Cannabidiol (CBD) and Δ⁹-tetrahydrocannabinol (Δ⁹-THC) are the most well-known phytocannabinoids in *Cannabis sativa*. Recent research has expanded the known homolog series of both CBD and THC, identifying butyl and heptyl homologs. This study aimed to identify and characterize a new series of phytocannabinoids that fill the gap between the known pentyl and heptyl homologs – those with a n-hexyl side chain. The researchers hypothesized that these would exist, despite the similar molecular formulas and weights to monomethyl ether derivatives of canonical phytocannabinoids like cannabigerol monomethyl ether (CBGM), cannabidiol monomethyl ether (CBDM), and Δ⁹-tetrahydrocannabinol monomethyl ether (Δ⁹-THCM). The identification of these hexyl homologs is crucial for understanding the full complexity of the cannabis plant's chemical profile, furthering our understanding of potential therapeutic applications and avoiding confusion with similar compounds in analytical studies. The expansion of the known cannabinoid series and the exploration of their unique biological activity are important for the cannabis research field, which has recently seen progress in both clinical and academic domains such as the introduction of Epidiolex for treating severe childhood epilepsies and the development of advanced analytical techniques for identifying new compounds. Despite advancements, the chemical landscape of cannabis remains complex, with nearly 150 phytocannabinoids currently known.

Previous research has identified butyl and heptyl homologs of CBD and THC, expanding the known cannabinoid series. The existence of monomethyl ether derivatives of canonical phytocannabinoids has also been established, namely CBDM, CBGM, and Δ⁹-THCM. However, the presence of hexyl derivatives of cannabinoids had not been previously reported until this research. Existing literature discussed the potential for cannabinoids with even numbers of carbons in the side chain to be artifacts of fungal ω-oxidation. The studies of cannabigerol monomethyl ether (CBGM), by researchers such as Yamauchi et al and Shoyama et al provided a foundation for understanding the related methylated cannabinoids, however, the presence and distinct characteristics of hexyl homologs were yet to be fully explored. The work of de Meijer et al on cannabinoid acid synthases (THCAS, CBDAS) and their differing affinities for CBGA alkyl homologues provided a theoretical basis for understanding the potential variations in concentrations of different homolog series.

The researchers used the FM2 medicinal cannabis variety. A 5g sample was extracted with ethanol (96%), and analyzed using UHPLC-HESI-Orbitrap mass spectrometry. To identify the decarboxylated forms, an ethanolic extract was heated. This UHPLC-HESI-Orbitrap analysis revealed peaks suggesting compounds with the molecular formula C₂₁H₃₀O₂, consistent with decarboxylated CBD and THC homologs. To distinguish between the hexyl homologs and the known methyl ether derivatives, the researchers performed a stereoselective synthesis of cannabidihexol (CBDH), Δ⁹-tetrahydrocannabihexol (Δ⁹-THCH), CBDM, and Δ⁹-THCM. The synthetic standards were used to confirm the identity of the compounds identified in the FM2 extract. The synthesis of the hexyl homologs involved a Wittig reaction, hydrogenation, and demethylation to obtain the appropriate resorcinol, which was then condensed with (1S,4R)-1-methyl-4-(prop-1-en-2-yl)cyclohex-2-enol. Careful monitoring of the reaction time was critical to isolate CBDH and Δ⁹-THCH separately and to prevent the formation of Δ⁸ isomers. The monomethyl ether derivatives were synthesized by methylation of CBD and CBG with dimethylsulfate. For the semi-quantitative analysis, calibration curves were constructed for CBDH, Δ⁹-THCH, CBDM, and CBGM. The in vivo antinociceptive activity of CBDH was assessed using the formalin test in mice. A separate semi-preparative HPLC was used to isolate the acidic precursor of CBDH (CBDHA) from the FM2 extract. The isolated CBDHA was then decarboxylated to obtain pure CBDH, for comparison with the synthetic standard. The UHPLC-HESI-Orbitrap analysis was performed using specific parameters for both positive and negative ionization modes, optimizing settings such as capillary temperature, vaporizer temperature, electrospray voltage, sheath and auxiliary gas flow, RF level of the S lens, scan range, AGC, injection time, and isolation window. Statistical analysis for the formalin test used two-way ANOVA and Bonferroni's post-hoc tests.

The UHPLC-HESI-Orbitrap analysis of the FM2 cannabis extract initially revealed three peaks (A, B, C) with a molecular formula suggesting acidic CBD and THC-type cannabinoids. After decarboxylation, three new peaks (Ad, Bd, Cd) appeared, and based on MS/MS spectra, these were identified as potential homologs of CBD and THC with a hexyl side chain. The researchers then conducted a stereoselective synthesis of CBDH, Δ⁹-THCH, CBDM, and Δ⁹-THCM to confirm the identities of the compounds found in the FM2 extract. Comparison of the retention time, molecular ion, and fragmentation spectra of the synthetic standards with those from the FM2 extract confirmed the presence of CBDH and CBDM. Δ⁹-THCH was also tentatively identified, although its low abundance and presence of interferents hindered definitive confirmation. No Δ⁹-THCM was detected. CBGM was also identified in the FM2 extract. Semi-quantification of these compounds revealed concentrations in the µg/g range, significantly lower than the main cannabinoids CBD and Δ⁹-THC (mg/g range). Specifically, CBDH and Δ⁹-THCH were found at 27 µg/g and 7 µg/g, respectively. The in vivo formalin test in mice showed that CBDH at doses of 1 and 2 mg/kg significantly reduced the late phase of formalin-induced nociceptive behavior, demonstrating antinociceptive activity. Higher doses (3 and 5 mg/kg) showed no significant effect. The NMR spectroscopic data of synthetic and extracted CBDH were perfectly superimposable, confirming the chemical structure.

The study successfully identified and characterized CBDH and Δ⁹-THCH, filling a gap in the known cannabinoid homolog series. The findings highlight the importance of high-resolution mass spectrometry for identifying minor cannabinoids in complex matrices. The distinct fragmentation patterns observed allowed for the differentiation between hexyl homologs and methyl ether derivatives, despite their similar molecular formulas. The antinociceptive activity of CBDH at low doses suggests potential therapeutic applications. The lack of effect at higher doses could indicate a complex pharmacodynamic profile involving multiple receptors or mechanisms of action, warranting further investigation. The differences in concentrations between various homolog series (butyl, hexyl, and heptyl) highlight the complex biosynthesis of cannabinoids in the cannabis plant, emphasizing the need for studies on the origin of these compounds. The discrepancies in the CBD to THC ratios across the different series further support the complex nature of cannabinoid biosynthesis and distribution.

This research adds to the growing knowledge of the cannabis cannabinoma, clarifying the potential confusion between phytocannabinoids with a 6-carbon alkyl chain and their methylated counterparts. The identification of CBDH and Δ⁹-THCH expands the known cannabinoid homolog series and reveals the antinociceptive potential of CBDH. Future research should explore the full pharmacological profile of CBDH, including its mechanism of action and potential therapeutic applications. Investigating the biosynthesis of these unusual cannabinoids would be important in understanding their origin and the broader metabolic pathways involved in cannabinoid production in the *Cannabis sativa* plant.

The study focused on a single cannabis variety (FM2). Therefore, the findings might not be generalizable to other varieties. The identification of Δ⁹-THCH was tentative due to low abundance and interferents. Further research is needed to confirm the presence and quantify this compound across different varieties. The semi-quantitative analysis was performed using an external standard method, which may introduce some inaccuracies. A more comprehensive investigation into the mechanisms of action of CBDH's antinociceptive effect would be valuable.