An-metastac agents and uses thereof

Abstract

The present invenon provides new chemical structures showing a highly potent an-invasive in vitro effect at nanomolar (nM) range, without showing a cytotoxic or an-proliferave effect at this range of concentraon. Moreover, our in vivo data strongly supports that these compounds, with their remarkable an-invasive effects, can be used as metastasis-targeng drugs, without affecng tumour growth or causing apparent systemic toxicity in vivo. These findings reveal a therapeuc window in which the compounds of the invenons exhibit robust an-invasive and an-migratory acvity at nanomolar (nM) concentraons, without affecng cytotoxicity or cell proliferaon. Furthermore, the an-invasive efficacy is significantly enhanced when combined with convenonal cytostac or cytotoxic drugs, which primarily target cell proliferaon or induce cell death.

Description

[0001] Anti-metastatic agents and uses thereof

[0002] TECHNICAL FIELD

[0003] The present invention relates to a novel class of compounds and to compositions comprising the same as well as their used as medicaments in the treatment of cancer.

[0004] STATE OF THE ART

[0005] Cancer is characterized by abnormal cellular proliferation and the potential to spread to other parts of the body. In solid tumours, the ability of cancer cells to spread is typically linked to local invasion and metastasis. The inhibition of cancer cells from spreading from the primary tumour to distant organs typically targets pathways involved in cell adhesion, migration, invasion, and the establishment of new metastatic sites. The mechanisms underlying proliferation are distinct from those related to cell migration and invasion, although there is often some overlap and interaction between these processes. Therefore, drug discovery efforts should focus on two strategies: one targeting proliferation and the other targeting metastasis1.

[0006] Metastases are responsible of about 90% of cancer deaths in solid tumours therefore there is a clear unmet clinical need to develop more effective treatments and improve the prognosis for patients with advanced cancer. The acquisition of an invasive phenotype is the key feature that distinguishes malignant tumours from benign ones2.

[0007] Tumour shrinkage is rarely complete or maintained and does not necessarily indicate an anti-metastatic effect. Medicinal chemists often concentrate on anti-proliferative agents because regulatory approval frequently requires evidence of tumour shrinkage. However, this approach underestimates the impact on cancer migration and invasion, leading to frustration among patients and oncologists due to the lack of effective anti-metastatic drugs3.

[0008] During carcinoma progression, epithelial cells can undergo a program named epithelial-to-mesenchymal transition (EMT), priming their migration from the primary tumour, their invasion, dissemination and metastasis formation. The loss of E-cadherin, a crucial tumour-suppressor protein responsible for maintaining cell-cell adhesion in epithelial tissue, is perhaps the most well-established hallmark of EMT. Indeed, loss of E-cadherin has been associated with EMT, increased invasiveness and metastasis. The loss of the cell-cell adhesion molecule E-cadherin is often associated with the progression from adenoma (benign tumour) to carcinoma (malignant tumour) in several types of cancer4,5 6.The E3 ubiquitin-ligase Haikai was the first posttranslational regulator of the E-cadherin stability, which mediates the ubiquitination and subsequent degradation of E-cadherin, leading to the disappearance of E-cadherin at cell-cell contact7. Haki regulates E-cadherin inducing EMT, thereby promoting cell migration and invasion across various cancers such as colon, gastric, lung, and hepatocellular carcinoma, among others. Moreover, Hakai is higher expressed in cancerous tissues compared to healthy tissues813. The higher expression of Hakai in several carcinomas together with its role in EMT, migratory and invasive capabilities underscore its significant contribution to tumour progression, highlighting its potential as a target for therapeutic intervention in combating cancer metastasis. Hakai is a new class of RING-finger type E3 ubiquitin-ligase, characterized by a novel phosphotyrosinebinding domain named HYB (Hakai pTyr-binding, where pTyr stands for phosphotyrosine), which targets E-cadherin. HYB is structurally different from other pTyr-binding domains of E3 ubiquitinligases. The uniqueness of the HYB region of Hakai is an attractive new drug target due to its novel structural features14.

[0009] By virtual screening using a library of 5M compounds, we identified a novel hit candidate, named Hakin-1 (Hakai inhibitor-1), that reduces Hakai-dependent ubiquitination and Hakai-dependent ubiquitination of E-cadherin. Hakin-1 inhibits cell proliferation, cell migration and invasion, and these effects were accompanied by the induction of epithelial markers and a reduction of mesenchymal markers in vitro. Hakin-1 inhibited carcinoma growth and tumour progression both in vitro and in vivo, in a mouse tumour xenograft model, without apparent systemic toxicity in mice15.

[0010] However, despite the effect observed for the previously reported Hakin-1 analogue, one objective of the present invention is developing a more potent and effective Hakai inhibitors that exhibit enhanced therapeutic effects with low toxicity. The present invention provides for such class of compounds, which includes enantiomers and pharmaceutically acceptable salts thereof and that, at the same time, represent excellent anti-cancer agents, effective in treating various cancers, including carcinomas.

[0011] References

[0012] 1. Steeg, P.S. (2016). Targeting metastasis. Nat Rev Cancer 16, 201-218. 10.1038 / nrc.2016.25.

[0013] 2. Steeg, P.S., and Theodorescu, D. (2008). Metastasis: a therapeutic target for cancer. Nat Clin Pract Oncol 5, 206-219. 10.1038 / ncponcl066.

[0014] 3. Anderson, R.L., Balasas, T., Callaghan, J., Coombes, R.C., Evans, J., Hall, J.A., Kinrade, S., Jones, D., Jones, P.S., Jones, R., et al. (2019). A framework for the development of effective anti- metastatic agents. Nat Rev Clin Oncol 16, 185-204. 10.1038 / s41571-018-0134-8.4. Kall uri, R., and Weinberg, R.A. (2009). The basics of epithelial-mesenchymal transition. J Clin Invest 119, 1420-1428. 10.1172 / JCI39104.

[0015] 5. Lamouille, S., Xu, J., and Derynck, R. (2014). Molecular mechanisms of epithelial-mesenchymal transition. Nat Rev Mol Cell Biol 15, 178-196. 10.1038 / nrm3758.

[0016] 6. Yang, J., Antin, P., Berx, G., Blanpain, C., Brabletz, T., Bronner, M., Campbell, K., Cano, A., Casanova, J., Christofori, G., et al. (2020). Guidelines and definitions for research on epithelial- mesenchymal transition. Nat Rev Mol Cell Biol. 10.1038 / s41580-020-0237-9.

[0017] 7. Fujita, Y., Krause, G., Scheffner, M., Zechner, D., Leddy, H., Behrens, J., Sommer, T., and Birchmeier, W. (2002). Hakai, a c-Cbl-like protein, ubiquitinates and induces endocytosis of the E-cadherin complex. Nat Cell Biol 4, 222-231. ncb758 [pii]10.1038 / ncb758.

[0018] 8. Zhou, W.J., Geng, Z.H., Chi, S., Zhang, W., Niu, X.F., Lan, S.J., Ma, L., Yang, X., Wang, L.J., Ding, Y.Q., and Geng, J.G. (2011). Slit-Robo signaling induces malignant transformation through Hakai-mediated E-cadherin degradation during colorectal epithelial cell carcinogenesis. Cell Res 21, 609-626. cr201117 [pii] 10.1038 / cr.2011.17.

[0019] 9. Weng, C.H., Chen, L.Y., Lin, Y.C., Shih, J.Y., Tseng, R.Y., Chiu, A.C., Yeh, Y.H., Liu, C., Lin, Y.T., Fang, J.M., and Chen, C.C. (2019). Epithelial-mesenchymal transition (EMT) beyond EGFR mutations per se is a common mechanism for acquired resistance to EGFR TKL Oncogene 38, 455-468. 10.1038 / S41388-018-0454-2.

[0020] 10. Figueroa, A., Kotani, H., Toda, Y., Mazan-Mamczarz, K., Mueller, E.C., Otto, A., Disch, L., Norman, M., Ramdasi, R.M., Keshtgar, M., et al. (2009). Novel roles of hakai in cell proliferation and oncogenesis. Mol Biol Cell 20, 3533-3542. 10.1091 / mbc.e08-08-0845. 11. Castosa, R., Martinez-Iglesias, O., Roca-Lema, D., Casas-Pais, A., Diaz-Diaz, A., Iglesias, P., Santamarina, I., Grana, B., Calvo, L., Valladares-Ayerbes, M., et al. (2018). Hakai overexpression effectively induces tumour progression and metastasis in vivo. Sci Rep 8, 3466.

[0021] 10.1038 / s41598-018-21808-w.

[0022] 12. Liu, M., Jiang, K., Lin, G., Liu, P., Yan, Y., Ye, T., Yao, G., Barr, M.P., Liang, D., Wang, Y., et al.

[0023] (2018). Ajuba inhibits hepatocellular carcinoma cell growth via targeting of -catenin and YAP signaling and is regulated by E3 ligase Hakai through neddylation. J Exp Clin Cancer Res 37, 165. 10.1186 / S13046-018-0806-3.

[0024] 13. Aparicio, L.A., Valladares, M., Blanco, M., Alonso, G., and Figueroa, A. (2012). Biological influence of Hakai in cancer: a 10-year review. Cancer Metastasis Rev 31, 375-386.

[0025] 10.1007 / S10555-012-9348-X.14. Mukherjee, M., Chow, S.Y., Yusoff, P., Seetharaman, J., Ng, C., Sinniah, S., Koh, X.W., Asgar, N.F., Li, D., Yim, D., et al. (2012). Structure of a novel phosphotyrosine-binding domain in Hakai that targets E-cadherin. EMBO J. emboj2011496 [pii]10.1038 / emboj.2011.496.

[0026] 15. Martinez-Iglesias, O., Casas-Pais, A., Castosa, R., Diaz-Diaz, A., Roca-Lema, D., Concha, A., Cortes, A., Gago, F., and Figueroa, A. (2020). Hakin-1, a New Specific Small-Molecule Inhibitor for the E3 Ubiquitin-Ligase Hakai, Inhibits Carcinoma Growth and Progression. Cancers (Basel) 12. 10.3390 / cancersl2051340.

