Fig. 2. Effect of R-group substitutions on sigma-1 receptor binding affinity. Affinity at sigma-1 receptors was determined by competition against [3H](+)-pentazocine as described in Materials and Methods. Sigma-1 receptor binding affinity was significantly decreased when R = NCS for all X-group substitutions. This pattern is most clearly illustrated for X = O substituted ligands, with CM572 (X = O, R = NCS) showing at least a 350-fold loss in sigma-1 receptor binding affinity compared with other R-group substitutions. Ki values for X = O substituted ligands at sigma-1 receptors were determined to be 28.0 ± 3.39*, >10,000, 22.2 ± 5.3, and 15.5 ± 2.4 nM for SN79 (X = O, R = COCH3), CM572 (X = O, R = NCS), CM458 (X = O, R = NO2), and CM571 (X = O, R = NH2), respectively. An accurate Ki value for CM572 (X = O, R = NCS) at sigma-1 receptors could not be determined due to insolubility above 1 mM; however, ∼50% of [3H](+)-pentazocine binding sites remained with 10,000 nM CM572 present. The competition curves shown are an average of four independent experiments for SN79, two independent experiments for CM572, three independent experiments for CM458, and three independent experiments for CM571. All experiments were performed in duplicate. The Ki values reported are an average ± S.D. of the individual Ki value from each independent experiment (*Kaushal et al., 2011).