Inhibitor Report for: Malaoxon

Syntheses of the enantiomers of malathion, malaoxon, and isomalathion are reported herein. Malathion enantiomers were prepared from (R)- or (S)-malic acid in three steps. Enantiomers of malathion were converted to the corresponding enantiomers of malaoxon in 52% yield by oxidation with monoperoxyphthalic acid, magnesium salt. The four isomalathion stereoisomers were prepared via two independent pathways using strychnine to resolve the asymmetric phosphorus moiety. The absolute configurations of the four stereoisomers of isomalathion were determined by X-ray crystallographic analysis of an alkaloid salt precursor. A high-performance liquid chromatography technique was developed to resolve the four stereoisomers of isomalathion, and to determine their stereoisomeric ratios.

The biomolecular reaction constants (ki), dissociation constants (Kd), and phosphorylation constants (kp) were determined for the enantiomers of malaoxon against rat brain acetylcholinesterase, and for the stereoisomers of isomalathion against rat brain acetylcholinesterase and electric eel acetylcholinesterase. (R)-Malaoxon was an 8.6-fold more potent anti-cholinesterase than (S)-malaoxon. Isomalathion stereoisomers with the R configuration at carbon were 3-13-fold stronger inhibitors than those with the S configuration. The isomalathion stereoisomers with the R configuration at phosphorus were 4.3-8.8-fold stronger inhibitors of rat brain acetylcholinesterase, yet 3.4-5.8-fold weaker inhibitors of electric eel acetylcholinesterase, than the isomalathion stereoisomers with the S configuration at phosphorus. The rat brain acetylcholinesterase spontaneous (k0 = approximately 13.0 x 10(-3) min-1) and oxime-mediated (koxime) = 51.0 x 10(-3) min-1) reactivation rate constants following inhibition by isomalathion stereoisomers with the R configuration at phosphorus were comparable to spontaneous (11.3 x 10(-3) min-1) and oxime-mediated (50.2 x 10(-3) min-1) reactivation rates obtained for (S)-isoparathion methyl. These data support a common phosphorylation mechanism, namely, the displacement of the thiosuccinyl moiety from isomalathion stereoisomers with the R configuration at phosphorus, and displacement of the p-nitrophenoxy ligand from (S)-isoparathion methyl to form the same O,S-dimethyl phosphorothiolated enzyme. Rat brain acetylcholinesterase inhibited by the isomalathion stereoisomers with the S configuration at phosphorus were refractory to reactivation, suggesting an alternate mechanism of inhibition, i.e., the loss of the methylthio ligand. Several mechanisms are proposed to account for the subsequent nonreactivation.(ABSTRACT TRUNCATED AT 250 WORDS)

Carboxylesterases (CbxE) can be inhibited by organophosphorus esters (OPs) without causing clinical evidence of toxicity. CbxE are thought to protect the critical enzyme acetylcholinesterase (AChE) from OP inhibition in animals. CbxE and AChE are both present in neuroblastoma cells, but, even though these cells have potential to be an in vitro model of OP toxicity, the effect of OPs on CbxE and the relationship of CbxE inhibition and AChE inhibition have not yet been examined in these cells. Therefore, this study examined concentration-related OP-induced inhibition of CbxE in human SH-SY5Y and mouse NB41A3 neuroblastoma cells with 11 active esterase inhibitors: paraoxon, malaoxon, chlorpyrifos-oxon, tolyl saligenin phosphate (TSP), phenyl saligenin phosphate (PSP), diisopropyl phosphorofluoridate (DFP), mipafox, dichlorvos, trichlorfon, dibutyryl dichlorovinyl phosphate (DBVP), and dioctyl dichlorovinyl phosphate (DOVP). All could inhibit CbxE, although the enzyme was less likely to be inhibited than AChE following exposure to 9 of the test compounds in the human cell line and to all 11 of the test compounds in the murine cell line. Species differences in concentration-related inhibitions of CbxE were evident. When cells were exposed first to an OP with a low IC50 toward CbxE (PSP), followed by an OP with high affinity for AChE (paraoxon or malaoxon), inhibitions of CbxE and AChE were additive. This indicated that CbxE did not protect AChE from OP-induced inhibition in this cell culture model.

