Synthesis of the Major Mammalian Metabolites of THCV

Article abstract of DOI:10.1021/acs.jnatprod.9b00831

A simple synthesis of the major oxidized metabolites in mammalian tissues of (-)-Δ9-tetrahydrocannabivarin (THCV) (1) has been accomplished by kinetic studies of allylic oxidation using SeO2 on botanically derived THCV with the aim to yield primary and secondary allylic alcohols concurrently. This synthetic approach led to the preparation of numerous THCV derivatives, including two new compounds, 8α-hydroxy-Δ9-tetrahydrocannabivarin (2) and 8β-hydroxy-Δ9-tetrahydrocannabivarin (3), and the known compounds 11-hydroxy-Δ9-tetrahydrocannabivarin (4) and Δ9-tetrahydrocannabivarin-11-oic acid (5), without affecting the C-10a stereogenic center in the natural precursor and without formation of tricyclic dibenzopyran derivatives. This simple synthetic methodology could be useful to investigate the pharmacological role of THCV metabolites at, among others, the endocannabinoid CB1 and CB2 receptors for which THCV reportedly acts as respectively a neutral antagonist and partial agonist.

Full text of DOI:10.1021/acs.jnatprod.9b00831

pubs.acs.org/jnp
Article
Synthesis of the Major Mammalian Metabolites of THCV
Francesco Tinto,* Rosaria Villano, Magdalena Kostrzewa, Alessia Ligresti, Hannah Straker,
and Emiliano Manzo
Cite This: https://dx.doi.org/10.1021/acs.jnatprod.9b00831
Read Online
sı
*
Metrics & More
Article Recommendations
Supporting Information
ACCESS
ABSTRACT: A simple synthesis of the major oxidized metabolites
in mammalian tissues of (−)-Δ9-tetrahydrocannabivarin (THCV)
(1) has been accomplished by kinetic studies of allylic oxidation
using SeO2 on botanically derived THCV with the aim to yield
primary and secondary allylic alcohols concurrently. This synthetic
approach led to the preparation of numerous THCV derivatives,
including two new compounds, 8α-hydroxy-Δ9-tetrahydrocannabi-
varin (2) and 8β-hydroxy-Δ9-tetrahydrocannabivarin (3), and the
known compounds 11-hydroxy-Δ9-tetrahydrocannabivarin (4) and
Δ9-tetrahydrocannabivarin-11-oic acid (5), without affecting the C-
10a stereogenic center in the natural precursor and without
formation of tricyclic dibenzopyran derivatives. This simple synthetic methodology could be useful to investigate the
pharmacological role of THCV metabolites at, among others, the endocannabinoid CB1 and CB2 receptors for which THCV
reportedly acts as respectively a neutral antagonist and partial agonist.
are not totally absent from the brain since they are expressed in
fter the discovery of the primary active constituent of
Amarijuana, (−)-trans-Δ -tetrahydrocannabinol (THC), in microglia,14 but their presence and role in the CNS have yet to
9
be fully elucidated.15 Unlike the other phytocannabinoids, little
is known about the pharmacokinetics of THCV in humans,
although a phase II trial of THCV in patients with type II
diabetes was recently completed (NCT02053272) by GW
Research Ltd. However, even less is known about the
pharmacokinetics of its oxidized metabolites. Additional
experiments are now required to establish whether THCV
metabolites can also interact with CB1 and CB2 receptors in
vitro, in various tests of functional activity, and in vivo. The
availability of synthesized THCV metabolites is therefore
critical for the analysis of their biological actions, and, although
some approaches have been reported16−19 for their de novo
synthesis, none of these have started from botanically derived,
stereochemically pure THCV. Cannabinoids are sensitive to
oxidative environments, and for this reason, the oxidized
metabolites are often prepared by complex de novo syntheses
that require expensive stereoselective reagents. With the
optimization of methods of isolation and purification of
products from botanical sources and the growing expansion of
C. sativa for medical purposes, it would be useful to design a
semisynthetic method starting from natural precursors that is
1964,1 a minor cannabinoid, (−)-trans-Δ9-tetrahydrocannabi-
varin (THCV) (1),2 was found in cannabis tincture in 1970.3
The only difference between these two cannabinoids is the
presence of a propyl group linked to the aromatic moiety in
THCV instead of a pentyl group in THC. Five different
numbering systems4 have been used for the phytocannabinoids
in general, but only two are used for the benzopyran
structures.5 Here we employed the dibenzopyran numbering
of the most widespread system used for THCV (1) and the
subsequent metabolites thereof (2−5) as shown in Scheme 1.
Since its discovery, the pharmacology of THCV has been
studied in order to consider its potential clinical use.6 This
phytocannabinoid is capable of interacting with the endocan-
nabinoid system via binding at the CB1 and CB2 receptors,
where it exerts either antagonistic or agonistic effects,
depending on its concentration. THCV appears to antagonize
the CB1 receptor at lower doses (3 mg kg−1 or less), whereas it
produces an agonist effect at higher doses (10 mg kg−1 or
more) in vivo.7 THCV behaves also in vitro as a potent
competitive antagonist of cannabinoid derivatives such as
CP55940 and R-(+)-WIN55212.8 With regard to the CB2
receptors, THCV has been defined as a partial agonist in vitro,7
and currently there is limited knowledge about its behavior on
CB2 receptors in vivo. The CB1 receptors are found in the
central nervous system (CNS) as well as many peripheral
tissues,9 while CB2 receptors are found primarily outside the
CNS in the human spleen,10 the tonsils,11 leukocytes,12 and
tissues associated with the immune system.13 CB2 receptors
Received: August 31, 2019
© XXXX American Chemical Society and
American Society of Pharmacognosy
https://dx.doi.org/10.1021/acs.jnatprod.9b00831
J. Nat. Prod. XXXX, XXX, XXX−XXX
A

Journal of Natural Products
pubs.acs.org/jnp
Article
Scheme 1. THCV (1) and Metabolites 8α-Hydroxy-Δ9-tetrahydrocannabivarin (2), 8β-Hydroxy-Δ9-tetrahydrocannabivarin
(3), 11-Hydroxy-Δ9-tetrahydrocannabivarin (4), and Δ9-Tetrahydrocannabivarin-11-oic acid (5)
a
Scheme 2. Preparation of Oxidized THCV Metabolites (2−4)
a
Reagents and conditions: (a) Ac2O in pyridine; (b) SeO2 in EtOH at reflux; (c) NaOH(aq).
