N-acyl-dopamines: novel synthetic CB1 cannabinoid-receptor ligands and inhibitors of anandamide inactivation with cannabimimetic activity in vitro and in vivo

Abstract

We reported previously that synthetic amides of polyunsaturated fatty acids with bioactive amines can result in substances that interact with proteins of the endogenous cannabinoid system (ECS). Here we synthesized a series of N-acyl-dopamines (NADAs) and studied their effects on the anandamide membrane transporter, the anandamide amidohydrolase (fatty acid amide hydrolase, FAAH) and the two cannabinoid receptor subtypes, CB1 and CB2. NADAs competitively inhibited FAAH from N18TG2 cells (IC50 = 19–100µM), as well as the binding of the selective CB1 receptor ligand, [3H]SR141716A, to rat brain membranes (Ki = 250–3900nM). The arachidonoyl (20:4 ω6), eicosapentaenoyl (20:5 ω3), docosapentaenoyl (22:5 ω3), α-linolenoyl (18:3 ω3) and pinolenoyl (5c,9c,12c 18:3 ω6) homologues were also found to inhibit the anandamide membrane transporter in RBL-2H3 basophilic leukaemia and C6 glioma cells (IC50 = 17.5–33µM). NADAs did not inhibit the binding of the CB1/CB2 receptor ligand, [3H]WIN55,212-2, to rat spleen membranes (Ki > 10µM). N-arachidonyl-dopamine (AA-DA) exhibited 40-fold selectivity for CB1 (Ki = 250nM) over CB2 receptors, and N-docosapentaenoyl-dopamine exhibited 4-fold selectivity for the anandamide transporter over FAAH. AA-DA (0.1–10µM) did not displace D1 and D2 dopamine-receptor high-affinity ligands from rat brain membranes, thus suggesting that this compound has little affinity for these receptors. AA-DA was more potent and efficacious than anandamide as a CB1 agonist, as assessed by measuring the stimulatory effect on intracellular Ca2+ mobilization in undifferentiated N18TG2 neuroblastoma cells. This effect of AA-DA was counteracted by the CB1 antagonist SR141716A. AA-DA behaved as a CB1 agonist in vivo by inducing hypothermia, hypo-locomotion, catalepsy and analgesia in mice (1–10mg/kg). Finally, AA-DA potently inhibited (IC50 = 0.25µM) the proliferation of human breast MCF-7 cancer cells, thus behaving like other CB1 agonists. Also this effect was counteracted by SR141716A but not by the D2 antagonist haloperidol. We conclude that NADAs, and AA-DA in particular, may be novel and useful probes for the study of the ECS.