[0027] BRIEF DESCRIPTION OF THE FIGURES AND TABLE 1

[0028] Figure 1. Chemical structures and synthetic routes of new compounds. (A-B) Chemical structures (A) and scheme of synthesis (B) of K-105 compound with molecular formula C17H19N5O (C-D) Chemical structures (C) and scheme of synthesis (D) of K-402 compound with molecular formula CigHiaCINgO.

[0029] Figure 2. Characterization of K-105 compound. (A) HPLC spectra and liquid chromatography-mass spectrometry (LC-MS) and (B)1H-NMR spectrum are shown.

[0030] Figure 3. Characterization of K-402 compound. (A) HPLC spectra and liquid chromatography-mass spectrometry (LC-MS) and (B)1H-NMR spectrum are shown.

[0031] Figure 4. New compound increase E-cadherin expression at cell-cell contacts in epithelial tumour cells. Immunofluorescence images and quantification of E-cadherin expression at cell-cell contacts in HT-29 human colon cancer cells treated with K-105 (A) and K-402 (B) compounds after 48h at indicated concentrations. Statistical quantification of E-cadherin staining was calculated using five photographs per well. The intensity of ten E-cadherin based cell-cell contacts were taken in every photograph (50 cell-cell contacts in total). The results are represented as the mean ± SD of staining intensity signal scoring per area of at least three independent experiments (*p < 0.05; *** p < 0.001; ****; p < 0.0001). Calibration and quantification of the images were obtained with a 20X objective and performed with ImageJ software. Scale bar, 50 pm.

[0032] Figure 5. Effect of selected compounds on cell viability in epithelial tumour cells. Colon tumour HCT116 cells were treated with increasing range of concentrations (20 pM, 50 pM, 100 pM) of K-105 (A) or K-402 (B) compounds for 48h and cell viability was measured by MTT assay. Assay was performedin 6 replicates and represented as mean ± SD of at least three independent experiments (*p < 0.05; ** p < 0.01; ***p < 0.001).

[0033] Figure 6. Effect of selected compounds on cell proliferation in epithelial tumour cells. HCT116 human colon cancer cells were treated with K-105 (A) or K-402 (B) at indicated concentrations for 48h and proliferation was measured in 6 replicates per condition by a BrdU assay as indicated in Material and Methods. Results are expressed as mean ± SD of three independent experiments (** p < 0.01).

[0034] Figure 7. Selected compounds reduce cell migration and cell invasion of colon epithelial tumour cells.

[0035] Migration and invasion assay in the human HCT116 colon cancer cell line was performed as described in Materials and Methods section. Cells were treated with K-105 and K-402 at indicated concentrations for 72h. Representative images for migration assays were taken using the 10X objective (left panel, A and C) and quantification of the photographed migrative cells are shown (right panel, A and C). The results are represented as the mean ± SD of staining intensity signal scoring per area of at least three independent experiments (****p < 0.0001). For invasion assays, dose response curve to drug treatment was represented and IC50 calculated (B and D). The means of at least three independent experiments are included.

[0036] Figure 8. K-402 compound reduces cell migration and cell invasion in a panel of colon cancer cell lines. Migration assay in human DLD-1, Lovo and HT-29 colon cancer cell lines and invasion assay in human DLD-1 and COLO320-DM colon cancer cell line were performed as described in Materials and Methods section. Cells were treated with K-402 at 200 nM for 72h. Quantification of the migrative (A) and invasive (B) cells is shown. The results are represented as the mean ± SD of staining intensity signal scoring per area of at least two independent experiments (*p < 0.05; ** p < 0.01; ***p < 0.001).

[0037] Figure 9. K-402 compound reduces cell migration and cell invasion in different human tumour cell lines. (A) Migration assay and (B) invasion assay was performed as previously described in Material and Methods section and quantification of the migrative and invasive cells is shown. Human cells analysed includes: N87 gastric cancer, A549 and H1299 lung adenocarcinoma, HepG2 hepatocellular carcinoma, PC3 prostatic adenocarcinoma and ACHN kidney adenocarcinoma. Cells were treated with K-402 at 200 nM for 72h. The results are represented as the mean ± SD of staining intensity signal scoring per area of at least two independent experiments (*p < 0.05; ** p < 0.01; ***p < 0.001, ****p < 0.0001).Figure 10. K-402 effect on primary tumour growth and lung metastasis in an orthotopic xenograft murine model of colorectal cancer. (A) Efficacy of K-402 on primary tumour growth. The weight (g) of the colon tumour xenografts in K-402-treated mice and vehicle-treated control mice bearing human HCT116-Luc2 colon cancer cells in an orthotopic colorectal cancer mouse model was measured (n = 8 tumours / group, ns = non-significant differences). (B) The number of mice with lung metastases is shown for the K-402-treated groups (10 mg / kg and 50 mg / kg) and the vehicle control group. Metastatic foci (>50 pm) were detected by immunohistochemical staining for human GAPDH in paraffin-embedded tumour sections.

[0038] Figure 11. K-402 reduces number and size of metastatic foci in the lung of an orthotopic mice model of colorectal cancer. (A) H&E staining of lung tissue sections from K-402-treated mice versus vehicle control mice for metastasis assessment in the orthotopic human colon tumour model (upper panel images). Immunohistochemical staining for human GAPDH was used to identify metastatic foci (>50 pm) in paraffin-embedded lung sections (lower panel images). Images were obtained with a 10x objective. Scale bar, 100 pm. (B-C) Quantification of the number (B) and size (C) of metastatic nodules. Each lung was divided into five distinct regions, and the number and size of metastatic lesions were quantified in each region to determine the extent of metastasis using automated analysis with Python. Statistically significant differences in the number and size of metastatic nodules were observed between the K-402-treated and vehicle control groups (One-way ANOVA). No statistically significant differences were found between the 10 mg / kg and 50 mg / kg treatment groups. Data are represented as scatter plots, with values expressed as means ± SD (*p < 0.05; **p < 0.01; ***p < 0.001).

[0039] Figure 12. K-402 reduces metastatic burden in the lung of an orthotopic mice model of colorectal cancer. (A) The metastatic burden for each tissue section was calculated based on the total area occupied by metastases. This burden was quantified and expressed in mm2, according to the methodology outlined in the Materials and Methods section. Statistically significant differences in metastatic burden were observed between the K-402-treated groups and the vehicle control group (One-way ANOVA). (B) The tumour burden is represented as a percentage (%). A 67.82% reduction in metastatic burden was observed with K-402 treatment at 10 mg / kg compared to the vehicle group, while a 71.28% reduction was observed at 50 mg / kg compared to the vehicle group (** p < 0.01).

[0040] Figure 13. K-402 recovers E-cadherin and reduces N-cadherin expression in primary tumors of an orthotopic mice model of colorectal cancer. The expression levels of E-Cadherin (A) and N-Cadherin (B) was analysed in colon tumour xenografts in K-402-treated mice (10 mg / ml and 50 mg / ml) andvehicle-treated control mice bearing human. A representative image per condition is shown (left panel, A and B) and the quantification, as described in Materials and Methods, was represented in violin plots, included in the right panel (A and B, ****p < 0.0001).

[0041] Figure 14. K-402 does not induce body weight reduction and preserves the intact cell morphology and tissue structure of the liver, kidney, and heart in mice. (A) Delta (A) body weight (g) changes during treatment showed no statistically significant differences in final versus initial body weight between the treated and vehicle control groups at the indicated concentrations (Two-way ANOVA). (B) H&E staining of liver (upper panel), heart (middle panel), and kidney (bottom panel) from mice treated with K-402 at 10 mg / kg, 50 mg / kg, or vehicle control are shown. Images were captured with a lOx objective. Scale bar, 100 pm.

[0042] Figure 15. Effect on cell invasion of the combination of K-402 compound and standard chemotherapeutic drugs. Invasion assay in the human HCT116 colon cancer cell line was performed as described in Materials and Methods. Cells were treated with K-402 in absence or presence of standard chemotherapeutic drugs including FOLFOX (A) or FOLFIRI (B) at indicated concentrations, 24 h before seeding cells into chambers. Representative images for invasion assays were taken 72 h after seeding into chambers, using 10X objective and quantification of the photographed invasive cells are shown. The results are represented as the mean ± SD of staining intensity signal scoring per area of at least three independent experiments (** p < 0.01; ****p < 0.0001).

[0043] Table 1. Characteristics of the selected compounds.

[0044]

[0045] (ns) no significative effect The methodology for table 1 is included in the example section (Immunofluorescence for E-cadherin recovery and cell viability to analyse cytotoxicity) and (proliferation, migration and invasion assays).SUMMARY OF THE INVENTION

[0046] The inventors have found new chemical structures showing a highly potent anti-invasive effect at nanomolar (nM) range, without showing a cytotoxic or anti-proliferative effect at this range of concentration. Our findings reveal a therapeutic window in which the compounds of the inventions exhibit robust anti-invasive and anti-migratory effects at nanomolar (nM) concentrations, without affecting cytotoxicity or cell proliferation. In fact, as shown in Table 1, we have a more effective compounds that exhibit increased potency and therapeutic effect on the inhibition of invasion and migration at nM concentration compared to previously reported compounds15, without affecting cytotoxicity in vitro. Importantly, the effect observed on cell migration in colon cancer cells was similar to that seen in other cancer cell lines, including lung adenocarcinoma (A549) and gastric adenocarcinoma (N87). This suggests that the compounds may consistently impact cell migration across various cancer types. Moreover, in vivo results using an orthotopic xenograft murine model of colorectal cancer confirm that K-402 exhibits robust antimetastatic efficacy without affecting tumour growth or causing apparent systemic toxicity. Finally, a synergistic effect was observed when the K-402 and FOLFOX were combined, significantly enhancing their effectiveness in inhibiting invasion in vitro. These findings strongly support the potential of these compounds as metastasis-targeting drugs, given their remarkable anti-invasive effects, and suggest they could enhance the efficacy of conventional cytostatic or cytotoxic drugs, which primarily target cell proliferation or induce cell death.

