Cannabis sativa has always been a controversial plant as it can be considered as a lifesaver for several pathologies including glaucoma and epilepsy, an invaluable source of nutrients, an environmentally friendly raw material for manufacturing and textiles, but it is also the most widely spread illicit drug in the world, especially among young adults.
Its peculiarity is its ability to produce a class of organic molecules called phytocannabinoids, which derive from an enzymatic reaction between a resorcinol and an isoprenoid group. The modularity of these two parts is the key for the extreme variability of the resulting product that has led to almost 150 different known phytocannabinoids. The precursors for the most commonly naturally occurring phytocannabinoids are olivetolic acid and geranyl pyrophosphate, which take part to a condensation reaction leading to the formation of cannabigerolic acid (CBGA). CBGA can be then converted into either tetrahydrocannabinolic acid (THCA) or cannabidiolic acid (CBDA) or cannabichromenic acid (CBCA) by the action of a specific cyclase enzyme. All phytocannabinoids are biosynthesized in the carboxylated form, which can be converted into the corresponding decarboxylated (or neutral) form by heat. The best known neutral cannabinoids are undoubtedly Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD), the former being responsible for the intoxicant properties of the cannabis plant, and the latter being active as antioxidant, anti-inflammatory, anti-convulsant, but also as antagonist of THC negative effects.
All these cannabinoids are characterized by the presence of an alkyl side chain on the resorcinyl moiety made of five carbon atoms. However, other phytocannabinoids with a different number of carbon atoms on the side chain are known and they have been called varinoids (with three carbon atoms), such as cannabidivarin (CBDV) and Δ9-tetrahydrocannabivarin (Δ9-THCV), and orcinoids (with one carbon atom), such as cannabidiorcol (CBD-C1) and tetrahydrocannabiorcol (THC-C1). Both series are biosynthesized in the plant as the specific ketide synthases have been identified.
Our research group has recently reported the presence of a butyl phytocannabinoid series with a four-term alkyl chain, in particular cannabidibutol (CBDB) and Δ9-tetrahydrocannabutol (Δ9-THCB), in CBD samples derived from hemp and in a medicinal cannabis variety. Since no evidence has been provided for the presence of plant enzymes responsible for the biosynthesis of these butyl phytocannabinoids, it has been suggested that they might derive from microbial ω-oxidation and decarboxylation of their corresponding five-term homologs.
The length of the alkyl side chain has indeed proved to be the key parameter, the pharmacophore, for the biological activity exerted by Δ9-THC on the human cannabinoid receptor CB1 as evidenced by structure-activity relationship (SAR) studies collected by Bow and Rimondi. In particular, a minimum of three carbons is necessary to bind the receptor, then the highest activity has been registered with an eight-carbon side chain to finally decrease with a higher number of carbon atoms. Δ8-THC homologs with more than five carbon atoms on the side chain have been synthetically produced and tested in order to have molecules several times more potent than Δ9-THC.
To the best of our knowledge, a phytocannabinoid with a linear alkyl side chain containing more than five carbon atoms has never been reported as naturally occurring. However, our research group disclosed for the first time the presence of seven-term homologs of CBD and Δ9-THC in a medicinal cannabis variety, the Italian FM2, provided by the Military Chemical Pharmaceutical Institute in Florence. The two new phytocannabinoids were isolated and fully characterized and their absolute configuration was confirmed by a stereoselective synthesis. According to the International Non-proprietary Name (INN), we suggested for these CBD and THC analogues the name “cannabidiphorol” (CBDP) and “tetrahydrocannabiphorol” (THCP), respectively. The suffix “-phorol” comes from “sphaerophorol”, common name for 5-heptyl-benzen-1,3-diol, which constitutes the resorcinyl moiety of these two new phytocannabinoids.
A number of clinical trials17,18,19 and a growing body of literature provide real evidence of the pharmacological potential of cannabis and cannabinoids on a wide range of disorders from sleep to anxiety, multiple sclerosis, autism and neuropathic pain20,21,22,23. In particular, being the most potent psychotropic cannabinoid, Δ9-THC is the main focus of such studies. In light of the above and of the results of the SAR studies14,15,16, we expected that THCP is endowed of an even higher binding affinity for CB1 receptor and a greater cannabimimetic activity than THC itself. In order to investigate these pharmacological aspects of THCP, its binding affinity for CB1 receptor was tested by a radioligand in vitro assay and its cannabimimetic activity was assessed by the tetrad behavioral tests in mice.
Isolation and characterization of natural CBDP and Δ9-THCP
In order to selectively obtain a cannabinoid-rich fraction of FM2, n-hexane was used to extract the raw material instead of ethanol, which carries other contaminants such as flavonoids and chlorophylls along with cannabinoids26. An additional dewaxing step at −20 °C for 48 h and removal of the precipitated wax was necessary to obtain a pure cannabinoids extract. Semi-preparative liquid chromatography with a C18 stationary phase allowed for the separation of 80 fractions, which were analyzed by LC-HRMS with the previously described method. In this way, the fractions containing predominantly cannabidiphorolic acid (CBDPA) and tetrahydrocannabipgorolic acid (THCPA) were separately subject to heating at 120 °C for 2 h in order to obtain their corresponding neutral counterparts CBDP and Δ9-THCP as clear oils with a >95% purity. The material obtained was sufficient for a full characterization by 1H and 13C NMR, circular dichroism (CD) and UV absorption.
