COMPOUNDS FOR PREVENTING MIGRATION OF CANCER CELLS

Abstract

The present invention relates to a composition for use in preventing migration of cancer cells in a subject known or suspected to suffer from cancer, the composition comprising at least one metal complex having the structure (I), formula (I), wherein M is a metal, preferably selected from the group consisting of copper, iron, manganese and zinc, X is X.sup.a or X.sup.b, wherein X.sup.a is selected from the group consisting of O, S and —N(R.sup.1)—, wherein R.sup.1 is H or alkyl, and wherein X.sup.b is a group forming a coordinate covalent bond to a second metal M′, preferably a group O, S or —N(R.sup.1)—, wherein M′ is preferably selected from the group consisting of copper, iron, manganese and zinc, and wherein M′ and M may be the same or different and are preferably the same, Z.sup.1 and Z.sup.2 are independently of each other a, substituted or unsubstituted, -Aryl-O—, -Aryl-N— or heteroaryl group, Y is Y.sup.a or Y.sup.b, wherein Y.sup.a is selected from the group consisting of H, alkyl, —OH, —SH, halogen, and —NR.sup.3R.sup.4, wherein R.sup.3 and R.sup.4, are independently of each other selected from H and alkyl, preferably R.sup.3 and R.sup.4 are both H, and wherein Y.sup.b is a group forming a coordinate covalent bond to M or M′, preferably Y.sup.b is a group O, S or —N(R.sup.Yb1R.sup.Yb2), wherein R.sup.Yb1 and R.sup.Yb1, are, independently of each other, H or alkyl, preferably H, and wherein n and m are integers, which are independently of each other, 0 or 1, Y.sup.2 is water or a halogen, and wherein Y.sup.3 is water or a halogen. The present invention further relates to a combined preparation comprising the aforesaid composition as well as to an in vitro method, for determining whether cancer cells are susceptible to immobilizing by the aforesaid compound.

##STR00001##

Claims

1-22. (canceled)

23. A method for preventing migration of cancer cells in a subject known or suspected to suffer from cancer with a composition comprising at least one metal complex having the structure (I): ##STR00088## wherein: M is a metal, preferably selected from the group consisting of copper, iron, manganese and zinc; X is X.sup.a or X.sup.b, wherein X.sup.a is selected from the group consisting of O, S and —N(R.sup.1)—, wherein R.sup.1 is H or alkyl, and wherein X.sup.b is a group forming a coordinate covalent bond to a second metal M′, preferably a group O, S or —N(R.sup.1)—, wherein M′ is preferably selected from the group consisting of copper, iron, manganese and zinc, and wherein M′ and M may be the same or different and are preferably the same; Z.sup.1 and Z.sup.2, are independently of each other a, substituted or unsubstituted, -Aryl-O—, -Aryl-N— or heteroaryl group; Y is Y.sup.a or Y.sup.b, wherein Y.sup.a is selected from the group consisting of H, alkyl, —OH, —SH, halogen, and —NR.sup.3R.sup.4, wherein R.sup.3 and R.sup.4, are independently of each other selected from H and alkyl, preferably R.sup.3 and R.sup.4 are both H, and wherein Y.sup.b is a group forming a coordinate covalent bond to M or M′, preferably Y.sup.b is a group O, S or —N(R.sup.Yb1R.sup.Yb2) wherein R.sup.Yb1 and R.sup.Yb1, are, independently of each other, H or alkyl, preferably H; n and m are integers, which are independently of each other, 0 or 1; Y.sup.2 is water or a halogen; and Y.sup.3 is water or a halogen.

