Cannabilactones: A novel class of CB2 selective agonists with peripheral analgesic activity

Article abstract of DOI:10.1021/jm070441u

The identification of the CB2 cannabinoid receptor has provided a novel target for the development of therapeutically useful cannabinergic molecules. We have synthesized benzo[c]chromen-6-one analogs possessing high affinity and selectivity for this recep

Full text of DOI:10.1021/jm070441u

J. Med. Chem. 2007, 50, 6493–6500
6493
Cannabilactones: A Novel Class of CB2 Selective Agonists with Peripheral Analgesic Activity
Atmaram D. Khanolkar,† Dai Lu,† Mohab Ibrahim,‡ Richard I. Duclos, Jr.,† Ganesh A. Thakur,† T. Phillip Malan, Jr.,‡
Frank Porreca,‡ Vijayabaskar Veerappan,† Xiaoyu Tian,† Clifford George,§ Damon A. Parrish,§ Demetris P. Papahatjis,| and
Alexandros Makriyannis*,†
Center for Drug DiscoVery, Northeastern UniVersity, 116 Mugar Hall, 360 Huntington AVenue, Boston, Massachusetts 02115-5000,
Departments of Anesthesiology and Pharmacology, UniVersity of Arizona College of Medicine, 1501 North Campbell AVenue,
Tucson, Arizona 85724-5114, NaVal Research Laboratory, Code 6030, 4555 OVerlook AVenue, Washington, DC 20375-5341, and Institute of
Organic and Pharmaceutical Chemistry, National Hellenic Research Foundation, 48 Vass. Constantinou, Athens 116-35 Greece
ReceiVed April 13, 2007
The identification of the CB2 cannabinoid receptor has provided a novel target for the development of
therapeutically useful cannabinergic molecules. We have synthesized benzo[c]chromen-6-one analogs
possessing high affinity and selectivity for this receptor. These novel compounds are structurally related to
cannabinol (6,6,9-trimethyl-3-pentyl-6H-benzo[c]chromen-1-ol), a natural constituent of cannabis with modest
CB2 selectivity. Key pharmacophoric features of the new selective agonists include a 3-(1′,1′-dimethylheptyl)
side chain and a 6-oxo group on the cannabinoid tricyclic structure that characterizes this class of compounds
as “cannabilactones.” Our results suggest that the six-membered lactone pharmacophore is critical for CB2
receptor selectivity. Optimal receptor subtype selectivity of 490-fold and subnanomolar affinity for the CB2
receptor is exhibited by a 9-hydroxyl analog 5 (AM1714), while the 9-methoxy analog 4b (AM1710) had
a 54-fold CB2 selectivity. X-ray crystallography and molecular modeling show the cannabilactones to have
a planar ring conformation. In vitro testing revealed that the novel compounds are CB2 agonists, while in
vivo testing of cannabilactones 4b and 5 found them to possess potent peripheral analgesic activity.
Introduction
inoid-related physiology and biochemistry. Ligands that can
selectively stimulate the CB2 receptor are expected to be largely
devoid of the central nervous system (CNS) psychotropic side
effects associated with the activation of CB1 receptors and
represent potentially useful medications1,13 for the treatment of
pain, inflammation, cancer proliferation, Alzheimer’s disease,
and other ailments related to cannabinoid physiology. The
structure–activity relationships (SARs) of cannabinoid analogs
for the CB2 receptor have recently received attention.14–17
Gareau et al.18 showed that when the C-1 phenolic hydroxyls
in (-)-∆8- and (-)-∆9,11-tetrahydrocannabinols (∆8-THC and
Cannabinoids are known to produce biochemical and phar-
macological effects by interacting with two well-characterized
G protein-coupled receptors, CB1a and CB2,1 although human
CB1 and CB2 share only a 44% homology.2,3 Also, CB1
exhibits high amino acid identity (97–99%)4–6 across the species
of human, rat, and mouse, while human CB2 displays only 81%
and 82% amino acid identity with rat7 and mouse,3 respectively.
The CB1 receptor was found to be localized primarily in the
brain, with the highest density in the cells of the basal ganglia,
cerebellum, and hippocampus.8 CB1 is also found in peripheral
tissues, including testis, eye, uterus, ovary, lungs, and heart.4,9,10
Conversely, CB2 is expressed predominantly in immune cells,
such as B-cells, monocytes, macrophages, and in several
peripheral organs, such as spleen, pancreas, thymus, lung, and
tonsil.10,11 The presence of CB2 receptor protein was recently
confirmed in the brain stem, cortex, and cerebellum of rat,
mouse, and ferret,12 however, the presence of CB2 receptor in
those tissues is only about 1.5% of that found in the spleen.
∆
9,11-THC) were replaced by methoxy groups, CB1 affinity is
dramatically reduced while its affinity for CB2 is affected only
slightly, thus leading to analogs possessing CB2 selectivity. Such
selectivity is also obtained by eliminating the C-1 phenolic
group, as 1-deoxy-11-hydroxy-DMH-∆8-THC shows signifi-
cantly higher CB2 selectivity when compared to its C-1 phenolic
analogue.19 The C-3 side chain length20 and the conformation15,21
of classical cannabinoid analogs were shown to affect cannab-
inoid receptor subtype selectivity. Additionally, Hanus et al.22
have reported a CB2 specific bicyclic agonist (+)-4-[4-(1,1-
dimethylheptyl)-2,6-dimethoxyphenyl]-6,6-dimethylbicyclo
[3.1.1]hept-2-en-2-methanol (HU-308), which was shown to
produce anti-inflammatory, hypotensive, and analgesic effects
that are blocked by the CB2 antagonist 5-(4-chloro-3-meth-
ylphenyl)-1-[(4-methylphenyl)methyl)-N-([1S,2S,4R]-1,3,3-
trimethylbicyclo[2.2.1]heptan-2-yl)-1H-pyrazole-3-carboxam-
ide (SR144528). Recently, we showed that the CB2 selective
agonist (R,S)-(2-iodo-5-nitrophenyl)-{1-[(1-methylpiperidin-2-
yl)methyl]-1H-indol-3-yl}-methanone (AM1241) also inhibits
nociception.23–27 Other CB2 selective ligands have been reported
and recently reviewed.28,29 Antinociception via peripheral CB2
receptors23–25,27,30–32 as well as via peripheral CB131–34 receptors
has been reported, and differentiation between these two
Since the discovery of CB1 and CB2, substantial medicinal
chemistry efforts have been directed toward the development
of high affinity and receptor-selective cannabinergic ligands that
would serve as pharmacological probes for studying cannab-
* To whom correspondence should be addressed Tel.: 1-617-373-4200.
Fax: 1-617-373-7493. E-mail: a.makriyannis@neu.edu.
†
Northeastern University.
University of Arizona College of Medicine.
Naval Research Laboratory.
National Hellenic Research Foundation.
‡
§
|
aAbbreviations: BSA, bovine serum albumin; CB1, cannabinoid receptor
1; CB2, cannabinoid receptor 2; CNS, central nervous system; DMH, 1,1-
dimethylheptyl; HEK, human embryonic kidney; 9-iodo-9-BBN, 9-iodo-
9-borabicyclo[3.3.1]nonane; MM, molecular mechanics; MPE, maximum
possible effect; SAR, structure–activity relationship; SEM, standard error
of the mean; THC, tetrahydrocannabinol; TME, Tris buffer (25 mM Tris,
5 mM MgCl2, 1 mM EDTA).
10.1021/jm070441u CCC: $37.00
2007 American Chemical Society
Published on Web 11/27/2007

6494 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26
Khanolkar et al.
Figure 1. General structures of cannabilactones and cannabinol
analogs.
Figure 2. Structures of the nonselective partial agonist (-)-5-(1,1-
dimethylheptyl)-2-[(1R,2R,5R)-5-hydroxy-2-(3-hydroxypropyl)cyclo-
hexyl]phenol (7) and the full agonist (+)-{(3R)-2,3-dihydro-5-methyl-
3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl}-(1-
naphthalenyl)-methanone (8).
