We describe here the synthesis and activity of a new series of oxime reactivators of cholinesterases (ChEs) that contain tertiary amine or imidazole protonatable functional groups. Equilibration between the neutral and protonated species at physiological pH enables the reactivators to cross the blood-brain barrier and distribute in the CNS aqueous space as dictated by interstitial and cellular pH values. Our structure-activity analysis of 134 novel compounds considers primarily imidazole aldoximes and N-substituted 2-hydroxyiminoacetamides. Reactivation capacities of novel oximes are rank ordered by their relative reactivation rate constants at 0.67 mm compared with 2-pyridinealdoxime methiodide for reactivation of four organophosphate (sarin, cyclosarin, VX, and paraoxon) conjugates of human acetylcholinesterase (hAChE). Rank order of the rates differs for reactivation of human butyrylcholinesterase (hBChE) conjugates. The 10 best reactivating oximes, predominantly hydroxyimino acetamide derivatives (for hAChE) and imidazole-containing aldoximes (for hBChE) also exhibited reasonable activity in the reactivation of tabun conjugates. Reactivation kinetics of the lead hydroxyimino acetamide reactivator of hAChE, when analyzed in terms of apparent affinity (1/K_ox) and maximum reactivation rate (k₂), is superior to the reference uncharged reactivators monoisonitrosoacetone and 2,3-butanedione monoxime and shows potential for further refinement. The disparate pH dependences for reactivation of ChE and the general base-catalyzed oximolysis of acetylthiocholine reveal that distinct reactivator ionization states are involved in the reactivation of ChE conjugates and in conferring nucleophilic reactivity of the oxime group.

We present a systematic structural optimization of uncharged but ionizable N-substituted 2-hydroxyiminoacetamido alkylamine reactivators of phosphylated human acetylcholinesterase (hAChE) intended to catalyze the hydrolysis of organophosphate (OP)-inhibited hAChE in the CNS. Starting with the initial lead oxime RS41A identified in our earlier study and extending to the azepine analog RS194B, reactivation rates for OP-hAChE conjugates formed by sarin, cyclosarin, VX, paraoxon, and tabun are enhanced severalfold in vitro. To analyze the mechanism of intrinsic reactivation of the OP-AChE conjugate and penetration of the blood-brain barrier, the pH dependence of the oxime and amine ionizing groups of the compounds and their nucleophilic potential were examined by UV-visible spectroscopy, ¹H NMR, and oximolysis rates for acetylthiocholine and phosphoester hydrolysis. Oximolysis rates were compared in solution and on AChE conjugates and analyzed in terms of the ionization states for reactivation of the OP-conjugated AChE. In addition, toxicity and pharmacokinetic studies in mice show significantly improved CNS penetration and retention for RS194B when compared with RS41A. The enhanced intrinsic reactivity against the OP-AChE target combined with favorable pharmacokinetic properties resulted in great improvement of antidotal properties of RS194B compared with RS41A and the standard peripherally active oxime, 2-pyridinealdoxime methiodide. Improvement was particularly noticeable when pretreatment of mice with RS194B before OP exposure was combined with RS194B reactivation therapy after the OP insult.

A series of eight double and triple mutants of mouse acetylcholinesterase (AChE; EC 3.1.1.7), with substitutions corresponding to residues found largely within the butyrylcholinesterase (BChE; EC 3.1.1.8) active-centre gorge, was analysed to compare steady-state kinetic constants for substrate turnover and inhibition parameters for enantiomeric methylphosphonate esters. The mutations combined substitutions in the acyl pocket (Phe295→Leu and Phe297→Ile) with the choline-binding site (Tyr337→Ala and Phe338→Ala) and with a side chain (Glu202→ Gln) N-terminal to the active-site serine, Ser203. The mutations affected catalysis by increasing Km and decreasing kcat, but these constants were typically affected by an order of magnitude or less, a relatively small change compared with the catalytic potential of AChE. To analyse the constraints on stereoselective phosphonylation, the mutant enzymes were reacted with a congeneric series of SP- and RP-methylphosphonates of known absolute stereochemistry. Where possible, the overall reaction rates were deconstructed into the primary constants for formation of the reversible complex and intrinsic phosphonylation. The multiple mutations greatly reduced the reaction rates of the more reactive SP-methylphosphonates, whereas the rates of reaction with the RP-methylphosphonates were markedly enhanced. With the phosphonates of larger steric bulk, the enhancement of rates for the RP enantiomers, coupled with the reduction of the SP enantiomers, was sufficient to invert markedly the enantiomeric preference. The sequence of mutations to enlarge the size of the AChE active-centre gorge, resembling in part the more spacious gorge of BChE, did not show an ordered conversion into BChE reactivity as anticipated for a rigid template. Rather, the individual aromatic residues may mutually interact to confer a distinctive stereospecificity pattern towards organophosphates.