heidelberg 2012 c isotopic chemistry: the most varied of careers.. bill lockley university of surrey...
TRANSCRIPT
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Heidelberg 2012
C
Isotopic chemistry: the most varied of careers..
Bill LockleyUniversity of Surrey
…with tritium & deuterium, the most versatile of the isotopes.
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Heidelberg 2012
Aims of the talk
Give some examples of the versatility of 3H and 2H
URL for slides at end of talk
Why do I say that D & T are so versatile?
How 3H contributes to problem solving in biochemistry
Give a mix of historical and scientific content
Including a Melvin Calvin talk in the symposium is novel.
Science content will increase as talk progresses!
Labelling methodology development for 3H & 2H
How 3H can contribute to successful pharma. projects
Current work at Surrey (time permitting)
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Heidelberg 2012
C
Tritium and deuterium the most versatile of the isotopes
There are ca. 1080 atoms in the observable universe
Four out of five of these are hydrogen
Nearly all organic compounds contain hydrogen
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Heidelberg 2012
H2O2 C2H5OH
Cu(NH3)4 SO4
CO2+ Thrust+ Steam =
1957 Schooldays: Rockets and catalysis
WJ S Lockley, G Hall, A Kinsella, R Hackett, G Crookes, D Taylor, unprosecuted results, 1958
Chemistry Physics Engineering
My first scientific team
H2O2C2H5OH
Cu(NH3)4SO4
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Heidelberg 2012
1966: First degree: more fireworks
Brian Iddon: Time to put some “flash, bang, wallop” back into chemistry lessons
Brian puts some flash, bang, wallop back into parliament
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Heidelberg 2012
1968-1971: Naïve PhD student
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Heidelberg 2012
O
O OH OH
OHOHOHOHOHOHR
W J S Lockley, T P Toube, B C L Weedon et al. Tet. lett, 471-474 (1970)
J. C. S. Perkin, 1848-1856 (1972).
Flavofungin: A polyene macrolide antifungal
OH OH
OHOHOHOHOHOH
OH
OH
O
O OH OH
OHOHOHOHOHOH
LiAlH4
Pt
Or
HI Red P
H2
323 amu
323 amu
211 amu
211 amu
85 amu
85 amu
Basil Weedon Trevor Toube
DD
323 amu213 amu
85 amu
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1971-72: Get more synthesis experience
Organic chemistry is the chemistry of carbon compounds. Biochemistry is the study of carbon compounds that wriggle.
N N O
R
RN
+N C O
R
R
R' R'
NN+
R
R
O
R'
R'
NN
O
R'
RR
R'
N
R
R
O
N3
Heat
Light
Walter Lwowski “If you want tritium work you need to go into biochemistry.”
Owen Weekes
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Heidelberg 2012
1972-77 Off to Liverpool!
www.sareenarts.com
Biochemistry, Botany, Beetles and Beatles!
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Heidelberg 2012
Biosynthesis is verycomplex: 3H can make it simpler!
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Heidelberg 2012
Carotenoids
Steroids
AcetateTrevor
Goodwin
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Bad Soden 2011
C
Plants resort to chemical warfare in the battle with insects
HughHeffyn Rees
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Heidelberg 2012
C
Moulting (ecdysis) is essential for insects
The process is triggered and controlled by steroid hormones which control an array of genes via binding to nuclear receptors
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Bad Soden 2011
C
These hormones are ecdysteroids
O
OH
OH
OH
OH
OH
H
OH
O
OH
OH
OH
OH
OH
H
O
OH
OH
OH
OH
OH
H
OH
They are made from cholesterol
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Heidelberg 2012
C
How can we use tritium to track the steroid A/B ring conversion reaction
in plants?
OH
Me
Me
OH
H
OOH
OHT
OH
T
OH
T
Synthesis of stereospecifically tritiated [4-3H]cholesterols, W J S Lockley, H H Rees, T W Goodwin, J. Lab. Comp. Radiopharm., 15, 413-423 (1978).
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Bad Soden 2011
C
Preparation of the labelled cholesterols.
