Bornyl- and isobornyl-Δ8-tetrahydrocannabinols: A novel class of cannabinergic ligands

Article abstract of DOI:10.1021/jm8005299

Structure-activity relationship studies of classical cannabinoid analogues have established that the C3 aliphatic side chain plays a pivotal role in determining cannabinergic potency. In earlier work, we provided evidence for the presence of subsites within the CB1 and CB2 cannabinoid receptor binding domains that can accommodate bulky conformationally defined substituents at the C3 alkyl side chain pharmacophore of classical cannabinoids. We have now extended this work with the synthesis of a series of Δ⁸-THC analogues in which bornyl substituents are introduced at the C3 position. Our results indicate that, for optimal interactions with both CB1 and CB2 receptors, the bornyl substituents need to be within close proximity of the tricyclic core of Δ⁸-THC and that the conformational space occupied by the C3 substituents influences CB1/CB2 receptor subtype selectivity.

Full text of DOI:10.1021/jm8005299

J. Med. Chem. 2008, 51, 6393–6399
6393
Bornyl- and Isobornyl-∆8-tetrahydrocannabinols: A Novel Class of Cannabinergic Ligands
Dai Lu, Jianxin Guo, Richard I. Duclos, Jr., Anna L. Bowman, and Alexandros Makriyannis*
Center for Drug DiscoVery, Northeastern UniVersity, 360 Huntington AVenue, 116 Mugar Life Sciences Building, Boston, Massachusetts 02115
ReceiVed May 7, 2008
Structure-activity relationship studies of classical cannabinoid analogues have established that the C3 aliphatic
side chain plays a pivotal role in determining cannabinergic potency. In earlier work, we provided evidence
for the presence of subsites within the CB1 and CB2 cannabinoid receptor binding domains that can
accommodate bulky conformationally defined substituents at the C3 alkyl side chain pharmacophore of
classical cannabinoids. We have now extended this work with the synthesis of a series of ∆8-THC analogues
in which bornyl substituents are introduced at the C3 position. Our results indicate that, for optimal interactions
with both CB1 and CB2 receptors, the bornyl substituents need to be within close proximity of the tricyclic
core of ∆8-THC and that the conformational space occupied by the C3 substituents influences CB1/CB2
receptor subtype selectivity.
Introduction
The classical cannabinoids found in cannabis have moderate
receptor binding affinities and signal transduction properties,
yet exhibit substantial potency in vivo.1,2 Phytocannabinoids
∆8- and ∆9-tetrahydrocannabinol (∆8-THCa and ∆9-THC) are
classical cannabinoids that have comparable potency3-6 and bind
almost equally4-6 to the two G protein-coupled cannabinoid
receptors7,8 CB19 and CB2.10 Structure-activity relationship
(SAR) studies of cannabinoid analogues with affinities for the
CB111-13 and CB212,14,15 receptors, including the utilization of
high affinity ligands with reactive functional groups suitable
for characterizing (ligand-assisted protein structure, LAPS) the
ligand binding domains of CB1 and CB2 subtypes,16-18 have
provided experimental three-dimensional structural information
about these membrane proteins in the absence of nuclear
magnetic resonance (NMR) or X-ray crystallographic structural
data. These SAR studies have identified the C1 phenolic
hydroxyl group and the C3 side chain as key pharmacophoric
features of classical cannabinoids. Athough the C1 phenolic
hydroxyl is not critical for CB2 affinity,12,15,19 the C3 side chain
pharmacophore of these ligands is a key factor in determining
receptor affinity and pharmacological potency for both CB1 and
CB2. Reviews of SAR on the C3 side chain of ∆9-THC, and
particularly with the relatively more stable ∆8-THC, have
suggested that optimal activity is obtained with a seven- or eight-
carbonchainsubstitutedwith1′,1′-or1′,2′-dimethylgroups.11,20-22
The flexibility of this side chain has hampered efforts to
elucidate the precise nature of the pharmacophoric conformation
of THC analogues with cannabinoid receptors. Recent studies
in which the C3 side chain carries aryl,23,24 cycloalkyl,25,26 or
other conformationally restricted moieties19,27 have increased
our understanding of the pharmacophoric features of this side
chain. Our earlier results on adamantyl cannabinoids28 suggested
the existence of distinct subsites within the CB1 and CB2
cannabinoid receptor binding domains occupied by bulky
conformationally restricted substituents at the C3 position.
Figure 1. Phytocannabinoid (-)-∆8-THC (1) was used as the prototype
to examine the steric effects of bulky, rigid, rotatable groups at C3 of
classical cannabinoids.
Exploration of the allowable conformational space for these side
chains has provided us with insights regarding the pharmacoph-
oric features required for CB1 and CB2 selectivities.
We have now examined the bornyl and isobornyl groups as
bulky hydrophobic C3 substituents on the prototypic classical
cannabinoid (-)-∆8-THC (1, Figure 1). The bornyl groups
derived from (+)-camphor were introduced at the C3 position
of ∆8-THC, either directly or with a methylene spacer. These
novel analogues were tested for their binding affinities for CB1
and CB2 receptors to further explore what conformational
characteristics of the cannabinoid C3 substituent are optimal
for interaction with the two receptor subtypes.
