Iodine-Promoted Aromatization of p -Menthane-Type Phytocannabinoids

Article abstract of DOI:10.1021/acs.jnatprod.7b00946

Treatment with iodine cleanly converts various p-menthane-type phytocannabinoids and their carboxylated precursors into cannabinol (CBN, 1a). The reaction is superior to previously reported protocols in terms of simplicity and substrate range, which inclu

Full text of DOI:10.1021/acs.jnatprod.7b00946

Note
pubs.acs.org/jnp
Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
Iodine-Promoted Aromatization of p‑Menthane-Type
Phytocannabinoids
Federica Pollastro,† Diego Caprioglio,† Patrizia Marotta,† Aniello Schiano Moriello,‡
Luciano De Petrocellis,‡ Orazio Taglialatela-Scafati,*,§ and Giovanni Appendino*,†
†Dipartimento di Scienze del Farmaco, Universita
̀
del Piemonte Orientale, Largo Donegani 2, 28100 Novara, Italy
‡Endocannabinoid Research Group, Institute of Biomolecular Chemistry, CNR, Via Campi Flegrei 34, 80078 Pozzuoli (NA), Italy
§Dipartimento di Farmacia, Universita
̀
di Napoli Federico II, Via Montesano 49, 80131 Napoli, Italy
S
* Supporting Information
ABSTRACT: Treatment with iodine cleanly converts various p-
menthane-type phytocannabinoids and their carboxylated precursors
into cannabinol (CBN, 1a). The reaction is superior to previously
reported protocols in terms of simplicity and substrate range, which
includes not only tricyclic tetrahydrocannabinols such as Δ9-THC (2a)
but also bicyclic phytocannabinoids such as cannabidiol (CBD, 3a).
Lower homologues from the viridin series (2c and 3c, respectively)
afforded cannabivarin (CBV), a non-narcotic compound that, when
investigated against a series of ionotropic (thermo-TRPs) biological end-
points of phytocannabinoids, retained the submicromolar TRPA1-
activating and TRPM8-inhibiting properties of CBN, while also potently
activating TRPV2. Treatment with iodine provides an easy access to CBN (1a) from crude extracts and side-cuts of the
purification of Δ9-THC and CBD from respectively narcotic Cannabis sativa (marijuana) and fiber hemp, substantially expanding
the availability of this compound and, in the case of fiber hemp, dissecting it from narcotic phytocannabinoids.
while the biological profile of its lower homologue cannabivarin
(CBV, 1b) is substantially unexplored.
annabinol (CBN, 1a) was first isolated by Easterfield at
1
CCambridge University in 1896 and remained for almost
four decades the only phytocannabinoid available in pure form.
Its tricyclic dibenzopyrane nature was first proposed by Cahn,2
and the full structure was eventually elucidated by Adams at
Urbana−Champaign in 1940.3 Adams also recognized its
artifact nature and its close relationship with the unstable and
more hydrogenated narcotic principle of marijuana, elusive at
that time and only identified two decades later as Δ9-
tetrahydrocannabinol (Δ9-THC, 2a) by Mechoulam in
Jerusalem.4 CBN is a partial agonist of the two cannabinoid
receptors (CB1 and CB2), retaining ca. 10% of the potency of
Δ9-THC for both these end-points.5 It was long considered of
minor interest in terms of bioactivity and mostly relevant as a
marker for the identification of narcotic Cannabis sativa L.
(Cannabaceae) in archeological plant samples due to its
exceptional stability and relationship with Δ9-THC.6
CBN (1a) shows similar polarity and chromatographic
behavior with Δ9-THC, and its purification from aged and
partially degraded marijuana samples is therefore complicated.
For this reason, CBN is more conveniently obtained by
aromatization of Δ9-THC and mixtures of hydrocannabinols.
The reaction was first reported by Adams using sulfur as the
oxidant under relatively harsh conditions (heating neat at ca.