quicker, more efficient, and “green”. We report here a simple
methodology to synthesize different oxidized metabolites of
THCV, including 8α-hydroxy-Δ9-tetrahydrocannabivarin (2),
8β-hydroxy-Δ9-tetrahydrocannabivarin (3), 11-hydroxy-Δ9-
tetrahydrocannabivarin (4), and Δ9-tetrahydrocannabivarin-
11-oic acid (5), in a limited number of steps starting from
purified THCV extracted from a THCV-rich strain of C. sativa.
Δ8- and Δ9-THC using an excess of SeO2 at 78 °C as described
by Ben-Zvi et al.20 When these experimental conditions were
evaluated using the major cannabinoid in C. sativa, a complex
mixture of hyperoxidized Δ8- and Δ9-THC metabolites was
obtained without achieving significant yields of the targeted
THC oxidized metabolites. In order to exploit the oxidizing
potential of SeO2 while avoiding the formation of unwanted
byproducts and increasing the overall yields, several exper-
imental conditions were attempted to selectively oxidize
THCV. Thus, oxidation of THCV with tert-butyl hydro-
peroxide (1.34 equiv) and SeO2 (0.55 equiv) in CH2Cl2 at
room temperature was evaluated, but without success. This
method failed to oxidize the allylic C-11 and led to a complex
mixture which confirmed the sensitivity of these molecules to
RESULTS AND DISCUSSION
■
For the preparation of the oxidized THCV metabolites (2−5),
botanical purified THCV (1), supplied by GW Research Ltd.,
was converted into its 1-O-acetyl derivative (6) using Ac2O in
pyridine followed by subsequent oxidation (Scheme 2). The
first attempt of this oxidation was carried out on a mixture of
B
https://dx.doi.org/10.1021/acs.jnatprod.9b00831
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
pubs.acs.org/jnp
Article
the common allylic oxidation conditions. Subsequently,
oxidation using SeO2 in EtOH at reflux (in the absence of
tert-butyl hydroperoxide) was attempted. This experiment was
designed to avoid oxidation at the C-10a stereogenic center of
(−)-Δ9-THCV at elevated temperatures and high concen-
trations of oxidants. Oxidation with only SeO2 (10 equiv) left
the starting material unchanged, indicating tert-butyl hydro-
peroxide as alternative hyperoxidant for these cannabinoids.
Subsequent attempts to increase the equivalents of SeO2 (30
equiv) proved to be too harsh. The THCV was completely
transformed into unwanted double-oxidized derivatives and
tricyclic dibenzopyran byproducts. (Figure 1).
TLC and HRESIMS data, and after 60 min many byproducts
were observed. The mass spectrum did not show any notable
peaks corresponding to the targeted products. The targeted
products were observed with 15 equiv of SeO2 in EtOH at
reflux over 75 min. Compound 6 was oxidized according to
this optimized strategy. The SeO2 was filtered, the EtOH was
evaporated, and the mixture was extracted with Et2O. Silica gel
and UHPLC purifications afforded the acetylated metabolites
11-hydroxy-Δ9-1-O-acetyltetrahydrocannabivarin (7, 10%),
8α-hydroxy-Δ9-1-O-acetyltetrahydrocannabivarin (8, 12%),
and 8β-hydroxy-Δ9-1-O-acetyltetrahydrocannabivarin (9,
20%) (Scheme 2). Subsequent saponification of the acetate
groups in alkaline conditions led to metabolite 11-hydroxy-Δ9-
tetrahydrocannabivarin (4) and the new compounds 8α-
hydroxy-Δ9-tetrahydrocannabivarin (2), and 8β-hydroxy-Δ9-
tetrahydrocannabivarin (3). All compounds were characterized
by NMR (1H/13C, COSY, and HSQC) and HRESIMS data.
The THCV derivatives were confirmed to be Δ9- instead of
Δ8- isomers, matching the same peculiar higher chemical shifts
for C-10, C-8, H-10, and H-8 as previously described in the
characterization of Δ9-THC and Δ8-THC derivatives.21,22 The
structure of compound 4 was elucidated through the HSQC
spectrum, showing the hydroxymethyl group at C-11, whereas
the hydroxymethine group was at C-8 in compounds 2 and 3
with the hydroxy groups in α- and β-orientations, respectively,
in close analogy with spectroscopic data of the botanical pentyl
derivatives 8α-hydroxy-Δ9-tetrahydrocannabinol and 8β-hy-
droxy-Δ9-tetrahydrocannabinol described by Radwan et al.21
In fact, the presence of a C-8 oxymethine carbon at δC 68.0,
corresponding to a proton resonance H-8 at δH 4.10 in the
HSQC spectrum, showed that 8α-hydroxy-Δ9-tetrahydro-
cannabivarin (2) is a hydroxylated Δ9-THCV derivative. The
NMR data of 8β-hydroxy-Δ9-tetrahydrocannabivarin (3) were
similar to those of compound 2 except for the chemical shifts
of H-8 at δH 4.30 and C-8 at δC 71.5. The higher frequency
shifts indicated the β-orientation as reported for the 8α- and
8β-hydroxy-Δ9-tetrahydrocannabinols.21
Figure 1. Examples of double-oxidized derivatives and tricyclic
dibenzopyran byproducts.
