Compounds and methods for development of Ret modulators

• US 20070049615A1
• Filed: 12/17/2004
• Published: 03/01/2007
• Est. Priority Date: 12/19/2003
• Status: Active Grant

• Current Assignee: Plexxikon Inc (Daiichi Sankyo Company Limited)
• Original Assignee: Plexxikon Inc (Daiichi Sankyo Company Limited)
• Inventors: Zhang, Jiazhong, West, Brian, Zhang, Chao, Mamo, Shumeye, Bremer, Ryan, Zhu, Yong, Kumar, Abhinav, Nespi, Marika, Krupka, Heike, Ibrahim, Prabha, Zuckerman, Rebecca, Habets, Gaston, Hurt, Clarence, Artis, Dean
• Granted Patent: US 7,504,509 B2
• Time in Patent Office: Days
• US Class Current: 514/300
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07D 471/04 Ortho-condensed systems

Abstract

Compounds active on Ret are described, as well as methods of using such compounds. Also described are crystal structures of Ret surrogates that were determined using X-ray crystallography. The use of such Ret surrogate crystals and strucural information can, for example, be used for identifying molecular scaffolds and for developing ligands that bind to and modulate Ret and for identifying improved ligands based on known ligands.

Claims

1. A compound having the chemical structure of Formula I, namely

2. The compound of claim 1, wherein said compound is a compound of Formula Ia, Ib, Ic, Id, or Ig.

3. The compound of claim 1, wherein X is S, and R⁴ or R⁵ is H.

4. The compound of claim 1, wherein X is S, and Y is O.

5. The compound of claim 1, wherein R⁴ or R⁵ is H, and R¹ or R² is H.

6. The compound of claim 1, wherein said compound is a compound listed in Table 1.

7. A pharmaceutical composition comprising

a compound according to claim 1;

and

a pharmaceutially acceptable carrier.

8. The pharmaceutical composition of claim 7, wherein said compound is a compound of Formula Ia, Ib, Ic, Id, or Ig.

9. The pharmaceutical composition of claim 7, wherein said compound is a compound listed in Table 1.

10. A method for treating a patient suffering from or at risk of a disease or condition for which Ret modulation provides a therapeutic benefit, comprising

administering to said patient a Ret modulator having a chemical structure of a compound according to claim 1.

11. The method of claim 10, wherein said compound is a compound of Formula Ia, Ib, Ic, Id, or Ig.

12. The method of claim 10, wherein said compound is a compound listed in Table 1.

13. The method of claim 10, wherein said compound is approved for administration to a human.

14. The method of claim 10, wherein said disease or condition is a Ret-mediated disease or condition.

15. The method of claim 10, wherein said disease or condition is selected from the group consisting of multiple endocrine neoplasia, type IIA (MEN2A), multiple endocrine neoplasia, type IIB (MEN2B), Hirschsprung disease (HSCR;

aganglionic megacolon), or medullary thyroid carcinoma (MTC), familial medullary thyroid carcinomas (FMTC), and papillary thyroid carcinomas (PTC).

16. A kit comprising a pharmaceutical composition comprising a compound according to claim 1.

17. The kit of claim 16, wherein said compound is a compound of Formula Ia, Ib, Ic, Id, or Ig.

18. The kit of claim 16, wherein said compound is a compound listed in Table 1.

19. The kit of claim 16, further comprising a written indication that said composition is approved for administering to a human.

20. The kit of claim 19, wherein said composition is approved for a medical indication selected from the group consisting of multiple endocrine neoplasia, type IIA (MEN2A), multiple endocrine neoplasia, type IIB (MEN2B), Hirschsprung disease (HSCR;

aganglionic megacolon), or medullary thyroid carcinoma (MTC), familial medullary thyroid carcinomas (FMTC), and papillary thyroid carcinomas (PTC).

21. A method for developing an improved modulator active on Ret, comprising

determining whether any of a plurality of test compounds of Formula I provides an improvement in one or more desired pharmacologic properties relative to a reference compound active on Ret;

and

selecting those compound(s) if any, that have an improvement in said desired pharmacologic property, thereby providing an improved modulator.

22. The method of claim 21, wherein said desired pharmacologic property is at least 10-fold greater activity on Ret than on FGFR1.

23. The method of claim 21, wherein said desired property is an IC 50 of less than 0.10 μ

M.

24. The method of claim 21, wherein said reference compound is a compound of Formula I.

25. The method of claim 21, wherein at least one derivative of said improved modulator is used as a test compound and said determining and selecting are repeated.

26. A method for developing ligands binding to Ret, comprising

identifying as molecular scaffolds one or more compounds that bind to a binding site of Ret or a Ret surrogate;

determining the orientation of at least one molecular scaffold in co-crystals with a Ret surrogate; and

identifying chemical structures of said molecular scaffolds, that, when modified, alter the binding affinity or binding specificity or both between the molecular scaffold and Ret; and

synthesizing a ligand wherein one or more of the chemical structures of the molecular scaffold is modified to provide a ligand that binds to Ret with altered binding affinity or binding specificity or both.

