestinal tract including the mouth (oral cancer), esophagus, stomach and small and large intestines (such as rectal cancer or colon cancer), skin cancer, breast (breast cancer), pancreatic cancer, lung cancer, head and neck cancer, liver cancer, ovarian cancer, cervical cancer, uterine cancer, gallbladder cancer, penile cancer and urinary bladder cancer (such as kidney cancer, prostate cancer or bladder cancer).

[0348] The following examples are only used to illustrate the present invention, but not to limit the present invention.

[0349] Example

[0350] Example 1 Synthesis

[0351] Any compound of formula (II), (III) or (IV) or derivative of compound #1 listed throughout this specification can be prepared according to the following general synthetic route, which is an improvement on the general method described by VV Zarubaev et al. (Bioorg. Med. Chem. 18 (2010) 839-848).

[0352]

[0353] Example 2

[0354] Materials and methods

[0355] Protein and ligand models. The X-ray crystal structure of the phosphotyrosine binding domain of Hakai (PDB 3VK6) was downloaded from the Protein Data Bank and the dimer was modeled using appropriate symmetry operations. Amino acid protonation was performed at a pH of 7.2 using the pdb2pqr server. The 3D models of the ligands were built using the Virtual Screening and Data Management Integrated Platform (VSDMIP) as described elsewhere. In brief, the initial 3D coordinates of each ligand were generated using CORINA [Sadowski, J.; Gasteiger, J.; Klebe, G. Comparison of Automatic Three-Dimensional Model Builders Using 639 X-Ray Structures. J. Chem. Inf. Comput. Sci. 1994, 34, 1000-1008 (DOI: 10.1021 / ci00020a039)]. Afterwards, various conformers were generated using ALFA[4], and the atomic partial charges calculated by MOPAC were assigned to each conformer by adopting the AM1 semiempirical model and the ESP method. All ligand models were stored in the VSDMIP database for use in different virtual screening campaigns.

[0356] Virtual screening. Ligands from the eMolecules catalog [https: / / www.emolecules.com / info / products-screening-compounds.html] were downloaded and processed as described in the previous section. Only molecules presenting a carboxylic acid moiety and / or a phosphate group capable of mimicking a phosphotyrosine residue were considered. Next, the selected molecules were embedded within the binding pocket of Hakai using CRDOCK by using the CRScore scoring function and the BFGS energy minimizer. The ligands were then ranked according to the predicted scores and the top 350 molecules were re-evaluated by using an in-house implementation of the HYDE scoring function. Finally, the best 20 molecules were visually inspected to select a final set of 6 molecules.

[0357] Binding pocket analysis. To better analyze the results of the virtual screening campaign, we used our in-house cGRILL software [6] to generate affinity maps within the binding pocket of the phosphotyrosine binding domain of Hakai based on hypothetical van der Waals, Coulomb, and hydrogen bonding interactions of atom probes. Possible positions of molecular recognition of phosphotyrosine residues were mapped using negatively charged receptor probes (=O) to help filter out docking solutions during visual inspection of the poses.

[0358] Plasmids, inhibitors, and antibodies

[0359] The pcDNA-Flag-Hakai, pBSSR-HA-ubiquitin, pSG-v-Src, and pcDNA-myc-E-cadherin plasmids were described previously. Compounds Hakin-1 [4-(5-{[2-(4-nitrophenyl)-2-oxoethyl]thio}-1H-tetrazol-1-yl)benzoic acid] and Hakin-5 [(2E,4E,8E)-7,13-dihydroxy-4,8,12-trimethyl-2,4,8-tetradecatrienoic acid] were obtained from ChemBridge Corporation and TimTec or Analyticon Discovery, respectively. The remaining analogs tested (ketophenyls A-1, A-7, A-8, A-9; ketoheteroaryls: A-23, A-25; cyclic amides: A-10, A-16; and benzamides: A-6.1) were obtained from Vitasmlab. The compounds were resuspended in DMSO (Sigma) at 100 mM for in vitro assays, while Hakin-1 was resuspended at 100 mM for in vivo assays. The highest concentration of DMSO was used as a vehicle control for the experiments. Figure 1 shown.

[0360] Cell culture

[0361] MDCK, HEK293T, HepG2, MCF7 and ACHN cells were cultured in Dulbecco's Modified Eagles Medium (DMEM). MDCK (Hakai-MDCK) stably expressing Hakai cells has been reported previously and grown in DMEM containing G418 (800 μg / ml). As mentioned above, different clones of Hakai-MDCK cells exhibited similar phenotypes and characteristics. LoVo cells and PC-3 cells were cultured in F-12K medium (Kaighn's Modification of Ham's F-12 Medium), and HT-29 cells were cultured in McCoy's 5a Medium Modified. 5637 cells were cultured in RPMI medium. All culture media were supplemented with 1% penicillin / streptomycin and 10% heat-inactivated fetal bovine serum (Fetal bovine serum, FBS) at 37°C in a humidified incubator with 5% CO2. Cells were tested for mycoplasma contamination once a month and used only 1-3 months after thawing. LoVo cells and HT29 cells were validated using the StemElite ID system (Promega). For phase contrast images, cultured cells were photographed using a Nikon Eclipse-TI microscope.

[0362] Ubiquitination assay

[0363] For ubiquitination assay, 750000 HEK293T cells were seeded in 6-well cell culture plates and transfected with 0.25 μg Src, 0.75 μg Flag-Hakai and 0.5 μg HA-ubiquitin with Lipofectamin 2000 (Invitrogen, Uk) after 24 h. 6 h after transfection, cells were treated with designated concentrations of Hakin-1, Hakin-5 or the remaining analogs tested for 36 h. Whole cell extracts were obtained in a lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl and 1% Triton X-100) containing 10 μg / ml leupeptin, 10 μg / ml aprotinin and 1 mM phenylmethanesulphonyl fluoride (PMSF) supplemented with 10 mM N-ethylmaleimide. Cells were harvested and western blotted using anti-HA antibodies to detect ubiquitination.

