research topic seminar - ccc/upcmld
TRANSCRIPT
Research Topic Seminar
Dr. Claire Coleman
The Chemistry and Biology ofWortmannin
Claire Coleman @ Wipf Group 1 3/13/2004
•Off white to pale yellow solid
•Hygroscopic/Light sensitive
•Small molecule natural product first isolated from culturefiltrates of Penicillium Wortmanni (1957)
•Later found to be a metabolite of a variety of Penicilliumand Myrothecium species
•Structure by chemical degradation and spectroscopicanalysis 1972
•X-ray analysis 1972
•Clinically used as an immunosuppressive and anti-inflammatory
•Commercially available AG Scientific 25 mg $ 330Claire Coleman @ Wipf Group 2 3/13/2004
Wortmannin and simple analogs were found to be potent anti-inflammatory agents 1970’s
Exhibited a high degree of toxicity-determined unsuitable forclinical development
20 years later Wortmannin was found to be a potent andselective inhibitor of PI 3-Kinase (IC50 = 4 nm)
Claire Coleman @ Wipf Group 3 3/13/2004
Phosphatidylinositol-3-kinase (PI-3-Kinase)
Important enzyme for intracellular signalling
Primary enzymatic activity of the PI-3-Kinases is the phosphorylation of inositol lipids at the 3 position
Different members of the PI-3-Kinase family generate differentlipid products (lipid secondary messengers)
Vesicle traffickingCell survivalProliferationCell migrationVesicle budding
Claire Coleman @ Wipf Group 4 3/13/2004
Signaling through PI 3-K lipid products and their targets. The lipid products of PI 3-K are indicatedat the top of the figure, and the cellular processes affected by these lipids are indicated at the bottom. Theblack ovals indicate the direct targets of each lipid, and the small boxes indicate the protein domains thatdirectly bind to them.
J. Biol. Chem. 1999, 274, 8347
Claire Coleman @ Wipf Group 5 3/13/2004
PI3-Kinase was initially purified and cloned as a heterodimericcomplex consisting of
110 kDa catalytic subunit now called p110a85 kDa regulatory/adaptor subunit p85 a
9 mammalian PI-3-kinases have been identified
Divided into 3 classes based on sequence homology andsubstrate preference in vitro
Claire Coleman @ Wipf Group 6 3/13/2004
Class I4 class I enzymesDivided into 2 subclasses (Iaand Ib) based on mechanism ofactivationFound in cytoplasm
Class II3 membersLeast understoodLarger than class I/IIIMembrane associated
Class IIITraffic of proteinsthrough the lysosome
Claire Coleman @ Wipf Group 7 3/13/2004
Wortmannin-an irreversible inhibitor of PI 3-Kinase
Nucleophilic attack at the electrophilic C-21 position of the furan ring
by Lys802 of p110 PI 3-Kinases
The Lysine residue resides in the ATP binding site of the p110
catalytic subunit--crucial role in the phosphotransfer reaction
OO
O
H
O
MeO AcO
NH2 Wortmannin ( 1)21
(p110 PI 3-kinase)K802
OO
O
H
MeO AcO
OHHN
(p110 PI 3-kinase)K802
A B
C D
OO
Claire Coleman @ Wipf Group 8 3/13/2004
Most protein kinase inhibitors developed for pharmaceutical application work by competing with
ATP bindingWortmannin binds more deeply in the ATP
binding pocket than ATPClaire Coleman @ Wipf Group 9 3/13/2004
Regulation of cell proliferation and the signaling pathways withincells that control it is important
If drugs can be developed to interfere with signal transductions thencancer cells may be made nonproliferating and new cancer therapiesmay emerge
Inhibitors of PI 3-kinase (an enzyme that functions within signaltransduction pathways) may be useful for new cancer and tumourtherapies
Claire Coleman @ Wipf Group 10 3/13/2004
PI 3-kinase Inhibitors other than Wortmannin
IC50 is 500 foldhigher thanwortmannin but iswidely used in cellbiology as it is muchmore stable insolution
