Development of Squaramides as Allosteric Modulators of the CB₁ Receptor: Synthesis, Computational Studies, Biological Characterization, and Effects against Cocaine-Induced Behavioral Sensitization and Reinstatement in Rats

Cannabinoid receptor type 1 (CB₁) negative allosteric modulators have emerged as an alternate approach to CB₁ orthosteric antagonists/inverse agonists for cocaine addiction treatment. This study explores aryl-alkyl squaramides as CB₁ allosteric modulators, featuring RTICBM-262 (3) with good in vitro potencies in CB₁ calcium mobilization, [³⁵S]GTPγS binding, and cAMP assays. Molecular modeling studies suggest 3 bound in a similar pocket as Org27569, forming π-stacking with key residues H154^2.41 and W241^4.50, and the potential C98-C107 disulfide bond had limited impact on its binding or receptor activation. ADME and in vivo pharmacokinetic studies suggest that 3 had reasonable metabolic stability, brain penetration, and selectivity against a panel of ∼ 50 targets but poor solubility and high protein binding. At 5.6 mg/kg (i.p.), 3 significantly attenuated both cocaine-seeking behavior specific to cue-induced reinstatement and cocaine-induced behavioral sensitization without altering locomotor activity. These results support squaramides as promising candidates for further investigation for cocaine addiction treatment.