Identification of a new cannabidiol n-hexyl homolog in a medicinal cannabis variety with an antinociceptive activity in mice: cannabidihexol

Abstract

The two most important and studied phytocannabinoids present in Cannabis sativa L. are undoubtedly cannabidiol (CBD), a non-psychotropic compound, but with other pharmacological properties, and Δ⁹-tetrahydrocannabinol (Δ⁹-THC), which instead possesses psychotropic activity and is responsible for the recreative use of hemp. Recently, the homolog series of both CBDs and THCs has been expanded by the isolation in a medicinal cannabis variety of four new phytocannabinoids possessing on the resorcinyl moiety a butyl-(in CBDB and Δ⁹-THCB) and a heptyl-(in CBDP and Δ⁹-THCP) aliphatic chain. In this work we report a new series of phytocannabinoids that fills the gap between the pentyl and heptyl homologs of CBD and Δ⁹-THC, bearing a n-hexyl side chain on the resorcinyl moiety that we named cannabidihexol (CBDH) and Δ⁹-tetrahydrocannabihexol (Δ⁹-THCH), respectively. However, some cannabinoids with the same molecular formula and molecular weight of CBDH and Δ⁹-THCH have been already identified and reported as monomethyl ether derivatives of the canonical phytocannabinoids, namely cannabigerol monomethyl ether (CBGM), cannabidiol monomethyl ether (CBDM) and Δ⁹-tetrahydrocannabinol monomethyl ether (Δ9-THCM). The unambiguously identification in cannabis extract of the n-hexyl homologues of CBD and Δ⁹-THC different from the corresponding methylated isomers (CBDM, CBGM and Δ⁹-THCM) was achieved by comparison of the retention time, molecular ion, and fragmentation spectra with those of the authentic standards obtained via stereoselective synthesis, and a semi-quantification of these cannabinoids in the FM2 medical cannabis variety was provided. Conversely, no trace of Δ⁹-THCM was detected. Moreover, CBDH was isolated by semipreparative HPLC and its identity was confirmed by comparison with the spectroscopic data of the corresponding synthetic standard. Thus, the proper recognition of CBDH, CBDM and Δ⁹-THCH closes the loop and might serve in the future for researchers to distinguish between these phytocannabinoids isomers that show a very similar analytical behaviour. Lastly, CBDH was assessed for biological tests in vivo showing interesting analgesic activity at low doses in mice.

Figure 1

Identification of compounds corresponding to the molecular formula C₂₃H₃₂O₄ in C. sativa FM2. (A) UHPLC-HRMS extracted ion chromatogram (EIC) for molecular formula C₂₃H₃₂O₄ in native FM2 and the relative fragmentation spectra, in negative ionization mode, for the identified peaks A (panel B), B (panel C) and C (panel D). (E) UHPLC-HRMS extracted ion chromatogram (EIC) for molecular formula C₂₂H₃₁O₂ in decarboxylated FM2 and the relative fragmentation spectra, in negative ionization mode, for the identified peaks Ad (panel F), Bd (panel G) and Cd (panel H).

Figure 2

MS/MS spectra library of CBD and Δ⁹-THC homologs by UHPLC-HESI-Orbitrap. Comparison of the high-resolution fragmentation spectra in positive (ESI+) mode for CBD (panel A) and Δ⁹-THC (panel B) homologs. The pale-yellow box point out the constant terpenic portion. The red lines highlight the shift of some fragments corresponding to the loss of a methylene portion (CH₂, m/z = 14) moving from CBDP (or Δ⁹-THCP) to CBDV (or Δ⁹-THCV).

Figure 3

Synthesis and UHPLC-HRMS identification of CBDH, CBDM, Δ⁹-THCH and Δ⁹-THCM, and in vivo activity of CBDH. (A) Scheme 1. Reagents and conditions: (a) triphenylphosphine (1.1 equiv.), toluene, reflux, 6 h, quant. yield; (b) valeraldehyde (1.5 equiv.), 0.1 M K₂CO₃ aq. (10 mL per mmol of 1), reflux, 5 h, 81% yield; (c) H-Cube ThalesNano H₂-Pd/C, EtOH, 30 °C, 20 bar, 1 mL/min, 91% yield; (d) BBr₃ 1 M in DCM (2.2 eq.), anhydrous DCM, N₂ atmosphere, − 15 °C → r.t, 24 h, quant. yield; (e) (1S,4R)-1-methyl-4-(prop-1-en-2-yl)cycloex-2-enol (0.9 equiv.), pTSA (0.1 equiv.), DCM, r.t., argon, 2 h, 17% yield for (−)-trans-CBDH and 20% yield for (−)-trans-Δ⁹-THCH. Scheme 2. Reagents and conditions: dimethylsulphate (0.5 equiv.), K₂CO₃ (1 equiv.), DMF, r.t. 62% yield for (−)-trans-CBDM and 57% yield for CBGM. Scheme 3. Reagents and conditions: p-TSA (0.1 equiv.), dry DCM, r.t., 1 h, 43% yield. (B) Superimposition of extracted UHPLC-HRMS ion chromatograms (EICs) of synthetic cannabinoid n-hexyl and monomethyl ether homologs. and relative fragmentation spectra, in positive ionization mode. EICs were chosen based on the exact mass calculated for C₂₃H₃₂O₄. (C) Effects of CBDH (1, 2, 3, and 5 mg/kg, i.p.) or vehicle in the formalin test in mice. The total time of the nociceptive response was measured every 5 min and expressed in min (see “Experimental” section). Data are represented as means ± SEM (n = 5–6). ^+,+++indicate statistically significant differences versus veh/form, p < 0.05 and p < 0.001, respectively. 2-way ANOVA followed by Bonferroni’s post hoc tests was used for statistical analysis.