O-Methyl Phytocannabinoids: Semi-synthesis, Analysis in Cannabis Flowerheads, and Biological Activity

Article abstract of DOI:10.1055/a-0883-5383

A general protocol for the selective mono-O-methylation of resorcinyl phytocannabinoids was developed. The availability of semisynthetic monomethyl analogues of cannabigerol, cannabidiol, and cannabidivarin (1a-3a, respectively) made it possible to quanti

Full text of DOI:10.1055/a-0883-5383

Original Papers
O-Methyl Phytocannabinoids: Semi-synthesis, Analysis in Cannabis
Flowerheads, and Biological Activity*
Authors
Diego Caprioglio1, Gianna Allegrone1, Federica Pollastro1, Stefano Valera1, Annalisa Lopatriello2, Juan A. Collado3,
Eduardo Munoz3, Giovanni Appendino1, Orazio Taglialatela-Scafati2
Affiliations
Correspondence
1
2
3
Dipartimento di Scienze del Farmaco, Università del
Piemonte Orientale, Novara, Italy
Prof. Giovanni Appendino
Dipartimento di Scienze del Farmaco, Università del
Piemonte Orientale
Dipartimento di Farmacia, Università di Napoli Federico II,
Napoli, Italy
Largo Donegani 2, 28100 Novara, Italy
Phone: + 390321375744, Fax: + 390321375744
giovanni.appendino@uniupo.it
Maimonides Biomedical Research Institute of Córdoba;
Department of Cellular Biology, Physiology and Immunol-
ogy, University of Córdoba; University Hospital Reina Sofía,
Córdoba, Spain
ABSTRACT
A general protocol for the selective mono-O-methylation of
resorcinyl phytocannabinoids was developed. The availability
of semisynthetic monomethyl analogues of cannabigerol,
cannabidiol, and cannabidivarin (1a–3a, respectively) made
it possible to quantify these minor phytocannabinoids in
about 40 different chemotypes of fiber hemp. No chemotype
significantly accumulated mono-O-methyl cannabidiol (2b) or
its lower homologue (3b), while at least three chemotypes
containing consistent amounts (≥ 400 mg/kg) of O-methyl-
cannabigerol (1b) were identified. O-Methylation of alkyl phy-
tocannabinoids (1b–3b) does not significantly change the ac-
tivity on peroxisome proliferator-activated receptors in con-
trast to what was reported for phenethyl analogues.
Key words
Cannabis sativa, Cannabaceae, meroterpenoids,
phytocannabinoids, O‑methylation, PPAR
received February 1, 2019
revised
March 18, 2019
accepted March 20, 2019
Bibliography
DOI https://doi.org/10.1055/a-0883-5383
Published online | Planta Med © Georg Thieme Verlag KG
Stuttgart · New York | ISSN 0032‑0943
Correspondence
Prof. Orazio Taglialatela-Scafati
Dipartimento di Farmacia, Università di Napoli Federico II
Via Montesano 49, 80131 Napoli, Italy
Phone: + 39081678509, Fax: + 39081678552
scatagli@unina.it
penyl moiety, but a certain degree of diversity is also present in
the resorcinyl core. Thus, in addition to the acidic precursors [1]
of neutral phytocannabinoids and their terpenyl esters [3], canna-
binoquinoids [4] and O-methyl phytocannabinoids have also been
reported [5,6].
