Novel selective and metabolically stable inhibitors of anandamide cellular uptake

Novel aromatic analogues of N-oleoylethanolamine and N-arachidonoylethanolamine (anandamide, AEA) were synthesized and, based on the capability of similar compounds to interact with proteins of the endocannabinoid and endovanilloid signaling systems, were tested on: (i) cannabinoid CB 1 and CB 2 receptors; (ii) vanilloid VR1 receptors; (iii) anandamide cellular uptake (ACU); and (iv) the fatty acid amide hydrolase (FAAH). The ( R)- and, particularly, the ( S)-1′-(4-hydroxybenzyl) derivatives of N-oleoylethanolamine and AEA (OMDM-1, OMDM-2, OMDM-3, and OMDM-4) inhibited to a varied extent ACU in RBL-2H3 cells ( K i ranging between 2.4 and 17.7 μM), the oleoyl analogues (OMDM-1 and OMDM-2, K i 2.4 and 3.0 μM, respectively) being 6- to 7-fold more potent than the arachidonoyl analogues (OMDM-3 and OMDM-4). These four compounds exhibited: (i) poor affinity for either CB 1 ( K i≥5 μM) or CB 2 ( K i>10 μM) receptors in rat brain and spleen membranes, respectively; (ii) almost no activity at vanilloid receptors in the intracellular calcium assay carried out with intact cells over-expressing the human VR1 ( ec 50≥10 μM); and (iii) no activity as inhibitors of FAAH in N18TG2 cell membranes ( K i>50 μM). The oleoyl- and arachidonoyl- N′-(4-hydroxy-3-methoxybenzyl)hydrazines (OMDM-5 and OMDM-6), inhibited ACU ( K i 4.8 and 7.0 μM, respectively), and were more potent as VR1 agonists ( ec 50 75 and 50 nM, respectively), weakly active as CB 1 receptor ligands ( K i 4.9 and 3.2 μM, respectively), and inactive as CB 2 ligands ( K i>5 μM) as well as on FAAH ( K i≥40 μM). In conclusion, we report two novel potent and selective inhibitors of ACU (OMDM-1 and OMDM-2) and one “hybrid” agonist of CB 1 and VR1 receptors (OMDM-6). Unlike other compounds of the same type, OMDM-1, OMDM-2, and OMDM-6 were very stable to enzymatic hydrolysis by rat brain homogenates.