ZUCLOPENTHIXOL HYDROCHLORIDE DERIVATIVES AND EBSELEN DERIVATIVES AS ERBB2 INHIBITORS

Abstract

The present invention relates to compounds of the following general formula (I) or (II): or a pharmaceutically acceptable salt and/or solvate thereof, for use in the treatment and/or in the prevention of ErbB2 dependent cancers, and pharmaceutical compositions containing such compounds.

##STR00001##

Claims

1. A method for preventing and/or treating ErbB2 dependent cancers, comprising: administering to a patient in need thereof an effective amount of a compound of following general formula (I): ##STR00014## or a pharmaceutically acceptable salt and/or solvate thereof, wherein: X is a sulfur atom or an oxygen atom; R.sub.1 is hydrogen atom, halo, CN, NO.sub.2, NO, CHO, NR.sub.7R.sub.8, CO.sub.2R.sub.9, SO.sub.2R.sub.10, SO.sub.2NR.sub.11R.sub.12, OR.sub.13, COR.sub.14, SR.sub.15, CONR.sub.16R.sub.17, SO.sub.2(O)R.sub.18 or a group selected from saturated (C.sub.1-C.sub.6)alkyl, unsaturated (C.sub.1-C.sub.6)alkyl and aryl, said group being optionally substituted with one or several groups selected from halo, CF.sub.3, CN and SO.sub.2NR.sub.19R.sub.20; R.sub.2 and R.sub.3 form together with the nitrogen atom to which they are chemically linked, an heterocycle or an heteroaryl group, optionally substituted with one or several groups selected from halo, CO.sub.2R.sub.21 and a (C.sub.1-C.sub.6)alkyl group optionally substituted with one or several groups selected from halo, OR.sub.22, SR.sub.23, S(O)R.sub.24, SO.sub.2R.sub.25, SO.sub.2NR.sub.26R.sub.27, OC(O)R.sub.28, NR.sub.29COR.sub.30, NR.sub.31CONR.sub.32R.sub.33, NR.sub.34C(O)OR.sub.35, CO.sub.2R.sub.36, CONR.sub.37R.sub.38, OCO.sub.2R.sub.39, OCONR.sub.40R.sub.41, COR.sub.42, NO.sub.2, CF.sub.3, and CN; R.sub.4, R.sub.5 and R.sub.6 are, independently of one another, hydrogen atom, halo, CN, NO.sub.2, NO, CHO, NR.sub.43R.sub.44, CO.sub.2R.sub.45, S(O)R.sub.46, SO.sub.2R.sub.47, SO.sub.2NR.sub.48R.sub.49, OCOR.sub.50, NR.sub.51COR.sub.52, NR.sub.53CO(O)R.sub.54, NR.sub.55CONR.sub.56R.sub.57, CO.sub.2R.sub.58, OR.sub.59, COR.sub.60, SR.sub.61, CONR.sub.62R.sub.63, OCONR.sub.64R.sub.65, SO.sub.2(O)R.sub.66, or a group selected from saturated (C.sub.1-C.sub.6)alkyl, unsaturated (C.sub.1-C.sub.6)alkyl and aryl, said group being optionally substituted with one or several groups selected from halo, CF.sub.3, CN, and SO.sub.2NR.sub.67R.sub.68; R.sub.7 to R.sub.68 are, independently of one another, a hydrogen atom or a (C.sub.1-C.sub.10)alkyl, aryl or aryl(C.sub.1-C.sub.6)alkyl group, said group being optionally substituted with one or several groups selected from halo, OH, CF.sub.3, CN and SO.sub.2NR.sub.69R.sub.70; with the proviso that R.sub.21 is not an hydrogen atom; R.sub.69 and R.sub.70 are independently of one another, a hydrogen atom or a (C.sub.1-C.sub.10)alkyl, aryl or aryl(C.sub.1-C.sub.6)alkyl group; and n is an integer selected from 1 to 6.

2. The method according to claim 1, wherein R.sub.1 is hydrogen atom, halo, CN, NO.sub.2, NO, CHO, NR.sub.7R.sub.8, CO.sub.2R.sub.9, SO.sub.2R.sub.10, SO.sub.2NR.sub.11R.sub.12, COR.sub.14, CONR.sub.16R.sub.17, SO.sub.2(O)R.sub.18 or a group selected from saturated (C.sub.1-C.sub.6)alkyl, unsaturated (C.sub.1-C.sub.6)alkyl and aryl, said group being optionally substituted with one or several groups selected from halo, CF.sub.3, CN and SO.sub.2NR.sub.19R.sub.20; R.sub.7 to R.sub.12, R.sub.14 and R.sub.16 to R.sub.20 being as defined in claim 1.

3. The method according to claim 1, wherein R.sub.1 is hydrogen atom, halo, CN, SO.sub.2NR.sub.11R.sub.12 or CF.sub.3; R.sub.11 and R.sub.12 being as defined in claim 1.

4. The method according to claim 1, wherein R.sub.2 and R.sub.3 form together with the nitrogen atom to which they are chemically linked, an heterocycle or an heteroaryl group, substituted with one or several (C.sub.1-C.sub.6)alkyl group optionally substituted with one or several groups Selected from OR.sub.22, SR.sub.23 , S(O)R.sub.24, SO.sub.2R.sub.25 , SO.sub.2NR.sub.26R.sub.27, OC(O)R.sub.28, NR.sub.29COR.sub.30, NR.sub.31CONR.sub.32R.sub.33, NR.sub.34C(O)OR.sub.35, CO.sub.2R.sub.36, CONR.sub.37R.sub.38, OCO.sub.2R.sub.39, OCONR.sub.40R.sub.41, COR.sub.42, NO.sub.2 and CN; R.sub.22 to R.sub.42 being as defined in claim 1.

5. The method according to claim 1, wherein R.sub.2 and R.sub.3 form together with the nitrogen atom to which they are chemically linked, an heterocycle or an heteroaryl group, substituted with one (C.sub.1-C.sub.6)alkyl group optionally substituted with one or several groups selected from OR.sub.22, SR.sub.23, S(O)R.sub.24, SO.sub.2R.sub.25, SO.sub.2NR.sub.26R.sub.27, OC(O)R.sub.28, OCO.sub.2R.sub.39 and COR.sub.42; and wherein R.sub.22 to R.sub.28, R.sub.39 and R.sub.42 are, independently of one another, a hydrogen atom or a (C.sub.1-C.sub.10)alkyl group.

6. The method according to claim 1, wherein R.sub.2 and R.sub.3 form together with the nitrogen atom to which they are chemically linked, an heterocycle or an heteroaryl group, substituted with one (C.sub.1-C.sub.6)alkyl group optionally substituted with one group selected from OR.sub.22 and OC(O)R.sub.28; and wherein R.sub.22, R.sub.28, and R.sub.31 to R.sub.33 are, independently of one another, a hydrogen atom or a (C.sub.1-C.sub.6)alkyl group.

7. The method according to claim 1, wherein R.sub.4, R.sub.5 and R.sub.6 are, independently of one another, hydrogen atom, halo or (C.sub.1-C.sub.6)alkyl.

8. The method according to claim 1, wherein X is a sulfur atom.

9. The method according to claim 1, wherein the compound is of following general formula (Ia): ##STR00015##

10. The method according to claim 1, wherein said compound is selected from the following compounds: ##STR00016## and pharmaceutically acceptable salt and/or solvate thereof.

