EVALUATION OF CANNABINOID RECEPTOR-BINDING AND IN-VIVO ACTIVITIES FORANANDAMIDE ANALOGS

Recent evidence implicates anandamide as the endogenous ligand for the cannabinoid receptor. One purpose of this study was to determine the structural requirements for anandamide's receptor interaction and the influence of phenylmethylsulfonyl fluoride (PMSF), an enzyme inhibitor , on receptor affinity. A second objective was evaluation of the corre lation between affinities of the analogs and in vivo pharmacological a ctivities. The ability of anandamide and analogs to displace [H-3]CP-5 5,940 eptyl)phenyl]-4-[3-hydroxylpropyl]cyclohexan-1-ol} was determine d by a filtration assay. Displacement curves for anandamide in the pre sence of PMSF produced a K-i of 89 +/- 10 nM; without PMSF the K-i inc reased to 5400 +/- 1600 nM. Anandamide analogs were evaluated for thei r ability to produce antinociception and hypomotility. The levels of s aturation of the anandamide structure were critical to receptor affini ty and in vivo potency, with complete saturation and hydroxyl substitu tion with a fluorine moiety resulting in a compound with increased pot ency in the spontaneous activity and antinociception assays. Substitut ion of the hydroxyl with a fluorine atom increased affinity only in th e presence of PMSF and reduced potency in the antinociception assay. E thanolamide substitution with bromobenzenesulfonamide produced an inac tive compound in all assays. Increasing the length of the N-substituen t by one or two carbons decreased receptor binding affinity and potenc y in the tail-flick assay only. Certain structural modifications, such as methylations, allowed the analogs to retain affinity without the a ddition of PMSF. Linear correlation between the behavioral and binding assays were performed, and the greatest correlation was obtained with compounds that were either very potent or inactive.