Research Topic Seminar - CCC/UPCMLD

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)


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


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


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


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


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


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


PI 3-kinase Inhibitors other than Wortmannin

IC50 is 500 foldhigher thanwortmannin but iswidely used in cellbiology as it is muchmore stable insolution


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


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



D ring modifications

Remote from furan ring Noticeable effects on inhibitor activityD ring is an important recognition element

10 fold increase in activity

D


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


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


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


Inhibitory activity increases with increasing lipophilic character

J. Med. Chem. 1996, 39, 5021-5024

C


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.


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%


1996 First Chemical Synthesis

Starting from commercially available hydrocortisone

Lengthy 35 step synthesis, 0.01% overall yield





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Research Topic Seminar - CCC/UPCMLD