Resorcinol derivatives: A novel template for the development of cannabinoid CB1/CB2 and CB2-selective agonists

Article abstract of DOI:10.1124/jpet.301.2.679

The role of the oxygen of the benzopyran substituent of Δ⁹-tetrahydrocannabinol in defining affinity for brain cannabinoid (CB₁) receptors is not well understood; however, it is known that opening the pyran ring can result in either increased potency and affinity, as in CP 55,940 [(-)-cis-3-[2-hydroxy-4(1,1-dimethyl-heptyl) phenyl]-trans-4-(3-hydroxy-propyl)cyclohexanol], or in an inactive cannabinoid, as in cannabidiol. In the present study, a series of bicyclic resorcinols that resemble cannabidiol were synthesized and tested in vitro and in vivo. Analysis of the structure-activity relationships of these analogs revealed several structural features that were important for maintaining CB₁ receptor recognition and in vivo activity, including the presence of a branched lipophilic side chain and free phenols as well as substitution of a cyclohexane as the second ring of these bicyclic cannabinoids. Many of these analogs exhibited CB₂ selectivity, particularly the dimethoxyresorcinol analogs, and this selectivity was enhanced by longer side chain lengths. Hence, unlike cannabidiol, these resorcinol derivatives had good affinity for CB₁ and/or CB₂ receptors as well as potent in vivo activity. These results suggest that the resorcinol series represent a novel template for the development of CB₂-selective cannabinoid agonists that have the potential to offer insights into similarities and differences between structural requirements for receptor recognition at CB₁ and CB₂ receptors.

Full text of DOI:10.1124/jpet.301.2.679

0022-3565/02/3012-679–689$7.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics
JPET 301:679–689, 2002
Vol. 301, No. 2
4650/979882
Printed in U.S.A.
Resorcinol Derivatives: A Novel Template for the Development
of Cannabinoid CB1/CB2 and CB2-Selective Agonists
JENNY L. WILEY, IRINA D. BELETSKAYA, EDWARD W. NG, ZONGMIN DAI, PETER J. CROCKER, ANU MAHADEVAN,
RAJ K. RAZDAN, and BILLY R. MARTIN
Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia (J.L.W., I.D.B., B.R.M.); and Organix, Inc.,
Woburn, Massachusetts (E.W.N., P.J.C., Z.D., A.M., R.K.R.)
Received October 15, 2001; accepted January 22, 2002
This article is available online at http://jpet.aspetjournals.org
ABSTRACT
The role of the oxygen of the benzopyran substituent of ⌬9- free phenols as well as substitution of a cyclohexane as the
tetrahydrocannabinol in defining affinity for brain cannabinoid second ring of these bicyclic cannabinoids. Many of these
(CB1) receptors is not well understood; however, it is known analogs exhibited CB2 selectivity, particularly the dime-
that opening the pyran ring can result in either increased po- thoxyresorcinol analogs, and this selectivity was enhanced by
tency and affinity, as in CP 55,940 [(Ϫ)-cis-3-[2-hydroxy-4(1,1- longer side chain lengths. Hence, unlike cannabidiol, these
dimethyl-heptyl)phenyl]-trans-4-(3-hydroxy-propyl)cyclohexa- resorcinol derivatives had good affinity for CB1 and/or CB2
nol], or in an inactive cannabinoid, as in cannabidiol. In the receptors as well as potent in vivo activity. These results sug-
present study, a series of bicyclic resorcinols that resemble gest that the resorcinol series represent a novel template for the
cannabidiol were synthesized and tested in vitro and in vivo. development of CB2-selective cannabinoid agonists that have
Analysis of the structure-activity relationships of these analogs the potential to offer insights into similarities and differences
revealed several structural features that were important for between structural requirements for receptor recognition at
maintaining CB1 receptor recognition and in vivo activity, in- CB1 and CB2 receptors.
cluding the presence of a branched lipophilic side chain and
At least five distinct classes of cannabinoids have been opening the pyran ring (as in CP 55,940) does not eliminate
identified: traditional tricyclic tetrahydrocannabinols [e.g.,
⌬9-tetrahydrocannabinol (THC)], synthetic bicyclic cannabi-
noids (e.g., CP 55,940; Little et al., 1988), aminoalkylindoles
(e.g., WIN 55,212; D’Ambra et al., 1992), endocannabinoids
(e.g., anandamide; Devane et al., 1992), and pyrazole antag-
onists (e.g., SR141716A; Rinaldi-Carmona et al., 1994). Al-
though the chemical structures of these cannabinoids differ
markedly, all of them contain at least one oxygen that is
hypothesized to be involved in the binding of these drugs to
brain cannabinoid (CB1) receptors. ⌬9-THC, the primary psy-
choactive constituent of the marijuana plant, and other tet-
rahydrocannabinols contain two oxygens: a phenolic hy-
droxyl at position 1 and an oxygen in a pyran ring on the
opposite side of the molecule (Fig. 1). The phenolic hydroxyl
group at position 1 interacts with the CB1 receptor through
hydrogen bonding with a lysine residue (Lys-192) (Song and
Bonner, 1996). The role of the oxygen of the benzopyran
substituent of ⌬9-THC is less clear; however, it is known that
binding or in vivo activity (Little et al., 1988). Furthermore,
in the absence of a phenolic hydroxyl, as in 1-deoxy analogs of
⌬8-THC, orientation of the cannabinoid molecule with re-
spect to the CB1 receptor may be inverted, and the pyran
oxygen may substitute as a substrate for hydrogen bonding
with Lys 192 (Huffman et al., 1996, 1999).
