COMPOUNDS THAT PARTICIPATE IN COOPERATIVE BINDING AND USES THEREOF

Abstract

The invention features compounds (e.g., macrocyclic compounds) capable of modulating biological processes, for example through binding to a presenter protein (e.g., a member of the FKBP family, a member of the cyclophilin family, or PIN1) and a target protein (e.g., a eukaryotic target protein such as a mammalian target protein or a fungal target protein or a prokaryotic target protein such as a bacterial target protein). These compounds bind endogenous intracellular presenter proteins, such as the FKBPs or cyclophilins, and the resulting binary complexes selectively bind and modulate the activity of intracellular target proteins. Formation of a tripartite complex among the presenter protein, the compound, and the target protein is driven by both protein-compound and protein-protein interactions, and both are required for modulation of the targeted protein's activity.

Claims

1. A macrocyclic compound comprising 14 to 40 ring atoms, said compound comprising: (a) a mammalian target protein interacting moiety; and (b) a presenter protein binding moiety; wherein said compound and a presenter protein form a complex that specifically binds to a target protein, and each of said compound and said presenter protein do not substantially bind to said target protein in the absence of forming said complex; or said compound and a presenter protein form a complex that binds to a target protein with at least 5-fold greater affinity than the affinity of each of said compound and said presenter protein to said target protein in the absence of forming said complex; or a pharmaceutically acceptable salt thereof.

2. The compound of claim 1, wherein said presenter protein binding moiety comprises 5 to 20 ring atoms.

3. The compound of claim 1 or 2, wherein said compound consists of 14 to 17 ring atoms.

4. The compound of claim 1 or 2, wherein said compound consists of 14 to 20 atoms.

5. The compound of claim 1 or 2, wherein said compound consists of 21 to 26 ring atoms.

6. The compound of any one of claims 1 to 5, wherein said presenter protein binding moiety comprises the structure of Formula I: ##STR00108## wherein n is 0 or 1; X.sup.1 and X.sup.3 are each independently O, S, CR.sup.3R4, or NR.sup.5; X.sup.2 is O, S, or NR.sup.5; R.sup.1, R.sup.2, R.sup.3, and R.sup.4 are each independently hydrogen, hydroxyl, optionally substituted amino, halogen, thiol, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 alkynyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.2-C.sub.6 heteroalkenyl, optionally substituted C.sub.2-C.sub.6 heteroalkynyl, optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.6-C.sub.10 aryl C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.9 heteroaryl, optionally substituted C.sub.2-C.sub.9 heteroaryl C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.9 heterocyclyl, or optionally substituted C.sub.2-C.sub.9 heterocyclyl C.sub.1-C.sub.6 alkyl, or any two of R.sup.1 R.sup.2, R.sup.3, or R.sup.4 are taken together with the atom or atoms to which they are bound to form an optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; each R.sup.5 is, independently, hydrogen, hydroxyl, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 alkynyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.2-C.sub.6 heteroalkenyl, optionally substituted C.sub.2-C.sub.6 heteroalkynyl, optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.6-C.sub.10 aryl C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.9 heteroaryl, optionally substituted C.sub.2-C.sub.9 heteroaryl C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.9 heterocyclyl, or optionally substituted C.sub.2-C.sub.9 heterocyclyl C.sub.1-C.sub.6 alkyl, or R.sup.5 and one of R.sup.1, R.sup.2, R.sup.3, or R.sup.4 are taken together with the atom or atoms to which they are bound to form an optionally substituted heterocyclyl or optionally substituted heteroaryl.

7. The compound of any one of claims 1 to 6, wherein said presenter protein binding moiety comprises the structure of any one of Formulae II-IV: ##STR00109## wherein o, and p are independently 0, 1, or 2; q is an integer between 0 and 7; r is an integer between 0 and 4; X.sup.4 and X.sup.5 are each, independently, absent, CH.sub.2, O, S, SO, SO.sub.2, or NR.sup.11; each R.sup.6 and R.sup.7 are independently hydrogen, hydroxyl, optionally substituted amino, halogen, thiol, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 alkynyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.2-C.sub.6 heteroalkenyl, optionally substituted C.sub.2-C.sub.6 heteroalkynyl, optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.6-C.sub.10 aryl C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.9 heteroaryl, optionally substituted C.sub.2-C.sub.9 heteroaryl C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.9 heterocyclyl, optionally substituted C.sub.2-C.sub.9 heterocyclyl C.sub.1-C.sub.6 alkyl, or R.sup.6 and R.sup.7 combine with the carbon atom to which they are bound to form CO; each R.sup.8 is, independently, hydroxyl, optionally substituted amino, halogen, thiol, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 alkynyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.2-C.sub.6 heteroalkenyl, optionally substituted C.sub.2-C.sub.6 heteroalkynyl, optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.6-C.sub.10 aryl C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.9 heteroaryl, optionally substituted C.sub.2-C.sub.9 heteroaryl C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.9 heterocyclyl, or optionally substituted C.sub.2-C.sub.9 heterocyclyl C.sub.1-C.sub.6 alkyl, or two R.sup.8 combine to form an optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally substituted C.sub.6-C.sub.10 aryl, or optionally substituted C.sub.2-C.sub.9 heteroaryl; R.sup.9 and R.sup.11 are, independently, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 alkynyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.2-C.sub.6 heteroalkenyl, optionally substituted C.sub.2-C.sub.6 heteroalkynyl, optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.6-C.sub.10 aryl C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.9 heteroaryl, optionally substituted C.sub.2-C.sub.9 heteroaryl C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.9 heterocyclyl, or optionally substituted C.sub.2-C.sub.9 heterocyclyl C.sub.1-C.sub.6 alkyl; R.sup.10 is optionally substituted C.sub.1-C.sub.6 alkyl; and R.sup.12 and R.sup.13 are each, independently, hydrogen, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 alkynyl, optionally substituted aryl, C.sub.3-C.sub.7 carbocyclyl, optionally substituted C.sub.6-C.sub.10 aryl C.sub.1-C.sub.6 alkyl, and optionally substituted C.sub.3-C.sub.7 carbocyclyl C.sub.1-C.sub.6 alkyl.

8. The compound of claim 7, wherein said presenter protein binding moiety comprises the structure of Formula V: ##STR00110## wherein R.sup.14 is hydrogen, hydroxyl, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 alkynyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.2-C.sub.6 heteroalkenyl, optionally substituted C.sub.2-C.sub.6 heteroalkynyl, optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.6-C.sub.10 aryl C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.9 heteroaryl, optionally substituted C.sub.2-C.sub.9 heteroaryl C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.9 heterocyclyl, optionally substituted C.sub.2-C.sub.9 heterocyclyl C.sub.1-C.sub.6 alkyl.

9. The compound of claim 8, wherein said presenter protein binding moiety comprises the structure of Formula VI or VII: ##STR00111## ##STR00112## wherein s and t are each, independently, an integer from 0 to 7; X.sup.6 and X.sup.7 are each, independently, O, S, SO, SO.sub.2, or NR.sup.19; R.sup.15 and R.sup.17 are each, independently, hydrogen hydroxyl, or optionally substituted C.sub.1-C.sub.6 alkyl; R.sup.16 and R.sup.18 are each, independently hydroxyl, optionally substituted amino, halogen, thiol, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 alkynyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.2-C.sub.6 heteroalkenyl, optionally substituted C.sub.2-C.sub.6 heteroalkynyl, optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.6-C.sub.10 aryl C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.9 heteroaryl, optionally substituted C.sub.2-C.sub.9 heteroaryl C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.9 heterocyclyl, or optionally substituted C.sub.2-C.sub.9 heterocyclyl C.sub.1-C.sub.6 alkyl; R.sup.19 is hydrogen, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 alkynyl, optionally substituted C.sub.6-C.sub.10 aryl, C.sub.3-C.sub.7 carbocyclyl, optionally substituted C.sub.6-C.sub.10 aryl C.sub.1-C.sub.6 alkyl, and optionally substituted C.sub.3-C.sub.7 carbocyclyl C.sub.1-C.sub.6 alkyl; and Ar is optionally substituted C.sub.6-C.sub.10 aryl or optionally substituted C.sub.2-C.sub.9 heteroaryl.

10. The compound of any one of claims 1 to 9, wherein said target interacting moiety comprises the structure of Formula IX: ##STR00113## wherein u is an integer from 1 to 20; and each Y is, independently, any amino acid, O, NR, S, S(O) SO.sub.2, or has the structure of any one of Formulae X-XIII: ##STR00114## wherein each R.sup.20 is, independently, hydrogen, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 alkynyl, optionally substituted aryl, C.sub.3-C.sub.7 carbocyclyl, optionally substituted C.sub.6-C.sub.10 aryl C.sub.1-C.sub.6 alkyl, and optionally substituted C.sub.3-C.sub.7 carbocyclyl C.sub.1-C.sub.6 alkyl or R.sup.19 combines with any R.sup.20, R.sup.21, R.sup.22, R.sup.23, R.sup.24, R.sup.25, R.sup.26, R.sup.27, R.sup.28, R.sup.29, or R.sup.30 to form an optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.2-C.sub.9 heterocyclyl, or optionally substituted C.sub.2-C.sub.9 heteroaryl; each R.sup.21 and R.sup.22 is, independently, hydrogen, halogen, optionally substituted hydroxyl, optionally substituted amino, or R.sup.20 and R.sup.21 combine to form O, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 alkynyl, optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.6-C.sub.10 aryl C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.9 heteroaryl, optionally substituted C.sub.2-C.sub.9 heteroaryl C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.9 heterocyclyl, or optionally substituted C.sub.2-C.sub.9 heterocyclyl C.sub.1-C.sub.6 alkyl, or R.sup.21 or R.sup.22 combines with any R.sup.20, R.sup.21, R.sup.22, R.sup.23, R.sup.24, R.sup.25, R.sup.26, R.sup.27, R.sup.28, R.sup.29, or R.sup.30 to form an optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.2-C.sub.9 heterocyclyl, or optionally substituted C.sub.2-C.sub.9 heteroaryl; each R.sup.23 R.sup.24, R.sup.25, and R.sup.26 is, independently, hydrogen, hydroxyl, or R.sup.22 and R.sup.23 combine to form O, or R.sup.23 R.sup.24, R.sup.25, or R.sup.26 combines with any R.sup.20, R.sup.21, R.sup.22, R.sup.23, R.sup.24, R.sup.25, R.sup.26, R.sup.27, R.sup.28, R.sup.29, or R.sup.30 to form an optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.2-C.sub.9 heterocyclyl, or optionally substituted C.sub.2-C.sub.9 heteroaryl; and each R.sup.27, R.sup.28, R.sup.29, and R.sup.30 is, independently, hydrogen, halogen, optionally substituted hydroxyl, optionally substituted amino, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 alkynyl, optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.6-C.sub.10 aryl C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.9 heteroaryl, optionally substituted C.sub.2-C.sub.9 heteroaryl C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.9 heterocyclyl, or optionally substituted C.sub.2-C.sub.9 heterocyclyl C.sub.1-C.sub.6 alkyl, or R.sup.27, R.sup.28, R.sup.29, or R.sup.30 combines with any R.sup.20, R.sup.21, R.sup.22, R.sup.23, R.sup.24, R.sup.25, R.sup.26, R.sup.27, R.sup.28, R.sup.29, or R.sup.30 to form an optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.2-C.sub.9 heterocyclyl, or optionally substituted C.sub.2-C.sub.9 heteroaryl.

11. The compound of claim 10, wherein the compound has the structure of any one of Formulae XIV-XVIII: ##STR00115##

12. The compound of any one of claims 1 to 11, wherein the portion of the molecule that comprises each ring atom that participates in binding to the target protein has a c Log P greater than 2.

13. The compound of any one of claims 1 to 12, wherein the portion of the molecule that comprises each ring atom that participates in binding to the target protein has a polar surface area less than 350 .

14. The compound of any one of claims 1 to 13, wherein the portion of the molecule that comprises each ring atom that participates in binding to the target protein does not have the structure: ##STR00116##

15. The compound of any one of claims 10 to 14, wherein at least one Y is an N-alkylated amino acid.

16. The compound of any one of claims 10 to 15, wherein at least one Y is a D-amino acid.

17. The compound of any one of claims 10 to 16, wherein at least one Y is a non-natural amino acid.

18. The compound of any one of claims 10 to 17, wherein at least one Y comprises a depsi-linkage.

19. The compound of any one of claims 1 to 18, wherein said compound does not comprise the structure: ##STR00117##

20. The compound of any one of claims 1 to 19, wherein said compound has a molecular weight between 400 and 2000 Daltons.

21. The compound of any one of claims 1 to 20, wherein said compound has an even number of ring atoms.

22. The compound of any one of claims 1 to 21, wherein said compound is cell penetrant.

23. The compound of any one of claims 1 to 22, wherein said compound is substantially pure.

24. The compound of any one of claims 1 to 23, wherein said compound is isolated.

25. The compound of any one of claims 1 to 24, wherein said compound is an engineered compound.

26. The compound of any one of claims 1 to 25, wherein said compound is non-naturally occurring.

27. The compound of any one of claims 1 to 26, wherein said complex binds to said target protein with at least 5-fold greater affinity than said complex binds to each of mTOR and/or calcineurin.

28. The compound of any one of claims 1 to 27, wherein said complex binds to said target protein with at least 5-fold greater affinity than the affinity of said compound to said target when said compound is not bound in a complex with said presenter protein.

29. The compound of any one of claims 1 to 28, wherein said complex binds to said target protein with at least 5-fold greater affinity than the affinity of said presenter protein to said target when said presenter protein is not bound in a complex with said compound.

30. The compound of any one of claims 1 to 29, wherein said complex inhibits the naturally occurring interaction between said target protein and a ligand that specifically binds said target protein.

31. The compound of any one of claims 1 to 30, wherein said presenter protein is a prolyl isomerase.

32. The compound of any one of claims 1 to 31, wherein said presenter protein is a member of the FKBP family, a member of the cyclophilin family, or PIN1.

33. The compound of claim 32, wherein said member of the FKBP family is FKBP12, FKBP12.6, FKBP25, or FKBP52.

34. The compound of claim 33, wherein said member of the cyclophilin family is PPIAL4A, PPIAL4B, PPIAL4C, PPIAL4D, or PPIAL4G.

35. The compound of any one of claims 1 to 34, wherein said mammalian target protein is a GTPase, GTPase activating protein, Guanine nucleotide-exchange factor, a heat shock protein, an ion channel, a coiled-coil protein, a kinase, a phosphatase, a ubiquitin ligase, a transcription factor, a chromatin modifier/remodeler, or a protein with classical protein-protein interaction domains and motifs.

36. A presenter protein/compound complex comprising a compound of any one of claims 1-35 and a presenter protein.

37. The complex of claim 36, wherein said presenter protein is a protein encoded by any one of the genes of Table 1.

38. The complex of claim 36, wherein said presenter protein is a prolyl isomerase.

39. The complex of claim 38, wherein said prolyl isomerase is a member of the FKBP family, a member of the cyclophilin family, or PIN1.

40. The complex of claim 38, wherein said member of the FKBP family is FKBP12, FKBP12.6, FKBP25, or FKBP4.

41. The complex of claim 38, wherein said member of the cyclophilin family is PP1A, CYPB, CYPC, CYP40, CYPE, CYPD, NKTR, SRCyp, CYPH, CWC27, CYPL1, CYP60, CYPJ, PPIL4, PPIL6, RANBP2, or PPWD1.

42. A pharmaceutical composition comprising a compound of any one of claims 1-35 and a pharmaceutically acceptable excipient.

43. The pharmaceutical composition of claim 42 in unit dosage form.

44. A method of modulating a target protein, said method comprising contacting said target protein with a modulating amount of a compound of any one of claims 1-35, a presenter protein/compound complex of any one of claims 36-41, or a composition of claim 42 or 43.

45. A method of modulating a target protein, said method comprising forming a presenter protein/compound complex of any one of claims 36-41 in a cell by contacting said cell with an effective amount of a compound of any one of claims 1-35 or a composition of claim 42 or 43.

46. A method of modulating a target protein, said method comprising contacting said target protein with a presenter protein/compound complex of any one of claims 36-41.

47. A method of inhibiting prolyl isomerase activity, said method comprising contacting a cell expressing said prolyl isomerase with a compound of any one of claims 1-35 or a composition of claim 42 or 43 under conditions that permit the formation of a complex between said compound and said prolyl isomerase, thereby inhibiting prolyl isomerase activity.

48. A method of forming a presenter protein/compound complex of claim 36 in a cell, said method comprising contacting a cell expressing said presenter protein with a compound of any one of claims 1-35 or a composition of claim 42 or 43 under conditions that permit the formation of a complex between said compound and said presenter protein.

49. A method for the preparation of a compound of any one of claims 1-35, said method comprising culturing a bacterial strain of the genus Streptomyces which has been modified to produce said compound and isolating said compound from the fermentation broth.

50. A method for the preparation of a compound of any one of claims 1-35, said method comprising culturing a bacterial strain of the genus Streptomyces under condition in which said strain produces said compound and isolating said compound from the fermentation broth.

51. A tripartite complex comprising (i) a mammalian target protein and (ii) a presenter protein/compound complex, said presenter protein/compound complex comprising a presenter protein and a macrocyclic compound of any one of claims 1-35.

52. The tripartite complex of claim 51, wherein said target protein does not have a traditional binding pocket.

53. The tripartite complex of claim 51 or 52, wherein said presenter protein/compound complex binds at a flat surface site on said target protein.

54. The tripartite complex of any one of claims 51 to 53, wherein said macrocyclic compound in said presenter protein/compound complex binds at a hydrophobic surface site on said target protein.

55. The tripartite complex of claim 54, wherein said hydrophobic surface site on said target protein comprises at least 50% hydrophobic residues.

56. The tripartite complex of any one of claims 51 to 55, wherein said presenter protein/compound complex binds to said target protein at a site of a naturally occurring protein-protein interaction between said target protein and a protein that specifically binds said target protein.

57. The tripartite complex of any one of claims 51 to 56, wherein said presenter protein/compound complex does not bind at an active site of said target protein.

58. The tripartite complex of any one of claims 51 to 56, wherein said presenter protein/compound complex binds at an active site of said target protein.

59. The tripartite complex of any one of claims 51 to 56, wherein said target protein is an undruggable target.

60. The tripartite complex of any one of claims 51 to 59, wherein the structural organization of said macrocyclic compound is substantially unchanged in said tripartite complex compared to said macrocyclic compound in said presenter protein/compound complex but not in said tripartite complex.

61. The tripartite complex of any one of claims 51 to 60, wherein at least 20% of the total buried surface area of said target protein comprises one or more atoms that participate in binding to said macrocyclic compound.

62. The tripartite complex of any one of claims 51 to 61, wherein at least 20% of the total buried surface area of said target protein comprises one or more atoms that participate in binding to said presenter protein.

63. The tripartite complex of any one of claims 51 to 62, wherein said macrocyclic compound contributes at least 10% of the total binding free energy.

64. The tripartite complex of any one of claims 51 to 63, wherein said presenter protein contributes at least 10% of the total binding free energy.

65. The tripartite complex of any one of claims 51 to 64, wherein at least 70% of binding interactions between one or more atoms of said macrocyclic compound and one or more atoms of said target protein are van der Waals interactions and/or r-effect interactions.

66. A compound collection comprising macrocyclic compounds consisting of 14 to 40 ring atoms, a mammalian target protein interacting moiety, and a presenter protein binding moiety, wherein said macrocyclic compounds, together with a presenter protein, form a complex that specifically binds to a target protein, and wherein said macrocyclic compounds and said presenter protein do not substantially bind to said target protein in the absence of forming said complex.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0297] FIG. 1 is a table of exemplary compounds of the invention.

[0298] FIG. 2 is a table of exemplary compounds of the invention.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0299] Small molecules are limited in their targeting ability because their interactions with the target are driven by adhesive forces, the strength of which is roughly proportional to contact surface area. Because of their small size, the only way for a small molecule to build up enough intermolecular contact surface area to effectively interact with a target protein is to be literally engulfed by that protein. Indeed, a large body of both experimental and computational data supports the view that only those proteins having a hydrophobic pocket on their surface are capable of binding small molecules. In those cases, binding is enabled by engulfment.

[0300] Nature has evolved a strategy that allows a small molecule to interact with target proteins at sites other than hydrophobic pockets. This strategy is exemplified by naturally occurring immunosuppressive drugs cyclosporine A, rapamycin, and FK506. The biological activity of these drugs involves the formation of a high-affinity complex of the small molecule with a small presenting protein. The composite surface of the small molecule and the presenting protein engages the target. Thus, for example, the binary complex formed between cyclosporin A and cyclophilin A targets calcineurin with high affinity and specificity, but neither cyclosporin A or cyclophilin A alone binds calcineurin with measurable affinity.

[0301] Many important therapeutic targets exert their function by complexation with other proteins. The protein/protein interaction surfaces in many of these systems contain an inner core of hydrophobic side chains surrounded by a wide ring of polar residues. The hydrophobic residues contribute nearly all of the energetically favorable contacts, and hence this cluster has been designated as a hotspot for engagement in protein-protein interactions. Importantly, in the aforementioned complexes of naturally occurring small molecules with small presenting proteins, the small molecule provides a cluster of hydrophobic functionality akin to a hotspot, and the protein provides the ring of mostly polar residues. In other words, presented small molecule systems mimic the surface architecture employed widely in natural protein/protein interaction systems.

[0302] Nature has demonstrated the ability to reprogram the target specificity of presented small moleculesportable hotspotsthrough evolutionary diversification. In the best characterized example, the complex formed between FK506 binding protein (FKBP) and FK506 targets calcineurin. However, FKBP can also form a complex with the related molecule rapamycin, and that complex interacts with a completely different target, TorC1. To date, no methodology has been developed to reprogram the binding and modulating ability of presenter protein/ligand interfaces so that they can interact with and modulate other target proteins that have previously been considered undruggable.

[0303] In addition, it is well established that some drug candidates fail because they modulate the activity of both the intended target and other non-intended proteins as well. The problem is particularly daunting when the drug binding site of the target protein is similar to binding sites in non-target proteins. The insulin like growth factor receptor (IGF-1R), whose ATP binding pocket is structurally similar to the binding pocket of the non-target insulin receptor (IR), is one such example. Small molecule development candidates that were designed to target IGF-1R typically have the unacceptable side effect of also modulating the insulin receptor. However, structural dissimilarities do exist between these two proteins in the regions surrounding the ATP binding pocket. Despite such knowledge, no methodology exists to date to take advantage of those differences and develop drugs that are specific to IGF-1R over IR.

[0304] The present invention features compounds (e.g., macrocyclic compounds) capable of modulating biological processes, for example through binding to a presenter protein (e.g., a member of the FKBP family, a member of the cyclophilin family, or PIN1) and a target protein. In some embodiments, the target and/or presenter proteins are intracellular proteins. In some embodiments, the target and/or presenter proteins are mammalian proteins. In some embodiments, provided compounds participate in tripartite presenter protein/compound/target protein complexes inside cells, e.g., mammalian cells. In some embodiments, provided compounds may be useful in the treatment of diseases and disorders such as cancer, inflammation, or infections.

Compounds

[0305] The invention features compounds (e.g., macrocyclic compounds) capable of modulating biological processes, for example through binding to a presenter protein (e.g., a member of the FKBP family, a member of the cyclophilin family, or PIN1) and a target protein (e.g., a eukaryotic target protein such as a mammalian target protein or a fungal target protein or a prokaryotic target protein such as a bacterial target protein). In brief, these compounds bind endogenous intracellular presenter proteins, such as the FKBPs and the resulting binary complexes selectively bind and modulate the activity of intracellular target proteins. Without wishing to be bound by any particular theory, we proposed that formation of a tripartite complex among the presenter protein, the compound, and the target protein is driven by both protein-compound and protein-protein interactions, and both are required for modulation (e.g., positive or negative modulation) of the targeted protein's activity. In some embodiments, the compounds of the invention re-program the binding of the presenter proteins to protein targets that either do not normally bind to the presenter protein or have binding that is greatly enhanced in the presence of the compound thereby resulting in the ability to modulate (e.g., positively or negatively modulate) the activity of these new targets.

[0306] As described herein, compounds of the invention include a presenter protein binding moiety and a target protein interacting moiety. In some embodiments, the presenter protein binding moiety and target protein interacting moiety are separate portions of the ring structure, e.g., they do not overlap. In some embodiments, the presenter protein binding moiety and target protein interacting moiety are connected to one another by linkers on one or both sides.

[0307] In some embodiments, compounds of the invention do not substantially bind to the target protein in the absence of forming a complex, as described herein. In some embodiments, a complex of a compound of the invention and a presenter protein binds to the target protein with at least 5-fold (at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 100-fold) greater affinity than the affinity of the compound to the target protein in the absence of forming a complex, as described herein. In certain embodiments, compounds of the invention do not substantially modulate the activity of the target protein in the absence forming a complex with a presenter protein. For example, in some embodiments, the compounds of the invention inhibit the activity of the target protein with an IC.sub.50 of greater than 10 M (e.g., greater than 20 M, greater than 50 M, greater than 100 M, greater than 500 M). Alternatively, compounds of the invention enhance the activity of the target protein with an AC.sub.50 of greater than 10 M (e.g., greater than 20 M, greater than 50 M, greater than 100 M, greater than 500 M). In certain embodiments, a complex of the compound and a presenter protein is at least 5-fold active (i.e., has a 5-fold lower IC.sub.50 or AC.sub.50) than the compound alone.

[0308] Compounds (e.g., macrocyclic compounds) of the invention generally bind strongly to the presenter protein. For example, in some embodiments, the compounds (e.g., macrocyclic compounds) of the invention bind to the presenter protein with a K.sub.D of less than 10 M (e.g., less than 5 M, less than 1 M, less than 500 nM, less than 200 nM, less than 100 nM, less than 75 nM, less than 50 nM, less than 25 nM, less than 10 nM) or inhibit the peptidyl-prolyl isomerase activity of the presenter protein, for example, with an IC.sub.50 of less than 1 M (e.g., less than 0.5 M, less than 0.1 M, less than 0.05 M, less than 0.01 M).

[0309] In some embodiments, the invention includes compounds having a structure according to formulae XIV to XVIII as described herein:

##STR00057## ##STR00058##

[0310] In certain embodiments, a compound has the structure of any of the compounds in FIG. 1 or FIG. 2.

[0311] In some embodiments, a compound is a natural compound (e.g., synthesized by an genetically unmodified bacterial strain). In some embodiments, a compound is a variant of a natural compound (e.g., a semi-synthetic compound). In some embodiments, a variant shares ring size with the reference natural compound. In some embodiments, a variant differs from the reference natural compound only by identity of one or more substituents (e.g., for at least one position, the variant has a different substitutent or set of substituents than is found at the corresponding position in an appropriate reference compound).