Results
Chemistry. The bornyl- and isobornyl-∆8-THC analogues
8a and 8b, respectively, were synthesized as shown in Scheme
1. The Grignard reagent prepared from 1-chloro-3,5-dimethoxy-
benzene (2) reacted with (+)-(1R)-camphor (3) to give a 2:1
mixture of adducts 4a:4b that was reduced with lithium/
ammonia to give a 3:1 mixture of 5-bornyl-1,3-dimethoxyben-
zene (5a) and 5-isobornyl-1,3-dimethoxybenzene (5b).29 Treat-
ment of the mixture with boron tribromide provided a
corresponding 3:1 mixture of the demethylated products,
5-bornylresorcinol (6a) and 5-isobornylresorcinol (6b). Follow-
ing a well-established protocol,30-32 condensation of the mixture
of resorcinols 6a and 6b with (+)-trans-p-mentha-2,8-dien-1-
ol (7) catalyzed by p-toluenesulfonic acid monohydrate gave a
3:1 mixture of the corresponding epimeric ∆8-tetrahydrocan-
nabinol analogues, which were separated by flash column
chromatography. The major product was bornyl-∆8-THC 8a,
which exhibited the characteristic “W” coupling between the
* To whom correspondence should be addressed. Phone: +1-617-373-
4200. Fax: +1-617-373-7493. E-mail: a.makriyannis@neu.edu.
a
Abbreviations: BSA, bovine serum albumin; Ki, inhibition constant;
LAPS, ligand-assisted protein structure; SAR, structure-activity relation-
ship; THC, tetrahydrocannabinol; TME buffer, 25 mM Tris, 5 mM MgCl2,
1 mM EDTA buffer; COSY, correlation spectroscopy; NOESY, nuclear
Overhauser enhancement spectroscopy.
10.1021/jm8005299 CCC: $40.75
2008 American Chemical Society
Published on Web 10/01/2008

6394 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Lu et al.
Scheme 1. Synthesis of Bornyl- and Isobornyl-∆8-THC (8a and 8b)a
a (a) Mg/THF, reflux, 8 h; (b) Li/NH3, THF, -78 °C, 6 h; (c) BBr3, CH2Cl2, 0 °C to rt; (d) p-TsOH·H2O, CHCl3, 65 °C, 6 h.
C2′-Hexo benzylic proton (δ 2.89) and C6′-Hexo. The minor
product, isobornyl-∆8-THC 8b exhibited the characteristic
apparent triplet for the C2′-Hendo benzylic proton (δ 2.72). 2D
NMR was employed to further characterize the structures of
8a and 8b. Nuclear Overhauser effects (NOE) were observed
between both C2-H and C4-H aromatics of the cannabinoid A
ring with the bornyl C1′-methyl, C3′-Hendo, C5′-Hendo, as well
as C6′-Hendo protons for the endo-adduct 8a. NOE was also
observed between C2′-Hexo and the syn-C7′-methyl of endo-
adduct 8a. NOEs were observed between the C2-H/C4-H
aromatics and C3′-Hexo, as well as between C2′-Hendo and C6′-
Hendo for the exo-adduct 8b (see Supporting Information).
The corresponding bornylmethyl- and isobornylmethyl-∆8-
THC analogues 8c and 8d, respectively, were synthesized as
shown in Scheme 2. The Grignard reagent prepared from 3,5-
dimethoxybenzylchloride (9) reacted with (+)-(1R)-camphor (3)
to give a 9.3:1 mixture of endo-adduct 4c to exo-adduct 4d.
The mixture was then treated with p-toluenesulfonic acid
monohydrate in CH2Cl2 to afford an 8:1 mixture of alkenes 10.
Hydrogenation of the mixture gave a 1:3.4 mixture of endo-
adduct 5c to the exo-adduct 5d, an observation that is consistent
with literature precedents for endo approach of larger reagents
to 2-methylidene-7,7-dimethylbicyclo[2.2.1]heptanes.33-35 The
mixture was inseparable by both flash chromatography and
chiral HPLC and was used without further separation. Dem-
ethylation of the mixture with BBr3 gave a corresponding
mixture of bornylmethylresorcinol 6c and isobornylmethylre-
sorcinol 6d that was also inseparable and was used directly in
the Friedel-Crafts type reaction with (+)-trans-p-mentha-2,8-
dien-1-ol (7). The corresponding 1:3.4 mixture of endo- to exo-
diastereomers was separated by chiral HPLC on a Chiralpak
AD column to afford bornylmethyl-∆8-THC (8c) and isobornyl-
methyl-∆8-THC (8d). The structures of 8c and 8d were
confirmed by 2D NMR. The NOESY spectrum of the minor
product, endo-adduct 8c, showed an NOE between the benzylic
C1′′ methylene and C6′-Hendo, while the NOESY spectrum of
the major product, exo-adduct 8d, clearly showed NOEs between
the benzylic C1′′ methylene and the isobornyl C1′-methyl and
syn-C7′-methyl groups. (see Supporting Information).
Receptor Binding Studies. Using purified rat forebrain
synaptosomes36 as a source of CB1 and using mouse spleen
membranes37 as a source of CB2, competition binding affinities
with radiolabeled (-)-5-(1,1-dimethylheptyl)-2-[(1R,2R,5R)-5-
hydroxy-2-(3-hydroxypropyl)-cyclohexyl]phenol (CP-55,940)
were performed as previously detailed28,38 for ∆8-THC (1) and
the newly synthesized analogues in which the phytocannabinoid
five-carbon side chain at C3 was replaced with bulky but
conformationally more defined bornyl 8a, isobornyl 8b, bornyl-
methyl 8c, and isobornylmethyl 8d substituents. Inhibition
constant values (IC50) determined from the respective displace-
ment curves were converted to Ki values (Table 1) according
to the reported method.39 As can be seen from the Ki values,
structural modifications of the C3 substituent affects cannabinoid
receptor affinities as well as CB1/CB2 selectivities. The bornyl-
∆8-THC analogue 8a exhibits robust affinities for both CB1
and CB2, exceeding those of ∆8-THC. Interestingly, the
isobornyl-∆8-THC epimer 8b exhibits 10-fold CB2 selectivity
due to a loss of binding affinity at the CB1 subtype, while the
binding affinity of this isobornyl epimer 8b to CB2 remained
comparable to that of the bornyl epimer 8a. The possibility exists

Bornyl- and Isobornyl-∆8-tetrahydrocannabinols
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6395
Scheme 2. Synthesis of Bornylmethyl- and Isobornylmethyl-∆8-THC (8c and 8d)a
a (a) (1) Mg/THF; (2) (+)-(1R)-camphor (3), reflux, 8 h. (b) p-TsOH·H2O, CH2Cl2, 45 °C, 1 h. (c) H2, 10% Pd/C, EtOH, rt. (d) BBr3, CH2Cl2, 0 °C to
rt. (e) p-TsOH·H2O, CHCl3, 65 °C, 6 h.
that the selectivities could be different within the same rodent
species; however, the comparisons made here between rCB1
and mCB2 are arguably relevant because of the close homology
between rat and mouse CB140,41 and CB242 receptors.
