Use of maleimide derivatives for preventing and treating leukemia

Abstract

The present invention is related to a compound of formula (I): a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, a metabolite thereof or a prodrug thereof; for use in a method for the treatment and/or prevention of leukemia, wherein X is selected from the group consisting of N—R.sup.1, O and S; R.sup.1 is selected from the group consisting of alkyl, cycloalkyl, aryl, arylalkyl and hydrogen; R.sup.2 is selected from the group consisting of indolyl, substituted indolyl, azaindolyl and substituted azaindolyl; and R.sup.3 is selected from the group consisting of aryl, substituted aryl, unsubstituted heteroaryl, heterocyclyl and substituted heterocyclyl. ##STR00001##

Claims

1. A method for arresting or reducing the development of leukemia or the clinical symptoms of leukemia or causing regression of leukemia or the clinical symptoms of leukemia, wherein the method comprises administering to a subject in need thereof a therapeutically effective amount of a compound of formula (I): ##STR00045## a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, a metabolite thereof or a prodrug thereof; wherein X is selected from the group consisting of N—R.sup.1 and O; R.sup.1 is alkyl or hydrogen; R.sup.2 is indolyl or substituted indolyl; and R.sup.3 is aryl or substituted aryl, wherein the substituted aryl consists of one, two or three substituents, wherein each and any of the substituents is individually and independently selected from the group consisting of fluoro, chloro, methyl, trifluoromethyl, vinyl, acetyl, acetamido, formyl, ethoxycarbonyl and dimethylamidocarbonyl.

2. The method of claim 1, wherein R.sup.2 comprises one, two, three, four, five or six substituents, whereby each and any of the substituents is individually and independently selected from the group comprising halogen, alkyl, alkenyl, alkynyl, acyl, formyl, cycloalkyl, aryl, haloalkyl, polyfluoroalkyl, alkylthio, arylthio, monoalkylamino, dialkylamino, monoarylamino, diarylamino, alkylarylamino, alkylimido, hydroxy, alkoxy, aryloxy, carboxyl, alkoxycarbonyl, aryloxycarbonyl, cyano, amino, amido, acylamino, nitro, alkylsulfinyl, arylsulfinyl, alkyl sulfonyl, aryl sulfonyl, alkylsulfinamido, arylsulfinamido, alkylsulfonamido and arylsulfonamido.

3. The method of claim 1, wherein X is N—R′, and wherein R.sup.1 is selected from the group consisting of alkyl and hydrogen.

4. The method of claim 3, wherein R.sup.1 is selected from the group consisting of methyl, butyl and hydrogen.

5. The method of claim 1, wherein X is O.

6. The method of claim 1, wherein R.sup.3 is selected from the group consisting of monocyclic aryl, substituted monocyclic aryl, bicyclic aryl, and substituted bicyclic aryl.

7. The method of claim 6, wherein R.sup.3 is selected from the group consisting of phenyl, substituted phenyl, naphthenyl, and substituted naphthenyl.

8. The method of claim 7, wherein each and any of the substituents is individually and independently selected from the group consisting of fluoro, chloro, methyl, trifluoromethyl, vinyl, acetyl, acetamido, methoxy, formyl, ethoxycarbonyl and dimethylamidocarbonyl.

9. The method of claim 8, wherein R.sup.3 is selected from the group consisting of phenyl and substituted phenyl, wherein substituted phenyl is phenyl consisting of one, two or three substituents, wherein each and any of the substituents is individually and independently selected from the group consisting of fluoro, chloro, methyl, trifluoromethyl, vinyl, acetyl, acetamido, methoxy, formyl, ethoxycarbonyl and dimethylamidocarbonyl.

10. The method of claim 7, wherein R.sup.3 is substituted phenyl and each and any of the substituents is individually any independently selected from the group consisting of alkyl, substituted alkyl, alkoxy, substituted alkoxy, alkynyl and halogen.

11. The method of claim 10, wherein alkyl is methyl or ethyl, substituted alkyl is halogen-substituted methyl or acetyl, alkoxy is ethoxy, and alkenyl is vinyl.

12. The method of claim 7, wherein the compound is of formula (IV) ##STR00046## wherein R.sup.5 is selected from the group consisting of alkyl, aminoalkyl, alkoxyalkyl, hydroxyalkyl, aryl and heteroaryl.

13. The method of claim 12, wherein R.sup.5 is methyl.

14. The method of claim 1, wherein R.sup.2 is a moiety of formula (VIa) ##STR00047## wherein R.sup.4 is selected from the group consisting of hydrogen, aryl, heteroaryl, alkyl, cycloalkyl, polyfluoroalkyl, arylalkyl and heteroarylalkyl, R.sup.6 is selected from the group consisting of alkyl and aryl, each and any of Y.sup.1, Y.sup.2, Y.sup.3 and Y.sup.4 is individually and independently selected from the group consisting of N and CR.sup.7, under the proviso that at least two of Y.sup.1, Y.sup.2, Y.sup.3 and Y.sup.4 are CR.sup.7, wherein each and any of R.sup.7 is individually and independently selected from the group consisting of hydrogen, halogen, alkyl, alkenyl, alkynyl, acyl, formyl, cycloalkyl, aryl, haloalkyl, polyfluoroalkyl, alkylthio, arylthio, monoalkylamino, dialkylamino, monoarylamino, diarylamino, alkyl aryl amino, alkylimido, hydroxy, alkoxy, aryloxy, carboxyl, alkoxycarbonyl, aryloxycarbonyl, cyano, amino, amido, acylamino, nitro, alkylsulfinyl, arylsulfinyl, alkyl sulfonyl, aryl sulfonyl, alkyl sulfinamido, aryl sulfinamido, alkyl sulfonamido and arylsulfonamido.

15. The method of claim 14, wherein each and any of Y.sup.1, Y.sup.2, Y.sup.3 and Y.sup.4 is CR.sup.7.

16. The method of claim 15, wherein R.sup.7 is hydrogen.

17. The method of claim 14, wherein R.sup.4 is selected from the group consisting of hydrogen, alkyl and benzyl, and/or wherein R.sup.6 is hydrogen or alkyl.

18. The method of claim 14, wherein each and any of Y.sup.1, Y.sup.2, Y.sup.3 and Y.sup.4 is individually and independently selected from the group consisting of N and CR.sup.7, under the proviso that one or two of Y.sup.1, Y.sup.2, Y.sup.3 and Y.sup.4 are N.

19. The method of claim 18, wherein R.sup.7 is hydrogen.

20. The method of claim 14, wherein R.sup.4 is selected from the group consisting of hydrogen, alkyl and benzyl, and/or wherein R.sup.6 is hydrogen or alkyl.

21. The method of claim 14, wherein the compound is of formula (VI) ##STR00048## wherein R.sup.5 is selected from the group consisting of alkyl, aminoalkyl, alkoxyalkyl, hydroxyalkyl, aryl and heteroaryl, or wherein the compound is of formula (V) ##STR00049##

22. The method of claim 21, wherein R.sup.5 is methyl.

23. The method claim 1, wherein the compound is selected from the group consisting of 1-Methyl-3,4-bis-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione; 1-Methyl-3-(2-methyl-1H-indol-3-yl)-4-(4-vinylphenyl)-1H-pyrrole-2,5-dione; 1-Methyl-3-(2-methyl-1H-indol-3-yl)-4-phenyl-1H-pyrrole-2,5-dione; 3-(4-Acetylphenyl)-1-methyl-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione (also referred to herein as PDA-66); 3-(2,6-Dimethylphenyl)-1-methyl-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione; 3-(3-Chlorophenyl)-1-methyl-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione; 3-(2,4-Dichlorophenyl)-1-methyl-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione; 1-Methyl-3-(2-methyl-1H-indol-3-yl)-4-(thiophen-3-yl)-1H-pyrrole-2,5-dione; 1-Methyl-3-(2-methyl-1H-indol-3-yl)-4-(pyridin-4-yl)-1H-pyrrole-2,5-dione; 1-Methyl-3-(2-methyl-1H-indol-3-yl)-4-(naphthalen-2-yl)-1H-pyrrole-2,5-dione; 3-(2,5-Dimethoxyphenyl)-1-methyl-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione; 1-Methyl-3-(2-methyl-1H-indol-3-yl)-4-(2-(trifluoromethyl)phenyl)-1H-pyrrole-2,5-dione; 3-(4-Fluorophenyl)-1-methyl-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione; 3-(5-Acetyl-2-fluorophenyl)-1-methyl-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione; N-(4-(1-Methyl-4-(2-methyl-1H-indol-3-yl)-2, 5-di oxo-2,5-dihydro-1H-pyrrol-3-yl)phenyl) acetamide; 3-(2-Methyl-1H-indol-3-yl)-4-phenylfuran-2, 5-dione; 3-(4-Acetylphenyl)-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2, 5-dione; 3-(2-Methyl-1H-indol-3-yl)-4-(naphthalen-2-yl)furan-2, 5-dione; 3-(2-Methyl-1H-indol-3-yl)-4-(naphthalen-2-yl)-1H-pyrrole-2, 5-dione; 3-(4-Acetylphenyl)-4-(2-methyl-1H-indol-3-yl)furan-2, 5-dione; and 3-(2-Methyl-1H-indol-3-yl)-4-phenyl-1H-pyrrole-2, 5-dione.

