Cannabimovone, a cannabinoid with a rearranged terpenoid skeleton from hemp

Article abstract of DOI:10.1002/ejoc.200901464

An investigation of the polar fractions from a nonpsychotropic variety of hemp (Cannabis sativa L.) afforded cannabimovone (6), a polar cannabinoid with a rearranged 2(3→4) abeo-terpenoid skeleton, biogenetically originating from the intramolecular aldolization of a 2′,3′-seco-menthanyl precursor. The structure of cannabimovone was elucidated by spectroscopic analysis, whereas attempts to mimic its biogenetic derivation from cannabidiol (2) gave only anhydrocannabimovone (12), the intramolecular oxy-Michael adduct of the crotonized version (11b) of the elusive natural products. Biological evaluation of cannabimovone (6) against metabotropic (CB₁, CB ₂) and ionotropic (TRPs) cannabinoid receptors showed a significant activity only for ionotropic receptors, especially TRPV1, whereas anhydrocannabimovone (12) exhibited strong activity at both, ionotropic and metabotropic cannabinoid receptors. Overall, the biological profile of anhydrocannabimovone (12) was somewhat similar to that of THC (4), suggesting a remarkable tolerance to constitutional and configurational changes.

Full text of DOI:10.1002/ejoc.200901464

FULL PAPER
DOI: 10.1002/ejoc.200901464
Cannabimovone, a Cannabinoid with a Rearranged Terpenoid Skeleton from
Hemp
Orazio Taglialatela-Scafati,*[a] Alberto Pagani,[b] Fernando Scala,[a]
Luciano De Petrocellis,[c] Vincenzo Di Marzo,[d] Gianpaolo Grassi,[e] and
Giovanni Appendino*[b]
Keywords: Hemp / Phytocannabinoids / Aldol reactions / Biomimetic synthesis / Cannabinoid receptors / Terpenoids
An investigation of the polar fractions from a nonpsycho- logical evaluation of cannabimovone (6) against meta-
tropic variety of hemp (Cannabis sativa L.) afforded cannabi- botropic (CB1, CB2) and ionotropic (TRPs) cannabinoid re-
movone (6), a polar cannabinoid with a rearranged 2(3Ǟ4)
abeo-terpenoid skeleton, biogenetically originating from the
intramolecular aldolization of a 2Ј,3Ј-seco-menthanyl precur-
sor. The structure of cannabimovone was elucidated by spec-
troscopic analysis, whereas attempts to mimic its biogenetic
ceptors showed a significant activity only for ionotropic re-
ceptors, especially TRPV1, whereas anhydrocannabimovone
(12) exhibited strong activity at both ionotropic and meta-
botropic cannabinoid receptors. Overall, the biological pro-
file of anhydrocannabimovone (12) was somewhat similar to
derivation from cannabidiol (2) gave only anhydrocannabi- that of THC (4), suggesting a remarkable tolerance to consti-
movone (12), the intramolecular oxy-Michael adduct of the
crotonized version (11b) of the elusive natural products. Bio-
tutional and configurational changes.
pericyclic cyclization [cannabichromene (CBC, 3)]. Further
elaboration involves formal intramolecular cyclization
either in a prototropic [tetrahydrocannabinol (THC, 4)] or
in a pericyclic way (cannabicyclol, 5).[4]
Introduction
With an inventory of over 500 secondary metabolites
identified so far, Cannabis sativa L. is one of the phyto-
chemically best characterized plant species.[1] The biomedi-
cal and agricultural relevance of hemp undoubtedly under-
lies the wealth of data on its constituents and their bio-
logical activities,[2] and cannabinoids, a class of unique mero-
terpenoids derived from the alkylation of olivetol with a
monoterpene unit, are the most typical constituents of can-
nabis.[3] Over 100 different cannabinoids have been de-
scribed,[1] basically derived from the geranyl derivative can-
nabigerol (CBG, 1, Scheme 1) either by oxidative proto-
tropic cyclization [cannabidiol (CBD, 2)] or by oxidative
[a] Dipartimento di Chimica delle Sostanze Naturali, Università di
Napoli Federico II,
Via Montesano 49, 80131 Napoli, Italy
Fax: +39-081-678552
E-mail: scatagli@unina.it
[b] Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche
e Farmacologiche, Università del Piemonte Orientale,
Viale Ferrucci 33, 28100 Novara, Italy
Fax: +39-0321-375621
E-mail: appendino@pharm.unipmn.it
[c] Endocannabinoid Research Group, Institute of Cybernetics,
Consiglio Nazionale delle Ricerche,
Scheme 1. Biogenetic relationships of the major phytocannabi-
noids.
