Plant material

FM2 cannabis variety is obtained from the strain CIN-RO produced by the Council for Agricultural Research and Economics (CREA) in Rovigo (Italy) and provided to the Military Chemical Pharmaceutical Institute (MCPI, Firenze, Italy) for breeding. FM2 inflorescence (batch n. 6A32/1) was supplied by the MCPI with the authorization of the Italian Ministry of Health (prot. n. SP/062). The raw plant material (10 g) was finely grinded and divided into two batches: one batch (500 mg) was extracted with 50 mL of ethanol 96% according to the procedure indicated by the monograph of Cannabis Flos of the German Pharmacopoeia56 and was analyzed by UHPLC-HESI-Orbitrap after proper dilution with acetonitrile (×100). The remaining 9.5 g were extracted following the protocol of Pellati et al. with some modifications26. Briefly, freeze-dried plant material was extracted with 400 mL of n-hexane for 15 min under sonication in an ice bath. Samples were centrifuged for 10 min at 2000 × g and the supernatants collected. The procedure was repeated twice more on the pellets. The combined supernatants were then dried under reduced pressure and resuspended in 10 mL of acetonitrile, filtered and used for the isolation of CBDPA and THCPA by semi-preparative liquid chromatography.

Isolation of natural CBDP and Δ9-THCP

Aliquots (1 mL) of the solution obtained as described in the ‘Plant Material’ section were injected in a semi-preparative LC system (Octave 10 Semba Bioscience, Madison, USA). The chromatographic conditions used are reported in the paper by Citti et al.11. The column employed was a Luna C 18 with a fully porous silica stationary phase (Luna 5 µm C18(2) 100 Å, 250 × 10 mm) (Phenomenex, Bologna, Italy) and a mixture of acetronitrile:0.1% aqueous formic acid 70:30 (v/v) was used as mobile phase at a flow rate of 5 mL/min. CBDPA and THCPA (retention time 19.0 min and 75.5 min respectively) were isolated as reported in our previous work11. The fractions containing CBDPA and THCPA were analyzed by UHPLC-HESI-Orbitrap. The fractions containing predominantly either one or the other cannabinoid were separately combined and dried on the rotavapor at 70 °C. Each residue was subject to decarboxylation at 120 °C for two hours in oven. An amount of about 0.6 mg of CBDP and about 0.3 mg of Δ9-THCP was obtained.

UHPLC-HESI-Orbitrap metabolomic analysis

FM2 extracts were analyzed on a Thermo Fisher Scientific Ultimate 3000 system equipped with a vacuum degasser, a binary pump, a thermostated autosampler, a thermostated column compartment and interfaced to a heated electrospray ionization source and a Q-Exactive Orbitrap mass spectrometer (UHPLC-HESI-Orbitrap). The parameters of the HESI source were set according to Citti et al.11: capillary temperature, 320 °C; vaporizer temperature, 280 °C; electrospray voltage, 4.2 kV (positive mode) and 3.8 kV (negative mode); sheath gas, 55 arbitrary units; auxiliary gas, 30 arbitrary units; S lens RF level, 45. Analyses were acquired using the Xcalibur 3.0 software (Thermo Fisher Scientific, San Jose, CA, USA) in full scan data-dependent acquisition (FS-dd-MS2) in positive (ESI+) and negative (ESI−) mode at a resolving power of 70,000 FWHM at m/z 200. A scan range of m/z 250–400, an AGC of 3e6, an injection time of 100 ms and an isolation window for the filtration of the precursor ions of m/z 0.7 were chosen as the optimal parameters for the mass analyzer. A normalized collision energy (NCE) of 20 was used to fragment the precursor ions. Extracted ion chromatograms (EIC) of the [M + H]+ and [M-H]− molecular ions were derived from the total ion chromatogram (TIC) of the FM2 extracts and matched with pure analytical standards for accuracy of the exact mass (5 ppm), retention time and MS/MS spectrum.

The chromatographic separation was carried out on a Poroshell 120 SB-C18 (3.0 × 100 mm, 2.7 µm, Agilent, Milan, Italy) following the conditions employed for our previous work11.

