NITROGEN-CONTAINING ANALOGS OF SALINOMYCIN, SYNTHESIS AND USE AGAINST CANCER STEM CELLS AND MALARIA

Abstract

The present invention concerns compounds of formula (I), enantiomers, mixture of enantiomers, diastereoisomers and mixture of diasteroisomers thereof formula (I): wherein at least one of W, X and Y is selected from the group consisting of —NR.sub.1R.sub.2; —NR.sub.3—(CH.sub.2).sub.n—NR.sub.4R.sub.5; —O—(CH.sub.2).sub.n—NR.sub.4R.sub.5; —NR.sub.3—(CH.sub.2).sub.n—N′R.sub.6R.sub.7R.sub.8; and —O—(CH.sub.2).sub.n—N′R.sub.6R.sub.7R.sub.8 and Z is a functional group capable of chelating iron salts. The present invention also concerns the compounds of formula (I) for use as a drug, in particular, in the treatment of cancer and malaria.

##STR00001##

Claims

1. A compound of formula (I), enantiomers, mixture of enantiomers, diastereoisomers and mixture of diasteroisomers thereof: ##STR00053## wherein: W is selected from the group consisting of ═O; —NR.sub.1R.sub.2; —NR.sub.3—(CH.sub.2).sub.n—NR.sub.4R.sub.5; —O—(CH.sub.2).sub.n—NR.sub.4R.sub.5; —NR.sub.3—(CH.sub.2).sub.n—N.sup.+R.sub.6R.sub.7R.sub.8 and —O—(CH.sub.2).sub.n—N.sup.+R.sub.6R.sub.7R.sub.8; X is selected from the group consisting of ═O, —OH; —NR.sub.1R.sub.2; —NR.sub.3—(CH.sub.2).sub.n—NR.sub.4R.sub.5; —O—(CH.sub.2).sub.n—NR.sub.4R.sub.5; —NR.sub.3—(CH.sub.2).sub.n—N.sup.+R.sub.6R.sub.7R.sub.8 and —O—(CH.sub.2).sub.n—N.sup.+R.sub.6R.sub.7R.sub.8, Y is selected from the group consisting of —OH; ═N—OH; —NR.sub.1R.sub.2, —NR.sub.3—(CH.sub.2).sub.n—NR.sub.4R.sub.5; —O—(CH.sub.2).sub.n—NR.sub.4R.sub.5, —NR.sub.3—(CH.sub.2).sub.n—N.sup.+R.sub.6R.sub.7R.sub.8 and —O—(CH.sub.2).sub.n—N.sup.+R.sub.6R.sub.7R.sub.8, R.sub.1 and R.sub.2, identical or different, are selected from the group consisting of H; (C.sub.1-C.sub.16)-alkyl; (C.sub.3-C.sub.16)-alkenyl; (C.sub.3-C.sub.16)-alkynyl; (C.sub.3-C.sub.16)-cycloalkyl; aryl; heteroaryl; (C.sub.1-C.sub.6)-alkyl-aryl; (C.sub.1-C.sub.6)-alkyl-heteroaryl; or R.sub.1 represents H and R.sub.2 represents OR.sub.9, where R.sub.9 is H, (C.sub.1-C.sub.6)-alkyl, aryl and (C.sub.1-C.sub.6)-alkyl-aryl; R.sub.3 is selected from the group consisting of H; (C.sub.1-C.sub.6)-alkyl; (C.sub.1-C.sub.6)-alkyl-aryl; R.sub.4 and R.sub.5, identical or different, are selected from the group consisting of H; (C.sub.1-C.sub.6)-alkyl; aryl and (C.sub.1-C.sub.6)-alkyl-aryl; R.sub.6, R.sub.7 and R.sub.8, identical or different, are selected from the group consisting of (C.sub.1-C.sub.6)-alkyl; aryl and (C.sub.1-C.sub.6)-alkyl-aryl; Z is a group such as OH; NHNR.sub.9R.sub.10, NHOC(O)R.sub.11; N(OH)—C(O)R.sub.11; OOH, SR.sub.12; 2-aminopyridine; 3-aminopyridine; —NR.sub.3—(CH.sub.2).sub.n—NR.sub.4R.sub.5; and —NR.sub.3—(CH.sub.2).sub.n—OH; where: R.sub.9 and R.sub.10, identical or different, are selected from the group consisting of H, (C.sub.1-C.sub.6)-alkyl, aryl and (C.sub.1-C.sub.6)-alkyl-aryl; R.sub.11 is selected from the group consisting of H; (C.sub.1-C.sub.16)-alkyl; (C.sub.3-C.sub.16)-alkenyl; (C.sub.3-C.sub.16)-alkynyl; aryl; heteroaryl; (C.sub.1-C.sub.6)-alkyl-aryl; (C.sub.1-C.sub.6)-alkyl-heteroaryl; R.sub.12 is selected from the group consisting of H; (C.sub.1-C.sub.16)-alkyl; (C.sub.3-C.sub.16)-alkenyl; (C.sub.3-C.sub.16)-alkynyl; aryl; heteroaryl; (C.sub.1-C.sub.6)-alkyl-aryl; (C.sub.1-C.sub.6)-alkyl-heteroaryl n=0,2,3,4, 5 or6, with the proviso that at least one of W, X and Y is selected from the group consisting of —NR.sub.1R.sub.2; —NR.sub.3—(CH.sub.2).sub.n—NR.sub.4R.sub.5; —O—(CH.sub.2).sub.n—NR.sub.4R.sub.5; —NR.sub.3—(CH.sub.2).sub.n—N.sup.+R.sub.6R.sub.7R.sub.8 and —O—(CH.sub.2).sub.n—N.sup.+R.sub.6R.sub.7R.sub.8.

2. The compound according to claim 1, wherein: R.sub.1 and R.sub.2, identical or different, are selected from the group consisting of H; (C.sub.1-C.sub.16)-alkyl; (C.sub.3-C.sub.16)-alkenyl; (C.sub.3-C.sub.16)-alkynyl; (C.sub.3-C.sub.16)-cycloalkyl; and (C.sub.1-C.sub.6)-alkyl-heteroaryl, R.sub.3 is selected from the group consisting of H; and (C.sub.1-C.sub.6)-alkyl; R.sub.4 and R.sub.5, identical or different, are selected from the group consisting of H; (C.sub.1-C.sub.6)-alkyl; and (C.sub.1-C.sub.6)-alkyl-aryl.