24. The method of claim 1, wherein the compound is 3-(4-acetylphenyl)-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione ##STR00050##

25. The method of claim 1, wherein the compound is 3-(4-acetylphenyl)-1-methyl-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione ##STR00051##

26. The method of claim 1, wherein leukemia is selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), refractory leukemia, resistant leukemia, FLT3-ITD-positive leukemia, any chronic leukemia, myelodysplasia, and lymphoma.

27. The method of claim 1, further comprising administering a chemotherapeutic agent.

28. The method of claim 27, wherein the chemotherapeutic agent is selected from the group comprising cytarabine, etoposide, mitoxantron, cyclophosphamide, retinoic acid, daunorubicin, doxorubicin, idarubicin, azacytidine, decitabine, a tyrosine-kinase inhibitor, a antineoplastic antibody, vincaalkaloids and steroids.

29. The method of claim 28, wherein the chemotherapeutic agent is a tyrosine-kinase inhibitor, wherein the tyrosine-kinase inhibitor is selected from the group comprising sorafenib, dasatinib, nilotinib, nelarabine and fludarabine or wherein the chemotherapeutic agent is Alemtuzumab.

Description

(1) The present invention is now further illustrated by reference to the following figures and examples from which further advantages, features, and embodiments may be taken, wherein

(2) FIG. 1 is a diagram showing the inhibition of GSK3β by compound PDA-66 at different concentrations;

(3) FIG. 2 is a set of bar diagrams showing the impact of compound PDA-66 on cell proliferation and metabolic activity of SEM cells, RS4;11 cells, Jurkat cells and Molt-4 cells;

(4) FIG. 3 is a set of light microscopic pictures indicating the effect of compound PDA-66 and DSMO on SEM cells and Jurkat cells;

(5) FIG. 4 is a set of bar diagrams showing the impact of compound PDA-66 on cell cycle distribution of SEM cells, RS4;11 cells, Jurkat cells and Molt-4 cells;

(6) FIG. 5A is a set of bar diagrams showing the impact of compound PDA-66 on apoptosis and necrosis of SEM cells, RS4;11 cells, Jurkat cells and Molt-4 cells;

(7) FIG. 5B shows the result of a Western blot analysis of the effect of compound PDA-66 on cleavage of Caspase 3, 7 and PRAP;

(8) FIG. 6A shows the result of a Western blot analysis of the effect of compound PDA-66 on the expression of pAktSer473, pAktThr308, Akt, pGSK3βSer9, β-Catenin and GAPDH;

(9) FIG. 6B shows the result of a Western blot analysis of the effect of compound PDA-66 on the expression of p4EBP-1Ser65, 4EBP1 and GAPDH in SEM cells, RS4;11 cells and Molt-4 cells; and

(10) FIG. 6C shows Table 1 indicating IC50 concentrations of compound PDA-66 in SEM cells, RS4;11 cells, Jurkat cells and Molt-4 cells.

EXAMPLES

(11) Abbreviations used in general procedures and examples are defined as follows: “HCl” for hydrochloric acid, “KOH” for potassium hydroxide, “NaHC0.sub.3” for sodium hydrocarbonate, “K.sub.2CO.sub.3” for potassium carbonate, “Na.sub.2SO.sub.4” for sodium sulfate, “CH.sub.2Cl.sub.2” for methylene chloride, “THF” for tetrahydrofuran, “EA” for ethyl acetate, “DMSO” for dimethylsulfoxide, “CDCl.sub.3” for deuterated chloroform, “TLC” for thin layer chromatography, “LiHMDS” for lithium hexamethyldisilazane, “Pd(OAc).sub.2” for palladium acetate.

(12) All reactions were carried out under argon atmosphere. Reactions were monitored by TLC analysis (pre-coated silica gel plates with fluorescent indicator UV254, 0.2 mm) and visualized with 254 nm UV light or iodine. Chemicals were purchased from Aldrich, Fluka, Acros, AlfaAsar, Strem and unless otherwise noted were used without further purification. All compounds were characterized by .sup.1H NMR, .sup.13C NMR, GC-MS, HRMS and IR spectroscopy. .sup.1H spectra were recorded on Bruker AV 300 and AV 400 spectrometers. .sup.13C NMR and .sup.19F NMR spectra were recorded at 75.5 MHz and 282 MHz respectively. Chemical shifts are reported in ppm relative to the center of solvent resonance. Melting points were determined on a digital SMP3 (Stuart). IR spectra were recorded on FT-IR ALPHA (Bruker) with Platinum-ATR (Bruker). EI (70 eV) mass spectra were recorded on MAT 95XP (Thermo ELECTRON CORPORATION). GC was performed on Agilent 6890 chromatograph with a 30 m HP5 column. HRMS was performed on MAT 95XP (EI) and Agilent 6210 Time-of-Flight LC/MS (ESI). GC-MS was performed on Agilent 5973 chromatograph Mass Selective Detector. All yields reported refer to isolated yields.

Example 1: Preparation 1—General Procedure for Condensation of Indole or Azaindoles Derivative with 3,4-dihalomaleimide Compound and Specific Compounds

(13) The (aza)indole derivative (10 mmol) was dissolved in dry THF (20 ml) and cooled under Argon to −20° C., before 21 ml of LiHMDS (1 M in THF) were slowly added. After stirring for 2 h at −20° C., a solution of 3,4-dihalomaleimide derivative (10 mmol) in THF (20 ml) was added to the lithiated (aza)indole solution all at once via syringe. After stirring additional 1 h at −20° C. (TLC control), the reaction mixture was carefully neutralized with 2N aq HCl and extracted with ethyl acetate (3×). The combined organics were washed with sat. aq NaHCO.sub.3, brine, and water. After drying over Na.sub.2SO.sub.4 and concentration, the crude material was crystallized from ether.

Example 1.1

3-Bromo-1-methyl-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione

(14) ##STR00020##

(15) Orange crystals; .sup.1H NMR (CDCl.sub.3) δ 2.48 (s, 3H), 3.19 (s, 3H), 7.18 (ddd, 1H), 7.20 (ddd, 1H), 7.31 (ddd, 1H), 7.48 (m, 1H), 8.48 (br.s, 1H); .sup.13C NMR (CDCl.sub.3) δ 14.3, 24.9, 102.0, 110.8, 120.5, 120.7, 120.8, 122.4, 126.4, 135.5, 137.6, 139.3, 166.4, 169.1; GC-MS (EI, 70 eV): m/z (%) 318 (100) [M.sup.+], 320 (96) [M.sup.+]; HRMS (EI): Cacld for C.sub.14H.sub.11O.sub.2N.sub.2Br: 317.99984; found: 317.99979; IR (ATR, cm.sup.−1): 3361, 3066, 1771, 1703, 1623, 1422, 1379, 990, 806, 749, 733, 656.

Example 1.2

3-Bromo-1-methyl-4-(1H-pyrrolo[2,3-b]pyridin-3-yl)-1H-pyrrole-2,5-dione

(16) ##STR00021##

(17) Preparation was performed using Grignard reagent. Orange crystals; .sup.1H NMR (DMSO-d.sub.6) δ 2.99 (s, 3H), 7.21 (ddd, 1H, J˜3.83, 5.31, 7.36 Hz), 8.20 (s, 1H), 8.31 (dd, 1H, J=1.53, 3.52 Hz), 8.33 (s, 1H), 12.68 (br.s, 1H); .sup.13C NMR (DMSO-d.sub.6) δ 24.6, 102.7, 114.8, 116.9, 117.0, 130.8, 131.2, 136.8, 144.0, 148.7, 166.4, 168.9; GC-MS (EI, 70 eV): m/z (%) 305 (58) [M.sup.+], 307 (57) [M.sup.+]; HRMS pos. (ESI): Calc for [M+H].sup.+, C.sub.12H.sub.9BrN.sub.3O.sub.2: 305.98727 and 307.98532; found: 305.98737 and 307.98544; IR (ATR, cm.sup.−1): 3079, 2742, 1764, 1707, 1584, 1488, 1440, 1419, 1384, 1287, 1167, 1141, 1101, 801, 778, 733, 628.

