Exploration of a fundamental substituent effect of α-ketoheterocycle enzyme inhibitors: Potent and selective inhibitors of fatty acid amide hydrolase

Abstract

A series of C4 substituted α-ketooxazoles were examined as inhibitors of the serine hydrolase fatty acid amide hydrolase in efforts that further define and generalize a fundamental substituent effect on enzyme inhibitory potency. Thus, a plot of the Hammett σ m versus -log K i provided a linear correlation (R 2 = 0.90) with a slope of 3.37 (ρ = 3.37) that is of a magnitude that indicates the electronwithdrawing character of the substituent dominates its effects (a one unit change in σ m provides a >1000-fold change in K i ).Fatty acid amide hydrolase (FAAH) 1,2 is the enzyme that serves to hydrolyze endogenous lipid amides 3,4 including anandamide (1a) 5 and oleamide (1b), 6 Figure 1. Its distribution is consistent with its role in degrading and regulating such signaling fatty acid amides at their sites of action. 3 Although it is a member of the amidase signature family of serine hydrolases, for which there a number of prokaryotic enzymes, it is currently the only characterized mammalian enzyme bearing the family's unusual Ser-Ser-Lys catalytic triad. 7,8 Due to the therapeutic potential of inhibiting FAAH 9 especially for the treatment of pain, 10 inflammation, 11 or sleep disorders, 12 there has been increasing interest in the development of selective and potent inhibitors of the enzyme. 9 Early studies shortly following the initial discovery and characterization of FAAH led to the demonstration that the endogenous sleepinducing molecule 2-octyl α-bromoacetoacetate is an effective FAAH inhibitor, 13 the disclosure of a series of nonselective, reversible inhibitors bearing an electrophilic ketone (e.g., trifluoromethyl ketone-based inhibitors), 14,15 and the reports of a set of irreversible inhibitors 16 (e.g., fluorophosphonates and sulfonyl fluorides). To date, two classes of inhibitors have been disclosed that provide opportunities for the development of inhibitors with therapeutic potential. One class is the reactive aryl carbamates and ureas 17-24 that irreversibly acylate a FAAH active site serine. 25 A second class is the α-ketoheterocycle-based inhibitors 26-29 that bind to FAAH via reversible hemiketal formation with an active site serine. Many of these latter competitive inhibitors are not only potent and extraordinarily selective for FAAH versus other mammalian serine hydrolases, but members of this class have been shown to be efficacious analgesics in vivo. 28In the course of these latter studies, we disclosed a fundamental substituent effect in which a well-defined correlation between the electronic character of a para substituent (Hammett σ p ) and the inhibitor potency (−log K i ) was observed. 27 Thus, the inhibitor potency was found to *Corresponding author: email boger@scripps.edu. Whereas the former para substituents are directly conjugated with the electrophilic carbonyl, the meta substituents would exert their effects through their inductive electron-withdrawing properties. Moreover and although intuitive expectations might suggest that such a nonconjugated substituent effect might...

X-ray Crystallographic Analysis of α-Ketoheterocycle Inhibitors Bound to a Humanized Variant of Fatty Acid Amide Hydrolase

“…In contrast and given its geometry, the FAAH Ser217 engages in a SerOH–π H-bond with the activating heterocycle. Thus, the role of the activating heterocycle is intrinsically different and this accounts for the remarkable and unanticipated substituent effects observed in our work,34,37,39 where the inhibitor potency actually increases, not decreases, with the addition of electron-withdrawing substituents representing effects that are not observed in the work of Edwards and others 46,47. True to our observations and in contrast to prior works, the heterocycle role is not one of preferential H-bonding stabilization of the tetrahedral adduct via interaction of its proximal basic nitrogen with a core catalytic residue.…”

Enol Carbamates as Inhibitors of Fatty Acid Amide Hydrolase (FAAH) Endowed with High Selectivity for FAAH over the Other Targets of the Endocannabinoid System

“…[19] Starting from activated ketones designed as putative active-site traps, and including chloroketones, a-ketoamides, a-ketoesters, trifluoromethylketones and diazoketones, [12] past development of FAAH inhibitors has led to the recent discovery of several classes of a-ketoheterocycles as potent and highly selective blockers of FAAH, compared to other serine hydrolases. [13,20] However, the systematic study of specificity towards FAAH over all the other well-defined ECS elements has been investigated only rarely (e.g., in the case of OL-135 only with respect to CB1R, CB2R and TRPV1). [21] Another class of FAAH inhibitors currently under investigation are the carbamate-based compounds.…”

Discovery and Development of Endocannabinoid-Hydrolyzing Enzyme Inhibitors

“…The most potent FAAH inhibitors in this series had an optimal C8-C12 chain length, -unsaturation introduced at the corresponding arachidonoyl 8,9 / 11,12 and oleoyl 8,9 / 9,10 location, and an oxazolpyridine moiety (for a representative examples: Table 2) [85]. Since this discovery, -ketoheterocyclic compounds have become one of the most widely investigated compound classes of FAAH inhibitors; with systematic modification of the alkyl chain and the core heterocycle [86][87][88][89][90][91][92][93] resulting in the synthesis of extremely potent compounds such as 7-phenyl-1- [91] with K i values of 4.7 and 0.75 nM, respectively. To date, OL-135 has been one of the most extensively studied FAAH inhibitors both in vitro and in vivo.…”