synthesis of lamellarin d a novel potent inhibitor of dna topoisomerase i wenhui hao march 16 th,...
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Synthesis of Lamellarin D
A Novel Potent Inhibitor of DNA Topoisomerase I
Wenhui Hao
March 16th , 2006
Outline
N
OH
HO
HO
O
O
MeO
MeO
MeO
Lamellarin D
1
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
•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
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 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
10
•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
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
N
N
O
O
OOHH3C
A B CD
E
Wall M et al J.Am.Chem.Soc 1966,88:3888-90 12
Topotecan (TPT)
N
N
O
O
OOHH3C
HO
NCH3
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
14
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
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
N
OH
HO
HO
O
O
MeO
MeO
MeO
Lamellarin D
1
16
•The main pentacyclic array is essentially planar
•The aromatic ring attached to C1 is rotated 90°to the main plane
Structure properties
N
OHHO
O
O
MeO
MeO
MeO
Lamellarin A
MeO
MeO
OH
17
A Growing Family --Three Groups
N
O
O
1
R4
R3
14
13
3
68R1
R2 9
2021
R6R5
5
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
OHHO
OX
MeO
LAM O, X=H LAM P, X=OH
N CO2Me
OHHO
R
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
19
Ishibashi’s Synthesis of LAM-D
NO
HO
MeO
MeO
HO
O
OH
OMe
NOEt
OBn
OH
O
O
Br
N
OEt
O
O
MeO
OBn
OMOM
Br
MeO
BnO
MeO
MeO
BnO
MeO
BnO
MeO
MeO
BnO1
2
3
4
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
N
CO2Me
OMOM
N
H
HO
OMOM
NH
O
H
OMOM
BrCH2CO2Et
N
O
OMOM
CO2Et
Br
Et3N, CH2CI2N
O
O
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
1) LDA, THF
2)
3
1
3
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
5 6 7a 7b
8 R=MOM9 R=H
10
33%, 3 steps
Model Study
7a:7b = 92:8
21
Ishibashi’s Synthesis of LAM-D
6-Benzyloxy-l-(4-benlzyloxy-3-methoxybenzyl)-7-methoxyisoquinoline(3)
CHO NO2
NO2
CO2H
OMe
NHN
BnO
MeO
BnO
MeO
BnO
MeO
BnO
MeO
BnO
MeO
BnO
MeO
BnO
BnO
MeOMeO
11 12 13
15
3
CH3NO2, NH4OAc, AcOH
81%MeONa, MeOH-CH2Cl2
74%
LiAlH4ether-THF
NH2
BnO
MeO
14
DCC, CH2Cl2, 76% 2steps
POCl3, Benzene
57%
OMe
OMe
O
22
Ishibashi’s Synthesis of LAM-D
Methyl 4-Benzyloxy-5-methoxy-2-methoxymethoxybenzoate(4)
OH
CO2Me
OMOM
CO2MeMeO
BnO
OH
CO2Me
BnO OH
CO2Me
BnO
Br
OH
CO2Me
BnO
MeO
16 17 18
194
BnBr, K2CO3
91% 91%
DMF-MeOH63%
MOMCl, t-BuOK, THF
94%
Br2, CHCl3
MeONa
HO
23
Ishibashi’s Synthesis of LAM-D
N
O
OMOM
CO2Et
OBn