When the bornyl or isobornyl groups are positioned further
from the aromatic A-ring by the introduction of a flexible C1′′
methylene link, affinities for CB1 and CB2 become comparable
to that of ∆8-THC (1). The endo-bornylmethyl analogue 8c and
its exo-epimer 8d are also nonselective.
conformers available and creating a greater accessible confor-
mational space. This analogue is less well tolerated in the CB1
receptor, implying that the CB1 binding pocket is unable to
accommodate the larger substituent. Extension to the corre-
sponding 3-bornylmethyl- and 3-isobornylmethyl-∆8-THC ana-
logues (8c and 8d, respectively) further increases the rotational
freedom of these bulky C3 substituents with a concomitant
increase in the number of low energy conformers expanding
the available conformational space.
Molecular Modeling. Among other considerations, the con-
formational space available to the novel analogues 8a-d offers
insight into the structural features required for CB1 and CB2
selectivity. Conformational scans were performed using a dihedral
drive around the C3-C2′ bonds of 8a and 8b as well as the
corresponding C3-C1′′ bonds of methylene analogues 8c and 8d
to explore the permissible rotations of the bulky substituents.
Rotation around the C1′′–C2′ bonds was also considered for
3-bornylmethyl-∆8-THC (8c) and 3-isobornylmethyl-∆8-THC (8d).
The dihedral angle was restrained at a value between 0° and 360°
in 1° steps, and minimization on the remaining geometric param-
eters was performed with the MM3* force field.43 Conformers
within 8 kcal mol-1 of the global minimum were retained. All
calculations were performed in Macromodel.44
Discussion
It is instructive to compare the present data with our earlier work
involving adamantyl substituents at the C3 position of ∆8-THC.28
These earlier results demonstrated that the cannabinoid side chain
pharmacophoric site of the CB1 and CB2 receptors is capable of
accommodating bulky substituents. However, the data also sug-
gested that this subsite has distinctive features for each of the two
receptors. The adamant-1′-yl analogue 11 (Table 1, Figure 2)
exhibited substantial CB1 selectivity. We postulated that the
adamant-1′-yl substituent has a favorable fit at the CB1 subsite,
allowing for optimal hydrophobic interactions with corresponding
CB1 amino acid residues while it engages in a looser suboptimal
fit at the CB2 subsite. Conversely, the adamant-2′-yl analogue,
which is capable of accessing a more expanded conformational
space, was CB2 selective.
3-Bornyl-∆8-THC (8a) has the smallest available conforma-
tional space of analogues 8a-d (Figure 2) and has high affinity
for both receptors with no subtype selectivity. The isobornyl
group of the corresponding C2′ epimer 8b has reduced steric
hindrance, thereby increasing the number of low energy
The bornyl group is also a rigid, compact, ten-carbon, hydro-
phobic, rotatable substituent. A comparison of the available

6396 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Lu et al.
Table 1. Affinities (Ki) of ∆8-THC Analogues for CB1 and CB2
Cannabinoid Receptorsa
Figure 2. Comparison of the conformational space for adamantyl and
bornyl side chains of various ∆8-THC analogues. Accessible conformers
within 8 kcal mol-1 of the global energy minimum for 3-(adamant-
1′-yl)-∆8-THC (11, green), 3-bornyl-∆8-THC (8a, magenta), 3-isobornyl-
∆8-THC (8b, orange), and 3-bornylmethyl-∆8-THC (8c, blue). The
accessible conformational space for 3-isobornylmethyl-∆8-THC (8d)
(not shown) is similar to that of 8c. The global minimum energy
conformer for each ligand is shown in stick display.
a Affinities for CB1 and CB2 were determined using rat brain (CB1) or
mouse spleen (CB2) membranes and [3H]CP55,940 as the radioligand
following previously described procedures.28,38 Ki values were obtained from
three independent experiments run in duplicate and are expressed as the
mean of the three values, 95% confidence limits are indicated in the
parenthesis. b Previously reported.28
This provides us with valuable stereochemical information that can
be used for the design of novel cannabinergic ligands with enhanced
affinities and CB1/CB2 selectivities.
conformational space for the novel terpene analogues 8a and 8b
suggests some interesting conclusions. We postulate that the bornyl
analogue 8a (Figure 2) can be accommodated optimally in both
receptors. However, the isobornyl group of analogue 8b accesses
a somewhat larger conformational space that can be optimally
accommodated only within the CB2 subsite. This epimer 8b is
not well tolerated in the spatially more restricted CB1 binding
pocket, which may account for its 10-fold CB2 selectivity.
However, steric arguments may not fully account for ligand
specificity and a combination of enthalpic and entropic reasoning
would ultimately explain ligand affinity for the receptor.
When the terpene substituent is separated by a methylene group
from the tricyclic cannabinoid structure as in 8c and 8d, the
available conformational space is significantly larger. Their interac-
tions with hydrophobic residues are impeded, resulting in reduced
affinities of 8d for both cannabinoid receptors and a complete loss
of subtype selectivities of 8c and 8d, as was the case for the
corresponding adamantyl methylene analogues.28 We thus postulate
that, in order to achieve subtype selectivity, the bulky C3
substituents not only need to be in close proximity to the tricyclic
core but also must have an appropriate allowable conformational
volume for an optimized interaction with each CB receptor.
We conclude that substituting the cannabinoid C3 side chain
with conformationally restricted rigid groups can be successfully
employed to probe the binding domains for the key cannabinoid
C3 side chain pharmacophore within the CB1 and CB2 receptors.