[0047] Thus, in a first aspect the present invention relates to compounds of formula (I)

[0048]

[0049] wherein:

[0050] Ri, R2, R3, R4 and R5are independently selected from H; -C1-4 alkyl including branched -C1-4 alkyl, optionally substituted with 1-3 fluorine atoms; -C1-4 alkenyl including -C1-4 branched alkenyl, optionally substituted with 1-3 fluorine atoms; cyclic alkyl, preferably -C3-4 cyclic alkyl (e.g., cyclopropyl), aryl and heteroaryl optionally substituted with 1-3 fluorine atoms; -OR'; -S(O)nR'; -OC(O)R'; -C(O)R'; -C(O)OR', -C(O)NR'2, -CN, NR'R' and halogen, preferably -Cl or -F; wherein if two of Ri, R2, R3, 4 and R5, are selected from -C1-4 alkyl, -C1-4 alkenyl, -OR', -S(O)nR', or NR'R', and are adjacent, they may be joined together to form a cyclic structure;

[0051] n = 0,1 or 2;

[0052] each R' is independently selected from H; -C1-4 alkyl including branched -C1-4 alkyl (e.g., isopropyl); -Ci-4 alkenyl including branched -C1-4 alkenyl; and cyclic alkyl, preferably -C3-4 cyclic alkyl (e.g., cyclopropyl); wherein each of these groups is optionally substituted with 1-3 fluorine atoms;

[0053] Rs and R7are independently selected from H; -C1-4 alkyl including branched -C1-4 alkyl; -C1-4 alkenyl including branched -C1-4 alkenyl; and cyclic alkyl, preferably -C3-4 cyclic alkyl (e.g., cyclopropyl); wherein each of these groups is optionally substituted with 1-3 fluorine atoms;

[0054] Rg Rg and Rio are independently selected from H; -C1-4 alkyl including branched -C1-4 alkyl; -C1-4 alkenyl including branched -Ci.4alkenyl; cyclic alkyl, preferably -C3-4 cyclic alkyl (e.g., cyclopropyl), wherein each of these groups is optionally substituted with 1-3 fluorine atoms; (N)mR"R" and -CN; wherein where two of Rg Rg and Rio, are independently selected from H, -C1-4 alkyl including branched -C1-4 alkyl; -C1-4 alkenyl including branched -C1-4 alkenyl, cyclic alkyl, preferably -C3-4 cyclic alkyl (e.g., cyclopropyl), wherein each of these groups is optionally substituted with 1-3 fluorine atoms, or (N)mR"R", and are adjacent, they may be joined together to form a cyclic structure including:

[0055]

[0056] m= 0,1, 2;

[0057] Rn is H or C1-4 alkyl optionally substituted with 1-3 fluorine atoms; and

[0058] each R" is independently selected from H; C1-4 alkyl including branched C1-4 alkyl; C1-4 alkenyl including branched C1-4 alkenyl; cyclic alkyl, preferably C3-4 cyclic alkyl (e.g., cyclopropyl), wherein each of these groups is optionally substituted with 1-3 fluorine atoms;or a stereoisomer or a pharmaceutically acceptable salt thereof;

[0059] preferably with the proviso that said compound is not lH-tetrazole-l-ethanol, 5-[(4-methylphenyl)amino]-a-[3-(trifluoromethyl)phenyl].

[0060] In particular, preferably with the proviso that said compound is not a compound having the following chemical structure:

[0061]

[0062] In particular, preferably with the proviso that said compound is not a compound having CAS Registry Number: 2995382-39-1.

[0063] In a second aspect of the present invention relates to pharmaceutical or veterinary compositions comprising therapeutically effective amounts of compounds of the first aspect of the invention and preferably adequate amounts of pharmaceutically acceptable excipients.

[0064] In a third aspect the present invention relates to the compounds of the first aspect of the invention and to the compositions of the second aspect of the invention for use as a medicament.

[0065] In a fourth aspect the present invention relates to the compounds of the first aspect of the invention and to the compositions of the second aspect of the invention for use in the treatment or prevention in an animal, including a human, of a cancer disease or disorder. In a fifth aspect the present invention relates to the use of the compounds of the first aspect of the invention for the manufacture of a medicament for the treatment or prevention in an animal, including a human, of a cancer disease or disorder.

[0066] DETAILED DESCRIPTION OF THE INVENTION

[0067] In a first aspect the present invention relates to compounds of formula (I) or a stereoisomer or a pharmaceutically acceptable salt thereof, wherein:

[0068] Ri, R2, R3, R4and R5are independently selected from H; C1-4 alkyl including branched C1-4 alkyl, optionally substituted with 1-3 fluorine atoms; C1-4 alkenyl including C1-4 branched alkenyl, optionally substitutedwith 1-3 fluorine atoms; cyclic alkyl, preferably Cg_4cyclic alkyl (e.g., cyclopropyl), aryl and heteroaryl optionally substituted with 1-3 fluorine atoms; -OR'; -S(O)nR'; -OC(O)R'; -C(O)R'; -C(O)OR', -C(O)NR'g, -CN, NR'R' and halogen, preferably Cl or F; wherein if two of Ri, Rg, Rg, R4and R5, are selected from Ci-4alkyl, Ci-4alkenyl, -OR', -S(O)nR', or NR'R', and are adjacent, they may be joined together to form a cyclic structure;

[0069] n = 0,1 or 2;

[0070] each R' is independently selected from H; Ci-4alkyl including branched Ci-4alkyl (e.g., isopropyl); Ci-4alkenyl including branched Ci-4alkenyl; and cyclic alkyl, preferably Cg_4cyclic alkyl (e.g., cyclopropyl); wherein each of these groups is optionally substituted with 1-3 fluorine atoms;

[0071] Rs and R7are independently selected from H; Ci-4alkyl including branched Ci-4alkyl; Ci-4alkenyl including branched Ci-4alkenyl; and cyclic alkyl, preferably Cg_4cyclic alkyl (e.g., cyclopropyl); wherein each of these groups is optionally substituted with 1-3 fluorine atoms;

[0072] Rg Rg and RM are independently selected from H; Ci-4alkyl including branched Ci-4alkyl; Ci-4alkenyl including branched Ci.4alkenyl; cyclic alkyl, preferably Cg_4cyclic alkyl (e.g., cyclopropyl), wherein each of these groups is optionally substituted with 1-3 fluorine atoms; (N)mR"R" and CN; wherein where two of Rg Rg and RM, are independently selected from H, Ci-4alkyl including branched Ci-4alkyl; Ci-4alkenyl including branched Ci-4alkenyl, cyclic alkyl, preferably Cg_4cyclic alkyl (e.g., cyclopropyl), wherein each of these groups is optionally substituted with 1-3 fluorine atoms, or (N)mR"R", and are adjacent, they may be joined together to form a cyclic structure including:

[0073]

[0074] m= 0,1, 2;

[0075] Rn is H or Ci-4alkyl optionally substituted with 1-3 fluorine atoms; and

[0076] each R" is independently selected from H; Ci-4alkyl including branched Ci-4alkyl; Ci-4alkenyl including branched Ci-4alkenyl; cyclic alkyl, preferably Cg_4cyclic alkyl (e.g., cyclopropyl), wherein each of these groups is optionally substituted with 1-3 fluorine atoms;

[0077] preferably with the proviso that said compound is not lH-tetrazole-l-ethanol, 5-[(4-methylphenyl)amino]-a-[3-(trifluoromethyl)phenyl].In particular, preferably with the proviso that said compound is not a compound having the following chemical structure:

[0078]

[0079] In particular, preferably with the proviso that said compound is not a compound having CAS Registry Number: 2995382-39-1.

[0080] An alkyl group refers to a saturated hydrocarbon group (containing only single bonds) derived from an alkane by removing one hydrogen atom. Alkyl groups can be straight, branched, or cyclic and may contain any number of carbon atoms. Examples include methyl (-CH3), and ethyl (-CH2CH3), and cyclohexyl (-CeHn). In the context of the present invention, the term "C1-4 alkyl" shall be understood as a straight or branched-chain alkyl group containing 1 to 4 carbon atoms. Examples include methyl (-CH3), ethyl (-CH2CH3), propyl (-CH2CH2CH3), isopropyl (-CH(CH3)2), and butyl (-CH2CH2CH2CH3).

[0081] An alkenyl group refers to a hydrocarbon group containing one or more carbon-carbon double bonds. These groups may be straight, branched, or cyclic and can vary in length. Examples include vinyl (-CH=CH2), allyl (-CH2CH=CH2), and cyclopentenyl (-C5H7). In the context of the present invention, the term "C1.4 alkenyl" shall be understood as a straight or branched-chain hydrocarbon group containing 1 to 4 carbon atoms and at least one double bond. Examples include ethenyl (vinyl, -CH=CH2), propenyl (-CH=CHCH3), and butenyl (-CH=CHCH2CH3).

[0082] A cyclic alkyl refers to a saturated hydrocarbon ring structure (a cycloalkane) containing only single bonds between carbon atoms. Examples include cyclopropyl (C3H5), cyclobutyl (C4H7), cyclopentyl (C5H9), and cyclohexyl (C6H ). As used herein, C3.4cyclic alkyl refers to a saturated monocyclic hydrocarbon radical having from three to four carbon atoms. Examples include, but are not limited to, cyclopropyl and cyclobutyl.

[0083] An aryl group refers to an aromatic hydrocarbon group containing one or more conjugated rings with delocalized n-electrons. Examples include phenyl (C6H5-) and naphthyl (C10H7-). These groups are typically derived from aromatic compounds such as benzene or naphthalene.A heteroaryl group refers to an aromatic ring structure containing at least one heteroatom (such as nitrogen, oxygen, or sulfur) as part of the ring system. Examples include pyridyl (C5H4N), thienyl (C4H3S), and furyl (C4H3O).

[0084] A halogen refers to an atom from Group 17 of the periodic table: fluorine (F), chlorine (Cl), bromine (Br), iodine (I), or astatine (At). Halogen substituents can be attached to carbon atoms in organic molecules, such as in fluoromethane (CH3F) or chloroethane (C2H5CI). In the present invention, fluorine (F), chlorine (Cl), and bromine (Br) atoms are preferred.