O OH
T
NaBT4
OH
DCC/DMSO
3α-T
OO
O
OOH
O T
NaBH4(T)
OH
T H
O
OT Cl
SOCl2/Pyr LiAlH4TLC sepn
MeOH
4α-T
Cl
O
O
O
OOH
SOCl2
Pyr.
LiAlH4(T)
OHT H
4β-T
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Bad Soden 2011
C
Overall mechanism in P. vulgare.
OH
Me
O
Me
H
OH H
H
H+
Me
OHO
H
HH
O2
O
OH
OH
OH
OH
OH
OH
HH H
W J S Lockley, H H Rees, T W Goodwin et al. Biochem J, 174, 397-404 (1978).
Only 3H and 2H can report such migrations
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Heidelberg 2012
Lucillia cuprina
Calliphora vicina
Schistocerca gregaria
Locusta migratoria
3H-studies showed that the mechanism in all these species was similar. They all use cholesterol to make their hormones
But [14C]mevalonate studieshad shown that insects cannot synthesise sterols de novo
So how do they get the cholesterol to make their hormones?
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Heidelberg 2012
OH
Alkyl
OH OHPlant sterol Desmosterol
Dealkylation
Enzymes
Cholesterol
Reduction
They get it from the plant sterols in their diet by removing the 24-alkyl
substituents
How do they do this?
Tenebrio molitor Tribolium confusum
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OO
OH
C14
T
(BH3)NaBT4
Hg2Cl2
OR
C14 I
H
H
H
Wittig route
5 Steps 4 Steps
OsO4/NaIO4
Heidelberg 2012
[25-3H,26-14C]Clionasterol
Preparation of [25-3H,26-14C]clionasterol
P J Pettler, W J S Lockley, H H Rees, T W Goodwin, Biochem. J., 174, 397-404 (1978)
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T
O
R
C+
O
T
Enzyme
ROH
T
OH
T
Enzyme+
Heidelberg 2012
Yet again 3H-labelling shows a key proton migration
But what about 2H-applications?
CH3CHO2H
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Heidelberg 2012
Biosynthesis of carotenoids
George Britton
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Heidelberg 2012
Putative cyclisation reaction
H
H+
H
H
H
+
Proton initiated cyclisation
Proton loss
Carotenoid (with incorporated proton from the medium)
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Heidelberg 2012
Cyclisation in Scenedesmus obliquus PG1 a green alga)
Grown on alcohol in the dark
Grown on CO2 in the light
Only produces acyclic carotenoids
Produces cyclic carotenoids
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Heidelberg 2012
The experiment is now simple
OH
OH
1. Grow scendesmus on alcohol in the dark in water
2. Spin down and replace the water with D2O
3. Switch on the light and feed in CO2
4. Extract the carotenoids, record MS & NMR
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Heidelberg 2012
MS of isolated β-carotene
Same result for all cyclic carotenoids isolated. No labelling seen for acyclics
0
10
20
30
40
50
60
70
80
90
100
M/Z
536
538
582m/z =536
De-novo synthesis
D DD D
W J S Lockley, G Britton, T W Goodwin, et al. Nature, 268, 81-81 (1977)
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Heidelberg 2012
NMR studies then revealed the complete cyclisation process.
C13 C+
C13
D
HC13
C13
C13
D
HC13
C13
C13
C13
H
H
D+
C13 C13 C13 C13
C13
C13
C13
C13
OH
OH
DD
[2,2’-2H2]Zeaxanthin
W J S Lockley, G Britton, T W Goodwin, et. al. Chem Commun, 655 (1977) 2H studies & 27 (1979), 2H &13C-studies.
LycopeneCarotene
2H Can identify interactions between a test system and its surrounding medium
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Heidelberg 2012
1977: Enough of playing around.. Get a Real Job!
“Successful projects are not about GANTT charts, resource allocation, or bean counting. They are about getting the best people, putting them in the right places, and giving them real responsibility.”
John Dixon.
“Put some chemistry into radiochemistry”
Dennis Smith,
Putting some real science into DMPK
DavidWilkinson
Family responsibilities!