[0312] In some embodiments, compounds of the invention (e.g., macrocyclic compounds of the invention) include 12 to 40 ring atoms (e.g., 12 to 20 ring atoms, 14 to 20 ring atoms, 17 to 25 ring atom, 21 to 26 ring atoms, 20 to 30 ring atoms, 25 to 35 ring atoms, 30 to 40 ring atoms). In some embodiments, such compounds include 19 ring atoms. In some embodiments, such compounds have an even number of ring atoms. In certain embodiments, at least 25% (e.g., at least 30%, at least 35%, at least 40%, at least 45%) of the atoms in the compound are included in a single or fused ring system.

[0313] In some embodiments, compounds of the invention include a ring (e.g., a macrocycle) whose ring atoms are selected from the group consisting of carbon atoms, hydrogen atoms, nitrogen atoms, oxygen atoms, sulfur atoms, phosphorous atoms, silicon atoms and combinations thereof; in some embodiments all ring atoms in the compound are selected from this group. In some embodiments, compounds of the invention include a ring (e.g., a macrocycle) whose ring atoms are selected only from the group consisting carbon atoms, hydrogen atoms, nitrogen atoms, oxygen atoms and combinations thereof; in some embodiments, all ring atoms in the compound are selected only from the group consisting carbon atoms, hydrogen atoms, nitrogen atoms, oxygen atoms and combinations thereof.

[0314] In certain embodiments, ring linkage in provided compounds (e.g., macrocyclic compounds) of the invention includes a ketone, an ester, an amide, an ether, a thioester, a urea, an amidine, or a hydrocarbon.

[0315] In some embodiments, a provided compound is non-peptidal. In certain embodiments, a provided compound includes one or more amino acid residues. In some embodiments, a provided compound includes only amino acid residues.

[0316] In some embodiments, the molecular weight of compounds of the invention is between 400 and 2000 daltons (e.g., 400 to 600 daltons, 500 to 700 daltons, 600 to 800 daltons, 700 to 900 daltons, 800 to 1000 daltons, 900 to 1100 daltons, 1000 to 1200 daltons, 1100 to 1300 daltons, 1200 to 1400 daltons, 1300 to 1500 daltons, 1400 to 1600 daltons, 1500 to 1700 daltons, 1600 to 1800 daltons, 1700 to 1900 daltons, 1800 to 2000 daltons, 400 to 1000 daltons, 1000-2000 daltons). In some embodiments, the molecular weight of compounds of the invention is less than 2000 daltons (e.g., less than 500 daltons, less than 600 daltons, less than 700 daltons, less than 800 daltons, less than 900 daltons, less than 1000 daltons, less than 1100 daltons, less than 1200 daltons, less than 1300 daltons, less than 1400 daltons, less than 1500 daltons, less than 1600 daltons, less than 1700 daltons, less than 1800 daltons, less than 1900 daltons).

[0317] In certain embodiments, molecule provided compound is hydrophobic. For example, in some embodiments, compounds have a c Log P of equal to or greater than 2 (e.g., equal to or greater than 2.5, equal to or greater than 3.0, equal to or greater than 3.5, equal to or greater than 4, equal to or greater than 4.5, equal to or greater than 5, equal to or greater than 5.5, equal to or greater than 6, equal to or greater than 6.5, equal to or greater than 7). Alternatively, in some embodiments, compounds have a c Log P of between 2 and 7 (e.g., between 2 and 4, between 3.5 and 4.5, between 4 and 5, between 4.5 and 5.5, between 5 and 6, between 5.5 and 6.5, between 6 and 7, between 4 and 7, between 4 and 6, between 4 and 5.5). A provided compound may also be characterized as hydrophobic by having low solubility in water. For example, in some embodiments, compounds have a solubility of greater than 1 M in water (e.g., greater than 1 M, greater than 2 M, greater than 5 M, greater than 10 M, greater than 20 M, greater than 30 M, greater than 40 M, greater than 50 M, greater than 75 M, greater than 100 M). Alternatively, in some embodiments, compounds have a solubility in water of between 1-100 M (e.g., 1-10 M, 5-10 M, 5-20 M, 10-50 M, 5-50 M, 20-100 M).

[0318] In some embodiments, compounds of the invention are cell penetrant (e.g., they are able to enter the intracellular domain of a cell without killing the cell and/or are capable of entering the intercellular domain when contacted with extracellular environs).

[0319] Compounds of the invention may or may not be naturally occurring. In some embodiments, compounds of the invention are not naturally occurring. In certain embodiments, compounds of the invention are engineered. An engineered compound is a compound whose design and/or production involves action of the hand of man (e.g., a compound prepared by chemical synthesis, a compound prepared by a cell that has been genetically manipulated relative to a reference wild type cell, a compound produced by a cell in culture conditions modified to enhance production of the compound).

[0320] In certain embodiments, a provided compound (e.g., macrocyclic compound) is not a compound described in in Benjamin et al. Nat. Rev. Drug. Discov. 2011, 10(11), 868-880 or Sweeney, Z. K. et al. J. Med. Chem. 2014, epub ahead of print, the structures of which are incorporated by reference and/or a compound having the structure:

[0321] Presenter Protein Binding Moiety

[0322] Compounds of the invention include a presenter protein binding moiety. This moiety includes the group of ring atoms (e.g., 5 to 20 ring atoms, 5 to 10 ring atoms, 10 to 20 ring atoms) and the moieties attached thereto (e.g., atoms within 20 atoms of a ring atom such as, atoms within 15 atoms of a ring atom, atoms within 10 atoms of a ring atom, atoms within 5 atoms of a ring atom) that participate in binding to a presenter protein such that a provided compound specifically binds to said presenter protein, for example, with a K.sub.D of less than 10 M (e.g., less than 5 M, less than 1 M, less than 500 nM, less than 200 nM, less than 100 nM, less than 75 nM, less than 50 nM, less than 25 nM, less than 10 nM) or inhibits the peptidyl-prolyl isomerase activity of the presenter protein, for example, with an IC.sub.50 of less than 1 M (e.g., less than 0.5 M, less than 0.1 M, less than 0.05 M, less than 0.01 M). In some embodiments, the presenter protein binding moiety does not encompass the entirety of atoms in a provided compound that interact with the presenter protein. In some embodiments, one or more atoms of the presenter protein binding moiety may be within the target protein interaction moiety (e.g., eukaryotic target protein interacting moiety such as a mammalian target protein interacting moiety or a fungal target protein interacting moiety or prokaryotic target protein interacting moiety such as a bacterial target protein interacting moiety). In certain embodiments, one or more atoms of the presenter protein binding moiety do not interact with the presenter protein.

[0323] In some embodiments, a presenter protein binding moiety includes a N-acyl proline moiety, a N-acyl-pipecolic acid moiety, a N-acyl 3-morpholino-carboxylic acid moiety, and/or a N-acyl piperzic acid moiety (e.g., with acylation on either nitrogen atom. In certain embodiments, a presenter protein binding moiety includes a N-acyl-pipecolic acid moiety. In some embodiments, a presenter protein binding moiety includes a N-acyl proline moiety. In certain embodiments, a presenter protein binding moiety includes a N-acyl 3-morpholino-carboxylic acid moiety. In some embodiments, a presenter protein binding moiety includes a N-acyl piperzic acid moiety.

[0324] In some embodiments, at least one atom of a presenter protein binding moiety participates in binding with one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen) of Tyr 27, Phe 37, Asp 38, Arg 41, Phe 47, Gln 54, Glu 55, Val 56, lie 57, Trp 60, Ala 82, Try 83, His 88, lie 92, and/or Phe 100 of FKBP12. In some embodiments, at least one at of a presenter protein binding moiety participates in binding with at least one (e.g., two, three, or four) of Arg 41, Gin 54, Glu 55, and/or Ala 82 of FKBP12.

[0325] In some embodiments, a presenter protein binding moiety has a structure according to Formula I-VIII:

##STR00059## ##STR00060## ##STR00061##

[0326] In some embodiments, a presenter protein binding moiety has a structure according to Formula Ia-IVa:

##STR00062##

[0327] In some embodiments, a presenter protein binding moiety includes or consists of the structure:

##STR00063## ##STR00064## ##STR00065##

or a stereoisomer thereof.

[0328] In certain embodiments, the presenter protein binding moiety is or includes the structure:

##STR00066##

[0329] Target Protein Interacting Moiety

[0330] Compounds of the invention include a target protein interacting moiety (e.g., a eukaryotic target protein interacting moiety such as a mammalian target protein interacting moiety or a fungal target protein interacting moiety or a prokaryotic target protein interacting moiety such as a bacterial target protein interacting moiety). This moiety includes the group of ring atoms (e.g., 5 to 20 ring atoms, 5 to 10 ring atoms, 10 to 20 ring atoms) and the moieties attached thereto (e.g., atoms within 20 atoms of a ring atom such as, atoms within 15 atoms of a ring atom, atoms within 10 atoms of a ring atom, atoms within 5 atoms of a ring atom) that when the compound is in a complex with a presenter protein, specifically bind to a target protein. In some embodiments, a target protein interacting moiety comprises a plurality of the atoms in the compound that interact with the target protein. In some embodiments, one or more atoms of a target protein interacting moiety may be within the presenter protein binding moiety. In certain embodiments, one or more atoms of a target protein interacting moiety do not interact with the target protein.

[0331] A target protein can bind to a ring atom in a target protein interacting moiety. Alternatively, a target protein can bind to two or more ring atoms in a target protein interacting moiety. In another alternative, a target protein bind can to a substituent attached to one or more ring atoms in a target protein interacting moiety. In another alternative, a target protein can bind to a ring atom in a target protein interacting moiety and to a substituent attached to one or more ring atoms in a target protein interacting moiety. In another alternative, a target protein binds to a group that mimics a natural ligand of a target protein and wherein the group that mimics a natural ligand of a target protein is attached to a target protein interacting moiety. In yet another alternative, a target protein binds to a presenter protein and the affinity of a target protein for a presenter protein in the binary complex is increased relative to the affinity of a target protein for a presenter protein in the absence of the complex. Binding in these examples is typically through, but not limited to non-covalent interactions of a target protein to a target protein interacting moiety.

[0332] In some embodiments, a target protein interacting moiety is hydrophobic. For example, in some embodiments, a target protein interacting moiety has a c Log P of equal to or greater than 2 (e.g., equal to or greater than 2.5, equal to or greater than 3, equal to or greater than 3.5, equal to or greater than 4, equal to or greater than 4.5, equal to or greater than 5, equal to or greater than 5.5, equal to or greater than 6, equal to or greater than 6.5, equal to or greater than 7). Alternatively, in some embodiments, a target protein interacting moiety has a c Log P of between 2 and 7 (e.g., between 2 and 4, between 2.5 and 4.5, between 3 and 5, between 3.5 and 5.5, between 4 and 6, between 4.5 and 6.5, between 5 and 7, between 3 and 6, between 3 and 5, between 3 and 5.5). A target protein interacting moiety may also be characterized as hydrophobic by having low polar surface area. For example, in some embodiments, a target protein interacting moiety has a polar surface area of less than 350 .sup.2 (e.g., less than 300 .sup.2, less than 250 .sup.2, less than 200 A.sup.2, less than 150 .sup.2, less than 125 .sup.2).

[0333] In some embodiments, a target protein interacting moiety comprises one or more hydrophobic pendant groups (e.g., one or more methyl, ethyl, isopropyl, phenyl, benzyl, and/or phenethyl groups). In some embodiments, the pendant groups comprise fewer than 30 total atoms (e.g., fewer than 25 total atoms, fewer than 20 total atoms, fewer than 15 total atoms, fewer than 10 total atoms.) Alternatively, in some embodiments, the pendant groups comprise between 10 and 30 total atoms (e.g., 10 to 20 total atoms, 15 to 25 total atoms, 20 to 30 total atoms). In certain embodiments the pendant groups have a molecular weight less than 200 daltons (e.g., less than 150 daltons, less than 100 daltons, less than 75 daltons, less than 50 daltons). Alternatively, in some embodiments, the pendant groups have a molecular weight between 50 to 200 daltons (e.g., 50 to 100 daltons, 75 to 150 daltons, 100 to 200 daltons).

[0334] In some embodiments, a target protein interacting moiety is hydrocarbon based (e.g., the moiety comprises mostly carbon-carbon bonds). In some embodiments, a target protein interacting moiety is hydrocarbon based and includes a linear bivalent C.sub.4-30 (e.g., C.sub.6-C.sub.20, C.sub.6-C.sub.15) aliphatic group consisting predominantly of carbon and hydrogen, optionally including one or more double bonds. In some embodiments, the bivalent aliphatic group can also be substituted with a group that mimics a natural ligand that binds to a target protein. Examples include phosphotyrosine mimics and ATP mimetics.

[0335] In some embodiments, a target protein interacting moiety is peptide based (e.g., the moiety comprises peptide bonds). In some embodiments, a target protein interacting moiety is peptide based and includes one or more (e.g., two, three, four, five, six, seven, or eight) alanine residues, one or more (e.g., two, three, four, five, six, seven, or eight) valine residues, one or more isoleucine (e.g., two, three, four, five, six, seven, or eight) residues, one or more leucine (e.g., two, three, four, five, six, seven, or eight) residues, one or more methionine (e.g., two, three, four, five, six, seven, or eight) residues, one or more phenylalanine (e.g., two, three, four, five, six, seven, or eight) residues, one or more (e.g., two, three, four, five, six, seven, or eight) tyrosine residues, one or more (e.g., two, three, four, five, six, seven, or eight) tryptophan residues, one or more (e.g., two, three, four, five, six, seven, or eight) glycine residues, and/or one or more (e.g., two, three, four, five, six, seven, or eight) proline residues. In some embodiments, a target protein interacting moiety is peptide based an includes one or more (e.g., two, three, four, five, six, seven, or eight) arginine residues or one or more (e.g., two, three, four, five, six, seven, or eight) lysine residues. In some embodiments, a target protein interacting moiety is peptide based and includes one or more (e.g., two, three, four, five, six, seven, or eight) non-natural amino acids, one or more (e.g., two, three, four, five, six, seven, or eight) D-amino acids, and/or one or more (e.g., two, three, four, five, six, seven, or eight) N-alkylated amino acids. In some embodiments, a target protein interacting moiety is peptide based and includes predominantly D-amino acids (e.g., at least 50% of the amino acids are D-amino acids, at least 75% of the amino acids are D-amino acids, 100% of the amino acids are D-amino acids). In certain embodiments, a target protein interacting moiety is peptide based and includes predominantly N-alkylated amino acids (e.g., at least 50% of the amino acids are N-alkylated amino acids, at least 75% of the amino acids are N-alkylated amino acids, 100% of the amino acids are N-alkylated amino acids). In certain embodiments, a target protein interacting moiety is peptide based and includes one or more (e.g., two, three, four, five, six, seven, or eight) depsi-linkages.

[0336] In some embodiments, a target protein interacting moiety (e.g., a eukaryotic target protein interacting moiety such as a mammalian target protein interacting moiety or a fungal target protein interacting moiety or a prokaryotic target protein interacting moiety such as a bacterial target protein interacting moiety) has a structure according to Formula IX:

##STR00067##

[0337] wherein u is an integer from 1 to 20; and

[0338] each Y is, independently, any amino acid, O, NR.sup.20, S, S(O), SO.sub.2, or has the structure of any one of Formulae X-XIII:

##STR00068##

[0339] wherein each R.sup.20 is, independently, hydrogen, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 alkynyl, optionally substituted aryl, C.sub.3-C.sub.7 carbocyclyl, optionally substituted C.sub.6-C.sub.10 aryl C.sub.1-C.sub.6 alkyl, and optionally substituted C.sub.3-C.sub.7 carbocyclyl C.sub.1-C.sub.6 alkyl or R.sup.19 combines with any R.sup.20, R.sup.21, R.sup.22, R.sup.23, R.sup.24, R.sup.25, R.sup.26, R.sup.27, R.sup.28, R.sup.29, or R.sup.30 to form an optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.2-C.sub.9 heterocyclyl, or optionally substituted C.sub.2-C.sub.9 heteroaryl;

[0340] each R.sup.21 and R.sup.22 is, independently, hydrogen, halogen, optionally substituted hydroxyl, optionally substituted amino, or R.sup.20 and R.sup.21 combine to form O, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 alkynyl, optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.6-C.sub.10 aryl C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.9 heteroaryl, optionally substituted C.sub.2-C.sub.9 heteroaryl C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.9 heterocyclyl, or optionally substituted C.sub.2-C.sub.9 heterocyclyl C.sub.1-C.sub.6 alkyl, or R.sup.21 or R.sup.22 combines with any R.sup.20, R.sup.21, R.sup.22, R.sup.23, R.sup.24, R.sup.25, R.sup.26, R.sup.27, R.sup.28, R.sup.29, or R.sup.30 to form an optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.2-C.sub.9 heterocyclyl, or optionally substituted C.sub.2-C.sub.9 heteroaryl;

[0341] each R.sup.23, R.sup.24, R.sup.25, and R.sup.26 is, independently, hydrogen, hydroxyl, or R.sup.23 and R.sup.24 combine to form O, or R.sup.23, R.sup.24, R.sup.25, or R.sup.26 combines with any R.sup.20, R.sup.21, R.sup.22, R.sup.23, R.sup.24, R.sup.25, R.sup.26, R.sup.27, R.sup.28, R.sup.29, or R.sup.30 to form an optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.2-C.sub.9 heterocyclyl, or optionally substituted C.sub.2-C.sub.9 heteroaryl; and

[0342] each R.sup.27, R.sup.28, R.sup.29, and R.sup.30 is, independently, hydrogen, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 alkynyl, optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.6-C.sub.10 aryl C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.9 heteroaryl, optionally substituted C.sub.2-C.sub.9 heteroaryl C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.9 heterocyclyl, or optionally substituted C.sub.2-C.sub.9 heterocyclyl C.sub.1-C.sub.6 alkyl, or R.sup.27, R.sup.28, R.sup.29, or R.sup.30 combines with any R.sup.20, R.sup.21, R.sup.22, R.sup.23, R.sup.24, R.sup.25, R.sup.26, R.sup.27, R.sup.28, R.sup.29, or R.sup.30 to form an optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.2-C.sub.9 heterocyclyl, or optionally substituted C.sub.2-C.sub.9 heteroaryl.

[0343] In some embodiments, a target protein interacting moiety has a structure according to Formula IXa:

##STR00069##

[0344] In certain embodiments, a target protein interacting moiety (e.g., a eukaryotic target protein interacting moiety such as a mammalian target protein interacting moiety or a fungal target protein interacting moiety or a prokaryotic target protein interacting moiety such as a bacterial target protein interacting moiety) does not include the structure:

##STR00070##

[0345] Linkers

[0346] The compounds of the invention include a linker (e.g., two linkers connecting the presenter protein binding moiety and target protein interacting moiety (e.g., a eukaryotic target protein interacting moiety such as a mammalian target protein interacting moiety or a fungal target protein interacting moiety or a prokaryotic target protein interacting moiety such as a bacterial target protein interacting moiety)). The linker component of the invention is, at its simplest, a bond, but may also provide a linear, cyclic, or branched molecular skeleton having pendant groups covalently linking two moieties.

[0347] In some embodiments, at least one atom of a linker participates in binding to the presenter protein and/or the target protein. In certain embodiments, at least one atom of a linker does not participate in binding to the presenter protein and/or the target protein.

[0348] Thus, linking of the two moieties is achieved by covalent means, involving bond formation with one or more functional groups located on either moiety. Examples of chemically reactive functional groups which may be employed for this purpose include, without limitation, amino, hydroxyl, sulfhydryl, carboxyl, carbonyl, carbohydrate groups, vicinal diols, thioethers, 2-aminoalcohols, 2-aminothiols, guanidinyl, imidazolyl, and phenolic groups.

[0349] The covalent linking of the two moieties may be effected using a linker that contains reactive moieties capable of reaction with such functional groups present in either moiety. For example, an amine group of a moiety may react with a carboxyl group of the linker, or an activated derivative thereof, resulting in the formation of an amide linking the two.

[0350] Examples of moieties capable of reaction with sulfhydryl groups include -haloacetyl compounds of the type XCH.sub.2CO (where X=Br, Cl, or I), which show particular reactivity for sulfhydryl groups, but which can also be used to modify imidazolyl, thioether, phenol, and amino groups as described by Gurd, Methods Enzymol. 11:532 (1967). N-Maleimide derivatives are also considered selective towards sulfhydryl groups, but may additionally be useful in coupling to amino groups under certain conditions. Reagents such as 2-iminothiolane (Traut et al., Biochemistry 12:3266 (1973)), which introduce a thiol group through conversion of an amino group, may be considered as sulfhydryl reagents if linking occurs through the formation of disulfide bridges.

[0351] Examples of reactive moieties capable of reaction with amino groups include, for example, alkylating and acylating agents. Representative alkylating agents include:

[0352] (i) -haloacetyl compounds, which show specificity towards amino groups in the absence of reactive thiol groups and are of the type XCH.sub.2CO (where X=Br, Cl, or I), for example, as described by Wong Biochemistry 24:5337 (1979);

[0353] (ii) N-maleimide derivatives, which may react with amino groups either through a Michael type reaction or through acylation by addition to the ring carbonyl group, for example, as described by Smyth et al., J. Am. Chem. Soc. 82:4600 (1960) and Biochem. J. 91:589 (1964);

[0354] (iii) aryl halides such as reactive nitrohaloaromatic compounds;

[0355] (iv) alkyl halides, as described, for example, by McKenzie et al., J. Protein Chem. 7:581 (1988);

[0356] (v) aldehydes and ketones capable of Schiff's base formation with amino groups, the adducts formed usually being stabilized through reduction to give a stable amine;

[0357] (vi) epoxide derivatives such as epichlorohydrin and bisoxiranes, which may react with amino, sulfhydryl, or phenolic hydroxyl groups;

[0358] (vii) chlorine-containing derivatives of s-triazines, which are very reactive towards nucleophiles such as amino, sufhydryl, and hydroxyl groups;

[0359] (viii) aziridines based on s-triazine compounds detailed above, e.g., as described by Ross, J. Adv. Cancer Res. 2:1 (1954), which react with nucleophiles such as amino groups by ring opening;

[0360] (ix) squaric acid diethyl esters as described by Tietze, Chem. Ber. 124:1215 (1991); and

[0361] (x) -haloalkyl ethers, which are more reactive alkylating agents than normal alkyl halides because of the activation caused by the ether oxygen atom, as described by Benneche et al., Eur. J. Med. Chem. 28:463 (1993).

[0362] Representative amino-reactive acylating agents include:

[0363] (i) isocyanates and isothiocyanates, particularly aromatic derivatives, which form stable urea and thiourea derivatives respectively;

[0364] (ii) sulfonyl chlorides, which have been described by Herzig et al., Biopolymers 2:349 (1964);

[0365] (iii) acid halides;

[0366] (iv) active esters such as nitrophenylesters or N-hydroxysuccinimidyl esters;

[0367] (v) acid anhydrides such as mixed, symmetrical, or N-carboxyanhydrides;

[0368] (vi) other useful reagents for amide bond formation, for example, as described by M. Bodansky, Principles of Peptide Synthesis, Springer-Verlag, 1984;

[0369] (vii) acylazides, e.g., wherein the azide group is generated from a preformed hydrazide derivative using sodium nitrite, as described by Wetz et al., Anal. Biochem. 58:347 (1974);

[0370] (viii) imidoesters, which form stable amidines on reaction with amino groups, for example, as described by Hunter and Ludwig, J. Am. Chem. Soc. 84:3491 (1962); and

[0371] (ix) haloheteroaryl groups such as halopyridine or halopyrimidine.

[0372] Aldehydes and ketones may be reacted with amines to form Schiff's bases, which may advantageously be stabilized through reductive amination. Alkoxylamino moieties readily react with ketones and aldehydes to produce stable alkoxamines, for example, as described by Webb et al., in Bioconjugate Chem. 1:96 (1990).

[0373] Examples of reactive moieties capable of reaction with carboxyl groups include diazo compounds such as diazoacetate esters and diazoacetamides, which react with high specificity to generate ester groups, for example, as described by Herriot, Adv. Protein Chem. 3:169 (1947). Carboxyl modifying reagents such as carbodiimides, which react through O-acylurea formation followed by amide bond formation, may also be employed.

[0374] It will be appreciated that functional groups in either moiety may, if desired, be converted to other functional groups prior to reaction, for example, to confer additional reactivity or selectivity. Examples of methods useful for this purpose include conversion of amines to carboxyls using reagents such as dicarboxylic anhydrides; conversion of amines to thiols using reagents such as N-acetylhomocysteine thiolactone, S-acetylmercaptosuccinic anhydride, 2-iminothiolane, or thiol-containing succinimidyl derivatives; conversion of thiols to carboxyls using reagents such as -haloacetates; conversion of thiols to amines using reagents such as ethylenimine or 2-bromoethylamine; conversion of carboxyls to amines using reagents such as carbodiimides followed by diamines; and conversion of alcohols to thiols using reagents such as tosyl chloride followed by transesterification with thioacetate and hydrolysis to the thiol with sodium acetate.

[0375] So-called zero-length linkers, involving direct covalent joining of a reactive chemical group of one moiety with a reactive chemical group of the other without introducing additional linking material may, if desired, be used in accordance with the invention.

[0376] More commonly, however, the linker will include two or more reactive moieties, as described above, connected by a spacer element. The presence of such a spacer permits bifunctional linkers to react with specific functional groups within either moiety, resulting in a covalent linkage between the two. The reactive moieties in a linker may be the same (homobifunctional linker) or different (heterobifunctional linker, or, where several dissimilar reactive moieties are present, heteromultifunctional linker), providing a diversity of potential reagents that may bring about covalent attachment between the two moieties.

[0377] Spacer elements in the linker typically consist of linear or branched chains and may include a C.sub.1-10 alkyl, C.sub.2-10 alkenyl, C.sub.2-10 alkynyl, C.sub.2-6 heterocyclyl, C.sub.6-12 aryl, C.sub.7-14 alkaryl, C.sub.3-10 alkheterocyclyl, C.sub.2-C.sub.100 polyethylene glycol, or C.sub.1-10 heteroalkyl.

[0378] In some instances, the linker is described by Formula V.

[0379] Examples of homobifunctional linkers useful in the preparation of conjugates of the invention include, without limitation, diamines and diols selected from ethylenediamine, propylenediamine and hexamethylenediamine, ethylene glycol, diethylene glycol, propylene glycol, 1,4-butanediol, 1,6-hexanediol, cyclohexanediol, and polycaprolactone diol.