[0085] In an embodiment of the different aspects of the present invention:

[0086] Ri, R2, R3, are selected from H; Ci-4alkyl including branched Ci-4alkyl; cyclic alkyl, preferably Ci-4cyclic alkyl; halogen; -SO2R' or -OR';

[0087] R' is independently selected from H; Ci-4alkyl including branched Ci-4alkyl (e.g., isopropyl); Ci-4alkenyl including branched Ci-4alkenyl; and cyclic alkyl, preferably Ci-4cyclic alkyl (e.g., cyclopropyl); wherein each of these groups is optionally substituted with 1-3 fluorine atoms;

[0088] R4, Rs, Re and R7are hydrogen;

[0089] Rg Rg and Rio are selected from H; Ci.4al kyl including branched Ci-4alkyl; Ci.4alkenyl including branched Ci-4 alkenyl; cyclic alkyl, preferably Ci-4cyclic alkyl (e.g., cyclopropyl); wherein each of these groups is optionally substituted with 1-3 fluorine atoms; (N)mR"R" and CN; wherein where two of Rg Rg and Rio, are independently selected from H; Ci-4alkyl including branched Ci-4alkyl; Ci.4alkenyl including branched Ci-4alkenyl; cyclic alkyl, preferably Ci-4cyclic alkyl (e.g., cyclopropyl), wherein each of these groups is optionally substituted with 1-3 fluorine atoms or (N)mR"R" and are adjacent, they may be joined together to form a cyclic structure including:

[0090]

[0091] Rn is H or methyl;

[0092] R" is independently selected from H; Ci-4alkyl including branched Ci-4alkyl; Ci-4alkenyl including branched Ci-4alkenyl; cyclic alkyl, preferably C3.4cyclic alkyl (e.g., cyclopropyl), wherein each of these groups is optionally substituted with 1-3 fluorine atoms; and

[0093] m= 0,1, 2.In another embodiment, the compounds of formula (I) or a stereoisomer or a pharmaceutically acceptable salt thereof, are selected from any one of the compounds selected from the list consisting of (it is noted that for each of the compounds shown in table 2 below R7 = H):

[0094] Table 2. List of selected compounds

[0095]

[0096] >

[0097]

[0098] It is noted that the isopropoxy group has the structure OCHfCHa)?.

[0099] In another embodiment, the compounds of formula (I) or a stereoisomer or a pharmaceutically acceptable salt thereof, are selected from any one of the lists consisting of:

[0100]

[0101] K-402

[0102] and

[0103] Preferably, the compound is

[0104]

[0105] K-105

[0106] or a stereoisomer or a pharmaceutically acceptable salt thereof.

[0107] Preferably, the compound is

[0108]

[0109] K-402

[0110] or a stereoisomer or a pharmaceutically acceptable salt thereof.

[0111] In a second aspect of the present invention relates to, preferably pharmaceutical or veterinary, compositions comprising therapeutically effective amounts of compounds of the first aspect of the invention and preferably adequate amounts of pharmaceutically acceptable excipients.

[0112] In an embodiment of the second aspect of the invention, the composition comprises:

[0113] a. a compound with anti-metastatic, or anti-invasive, capabilities selected from the list of compounds of the first aspect of the invention, including any of the preferred embodiments; and

[0114] b. a compound with anti-proliferative or cytotoxic effects.

[0115] As used herein, a compound is considered anti-metastatic if it reduces or inhibits the spread (metastasis) of malignant or undesirable cells from their primary site of origin to distant tissues ororgans. This typically involves disrupting the processes by which tumor cells detach, migrate through the bloodstream or lymphatic system, and colonize new sites.

[0116] Non-limiting methods to Evaluate Anti-Metastatic Effects:

[0117] • Transwell Migration Assays (Boyden Chamber): Cells are placed in the upper compartment of a chamber separated by a porous membrane. A chemotactic agent in the lower compartment attracts migrating cells. A decrease in the number of cells that migrate to the lower compartment suggests anti-metastatic activity.

[0118] • Wound Healing (Scratch) Assay: A "scratch" is created in a confluent cell monolayer. The rate of cell migration into the scratched area is monitored microscopically. Compounds that slow the closure of the wound display anti-migratory and potentially anti-metastatic effects. • In Vivo Metastasis Models: In animal studies (e.g., xenograft models in mice), tumor cells are injected orthotopically or intravenously, and the formation of secondary tumors is monitored. A reduction in tumor spread to distant organs indicates anti-metastatic potential.

[0119] As used herein, a compound exhibits anti-invasive properties if it prevents or decreases the infiltration of malignant cells into surrounding tissues, thereby hindering local expansion and tissue destruction. Non-limiting methods to Evaluate anti-invasive properties:

[0120] • Matrigel Invasion Assay: Similar to the Transwell Migration Assay, but the porous membrane is coated with an extracellular matrix substitute (e.g., Matrigel). The ability of cells to degrade and traverse the matrix layer reflects their invasive potential. A compound that reduces the number of cells passing through the membrane is considered anti-invasive.

[0121] • 3D Spheroid Invasion Assay: Tumor spheroids embedded in a 3D matrix (e.g., collagen or Matrigel) are tracked to see how far cells spread outward from the spheroid core. Compounds that limit invasion into the surrounding matrix have anti-invasive activity.

[0122] • Protease Activity Assays: Invasive cells often secrete enzymes such as matrix metalloproteinases (MMPs). Measuring MMP levels or activity after treatment can indicate whether a compound inhibits the invasiveness of the cancer cells.

[0123] As used herein, a compound is deemed anti-proliferative if it inhibits or suppresses the growth and multiplication (proliferation) of cells, especially cancerous or otherwise pathological cells. Such compounds may interfere with cell cycle progression, DNA replication, or other mechanisms essential for cell division.

[0124] Non-limiting methods to Evaluate Anti-Proliferative Effects:• MTT, MTS, or XTT Viability Assays: These colorimetric assays measure metabolic activity as a proxy for cell number. Reduced signal indicates diminished proliferation or viability.

[0125] • Cell Counting and Doubling Time: Traditional cell counting (manual or automated) can determine changes in growth rates or doubling times in the presence of a test compound. • BrdU (Bromodeoxyuridine) Incorporation: BrdU is a thymidine analog incorporated into newly synthesized DNA during cell division. Reduced BrdU incorporation indicates decreased cell proliferation.

[0126] • Clonogenic (Colony Formation) Assay: Cells are plated at low density, and the number of colonies formed after treatment is measured. Fewer colonies suggest inhibition of long-term proliferative capacity.

[0127] As used herein, a compound is cytotoxic if it is toxic to cells, typically causing damage to cell membranes or critical intracellular components. Cytotoxic effects often lead to cell death or irreversible impairment of essential cellular functions.

[0128] Non-limiting methods to Evaluate Cytotoxic Effects:

[0129] • LDH Release Assay: Cytotoxicity often results in damage to the cell membrane, causing intracellular enzymes like lactate dehydrogenase (LDH) to leak into the culture medium. Higher LDH levels in the supernatant indicate increased cell death.

[0130] • Flow Cytometry (Annexin V / Propidium Iodide Staining): Annexin V detects phosphatidylserine externalization (early apoptosis), and PI detects loss of membrane integrity (late apoptosis or necrosis). This method distinguishes between live, early-apoptotic, and late- apoptotic / necrotic cells.

[0131] • TUNEL Assay: Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) detects DNA fragmentation associated with apoptosis, indicating cytotoxicity leading to programmed cell death.

[0132] • Microscopy-Based Morphological Assessments: Changes such as cell rounding, blebbing, and detachment from the plate can be indicative of cytotoxic effects.

[0133] In the context of the present invention, a compound considered anti-invasive— meaning it impedes the infiltration of malignant cells into surrounding tissues— can also plausibly be regarded as anti-metastatic. This is because the local invasion of tumor cells into adjacent tissues is one of the preliminary steps in the metastatic cascade; thus, inhibiting cancer cell invasion often correlates with a reduction in the capacity of these cells to disseminate and colonize distant organs.In another embodiment of the second aspect of the invention, the compound with anti-metastatic capabilities is a compound of formula (I) or a stereoisomer or a pharmaceutically acceptable salt thereof, selected from any one of the list consisting of:

[0134]

[0135] K-402

[0136] and

[0137] In another embodiment, the compound with anti-proliferative or cytotoxic effects is selected from the list consisting of

[0138] 5-Fluorouracil (5-FU) and Derivatives

[0139] 5-Fluorouracil (5-FU)

[0140] Capecitabine

[0141] Tegafur

[0142] S-l (Tegafur + Gimeracil + Oteracil)

[0143] Floxuridine

[0144] Carmofur

[0145] Platinum-Based Compounds (Platin Derivatives)

[0146] Cisplatin

[0147] Carboplatin

[0148] Oxaliplatin

[0149] NedaplatinSatraplatin

[0150] Lobaplatin

[0151] Leucovorin and Related Agents

[0152] Leucovorin

[0153] Levoleucovorin

[0154] Methotrexate

[0155] Raltitrexed

[0156] Combination Agents Used in 5-FU / Platin Regimens Irinotecan

[0157] Docetaxel

[0158] Paclitaxel

[0159] Bevacizumab

[0160] Cetuximab

[0161] Panitumumab

[0162] Additional Compounds

[0163] Trifluridine / Tipiracil

[0164] Pemetrexed

[0165] Gemcitabine

[0166] Etoposide

[0167] Vinorelbine

[0168] Aflibercept

[0169] Natural and Targeted Compounds

[0170] Resveratrol

[0171] Curcumin

[0172] SorafenibZiv-aflibercept

[0173] In another embodiment, the compound with anti-proliferative effects is 5-fluorouracil (5-FU), platin or a platin derivative or leucovorin or any combination thereof, optionally in the presence of oxaliplatin. In a third aspect the present invention relates to the compounds of the first aspect of the invention and to the compositions of the second aspect of the invention for use as a medicament or for use in therapy.

[0174] In a fourth aspect the present invention relates to the compounds of the first aspect of the invention and to the compositions of the second aspect of the invention for use in the treatment or prevention in an animal, including a human, of a cancer disease or disorder. In a fifth aspect the present invention relates to the use of the compounds of the first aspect of the invention for the manufacture of a medicament for the treatment or prevention in an animal, including a human, of a cancer disease or disorder.

[0175] In particular embodiments of the fourth, or fifth aspects of the present invention the compounds of the first aspect of the invention or the compositions of the second aspect of the invention, are used for ameliorating, inhibiting or reducing metastatic spread in a subject in need thereof.

[0176] In particular embodiments of the fourth, or fifth aspects of the present invention the compositions of the second aspect of the invention, are used for ameliorating, inhibiting or reducing metastatic spread in a subject in need thereof or for ameliorating, inhibiting or reducing tumour proliferation (cytostatic action) or inducing cytotoxic (cell death) in a subject in need thereof.