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O
O
Na
H
Chromone 2-carboxylates : annexin-A1 releasing agents.
(leukotriene supressors)
Heidelberg 2012
This site would be good for labelling the whole class. But how?
Needed the labelled compounds for immunoassay development, human DMPK and mechanism studies
Model
O
O
O
NaO
R
R'
R''
R'''
H
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1978 on : Learning about metal catalysed isotope exchange.
Heidelberg 2012
Tony Evans
Derek Brundish
Phil Williams
Mervyn Long
John Jones
N F Myasoedov
CharlesKemball
John Garnett
VictorGold
A E Shilov
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How to control metal catalysis to get regioselectivity?
Heidelberg 2012
OOH OO
H
D
D
DR R
HFBA
Pd black
D2O
145 oC
OONa OO
Na
DD
R R
D2O
1% Pd/C
145 oC
97% meta/para at 2hand 53% D
99% ortho at 18h and 60%D
To get labelling β-to carboxyl must maximise the interaction
of the carboxyl with the catalytic centre.
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Maximising the catalyst interaction.
Heidelberg 2012
Game on!
Mervyn Long’s work
D
D
D
DD
D
CH3CO2D
RhCl3 (H2O)3
D2O
OONa OO
Na
DD
R
RhCl3 (H2O)3
D2O
DMF 107o
Maximise interaction with the metal centre
OOH OO
H
D
D
D
DD
RCH3CO2D
RhCl3 (H2O)3
D2O
Try it with the model
>99% ortho
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A general method
Heidelberg 2012
O
O
CO2H
H*
S
O
CO2H
H*
NH
O
CO2H
H*
CO2H
H*
H*
CO2H
H*
H*
CO2HN
H*
H*
O CO2H
H*
O CO2H
H*
H*S CO2H
H*
CO2Na
H* CO2Na
H*
Carboxylates
NH2O
H*H*
NH2
H* H*
NH
O
H* H*
NNH
H*H*
NH
NN
N
H*H*
N
H* H*
Non-carboxylates
Only worked when the directing group geometry allowed a 5-ring
with the Rh
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First example of use. Excretion of sodium cromoglycate
Heidelberg 2012
O
O
OH
O
O
OH
O
O
O
O
OH
Sodium cromoglycateIntalR
W J S Lockley, D J Wilkinson, J. Lab. Comp. Radiopharm., 21, 363-373 (1984).
O
O
OH
O
O
OH
O
O
O
O
OH
Sodium cromoglycateIntalR
Now of interest in a range of new medical conditions
O
O
OH
O
O
OH
O
O
O
O
OH
TTRhCl3(H2O)3
T2O / DMF
Compound not metabolisedNo known drug interactions
Why? How to maintain thisin other follow on drugs
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Microautoradiograph: rat liver
Heidelberg 2012
Explains: No metabolism, lack of adverse drug interactions
W J S Lockley, A Murphy, unpublished results (1983). See also M J Potchoiba, M R Nocerini, Drug Met Disp, 32, 1190-1198 (2004).
Tritium can track behaviour at the cellular level.
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Heidelberg 2012
1982. 1st Kansas City Isotope Symposium
John Jones
“The geometry of the substrates suggest this type of cyclic metallated intermediate. However I have no kinetic data in support as yet”
“ About that kinetic data….
Alex Susan, Bill Duncan
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Heidelberg 2012
First of a series of joint Ph.D. students
David Hesk
Application to the labelling of 25 common drugs with tritium
Probenecid, nedocromil, cromolyn, etenzamide, phenacetin, niclosamide, sulfanitranantipyrine, paracetamol, phenylbutazone, metoclopramide, aminopyrine, cinchocaine
acecainide, labetalol, mefenamate, phthalylsulfacetamide, phthalylsulfathiazole,hippurate, flufenamate, procainamide, alpidem, zolpidem, flecainide, sulpiride
J Pharm Sci, 80, 887-890 (1991).