24. The method of claim 23, wherein the complex is a mononuclear or binuclear complex or a mixture thereof.

25. The method of claim 23, wherein Y is Y.sup.a and wherein the complex has one of the following structures: ##STR00089## preferably wherein: M and M′ are metals, preferably selected from the group consisting of copper, iron, manganese and zinc, wherein M′ and M may be the same or different and are preferably the same; X.sup.b is a group O, S or —N(R.sup.1)—, wherein R.sup.1 is H or alkyl; Z.sup.1 and Z.sup.2, are independently of each other a, substituted or unsubstituted, -Aryl-O—, -Aryl-N— or heteroaryl group; Y.sup.b is a group O, S or —N(R.sup.Yb1R.sup.Yb2), wherein R.sup.Yb1 and R.sup.Yb1 are, independently of each other, H or alkyl, preferably H; Y.sup.2 is water or a halogen; y.sup.2* is a solvent molecule or a halogen, preferably water, methanol or a halogen, more preferably, water, methanol or —Cl; Y.sup.3 is water or a halogen; Y.sup.3* is a solvent molecule or a halogen, preferably water, methanol or a halogen, more preferably, water, methanol or —Cl; n and m are integers, which are independently of each other, 0 or 1; n* and m* are integers, which are independently of each other, 0 or 1; and q is an integer of from 2 to 5, preferably q is 2 or 3, more preferably 2.

26. The method of claim 23, wherein Y is Y.sup.b and wherein the complex has the structure: ##STR00090##

27. The method of claim 23, wherein M and M′ are, independently of each other selected from the group consisting of Fe(III), Cu(II), Mn(II) and Zn(II), with Fe(III) and Cu(II) being particularly preferred.

28. The method of claim 23, wherein the complex has a structure selected from the group consisting of: ##STR00091##

29. The method of claim 23, wherein the complex has a structure selected from the group consisting of: ##STR00092##

30. The method of claim 23, wherein the complex has as structure selected from the group consisting of: ##STR00093## and mixtures of two or more thereof.

31. A method for preventing migration of cancer cells in a subject known or suspected to suffer from cancer with a composition comprising a metal complex, wherein the metal complex is obtained or obtainable by reacting a metal salt comprising a metal ion M* with a ligand having the structure (I*): ##STR00094## wherein: X* is selected from the group consisting of —OH, SH, and —N(R.sup.1)(R.sup.2)—, wherein R.sup.1 is H or alkyl, and wherein R.sup.2 is H or alkyl; Z.sup.1* and Z.sup.2* are independently of each other, a substituted aryl or a, substituted or unsubstituted, heteroaryl group; wherein the substituted aryl group is preferably substituted with an hydroxyl or amine group; Y* is selected from the group consisting of H, alkyl, —OH, —SH, halogen and —NR.sup.3R.sup.4; and wherein M* is selected from the group consisting of copper, iron, and manganese.

32. The method of claim 31, wherein the ligand is selected from the group consisting of. ##STR00095##

33. The method of claim 23, wherein the preventing migration of cancer cells is preventing metastasis, tissue invasion, and/or relapse.

34. The method of claim 23, wherein the composition is administered before tumor removal and/or wherein the composition is administered at a site of tumor removal.

35. The method of claim 23, wherein the cancer is brain cancer, colorectal cancer, breast cancer, pancreatic cancer, lung cancer, bladder cancer, prostate cancer, or ovarian cancer, preferably is brain cancer, more preferably glioma.

36. A method of claim 23, wherein the composition is combined with a cancer therapeutic agent.

37. A method, preferably an in vitro method, for determining whether cancer cells are susceptible to immobilizing by a composition as specified in claim 23, comprising contacting the cancer cells with the composition and determining cancer cells to be susceptible to immobilizing by the compound in case the cancer cells are found to be immobilized.

38. The method of claim 23, comprising contacting cancer cells with the compound as specified in claim 23, and thereby preventing migration of cancer cells.

39. A method for treating cancer, comprising the steps of the method for preventing migration of cancer cells according to claim 38, and administering at least one anticancer therapy.

40. The method of claim 39, wherein the anticancer therapy is selected from the list consisting of (i) radiotherapy, (ii) chemotherapy, (iii) anti-hormone therapy, (iv) targeted therapy, (v) immunotherapy, and (vi) any combination of (i) to (v).