Owing to the high sensitivity of the THCV structure to the
oxidizing conditions observed in these attempts, the allylic
oxidation reaction was modified by changing the SeO2
concentration and reaction time while studying the reaction
kinetics using mass spectrometry. These experiments permitted
the design of an optimized methodology to produce THCV
metabolites with retention of the C-10a stereocenter (2−4,
Scheme 1). The mass spectrometry and kinetic studies were
carried out on two different allylic oxidations using 15 and 30
equiv of SeO2 in EtOH at reflux. These reactions were
monitored by taking aliquots every 30 min over 4 h, followed
by HRESIMS analysis. Initially, 15 equiv of SeO2 was used, and
after 60 min THCV was observed unaltered. However, at 90
min, it was completely consumed and transformed into
multiple oxidation products as assessed by TLC and
HRESIMS data. The mass spectrum of this sample was
consistent with the three oxidized compounds 2−4 and some
byproducts with higher masses. In the second experiment using
30 equiv of SeO2, THCV was consumed after 30 min based on
With the aim of obtaining Δ9-tetrahydrocannabivarin-11-oic
acid (5),23 compound 7 was oxidized to the corresponding
aldehyde (10) using MnO2 (Scheme 3). This mild oxidant was
chosen to obtain the aldehyde (10) instead of directly the
carboxylic acid to avoid possible byproduct formation using
a
Scheme 3. Preparation of Oxidized THCV Metabolites (5)
a
Reagents and conditions: (a) MnO2 in Et2O; (b) NaClO2; (c) NaOH(aq).
C
https://dx.doi.org/10.1021/acs.jnatprod.9b00831
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
pubs.acs.org/jnp
Article
a
Table 1. CB1R and CB2R Affinity Values
IC50 on CB1, nM (m SD, Ki on CB1, nM (m SD, IC50 on CB2, nM (m SD, Ki on CB2, nM (m SD,
sample
n = 3)
n = 3)
n = 3)
n = 3)
Δ9-THCV
13.1 4.1
1681.5 164.5
1.5 0.5
186.8 78.3
17.6 3.9
3715.0 287.1
2.8 0.6
589.7 45.6
Δ9-tetrahydrocannabivarin-11-oic acid
(5)
11-hydroxy-Δ9-tetrahydrocannabivarin
22.7 6.7
230.1 23.3
>10 000
2.5 0.5
25.6 2.6
>10 000
29.8 8.3
807.7 17.5
>10 000
4.7 1.3
128.2 2.8
>10 000
(4)
8α-hydroxy-Δ9-
tetrahydrocannabivarin (2)
8β-hydroxy-Δ9-tetrahydrocannabivarin
(3)
a
Data represent mean values for three separate experiments performed in duplicate and are expressed as IC50 (the concentration of competing
ligand that displaces 50% of the specific binding of the radioligand) and Ki (the concentration of the competing ligand that will bind to half the
binding sites at equilibrium in the absence of radioligand or other competitors).
instrument (1H 400; 13C 101 MHz). Samples for NMR spectroscopic
stronger oxidants. The method led to the isolation and
analysis were dissolved in the appropriate solvent; spectra in CDCl3:
characterization of this important compound, which can be
δH 7.26, δC 77.0; methanol-d4: δH 3.34, δC 49.0. HRESIMS data were
acquired on a Micromass Q-TOF Micro coupled with an HPLC
Waters Alliance 2695. TLC plates (Kieselgel 60 F254) and silica gel
powder (Kieselgel 60, 0.063−0.200 mm) were from Merck
(Darmstadt, Germany). All the reagents and solvents were purchased
from Sigma-Aldrich (Steinheim, Germany) and used without further
purification.
useful for future derivatizations at C-11. The subsequent
Pinnick oxidation24 oxidized aldehyde 10 to the corresponding
acetylated carboxylic acid (11), which gave Δ9-tetrahydro-
cannabivarin-11-oic acid (5) in high yield (98%) after
hydrolysis under alkaline conditions.
In conclusion, we developed a simple methodology for the
synthesis of THCV oxidized metabolites starting from
botanical sources and with retention of absolute configuration.
The controlled approach uses an optimized allylic oxidation
methodology as key step and permitted synthesis of four
metabolites, 2−5, two of which, 8α-hydroxy-Δ9-tetrahydro-
cannabivarin (2) and 8β-hydroxy-Δ9-tetrahydrocannabivarin
(3), are new. This method requires only three steps to access
the hydroxylated derivatives and five steps for the correspond-
ing carboxylic acid, starting from the pure Δ9-THCV extracted
from Cannabis sativa. This chemical approach may be used for
semisynthesis of cannabinoid derivatives to form a range of
oxidation products. Moreover, it was investigated whether
these compounds are able to bind CB1 and CB2 receptors. In
particular, the new molecules were evaluated in parallel with
THCV as reference compound, in a competition-binding assay.
