SMALL MOLECULE COMPOUNDS FOR STEM CELL DIFFERENTIATION

• US 20100159596A1
• Filed: 09/16/2009
• Published: 06/24/2010
• Est. Priority Date: 09/17/2008
• Status: Active Grant

• Current Assignee: Human Biomolecular Research Institute, Sanford Burnham Prebys Medical Discovery Institute
• Original Assignee: Burnham Institute For Medical Research, Human Biomolecular Research Institute
• Inventors: Mercola, Mark, Cashman, John, Dawson, Marcia
• Granted Patent: US 9,012,217 B2
• Time in Patent Office: Days
• US Class Current: 435/377
• CPC Class Codes: C07D 215/54 attached in position 3
• CPC Class Codes: C07D 235/06 with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached in position 2
• CPC Class Codes: C07D 279/20 with hydrogen atoms directly attached to the ring nitrogen atom
• CPC Class Codes: C07D 279/22 with carbon atoms directly attached to the ring nitrogen atom
• CPC Class Codes: C07D 279/28 with other substituents attached to the ring system
• CPC Class Codes: C07D 401/04 directly linked by a ring-member-to-ring-member bond
• CPC Class Codes: C07D 401/10 linked by a carbon chain containing aromatic rings
• CPC Class Codes: C07D 401/12 linked by a chain containing hetero atoms as chain links
• CPC Class Codes: C07D 401/14 containing three or more hetero rings
• CPC Class Codes: C07D 403/12 linked by a chain containing hetero atoms as chain links
• CPC Class Codes: C07D 405/04 directly linked by a ring-member-to-ring-member bond
• CPC Class Codes: C07D 405/12 linked by a chain containing hetero atoms as chain links
• CPC Class Codes: C07D 409/04 directly linked by a ring-member-to-ring-member bond
• CPC Class Codes: C07D 491/04 Ortho-condensed systems
• CPC Class Codes: C12N 5/0603 Embryonic cells production of embryos, nuclear transfer A01K67/027; Embryoid bodies
• 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 5/0623 Stem cells

Abstract

Methods and small molecule compounds for stem cell differentiation are provided. One example of a class of compounds that may be used is represented by the compound having the structure IA or IB in the form of free base or a pharmaceutically acceptable salt, hydrate, solvate or N-oxide thereof:

R₁ is independently hydrogen or (C₁-C₆)alkyl; R₂ is independently hydrogen, (C₁-C₆)alkyl, aryl, or heteroaryl; R_2′ is independently hydrogen, (C₁-C₆)alkyl, CF₃ or C₂F₅; R₃ is independently (C₁-C₆)alkyl, aryl, 2-tetrahydrofurylmethyl, an aliphatic tertiary amine, or 4-methoxybenzyl; or R₂ and R₃ may be joined together to form a 5 or 6 member ring lactone; R₄ is independently hydrogen, (C₁-C₆)alkyl, a 2- or 4-R₅-substituted aromatic ring selected from a 4-R₅-phenyl or a 2-R₅-5-pyridyl, aryl, heteroaryl, aliphatic tertiary amine or halogen; and R₅, R_5′, R₆, R_6′, R₇, R_7′, are each independently hydrogen, (C₁-C₆)alkyl, aryl, optionally substituted phenyl, heteroaryl, a heterocyclic ring, an aliphatic tertiary amine, or halogen.

Claims

1. A compound of structure IA or IB in the form of a free base or a pharmaceutically acceptable salt, hydrate, solvate or N-oxide thereof:

wherein;

R₁ is independently hydrogen or a moiety forming a salt;

R₂ is independently hydrogen, (C₁-C₆)alkyl, aryl, or heteroaryl;

R_2′

, is independently hydrogen, (C₁-C₆)alkyl, CF₃ or C₂F₅;

R₃ is independently (C₁-C₆)alkyl optionally substituted by an amine, aryl, 2-tetrahydrofurylmethyl, an aliphatic tertiary amine, or 4-methoxybenzyl;

or R₂ and R₃ may be joined together to form a 5 or 6 member ring lactone;

R₄ is independently hydrogen, (C₁-C₆)alkyl, a 2- or 4-R₈-substituted aromatic ring selected from a phenyl, pyridyl, aryl, heteroaryl;

R₅, R_5′

, R₆, R_6′

, R₇, R_7′

are each independently hydrogen, (C₁-C₆)alkyl, aryl, optionally substituted phenyl, heteroaryl, a heterocyclic ring, an aliphatic tertiary amine, or halogen; and

R₈ is independently hydrogen, (C₁-C₆)alkyl, alkoxy, CF₃, aliphatic tertiary amine, cyano or halogen.