250 °C).10 More recently, chloranyl (tetrachloro-1,4-benzoqui-
none) was found to selectively oxidize Δ9-THC to CBN while
leaving other isomeric tetrahydrocannabinols unaffected,11 and
a dehydrogenation protocol based on refluxing a chloroform
solution of Δ9-THC with selenium dioxide and trimethylsilyl
polyphosphate (prepared from P4O10 and hexamethyldisilox-
ane) was also described.12 We have developed a mild protocol
to convert p-menthane phytocannabinoids, including cannabi-
diol (CBD, 3a), to CBN, extending its availability to non-
narcotic cannabis biomasses, as well as to crude extracts, side-
cuts, and mother liquors from the purification of the major
medicinal phytocannabinoids (Δ9-THC and CBD).
On the other hand, CBN shows a pharmacology that goes
substantially beyond its affinity for cannabinoid receptors.
Thus, CBN shows antibacterial activity on multi-drug-resistant
Staphylococcus aureus (MDRSA) comparable to that of Δ9-
THC (2a) and cannabidiol (CBD, 3a),7 potently activates and
desensitizes TRPA1 in a noncovalent fashion,8 and in animal
experiments induces a remarkable prolongation of sleeping
time.9 Despite its recognized pharmacological potential5 and its
availability in pure form from over a century, CBN (1a)
remains paradoxically underinvestigated in terms of bioactivity,5
Special Issue: Special Issue in Honor of Susan Horwitz
Received: November 9, 2017
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.7b00946
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Note
activator of TRPA1 (EC50 = 0.18 0.02 μM)8 and inhibitor
of TRPM8 (IC50 = 0.21 0.05 μM).8 The activity against these
two targets was fully retained by its lower homologue CBV
(1b) (EC50 = 0.20 0.09 μM for the activation of TRPA1 and
IC50 = 0.50 0.05 μM for the inhibition of TRPM8). On the
other hand, activity on TRPV2 was significantly increased
(EC50 = 3.1 0.10 μM for CBV vs 19 3.7 μM for CBN) and,
to a lesser extenct, also that on TRPV4 (EC50 = 7.3 0.50 μM
for CBV vs 16.1 4.5 μM for CBN). These observations are
interesting, since potent noncovalent activators of TRPA1 are
rare,19 while few modulators of TRPV2, a clinically validated
cardiovascular target,20 have been reported within both natural
and synthetic products.21 Differences for the modulation of
TRPV3 were less significant (EC50 = 3.5 0.60 μM for CBV vs
5.3 2.7 μM for CBN), while both CBN and CBV showed
only marginal activity against the capsaicin receptor TRPV1
(EC50 = 6.6 0.10 μM for CBV vs 6.2 3.7 μM for CBN).
The spectroscopic characterization of CBV is reported in the
Experimental Section. Apart from having two less carbons in
the alkyl chain, no other significant spectroscopic difference
with CBN21 was detected.
A combination of iodine and dichlorodicyanobenzoquinone
(DDQ) was recently reported to aromatize p-menthadienes or
p-menthene alcohols to their corresponding fully aromatic p-
cymene derivatives via an iterative addition−elimination
mechanism and a final oxidative aromatization.13 The high
yields of the reaction prompted us to evaluate the possibility of
applying these conditions also to the aromatization of Δ9-THC
(2a). As a p-menthene derivative, 2a lacks one double bond or
double-bond equivalent to achieve aromatization via the
addition−elimination mechanism operating in p-menthadienes
and p-menthene alcohols. Nevertheless, Δ9-THC shows an
inherent tendency to aromatize, which we hoped to foster
through the agency of the iodine−DDQ pair. In the event,
DDQ turned out to be redundant, and refluxing in toluene with
two equivalents of iodine was sufficient for the spot-to-spot
conversion of Δ9-THC (2a) into CBN (1a). After workup and
chromatographic purification, a rewarding 70% yield was
obtained.
From a mechanistic standpoint, the aromatization of Δ9-
THC, a 2π-system, is somewhat surprising, owing to its low
unsaturation.13 The reaction presumably involves the formation
of an intermediate 4π-system by addition of iodine and the
elimination of two molecules of hydrogen iodide (Scheme 1).
