The anandamide membrane transporter. Structure–activity relationships of anandamide and oleoylethanolamine analogs with phenyl rings in the polar head group region Vincenzo Di Marzo, a Alessia Ligresti, a Enrico Morera, b Marianna Nalli b and Giorgio Ortar b, * a Endocannabinoid Research Group, Institute of Biomolecular Chemistry, Consiglio Nazionale delle Ricerche, Via Campi Flegrei 34, 80078 Pozzuoli, Napoli, Italy b Dipartimento di Studi Farmaceutici, Universita ` di Roma ÔLa SapienzaÕ, Piazzale A. Moro 5, 00185 Roma, Italy Received 19 March 2004; accepted 13 July 2004 Available online 7 August 2004 Abstract—A new series of anandamide and N-oleoylethanolamine analogs, most of which with aromatic moieties in the head group region, has been synthesized and evaluated as inhibitors of anandamide uptake. Some of them efficaciously inhibit the uptake process with K i values in the low micromolar range (2.4–21.2 lM). Strict structural requisites are needed to observe a significant inhibition and in no case inhibition of fatty acid amidohydrolase overlaps with inhibition of anandamide uptake. Ó 2004 Elsevier Ltd. All rights reserved. 1. Introduction The endogenous cannabinoid system (ECS) 1 comprises two G-protein coupled receptors, CB 1 2 and CB 2 , 3 their endogenous ligands [anandamide (AEA), 4 2-arachido- noylglycerol (2-AG), 5 2-arachidonyl glyceryl ether (2- AGE, noladin ether) 6 and O-arachidonoylethanolamine (virodhamine)] 7 and an endocannabinoid inactivation system. AEA and 2-AG are inactivated via a cellular up- take, facilitated by one or more transporters, 8 followed by enzymatic hydrolysis, which is catalyzed by a fatty acid amide hydrolase (FAAH) 9 and, in the case of 2- AG, also by monoacylglycerol lipases. 10 The putative AEA transporter (AMT) has not yet been cloned and its nature is still controversial, 11,12 although consider- able evidence, including selective inhibition by various structural analogs of AEA, points to its existence. 13–22 The well established involvement of ECS in the regula- tion of a wide series of both central and peripheral pro- cesses has stimulated an enormous interest in the potential therapeutic applications of compounds acting on the proteins that constitute the ECS. 23–30 In particu- lar, AEA transport inhibitors could offer a therapeutic approach to a variety of diseases including pain, 31 multi- ple sclerosis, 32 HuntingtonÕs chorea 33 in which elevation of AEA levels would seem to afford more favorable responses and fewer undesirable side-effects than direct activation of CB 1 receptors by agonists. Studies of structure–activity relationships concerned with the AMT and its substrates/inhibitors represent therefore an area of considerable interest. A large num- ber of modifications on the three potential pharmaco- phore moieties of AEA, the hydrophobic cis-tetraene side chain, the carboxamido group and the polar head group, has been already carried out 34 and resulted in the identification of a number of potent inhibitors such as AM404, 35 some N-acylvanillylamides (N-AVAMs), 36 VDM11, 37 UCM707, 15 3-pyridinylarachidonylamide 38 and SKM4451. 39 However, AM404 also activates vanil- loid (TRPV1) receptors, inhibits FAAH 37,40 and is not very stable to enzymatic hydrolysis; N-AVAMs are also agonists at TRPV1 and CB 1 receptors, while VDM11, although almost inactive at these receptors and at FAAH, is hydrolytically instable. 37 Finally, 3-pyridinyl- arachidonylamide also has very high affinity for FAAH. 38 The identification of new potent, selective 0968-0896/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmc.2004.07.026 Keywords: Endocannabinoids; Anandamide transporter; Anandamide transporter inhibitors; Structure–activity relationships. * Corresponding author. Tel.: +39-649913612; fax: +39-6491491; e-mail: giorgio.ortar@uniroma1.it Bioorganic & Medicinal Chemistry 12 (2004) 5161–5169