Scheme 1a
5. Biphenyls 3a and 3b were prepared by coupling 4-substituted
2-(N,N-diisopropylcarboxamido)phenylboronic acids 2a and 2b,
respectively, with 2-bromo-5-(1′,1′-dimethylheptyl)-1,3-dimeth-
oxybenzene45 using tetrakis(triphenylphosphine)palladium and
barium hydroxide (Scheme 1). The boronic acids were prepared
by ortho lithiation followed by boronation of the appropriate
N,N-diisopropylbenzamides 1a46 and 1b47 using the method
described by Snieckus et al.43 The biphenyls 3a and 3b were
demethylated with boron tribromide or 9-iodo-9-BBN,48 which
was utilized for the selective demethylations of the two methoxy
groups of the electron-rich aromatic ring without cleaving the
aryl methyl ether in the benzamide ring of biphenyl 3b. Without
purification, refluxing of the biphenyl resorcinol intermediates
in glacial acetic acid gave the corresponding cannabilactones
4a and 4b via intramolecular cyclization in 36% and 61% overall
yields, respectively. The methoxy group of cannabilactone 4b
was cleaved with boron tribromide to give cannabilactone 5 in
72% yield. Cannabilactones 4a, 4b, and 5 were converted to
their 6,6-dimethyl analogs by treatment with methylmagnesium
bromide followed by cyclization in the presence of p-toluene-
sulfonic acid monohydrate49 to give the corresponding cannab-
inol analogs 6a,38 6b, and 6c.
a Reagents and conditions: (a) i. sec-BuLi, TMEDA, THF, -78 °C; ii.
B(OCH3)3; iii. H3O+; (b) 2-bromo-5-(1′,1′-dimethylheptyl)-1,3-dimethoxy-
benzene, Pd(PPh3)4, Ba(OH)2, DME, H2O; (c) BBr3 or 9-iodo-9-BBN,
CH2Cl2; (d) AcOH, reflux; (e) BBr3, CH2Cl2; (f) CH3MgBr, THF; (g)
p-TsOH·H2O, CHCl3, RT.
mechanisms is dependent on the specific analgesic test and the
cannabinoid being used.
In a recently published patent application,35 we reported a
new class of CB2 selective ligands based on structural modifica-
tions of cannabinol, which is weakly CB2 selective.2,36–38 The
key pharmacophore of these benzo[c]chromen-6-ones is a
carbonyl group in place of the 6,6-dimethyl moiety of the
classical cannabinoid tricyclic structure. This class, whose
synthesis, pharmacophoric characterization, and pharmacological
evaluation are reported here, we refer to as “cannabilactones.”
Cannabilactones 4a,37 4b,35 and 535 (Figure 1 and Scheme 1)
were synthesized in our laboratory using the Suzuki coupling
reaction between arylboronic acids and aryl bromides as the
key synthetic step. This synthetic approach also provides a new
route for the synthesis of classical cannabinol analogs.
Results and Discussion
CB1 and CB2 Receptor Binding Studies. The affinities
of analogs 4a, 4b, 5, 6a, 6b, and 6c for the CB1 and CB2
cannabinoid receptors were determined by a standard competi-
tive radioligand displacement assay using tritiated (-)-5-(1,1-
dimethylheptyl)-2-[(1R,2R,5R)-5-hydroxy-2-(3-hydroxypropyl)
cyclohexyl]phenol ([3H]CP55,940, 7; see Figure 2).50 Rat brain
synaptosomal and mouse spleen membranes were used as the
sources for CB1 and CB2 receptors, respectively. As can be
seen from the data in Table 1, the cannabilactones 4a, 4b, and
5 exhibit considerably higher selectivities for the CB2 receptor
relative to their corresponding 6,6-dimethyl analogs 6a, 6b, and
6c, which bind with comparable affinities to both CB1 and CB2
in our assays. Compound 4a was previously reported to have
moderate selectivity for the CB2 receptor.37 While our binding
data for the CB1 receptor show good correspondence with this
report, our Ki value for the CB2 receptor is lower, and this
discrepancy between the two laboratories is due to the differ-
ences in CB2 preparations (mouse spleen versus COS-7 cells
expressing hCB2). Substitution of the C-9 methyl group in 4a
by a methoxy group in 4b substantially reduces the CB1 affinity
of 4b, which exhibits over 50-fold CB2 selectivity. However,
the most striking result was observed with the C-9 phenolic
hydroxyl analog 5, which exhibits subnanomolar CB2 receptor
affinity (Ki, CB1 ) 400 nM; Ki, CB2 ) 0.82 nM) and nearly
500-fold selectivity.
Chemistry. The previously reported syntheses of substituted
benzo[c]chromen-6-ones involved von Pechmann condensation
reactions between an appropriately substituted 1-oxocyclohex-
ane-2-carboxylate and 5-alkylresorcinol followed by oxidation
of the tetrahydrobenzopyranone intermediates.37,39,40 However,
the oxidation required harsh reaction conditions including high
temperatures and prolonged reaction times. The reactions
generally gave low yields of multiple products that required
extensive purifications. More importantly, this route is not
suitable for the synthesis of functionalized cannabinol analogs.
In an effort to develop a high yield approach of more general
applicability, we chose the Suzuki reaction,41,42 which has
alreadybeenusedforthesynthesisofbenzo[c]chromen-6-ones,43,44
as the key step in the synthesis of cannabilactones 4a, 4b, and

Cannabilactones: CB2 SelectiVe Agonists
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26 6495
Stimulation of [35S]GTPγS
Table 1. Cannabinoid Receptor Binding and Stimulation of [35S]GTPγS Binding
Kia (nM)
analogue (9-substituent)
rCB1
mCB2
CB1/CB2
bindingb (percent)
4a (Me)
39 (34, 44)
3.1 (2.6, 3.8)
6.7 (5.5, 8.1)
0.82 (0.66, 1.0)
1.1 (0.89, 1.2)
5.9 (5.0, 6.9)
4.8 (3.8, 5.5)
NDc
13
54
56 ( 21
50 ( 8
55 ( 17
NDc
4b (OMe) AM1710
5 (OH) AM1714
6a (Me)
6b (OMe) AM1715
6c (OH) AM4768
8 WIN55,212–2
360 (330, 390)
400 (340, 450)
0.95 (0.81, 1.2)
5.4 (4.9, 6.0)
2.6 (2.0, 3.4)
NDc
490
0.86
0.92
0.54
NDc
42 ( 15
NDc
56 ( 10
a Rat brain synaptosomal and mouse spleen membranes, respectively, were used as sources for rCB1 and mCB2 for the receptor binding assays with
b
[3H]CP55,940 (7) as the radioligand. The values in the parentheses indicate the 95% confidence limits.
[
35S]GTPγS binding using mouse spleens with
standard deviations. c ND: Not determined.
After this study was completed, we became aware that the
CB2 selectivities of 4b and 5 are considerably reduced from
54-fold to 4.0-fold and from 490-fold to 8.5-fold, respectively,
when these compounds were tested using a human CB2 receptor
preparation from HEK cells using [3H]CP55,940 (7). This
difference between mouse and human CB2 was also observed
by another laboratory.51 While these data confirmed the value
of the above ligands as excellent pharmacological probes in
experiments involving rodents or rodent-derived tissues, the
usefulness of these cannabilactones as potential human medica-
tions remains to be further investigated. These species-based
differences in CB2 affinity data suggest that cannabilactone
analogs interact with the mouse and human CB2 receptors
through different binding motifs. Currently, we are pursuing
this observation through the generation of suitable receptor
mutations, and these data should provide interesting information
pertaining to CB2 receptor structure. We are also pursuing
additional SAR work aimed at developing cannabilactone
analogs with robust CB2 selectivities for both rodent and human
receptors.