The mono-O-methyl derivatives of CBG (1a) (▶ Fig. 1) and CBD
(2a) (▶ Fig. 1) were first isolated as Beam test negative constitu-
ents of a Japanese chemotype of C. sativa L. (Minamishihara num-
ber 1) [5, 6], and were next also identified as trace constituents of
Introduction
Phytocannabinoids, the hallmark secondary metabolites of Can-
nabis (Cannabis sativa L., Cannabaceae), are meroterpenoids re-
sulting from the convergence of mevalonate and polyketide path-
ways [1]. Their structure is based on a resorcinyl core para-substi-
tuted with a monoterpenyl and pentyl groups, although com-
pounds with a different prenyl moiety (e.g., sesquiterpenyl [1, 2])
or with a shortened alkyl group (methyl, propyl, or, more rarely,
ethyl and butyl) are also present in the plant [1], and a recent re-
view has listed almost 150 Cannabis phytocannabinoids [1]. This
impressive chemodiversity is mostly the result of changes (cycli-
zations, rearrangements, oxidations, additions, etc.) in the ter-
*
Dedicated to Professor Dr. Cosimo Pizza 70th birthday in recognition of
his outstanding contribution to natural product research.
Caprioglio D et al. O-Methyl Phytocannabinoids: Semi-synthesis, … Planta Med

Original Papers
ABBREVIATIONS
CB1-CB2
CBD
cannabinoid receptors 1 and 2
cannabidiol
CBDV
CBG
cannabidivarin
cannabigerol
GCC
gravity column chromatography
limit of quantitation
LOQ
▶ Fig. 1 Chemical structures of CBG (1a), CBD (2a), and CBDV (3a)
and their corresponding O-methyl analogues (1b–3b).
PE
petroleum ether
PPARs
TBAF
peroxisome proliferator-activated receptors
tetrabutylammonium fluoride
tBDMS‑Cl t-butyldimethylsilyl chloride
TMSCHN2 trimethylsilyldiazomethane
TRP
transient receptor potential
some Western chemotypes of Cannabis [1]. Just like their resor-
cinyl analogues, O-methyl CBG (1b) and O-methyl CBD (2b) are
not narcotic [5,6], but little is known about their biological profile,
especially their activity on other targets of the biological space of
phytocannabinoids, which includes not only metabotropic recep-
tors (CB1 and CB2), but also ionotropic receptors (thermo-TRPs),
transcription factors (PPAR-α and -γ), and various enzymes in-
volved in the metabolism of eicosanoids and endocannabinoids
[7]. Our interest for this class of minor phytocannabinoids was fos-
tered by the observation that mono-O-methylation of phenethyl
phytocannabinoids (amorfrutinoids) promotes PPAR-γ activity
[8], and a similar increase of potency was observed for the inhibi-
tion of 15-lipooxygenase (15-LO) when the monomethyl ether of
CBD (2b) was compared to its parent resorcinol (CBD, 2a) [1].
There is growing evidence that the phytocannabinoid structural
motif is a privileged structure for bioactivity [1], and we have
therefore developed a semi-synthesis of mono-O-methyl phyto-
cannabinoids to gain further information about their biological
profile and to quantify their occurrence in various chemotypes of
fiber hemp.
▶ Fig. 2 Chemical conversion of CBG (1a) into the corresponding
O-methyl analogue (1b). A parallel procedure was also applied to
CBD (2a) and CBDV (3a).
served that the formation of these compounds could not be
avoided even with substoichiometric amounts of methylating re-
agents [5, 9]. This observation can be rationalized considering
that the electron-donating properties of the O-alkyl methyl seem-
ingly increase the reactivity of the other resorcinyl hydroxyl, mak-
ing the monoalkylated product more reactive than the starting di-
phenol. The selective mono-O-methylation observed for the alkyl
esters of acidic phytocannabinoids can be related to the presence
of a strong intramolecular hydrogen bonding between the ester
carbonyl and the ortho-hydroxyl group, with a resulting decrease
of reactivity compared to the non-hydrogen-bonded hydroxyl
group [9].
In sharp contrast with the methylation, silylation with the bulky
reagent tBDMS‑Cl was highly chemoselective, exclusively provid-
ing the mono-protected resorcinols 1c–3c from 1a–3a, respec-
tively (▶ Fig. 2). “Slenderer” silylating agents like triethylsilylchlor-
ide and trimethylsilyl chloride were less selective, providing, to-
gether with the O-monosilylated derivatives, the major reaction
products, and also significant amounts of the O-disilylated ana-
logues. Methylation with TMSCHN2 [10] of 1c–3c cleanly afforded
1d–3d (▶ Fig. 2), and subsequent deprotection (TBAF) was un-
eventful, providing 1b–3b in an excellent overall yield (▶ Fig. 2).