Br
MeO
MeO
MeO
BnO
BnO
BrCH2CO2Et
21
N
O
OMOM
OBn
NH
O
OMOM
N
OBn
MeO
MeO
MeO
MeOMeO
MeO
BnO
BnO
BnO
BnO
MeO
BnO
BnO
MeO3
20a 20b
LDA, THF, 63%
OMOM
CO2MeMeO
BnO
4
24
Ishibashi’s Synthesis of LAM-D
4% 14 steps
N
O
OMOM
CO2Et
OBn
Br
NO
O
OBn
OMe
MeO
MeO
MeO
MeO
MeO
BnO
BnO
21
N
O
OH
CO2Et
OBn
Br
MeO
MeO
MeO
BnO
BnO
2
HCl, MeOH
22
BnO
BnO
34% 3 steps
H2, Pd/C, EtOAc
82%
NO
O
OH
OMe
MeO
MeO
LAM-D
HO
HO
Et3N, CH2Cl2
25
26
Activity of Lam D and Lam 11
N
H
O
O
HMeO
MeO
MeO
MeO
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
N
OH
HO
HO
O
O
MeO
MeO
MeO
Lam D IC50(¦Ìm) 0.0105
1
8
9
14
13
2021
26
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
N
OH
HO
HO
O
O
MeO
MeO
MeO
Lam D IC50(¦Ìm) 0.0105
1
8
9
14
13
2021
N
O
O
HOH
MeO
MeO
Lam 3 0.0395
HO
HO
N
O
O
OHMeO
MeO
Lam 6 0.0380
HO
HO
H
N
O
O
OH
MeO
Lam 7 0.0700HO
H
MeO
MeO
27
28
Effect of OH at C-20
N
O
O
HMeO
MeO
HO
Lam 4 0.8500
HO
MeO
N
O
O
OHMeOMeO
MeO
MeO
Lam 5 2.5
MeO
N
O
O
MeO
MeO
Lam 12 >100
MeO OO
MeO
N
OH
HO
HO
O
O
MeO
MeO
MeO
Lam D IC50(¦Ìm) 0.0105
1
8
9
14
13
2021
28
29
Effect of OH at C-8
N
O
O
OHMeOMeO
MeO
MeO
Lam 5 2.5
MeO
C-8 OH, lacks C-14 OH, maintains high activity
Methylation of OH at C-8 , C-14
decrease activity
N
OH
HO
HO
O
O
MeO
MeO
MeO
Lam D IC50(¦Ìm) 0.0105
1
8
9
14
13
2021
N
O
O
OH
MeO
Lam 7 0.0700HO
H
MeO
MeO
29
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
N
O
O
AcO
OO
NBr
+Pd(PPh3)4 , CuI
Et3 N, 99%
AcO HO
18oC, 4h
O
1 2 3 4
5CCH2BrO
K2CO3, MeOH
BrCH2COBr CH2Cl291% 2 steps
67
i. Et3N, THF, 66C, 4h
ii. DDQ, CH2Cl2,
92% 2 stepsTHF
N
30
31
OMe
OH
CHO
OMe
Oi-Pr
CHO
OMe
Oi-Pr
Br
Br
OMe
Oi-Pr
OMe
Oi-Pr
CHO
I
12 3
54
OMe
Oi-Pr
OMe
Oi-Pr
OHC
i-PrBr, K2CO3, DMF
CBr4, Zn0-25oC, 4h
n-BuLi, THF
AgOCOCF3, I2, CH2Cl2
Pd(PPh3)4, CuI, NEt3
PPh3, CH2Cl2
6
94% 95%
80% 2 steps
66%
Christian P. Ridley, et al. Bioorg. Med. Chem., 2002, 10: 3285-3290.
Application of Banwell’s Approach
31
32
N
MeO
MeO
OMe
Oi-Pr
ON
O
I
i-PrO
MeO
MeO
MeO 8
i. MCPBA, KHCO3, CH2Cl2
ii. NH3, CH2Cl2/MeOH(1:1)
iii. ICH2COOH, DCC, DMAP, CH2Cl2
ClCH2CH2Cl
OMe
Oi-Pr
OI
O
i-PrO
MeO
7
OMe
Oi-Pr
i-PrO
MeO
OHC
89%
98%
90%
6
Application of Banwell’s Approach
32
33
OMe
Oi-Pr
ON
O
I
i-PrO
MeO
MeO
MeO
N
O
O
Oi-PrMeOMeO
MeO
MeO
i-PrO
N
O
O
Oi-PrMeOMeO
MeO
MeO
i-PrO
N
O
O
OHHO
HO
HO
HO
HO
8
11 Lam H
E t3N
54% 2 steps
DDQ, CH2Cl2/EtOH(1:1)
93%
BBr3, CHCl3
88%
9 Lam U diisopropyl ether
10 Lam ¦Á diisopropyl ether
Application of Banwell’s Approach
17% 12 steps33
34
Identification of LAM-D as an Inhibitor of TopoI