[0177] In particular embodiments of the fourth, or fifth aspects of the present invention the compounds of the first aspect of the invention or the compositions of the second aspect of the invention, the cancer is a carcinoma.

[0178] In particular embodiments of the fourth, or fifth aspects of the present invention the compounds of the first aspect of the invention or the compositions of the second aspect of the invention, the cancer or carcinoma is selected from the list consisting of tumours arising from epithelial layers of the gastrointestinal track including month (oral cancer), esophagus, stomach, and small and large intestines (such as rectal or colon cancer), skin cancer, mammary gland (breast cancer), pancreas cancer, lung cancer, head and neck cancer, liver cancer, ovary cancer, cervix cancer, uterus cancer,gallbladder cancer, penile cancer, and urinary bladder cancer (such as renal, prostate or bladder cancer).

[0179] According to another aspect of the present invention, the compounds of formula (I) may be prepared in accordance with the information provided in the examples and drawings.

[0180] As used herein the term pharmaceutically acceptable salt designates any salt which, upon administration to the patient is capable of providing (directly or indirectly) a compound as described herein. For instance, pharmaceutically acceptable salts of compounds provided herein are synthesized from the parent compound, which contains a basic or acidic moiety, by conventional chemical methods. Generally, such salts are, for example, prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent or in a mixture of both. Generally, non-aqueous media like ether, ethyl acetate, ethanol, 2-propanol or acetonitrile are preferred. Examples of the acid addition salts include mineral acid addition salts such as, for example, hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, phosphate, and organic acid addition salts such as, for example, acetate, trifluoroacetate, maleate, fumarate, citrate, oxalate, succinate, tartrate, malate, mandelate, methanesulfonate and p-toluenesulfonate. Examples of the alkali addition salts include inorganic salts such as, for example, sodium, potassium, calcium and ammonium salts, and organic alkali salts such as, for example, ethylenediamine, ethanolamine, A / . / V-dialkylenethanolamine, triethanolamine and basic aminoacids salts. As used herein the term stereoisomers designates molecules that have the same molecular formula and sequence of bonded atoms (constitution) but differ in the three- dimensional orientations of their atoms in space.

[0181] Throughout the description and claims the word "comprise" and variations of the word, are not intended to exclude other technical features, additives, components, or steps. Furthermore, the word "comprise" encompasses the case of "consisting of". Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention.

[0182] The following examples are provided by way of illustration, and they are not intended to be limiting of the present invention. Furthermore, the present invention covers all possible combinations of particular and preferred embodiments described herein.EXAMPLES

[0183] MATERIALS AND METHODS

[0184] Synthesis of K-105

[0185] Synthesis of K-105 was carried out by the route shown in figure 1, synthetic methodology as follow:

[0186] Step A: to the solution of isothiocyanate (100 g, 0.670 mol) in 1 L of carbon tetrachloride gaseous chlorine was added for 18 h at 0 °C. Then the solution was allowed to rt, evaporated at reduced pressure, the crude product obtained was purified by distillation at 20 mm Hg. (b.p. = 125 °C). Yield 70 g (56%).

[0187] Step B: the solution of sodium azide (24.7 g, 380 mmol) and tetra-n-butylammonium bromide (5.48 g, 17 mmol) in water (80 mL) was added to a solution of 1 (47 g, 250 mmol) in toluene (400 mL) and the whole was stirred at rt. The organic layer was monitored by tic until the starting material had disappeared. The aqueous layer was saturated with NaCI and the organic layer was separated off; the aqueous layer was extracted with more toluene. The combined organic extracts were dried (Na2SO4) and filtered. The filtrate was rotary evaporated at about 35 °C until crystals started to form. The crude product was purified by column chromatography (DCM-MTBE as eluent) to obtain the desired product (18.4 g, 38 %). M.p. = 101-102 °C.

[0188] Step C: to the solution of chlorotetrazole 2 (1.94 g, 0.01 mol) in DMF (50 ml) the corresponding aminoalcohol 3 (1.80 g, 0.012 mol) and DIPEA (2.61 ml, 0.015 mol) were subsequently added. The resulting mixture was stirred for 12 h at 110 °C. After the completion of the reaction the solution was cooled to rt, concentrated in vacuo, the residue was diluted with 100 ml of water and extracted with 100 ml of DCM. The organic phase was separated and evaporated; the resulting crude product (about 2.7 g of the crude) was purified by column chromatography (DCM-MTBE as eluent) to obtain the desired product (0.91 g, 30 %).

[0189] Synthesis of K-402

[0190] Synthesis of K-402 was carried out by the route shown in figure 1, synthetic methodology as follow:

[0191] Step A: to a solution of 1 (20.0 g, 116.5 mmol, 1 eq.) and DBU (88.87 g, 0.582 mol, 5 eq.) in THF (300 mL) was added TBDMSCI (22.83 g, 0.151 mol, 1.3 eq.) portion wise at 0 °C under argon atmosphere. The reaction mixture was then stirred for 12 hr at r.t. After completion of the reaction, the volatiles were evaporated in vacuo and the residue was taken up with water (250 ml) and extracted with MTBE (2*150 ml). The combined organic layer was washed with water (50 ml*2), brine, dried over Na2SO4 and evaporated in vacuo to afford 2 (28.2 g, ~85% purity by1H-NMR, 84.6 % yield)Step B: to a solution compound 2 (28.2 g, 98.6 mmol, 1 eq.) and TEA (29.88 g, 296 mmol, 3eq.) in THF (400 mL) was added a solution of 4-isothiocyanatobenzonitrile (15.8 g, 296 mmol, 1 eq.) in THF (100 ml) dropwise at 0 °C under argon atmosphere. The reaction mixture was then stirred for 12 hr at r.t. After completion of the reaction, the mixture was evaporated under reduced pressure and the residue was mixed with MTBE / Hexane (v / v=l / 4, 300 ml). After stirring for 20 min the resulting precipitate was filtered, washed with MTBE / Hexane (v / v=l / 4, 400 ml) and dried in vacuo to afford thiourea 3 (35.0 g, ~95 % by 1H-NMR, 79.5% yield).

[0192] Step C: IBX (35.77 g, 127.7 mmol, 1.2 eq.) was added in portions to a solution of compound 3 (35.0 g, 78.4 mmol, 1 eq.) and TEA (23.77 g, 235.4 mmol, 3 eq.) in DMF (140 mL) at 0°C for 30 min. Then, Sodium azide (15.3 g, 235.4 mmol, 3 eq.) was added and resulting mixture was stirred at 90 °C for 12 hr under inert atmosphere. After completion of the reaction, the volatiles were evaporated in vacuo, the residue was taken up with water (400 ml) and extracted with MTBE (2*150 ml). The combined organic layer was washed with water (50 ml*3), brine, dried over Na2SO4 and evaporated in vacuo to give ~45 g of crude product, which was purification by column chromatography on silica gel using Hexane / MTBE gradient (10-100% MTBE) to afford 4 (8.0 g, 17.58 mmol, 85% purity by LCMS, 22.4% yield).

[0193] Step D: 10% HCI water solution (60 ml) was added to a solution of compound 4 (8.0 g, 17.58 mmol, 1 eq.) in THF (120 mL) at r.t. under argon atmosphere. The reaction mixture was then stirred for 12 hr at r.t. After completion of the reaction, the volatiles were evaporated in vacuo, the residue was taken up with water (100 ml) and extracted with EtOAc (2*40 ml). The combined organic layer was washed with water (30 ml*2), brine, dried over Na2SO4 and evaporated under reduced pressure. The residue was triturated with MTBE (20 ml). White precipitate was formed, filtered, washed with MTBE and dried in vacuo to afford K-402 (2.2 g, 98% purity by1H- NMR / LCMS, 36.7 % yield).

[0194] Cell culture and treatments

[0195] Human colon adenocarcinoma HT-29 cell line was cultured in McCoy's 5A Medium. Human colon adenocarcinoma HCT116, human lung adenocarcinoma A549 and human gastric adenocarcinoma N87 cell lines were cultured in and Dulbecco's Modified Eagle's Medium (DMEM). All culture media were supplemented with 1% penicillin / streptomycin and 10% of heat-inactivated fetal bovine serum (FBS) at 379C in a humidified incubator with 5% CO2. Cells were treated in every experiment with indicated compounds or DMSO as control at the specific concentrations, and in presence or absence of FOLFOX, a combination chemotherapy regimen that is used to treat colorectal cancer including 5-fluorouracil, leucovorin and oxaliplatin (5-Fu:leucovorin:oxaliplatin = 25:5:1), where the indicated concentration (2pM) correspond to the final concentration of 5-Fu. Cells were monthly tested for mycoplasma contamination and used only for 1-3 months after defrosted.Immunofluorescence

[0196] For immunofluorescence assays, 12.5 xlO3HT-29 cells were grown for 24h on glass coverslips and treated with the indicated compounds for 48h. Cells were fixed with 4% PFA for 40 min, permeabilized with 0.25% Triton X-100 for 15 min and incubated with anti-human E-cadherin (DECMA-1, rat monoclonal, #abll512, Abeam) for 2h. Coverslips were incubated with Goat anti-Rat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor® 488 conjugate (#A-11006, LifeTech) for lh. Finally, coverslips were mounted with Prolong Gold antifade reagent (LifeTech, UK) and images were taken in epifluorescence microscope (Olympus) using 20X objective.

[0197] Viability assays

[0198] For viability assays, 6.5 xlO3HCT116 cells were seeded per well into a 96-well plate. After 24h cells were treated with the indicated inhibitors for 48h, and a MTT colorimetric cell viability assay was performed following manufacturer's instructions (Sigma Aldrich, St Louis, MO). Absorbance was measured at 570 and 630 nm using a Multiskan Plus Reader (Nanoquant Infinite M200 Tecan Trading AG, Switzerland). Represented data are the mean ± SD of at least three independent experiments with six replicates per condition.

[0199] Proliferation assays

[0200] For BrdU assays, 6.5 xlO3HCT116 cells were plated per well into a 96-well plate. After 24 h, cells were treated with the indicated inhibitors for 48 h. Then, cells were treated with 10 pM BrdU for 2 h. BrdU incorporation into newly synthesized DNA was measured using a cell proliferation colorimetric immunoassay kit according to the manufacturer's instructions (Roche, Switzerland). Represented data are the mean ± SD of at least three independent experiments with six replicates per condition.