Stereoselective synthesis of CBDP and Δ9-THCP
(-)-trans-Cannabidiphorol ((-)-trans-CBDP) and (-)-trans-Δ9-tetrahydrocannabiphorol ((-)-trans-Δ9-THCP) were stereoselectively synthesized as previously reported for the synthesis of (-)-trans-CBDB and (-)-trans-Δ9-THCB homologs11,12,24. Accordingly, (-)-trans-CBDP was prepared by condensation of 5-heptylbenzene-1,3-diol with (1 S,4 R)-1-methyl-4-(prop-1-en-2-yl)cycloex-2-enol, using pTSA as catalyst, for 90 min.
Longer reaction time did not improve the yield of (-)-trans-CBDP because cyclization of (-)-trans-CBDP to (-)-trans-Δ9-THCP and then to (-)-trans-Δ8-THCP occurred. 5-heptylbenzene-1,3-diol was synthesized first as reported in the Supporting Information (Supplementary Fig. SI-1). The conversion of (-)-trans-CBDP to (-)-trans-Δ9-THCP using diverse Lewis’ acids, as already reported in the literature for the synthesis of the homolog Δ9-THC27,28,29, led to a complex mixture of isomers which resulted in an arduous and low-yield isolation of (-)-trans-Δ9-THCP by standard chromatographic techniques.
Therefore, for the synthesis of (-)-trans-Δ9-THCP, its regioisomer (-)-trans-Δ8-THCP was synthesized first by condensation of 5-heptylbenzene-1,3-diol with (1 S,4 R)-1-methyl-4-(prop-1-en-2-yl)cycloex-2-enol, as described above, but the reaction was left stirring for 48 hours. Alternatively, (-)-trans-CBDP could be also quantitatively converted to (-)-trans-Δ8-THCP in the same conditions. Hydrochlorination of the Δ8 double bond of (-)-trans-Δ8-THCP, using ZnCl2 as catalyst, allowed to obtain (-)-trans-HCl-THCP, which was successively converted to (-)-trans-Δ9-THCP in 87% yield by selective elimination on position 2 of the terpene moiety using potassium t-amylate as base (Fig. 2a).
Synthesis and spectroscopic characterization of (-)-trans-CBDP and (-)-trans-Δ9-THCP. (a) Reagents and conditions: (a) 5-heptylbenzene-1,3-diol (1.1 eq.), pTSA (0.1 eq.), CH2Cl2, r.t., 90 min.; (b) 5-heptylbenzene-1,3-diol (1.1 eq.), pTSA (0.1 eq.), DCM, r.t., 48 h; (c) pTSA (0.1 eq.), DCM, r.t., 48 h; (d) ZnCl2 (0.5 eq.), 4 N HCl in dioxane (1 mL per 100 mg of Δ8-THCP), dry DCM, argon, 0 °C to r.t., 2 h. (e) 1.75 M potassium t-amylate in toluene (2.5 eq.), dry toluene, argon, −15 °C, 1 h. (b–g) Superimposition of 1H, 13C NMR and CD spectra for natural (red line) and synthesized (blue line) (-)-trans-CBDP (b–d) and (-)-trans-Δ9-THCP (e–g).
The chemical identification of synthetic (-)-trans-CBDP and (-)-trans-Δ9-THCP, and their unambiguous 1H and 13C assignments were achieved by NMR spectroscopy (Supplementary Table SI-1,2 and Supplementary Fig. SI-2,3). Since (-)-trans-CBDP and (-)-trans-Δ9-THCP differ from the respective homologs (CBD, CBDB, CBDV, Δ9-THC, Δ9-THCB and Δ9-THCV) solely for the length of the alkyl chain on the resorcinyl moiety, no significant differences in the proton chemical shifts of the terpene and aromatic moieties were observed for CBD and Δ9-THC homologs. The perfect match in the chemical shift of the terpene and aromatic moieties between the synthesized (-)-trans-CBDP and (-)-trans-Δ9-THCP and the respective homologues11,24,30, combined with the mass spectra and fragmentation pattern, allowed us to unambiguously confirm the chemical structures of the two new synthetic cannabinoids. The trans (1R,6R) configuration at the terpene moiety was confirmed by optical rotatory power. The new cannabinoids (-)-trans-CBDP and (-)-trans-Δ9-THCP showed an [α]D20 of −145° and −166°, respectively, in chloroform. The [α]D20 values were in line with those of the homologs11,31, suggesting a (1R,6R) configuration for both CBDP and Δ9-THCP. A perfect superimposition between the 1H (Fig. 2b,e) and 13C NMR spectra (Fig. 2c,f) and the circular dichroism absorption (Fig. 2d,g) of both synthetic and extracted (-)-trans-CBDP and (-)-trans-Δ9-THCP was observed, confirming the identity of the two new cannabinoids identified in the FM2 cannabis variety.
