Small-Molecule Probes DOI: 10.1002/ange.201200467 Chemical Probes for the Recognition of Cannabinoid Receptors in Native Systems** Lidia Martín-Couce, Mar Martín-Fontecha, Óscar Palomares, Leyre Mestre, Arnau Cordomí, Miriam Hernangomez, Sara Palma, Leonardo Pardo, Carmen Guaza, María L. López- Rodríguez,* and Silvia Ortega-GutiØrrez* Dedicated to Professor Miguel ngel Miranda on the occasion of his 60th birthday The endogenous cannabinoid system (ECS) regulates a broad number of physiological processes, and perturbations in its normal functioning are linked to many disorders. [1] In this respect, the development of high-sensitivity and high- throughput analytical tools that afforded a broader view of the ECS would be highly valuable. As such, small-molecule fluorescent probes could provide dynamic information con- cerning the direct spatial and temporal expression levels of cannabinoid receptors as has been recently reported for some specific classes of enzymes. [2] Accordingly, the development of small-molecule probes that are able to recognize cannabinoid receptors is an area of current interest, because they could complement and even overcome some of the drawbacks of the available antibodies. [3] Recent attempts toward this goal have been mainly focused on the CB 2 receptor (CB 2 R = can- nabinoid receptor type 2). However, these probes show moderate affinities [K i = (260–387) nm] , and their application in native systems is limited. [4] Similarly, endocannabinoid- based probes display modest affinities for CB 1 and CB 2 receptors [(84.7–450) nm], [5] a fact that could limit their use in complex systems. Therefore, we focused our efforts on the synthetic high-affinity cannabinoid ligands HU210 [K i (CB 1 R) = 0.061 nm, K i (CB 2 R) = 0.52 nm], [6] and HU308 [K i (CB 1 R) > 10 000 nm, K i (CB 2 R) = 22.7 nm] [7] (Scheme 1). Among the different tags, biotin was selected owing to its versatility for detection by a variety of readily available (strept)avidin conjugates. A closer look to the structure of these ligands revealed that the most straightforward possibil- ity was to attach the tag at the free hydroxy groups (Scheme 1). Ligand HU210 was prepared as previously described. [8] For the introduction of the tag, we made some attempts to selectively acylate the allylic hydroxy group using either Mitsunobu conditions [9] or the HfCl 4 ·2 THF catalyst. [10] How- ever, none of them gave good yields in our hands and we carried out the reaction between ligand HU210 and N- (+)-biotinyl-6-aminohexanoic acid in the presence of 1-ethyl- 3-(3-dimethylaminopropyl)carbodiimide and 4-dimethylami- nopyridine. Under these conditions, formation of bisacylated product was not observed, starting material was partly recovered, and compounds 1 and 2 were obtained after separation by column chromatography with 25 % and 14 % yields, respectively. Ligand HU308 was obtained as previously described [7] from commercially available a-pinene. Esterifi- cation of ligand HU308 with N-(+)-biotinyl-6-aminohexanoic acid gave derivative 3 in 55 % yield. Compounds 1–3 were Scheme 1. Structures of the synthetic cannabinoid ligands HU210 and HU308 and of synthesized probes 1–3. [*] Dr. L. Martín-Couce, Prof. Dr. M. Martín-Fontecha, Prof. Dr. M. L. López-Rodríguez, Dr. S. Ortega-GutiØrrez Department of Organic Chemistry Universidad Complutense de Madrid 28040 Madrid (Spain) E-mail: mluzlr@quim.ucm.es siortega@quim.ucm.es Dr. Ó. Palomares, S. Palma Department of Biochemistry and Molecular Biology Universidad Complutense de Madrid 28040 Madrid (Spain) Dr. L. Mestre, Dr. M. Hernangomez, Dr. C. Guaza Cajal Institute, CSIC Dr. Arce 37, 28002 Madrid (Spain) Dr. A. Cordomí, Prof. Dr. L. Pardo Universitat Autònoma de Barcelona 08193 Bellaterra, Barcelona (Spain) [**] This work was supported by grants from the Spanish Ministerio de Economía y Competitividad (MINECO, SAF2010-22198, SAF2010- 17501, and SAF2008-04053, and predoctoral fellowship to L.M.-C.) and Comunidad de Madrid (S2010/BMD-2353). The authors thank the Spanish Society of Medicinal Chemistry (SEQT) and Glaxo- SmithKline for the young researcher award to L.M.-C. S.O.-G. and O.P. are Ramon y Cajal Scholars funded by MINECO and the European Social Fund. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/anie.201200467. . Angewandte Zuschriften 7002  2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. 2012, 124, 7002 –7005