Scheme 1. Possible Mechanism for the Iodine-Induced
Aromatization of Δ9-THC (2a) to CBN (1a) (R = C5H11)
The reaction was also successful with the acidic precursor of
Δ9-THC (THCA-A or pre-THC, 2b), with decarboxylation,
sluggish at this temperature in solvent phase, being presumably
accelerated by the formation of HI in the dehalogenative step.14
Since cannabidiol (3a) is turned by acids into a mixture of
isomeric tetrahydrocannabinols,15 we next wondered if the
aromatization could be directly applied also to CBD (3a). In
the event, an excellent yield was obtained from both CBD (3a)
(72%) and its acidic precursor (3b), telescoping into a single-
step decarboxylation, cyclization, and aromatization. Starting
from the viridins 2c and 3c, the lower homologues of
respectively Δ9-THC and CBD, cannabivarin (CBV, 1b), the
C3 lower homologue of CBN (1a), was obtained. This
compound was first isolated by Markus from a Nepalese
sample of hashish in 1971,16 but has been substantially
overlooked in the literature in terms of both spectroscopic
characterization and bioactivity profile. The paucity of
bioactivity data is surprising, given the current interest in
divarinol (5-propylresorcinol)-type phytocannabinoids as neg-
ative modulators of the endocannabinoid system.17 CBN has
been reported to retain a significant (10%) activity in binding
assays on CB1 and CB2 (EC50 = 75 nM and 73 nM,
respectively),18 while CBV was only marginally active (EC50
= 565 nM and 4780 nM, respectively) in these assays.18 On the
other hand, nothing is known on the activity of CBV on
thermo-TRPs, another major class of phytocannabinoid
targets,8 and it was therefore interesting to compare its activity
to that of CBN, a potent, sub-micromolar, noncovalent
This generates a diene (5) that could then undergo conjugate
1,4-addition of iodine, affording the allyl diiodide 6, which
eventually aromatizes by loss of two further units of HI. The
generation of a strong acid is presumably also responsible for
the cyclization of CBD to THC via a shift of the exocyclic
double bond to an endocyclic location, a reaction similar to that
reported for the conversion of limonene into p-cymene.13
Remarkably, under these acidic conditions, reversible retro-
Friedel-Craft fragmentation of CBD did not take place,15 and
the para-relationship between the alkyl and the isoprenyl
substituents of the resorcinyl core was maintained. Retro-
Friedel-Craft reaction is, indeed, responsible for scrambling of
the relationship between the alkyl and the terpenyl residues,
with the formation of abnormal cannabinoids during the acidic
treatment of the native phytocannabinoids.23 After partition
between petroleum ether and acetonitrile to remove fats and
waxes, the aromatization reaction could also be applied to crude
extracts from fiber hemp without any previous isolation of
purified phytocannabinoids (see Experimental Section). The
reaction with iodine makes it therefore possible to obtain CBN
from non-narcotic C. sativa sources, overcoming the regulatory
B
DOI: 10.1021/acs.jnatprod.7b00946
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Note
phytocannabinoid was dissolved in toluene (100 mL), and iodine
(200 mg) was added. The solution was then refluxed, following the
course of the reaction by TLC (petroleum ether/EtOAc, 9:1; Rf 3d =
0.05, Rf 1b = 0.35). Additional amounts of iodine (100 and 150 mg)
were added after respectively 30 and 40 min. After refluxing for 15 min
from the last addition, the reaction was worked up as described for the
reaction with CBD (3a). Purification by GCC on silica gel with
petroleum ether as eluant afforded 25 mg of CBN (1a)22 and 150 mg
of cannabivarin (1b).
issues associated with obtaining this compound from aged
marijuana extracts or from Δ9-THC. Hopefully, this could
foster studies on the pharmacology and clinical potential of
CBN (1a), the first phytocannabinoid to be isolated and
elucidated structurally.
