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.
Synthetic procedure a for HHC. CBD (0.7mmol) 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).
(9 S )-6,6,9-trimethyl-3-pentyl-6a,7,8,9,10,10a-hexahydro-6 H -benzo[ c ]chromen-1-ol ((9 S )-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.
(9 R )-6,6,9-trimethyl-3-pentyl-6a,7,8,9,10,10a-hexahydro-6 H -benzo[ c ]chromen-1-ol ((9 R )-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°C23,25. 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 20-40-10023,25. 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 inflorescence21. 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. Male C57BL6/J mice (7 weeks old; n = 4) received (9R)-HHC (10 mg/kg) or vehicle (1:1:18; EtOH:Kolliphor EL:0.9% saline) by i.p. administration. Hypomotility (open field test), hypothermia (body temperature), antinociceptive (hot plate test), and cataleptic (bar test) effects were evaluated using the procedures reported by Metna-Laurent et al.25. The same animals were used in all four behavioral tests. Statistical analysis was performed using the one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test.
Body temperature. After immobilization of the mouse the probe was gently inserted 1 cm into the rectum and the temperature was registered after stabilization. The probe was cleaned with 70% EtOH and dried with a paper towel in between each mouse.
Open field. Behavioral assays of locomotor activity were performed 30 min after drug (or vehicle) injection. The apparatus was cleaned before each behavioral session with a 70% EtOH solution. Naϊve mice were randomly assigned to a treatment group. Behaviors were recorded, stored, and analyzed using an automated behavioral tracking system (Any-maze, Video-tracking software by Ugo Basile). Mice were placed in an open field test (OFT) arena (l×w×h: 44×44×30 cm) and their ambulatory activity (total distance travelled in centimeters) was recorded for 15 min and analyzed.
Bar test. The bar consisted of a 40 cm in length and 0.4 cm in diameter glass rod, which was horizontally elevated by 5 cm above the surface. Both forelimbs of the mouse were positioned on the bar and its hind legs on the floor of the cage, ensuring that the mouse was not lying down on the floor. The chronometer 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 time duration each mouse held the elevated bar by both its forelimbs (latency for moving in seconds).
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.
Animals. Male C57BL/6 mice (Charles River, Italy) weighing 18–20 g were used for the tetrad experiments. A 12 h light/dark cycle starting light at 6:00 A.M., a constant temperature of 20–22°C, and a 55–60% humidity were maintained for at least 1 week before the beginning of the experiments. Each cage housed three mice with chow and tap water available ad libitum. The experimental procedures employed for the work presented herein were approved by the Animal Ethics Committee of the University of Campania “L. Vanvitelli” (Naples, Italy). Animal care and welfare were entrusted to adequately trained personnel in compliance with Italian (D.L. 116/92) and European Commission (O.J. of E.C. L358/1, 18/12/86) regulations on the protection of animals used for research purposes. All efforts were made to minimize animal numbers and avoid unnecessary suffering during the experiments.