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The Epothilones

The Epothilones. Brian Lucas October 16, 2003. Epothiwhat?. Epo xide, thi azo l e, ket one = epothilone. Epothilone A, R = H Epothilone B, R = CH 3. Overview . The epothilones are extremely cytotoxic agents with a similar mode of action to Taxol®

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The Epothilones

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  1. The Epothilones Brian Lucas October 16, 2003

  2. Epothiwhat? Epoxide, thiazole, ketone = epothilone Epothilone A, R = H Epothilone B, R = CH3

  3. Overview • The epothilones are extremely cytotoxic agents with a similar mode of action to Taxol® • Epo B is more active than Taxol (in vitro) • Comparatively simple in structure Taxol Epothilone B

  4. Overview • The epothilones are active against Taxol resistant cancer cell lines • The epothilone scaffold is easier to derivatize than Taxol • The epothilones are more water soluble than Taxol Taxol Epothilone B

  5. Outline • Discovery and Background • Mode of Action • Biosynthesis • Initial Synthetic Efforts • Selected Total Syntheses • Structure Activity Relationships (SAR) • In vivo Studies and Phase I/II Clinical Results

  6. Discovery • First isolated from the common soil bacteria Sorangium cellulosum as early as 1987 by Höfle and Reichenbach (GBF) • Exhibited a narrow antifungal spectrum (against Mucor hiemalis only) • Found to be too toxic for use as an antifungal Höfle, G. Bedorf, N.; Gerth, K.; Reichenbach, H. (GBF), DE-B 4138042, 1993, [Chem. Abstr.1993, 120, 52841] Nicolaou, K.C.; Roshangar, F.; Vourloumis, D., Angew. Chem. Int. Ed.1998, 37, 2014.

  7. Cytotoxicity • Bollag (Merck) discovered taxol-like cytotoxicity • Epo A and B were competitive inhibitors of taxol binding • IC50 values comparable (Epo A) or better (Epo B) than Taxol • Active against Pgp MDR (“Taxol refractory”) cells Bollag, D.M.; McQueney, P.A.; Zhu, J.; Hensens, O.; Koupal, L.; Leisch, J.; Goetz, M.; Lazarides, E.; Woods, C.M. Cancer Res.1995, 55, 2325.

  8. Comparison of IC50 Values [nM] The epothilones retain activity in taxol-resistant cancer cell lines Altmann, K.H. Mini-Reviews in Medicinal Chemistry2003, 3, 149. Altmann, K.H.; Wartmann, M.; O’Reilly, T. Biochim. Biophys. Acta2000, 1470, M79

  9. Mode of Action • Like Taxol, the epothilones are microtubule stabilizers • Both bind to β tubulin • Competitive binding suggests same binding site • Epo B efficacy against tubulin-mutated cell lines suggests different interactions Bollag, D.M.; McQueney, P.A.; Zhu, J.; Hensens, O.; Koupal, L.; Leisch, J.; Goetz, M.; Lazarides, E.; Woods, C.M. Cancer Res.1995, 55, 2325.

  10. Epothilone B / Taxol Mode I Carbon:Taxol, Epothilone B Nitrogen,Oxygen Giannakakou, P.; Gussio, R.; Nogales, E. Downing, K.H.; Zaharevitz, D.; Bollubuck, B.; Poy, G.; Sackett, D.; Nicolaou, K.C. Fojo, T. Proc. Natl. Acad. Sci. USA, 2000, 97, 2904

  11. Epothilone B / Taxol Mode II Carbon:Taxol, Epothilone B Nitrogen,Oxygen Giannakakou, P.; Gussio, R.; Nogales, E. Downing, K.H.; Zaharevitz, D.; Bollubuck, B.; Poy, G.; Sackett, D.; Nicolaou, K.C. Fojo, T. Proc. Natl. Acad. Sci. USA, 2000, 97, 2904

  12. Formation of Microtubules Nicolaou, K.C.; Roshangar, F.; Vourloumis, D., Angew. Chem. Int. Ed.1998, 37, 2014.

  13. Stabilized Microtubules BlockMitosis Nicolaou, K.C.; Roshangar, F.; Vourloumis, D., Angew. Chem. Int. Ed.1998, 37, 2014.