Pozzuoli (NA), Italy
[d] Endocannabinoid Research Group, Institute of Biomolecular
Chemistry, Consiglio Nazionale delle Ricerche,
Pozzuoli (NA), Italy
Most phytocannabinoids are derived from the dehydro-
genation or oxidative modification of these five primary ter-
penoid skeletons, so structural variation within these com-
pounds is limited.[1]
[e] CRA-CIN,
Viale G. Amendola 82, 45100 Rovigo, Italy
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.200901464.
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O. Taglialatela-Scafati, G. Appendino et al.
FULL PAPER
In past centuries, the unmatched excellence of some Ital- Table 1. 13C (125 MHz) and 1H (500 MHz) NMR data for cannabi-
ian varieties of fiber hemp was recognized worldwide.[5] Re-
movone (6) in CDCl3.
markably, there is only limited phytochemical information
available for these plants, which have so far been investi-
gated mostly for the presence of THC and some of the
major cannabinoids.[5] While investigating the phenolic
fraction from an industrial Italian cultivar of hemp rich in
CBD (2), we isolated a cannabinoid with an unprecedented
abeo-menthane terpenoid structure.[6] We named this com-
pound cannabimovone (6, Figure 1), and here we present
details on its structure elucidation, its attempted biomime-
tic preparation from CBD (2), and its biological profile
against a series of metabotropic and ionotropic phytocan-
nabinoid targets.[7]
Position
δH (mult., J in [Hz])
δC
1/5
2/4
3
6
1Ј
2Ј
3Ј
4Јa
4Јb
5Ј
155.4 (C)
109.0 (CH)
144.1 (C)
110.0 (C)
48.4 (CH)
77.9 (CH)
58.0 (CH)
31.2 (CH2)
6.18 (s)
3.52 (dd, 11.3, 8.9)
4.73 (dd, 8.9, 6.8)
3.03 (ddd, 11.2, 6.8, 6.8)
2.13 (m)
2.04 (m)
3.35 (ddd, 11.3, 11.3, 9.2)
45.3 (CH)
146.3 (C)
110.4 (CH2)
6Ј
7Јa
7Јb
8Ј
4.71 (br. s)
4.63 (br. s)
1.70 (s)
20.0 (CH3)
211.1 (C)
9Ј
10Ј
1ЈЈ
2ЈЈ
3ЈЈ
4ЈЈ
5ЈЈ
2.27 (s)
2.41 (t, 7.6)
1.55 (m)
1.30 (m)
1.27 (m)
29.5 (CH3)
35.5 (CH2)
30.7 (CH2)
31.5 (CH2)
22.7 (CH2)
14.3 (CH3)
0.87 (t, 7.0)
spectrum associated the two singlets at δH = 4.71 and
4.63 ppm with the same carbon at δC = 110.4 ppm, im-
plying the presence of an olefinic methylene moiety.
Figure 1. Structure of cannabimovone (6).
Results and Discussion
Cannabimovone (6, Figure 1) was isolated as an optically
active, light yellow, amorphous solid from a polar fraction
of an acetone extract of a CBD-rich nonpsychotropic vari-
ety of C. sativa derived from the ancient Italian variety
“Carmagnola”.[5] Its ESI (positive ions) mass spectrum ex-
hibited pseudomolecular ions at m/z 347 ([M + H]+) and
369 ([M + Na]+). These data, along with 1D NMR spectro-
scopic data, pointed to the molecular formula C21H30O4,
Figure 2. COSY (in bold) and key HǞC HMBC correlations ob-
served in cannabimovone (6).
The 2D NMR HMBC spectrum (see Figure 2) provided
indicative of seven degrees of unsaturation, as confirmed by critical information with which the structure of cannabimo-
HREIMS. The 1H NMR spectrum of 6 (500 MHz, CDCl3, vone (6) could be pieced together. In particular, H3-10Ј
2,3
Table 1) displayed two relatively downfield-shifted methyl showed
J
cross-peaks with the ketone carbonyl and
H,C
singlets (δH = 1.70 and 2.27 ppm) and a methyl triplet at δH with C-3Ј, showing the presence of an acetyl group on the
= 0.87 ppm. In addition to these signals, the proton spec- cyclopentane ring. An isopropenyl group was also linked to
trum was completed by a 2H sharp singlet at δH = 6.18 ppm the five-membered ring on the basis of the HMBC cross-
and two 1H broad singlets at δH = 4.71 and 4.63 ppm, four peaks of H3-8Ј with C-5Ј, C-6Ј, and C-7Ј. Finally, cross-
multiplets between δH = 3.00 and 4.80 ppm, and a series of peaks of H-1Ј with C-6 and with the oxygen-bearing carbon
signals in the δH = 1.20–2.50 ppm region. The 13C NMR atoms C-1/C-5 (δC = 155.4 ppm) and of H-1ЈЈ with C-3 and
spectrum of 6 (125 MHz, CDCl3, Table 1) showed 19 dis- with the protonated C-2/C-4 (δC = 109.0 ppm) aromatic
tinct resonances, including a ketone carbonyl at δC
=
pair completely defined the planar structure of cannabimo-
211.1 ppm and six signals in the sp2 region. Both the pres- vone (6).
ence of a 2H singlet at δH = 6.18 ppm in the 1H NMR
The five-membered ring of this compound features four
spectrum and the overlap of two sets of carbon resonances stereogenic centers, the relative orientation of which was as-
in the 13C NMR spectrum could be explained by the pres- sessed on the basis of the 2D NMR ROESY spectrum. The
ence of a symmetrically tetrasubstituted phenyl ring.