[0364] Immunoprecipitation

[0365] For immunoprecipitation experiments, 293 cells were transfected with 3 μg Src, 4 μg Flag-Hakai, 2 μg Ha-ubiquitin and 3 μg E-cadherin with Lipofectamin 2000 (Invitrogen, UK). 24 h after transfection, cells were lysed for 20 min in 1 ml 1% Triton X-100 lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl and 1% Triton X-100) supplemented with 10 mM N-ethylmaleimide and 2.5 mM sodium orthovanadate and containing 10 μg / ml leupeptin, 10 μg / ml aprotinin and 1 mM phenylmethylsulfonyl fluoride (PMSF). After centrifugation at 18,000 g for 10 min, the supernatant was immunoprecipitated with 2 μg of anti-E-cadherin antibody bound to 60 μl of ProteinGPLUS-agarose beads for 2 h, followed by SDS-polyacrylamide gel electrophoresis (PAGE) and Western immunoblotting with the indicated antibodies as described previously.

[0366] Viability assay

[0367] For cytotoxicity assays, cells were plated at 1 × 10 4 Each well was inoculated into a 96-well plate. After 24 h, cells were treated with the specified inhibitor for 72 h and MTT colorimetric cell viability assay was performed according to the manufacturer's instructions (Sigma Aldrich, StLouis, MO). The absorbance was measured at 570 nm and 630 nm using a Multiskan Plus Reader (Nanoquant Infinite M200 Tecan Trading AG, Switzerland). Dose-response curves were designed using GraphPad Prism software and half-maximal inhibitory concentration (IC50) values ​​were calculated. The data shown are mean ± SEM of at least three independent experiments, with six replicates for each condition.

[0368] Western blotting and immunofluorescence

[0369] For western blot analysis, cells were treated with the indicated inhibitors for 48 h, and whole-cell extracts were obtained as described previously. 20 μg of lysate was resolved on 10% polyacrylamide SDS-PAGE and then subjected to western blot analysis as described previously. For immunofluorescence assays, cells were grown on glass coverslips for 24 h and treated with the indicated inhibitors for 48 h. Cells were fixed with 4% PFA for 15 min, permeabilized with 0.5% Triton X-10 and incubated with E-cadherin antibody for 2 h. Coverslips were incubated with fluorescein-tagged secondary antibodies (Dakopatts, Sweden) for 1 h. Coverslips were finally fixed with ProLong Gold antifade reagent (LifeTech, UK) and images were captured in an epifluorescence microscope (Olympus) using a 40x objective. Figure 22-23 E-cadherin immunofluorescence and DNA content analysis were performed on HT-29 human colorectal adenocarcinoma cell line cultured in 384-well plates at a density of 3,000 cells per well in a total volume of 25 μl. After staining was completed, four images (2x2 array) of each well were taken using ValaSciences' SIMTM with a 20x objective. Each image was acquired as a SIM Synthetic Focus with a z-stack range of 6 μm and a step size of 1 μm. The SIM Synthetic Focus feature will compile a "best focus" image from multiple images. For Hoechst 33342 staining, one set of SIM Synthetic Focus images were taken in the DAPI channel, and for anti-E-cadherin-AlexaFluor488 labeling, another set of images were taken in the FITC channel, and cell imaging was performed by the Structured Illumination Microscopy (SIMTM) platform, as well as by Vala's The above images were analyzed using the intercellular connectivity algorithm to quantify the expression level and intracellular location of E-cadherin. The software can also measure relative DNA content by analyzing the total integrated intensity (TII) of the nuclear image (Ni) on the nuclear mask (Nm). This analysis was performed by Vala Science. 20 compounds from were assayed in a "blind" manner, the first assay was performed in 6 replicate wells at 2 test concentrations, and the second assay was performed in 3 replicates.

[0370] Proliferation assay

[0371] For BrdU assay, the indicated cells were plated at 1 × 10 4100 μg / well were plated in 96-well plates. After 24 h, cells were treated with the specified inhibitor for 48 h. Three independent experiments were repeated six times for each condition. Cells were treated with 10 mM BrdU for 2 h. According to the manufacturer's instructions (Roche, Switzerland), BrdU incorporation in newly synthesized DNA was measured using a cell proliferation colorimetric immunoassay kit. Results are expressed as mean ± standard deviation. Results are expressed as percentage of positive cells (mean ± standard deviation) of three independent experiments.

[0372] Soft agar colony formation assay

[0373] In a 12-well plate, 5 × 10 3 MDCK and MDCK-Hakai cells / well or 12×10 3 Soft agar colony formation assay was performed in triplicate at a density of 100 HT29 cells / well. Cells were seeded in a culture medium with 0.5% low melting point agarose covered on a 0.75% low melting point agarose layer (Lonza Rockland, Maine, the United States). Cells were treated with specified inhibitors and DMSO was used as a solvent. Treatment was updated every 3 days, and colony counts were quantified after 21 days (for MDCK and MDCK-Hakai cells) or 28 days (for HT29 cells). Five randomly selected visual fields under each condition were taken using a Nikon Eclipse-TI microscope (objective lens 4 times) for quantification. The experiment was performed in triplicate and repeated three times. Data are represented by mean ± standard deviation.

