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 Δ 9-tetrahydrocannabinol (Δ 9-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 Δ 9-THCB) and a heptyl-(in CBDP and Δ 9-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 Δ 9-THC, bearing a n-hexyl side chain on the resorcinyl moiety that we named cannabidihexol (CBDH) and Δ 9-tetrahydrocannabihexol (Δ 9-THCH), respectively. However, some cannabinoids with the same molecular formula and molecular weight of CBDH and Δ 9-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 Δ 9-tetrahydrocannabinol monomethyl ether (Δ9-THCM). The unambiguously identification in cannabis extract of the n-hexyl homologues of CBD and Δ 9-THC different from the corresponding methylated isomers (CBDM, CBGM and Δ 9-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 Δ 9-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 Δ 9-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. Cannabis research has made great progresses in the latest years in both clinical and academic field. For example, new cannabis based drugs, like Epidiolex, have been placed on the market for the treatment of severe forms of infant epilepsy not responding to conventional therapies 1. In the academic research, new insights on cannabis chemistry have been disclosed thanks to the high-performing technological platforms for the identification of OPEN

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
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Identication of a new
cannabidiol n‑hexyl homolog
in a medicinal cannabis variety
with an antinociceptive activity
in mice: cannabidihexol
Pasquale Linciano1,6, Cinzia Citti1,2,3,6, Fabiana Russo1, Francesco Tolomeo2,
Aldo Lagan3,4, Anna Laura Capriotti4, Livio Luongo5, Monica Iannotta5, Carmela Belardo5,
Sabatino Maione5, Flavio Forni1, Maria Angela Vandelli1, Giuseppe Gigli3 &
Giuseppe Cannazza 1,3*
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 lls 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 identied 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
identication in cannabis extract of the n‑hexyl homologues of CBD and Δ9‑THC dierent 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‑quantication 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 conrmed 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.
Cannabis research has made great progresses in the latest years in both clinical and academic eld. For example,
new cannabis based drugs, like Epidiolex, have been placed on the market for the treatment of severe forms of
infant epilepsy not responding to conventional therapies1. In the academic research, new insights on cannabis
chemistry have been disclosed thanks to the high-performing technological platforms for the identication of
OPEN
              
Italy.            
      Department of Chemistry, Sapienza University of Rome,
              
   
and Cinzia Citti. *
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new compounds2–6. Although there is still much to do in the cannabis chemistry research, almost 150 phyto-
cannabinoids can be count on the most updated inventory7. Our most recent works have disclosed the exist-
ence of new phytocannabinoids series besides those of the orcinoids, varinoids and olivetoids, belonging to the
cannabidiol (CBD) and Δ9-tetrahydrocannabinol (Δ9-THC) type cannabinoids4,8. e new series of phytocan-
nabinoids share the terpenophenolic core of CBD and Δ9-THC and dier for the length of the linear alkyl side
chain; specically, cannabidibutol (CBDB), and Δ9-tetrahydrocannabutol (Δ9-THCB) have a n-butyl side chain8,
whereas cannabidiphorol (CBDP) and Δ9-tetrahydrocannabiphorol (Δ9-THCP) have a n-heptyl side chain4. e
discovery of new phytocannabinoids, which were both directly isolated from the plant and synthetically prepared
in the lab, has opened new gaps on their still unexplored biological activity, making us wondering about their
pharmacological eects on humans.
To further complicate the already intricate scenario, we report a new series of phytocannabinoids that lls the
gap between the pentyl and heptyl homologs of CBD and Δ9-THC, bearing a n-hexyl side chain on the resorcinyl
moiety. At the best of our knowledge and according to the literature, no case of hexyl derivatives of cannabinoid
has been reported so far. Conversely, cannabinoids with the same molecular formula and molecular weight have
been classied as monomethyl ether derivatives of canonical phytocannabinoids, namely cannabigerol monome-
thyl ether (CBGM), cannabidiol monomethyl ether (CBDM) and Δ9-tetrahydrocannabinol monomethyl ether
(Δ9-THCM)9. Whilst CBGM and CBDM have been already isolated and characterized10,11, Δ9-THCM has been
detected in cannabis smoke12 and some authors reported that it is present in the plant, but they were not able
to isolate it due to chromatographic issues13. Our ndings on the presence of the hexyl homologs of CBD and
Δ9-THC, which we named cannabidihexol (CBDH) and Δ9-tetrahydrocannabihexol (Δ9-THCH) respectively,
were supported by the stereoselective synthesis of the corresponding pure standards that are found in the plant
prior to decarboxylation.
Results
Identication of CBD and Δ9‑THC hexyl homologs by UHPLC‑HESI‑Orbitrap. In the attempt to
provide a complete characterization of the FM2 cannabis variety, we noticed the presence of two major peaks
at 18.13 and 20.21, and a minor one at 21.46min, corresponding to the molecular formula C23H32O4, suggest-
ing the presence of the carboxylic group. e analysis of the fragmentation spectra in negative ionization mode
conrmed this hypothesis but showed three dierent fragmentation patterns (Fig.1A). e two major peaks A
and B presented very similar spectra diering only for the relative intensity of the fragments, whereas the minor
peak C showed very poor fragmentation. Peak A at 18.13min could be associated to a CBDA-like molecule
(Fig.1B), while peak B at 20.21min had lower intensity for the fragment corresponding to [M–H2O]− at m/z 353
and presented a new fragment at m/z 178, not found in the other spectra (Fig.1C). Peak C at 21.46min showed
a THCA-like fragmentation characterized by the very low intensity of the fragment [M–H2O]− and the absence
of other major fragments besides the one corresponding to [M-CO2]− at m/z 327 (Fig.1D).
