Total Synthesis of (-)-Balanol A potent Protein Kinase C inhibitor and Analogues

1. Total Synthesis of (-)-BalanolA potent Protein Kinase C inhibitorand Analogues

2. Protein Kinase C • Protein Kinase C (PKC) is a family of Ser/Thr specific kinases • Play a role in cellular control • Lipid dependant • Target of phorbol esters (tumor promoters) • Sphinx was formed to exploit PKC • Robotic assays with 9 expressed PKC human isoforms • Natural Products and MedChem groups

3. PKC Down Stream effects

4. PKC & Phorbolester Phorbolester Phorbolester (PE) is a natural product tumor promoter. Although PE is not tumorigenic by itself, it substantially enhances the tumorigenic potency of tumorigenic agents. PKC is the target of PE which acts as a super diacyl glycerol surrogate leading to enhanced levels of activated PKC. The correlation of tumor promoter (PE) and PKC activity suggested that PKC inhibitors might have anti-cancer properties.

5. (-)-Balanol • Natural Products Screening • Verticillium balanoides (fungus) • Very potent inhibitor of PKC's • IC50 4-9 nM in PKC a, b, g, d, e & h • Bioassay guided isolation • C28H26N2O10, m/z 551.1685 • Structure by 2-D NMR • Chirality by x-ray of derivative • Binds at the ATP binding site Kulanthaivel, P.; Hallock, Y. F.; Boros, C.; Hamilton, S. M.; Janzen, W. P.; Ballas, L. M.; Loomis, C. R.; Jiang, J. B.; Katz, B.; Steiner, J. R.; Clardy, J. J. Am. Chem. Soc.1993, 115, 6452-6453.

6. (-)-Balanol Retrosynthesis Benzophenone Azapine

7. (-)-Balanol Benzophenone

8. (-)-Balanol Benzophenone

9. (-)-Balanol Benzophenone John Lampe, Kelly Biggers

10. (-)-Balanol Benzophenone

11. (-)-Balanol Racemic Azepane Two Efficient Synthesis of (±)-anti-N-Benzyl-3-amino-4-hydroxyhexahydroazepine. Hong Hu, G. Erik Jagdmann, Philip F. Hughes and Jeffrey B. Nichols. Tet. Lett., 1995, 3659.

12. (±)-Balanol

13. (-)-Balanol Azepane Resolution

14. (-)-Balanol Chiral Azepane threo-3-Hydroxylysine (+)-(2S,3R)-3-Hydroxylysine Two Chiral Syntheses of Threo-3-Hydroxylysine. Philip F. Hughes, Shelley H. Smith and John Olson. J. Org. Chem.1994, 59, 5799.

15. (-)-Balanol threo-3-Hydroxylysine T. Hayashi 75% after recrystallization 19:1 diastereomeric ratio 90%, 75% after recrystallization

16. (-)-Balanol (+)-(2S,3R)-3-Hydroxylysine

17. Two Chiral Syntheses of Threo-3-Hydroxylysine. Philip F. Hughes, Shelley H. Smith and John Olson. J. Org. Chem.1994, 59, 5799.

18. (-)-Balanol threo-3-Hydroxylysine chiral purity >99% >99%

19. (-)-Balanol Chiral Azepane Reduction by-product if basic workup is omitted

20. (-)-Balanol Total Synthesis of (-)- and (+)-Balanol. John W. Lampe, Philip F. Hughes, Christopher K. Biggers, Shelley H. Smith and Hong Hu. J. Org. Chem.1996, 61, 4572.

21. (-)-Balanol SAR Problem: Balanol shows no inhibitory activity in any assays for kinase inhibition or cellular proliferation. Diagnosis: Balanol, though stable to plasma, is too polar to enter cells. Direction: Find less polar compounds with comparable PKC inhibitory activity, which also show cellular activity 1/10 on removal • Neutrophil Assay • measures inhibition of phorbol-induced superoxide generation in human neutrophils • serves as a model system for studying cellular responses that involve PKC.

