PI3K/AKT/mTOR inhibitors and pharmaceutical uses thereof

Abstract

The invention relates to new PI3K/AKT/m TOR inhibitors and their use for the prevention and/or the treatment of a disease selected from the group consisting of: inflammatory diseases, autoimmune diseases, neurodegenerative diseases, cancers, transplant rejection, diseases characterized by a premature aging and tuberous sclerosis.

Claims

1. A compound having one of the following formulae: ##STR00083## or its pharmaceutically acceptable salts, hydrates or hydrated salts or its polymorphic crystalline structures, racemates, diastereomers or enantiomers.

2. The compound according to claim 1, having the following formula: ##STR00084## or its pharmaceutically acceptable salts, hydrates or hydrated salts or its polymorphic crystalline structures, racemates, diastereomers or enantiomers.

3. A method of treatment of a disease selected from the group consisting of: inflammatory diseases, autoimmune diseases, neurodegenerative diseases, cancers, transplant rejection and diseases characterized by a premature aging comprising administering a pharmaceutical acceptable amount of a compound of formula (I) as defined in claim 1 to a patient in need thereof.

4. The method according to claim 3, wherein the disease is selected among cancers.

5. The method according to claim 4, wherein the cancer is breast cancer.

6. A method of treatment of tuberous sclerosis comprising administering a pharmaceutical acceptable amount of a compound having formula (I): ##STR00085## wherein: X is O or S; R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are independently selected from the group consisting of: H, (C.sub.1-C.sub.10)alkyl, (C.sub.1-C.sub.10)alkoxyl, (C.sub.2-C.sub.10)alkenyl, (C.sub.2-C.sub.10)alkynyl, OH, a fluorine atom, a bromine atom, a iodine atom, O(C.sub.1-C.sub.10)alkylene-NHCO(C.sub.1-C.sub.10)alkylene-(C.sub.5-C.sub.10)heterocycloalkyl and O(C.sub.1-C.sub.10)alkylene-NHCSNHR with R being: ##STR00086## wherein: R.sub.2 and R.sub.3 may form together with the carbon atoms to which R.sub.2 and R.sub.3 are attached to form a (C.sub.6-C.sub.10)aryl group; and R and the (C.sub.5-C.sub.10)heterocycloalkyl are optionally substituted by at least one substituent selected from OH and O; EWG is chosen selected from the group consisting of: NO.sub.2, CHO, COR, CN, CNOH, CONHR, CONRR and COOR; R and R being independently from each other chosen from (C.sub.1-C.sub.10)alkyl groups; R.sub.5 is a (C.sub.6-C.sub.10)aryl, a (C.sub.5-C.sub.10)heteroaryl group, a (C.sub.3-C.sub.10)cycloalkyl or a (C.sub.3-C.sub.10)heterocycloalkyl group; said aryl and heteroaryl being optionally substituted by at least one substituent independently chosen from halogen, (C.sub.1-C.sub.10)alkoxyl and nitro; R.sub.5 being different from the group: ##STR00087## and wherein when EWG is COOMe, one of R.sub.1, R.sub.2, R.sub.3 or R.sub.4 is different from H; and provided that the compound of formula (I) is not: ##STR00088## or its pharmaceutically acceptable salts, hydrates or hydrated salts or its polymorphic crystalline structures, racemates, diastereomers or enantiomers to a patient in need thereof.

7. The method of claim 6, wherein EWG is NO.sub.2 or CHO.

8. The method of claim 6, wherein R.sub.1 is H.

9. The method of claim 6, wherein R.sub.2 is H, (C.sub.2-C.sub.10)alkynyl, Br, F, I, OH, O(C.sub.1-C.sub.10)alkylene-NHCO(C.sub.1-C.sub.10)alkylene-(C.sub.5-C.sub.10)heterocycloalkyl or O(C.sub.1-C.sub.10)alkylene-NHCSNHR with R being: ##STR00089## wherein: R and the (C.sub.5-C.sub.10)heterocycloalkyl group are optionally substituted by OH or O; or R.sub.2 forms with R.sub.3 together with the carbon atoms to which R.sub.2 and R.sub.3 are attached to form a (C.sub.6-C.sub.10)aryl.

10. The method of claim 6, wherein R.sub.3 is H, (C.sub.1-C.sub.10)alkoxyl or R.sub.3 forms with R.sub.2 together with the carbon atoms to which R.sub.3 and R.sub.2 are attached to form a (C.sub.6-C.sub.10)aryl.

11. The method of claim 6, wherein R.sub.4 is H, halogen or (C.sub.1-C.sub.10)alkoxyl.

12. The method of claim 6, wherein R.sub.5 is a possibly substituted phenyl or a tetrahydropyranyl group.

13. A compound having the following formula (i): ##STR00090## wherein X is O or S and Ri is selected from the group consisting of H, (C.sub.1-C.sub.10)alkyl, (C.sub.1-C.sub.10)alkoxyl, (C.sub.2-C.sub.10)alkenyl, (C.sub.2-C.sub.10)alkynyl, OH, a fluorine atom, a bromine atom and a iodine atom; or its pharmaceutically acceptable salts, hydrates or hydrated salts or its polymorphic crystalline structures, racemates, diastereomers or enantiomers.

14. The compound according to claim 13, where Ri is selected from the group consisting of H, a fluorine atom, a bromine atom or a iodine atom.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1 and FIG. 6 show the percentage of cell death of leukemic T-cells Jurkat, versus the concentration of some compounds of formula (I).

(2) FIGS. 2, 3, 4 and 5 show the percentage of cell death of leucemic T-cells CEM, versus the concentration of some compounds of formula (I).

(3) FIG. 7 and FIG. 8 show the PI3K activity, represented by the level of AKT phosphorylation, versus the time of incubation of CEM cells with compounds of formula (I) at a concentration of 10 M.

(4) FIG. 9 and FIG. 10 show the percentage of cell death of triple-negative breast cancer cells (respectively MDA-MB-468 and MDA-MB-231 cell lines), versus the concentration of some compounds of formula (I).

(5) FIG. 11 shows a part of the kinome perfomed by KinaseProfiler with compound 25 at 1 M. It shows the selectivity of compound 25 for the mTOR protein regarding 243 human kinases.

(6) FIG. 12 shows the binding of compound 48 to mTOR and its inhibitory activity. FIG. 12A shows the inhibition of the phosphorylation of AKT at serine 473 and of 4EBP1 at threonine 37/46. FIG. 12B shows the binding of compound 48 to the protein mTOR.

(7) FIG. 13 shows the in vivo tolerance of compound 25 in mice. FIG. 13A shows the body weight of the tested mice in grams versus the time in days. FIG. 13B shows the percentage of survival of the tested mice versus the time in days.

(8) FIG. 14 shows the cytotoxic activity of the compounds of the invention on TSC2.sup./ and TSC2.sup.+/+ cells. FIG. 14A shows the cell number of TSC2.sup./ (white circles) and TSC2.sup.+/+ (black circles) obtained in culture over time (in hours). FIG. 14B shows the percentage of cell death (metabolic activity) of TSC2.sup./ (white circles) and TSC2.sup.+/+ (black circles) versus the concentration of compound 25 in M. FIG. 14C shows the percentage of cell death (metabolic activity) of TSC2.sup./ (white circles) and TSC2.sup.+/+ (black circles) versus the concentration of rapamycin in M.

(9) FIG. 15 shows the results of an immunoblot demonstrating that compound 25 inhibits mTORC1 activity in TSC2.sup./ cells.

(10) FIG. 16 shows the results of an immunoblot demonstrating that compound 25 inhibits mTORC1 and mTORC2 activities.

(11) FIG. 17 shows that compound 25 inhibits cell migration of MDA-MB-231 cells (FIG. 17B) and of BT549 cells (FIG. 17A).

(12) The following examples show the improved PI3K/AKT/mTOR inhibition and the increased cytotoxic effect of the compounds of formula (I). Some preparative examples are also given below, without limitation of the present invention.

DETAILED DESCRIPTION

Preparative Examples

1. Preparation of Compounds 1 to 40

(13) General Procedure to Prepare Compounds of Formula (I) Wherein EWG is NO.sub.2:

(14) ##STR00060##

(15) The mixture of nitrostyrene derivative (1 mmol), salicylaldehyde compound (1 mmol) and pipecolic acid (0.2 mmol) in 1.5 mL of dry toluene was heated at 80-100 C. for 24-72 h under nitrogen atmosphere (conversion followed by TLC). After cooling to the room temperature, the mixture was charged directly on the silica gel column for the separation to give desired chromene (yields=40-75%). Room temperature is comprised between 18 C. and 25 C.

(16) R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5 and X are defined above.

(17) General Procedure to Prepare Compounds of Formula (I) Wherein EWG is CHO:

(18) ##STR00061##

(19) The mixture of cinnamaldehyde derivative (1 mmol), salicylaldehyde compound (1 mmol) and 1,1,3,3-Tetramethylguanidine (0.2 mmol) in 1.5 mL of dry toluene was heated at 80-100 C. for 48-72 h under nitrogen atmosphere (conversion followed by TLC). After cooling to room temperature, the mixture was charged directly on the silica gel column for the separation to give desired chromene (yields=30-55%). Room temperature is comprised between 18 C. and 25 C.