In order to identify these compounds, but being unable to isolate acidic species, we moved to work on the
decarboxylated forms of such cannabinoids. erefore, the ethanolic extract of FM2 was heated and the new
mixture was analysed employing the same conditions by UHPLC-HESI-Orbitrap. As expected, in place of the
previously detected peaks, three new peaks appeared at dierent retention times, 18.62, 20.62, and 20.77min,
with the molecular formula C22H32O2 corresponding exactly to the loss of a CO2 molecule. Figure1E shows a
second chromatogram with the decarboxylated compounds Ad, Bd, and Cd. Surprisingly, peaks Bd and Cd had
inverted elution order and peaks Ad and Cd presented superimposable fragmentation spectra in positive ioniza-
tion mode with the same pattern as CBD and THC. Moreover, peaks Ad and Bd were very similar and diered
for the relative intensity of the molecular ion [M+H]+ at m/z 329 and the base peak at m/z 207. We concluded
that peak A and B could be acidic CBD-type cannabinoids, whereas peak C could be an acidic THC-type can-
nabinoid (Fig.1F–H).
According to the literature, cannabinoids with such molecular formula and molecular ions are reported
as monomethyl ethers of CBDA and THCA, named cannabidiolic acid monomethyl ether (CBDMA) and
tetrahydrocannabinolic acid monomethyl ether (THCMA). Similarly, cannabinoids with molecular formula
C22H32O2 could be the corresponding decarboxylated derivatives, the already known cannabidiol monomethyl
ether (CBDM) and the putative tetrahydrocannabinol monomethyl ether (THCM). However, we found three
peaks corresponding to the same formula but dierent MS2 spectra. By a comparison with other CBD and
THC homologs present in our spectral library, such as cannabidivarin (CBDV), cannabidibutol (CBDB), can-
nabidiphorol (CBDP), Δ9-tetrahydrocannabivarin (Δ9-THCV), Δ9-tetrahydrocannabutol (Δ9-THCB), and Δ9-
tetrahydrocannabiphorol (Δ9-THCP), we were able to putatively identify two new homologs of CBD and THC
with a hexyl side chain. As shown in Fig.2, the new compounds dier exactly by a –CH2 unit (14 amu) from
the corresponding pentyl (CBD and Δ9-THC) and the recently identied heptyl homologs (CBDP and THCP),
not only for the molecular ion [M+H]+ but also for all fragments. For both CBD (Fig.2A) and THC (Fig.2B)
homologs, it was evident that the molecular ion [M+H]+ and the base peak inverted their relative intensity as
the length of the side chain increased from the propyl to the heptyl homologs, most likely due to the increasing
stability of the molecular ion.
In order to conrm the identity of these two new cannabinoids and unambiguously identify peak Bd, a
stereoselective synthesis of the putatively identied cannabinoids, which for sake of simplicity and consistency
were called cannabidihexol (CBDH) and Δ9-tetrahydrocannabihexol (Δ9-THCH), and the monomethyl ether
derivatives of CBD and Δ9-THC (CBDM and Δ9-THCM) was performed.
Identication of CBGM by UHPLC‑HESI‑Orbitrap. Given the results obtained for CBD and Δ9-THC,
we hypothesized the presence in the FM2 variety of the hexyl homolog of CBG, which has a molecular formula
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C22H34O2 and [M+H]+ ion at m/z 331.2632. Only one peak resulted from the specic ion search, thus instill-
ing the doubt about its identity as hexyl or methyl ether derivative. e fragmentation spectrum of its acidic
precursor in the FM2 native extract showed a pattern dierent from that of cannabigerolic acid (CBGA), for
which the analytical standard was available, and from those reported in the literature for the other series of CBG
like cannabigerovarin (CBGV)14 and cannabigerobutol (CBGB)9. e match of retention time (20.98min) and
fragmentation spectrum with those of synthetic CBGM and the comparison with spectral data reported in the
literature for the same cannabinoid conrmed that the additional methyl group was attached to the oxygen of
the resorcinyl moiety and was not part of the alkyl side chain.
Stereoselective synthesis of CBDH, Δ9‑THCH and monomethyl derivatives CBDM, Δ9‑THCM
and CBGM. e stereoselective synthesis of (−)-trans-cannabidihexol ((−)-trans-CBDH) and (−)-trans-
Δ9-tetrahydrocannabihexol ((−)-trans-Δ9-THCH) was performed as previously reported for the synthesis
Figure1. Identication 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 identied 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 identied peaks
Ad (panel F), Bd (panel G) and Cd (panel H).
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of the corresponding homologs (−)-trans-CBDB, (−)-trans-CBDP, (−)-trans-Δ9-THCB and (−)-trans-Δ9-
THCP2–4,8. e appropriate 5-hexylbenzene-1,3-diol (4) was prepared rst as reported in Fig.3A, Scheme1.
(3,5-dimethoxybenzyl)triphenylphosphonium bromide (1) was easily prepared in quantitative yield by reac-
tion of 1-(bromomethyl)-3,5-dimethoxybenzene with triphenylphosphine in reuxing toluene for 6h. Wittig’s
reaction between 1 and valeraldehyde in 0.1M K2CO3 aqueous solution at reux for 24h gave 1-(hex-1-en-
1-yl)-3,5-dimethoxybenzene (2) as a 55:45 E/Z mixture which was hydrogenated using the alesNano H-Cube
ow reactor, to give the corresponding 1-hexyl-3,5-dimethoxybenzene (3) in 91% yield. e demethylation
performed using BBr3 in anhydrous DCM, overnight at room temperature and under nitrogen atmosphere,
gave 5-hexyl-resorcinol (4) in quantitative yield. In our previous works CBDB and CBDP were synthesized rst
by condensation of the appropriate resorcinol with (1S,4R)-1-methyl-4-(prop-1-en-2-yl)cycloex-2-enol, using
pTSA as catalyst and stopping the reaction before CBDs evolve to THCs in the same conditions. Conversely,
for the selective synthesis of (−)-trans-Δ9-THCB and (−)-trans-Δ9-THCP a longer procedure was adopted. e
appropriate resorcinol was condensed with (1S,4R)-1-methyl-4-(prop-1-en-2-yl)cycloex-2-enol in the same
condition described above for a longer reaction time (usually 48h). In this way, the CBDs were quantitatively
converted into the corresponding Δ8-THCs. Hydrochlorination of the Δ8 double bond of (−)-trans-Δ8-THCs,
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. e pale-yellow box point out the constant terpenic portion. e red lines highlight the shi 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).