Summary
The results from our earlier work and this study revealed that
rigid bulky groups at C3 in the direct proximity of tricyclic
classical cannabinoid structures can be accommodated within
the CB1 and CB2 binding sites. Substituting bulky aliphatic
substituents for the n-pentyl chain at the 3-position of the
classical phytocannabinoid ∆8-THC (1) gave analogues with
higher affinities for each of the cannabinoid receptors. The
affinities and selectivities of these novel ligands for the receptor
subtypes may be explained by the conformational space
occupied by the bulky C3 substituents with respect to the
tricyclic cannabinoid structure. The novel isobornyl analogue
8b was found to have 10-fold CB2 selectivity.
Materials and Experimental Procedures
General Synthetic Methods. (+)-trans-p-Mentha-2,8-dien-1-ol
(7) was purchased from Firmenich Inc., Princeton, NJ. All other
reagents and solvents were purchased from Sigma-Aldrich, Milwaukee,
WI, and used without further purification. All reactions were performed
with magnetic stirring under a static argon or nitrogen atmosphere in
flame-dried glassware using scrupulously dry solvents. Organic phases
were dried over Na2SO4, rotary evaporated under reduced pressure,
and flash column chromatography employed silica gel 60 (230-400

Bornyl- and Isobornyl-∆8-tetrahydrocannabinols
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20 6397
mesh, Selecto Scientific Inc., Suwanee, GA). Semipreparative chiral
HPLC used a Chiralpak AD column (250 mm × 10 mm, Chiral
Technologies Inc., Exton, PA). All compounds were demonstrated to
be homogeneous by analytical thin-layer chromatography on precoated
silica gel TLC aluminum plates (Whatman, UV254, layer thickness 250
µm), and chromatograms were visualized under ultraviolet light or by
phosphomolybdic acid staining. Melting points were determined on
an Electrothermal capillary melting point apparatus and are uncorrected.
1H NMR spectra were recorded on Bruker DMX-500 spectrometer
operating at 500 MHz. All NMR spectra were recorded using CDCl3
as solvent, and chemical shifts are reported in ppm relative to
tetramethylsilane as an internal standard with multiplicities indicated
as b (broadened), s (singlet), d (doublet), t (triplet), q (quartet), m
(multiplet). Specific rotations were measured on a Rudolph Autopol
II polarimeter in a 1.00 dm cell. Low and high resolution mass spectra
were performed at the School of Chemical Sciences, University of
Illinois at UrbanasChampaign or were recorded on a Hewlett-Packard
6890 GC/MS instrument at the Center for Drug Discovery. Elemental
analyses were performed at Baron Consulting Co., Milford, CT.
General Procedure A: Preparation of 5-Alkylresorcinols (6a-6d)
from 5-Alkyl-1,3-dimethoxybenzenes (5a-5d). A solution of boron
tribromide (2.1 mL of 1.0 M) in CH2Cl2 was added dropwise to a
stirred solution of 2.0 mmol of 5-alkyl-1,3-dimethoxybenzenes
(5a-5d) in 20 mL of CH2Cl2 at 0 °C. The reaction mixture was
then stirred at 0 °C for 2 h and allowed to warm to room temperature
over a period of time ranging between 6 and 16 h. Upon completion,
the reaction mixture was cooled in an ice bath and cold water was
added cautiously. The organic layer was separated and washed with
H2O, brine, and dried. Filtration, solvent removal, and purification
by flash column chromatography (1:2 acetone/petroleum ether)
provided 5-alkylresorcinols (6a-6d).
225 mg of a crude 3:1 mixture of endo-adduct 8a to exo-adduct
8b. The crude product was chromatographed (10:90 Et2O/petroleum
ether) to give 95 mg (0.25 mmol, 60%) of 8a as a white solid
followed by 32 mg (0.084 mmol, 20%) of 8b as a white solid. 8a
(endo-adduct, major isomer); mp 84-85 °C; H NMR δ 6.30 (d,
J2,4 ) 1.6 Hz, 1H, 4), 6.14 (d, J2,4 ) 1.6 Hz, 1H, 2), 5.42-5.45
(m, 1H, 8), 4.70 (s, 1H, OH), 3.19 (dd, J10R,10ꢀ ) 17.0 Hz, J10R,10a
1
) 4.3 Hz, 1H, 10R), 2.89 (ddd, J2′exo,3′exo ) 11.6 Hz, J2′exo,3′endo
)
5.4 Hz, 4J2′exo,6′exo ) 2.4 Hz, 1H, benzylic 2′exo), 2.71 (ddd, J10a,10ꢀ
) 11.1 Hz, J10a,6a ) 11.1 Hz, J10a,10R ) 4.3 Hz, 1H, 10a), 2.12-2.16
(m, 1H, 7ꢀ), 2.09 (dddd, J2′exo,3′exo ) 11.6 Hz, J3′exo,3′endo ) 13.3
Hz, J3′exo,4′ ) 4.5 Hz,4J3′exo,5′exo ) 3.3 Hz, 1H, 3′exo), 1.85-1.92
(m, 1H, 10ꢀ), 1.79-1.86 (m, 2H, 6a,7R), 1.74-1.81 (m, 1H, 5′exo),