In contrast to the high binding affinity of CP 55,940 and
other similar pyran ring open analogs, the natural product
cannabidiol is also a pyran ring open compound, but it does
not bind to CB1 or CB2 receptors nor does it have a cannabi-
noid profile of effects in vivo. Even the 1Ј,1Ј-dimethylheptyl
analog of cannabidiol binds very poorly to the CB1 receptor
(R. K. Razdan, unpublished observations). This intriguing
feature of cannabidiol prompted us to examine the structure-
activity relationship of resorcinol derivatives, which could be
considered as cannabidiol analogs.
After our work on the resorcinol series was initiated,
Hanusˇ et al. (1999) published the synthesis and activity of
HU-308, a dimethoxyresorcinol derivative that is a CB2-se-
lective agonist. The transmembrane regions of CB2 receptors
(areas involved in ligand recognition) exhibit 68% homology
Research supported by National Institute on Drug Abuse Research Grants
DA-03672 and DA-05488 and Training Grant DA-07027.
ABBREVIATIONS: THC, tetrahydrocannabinol; CP 55,940, (Ϫ)-cis-3-[2-hydroxy-4(1,1-dimethyl-heptyl)phenyl]-trans-4-(3-hydroxy-propyl)cyclo-
hexanol; MPE, maximal possible antinociceptive effect; CB1, brain cannabinoid; THF, tetrahydrofuran; TLC, thin layer chromatography; DMH,
dimethylheptyl; DM, dimethyl.
679

680
Wiley et al.
chemical structure contained two hydroxyl substituents po-
sitioned with a single intervening carbon on a benzene ring.
For most of the bicyclic resorcinols presented here, the sec-
ond cyclic substituent is attached at the intermediate carbon.
Materials and Methods
Subjects. Male Institute for Cancer Research (ICR) mice (25–32
g), obtained from Harlan (Indianapolis, IN), were housed in groups of
five. All animals were kept in a temperature-controlled (20–22°C)
environment with a 12-h light/dark cycle (lights on at 7 AM). Sepa-
rate mice were used for testing each dose of each experimental
compound in the in vivo behavioral procedures. Brain tissue for
binding studies was obtained from male Sprague-Dawley rats (150–
200 g) purchased from Harlan.
Apparatus. Measurement of spontaneous activity in mice oc-
curred in standard activity chambers interfaced with a Digiscan
animal activity monitor (Omnitech Electronics, Inc., Columbus, OH).
A standard tail-flick apparatus and a digital thermometer (Fisher
Scientific, Pittsburgh, PA) were used to measure antinociception and
rectal temperature, respectively.
Fig. 1. Chemical structures of ⌬9-THC, CP 55,940, and cannabidiol.
with those of CB1 receptors (Munro et al., 1993). Showalter et
Compounds. Resorcinols were synthesized in our laboratories
al. (1996) reported a high positive correlation (r ϭ 0.82) (Organix, Inc., Woburn, MA) according to the procedure specified
below and were suspended in a vehicle of absolute ethanol, Emul-
phor-620 (Rhone-Poulenc, Inc., Princeton, NJ), and saline in a ratio
of 1:1:18. Experimental compounds were administered to the mice
i.v. in the tail vein at a volume of 0.1 ml/10 g.
Analogs O-1376 and O-1532 listed in Table 1 were synthesized as
previously described (Mahadevan et al., 2000). Analog O-1601 was
synthesized from 1-deoxy-9-carbomethoxy cannabinol dimethylhep-
tyl analog (Mahadevan et al., 2000) by lithium/liquid ammonia re-
duction as described for the preparation of O-1376. The compounds
listed in Tables 2 and 3 were prepared using a three-step sequence
between binding affinities at these two cannabinoid receptors
for cannabinoids in various classes. Given these findings, it is
not surprising that some of the structural features of the
tetrahydrocannabinols that enhance affinity for CB1 recep-
tors also increase binding to CB2 receptors. For example,
addition of a 1Ј,1Ј-dimethyl group to the lipophilic C3 side
chain of ⌬8-THC results in higher affinities for both types of
cannabinoid receptors compared with a nonbranched chain of
identical length (Showalter et al., 1996). Several previous
studies have explored the role of oxygen in CB2 binding. (Fig. 2). The 2-lithio derivative of 1,3-dimethoxy-5-(1Ј,1Ј-dimethyl-
Synthesis of a series of ⌬8-THC analogs in which the phenolic
heptyl)resorcinol was prepared using n-BuLi/hexane in THF (step 1).
hydroxyl at position 1 was removed (deoxy-⌬8-THC analogs)
It was condensed with the appropriate ketone to give the tertiary
alcohol (step 2), which upon treatment with trifluoroacetic acid/
or replaced with a methoxyl resulted in analogs with selec-
Et3SiH gave the dimethoxy precursors (step 3). Demethylation with
tivity for CB2 receptors (Gareau et al., 1996; Huffman et al.,
BBr3/CH2Cl2 gave the target compounds (Crocker et al., 1999). The
1996, 1999). Incorporation of an oxygen into a fourth ring
general procedure is illustrated in Fig. 2 and described below.
attached at C1 also increased CB2 selectivity, suggesting
possible differences in the interaction of oxygen in the bind-
ing pockets of CB1 and CB2 receptors (Reggio et al., 1997). In
the present study, we examined structure-activity relation-
To a solution of the resorcinol (5 mmol) in 25 ml of dry THF was
added a 2.5 M solution of n-BuLi in hexane (5.5 mmol) at 0°C with
stirring in N2. After additional stirring for 1 h at 0°C, a solution of
the ketone (7.5 mmol) in 3 ml of dry THF was added all at once. The
ships of a series of bicyclic resorcinols in which the core solution was stirred for 0.5 h at 0°C and then for 18 h at 23°C. The
TABLE 1
CB1 and CB2 binding affinities and pharmacological effects of phenols
The Ki values are presented as means Ϯ S.E.M. All ED50 values are expressed as micromoles per kilogram (with 95% confidence limits in parentheses). For compounds that
failed to produce either maximal or dose-related effects, the percent effect at the highest dose a(milligrams per kilogram, in parentheses) is provided.
