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

Wenhui Hao March 16 th , 2006

Outline

HO MeO OH MeO MeO HO

1

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

2

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

Cancer Treatment

•

Surgery

•

Radiation

•

Chemotherapy : Alkylating agents Antimetabolites Plant alkaloids Antitumour agents Topoisomerase inhibitors

4

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

DNA Structure

DNA Double Strand Helix

6

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

Topoisomerase I Activity

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

8

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

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

10

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

Camptothecin (CPT) and Its Analogs

A B N C N D O E O H 3 C OH O 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

Topotecan (TPT)

HO CH 3 N CH 3 N H 3 C N O O OH O

•

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

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

Bart Staker

et al

PNAS Vol. 99, No.24

2002

,15387-15392 14

Mechanism of Topo I Inhibitor- TPT

Hydrogen bond contact to the active site of Topo I and phosphotyrosine Free-OH displaced 8 Ǻ from phosphotyrosine of Topo I

15

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 MeO HO MeO

1

MeO N HO Lamellarin D

Raymond J. Andenen

et al

J. Am. Chem. Soc.

1985

, 107

,

5492-5495

O O OH

16

Structure properties

HO MeO MeO MeO MeO N OH O O MeO Lamellarin A OH •

The main pentacyclic array is essentially planar

•

The aromatic ring attached to C1 is rotated 90

°

to the main plane

17

A Growing Family --Three Groups

Fused: S or D

R 4

14

R 3

13

R 2

9

R 1

8

Open Chain

R 5

21 20

R 6

1

O

6

N

3 5

R N O CO 2 Me

*

35 lamellarins have been isolated, from ascidian and sponge species * A pentacyclic core, variation from hydroxy, methoxy substitution X O N CO 2 Me MeO OH HO LAM Q, R=H LAM R, R=p-HOC 6 H 4 HO OH LAM O, X=H LAM P, X=OH

18

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

Ishibashi’s Synthesis of LAM-D

N-ylide- mediated pyrrole ring formation of a quaternary ammonium salt followed by lactonization HO MeO MeO HO

1

BnO N O O MeO MeO OMe OH BnO N Br O OEt O OH MeO

2

OBn BnO MeO MeO BnO

3

Br O OEt N MeO O OMOM MeO OBn

4 Fumito Ishibashi

et al

. Tetrahedron,

1997

, 53(17): 5951-5962 20

Model Study

MeO MeO MeO MeO

5

N

2)

1) LDA, THF CO 2 Me

6

OMOM MeO MeO MeO MeO

7a

H

1

N H

3

O OMOM

7a:7b = 92:8

MeO MeO MeO MeO

7b

N

3

H H O OMOM BrCH 2 CO 2 Et MeO MeO MeO MeO

10

N O O Et 3 N, CH 2 CI 2 33%, 3 steps Br MeO MeO MeO MeO 8 R=MOM 9 R=H N O CO 2 Et OMOM

21

Ishibashi’s Synthesis of LAM-D

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

11

BnO MeO MeO BnO CHO CH 3 NO 2, NH 4 OAc, AcOH 81%

3

BnO MeO

12

NO 2 MeONa, MeOH-CH 74% 2 Cl 2 OMe BnO MeO

13

NO 2 N POCl 3 , Benzene 57% LiAlH 4 ether-THF BnO MeO MeO BnO

15

O OMe BnO NH MeO CO 2 H BnO DCC, CH 2 Cl 2 , 76% 2steps MeO

14

OMe NH 2

22

Ishibashi’s Synthesis of LAM-D

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

16

OH CO 2 Me BnBr, K 2 CO 3 91% BnO

17

OH CO 2 Me Br 2 , CHCl 3 91% BnO Br OH

18

CO 2 Me 63% MeONa DMF-MeOH BnO MeO

4

OMOM CO 2 Me MOMCl, t-BuOK, THF 94% BnO MeO

19

OH CO 2 Me

23

Ishibashi’s Synthesis of LAM-D

BnO MeO MeO BnO

3

BrCH 2 CO BnO OMOM N MeO CO 2 Me

4

LDA, THF, 63% BnO MeO MeO BnO N O OMOM MeO

20a

OBn 2 Et BnO MeO MeO BnO Br N O CO 2 Et OMOM MeO

21

OBn BnO MeO MeO BnO N H O OMOM MeO

20b

OBn

24

Ishibashi’s Synthesis of LAM-D

HO MeO MeO HO BnO MeO MeO BnO N O Br CO 2 Et OMOM MeO

21

OBn HCl, MeOH BnO N O O OMe OH H 2 , Pd/C, EtOAc 82% MeO MeO BnO LAM-D 4% 14 steps BnO MeO MeO BnO Br N O CO 2 Et OH MeO

