Chemicals and reagents
Ethanol 96%, acetonitrile (ACN), and formic acid (FA) were all LC–MS grade and were bought from Sigma Aldrich (USA). Ultrapure water was obtained with a water purification system (Direct-Q 3UV, Merck Millipore, Milan, Italy). Chemicals and solvents employed in the synthetic process were reagent grade and used without further purification. The following abbreviations for common organic solvents were adopted: diethyl ether (Et2O); dichloromethane (CH2Cl2); cyclohexane (CE); chloroform (CDCl3); ethanol (EtOH). Stock solutions (1 mg/mL) of certified reference cannabinoid standards of CBDA, Δ9-THCA, CBGA, CBCA, CBD, CBG, CBC, CBN, as well as of Δ9-THC and Δ8-THC (500 µg/mL) were bought from Cayman Chemical (Ann Arbor, Michigan, USA). Stock solutions (1 mg/mL) of CBDH, Δ9-THCH, (9R)-HHC and (9S)-HHC were obtained by properly diluting the pure compounds in-house synthesized.
Plant material
Samples of hemp biomass (HHC-1 and HHC-2), HHC hashish (HHC-3) and pure HHC were purchased from Baked Bologna (Bologna, Italy). The use of Cannabis sativa L. plants with THC levels below 0.5% in the present study complies with the Italian guidelines according to the Law 242/2016 and to the common agricultural policy (art. 38–44) of the European Union Treaty (GUCE 26/10/2012).
Synthetic procedure a for HHC
CBD (0.7 mmol) was dissolved in 20 mL of absolute EtOH containing 0.05% HCl and refluxed for 2 h. The resulting crude was neutralized with a saturated solution of Na2CO3 and subject to hydrogenation with an H-Cube ThalesNano flow reactor (Budapest, Hungary) according to the following experimental conditions: 3 mm 10% Pd/C cartridge, 30 °C, 20 bar, 1 mL/min. The crude product mixture showed a 57:43 S/R HHC mixture along with other by-products. The solvent was evaporated and the crude of the reaction was purified with semipreparative HPLC–UV (Coloumn Luna 5 μm C18, 100 Å, 250 × 10 mm-Phenomenex, Bologna, Italy). An isocratic elution was employed with mobile phase 80% ACN (with 0.1% FA) and 20% MilliQ water (with 0.1% FA) at a flow rate of 7.5 mL/min. The UV trace was followed at 228 nm and the compounds of interest were obtained with a purity higher than 95% (12 mg for (9R)-HHC and 8 mg for (9S)-HHC).
Synthetic procedure b for HHC
To a solution of CBD (0.7 mmol) in 15 mL of anhydrous CH2Cl2, pTSA (10 mg, 10% mol) was added at room temperature, under a nitrogen atmosphere. The reaction was stirred in the same conditions for 48 h. After that, the mixture was diluted with Et2O and washed with a saturated solution of NaHCO3. The organic layer was collected, washed with brine, dried over anhydrous Na2SO4 and concentrated. The obtained crude was subject to hydrogenation as described for the synthetic procedure b to give a crude product with a 61:39 R/S HHC mixture The two epimers were purified with semi-preparative HPLC as reported above (11 mg for (9S)-HHC and 8 mg for (9R)-HHC).
NMR characterization of HHC
NMR spectra were recorded either on a Bruker 400 (working at 400.134 MHz for 1H and 100.62 MHz for 13C) or a Bruker 600 spectrometer (working at 600.130 MHz for 1H and 150.902 MHz for 13C). Monodimensional spectra were acquired with a spectral width of 8278 Hz (for 1H NMR) and 23.9 kHz (for 13C NMR), a relaxation delay of 1 s and a number of transients of 32 and 1024 for 1H NMR and 13C NMR, respectively. NMR spectra were acquired in CDCl3 and chemical shifts (δ) were registered in ppm with respect to that of the residual solvent (δ = 7.26 ppm for 1H and δ = 77.20 ppm for 13C); coupling constants are reported in Hz, splitting patterns are expressed as singlet (s), doublet (d), triplet (t), quartet (q), double doublet (dd), quintet (qnt), multiplet (m), broad signal (b).
