Original article Carbamoyl tetrazoles as inhibitors of endocannabinoid inactivation: A critical revisitation Giorgio Ortar a , Maria Grazia Cascio b,c , Aniello Schiano Moriello b , Mercedes Camalli d , Enrico Morera a , Marianna Nalli a , Vincenzo Di Marzo b, * a Dipartimento di Studi Farmaceutici, Universita ` di Roma ‘La Sapienza’, piazzale Aldo Moro 5, 00185 Roma, Italy b Endocannabinoid Research Group, Institute of Biomolecular Chemistry, Consiglio Nazionale delle Ricerche, via dei Campi Flegrei 34, 80078 Pozzuoli (Napoli), Italy c Dipartimento di Scienze Farmaceutiche, Universita ` degli Studi di Salerno, via Ponte Don Melillo, 84084 Fisciano (SA), Italy d Istituto di Cristallografia, Consiglio Nazionale delle Ricerche, Area della Ricerca Roma 1, 00016 Monterotondo Stazione, Roma, Italy Received 24 November 2006; accepted 28 February 2007 Available online 19 March 2007 Abstract We have synthesized a series of 18 1,5- and 2,5-disubstituted carbamoyl tetrazoles, including LY2183240 (1) and LY2318912 (7), two compounds previously described as potent inhibitors of the cellular uptake of the endocannabinoid anandamide, and their regioisomers 2 and 8. We confirm that compound 1 is a potent inhibitor of both the cellular uptake and, like the other new compounds synthesized here, the enzymatic hydrolysis of anandamide. With the exception of 9, 12, 15, and the 2,5-regioisomer of LY2183240 2, the other compounds were all found to be weakly active or inactive on anandamide uptake. Several compounds also inhibited the enzymatic hydrolysis of the other main endocannabinoid, 2-arachidonoylglycerol, as well as its enzymatic release from sn-1-oleoyl-2-arachidonoyl-glycerol, at submicromolar concentrations. Four of the novel compounds, i.e. 3, 4, 17, and 18, inhibited anandamide hydrolysis potently (IC 50 ¼ 2.1e5.4 nM) and selectively over all the other targets tested (IC 50  10 mM), thus representing new potentially useful tools for the inhibition of fatty acid amide hydrolase. Ó 2007 Elsevier Masson SAS. All rights reserved. Keywords: Endocannabinoids; Anandamide cellular uptake inhibitors; Fatty acid amide hydrolase inhibitors; Carbamoyl tetrazoles 1. Introduction Endocannabinoids, i.e. endogenous agonists of CB 1 and CB 2 cannabinoid receptors [1,2], are involved in an extraordi- narily large number of physiological and pathological condi- tions in mammals [3,4]. Increasing evidence suggests that the modulation of endocannabinoid levels might be of valu- able therapeutic relevance in the treatment of a variety of path- ological conditions including anorexia, anxiety, inflammation, nausea and emesis, neuropsychiatric disorders, pain and spas- ticity [5]. The levels of the two main endocannabinoids, anan- damide (AEA) [6] and 2-arachidonoylglycerol (2-AG) [7,8], are regulated by specific biosynthetic and degradative path- ways, most of which have been now fully characterized and the relevant enzymes cloned [9]. The termination of the action of AEA is regulated by its cellular uptake, followed by intra- cellular hydrolysis by fatty acid amide hydrolase (FAAH). The mechanism of AEA transport, first characterized in 1994 by Di Marzo et al. [10], has been proposed to be an ATP-indepen- dent process of facilitated diffusion. However, a protein able to specifically bind AEA and facilitate its migration across the cell membrane has not yet been identified, its existence is still controversial, and alternative hypotheses have been pro- posed, including passive diffusion, endocytosis, and intracellu- lar sequestration [11e17]. Much of the discussion in recent years has been centred on the issue of whether or not AEA uptake is driven uniquely by the FAAH-catalyzed hydrolysis of the compound. * Corresponding author. Tel.: þ39 081 8675093; fax: þ39 081 8041770. E-mail address: vdimarzo@icmib.na.cnr.it (V. Di Marzo). 0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejmech.2007.02.023 Available online at www.sciencedirect.com European Journal of Medicinal Chemistry 43 (2008) 62e72 http://www.elsevier.com/locate/ejmech