Ki (nM)
ED50
TF
ID
R
R1
CB1/CB2
CB1
CB2
SA
RT
1
2
3
O-1376
O-1532
O-1601
CH3
DMH
Dimethylbutyl
DMH
33 Ϯ 4
876 Ϯ 18
5 Ϯ 0.6
3 Ϯ 0.4
113 Ϯ 21
3 Ϯ 0.4
11
8
2
8.5 (5–16)
32% (30)
1.1 (0.8–1.4)
5.7 (3–10)
7% (30)
1.1 (0.8–1.4)
2.3 (1–5)
Ϫ0.4 (30)
1.6 (1.4–2.2)
CH3
CH2OH
SA, suppression of spontaneous activity; RT, rectal temperature; TF, tail flick.

Cannabinoid Resorcinols
681
TABLE 2
Pharmacological effects and cannabinoid receptor binding affinities of bicyclic resorcinols
The Ki values are presented as means Ϯ S.E.M. All ED50 values are expressed as micromoles per kilogram (with 95% confidence limits in parentheses). For compounds that
failed to produce either maximal or dose-related effects, the percent effect at the highest dose (milligrams per kilogram, in parentheses) is provided.
Ki (nM)
ED50
TF
ID
R
R1
R2
CB1/CB2
14
CB1
CB2
SA
RT
4
5
O-1424
DMH
H
H
95 Ϯ 6
7 Ϯ 0.4
27 (13–56)
13 (9–23)
13 (10–20)
O-1422
DMH
11 Ϯ 2
1.5 Ϯ 0.1
7
0.1 (0.02–0.6)
0.6 (0.5–1.1)
0.6 (0.5–0.7)
6
7
8
O-1656
O-1660
O-1425
DMH
DMH
DMH
H
H
H
18 Ϯ 1
7 Ϯ 1
2 Ϯ 0.2
3 Ϯ 0.8
12 Ϯ 2
9
2
1.5 (0.4–7.0)
3.5 (3.2–3.8)
17 (10–29)
1.2 (0.9–1.5)
2.4 (1.9–3.0)
15 (10–24)
0.6 (0.1–8.7)
4.6 (2.4–9.2)
13 (10–19)
153 Ϯ 17
13
9
O-1661
DMH
H
138 Ϯ 4
28 Ϯ 12
5
24 (13–42)
14 (9–20)
24 (17–34)
10
11
12
O-1662
O-1423
O-2010
DMH
DMH
H
H
H
Ͼ10,000
97 Ϯ 5
5,424 Ϯ 1,103
28 Ϯ 5
87% (30)
12 (8–20)
Ϫ18% (30)
30% (30)
9 (7–13)
9% (30)
Ϫ3 (30)
3
9 (6–15)
Ϫ0.4 (30)
C6H13
9,515 Ϯ 332
NT
SA, suppression of spontaneous activity; RT, rectal temperature; TF, tail flick; NT, not tested.
reaction was worked up by the addition of saturated NH4Cl solution purified by chromatography, generally using hexane/ethyl acetate
and extracted with ether. After washing (H2O) and drying (Na2SO4), mixtures. In the case of O-1662 (Table 2), the corresponding tertiary
the solvent was evaporated to give the crude tertiary alcohol, which alcohol, upon treatment with CF3COOH/Et3SiH, gave the unsatur-
was used as such in the subsequent reaction. A solution of the ated compound (dehydrated but not reduced), which on catalytic
tertiary alcohol (5 mmol) in 10 ml of dry CH2Cl2 was treated with reduction (PtO2/C/H2) in acetic acid gave the desired dimethoxy
CF3COOH (27.5 mmol) followed by Et3SiH (12.5 mmol). The solution precursor. The final compound was purified by chromatography us-
was stirred in N2 for 1 h or more (followed by TLC) and then ing a 5% Et3NH2/EtOAc mixture. The unsaturated analog O-1423
quenched by the addition of saturated NaHCO3 solution. The organic (Table 2) was prepared by treatment of the corresponding tertiary
layer was separated and after washing (H2O) and drying gave the alcohol with CF3COOH alone in CH2Cl2, followed by demethylation.
crude dimethoxy precursor of the target compound. This material In Table 3, compounds O-1797A and O-1798B were diastereomeric
was used as such for the demethylation step. Treatment of the
mixtures and showed as two distinct spots in TLC, which were
dimethoxy precursor as a solution in dry CH2Cl2 at 0°C with three separated by column chromatography on silica gel and eluting with
equivalents of 1 N BBr3 solution in CH2Cl2, using the standard hexane/ethyl acetate mixtures (10:1 to 5:1). O-1657 was a sample of
procedure and workup, gave the crude target compound, which was the mixture of diastereomers O-1797A and O-1798B. The dimethoxy

682
Wiley et al.