EXPERIMENTAL SECTION
General Experimental Procedures. IR spectra were registered
on an Avatar 370 FT-IR Techno-Nicolet apparatus. H (500 MHz)
■
1
Cannabivarin (1b): orange oil; H NMR (CD3OD, 500 MHz) δH
1
8.33 (1H, s, H-10), 7.12 (1H, d, J = 7.8 Hz, H-7), 7.01 (1H, d, J = 7.8
Hz, H-8), 6.35 (1H, s, H-4), 6.26 (1H, s, H-2), 2.44 (2H, t, J = 7.5 Hz,
H-1′), 2.34 (3H, s, H-11), 1.62 (2H, sextet, J = 7.5 Hz, H-2′), 1.53
(6H, s, H-12, H-13), 0.94 (3H, t, J = 7.5 Hz, H-3′); 13C NMR (CDCl3,
125 MHz) δC 155.0 (C-1), 154.4 (C-4a), 143.9 (C-3), 143.6 (C-10a),
136.4 (C-6a), 136.0 (C-9), 126.9 (C-8), 126.7 (C-10), 121.8 (C-7),
109.3 (C-4), 108.9 (C-2), 108.6 (C-10b), 76.6 (C-6), 37.4 (C-1′), 26.0
(C-12, C-13), 24.1 (C-2′), 20.1 (C-11), 12.7 (C-3′); ESIMS m/z 305
[M + Na]+; HRESIMS m/z [M + Na]+ 305.1519 (C19H22O2Na
requires 305.1517).
and 13C (125 MHz) NMR spectra were measured on Varian INOVA
NMR spectrometers. Chemical shifts were referenced to the residual
solvent signal (CD3OD: δH = 3.34, δC = 49.0). Homonuclear 1H
connectivities were determined by the COSY experiment. One-bond
heteronuclear 1H−13C connectivities were determined with the HSQC
experiment. Two- and three-bond 1H−13C connectivities were
determined by gradient 2D HMBC experiments optimized for a 2,3J
= 9 Hz. Low- and high-resolution ESIMS were obtained on an LTQ
OrbitrapXL (Thermo Scientific) mass spectrometer.
Reactions were monitored by TLC on Merck 60 F254 (0.25 mm)
plates and visualized by staining with 5% H2SO4 in ethanol and
heating. Organic phases were dried with Na2SO4 before evaporation.
Chemical reagents and solvents were purchased form Sigma-Aldrich
(Germany) and were used without any further purification unless
stated otherwise. Petroleum ether with a boiling point of 40−60 °C
was used. Silica gel 60 (70−230 mesh) used for gravity column
chromatography (GCC) and RP C18 (Nucleodur C18 HTec) used for
vacuum filtration were purchased from Macherey-Nagel. Flash
chromatography was carried out on Biotage SP-1 equipment, using
Biotage Snap Cartrige KP-C18-HS, 60 g (particle size 25 μm). For all
fractions obtained with Biotage SP-1, a liquid−liquid partition between
EtOAc/brine and anhydrification with Na2SO4 was applied before
complete evaporation.
Plant Material. The CBDV (3c)-rich Cannabis sativa strain used in
this study was obtained from a greenhouse cultivation at CRA-CIN,
Rovigo (Italy), where a voucher specimen is kept, and was supplied by
Dr. Gianpaolo Grassi (CRA-CIN, Rovigo, Italy). The manipulation of
the plant material and of the narcotic phytocannabinoids was done in
accordance with their legal status (Authorization SP/101 of the
Ministero della Salute, Rome, Italy).
Isolation of Cannabidivarin (3c, CBDV). Dried aerial parts of C.
sativa (flowers and leaves, 156 g) were extracted with acetone (3 × 2
L) in a 10 L vertical percolator. Evaporation of the solvent left a black
gum (11.6 g, 7.4%). The latter was then dissolved with heating (40
°C) in the minimal amount of methanol and then charged on a bed of
35 g of RP-C18 (ratio extract/RP-C18 silica gel 1:3) packed with
methanol in a sintered filtration funnel (9 × 15 cm) with vacuum side
arm. Washing with methanol (200 mL) under a vacuum gave 9.8 g of a
dark green gum, which was decarboxylated by heating at 130 °C under
stirring. Fractionation by GCC on silica gel (400 g, 20 mL fractions)
by using a petroleum ether/EtOAc gradient (from 90:10 to 20:80)
gave 2.71 g of crude CBDV, which was purified by flash column
chromatography on a Snap Cartrige KP-C18-HS (60 g, 15 mL volume
fractions) using a methanol/acidic water (phosphate buffer, pH = 3)
gradient, from 70:30 to 95:05. Crystallization with ether afforded 3c
(963 mg) as a white powder.24
Thermo-TRPs (TRPV1, TRPV2, TRPV3, TRPV4, TRPM8,
TRPA1) Receptor Assays. HEK-293 cells stably overexpressing
recombinant rat TRPA1, TRPM8, TRPV2-4, and TRPM8 or human
TRPV1 were selected by G-418 (geneticin; 600 μg mL−1), grown on
100 mm diameter Petri dishes as monolayers in minimum essential
medium supplemented with nonessential amino acids, 10% fetal
bovine serum, and 2 mM glutamine, and maintained under 5% CO2 at
37 °C. Stable expression of each channel was checked by quantitative
real-time PCR. The effect of the substances on intracellular Ca2+
concentration ([Ca2+]i) was determined using Fluo-4, a selective
intracellular fluorescent probe for Ca2+. Toward this aim, on the day of
the experiment, cells overexpressing the TRP channels were loaded for
1 h in the dark at room temperature with the methyl ester Fluo4-AM
(4 μM in dimethyl sulfoxide containing 0.02% Pluronic F-127,
Invitrogen) in minimum essential medium without fetal bovine serum.
