MCL-1 MODULATING COMPOUNDS FOR CANCER TREATMENT

Abstract

The invention relates to compounds of formula (I), and to their therapeutic use in the treatment of cancer:

##STR00001## Wherein Y.sub.1, Y.sub.2, Ar.sub.1, Ar.sub.2, R.sub.1, R.sub.2, i, j, k, l are as defined in claim 1.

Claims

1. A pharmaceutical composition comprising a compound of formula (I): ##STR00042## Wherein: Y.sub.1, Y.sub.2 are NC; Ar.sub.1, Ar.sub.2 are each independently selected from C.sub.6-C.sub.10 aryl or a 5 to 7 membered heteroaryl, said aryl and heteroaryl groups being optionally substituted by one to three R.sub.3 groups provided that: Ar.sub.1, Ar.sub.2 cannot both identically represent either a 4-pyridyl, an unsubstituted 2 or 3-thiophenyl, or a 3,4-dimethoxyphenyl or a 3,4,5-trimethoxyphenyl, i and j are independently 0 or 1, provided that: i+j1; and when none of Y.sub.1, Y.sub.2 is S, then i+j=2; and R.sub.1, R.sub.2, are at each occurrence, independently selected from, C.sub.1-C.sub.6 alkyl, C.sub.6-C.sub.10 aryl, (C.sub.6-C.sub.10)aryl(C.sub.1-C.sub.6)alkyl, (C.sub.6-C.sub.10)aryl(C.sub.2-C.sub.6)alkenyl, (C.sub.6-C.sub.10)arylcarbonyl, (C.sub.6-C.sub.10)aryl(C.sub.1-C.sub.6)alkylcarbonyl, C(O)H, COOH, OH said alkyl groups being optionally substituted by OH; k and l are independently 0, 1; R.sub.3 is, at each occurrence, independently selected from C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, OH, C(O)H, (CH.sub.2).sub.nCO.sub.2H, (CH.sub.2).sub.pCN, (CH.sub.2).sub.qC(N(OH))NH.sub.2, I, Cl, Br, F, C.sub.6-C.sub.10 aryl, and a 5 to 7 membered heteroaryl, (C.sub.6-C.sub.10)aryl(C.sub.1-C.sub.6)alkyl, (C.sub.6-C.sub.10)aryl(C.sub.2-C.sub.6)alkenyl, said alkyl groups being optionally substituted by OH; n is 0, 1, 2, 3; p is 0, 1, 2, 3; q is 0, 1, 2, 3; with the exclusion of the following compounds: 2-(pyridin-3-yl)-5-(5-(pyridin-3-yl)-3-styrylpyridin-2-yl)pyridine 3-(5-methyl-6-(5-methyl-6-(pyridin-3-yl)pyridin-3-yl)pyridin-3-yl)pyridine 3-(6-(5-methyl-6-(pyridin-3-yl)pyridin-3-yl)pyridin-3-yl)pyridine and the pharmaceutically acceptable salts thereof, in admixture with at least one pharmaceutically acceptable excipient or carrier.

2. The pharmaceutical composition of claim 1, wherein Ar.sub.1 and/or Ar.sub.2 are selected from phenyl, pyridyl, pyrimidyl, imidazolyl, pyrazolyl, thiophenyl, triazolyl, in particular from phenyl, 3-pyridyl, 5-pyrimidyl, 2-imidazolyl, 3-pyrazolyl, 2-thiophenyl, 5-triazolyl.

3. The pharmaceutical composition of claim 2, wherein at least one of Ar.sub.1, Ar.sub.2 is a 5 to 7 membered heteroaryl containing a nitrogen atom, preferably pyridyl, notably 3-pyridyl.

4. The pharmaceutical composition of claim 2, wherein Ar.sub.1 is 3-pyridyl or phenyl.

5. The pharmaceutical composition of claim 2, wherein Ar.sub.2 is 3-pyridyl or phenyl.

6. The pharmaceutical composition of claim 1, wherein R.sub.1, R.sub.2 are independently selected from C.sub.1-C.sub.6 alkyl, (C.sub.6-C.sub.10)aryl(C.sub.2-C.sub.6)alkenyl, preferably from methyl and styryl.

7. The pharmaceutical composition of claim 1, wherein R.sub.1 is 5-methyl.

8. The pharmaceutical composition of claim 1, wherein R.sub.2 is 5-styryl.

9. The pharmaceutical composition of claim 1, selected from compounds of formula (Ia): ##STR00043## Wherein: X.sub.1, X.sub.2, are at each occurrence, independently selected from C or N.

10. The pharmaceutical composition of claim 1, wherein the compound of formula (I) is selected from: 5,6-di(pyridin-3-yl)-5-methyl-3-((E)-styryl)-2,3-bipyridine 5,6-di(pyridin-3-yl)-3,5-bis-((E)-styryl)-[2,3;6,3]terpyridine 3,5,5-trimethyl-5,6-diphenyl-[2,3;6,3]-terpyridine 5-bromo-3,5-dimethyl-6-(3-methyl-4-pyridin-3-ylphenyl)-3,2-bipyridine 5-(2-methyl-4-pyridin-3-ylphenyl)-3,5-dimethyl-6-(2-methyl-4-pyridin-3-ylphenyl)-3,2-bipyridine 2-(5-methyl-6-(pyridin-3-yl)pyridin-3-yl)-5-phenyl-3-styrylpyridine 2-(5-methyl-6-phenylpyridin-3-yl)-5-(pyridin-3-yl)-3-styrylpyridine 5-(3-benzylpyridin-2-yl)-2-(5-benzylpyridin-3-yl)pyridine

11. The pharmaceutical composition of claim 1, further comprising a Bcl-x.sub.L inhibitor, notably a BH3-mimetic inhibitor, such as 2-amino-6-bromo--cyano-3-(ethoxycarbonyl)-4H-1-benzopyran-4-acetic acid ethyl ester (HA 14-1) or 4-[4-[[2-(4-chlorophenyl)phenyl]methyl]piperazin-1-yl]-N-[4-[[(2R)-4-(dimethylamino)-1-phenylsulfanylbutan-2-yl]amino]-3-nitrophenyl] sulfonylbenzamide (ABT-737).

12. A combination comprising a compound of formula (I) according to claim 1, in combination with a Bcl-.sub.XL inhibitor, notably a BH3-mimetic inhibitor, such as HA 14-1 or ABT-737.

13. A compound of formula (I) according to claim 1, for use in the treatment of cancer, notably of hematologic malignancies, such as lymphoma, leukemia, multiple myeloma, and/or of solid tumors such as ovarian cancers, mesothelioma, melanoma, pancrea, lung, breast, kidney and liver cancers.