3. The compound according to claim 1 or 2, wherein R.sub.1 is H and R.sub.2 is selected from the group consisting of (C.sub.1-C.sub.16)-alkyl, advantageously (C.sub.3-C.sub.14)-alkyl; (C.sub.3-C.sub.16)-alkenyl, advantageously (C.sub.3-C.sub.5)-alkenyl; (C.sub.3-C.sub.16)-alkynyl, advantageously (C.sub.3-C.sub.5)-alkynyl; (C.sub.3-C.sub.16)-cycloalkyl, advantageously (C.sub.3-C.sub.6)-cycloalkyl; and (C.sub.1-C.sub.6)-alkyl-heteroaryl advantageously, CH.sub.2-pyridinyl.

4. The compound of formula (I) according to any of claims 1 to 3, wherein Z is OH or NHOH, advantageously OH.

5. The compound according to any of claims 1 to 4, wherein W, X and Y, identical or different, are selected from the group consisting of —NR.sub.1R.sub.2; —NR.sub.3—(CH.sub.2).sub.n—NR.sub.4R.sub.5; —O—(CH.sub.2).sub.n—NR.sub.4R.sub.5; —NR.sub.3—(CH.sub.2).sub.n—N.sup.+R.sub.6R.sub.7R.sub.8; and —O—(CH.sub.2).sub.n—N.sup.+R.sub.6R.sub.7R.sub.8; R.sub.1 to R.sub.8 and n being as previously defined.

6. The compound according to any of claims 1 to 4, wherein two of X, Y or Z, identical or different, are selected from the group consisting of —NR.sub.1R.sub.2; —NR.sub.3—(CH.sub.2).sub.n—NR.sub.4R.sub.5; —O—(CH.sub.2).sub.n—NR.sub.4R.sub.5; —NR.sub.1—(CH.sub.2).sub.n—N.sup.+R.sub.6R.sub.7R.sub.8; and —O—(CH.sub.2).sub.n—N.sup.+R.sub.6R.sub.7R.sub.8; R.sub.1 to R.sub.8 and n being as previously defined.

7. The compound of formula (I) according to any of claims 1 to 4, wherein one of X, Y or Z is selected from the group consisting of —NR.sub.1R.sub.2; —NR.sub.3—(CH.sub.2).sub.n—NR.sub.4R.sub.5; —O—(CH.sub.2).sub.n—NR.sub.4R.sub.5; —NR.sub.1—(CH.sub.2).sub.n—N.sup.+R.sub.6R.sub.7R.sub.8; and —O—(CH.sub.2).sub.n—N.sup.+R.sub.6R.sub.7R.sub.8, R.sub.1 to R.sub.8 and n being as previously defined.

8. The compound of formula (I) according to claim 7, wherein X is selected from the group consisting of —NR.sub.1R.sub.2; —NR.sub.3—(CH.sub.2).sub.n—NR.sub.4R.sub.5, —O—(CH.sub.2).sub.n—NR.sub.4R.sub.5; —NR.sub.1—(CH.sub.2).sub.n—N.sup.+R.sub.6R.sub.7R.sub.8; and —O—(CH.sub.2).sub.n—N.sup.+R.sub.6R.sub.7R.sub.8, advantageously —NR.sub.1R.sub.2; —NR.sub.3—(CH.sub.2).sub.n—NR.sub.4R.sub.5 and —O—(CH.sub.2).sub.n—NR.sub.4R.sub.5 and Y is OH; R.sub.1 to R.sub.8 and n being as previously defined.

9. The compound of formula (I) according to claim 7, wherein X is selected from the group consisting of ═O and OH, advantageously OH and Y is selected from the group consisting of —NR.sub.1R.sub.2; —NR.sub.3—(CH.sub.2).sub.n—NR.sub.4R.sub.5; —O—(CH.sub.2).sub.n—NR.sub.4R.sub.5; —NR.sub.1—(CH.sub.2).sub.n—N.sup.+R.sub.6R.sub.7R.sub.8; and —O—(CH.sub.2).sub.n—N.sup.+R.sub.6R.sub.7R.sub.8, advantageously —NR.sub.1R.sub.2; —NR.sub.3—(CH.sub.2).sub.n—NR.sub.4R.sub.5 and —O—(CH.sub.2).sub.n—NR.sub.4R.sub.5, R.sub.1 to R.sub.8 and n being as previously defined.

10. The compound according to claim 9, wherein X is OH, Z is OH and Y is NR.sub.1R.sub.2 where R.sub.1 is H and R.sub.2 is selected from the group consisting of (C.sub.1-C.sub.16)-alkyl, advantageously (C.sub.8-C.sub.14)-alkyl; (C.sub.3-C.sub.16)-alkenyl, advantageously (C.sub.3-C.sub.5)-alkenyl; (C.sub.3-C.sub.16)-alkynyl, advantageously (C.sub.3-C.sub.5)-alkynyl and (C.sub.3-C.sub.16)-cycloalkyl, advantageously (C.sub.3-C.sub.6)-cycloalkyl.

11. The compound according to claim 1, selected from the group consisting of: ##STR00054## ##STR00055## ##STR00056## ##STR00057## ##STR00058## ##STR00059## ##STR00060##

12. The compound according to any of the preceding claims for use as a drug.

13. The compound for use according to claim 12 in the treatment of cancer, advantageously breast cancer.

14. The compound for use according to claim 12 in the prevention of cancer relapse and/or metastases.

15. The compound for use according to claim 12 for the treatment of malaria.

16. A pharmaceutical composition comprising at least a compound of formula (I) according to any one of claims 1 to 11, a pharmaceutically acceptable salt, solvate or hydrate thereof, and at least one pharmaceutically acceptable excipient, advantageously for use in the treatment of cancer, such as breast cancer.

17. The pharmaceutical composition according to claim 16, further comprising another anticancer drug, advantageously Adriamycin and Cyclophosphamide or Docetaxel.

18. A pharmaceutical product comprising: a) the compound of formula (I) according to any of claims 1 to 11, and b) another chemotherapy compound, such as Adriamycin and Cyclophosphamide or Docetaxel, as combination product for simultaneous, separate or staggered use as a medicament, in particular in the treatment of cancer, advantageously breast cancer.

Description

DESCRIPTION DES FIGURES

[0174] FIG. 1 represents viability of HMLER CD24− cells (full line) and HMLER CD24+ cells (dotted line) at different concentration of salinomycine (1A), AM5 (16), AM9 (10) or AM13 (1D).

[0175] The Y axis represents cell viability and is expressed as percentage.

[0176] The X axis represents the concentration of each product in μM. The concentrations used are, from left to right from the intersection between the X and Y axes: 0.0001 μM; 0.001; 0.01 μM; 0.1 μM, 1 μM and 10 μM. Each dot on the line represents measured cell viability at the corresponding concentration.

