Treatment of Pluripotent Cells

• US 20140080210A1
• Filed: 11/20/2013
• Published: 03/20/2014
• Est. Priority Date: 04/24/2008
• Status: Active Grant

• Current Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Original Assignee: Janssen Biotech, Inc. (Johnson & Johnson)
• Inventors: Davis, Janet, Liu, Jiajian
• Granted Patent: US 9,845,460 B2
• Time in Patent Office: Days
• US Class Current: 435/377
• CPC Class Codes: A61K 31/00 Medicinal preparations containing organic active ingredients
• CPC Class Codes: C12N 2501/105 Insulin-like growth factors [IGF]
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/117 Keratinocyte growth factors (KGF-1, i.e. FGF-7; KGF-2, i.e. FGF-12)
• CPC Class Codes: C12N 2501/119 Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
• CPC Class Codes: C12N 2501/16 Activin; Inhibin; Mullerian inhibiting substance
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/48 Regulators of apoptosis
• CPC Class Codes: C12N 2501/70 Enzymes
• CPC Class Codes: C12N 2506/00 Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/0676 Pancreatic cells

Abstract

The present invention is directed to methods to treat pluripotent cells, whereby the pluripotent cells can be efficiently expanded in culture and differentiated by treating the pluripotent cells with an inhibitor of GSK-3B enzyme activity.

Claims

1. A method to expand and differentiate pluripotent cells, comprising the steps of:

a. Culturing pluripotent cells, and

b. Treating the pluripotent cells with an inhibitor of GSK-3B enzyme activity.

2. The method of claim 1, wherein the pluripotent cells are embryonic stem cells.

3. The method of claim 1, wherein the pluripotent cells are cells expressing pluripotency markers derived from embryonic stem cells.

4. The method of claim 3, wherein the cells expressing pluripotency markers express at least one of the following pluripotency markers selected from the group consisting of:

ABCG2, cripto, FoxD3, Connexin43, Connexin45, Oct4, SOX-2, Nanog, hTERT, UTF-1, ZFP42, SSEA-3, SSEA-4, Tra1-60, and Tra1-81.

5. The method of claim 1, wherein the pluripotent cells are differentiated into cells expressing markers characteristic of the definitive endoderm lineage.

6. The method of claim 1, wherein the pluripotent cells are treated with the inhibitor of GSK-3B enzyme activity for about one to about 72 hours.

7. The method of claim 1, wherein the pluripotent cells are treated with the inhibitor of GSK-3B enzyme activity for about 12 to about 48 hours.

8. The method of claim 1, wherein the pluripotent cells are treated with the inhibitor of GSK-3B enzyme activity for about 48 hours.

9. The method of claim 1, wherein the inhibitor of GSK-3B enzyme activity is used at a concentration of about 100 nM to about 100 μ

M.

10. The method of claim 1, wherein the inhibitor of GSK-3B enzyme activity is used at a concentration of about 1 μ

M to about 10 μ

M.

11. The method of claim 1, wherein the inhibitor of GSK-3B enzyme activity is used at a concentration of about 10 μ

M.

12. The method of claim 1, wherein the inhibitor of GSK-3B enzyme activity is a compound of the Formula (I):

13. The method of claim 12, wherein R₁ is phenyl, substituted phenyl wherein the phenyl substituents are selected from the group consisting of C_1-5alkyl, halogen, nitro, trifluoromethyl and nitrile, or pyrimidinyl.

14. The method of claim 12, wherein R₂ is phenyl, substituted phenyl wherein the phenyl substituents are selected from the group consisting of C_1-5alkyl, halogen, nitro, trifluoromethyl and nitrile, or pyrimidinyl which is optionally C_1-4alkyl substituted, and at least one of R₁ and R₂ is pyrimidinyl.

15. The method of claim 12, wherein R₃ is hydrogen, 2-(trimethylsilyl)ethoxymethyl, C_1-5alkoxycarbonyl, aryloxycarbonyl, arylC_1-5alkyloxycarbonyl, arylC_1-5alkyl, substituted arylC_1-5alkyl wherein the one or more aryl substituents are independently selected from the group consisting of C_1-5alkyl, C_1-5alkoxy, halogen, amino, C_1-5alkylamino, and diC_1-5alkylamino, phthalimidoC_1-5alkyl, aminoC_1-5alkyl, diaminoC_1-5alkyl, succinimidoC_1-5alkyl, C_1-5alkylcarbonyl, arylcarbonyl, C_1-5alkylcarbonylC_1-5alkyl and aryloxycarbonylC_1-5alkyl.

16. The method of claim 12, wherein R₄ is -(A)-(CH₂)_q—

X.

17. The method of claim 16, wherein A is vinylene, ethynylene or

18. The method of claim 17, wherein R₅ is selected from the group consisting of hydrogen, C_1-5alkyl, phenyl and phenylC_1-5alkyl.

19. The method of claim 16, wherein q is 0-9.

20. The method of claim 16, wherein X is selected from the group consisting of hydrogen, hydroxy, vinyl, substituted vinyl wherein one or more vinyl substituents are each selected from the group consisting of fluorine, bromine, chlorine and iodine, ethynyl, substituted ethynyl wherein the ethynyl substituents are selected from the group consisting of fluorine, bromine chlorine and iodine, C_1-5alkyl, substituted C_1-5alkyl wherein the one or more alkyl substituents are each selected from the group consisting of C_1-5alkoxy, trihaloalkyl, phthalimido and amino, C_3-7cycloalkyl, C_1-5alkoxy, substituted C_1-5alkoxy wherein the alkyl substituents are selected from the group consisting of phthalimido and amino, phthalimidooxy, phenoxy, substituted phenoxy wherein the one or more phenyl substituents are each selected from the group consisting of C_1-5alkyl, halogen and C_1-5alkoxy, phenyl, substituted phenyl wherein the one or more phenyl substituents are each selected from the group consisting of C_1-5alkyl, halogen and C_1-5alkoxy, arylC_1-5alkyl, substituted arylC_1-5alkyl wherein the one or more aryl substituents are each selected from the group consisting of C_1-5alkyl, halogen and C_1-5alkoxy, aryloxyC_1-5alkylamino, C_1-5alkylamino, diC_1-5alkylamino, nitrile, oxime, benxyloxyimino, C_1-5alkyloxyimino, phthalimido, succinimido, C_1-5alkylcarbonyloxy, phenylcarbonyloxy, substituted phenylcarbonyloxy wherein the one or more phenyl substituents are each selected from the group consisting of C_1-5alkyl, halogen and C_1-5alkoxy, phenylC_1-5alkylcarbonyloxy wherein the one or more phenyl substituents are each selected from the group consisting of C_1-5alkyl, halogen and C_1-5alkoxy, aminocarbonyloxy, C_1-5alkylaminocarbonyloxy, diC_1-5alkylaminocarbonyloxy, C_1-5alkoxycarbonyloxy, substituted C_1-5alkoxycarbonyloxy wherein the one or more alkyl substituents are each selected from the group consisting of methyl, ethyl, isopropyl and hexyl, phenoxycarbonyloxy, substituted phenoxycarbonyloxy wherein the one or more phenyl substituents are each selected from the group consisting of C_1-5alkyl, C_1-5alkoxy and halogen, C_1-5alkylthio, substituted C_1-5alkylthio wherein the alkyl substituents are selected from the group consisting of hydroxy and phthalimido, C_1-5alkylsulfonyl, phenylsulfonyl, substituted phenylsulfonyl wherein the one or more phenyl substituents are each selected from the group consisting of bromine, fluorine, chloride, C_1-5alkoxy and trifluoromethyl;

with the proviso that if A is

21. The method of claim 12, wherein R₁ is substituted phenyl and R₂ is pyrimidin-3-yl.

22. The method of claim 12, wherein R₁ is 4-fluorophenyl.

23. The method of claim 12, wherein R₃ is hydrogen, arylC_1-5alkyl, or substituted arylC_1-5alkyl.

24. The method of claim 12, wherein R₃ is hydrogen or phenylC_1-5alkyl.

25. The method of claim 16, wherein A is ethynylene and q is 0-5.

26. The method of claim 16, wherein X is succinimido, hydroxy, methyl, phenyl, C_1-5alkylsulfonyl, C_3-6cycloalkyl, C_1-5alkylcarbonyloxy, C_1-5alkoxy, phenylcarbonyloxy, C_1-5alkylamino, diC_1-5alkylamino or nitrile.

27. The method of claim 12, wherein the compound of the Formula I is 4-(4-fluorophenyl)-2-(4-hydroxybutyn-1-yl)-1-(3-phenylpropyl)-5-(4-pyridyl)imidazole.