A semi-quantitative analysis of Δ9-THC and CBD and their heptyl analogs CBDP and Δ9-THCP was achieved using a calibration curve with an external standard. A stock solution of CBD and Δ9-THC, CBDP and Δ9-THCP (1 mg/mL) was properly diluted to obtain five non-zero calibration points at the final concentrations of 50, 100, 250, 500, and 1000 ng/mL for CBD and Δ9-THC and of 1, 5, 10, 25, and 50 ng/mL for CBDP and Δ9-THCP. A standard solution of Δ9-THC-d 3 was added at each calibration standard at a final concentration of 50 ng/mL. The linearity was assessed by the coefficient of determination (R2), which was greater than 0.993 for each analyte.

Synthetic procedure

All reagents and solvents were employed as purchased without further purification unless otherwise specified. The following abbreviations for common organic solvents have been used herein: diethyl ether (Et 2 O); dichloromethane (DCM); cyclohexane (CE). Reaction monitoring was performed by thin-layer chromatography on silica gel (60F-254, E. Merck) and checked by UV light, or alkaline KMnO 4 aqueous solution57,58,59. Reaction products were purified, when necessary, by flash chromatography on silica gel (40−63 μm) with the solvent system indicated. NMR spectra were recorded on a Bruker 400 or Bruker 600 spectrometer working respectively at 400.134 MHz and 600.130 MHz for 1H and at 100.62 MHz or 150.902 MHz for 13C. Chemical shifts (δ) are in parts per million (ppm) and they were referenced to the solvent residual peaks (CDCl 3 δ = 7.26 ppm for proton and δ = 77.20 ppm for carbon); coupling constants are reported in hertz (Hz); splitting patterns are expressed with the following abbreviations: singlet (s), doublet (d), triplet (t), quartet (q), double doublet (dd), quintet (qnt), multiplet (m), broad signal (b). 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 32 and 1024 number of transients for 1H-NMR and 13C-NMR, respectively12. The COSY spectra were recorded as a 2048 × 256 matrix with 2 transients per t1 increment and processed as a 2048 × 1024 matrix; the HSQC spectra were collected as a 2048 × 256 matrix with 4 transients per t1 increment and processed as a 2048 × 1024 matrix, and the one-bond heteronuclear coupling value was set to 145 Hz; the HMBC spectra were collected as a 4096 × 256 matrix with 16 transients per t1 increment and processed as a 4096 × 1024 matrix, and the long-range coupling value was set to 8 Hz12. Circular dichroism (CD) and UV spectra were acquired on a Jasco (Tokyo, Japan) J-1100 spectropolarimeter using a 50 nm/min scanning speed. Quartz cells with a 10 mm path length were employed to record spectra in the 500–220 nm range12. Optical rotation (λ) was measured with a Polarimeter 241 (cell-length 100 mm, volume 1 mL) from Perkin-Elmer (Milan, Italy). The synthetic procedures described below were adjusted from previously published works12,57.

Synthesis of (1′R,2′R)-4-heptyl-5′-methyl-2′-(prop-1-en-2-yl)-1′,2′,3′,4′-tetrahydro-[1,1′-biphenyl]-2,6-diol, (-)-trans-CBDP

(1 S,4 R)-1-methyl-4-(prop-1-en-2-yl)cycloex-2-enol (146 mg, 0.96 mmol, 0.9 eq.), solubilized in 15 mL of anhydrous DCM, was added over a period of 20 min to a stirred solution of 5-heptylbenzene-1,3-diol (222 mg, 1.07 mmol, 1 eq.) and p-toluenesulfonic acid (20 mg, 0.11 mmol, 0.1 eq.) in anhydrous DCM (15 mL) at room temperature and under a positive pressure of argon. After stirring in the same conditions for 1 h, the reaction was quenched with 10 mL of a saturated aqueous solution of NaHCO 3 . The mixture was partitioned between Et 2 O and water. The organic layer was separated and washed with brine, dried with anhydrous Na 2 SO 4 and evaporated. The residue was chromatographed (ratio crude:silica 1/120, eluent: CE:DCM 8/2). All the chromatographic fractions were analyzed by HPLC-UV and UHPLC-HESI-Orbitrap and only the fractions containing exclusively CBDP were concentrated to give 76 mg of a colorless oil (23% yield, purity > 99%).