1.71 (s, 3H, 9-CH3), 1.70 (dd, J3′exo,4′ ) 4.5 Hz, J4′,5′exo ) 4.5 Hz,
1H, 4′), 1.41-1.47 (m, 2H, 3′endo, 6′endo), 1.38 (s, 3H, 6-ꢀ-CH3),
1.30 (ddd, J5′endo,5′exo ) 12.4 Hz, J5′endo,6′endo ) 9.5 Hz, J5′endo,6′exo
) 4.6 Hz, 1H, 5′endo), 1.13 (dddd, J6′exo,6′endo ) 12 Hz, J6′exo,5′exo
4
) 11 Hz, J6′exo,5′endo ) 4.6 Hz, J2′exo,6’exo ) 2.4 Hz, 1H, 6′exo),
1.11 (s, 3H, 6-R-CH3), 0.98 (s, 3H, syn-7′-CH3), 0.90 (s, 3H, anti-
22
7′-CH3), 0.75 (s, 3H, 1′-CH3); [R]D -220° (c 0.223, CH2Cl2);
MS m/z 380 (M+); Anal. (C26H36O2 ·1/2H2O) C, H. 8b (exo-adduct,
1
minor isomer); mp 86-88 °C; H NMR δ 6.34 (d, J2,4 ) 1.3 Hz,
1H, 4), 6.15 (d, J2,4 ) 1.3 Hz, 1H, 2), 5.41-5.44 (m, 1H, 8), 4.63
(s, 1H, OH), 3.17 (dd, J10R,10ꢀ ) 17.0 Hz, J10R,10a ) 4.2 Hz, 1H,
10R), 2.72 (dd, J2′endo,3′endo ) 8.5 Hz, J2′endo,3′exo ) 8.5 Hz, 1H,
benzylic 2′endo), 2.68 (ddd, J10a,10ꢀ ) 11.1 Hz, J10a,6a ) 11.1 Hz,
J10a,10R ) 4.4 Hz, 1H, 10a), 2.07-2.23 (m, 2H, 3′exo, 7ꢀ),
1.84-1.92 (m, 1H, 10ꢀ), 1.74-1.85 (m, 4H, 6a, 7R, 4′, 5′exo),
1.70 (s, 3H, 9-CH3), 1.53-1.64 (m, 2H, 3′endo, 6′exo), 1.37 (s,
3H, 6-ꢀ-CH3), 1.22-1.30 (m, 2H, 5′endo, 6′endo), 1.09 (s, 3H,
6-R-CH3), 0.82 (s, 6H, syn-7′-CH3, anti-7′-CH3), 0.79 (s, 3H, 1′-
24
CH3); [R]D -200° (c 0.223, CH2Cl2); MS m/z 380 (M+); Anal.
General Procedure B: Synthesis of (-)-∆8-Tetrahydrocannab-
inol Analogues (8a-8d). A mixture of 1.0 mmol of 5-alkylresor-
cinols (6a-6d), 1.1 mmol of (+)-trans-p-mentha-2,8-dien-1-ol (7),
and 0.1 mmol of p-toluenesulfonic acid monohydrate in 15 mL of
anhydrous CHCl3 was stirred and heated at 65 °C for 6 h. The
reaction was monitored by TLC (15:85 EtOAc/petroleum ether).
Upon completion, the reaction mixture was cooled and diluted with
10 mL of CH2Cl2 and stirred with 10 mL of saturated aqueous
NaHCO3 solution for 15 min. The organic phase was then separated
and washed with H2O, brine, and then dried. Filtration, concentra-
tion, and purification by flash column chromatography (1:10 EtOAc/
petroleum ether) provided the ∆8-tetrahydrocannabinol analogues
8a-8d.
(C26H36O2 ·1/2H2O) C, H.
2′-(3,5-Dimethoxybenzyl)-1′,7′,7′-trimethylbicyclo[2.2.1]heptan-
2′-ols (4c and 4d). A solution of 3,5-dimethoxybenzyl chloride (9)
(1.02 g, 5.46 mmol) in anhydrous Et2O (25 mL) was added
dropwise to a flask containing magnesium (132 mg, 5.43 mmol)
over a period of 1.5 h. The reaction mixture was heated with a 50
°C oil bath for 1 h, and then a solution of (+)-(1R)-camphor (3)
(760 mg, 4.99 mmol) in 25 mL of Et2O was added dropwise. The
reaction mixture was refluxed for 2 h and was then cooled to room
temperature and treated cautiously with saturated aqueous NH4Cl
(12 mL). The Et2O phase was separated and washed with water,
brine, and then dried. Removal of solvent gave 1.80 g of crude
product, which was chromatographed (20:80 acetone/petroleum
ether) to afford 1.25 g (4.11 mmol, 82%) of a 9.3:1 mixture of
2′-(3,5-Dimethoxyphenyl)-1′,7′,7′-trimethylbicyclo[2.2.1]heptan-
2′-ols (4a and 4b). See ref 29.
2′-(3,5-Dimethoxyphenyl)-1′,7′,7′-trimethylbicyclo[2.2.1]hepta-
1
endo-adduct 4c to exo-adduct 4d as a viscous liquid. H NMR δ
4c (endo-adduct, major isomer) 6.43 (d, J ) 2.1 Hz, 2H), 6.36 (t,
J ) 2.1 Hz, 1H), 3.78 (s, 6H), 2.77 (d, J ) 13.0 Hz, 1H), 2.73 (d,
J ) 13.0 Hz, 1H), 2.30 (bs, 1H), 1.82-1.86 (m, 1H), 1.74-1.78
(m, 2H), 1.65 (d, J ) 13.0 Hz, 1H), 1.57 (d, J ) 13.0 Hz, 1H),
1.45-1.49 (m, 1H), 1.11-1.15 (m, 1H), 1.08 (s, 3H), 0.87 (s, 3H),
0.83 (s, 3H); 4d (exo-adduct, minor isomer, partial data) 6.33 (d, J
) 1.6 Hz, 2H), 6.29 (t, J ) 1.6 Hz, 1H), 3.77 (s, 6H), 2.77 (d, J
) 13.0 Hz, 1H), 2.73 (d, J ) 13.0 Hz, 1H), 2.33-2.38 (m, 1H),
2.09 (t, 1H), 1.91-1.98 (m, 1H), 1.76-1.81 (m, 2H), 1.60-1.64
(m, 1H), 0.96 (s, 3H), 0.91 (s, 3H), 0.83 (s, 3H); MS m/z 304 (M+).
2′-(3,5-Dimethoxybenzylidene)-1′,7′,7′-trimethylbicyclo[2.2.1]-
heptanes (10). The mixture (880 mg, 2.89 mmol) of 4c and 4d
and 100 mg of p-toluenesulfonic acid monohydrate in 15 mL of
anhydrous CH2Cl2 was stirred and heated at 45 °C for 1 h. The
reaction mixture was cooled and washed with saturated aqueous
NaHCO3 solution, water, brine, and then dried. Removal of solvent
gave 820 mg of crude product as a liquid, which was chromato-
graphed (10:90 Et2O/petroleum ether) to afford 790 mg (2.76 mmol,
96%) of 10 as a liquid that was a 1:8 mixture of cis and trans
nes (5a and 5b). See ref 29.