Example 1.3

1-Methyl-3,4-bis-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione

(18) ##STR00022##

(19) Red crystals; .sup.1H NMR (DMSO-d.sub.6) δ 1.97 (s, 3H), 1.98 (s, 3H), 3.05 (s, 3H), 6.75 (br.t, 2H, J=7.41 Hz), 6.95 (ddd, 2H, J˜3.83, 5.31, 7.36 Hz), 7.03 (br.d, 2H, J=7.90 Hz), 7.23 (br.d, 2H, J=8.09 Hz), 11.29 (br.s, 2H); .sup.13C NMR (DMSO-d.sub.6) δ 13.0, 23.9, 103.3, 110.7, 119.2, 119.4, 120.8, 126.6, 131.2, 135.5 (2C), 137.3, 170.4, 171.3; GC-MS (EI, 70 eV): m/z (%) 369 (100) [M.sup.+]; HRMS (EI): Cacld for C.sub.23H.sub.19O.sub.2N.sub.3: 369.14718; found 369.14705; IR (ATR, cm.sup.−1): 3383, 3307, 1755, 1692, 1456, 1435, 1377, 1239, 1049, 1022, 1003, 747, 737, 693.

Example 2: Preparation 2—General Procedure for Suzuki Coupling and Specific Compounds

(20) In an Ace-pressure tube into a solution of (aza)indolylmaleimide derivative (1 mmol) and corresponding boronic acid (1.5 mmol) in dimethoxyethane (3 ml) were added K.sub.2CO.sub.3 (1M in water, 3 ml), Pd(OAc).sub.2 (2 mol %) and ligand (2.5 mol %) under argon atmosphere. The pressure tube was fitted with a Teflon cap and heated at 100° C. (TLC control). The mixture was cooled to room temperature and diluted with ethyl acetate. The organic layer was washed with sat. aq ammonium chloride (2×30 mL) and water. After drying over Na.sub.2SO.sub.4 and removal of the solvent in vacuum, the coupling product was isolated by column chromatography in heptane/ethyl acetate.

Example 2.4

1-Methyl-3-(2-methyl-1H-indol-3-yl)-4-(4-vinylphenyl)-1H-pyrrole-2,5-dione

(21) ##STR00023##

(22) Red-orange crystals; .sup.1H NMR (CDCl.sub.3) δ 2.14 (s, 3H), 3.17 (s, 3H), 5.25 (dd, 1H, J=0.66, 10.89 Hz), 5.72 (dd, 1H, J=0.70, 17.61 Hz), 6.63 (dd, 1H, J=10.88, 17.62 Hz), 6.96 (m, 1H), 7.09 (m, 2H), 7.23 (m, 1H), 7.27 (m, 2H), 7.53 (m, 2H), 8.32 (br s, 1H); .sup.13C NMR (CDCl.sub.3) δ 13.7, 24.2, 103.0, 110.5, 115.0, 120.3, 120.5, 122.0, 126.1 (2C), 126.5, 129.5 (2C), 129.6, 132.7, 133.7, 135.7, 136.2, 136.8, 138.1, 171.2, 171.6; GC-MS (EI, 70 eV): m/z (%) 342 (100) [M.sup.+]; HRMS (EI): Cacld for C.sub.22H.sub.18O.sub.2N.sub.2: 342.13628; found: 342.13618; IR (ATR, cm.sup.−1): 3380, 3053, 2920, 1745, 1689, 1456, 1428, 1383, 1235, 990, 903, 847, 814, 741, 656.

Example 2.5

1-Methyl-3-(2-methyl-1H-indol-3-yl)-4-phenyl-1H-pyrrole-2,5-dione

(23) ##STR00024##

(24) Red crystals; .sup.1H NMR (CDCl.sub.3) δ 2.14 (s, 3H), 3.20 (s, 3H), 6.97 (ddd, 1H), 7.11 (m, 2H), 7.22 (ddd, 1H), 7.27 (m, 3H), 7.55 (m, 2H), 8.33 (br.s, 1H); .sup.13C NMR (CDCl.sub.3) δ 13.6, 24.2, 102.8, 110.5, 120.3, 120.5, 122.0, 126.5, 128.4 (2C), 129.1, 129.3 (2C), 130.2, 133.2, 134.1, 135.7, 136.8, 171.2, 171.5; GC-MS (EI, 70 eV): m/z (%) 316 (100) [M.sup.+]; HRMS (EI): Cacld for C.sub.20H.sub.16O.sub.2N.sub.2 316.12063; found: 316.12091; IR (ATR, cm.sup.−1): 3426, 3381, 3052, 1759, 1690, 1618, 1435, 1422, 1382, 1234, 1002, 989, 938, 786, 752, 736, 693.

Example 2.6

3-(4-Acetylphenyl)-1-methyl-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione (PDA-66)

(25) ##STR00025##

(26) Red crystals; .sup.1H NMR (CDCl.sub.3) δ 2.18 (s, 3H), 2.57 (s, 3H), 3.21 (s, 3H), 6.94 (ddd, 1H, J˜0.99, 7.05, 8.00 Hz), 7.03 (ddd, 1H), 7.11 (ddd, 1H, J 1.15, 7.05, 8.11 Hz), 7.25 (dd, 1H, J˜0.41, 8.11 Hz), 7.67 (ddd, 2H, J˜1.72, 3.63, 8.61 Hz), 7.84 (ddd, 2H, J˜1.85, 3.70, 8.61 Hz), 8.57 (br.s, 1H); .sup.13C NMR (CDCl.sub.3) δ 13.8, 24.3, 26.6, 102.5, 110.7, 120.1, 120.6, 122.2, 126.2, 128.2 (2C), 129.5 (2C), 131.9, 134.9, 135.0, 135.8, 136.6, 137.6, 170.8, 171.1, 197.8; GC-MS (EI, 70 eV): m/z (%) 358 (100) [M.sup.+]; HRMS (EI): Cacld for C.sub.22H.sub.18O.sub.3N.sub.2: 358.13119; found: 358.131088; IR (ATR, cm.sup.−1): 3339, 3058, 2923, 1762, 1692, 1678, 1427, 1407, 1383, 1358, 1265, 1234, 990, 846, 817, 742.

Example 2.7

3-(2,6-Dimethylphenyl)-1-methyl-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione

(27) ##STR00026##

(28) Orange crystals; .sup.1H NMR (CDCl.sub.3) δ 1.97 (d, 3H, J=0.88), 2.08 (s, 6H), 3.22 (s, 3H), 7.00 (ddd, 1H), 7.01 (d, 2H), 7.10 (ddd, 1H), 7.12 (ddd, 1H), 7.19 (ddd, 1H), 7.25 (ddd, 1H), 8.21 (br.s, 1H); .sup.13C NMR (CDCl.sub.3) δ 13.2, 20.7 (2C), 24.4, 103.6, 110.3, 119.9, 120.6, 122.1, 126.8, 128.0 (2C), 128.8, 129.5, 135.4, 136.1, 136.9, 137.0 (2C), 137.1, 171.0, 171.2; GC-MS (EI, 70 eV): m/z (%) 344 (100) [M.sup.+]; HRMS (ED: Cacld for C.sub.22H.sub.20O.sub.2N.sub.2: 344.15193; found: 344.15175; IR (ATR, cm.sup.−1): 3342, 2951, 1763, 1689, 1433, 1381, 1229, 987, 739, 665.

Example 2.8

3-(3-Chlorophenyl)-1-methyl-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione

(29) ##STR00027##

(30) Red crystals; .sup.1H NMR (CDCl.sub.3) δ 2.20 (s, 3H), 3.20 (s, 3H), 6.98 (ddd, 1H, J˜1.03, 7.0, 8.03 Hz), 7.05 (br.d, 1H, J˜7.52 Hz), 7.13 (ddd, 1H, J˜1.23, 7.0, 8.14 Hz), 7.18 (br.t, 1H, J=7.91 Hz), 7.25 (m, 2H), 7.40 (ddd, 1H, J˜1.26, 2.72, 7.84 Hz), 7.62 (br.t, 1H, J=1.80 Hz), 8.36 (br.s, 1H); .sup.13C NMR (CDCl.sub.3) δ 13.8, 24.3, 102.6, 110.6, 120.3, 120.7, 122.2, 126.2, 127.5, 129.1, 129.2, 129.6, 131.9, 132.1, 134.2, 134.3, 135.85, 137.2, 170.8, 171.1; GC-MS (EI, 70 eV): m/z (%) 350 (100) [M.sup.+]; HRMS (EI): Cacld for C.sub.20H.sub.15O.sub.2N.sub.2Cl: 350.08166; found: 350.08115; IR (ATR, cm.sup.−1): 3350, 3068, 2909, 1764, 1689, 1433, 1383, 1235, 991, 743, 735, 715, 683.

