Knowledge Bank

Organophosphorus (OP) compounds are a class of toxic compounds that are responsible for many toxic exposures and deaths annually. OP compounds inhibit acetylcholinesterase (AChE), an enzyme responsible for the hydrolysis of the neurotransmitter acetylcholine (ACh). OP compounds phosphylate the Ser203 residue of AChE, disabling the hydrolysis of ACh, leading to a cholinergic crisis where death can occur in severe cases. Oximes have historically been used to return the native enzyme from the inhibited enzyme in a process known as reactivation. However, a second step after OP inhibition can occur, known as aging, where spontaneous O-dealkylation occurs. Once aging happens, there are no approved therapeutics to recover the native enzyme. Oximes are also not capable of crossing the blood-brain barrier, and thus are not able to treat OP-inhibited AChE in the central nervous system (CNS) or any symptoms, like seizures, that can arise in the CNS.

Previously, our lab has demonstrated effective (>20%) reactivation and resurrection using quinone methide precursors (QMPs) with varying substituents at the 4-position of a phenol framework. One library of such QMPs include dimethoxyindanone-linked QMPs; dimethoxyindanone is a cholinesterase inhibitor with a high affinity for AChE, specifically its peripheral binding site (PAS). These compounds performed significant reactivation of ethyl paraoxon (EP)-inhibited AChE. To expand further on this knowledge, indanone-linked QMP precursors were synthesized with both varying substituents at the 5- and 6-positions and with different lengths of the alkyl chains. In the future, these compounds will undergo cross-electrophile coupling (XEC) to both phenol-based and pyridyl-based QMPs.

Our lab has shown that 4-amidophenol QMPs are effective reactivators and resurrectors of OP-inhibited and OP-aged cholinesterases. Due to their strong activity, carbamate QMPs were hypothesized to perform well due to the similarities between amides and carbamates. The synthesis and biochemical screenings of carbamate QMPs are detailed in this report. A total of 33 carbamate QMPs were synthesized with a variety of carbamate side chains and amine leaving groups. These compounds were tested for reactivation potential against OEt-inhibited butyrylcholinesterase (BChE) and AChE as well as resurrection potential against EP-aged AChE. Four (4) compounds showed significant activity in BChE. In AChE, five (5) compounds performed significant reactivation, and of these 5 compounds, two (2) showed significant resurrection activity.

Also detailed in this report are biochemical in vitro screenings for reactivation of OP-inhibited BChE using 4-amidophenols. In these studies, 44 compounds were tested against 4 different OP surrogates and results varied significantly between differing inhibited forms. In OiBu-inhibited BChE, 30 of the 44 QMPs synthesized performed significant reactivation, with native enzyme activity as high as 60%. In OEt-inhibited BChE, 10 compounds were significant reactivators, with the top compound reaching 43% native enzyme activity.

Overall, this report details the synthesis and screenings of a wide variety of QMPs capable of the reactivation and resurrection of various OP-inhibited and OP-aged forms of AChE and BChE.