2

OBn Et 3 N, CH 2 Cl 2 34% 3 steps N O O

22

OMe OBn

25

Activity of Lam D and Lam 11

MeO MeO MeO H

HO MeO OH

14 21 20

MeO MeO HO

13 1

O

9

N O

8

Lam D IC 50 (¦Ìm) 0.0105

MeO O N O Lam 11

Cytotoxicities against tumor Cell Lines, IC 50 ( μ M) compound Hella XC lamellarin D Lam 11 mitomycin C 0.0105

5.7

68.0

0.0124

5.6

ND a

H 26 26

Structure-Activity Relationship Study of Lamellarin Derivatives HO MeO OH

14 21 20

MeO MeO HO

13 1

O

9

N O

8

Lam D IC 50 (¦Ìm) 0.0105

MeO MeO HO HO N H O O OH HO MeO H Lam 3 0.0395

MeO OH OH H MeO MeO O MeO N N O HO HO Lam 7 0.0700

Lam 6 0.0380

Fumito Ishibashi

et al

. J. Nat. Prod . 2002 , 65

,

500-504

O O

OH at C-8 C-20 essential OH at C-14 MeO at C-13 , C-21 less important 27 27

Effect of OH at C-20

HO MeO OH

14 21 20

MeO MeO HO

13 1

O

9

N O

8

Lam D IC 50 (¦Ìm) 0.0105

MeO MeO MeO MeO MeO N O O Lam 5 2.5

OH HO MeO H MeO MeO N O O HO Lam 4 0.8500

MeO O O MeO MeO N MeO Lam 12 >100 O O

28 28

Effect of OH at C-8

HO MeO OH

14 21 20

MeO MeO HO

13 1

O

9

N O

8

Lam D IC 50 (¦Ìm) 0.0105

MeO MeO OH MeO MeO N MeO H Lam 5 2.5

MeO O O OH MeO MeO HO N Lam 7 0.0700

O O Methylation of OH at C-8 , C-14 decrease activity C-8 OH, lacks C-14 OH, maintains high activity

29 29

Banwell’s Synthesis Lamellarin Parent Ring System An intramolecular [3 + 2] cycloaddition between an isoquinoline-based azomethine ylide and a tethered tolan

1 +

AcO

2

Pd(PPh 3 ) 4 , CuI Et 3 N, 99%

3

AcO K 2 CO 3 , MeOH 18 o C, 4h

4

HO BrCH 2 COBr CH 2 Cl 2 91% 2 steps N

7

O O i. Et 3 N, THF, 66C, 4h ii. DDQ, CH 2 Cl 2 , 92% 2 steps

6

N O Br O THF N

5

O O CCH 2 Br

Martin Banwell,

et al

. Chem. Commun.

1997

: 2259-2260 30 30

Application of Banwell’s Approach

OMe OH i-PrBr, K 2 CO 3 , DMF CHO

1

OMe Oi-Pr AgOCOCF 3 , I 2 , CH 2 Cl 2 94% CBr 4 , Zn PPh 3 , CH 2 Cl 2 CHO

2

0-25 o C, 4h OMe Oi-Pr 95% n-BuLi, THF

I

OMe CHO

3

Oi-Pr OMe Oi-Pr Pd(PPh 3 ) 4 , CuI, NEt 3 66% OHC 80% 2 steps Br

4

Br

5

OMe Oi-Pr Oi-Pr OMe

6 Christian P. Ridley,

et al

. Bioorg. Med. Chem.,

2002

, 10: 3285-3290.

31 31

Application of Banwell’s Approach

i-PrO MeO

6

OHC OMe Oi-Pr i-PrO i. MCPBA, KHCO 3 , CH 2 Cl 2 89% ii. NH 3 , CH 2 Cl 2 /MeOH(1:1) 98% iii. ICH 2 COOH, DCC, DMAP, CH 2 Cl 2 90% MeO MeO MeO ClCH 2 CH 2 Cl N i-PrO MeO MeO MeO

8

N O O I OMe Oi-Pr I O O

7

OMe Oi-Pr

32 32

Application of Banwell’s Approach

i-PrO MeO MeO MeO

8

N O O I OMe Oi-Pr E t 3 N 54% 2 steps MeO Oi-Pr MeO i-PrO O MeO N O MeO 9 Lam U diisopropyl ether DDQ, CH 2 Cl 2 /EtOH(1:1) 93% HO HO HO HO HO N 11 Lam H O O OH MeO Oi-Pr MeO BBr 3 , CHCl 3 88% i-PrO O MeO N O MeO 10 Lam ¦Á diisopropyl ether 17% 12 steps