TABLE 3
In vitro and in vivo cannabinoid effects of bicyclic resorcinols with methylated cyclohexane
The Ki values are presented as means Ϯ S.E.M. All ED50 values are expressed as micromole per kilogram (with 95% confidence limits in parentheses). For compounds that
failed to produce either maximal or dose-related effects, the percent effect at the highest dose (mg/kg; in parentheses) is provided.
Ki (nM)
ED50
TF
ID
R
R1
CB1/CB2
16
CB1
CB2
SA
RT
13
14
O-1658
DMH
DMH
16 Ϯ 2
1 Ϯ 0.3
0.2 (0.1–0.3)
0.3 (0.2–0.3)
0.3 (0.27–0.5)
O-1659
45 Ϯ 1
5 Ϯ 0.9
9
4.8 (3–9)
3.9 (3–6)
3.3 (2–5)
15
16
O-1663
O-1657
DMH
DMH
144 Ϯ 22
9 Ϯ 2
16
17
32% (30)
7% (30)
Ϫ2.2 (30)
14 Ϯ 0.5
0.8 Ϯ 0.04
0.3 (0.3–0.5)
0.6 (0.5–1)
0.9 (0.7–1.1)
17
18
O-1797A
O-1798B
DMH
DMH
5 Ϯ 0.6
4 Ϯ 0.6
0.4 Ϯ 0.03
0.5 Ϯ 0.07
12
8
0.5 (0.4–0.6)
0.2 (0.03–1.2)
1.1 (0.8–1.5)
1.0 (0.7–1.6)
0.7 (0.6–1.0)
0.6 (0.5–0.7)
19
O-1826
DMH
40 Ϯ 11
0.8 Ϯ 0.05
50
2.7 (2.1–3.9)
2.4 (1.8–3.3)
3.6 (2.7–4.5)
20
21
O-1890
O-1871
Dimethylbutyl
DMH
96 Ϯ 4
2 Ϯ 0.3
13 Ϯ 1
7
7
69 (55–90)
Ͻ1.0a
48 (31–69)
72 (45–114)
1.3 (0.3–4.3)
0.3 Ϯ 0.01
2.3 (2.0–2.6)
SA, suppression of spontaneous activity; RT, rectal temperature; TF, tail flick.
a This dose (␮mol/kg) produced Ͼ50% inhibition and was the lowest dose tested.
compounds listed in Tables 4 and 5 were prepared (Fig. 2) from the
gave the target compound O-2122. In the preparation of O-2090, the
2-lithio derivative of 1,3-dimethoxy-5-(1Ј,1Ј-dimethylheptyl)resor- corresponding diethoxyresorcinol derivative of step 1 was used in
cinol and the appropriate ketones using BuLi, as in the preparation place of the 2-lithio derivative of 1,3-dimethoxy-5-(1Ј,1Ј-dimethyl-
of the tertiary alcohol, and isolating and purifying the compounds by heptyl)resorcinol. All compounds showed appropriate 1H NMR pro-
chromatography (ethyl acetate/hexane mixtures). Deprotection of files (Jeol Eclipse 300 MHz; Jeol USA, Inc., Peabody, MA) and were
O-2092 was carried out by treatment with 10% HCl in an ether/THF characterized on the basis of their 1H NMR profiles, TLC, and ele-
(5:4) mixture for 0.5 h at 23°C to give a mixture of O-2115 (major)
mental analyses. The general profile of resorcinols (Tables 2 and 3) is
and the dehydrated compound O-2114 (minor). Sodium borohydride illustrated by the 1H NMR profile of O-1797A: (CDCl3) ␦, 6.26 (s, 2H),
reduction of O-2115 furnished a mixture of diastereomeric com- 4.64 (s, 2H, D2O exchangeable), 3.4 to 3.2 (m, 1H), 2.4 to 1.2 (m, 25H),
pounds, which were separated by column chromatography on silica 1.1 (d, J ϭ 5.9 Hz, 3H), and 0.86 (t, 3H). The dimethoxyresorcinols
gel and eluting with hexane/ethyl acetate mixtures (5:1 to 3:1) to give (Tables 4 and 5) showed an additional peak at ␦, 3.85 region (s, 6H)
the target compounds O-2116A and O-2117B. Separation of O-1966A for the methoxyl groups and the multiplet for the benzylic methine at
and O-1967B from a diastereomeric mixture was undertaken simi- ␦, 3.4 to 3.2 was absent.
larly, by eluting with hexane followed by 99% hexane/1% ethyl ace-
Mouse Behavioral Procedures. Prior to testing in the behav-
tate mixture. Epoxidation of O-2114 followed by NaBH4 reduction ioral procedures, mice were acclimated to the experimental setting

Cannabinoid Resorcinols
683
Fig. 2. Scheme for synthesis of resorcinol analogs. a, n-BuLi, hexane/THF, 0°C, 1 h; ketone, THF, 0°C, 0.5 h to 23°C, 18 h, 80 to 90%. b, CF3COOH,
ET3SiH, CH2Cl2, 23°C, 1 h. c, BBr3/CH2Cl2, 0°C to 23°C, 18 h. Overall yield (a to c), ϳ50 to 80%. d, 10% HCl, ether/THF (5:4), 23°C, 0.5 h; yield O-2115
(41%) and O-2114 (19%). e, m-chloroperbenzoic acid, CH2Cl2/H2O (4:3), 23°C, 10 min. f, NaBH4, CH2Cl2/MeOH (1:1), 23°C, 18 h, ϳ90%. Overall yield
(e and f), ϳ50%.