After the loading, cells were washed twice in Tyrode’s buffer (145 mM
NaCl, 2.5 mM KCl, 1.5 mM CaCl2, 1.2 mM MgCl2, 10 mM D-glucose,
and 10 mM HEPES, pH 7.4), resuspended in Tyrode’s buffer, and
transferred (about 100 000 cells) to the quartz cuvette of the
spectrofluorimeter (PerkinElmer LS50B; PerkinElmer Life and
Analytical Sciences, Waltham, MA, USA) under continuous stirring.
[Ca2+]i was determined before and after the addition of various
concentrations of test compounds by measuring cell fluorescence at 25
°C (λEX = 488 nm, λEM = 516 nm). Curve fitting (sigmoidal dose−
response variable slope) and parameter estimation were performed
with GraphPad Prism (GraphPad Software Inc., San Diego, CA, USA).
Potency was expressed as the concentration of test substances exerting
a half-maximal agonist effect (i.e., half-maximal increases in [Ca2+]i
(EC50), calculated by using GraphPad). The efficacy of the agonists
was first determined by normalizing their effect to the maximum Ca2+
influx effect on [Ca2+]i observed with application of 4 μM ionomycin
(Sigma). The increases in fluorescence in wild-type HEK293 cells (i.e.,
not transfected with any construct) were used as a baseline and
subtracted from the values obtained from transfected cells. The effects
of TRPA1 agonists are expressed as a percentage of the effect obtained
with 100 μM allyl isothiocyanate (AITC). In the case of TRPM8, the
experiments were carried out at 22 °C with a Fluorescence Peltier
System (PTP-1, PerkinElmer). Antagonist/desensitizing behavior was
evaluated against capsaicin (0.1 μM) for TRPV1, icilin (0.25 μM) for
TRPM8, AITC (100 μM) for TRPA1, lysophosphatidylcholine (3
μM) for TRPV2, and 10 nM GSK1016790A for TRPV4 by adding the
compounds in the quartz cuvette 5 min before stimulation of cells with
agonists. In the case of TRPV3, rat TRPV3-expressing HEK-293 cells
were first sensitized with the nonselective agonist 2-aminoethox-
ydiphenyl borate (100 μM). Antagonist/desensitizing behavior was
evaluated against thymol (100 μM). Data are expressed as the
concentration exerting a half-maximal inhibition of agonist [Ca2+]i
increasing effect (IC50), which was calculated again using GraphPad
Prism software. The effect on [Ca2+]i exerted by the agonist alone was
taken as 100%. All determinations were at least performed in triplicate.
Aromatization of p-Menthane Cannabinoids: Reaction of
CBD (3a) and with a CBDV (3c)-Rich Crude Extract as
Exemplificative. Reaction with CBD (3a). To a solution of CBD
(3a, 100 mg, 0.32 mmol) in toluene (20 mL) was added iodine (162
mg, 0.64 mmol, 2 molar equiv). The solution was refluxed, following
its course by TLC (petroleum ether/EtOAc, 9:1, Rf 3a = 0.55, Rf 1a =
0.45). After 60 min, the reaction was worked up by cooling to room
temperature and sequentially washed with 5% Na2S2O3 and brine.