The NMR spectra of the mono-methylated phytocannabinoids
1b–3b were fully assigned by using 2D experiments and are listed
in the Materials and Methods section. The most evident effect of
O-methylation on NMR resonances was the expected downfield
shift experienced by the O-methylated carbons compared to the
OH- linking ones. Moreover, O-methylation could increase the
steric hindrance to the rotation of the phenyl-cyclohexenyl bond
in 2b/3b compared to 2a/3a. Thus, in addition to the obvious elec-
tronic effects, this could explain the slight splitting in the proton
resonances of H-3 and H-5 (δ 6.22 and 6.20, in CD3OD) in 2b/3b
Results and Discussion
Although Cannabis produces almost 150 phytocannabinoids [1],
many of them are only available in minute amounts by isolation,
therefore, synthesis or semi-synthesis are the only option to try
to advance their medicinal potential. Over the years, we have iso-
lated the monomethyl ethers of CBG (1a), CBD (2a), and CBDV
(3a) from some European chemotypes of fiber hemp in yields un-
attractive to sustain a medicinal chemistry effort (maximum
40 mg/kg). To increase the availability of these compounds, we
decided to investigate the selective monomethylation of their cor-
responding, and much more easily available, resorcinol analogues
[CBG (1a), CBD (2a), and CBDV (3a)] (▶ Fig. 1). The methylation
of phytocannabinoids has already been investigated previously
[5, 9], but a general and chemoselective protocol for the mono-
methylation is still lacking. Indeed, treatment with diazomethane
or other common methylating agents (methyl iodide, dimethyl-
sulfate) and bases affords reaction mixtures containing major, or
exclusive, amounts of the dimethylated products. It has been ob-
Caprioglio D et al. O-Methyl Phytocannabinoids: Semi-synthesis, … Planta Med

compared to their coincidence in the non-methylated 2a/3a (δ
6.07, in CD3OD) [11].
3a show a symmetry plane bisecting the resorcinyl hydroxyls, it is
tempting to assume that a change in one hydroxyl can be com-
pensated, at least for the interaction with PPARs, by the other
one. On the other hand, in phenethyl phytocannabinoids, the
presence of an aromatic ring in the alkyl substituent could bias
the conformation of the resorcinyl ring, desymmetrizing, in terms
of bioactivity, the two phenolic hydroxyls.
In conclusion, O-methylation is the most common modifica-
tion of the resorcinyl core of phytocannabinoids, and seems more
common in compounds, like CBG (1a), having a non-cyclized iso-
prenyl residue compared with compounds like CBD (2a) where
this moiety is cyclized. A systematic survey of about 40 cultivars
identified that a couple of them produce considerable amounts
of 1b. Overall, the concentration of O-methyl phytocannabinoids
in Cannabis flowerheads is highly variable and of chemotaxonom-
ic relevance for a better characterization of Cannabis cultivars.
From a biological standpoint, O-methylation of alkyl phytocanna-
binoids does not significantly alter the activity on PPARs, in con-
trast to what has been reported for phenethyl analogues.
