synthesis of lamellarin d a novel potent inhibitor of dna topoisomerase i wenhui hao march 16 th,...

Synthesis of Lamellarin D

A Novel Potent Inhibitor of DNA Topoisomerase I

Wenhui Hao

March 16th , 2006

Outline

Lamellarin D

Biological activities

Structure-activity relationship

Identification of LAM-D as an

inhibitor of Topo I

Three synthetic routes

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

Cancer Treatment

•Surgery

•Radiation

•Chemotherapy : Alkylating agents

Antimetabolites

Plant alkaloids

Antitumour agents

Topoisomerase inhibitors

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

DNA Structure

DNA Double Strand Helix

•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

Topoisomerase I Activity

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

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

Topo Inhibitors Mechanism of Action

Covalent binding to

double-stranded DNACleavable 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

•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

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

OOHH3C

A B CD

Wall M et al J.Am.Chem.Soc 1966,88:3888-90 12

Topotecan (TPT)

OOHH3C

CH3 •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

Bart Staker et al PNAS Vol. 99, No.24 2002,15387-15392

Crystal Structures of Topo I-DNA-TPT Complex

Topo 70-DNA Binary Complex Topo 70-DNA-Topotecan Ternary Complex

3.6 Ǻ 7.2 Ǻ

Mimic bp Extends bp distance

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

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

Lamellarin D

•The main pentacyclic array is essentially planar

•The aromatic ring attached to C1 is rotated 90°to the main plane

Structure properties

Lamellarin A

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

N CO2Me

LAM O, X=H LAM P, X=OH

N CO2Me

LAM Q, R=H LAM R, R=p-HOC6H418

Biological ActivitiesCommon activities

Inhibition of cell division

Cytotoxicity

Immunomodulatory activity

Recent findings

Lamellarin D :

Antitumor activity against MDR cell lines

Selective cytotoxicity for prostate cancer cells

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

BrCH2CO2Et

Et3N, CH2CI2N

1) LDA, THF

5 6 7a 7b

8 R=MOM9 R=H

33%, 3 steps

Model Study

7a:7b = 92:8

6-Benzyloxy-l-(4-benlzyloxy-3-methoxybenzyl)-7-methoxyisoquinoline(3)

CHO NO2

MeOMeO

11 12 13

CH3NO2, NH4OAc, AcOH

81%MeONa, MeOH-CH2Cl2

LiAlH4ether-THF

DCC, CH2Cl2, 76% 2steps

POCl3, Benzene

Methyl 4-Benzyloxy-5-methoxy-2-methoxymethoxybenzoate(4)

CO2MeMeO

BnO OH

16 17 18

BnBr, K2CO3

91% 91%

DMF-MeOH63%

MOMCl, t-BuOK, THF

Br2, CHCl3

BrCH2CO2Et

MeOMeO

20a 20b

LDA, THF, 63%

CO2MeMeO

4% 14 steps

HCl, MeOH

34% 3 steps

H2, Pd/C, EtOAc

Et3N, CH2Cl2

Activity of Lam D and Lam 11

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

Lam D IC50(¦Ìm) 0.0105

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

Lam D IC50(¦Ìm) 0.0105

Lam 3 0.0395

Lam 6 0.0380

Lam 7 0.0700HO

Effect of OH at C-20

Lam 4 0.8500

OHMeOMeO

Lam 5 2.5

Lam 12 >100

MeO OO

Lam D IC50(¦Ìm) 0.0105

Effect of OH at C-8

OHMeOMeO

Lam 5 2.5

C-8 OH, lacks C-14 OH, maintains high activity

Methylation of OH at C-8 , C-14

decrease activity

Lam D IC50(¦Ìm) 0.0105

Lam 7 0.0700HO

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

+Pd(PPh3)4 , CuI

Et3 N, 99%

AcO HO

18oC, 4h

1 2 3 4

5CCH2BrO

K2CO3, MeOH

BrCH2COBr CH2Cl291% 2 steps

i. Et3N, THF, 66C, 4h

ii. DDQ, CH2Cl2,

92% 2 stepsTHF

i-PrBr, K2CO3, DMF

CBr4, Zn0-25oC, 4h

n-BuLi, THF

AgOCOCF3, I2, CH2Cl2

Pd(PPh3)4, CuI, NEt3

PPh3, CH2Cl2

94% 95%

80% 2 steps

Christian P. Ridley, et al. Bioorg. Med. Chem., 2002, 10: 3285-3290.