  14. Epothilone Biosynthesis

  15. Biosynthesis • Produced by the myxobacterium Sorangium cellulosum So Ce90 • ~20 mg/L 2:1 Epo A : Epo B • The So Ce90 genome sequenced: • Nine modules of polyketide synthase (PKS) • One nonribosomal peptide synthetase (NRPS) • Cytochrome P450 monooxygenase (epoxidation) Molnar, I.; Schupp, T.; Ono, M.; Zirkle, R.E.; Milnamow, M.; Nowak-Thompson, B.; Engel, N.; Toupet, C.; Stratmann, A.; Cyr, D.D.; Gorlach, J.; Mayo, J.M.; Hu, A.; Goff, S.; Schmid, J. Ligon, J.M. Chem. Biol.2000, 7, 97. Tang, L.; Shah, S.; Chung, L.; Carney, J.; Katz, L.; Kholsa, C.; Julien, B. Science2000, 287, 640

  16. Epothilone Gene Cluster Walsh, C.T.; O’Connor, S.E.; Schneider, T.L. J. Ind. Microbiol. Biotechnol.2003, 30, 448.

  17. Post Assembly Line Epoxidation Boddy, C.N.; Scheider, T.L.; Hotta, K.; Walsh, C.T.; Khosla, C. J. Am. Chem. Soc.2003, 125, 3428 Crystal Structure of Epo D and Epo B bound Cytochrome P450EpoK : Nagano, S.; Huiyang, L.; Shimizu, H.; Nishida, Ogura, H.; Ortiz de Montellan, P.R.; Poulos, T.L. JBC Papers in Press, 2003.

  18. Cloning and Heterologous Expression • The epothilone gene cluster has been expressed in Streptomyces coelicolor CH999. • Doubling time of 2h vs. 16h • Expression in Myxococcus xanthus • 1:10 Epo A: Epo B “Large Scale” • Reengineering of the biosynthetic pathway can lead to novel epothilones Tang, L.; Shah, S.; Chung, L.; Carney, J.; Katz, L.; Kholsa, C.; Julien, B. Science, 2000, 287, 640 Arslanian, R.L.; Parker, C.D.; Wang, P.K.; McIntire, J.R.; Lau, J.; Starks, C.; Licari, P.J. J. Nat. Prod.2002, 65, 570. O’Connor, S.E.; Walsh, C.T.; Liu, F. Angew. Chem. Int. Ed. 2003, 42, 3917.

  19. Initial Synthetic Efforts Höfle, G.; Bedorf, N.; Steinmetz, H.; Schomburg, D.; Gerth, K.; Reichenbach, H. Angew. Chem. Int. Ed.1996, 35, 1567.

  20. The Race is On EpoA Oct. 17, 1996 EpoA Nov. 25, 1996 EpoA Dec 28, 1996

  21. Retrosyntheses All chose a late-stage epoxidation Key issues: E / Z isomers, stereochemistry, epoxidation selectivity

  22. Danishefsky’s Macroaldolization Balog, A.; Meng, D.; Kamenecka, T.; Bertinato, P.; Su, D.-S., Sorensen, E.J.; Danishefsky, S.J. Angew. Chem. Int. Ed.1996, 35, 2801

  23. Nicolaou’s Macrolactonization Nicolaou, K.C.; Sarabia, F.; Ninkovic, S.; Yang, Z. Angew. Chem. Int. Ed.1997, 36, 525. Nicolaou, K.C.; Ninkovic, S.; Sarabia, F.; Vourloumis, D.; He, Y.; Vallberg, H.; Finlay, M.R.V.; Yang, Z. J.Am. Chem. Soc.1997, 119, 7974.