Example 2.9

3-(2,4-Dichlorophenyl)-1-methyl-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione

(31) ##STR00028##

(32) Orange crystals; .sup.1H NMR (CDCl.sub.3) δ 2.11 (s, 3H), 3.21 (s, 3H), 6.99 (ddd, 1H, J˜1.03, 7.0, 8.03 Hz), 7.10 (ddd, 1H, J˜0.93, 7.09, 8.23 Hz), 7.18 (m, 2H), 7.19 (d, 2H, J˜1.20 Hz), 7.40 (br.t, 1H, J=1.15 Hz), 8.41 (br.s, 1H); .sup.13C NMR (CDCl.sub.3) δ 13.5, 24.4, 103.1, 110.6, 119.7, 120.8, 122.2, 126.7, 127.3, 128.5, 130.1, 132.1, 132.4, 134.7, 135.5, 137.5, 137.6, 170.1, 170.6; GC-MS (EI, 70 eV): m/z (%) 384 (100) [M.sup.+]; HRMS (EI): Cacld for C.sub.20H.sub.14O.sub.2N.sub.2Cl.sub.2: 384.04268; found: 384.04261; IR (ATR, cm.sup.−1): 3358, 3064, 2949, 1756, 1687, 1436, 1386, 1228, 992, 857, 810, 778, 741, 673, 666.

Example 2.10

1-Methyl-3-(2-methyl-1H-indol-3-yl)-4-(thiophen-3-yl)-1H-pyrrole-2,5-dione

(33) ##STR00029##

(34) Red crystals; .sup.1H NMR (CDCl.sub.3) δ 2.27 (s, 3H), 3.18 (s, 3H), 7.01 (ddd, 1H, J˜1.07, 7.07, 8.05 Hz), 7.12 (ddd, 1H), 7.137 (dd, 1H, J˜3.02, 5.15 Hz), 7.14 (ddd, 1H), 7.19 (dd, 1H, J=1.22, 5.17 Hz), 7.25 (dt, 1H, J=0.91, 8.06 Hz), 8.11 (dd, 1H, J=1.24, 2.95 Hz), 8.42 (br.s, 1H); .sup.13C NMR (CDCl.sub.3) δ 13.5, 24.2, 102.9, 110.6, 120.1, 120.5, 122.0, 125.1, 126.7, 127.5, 129.2, 130.0, 130.2, 130.5, 135.7, 136.7, 171.5, 171.6; GC-MS (EI, 70 eV): m/z (%) 322 (100) [M.sup.+]; HRMS (EI): Cacld for C.sub.18H.sub.14O.sub.2N.sub.2S: 322.07705; found: 322.07631; IR (ATR, cm.sup.−1): 3391, 3102, 1756, 1689, 1624, 1438, 1410, 1382, 1334, 1228, 1071, 1003, 989, 820, 804, 790, 752, 737, 653.

Example 2.11

1-Methyl-3-(2-methyl-1H-indol-3-yl)-4-(pyridin-4-yl)-1H-pyrrole-2,5-dione

(35) ##STR00030##

(36) Red crystals; .sup.1H NMR (CDCl.sub.3) δ 2.28 (s, 3H), 3.21 (s, 3H), 6.97 (m, 2H), 7.14 (ddd, 1H, J˜3.58, 4.69, 8.23 Hz), 7.30 (dt, 1H, J˜0.7, 8.15 Hz), 7.46 (2dd, 2H, J˜1.59, 4.57 Hz), 8.53 (2dd, 2H, J˜1.57, 4.62 Hz), 8.71 (br.s, 1H); .sup.13C NMR (CDCl.sub.3) δ 14.1, 24.4, 102.5, 110.8, 120.3, 120.9, 122.5, 123.3 (2C), 125.9, 139.9, 135.9, 136.5, 137.9, 138.1, 149.8 (2C), 170.3, 170.6; GC-MS (EI, 70 eV): m/z (%) 317 (100) [M.sup.+]; HRMS (EI): Cacld for C.sub.19H.sub.15O.sub.2N.sub.3: 317.11588; found: 317.11635; IR (ATR, cm.sup.−1): 3342, 2923, 1765, 1694, 1456, 1428, 1383, 1237, 990, 813, 742, 656.

Example 2.12

1-Methyl-3-(2-methyl-1H-indol-3-yl)-4-(naphthalen-2-yl)-1H-pyrrole-2,5-dione

(37) ##STR00031##

(38) Red crystals; .sup.1H NMR (CDCl.sub.3) δ 2.09 (s, 3H), 3.24 (s, 3H), 6.95 (ddd, 1H, J˜1.04, 7.14, 8.12 Hz), 7.11 (ddd, 1H, J˜1.11, 7.13, 8.18 Hz), 7.20 (dd, 1H, J˜0.5, 8.12 Hz), 7.25 (dd, 1H, J˜<0.5, 8.07 Hz), 7.44 (dd, 1H, J˜1.68, 8.58 Hz), 7.48 (m, 2H), 7.59 (br.d, 1H, J˜8.71 Hz), 7.74 (m, 1H), 7.83 (m, 1H), 8.33 (br.s, 2H); .sup.13C NMR (CDCl.sub.3) δ 13.7, 24.3, 103.1, 110.5, 120.4, 120.6, 122.1, 125.8, 126.3, 126.8, 127.1, 127.6, 127.7, 127.8, 128.9, 130.0, 133.0, 133.18, 133.22, 133.8, 135.7, 136.9, 171.2, 171.6; GC-MS (EI, 70 eV): m/z (%) 366 (100) [M.sup.+]; HRMS (ED: Cacld for C.sub.24H.sub.18O.sub.2N.sub.2: 366.13628; found: 366.13581; IR (ATR, cm.sup.−1): 3345, 3055, 2946, 1759, 1689, 1425, 1381, 1226, 989, 816, 737, 660.

Example 2.13

3-(2,5-Dimethoxyphenyl)-1-methyl-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione

(39) ##STR00032##

(40) Deep orange crystals; .sup.1H NMR (CDCl.sub.3) δ 2.05 (s, 3H), 3.19 (s, 3H), 3.36 (s, 3H), 3.66 (s, 3H), 6.75 (br.d, 1H, J˜8.79 Hz), 6.81 (br.d, 1H, J˜2.57 Hz), 6.84 (dd, 1H, J˜3.05, 8.80 Hz), 6.94 (ddd, 1H, J˜1.13, 7.08, 8.02 Hz), 7.05 (ddd, 1H, J˜1.07, 7.15, 8.02 Hz), 7.14 (br.d, 1H, J˜8.11 Hz), 7.18 (br.d, 1H, J˜7.94 Hz), 8.38 (br.s, 1H); .sup.13C NMR (CDCl.sub.3) δ 13.3, 24.2, 55.78, 55.84, 104.0, 110.2, 112.7, 115.9, 116.2, 119.9, 120.3, 120.6, 121.8, 127.0, 133.3, 135.5, 135.6, 136.6, 151.9 (2C), 153.4 (2C), 171.0, 171.3; GC-MS (EI, 70 eV): m/z (%) 376 (100) [M.sup.+]; HRMS (EI): Cacld for C.sub.22H.sub.20O.sub.4N.sub.2: 376.14176; found: 376.14113; IR (ATR, cm.sup.−1): 3338, 2924, 1750, 1689, 1427, 1383, 1273, 1237, 1212, 1049, 1018, 997, 823, 760, 746, 724, 667.

Example 2.14

1-Methyl-3-(2-methyl-1H-indol-3-yl)-4-(2-(trifluoromethyl)phenyl)-1H-pyrrole-2,5-dione

(41) ##STR00033##

(42) Orange crystals; .sup.1H NMR (Aceton-d.sub.6) δ 2.20 (s, 3H), 3.11 (s, 3H), 6.86 (ddd, 1H, J˜1.06, 7.13, 8.07 Hz), 7.00 (ddd, 1H, J˜1.16, 7.16, 8.13 Hz), 7.19 (br.d, 1H, J 7.95 Hz), 7.27 (ddd, 1H), 7.37 (m, 1H), 7.55 (m, 2H), 7.76 (m, 1H), 10.55 (br.s, 1H); .sup.13C NMR (Aceton-d.sub.6) δ 13.1, 24.1, 102.3 (d, J=4.55 Hz), 111.2 (d, J=5.13 Hz), 120.0, 120.2, 121.9, 124.9 (q, J=272.93 Hz), 127.7 (q, J=4.42 Hz), 127.9 (d, J=3.68 Hz), 129.6 (q, J=30.37 Hz), 129.9 (d, J=1.79 Hz), 130.0, 132.5 (2C), 135.4, 136.5 (d, J=15.20 Hz), 137.5, 138.4 (d, J=14.52 Hz), 170.87, 170.93; .sup.19F NMR (CDCl.sub.3) δ −57.57 (s); GC-MS (EI, 70 eV): m/z (%) 384 (100) [M.sup.+]; HRMS (EI): Cacld for C.sub.21H.sub.15O.sub.2N.sub.2F.sub.3: 384.10801; found: 384.10765; IR (ATR, cm.sup.−1): 3365, 3080, 1768, 1694, 1445, 1385, 1315, 1163, 1118, 1036, 991, 764, 742, 657.