The availability of reference standards of O-methyl cannabi-
noids made it possible to quantify their presence in a wide selec-
tion of chemotypes of fiber hemp registered in the EU or under
development in breeding centers. Because of this, plant samples
(flowerheads) were extracted with dichloromethane, and the
crude extracts were directly analyzed by GC‑MS using tribenzyl-
amine as the internal standard. This procedure was designed to
exclude any extraction/chromatographic artifact formation of
the O-methyl derivatives of the phytocannabinoids. Cannabis
samples contain mixtures of native phytocannabinoids (acidic
phytocannabinoids), and their decarboxylated version (neutral
phytocannabinoids) formed on storage, but, under GC conditions,
acidic cannabinoids (= pre-cannabinoids) are decarboxylated,
thus simplifying the analysis. CBG (1a) and CBD (2a) are typical
constituents of fiber chemotypes of Cannabis (hemp), and we
comparatively analyzed the concentration of these compounds
and their O-methyl derivatives in a series of chemotypes regis-
tered in the EU (Eletta Campana, Fibranova, Tiborszallasi,
Bialobrzeskie, Beniko) or under development in breeding centers
(▶ Table 1). All these plants accumulated significant amounts of
CBD (samples 1–8), CBDV (samples 9–23), or CBG (samples 24–
29), but none of them accumulated monomethyl phytocannabi-
noids as the major constituent. Nevertheless, some chemotypes
contained significant (> 100 mg/kg) amounts of O-methyl phyto-
cannabinoids, with the highest concentration (490 mg/kg) ob-
served in the chemotype GBC-1.
O-Methylation of phytocannabinoids presumably occurs via an
S-adenosylmethionine transfer reaction and is more common in
phenethyl phytocannabinoids compared to alkyl phytocannabi-
noids. Nothing is known on the enzyme(s) responsible for this ac-
tivity, but CBG seems to be a better substrate than CBD, since
higher amounts of the O-methyl derivative occur with the former
compared to the latter, while phytocannabinoids devoid of two
free resorcinyl hydroxyls, like Δ9-THC and CBC, do not seem to be
substrates for this enzymatic activity, since O-methyl derivatives
of these compounds have never been reported as natural prod-
ucts.
Very little is known about the biological profile of O-methyl
phytocannabinoids from C. sativa. Their parent compounds [CBG
(1a), CBD (2a) and CBDV (3a)] do not significantly modulate the
activity of cannabinoid receptors, but they can, nevertheless,
modulate the endocannabinoid system by allosteric modulation
of the receptors (CBD) [7] or by interaction with the enzymes in-
volved in the synthesis and degradation of endocannabinoids [7].
In addition, CBG is also the electron-donating properties powerful
antagonist of the menthol receptor TRPM8 [12], while both com-
pounds can also modestly interact with the transcription factor
PPAR-γ. Since we had enough O-monomethyl derivative phyto-
cannabinoids (1b–3b) on hand, we tested them on this endpoint,
revealing that the activity remained modest and not significantly
different from the parent compounds 1a–3a. Similar results were
obtained for the interaction with PPAR-α. These observations are
in contrast with data reported for phytocannabinoid analogues
showing a phenethyl group in place of the alkyl one, for which O-
methylation increased the potency toward PPAR-γ [8]. Since 1a–
Materials and Methods
General experimental procedures
IR spectra were registered on an Avatar 370 FT‑IR Techno-Nicolet
apparatus. 1H (500 MHz) and 13C (125 MHz) NMR spectra were
measured on Varian INOVA 500 MHz NMR spectrometers. Chemi-
cal shifts were referenced to the residual solvent signal (CD3OD:
1
δH = 3.34, δC = 49.2). Homonuclear H connectivities were deter-
1
mined by the COSY experiment. One-bond heteronuclear H-13
C
connectivities were determined with the HSQC experiment. Two-
and three-bond 1H-13C connectivities were determined by gra-
dient 2D HMBC experiments optimized for a 2,3J = 9 Hz. Low- and
high-resolution ESIMS were obtained on an LTQ OrbitrapXL (Ther-
mo Scientific) mass spectrometer.
Reactions were monitored by TLC on Merck 60 F254 (0.25 mm)
plates, visualized by staining with 5% H2SO4 in ethanol and heat-
ing. Organic phases were dried with Na2SO4 before evaporation.
Chemical reagents and solvents were purchased form Sigma-Al-
drich and were used without any further purification unless stated
otherwise. PE with a boiling point between 40–60°C was used.