14. The compound of formula (I) for use of claim 13, wherein the compound of formula (I) is administered together with a Bcl-.sub.XL inhibitor, notably a BH3-mimetic inhibitor, such as 2-amino-6-bromo--cyano-3-(ethoxycarbonyl)-4H-1-benzopyran-4-acetic acid ethyl ester (HA 14-1), 4-[4-[[2-(4-chlorophenyl)phenyl]methyl]piperazin-1-yl]-N-[4-[[(2R)-4-(dimethylamino)-1-phenylsulfanylbutan-2-yl]amino]-3-nitrophenyl] sulfonylbenzamide (ABT-737) or 4-[4-[[2-(4-chlorophenyl)-5,5-dimethylcyclohexen-1-yl]methyl]piperazin-1-yl]-N-[4-[[(2R)-4-morpholin-4-yl-1-phenylsulfanylbutan-2-yl]amino]-3-(trifluoromethylsulfonyl)phenyl]sulfonylbenzamide (ABT-263).

15. A compound of formula (I) according to claim 13, for use for inducing apoptose mediated by Mcl-.sub.1 protein.

16. A compound of formula (I): ##STR00044## Wherein: Y.sub.1, Y.sub.2, Ar.sub.1, Ar.sub.2, R.sub.1, R.sub.2, i, j, k, l are as defined in claim 1, Provided that, when k=l=1: Then at least one of R.sub.1, R.sub.2 is different from H; and if one of R.sub.1, R.sub.2 is CH.sub.3, then the other is not H or CH.sub.3, With the exclusion of the following compounds: 2-(pyridin-3-yl)-5-(5-(pyridin-3-yl)-3-styrylpyridin-2-yl)pyridine 3-(5-methyl-6-(5-methyl-6-(pyridin-3-yl)pyridin-3-yl)pyridin-3-yl)pyridine 3-(6-(5-methyl-6-(pyridin-3-yl)pyridin-3-yl)pyridin-3-yl)pyridine And the pharmaceutically acceptable salts thereof.

17. The compound of formula (I) of claim 16, wherein one of R.sub.1 is methyl and R.sub.2 is styryl.

18. The compound of formula (I) of claim 17, wherein R.sub.1 is 5-methyl and R.sub.2 is 5-styryl.

Description

DESCRIPTION OF THE DRAWINGS

[0181] FIG. 1 shows the effect of Pyridoclax and ABT-737 on Mcl-1/Puma and Noxa/Mcl-1 interactions in BRET assay (FIGS. 1A and 1B, respectively); Pyridoclax is able to disrupt both Mcl-1/Puma and Noxa/Mcl-1 interactions whereas ABT-737 is not able to modify these interactions.

[0182] FIG. 2 shows the effect of Pyridoclax on IGROV1-R10 ovarian cancer cells, alone or associated to a Bcl-x.sub.L targeting siRNA (siXL1). Cells were transfected with 10 nM siRNA for 48 h before a 24 h (A, B, C, D) or 2, 4, 6 h (B) exposure to 25 M Pyridoclax. [A] Cellular morphology, DNA content histograms and Annexin V/propidium iodide bi-parametric histograms. [B]: Trypan blue exclusion assay. [C]: Bcl-x.sub.L expression and PARP and caspase 3 cleavage assessed by western blot. [D]: Nuclear morphology studied after DAPI staining (top) and cellular morphology studied by electron microscopy. [E]: Short time effects of the association Pyridoclax/siXL1 (assessed 2, 4 and 6 h after the beginning of the exposure to Pyridoclax). Cellular morphology and DNA content histograms (left panel) and PARP and caspase 3 cleavage assessed by western blot (right panel).

[0183] FIG. 3 illustrates the effect of Pyridoclax alone or associated to a Bcl-x.sub.L targeting siRNA (siXL1) on ovarian, lung and mesothelial cancer cell lines. Cells were transfected with 10 nM siRNA for 48 h before a 24 h exposure to 25 M Pyridoclax. Cell detachment and sub-G1 fraction proportion on DNA histograms were assessed by the observation of cellular morphology and by flow cytometry, respectively, in ovarian carcinoma cells [A] and lung or mesothelioma cancer cells [B].

[0184] FIG. 4 represents the effect of the association of Pyridoclax with ABT-737 on chemoresistant ovarian cancer cells IGROV1-R10 (top) and SKOV3 (bottom). Cells were concomitantly (A, E) or sequentially (B) exposed to 25 M Pyridoclax and 5 M ABT-737, and cellular effects were assessed either longitudinally (A, B, E) or after 24 h (C, D, F). [A, B, E] real time cellular activity assessed by impedancmetry (xCELLigence technology), after concomitant exposure (A, E) or sequential exposure (B; 24 h Pyridoclax, then ABT-737). [C]: Cellular morphology and DNA content histograms after a 24 h concomitant exposure. [D and F]: PARP and caspase 3 cleavage after a 24 h concomitant exposure.

EXAMPLES

I. Synthesis of Compounds of Formula (I)

[0185] Material and method are described below.

[0186] Material

[0187] Commercial reagents were used as received without additional purification. Melting points were determined on a Kofler heating bench. IR spectra were recorded on a Perkin Elmer BX FT-IR spectrophotometer. The band positions are given in reciprocal centimeters (cm.sup.1). .sup.1H NMR (400 MHz) and .sup.13C NMR (100 MHz) spectra were recorded on a JEOL Lambda 400 spectrometer. Chemical shifts are expressed in parts per million downfield from tetramethylsilane as an internal standard and coupling constants in Hertz. Chemical shift are reported in part per million (ppm) relative to the solvent resonance. Chromatography was carried out on a column using flash silica gel 60 Merck (0.063-0.200 mm) as the stationary phase. The during solvent indicated for each purification was determined by thin layer chromatography (TLC) performed on 0.2 mm precoated plates of silica gel 60F-264 (Merck) and spots were visualized using an ultraviolet-light lamp. Elemental analyses for new compounds were performed and the data for C, H, and N were within 0.4 of the theoretical values for all final compounds.

[0188] Methods

[0189] (Het)aromatic oligosystems of the invention were synthesized according to the same procedure as that used to obtain MR29072 from 4-bromo-2-hydroxypyridine 1, trans-phenylvinylboronic acid 4 and 6-bromo-S-methylpyridin-3-ylboronic acid 7, and which is set out in scheme 1 and in example 1 below.

##STR00021## ##STR00022##

Example 1

5,6-di(pyridin-3-yl)-5-methyl-3-((E)-styryl)-2,3-bipyridine (MR 29072)

[0190] To 5-bromo-2-hydroxypyridine 1 (5 g, 29 mmol) was added solid N-Iodosuocinimide (7.1 g, 32 mmol) in acetonitrile (120 mL). The solution was stirred at reflux fir 4 hours and followed by TLC. The solution was cooled to room temperature, filtered and washed with methanol, 5-bromo-2-hydroxy-3-iodopyridine 2 was obtained as a pink solid (yield: 83%).

[0191] 2 (9 g, 30 mmol) was dissolved in phenylphosphonic dichloride 90% (100 mL, 0.3 mol). The mixture was stirred and heated (160 C.) for 4 hours and followed by TLC. At room temperature, it was introduced drops in a vial of 1 L filled with water and cooled at 0 C. The solution was neutralized by addition of NH.sub.4OH solution. The mixture was extracted in ethyl acetate. The product was obtained as a white solid, 5-bromo-2-chloro-3-iodopyridine 3 (yield: 82%).