[0177] FIG. 2 is a representative phase contrast photomicrographs of mammospheres formed after 11 days in the absence of any added compound (Control) or in the presence of a defined amount of salinomycine, AM5, AM9 or AM13. Sal analogues (AM5, AM13) reduced the number and the size of mammosphere at low nanomolar concentrations. The size of the mammospheres is correlated with progenitor cell proliferation, whereas the number of mammospheres formed after serial passages at clonal density is correlated with the self-renewal capacity of primitive Cancer Stem Cells. A smaller mass indicates cell death and regression of the mammosphere.

[0178] FIG. 3A is a representative phase contrast photomicrographs of mammospheres formed after 7 or 14 days in the absence of any added compound (Not treated) or in the presence of a defined amount of salinomycine, AM5, Taxol or the combination of AM5 and Taxol. A smaller mass indicates cell death and regression of the mammosphere.

[0179] FIG. 3B represents the quantification of the number and the size of mammosphere. The combination of AM5 at 15 nM and Taxol at 5 nM decrease the number and the size of mammosphere with an improved efficacy than AM5 alone at 15 nM or 5 nM.

[0180] FIG. 4 represents MCF-7 tumor-growth curves of compound-treated mice (3 mg per kg body weight per day, intra peritoneal injection, n=5). Non-lethal injections of an active Sal analogue (AM5) inhibits breast cancer tumor growth in mice (n=5; error bars, s.e.m).

[0181] FIG. 5: Salinomycine analogue—induced cell death is inhibited by the ROS scavenger N-acetylcysteine (NAC). Cell lines were incubated with or without 500 nM of Sal analogues for 48 h. Apoptosis was evaluated by Annexin V-FITC and PI staining, and FACS analysis. All data are expressed as means±s.d. from three individual experiments (*; P<0.05)

[0182] FIG. 6: Lysosomal iron mediates Sal analogues-activated cell death signaling. Sal analogue-induced cell death is inhibited by lysosomal iron chelator deferoxamine mesylate (DFO). Cells were treted as in (c) with or without the indicated concentration of DFO for 48 h. Apoptosis was evaluated as in c.

[0183] FIG. 7 represents the quantification of the number of mammospheres counted in experiments shown in FIG. 9. The number of mammospheres formed after serial passages at clonal density is correlated with the self-renewal capacity of primitive Cancer Stem Cells. A smaller mass indicates cell death and regression of the mammosphere. As compared to untreated cells or cells treated with salinomycine, only AM5 and more efficiently AM23 reduced the number of mammospheres.

[0184] FIG. 8 represents the quantification of the size of mammospheres counted in experiments shown in FIG. 9. The size of the mammosphere is only reduced by the highest dose of AM23 whereas salinomycine, AM5 and lower dose of AM23 do not alter the tumor diameters.

[0185] FIG. 9 represents images of the third generation of mammospheres formed from individual HMLER CD24low cells treated during 7 days with the indicated drug. The size of the mammospheres is correlated with progenitor cell proliferation capacity, whereas the number of mammospheres formed after serial passages at clonal density is correlated with the self-renewal capacity of primitive Cancer Stem Cells. A smaller mass indicates cell death and regression of the mammosphere.

[0186] FIG. 10 is an ORTEP drawing of the X-Ray structure of AM23.

[0187] FIG. 11 represents FACS analyses of ROS in HMLER CD24low cells treated with salinomycine, AM5 and AM23.

[0188] FIG. 12 is a graph representing the influence of salinomycine, AM5, AM13 and AM9 on intracellular sodium concentration

[0189] FIG. 13 represents the evaluation of the prevention of tumor growth in MCF-7 xenograft-bearing mice.

[0190] FIG. 14 represents a comparative H&E staining images of peripheral tissues of mice treated as in FIG. 13, representative of five biological replicates (scale bar, 100 μm).

[0191] FIG. 15 is a graph representing the mice body-weight during treatment with AM5. Error bars for mice body weight represent s.d. and correspond to correspond to five animals per group.

[0192] FIG. 16 is a graphic representation of percent of dead cells (DIOC6(3) negative/DAPI positive or negative)

[0193] FIG. 17 represents a FACS analysis of ROS in cells treated for 48 h.

EXEMPLES

Example 1

Synthesis of Compound of Formula (I)

[0194] Preparation of Oxidized Salinomycin Acid (oxo-Sal-H) 2:

##STR00027##

[0195] Salinomycin sodium (2.00 g, 2.587 mmol) was dissolved in 250 mL DCM and mangandioxide was added (9.00 g, 103.5 mmol, 40 eq). The suspension was stirred over night at room temperature. After complete conversion of starting material the mixture was filtrated on celite. The filtrate was extracted with 15 mM aqueous H2SO4 solution, dried on MgSO4 and concentrated to give product 2 (1,71 g, 2.28 mmol, 96%) as pure and white foam without any further purification.

[0196] 1H NMR (CDCl3, 500 MHz, rt): 0.64-0.72 (6H, m), 0.72-0.82 (6H, m), 0.83-0.98 (12H, m), 1.04-1.17 (4H, m), 1.19-1.27 (2H, m), 1.30-1.57 (12H,m), 1.59-2.05 (14H, m), 2.43-2.60 (2H, m), 2.63-2.73 (1H, m) 2.76-2.88 (1H, m), 3.38-3.52 (1H, m), 3.66 (1H, d, J=9.6 Hz), 3.76 (1H, d, J=10.2 Hz), 3.88-4.04 (2H, m), 4.11-4.22 (1H, m), 6.20 (1H, d, J=10.7), 7.12 (1H, d, J=10.7).

[0197] 13C NMR (CDCl3, 500 MHz, rt): 6.6, 7.0, 11.3, 12.1, 12.6, 13.1, 14.2, 15.5, 16.0, 17.6, 19.8, 20.7, 22.9, 26.4, 27.2, 28.3, 28.6, 32.1, 32.2, 33.2, 34.2, 35.5, 38.4, 40.2, 50.3, 51.6, 55.7, 67.7, 69.6, 71.0,73.2, 75.8, 76.5, 76.7, 90.0, 98.0, 105.3, 107.1, 142.3, 183.2, 187.9, 217.9.

[0198] HRMS (ESI) m/z: Calculated for C42H68NaO11+[M+Na+] 771.4654, found: 771.4560.

[0199] Methylation of oxo-Sal-H to Form oxo-Sal-Me 3:

##STR00028##

[0200] In a flame dried and Ar-flushed schlenk flask 2 (100 mg, 0.134 mmol) was introduced and dissolved in anhydrous DMF (3 mL). Cesium carbonate (56.5 mg, 0.174 mmol, 1.3 eq) was added followed by methyl iodide (11 μL, 0.174 mmol, 1.3 eq) and the solution was stirred for 24 h at room temperature. After completion of the reaction the solvent was removed, the residue was taken up in DCM and the solution was extracted with 15 mM aqueous H2SO4 solution, saturated NaHCO3 solution, water, brine and dried over MgSO4. The solution was filtred, concentrated and purified on silica gel with a CombiFlash using DCM/MeOH 10/0.2. The pure product 3 (96.5 mg, 0.126 mmol, 95%) was isolated as a white foam.