(20) R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5 and X are defined above. The starting products used were obtained as follows:

(21) TABLE-US-00001 Corresponding com- Compounds of formula (III) pounds of formula (I) 1. marketed by Aldrich CAS [148-53-8] 13 to 16 and 35. marketed by Aldrich CAS [492-88-6] 2, 25 to 27 and 39. marketed by Aldrich CAS [90-59-5] 3. marketed by Aldrich CAS [673-22-3] 6, 22 to 24 and 40. marketed by Aldrich CAS [5034-74-2] 7, 9 to 11, 33, 34 and 36. marketed by Aldrich CAS [1761-61-1] 8, 18 to 20 and 37. marketed by Aldrich CAS [1829-34-1] 5. prepared according to B. Legoin et al., Eur. J. Org. Chem. 2010, 5503-5508. 0 32. prepared according to Y. Xu et al., Chem. Eur. J., 2010, 16, 12898-12903. Corresponding com- Compounds of formula (II) pounds of formula (I) 1 to 3 and 5 to 8. marketed by Aldrich CAS [706-08-1] 9, 13, 18, 22, 25 and 32. marketed by Aldrich CAS [5153-71-9] 10, 14, 19, 23 and 26. marketed by Aldrich CAS [706-07-0] 11, 15, 20, 24 and 27. prepared according to J. A. Burkhard et al., Angew. Chem. Int. Ed. 2011, 50, 5379- 5382. 33, 35. marketed by Aldrich CAS [104-55-2] 34. marketed by Aldrich CAS [51791-26-5] 16. marketed by Aldrich CAS [3156-34-1] 36, 37, 39 and 40. prepared according to D. A. DiRocco, T. Rovis, J. Am. Chem. Soc., 2011, 133, 10402-10405.

2. Preparation of Compounds 41 to 63

Preparation of Compounds 50 and 51

Dimethylthiocarbamic Acid O-(4-Bromo-2-Formylphenyl) Ester

(22) ##STR00079##

(23) To a solution of 5-bromosalicylaldehyde (5.0 g, 25.0 mmol) in dry acetonitrile (25 mL) was added potassium carbonate (13.75 g, 100.0 mmol, 4.0 eq.) at room temperature. After 10 minutes dimethylthiocarbamoyl chloride (3.7 g, 30.0 mmol, 1.2 eq.) was added and the mixture was heated at reflux for 4 h. Then the reaction mixture was then cooled to room temperature, diluted with EtOAc and washed with a saturated aqueous NaHCO.sub.3 solution. The combined organic layers were washed with brine, dried over MgSO.sub.4, filtered and the filtrate concentrated under reduced pressure. The title compound was purified by column chromatography on silica gel using PE/EtOAc 9/1 to 7/3 as eluent affording a yellow solid (5.5 g, 77%). Mp=144-146 C. .sup.1H-NMR (CDCl.sub.3, 500 MHz) 3.42 (s, 3H), 3.47 (s, 3H), 7.03 (d, J=8.5, 1H), 7.72 (dd, J=8.5, 2.2, 1H), 8.02 (d, J=2.2, 1H), 9.99 (s, 1H). .sup.13C-NMR (CDCl.sub.3, 125 MHz) 39.0, 43.5, 119.9, 126.3, 130.5, 132.1, 137.6, 154.3, 186.7, 186.7.

Dimethylthiocarbamic acid S-(4-bromo-2-formylphenyl) ester

(24) ##STR00080##

(25) Dimethylthiocarbamic acid O-(4-bromo-2-formylphenyl) ester (5.4 g, 18.7 mmol) was heated neat at 150 C. for 15 h then cooled to room temperature. The title compound was purified by column chromatography on silica gel using PhMe/Et.sub.2O 95/5 to 90/10 as eluent affording a yellow solid (1.1 g, 20%). Mp=116-118 C. .sup.1H-NMR (CDCl.sub.3, 300 MHz) 3.03 (s, 3H), 3.16 (s, 3H), 7.42 (d, J=8.2, 1H), 7.70 (dd, J=8.2, 2.4, 1H), 8.14 (d, J=2.4, 1H), 10.25 (s, 1H). .sup.13C-NMR (CDCl.sub.3, 75 MHz) 37.1, 37.3, 124.9, 131.1, 131.6, 136.5, 138.7, 138.9, 164.6, 189.8.

6-Bromo-2-(4-bromophenyl)-3-nitro-2H-thiochromene (50)

(26) To a solution of dimethylthiocarbamic acid S-(4-bromo-2-formylphenyl) ester (200 mg, 0.69 mmol) in methanol (4.3 mL) was added an aqueous NaOH solution (0.8 M, 4.3 mL, 3.45 mmol, 5 eq.) at room temperature. After 2 h, a 10% (w/v) aqueous citric acid solution was added followed by water and the mixture was extracted with EtOAc. The combined organic layers were washed with brine, dried over MgSO.sub.4, filtered and the filtrate concentrated under reduced pressure. The resulting crude thiophenol was dissolved in dry toluene and 1-bromo-4-(2-nitrovinyl)benzene (158 mg, 1 eq., 0.69 mmol) was added followed by pipecolic acid (45 mg, 0.5 eq., 0.35 mmol). The reaction mixture was heated at 100 C. for 12 h then cooled to room temperature. The solvent was removed under reduce pressure and the title compound was purified by column chromatography on silica gel using PE/PhMe 4/1 as eluent affording a yellow solid (115 mg, 39%).

Dimethylthiocarbamic acid O-(2,4-dibromo-6-formylphenyl) ester

(27) ##STR00081##

(28) To a solution of 3,5-dibromosalicylaldehyde (1.2 g, 4.3 mmol) in dry tetrahydrofurane (10 mL) was added sodium hydride (60% in oil, 0.19 g, 4.7 mmol, 1.1 eq.) at 0 C. After 15 minutes at 0 C. and 1 h at room temperature a solution of dimethylthiocarbamoyl chloride (0.65 g, 5.2 mmol, 1.2 eq.) in dry tetrahydrofurane (2 mL) was added at 0 C. and the mixture was stirred at room temperature for 12 h. Then a saturated aqueous NH.sub.4Cl solution was added and the mixture was extracted with EtOAc. The combined organic layers were washed with brine, dried over MgSO.sub.4, filtered and the filtrate concentrated under reduced pressure. The title compound was purified by column chromatography on silica gel using PE/Et.sub.2O 9/1 to 7/3 as eluent affording a yellow solid (0.4 g, 25%). .sup.1H-NMR (CDCl.sub.3, 500 MHz) 3.48 (s, 3H), 3.49 (s, 3H), 7.96 (d, J=2.4, 1H), 7.99 (d, J=2.4, 1H), 9.94 (s, 1H). .sup.13C-NMR (CDCl.sub.3, 125 MHz) 39.2, 43.8, 119.6, 120.2, 130.8, 132.3, 140.4, 151.9, 185.1, 186.2.

Dimethylthiocarbamic acid S-(2,4-dibromo-6-formylphenyl) ester

(29) ##STR00082##

(30) Dimethylthiocarbamic acid O-(2,4-dibromo-6-formylphenyl) ester (0.26 g, 0.71 mmol) was heated neat at 150 C. for 15 h then cooled to room temperature. The title compound was purified by column chromatography on silica gel using PhMe/Et.sub.2O 9/1 as eluent affording a yellow solid (0.17 g, 53%). Mp=134-136 C. .sup.1H-NMR (CDCl.sub.3, 500 MHz) 3.04 (s, 3H), 3.22 (s, 3H), 8.06 (d, J=2.2, 1H), 8.09 (d, J=2.2, 1H), 10.25 (s, 1H). .sup.13C-NMR (CDCl.sub.3, 125 MHz) 37.3, 37.4, 125.2, 130.7, 132.6, 133.0, 140.0, 140.9, 163.1, 189.7.

6,8-Dibromo-2-(4-bromophenyl)-3-nitro-2H-thiochromene (51)

(31) To a solution of dimethylthiocarbamic acid S-(2,4-dibromo-6-formylphenyl) ester (160 mg, 0.43 mmol) in methanol (2.7 mL) was added an aqueous NaOH solution (0.8 M, 2.7 mL, 2.15 mmol, 5 eq.) at room temperature. After 2 h, a 10% (w/v) aqueous citric acid solution was added followed by water and the mixture was extracted with EtOAc. The combined organic layers were washed with brine, dried over MgSO.sub.4, filtered and the filtrate concentrated under reduced pressure. The resulting crude thiophenol was dissolved in dry toluene and 1-bromo-4-(2-nitrovinyl)benzene (98 mg, 1 eq., 0.43 mmol) was added followed by pipecolic acid (28 mg, 0.5 eq., 0.22 mmol). The reaction mixture was heated at 100 C. for 12 h then cooled to room temperature. The solvent was removed under reduce pressure and the title compound was purified by column chromatography on silica gel using PE/PhMe 4/1 as eluent followed by a preparative thin layer chromatography using PE/PhMe 3/2 as eluent affording a yellow solid (40 mg, 19%).

Preparation of Compounds 44 and 52

(32) To a solution of salicylaldehyde derivative was placed in dry toluene and 1-bromo-4-(2-nitrovinyl)benzene (1.1 eq.) was added followed by tetramethylguanidine (0.5 eq.) and benzoic acid (0.5 eq.). The reaction mixture was heated at 100 C. for 16 h then cooled to room temperature. The solvent was removed under reduce pressure and the title compound was purified by column chromatography on silica gel using PhMe/Et.sub.2O 7/3 as eluant affording a yellow solid.

