The synthesis and evaluation of a refined series of α-ketoheterocycles based on the oxazole 2 (OL-135) incorporating systematic changes in the central heterocycle bearing a key set of added substituents are described. The nature of the central heterocycle, even within the systematic and minor perturbations explored herein, significantly influenced the inhibitor activity: 1,3,4-oxadiazoles and 1,2,4-oxadiazoles 9 > tetrazoles, the isomeric 1,2,4-oxadiazoles 10, 1,3,4-thiadiazoles > oxazoles including 2 > 1,2-diazines > thiazoles > 1,3,4-triazoles. Most evident in these trends is the observation that introduction of an additional heteroatom at position 4 (oxazole numbering, N > O > CH) substantially increases activity that may be attributed to a reduced destabilizing steric interaction at the FAAH active site. Added heterocycle substituents displaying well defined trends may be utilized to enhance the inhibitor potency and, more significantly, to enhance the inhibitor selectivity. These trends, exemplified herein, emerge from both enhancements in the FAAH activity and simultaneous disruption of binding affinity for competitive off-target enzymes.

A series of alpha-ketooxazoles containing conformational constraints in the flexible C2 acyl side chain of 2 (OL-135) and representative oxazole C5 substituents were prepared and examined as inhibitors of fatty acid amide hydrolase (FAAH). Exceptionally potent and selective FAAH inhibitors emerged from the series (e.g., 6, Ki = 200 and 260 pM for rat and rhFAAH). With simple and small C5 oxazole substituents, each series bearing a biphenylethyl, phenoxyphenethyl, or (phenoxymethyl)phenethyl C2 side chain was found to follow a well-defined linear relationship between -log Ki and Hammett sigmap of a magnitude (rho = 2.7-3.0) that indicates that the substituent electronic effect dominates, confirming its fundamental importance to the series and further establishing its predictive value. Just as significantly, the nature of the C5 oxazole substituent substantially impacts the selectivity of the inhibitors whereas the effect of the C2 acyl chain was more subtle but still significant even in the small series examined. Combination of these independent features, which display generalized trends across a range of inhibitor series, simultaneously improves FAAH potency and selectivity and can provide exquisitely selective and potent FAAH inhibitors.

A series of α-ketooxazoles containing conformational constraints in the C2 acyl side chain of 2 (OL-135) were examined as inhibitors of fatty acid amide hydrolase (FAAH). Only one of the two possible enantiomers displayed potent FAAH inhibition (S vs R enantiomer), and their potency is comparable or improved relative to 2, indicating that the conformational restriction in the C2 acyl side chain is achievable. A cocrystal X-ray structure of the -ketoheterocycle 12 bound to a humanized variant of rat FAAH revealed its binding details, confirmed that the (S)-enantiomer is the bound active inhibitor, shed light on the origin of the enantiomeric selectivity, and confirmed that the catalytic Ser241 is covalently bound to the electrophilic carbonyl as a deprotonated hemiketal. Preliminary in vivo characterization of the inhibitors 12 and 14 is reported demonstrating that they raise brain anandamide levels following either intraperitoneal (i.p.) or oral (p.o.) administration indicative of effective in vivo FAAH inhibition. Significantly, the oral administration of 12 caused dramatic accumulation of anandamide in the brain, with peak levels achieved between 1.5–3 h and these elevations were maintained over 9 h. Additional studies of these two representative members of the series (12 and 14) in models of thermal hyperalgesia and neuropathic pain are reported, including the demonstration that 12 administered orally significantly attenuated mechanical (>6 h) and cold (>9 h) allodynia for sustained periods consistent with its long acting effects in raising the endogenous concentration of anandamide.

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...

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