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allowed to obtain (−)-trans-HCl-THCs, which were successively converted to (−)-trans-Δ9-THCs by selective
elimination on position 2 of the terpene moiety using potassium t-amylate as base. Although this procedure
allowed to selectively prepare (−)-trans-Δ9-THCs, it has the inconvenience to be time consuming, and with
low atom economy. Because the conversion of CBDs to Δ8-THCs passes through the formation of Δ9-THCs
rst, for the synthesis of (−)-trans-CBDH and (−)-trans-Δ9-THCH we evaluated the possibility to stop the reac-
tion before Δ9-THCH starts to convert into Δ8-THCH. erefore, 5-hexyl-resorcinol (4) was condensed with
(1S,4R)-1-methyl-4-(prop-1-en-2-yl)cycloex-2-enol using pTSA as catalyst and the progression of the reaction
was monitored every 15min by HPLC–UV/Vis. Aer approximately 2h, almost the 50% of (−)-trans-CBDH
converted in (−)-trans-Δ9-THCH, but no traces of Δ8-THCH were detected. e reaction was therefore stopped
and (−)-trans-CBDH and (−)-trans-Δ9-THCH were puried as reported in Material and Methods section.
(−)-trans-CBDH and (−)-trans-Δ9-THCH were obtained in 17% and 20% yield, respectively. e total yield of
the two phytocannabinoids was 37%, which is in line with the yield obtained for the sole synthesis of CBDB or
CBDP using the same procedure, but quenching the reaction aer the consumption of the starting materials and
before that CBDs started to isomerize into THCs (usually 30min–1h). erefore, strictly monitoring the con-
densation of the appropriate resorcinol with (1S,4R)-1-methyl-4-(prop-1-en-2-yl)cycloex-2-enol it is possible
to prepare in one-pot reaction both (−)-trans-CBDs and (−)-trans-Δ9-THCs, avoiding cumbersome and longer
procedure to selectively prepare the (−)-trans-Δ9-THCs, without the awkward formation of their Δ8 isomers. e
synthesis of the monomethyl ether derivatives of CBD, THC and CBG is reported in Fig.3A, Scheme2. (−)-trans-
CBDM and CBGM were easily prepared by methylation of the commercially available CBD and CBG by reaction
with 0.5 equivalents of dimethylsulfate, in DMF at room temperature, using K2CO3 as base (Fig.3A, Scheme3).
In contrast (−)-trans-Δ9-THCM was prepared from (−)-trans-CBDM, through cyclization catalyzed by pTSA
(Fig.3A, Scheme2). e chemical identication of synthetic (−)-trans-CBDH, (−)-trans-Δ9-THCH, (−)-trans-
CBDM, (−)-trans-Δ9-THCM and CBGM, and their unambiguous 1H and 13C assignments were achieved by
NMR spectroscopy (FigureSI-1–5, Supporting Information). In particular for (−)-trans-CBDH and (−)-trans-
Δ9-THCH, as already stated during the synthesis of (−)-trans-CBDB, (−)-trans-CBDP, (−)-trans-Δ9-THCB and
(−)-trans-Δ9-THCP, and by comparison with the well-known homologs (CBD, CBDV, Δ9-THC, and Δ9-THCV)
no signicant dierences in the proton and carbon chemical shis of the terpene and aromatic moieties were
observed among CBD and Δ9-THC homologs. e sole exception observed regards the integration of the multi-
plet in the range 1.4–1.2ppm in the 1H spectra and the number of carbon signals in the range 20–30ppm of the
13C spectra, corresponding to the central methylene units of the alkyl chain on the resorcinyl moiety. e perfect
match in the chemical shi of the terpene and aromatic moieties between the synthesized (−)-trans-CBDH and
(−)-trans-Δ9-THCH and the respective homologues2–4,8, combined with the mass spectra and fragmentation
pattern, allowed us to unambiguously conrm the chemical structures of the two new synthetic cannabinoids.
e trans (1R,6R) conguration at the terpene moiety was conrmed by optical rotatory power. e new can-
nabinoids (−)-trans-CBDH, (−)-trans-Δ9-THCH, (−)-trans-CBDM and (−)-trans-Δ9-THCM showed an [α]D20
of − 146°, − 166°, − 113° and − 161°, respectively, in chloroform. e [α]D20 values were in line with those of the
homologs2,8,15, suggesting a (1R,6R) conguration for the four phytocannabinoids. Lastly, the perfect superim-
position between the 1H and 13C NMR spectra of both synthetic and extracted (−)-trans-CBDH was observed,
conrming the identity of the new cannabinoids identied in the FM2 cannabis variety (FigureSI-6, Supporting
Information).
A comparison of the retention time, molecular ion and fragmentation spectra of each pure synthesized
standard with those found in FM2 led us to conclude that the rst peak Ad could be assigned to CBDH and the
second one Bd to CBDM (Fig.3B). e third peak Cd could most likely be associated to Δ9-THCH although its
very low abundance and the presence of other interferents in the fragmentation spectrum from the FM2 extract
did not allow an unambiguous assignment of its chemical structure (Fig.3B). Moreover, no trace of Δ9-THCM
was found. Fragmentation in negative ionization mode helped us to distinguish between CBDH and Δ9-THCH,
which were identical in positive ionization mode, whereas no ionization was obtained in negative mode for Δ9-
THCM due to the lack of free hydroxyl groups to be deprotonated (Fig.3B). Conrmation of the identication of
CBDH was achieved by isolation of pure fractions from the FM2 extract containing the acidic precursor CBDHA
by semipreparative liquid chromatography. e pure compound was decarboxylated by heat and analysed by
UHPLC-HESI-Orbitrap. Unfortunately, it was not possible to isolate fractions of FM2 containing THCHA due to
its very low abundance. However, the stereoselective synthesis of Δ9-THCH allowed to assign a certain chemical
structure to the corresponding peak in the FM2 sample.
Semi‑quantication of CBDH and Δ9‑THCH in the FM2 extract. anks to the synthetically pre-
pared analytical standards of CBDH, Δ9-THCH, CBDM and CBGM, we were able to provide a semi-quanti-
cation of these cannabinoids in the FM2 cannabis variety by building the corresponding calibration curves.
e results of concentration were in the order of the µg/g, while the main cannabinoids CBD and Δ9-THC were
in the order of the mg/g (56 and 39mg/g respectively). In particular, the hexyl homologs of CBD and Δ9-THC
resulted 27µg/g and 7µg/g, while the methyl ether derivatives CBDM and CBGM were 50µg/g and 102µg/g.