Preparation of Compounds 55 and 56

(33) To a solution of carboxylic acid 53 or 54 in methanol, thionyl chloride (1.6 eq.) was added. The reaction mixture was heated at reflux for 20 h then cooled to room temperature. The solvent was removed under reduce pressure and the title compound was purified by column chromatography on silica gel using PhMe/PE 3/1 as eluant.

Preparation of Compounds 57 and 58

(34) To a solution of carboxylic acid 53 or 54 in methylene chloride in the presence of one drop of DMF, oxalyle chloride (1.15 eq.) was added. The reaction mixture was stirred 20 h at room temperature and Me.sub.2NH.HCl (2 eq.) and Et.sub.3N (5 eq.) were added. The reaction mixture was stirred 4 h at room temperature and after addition of HCl 1M, the aqueous phase was extracted with EtOAc. The combined organic layers were washed with brine, dried over MgSO.sub.4, filtered and the filtrate concentrated under reduced pressure. The title compound was purified by column chromatography on silica gel using CH.sub.2Cl.sub.2/AcOEt 8/2 as eluant.

Preparation of Compounds 59 and 60

(35) To a solution of aldehyde 44 or 52 in methanol, were successively added hydroxylamine chlorhydrate (5 eq.) and triethylamine (5 eq.). The reaction mixture was stirred 16 h at room temperature. After addition of HCl 1M the solution was extracted with EtOAc. The combined organic layers were washed with brine, dried over MgSO.sub.4, filtered and the filtrate concentrated under reduced pressure. The title compound was purified by column chromatography on silica gel using PhMe/PE 7/3 as eluant.

Preparation of Cyano 61 and 62

(36) A solution of aldehyde 44 or 52 and NH.sub.2OH.HCl (1.5 eq.) in DMSO was stirred at 100 C. for 20 h. After addition of water, the solution was extracted with Et.sub.2O. The combined organic layers were washed with brine, dried over MgSO.sub.4, filtered and the filtrate concentrated under reduced pressure. The title compound was purified by column chromatography on silica gel using PE/AcOEt 85/15 as eluant.

(37) Analytic Data:

8-ethoxy-2-(4-fluoropheneyl)-3-nitro-3,4-dihydro-2H-chromene (compound A)

(38) .sup.1H NMR (CDCl.sub.3, 500 MHz): (ppm)=1.36 (s, 3H, J=7.1 Hz), 3.97-4.08 (m, 2H, J=7.1 Hz), 6.64 (s, 1H), 6.93-6.99 (m, 5H), 7.37 (d, 1H; J=5.2 Hz), 7.38 (d, 1H, J=5.2 Hz), 8.03 (s, 1H).

(39) .sup.13C NMR (CDCl.sub.3, 125 MHz): (ppm)=164.2, 162.2, 148.0, 143.0, 141.3, 132.6, 129.5, 128.9, 128.8, 122.6, 122.2, 118.9, 118.7, 115.8, 115.6, 65.2, 14.7.

2-(4-fluorophenyl)-8-methoxy-3-nitro-3,4-dihydro-2H-chromene (1)

(40) .sup.1H NMR (CDCl.sub.3, 500 MHz): (ppm)=4.11 (s, 3H), 6.93 (s, 1H), 7.24-7; 30 (m, 5H), 7.66 (d, 2H), 7.69 (d, 1H), 8.33 (s, 1H).

(41) .sup.13C NMR (CDCl.sub.3, 125 MHz): (ppm)=164.3, 162.3, 148.7, 142.5, 141.3, 132.6, 129.3, 129.0, 128.9, 122.6, 122.1, 118.6, 115.9, 115.7, 56.3.

6,8-dibromo-2-(4-fluorophenyl)-3-nitro-3,4-dihydro-2H-chromene (2)

(42) .sup.1H NMR (CDCl.sub.3, 500 MHz) (ppm)=6.68 (s, 1H), 7.02 (m, 2H), 7.34 (d, 1H), 7.36 (d, 1H), 7.40 (s, 1H), 7.65 (s, 1H), 7.94 (s, 1H).

(43) .sup.13C NMR (CDCl.sub.3, 125 MHz) (ppm)=164.4, 162.4, 149.3, 142.5, 139.1, 131.6, 128.9, 128.8, 127.4, 120.6, 116.9, 116.2, 116.0, 114.8, 112.4.

2-(4-fluorophenyl)-7-methoxy-3-nitro-3,4-dihydro-2H-chromene (3)

(44) .sup.1H NMR (CDCl.sub.3, 500 MHz) (ppm)=6.39 (d, 1H), 6.53 (s, 1H), 6.57 (dd, 1H), 7.0 (m, 2H), 7.24 (d, 1H), 7.34-7.36 (m, 2H), 8.04 (s, 1H).

(45) .sup.13C NMR (CDCl.sub.3, 125 MHz) (ppm)=165.2, 164.2, 162.3, 155.4, 138.2, 133.0, 131.8, 129.9, 129.0, 115.9, 115.8, 110.0, 109.9, 102.3, 55.7.

2-(4-fluorophenyl)-3-nitro-3,4-dihydro-2H-benzo[g]chromene (5)

(46) .sup.1H NMR (CDCl.sub.3, 300 MHz) (ppm)=6.49 (s, 1H), 6.90-6.97 (m, 4H), 7.23-7.31 (m, 5H), 7.99 (s, 1H).

(47) .sup.13C NMR (CDCl.sub.3, 75 MHz) (ppm)=164.9, 161.6, 153.3, 141.0, 134.5, 132.8, 132.7, 130.5, 129.4, 129.1, 128.9, 128.5, 127.1, 122.7, 117.8, 117.3, 116.0, 115.7.

6-bromo-2-(4-fluorophenyl)-8-methoxy-3-nitro-3,4-dihydro-2H-chromene (6)

(48) .sup.1H NMR (CDCl.sub.3, 300 MHz) (ppm)=3.76 (s, 3H), 6.59 (s, 1H), 6.94-7.03 (m, 4H), 7.49 (d, 1H), 7.53 (d, 1H), 7.92 (s, 1H).

6-bromo-2-(4-fluorophenyl)-3-nitro-3,4-dihydro-2H-chromene (7)

(49) .sup.1H NMR (CDCl.sub.3, 300 MHz) (ppm)=6.54 (s, 1H), 6.75 (d, 1H, J=8.6 Hz), 7.00 (t, 2H, J=8.6 Hz), 7.32 (d, 1H, J=5.2 Hz), 7.35 (d, 1H, J=5.2 Hz), 7.45 (d, 1H, J=2.3 Hz), 7.96 (s, 1H).

8-bromo-2-(4-fluorophenyl)-3-nitro-3,4-dihydro-2H-chromene (8)

(50) .sup.1H NMR (CDCl.sub.3, 300 MHz) (ppm)=6.60 (s, 1H), 6.81 (t, 1H), 6.90 (t, 2H), 7.20 (dd, 1H), 7.27-7.30 (m, 2H), 7.44 (dd, 1H), 7.94 (s, 1H).

6-bromo-2-(4-bromo-phenyl)-3-nitro-2H-chromene (9)

(51) .sup.1H NMR (CDCl.sub.3, 300 MHz) (ppm)=6.52 (s, 1H), 6.77 (d, 1H, J=8.6 Hz), 7.23 (d, 2H, J=8.6 Hz), 7.45 (m, 4H), 7.61 (dd, 1H, J=6.1 and 2.9 Hz), 7.97 (s, 1H).

(52) .sup.13C NMR (CDCl.sub.3, 75 MHz) (ppm)=73.8, 114.8, 119.1, 119.6, 124.0, 128.0, 128.7, 132.2, 132.5, 135.2, 136.9, 141.7, 152.2.

6-bromo-2-(4-chloro-phenyl)-3-nitro-2H-chromene (10)

(53) .sup.1H NMR (CDCl.sub.3, 300 MHz) (ppm)=6.54 (s, 1H), 6.77 (d, 1H, J=8.6 Hz) 7.29-7.32 (m, 4H), 7.39-7.46 (m, 2H), 7.97 (s, 1H).

(54) .sup.13C NMR (CDCl.sub.3, 75 MHz) (ppm)=73.7, 114.7, 119.0, 119.5, 127.9, 128.3, 128.4, 129.2, 129.8, 130.2, 132.4, 134.7, 135.7, 136.8, 137.6, 141.6, 152.0.

6-bromo-2-(4-nitro-phenyl)-3-nitro-2H-chromene (11)

(55) .sup.1H NMR (CDCl.sub.3, 300 MHz) (ppm)=6.66 (s, 1H), 6.81 (d, 1H, J=8.6 Hz), 7.43-7.56 (m, 4H), 8.02 (s, 1H), 8.21 (m, 2H).

(56) .sup.13C NMR (CDCl.sub.3, 75 MHz) (ppm)=73.2, 115.2, 119.0, 119.3, 124.2, 128.0, 128.6, 132.7, 137.2, 141.0, 142.9, 148.5, 152.0.

2-(4-bromo-phenyl)-8-ethoxy-3-nitro-2H-chromene (13)

(57) .sup.1H NMR (CDCl.sub.3, 300 MHz) (ppm)=1.39 (t, 3H, J=6.9 Hz), 4.04 (q, 2H, J=7.1 Hz), 6.62 (s, 1H), 6.69 (m, 3H), 7.28 (m, 2H), 7.44 (dd, 2H, J=8.4 and 1.6 Hz), 8.03 (s, 1H).