11. A method for preventing and/or treating ErbB2 dependent cancers, comprising: administering to a patient in need thereof an effective amount of a compound of formula (II): ##STR00017## or a pharmaceutically acceptable salt and/or solvate thereof, wherein: Y is SeO; R.sub.1 and R.sub.2 are, independently of one another, H, halo, (C.sub.1-C.sub.6)alkyl, CN, CF.sub.3, CHO, CO.sub.2R.sub.4, SO.sub.2R.sub.5, SO.sub.2NR.sub.6R.sub.7, COR.sub.8, CONR.sub.9R.sub.10 or SO.sub.2OR.sub.11; R.sub.3 is H, halo, (C.sub.1-C.sub.6)alkyl, OR.sub.12, NR.sub.13R.sub.14, SR.sub.15, S(O)R.sub.16, SO.sub.2R.sub.17, SO.sub.2NR.sub.18R.sub.19, OCOR.sub.20, NR.sub.21COR.sub.22, NR.sub.23CONR.sub.24R.sub.25, NR.sub.26C(O)OR.sub.27, CO.sub.2R.sub.28, CONR.sub.29R.sub.30, OCO.sub.2R.sub.31, OCONR.sub.32R.sub.33, COR.sub.34, nitro (NO.sub.2) or cyano (CN); and R.sub.4 to R.sub.34 are, independently of one another, H or a (C.sub.1-C.sub.6)alkyl, aryl or aryl(C.sub.1-C.sub.6)alkyl group, said group being optionally substituted with one or several groups selected from halo.

12. The method according to claim 11, wherein R.sub.1 is H and R.sub.2 is H, halo, (C.sub.1-C.sub.6)alkyl, CN or CF.sub.3.

13. The method according to claim 11, wherein R.sub.3 is H, halo or (C.sub.1-C.sub.6)alkyl.

14. The method according to claim 11, wherein said compound is the following compound: ##STR00018## or a pharmaceutically acceptable salt and/or solvate thereof.

15. (canceled)

16. The method according to claim 1, for preventing and/or treating ErbB2 dependent cancer metastasis.

17. The method according to claim 1, wherein said ErbB2 dependent cancer is lung cancer, ovarian cancer, stomach cancer, bladder cancer, uterine cancer, pancreas cancer, liver cancer, kidney cancer, gastroeosophageal cancer, gastric cancer, colorectal cancer, female genital tract cancer, endometrial cancer, anal cancer, breast cancer or neurofibroma.

18. The method according to claim 11, for preventing and/or treating ErbB2 dependent cancer metastasis.

19. The method according to claim 11, wherein said ErbB2 dependent cancer is lung cancer, ovarian cancer, stomach cancer, bladder cancer, uterine cancer, pancreas cancer, liver cancer, kidney cancer, gastroeosophageal cancer, gastric cancer, colorectal cancer, female genital tract cancer, endometrial cancer, anal cancer, breast cancer or neurofibroma.

20. The method according to claim 17, wherein said ErbB2 dependent cancer is ovarian cancer, pancreas cancer, gastroeosophageal cancer, gastric cancer, colorectal cancer, endometrial cancer, anal cancer or neurofibroma.

21. The method according to claim 17, wherein said ErbB2 dependent cancer is ovarian cancer, gastric cancer or breast cancer.

22. The method according to claim 19, wherein said ErbB2 dependent cancer is ovarian cancer, pancreas cancer, gastroeosophageal cancer, gastric cancer, colorectal cancer, endometrial cancer, anal cancer or neurofibroma.

23. The method according to claim 19, wherein said ErbB2 dependent cancer is ovarian cancer, gastric cancer or breast cancer.

24. The method according to claim 3, wherein halo is Cl or F.

25. The method according to claim 12, wherein R.sub.2 is H, halo or (C.sub.1-C.sub.6)alkyl.

26. The method according to claim 13, wherein R.sub.3 is H.

Description

BRIEF SUMMARY OF THE FIGURES

[0180] FIG. 1. represents the inhibition of (A) FERM/ErbB2 or (B) FERM/CD44 interaction by compound ZU; (C) the inhibition of FERM/ErbB2 by comparative compound lapatinib measured by Alphascreen experiments after 24 h.

[0181] FIG. 2. represents the effect of (A) compound ZU and (B) compound EB1 on ErbB2 activation measured by dot blot analysis of ErbB2 phosphorylation in SKBR3 cells.

[0182] FIG. 3. represents the specific inhibition of the ErbB2-dependent cell proliferation of (A) SKBR3 and (B) BT474 by ZU measured by MTT assays as compared to an absence of inhibition of the proliferation of the ErbB2-independent (C) MCF7 cell line (D) MDA-MB-231 cell line-and the normal endothelial cell line (E) HBMEC (unpaired T-test, *p<0.05, significantly different from control).

[0183] FIG. 4. represents the specific inhibition of the ErbB2-dependent cell proliferation of (A) SKBR3 and (B) BT474 by EB1 measured by MTT assays as compared to an absence of inhibition of the proliferation of the ErbB2-independent (C) MCF7 cell line (D) MDA-MB-231 cell line-and the normal endothelial cell line (E) HBMEC (unpaired T-test, **p<0.01, ***p<0.001, significantly different from control).

[0184] FIG. 5. represents the inhibition of the anchorage-independent growth of SKBR3 (A-B) or BT474 (C-D) cells by compound ZU and compound EB1 measured in soft agar assays. AG1478, a tyrosine kinase inhibitor of the EGFR family members was used as a control. (A) and (C) display representative photographs of the colonies; (B) and (D) show the colony mean area measured for each treatment condition (5B : One way ANOVA F(3,6)=227.5; Dunnett's Post Hoc Test, *** p<0.001, ****p<0.0001 ; 5D, One way ANOVA F(1,10)=14.03; Dunnett's Post Hoc Test * p<0.01).

[0185] FIG. 6. represents the inhibition by compound ZU of the rate of BT474 tumor proliferation in vivo in orthotopic xenografts.

[0186] FIG. 7. represents the inhibition by compound ZU of the rate of BT474 tumor proliferation in vivo in orthotopic xenografts. (A), (B) and (C) show the quantification of the tumor volume (mm.sup.3) of each mouse injected with saline, 4 mg/kg and 5 mg/kg of ZU respectively; while (D) and (E) show the normalized tumor volume (expressed as a % of tumor volume measured at day 19 (d19) post graft) of mice injected with saline and 4 mg/kg of ZU and saline and 5 mg/kg of ZU respectively. (G) displays pictures of tumors from each group. (F) and (H) display quantification of the tumor weight and body weight respectively.

[0187] FIG. 8. represents the interaction of compound ZU with ErbB2 juxtamembrane peptide as measured by SPR experiments at 50 and 10 M.

[0188] FIG. 9. represents the effect of compound ZU cis (C)/trans (T) conformation, or a mixture C/T (50/50) on the specific inhibition of the ErbB2-dependent cell proliferation of (A) SKBR3 and (B) BT474 and (C) HBMEC as a control measured by MTT assays.

[0189] FIG. 10. represents the specific inhibition of overexpressed, mutated (V659E) or truncated (p95, DeltaEBM) forms of ErbB2 by compound ZU measured by western blot analysis of ErbB2 phosphorylation in transfected HBMECs cells. The upper line displays representative western-blots and the lower histograms displays their respective quantification for each ErbB2 form.