No Δ9-THCM was detected in the FM2.
Eects of CBDH on the formalin test in mice. Formalin paw injection is a solid and widely used model
of nociception with high face validity when tested with analgesic drugs. A nociceptive response to subcutaneous
formalin induced an early, short-lasting rst phase (0–7min) followed by a quiescent period, and then a second,
prolonged phase (15–60min) of tonic hyperalgesia (Fig.3C). In the tonic phase, two-way ANOVA revealed that
CBDH (1, 2mg/kg, i.p.) signicantly reduced the late phase of the formalin-induced nocifensive behavior when
compared to the vehicle-treated group (treatment F(4,288) = 17.32, P < 0.0001, time F(12,288 ) = 67.80, P < 0.0001 and
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interaction F(48,288) = 3.02, P < 0.0001); also, the dose of 2mg/kg had a signicant antinociceptive eect as com-
pared to the vehicle group. e doses of 3 and 5mg/kg had no eect on the formalin test (Fig.3C).
Discussion
e comprehensive characterization of the chemical prole of a cannabis variety is a rather arduous task as
the analytical tools in the chemist’s hand are not able to cover such a broad range of compounds. However, the
high sensitivity and selectivity of the high-resolution mass spectrometry, for example those achieved with the
Orbitrap technology, can enable the identication of a reasonable number of molecules, even when present in
very small traces. is approach allowed for the identication of new series of cannabinoids, CBD and THC
homologs, with dierent lengths of the alkyl side chain, which were recently reported by our group2–4,8. e
present work expanded the scope of cannabinoids identication completing the series of homologs with dier-
ent alkyl side chain from three to seven methylene units. Up to now, only cannabinoids with an odd number of
carbon atoms on the side chain have been reported and those with an even number of carbon atoms have been
supposed to be artifacts derived from fungal ω-oxidation of their corresponding homologs7. e investigation
of the origin of these species, such as those with a butyl and hexyl side chain, is beyond the scope of this work,
but, although surprising, it is certain that such cannabinoids are actually present in a medicinal cannabis vari-
ety. e literature reports the existence of monomethyl ether derivatives of the canonical pentyl cannabinoids
to justify the presence of compounds bearing an additional methyl group. Although on one side the structural
identity of such derivatives was conrmed, our ndings pointed out a new series of cannabinoids with the same
molecular formula of the monomethyl ethers but with a dierent arrangement. eir origin, whether it is from
the plant or from microorganisms, should be investigated as this might disclose new insights in the cannabis
biochemistry. It is certainly important to underline that it is very easy to confuse CBDH and CBDM, as well as
Δ9-THCH and Δ9-THCM. However, the match of the high-resolution fragmentation patterns with their pure
synthetic standards was determinant to assign the respective chemical structure. is work might serve in the
future for any researcher to distinguish between two species that show a very similar analytical behaviour. In a
similar way, the methyl ether derivative of CBG was also identied (CBGM).
It is worth noting that no Δ9-THCM was detected in the FM2 variety. On the other hand, both CBDM and
CBGM showed a high peak as well as their native precursors CBDMA and CBGMA. Achieved results are in
accordance with what reported by Lumır Ondrej Hanus etal., which showed that the cannabigerol monomethyl
ether is always presents in greater quantities than its products, THCMA and CBDMA7,16.
de Meijer etal. demonstrated that the cannabinoid acid synthases (THCAS, CBDAS) show a dierent anity
for CBGA alkyl homologues. is concept would explain the achieved results. CBDAS could be competitively
stronger than THCAS when the substrate is CBGMA17,18.
e comparison of the results obtained for the concentrations of unorthodox cannabinoids in the FM2 variety
suggested that there is no relationship between the dierent series as the same CBD to THC ratio is not respected.
Considering our recent work on the heptyl derivatives of CBD and Δ9-THC, CBDP and Δ9-THCP were found in
the FM2 at the concentration of 243µg/g and 29µg/g4; whereas, in the same cannabis variety, the butyl series of
CBD and THC was found at the concentration of 500µg/g and 400µg/g for CBDB and Δ9-THCB respectively8.
In this work, we found 27µg/g and 7µg/g for CBDH and Δ9-THCH respectively. However, this data should be
veried considering a larger number of dierent varieties in order to provide a reliable statistical signicance.
e CBDH could have pleiotropic mechanisms of action through which it can exert its pharmacological
eect. We found that the doses of the 1 and 2mg/Kg signicantly reduced the late phase of the formalin-induced
nocifensive behavior, whereas the higher doses 3 and 5mg/Kg were uneective. is could be due, at least in
part, assuming that at these doses CBDH can activate receptor facilitating nociception such as TRPV1 or other
channels. On the other hand, we can speculate that CBDH at the higher doses could block receptors involved in
antinociception such as CB1 or CB2. However, further pharmacological studies are needed to better investigate
the pharmacodynamics prole of this interesting compound.
Another piece of knowledge towards understanding Cannabis Sativa L. cannabinoma has been added with
this work. In particular, clarity has been made about the possible confusion between phytocannabinoids with a
6-term alkyl chain (CBDH, THCH) and those with a methylated resorcinolic hydroxyl group (CBDM, THCM).
Furthermore, two new phytocannabinoids CBDH and THCH have been identied in the FM2 variety by
Figure3. Synthesis and UHPLC-HRMS identication 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,
reux, 6h, quant. yield; (b) valeraldehyde (1.5 equiv.), 0.1M K2CO3 aq. (10mL per mmol of 1), reux, 5h,
81% yield; (c) H-Cube alesNano 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) Eects of CBDH (1, 2, 3, and 5mg/kg, i.p.) or vehicle in the formalin test in mice. e 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 signicant dierences 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.
◂
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comparison with their respective authentic synthesized compounds. In particular, CBDH has been isolated and
its pharmacological activity has been evaluated invivo in mice. At extremely low doses (1mg/kg) it showed an
interesting nocifensive activity. However, the CBDH concentration of 27μg/g found in the FM2 variety is too
low to exert the pharmacological eect but it is not excluded that other cannabis varieties may contain higher
concentrations. More in-depth pharmacological studies are currently underway to clarify the mechanism of
action of this new phytocannabinoid.