Claire Coleman @ Wipf Group 11 3/13/2004
The furan ring in Wortmannin is essential for biological activity
Chemists at Eli Lilly performed SAR on Wortmannin analogs to probe the structural requirements necessary for PI 3-Kinase inhibition
Made electronic/steric changes that influenced the elctrophilicityof the C-21 centreClaire Coleman @ Wipf Group 12 3/13/2004
O
O
OO
AcO
O
MeO
H
Wortmannin (1)
NuH
O
O
HOO
AcO
O
MeO
H
Nu
Nu
Et2N 80
NH2 >>500
MeNH >>500
EtNH >>500
n-PrNH >>500
n-BuS 52
PI 3-KIC50 (nM)
1 and 6 retain activity but greatly reduced
Claire Coleman @ Wipf Group 13 3/13/2004
Primary (Z) vssecondary amine(E) adducts
Difference in reactivity suggested to be differencein orientation of the enamine in relation to thelactone carbonyl
E configuration mimics wortmannin in ability toaccept a nucleophile
Z--steric problemsClaire Coleman @ Wipf Group 14 3/13/2004
Introduction of methyl groupat C-21No inhibition up to 500 nM
No reaction with diethylamineClaire Coleman @ Wipf Group 15 3/13/2004
When the furan is replaced by a pyran ring it has only moderate activity--pyran is not planar/thermodynamics
Wanted to prepare4,21-cyclopropyl wortmannin but isolated a ring expansion productinstead
Reacts with xs ylide to give a the desired product as a mixtureof diasterisomers
Claire Coleman @ Wipf Group 16 3/13/2004
Claire Coleman @ Wipf Group 17 3/13/2004
D ring modifications
Remote from furan ring Noticeable effects on inhibitor activityD ring is an important recognition element
10 fold increase in activity
D
Claire Coleman @ Wipf Group 18 3/13/2004
B ring modifications
Have been limited
Deconjugated derivative remained active
Aromatized derivative lost activity
O
O
O
OO
MeO
H MeO
O
O
OHOH
Me
IC50 = 54 nM IC50 = 4600 nM
B B
Claire Coleman @ Wipf Group 19 3/13/2004
A ring modifications
O
O
OO
AcO
O
MeO
H
Wortmannin (1)
1. HCl, MeOH
2. NaOH MeOH/THF
O
O
OOH
AcO
O
H
1000 fold loss of activity and no toxicity
Bioorg. Med. Chem. Lett. 1995, 5, 1183
A
Claire Coleman @ Wipf Group 20 3/13/2004
C ring modifications
Removal of C-11 acetoxy group
J. Med. Chem. 1996, 39,1106
54 nM 17 nM 4600 nM
Anti-tumour activity(Wortmannin 4.2 nM)
C C C
Claire Coleman @ Wipf Group 21 3/13/2004
Inhibitory activity increases with increasing lipophilic character
J. Med. Chem. 1996, 39, 5021-5024
C
Claire Coleman @ Wipf Group 22 3/13/2004
Wortmannin beyond anti-proliferation studies
•Radiation sensitizer (but maysensitize normal cells over tumourcells) Rad. Res. 2001, 155, 826.
•Alzheimers disease-alters effectson the metabolism of theAlzheimers amyloid precursorprotein (APP) J. Neurochem,1999, 73, 2316.
•Treatment of type I osteporosis-ability to inhibit osteoclasts fromresorbing bone Pharm. Exp. Ther.1995, 277, 543.
Claire Coleman @ Wipf Group 23 3/13/2004
Synthetic Efforts towards Wortmannin
J. Org. Chem. 1992, 57, 4888
First synthesis of the furancyclohexadiene lactone subunit1992
OO
1. O
O
O
185 0C
2. Red-Al
OH
HO +
26%
OH
HO
13%
1. Pivaloyl chloridepyridine, -30 0C
2. TBSPS-Cl3.OsO4/nmo
OTBSPS
PivO
HOOH
OTBSPS
MsO
OO
1.OMeOMe PPTS
2. MELI3. MSCL, NEt3
87%
OTBSPS
OHC
OO
1. NaCN DMSO2. DIBAL
77%
1. TBAF2. PDC, 3A MS3. PPTS1:1 MeOH/H2O
77%HOOH
OODESS-MARTINPERIODINANE
HOO
OOHC(NMe2)3
32%, 2 steps
O
OO
Me2NHO
Cu(OAc)2
O
OO
Me2NHO
O
OO
O
HCl
42%
Claire Coleman @ Wipf Group 24 3/13/2004
1996 First Chemical Synthesis
Starting from commercially available hydrocortisone
Lengthy 35 step synthesis, 0.01% overall yield
Claire Coleman @ Wipf Group 25 3/13/2004
Claire Coleman @ Wipf Group 26 3/13/2004
Claire Coleman @ Wipf Group 27 3/13/2004
Claire Coleman @ Wipf Group 28 3/13/2004