[0192] To a reaction vessel (100 mL) in a nitrogen environment containing 3 (5 g, 15.7 mmol) were added trans-phenylvinylboronic acid 4 (2.7 g, 18 mmol), tetrakis triphenylphosphine (907 mg, 0.8 mmol), sodium carbonate (4.2 g, 39 mmol) in 1,4-dioxane (100 mL). The mixture was stirred at reflux for 24 hours until consumption of starting material followed by TLC. The product was cooled to room temperature; it was filtered on celite. The solution was dried on MgSO.sub.4, filtered and evaporated. The residue was purified by chromatography (c-hexane:ethyl acetate=99:1, then 98:2) to afford 5-bromo-2-chloro-3-((E)-styryl)-pyridine S (yield: 93%).

[0193] To 5 (200 mg, 0.7 mmol) were added sodium iodide (1 g, 6.8 mmol), acetyl chloride (0.07 mL, 1 mmol), and acetonitrile (10 mL). The solution was stirred under microwaves irradiation for 1 hour at 100 C. At room temperature, the mixture was neutralized by a NaHCO.sub.3 solution. After an extraction and a wash with sodium bisulfite solid/water, the organic layer was dried with MgSO.sub.4, filtered and evaporated. The product was purified by chromatography (c-hexane:ethyl acetate=98:2, then 95:5) to achieve at 5-bromo-2-iodo-3-((E)-styryl)-pyridine 6 (yield: 84%).

[0194] To a reaction vessel (100 mL) in a nitrogen atmosphere containing 6 (1.4 g, 3.6 mmol) were added 6-bromo-5-methylpyridin-3-ylboronic acid 7 (978 mg, 4.5 mmol), tetrakis triphenylphosphine (210 mg, 0.18 mmol), potassium phosphate (2.1 g, 9 mmol) in 1,2-dimethoxyethane (30 mL). The mixture was stirred at reflux for 20 hours until consumption of starting material followed by TLC. At room temperature, the solution was extracted with ethyl acetate. The organic layer was dried on MgSO.sub.4, filtered and evaporated. The residue was purified by chromatography (c-hexane:ethyl acetate=95:5, then 9:1 and 8:2) and 5,6-dibromo-5-methy-3-((E)-styryl)-2,3-bipyridine 8 (yield: 86%) was obtained.

[0195] To a reaction vessel (100 mL) in a nitrogen atmosphere containing 8 (320 mg, 0.7 mmol) were added pyridin-3-yl boronic acid 9 (156 mg, 1.7 mmol), tetrakis triphenylphosphine (78 mg, 0.07 mmol), sodium carbonate (355 mg, 3.35 mmol) in 1,4-dioxane (20 mL). The mixture was stirred at reflux for 24 hours until consumption of starting material followed by TLC. At room temperature, the suspension was filtered on celite and the solution was extracted with ethyl acetate. The organic layer was dried on MgSO.sub.4, filtered and evaporated. The product was purified by chromatography (c-hexane:ethyl acetate=8:2, then 7:3 and 50:50) to achieve 5,6-di(pyridin-3-yl)-5-methyl-3-((E)-styryl)-2,3-bipyridine MR29072 (yield: 86%) as a white solid (Mp 158 C.). IR (KBr): 2957, 1727, 1575, 1274, 1125, 1014, 967, 801, 770, 688 cm.sup.1. .sup.1H NMR (400 MHz, CDCl.sub.3): 8.98 (d, .sup.4J=1.9 Hz, 1H), 8.91 (d, .sup.4J=1.9 Hz, 1H), 8.87 (m, 2H), 8.72 (dd, .sup.3J=4.9 Hz, .sup.4J=1.9 Hz, 2H), 8.68 (dd, .sup.3J=4.9 Hz, .sup.4J=1.9 Hz, 1H), 8.24 (d, .sup.4J=1.9 Hz, 1H), 8.00 (m, 3H), 7.48 (d, .sup.3J=7.8 Hz, 3H), 7.44 (dd, .sup.3J=7.8 Hz, .sup.4J=1.9 Hz, 1H), 7.36 (d, .sup.3J=7.8 Hz, 1H), 7.31 (d, .sup.3J=7.8 Hz, 1H), 7.28 (d, .sup.3J=16.6 Hz, 1H), 7.23 (di, .sup.3J=16.6 Hz, 1H), 2.51 (s, 3H). .sup.13C NMR (100 MHz, CDCl.sub.3): 155.3, 153.4, 149.9, 149.6, 149.2, 148.2, 148.0, 146.9, 139.8, 136.5, 136.3, 135.8, 134.4, 134.1, 133.1, 133.0, 132.9, 132.6, 132.0, 131.1, 128.8 (2C), 128.5, 126.8 (2C), 124.6, 123.8, 123.2, 20.0. LCMS (EI): m/z (%)=[M+H].sup.+ theoretical: 427.53, experimental: 427.32. Anal. Calcd for C.sub.29H.sub.22N.sub.4: C, 81.67; H, 5.20; N, 13.14. Found: C, 81.65; H, 5.25; N, 13.23.

[0196] This first procedure is applied to compounds 3-methyl-5-(3-(E)-styryl-5-(thiophen-3-yl)pyridin-2-yl)-2-(thiophen-3-yl)pyridine (MR31322), 3-methyl-2-(3-methylthiophen 2-yl)-5-(5-(3-methylthiophen-2-yl)-3-(E)-styrylpyridin-2-yl)pyridine (MR31336), 3-methyl-5-(3-(E)-styryl-5-(thiophen-2-yl)pyridin-2-yl)-2-(thiophen-2-yl)pyridine (MR31321), 2-(5-methyl-6-(1H-pyrazol-5-yl)pyridin-3-yl)-5-(1H-pyrazol-5-yl)-3-styrylpyridine (MR31363), 5-(2-chloro-1-methyl-1H-imidazol-5-yl)-2-(6-(2-chloro-1-methyl-1H-imidazol-5-yl)-5-methylpyridin-3-yl)-3-styrylpyridine (MR31351), 3-methyl-2-(4-cyanophenyl)-5-(5-(4-cyanophenyl)-3-styrylpyridin-2-yl)pyridine MR30854, 5-(3,4,5-trimethoxyphenyl)-2-(6-(3,4,5-trimethoxyphenyl)-5-methylpyridin-3-yl)-3-styrylpyridine (MR30847), 2-(3,4-dimethoxyphenyl)-5-(5-(3,4-dimethoxyphenyl)-3-styrylpyridin-2-yl)-3-methylpyridine (MR30846), 4-(3-methyl-5-(5-(pyridin-4-yl)-3-styrylpyridin-2-yl)pyridin-2-yl)pyridine (MR31350) 3-methyl-2-phenyl-5-(5-phenyl-3-styrylpyridin-2-yl)pyridine (MR30814), 5-(3-methyl-5-(5-(pyrimidin-5-yl)-3-styrylpyridin-2-yl)pyridin-2-yl)pyrimidine (MR31361)

[0197] From 5,6-dibromo-5-methyl-3-((E)-styryl)-2,3-bipyridine 8 introduced in a reaction vessel (100 mL) in a nitrogen atmosphere with thiophene-3-boronic acid, 3-methyl-thiophene-2-boronic acid, thiophene-2-borinic acid, 1-(tetrahydro-2H-pyran-2-yl) 5-(4,4,5,5-tetramethyl-1,3,2-dioxoborolan-2-yl)-1H-pyrazole, 2-chloro-1-methyl-5-(4,4,5,5-tetramethyl-)-1,3,2-dioxaborolan-2-yl)-1H-imidazole, 4-cyano phenylboronic acid, 3,4,5-trimethoxyphenylboronic acid, 3,4-dimethoxyphenylboronic acid, 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine, benzeneboronic acid, pyrimidin-5-yl boronic acid, tetrakis triphenylphosphine, sodium carbonate in 1,4-dioxane (20 mL), we respectively obtained MR31322, MR31336, MR31321. MR31363, MR31351, MR30854, MR30847, MR30846, MR31350, MR30814, MR31361.