(ambient temperature 22–24°C) overnight. Preinjection control val- cient quantity of buffer to bring the total incubation volume to 1 ml.
ues were determined for rectal temperature and tail-flick latency (in Nonspecific binding was determined by the addition of 1 ␮M unla-
seconds). Five min after i.v. injection with an experimental com- beled CP 55,940. Following incubation at 30°C for 1 h, binding was
pound or vehicle, mice were placed in individual activity chambers, terminated by addition of ice-cold buffer and vacuum filtration
and spontaneous activity was measured for 10 min. Activity was through pretreated filters in a 12-well sampling manifold (Millipore
measured as total number of interruptions of 16 photocell beams per Corp., Bedford, MA). After washing, filters were placed into plastic
chamber during the 10-min test and expressed as percent inhibition scintillation vials (Packard Instrument Co., Inc., Downers Grove, IL)
of activity of the vehicle group. Tail-flick latency was measured at 20 and shaken. The quantity of radioactivity present was determined by
min postinjection. Maximum latency of 10 s was used. Antinocicep- liquid scintillation spectrometry.
tion was calculated as the percent maximal possible effect {%MPE ϭ
CB2 Binding Procedure. Human CB2 cDNA was provided by
[(test-control latency)/(10-control)] ϫ 100}. Control latencies typi- Dr. Sean Munro (MRC Laboratory of Molecular Biology, Cambridge,
cally ranged from 1.5 to 4.0 s. At 30 min postinjection, rectal tem- England) and was expressed in Chinese hamster ovary cells as
perature was measured. This value was expressed as the difference previously described (Showalter et al., 1996). Briefly, transfected
between control temperature (before injection) and temperatures CB2 Chinese hamster ovary cell lines were maintained in Dulbecco’s
following drug administration (⌬oC). Different mice (n ϭ 5–6 per modified Eagle’s medium (Invitrogen, Carlsbad, CA) to maintain
dose) were tested for each dose of each compound. Each mouse was selective pressure of stable transformants and 10% fetal clone II
tested in each of the three procedures.
(Hyclone Laboratories, Inc., Logan, UT) plus 0.3 to 0.5 mg/ml G418
CB1 Binding Procedure. The methods used for tissue prepara- (to maintain selective pressure) under 5% CO2 at 37°C. When con-
tion and binding have been described previously (Compton et al., fluent, cells were harvested with 1 mM EDTA in phosphate-buffered
1993) and are similar to those described by Devane et al. (1988). All saline and centrifuged at 1000g for 5 min at 4°C. The supernatant
assays, as described briefly below, were performed in triplicate, and was saved, and the P1 pellet was resuspended in centrifugation
the results represent the combined data from three to six individual buffer. Homogenization and centrifugation were repeated twice, and
experiments.
the combined supernatant fractions were centrifuged at 40,000g for
Following decapitation and rapid removal of the brain, whole 30 min at 4°C. The P2 pellet was resuspended in centrifugation
brain was homogenized and centrifuged. The resulting pellet was buffer 2 (50 mM Tris HCl, 1 mM EDTA, and 3 mM MgCl2, pH 7.4) to
termed P1. The supernatant was saved and combined with the two
a protein concentration of approximately 2 mg/ml. Protein concen-
subsequent supernatants obtained from washing the P1 pellet. The trations were determined by the method of Bradford (1976) using
combined supernatant fractions were centrifuged, resulting in the P2 Bio-Rad protein assay (Bio-Rad, Hercules, CA) and bovine serum
pellet. After further incubation and centrifuging, this pellet was albumin standards (fatty acid free; Sigma-Aldrich). The membrane
resuspended in assay buffer to a protein concentration of approxi- preparation was divided into amounts that were convenient for bind-
mately 2 mg/ml. The membrane preparation was quickly frozen in a ing assays, frozen rapidly in dry ice, and stored at Ϫ80°C.
bath solution of dry ice and 2-methylbutane (Sigma-Aldrich, St.
Binding was initiated by the addition of 50 ␮g of quickly thawed P2
Louis, MO), then stored at Ϫ80°C for no more than 2 weeks. Prior to membranes to test tubes containing [3H]CP 55,940 (final reaction
performing a binding assay, an aliquot of frozen membrane was concentration, 0.5 nM), an appropriate concentration of unlabeled
rapidly thawed, and protein values were determined by the method CP 55,940 or test compound, and sufficient quantity of assay buffer
of Bradford (1976).
(50 mM Tris-HCl, 1 mM EDTA, 3 mM MgCl2, and 5 mg/ml bovine
Binding was initiated by the addition of 150 ␮g of P2 membrane to serum albumin, pH 7.4) to bring the total incubation volume to 0.5
test tubes containing 1 nM [3H]CP 55,940 (79 Ci/mmol) and a suffi- ml. The concentration of [3H]CP 55,940 in saturation studies ranged

684
Wiley et al.
TABLE 4
CB1 and CB2 binding affinities of dimethoxy-dimethylheptyl resorcinol analogs
The Ki values are presented as means Ϯ S.E.M.