[0201] (The oxidation-methylation procedure can be inversed, the yields in both steps do not change so much.)

[0202] 1H NMR (CDCl3, 300 MHz, rt): 0.63-0.72 (9H, m), 0.72-0.77 (3H, d, J=7.0 Hz), 0.80-0.89 (12H, m), 1.06-1.17 (7H, m), 1.17-1.21 (2H, m), 1.21-1.32 (3H, m), 1.32-1.46 (8H, m), 1.46-1.58 (3H, m), 1.60-1.80 (4H, m), 1.80-1.94 (2H, m), 1.98-2.12 (2H, m), 2.48-2.56 (1H, m), 2.58-2.68 (1H, m), 2.81-2.99 (2H, m), 3.26-3.32 (1H, m), 3.51 (1H, dd, J=9.8 Hz, 1.5 Hz), 3.59-3.73 (2H, m), 3.70 (3H, s, OMe), 3.85-3.97 (2H, m), 6.16 (1H, d, J=10.7 Hz), 7.18 (1H, d, 10.7 Hz).

[0203] 13C NMR (CDCl3, 300 MHz, rt): 6.6, 7.2, 11.1, 11.9, 12.1, 14.0, 15.0, 17.9, 18.7, 19.8, 20.8, 22.6, 22.7, 26.3, 28.1, 29.2, 29.7, 30.3, 34.26, 34.30, 34.4, 36.6, 39.2, 39.9, 47.7, 49.1, 52.7, 57.5, 70.0, 71.2, 71.8, 72.3, 75.1, 77.1, 77.4, 88.7, 97.6, 105.5, 127.3, 144.2, 176.6, 190.9, 214.3.

[0204] HRMS (ESI) m/z: calculated for C43H70NaO11+ [M+Na+] 785.4810, found: 785.4807.

[0205] Procedure for Reductive Amination Reactions on 2 or 3:

[0206] 100 mg of starting material 2 was dissolved in 3 ml MeOH, the primary amine was added (10 eq.), followed by AcOH (50 μL). The solution was stirred one hour at room temperature before CeCl3.7H2O was added. A solution of NaBH3CN (1.05-1.3 eq) in 2 mL of MeOH was added very slowly with the help of a syringe pump over a period of 8 h at room temperature. After further 4 h stirring at room temperature, a sample was taken out of the reaction mixture and a miniwork-up was done, followed by TLC. If starting material was not fully consumed, some more NaBH3CN in MeOH was slowly added until full conversion was visible. Then, a aqueous solution of 15 mM H2SO4 (2-4 mL) was carefully added, followed by DCM. The layers were separated and the aqueous layer was extracted 2 times with DCM. The combined organic layers were washed with aqueous 15 mM H2SO4, sat. aqueous NaHCO3 solution, water and brine. The solution was dried over MgSO4 and concentrated, before purification with a Combi Flash, using gradually 1 to 3% MeOH in DCM on silica gel. Most of side products could be removed by this step. For the final purification the product was purified by HPLC on C18-reversed phase column. Elution gradient: 50%/50% ACN/H2O (both with 0.1% formic acid) to 100% ACN within 12 min, 10-20 min 100% ACN (depending on polarity of products and side products). Amines eluted at around 60-90% ACN (AM5: 60%, AM9: 70%, AM13: 90-100%). Detection with UV detector at a wavelength of 217 nm.

##STR00029##

[0207] Sal-Propargylamine was prepared using 103 mg of 2 (0.134 mmol), 86 μL (1.34 mmol, 10 eq) propargyl amine, 11 mg (0.174 mmol, 1.3 eq) NaBH3CN, 50.0 mg (0.134 mmol, 1 eq) of CeCl3.7H2O and 50 μL acetic acid in 8 mL of MeOH. After purification with CombiFlash and HPLC 25 mg (0.032 mmol, 24%) of pure product could be isolated as colorless foam.

[0208] 1H NMR (CDCl3, 600 MHz, 5° C.): 0.66 (3H, d, J=7.2 Hz, C39H3), 0.71 (3H, d, J=6.6 Hz, C34H3), 0.75 (3H, dd, J=J=7.8 Hz, C37H3), 0.77 (3H, d, J=7.2 Hz, C38H3), 0.85 (3H, d, J=6.6 Hz, C35H3), 0.86 (3H, m, C32H3), 0.88 (3H, m, C40H3), 0.90 (3H, m, C42H3), 1.13 (1H, dd, J=J=13.8 Hz, C15H), 1.21 (3H, d, J=7.2 Hz, C30H3), 1.25-1.38 (8H, m, C41H, C31H2, C36H, C33H3, C4H), 1.39-1.48 (3H, m, C5H, C8H, C41H), 1.49-1.55 (1H, m, C26H), 1.55-1.63 (3H, m, C26H, C27H2), 1.63-1.71 (3H, m, C14H, C15H, C16H), 1.71-1.80 (3H, m, C6H, C23H, C5H), 1.80-1.91 (2H, m, C36H, C4H), 1.91-2.00 (2H, C22H2), 2.09-2.16 (1H, m, C23H), 2.36-2.40 (1H, s, ≡CH), 2.55-2.64 (2H, m, C12H, C10H), 2.84-2.91 (1H, dt, C2H), 3.53 (1H, m, C13H), 3.57-3.63 (2H, C25H, m, C7H), 3.81-3.85 (1H, m, C29H), 3.91-4.01 (2H, m, C3H, C20H), 4.15 (1H, d, J=10.2 Hz, C9H), 4.28 (2H, bs, NHCH2), 6.28 (1H, m, C19H), 6.44 (1H, d, J=9.6 Hz, C18H).

[0209] 13C NMR (CDCl3, 600 MHz, 5° C.): 6.5 (C32), 7.1 (C39), 11.2 (C40), 12.2 (C38), 12.5 (C42), 13.3 (C37), 14.5 (C30), 15.6 (C34), 16.7 (C36), 17.6 (C35), 20.0 (C4), 21.8 (C26), 22.9 (C41), 25.0 (C33), 26.4 (C5), 28.1 (C6), 28.8 (C27), 30.7 (C31), 30.8 (C23), 32.3 (C14), 36.1 (C8), 37.1 (NHCH2), 37.6 (C15), 39.0 (C16), 40.0 (C22), 49.2 (C2), 50.0 (C10), 53.0 (C20), 55.2 (C12), 68.9 (C9), 71.1 (C28), 71.5 (C7), 72.9 (C25), 75.5 (C3), 75.8 (C13), 76.3 (≡CH), 76.9 (C29), 77.3 (≡C—), 88.6 (C24), 98.6 (C17), 105.6 (C21), 125.8 (C19), 132.2 (C18), 180.8 (C1), 216.1 (C11).