41. A complex having the structure: ##STR00096## wherein: M and M′ are metals, preferably selected from the group consisting of copper, iron, manganese and zinc, wherein M′ and M may be the same or different and are preferably the same; X.sup.b is a group O, S or —N(R.sup.1)—, wherein R.sup.1 is H or alkyl; Z.sup.1 and Z.sup.2, are independently of each other a, substituted or unsubstituted, -Aryl-O—, -Aryl-N— or heteroaryl group; Y.sup.b is a group O, S or —N(R.sup.Yb1R.sup.Yb2), wherein R.sup.Yb1 and R.sup.Yb1 are, independently of each other, H or alkyl, preferably H; Y.sup.2 is water or a halogen; Y.sup.2* is a solvent molecule or a halogen, preferably water, methanol or a halogen, more preferably, water, methanol or —Cl; Y.sup.3 is water or a halogen; Y.sup.3* is a solvent molecule or a halogen, preferably water, methanol or a halogen, more preferably, water, methanol or —Cl; n and m are integers, which are independently of each other, 0 or 1; n* and m* are integers, which are independently of each other, 0 or 1; and q is an integer of from 2 to 5, preferably q is 2 or 3, more preferably 2.

42. The complex of claim 41, having the structure: ##STR00097## preferably wherein M and M′ are copper, Y.sup.b is NH.sub.2, X.sup.b is 0, m* and n* are both 1, wherein Y.sup.2* and Y.sup.3* are Cl, and q is an integer in the range of from 2 to 5, preferably 2 or 3, more preferably 2.

Description

FIGURE LEGENDS

[0262] FIG. 1: Synthesis scheme of the ligand.

[0263] FIG. 2: Synthesis scheme of the complexes (FeL and CuL).

[0264] FIG. 3: Synthesis route for ligand (8).

[0265] FIG. 4: FeL and CuL complexes inhibit migration and induce mesenchymal-epithelial transition (MET) in H4 glioma cells. (A) Migration of H4 cells after 24 h of incubation with 25 μM of FeL and CuL assessed with the transwell migration assay. (B) Cell cycle analysis and (C) expression of EMT marker genes in H4 cells under those same conditions. The results were calculated from three independent experiments and are given as the mean±S.E.M. Statistical significance was calculated using one-way ANOVA, followed by Dunnet's test ((*P<0.05, **P<0.01).

[0266] FIG. 5: FeL and CuL complexes inhibit H4 spheroids invasion. Viability (A) and growth (B) of H4 spheroids after 24 h and 72 h of incubation with 25 μM of FeL and CuL. Invasion of H4 spheroids after 24 h and 72 h of incubation with 25 μM of FeL and CuL without (C) or with irradiation with 6 Gy X-rays (D). Scale bars, 500 μm; (E) and (F) are quantifications of the relative sizes of spheroids in the experiments shown in (C) and (D), respectively.

[0267] FIGS. 5 G and H: Invasion of H4 spheroids after 24 h and 72 h of incubation with 25 μM of FeL and CuL and 12.5 μM and 25 μM of the copper complex obtained according to example 6 (“CuL.sub.2”).

[0268] FIG. 6: Reduction of U87-MG cell line invasion of Matrigel™ by compounds of the invention in a spheroid invasion model; reduction in % compared to DMSO control was at 24H: 6.9% for FeL and 39.1% for CuL, and for 48H: 16.3% for FeL and 44.0% for CuL (average of three independent assays using 2-4 spheroids per condition per assay in each case).

[0269] FIG. 7: Reduction of U87-MG cell line invasion of Matrigel™ by compounds of the invention in a spheroid invasion model; reduction in % compared to control for the copper complex obtained according to example 6 (“CuL.sub.2”) and CuL (average of three independent assays using 2-3 spheroids per condition per assay in each case).

[0270] The following Examples shall merely illustrate the invention. They shall not be construed, whatsoever, to limit the scope of the invention.