Ki (nM)
ID
R
CH2OH
CB1/CB2
CB1
CB2
22
23
HU-308a
Ͼ10,000
23 Ϯ 4
O-1999
Ͼ10,000
466 Ϯ 110
24
25
O-1964
O-1965
Ͼ10,000
Ͼ10,000
911 Ϯ 116
Ͼ10,000
26
O-1962
Ͼ10,000
342 Ϯ 22
27
28
O-2092
O-2122
4,581 Ϯ 312
3,758 Ϯ 184
126 Ϯ 12
36
4
1,065 Ϯ 107
29
30
31
O-2114
O-2115
O-2123
8,442 Ϯ 954
4,572 Ϯ 173
1,731 Ϯ 117
1,773 Ϯ 184
346 Ϯ 49
5
13
14
125 Ϯ 14
a Values from Hanusˇ et al., 1999. Note: binding ligand, [3H]HU-243, was different from that used in present study.
from 50 to 10,000 pM. Nonspecific binding was determined by the
were prepared by air drying (12 h) inverted borosilicate tubes after
addition of 1 ␮M unlabeled CP 55,940. CP 55,940 and all cannabi- two rinses with a 0.1% solution of AquaSil (Pierce Chemical, Rock-
noid analogs were prepared by suspension in assay buffer from 1 ford, IL). The GF/C glass-fiber filters (2.4 cm; Baxter, McGaw Park,
mg/ml ethanolic stock without evaporation of the ethanol (final con- IL) were pretreated in a 0.1% solution of pH 7.4 polyethylenimine
centration, no more than 0.4%). In competition studies, analog con- (Sigma-Aldrich) for at least 6 h.
centrations ranged from 0.1 nM to 10 ␮M. After incubation at 30°C
Data Analysis. Based on data obtained from numerous previous
for 1 h, binding was terminated by the addition of 2 ml of ice-cold studies with cannabinoids, maximal cannabinoid effects in each pro-
wash buffer (50 mM Tris-HCl and 1 mg/ml bovine serum albumin) cedure were estimated as follows: 90% inhibition of spontaneous
and vacuum filtration through pretreated filters in a 12-well sam- activity, 100% MPE in the tail-flick procedure, and Ϫ6°C change in
pling manifold (Millipore). Reaction vessels were washed once with 2 rectal temperature. ED50 was defined as the dose at which half-
ml of ice-cold wash buffer. Filters were placed into 7-ml plastic maximal effect occurred. For compounds that produced one or more
scintillation vials (RPI Corp., Mount Prospect, IL) with 4 ml of cannabinoid effect, ED50 was calculated separately using least-
Budget-Solve (RPI Corp.). After shaking for 30 min, the radioactivity squares linear regression on the linear part of the dose-effect curve
present was determined by liquid scintillation spectrometry. Three for each measure in the mouse tetrad, plotted against log10 transfor-
reaction vessels were used for each drug concentration in each assay.
The results represent the combined data of three independent ex- cies were expressed as millimoles per kilogram.
periments. All assays were performed in siliconized test tubes, which Pearson product-moment correlation coefficients (with associated
mation of the dose. For the purposes of potency comparison, poten-

Cannabinoid Resorcinols
685
TABLE 5
CB1 and CB2 binding affinities of hydroxylated dimethoxy-dimethylheptyl resorcinols
The Ki values are presented as means Ϯ S.E.M.
Ki (nM)
ID
R
R1
R2
CB1/CB2
CB1
CB2
32
33
34
O-2072
OCH3
DMH
5,820 Ϯ 662
105 Ϯ 19
55
220
15
O-1966A
O-1967B
OCH3
OCH3
DMH
DMH
5,055 Ϯ 984
1,716 Ϯ 105
23 Ϯ 2.1
111 Ϯ 8
35
36
37
38
O-2121
OCH3
OCH3
OCH3
OCH3
DMH
DMH
DMH
DMH
1,990 Ϯ 77
3,932 Ϯ 483
Ͼ10,000
101 Ϯ 14
190 Ϯ 17
20
21
O-2116A
O-2117B
O-2068
1,561 Ϯ 70
161 Ϯ 24
7,515 Ϯ 721
47
39
O-2139
OCH3
CH3
Ͼ10,000
Ͼ10,000
40
41
O-2090
O-2091
OC2H5
OCH3
DMH
DMH
8,810 Ϯ 422
3,201 Ϯ 141
858 Ϯ 43
64 Ϯ 8
10
50
significance tests) were calculated between CB1 binding affinity (ex- a phenolic hydroxyl and lipophilic side chain in the same
pressed as log Ki) and in vivo potency for each measure (expressed as
log ED50 in millimoles per kilogram) for all active cannabinoid com-
pounds that bound to the CB1 receptor. The Pearson product-mo-
ment correlation provided a measure of the strength and direction of
relationship between each pair of quantitative variables. In addition,
multiple linear regression was used to calculate the overall degree of
relationship between CB1 binding affinity and potency in the mouse
measures for all active cannabinoids. A correlation between CB1 and
CB2 binding affinities was calculated for all compounds that had a
measurable Ki for CB1 and CB2 binding (Ki Ͻ 10,000 nM). Ki values
for CB1 and CB2 binding were obtained from the Scatchard displace-
ment analysis program of the KELL software package (Biosoft, Mill-
town, NJ).
orientation as in cannabinol. In addition, the pyran oxygen is
absent, and the analogs have substituents in the phenyl ring
(ring C) of cannabinol. Two of the analogs (O-1376 and
O-1601) have a dimethylheptyl side chain; each possess good
CB1 and CB2 binding affinities and in vivo activity. O-1601,
the more potent of the two active compounds, had a hydroxy-
methyl group in the phenyl ring. This substitution increased
CB1 affinity and in vivo potencies compared with O-1376 but
did not affect affinity for CB2 receptors. A similar effect was
observed in the cannabinol series, where the substitution of a
hydroxymethyl group for a methyl at C-9 in cannabinol in-
creased binding affinity and potency (Mahadevan et al.,
2000). Shortening the side chain of O-1376 to dimethylbutyl
(O-1532) markedly decreased affinity for both receptors and
Results
The CB1 and CB2 binding affinities for substituted biphe- resulted in loss of in vivo activity.