[0380] In some embodiments, the linker is a bond or a linear chain of up to 10 atoms, independently selected from carbon, nitrogen, oxygen, sulfur or phosphorous atoms, wherein each atom in the chain is optionally substituted with one or more substituents independently selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, chloro, iodo, bromo, fluoro, hydroxyl, alkoxy, aryloxy, carboxy, amino, alkylamino, dialkylamino, acylamino, carboxamido, cyano, oxo, thio, alkylthio, arylthio, acylthio, alkylsulfonate, arylsulfonate, phosphoryl, and sulfonyl, and wherein any two atoms in the chain may be taken together with the substituents bound thereto to form a ring, wherein the ring may be further substituted and/or fused to one or more optionally substituted carbocyclic, heterocyclic, aryl, or heteroaryl rings.

[0381] In some embodiments, the linker has the structure of Formula XIX:

A.sup.1-(B.sup.1).sub.a(C.sup.1).sub.b(B.sup.2).sub.c-(D)-(B.sup.3).sub.d(C.sup.2).sub.e(B.sup.4).sub.f-A.sup.2 Formula XIX

[0382] where A.sup.1 is a bond between the linker and presenter protein binding moiety; A.sup.2 is a bond between the mammalian target interacting moiety and the linker; B.sup.1, B.sup.2, B.sup.3, and B.sup.4 each, independently, is selected from optionally substituted C.sub.1-C.sub.2 alkyl, optionally substituted C.sub.1-C.sub.3 heteroalkyl, O, S, and NR.sup.N; R.sup.N is hydrogen, optionally substituted C.sub.1-4 alkyl, optionally substituted C.sub.3-4 alkenyl, optionally substituted C.sub.2-4 alkynyl, optionally substituted C.sub.2-6 heterocyclyl, optionally substituted C.sub.6-12 aryl, or optionally substituted C.sub.1-7 heteroalkyl; C.sup.1 and C.sup.2 are each, independently, selected from carbonyl, thiocarbonyl, sulphonyl, or phosphoryl; a, b, c, d, e, and f are each, independently, 0 or 1; and D is optionally substituted C.sub.1-10 alkyl, optionally substituted C.sub.2-10 alkenyl, optionally substituted C.sub.2-10 alkynyl, optionally substituted C.sub.2-6 heterocyclyl, optionally substituted C.sub.6-12 aryl, optionally substituted C.sub.2-C.sub.10 polyethylene glycol, or optionally substituted C.sub.1-10 heteroalkyl, or a chemical bond linking A.sup.1-(B.sup.1).sub.a(C.sup.1).sub.b(B.sup.2).sub.c to (B.sup.3).sub.d(C.sup.2).sub.e(B.sup.4).sub.f-A.sup.2.

Cross-Linking Groups

[0383] In some embodiments, compounds of the invention include a cross-linking group. A cross-linking group refers to a group comprising a reactive functional group capable of chemically attaching to specific functional groups (e.g., primary amines, sulfhydryls) on proteins or other molecules. Examples of cross-linking groups include sulfhydryl-reactive cross-linking groups (e.g., groups comprising maleimides, haloacetyls, pyridyldisulfides, thiosulfonates, or vinylsulfones), amine-reactive cross-linking groups (e.g., groups comprising esters such as NHS esters, imidoesters, and pentafluorophenyl esters, or hydroxymethylphosphine), carboxyl-reactive cross-linking groups (e.g., groups comprising primary or secondary amines, alcohols, or thiols), carbonyl-reactive cross-linking groups (e.g., groups comprising hydrazides or alkoxyamines), and triazole-forming cross-linking groups (e.g., groups comprising azides or alkynes).

[0384] Exemplary cross-linking groups include 2-pyridyldisulfide, 4-pyridyldisulfide iodoacetyl, maleimides, thioesters, alkyldisulfides, alkylamine disulfides, nitrobenzoic acid disulfide, anhydrides, NHS esters, aldehydes, alkyl chlorides, alkynes, Michael acceptor groups (e.g., ,-unsubstituted ketones or sulfones), epoxides, heteroaryl nitriles, and azides.

[0385] Compound Characteristics

[0386] Pharmacokinetic Parameters

[0387] Preliminary exposure characteristics of the compounds can be evaluated using, e.g., an in vivo Rat Early Pharmacokinetic (EPK) study design to show bioavailability. For example, Male Sprague-Dawley rats can be dosed via oral (PO) gavage in a particular formulation. Blood samples can then be collected from the animals at 6 timepoints out to 4 hours post-dose. Pharmacokinetic analysis can then performed on the LC-MS/MS measured concentrations for each timepoint of each compound.

[0388] Cell Permeability

[0389] In some embodiments, the compound is cell penetrant. To determine permeability of a compound any method known in the art may be employed such as a Biosensor assay as described herein.

Proteins

[0390] Presenter Proteins

[0391] Presenter proteins can bind a small molecule to form a complex, which can bind to and modulate the activity of a target protein (e.g., a eukaryotic target protein such as a mammalian target protein or a fungal target protein or a prokaryotic target protein such as a bacterial target protein). In some embodiments, the presenter protein is a mammalian presenter protein (e.g., a human presenter protein). In some embodiments, the presenter protein is a fungal presenter protein. In certain embodiments, the presenter protein is a bacterial presenter protein. In some embodiments, the presenter protein is a plant presenter protein. In some embodiments, the presenter protein is a relatively abundant protein (e.g., the presenter protein is sufficiently abundant that participation in a tripartite complex does not materially negatively impact the biological role of the presenter protein in a cell and/or viability or other attributes of the cell). In some embodiments, the presenter protein is more abundant than the target protein. In certain embodiments, the presenter protein is a protein that has chaperone activity within a cell. In some embodiments, the presenter protein has multiple natural interaction partners within a cell. In certain embodiments, the presenter protein is one which is known to bind a small molecule to form a binary complex that is known to or suspected of binding to and modulating the biological activity of a target protein. Immunophilins are a class of presenter proteins which are known to have these functions and include FKBPs and cyclophilins. In some embodiments, a reference presenter protein exhibits peptidyl prolyl isomerase activity; in some embodiments, a presenter protein shows comparable activity to the reference presenter protein. In certain embodiments, the presenter protein is a member of the FKBP family (e.g., FKBP12, FKBP12.6, FKBP13, FKBP19, FKBP22, FKBP23, FKBP25, FKBP36, FKBP38, FKBP51, FKBP52, FKBP60, FKBP65, and FKBP133), a member of the cyclophilin family (e.g., PP1A, CYPB, CYPC, CYP40, CYPE, CYPD, NKTR, SRCyp, CYPH, CWC27, CYPL1, CYP60, CYPJ, PPIL4, PPIL6, RANBP2, PPWD1, PPIAL4A, PPIAL4B, PPIAL4C, PPIAL4D, or PPIAL4G), or PIN1. The FKBP family is a family of proteins that have prolyl isomerase activity and function as protein folding chaperones for proteins containing proline residues. Genes that encode proteins in this family include AIP, AIPL1, FKBP1A, FKBP1B, FKBP2, FKBP3, FKBP4, FKBP5, FKBP6, FKBP7, FKBP8, FKBP9, FKBP9L, FKBP10, FKBP11, FKBP14, FKBP15, and LOC541473.

[0392] The cyclophilin family is a family of proteins that bind to cyclosporine. Genes that encode proteins in this family include PPIA, PPIB, PPIC, PPID, PPIE, PPIF, PPIG, PPIH, SDCCAG-10, PPIL1, PPIL2, PPIL3, PPIL4, P270, PPWD1, and COAS-2. Exemplary cyclophilins include PP1A, CYPB, CYPC, CYP40, CYPE, CYPD, NKTR, SRCyp, CYPH, CWC27, CYPL1, CYP60, CYPJ, PPIL4, PPIL6, RANBP2, PPWD1, PPIAL4A, PPIAL4B, PPIAL4C, PPIAL4D, and PPIAL4G.

[0393] In some embodiments, a presenter protein is a chaperone protein such as GRP78/BiP, GRP94, GRP170, calnexin, calreticulin, HSP47, ERp29, Protein disulfide isomerase (PDI), and ERp57.

[0394] In some embodiments, a presenter protein is an allelic variant or splice variant of a FKBP or cyclophilin disclosed herein.

[0395] In some embodiments, a presenter protein is a polypeptide whose amino acid sequence i) shows significant identity with that of a reference presenter protein; ii) includes a portion that shows significant identity with a corresponding portion of a reference presenter protein; and/or iii) includes at least one characteristic sequence found in presenter protein. In many embodiments, identity is considered significant for the purposes of defining an presenter protein if it is above 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher. In some embodiments, the portion showing significant identity has a length of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 300, 350, 450, 500, 550, 600 amino acids or more.

[0396] Representative presenter proteins are encoded by the genes or homologs thereof listed in Table 1; in some embodiments, a reference presenter protein is encoded by a gene set forth in Table 1. Also, those of ordinary skill in the art, referring to Table 3, can readily identify sequences that are characteristic of presenter proteins generally, and/or of particular subsets of presenter proteins.

TABLE-US-00001 TABLE 1 Genes that Encode Selected Presenter Proteins Uniprot Accession Gene Name Number AIP O00170 AIPL1 Q9NZN9 FKBP1A P62942 FKBP1B P68106 FKBP2 P26885 FKBP3 Q00688 FKBP4 Q02790 FKBP5 Q13451 FKBP6 O75344 FKBP7 Q9Y680 FKBP8 Q14318 FKBP9 O95302 FKBP9L Q75LS8 FKBP10 Q96AY3 FKBP11 Q9NYL4 FKBP14 Q9NWM8 FKBP15 Q5T1M5 LOC541473 PPIA Q567Q0 PPIB P23284 PPIC P45877 PPID Q08752 PPIE Q9UNP9 PPIG Q13427 PPIH O43447 PPIL1 Q9Y3C6 PPIL2 Q13356 PPIL3 Q9H2H8 PPIL4 Q8WUA2 PPIL5 Q32Q17 PPIL6 Q8IXY8 PPWD1 Q96BP3

[0397] Target Proteins

[0398] A target protein (e.g., a eukaryotic target protein such as a mammalian target protein or a fungal target protein or a prokaryotic target protein such as a bacterial target protein) is a protein which mediates a disease condition or a symptom of a disease condition. As such, a desirable therapeutic effect can be achieved by modulating (inhibiting or increasing) its activity. Target proteins useful in the complexes and methods of the invention include those which do not naturally associate with a presenter protein, e.g., those which have an affinity for a presenter protein in the absence of a binary complex with a compound of the invention of greater than 1 M, preferably greater than 5 M, and more preferably greater than 10 M. Alternatively, target proteins which do not naturally associate with a presenter protein are those which have an affinity for a compound of the invention in the absence of a binary complex greater than 1 M, preferably greater than 5 M, and more preferably greater than 10 M. In another alternative, target proteins which do not naturally associate with a presenter protein are those which have an affinity for a binary complex of cyclosporine, rapamycin, or FK506 and a presenter protein (e.g., FKBP) of greater than 1 M, preferably greater than 5 M, and more preferably greater than 10 M. In yet another alternative, target proteins which do not naturally associate with a presenter protein are those which are other than calcineurin or mTOR. The selection of suitable target proteins for the complexes and methods of the invention may depend on the presenter protein. For example, target proteins that have low affinity for a cyclophilin may have high affinity for an FKBP and would not be used together with the latter.

[0399] Target proteins can be naturally occurring, e.g., wild type. Alternatively, a target protein can vary from the wild type protein but still retain biological function, e.g., as an allelic variant, a splice mutant or a biologically active fragment.

[0400] In some embodiments, a target protein is a transmembrane protein. In some embodiments, a target protein has a coiled coil structure. In certain embodiments, a target protein is one protein of a dimeric complex.

[0401] In some embodiments, a target protein of the invention includes one or more surface sites (e.g., a flat surface site) characterized in that, in the absence of forming a presenter protein/compound complex, small molecules typically demonstrate low or undetectable binding to the site(s). In some embodiments, a target protein includes one or more surface sites (e.g., a flat surface site) to which, in the absence of forming a presenter protein/compound complex, a particular small molecule (e.g., the compound) shows low or undetectable binding (e.g., binding at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100 fold or more lower than that observed with a presenter protein/compound complex involving the same compound). In some embodiments, a target protein has a surface characterized by one or more sites (and, in some embodiments, an entire surface) that lack(s) any a traditional binding pocket, for example, a cavity or pocket on the protein structure with physiochemical and/or geometric properties comparable to proteins whose activity has been modulated by one or more small molecules. In certain embodiments, a target protein has a traditional binding pocket and a site for a protein-protein interaction. In some embodiments, a target protein is an undruggable target, for example, a target protein is not a member of a protein family which is known to be targeted by drugs and/or does not possess a binding site that is expected (e.g., according to art-accepted understanding, as discussed herein) to be suitable for binding to a small molecule.

[0402] In some embodiments, the target protein is a GTPase such as DIRAS1, DIRAS2, DIRAS3, ERAS, GEM, HRAS, KRAS, MRAS, NKIRAS1, NKIRAS2, NRAS, RALA, RALB, RAP1A, RAP1B, RAP2A, RAP2B, RAP2C, RASD1, RASD2, RASL10A, RASL10B, RASL11A, RASL11B, RASL12, REM1, REM2, RERG, RERGL, RRAD, RRAS, RRAS2, RHOA, RHOB, RHOBTB1, RHOBTB2, RHOBTB3, RHOC, RHOD, RHOF, RHOG, RHOH, RHOJ, RHOQ, RHOU, RHOV, RND1, RND2, RND3, RAC1, RAC2, RAC3, CDC42, RAB1A, RAB1B, RAB2, RAB3A, RAB3B, RAB3C, RAB3D, RAB4A, RAB4B, RAB5A, RAB5B, RAB5C, RAB6A, RAB6B, RAB6C, RAB7A, RAB7B, RAB7L1, RAB8A, RAB8B, RAB9, RAB9B, RABL2A, RABL2B, RABL4, RAB10, RAB11A, RAB11B, RAB12, RAB13, RAB14, RAB15, RAB17, RAB18, RAB19, RAB20, RAB21, RAB22A, RAB23, RAB24, RAB25, RAB26, RAB27A, RAB27B, RAB28, RAB2B, RAB30, RAB31, RAB32, RAB33A, RAB33B, RAB34, RAB35, RAB36, RAB37, RAB38, RAB39, RAB39B, RAB40A, RAB40AL, RAB40B, RAB40C, RAB41, RAB42, RAB43, RAP1A, RAP1B, RAP2A, RAP2B, RAP2C, ARF1, ARF3, ARF4, ARF5, ARF6, ARL1, ARL2, ARL3, ARL4, ARL5, ARL5C, ARL6, ARL7, ARL8, ARL9, ARL10A, ARL10B, ARL10C, ARL11, ARL13A, ARL13B, ARL14, ARL15, ARL16, ARL17, TRIM23, ARL4D, ARFRP1, ARL13B, RAN, RHEB, RHEBL1, RRAD, GEM, REM, REM2, RIT1, RIT2, RHOT1, or RHOT2. In some embodiments, the target protein is a GTPas activating protein such as NF1, IQGAP1, PLEXIN-B1, RASAL1, RASAL2, ARHGAP5, ARHGAP8, ARHGAP12, ARHGAP22, ARHGAP25, BCR, DLC1, DLC2, DLC3, GRAF, RALBP1, RAP1GAP, SIPA1, TSC2, AGAP2, ASAP1, or ASAP3. In some embodiments, the target protein is a Guanine nucleotide-exchange factor such as CNRASGEF, RASGEF1A, RASGRF2, RASGRP1, RASGRP4, SOS1, RALGDS, RGL1, RGL2, RGR, ARHGEF10, ASEF/ARHGEF4, ASEF2, DBS, ECT2, GEF-H1, LARG, NET1, OBSCURIN, P-REX1, P-REX2, PDZ-RHOGEF, TEM4, TIAM1, TRIO, VAV1, VAV2, VAV3, DOCK1, DOCK2, DOCK3, DOCK4, DOCK8, DOCK10, C3G, BIG2/ARFGEF2, EFA6, FBX8, or GEP100. In certain embodiments, the target protein is a protein with a protein-protein interaction domain such as ARM; BAR; BEACH; BH; BIR; BRCT; BROMO; BTB; C1; C2; CARD; CC; CALM; CH; CHROMO; CUE; DEATH; DED; DEP; DH; EF-hand; EH; ENTH; EVH1; F-box; FERM; FF; FH2; FHA; FYVE; GAT; GEL; GLUE; GRAM; GRIP; GYF; HEAT; HECT; IQ; LRR; MBT; MH1; MH2; MIU; NZF; PAS; PB1; PDZ; PH; POLO-Box; PTB; PUF; PWWP; PX; RGS; RING; SAM; SC; SH2; SH3; SOCS; SPRY; START; SWIRM; TIR; TPR; TRAF; SNARE; TUBBY; TUDOR; UBA; UEV; UIM; VHL; VHS; WD40; WW; SH2; SH3; TRAF; Bromodomain; or TPR. In some embodiments, the target protein is a heat shock protein such as Hsp20, Hsp27, Hsp70, Hsp84, alpha B crystalline, TRAP-1, hsf1, or Hsp90. In certain embodiments, the target protein is an ion channel such as Cav2.2, Cav3.2, IKACh, Kv1.5, TRPA1, NAv1.7, Nav1.8, Nav1.9, P2X3, or P2X4. In some embodiments, the target protein is a coiled-coil protein such as geminin, SPAG4, VAV1, MAD1, ROCK1, RNF31, NEDP1, HCCM, EEA1, Vimentin, ATF4, Nemo, SNAP25, Syntaxin 1a, FYCO1, or CEP250. In certain embodiments, the target protein is a kinase such as ABL, ALK, AXL, BTK, EGFR, FMS, FAK, FGFR1, 2, 3, 4, FLT3, HER2/ErbB2, HER3/ErbB3, HER4/ErbB4, IGF1R, INSR, JAK1, JAK2, JAK3, KIT, MET, PDGFRA, PDGFRB, RET RON, ROR1, ROR2, ROS, SRC, SYK, TIE1, TIE2, TRKA, TRKB, KDR, AKT1, AKT2, AKT3, PDK1, PKC, RHO, ROCK1, RSK1, RKS2, RKS3, ATM, ATR, CDK1, CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK8, CDK9, CDK10, ERK1, ERK2, ERK3, ERK4, GSK3A, GSK3B, JNK1, JNK2, JNK3, AurA, ARuB, PLK1, PLK2, PLK3, PLK4, IKK, KIN1, cRaf, PKN3, c-Src, Fak, PyK2, or AMPK. In some embodiments, the target protein is a phosphatase such as WIP1, SHP2, SHP1, PRL-3, PTP1B, or STEP. In certain embodiments the target protein is a ubiquitin ligase such as BMI-1, MDM2, NEDD4-1, Beta-TRCP, SKP2, E6AP, or APC/C. In some embodiments, the target protein is a chromatin modifier/remodeler such as a chromatin modifier/remodeler encoded by the gene BRG1, BRM, ATRX, PRDM3, ASH1 L, CBP, KAT6A, KAT6B, MLL, NSD1, SETD2, EP300, KAT2A, or CREBBP. In some embodiments, the target protein is a transcription factor such as a transcription factor encoded by the gene EHF, ELF1, ELF3, ELF4, ELF5, ELK1, ELK3, ELK4, ERF, ERG, ETS1, ETV1, ETV2, ETV3, ETV4, ETV5, ETV6, FEV, FLI1, GAVPA, SPDEF, SPI1, SPIC, SPIB, E2F1, E2F2, E2F3, E2F4, E2F7, E2F8, ARNTL, BHLHA15, BHLHB2, BHLBHB3, BHLHE22, BHLHE23, BHLHE41, CLOCK, FIGLA, HAS5, HES7, HEY1, HEY2, ID4, MAX, MESP1, MLX, MLXIPL, MNT, MSC, MYF6, NEUROD2, NEUROG2, NHLH1, OLIG1, OLIG2, OLIG3, SREBF2, TCF3, TCF4, TFAP4, TFE3, TFEB, TFEC, USF1, ARF4, ATF7, BATF3, CEBPB, CEBPD, CEBPG, CREB3, CREB3L1, DBP, HLF, JDP2, MAFF, MAFG, MAFK, NRL, NFE2, NFIL3, TEF, XBP1, PROXI, TEAD1, TEAD3, TEAD4, ONECUT3, ALX3, ALX4, ARX, BARHL2, BARX, BSX, CART1, CDX1, CDX2, DLX1, DLX2, DLX3, DLX4, DLX5, DLX6, DMBX1, DPRX, DRGX, DUXA, EMX1, EMX2, EN1, EN2, ESX1, EVX1, EVX2, GBX1, GBX2, GSC, GSC2, GSX1, GSX2, HESX1, HMX1, HMX2, HMX3, HNF1A, HNF1B, HOMEZ, HOXA1, HOXA10, HOXA13, HOXA2, HOXAB13, HOXB2, HOXB3, HOXB5, HOXC10, HOXC11, HOXC12, HOXC13, HOXD11, HOXD12, HOXD13, HOXD8, IRX2, IRX5, ISL2, ISX, LBX2, LHX2, LHX6, LHX9, LMX1A, LMX1B, MEIS1, MEIS2, MEIS3, MEOXI, MEOX2, MIXL1, MNX1, MSX1, MSX2, NKX2-3, NKX2-8, NKX3-1, NKX3-2, NKX6-1, NKX6-2, NOTO, ONECUT1, ONECUT2, OTX1, OTX2, PDX1, PHOX2A, PHOX2B, PITX1, PITX3, PKNOX1, PROP1, PRRX1, PRRX2, RAX, RAXL1, RHOXF1, SHOX, SHOX2, TGIF1, TGIF2, TGIF2LX, UNCX, VAX1, VAX2, VENTX, VSX1, VSX2, CUX1, CUX2, POU1F1, POU2F1, POU2F2, POU2F3, POU3F1, POU3F2, POU3F3, POU3F4, POU4F1, POU4F2, POU4F3, POU5F1P1, POU6F2, RFX2, RFX3, RFX4, RFX5, TFAP2A, TFAP2B, TFAP2C, GRHL1, TFCP2, NFIA, NFIB, NFIX, GCM1, GCM2, HSF1, HSF2, HSF4, HSFY2, EBF1, IRF3, IRF4, IRF5, IRF7, IRF8, IRF9, MEF2A, MEF2B, MEF2D, SRF, NRF1, CPEB1, GMEB2, MYBL1, MYBL2, SMAD3, CENPB, PAX1, PAX2, PAX9, PAX3, PAX4, PAX5, PAX6, PAX7, BCL6B, EGR1, EGR2, EGR3, EGR4, GLIS1, GLIS2, GLI2, GLIS3, HIC2, HINFP1, KLF13, KLF14, KLF16, MTF1, PRDM1, PRDM4, SCRT1, SCRT2, SNAI2, SP1, SP3, SP4, SP8, YY1, YY2, ZBED1, ZBTB7A, ZBTB7B, ZBTB7C, ZIC1, ZIC3, ZIC4, ZNF143, ZNF232, ZNF238, ZNF282, ZNF306, ZNF410, ZNF435, ZBTB49, ZNF524, ZNF713, ZNF740, ZNF75A, ZNF784, ZSCAN4, CTCF, LEF1, SOX10, SOX14, SOX15, SOX18, SOX2, SOX21, SOX4, SOX7, SOX8, SOX9, SRY, TCF7L1, FOXO3, FOXB1, FOXC1, FOXC2, FOXD2, FOXD3, FOXG1, FOXI1, FOXJ2, FOXJ3, FOXK1, FOXL1, FOXO1, FOXO4, FOXO6, FOXP3, EOMES, MGA, NFAT5, NFATC1, NFKB1, NFKB2, TP63, RUNX2, RUNX3, T, TBR1, TBX1, TBX15, TBX19, TBX2, TBX20, TBX21, TBX4, TBX5, AR, ESR1, ESRRA, ESRRB, ESRRG, HNF4A, NR2C2, NR2E1, NR2F1, NR2F6, NR3C1, NR3C2, NR4A2, RARA, RARB, RARG, RORA, RXRA, RXRB, RXRG, THRA, THRB, VDR, GATA3, GATA4, or GATA5; or C-myc, Max, Stat3, androgen receptor, C-Jun, C-Fox, N-Myc, L-Myc, MITF, Hif-1alpha, Hif-2alpha, Bcl6, E2F1, NF-kappaB, Stat5, or ER(coact). In certain embodiments, the target protein is TrkA, P2Y14, mPEGS, ASK1, ALK, Bcl-2, BCL-XL, mSIN1, RORt, IL17RA, eIF4E, TLR7R, PCSK9, IgE R, CD40, CD40L, Shn-3, TNFR1, TNFR2, IL31RA, OSMR, IL12beta1,2, Tau, FASN, KCTD 6, KCTD 9, Raptor, Rictor, RALGAPA, RALGAPB, Annexin family members, BCOR, NCOR, beta catenin, AAC 11, PLD1, PLD2, Frizzled7, RaLP, MLL-1, Myb, Ezh2, RhoGD12, EGFR, CTLA4R, GCGC (coact), Adiponectin R2, GPR 81, IMPDH2, IL-4R, IL-13R, IL-1R, IL2-R, IL-6R, IL-22R, TNF-R, TLR4, Nrlp3, or OTR.

Complexes

[0403] Presenter Protein/Compound Complexes

[0404] In naturally occurring protein-protein interactions, the binding event is driven largely by hydrophobic residues on flat surface sites of the two proteins, in contrast to many small molecule-protein interactions which are driven by interactions between the small molecule in a cavity or pocket on the protein. The hydrophobic residues on the flat surface site form hydrophobic hot spots on the two interacting proteins wherein most of the binding interactions between the two proteins are van der Waals interactions. Small molecules may be used as portable hotspots for proteins which are lacking one (e.g., presenter proteins) through the formation of complexes (e.g., a presenter protein/compound complex) to participate in pseudo protein-protein interactions (e.g., forming a tripartite complex with a target protein).

[0405] Many mammalian proteins are able to bind to any of a plurality of different partners; in some cases, such alternative binding interactions contribute to biological activity of the proteins. Many of these proteins adapt the inherent variability of the hot spot protein regions to present the same residues in different structural contexts. More specifically, the protein-protein interactions can be mediated by a class of natural products produced by a select group of fungal and bacterial species. These molecules exhibit both a common structural organization and resultant functionality that provides the ability to modulate protein-protein interaction. These molecules contain a presenter protein binding moiety that is highly conserved and a target protein interacting moiety that exhibits a high degree of variability among the different natural products. The presenter protein binding moiety confers specificity for the presenter protein and allows the molecule to bind to the presenter protein to form a binary complex; the mammalian target protein interacting moiety confers specificity for the target protein and allows the binary complex to bind to the target protein, typically modulating (e.g., positively or negatively modulating) its activity.

