Synthesis of Lamellarin D

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Synthesis of Lamellarin D
A Novel Potent Inhibitor of DNA
Topoisomerase I


Wenhui Hao
March 16th , 2006

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Outline

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

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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

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Cancer Treatment

• Surgery
• Radiation
• Chemotherapy Alkylating agents
• Antimetabolites
  
• Plant alkaloids
  
• Antitumour agents
  
• Topoisomerase
  inhibitors

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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

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DNA Structure
DNA Double Strand
Helix
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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 3.4?

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Topoisomerase I Activity
Topo I 100 KD monomeric protein ,breaks single
strands , by cleaving a phosphodiester bond form
a phosphotyrosine topoI-DNA complex
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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
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Topo Inhibitors Mechanism of Action
Covalent binding to double-stranded DNA
Cleavable complex 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
Uncoiling of double-strande DNA , prevents
resealing
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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
•  

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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
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Wall M et al J.Am.Chem.Soc 1966,883888-90
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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

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John Nitiss Nurrent Opinion In Investigational
Drugs 2002, 3 (10) 1512-1516
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Crystal Structures of Topo I-DNA-TPT Complex
Topo 70-DNA Binary Complex
Topo 70-DNA-Topotecan Ternary Complex
7.2 ?
3.6 ?
Mimic bp Extends bp distance
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Bart Staker et al PNAS Vol. 99, No.24
2002,15387-15392
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Mechanism of Topo I Inhibitor- TPT
Hydrogen bond contact to the active site of Topo
I and phosphotyrosine Free-OH displaced 8?
from phosphotyrosine of Topo I
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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
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Structure properties

• The main pentacyclic array is essentially planar
• The aromatic ring attached to C1 is rotated 90to
  the main plane

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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
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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

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Ishibashis 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
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Model Study
7a7b 928
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Ishibashis Synthesis of LAM-D
6-Benzyloxy-l-(4-benlzyloxy-3-methoxybenzyl)-7-met
hoxyisoquinoline(3)
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Ishibashis Synthesis of LAM-D
Methyl 4-Benzyloxy-5-methoxy-2-methoxymethoxybenzo
ate(4)
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Ishibashis Synthesis of LAM-D
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Ishibashis Synthesis of LAM-D
4 14 steps
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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
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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
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Fumito Ishibashi et al. J. Nat. Prod. 2002, 65,
500-504
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Effect of OH at C-20
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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
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Banwells Synthesis- Lamellarin Parent Ring
System
An intramolecular 3 2 cycloaddition between
an isoquinoline-based azomethine ylide and a
tethered tolan
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Martin Banwell, et al. Chem. Commun. 1997
2259-2260
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Application of Banwells Approach
Christian P. Ridley, et al. Bioorg. Med. Chem.,
2002, 10 3285-3290.
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Application of Banwells Approach
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Application of Banwells Approach
17 12 steps
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Identification of LAM-D as an Inhibitor of TopoI
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Michael Facompre et al. Cancer Research 2003,
63,7392-7399
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DNA Relaxation Experiment Topo I Inhibition
Efficacy
d
a
b
c
Ncknicked form II,single-strand break
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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

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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
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Topo I Inhibition Site Selectivity
CPT specific


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Topo I Inhibition Site Selectivity
LAM D specific

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Molecular Modeling
Theoretical model of LAM-D covalently bound to
topoisomerase IDNA complex.
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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
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Olsen-Plas Open Chain-Modular Synthetic Route
to Lamellarins
N-alkylation with p-toluenesulfonate and
intramolecular Heck cyclization from Methyl
pyrrole-2-carboxylate to Scaffold 1
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Christian A. Olsen, et al. Tetrahedron Letters,
2005, 46 2041-2044
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Olsen-Plas Open Chain-Modular Synthetic Route
to Lamellarins
Synthesis of open chain analogues
For all compounds R1Oi-Pr,R2OMe
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Olsen-Plas Open Chain-Modular Synthetic Route
to Lamellarins
35 9 steps
27 9 steps
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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.2005,708231-8234
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Modular Synthesis of Lamellarin D
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Modular Synthesis of Lamellarin D
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Modular Synthesis of Lamellarin D
9 13 steps
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Comparison of Three Synthesis
1. Ishibashis N-ylide approach Prepared
and evaluated 10 derivatives   Lam D 14
steps, overall yield 4   Ring substitution
limited 2. Banwells Intermolecular 32 approach
  Most direct method to the lamellarins
  12 steps, overall yield 17  
Prepared Lam D and Lam 501   3. Olsen-Plas Open
chain-Modular synthesis approach   More
flexible, effective method   Open chain
analogues  9 steps, 27-35 yield   Lam D
13 steps, overall yield 9
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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
• Ishibashis synthesis
• Banwells synthesis
• Olsen-Plas synthesis

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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
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