Example 2

3-methyl-5-(3-(E)-styryl-5-(thiophen-2-yl)pyridin-2-yl)-2-(thiophen-2-yl)pyridine (MR31321)

[0198] .sup.1H-NMR (CDCl.sub.3) 8.90 (d, 1H, H6, J=1.96 Hz), 8.73 (d, 1H, H6, J=1.96 Hz), 8.18 (d, 1H, H4, J=1.96 Hz), 7.95 (d, 1H, H4, J=1.96 Hz), 7.58 (d, 1H, H3, J=3.87 Hz), 7.49-7.46 (m, 4H, 2H ortho Ph, H5 and H5), 7.43 (d, 1H, H3, J=3.87 Hz), 7.38-7.34 (dd, 2H meta Ph, J=7 Hz), 7.30 (m, 1H para Ph, J=7 Hz), 7.20 (s, 2H, CHCH), 7.19-7.17 (m, 2H, H4 and H4), 2.68 (3H, CH.sub.3).

[0199] MS(EI): 437[M+]*.

Example 3

3-methyl-5-(3-(E)-styryl-5-(thiophen-3-yl)pyridin-2-yl)-2-(thiophen-3-yl)pyridine (MR31322)

[0200] .sup.1H-NMR (CDCl.sub.3) 8.89 (d, 1H, H6, J=1.92 Hz), 8.77 (d, 1H, H6, J=1.92 Hz), 8.21 (d, 1H, H4, J=1.92 Hz), 7.96 (1H, H4, J=1.92 Hz), 7.68-7.66 (dd, 2H, H5 and H5, J=2.92 Hz, J=7.4 Hz), 7.57 (d, 1H, H para Ph, J=7 Hz) 7.51 (d, 2H, H2 and H5, J=2.92 Hz), 7.47 (d, 2H ortho Ph, J=7 Hz), 7.43-7.41 (dd, 2H, H4 and H4, J=2.92 Hz), 7.38-7.34 (dd, 2H meta Ph, J=7 Hz), 7.26-7.21 (m, 2H, CHCH), 2.68 (s, 3H, CH.sub.3).

[0201] MS(EI): 437 [M+]*.

Example 4

3-methyl-2-(3-methylthiophen-2-yl)-5-(5-(3-methylthiophen-2-yl)-3-(E)-styrylpyridin-2-yl)pyridine (MR31336)

[0202] .sup.1H-NMR (CDCl.sub.3): 8.68 (d, 1H, H6, J=1.92 Hz), 8.59 (d, 1H, H6, J=1.92 Hz), 8.07 (d, 1H, H1, J=1.92 Hz), 7.95 (d, 1H, H4, J=1.92 Hz), 7.52-7.28 (m, 7H), 7.25-7.6.95 (m, 4H), 2.41 (s, 3H, CH.sub.3), 2.16 (s, 3H, CH3), 2.03 (s, 3H, CH.sub.3).

[0203] MS(EI): 466 [M++H, 100].

Example 5

2-(5-methyl-6-(1H-pyrazol-5-yl)pyridin-3-yl)-5-(1H-pyrazol-5-yl)-3-styrylpyridine (MR31363)

[0204] .sup.1H-NMR (d.sub.6-DMSO): 9.08 (d, 1H, J=1.7 Hz), 8.63 (d, 1H, J=1.7 Hz), 8.58 (d, 1H, J=1.7 Hz), 7.98 (d, 1H, J=1.6 Hz), 7.86 (d, 1H, J=1.5 Hz), 7.76 (bs, 1H), 7.55 (d, 2H, J=7.8 Hz), 7.45 (d, 1H, CHCH, J=16.4 Hz), 7.40-7.36 (m, 2H), 7.31-7.27 (m, 1H), 7.22 (d, 1H, CHCH, J=16.4 Hz), 7.02 (d, 1H, J=2.16), 6.86 (m, 1H), 6.53 (bs, 1H), 2.65 (s, 3H, CH.sub.3).

[0205] MS(EI): 405.60 [M.sup.+]*.

Example 6

5-(2-chloro-1-methyl-1H-imidazol-5-yl)-2-(6-(2-chloro-1-methyl-1H-imidazol-5-yl)-5-methylpyridin-3-yl)-3-styrylpyridine (MR31351)

[0206] .sup.1H-NMR (CDCl.sub.3): 8.82 (d, 1H, J=2.2 Hz), 8.66 (di, 1H, J=2.2 Hz), 8.04 (d, 1H, J=2.2 Hz), 7.99 (d, 1H, J=2.2 Hz), 7.39 (d, 2H, J=7.3 Hz), 7.32-7.25 (m, 3H), 7.18 (s, 1H), 7.14 (d, 1H, J=16 Hz), 7.12 (s, 1H), 7.10 (d, 1H, J=16 Hz), 3.69 (s, 3H, CH.sub.3), 3.65 (s, 3H, CH.sub.3), 2.47 (s, 3H, CH.sub.3).

[0207] MS(EI): 501.13 [M.sup.+]*, 503.12 [M.sup.++2]*, 505.32 [M.sup.++4]*.

Example 7

3-methyl-2-(4-cyanophenyl)-5-(5-(4-cyanophenyl)-3-styrylpyridin-2-yl)pyridine (MR30854)

[0208] .sup.1H NMR (400 MHz, CDCl.sub.3): 8.88 (di, J=2.2, 1H, H2), 8.85 (d, J=2.2, 1H, H6), 8.24 (d, J=2.2, 1H, H4), 8.04 (d, J=2.2, 1H, H4), 7.79 (dd, J=8.3, 1.9, 4H), 7.74 (dd, J=8.5, 2.0, 4H), 7.45 (d, J=8.0, 2H, Hstyr), 7.35 (d, J=8.1, 2H, Hstyr), 7.31 (d, J=7.3, 1H, Hstyr), 7.17 (d, J=16.3, 2H, CHCH), 2.49 (s, 3H, CH.sub.3).