The binding affinities for human recombinant CB1 and CB2
receptors are collated in Table 1. Nearly all compounds
displayed binding affinity for CBRs, with Ki values for CB1
spanning 2 orders of magnitude (1.45 to 186.8 nM) and with
Ki values for CB2 spanning 3 orders of magnitude (17.6 to
3715.0 nM). Only 11-hydroxy-Δ9-tetrahydrocannabivarin (4)
showed a pattern comparable to THCV with similar affinity to
both subtypes, while 8α-hydroxy-Δ9-tetrahydrocannabivarin
(2) and Δ9-tetrahydrocannabivarin-11-oic acid (5) showed
higher affinity for CB1R compared to CB2R (Ki values 25.6
versus 128.2, and 186.8 versus 589.7, respectively). Moreover,
the chiral metabolite 8β-hydroxy-Δ9-tetrahydrocannabivarin
(3) was devoid of receptor affinity (Ki > 10 000 nM),
suggesting distinct interactions of the two stereoisomers with
the binding pocket. Further studies will elucidate how they
activate cannabinoid receptors.
1-O-Acetyl-Δ9-tetrahydrocannabivarin (6). (−)-Δ9-THCV
(1), obtained from GW Pharmaceutical Company (London, UK)
(100 mg, 0.35 mmol), was dissolved in pyridine (2 mL) and Ac2O (2
mL), and the mixture was stirred for 18 h at room temperature. The
solution was poured onto iced water (20 mL) and extracted with
Et2O (3 × 15 mL). The combined organic extracts were washed with
1 N HCl, aqueous NaHCO3, and brine, dried on MgSO4, and filtered.
Removal of the solvents under reduced pressure afforded an oily
1
residue that by H NMR data was proven to be 6 (111 mg, 0.34
mmol, ∼99%): 1H NMR (CDCl3, 400 MHz) δH 6.55 (1H, d, J = 1.6
Hz, H-2), 6.40 (1H, d, J = 1.6 Hz, H-4), 5.98 (1H, q, J = 1.6 Hz, H-
10), 3.10−3.04 (1H, dm, J = 10.9 Hz, H-10a), 2.48 (2H, t, J = 7.8 Hz,
H-1′), 2.28 (3H, s, H-2″), 2.14 (2H, m, H-8), 1.90 (1H, m, H-7),
1.70 (1H, m, H-6a), 1.67 (3H, s, H-11), 1.62 (2H, m, H-2′), 1.40
(3H, s, H-13), 1.39 (1H, m, H-7), 1.09 (3H, s, H-12), 0.91 (3H, t, J =
7.2 Hz, H-3′); 13C NMR (CDCl3, 101 MHz) δC 122.4 (CH, C-10),
114.6 (CH, C-4), 113.2 (CH, C-2), 44.8 (CH, C-6a), 37.2 (CH2, C-
1′), 33.6 (CH, C-10a), 30.5 (CH2, C-8), 27.0 (CH3, C-13), 24.3
(CH2, C-7), 23.8 (CH3, C-11), 23.2 (CH2, C-2′), 20.7 (CH3, C-2″),
18.8 (CH3, C-12), 13.3 (CH3, C-3′); HRMS (ESI) m/z 351.2035 [M
+ Na]+ (calcd 351.1931 for C21H28O3).
11-Hydroxy-Δ9-1-O-acetyltetrahydrocannabivarin (7). Com-
pound 6 (111 mg, 0.34 mmol) was dissolved in EtOH (5 mL), SeO2
(566 mg, 5.1 mmol) was added, and the reaction was stirred at reflux
for 75 min. The mixture was filtered, the EtOH was removed under
reduced pressure, and the residue was diluted with H2O (20 mL) and
extracted with Et2O (3 × 15 mL). The combined organic extracts
were dried on Na2SO4 and filtered. Removal of the solvent under
reduced pressure afforded a residue, which was chromatographed on
silica gel (30% ether/light petroleum ether) to give 61 mg of the
mixture (7, 8, 9). Compound 7 was isolated with UHPLC isocratic
chromatography (silica column, normal phase, eluent isopropanol/n-
hexane, 5:95 v/v; flow rate = 3 mL/min; λ = 220 nm), at a retention
time 16.5 min (11 mg, 0.034 mmol, 10%). TLC (50% Et2O/light
1
petroleum ether, Rf = 0.18); H NMR (CDCl3, 400 MHz) δH 6.55
(1H, d, J = 1.4 Hz, H-2), 6.41 (1H, d, J = 1.4 Hz, H-4), 6.31 (1H, H-
10), 4.02 (2H, s, H-11), 3.16−3.10 (1H, dm, J = 11.4 Hz, H-10a),
2.48 (2H, t, J = 7.4 Hz, H-1′), 2.28 (3H, s, H-2″), 2.26 (2H, m, H-8),
2.01−1.94 (1H, m, H-7), 1.70 (1H, m, H-6a), 1.60 (2H, m, H-2′),
1.42 (3H, s, H-13), 1.40 (1H, m, H-7), 1.09 (3H, s, H-12), 0.91 (3H,
t, J = 7.2 Hz, H-3′); 13C NMR (CDCl3, 101 MHz) δC 123.8 (CH, C-
10), 114.7 (CH, C-4), 113.1 (CH, C-2), 66.4 (CH2, C-11), 44.9 (CH,
C-6a), 36.8 (CH2, C-1′), 33.5 (CH, C-10a), 26.5 (CH3, C-13), 26.1
(CH2, C-8), 24.3 (CH2, C-7), 23.2 (CH2, C-2′), 20.7 (CH3, C-2″),
EXPERIMENTAL SECTION
■
General Experimental Procedures. The structures of products
and synthetic intermediates were assigned using 1H, 13C, COSY,
1
HSQC, and HMBC NMR spectra. H, 13C, and 2D NMR spectra
were acquired by the NMR Service of the Institute of Biomolecular
Chemistry of the National Council of Research (ICB-CNR) and
recorded on a Bruker DRX-600 spectrometer, equipped with a TCI
CryoProbe, fitted with a gradient along the Z-axis and on a Bruker
D
https://dx.doi.org/10.1021/acs.jnatprod.9b00831
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
pubs.acs.org/jnp
Article
18.8 (CH3, C-12), 13.3 (CH3, C-3′); HRMS (ESI) m/z 367.0705 [M
+ Na]+ (calcd 367.1880 for C21H28O4).
