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

The study addresses the chemical diversity of phytocannabinoids in Cannabis sativa L., focusing on expanding the known homolog series of CBD- and THC-type cannabinoids. Prior work identified butyl (CBDB, Δ⁹-THCB) and heptyl (CBDP, Δ⁹-THCP) homologs. This work hypothesizes and investigates the existence of hexyl side-chain homologs—cannabidihexol (CBDH) and Δ⁹-tetrahydrocannabihexol (Δ⁹-THCH)—which would bridge the gap between pentyl and heptyl derivatives. A key challenge is distinguishing these from monomethyl ether isomers (CBDM, CBGM, THCM) that share the same molecular formula. The purpose is to unambiguously identify and semi-quantify the hexyl homologs in a medicinal cannabis variety (FM2) through high-resolution mass spectrometry corroborated by stereoselective synthesis, confirm structural identity by isolation and NMR, and provide an initial assessment of in vivo antinociceptive activity for CBDH. This is significant for accurate phytocannabinoid profiling and for exploring potential pharmacological properties of newly identified cannabinoids.

The paper situates its work within a growing catalog of roughly 150 phytocannabinoids and recent discoveries of CBD/THC homolog series differing by alkyl side-chain length. Previous identifications include CBDB and Δ⁹-THCB (n-butyl) and CBDP and Δ⁹-THCP (n-heptyl). Compounds with even-numbered side chains have been rare and sometimes considered artifacts of ω-oxidation. The literature also reports monomethyl ether derivatives with the same formulas as the putative hexyl homologs: CBGM, CBDM, and THCM; CBGM and CBDM have been isolated, while THCM has been detected in smoke but not isolated from plant material due to chromatographic issues. Prior analytical frameworks employing UHPLC-HRMS have enabled detection and tentative identification of numerous cannabinoids, and enzyme specificity studies (THCAS, CBDAS) suggest differing affinities for CBGA alkyl homologues, potentially influencing observed cannabinoid profiles.

Plant material: FM2 cannabis variety (strain CIN-RO; Military Chemical Pharmaceutical Institute, Florence, Italy) was processed under authorization. Two 5 g packs of inflorescences were ground. A 500 mg aliquot was extracted with 50 mL of 96% ethanol (per German Pharmacopoeia Cannabis Flos) for UHPLC-HESI-Orbitrap analysis. The remaining 9.5 g were freeze-dried, sonicated with n-hexane (3×, 15 min, ice bath), centrifuged, combined supernatants dried, resuspended in acetonitrile, filtered, and fractionated by semipreparative LC to isolate acidic cannabinoid species. Isolation of natural CBDH: Semi-preparative LC (Luna 5 µm C18(2) 100 Å, 250×10 mm; acetonitrile:0.1% formic acid in water 70:30 v/v; 5 mL/min) generated 80 fractions over 80 min. Fractions containing cannabidihexolic acid (CBDHA; RT 13.0 min) were pooled, dried, and decarboxylated at 120 °C for 2 h to yield ~0.3 mg CBDH. UHPLC-HESI-Orbitrap analysis: Thermo Ultimate 3000 UHPLC coupled to HESI-Q-Exactive Orbitrap. HESI settings: capillary 320 °C, vaporizer 280 °C, 4.2 kV (ESI+), 3.8 kV (ESI−), sheath/aux gas 55/30, S-lens RF 45. Acquisition: FS-dd-MS2 in positive and negative mode, 70,000 FWHM at m/z 200, scan range m/z 250–400, AGC 3e6, IT 100 ms, isolation window m/z 0.7, NCE 20 eV. Chromatography on Poroshell 120 SB-C18, 3.0×100 mm, 2.7 µm. Exact masses ([M+H]+ and [M−H]−), retention times, and MS/MS spectra were matched to synthesized standards within 5 ppm tolerance. Decarboxylation study: Ethanolic FM2 extract was heated to convert acidic cannabinoids, enabling analysis of neutral forms and comparison of retention times and MS2 patterns. Stereoselective synthesis: 5-hexylbenzene-1,3-diol was synthesized via Wittig olefination of (3,5-dimethoxybenzyl)triphenylphosphonium bromide with valeraldehyde, hydrogenation to 1-hexyl-3,5-dimethoxybenzene, and BBr3 demethylation to 5-hexyl-resorcinol. Condensation with (1S,4R)-1-methyl-4-(prop-1-en-2-yl)cyclohex-2-enol using p-TSA in DCM at r.t. was monitored by HPLC-UV/Vis; quenching at ~2 h provided (-)-trans-CBDH and (-)-trans-Δ⁴-THCH in one pot (17% and 20% yields; total 37%). Monomethyl ethers (-)-trans-CBDM and CBGM were prepared by partial methylation (dimethyl sulfate, K2CO3, DMF, r.t.), and (-)-trans-THCM by p-TSA-catalyzed cyclization of CBDM. Structures and stereochemistry were confirmed by 1H/13C NMR, HRMS, and optical rotation. Semi-quantification: External calibration with five-point curves: CBD and Δ⁹-THC (50–1000 ng/mL), CBDH, Δ⁹-THCH, CBDM, CBGM (5–250 ng/mL). Linearity R2 > 0.992. Concentrations in FM2 were calculated for targeted analytes. In vivo formalin test: Male C57BL/6J mice (6–8 weeks) received CBDH (1, 2, 3, 5 mg/kg, i.p.) or vehicle 20 min before subcutaneous formalin (1.25%, 30 µL) in the hind paw. Nocifensive behavior (time spent) was recorded every 5 min for 60 min. Two-way ANOVA with Bonferroni post hoc assessed effects on acute (0–7 min) and tonic phases (15–60 min). Ethical approvals and housing conditions are detailed.

