1 be 10988 : an inhibitor of dna –topoisomerase ii indira thapa november 24, 2005
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BE 10988 : An Inhibitor of BE 10988 : An Inhibitor of DNA –Topoisomerase IIDNA –Topoisomerase II
Indira ThapaIndira ThapaNovember 24, 2005November 24, 2005
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DNADNA
Sugar-phosphate backbone
Bases
H-bonds
Double helical structure
Store genetic information
Uncontrolled cell growth-Cancer
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DNA - A Molecular Target for DNA - A Molecular Target for Cancer TherapeuticsCancer Therapeutics
DNA-DNA crosslinker e.g. mitomycin C Intercalator e.g. ellipticine Double-stranded break e.g. bleomycin Develop drug resistance and mutagenesis
DNA binding during replication - Topoisomerase II
New anticancer drug target
Protein-DNAcomplex
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OverviewOverview
DNA Replication and Supercoiling
Topoisomerase II - A nuclear enzyme -Importance and functions in cellular processes -Enzymatic action mechanism & interaction with inhibitor
BE 10988 -Biological properties -Total synthesis
Structure Activity Relationship (SAR) Studies
Summary
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DNA ReplicationDNA Replication
DNA helix rotation during replication
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Supercoil formed during replication
DNA Supercoiling DNA Supercoiling
Supercoiling - coiling of coils
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Topoisomerase II (Topo II )Topoisomerase II (Topo II )
FUNCTIONS: transiently breaks and reseals double stranded DNA simultaneously, and allows the passage of separate double helical strand through the break site.
Eukaryotic topoisomerases - type I and type II
Homologous dimer
Separate intact DNA
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Mechanism of Action of Topoisomerase IIMechanism of Action of Topoisomerase II
“Cleavable complex”
N-terminal
C-terminal
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Action of InhibitorAction of Inhibitor
Inhibitor stabilizes“Cleavable complex”
Permanent DNA double strand break
Cell Death
Blocks re-ligation
inhibitor
inhibitor
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Interest in Topo II InhibitorsInterest in Topo II Inhibitors
Without Topo II DNA cannot replicate normally
Inhibitors of topoisomerase II have been used as anticancer drugs
Proliferating tumour cells show high levels of topo II activity and this proliferative activity has in turn been associated with increased tumour cell
sensitivity to topoisomerase II – interactive drugs.
Selective inhibition of topoisomerase II in cancer cells could lead to a new approach to cancer therapy
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BE 10988BE 10988
Isolated from the culture broth of a strain of actinomycetes in 1991
Potent topoisomerase II inhibitor
H2N
NMe
O
O
NS
NH2
O
Shizuri, Y. et. al. J. Antibiot. 1991, 44, 486-491
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Biological PropertiesBiological Properties
Shizuri, Y. et. al. J. Antibiot. 1991, 44, 486-491
Relaxed Plasmid DNA
H2N
NMe
O
O
NS
NH2
O
Showed growth inhibitory activity against resistant mouse tumour cells
Topoisomerase II-DNA complex formed
Inhibited the relaxation of plasmid DNA
SupercoiledPlasmid DNA
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First Total Synthesis-RetrosynthesisFirst Total Synthesis-Retrosynthesis
Moody, C. J.; Jones, G. B.; J. Chem. Soc. Perkin Trans. 1, 1989, 2455 Moody, C. J.; Swann, E.; J. Chem. Soc. Perkin Trans. 1, 1993, 2561
N
NS
Me
H2N
NH2
O
O
O
N
NH2S
Me
MeOBnO
N
Me
MeOBnOOH O
OH
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First Total SynthesisFirst Total Synthesis
OH O
OH
OH O
OMeH2SO4, MeOH
97%
OH O
OMeCl2CHOMeTiCl4; CH2Cl2 H
O
0 oC, 77%
Moody, C. J.; Jones, G. B.; J. Chem. Soc. Perkin Trans. 1, 1989, 2455 Moody, C.J.; Swann, E.; J. Chem. Soc. Perkin Trans. 1, 1993, 2561
BrDMFNaH
OBnO
OMeH
O
83%
BnO O
OMeHO m-CPBA
H3O, 96%
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BnO O
OMeHO
Me2SO4 K2CO3
BnO O
OMeMeO
71%
LiAlH4 BnO O
HMeO
1.