Figure 3. Peripheral antinociceptive effect of 9-methoxy cannabilac-
tone 4b (triangles) and 9-hydroxy cannabilactone 5 (circles). Com-
pounds were dissolved in DMSO and injected subcutaneously into the
dorsal surface of the tested hindpaw (intrapaw) 20 min before
nociceptive testing. Nociception was assessed by measuring withdrawal
latency to radiant heat as described by Hargreaves et al.59 Data are
expressed as a percent of the maximum possible effect (%MPE)
calculated by the formula: %MPE ) (WL - CL)/(CO - CL), where
WL is the withdrawal latency obtained experimentally, CL is the control
(baseline) value before drug administration, and CO is the cutoff value
(a 40 s cutoff was used to prevent tissue damage). Dose–response curves
were generated and the A50 values (doses producing 50% MPE) were
calculated as described by Tallarida and Murray.60 Data expressed as
mean ( SEM. N ) 6 per group.
Functional Characterization. The functional potencies of
cannabilactones 4a, 4b, and 5 on the CB2 receptor were assessed
with a [35S]GTPγS binding assay, as described previously,52,53
with a mouse spleen membrane preparation. All compounds
were found to act as agonists and to stimulate [35S]GTPγS
binding at a concentration of 1 µM to an extent comparable to
that of (+)-{(3R)-2,3-dihydro-5-methyl-3-(4-morpholinylmeth-
yl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl}-(1-naphthalenyl)-meth-
anone (WIN55,212-2, 8), a prototypic, although only modestly
selective, CB2 receptor agonist.36 Furthermore, the functional
potencies of 4a, 4b, and 5 on the CB2 receptor to decrease
forskolin-stimulated cAMP were assayed as previously de-
scribed.53 These cannabilactones were confirmed to be agonists
with functional potencies that were within a factor of 2-3 of
the EC50 of WIN55,212-2 (8).
In Vivo Cannabinergic Evaluation. The cannabilactone
agonists were evaluated in vivo for antinociceptic activity in
mice as previously described.23 Cannabilactones 4b and 5 were
administered into the dorsal surface of the hindpaw of male
Sprague–Dawley rats, where they produced significant anti-
nociceptive effects (Figure 3). The A50 (dose producing a 50%
antinociceptive effect) was 1.6 mg/kg (95% confidence limits
) 0.8–3.3 mg/kg) for 4b and 4.5 mg/kg (95% confidence limits
) 2.2–9.0 mg/kg) for 5. It is interesting to note that although
cannabilactones 4b and 5 exhibit similar potency in the
[35S]GTPγS in vitro functional assay, 4b exhibits higher in vivo
potency. It can be argued that this may be due to differences in
their respective pharmacokinetic profiles.
ceptive effects of 5 were not affected by the CB1 selective
antagonist 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-
N-1-piperidinyl-1H-pyrazole-3-carboxamide (9)54 but were
completely antagonized by the CB2 selective antagonist {6-
iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl}-(4-
methoxyphenyl)-methanone (10;55 Figure 4). These findings
provide an in vivo confirmation of the CB2 receptor agonist
activity of these compounds and confirm previous findings, using
other structural classes of cannabinoid receptor agonists, that
CB2 receptor activation produces analgesia.22–27,30–32,56
Finally, 5 did not affect ambulation when administered
systemically (intraperitoneally), as assessed by performance on
the rotarod apparatus. In contrast, the mixed CB1/CB2 receptor
agonist WIN55,212-2 (8) significantly impaired performance
on the rotarod apparatus (Figure 5), and this is believed to be
mediated by CB1 receptors in the CNS.23 Our in vivo findings
suggest that CB2 receptor selective agonists may be free of CNS
side effects typically produced by cannabinoids. This is
consistent with the very recent observation that CB2 receptor
activation alone is not sufficient to generate some typical CNS
effects associated with CB1 receptor activation.12
Molecular Modeling and X-ray Crystallography. The
presence of a carbonyl group at C-6 of the cannabinoid tricyclic
ring system appears to be responsible for the reduced affinities
of these cannabilactones for the CB1 receptors. To better
understand the molecular basis for the observed CB2 selectivities
The CB2 selectivity of the above in vivo response was
confirmed by using CB1 and CB2 antagonists whose respective
selectivities are over 100-fold.16 We showed that the antinoci-

6496 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26
Khanolkar et al.
Figure 4. The peripheral antinociceptive effects of 9-hydroxy cannabilactone 5 (3 mg/kg, intrapaw) were not affected by the CB1 selective antagonist
1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide (9; 1 mg/kg, intrapaw) but were antagonized by the
CB2 selective antagonist {6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl}-(4-methoxyphenyl)-methanone (10; 1 mg/kg, intrapaw). All
drugs were administered 20 min before nociceptive testing. Data are expressed as mean ( SEM. Groups were compared using ANOVA followed
by pairwise comparisons using Student’s t-test. *P < 0.05 compared to 5 alone. N ) 6 per group.
Figure 5. Effect of WIN55,212-2 (8; 3.3 mg/kg) and 9-hydroxy
cannabilactone 5 (3.3 mg/kg) compared to baseline (BL) containing
only vehicle on ambulation, as assessed by performance on the rotarod
apparatus (Columbus Instruments International, Columbus, OH).
Animals were trained until they could remain on the device for a
Figure 6. Comparison of minimum energy conformations of 6-oxo
cannabilactone 5 and 6,6-dimethyl cannabinol 6c tricyclic analogs
presented in green and blue, respectively. Oxygen atoms are presented
duration of 180 s at a speed of 10 rpm. They were again tested 15 min
in red. The C-3 dimethylheptyl side chains are partially displayed. The
after i.p. administration of WIN55,212-2 (8) or 9-hydroxy cannabilac-
aromatic A rings of the two molecules were superimposed with the
1-hydroxyl group tilted furthest from the viewer. The dihedral angle
between the C rings of 5 and 6c is about 29.3°. Molecular mechanics
(MM) calculations were conducted on a Silicon Graphics Fuel
tone 5 and the time they were able to remain on the device until falling
was recorded. A maximal cutoff time of 180 s was used. Data are
expressed as mean ( SEM; *P < 0.05 compared to vehicle (DMSO).
N ) 6 per group.
workstation using Insight II Discover molecular dynamics package
(Accelrys, San Diego, CA). Atomic potentials and charges were
assigned using the cvff force field. Conformations were geometrically
optimized using a distance-dependent dielectric constant mimicking an
aqueous environment. Energy minimizations were performed until the
maximum root-mean-square (rms) derivative was less than 0.001 kcal
of the novel cannabilactones when compared to their corre-
sponding 6,6-dimethyl cannabinol analogs, we have compared
the conformations of 5 and 6c using molecular mechanics and
dynamics simulations. Our computational study shows that the
presence of the six-membered lactone ring of 5 leads to a
conformation in which all three rings are coplanar (Figure 6, in
green). This is in agreement with the X-ray crystal structures
of cannabilactones 4a (Figure 7) and 4b (Figure 8). Conversely,
the pyran ring of 6,6-dimethyl analogue 6c is in a puckered
conformation (Figure 6, in blue). It can be seen that when the
A rings of the two ligands are superimposed, the C ring of 6c
is positioned approximately 30° above that of 5 directing the
respective 9-substituents of cannabilactones and cannabinols in
different orientations.
mol-1 Å-1
.
differently within the CB1 receptor and exhibit unfavorable
steric interactions with the CB1 side chain subsite.
Conclusion
Our data reveal that the lactone functionality plays a key role
in the observed CB2 selectivity of cannabilactones. Cannabi-
lactone analogs 4a, 4b, and 5 bind with high affinities to the
CB2 receptor and exhibit much lower affinities for CB1. The
data also show that the presence of a phenolic hydroxyl group
at C-9 enhances binding to the CB2 receptor, while the presence
of a methyl group at C-9 enhances binding to the CB1 receptor.