[0201] Migration and invasion assays

[0202] For migration and invasion assays, cells were seeded with serum free media in a cell migration chamber (QCM™ Laminin Migration Assay 24-Well-colorimetric ECM220, Merk millipore) or cell invasion chamber (CytoSelect™ 24-Well Cell Invasion Assay CBA110, Cell Biolabs), respectively. Each insert was treated at time 0, 24 and 48 hours with indicated compounds or DMSO as control. For combination treatments in invasion assays, cells were treated once in presence or absence of compounds and / or chemotherapy 24 h before seeding 105cells in chambers. 72 hours after seeding, invasive or migratory cells reaching the lower chamber that contains 10% FBS were then fixed and stained with crystal violet (Sigma Aldrich, St Louis, MO) following the manufacturer's specifications. Ten photographs werecapture per well using an inverted microscope (Nikon) and the area occupied by the stained invasive cells area was measured. Results are expressed as mean ± SD of at least three independent experiments.

[0203] Orthotopic xenograft murine model of colorectal cancer

[0204] Female athymic Nude-Foxnlnu mice, 5 weeks old, were purchased from Inotiv. The animals were maintained in laminar flow rooms at constant temperature (20-249C) and humidity with 5 animals per cage and an inspection was performed before transplantation to ensure their suitability for the study. All procedures related to animal handling, care, and treatment in this study were performed according to the guidelines approved by the Ethical Committee of Animal Experimentation of the Parc Cienti'fic de Barcelona (PCB). The procedure applied was number 23049-P4 approved by the Generalitat de Catalunya. The animal procedure was performed by Xenopat. C. bovis and mycoplasma-free human colon cancer cell line HTC116-Luc2 (3xl06in 100 pl PBS + 100 ul Matrigel), obtained from the American Type Culture Collection (ATCC CCL-247-LUC2), were injected subcutaneously into 5 athymic mice. Once tumours reached the desired volume, they were excised, fragmented and implanted orthotopically into the colon of 24 mice to generate an orthotopic model of CRC. When tumours were well established (score of approximately 0.5; 50 mm3considered), mice with orthotopic tumours were randomized, and assigned in three groups of 8 animals to receive the different treatments. Palpation for all mice was performed once / week. When tumours reached a mean palpation scoring 0.5, bidaily administration of oral compound K-402 was conducted at doses of 10 mg / kg and 50 mg / kg, each in groups of eight animals. An additional group of eight animals received vehicle control consisting of 2.5% DMSO, 30% PEG300, 5% T80, and water. In vivo bioluminescent imaging (BLI) of all mice was performed three times to monitor the experiment. Ex vivo bioluminescence imaging (BLI) of all lungs was performed post-sacrifice. For BLI, mice were injected 10 ml / kg intraperitoneal D-luciferin (15mg / ml) and images acquired within 10 minutes using IVIS ( IVIS Spectrum In Vivo Imaging System, PerkinElmer). Mice were sacrificed 23 days after treatment initiation. Tumours, lungs, kidneys, livers and hearts were collected and fixed in 4% PFA and embedded in paraffin blocks for histology and / or immunohistochemistry analyses.

[0205] Histology and Immunohistochemistry

[0206] Tumours and tissues were embedded in paraffin, sectioned at 4 pM and then deparaffinised and hydrated for a standard Haematoxylin and Eosin (H&E) staining or immunohistochemistry, as previously described

[0015] , For immunohistochemistry, endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 40 min. Antigen retrieval was carried by heating the samples (21001

[0207] Retriever; PickCell Laboratories, Amsterdam, The Netherlands) in EDTA buffer (Agilent, Santa Clara, CA, USA). Then, endogenous peroxidase activity was then blocked a second time using Peroxidase Blocking Reagent (Agilent). Sections were blocked and permeabilized with 0.2% BSA and 0.1% Tx-100 for 1 h and incubated with the indicated primary antibody: human GAPDH (abl28915, Abeam), E-Cadherin (ab40772, Abeam), N-Cadherin (abl8203, Abeam), overnight at 4 °C in a wet chamber. Slides were incubated for 1 h at room temperature and the secondary antibody and detection was carried out using DAB (Dako Real Envision kit, Agilent) according to manufacturer instructions. Finally, nuclei were counterstained with Gill's haematoxylin and mounted with DePeX. For the detection and quantification of metastatic foci in the lungs, five sections spanning the entire lung from each mouse were taken. Number and size of metastatic foci in the lung were quantified to determine the extent of metastasis by an automated analysis using Python. A representative image is shown for each condition, and the represented results are shown as scatter plots with mean ± SD. For the quantification of E-Cadherin and N-Cadherin expression levels, total intensity of the specific antibody signal was measured in five sections of the primary tumour from each mouse using an automated Python-based image analysis pipeline. Images were acquired using an Olympus microscope. Representative images are shown for each condition, and results are presented as violin plots.

[0208] Statistical analysis

[0209] Statistical analyses were carried out by using GraphPad Prism software. Statistical significance of data was determined with ANOVA with the Bonferroni test or Kruskal-Wallis with the Tukey correction test. Significance among the experimental groups indicated in the figures is shown as * p < 0.05, ** p < 0.01 and *** p < 0.001****p < 0.0001. Results obtained are expressed as mean ± SD as indicated.

[0210] RESULTS

[0211] New novel compounds were synthetized.

[0212] Compounds K-105 [l-(3-methylphenyl)-2-{5-[(4-methylphenyl)amino]-lH-l,2,3,4-tetrazol-l-yl}ethan- l-ol] and K-402 [4-({l-[2-(3-chlorophenyl)-2-hydroxyethyl]-lH-l,2,3,4-tetrazol-5-yl}amino)benzonitrile] were synthetized as described in Material and Methods. Compounds were resuspended in DMSO at 10 mM for in vitro assays. The highest concentration of DMSO was used as the vehicle control for the experiments. Chemical structure (A-C) and synthetic routes (B-D) of K-105 and K-402 are shown in Figure 1, respectively. The HPLC spectra and liquid chromatography-mass spectrometry (LC-MS) and1H-RMN spectrum of K-105 and K-402 compounds are included in Figures 2 and 3, respectively.Effect of novel compounds on E-cadherin recovery in tumour culture cells

[0213] It was previously reported the effect of Hakai as an E3 ubiquitin-ligase on the E-cadherin ubiquitination, endocytosis and degradation, leading the alteration of cell-cell contacts. Given the previously reported role of Hakai on the E-cadherin degradation, we aim to analyse the effect of novel compounds analogues on the recovery of E-cadherin at cell-cell contacts. By immunofluorescence assays, we observed a significant increase of E-cadherin levels at cell-cell contacts in HT-29 colon cancer cells under the treatment with K-105, and K-402 compounds (Figure 4 A-B). The increase of E-cadherin recovery at cell-cell contacts is statistically significant at 20 pM and 2 pM.

[0214] Effect of novel compounds on cell viability and cell proliferation in tumour culture cells

[0215] We also analysed the effect of K-105 and K-402 compounds on cell viability. As shown in Figure 5, the effect on cell viability was statistically significant at 100 pM for K-105, while no statistically significant differences were detected at 50 pM or lower concentrations tested. On the other hand, the cytotoxic impact of the K-402 compound increased in a dose-dependent manner, showing statistically significant differences at 50 pM and 100 pM, and no statistically significant differences were detected at lower concentrations tested. In contrast, when we analysed the effect of K-105 and K-402 compounds on cell proliferation, the only compound that decreases cell proliferation was K-402 at the highest concentration tested of 100 pM, (Figure 6), without having any effect at lower concentrations. Therefore, taken together this data indicate that cell proliferation or viability is not affected by these compounds at 20 pM or lower concentrations.

[0216] Effect of novel compounds on cell migration and cell invasion in tumour culture cells

[0217] The loss of the cell-cell adhesion molecule E-cadherin is associated with the presence of migratory and invasive capabilities. Considering Hakai's reported effect on E-cadherin degradation and the subsequent increase in cell migration and invasion, we aim to determine the effect of K-105 and K-402 molecules on cell migration and invasion in epithelial tumour cells. As shown in Figure 7, statistically significant differences on cell migration under the treatment of K-105 and K-402 compounds are observed at concentration of not only 2 pM but also 200 nM in HCT116 human colon cancer cell line. Moreover, we analysed the effect of K-105 and K-402 compounds on cell invasion capacity. The IC5o values for these compounds were determined from the dose-response curve of this invasion assay. Dose-response curve slope helps to predict therapeutic potency in cell invasion under the treatment of the compounds, showing a very potent effect for both compounds on cell invasion ( I C5o 363 pM for K-105 and IC5o 6 nM for K-402). These data show a highly potent anti-invasive effect for both compounds at nanomolar (nM) range, without showing a cytotoxic or anti-proliferative effect at thisrange of concentration. Our findings reveal a therapeutic window in which both compounds exhibit robust anti-invasive and anti-migratory effects at nanomolar (nM) concentrations, without affecting cytotoxicity or cell proliferation. In conclusion, as shown in Table 1, we have a more effective compounds that exhibit increased potency and therapeutic effect on the inhibition of invasion and migration at nM concentration compared to our previous reported compounds15, without affecting cytotoxicity. We extended our results on cell migration and invasion using additional colon cancer cell lines (Figure 8), and other human cancer cells (Figure 9), including gastric cancer (N87), lung adenocarcinoma (A549 and H1299), hepatocellular carcinoma (HepG2), prostatic adenocarcinoma (PC3) and kidney adenocarcinoma (ACHN). As shown, statistically significant differences were confirmed under the treatment of K-402 compound at 200 nM further highlighting its broad impact on cell migration and invasion across different types of cancer.