[0374] Migration and invasion assays

[0375] For invasion assays, cells were treated with Hakin-1 or DMSO as a vehicle for 48 h and 1% FBS was used for the last 24 h. 5 MDCK, MDCK-Hakai or LoVo cells were seeded in a cell invasion chamber containing medium containing 2% FBS (Cell Invasion Assay Kit, Chemicon International). After 72 h (for MDCK and MDCK-Hakai invasion) and 16 h (for LoVo cells), the invasive cells that invaded to the lower chamber containing 30% FBS were fixed and stained with crystal violet (Sigma Aldrich, StLouis, MO) according to the manufacturer's instructions. For migration assays, HT29 cells were cultured with Hakin-1 or DMSO as a solvent for 48 h and treated with serum-free medium for the last 24 h. 3 × 10 cells were seeded in a cell migration chamber containing serum-free medium. 5HT29 cells (cell migration kit, Millipore, Bedford, MA). After 16 h, the migrated cells in the lower chamber containing 30% FBS serum were stained with crystal violet and counted according to the manufacturer's instructions. For invasion and migration assays, cells were counted in five fields of view photographed with an Olympus microscope using a 20x objective, and the experiments were performed in triplicate for each condition, and at least three experiments were repeated. The results are expressed as mean ± standard deviation.

[0376] Tumor xenograft models

[0377] Xenograft experiments were performed at the Experimental Surgery Unit–Technological Training Center of INIBIC in accordance with European Community legislation (86 / 609 / EEC) and Spanish legislation (RD53 / 2013). The experiments were approved by the Ethics Committee for Animal Experimentation of Xerencia de Xestion Integrada da (XXIAC). Mice were kept on a 12h / 12h light / dark cycle with water and food available ad libitum. Six-week-old athymic nu / nu mice were randomized. One million MDCK cells resuspended in serum- and antibiotic-free DMEM were inoculated subcutaneously into both flanks of the animals in two groups of three. The same number of Hakai-MDCK cells were injected into the animals in two groups of four. Twenty days after inoculation, tumors in Hakai-MDCK were palpable. Then, half of the animals were treated with Hakin-1 (5 mg / kg) every 3 days, and the other half were treated with the same concentration of DMSO. Tumor length (L) and width (W) were measured with electronic calipers, and tumor growth was monitored twice a week. Tumor volume was calculated as pLW2 / 6. After forty days of inoculation, animals were sacrificed. Tumors, lungs, kidneys, and livers were collected and fixed in 4% PFA and embedded in paraffin blocks for histological and / or immunohistochemical (IHC) analysis.

[0378] Histology and immunohistochemistry

[0379] As previously described, tumors and tissues were dewaxed, rehydrated, and stained with hematoxylin and eosin (H&E). Tumor sections (4 μm) were also dewaxed and hydrated for immunohistochemistry. Antigen retrieval was performed by heating samples in citrate buffer (DakoREAL, Denmark) or EDTA buffer (2100Retriever; PickCell Laboratories). Endogenous peroxidase activity was then blocked with peroxidase blocker (DakoCytomation, Denmark). Samples were blocked and permeabilized with 0.2% BSA and 0.1% Tx-100 for 1 hour and incubated with the designated primary antibodies overnight at 4°C in a wet chamber. Slides were incubated with secondary antibodies at room temperature for 1 hour and detected using DAB (DakoReal Envision kit) according to the manufacturer's instructions. Finally, nuclei were counterstained with Gill's hematoxylin and fixed with DePeX. Photos were taken with an Olympus microscope. Five pictures were taken for each animal, and the images were quantified using the Image J program, and the results shown are shown as mean ± SEM. The number of mitosis was counted in the sections stained with H&E. In this case, ten pictures were taken for each tumor using an Olympus BX50 microscope (objective 40 times), and the number of mitosis was manually counted. The results are expressed as mean ± SEM, and representative pictures of each condition are shown.

[0380] Quantification of lung metastases from in vivo mouse models

[0381] Real-time PCR was used to study the presence of metastasis in the mouse lungs. Quantification was performed using primers for the HA epitope present in the ectopic HA-tagged Hakai expressed in MDCK-Hakai cells and Hakai (5′-TCTGGGACGTCGTATGGGTA-3′; 5′-TTCTTCATCACCTTGCGGG-3′). Primers for mouse apolipoprotein B (apob) (5′-CGTGGGCTCCAGCATTCTA-3′; 5′-TCACCAGTCATTTCTGCCTTTG-3′) were used as endogenous controls. The MDCK cell line was used as a negative control. Lung DNA was extracted from 10-15 sections of paraffin blocks (4 μm) using the QIAamp DNAMini kit (Qiagen). Using the LightCycler 480 real-time light cycler (Roche), quantitative PCR was performed in triplicate technically.

[0382] DNA was amplified and quantified. Relative DNA levels were determined by 2 -ΔΔCt Method calculation.

[0383] In vivo TUNEL assay

[0384] Tissue sections from tumors were dewaxed and rehydrated using standard protocols. The slides were rinsed twice with PBS and treated with citrate buffer (DakoREAL, Denmark) in a microwave at 350W for 5 minutes. Tissue sections were then analyzed using a fluorescein in situ cell death detection kit (Cell Death Detection Kit, Fluorescein) (Roche) according to the manufacturer's instructions. The slides were then incubated in the dark with Hoechst for 5 minutes. The reaction was observed using a 20x objective lens under an epifluorescence Olympus microscope. Five representative photos were taken for each section. The percentage of positive cells was calculated, and the results were expressed as mean ± SEM.

[0385] Hepatocyte stability

[0386] Test compounds (final substrate concentration, 3 μM; final DMSO concentration, 0.25%) were pre-incubated at 37°C before addition of cryopreserved hepatocyte suspension (final cell density, 0.5 x 10 6 Live cells / mL, in Williams E medium supplemented with 2mM L-glutamine and 25mM HEPES) to start the reaction. The reaction was terminated with acetonitrile containing internal standard at appropriate time points (0, 10, 20, 40, 60 and 120 minutes). The termination plate was centrifuged at 2500rpm for 30 minutes at 4°C to precipitate protein. After the hepatocyte stability assay, the sample was used for metabolite analysis. The hepatocyte stability of mice and humans was performed by incubating the test compound (3 μM) with a frozen hepatocyte suspension. During the 60-minute experiment, samples were taken out at 6 time points, and the remaining test compound at each time point was analyzed using LC-MS / MS. Then, the data was analyzed and the intrinsic clearance (CLint) and half-life (t1 / 2) with standard error were reported.