2. The compound of structure IA or IB of claim 1, having formula I-14:

3. A compound of structure II in the form of a free base or a pharmaceutically acceptable salt, hydrate, solvate or N-oxide thereof:

wherein;

A is independently NH, O, NHCH₂, NHCO, COO, or bond;

B is independently an aromatic ring or a saturated 4, 5, 6 or 7 member ring optionally containing a heteroatom such as N or O;

D is independently N or CH,

E is independently N, CH, C—

R₁, C—

NH₂, C═

O or C—

N(R₁)₂;

F is independently N or C,

G is independently phenyl or cycloalkyl optionally substituted by 1 to 5 R₂;

I is independently N or CH;

R₁ is independently hydrogen or (C₁-C₆)alkyl; and

R₂ is independently hydrogen, (C₁-C₆)alkyl, (C₁-C₆)alkoxy, (C₁-C₆)thioalkoxy, hydroxy, halogen, CF₃, or C₂F₅.

4. The compound of structure II of claim 3, having formula:

wherein;

R is independently hydrogen, methyl, amino or aminoalkyl;

R₁ is independently hydrogen, hydroxyl, pyridyl, methyl, trifluoromethyl, methoxy or methylthio, with the further proviso that when R₁ is a methoxy group in position 2 to the benzimidazole ring, a further methyl group in position 6 to the benzimidazole is optionally present;

R₂ is independently hydrogen, phenyl, benzyl, methoxy, methyl or halogen.

5. The compound of structure II of claim 3, having structure IIA, IIB, IIC, IID, IIE, IIF, IIG, IIH, IIJ, IIK, IIL, or IIM in the form of a free base or a pharmaceutically acceptable salt, hydrate, solvate or N-oxide thereof:

6. The compound of structure II of claim 3, having formula 15-37:

7. A compound of structure I or II in the form of a free base or a pharmaceutically acceptable salt, hydrate, solvate or N-oxide thereof:

wherein;

A is independently S, O or (CH₂)_n, and n is independently an integer from 0 to 2;

B and D are each independently alkyl, aryl, halo, SCH₃, or CF₃;

E is independently (CH₂)_m, and m is independently an integer from 0 to 5;

F is independently a primary or secondary alkyl amine, cyclic amine, an aryl amine or aliphatic cyclic amine;

G and H are each independently alkyl, aryl, halo, SCH₃, or CF₃;

I is independently (CH₂)_m, and m is independently an integer from 0 to 5; and

J is independently a primary or secondary alkyl amine, cyclic amine, an aryl amine or aliphatic cyclic amine.

8. The compound of structure I of claim 7, having structure III:

wherein;

R₁ is independently hydrogen, CH₃, SCH₃, or CF₃;

X is independently CH, N, or O; and

Y is independently S, O, (CH₂)_n, where n is an integer from 0 to 2.

9. The compound of structure I of claim 7, having structure IIIa:

wherein;

R₁ is independently CF₃ or chloro; and

R₂ is independently an alkyl amine, a cyclic amine, an aliphatic cyclic amine, or an alkyl amide.

10. The compound of claim 9, having formula 38-43:

11. A compound of structure V, VI or VII in the form of free base or a pharmaceutically acceptable salt, hydrate, solvate or N-oxide thereof:

wherein for compound V;

X is independently a bond, CH₂, or CHR₇;

R₁ is independently methyl, ethyl, (C₁-C₆)alkyl, halogen, phenyl, methoxy, phenoxy, nitro, trifluoromethyl, or alkylamino;

R₂ is independently methyl, ethyl, phenyl, (C₁-C₆)alkyl, trifluoromethyl, or halogen;

R₃ is independently methyl, ethyl, (C₁-C₆)alkyl, phenyl, or aryl;