Conversion of the 6-oxo groups of the cannabilactones to the
corresponding 6,6-dimethyl analogs results in complete loss of
CB2 selectivity, as these cannabinol analogs 6a, 6b, and 6c
exhibit nearly equal high affinities for both cannabinoid receptor
subtypes. Cannabilactone analogs have distinctly different
interactions with the mouse and human CB2 receptors. In our
The structural differences between the cannabilactone analogs
and their 6,6-dimethyl congeners should account for the lower
affinities of this new class of ligands in the CB1 receptor while
both classes interact equally well with CB2. This can be
explained by invoking that cannabilactone analogs have (a) a
carbonyl group that may engage in unfavorable interactions with
hydrophobic residues of the CB1 binding site; (b) reduced
opportunity for hydrogen bonding through their 9-OH or
9-OCH3 substituents; and (c) C-3 side chains that are oriented

Cannabilactones: CB2 SelectiVe Agonists
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26 6497
of Chemical Sciences, University of Illinois at Urbana–Champaign.
Elemental analyses were performed by Baron Consulting Co.,
Milford, CT.
2-Diisopropylcarbamoyl-5-methylphenylboronic Acid (2a). To
a solution of sec-BuLi (42 mL of a 1.3 M solution, 55 mmol) and
1,2-bis(dimethylamino)ethane (TMEDA; 7.5 mL, 55 mmol) in
anhydrous THF (300 mL) at -78 °C under an argon atmosphere
was added dropwise a solution of N,N-diisopropyl-4-methylben-
zamide46 (1a; 10.0 g, 45.6 mmol) in THF (40 mL). The reaction
mixture was stirred for 60 min and then trimethyl borate (14.9 mL,
132 mmol) was added over a period of 30 min. The reaction mixture
was allowed to warm to room temperature over 12 h and was then
cooled to 0 °C and acidified to pH 6.5 with 5% aqueous HCl. The
THF was removed, and the product was extracted with 150 mL of
CH2Cl2. The organic phase was separated, washed with water and
brine, and dried. The solvent was removed to give a solid that was
recrystallized from Et2O to afford 11.4 g, (43.3 mmol, 95%) of 2a
Figure 7. The molecular structure and numbering scheme for 9-methyl
cannabilactone 4a with displacement ellipsoids drawn at the 30%
probability level.
1
as a white solid: mp 134–136 °C; H NMR (CD3COCD3) δ 7.72
(d, J ) 8.1 Hz, 1H), 7.24 (s, 1H), 7.19 (d, J ) 8.1 Hz, 1H), 5.00
(m, 1H), 4.86 (s, 2H, OH), 3.98 (m, 1H), 2.41 (s, 3H), 1.57 (d, J
) 5.5 Hz, 6H), 1.45 (d, J ) 5.5 Hz, 6H); MS m/z 263 (M+).
2-Diisopropylcarbamoyl-5-methoxyphenylboronic Acid (2b).
Compound 2b was prepared from 11.8 g (50.1 mmol) N,N-
diisopropyl-4-methoxybenzamide47 (1b) as described for 2a to give
13.4 g (48.0 mol, 96%) of 2b as a white solid: mp 125–127 °C; 1H
NMR (CD3OD) δ 7.83 (d, J ) 8.2 Hz, 1H), 7.10 (d, J ) 1.9 Hz,
1H), 6.94 (dd, J ) 1.9 Hz, 8.2 Hz, 1H), 5.00–5.08 (m, 1H),
3.96–4.04 (m, 1H), 3.89 (s, 3H), 1.57 (d, J ) 5.9 Hz, 6H), 1.46 (d,
J ) 5.8 Hz, 6H); MS m/z 279 (M+).
2-(N,N-Diisopropylcarboxamido)-5-methyl-2′,6′-dimethoxy-
4′-(1′′,1′′-dimethylheptyl)biphenyl (3a). Argon was bubbled through
a mixture of boronic acid 2a (0.185 g, 0.703 mmol), 2,6-dimethoxy-
4-(1′,1′-dimethylheptyl)bromobenzene45 (0.22 g, 0.64 mol),
Ba(OH)2 ·8H2O (0.303 g, 0.96 mmol), 0.7 mL of water, and 5 mL
of dimethoxyethane for 10 min. The Pd(PPh3)4 (74 mg, 0.064 mmol)
catalyst was added to the mixture while argon bubbling was
maintained through the mixture, and degassing was continued for
an additional 5 min. The reaction mixture was microwaved for 15
min at 110 °C in a CEM Discover apparatus. Then the mixture
was cooled to room temperature and filtered through a short celite
pad. The filtrate was concentrated and Et2O was added. The ether
solution was washed with water and brine and dried, and solvent
removal gave crude 3a as a light yellow solid. The crude product
was chromatographed (acetone/petroleum ether, 20:80) to afford
0.205 g (0.426 mmol, 67%) of biphenyl 3a as a white solid: mp
118–120 °C; Rf 0.36 (Et2O/petroleum ether, 50:50); 1H NMR δ
7.22 (d, J ) 8.0 Hz, 1H), 7.14 (dd, J ) 8.0 Hz, 1.0 Hz, 1H), 7.06
(d, J ) 1.0 Hz, 1H), 6.52 (s, 1H), 6.51 (s, 1H), 3.72 (s, 3H), 3.70
(s, 3H), 3.66–3.71 (m, 1H), 3.18–3.20 (m, 1H), 2.37 (s, 3H),
1.58–1.61 (m, 2H), 1.46 (d, J ) 6.5 Hz, 3H), 1.30 (s, 6H), 1.19–1.26
(m, 6H), 1.04–1.12 (m, 5H, especially, 1.08 d, J ) 6.5 Hz, 3H),
0.91 (d, J ) 6.5 Hz, 3H), 0.86 (t, J ) 7.0 Hz, 3H), 0.57 (d, J ) 6.5
Hz, 3H). Exact mass calculated for C31H47NO3, 481.7079; found,
481.7082.
Figure 8. The molecular structure and numbering scheme for 9-meth-
oxy cannabilactone 4b with displacement ellipsoids drawn at the 30%
probability level.
functional assays, all compounds were found to act as agonists,
with activities comparable to WIN55,212-2 (8). The analgesic
action by this class of compounds in vivo, without central
nervous system side effects, represents a promising approach
to the clinical treatment of pain.
Materials and Experimental Procedures
General Synthetic Methods. All reagents and solvents were
purchased from Aldrich (Milwaukee, WI) unless specified otherwise
and used without further purification. All anhydrous reactions
were performed under a static argon or nitrogen atmosphere in
flame-dried glassware using anhydrous solvents. Organic phases
were dried over MgSO4, rotary evaporated under reduced pressure,
and flash column chromatography used silica gel 60 (230–400 mesh,
Selecto Scientific Inc., Suwanee, GA). Purity of the intermediates
and final compounds was established by analytical TLC on
precoated aluminum silica gel plates (Whatman, UV254, layer
thickness 250 µm), and chromatograms were visualized under
ultraviolet light and by phosphomolybdic acid straining. Melting
points were determined on a capillary Electrothermal melting point
apparatus and are uncorrected. 1H NMR spectra were recorded on
a Bruker DMX-500 spectrometer operating at 500 MHz. All NMR
spectra were recorded using CDCl3 as solvent unless otherwise
stated, and chemical shifts are reported in ppm relative to
tetramethylsilane as internal standard. Multiplicities are indicated
as br (broadened), s (singlet), d (doublet), t (triplet), q (quartet),
and m (multiplet), and coupling constants (J) are reported in hertz
(Hz). Mass spectra were recorded on a Hewlett-Packard 6890 GC/
MS instrument at the School of Pharmacy, University of Con-
necticut. High-resolution mass spectra were performed at the School
2-(N,N-Diisopropylcarboxamido)-5,1′,6′-trimethoxy-4′-(1′′,1′′-
dimethylheptyl)biphenyl (3b). Biphenyl 3b was prepared from
boronic acid 2b (0.89 g, 3.2 mmol), 2,6-dimethoxy-4-(1′,1′-
dimethylheptyl)bromobenzene (1.00 g, 2.91 mmol), Ba(OH)2 ·8H2O
(1.38 g, 4.37 mmol), and Pd(PPh3)4 (336 mg, 0.291 mmol) as
described for 3a to give 1.32 g (2.65 mmol, 83%) of 3b as a white
1
solid: mp 103–104 °C; Rf 0.28 (Et2O/petroleum ether, 50:50); H
NMR δ 7.28 (d, J ) 8.5 Hz, 1H), 6.87 (dd, J ) 8.5 Hz, 2.5 Hz,
1H), 6.79 (d, J ) 2.5 Hz, 1H), 6.53 (s, 1H), 6.52 (s, 1H), 3.80 (s,
3H), 3.72 (s, 3H), 3.71 (s, 3H), 3.52 (m, 1H), 3.15 (m, 1H),
1.58–1.62 (m, 2H), 1.46 (d, J ) 6.5 Hz, 3H), 1.18–1.35 (m, 12 H,
especially, 1.30, s, 6H), 1.05–1.11 (m, 5H, especially, 1.08, d, J )
6.5 Hz, 3H), 0.90 (d, J ) 6.5 Hz, 3H), 0.86 (t, J ) 7.2 Hz, 3H),
0.56 (d, J ) 6.5 Hz, 3H). Exact mass calculated for C31H47NO4,
497.7092; found, 497.7095.