A growing body of evidence indicates that young animals exhibit an increased susceptibility to the lethal effects of cholinesterase (ChE)-inhibiting insecticides. Our laboratory is engaged in defining factors which may explain this age-related sensitivity. This report includes results from experiments designed to compare the developmental profiles, kinetic parameters and intrinsic (i.e. in vitro) sensitivity of developing male rat brain acetylcholinesterase (AChE) activity to carbamate and organophosphorus anticholinesterases. Total ChE activity in whole brain for each age was composed of about 90% AChE and 10% butyrylcholinesterase (BCHE) activity for the six ages examined. Brain AChE activity showed an age-related increase in Vmax until postnatal day 17 with no change in Km (average of all six ages approximately equal to 72 microM). Optimal substrate (acetylthiocholine) concentration for each age was 1 mM, and there was substrate inhibition (approximately 10%) at 2.5 mM. IC50s (the concentration of compound that inhibits 50% of the AChE activity in 30 min at 26 degrees C) defined concomitantly for postnatal day 4 and adult brain AChE using either aldicarb, carbaryl, chlorpyrifos-oxon or malaoxon were virtually identical at both ages with average IC50 values being: aldicarb = 2.4 microM, carbaryl = 1.7 microM, chlorpyrifos-oxon = 4.9 nM and malaoxon = 140 nM. In summary, AChE in young and adult brain differs mostly in specific activity while the Km(s), substrate profiles, and in vitro sensitivity to selected anticholinesterase insecticides are not different. Therefore, these data support the hypothesis that the greater sensitivity of the young animals to anticholinesterase pesticides is not due to the greater sensitivity of the target molecule AChE to these inhibitors.

Syntheses of the enantiomers of malathion, malaoxon, and isomalathion are reported herein. Malathion enantiomers were prepared from (R)- or (S)-malic acid in three steps. Enantiomers of malathion were converted to the corresponding enantiomers of malaoxon in 52% yield by oxidation with monoperoxyphthalic acid, magnesium salt. The four isomalathion stereoisomers were prepared via two independent pathways using strychnine to resolve the asymmetric phosphorus moiety. The absolute configurations of the four stereoisomers of isomalathion were determined by X-ray crystallographic analysis of an alkaloid salt precursor. A high-performance liquid chromatography technique was developed to resolve the four stereoisomers of isomalathion, and to determine their stereoisomeric ratios.

The biomolecular reaction constants (ki), dissociation constants (Kd), and phosphorylation constants (kp) were determined for the enantiomers of malaoxon against rat brain acetylcholinesterase, and for the stereoisomers of isomalathion against rat brain acetylcholinesterase and electric eel acetylcholinesterase. (R)-Malaoxon was an 8.6-fold more potent anti-cholinesterase than (S)-malaoxon. Isomalathion stereoisomers with the R configuration at carbon were 3-13-fold stronger inhibitors than those with the S configuration. The isomalathion stereoisomers with the R configuration at phosphorus were 4.3-8.8-fold stronger inhibitors of rat brain acetylcholinesterase, yet 3.4-5.8-fold weaker inhibitors of electric eel acetylcholinesterase, than the isomalathion stereoisomers with the S configuration at phosphorus. The rat brain acetylcholinesterase spontaneous (k0 = approximately 13.0 x 10(-3) min-1) and oxime-mediated (koxime) = 51.0 x 10(-3) min-1) reactivation rate constants following inhibition by isomalathion stereoisomers with the R configuration at phosphorus were comparable to spontaneous (11.3 x 10(-3) min-1) and oxime-mediated (50.2 x 10(-3) min-1) reactivation rates obtained for (S)-isoparathion methyl. These data support a common phosphorylation mechanism, namely, the displacement of the thiosuccinyl moiety from isomalathion stereoisomers with the R configuration at phosphorus, and displacement of the p-nitrophenoxy ligand from (S)-isoparathion methyl to form the same O,S-dimethyl phosphorothiolated enzyme. Rat brain acetylcholinesterase inhibited by the isomalathion stereoisomers with the S configuration at phosphorus were refractory to reactivation, suggesting an alternate mechanism of inhibition, i.e., the loss of the methylthio ligand. Several mechanisms are proposed to account for the subsequent nonreactivation.(ABSTRACT TRUNCATED AT 250 WORDS)