[0190] FIG. 11. represents the absence of inhibition of the ligand-dependent ErbB2 activation by compound ZU and compound EB1 measured by western blot analysis in HBMECs cells. Briefly, HBMECs cells were stimulated 5 min with EGF or HRG to induce heterodimeric activation of ErbB2 with EGFR or ErbB3 respectively in the absence or presence of (A) compound ZU, (B) compound EB1, for lh or 24 h. (C) As a control AG1478 strongly blocked the ligand-dependent ErbB2 activation.

[0191] FIG. 12. represents the inhibition by compound EB1 of the rate of BT474 tumor proliferation in vivo in orthotopic xenografts. (A), (B) and (C) show the quantification of the tumor volume (mm.sup.3) of each mouse injected twice a day (2D) 5 days a week for two weeks then once a day (1D) for 3 days a week with saline, 3 mg/kg and 5 mg/kg of EB1 respectively; while (D) and (E) show the normalized tumor volume (expressed as a % of tumor volume measured at day 19 (d19) post graft) of mice injected with saline and 3 mg/kg of EB1 and saline and 5 mg/kg of EB1 respectively (Point by point comparison with controls using the Dunnett's test: * P<0.05, **P<0.01). (F) displays pictures of tumors from each group. (G) and (H) display quantification of the tumor weight and body weight respectively.

[0192] FIG. 13. represents the effect of (A) compound ZU, (B) compound EB1 and (C) AG1478 on ErbB2 activation measured by dot blot analysis of ErbB2 phosphorylation in N87 cells.

[0193] FIG. 14. represents the specific inhibition of the ErbB2-dependent cell proliferation of (A) N87 by ZU measured by MTT assays as compared to an absence of inhibition of the proliferation of the ErbB2-independent (B) MDA-MB-231 cell line.

[0194] FIG. 15. represents the inhibition of the anchorage-independent growth of N87 (A-B) by compound ZU measured in soft agar assays. AG1478, a tyrosine kinase inhibitor of the EGFR family members was used as a control. (A) displays representative photographs of the colonies; (B) shows the colony mean area measured for each treatment condition (One way ANOVA: F(3, 6)=170.9, Dunnet's Post Hoc Test, ****p<0.0001)

[0195] FIG. 16. represents the effect of (A) compound ZU and (B) AG1478 on ErbB2 activation measured by dot blot analysis of ErbB2 phosphorylation in SKOV3 cells.

[0196] FIG. 17. represents the specific inhibition of the ErbB2-dependent cell proliferation of (A) SKOV3 by ZU measured by MTT assays as compared to an absence of inhibition of the proliferation of the ErbB2-independent (B) MDA-MB-231 cell line.

[0197] FIG. 18. represents the inhibition of the anchorage-independent growth of SKOV3 (A-B) by compound ZU measured in soft agar assays. AG1478, a tyrosine kinase inhibitor of the EGFR family members was used as a control. (A) displays representative photographs of the colonies; (B) shows the colony mean area measured for each treatment condition.

[0198] FIG. 19. represents the effect of compound ZU, compound EB1 on the activation of (A) WT, (B) V777L and (C) V8421 ErbB2 measured by western blot analysis of ErbB2 phosphorylation.

[0199] FIG. 20. represents the specific inhibition of the (A) WT, (B) V777L and (C) V842I ErbB2-dependent cell proliferation by ZU measured by MTT assays as compared to an absence of inhibition of the proliferation of the ErbB2-independent (D) non transfected cells.

[0200] FIG. 21. represents the specific inhibition of the (A) WT, (B) V777L and (C) V842I ErbB2-dependent cell proliferation by EB1 measured by MTT assays as compared to an absence of inhibition of the proliferation of the ErbB2-independent (D) non transfected cells

EXAMPLES

[0201] The Following Abbreviations have been used in the Following Examples. [0202] a.a.: Amino acid [0203] AdoMet: S-Adenosyl-L-methionine [0204] ATP: Adenosine triphosphate [0205] BSA: Bovine Serum Albumin [0206] CMV: Cytomegalovirus [0207] DCM: Dichloromethane [0208] DIAD: Diisopropyl azodicarboxylate [0209] DiPEA: N,N-Diisopropylethylamine [0210] DMF: Dimethylformamide [0211] DMSO: Dimethylsulfoxide [0212] DNA: Deoxyribonucleic acid [0213] EDTA: Ethylenediaminetetraacetic acid [0214] ESI: Electrospray ionisation [0215] HEPES: 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid [0216] HPLC: High Performance Liquid Chromatography [0217] HRMS: High Resolution Mass Spectrometry [0218] MW: Microwave [0219] ND: Not determined [0220] NMR: Nuclear Magnetic Resonance [0221] PBS: Phosphate buffered saline [0222] PBST: Phosphate buffered saline+Tween-20 [0223] RPMI: Roswell Park Memorial Institute medium [0224] RT: Room temperature [0225] SAH: S-Adenosyl-L-homocysteine [0226] SAM: S-Adenosyl-L-methionine [0227] TEA: Triethylamine [0228] TFA: Trifluoroacetic acid [0229] Tris: Tris(hydroxymethyl)aminomethane

[0230] I. Synthesis of the Compounds According to the Invention

Example 1

Zuclopentixol Derivatives

Preparation of ZU3:

9-(3-(4-(2-hydroxyethyl)piperazinyl)propylidene)-thioxanthene

[0231] ##STR00006## [0232] To a solution of 9-oxothioxanthene (1.0 equiv.) in THF at reflux were added a solution of cyclopropylmagnesium bromide in THF (1.0 equiv.) and stirred during 2 hours. The mixture was cooled down at room temperature and a solution of hydrogen bromide in acetic acid (4 eq.) was added and stirred at room temperature. The reaction mixture was concentrated in vacuo and purified by silica gel column chromatography to obtain 9-(3bromopropylidene)thioxanthene in 30% yield. [0233] Then, to a solution of 9-(3bromopropylidene)thioxanthene in acetonitrile at reflux was added N-(2-hydroxyethyl)piperazine (1,5 eq.), potassium iodide (0.1 eq.) and potassium carbonate (3 eq.). The mixture was stirred at reflux then concentrated in vacuo and purified by silica gel column chromatography to afford ZU3 with 98% purity (HPLC). HPLC analysis (BEH C18 type, mobile phase: H2O/acetonitrile (HCOOH 0.1%)): t.sub.R=1.68 min.

Preparation of ZUf:

1-(3-(9H-thioxanthen-9-ylidene)propyl)piperidine-4-carboxylic acid), ZU4 (9-(3-(4-(ethylacetate)piperidine)propylidene)-thioxanthene

[0234] ##STR00007## [0235] To a solution of 9-(3-bromopropylidene)thioxanthene in acetonitrile at reflux was added N-ethylacetate piperidine (1,5 eq.), potassium iodide (0.1 eq.) and potassium carbonate (3 eq.). The mixture was stirred at reflux then concentrated in vacuo and purified by silica gel column chromatography to afford ZU4 as a brown oil with a purity of 97% in HPLC analysis [0236] HPLC analysis (BEH C18 type, mobile phase: H2O/acetonitrile (HCOOH 0.1%)): t.sub.R=2.07 min. [0237] The compound ZU4 was stirred during 2 hours at reflux in a mixture of THF and a solution of NaOH in water. After phase separation, the aqueous layer was extracted twice by diethyl ether. The global organic layer was, then, washed by a saturated solution of NaCl, dried over MgSO.sub.4, filtered and concentrated in vacuo to afford ZUf HPLC analysis (BEH C18 type, mobile phase: H2O/acetonitrile (HCOOH 0.1%)): t.sub.R=2.24 min.