R₄ is independently methyl, ethyl, (C₁-C₆)alkyl, phenyl, or aryl;

R₅ is independently methyl, ethyl, (C₁-C₆)alkyl, phenyl, or aryl;

R₆ is independently methyl, ethyl, (C₁-C₆)alkyl, phenyl, or aryl, wherein R₅ and R₆ may be joined via a ring;

R₇ is independently methyl, ethyl, propyl, (C₁-C₆)alkyl, phenyl, or benzyl; and

m is independently 0-4 methylene units;

wherein for compound VI;

X is independently a bond, CH₂, or CHR₁₂;

R₈ is independently methyl, ethyl, (C₁-C₆)alkyl, halogen, phenyl, methoxy, phenoxy, nitro, trifluoromethyl, or alkylamino,

R₉ is independently methyl, ethyl, phenyl, (C₁-C₆)alkyl, trifluoromethyl, or halogen;

R₁₀ is independently methyl, ethyl, (C₁-C₆)alkyl, phenyl, or aryl;

R₁₁ is independently methyl, ethyl, (C₁-C₆)alkyl, phenyl, or aryl, wherein R₁₀ and R₁₁ can be joined via a ring;

R₁₂ is independently methyl, ethyl, propyl, (C₁-C₆)alkyl, phenyl, or benzyl; and

m is independently 0-4 methylene units; and

wherein for compound VII;

X is independently a bond, CH₂, or CHR₁₇;

R₁₃ is independently methyl, ethyl, (C₁-C₆)alkyl, halogen, phenyl, methoxy, phenoxy, nitro, trifluoromethyl, or alkylamino;

R₁₄ is independently methyl, ethyl, phenyl, (C₁-C₆)alkyl, trifluoromethyl, or halogen;

R₁₅ is independently methyl, ethyl, (C₁-C₆)alkyl, phenyl, or aryl;

R₁₆ is independently methyl, ethyl, (C₁-C₆)alkyl, phenyl, or aryl, wherein R₁₅ and R₁₆ may be joined via a ring;

R₁₇ is independently methyl, ethyl, propyl, (C₁-C₆)alkyl, phenyl, or benzyl;

R₁₈ is independently methyl, ethyl, propyl, (C₁-C₆)alkyl, phenyl, or benzyl; and

m is independently 0-4 methylene units.

12. A method for producing differentiated cells from stem cells, comprising contacting the stem cells with a compound of structure IA or IB in the form of a free base or a pharmaceutically acceptable salt, hydrate, solvate or N-oxide thereof:

wherein;

R₁ is independently hydrogen or a moiety forming a salt;

R₂ is independently hydrogen, (C₁-C₆)alkyl, aryl, or heteroaryl;

R_2′

is independently hydrogen, (C₁-C₆)alkyl, CF₃ or C₂F₅;

R₃ is independently (C₁-C₆)alkyl, aryl, 2-tetrahydrofurylmethyl, an aliphatic tertiary amine, or 4-methoxybenzyl;

or R₂ and R₃ may be joined together to form a 5 or 6 member ring lactone;

R₄ is independently hydrogen, (C₁-C₆)alkyl, a 2- or 4-R₅-substituted aromatic ring selected from a 4-R₅-phenyl or a 2-R₅-5-pyridyl, aryl, heteroaryl, aliphatic tertiary amine or halogen; and

R₅, R_5′

, R₆, R_6′

, R₇, R_7′

, are each independently hydrogen, (C₁-C₆)alkyl, aryl, optionally substituted phenyl, heteroaryl, a heterocyclic ring, an aliphatic tertiary amine, or halogen.

13. The method of claim 12, wherein the compound of structure IA and IB has formula 1-14:

14. The method of claim 12, wherein contacting the stem cells with a compound of structure IA or IB is from about 24 hours to about 192 hours.

15. The method of claim 14, wherein contacting the stem cells with a compound of structure IA or IB is from about 48 hours to about 144 hours.

16. The method of claim 12, wherein the differentiated cells are cardiomyocytes.

17. The method of claim 12, further comprising contacting the cells with Activin A.

18. The method of claim 12, wherein the cells differentiate to mesoderm.

19. The method of claim 12, further comprising contacting the cells with a Wnt protein.

20. The method of claim 19, wherein the Wnt protein is Wnt3a.

21. The method of claim 12, wherein the stem cells are embryonic stem cells, induced pluripotent stem cells or adult stem cells.