8β-Hydroxy-Δ9-tetrahydrocannabivarin (3). Compound 3 (20
mg, 0.066 mmol, 94%) was synthesized starting from compound 9
(22 mg, 0.070 mmol) with the same procedure reported for
8α-Hydroxy-Δ9-1-O-acetyltetrahydrocannabivarin (8). Com-
pound 8 was isolated with UHPLC isocratic chromatography (silica
column, normal phase, eluent isopropanol/n-hexane, 5:95 v/v; flow
rate = 3 mL/min; λ = 220 nm), at retention time 14.5 min (13 mg,
0.042 mmol, 12%). TLC (50% Et2O/light petroleum ether, Rf =
0.20); 1H NMR (CDCl3, 400 MHz) δH 6.55 (1H, d, J = 1.4 Hz, H-2),
6.41 (1H, d, J = 1.4 Hz, H-4), 6.24 (1H, H-10), 4.07 (1H, s, H-8),
2.98 (1H, dm, J = 11.1 Hz, H-10a), 2.48 (2H, t, J = 7.4 Hz, H-1′),
2.28 (3H, s, H-2″), 2.01 (1H, dm, J = 13.6 Hz, H-7), 1.83 (1H, brs,
H-6a), 1.60 (2H, m, H-2′), 1.55 (3H, m, H-11), 1.42 (3H, s, H-13),
1.38−1.33 (1H, m, H-7), 1.10 (3H, s, H-12), 0.91 (3H, t, J = 7.2 Hz,
H-3′); 13C NMR (CDCl3, 101 MHz) δC 126.4 (CH, C-10), 114.7
(CH, C-4), 113.1 (CH, C-2), 67.7 (CHOH, C-8), 40.0 (CH, C-6a),
37.1 (CH2, C-1′), 34.4 (CH, C-10a), 34.3 (CH2, C-7), 26.7 (CH3, C-
13), 23.6 (CH2, C-2′), 20.4 (CH3, C-2″) 20.2 (CH3, C-11), 19.1
(CH3, C-12), 13.3 (CH3, C-3′); HRMS (ESI) m/z 367.0705 [M +
Na]+ (calcd 367.1880 for C21H28O4).
1
compound 4: H NMR (CDCl3, 400 MHz) δH 6.52 (1H, brq, J =
1.5 Hz, H-10), 6.26 (1H,d, J = 1.4 Hz, H-2), 6.12 (1H,d, J = 1.4, H-
4), 4.30 (1H, brt, J = 7.0 Hz, H-8), 3.31 (1H, dm, J = 10.6 Hz, H-
10a), 2.42 (2H, dd, J = 6.6, 8.4 Hz, H-1′), 2.35 (1H, m, H-7), 1.79
(1H, m, overlapped, H-6a), 1.78 (3H, s, H-11), 1.60 (2H, m, H-2′),
1.41 (3H, s, H-13), 1.39 (1H, m, H-7), 1.12 (3H, s, H-12), 0.91 (3H,
t, J = 7.2 Hz, H-3′); 13C NMR (CDCl3, 101 MHz) δC 127.8 (CH, C-
10), 109.8 (CH, C-4), 107.1 (CH, C-2), 71.5 (CHOH β form, C-8),
45.5 (CH, C-6a), 37.1 (CH2, C-1′), 35.3 (CH2, C-7), 33.6 (CH, C-
10a), 26.7 (CH3, C-13), 23.4 (CH2, C-2′), 18.9 (CH3, C-11), 18.8
(CH3, C-12), 13.3 (CH3, C-3′); HRMS (ESI) m/z 325.2298 [M +
Na]+ (calcd 325.1774 for C19H26O3).
11-Oxo-Δ9-1-O-acetyltetrahydrocannabivarin (10). A solu-
tion of compound 7 (11 mg, 0.034 mmol) was added to anhydrous
Et2O (1 mL) at 0 °C and treated with activated MnO2 (96%, 89 mg,
1.02 mmol). The reaction mixture was stirred for 1 h at 0 °C and
overnight at rt. The mixture was filtered through Celite, and the filter
pad was washed with Et2O. The combined filtrates were dried with
Na2SO4, and removal of the solvents under reduced pressure afforded
pure compound 10 (11 mg, 0.033 mmol, 94%): TLC (50% Et2O/
8β-Hydroxy-Δ9-1-O-acetyltetrahydrocannabivarin (9). Com-
pound 9 was isolated with UHPLC isocratic chromatography (silica
column, normal phase, eluent isopropanol/n-hexane, 5:95 v/v; flow
rate = 3 mL/min; λ = 220 nm) at retention time 12.0 min (22 mg,
0.070 mmol, 20%). TLC (50% Et2O/light petroleum ether, Rf =
0.20); 1H NMR (CDCl3, 400 MHz) δH 6.55 (1H, d, J = 1.4 Hz, H-2),
6.41 (1H, d, J = 1.4 Hz, H-4), 6.15 (1H, brs, H-10), 4.29 (1H, s, H-
8), 3.18 (1H, dm, J = 11.1 HZ, 1 Hz, H-10a), 2.48 (2H, t, J = 7.4 Hz,
H-1′), 2.32 (1H, m, H-7), 2.28 (3H, s, H-2″), 1.81 (1H, m, H-6a),
1.77 (3H, s, H-11), 1.60 (2H, m, H-2′), 1.40 (3H, s, H-13), 1.36 (1H,
m, H-7), 1.11 (3H, s, H-12), 0.91 (3H, t, J = 7.2 Hz, H-3′); 13C NMR
(CDCl3, 101 MHz) δC 125.7 (CH, C-10), 114.4 (CH, C-4), 113.0
(CH, C-2), 71.1 (CHOH, C-8), 45.2 (CH, C-6a), 36.7 (CH2, C-1′),
34.8 (CH2, C-7), 33.7 (CH, C-10a), 26.1 (CH3, C-13), 23.0 (CH2, C-
2′), 20.5 (CH3, C-2″), 18.5 (CH3, C-12), 18.4 (CH3, C-11), 13.0
(CH3, C-3′); HRMS (ESI) m/z 367.0705 [M + Na]+ (calcd 367.1880
for C21H28O4).