nyl analogs are shown in Table 1. These compounds contain Table 2 presents binding and in vivo data for a series of two

686
Wiley et al.
cyclic ring-substituted-5-dimethylheptyl resorcinols. Manip- showed decreased affinity for CB1 receptors compared with
ulation of the size of the cyclic structure attached at position
2 of the resorcinol ring resulted in changes in binding affin-
ities and potencies. Substitution of a cyclopentane ring (O-
1424) resulted in moderate affinity for the CB1 receptor, with
excellent affinity for the CB2 receptor. Although this com-
pound was active in all three in vivo assays, potency was
relatively poor. In addition, potencies across the measures
were not equal; i.e., potency for reducing spontaneous activ-
ity was approximately half that for producing antinociceptive
and hypothermic effects. Increasing ring size to a cyclohex-
ane (O-1422), cycloheptane (O-1656), or adamantyl (O-1660)
improved affinity 5- to 14-fold for both cannabinoid receptors
and greatly increased potencies in vivo. Substitution of a
sulfur for a carbon in a cyclohexane ring (O-1425) decreased
CB1 affinity by 14-fold and CB2 affinity by 8-fold (compared
with O-1422) as well as reducing in vivo potencies. Similarly,
sulfur substitution in a cyclopentane ring (O-1661) also at-
tenuated binding to both cannabinoid receptors. When a
methylated nitrogen (O-1662) was inserted into the cyclohex-
ane ring in the same position as the sulfur of O-1425, binding
to CB1 receptors did not occur. In addition, CB2 binding was
drastically decreased, and the compound was not fully active
in vivo. In contrast, placing a double bond in the cyclohexane
ring (O-1423) decreased affinities and potencies, but the com-
pound remained active. However, moving the lipophilic side
chain of O-1422 from C-5 to C-4 and replacing the dimethyl-
heptyl with an n-hexyl chain (O-2010) produced a 865-fold
decrease in CB1 affinity and a loss of activity in vivo.
Table 3 shows results of tests with cyclohexane-substituted
resorcinols in which the position of the substituent at the
cyclohexane ring attached to the core resorcinol was varied.
All compounds were diastereomeric mixtures. All of these
analogs had high (Ki ϭ 2 nM) to moderate (Ki ϭ 144 nM)
affinity for CB1 receptors and were CB2-selective (Ki range ϭ
0.3–13 nM). Methylation at the 2-position of the cyclohexane
ring (O-1658) did not dramatically alter affinity for either
cannabinoid receptor or in vivo potencies compared with the
corresponding cannabinoid with a nonmethylated cyclohex-
ane (O-1422 in Table 2). Moving the methyl to position 4 of
the cyclohexane ring (O-1659) decreased affinity for both
cannabinoid receptors by about 5-fold and produced an even
greater decrease (11- to 24-fold) in potencies in vivo. Substi-
tuting a phenyl group for the methyl at this same position
(O-1663) resulted in 2- to 3-fold decreases in CB2 and CB1
affinities, respectively, and a loss of activity in vivo. In the
next five analogs shown in Table 3, the methyl was attached
at position 3 of the cyclohexane ring. O-1657 exhibited CB1
and CB2 affinities that were similar to those of O-1658;
however, the profiles of in vivo potencies differed. Whereas
the two analogs showed approximately equal potencies in
suppressing spontaneous activity, O-1658 was twice as po-
tent in producing antinociception and three times as potent
in reducing body temperature. As described under Materials
and Methods, compound O-1657 was separated into two dis-
tinct entities, which were designated O-1797A and O-1798B.
These analogs were still mixtures. Affinities of O-1797A and
O-1798B were two to three times greater than those of
O-1657. Although potencies of these isomers for suppression
of locomotor activity and hypothermia were not notably dif-
ferent from those of O-1657, antinociceptive potencies were
O-1657; however, affinity for CB2 receptors was identical for
both compounds. Not surprisingly given its decreased CB1
affinity, O-1826 was less potent than O-1657 in vivo. Substi-
tution of a dimethylbutyl for the dimethylheptyl side chain at
C5 of the resorcinol component (O-1890) decreased affinities
for both cannabinoid receptors. This compound was active in
vivo, although potency was notably low for all measures. In
contrast, addition of a gem-dimethyl group at the 3-position
of the cyclohexane ring, with retention of the dimethylheptyl
side chain of the resorcinol component (O-1871), resulted in
the best CB1 and CB2 affinities of this series. Given its higher
CB1 binding affinity, in vivo potencies for this compound
were lower than expected, although the lack of pharmacoki-
netics assessments tempers this conclusion somewhat.
To develop CB2-selective ligands, we examined cyclic ring-
substituted dimethoxyresorcinols. The CB1 and CB2 binding
affinities of these analogs are shown in Tables 4 and 5.
Although most of the compounds shown in Tables 4 and 5
possessed a dimethylheptyl side chain, all had poor CB1
affinity; hence, they were not tested in vivo. The bicyclic
structure of O-1999 (Table 4) was almost identical to that of
O-1657 (Table 3), an analog with good CB1 and CB2 affinities
and potent in vivo effects. Both compounds had a dimethyl-
heptyl side chain attached to the 5-position of a resorcinol
core that was attached at position 2 to a cyclohexane ring.