10
10.5
11
11.5
12
12.5
13
13.5
0 20 40 60 80
Time (Hours)
In (D
PM
t-DP
M in
finity
)
p-Nitro
p-Chloro
p-Methyl
Kinetics and mechanism of Rh and Ru catalysed exchange
OO
H (T)
Ru(acac)2 ss
OO Ru(acac)2s
H(T)
OO
Ru(acac)2
HOT
OHO
T(H)
s
s
ss
Synth Appl Isotop.Label Compds 1991, Eds Buncel, Kabalka, 600-603 (1992)
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Heidelberg 2012
1990-0n: Applying parallel analysis
M e d i c i n a l C h e m i s t r yR a d i o c h e m i s t r y , W J S L o c k l e y
VV e r s a t i l ee r s a t i l e AA u t o m a t e du t o m a t e d SS a m p l e a m p l e TT r a n s p o r tr a n s p o r t
D i r e c t I n j e c t i o n N M R
Mike Bernstein
Medicinal ChemistryRadiochemistry, W J S Lockley
HPLC/MS 96 Well System, 2 min analysis per well
AndyWright
MarkJones
Barry McAuley
32 position deuterogenator
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Medicinal ChemistryRadiochemistry, W J S Lockley
Reaction optimisation
HO2C
H
H
H H H
8.1 7.7
Medicinal ChemistryRadiochemistry, W J S Lockley
H-NMR (8.1 ppm) D-NMR (8.1ppm)
15
30
60
120
240
0
360
480
Time(min)
0.125 0.25 0.5 0.125 0.25 0.5Mol Cat.
Mol ratio, temperature, time...
406080 406080 406080 406080 406080 406080Temp.
W J S Lockley et al., Synth. Appl. Isotop. Lab. Comp 2000, Proc. 7th Int. Symp, John Wiley (2001).
W J S Lockley, S P Thompson, D J Wilkinson et al., Tetrahedron letters, 41, 2705-2708 (2000)
Rapid catalyst screening & optimisation
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Heidelberg 2012
A simple apparatus for reductive labelling with isotopic hydrogen at atmospheric pressure, W J S Lockley, J. Lab. Comp. Radiopharm., 24, 1509-1515 (1987).
Tritium handling improves
David WilkinsonNaBH4 + 2H2O = NaBO2 + 4H2
Access to H2, HD, D2, HT, DT
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1980-Onwards: Rapid progress in tritium chemistry
Heidelberg 2012
Active tritides: Hendrik Andres, along with the NTLF group.
Stereospecific 3H-syntheses: Rolf Voges: Tom Moenius et. al.
Overall Result: Most compounds can now be labelled with tritium rapidly, in timescales commensurate
with industrial research projects
Ortho-exchange: Dick Heys, Art Shu, David Hesk, Rhys Salter, John Herbert, Goran Nilsson and many, many others
Solid state exchange: Myasoedov group
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Bad Soden 2011
RhClCl
O OCl
OHH
H
HH
H
1982
P
Ir+
PH
H O
O
BF4-
1992
P(IV)
N(IV)
Ir+
PF6-
1995
Fe
P(IV)
P(IV) Ir
OOPF6-2001
O
OIr
+
F F
F
F
F F
F
2000
CH2Cl2
Ir+
P
Polystyrene
P F6P-
2004
N
N
R
P(IV)
RR
Ir+
PF6-
2007
Development of ortho-3H exchange catalysts
WJS Lockley, et al. Tet Lett, 45 8621-8623 (2004).
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Heidelberg 2012
Reagents React Wash Store Use
Solid-phase catalyst: preparation & use
P
Polystyrene
P
P(IV)
N(IV)
Ir+
CH2Cl2
Ir+
P
Polystyrene
P
F6P-
F6P-
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Heidelberg 2012
Cleaner reactions.
N
NH
O
R
R'
13% 66%
N
O
OH
O
53% 98.5%
Crabtree’s catalyst Solid-phase catalystCompound
Hickey MJ, Kingston LP, Mather AN, Lockley WJS, Wilkinson DJ. J Label Compd Radiopharm, 49, (4) 2006, 388-389.