Preparation of ZUS:

(Z)-2-(4-(3-(2-chloro-9H-thioxanthen-9-ylidene)propyl)piperazin-1-yl)ethylacetate

[0238] ##STR00008## [0239] To a solution of ZU (4-[3-(2-chloro-9H-thioxanthen-9-ylidene)propyl]-1-piperazineethanol) (1 eq.) in dichloromethane was added acetic anhydride (1.5 eq.), 4-dimthylaminopyridine (0,1 eq.) and trimethylamine (1 eq.). The mixture was stirred at room temperature and then concentrated in vacuo to afford ZU5 as a yellow oil with 97% of purity (HPLC). HPLC analysis (BEH C18 type, mobile phase: H2O/acetonitrile (HCOOH 0.1%)):t.sub.R=2.44 min

Preparation of ZUe and ZU6:

1-(3-(9H-xanthen-9-ylidene)propyl)piperidine-4-carboxylic acid and ethyl 1-(3-(9H-xanthen-9-ylidene)propyl)piperidine-4-carboxylate

[0240] ##STR00009## [0241] To a solution of 9-oxoxanthene (1.0 equiv.) in THF at reflux were added a solution of cyclopropylmagnesium bromide in THF (1.0 equiv.) and stirred during 2 hours. The mixture was cooled down at room temperature and a solution of hydrogen bromide in acetic acid (4 eq.) was added and stirred at room temperature. The reaction mixture was concentrated in vacuo and purified by silica gel column chromatography to obtain 9-(3bromopropylidene)-oxoxanthene. [0242] Then, to a solution of 9-(3bromopropylidene)-oxoxanthene in acetonitrile at reflux was added Ethyl 4-piperidinecarboxylate (1,5 eq.), potassium iodide (0.1 eq.) and potassium carbonate (3 eq.). The mixture was stirred at reflux then concentrated in vacuo and purified by silica gel column chromatography to obtain ZU6 ZU6 is then dissolved in a mixture of THF and a solution of NaOH in water. After phase separation, the aqueous layer was extracted twice by diethyl ether. The global organic layer was, then, washed by a saturated solution of NaCl, dried over MgSO.sub.4, filtered and concentrated in vacuo to afford ZUe as a white solid with a purity up to 97% in HPLC.

Preparation of ZUc:

(Z)-2-(4-(3-(2-(trifluoromethyl)-9H-thioxanthen-9-ylidene)propyl)piperazin-1-yl) ethanamine

[0243] ##STR00010## [0244] To a solution of ZU1 (2- [4- [3 - [2-(trifluoromethyl)thio xanthen-9-ylidene]propyl]piperidin-1-yl] ethanol) (1 eq.) in THF was added diethylazodicarboxylate, phtalimide and triphenylphosphine. The solution was stirred at room temperature during 3 hours and then concentrated in vacuo. The crude oil was then dissolved in ethanol, hydrazine was added and the mixture was stirred at reflux during 2 hours. The crude product obtained after concentration was purified via a reversed phase chromatography using HCl as buffer to afford the compound ZUc as an hydrochloride salt (orange solid). [M+H].sup. (ESI+): 434. HPLC analysis (BEH C18 type, mobile phase: H2O/acetonitrile (HCOOH 0.1%)):t.sub.R=2.04 min

Preparation of ZUd:

(Z)-1-(2-fluoroethyl)-4-(3-(2-(trifluoromethyl)-9H-thioxanthen-9-ylidene)propyl) piperazine

[0245] ##STR00011## [0246] To a solution of flupenthixol in dichloromethane was added at 10 C. diethylaminosulfur trifluoride. The mixture was then stirred at room temperature. The crude product was purified via a reversed phase chromatography using HCl as buffer to afford the compound ZUd as a hydrochloride salt (orange solid) with 97% purity in HPLC. HPLC analysis (BEH C18 type, mobile phase: H2O/acetonitrile (HCOOH 0.1%)): t.sub.R=3.59 min. [0247] Compounds ZU, ZUa, ZUb, ZU1, ZU2
The Following Compounds can be easily Found in Commerce:

##STR00012##

Example 2

Ebselen Oxide Derivatives

[0248] The Following Compounds can be easily Found in Commerce:

##STR00013##

[0249] II. Biological Tests of the Compounds According to the Invention

Example 3

Inhibition of FERM/ErbB2 interaction and determination of IC.SUB.so .valuesin AlphaScreen (Amplified Luminescent Proximity Homogenous Assay)

[0250] To validate that the Zuclopenthixol hydrochloride (ZU) and its derivatives interact with the juxtamembrane domain of ErbB2, we first analyzed in AlphaScreen their efficiency to inhibit FERM/ErbB2 interaction in a dose-dependent manner and determined IC.sub.50 value. Furthermore, to test the selectivity of those compounds, we tested their ability to disrupt the interaction between the FERM domain and the known ERM binding motif contained in the juxtamembrane region of CD44 as a control.

METHOD:

[0251] Reagents: AlphaScreen technology was used to assess the interaction between the Ezrin binding motif (EBM) contained in the juxtamembrane portions of ErbB2 or CD44 with the Ezrin FERM domain. For this purpose the Ezrin FERM domain in fusion with Glutathione-S-Transferase (GST) has been coupled to GSH-coated acceptor beads. Biotinylated ErbB2 peptide encoding the Ezrin binding motif (amino acid 674 to 689: biotin-ILIKRRQQKIRKYTMRRL, 26 aa) or the juxtamembrane region of CD44 (biotin-NSRRRCGQKKKLVINSG), for the counter screen have been synthesized and coupled to streptavidin-covered donor beads. The AlphaScreen reagents (Glutathione-coated Acceptor beads and streptavidin-coated Donor beads) were obtained from PerkinElmer.

[0252] Competition assay: The reaction was performed using white 384-well Optiplates (PerkinElmer, Whalham, Mass., USA) in 20 l (total reaction volume) in a reaction buffer containing PBS, pH 7.4, 5 mM MgCl.sub.2 and 0,02% CHAPS. 2,5 L, of the compounds (0-50 M) were transferred to the 384-well Optiplates containing 2,5 l buffer and 5 l of a mix solution containing 0,625 M GST-FERM and 20 nM biotin-EBM or biotin-CD44 was added for 30 min at room temperature. 10 1 of a mix solution containing 20 g/ml Glutathione-coated Acceptor beads and 20 g/ml streptavidin-coated Donor beads was then added to the wells and incubation was further proceeded for 40 min or overnight in the dark and at room temperature. Light signal was detected with the EnVision multilabel plate reader (PerkinElmer). All experiments involving AlphaScreen beads were performed under subdued lighting.