6498 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26
Khanolkar et al.
3-(1′,1′-Dimethylheptyl)-1-hydroxy-9-methyl-6H-benzo[c]-
chromene-6-one (Cannabilactone 4a). A solution of 3a (0.241 g,
0.500 mmol) in 10 mL of anhydrous CH2Cl2 was cooled to -78
°C and BBr3 (1.38 mL of a 1.0 M CH2Cl2 solution, 1.38 mmol)
was added dropwise. The reaction mixture was stirred and allowed
to warm to room temperature overnight. The mixture was cooled
to 0 °C, carefully quenched with 1 mL of dry MeOH, and allowed
to warm to room temperature over 2 h. Then 5 mL of 5% aqueous
HCl was added, the product extracted with CH2Cl2, and the solution
dried. The CH2Cl2 was removed to give the demethylated inter-
mediate as a yellow foam, which was dissolved in 5 mL of glacial
acetic acid. The reaction mixture was refluxed for 5 h and then
cooled to room temperature. Water was added cautiously to the
mixture at 0 °C. Diethyl ether was added, and the ether solution
was washed with water, 15% aqueous NaHCO3, water, and brine
and then dried. Filtration and removal of solvent gave a crude solid
that was chromatographed (acetone/petroleum ether, 30:70) to give
0.100 g (0.284 mmol, 57%) of 4a37 as a white solid: mp 186–188
°C (lit.37 mp 184–185 °C); Rf 0.57 (Et2O/petroleum ether, 50:50);
1H NMR δ 8.80 (s, 1H), 8.31 (d, J ) 8.5 Hz, 1H), 7.34 (dd, J )
8.5 Hz, 1.0 Hz, 1H), 6.95 (d, J ) 2.0 Hz, 1H), 6.70 (d, J ) 2.0 Hz,
1H), 5.96 (bs, 1H), 2.54 (s, 3H), 1.57–1.60 (m, 2H), 1.29 (s, 6H),
1.16–1.25 (m, 6H), 1.02–1.09 (m, 2H), 0.83 (t, J ) 7.0 Hz, 3H);
MS m/z 352 (M+). An X-ray crystal structure was obtained for 4a,
see Figure 6 and Supporting Information.
refluxed for 1.5 h. The reaction was cooled to room temperature
and quenched by the addition of 20 mL of saturated aqueous NH4Cl.
The THF was removed and the residue was dissolved in anhydrous
Et2O (50 mL). The ether solution was washed with water and brine
and dried, and solvent removal gave the crude intermediates that
were used without further purification in the subsequent cyclization
reactions. The intermediates were dissolved in anhydrous CHCl3
(10–15 mL) and 60 mg of p-toluenesulfonic acid monohydrate was
then added under an argon atmosphere. The reaction mixture was
stirred at room temperature for 6–8 h and then treated with 10 mL
of water. The organic phase was separated and washed with water,
15% aqueous NaHCO3, water, and brine and then dried. Solvent
removal gave the crude product that was chromatographed (acetone/
petroleum ether, 10:90) to give the cannabinol analogs 6 in yields
of 63–74%.
3-(1′,1′-Dimethylheptyl)-6,6,9-trimethyl-6H-benzo[c]chromene-
1-ol (Cannabinol Analog 6a)37,38 This compound was identical
to reported 6a: mp 97–98 °C (lit.37 mp 95–98 °C).
6,6-Dimethyl-3-(1′,1′-dimethylheptyl)-9-methoxy-6H-benzo[c]
chromene-1-ol (Cannabinol Analog 6b). White solid, 140 mg
(0.287 mmol, 71%); mp 60–61 °C; Rf 0.22 (Et2O/petroleum ether,
10:90); 1H NMR δ 7.99 (d, J ) 2.5 Hz, 1H), 7.15 (d, J ) 8.5 Hz,
1H), 6.80 (dd, J ) 2.5 Hz, 8.5 Hz, 1H), 6.56 (d, J ) 1.6 Hz, 1H),
6.40 (d, J ) 1.6 Hz, 1H), 5.16 (s, 1H), 3.84 (s, 3H), 1.57 (s, 6H),
1.52–1.55 (m, 2H), 1.25 (s, 6H), 1.21–1.24 (m, 2H), 1.16–1.20 (m,
4H), 1.06–1.10 (m, 2H), 0.83 (t, J ) 2.5 Hz, 3H). Exact mass
calculated for C25H34O3, 382.5357; found, 382.5361. Anal.
(C25H34O3 ·1/3H2O) C, H: calcd, C% 77.28, H% 8.99; found, C%
77.20, H% 8.40.
6,6-Dimethyl-3-(1′,1′-dimethylheptyl)-6H-benzo[c]chromene-
1,9-diol (Cannabinol Analog 6c). White solid, 14.7 mg (3.99 ×
10-5 mol, 74%); mp 163–164 °C; Rf 0.63 (EtOAc/petroleum ether,
25:75); 1H NMR δ 7.91 (d, J ) 2.4 Hz, 1H), 7.11 (d, J ) 8.2 Hz,
1H), 6.73 (dd, J ) 2.4 Hz, 8.2 Hz, 1H), 6.55 (d, J ) 1.8 Hz, 1H),
6.38 (d, J ) 1.8 Hz, 1H), 5.22 (bs, 1H), 4.86 (bs, 1H), 1.59 (s,
6H), 1.51–1.55 (m, 2H), 1.24 (s, 6H), 1.16–1.23 (m, 6H), 1.03–1.11
(m, 2H), 0.83 (t, J ) 7.0 Hz, 3H). Exact mass calculated for
C24H32O3, 368.2351; found, 368.2355. Anal. (C24H32O3 ·1/2H2O)
C, H.
3-(1′,1′-Dimethylheptyl)-1-hydroxy-9-methoxy-6H-benzo[c]-
chromene-6-one (Cannabilactone 4b). A solution of 3b (0.500
g, 1.00 mmol) in 10 mL of anhydrous CH2Cl2 was cooled to 0 °C
and 3 mL of 9-iodo-9-BBN (4.0 mL of a 1.0 M in hexane, 4.0
mmol) was added dropwise. The reaction mixture was stirred at 0
°C for 4 h. The CH2Cl2 was removed and the residue was dissolved
in anhydrous Et2O (30 mL). The mixture was then treated with 4
mL of ethanolamine solution (1.0 M in ether). The reaction mixture
was stirred for 40 min and filtered through a short celite column.
The filtrate was concentrated and dissolved in 5 mL of glacial acetic
acid. The reaction mixture was refluxed for 5 h and then cooled to
room temperature. Water was added cautiously to the mixture at 0
°C. Diethyl ether was added and the ether solution was washed
with water, 15% aqueous NaHCO3, water, and brine and then dried.