27. The method of claim 26, wherein said molecular scaffold is a weak binding compound.

28. The method of claim 26, wherein said molecular scaffold binds to a plurality of kinases.

29. The method of claim 26, wherein said molecular scaffold includes the core structure of Formula I.

30. A method for developing ligands specific for Ret, comprising

identifying a compound that binds to a plurality of tyrosine kinases;

and

determining whether a derivative of said compound has greater specificity for Ret than said compound.

31. The method of claim 30, wherein said compound binds to Ret with an affinity at least 10-fold greater than for binding to any of said plurality of tyrosine kinases.

32. The method of claim 31, wherein said compound interacts with at least one conserved Ret active site residue.

33. The method of claim 30, wherein said compound binds weakly to said plurality of tyrosine kinases.

34. The method of claim 30, wherein said plurality of tyrosine kinases comprises Ret and FGFR1.

35. The method of claim 30, wherein said plurality of tyrosine kinases comprises Ret, FGFR1, FGFR2, FGFR3, and FGFR4.

36. The method of claim 30, wherein said compound includes the core structure of Formula I.

37. A method for obtaining a crystal of Ret surrogate, comprising subjecting Ret surrogate protein at 5-20 mg/ml to crystallization condition substantially equivalent to 10-20% PEG 3350, 0.1M Hepes pH 6.5, 0.2M (NH₄)₂SO₄, 10% ethylene glycol at 4°

C.

38. The method of claim 37, further comprising optimizing said crystallization condition.

39. The method of claim 37, wherein said Ret surrogate is derived from FGFR1 and comprises the amino acid substitutions present in Ret surrogate 2.

40. A co-crystal of Ret surrogate and a Ret binding compound.

41. The co-crystal of claim 40, wherein said binding compound interacts with at least one conserved Ret active site residue.

42. The co-crystal of claim 40, wherein said binding compound is a compound of Formula I.

43. The co-crystal of claim 40, wherein said co-crystal is in an X-ray beam.

44. The co-crystal of claim 40, wherein said binding compound includes the core structure of Formula I.

45. A method for determining a structure of a kinase, comprising

creating a homology model from an electronic representation of a Ret surrogate structure containing representations of atomic coordinates as in any of Tables 3, 4, and 5.

46. The method of claim 45, wherein said creating comprises

identifying conserved amino acid residues between Ret and said kinase;

transferring the atomic coordinates of a plurality of conserved amino acids in said Ret surrogate structure to the corresponding amino acids of said kinase to provide a rough structure of said kinase; and

constructing structures representing the remainder of said kinase using electronic representations of the structures of the remaining amino acid residues in said kinase.

47. The method of claim 46, further comprising fitting said homology model to low resolution x-ray diffraction data from one or more crystals of said kinase.

48. The method of claim 46, wherein the coordinates of conserved residues shown in any of Tables 3, 4, and 5 are utilized.

49. An electronic representation of a co-crystal structure of Ret or Ret surrogate binding site with a binding compound, wherein said crystal structure includes representations of atomic coordinates corresponding to binding site atoms of said '"'"'Ret or Ret surrogate.

50. The electronic representation of claim 49, wherein said binding compound is a compound of Formula I.

51. The electronic representation of claim 49, comprising a schematic representation.

52. The electronic representation of claim 49, wherein atomic coordinates for a Ret surrogate binding site comprising the amino acid sequence of Ret are used.

53. The electronic representation of claim 49, wherein said Ret surrogate consists essentially of a kinase domain.

54. The electronic representation of claim 49, wherein said binding compound includes a core structure different from the core structure of Formula I.

55. The electronic representation of claim 49, comprising a binding site surface contour.

56. The electronic representation of claim 49, comprising representations of the binding character of a plurality of conserved amino acid residues.

57. A method for developing a biological agent, comprising

analyzing a Ret surrogate crystal structure and identifying at least one sub-structure for forming a said biological agent.

58. The method of claim 57, wherein said substructure comprises a Ret epitope, and said method further comprises developing antibodies against said epitope.

59. The method of claim 57, wherein said sub-structure comprises a mutation site expected to provide altered Ret activity, and said method further comprises creating a mutation at said site in Ret thereby providing a modified Ret.

60. The method of claim 57, wherein said sub-structure comprises an attachment point for attaching a separate moiety.

61. The method of claim 60, wherein said separate moiety is selected from the group consisting of a peptide, a polypeptide, a solid phase material, a linker, and a label.

62. The method of claim 60, further comprising attaching said separate moiety.

63. A method for identifying potential Ret binding compounds, comprising

fitting at least one electronic representation of a compound in an electronic representation of a Ret or Ret surrogate binding site of a structure of Ret surrogate complexed with a binding compound.

64. The method of claim 63, wherein said electronic representation of a Ret or Ret surrogate binding site is defined by atomic structural coordinates set forth in any of Tables 1-3.

65. The method of claim 63, comprising

removing a computer representation of said compound complexed with Ret or Ret surrogate and fitting a computer representation of a compound from a computer database with a computer representation of the active site of Ret or Ret surrogate;

and

identifying compounds that best fit said active site based on favorable geometric fit and energetically favorable complementary interactions as potential binding compounds.