(58) .sup.13C NMR (CDCl.sub.3, 75 MHz) (ppm)=14.7, 65.0, 73.2, 118.5, 118.8, 122.1, 122.7, 123.4, 128.5, 129.6, 131.8, 135.7, 141.0, 142.8, 147.9.

2-(4-chloro-phenyl)-8-ethoxy-3-nitro-2H-chromene (14)

(59) .sup.1H NMR (CDCl.sub.3, 300 MHz) (ppm)=1.38 (t, 3H, J=7 Hz), 3.97-4.08 (m, 2H), 6.63 (s, 1H), 6.91-6.96 (m, 3H), 7.25-7.34 (m, 4H), 8.03 (s, 1H).

(60) .sup.13C NMR (CDCl.sub.3, 75 MHz) (ppm)=14.7, 65.0, 73.1, 118.6, 118.9, 122.2, 122.6, 128.3, 129.0, 129.6, 135.3, 141.1, 142.9, 148.0.

2-(4-nitro-phenyl)-8-ethoxy-3-nitro-2H-chromene (15)

(61) .sup.1H NMR (CDCl.sub.3, 300 MHz) (ppm)=1.41 (t, 3H, J=7.0 Hz), 4.06 (m, 2H), 6.74 (s, 1H), 6.95 (m, 3H), 7.58 (d, 2H, J=8.5 Hz), 8.07 (s, 1H), 8.15 (d, 2H, J=8.9 Hz).

(62) .sup.13C NMR (CDCl.sub.3, 75 MHz) (ppm)=14.7, 64.9, 72.6, 118.4, 118.6, 122.2, 123.1, 123.9, 127.7, 130.1, 140.5, 142.4, 143.7, 147.9, 148.28.

2-(2-chloro-phenyl)-8-ethoxy-3-nitro-2H-chromene (16)

(63) .sup.1H NMR (CDCl.sub.3, 300 MHz) (ppm)=1.29 (t, 3H, J=7.0 Hz), 3.94 (q, 2H, J=3.6 Hz), 7.00 (m, 3H), 7.11-7.27 (m, 3H), 7.28 (m, 2H), 7.49 (dd, 1H, J=1.0, 7.9 Hz), 8.17 (s, 1H).

(64) .sup.13C NMR (CDCl.sub.3, 75 MHz) (ppm)=14.7, 65.8, 70.8, 118.9, 120.6, 122.5, 122.6, 127.0, 128.0, 130.5, 130.6, 130.9, 133.0, 134.5, 140.1, 143.2, 148.14.

8-bromo-2-(4-bromo-phenyl)-3-nitro-2H-chromene (18)

(65) .sup.1H NMR (CDCl.sub.3, 300 MHz) (ppm)=6.67 (s, 1H), 6.92 (t, 1H, J=7.9 Hz), 7.29 (m, 2H), 7.46 (m, 2H), 7.45-7.61 (m, 2H), 8.03 (s, 1H).

(66) .sup.13C NMR (CDCl.sub.3, 75 MHz) (ppm)=72.8, 111.5, 119.4, 123.7, 123.8, 128.5, 128.9, 129.6, 132.1, 135.2, 137.5, 141.5, 150.1.

8-bromo-2-(4-chloro-phenyl)-3-nitro-2H-chromene (19)

(67) .sup.1H NMR (CDCl.sub.3, 300 MHz) (ppm)=6.69 (s, 1H), 6.93 (t, 1H, J=7.7 Hz), 7.28-7.35 (m, 5H), 7.56 (dd, 1H, J=6.5 and 1.5 Hz), 8.03 (s, 1H).

(68) .sup.13C NMR (CDCl.sub.3, 75 MHz) (ppm)=73.7, 111.4, 119.3, 123.6, 128.2, 128.8, 129.0, 129.50, 134.6, 135.5, 137.5, 141.5, 150.0.

8-bromo-2-(4-nitro-phenyl)-3-nitro-2H-chromene (20)

(69) .sup.1H NMR (CDCl.sub.3, 300 MHz) (ppm)=6.80 (s, 1H), 6.94 (t, 1H, J=7.0 Hz), 7.31 (dd, 1H, J=7.0 and 1.0 Hz), 7.59 (m, 3H), 8.07 (s, 1H), 8.20 (d, 2H, J=7.0 Hz).

(70) .sup.13C NMR (CDCl.sub.3, 75 MHz) (ppm)=73.3, 111.5, 119.2, 124.1, 124.5, 127.8, 129.4, 129.7, 129.8, 136.0, 137.8, 143.0, 149.9.

6-bromo-2-(4-bromo-phenyl)-8-methoxy-3-nitro-2H-chromene (22)

(71) .sup.1H NMR (CDCl.sub.3, 300 MHz) (ppm)=3.82 (s, 3H), 6.60 (s, 1H), 7.04 (d, 1H, J=2.1 Hz), 7.08 (d, 1H, J=2.1 Hz), 7.24 (td, 2H, J=2.2 and 8.3 Hz), 7.47 (td, 2H, J=2.2 and 8.6 Hz), 7.95 (s, 1H).

(72) .sup.13C NMR (CDCl.sub.3, 75 MHz) (ppm)=56.5, 73.6, 111.4, 119.7, 119.5, 123.9, 128.0, 128.5, 129.5, 132.1, 135.1, 141.7, 149.3.

6-Bromo-2-(4-chloro-phenyl)-8-methoxy-3-nitro-2H-chromene (23)

(73) .sup.1H NMR (CDCl.sub.3, 300 MHz) (ppm)=3.82 (s, 3H), 6.60 (s, 1H), 7.08 (dd, 2H, J=13.1 and 2.0 Hz); 7.3 (s, 4H), 7.95 (s, 1H).

6-Bromo-2-(4-nitro-phenyl)-8-methoxy-3-nitro-2H-chromene (24)

(74) .sup.1H NMR (CDCl.sub.3, 300 MHz) (ppm)=3.86 (s, 3H) 6.75 (s, 1H), 7.07 (d, 1H, J=2.1 Hz), 7.11 (d, 1H, J=2.1 Hz), 7.55 (td, 2H, J=2.0 and 8.5 Hz), 7.99 (s, 1H), 8.19 (td, 2H J=2.0 and 8.9 Hz).

6,8-dibromo-2-(4-bromo-phenyl)-3-nitro-2H-chromene (25)

(75) .sup.1H NMR (CDCl.sub.3, 300 MHz) (ppm)=6.67 (s, 1H), 7.24 (m, 2H), 7.40 (d, 1H, J=2.2 Hz), 7.48 (m, 2H, J=8.5 and 1.95 Hz), 7.67 (d, 1H, J=2.2 Hz), 7.95 (s, 1H).

(76) .sup.13C NMR (CDCl.sub.3, 75 MHz) (ppm)=73.9, 112.4, 114.8, 120.6, 124.1, 127.5, 128.4, 129.5, 121.5, 132.2, 132.7, 134.6, 139.1, 142.2, 149.2.

6,8-dibromo-2-(4-chloro-phenyl)-3-nitro-2H-chromene (26)

(77) .sup.1H NMR (CDCl.sub.3, 300 MHz) (ppm)=6.68 (s, 1H), 7.41 (d, 1H, J=2.0 Hz), 7.43 (d, 2H, J=8.0 Hz), 7.49 (m, 2H, J=8.0 Hz), 7.67 (d, 1H, J=2.0 Hz), 7.98 (s, 1H).

6,8-dibromo-2-(4-nitro-phenyl)-3-nitro-2H-chromene (27)

(78) .sup.1H NMR (CDCl.sub.3, 300 MHz) (ppm)=8.28 (d, 2H, J=8.3 Hz), 8.00 (s, 1H), 7.70 (dd, 1H, J=1.0 and 2.2 Hz), 7.58 (td, 2H, J=2.0 and 8.5 Hz), 7.44 (d, 1H, J=2.2 Hz), 6.80 (s, 1H).

(79) .sup.13C NMR (CDCl.sub.3, 75 MHz) (ppm)=73.4, 112.4, 115.3, 120.3, 124.2, 127.8, 128.1, 131.8, 139.5, 142.4, 148.98.

2-(4-bromo-phenyl)-6-ethynyl-3-nitro-2H-chromene (32)

(80) .sup.1H NMR (CDCl.sub.3, 300 MHz) (ppm)=3.06 (s, 1H), 6.54 (s, 1H), 6.82 (d, 1H, J=6.0 Hz), 7.23 (m, 2H), 7.45 (m, 4H), 8.00 (s, 1H).

(81) .sup.13C NMR (CDCl.sub.3, 75 MHz) (ppm)=155.4, 141.3, 137.9, 135.4, 133.9, 132.1, 128.7, 128.5, 124.0, 117.8, 117.5, 116.7, 81.8, 73.9.

6-bromo-2-phenyl-2H-chromene-3-carbaldehyde (33)

(82) .sup.1H NMR (CDCl.sub.3, 300 MHz) (ppm)=6.32 (s, 1H), 6.76 (d, 1H, J=8.2 Hz), 7.27-7.32 (m, 5H), 7.34-7.38 (m, 3H), 9.65 (s, 1H).

6-bromo-2-(4-fluoro-phenyl)-2H-chromene-3-carbaldehyde (34)

(83) .sup.1H NMR (CDCl.sub.3, 300 MHz) (ppm)=6.29 (s, 1H), 6.76 (d, 1H, J=8.8 Hz), 6.97 (m, 2H), 7.27-7.32 (m, 3H), 7.38 (m, 3H), 9.65 (s, 1H).