[0387] Metabolite analysis

[0388] Samples from the hepatocyte stability assay were used for metabolite analysis. The 10- and 20-minute samples were compared to the 0-minute control sample to identify metabolites formed. Metabolites have been shown as extracted ion chromatograms (XICs) and representative mass spectra. Areas and percentages of parent and metabolites were calculated using the XIC data; it was assumed that each metabolite had the same ionization efficiency and that metabolite sensitivity was not affected by biotransformation. The m / z of each metabolite in each species and its associated ppm error are shown starting from the time point showing the maximum peak area. For precision, a ppm error of less than 5 is desirable; when the response of the observed metabolites is low, the ppm error may be greater than 5. When a metabolite is referred to as potentially oxidized, it can refer to potential hydroxylation, epoxidation, or oxide formation. Representative collision-induced dissociation (CID) mass spectra of the metabolites found were obtained. Structural elucidation was then performed on the observed metabolites. Data were processed using Metabolynx XS (Waters Ltd). When compound structures were provided, data were processed using mass defect filtering 1 and dealkylation tools 2; if structures were not provided, only mass defect filtering was used. Mass defect filtering exploits the chemical property that the mass defect (i.e., the fraction of the observed m / z) of a metabolite does not change significantly when a compound is metabolized, thus distinguishing drug-related substances from endogenous substances. In addition to mass defect filtering, a dealkylation tool was used to take into account that the mass defect may change when a molecule undergoes metabolic cleavage (e.g., at an amine group). After this initial processing, the data were compared to a control sample to determine whether a range of expected metabolites were observed. Finally, the same data set was processed using a 1a.mu window across the entire data acquisition range to look for non-predicted or unexpected metabolites. After automated Metabolynx XS processing, the data were reviewed to confirm the validity of the detected metabolites and to ensure that the observed metabolites were correctly assigned. The elemental formula of the observed ions and compounds is reported, as well as the parts per million (ppm) error of the measured ions compared to the theoretical mass of the ions. After structural elucidation, collision-induced dissociation data generally do not give the exact location of the biotransformation. Therefore, the structure is depicted in Markush format3, with shaded areas indicating potential modification sites. Hepatocyte stability, metabolic profiling, and test compound analysis were performed by Cyprotex Discovery.

[0389] Statistical analysis

[0390] The Shapiro-Wilk test was used to investigate normal distribution, and the Levene test was used to assess homogeneity of variance. The statistical significance of the data was determined using ANOVA with Bonferroni's test or Kruskal-Wallis with Tukey's correction test. The significance between the experimental groups shown in the figures is shown as *p<0.05, **p<0.01 and ***p<0.001. As indicated, the results obtained are expressed as mean ± standard deviation or mean ± SEM. Survival graphs in xenograft assays were analyzed using GraphPad Prism software, and p values ​​were calculated using the Breslow test. The results are expressed as the fold change of the values ​​obtained for treated cells compared to untreated cells. The data generated by the CyteSeer algorithm were analyzed using analysis of variance with GraphPad Prism 5, followed by Dunnett's post hoc multiple comparison test for statistical significance of each dose compared to the vehicle control. Abbreviations / symbols: ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001. In the bar graphs, the bars represent the mean of 6 samples, and the error bars represent the standard deviation of the mean.

[0391] List of antibodies useful in practicing the invention.

[0392]

[0393] 1.2 Results

[0394] Identification of putative selective Hakai inhibitors

[0395] To find candidate molecules with the required potential to inhibit Hakai, we designed a virtual screening workflow based on the available structural information and the properties of the phosphotyrosine binding pocket, the exploration of which was carried out with the help of affinity probes. As a first step, we only considered molecules in the chemical library that displayed negatively charged carboxylates or phosphates that would complement the highly positive molecular electrostatic potential of the binding pocket. The selected molecules were then docked into the Hakai dimer to evaluate all possible binding poses and then ranked using the HYDE post-processing scoring function to estimate the interaction energy of the hypothetical Hakai-inhibitor complex. The top 20 molecules were visually inspected and two of them were selected for subsequent experimental validation, namely Hakin-1 and Hakin-5 ( Figure 1 a. According to our binding mode model, the benzoate moiety present in Hakin-1 would be a replacement for phosphotyrosine ( Figure 1b, top). The carboxylate group is located in a region that is highly favorable for negatively charged probes, as estimated by our affinity maps. This region is lined by residues Lys-126, Tyr-176, His-185, and Arg-189, while the benzene ring will be sandwiched between the guanidinium group and the side chains of Arg-174 and Arg-189. The rest of the molecule will be able to establish hydrogen bonds with the side chains of the Arg-174 residue from both monomers, as well as with the Gln-170 backbone, while maintaining the desired shape. Although Hakin-5 ( Figure 1 b, bottom) bears a carboxylate group and presents the same number of potential hydrogen-bonding interactions, but lacks the benzene ring that could mimic phosphotyrosine.