2. BaMnO473%
MeO2CCH2N3NaOMe, MeOH
-15 oC
BnOMeO CO2Me
N3
80%
Xylene, reflux
BnO
MeO
NH
CO2Me96%
First Total SynthesisFirst Total Synthesis H2N
NMe
O
O
NS
NH2
O
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BnOMeO
NH
CO2Me ether-THF
BnOMeO
NH
CH2OHLiAlH4
99%
BaMnO4CH2Cl2reflux
BnOMeO
NH
CHO
61%
[Rh(Ph3P)2 CO]ClPh2P(CH2)3PPh2Mesitylene, 82%
BnOMeO
NH
First Total SynthesisFirst Total Synthesis
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BnOMeO
NH
KH , MeI
DMF 91%
BnOMeO
NMe
97%
OBnMeO
NMe
NHSO2ClO
Cl SO
ON C O
Et2O
Bu3SnH, AIBN
PhH 88%
OBnMeO
NMe
NH2
O
First Total SynthesisFirst Total Synthesis H2N
NMe
O
O
NS
NH2
O
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First Total SynthesisFirst Total Synthesis
OBnMeO
NMe
NH2
O
PhH 94%
OBnMeO
NMe
NH2
S
Fremy's saltNaH2PO4
MeO
NMe
NS
OEt
OO
O 65%Acetone
BrCH2COCO2Et
EtOH, reflux
OHMeO
NMe
NS
OEt
O
MeO P OMePS
S
S
S
H2N
NMe
O
O
NS
NH2
O
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First Total SynthesisFirst Total Synthesis
MeO
NMe
NS
OEt
OO
O
Liq. NH3H2N
NMe
NS
NH2
OO
O72%
H2N
NMe
NS
NH2
OO
O28% overall yield
OH O
OH
BE 10988
20Nicolaou, K. C. et al. J. Am. Chem. Soc. 2002, 124, 2221-2232
Nicolaou’s Synthesis Synthesis
H2N
NMe
NS
NH2
OO
O
Formylation
NMe
HO
BzHN
+
HSCO2Me
NH3 Cl
DMPMediatedOxidation
Retrosynthetic - AnalysisRetrosynthetic - Analysis
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NMe
H2N
NMe
BzHN
CH2Cl2, 0 25 oC100%
BzCl , Et3N
H2N
NMe
O
O
NS
NH2
O
POCl3DMF -20 0 oC
100%
NMe
BzHNCHO
Nicolaou’s Synthesis
HSCO2Me
NH3 Clpyridine
1.
2.
BzHN
NMe
NS
OMe
O
85%
MnO2Pyr / PhH
Nicolaou, K. C. et al. J. Am. Chem. Soc. 2002, 124, 2221-2232
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BzHN
NMe
NS
OMe
O
DMP/ H2O
CH2Cl2, 25 oC
BzHN
NMe
NS
OMe
OO
O67%
H2N
NMe
O
O
NS
NH2O
IO
O
AcO
OOAc
O
OI O
OAcOH2O
DMP Ac-IBX
Nicolaou’s Synthesis
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N
NMe
NS
OMe
O
I OOAcO
O OAcC
O
H3C
HO
Ph
-AcOH N
N
O
I
O
O
OAc
MePh
AcO
O
I O OAcO
N
NMe
O
PhH
I O O
O
AcOOAc
I O OAcO
NN
S
O
Ph
O
O-Imidoquinone Intermediate
I O OAcO
NMe
O
OMe
Proposed Mechanism
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I O OAcO
NMe
NS
OMe
OHN
O
O
Op-quinone
IO
O
AcO O
N
N
O
PhO
Me
pp-quinone Formation-quinone Formation
OIHO
O
AcON
HN
O
PhO
H
OAc
Me
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HH22OO1818 Isotope Labeling Studies Isotope Labeling Studies
I O OAcOAcO
OAc
H218O (2.0 eq)
CH2Cl2, 25 oCUltrasound,1 min
I O O18OAcO
I O OAcOAcOOAc
(2.0 eq)Ac-IBX18O
DMP(2.0 eq)
(4.0 eq)DMP
18O
18O
p-quinone
HN
OR
HN
OR Ac-IBX18O
DMP
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BE 10988 – Nicolaou’s SynthesisBE 10988 – Nicolaou’s Synthesis
liq.NH3
THF, -78 25 oC
H2N
NMe
NS
NH2
OO
O
24% overall
BzHN
NMe
NS
OMe
OO
O
BE 10988
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Summary : Total SynthesisSummary : Total Synthesis
OH O
OHH2N
NMe
O
O
NS
NH2
O
18 steps
28% overall yield
Moody’s synthesis: 1993
Nicolaou’s synthesis: 2002
NMe
H2N
24% overall yield
5 steps H2N
NMe
O
O
NS
NH2
O
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SAR StudiesSAR Studies
H2N
NMe
O
O
NS
NH2
O
Quinone system
Thiazole moeity
Which part of the molecule is essential ?
