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Synthesis of Lamellarin D A Novel Potent Inhibitor of DNA

Synthesis of Lamellarin D A Novel Potent Inhibitor of DNA Topoisomerase I. Wenhui Hao March 16 th , 2006. Outline. Biological activities Structure-activity relationship Identification of LAM-D as an inhibitor of Topo I

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Synthesis of Lamellarin D A Novel Potent Inhibitor of DNA

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  1. Synthesis of Lamellarin D A Novel Potent Inhibitor of DNA Topoisomerase I Wenhui Hao March 16th , 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 • Maintainingthe 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. Base Pairs • 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 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 Cleavablecomplex by binding to DNA-Topo I or II Replication halted at Topo-DNA complex stage Replication fork collides with trapped complex double strand breaks and cell death Uncoilingof double-strande DNA , prevents resealing 10

  11. Advantages of Topo I Inhibitors • 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 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 12 Wall M et al J.Am.Chem.Soc 1966,88:3888-90

  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 13 John Nitiss Nurrent Opinion In Investigational Drugs 2002, 3 (10) :1512-1516

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

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

  16. Marine Alkaloid- Lamellarins • Isolated in 1985 from a Lamellaria sp. • of marine prosobranchmollusc • 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. Structure properties • The main pentacyclic array is essentially planar • The aromatic ring attached to C1 is rotated 90°to the main plane 17

  18. A Growing Family --Three Groups Fused: S or D * 35 lamellarins have been isolated, from ascidian and sponge species * A pentacyclic core, variation from hydroxy, methoxy substitution Open Chain 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 N-ylide- mediated pyrrole ring formation of a quaternary ammonium salt followed by lactonization Fumito Ishibashi et al. Tetrahedron, 1997, 53(17): 5951-5962 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. Activity of Lam D and Lam 11 Cytotoxicities against tumor Cell Lines, IC50 (μM)   compound Hella XC lamellarin D 0.0105 0.0124 Lam 11 5.7 5.6 mitomycin C 68.0 NDa 26

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

  28. Effect of OH at C-20 28

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

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

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

  32. Application of Banwell’s Approach 32

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

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

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

  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. 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. Topo I Inhibition: Site Selectivity CPT specific 38

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

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

  41. Summary of the Study of SAR Essential 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 Essential 41

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

  43. Olsen-Pla’s Open Chain-Modular Synthetic Route to Lamellarins Synthesis of open chain analogues For all compounds R1=Oi-Pr,R2=OMe 43

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

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

  46. Modular Synthesis of Lamellarin D 46

  47. Modular Synthesis of Lamellarin D 47

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

  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 yield9% 49

  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

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