Methods
Plant material. FM2 cannabis variety is produced from the strain CIN-RO bred by the Council for Agricul-
tural Research and Economics (CREA) in Rovigo (Italy) and supplied to the Military Chemical Pharmaceutical
Institute (MCPI, Firenze, Italy). Experiments on FM2 inorescence (batch n. 6A32/1) were performed with the
authorization of the Italian Ministry of Health (prot. n. SP/062). Two 5g packs were nely grinded (< 2mm
particle size) and divided into two batches: 500mg were extracted with 50mL of ethanol 96% according to
the procedure reported in the monograph of Cannabis Flos of the German Pharmacopoeia19 and analyzed by
UHPLC-HESI-Orbitrap without further dilution. e remaining 9.5g were treated according to the protocol of
Pellati etal. with minor changes20. Briey, freeze-dried plant material was sonicated with 400mL of n-hexane for
15min in an ice bath. Aer centrifugation for 10min at 2000 × g the pellets were discarded. e same procedure
was repeated twice more. e supernatants were then dried under reduced pressure and resuspended in 10mL
of acetonitrile, ltered and passed through a semi-preparative liquid chromatography for the isolation of the
acidic species of the cannabinoids of interest.
Isolation of natural CBDH. A semi-preparative LC system (Octave 10 Semba Bioscience, Madison, USA)
was used to separate the FM2 mixture into 80 fractions in a total run time of 80min. e chromatographic con-
ditions used are reported in the paper by Citti etal.4. A Luna C18 with a fully porous silica stationary phase (Luna
5µm C18(2) 100Å, 250 × 10mm) (Phenomenex, Bologna, Italy) was the column employed and a mixture of
acetronitrile:0.1% aqueous formic acid 70:30 (v/v) was used as mobile phase at a constant ow rate of 5mL/min.
e fractions containing CBDHA (retention time 13.0min) was isolated as reported in our previous work2. e
fractions containing CBDHA (13.0min) were analyzed by UHPLC-HESI-Orbitrap and dried on the rotavapor
at 70°C. e residue was placed in an oven at 120°C for 2h to achieve decarboxylation. An amount of about
0.3mg of CBDH was obtained.
UHPLC‑HESI‑Orbitrap metabolomic analysis. Analyses on FM2 extracts were performed on a ermo
Fisher Scientic Ultimate 3000 provided with a vacuum degasser, a binary pump, a thermostated autosampler, a
thermostated column compartment and interfaced to a heated electrospray ionization source and a Q-Exactive
Orbitrap mass spectrometer (UHPLC-HESI-Orbitrap). e HESI and Orbitrap parameters were set following
our previous work4. Briey, the capillary temperature was set at 320°C, the vaporizer temperature at 280°C, the
electrospray voltage at 4.2kV (for the positive ionization mode) and 3.8kV (for the negative mode), the sheath
gas and the auxiliary gas at 55 and 30 arbitrary units respectively, the RF level of the S lens at 45. Analyses were
acquired in full scan data-dependent acquisition (FS-dd-MS2) in positive and negative mode with a resolving
power of 70,000 FWHM and m/z of 200 using the Xcalibur 3.0 soware (ermo Fisher Scientic, San Jose, CA,
USA). For the Orbitrap mass analyzer, a scan range of m/z 250–400, an AGC of 3e6, an injection time of 100ms
and an isolation window of m/z 0.7 were chosen as the optimal parameters. e collision energy for the frag-
mentation of the molecular ions was set at 20eV. e exact masses of the [M+H]+ and [M−H]− molecular ions
were extracted from the total ion chromatogram (TIC) of the FM2 extracts and matched with pure analytical
standards for accuracy of the exact mass (5ppm), retention time and MS/MS spectrum.
e chromatographic separation was carried out on a core shell C18 stationary phase (Poroshell 120 SB-C18,
3.0 × 100mm, 2.7µm, Agilent, Milan, Italy) following the conditions employed for our previous work4.
A semi-quantitative analysis of Δ9-THC and CBD, their hexyl homologs CBDH and Δ9-THCH, and the
methyl ether derivatives of CBD and CBG, CBDM and CBGM, was carried out using a calibration curve with
the external standard method. A stock solution of CBD and Δ9-THC (1mg/mL) was properly diluted to obtain
ve non-zero calibration points at the nal concentrations of 50, 100, 250, 500 and 1000ng/mL; a stock solution
of CBDH, CBDM, Δ9-THCH, CBDM and CBGM was diluted to obtain the nal concentrations of 5, 25, 50,
100 and 250ng/mL. e linearity was assessed by the coecient of determination (R2), which was greater than
0.992 for each analyte.
Synthetic procedure. e reagents and the solvents used for the synthesis of the analytical standards were
purchased from Sigma-Aldrich and VWR, respectively. In the synthetic procedures, the solvents were abbrevi-
ated as following: acetonitrile (ACN); chloroform (CHCl3); cyclohexane (CE); dichloromethane (DCM); diethyl
ether (Et2O); dimethyl sulfoxide (DMSO); ethyl acetate (AcOEt). e reactions were monitored by thin-layer
chromatography (TLC) using 60F-254 silica gel plates (from Merck) and inspected with UV lamp, or alka-
line KMnO4 stain. Purication of the synthesized products was performed by ash chromatography on silica
gel (40–63μm). e mobile phase is specied in the respective following monographies. NMR spectra were
recorded on a Bruker 400 (at 400.134MHz for 1H and 100.62MHz for 13C) or on a Bruker 600 (at 600.130MHz
for 1H and 150.902MHz for 13C) spectrometer. Chemical shis (δ) are reported in parts per million (ppm) and
referenced to the solvent residual peaks. Coupling constants are reported in hertz (Hz) and the splitting pattern
is reported as: singlet (s), doublet (d), triplet (t), quartet (q), double doublet (dd), quintet (quin), multiplet (m),
broad signal (b). Monodimensional and bidimensional spectra were acquired using the same parameters previ-
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ously reported3–5,8. Optical rotation (α) was acquired with a Polarimeter 240C from Perkin–Elmer (Milan, Italy),
using a cell with a length of 100mm, and a volume 1mL.
