T. W. Schultz Presented at the Logan Workshop March 23-24, 2010

 Background  Method & Data Base  Reactions & Coverage  What We are Currently Doing

 No consensus in number > 25 < 50 (40)  Vary in preferred target moiety  Typically 45% -SH and 45% -NH 2  Vary in structural domain  Simple: isothiocyanate RN=C=S  Complicated: Michael addition  Complex: S N Ar addition

 In order of increasing hardness include:  Thiol-group of cysteine  S-atom of methionine  Primary amino-group of lysine  Secondary amino-group of histidine

CH 2 =CH- k(thiol) K(amine) ______________________________________________________ C(=O)OMe C#N C(=O)NH 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.

 Use in a similar context to in vitro or in silico  Quantitative, rapid, inexpensive experiments with model nucleophiles  Verify reaction-based rules of reactivity  Define the chemical space of a reaction  Provides a measure of relative potency  Useful in refining categories and modeling

 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 (O’Brien Assay)  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 (Gerberick Assay)

 Quantitative with kinetics-linked endpoint  Simple, rapid, repeatable, and inexpensive  Cysteine-based thiol target  Depletion-based (% free thiol)  Analyses by  Concentration-Response (RC 50 )  Full kinetic

 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

 Initial concentration of electrophile that gives a half-life of 120 minutes  Good if electrophile is in excess  Adequate if concentrations of electrophile and GSH are similar  Poor if GSH is in excess; RC 50 values are then extrapolated

 > 2,600 individual assays  ≈ 1,000 separate structure  > 25 different organic reactions (mechanisms)  > 300 not reactive because of structure  < 100 not reactive because of solubility  < 50 not reactive because of color interference

 Highly relevant with multiple domains  1) Michael Addition  2) Nucleophilic substitution (N-sub) of haloaliphatics  3) N-sub of haloaromatics (S N Ar)

 1) Michael Addition > 250 compounds (cpds)  2) Pre-Michael Addition > 50 cpds  3) N-sub of Haloaliphatics > 150 cpds  4) N-sub of Haloaromatics > 125 cpds

 Highly relevant with simple domains  1) disulfide exchange  2) O-heterocyclic ring opening  3) N-sub of alkyl sulfates & sulfonates  4) nitroso- & N-oxides  5) disulfide formation

 1) disulfide exchange, >10 cpds  2) O-heterocyclic ring opening, 20 cpds  3) N-sub of alkyl sulfates, 5 cpds  4) N-sub of alkyl sulfonates, >10 cpds  5) nitroso-compounds, >5 cpds  6) N-oxides, 10 cpds  All demonstrate GSH reactivity & are related to sensitization

 Less relevant  1) arenesulfinic acid substitution  2) azomethyne addition  3) thiocyanate addition  4) mercury thiolate formation  5) others

 Other chemical classes  1) unsaturated alcohols  2) secondary amines  3) dialkyl acetals  4) lactates  5) anhydrides (hydrolysis)  6) aldehydes (Schiff-base formers)  7) diones (cycloaddition to diamines)

only ,  -unsaturates are reactive CompoundsRC 50 (mM) _______________________________________________ C=CC(O)C n  C#CC(O)C n  1.0 – 3.0 OCC=CC n  OCC#CC n  pentyn-3-ol 1.3, CH 3 -1-pentyn-3-ol 16, 13 3,4-CH 3 -1-pentyn-3-ol NR at 50mM

Base Structure and Special Features R 1 C(X)YR 2 Y = C 6 H 5 > C#C > C=C, etc X = I > Br > Cl > F R 1 = H > C n H (2n +1) R 2 no effect

Base Structure and Special Features XC 6 H 3 Y 2 Y = NO 2, > in-ring-N > CHO > CN X = F > Cl > Br > I Position of leaving group in relationship to activity groups effects potency

Thank you