28. The method of claim 1, wherein the inhibitor of GSK-3B enzyme activity is a compound of the Formula (II):

29. The method of claim 28, wherein R is selected from the group consisting of R_a, —

C_1-8alkyl-R_a, —

C_2-8alkenyl-R_a, —

C_2-8alkynyl-R_a and cyano.

30. The method of claim 29, wherein R_a is selected from the group consisting of cycloalkyl, heterocyclyl, aryl and heteroaryl.

31. The method of claim 28, wherein R¹ is selected from the group consisting of hydrogen, —

C_1-8alkyl-R⁵, —

C_2-8alkenyl-R⁵, —

C_2-8alkynyl-R⁵, —

C(O)—

(C_1-8)alkyl-R⁹, —

C(O)-aryl-R⁸, —

C(O)—

O—

(C_1-8)alkyl-R⁹, —

C(O)—

O-aryl-R⁸, —

C(O)—

NH(C_1-8alkyl-R⁹), —

C(O)—

NH(aryl-R⁸), —

C(O)—

N(C_1-8alkyl-R⁹)₂, —

SO₂—

(C_1-8)alkyl-R⁹, —

SO₂-aryl-R⁸, -cycloalkyl-R⁶, -heterocyclyl-R⁶, -aryl-R⁶ and -heteroaryl-R⁶;

wherein heterocyclyl and heteroaryl are attached to the azaindole nitrogen atom in the one position via a heterocyclyl or heteroaryl ring carbon atom.

32. The method of claim 31, wherein R⁵ is 1 to 2 substituents independently selected from the group consisting of hydrogen, —

O—

(C_1-8)alkyl, —

O—

(C_1-8)alkyl-OH, —

O—

(C_1-8)alkyl-O—

(C_1-8)alkyl, —

O—

(C_1-8)alkyl-NH₂, —

O—

(C_1-8)alkyl-NH(C_1-8alkyl), —

O—

(C_1-8)alkyl-N(C_1-8alkyl)₂, —

O—

(C_1-8)alkyl-S—

(C_1-8)alkyl, —

O—

(C_1-8)alkyl-SO₂—

(C_1-8)alkyl, —

O—

(C_1-8)alkyl-SO₂—

NH₂, —

O—

(C_1-8)alkyl-SO₂—

NH(C_1-8alkyl), —

O—

(C_1-8)alkyl-SO₂—

N(C_1-8alkyl)₂, —

O—

C(O)H, —

O—

C(O)—

(C_1-8)alkyl, —

O—

C(O)—

NH₂, —

O—

C(O)—

NH(C_1-8alkyl), —

O—

C(O)—

N(C_1-8alkyl)₂, —

O—

(C_1-8)alkyl-C(O)H, —

O—

(C_1-8)alkyl-C(O)—

(C_1-8)alkyl, —

O—

(C_1-8)alkyl-CO₂H, —

O—

(C_1-8)alkyl-C(O)—

O—

(C_1-8)alkyl, —

O—

(C_1-8)alkyl-C(O)—

NH₂, —

O—

(C_1-8)alkyl-C(O)—

NH(C_1-8alkyl), —

O—

(C_1-8)alkyl-C(O)—

N(C_1-8alkyl)₂, —

C(O)H, —

C(O)—

(C_1-8)alkyl, —

CO₂H, —

C(O)—

O—

(C_1-8)alkyl, —

C(O)—

NH₂, —

C(NH)—

NH₂, —

C(O)—

NH(C_1-8alkyl), —

C(O)—

N(C_1-8alkyl)₂, —

SH, —

S—

(C_1-8)alkyl, —

S—

(C_1-8)alkyl-S—

(C_1-8)alkyl, —

S—

(C_1-8)alkyl-O—

(C_1-8)alkyl, —

S—

(C_1-8)alkyl-O—

(C_1-8)alkyl-OH, —

S—

(C_1-8)alkyl-O—

(C_1-8)alkyl-NH₂, —

S—

(C_1-8)alkyl-O—

(C_1-8)alkyl-NH(C_1-8alkyl), —

S—

(C_1-8)alkyl-O—

(C_1-8)alkyl-N(C_1-8alkyl)₂, —

S—

(C_1-8)alkyl-NH(C_1-8alkyl), —

SO₂—

(C_1-8)alkyl, —

SO₂—

NH₂, —

SO₂—

NH(C_1-8alkyl), —

SO₂—

N(C_1-8alkyl)₂, —

N—

R⁷, cyano, (halo)_1-3, hydroxy, nitro, oxo, -cycloalkyl-R⁶, -heterocyclyl-R⁶, -aryl-R⁶ and -heteroaryl-R⁶.

33. The method of claim 31, wherein R⁶ is 1 to 4 substituents attached to a carbon or nitrogen atom independently selected from the group consisting of hydrogen, —

C_1-8alkyl, —

C_2-8alkenyl, —

C_2-8alkynyl, —

C(O)H, —

C(O)—

(C_1-8)alkyl, —

CO₂H, —

C(O)—

O—

(C_1-8)alkyl, —

C(O)—

NH₂, —

C(NH)—

NH₂, —

C(O)—

NH(C_1-8alkyl), —

C(O)—

N(C_1-8)alkyl)₂, —

SO₂—

(C_1-8)alkyl, —

SO₂—

NH₂, —

SO₂—

NH(C_1-8alkyl), —

SO₂—

N(C_1-8alkyl)₂, —

(C_1-8)alkyl-N—

R⁷, —

(C_1-8)alkyl-(halo)_1-3, —

(C_1-8)alkyl-OH, -aryl-R⁸, —

(C_1-8)alkyl-aryl-R⁸ and —

(C_1-8)alkyl-heteroaryl-R⁸;

with the proviso that, when R⁶ is attached to a carbon atom, R⁶ is further selected from the group consisting of —

C_1-8alkoxy, —

(C_1-8)alkoxy-(halo)_1-3, —

SH, —

S—

(C_1-8)alkyl, —

N—

R⁷, cyano, halo, hydroxy, nitro, oxo and -heteroaryl-R⁸.

34. The method of claim 33, wherein R⁷ is 2 substituents independently selected from the group consisting of hydrogen, —

C_1-8alkyl, —

C_2-8alkenyl, —

C_2-8alkynyl, —

(C_1-8)alkyl-OH, —

(C_1-8)alkyl-O—

(C_1-8)alkyl, —

(C_1-8)alkyl-NH₂, —

(C_1-8)alkyl-NH(C_1-8alkyl), —

(C_1-8)alkyl-N(C_1-8alkyl)₂, —

(C_1-8)alkyl-S—

(C_1-8)alkyl, —

C(O)H, —

C(O)—

(C_1-8)alkyl, —

C(O)—

O—

(C_1-8)alkyl, —

C(O)—

NH₂, —

C(O)—

NH(C_1-8alkyl), —

C(O)—

N(C_1-8alkyl)₂, —

SO₂—

(C_1-8)alkyl, —

SO₂—

NH₂, —

SO₂—

NH(C_1-8alkyl), —

SO₂—

N(C_1-8alkyl)₂, —

C(N)—

NH₂, -cycloalkyl-R⁸, —

(C_1-8)alkyl-heterocyclyl-R⁸, -aryl-R⁸, —

(C_1-8)alkyl-aryl-R⁸ and —

(C_1-8)alkyl-heteroaryl-R⁸.

35. The method of claim 31, wherein R⁸ is 1 to 4 substituents attached to a carbon or nitrogen atom independently selected from the group consisting of hydrogen, —

C_1-8alkyl, —

(C_1-8)alkyl-(halo)_1-3 and —

(C_1-8)alkyl-OH;

with the proviso that, when R⁸ is attached to a carbon atom, R⁸ is further selected from the group consisting of —

C_1-8alkoxy, —

NH₂, —

NH(C_1-8alkyl), —

N(C_1-8alkyl)₂, cyano, halo, —

(C_1-8)alkoxy-(halo)_1-3, hydroxy and nitro.

36. The method of claim 31, wherein R⁹ is 1 to 2 substituents independently selected from the group consisting of hydrogen, —

C_1-8alkoxy, —

NH₂, —

NH(C_1-8alkyl), —

N(C_1-8alkyl)₂, cyano, (halo)_1-3, hydroxy and nitro.