Results:

[0253] The results of these tests obtained with the compounds of the invention are indicated in Table 1 below:

TABLE-US-00001 TABLE 1 Competition assay FERM-ErbB2 interaction Max inhibition, % Compound 20 h ZU 87.2 ZU1 57 ZU2 16 ZU3 28 ZU4 36 ZU5 41 ZUa 3.9 ZUb 0 ZUc 45 ZUd 54 ZUf 11

[0254] ZU inhibited FERM/ErbB2 interaction in a dose-dependent manner by 72% at lh with an IC.sub.50=50 M, whereas it did not interfere with FERM/CD44 interaction (FIGS. 1A, 1B). This inhibitory effect was persistent at 24 h (>85% inhibition) with IC.sub.50=9.8 M. At 24 h, ZU also inhibited FERM/CD44 interaction, however to a lesser extent than FERM/ErbB2 interaction (53.32 vs. 87.2%).

[0255] ZU1, ZU2, ZU3, ZU4 and ZU5 also inhibited FERM/ErbB2 interaction in a specific and dose-dependent manner (Table 1). On the contrary, compounds ZUb, lacking a double bond between the heterocyclic groups and ZUa lacking a heterocyclic group, do not significantly inhibited FERM/ErbB2 interaction.

[0256] EB1 efficiently inhibited FERM/ErbB2 interaction in a dose-dependent manner by 43% at 1 h, without significantly altering FERM/CD44 interaction at low doses (FIGS. 1C, 1D).

[0257] Hence, these compounds selectively disrupted the FERM/ErbB2 interaction and therefore represent very attractive compounds.

[0258] Moreover, as a comparative compound, lapatinib did not inhibit FERM/ErbB2 interaction.

Example 4

Inhibition of ErbB2 activation and determination of IC.SUB.50 .valuesIn cellulo

[0259] The ability of ZU and EB1 and their derivatives to inhibit ErbB2 activity in cellulo on a commonly used ErbB2-overexpressing breast cancer cell line SKBR3 was analyzed. SKBR3 were treated 24 h with increasing concentrations of the compounds (0.625-80 M) and ErbB2 phosphorylation was addressed by dot blot analysis using an anti-phosphotyrosine antibody (clone 4G10). Quantifications show the result of two independent experiments.

Results:

[0260] The results of these tests obtained are indicated in Table 2 below:

TABLE-US-00002 TABLE 2 ErbB2 activation 24 h Compound Max inhibition, % EC.sub.50, M ZU 93.22 22.39 ZU1 88.58 19.05 ZU2 74.17 31.62 ZU3 75.12 32.36 ZU4 27.48 47.86 ZU5 97.49 43.65 ZU6 36.23 35.48 ZUc 90.80 5.13 ZUd 51.35 20.42 ZUe 0 / ZUf 0 / EBa 15.55 40.74 EB1 66.86 23.44 EB2 43.81 18.20 EB3 66.41 19.50

[0261] ZU strongly decreased ErbB2 phosphorylation in a dose-dependent manner, reaching a maximum inhibition of 90% with an IC.sub.50=20 M (FIG. 2A).

[0262] ZU derivatives such as ZU1, ZU2, ZU3, ZU5, ZUc and ZUd potently decreased ErbB2 phosphorylation in a dose-dependent manner (reaching a maximum of 75 to 95% inhibition with IC.sub.50 varying from 5 to 35 M) (Table 2). On the contrary, ZU4 and ZU6 exhibit a lower decrease of the ErbB2 phosphorylation. ZUf and ZUe had no effect on ErbB2 activation.

[0263] EBa only slightly reduced ErbB2 phosphorylation by 15% with concentration of 10 M. EB1 was more efficient in preventing ErbB2 activation than EBa, as it reduced ErbB2 phosphorylation in a dose-dependent manner, reaching a maximum inhibition of 70% with an IC.sub.50=20 M (FIG. 2B).

[0264] EB2 and EB3 decreased ErbB2 phosphorylation in a dose-dependent manner reaching a maximum of 43 to 66% inhibition with IC.sub.50 varying from 18 to 20 M.

Example 5

Inhibition of ErbB2-Dependent Cell Proliferation

[0265] We then analyzed the ability of ZU and EB1 and their derivatives to decrease the ErbB2-dependent cell proliferation. We used the ErbB2-overexpressing human breast cancer cell lines SKBR3 and BT474, as well as two non ErbB2-dependent breast cancer cell lines MCF7 and MDA-MB-231 and normal human endothelial cells (HBMEC) as a control, to discard compounds presenting non-specific toxic effects.

Method:

[0266] SKBR3, BT474, MCF-7, MDA-MB-231 and HBMEC cells were treated with the compounds as indicated, and cell proliferation was determined each day during 3 days using an MTT assay. Non treated cells were included as a negative control.

Results:

[0267] The results of these tests obtained are indicated in Table 3 below:

TABLE-US-00003 TABLE 3 Cell proliferation at d 3 SKBR3 BT474 HBMEC Max EC.sub.50, Max EC.sub.50, Max EC.sub.50, inhibition, % M inhibition, % M inhibition, % M ZU 46.2 14.1 31.5 6.8 4.7 ND ZU1 51.12 5.13 79.67 3.1 4.7 ND ZU3 32.6 15.5 31.5 4.2 0 ND ZU5 28.2 3.5 30.8 17.8 24.5 21.9 ZUc 78.9 0.34 94.9 0.23 68.8 0.14 ZUd 86.4 6.7 86 16.5 90 16.3 EB1 96.7 7.4 100 5.5 94.4 99.15 EB2 34.1 9.3 20 11.7 29.5 11.5 EB3 63.5 8.9 82.7 6.9 75.1 2770

[0268] Treatment with 5 M ZU reduced by 50% SKBR3 and BT474 proliferation at day 2 and 3, with an IC.sub.50 of 14 M and 7 M respectively, without any effect on HBMEC or on MCF7 or MDA-MB-231 proliferation (FIG. 3, unpaired T-test, *p<0.05, significantly different from control).

[0269] Treatment with 5 M ZU1 reduced SKBR3 and BT474 proliferation at day 2 and 3 by 30 to 50%, without any effect on HBMEC proliferation. This inhibition could be further enhanced to 80 to 95% when treating cells with 10 M ZU1.

[0270] Treatment with 20 M ZU3 decreased SKBR3 and BT474 proliferation by 30% without any effect on HBMEC cell proliferation.

[0271] Treatment with 1 and 5 M ZU5 reduced SKBR3 and BT474 proliferation by 60% and 33% respectively, at day 3, without any effect on HBMEC proliferation.

[0272] Treatment with 0.5, 1 and 2 M ZUc reduced SKBR3 and BT474 proliferation from 50% to 95%, at day 3, but also inhibited HBMEC proliferation from 60% to 85% at day 3.

[0273] Treatment with 20 M ZUd reduced SKBR3 and BT474 proliferation by 86%, at day 3, but also inhibited HBMEC proliferation by 90% at day 3.

[0274] Furthermore, treatment with 10 M EB1 potently inhibited the proliferation of both SKBR3 and BT474 at day 2 and 3, reaching a 95 to 100% inhibition at a IC.sub.50 of 7,5 and 5,5 M respectively, without any effect on HBMEC or on MCF7 or MDA-MB-231 proliferation (FIG. 4, unpaired T-test, * *p<0.01, * * *p<0.001, significantly different from control).

[0275] Treatment with 5 M EB2 did not significantly decreased SKBR3 proliferation but reduced BT474 proliferation at day 2 and 3 by 30 to 35%, without any effect on HBMEC cell proliferation.

[0276] Treatment with 7 M EB3 did not significantly decreased SKBR3 proliferation but significantly reduced BT474 proliferation by 30% at day 2, without any effect on HBMEC cell proliferation.

