Synthesis and SAR Studies of 2-Oxoquinoline Derivatives as CB2 Receptor Inverse Agonists Katri H. Raitio, ², * Juha R. Savinainen, ‡ Jouko Vepsa ¨la ¨inen, § Jarmo T. Laitinen, ‡ Antti Poso, ² Tomi Ja ¨rvinen, ² and Tapio Nevalainen ² Department of Pharmaceutical Chemistry, Department of Physiology, and Department of Chemistry, UniVersity of Kuopio, P.O. Box 1627, FI-70211 Kuopio, Finland ReceiVed September 6, 2005 The highly CB2 selective cannabinoid receptor inverse agonist, 7-methoxy-2-oxo-8-pentyloxy-1,2- dihydroquinoline-3-carboxylic acid N-benzo[1,3]dioxol-5-ylmethyl)amide (JTE-907; 9b), served as the lead compound for investigating the structure-activity relationships of its analogues and in the search for more potent and effective CB2 receptor inverse agonists. A series of aromatic amides of 7-methoxy-2-oxo-8- pentyloxy-1,2-dihydroquinoline-3-carboxylic acid 6 was synthesized, and the CB2 receptor activities of the compounds were determined by a [ 35 S]GTP γ S-binding assay using membranes of CHO cells stably transfected with the human CB2 receptor. As a result, all the compounds were defined as full CB2 receptor inverse agonists, and additionally, except for two 3,4-dihydroxyphenylalkylamides, they were found to be equally potent as SR144528. Introduction The peripheral cannabinoid receptor CB2 was molecularly identified in 1993 by Munro et al., 1 and their work in determining the distribution pattern of CB2 was subsequently complemented by Galie `gue et al. 2 The two signaling pathways utilized by the CB2 receptor are inhibition of adenylate cyclase through activation of the G i/o protein 3 and activation of mitogen- activated protein (MAP) kinase resulting in induction of the growth-related gene Krox-24 expression. 4 The immunomodulatory properties of the cannabinoids present in the plant Cannabis satiVa have been proposed to be mediated via the CB2 receptor. This hypothesis is supported by the abundant expression of CB2 in cells of the immune system, such as those in spleen, tonsils, and thymus, natural killer cells, T cells, and B cells. 2 Therefore, modification of immune functions by CB2 receptor ligands has become a focus of particular interest. In drug development, the mainly peripheral distribution of CB2 suggests that CB2-selective ligands will not have cannabinoid-related psychoactive side effects, i.e., drugs can be selectively targeted at the peripheral cannabinoid receptor. 5 The CB2 selective ligands developed so far have recently been reviewed by Raitio et al. 6 This study stems from the Japan Tobacco invention of the CB2 selective inverse agonist 9b (JTE-907), which binds to the CB2 receptor with nanomolar affinity (K i 35.9 nM) and possesses antiinflammatory properties in vivo. 7 The concept of inverse agonism can be explained with the prevailing two-state receptor model theory, according to which the G-protein coupled receptors may fluctuate between active (R*) and inactive (R) states. Inverse agonists possess higher affinity to the R form, agonists to R* and neutral agonists bind with similar affinity to both receptor states. Therefore, inverse agonists decrease the proportion of R* in the receptor population, and this results in reduced constitutive activation of G-proteins by receptors in the absence of agonist. 8,9 9b contains several functionalities which might be important for its action. Based on the affinity data presented in the patent, 10 the selected backbone structure was considered as one of the most potential starting points for nontraditional cannabinoid receptor ligand development with a special focus on CB2 selectivity. In this study we have improved the previously published synthesis pathway 10 for compound 6 (see Scheme 1) and coupled it with a series of phenylalkylamines carrying variable substitu- tion patterns to produce a series of amides. Also the effect of inversion of the amide bond has been studied, since we also prepared the 9b inverse amide 9a (Scheme 2). CB2 receptor activities of these compounds were determined by using a functional assay monitoring G protein activation, assessed by [ 35 S]GTP γ S binding to Chinese hamster ovary (CHO) cell membranes stably expressing the human CB2 receptor (hCB2). Our previous study has established that the CB2 receptor is constitutively active in this model and therefore allows detection of agonist, neutral antagonist, and inverse agonist ligand activities. 11 As far as we are aware, these results have permitted us to derive the first structure-activity relationships of 2-oxo- quinoline derivatives as CB2 receptor inverse agonists. Results and Discussion Chemistry. The synthesis pathway for 6 is presented in Scheme 1. Nitration of isovanillin 1 was carried out with nitronium tetrafluoroborate 12 since the typical nitration reagent (HNO 3 /H 2 SO 4 ) used in the patent 10 only gave 2 as a side product with approximately 10% yield. The phenolic hydroxyl group of 2 was alkylated with pentylbromide by using the Williamson ether synthesis 10 with satisfactory yields. The reduction of the nitro group with iron powder in EtOAc/AcOH/H 2 O-mixture with a catalytic amount of HCl 13 was found to be a rapid and efficient method to reduce the nitro group prior to the aldehyde group. Reaction of 4 with dimethylmalonate in the presence of piperidine and AcOH using EtOH as solvent produced the transesterified 2-oxoquinoline as an ethyl ester 5 at high yield. Hydrolyzation of 5 in aqueous EtOH/2M HCl yielded the directly crystallized carboxylic acid 6 with high purity and at relatively high yield. 14 As presented in Scheme 2, compound 9a, the inverse amide of 9b, was prepared from the acid chloride of 6 with concen- trated NH 4 OH in dioxane. 15 The amide was subjected to * To whom correspondence should be addressed. Tel: +358-17-163662. Fax: +358-17-162456. E-mail: Katri.Raitio@uku.fi. ² Department of Pharmaceutical Chemistry. ‡ Department of Physiology. § University of Kuopio. 2022 J. Med. Chem. 2006, 49, 2022-2027 10.1021/jm050879z CCC: $33.50 © 2006 American Chemical Society Published on Web 02/22/2006