37. The method of claim 28, wherein R² is one substituent attached to a carbon or nitrogen atom selected from the group consisting of hydrogen, —

C_1-8alkyl-R⁵, —

C_2-8alkenyl-R⁵, —

C_2-8alkynyl-R⁵, —

C(O)H, —

C(O)—

(C_1-8)alkyl-R⁹, —

C(O)—

NH₂, —

C(O)—

NH(C_1-8alkyl-R⁹), —

C(O)—

N(C_1-8alkyl-R⁹)₂, —

C(O)—

NH(aryl-R⁸), —

C(O)-cycloalkyl-R⁸, —

C(O)-heterocyclyl-R⁸, —

C(O)-aryl-R⁸, —

C(O)-heteroaryl-R⁸, —

CO₂H, —

C(O)—

O—

(C_1-8)alkyl-R⁹, —

C(O)—

O-aryl-R⁸, —

SO₂—

(C_1-8)alkyl-R⁹, —

SO₂-aryl-R⁸, -cycloalkyl-R⁶, -aryl-R⁶ and —

(C_1-8)alkyl-N—

R²;

with the proviso that, when R² is attached to a carbon atom, R² is further selected from the group consisting of —

C_1-8alkoxy-R⁵, —

N—

R², cyano, halogen, hydroxy, nitro, oxo, -heterocyclyl-R⁶ and -heteroaryl-R⁶.

38. The method of claim 28, wherein R³ is 1 to 3 substituents attached to a carbon atom independently selected from the group consisting of hydrogen, —

C_1-8alkyl-R¹⁰, —

C_2-8alkenyl-R¹⁰, —

C_2-8alkynyl-R¹⁰, —

C_1-8alkoxy-R¹⁰, —

C(O)H, —

C(O)—

(C_1-8)alkyl-R⁹, —

C(O)—

NH₂, —

C(O)—

NH(C_1-8alkyl-R⁹), —

C(O)—

N(C_1-8alkyl-R⁹)₂, —

C(O)-cycloalkyl-R⁸, —

C(O)-heterocyclyl-R⁸, —

C(O)-aryl-R⁸, —

C(O)-heteroaryl-R⁸, —

C(NH)—

NH₂, —

CO₂H, —

C(O)—

O—

(C_1-8)alkyl-R⁹, —

C(O)—

O-aryl-R⁸, —

SO₂—

(C_1-8)alkyl-R⁹, —

SO₂-aryl-R⁸, —

N—

R⁷, cyano, halogen, hydroxy, nitro, -cycloalkyl-R⁸, -heterocyclyl-R⁸, -aryl-R⁸ and -heteroaryl-R⁸.

39. The method of claim 38, wherein R¹⁶ is 1 to 2 substituents independently selected from the group consisting of hydrogen, —

NH₂, —

NH(C_1-8alkyl), —

N(C_1-8alkyl)₂, cyano, (halo)_1-3, hydroxy, nitro and oxo.

40. The method of claim 28, wherein R⁴ is 1 to 4 substituents attached to a carbon atom independently selected from the group consisting of hydrogen, —

C_1-8alkyl-R¹⁰, —

C_2-8alkenyl-R¹⁰, —

C_2-8alkynyl-R¹⁰, —

C_1-8alkoxy-R¹⁰, —

C(O)H, —

C(O)—

(C_1-8)alkyl-R⁹, —

C(O)—

NH₂, —

C(O)—

NH(C_1-8alkyl-R⁹), —

C(O)—

N(C_1-8alkyl-R⁹)₂, —

C(O)-cycloalkyl-R⁸, —

C(O)-heterocyclyl-R⁸, —

C(O)-aryl-R⁸, —

C(O)-heteroaryl-R⁸, —

C(NH)—

NH₂, —

CO₂H, —

C(O)—

O—

(C_1-8)alkyl-R⁹, —

C(O)—

O-aryl-R⁸, —

SH, —

S—

(C_1-8)alkyl-R¹⁰, —

SO₂—

(C_1-8)alkyl-R⁹, —

SO₂-aryl-R⁸, —

SO₂—

NH₂, —

SO₂—

NH(C_1-8alkyl-R⁹), —

SO₂—

N(C_1-8alkyl-R⁹)₂, —

N—

R⁷, cyano, halogen, hydroxy, nitro, -cycloalkyl-R⁸, -heterocyclyl-R⁸, -aryl-R⁸ and -heteroaryl-R⁸.

41. The method of claim 40, wherein R¹⁰ is 1 to 2 substituents independently selected from the group consisting of hydrogen, —

NH₂, —

NH(C_1-8alkyl), —

N(C_1-8alkyl)₂, cyano, (halo)_1-3, hydroxy, nitro and oxo.

42. The method of claim 28, wherein Y and Z are independently selected from the group consisting of O, S, (H,OH) and (H,H);

with the proviso that one of Y and Z is O and the other is selected from the group consisting of O, S, (H,OH) and (H,H); and

pharmaceutically acceptable salts thereof.

43. The method of claim 28, wherein R is selected from the group consisting of R_a, —

C_1-4alkyl-R_a, —

C_2-4alkenyl-R_a, —

C_2-4alkynyl-R_a and cyano.

44. The method of claim 29, wherein R_a is selected from the group consisting of heterocyclyl, aryl and heteroaryl.

45. The method of claim 29, R_a is selected from the group consisting of dihydro-pyranyl, phenyl, naphthyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, pyridinyl, azaindolyl, indazolyl, benzofuryl, benzothienyl, dibenzofuryl and dibenzothienyl.

46. The method of claim 28, wherein R¹ is selected from the group consisting of hydrogen, —

C_1-4alkyl-R⁵, —

C_2-4alkenyl-R⁵, —

C_2-4alkynyl-R⁵, —

C(O)—

(C_1-4)alkyl-R⁹, —

C(O)-aryl-R⁸, —

C(O)—

O—

(C_1-4)alkyl-R⁹, —

C(O)—

O-aryl-R⁸, —

C(O)—

NH(C_1-4alkyl-R⁹), —

C(O)—

NH(aryl-R⁸), —

C(O)—

N(C_1-4alkyl-R⁹)₂, —

SO₂—

(C_1-4)alkyl-R⁹, —

SO₂-aryl-R⁸, -cycloalkyl-R⁶, -heterocyclyl-R⁶, -aryl-R⁶ and -heteroaryl-R⁶;

wherein heterocyclyl and heteroaryl are attached to the azaindole nitrogen atom in the one position via a heterocyclyl or heteroaryl ring carbon atom.

47. The method of claim 28, wherein R¹ is selected from the group consisting of hydrogen, —

C_1-4alkyl-R⁵, -aryl-R⁶ and -heteroaryl-R⁶;

wherein heteroaryl is attached to the azaindole nitrogen atom in the one position via a heteroaryl ring carbon atom.

48. The method of claim 28, wherein R¹ is selected from the group consisting of hydrogen, —

C_1-4alkyl-R⁵ and -naphthyl-R⁶.

49. The method of claim 31, wherein R⁵ is 1 to 2 substituents independently selected from the group consisting of hydrogen, —

O—

(C_1-4alkyl, —

O—

(C_1-4alkyl-OH, —

O—

(C_1-4alkyl-O—

(C_1-4alkyl, —

O—

(C_1-4)alkyl-NH₂, —

O—

(C_1-4alkyl-NH(C_1-4alkyl), —

O—

(C_1-4alkyl-N(C_1-4alkyl)₂, —

O—

(C_1-4alkyl-S—

(C_1-4alkyl, —

O—

(C_1-4)alkyl-SO₂—

(C_1-4alkyl, —

O—

(C_1-4alkyl-SO₂—

NH₂, —

O—

(C_1-4alkyl-SO₂—

NH(C_1-4alkyl), —

O—

(C_1-4alkyl-SO₂—

N(C_1-4alkyl)₂, —

O—

C(O)H, —

O—

C(O)—

(C_1-4alkyl, —

O—

C(O)—

NH₂, —

O—

C(O)—

NH(C_1-4alkyl), —

O—

C(O)—

N(C_1-4alkyl)₂, —

O—

(C_1-4alkyl-C(O)H, —

O—

(C_1-4alkyl-C(O)—

(C_1-4)alkyl, —

O—

(C_1-4alkyl-CO₂H, —

O—

(C_1-4)alkyl-C(O)—

O—

(C_1-4)alkyl, —

O—

(C_1-4)alkyl-C(O)—

NH₂, —

O—

(C_1-4alkyl-C(O)—

NH(C_1-4alkyl), —

O—

(C_1-4alkyl-C(O)—

N(C_1-4alkyl)₂, —

C(O)H, —

C(O)—

(C_1-4alkyl, —

CO₂H, —

C(O)—

O—

(C_1-4alkyl, —

C(O)—

NH₂, —

C(NH)—

NH₂, —

C(O)—

NH(C_1-4alkyl), —

C(O)—

N(C_1-4alkyl)₂, —

SH, —

S—

(C_1-4alkyl, —

S—

(C_1-4alkyl-S—

(C_1-4)alkyl, —

S—

(C_1-4alkyl-O—

(C_1-4)alkyl, —

S—

(C_1-4alkyl-O—

(C_1-4)alkyl-OH, —

S—

(C_1-4alkyl-O—

(C_1-4)alkyl-NH₂, —

S—

(C_1-4alkyl-O—

(C_1-4)alkyl-NH(C_1-4alkyl), —

S—

(C_1-4alkyl-O—

(C_1-4)alkyl-N(C_1-4alkyl)₂, —

S—

(C_1-4alkyl-NH(C_1-4alkyl), —

SO₂—

(C_1-4alkyl, —

SO₂—

NH₂, —

SO₂—

NH(C_1-4alkyl), —

SO₂—

N(C_1-4alkyl)₂, —

N—

R⁷, cyano, (halo)_1-3, hydroxy, nitro, oxo, -cycloalkyl-R⁶, -heterocyclyl-R⁶, -aryl-R⁶ and -heteroaryl-R⁶.

