ABSTRACT
Isosorbide-2-carbamate-5-esters are highly potent and selective butyrylcholinesterase inhibitors with potential utility in the treatment of Alzheimer's Disease (AD). They are stable in human plasma but in mouse plasma they undergo hydrolysis at the 5-ester group potentially attenuating in vivo potency. In this paper we explore the role of the 5-position in modulating potency. The focus of the study was to increase metabolic stability while preserving potency and selectivity. Dicarbamates and 5-keto derivatives were markedly less potent than the ester class. The 2-benzylcarbamate-5-benzyl ether was found to be potent (IC(50) 52 nM) and stable in the presence of mouse plasma and liver homogenate. The compound produces sustained moderate inhibition of mouse butyrylcholinesterase at 1mg/kg, IP.
Subject(s)
Butyrylcholinesterase/metabolism , Cholinesterase Inhibitors/metabolism , Cholinesterase Inhibitors/pharmacology , Isosorbide/metabolism , Isosorbide/pharmacology , Alzheimer Disease/drug therapy , Animals , Cholinesterase Inhibitors/chemistry , Humans , Isosorbide/chemistry , Mice , Molecular Structure , Plasma/metabolism , Structure-Activity RelationshipABSTRACT
Isosorbide-2-benzyl carbamate-5-benzoate is a highly potent and selective BuChE inhibitor. Meanwhile, isosorbide-2-aspirinate-5-salicylate is a highly effective aspirin prodrug that relies on the salicylate portion to interact productively with human BuChE. By integrating the salicylate group into the carbamate design, we have produced isosorbide-2-benzyl carbamate-5-salicylate, an inhibitor of high potency (150 pM) and selectivity for human BuChE over AChE (666000) and CES2 (23000). Modeling and mutant studies indicate that it achieves its exceptional potency because of an interaction with the polar D70/Y332 cluster in the PAS of BuChE in addition to pseudosubstrate interactions with the active site.
Subject(s)
Anions/metabolism , Butyrylcholinesterase/chemistry , Carbamates/chemistry , Cholinesterase Inhibitors/pharmacology , Isosorbide/analogs & derivatives , Isosorbide/chemistry , Salicylates/pharmacology , Anions/chemistry , Binding Sites , Butyrylcholinesterase/blood , Butyrylcholinesterase/genetics , Carbamates/metabolism , Cholinesterase Inhibitors/chemical synthesis , Cholinesterase Inhibitors/chemistry , Humans , Inhibitory Concentration 50 , Intestines/drug effects , Intestines/enzymology , Isosorbide/chemical synthesis , Isosorbide/metabolism , Isosorbide/pharmacology , Male , Microsomes, Liver/drug effects , Microsomes, Liver/enzymology , Models, Molecular , Molecular Structure , Mutation/genetics , Salicylates/chemical synthesis , Salicylates/chemistry , Structure-Activity RelationshipABSTRACT
In this study, we report the SAR and characterization of two groups of isosorbide-based cholinesterase inhibitors. The first was based directly on the clinically used nitrate isosorbide mononitrate (ISMN) retention of the 5-nitrate group and introduction of a series of 2-carbamate functionalities. The compounds proved to be potent and selective inhibitors of human plasma butyrylcholinesterase ( huBuChE). In the second group, the nitrate ester was removed and replaced with a variety of alkyl and aryl esters. These generally exhibited nanomolar potency with high selectivity for BuChE over acetylcholinesterase (AChE). The most potent and selective compound was isosorbide-2-benzyl carbamate-5-benzoate with an IC 50 of 4.3 nM for BuChE and >50000 fold selectivity over human erythrocyte AChE. Inhibition with these compounds is time-dependent, competitive, and slowly reversible, indicating active site carbamylation.
Subject(s)
Butyrylcholinesterase/metabolism , Carbamates/chemistry , Cholinesterase Inhibitors/chemical synthesis , Cholinesterase Inhibitors/pharmacology , Esters/chemical synthesis , Esters/pharmacology , Isosorbide/chemistry , Binding Sites , Cholinesterase Inhibitors/chemistry , Esters/chemistry , Humans , Models, Molecular , Molecular Structure , Structure-Activity RelationshipABSTRACT
We report herein that a variety of isosorbide di-esters, previously reported to be novel substrates for butyrylcholinesterase (BuChE, EC 3.1.1.8), are in fact inhibitors of the homologous enzyme acetylcholinesterase (AChE), with IC(50) values in the micromolar range. In vitro studies show that they are mixed inhibitors of the enzyme, and thus the ternary enzyme-inhibitor-substrate complex can form in acetylcholinesterase. This is rationalised by molecular modelling which shows that the compounds bind in the mid-gorge area. In this position, simultaneous substrate binding might be possible, but the hydrolysis of this substrate is prevented. The di-esters dock within the butyrylcholinesterase gorge in a very different manner, with the ester sidechain at the 5-position occupying the acyl pocket at residues Leu286 and Val288, and the 2-ester binding to Trp82. The carbonyl group of the 2-ester is susceptible to nucleophilic attack by Ser198 of the catalytic triad. The larger residues of the acyl pocket in acetylcholinesterase prevent binding in this manner. The results complement each other and explain the differing behaviours of the esters in the cholinesterase enzymes. These findings may prove very significant for future work.
Subject(s)
Acetylcholinesterase/drug effects , Cholinesterase Inhibitors/pharmacology , Isosorbide/pharmacology , Cholinesterase Inhibitors/chemistry , Chromatography, High Pressure Liquid , Esters , Isosorbide/chemistry , Models, MolecularABSTRACT
Reported here is the synthesis and SAR of novel group of highly potent and selective inhibitors of human plasma butyrylcholinesterase (BuChE; EC 3.1.1.8). The design is based on the discovery that isosorbide 2-esters are hydrolysed by BuChE at exceptionally rapid rates. Two families of carbamates were synthesised in which the vulnerable 2-ester was replaced with a carbamate or reversed carabamate. Several compounds in one of the families are among the most potent and selective BuChE inhibitors reported.