[0406] These natural products are presented by presenter proteins, such as FKBPs and cyclophilins and act as diffusible, cell-penetrant, orally bio-available adaptors for protein-protein interactions. Examples include well known and clinically relevant molecules such as Rapamycin (Sirolimus), FK506 (Tacrolimus), and Cyclosporin. In brief, these molecules bind endogenous intracellular presenter proteins, the FKBPs e.g. rapamycin and FK506 or cyclophilins e.g. diluents, and the resulting binary complexes of presenter protein-bound molecules selectively bind and inhibit the activity of intracellular target proteins. Formation of a tripartite complex between the presenter protein, the molecule, and the target protein is driven by both protein-molecule and protein-protein interactions and both are required for inhibition of the target protein. In the example of the FKBP-rapamycin complex, the intracellular target is the serine-threonine kinase mTOR, whereas for FKBP-FK506 complex, the intracellular target is the phosphatase calcineurin. Of particular interest in the preceding two examples, FKBP12 is utilized as a partner presentation protein by both the rapamycin and FK506 presentation ligands. Moreover, the sub-structure components of rapamycin and FK506 responsible for binding to FKBP12 are closely related structurally, i.e. the so-called Conserved Region, but it is the dramatic structural differences between rapamycin and FK506 in the non FKBP12-binding regions, i.e. the Variable Region, that results in the specific targeting of two distinct intracellular proteins, mTOR and calcineurin, respectively. In this fashion, the Variable Regions of rapamycin and FK506 are serving as contributors to the binding energy necessary for enabling presenter protein-target protein interaction.

[0407] In some embodiments, a presenter protein/compound complexes of the invention bind to a target protein with at least 5-fold (e.g., at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 100-fold) greater affinity than the complex binds to each of mTOR and/or calcineurin.

[0408] In some embodiments, a presenter protein/compound complexes of the invention bind to a target protein with at least 5-fold (e.g., at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 100-fold) greater affinity than the affinity of the compound to a target protein when the compound is not bound in a complex with a presenter protein.

[0409] In certain embodiments, a presenter protein/compound complexes of the invention bind to a target protein with at least 5-fold (e.g., at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 100-fold) greater affinity than the affinity of the presenter protein to a target protein when the presenter protein is not bound in a complex with a compound.

[0410] In some embodiments, a presenter protein/compound complexes of the invention inhibit a naturally occurring interaction between a target protein and a ligand, such as a protein or a small molecule that specifically binds to the target protein.

[0411] In certain embodiments, when the presenter protein is a prolyl isomerase, the prolyl isomerase activity is inhibited by formation of the presenter protein/compound complex. In some embodiments of the presenter protein/compound complexes of the invention, the compound specifically binds to said presenter protein with a K.sub.D of less than 10 M (e.g., less than 5 M, less than 1 M, less than 500 nM, less than 200 nM, less than 100 nM, less than 75 nM, less than 50 nM, less than 25 nM, less than 10 nM) or inhibits the peptidyl-prolyl isomerase activity of the presenter protein, for example, with an IC.sub.50 of less than 1 M (e.g., less than 0.5 M, less than 0.1 M, less than 0.05 M, less than 0.01 M).

[0412] Tripartite Complexes

[0413] The vast majority of small molecule drugs act by binding a functionally important site on a target protein, thereby modulating (e.g., positively or negatively modulating) the activity of that protein. For example, the cholesterol-lowering drugs statins bind the enzyme active site of HMG-CoA reductase, thus preventing the enzyme from engaging with its substrates. The fact that many such drug/target interacting pairs are known may have misled some into believing that a small molecule modulator could be discovered for most, if not all, proteins provided a reasonable amount of time, effort, and resources. This is far from the case. Current estimates hold that only about 10% of all human proteins are targetable by small molecules. The other 90% are currently considered refractory or intractable toward small molecule drug discovery. Such targets are commonly referred to as undruggable. These undruggable targets include a vast and largely untapped reservoir of medically important human proteins. Thus, there exists a great deal of interest in discovering new molecular modalities capable of modulating the function of such undruggable targets.

[0414] The present invention encompasses the recognition that small molecules are typically limited in their targeting ability because their interactions with the target are driven by adhesive forces, the strength of which is roughly proportional to contact surface area. Because of their small size, the only way for a small molecule to build up enough intermolecular contact surface area to effectively interact with a target protein is to be literally engulfed by that protein. Indeed, a large body of both experimental and computational data supports the view that only those proteins having a hydrophobic pocket on their surface are capable of binding small molecules. In those cases, binding is enabled by engulfment. Not a single example exists of a small molecule binding with high-affinity to a protein outside of a hydrophobic pocket.

[0415] Nature has evolved a strategy that allows a small molecule to interact with target proteins at sites other than hydrophobic pockets. This strategy is exemplified by the naturally occurring immunosuppressive drugs cyclosporine A, rapamycin, and FK506. The activity of these drugs involves the formation of a high-affinity complex of the small molecule with a small presenting protein. The composite surface of the small molecule and the presenting protein then engages the target. Thus, for example, the binary complex formed between cyclosporine A and cyclophilin A targets calcineurin with high affinity and specificity, but neither cyclosporine A or cyclophilin A alone binds calcineurin with measurable affinity.

[0416] Many important therapeutic targets exert their function by complexation with other proteins. The protein/protein interaction surfaces in many of these systems contain an inner core of hydrophobic side chains surrounded by a wide ring of polar residues. The hydrophobic residues contribute nearly all of the energetically favorable contacts, and hence this cluster has been designated as a hotspot for engagement in protein-protein interactions. Importantly, in the aforementioned complexes of naturally occurring small molecules with small presenting proteins, the small molecule provides a cluster of hydrophobic functionality akin to a hotspot, and the protein provides the ring of mostly polar residues. In other words, presented small molecule systems mimic the surface architecture employed widely in natural protein/protein interaction systems.

[0417] Compounds (e.g., macrocyclic compounds) of the invention are capable of modulating biological processes, for example through binding to a presenter protein (e.g., a member of the FKBP family, a member of the cyclophilin family, or PIN1) to form a presenter protein/compound complex as described above which binds to a target protein to form a tripartite complex. The formation of these tripartite complexes allow for modulation of proteins that do not have traditional binding pockets and/or are considered undruggable. The presenter protein/compound complexes are able to modulate biological processes through cooperative binding between the compound and the presenter protein. Both the compound and presenter protein have low affinity for the target protein alone, but the presenter protein/compound complex has high affinity for the target protein. Cooperative binding can be determined by measurement of the buried surface area of the target protein that includes atoms from the compound and/or presenter protein and/or by measurement of the free binding energy contribution of the compound and/or presenter protein. Binding is considered cooperative if at least one atom from each of the compound and presenter protein participates in binding with the target protein.

[0418] The binding of a presenter protein/compound complex and a target protein is achieved through formation of a combined binding site including residues from both the presenter protein and compound that allow for increased affinity that would not be possible with either the presenter protein or compound alone. For example at least 20% (e.g., at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%) of the total buried surface area of the target protein in the tripartite complex includes one or more atoms that participate in binding to the compound and/or at least 20% (e.g., at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%) of the total buried surface area of the target protein in the tripartite complex includes one or more atoms that participate in binding to the presenter protein. Alternatively, the compound contributes at least 10% (e.g., at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%) of the total binding free energy of the tripartite complex and/or the presenter protein contributes at least 10% (e.g., at least 20% at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%) of the total binding free energy of the tripartite complex.

[0419] In some embodiments, a presenter protein/compound complex binds at a flat surface site on a target protein. In some embodiments, a compound (e.g., macrocyclic compound) in a presenter protein/compound complex binds at a hydrophobic surface site on a target protein, e.g., a site that includes at least 50% hydrophobic residues. In some embodiments, at least 70% of the binding interactions between one or more of the atoms of a compound and one or more atoms of a target protein are van der Waals and/or -effect interactions. In certain embodiments, a presenter protein/compound complex binds to a target protein at a site of a naturally occurring protein-protein interaction between a target protein and a protein that specifically binds the target protein. In some embodiments, a presenter protein/compound complex does not bind at an active site of a target protein. In some embodiments, a presenter protein/compound complex binds at an active site of a target protein.

[0420] A characteristic of compounds of the invention that form tripartite complexes with a presenter protein and a target protein is a lack of major structural reorganization in the presenter protein/compound complex compared to the tripartite complex. This lack of major structural reorganization results in a low entropic cost to reorganize into a configuration favorable for the formation of the tripartite complex once the presenter protein/compound complex has been formed. For example, threshold quantification of RMSD can be measured using the align command in PyMOL version 1.7rc1 (Schrdinger LLC). Alternatively, RMSD can be calculated using the ExecutiveRMS parameter from the algorithm LigAlign (J. Mol. Graphics and Modelling 2010, 29, 93-101). In some embodiments, the structural organization of the compound (i.e., the average three dimensional configuration of the atoms and bonds of the molecule) is substantially unchanged in the tripartite complex compared to the compound when in the presenter protein/compound complex before binding to the target protein. For example, the root mean squared deviation (RMSD) of the two aligned structures is less than 1.

Utility and Administration

[0421] Compounds and presenter protein/compound complexes described herein are useful in the methods of the invention and, while not bound by theory, are believed to exert their desirable effects through their ability to modulate (e.g., positively or negatively modulate) the activity of a target protein (e.g., a eukaryotic target protein such as a mammalian target protein or a fungal target protein or a prokaryotic target protein such as a bacterial target protein), through interaction with presenter proteins and the target protein.

Kits

[0422] In some embodiments, the present invention relates to a kit for conveniently and effectively carrying out the methods in accordance with the present invention. In general, the pharmaceutical pack or kit comprises one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Such kits are especially suited for the delivery of solid oral forms such as tablets or capsules. Such a kit preferably includes a number of unit dosages, and may also include a card having the dosages oriented in the order of their intended use. If desired, for instance if the subject suffers from Alzheimer's disease, a memory aid can be provided, for example in the form of numbers, letters, or other markings or with a calendar insert, designating the days in the treatment schedule in which the dosages can be administered. Alternatively, placebo dosages, or calcium dietary supplements, either in a form similar to or distinct from the dosages of the pharmaceutical compositions, can be included to provide a kit in which a dosage is taken every day. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceutical products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

Pharmaceutical Compositions

[0423] For use as treatment of human and animal subjects, the compounds of the invention can be formulated as pharmaceutical or veterinary compositions. Depending on the subject to be treated, the mode of administration, and the type of treatment desirede.g., prevention, prophylaxis, or therapythe compounds are formulated in ways consonant with these parameters. A summary of such techniques is found in Remington: The Science and Practice of Pharmacy, 21.sup.st Edition, Lippincott Williams & Wilkins, (2005); and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York, each of which is incorporated herein by reference.

[0424] Compounds described herein may be present in amounts totaling 1-95% by weight of the total weight of the composition. The composition may be provided in a dosage form that is suitable for intraarticular, oral, parenteral (e.g., intravenous, intramuscular), rectal, cutaneous, subcutaneous, topical, transdermal, sublingual, nasal, vaginal, intravesicular, intraurethral, intrathecal, epidural, aural, or ocular administration, or by injection, inhalation, or direct contact with the nasal, genitourinary, reproductive or oral mucosa. Thus, the pharmaceutical composition may be in the form of, e.g., tablets, capsules, pills, powders, granulates, suspensions, emulsions, solutions, gels including hydrogels, pastes, ointments, creams, plasters, drenches, osmotic delivery devices, suppositories, enemas, injectables, implants, sprays, preparations suitable for iontophoretic delivery, or aerosols. The compositions may be formulated according to conventional pharmaceutical practice.

[0425] In general, for use in treatment, compounds described herein may be used alone, or in combination with one or more other active agents. An example of other pharmaceuticals to combine with the compounds described herein would include pharmaceuticals for the treatment of the same indication. Another example of a potential pharmaceutical to combine with compounds described herein would include pharmaceuticals for the treatment of different yet associated or related symptoms or indications. Depending on the mode of administration, compounds will be formulated into suitable compositions to permit facile delivery. Each compound of a combination therapy may be formulated in a variety of ways that are known in the art. For example, the first and second agents of the combination therapy may be formulated together or separately. Desirably, the first and second agents are formulated together for the simultaneous or near simultaneous administration of the agents.

[0426] Compounds of the invention may be prepared and used as pharmaceutical compositions comprising an effective amount of a compound described herein and a pharmaceutically acceptable carrier or excipient, as is well known in the art. In some embodiments, a composition includes at least two different pharmaceutically acceptable excipients or carriers.

[0427] Formulations may be prepared in a manner suitable for systemic administration or topical or local administration. Systemic formulations include those designed for injection (e.g., intramuscular, intravenous or subcutaneous injection) or may be prepared for transdermal, transmucosal, or oral administration. A formulation will generally include a diluents as well as, in some cases, adjuvants, buffers, preservatives and the like. Compounds can be administered also in liposomal compositions or as microemulsions.

[0428] For injection, formulations can be prepared in conventional forms as liquid solutions or suspensions or as solid forms suitable for solution or suspension in liquid prior to injection or as emulsions. Suitable excipients include, for example, water, saline, dextrose, glycerol and the like. Such compositions may also contain amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, such as, for example, sodium acetate, sorbitan monolaurate, and so forth.

[0429] Various sustained release systems for drugs have also been devised. See, for example, U.S. Pat. No. 5,624,677, which is herein incorporated by reference.

[0430] Systemic administration may also include relatively noninvasive methods such as the use of suppositories, transdermal patches, transmucosal delivery and intranasal administration. Oral administration is also suitable for compounds of the invention. Suitable forms include syrups, capsules, and tablets, as is understood in the art.

[0431] Each compound of a combination therapy, as described herein, may be formulated in a variety of ways that are known in the art. For example, the first and second agents of the combination therapy may be formulated together or separately.

[0432] The individually or separately formulated agents can be packaged together as a kit. Non-limiting examples include, but are not limited to, kits that contain, e.g., two pills, a pill and a powder, a suppository and a liquid in a vial, two topical creams, etc. The kit can include optional components that aid in the administration of the unit dose to subjects, such as vials for reconstituting powder forms, syringes for injection, customized IV delivery systems, inhalers, etc. Additionally, the unit dose kit can contain instructions for preparation and administration of the compositions. The kit may be manufactured as a single use unit dose for one subject, multiple uses for a particular subject (at a constant dose or in which the individual compounds may vary in potency as therapy progresses); or the kit may contain multiple doses suitable for administration to multiple subjects (bulk packaging). The kit components may be assembled in cartons, blister packs, bottles, tubes, and the like.

[0433] Formulations for oral use include tablets containing the active ingredient(s) in a mixture with non-toxic pharmaceutically acceptable excipients. These excipients may be, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starches including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate); granulating and disintegrating agents (e.g., cellulose derivatives including microcrystalline cellulose, starches including potato starch, croscarmellose sodium, alginates, or alginic acid); binding agents (e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, carboxymethylcellulose sodium, methylcellulose, hydroxypropyl methylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol); and lubricating agents, glidants, and antiadhesives (e.g., magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenated vegetable oils, or talc). Other pharmaceutically acceptable excipients can be colorants, flavoring agents, plasticizers, humectants, buffering agents, and the like.

[0434] Two or more compounds may be mixed together in a tablet, capsule, or other vehicle, or may be partitioned. In one example, the first compound is contained on the inside of the tablet, and the second compound is on the outside, such that a substantial portion of the second compound is released prior to the release of the first compound.

[0435] Formulations for oral use may also be provided as chewable tablets, or as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluents (e.g., potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate or kaolin), or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin, or olive oil. Powders, granulates, and pellets may be prepared using the ingredients mentioned above under tablets and capsules in a conventional manner using, e.g., a mixer, a fluid bed apparatus or a spray drying equipment.

[0436] Dissolution or diffusion controlled release can be achieved by appropriate coating of a tablet, capsule, pellet, or granulate formulation of compounds, or by incorporating the compound into an appropriate matrix. A controlled release coating may include one or more of the coating substances mentioned above and/or, e.g., shellac, beeswax, glycowax, castor wax, carnauba wax, stearyl alcohol, glyceryl monostearate, glyceryl distearate, glycerol palmitostearate, ethylcellulose, acrylic resins, dl-polylactic acid, cellulose acetate butyrate, polyvinyl chloride, polyvinyl acetate, vinyl pyrrolidone, polyethylene, polymethacrylate, methylmethacrylate, 2-hydroxymethacrylate, methacrylate hydrogels, 1,3 butylene glycol, ethylene glycol methacrylate, and/or polyethylene glycols. In a controlled release matrix formulation, the matrix material may also include, e.g., hydrated methylcellulose, carnauba wax and stearyl alcohol, carbopol 934, silicone, glyceryl tristearate, methyl acrylate-methyl methacrylate, polyvinyl chloride, polyethylene, and/or halogenated fluorocarbon.

[0437] The liquid forms in which the compounds and compositions of the present invention can be incorporated for administration orally include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.

[0438] Generally, when administered to a human, the oral dosage of any of the compounds of the combination of the invention will depend on the nature of the compound, and can readily be determined by one skilled in the art. Typically, such dosage is normally about 0.001 mg to 2000 mg per day, desirably about 1 mg to 1000 mg per day, and more desirably about 5 mg to 500 mg per day. Dosages up to 200 mg per day may be necessary.

[0439] Administration of each drug in a combination therapy, as described herein, can, independently, be one to four times daily for one day to one year, and may even be for the life of the subject. Chronic, long-term administration may be indicated.

[0440] The following Examples are intended to illustrate the synthesis of a representative number of compounds and the use of these compounds for the induction of chemotaxis and antifungal activity. Accordingly, the Examples are intended to illustrate but not to limit the invention. Additional compounds not specifically exemplified may be synthesized using conventional methods in combination with the methods described herein.

EXAMPLES

Example 1. General Fermentation and Isolation Protocols

[0441] Compounds synthesized by bacterial strains may be fermented and isolated using the following general protocol:

[0442] General Fermentation Protocol

[0443] Strains: Bacterial strains such as Streptomyces malaysiensis DSM41697, other producing species or genetically modified derivatives producing FKBP ligands (Example: F1, F2, F3 or structurally similar compounds and their analogs) were propagated aseptically on a solid medium (Example: ISP4).

[0444] Working cell bank: Spores or mycelia derived from the cultures grown on a solid medium plate at 30 C. for 3-14 d were used to inoculate a liquid culture (Example: 40 ml ATCC172 liquid medium in an 250 ml Erlenmeyer flask). The culture was incubated with shaking at 30 C. for 2-3 d. The resulting cell suspension was mixed with sterile 50% glycerol giving a mixture containing a final concentration of 15-25% glycerol. Aliquots (about 1 ml) of glycerol-mycelia mixture were stored at 80 C. in sterile cryovials until further use.

[0445] Primary seed culture: Primary seed cultures (Example: 40 mL ATCC172 medium in a 250 mL Erlenmeyer flask) were inoculated with 1 mL working cell bank suspension. Cultures were incubated on a shaker with a 2-inch throw at 200-220 rpm for 2-3 d at 30 C.

[0446] Secondary seed culture: Secondary seed cultures (Example: 100-200 mL ATCC172 in an 500 mL Erlenmeyer flask) were inoculated with the primary seed cultures (5% v/v) and incubated as described above with various incubation periods of time (Example: 18-48 h).

[0447] Production fermentation in flasks: Production fermentation was done in a 1.8 L Fernbach or Erlenmeyer flask containing 0.5 L production medium supporting biosynthesis of these compounds (Example: Medium 8430 or its derivatives). The culture was inoculated with a seed culture prepared as described above at 2-5% (v/v), and incubated as described above conditions for 3-7 d.

[0448] Production fermentation in bioreactors: Production fermentation was done in a bioreactor (7.5 L capacity, New Brunswick Scientific, NJ, USA) controlled by a BioFlo 300 module. The bioreactor containing 5 L of sterilized medium (Example: 8430 and its derivatives) was inoculated with a seed culture (2-5%, v/v) and incubated for 3-7 d with or without controlled parameters such as dissolved oxygen amounts (Example: 10-50%), propeller speed (Example 200-500 rpm), pH (Example: pH 4.5-7.0), temperature (Example: 25-35 C.), and nutrient feeding when appropriate.

TABLE-US-00002 ISP4 (per liter) Soluble Starch 10.0 g Dipotassium Phosphate 1.0 g Magnesium Sulfate USP 1.0 g Sodium Chloride 1.0 g Ammonium Sulfate 2.0 g Calcium Carbonate 2.0 g Ferrous Sulfate 1.0 mg Manganous Chloride 1.0 mg Zinc Sulfate 1.0 mg Agar 20.0 g

TABLE-US-00003 TABLE 2 ATCC #172 media (per liter) Yeast extract 5 g Difco Soluble Starch 20 g Dextrose 10 g NZ Amine A 5 g Calcium Carbonate 3 g Add distilled water to 1000 mL, no pH adjustment.

TABLE-US-00004 TABLE 3 8430 Medium Component Amount Pharmamedia or Proflo powder 10 g (ADM) D-Mannitol 20 g Yeast extract 1.0 g KH.sub.2PO.sub.4 0.10 g MES buffer, hemi-Na+ salt 20.67 g (adjust media to (100 mM final) pH 6.5 final with 5N NaOH) MgSO.sub.4-7H.sub.2O (Anh.) 0.05 g/L CaCl.sub.2-2H.sub.2O 0.02 g/L R2 trace elements solution* 2 mL Add distilled water to 1000 mL. Proflo oil containing dominantly oleate and palmitate was added (4 mL/L) as an antifoam agent.

TABLE-US-00005 TABLE 4 * R2 trace element solution. Amount Element (mg/L) ZnSO.sub.47H.sub.2O 40 FeCl.sub.36H.sub.2O 200 CuCl.sub.22H.sub.2O 10 MnCl22 H.sub.2O 10 Na.sub.2B.sub.4O.sub.710H.sub.2O 10 (NH.sub.4).sub.6Mo.sub.7O.sub.244H.sub.2O 10

[0449] General Isolation Protocol

[0450] Fermentation broth of a strain producing specific compounds was separated to supernatant and microbial pellets by centrifugation. Target compounds in the supernatant can be extracted either with partition extraction using water-immiscible solvents such as dichloromethane (DCM), ethyl acetate (EtOAc), etc or with solid phase extraction by mixing with non-polar resins such as HP20, HP20ss, etc. The target compounds in the pellets can be extracted repeatedly (4) using ethyl EtOAc-methanol (9:1, v/v). The microbial extracts are pooled in preparation for concentrating in vacuo. To this extract can be added the material eluted from the HP20 beads (using organic solvents such as methanol (MeOH), DCM, acetonitrile, isopropanol (IPA), etc) and/or the organic phase of the liquid/liquid extraction of the original supernatant.

[0451] The combined extracts are filtered through Celite and dried in vacuo yielding a primary crude and this material is weighed. The primary crude is dissolved in minimal 100% MeOH or a mixture of DCM and tetrahydrofuran (THF). To this a binding medium such as silica gel powder is added to the flask and re-dried in vacuo for normal-phase silica gel column chromatography. The ratio of crude to silica gel in the column bed is preferably ca. 1:5 (wt/wt). The crude material can be fractionated over a RediSep Normal-phase Silica Flash Column using step gradients, linear gradients or isocratic elution conditions. Elution solvents can include hexane, heptane, ethyl acetate, ethanol, acetone, isopropanol, or other organic solvents, or combination. Fractions with enriched target compound(s) are pooled and dried for further purification after LC/MS analysis and/or Thin Layer Chromatography (TLC) analysis.

[0452] Further purification could be achieved via normal-phase or specific prep-HPLC columns such as Waters Spherisorb CN, Waters Prep Silica, or Kromacil 60-5DIOL. Elution solvents can also include hexane, heptane, ethyl acetate, ethanol, acetone, isopropanol, or other organic solvents, or combination. Fractions with enriched or pure target compound(s) are pooled and dried for further workup after LC/MS analysis and/or Thin Layer Chromatography (TLC) analysis.

[0453] Additional purification could be achieved various reverse-phase prep-HPLC depending on the complexity of the enriched material and target compounds' properties such as polarity, solubility, etc. Reverse-phase Prep-HPLC columns employed for separation include Waters Sunfire Prep C18 OBD, Waters Xbridge Prep C18 OBD, Kromacil C4, Thermo Acclaim Polar Advantage 2, and Phenomenex Luna C18. Common solvent systems are a mixture of water and acetonitrile or methanol without or with 0.1% formic acid or 0.01% trifluoroacetic acid modifiers or 25 mM ammonium formate buffer. The elution mode can be either linear gradient or isocratic. Fractions with pure target compound(s) are pooled and dried for further workup after LC/MS analysis and/or Thin Layer Chromatography (TLC) analysis.

[0454] Fractions containing pure compounds are subjected to workup and drying process to obtain pure solid material. Certain target compounds can be extracted with ethyl acetate or dichloromethane from aqueous matrix after reverse-phase column chromatographic purification. Solvent removal and drying techniques include rotavap, speedvac, and lyophilization. Purity and chemical structure of purified target compounds are determined by LC-MS (/MS) and NMR techniques.

Example 2. Isolation of F2 and F3

[0455] 10 L fermentation broth of Streptomyces malaysiensis (NRRL B-24313; ATCC BAA-13; DSM 41697; JCM 10672; KCTC 9934; NBRC 16446; CGMCC 4.1900; IFO 16448) producing F1 (target mass 595), F2 (target mass 609) and Compound 3 (target mass 623) was separated by centrifugation. F1 and F2 are present in both the clarified broth and microbial pellets. Target compounds in the supernatant were extracted once with EtOAc at a ratio of volume (1:1, v/v). The pellets were extracted 3 times with 1.5 L of EtOAc-MeOH (9:1, v/v) stirring with an overhead stirrer for 1 h-1.5 h for each extraction. The organic extracts were filtered through Celite. The combined filtrates were evaporated at 35 C. until dryness to afford ca. 30 g of crude extract. The residue was then dissolved in 90 mL of DCM-THF (80:20, v/v), and to this 60 g of silica gel were added and dried in vacuo at 35 C. The dried residue/silica mixture was loaded onto a 120 g RediSep silica gold cartridge. Compounds were eluted with 100% heptane to heptane-EtOAc (6:4, v/v) with a linear gradient over 30 min at 85 mL/min and collected with 50 mL per fraction on a Teledyne ISCO Combiflash Rf instrument.