MikeHickey
LeeKingston
David Wilkinson
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OCF3
O
CF3
Ir
Iridium 1,3-dionate catalysis
OHO
H*H*
NH2O
H*H*
R R
NH2
H* H*
SO O
NH2
H*H*
NH*
DMA or DMF
RT-1000C, H*2O
NH2
H*H*
NHR'
H*H*
R
NH2
H*H*NH*
R R
R
N
H* H*
H*2, Ambient T and P
One step synthesis but now available
from Sigma/Aldrich & Strem
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Labelling piperidines, piperazinesand other secondary amines
Cl(II)
RuRu
Cl(II)
Cl
Cl
O
O
O
O
O
O
N
NH
H*
H*H*
H*
NH
H*
H*H*
H*
NH
H*
H*H*
H*
NH
H* H*
H*
N
N
NH
CF3
H*
H*H*
H*
Tetrahedron Letters, 46 , 4291-4293 (2005).
Substrate (0.03mmol)Catalyst (0.01mmol)D2O (10ul)DMSO (50ul)150oC/3h
NH
R'
R RNH
H*
H*H*
H* R'
H*2O
DMSO
Traci Smith
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-Labelling of N-heteroaromatics
NN
D(T)
D(T)
D(T)
D(T)
N
N
D D
N
D
D
N
O
D D
N
N
D
D
StathisAlexakis
Tetrahedron Letters, 47, 5025-5028 (2006).
N NH* H*
H*2Rh
THF
RT, 2h
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Heidelberg 2012
Metal-catalysed Isotope Exchange
W J S Lockley & J R Heys, Special Issue of the J Label Comp Radiopharm, 2010, 53, 634-751
Ir catalysis, Hesk, Lockley, Salter, Herbert, Nilsson, Kerr.
Ru/Rh catalysis, Hesk, Lockley.
Pd/Pt catalysis, Derdau, Atzrodt, Megawa, Sajiki, Myasoedov et al.
Ni & Other metals, Heys, Lockley.
Industrial applications, Hesk & Lavey, Chappelle & Hawes, Filer, Wilkinson et al..
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Heidelberg 2012
What has driven these developments in 3H-chemistry in Pharma?
Need for radioligands: screening, receptor isolation
Complex agents not amenable to 14C-synthesis
More potent agents: 14C impractical
Need for comparator agents: Standards, competitor agents
Need for early information in drug projects
Need for high quality information in drug projects
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The optimist’s paradise
All projects ideas start here:
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Project Leader: Self-Image
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The Wood of Ignorance
But real projects start here:
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Out from the Wood of Ignorance?
Crossing the chasm of bioavalabilty!
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And have to change direction?
Climbing the Pass of Protein Binding!
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And then change direction again?
Avoiding the deserts of receptor polymorphism
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And again!
Negotiating the hERG channel
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And once more!
Dealing with tricky active transport mechanisms
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And so on!
Hoping there are no false steps
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Project Leader: Observed Image
But..Why does this happen ?
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A little probability theory
The overall probability of several independent simultaneous or successive probabilities is the product of the individual probabilities.
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Very high quality data is needed at key decision points because errors can multiply.
First Project Decision
First Project Decision
Good choice ofwhich seriesto progress (But 10% of
structures will hit a toxic receptor)
Second Project
Decision
Screen parent for unwanted activity(but 20% of series
will have a metabolite with
the activity)
Third Project Decision
PK/PD modelling for Tox. studiesleads to serious
under/over exposure 20%
of time
Fourth Project Decision
Fourth Project Decision
PK/PD prediction to man from best
models is “only” 80% accurate
(20% unsuitable for man)
Probability of success = 0.9 x 0.8 x 0.8 x 0.8 = 0.46
After four typical decisions there is already more chance of failure than of success
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3H-Studies can often provide highly reliable information for project decisions
Our assumptionswere wrong !!
We’ll have to Rethink!
More teaanyone?