Synthesis of(3,5‑dimethoxybenzyl)triphenylphosphonium bromide (1). Triphenylphosphine (6.3g, 23.8mmol,
1.1 equiv.) was added to a stirred solution of 1-(bromomethyl)-3,5-dimethoxybenzene (5.0g, 21.6mmol, 1
equiv.), in 30mL of toluene and reuxed for 6h. Aer standing at room temperature overnight, the precipitate
formed was collected by ltration, washed with diethyl ether and dried to give 10.4g of a white solid (quant.
yield).
1H-NMR (400MHz, CDCl3) δ 7.78–7.76 (m, 9H), 7.66–7.63 (m, 6H), 6.35 (t, 2H, J = 2.3Hz), 6.30 (q, 1H,
J = 2.3Hz), 5.32 (d, 2H, J = 14.3Hz), 3.54 (s, 6H).
Synthesis of (E/Z)‑1‑(hexyl‑1‑en‑1‑yl)‑3,5‑dimethoxybenzene (2). Valeraldehyde (0.48 mL, 4.56 mmol, 1.5
equiv.) was added to a stirred suspension of 1 (1.5g, 3.04mmol, 1 equiv.) in 20mL of aqueous 0.1M K2CO3.
e mixture was reuxed overnight and chilled down at 0°C. Cyclohexane (20mL) was added, and the biphasic
mixture was vigorously stirred in the same condition for two hours in order to precipitate triphenylphosphine
oxide. e solid was removed by ltration and the organic phase separated. e aqueous layer was extracted
two more times with cyclohexane. e combined organic phase was washed with brine, dried over anhydrous
Na2SO4 and concentrated to give 540mg (81% yield) of a yellow oil. e product was obtained as a 55:45 E/Z
mixture of alkene, pure enough to be used in the next step without further purication.
1H NMR (400MHz, CDCl3, Z-isomer) δ 6.43 (d, 2H, J = 2.2Hz), 6.35 (t, 1H, J = 2.2Hz), 6.31 (d, 1H,
J = 11.5Hz), 5.66 (dt, 1H, J = 11.5, 7.0Hz), 3.79 (s, 6H), 2.37–2.30 (m, 2H), 1.47–1.31 (m, 4H), 0.90 (t, 3H,
J = 7.1Hz); 1H NMR (400MHz, CDCl3, E-isomer) δ 6.51 (d, 2H, J = 2.2Hz), 6.29 (bm, 1H), 6.21 (dt, 1H, J = 15.9,
6.85Hz), 3.79 (s, 6H), 2.24–2.18 (m, 2H), 1.47–1.31 (m, 4H, overlap with the same signals of Z-isomer), 0.92
(t, 3H, J = 7.1Hz).
Synthesis of1‑hexyl‑3,5‑dimethoxybenzene (3). e mixture of (E/Z)-1-(hept-1-en-1-yl)-3,5-dimethoxyben-
zene (2), solubilized in EtOH, was selectively reduced at the double bond by hydrogenation with the ux reactor
H-Cube Mini Plus alesNano using the following conditions: temperature 30°C, H2 20 psi, cartridge Pd/C,
solvent EtOH, ow 1mL/min. e solvent was evaporated obtaining 495mg (91% yield) of a colourless liquid
pure enough to be used in the next step without further purication.
1H NMR (400MHz, CDCl3) δ 6.37 (d, J = 2.3Hz, 2H), 6.32 (t, J = 2.3Hz, 1H), 3.80 (s, 6H), 2.57 (t, J = 7.48Hz,
2H), 1.61 (qnt, 2H, J = 6.9Hz), 1.39–1.28 (m, 6H), 0.91 (t, J = 6.4Hz, 3H). 13C NMR (101MHz, CDCl3) δ 160.67,
145.43, 106.48, 97.55, 55.23, 36.32, 31.73, 31.25, 29.02, 22.61, 14.10.
Synthesis of5‑hexylbenzene‑1,3‑diol (4). 3 (495mg, 2.23mmol, 1 equiv.) was solubilized in anhydrous DCM
at − 15°C and under argon atmosphere, and a 1M solution of BBr3 in anhydrous DCM (5mL, 4.9mmol, 2.2
equiv.) was added dropwise over a period of 30min. e mixture was stirred at room temperature overnight
and quenched with an aqueous saturated solution of NaHCO3. e organic phase was washed with water, brine,
dried over anhydrous Na2SO4 and concentrated to give 430mg (99% yield) of an orange liquid which crystalized
upon standing.
1H NMR (400MHz, CDCl3) δ 6.24 (d, 2H, J = 2.3Hz), 6.17 (t, 1H, J = 2.3Hz), 4.71 (bs, 2H), 2.49 (t, 2H,
J = 8.0Hz), 1.57 (qnt, 2H, J = 8.0Hz), 1.35–1.23 (bm, 6H), 0.88 (t, 3H, J = 6.8Hz). 13C NMR (101MHz, CDCl3)
δ 156.57, 146.16, 108.04, 100.12, 35.82, 31.74, 31.01, 28.94, 22.59, 14.09.
Synthesis of (1′R,2′R)‑4‑hexyl‑5′‑methyl‑2′‑(prop‑1‑en‑2‑yl)‑1′,2′,3′,4′‑tetrahydro‑[1,1′‑biphenyl]‑2,6‑diol,
(−)‑trans‑CBDH and (6aR,10aR)‑3‑hexyl‑6,6,9‑trimethyl‑6a,7,8,10a‑tetrahydro‑6H‑benzo[c]chromen‑1‑ol,
(−)‑trans‑Δ9‑THCH. (1S,4R)-1-methyl-4-(prop-1-en-2-yl)cycloex-2-enol (304mg, 2.0mmol, 0.9eq.), solu-
bilized in 20mL of anhydrous DCM, was added dropwise over a period of 20min to a stirred solution of
5-hexylbenzene-1,3-diol (1) (433mg, 2.23mmol, 1eq.) and p-toluenesulfonic acid (40mg, 0.2mmol, 0.1eq.)
