A Novel Phytocannabinoid Isolated From Cannabis sativa L. With An In Vivo Cannabimimetic Activity Higher Than Δ9-tetrahydrocannabinol: Δ9-Tetrahydrocannabiphorol

(-)-Trans-Δ9-tetrahydrocannabinol (Δ9-THC) is the main compound responsible for the intoxicant activity of Cannabis sativa L. The length of the side alkyl chain influences the biological activity of this cannabinoid. In particular, synthetic analogues of Δ9-THC with a longer side chain have shown cannabimimetic properties far higher than Δ9-THC itself. In the attempt to define the phytocannabinoids profile that characterizes a medicinal cannabis variety, a new phytocannabinoid with the same structure of Δ9-THC but with a seven-term alkyl side chain was identified.

The natural compound was isolated and fully characterized and its stereochemical configuration was assigned by match with the same compound obtained by a stereoselective synthesis. This new phytocannabinoid has been called (-)-trans-Δ9-tetrahydrocannabiphorol (Δ9-THCP). Along with Δ9-THCP, the corresponding cannabidiol (CBD) homolog with seven-term side alkyl chain (CBDP) was also isolated and unambiguously identified by match with its synthetic counterpart. The binding activity of Δ9-THCP against human CB1 receptor in vitro (Ki=1.2nM) resulted similar to that of CP55940 (Ki=0.9nM), a potent full CB1 agonist. In the cannabinoid tetrad pharmacological test, Δ9-THCP induced hypomotility, analgesia, catalepsy and decreased rectal temperature indicating a THC-like cannabimimetic activity.
The presence of this new phytocannabinoid could account for the pharmacological properties of some cannabis varieties difficult to explain by the presence of the sole Δ9-THC.

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 resorcinol 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)7. 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 homolog.
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 resorcinol moiety of these two new phytocannabinoids.
A number of clinical trials 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 pain20–23. 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 studies, 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.
Identifcation of cannabidiphorol (CBDP) and Δ9-tetrahydrocannabiphorol (Δ9-THCP) by liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS).

The FM2 ethanolic extract was analyzed by an analytical method recently developed for the cannabinoid profiling of this medicinal cannabis variety. As the native extract contains mainly the carboxylated forms of phytocannabinoids as a consequence of a cold extraction25, part of the plant material was heated to achieve decarboxylation where the predominant forms are neutral phytocannabinoids.

The advanced analytical platform of ultra-high performance liquid chromatography coupled to high resolution Orbitrap mass spectrometry was employed to analyze the FM2 extracts and study the fragmentation spectra of the analytes under investigation. The precursor ions of the neutral derivatives cannabidiphorol (CBDP) and Δ9-tetrahydrocannabiphorol (Δ9-THCP), 341.2486 for the [M-H]− and 343.2632 for the [M+H]+, showed an elution time of 19.4 min for CBDP and 21.3 min for Δ9-THCP (Fig. 1a).
Their identification was confirmed by the injection of a mixture (5 ng/mL) of the two chemically synthesized CBDP and Δ9-THCP (Fig. 1b) as it will be described later. As for their carboxylated counterpart, the precursor ions of the neutral forms CBDP and Δ9-THCP break in the same way in ESI+mode, but they show a different fragmentation pattern in ESI− mode. Whilst Δ9-THCP shows only the precursor ion [M-H]− (Fig. 1d), CBDP molecule generates the fragments at m/z 273.1858 corresponding to a retro Diels-Alder reaction, and 207.1381
corresponding to the resorcinol moiety after the break of the bond with the terpenoid group (Fig. 1c). It is noteworthy that for both molecules, CBDP and Δ9-THCP, each fragment in both ionization modes differ exactly by an ethylene unit (CH2)2 from the corresponding five-termed homologs CBD and THC.

Moreover, the longer elution time corroborates the hypothesis of the seven-termed phytocannabinoids considering the higher lipophilicity of the latter.

Source: https://www.nature.com/articles/s41598-019-56785-1 December 2019

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