ROESY cross-peaks of H-3Ј/H-1Ј and H-2Ј/H-5Ј indicated
The combined inspection of 2D NMR COSY and HSQC the cis orientation of these protons. The spatial proximity
spectra indicated two spin systems (highlighted in bold in of H-3Ј with H-7Ј indicated their trans relative orientation.
Figure 2): the first one was a typical Cannabis phenyl-linked
A plausible biogenetic derivation of cannabimovone be-
pentyl moiety, whereas the second was attributed to a tetra- gins with CBD (2), the major constituent of the extract,
substituted cyclopentyl moiety bearing an oxymethine car- and involves stereoselective dihydroxylation of the endo-
bon (δH = 4.73 ppm, δC = 77.9 ppm). Moreover, the HSQC cyclic double bond, followed by oxidative cleavage of the
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Hemp Cannabinoid with a Rearranged Terpenoid Skeleton
Scheme 2. Attempted biomimetic synthesis of cannabimovone.
glycol system and stereoselective aldolization of the re- groups. Periodate cleavage of 8a afforded the keto-aldehyde
sulting dicarbonyl intermediate (Scheme 2). In view of the 9, which proved rather resistant to aldolization, presumably
plausibility of this scheme, it was assumed that the absolute because of steric strain in its aldolization product 10, in
configuration of 6 should be the same as that of its precur- which the cyclopentane ring features four adjacent substitu-
sor.
ents. Despite considerable experimentation, only the cro-
Only minute amounts of cannabimovone (6) were ob- tonized analogue of the natural product (11a) could be ob-
tained by isolation, and so it was attempted to synthesize tained in acidic medium, whereas under basic conditions
cannabimovone from its easily available putative precursor. (pyrrolidine) crotonization was accompanied by loss of the
CBD was degraded during dihydroxylation, due to the high phenolic acetates and eventual formation of the intramolec-
sensitivity of its resorcinyl moiety to oxidation,[8] but pro- ular oxy-Michael adduct 12 (anhydrocannabinovone) as the
tection of the two phenolic hydroxy groups made the reac- only reaction product. The Michael addition was com-
tion manageable. Excellent chemoselectivity has been re- pletely stereoselective, affording exclusively the isomer with
ported in the epoxidation of CBD,[9] and we were therefore a trans relationship between the oxygen bridge and the ace-
confident that the dihydroxylation of its acetate (7) could tyl group. The fast Michael trapping of 11b was further
proceed with preferential attack of the endocyclic double attested to by the impossibility of deacetylation of the en-
bond. Considerable efforts were in fact required to steer one 11a without formation of the tricyclic compound 12.
dihydroxylation chemoselectively to the endocyclic double Eventually, under both acidic and basic conditions, isola-
bond, and the reaction course was strongly dependent on tion of the natural product from the aldolization mixture
the protocol employed.
remained elusive. While not disproving the biogenetic pro-
The best results in terms of chemoselectivity were ob- posal for the formation of cannabimovone (6), these results
served with the Upjohn protocol (cat. OsO4/NMMO), point to the existence of considerable steric tension in the
whereas the asymmetric dihydroxylation (AD-mix-α or natural product, consistently with the surprising stability of
AD-mix-β) was poorly selective, affording almost equi- the keto-aldehyde 9 toward aldolization. Compound 12
molar mixtures of the endocyclic and the exocyclic diols. could not be isolated from the extract that yielded 6, tes-
Conversely, excellent stereoselectivity was observed in the tifying to the mildness of the isolation scheme employed
attack at the endocyclic double bond, with exclusive attack during the fractionation step.
trans to the olivetyl moiety.[10] The diol 8a was surprisingly
Phytocannabinoids can interact with a host of meta-
stable with respect to acyl migration, an event that would botropic (CB1 and CB2) and ionotropic (TRPV1, TRPM8,
have required remodulation of the synthesis, with a change and TRPA1) targets[7] and in view of the structural novelty
from ester to ether protection for the phenolic hydroxy of cannabimovone (6) it was interesting to assess its profile
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O. Taglialatela-Scafati, G. Appendino et al.