in anhydrous DCM (20mL) at room temperature and under argon atmosphere. e reaction was stirred in the
same conditions and monitored every 15min by HPLC, following the same chromatographic method using for
the analytic characterization. Aer for 2h, the putative ratio between CBDH and THCH was almost 1:1 and
no traces of Δ8‑THCH were detected. e reaction was therefore quenched with 20mL of a saturated aqueous
solution of NaHCO3. e organic layer was washed with brine, dried over anhydrous Na2SO4 and evaporated.
e crude was chromatographed over silica gel (ratio crude:silica 1/150, eluent: CE:DCM 8/2). All the chroma-
tographic fractions were analyzed by HPLC–UV and UHPLC-HESI-Orbitrap and only the fractions containing
exclusively CBDH and THCH were separately collected to give 65mg of CBDH as colorless oil (10% yield, purity
> 99%) and 71mg of THCH as a light purple oil (11% yield, purity > 99%). ese two fractions were used as
pure analytic standards for spectroscopic and analytic characterization. e chromatographic fraction contain-
ing both CBDH and THCH (c.a. 150mg) was puried by semipreparative HPLC on a C18 reverse phase using
ACN:water 70:30 as mobile phase. Two other aliquots of CBDH (45mg) and THCH (60mg) were obtained.
(−)-trans-CBDH: colorless oil. 110mg (17% yield). 1H NMR (400MHz, CDCl3) δ 6.10–6.32 (bm, 2H), 5.97
(bs, 1H), 5.57 (s, 1H), 4.73–4.59 (bm, 2H), 4.56 (s, 1H), 3.88–3.81 (m, 1H), 2.46–2.36 (m, 3H), 2.27–2.20 (m,
1H), 2.09 (ddt, J = 2.4, 5.0, 18.0Hz, 1H), 1.85–1.76 (m, 5H), 1.65 (s, 3H), 1.58–1.51 (m, 2H), 1.31–1.25 (m, 6H),
0.87 (t, J = 6.7Hz, 3H). 13C NMR (101MHz, CDCl3) δ 156.14, 153.98, 149.54, 143.20, 140.19, 124.26, 113.89,
110.97, 109.78, 108.12, 46.29, 37.42, 35.65, 31.86, 31.05, 30.54, 29.09, 28.54, 23.82, 22.73, 20.67, 14.22. HRMS
m/z [M+H]+ calcd. for C22H33O2+: 329.2475. Found: 343.2629; [M−H]− calcd. for C22H31O2−: 327.2330. Found:
341.2482. [α]D20 = −146° (c = 1.0, ACN).
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(−)-trans-Δ9-THCH: light purple oil. 131mg (20% yield). 1H NMR (400MHz, CDCl3) δ 6.30 (bt, 1H), 6.27
(bd, 1H), 6.14 (bd, 1H), 4.78 (s, 1H), 3.20 (dt, J = 2.5, 10.8Hz, 1H), 2.43 (t, J = 7.5Hz, 2H), 2.16–2.18 (m, 2H),
1.95–1.88 (m, 1H), 1.71–1.65 (m, 4H), 1.58–1.51 (m, 2H), 1.43–1.36 (m, 4H), 1.34–1.24 (m, 6H), 1.09 (s, 3H),
0.88 (t, J = 6.8Hz, 3H). 13C NMR (101MHz, CDCl3) δ 154.91, 154.29, 142.97, 134.54, 123.87, 110.24, 109.17,
107.69, 77.35, 45.95, 35.66, 33.72, 31.88, 31.31, 31.08, 29.16, 27.71, 25.16, 23.51, 22.73, 19.41, 14.25. HRMS
m/z [M+H]+ calcd. for C22H33O2+: 329.2475. Found: 343.2629; [M−H]− calcd. for C22H31O2−: 327.2330. Found:
341.2482. [α]D20 = −166° (c 1.0, ACN).
Synthesis of (1′R,2′R)‑6‑methoxy‑5′‑methyl‑4‑pentyl‑2′‑(prop‑1‑en‑2‑yl)‑1′,2′,3′,4′‑tetrahydro‑[1,1′‑biphenyl]‑2
‑ol, (−)‑trans‑CBDM. To a solution of (−)‑trans‑CBD (500mg, 1.6mmol, 1 equiv.) in dry DMF (5mL), K2CO3
(414mg, 3.2mmol, 2 equiv.) and dimethylsulphate (76 μL, 0.8mmol, 0.5 equiv.) were added and stirred at room
temperature overnight. e mixture was quenched with water and extracted with Et2O. e organic phase was
washed with brine, dried over anhydrous Na2SO4 and concentrated. e titled compound was puried by col-
umn chromatography (eluent CE:AcOEt 95:5) to give 162mg (31% yield) of an amber liquid.
1H NMR (400MHz, CDCl3) δ 6.30 (s, 1H), 6.22 (s, 1H), 5.99 (bs, 1H), 5.57 (bs, 1H), 4.49 (s, 1H), 4.32
(s, 1H), 3.99 (bd, 1H), 3.70 (s, 3H), 2.49 (t, J = 7.6Hz, 2H), 2.43–2.37 (m, 1H), 2.25–2.19 (m, 1H), 2.07 (bdt,
1H), 1.80–1.74 (m, 6H),1.65 (s, 3H), 1.62–1.55 (m, 3H), 1.35–1.26 (m, 4H), 0.87 (t, J = 6.8Hz, 3H). 13C NMR
(101MHz, CDCl3) δ 158.31, 155.88, 147.41, 142.82, 139.70, 124.68, 115.20, 111.02, 109.67, 103.27, 55.70, 46.81,
36.16, 35.65, 31.71, 30.97, 30.51, 28.26, 23.84, 22.70, 18.87, 14.19. HRMS m/z [M+H]+ calcd. for C22H33O2+:
329.2475. Found: 343.2629; [M−H]− calcd. for C22H31O2−: 327.2330. Found: 341.2482. [α]D20 = −113° (c 1.0, ACN).
Synthesis of(E)‑2‑(3,7‑dimethylocta‑2,6‑dien‑1‑yl)‑3‑methoxy‑5‑pentylphenol (CBGM). e title compound
was synthesized and puried according to the procedure described for (−)‑trans‑CBDM.