66. The method of claim 63, comprising

modifying a computer representation of said compound complexed with Ret or Ret surrogate by the deletion or addition or both of one or more chemical groups;

fitting a computer representation of a compound from a computer database with a computer representation of the active site of Ret or Ret surrogate; and

identifying compounds that best fit said active site based on favorable geometric fit and energetically favorable complementary interactions as potential binding compounds.

67. The method of claim 63, comprising

removing a computer representation of a compound complexed with Ret or Ret surrogate and;

and

searching a database for compounds having structural similarity to said compound using a compound searching computer program or replacing portions of said compound with similar chemical structures using a compound construction computer program.

68. The method of claim 63, wherein said compound complexed with Ret surrogate is a compound of Formula I.

69. The method of claim 63, wherein said fitting comprises determining whether a said compound will interact with one or more of conserved Ret active site residues.

70. A method for attaching a Ret binding compound to an attachment component,

comprising

identifying energetically allowed sites for attachment of a said attachment component on a kinase binding compound;

and

attaching said compound or derivative thereof to said attachment component at said energetically allowed site.

71. The method of claim 70, wherein said attachment component is a linker for attachment to a solid phase medium, and said method further comprises attaching said compound or derivative to a solid phase medium through a linker attached at a said energetically allowed site.

72. The method of claim 70, wherein said kinase comprises conserved residues matching at least one conserved Ret active site residues.

73. The method of claim 71, wherein said linker is a traceless linker.

74. The method of claim 70, wherein said kinase binding compound or derivative thereof is synthesized on a said linker attached to said solid phase medium.

75. The method of claim 74, wherein a plurality of said compounds or derivatives are synthesized in combinatorial synthesis.

76. The method of claim 70, wherein attachment of said compound to said solid phase medium provides an affinity medium.

77. The method of claim 70, wherein said attachment component comprises a label.

78. The method of claim 77, wherein said label comprises a fluorophore.

79. A modified compound, comprising

a Ret binding compound, with a linker moiety attached thereto at an energetically allowed site for binding of said modified compound to Ret.

80. The compound of claim 79, whereins said linker is attached to a solid phase.

81. The compound of claim 79, wherein said linker comprises or is attached to a label.

82. The compound of claim 79, wherein said linker is a traceless linker.

83. A method for developing ligands binding to Ret, comprising

identifying as molecular scaffolds one or more compounds that bind to a binding site of Ret;

determining the orientation of at least one molecular scaffold in co-crystals with Ret or Ret surrogate; and

identifying chemical structures of said molecular scaffolds, that, when modified, alter the binding affinity or binding specificity or both between the molecular scaffold and Ret; and

synthesizing a ligand wherein one or more of the chemical structures of the molecular scaffold is modified to provide a ligand that binds to Ret with altered binding affinity or binding specificity or both.

84. The method of claim 83, wherein said molecular scaffold is a weak binding compound.

85. The method of claim 83, wherein said molecular scaffold binds to a plurality of kinases.

86. The method of claim 83, wherein said molecular scaffold includes the core structure of Formula I.

87. A method for developing ligands specific for Ret, comprising

identifying a compound that binds to a plurality of kinases;

and

determining whether a derivative of said compound has greater specificity for Ret than said compound.

88. The method of claim 87, wherein said compound binds to Ret with an affinity at least 10-fold greater than for binding to any of said plurality of kinases.

89. The method of claim 87, wherein said compound interacts with at least one conserved Ret active site residue.

90. The method of claim 87, wherein said compound binds weakly to said plurality of kinases.

91. The method of claim 87, wherein said plurality of kinases comprises Ret and FGFR1.

92. The method of claim 87, wherein said plurality of kinases comprises Ret, FGFR1, FGFR2, FGFR3, and FGFR4.

93. An electronic representation of a crystal structure of Ret surrogate, containing atomic coordinate representations corresponding to Ret binding site atoms.

94. The electronic representation of claim 93, comprising a schematic representation.

95. The electronic representation of claim 93, wherein said Ret surrogate comprises a Ret binding site sequence.

96. A FGFR-based homology model for Ret, comprising an atomic coordinate set derived by replacing FGFR amino acids with corresponding Ret residues.

97. A method for modeling binding of a compound in Ret kinase binding site, comprising modeling binding of a said compound in binding site of a Ret surrogate.

98. The method of claim 97, wherein said Ret surrogate comprises a FGFR sequence substituted with at least 6 corresponding Ret amino acids.

99. A Ret surrogate protein, comprising a FGFR kinase domain sequence modified by the substitution of at least 4 binding site amino acid residues to amino acids present at the corresponding sites in Ret.

100. The Ret surrogate protein of claim 99, wherein said Ret surrogate comprises the amino acid substitutions of Ret Surrogate 1.

101. The Ret surrogate protein of claim 99, wherein said Ret surrogate comprises the amino acid substitutions of Ret Surrogate 2.

102. The Ret surrogate protein of claim 99, wherein said Ret surrogate comprises the substitution of Ret binding site sequence for corresponding FGFR binding site sequence.