22. A method for producing differentiated cells from stem cells, comprising contacting the stem cells with a compound of structure II in the form of a free base or a pharmaceutically acceptable salt, hydrate, solvate or N-oxide thereof:

wherein;

A is independently NH or NHCH₂;

B is independently an aromatic ring or a saturated 4, 5, 6 or 7 member ring optionally containing a heteroatom such as N or O;

D is independently N or CH,

E is independently N, CH, C—

R₁, C—

NH₂, or C—

N(R₁)₂;

F is independently N or CH,

G is independently phenyl or cyclohexyl optionally substituted by 1 to 5 R₂;

I is independently N or C;

R₁ is independently hydrogen or (C₁-C₆)alkyl; and

R₂ is independently hydrogen, (C₁-C₆)alkyl, (C₁-C₆)alkoxy, (C₁-C₆)thioalkoxy, hydroxy, halogen, CF₃, or C₂F₅.

23. The method of claim 22, wherein the compound of structure II has formula:

wherein;

R is independently hydrogen, methyl, or amino;

R₁ is independently hydrogen, hydroxyl, pyridyl, methyl, trifluoromethyl, methoxy or methylthio, with the further proviso that when R₁ is a methoxy group in position 2 to the benzimidazole ring, a further methyl group in position 6 to the benzimidazole is optionally present;

R₂ is independently hydrogen, phenyl, benzyl, methoxy, methyl or halogen.

24. The method of claim 22, wherein the compound of structure II has structure IIA, IIB, IIC, IID, IIE, IIF, IIG, IIH, IIJ, IIK, IIL, or IIM in the form of a free base or a pharmaceutically acceptable salt, hydrate, solvate or N-oxide thereof:

25. The method of claim 22, wherein the compound of structure II has formula 15-39:

26. The method of claim 22, further comprising a step of maintaining contact between for a period of time between about 24 hours and about 192 hours.

27. The method of claim 26, wherein the period of contact is between about 48 hours and about 144 hours.

28. The method of claim 22, wherein the differentiated cells are cardiomyocytes.

29. The method of claim 22, further comprising contacting the cells with Activin A.

30. The method of claim 22, wherein the cells differentiate to mesoderm.

31. The method of claim 22, further comprising contacting the cells with a Wnt protein.

32. The method of claim 31, wherein the Wnt protein is Wnt3a.

33. The method of claim 22, wherein the stem cells are embryonic stem cells, induced pluripotent stem cells or adult stem cells.

34. A method for producing differentiated cells from stem cells, comprising contacting the stem cells with a compound of structure I or II in the form of a free base or a pharmaceutically acceptable salt, hydrate, solvate or N-oxide thereof:

wherein;

A is independently S, O or (CH₂)_n, and n is independently an integer from 0 to 2;

B and D are each independently alkyl, aryl, halo, SCH₃, or CF₃;

E is independently (CH₂)_m, and m is independently an integer from 0 to 5;

F is independently a primary or secondary alkyl amine, cyclic amine, an aryl amine or aliphatic cyclic amine;

G and H are each independently alkyl, aryl, halo, SCH₃, or CF₃;

I is independently (CH₂)_m, and m is independently an integer from 0 to 5; and

J is independently a primary or secondary alkyl amine, cyclic amine, an aryl amine or aliphatic cyclic amine.

35. The method of claim 34, wherein the compound of structure I has structure III:

wherein;

R₁ is independently hydrogen, CH₃, SCH₃, or CF₃;

X is independently CH, N, or O; and

Y is independently S, O, (CH₂)_n, where n is an integer from 0 to 2.

36. The method of claim 34, wherein the compound of structure I has structure III:

wherein;

R₁ is independently CF₃ or chloro; and

R₂ is independently an alkyl amine, a cyclic amine, an aliphatic cyclic amine, or an alkyl amide.

37. The method of claim 34, wherein the compound of structure I has formula 38-43:

38. The method of claim 34, further comprising a step of maintaining contact between for a period of time between about 24 hours and about 192 hours.