1H NMR (400 MHz, CDCl 3 ) δ 6.10–6.30 (m, 2 H), 5.97 (bs, 1 H), 5.57 (s, 1 H), 4.66 (s, 1 H), 4.66 (bs, 1 H), 4.56 (s, 1 H), 3.89–3.81 (m, 1 H), 2.52–2.35 (m, 3 H), 2.24 (td, J = 6.1, 12.7 Hz, 1 H), 2.09 (ddt, J = 2.4, 5.1, 17.9 Hz, 1 H), 1.89–1.74 (m, 5 H), 1.65 (s, 3 H), 1.55 (qnt, J = 7.6 Hz, 2 H), 1.28 (td, J = 4.7, 8.2, 9.0 Hz, 8 H), 0.87 (t, J = 6.7 Hz, 3 H). 13C NMR (101 MHz, CDCl 3 ) δ 156.27, 154.09, 149.56, 143.23, 140.22, 124.30, 113.93, 111.01, 109.91, 108.26, 46.33, 37.46, 35.70, 31.99, 31.14, 30.59, 29.43, 29.35, 28.60, 23.86, 22.84, 20.71, 14.29. HRMS m/z [M + H]+ calcd. for C 23 H 35 O 2 +: 343.2632. Found: 343.2629; [M-H]− calcd. for C 23 H 33 O 2 −: 341.2475. Found: 341.2482. [α] D 20 = −146° (c 1.0, ACN).

Synthesis of (6aR,10aR)-3-heptyl-6,6,9-trimethyl-6a,7,10,10a-tetrahydro-6H-benzo[c]chromen-1-ol, (-)-trans-Δ 8 -THCP

The set-up of the reaction for the synthesis of (-)-trans-Δ8-THCP was performed as described for (-)-trans-CBDP and the resulting mixture was stirred at room temperature for 48 h. The mixture was diluted with Et 2 O, and washed with a saturated solution of NaHCO 3 (10 mL). The organic layer was collected, washed with brine, dried (anhydrous Na 2 SO 4 ) and concentrated. After purification over silica gel (ratio crude:silica 1/150, eluent: CE:Et 2 O 95/5) 315 mg of a colorless oil (46% yield) were obtained.

1H NMR (400 MHz, CDCl 3 ) δ 6.28 (d, J = 1.6 Hz, 1 H), 6.10 (d, J = 1.6 Hz, 1 H), 5.46–5.39 (m, 1 H), 4.78 (s, 1 H), 3.20 (dd, J = 4.5, 16.0 Hz, 1 H), 2.70 (td, J = 4.7, 10.8 Hz, 1 H), 2.44 (td, J = 2.3, 7.4 Hz, 2 H), 2.21–2.10 (m, 1 H), 1.92–1.76 (m, 3 H), 1.70 (s, 3 H), 1.63–1.52 (m, 2 H), 1.38 (s, 3 H), 1.30 (tt, J = 4.3, 9.4, 11.8 Hz, 8 H), 1.11 (s, 3 H), 0.88 (t, J = 7.0 Hz, 3 H).

Synthesis of (6aR,10aR)-3-heptyl-9-chloro-6,6,9-trimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-ol (HCl-THCP)

1 N ZnCl 2 in Et 2 O (440 µL, 0.44 mmol, 0.5 eq.) was added to a stirred solution of Δ8-THCP (300 mg, 0.87 mmol, 1 eq.) in 20 mL of anhydrous DCM, at room temperature and under nitrogen atmosphere. After 30 min, the reaction was cooled at 0 °C and 2 mL of 4 N HCl in dioxane was added. The resulting mixture was stirred at room temperature, overnight and then diluted with Et 2 O. The organic layer was collected and washed, in sequence, with an aqueous saturated solution of NaHCO 3 and brine. After dehydration with anhydrous Na 2 SO 4 , the organic phase was concentrated to give 305 mg (93% yield) of a yellowish oil, pure enough to be used in the next step without further purification.

