Arachidonylethanolamide, an arachidonic acid derivative in porcine brain, was identified in a screen for endogenous ligands for the cannabinoid receptor. The structure of this compound, which has been named "anandamide," was determined by mass spectrometry and nuclear magnetic resonance spectroscopy and was confirmed by synthesis. Anandamide inhibited the specific binding of a radiolabeled cannabinoid probe to synaptosomal membranes in a manner typical of competitive ligands and produced a concentration-dependent inhibition of the electrically evoked twitch response to the mouse vas deferens, a characteristic effect of psychotropic cannabinoids. These properties suggest that anandamide may function as a natural ligand for the cannabinoid receptor.

We investigated the pharmacology of three novel compounds, Org 27569 (5-chloro-3-ethyl-1H-indole-2-carboxylic acid [2-(4-piperidin-1-yl-phenyl)-ethyl]-amide), Org 27759 (3-ethyl-5-fluoro-1H-indole-2-carboxylic acid [2-94-dimethylamino-phenyl)-ethyl]-amide), and Org 29647 (5-chloro-3-ethyl-1H-indole-2-carboxylic acid (1-benzyl-pyrrolidin-3-yl)-amide, 2-enedioic acid salt), at the cannabinoid CB 1 receptor. In equilibrium binding assays, the Org compounds significantly increased the binding of the CB 1 receptor agonist, indicative of a positively cooperative allosteric effect. The same compounds caused a significant, but incomplete, decrease in the specific binding of the CB 1 receptor inverse agonist studies also validated the allosteric nature of the Org compounds, because they all significantly decreased radioligand dissociation. These data suggest that the Org compounds bind allosterically to the CB 1 receptor and elicit a conformational change that increases agonist affinity for the orthosteric binding site. In contrast to the binding assays, however, the Org compounds behaved as insurmountable antagonists of receptor function; in the reporter gene assay, the guanosine 5Ј-O-(3-[35 S]thio)triphosphate binding assay and the mouse vas deferens assay they elicited a significant reduction in the E max value for CB 1 receptor agonists. The data presented clearly demonstrate, for the first time, that the cannabinoid CB 1 receptor contains an allosteric binding site that can be recognized by synthetic small molecule ligands.Mammalian tissues express at least two types of cannabinoid receptor, CB 1 and CB 2 , both G protein-coupled (for review, see Howlett et al., 2002). CB 1 receptors are found predominantly at central and peripheral nerve terminals where they mediate inhibition of transmitter release. Endogenous ligands for these receptors also exist. These "endocannabinoids" are all eicosanoids, prominent examples including arachidonoylethanolamide (anandamide) and 2-arachidonoyl glycerol, both of which are synthesized on demand, removed from their sites of action by tissue uptake processes and metabolized by intracellular enzymes (Pertwee and Ross,

1 We have tested our prediction that AM630 is a CB 2 cannabinoid receptor ligand and also investigated whether L759633 and L759656, are CB 2 receptor agonists. 2 Binding assays with membranes from CHO cells stably transfected with human CB 1 or CB 2 receptors using [ 3 H]-CP55940, con®rmed the CB 2 -selectivity of L759633 and L759656 (CB 2 /CB 1 a nity ratios=163 and 414 respectively) and showed AM630 to have a K i at CB 2 receptors of 31.2 nM and a CB 2 /CB 1 a nity ratio of 165. 3 In CB 2 -transfected cells, L759633 and L759656 were potent inhibitors of forskolin-stimulated cyclic AMP production, with EC 50 values of 8.1 and 3.1 nM respectively and CB 1 /CB 2 EC 50 ratios of 41000 and 43000 respectively. 4 AM630 inhibited [ 35 S]-GTPgS binding to CB 2 receptor membranes (EC 50 =76.6 nM), enhanced forskolin-stimulated cyclic AMP production in CB 2 -transfected cells (5.2 fold by 1 mM), and antagonized the inhibition of forskolin-stimulated cyclic AMP production in this cell line induced by CP55940. 5 In CB 1 -transfected cells, forskolin-stimulated cyclic AMP production was signi®cantly inhibited by AM630 (22.6% at 1 mM and 45.9% at 10 mM) and by L759633 at 10 mM (48%) but not 1 mM. L759656 (10 mM) was not inhibitory. AM630 also produced a slight decrease in the mean inhibitory e ect of CP55940 on cyclic AMP production which was not statistically signi®cant. 6 We conclude that AM630 is a CB 2 -selective ligand that behaves as an inverse agonist at CB 2 receptors and as a weak partial agonist at CB 1 receptors. L759633 and L759656 are both potent CB 2 -selective agonists.

Four chiral congeners of arachidonylethanolamide (anandamide) have been synthesized and evaluated for (a) their ability to bind to the cannabinoid receptor in rat forebrain membranes and (b) their pharmacological potency as measured by the compounds' ability to inhibit electrically-evoked contractions of the mouse vas deferens. The lead analog was also tested for its potency in vivo. Of the analogs tested, (R)-(+)-arachidonyl-1'-hydroxy-2'-propylamide [(R)-methanandamide] exhibited the highest affinity for the cannabinoid receptor with a Ki of 20 +/- 1.6 nM, 4-fold lower than that of anandamide (Ki = 78 +/- 2 nM). Moreover, determination of the cannabinoid binding affinity in the presence and absence of the protease inhibitor phenylmethanesulfonyl fluoride (PMSF) revealed that (R)-methanandamide possesses a remarkable stability to aminopeptidase hydrolysis. Pharmacological studies on mouse isolated vasa deferentia demonstrated that all four analogs produce concentration-related inhibition of the twitch response and the order of potency is the same as the rank order of the affinities of these agonists for cannabinoid binding sites. Furthermore, experiments with mice have demonstrated that (R)-methanandamide also possesses cannabimimetric properties in vivo, as established by the four tests of hypothermia, hypokinesia, ring immobility, and antinociception.