Heidelberg 2012

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Isotopic chemistry: the most varied of careers..

Bill LockleyUniversity of Surrey

…with tritium & deuterium, the most versatile of the isotopes.

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)

Heidelberg 2012

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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

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

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

Heidelberg 2012

1968-1971: Naïve PhD student

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

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

Heidelberg 2012

1972-77 Off to Liverpool!

www.sareenarts.com

Biochemistry, Botany, Beetles and Beatles!

Heidelberg 2012

Biosynthesis is verycomplex: 3H can make it simpler!

Heidelberg 2012

Carotenoids

Steroids

AcetateTrevor

Goodwin

Bad Soden 2011

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Plants resort to chemical warfare in the battle with insects

HughHeffyn Rees

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

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

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).

Bad Soden 2011

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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

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

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?

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

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)

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

Heidelberg 2012

Biosynthesis of carotenoids

George Britton

Heidelberg 2012

Putative cyclisation reaction

H

H+

H

H

H

+

Proton initiated cyclisation

Proton loss

Carotenoid (with incorporated proton from the medium)

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

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

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)

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

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!

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

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

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.

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

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

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

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.

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

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)

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

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

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

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

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).

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-

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

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

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

-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

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..

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

The optimist’s paradise

All projects ideas start here:

Project Leader: Self-Image

The Wood of Ignorance

But real projects start here:

Out from the Wood of Ignorance?

Crossing the chasm of bioavalabilty!

And have to change direction?

Climbing the Pass of Protein Binding!

And then change direction again?

Avoiding the deserts of receptor polymorphism

And again!

Negotiating the hERG channel

And once more!

Dealing with tricky active transport mechanisms

And so on!

Hoping there are no false steps

Project Leader: Observed Image

But..Why does this happen ?

A little probability theory

The overall probability of several independent simultaneous or successive probabilities is the product of the individual probabilities.

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

3H-Studies can often provide highly reliable information for project decisions

Our assumptionswere wrong !!

We’ll have to Rethink!

More teaanyone?

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

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

So what other projects are ongoing at Surrey at the

moment?

Heidelberg 2012

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

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

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.

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

Heidelberg 2012

C

CoLogica isotopic chemistry database

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!

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”.

ADD in the URL

Bad Soden 2011

The first rule of chemistry?

Don’t lick the spatula!Hannah Rosa Jayne Lockley (age 10).