1H NMR (400 MHz, CDCl 3 ) δ 6.24 (d, J = 1.7 Hz, 1 H), 6.07 (d, J = 1.6 Hz, 1 H), 4.94 (s, 1 H), 3.45 (dd, J = 2.9, 14.4 Hz, 1 H), 3.05 (td, J = 2.9, 11.3 Hz, 1 H), 2.42 (td, J = 1.5, 7.4 Hz, 2 H), 2.20–2.12 (m, 1 H), 1.80–1.71 (m, 1 H), 1.66 (s, 4 H), 1.60–1.51 (m, 2 H), 1.49–1.42 (m, 1 H), 1.38 (s, 3 H), 1.34–1.18 (m, 10 H), 1.13 (s, 3 H), 0.87 (t, J = 6.6 Hz, 3 H). ESI-MS m/z [M + H] + calcd. for C 23 H 36 35[Cl]O 2 +: 379.2. Found: 379.4. Calcd. for C 23 H 36 37[Cl]O 2 +: 381.2. Found: 381.3.

Synthesis of (6aR,10aR)-3-heptyl-6,6,9-trimethyl-6a,7,8,10a-tetrahydro-6H-benzo[c]chromen-1-ol, (-)-trans-Δ9-THCP.

HCl-THCP (305 mg, 0.82 mmol, 1 eq.) was solubilized in 10 mL of anhydrous toluene and cooled at −15 °C. 1.75 N potassium t-amylate in toluene (1.17 mL, 2.05 mmol, 2.5 eq.) was added dropwise with a syringe to the first solution under a positive pressure of argon. The mixture was stirred in the same condition for 15 min and then at 60 °C for 1 h. After cooling at room temperature, the reaction was quenched with a 1% solution of ascorbic acid and diluted with Et 2 O. The organic layer was washed with brine, dried over anhydrous Na 2 SO 4 and concentrated. The residue was chromatographed (ratio crude/silica 1:300, hexane:i-propyl ether 9/1) to give 232 mg of a greenish oil (83% yield). 50 mg of (-)-trans-Δ9-THCP were further purified by semipreparative HPLC to prepare a pure analytic standard (purity > 99.9%).

1H NMR (600 MHz, CDCl 3 ) δ 6.30 (t, J = 2.0 Hz, 1 H), 6.27 (d, J = 1.6 Hz, 1 H), 6.14 (d, J = 1.5 Hz, 1 H), 4.75 (s, 1 H), 3.20 (dt, J = 2.5, 10.8 Hz, 1 H), 2.43 (dd, J = 6.4, 8.9 Hz, 2 H), 2.22–2.11 (m, 2 H), 1.97–1.87 (m, 1 H), 1.69–1.65 (m, 4 H), 1.58–1.50 (m, 2 H), 1.43–1.37 (m, 4 H), 1.34–1.21 (m, 8 H), 1.09 (s, 3 H), 0.87 (t, J = 6.6 Hz, 3 H). 13C NMR (151 MHz, CDCl 3 ) δ 154.97, 154.34, 143.02, 134.59, 123.92, 110.30, 109.22, 107.72, 77.38, 46.01, 35.72, 33.78, 31.99, 31.37, 31.16, 29.50, 29.38, 27.77, 25.22, 23.55, 22.87, 19.47, 14.29. HRMS m/z [M + H]+ calcd. for C 23 H 35 O 2 +: 343.2632. Found: 343.2633; [M-H]− calcd. for C 23 H 33 O 2 −: 341.2475. Found: 341.2481. [α] D 20 = −166° (c 1.0, ACN).

Binding at CB 1 and CB 2 Receptors

The binding affinity of (-)-trans-Δ9-THCP against human CB 1 and CB 2 receptors was assessed by Eurofins Discovery using a radioligand binding assay. Ten concentrations of the phytocannabinoid from 1 nM to 30 µM were tested in duplicate. [3H]CP55940 (at 2 nM, K d = 2.4 nM) and [3H]WIN 55212-2 (at 0.8 nM, K d = 1.5 nM) were used as specific radioligand for hCB 1 and hCB 2 , respectively60,61. Equation 1 was employed to calculate the percent inhibition (%in) of control specific binding obtained in the presence of the tested compounds.