Example 2.15

3-(4-Fluorophenyl)-1-methyl-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione

(43) ##STR00034##

(44) Orange crystals; .sup.1H NMR (CDCl.sub.3) δ 2.21 (s, 3H), 3.20 (3, 3H), 6.96 (m, 3H), 7.03 (dd, 1H, J˜0.35, 7.75 Hz), 7.12 (ddd, 1H, J˜1.24, 6.93, 8.13 Hz), 7.25 (ddd, 1H), 7.59 (ddt, 2H, J˜2.90, 5.50, 8.48 Hz), 8.35 (br.s, 1H); .sup.13C NMR (CDCl.sub.3) δ 13.7, 24.2, 102.6, 110.6, 115.4, 115.7, 120.2, 120.6, 122.1, 126.22 (d, J˜3.60 Hz), 126.3, 131.4, 131.5, 132.87 (d, J˜1.07 Hz), 132.0, 135.8, 136.9, 162.89 (d, J=251.81 Hz), 171.1, 171.5; .sup.19F NMR (CDCl.sub.3) δ −109.8 (s); HRMS (EI): Cacld for C.sub.20H.sub.15O.sub.2N.sub.2F: 334.11121; found: 334.11137; IR (ATR, cm.sup.−1): 3380, 3042, 1755, 1700, 1600, 1508, 1458, 1427, 1379, 1232, 1159, 996, 841, 813, 750, 731, 657.

Example 2.16

3-(5-Acetyl-2-fluorophenyl)-1-methyl-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione

(45) ##STR00035##

(46) Deep red crystals; .sup.1H NMR (Aceton-d.sub.6) δ 2.29 (s, 3H), 2.49 (s, 3H), 3.12 (s, 3H), 6.80 (ddd, 1H, J˜1.06, 7.15, 8.37 Hz), 7.00 (m, 2H), 7.16 (dd, 1H, J=8.69, 9.51 Hz), 7.30 (dd, 1H, J=0.82, 8.69 Hz), 8.01 (ddd, 1H, J=2.34, 4.90, 8.57 Hz), 8.17 (dd, 1H, J=2.23, 6.79 Hz), 10.65 (br.s, 1H); .sup.13C NMR (Aceton-d.sub.6) δ 13.4, 24.1, 26.3, 103.5, 111.4, 116.6 (d, J=22.4 Hz), 119.9, 120.1 (d, J=16.2 Hz), 120.4, 122.0, 127.4, 128.7 (d, J=2.5 Hz), 131.8 (d, J=9.6 Hz), 133.0 (d, J=4.6 Hz), 134.2 (d, J=3.4 Hz), 136.7, 138.4, 138.9, 163.4 (d, J=259.4 Hz), 170.6, 170.8, 195.9; .sup.19F NMR (Aceton-d.sub.6) δ −102.9 (m); GC-MS (EI, 70 eV): m/z (%) 376 (100) [M.sup.+]; HRMS pos. (ESI): Calc for [M+H].sup.+, C.sub.22H.sub.18FN.sub.2O.sub.3: 377.1296; found: 377.1302; HRMS pos. (ESI): Calc for [M+Na].sup.+, C.sub.22H.sub.17FN.sub.2NaO.sub.3: 399.11154; found: 399.11152; IR (ATR, cm.sup.−1): 3351, 1689, 1645, 1602, 1439, 1386, 1353, 1250, 1223, 828, 778, 742, 630, 568, 436, 408.

Example 2.17

N-(4-(1-Methyl-4-(2-methyl-1H-indol-3-yl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl)phenyl)acetamide

(47) ##STR00036##

(48) Orange crystals; .sup.1H NMR (Aceton-d.sub.6) δ 2.05 (s, 3H), 2.27 (s, 3H), 3.07 (s, 3H), 6.83 (ddd, 1H, J˜0.98, 6.93, 8.06 Hz), 7.00 (d, 1H, J=7.60 Hz), 7.02 (ddd, 1H), 7.32 (ddd, 1H, J˜1.00, 2.09, 7.76 Hz), 7.53 (m, 4H), 9.27 (br.s, 1H), 10.59 (br.s, 1H); .sup.13C NMR (Aceton-d.sub.6) δ 13.3, 23.8, 24.0, 103.0, 111.3, 118.8 (2C), 120.1, 120.5, 121.8, 125.9, 127.2, 130.6 (2C), 132.6, 133.8, 136.9, 137.9, 140.7, 168.8, 171.4, 171.9; GC-MS (EI, 70 eV): m/z (%) 373 (100) [M.sup.+]; HRMS pos. (ESI): Calc for [M+H].sup.+, C.sub.22H.sub.20N.sub.3O.sub.3: 374.14992; found: 374.15012; HRMS pos. (ESI): Calc for [M+Na].sup.+, C.sub.22H.sub.19N.sub.3NaO.sub.3: 396.13186; found: 396.13226; IR (ATR, cm.sup.−1): 3379, 1675, 1582, 1505, 1424, 1403, 1386, 1365, 1310, 1237, 1179, 851, 815, 750, 653, 585, 567, 556, 532, 434, 379.

Example 3: Preparation 3—General Procedure for Preparation of Compounds of Formula (II) and (III) and Specific Compounds

(49) Step 1. The mixture of compound of formula (I) (1 mmol) wherein X is N—R.sup.1, and 100 ml of 10% aq KOH was heated at 140° C. until the mixture become homogenous (10 to 30 min, TLC control). Then the solution was cooled and acidified with 2N aq HCl, until precipitate was formed, which was collected, dried and recrystallized to give nearly quantitatively cyclic anhydride of formula (II).

(50) Step 2. Compound of formula (II) (1 mmol) was heated with ammonium acetate (100 mmol) at 140° C. until the mixture become homogenous (TLC control). The mixture was cooled down, water was added, and the mixture was extracted with ethyl acetate. The combined organics were washed with water, dried over Na.sub.2SO.sub.4 and concentrated. The crude material was crystallized from ether. The product of formula (III) was isolated by column chromatography in heptane/ethyl acetate.

Example 3.18

3-(2-Methyl-1H-indol-3-yl)-4-phenylfuran-2,5-dione

(51) ##STR00037##

(52) Red crystals; .sup.1H NMR (Aceton-d.sub.6) δ 2.31 (s, 3H), 6.86 (ddd, 1H, J 1.03, 7.06, 8.08 Hz), 7.02 (ddd, 1H), 7.07 (ddd, 1H, J˜1.14, 7.17, 8.21 Hz), 7.36 (m, 4H), 7.60 (m, 2H), 10.83 (br.s, 1H); .sup.13C NMR (Aceton-d.sub.6) δ 13.4, 102.2, 111.6, 120.6 (2C), 122.4, 126.7, 128.9 (2C), 129.9 (2C), 130.2, 130.4, 134.9, 136.1, 136.9, 139.7, 166.1, 166.3; GC-MS (EI, 70 eV): m/z (%) 303 (52) [M.sup.+]; HRMS (EI): Calc for C.sub.19H.sub.13O.sub.3N: 303.08899; found: 303.08861; IR (ATR, cm.sup.−1): 3350, 2921, 2852, 1825, 1749, 1618, 1456, 1423, 1252, 902, 741, 726, 693, 671, 635, 622, 564, 531.

Example 3.19

3-(2-Methyl-1H-indol-3-yl)-4-phenyl-1H-pyrrole-2,5-dione

(53) ##STR00038##

(54) Red crystals; .sup.1H NMR (Aceton-d.sub.6) δ 2.24 (s, 3H), 6.83 (ddd, 1H, J˜1.01, 7.08, 8.01 Hz), 7.02 (ddd, 1H), 7.03 (d, 1H, J=7.58 Hz), 7.27 (m, 3H), 7.31 (ddd, 1H), 7.54 (m, 2H), 9.83 (br.s, 1H), 10.56 (br.s, 1H); .sup.13C NMR (Aceton-d.sub.6) δ 13.2, 102.8, 111.2, 120.1, 120.5, 121.8, 127.3, 128.6 (2C), 129.3, 130.0 (2C), 131.3, 134.8, 134.9, 136.8, 138.0, 171.7, 172.2; GC-MS (EI, 70 eV): m/z (%) 302 (100) [M.sup.+]; HRMS (EI): Calc for C.sub.19H.sub.14O.sub.2N.sub.2: 302.10498; found: 302.105426; IR (ATR, cm.sup.−1): 3379, 3205, 3065, 2917, 2764, 1764, 1704, 1598, 1451, 1423, 1335, 1289, 1278, 1227, 1013, 993, 770, 754, 729, 719, 690.