[0456] By TLC, F2 enriched fractions were eluted at 20% to 30% EtOAc in heptane. The pooled fraction was then concentrated at 35 C. to provide 900 mg of enriched F2 material which was further re-purified on a silica gel cartridge. Ca. 1 mL of DCM was used to dissolve the fraction and 1.8 g of silica gel was added. The dried mixture was loaded onto a 80 g RediSep silica gold cartridge. Compounds were eluted with 100% heptane to heptane-EtOAc (6:4, v/v) with a linear gradient over 30 min at 60 mL/min and collected with 50 mL per fraction. By TLC, pure fractions 25-28 were combined for solvent removal in vacuo at 35 C. to obtain 300 mg of pure F2 (beta-form) for structure elucidation and biological tests.

[0457] F2: .sup.1H NMR (500 MHz, Benzene-d.sub.6) 7.20-7.13 (m, 4H), 7.0-7.05 (m, 1H), 5.82 (s, 1H), 5.79-5.69 (m, 2H), 5.51 (m, 1H), 5.46-5.35 (m, 3H), 4.60 (d, J=12 Hz, 1H), 3.98-3.90 (m, 1H), 3.63 (dqd, J=13, 6.5, 3.0 Hz, 1H), 3.22 (d, J=3.6 Hz, 1H), 3.07 (td, J=12, 2.8 Hz, 1H), 3.00 (t, J=9.9 Hz, 1H), 2.93 (dd, J=13, 4.4 Hz, 1H), 2.63-2.54 (m, 3H), 2.20 (d, J=13 Hz, 1H), 2.11-2.03 (m, 1H), 1.99-1.86 (m, 2H), 1.79-1.71 (m, 1H), 1.68-1.60 (m, 1H), 1.51-1.47 (m, 1H), 1.45 (d, J=6.6 Hz, 3H), 1.37 (m, 4H), 1.31 (m, 1H), 1.30 (d, J=6.6 Hz, 3H), 1.29-1.22 (m, 2H), 1.16-1.08 (m, 1H), 1.04-0.94 (m, 1H), 0.82 (t, J=7.4 Hz, 3H), 0.69 (d, J=6.7 Hz, 3H). .sup.13C NMR (125 MHz, Benzene-d.sub.6) 209.9, 169.7, 167.5, 141.3, 132.2, 129.6, 129.4, 128.7, 128.0, 127.7, 126.4, 98.2, 79.7, 75.5, 71.1, 51.9, 46.9, 44.2, 44.0, 40.4, 36.2, 35.3, 35.3, 35.2, 34.0, 33.3, 25.4, 25.3, 22.5, 21.1, 17.4, 17.1, 11.6, 9.7. HR-MS [M+Na].sup.+: calc [C.sub.36H.sub.51NO.sub.7+Na].sup.+ 632.3563, obs 632.3569.

[0458] By TLC and LC-MS analysis, Compound 3 enriched fractions were eluted at 30% to 40% EtOAc in heptane. The pooled fraction was then concentrated at 35 C. to provide 500 mg of enriched Compound 3 material which was further re-purified by reverse-phase prep-HPLC on a Thermo Polar Advantage II column (5 m, 25021.2 mm). Prep-HPLC conditions included 70% acetonitrile in water plus 0.1% formic acid, isocratic elution mode at 15 mL/min, 254 nm. The enriched Compound 3 sample was dissolved in 10 mL methanol for repeatable 10 injections. Target Compound 3 peak at 23.5 minute was collected. After extraction with EtOAc from prep-HPLC pooled fractions and organic solvent removal in vacuo, 250 mg of pure Compound 3 were obtained. Its chemical structure was subsequently determined by various LC-MS and NMR techniques.

[0459] F3: .sup.1H NMR (500 MHz, Benzene-d.sub.6, 1:1 mixture of rotamers) 7.30 (m, 1H), 7.20-7.10 (m, 6H), 7.10-7.06 (m, 3H), 7.00 (m, 2H), 5.65-5.55 (m, 2H), 5.45 (m, 1H), 5.25-5.15 (m, 2H), 4.98 (dd, J=15, 7.3 Hz, 1H), 4.89 (dd, J=8.9, 5.0 Hz, 1H), 4.67 (dd, J=15, 8.8 Hz, 1H), 4.45 (m, 2H), 4.20 (m, 1H), 4.13 (m, 1H), 3.87 (m, 1H), 3.57 (m, 2H), 3.35-3.05 (m, 3H), 2.72 (m, 2H), 2.65-2.50 (m, 2H), 2.50-2.30 (m, 6H), 2.08 (m, 1H), 1.93 (m, 1H), 1.80-0.90 (m, 50H) [1.71 (d, J=6.8 Hz, 3H), 1.54 (d, J=6.8 Hz, 3H)], 1.24 (d, J=6.5 Hz, 3H), 1.18 (d, J=6.6 Hz, 3H), 1.08 (d, J=6.8 Hz, 3H), 1.01 (m, J=6.7 Hz, 3H)], 0.73 (t, J=7.5 Hz, 3H), 0.69 (t, J=7.5 Hz, 3H). .sup.13C NMR (125 MHz, Benzene-d.sub.6) 201.5, 199.9, 197.8, 191.6, 170.4, 169.5, 166.8, 166.6, 145.4, 144.8, 140.6, 140.5, 133.9, 131.3, 129.7, 129.4, 129.3, 128.8, 128.8, 128.4, 128.3, 126.6, 126.5, 126.0, 100.0, 99.3, 80.6, 78.2, 73.1, 72.4, 71.7, 70.6, 57.1, 52.6, 51.9, 51.1, 45.8, 45.4, 44.2, 42.5, 42.2, 39.8, 35.8, 35.7, 35.6, 34.1, 33.6, 33.4, 29.9, 29.9, 29.3, 28.2, 27.4, 27.1, 25.1, 25.1, 22.3, 22.2, 21.3, 21.2, 16.6, 16.2, 14.6, 13.7, 11.2, 11.1, 10.6, 9.5. HR-MS [M+H].sup.+: calc [C.sub.36H.sub.49NO.sub.8+H].sup.+ 624.3536, obs 624.3547.

Example 3. Isolation of F22

[0460] 10 L of fermentation broth produced from a recombinant strain S1806 were centrifuged to obtain the pellets and supernatant. The pellets were extracted 3 times with 1.5 L of EtOAc-MeOH (9:1, v/v). The organic solvents were combined and concentrated in vacuo to obtain 1.8 g of crude extract. To this, 2 mL of Heptane-THF (4:1, v/v) was added to dissolve and 2 g of Celite were then added to obtain the dried mixture after removal of solvents on a rotavapor at 30 C. The dried residue/celite mixture was loaded onto a 40 g RediSep silica gold cartridge for column chromatography. Compounds were fractionated with a linear gradient elution from 100% n-heptane to 40% EtOAc in heptane (v/v) over 25 min at 20 mL/min collected with 50 mL per fraction. F22 (target mass 607) was primarily enriched in Fraction 14 identified by LC-MS analysis. Fraction 14 was then dried in vacuo at 30 C. to afford 17.8 mg solid material which was further purified by prep-HPLC on a Thermo Polar Advantage II column (5 m, 25021.2 mm). Prep-HPLC conditions included 90% acetonitrile in water plus 0.1% formic acid, isocratic elution mode at 15 mL/min, 254 nm. The sample was dissolved in 1.78 mL methanol for repeatable 5 injections. Target F22 peak at 11.5 minute was collected. After solvent removal in vacuo, 3.64 mg of pure F22 was obtained. Its chemical structure was subsequently determined by various LC-MS/MS and NMR techniques.

Example 4. Synthesis of Selected Compounds

Instrumentation:

[0461] Purification was performed on HPLC preparative using Agilent SD-1 system.
Electrospray LC/MS analysis was performed using an Agilent 1260 Infinity system equipped with an Agilent 1260 series LC pump. The methods used were:

Analytical HPLC Method 1:

[0462] Agilent Zorbax Extend C-18 reverse phase column (2.150 mm), 1.8 m:

Solvent A: Water+0.1% Formic Acid

Solvent B: Acetonitrile+0.1% Formic Acid

[0463] Flow rate: 0.5 mL/min
Injection volume: 5 L
Column temperature: 400C

Gradient:

[0464]

TABLE-US-00006 Time, min % A % B 0 95 5 3 30 70 10 0 100 13 0 100 14 95 5 16 95 5

Analytical HPLC Method 2:

ThermoScientific Acclaim, Polar Advantage II, 4.6150 mm, 5 m

Solvent A: Water+0.1% Formic Acid

Solvent B: Acetonitrile+0.1% Formic Acid

[0465] Flow rate: 0.8 mL/min
Injection volume: 5 L
Column temperature: 400C

Isocratic:

[0466]

TABLE-US-00007 Time, min % A % B 0 20 80 6 20 80 7 5 95 9 5 95 10 20 80 12 20 80
Electrospray UHPLC/MS was performed using an Agilent 1290 Infinity system equipped with an Agilent 1290 series LC pump. The columns used were the same.

Analytical UHPLC Method 1:

[0467] Agilent Zorbax Extend C-18 reverse phase column (2.150 mm), 1.8 m:

Solvent A: Water+0.1% Formic Acid

Solvent B: Acetonitrile+0.1% Formic Acid

[0468] Flow rate: 0.5 mL/min
Injection volume: 5 L
Column temperature: 400C

Gradient:

[0469]

TABLE-US-00008 Time, min % A % B 0 95.24 4.76 5.21 30.19 69.81 9.66 9.04 90.96 10.5 0 100 11.5 0 100 12 95.24 4.76 13 95.24 4.76

Purification Method A:

[0470] Performed using an ACCLAIM Polar Advantage II (21.2250 mm) column. Flow rate 17 mL/min, isocratic 70% B. Solvent A was 0.1% aqueous formic acid, solvent B was 100% acetonitrile containing 0.1% formic acid.

Synthesis of F11

Synthesis of (2S)-1-((4R,7S)-7-((2R,3S,4R,11S,12R)-12-benzyl-3,11-dihydroxy-4-methyltetradecan-2-yl)-2-hydroxy-4-methyl-3-oxooxepane-2-carbonyl)piperidine-2-carboxylic acid C-11 Lactone. F11

[0471] ##STR00071##

[0472] To a mixture of (2S)-1-((4R,7S)-7-((2R,3S,4R,6E,9E,11R,12R)-12-benzyl-3,11-dihydroxy-4-methyltetradeca-6,9-dien-2-yl)-2-hydroxy-4-methyl-3-oxooxepane-2-carbonyl)piperidine-2-carboxylic acid C-11 lactone (5 mg, 8.2 umol) and 10% palladium on carbon (2 mg) and a stirrer bead under nitrogen was added ethyl acetate (1 mL). The flask was charged with hydrogen and stirred vigorously for 1.5 hr. The atmosphere of hydrogen was replaced with nitrogen and the reaction filtered through celite. The celite pad was washed with more ethyl acetate and the solvent evaporated in vacuo. The residue was purified by chromatography on silica gel, gradient elution ethyl acetate: hexanes 40:60 to 100:0 to afford the title compound.

[0473] 1H NMR (CDCl3, 500 MHz): 7.28 (m, 2H), 7.19 (m, 1H), 7.13 (d, J=6.98 Hz, 2H), 5.65 (s, 1H), 5.26 (d, J=4.92 Hz, 1H), 5.11 (m, 1H), 4.67 (d, J=13.02 Hz, 1H), 4.02 (dd, J=10.67, 1.13 Hz, 1H), 3.35 (m, 1H), 3.23-3.10 (m, 2H), 2.72 (dd, J=13.85, 5.50 Hz, 1H), 2.50 (dd, J=13.93, 9.25 Hz, 1H), 2.39 (m, 1H), 1.95-1.73 (m, 5H), 1.71-1.15 (m, 25H), 1.03 (d, J=6.71 Hz, 3H), 0.85 (t, J=7.42 Hz, 3H), 0.79 (d, J=6.82 Hz, 3H) ppm.

[0474] 13C NMR (CDCl3, 500 MHz): 210.5, 170.3, 167.4, 140.5, 129.0, 128.3, 126.0, 97.8, 79.1, 76.9, 71.1, 52.0, 45.9, 43.9, 43.5, 39.9, 36.3, 35.1, 33.1, 32.3, 31.9, 29.1, 27.9, 27.1, 25.8, 25.1, 23.4, 22.1, 21.1, 20.0, 17.0, 16.6, 11.5, 8.9 ppm.

[0475] MS (ESI): calculated for (C36H55NO7+H)+ 614.4057, found 614.4066.

Synthesis of F24

Synthesis of (S)-1-(2-((2R,3R,6S)-6-((2R,3R,4S,6E,9E,11R,12R)-12-benzyl-3,11-dihydroxy-4-methyl-5-oxotetradeca-6,9-dien-2-yl)-2-hydroxy-3-methyltetrahydro-2H-pyran-2-yl)-2-oxoacetyl)piperidine-2-carboxylic acid C-11 Lactone

[0476] ##STR00072##

[0477] To a solution of F3 (24.2 mg, 36.7 mol) in ethyl acetate (1 mL) under nitrogen was added 10% Pd/C (12 mg, 50% w/w). The flask was charged with hydrogen and the suspension was stirred at room temperature for 30 min. The hydrogen was replaced with nitrogen and the reaction mixture was then filtered through celite. The filtrate was concentrated under vacuum to give 24 mg of crude product, of which, a portion was purified using Method A to afford the tetrahydro WDB-003 as a white solid (11 mg, 47.8%). TLC: (50/50 heptane/ethyl acetate) Rf=0.45.

[0478] .sup.1H NMR (400 MHz, C.sub.6D.sub.6, 1:0.3 mixture of rotamers, asterisk (*) denotes peaks associated with the minor isomer) 7.25-7.0 (m, 5H), 6.18* (s, 1H), 5.33-5.28 (m, 2H), 5.11* (d, J=12 Hz, 1H), 4.92* (m, 1H), 4.45* (d, J=12 Hz, 1H), 4.24 (m, 1H), 4.06* (td, J=8 Hz, 1H), 3.40 (dd, J=4 Hz, 1H), 3.88 (t, J=8 Hz, 1H), 3.65 (d, J=12 Hz, 1H), 3.30 (td, J=12 Hz, 1H), 3.02* (td, J=12, 4 Hz, 1H), 2.84* (m, 1H), 2.74 (dd, 16, 8 Hz, 1H), 2.65 (q, 8 Hz, 1H), 2.61-2.48 (m, 2H), 2.38-2.09 (m, 5H), 1.73-1.54 (m, 6H), 1.47-1.02 (m, 28H), 0.90 (m, 4H), 0.80 (t, J=8 Hz, 3H), 0.73* (t, J=8 Hz, 3H) ppm.

[0479] .sup.13C NMR (400 MHz, C.sub.6D.sub.6) : 212.59, 197.51, 170.64, 166.70, 140.93, 129.39, 128.77, 128.17, 127.94, 126.43, 99.30, 76.54, 72.64, 71.61, 52.46, 51.24, 46.46, 45.30, 41.62, 40.82, 36.20, 35.31, 32.09, 29.96, 29.47, 27.67, 26.17, 25.71, 24.92, 22.57, 21.78, 21.59, 16.59, 13.68, 11.49, 10.37 ppm. MS (ESI): calculated for (C.sub.36H.sub.53NO.sub.8+Na).sup.+ 650.37, found 650.3.

Synthesis of F25

Synthesis of (2S)-1-((4R,7S)-7-((2R,3R,4S,11 S,12R)-12-benzyl-3,11-dihydroxy-4-methyl-5-oxotetradecan-2-yl)-2-hydroxy-4-methyl-3-oxooxepane-2-carbonyl)piperidine-2-carboxylic acid C-11 Lactone

[0480] ##STR00073##

[0481] tert-Butyldimethylsilyl trifluoromethanesulfonate (6.9 L, 30.1 mol) was added by syringe to an ice-cooled solution of (S)-1-(2-((2R,3R,6S)-6-((2R,3R,4S,6E,9E,11R,12R)-12-benzyl-3,11-dihydroxy-4-methyl-5-oxotetradeca-6,9-dien-2-yl)-2-hydroxy-3-methyltetrahydro-2H-pyran-2-yl)-2-oxoacetyl)piperidine-2-carboxylic acid C-11 lactone-F24 (18.8 mg, 30.1 L) and triethylamine (4.0 L, 30.1 L) in dichoromethane (2 mL) under nitrogen. The resulting solution was stirred at 0 C. for 15 min and was then allowed to warm to room temperature for 2 h. The reaction was cooled to 0 C. and a second portion of triethylamine (4.0 L, 30.1 L) and tert-butyldimethylsilyl trifluoromethanesulfonate (6.9 L, 30.1 mol) was added. The reaction was again allowed to warm to room temperature and stirred under nitrogen 16 h. Dichloromethane (10 mL) and 0.5 M aqueous sodium bicarbonate solution (10 mL) were added and the organic layer was separated and washed with 5% brine solution (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated in vacuo. The crude product was purified using method A to afford the starting material as a white solid (2.52 mg) and the title compound (4.51 mg) as a white solid.

[0482] .sup.1H NMR (400 MHz, C.sub.6D.sub.6) 7.26-7.16 (m, 4H), 7.07 (tt, J=6.4, 2 Hz, 1H), 5.58 (s, 1H), 5.39 (d, J=4.8 Hz, 1H), 5.17 (m, 1H), 4.67 (d, J=12.4 Hz, 1H), 4.29 (d, J=10.8 Hz, 1H), 3.42 (m, 1H), 3.06 (td, J=11.2, 2.8 Hz, 1H), 2.92 (t, J=10 Hz, 1H), 2.85 (m, 1H), 2.75 (s, 1H), 2.66 (dd, J=14, 5.6 Hz, 1H), 2.52 (dd, J=14, 9.2 Hz, 1H), 2.35 (m, 1H), 2.28 (m, 1H), 1.84 (m, 2H), 1.68-1.59 (m, 2H), 1.46-1.06 (m, 23H), 0.88 (m, 4H), 0.82 (t, 3H, J=7.2 Hz) ppm.

[0483] .sup.13C NMR (400 MHz, C.sub.6D.sub.6) : 226.15, 210.53, 209.8, 179.03, 167.59, 140.84, 129.40, 128.77, 128.18, 127.9, 126.45, 98.16, 79.21, 76.85, 70.48, 52.20, 46.07, 44.46, 43.88, 42.76, 36.67, 35.30, 35.14, 32.88, 30.53, 27.94, 25.49, 25.32, 24.57, 22.39, 21.36, 20.72, 16.98, 15.16, 11.68, 9.08 ppm.

[0484] MS (ESI): calculated for (C.sub.36H.sub.53NO.sub.8+Na).sup.+ 650.37, found 650.3.

Example 5. Synthesis of Cyclosporine Analogs

[0485] General Protocol

[0486] Over 2,000 analogs of cyclosporine have been made using solution-phase peptide synthesis, for example according to the method of Li et al. J. Org. Chem 2000(65), 2951. In the amino acid sequence of cyclosporine: cyclo-(D-Ala.sup.8-MeLeu.sup.9-MeLeu.sup.10-MeVal.sup.11-MeLeu.sup.1-Nva.sup.2-Sar.sup.3-MeLeu.sup.4-Val.sup.5-MeLeu.sup.6-Ala.sup.7), the polypeptide stretch from D-Ala.sup.8 to Sar.sup.3 can be considered the constant region responsible for the majority of binding with cyclophilin A. Therefore cyclosporine analogues that preserve cyclophilin binding can be made by synthesis of a tetrapeptide surrogate for the MeLeu.sup.4-Val.sup.5-MeLeu.sup.6-Ala.sup.7 fragment, then elongation and cyclization.

[0487] In a specific example for the synthesis of cyclo-(D-Ala.sup.8-MeLeu.sup.9-MeLeu.sup.10-MeVal.sup.11-MeLeu.sup.1-Nva.sup.2-Sar.sup.3-Gly.sup.4-Gly.sup.5-Gly.sup.6-Gly.sup.7), Fmoc-Gly-OH and Ala-OBzl are coupled in the presence of 2,6-lutidine and BDMP (5-(1H-benzotriazol-1-yloxy)-3,4-dihydro-1-methyl 2H-pyrrolium hexachloroantimonate) to yield Fmoc-Gly-Gly-OBzl. Removal of the Fmoc group with diethylamine followed by coupling with Fmoc-Gly-OH (promoted by BDMP) yields Fmoc-Gly-Gly-Gly-OBzl. Another iteration of Fmoc removal and Fmoc-Gly-OH coupling yields Fmoc-Gly-Gly-Gly-Gly-OBzl. This suitably protected tetrapeptide can be elaborated to the cyclosporine analog cyclo-(D-Ala.sup.8-MeLeu.sup.9-MeLeu.sup.10-MeVal.sup.11-MeLeu.sup.1-Nva.sup.2-Sar.sup.3-Gly.sup.4-Gly.sup.5-Gly.sup.6-Gly.sup.7) in accordance with the methods provided by Li et al.

Example 6. Synthesis of Cyclic Peptide Compounds of the Invention

[0488] General Protocol

[0489] The general method described by Ishizawa et al. J. Am. Chem. Soc. 2013(135), 5433 can be used. A synthetic constant region is prepared wherein the ends terminate in a carboxylic acid, and a (2-chloroacetamido)-acylated amine (in either orientation). Subsequently, a peptidic variable region is prepared using standard Fmoc solid phase peptide synthesis (SPPS), starting from Fmoc-Gly-Wang resin. A cysteine residue is incorporated at an internal position so as to effect later macrocyclization. The linear polypeptide is coupled with the synthetic constant region, then cleaved from the resin using trifluoroacetic acid. To promote macrocyclization, the peptide is treated with triethylamine in DMSO.

Example 7. Binding of Compounds to Cyclophilin A

[0490] The binding of compounds of the invention to Cyclophilin A can be determined using the following protocol.

[0491] General Protocol

[0492] This protocol utilizes Perkin Elmers AlphaLISA technology platform to detect cyclosporine analogues by measuring the inhibition of binding of biotinylated Cyclosporin A to FLAG tagged Cyclophilin A.

[0493] Reagents:

[0494] 10TBST Buffer (Boston BioProducts IBB-181), Biotinylated Cyclosporin A (in-house), FLAG tagged Cyclophilin A (in-house); anti-FLAG Donor beads (PerkinElmer AS103) and Streptavidin Acceptor beads (PerkinElmer AL125); Compounds in DMSO (in-house), Cyclosporin A (LC Labs Cat #C-6000).

[0495] Equipment:

[0496] Biotek Synergy2, Janus MTD Head pipettor, Eppendorf Repeat Pipettor

[0497] Supplies:

[0498] White 96-well Corning area plates (Cat #3642), 96-well polypropylene full skirt (180 ul) PCR plates, 96-well Viaflow P20 tips for Janus MTD Head pipettor.

[0499] Experimental Protocol/Description of Assay:

[0500] Add 20 uL of 6 nM Biotinylated CsA working stock to each well of the 96-well plate. Add 1 uL of test compound (100% DMSO) to each well of the plate using the Janus MTD head and P20 tips (except control wells). Add 1 uL of DMSO to negative control wells and 1 uL of 500 uM Cyclosporin A solution to positive control wells. In the dark, add 20 uL of combined Donor/Acceptor beads to each well. Incubate in the dark for 30 minutes at room temperature. In the dark, add 10 uL of 25 nM Flag tagged CypA working stock to each well. Incubate in the dark for 60 minutes at room temperature. Protect plate from light until reading on Biotek Synergy2 Plate Reader; Alphalisa 96-well protocol (680 excitation/615 emission).

[0501] Results:

[0502] The binding affinity of 104 cyclosporine analogs for cyclophilin A was determined as shown in Table 5.

TABLE-US-00009 TABLE 5 Cyclophilin A Binding of Cyclosporine Analogs IC50 value % inhibition of of CsA CsA binding to binding to # % signal Cyclophilin A Cyclophilin A C1 0.56 99.44 <2 uM C2 5.25 94.75 <2 uM C3 13.43 86.57 <2 uM C4 4.12 95.88 <2 uM C5 9.09 90.91 <2 uM C6 7.29 92.71 <2 uM C7 2.65 97.35 <2 uM C8 9.44 90.56 <2 uM C9 0.11 99.89 <2 uM C10 0.56 99.44 <2 uM C11 6.27 93.73 <2 uM C12 3.22 96.78 <2 uM C13 2.70 97.30 <2 uM C14 9.36 90.64 <2 uM C15 2.93 97.07 <2 uM C16 2.56 97.44 <2 uM C17 17.17 82.83 <2 uM C18 2.12 97.88 <2 uM C19 16.79 83.21 <2 uM C20 2.77 97.23 <2 uM C21 31.08 68.92 <2 uM C22 5.97 94.03 <2 uM C23 1.28 98.72 <2 uM C24 6.65 93.35 <2 uM C25 0.37 99.63 <2 uM C26 3.92 96.08 <2 uM C27 5.27 94.73 <2 uM C28 1.06 98.94 <2 uM C29 1.23 98.77 <2 uM C30 0.06 99.94 <2 uM C31 0.03 100.03 <2 uM C32 0.14 99.86 <2 uM C33 0.08 100.08 <2 uM C34 0.01 100.01 <2 uM C35 0.89 99.11 <2 uM C36 2.08 97.92 <2 uM C37 1.56 98.44 <2 uM C38 1.57 98.43 <2 uM C39 3.78 96.22 <2 uM C40 0.40 99.60 <2 uM C41 0.73 99.27 <2 uM C42 1.26 98.74 <2 uM C43 0.29 99.71 <2 uM C44 0.07 100.07 <2 uM C45 0.03 100.03 <2 uM C46 1.04 98.96 <2 uM C47 4.29 95.71 <2 uM C48 15.72 84.28 <2 uM C49 19.41 80.59 <2 uM C50 6.06 93.94 <2 uM C51 3.38 96.62 <2 uM C52 7.40 92.60 <2 uM C53 0.01 100.01 <2 uM C54 0.90 99.10 <2 uM C55 32.88 67.12 <2 uM C56 2.44 97.56 <2 uM C57 0.15 99.85 <2 uM C58 3.28 96.72 <2 uM C59 11.76 88.24 <2 uM C60 52.68 47.32 <2 uM C61 14.38 85.62 <2 uM C62 27.12 72.88 <2 uM C63 4.64 95.36 <2 uM C64 28.50 71.50 <2 uM C65 0.27 99.73 <2 uM C66 10.95 89.05 <2 uM C67 40.86 59.14 <2 uM C68 20.46 79.54 <2 uM C69 0.03 99.97 <2 uM C70 0.05 99.95 <2 uM C71 0.05 100.05 <2 uM C72 2.78 97.22 <2 uM C73 0.01 99.99 <2 uM C74 4.92 95.08 <2 uM C75 3.88 96.12 <2 uM C76 5.09 94.91 <2 uM C77 0.44 99.56 <2 uM C78 1.42 98.58 <2 uM C79 3.24 96.76 <2 uM C80 3.35 96.65 <2 uM C81 0.76 99.24 <2 uM C82 0.77 99.23 <2 uM C83 0.65 99.35 <2 uM C84 6.81 93.19 <2 uM C85 0.87 99.13 <2 uM C86 17.23 82.77 <2 uM C87 4.53 95.47 <2 uM C88 5.94 94.06 <2 uM C89 3.10 96.90 <2 uM C90 6.46 93.54 <2 uM C91 5.17 94.83 <2 uM C92 9.00 91.00 <2 uM C93 1.28 98.72 <2 uM C94 3.71 96.29 <2 uM C95 6.90 93.10 <2 uM C96 5.00 95.00 <2 uM C97 6.42 93.58 <2 uM C98 4.70 95.30 <2 uM C99 2.59 97.41 <2 uM C100 4.28 95.72 <2 uM C101 0.15 99.85 <2 uM C102 0.70 99.30 <2 uM C103 13.65 86.35 <2 uM C104 0.99 99.01 <2 uM

Example 8. Binding of Compounds to FKBP12

[0503] The binding of compounds of the invention to FKBP12 can be determined using the following protocol.