Example 8

5-(3,4,5-trimethoxyphenyl)-2-(6-(3,4,5-trimethoxyphenyl)-5-methylpyridin-3-yl)-3-styrylpyridine (MR30847)

[0209] .sup.1H NMR (400 MHz, CDCl.sub.3: 8.83 (s, 1H, H6), 8.80 (s, 1H, H2), 8.16 (s, 1H, H4), 7.99 (s, 1H, H4), 7.49 (d, J=7.1, 2H, Hstyr), 7.35 (dd, J=7.6, 6.8, 2H, Hstyr), 7.31 (d, J=7.3, 1H, Hstyr), 7.28 (d, J=15.6, 1H, CHCH), 7.21 (d, J=15.6, 1H, CHCH), 6.86 (s, 2H), 6.84 (s, 2H), 3.99 (s, 6H, CH.sub.3O-meta), 3.93 (s, 3H, CH.sub.3O-para), 3.91 (s, 6H, CH.sub.3O-meta), 3.82 (s, 3H, CH.sub.3O-para), 2.50 (s, 3H, CH.sub.3).

Example 9

2-(3,4-dimethoxyphenyl)-5-(5-(3,4-dimethoxyphenyl)-3-styrylpyridin-2-yl)-3-methylpyridine (MR301846)

[0210] .sup.1H NMR (400 MHz, CDCl.sub.3): 8.84 (s, 1H, H6), 8.80 (s, 1H, H2), 8.17 (s, 1H, J=8.3, 1H, Hstyr), 7.28 (d, J=16.4, 1H, CHCH), 7.27-7.21 (m, 4H), 7.18 (d, J=15.9, 1H, CHCH), 7.04 (d, J=8.3, 1H), 6.98 (d, J=8.3, 1H), 4.01 (s, 3H, CH.sub.3O-meta), 3.97 (s, 9H), 2.50 (s, 3H, CH.sub.3).

Example 10

4-(3-methyl-5-(5-(pyridin-4 yl)-3-styrylpyridin-2-yl)pyridin-2-yl)pyridine (MR31350)

[0211] .sup.1H-NMR (CDCl.sub.3): 8.85 (d, 1H, J=2.2 Hz), 8.80 (d, 1H, J=1.9 Hz), 8.73-8.71 (dd, 2H, J=1.7 Hz, J=4.5 Hz), 8.70-8.68 (dd, 2H, J=1.7 Hz, J=4.5 Hz), 8.21 (d, 1H, J=2.2 Hz), 7.97 (d, 1H, J=1.9 Hz), 7.58-7.57 (dd, 2H, J=1.7 Hz, J=4.5 Hz), 7.48-7.47 (dd, 2H, J=1.7 Hz, J=4.5 Hz), 7.41 (d, 2H, J=6.8 Hz), 7.32-7.29 (m, 2H), 7.26-7.25 (m, 1H), 7.23-7.16 (m, 2H, CHCH), 2.43 (s, 3H, CH3).

[0212] MS (EI): 427.37 [M.sup.+]*

Example 11

3-methyl-2-phenyl-5-(5-phenyl-3-styrylpyridin-2-yl)pyridine (MR30814)

[0213] .sup.13C NMR (100 MHz, CDCl.sub.3): 158.4, 153.1, 146.9, 146.2, 140.3, 139.7, 137.5, 136.8, 135.9, 133.3, 132.5 (2C), 131.7, 130.6, 129.3 (2C), 129.2 (2C), 128.8 (2C), 128.3 (2C), 128.2 (2C), 128.1, 127.2 (2C), 126.8 (2C), 125.4, 20.1.

[0214] LCMS (ESI) (m/z): 424.55; [M+H.sup.+] 425.27.

Example 12

5-(3-methyl-5-(5-(pyrimidin-5-yl)-3-styrylpyridin-2-yl)pyridin-2-yl)pyrimidine (MR31361)

[0215] .sup.1H-NMR (CDCl.sub.3): 9.33 (s, 1H), 9.30 (s, 1H), 9.10 (s, 2H), 9.07 (s, 2H), 8.90 (d, 1H, J=1.9 Hz), 8.88 (d, 1H J=2.2 Hz), 8.25 (d, 1H, J=2.2 Hz), 8.07 (d, 1H, J=1.9 Hz), 7.48 (d, 2H, J=7 Hz), 7.40-7.32 (m, 3H), 7.27 (d, 1H, J=16 Hz, CHCH), 7.22 (d, 1H, J=16 Hz, CHCH), 2.53 (s, 3H, CH.sub.3).

[0216] MS(EI): 429.58 [M.sup.+]*.

[0217] A second and a third procedure (Scheme 2) were applied to compounds 3-methyl-5-(5-phenyl-3-styrylpyridin-2-yl)-2-(pyridin-3-yl)pyridine (MR31348), 3-(6-(5-methyl-6-phenylpyridin-3-yl)-5-styrylpyridin-3-yl)pyridine (MR131349), 3-(3-methyl-5-(5-(pyridin-3-yl)-3-styrylpyridin-2-yl)pyridin-2-yl)phenol (MR31364), 3-(3 methyl-5-(5-(pyridin-3-yl)-3-styrylpyridin-2-yl)pyridin-2-yl)phenol (MR31366), (1Z)N-hydroxy-2-(3-(3-methyl-5-(5-(pyridin-3-yl)-3-styrylpyridin-2-yl)pyridin-2-yl)phenyl)acetamidine (MR31367), 5-(3,4-dimethoxyphenyl)-2-(6-(3,4,5-trimethoxyphenyl)-5-methylpyridin-3-yl)-3-styrylpyridine (MR30849), 5-(3,4,5-trimethoxyphenyl)-2-(6-(3,4-dimethoxyphenyl)-5-methylpyridin-3-yl)-3-styrylpyridine (MR30850).

##STR00023## ##STR00024##

Example 13

5-(3,4,5-trimethoxyphenyl)-2-(6-(3,4-dimethoxyphenyl)-5-methylpyridin-3-yl)-3-styrylpyridine (MR30650)

[0218] .sup.1H NMR (400 MHz, CDCl.sub.3): 8.83 (d, J=2.2, 1H, H6), 8.80 (d, J=2.2, 1H, H2), 8.16 (d, J=2.4, 1H, H4), 7.98 (d, J=1.7, 1H, H4), 7.49 (d, J=7.1, 2H, Ha), 7.36 (dd, J=7.6, 7.1, 2H, Hb), 7.30 (d, J=6.8, 1H, Hc), 7.21 (d, J=17.8, 1H, CHCH), 7.20 (d, J=17.8, 1H, CHCH), 7.20 (d, J=8.3, 1H), 7.19 (di, J=8.0, 1H), 6.98 (d, J=8.3, 1H), 6.86 (s, 2H), 3.99 (s, 6H), 3.97 (s, 3H), 3.96 (s, 3H), 3.94 (s, 3H), 2.50 (s, 3H, CH.sub.3).

Example 14

5-(3,4-dimethoxyphenyl)-2-(6-(3,4,5-trimethoxyphenyl)-5-methylpyridin-3-yl)-3-styrylpyridine (MR30649)

[0219] .sup.1H NMR (400 MHz, CDCl.sub.3): 8.84 (d, J=2.2, 1H, H6), 8.79 (d, J=2.2, 1H, H2), 8.17 (d, J=2.2, 1H, H4), 7.99 (d, J=2.0, 1H, H4), 7.48 (d, J=7.3, 2H, Hstyr), 7.35 (dd, J=7.8, 6.8, 2H, Hstyr), 7.30 (d, J=7.1, 1H, Hstyr), 7.27 (d, J=2.2, 1H), 7.27 (d, J=16, 1H, CHCH), 7.23 (d, J=16.2, 1H, CHCH), 7.19 (d, J=2.2, 1H), 6.84 (s, 3H), 4.01 (s, 3H), 3.97 (s, 3H), 3.93 (s, 6H), 3.91 (s, 3H), 2.50 (s, 3H, CH.sub.3).