Example 3.20

3-(2-Methyl-1H-indol-3-yl)-4-(naphthalen-2-yl)furan-2,5-dione

(55) ##STR00039##

(56) Orange crystals; .sup.1H NMR (CDCl.sub.3) δ 2.30 (s, 3H), 6.81 (ddd, 1H), 7.05 (ddd, 2H), 7.37 (ddd, 1H), 7.53 (m, 3H), 7.75 (d, 1H, J˜8.62 Hz), 7.87 (m, 2H), 8.36 (s, 1H), 10.86 (s, 1H); .sup.13C NMR (CDCl.sub.3) δ 13.3, 102.5, 111.6, 120.6, 120.7, 122.4, 126.0, 126.9, 127.2, 127.7, 128.16, 128.24, 128.4, 129.3, 130.7, 133.5, 134.1, 134.7, 136.1, 136.9, 139.9, 166.1, 166.4; MS (EI): m/z (%) 353 (650) [M.sup.+]; HRMS pos. (ESI): Calc for [M+H].sup.+, C.sub.23H.sub.16NO.sub.3: 354.11247; found: 354.11221; HRMS pos. (ESI): Calc for [M+Na].sup.+, C.sub.23H.sub.15NNaO.sub.3: 376.09441; found: 376.09419; IR (ATR, cm.sup.−1): 3366, 2926, 1757, 1460, 1428, 1259, 1242, 1222, 1158, 910, 784, 766, 737, 590, 554, 475.

Example 3.21

3-(2-Methyl-1H-indol-3-yl)-4-(naphthalen-2-yl)-1H-pyrrole-2,5-dione

(57) ##STR00040##

(58) Orange crystals; .sup.1H NMR (Aceton-d.sub.6) δ 2.24 (s, 3H), 6.77 (ddd, 1H, J˜1.04, 7.12, 8.07 Hz), 7.00 (ddd, 1H, J 1.15, 7.10, 8.07 Hz), 7.08 (d, 1H, J=8.0 Hz), 7.33 (ddd, 1H, J=0.85, 8.08), 7.48 (m, 3H), 7.66 (d, 1H, J=8.8 Hz), 7.80 (m, 1H), 7.84 (m, 1H), 8.30 (d, 1H, J=0.72 Hz), 9.90 (br.s, 1H), 10.60 (br.s, 1H); .sup.13C NMR (Aceton-d.sub.6) δ 13.3, 103.1, 111.3, 120.2, 120.5, 121.9, 126.6, 126.9, 127.5 (2C), 128.0, 128.1, 128.9, 129.1, 130.4, 133.6, 133.7, 134.5, 135.0, 136.8, 138.3, 171.7, 172.3; MS (EI): m/z (%) 352 (100) [M.sup.+]; HRMS (EI): Calc for C.sub.23H.sub.16O.sub.2N.sub.2: 352.12063; found: 352.120553; IR (ATR, cm.sup.−1): 3379, 3209, 3062, 2959, 2925, 2738, 1762, 1702, 1621, 1457, 1426, 1329, 1290, 1222, 1034, 993, 858, 826, 786, 754, 742, 715, 670, 662.

Example 3.22

3-(4-Acetylphenyl)-4-(2-methyl-1H-indol-3-yl)furan-2,5-dione

(59) ##STR00041##

(60) Red-orange crystals; .sup.1H NMR (CDCl.sub.3) δ 2.33 (s, 3H), 2.56 (s, 3H), 6.86 (ddd, 1H, J˜1.0, 7.04, 8.08 Hz), 6.99 (br. d, 1H, J˜8.0 Hz), 7.07 (ddd, 1H, J˜1.22, 7.02, 8.12 Hz), 7.34 (ddd, 1H, J˜0.80, 0.84, 8.10), 7.72 (ddd, 2H), 7.94 (ddd, 2H), 10.98 (br.s, 1H); .sup.13C NMR (CDCl.sub.3) δ 13.5, 26.4, 102.3, 111.7, 120.6, 120.8, 122.5, 126.5, 128.6 (2C), 130.1 (2C), 133.3, 134.5, 136.9, 137.5, 138.0, 140.4, 165.9, 166.0, 197.2; GC-MS (EI, 70 eV): m/z (%) 345 (87) [M.sup.+]; HRMS (EI): Calc for C.sub.21H.sub.15O.sub.4N: 345.09956; found: 345.09942; IR (ATR, cm.sup.−1): 3233, 2921, 2852, 1759, 1671, 1460, 1252, 1186, 1112, 924, 831, 747, 731, 628, 591, 578, 516, 456, 434, 416.

Example 3.23

3-(4-Acetylphenyl)-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione

(61) ##STR00042##

(62) Orange crystals; .sup.1H NMR (Aceton-d.sub.6) δ 2.28 (s, 3H), 2.53 (s, 3H), 6.82 (ddd, 1H, J˜1.03, 7.07, 8.03 Hz), 6.99 (br. d, 1H, J˜7.40 Hz), 7.02 (ddd, 1H, J˜1.12, 7.07, 8.11 Hz), 7.33 (ddd, 1H, J˜0.81, 0.95, 8.08), 7.66 (ddd, 2H), 7.87 (ddd, 2H), 9.93 (br.s, 1H), 10.67 (br.s, 1H); .sup.13C NMR (Aceton-d.sub.6) δ 13.4, 26.4, 102.8, 111.4, 120.3, 120.5, 122.0, 127.1, 128.4 (2C), 130.2 (2C), 133.3, 135.9, 136.3, 136.9, 137.2, 138.7, 171.4, 171.8, 197.2; GC-MS (EI, 70 eV): m/z (%) 344 (100) [M.sup.+]; HRMS (ED: Calc for C.sub.21H.sub.16O.sub.3N.sub.2: 344.11554; found: 344.11495; IR (ATR, cm.sup.−1): 3343, 3296, 3057, 1757, 1699, 1676, 1428, 1343, 1262, 1230, 740, 666, 638, 595, 460, 409.

Example 3.24

3-(4-Acetylphenyl)-4-(1,2-dimethyl-1H-indol-3-yl)-1-methyl-1H-pyrrole-2,5-dione

(63) ##STR00043##

(64) Dark red crystals; .sup.1H NMR (DMSO-d.sub.6) δ 2.18 (s, 3H), 2.52 (s, 3H), 3.03 (s, 3H), 3.71 (s, 3H), 6.84 (ddd, 1H), 6.93 (br.d, 1H, J 7.54 Hz), 7.08 (ddd, 1H, J˜1.06, 7.08, 8.14 Hz), 7.45 (br.d, 1H, J 8.25 Hz), 7.56 (br.d, 2H, J 8.50 Hz), 7.86 (br.d, 2H, J 8.50 Hz); .sup.13C NMR (DMSO-d.sub.6) δ 12.3, 24.2, 26.8, 29.9, 101.3, 109.9, 119.7, 119.9, 121.3, 125.1, 128.1 (2C), 129.3 (2C), 131.6, 134.6, 135.1, 136.2, 137.1, 139.6, 170.4, 170.7, 197.5; GC-MS (EI, 70 eV): m/z (%) 372 (100) [M.sup.+]; HRMS pos. (ESI): Calc for [M+H].sup.+, C.sub.23H.sub.21N.sub.2O.sub.3: 373.15467; found: 373.15473; IR (ATR, cm.sup.−1): 3433, 2915, 1759, 1680, 1599, 1433, 1404, 1382, 1359, 1265, 1240, 957, 849, 829, 749, 738, 726, 595, 546, 439.