Application of Banwell’s Approach

i. MCPBA, KHCO3, CH2Cl2

ii. NH3, CH2Cl2/MeOH(1:1)

iii. ICH2COOH, DCC, DMAP, CH2Cl2

ClCH2CH2Cl

Oi-PrMeOMeO

11 Lam H

54% 2 steps

DDQ, CH2Cl2/EtOH(1:1)

BBr3, CHCl3

9 Lam U diisopropyl ether

10 Lam ¦Á diisopropyl ether

17% 12 steps33

Identification of LAM-D as an Inhibitor of TopoI

Michael Facompre et al. Cancer Research 2003, 63,7392-7399

LAM-501

DNA Relaxation Experiment – Topo I Inhibition Efficacy

Nck:nicked form II,single-strand break35

Detectation of the Extents of Cleavage

LAM D induced dose dependent stimulation

of DNA cleavage by topo I

Equally effective at 2 μM70% of the DNA

single-strand breaks

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

CPT specific

LAM D specific

Molecular Modeling

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

Summary of the Study of SAR

202114

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 Essential

Olsen-Pla’s Open Chain-Modular

Synthetic Route to Lamellarins

Christian A. Olsen, et al. Tetrahedron Letters, 2005, 46: 2041-2044

N CO2MeR2

CO2MeNH

R1 = Oi-Pr, R2 = OMe

N-alkylation with p-toluenesulfonate and intramolecular Heck cyclization from Methyl pyrrole-2-carboxylate to Scaffold 1

For all compounds

R1=Oi-Pr,R2=OMe

N CO2Me

NCO2MeR2

NBS, THF

CO2MeNH

Pd(PPh3)4, NaOAc

NaH, DMF

Synthesis of open chain analogues

NCO2MeR2

N CO2MeR2

B(OH)2

NCO2MeR2

AlCl3,CH2Cl26

NCO2MeR2

4Pd(PPh3)4, Na2CO3, DMF

Pd(PPh3)4, Na2CO3, DMF

NCO2MeR2

78% 2 steps

AlCl3,CH2Cl2

62% 2 steps

27% 9 steps

35% 9 steps

N CO2Me

Lamellarin D

Daniel Pla, et al. J.Org.Chem.2005,70:8231-8234

Modular Synthesis of Lamellarin D

Two sequential and regio-selective bromination and cross-coupling reactions using different substituted arylboronic ester

N CO2Me

BrN CO2Me

TsOi-PrO

Pd(PPh3)4 Na2CO3 DMF 78%

N CO2MeMeO

i-PrO5

NBS, THF

N CO2Me

i-PrO7

1)NaH, DMF

2)PdCI2(PPh3)2,PPh3, K2CO3

NCO2Me

N CO2Me

i-PrO 7

Pd(PPh3)4, , DMF 87%

i-PrO MeO

NBS THF

i Pr-Br,K2CO3,DMF

DDQ, CHCI3, MWMeO

NaH, THFMeO

Lamellarin D

i-PrOMeO

AlCl3, CH2Cl2

38% 3 steps

9% 13 steps

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%

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

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

synthesis of lamellarin d a novel potent inhibitor of dna topoisomerase i wenhui hao march 16 th,...

cells topo

inhibitor of topo

cleavage dnatopo

inhibition of topo

dna binary complex topo

active site of topo

crystal structures of

dnatpt complextopo

Documents

topoisomerase i-mediated integration of hepadnavirus

an introduction to the model verifier verds wenhui zhang...

contribution of c-erbb-2and topoisomerase iiato...

methanopyrus kandleri topoisomerase v contains three...

topoisomerase ii minimizes dna entanglements by...

alkylating agents platinating agents antimetabolites...

topoisomerase ii regulates yeast genes with singular .......

irinotecan - topoisomerase poison

glycosylated flavones as selective inhibitors of...

functional cooperation between topoisomerase i and single...

arrest of replication forks by drug-stabilized topoisomerase...

topoisomerase inhibiton by gold iii

a type ib topoisomerase with dna repair activities

topoisomerase 1 inhibition reversibly impairs …expression...

cellular distribution of mammalian dna topoisomerase ii is

the catalytic topoisomerase ii inhibitor dexrazoxane...

effects of quinolone derivatives on eukaryotic topoisomerase...

topoisomerase ii is required for the production of long...

topoisomerase inhibitors unsilence the dormant allele of...

wenhui wang 1 & cheng- zhi zou 2 1 imsg at...