22. Improved BenzophenoneSynthesis Sean Hollinshead, Jeffrey B. Nichols, Joseph Wilson J. Org. Chem.1994, 59, 6703.

23. Better Same Worse Lethal Azapine replacements PKC inhibition vs. Balanol d, h 2x d, h syn b Lai, Y.-S.; Mendoza, J. S.; Jagdmann, G. E., Jr.; Menaldino, D. S.; Biggers, C. K.; Heerding, J. M.; Wilson, J. W.; Hall, S. E.; Jiang, J. B.; Janzen, W. P.; Ballas, L. W. J. Med. Chem.1997, 40, 226.

24. Better Same Worse Lethal Benzamide replacements PKC inhibition vs. Balanol b, d Hu, H,; Mendoza, J. S.; Lowden, C.T.; Ballas, L. M.; Janzen, W. P. Biorg. Med. Chem. Lett.1997, 9, 1873.

25. Better Same Worse Lethal Benzophenone replacements PKC inhibition vs. Balanol * 0.5 uM potent PKA * 1.2 uM PKA * 9 uM All Lethal John W. Lampe, Christopher K. Biggers, Jean M. Defauw, Robert J. Foglesong, Steven E. Hall, Julia M. Heerding, Sean P. Hollinshead, Hong Hu, Philip F. Hughes, G. Erik Jagdmann, Jr., Mary George Johnson, Yen-Shi Lai, Christopher T. Lowden, Michael P. Lynch, José S. Mendoza, Marcia M. Murphy, Joseph W. Wilson, Lawrence M. Ballas, Kiyomi Carter, James W. Darges, Jefferson E. Davis, Frederick R. Hubbard, and Mark L. Stamper. J. Med. Chem.; 2002;45(12) 2624.

26. Better Same Worse Lethal Benzophenone replacements PKC inhibition vs. Balanol * * No Cellular activity

27. Better Same Worse Lethal Benzophenone Esterreplacements PKC inhibition vs. Balanol d, h Jagdmann, G. E., Jr.; Defauw, J. M.; Lai, Y.-S.; Crane, H. M.; Hall, S. E.; Buben, J. A.; Hu, H.; Gosnell, P. A. Biorg. Med. Chem. Lett.1995, 5, 2015.

28. Better Same Worse Lethal Azapine and benzamide replacements PKC inhibition vs. Balanol b, d, h * * R * * Defauw, J. M.; Murphy, M. M.; Jagdmann, G. E., Jr.; Hu, H.; Lampe, J. W.; Hollinshead, S. P.; Mitchell, T. J.; Crane, H. M.; Heerding, J. M.; Mendoza, J. S.; Davis, J. E.; Dargess, J. W.; Hubbard, F. R.; Hall, S. E. J. Med. Chem.1996, 39, 5215.

29. Cellular ActivityProdrugs? POM =

30. Cellular ActivityProdrugs? Defauw, J. M.; Murphy, M. M.; Jagdmann, G. E., Jr.; Hu, H.; Lampe, J. W.; Hollinshead, S. P.; Mitchell, T. J.; Crane, H. M.; Heerding, J. M.; Mendoza, J. S.; Davis, J. E.; Dargess, J. W.; Hubbard, F. R.; Hall, S. E. J. Med. Chem.1996, 39, 5215.

31. (-)-Balanol SAR Greatest impact on cellular penetration Benzophenone was very sensitive to change Sensitive to change but tolerant of a wide variety of changes p-Hydroxybenzamide was very sensitive to change Final analysis: No compelling cellular anti-proliferation activity

32. Cyclic AMP-dependantProtein Kinase Balanol ATP & peptide inhibitor

33. Cyclic AMP-dependantProtein Kinase Balanol ATP & peptide inhibitor Narayana, N., Diller, T.C., Koide, K., Bunnage, M.E., Nicolaou, K.C., Brunton, L.L., Xuong, N.H., Ten Eyck, L.F., Taylor, S.S. Crystal structure of the potent natural product inhibitor balanol in complex with the catalytic subunit of cAMP-dependent protein kinase. Biochemistry,1999 v38 pp.2367-2376 ,

