T. W. Schultz Presented at the McKim Conference September 17, 2008

T. W. Schultz

Presented at the McKim Conference

September 17, 2008

MolecularInitiating Events

Speciation,MetabolismReactivity

Etc.

In Vitro and

System Effects

In VivoAdverse

Outcomes

ParentChemical

The Toxicity Pathway The Toxicity Pathway Framework for Predicting Framework for Predicting

HazardHazard

Up-Stream Down-Stream CHEMISTRY BIOLOGY Structure-Activity Levels of Organization

Human Health Hazards Sensitization - skin and respiratory Acute toxicity - inhalation gas or vapors Chromosomal aberrations Developmental toxicity Selected organ-specific effects

Aquatic Toxicity Excess toxicity for aquatic organisms

No consensus in number > 25 < 50 (40)Vary in target moiety

Typically 45% -SH and 45% -NH2

Vary in structural domain Simple: isothiocyanate RN=C=S More complex: Michael addition Most complex: SNAr addition

Michaeladdition

SN2

SNAr

Reactive Potency

In vitroeffects

Mechanisms of

ProteinBinding

In vivosensitization

HazardAssessme

nt Endpoints

In silicomodeling

In ChemicoMeasurem

ent

In VitroMeasureme

nts

CH2=CH- k(thiol) K(amine) ______________________________________________________

C(=O)OMe 0.011 0.00076

C#N 0.0027 0.00020

C(=O)NH2 0.00046 0.000026

Different nucleophiles can differ in their absolute reactivity towards a given

electrophile, but relative reactivity is well correlated over a range of nucleophiles

within the same mechanism.

In silico; qualitative, pre-screen 1000’s of compounds

In chemico; depletion-based quantitative, screen 100’s of compounds

Adduct identification; LC-MS-MS, 10’s of compounds

Use in a similar context to in vitro or in silico

Quantitative, rapid, inexpensive experiments with model nucleophiles

Verify mechanism-based rules of reactivity

Define the applicability domain of a reactive mechanism

Provides a measure of relative potency

Protocol (Schultz et al., 2005) is quantitative, rapid, inexpensive, abiotic, and spectrophotometric-based

Depletion method; measures % free thiol with GSH as the model nucleophile

Endpoint is RC50 (50% reactive concentration; mM) is calculated by probit analysis of concentrations-response data after 2-hrs

RC50 (mM) Date Lot Operator_____________________________________________________________

0.094 02-10-05 1 A 0.081 02-18-05 1 A 0.085 02-18-05 1 B 0.086 02-19-05 1 B 0.092 03-03-05 1 A 0.074 03-06-05 1 A 0.084 03-14-05 1 A 0.085 03-21-05 1 A 0.088 12-21-06 2 C 0.068 01-03-07 2 D 0.100 01-05-07 2 D

Readily available

Concentration can be analyzed by simple methods

Odorless, non-hazardous

Water soluble but NOT readily soluble in organic solvents

Does not lead itself to HPLC

Depletion of reactant or formation of product

Full Kinetics- measured at several time intervals with several initial concentrations of electrophile (100 chemicals)

Partial Kinetics- measured at several time intervals with one initial concentrations of electrophile

Concentration giving 50% reaction in a fixed time- measured at one time with several initial concentrations of electrophile (1000 chemicals)

Extent of reaction after a fixed time- measured at one time with one initial concentrations of electrophile

highly relevant with complex domains1) Michael Addition,2) Nucleophilic substitution (N-sub) of

haloaliphatics,3) N-sub of haloaromatics (SNAr).

Base Structure and Special Features

X = -CHO, -COR,

-CN, etc.

XC=CXC#C

GSH & LLNA DATA

O

OO

ClO

OO

O

OO

O

RC50 = 0.05

RC50 = 0.05 RC50 = 0.03

RC50 = 0.09 RC50 = 0.02

EC3 = 0.01

TYPE STRUCTURE RC50 (mM)

Acetylenedicarboxylates RC(=O)C#CC(=O)R 0.025

Propiolates C#CC(=O)OR 0.1

trans-Vinylene dicarboxylates RC(=O)C=CC(=O)R 0.2

Acrylates C=CC(=O)CR 0.8

cis-Vinylene dicarboxylates RC(=O)C=CC(=O)R 2.0

Methacrylates C=C(C)C(=O)CR 30.0

Base Structure and Special Features

R1C(X)YR2

Y = C6H5 > C#C > C=C, etc

X = I > Br > Cl > F

R1 = H > CnH(2n +1)

R2 no effect

Base Structure and Special Features

XC6H3Y2

Y = NO2, > in-ring-N > CHO > CN

X = F > Cl > Br > IPosition of leaving group in relationship to activity groups effects potency

Substance RC50 (mM) Sensitizer_______________________________________________1-F-2,4-dinitrobenzene 0.07 +1-Cl-2,4-dinitrobenzene 1.5 +1-Br-2,4-dinitrobenzene 1.2 +1,3-Cl-4,6-dinitrobenzene 0.24 +1,5-Cl-2,3-dinitrobenzene 0.20 +1,2-dinitrobenzene NRAS -2,4-F-1-nitrobenzene NRAS -2,4-Cl-1-nitrobenzene NRAS -1-Br-4-Cl-2-nitrobenzene NRAS -1,2,3-Cl-5-nitrobenzene NRAS -

F

NO2

NO2

F

NO2

F

F

NO2

F

FF

NO2

F F

F

1.4 0.6 3.40.07

1) Michael Addition (largely completed),

2) Pre-Michael Addition (nearly completed),

3) N-sub of Haloaliphatics (largely completed),

4) N-sub of Haloaromatics (just started).

relevant with simple domains

1) disulfide exchange; 2) disulfide formation, 3) O-heterocyclic ring opening, 4) N-sub of alkyl sulfates &

sulfonates, 5) nitroso-binding.

1) disulfide exchange, (5 compounds) 2) disulfide formation, (5 compounds) 3) O-heterocyclic ring opening, (5 compounds) 4) N-sub of alkyl sulfates, (3 compounds) 5) N-sub of alkyl sulfonates, (3 compounds) 6) nitroso-binding (3 compounds). All demonstrate GSH reactivity and

are related to sensitization

less relevant

1) arycarboxylate aminolysis,2) electrostatic interactions,3) mercury thiolate formation,4) oxime group condensation,5) others.

Applies computational methods to filling data gaps

By facilitating the selection of chemical analogues and grouping chemicals into categories

Integrates (among other things) mechanisms of action with categories

Among the best characterized method, which integrates mechanisms of action into grouping chemicals is the profiler bases on Protein-binding.

Currently covers 38 different mechanismVary in specificityVary in completenessStructural Alerts for mechanisms tend to be

qualitative in nature Michael addition alerts include the extreme

sensitizer p=benzoquinone, the moderate sensitizer ethyl acrylate, and the very weak sensitizer methyl methacrylate.

Extremely fast: quinones, propiolates , 1-alken-3-ones

Fast: acrylates, 2-alkenals, 3-alken-2-ones

Moderately Fast: alkyl 2-alkynoates

Slow: tiglates

Very Slow: methacryates

Non-Reactive: non-,-unsaturated

≈ 1500 substances on the List of Flavor and Fragrance Related Substances

≈1300 discrete substances of which: 79 Fast- to moderate-reacting Michael-acceptors; 19 Slow-reacting Michael-acceptors; 57 Schiff-base aldehydes; 29 Acetals; 15 Disulfide formers; 11 Cyclic addition diones; 9 Disulfide exchangers; 3 O-heterocyclic ring openers.

>40 pro-electrophiles

Thank you.