[0396] Effects of Hakin-1 inhibitors on Hakai-induced ubiquitination

[0397] We first investigated the effect of Hakin-1 inhibitor on ubiquitination induced by the E3 ubiquitin ligase Hakai by using cultured tumor cells. 293T cells were transfected with Src, Hakai, and ubiquitin in the presence of Hakin-1 inhibitor or DMSO as a control. Hakin-1 strongly reduced ubiquitination mediated by Hakai in a dose-dependent manner ( Figure 1 c), and no effect was observed on the level of Hakai protein. However, when Hakai was not overexpressed, Hakin-1 did not affect ubiquitination, confirming that Hakin-1 reduces ubiquitination in a Hakai-dependent manner ( Figure 1 d). Furthermore, when cells were treated in the presence of Hakin-5, another Hakai inhibitor identified by virtual screening, no effect on Hakai-mediated ubiquitination was detected ( Figure 1 e), further supporting the specific effect of Hakin-1 on Hakai-induced ubiquitination. Finally, we observed a reduction in Hakai-dependent ubiquitination of the E-cadherin complex when cells were treated with Hakin-1 ( Figure 1 f). Collectively, our data suggest that Hakin-1 inhibits Hakai-mediated ubiquitination without affecting Hakai protein levels.

[0398] Inhibition of Hakai by Hakin-1 activates epithelial differentiation of tumor cells

[0399] Next, we investigated the effect of Hakin inhibition on cell viability of cancer cells. To this end, we generated dose-response curves by using Hakin inhibitors in several epithelial cells, as we have previously reported. First, we analyzed the cytotoxic effect of Hakin-1 on HT-29 and LoVo colon tumor cell lines, showing a significant inhibitory response ( Figure 2 a). We extended our studies by using the normal epithelial MDCK cell line, which has been widely used as an in vitro model system to study EMT. As previously described, Hakai overexpression in MDCK cells (Hakai-MDCK) induces a fibroblast-like phenotype and the disappearance of E-cadherin-based cell-cell contacts. When these cell lines were treated with Hakin-1 or Hakin-5 inhibitors, two tested clones of the Hakai-MDCK cell line were more sensitive to Hakin-1 and Hakin-5 treatment compared to the more resistant normal epithelial MDCK ( Figure 2 b). These results further suggest that Hakin-1 may be particularly effective against cancer cells that overexpress Hakai, as seen in human colon cancer, where Hakai is significantly increased compared to adjacent normal tissue. Given the previously reported role of Hakai in epithelial-mesenchymal transition, we next analyzed the effect of Hakin-1 on tumor cell phenotype. Using phase contrast, we observed that Hakin slightly induced the epithelial phenotype of HT-29 and LoVo cells; however, it is important to note that both cell lines already display an epithelial morphology ( Figure 2 c). In addition, we tested the effects of Hakin-1 and Hakin-5 inhibitors on the mesenchymal phenotype of Hakai-transformed MDCK cells. We observed the induction of an epithelial phenotype with increased cell-cell contacts accompanied by a decrease in protrusion formation. In contrast, no effects were observed when epithelial MDCK cells were treated with specific inhibitors ( Figure 2 d). Given the reported role of Hakai in altering cell-cell contacts during EMT through its molecular effects on E-cadherin ubiquitination, endocytosis, and degradation, we further investigated the effects of Hakin-1 inhibitors on EMT reversal. Figure 3 As shown in a, by western blotting, we observed that Hakin-1 was able to increase E-cadherin levels in HT-29 cells, while the mesenchymal vimentin marker was reduced. We also analyzed the effect of Hakin-1 on cortical actin, another reported substrate of Hakai, confirming the effect of Hakin-1 on increasing expression levels. We also confirmed these results by using another epithelial tumor cell line, LoVo cells ( Figure 3 b). In addition, because the loss of E-cadherin at cell-cell contacts is considered a hallmark of EMT, we investigated the expression of E-cadherin by immunofluorescence, showing a significant increase in E-cadherin levels at cell-cell contacts in both HT-29 and LoVo cells ( Figure 3 c) However, when Hakin-5 was used, no effect on the protein level of E-cadherin was detected by Western blotting or immunofluorescence ( Figure 8). Finally, to test the effects of Hakin-1, mesenchymal Hakai-MDCK cells were also used. Hakai-MDCK cells do not express basal protein levels of E-cadherin, so no restoration was detected under Hakin-1 treatment. Taken together, these results suggest that Hakin-1 induces epithelial differentiation in different tumor epithelial cells, accompanied by a reduction in mesenchymal markers in vivo.

[0400] Hakin-1 inhibits proliferation, oncogenic potential and invasion in cultured tumor cells

[0401] We next characterized the role of Hakin-1 in tumor cell lines using standard proliferation and soft agar colony formation assays. Given that Hakai affects not only cell-cell contacts but also the proliferation of fibroblasts and epithelial cells, we decided to determine the possible effects of Hakin-1 in cell proliferation. Although no effect was seen using a Hakin-5 inhibitor ( Figure 4 b), but Hakin-1 reduced cell proliferation in HT29 and LoVo cell lines ( Figure 4 a), further supporting the antitumor effect of Hakin-1 through its control of cell proliferation. We extended our analysis by using Hakai-MDCK cells in comparison with normal epithelial MDCK cells. As previously reported, we demonstrated that Hakai-transformed MDCK cells strongly increased cell proliferation compared with normal MDCK cells ( Figure 4 c). Interestingly, Hakin-1 was able to inhibit the proliferation of Hakai-MDCK cells, whereas no effect was detected in non-transformed epithelial MDCK cells ( Figure 4 c). Hakin-1 also inhibits cell proliferation in other epithelial cell lines, such as breast cancer MCF7 cells, prostate cancer PC-3 cells, bladder cancer 5637 cells, liver cancer HepG2 cells, and kidney cancer ACHN cells ( Fig. 9 ). These results suggest that Hakin-1 can act as an antiproliferative agent when high levels of Hakai are expressed (e.g., as occurs in human adenocarcinomas). By using a soft agar colony formation assay, we evaluated the effects of Hakin-1 in HT-29 and Hakai-MDCK tumor cell lines, showing strong inhibition of colony formation in both cell lines ( Figure 4 d). As we have previously published, no colony formation was detected when non-transformed MDCK cells were treated with Hakin-1. The EMT process is characterized by the acquisition of migration and invasion capabilities. We demonstrated that Hakin-1 strongly reduced cell invasion in LoVo cells ( Figure 5a). In addition, although no cell invasion was detected in MDCK cells, Hakai overexpression induced the ability to invade in normal epithelial MDCK cells, and Hakin-1 also reduced cell invasion in these Hakai-MDCK cells ( Figure 5 b). Finally, given that HT-29 cells are unable to invade under these experimental conditions, we tested the effect of Hakin-1 on cell motility, showing a significant reduction in cell migration under Hakin-1 treatment ( Figure 5 c). All these findings support the antitumor role of Hakin-1 through its effects on cell proliferation, oncogenic potential, cell motility and invasion.