Example 15

(1Z)N-hydroxy-2-(3-(3-methyl-5-(5-(pyridin-3-yl)-3-styrylpyridin-2-yl)pyridin-2-yl)phenyl)acetamidine (MR31367)

[0220] .sup.1H-NMR (CDCl.sub.3): 8.98 (d, 1H, J=1.96 Hz), 8.87 (d, 1H, J=2.2 Hz), 8.82 (d, 1H, J=2.2 Hz), 8.72 (d, 1H, J=1.96 Hz), 8.24 (d, 1H, J=2.2 Hz), 8.02-8.00 (m, 2H), 7.55-7.43 (m, 7H), 7.36-7.20 (m, 4H), 4.58 (bs, 1H, NH.sub.2), 3.56 (s, 2H, CH.sub.2), 2.46 (s, 3H, CH.sub.3).

[0221] MS(EI): 498.55 [M.sup.+]*

Example 16

3-(3-methyl-5-(5-(pyridin-3-yl)-3-styrylpyridin-2-yl)pyridin-2-yl)phenol (MR31366)

[0222] .sup.1H-NMR (CD.sub.3OD): 10.5 (bs, 1H, COOH), 9.02 (d, 1H, J=1.96 Hz), 8.90 (d, 1H, J=2.2 Hz), 8.68 (di, 1H, J=2.2 Hz), 8.66-8.64 (dd, 1H, J=1.2 Hz, J=4.6 Hz), 8.56 (d, 1H, J=2.2 Hz), 8.33-8.31 (dd, 1H, J=1.3 Hz, J=4.6 Hz), 8.07 (d, 1H, J=2.2 Hz), 7.63-7.62 (m, 1H), 7.53-7.48 (m, 5H), 7.37-7.33 (m, 2H), 7.29-7.21 (m, 2H), 3.71 (s, 2H, CH.sub.2), 2.44 (s, 3H, CH.sub.3)

[0223] MS(EI): 484.54 [M.sup.+]*

Example 17

3-(3-methyl-5-(5-(pyridin-3-yl)-3-styrylpyridin-2-yl)pyridin-2-yl)phenol (MR31364)

[0224] .sup.1H-NMR (CDCl.sub.3): 8.98 (d, 1H, J=1.96 Hz), 8.86 (d, 1H, J=1.96 Hz), 8.78 (d, 1H, J=1.96 Hz), 8.72-8.71 (m, 1H) 8.26 (d, 1H, J=2.2 Hz), 8.03-7.99 (m, 3H), 7.51-7.46 (m, 3H), 7.35-7.32 (m, 2H), 7.31-7.20 (m, 4H), 7.07 (d, 1H, J=7.56 Hz), 6.93 (s, 1H), 6-84-6.81 (dd, 1H, J=1.6 Hz, J=7.56 Hz), 2.45 (s, CH.sub.3), 1.77 (bs, 1H, OH)

[0225] MS(EI): 442.41 [M.sup.+]*

Example 18

3-(6-(5-methyl-6-phenylpyridin-3-yl)-5-styrylpyridin-3-yl)pyridine (MR31349)

[0226] .sup.1H-NMR (CDCl.sub.3): 8.90 (d, 1H, J=1.9 Hz), 8.79 (d, 1H, J=2.2 Hz), 8.75 (d, 1H, J=2.2 Hz), 8.64-8.62 (dd, 1H, J=1.2 Hz, J=4.7 Hz), 8.15 (d, 1H, J=2.2 Hz), 7.94-7.91 (m, 21H), 7.55 (d, 2H, J=7 Hz), 7.44-7.33 (m, 6H), 7.29-7.26 (m, 2H), 7.23-7.19 (m, 2H), 7.14 (d, 1H, J=16 Hz, CHCH), 2.42 (s, 3H, CH3).

[0227] MS(EI): 426.42 [M.sup.+]*

Example 19

3-methyl-5-(5-phenyl-3-styrylpyridin-2-yl)-2-(pyridin-3-yl)pyridine (MR31348)

[0228] .sup.1H-NMR (CDCl.sub.3): 8.90 (d, 1H, J=1.9 Hz), 8.88 (d, 1H, J=2.2 Hz), 8.86 (d, 1H, J=1.9 Hz), 8.68-8.67 (dd, 1H, J=1.7 Hz, J=4.8 Hz), 8.24 (d, 1H, J=1.9 Hz), 8.03 (d, 1H, J=1.7 Hz), 7.98-7.97 (m, 1H), 7.72 (d, 2H, J=8.3 Hz), 7.57-7.53 (m, 2H), 7.49-7.46 (m, 4H), 7.38-7.34 (m, 2H), 7.31-7.29 (m, 1H), 7.27 (d, 1H, J=16.6 Hz), 7.22 (d, 1H, J=16.6 Hz), 2.50 (s, 3H, CH3).

[0229] MS(EI): 426.58 [M.sup.+]*

II. Biological Activity of Compounds of Formula (I)

[0230] II.A. Materials & Methods

[0231] Tested Compounds

[0232] (Het)aromatic oligosystems are synthesized as described in Example 1 and purified by chromatography (column using flash silica gel 60 Merck [0.063-0.200 mm] as the stationary phase).

[0233] ABT-737 was obtained from Selleckchem (Houston, Tex., USA) and dimethylsulfoxide (DMSO) from Sigma-Aldrich (Saint-Quentin Fallavier, France).

[0234] These compounds were commonly stored as stock solutions in DMSO at 20 C.

[0235] Cell Culture

[0236] Human ovarian carcinoma OAW42 cell line was established from a human ovarian adenocarcinoma and was obtained from ECACC (Sigma-Aldrich, Saint Quentin Fallavier, France). It was grown in DMEM medium supplemented with 4500 mg/l glucose, 2 mM Glutamax, 1 mM sodium pyruvate, 10% fetal calf serum, 33 mM sodium bicarbonate (Gibco BRL, Lyon, France) and 20 IU/I recombinant human insulin (Lilly, Suresnes, France).

[0237] Human ovarian carcinoma SKOV3 cell line was established from a human ovarian adenocarcinoma and was obtained from American Type Culture Collection (Manassas, Va., USA), as well as the human malignant mesothelioma cell line NCI-H28 and the human lung carcinoma cell line A549.

[0238] IGROV1 cell line was kindly provided by Dr. J. Bnard (Institut G. Roussy, Villejuif, France). These cell lines were grown in RPMI 1640 medium supplemented with 2 mM Glutamax, 25 mM HEPES, 10% fetal calf serum, and 33 mM sodium bicarbonate (Fisher Scientific, Illkirch, France),

[0239] In vitro chemoresistant model of IGROV1 (IGROV1-R10) and OVW42 (OAW42-R) cell lines were obtained previously by mimicking a clinical protocol of cisplatin administration [Poulain et al. (1998) Int J Cancer 78, 454-463; Villedieu et al., (2007) Gynecol Oncol. 10(1), 31-44].