After drying, the organic phase was evaporated, and the residue was
purified by GCC on silica gel with petroleum ether as eluant to afford
72 mg (72%) of 1a22 as a pale yellow oil.
Reaction with a CBDV (3c)-Rich Extract. The plant material was
extracterd and defatted as described for the isolation of CBDV. The
crude extract (2 g) containing CBDVA (3d) as its major
C
DOI: 10.1021/acs.jnatprod.7b00946
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Journal of Natural Products
Note
Statistical analysis of the data was performed by analysis of variance at
each point using ANOVA followed by Bonferroni’s test.
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ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.7b00946.
1H and 13C NMR spectra for CBV (1b) and efficacy,
potency, and IC50 values for the activity of 1b toward
thermo-TRPs (PDF)
AUTHOR INFORMATION
Corresponding Authors
*Tel: +39-081-678509. Fax: +39-081678552. E-mail: scatagli@
unina.it (O. Taglialatela-Scafati).
■
*Tel: +39-0321-375744. Fax: +39-0321375744. E-mail:
giovanni.appendino@uniupo.it (G. Appendino).
ORCID
Orazio Taglialatela-Scafati: 0000-0001-8010-0180
Giovanni Appendino: 0000-0002-4170-9919
Notes
(13) Domingo, V.; Prieto, C.; Silva, L.; Rodilla, J. M.; Quílez del
Moral, J. F.; Barrero, A. F. J. Nat. Prod. 2016, 79, 831−837.
(14) Veress, T.; Szanto, J. I.; Leisztner, L. J. Chromatogr. 1990, 520,
339−347.
(15) Adams, R.; Cain, K.; McPhee, D.; Wearn, R. B. J. Am. Chem. Soc.
1941, 63, 2009−2013.
(16) Markus, F. W. H. M. Nature 1971, 232, 679−680.
(17) McPartland, J. M.; Duncan, M.; Di Marzo, V.; Pertwee, R. G. Br.
J. Pharmacol. 2015, 172, 737−753.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
(18) Husni, A. S.; McCurdy, C. R.; Radwan, M. M.; Ahmed, S. A.;
Slade, D.; Ross, S. A.; ElSohly, M. A.; Cutler, S. J. Med. Chem. Res.
2014, 23, 4295−4300.
■
We are grateful to C. Monti and A. Cuadari for their help in the
isolation of phytocannabinoids from plant sources and their
help throughout this work, to Dr. G. Grassi (CREA, Rovigo)
for supplying the Cannabis strain used for the isolation of
CBDV, and to Vivacell Spain for financial support.
(19) Del Prete, D.; Caprioglio, D.; Appendino, G.; Minassi, A.;
Schiano-Moriello, A.; Di Marzo, V.; De Petrocellis, L. Bioorg. Med.
Chem. Lett. 2015, 25, 1009−1011.
(20) Matsumura, T.; Matsui, M.; Iwata, Y.; Asakura, M.; Saito, T.;
Fujimura, H.; Sakoda, S. Intern. Med. 2017, DOI: 10.2169/internal-
medicine.8651-16.
DEDICATION
■
Dedicated to Dr. Susan Band Horwitz, of Albert Einstein
College of Medicine, Bronx, NY, for her pioneering work on
bioactive natural products.
(21) Korolkova, Y.; Makarieva, T.; Tabakmakher, K.; Shubina, L.;
Kudryashova, E.; Andreev, Y.; Mosharova, I.; Lee, H. S.; Lee, Y. J.;
Kozlov, S. Mar. Drugs 2017, 15, 87−97.
(22) Choi, Y. H.; Hazekamp, A.; Peltenburg-Looman, A. M.;
Fred
Anal. 2004, 15, 345−354.
(23) Hanus, L. O.; Meyer, S. M.; Munoz, E.; Taglialatela-Scafati, O.;
Appendino, G. Nat. Prod. Rep. 2016, 33, 1357−1392.
(24) Turner, C. E.; Cheng, P. C.; Lewis, G. S.; Russell, M. H.;
Sharma, G. K. Planta Med. 1979, 37, 217−225.
́ ́
erich, M.; Erkelens, C.; Lefeber, A. W.; Verpoorte, R. Phytochem.
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