39. The method of claim 38, wherein the period of contact is between about 48 hours and about 144 hours.

40. The method of claim 34, wherein the differentiated cells are cardiomyocytes.

41. The method of claim 34, further comprising contacting the cells with Activin A.

42. The method of claim 34, wherein the cells differentiate to mesoderm.

43. The method of claim 34, further comprising contacting the cells with a Wnt protein.

44. The method of claim 43, wherein the Wnt protein is Wnt3a.

45. The method of claim 34, wherein the stem cells are embryonic stem cells, induced pluripotent stem cells or adult stem cells.

46. A method for producing differentiated cells from stem cells, comprising contacting the stem cells with a compound of structure V, VI or VII in the form of free base or a pharmaceutically acceptable salt, hydrate, solvate or N-oxide thereof:

wherein for compound V;

X is independently a bond, CH₂, or CHR₇;

R₁ is independently methyl, ethyl, (C₁-C₆)alkyl, halogen, phenyl, methoxy, phenoxy, nitro, trifluoromethyl, or alkylamino;

R₂ is independently methyl, ethyl, phenyl, (C₁-C₆)alkyl, trifluoromethyl, or halogen;

R₃ is independently methyl, ethyl, (C₁-C₆)alkyl, phenyl, or aryl;

R₄ is independently methyl, ethyl, (C₁-C₆)alkyl, phenyl, or aryl;

R₅ is independently methyl, ethyl, (C₁-C₆)alkyl, phenyl, or aryl;

R₆ is independently methyl, ethyl, (C₁-C₆)alkyl, phenyl, or aryl, wherein R₅ and R₆ may be joined via a ring;

R₇ is independently methyl, ethyl, propyl, (C₁-C₆)alkyl, phenyl, or benzyl; and

m is independently 0-4 methylene units;

wherein for compound VI;

X is independently a bond, CH₂, or CHR₁₂;

R₈ is independently methyl, ethyl, (C₁-C₆)alkyl, halogen, phenyl, methoxy, phenoxy, nitro, trifluoromethyl, or alkylamino,

R₉ is independently methyl, ethyl, phenyl, (C₁-C₆)alkyl, trifluoromethyl, or halogen;

R₁₀ is independently methyl, ethyl, (C₁-C₆)alkyl, phenyl, or aryl;

R₁₁ is independently methyl, ethyl, (C₁-C₆)alkyl, phenyl, or aryl, wherein R₁₀ and R₁₁ can be joined via a ring;

R₁₂ is independently methyl, ethyl, propyl, (C₁-C₆)alkyl, phenyl, or benzyl; and

m is independently 0-4 methylene units; and

wherein for compound VII;

X is independently a bond, CH₂, or CHR₁₇;

R₁₃ is independently methyl, ethyl, (C₁-C₆)alkyl, halogen, phenyl, methoxy, phenoxy, nitro, trifluoromethyl, or alkylamino;

R₁₄ is independently methyl, ethyl, phenyl, (C₁-C₆)alkyl, trifluoromethyl, or halogen;

R₁₅ is independently methyl, ethyl, (C₁-C₆)alkyl, phenyl, or aryl;

R₁₆ is independently methyl, ethyl, (C₁-C₆)alkyl, phenyl, or aryl, wherein R₁₅ and R₁₆ may be joined via a ring;

R₁₇ is independently methyl, ethyl, propyl, (C₁-C₆)alkyl, phenyl, or benzyl;

R₁₈ is independently methyl, ethyl, propyl, (C₁-C₆)alkyl, phenyl, or benzyl; and

m is independently 0-4 methylene units.

47. The method of claim 46, further comprising a step of maintaining contact between for a period of time between about 24 hours and about 192 hours.

48. The method of claim 47, wherein the period of contact is between about 48 hours and about 144 hours.

49. The method of claim 46, wherein the differentiated cells are cardiomyocytes.

50. The method of claim 46, further comprising contacting the cells with Activin A.

51. The method of claim 46, wherein the cells differentiate to mesoderm.

52. The method of claim 46, further comprising contacting the cells with a Wnt protein.

53. The method of claim 52, wherein the Wnt protein is Wnt3a.

54. The method of claim 46, wherein the stem cells are embryonic stem cells, induced pluripotent stem cells or adult stem cells.