[0277] These results indicated that both ZU and EB1 and their derivatives ZU1, ZU2, ZU3, ZU5, EB2 and EB3 have a potent and selective inhibitory effect on human breast cancer cells overexpressing ErbB2. On the contrary, ZUc and ZUd exhibit non-specific toxic effects, since they also inhibit the proliferation of control cells.

Example 6

Inhibition of the Colony Formation of SKBR3 and of BT474 in a Soft Agar Assay

[0278] To confirm the potent effect of ZU and EB1, we analyzed their efficiency to inhibit the colony formation of SKBR3 or BT474 cells in a soft agar assay.

Method:

[0279] A bottom layer of 0.8% agarose in DMEM supplemented with 20% SVF and penicillin/streptomycin was added to 24 well plates before seeding 25.10.sup.3 SKBR3 or BT474 cells/well in a 0.6% agarose top layer. Cells were left untreated or treated with 5 or 10 M ZU or EB1 or 5 M AG1478 (a non-specific ErbB2 kinase inhibitor) as a positive control. Treatments were renewed 3 times a week. After 6 weeks, the number of colonies and their size were quantified using image J software. The colony formation is illustrated for each condition. Data are presented as meanSEM.

Results

[0280] The results of these tests are shown in FIGS. 5A to 5D.

[0281] We observed that treatment with both ZU (FIG. 5A and 5B : One way ANOVA F(3,6)=227.5; Dunnett's Post Hoc Test, *** p<0.001, ****p<0.0001) and EB1 (FIG. 5C and 5D : One way ANOVA F(1,10)=14.03; Dunnett's Post Hoc Test *p<0.01) compounds inhibited the anchorage-independent growth of SKBR3 and BT474, with a potent action on the size of the colony formed.

Example 7

Inhibition of the Development In Vivo of Human Breast Cancer BT474 Cells

[0282] We addressed the ability of ZU to inhibit the development in vivo of human breast cancer BT474 cells orthotopically implanted in the mammary fat pad of immunodeficient NOG mice, in the presence of estradiol supplement, as this constitutes the more relevant system comparable to the human situation to address the access of these molecules to tumors cells inside their organ of origin and their potential effect on tumoral dissemination.

Method, First Set of Experiments:

[0283] 5.10.sup.6 BT474 cells were implanted orthotopically in the mammary fat pad of NOD.Cg-Prkdc scid/J mice, in the presence of estradiol supplement. After 4 weeks, mice were injected with ZU (N=10, 5 mg/kg per day for 5 days a week, followed by 3 mg/kg for 5 days a week during three weeks for N=6) or vehicle (N=10, 10% DMSO in PBS). Mice weight and tumor volume were measured 3 and 2 times a week respectively.

Results, First Set of Experiments:

[0284] Injection of ZU (5 mg/kg per day for 5 days a week, 10 mice per group, followed by 3 mg/kg for 5 days a week during three weeks for 6 mice) completely blocked tumor progression in vivo, in comparison to the tumor progression in mice treated with the vehicle (10 mice per group, 10% DMSO in PBS) as a control (FIG. 6).

[0285] This experiment confirmed the potent inhibitory effect of ZU on human breast cancer cells overexpressing ErbB2. Furthermore, as BT474 cells have a known Trastuzumab-resistance status, this experiment shows the efficacy of ZU to overcome Trastuzumab-resistance.

[0286] Nonetheless, at 5 mg/kg, 3 mice out of 10 were dizzy or sleepy for long, lost weight and finally died after several injections. When switched at 3 mg/kg, the treatment was well supported by the mice.

Method, Second Set of Experiments:

[0287] Drug Administration Schedules were Slightly Changed to Overcome the Potential Side Effects Detected with the 5 mg/kg Dose.

[0288] 5.10.sup.6 BT474 cells were implanted orthotopically in the mammary fat pad of NOD.Cg-Prkdc scid/J mice, in the presence of estradiol supplement. Nineteen days after implantation, treatment began and mice were injected i.p. with 5 m/kg ZU (5 mg/kg per day for 3 days a week, during three weeks for N=9), 4 mg/kg ZU (5 days a week during three weeks, N=8) or vehicle (N=9, 10% DMSO in PBS). Mice weight and tumor volume were measured 3 and 2 times a week respectively.

Results, Second Set of Experiment:

[0289] Injection of ZU (5 mg/kg per day, 3 days a week, or 4 mg/kg, 5 days a week) dramatically reduced tumor progression in vivo, in comparison to the tumor progression in mice treated with the vehicle as a control (FIG. 7). Of, note using this administration scheme, treatment is well tolerated.

[0290] Two-way ANOVA: interaction between time and ZU treatment F(16,207)=8.309, P<0.0001, ZU treatment effect F(2,207)=15.97, P<0.0001, time effect F(8,207)=115.9, P<0.0001. Point by point comparison with controls using the Bonferroni posttest: ** P<0.01, ***P<0.001. Quantification of the tumor weight (F). Student T test * P<0,05. Pictures of tumors from each group (G) and body weight (H).

[0291] This experiment confirmed the potent inhibitory effect of ZU on human breast cancer cells overexpressing ErbB2. Furthermore, as BT474 cells have a known trastuzumab-resistance status, this experiment shows the efficacy of ZU to overcome trastuzumab-resistance.

Example 8

Surface Plasmon Resonance Assays

[0292] We analyzed the ability of ZU to directly bind to the juxtamembrane domain of ErbB2 using Surface Plasmon Resonance assays on a Biacore T200. For that, we compared the affinity of the compounds for a peptide containing the Ezrin binding motif ILIKRRQQKIRKYTMRRL of ErbB2 immobilized on sensorchips, which was reflected by the amplitude of the SPR response.

Method:

[0293] Compound ZU and EB1 were validated for their interaction with peptide encoding the juxtamembrane region of ERBB2 using a Biacore T200 (IECB, Bordeaux). Biotinylated peptide encoding the juxtamembrane region of ERBB2 (biotin-ILIKRRQQKIRKYTMRRL) has been immobilized on Streptavidin-coated sensor chips (Series S sensor chip SA, GE Healthcare). Compound ZU in PBS, 0.02% tween20 buffer was used as analyte, and the Ezrin FERM domain has been used as a positive control.

Results:

[0294] Both Zuclopenthixol (ZU) and Ebselen oxide (EB1) bound to the immobilized peptide, therefore confirming the molecular interaction between these compounds and ErbB2 (FIG. 8).

Example 9

ZU Conformation Tests

[0295] Zuclopenthixol is in a cis conformation. To evaluate the importance of this conformation for the biological effect of this molecule on ErbB2, we tested the effects of a mix of cis/trans isomers (50:50), as well as of a pure trans isomer of Zuclopenthixol.

Method:

[0296] A solution of ZU in cis conformation was mixed to a solution of ZU in trans conformation (1:1) to obtain ZU in a 50/50 cis/trans conformation and their respective effects on ErbB2 activation and ErbB2-dependent cell proliferation were analyzed as previously by dot blot analysis using an anti-phosphotyrosine antibody (clone 4G10) and by MTT assays in the ErbB2-overexpressing breast cancer cell line SKBR3, BT474, or HBMEC as a control.

Results:

[0297] The results of these tests are indicated in FIGS. 9A to 9C.