50. The method of claim 31, wherein R⁵ is 1 to 2 substituents independently selected from the group consisting of hydrogen, —

O—

(C_1-4alkyl, —

N—

R⁷, hydroxy and -heteroaryl-R⁶.

51. The method of claim 31, wherein R⁵ is 1 to 2 substituents independently selected from the group consisting of hydrogen, —

O—

(C_1-4)alkyl, —

N—

R⁷, hydroxy, -imidazolyl-R⁶, -triazolyl-R⁶ and -tetrazolyl-R⁶.

52. The method of claim 31, wherein R⁶ is 1 to 4 substituents attached to a carbon or nitrogen atom independently selected from the group consisting of hydrogen, —

C_1-4alkyl, —

C_2-4alkenyl, —

C_2-4alkynyl, —

C(O)H, —

C(O)—

(C_1-4alkyl, —

CO₂H, —

C(O)—

O—

(C_1-4alkyl, —

C(O)—

NH₂, —

C(NH)—

NH₂, —

C(O)—

NH(C_1-4alkyl), —

C(O)—

N(C_1-4)alkyl)₂, —

SO₂—

(C_1-4alkyl, —

SO₂—

NH₂, —

SO₂—

NH(C_1-4alkyl), —

SO₂—

N(C_1-4alkyl)₂, —

(C_1-4)alkyl-N—

R⁷, —

(C_1-4)alkyl-(halo)_1-3, —

(C_1-4alkyl-OH, -aryl-R⁸, —

(C_1-4)alkyl-aryl-R⁸ and —

(C_1-4)alkyl-heteroaryl-R⁸;

with the proviso that, when R⁶ is attached to a carbon atom, R⁶ is further selected from the group consisting of —

C_1-4alkoxy, —

(C_1-4)alkoxy-(halo)_1-3, —

SH, —

S—

(C_1-4)alkyl, —

N—

R⁷, cyano, halo, hydroxy, nitro, oxo and -heteroaryl-R⁸.

53. The method of claim 31, wherein R⁶ is hydrogen.

54. The method of claim 33, wherein R⁷ is 2 substituents independently selected from the group consisting of hydrogen, —

C_1-4alkyl, —

C_2-4alkenyl, —

C_2-4alkynyl, —

(C_1-4)alkyl-OH, —

(C_1-4alkyl-O—

(C_1-4)alkyl, —

(C_1-4alkyl-NH₂, —

(C_1-4)alkyl-NH(C_1-4alkyl), —

(C_1-4)alkyl-N(C_1-4alkyl)₂, —

(C_1-4)alkyl-S—

(C_1-4)alkyl, —

C(O)H, —

C(O)—

(C_1-4alkyl, —

C(O)—

O—

(C_1-4alkyl, —

C(O)—

NH₂, —

C(O)—

NH(C_1-4alkyl), —

C(O)—

N(C_1-4alkyl)₂, —

SO₂—

(C_1-4alkyl, —

SO₂—

NH₂, —

SO₂—

NH(C_1-4alkyl), —

SO₂—

N(C_1-4alkyl)₂, —

C(N)—

NH₂, -cycloalkyl-R⁸, —

(C_1-4)alkyl-heterocyclyl-R⁸, -aryl-R⁸, —

(C_1-4)alkyl-aryl-R⁸ and —

(C_1-4)alkyl-heteroaryl-R⁸.

55. The method of claim 33, wherein R⁷ is 2 substituents independently selected from the group consisting of hydrogen, —

C_1-4alkyl, —

C(O)H, —

C(O)—

(C_1-4alkyl, —

C(O)—

O—

(C_1-4)alkyl, —

SO₂—

NH₂, —

SO₂—

NH(C_1-4alkyl) and —

SO₂—

N(C_1-4alkyl)₂.

56. The method of claim 31, wherein R⁸ is 1 to 4 substituents attached to a carbon or nitrogen atom independently selected from the group consisting of hydrogen, —

C_1-4alkyl, —

(C_1-4)alkyl-(halo)_1-3 and —

(C_1-4)alkyl-OH;

with the proviso that, when R⁸ is attached to a carbon atom, R⁸ is further selected from the group consisting of —

C_1-4alkoxy, —

NH₂, —

NH(C_1-4alkyl), —

N(C_1-4alkyl)₂, cyano, halo, —

(C_1-4)alkoxy-(halo)_1-3, hydroxy and nitro.

57. The method of claim 31, wherein R⁸ is hydrogen.

58. The method of claim 31, wherein R⁹ is 1 to 2 substituents independently selected from the group consisting of hydrogen, —

C_1-4alkoxy, —

NH₂, —

NH(C_1-4alkyl), —

N(C_1-4alkyl)₂, cyano, (halo)_1-3, hydroxy and nitro.

59. The method of claim 31, wherein R⁹ is hydrogen.

60. The method of claim 28, wherein R² is one substituent attached to a carbon or nitrogen atom selected from the group consisting of hydrogen, —

C_1-4alkyl-R⁵, —

C_2-4alkenyl-R⁵, —

C_2-4alkynyl-R⁵, —

C(O)H, —

C(O)—

(C_1-4)alkyl-R⁹, —

C(O)—

NH₂, —

C(O)—

NH(C_1-4alkyl-R⁹), —

C(O)—

N(C_1-4alkyl-R⁹)₂, —

C(O)—

NH(aryl-R⁸), —

C(O)-cycloalkyl-R⁸, —

C(O)-heterocyclyl-R⁸, —

C(O)-aryl-R⁸, —

C(O)-heteroaryl-R⁸, —

CO₂H, —

C(O)—

O—

(C_1-4alkyl-R⁹, —

C(O)—

O-aryl-R⁸, —

SO₂—

(C_1-4alkyl-R⁹, —

SO₂-aryl-R⁸, -cycloalkyl-R⁶, -aryl-R⁶ and —

(C_1-4)alkyl-N—

R⁷;

with the proviso that, when R² is attached to a carbon atom, R² is further selected from the group consisting of —

C_1-4alkoxy-R⁵, —

N—

R⁷, cyano, halogen, hydroxy, nitro, oxo, -heterocyclyl-R⁶ and -heteroaryl-R⁶.

61. The method of claim 28, wherein R² is one substituent attached to a carbon or nitrogen atom selected from the group consisting of hydrogen, —

C_1-4alkyl-R⁵, —

C_2-4alkenyl-R⁵, —

C_2-4alkynyl-R⁵, —

CO₂H, —

C(O)—

O—

(C_1-4alkyl-R⁹, -cycloalkyl-R⁶, -aryl-R⁶ and —

(C_1-4)alkyl-N—

R⁷;

with the proviso that, when R² is attached to a nitrogen atom, a quaternium salt is not formed; and

, with the proviso that, when R² is attached to a carbon atom, R² is further selected from the group consisting of —

C_1-4alkoxy-R⁵, —

N—

R⁷, cyano, halogen, hydroxy, nitro, oxo, -heterocyclyl-R⁶ and -heteroaryl-R⁶.

62. The method of claim 28, wherein R² is one substituent attached to a carbon or nitrogen atom selected from the group consisting of hydrogen, —

C_1-4alkyl-R⁵ and -aryl-R⁶;

with the proviso that, when R² is attached to a nitrogen atom, a quaternium salt is not formed; and

, with the proviso that when R² is attached to a carbon atom, R² is further selected from the group consisting of —

N—

R⁷, halogen, hydroxy and -heteroaryl-R⁶.