5-(1′,7′,7′-Trimethylbicyclo[2.2.1]hept-2′-yl)resorcinols (6a and
6b). A 3:1 mixture of 6a and 6b (170 mg, 0.69 mmol, 79%) was
prepared from 240 mg (0.87 mmol) of the mixture of 5a and 5b
following general procedure A to give a white solid. The product
was determined to be a 3:1 mixture of endo- to exo-adducts by
NMR: mp 52-55 °C. 1H NMR δ 6a (endo-adduct, major isomer)
6.38 (d, J ) 2.0 Hz, 2H), 6.22 (t, J ) 2.0 Hz, 1H), 4.95 (bs, 2H),
2.92 (ddd, J ) 9.0, 5.0, 2.0 Hz, 1H, benzylic 2exo), 2.08-2.13
(m, 1H), 1.73-1.78 (m, 1H), 1.71 (t, J ) 4.0 Hz, 1H), 1.36-1.43
[m, 2H, especially 1.41 (dd, J ) 13.0, 5.5 Hz, 1H)], 1.24-1.30
(m, 1H), 1.11-1.17 (m, 1H), 0.99 (s, 3H), 0.91 (s, 3H), 0.74 (s,
3H); 6b (exo-adduct, minor isomer) 6.35 (d, J ) 2.0 Hz, 2H), 6.16
(t, J ) 2.0 Hz, 1H), 4.91 (bs, 2H), 2.77 (dd, J ) 8.5, 8.5 Hz, 1H,
benzylic 2endo), 2.13-2.17 (m, 1H), 1.77-1.83 (m, 2H), 1.56-1.64
(m, 2H), 1.26-1.30 (m, 2H), 0.82 (s, 3H), 0.80 (s, 3H), 0.78 (s,
3H). MS m/z 246 (M+).
(6aR-trans)-3-(endo-1′,7′,7′-Trimethylbicyclo[2.2.1]hept-2′-yl)-
6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran-1-
ol (3-Bornyl-∆8-THC, 8a) and (6aR-trans)-3-(exo-1′,7′,7′-Tri-
methylbicyclo[2.2.1]hept-2′-yl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-
6H-dibenzo[b,d]pyran-1-ol (3-Isobornyl-∆8-THC, 8b). A 3:1 mixture
of 6a and 6b (160 mg, 0.42 mmol) was condensed with (+)-trans-
p-2,8-menthadien-1-ol (7) following general procedure B to afford
1
products by NMR. H NMR δ major isomer 6.53 (d, J ) 1.9 Hz,
2H), 6.15 (d, J ) 1.9 Hz, 1H), 6.01 (bs, 1H), 3.79 (s, 6H), 2.71
(bd, J ) 16.4 Hz, 1H), 2.24 (dd, J ) 16.4, 1.4 Hz, 1H), 1.87 (t, J
) 4.2 Hz, 1H), 1.79-1.84 (m, 1H), 1.70 (dt, J ) 12.0, 3.9 Hz,
1H), 1.30-1.36 (m, 1H), 1.19-1.24 (m, 1H), 1.03 (s, 3H), 0.93

6398 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 20
Lu et al.
(s, 3H), 0.76 (s, 3H); minor isomer 6.33 (d, J ) 1.6 Hz, 2H), 6.28
(d, J ) 1.6 Hz, 1H), 6.01 (bs, 1H), 3.77 (s, 6H), 2.31-2.38 (m,
1H), 2.08 (t, J ) 4.5 Hz, 1H), 1.92-1.97 (m, 1H), 1.79-1.83 (m,
1H), 1.65-1.70 (m, 1H), 1.35-1.41 (m, 1H), 1.19-1.27 (m, 1H),
0.95 (s, 3H), 0.91 (s, 3H), 0.83 (s, 3H); MS m/z 286 (M+).
2′-(3,5-Dimethoxybenzyl)-1′,7′,7′-trimethylbicyclo[2.2.1]hepta-
nes (5c and 5d). The cis/trans mixture of 10 (370 mg, 1.29 mmol)
and 25 mg of 10% Pd/C in 15 mL of anhydrous ethanol was
hydrogenated at atmospheric pressure with stirring. Upon comple-
tion of the reaction, the mixture was filtered and concentrated. The
crude product was chromatographed (10:90 acetone/petroleum
ether) to afford 342 mg (1.19 mmol, 92%) of a mixture of 5c and
5d. The product was determined to be a 1:3.4 mixture of endo- to
exo-adducts by NMR. 1H NMR δ 5c (endo-adduct, minor isomer,
partial data) 6.35 (d, J ) 1.9 Hz, 2H), 6.33 (t, J ) 1.9 Hz,
1H), 3.77 (s, 6H), 2.67 (dd, J ) 13.8, 3.0 Hz, 1H), 2.34-2.38 (m,
1H), 1.85-1.97 (m, 2H), 1.65-1.75 (m, 2H), 1.51-1.55 (m, 1H),
1.31-1.37 (m, 1H), 1.05-1.15 (m, 1H), 0.86 (s, 3H), 0.84 (s, 3H),
0.82 (s, 3H); 5d (exo-adduct, major isomer) 6.31 (d, J ) 1.8 Hz,
2H), 6.29 (t, J ) 1.8 Hz, 1H), 3.78 (s, 6H), 2.82 (dd, J ) 13.0, 4.6
Hz, 1H), 2.34 (dd, J ) 13.0, 12.2 Hz, 1H), 1.74-1.78 (m, 1H),
1.67-1.72 (m, 1H), 1.65 (t, J ) 3.7 Hz, 1H), 1.51-1.58 (m, 2H),
1.27 (dd, J ) 12.5, 9.3 Hz, 1H), 1.09-1.16 (m, 2H), 0.97 (s, 3H),
0.90 (s, 3H), 0.84 (s, 3H); MS m/z 288 (M+).