FULL PAPER
of activity toward these end-points. The cyclized analogue
(anhydrocannabimovone, 12) and the oxidized p-menthane
precursor (dihydroxy-CBD, 8b) of the natural product were
also investigated. Cannabimovone (6) and dihydroxy-CBD
(8b) were devoid of significant affinity for either CB1 or
CB2 (Ki Ͼ 10 µ) receptors, but anhydrocannabimovone
(12) showed high affinity for both receptors (Ki ca.
100 nM). On the other hand, cannabimovone (6) was a full,
albeit weak, TRPV1 agonist (EC50 = 6.4 µ), more potent
than 8b (EC50 = 17.1 µ) and more efficacious than 12. It
also activated TRPA1 (EC50 = 6.3 µ). In comparison, 12
was more potent (EC50 = 1.7 µ) and efficacious toward
Experimental Section
General: Optical rotations (CHCl3) were measured at 589 nm with
a Perkin–Elmer 192 polarimeter with a sodium lamp (λ = 589 nm)
and a 10 cm microcell. 1H (500 MHz) and 13C (125 MHz) NMR
spectra were measured with a Varian INOVA spectrometer. Chemi-
cal shifts were referenced to the residual solvent signal (CDCl3: δH
1
= 7.26 ppm, δC = 77.0 ppm). Homonuclear H connectivities were
determined by COSY experimentation. One-bond heteronuclear
1H–13C connectivities were determined by HSQC experiments.
Through-space 1H connectivities were determined by use of a
ROESY experiment with a mixing time of 500 ms. Two- and three-
1
bond H–13C connectivities were determined by means of gradient
2D HMBC experiments optimized for 2,3J = 9 Hz. Low- and high-
resolution EIMS spectra (70 eV) were performed with a VG Pro-
spec (Fisons) mass spectrometer. ESIMS spectra were performed
with a LCQ Finnigan MAT mass spectrometer. Silica gel 60 (70–
230 mesh) was used for gravity column chromatography. Reactions
were monitored by TLC on Merck plates (60 F254, 0.25 mm), which
were visualized by UV inspection and/or staining with H2SO4 in
ethanol (5%) and heating. Organic phases were dried with Na2SO4
before evaporation.
TRPA1, whereas 8b exhibited weaker potency (EC50
=
15.4 µ) and efficacy than the natural product. Finally, of
the three compounds investigated, only 12, like several
plant cannabinoids,[7] exhibited significant functional an-
tagonist activity at TRPM8 channels (IC50 = 1.83 µ).
Taken together, these observations suggest that cannabimo-
vone (6) has a biological profile similar to that of its bioge-
netic precursor CBD, with modest affinity for metabotropic
cannabinoid (CB1, CB2) receptors and relatively good ac-
tivity at TRPV1. On the other hand, anhydrocannabimo-
vone (12) exhibited potent cannabinoid activity at both
CB1 and CB2 receptors, as well as at TRPA1 channels and
TRPM8, mimicking to some extent the biological profile of
THC (4).[7] Indeed, as highlighted in Scheme 3, the tricyclic
cannabinoid skeleton of anhydrocannabimovone (12) shows
Plant Material: C. sativa, derived from a greenhouse cultivation at
CRA-CIN, Rovigo (Italy), where a voucher specimen is kept, was
collected in November 2008. The isolation and manipulation of
all cannabinoids was done in accordance with their legal status
(Authorization SP/101 of the Ministero della Salute, Rome, Italy).
Extraction and Isolation of Cannabimovone (6): Dried flowerheads
of C. sativa (500 g) were heated at 120 °C in a ventilated oven for
a certain structural similarity with THC (4), suggesting a 2.5 h to decarboxylate pre-cannabinoids. After cooling to room
temp., the plant material was extracted with acetone (2ϫ10 L). Re-
moval of the solvent left a gummy residue that was partitioned
between 1:1 aqueous methanol (1 L) and petroleum ether (1 L).
The defatted polar phase was concentrated and extracted with
CH2Cl2. The organic phase was dried (Na2SO4) and evaporated to
afford a black gum (10 g), which was purified by flash chromatog-
raphy on RP-18 silica gel (Biotage equipment, 250 mL column, lin-
ear gradient, from methanol water 55:45 to 90:10). Overall, five
fractions were collected. The more polar one was further fraction-
ated by gravity column chromatography on silica gel, with use of
acidified (0.5% HOAc) petroleum ether/EtOAc mixtures. After
four chromatographic steps, crude cannabimovone (10 mg) was ob-
tained from a fraction directly eluted after the stilbenoid canni-
prene. The crude fraction was further purified by HPLC (eluent
n-hexane/EtOAc 7:3) to provide pure cannabimovone (6, 7.0 mg,
14 ppm based on dried plant material).
surprising preservation of the biological profile through
changes of constitution and configuration.
Cannabimovone (6): Colorless, amorphous solid. [α]2D2 = –10 (c =
0.07, CHCl3). 1H NMR (500 MHz, CDCl3): see Table 1. 13C NMR
(125 MHz, CDCl3): see Table 1. ESI-MS: m/z = 347 [M + H]+, 369
[M + Na]+. HREIMS calcd. for C21H30O4 [M]+ 346.2144; found
346.2148.