63. The method of claim 28, wherein R³ is 1 to 3 substituents attached to a carbon atom independently selected from the group consisting of hydrogen, —

C_1-4alkyl-R¹⁰, —

C_2-4alkenyl-R¹⁰, —

C_2-4alkynyl-R¹⁰, —

C_1-4alkoxy-R¹⁰, —

C(O)H, —

C(O)—

(C_1-4alkyl-R⁹, —

C(O)—

NH₂, —

C(O)—

NH(C_1-4alkyl-R⁹), —

C(O)—

N(C_1-4alkyl-R⁹)₂, —

C(O)-cycloalkyl-R⁸, —

C(O)-heterocyclyl-R⁸, —

C(O)-aryl-R⁸, —

C(O)-heteroaryl-R⁸, —

C(NH)—

NH₂, —

CO₂H, —

C(O)—

O—

(C_1-4alkyl-R⁹, —

C(O)—

O-aryl-R⁸, —

SO₂—

(C_1-8)alkyl-R⁹, —

SO₂-aryl-R⁸, —

N—

R⁷, —

(C_1-4)alkyl-N—

R⁷, cyano, halogen, hydroxy, nitro, -cycloalkyl-R⁸, -heterocyclyl-R⁸, -aryl-R⁸ and -heteroaryl-R⁸.

64. The method of claim 28, wherein R³ is one substituent attached to a carbon atom selected from the group consisting of hydrogen, —

C_1-4alkyl-R¹⁰, —

C_2-4alkenyl-R¹⁰, —

C_2-4alkynyl-R¹⁰, —

C_1-4alkoxy-R¹⁰, —

C(O)H, —

CO₂H, —

NH₂, —

NH(C_1-4alkyl), —

N(C_1-4alkyl)₂, cyano, halogen, hydroxy and nitro.

65. The method of claim 28, wherein R³ is one substituent attached to a carbon atom selected from the group consisting of hydrogen, —

C_1-4alkyl-R¹⁰, —

NH₂, —

NH(C_1-4alkyl), —

N(C_1-4alkyl)₂, halogen and hydroxy.

66. The method of claim 28, wherein R⁴ is 1 to 4 substituents attached to a carbon atom independently selected from the group consisting of hydrogen, —

C_1-4alkyl-R¹⁰, —

C_2-4alkenyl-R¹⁰, —

C_2-4alkynyl-R¹⁰, —

C_1-4alkoxy-R¹⁰, —

C(O)H, —

C(O)—

(C_1-4alkyl-R⁹, —

C(O)—

NH₂, —

C(O)—

NH(C_1-4alkyl-R⁹), —

C(O)—

N(C_1-4alkyl-R⁹)₂, —

C(O)-cycloalkyl-R⁸, —

C(O)-heterocyclyl-R⁸, —

C(O)-aryl-R⁸, —

C(O)-heteroaryl-R⁸, —

C(NH)—

NH₂, —

CO₂H, —

C(O)—

O—

(C_1-4alkyl-R⁹, —

C(O)—

O-aryl-R⁸, —

SH, —

S—

(C_1-4alkyl-R¹⁰, —

SO₂—

(C_1-4alkyl-R⁹, —

SO₂-aryl-R⁸, —

SO₂—

NH₂, —

SO₂—

NH(C_1-4alkyl-R⁹), —

SO₂—

N(C_1-4alkyl-R⁹)₂, —

N—

R⁷, cyano, halogen, hydroxy, nitro, -cycloalkyl-R⁸, -heterocyclyl-R⁸, -aryl-R⁸ and -heteroaryl-R⁸.

67. The method of claim 28, wherein R⁴ is 1 to 4 substituents attached to a carbon atom independently selected from the group consisting of hydrogen, —

C_1-4alkyl-R¹⁰, —

C_2-4alkenyl-R¹⁰, —

C_2-4alkynyl-R¹⁰, —

C_1-4alkoxy-R¹⁰, —

C(O)H, —

CO₂H, —

NH₂, —

NH(C_1-4alkyl), —

N(C_1-4alkyl)₂, cyano, halogen, hydroxy, nitro, -cycloalkyl, -heterocyclyl, -aryl and -heteroaryl.

68. The method of claim 28, wherein R⁴ is 1 to 4 substituents attached to a carbon atom independently selected from the group consisting of hydrogen, C_1-4alkyl-R¹⁰, C_1-4alkoxy-R¹⁰, —

NH₂, —

NH(C_1-4alkyl), —

N(C_1-4alkyl)₂, halogen and hydroxy.

69. The method of claim 28, wherein R⁴ is 1 to 4 substituents attached to a carbon atom independently selected from the group consisting of hydrogen, C_1-4alkyl-R¹⁰, C_1-4alkoxy-R¹⁰, —

NH₂, —

NH(C_1-4alkyl), —

N(C_1-4alkyl)₂, chlorine, fluorine and hydroxy.

70. The method of claims 38 and 41, wherein R¹⁰ is 1 to 2 substituents independently selected from the group consisting of hydrogen, —

NH₂, —

NH(C_1-4alkyl), —

N(C_1-4alkyl)₂, cyano, (halo)_1-3, hydroxy, nitro and oxo.

71. The method of claims 38 and 41, wherein R¹⁰ is 1 to 2 substituents independently selected from the group consisting of hydrogen and (halo)_1-3.

72. The method of claims 38 and 41, wherein R¹⁰ is 1 to 2 substituents independently selected from the group consisting of hydrogen and (fluoro)₃.

73. The method of claim 28, wherein Y and Z are independently selected from the group consisting of O, S, (H,OH) and (H,H);

with the proviso that one of Y and Z is O and the other is selected from the group consisting of O, S, (H,OH) and (H,H).

74. The method of claim 28, wherein Y and Z are independently selected from the group consisting of O and (H,H);

with the proviso that one of Y and Z is O, and the other is selected from the group consisting of O and (H,H).

75. The method of claim 28, wherein Y and Z are independently selected from O.

76. The method of claim 28, where the compound of the Formula II is 3-[1-(3-hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-[2-(trifluoromethyl)phenyl]-1H-pyrrole-2,5-dione.

77. The method of claim 28, where the compound of the Formula II is 3-[1-(3-hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-(1-methyl-1H-pyrazol-3-yl)-1H-pyrrole-2,5-dione.

78. The method of claim 28, where the compound of the Formula II is 3-[1-(3-hydroxy-propyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-pyrazin-2-yl-pyrrole-2,5-dione.

79. The method of claim 28, where the compound of the Formula II is 3-(2,4-dimethoxy-pyrimidin-5-yl)-4-[1-(3-hydroxy-propyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-pyrrole-2,5-dione.

80. The method of claim 28, where the compound of the Formula II is 4-{3-[4-(2,4-dimethoxy-pyrimidin-5-yl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl]-pyrrolo[2,3-b]pyridin-1-yl}-butyronitrile.

81. The method of claim 28, where the compound of the Formula II is 4-{3-[4-(1-methyl-1H-pyrazol-3-yl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl]-pyrrolo[2,3-b]pyridin-1-yl}-butyronitrile.

82. The method of claim 28, where the compound of the Formula II is 3-(2,4-dimethoxy-pyrimidin-5-yl)-4-(1-phenethyl-1H-pyrrolo[2,3-b]pyridine-3-yl)-pyrrole-2,5-dione.

83. The method of claim 1, wherein the inhibitor of GSK-3B enzyme activity is a compound of the Formula (III):

84. The method of claim 83, wherein A and E are independently selected from the group consisting of a hydrogen substituted carbon atom and a nitrogen atom;

wherein

85. The method of claim 83, wherein Z is selected from O;

alternatively, Z is selected from dihydro;

wherein each hydrogen atom is attached by a single bond.

86. The method of claim 83, wherein R₄ and R₅ are independently selected from C_1-8alkyl, C_2-8alkenyl and C_2-8alkynyl optionally substituted with oxo.