Silica gel 60 (70–230 mesh) used for GCC was purchased from
Macherey-Nagel.
O-Silyl cannabinoids: synthesis of O-tert-butyldimer-
thylsilyl cannabigerol (1c) as exemplificative
To a stirred solution of CBG (1a, 1.19 g, 3.8 mmol) in dry CH2Cl2
(10 mL), imidazole (1.02 g, 15.0 mmol, 4 mol. equivalents) and
tBDMS‑Cl (1 M in CH2Cl2, 4.5 mL, 4.5 mmol, 1.2 mol. equivalents)
were sequentially added. The solution was stirred at room tem-
perature overnight, then quenched by the addition of 2 M H2SO4
(20 mL) and CH2Cl2 (10 mL). The organic layer was washed with
brine (3 × 10 mL) then dried, filtered, and evaporated. The residue
was purified by GCC on silica gel (PE/CH2Cl2 9:1 as the eluent) to
afford 1c as a brown oil (667 mg, 52%). Under the same condi-
tions, the yield was 54% for O-tBDMS-cannabidiol (2c) and 66%
for O-tBDMS-cannabivarin (3c).
Caprioglio D et al. O-Methyl Phytocannabinoids: Semi-synthesis, … Planta Med

Original Papers
▶ Table 1 Concentration of mono-O-methyl phytocannabinoids in different chemotypes of fiber hemp.
N°
Samples
Chemotype
1b
2b
3b
µg/g SD
µg/g SD
µg/g SD
1
2
Kompolti
Ferimon
Fedora17
Felina 32
Epsilon 68
Futura 75
Charmaeleon
Markant
Ivor
CBD
125.35 10.21
5.42 0.43
9.52 0.76
6.38 0.35
< LOQ
15.55 1.32
3.54 0.16
2.46 0.17
< LOQ
nd
CBD
nd
3
CBD
nd
4
CBD
nd
5
CBD
nd
nd
6
CBD
nd
nd
nd
7
CBD
6.71 0.44
< LOQ
nd
nd
8
CBD
< LOQ
nd
9
CBD
< LOQ
nd
nd
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
Delta Ilosa
Carma monoica
Carmagnola
Carmaleonte
Denise
CBD
12.29 0.20
15.92 1.11
20.14 1.00
nd
< LOQ
nd
CBD
2.29 0.15
2.38 0.14
nd
nd
CBD
nd
CBD
nd
CBD
nd
< LOQ
nd
Uso 31
CBD
nd
< LOQ
nd
Eletta Campana
Fibranova
Fibrol
CBD
293.31 15.59
157.93 0.52
23.43 1.31
40.67 3.43
62.89 4.89
195.31 9.44
< LOQ
50.32 0.28
475.25 5.42
5.84 0.22
19.73 1.31
22.81 2.02
84.16 0.16
< LOQ
nd
CBD
nd
CBD
nd
Tiborszallasi
KC Dora
Monoica
Bialorbrzeskie
Beniko
CBD
nd
CBD
nd
CBD
nd
CBD
nd
CBD
< LOQ
2.28 0.11
81.81 1.51
30.14 1.69
84.22 4.21
14.69 0.58
101.88 4.11
175.23 2.59
12.75 0.61
57.28 1.17
69.35 1.66
64.69 0.19
24.68 0.22
84.96 2.31
100.72 2.51
2.00 0.05
nd
GBC-1
CBDV
CBDV
CBDV
CBDV
CBDV
CBDV
CBDV
CBD
491.91 1.78
68.11 2.41
333.61 2.60
57.37 2.83
339.70 6.90
16.20 0.22
20.22 0.73
209.97 5.33
469.00 27.8
177.37 6.03
83.27 1.41
400.15 6.68
197.98 3.35
31.21 0.52
26.98 0.92
7.20 0.27
48.71 0.29
4.46 0.18
19.66 0.24
4.62 0.10
4.87 0.14
7.53 0.08
18.28 0.40
9.71 0.14
9.13 0.10
18.01 0.20
19.01 0.28
< LOQ
GBC-2
GBC-3
GBC-4
GBC-5
GBC-6
GBC-7
GBC-8
GBC-9
CBDV
CBDV
CBDV
CBDV
CBDV
CBG
GBC-10
GBC-11
GBC-14
GBC-16
GBC-18
nd = not detected
O-Methyl-O-silyl cannabinoids: synthesis of
O-tert-butyldimethylsilyl-O-methylcannabigerol (1d)
as exemplificative
(20 mL) and CH2Cl2 (20 mL). The organic layer was washed with
brine (3 × 10 mL) dried, filtered, and evaporated. The residue was
purified by GCC on silica gel (PE as the eluent) to afford 1d as a
colorless oil (589 mg, 98%). Under the same conditions, the yield
was also > 90% for 2-O-tBDMS‑6-OMe-cannabidiol (2d) and 2-O-
tBDMS‑6-OMe-cannabivarin (3d).