34. Acknowledgments • Analytical group Tom Mitchell Carolyn Schwarz

35. Parallel Synthesis • As Practiced at Lilly RTP • Technologies • Reactors • Examples

36. Reactors • Solid Phase Chemistry Reactor • Plate in a Plate Clamp Strip Caps used to seal reaction after reagent addition Plate removed from clamp for resin washing Plate Bottom acts as a 96-way valve H.V. Meyers, G.J. Dilley, T.L. Durgin, et al Molecular Diversity1995, 1, 13-20

37. Examples • Solid Phase • Solution Phase • Sequential reactions • Multi-Component Reactions

38. Factor Xa inhibitor SAR SAR IC50 = 1.5 uM IC50 = 40 nM IC50 = 2 nM Target: Human Factor Xa Description: Factor Xa (fXa) produces thrombin by activating prothrombin during blood clotting whether coagulation is triggered by tissue factor or by blood-contact mechanisms. Indication: Anticoagulant and antithrombotic Wiley, Michael R. et al, J. Med. Chem. 2000, 43, 859-872, 873-882, 883-899

39. Ion Channel Inhibitor Target: Ligand-gated potassium channel receptors Description: Screen used to discover molecules that inhibit potassium transport across membranes involved in atrial fibrillation Indication: Anti-arrhythmics Discovery of potassium channel inhibitors from this library was based on a collaborative screening effort between Lilly and ICAgen Castle, Neil Alexander; Hollinshead, Sean Patrick; Hughes, Philip Floyd; Mendoza, Jose Serafin; Wilson, Joseph Wendell; Amato, George; Beaudoin, Serge; et al. Preparation of N-indanylbenzenesulfonamides and Analogues as Potassium Channel Blockers. U.S. patent number 6,083,986

40. M1 Agonist (R,R) Product of residual DMF reacting with sulfonyl chloride to give the Vilsmeier product. Target: M1 subtype of the muscarinic acetylchloine receptor Description: Stimulated muscarinic receptors initiate a release of arachidonic acid. CHO cells transfected with these muscarinic M1 receptors are prelabeled with tritiated arachidonic acid to provide a signal of muscarinic agonist activity Indication: Cognitive impairment associated with Alzheimer's Disease

41. MRP Inhibitor Problem: M+1+ light by 2 in the Mass Spect NMR showed rotamers Target: Cell based assay to discover molecules that interact with the multidrug resistance protein (MRP1) Description: Cancer cells transfected with the MRP protein show resistance to anticancer agents,such as doxorubicin, because it pumps these agents out of the cell. When an MRP inhibitor is introduced, this pump shuts down therefore lowering the cell line's resistance to oncolytics Indication: Cancer B.H. Norman, J.M. Gruber, S.P. Hollinshead, J.W. Wilson, et al., Bioorganic and Medicinal Chemistry Letters, 883, 12, 2002

42. MRP Inhibitor 2 1 Further SAR 2 EC50 = 1.13 mM in MRP1-transfected HeLa-T5 cells Selectivity demonstrated over P-gp expressing vincristine resistant cells 1 Ramachandran plot of the rotation of the two scaffold C-N bonds

43. Reactors • Solution Phase Chemistry Reactors • Microtitier Format • Various Sizes – Disposable Glassware • Reactor Vessels held at Top • Add functionality (complexity) as needed

44. Solution Library 3072 Compounds Single isomer > 95%

45. FTase Inhibitor Further SAR IC50 = 1.9 nM for the enanatiomer shown IC50 = 420 nM Competitive Inhibitor Target: Farnesyltransferase (FTase), an enzyme that catalyzes farnesylation of proteins ending with the CAAX motif Description: FTase inhibitors inhibit anchorage-independent growth of a variety of transformed cells. Indication: Cancer Discovery of farnesyltransferas inhibitors from this library was based on a collaborative screening effort between Eli Lilly and Kyowa Hakko Kogyo Co. Discovery and Structure-Activity Relationships of Novel Piperidine Inhibitors of Farnesyltransferase. Shinji Nara, Rieko Tanaka, Jun Eishima, Mitsunobu Hara, Yuichi Takahashi, Shizuo Otaki, Robert J. Foglesong, Philip F. Hughes, Shelley Turkington, and Yutaka Kanda. J. Med. Chem.; 2003, 46, 2467-2473.