5-(1′,7′,7′-Trimethylbicyclo[2.2.1]hept-2′-ylmethyl)resorcinols (6c
and 6d). A 1:3.4 mixture by NMR of 6c and 6d (218 mg, 0.837
mmol, 88%) as a white solid was prepared from 274 mg (0.950
mmol) of the mixture of 5c and 5d following general procedure A.
1H NMR δ 6c (endo-adduct, minor isomer, partial data) 6.26 (d, J
) 1.9 Hz, 2H), 6.19 (t, J ) 1.9 Hz, 1H), 5.62 (bs, 2H), 2.61 (dd,
J ) 12.8, 2.9 Hz, 1H), 2.28-2.32 (m, 1H), 1.83-1.93 (m, 2H),
1.68-1.74 (m, 1H), 1.49-1.57 (m, 2H), 1.30-1.36 (m, 1H),
0.94-0.97 (m, 1H), 0.86 (s, 3H), 0.83 (s, 3H), 0.80 (s, 3H); 6d
(exo-adduct, major isomer) 6.23 (d, J ) 1.7 Hz, 2H), 6.19 (t, J )
1.7 Hz, 1H), 5.62 (bs, 2H), 2.75 (dd, J ) 13.3, 4.6 Hz, 1H), 2.28
(dd, J ) 13.3, 12.7 Hz, 1H), 1.66-1.76 (m, 2H), 1.64 (t, J ) 4.0
Hz, 1H), 1.49-1.58 (m, 2H), 1.26 (dd, J ) 9.3, 9.2 Hz, 1H),
1.07-1.16 (m, 2H), 0.95 (s, 3H), 0.89 (s, 3H), 0.83 (s, 3H); MS
m/z 260 (M+).
2.73 (dd, Jgem ) 13.6 Hz, Jvic 4.3 Hz, 1H, benzylic 1′′-Ha), 2.68 (ddd,
J10a,10ꢀ ) 10.9 Hz, J10a,6a ) 10.9 Hz, J10a,10R ) 4.5 Hz, 1H, 10a), 2.22
(dd, Jgem ) 13.6 Hz, Jvic 11.8 Hz, 1H, benzylic 1′′-Hb), 2.14 (bdd,
J7R,7ꢀ ) 12 Hz, J7R,8 ) 4 Hz, 1H, 7ꢀ), 1.63-1.90 [m, 8H,
6a,7R,10ꢀ,2′endo,5′exo, especially 1.70 (s, 3H, 9-CH3)], 1.63 (dd,
J4′,3′exo ) 4 Hz, J4′,5′exo ) 4 Hz, 1H, 4′), 1.47-1.57 (m, 2H, 3′exo,6′exo),
1.37 (s, 3H, 6-ꢀ-CH3), 1.30 (dd, J3′endo,3′exo ) 12.6 Hz, J3′endo,2′endo
)
9.3 Hz, 1H, 3′endo), 1.05-1.14 [m, 5H, 5′endo, 6′endo, especially
1.10 (s, 3H, 6-R-CH3)], 0.94 (s, 3H, syn-7′-CH3), 0.88 (s, 3H, 1′-CH3),
22
0.83 (s, 3H, anti-7′-CH3); [R]D -290° (c 0.223, CH2Cl2); MS m/z
394 (M+); HRMS exact mass calculated for C27H38O2, 394.2872,
found, 394.2870; Anal. (C27H38O2 ·1/2H2O), C, H.
Rat Brain CB1 Membrane Preparation. Rat forebrain membrane
microsomes were prepared from frozen rat brains by the method of
Dodd et al.36 Fifteen frozen rat brains (Pel-Freez, no. 56004-2, Rogers,
AR), stored at -80 °C, were placed in a plastic dish and allowed to
partially thaw so that the cerebellum could be removed with a spatula
and discarded. The remaining brain tissue was homogenized in 40
mL of ice-cold homogenization buffer (0.32 M sucrose, 10 mM Tris
base, 5 mM EDTA, pH 7.4) in two installments. All tissues and
homogenates were kept on ice to prevent tissue degradation. The
homogenate was decanted into prechilled tubes for centrifugation at 4
°C and 3700g for 10 min. The supernatants were pooled, kept on ice,
and the total volume brought to 125 mL with ice-cold homogenization
buffer. The supernatant was aliquoted (12 mL) into 10 prechilled
centrifuge tubes (24 mL). Using a syringe and needle, 10 mL of cold
1.2 M sucrose in TME buffer (25 mM Tris base, 5 mM MgCl2, 1
mM EDTA, pH 7.4) was carefully layered at the bottom of each
centrifuge tube, and the tubes were carefully balanced with cold
homogenization buffer added to the top layer. These tubes were
centrifuged in a 4 °C ultracentrifuge for 35 min at 245000g. The
resulting layer at the interface was carefully collected. The total volume
was brought to 105 mL with ice-cold homogenization buffer and
aliquoted in eight centrifuge tubes (12 mL each). Using a syringe and
needle again, 10 mL of cold 0.8 M sucrose in TME buffer was carefully
layered at the bottom of each centrifuge tube, the tubes carefully
balanced with cold homogenization buffer added to the top layer,
followed by ultracentrifugation as described above. After discarding
the resulting supernatant, the pellets were resuspended in ice-cold TME,
pooled (total volume of 6 mL), and gently homogenized by hand. This
membrane suspension was aliquoted into silanized Eppendorf tubes
and flash frozen in liquid nitrogen, followed by storage at -80 °C
until use within 2 months. One of the aliquoted samples was used for
protein determination using a Bio-Rad (500-0006) Bradford protein
assay kit.