[0402] In vivo antitumor effects of Hakin-1 in tumor xenografts

[0403] The migratory and invasive capabilities acquired during EMT are key events in the formation of distant metastases, and targeting these events is therefore an ideal approach for cancer therapy. Since we have demonstrated that Hakin-1 effectively inhibits cell proliferation, oncogenic potential, and cell invasion and motility in cell culture, we decided to study the efficacy of Hakin-1 in inhibiting pre-existing tumors in vivo. To this end, MDCK and Hakai-MDCK cells were injected subcutaneously into the flanks of nude mice. As previously reported, Hakai-MDCK cells form primary tumors, while parental MDCK cells cannot. Hakin-1 showed a potent effect in inhibiting the growth of xenograft tumors in vivo ( Figure 6 a). Morphologically, xenograft tumor cells displayed an undifferentiated spindle-shaped phenotype, large nuclei, and reduced cytoplasmic size. This morphology was strongly altered by Hakin-1 treatment, showing an induction of tumor differentiation and an increase in cytoplasmic size ( Figure 6 b). In addition, we found tumor cell infiltration in the blood vessels, whereas no infiltration was detected in Hakin-1-treated xenograft tumors ( Figure 6 c). In addition, analysis of two proliferation markers (Ki67 and mitotic index) showed that Hakin-1 significantly reduced the number of Ki67-positive cells and the mitotic index ( Figure 6 de), while no effect on cell apoptosis was detected ( Fig.10 ), which further emphasizes the inhibitory effect of Hakin-1 on cell proliferation in vivo. We also visualized blood vessels in tumor sections by immunohistochemistry using the CD31 angiogenesis marker. A strong reduction in the number of blood vessels in Hakin-1-treated xenograft tumors was quantified compared to untreated tumors, indicating its inhibitory effect on angiogenesis ( Figure 6f). Interestingly, no damage was observed in the lung and kidney tissues of Hakin-1-treated nude mice, showing normal morphological structures, which supports that Hakin-1 treatment inhibits tumor growth in nude mice and is apparently free of systemic toxicity ( Fig.11 ).

[0404] Hakin-1 treatment reduces N-cadherin mesenchymal markers and micrometastasis formation in lung tumor xenografts in vivo

[0405] We further evaluated the in vivo effects of Hakin-1 on the reversal of EMT, a key process in tumor progression and cell invasion. First, we confirmed that Hakai protein expression levels were not affected by Hakin-1 in xenograft tumors of nude mice ( Figure 7 a), which further supports previous in vitro results showing that Hakin-1 inhibits Hakai activity through its action on its ubiquitin ligase activity without altering Hakai protein expression ( Figure 1 ). As expected, given the complete loss of E-cadherin in Hakai-MDCK cells, no E-cadherin was detected in Hakai-MDCK xenograft tumors in nude mice in the presence or absence of Hakin-1 ( Figure 7 b) However, when we investigated the expression levels of the mesenchymal marker N-cadherin, another hallmark of EMT, we found that N-cadherin expression was strongly reduced in tumor xenografts treated with Hakin-1 compared to DMSO-treated mice ( Figure 7 c). These data further support that Hakin-1 inhibits mesenchymal-type tumor cells by reducing the N-cadherin mesenchymal marker. Given that E-cadherin, the best described target of Hakai, is completely absent in Hakai-MDCK mesenchymal tumors, we also extended our studies by analyzing another described target of Hakai, cortical actin. Cortical actin is a cytoskeletal protein and one of the major substrates of Src kinase. Interestingly, cortical actin expression was only detected in the cytoplasm of Hakai-MDCK xenograft tumors and its expression was restored by Hakin-1 treatment ( Figure 7 d), which further supports that Hakin-1 can inhibit the induction of ubiquitination and degradation of cortical actin by Hakai. To determine whether Hakin-1 can affect cancer metastasis, lung tissues from nude mice were analyzed by H&E staining; however, no distant metastasis was detected under the experimental conditions ( Figure 7e). Therefore, in order to detect the possible presence of micrometastases, we performed quantitative PCR by analyzing the presence of Hakai-MDCK DNA in the lungs. To this end, the presence of HA-tagged Hakai in Hakai-MDCK cells was measured by using two different specific primers (one designed for the HA epitope and the second primer designed for Hakai). Hakin-1 led to a significant reduction in the micrometastases detected in the lungs of Hakai-MDCK xenograft mice compared to control DMSO-treated mice, while no micrometastases were detected in the lungs of mice injected with MDCK ( Figure 7 f). These results highlight that Hakin-1 inhibits metastasis to the lung in vivo.

