RETRO-DIELS-ALDER REACTION - POSSIBLE INVOLVEMENT IN THE METABOLIC-ACTIVATION OF YCLO[2.2.1]HEPTA2(3),5(6)-DIENE-2,3-DICARBOXYLATES AND A PHOSPHONATE ANALOG

A discontinuous structure-activity relationship signaled a change in m ode of action and led to the discovery of a possible novel metabolic a ctivation mechanism. The toxicity of the herbicide endothal (exo,exo-7 -oxabicyclo[2.2.1]heptane-2,3 acid) to mice (ip LD(50) = 14 mg/kg) is attributed to the inhibition of protein phosphatase 2A (PP2A) at the c antharidin binding site. The potency is reduced by the introduction of a 2,3- or 5,6-double bond. Surprisingly, high toxicity (ip LD(50)'s = 15-50 mg/kg) is restored in oxabicyclohepta-2(3),5(6)-dienes substitu ted in the 2- and 3-positions with bis(methyl carboxylate), bis(ethyl carboxylate), and diethyl phosphonate/ethyl carboxylate, whereas the d icarboxylic acid, bis(tert-butyl carboxylate), and bis(dimethyl phosph onate) are inactive. The diene adducts do not inhibit the cantharidin binding site of PP2A. Two observations provided an alternative working hypothesis that the active but not the inactive diene adducts are pro toxicants: GC analyses revealed that selected bicyclic dienes readily undergo thermal dissociation by retro-Diels-Alder reactions to liberat e disubstituted acetylenes; the liberated acetylenes have mouse ip LD( 50)'s of 8-25 mg/kg. Apparent exceptions to this hypothesis are that b icyclic dienes with bis(tert-butyl carboxylate) and bis(dimethyl phosp honate) substituents are not toxic, yet their corresponding acetylenes are quite toxic. These apparent anomalies are resolved by finding tha t only the toxic bicyclic dienes readily react with albumin and 4-nitr obenzenethiol and that their low-toxicity analogs are much less reacti ve. Albumin can be replaced by hemoglobin but not by myoglobin or chym otrypsin in reaction with a bicyclic diene indicating the importance o f the free thiol group. Diethyl oxabicycloheptadienedicarboxylate read ily reacts with GSH to give two products, which are also formed from t he corresponding acetylene, identified as the cis and trans isomers of the GSH-acetylene conjugate. This is the first proposal, to our knowl edge, that a retro-Diels-Alder-type reaction is involved in the metabo lic activation of a toxicant.