Michael Facompre et al. Cancer Research 2003, 63,7392-7399
N
OH
HO
HO
O
O
MeO
MeO
MeO
LAM-D
1
N
OH
HO
HO
O
O
MeO
MeO
MeO
LAM-501
34
35
DNA Relaxation Experiment – Topo I Inhibition Efficacy
c
Nck:nicked form II,single-strand break35
a
d
b
36
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
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
N
OH
HO
HO
O
O
MeO
MeO
MeO
LAM-D
8
9
202114
13
5
6
6
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
41
42
Olsen-Pla’s Open Chain-Modular
Synthetic Route to Lamellarins
Christian A. Olsen, et al. Tetrahedron Letters, 2005, 46: 2041-2044
N
R2R1
R2
R1
R2
R1
CO2Me
N CO2MeR2
R1
XBrR2
R1
CO2MeNH
R1 = Oi-Pr, R2 = OMe
1
N-alkylation with p-toluenesulfonate and intramolecular Heck cyclization from Methyl pyrrole-2-carboxylate to Scaffold 1
42
43
For all compounds
R1=Oi-Pr,R2=OMe
Olsen-Pla’s Open Chain-Modular
Synthetic Route to Lamellarins
R1
R2
Br
R2
R1
Br
N CO2Me
NCO2MeR2
R1
NCO2MeR2
R1
Br
NCO2MeR2
R1
BrBr
TsO
101
3
5
NBS, THF
CO2MeNH
2
Pd(PPh3)4, NaOAc
1
95%
4
5
3
NaH, DMF
50%
94%
92%
43
Synthesis of open chain analogues
44
NCO2MeR2
R1
N CO2MeR2
R1
B(OH)2
HO
B
O
O
iPrO
MeO
NCO2MeR2
NCO2MeR2
HO
OH
10
11
HO
HO
AlCl3,CH2Cl26
7
NCO2MeR2
R1
Br
4Pd(PPh3)4, Na2CO3, DMF
Pd(PPh3)4, Na2CO3, DMF
NCO2MeR2
R1
Br Br
5
78% 2 steps
AlCl3,CH2Cl2
62% 2 steps
HO8
iPrO
OH
OMe
MeO
HO
MeO
OMe
9
27% 9 steps
35% 9 steps
Olsen-Pla’s Open Chain-Modular
Synthetic Route to Lamellarins
44
45
N
O
O
OH
HO
HO
N CO2Me
N
OH
HO
HO
OMe
CO2Me
N CO2Me
HO
HO
MeO
MeO
MeO
MeO
MeO
MeO
i-PrO
MeO
MeO
2
3
1
Lamellarin D
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
N CO2Me
BrN CO2Me
HO
B
O
O
H
TsOi-PrO
MeO
MeO
i-PrO
MeO
Pd(PPh3)4 Na2CO3 DMF 78%
4
1
N CO2MeMeO
i-PrO5
Br
NBS, THF
6
N CO2Me
HO
MeO
MeO
i-PrO7
1)NaH, DMF
2)PdCI2(PPh3)2,PPh3, K2CO3
50%
94%
1
3
Modular Synthesis of Lamellarin D
46
47
NCO2Me
i-PrO
MeO
MeO
i-PrO
8
N CO2Me
BrMeO
MeO
i-PrO
i-PrO
9
N CO2Me
HO
MeO
MeO
i-PrO 7
B
O
O
Oi-Pr
MeO
Pd(PPh3)4, , DMF 87%
i-PrO
10
N
Oi-Pr
CO2Me
Oi-Pr
MeO
MeO
i-PrO
i-PrO MeO
11
NBS THF
90%
i Pr-Br,K2CO3,DMF
84%
K2CO3
Modular Synthesis of Lamellarin D
47
48
N
Oi-Pr
i-PrO
i-PrO
CO2Me
Oi-Pr
N
OH
HO
HO
O
O
DDQ, CHCI3, MWMeO
MeO
MeO
NaH, THFMeO
MeO
MeO
Lamellarin D
N
Oi-Pr
CO2Me
Oi-Pr
MeO
MeO
i-PrO
i-PrOMeO
1112
N
OH
CO2Me
OH
MeO
MeO
MeOHO
HO
AlCl3, CH2Cl2
13
38% 3 steps
9% 13 steps
Modular Synthesis of Lamellarin D
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 yield 9%
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
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