Opportunities for Measuring Amine Reactivity Under LINK

1. Mark Cronin Liverpool John Moores University England Opportunities for Measuring Amine Reactivity Under LINK

2. 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 ?

3. 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...

4. 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

5. Liverpool John Moores University (co-ordinator) • Sub-contractors: Schultz Group LLC, Dave Roberts PLC, FRAME

6. Current Link Project • Purpose is to develop ITS for fish acute toxicity and skin sensitisation • Allowed us to develop reactivity concepts • www.inchemicotox.org

7. 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

8. 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.

9. 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.

10. 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.

11. 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

12. Amine Assay: Philosophy • Concentrate on experimental determination (not ITS development) • Develop assay at Liverpool (12 month post-doc) • Expert advise on chemical selection

13. 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

14. 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 SNArelectrophiles • 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

15. 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 • a-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

16. 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 • a-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

17. 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

18. Existing partners • Unilever • Procter and Gamble • Marks and Spencer • New Partners • Givaudan • Dupont

19. 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