[0210] HRMS (ESI) m/z: Calculated for C45H74NO10+ [M+H+] 788.5307, found: 788.5304.

##STR00030##

[0211] Me-Sal-propargylamine was prepared using 106 mg of 3 (0.139 mmol), 89 μL (1.39 mmol, 10 eq) propargyl amine, 9.6 mg (0.153 mmol, 1.1 eq) NaBH3CN, 51.8 mg (0.138 mmol, 1 eq) of CeCl3.7H2O and 50 μL acetic acid in 8 mL of MeOH. After purification with CombiFlash and HPLC 25 mg (0.031 mmol, 22%) of pure product could be isolated as colorless foam.

[0212] 1H NMR (CDCl3, 500 MHz, 5° C.): 0.72 (3H, d, J=6.9 Hz), 0.75-0.87 (11H, m), 0.88-0.99 (9H, m), 1.07 (1H, ddd, J=13.1 Hz, 13.1 Hz, 12.1 Hz), 1.20-1.68 (21H, m), 1.70-2.05 (8 Hz, m), 2.10-2.26 (3H, m), 2.30-2.41 (2H, m), 2.69-2.74 (1H, m), 3.04 (1H, dt, J=10.8 Hz, 4.1 Hz), 3.47-3.72 (4H, m), 3.80-3.88 (1H, m), 3.90 (3H, s), 4.02-4.09 (2H, m), 6.01-6.08 (2H, m).

[0213] 13C NMR (500 MHz, CDCl3, 5° C.): 6.5, 7.4, 11.0, 12.0, 13.2, 13.9, 14.7, 15.7, 17.5, 19.7, 22.2, 22.7, 25.5, 26.2, 28.0, 29.0, 30.6, 30.7, 32.9, 36.4, 37.1 (2C), 38.6, 38.7, 40.3, 48.0, 48.6, 52.6, 55.2, 56.6, 69.2, 71.0, 71.7, 73.9, 75.1, 76.9, 77.3, 80.1, 88.0, 98.6, 102.0, 108.3, 123.2, 130.5, 176.2, 214.0.

[0214] HRMS (ESI) m/z: calculated for C46H76NO10+ [M+H+] 802.5464, found: 802.5465.

##STR00031##

[0215] Sal-Dodecylamine was prepared using 103 mg of 2 (0.138 mmol), 255.8 mg (1.38 mmol, 10 eq) dodecyl amine, 9 mg (0.145 mmol, 1.05 eq) NaBH3CN, 51.4 mg (0.138 mmol, 1 eq) of CeCl3.7H2O and 20 μL acetic acid in 8 mL of MeOH. After purification with CombiFlash and HPLC 14 mg (0.0152 mmol, 11%) of pure product could be isolated as colorless foam.

[0216] 1H NMR (CDCl3, 500 MHz, 5° C.): 0.67 (3H, d, J=6.9 Hz), 0.66-0.77 (9H, m), 0.78-0.90 (15H, m), 1.10-1.51 (34H, m), 1.51-1.78 (9H, m), 1.80-2.10 (6H, m), 2.50-2.60 (2H, m), 2.77-2.87 (1H, m), 3.30-3.62 (5H, m), 3.68-3.80 (2H, m), 3.97-4.04 (1H, m), 4.20-4.30 (1H, m), 6.32-6.42 (2H, m).

[0217] 13C NMR (500 MHz, CDCl3, 5° C.): 6.5, 7.1, 11.3, 12.3, 12.8, 13.1, 14.3, 14.4, 15.5, 16.5, 17.6, 20.3, 21.8, 22.81, 22.84, 24.5, 26.4, 26.7, 27.3, 28.2, 29.0, 29.5, 29.6, 29.76, 29.81 (4C), 30.5, 31.0, 32.0, 32.2, 35.8, 37.7, 39.0, 40.6, 48.6, 49.3, 50.5, 55.0, 55.1, 71.05, 71.14, 73.0, 75.5, 76.4, 77.0, 88.8, 99.0, 106.5, 128.0, 130.9, 204.7, 214.8.

[0218] HRMS (ESI) m/z: calculated for C54H96NO10+ [M+H+] 918.7029, found: 918.7034.

##STR00032##

[0219] Sal-cyclopropylamine was prepared using 100 mg of 2 (0.133 mmol), 94 μL (1.33 mmol, 10 eq) cyclopropyl amine, 11 mg (0.17 mmol, 1.3 eq) NaBH.sub.3CN, 50.0 mg (0.134 mmol, 1 eq) of CeCl.sub.3.7H2O and 50 μL acetic acid. AM23 was obtained as a colorless foam (44 mg, 42%).

[0220] .sup.1H NMR (CDCl.sub.3, 500 MHz, 278 K) δ 0.47-0.58 (2H, m), 0.69 (3H, J=10.0 Hz), 0.71-0.78 (9H, m), 0.78-0.95 (14H, m), 1.12-1.50 (16H, m), 1.50-1.75 (5H, m), 1.76-1.91 (4H, m), 2.02-2.20 (2H, m), 2.60 (1H, d, J=10.5 Hz), 2.62-2.68 (1H, m), 2.70-2.78 (1H, m), 2.78-2.88 (1H, m), 3.37 (1H, s), 3.53-3.80 (3H, m), 3.82-3.89 (1H, m), 4.08 (1H, d, J=9.5 Hz), 5.15 (2H, br s), 6.13 (2H, s).

[0221] .sup.13C NMR (CD.sub.3CN, 125 MHz, 278 K) δ 5.9, 6.4, 6.9, 7.7, 12.0, 12.4, 13.6, 13.7, 15.2, 16.1, 17.4, 17.9, 21.0, 22.7, 24.0, 25.7, 27.3, 29.1, 30.1, 31.8, 31.9, 33.4, 36.8, 39.0, 39.5, 41.2, 49.0, 50.4, 56.7, 57.6, 69.5, 71.5, 72.4, 74.7, 76.0, 77.1, 77.9, 89.7, 99.9, 107.9, 126.0, 130.8, 178.8, 214.7.

[0222] HRMS (ESI) m/z: calculated for C.sub.45H.sub.75NO.sub.10.sup.+ [M+H.sup.+] 789.5385, found: 789.5381. FIG. 10 shows the 3D-structure of compound AM23 which confirms unambiguously the crystalline structure and the stereochemistry of AM23.