Filtration and removal of solvent gave a solid crude product that
was chromatographed (EtOAc/hexanes, 20:80) to give 0.281 mg
(0.763 mmol, 76%) of 4b as a white solid: mp 149–151 °C; Rf
Radioligand Binding Assay. Forebrain synaptosomal mem-
branes were prepared from frozen rat brains by the method of Dodd
et al.57 and were used to assess the affinities of the novel analogs
for the CB1 binding sites, while affinities for the CB2 sites were
measured using a membrane preparation from frozen mouse spleen
using a similar procedure.50 The displacement of specifically
tritiated CP55,940 (7) from these membranes was used to determine
the IC50 values for the test compounds. The radioligand binding
assay was conducted on 96-well microfilter plates, as previously
described.50 Briefly, 100 µL of cannabinergic ligand (at eight
different concentrations) in DMSO, 50 µL of rat brain or mouse
spleen membrane preparation (40–50 µg protein), and 50 µL of
[3H]CP55,940 (7; 3.08 nM) in TME (25 mM Tris, 5 mM MgCl2,
1 mM EDTA) buffer containing 0.1% BSA was incubated for 1 h
at 30 °C. For the nonspecific binding control, 100 µL of 200 nM
CP55,940 (7) 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
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 normal-
ized data from three independent experiments were combined and
analyzed using a four-parameter logistic equation to yield IC50
values that were converted to Ki values using the assumptions of
Cheng and Prussoff.58
1
0.44 (Et2O/petroleum ether, 50:50); H NMR δ 8.52 (d, J ) 2.5
Hz, 1H), 8.35 (d, J ) 8.5 Hz, 1H), 7.05 (dd, J ) 2.5 Hz, 8.5 Hz,
1H), 6.94 (d, J ) 1.8 Hz, 1H), 6.71 (d, J ) 1.8 Hz, 1H), 6.12 (bs,
1H), 3.97 (s, 3H), 1.56–1.60 (m, 2H), 1.28 (s, 6H), 1.18–1.24 (m,
6H), 1.02–1.08 (m, 2H) 0.82 (t, J ) 7.0 Hz, 3H). Anal. (C23H28O4)
C, H. An X-ray crystal structure was obtained for 4b, see Figure 6
and Supporting Information.
1,9-Dihydroxy-3-(1′,1′-dimethylheptyl)-6H-benzo[c]chromene-
6-one (Cannabilactone 5). To a solution of 4b (700 mg, 1.99
mmol) in 40 mL of anhydrous CH2Cl2 was added 3.80 mL (3.80
mmol) of boron tribromide solution (1.0 M in CH2Cl2) dropwise
at room temperature. The mixture was stirred at room temperature
for 15 min and then refluxed for 12 h. The reaction mixture was
then cooled to room temperature and treated with water dropwise.
The organic phase was separated, washed with water, 15% aqueous
NaHCO3, water, and brine, and then dried. Solvent removal gave
a crude solid product that was chromatographed (Et2O/petroleum
ether, 50:50) to give 510 mg (1.44 mmol, 72%) of 5 as an off-
white foam: mp 103–108 °C; Rf 0.28 (Et2O/petroleum ether,
50:50); 1H NMR δ 8.53 (d, J ) 2.2 Hz, 1H), 8.28 (dd, J ) 8.6 Hz,
2.2 Hz, 1H), 7.02 (d, J ) 8.6 Hz, 1H), 6.86 (d, J ) 1.4 Hz, 1H),
6.75 (d, J ) 1.4 Hz, 1H), 6.20 (bs, 1H), 6.05 (bs, 1H), 1.49–1.56
(m, 2H), 1.27 (s, 6H), 1.12–1.22 (m, 6H), 0.98–1.05 (m, 2H), 0.82
(t, J ) 7.1 Hz, 3H). Anal. (C22H26O4 ·1/2H2O) C, H.
General Procedure for Preparation of Cannabinol Analogs
6. To a solution of cannabilactone (1.0 mmol) in anhydrous THF
(20 mL) was added methylmagnesium iodide (1.66 mL, 3.0 M in
Et2O) at room temperature under an argon atmosphere. The reaction
mixture was stirred at room temperature for 30 min and then
[35S]GTPγS Binding Assay. This assay was performed as
reported,52,53 except using a mouse spleen preparation. The mouse
spleen was lysed in a cell disruption bomb, centrifuged twice at
1000 g and 175000 g for 10 min each, and finally resuspended in
TME buffer with 0.1% BSA to a protein concentration of 0.6 mg/

Cannabilactones: CB2 SelectiVe Agonists
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26 6499
(8) Herkenham, M.; Lynn, A. B.; Little, M. D.; Johnson, M. R.; Melvin,
L. S.; de Costa, B. R.; Rice, K. C. Cannabinoid receptor localization
in brain. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 1932–1936.
(9) Straiker, A.; Stella, N.; Piomelli, D.; Mackie, K.; Karten, H. J.;
Maguire, G. Cannabinoid CB1 receptors and ligands in vertebrate
retina: Localization and function of an endogenous signaling system.
Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 14565–14570.
(10) Galiègue, S.; Mary, S.; Marchand, J.; Dussossoy, D.; Carrière, D.;
Carayon, P.; Bouaboula, M.; Shire, D.; Le Fur, G.; Casellas, P.
Expression of central and peripheral cannabinoid receptors in human
immune tissues and leukocyte subpopulations. Eur. J. Biochem. 1995,
232, 54–61.
mL. The cell membrane preparations (24–40 µg) were incubated
for 2 h at 30 °C with compounds at 1 µM, 30 µM GDP, and 0.05
nM [35S]GTPγS to a final volume of 250 µL. Nonspecific binding
was assessed in the presence of 10 µM nonradiolabeled GTPγS.
Postincubation, the wells were filtered using GF/B filters (Perkin-
Elmer) and washed. Bound [35S]GTPγS was determined using a
Packard Topcount scintillation counter and results were analyzed
using Prizm software (GraphPad Software, Inc.).
cAMP Assay. The cAMP assay was performed as previously
described.53
CannabinoidMediatedAntinociception.Animals.MaleSprague–
Dawley rats (Harlan, Indianapolis, IN) 200–300 g at time of testing,
were maintained in a climate-controlled room on a 12 h light/dark
cycle (lights on at 06:00 h) and food and water were available ad
libitum. All of the testing was performed in accordance with the
policies and recommendations of the International Association for
the Study of Pain (IASP) and the National Institutes of Health (NIH)
guidelines for the handling and use of laboratory animals and
received approval from the Institutional Animal Care and Use
Committee (IACUC) of the University of Arizona.
In Vivo Drug Administration. All drugs were dissolved in
dimethyl sulfoxide (DMSO) and were injected subcutaneously in
the plantar surface of the hindpaw in a total volume of 50 µL.
DMSO given in hindpaw at this volume had no effect. The method
of Hargreaves et al.59 was employed to assess paw-withdrawal
latency to a thermal nociceptive stimulus. Rats were allowed to
acclimate within a plexiglass enclosure on a clear glass plate
maintained at 30 °C. A radiant heat source (i.e., high intensity
projector lamp) was activated with a timer and focused onto the
plantar surface of the hindpaw. Paw-withdrawal latency was
determined by a photocell that halted both lamp and timer when
the paw was withdrawn. The latency to withdrawal of the paw from
the radiant heat source was determined both before and after drug
or vehicle administration. A maximal cutoff of 40 s was employed
to prevent tissue damage.
(11) Bouaboula, M.; Rinaldi, M.; Carayon, P.; Carillon, C.; Delpech, B.;
Shire, D.; Le Fur, G.; Casellas, P. Cannabinoid-receptor expression
in human leukocytes. Eur. J. Biochem. 1993, 214, 173–180.
(12) 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. Identification and functional characterization of brainstem
cannabinoid CB2 receptors. Science 2005, 310, 329–332.
(13) Pacher, P.; Batkai, S.; Kunos, G. The endocannabinoid system as an
emerging target of pharmacotherapy. Pharmacol. ReV. 2006, 58, 389–
462.
(14) Reggio, P. Pharmacophores for ligand recognition and activation/
inactivation of the cannabinoid receptors. Curr. Pharm. Des. 2003, 9,
1607–1633.