87. The method of claim 83, wherein R₂ is selected from the group consisting of —

C_1-8alkyl-, —

C_2-8alkenyl-, —

C_2-8alkynyl-, —

O—

(C_1-8)alkyl-O—

, —

O—

(C_2-8)alkenyl-O—

, —

O—

(C_2-8)alkynyl-O—

, —

C(O)—

(C_1-8)alkyl-C(O)—

(wherein any of the foregoing alkyl, alkenyl and alkynyl linking groups are straight carbon chains optionally substituted with one to four substituents independently selected from the group consisting of C_1-8alkyl, C_1-8alkoxy, C_1-8alkoxy(C_1-8)alkyl, carboxyl, carboxyl(C_1-8)alkyl, —

C(O)O—

(C_1-8)alkyl, —

C_1-8alkyl-C(O)O—

(C_1-8)alkyl, amino (substituted with a substituent independently selected from the group consisting of hydrogen and C_1-4alkyl), amino(C_1-8)alkyl (wherein amino is substituted with a substituent independently selected from the group consisting of hydrogen and C_1-4alkyl), halogen, (halo)_1-3(C_1-8)alkyl, (halo)_1-3(C_1-8)alkoxy, hydroxy, hydroxy(C_1-8)alkyl and oxo; and

, wherein any of the foregoing alkyl, alkenyl and alkynyl linking groups are optionally substituted with one to two substituents independently selected from the group consisting of heterocyclyl, aryl, heteroaryl, heterocyclyl(C_1-8)alkyl, aryl(C_1-8)alkyl, heteroaryl(C_1-8)alkyl, spirocycloalkyl and spiroheterocyclyl (wherein any of the foregoing cycloalkyl, heterocyclyl, aryl and heteroaryl substituents are optionally substituted with one to four substituents independently selected from the group consisting of C_1-8alkyl, C_1-8alkoxy, C_1-8alkoxy(C_1-8)alkyl, carboxyl, carboxyl(C_1-8)alkyl, amino (substituted with a substituent independently selected from the group consisting of hydrogen and C_1-4alkyl), amino(C_1-8)alkyl (wherein amino is substituted with a substituent independently selected from the group consisting of hydrogen and C_1-4alkyl), halogen, (halo)_1-3(C_1-8)alkyl, (halo)_1-3(C_1-8)alkoxy, hydroxy and hydroxy(C_1-8)alkyl; and

, wherein any of the foregoing heterocyclyl substituents are optionally substituted with oxo)), cycloalkyl, heterocyclyl, aryl, heteroaryl (wherein cycloalkyl, heterocyclyl, aryl and heteroaryl are optionally substituted with one to four substituents independently selected from the group consisting of C_1-8alkyl, C_1-8alkoxy, C_1-8alkoxy(C_1-8)alkyl, carboxyl, carboxyl(C_1-8)alkyl, amino (substituted with a substituent independently selected from the group consisting of hydrogen and C_1-4alkyl), amino(C_1-8)alkyl (wherein amino is substituted with a substituent independently selected from the group consisting of hydrogen and C_1-4alkyl), halogen, (halo)_1-3(C_1-8)alkyl, (halo)_1-3(C_1-8)alkoxy, hydroxy and hydroxy(C_1-8)alkyl; and

, wherein heterocyclyl is optionally substituted with oxo), —

(O—

(CH₂)_1-6)_0-5—

O—

, —

O—

(CH₂)_1-6—

O—

(CH₂)_1-6—

O—

, —

O—

(CH₂)_1-6—

O—

(CH₂)_1-6—

O—

(CH₂)_1-6—

O—

, —

(O—

(CH₂)_1-6)_0-5—

NR₆—

, —

O—

(CH₂)_1-6—

NR₆—

(CH₂)_1-6—

O—

, —

O—

(CH₂)_1-6—

O—

(CH₂)_1-6—

NR₆—

, —

(O—

(CH₂)_1-6)_0-5—

S—

, —

O—

(CH₂)_1-6—

S—

(CH₂)_1-6—

O—

, —

O—

(CH₂)_1-6—

O—

(CH₂)_1-6—

S—

, —

NR₆—

, —

NR₆—

NR₇—

, —

NR₆—

(CH₂)_1-6—

NR₇—

, —

NR₆—

(CH₂)_1-6—

NR₇—

(CH₂)_1-6—

NR₈—

, —

NR₆—

C(O)—

, —

C(O)—

NR₆—

, —

C(O)—

(CH₂)_0-6—

NR₆—

(CF₂)_0-6—

C(O)—

, —

NR₆—

(CH₂)_0-6—

C(O)—

(CH₂)_1-6—

C(O)—

(CH₂)_0-6—

NR₇—

, —

NR₆—

C(O)—

NR₇—

, —

NR₆—

C(NR₇)—

NR₈—

, —

O—

(CH₂)_1-6—

NR₆—

(CH₂)_1-6—

S—

, —

S—

(CH₂)_1-6—

NR₆—

(CH₂)_1-6—

O—

, —

S—

(CH₂)_1-6—

NR₆—

(CH₂)_1-6—

S—

, —

NR₆—

(CH₂)_1-6—

S—

(CH₂)_1-6—

NR₇— and

—

SO₂—

(wherein R₆, R₇ and R₈ are independently selected from the group consisting of hydrogen, C_1-8alkyl, C_1-8alkoxy(C_1-8)alkyl, carboxyl(C_1-8)alkyl, amino(C_1-8)alkyl (wherein amino is substituted with a substituent independently selected from the group consisting of hydrogen and C_1-4alkyl), hydroxy(C_1-8)alkyl, heterocyclyl(C_1-8)alkyl, aryl(C_1-8)alkyl and heteroaryl(C_1-8)alkyl (wherein the foregoing heterocyclyl, aryl and heteroaryl substituents are optionally substituted with one to four substituents independently selected from the group consisting of C_1-8alkyl, C_1-8alkoxy, C_1-8alkoxy(C_1-8)alkyl, carboxyl, carboxyl(C_1-8)alkyl, amino (substituted with a substituent independently selected from the group consisting of hydrogen and C_1-4alkyl), amino(C_1-8)alkyl (wherein amino is substituted with a substituent independently selected from the group consisting of hydrogen and C_1-4alkyl), halogen, (halo)_1-3(C_1-8)alkyl, (halo)_1-3(C_1-8)alkoxy, hydroxy and hydroxy(C_1-8)alkyl; and

, wherein heterocyclyl is optionally substituted with oxo));

with the proviso that, if A and E are selected from a hydrogen substituted carbon atom, then R₂ is selected from the group consisting of —

C_2-8alkynyl-, —

O—

(C_1-8)alkyl-O—

, —

O—

(C_2-8)alkenyl-O—

, —

O—

(C_2-8)alkynyl-O—

, —

C(O)—

(C_1-8)alkyl-C(O)—

(wherein any of the foregoing alkyl, alkenyl and alkynyl linking groups are straight carbon chains optionally substituted with one to four substituents independently selected from the group consisting of C_1-8alkyl, C_1-8alkoxy, C_1-8alkoxy(C_1-8)alkyl, carboxyl, carboxyl(C_1-8)alkyl, —

C(O)O—

(C_1-8)alkyl, —

C_1-8 alkyl-C(O)O—

(C_1-8)alkyl, amino (substituted with a substituent independently selected from the group consisting of hydrogen and C_1-4alkyl), amino(C_1-8)alkyl (wherein amino is substituted with a substituent independently selected from the group consisting of hydrogen and C_1-4alkyl), halogen, (halo)_1-3(C_1-8)alkyl, (halo)_1-3(C_1-8)alkoxy, hydroxy, hydroxy(C_1-8)alkyl and oxo; and

, wherein any of the foregoing alkyl, alkenyl and alkynyl linking groups are optionally substituted with one to two substituents independently selected from the group consisting of heterocyclyl, aryl, heteroaryl, heterocyclyl(C_1-8)alkyl, aryl(C_1-8)alkyl, heteroaryl(C_1-8)alkyl, spirocycloalkyl and spiroheterocyclyl (wherein any of the foregoing cycloalkyl, heterocyclyl, aryl and heteroaryl substituents are optionally substituted with one to four substituents independently selected from the group consisting of C_1-8alkyl, C_1-8alkoxy, C_1-8alkoxy(C_1-8)alkyl, carboxyl, carboxyl(C_1-8)alkyl, amino (substituted with a substituent independently selected from the group consisting of hydrogen and C_1-4alkyl), amino(C_1-8)alkyl (wherein amino is substituted with a substituent independently selected from the group consisting of hydrogen and C_1-4alkyl), halogen, (halo)_1-3(C_1-8)alkyl, (halo)_1-3(C_1-8)alkoxy, hydroxy and hydroxy(C_1-8)alkyl; and

, wherein any of the foregoing heterocyclyl substituents are optionally substituted with oxo)), cycloalkyl (wherein cycloalkyl is optionally substituted with one to four substituents independently selected from the group consisting of C_1-8alkyl, C_1-8alkoxy, C_1-8alkoxy(C_1-8)alkyl, carboxyl, carboxyl(C_1-8)alkyl, amino (substituted with a substituent independently selected from the group consisting of hydrogen and C_1-4alkyl), amino(C_1-8)alkyl (wherein amino is substituted with a substituent independently selected from the group consisting of hydrogen and C_1-4alkyl), halogen, (halo)_1-3(C_1-8)alkyl, (halo)_1-3(C_1-8)alkoxy, hydroxy and hydroxy(C_1-8)alkyl), —