Exemple 2

IC.SUB.50 .Assessment

[0223] Cell viability assay was carried out by plating 1000 cells per well in 96-well plates. NAC (2 mM, A9165 Sigma) or DFO (1 mM) were pretreated 2 hours prior to the compound treatment. CellTiter-Blue® Reagent (Promega; G3582) (20 μl/well) was added after 24, 48, or 72 hours treatment and cells were incubated for 1 hour before recording fluorescence (560(20)Ex/590(10)Em) using a Perkin Elmer Wallac 1420 Victor2 Microplate Reader.

TABLE-US-00001 Results: Compound ID Compound CD24− CD24+ Salinomycine [00033] 1.56 10.97 AM6 [00034] 6.18 12.77 AM7 [00035] 7.25 12.52 AM5 [00036] 0.0849 3.5147 AM9 [00037] 11.756 22 AM10 [00038] 10.012 19.74 AM8 [00039] 1.02 8.06 AM11 [00040] 0.8 6.7 AM12 [00041] 1.79 6.92 AM13 [00042] 0.0727 3.191528 AM16 [00043] 0.30 1.17 AM17 [00044] 0.1 2 AM18 [00045] 0.1 2.5 AM21 [00046] 0.34 6.50 AM22 [00047] 0.30 1.53 AM23 [00048] 0.043 1.20 AM24 [00049] 0.107 1.40 AM25 [00050] 0.108 1.36 AM26 [00051] 1.149 10.09 AM28 [00052] 6.43 5.83

[0224] The above results show that the compounds not containing an amine functionality at the 20-position have lower potency than salinomycine against cancer stem cells.

[0225] Introducing an amine function at position 20 of salinomycine results in a significantly improved activity against CD24 cells (AM 5, AMB, AM11, AM12, AM13 and AM23), up to an 18 fold improvement.

[0226] Replacement of the carboxylic acid functionality in the 1-position of salinomycine with an ester group results in compounds with lower efficiency than salinomycine (AM9 and AM10).

[0227] These results demonstrate that both the amine and a functional group capable of chelating iron, such as a carboxylic acid are necessary for improved activity. It is contemplated that the presence of these two functional groups help iron coordination, thereby favoring the Fenton reaction in the lysosomes.

[0228] The compounds of formula (I) are therefore useful for the treatment of cancer and/or the prevention of cancer relapse and/or metastases.

Example 3

Effect of AM5, AM9 and AM13 on the Proliferation of HMLER CD24− Cells

[0229] AM5, AM9, AM13 and salinomycine were assessed for their capability to inhibit cell proliferation and formation of mammospheres.

[0230] The results are presented in FIG. 2.

[0231] At 30 nM, AM 5 and AM13 inhibit cell proliferation with a ten-fold improved efficacy in comparison with salinomycine.

[0232] In contrast, AM 9 did not inhibit cell proliferation, even at 500 nM.

[0233] These results thus indicate that the compounds of formula (I) according to the present invention are capable of inhibiting the formation of mammospheres more efficiently than salinomycine.

Example 4

Effect of AM5, Taxol and Combination Thereof on the Proliferation of HMLER CD24− Cells

[0234] AM5, Taxol and a combination of AM5 and Taxol were also assessed for their capability to inhibit cell proliferation and formation of mammospheres.

[0235] The results are presented in FIG. 3.

[0236] The combination of AM5 at 15 nM and Taxol at 5 nM inhibits cell proliferation and mammosphere formation with an improved efficacy than AM5 alone at 15 nM or 5 nM.

Example 5

Effect of AM5 on Xenograft Tumor Formation

[0237] Human breast cancer cell line MCF-7 cells cultures were collected, enzymatically dissociated, washed in PBS, and resuspended in PBS/Matrigel mixture (1:1 volume). 0.1 ml of this mixture was then implanted in the mammary fat pad of 5-week-old female AthymicNude-Fox1nu mice (Harlan, France). The mice were maintained in individually-ventilated cages (Tecniplast, France) under constant temperature and humidity; all experiments were performed under laminar flow (Tecniplast France). The mice received estradiol supplementation (0.4 mg/kg) in the same day and 7th day from cell injection, and were observed and palpated for tumor appearance. The mice received Salinomycine analogue (here AM5, 3 mg per kg body weight per day, intra peritoneal injection) every 5 opened days of the week for 33 days. Tumor growth was measured weekly using calipers. Tumor volume was determined using the standard formula: L×W2×0.52, where L and W are the longest and shortest diameters, respectively. All animal work was done according to the Guidelines of the United Kingdom Coordinating Committee on Cancer Research.

[0238] The results are presented in FIG. 4.

[0239] Following AM5 treatment, the tumor volume and tumor weight were lower.

[0240] These results are consistent with the in vitro assay and indicate that the compounds of formula (I) according to the present invention are capable of inhibiting the tumor formation in nude mice.

Example 6

Salinomycin and Active Analogues Trigger Cell Death Through Lysosomal Fenton Catalysis

[0241] Salinomycine analogue-induced cell death is inhibited by the ROS scavenger N-acetylcysteine (NAC). Cell lines were incubated with or without 500 nM of Sal analogues for 48 h. Apoptosis was evaluated by Annexin V -FITC and PI staining, and FACS analysis.

[0242] The results are presented in FIG. 5. All data are expressed as means±s.d. from three individual experiments (*; P<0.05)

[0243] Data indicate that Salinomycine and AM5 induce cell death through lysosomal ROS production.

Exemple 7

Lysosomal Iron Mediates Salinomycine Analogues-Activated Cell Death Signaling

[0244] Salinomycine analogue-induced cell death is inhibited by lysosomal iron chelator deferoxamine mesylate (DFO). Cells were treated as in Example 6 with or without the indicated concentration of DFO for 48 h. Apoptosis was evaluated as in Example 6.

[0245] The results are presented in FIG. 6.

[0246] Data indicate that lysosomal iron mediated Salinomycine analogues activated cell death signaling.

Example 8

Effect of AM5 and AM23 on the Proliferation of HMLER CD24− Cells

[0247] AM5 and AM23 and salinomycine were assessed for their capability to inhibit cell proliferation and formation of mammospheres.

[0248] The results are presented in FIGS. 7 to 9.

[0249] At 30 nM, AM 5 and AM23 inhibit cell proliferation with a ten-fold improved efficacy in comparison with salinomycine.

[0250] These results thus indicate that the compounds of formula (I) according to the present invention are capable of inhibiting the formation of mammospheres more efficiently than salinomycine.