1
light petroleum ether, Rf = 0.60); H NMR (CDCl3, 400 MHz) δH
9.46 (1H, s, H-11), 6.97 (1H, brd, J = 1.1 Hz, H-10), 6.59 (1H, d, J =
1.1 Hz, H-2), 6.47 (1H, d, J = 1.2 Hz, H-4), 5.02 (2H, m, H-8), 3.35
(1H, dm, J = 11.4 Hz, H-10a), 2.51 (2H, m, H-1′), 2.30 (1H, m, H-
7), 2.29 (3H, s, H-2″), 2.05 (1H, m, H-6a) 1.74 (1H, m,), 1.61 (2H,
m, H-2′), 1.43 (3H, s, H-13), 1.42 (1H, m, H-7), 1.14 (3H, s, H-12),
0.92 (3H, t, J = 7.2 Hz, H-3′); 13C NMR (CDCl3, 101 MHz) δC 192.8
(COH, C-11), 124.7 (CH, C-10), 115.0 (CH, C-4), 113.4 (CH, C-2),
44.0 (CH, C-6a), 36.8 (CH2, C-1′), 34.9 (CH, C-10a), 33.3 (CH2, C-
8), 29.6 (CH3, C-13), 23.5 (CH2, C-2′), 22.5 (CH2, C-7), 18.5 (CH3,
C-12), 13.0 (CH3, C-3′); HRMS (ESI) m/z 365.1926 [M + Na]+
(calcd 365.1723 for C21H26O4).
1-O-Acetyl-Δ9-tetrahydrocannabivarin-11-oic acid (11).
NaClO2 (80% pure 5.5 mg, 0.05 mmol) was added to a stirred
mixture of 10 (4 mg, 0.012 mmol), 2-methyl-2-butene (0.03 mL, 0.28
mmol), and a saturated aqueous solution of KH2PO4 (14 μL) in t-
BuOH (500 μL). The mixture was stirred at room temperature
overnight. Water was added (2 mL), and the mixture was extracted
with EtOAc (3 × 5 mL). The organic phase was washed with brine,
dried over Na2SO4, and filtered. Removal of the solvent under
reduced pressure gave compound 11 (3.9 mg, 0.011 mmol, 92%): 1H
NMR (CDCl3, 400 MHz) δH 7.73 (1H, brd, J = 2.0 Hz, H-10), 6.57
(1H, d, J = 1.1 Hz, H-2), 6.46 (1H, d, J = 1.2 Hz, H-4), 3.26 (1H, dm,
J = 11.4 Hz, H-10a), 2.60−2.40 (3H, m, overlapped, H-1′, H-7), 2.30
(3H, s, H-2″), 2.05 (1H, m, H-6a), 1.61 (2H, m, H-2′), 1.43 (3H, m,
H-13), 1.42 (1H, m overlapped, H-7), 1.12 (3H, s, H-12), 0.92 (3H,
t, J = 7.2 Hz, H-3′); HRMS (ESI) m/z 381.1871 [M + Na]+ (calcd
381.1673 for C21H26O5).
11-Hydroxy-Δ9-tetrahydrocannabivarin (4). Compound 7 (11
mg, 0.034 mmol) was dissolved in MeOH (2 mL), 1 M NaOH (1
mL) was added, and the reaction mixture was stirred for 1 h at rt. The
solution was acidified with 1 M HCl (pH ∼2) and extracted with
Et2O (3 × 5 mL). The combined organic extracts were washed with
brine, dried on Na2SO4, and filtered. Removal of the solvents under
reduced pressure afforded the pure compound 4 (10 mg, 0.033 mmol,
1
97%): TLC (50% Et2O/light petroleum ether, Rf = 0.13); H NMR
(CDCl3, 400 MHz) δH 6.67 (1H, brd, J = 1.1 Hz, H-10), 6.27 (1H, d,
J = 1.1 Hz, H-2), 6.12 (1H, d, J = 1.2 Hz, H-4), 4.03 (2H, s, H-11),
3.26 (1H, dm, J = 11.4 Hz, H-10a), 2.42 (2H, m, H-1′), 2.29 (2H, m,
H-8), 1.99 (1H, dm, H-7), 1.71 (1H, m, H-6a), 1.58 (2H, m, H-2′),
1.43 (1H, m, H-7), 1.42 (3H, s, H-13), 1.11 (3H, s, H-12), 0.91 (3H,
t, J = 7.2 Hz, H-3′); 13C NMR (CDCl3, 101 MHz) δC 125.5 (CH, C-
10), 109.5 (CH, C-4), 106.7 (CH, C-2), 66.7 (CH2OH, C-11), 45.0
(CH, C-6a), 36.9 (CH2, C-1′), 32.8 (CH, C-10a), 26.7 (CH3, C-13),
26.0 (CH2, C-8), 24.0 (CH2, C-7), 23.1 (CH2, C-2′), 18.5 (CH3, C-
12), 13.1 (CH3, C-3′); HRMS (ESI) m/z 325.2298 [M + Na]+ (calcd
325.1774 for C19H26O3).
