Opportunities for Measuring Amine Reactivity Under LINK

Mark CroninLiverpool John Moores University

England

InSilicoTox Project EU funded Marie Curie project 2006-2010 48 months post-doc funded at Liverpool

Modelling reactive toxicity endpoints and investigating mechanisms

Molecular Orbital models of reactivity Database: chemical reactivity data for 802

electrophiles (52 classes / 57 mechanisms of protein and DNA binding) and their reactions with 99 reference nucleophiles. 2728 entries Submitted to Chemical Reviews So what ?

OSIRIS ITS

OSIRIS is an EU project (2008-2012) to develop ITS for REACH

ITS web-tool – weight of evidence based

The time is now to show utility of Categories Reactivity Workflows...

The Current Defra LINK Project U.K. Department for Environment, Food and Rural

Affairs LINK project under the Sustainable Arable funding

scheme Came about as a result of a smaller project, to

support UK efforts for REACH LINK requires a consortium of industrial partners to

provide “input” which is then matched by Defra

Liverpool John Moores University (co-ordinator)

Sub-contractors: Schultz Group LLC, Dave Roberts PLC, FRAME

Current Link Project Purpose is to develop ITS for fish acute toxicity

and skin sensitisation

Allowed us to develop reactivity concepts

www.inchemicotox.org

A Possible One Year Extension Important: Funding is NOT agreed – although Defra

are very keen to fund the science

Aim: to develop an amine-based reactivity to model hard nucleophiles To provide a simple assay for reactivity

assessment To define domains

Start-date November 2010

Nora – 8 hours Hard Nu is fine, but it is different to what we would

like to do.

We want to finalise the soft spectrum for those chemicals which are NH2 reactive, [this is why it is butylamine.]

Maybe the aim should be 2 fold - to finish the soft GSH and then the other one is the hard Nu.

Further Comments – DWR 46 mins What exactly do we want this hard nucleophile to do?   Tell us how reactive hard electrophiles are... Preferably quantitatively, i.e. rate constants or equivalents like RC50 values consistent with the

kinetic definition.. Preferably usable in non-aqueous media... a) because a lot of electrophiles are water insoluble... b) a large number of electrophiles don't react well in water, even if soluble (eg SB

electrophiles)... Preferably able to react with electrophiles from all the major domains...  (And, for PR/spin purposesmake it as biological-looking as possible)... (And be cheap and readily available in large quantities)   The last two are of course contradictory.   Butylamine can do all these, except the last but one.  

Lysine is just butylamine with a carboxyl group and an amino group on the delta carbon. Acetylate the NH2 group and convert the CO2H group to its amide and you have N2NCH2CH2CH2CH(NHCOMe)CONH2, which will react like butylamine, work in non-aqueous solvents (if not the acetate can be replace by higher alkanoate), can be bought, but more pricey than butylamine, and looks a bit more biological

  2-pyridone has a lot of the advantages of butylamineSB and acyl

transfer may be problematic.   4-nitrobenzylpyridine - brilliant for quantifying by uv/visible. Very

good for SN2. But may not be hard enough) to be the complete answer.

 

Overall - unless butylamine or a similar version of it is the one chosen, there may not be a suitable one-size-fits-all hard nucleophile, and it would be better to choose the best one according to the mechanistic domain

Another simple and genuinely hard nucelophile would be p-nitrophenol, used at a pH at which it is fully ionised (its pKa ia just over 7), or in non-aqueous medium, eg ethanol, as its sodium salt. Very good to follow by uv. Again, won't work for all mechanistic domains

Amine Assay: Philosophy

Concentrate on experimental determination (not ITS development)

Develop assay at Liverpool (12 month post-doc)

Expert advise on chemical selection

Experimental Details: Basics

Butylamine / 2-pyridone as the nucleophile as opposed to a peptide Investigate correlations between the two Butylamine may be more appropriate for some

electrophiles – particularly the Schiff base domain Butylamine in excess to make psuedo 1st order (5:1) An organic solvent e.g. ethanol (0.1 molar)

Schiff base domain – reaction produces water Vary solvents and / or nucleophile if required, but the

purpose is to keep the assay as simple as possible

Experimental Details: Determination

Where possible we will use the speedy but lowest-tech option of following the reaction by titrating (with HCl) samples to measure remaining BuNH2 Usually good for SN2 and SNAr electrophiles 

  If titration not possible - a spectroscopic method (maybe

UV) to follow disappearance of the electrophile (e.g. disappearance of the double bond in a Michael acceptor)  

  Otherwise chromatography e.g. HPLC

Potential Initial Test Set

Michael acceptors for which we have both Natsch and Schultz reactivity data

2-Hydroxyethyl acrylate Diethyl maleate Cinnamic aldehyde Benzylidenacetone 4-Vinyl-pyridine Trans-2-Hexenal Benzyl cinnamate -Methyl cinnamic aldehyde trans, trans-2.4-Heptadienal Ethylene glycol dimethacrylate

Some compounds expected to be non-reactive

• Diethyl succinate

• Cinnamic alcohol

• Ethylene glycol diacetate

 

Some compounds which may be activated by oxidation

• Isoeugenol

• Hydroquinone

Potential Initial Test Set

Michael acceptors for which we have both Natsch and Schultz reactivity data

2-Hydroxyethyl acrylate Diethyl maleate Cinnamic aldehyde Benzylidenacetone 4-Vinyl-pyridine Trans-2-Hexenal Benzyl cinnamate -Methyl cinnamic aldehyde trans, trans-2.4-Heptadienal Ethylene glycol dimethacrylate

Some compounds expected to be non-reactive

• Diethyl succinate

• Cinnamic alcohol

• Ethylene glycol diacetate

 

Some compounds which may be activated by oxidation

• Isoeugenol

• Hydroquinone

Reactive with soft SH nucleophiles – investigate correlations

Potential Further Test Set Extend the test set to compounds where amine

reactivity is important A selection of aldehydes and activated ketones

from Aptula AO (2006) Chem. Res. Toxicol. 19: 1228-1233.

Acyl transfer agents e.g. dihydrocoumarin Aim is a method (protocol/approach) to determine

reactivity to harder nucleophiles Demonstration that this hard reactivity data gives

rise to robust categories and read across

Existing partners Unilever Procter and Gamble Marks and Spencer

New Partners Givaudan Dupont

Acknowledgements This project was sponsored by Defra through the

Sustainable Arable Link Programme European Union 6th Framework OSIRIS Integrated

Project (GOCE-037017-OSIRIS) CAESAR Specific Targeted Project (SSPI-022674-

CAESAR) European Chemicals Agency (EChA) Service Contract

No. ECHA/2008/20/ECA.203 InSilicoTox Marie Curie Project (MTKD-CT-2006-42328)

www.inchemicotox.org