Example 9

IC50 of Salinomycin, of AM5 and of AM23 on Breast Cell Lines

[0251] Table 1 below represents the IC50 of Salinomycin (Sal) and its derivatives AM5 and AM23 for a wide range of the breast cell lines.

[0252] The cells were seeded in a 6-well plate at density 5.105 cells/well and cultured overnight. The cells were then treated with various concentration (15, 30, 100, 500, 1000 and 10.000 nM) of salinomycine, AM5 and AM23 for 72 h, 96 h and 108 h. After treatment, cell death was quantified using Annexin V-FITC/Propidium Iodide (PI) assay according to the manufacturer's protocol (FITC Annexin V Apoptosis Detection Kit II, 556570, BD Pharmingen™) and analyzed by a LSRFortessa™ flow cytometer (BD Bioscience, San Jose, Calif.). The data were processed using Cell Quest software (BD Biosciences). Dose-response cell death curves were determined for indicated time.

[0253] For tumor cells, the cells are classified in function of their sensitivity of drugs. Here, the most sensitive cells are incubated for 72 h, the middle sensitive cells for 96 h and the less sensitive or resistant cells are incubated for 108 h with drug at several concentrations.

[0254] Concentrations of 30 nM, 500 nm and 1 μM were used for determining the IC50 of drugs.

[0255] Intervals ]130-500 nM] mean that IC50 is included in this interval with exclusion of the valor 30 nM.

TABLE-US-00002 TABLE 1 Tumor cells Sal AM5 AM23 No tumoral HBL100 >1 μM >1 μM >1 μM Immortalized HMLE W2 >1 μM >1 μM 1 μM 1rst sensibility HMLER ID2 >1 μM >1 μM <30 nM (72 h) HMLER [30-500] nM <30 nM <30 nM CD24low HMLER GFP 1 μM 1 μM 30 nM HMLER [30-500] nM [30-500] nM 30 nM shECAD MCF-7 [30-500] nM [30-500] nM ]30-500] nM Zr75.1 500 nM [30-500] nM ]30-500] nM 2nd sensibility MDA-MB-361 500 nM 500 nM 1 μM (96 h) MDA-MB-134 [30-500] nM [30-500] nM >1 μM MDA-MD-157 1 μM 500 nM [30-500] nM MDA-MB-231 >1 μM >1 μM >1 μM BT474 >1 μM >1 μM >1 μM 3rd sensibility Hs528T ND ND ND (108 h) BT20 500 nM [30-500] nM [30-500] nM SW620 [500 nM-1 μM] [30-500] nM [30-500] nM SW480 [30-500] nM [30-500] nM [30-500] nM Resistant BT549 >1 μM >1 μM >1 μM (108 h) T47D >1 μM >1 μM >1 μM

[0256] The SW620 and SW480 cell lines are from colon tumors. Table 2 describes the essential specificities of each cell line:

TABLE-US-00003 TABLE 2 Name Essential specificities HBL100 Human mammary epithelial cell line obtained from primary cultures of cells derived from an early lactation sample of human milk (from ATCC). HMLE W2 Human mammary epithelial cell line infected with a retrovirus carrying hTERT, SV40 (R. A. Weinberg, Whitehead Institute, Massachusetts Institute of Technology, USA) HMLER ID2 Human mammary epithelial cell line infected with a retrovirus carrying hTERT, SV40 and the oncogenic allele HrasV12 (R. A. Weinberg, Whitehead Institute, Massachusetts Institute of Technology, USA) HMLER HMLER CD44high/CD24low not expressing E-cadherin and expressing CD24low Vimentin (was obtained from A. Puisieux INSERM) HMLER shGFP HMLER cells expressing a control shRNA (shCtrl). Generated by (ctrl) infection with retrovirus encoding the pWZL-GFP plasmid. (R. A. Weinberg, Whitehead Institute, Massachusetts Institute of Technology, USA) HMLER transformed HMLER breast cancer cells displaying a short hairpin RNA shECAD (shRNA)-mediated inhibition of the human CDH1 gene, which encodes E-cadherin. Generated by infection with retrovirus encoding the pWZL- GFP plasmid. (R. A. Weinberg, Whitehead Institute, Massachusetts Institute of Technology, USA) MCF-7 Human ductal breast epithelial tumor cell line classified in Estrogen/Progesteron Receptor (ER/PR) positive group and luminal A (from ATCC). Zr75.1 Human ductal breast epithelial tumor cell line, classified in Estrogen/Progesteron Receptor (ER/PR) and HER-2 positive group and luminal A (from ATCC). MDA-MB-361 Human ductal breast epithelial tumor cell line, classified in Progesteron Receptor (PR) and HER-2 positive group and luminal B (from ATCC). These cells were isolated from a metastatic site in the brain. MDA-MB-134 Human ductal breast epithelial tumor cell line classified in Estrogen/Progesteron Receptor (ER/PR) positive group and luminal B (from ATCC). MDA-MD-157 Human ductal breast epithelial tumor cell line, classified in Estrogen/Progesteron Receptor (ER/PR) and HER-2 negative group and Basal (from ATCC). MDA-MB-231 Human ductal breast epithelial tumor cell line, classified in Estrogen/Progesteron Receptor (ER/PR) and HER-2 negative group and Basal (from ATCC). BT474 Human ductal breast epithelial tumor cell line, classified in Progesteron Receptor (PR) and HER-2 positive group and luminal B (from ATCC). Hs578T Human ductal breast epithelial tumor cell line, classified in Estrogen/Progesteron Receptor (ER/PR) and HER-2 negative group and Basal (from ATCC). BT20 Human ductal breast epithelial tumor cell line, classified in Estrogen/Progesteron Receptor (ER/PR) and HER-2 negative group and Basal (from ATCC). SW620 colon tumor cells; derived from metastatic site: lymph node (from ATCC). SW480 colon tumor cells; derived from a primary adenocarcinoma of the colon (from ATCC). BT549 Human ductal breast epithelial tumor cell line, classified in Estrogen/Progesteron Receptor (ER/PR) and HER-2 negative group and Basal (from ATCC). T47D Human ductal breast epithelial tumor cell line classified in Estrogen/Progesteron Receptor (ER/PR) positive group and luminal A (from ATCC).

[0257] These results indicate that AM5 and AM23 have a IC50 compared to Salinomycin similar or better depending on the cells.