(9S)-6,6,9-trimethyl-3-pentyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-ol ((9S)-HHC)
1H NMR (400 MHz, CDCl3) δ 6.25 (s, 1H, C4), 6.07 (s, 1H, C2), 4.61 (s, 1H, OH), 2.92–2.79 (m, 1H, C10α), 2.67 (td, J = 11.4, 2.9 Hz, 1H, C10a), 2.46–2.38 (m, 2H, C1′), 2.11 (m, 1H, C9), 1.68–1.61 (m, 2H, C8-C7), 1.58–152 (m, 2H, C2′), 1.46–1.44 (m, 1H, C6a) 1.36 (s, 3H, C12), 1.33–1.28 (s, 6H, C4′-C3′-C10β), 1.13 (d, J = 7.2 Hz, 3H, C11), 1.09 (s, 3H, C13), 0.88 (t, J = 7.0 Hz, 3H, C5′). 13C NMR (101 MHz, CDCl3) δ 155.38, 154.77, 142.65, 110.63, 110.19, 107.79, 77.03, 50.09, 36.33, 35.59, 32.40, 31.77, 30.76, 29.49, 28.06, 27.76, 27.10, 23.26, 22.73, 19.30, 18.98, 14.20.
(9R)-6,6,9-trimethyl-3-pentyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-ol ((9R)-HHC)
1H NMR (600 MHz, CDCl3) δ 6.25 (s, 1H, C4), 6.08 (s, 1H, C2), 4.64 (s, 1H, OH), 3.03 (d, J = 12.9 Hz, 1H, C10α), 2.49–2.37 (m, 3H, C1’-C10a), 1.87–1.80 (m, 2H, C8-1H, C7-1H), 1.67–1.60 (m, 1H, C9), 1.59–1.52 (m, 2H, C 2’), 1.46–1.44 (m, 1H, C6a), 1.36 (s, 3H, C12), 1.34–1.24 (m, 4H, C4’-C3’), 1.16–1.07 (m, 2H, C8-1H, C7-1H), 1.06 (s, 3H, C13) 0.94 (d, J = 6.6 Hz, 3H, C11), 0.88 (t, J = 7.0 Hz, 3H, C5’), 0.78 (m, 1H, C10β). 13C NMR (151 MHz, CDCl3) δ 155.15, 154.83, 142.74, 110.44, 110.23, 107.77, 77.14, 49.30, 39.16, 35.71, 35.58, 33.03, 31.75, 30.76, 28.24, 27.94, 22.78, 22.73, 19.22, 14.21.
HPLC–UV-HRMS analysis
A Vanquish Core system (Thermo Fisher Scientific, Waltham, Massachusetts, USA) with binary pump, vacuum degasser, thermostated autosampler and column compartment, and diode array detector (DAD) was interfaced to an Exploris 120 Orbitrap mass analyzer with a heated electrospray ionization source (HESI). The chromatographic separation was achieved on a Poroshell 120 EC C18 (100 × 3.0 mm, 2.7 µm) (Agilent, Milan, Italy) with an isocratic elution at 70% ACN for 20 min and a washing step at 98% ACN for 3 min. The column was re-equilibrated at 70% ACN for further 3 min for a total run time of 26 min at a constant flow rate of 0.5 mL/min.
The HESI parameters were optimized in previous works for cannabinoids: spray voltage 4200 kV and 3800 kV for HESI + and HESI- mode respectively, sheath gas 70 au, auxiliary gas 5 au, sweep gas 0.5 au, ion transfer tube temperature 390 °C and vaporizer temperature 150°C26,29. The mass analyzer was operated in both full scan (FS) and data-dependent acquisition (DDA) mode. In FS mode the resolution was set at 60,000 FWHM (full width at half maximum), the RF lens level at 70%, the maximum injection time 54 ms, the m/z range at 150–750, and the isolation window at m/z 1.2. In DDA mode the resolution was set at 30,000 FWHM, the maximum injection time at 22 ms, the m/z range at 50–750, the isolation window at m/z 1.2, and the stepped normalized collision energy (NCE) at 20–40-10026,29. The injection volume was 5 µL. The analyses were acquired with Xcalibur 3.0 and processed using Chromeleon 7 for the UV traces and TraceFinder 54.0 for the MS traces (all from Thermo Fisher Scientific).
Calibration standards and sample preparation and cannabinoids quantification
Calibration solutions of all phytocannabinoid standards were prepared by diluting the stock solutions with ACN to get the final concentrations indicated in Table S1. Each dilution was run in triplicate and the calibration curves were built using both UV and MS data. Area of the peaks for each analyte was plotted against nominal concentration and the back-calculated concentration was checked to be within 15% of the nominal value. Samples of hemp inflorescence and HHC hashish were extracted using the method reported in the German Pharmacopoeia for the extraction of phytocannabinoids from cannabis inflorescence24. The extracts HHC-1 and HHC-2 from hemp inflorescence were analysed after 100 × dilution with mobile phase, while the extract HHC-3 from HHC hashish was 1000 × diluted. The sample of pure HHC (HHC-4) was injected at the concentration of 10 µg/mL obtained by dissolving 10 mg of the sample in 1 mL of ACN and preparing serial 10 × dilutions with mobile phase up to the desired concentration.