[0406] Effects of Hakin-1 analogs on cytotoxicity and Hakai-induced ubiquitination

[0407] Next, we investigated the effects of the selected analogs on HT29 colon cancer cells. First, we analyzed the cytotoxic effects of the following analogs on the HT-29 colon tumor cell line: ketophenyls: A-1, A-7, A-8, and A-9; ketoheteroaryls: A-23 and A-25; cyclic amides: A-10 and A-16; and benzamides: A-6.1. Significant inhibitory responses were shown for ketophenyls A-7, A-8, A-9 and ketoheteroaryls A-23, A-25, but no cytotoxic effects were detected by the effects of ketophenyls A-1, cyclic amides A-10 and A-16, and benzamides A-6.1 ( Fig.12 ). We then investigated the effect of specifically selected analogs on ubiquitination induced by the E3 ubiquitin ligase Hakai by using cultured tumor cells. 293T cells were transfected with Src, Hakai, and ubiquitin in the presence of specific analog inhibitors or DMSO as a control. By using the ketophenyl A-7 analog, a significant reduction in ubiquitination mediated by Hakai was observed in a dose-dependent manner ( Fig.13 ), showed an effect on the reduction of Hakai activity without affecting Hakai protein levels. On the other hand, the analog ketophenyl class A-9 reduced Hakai-mediated ubiquitination at the tested concentrations, but reduced Hakai protein levels. In addition, cyclic amides A-10 and A-16 slightly reduced Hakai-mediated ubiquitination without affecting protein levels. Finally, low concentrations of ketoheteroaryl class A-23 were also detected to inhibit Hakai activity.

[0408] 2.3. Other results

[0409] Metabolite analysis report of Hakin-1 in cryopreserved hepatocytes

[0410] In an in vivo model, we observed a strong antitumor effect by using low concentrations of Hakin-1 (5 mg / kg / three days per week), while in vitro antitumor effects were observed using very high concentrations (100-200 μM). Therefore, we decided to analyze whether intermediate metabolites from Hakin-1 could be identified and whether the intermediate metabolites were involved in the antitumor effect. To address this point, we performed metabolite profiling of Hakin-1 in mouse cryopreserved hepatocytes to determine whether any metabolites could be formed. Metabolite profiling of Hakin-1 was performed in mouse cryopreserved hepatocytes using a 10 minute sample and a 20 minute sample compared to a 0 minute control sample. The identified metabolites of Hakin-1, named MKH1, can be found in Table 1, with extended data in Tables 2 to 3. Representative chromatograms and spectra of Hakin-1 and the reported metabolites as well as the proposed assignment of the parent (Hakin-1) and the metabolite (named MKH1) can be seen in Figure 15-19 Our results showed that only one metabolite was identified in mouse hepatocyte samples, and a structural elucidation and synthetic pathway have been provided for this metabolite (Tables 1 and Fig. 20 Interestingly, only 5.99% of Hakin-1 remained in the mouse cryopreserved hepatocyte samples after 20 minutes compared to the 0-minute control sample (97.2%), while 94% of MKH1 was present at 20 minutes compared to the 0-minute control sample (2.72%). We then decided to determine the stability of Hakin-1 and MKH1 in cryopreserved hepatocytes from humans and mice to compare their stabilities. The results showed that the half-life of Hakin-1 in mice (T 1 / 2 ) was 5.92 minutes, while in humans 77.3% of the compound was still present after 60 minutes (estimated half-life of 140 minutes). In the case of the metabolite (MKH1), the compound was stable after 60 minutes, in fact, about 95%-100% of the compound was still present in both mouse and human species. These data indicate that the identified metabolite MKH1 is more stable than the Hakin-1 parent compound, suggesting that MKH1 could be involved in the anti-tumor effects observed in vivo.

[0411] Effects of newly selected compounds on E-cadherin expression and cell cycle analysis

[0412] Given the antitumor effects of Hakin-1 observed in vitro and in vivo, we decided to select analogs with the goal of improving the potency and / or efficacy of Hakin-1. To this end, in collaboration with medicinal chemists, several compounds were selected based on their potential to form the same molecular interactions with the target protein but with a new structure ( Fig.21), and the effect on E-cadherin expression was analyzed by immunofluorescence and quantified after 48 h of treatment with each compound.

[0413] We first used the HT-29 colon cancer cell line and imaged the cells using Vala's structured illumination microscopy platform and analyzed the The cell-cell junction algorithm was used to quantify the expression of E-cadherin at cell-cell contacts, thereby analyzing the effects of K1 and K2 on E-cadherin expression. Fig. 22 As shown in A, significantly increased E-cadherin levels were observed by immunofluorescence of compounds K1 and K2. Fig. 22 B includes quantification of immunofluorescence images. The results show that treatment with 100 μM K1 compound significantly increased E-cadherin levels at cell-cell contacts compared to DMSO vehicle control. Significant differences were also detected for K2 compound. As a positive control, we used nocodazole, a microtubule polymerization inhibitor, which significantly increased E-cadherin expression levels by 32%, 42%, and 47% at concentrations of 250 ng / ml, 500 ng / ml, or 1000 ng / ml for 24 h (P < 0.0001 for all three conditions) ( Fig.23 In addition, the effects of the compounds on cell cycle analysis were analyzed ( Fig.24 ). After 48 hours of treatment with 100 μM of K1 and K2 compounds, cells were arrested in the G0 / G1 phase, thus reducing the cell number ( Fig.24 Using nocodazole as a positive control, a sharp shift to G2 / M phase arrest was shown compared to the DMSO vehicle control ( Fig.25 ). In conclusion, K1 and K2 are novel analogs that increase the levels of E-cadherin at cell-cell contacts and arrest cells in the G0 / G1 phase compared to DMSO controls.

[0414] Then, the effect of the identified Hakin-1 metabolite (MKH1) on E-cadherin expression was also investigated by immunofluorescence, showing that E-cadherin expression was significantly increased, even at low concentrations (50 μM, 20 μM) ( Fig.26 In addition, K8 and K33 were tested by immunofluorescence ( Figure 27-28 Our results showed that K8 and K33 increased E-cadherin levels at cell-cell contacts compared with DMSO vehicle controls ( Figure 27-28 ). K1 was used as a positive control. Fig.29 In this study, we showed that MKH1 and K8 compounds inhibited cell proliferation in human colon cancer cell lines, as we have already shown for Hakin-1 ( Figure 4 ) and K1( Fig.24 ) as displayed.

