Reversible Competitive α-Ketoheterocycle Inhibitors of Fatty Acid Amide Hydrolase Containing Additional Conformational Constraints in the Acyl Side Chain: Orally Active, Long-Acting Analgesics

Abstract

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.

Computational insights into function and inhibition of fatty acid amide hydrolase

“…These inhibitors, however, were not selective, inhibiting several other serine hydrolases too. Later, several reversible inhibitors were discovered, including new trifluoromethyl ketones, a-ketoesters, a-ketoamides, and the a-ketoheterocycles, including the highly selective OL135 (4 e Scheme 3) [34]. Very recently, a new series of a-ketoheterocycles compounds, as derivatives of OL135 (for example, compound 5 e Scheme 3), have been described as irreversibly targeting a cysteine in the FAAH catalytic site (Cys269) of the enzymatic cavity, while also forming a reversible covalent bond with Ser241 [17].…”

Pyrazole phenylcyclohexylcarbamates as inhibitors of human fatty acid amide hydrolases (FAAH)

“…The chemical structures of a number of FAAH inhibitors are increases the analgesic and hypothermic activity of anandamide [11]. The compound was found to significantly elevate brain anandamide levels (approximately 5-fold) and produce antinociceptive effects for over 9 h [12]. Several carbamate inhibitors of FAAH have been described in the scientific literature [13].…”

α-Ketoheterocycle inhibitors of fatty acid amide hydrolase: Exploration of conformational constraints in the acyl side chain

“…In the most recent of these studies, we disclosed 63,64 a series of inhibitors including 3 with added conformational constraints in the flexible C2 acyl side chain of 2 (Figure 2). This latest series showed improved or comparable enzyme inhibition potency relative to 2 , indicating that removing many of the rotatable bonds in the C2 acyl side chain can simultaneously enhance target binding affinity and improve the drug-like characteristics of the inhibitor.…”

“…Although many may be under the impression such inhibitors bearing electrophilic carbonyls may be metabolically labile, we have found that they are subject to competitive reduction/reoxidation metabolism that sets up a steady-state equilibrium between the two states (ketone/alcohol) with the true in vivo fate of the candidate inhibitors being determined by other features of the molecule. 63 Moreover, the added conformational constraints in the C2 acyl chain and the increased steric hindrance surrounding the electrophilic ketone both slow the rate of equilibration and improve the ketone/alcohol ratio in vivo. 63 As a result, the lead 1,2,3,4-tetrahydronaphthalene analogue 3 exhibited robust, long acting analgesic activity when administered orally in mouse models of thermal hyperalgesia and neuropathic pain in preliminary in vivo studies.…”