8-ethoxy-2-phenyl-2H-chromene-3-carbaldehyde (35)

(84) .sup.1H NMR (CDCl.sub.3, 300 MHz) (ppm)=1.37 (t, 3H, J=7.0 Hz), 4.04 (q, 2H, J=3.9) 6.43 (s, 1H) 6.86 (m, 2H), 6.92 (m, 1H, J=5.3 Hz), 7.25 (m, 3H), 7.35 (m, 2H), 7.38 (s, 1H); 9.67 (s, 1H).

6-bromo-3-nitro-2-(tetrahydro-pyran-4-yl)-2H-chromene (36)

(85) .sup.1H NMR (CDCl.sub.3, 300 MHz) (ppm)=1.45 (m, 2H), 1.68 (m, 2H), 2.07 (m, 1H); 3.27 (m, 2H), 3.95 (m, 2H); 5.48 (d, 1H, J=6.4 Hz), 6.85 (d, 1H, J=8.6 Hz), 7.40 (d, 1H, J=2.3 Hz), 7.45 (dd, 1H, J=8.6 and 2.3 Hz), 7.79 (s, 1H).

8-bromo-3-nitro-2-(tetrahydro-pyran-4-yl)-2H-chromene (37)

(86) .sup.1H NMR (CDCl.sub.3, 300 MHz) (ppm)=1.45 (m, 2H); 1.83 (m, 2H), 2.07 (m, 1H), 3.28 (m, 2H), 3.97 (m, 2H), 5.60 (d, 1H, J=6.7 Hz), 6.91 (t, 1H, J=7.7 Hz), 7.24 (dd, 1H, J=6.2 and 1.4 Hz), 7.58 (dd, 1H, J=6.5 and 1.5 Hz), 7.86 (s, 1H).

6,8-dibromo-3-nitro-2-(tetrahydro-pyran-4-yl)-2H-chromene (39)

(87) .sup.1H NMR (CDCl.sub.3, 300 MHz) (ppm)=1.43 (m, 2H), 1.76 (m, 2H), 2.05 (m, 1H, H-5), 3.28 (m, 2H), 3.98 (m, 2H), 5.59 (d, 1H, J=6.6 Hz), 7.37 (d, 1H, J=2.2 Hz), 7.71 (d, 1H, J=2.2 Hz), 7.78 (s, 1H).

6-bromo-8-methoxy-3-nitro-2-(tetrahydro-pyran-4-yl)-2H-chromene (40)

(88) .sup.1H NMR (CDCl.sub.3, 300 MHz) (ppm)=1.49 (m, 2H), 1.72 (m, 2H), 2.04 (m, 1H), 3.26 (m, 2H), 3.89 (s, 3H); 3.98 (m, 2H), 5.53 (d, 1H, J=6.86 Hz), 7.04 (d, 1H, J=2.1 Hz), 7.07 (d, 1H, J=2.1 Hz), 7.78 (s, 1H).

6-Bromo-2-(4-bromophenyl)-2H-chromene-3-carbaldehyde (44)

(89) Mp=144-146 C. .sup.1H-NMR (CDCl.sub.3, 300 MHz) 6.27 (s, 1H), 6.76 (d, J=8.5, 1H), 7.19 (d, J=8.5, 2H), 7.35-7.40 (m, 4H), 7.43 (s, 1H), 9.66 (s, 1H). .sup.13C-NMR (CDCl.sub.3, 75 MHz) 73.7, 113.9, 119.0, 121.5, 123.0, 128.5, 131.5, 131.8, 134.0, 136.2, 137.4, 139.2, 153.4, 189.6.

2-(4-Bromo-phenyl)-3-nitro-2H-chromen-6-ol (45)

MMJ

(90) .sup.1H-NMR (300 MHz, CDCl.sub.3) 6.47 (s, 1H), 6.71-6.86 (m, 2H), 6.80 (s, 1H), 7.22 (d, J=8.5, 2H), 7.44 (d, J=8.5, 2H), 7.97 (s, 1H). .sup.13C-NMR (75 MHz, CDCl.sub.3) 73.2, 115.6, 118.1, 118.4, 121.6, 123.5, 128.7, 129.6, 131.9, 135.6, 141.3, 146.6, 151.6.

Compound (47)

MJ/4/140-1

(91) .sup.1H NMR (500 MHz, CDCl.sub.3) 1.39-1.47 (m, 2H), 1.62-1.74 (m, 4H), 1.93-2.02 (m, 2H), 2.21 (t, J=7.4, 2H), 2.68 (d, J=12.8, 1H), 2.84-2.88 (dd, J=4.7, 12.8, 1H), 3.08-3.14 (m, 1H), 3.38-3.45 (m, 2H), 3.96 (t, J=6.0, 2H), 4.24-4.27 (m, 1H), 4.41-4.45 (m, 1H), 5.57 (bs, 1H), 6.44 (t, J=5.6, 1H), 6.47 (s, 1H), 6.53 (bs, 1H), 6.77 (d, J=8.6, 1H), 6.85 (t, J=2.7, 1H), 6.88 (dd, J=2.9, 8.6, 1H), 7.21 (d, J=8.4, 2H), 7.43 (d, J=8.4, 2H), 8.02 (s, 1H). .sup.13C NMR (125 MHz, CDCl.sub.3) 25.6, 28.0, 28.1, 29.1, 35.8, 36.9, 40.5, 55.6, 60.3, 61.8, 66.7, 73.3, 114.6, 114.6, 118.1, 118.4, 121.4, 121.4, 123.5, 128.7, 129.7, 131.9, 135.6, 141.2, 147.2, 154.0, 163.9, 173.6.

Compound (48)

MJ/4/188-2

(92) 1H NMR (500 MHz, CDCl3) 1.38-1.44 (m, 2H), 1.60-1.74 (m, 4H), 1.94-1.99 (m, 2H), 2.21 (t, J=7.2, 2H), 2.68 (d, J=12.7, 1H), 2.84-2.88 (m, 1H), 3.09-3.13 (m, 1H), 3.38-3.41 (m, 2H), 3.94 (t, J=5.8, 2H), 4.24-4.27 (m, 1H), 4.41-4.45 (m, 1H), 5.51 (bs, 1H), 6.41 (bs, 1H), 6.53 (bs, 1H), 6.60 (s, 1H), 6.84 (t, J=2.2, 1H), 7.11 (d, J=2.0, 1H), 7.25 (dd, J=2.2, 8.6, 2H), 7.44 (d, J=8.6, 2H), 8.02 (s, 1H). 13C NMR (125 MHz, CDCl3) 25.5, 27.9, 28.0, 29.0, 35.8, 36.5, 40.5, 55.5, 60.1, 61.6, 66.7, 73.4, 111.5, 114.5, 114.5, 119.5, 123.6, 123.7, 123.7, 128.4, 129.2, 129.2, 131.9, 135.0, 142.0, 142.0, 144.0, 154.0, 163.9, 173.4.

Compound (49)

MJ/4/191-1

(93) .sup.1H NMR (500 MHz, CDCl.sub.3) 1.37-1.47 (m, 4H), 1.57-1.63 (m, 2H), 1.69-1.74 (m, 2H), 3.50-3.53 (m, 2H), 3.95 (t, J=6.2, 2H), 6.56 (dd, J=2.2, 8.6, 2H), 6.60 (d, J=8.6, 2H), 6.67 (d, J=2.2, 2H), 6.72 (s, 1H), 7.17 (d, J=8.2, 1H), 7.26 (d, J=2.7, 1H), 7.28 (d, J=2.7, 1H), 7.34 (d, J=8.5, 2H), 7.57 (d, J=8.5, 2H), 7.71-7.74 (m, 1H), 8.07 (bs, 1H), 8.22 (s, 1H), 8.38 (s, 1H), 9.85 (bs, 1H), 10.11 (s, 2H). .sup.13C NMR (125 MHz, DMSO) 25.1, 26.0, 28.2, 28.4, 30.7, 35.7, 68.3, 72.8, 82.9, 102.1, 109.6, 110.2, 112.4, 115.8, 119.9, 122.7, 122.8, 128.9, 129.0, 129.7, 131.7, 135.3, 141.6, 142.8, 151.7, 153.8, 159.3, 162.2, 168.4.

6-Bromo-2-(4-bromophenyl)-3-nitro-2H-thiochromene (50)

(94) .sup.1H-NMR (CDCl.sub.3, 300 MHz) 5.50 (s, 1H), 7.05 (d, J=8.6, 2H), 7.13 (d, J=8.4, 1H), 7.36 (d, J=8.6, 2H), 7.44 (dd, J=8.4, 2.2, 1H), 7.61 (d, J=2.2, 1H), 8.13 (s, 1H). .sup.13C-NMR (CDCl.sub.3, 75 MHz) 39.2, 119.7, 122.7, 127.8, 128.7, 129.8, 130.6, 131.0, 132.1, 134.2, 135.1, 138.3, 144.0.

6,8-Dibromo-2-(4-bromophenyl)-3-nitro-2H-thiochromene (51)

(95) .sup.1H-NMR (CDCl.sub.3, 300 MHz) 5.59 (s, 1H), 7.06 (d, J=8.5, 2H), 7.38 (d, J=8.6, 2H), 7.57 (d, J=1.9, 1H), 7.71 (d, J=1.9, 1H), 8.10 (s, 1H). .sup.13C-NMR (CDCl.sub.3, 75 MHz) 40.1, 119.2, 122.1, 122.9, 127.9, 130.5, 130.8, 132.3, 133.1, 133.3, 138.1, 138.2, 144.1.