Bailly. C. et. al. Bioorg. Med. Chem. Lett. 9, 1999 2025-2030
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Analogues of BE 10988Analogues of BE 10988
NMe
CH3O
CH3O
NS
NH2
O
H
NMe
O
O
NS
NH2
O
H
NMe
CH3O
CH3O
CONH(CH)2N OH
NMe
O
O
CONH(CH)2N OH
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3344
Bailly. C. et. al. Bioorg. Med. Chem. Lett. 9, 1999, 2025-2030
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Growth Growth Inhibitory Activity
1
NMe
CH3O
CH3O
NS
NH2
O
H
2
NMe
O
O
NS
NH2
O
H
Mouse leukemia cells
Mouse leukemia cells
+
+
IC 50 (µM)
> 10
0.8
Indolequinone skeleton is essential to the activity
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Growth Inhibitory ActivityGrowth Inhibitory Activity
3
NMe
CH3O
CH3O
CONH(CH)2N OH
4
NMe
O
O
CONH(CH)2N OH
+
+
Mouse Leukemia cells
Mouse leukemia cells
IC 50 (µM)
> 100
6.5
Thiazole ring is also important for the activity
Analogue 4 is 8 fold less active than analogue 2
2
NMe
O
O
NS
NH2
O
H
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Reaction Catalyzed By Topo II
Decatenation
Catenated circular DNA
Decatenated circular DNA
prokaryotic system
simple plasmid DNA
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Topo II Inhibition: Decatenation AssayTopo II Inhibition: Decatenation Assay
2
NMe
O
O
NS
NH2
O
H
4
NMe
O
O
CONH(CH)2N OH
KDNA (catenated circular DNA)thiazolylindolequinone
Morpholino derivative
SDS Proteinase K
KDNA
SDS Proteinase K
Standard for comparision
Ele
ctro
ph
ores
is
EtoposideEtoposide
Topoisomerase II
Topoisomerase II
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Topo II inhibition: Decatenation AssayTopo II inhibition: Decatenation Assay
2
NMe
O
O
NS
NH2
O
H
4
NMe
O
O
CONH(CH)2N OH
50µM
EffectiveEffective
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2
NMe
O
O
NS
NH2
O
H
Linear DNA
Circular plasmid DNA
DNA Cleavage AssayDNA Cleavage Assay
4
NMe
O
O
CONH(CH)2N OH
Drug inhibits re-ligation of DNA once the double helix is cleaved by the enzyme
2
NMe
O
O
NS
NH2
O
H
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H2N
NMe
O
O
NS
NH2
O
Summary : SAR StudiesSummary : SAR Studies
Quinone system is essential to activity
Thiazole moeity plays a majorrole in topoisomerase IIinhibition
Analogue 2 is weak inhibitor of topoisomerase II, suggests that - NH2 substituent at quinone ring is also important for activity
Required
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DNA BindingDNA Binding
Non-specific binding
Analogues do not intercalate into DNA
Study supports the fact that BE 10988 does not bind DNA covalently
Further studies have Shown:
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Conclusions Conclusions
Two total syntheses of BE 10988
SAR studies of BE 10988 using four different analogues
More detailed mechanistic studies need to be done on BE 10988
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AcknowledgementsAcknowledgements
Dr. Robert Ben
Ben Research GroupVincent BouvetFrank CeaseSuhuai LiuPawel CzechuraElisabeth von MoosRoger TamJennifer ChaytorJessica JackmanNicole Le GrandAleksandra Paliga
Alison Lemay and …You
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Mechanistic StudiesHN
ODMP (2.eq)
Ac-IBX18O (2.eq)
N
OO
O-imidoquinone
18O
18O
O
N
O
18 H
H
H+
18
p-quinone
Anilide
NH
The new oxygen atom in the reaction is derived from Ac-IBX.
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2
NMe
O
O
NS
NH2
O
4
NMe
O
O
CONH(CH)2N O Compds 2 & 4 in presence of DNA
DNA Binding DNA Binding
Non –specific binding of drugs to DNA
Drugs trigger double stranded DNA cleavage via topoisomerase II
Compds 2 & 4
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inhibitor
inhibitor
inhibitor
inhibitor
Formation of Ternary ComplexFormation of Ternary Complex
inhibitor
Route 2
Route 1 Route 3
Fortune JM, et. al,; Prog Nucleic acid Res Mol Bio 64:221-253
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OBnMeO
NH
Oxidative addition or C-H Insertion
Rh
PPh2(CH2)3PPh2
-2PPh3
MeO
NH
OBn
MeO
NH
OBn
Ph3P
Ph3P CO
Cl
RhCO
Cl
P
P
PhPh
PhPh
HR
O
Rh
Cl
P
P
PhPh
PhPh
H R
RhCO
Cl
P
P
PhPh
Ph Ph
- CO
CO
= R
Migratory insertion & decarbonylaion
Reductive elimination O
H
DecarbonylationDecarbonylation