[0504] General Protocol

[0505] This protocol utilizes Perkin Elmers AlphaLISA technology platform to detect FKBP binders by measuring the inhibition of binding of biotinylated FK506 to FLAG tagged FKBP12.

[0506] Reagents:

[0507] 10TBST Buffer (Boston BioProducts IBB-181), Biotinylated FK506 (in-house), FLAG tagged FKBP (in-house); anti-FLAG Donor beads (PerkinElmer AS103) and Streptavidin Acceptor beads (PerkinElmer AL125); Compounds in DMSO (in-house), FK506.

[0508] Equipment:

[0509] Biotek Synergy2, Janus MTD Head pipettor, Eppendorf Repeat Pipettor

[0510] Supplies:

[0511] White 96-well Corning area plates (Cat #3642), 96-well polypropylene full skirt (180 ul) PCR plates, 96-well Viaflow P20 tips for Janus MTD Head pipettor.

[0512] Experimental Protocol/Description of Assay:

[0513] Add 20 uL of 12.5 nM FKBP-FLAG working stock to each well of the 96-well plate. Add 1 uL of test compound (100% DMSO) to each well of the plate using the Janus MTD head and P20 tips (except control wells). Add 1 uL of DMSO to negative control wells and 1 uL of 50 uM FK506 solution to positive control wells. In the dark, add 20 uL of combined Donor/Acceptor beads to each well. Incubate in the dark for 30 minutes at room temperature. In the dark, add 10 uL of 5 nM biotinylated FK506 working stock to each well. Incubate in the dark for 60 minutes at room temperature. Protect plate from light until reading on Biotek Synergy2 Plate Reader; Alphalisa 96-well protocol (680 excitation/615 emission).

[0514] Results:

[0515] The FKBP12 binding for selected compounds was determined as shown in Table 6.

TABLE-US-00010 TABLE 6 FKBP12 Binding Binding affinity to FKBP12 # (by displacement of FK506) F1 355 nM F2 1.5 nM F3 0.34 nM F4 4.8 nM F5 18.8 nM F6 60 nM F7 26 nM F8 10.6 F9 0.3 nM F10 21.1 nM F11 0.51 nM F12 36.3 nM F13 33.6 nM F14 1800 nM F15 1930 nM F16 67.5 nM F17 0.26 nM F18 229 nM F19 0.22 nM F20 0.95 nM F21 1.1 nM F22 785 nM F23 4690 nM F24 0.21 nM

Example 9. SPR Protocol to Measure Binding of a Compound to FKBP12

[0516] This protocol utilizes Surface Plasmon Resonance (SPR) as a method to determine kinetics (K.sub.D, K.sub.a, K.sub.d) for the binding of compound (analyte) to immobilized FKBP12 (ligand).

[0517] Reagents:

[0518] Compound in 100% DMSO (in-house), 10HBS-P+ buffer (GE Healthcare BR-1006-71), Assay buffer (1HBS-P+ buffer, 1% DMSO), 12HIS tagged FKBP12 (in-house).

[0519] Equipment:

[0520] BIACORE X100 (GE Healthcare)

[0521] Supplies:

[0522] NTA Sensor chip (GE Healthcare BR-1000-34)

[0523] Experimental Protocol:

[0524] Experiments are performed at 25 C. Stock solution of 12HIS tagged FKBP12 is diluted to 100 nM in assay buffer (1% DMSO final). Approximately 500-600 RU of FKBP12 is immobilized on one of two flow cells of an activated NTA chip. The second flow cell is not activated as a reference for non-specific interaction of the analyte to the sensor chip. Various concentrations of compound (1 nM-1 M range), serially diluted into the same assay buffer (1% DMSO final), are injected onto the FKBP12 surface and reference surface at a flow rate of 10 l/min. The surface is regenerated between analyte injections with 350 mM EDTA.

[0525] Data Fitting:

[0526] The BiaEvaluation software program is used for data fitting. All data is reference subtracted against both the reference flow cell and a buffer injection. For kinetic analyses, data is locally fit to a 1:1 interaction model.

TABLE-US-00011 TABLE 7 FKBP12 Binding Data SPR affinity to # FKBP12: K.sub.D F1 71.5 nM F2 12.6 nM F3 1.3 nM F4 23.1 nM F5 7 nM F6 141.2 nM F7 120 nM F8 66 nM F9 0.23 nM F10 17.2 nM F11 21.5 nM F12 21.2 nM F13 105.7 nM F16 290 nM F17 0.2 nM

Example 10. Determination of Binding of F2 and F11 to FKBP12 by SPR

[0527] This protocol utilizes Surface Plasmon Resonance (SPR) as a method to determine kinetics (K.sub.D, K.sub.a, K.sub.d) for the binding of F2 and F11 (analyte) to immobilized FKBP12 (ligand).

[0528] Reagents:

[0529] F2 and F11 in 100% DMSO (in-house), 10HBS-P+ buffer (GE Healthcare BR-1006-71), Assay buffer (1HBS-P+ buffer, 1% DMSO), 12HIS tagged FKBP12 (in-house).

[0530] Equipment:

[0531] BIACORE X100 (GE Healthcare)

[0532] Supplies:

[0533] NTA Sensor chip (GE Healthcare BR-1000-34)

[0534] Experimental Protocol:

[0535] Experiments are performed at 25 C. Stock solution of 12HIS tagged FKBP12 is diluted to 100 nM in assay buffer (1% DMSO final). Approximately 500-600 RU of FKBP12 is immobilized on one of two flow cells of an activated NTA chip. The second flow cell is not activated as a reference for non-specific interaction of the analyte to the sensor chip. Various concentrations of F2 or F11 (1 nM-1 M range), serially diluted into the same assay buffer (1% DMSO final), are injected onto the FKBP12 surface and reference surface at a flow rate of 10 l/min. The surface is regenerated between analyte injections with 350 mM EDTA.

[0536] Data Fitting:

[0537] The BiaEvaluation software program is used for data fitting. All data is reference subtracted against both the reference flow cell and a buffer injection. For kinetic analyses, data is locally fit to a 1:1 interaction model.

[0538] Results:

[0539] The values for binding of F2 to FKBP12 are: K.sub.a (1/Ms): 4.5010.sup.4; K.sub.d (1/S): 5.9410.sup.4; and K.sub.D: 13.2 nM.

[0540] The values for binding of F11 to FKBP12 are: K.sub.a (1/Ms): 5.6710.sup.5; K.sub.d (1/s): 8.810.sup.3; and K.sub.D: 15.6 nM.

Example 11. Determination of Cell Permeability of Compounds

[0541] Cell permeability of compounds can be determined using the following protocol.

[0542] General Protocol

[0543] This protocol utilizes a modified FKBP or cyclophilin destabilizing mutant to determine the bioactivity of FKBP binding compounds or cyclophilin binding compounds in whole cell assay.

[0544] Reagents:

[0545] DMEM, DMEM without Phenol Red, 10% FBS, 1 Sodium Pyruvate, 1 Glutamax. Add 125 l of media with compound per well.

[0546] Equipment:

[0547] Biotek Synergy2, Janus MTD Head pipettor, Eppendorf Repeat Pipettor

[0548] Supplies:

[0549] White 96-well Corning area plates (Cat #3642), 96-well polypropylene full skirt (180 ul) PCR plates, 96-well Viaflow P20 tips for Janus MTD Head pipettor.

[0550] Experimental Protocol/Description of Assay:

[0551] Plate HeLa-FKBP12 cells (for FKBP binding compounds) or HeLa-CyclophilinA cells (for cyclophilin binding compounds) and seed overnight at 5 k/well (approximately 18 hrs.). Using a multi-channel pipet, take out the old media and add 125 ul of new media with compounds. Compounds are diluted using DMEM without Phenol Red, 10% FBS, 1 Sodium Pyruvate, 1 Glutamax. Add 125 L of media with compound per well. Cells are treated with compounds at concentration: 30, 10, 3.33, 1.11, 0.37, 0.12, 0.04 and 0.013 uM. Time points are taken at 72 hrs and plate read using plate reader with excitation/emission: 575/620.

[0552] Calculation:

[0553] Cell binding/permeability is calculated in fold-change (Total RFU of treated samples/total RFU of DMSO treated samples or total RFU above background (Total RFU minus total RFU of DMSO treated samples).

[0554] Results:

[0555] Cell permeability data was gathered for selected compounds as shown in Table 8.

TABLE-US-00012 TABLE 8 Biosensor Permeability Biosensor Permeability # IC50 value C33 <3uM C34 <3uM C35 >3uM C36 <3uM C37 >3uM C38 <3uM C39 <3uM C40 <3uM C41 >3uM C42 >3uM C43 <3uM C44 <3uM F2 <1uM F4 <1uM F6 <1uM F9 <1uM F10 >1uM F12 >1uM F13 >1uM F14 >1uM F16 >1uM F17 <1uM F18 <1uM F19 <1uM F20 <1uM F21 <1uM F22 >1uM F23 >1uM

Example 12. Binding of a Presenter Protein/Compound Complex to a Target Protein

[0556] The binding of a presenter protein/compound complex of the invention to a target protein can be determined using the following protocol.

[0557] General Protocol for Cyclophilin A Complexes

[0558] This protocol utilizes Perkin Elmers AlphaLISA technology platform to detect cyclosporine analogues by measuring the binding of 6HIS tagged target protein+FLAG tagged Cyclophilin A and cyclosporine compound.

[0559] Reagents:

[0560] 10TBST Buffer (Boston BioProducts IBB-181), MgCl.sub.2 (Sigman), 6HIS tagged target protein (in-house), FLAG tagged Cyclophilin A (in-house); anti-FLAG Donor beads (PerkinElmer AS103) and Streptavidin Acceptor beads (PerkinElmer AL125); Compounds in DMSO (in-house), Cyclosporin A (LC Labs Cat #C-6000).

[0561] Equipment:

[0562] Biotek Synergy2, Janus MTD Head pipettor, Eppendorf Repeat Pipettor.

[0563] Supplies:

[0564] White 96-well Corning area plates (Cat #3642), 96-well polypropylene full skirt (180 ul) PCR plates, 96-well Viaflow P20 tips for Janus MTD Head pipettor.

[0565] Experimental Protocol/Description of Assay:

[0566] Add 20 uL of 250 nM 6HIS tagged target protein working stock to each well of the 96-well plate. Add 1 uL of test compound (100% DMSO) to each well of the plate using the Janus MTD head and P20 tips (except control wells). Add 1 uL of DMSO to control wells. In the dark, add 20 uL of combined Donor/Acceptor beads to each well. Incubate in the dark for 30 minutes at room temperature. In the dark, add 10 uL of 10 uM Flag tagged CypA working stock to each well. Incubate in the dark for 60 minutes at room temperature. Protect plate from light until reading on Biotek Synergy2 Plate Reader; Alphalisa 96-well protocol (680 excitation/615 emission).

[0567] General Protocol for FKBP12 Complexes

[0568] This protocol utilizes Perkin Elmers AlphaLISA technology platform to detect compounds by measuring the binding of 6HIS tagged target protein+FLAG tagged FKBP12 and FKBP binding compound.

[0569] Reagents:

[0570] 10TBST Buffer (Boston BioProducts IBB-181), MgCl.sub.2 (Sigman), 6HIS tagged target protein (in-house), FLAG tagged FKBP12 (in-house); anti-FLAG Donor beads (PerkinElmer AS103) and Streptavidin Acceptor beads (PerkinElmer AL125); Compounds in DMSO (in-house), FK506.

[0571] Equipment:

[0572] Biotek Synergy2, Janus MTD Head pipettor, Eppendorf Repeat Pipettor.

[0573] Supplies:

[0574] White 96-well Corning area plates (Cat #3642), 96-well polypropylene full skirt (180 ul) PCR plates, 96-well Viaflow P20 tips for Janus MTD Head pipettor.

[0575] Experimental Protocol/Description of Assay:

[0576] Add 20 uL of 250 nM 6HIS tagged target protein working stock to each well of the 96-well plate. Add 1 uL of test compound (100% DMSO) to each well of the plate using the Janus MTD head and P20 tips (except control wells). Add 1 uL of DMSO to control wells. In the dark, add 20 uL of combined Donor/Acceptor beads to each well. Incubate in the dark for 30 minutes at room temperature. In the dark, add 10 uL of 10 uM Flag tagged FKBP12 working stock to each well. Incubate in the dark for 60 minutes at room temperature. Protect plate from light until reading on Biotek Synergy2 Plate Reader; Alphalisa 96-well protocol (680 excitation/615 emission).

Example 13. Determination of Binding of Presenter Protein/Compound Complexes to Target Proteins by SPR

[0577] This protocol utilizes Surface Plasmon Resonance (SPR) as a method to determine kinetics (K.sub.D, K.sub.a, K.sub.d) for the binding of mammalian target protein (analyte) to immobilized FKBP12-compound binary complex (ligand).

[0578] Reagents:

[0579] Compound in 100% DMSO (in-house), 10HBS-P+ buffer (GE Healthcare BR-1006-71), Assay buffer (1HBS-P+ buffer, 1% DMSO, 1 M F2), 12HIS tagged FKBP12 (in-house), mammalian target protein (in-house).

[0580] Equipment:

[0581] BIACORE X100 (GE Healthcare) Supplies:

[0582] NTA Sensor chip (GE Healthcare BR-1000-34)

[0583] Experimental Protocol:

[0584] Experiments are performed at 25 C. Stock solution of 12HIS tagged FKBP12 is diluted to 100 nM in assay buffer containing 1 M compound (1% DMSO final). Approximately 200-400 RU of FKBP12 is immobilized on one of two flow cells of an activated NTA chip. The second flow cell is not activated as a reference for non-specific interaction of the analyte to the sensor chip. Various concentrations of target protein (1 nM-1 M range), serially diluted into the same assay buffer containing 1 M compound (1% DMSO final), are injected onto the FKBP12 surface and reference surface at a flow rate of 10 l/min. The surface is regenerated between analyte injections with 350 mM EDTA.

[0585] Data Fitting: The BiaEvaluation software program is used for data fitting. All data is reference subtracted against both the reference flow cell and a buffer injection. For kinetic analyses, data is locally fit to a 1:1 interaction model.

Example 14. Determination of Binding of FKBP12/F2 Complex to CEP250 by SPR

[0586] This protocol utilizes Surface Plasmon Resonance (SPR) as a method to determine kinetics (K.sub.D, K.sub.a, K.sub.d) for the binding of CEP250 (analyte) to immobilized FKBP12-F2 binary complex (ligand).

[0587] Reagents:

[0588] F2 in 100% DMSO (in-house), 10HBS-P+ buffer (GE Healthcare BR-1006-71), Assay buffer (1HBS-P+ buffer, 1% DMSO, 1 M F2), 12HIS tagged FKBP12 (in-house), CEP250.sub.29.2 (residues 1982-2231) and CEP250.sub.11.4 (residues 2134-2231) (in-house).

[0589] Equipment:

[0590] BIACORE X100 (GE Healthcare)

[0591] Supplies:

[0592] NTA Sensor chip (GE Healthcare BR-1000-34)

[0593] Experimental Protocol:

[0594] Experiments are performed at 25 C. Stock solution of 12HIS tagged FKBP12 is diluted to 100 nM in assay buffer containing 1 M F2 (1% DMSO final). Approximately 200-400 RU of FKBP12 is immobilized on one of two flow cells of an activated NTA chip. The second flow cell is not activated as a reference for non-specific interaction of the analyte to the sensor chip. Various concentrations of CEP250 (1 nM-1 M range), serially diluted into the same assay buffer containing 1 M F2 (1% DMSO final), are injected onto the FKBP12 surface and reference surface at a flow rate of 10 l/min. The surface is regenerated between analyte injections with 350 mM EDTA.

[0595] Data Fitting:

[0596] The BiaEvaluation software program is used for data fitting. All data is reference subtracted against both the reference flow cell and a buffer injection. For kinetic analyses, data is locally fit to a 1:1 interaction model.

[0597] Results:

[0598] The k values for the binding of the FKBP12/F2 complex to CEP250.sub.11.4 and CEP250.sub.29.2 are: K.sub.a (1/Ms): 5.7110.sup.5; K.sub.d (1/s): 3.0910.sup.3; and K.sub.D: 5.4 nM and K.sub.a (1/Ms): 3.1110.sup.5; K.sub.d (1/s): 9.2510.sup.5; and K.sub.D: 0.29 nM, respectively.

Example 15. Determination of Binding of Presenter Protein/Compound Complexes to Target Proteins by ITC

[0599] General Protocol

[0600] This protocol utilizes Isothermal Titration Calorimetry (ITC) to directly measure the heat change associated with binding of presenter protein (e.g. FKBP, cyclophilin)-compound binary complexes to target proteins. Measurement of the heat change allows accurate determination of association constants (K.sub.a), reaction stoichiometry (N), and the change in binding enthalpy (H).

[0601] Reagents:

[0602] Compounds in 100% DMSO (in-house), Protein Buffer (10 mM HEPES, pH 7.5, 75 mM NaCl, 0.5 mM TCEP), assay buffer (protein buffer+1% DMSO), presenter protein (e.g. FKBP, cyclophilin) (in-house), target protein (in-house).

[0603] Equipment:

[0604] MicroCal ITC.sub.200 (GE Healthcare)

[0605] Experimental Protocol:

[0606] presenter protein (e.g. FKBP, cyclophilin) stock solution is diluted to 10 M in assay buffer (1% DMSO final). Compound is added to presenter protein to 20 M (1% DMSO final), and binary complex is filled into the reaction cell of the ITC device after 5-10 min pre-incubation time. Target protein stocks are diluted to 50 M in assay buffer and supplemented with 20 M compound (1% DMSO final) before being filled into the injection syringe. A control experiment in the absence of compound is also run to determine the heat associated with operational artifacts and the dilution of titrant as it is injected from the syringe into the reaction cell. Data collection and analysis are as described for binding of FKBP12-F2 and FKBP12-F11 binary complexes to CEP250.

Example 16. Determination of Binding of FKBP12/F2 and FKBP12/F2 Complexes to CEP250 by ITC

[0607] This protocol utilizes Isothermal Titration Calorimetry (ITC) to directly measure the heat change associated with binding of FKBP12-F2 and FKBP12-F11 binary complexes to CEP250. Measurement of the heat change allows accurate determination of association constants (K.sub.a), reaction stoichiometry (N), and the change in binding enthalpy (AH).

[0608] Reagents:

[0609] F2 and F11 in 100% DMSO (in-house), Protein Buffer (10 mM HEPES, pH 7.5, 75 mM NaCl, 0.5 mM TCEP), assay buffer (protein buffer+1% DMSO), FKBP12 (in-house), CEP250.sub.29.4 (residues 1982-2231) and CEP250.sub.11.4 (residues 2134-2231) (in-house).

[0610] Equipment:

[0611] MicroCal ITC2.sub.00 (GE Healthcare)

[0612] Experimental Protocol:

[0613] FKBP12 stock solution is diluted to 10 M in assay buffer (1% DMSO final). Compound is added to FKBP12 to 20 M (1% DMSO final), and binary complex is filled into the reaction cell of the ITC device after 5-10 min pre-incubation time. CEP250 protein stocks are diluted to 50 M in assay buffer and supplemented with 20 M compound (1% DMSO final) before being filled into the injection syringe. A control experiment in the absence of compound is also run to determine the heat associated with operational artifacts and the dilution of titrant as it is injected from the syringe into the reaction cell. More detailed experimental parameters are shown in Tables 11 and 12, below:

TABLE-US-00013 TABLE 9 ITC Experimental Parameters Experimental device: MicroCal iT.sub.200 (GE Healthcare) sample cell volume [l] 270 injector volume [l] 40 Experimental parameters Total #of Injections 19 Cell Temperature [ C.] 25 Reference Power [Cal/s] 5 Initial Delay [s] 200 Stirring Speed [rpm] 750 Injection parameters Volume [l] 2 Duration [s] 4 Spacing [s] 170-200 Filter Period [s] 5 Feedback Mode/Gain high

TABLE-US-00014 TABLE 10 Protein and Ligand Concentrations for ITC Final protein and ligand concentrations DMSO assay cell content syringe content ligand conc. [%] FKBP12, 10 M CEP250.sub.29.4, 50 M none 1.0 FKBP12, 10 M CEP250.sub.11.4, 50 M none 1.0 FKBP12, 10 M CEP250.sub.29.4, 118 M F2, 20 M 1.0 FKBP12, 10 M CEP250.sub.29.4, 118 M F11, 20 M 1.0 FKBP12, 10 M CEP250.sub.11.4, 68 M F2, 20 M 1.0 FKBP12, 10 M CEP250.sub.11.4, 68 M F11, 20 M 1.0

[0614] Data Fitting: Data were fitted with the Origin ITC200 software according to the following procedure: [0615] 1) Read raw data [0616] 2) In mRawITC: adjust integration peaks and baseline, integrate all peaks [0617] 3) In Delta H-data control: remove bad data (injection #1 and other artifacts), subtract straight line (background subtraction) [0618] 4) In Delta H-model fitting: select one set of sites model, perform fitting with Levenberg-Marquardt algorithm until Chi Square is not reduced further, finish with done (parameters N, K.sub.a and H are calculated based on fitting)

[0619] ITC measurements for the binding of FKBP12-F2 and FKBP12-F11 binary complexes to CEP250 are summarized in Table 11 below.

TABLE-US-00015 TABLE 11 ITC Measurements H T*S G cell syringe T Kd [kJ*mol [kJ*mol [kJ*mol Experiment content content ligand [K] N [M]* mol1]** mol1]*** mol1]**** 3 FKBP12, CEP25029.4, none 298 ND ND ND ND ND 10 M 118 M 4 FKBP12, CEP25011.4, none 298 ND ND ND ND ND 10 M 68 M 5 FKBP12, CEP25029.4, F2, 298 0.50 0.19 52.21 13.80 38.41 10 M 118 M 20 M 6 FKBP12, CEP25029.4, F11, 298 0.57 0.36 58.48 21.73 36.74 10 M 118 M 20 M 7 FKBP12, CEP25011.4, F2, 298 0.56 0.07 49.37 8.62 40.75 10 M 68 M 20 M 8 FKBP12, CEP25011.4, F11, 298 0.54 0.08 47.78 7.41 40.36 10 M 68 M 20 M *K.sub.d (calculated from K.sub.a = 1/K.sub.d) **H ***T*S (calculated from equation (TS = G H) ****G = RT In K.sub.a = RT In K.sub.d

[0620] Results:

[0621] Overall, the data for FKBP12-F2 and FKBP12-F11 binary complexes binding to CEP250.sub.11.4 and CEP250.sub.29.4 show similar interaction parameters. K.sub.d values were similar for all combinations. All interactions show an almost identical thermodynamic profile in which binding is characterized by a purely enthalpic binding mode (T*S term is positive and does not contribute to the Gibbs free energy). Binding stoichiometries for all interactions were N=0.5-0.6 and support a 1:2 binding ratio for 1 CEP250 homodimer binding to 2 FKBP12 molecules, as evidenced in the crystal structure of CEP250.sub.11.4/F2/FKBP12.

Example 17. Crystallographic Structural Determination of Tertiary Complexes

[0622] General Protocol

[0623] This protocol describes the crystallization and structure determination method for structures of specific FKBP12-compound-target protein ternary complexes.

[0624] Reagents:

[0625] Compound in 100% DMSO (in-house), FKBP12 (in-house), and mammalian target protein (in-house).

[0626] Equipment:

[0627] Superdex 200 (GE Healthcare)

[0628] Experimental Protocol:

[0629] A 3:1 molar excess of compound is added to FKBP12 in 12.5 mM HEPES pH 7.4, 75 mM NaCl buffer, and incubated overnight at 4 C. A 3:1 molar excess of FKBP12-compound binary complex is added to target protein and incubated at 4 C. overnight to complete ternary complex formation. Pure ternary complex is isolated by gel filtration purification on a Superdex 200 column in 12.5 mM HEPES pH 7.4, 75 mM NaCl. Purified complex (at 10-20 mg/ml) is subjected to crystallization at 22 C. using sitting drop vapor diffusion using various buffers, surfactants and salt solutions. For data collection, crystals are transferred to a solution containing mother liquor supplemented with 20-25% glycerol, and then frozen in liquid nitrogen. Diffraction datasets are collected at the Advanced Photon Source (APS) and processed with the HKL program. Molecular replacement solutions are obtained using the program PHASER in the CCP4 suite, using the published structure of FKBP12 (PDB-ID 1FKD) as a search model. Subsequent model building and refinement are performed according to standard protocols with the software packages CCP4 and COOT.

Example 18. Crystallographic Structural Determination of Tertiary Complexes of FKBP12/F2 and FKBP12/F11 Complexes with CEP250

[0630] This protocol describes the crystallization and structure determination method for structures of FKBP12-Compound 2-CEP250 and FKBP12-F11-CEP250 ternary complexes.

[0631] Reagents:

[0632] F2 and F11 in 100% DMSO (in-house), FKBP12 (in-house), and CEP250.sub.11.4 (residues 2134-2231) (in-house).