Example 10

Influence of AM23 in ROS Inducement

[0258] Reactive Oxygen Species (ROS) levels were measured by flow cytometry or by confocal scanning immunofluorescence microscopy using CM-H2DCF-DA (C6827, invitrogen). Briefly, U2OS and HMLER CD24low cells were treated as indicated in FIG. 11 (30 nM, 500 nM or 1 μM of salinomycine, AM5 or AM23 or untreated during 48 h). Then, these cells were trypsined and incubated with 5 μM CM-H2DCF-DA at 37° C. for 40 min, washed once with PBS and were counterstained with DAPI (0.5 μg/ml) to exclude non-viable cells. The mean fluorescence intensity was determined as ROS production by flow cytometry with LSRFortessa™ cytometer (BD Bioscience, San Jose, Calif.). For immunofluorescence microscopy analysis, cells were seeded on coverslips and were treated with salinomycine derivatives (injection in culture medium then treatment during 24 h, 48 h and 72 h). LysoTracker® Red DND-99 (L-7528, Life technologies) was used to visualize lysosomes. Then, cells were fixed with 4% PFA/PBS. DAPI was used to visualize nuclear DNA. Cell images were obtained using a Deltavision real-time microscope (Applied Precision) or an ApoTome.2 microscope (Zeiss). ImageJ was used for further image processing. As shown in FIG. 11, AM23 induces ROS in HMLER CD24low cells.

Example 11

Intracellular Sodium Measurement and Tumor Growth in MCF-7 Xenograft-Bearing Mice

[0259] Intracellular sodium measurement: Sodium and potassium buffers (10 mM HEPES, 1 mM CaCl.sub.2, 1 mM MgCl.sub.2, 130 mM Sodium-D-Gluconate or Potassium-D-Gluconate, 30 mM NaCl/KCl) were mixed at different ratios to produce five buffers with various sodium concentrations (0, 20, 40, 80, 160 mM). Nigericin (N7143, Sigma, 10 μM) and monensin (M5273, Sigma, 10 μM) were used to equilibrate the intracellular sodium concentration and establish a calibration curve. HMLER CD24low cells were harvested and re-suspended in ECS buffer (15 mM HEPES, 5.4 mM KCl, 140 mM NaCl, 10 mM Glucose, 1 mM MgCl.sub.2, 1.8 mM CaCl.sub.2, 0.1% BSA, pH 7.6) containing 10 μM of the sodiumspecific probe (SBFI-AM, S-1263, Molecular Probes®) and 0.2% Pluronic F-127 (P2443, Sigma) and were incubated for 1 h in the dark at 37° C. Then, cells were washed to remove excess dye and incubated for an additional 30 min in ECS buffer. Cells were introduced into a 96-well plate (1000 cells/well) and treated with salinomycine derivatives in a concentration ranging from 0.03 to 20 μm (AM5: 0.120 μM; AM13: 0.120 μM; AM9: 20 μM; salinomycine: 20 μM and 1 μM) during 5 min. Each well was sequentially excited at 340 and 370 nm and emission was recorded at 500 nm. The spectral response of SBFI upon sodium binding was assessed by excitation ratio measurement (340/370 nm). Measurements were performed on a Perkin Elmer Wallac 1420 Victor2 Microplate Reader at 37° C.

[0260] Xenograft tumor formation experiments: MCF-7 cell cultures were collected, enzymatically dissociated, washed with PBS, and re-suspended in a PBS/Matrigel mixture (1:1 v/v). The mixture (0.1 mL) was then implanted in the mammary fat pad of 5-week-old female AthymicNude-Fox1nu mice bilaterally (Harlan, France). Mice were maintained in individually-ventilated cages (Tecniplast, France) under constant temperature and humidity. All experiments were performed under laminar flow (Tecniplast France). Mice received estradiol supplementation (0.4 mg/kg) the same day and 7 days from cell injection, and were observed and palpated for tumor appearance. Mice were treated with AM5 or Paclitaxel (3 mg/kg body weight/day) by means of intraperitoneal injections every 5 opened days of the week. Tumor growth was measured weekly using calipers. Tumor volume was determined using the standard formula: L×W2×0.52, where L and W are the longest and shortest diameters, respectively. All animal studies were approved by the Direction des services Vétérinaires, Préfecture de Police, Paris, France (authorization number A75-14-08) and the ethical committee (number 34) of Paris Descartes University. No randomization was used and experimenters were blinded to drug treatments and tissue analyses.

[0261] While salinomycine induced a fast increase in intracellular sodium using a dose as high as twenty times the IC50 value, salinomycine derivatives had no effect on sodium transport at doses effective against the proliferation of HMLER CD24low cells (FIG. 12). This data challenged the idea that salinomycine selectively affects the maintenance of CSCs by directly altering membrane potentials. In contrast, AM9 was ineffective in these assays validating the carboxylate as a required motif to alter CSC maintenance, and paclitaxel alone was poorly effective against tumorsphere formation.

[0262] Moreover, AM5 prevented tumor growth in MCF-7 xenograft-bearing mice (FIG. 13).

Example 12

Toxicity Assessment

[0263] Histology. Organs from mice were removed at time of sacrifice. For morphological analyses, organs were fixed with 4% paraformaldehyde, paraffin embedded, and 4-μm sections were stained with hematoxylin and eosin (H&E). Sections were scanned at high resolution using a slide scanner (NanoZoomer 2.0-HT, Hamamatsu, Massy, France). Representative images are shown in FIG. 14.

[0264] No generic toxicity was observed upon treatment with an effective dose of AM5 as observed from the integrity of peripheral tissues and a constant body weight throughout treatment (FIG. 15).

[0265] All the samples of the lung from both untreated and treated groups showed minimal to moderate multifocal macrophages aggregates in the alveoli. This finding is poorly significant and commonly observed in mice. In 4 mice (2 untreated, 2 treated), extra-pulmonary, interstitial mononuclear cells infiltrates were observed. Most likely poorly significant, not treatment related.

[0266] In untreated mouse, a focal sub-pleural pulmonary densification was noticed with interstitial fibrosis and atypical cell infiltrates evoking tumor cells (metastasis). A large artefact on the lesion (tissue fold) interfered with the analysis

[0267] No significant changes were observed on kidney.

Example 13

[0268] MDA-MB-231 cells were cultivated with or without cathepsine B inhibitor (COA74-Me, 30 μM), and/or salinomycine, AM5, or AM23 (500 nM) at indicated duration (48 h, 96 h, 108 h). From the treatment, dead cells were assessed by DIOC6(3)/DAPI test and analyzed by flow cytometry. Graphic representation of percent of dead cells (DIOC6(3) negative/DAPI positive or negative) is shown in FIG. 16. Cathepsine B inhibition and salinomycine, AM5 or AM23 treatment combine themselves to induce the death of human breast cancer cell line MDA-MB-231.

[0269] FACS analysis of ROS in cells treated for 48 h is represented in FIG. 17. Pharmacological inhibition of Cathepsin B prevents AM5 from inducing ROS production in HMLER CD24low cells.