Binding affinity at human CB1 and CB2 receptors
The binding affinity of (-)-trans-Δ9-THCP against purified human CB1 and CB2 receptors was determined in a radioligand binding assay, using [3H]CP55940 or [3H]WIN 55212-2 as reference compounds, and dose-response curves were determined (Fig. 3a,b). (-)-trans-Δ9-THCP binds with high affinity to both human CB1 and CB2 receptors with a Ki of 1.2 and 6.2 nM, respectively. (-)-trans-Δ9-THCP resulted 33-times more active than (-)-trans-Δ9-THC (Ki = 40 nM), 63-times more active than (-)-trans-Δ9-THCV (Ki = 75.4 nM) and 13-times more active than the newly discovered (-)-trans-Δ9-THCB (Ki = 15 nM) against CB1 receptor12,14. Moreover, the new identified (-)-trans-Δ9-THCP resulted about 5- to 10-times more active against CB2 receptor (Ki = 6.2 nM), in contrast with (-)-trans-Δ9-THC, (-)-trans-Δ9-THCB and (-)-trans-Δ9-THCV, which instead showed a comparable binding affinity with a Ki ranging from 36 to 63 nM (Fig. 3a)12,14.
In vitro activity and docking calculation of Δ9-THCP. (a) Binding affinity (Ki) of the four homologues of Δ9-THC against human CB1 and CB2 receptors. (b) Dose-response studies of Δ9-THCP against hCB1 (in blue) and hCB2 (in grey). All experiments were performed in duplicate and error bars denote s.e.m. of measurements. (c) Docking pose of (-)-trans-Δ9-THCP (blue sticks), in complex with hCB1 receptor (PDB ID: 5XRA, orange cartoon). Key amino acidic residues are reported in orange sticks. H-bonds are reported in yellow dotted lines. Heteroatoms are color-coded: oxygen in red, nitrogen in blue and sulphur in yellow. (d) Binding pocket of hCB1 receptor, highlighting the positioning of the heptyl chain within the long hydrophobic channel of the receptor (yellow dashed line). The side hydrophobic pocket is bordered in magenta. Panels c and d were built using Maestro 10.3 of the Schrödinger Suite.
Dose-dependent effects of Δ9-THCP administration (2.5, 5, or 10 mg/kg, i.p.) on the tetrad phenotypes in mice in comparison to vehicle. (a) Time schedule of the tetrad tests in minutes from Δ9-THCP or vehicle administration. (b,c) Locomotion decrease induced by Δ9-THCP administration in the open field test. (d) Decrease of body temperature after Δ9-THCP administration; the values are expressed as the difference between the basal temperature (i.e., taken before Δ9-THCP or vehicle administration) and the temperature measured after Δ9-THCP or vehicle administration. (e) Increase in the latency for moving from the catalepsy bar after Δ9-THCP administration. (f) Increase in the latency after the first sign of pain shown by the mouse in the hot plate test following Δ9-THCP administration. Data are represented as mean ± SEM of 5 mice per group. * indicate significant differences compared to 0 (vehicle injection), respectively. *p < 0.05, **p < 0.01, ***p < 0.001 versus Δ9-THCP 0 mg/kg (vehicle). The Kruskall-Wallis test followed by Dunn’s post hoc tests were performed for statistical analysis.
Up to now, almost 150 phytocannabinoids have been detected in cannabis plant7,41,42, though most of them have neither been isolated nor characterized. The well-known CBD and Δ9-THC have been extensively characterized and proved to possess interesting pharmacological profiles43,44,45,46,47, thus the attention towards the biological activity of their known homologs like CBDV and Δ9-THCV has recently grown as evidenced by the increasing number of publications per year appearing on Scopus. Other homologs like those belonging to the orcinoid series are scarcely investigated likely due to their very low amount in the plant that makes their isolation very challenging. In recent years, the agricultural genetics research has made great progresses on the selection of rare strains that produce high amounts of CBDV, CBG and Δ9-THCV48,49,50, thus it would not be surprising to see in the near future cannabis varieties rich in other minor phytocannabinoids. This genetic selection would enable the production of extracts rich in a specific phytocannabinoid with a characteristic pharmacological profile. For this reason, it is important to carry out a comprehensive chemical profiling of a medicinal cannabis variety and a thorough investigation of the pharmacological activity of minor and less known phytocannabinoids.
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 C18 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.
These authors contributed equally: Cinzia Citti and Pasquale Linciano.
G.C. developed and supervised the project, C.C. and P.L. conceived the experiments plan and drafted the manuscript, C.C. and F.R. carried out the UHPLC-HRMS analyses, P.L. performed the stereoselective syntheses and characterization, P.L. and G.G. performed the docking simulations, L.L., M.I. and S.M. performed the in vivo tetrad tests, A.L. and A.L.C. developed the semi-quantification method, F.F. and M.A.V. analyzed the binding assay data. All authors reviewed the manuscript.