[0415] Effects of selected compounds on invasion and cell viability of tumor cultured cells

[0416] Next, we investigated the effects of selected compounds K1, MKH1, and K8 on cell invasion compared to Hakin-1. We used the following concentrations (1 μM, 10 μM, 50 μM) and DMSO as a control to test the effects. A potent inhibitory effect on cell invasion was detected using these inhibitors ( Fig.30 ). Interestingly, Hakin-1 and K1 showed a significant reduction in invasion assays between 1-10 μM, whereas MKH1 and K8 achieved 50% inhibition at approximately 1 μM, emphasizing that MKH1 and K8 have a higher potency compared to Hakin 1 and K1. In addition, the cytotoxic effects of these compounds were investigated in HCT116 human colon cancer cells and HEK293T non-cancer cells. We used the concentrations tested in the previous assay. Interestingly, as Fig.31 As observed in the literature, no cytotoxic effects of K1 and MKH1 were detected in either human colon cancer cells or human non-cancer cells at the tested concentrations. In contrast, K8 showed an inhibitory response in cancer cells, while no effect was detected in non-tumor cells. These results further emphasize that K1, K8, and MKH1 increase E-cadherin expression, reduce cell proliferation and cell invasion, while having no cytotoxic effects in non-tumor cells.

[0417] Effects of K1, MKH1 and K8 inhibitors on Hakai-induced ubiquitination

[0418] Finally, we investigated the effects of K1, MKH1, and K8 inhibitors on ubiquitination induced by the E3 ubiquitin ligase Hakai. HEK293T cells were transfected with Hakai and ubiquitin in the presence of the selected inhibitors or DMSO as a control as described in Materials and Methods. Fig.32 As shown, K1 and MKH1 reduced ubiquitination at 20 μM and 100 μM, whereas K8 reduced ubiquitination at 20 μM.

[0419] Collectively, our data indicate that the tested compounds displayed antitumor activity by inhibiting Hakai-mediated ubiquitination, increasing E-cadherin expression at cell-cell contacts, and reducing cell invasion without cytotoxic effects in non-tumor epithelial cells.

[0420] Table 1. Summary of Hakin-1 metabolites in hepatocyte stability assay samples

[0421]

[0422] Table 2. Summary of the areas and compound percentages of Hakin-1 and MKH1 metabolites in mouse hepatocyte samples at 0 min (control) and 10 and 20 min

[0423]

[0424] Table 3. Summary of metabolites observed in mouse hepatocyte samples at 20 minutes

[0425]

Claims

1. A compound selected from any one of the following list: a. Compounds having the following chemical structure: b. Compounds of formula (II) and pharmaceutically acceptable salts thereof: in: -A represents a group selected from C1-C4 alkyl, phenyl; wherein any one of these groups is unsubstituted or substituted by 1, 2 or 3 groups independently selected from the following: a) Halogen atoms, hydroxyl groups, -N(R a )R b 、-C(=O)R a 、-C(=O)OR a ; b) a linear or branched C1-C6 alkyl group; -R a and R b each independently represents a methyl group; -y is 0 or 1; -R 1 and R 2 each independently represents a group selected from hydrogen or aryl; - z is an integer selected from 0 or 2; -R 3 represents a halogen atom or -OR a A group in which R a represents a methyl group; wherein the group R 3 If present, replaces the one present in R 3 a hydrogen atom of one of the CH groups in the attached benzene ring; Wherein, the compound is not: c. Compounds of formula (III) and pharmaceutically acceptable salts thereof: in: -A represents a group selected from hydrogen and C1-C4 alkyl, wherein the C1-C4 alkyl is unsubstituted or substituted by 1, 2 or 3 groups independently selected from the following: a)-OR a , where R a represents a methyl group; b) C3-C6 cycloalkyl; c) phenyl or C5-C6 heteroaryl, each of which is unsubstituted or substituted by a halogen atom, a cyano group, a C1-C3 alkyl group or a cyclopropyl group; -y is 0; -B is a nitrogen atom or an oxygen atom; -R 1 is present only when B is a nitrogen atom and is represented by a group selected from hydrogen or methyl; -R 2 and R 3 represents hydrogen; - z is an integer selected from 0 or 2; -R 4 represents a chlorine atom or -OR a A group in which R a represents a methyl group; wherein the group R 4 If present, replaces the one present in R 4 a hydrogen atom of one of the CH groups in the attached benzene ring; Wherein, the compound is not:

2. The compound according to claim 1, wherein The compounds are those of list a).

3. The compound according to claim 1, wherein The compound is a compound of formula (II) in list b).

4. The compound according to claim 1, wherein The compound is a compound of formula (III) in list c).

5. The compound according to claim 3, wherein the compound is selected from the following list, or a pharmaceutically acceptable salt thereof:

6. The compound according to claim 4, wherein the compound is selected from the following list, or a pharmaceutically acceptable salt thereof:

7. Use of the compound according to claim 2 for preparing a drug for treating cancer, wherein: The cancer is selected from the list consisting of rectal or colon cancer, breast cancer, prostate cancer, bladder cancer, liver cancer and kidney cancer.

8. Use of the compound according to claim 3 or 4 for preparing a drug for treating cancer, wherein: The cancer is selected from the list consisting of rectal or colon cancer, breast cancer, prostate cancer, bladder cancer, liver cancer and kidney cancer.

9. Use of the compound according to claim 5 or 6 for preparing a drug for treating cancer, wherein: The cancer is selected from the list consisting of rectal or colon cancer, breast cancer, prostate cancer, bladder cancer, liver cancer and kidney cancer.

10. A pharmaceutical composition comprising the compound according to claim 2 or a pharmaceutically acceptable salt thereof.

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