- UHPLC-HESI-Orbitrap of native FM2 revealed three acidic peaks (A: 18.13 min; B: 20.21 min; C: 21.46 min) with formula C23H32O4 and distinct MS2 fragmentation. After decarboxylation, three neutral peaks appeared (Ad: 18.62–18.80 min; Bd: 20.62–20.91 min; Cd: 20.75–20.77 min) with formula C22H30O2. - Comparison with spectral libraries and synthesized standards established identities: Peak A/Ad = CBDH; Peak B/Bd = CBDM; Peak C/Cd = Δ⁴/Δ⁹-THCH (assignment supported by synthesis; low abundance precluded full confirmation from plant extract alone). Δ⁴/Δ⁹-THCM was not detected. - CBGM was identified by matching retention time (≈20.98 min) and MS2 with the synthetic standard; data were consistent with literature for CBGM. - Isolation of natural CBDH (via acidic precursor CBDHA) and superimposable NMR/HRMS with the synthetic standard confirmed structure. - Semi-quantification in FM2 (µg/g): CBDH 27; Δ⁹-THCH 7; CBDM 50; CBGM 102. Major cannabinoids were in mg/g: CBD 56; Δ⁹-THC 39. No THCM detected. - In vivo antinociception: CBDH significantly reduced formalin-induced nocifensive behavior in the tonic phase at 1 and 2 mg/kg i.p. (two-way ANOVA: treatment F(4,288)=17.32, P<0.0001; time F(12,288)=67.80, P<0.0001; interaction F(4,8,288)=3.02, P<0.0001). Doses 3 and 5 mg/kg were ineffective. Effects were not evident in the acute phase.

High-resolution mass spectrometry (Orbitrap) enabled detection and differentiation of cannabinoids with identical molecular formulas by leveraging retention times and MS/MS fragmentation, but definitive assignment required synthesized standards. This study expands the homolog series for CBD and THC to include n-hexyl side chains, filling the gap between pentyl and heptyl derivatives and demonstrating that even-numbered side chains can occur naturally in a medicinal cannabis variety. The work also clarifies potential confusion between hexyl homologs (CBDH, THCH) and monomethyl ethers (CBDM, THCM) due to identical formulas but distinct fragmentation patterns and ionization behavior (e.g., lack of negative-mode ionization for monomethyl ethers). The absence of THCM in FM2, alongside substantial levels of CBDM and CBGM (and their acidic precursors), aligns with prior observations that CBGM can be more abundant than its downstream products. Differential enzyme affinities (CBDAS vs. THCAS) for CBGA alkyl homologues may influence observed distributions. Concentrations of unconventional cannabinoids did not mirror the CBD:THC ratio across side-chain series (butyl, hexyl, heptyl), suggesting independent biosynthetic or transformation pathways and underscoring the need for broader varietal surveys. Pharmacologically, CBDH exhibited antinociceptive effects at low doses in the tonic phase of the formalin test, while higher doses were ineffective, hinting at complex, possibly pleiotropic mechanisms (e.g., dose-dependent engagement of TRPV1 or modulation of CB1/CB2). Further receptor profiling and mechanistic studies are warranted.

This study identifies and validates two new phytocannabinoids in Cannabis sativa L. FM2—cannabidihexol (CBDH) and Δ⁹-tetrahydrocannabihexol (Δ⁹-THCH)—through UHPLC-HESI-Orbitrap analysis corroborated by stereoselective synthesis, isolation, and spectroscopic confirmation. It distinguishes these hexyl homologs from monomethyl ether isomers (CBDM, CBGM, THCM), provides semi-quantitative levels in plant material, and reports in vivo antinociceptive activity of CBDH at low doses in mice. The findings expand the cannabinoid homolog series and offer analytical benchmarks to avoid misidentification. Future work should investigate the biosynthetic origins of even-numbered side-chain cannabinoids (plant vs. microbial transformation), extend quantitative surveys across diverse cannabis varieties, comprehensively profile receptor interactions and mechanisms underlying CBDH’s analgesic effects, and attempt isolation/confirmation of THCH directly from plant matrices where abundance permits.

- Δ⁹/Δ⁴-THCH was of very low abundance in FM2; its assignment relied on comparison with a synthesized standard, and isolation of its acidic precursor (THCHA) from plant was not achieved. - Δ⁹/Δ⁴-THCM was not detected; conclusions regarding its absence are limited to the FM2 batch analyzed. - Semi-quantification was performed on a single cannabis variety and batch; broader generalization requires analysis of multiple varieties and replicates. - The in vivo assessment was limited to the formalin pain model without mechanistic receptor studies; dose-response showed non-linear effects, and pharmacodynamics remain to be elucidated.