Scheme 3. Structural similarity between anhydrocannabimovone
(12) and THC (4).
Diacetylcannabidiol (7): Acetic anhydride (188.0 µL, 6 equiv.) and
DMAP (19.5 mg, 0.5 equiv.) were added to a solution of cannabi-
diol (2, 100 mg, 0.32 mmol) in pyridine (300 µL). After having been
stirred at room temperature for 16 h, the reaction mixture was
worked up with methanol (200 µL) to destroy excess acetic anhy-
dride, and washed five times with H2SO4 (2 ) and with brine. The
organic phase was dried with Na2SO4 and filtered, and the solvents
were evaporated. The residue was purified by column chromatog-
raphy on silica gel (hexane/EtOAc 98:2) to afford 1,5-diacetylcan-
nabidiol (7, 54 mg, 0.14 mmol, 44% yield) as a viscous, colorless
Conclusions
Cannabis is one of the most thoroughly investigated
plants in terms of secondary metabolites,[1] and the isola-
tion of a cannabinoid of a novel skeletal type highlights
the potential of modern techniques to expand the current
inventory of natural products substantially, affording, even
from well known “old” plants, new chemotypes useful to
explore uncharted areas of the druggable space.
1
oil. H NMR (500 MHz, CDCl3): δH = 6.86, 6.82 (2ϫs, 2 H, 2-H,
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Hemp Cannabinoid with a Rearranged Terpenoid Skeleton
4-H), 5.56 (br. s, 1 H, 2Ј-H), 4.67 (br. s, 1 H, 9Јa-H), 4.61 (br. s, 1
H, 9Јb-H), 3.92 (m, 1 H, 1Ј-H), 3.20 (m, 1 H, 6Ј-H), 2.51 (m, 2 H,
1ЈЈ-H), 2.32 (s, 6 H, 2ϫOAc), 2.21 (m, 2 H, 4Ј-H), 1.84 (m, 2 H,
5Ј-H), 1.80 (s, 3 H, 7Ј-H), 1.68 (s, 3 H, 10Ј-H), 1.56 (q, J = 7.6 Hz,
2 H, 2ЈЈ-H), 1.30 (overlapped signals, 4 H, 3ЈЈ-H, 4ЈЈ-H), 0.88 (t, J
= 7.0 Hz, 3 H, 5ЈЈ-H) ppm. ESI-MS: m/z = 421 [M + Na]+.
0.23 mmol) in THF (1.0 mL). After having been stirred at room
temperature for 12 h, the reaction mixture was worked up by di-
lution with CH2Cl2 and sequential washing with satd. NaHCO3
and brine. The organic phase was dried with Na2SO4, filtered, and
concentrated, and the residue was purified by gravity column
chromatography on silica gel (hexane/EtOAc 9:1 as eluent) to af-
ford 11a (43 mg, 0.11 mmol, 48% yield) as a yellowish, amorphous
solid. [α]2D2 = –52 (c = 0.10, CHCl3). 1H NMR (500 MHz, CDCl3):
δH = 6.75 (s, 2 H, 2-H, 4-H), 6.55 (d, J = 1.5 Hz, 1 H, 2Ј-H), 4.74
(s, 1 H, 7Јa-H), 4.73 (s, 1 H, 7Јb-H), 4.16 (dd, J = 7.5, 1.5 Hz, 1
H, 1Ј-H), 3.18 (ddd, J = 1.5, 7.5, 7.3, 2.5 Hz, 1 H, 5Ј-H), 2.91 (dd,
J = 12.1, 7.3 Hz, 1 H, 4Јa-H), 2.56 (overlapped signal, 1 H, 4Јb-
H), 2.55 (overlapped signal, 2 H, 1ЈЈ-H), 2.30 (s, 3 H, 10Ј-H), 2.17
(s, 6 H, 2ϫOAc), 1.70 (s, 3 H, 8Ј-H), 1.60 (m, 2 H, 2ЈЈ-H), 1.32
(m, 4 H, 3ЈЈ-H, 4ЈЈ-H), 0.88 (t, J = 7.3 Hz, 3 H, 5ЈЈ-H) ppm. 13C
NMR (125 MHz, CDCl3): δC = 196.9 (s, 9Ј-C), 169.2 (s, 2ϫOAc),
150.1 (s, 1-C, 5-C), 146.1 (s, 6Ј-C), 144.2 (s, 3-C), 143.7 (s, 3Ј-C),
123.9 (d, 2Ј-C), 123.7 (d, 2-C, 4-C), 121.0 (s, 6-C), 111.6 (t, 7Ј-C),
52.8 (d, 5Ј-C), 48.2 (d, 1Ј-C), 36.2 (t, 4Ј-C), 35.7 (t, 1ЈЈ-C), 31.7 (t,
3ЈЈ-C), 31.0 (t, 2ЈЈ-C), 29.2 (q, 1Ј-C), 23.8 (t, 4ЈЈ-C), 20.5 (q, 8Ј-C),
20.4 (2ϫOAc), 14.3 (q, 5ЈЈ-C) ppm. ESI-MS: m/z = 435 [M +
Na]+.