33 33

Identification of LAM-D as an Inhibitor of TopoI HO MeO OH MeO

1

MeO N HO HO LAM-D MeO O O OH MeO O MeO N O HO LAM-501

Michael Facompre

et al

. Cancer Research

2003

, 63,7392-7399 34 34

DNA Relaxation Experiment – Topo I Inhibition Efficacy

d

Nck:nicked form II,single-strand break

c a b

35 35

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 36

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 37

Topo I Inhibition: Site Selectivity

CPT specific

38 38

Topo I Inhibition: Site Selectivity

LAM D specific

39 39

Molecular Modeling

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

40 40

Summary of the Study of SAR

Essential HO

14

MeO

13

MeO

9

Essential HO

8

MeO

21

OH

20

O

6

N

5

O

6

LAM-D 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

41 41

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 R 2 R 1 R 2 R 2 R 1 R 1 N CO 2 Me R 2 N CO 2 Me R 1 1 R 1 = Oi-Pr, R 2 = OMe R 2 Br X N H CO 2 Me R 1

Christian A. Olsen,

et al.

Tetrahedron Letters,

2005

, 46: 2041-2044 42 42

Olsen Pla’s Open Chain-Modular Synthetic Route to Lamellarins Synthesis of open chain analogues R 2 N H R 1 CO 2 Me TsO

3

Br NaH, DMF 50% R 2 R 1 N CO 2 Me 94% R 2

10 1

N

3

CO 2 Me

5

R 1

1

NBS, THF 92% Br

2

R 2 R 1 R 2 R 1 Br

5

N CO 2 Me Pd(PPh 3 ) 4 , NaOAc 95%

4

Br Br N CO 2 Me For all compounds R 1 =Oi-Pr,R 2 =OMe

43 43

Olsen Pla’s Open Chain-Modular Synthetic Route to Lamellarins R 2 R 1 R 2 R 1 iPrO i PrO Br

4

N CO 2 Me B(OH) 2

6

Pd(PPh 3 ) 4 , Na 2 CO 3 , DMF R 2 R 1

8

N CO 2 Me AlCl 3, CH 2 Cl 2 R 2 78% 2 steps HO HO

10

N CO 2 Me 35% 9 steps Br

5

HO HO N Br CO 2 Me MeO

7

B O O Pd(PPh 3 ) 4 , Na 2 CO 3 , DMF MeO R 2 R 1

9

N OH CO 2 Me OMe AlCl 3, CH 2 Cl 2 MeO R 2 62% 2 steps HO HO

11

N OH OMe CO 2 Me 27% 9 steps

44 44

Modular Synthesis of Lamellarin D Two sequential and regio-selective bromination and cross-coupling reactions using different substituted arylboronic ester MeO HO MeO MeO N HO Lamellarin D O O OH MeO i-PrO

1 Daniel Pla,

et al

. J.Org.Chem.

2005

,70:8231-8234

N CO 2 Me MeO HO OH OMe MeO HO HO

2

N CO 2 Me MeO MeO N CO 2 Me HO

3 45 45

Modular Synthesis of Lamellarin D N H CO 2 Me i-PrO TsO MeO

4

Br 1)NaH, DMF 50% 2)PdCI 2 (PPh 3 ) 2 ,PPh 3 , K 2 CO 3 MeO i-PrO

1 1

N

3

CO 2 Me HO MeO MeO i-PrO

7

94% NBS, THF HO MeO

6

B O O MeO N CO 2 Me Pd(PPh 3 ) 4 Na 2 CO 3 DMF 78% i-PrO Br

5

N CO 2 Me

46 46

Modular Synthesis of Lamellarin D HO i-PrO MeO MeO i-PrO i-PrO i-PrO MeO MeO

7 11

N CO 2 Me MeO Oi-Pr N Oi-Pr CO 2 Me i Pr-Br,K 2 CO 3 ,DMF 90% i-PrO MeO

10

Oi-Pr B O O Pd(PPh 3 ) 4 , , DMF 87% MeO MeO N CO i-PrO

8

84% i-PrO NBS THF 2 Me MeO Br MeO N CO 2 Me i-PrO

9 47 47

Modular Synthesis of Lamellarin D i-PrO MeO MeO i-PrO

11

MeO N Oi-Pr Oi-Pr CO 2 Me DDQ, CHCI3, MW i-PrO MeO MeO i-PrO MeO Oi-Pr N Oi-Pr CO 2 Me

12

AlCl 3 , CH 2 Cl 2 HO MeO MeO MeO N HO Lamellarin D O O OH NaH, THF 38% 3 steps 9% 13 steps HO MeO MeO HO

13

MeO N OH OH CO 2 Me

48 48

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 49

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 50

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 51