BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 231, 217–221 (1997) ARTICLE NO. RC976072 Fatty Acid Sulfonyl Fluorides Inhibit Anandamide Metabolism and Bind to the Cannabinoid Receptor D. G. Deutsch,* ,1 S. Lin,† W. A. G. Hill,† K. L. Morse,† D. Salehani,* G. Arreaza,* R. L. Omeir,* and A. Makriyannis† ,1 *Department of Biochemistry and Cell Biology, State University of New York at Stony Brook, Stony Brook, New York 11794; and †Department of Medicinal Chemistry, School of Pharmacy, University of Connecticut, Storrs, Connecticut 06269 Received December 30, 1996 named CB1 (1-4). Arachidonoyl ethanolamide (anan- Arachidonoyl ethanolamide (anandamide) is an en- damide), homo-g-linolenyl ethanolamide, and docosa- dogenous ligand for cannabinoid receptors (CB1, CB2) tetraenyl ethanolamide are naturally occurring brain and a putative neurotransmitter. Phenylmethylsulfo- constituents that bind to CB1 (5-7). Converging lines nyl fluoride (PMSF) is an inhibitor of the enzyme (an of evidence suggest that anandamide satisfies the es- amidase) which hydrolyzes anandamide to arachi- sential criteria of a neurotransmitter or intracellular donic acid and ethanolamine. We report here that fatty messenger for the central cannabinoid receptor (8-10). acid sulfonyl fluorides are potent inhibitors of anan- Soon after the discovery of anandamide, an enzyme damide metabolism. In order to investigate the SAR of (E.C. 3.5.1.4, E.C. 3.5.1.60, called anandamide ami- these anandamide amidase inhibitors we tested a se- dase, amidohydrolase, fatty acid amide hydrolase, N- ries of fatty acid (C12 to C20) sulfonyl fluorides both arachidonoyl ethanolamine deacylase) responsible for as inhibitors of anandamide degradation and as li- its hydrolysis was described (11-13) and cloned (14). gands for the central cannabinoid receptor (CB1). Anandamide is the preferred substrate for this enzyme AM374 (palmitylsulfonyl fluoride, C16) was approxi- although it reacts with a variety of other fatty acid mately 20 times more potent than PMSF and 50 times ethanolamides (15,16) and fatty acid amides including more potent than arachidonyltrifluoromethyl ketone in preventing the hydrolysis of anandamide in brain oleamide, a putative sleep factor (17). A series of ’’tran- homogenates. AM374 was over a thousand-fold more sition-state’’ inhibitors (trifluoromethyl ketone, a-keto effective than PMSF in inhibiting the amidase in cul- ester, and a-keto amide derivatives) were synthesized tured cells. The C12 to C18 sulfonyl fluoride analogs and tested in vitro as amidase inhibitors (18,19). The were equipotent as inhibitors of the amidase and the trifluoromethyl ketones (e.g., arachidonyltrifluoro- reverse reaction (the synthase) with nanomolar IC 50 methyl ketone) were found to inhibit anandamide hy- values. These compounds generally showed decreas- drolysis at low micromolar concentrations. Phenyl- ing affinity for the CB1 receptor as the chain length methylsulfonyl fluoride (PMSF) a non-selective in- increased; thus, C12 sulfonylfluoride had an IC 50 of 18 hibitor of serine proteases was found to act as an nM and C20 sulfonylfluoride had an IC 50 of 78 mM. The irreversible inhibitor of this amidase and its inclusion C14, C16, and C18 sulfonyl fluorides showed high selec- in receptor binding assays, or in in vitro assays, in- tivity for the amidase over the CB1 receptor and thus creases the apparent potency of anandamide are potentially useful selective anandamide amidase (11,15,16,20-27). However, the relatively low activity inhibitors.  1997 Academic Press of the above inhibitors severely limits their effective- ness as biochemical and pharmacological tools or as therapeutic agents. In the present communication, a series of fatty acid sulfonyl fluorides were tested as D 9 -Tetrahydrocannabinol (THC), the psychoactive inhibitors of anandamide metabolism and were found marijuana plant-derived cannabinoid and numerous to be potent and specific. synthetic analogs bind to a specific brain receptor, MATERIALS AND METHODS 1 Corresponding authors. Dale G. Deutsch, e-mail: ddeutsch@ ccmail.sunysb.edu; Fax: 516 632 8575. Alexandros Makriyannis, Synthesis of PMSF analogs. The fatty acid sulfonyl fluorides were synthesized from the respective alkyl bromides. Details of the syn- e-mail: makriyan@uconn.vm.uconn.edu; Fax: 860 486 3089. 0006-291X/97 $25.00 Copyright  1997 by Academic Press All rights of reproduction in any form reserved. 217