1,5-Diacetyl-2Ј,3Ј-dihydroxycannabidiol (8a): OsO4 (cat., 25.4 µL of
a 5.0% toluene solution) and N-methylmorpholine oxide (NMMO,
117 mg, 4 mol. equiv.) were added to a solution of 1,5-diacetylcan-
nabidiol (7, 100 mg, 0.25 mmol) in acetone/water (4:1, 800 µL). Af-
ter the system had been stirred at room temperature for 6 h, further
N-methylmorpholine oxide (117 mg, 4 equiv.) was added, and after
6 h the reaction mixture was worked up by dilution with EtOAc
and sequential washing with H2SO4 (2 ), satd. NaHCO3, and
brine. After drying (Na2SO4), the organic phase was filtered and
concentrated, and the residue was purified by gravity column
chromatography on silica gel (hexane/EtOAc 9:1Ǟ8:2 as eluent)
to afford 1,5-diacetyl-2Ј,3Ј-dihydroxycannabidiol (8a, 54 mg,
0.12 mmol, 50% yield) as an amorphous, white solid. 1H NMR
(500 MHz, CDCl3): δH = 6.87, 6.83 (2ϫs, 2 H, 2-H, 4-H), 4.68 (br.
s, 1 H, 9Јa-H), 4.62 (br. s, 1 H, 9Јb-H), 3.67 (br. d, J = 8.5 Hz, 1
H, 2Ј-H), 3.20 (t, J = 8.5 Hz, 1 H, 1Ј-H), 2.60 (ddd, J = 8.5, 8.5,
2.1 Hz, 1 H, 6Ј-H), 2.55 (t, J = 7.0 Hz, 2 H, 1ЈЈ-H), 2.40 (s, 3 H,
OAc), 2.36 (s, 3 H, OAc), 1.84 (m, 1 H, 5Јa-H), 1.68 (s, 3 H, 10Ј-
H), 1.65 (m, 1 H, 5Јb-H), 1.50 (m, 2 H, 2ЈЈ-H), 1.40 (m, 1 H, 4Јa-
H), 1.36 (overlapped signals, 1 H, 4Јb-H), 1.32 (m, 4 H, 3ЈЈ-H, 4ЈЈ-
H), 1.29 (s, 3 H, 7Ј-H), 0.88 (t, J = 7.3 Hz, 3 H, 5ЈЈ-H) ppm. ESI-
MS: m/z = 453 [M + Na]+.
Anhydrocannabimovone (12): Pyrrolidine (60 µL, 13 equiv.) was
added to a solution of 11a (100 mg, 0.24 mmol) in CH2Cl2
(1.0 mL). After having been stirred at room temperature for 12 h,
the reaction mixture was worked up by dilution with brine and
extraction with CH2Cl2. The organic phase was dried with Na2SO4,
filtered, and concentrated, and the residue was purified by gravity
column chromatography on silica gel (hexane/EtOAc 9:1 as eluent)
to yield 12 (63 mg, 80%) as a yellow, amorphous solid. [α]2D2 = –17
(c = 0.02, CHCl3). 1H NMR (500 MHz, CDCl3): δH = 6.15 (s, 1
H, 4-H), 6.11 (s, 1 H, 2-H), 5.54 (dd, J = 3.5, 3.5 Hz, 1 H, 2Ј-H),
Compound 8a was deacetylated by treatment with 10 equimolecu-
lar of pyrrolidine in CH2Cl2, to afford 8b (80% yield) as an
1
amorphous solid. H NMR (500 MHz, CDCl3): δH = 6.28 (br. s, 2
H, 2-H, 4-H), 4.66 (br. s, 1 H, 9Јa-H), 4.61 (br. s, 1 H, 9Јb-H), 3.69
(br. d, J = 8.5 Hz, 1 H, 2Ј-H), 3.16 (t, J = 8.5 Hz, 1 H, 1Ј-H), 2.61 4.87 (s, 1 H, 7Јa-H), 4.71 (s, 1 H, 7Јb-H), 4.68 (br. s, 1 H, OH),
(ddd, J = 8.5, 8.5, 2.1 Hz, 1 H, 6Ј-H), 2.47 (t, J = 7.0 Hz, 2 H, 1ЈЈ- 3.93 (dd, J = 3.5, 1.5 Hz, 1 H, 1Ј-H), 3.23 (m, 1 H, 3Ј-H), 2.85 (br.