Example 3.25

3-(4-Acetylphenyl)-1-methyl-4-(1H-pyrrolo[2,3-b]pyridin-3-yl)-1H-pyrrole-2,5-dione

(65) ##STR00044##

(66) Yellow crystals; .sup.1H NMR (DMSO-d.sub.6) δ 2.57 (s, 3H), 3.04 (s, 3H), 6.66 (dd, 1H, J˜0.89, 8.00 Hz), 6.80 (dd, 1H, J˜4.74, 7.96 Hz), 7.55 (br.d, 2H, J˜8.27 Hz), 7.93 (br.d, 2H, J˜8.20 Hz), 8.11 (s, 1H), 8.18 (br.d, 2H, J˜3.70 Hz), 12.55 (br.s, 1.H); .sup.13C NMR (DMSO-d.sub.6) δ 24.1, 26.8, 102.8, 116.28, 116.34, 128.0 (2C), 128.4, 129.1, 129.9 (2C), 131.8, 132.5, 134.8, 136.5, 143.7, 149.0, 170.6, 170.8, 197.5; GC-MS (EI, 70 eV): m/z (%) 345 (100) [M.sup.+]; HRMS pos. (ESI): Calc for [M+H].sup.+, C.sub.20H.sub.16N.sub.3O.sub.3: 346.11862; found: 346.11828; IR (ATR, cm.sup.−1): 3025, 2873, 2817, 1756, 1695, 1677, 1440, 1421, 1385, 1289, 1269, 1229, 1090, 814, 776, 750, 645, 596, 514.

Example 4: GSK3β Kinase Activity Assay

(67) The kinase activity assay was performed as previously described by Schmole et. al., 2010 (Schmole et al., 2010, Novel indolylmaleimide acts as GSK-3beta inhibitor in human neural progenitor cells. Bioorganic medicinal chemistry, 18, 6785-95). Briefly, recombinant human GSK3β (Biomol, Hamburg, Germany) was incubated with its substrate phospho glycogen synthase peptide 2 (pGS2) (Millipore, Billerica, USA), ATP (Cell Signaling, Frankfurt am Main, Germany) and different concentrations of PDA-66 for 30 min at 30° C. After addition of Kinase-Glo (Promega, Mannheim, Germany) and 10 min of incubation at room temperature the luminescence signal was measured with a Glomax 96 microplate reader (Promega). More specifically, recombinant human GSK3β was incubated with pGS2, ATP and different concentrations of PDA-66. Kinase activity was significantly inhibited at concentrations between 0.25 μM and 1 μM of PDA-66. Results are displayed as the mean±SD of two independent experiments. In each experiment the concentrations of PDA-66 and the control were tested with 8 replicates. * Significant treatment effect vs. DMSO control, α=0.05.

(68) PDA-66 Inhibits Kinase Activity of Recombinant GSK3β

(69) The effect of PDA-66 on the enzyme activity of GSK3β was determined by incubation of the enzyme with a specific substrate, PDA-66 and ATP. With growing inhibitory effect there is more ATP present after the incubation. In a second step remaining ATP is converted to a luminescence signal, which is inversely proportional to the enzyme activity. The analysis of kinase activity of GSK3β in our study shows a bell shaped dose response relationship (FIG. 1). The incubation with 0.25-1 μM of PDA-66 led to significant increase of luminescence signal and therefore an inhibition of the enzyme, whereas 10 μM seems to enhance enzyme activity.

Example 5: Treatment of ALL Cell Lines with PDA-66

(70) The human B-ALL cell lines SEM, RS4;11 and the human T-ALL cell lines Jurkat and Molt-4 were purchased from DSMZ (Germany) and cultured according to manufacturer's protocol. The corresponding medium was supplemented with 10% heat-inactivated fetal bovine serum (PAA, Pasching, Austria) and 1% penicillin and streptomycin (Biochrom AG, Berlin, Germany). The Molt-4 cells were cultured with medium supplemented with 20% heat-inactivated fetal bovine serum. All cells were maintained at 37° C. in 5% CO.sub.2. Cells (5×10.sup.5/well) were seeded in 24 well plates (Nunc, Langenselbold, Germany) and incubated for up to 72 h with PDA-66. Treated cells were harvested after 4, 24, 48 and 72 h and used for further analyses.

(71) Cell counts were determined using the trypan blue staining. Metabolic activity was analyzed by using tetrazolium compound WST-1 (Roche, Mannheim, Germany). In brief, triplicates of cells (5×10.sup.4/well) were seeded in 96 well plates, treated with PDA-66 and incubated with 15 μl WST-1 for up to 4 h. The mitochondrial dehydrogenases reduce WST-1 to soluble formazan and cause a change of color, which correlates with the amount of metabolically active cells. Absorbance at 450 nm and a reference wavelength at 620 nm were determined by an ELISA Reader (Anthos, Krefeld, Germany). The absorbance of culture medium with supplemented WST-1 in the absence of cells was used as background control.

(72) PDA-66 Inhibits Proliferation and Metabolic Activity of ALL Cells

(73) The influence of PDA-66 on proliferation in ALL cell lines SEM, RS4;11, Jurkat and Molt-4 was analyzed by incubation with different concentrations of the drug (0.001 μM to 10 μM). Metabolic activity was determined using WST-1 assay. The proliferation and metabolic activity of all cell lines was suppressed significantly at higher concentrations. Results are displayed as the mean±SD of three independent experiments. * Significant treatment effect vs. DMSO control, α=0.05. Results are summarized in FIG. 2, whereby the individual nine bars for each set of experiments represent, from left to right, DMSO, 0.001 μM, 0.01 μM, 0.1 μM, 0.25 μM, 0.5 μM, 1.0 μM, 5 μM and 10 μM. After 48 h of incubation an inhibition of proliferation could be observed, but was more distinct after 72 h. There was a significant inhibition after 72 h in all cell lines at a concentration of 0.5 μM PDA-66.

(74) Similar results could be detected in WST-1 assay. After 72 h of incubation the metabolic activity was significantly decreased in all cell lines at a concentration of 0.5 μM PDA-66. At this concentration the metabolic activity decreased to 35.7±8.3% in SEM, 33.3±4.4% in RS4;11, 66.7±8% in Jurkat and 35.5±17% in Molt-4 cells compared to control cells treated with DMSO. In WST-1 assay the IC50 concentrations for PDA-66 in all four cell lines where determined (Table 1). The IC50 values range from 0.41 μM in SEM cells to 1.28 μM in Jurkat cells after 72 h of incubation.

(75) The incubation of ALL cell lines with higher dosages of PDA-66 (0.5 μM or more) led to a decrease in cell numbers i.e. below the amount of seeded cells (5×10.sup.5). This result indicates not only an influence on proliferation but also an induction of cell death.

Example 6: May Grunwald-Giemsa Staining

(76) Cytospins of SEM and Jurkat cells were stained with May Grunwald-Giemsa Staining after 48 h of incubation with 1 μM PDA-66 and DMSO, respectively

(77) After treatment with 1 μM PDA-66 3×10.sup.4 cells were brought onto object slides with Cytospin 3 centrifuge (Shandon, Frankfurt/Main, Germany). Subsequently cells were stained using May-Grunwald-Giemsa staining. Briefly, slides were incubated in May-Grunwald solution (Merck, Darmstadt, Germany) for 6 min, then washed with tap water, incubated in Giemsa solution (Merck, Darmstadt, Germany) for 20 min and washed in tap water again. After letting the slides dry the cells were analyzed by Nikon Eclipse E 600 light microscope and pictures were taken with NIS Elements software (Nikon, Düsseldorf, Germany).

(78) PDA-66 Influences Morphology of ALL Cells

(79) The analysis via light microscopy showed an influence on the morphology of all four cell lines. After treatment an increased amount of cells with chromatin condensation (black arrow a in FIG. 3) and karyorrhexis (black arrow b in FIG. 3) could be observed along with more cell debris. In contrast to DMSO treated control cells the incubation of PDA-66 led to condensation of chromatin in the nucleus, karyorrhexis and an increasing amount of vacuoles and cell debris after 48 h of treatment (FIG. 3). Condensated chromatin might hint to induction of apoptosis on the one hand or cell cycle arrest on the other hand.

Example 7: Cell Cycle Analysis

(80) After treatment cells were harvested and washed twice in PBS. Cells were fixed with 70% ethanol and incubated with 1 mg/ml Ribonuclease A (Sigma-Aldrich, St. Louis, USA) for 30 min at 37° C. After washing the cells twice in PBS, they were stained with PI (50 μg/ml) and DNA content was determined by flow cytometry. All cell lines were incubated with PDA-66 and cell cycle distribution was determined using Propidium iodide staining.

(81) The treatment with PDA-66 influenced the four cell lines differently. Results are displayed in FIG. 4, whereby the individual four bars for each set of experiments represent, from left to right, DMSO, 0.25 μM, 0.5 μM and 1.0 μM. G2 arrest could be detected in RS4;11 and Molt-4 cells after 48 h of treatment. Treatment of Jurkat cells induced a decrease of cells in G0/G1 phase in favor of cells in S phase. Results are displayed as the mean±SD of three independent experiments. * Significant treatment effect vs. DMSO control, α=0.05.