[0240] Cells were maintained at 37 C. in a 5% CO.sub.2 humidified atmosphere and split twice a week by trypsinization.

[0241] Treatments

[0242] Exponentially growing cells were transfected by siRNA as described below, and after 48 h, cells were continuously exposed to (Het)aromatic oligosystems (10, 25 or 50 M) dissolved in DMSO (<0.1% of total volume) for 4 to 24 supplementary hours.

[0243] Gene Silencing

[0244] siRNAs were synthesized and annealed by Eurogentec (Liege, Belgium). Sequences were as follows:

[0245] Bcl-x.sub.L siRNA antisense (siXL1): 5-auuggugagucggaucgcatt-3 (SEQ. ID. No1);

[0246] Mcl-1 siRNA (siMCL1): 5-gugccuuuguggcuaacatt-3 (SEQ. ID. No2):

[0247] Control siRNA (siCONT): 5-gacguaaacggccacaagutt-3 (SEQ. ID. No3).

[0248] The control siRNA does not bear any homology with any relevant human genes. Cells were seeded in 25 cm.sup.2 flasks the day before to reach 30-50% confluency at the time of transfection. The transfection INTERFERin reagent (Polyplus Transfection, Strasbourg. France) was added to siRNA diluted in Opti-MEM reduced serum medium (Invitrogen, Cergy-Pontoise, France) and complexes formation was allowed to proceed for 15 min at RT before being applied to cells. The final siRNA concentration in the flasks was 20 nM.

[0249] BRET Assay

[0250] Hela cells were seeded on 6-well plates and transfected with 200 ng/well of plasmid pRLuc-Bax, pRLuc-Puma or pRLuc-Noxa coding for BRET donors and 1 g/well of peYFP-Bcl-x.sub.L or peYFP-Mcl-1 coding for BRET acceptors (or with pCMV-Bcl-x.sub.L or pCMV-Mcl-1 for control). Twenty-four hours after transfection, cells were trypsinized and re-seeded into white 96 well plate flat bottom, incubated for another day, and then treated with drugs for 16 hours at 10 M. Light emission at 485 nm and 530 nm was measured consecutively using the Mithras fluorescence-luminescence detector LB 940 (Berthold) after adding the luciferase substrate, coelenterazine H (Uptima) at a final concentration of 5 M. BRET ratios were calculated as described [Terrillon et al. (2003) Mol Endocrinol 17, 677-691, Vo et al. (2012) Eur J Med Chem., 286-93].

[0251] Real-Time Cellular Activity Assay

[0252] Compound-mediated cytotoxicity was monitored using Real-Time Cell Analyzer multi-plate (RTCA MP) Instrument, xCELLigence System (Roche Applied Science, Mannheim, Germany). This system monitors cellular events in real time measuring electrical impedance across interdigitated micro-electrodes integrated on the bottom of tissue culture E-plates View (Roche). The increase in the number and size of cells attached to the electrode sensors leads to increased impedance, from which derive the Cell Index values (CI) displayed at the plot. Thus, this index reflects changes in cell viability as described by Ke et al. (2011, Methods Mol Biol 740, 33-43). Briefly, 96-well E-Plate were seeded with 310.sup.3 cells/well and placed onto the RTCA MP located inside a tissue culture incubator, where cells were left to grow for 24 h before treatment. Impedance was continuously measured until the end of the treatment. Standard deviations of well replicates were analyzed with the RTCA Software.

[0253] Apoptosis Assays

[0254] Morphological Characterization of Apoptotic Cells by Nuclear Staining with DAPI

[0255] After treatment, both detached and adherent cells were pooled after trypsinization, applied to a polylysine-coated glass slide by cytocentrifugation and fixed with a solution of ethanol/chloroform/acetic acid (6:3:1). The preparations were then incubated for 15 min at room temperature with 1 g/ml DAPI solution (Boehringer Mannheim-Roche, Mannheim, Germany), washed in distillated water, mounted under a coverslip in Mowiol (Calbiochem) and analyzed under a fluorescence microscope (BX51, Olympus, Rungis, France).

[0256] Cell Cycle Analysis by Flow Cytometry

[0257] Adherent and floating cells were pooled, washed with 1PBS and centrifuged at 200 g for 5 min before staining by Annexin V, propidium iodide or both, as recommended by the manufacturer (Roche Diagnostic, Indianapolis, USA). Briefly, 100 l of Annexin VFlit or propidium iodide or both were added on the cells pellet (10.sup.6 cells) and incubated 15 minutes at room temperature in obscurity. 500 l of sample buffer was then added on the suspensions that were thereafter analyzed using a Gallios flow cytometer (Beckman Coulter, Roissy, France) and cell cycle distribution was determined using Kaluza acquisition software (Beckman Coulter).

[0258] Preparation of Cell Extracts and Western Blot Analysis

[0259] Cells were rinsed with ice-cold PBS, suspended in a lysis buffer [RIPA:NaCl 150 mM, Tris (pH 8) 50 mM, Triton X100 1%, PMSF 4 mM, EDTA 5 mM, NaF 10 mM, NaPPi 10 mM, Na3OV4 1 mM, aprotinin 0.5 l/ml and 4.6 ml ultra pure water] and incubated on ice for 30 minutes. Lysates were collected after centrifugation (13200 g, 10 min, 4 C.) and protein concentrations were determined using the Bradford assay (Bio-Rad, Hercules, USA). 20 g of protein were separated by SDS-PAGE on a 4-12% gradient polyacrylamide gel (Invitrogen, Cergy-Pontoise, France) and transferred to Hybond-PVDF membranes (Amersham, Orsay, France). After blocking non-specific binding sites for 1 hour at RT by 5% (w/v) non-fat dry milk in TBS with 0.1% (v/v) Tween20 (T-TBS), the membranes were incubated overnight at 4 C. with the following rabbit monoclonal antibody: PARP, caspase-3 and Bcl-x.sub.L, Bim (Cell Signaling Technology, Ozyme, Saint-Quentin-en-Yvelines, France), Mcl-1 (Santa Cruz, Le Perray-en-Yvelines, France), HSP-70, Noxa (Calbiochem, Fontenay-sous-Bois, France), (Cell Signalling) and Actin (Sigma-Aldrich, Saint-Quentin Fallavier, France). Membranes were then washed with T-TBS and incubated for 1 hour with the appropriate horseradish peroxidase-conjugated anti-rabbit or anti-mouse (Amersham, Orsay, France) secondary antibodies. Revelation was done using a luminescent Image Analyzer (GE Healthcare, Orsay, France).

[0260] Transmission Electron Microscopy

[0261] Cells were fixed with 2.5% glutaraldehyde in PBS buffer, included in agar, rinsed in Sorensen's buffer, post-fixed in osmium tetroxide 1% in Sorensen's buffer, deshydrated in ethanol and embedded in EPON resin. Ultrathin sections were cut and stained with uranyl acetate and lead citrate and examined using a JEOL1011 transmission electron microscope.

