1. Synthesis of Lamellarin D A Novel Potent Inhibitor of DNA Topoisomerase I Wenhui Hao March 16 th, 2006

2. Outline Biological activities Structure-activity relationship Identification of LAM-D as an inhibitor of Topo I Three synthetic routes 2

3. Background Cancer Normal cells-- new cell growth balance with old cells die Cancer cells-- loss of normal growth control loss of ability to undergo programmed cell death 3

4. Cancer Treatment Surgery Radiation Chemotherapy : Alkylating agents Antimetabolites Plant alkaloids Antitumour agents Topoisomerase inhibitors 4

5. Topoisomerases Maintaining the topographic structure of circular DNA Topo I: transient single-strand break (Lam D) Topo II: double-strand break Breaking--Uncoiling--Replication DNA helix 5

6. DNA Structure DNA Double Strand Helix 6

7. A,T, G, C bases can extend away from chain stack at top each other dA-dT, dG-dC base pairs are the same length Occupy the same space The distance between the two bps is 3.4Ǻ Base Pairs 7

8. Topoisomerase I Activity Topo I : 100 KD monomeric protein,breaks single strands, by cleaving a phosphodiester bond form a phosphotyrosine topoI-DNA complex 8

9. Topoisomerase I Activity Religation is faster than cleavage → DNA-Topo I complex concentration remains low Drugs stabilize the complex and Block DNA religation, converting Topo I into a DNA damaging agent 9

10. Topo Inhibitors Mechanism of Action Covalent binding to double-stranded DNA Cleavable complex by binding to DNA-Topo I or II Uncoiling of double-strande DNA, prevents resealing Replication halted at Topo-DNA complex stage Replication fork collides with trapped complex double strand breaks and cell death 10

11. Activity does not change with growth of the cells Topo I levels in tumor specimens are higher than normal tissues and Topo II making inhibition of Topo I an attractive target for anticancer agents Significant activity against a broad range of tumors Advantages of Topo I Inhibitors 11

12. Camptothecin (CPT) and Its Analogs First isolated from the Chinese tree Camptotheca acuminata, Nyssaceae. in 1966 CPTs inhibit Topo I as cytotoxic agents Clinical test against colon, ovarian cancers Serious side effects, poor water solubility Wall M et al J.Am.Chem.Soc 1966,88:3888-90 12

13. Topotecan (TPT) Water-soluble CPT derivative Significant activity against tumor cell lines (breast, lung ) Stabilizes DNA-drug-Topo I complex and inhibits Topo I function causing DNA strand breakage. Approved in 1996, first Topo I inhibitor treating ovarian cancer John Nitiss Nurrent Opinion In Investigational Drugs 2002, 3 (10) :1512-1516 13

14. Bart Staker et al PNAS Vol. 99, No.24 2002,15387-15392 Crystal Structures of Topo I-DNA-TPT Complex Topo 70-DNA Binary ComplexTopo 70-DNA-Topotecan Ternary Complex 3.6 Ǻ 7.2 Ǻ Mimic bp Extends bp distance 14

15. Hydrogen bond contact to the active site of Topo I and phosphotyrosine Free-OH displaced 8Ǻ from phosphotyrosine of Topo I Mechanism of Topo I Inhibitor- TPT 15

16. Marine Alkaloid- Lamellarins Isolated in 1985 from a Lamellaria sp. of marine prosobranch mollusc Lam A,B,C,D were obtained C and D inhibition of cell division A and B were inactive Raymond J. Andenen et al J. Am. Chem. Soc. 1985, 107, 5492-5495 16

17. The main pentacyclic array is essentially planar The aromatic ring attached to C1 is rotated 90°to the main plane Structure properties 17

18. A Growing Family --Three Groups Open Chain Fused: S or D * 35 lamellarins have been isolated, from ascidian and sponge species * A pentacyclic core, variation from hydroxy, methoxy substitution 18

19. Biological Activities Common activities Inhibition of cell division Cytotoxicity Immunomodulatory activity Recent findings Lamellarin D : Antitumor activity against MDR cell lines Selective cytotoxicity for prostate cancer cells 19

20. Ishibashi’s Synthesis of LAM-D Fumito Ishibashi et al. Tetrahedron, 1997, 53(17): 5951-5962 N-ylide- mediated pyrrole ring formation of a quaternary ammonium salt followed by lactonization 20

21. Model Study 7a:7b = 92:8 21

22. Ishibashi’s Synthesis of LAM-D 6-Benzyloxy-l-(4-benlzyloxy-3-methoxybenzyl)-7-methoxyisoquinoline(3) 22

23. Ishibashi’s Synthesis of LAM-D Methyl 4-Benzyloxy-5-methoxy-2-methoxymethoxybenzoate(4) 23

24. Ishibashi’s Synthesis of LAM-D 24

25. Ishibashi’s Synthesis of LAM-D 4% 14 steps 25

26. 26 Activity of Lam D and Lam 11 Cytotoxicities against tumor Cell Lines, IC 50 ( μ M) compound Hella XC lamellarin D 0.0105 0.0124 Lam 11 5.7 5.6 mitomycin C 68.0 ND a 26

