Pergamon Bioorganic &Medicinal Chemistry Letters 8 (1998) 613-618 BIOORGANIC & MEDICINAL CHEMISTRY LETIERS AN ENDOGENOUS SLEEP-INDUCING COMPOUND IS A NOVEL COMPETITIVE INHIBITOR OF FATTY ACID AMIDE HYDROLASE Matthew P. Patricelli, Jean E. Patterson, Dale L. Boger,” and Benjamin F. Cravattb* Skaggs Institutefor Chemical Biology and the Departments of “ Chemistry, and hCell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, L.u Jolla, California, 92037, U.S.A. Received 23 January 1998; accepted 2 February 1998 Abstract: 2-Octyl y-bromoacetoacetate (OyBr), an endogenous compound originally isolated from human cerebrospinal fluid (CSF), has previously been demonstrated to increase REM sleep duration in cats. Based on the chemical structure of OyBr and its reported sleep-inducing effects, we synthesized OyBr along with chemically related analogs and tested these compounds as inhibitors of fatty acid amide hydrolase (FAAH), a brain enzyme that degrades neuromodulatory fatty acid amides. OyBr was found to competitively inhibit FAAH activity with IC,, and K, values of 2.6 FM and 0.8 PM, respectively [for the (R)-enantiomer of OyBr (l)]. A set of synthetic analogs of OyBr was examined to define the structural features required for FAAH inhibition and inhibitor potencies were assessed at pH 9.0 (near the pH optimum of FAAH) and pH 7.0. Interestingly, at pH 7.0 the y- halo P-keto ester inhibitors proved to be significantly more potent than the trifluoromethyl ketone of oleic acid, one of the most potent FAAH inhibitors described to date. This study supports the possibility that OyBr may be a physiological regulator of FAAH activity and fatty acid amide levels in vivo. Additionally, the characterization of y-halo P-keto esters as powerful FAAH inhibitors near physiological pH may aid in future studies of the enzymology and biological properties of FAAH. 0 1998 Published by Elsevier Science Ltd. All rights reserved. Fatty acid amides represent a growing family of biologically active lipids implicated in a diverse range of cellular and physiological processes. l-3 Members of the fatty acid amide family include anandamide (arachidonoyl ethanolamide), an endogenous ligand for the CB 1 cannabinoid receptor! and oleamide [9(Z)octadecenamide], a sleep-inducing compound isolated from the cerebrospinal fluid of sleep-deprived cats.‘.4 Consistent with its proposed role as an endogenous cannabinoid, anandamide has been shown to elicit several physiological effects common to cannabinoid agonists.’ Oleamide has been shown to act in a structurally specific manner to induce physiological sleep in rats’ and potentiate 5-HT receptor responses to serotonin.6.7 Both anandamide’ and oleamide’ have been found to block gap junction communication in glial cells. Anandamide and oleamide are rapidly degraded to their corresponding inactive acids by a membrane- bound enzyme activity, fatty acid amide hydrolase (FAAH).‘O Recent studies have suggested that manipulation of FAAH activity in vivo can affect both the level and duration of the responses elicited by its fatty acid amide substrates. Addition of FAAH inhibitors to neuroblastoma cell cultures increased the amounts of anandamide released,” and FAAH-resistant analogs of anandamide have been shown to induce prolonged inhibition of motor activity.‘* We identified 2-octyl y-bromoacetoacetate (OyBr, l), an endogenous compound originally isolated from human cerebrospinal fluid,‘3 as a possible FAAH regulator based on its physiological and chemical properties. OyBr has been found in rat cortex, pituitary, and retinal tissues,14 and was shown to induce REM *Corresponding Author email: cravatt@scripps.edu 0960-894X/98/$19.00 0 1998 Published by Elsevier Science Ltd. All rights reserved. PII: SO960-894X(98)00073-0