EXAMPLE 1: SYNTHESIS OF COMPLEX CUL AND FEL

[0271] Synthesis of compounds FeL and CuL described previously on the following publications: Horn, A. et al. Synthesis, crystal structure and properties of dinuclear iron(III) complexes containing terminally coordinated phenolate H2O/OH-groups as models for purple acid phosphatases: efficient hydrolytic DNA cleavage. Inorganica Chim. Acta 358, 339-351 (2005); Horn Jr., A. et al. Synthesis, molecular structure and spectroscopic, electrochemical and magnetic properties of a new dinuclear iron complex containing, μ-sulfate-di-μ-alkoxo bridges: evaluating the influence of the sulfate bridge on the physicochemical properties of the di-μ-alkoxo-diiron unit. J. Braz. Chem. Soc. 17, 1584-1593 (2006); and Fernandes, C. et al. Synthesis, characterization and antibacterial activity of FeIII, CoII, CuII and ZnII complexes probed by transmission electron microscopy. J. Inorg. Biochem. 104, 1214-1223 (2010).

EXAMPLE 2: SYNTHESIS OF 2-OXIRANYLMETHYL-ISOINDOLE-1,3-DIONE (3)

[0272] 16.0 g (86.7 mmol) of compound (2) in 45 mL (573.9 mmol) of epichlorohydrin (3) were placed in a 125 mL flask. The reaction was refluxed for 24 hours and then the excess of compound 3 was removed by vacuum distillation. A white solid was obtained which was then treated with hot methanol and filtered while still hot. The solution obtained was brought to the refrigerator for 24 hours to obtain 11.0 g (mmol) of the product (4). White solid; yield of 63%; .sup.1H RMN (CDCl.sub.3, 500 MHz/ppm): δ 7.88 (dd, J=5.5 Hz, 3.0 Hz, 2H, CH.sub.arom.), 7.83 (dd, J=5.5, 3.0 Hz, 2H, CH.sub.arom.), 3.89 (dd, J=14.53, 5.1 Hz, 1H, CH.sub.2), 3.83 (dd, J=14.53, 5.1 Hz, 1H, CH.sub.2), 3.26-3.21 (m, 1H, CH), 2.81 (t, J=4.6 Hz, 1H, CH.sub.2), 2.65 (dd, J=4.6, 2.6 Hz, 1H, CH.sub.2). .sup.13C RMN (CDCl.sub.3, 125 MHz/ppm): δ 167.9 (2×C), 134.1 (2×CH), 131.9 (2×C), 123.4 (2×CH), 49.0 (CH), 46.1 (CH.sub.2), 39.6 (CH.sub.2).

EXAMPLE 3: SYNTHESIS OF 2-{[(PYRIDIN-2-YLMETHYL)-AMINO]-METHYL}-PHENOL (6)

[0273] 4.35 mL (41.0 mmol) of 2-hidroxibenzaldehyde and 4.22 mL (41.0 mmol) of 2-aminemethilpyridine were added in a 125 mL flask in 50 mL of methanol. The reaction was stirred for 30 min. at room temperature. Then, 1.52 g (41.0 mmol) of Sodium borohydride were added slowly in an ice bath and the reaction was stirred for 24 hours more. The reaction was concentrated and extracted with dichloromethane x brine. The organic phase was treated with sodium sulfate anhydrous, providing an orange oil, and left in a beaker for crystallization. The obtained solid was macerated, washed with cold isopropanol, vacuum filtered and dried in the desiccator. Obtained 7.00 g (32.6 mmol) of white solid with yield of 80%. .sup.1H RMN (CDCl.sub.3, 500 MHz/ppm): δ 8.58 (s, 1H, CH.sub.arom.), 7.66 (d, J=7.6, 1.7 Hz, 1H, CH.sub.arom.), 7.23-7.18 (m, 3H, CH.sub.arom.), 6.97 (d, J=7.30 Hz, 1H, CH.sub.arom.), 6.86 (dd, J=8.1, 0.6 Hz, 1H, CH.sub.arom.), 6.78 (dt, J=7.3, 0.6 Hz, 1H, CH.sub.arom.), 4.01 (s, 2H, CH.sub.2), 3.93 (s, 2H, CH.sub.2). .sup.13C RMN (CDCl.sub.3, 125 MHz/ppm): δ 158.2 (C), 157.8 (C), 149.5 (CH), 136.7 (CH), 128.8 (CH), 128.6 (CH), 122.7 (C), 122.5 (CH), 119.1 (CH), 116.5 (2×CH), 53.1 (CH.sub.2), 51.9 (CH.sub.2).