To a stirred solution of 1c (500 mg, 1.2 mmol) in methanol (5 mL),
trimethylsilyl diazomethane (2 M in ether, 11.6 mL, 23.2 mmol,
20 mol. equivalents) was added. The solution was stirred at room
temperature overnight, and then quenched with sat. NaHCO3
Caprioglio D et al. O-Methyl Phytocannabinoids: Semi-synthesis, … Planta Med

Final desylyation. Synthesis of O-methylcannabigerol
Analysis of O-methylcannabinoids
(1b) as exemplificative
in Cannabis flowerheads
To a stirred solution of 1d (500 mg, 1.1 mmol) in tetrahydrofuran
(THF) (5 mL), acetic acid (65 µL, 1.1 mmol, 1 mol. equivalent) and
TBAF were (1 M in THF, 1.1 mL, 1.1 mmol, 1 mol. equivalent) se-
quentially added. The solution was stirred at room for 10 min,
and then quenched with EtOAc (20 mL) and brine (20 mL). The or-
ganic layer was washed with brine (2 × 10 mL), dried, and evapo-
rated. The residue was purified by GCC on silica gel (PE:EtOAc
95:5 as the eluent) to afford 1b as a pale yellow oil (352 mg,
94%). Under the same conditions, the yield was also > 90% for 2b
and 3b.
Powdered plant material (1.0 g) was extracted with CH2Cl2
(2 × 10 mL). After evaporation to constant weight, a sample of
the extract (5.0 mg) was diluted with CH2Cl2 (500 µL) and a solu-
tion of tribenzylamine (100 mg/mL) was added as the internal
standard. GC analysis was carried out on a Trace GC apparatus
coupled to a Polaris Q ion trap mass spectrometer (Thermo Finni-
gan). The gas chromatograph was operated in split mode using a
1-µL injection with the injector set and was maintained at 270°C.
Helium was used as the carrier gas at a flow rate of 1.0 mL/min.
The separation was performed on a TG-5MS capillary column
(30 m, 0.25 mm I. D., 0.25 mm thickness) (Thermo Fisher Scien-
tific). The oven column temperature was programmed as follow:
the initial temperature of 150°C was maintained for 2 min and
was next increased from 150 to 270°C at a rate of 5°C/min, even-
tually staying at 270°C for 15 min. Electron ionization was
operated at 70 eV. The transfer line and ion source were kept at
270°C and 250°C respectively. The MS was used in full scan (33–
350 m/z) and tandem MS/MS. The selected parent/daughter ions
transitions were: m/z 287 → 210 for 1b, m/z 245 → 188 and m/z
245 → 174 for 2b, and m/z 287 → 196 for the internal standard.