Quantification of cannabinoids was accomplished with both UV and MS data. The UV chromatograms were extracted at 228 nm. The exact m/z of the precursor ion in both HESI + and HESI- mode was extracted with a 5-ppm error from the HRMS TIC and used for calibration.
Tetrad test
In the experiment, male C57BL6/J mice at 7 weeks old were used (n = 4). They were divided into two groups: one group received (9R)-HHC at a dose of 10 mg/kg dissolved in a vehicle (1:1:18; EtOH:Kolliphor EL:0.9% saline) via intraperitoneal (i.p.) administration, and the other group received only the vehicle as a control. The same animals were used for all four behavioural tests.
The effects of (9R)-HHC on hypomotility (measured using the open field test), hypothermia (measured by monitoring body temperature), antinociception (evaluated using the hot plate test), and catalepsy (assessed through the bar test) were assessed. These tests were performed following the procedures described by Metna-Laurent et al.30.
Statistical analysis of the data was conducted using the one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test.
Body temperature. The measurement of body temperature was performed after immobilizing the mouse. A probe was gently inserted 1 cm into the rectum, and the temperature was recorded once it stabilized. The probe was cleaned with 70% ethanol and dried with a paper towel between each mouse to prevent cross-contamination.
Open field. The open field test was conducted 30 min after administering the drug (or vehicle). The apparatus used for the test was cleaned with a 70% ethanol solution before each behavioural session. Naïve mice were randomly assigned to different treatment groups. The mice were placed in an open field arena (dimensions: length × width × height: 44 × 44 × 30 cm), and their ambulatory activity (total distance travelled in cm) was recorded and analyzed for a duration of 15 min. An automated behavioural tracking system called Any-maze, specifically the Video-tracking software by Ugo Basile, was used to record and analyze the behaviours.
Bar test. The bar test was conducted to assess catalepsy. A glass rod measuring 40 cm in length and 0.4 cm in diameter was horizontally elevated 5 cm above the surface. The mouse’s forelimbs were positioned on the bar while the hind legs remained on the floor of the cage, ensuring that the mouse was not lying down on the floor. The chronometer was started when the mouse held onto the bar with both forelimbs, and it was stopped when the mouse descended from the bar (i.e., when the two forepaws touched the floor) or after 5 min (cut-off time). Catalepsy was measured as the duration in seconds that the mouse held the elevated bar.
Hot plate. Each mouse was placed on a hot plate (Ugo Basile), which was kept at the constant temperature of 52 °C. Licking of the hind paws or jumping were considered as a nociceptive response (NR) and the latency was measured in s 85 min after drug or vehicle administration, taking a cut-off time of 30 s to prevent tissue damage. The hot plate test was performed to evaluate antinociceptive effects. Each mouse was placed on a hot plate set at a constant temperature of 52 °C. The licking of hind paws or jumping were considered nociceptive responses (NR), and the latency to respond was measured in seconds. The measurement was taken 85 min after drug or vehicle administration, with a cut-off time of 30 s to prevent tissue damage.
Animals
In the described experiments, male C57BL/6 mice from Charles River (Italy) were used. The mice weighed between 18 and 20 g. The mice were acclimated to the laboratory conditions for at least 1 week before the start of the experiments. The laboratory maintained a 12-h light/dark cycle with the lights turning on at 6:00 A.M. The temperature in the facility was maintained at 20–22 °C, and the humidity was kept at 55–60%. The mice were housed in cages with three mice per cage. They had access to standard chow and tap water ad libitum, meaning they could eat and drink freely.
The experimental procedures conducted in this study received ethical approval from the Animal Ethics Committee of the University of Campania “L. Vanvitelli” in Naples, Italy. The specific protocol number for the experiments was 24/2023-PR. All the experiments were conducted in accordance with the regulations outlined by the Italian law (D.L. 116/92) and the European Commission (O.J. of E.C. L358/1, 18/12/86) regarding the protection of animals used for research purposes. The experimental methods described in the study also followed the ARRIVE guidelines, which provide recommendations for reporting animal research31. Animal care and welfare were the responsibility of trained personnel who adhered to the relevant Italian and European regulations. Every effort was made to minimize the number of animals used in the experiments and to prevent any unnecessary suffering or harm to the animals during the course of the study. (Fig. S1).