EXAMPLE 4: SYNTHESIS OF 2-{2-HYDROXY-3-[(2-HYDROXY-BENZYL)-PYRIDIN-2-YLMETHYL-AMINO]-PROPYL}-ISOINDOLE-1,3-DIONE (7)

[0274] 5.54 g (25.8 mmol) of product 6 and 5.24 g (25.8 mmol) of product 3 were added in 50 mL of methanol in a 125 mL Ambar flask and the reaction was stirred at room temperature for 96 hours. The solid precipitated and was vacuum filtered, washed with cold isopropanol and dried in the desiccator. Obtained 7.55 g (18.00 mmol) of white solid with yield of 70%. .sup.1H RMN (CDCl.sub.3, 500 MHz/ppm): δ 8.53 (ddd, J=4.88, 2.59, 0.76 Hz, 1H, CH.sub.arom.), 7.81-7.77 (m, 2H, CH.sub.arom.), 7.77-7.70 (m, 2H, CH.sub.arom.), 7.62 (m, 1H, CH.sub.arom.), 7.19-7.16 (m, 1H, CH.sub.arom.), 7.13-7.07 (m, 2H, CH.sub.arom.), 6.97 (dd, J=7.47, 1.52 Hz, 1H, CH.sub.arom.), 6.75 (dd, J=8.09, 1.07 Hz, 1H, CH.sub.arom.), 6.71 (dt, J=7.47, 1.21 Hz, 1H, CH.sub.arom.), 4.20-4.14 (m, 1H, CH), 4.02 (d, J=15.4, 1H, CH.sub.2), 3.93 (d, J13.4, 1H, CH.sub.2), 3.86 (d, J15.4, 1H, CH.sub.2), 3.72-3.68 (m, 2H, CH.sub.2), 2.75-2.71 (m, 2H, CH.sub.2), 3.65-3.60 (m, 1H, CH.sub.2). .sup.13C RMN (CDCl.sub.3, 125 MHz/ppm): δ 181.5 (2×C), 181.2 (2×C), 161.0 (C), 156.0 (C), 148.9 (CH), 137.3 (CH), 134.1 (2×CH), 129.3 (2×CH), 123.4 (3×CH), 122.7 (C), 122.6 (CH), 119.2 (CH), 116.7 (CH), 67.2 (CH), 58.5 (2×CH.sub.2), 58.0 (CH.sub.2), 42.2 (CH.sub.2).

EXAMPLE 5: SYNTHESIS OF 2-{[(3-AMINO-2-HYDROXY-PROPYL)-PYRIDIN-2-YLMETHYL-AMINO]-METHYL}-PHENOL (8)