[0218] Effect of K-402 in primary tumour and metastasis in an orthotopic xenograft murine model of colorectal cancer

[0219] The effect of K-402 against the primary tumour and metastasis were evaluated in an orthotopic xenograft murine model of colorectal cancer. Human colon cancer cell line HCT116-Luc2 were orthotopically implanted into the colons of the mice. Six days after implantation, tumours were palpable (mean palpation score of approximately 0.5), and an in vivo I VIS for all mice was performed to confirm tumour viability. Once tumours reached a score of 0.5, tumour-bearing mice were treated with indicated doses starting on day 1, and all mice continued receiving treatments thereafter. From that point onward, all mice received twice-daily treatments with different doses of K-402 (10 mg / kg and 50 mg / kg) or the vehicle as a control. At necropsy, all animals were opened, and tumours and different organs were collected. K-402 did not show statistically significant differences in tumour growth at the evaluated doses (10 or 50 mg / kg) compared to vehicle group (Figure 10A). For histological examination of metastasis, the lung, and liver were collected and H&E staining was performed (Figure 10B). Mice free of metastasis were detected in 50% of the mice (4 of 8) treated with K-402 at 50 mg / kg, and in 25% of the mice (2 of 8) treated with K-402 at 10 mg / kg, whereas no metastatic foci were detected in the liver. Haematoxylin & Eosin staining and immunohistochemistry against human GAPDH were used to detect and quantify the number and size of the human colon metastatic foci in the lung of the mice (Figure 11A). Significant differences of metastatic foci number and size were found under K-402 treatment (Figure 11B-C). Importantly, a 70% reduction in metastatic burden, calculated based on the total area occupied by metastases for each tissue section, was observed following K-402 treatment (Figure 12). Consistent with these findings, immunohistochemical analysis of epithelial-mesenchymal transition markers in primary tumours confirmed a significantrecovery of E-Cadherin expression levels in K-402-treated mice (Figure 13A), accompanied by a significant reduction in N-Cadherin expression (Figure 13B). Notably, K-402 did not cause a reduction in body weight compared to the vehicle group (Figure 14A), and no apparent damage was observed in liver, kidney, or heart, all of which maintained a normal morphological structure (Figure 14B). Taken together, these results reinforce that K-402 exhibits robust antimetastatic efficacy without affecting tumour growth or causing apparent systemic toxicity in vivo.

[0220] Effect of novel K-402 compound alone and / or in combination with standard chemotherapeutic drugs.

[0221] As previously mentioned, cancer is characterized not only by abnormal cellular proliferation but also by the potential of cells to metastasize. In solid tumours, the ability of cancer cells to metastasize is typically associated with their capacity for local invasion and distant spread. Given that the mechanisms underlying cell invasion are distinct to those related to proliferation, our value proposition is to combine the proposed novel compounds, that exhibit a potent anti-invasive effect at nanomolar concentration, without affecting cytotoxicity or cell proliferation at these concentrations, together with conventional cytostatic or cytotoxic drugs, that primarily target cell proliferation or induce cell death. In order to do so, we combined the K-402 compound together with FOLFOX or FOLFIRI, a frequently standard-of-care chemotherapeutic agents used at various stages in colorectal cancer. We aim to investigate whether the use of these combination therapies (K-402 plus FOLFOX or FOLFIRI) could influence the anti-invasive capabilities of K-402 compound. We analysed the anti-invasive effect on HCT116 tumour cells previously treated once in presence or absence of K-402 and / or indicated chemotherapy for 24 h before seeding cells into chambers. After 72 hours in the chamber, we analysed the effect on cellular invasion. K-402 showed an anti-invasive effect in a concentration dependent manner, at 20 nM and 200 nM. Moreover, this effect is maintained, at least, 96 h after treatment (Figure 15). Importantly, a synergistic effect was observed when combining K-402 with FOLFOX or FOLFIRI chemotherapy, improving its effectiveness. This data strongly supports the notion that these compounds, with their remarkable anti-invasive effects, can be used as metastasis-targeting drugs. Furthermore, enhanced anti-invasive effectiveness is achieved when they are combined with conventional cytostatic or cytotoxic drugs, which primarily target cell proliferation or induce cell death.

[0222] Support for the compounds shown in the Markush formula I.

[0223] Table 3. Inhibition of cell invasion of the compounds of formula I.

[0224]

[0225]

[0226] As shown in Table 3, not only compounds K-402 or K-105 NS exhibit increased potency and therapeutic effect on the inhibition of invasion and migration at nM concentration compared to previously reported compounds (please refer to table 1), but further compounds falling within the scope of Markush formula I such as K-414, K-415, K-416, K-505 and K-507 also exhibit increased potency and therapeutic effect on the inhibition of invasion at nM concentrations. All of these compounds exhibit robust antimetastatic efficacy without affecting tumour growth or causing apparent systemic toxicity.CLAUSES

[0227] 1. A compound of formula (I)

[0228]

[0229] wherein:

[0230] Ri, R2, R3, R4 and R5are independently selected from H, C1.4 alkyl, C1-4 alkenyl, including branched alkyl, alkenyl and cyclic alkyl, aryl, heteroaryl optionally substituted with 1-3 fluorine atoms, -OR', -S(O)nR', -OC(O)R', -C(O)R', -C(O)OR' and -C(O)NR'2, -CN, NR'R' and halogen; wherein if two of Ri, R2, R3, 4 and R5, are selected from C1-4 alkyl, C1-4 alkenyl, -OR', -S(O)nR', or NR'R', and are adjacent, they may be joined together to form a cyclic structure;

[0231] n = 0,1 or 2;

[0232] each R' is independently selected from H, C1-4 alkyl including branched alkyl, alkenyl and cyclic alkyl optionally substituted with 1-3 fluorine atoms;

[0233] Rs and R7are selected from H, alkyl including branched alkyl, alkenyl and cyclic alkyl optionally substituted with 1-3 fluorine atoms

[0234] Rg Rg and Rio are selected from H, C1-4 alkyl including branched alkyl, alkenyl and cyclic alkyl optionally substituted with 1-3 fluorine atoms, (N)mR"R" and CN; wherein where two of Rg Rg and Rio, are selected from H, C1-4 alkyl including branched alkyl, alkenyl and cyclic alkyl optionally substituted with 1-3 fluorine atoms or (N)mR"R", and are adjacent, they may be joined together to form a cyclic structure including:

[0235]

[0236] m= 0,1, 2;

[0237] Rn is H or C1-4 alkyl optionally substituted with 1-3 fluorine atoms; and

[0238] each R" is independently selected from H, C1-4 alkyl including branched alkyl, alkenyl and cyclic alkyl optionally substituted with 1-3 fluorine atoms;

[0239] and pharmaceutically acceptable salts thereof.

[0240] 2. The compound according to clause 1,

[0241] wherein:

[0242] Ri, R2, R3, are selected from H, C1-4 alkyl including branched alkyl and cyclic alkyl, halogen, SO2R' or OR'; R' is independently selected from H, C1-4 alkyl including branched alkyl, alkenyl and cyclic alkyl optionally substituted with 1-3 fluorine atoms;

[0243] R4, Rs, Re and R7are hydrogen;

[0244] Rg Rg and Rio are selected from H, C1-4 alkyl including branched alkyl, alkenyl and cyclic alkyl optionally substituted with 1-3 fluorine atoms, (N)mR"R" and CN; wherein where two of Rg Rg and Rio, are selected from H, C1-4 alkyl including branched alkyl, alkenyl optionally substituted with 1-3 fluorine atoms or (N)mR"R" and are adjacent, they may be joined together to form a cyclic structure including:

[0245]

[0246] Rn is H or Methyl;

[0247] R" is H or C1-4 alkyl including branched alkyl, cyclic alkyl optionally substituted with 1-3 fluorine atoms; and

[0248] m= 0,1, 2.3. The compound according to any one of clauses 1 or 2, wherein the compound is selected from any one from the list consisting of:

[0249]

[0250]

[0251] or any pharmaceutically acceptable salts thereof.

[0252] 4. The compound according to clause 3, wherein the compound is selected from any one from the list consisting of: K-402, K-105, K-414, K-415, K-416, K-505 and K-507, or any pharmaceutically acceptable salts thereof.

[0253] 5. The compound according to clause 3, wherein the compound is selected from any one from the list consisting of: K-402, K-105, and K-505, or any pharmaceutically acceptable salts thereof.

[0254] 6. The compound according to clause 4, wherein the compound is

[0255]

[0256] K-105

[0257] or any pharmaceutically acceptable salts thereof.

[0258] 7. The compound according to clause 4, wherein the compound is

[0259]

[0260] or any pharmaceutically acceptable salts thereof.

[0261] 8. The compound according to clause 4, wherein the compound is K-505 or any pharmaceutically acceptable salts thereof.

[0262] 9. A composition of matter comprising:

[0263] a. a compound with anti-metastatic, or anti-invasive, capabilities selected from the list of compounds of any one of clauses 1 to 8; and

[0264] b. a compound with anti-proliferative or cytotoxic effects.

[0265] 10. The composition according to clause 9, wherein the compound with anti-proliferative effects is 5-fluorouracil (5-FU), platin or a platin derivative or leucovorin or any combination thereof, optionally in the presence of oxaliplatin.

[0266] 11. A pharmaceutical composition comprising a compound as defined in any one of clauses 1 to 8 or the composition as defined in any one of clauses 9 to 10, together with one or more pharmaceutically acceptable carriers or excipients.

[0267] 12. The compound of any one of clauses 1 to 8, the composition of any one of clauses 9 to 10 or the pharmaceutical composition of clause 11, for use in therapy.

[0268] 13. The compound of any one of clauses 1 to 8, the composition of any one of clauses 9 to 10 or the pharmaceutical composition of clause 11, for use in cancer, preferably a cancer carcinoma.

[0269] 14. The compound of any one of clauses 1 to 8, the composition of any one of clauses 9 to 10 or the pharmaceutical composition of clauses 11, for use according to clause 13, for inhibiting orreducing metastatic spread in a subject in need thereof; or for inhibiting tumour proliferation and inhibiting or reducing metastatic spread in a subject in need thereof.

[0270] The compound of any one of clauses 1 to 8, the composition of any one of clauses 9 to 10 or the pharmaceutical composition of clause 11, for use according to any one of clauses 13 or 14, wherein the cancer or carcinoma is selected from the list consisting of tumours arising from epithelial layers of the gastrointestinal track including month (oral cancer), esophagus, stomach, and small and large intestines (such as rectal or colon cancer), skin cancer, mammary gland (breast cancer), pancreas cancer, lung cancer, head and neck cancer, liver cancer, ovary cancer, cervix cancer, uterus cancer, gallbladder cancer, penile cancer, and urinary bladder cancer (such as renal, prostate or bladder cancer).