Δ9-Tetrahydrocannabivarin-11-oic acid (5). Compound 5 was
prepared from 11 (3.9 mg, 0.011 mmol) using the same procedure
reported for compound 4. The pure compound 5 was isolated with
UHPLC isocratic chromatography (RP-18 analytical column, eluent
MeOH/H2O/TFA, 80:20:0.1 v/v; flow rate = 1 mL/min; λ = 220
nm) at retention time 9.5 min (3.47 mg, 0.011 mmol, 98%). TLC
(70% Et2O/light petroleum ether, Rf = 0.28); 1H NMR (CDCl3, 400
MHz) δH 8.08 (1H, brd, J = 1.6 Hz, H-10), 6.28 (1H, d, J = 1.4 Hz,
H-2), 6.13 (1H, d, J = 1.4 Hz, H-4), 3.38 (1H, dm, J = 11.1 Hz, H-
10a), 2.55 (1H, m, H-7), 2.43 (2H, t, J = 7.4 Hz, H-1′), 2.02 (1H, m,
H-6a), 1.59 (2H, m, H-2′), 1.44 (3H, s, H-13), 1.41 (1H, m,
overlapped, H-7), 1.11 (3H, s, H-12), 0.91 (3H, t, J = 7.2 Hz, H-3′);
13C NMR (CDCl3, 101 MHz) δC 144.1 (CH, C-10), 109.4 (CH, C-
4), 106.8 (CH, C-2), 43.5 (CH, C-6a), 36.8 (CH2, C-1′), 33.9 (CH,
C-10a), 26.1 (CH3, C-13), 24.4 (CH2, C-8), 23.4 (CH2, C-7), 23.3
(CH2, C-2′), 18.3 (CH3, C-12), 13.0 (CH3, C-3′); HRMS (ESI) m/z
339.2953 [M + Na]+ (calcd 339.1567 for C19H24O4).
In Vitro Pharmacological Evaluation. Competition Binding
Assay. Membranes from HEK-293 cells overexpressing the respective
8α-Hydroxy-Δ9-tetrahydrocannabivarin (2). Compound 2 (12
mg, 0.040 mmol, 95%) was synthesized starting from compound 8
(13 mg, 0.042 mmol) with the same procedure reported for
1
compound 4; H NMR (CDCl3, 400 MHz) δH 6.66 (1H, brs, H-
10), 6.28 (1H, s, H-2), 6.11 (1H,d, J = 1.1, H-4), 4.10 (1H, d, H-8),
3.14 (1H, dm, H-10a), 2.42 (2H, dd, J = 6.6, 8.4 Hz, H-1′), 2.04 (1H,
dm, H-7), 1.84 (3H, s, overlapped, H-11), 1.83 (1H, m, H-6a), 1.64
(2H, m, H-2′), 1.58 (1H, m, H-7), 1.43 (3H, s, H-13), 1.10 (3H, s, H-
12), 0.92 (3H, t, J = 7.2 Hz, H-3′); 13C NMR (CDCl3, 101 MHz) δC
127.6 (CH, C-10), 109.9 (CH, C-4), 107.1 (CH, C-2), 68.0 (CHOH
α form, C-8), 40.1 (CH, C-6a), 37.2 (CH2, C-1′), 34.4 (CH2, C-7),
34.3 (CH, C-10a), 27.1 (CH3, C-13), 24.4 (CH2, C-2′), 20.2 (CH3,
C-11), 19.3 (CH3, C-12), 13.5 (CH3, C-3′); HRMS (ESI) m/z
325.2298 [M + Na]+ (calcd 325.1774 for C19H26O3).
E
https://dx.doi.org/10.1021/acs.jnatprod.9b00831
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
pubs.acs.org/jnp
Article
human recombinant CB1R (Bmax = 2.5 pmol/mg protein) and human
recombinant CB2R (Bmax = 4.7 pmol/mg protein) were incubated
with [3H]-CP-55,940 (0.14 nM/Kd = 0.18 nM and 0.084 nM/Kd =
0.31 nM respectively for CB1R and CB2R) as the high-affinity ligand.
Competition curves were generated by displacing [3H]-CP-55,940
with increasing concentrations of compounds (0.1 nM to 10 μM).
Nonspecific binding was defined by 10 μM WIN55,212-2 as the
heterologous competitor (Ki values 9.2 and 2.1 nM respectively for
CB1R and CB2R). IC50 values were determined for compounds
showing >50% displacement at 10 μM. All compounds were tested
following the procedure described by the manufacturer (PerkinElmer,
Milan, Italy). Displacement curves were generated by incubating
drugs with [3H]-CP-55,940 for 90 min at 30 °C. Ki values were
calculated by applying the Cheng−Prusoff equation to the IC50 values
(obtained by GraphPad) for the displacement of the bound
radioligand by increasing concentrations of the test compound.
Data represent mean values of three independent experiments
performed in duplicate and are expressed as the average of IC50 and
Ki (nM) standard deviation.
REFERENCES
■
(1) Gaoni, Y.; Mechoulam, R. J. Am. Chem. Soc. 1964, 86, 1646−
1647.
(2) Merkus, F. W. H. M. Nature 1971, 232, 579−580.
(3) Gill, E. W.; Paton, W. D. M.; Pertwee, R. G. Nature 1970, 228,
134−136.
(4) ElSohly, M. A.; Slade, D. Life Sci. 2005, 78, 539−548.
(5) Grotenhermen, F. Clin. Pharmacokinet. 2003, 42, 327−360.
(6) Formukong, E. A.; Evans, A. T.; Evans, F. Phytother. Res. 1989, 3,
219−231.
(7) Pertwee, R. G. Br. J. Pharmacol. 2008, 153, 199−215.