Calibration curves were 2–2000 ppm for 1b and 0.1–100 ppm for
2b. The LOQ was 1 ppm for 1b and 0.1 ppm for 2b. The quantity
(µg) of analyte per gram of each sample was calculated based on
the concentration of analytes in the extracts, derived by interpola-
tion with their respective calibration curves.
O-Methylcannabigerol (1b): Pale yellow oil. [α]D = 0 (c = 0.2 in
CH3OH). IR (KBr) cm−1 3429, 1616, 1588, 1451, 1424, 1212,
1163, 1099; 1H NMR (CD3OD, 400 MHz) δ 6.26 (2H, s, H-3–5),
5.17 (1H, t, 7.2 Hz H-2′), 5.05 (1H, t, 6.8 Hz H-6′), 3.75 (3H, s,
OMe), 3.25 (2H, d, 7.2 Hz, H-1′), 2.48 (2H, t, 7.5 Hz H-1′′), 2.04
(2H, q, 7.7 Hz, H-5′), 1.93 (2H, t, 7.7 Hz, H-4′), 1.74 (3H, s, H-
10′), 1.60 (3H, s, H-8′), 1.60 (3H, s, H-9′), 1.59 (2H, overlapped,
H-2′′), 1.34 (4H, m, H-3′′-4′′), 0.91 (3H, t, 6.9 Hz H-5′′). 13C NMR
(CD3OD, 100 MHz): δ 159.6 (C-6), 151.5 (C-2), 142.6 (C-4), 134.4
(C-3′), 131.9 (C-7′), 125.5 (C-6′), 124.9 (C-2′), 115.0 (C-1), 109.2
(C-5), 103.7 (C-3), 56.0 (OMe-6), 40.9 (C-4′), 37.1 (C-1′′), 32.7
(C-3′′), 32.4 (C-2′′), 27.7 (C-5′), 25.9 (C-9′), 23.6 (C-1′), 22.9 (C-
4′′), 17.7 (C-8′), 16.2 (C-10′), 14.4 (C-5′′). HR-ESIMS: m/z [M–H]−
329.2469 (calcd. for C22H33O2, 329.2475).
O-Methylcannabidiol (2b): Pale yellow oil. [α]2D5 = + 110 (c = 0.2
1
in CH3OH). H NMR (CD3OD, 500 MHz) δ 6.22 (1H, s, H-5), 6.20
Peroxisome proliferator-activated receptor
activity assays
(1H, s, H-3), 5.21 (1H, s, H-2′), 4.40 (2H, s, H-9′), 3.93 (1H, d,
9.8 Hz, H-3′), 3.68 (3H, s, -OCH3), δ 2.89 (1H, td, 10.7 Hz,
4.7 Hz, H-4′), 2.45 (2H, t, 7.7 Hz, H-1′′), 2.19 (1H, m, H-6′a),
1.99 (1H, bd, 16.5 Hz, H-6′b), 1.73 (2H, m, H-5′), 1.66 (3H, s, H-
10′), 1.60 (3H, s, H-7′), 1.57 (2H, m, H-2′′), 1.33 (4H, m, H-3′′-4′
′), 0.90 (3H, t, 6.7 Hz, H-5′′); 13C NMR (CD3OD, 125 MHz) δ 159.6
(C-6), 156.5 (C-2), 150.4 (C-8′), 142.9 (C-4), 127.4 (C-2′), 112.0
(C-1), 110.4 (C-9′), 109.9 (C-5), 105.4 (C-3), 56.1 (OMe), 46.5
(C-4′), 37.4 (C-3′), 37.0 (C-1′′), 32.6 (C-3′′), 32.1 (C-2′′), 31.7
(C-5′), 30.8 (C-6′), 23.7 (C-7′), 23.6 (C-4′′), 19.4 (C-10′), 14.4
(C-5′′). HRESIMS m/z [M–H]− 327.2321 (calcd. for C22H31O2,
327.2324).