6,8-Dibromo-2-(4-bromophenyl)-2H-chromene-3-carbaldehyde (52)

(96) .sup.1H-NMR (CDCl.sub.3, 300 MHz) 6.42 (s, 1H), 7.19 (d, J=8.2, 2H), 7.33 (s, 1H), 7.34 (d, J=2.2, 1H), 7.42 (d, J=8.2, 2H), 7.65 (d, J=2.2, 1H), 9.72 (s, 1H). .sup.13C-NMR (CDCl.sub.3, 75 MHz) 74.1, 112.2, 114.0, 122.6, 123.1, 128.2, 130.6, 131.8, 134.8, 136.8, 138.5, 138.6, 150.4, 189.3.

6-bromo-2-(4-bromophenyl)-2H-chromene-3-carboxylic acid (53)

(97) Mp=208-210 C. .sup.1H-NMR (DMSO-d.sub.6, 300 MHz) 6.23 (s, 1H), 6.78 (d, J=8.6, 1H), 7.22-7.32 (m, 2H), 7.38 (dd, J=8.6, 2.5, 1H), 7.50-7.60 (m, 2H), 7.66 (d, J=2.5, 1H), 7.74 (s, 1H). .sup.13C-NMR (DMSO-d.sub.6, 75 MHz) 74.0, 112.9, 118.6, 122.1, 122.5, 125.9, 129.3, 131.1, 131.9, 131.6, 134.4, 137.7, 151.6, 165.3.

6,8-dibromo-2(4-bromophenyl)-2H-chromene-3-carboxylic acid (54)

(98) Mp=256-258 C. .sup.1H-NMR (DMSO-d.sub.6, 300 MHz) 7.71 (d, J=2.3, 1H), 6.37 (s, 1H), 7.29 (d, J=8.4, 2H), 7.56 (d, J=8.5, 2H), 7.71 (d, J=2.3, 1H), 7.73-7.78 (m, 2H). .sup.13C-NMR (DMSO-d.sub.6, 75 MHz) 75.1, 111.5, 113.8, 122.8, 124.1, 127.3, 129.6, 131.2, 131.5, 132.2, 136.8, 137.8, 149.1, 165.5.

Methyl-6-Bromo-2-(4-bromophenyl)-2H-chromene-3-carboxylate (55)

(99) Mp=118-120 C. .sup.1H-NMR (CDCl.sub.3, 300 MHz) 3.77 (s, 3H), 6.22 (s, 1H), 6.67 (d, J=7.9, 1H), 7.21 (d, J=8.5, 2H), 7.27-7.31 (m, 2H), 7.41 (d, J=8.5, 2H), 7.59 (s, 1H). .sup.13C-NMR (CDCl.sub.3, 75 MHz) 52.2, 74.7, 113.7, 118.7, 122.0, 123.1, 125.1, 128.9, 131.1, 131.8, 132.2, 134.9, 137.5, 152.3, 164.7.

Methyl 6,8-Dibromo-2-(4-bromophnyl)-2H-chromne-3-carboxylate (56)

(100) .sup.1H-NMR (CDCl.sub.3, 300 MHz) 3.81 (s, 3H), 6.36 (s, 1H), 7.21-7.31 (m, 3H), 7.38-7.48 (m, 2H), 7.55 (d, J=2.2, 1H), 7.57 (s, 1H). .sup.13C-NMR (CDCl.sub.3, 75 MHz) 52.4, 75.0, 111.9, 113.9, 123.1, 123.2, 126.1, 128.7, 130.2, 131.8, 131.9, 137.0, 137.3, 149.4, 164.5.

6-Bromo-2-(4-bromophenyl)-N,N-dimethyl-2H-chromene-3-carboxamide (57)

(101) .sup.1H-NMR (CDCl.sub.3, 500 MHz) 2.96 (s, 6H), 6.18 (s, 1H), 6.54 (s, 1H), 6.71 (d, J=8.6, 1H), 7.19 (d, J=2.4, 1H), 7.27 (d, J=1.3, 3H), 7.44 (d, J=8.4, 2H). .sup.13C-NMR (CDCl.sub.3, 125 MHz) 35.2, 38.5, 76.8, 113.5, 118.1, 121.8, 122.9, 123.2, 128.6, 130.0, 130.4, 131.8, 133.4, 137.9, 151.6, 167.8.

6,8-Bromo-2-(4-bromophenyl)-N,N-dimethyl-2H-chromene-3-carboxamide (58)

(102) .sup.1H-NMR (CDCl.sub.3, 500 MHz) 2.99 (s, 6H), 6.29 (s, 1H), 6.56 (s, 1H), 7.15 (d, J=2.2, 1H), 7.22-7.34 (m, 2H), 7.44 (d, J=8.5, 2H), 7.52 (d, J=2.2, 1H). .sup.13C-NMR (CDCl.sub.3, 125 MHz) 35.3, 38.5, 111.3, 113.7, 122.9, 123.0, 123.1, 128.4, 129.2, 131.3, 131.8, 136.0, 137.3, 148.6, 162.3, 167.5.

6-Bromo-2-(4-bromophenyl)-2H-chromene-3-carbaldehyde oxime (59)

(103) Mp=158-160 C. .sup.1H-NMR (DMSO-d.sub.6, 500 MHz) 6.28 (s, 1H), 6.74 (d, J=8.7, 1H), 7.09 (s, 1H), 7.29 (dd, J=8.5, 3.2, 3H), 7.47 (d, J=2.4, 1H), 7.49-7.57 (m, 2H), 7.97 (s, 1H), 11.44 (s, 1H). .sup.13C-NMR (DMSO-d.sub.6, 125 MHz) 74.3, 113.5, 119.0, 122.4, 124.3, 126.0, 130.0, 130.0, 130.2, 132.0, 133.0, 137.8, 147.6, 151.4.

6,8-Bromo-2-(4-bromophenyl)-2H-chromene-3-carbaldehyde oxime (60)

(104) Mp=214-216 C. .sup.1H-NMR (DMSO-d.sub.6, 500 MHz) 6.43 (s, 1H), 7.10 (s, 1H), 7.30 (d, J=8.4, 2H), 7.52 (dd, J=17.8, 5.4, 3H), 7.62 (d, J=2.3, 1H), 8.01 (s, 1H), 11.57 (s, 1H). .sup.13C-NMR (DMSO-d.sub.6, 125 MHz) 75.0, 111.4, 113.8, 122.6, 125.5, 125.5, 129.5, 129.9, 131.1, 132.0, 134.9, 137.4, 147.4, 148.3.

6-Bromo-2-(4-bromophenyl)-2H-chromene-3-carbonitrile (61)

(105) Mp=136-138 C. .sup.1H-NMR (CDCl.sub.3, 500 MHz) 5.88 (s, 1H), 6.75 (d, J=8.6, 1H), 7.23 (s, 1H), 7.27 (d, J=2.3, 1H), 7.31 (d, J=8.4, 2H), 7.37 (dd, J=8.7, 2.4, 1H), 7.50-7.56 (m, 2H). .sup.13C-NMR (CDCl.sub.3, 125 MHz) 75.6, 107.8, 114.5, 116.1, 118.8, 120.7, 124.0, 128.8, 130.6, 132.3, 135.7, 135.7, 137.1, 151.9.

6,8-Dibromo-2-(4-bromophenyl)-2H-chromene-3-carbonitrile (62)

(106) Mp=196-198 C. .sup.1H-NMR (CDCl.sub.3, 500 MHz) 6.01 (s, 1H), 7.23 (d, J=1.1, 2H), 7.33 (d, J=8.5, 2H), 7.54 (d, J=8.5, 2H), 7.63 (d, J=2.2, 1H). .sup.13C-NMR (CDCl.sub.3, 125 MHz) 75.8, 108.6, 112.1, 114.6, 115.8, 121.6, 124.1, 128.5, 129.8, 132.3, 135.2, 136.6, 138.3, 148.9.

8-Bromo-2-(4-bromo-phenyl)-3-nitro-2H-chromene-6-ol (63)

MJ/4/180-2

(107) .sup.1H NMR (500 MHz, CDCl.sub.3) 5.16 (bs, 1H), 6.61 (s, 1H), 6.77 (d, J=2.9, 1H), 7.08 (d, J=2.9, 1H), 7.24 (d, J=8.5, 2H), 7.45 (d, J=8.5, 2H), 8.05 (s, 1H). .sup.13C NMR (125 MHz, CDCl.sub.3) 73.5, 111.6, 115.5, 119.7, 123.7, 124.4, 128.4, 128.7, 132.0, 135.0, 142.3, 144.1, 151.0.

EXAMPLES

Example 1

Cytotoxic Effect of the Compounds of Formula (I)

(108) It has been shown that the Jurkat and CEM cell lines are addicted to the PI3K/AKT/mTOR signaling pathway to survive and to proliferate (Beneteau, M. et al. Localization of Fas/CD95 into the lipid rafts on down-modulation of the phosphatidylinositol 3-kinase signaling pathway. Molecular cancer research: MCR 6, 604-613, (2008); Pizon, M. et al. Actin-independent exclusion of CD95 by PI3K/AKT signalling: Implications for apoptosis. European journal of immunology 41, (2011)).