[0275] 7.55 g (18.0 mmol) of product 7 and 1.81 g (36.00 mmol) of hydrazine monohydrate were added in 50 mL of ethanol in a 125 mL flask, and the reaction was refluxed for 10 minutes. When a white insoluble product was observed, the solution was acidified until pH 4.0 with HCl .sub.(conc.) and vacuum filtered. Then the solution was concentrated, basified with NaOH 5M until pH 10.0 and successive extractions with DCM were done. The organic phase was treated with sodium sulfate sodium sulfate anhydrous, providing 3.37 g of an orange oil with yield of 65%. IR (KBr, cm.sup.−1): 3479, 3410, 3321, 3209, 2829, 1676, 1653, 1489, 1411, 758. .sup.1H RMN (CDCl.sub.3, 500 MHz/ppm): δ 8.57 (d, J=4.28 Hz, 1H, CH.sub.arom.), 7.64 (dt, J=7.7, 1.1 Hz, 1H, CH.sub.arom.), 7.22-7.13 (m, 3H, CH.sub.arom.), 6.99 (dd, J=7.5, 1.1 Hz, 1H, CH.sub.arom.), 6.83 (dd, J=7.7, 1.1 Hz, 1H, CH.sub.arom.), 6.76 (dt, J=7.7, 1.1 Hz, 1H, CH.sub.arom.), 4.00 (d, J=15.3 Hz, 1H, CH.sub.2), 3.89 (m, 1H, CH.sub.2), 3.85 (d, J=15.3 Hz, 1H, CH.sub.2), 3.83-3.77 (m, 1H, CH), 3.69 (d, J=13.6 Hz, 1H, CH.sub.2), 2.74-2.56 (m, 4H, CH.sub.2). .sup.13C RMN (CDCl.sub.3, 125 MHz/ppm): δ 157.6 (C), 157.5 (C), 150.0 (CH), 137.4 (CH), 129.7 (CH), 129.2 (CH), 123.4 (CH), 122.6 (C), 122.6 (CH), 119.2 (CH), 116.7 (CH), 69.5 (CH), 59.0 (CH.sub.2), 58.4 (CH.sub.2), 57.9 (CH.sub.2), 45.6 (CH.sub.2).

EXAMPLE 6: SYNTHESIS OF COMPLEX BETWEEN LIGAND 8 AND CuCL.SUB.2 .(“CuL.SUB.2.”)

[0276] 0.287 g (1.00 mmol) of compound 8 in 10 mL of isopropanol were stirred in reflux for 10 minutes, then 0.170 g (1.00 mmol) of CuCl.sub.2 were added. H.sub.2O solubilized in 10 mL of isopropanol was added in the reaction and refluxed for 2 hours. The reaction medium was cooled to room temperature and the precipitate formed was vacuum filtered. Elemental Analysis (% CHN) found: C 39.49 H 5.31 N 7.85. Mass ESI+(m/z): 554.0997; 697.1631; 796.0520. The complex is water soluble, such as in a concentration of 1 mM.

EXAMPLE 7: CELL CULTURE

[0277] Human brain neuroglioma (H4) cells (ATCC, Manassas, Va., USA) were grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) and 1% Penicillin/Streptomycin. Human brain glioblastoma (U-87 MG) cells were grown in Minimum Essential Medium (MEM) supplemented with 10% Fetal Bovine Serum (FBS) and 1% Penicillin/Streptomycin. All medium and supplements were obtained from Gibco™ Invitrogen. The cell lines were cultured continuously as a monolayer at 37° C. and 5% of C02 for no more than 20 passages at a time after resuscitation.

EXAMPLE 8: CELL CYCLE ASSAY

[0278] For the cell cycle assay, 7×105 cells were seeded in 75 cm.sup.2 flasks and incubated for 24 hours at 37° C. The medium was removed and cells were washed once with DPBS before 10.5 mL of fresh medium, solutions of 24, 16 and 8 μM of FeL and CuL, or a solution of 0.125% of DMSO (the concentration relative to the highest compound's concentration used—24 μM) were added to the flasks. The flasks were incubated for an additional 24 hours, after which cells were detached, washed with PBS, and 10.sup.6 cells were fixed through drop by drop addition of 70% cold ethanol (v/v in DPBS) under gently vortexing. Samples were stored at 4° C. for 24 hours, centrifuged and the supernatant was removed. Subsequently, 250 μL of RNase A (10 mg/mL in PBS; Sigma Aldrich, St. Louis, Mo., USA) were added to each sample, which was then incubated at room temperature for 30 min and washed twice with DPBS. In the dark, each sample was stained with 20 μg/mL of propidium iodide (PI) (eBioscience, San Diego, Calif., USA) for 15 min before being analyzed using a flow cytometer (BD FACS CANTO™ II).