(15) Lu, D.; Meng, Z.; Thakur, G. A.; Fan, P.; Steed, J.; Tartal, C. L.;
Hurst, D. P.; Reggio, P. H.; Deschamps, J. R.; Parrish, D. A.; George,
C.; Järbe, T. U. C.; Lamb, R. J.; Makriyannis, A. Adamantyl
cannabinoids: A novel class of cannabinergic ligands. J. Med. Chem.
2005, 48, 4576–4585.
(16) Thakur, G. A.; Nikas, S. P.; Li, C.; Makriyannis, A. Structural
requirements for cannabinoid receptor probes. Handb. Exp. Pharmacol.
2005, 168, 209–246.
(17) Raitio, K. H.; Salo, O. M. H.; Nevalainen, T.; Poso, A.; Järvinen, T.
Targeting the cannabinoid CB2 receptor: Mutations, modeling and
development of CB2 selective ligands. Curr. Med. Chem. 2005, 12,
1217–1237.
(18) Gareau, Y.; Dufresne, C.; Gallant, M.; Rochette, C.; Sawyer, N.;
Slipetz, D. M.; Tremblay, N.; Weech, P. K.; Metters, K. M.; Labelle,
M. Structure-activity relationships of tetrahydrocannabinol analogues
on human cannabinoid receptors. Bioorg. Med. Chem. Lett. 1996, 6,
189–194.
(19) Huffman, J. W.; Yu, S.; Showalter, V.; Abood, M. E.; Wiley, J. L.;
Compton, D. R.; Martin, B. R.; Bramblett, R. D.; Reggio, P. H.
Synthesis and pharmacology of a very potent cannabinoid lacking a
phenolic hydroxyl with high affinity for the CB2 receptor. J. Med.
Chem. 1996, 39, 3875–3877.
(20) Huffman, J. W.; Miller, J. R. A.; Liddle, J.; Yu, S.; Thomas, B. F.;
Wiley, J. L.; Martin, B. R. Structure-activity relationships for 1′,1′-
dimethylalkyl-∆8-tetrahydrocannabinols. Bioorg. Med. Chem. 2003,
11, 1397–1410.
(21) Khanolkar, A. D.; Lu, D.; Fan, P.; Tian, X.; Makriyannis, A. Novel
conformationally restricted tetracyclic analogs of ∆8-tetrahydrocan-
nabinol. Bioorg. Med. Chem. Lett. 1999, 9, 2119–2124.
(22) Hanus, L.; Breuer, A.; Tchilibon, S.; Shiloah, S.; Goldenberg, D.;
Horowitz, M.; Pertwee, R. G.; Ross, R. A.; Mechoulam, R.; Fride, E.
HU-308: A specific agonist for CB2, a peripheral cannabinoid receptor.
Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 14228–14233.
(23) Malan, T. P., Jr.; Ibrahim, M. M.; Deng, H.; Liu, Q.; Mata, H. P.;
Vanderah, T.; Porreca, F.; Makriyannis, A. CB2 cannabinoid receptor-
mediated peripheral antinociception. Pain 2001, 93, 239–245.
(24) Ibrahim, M. M.; Deng, H.; Zvonok, A.; Cockayne, D. A.; Kwan, J.;
Mata, H. P.; Vanderah, T. W.; Lai, J.; Porreca, F.; Makriyannis, A.;
Malan, T. P. Jr. Activation of CB2 cannabinoid receptors by AM1241
inhibits experimental neuropathic pain: Pain inhibition by receptors
not present in the CNS. Proc. Natl. Acad. Sci. U.S.A. 2003, 100,
10529–10533.
Acknowledgment. This research work was supported by
Grants DA-7215, DA-3801, DA-152, DA-9158 (Center for Drug
Discovery, Northeastern University), DA-0355 (The University
of Arizona College of Medicine) from the National Institute on
Drug Abuse, and by the Office of Naval Research. We also
thank Joy Erickson for the biochemical assays, Ruoxi Lan who
prepared 9, and Fenmei Yao who prepared 10.
Supporting Information Available: Elemental analyses data
and X-ray diffraction data for 4a and 4b. This material is available
free of charge via the Internet at .
References
(1) Lambert, D. M.; Fowler, C. J. The endocannabinoid system: Drug
targets, lead compounds, and potential therapeutic applications. J. Med.
Chem. 2005, 48, 5059–5087.
(2) Munro, S.; Thomas, K. L.; Abu-Shaar, M. Molecular characterization
of a peripheral receptor for cannabinoids. Nature 1993, 365, 61–65.
(3) Shire, D.; Calandra, B.; Rinaldi-Carmona, M.; Oustric, D.; Pessègue,
B.; Bonnin-Cabanne, O.; Le Fur, G.; Caput, D.; Ferrara, P. Molecular
cloning, expression and function of the murine CB2 peripheral
cannabinoid receptor. Biochim. Biophys. Acta 1996, 1307, 132–136.
(25) Hohmann, A. G.; Farthing, J. N.; Zvonok, A. M.; Makriyannis, A.
Selective activation of cannabinoid CB2 receptors suppresses hyper-
algesia evoked by intradermal capsaicin. J. Pharmacol. Exp. Ther.
2004, 308, 446–453.
(4) Gérard, C. M.; Mollereau, C.; Vassart, G.; Parmentier, M. Molecular
cloning of a human cannabinoid receptor which is also expressed in
testis. Biochem. J. 1991, 279, 129–134.
(5) Chakrabarti, A.; Onaivi, E. S.; Chaudhuri, G. Cloning and sequencing
of a cDNA encoding the mouse brain-type cannabinoid receptor
protein. DNA Sequence 1995, 5, 385–388.
(6) Abood, M. E.; Ditto, K. E.; Noel, M. A.; Showalter, V. M.; Tao, Q.
Isolation and expression of a mouse CB1 cannabinoid receptor gene.
Comparison of binding properties with those of native CB1 receptors
in mouse brain and N18TG2 neuroblastoma cells. Biochem. Pharma-
col. 1997, 53, 207–214.
(7) Griffin, G.; Tao, Q.; Abood, M. E. Cloning and pharmacological
characterization of the rat CB2 cannabinoid receptor. J. Pharmacol.
Exp. Ther. 2000, 292, 886–894.
(26) Ibrahim, M. M.; Porreca, F.; Lai, J.; Albrecht, P. J.; Rice, F. L.;
Khodorova, A.; Davar, G.; Makriyannis, A.; Vanderah, T. W.; Mata,
H. P.; Malan, T. P. Jr. CB2 cannabinoid receptor activation produces
antinociception by stimulating peripheral release of endogenous
opioids. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 3093–3098.
(27) Ibrahim, M. M.; Rude, M. L.; Stagg, N. J.; Mata, H. P.; Lai, J.;
Vanderah, T. W.; Porreca, F.; Buckley, N. E.; Makriyannis, A.; Malan,
T. P. Jr. CB2 cannabinoid receptor mediation of antinociception. Pain
2006, 122, 36–42.
(28) Huffman, J. W. CB2 receptor ligands. Mini-ReV. Med. Chem. 2005,
5, 641–649.

6500 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26
Khanolkar et al.
(29) Lu, D.; Vemuri, K.; Duclos, R. I., Jr.; Makriyannis, A. The Cannab-
inergic system as a target for anti-inflammatory therapies. Curr. Top.
Med. Chem. 2006, 6, 1401–1426.
(30) Nackley, A. G.; Makriyannis, A.; Hohmann, A. G. Selective activation
of cannabinoid CB2 receptors suppresses spinal Fos protein expression
and pain behavior in a rat model of inflammation. Neuroscience 2003,
119, 747–757.
(31) Nackley, A. G.; Suplita, R. L., II; Hohmann, A. G. A peripheral
cannabinoid mechanism suppresses spinal Fos protein expression and
pain behavior in a rat model of inflammation. Neuroscience 2003,
117, 659–670.
in the metalations of N,N-dialkyltoluamides. J. Org. Chem. 1973, 38,
1668–1674.
(47) Alonso, E.; Ramón, D. J.; Yus, M. Naphthalene-catalysed lithiation
of N,N-diisopropylbenzamide and its methoxy derivatives. Tetrahedron
1998, 54, 13629–13638.