(8) Thomas, A.; Stevenson, L. A.; Wease, K. N.; Price, M. R.; Baillie,
G.; Ross, R. A.; Pertwee, R. G. Br. J. Pharmacol. 2005, 146, 917−926.
(9) Barth, F. Annu. Rep. Med. Chem. 2005, 40, 103−118.
(10) Rayman, N.; Lam, K. H.; Van der Holt, B.; Koss, C.; Van
Leeuwen, J.; Budel, L. M.; Mulder, A. H.; Sonneveld, P.; Delwel, R.
Eur. J. Haematol. 2011, 86, 466−476.
(11) Carayon, P.; Marchand, J.; Dussossoy, D.; Derocq, J. M.; Jbilo,
O.; Bord, A.; Bouaboula, M.; Galiegue, S.; Mondiere, P.; Penarier, G.;
̀
̀
́
Le Fur, G.; Defrance, T.; Casellas, P. Blood 1998, 92, 3605−3615.
ASSOCIATED CONTENT
* Supporting Information
̀
̀
■
(12) Galiegue, S.; Mary, S.; Marchand, J.; Dussossoy, D.; Carriere,
D.; Carayon, P.; Bouaboula, M.; Shire, D.; Le Fur, G.; Casellas, P. Eur.
J. Biochem. 1995, 232, 54−61.
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jnatprod.9b00831.
(13) Ashton, J. C.; Glass, M. Curr. Neuropharmacol. 2007, 5, 73−80.
(14) Klegeris, A.; Bissonnette, C. J.; McGeer, P. L. Br. J. Pharmacol.
2003, 139, 775−786.
1H NMR, COSY, HSQC edited spectral data of
compounds 2−11 and structures of the known
compounds (PDF)
(15) Van Sickle, M. D.; Duncan, M.; Kingsley, P. J.; Mouihate, A.;
Urbani, P.; Mackie, K.; Stella, N.; Makriyannis, A.; Piomelli, D.;
Davison, J. S.; Marnett, L. J.; Di Marzo, V.; Pittman, Q. J.; Patel, K.
D.; Sharkey, K. A. Science 2005, 310, 329−332.
AUTHOR INFORMATION
Corresponding Author
(16) Karlsen, M.; Liu, H.; Johansen, J. E.; Hoff, B. H. Molecules
2014, 19, 13526−13540.
■
(17) Mechoulam, R.; Ben-Zvi, Z.; Agurell, S.; Nilsson, I. M.; Nilsson,
J. L. G.; Edery, H.; Grunfeld, Y. Experientia 1973, 29, 1193−1195.
(18) Razdan, R. K.; Uliss, D. B.; Dalzell, H. C.; Hashish. J. Am.
Chem. Soc. 1973, 95, 2361−2362.
Francesco Tinto − Institute of Biomolecular Chemistry,
National Research Council of Italy, Pozzuoli, NA 80078, Italy;
orcid.org/0000-0002-7818-591X; Email: francesco.tinto@
icb.cnr.it
(19) Nikas, S. P.; Thakur, G. A.; Parrish, D.; Alapafuja, S. O.;
Huestis, M. A.; Makriyannis, A. Tetrahedron 2007, 63 (34), 8112−
8123.
Authors
Rosaria Villano − Institute of Biomolecular Chemistry, National
Research Council of Italy, Pozzuoli, NA 80078, Italy
Magdalena Kostrzewa − Institute of Biomolecular Chemistry,
National Research Council of Italy, Pozzuoli, NA 80078, Italy;
Institute of Genetics and Biophysics, National Research Council
of Italy, Naples 80078, Italy
Alessia Ligresti − Institute of Biomolecular Chemistry, National
Research Council of Italy, Pozzuoli, NA 80078, Italy;
orcid.org/0000-0003-1787-3900
Hannah Straker − GW Pharmaceuticals, Kent ME9 8AG,
United Kingdom
Emiliano Manzo − Institute of Biomolecular Chemistry,
National Research Council of Italy, Pozzuoli, NA 80078, Italy;
orcid.org/0000-0002-7394-020X
(20) Ben-Zvi, Z.; Mechoulam, R. Tetrahedron Lett. 1970, 6, 4495−
4497.
(21) Radwan, M. M.; ElSohly, M. A.; El-Alfy, A. T.; Ahmed, S. A.;
Slade, D.; Husni, A. S.; Manly, S. P.; Wilson, L.; Seale, S.; Cutler, S. J.;
Ross, S. A. J. Nat. Prod. 2015, 78, 1271−1276.
(22) Choi, Y. H.; Hazekamp, A.; Peltenburg-Looman, A. M. G.;
́
́
Frederich, M.; Erkelens, C.; Lefeber, A. W. M.; Verpoorte, R.
Phytochem. Anal. 2004, 15, 345−354.
(23) Elsohly, M. A.; Feng, S.; Murphy, T. P.; Warrington, A. W.;
Ross, S.; Nimrod, A.; Mehmedic, Z.; Fortner, N. J. Anal. Toxicol.
2001, 25, 476−480.
(24) Bal, B. S.; Childers, W. E.; Pinnick, H. W. Tetrahedron 1981, 37,
2091−2096.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jnatprod.9b00831
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank GW Research Ltd. for the supply of Δ9-
THCV and for financial support. M.K. is a recipient of a
scholarship from the INCIPIT PhD program, which is
cofunded by the COFUND scheme Marie Skłodowska-Curie
Actions. We also thank Professor Vincenzo Di Marzo for his
critical analysis of the data and review of the manuscript.
F
https://dx.doi.org/10.1021/acs.jnatprod.9b00831
J. Nat. Prod. XXXX, XXX, XXX−XXX