EXAMPLE 9: TRANSWELL MIGRATION ASSAY

[0279] Cells starved overnight were detached and seeded onto cell culture inserts in 24-well plates (Millipore transwell PET filters, 8 μm pore; Merck, Kenilworth, N.J., USA) at a density of 1.0×10.sup.4 cells in 150 μL of FBS-free medium, or FBS-free medium containing 0.125% DMSO, 25 μM of FeL or 25 μM of CuL. The lower transwell chambers were filled with 600 μL of media without FBS (negative control) or with medium containing 10% FBS. After 24 h of incubation at 37° C., the inserts were washed with DPBS, fixed with 4% paraformaldehyde, washed again, and stained with 1 g/mL of Hoechst 33342 (Thermo Scientific, Waltham, Mass., USA) for 20 min at room temperature. Cells were then imaged at a 200× amplification on a confocal microscope (Zeiss LSM 710). Seven random fields were photographed per insert, with at least two inserts being analyzed for each condition per experiment. The results shown were calculated based on three independent experiments.

EXAMPLE 10: SPHEROIDS VIABILITY ASSAY

[0280] For spheroids formation, 2.5×10.sup.3 cells were seeded in 100 μL/well in 96-well plates coated with 1.5% agarose (w/v in PBS). After 1 day of incubation, spheroids were fully formed, and 100 μL of fresh medium or medium with DMSO or the compounds was added to a final concentration of 0.125% and 25 μM, respectively. Cells were incubated for 24 h or 72 h at 37° C. before cell viability was estimated using the CellTiter-Glo® 3D assay (Promega, Madison, Wis., USA) according to the manufacturer's instructions. Luminescence was read in a CLARIOstar® microplate reader (BMG LABTECH). In addition, spheroids' viability was also estimated based on spheroids' growth. For that, the total area of each spheroid was determined using the INSIDIA macro in FIJI, and then normalized to the area of the spheroid at day 0 (to account for possible differences in the spheroids initial size) and to the size of the untreated spheroids at each time point (to assess the effect of the DMSO and the compounds on spheroid growth).

EXAMPLE 11: SPHEROID INVASION ASSAY

[0281] Each one-day old spheroid, formed as described above, was collected into a tube, washed once with FBS-free medium, and resuspended in 40 μl of a 4.5 mg/ml Matrigel™ (Corning #356231) solution in FBS-free medium. Then, each spheroid-containing suspension was spotted onto the centre of a well of a 24-wells plate and incubated as a hanging drop for 1 h until the matrigel had polymerized. Complete medium, complete medium with 0.125% DMSO, or complete medium containing 24 μM of the compounds were added and the spheroids were incubated for 24 h at 37° C. before being irradiated (or not as a control) with 6 Gy X-rays on a Faxitron MultiRad225. Images of spheroids and invading cells were acquired immediately after embedment and every 24 h after that, using an Eclipse Ts2 microscope (Nikon). At each time point (24 h, 48 h, and 72 h) the total area of the spheroid and invading cells was determined as described above.

EXAMPLE 12: FEL AND CUL COMPLEXES INHIBIT MIGRATION THROUGH INDUCTION OF MESENCHYMAL-EPITHELIAL TRANSITION (MET) IN GLIOMA CELLS

[0282] The effect of FeL and CuL on the migration of H4 cells was thus investigated by the transwell assay. The number of cells migrated to the bottom of the membrane revealed that both compounds can clearly inhibit the migratory ability of H4 cells (FIG. 4A). To investigate to what extent this observation was related to cell proliferation or cell cycle arrest induction, the effects of the compounds on the cell cycle of H4 cells were investigated by flow cytometry. While FeL showed no effect on the cell cycle of H4 cells, CuL induced a significant decrease in the G0/G1 phase of the cycle (*P<0.05), with a concomitant increase in the % of cells in the S and G2/M phases (of about 7.7 and 6.2%, respectively) that was, however, statistically not significant (FIG. 4B). Regardless, despite the fact that exit from the S phase of the cycle, as well as entry or exit of the G2/M was slightly affected in response to CuL, this difference does not justify the significant difference observed in cells' migration upon exposure to the compound. As such, looking for another possible explanation, we next analyzed the expression of several EMT markers in the FeL/CuL treated cells by qPCR. The results evidenced that treatment with the compounds is accompanied by a statistically significant increase in expression of E-cadherin, and a slight reduction of vimentin (FIG. 4C). The expression of the EMT-related transcription factor snail was found to also be decreased upon treatment with CuL. This expression profile is consistent with the hypothesis that cells treated with FeL and CuL experienced a MET transition, which should originate cells with a less motile phenotype, and is in accordance with the decreased migratory ability observed in compound-treated cells, demonstrating that the compounds do possess anti-metastatic properties.