[0298] Pure cis, or mix of cis/trans isomers of Zuclopenthixol all similarly inhibited ErbB2 activation in SKBR3. However, at 10 M the trans isomer had no significant inhibitory effect on the proliferation of the SKBR3 and BT474 cell lines (FIGS. 9B).

[0299] These results demonstrate that the cis conformation of Zuclopenthixol is particularly advantageous to ensure a specific inhibition of ErbB2.

Example 10

ZU Specifically Inhibits Overexpressed, Mutated or Truncated Forms of ErbB2

Method:

[0300] HBMECs cells were transfected with plasmids encoding WT, EBM (a form of ErbB2 carrying mutations in the juxtamembrane domain and unable to bind to the Ezrin FERM domain), V659E or p95 ErbB2 and treated with 0, 5, 10 or 20 M ZU for 24 h. ErbB2 activation was then analyzed by western blot analysis using a phospho-ErbB2-specific antibody (pY1248) and a tubulin antibody as a loading control. Histograms show optical density quantification.

Results:

[0301] ZU decreased the activation of WT, V659E or p95 ErbB2 without any effect on EBM ErbB2 mutant (FIG. 10).

[0302] These results demonstrate that ZU activity is mediated by the EBM motif in the juxtamembrane region of ErbB2, therefore confirming the molecular mechanism by which ZU inhibits ErbB2. Moreover, these results also demonstrate the activity of ZU on V659E and p95 ErbB2 that confer aggressiveness to the breast tumors and/or resistance to the actual treatments and therefore are associated with bad prognosis.

Example 11

ZU, EBa and their Derivatives do not Affect the Ligand-Dependent Activation of ErbB2

[0303] In physiological conditions, ErbB2 activation occurs in heterodimer with the other ErbB family members, such as the EGFR in response to EGF stimulation or ErbB3 in response to heregulin (HRG) stimulation. We therefore addressed whether ZU, EB1 and their derivatives also affected the ligand-dependent physiological ErbB2 activation.

Method:

[0304] 16 h-starved HBMECs cells were pre-treated or not for 1 h or 24 h with ZU, EBa and their derivatives or AG1478 before EGF (50 ng/mL) or HRG1 (100 ng/mL) stimulation for 5 minutes. The activation of EGFR/ErBB2 or ErbB3/ErbB2 heterodimers-dependent signalling pathways was then analysed by western blot experiments using pAkt, Akt, .sub.PERK, ERK or 4G10 antibodies.

Results:

[0305] ZU, EBa and their derivatives did not block the ligand-dependent ErbB2 activation induced by EGF or HRG stimulation whereas AG1478 did (FIG. 11).

[0306] These results demonstrate that ZU and their derivatives specifically block the ligand-independent activation of ErbB2. Therefore they will not interfere with physiological ErbB2 activation.

Example 12

Inhibition of the Development In Vivo of Human Breast cancer BT474 Cells by EB1

[0307] We addressed the ability of EB1 to inhibit the development in vivo of human breast cancer BT474 cells orthotopically implanted in the mammary fat pad of immunodeficient NOG mice, as we did with ZU.

Method:

[0308] 5.10.sup.6 BT474 cells were implanted orthotopically in the mammary fat pad of NOD.Cg-Prkdc scid/J mice, in the presence of estradiol supplement. After 4 weeks, mice were injected with EB1 3 mg/kg (N=8), 5 mg/kg (N=6) or vehicle (N=8, 10% DMSO in PBS) twice a day for 5 days a week (2D), during 2 weeks then once per day (1D) during 3 days. Mice weight and tumor volume were measured 3 and 2 times a week respectively.

Results:

[0309] The result is shown in FIG. 12 (A-H). EB1 was able to decrease the tumor growth in vivo at 3 mg/kg but this was more significant at 5 mg/kg as assessed by tumor volume measurement as well as tumor weight. The treatment was well tolerated as shown by the body weight. As BT474 cells have a known trastuzumab-resistance status, EB1 can therefore overcome trastuzumab resistance. [0310] FIG. 12E: Two-way ANOVA: interaction between time and EB1 treatment F(16,128)=2.797, P=0.0007, P2 treatment effect F(2,16)=3.196, P=0.068, time effect F(8,128)=23.69, P<0.0001. Point by point comparison with controls using the Dunnett's test: * P<0.05, **P<0.01.

Example 13

Inhibition of ErbB2 Activation in N87 Gastric Carcinoma Cell Line

[0311] ErbB2 has also been shown to be overexpressed in other types of cancers such as ovarian, endometrial, salivary gland, gastric or colorectal cancers. [0312] We therefore wanted to test the effect of ZU and EB1 to inhibit ErbB2 in other types of ErbB2-overexpressiong cancers. For this, we first used N87 a gastric carcinoma cell line overexpressing ErbB2 and analyzed the effect of ZU and EB1 on ErbB2 activation.

Method:

[0313] The phosphotyrosine content in the ErbB2-overexpressing gastric cancer cell line N87 was analyzed by dot blot using an anti-phosphotyrosine antibody (clone 4G10). As in these cells, ErbB2 is the main phosphorylated protein, decreased in total phosphotyrosine content mainly reflected inhibition of ErbB2 activity. As a control, the non-specific ErbB2 kinase inhibitor AG1478 was used. Quantifications show the result of two independent experiments.

Results:

[0314] The result is shown in FIG. 13 (A-C). ZU and EB1 were both able to potently decrease ErbB2 activation in N87 cell line.

Example 14

Inhibition of ErbB2-Dependent Proliferation of N87 Gastric Carcinoma Cell Line by ZU

[0315] We then examined ZU ability to inhibit ErbB2-dependent N87 cell proliferation.

Method:

[0316] The ErbB2-overexpressing gastric cancer cell line N87 and MDAMB-231, a non ErbB2-overexpressing breast cancer cell line were treated with 3, 5 or 7 M of ZU and cell proliferation was determined each day during 3 days using an MTT assay. Non treated cells were included as a negative control.

Results:

[0317] The result is shown in FIG. 14 (A-B). ZU 3, 5 and 7 M induced a 35% decrease of the proliferation of N87 gastric cancer cell line at day 3 whereas there was no effect on MDAMB-231 control cell line.

Example 15

Inhibition of the Colony Formation of N87 in a Soft Agar Assay

[0318] To confirm the potent effect of ZU, we analyzed their efficiency to inhibit the colony formation of N87 cells in a soft agar assay.

Method:

[0319] A bottom layer of 0.8% agarose in DMEM supplemented with 20% SVF and penicillin/streptomycin was added to 24 well plates before seeding 15.10.sup.3 N87 cells/well in a 0.6% agarose top layer. Cells were left untreated or treated with 5 or 10 M ZU or 5 M AG1478 (a non-specific ErbB2 kinase inhibitor) as a positive control. Treatments were renewed 3 times a week. After 6 weeks, the number of colonies and their size were quantified using image J software. The colony formation is illustrated for each condition. Data are presented as meanSEM (One way ANOVA: F(3, 6)=170.9; Dunnett's Post Hoc Test **** p<0.0001).

Results:

[0320] The result is shown in FIG. 15 (A-B). ZU induced a 50% inhibition of N87 microcolony formation.

Example 16

Inhibition of ErbB2 Activation in SKOV3 Ovarian Carcinoma Cell Line

[0321] We then wanted to test the effect of ZU to inhibit ErbB2 in SKOV3, an ovarian carcinoma cell line overexpressing ErbB2.

