title: Novel Organophosphate Ligand O-(2-Fluoroethyl)-O-(p-Nitrophenyl)Methylphosphonate: Synthesis, Hydrolytic Stability and Analysis of the Inhibition and Reactivation of Cholinesterases.
abstract: The organophosphate O-(2-fluoroethyl)-O-(p-nitrophenyl) methyphosphonate 1 is the first-in-class, fluorine-18 radiolabeled organophosphate inhibitor ([18F]1) of acetylcholinesterase (AChE). In rats, [18F]1 localizes in AChE rich regions of the brain and other tissues where it likely exists as the (CH3)(18FCH2CH2O)P(O)-AChE adduct (ChE-1). Characterization of this adduct would define the inhibition mechanism and subsequent postinhibitory pathways and reactivation rates. To validate this adduct, the stability (hydrolysis) of 1 and ChE-1 reactivation rates were determined. Base hydrolysis of 1 yields p-nitrophenol and (CH3) (FCH2CH2O)P(O)OH with pseudo first order rate constants (kobsd) at pH 7.4 (PBS) of 3.25 × 10-4min-1(t1/2= 35.5 h) at 25 °C and 8.70 × 10-4min-1(t1/2= 13.3 h) at 37 °C. Compound 1 was a potent inhibitor of human acetylcholinesterase (HuAChE; ki= 7.5 × 105M-1min-1), electric eel acetylcholinesterase (EEAChE) (ki= 3.0 × 106M-1min-1), and human serum butyrylcholinesterase (HuBChE; 1.95 × 105M-1min-1). Spontaneous and oxime-mediated reactivation rates for the (CH3) (FCH2CH2O)P(O)-serine ChE adducts using 2-PAM (10 μM) were (a) HuAChE 8.8 × 10-5min-1(t1/2= 131.2 h) and 2.41 × 10-2min-1(t1/2= 0.48 h), (b) EEAChE 9.32 × 10-3min-1(t1/2= 1.24 h) and 3.33 × 10-2min-1(t1/2= 0.35 h), and (c) HuBChE 1.16 × 10-4min-1(t1/2= 99.6 h) and 4.19 × 10-2min-1(t1/2= 0.27 h). All ChE-1 adducts undergo rapid and near complete restoration of enzyme activity following addition of 2-PAM (30 min), and no aging was observed for either reactivation process. The fast reactivation rates and absence of aging of ChE-1 adducts are explained on the basis of the electron-withdrawing fluorine group that favors the nucleophilic reactivation processes but disfavors cation-based dealkylation aging mechanisms. Therefore, the likely fate of radiolabeled compound 1 in vivo is the formation of (CH3)(FCH2CH2O)P(O)-serine adducts and monoacid (CH3)(FCH2CH2O)P(O)OH from hydrolysis and reactivation.
authors.affiliation: Department of Biomedical and Pharmaceutical Sciences, University of Montana , Missoula, Montana 59812, United States.
authors.forename: Chih-Kai
authors.initials: CK
authors.lastname: Chao
authors.affiliation: Department of Biomedical and Pharmaceutical Sciences, University of Montana , Missoula, Montana 59812, United States.|Center for Neuromolecular Research, Drug Discovery Division, Southern Research Institute , Birmingham, Alabama 35205, United States.
authors.forename: S Kaleem
authors.initials: SK
authors.lastname: Ahmed
authors.affiliation: Department of Biomedical and Pharmaceutical Sciences, University of Montana , Missoula, Montana 59812, United States.|Center for Neuromolecular Research, Drug Discovery Division, Southern Research Institute , Birmingham, Alabama 35205, United States.
authors.forename: John M
authors.initials: JM
authors.lastname: Gerdes
authors.affiliation: Department of Biomedical and Pharmaceutical Sciences, University of Montana , Missoula, Montana 59812, United States.
authors.forename: Charles M
authors.initials: CM
authors.lastname: Thompson
mesh_terms: D000818:Animals; D066241:Carbon-13 Magnetic Resonance Spectroscopy; D002800:Cholinesterase Inhibitors / Q000494:pharmacology*; D002801:Cholinesterase Reactivators / Q000494:pharmacology*; D002802:Cholinesterases / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D008024:Ligands; D009943:Organophosphorus Compounds / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology*; D066244:Proton Magnetic Resonance Spectroscopy; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D002800:Cholinesterase Inhibitors; D002801:Cholinesterase Reactivators; D008024:Ligands; D009943:Organophosphorus Compounds; C032627:methylphosphonic acid; D002802:Cholinesterases