Claims

1. CLAIMS1. A compound of formula (I)wherein:Ri, R2, R3, R4and R5are independently selected from H; C1-4 alkyl including branched Ci-4alkyl, optionally substituted with 1-3 fluorine atoms; C1-4 alkenyl including C1-4 branched alkenyl, optionally substituted with 1-3 fluorine atoms; cyclic alkyl, preferably C3-4 cyclic alkyl (e.g., cyclopropyl), aryl and heteroaryl optionally substituted with 1-3 fluorine atoms; -OR'; -S(O)nR'; -OC(O)R'; -C(O)R'; -C(O)OR', -C(O)NR'2, -CN, NR'R' and halogen, preferably Cl or F; wherein if two of Ri, R2, R3, R4 and R5, are selected from Ci- 4 alkyl, C1-4 alkenyl, -OR', -S(O)nR', or NR'R', and are adjacent, they may be joined together to form a cyclic structure;n = 0,1 or 2;each R' is independently selected from H; C1-4 alkyl including branched C1-4 alkyl (e.g., isopropyl); C1-4 alkenyl including branched C1-4 alkenyl; and cyclic alkyl, preferably C3-4 cyclic alkyl (e.g., cyclopropyl); wherein each of these groups is optionally substituted with 1-3 fluorine atoms;Rs and R7are independently selected from H; C1-4 alkyl including branched C1-4 alkyl; C1-4 alkenyl including branched C1-4 alkenyl; and cyclic alkyl, preferably C3-4 cyclic alkyl (e.g., cyclopropyl); wherein each of these groups is optionally substituted with 1-3 fluorine atoms;Rg Rg and RM are independently selected from H; Ci-4alkyl including branched Ci-4alkyl; Ci-4alkenyl including branched Ci.4alkenyl; cyclic alkyl, preferably Cg.4cyclic alkyl (e.g., cyclopropyl), wherein each of these groups is optionally substituted with 1-3 fluorine atoms; (N)mR"R" and CN; wherein two of Rg Rg and RM, may be joined together to form a cyclic structure including:m= 1;Rn is H or Ci-4 alkyl optionally substituted with 1-3 fluorine atoms; andR" is independently selected from H; Ci-4alkyl including branched Ci-4alkyl; Ci-4alkenyl including branched Ci-4alkenyl; cyclic alkyl, preferably Cg-4cyclic alkyl (e.g., cyclopropyl), wherein each of these groups is optionally substituted with 1-3 fluorine atoms; orany pharmaceutically acceptable salts thereof;with the proviso that said compound is not lH-tetrazole-l-ethanol, 5-[(4-methylphenyl)amino]-a-[3-(trifluoromethyl)phenyl],2. The compound according to claim 1,wherein:Ri, R?, Rg, are selected from H; Ci-4alkyl including branched Ci-4alkyl; cyclic alkyl, preferably Ci-4cyclic alkyl; halogen; SO2R' or OR';R' is independently selected from H; Ci-4alkyl including branched Ci-4alkyl (e.g., isopropyl); Ci-4alkenyl including branched Ci-4alkenyl; and cyclic alkyl, preferably Cg.4cyclic alkyl (e.g., cyclopropyl); wherein each of these groups is optionally substituted with 1-3 fluorine atoms;R4, R5, RG and R7are hydrogen;Rg Rg and RM are selected from H; Ci.4al kyl including branched Ci-4alkyl; Ci.4alkenyl including branched Ci-4 alkenyl; cyclic alkyl, preferably Cg.4cyclic alkyl (e.g., cyclopropyl); wherein each of these groups is optionally substituted with 1-3 fluorine atoms; (N)mR"R" and CN; wherein two of Rg Rg and RM, may be joined together to form a cyclic structure including:Rn is H or Methyl;R" is independently selected from H; Ci-4alkyl including branched Ci-4alkyl; Ci-4alkenyl including branched Ci-4alkenyl; cyclic alkyl, preferably C3.4cyclic alkyl (e.g., cyclopropyl), wherein each of these groups is optionally substituted with 1-3 fluorine atoms; andm= 1; orany pharmaceutically acceptable salts thereof.

3. The compound according to any one of claims 1 or 1, wherein the compound is selected from any one of the list consisting of:>or any pharmaceutically acceptable salts thereof.

4. The compound according to claim 3, wherein the compound is selected from any one from the list consisting of: K-402, K-105, K-414, K-415, K-416, K-505 and K-507, or any pharmaceutically acceptable salts thereof.

5. The compound according to claim 3, wherein the compound is selected from any one from the list consisting of: K-402, K-105, and K-505, or any pharmaceutically acceptable salts thereof.

6. The compound according to claim 4, wherein the compound isK-105or any pharmaceutically acceptable salts thereof.

7. The compound according to claim 4, wherein the compound isor any pharmaceutically acceptable salts thereof.

8. The compound according to claim 4, wherein the compound is K-505 or any pharmaceutically acceptable salts thereof.

9. A composition of matter comprising:a. a compound with anti-metastatic, or anti-invasive, capabilities selected from the list of compounds of any one of claims 1 to 8; andb. a compound with anti-proliferative or cytotoxic effects.

10. The composition according to claim 9, wherein the compound with anti-proliferative effects is 5-fluorouracil (5-FU), platin or a platin derivative or leucovorin or any combination thereof, optionally in the presence of oxaliplatin.

11. A pharmaceutical composition comprising a compound as defined in any one of claims 1 to 8 or the composition as defined in any one of claims 9 to 10, together with one or more pharmaceutically acceptable carriers or excipients.

12. A compound of formula (I)wherein:Ri, R2, R3, R4and R5are independently selected from H; C1-4 alkyl including branched Ci-4alkyl, optionally substituted with 1-3 fluorine atoms; C1-4 alkenyl including C1-4 branched alkenyl, optionally substituted with 1-3 fluorine atoms; cyclic alkyl, preferably C3-4 cyclic alkyl (e.g., cyclopropyl), aryl and heteroaryl optionally substituted with 1-3 fluorine atoms; -OR'; -S(O)nR'; -OC(O)R'; -C(O)R'; -C(O)OR', -C(O)NR'2, -CN, NR'R' and halogen, preferably Cl or F; wherein if two of Ri, R2, R3, 4 and R5, are selected from Ci-4 alkyl, C1-4 alkenyl, -OR', -S(O)nR', or NR'R', and are adjacent, they may be joined together to form a cyclic structure;n = 0,1 or 2;each R' is independently selected from H; C1-4 alkyl including branched C1-4 alkyl (e.g., isopropyl); C1-4 alkenyl including branched C1-4 alkenyl; and cyclic alkyl, preferably C3-4 cyclic alkyl (e.g., cyclopropyl); wherein each of these groups is optionally substituted with 1-3 fluorine atoms;Rs and R7are independently selected from H; C1-4 alkyl including branched C1-4 alkyl; C1-4 alkenyl including branched C1-4 alkenyl; and cyclic alkyl, preferably C3-4 cyclic alkyl (e.g., cyclopropyl); wherein each of these groups is optionally substituted with 1-3 fluorine atoms;Rg Rg and RM are independently selected from H; C1-4 alkyl including branched C1-4 alkyl; C1-4 alkenyl including branched C1.4 alkenyl; cyclic alkyl, preferably C3-4 cyclic alkyl (e.g., cyclopropyl), wherein each of these groups is optionally substituted with 1-3 fluorine atoms; (N)mR"R" and CN; wherein two of Rg Rg and RM, may be joined together to form a cyclic structure including:m= 1;Rn is H or C1-4 alkyl optionally substituted with 1-3 fluorine atoms; andR" is independently selected from H; C1-4 alkyl including branched C1-4 alkyl; C1-4 alkenyl including branched C1-4 alkenyl; cyclic alkyl, preferably C3-4 cyclic alkyl (e.g., cyclopropyl), wherein each of these groups is optionally substituted with 1-3 fluorine atoms;or any pharmaceutically acceptable salts thereof;for use in therapy.

13. A compound for use according to claim 12,wherein:Ri, R2, R3, are selected from H; C1-4 alkyl including branched C1-4 alkyl; cyclic alkyl, preferably C1-4 cyclic alkyl; halogen; SO2R' or OR';R' is independently selected from H; C1-4 alkyl including branched C1-4 alkyl (e.g., isopropyl); C1-4 alkenyl including branched C1-4 alkenyl; and cyclic alkyl, preferably C3-4 cyclic alkyl (e.g., cyclopropyl); wherein each of these groups is optionally substituted with 1-3 fluorine atoms;R4, Rs, Re and R7are hydrogen;Rg Rg and RM are selected from H; C1-4 alkyl including branched C1-4 alkyl; CMalkenyl including branched C1-4 alkenyl; cyclic alkyl, preferably C3-4 cyclic alkyl (e.g., cyclopropyl); wherein each of these groups is optionally substituted with 1-3 fluorine atoms; (N)mR"R" and CN; wherein two of Rg Rg and RM, may be joined together to form a cyclic structure including:Rn is H or Methyl;R" is independently selected from H; C1-4 alkyl including branched C1-4 alkyl; C1-4 alkenyl including branched C1-4 alkenyl; cyclic alkyl, preferably C3-4 cyclic alkyl (e.g., cyclopropyl), wherein each of these groups is optionally substituted with 1-3 fluorine atoms; andm= 1;or any pharmaceutically acceptable salts thereof.

14. A compound for use according to claim 12, wherein the compound is selected from any one of the list consisting of:>15. The compound as defined in any one of claims 12 to 14, for use in cancer, preferably a cancer carcinoma.

16. The compound as defined in any one of claims 12 to 14, for use according to claim 15, for inhibiting or reducing metastatic spread in a subject in need thereof; or for inhibiting tumour proliferation and inhibiting or reducing metastatic spread in a subject in need thereof.

17. The compound as defined in any one of claims 12 to 14, for use according to any one of claims 15 or 16, wherein the cancer or carcinoma is selected from the list consisting of tumours arising from epithelial layers of the gastrointestinal track including month (oral cancer), esophagus, stomach, and small and large intestines (such as rectal or colon cancer), skin cancer, mammary gland (breast cancer), pancreas cancer, lung cancer, head and neck cancer,liver cancer, ovary cancer, cervix cancer, uterus cancer, gallbladder cancer, penile cancer, and urinary bladder cancer (such as renal, prostate or bladder cancer).

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