New Structural Scaffolds for Centrally Acting Oxime Reactivators of Phosphylated Cholinesterases

Abstract

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 (k 2 ), 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.It has become increasingly apparent that efficient reinstatement of CNS acetylcholinesterase (AChE) 2 activity inhibited in organophosphate (OP)-intoxicated individuals is required for sustained symptom recovery. In particular, nerve agent OPs already used by terrorists, but also active metabolites of OP-based pesticides, readily cross the bloodbrain barrier (BBB). The exposure to OP doses close to lethality results in initial severe motor convulsions and epileptic seizures. Accumulating evidence points to these seizure events being linked to irreversible long term compromise of cognitive functions and alteration of CNS electrical excitability. Once accumulated into hydrophobic sites, OPs that do enter the CNS are retained and partition slowly back into the circulation. For example, victims of Tokyo subway nerve gas attack in 1995 were found to suffer from both short and long term symptoms of OP exposure (1-4). Accordingly, comprehensive protection from and treatment of OP intoxication to minimize the longer term consequences require administration of antidotes capable of reactivating OP-inhibited AChE in the CNS. Current therapy directed to reactivating inhibited AChE is limited to the peripheral circulation because commonly used quaternary pyridinium aldoxime reactivators do not cross the BBB at ...

Refinement of Structural Leads for Centrally Acting Oxime Reactivators of Phosphylated Cholinesterases

“…The data presented herein are based on our recent revelation that N-substituted 2-hydroxyimino acetamidoalkyl amines, although devoid of a permanent cationic charge, can efficiently reactivate OP conjugated hAChE in vitro (1). Formation of protonation equilibria around two ionizable groups in those oxime structures, an oxime group and an additional amine group, results in coexistence of charged, zwitterionic, and uncharged reactivator species around physiological pH values.…”

“…OP-hAChE conjugates were prepared, and oxime reactivation was performed (at 37°C in 0.1 M sodium phosphate buffer, pH 7.4, containing 0.01% BSA) as described earlier (1,16). The first order reactivation rate constant (k obs ) for each oxime ϩ OP conjugate combination was calculated by nonlinear regression (19).…”

“…S2-S6 and Tables S1-S6. 1 Both authors contributed equally to this work. 2 To whom correspondence should be addressed: SSPPS, UCSD, 9500 Gilman Drive, La Jolla, CA 92093-0657.…”

The Lock is the Key: Development of Novel Drugs through Receptor Based Combinatorial Chemistry

“…111 Therefore, attempts have been made to develop centrally acting reactivators using click-chemistry approach. 112,113 The AChE related enzyme butyrylcholinesterase (BChE) is present in the plasma in high concentrations and differs in the amino acid composition. 114,115 BCh-E is capable of hydrolyzing a variety of esters and plays an important role in the bioconversion of carbamates and other ester-based prodrugs.…”

Enzyme-catalyzed cascade synthesis of hydroxyiminoacetamides

“…The final reaction was the formation of an amide bond between 3-azido-1-phenylpropylamine (5) and ethyl glyoxylate oxime, which was previously described for similar compounds. 7 However, as in numerous other cases, amide bond formation turned out to represent a significant challenge.…”