A novel bioactivation mechanism of phosphoramidate insecticides

In order to elucidate the activation mechanism of phosphoramidate insecticides, 2,4-dichlorophenyl methyl N-alkylphosphoramidates (alkyl: methyl, ethyl, propyl, isoproppyl, and butyl) were chosen as model compounds and compared with isofenphos oxon. Although they showed a potent insecticidal activity against susceptible housefly except for butyl homologue, their in vitro acetylcholinesterase (AChE) inhibitory activity was very poor N-Desalkyl compound has been known as an activated form. When bovine erythrocyte AChE inhibition of N-isopropyl homologue and isofenphos oxon were carried out in rat liver microsomal system, they gave a strong inhibitory activity under the oxidative condition with NADPH, but, in the presence of SKF-525A or piperonyl butoxide as a mixed function oxidase (MFO) inhibitor, isofenphos oxon lost all the inhibitory activity. On the other hand, N-isopropyl homologue partly retained its AChE activity and completely lost the activity by the addition of methimazole as a flavin-containing monooxygenase (FMO) inhibitor. Comparison of these data with insecticidal activity suggested the involvement of another bioactivation mechanism. Peracid oxidation as a model of biological oxidation resulted in the production of des-N-alkyl compound (HO-PO) in addition to N-desalkyl compound (H2N-PO), which suggested the structure of the activated form as an oxidized form of phosphoramide moiety of the starting compound. Three structures were considered from a computational chemistry point of view. N-Oxide structure (RNH(O)-PO) is isomerized to O-aminophosphate structure (RNHO-PO) depending on the alkyl substituent on the nitrogen atom, and the latter is the intermediate to give des-N-alkyl compound, which has no AChE inhibitory action. The formation of O-aminophosphate structure from the adduct of the starting compound with peracid was analyzed using model compounds by the theory of Frontier orbital. Also, O-aminophosphate structure might be produced via phosphorus oxyoxonate structure (RNH-P(OO)). The possibility of hydroxylamine structure (RN(OH)-PO) to give N-oxide or O-aminophosphate structure was small if any in this model phosphoramidate compounds. In any way, O-aminophosphate structure is most probable as a novel activated form mainly formed by FMO or peracid.