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Heidelberg 2012
The Rapid 3H-Labelling Approach: High Quality Information for Project Decision Making
David Wilkinson
LeeKingston
AndyMather
MikeHickey
MandyCoombs
Carbon-14
Tritium
Iodine-125
St. Isotope
Metabolites
19892000
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Bad Soden 2011
AndrewMcEwan
RayCooke
W. J. S. Lockley, A. McEwen, R. Cooke, J. Label Compd. Radiopharm 2012, 55
235–257
“Tritium: a coming of age for drug discovery and development ADME studies”
Modern synthesis and analysis of 3H-labelled compounds
Properties of the isotope: deciding on the appropriate 3H-labelled compound
Current examples from Discovery ADME
Examples from Development ADME
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So what other projects are ongoing at Surrey at the
moment?
Heidelberg 2012
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Heidelberg 2012W J S Lockley, O R Steward, J Label Compd Radiopharm, 53, 453-454 (2010)
and references therein..
Using NMR and computational approaches to explore amide group rotation.
Anomericinteraction
Dipoleinduced-dipole
Hydrogen-bonding
R
N OH
H D
R
NH O:
H D
R
N OH
H D
BrendanHowlin
OllieSteward
D&T can help to investigate fundamental molecular energetics
R
NH O
HD
R
NH O
DH
Richard Lewis
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β-elimination
In-chainlabelling
Pd Pd Pd Pd Pd PdH
H
H
R
D
D
Pd Pd Pd Pd Pd PdH
HH
RD
D
R
D(H)
H
H
β-eliminationTerminal labelling
RD
D
Pd Pd Pd Pd Pd Pd
H
H
H
R D
D
Pd Pd Pd Pd Pd Pd
H
H
D
R H
D
RD(H)
H(D)
H
Pd Pd Pd Pd Pd PdHH
HR
DD
HH
HR
Pd
Heidelberg 2012
Primary Pdalkyl
Secondary Pdalkyl
Л-Complex
Using D and T to characterise hydrogenation mechanisms
W J S Lockley, E Alexakis: unpublished results (ATHENA consortium)
DavidJackson
StathisAlexakis
D/T analysis of recovered starting material allows the classification of GpVIII metal catalysts according to mechanism
Di-σ-bondedcomplex
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Heidelberg 2012
NAIC & IsoPat2 (Mod 21): Software to facilitate the analysis of heavy isotopes by MS.
Original program: C. C. Gruber, G. Oberdorfer, C. V. Voss, J. M. Kremsner, C. O Kappe, W. Kroutil, J. Org. Chem. 2007, 72, 5778-5783. Modification as yet unpublished
BrendanHowlin
KostasSfyrakis
K N Barakzai, B J Howlin, W J S Lockley, K Sfyrakis, J Label Compd Radiopharm, 52, 559-562 (2009).
W.J.S. Lockley. Appl. Radiat. Isotopes. 37 I-II 1986
Use deconvolution to remove MS peaks associated with naturally occurring heavy isotopes.
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LMSP: Modelling of isotopic exchange via MS isotope distribution functions
Heidelberg 2012
N
N
N
N
D
D
D
D
D2
Rh black
THF
18h
IDF = (fH1+fD1)a × (fH2+fD2)b …. × (fHn+fDn)m
Gives insight into the mechanism of labelling processes and kinetics of labelling at various
molecular sites.
Lockley WJS, Sherhod R, J Label Compd Radiopharm, 51, 258 (2008).
RichardSherhod
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Heidelberg 2012
C
CoLogica isotopic chemistry database
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Heidelberg 2012
Isotopic chemistry: In what other career could one study such varied subjects and still find compelling
challenges four decades later?
When will the
fun ever stop!
I can’t believe
they pay me to
do this!
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Heidelberg 2012
My family for their tolerance of my frequent absences.
Collaborators from Surrey, Liverpool & Glasgow.
To yourselves for your patience & interest
Thanks to:
Many IIS colleagues throughout the world.
Finally, to the committee and organisers for this much valued and quite unexpected award
Science is about teamwork: My research has taken place in a rich network of colleagues, friends, mentors & students – Apologies to all those whose
projects and collaborations were not mentioned
Radiochemists at AstraZeneca & other pharma companies
My daughter for teaching me the “first rule of chemistry”.
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ADD in the URL
Bad Soden 2011
The first rule of chemistry?
Don’t lick the spatula!Hannah Rosa Jayne Lockley (age 10).