Method:

[0322] The phosphotyrosine content in the ErbB2-overexpressing ovarian cancer cell line SKOV3 was analyzed by dot blot using an anti-phosphotyrosine antibody (clone 4G10). As in these cells, ErbB2 is the main phosphorylated protein, decreased in total phosphotyrosine content mainly reflected inhibition of ErbB2 activity. As a control, the non-specific ErbB2 kinase inhibitor AG1478 was used. Quantifications show the result of two independent experiments.

Results:

[0323] The result is shown in FIG. 16 (A-B). ZU potently decreases ErbB2 activation in SKOV3 cell line.

Example 17

Inhibition of ErbB2-Dependent Proliferation of SKOV3 Ovarian Carcinoma Cell Line by ZU

[0324] As it was reported that SKOV3 exhibited a strong non ErbB2-dependent cell proliferation in the presence of serum, we decrease the serum content of the culture medium to 1% and examined ZU ability to inhibit ErbB2-dependent SKOV3 cell proliferation.

Method:

[0325] The ErbB2-overexpressing ovarian cancer cell line SKOV3 and MDAMB-231, a non ErbB2-overexpressing breast cancer cell line were treated with 3, 5 or 7 M of ZU and cell proliferation was determined each day during 3 days using an MTT assay. Non treated cells were included as a negative control.

Results:

[0326] The result is shown in FIG. 17 (A-B). ZU 3, 5 and 7 M induced a 50% decrease of the proliferation of SKOV3 ovarian carcinoma cell line at days 2 and 3 whereas there was no effect on MDAMB-231 control cell line. [0327] Altogether these results show that ZU can actively block ErbB2 activation and ErbB2-dependent cell proliferation in the SKOV3 ovarian carcinoma cell line.

Example 18

Inhibition of the Colony Formation of SKOV3 in a Soft agar Assay

[0328] To confirm the potent effect of ZU, we analyzed their efficiency to inhibit the colony formation of SKOV3 cells in a soft agar assay.

Method:

[0329] A bottom layer of 0.8% agarose in DMEM supplemented with 2% SVF and penicillin/streptomycin was added to 24 well plates before seeding 15.10.sup.3 SKOV3 cells/well in a 0.6% agarose top layer. Cells were left untreated or treated with 5 or 10 M ZU or 5 M AG1478 (a non-specific ErbB2 kinase inhibitor) as a positive control. Treatments were renewed 3 times a week. After 6 weeks, the number of colonies and their size were quantified using image J software. The colony formation is illustrated for each condition. Data are presented as meanSEM

Results:

[0330] The result is shown in FIG. 18 (A-B). ZU totally inhibited the formation of SKOV3 microcolonies. [0331] Altogether these results show that ZU and EB1 can actively block Erbb2 activation and ErbB2-dependent cell proliferation in other ErbB2-overexpressing cancers such as gastric or ovarian cancers.

Example 19

Inhibition of the Activation of Erbb2 Mutated in the Kinase Domain

[0332] Activating mutations of ErbB2 located in its kinase domain have been reported in ErbB2-dependent cancers. [0333] We therefore wanted to test the effect of ZU and EB1 to inhibit some of these mutated forms of ErbB2. We used ErbB2 carrying Val 777 to Leu mutation that was notably reported in cases of breast, colorectal and anal cancers as well as in neurofibroma. Of note, V777L mutated form was found as mediating resistance to trastuzumab in breast cancer. We also used ErbB2 carrying Val 842 to Ileu mutation that was reported in cases of colorectal, endometrial, gastroesophageal, ovarian and pancreatic cancers. We therefore analyzed their effect on V777L and V842I ErbB2 activation.

Method:

[0334] HBMECs cells were transfected with plasmids encoding WT, V777L or V842I and treated with 0, 5, 10 or 20 M ZU or EB1 for 48 h. ErbB2 activation was then analyzed by western blot analysis using a phospho-ErbB2-specific antibody (pY1248) and a total ErbB2 antibody. Histograms show optical density quantification.

Results:

[0335] The result is shown in FIG. 19 (A-C). ZU and EB1 were both able to potently decrease ErbB2 WT, V777L and V842I activation.

Example 20

Inhibition of the V777L and V842I Erbb2-Dependent Cell Proliferation by ZU

[0336] We then examined ZU ability to inhibit V777L and V842I ErbB2-dependent cell proliferation.

Method:

[0337] The WT, V777L or V842I ErbB2-transfected cells were treated with 5 or 10 M of ZU and cell proliferation was determined each day during 3 days using an MTT assay. Non transfected cells were included as a negative control.

Results:

[0338] The result is shown in FIG. 20 (A-D). ZU 10 M induced 50%, 35% and 65% decrease of the proliferation of WT, V777L and V842I transfected HBMECat day 3, respectively, whereas there was no effect on non-transfected HBMECs cells.

Example 21

Inhibition of the V777L and V842I Erb2-Dependent Cell Proliferation by EB1

[0339] We then examined EB1 ability to inhibit V777L and V842I ErbB2-dependent cell proliferation.

Method:

[0340] The WT, V777L or V842I ErbB2-transfected cells were treated with 5 or 10 M of EB1 and cell proliferation was determined each day during 3 days using an MTT assay. Non transfected cells were included as a negative control.

Results:

[0341] The result is shown in FIG. 21 (A-D). EB1 5M induced 60%, 70% and 55% decrease of the proliferation of WT, V777L and V842I transfected HBMEC at day 3, respectively. This effect reached 95%, 85% and 90% inhibition at 10 M whereas there was no effect on non-transfected HBMECs cells. [0342] Altogether these results show that ZU and EB1 can actively block ErbB2 activation and ErbB2-dependent cell proliferation of V777L and V842I mutated forms of ErbB2 which are notably found in breast, colorectal, anal cancers, neurofibroma, endometrial, gastroesophageal, ovarian and pancreatic cancers. Interestingly, as mentioned above, V777L mutation was found as mediating resistance to trastuzumab in breast cancer.

[0343] III-Conclusion

[0344] Altogether, these data unraveled the identification of two novel families of molecules that selectively inhibit HER2 activation by a mechanism which differs from the one of trastuzumab and lapatinib: i.e. binding to the juxtamembrane domain of ErbB2.

[0345] In conclusion, we identified 2 families of compounds exhibiting key features: [0346] Direct interaction with the juxtamembrane domain of ErbB2 [0347] Specific inhibition of ErbB2 phosphorylation [0348] Specific inhibition of ErbB2-dependent cell proliferation, both in vitro and in vivo on the development of orthotopic tumors.

[0349] Of note this inhibition is observed in vitro for compound concentrations below 15 M above which nonspecific toxicity is noticed whatever the ErbB2 status of the cells.

[0350] These compounds inhibit ErbB2 activation by a mechanism which differs from the one of trastuzumab and lapatinib and efficiently blocks the activation of trastuzumab-resistant cell lines.

[0351] These novel molecules provide alternative treatment to ErbB2-dependent cancers. These molecules can be used as well in combinatory treatments and also to maximize the clinical benefit from immune therapies directed to extracellular part of ErbB2 (e.g. trastuzumab) or from inhibitor of tyrosine kinase based therapies (e.g. lapatinib), consequently allowing reducing the doses of the drug required and their associated toxicity and preventing or delaying resistance and metastases spreading.