(109) Protocol:

(110) Leukemic T-cell lines Jurkat and CEM were incubated for 24 hours (see FIGS. 1, 2, 3, 4 and 5) or for 20 hours (see FIG. 6) with compounds of formula (I) and with compound A, at the indicated concentrations and cell death was assessed using the viability assay MTT, which estimates the metabolic state of the cell.

(111) Results:

(112) The results are shown in FIGS. 1, 2, 3, 4, 5 and 6. The compounds of formula (I) trigger a strong cell death signal in CEM and Jurkat cells as measured by MTT assay.

(113) In FIGS. 1 and 2, compound A shows a lower percentage of cell death than most of the compounds of formula (I).

(114) In FIGS. 3, 4 and 5, LY294002 shows a much lower cell death percentage than the compounds of formula (I).

(115) These results demonstrate the strong cytotoxic activity of the compounds of formula (I), in comparison with other PI3K/AKT/mTOR pathway inhibitors such as compound A and LY294002.

Example 2

PI3K/AKT/mTOR Pathway Inhibitory Activity of the Compounds of Formula (I)

(116) Protocol:

(117) The CEM cell was incubated with 10 M of each mentioned compound of formula (I) for the indicated times and then cells were lysed and 100 g of protein was loaded per line and resolved by SDS-PAGE. The levels of AKT phosphorylation (hallmark of PI3K activation) and total AKT (loading control) were analyzed by Western blot and the amount of AKT phosphorylation relative to the total amount of protein AKT was quantified by densitometric analysis, the intensity of each band was scanned and the level of AKT phosphorylation was reported to the amount of whole AKT and the percentage of phosphorylated-AKT was depicted.

(118) Results:

(119) As shown in FIGS. 7 and 8 the compounds of formula (I) are very strong inhibitors of the PI3K/AKT activity, in particular the compounds 9, 10, 13, 14, 18, 19 and 25 (see FIG. 8). The other tested compounds also show a strong inhibitory activity (see FIG. 7).

(120) It has to be noted that compound A, which has been described in prior art as PI3K inhibitor shows a very weak inhibitory activity on PI3K, even no inhibitory activity (see FIGS. 7 and 8). Moreover, as shown in FIG. 8, compounds of formula (I) show a stronger inhibitory activity on the PI3K/AKT/mTOR pathway than LY294002.

(121) The results confirm that the compounds of the invention strongly inhibit the PI3K/AKT/mTOR pathway and are more potent than other PI3K/AKT/mTOR pathway inhibitors such as compound A and LY294002, especially much more potent than compound A.

Example 3

In Cellulo IC50 of Compounds of Formula (I)

(122) Protocol:

(123) CEM cells were incubated for 2 hours with the indicated concentrations of each mentioned compound of formula (I). Cells were then lysed and 100 g of protein was loaded per line in an SDS-PAGE. Bands from AKT phosphorylation and whole AKT observed by Western blot were scanned and quantified by densitometric analysis. Based on these values, an in cellulo IC.sub.50 (half maximal inhibitory concentration) was measured for each compound of formula (I).

(124) The obtained IC.sub.50 of the tested compounds are mentioned below in Table 1:

(125) TABLE-US-00002 TABLE 1 Compound In vivo IC.sub.50 (M) 9 0.4 10 2 13 8 14 10 18 1.5 19 >10 25 3 LY294002 >10

(126) It is noteworthy that among the tested compounds, the most effective reagent designated compound 9, possesses an in cellulo IC.sub.50 at 0.4 M, which is much more efficient than the LY294002 IC.sub.50, which is higher than 10 M.

Example 4

Compounds of Formula (I) as Potent Inducers of Cell Death in Triple Negative Breast Cancer Cells

(127) Protocol:

(128) The Triple-negative tumor cell lines MDA-MB-231 and MDA-MB-468 were incubated with the indicated concentrations of each mentioned compound of formula (I) for 24 hours and then cell death was assessed using MTT assay.

(129) Results:

(130) In contrast to LY294002, the compounds of formula (I) implement a strong cell death signal in triple-negative breast tumor cells MDA-MB-231 and MDA-MB-468 (see FIGS. 9 and 10). These results confirm that compounds of formula (I) are of interest to prevent and/or treat breast cancers, especially triple-negative breast cancer.

Example 5

Compounds of Formula (I) as Potent Inducers of Cell Death in Non-Triple Negative Breast Cancer Cells

(131) Protocol:

(132) The ER-positive breast tumor cell lines MCF-7 and T47-D and the HER2 positive breast tumor cell lines MDA-MB-453 and BT474 (also ER-positive) were incubated with the indicated concentration of each mentioned compound of formula (I) for 24 hours and then cell death was assessed using MTT assay.

(133) Results:

(134) The results are shown in Tables below:

(135) For each cell line, the percentage of cell death (mean of four experiments for cell lines MCF-7 and T47-D, and two experiments for cell lines MDA-MB-453 and BT474) is given in view of the increasing concentration of each tested compound of the invention.

(136) TABLE-US-00003 Cell line MCF-7(ER-positive) percentage percentage percentage percentage of cell death of cell death of cell percentage of cell death with with death with percentage of of cell Concentration with compound compound compound cell death with death with (M) compound 9 10 19 25 compound 7 LY294002 50 80.16 81.85 84.65 94.09 95.55 18.89 25 52.18 49.08 52.14 76.72 79.7 13.51 12.5 32.14 24.06 25.75 63.54 38.7 11.067 6.25 0 0 0 47.86 10.88 11.53

(137) TABLE-US-00004 Cell line T47-D (ER-positive) percentage percentage of cell of cell death percentage death with percentage of cell percentage of with percentage of of cell death Concentration compound death with cell death with compound cell death with with (M) 9 compound 10 compound 18 25 compound 7 LY294002 50 93.62 92.93 78.28 91.25 88.3 55.69 25 91.2 91.71 23.71 90.23 92.07 50.01 12.5 92.97 87.84 0 91.27 85.1 40.42 6.25 20.53 4.62 0 85.31 34.43 32.33

(138) TABLE-US-00005 Cell line MDA-MB-453 (HER2 positive) percentage percentage of cell percentage of cell death percentage death with percentage of cell percentage of of cell death with percentage of of cell death Concentration compound death with cell death with with compound cell death with with (M) 9 compound 10 compound 18 compound 19 25 compound 7 LY294002 50 98.67 99.07 100 101.13 100.76 100.25 86.55 25 97.49 98.46 93.72 98.05 99.35 102.46 76.61 12.5 87.47 93.07 81.73 91.89 95.09 97.05 53.74 6.25 71.22 76.17 45.963 53.93 89.213 65.61 17.45 3.125 53.8 70.42 0 24.69 85.57 52.93 0 1.5625 4.67 5.06 0 0 81 13.84 0 0.78125 7.56 7.8 3.17 0 9.09 0 0 0.390625 1.52 6.6 3.17 0 0 1.13 0

(139) TABLE-US-00006 Cell line BT474 (ER-positive/HER2-positive) percentage percentage percentage percentage percentage of cell death of cell death of cell death of cell death of cell death Concentration with with with with with (M) compound 9 compound 10 compound 25 compound 7 LY294002 50 93.62 93.84 96.81 96.07 77.2 25 88.01 69.52 90.49 91.87 62.79 12.5 71.4 40.42 95.61 55.3 48.7 6.25 25.95 0 75.54 44.85 35.35

(140) The percentages of cell death induced by the compounds of formula (I) are higher than the percentages of cell death induced by LY294002 with concentrations from 6.25 to 50 M. In some cases, the percentages of cell death induced by the compounds of formula (I) are higher than the percentages of cell death induced by LY294002 with concentrations from 0.390625 to 50 M.

(141) The compounds of formula (I) implement a strong cell death signal on tumor cells MCF-7, T47-D, BT474 and MDA-MB-453. These results confirm that compounds of formula (I) are of interest to prevent and/or treat breast cancers.

Example 6

Compounds of Formula (I) Inhibit the PI3K/Akt/mTOR Pathway and Bind to mTOR

(142) FIG. 12A:

(143) The inhibition of the phosphorylation level of AKT at serine 473, target of mTORC2, and 4EBP1 (also called Eukaryotic translation initiation factor 4E-binding protein 1) at threonines 37/46, which are targets of mTORC1, by compound 48 (compound 25 linked to biotin) was assessed by immunoblotting. Total AKT and 4EBP1 were added as loading controls.

(144) These results show that the phosphorylation of AKT and 4EBP1 decreases with the increase in the compound 48 concentrations. These results show that the compounds of formula (I) inhibit the PI3K/AKT/mTOR pathway by inhibiting the phosphorylation of mTOR downstream effectors such as AKT and 4EBP1.

(145) FIG. 12B:

(146) Compounds 25 and 48 (compound 25 linked to biotin) (1 M) were incubated with CEM leukemic T cells for indicated times. Then cells were lyzed in RIPA buffer and biotin was immunoprecipitated using streptavidin-coated magnetic beads. Beads were washed and the presence of mTOR was analyzed in compound 48-associated complex by Immunoblotting.

(147) The results show that compound 48 binds to mTOR. Therefore, the compounds of formula (I) target the mTOR protein in tumor cell lines.

Example 7

Toxicity of the Compounds of the Invention in Vivo

(148) The in vivo toxic activity of compound 25 was studied as follows.

