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 × 10−4 min−1 (t1/2 = 35.5 h) at 25 °C and 8.70 × 10−4 min−1 (t1/2 = 13.3 h) at 37 °C. Compound 1 was a potent inhibitor of human acetylcholinesterase (HuAChE; ki = 7.5 × 105 M−1 min−1), electric eel acetylcholinesterase (EEAChE) (ki = 3.0 × 106 M−1 min−1), and human serum butyrylcholinesterase (HuBChE; 1.95 × 105 M−1 min−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 × 10−5 min−1 (t1/2 = 131.2 h) and 2.41 × 10−2 min−1 (t1/2 = 0.48 h), (b) EEAChE 9.32 × 10−3 min−1 (t1/2 = 1.24 h) and 3.33 × 10−2 min−1 (t1/2 = 0.35 h), and (c) HuBChE 1.16 × 10−4 min−1 (t1/2 = 99.6 h) and 4.19 × 10−2 min−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.

An improved radiosynthesis of O-(2-[18 F]fluoroethyl)-O-(p -nitrophenyl)methylphosphonate: A first-in-class cholinesterase PET tracer

“…Incorporation of fluorine at the methyl or alpha carbon of the ethoxy substituent would increase hydrolytic reactivity at phosphorus atom and ester group, respectively, and were dismissed as possible fluorine atom introduction sites leaving the beta ethyl position as the sole option for positron atom labeling. The suitable viability of this beta ethyl position strategy has recently been further confirmed by cold ligand in vitro cholinesterase studies …”

“…Although a number of radiotracers and imaging approaches to investigate AChE have been reported, there are very few reports of OP PET imaging radiotracers that are based on the covalent modification of this target. Tracer [ 18 F]1 is the first‐in‐class example of an OP agent that simulates the mechanism of inhibition of OP insecticides and chemical warfare agents, specifically, phosphonylation of the active site serine . For this reason, radioligand [ 18 F]1 is of strategic importance to evaluate in vivo tissue exposures to OP compounds and to interrogate new and existing countermeasures to OP toxicity.…”

“…The resultant fluoro‐VX surrogate, O‐(2‐fluoroethyl)‐O‐( p ‐nitrophenyl) methylphosphonate, uses a p ‐nitrophenoxy leaving group and acts similarly in vitro and in vivo to VX without handling concerns . The fluorine tracer atom is strategically positioned 4 bonds from the reactive phosphorus atom to minimize steric or electronic perturbation . Injection of high specific activity O‐(2‐[ 18 F]fluoroethyl)‐O‐( p ‐nitrophenyl) methylphosphonate, [ 18 F]1 , in rats shows localization of radioactivity in AChE‐rich regions of brain and other tissues where it likely exists as (CH 3 )( 18 FCH 2 CH 2 O)P(O)‐AChE and other esterase adducts …”

Radiosynthesis of O‐(1‐[18F]fluoropropan‐2‐yl)‐O‐(4‐nitrophenyl)methylphosphonate: A novel PET tracer surrogate of sarin

“…The PNP group effectively mimics the leaving group in the CWA preserving the mechanism of inhibition forming the O‐(2‐fluoro‐ethyl) methylphosphonate‐AChE adduct thereby leaving the [ 18 F]‐OP attached to AChE. The rate of AChE inhibition by the VX and sarin PNP surrogates was slightly less than VX and sarin, but the presence of a fluorine on the alkoxy ester group reduced the rate of aging while favoring reactivation . Thus, these surrogate compounds labeled with fluorine‐18 furnish stable OP‐AChE adducts that can aptly report on pools of active AChE in small animal models …”

“…Previously, a novel radiolabeled OP surrogate based upon the CWA compound VX was prepared by replacing the β‐aminothiol leaving group with a p ‐nitrophenyl (X = PNP) and the addition of a fluorine‐18 atom to the ethyl ester side chain (R = CH 2 CH 2 F; Scheme ) providing an OP‐based PET imaging tracer . The PNP group effectively mimics the leaving group in the CWA preserving the mechanism of inhibition forming the O‐(2‐fluoro‐ethyl) methylphosphonate‐AChE adduct thereby leaving the [ 18 F]‐OP attached to AChE.…”