In this study, we synthesized both (S)- and (R)-enantiomers of synthetic cannabinoids to evaluate their pharmacological properties. All target compounds were reported in an EMCDDA-Europol joint report to be NPS sold and recreationally used in Europe [12, 13]. NMR data for the enantiomers were similar, as shown in the section on materials and methods above, and the results are in good agreement with the values expected for their structures.

Figure 3 shows the extracted ion chromatograms for the enantiomers of the target compounds (1 μg/mL solution for both enantiomers) obtained by LC–HR-MS using the method that we developed previously [3]. By the previously developed method, all of the enantiomers of the tested compounds were separated, and no enantio-impurities were found in their chromatograms, except for (S) APP-CHMINACA. Approximately less than 2% of (R) APP-CHMINACA was confirmed in the extracted ion chromatogram of (S) APP-CHMINACA as shown in Fig. 3. However, because (R)-enantiomer was much less effective than (S)-enantiomer to both CB₁ and CB₂ receptors, it is suspected that the effect of contaminated (R)-enantiomer on the EC₅₀ value was almost negligible.

Table 1 shows the results of the [³⁵S]GTPγS binding assays performed in this study. Each compound functioned as both a CB₁ and a CB₂ receptor agonist, except for (R) MDMB-FUBICA. The EC₅₀ values of (S)-enantiomers for the CB₁ receptors were at least one-fifth of or less than those of (R)-enantiomers, which indicates that (S)-enantiomers are more potent CB₁ receptor agonists than (R)-enantiomers. The (S)-enantiomer of APP-CHMINACA could activate CB₁ at a concentration 134 times lower than that for the (R)-enantiomer. For MDMB-FUBICA, the CB₁ receptor activity was not confirmed for the (R)-enantiomer even at the maximum tested concentration, but the EC₅₀ of the (S)-enantiomer was 9.72 × 10⁻⁹ M; the difference in the EC₅₀ levels between the enantiomers of MDMB-FUBICA to activate the CB₁ receptor was more than 10,000-fold, which indicates that the activities for the CB₁ receptor are markedly different depending on the chirality of the compounds. In contrast, we could not observe a clear relevance between EC₅₀ values for the CB₂ receptor and chirality of the compounds. (R) APP-CHMINACA and (R) MDMB-FUBICA lacked the potential to be CB₁ receptor agonists, but both of their EC₅₀ values for the CB₂ receptor were less than 10⁻⁶ M; all of the target compounds were strong agonists for the CB₂ receptor, irrespective of their chirality.

As for the CB₁/CB₂ selectivity, (R)-enantiomers tended to be CB₂-selective and were at least 20-fold more active than for CB₁ for all of the tested compounds. Interestingly, (R) MDMB-FUBICA functioned only on the CB₂ receptor as an agonist, while showing no CB₁ activity as mentioned above, although (S) MDMB-FUBICA could activate both of these receptors at nanomolar levels. In several preclinical studies, compounds such as JWH-133 and HU308 were used as CB₂-selective ligands [14]. More recently, the synthesis and evaluation of novel CB₂-selective agonists have been repeatedly reported. In these pharmacological or medicinal chemistry studies seeking CB₂ agonists, the most selective compound showed a selective index of CB₁/CB₂ of only 100- to 1000-fold. In this study, (R) MDMB-FUBICA was shown to have potency to activate the CB₂ receptor signal with an EC₅₀ value of 3.1 × 10⁻⁹ M, but had no [³⁵S]GTPγS binding activity for the CB₁ receptor, even at 1.0 × 10⁻⁴ M, which corresponds to a selective index of more than 3.2 × 10⁴-fold.

The metabolism of MDMB-FUBICA enantiomers has not been reported yet; thus it remains unclear whether (R) MDMB-FUBICA can mimic the function of the CB₂ receptor agonist when applied to an animal model to evaluate the effect on the central nervous system. Recently, it was reported that the (S)-enantiomer was mainly detected in herbal products containing chiral carboxamide-type synthetic cannabinoids [3, 15]. The cannabimimetic activities of each enantiomer presented in this study may be helpful to precisely recognize forensic cases associated with these synthetic cannabinoids when evaluated with the quantitative data of a specimen analyzed after enantiomeric separation.