(82) SEM cells showed a significant increase in the amount of cells in G0/G1 after 48 h of incubation with 0.5 μM (DMSO control: 62.8±2.8%; 0.5 μM PDA-66: 69.3±2.7%) whereas 1 μM did not affect the cell cycle significantly. RS4;11 and Molt-4 cells were characterized by a significant G2 arrest 48 h after treatment with 1 μM PDA-66. The amount of RS4;11 and Molt-4 cells in G2 phase increased from 20.1±3.9% and 21.9±4.9% after incubation with DMSO to 42.1±4.4% and 41.0±5.8% after PDA-66 treatment. This was associated with a significant decrease in G0/G1 phase (RS4;11 and Molt-4: 65.7±2.1% and 63.7±6.6% in control; 47.3±2.7% and 45.0±7.3% after treatment with 1 μM PDA-66). On the other hand smaller dosages led to significant increase of cells in G0/G1 phase. Jurkat cells showed a significant decrease in G0/G1 phase (from 60.0±3.7% in control to 47.3±4.3% with PDA-66) and an increase in S phase (from 14.6±1.5% in control to 20.0±1.1% with PDA-66) after incubation with 1 μM PDA-66.

Example 8: Analyses of Apoptosis and Necrosis

(83) Apoptosis and necrosis were analyzed by staining the cells with Annexin V FITC (BD Biosciences, Heidelberg, Germany) and propidium iodide (PI) (Sigma Aldrich, St. Louis, USA). Results were assessed by flow cytometry.

(84) (A) Cells were treated with PDA-66 for up to 72 h and stained with Annexin V FITC and Propidium iodide (PI). Rates of early apoptotic (FITC.sup.+, PI.sup.−) and late apoptotic and necrotic (FITC.sup.+, PI.sup.+) cells were measured by flow cytometry. Significant induction of apoptosis could be observed in all cell lines after 48 h of incubation as well as tendential induction of necrosis at both points of time. Results are displayed as the mean±SD of three independent experiments. * Significant treatment effect vs. DMSO control, α=0.05.
(B) Induction of apoptosis was confirmed by Western blot. Cells were treated with different concentrations of PDA-66 and total cell lysates (25 μg) were analyzed by Western blot to detect cleavage of Caspase 3, 7 and PARP. GAPDH was used as loading control. Exemplary results of SEM cells are displayed.

(85) More specifically, 5×10.sup.5 cells were harvested and washed twice (180 g, 10 min, 4° C.) with PBS. After resuspending the cells in 100 μl of binding buffer (1×) 4 μl of Annexin V FITC were added and incubated for 15 min at room temperature. Following addition of 400 μl binding buffer for a final volume of 500 μl the cells were stained with PI (0.6 μg/ml) immediately before measurement. As controls unstained and single stained cells were included in each experiment. Measurements were performed using FACSCalibur (Becton and Dickinson, Heidelberg, Germany) and data analyses were carried out with CellQuest software (Becton and Dickinson, Heidelberg, Germany).

(86) PDA-66 Induces Apoptosis

(87) Additionally, the effect of PDA-66 on apoptosis was determined by and western blot. For protein extraction cells were washed twice in PBS and lysed with RIPA buffer (50 mM Tris HCl pH 7.4; 150 mM NaCl; 0.1% SDS and 1% NP40) including protease and phosphatase inhibitors (Roche Applied Science, Mannheim, Germany). Samples were incubated for 20 min at 4° C. and frozen at −20° C. Cell extracts were thawed and centrifuged at 12000 g for 10 min at 4° C. Total protein concentration of supernatants was determined using Bio-Rad Protein Assay (Bio-Rad, München, Germany).

(88) Equal amounts of protein samples were separated by SDS-polyacrylamid gel (8% or 15%) electrophoresis and transferred onto a PVDF membrane (Amersham Biosciences, Buckinghamshire, UK). Membranes were blocked in 5% milk or 5% BSA and incubated at 4° C. overnight with the following polyclonal antibodies: rabbit anti-cleaved caspase 3, rabbit anti-caspase 3, rabbit anti-cleaved PARP, rabbit anti-cleaved caspase 7, rabbit anti-caspase 7 (all Cell Signaling Frankfurt/Main, Germany. Blots were incubated with mouse anti-GAPDH (Invitrogen, Carlsbad, USA) as loading control. Specific horseradish peroxidase-conjugated secondary antibodies (anti-mouse or anti-rabbit) were used. Signals were detected with ECL Plus reagent (Amersham Biosciences; Buckinghamshire, UK) and a CCD camera (Kodak Digital Science Image Station 440CF, Rochester, USA).

(89) After 48 h of incubating the cells with 1 μM PDA-66 all cell lines showed a significant increase in apoptosis compared to control cells (SEM: 2.1±0.9% to 10.5±1.3%; RS4;11: 2.5±0.7% to 7.4±1.1%; Jurkat: 3.8±0.6% to 8.3±1.9%; Molt-4: 3.7±1.2% to 16.3±5.1%). After 72 h a similar tendency could be observed, but only deviations in SEM and Molt-4 cells where significant (SEM: 1.3±0.4% to 5.6±1.6%; RS4;11: 2.1±0.9% to 6.4±3.6%; Jurkat: 4.7±1.9% to 6.1±0.7%; Molt-4: 4.9±1.9% to 20.1±6.6%). All cells showed a tendential increase in necrosis after 48 and 72 h of incubation with 1 μM PDA-66. After 72 h of incubation necrosis rate rose in SEM cells from 3.1±1.6% to 27.8±5.81%, in RS4;11 cells from 6.1±0.8% to 26.5±10.2%, in Jurkat cells from 5.7±3.5% to 28.0±13.4% and in Molt-4 cells from 11.7±3.6% to 46.7±15.6% (FIG. 5A, whereby the individual four bars for each set of experiments represent, from left to right, DMSO, 0.25 μM, 0.5 μM and 1.0 μM).

(90) Analysis via Western blot showed an apoptosis induction in all cell lines. Treatment with PDA-66 induced cleavage of caspases 3 and 7 and PARP 48 h after addition of PD066. In FIG. 5B results of SEM cells are displayed exemplarily.

Example 9: Influence on PI3K/Akt and Wnt-Pathway

(91) After treatment with PDA-66 and DMSO, respectively, cells were lyzed and protein expression analyzed with Western blot.

(92) Protein extraction and Western blot was performed as described above. Following polyclonal antibodies were used: rabbit anti-cleaved caspase 3, rabbit anti-caspase 3, rabbit anti-cleaved PARP, rabbit anti-PARP, rabbit anti-cleaved caspase 7, rabbit anti-caspase 7, rabbit anti-pAktThr308, rabbit anti-pAktSer473, rabbit anti-Akt, rabbit anti-β-catenin, rabbit anti-pGSK3βSer9, rabbit anti-GSK3β, rabbit anti-p4EBP-1Ser65 and rabbit anti-4EBP-1 (all Cell Signaling Frankfurt/Main, Germany). Blots were incubated with mouse anti-GAPDH antibody (Invitrogen, Carlsbad, USA) as loading control.

(93) PDA-66 Influences Protein Expression of 4EBP-1, but not β-Catenin or GSK3β

(94) To characterize the effects of PDA-66 on PI3K/Akt and Wnt/β-catenin pathway we performed Western blot analysis. As may be taken from FIG. 6A, no influence on expression of total GSK3β and the total form of Akt could be noticed. A small decrease of pGSK3βSer9 was observed, but no influence on the amount of β-catenin. A slight increase of pAktThr308 was not confirmed by an increase of pAktSer473. Exemplary results of SEM cells are displayed. As may be taken from FIG. 6B, in SEM and RS4;11 cells a decrease of 4 EBP-1 and p4EBP-1Ser65 was detectable, in contrast Molt-4 cells showed an increased expression of p4EBP-1Ser65 after PDA-66 treatment.

(95) More specifically, the incubation with PDA-66 showed no influence on the expression of β-catenin, total GSK3β and total Akt (FIG. 6). However, an increase of pAktThr308 could be detected in SEM and Jurkat cells after an incubation of 24 h, which was not confirmed by a rise of pAktSer473. Furthermore, in SEM cells a slight decrease of pGSK3βSer9 was observed after 4 h. However, no influence on the total form of β-catenin was detectable. This would not account for an increase of GSK3β activation. Nevertheless, there was an influence of PDA-66 on the expression of 4EBP-1 and p4EBP-1Ser65. SEM, RS4;11 and Jurkat cells showed a decrease of the phosphorylated as well as the total form of 4EBP-1 after an incubation of 4 and 24 h. In contrast, Molt-4 cells displayed an increase of the phosphorylated form to these points of time.

(96) The features of the present invention disclosed in the specification, the claims, the sequence listing and/or the drawings may both separately and in any combination thereof be material for realizing the invention in various forms thereof.