[0633] Equipment:

[0634] Superdex 200 (GE Healthcare)

[0635] Experimental Protocol:

[0636] A 3:1 molar excess of F2 or F11 is added to FKBP12 in 12.5 mM HEPES pH 7.4, 75 mM NaCl buffer, and incubated overnight at 4 C. A 3:1 molar excess of FKBP12-F2 or FKBP12-F11 binary complex is added to CEP250.sub.11.4 and incubated at 4 C. overnight to complete ternary complex formation. Pure ternary complex is isolated by gel filtration purification on a Superdex 200 column in 12.5 mM HEPES pH 7.4, 75 mM NaCl. Purified complex (at 10-20 mg/ml) is subjected to crystallization at 22 C. using sitting drop vapor diffusion. FKBP12-F2-CEP250 crystals grow in a well solution containing 0.2 M sodium malonate, 0.1 M HEPES 7.0, 21% PEG 3350. FKBP12-F11-CEP250 crystals grow in a well solution containing 0.1 M Tris pH 8.5, 0.2 M trimethylamine N-oxide, 22-24% PEG2000 MME. For data collection crystals are transferred to a solution containing mother liquor supplemented with 20-25% glycerol, and then frozen in liquid nitrogen. Diffraction datasets are collected at the Advanced Photon Source (APS) and processed with the HKL program. Molecular replacement solutions are obtained using the program PHASER in the CCP4 suite, using the published structure of FKBP12 (PDB-ID 1FKD) as a search model. Subsequent model building and refinement are performed according to standard protocols with the software packages CCP4 and COOT.

[0637] Results:

[0638] Overall structure of FKBP12-F2-CEP250: In the structure of FKBP12 with CEP250 in complex with F2, two FKBP12 monomers are bound to a homodimer of CEP250. The two CEP250 monomers form a coiled-coil structure. There are four hetero dimers on the asymmetric unit with basically the same overall conformation. The model comprises residues Met1 to Glu108 of FKBP12 and Asp2142 to His2228 of CEP250. The electron density shows an unambiguous binding mode for the ligand F2, including the orientation and conformation of the ligand.

[0639] The CEP250 residues involved in binding F2 are L2190, Q2191, V2193, A2194, M2195, F2196, L2197, and Q2198. The CEP250 residues involved in binding to FKBP12 are A2185, S2186, S2189, Q2191, M2195, Q2198, V2201, L2202, R2204, D2205, S2206, Q2208, Q2209, and Q2212.

[0640] The total buried surface area of the ternary complex is 1759 .sup.2. The total buried surface area of CEP250 is 865 .sup.2 of which 663 .sup.2 is contributed by FKBP12 and 232 .sup.2 is contributed by F2.

[0641] 100% of the binding interactions in the ternary complex between F2 and CEP250 are van der Waals or pi-pi interactions. By comparison, 100% of the binding interactions between rapamycin and mTOR are van der Waals or pi-pi interactions, and 89% of the binding interactions between FK506 and calcineurin are van der Waals or pi-pi interactions while 11% are hydrogen bonds (two H-bonds from C13 and C15 OMe to Trp 352 NH).

[0642] Overall structure of FKBP12-F11-CEP250: In the structure of FKBP12 with CEP250 in complex with F11, one FKBP12 monomer is bound to a homodimer of CEP250. The two CEP250 monomers form a coiled-coil structure. The crystals contain a heterotrimer (one FKBP12 and two CEP250) in the asymmetric unit. The model comprises residues Met1 to Glu108 of FKBP12 and Ser2143 to His2228 of CEP250. One short loop region of FKBP12 (18-19) is not fully defined by electron density and is not included in the model. The electron density shows an unambiguous binding mode for the ligand F11, including the orientation and conformation of the ligand.

[0643] The CEP250 residues involved in binding F2 are L2190, Q2191, V2193, A2194, M2195, F2196, L2197, and Q2198. The CEP250 residues involved in binding to FKBP12 are Q2182, A2185, S2186, S2189, Q2191, M2195, Q2198, V2201, L2202, R2204, D2205, S2206, Q2208, Q2209, and Q2212.

[0644] The total buried surface area of the ternary complex is 1648 .sup.2. The total buried surface area of CEP250 is 831 .sup.2 of which 590 .sup.2 is contributed by FKBP12 and 241 .sup.2 is contributed by F2.

Statistics of the final structures are listed in Table 12 and 13 below.

TABLE-US-00016 TABLE 12 FKBP12-F2-CEP250 Ligand F2 Resolution [] 136.05-2.20 Number of reflections (working/test) 50564/2723 R.sub.cryst [%] 20.8 R.sub.free [%].sup.2 25.6 Total number of atoms: Protein 6146 Water 226 Ligan 176 PEG 273 Magnesium 1 Maltose 7 Deviation from ideal geometry: .sup.3 Bond lengths [] 0.007 Bond angles [] 1.17 Bonded B's [.sup.2].sup.4 5.4 Ramachandran plot: .sup.5 Most favoured regions [%] 94.6 Additional allowed regions [%] 4.8 Generously allowed regions [%] 0.6 Disallowed region [%] 0.0 .sup.1Values as defined in REFMAC5, without sigma cut-off .sup.2Test-set contains 2.4% of measured reflections .sup.3 Root mean square deviations from geometric target values .sup.4Calculated with MOLEMAN .sup.5 Calculated with PROCHECK

TABLE-US-00017 TABLE 13 FKBP12-F11-CEP250 Ligand F11 Resolution [] 72.84-2.10 Number of reflections (working/test) 16398/877 R.sub.cryst [%] 24.2 R.sub.free [%].sup.2 29.9 Total number of atoms: Protein 2224 Water 31 Ligand 44 PEG 14 Deviation from ideal geometry: .sup.3 Bond lengths [] 0.008 Bond angles [] 1.07 Bonded B's [.sup.2].sup.4 3.0 Ramachandran plot: .sup.5 Most favoured regions [%] 93.5 Additional allowed regions [%] 4.9 Generously allowed regions [%] 1.6 Disallowed region [%] 0.0 .sup.1Values as defined in REFMAC5, without sigma cut-off .sup.2Test-set contains 5.1% of measured reflections .sup.3 Root mean square deviations from geometric target values .sup.4Calculated with MOLEMAN .sup.5 Calculated with PROCHECK

Example 19. Synthesis of Compounds Including Cross-Linking Groups

Synthesis of Acrylamide-Containing Cyclosporine Analogs

[0645] ##STR00074##

[0646] All reagents and solvents were purchased from Sinopharm Chemical Reagent Co. Ltd. Fmoc-amino acids, HATU, HOAT, H-Ala-2-Cl-(Trt) resin (0.36 mmol/g), H-Leu-2-Cl-(Trt) resin (0.30 mmol/g), H-Phe-2-Cl-(Trt) resin (0.35 mmol/g) and H-Thr(tBu)-2-Cl-(Trt) resin (0.36 mmol/g) were purchased from GL Biochem (Shanghai) Ltd.

[0647] The coupling of linear peptides was carried out using standard Fmoc SPSS procedure on automated synthesizer.

General Method A:

[0648] The linear peptides were synthesized by using a TETRAS synthesizer with a scale of 0.025 mmol of resin. A general protocol is as follows: 2NMP, 30 s; 120% (vol/vol) piperidine in NMP, 15 min; 5NMP, 30 s; amino acids (3 eq) solution in NMP was added into the vessel containing resin followed by the addition of a solution of HATU and DIEA in DMF respectively, coupling for 45 min; 3NMP, 30 s. A double coupling strategy was applied for all the amino acids.

General Method B: Attaching of Boc-7mer

[0649] The coupling was accomplished on TETRAS synthesizer by using the same general protocol as for amino acid coupling except that the amount of Boc-7mer is 1.5 equivalents. Only one coupling was needed.

General Method C: Removal of ivDde Protecting Group.

[0650] The removal of ivDde protecting group on Dap side chain was accomplished on TETRAS synthesizer. General protocol: A solution of 20% (vol/vol) hydrazine monohydrate in NMP was added in the vessel containing the resin. The vessel was shook for 30 min. The resin was drained and rinsed with 55 mL (30 s) of NMP.

General Method D: Attaching of Acrylic Acid on Side-Chain Amino Group of Dap.

[0651] The coupling was accomplished on TETRAS synthesizer by using the same general protocol as for amino acid coupling. A double coupling strategy was applied.

General Method E: Deprotection of Side Chain Protecting Groups and Final Cleavage from Resin.

[0652] Global deprotection and cleavage from resin was achieved by TFA cocktails for 1-2 hr at room temperature. The cleavage cocktails (TFA/TIPS/H.sub.2O, 95/2.5/2.5) or (TFA/DCM/TIPS, 40:60:1) can be used for the final cleavage. Most solvent was removed under reduced pressure and the residue was concentrated under vacuum to remove the trace solvent. The resulting residue was used in the next cyclization directly without further purification.

General Method F: Cyclization of Linear Peptides

[0653] The crude linear peptide was dissolved in dry DCM to get the final concentration of 0.1 M. Then HATU (3 eq), HOAt (3 eq) and DIEA (6 eq) was added. The reaction mixture was stirred overnight and then monitored by ESI-LCMS. The solvent was concentrated under reduced pressure and the residue was dissolved in NMP and purified by preparative HPLC. Cyclo-peptides were identified by ESI-LCMS.

Reversed-Phase HPLC.

[0654] Accucore C18 column (2.6 m, 2.1 mm50 mm) with a 1 mL/min flow rate was used for analytical RP-HPLC. Xselect Peptide CSH column (5 um, 19 mm150 mm) was used for preparative RP-HPLC. Mobile phase A: water (0.1% formic acid), Mobile phase B: ACN; Flow rate: 20 mL/min; Gradient: 25% B to 95% B in 16 min.

Synthesis of Cyclo[7mer-NMALA-Dap-d-nmala-LEU]:

##STR00075##

[0655] The linear peptide-chain assembly was performed using the general methods described above with 850 mg of H-Leu-2-Cl-(Trt) resin (0.3 mmol/g, 0.025 mmol scale). D-N-methyl Ala, Dap and L-N-methyl Ala residue were attached using general method A. Then Boc-7mer was assembled with general method B. The removal of side-chain ivDde protecting group was accomplished by using general method C. Then acrylic acid was attached on the free amino group on the side chain of Dap with general method D. After global deprotection and cleavage from the resin in one step (general method E), the crude linear peptide was submitted to the cyclization (general method F). After final preparative HPLC, 17 mg of final compound was obtained as a white solid (56.6% calculated by resin loading). ESI-MS: [M+1].sup.+=1202, [M+Na].sup.+=1224, [M/2+1].sup.+=602.

Synthesis of Cyclo[7mer-NMALA-Dap-d-nmala-PHE]:

##STR00076##

[0656] 2.8 mg of final compound was obtained as a white solid (9.1% calculated by resin loading) with the similar method as the synthesis of cyclo[7mer-NMALA-Dap-d-nmala-LEU]. ESI-MS: [M+1].sup.+=1236, [M+Na].sup.+=1258, [M/2+1].sup.+=619.

Synthesis of Cyclo[7mer-NMALA-Dap-d-nmala-THR]:

##STR00077##

[0657] 3.4 mg of final compound was obtained as a white solid (11.4% calculated by resin loading) with the similar method as the synthesis of cyclo[7mer-NMALA-Dap-d-nmala-LEU]. ESI-MS: [M+1].sup.+=1190, [M+Na].sup.+=1212, [M/2+1].sup.+=596.

Synthesis of Acrylamide-Containing FKBP12 Ligands

[0658] ##STR00078##

Boc-3mer

[0659] All reagents and solvents were purchased from Sinopharm Chemical Reagent Co. Ltd. Fmoc-amino acids, HATU, HOAT, 2-Cl-(Trt)-Cl resin (0.9 mmol/g based on active site) and Ala loaded 2-Cl-(Trt)-Cl resin (0.36 mmol/g) were purchased from GL Biochem (Shanghai) Ltd.

[0660] To attach the first amino acid on 2-Cl-(Trt)-Cl resin.

[0661] In a vessel for SPSS, 5 g of resin was swelled in 50 mL of dry NMP for 40 min. Drain the resin and rinse it with DCM (530 mL) then with NMM 2%/DCM (330 mL). A solution of Fmoc-Dap(ivDde)-OH (3.0 mmol, 1.6 g) with NMM (4 mmol, 400 mg) in 50 mL of DCM was added. The vessel was shook overnight. Then 2 mL of a solution of 25% NMM/MeOH was added and the vessel was shook for one more hour. The resin was drained and rinsed with DCM, NMP, MeOH and EtOH (350 mL each). The resin was dried under vacuum at room temperature.

[0662] The loading was measure by Fmoc cleavage photometric measurement (0.32 mmol/g). The coupling of linear peptides was carried out using standard Fmoc SPSS procedure on an automated synthesizer.

General Method A:

[0663] The linear peptides were synthesized by using a TETRAS synthesizer with a scale of 0.025 mmol of resin. A general protocol as follow: 2NMP, 30 s; 120% (vol/vol) piperidine in NMP, 15 min; 5NMP, 30 s; amino acids (3 eq) solution in NMP was added into the vessel containing resin followed by the addition of a solution of HATU and DIEA in DMF respectively, coupling for 45 min; 3NMP, 30 s. A double coupling strategy was applied for all the amino acids.

General method B: Attaching of Boc-3mer

[0664] The coupling was accomplished on TETRAS synthesizer by using the same general protocol as for amino acids coupling except that the amount of Boc-3mer is 1.5 equivalents. Only once coupling was needed.

General Method C: Removal of ivDde Protecting Group.

[0665] The removal of ivDde protecting group on Dap side chain was accomplished on TETRAS synthesizer. General protocol: A solution of 20% (vol/vol) hydrazine monohydrate in NMP was added in the vessel containing the resin. The vessel was shook for 30 min. The resin was drained and rinsed with 55 mL (30 s) of NMP.

General Method D: Attaching of Acrylic Acid on Side-Chain Amino Group of Dap.

[0666] The coupling was accomplished on TETRAS synthesizer by using the same general protocol as for amino acids coupling. A double coupling strategy was applied.

General Method E: Deprotection of Side Chain Protecting Groups and Final Cleavage from Resin.

[0667] Global deprotection and cleavage from resin was achieved by TFA cocktails for 1-2 hr at room temperature. The cleavage cocktails (TFA/TIPS/H.sub.2O, 95/2.5/2.5) or (TFA/DCM/TIPS, 40:60:1) can be used for the final cleavage. Most solvent was removed under reduced pressure and the residue was concentrated under vacuum to remove the trace solvent. The resulting residue was used in the next cyclization directly without further purification.

General Method F: Cyclization of Linear Peptides

[0668] The crude linear peptide was dissolved in dry DCM to get the final concentration of 0.1 M. Then HATU (3 eq), HOAt (3 eq) and DIEA (6 eq) was added. The reaction mixture was stirred overnight and then monitored by ESI-LCMS. The solvent was concentrated under reduced pressure and the residue was dissolved in NMP and purified by preparative HPLC. Cyclo-peptides were identified by ESI-LCMS.

Reversed-Phase HPLC.

[0669] Accucore C18 column (2.6 m, 2.1 mm50 mm) with a 1 mL/min flow rate was used for analytical RP-HPLC. Xselect Peptide CSH column (5 um, 19 mm150 mm) was used for preparative RP-HPLC. Mobile phase A: water (0.1% formic acid), Mobile phase B: ACN; Flow rate: 20 mL/min; Gradient: 25% B to 95% B in 16 min.

Synthesis of Cyclo[diamine-Dap-ALA-ALA]:

[0670] ##STR00079##

[0671] The linear peptide-chain assembly was performed using the general methods described above with 700 mg of H-Ala-2-Cl-(Trt) resin (0.025 mmol scale). Ala and Dap residue was attached by using general method A. Then Boc-3mer was assembled with general method B. The removal of side-chain ivDde protecting group was accomplished by using general method C. Then acrylic acid was attached on the free amino group on the side chain of Dap with general method D. After global deprotection and cleavage from the resin in one step (general method E), the crude linear peptide was submitted to the cyclization (general method F). After final preparative HPLC, 3.1 mg of final compound was obtained as a white solid (14.5% calculated by resin loading). ESI-MS: [M+1].sup.+=857, [M+Na].sup.+=879.

Synthesis of Cyclo[diamine-ALA-ALA-Dap]

[0672] ##STR00080##

[0673] 2.2 mg of final compound was obtained as a white solid (10.3% calculated by resin loading) with the similar method as the synthesis of cyclo[diamine-Dap-ALA-ALA]. ESI-MS: [M+1].sup.+=857, [M+Na].sup.+=879.

Synthesis of Cyclo[diamine-Dap-ALA]:

[0674] ##STR00081##

[0675] 2.7 mg of final compound was obtained as a white solid (12.6% calculated by resin loading) with the similar method as the synthesis of cyclo[diamine-Dap-ALA-ALA]. ESI-MS: [M+1].sup.+=786.

Synthesis of Sanglefehrin Analogs:

[0676] ##STR00082##

[0677] Method A may be used to prepare compounds of Formula IV as shown below in Scheme 1.

##STR00083##

where R.sup.7, R.sup.8, Z.sup.5, and Z.sup.6 are as previously defined, PG.sup.1 is a suitable amine protecting group including but not limited to Boc, Cbz, Alloc, and Fmoc, and X is either OH, Cl, F, or some other group suitable for displacement or activation followed by displacement.

[0678] In a typical procedure, protected amine IV is reacted with an appropriate reagent familiar to those skilled in the art to remove the protecting group PG.sup.1. The isolated crude product may be reacted with acylating agent Z.sup.5C(O)X in the presence of standard coupling agents (e.g., EDC/HOBT, DCC, PyBOP, PyBROP, HATU, HBTU, COMU) known to those skilled in the art. The acid and amine coupling partners are combined with the coupling agent in an organic solvent (including but not limited to DMF, dichloromethane, acetonitrile, and tetrahydrofuran) in the presence of a base (including but not limited to DIPEA, triethylamine, and NMM) at room temperature or slightly elevated temperature.

[0679] Alternatively, the deprotected amine may be directly reacted with the acylating reagent Z.sup.5C(O)X when X is a halogen in a suitable solvent (including but not limited to DMF, dichloromethane, DME, acetonitrile, and tetrahydrofuran) in the presence of base (including but not limited to pyridine, DIPEA, triethylamine, and NMM) in the temperature range of 78 C. to about 120 C., preferably between 20 C. and 50 C.

[0680] Compounds of Formula IVb of Scheme 1 may be prepared as shown below in Scheme 2.

##STR00084##

where R.sup.7, R.sup.8, and Z.sup.6 are as previously defined and PG.sup.1 and PG.sup.2 are each independently suitable amine protecting groups including but not limited to Boc, Cbz, Alloc, and Fmoc.

[0681] In a typical procedure, protected amine IVc is reacted with the appropriate reagent familiar to those skilled in the art to remove the protecting group PG.sup.2 to produce the corresponding amine, which is then reacted with the appropriate amino acid in the presence of standard coupling agents (e.g., EDC/HOBT, DCC, PyBOP, PyBROP, HATU, HBTU, COMU) known to those skilled in the art. The acid and amine coupling partners are combined with the coupling agent in an organic solvent (including but not limited to DMF, dichloromethane, acetonitrile, and tetrahydrofuran) in the presence of a base (including but not limited to DIPEA, triethylamine, and NMM) at room temperature or slightly elevated temperature to deliver compounds of Formula IVb.

[0682] Compounds of Formula IVc of Scheme 2 may be prepared as shown below in Scheme 3.

##STR00085##

[0683] where R.sup.7 and Z.sup.6 are as previously defined and PG.sup.2 is a suitable amine protecting group including but not limited to Boc, Cbz, Alloc, and Fmoc.

[0684] In a typical procedure, protected amine IVd is reacted with the piperazic acid derivative IVe in the presence of standard coupling agents (e.g., EDC/HOBT, DCC, PyBOP, PyBROP, HATU, HBTU, COMU) known to those skilled in the art. The acid and amine coupling partners are combined with the coupling agent in an organic solvent (including but not limited to DMF, dichloromethane, acetonitrile, and tetrahydrofuran) in the presence of a base (including but not limited to DIPEA, triethylamine, and NMM) at room temperature or slightly elevated temperature.

[0685] Alternatively, compounds of Formula IVb may be synthesized from compounds of formula II-B as described in Scheme 4.

##STR00086##

[0686] where R.sup.7, R.sup.8, and Z.sup.6 are as previously defined, PG.sup.1 is a suitable amine protecting group including but not limited to Boc, Cbz, Alloc, and Fmoc, and PG.sup.3 is an alkyl or aryl group included but not limited to methyl, ethyl, benzyl, allyl, phenyl, and the like that can be removed by methods known to those skilled in the art.

[0687] In a typical procedure, compound IVe is reacted at room temperature under hydrolysis conditions using base (e.g., lithium hydroxide, sodium hydroxide, sodium carbonate) in a solvent system comprised of organic (e.g., methanol, THF, dioxane) with or without water. It will be appreciated by those skilled in the art that, depending on the identity of PG.sup.3, removal of PG.sup.3 can also occur under hydrogenolysis conditions using an appropriate catalyst, or under de-allylation conditions using a palladium catalyst, for example Pd(PPh.sub.3).sub.4 and a basic scavenger (e.g., piperidine, morpholine, piperidine) Following deprotection to give the resulting carboxylic acid, Z.sup.6 may be introduced in the presence of standard coupling agents (e.g., EDC/HOBT, DCC, PyBOP, PyBROP, HATU, HBTU, COMU) known to those skilled in the art. The carboxylic acid and the coupling partners are combined with the coupling agent in an organic solvent (including but not limited to DMF, dichloromethane, acetonitrile, and tetrahydrofuran) in the presence of a base (including but not limited to DIPEA, triethylamine, and NMM) at room temperature or slightly elevated temperature to give product IVb.

[0688] Alternatively, compounds of Formula IVb may be synthesized as shown below in Scheme 5.

##STR00087##

[0689] where R.sup.7, R.sup.8, and Z.sup.6 are as previously defined and PG.sup.1 is a suitable amine protecting group including but not limited to Boc, Cbz, Alloc, and Fmoc.

[0690] In a typical procedure, protected amine VI is reacted with reagent V in the presence of standard coupling agents (e.g., EDC/HOBT, DCC, PyBOP, PyBROP, HATU, HBTU, COMU) known to those skilled in the art. The acid and amine coupling partners are combined with the coupling agent in an organic solvent (including but not limited to DMF, dichloromethane, acetonitrile, and tetrahydrofuran) in the presence of a base (including but not limited to DIPEA, triethylamine, NMM) at room temperature or slightly elevated temperature.

Synthesis of Intermediate I-8

[0691] ##STR00088## ##STR00089## ##STR00090##

[0692] To a room temperature solution of Intermediate I-1 (2.00 g, 7.11 mmol) in DMF (13 m) was added cesium carbonate (4.75 g, 14.58 mmol) and benzyl bromide (2.49 g, 14.58 mmol, 1.73 mL) at 25 C. The reaction mixture was stirred for two hours and then diluted with ethyl acetate (100 mL) and washed with brine (50 mL3). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a crude residue. The crude product was purified by column chromatography with silica gel (eluent: petroleum ether/ethyl acetate.fwdarw.20:1 to 10:1) to afford Intermediate I-2 (3.20 g, 96% yield) as colorless oil. .sup.1H NMR (400 MHz, CDCl.sub.3) 7.45-7.30 (m, 10H), 7.20-7.10 (m, 1H), 6.95-6.85 (m, 1H), 6.74 (s, 1H), 6.70-6.60 (m, 1H), 5.20-5.10 (m, 2H), 4.99 (s, 2H), 4.70-4.60 (m, 1H), 3.15-3.05 (m, 2H), 1.43 (s, 9H). ESI-MS m/z=484.1 [M+Na].sup.+; Calculated MW: 461.55.

[0693] To a solution of Intermediate I-2 (3.20 g, 6.93 mmol) in tetrahydrofuran (15 mL) was added lithium hydroxide (1 M in water, 10 mL) at 0 C. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was adjusted to pH6 with HCl (1 M in water) at 0 C. and extracted with ethyl acetate (100 mL3). The combined organic layer was washed with brine (20 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure to give a crude residue. The crude product was dissolved in aqueous sodium bicarbonate (7 mL) and extracted with MTBE (100 mL3). The aqueous layer was adjusted to pH6 with hydrochloric acid (1 M in H.sub.2O) and extracted with ethyl acetate (50 mL3). The combined organic layers were washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to afford Intermediate I-3 (2.27 g, 88% yield) as a colorless oil. .sup.1HNMR (400 MHz, CDCl.sub.3) 7.45-7.30 (m, 5H), 7.25-7.18 (m, 1H), 6.90-6.85 (m, 1H), 6.83-6.75 (m, 2H), 5.05 (s, 2H), 4.94 (d, J=8.00 Hz, 1H), 4.60-4.50 (m, 1H), 3.20-3.12 (m, 1H), 3.10-3.00 (m, 1H), 1.43 (s, 9H). ESI-MS m/z=394.3 [M+Na].sup.+; Calculated MW: 371.43.

[0694] To a solution of Intermediate I-3 (888 mg, 2.39 mmol) in dichloromethane (15 mL) was added N-methyl morpholine (967 mg, 9.56 mmol), HOBt (65 mg, 478 umol), (S)-methyl hexahydropyridazine-3-carboxylate as the TFA salt (890 mg, 2.39 mmol), and EDCI (917 mg, 4.78 mmol) at 0 C. The reaction mixture was stirred at 0 C. for 1 hour. The reaction mixture was diluted with dichloromethane (50 mL) and adjusted to pH6 with 5% aqueous citric acid. The aqueous layer was extracted with dichloromethane (20 mL2). The combined organic layers were washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a crude product. The crude product was purified by column chromatography with silica gel (eluent: petroleum ether/ethyl acetate.fwdarw.10:1, 5:1 to 3:1) to afford Intermediate I-4 (910 mg, 77% yield) as colorless oil. .sup.1HNMR (400 MHz, CDCl.sub.3) 7.50-7.35 (m, 5H), 7.20-7.13 (m, 1H), 6.90-6.80 (m, 3H), 5.60-5.50 (m, 1H), 5.30-5.20 (m, 1H), 5.05-5.00 (m, 2H), 4.40-4.30 (m, 1H), 3.65 (s, 3H), 3.55 (d, J=11.20 Hz, 1H), 3.00-2.93 (m, 1H), 2.90-2.80 (m, 1H), 2.75-2.65 (m, 1H), 2.35-2.25 (m, 1H), 1.80-1.72 (m, 2H), 1.42 (s, 9H). ESI-MS m/z=520.1 [M+Na].sup.+. Calculated MW: 497.58.

[0695] To a solution Intermediate I-4 (450 mg, 904 umol) in ethyl acetate (4 mL) was added hydrochloric acid in ethyl acetate (4 M, 8.00 mL) at 0 C. The reaction mixture was stirred at 25 C. for 1 hour. The reaction mixture was concentrated under reduced pressure to afford the HCl salt of Intermediate I-5 (390 mg, 100% yield) as light yellow solid and used for next step without purification. ESI-MS m/z=398.0 [M+H].sup.+, 420.0 [M+Na].sup.+, Calculated MW: 397.47.

