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.

Analogues of the biaryl pyrazole N-(piperidinyl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716; 5) were synthesized to investigate the structure-activity relationship (SAR) of the aminopiperidine region. The structural modifications include the substitution of alkyl hydrazines, amines, and hydroxyalkylamines of varying lengths for the aminopiperidinyl moiety. Proximity and steric requirements at the aminopiperidine region were probed by the synthesis of analogues that substitute alkyl hydrazines of increasing chain length and branching. The corresponding amide analogues were compared to the hydrazides to determine the effect of the second nitrogen on receptor binding affinity. The N-cyclohexyl amide 14 represents a direct methine for nitrogen substitution for 5, reducing the potential for heteroatom interaction, while the morpholino analogue 15 adds the potential for an additional heteroatom interaction. The series of hydroxyalkyl amides of increasing chain length was synthesized to investigate the existence of additional receptor hydrogen binding sites. In displacement assays using the cannabinoid agonist [(3)H](1R,3R,4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl) cyclohexan-1-ol (CP 55 940; 2) or the antagonist [(3)H]5, 14 exhibited the highest CB(1) affinity. In general, increasing the length and bulk of the substituent was associated with increased receptor affinity and efficacy (as measured in a guanosine 5'-triphosphate-gamma-[(35)S] assay). However, in most instances, receptor affinity and efficacy increases were no longer observed after a certain chain length was reached. A quantitative SAR study was carried out to characterize the pharmacophoric requirements of the aminopiperidine region. This model indicates that ligands that exceed 3 A in length would have reduced potency and affinity with respect to 5 and that substituents with a positive charge density in the aminopiperidine region would be predicted to possess increased pharmacological activity.

The aminoalkylindoles (AAIs) are agonists at both the cannabinoid CB1 and CB2 receptors. To determine whether the s-trans or s-cis form of AAIs is their receptor-appropriate conformation, two pairs of rigid AAI analogues were studied. These rigid analogues are naphthylidene-substituted aminoalkylindenes that lack the carbonyl oxygen of the AAIs. Two pairs of (E)- and (Z)-naphthylidene indenes (C-2 H and C-2 Me) were considered. In each pair, the E geometric isomer is intended to mimic the s-trans form of the AAIs, while the Z geometric isomer is intended to mimic the s-cis form. Complete conformational analyses of two AAIs, pravadoline (2) and WIN-55, 212-2 (1), and of each indene were performed using the semiempirical method AM1. S-trans and s-cis conformations of 1 and 2 were identified. AM1 single-point energy calculations revealed that when 1 and each indene were overlayed at their corresponding indole/indene rings, the (E)- and (Z)-indenes were able to overlay naphthyl rings with the corresponding s-trans or s-cis conformer of 1 with an energy expense of 1.13/0.69 kcal/mol for the C-2 H (E/Z)-indenes and 0.82/0.74 kcal/mol for the C-2 Me (E/Z)-indenes. On the basis of the hypothesis that aromatic stacking is the predominant interaction of AAIs such as 1 at the CB receptors and on the demonstration that the C-2 H (E/Z)- and C-2 Me (E/Z)-indene isomers can mimic the positions of the aromatic systems in the s-trans and s-cis conformers of 1, the modeling results support the previously established use of indenes as rigid analogues of the AAIs. A synthesis of the naphthylidene indenes was developed using Horner-Wittig chemistry that afforded the Z isomer in the C-2 H series, which was not produced in significant amounts from an earlier reported indene/aldehyde condensation reaction. This approach was extended to the C-2 Me series as well. Photochemical interconversions in both the C-2 H and C-2 Me series were also successful in obtaining the less favored isomer. Thus, the photochemical process can be used to provide quantities of the minor isomers C-2 H/Z and C-2 Me/E. The CB1 and CB2 affinities as well as the activity of each compound in the twitch response of the guinea pig ileum (GPI) assay were assessed. The E isomer in each series was found to have the higher affinity for both the CB1 and CB2 receptors. In the rat brain membrane assay versus [3H]CP-55,940, the Ki's for the C-2 H/C-2 Me series were 2.72/2.89 nM (E isomer) and 148/1945 nM (Z isomer). In membrane assays versus [3H]SR141716A, a two-site model was indicated for the C-2 H/C-2 Me (E isomers) with Ki's of 10. 8/9.44 nM for the higher-affinity site and 611/602 nM for the lower-affinity site. For the Z isomers, a one-site model was indicated with Ki's of 928/2178 nM obtained for the C2 H/C-2 Me analogues, respectively. For the C-2 H/C-2 Me series, the CB2 Ki's obtained using a cloned cell line were 2.72/2.05 nM (E isomer) and 132/658 nM (Z isomer). In the GPI assay, the relative order of potency was C-2 H E > C-2 Me E > C-2 H Z > C-2 Me Z. The C-2 H E...