Synthesis and In Vitro and In Vivo Inhibition Potencies of Highly Relevant Nerve Agent Surrogates

Abstract

Four nonvolatile nerve agent surrogates, 4-nitrophenyl ethyl dimethylphosphoramidate (NEDPA, a tabun surrogate), 4-nitrophenyl ethyl methylphosphonate (NEMP, a VX surrogate), and two sarin surrogates, phthalimidyl isopropyl methylphosphonate (PIMP) and 4-nitrophenyl isopropyl methylphosphonate (NIMP), were synthesized and tested as acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitors. These surrogates were designed to phosphorylate cholinesterases with the same moiety as their respective nerve agents, making them highly relevant for the study of cholinesterase reactivators. Surrogates were characterized by liquid chromatography-mass spectrometry and nuclear magnetic resonance. NEMP, PIMP, and NIMP were potent inhibitors of rat brain, skeletal muscle, diaphragm, and serum AChE as well as human erythrocyte AChE and serum BuChE in vitro. PIMP was determined to degrade quickly in aqueous solution, making it useful for in vitro assays only, and NEDPA was not a potent inhibitor of AChE or BuChE in vitro; therefore, these two surrogates were not tested in subsequent in vivo studies. Sublethal dosages (yielding about 80% brain AChE inhibition) were determined for both the stable sarin surrogate, NIMP (0.325 mg/kg ip), and the VX surrogate, NEMP (0.4 mg/kg ip), in adult male rats. Time course studies indicated the time to peak brain AChE inhibition for both NIMP and NEMP to be 1 h postexposure. Both surrogates yielded severe cholinergic signs. These dosages did not require the addition of atropine to prevent lethality, and the rate of AChE aging was slow, making these surrogates useful for reactivation studies both in vitro and in vivo. The surrogates synthesized in this study are potent yet safer to test than nerve agents and are useful tools for initial screening of nerve agent oxime therapeutics.

Comparison of inhibition kinetics of several organophosphates, including some nerve agent surrogates, using human erythrocyte and rat and mouse brain acetylcholinesterase

“…The inhibitory potency can be influenced by the leaving group attached to the isopropyl methylphosphonate base structure. PIMP was developed as a less stable sarin surrogate to be used in in vitro assays to prevent significant reinhibition of reactivated AChE (Meek et al, 2012). Although it degrades within about 15 min in aqueous solution, PIMP was a more potent inhibitor than NIMP, indicating that the optimal leaving group is a phthalimidyl substituent which is 1.25-fold more potent than when the leaving group is a nitrophenyl substituent.…”

“…The synthesis reaction was as described by Meek et al (2012). Briefly, triethylamine in benzene was slowly added to a mixture of methylphosphonic dichloride and 4-nitrophenol in benzene (80 ml) at room temperature.…”

“…It would be beneficial to have chemicals that behave similarly to the nerve agents but are less toxic in order to allow laboratories that are not authorized to use the actual nerve agents to conduct research relevant to nerve agents. Therefore our laboratories developed surrogates for sarin and VX (Meek et al, 2012) and more recently to cyclosarin which substitute the nerve agent leaving group with another moiety ( i.e. , 4-nitrophenyl, 3,5,6-trichloro-2-pyridinyl, or phthalimidyl) (Fig.…”

The effect of PON1 enhancers on reducing acetylcholinesterase inhibition following organophosphate anticholinesterase exposure in rats

“…Organophosphate substrate synthesis: Surrogates of sarin (nitrophenyl isopropyl methylphosphonate; NIMP) and VX (nitrophenyl ethyl methylphosphonate; NEMP) were synthesized using established methods (Meek et al, 2012).…”

“…From our previous in vitro study (Chambers et al, 2015), a subset of the most efficacious enhancers was selected for these in vivo tests. The OPs selected as substrates for PON1 were surrogates for the nerve agents sarin and VX that our laboratories have synthesized and used in AChE reactivation studies (Meek et al, 2012;Chambers et al, 2013) and in the in vitro PON1 enhancer studies (Chambers et al, 2015). The goal of the present study was to determine if nucleophiles that can enhance PON1-mediated detoxication of OPs in vitro can provide protection from circulating toxic OPs in an in vivo system, thereby identifying compounds with the potential for being developed into nerve agent therapeutics possessing a novel mechanism of action.…”