  24. Schinzer’s RCM Schinzer, D.; Limberg, A.; Bauer, A.; Bohm, O.M.; Cordes, M. Angew. Chem. Int. Ed.1997, 36, 523.

  25. Nicolaou’s RCM Yang, Z.; He, Y.; Vourlumis, D.; Vallberg, H.; Nicolaou, K.C. Angew. Chem. Int. Ed.1997, 36, 166. Nicolaou, K.C.; He., Y.; Vourloumis, D.; Vallberg, H.; Roschanger, F.; Sarabia, F.; Ninkovic, S.; Yang, Z.; Trujillo, J.I. J. Am. Chem. Soc. 1997, 119, 7960.

  26. Danishefsky’s RCM Meng, D.; Bertinato, P.; Balog, A.; Su, D.-S.; Kameneka, T.; Sorensen, E.J.; Danishefsky, S.J. J. Am. Chem. Soc.1997, 119, 10073 Meng, D.; Su, D.-S.; Balog, A.; Bertinato, P.; Sorensen, E.J.; Danishefsky, S.J.; Zheng, Y.H.; Chou, T.C.; He, L.; Horwitz, S.B. J. Am. Chem. Soc.1997, 119, 2733.

  27. Substituent Effects on RCM Meng, D.; Bertinato, P.; Balog, A.; Su, D.-S.; Kameneka, T.; Sorensen, E.J.; Danishefsky, S.J. J. Am. Chem. Soc.1997, 119, 10073 Meng, D.; Su, D.-S.; Balog, A.; Bertinato, P.; Sorensen, E.J.; Danishefsky, S.J.; Zheng, Y.H.; Chou, T.C.; He, L.; Horwitz, S.B. J. Am. Chem. Soc.1997, 119, 2733.

  28. Overall # of Steps / Yields

  29. Summary of Initial Synthesesof Epothilone A • Remarkably short period of time from Höfle’s crystal structure to first syntheses • Numerous synthetic challenges were identified: • Z:E selectivity about C12-C13 • RCM problematic • Epoxidation yields / selectivity moderate • Stereochemical outcome of aldol reactions highly dependent on substrate

  30. Selected Total Syntheses

  31. More Epothilone Syntheses • Epo B Danishefsky (1997) • Subsequent syntheses by Nicolaou, Schinzer, Grieco, Mulzer, White, Sinha- Lerner, Panek, Shibasaki, Carreira, Ley, Taylor

  32. Mulzer’s “Early Epoxide” Route Martin, H.J.; Drescher, M.; Mulzer, J. Angew. Chem. Int. Ed. 2000, 39, 581.

  33. Mulzer’s “Early Epoxide” Route Martin, H.J.; Drescher, M.; Mulzer, J. Angew. Chem. Int. Ed. 2000, 39, 581.

  34. Reductive DIBAL-H (neutral) L-Selectride (ionic) Zn (metallic) Oxidative OsO4 / NaIO4 NaOCl2 Basic TBAF DMAP LDA Enolates Electrophilic Acyl Chloride Epoxide Stability Martin, H.J.; Drescher, M.; Mulzer, J. Angew. Chem. Int. Ed. 2000, 39, 581.

  35. Ley’s Resin Route to Epo C Storer, R.I.; Takemoto, T.; Jackson, P.S.; Ley, S.V. Angew. Chem. Int. Ed.2003, 42, 2521.

  36. Ley - Fragment A Storer, R.I.; Takemoto, T.; Jackson, P.S.; Ley, S.V. Angew. Chem. Int. Ed.2003, 42, 2521.

  37. Ley – Fragment B Storer, R.I.; Takemoto, T.; Jackson, P.S.; Ley, S.V. Angew. Chem. Int. Ed.2003, 42, 2521.

  38. Ley – Fragment C Storer, R.I.; Takemoto, T.; Jackson, P.S.; Ley, S.V. Angew. Chem. Int. Ed.2003, 42, 2521.

  39. Ley - Convergence Storer, R.I.; Takemoto, T.; Jackson, P.S.; Ley, S.V. Angew. Chem. Int. Ed.2003, 42, 2521.

  40. Ley’s Resin Route • 29 total steps • 17 step longest linear sequence from commercially available materials • 1 column • Most complex natural product built by these techniques