Human embryonic kidney epithelial cells 293 T cells were ob-
tained from the American Type Culture Collection (CRL-3216)
and cultured in DMEM supplemented with 10% fetal calf serum
and antibiotics. To analyze PPAR transcriptional activities, HEK-
293 T cells were cultured in 24-well plates (2 × 104 cells/well)
and transiently co-transfected with either GAL4-PPARγ (50 ng) or
GAL4-PPARα (50 ng) vectors together with the luciferase reporter
vectors GAL4-luc (Firefly luciferase) (50 ng) and pRL‑CMV (Renilla
luciferase) (100 ng) using Roti-Fect (Carl Roth). Twenty hours after
transfection, the cells were stimulated with increasing concentra-
tions of the compounds (1, 5, 10, 25, and 50 µM) for 6 h and lucif-
erase activities were quantified using a Dual-Luciferase Assay
(Promega). Rosiglitazone (1 µM) (Cayman Chemical) was used as
a positive control for PPARγ activation (50-fold induction over ba-
sal activity) and WY14643 (5 µM) (Tocris Bioscience) was used as a
positive control for PPARα activation (60-fold induction over basal
activity). Test compounds and control stocks were prepared in
DMSO, and the final concentration of the solvent was always less
than 0.5% vol/vol. The plasmids GAL4-PPARγ and GAL4-PPARγ
were obtained from Prof. Christopher Sinal (Dalhousie University,
Canada).
O-Methylcannabivarin (3b): Pale yellow oil. [α]2D5 = + 102 (c = 0.2
in CH3OH). IR (KBr) cm−1 3433, 1615, 1589, 1456, 1429, 1211,
1
1168, 1101 H NMR (CD3OD, 500 MHz) δ 6.22 (1H, s, H-5), 6.20
(1H, s, H-3), 5.21 (1H, s, H-2′), 4.40 (2H, s, H-9′), 3.93 (1H, d,
9.8 Hz, H-3′), 3.68 (3H, s, -OCH3), δ 2.89 (1H, td, 10.7 Hz,
4.7 Hz, H-4′), 2.44 (2H, t, 7.7 Hz, H-1′′), 2.18 (1H, m, H-6′a),
1.99 (1H, bd, 16.5 Hz, H-6′b), 1.74 (2H, m, H-5′), 1.66 (3H, s, H-
10′), 1.60 (3H, s, H-7′), 1.60 (2H, overlapped, H-2′′), 0.92 (3H, t,
7.7 Hz, H-3′′); 13C NMR (CD3OD, 125 MHz) δ 159.6 (C-6), 156.5
(C-2), 150.4 (C-8′), 142.7 (C-4), 127.4 (C-2′), 112.0 (C-1), 110.4
(C-9′), 109.9 (C-5), 105.4 (C-3), 56.1 (OMe), 46.5 (C-4′), 39.1 (C-
1′′), 37.4 (C-3′), 31.7 (C-5′), 30.8 (C-6′), 25.5 (C-2′′), 23.7 (C-7′),
19.4 (C-10′), 14.1 (C-3′′). HRESIMS m/z [M–H]− 299.2007 (calcd.
for C20H27O2, 299.2011).
Acknowledgements
We are grateful to MIUR for financial support to the groups in Novara
and Naples (PRIN2017, Project 2017WN73PL, Bioactivity-directed
exploration of the phytocannabinoid chemical space).
Caprioglio D et al. O-Methyl Phytocannabinoids: Semi-synthesis, … Planta Med

Original Papers
[6] Shoyama Y, Kuboe K, Nishioka I, Yamaguchi T. Cannabidiol monomethyl
ether. A new neutral cannabinoid. Chem Pharm Bull 1972; 20: 2072
Conflict of Interest
[7] Izzo AA, Borrelli F, Capasso R, Di Marzo V, Mechoulam R. Non-psycho-
tropic plant cannabinoids: new therapeutic opportunities from an an-
cient herb. Trends Pharmacol Sci 2009; 10: 515–527
The authors declare no conflict of interest.
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