Synthesis and Biological Evaluation of Bivalent Ligands for the Cannabinoid 1 Receptor

Abstract

Dimerization or oligomerization of many G protein-coupled receptors, including the CB1 receptor, is now widely accepted and may have significant implications towards medications development targeting these receptor complexes. A library of bivalent ligands composed of two identical CB1 antagonist pharmacophores derived from SR141716 linked by spacers of various lengths were developed. The affinities of these bivalent ligands at CB1 and CB2 receptors were determined using radiolabeled binding assays. Their functional activities were measured using GTP-γ-S accumulation and intracellular calcium mobilization assays. The results suggest that the nature of the linker and its length are crucial factors for optimum interactions of these ligands at CB1 receptor binding sites. Finally, selected bivalent ligands (5d and 7b) were able to attenuate the antinociceptive effects of the cannabinoid agonist CP55,940 in a rodent tail-flick assay. These novel compounds as probes will enable further evaluation of CB1 receptor dimerization and oligomerization, its functional significance, and may prove useful in the development of new therapeutic approaches to G protein-coupled receptor mediated disorders.

AB-CHMINACA, AB-PINACA, and FUBIMINA: Affinity and Potency of Novel Synthetic Cannabinoids in Producing Δ9-Tetrahydrocannabinol–Like Effects in Mice

“…Transfected cell membrane preparations with human CB 1 (hCB 1 ) and human CB 2 (hCB 2 ) receptors (PerkinElmer, Waltham, MA) isolated from a HEK-293 expression system were used for cannabinoid-binding assays, as previously described (Zhang et al, 2010). Binding was initiated with the addition of 40 fmol cell membrane proteins to polypropylene assay tubes containing 0.62 nM [ 3 H]CP55,940 (approximately 130 Ci/mmol), a test compound (for displacement studies), and a sufficient quantity of buffer A [50 mM Tris•HCl, 1 mM EDTA, 3 mM MgCl 2 , 5 mg/ml bovine serum albumin (BSA), pH 7.4] to bring the total incubation volume to 0.5 ml.…”

Design, synthesis and in vitro evaluation of novel uni- and bivalent ligands for the cannabinoid receptor type 1 with variation of spacer length and structure

“…Reference compound AM251 described as an inverse agonist at the hCB 1 R showed in this assay an EC 50 value of 70 nM, which is as potent as rimonabant (EC 50 20 Possible causes can be different assay conditions (human cells vs Sf9 cells, GTPase measurements), but also differences in ligand specific receptor conformations as in the functional assay a hCB 1 R-Ga i2 fusion system co-expressing Gb 1 c 2 and RGS4 was used. 26 The potency of intermediate 5b with a spacer of 4 methylene units is in the micromolar range (EC 50 = 2.8 lM).…”

“…A significant amount of research has been pursued applying the bivalent approach to GPCR ligands, 15,19 but for the huge majority of bivalent GPCR ligands simple hydrocarbon chains with a connecting heteroatom, like amine function have been applied. In 2010, the first bivalent ligand approach on CB 1 Rs was pursued by Thomas et al 20 Applying rimonabant as the parent structure, two series of bivalent ligands with hydrophobic alkylene chains at a secondary or tertiary amine were synthesized and evaluated in radioligand binding studies and in a functional assay. The compound with highest binding affinity, compound 19 (Scheme 4), has a linker consisting of 15 atoms, and potency in the functional assay was not as high as for the parent structure.…”

“…Also we chose rimonabant as the potent parent structure due to its well described structure-activity relationships, high affinity and excellent selectivity over CB 2 R and the already described data of bivalent rimonabant-based structures with simple linkers. 20,21 Since the binding properties of rimonabant are hard to surpass, even lower affinities and decreased selectivities of bivalent ligands can provide first insights into the consequences of modified linker structures. The best length of the spacers in terms of affinity at CB 1 R is 15 atoms.…”

“…The best length of the spacers in terms of affinity at CB 1 R is 15 atoms. 20 Therefore, a series of bivalent ligands with 15 atoms or a distance equivalent to a 15 atom chain was synthesized first. For comparison, ligands with longer linkers, that is approximately 23 atoms, were also synthesized since there is evidence that such a long spacer length can enable bridging two receptors.…”