H), 1.87 (m, 1 H, 5Јa-H), 1.71 (s, 3 H, 10Ј-H), 1.62 (m, 1 H, 5Јb- s, 1 H, 5Ј-H), 2.46 (d, J = 7.0 Hz, 2 H, 1ЈЈ-H), 2.32 (s, 3 H, 10Ј-
H), 1.51 (m, 2 H, 2ЈЈ-H), 1.41 (m, 1 H, 4Јa-H), ca. 1.35 (m, 1 H,
4Јb-H), 1.32 (m, 4 H, 3ЈЈ-H, 4ЈЈ-H), 1.26 (s, 3 H, 7Ј-H), 0.89 (t, J
= 7.3 Hz, 3 H, 5ЈЈ-H) ppm. ESI-MS: m/z = 369 [M + Na]+.
H), 2.07 (m, 1 H, 4Јa-H), 1.84 (s, 3 H, 8Ј-H), 1.81 (m, 1 H, 4Јb-
H), 1.54 (m, 2 H, 2ЈЈ-H), 1.29 (m, 4 H, 3ЈЈ-H, 4ЈЈ-H), 0.89 (t, J =
7.3 Hz, 3 H, 5ЈЈ-H) ppm. 13C NMR (125 MHz, CDCl3): δC = 206.1
(s, 9Ј-C), 161.4 (s, 1-C), 157.9 (s, 5-C), 148.3 (s, 6Ј-C), 145.5 (s, 3-
C), 115.7 (s, 6-C), 109.7 (t, 7Ј-C), 108.2 (d, 4-C), 102.3 (d, 2-C),
91.2 (d, 2Ј-C), 57.4 (d, 3Ј-C), 56.0 (d, 1Ј-C), 50.6 (d, 5Ј-C), 35.7 (t,
1ЈЈ-C), 32.3 (t, 3ЈЈ-C), 31.1 (t, 2ЈЈ-C), 28.5 (q, 10Ј-C), 27.4 (t, 4Ј-C),
23.6 (t, 4ЈЈ-C), 20.7 (q, 8Ј-C), 14.3 (q, 5ЈЈ-C) ppm. ESI-MS: m/z =
351 [M + Na]+.
Periodate Cleavage of 1,5-Diacetyl-2Ј,3Ј-dihydroxycannabidiol (8a):
Sodium periodate (214 mg, 5.0 equiv.) was added to a solution of
1,5-diacetyl-2Ј,3Ј-dihydroxycannabidiol (8a, 100 mg, 0.23 mmol) in
toluene/THF/H2O (1:1:1, 1.0 mL). After having been stirred at
room temperature for 16 h the reaction mixture was worked up by
dilution with EtOAc and washing with satd. NaHCO3 and brine.
After drying (Na2SO4), filtration, and concentration, the residue
was purified by gravity column chromatography on silica gel (hex-
ane/EtOAc 9:1) to afford compound 9 (56 mg, 56%) as a colorless
oil. 1H NMR (500 MHz, CDCl3): δH = 9.72 (s, 1 H, 2Ј-H), 6.18 (s,
2 H, 2-H, 4-H), 4.68 (br. s, 1 H, 9Јa-H), 4.52 (br. s, 1 H, 9Јb-H),
3.80 (m, 1 H, 1Ј-H), 3.52 (m, 1 H, 6Ј-H), 2.77 (m, 1 H, 4Јa-H), 2.56
(t, J = 7.6 Hz, 2 H, 1ЈЈ-H), 2.41 (m, 1 H, 4Јb-H), 2.16 (s, 3 H, 7Ј-
H), 2.07 (s, 6 H, 2ϫOAc), 1.78 (m, 2 H, 5Ј-H), 1.72 (s, 3 H, 10Ј-
H), 1.58 (q, J = 7.6 Hz, 2 H, 2ЈЈ-H), 1.30 (overlapped signal, 2 H,
3ЈЈ-H), 1.28 (overlapped signal, 2 H, 4ЈЈ-H), 0.89 (t, J = 7.0 Hz, 3
H, 5ЈЈ-H) ppm. 13C NMR (125 MHz, CDCl3): δC = 209.2 (s, 2Ј-C),
198.9 (s, 3Ј-C), 169.4 (s, OAc), 150.5 (s, 1-C), 150.5 (s, 5-C), 145.2
(s, 8Ј-C), 143.8 (s, 3-C), 121.4 (t, 9Ј-C), 117.9 (s, 6-C), 115.6 (d, 2-
C), 115.6 (d, 4-C), 54.7 (d, 1Ј-C), 45.0 (d, 6Ј-C), 42.3 (t, 4Ј-C), 36.1
(t, 1ЈЈ-C), 32.1 (t, 3ЈЈ-C), 30.9 (t, 2ЈЈ-C), 29.8 (q, 7Ј-C), 25.6 (t, 5Ј-
C), 22.2 (t, 4ЈЈ-C), 21.0 (q, OAc), 17.8 (q, 10Ј-C), 14.3 (q, 5ЈЈ-
C) ppm. ESI-MS: m/z = 451 [M + Na]+.