  41. Danishefsky’s “Scalable” Synthesis Chappell, M.D.; Stachel, S.; Lee, C.B.; Danishefsky, S.J. Org. Lett.2000, 2, 1633. Lee, C.B.; Wu, Z.; Zhang, F.; Chappell, M.D.; Stachel, S.J.; Chou, T.-C.; Guan, Y.; Danishefsky, S.J., J. Am. Chem. Soc.2001, 123, 5249.

  42. Subunit A Chappell, M.D.; Stachel, S.; Lee, C.B.; Danishefsky, S.J. Org. Lett.2000, 2, 1633. Lee, C.B.; Wu, Z.; Zhang, F.; Chappell, M.D.; Stachel, S.J.; Chou, T.-C.; Guan, Y.; Danishefsky, S.J., J. Am. Chem. Soc.2001, 123, 5249.

  43. Subunit B Chappell, M.D.; Stachel, S.; Lee, C.B.; Danishefsky, S.J. Org. Lett.2000, 2, 1633. Lee, C.B.; Wu, Z.; Zhang, F.; Chappell, M.D.; Stachel, S.J.; Chou, T.-C.; Guan, Y.; Danishefsky, S.J., J. Am. Chem. Soc.2001, 123, 5249.

  44. Subunit C / BC coupling Chappell, M.D.; Stachel, S.; Lee, C.B.; Danishefsky, S.J. Org. Lett.2000, 2, 1633. Lee, C.B.; Wu, Z.; Zhang, F.; Chappell, M.D.; Stachel, S.J.; Chou, T.-C.; Guan, Y.; Danishefsky, S.J., J. Am. Chem. Soc.2001, 123, 5249.

  45. Final Convergence Chappell, M.D.; Stachel, S.; Lee, C.B.; Danishefsky, S.J. Org. Lett.2000, 2, 1633. Lee, C.B.; Wu, Z.; Zhang, F.; Chappell, M.D.; Stachel, S.J.; Chou, T.-C.; Guan, Y.; Danishefsky, S.J., J. Am. Chem. Soc.2001, 123, 5249.

  46. Completion of Epo D 23 – 25 total steps 16 (13) step longest linear sequence 6.2% (17%) overall yield Chappell, M.D.; Stachel, S.; Lee, C.B.; Danishefsky, S.J. Org. Lett.2000, 2, 1633. Lee, C.B.; Wu, Z.; Zhang, F.; Chappell, M.D.; Stachel, S.J.; Chou, T.-C.; Guan, Y.; Danishefsky, S.J., J. Am. Chem. Soc.2001, 123, 5249.

  47. Epothilone B in vivo

  48. Epothilone B in vivo (MSK) • Found to be toxic in non-tumor-bearing nude mouse models • 0.6 mg/kg/day x 4 given i.p. resulted in 8/8 deaths (days 5-7) MX-1 xeno graft Chou, T.C.; Zhang, X.-G.; Balog, A.; Shu, D.-S.; Meng, D.; Savin, K.A.; Bertino, J.R.; Danishefsky, S.J. Proc. Nat. Acad. Sci. USA 199895, 9642.

  49. Epothilone B in vivo (Novartis) • Significant tumor regression in Pgp MDR cells (HCT-15, KB-8511) • relatively narrow therapeutic window • t½≈ 40 min in mouse plasma • stability in human plasma is much greater Altmann, K.H., Wartmann, KM.; O’Reilly, T. Biochem. Biophys. Acta. 2000, 1470, M79

  50. Structure-Activity Relationships

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