[0696] To a solution of Intermediate I-5 (195 mg, 899 umol) in dichloromethane (5.00 mL) was added N-methylmorpholine (273 mg, 2.70 mmol), HOBt (24.29 mg, 179.75 umol), the HCl salt of (S)-methyl 1-((S)-2-amino-3-(3-(benzyloxy)phenyl)propanoyl)hexahydropyridazine-3-carboxylate (390 mg, 899 umol) and EDCI (345 mg, 1.80 mmol) at 0 C. The reaction mixture was stirred at 0 C. for 1 hour. The reaction mixture was diluted with dichloromethane (20 mL) and adjusted to pH6 with 5% aqueous critic acid. The aqueous layer was extracted with dichloromethane (20 mL2). The combined organic layers were washed with brine (20 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a crude product. The crude product was purified by column chromatography with silica gel (eluent: petroleum ether/ethyl acetate.fwdarw.5:1, 2:1, 1:1) to afford Intermediate I-6 (750 mg, 70% yield) as a white solid. .sup.1H NMR (400 MHz, CDCl.sub.3) 7.50-7.35 (m, 5H), 7.23-7.15 (m, 1H), 6.90-6.80 (m, 3H), 6.13-6.08 (m, 1H), 5.83-5.73 (m, 1H), 5.10-5.00 (m, 3H), 4.30-4.20 (m, 1H), 4.00-3.90 (m, 1H), 3.65 (s, 3H), 3.50 (d, J=11.20 Hz, 1H), 3.05-2.97 (m, 1H), 2.93-2.83 (m, 1H), 2.80-2.70 (m, 1H), 2.35-2.25 (m, 1H), 2.15-2.10 (m, 1H), 1.75-1.65 (m, 4H), 1.45 (s, 9H), 0.94 (d, J=6.80 Hz, 3H), 0.88 (d, J=6.80 Hz, 3H). ESI-MS m/z=597.1 [M+H].sub.+. Calculated MW: 596.71.

[0697] To a solution of Intermediate I-6 (3.00 g, 6.03 mmol) in methanol (300 mL) was added palladium on carbon (2 grams, 10% loading) under a nitrogen atmosphere. The suspension was degassed and purged with hydrogen gas and the mixture was stirred under hydrogen (1 atm) at 20 C. for 3 hours. The mixture was filtered and concentrated under reduced pressure to give Intermediate 1-7 (2.3 g, 5.64 mmol, 94% yield) as a white solid. ESI-MS m/z=430.1 [M+Na].sup.+. Calculated MW: 407.46.

[0698] To a mixture of Intermediate I-7 (2.3 g, 5.64 mmol) in dioxane (10 mL) was added hydrochloric acid in dioxane (4 M, 30 mL) in one portion at 20 C. The mixture was stirred at 20 C. for 3 hours. The mixture was adjusted to pH 7-8 with saturated aqueous NaHCO.sub.3 and extracted with ethyl acetate (100 mL3). The combined organic phase was washed with brine (100 mL2), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to give Intermediate I-8 (1.3 g, 3.63 mmol, 64% yield, 86% purity) as a light yellow solid. .sup.1H NMR (400 MHz, CDCl.sub.3) 7.15-7.02 (m, 1H), 6.75-6.5 (m, 3H), 4.90-4.77 (m, 1H), 4.50-4.37 (m, 1H), 4.00-3.90 (m, 1H), 3.80-3.70 (m, 1H), 3.68-3.65 (m, 3H), 2.95-2.80 (m, 1H), 2.77-2.62 (m, 2H), 2.50-2.35 (m, 1H), 1.97-1.85 (m, 1H), 1.82-1.70 (m, 1H), 1.60-1.45 (m, 1H), 1.42-1.28 (m, 1H). ESI-MS m/z=308.2 [M+Na].sup.+. Calculated MW: 307.34.

Synthesis of Intermediate I-11

[0699] ##STR00091##

[0700] To a solution of 2-(dimethylamino)ethanethiol I-9 (500 mg, 3.53 mmol) in methanol (10 mL) was added a solution of 1,2-di(pyridin-2-yl)disulfide (1.17 g, 5.3 mmol) in methanol (10 mL) at 0 C. The mixture was stirred at 25 C. for 15 hours. The reaction mixture was concentrated in vacuo. The residue was purified by silica gel column (eluent: petroleum ether/ethyl acetate.fwdarw.3:1, 1:1, then dichloromethane/ethyl acetate.fwdarw.2:1, then dichloromethane/methanol.fwdarw.10:1) which was combined with a previous batch to afford Intermediate I-10 (1.05 g, 58% yield) as light yellow solid. .sup.1H NMR (400 MHz, CD.sub.3OD) 8.56 (d, J=4.0 Hz, 1H), 7.85-7.75 (m, 1H), 7.69 (d, J=8.0 Hz, 1H), 7.36-7.26 (m, 1H), 3.48-3.40 (m, 2H), 3.28-3.20 (m, 2H), 2.93 (s, 6H). ESI-MS m/z=214.9 [M+H]+. Calculated MW: 214.35.

[0701] To a solution of 4-mercaptobutanoic acid (50 mg, 416 umol) in methanol (1 mL) was added a solution of Intermediate I-10 (125 mg, 499 umol) in methanol (1.5 mL) at 25 C. The mixture was stirred at 25 C. for 15 hours. The reaction mixture was concentrated at low pressure. The residue was purified by column chromatography with silica gel (eluent: petroleum ether/ethyl acetate.fwdarw.2:1, 1:1, then dichloromethane/methanol.fwdarw.10:1) to afford Intermediate I-11 (80 mg, 86% yield) as a white gum. .sup.1H NMR (400 MHz, CD.sub.3OD) 3.55-3.45 (m, 2H), 3.08-3.00 (m, 2H), 2.93 (s, 6H), 2.83 (t, J=7.2 Hz, 2H), 2.43 (t, J=7.2 Hz, 2H), 2.05-1.94 (m, 2H).

Synthesis of Intermediate I-15

[0702] ##STR00092##

[0703] To a solution of tert-butyl 2-mercaptoacetate I-12 (800 mg, 5.4 mmol) in N,N-dimethylformamide (15 mL) was added potassium carbonate (1.49 g, 10.8 mmol) and 1-bromo-2-chloroethane (2.32 g, 16.2 mmol). The mixture was stirred at 25 C. for 2 hours. The mixture was diluted with ethyl acetate (100 mL) and washed with water (50 mL3). The organic layer was dried over anhydrous sodium sulfate and concentrated to give Intermediate I-13 (1.00 g, 79% yield) as a colorless oil. .sup.1H NMR (400 MHz, CDCl.sub.3) 3.64-3.71 (m, 2H), 3.14-3.17 (m, 2H), 2.95-3.01 (m, 2H), 1.47 (s, 9H).

[0704] To a solution of Intermediate I-13 (1.00 g, 4.75 mmol) in dichloromethane (10 mL) was added a solution of m-CPBA (4.61 g, 21.4 mmol, 80% purity) in dichloromethane (10 mL) at 0 C. The mixture was warmed to room temperature stirred for 2 hours. The mixture was diluted with dichloromethane (80 mL) and washed with saturated aqueous sodium sulfite (50 mL) and saturated aqueous sodium bicarbonate (50 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated to give a crude residue that was purified by silica gel chromatography (eluent: petroleum ether/ethyl acetate.fwdarw.5/1) to give Intermediate I-14 (700 mg, 60% yield) as a colorless oil. .sup.1H NMR (400 MHz, CDCl.sub.3) 3.99 (s, 2H), 3.90-3.95 (m, 2H), 3.69-3.75 (m, 2H), 1.50-1.53 (m, 9H).

[0705] To a solution of Intermediate I-14 (650 mg, 2.68 mmol) in dichloromethane (12 mL) was added trifluoroacetic acid (6.00 mL). The mixture was stirred at 45 C. for 2 hours. Then the mixture was concentrated to give Intermediate I-15 (360.00 mg, 72% yield) as a white solid. .sup.1H NMR (400 MHz, DMSO-d6) 4.34 (s, 2H), 3.89-4.01 (m, 2H), 3.71-3.81 (m, 2H).

Synthesis of Compound-1

[0706] ##STR00093##

[0707] To a solution of the HCl salt of Intermediate I-8 (31 mg, 119 umol) and Intermediate I-11 (44 mg, 99 umol) in dichloromethane (1.5 mL) was added HOBt (1.34 mg, 9.9 umol), N-methylmorpholine (38.2 L) and EDCI (27 mg, 139 umol). The mixture was stirred at 25 C. for one hour. The reaction mixture was diluted with dichloromethane (10 mL) and washed with water (5 mL). The aqueous layer was extracted with dichloromethane (5 mL2). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by reverse phase HPLC (HCl additive) and repurified using reverse phase HPLC (HCOOH additive) and lyophilized to afford Compound-1 (5.8 mg, 9% yield) as a light yellow oil. .sup.1H NMR (400 MHz, MeOD) 8.53 (br.s., 1H), 7.12-7.04 (m, 1H), 6.72-6.62 (m, 3H), 5.75-5.65 (m, 1H), 4.20-4.10 (m, 2H), 3.70 (s, 3H), 3.08-3.00 (m, 2H), 2.95-2.76 (m, 5H), 2.76-2.70 (m, 2H), 2.60 (s, 6H), 2.42-2.32 (m, 3H), 2.10-1.98 (m, 3H), 1.85-1.70 (m, 2H), 1.55-1.40 (m, 2H), 1.02-0.85 (m, 6H). ESI-MS m/z=612.1 [M+H].sup.+, 634.5 [M+Na].sup.+. Calculated MW: 611.82.

Synthesis of Compound-2

[0708] ##STR00094##

[0709] A solution of Intermediate I-8 (27 mg, 60 umol) in dichloromethane was treated with N,N-diisopropylethylamine (24 mg, 180 umol), Intermediate I-16 (14 mg, 60 umol), HOBt (1.6 mg, 2 umol) and then finally EDCI (18 mg, 90 umol). After stirring for 15 hours, the solution was poured into water and ethyl acetate. The layers were separated and the aqueous layer was extracted with ethyl acetate. The organics were dried over magnesium sulfate, filtered, and the solvent removed was in vacuo. Purification by reverse phase HPLC (acetonitrile in water with 0.1% formic acid) and lyophilization provided Compound-2 (16 mg, 42% yield) as a white solid. .sup.1H NMR (400 MHz, CDCl.sub.3) 8.47 (d, 1H), 8.16 (br s, 1H), 7.69-7.66 (m, 1H), 7.63-7.60 (m, 1H), 7.13 (app t, 1H), 7.09-7.05 (m, 1H), 5.85-5.79 (m, 1H), 4.72 (m, 1H), 4.31 (br s, 1H), 3.70 (s, 3H), 3.42 (d, 1H), 3.00 (dd, 1H), 2.87-2.78 (m, 3H), 2.52-2.43 (m, 2H), 2.28 (br s, 1H), 2.14-2.04 (m, 3H), 1.83-1.73 (m, 2H), 1.62-143 (m, 5H), 0.95 (d, 3H), 0.90 (d, 3H). ESI-MS m/z=617.9 [M+H].sup.+. Calculated MW: 617.78.

Synthesis of Compound-3

[0710] ##STR00095##

[0711] To a solution of the HCl salt of Intermediate I-15 (31.8 mg, 0.17 mmol), HOBt (2.3 mg, 0.017 mmol) and NMM (54 L, 0.54 mmol) in DCM (0.75 mL) was added the HCl salt of 1-8 (86 mg, 0.17 mmol,) and EDCI (76 mg, 0.34 mmol) at room temperature. The reaction mixture was stirred at room temperature for 2 hours. The mixture was diluted with dichloromethane (2 mL), washed with citric acid (pH3, 2 mL), saturated aqueous sodium bicarbonate (2 mL), and saturated aqueous sodium chloride (2 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure at 15 C. to give a product as yellow oil. The residue was purified by prep-TLC with silica gel (eluent: DCM/MeOH.fwdarw.10:1) followed by preparative HPLC (column: Phenomenex Gemini C18 25050 10 u; mobile phase: [water (0.225% FA)-ACN]; B %: 26%-56%, 11.2 min) to afford Compound-3 (8.5 mg, 8% yield) as white solid. .sup.1H NMR (400 MHz, CDCl.sub.3) 9.02 (s, 1H), 8.80 (s, 1H), 8.42 (s, 1H), 7.01-7.08 (m, 1H), 6.82 (dd, J=16.3, 9.9 Hz, 1H), 6.74 (d, J=7.5 Hz, 1H), 6.64-6.70 (m, 2H), 6.36 (d, J=16.5 Hz, 1H), 6.12-6.16 (m, 2H), 4.81-4.85 (m, 1H), 4.40-4.46 (m, 2H), 4.21-4.25 (m, 1H), 4.09-4.13 (m, 1H), 3.57 (s, 3H), 2.81-3.08 (m, 2H), 2.63-2.65 (m, 1H), 2.15-2.19 (m, 1H), 1.21-1.60 (m, 4H), 0.88-0.92 (m, 6H). ESI-MS m/z=539.1 [M+H]+. Calculated MW: 538.61.

Synthesis of Compound-4

[0712] ##STR00096##

[0713] Compound-4 was prepared as described in the preparation of Compound-3 by using the HCl salt of Intermediate I-8 (34 mg, 77 umol) and 2-(3-methyl-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetic acid 1-17 (13 mg, 77 umol) as starting materials. The resulting crude product was combined with previously synthesized crude material and purified to give Compound-4 (29.0 mg, 51.28 umol, 45% yield) as a white solid following lyophilization. .sup.1H NMR (400 MHz, CDCl.sub.3) 8.55-8.40 (s, 1H), 8.20-8.07 (m, 1H), 7.10-6.97 (m, 2H), 6.80-6.73 (m, 1H), 6.73-6.63 (m, 1H), 6.63-6.50 (m, 1H), 6.47-6.37 (m, 1H), 5.90-5.75 (m, 1H), 4.80-4.67 (m, 1H), 4.35-4.15 (m, 3H), 3.70-3.57 (m, 3H), 3.45-3.30 (d, 1H), 3.10-3.00 (m, 1H), 3.00-2.70 (m, 2H), 2.20-2.00 (m, 5H), 1.85-1.60 (m, 2H), 1.60-1.45 (m, 1H), 1.45-1.30 (m, 1H), 1.05-0.80 (m, 6H). ESI-MS m/z=558.3 [M+H]+; Calculated MW: 557.60.

Synthesis of Compound-5

[0714] ##STR00097##

[0715] Compound-5 was prepared as described in the preparation of Compound-3 by using the HCl salt of Intermediate I-8 (34 mg, 77 umol) and 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetic acid 1-18 (13 mg, 77 umol) as starting materials to give Compound-5. ESI-MS m/z=544.0 [M+H]+; Calculated MW: 543.58.

Synthesis of Compound-6

[0716] ##STR00098##

[0717] Compound-6 was prepared starting with Intermediate 1-19 to give Compound-6. ESI-MS m/z=631.0 [M+H].sup.+; Calculated MW: 630.82.

Synthesis of Compounds of Formula IVf

[0718] Synthesis of compounds of Formula IVf may be synthesized as shown below in Scheme 6.

##STR00099##

[0719] where R.sup.7, R.sup.8, and Z.sup.6 are as previously defined, PG.sup.1 is a suitable amine protecting group including but not limited to Boc, Cbz, Alloc, and Fmoc, and PG.sup.4 is an alkyl or aryl group included but not limited to methyl, ethyl, benzyl, allyl, and phenyl that can be removed by methods known to those skilled in the art.

[0720] In a typical procedure, compound IVd is reacted with reagent IVg in the presence of standard coupling agents (e.g., EDC/HOBT, DCC, PyBOP, PyBROP, HATU, HBTU, COMU) familiar to those skilled in the art. The acid and amine coupling partners are combined with the coupling agent in an organic solvent (including but not limited to DMF, dichloromethane, acetonitrile, and tetrahydrofuran) in the presence of a base (including but not limited to DIPEA, triethylamine, and NMM) at room temperature or slightly elevated temperature. Following amide formation, the protecting group PG.sup.4 is then removed using conditions described for removing of PG.sup.3 of compound of formula IVe (Scheme 4).

##STR00100##

[0721] Method B may be used to prepare compounds of Formula IIa as shown below in Scheme 7.

##STR00101##

wherein R.sup.1, R.sup.2, R.sup.3, X.sup.1, X.sup.2, Z.sup.1 and Z.sup.2 are as previously defined.

[0722] In a typical procedure, carboxylic acid IIc is reacted with intermediate XI in the presence of standard coupling agents (e.g., EDC/HOBT, DCC, PyBOP, PyBROP, HATU, HBTU, COMU) known to those skilled in the art. The acid IIc and coupling partner is combined with the coupling agent in an organic solvent (including but not limited to DMF, dichloromethane, DCE, acetonitrile, and tetrahydrofuran) in the presence of a base (including but not limited to DIPEA, triethylamine, and NMM) at room temperature or slightly elevated temperature.

[0723] Compounds of Formula IIc may be synthesized as shown below in Scheme 8.

##STR00102##

where R.sup.1, R.sup.2, R.sup.3, X.sup.1, and Z.sup.2 are as previously defined, PG.sup.5 is an alkyl or aryl group included but not limited to methyl, ethyl, benzyl, allyl, and phenyl that can be removed by methods known to those skilled in the art, and LG.sup.1 is a group such as OH that can be activated and displaced by the amine of compound lie. Alternatively, LG.sup.1 may be an appropriate halide (such as F, Cl, and Br) that can be displaced by a nucleophile.

[0724] In a typical procedure, lie is reacted with IIf (LG.sup.1=halide) in the presence of a suitable base (including but not limited to pyridine, triethylamine, DIPEA, and NMM) in an appropriate solvent (including but not limited to THF, DCM, and DMF) at temperatures ranging from 78 C. to 120 C., but optimally from 20 C. to 50 C. Alternatively, if LG.sup.1 is OH, XII is reacted with reagent IIf in the presence of standard coupling agents (e.g., EDC/HOBT, DCC, PyBOP, PyBROP, HATU, HBTU, COMU) known to those skilled in the art. The acid and amine coupling partners are combined with the coupling agent in an organic solvent (including but not limited to DMF, dichloromethane, acetonitrile, and tetrahydrofuran) in the presence of a base (including but not limited to DIPEA, triethylamine, and NMM) at room temperature or slightly elevated temperature. It will be appreciated by those skilled in the art that compound IIf (LG=halide), may be readily made from the corresponding carboxylic acid by treatment with a suitable halogenating reagent. Following amide formation between IIe and IIf, the protecting group PG.sup.5 is then removed using conditions described for removing of PG.sup.3 of compound of formula IVe (scheme 4) to give compound IIc.

[0725] Method B-2 may be used to prepare compounds of Formula IIa as shown below in Scheme 9.

##STR00103##

where R.sup.1, R.sup.2, R.sup.3, X.sup.1, X.sup.2, Z.sup.1 and Z.sup.2 are as previously defined. The reaction may be conducted under conditions described for coupling reaction between compound IIg and compound IIf (scheme 8) Compounds of Formula IIg may be prepared as shown below in Scheme 11.

##STR00104##

[0726] where R.sup.3, X.sup.1, X.sup.2, Z.sup.1 are as previously defined and PG.sup.6 is a suitable amine protecting group including but not limited to Boc, Cbz, Alloc, and Fmoc.

[0727] In a typical procedure, carboxylic acid IIg is reacted with an appropriate coupling partner containing a X.sup.2Z.sup.1 moiety in the presence of standard coupling agents (e.g., EDC/HOBT, DCC, PyBOP, PyBROP, HATU, HBTU, COMU) known to those skilled in the art. The acid and the coupling partner are combined with the coupling agent in an organic solvent (including but not limited to DMF, dichloromethane, acetonitrile, and tetrahydrofuran) in the presence of a base (including but not limited to DIPEA, triethylamine, and NMM) at room temperature or slightly elevated temperature. PG.sup.6 is then removed by reacting the resulting intermediate with the appropriate reagent familiar to those skilled in the art.

Synthesis of Compound-7

[0728] ##STR00105##

[0729] To a solution of Intermediate I-24 (18 mg, 0.085 mmol) in tetrahydrofuran (1.2 mL) was added N-methylmorpholine (10 L, 0.085 mmol). The solution was cooled to 20 C. and then isobutylchloroformate (11 L, 0.085 mmol) was added dropwise. The solution was immediately warmed to 0 C. After stirring for 45 minutes at 0 C., the TFA salt of Intermediate I-23 (37 mg, 0.057 mmol) was mixed with N-methylmorpholine (7 L, 0.057 mmol) in anhydrous tetrahydrofuran (0.5 mL) and added dropwise to the 0 C. solution of mixed anhydride over the course of 5 minutes. The resulting solution was stirred for 1.5 hours at 0 C., at which point methanol (1 mL) was added, and the solution was immediately warmed to room temperature and stirred for 5 minutes. The solution was diluted with dichloromethane (75 mL) and saturated aqueous sodium bicarbonate (50 mL). The layers were separated and the aqueous layer was extracted with dichloromethane (230 mL). The organics were combined, washed with brine (20 mL), dried over magnesium sulfate, filtered, and the solvent was removed in vacuo. Purification by silica gel chromatography (0-80% ethyl acetate in hexanes) provided Compound 7 as a white foam (20 mg, 50% yield). .sup.1H NMR (400 MHz, CDCl.sub.3) 9.49 (s, 1H), 8.55 (d, 1H), 7.77 (app t, 1H), 7.71-7.65 (m, 2H), 7.60-7.58 (m, 1H), 7.20-7.17 (m, 1H), 7.01 (d, 1H), 6.78-6.76 (m, 1H) 6.70-6.67 (m, 2H), 5.77 (dd, 1H), 5.33 (d, 1H), 3.86 (s, 3H), 3.85 (s, 3H), 3.35 (d, 1H), 3.25 (t, 2H), 3.15 (dt, 1H), 2.92 (t, 2H), 2.64-2.50 (m, 2H), 2.38 (d, 1H), 2.29-2.15 (m, 1H), 2.10-2.00 (m, 1H), 1.78-1.63 (m, 3H), 1.53-1.37 (m, 3H), 1.22 (s, 6H), 0.89 (t, 3H, rotamer 1), 0.80 (t, 3H, rotamer 2). ESI-MS m/z=722.0 [M+H].sup.+, 744.0 [M+Na].sup.+; Calculated MW: 721.93.

Synthesis of Compound-8

[0730] ##STR00106##

[0731] To a solution Compound 7 (22 mg, 0.031 mmol) in dichloromethane (0.5 mL) was added triethylamine (62 L, 0.55 mmol) followed by 2-(dimethylamino)ethanethiol (0.mL, 0.0500 mmol) (4.8 mg, 0.046 mmol). After stirring at room temperature for 30 minutes, the solution was poured into dichloromethane (30 mL) and saturated aqueous sodium bicarbonate (30 mL). The layers were separated and the aqueous layer was extracted with dichloromethane (220 mL). The organics were dried over magnesium sulfate, filtered, and the solvent was removed in vacuo. Purification by silica gel chromatography (0-10% methanol in dichloromethane) provided impure material that was repurified by reverse phase HPLC (acetonitrile in water with 0.1% formic acid) to give the formic acid salt of Compound-8 (7.1 mg, 21% yield) as a white solid. .sup.1H NMR (400 MHz, CDCl.sub.3) (12.53 (br s, 1H), 9.50 (s, 1H), 8.00 (s, 1H), 7.75 (d, 1H), 7.68 (s, 1H), 7.29 (t, 1H), 7.02 (d, 1H), 6.79-6.77 (m, 1H), 6.70-6.67 (m, 2H), 5.76 (dd, 1H), 5.29 (d, 1H), 3.86 (s, 3H), 3.85 (s, 3H), 3.36-3.27 (m, 2H), 3.23-3.11 (m, 4H), 2.90 (t, 2H), 2.79-2.71 (m, 8H), 2.62-2.50 (m, 2H), 2.55 (d, 1H), 2.28-2.17 (m, 1H), 2.12-2.03 (m, 1H), 1.72-1.61 (m, 3H), 1.49-1.36 (m, 3H), 1.22 (s, 3H), 1.21 (s, 3H), 0.88 (t, 3H, rotamer 1), 0.79 (t, 3H, rotamer 2). ESI-MS m/z=716.1 [M+H].sup.+; Calculated MW: 715.97.

Synthesis of Compound-9

[0732] ##STR00107##

[0733] To a room temperature solution of Intermediate 1-23 (15 mg, 0.029 mmol), 3-(2,5-dioxopyrrol-1-yl)propanoic acid (5.8 mg, 0.034 mmol), and DMAP (0.4 mg, 0.003 mmol), in dichloromethane was added N,N-dicyclohexylcarbodiimide (9.4 mg, 0.046 mmol). After stirring at room temperature for 15 hours, the resulting solids were filtered through a syringe filter and washed with dichloromethane. The solvent was removed in vacuo and the resulting residue was brought up in ethyl acetate. After cooling to 20 C., the suspension was filtered through a syringe filter and washed with cold ethyl acetate. The filtrate was diluted with ethyl acetate (30 mL) and water (30 mL). The layers were separated and the aqueous layer was extracted with ethyl acetate (220 mL). The organics were dried over magnesium sulfate, filtered, and the solvent was removed in vacuo. Purification by silica gel chromatography (0-90% ethyl acetate in hexanes) provided Compound-9 (11 mg, 60% yield) as an oil. .sup.1H NMR (400 MHz, CDCl.sub.3) (8.25 (br s, 1H), 7.79 (d, 1H), 7.38 (s, 1H), 7.29 (t, 1H), 6.98 (d, 1H), 6.82 (s, 2H), 6.78-6.76 (m, 1H), 6.70-6.66 (m, 2H), 5.83 (dd, 1H), 5.37 (d, 1H), 4.43 (d, 1H), 4.37 (d, 1H), 3.86 (s, 3H), 3.85 (s, 3H), 3.32 (d, 1H), 2.96 (t, 1H), 2.55 (t, 2H), 2.35 (d, 1H), 2.29-2.15 (m, 1H), 2.11-2.01 (m, 1H), 1.81-1.60 (m, 4H), 1.49-1.42 (m, 3H), 1.27 (s, 3H), 1.25 (s, 3H), 0.93 (t, 3H, rotamer 1), 0.82 (t, 3H, rotamer 2). ESI-MS m/z=662.0 [M+H].sup.+; Calculated MW: 661.75.

[0734] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth.

[0735] All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.