(48) Fürstner, A.; Seidel, G. Shortcut syntheses of naturally occurring
5-alkylresorcinols with DNA-cleaving properties. J. Org. Chem. 1997,
62, 2332–2336.
(49) Novák, J.; Salemink, C. A. Cannabis. Part 27. Synthesis of 8-, 10-,
and 11-oxygenated cannabinols. J. Chem. Soc., Perkin Trans. 1 1983,
2867–2871.
(32) Gutierrez, T.; Farthing, J. N.; Zvonok, A. M.; Makriyannis, A.;
Hohmann, A. G. Activation of peripheral cannabinoid CB1 and CB2
receptors suppresses the maintenance of inflammatory nociception:
A comparative analysis. Br. J. Pharmacol. 2007, 150, 153–163.
(33) Dogrul, A.; Gul, H.; Akar, A.; Yildiz, O.; Bilgin, F.; Guzeldemir, E.
Topical cannabinoid antinociception: Synergy with spinal sites. Pain
2003, 105, 11–16.
(34) Choong, K.-C.; Su, X.; Urban, M. O. Effect of CP55,940 on
mechanosensory spinal neurons following chronic inflammation.
Neurosci. Lett. 2007, 414, 105–109.
(35) Makriyannis, A.; Khanolkar, A. Peripheral cannabinoid receptor (CB2)
selective ligands (University of CT, U.S.A.). WO Patent 2001/28329
A1, 2001;pp 33.
(36) Showalter, V. M.; Compton, D. R.; Martin, B. R.; Abood, M. E.
Evaluation of binding in a transfected cell line expressing a peripheral
cannabinoid receptor (CB2): identification of cannabinoid receptor
subtype selective ligands. J. Pharmacol. Exp. Ther. 1996, 278, 989–
999.
(50) Khanolkar, A. D.; Abadji, V.; Lin, S.; Hill, W. A. G.; Taha, G.;
Abouzid, K.; Meng, Z.; Fan, P.; Makriyannis, A. Head group analogs
of arachidonylethanolamide, the endogenous cannabinoid ligand.
J. Med. Chem. 1996, 39, 4515–4519.
(51) Mukherjee, S.; Adams, M.; Whiteaker, K.; Daza, A.; Kage, K.; Cassar,
S.; Meyer, M.; Yao, B. B. Species comparison and pharmacological
characterization of rat and human CB2 cannabinoid receptors. Eur.
J. Pharmacol. 2004, 505, 1–9.
(52) Lin, S.; Khanolkar, A. D.; Fan, P.; Goutopoulos, A.; Qin, C.;
Papahadjis, D.; Makriyannis, A. Novel analogues of arachidonyl-
ethanolamide (anandamide): Affinities for the CB1 and CB2 cannab-
inoid receptors and metabolic stability. J. Med. Chem. 1998, 41, 5353–
5361.
(53) McLaughlin, P. J.; Qian, L.; Wood, J. T.; Wisniecki, A.; Winston,
K. M.; Swezey, L. A.; Ishiwari, K.; Betz, A. J.; Pandarinathan, L.;
Xu, W.; Makriyannis, A.; Salamone, J. D. Suppression of food intake
and food-reinforced behavior produced by the novel CB1 receptor
antagonist/inverse agonist AM 1387. Pharmacol., Biochem. BehaV.
2006, 83, 396–402.
(54) Lan, R.; Liu, Q.; Fan, P.; Lin, S.; Fernando, S. R.; McCallion, D.;
Pertwee, R.; Makriyannis, A. Structure-activity relationships of
pyrazole derivatives as cannabinoid receptor antagonists. J. Med.
Chem. 1999, 42, 769–776.
(55) Ross, R. A.; Brockie, H. C.; Stevenson, L. A.; Murphy, V. L.;
Templeton, F.; Makriyannis, A.; Pertwee, R. G. Agonist-inverse
agonist characterization at CB1 and CB2 cannabinoid receptors of
L759633, L759656, and AM630. Br. J. Pharmacol. 1999, 126, 665–
672.
(56) Zimmer, A.; Zimmer, A. M.; Hohmann, A. G.; Herkenham, M.;
Bonner, T. I. Increased mortality, hypoactivity, and hypoalgesia in
cannabinoid CB1 receptor knockout mice. Proc. Natl. Acad. Sci. U.S.A.
1999, 96, 5780–5785.
(57) Dodd, P. R.; Hardy, J. A.; Oakley, A. E.; Edwardson, J. A.; Perry,
E. K.; Delaunoy, J.-P. A rapid method for preparing synaptosomes:
Comparison with alternative procedures. Brain Res. 1981, 226, 107–
118.
(37) Rhee, M.-H.; Vogel, Z.; Barg, J.; Bayewitch, M.; Levy, R.; Hanuš,
L.; Breuer, A.; Mechoulam, R. Cannabinol derivatives: Binding to
cannabinoid receptors and inhibition of adenylylcyclase. J. Med. Chem.
1997, 40, 3228–3233.
(38) Mahadevan, A.; Siegel, C.; Martin, B. R.; Abood, M. E.; Beletskaya,
I.; Razdan, R. K. Novel cannabinol probes for CB1 and CB2
cannabinoid receptors. J. Med. Chem. 2000, 43, 3778–3785.
(39) Adams, R.; Baker, B. R. Structure of cannabinol. V. A second method
of synthesis of cannabinol. J. Am. Chem. Soc. 1940, 62, p. 2401.
(40) Ghosh, R.; Todd, A. R.; Wilkinson, S. Cannabis indica. Part V. The
synthesis of cannabinol. J. Chem. Soc. 1940, 1393–1396.
(41) Watanabe, T.; Miyaura, N.; Suzuki, A. Synthesis of sterically hindered
biaryls via the palladium-catalyzed cross-coupling reaction of aryl-
boronic acids or their esters with haloarenes. Synlett 1992, 207–210.
(42) Miyaura, N.; Suzuki, A. Palladium-catalyzed cross-coupling reactions
of organoboron compounds. Chem. ReV. 1995, 95, 2457–2483.
(43) Alo, B. I.; Kandil, A.; Patil, P. A.; Sharp, M. J.; Siddiqui, M. A.;
Snieckus,V.;Josephy,P.D.Sequentialdirectedorthometalation-boronic
acid cross-coupling reactions. A general regiospecific route to oxygen-
ated dibenzo[b,d]pyran-6-ones related to ellagic acid. J. Org. Chem.
1991, 56, 3763–3768.
(44) Ku, Y.-Y.; Grieme, T.; Raje, P.; Sharma, P.; Morton, H. E.; Rozema,
M.; King, S. A. A practical and scaleable synthesis of A-224817.0, a
novel nonsteroidal ligand for the glucocorticoid receptor. J. Org. Chem.
2003, 68, 3238–3240.
(45) Srebnik, M.; Mechoulam, R.; Yona, I. Halogenation of phenols and
phenyl ethers with potassium halides in the presence of 18-crown-6
on oxidation with m-chloroperbenzoic acid. J. Chem. Soc., Perkin.
Trans. 1 1987, 1423–1427.
(58) Cheng, Y.-C.; Prusoff, W. H. Relationship between the inhibition
constant (KI) and the concentration of inhibitor which causes 50%
inhibition (I50) of an enzymatic reaction. Biochem. Pharmacol. 1973,
22, 3099–3108.
(59) Hargreaves, K.; Dubner, R.; Brown, F.; Flores, C.; Joris, J. A new
and sensitive method for measuring thermal nociception in cutaneous
hyperalgesia. Pain 1988, 32, 77–88.
(60) Tallarida, R. J.; Murray, R. B. Manual of Pharmacological Calcula-
tions with Computer Programs., 2nd ed.; Springer: New York, 1987;
pp 150.
(46) Ludt, R. E.; Griffiths, J. S.; McGrath, K. N.; Hauser, C. R. Comparison
of the synthetic utility of n-butyllithium and lithium diisopropylamide
JM070441U