II.B. RESULTS

[0262] II.B.1. Activity of Pyridoclax (MR29072)

[0263] Pyridoclax Disrupts Mcl-1/Puma Interaction

[0264] As presented on the FIGS. 1A and 1B, Pyridoclax is able to disrupt both Mcl-1/Puma and Mcl-1/Noxa interactions in a cellular assay (BRET) performed in Hela cells. Whereas ABT-737 is not able to modify this interaction, Pyridoclax induced a drastic inhibition of Mcl-1/Puma interaction (about 50%).

[0265] Effect of Pyridoclax as Single Agent or Associated to siRNA-Mediated Bcl-x.sub.L Inhibition

[0266] To demonstrate the interest of Pyridoclax as a Mcl-1 inhibitor, a model of selective addiction to both Bcl-x.sub.L, and Mcl-1 in which Bcl-x.sub.L expression is silenced by RNA interference 48 h before exposure has been used. The ovarian carcinoma cell line IGROV1-R10 is chosen to conduct these assays since it has been previously demonstrated that this cell line was highly sensitive to the concomitant inhibition of Bcl-x.sub.L and Mcl-1 (Brotin et al. (2010) Int J Cancer 126, 885-895), but remained viable when only one of these targets was inhibited.

[0267] As expected, neither Pyridoclax nor the Bcl-x.sub.L targeting siRNA (siXL1) induced massive cell death on their own. A slowed down proliferation is observed, but neither cell detachment, nor strong sub-G1 peak, caspase 3 activation and condensed or fragmented nuclei were observed (FIGS. 2A, B, C) in these conditions.

[0268] In contrast, their association led to a massive cell death, as demonstrated by a strong cell detachment, by the appearance of a strong sub-G1 Peak on the DNA content histogram (over 50%) and of a 60% fraction of annexin V positive cells (FIG. 2A). Moreover, the viability evaluation showed that this association led to a drastic decrease of the number of viable cells and to a concomitant increase of dead cells (FIG. 2B) and western blot showed a complete cleavage of PARP and caspase 3. DAPI staining and electron microscopy showed that this association led to nuclear condensations and fragmentations (only when the two agent were combined), highly evocative of apoptotic cell death (FIG. 2D).

[0269] These effects are optimal after exposure to a concentration of 25 M of Pyridoclax, but are also observed in a lower extend in response to 10 M (data not shown).

[0270] The kinetic study of the effect of this combination showed that apoptosis was observed as soon as 2 to 4 h after the beginning of the exposure (37% of events in sub-G1 fraction after 4 h), this observation being compatible with a pharmacologic Mcl-1 inhibition through BH3-mimetic activity (FIG. 2E).

[0271] Altogether, these elements show that Pyridoclax strongly sensitizes ovarian cancer chemoresistant IGROV1-R10 cells to Bcl-x.sub.L targeting siRNA, their combination leading to massive apoptosis.

[0272] Pyridoclax Sensitizes Various Cancer Cell Types to Bcl-x.sub.L Targeting siRNA

[0273] The effect of the combination of Pyridoclax with siXL1 in other ovarian carcinoma cell lines (FIG. 3A) as well as in other cancer cell types (FIG. 3B) has then been studied.

[0274] A similar response to this association is observed in all ovarian carcinoma cell lines, as well as in lung carcinoma (A549) and mesothelioma (NCI-H28 and MSTO-211H) cell lines.

[0275] Pyridoclax Sensitizes Chemoresistant Ovarian Cancer Cells to ABT-737

[0276] ABT-737 being yet one of the most potent Bcl-x.sub.L inhibiting BH3-mimetic molecule, and the response to ovarian cancer cells being conditioned by the inhibition of Mcl-1, the effect of the combination of ABT-737 with Pyridoclax has been evaluated (FIG. 4). As described previously with siXL1, it is observed that neither ABT-737 nor Pyridoclax induced cell death as single agents, whereas their combination led to massive apoptotic cell death in both IGROV1-R10 and SKOV3 chemoresistant ovarian cancer cell lines. Indeed, when the two molecules were combined, cellular activity was drastically decreased in both cell lines as assessed by impedancemetry (xCELLigence technology) (FIGS. 4A and 4D); furthermore a strong cell detachment and an important sub-G1 fraction are observed (FIG. 4B) as well as complete PARP and caspase 3 cleavages (FIGS. 4C and 4D right panel). It should be noticed that these effects were similar in concomitant exposure experiments and in sequential exposure (Pyridoclax 24 h, then ABT-737). Moreover, the observed apoptosis was quasi-immediate as soon as the cells are exposed to Pyridoclax and ABT-737, arguing in favor of a BH3-mimetic activity.

[0277] ILB.2. Activity of Other Compounds of the Invention

[0278] Compounds selected on their capability to disrupt the Mcl-1/Puma interaction in BRET assay have then been tested to assess their activity on cell morphology, on cell cycle, on PARP cleavage and on nuclear morphology; results are presented in Table I below:

TABLE-US-00001 TABLE I Molecule + Bcl-xL targeting siRNA Cell Sub- PARP Apoptotic nuclear Molecule identification detachment G1 (%) cleavage features 29072 or Pyridoclax [00025] +++ 53.6 +++ +++ 30814 [00026] 37.2 + + 31349 [00027] ++ 59.2 +++ +++ 31348 [00028] +++ 58.8 +++ +++ 30846 [00029] 33.1 nd + 30847 [00030] 22 nd + 30854 [00031] 24.3 nd + 31336 [00032] ++ 48.8 nd ++ 31351 [00033] +++ 56.7 nd +++ 31361 [00034] ++ 35.2 nd ++ 31363 [00035] +++ 63.7 nd +++ 30849 [00036] 19.9 nd + 30850 [00037] 20.2 nd + 30820 [00038] ++ 57.8 + ++ 31364 [00039] ++ 39.6 nd ++ 31366 [00040] +++ 51 nd ++ 31367 [00041] +++ 52.7 nd +++

[0279] Assessment of the Different Criteria:

[0280] Cell Detachment: [0281] means no difference between untreated cells and those treated with the tested compound; [0282] means that very few cells were detached from the support (less than 10%); [0283] + means that about 20% of cells were detached from the support; [0284] ++ means that about half of the cells were detached from the support; [0285] +++ means that majority of the cells are detached from the support.

[0286] PARP Cleavage: [0287] + means that a little band corresponding to the 85 kDa cleaved form of PARP is observable on the western blot. This band is usually absent or weak when cells are untreated, the only band observable being thus the 110 kDa band; [0288] ++ means that a band corresponding to the 85 kDa cleaved form of PARP is clearly observable on the western blot. An uncleaved band (110 kDa) usually coexists with the cleaved band; [0289] +++ means that PARP in nearly completely cleaved. The 100 kDa band has often disappeared to the benefit of 85 kDa form.

[0290] Apoptotic Nuclear Features: [0291] + means a few condensed or fragmented nuclei are observable after DAPI staining; [0292] ++ means that numerous condensed or fragmented nuclei are observable after DAPI staining (20-50%); [0293] +++ means that most of the nuclei are condensed or fragmented.