TRPV1, TRPM8, TRPA1 Receptor Assays: HEK293 (human em-
bryonic kidney) cells were grown as monolayers in minimum essen-
tial medium supplemented with nonessential amino acids, fetal calf
serum (10%), and glutamine (2 m), maintained under CO2 (5%)
at 37 °C, and plated on Petri dishes (100 mm diameter). The cells
were transfected at approximately 80% confluence with Lipofecta-
mine 2000 (Invitrogen, Carlsbad, CA) with the aid of a plasmid
pcDNA3 (Invitrogen) containing human TRPV1-cDNA, rat
TRPA1-cDNA, or rat TRPM8-cDNA according to the manufac-
turer’s protocol. Stably transfected clones were selected by use of
G-418 (Geneticin, 600 µgmL–1). The effect of the substances on
[Ca2+]i was determined by use of Fluo-4, a selective intracellular
fluorescent probe for Ca2+. For this purpose, on the day of the
experiment, cells overexpressing the TRPV1, or the TRPM8, or the
TRPA1 channels were loaded for 1 h at room temperature with the
methyl ester Fluo4-AM (4 µ, Invitrogen) in minimum essential
medium without fetal bovine serum. After the loading, cells were
washed twice in Tyrode’s buffer [NaCl (145 m), KCl (2.5 m),
CaCl2 (1.5 m), MgCl2 (1.2 m), -glucose (10 m), and HEPES
Crotonization of the seco-Cannabinoid 9: p-Toluenesulfonic acid
(PTSA, 394 mg, 1.0 equiv.) was added to a solution of 9 (100 mg,
Eur. J. Org. Chem. 2010, 2067–2072
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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2071

O. Taglialatela-Scafati, G. Appendino et al.
FULL PAPER
(10 m), pH 7.4], resuspended in Tyrode’s buffer, and transferred
(50–60000 cells) to the quartz cuvette of the spectrofluorimeter
(Perkin–Elmer LS50B; Perkin–Elmer Life and Analytical Sciences,
Waltham, MA) with continuous stirring. Intracellular Ca2+ concen-
tration ([Ca2+]i) was determined before and after the addition of
various concentrations of test compounds by measuring cell fluo-
rescence 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). 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 with use of GraphPad®. The
efficacies of the agonists were first determined by normalizing their
effects to the maximum Ca2+ influx effect on [Ca2+]i observed with
application of ionomycin (Sigma, 4 µ). The effects of TRPA1 ago-
nists are expressed as percentages of the effect obtained with allyl
isothiocyanate (100 µ). In the case of TRPM8 the experiments
were carried out at 22 °C with a Fluorescence Peltier System (PTP-
1, Perkin–Elmer): varying doses of the compounds were added di-
rectly to the quartz cuvette with stirring 5 min before stimulation
of cells with icilin (0.25 µ). Data were expressed as the concentra-
tions exerting half-maximal inhibition of agonist [Ca2+]i increasing
effect (IC50), which was again calculated with the aid of GraphPad
Prism® software. The effect on [Ca2+]i exerted by icilin (0.25 µ)
was taken as 100%. All determinations were performed at least in
triplicate. Statistical analysis of the data was performed by analysis
of variance at each point with the aid of ANOVA, followed by
Bonferroni’s test. At higher concentrations (Ͼ10 µ), compound
12 caused an effect on [Ca2+]i in cells transfected both with and
without any TRP construct, and the values obtained from the latter
were taken as baselines to be subtracted from the former.
equation. Data are represented as means ϮSEMs of at least n = 3
experiments.
Supporting Information (see also the footnote on the first page of
this article): Selected NMR spectra.
Acknowledgments
We gratefully thank Aniello Schiano Moriello (for TRP receptor
assays) and Marco Allarà (for CB1 and CB2 receptor binding as-
says). Mass and NMR spectra were recorded at the “Centro di
Servizio Interdipartimentale di Analisi Strumentale”, Università di
Napoli “Federico II”. The assistance of the staff is acknowledged.
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CB1 and CB2 Receptor Binding Assays: Membranes harvested from
human recombinant CB1 (Bmax = 2.5 pmol per mg protein) or CB2
(Bmax = 4.7 pmol per mg protein) receptor transfected HEK-293
cells were incubated with the high-affinity ligand [3H]-CP-55,940
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with [3H]-CP-55,940 for 90 min at 30 °C to generate displacement
curves. IC50 values of the test compounds for the displacement of
the bound radioligand were obtained by use of GraphPad Prism®
and used to calculate Ki values with the aid of the Cheng–Prusoff
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Received: December 15, 2009
Published Online: February 11, 2010
2072
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© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2010, 2067–2072