(6aR-trans)-3-(endo-1′,7′,7′-Trimethylbicyclo[2.2.1]hept-2′-ylm-
ethyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran-
1-ol (3-Bornylmethyl-∆8-THC, 8c) and (6aR-trans)-3-(exo-1′,7′,7′-
Trimethylbicyclo[2.2.1]hept-2′-ylmethyl)-6a,7,10,10a-tetrahydro-
6,6,9-trimethyl-6H-dibenzo[b,d]pyran-1-ol (3-Isobornylmethyl-∆8-
THC, 8d). The 1:3.4 mixture of 6c and 6d (180 mg, 0.691 mmol)
was condensed with (+)-trans-p-2,8-menthadien-1-ol (7) following
general procedure B to afford 185 mg (0.469 mmol, 68%) of a 1:3.4
mixture by NMR of endo-adduct 8c to exo-adduct 8d. The product
mixture was then separated by semipreparative chiral HPLC (Chiralpak
AD, 250 mm × 10 mm, 5:95 2-propanol/hexane, 2 mL/min) that
afforded the major isomer 8d as a white solid, with a retention time
of 18.28 min, and the minor isomer 8c as a white solid, with a retention
time of 19.75 min. 8c (endo-adduct, minor product) mp 76-77 °C;
1H NMR δ 6.28 (d, J2,4 ) 1.9 Hz, 1H, 4), 6.10 (d, J2,4 ) 1.9 Hz, 1H,
2), 5.41-5.44 (m, 1H, 8), 4.64 (bs, 1H, OH), 3.18 (dd, J10R,10ꢀ ) 16.5
Hz, J10R,10a ) 4.4 Hz, 1H, 10R), 2.69 (ddd, J10a,10ꢀ ) 10.9 Hz, J10a,6a
) 10.9 Hz, J10a,10R ) 4.4 Hz, 1H, 10a), 2.56 (dd, Jgem ) 13.4 Hz, Jvic
) 1.6 Hz, 1H, benzylic 1′′-Ha), 2.26 (dd, Jgem ) 13.4 Hz, Jvic ) 10.1
Hz, 1H, benzylic 1′′-Hb), 2.10-2.18 (m, 1H, 7ꢀ), 1.75-1.95 (m, 5H,
2′exo,3′exo,6a,7R,10ꢀ), 1.65-1.75 (m, 1H, 5′exo), 1.70 (s, 3H, 9-CH3),
1.54-1.63 (m, 2H, 4′,6′endo), 1.37 (s, 3H, 6-ꢀ-CH3), 1.32 (dddd,
J6′exo,6′endo ) 12 Hz, J6′exo,5′exo ) 12 Hz, J6′exo,5′endo ) 4.9 Hz, 4J2′exo,6′exo
) 1.2 Hz, 1H, 6′exo), 1.11 (ddd, J5′endo,5′exo ) 12 Hz, J5′endo,6′endo ) 10
Hz, J5′endo,6′exo ) 4.9 Hz, 1H, 5′endo), 1.10 (s, 3H, 6-R-CH3), 0.82-0.89
(m, 1H, 3′endo), 0.86 (s, 3H, anti-7′-CH3), 0.84 (s, 3H, syn-7′-CH3),
Mouse Spleen CB2 Membrane Preparation. Membrane mi-
crosomes with CB2 receptors were prepared from whole frozen
mouse spleens (Pel-Freez no. 55049-2) according to the procedure
detailed above for rat brain.
Competitive Binding Assay. Rat brain membrane and mouse
spleen membrane preparations were used to assess the affinities of
the novel analogues for CB1 and CB2 binding, respectively. The
displacement of specifically tritiated CP55,940 from these membrane
preparations was used to determine the IC50 values for the ∆8-THC
(1) and analogues 8a-8d. The [3H]CP55,940 binding assay was
conducted on 96-well microfilter plates as previously described.28,38
Briefly, 100 µL of cannabinergic ligand (at eight different concentra-
tions) in DMSO, 50 µL of rat brain or mouse spleen membrane
preparation (40-50 µg protein), and 50 µL of [3H]CP55,940 (3.08
nM) in TME (25 mM Tris, 5 mM MgCl2, 1 mM EDTA) buffer
containing 0.1% bovine serum albumin (BSA) was incubated for 1 h
at 30 °C. For the nonspecific binding control, 100 µL of 200 nM
CP55,940 was used and 100 µL of TME buffer containing 0.1% BSA
was used for the total binding control. The competitive reaction was
terminated by rapid filtration through a Packard Filtermate harvester
and Whatman GF/B unifilter-96 plates, and an ice-cold TME wash
buffer containing 0.5% BSA was used. Radioactivity was detected
using MicroScint 20 scintillation cocktail added to the dried filter plates
and was counted using a Packard Instruments Topcount microplate
scintillation counter. The normalized data from three independent
experiments were combined and analyzed using a four-parameter
21
0.80 (s, 3H, 1′-CH3); [R]D -180° (c 0.223, CH2Cl2); MS m/z 394
(M+); HRMS exact mass calculated for C27H38O2, 394.2872, found,
394.2868; Anal. (C27H38O2 ·1/4H2O), C, H. 8d (exo-adduct, major
1
isomer) mp 78-80 °C; H NMR δ 6.25 (d, J2,4 ) 1.7 Hz, 1H, 4),
6.08 (d, J2,4 ) 1.7 Hz, 1H, 2), 5.43 (bd, J7R,8 ) 4 Hz, 1H, 8), 4.65 (bs,
1H, OH), 3.17 (dd, J10R,10ꢀ ) 16.4 Hz, J10R,10a ) 4.5 Hz, 1H, 10R),

Bornyl- and Isobornyl-∆8-tetrahydrocannabinols
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Acknowledgment. This work was supported by grants DA-
7215, DA-3801, DA-152, DA-9158, and DA24842 from the
National Institute on Drug Abuse. We thank Pusheng Fan and
JodiAnne Wood for the receptor binding assays.
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Supporting Information Available: Elemental analysis results
1
for compounds 8a-8d. H NMR, COSY, and NOESY (500 ms
mixing time) spectra for compounds 8a-8d in CDCl3 solutions.
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