(O—

(CH₂)_1-6)_1-5—

O—

, —

O—

(CH₂)_1-6—

O—

(CH₂)_1-6—

O—

, —

O—

(CH₂)_1-6—

O—

(CH₂)_1-6—

O—

(CH₂)_1-6—

O—

, —

(O—

(CH₂)_1-6)_1-5—

NR₆—

, —

O—

(CH₂)_1-6—

NR₆—

(CH₂)_1-6—

O—

, —

O—

(CH₂)_1-6—

O—

(CH₂)_1-6—

NR₆—

, —

(O—

(CH₂)_1-6)_0-5—

S—

, —

O—

(CH₂)_1-6—

S—

(CH₂)_1-6—

O—

, —

O—

(CH₂)_1-6—

O—

(CH₂)_1-6—

S—

, —

NR₆—

NR₇—

, —

NR₆—

(CH₂)_1-6—

NR₇—

, —

NR₆—

(CH₂)_1-6—

NR₇—

(CH₂)_1-6—

NR₈—

, —

NR₉—

C(O)—

, —

C(O)—

NR₉—

, —

C(O)—

(CH₂)_0-6—

NR₆—

(CH₂)_0-6—

C(O)—

, —

NR₆—

(CH₂)_0-6—

C(O)—

(CH₂)_1-6—

C(O)—

(CH₂)_0-6—

NR₇—

, —

NR₆—

C(O)—

NR₇—

, —

NR₆—

C(NR₇)—

NR₈—

, —

O—

(CH₂)_1-6—

NR₆—

(CH₂)_1-6—

S—

, —

S—

(CH₂)_1-6—

NR₆—

(CH₂)_1-6—

O—

, —

S—

(CH₂)_1-6—

NR₆—

(CH₂)_1-6—

S— and

—

NR₆—

(CH₂)_1-6—

S—

(CH₂)_1-6—

NR₇—

(wherein R₆, R₇ and R₈ are independently selected from the group consisting of hydrogen, C_1-8alkyl, C_1-8alkoxy(C_1-8)alkyl, carboxyl(C_1-8)alkyl, amino(C_1-8)alkyl (wherein amino is substituted with a substituent independently selected from the group consisting of hydrogen and C_1-4alkyl), hydroxy(C_1-8)alkyl, heterocyclyl(C_1-8)alkyl, aryl(C_1-8)alkyl and heteroaryl(C_1-8)alkyl (wherein the foregoing heterocyclyl, aryl and heteroaryl substituents are optionally substituted with one to four substituents independently selected from the group consisting of C_1-8alkyl, C_1-8alkoxy, C_1-8alkoxy(C_1-8)alkyl, carboxyl, carboxyl(C_1-8)alkyl, amino (substituted with a substituent independently selected from the group consisting of hydrogen and C_1-4alkyl), amino(C_1-8)alkyl (wherein amino is substituted with a substituent independently selected from the group consisting of hydrogen and C_1-4alkyl), halogen, (halo)_1-3(C_1-8)alkyl, (halo)_1-3(C_1-8)alkoxy, hydroxy and hydroxy(C_1-8)alkyl; and

, wherein heterocyclyl is optionally substituted with oxo); and

, wherein R₉ is selected from the group consisting of C_1-8alkyl, C_1-8alkoxy(C_1-8)alkyl, carboxyl(C_1-8)alkyl, amino(C_1-8)alkyl (wherein amino is substituted with a substituent independently selected from the group consisting of hydrogen and C_1-4alkyl), hydroxy(C_1-8)alkyl, heterocyclyl(C_1-8)alkyl, aryl(C_1-8)alkyl and heteroaryl(C_1-8)alkyl (wherein the foregoing heterocyclyl, aryl and heteroaryl substituents are optionally substituted with one to four substituents independently selected from the group consisting of C_1-8alkyl, C_1-8alkoxy, C_1-8alkoxy(C_1-8)alkyl, carboxyl, carboxyl(C_1-8)alkyl, amino (substituted with a substituent independently selected from the group consisting of hydrogen and C_1-4alkyl), amino(C_1-8)alkyl (wherein amino is substituted with a substituent independently selected from the group consisting of hydrogen and C_1-4alkyl), halogen, (halo)_1-3(C_1-8)alkyl, (halo)_1-3(C_1-8)alkoxy, hydroxy and hydroxy(C_1-8)alkyl; and

, wherein heterocyclyl is optionally substituted with oxo)).

88. The method of claim 83, wherein R₁ and R₃ are independently selected from the group consisting of hydrogen, C_1-8alkyl, C_2-8alkenyl, C_2-8alkynyl (wherein alkyl, alkenyl and alkynyl are optionally substituted with a substituent selected from the group consisting of C_1-8alkoxy, alkoxy(C_1-8)alkyl, carboxyl, carboxyl(C_1-8)alkyl, amino (substituted with a substituent independently selected from the group consisting of hydrogen and C_1-4alkyl), amino(C_1-8)alkyl (wherein amino is substituted with a substituent independently selected from the group consisting of hydrogen and C_1-4alkyl), (halo)_1-3, (halo)_1-3(C_1-8)alkyl, (halo)_1-3 (C_1-8)alkoxy, hydroxy, hydroxy(C_1-8)alkyl and oxo), C_1-8alkoxy, C_1-8alkoxycarbonyl, (halo)_1-3(C_1-8)alkoxy, C_1-8alkylthio, aryl, heteroaryl (wherein aryl and heteroaryl are optionally substituted with a substituent selected from the group consisting of C_1-8alkyl, C_1-8alkoxy, alkoxy(C_1-8)alkyl, carboxyl, carboxyl(C_1-8)alkyl, amino (substituted with a substituent independently selected from the group consisting of hydrogen and C_1-4alkyl), amino(C_1-8)alkyl (wherein amino is substituted with a substituent independently selected from the group consisting of hydrogen and C_1-4alkyl), halogen, (halo)_1-3(C_1-8)alkyl, (halo)_1-3(C_1-8)alkoxy, hydroxy and hydroxy(C_1-8)alkyl), amino (substituted with a substituent independently selected from the group consisting of hydrogen and C_1-4alkyl), cyano, halogen, hydroxy and nitro;

and pharmaceutically acceptable salts thereof.

89. The method of claim 83, wherein the compound of the Formula (III) is 6,7,9,10,12,13,15,16-octahydro-23H-5,26:

17,22-dimetheno-5H-dipyrido[2,3-k;

3′

,2′

-q]pyrrolo[3,4-n][1,4,7,10,19]trioxadiazacyclohenicosine-23,25(24H)-dione.

90. The method of claim 83, wherein the compound of the Formula (III) is 10,11,13,14,16,17,19,20,22,23-decahydro-9,4:

24,29-dimetheno-1H-dipyrido[2,3-n;

3′

,2′

-t]pyrrolo[3,4-q][1,4,7,10,13,22]tetraoxadiazacyclotetracosine-1,3(2H)-dione.

91. The method of claim 83, wherein the compound of the Formula (III) is 10,11,13,14,16,17,19,20,22,23,25,26-dodecahydro-9,4:

27,32-dimetheno-1H-dipyrido[2,3-q;

3′

,2′

-w]pyrrolo[3,4-t][1,4,7,10,13,16,25]pentaoxadiazacycloheptacosine-1,3(2H)-dione.

92. The method of claim 83, wherein the compound of the Formula (III) is 6,7,9,10,12,13-hexahydro-20H-5,23:

14,19-dimetheno-5H-dibenzo[h,n]pyrrolo[3,4-k][1,4,7,16]dioxadiazacyclooctadecine-20,22(21H)-dione.

93. The method of claim 83, wherein the compound of the Formula (III) is 6,7,9,10,12,13,15,16-octahydro-23H-5,26:

17,22-dimetheno-5H-dibenzo[k,q]pyrrolo[3,4-n][1,4,7,10,19]trioxadiazacycloheneicosine-23,25(24H)-dione.

94. The method of claim 83, wherein the compound of the Formula (III) is 10,11,13,14,16,17,19,20,22,23-decahydro-9,4:

24,29-dimetheno-1H-dibenzo[n,t]pyrrolo[3,4-q][1,4,7,10,13,22]tetraoxadiazacyclotetracosine-1,3(2H)-dione.