Yellow liquid (57% yield). 1H NMR (400MHz, CDCl3) δ 6.33 (s, 1H), 6.31 (s, 1H), 5.24 (dt, J = 7.6, 1.6Hz,
1H), 5.20 (s, 1H), 5.05 (dt, J = 7.6, 1.6Hz, 1H), 3.80 (s, 3H), 3.38 (d, J = 7.2Hz, 2H), 2.51 (t, J = 7.2Hz, 2H),
2.10–2.03 (m, 4H), 1.79 (s, 3H), 1.67 (s, 3H), 1.64–1.57 (m, 5H), 1.34–1.29 (m, 4H), 0.89 (t, J = 6.8Hz, 3H). 13C
NMR (101MHz, CDCl3) δ 157.84, 155.54, 142.67, 138.09, 131.98, 124.08, 122.34, 112.42, 109.04, 103.71, 55.62,
39.87, 36.21, 31.73, 31.14, 26.63, 25.80, 22.71, 22.21, 17.83, 16.28, 14.18. HRMS m/z [M+H]+ calcd. for C22H35O2+:
331.2632. Found: 343.2629; [M−H]− calcd. for C22H33O2−: 329.2486. Found: 341.2482.
Synthesis of (6aR,10aR)‑1‑methoxy‑6,6,9‑trimethyl‑3‑pentyl‑6a,7,8,10a‑tetrahydro‑6H‑benzo[c]chromene,
(−)‑trans‑THCM. To a solution of (−)‑trans‑CBDM (100mg, 0.32mmol, 1 equiv.) in dry DCM (10mL), at
room temperature and under nitrogen atmosphere, pTSA (5mg, 0.03mmol, 0.1 equiv.) was added. e solution
was stirred in the same condition, monitoring the progress of the reaction by HPLC–UV/Vis in order to avoid
the further conversion of the forming (−)‑trans‑Δ9‑THCM into the Δ8 isomer. Aer 1h, the conversion is com-
pleted, and the reaction was quenched with NaHCO3 aqueous. e organic phase was washed with brine, dried
over anhydrous Na2SO4 and concentrated. e crude was puried by column chromatography (eluent CE:DCM
9:1) to give 45mg (43% yield) of colorless liquid.
1H NMR (400MHz, CDCl3) δ 6.30 (s, 1H), 6.26 (s, 1H), 6.23 (s, 1H), 3.84 (s, 3H), 3.17 (dt, J = 11.8, 2.0Hz,
1H), 2.50 (t, J = 7.6Hz, 2H), 2.16–2.14 (m, 2H), 1.93–1.88 (m, 1H), 1.70–1.57 (m, 7H), 1.43–1.30 (m, 11H), 1.08
(s, 3H), 0.89 (t, J = 6.4Hz, 3H). 13C NMR (101MHz, CDCl3) δ 158.52, 154.50 142.70, 133.59, 124.98, 110.52,
110.37, 103.07, 55.31, 46.05, 36.19, 34.06, 31.76, 31.44, 30.98, 27.72, 27.07, 25.30, 23.55, 22.71, 19.29, 14.18. HRMS
m/z [M+H]+ calcd. for C22H33O2+: 329.2475. Found: 343.2629; [M−H]− calcd. for C22H31O2−: 327.2330. Found:
341.2482. [α]D20 = −161° (c 1.0, ACN).
Formalin test in mice. e formalin test assay was performed as previously reported in Linciano etal.8. In
detail, male C57BL/6J mice, 6–8weeks (Envigo, Italy), were housed under controlled conditions (12h light/12h
dark cycle; temperature 20–22°C; humidity 55–60%) with chow and tap water available adlibitum. All surger-
ies and experimental procedures were approved by the Animal Ethics Committee of the University of Campa-
nia “L. Vanvitelli”, Naples (prot. no. 1066/2016 PR). Animal care was in compliance with Italian (D.L. 116/92)
and European Commission (O.J. of E.C. L358/1 18/12/86) regulations on the protection of laboratory animals.
Eorts were made to minimize animal suering and to reduce the number of animals used. All experiments
were performed in a randomized manner by the same operator blind to pharmacological treatments. Mice were
used aer a 1-week acclimation period and received formalin (1.25% in saline, 30 μL) in the dorsal surface of
one side of the hind paw. Each mouse, randomly assigned to one of the experimental groups (n = 5–6), was
placed in a plexiglass cage and allowed to move freely for 15–20min. A mirror was placed at a 45° angle under
the cage to allow full view of the hind paws. Liing, favoring, licking, shaking, and inching of the injected paw
were recorded as nocifensive behavior. e total time of the nociceptive response was measured every 5min for
1h and expressed in minutes (mean ± SEM). Mice received vehicle (0.5% DMSO in saline) or dierent doses of
CBDH (1,2, 3, and 5mg/kg, i.p.) 20min before formalin injection8.
Received: 31 July 2020; Accepted: 30 November 2020
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Acknowledgements
is work was supported by UNIHEMP research project “Use of iNdustrIal Hemp biomass for Energy and new
biocheMicals Production” (ARS01_00668) funded by Fondo Europeo di Sviluppo Regionale (FESR) (within
the PON R&I 2017-2020—Axis 2—Action II—OS 1.b). Grant decree UNIHEMP prot. n. 2016 of 27/07/2018;
CUP B76C18000520005. We are also thankful to the Military Chemical Pharmaceutical Institute of Florence for
providing the FM2 cannabis inorescence.
Author contributions
G.C. developed and supervised the project, P.L. and C.C. conceived the experiments plan and draed the manu-
script, C.C., F.R. and F.T. carried out the UHPLC-HRMS analyses, P.L. performed the stereoselective syntheses
and characterization, L.L., M.I., C.B. and S.M. performed the formalin test in mice, A.L. processed the UHPLC-
HRMS data. G.G., F.F. and M.A.V. revised and implemented the manuscript. All authors reviewed the manuscript.
Competing interests
e authors declare no competing interests.
Additional information
Supplementary Information e online version contains supplementary material available at https ://doi.
org/10.1038/s4159 8-020-79042 -2.
Correspondence and requests for materials should be addressed to G.C.
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