Chromenopyrazole as Versatile Cannabinoid Scaffold: from CB1 towards CB2 Selectivity

Paula Morales1, Maria Gómez-Cañas2, Dow P. Hurst3, Moisés Garcia-Arencibia2, Javier Fernández-Ruiz2, Patricia Reggio3, Pilar Goya1, Nadine Jagerovic1. 1Consejo Superior de Investigaciones Científicas, Instituto de Química Médica, Madrid, Spain, 2Facultad de Medicina, Universidad Complutense de Madrid, Departamento de Bioquímica y Biología Molecular. Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Ins, Madrid, Spain, 3Department of Chemistry and Biochemistry, University of North Carolina Greensboro, Greensboro, USA

Background. A series of chromenopyrazoles has been described by us as non-psychoactive and selective CB1 cannabinoid agonists with peripheral antinociceptive properties (Cumella J et al, ChemMedChem 7:452, 2012).

Purpose. In an attempt to target the CB2 type receptor, we propose different structural modifications of the chromenopyrazole scaffold. Structural features required for CB1/CB2 affinity and selectivity are explored by molecular modelling. Affinity of new compounds is evaluated using competitive binding assays. Then, compounds with the highest CB2 affinity profiles were tested in CB2-mediated functional assays using BV2 cultured cells (a mouse microglia cell line).

Methods.

Synthesis. The 7-alkyl-1,4-dihydro-4,4-dimethylchromenopyrazol-9-oles were synthesized from the corresponding resorcinol following the procedure described in Cumella J et al. The preparation of the 9-alkoxy-7-alkyl-1,4-dihydro-4,4-dimethylchromenopyrazoles was achieved by alkylation of the corresponding chromenopyrazol-9-oles.

Molecular modelling. Conformational analysis of selected new chromenopyrazoles was first performed to determine the global minimum-energy conformation. Then, they were docked by using models of active state CB1 (CB1R*) and CB2 (CB2R*) receptors (Pei Y et al, Chem Biol 15: 1207, 2008; Hurst DP et al, J Biol Chem 285:17954, 2010) that include extracellular and intracellular loops and N and C terminus.

Pharmacological evaluation. The affinity of the new compounds was evaluated measuring their ability to displace [3H]CP55940 from human cannabinoid CB1 and CB2 receptors transfected into HEK293 EBNA cells. The effects of the selected CB2 chromenopyrazole ligands on lipopolysaccharide (LPS)-induced production of prostaglandin E2 (PGE2) were examined in BV2 microglia. The PGE2 release was measured using an ELISA kit. This CB2-mediated functional assay was performed on new compounds alone or in combination with WIN 55,212,2 or SR144528. (Oh YT et al, Neurosci Lett 474:148, 2010).

Results. A new library of chromenopyrazoles was synthesized, characterized, and tested at cannabinoid CB1 and CB2 receptors by radioligand binding and functional activity assays. Fine-tuning of affinity and selectivity was achieved by structural modifications performed on the scaffold (figure 1). The docking studies using the CB1 and CB2 receptors models (CB1R* and CB2R*) validate the structure activity relationships. The different ligand-receptor interactions provide detailed structural information.

Figure 1. Examples of chromenopyrazoles.

Conclusion. Chromenopyrazoles were found to constitute a versatile scaffold for obtaining potent cannabinoid receptor ligands with selectivity at either CB1 or CB2 receptor types.

Supported by grants MICINN SAF2012-40075, SAF2009-12422, and CAM S2011/BMD-2308.