EXAMPLE 13: COMPLEXES OF THE PRESENT INVENTION INHIBIT 3D SPHEROIDS INVASION

[0283] Several 3D cellular models have been developed that present a level of complexity which is much closer and more representative of several aspects of tumor tissues than the ones shown by monolayer cell cultures. In particular, matrix-embedded 3D cultures have been more and more applied to investigate tumor migration and invasion. As such, in order to try to better estimate the clinical translational potential of the compounds under study, we extended our studies to H4 multicellular spheroids, which are expected to better recapitulate in vivo tumor properties. For that purpose, spheroids generated in agarose-coated plates were first treated with FeL or CuL for up to 24 h or 72 h. Then, cell viability was assessed using the CellTiter-Glo® 3D assay, while spheroid size and growth was accompanied using classical bright field microscope. Surprisingly, incubation with FeL increased cellular viability (FIG. 5A), both after 24 and 72 h of incubation. This increase in viability was accompanied by an increase in spheroid size after 72 h of incubation (FIG. 5B). In contrast, CuL induced a decrease in viability as early as after 24 h of incubation, along with a concomitant decrease in spheroid size (FIGS. 5A and 5B).

[0284] Next, we observed that the compounds FeL and CuL and CuL.sub.2 were able to interfere with the invasive behavior exhibited by H4 cells embedded in matrigel, both without and after irradiation with 6 Gy X-rays (FIGS. 5C, E and 5D, F, G and H respectively). CuL and CuL.sub.2 in particular completely eliminated H4 cells' ability to invade the matrigel matrix, an effect that, in the case of CuL, cannot be attributed solely to the slight decrease in viability found to occur following incubation with this compound. FeL also displayed an ability to inhibit the invasive behavior of H4 cell. The results obtained in the 3D invasion assays thus clearly demonstrate that the compounds possess an anti-metastatic effect not only in monolayer cells, but also in the more representative spheroids model.

EXAMPLE 14: REDUCTION OF U87-MG CELL LINE INVASION OF MATRIGEL™

[0285] The effect of the compounds of the invention on the invasion into Matrigel™ by the highly invasive glioma cell line U87-MG cell line was determined in a spheroid invasion model in analogy to the above. For spheroids formation, 2.0×10.sup.3 cells were seeded in 100 μL/well in Nunclon™ Sphera™ Microplates. After 3 days of incubation, spheroids were fully formed and were embedded in 4.5 mg/ml Matrigel™ as described above. Invasion was assessed at 24 h, 48 h and 72 h after embedment, in the absence of irradiation. As shown in FIG. 6 and FIG. 7, invasion by this cell line is also inhibited by the compounds of the invention.

LITERATURE CITED

[0286] BR 10 2014 022630 A2 [0287] BR 10 2014 017397 A2 [0288] Chen, et al. (2017), J. Pharmacol. Sci. 134, 59-67 [0289] Fernandes et al. (2010) J. Inorg. Biochem. 104, 1214-1223 [0290] Gu et al. (2019), Eur. J. Med. Chem. 164, 654-664 [0291] He et al. (2017), J. Organomet. Chem. 842, 82-92 [0292] Horn et al. (2005) Inorganica Chim. Acta 358, 339-351 [0293] Horn Jr. et al. (2006) J. Braz. Chem. Soc. 17, 1584-1593