$$ \% in=100-(\frac{measured\,specific\,binding}{control\,specific\,binding}\ast 100)$$ (1)

A non-linear regression analysis of the competition curves generated with mean replicate values (Eq. 2) was used to calculate the IC 50 values (concentration causing a half-maximal inhibition of control specific binding)62.

$$Y=D+[\frac{A-D}{1+(\frac{C}{{C}_{50}})nH}]$$ (2)

Where Y is the specific binding, A is the left asymptote of the curve, D is the right asymptote f the curve, C is the compound concentration, C 50 is the IC 50 value and nH is the slope factor. This analysis was carried out using a software developed at Cerep (Hill software) and validated by comparing the data with that generated by the commercial software SigmaPlot 4.0 for Windows (1997 by SPSS Inc.). The inhibition constants (K i ) were determined using the Cheng Prusoff equation (Eq. 3):

$$Ki=\frac{I{C}_{50}}{(1+\frac{L}{{K}_{D}})}$$ (3)

where L is the concentration of the radioligand, and K D is the affinity of the radioligand for the receptor.

The data obtained for CP 55940 (CB 1 IC 50 = 1.7 nM, CB 1 K i = 0.93 nM) and WIN 55212-2 (CB 2 IC 50 = 2.7 nM, CB 2 K i = 1.7 nM) were in accordance with the values reported in literature60,61.

Docking simulation

The prediction of the binding mode of Δ9-THCP in complex with human CB 1 receptor was performed using Maestro 10.3 of the Schrödinger Suite63. The crystallographic structure of the active conformation of CB 1 in complex with AM11542 (PDB ID: 5XRA) was downloaded from the Protein Data Bank and was used as reference for docking calculation. The protein was prepared using the Protein Preparation Wizard module64. The chemical structure of (-)-trans-Δ9-THCP was sketched with ChemDraw 12.0 and converted from 2D to 3D with the LigPrep utility65. Five conformations per ligand were initially generated, and appropriate ionization state and tautomers were evaluated for each conformation at physiological pH66,67. Afterwards, ligand conformations were minimized with the OPLS_2005 force field. Rigid docking was performed in extra precision mode with Glide version 6.868.

Tetrad test

Male C57BL6/J mice (7 weeks old; n = 5) were treated with Δ9-THCP (10, 5 and 2.5 mg/kg) or vehicle (1:1:18; ethanol:Kolliphor EL:0.9% saline) by i.p. administration. Mice were evaluated for hypomotility (open field test), hypothermia (body temperature), antinociceptive (hot plate test), and cataleptic (bar test) effects, using the procedures of the tetrad tests as reported by Metna-Laurent et al.69. The same animals were used in all four behavioral tests. Statistical analysis was performed using the Kruskall-Wallis test and Dunn’s post hoc tests.

Body temperature

The mouse was immobilized and the probe gently inserted for 1 cm into the rectum until stabilization of temperature. Between each mouse the probe was cleaned with 70% ethanol and dried with paper towel.

Open field

The open field test was used for the evaluation of motor activity. Behavioral assays were performed 30 min after drug (or vehicle) injection. The apparatus was cleaned before each behavioral session by a 70% ethanol solution. Naϊve mice were randomly assigned to a treatment group. Behaviors were recorded, stored, and analyzed using an automated behavioral tracking system (Smart v3.0, Panlab Harvard Apparatus). Mice were placed in an OFT arena (l × w × h: 44 cm × 44 cm × 30 cm), and ambulatory activity (total distance travelled in centimeter) was recorded for 15 min and analyzed.

Bar test

The bar test was used for the evaluation of catalepsy. The bar was 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 when 10 min had elapsed (i.e., 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

The hot plate test was performed to assess changes in the nociception. On the day of the experiment each mouse was placed on a hot plate (Ugo Basile) that was kept at a 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 seconds 85 minutes after drug or vehicle administration, taking a cut-off time of 30 or 60 s in order to prevent tissue damage.

Animals

Male C57BL/6 mice (Charles River, Italy) of 18–20 g weight were used for the tetrad experiments. A 12 h light/dark cycle with light on 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 beginning the experiments. Mice were housed three per cage 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. 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.