Novel Analogues of Arachidonylethanolamide (Anandamide):  Affinities for the CB1 and CB2 Cannabinoid Receptors and Metabolic Stability

Abstract

Several analogues of the endogenous cannabinoid receptor ligand arachidonylethanolamide (anandamide) were synthesized and evaluated in order to study (a) the structural requirements for high-affinity binding to the CB1 and CB2 cannabinoid receptors and (b) their hydrolytic stability toward anandamide amidase. The series reported here was aimed at exploring structure-activity relationships (SAR) primarily with regard to stereoelectronic requirements of ethanolamido headgroup for interaction with the cannabinoid receptor active site. Receptor affinities, reported as Ki values, were obtained by a standard receptor binding assay using [3H]CP-55,940 as the radioligand, while stability toward the amidase was evaluated by comparing the Ki of each analogue in the presence and absence of phenylmethanesulfonyl fluoride (PMSF), a serine protease blocker and inhibitor of anandamide amidase. Introduction of a methyl group in the 1'- and 2'-positions or substitution of the ethanolamido headgroup with a butylamido group gave analogues with vastly improved biochemical stability. This is accomplished in some cases with increased receptor affinity. Conversely, oxazolyl and methyloxazolyl headgroups led to low-affinity analogues. Substitution of the hydroxyl group with electronegative substituents such as fluoro, chloro, allyl, and propargyl groups significantly increased receptor affinity but did not influence the biochemical stability. The 2'-chloro analogue of anandamide was found to have the highest affinity for CB1. Additionally, reversing the positions of the carbonyl and NH in the amido group produces retro-anandamides possessing considerably higher metabolic stability. Replacement of the arachidonyl tail with oleyl or linoleyl results in analogues with low affinities for both receptors. All of the analogues in this study showed high selectivity for the CB1 receptor over the peripheral CB2 receptor. The most potent analogues were tested for their ability to stimulate the binding of [35S]GTPgammaS to G-proteins and were shown to be potent cannabimimetic agonists. The results are discussed in terms of pharmacophoric features affecting receptor affinity and enzymatic stability.

The Endocannabinoid System in Human Keratinocytes

“…These saturation curves were very close to those obtained with mouse brain membranes (Fig. 1A), a positive control for CB1R (35,36) (Fig. 1B).…”

Discriminative stimulus functions in rats of AM1346, a high-affinity CB1R selective anandamide analog

“…However, the previous assessment of the duration of action for AM1346 was less than ideal because of limited drug availability at the time of conducting that study. Metabolically stable AEA analogs are expected to be more resistant to degradation by the enzyme FAAH compared to AEA (Cravatt et al 1996;Lin et al 1998). Thus, the long duration of action of AM1346 can be attributed, at least partly, to the ligand being a poor substrate for FAAH (Khanolkar and Makriyannis 1999).…”

A biosynthetic pathway for anandamide

“…Quantitation was performed by SRM in the positive ion mode using [ Radioligand Binding. The binding of pAEA and AEA to mouse brain cannabinoid receptors was assessed as described (28), except that the membranes were treated with PMSF (28). The membranes were subsequently treated with or without 1 mM NaVO 3 for 1 h on ice before the initiation of the assay.…”