(149) Mice received repeated intravenous injections of compound 25 (10 mg/kg) or vehicle. The results show that treatment with compound 25 did not exhibit toxicity in mice: the body weight of the tested mice was maintained and all mice survived at 7 days (see FIGS. 13A and 13B).

Example 8

Cytotoxic Activity of the Compounds of the Invention on TSC2/ and TSC2+/+ Cells

(150) AML (AngioMyoLipoma) cells from a patient (generous gift from Dr M. Pende, Paris) were enriched for TSC2.sup./ cells, as a model of tuberous sclerosis. These cells have been reconstituted with wild type TSC2 (TSC2.sup.+/+). Cell proliferation was assessed in cells from angiomyolipomas and their counterparts reconstituted with wild type TSC2. FIG. 14A shows the cell number obtained over time.

(151) Cell viability was assessed by MTT assay. Cells were incubated for 16 hours in a 1% fetal calf serum-containing medium supplemented with the indicated concentrations of the two mTOR inhibitors, compound 25 or rapamycin (see FIGS. 14B and 14C). Data represent mean and standard deviation of three independent experiments.

(152) These results show that TSC2.sup./ cells are more sensitive to compound 25 than wild type TSC2.sup.+/+. Moreover, the compound 25 of the invention is more efficient to kill TSC2.sup./ cells than rapamycin, which is used in the treatment of tuberous sclerosis.

(153) Thus, the compounds of the invention are useful to treat tuberous sclerosis and are more efficient than rapamycin.

Example 9

The Compounds of the Invention Inhibit mTORC1 Activity in TSC2/ Cells

(154) AML cells (1.10.sup.6 cells) deficient for TSC2 (TS2.sup./) or reconstituted with wild type TSC2 (TSC2.sup.+/+) (generous gift from Dr M. Pende, Paris) were treated or untreated for indicated times with 10 M of compound 25 or rapamycin and then cells were lyzed. 100 g of protein was loaded and resolved by SDS-PAGE and indicated immunoblots were performed. Total S6 and -actin serve as loading controls. S6 is phosphorylated by p70S6K on its serine 240 and 244. 4EBP1 is phosphorylated by mTORC1 on its Threonine at positions 37 and 46. p70S6K and 4EBP1 are direct substrates of mTORC1. mTORC1-driven phosphorylation of 4EBP1 can be monitored by the appearance of a high molecular weight band which disappears in presence of compound 25 or Rapamycin (see FIG. 15). Of note, restoration of TS2 expression in AML cells reduces the basal level of S6 and 4EBP1 phosphorylation.

(155) These results show that compounds of the invention such as compound 25, inhibit mTORC1 activity in TSC2.sup./ cells.

Example 10

Competitive Inhibition of mTOR by the Compounds of the Invention

(156) Unlike rapamycin, which when bound to FKBP12, interacts with and inhibits the kinase activity of mTORC1, competitive mTOR inhibitors target both mTORC1 and mTORC2.

(157) CEM cells (1.Math.10.sup.6 cells) were incubated with indicated concentrations of compound 25 for 2 hours and lysates of the cells were subjected to Western blot analysis. Inhibitory activity of compound 25 on mTORC1 substrates p70S6K-Thr389 and 4EBP1-Thr37 and 46 and on mTORC2 substrate Akt-Ser473 and on PDK1 substrate Akt-Thr308 was evaluated by immunoblotting. Total 4EBP1, p70S6K, Akt and 1-actin serve as loading controls. The compounds of the invention and more particularly compound 25 may be competitive inhibitors of mTOR because they inhibit mTORC1 (4EBP1 and p70S6K) and mTORC2 substrates (AKT at S473) as shown in FIG. 16.

Example 11

Prevention of Cell Migration by the Compounds of the Invention in Triple Negative Breast Cancer (TNBC) Cells

(158) CD95L (also known as FasL) belongs to the TNF (Tumor Necrosis Factor) family and is the ligand for the death receptor CD95 (Fas/APO1). This transmembrane cytokine can be cleaved by metalloproteases, to produce a soluble ligand. This naturally-processed CD95L (cl-CD95L) in patients affected by triple negative breast cancer triggers cancer cell migration and by doing so, enhances the risk of metastatic dissemination in these patients. Unlike membrane-bound-CD95L, cl-CD95L fails to induce apoptosis and instead promotes the formation of an atypical receptosome herein designated Motility-Inducing Signaling Complex (MISC).

(159) MISC formation leads to the induction of the pro-oncogenic phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling pathway.

(160) TNBC (MDA-MB-231 and BT549) cell lines were pre-incubated for 1 hour in presence or absence of non-cytotoxic amount of compound 25 (1 M) and then treated or untreated with CD95L (100 ng/ml) for 24 hours. Cell migration was analyzed using Boyden chamber assay. Migrating Giemsa-stained cells were lysed and absorbance was measured at a wavelength of 560 nm. Values represent the meansSEM of three independently performed experiments. *p<0.05 as calculated using two-tailed Mann-Whitney test.

(161) The results show that 1 M of compound 25 was sufficient to abrogate the migration of the tumor cells stimulated with the pro-migratory factor CD95L (see FIG. 17).

(162) Materiel and Methods of the Examples:

(163) Antibodies and Other Reagents

(164) LY294002 and Wortmannin were purchased from Calbiochem (Merck Chemicals Ltd., Nottingham, UK). Anti-mTOR, anti-4EBP, anti-phospho-4EBP, anti-AKT and anti-phospho-AKT antisera were from Cell Signaling Technology, Inc (Boston, Mass., USA).

(165) Cell Lines

(166) The human leukemic T-cell lines Jurkat and CEM and the lymphoma T-cell lines H9 were cultured in RPMI supplemented with 8% (v/v) heat-inactivated FCS and 2 mM L-glutamine at 37 C. in a 5% CO.sub.2 incubator. The human breast cancer cell lines BT549, BT474, MDA-MB-231, MDA-MB-468, MDA-MB-453, T47D, and MCF7 were cultured in DMEM supplemented with 8% v/v heat-inactivated fetal calf serum (FCS) and 2 mM L-glutamine at 37 C. in a 5% CO.sub.2 incubator. All cells were from American Type Culture Collection (ATCC, LGC Standards, Molsheim, France).

(167) Compound 25-Biotine (Compound 48) Immunoprecipitation

(168) CEM cells (10.sup.7 cells) were pre-incubated for indicated times with 1 M of compound 25 or compound 48, washed with PBS and lysed using RIPA buffer [50 mM Tris pH7.4, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, 150 mM NaCl, 2 mM EDTA, inhibitors of protease and phosphatase (Sigma)]. Next, compound 48 was immunoprecipitated using streptavidin-coated magnetic beads (Ademtech, Bordeaux, France) and after extensive washing, the immune complex was resolved by SDS-PAGE and mTOR was revealed by western blot.

(169) Immunoblots Excepted for immunoprecipitation experiments in which cells were lyzed with RIPA buffer, cells were lyzed for 30 min at 4 C. in Lysis buffer (25 mM HEPES pH 7.4, 1% v/v Triton X-100, 150 mM NaCl, 2 mM EGTA supplemented with a mix of protease inhibitors). Protein concentration was determined by the bicinchoninic acid method (Pierce, Rockford, Ill., USA) according to the manufacturer's protocol. Proteins were resolved by 8, 10 or 12% SDS-PAGE and transferred to a nitrocellulose membrane (GE Healthcare, Buckinghamshire, UK). The membrane was blocked 15 min with TBST (50 mM Tris, 160 mM NaCl, 0.05% v/v Tween 20, pH 7.4) containing 5% w/v dried skimmed milk (TBS). Primary antibody was incubated overnight at 4 C. in TBS. The membrane was intensively washed (TBST) and then the peroxydase-labeled anti-mouse IgG1 or IgG2a (CliniSciences, Nanterre, France) was added for 45 min. Proteins were visualized with the enhanced chemiluminescence substrate kit (ECL RevelBIOt, Ozyme, Saint Quentin en Yvelines, France).

(170) Cell Death Assays

(171) Cell viability was assessed using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) viability assay (1). In brief, cells (410.sup.4 per well) were cultured for 20 h in flat-bottomed 96 well plates with various concentrations of the apoptosis inducer. Then 0.015 ml of MTT (5 mg/ml in PBS) was added to each well and incubated for 4 h at 37 C. Formazan salt precipitats were dissolved by adding 0.115 ml of isopropyl alcohol containing 1% formic acid (v/v), and the absorbance was measured at 570 nm.

(172) In Vitro Motility Assays

(173) After membrane hydration of Boyden chambers (Millipore, Molsheim, France) containing 8 m pore membranes, 10.sup.5 cells were added to the top chamber. The bottom chamber was filled with low serum (1%)-containing medium in the presence or absence of cl-CD95L (100 ng/ml). Breast cancer cells were incubated for 24 h. To quantify invasion, cells were fixed with methanol and stained with Giemsa. Stained cells were then removed from the top-side of the membrane using a cotton-tipped swab and five representative pictures for each insert were taken of the invading cells from the reverse side. For each experiment, invading cells were lysed and absorbance at 560 nm was measured.

REFERENCE

(174) Weichert H, Blechschmidt I, Schroder S, Ambrosius H. The MTT-assay as a rapid test for cell proliferation and cell killing: application to human peripheral blood lymphocytes (PBL). Allerg Immunol (Leipz). 1991; 37:139-44.