95. The method of claim 83, wherein the compound of the Formula (III) is Compound 1a.

96. The method of claim 83, wherein the compound of the Formula (III) is 3-[1-[3-[(2-hydroxyethyl)methylamino]propyl]-1H-indazol-3-yl]-4-[1-(3-pyridinyl)-1H-indol-3-yl]-1H-pyrrole-2,5-dione.

97. The method of claim 83, wherein the compound of the Formula (III) is 3,5-dichloro-N-[3-chloro-4-[(3,4,12,12a-tetrahydro-1H-[1,4]thiazino[3,4-c][1,4]benzodiazepin-11(6H)-yl)carbonyl]phenyl]-benzamide.

98. The method of claim 83, wherein the compound of the Formula (III) is 3-[1-(2-hydroxy-ethyl)-1H-indol-3-yl]-4-(1-pyridin-3-yl-1H-indol-3-yl)-pyrrole-2,5-dione.

99. The method of claim 83, wherein the compound of the Formula (III) is 3-(2-methoxy-phenyl)-4-(1-pyridin-3-yl-1H-indol-3-yl)-pyrrole-2,5-dione.

100. The method of claim 83, wherein the compound of the Formula (III) is 6-[[2-[[4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile.

101. The method of claim 83, wherein the compound of the Formula (III) is 3-(5-chloro-1-methyl-1H-indol-3-yl)-4-[1-(3-imidazol-1-yl-propyl)-1H-indazol-3-yl]-pyrrole-2,5-dione.

102. The method of claim 83, wherein the compound of the Formula (III) is 3-(5-chloro-1-methyl-1H-indol-3-yl)-4-[1-(3-[1,2,3]triazol-1-yl-propyl)-1H-indazol-3-yl]-pyrrole-2,5-dione.

103. The method of claim 83, wherein the compound of the Formula (III) is 3-[1-(3-hydroxy-propyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-(1-methyl-1H-pyrazol-3-yl)-pyrrole-2,5-dione.

104. The method of claim 83, wherein the compound of the Formula (III) is Compound 10a.

105. The method of claim 83, wherein the compound of the Formula (III) is 3-[1-(3-hydroxy-3-methyl-butyl)-1H-indazol-3-yl]-4-(1-pyridin-3-yl-1H-indol-3-yl)-pyrrole-2,5-dione.

106. The method of claim 83, wherein the compound of the Formula (III) is 3-[1-(2-hydroxy-ethyl)-1H-indazol-3-yl]-4-(1-pyrimidin-5-yl-1H-indol-3-yl)-pyrrole-2,5-dione.

107. The method of claim 83, wherein the compound of the Formula (III) is 3-[1-(2-hydroxy-ethyl)-1H-indol-3-yl]-4-(1-pyrimidin-5-yl-1H-indol-3-yl)-pyrrole-2,5-dione.

108. The method of claim 83, wherein the compound of the Formula (III) is (11Z)-8,9,10,13,14,15-hexahydro-2,6:

17,21-di(metheno)pyrrolo[3,4-h][1,15,7]dioxazacyclotricosine-22,24(1H,23H)-dione.

109. The method of claim 83, wherein the compound of the Formula (III) is 3-(5-chloro-1-pyridin-3-yl-1H-indol-3-yl)-4-[1-(3-hydroxy-propyl)-1H-indazol-3-yl]-pyrrole-2,5-dione.

110. The method of claim 83, wherein the compound of the Formula (III) is 3-(2-methoxy-phenyl)-4-[1-(3-methoxy-propyl)-1H-pyrrolo[3,2-c]pyridin-3-yl]-pyrrole-2,5-dione.

111. The method of claim 83, wherein the compound of the Formula (III) is 3-[1-(3-hydroxy-propyl)-1H-indazol-3-yl]-4-[1-(tetrahydro-pyran-4-yl)-1H-indol-3-yl]-pyrrole-2,5-dione.

112. The method of claim 83, wherein the compound of the Formula (III) is 2-{3-[4-(5-chloro-1-methyl-1H-indol-3-yl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl]-indazol-1-yl}-N-(2-hydroxy-ethyl)-acetamide.

113. The method of claim 83, wherein the compound of the Formula (III) is 4-(3-chloro-phenyl)-6-(3-dimethylamino-propyl)-5,6-dihydro-4H-2,4,6-triaza-cyclopenta[c]fluorine-1,3-dione.

114. The method of claim 83, wherein the compound of the Formula (III) is 14-ethyl-6,7,9,10,13,14,15,16-octahydro-12H,23H-5,26:

17,22-dimethenodibenzo[k,q]pyrrolo[3,4-n][1,4,7,10,19]dioxatriazacycloheneicosine-23,25(24H)-dione.

115. The method of claim 83, wherein the compound of the Formula (III) is 14-benzyl-6,7,9,10,13,14,15,16-octahydro-12H,23H-5,26:

17,22-di(metheno)dibenzo[k,q]pyrrolo[3,4-n][1,4,7,10,19]dioxatriazacyclohenicosine-23,25(24H)-dione.

116. The method of claim 83, wherein the compound of the Formula (III) is 3-(1-{2-[2-(2-hydroxy-ethoxy)-ethoxy]-ethyl}-1H-indol-3-yl)-4-[1-(2-hydroxy-ethyl)-1H-indol-3-yl]-pyrrole-2,5-dione.

117. The method of claim 83, wherein the compound of the Formula (III) is 6,7,8,9,10,11,12,13-octahydro-8,11-dimethyl-5,23:

14,19-dimetheno-20H-dibenzo[k,q]pyrrolo[3,4-n][1,4,7,10]tetraazacyclooctadecine-20,22 (21H)-dione.

118. The method of claim 83, wherein the compound of the Formula (III) is 7,8,9,10,12,13,16,17,18,19-decahydro-8,17-dimethyl-15H,26H-5,29:

20,25-dimetheno-6H-dibenzo[k,q]pyrrolo[3,4-n][1,4,7,10,19,22]dioxatetraazacyclotetracosine-26,28(27H)-dione.

119. The method of claim 83, wherein the compound of the Formula (III) is 14-(2-furylmethyl)-6,7,9,10,13,14,15,16-octahydro-12H,23H-5,26:

17,22-di(metheno)dibenzo[k,q]pyrrolo[3,4-n][1,4,7,10,19]dioxatriazacyclohenicosine-23,25(24H)-dione.

120. The method of claim 83, wherein the compound of the Formula (III) is 14-(2-thienylmethyl)-6,7,9,10,13,14,15,16-octahydro-12H,23H-5,26:

17,22-di(metheno)dibenzo[k,q]pyrrolo[3,4-n][1,4,7,10,19]dioxatriazacyclohenicosine-23,25(24H)-dione.

121. The method of claim 83, wherein the compound of the Formula (III) is 14-(1-naphthylmethyl)-6,7,9,10,13,14,15,16-octahydro-12H,23H-5,26:

17,22-di(metheno)dibenzo[k,q]pyrrolo[3,4-n][1,4,7,10,19]dioxatriazacyclohenicosine-23,25(24H)-dione.

122. The method of claim 83, wherein the compound of the Formula (III) is 14-(pyridin-4-ylmethyl)-6,7,9,10,13,14,15,16-octahydro-12H,23H-5,26:

17,22-di(metheno)dibenzo[k,q]pyrrolo[3,4-n][1,4,7,10,19]dioxatriazacyclohenicosine-23,25(24H)-dione.

123. The method of claim 83, wherein the compound of the Formula (III) is 3-[1-(2-{2-[2-(1,2,3,4-tetrahydro-naphthalen-1-ylamino)-ethoxy]-ethoxy}-ethyl)-1H-indol-3-yl]-4-{1-[2-(1,2,3,4-tetrahydro-naphthalen-1-ylamino)-ethyl]-1H-indol-3-yl}-pyrrole-2,5-dione.

124. The method of claim 83, wherein the compound of the Formula (III) is 3-[1-(3-dimethylamino-phenyl)-1H-indol-3-yl]-4-[1-(2-hydroxy-ethyl)-1H-indazol-3-yl]-pyrrole-2,5-dione.

125. The method of claim 83, wherein the compound of the Formula (III) is 3-[5-chloro-1-(6-dimethylamino-pyridin-3-yl)-1H-indol-3-yl]-4-[1-(2-hydroxy-ethyl)-1H-indazol-3-yl]-pyrrole-2,5-dione.

126. The method of claim 83, wherein the compound of the Formula (III) is 5-(5-chloro-3-{4-[1-(2-hydroxy-ethyl)-1H-indazol-3-yl]-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl}-indol-1-yl)-nicotinic acid methyl ester.