Synthesis and pharmacological activity of the epimers of hexahydrocannabinol (HHC)

Abstract

Cannabis is a multifaceted plant with numerous therapeutic properties on one hand, and controversial psychotropic activities on the other hand, which are modulated by CB1 endocannabinoid receptors. Δ9-Tetrahydrocannabinol (Δ9-THC) has been identified as the main component responsible for the psychotropic effects, while its constitutional isomer cannabidiol (CBD) has shown completely different pharmacological properties. Due to its reported beneficial effects, Cannabis has gained global popularity and is openly sold in shops and online. To circumvent legal restrictions, semi-synthetic derivatives of CBD are now frequently added to cannabis products, producing "high" effects similar to those induced by Δ9-THC. The first semi-synthetic cannabinoid to appear in the EU was obtained through cyclization and hydrogenation of CBD, and is known as hexahydrocannabinol (HHC). Currently, there is limited knowledge regarding HHC, its pharmacological properties, and its prevalence, as it is not commonly investigated in routine toxicological assays. In this study, synthetic strategies were explored to obtain an excess of the active epimer of HHC. Furthermore, the two epimers were purified and individually tested for their cannabinomimetic activity. Lastly, a simple and rapid chromatographic method employing a UV detector and a high-resolution mass spectrometer was applied to identify and quantify up to ten major phytocannabinoids, as well as the HHC epimers, in commercial cannabis samples.

Figure 1

Chemical structure of the analyzed cannabinoids.

Figure 2

Synthesis of HHC. Step 1a-b: Cyclization of CBD. Step 2: Hydrogenation of the crude reaction mixture.

Figure 3

HPLC–UV-HRMS/MS and NMR characterization of the isolated (9S)-HHC and (9R)-HHC. HPLC–UV trace of (9S)-HHC (a) and (9R)-HHC (b) with the respective UV spectrum in the boxes; HRMS/MS pattern in HESI + mode of (9S)-HHC (c) and (9R)-HHC (d) (the discriminant fragment is circled in blue and red for (9S)-HHC and (9R)-HHC respectively); HRMS/MS pattern in HESI- mode of (9S)-HHC (e) and (9R)-HHC (f); discriminant chemical shifts in the ¹H NMR spectra of (9S)-HHC (blue) and (9R)-HHC (red) (g).

Figure 4

HPLC–UV chromatogram of a cannabinoid standard mixture. HPLC–UV chromatogram of a standard mixture containing ten phytocannabinoids (CBDA, CBGA, CBG, CBD, CBN, Δ⁹-THC, Δ⁸-THC, CBC, THCA, and CBCA) and the two HHC epimers at the concentration of 10 µg/mL.

Figure 5

Dose-dependent effects of (9R)-HHC and (9S)-HHC administration (10 mg/kg, i.p.) on the tetrad behavioural tests in mice in comparison to vehicle. Time schedule of the tetrad tests in min from (9R)-HHC, (9S)-HHC or vehicle administration (a). Locomotion decrease induced by (9R)-HHC administration in the open field test (b, f). Decrease of body temperature after (9R)-HHC administration (e); the values are expressed as the difference between the basal temperature (i.e., taken before (9R)-HHC or vehicle administration) and the temperature measured after (9R)-HHC or vehicle administration. Increase in the latency for moving from the catalepsy bar after (9R)-HHC administration (c). Increase in the latency after the first sign of pain shown by the mouse in the hot plate test following (9R)-HHC administration (d). Data are represented as the mean ± SEM of 4 mice per group. * indicates significant differences compared to vehicle injection, *p < 0.05 Tukey’s test.