27. 27 Structure-Activity Relationship Study of Lamellarin Derivatives Fumito Ishibashi et al. J. Nat. Prod. 2002, 65, 500-504 OH at C-8 C-20 essential OH at C-14 MeO at C-13, C-21 less important 27

28. 28 Effect of OH at C-20 28

29. 29 Effect of OH at C-8 C-8 OH, lacks C-14 OH, maintains high activity Methylation of OH at C-8, C-14 decrease activity 29

30. 30 Banwell’s Synthesis- Lamellarin Parent Ring System An intramolecular [3 + 2] cycloaddition between an isoquinoline-based azomethine ylide and a tethered tolan Martin Banwell, et al. Chem. Commun. 1997: 2259-2260 30

31. 31 Christian P. Ridley, et al. Bioorg. Med. Chem., 2002, 10: 3285-3290. Application of Banwell’s Approach 31

32. 32 Application of Banwell’s Approach 32

33. 33 Application of Banwell’s Approach 17% 12 steps 33

34. 34 Identification of LAM-D as an Inhibitor of TopoI Michael Facompre et al. Cancer Research 2003, 63,7392-7399 34

35. 35 DNA Relaxation Experiment – Topo I Inhibition Efficacy c Nck:nicked form II,single-strand break 35 a d b

36. 36 Detectation of the Extents of Cleavage LAM D induced dose dependent stimulation of DNA cleavage by topo I Equally effective at 2 μM 70% of the DNA single-strand breaks 36

37. 37 Topo I Inhibition: Site Selectivity Cleavage of DNA fragment by Topo I (increasing concentrations of LAM-D) Common site Side numbers of gels show nucleotide positions determined with reference to guanine(G) tracks 37

38. 38 Topo I Inhibition: Site Selectivity CPT specific 38

39. 39 Topo I Inhibition: Site Selectivity LAM D specific 39

40. 40 Molecular Modeling Theoretical model of LAM-D covalently bound to topoisomerase I–DNA complex. 40

41. 41 Summary of the Study of SAR Planar conformation of LAM-D suited for intercalation into DNA OH at C-8, C-20 : Essential OH at C-14, MeO at C-13, C-21 : Less important Essential 41

42. 42 Olsen-Pla’s Open Chain-Modular Synthetic Route to Lamellarins Christian A. Olsen, et al. Tetrahedron Letters, 2005, 46: 2041-2044 N-alkylation with p-toluenesulfonate and intramolecular Heck cyclization from Methyl pyrrole-2-carboxylate to Scaffold 1 42

43. 43 For all compounds R 1 =Oi-Pr,R 2 =OMe Olsen-Pla’s Open Chain-Modular Synthetic Route to Lamellarins 43 Synthesis of open chain analogues

44. 44 27% 9 steps 35% 9 steps Olsen-Pla’s Open Chain-Modular Synthetic Route to Lamellarins 44

45. 45 Daniel Pla, et al. J.Org.Chem.2005,70:8231-8234 Modular Synthesis of Lamellarin D 45 Two sequential and regio-selective bromination and cross-coupling reactions using different substituted arylboronic ester

46. 46 Modular Synthesis of Lamellarin D 46

47. 47 Modular Synthesis of Lamellarin D 47

48. 48 9% 13 steps Modular Synthesis of Lamellarin D 48

49. 49 Comparison of Three Synthesis 1. Ishibashi’s N-ylide approach · Prepared and evaluated 10 derivatives · Lam D: 14 steps, overall yield 4% · Ring substitution limited 2. Banwell’s Intermolecular 3+2 approach · Most direct method to the lamellarins · 12 steps, overall yield 17% · Prepared Lam D and Lam 501 3. Olsen-Pla’s Open chain-Modular synthesis approach · More flexible, effective method · Open chain analogues: 9 steps, 27-35% yield · Lam D: 13 steps, overall yield 9% 49

50. 50 Conclusion A novel class of marine alkaloids – Lamellarins isolated Lamellarin D Identified as a lead candidate for Topo I targeted antitumor agent Structure-activity relationship studied Three different synthetic methods compared Ishibashi’s synthesis Banwell’s synthesis Olsen-Pla’s synthesis 50

51. 51 Acknowledgment Dr.Wang Hadizad Tayebeh Dr. Jane Gao Shidi Xun Dr. Hongding Tang Xun Sun Dr. Xianzhen Li Xianguo Wu Yuxing Cui Ying Xiong Gaetan LeClair 51