Each compound had a methyl group at the 3-position of the
cyclohexane ring. The major structural difference between
the two compounds was that O-1999 was a dimethoxy deriv-
ative of the resorcinol O-1657. This structural change from a
phenol to a methoxy derivative resulted in complete loss of
affinity for CB1 receptors and an almost 600-fold reduction in
affinity for CB2 receptors. Similarly, the other analogs that
were dimethoxy derivatives of the corresponding resorcinols
had poor affinity for CB1 receptors (Ki ranged from 1716 to Ͼ
10,000) regardless of the cyclic ring substitution at position 2.
In contrast, CB2 binding affinities for some of these analogs
remained high, as described in more detail below.
Table 4 presents binding data for two cyclic ring-substi-
tuted dimethoxy-resorcinol-dimethylheptyl analogs that con-
tain at least one oxygen inserted into or attached to the
nonresorcinol cyclohexane ring. Compared with O-1999,
which did not contain an oxygen in the cyclohexane ring,
conversion of the cyclohexane ring to a pyran ring (O-1964)
decreased CB2 affinity almost 2-fold without effect on CB1
binding. Further addition of a double bond at position 3 of the
pyran ring resulted in O-1965, which did not bind to either
cannabinoid receptor. In contrast, the introduction of a ter-
tiary hydroxyl group at C-4 of the pyran ring (O-1962) in-
creased CB2 affinity by 3-fold. Adding additional oxygens,
such as a ketol group attached at C-4 to the point of attach-
ment of the dimethoxyresorcinol substituent (O-2092), also
increased CB2 affinity whereas adding an oxygen as an ep-
oxide (O-2122) decreased it. The presence of a ketone group
at C-4 of the cyclohexane ring and having unsaturation in the
ring (O-2114) resulted in a compound with poor affinity for
either cannabinoid receptor; however, if a tertiary hydroxyl
group was added at the site of dimethoxyresorcinol attach-
ment (O-2115), CB2 affinity improved. Retention of the ter-
tiary hydroxyl, methylation at position 5, and the presence of
reduced by about half. The 3S isomer of this series (O-1826) a ketone at position 3 of the cyclohexane ring increased

Cannabinoid Resorcinols
687
affinity for both receptors and resulted in a compound (O-
2123) with the best CB2 affinity (Ki ϭ 125 nM) in this series.
Table 5 shows CB1 and CB2 affinities for two cyclic ring-
substituted dimethoxy-resorcinol-dimethylheptyl analogs in
which the ring size and the position of the methyl or hydroxyl
substituent on the cyclohexane ring are varied. The first
analog (O-2072) contains one hydroxyl attached to the cyclo-
hexane at the same position at which the resorcinol core is
attached. This compound is CB2-selective. Although it had
poor affinity for CB1 receptors, it bound with moderate affin-
ity to CB2 receptors. Introduction of a methyl substituent in
the 3-position of the cyclohexane ring gave a diastereomeric
mixture from which two distinct entities were separated by
careful chromatography. These analogs (O-1966A and
O-1967B) were still mixtures. This substitution resulted in a
5-fold increase in affinity for CB2 receptors with continued
poor affinity for CB1 receptors. However, one of these isomers
(O-1966A) showed the best CB2 selectivity (225-fold) in the
series and had high binding affinity for the CB2 receptor (Ki
ϭ 22.5 nM). Addition of an extra hydroxyl group to the
cyclohexane ring (O-2121) reduced both selectivity and bind-
ing affinity for the CB2 receptor comparable with those ob-
tained with O-1967B. Removal of the methyl at position 3
and addition of a hydroxyl at position 4 resulted in two
diastereomeric mixtures that could be separated, which were
designated as O-2116A and O-2117B. Both of these isomers
had poor affinity for CB1 receptors, but although the B iso-
Fig. 3. Scatterplots and regression lines of log CB1 Ki plotted against log
CB2 Ki (top left) and log ED50 for each of the three in vivo tests. SA,
spontaneous activity (top right); MPE, percent maximal possible antino-
ciceptive effect (bottom right); RT, change in rectal temperature (bottom
right).
mer also had poor affinity for CB2 receptors, the A isomer receptor recognition and activation in traditional cannabi-
bound to CB2 receptors with moderate affinity. Attachment noids similarly alter binding and activity in this resorcinol
of a gem-dimethyl group to position 3 of O-2072 (i.e., O-2068) series. Previous research has shown that the length and
did not significantly alter affinities for CB1 or CB2 receptors; branching of a lipophilic substituent is important for CB1
however, replacement of the dimethylheptyl group of O-2068 receptor recognition in all of the major cannabinoid agonist
with a methyl group (O-2139) produced loss of affinity at both classes, including tetrahydrocannabinols and bicyclic canna-
receptors. Changing the dimethyoxy groups of the resorcinol binoids (Compton et al., 1993), indole-derived cannabinoids
by adding diethoxy groups (O-2090) drastically decreased (Wiley et al., 1998), and anandamides (Ryan et al., 1997;
affinities for CB1 and CB2 receptors (compare O-2090 with Seltzman et al., 1997). In the tricyclic and bicyclic series, a
O-1966A or O-1967B). Enlarging the cyclohexane ring in 1Ј,1Ј-dimethylheptyl side chain is optimal (Compton et al.,
O-2072 to a cycloheptane ring (O-2091) resulted in little 1993) and is contained in most of the resorcinols presented
change in affinity for CB1 receptors and an almost 2-fold here. Reducing the length of this substituent resulted in a
increase in CB2 affinity.
concomitant elimination or decrease in CB1 receptor recog-
Multiple regression analysis of binding affinity (Y ϭ log nition, as occurs in other cannabinoid series with similar
CB1 Ki) and potency for each mouse measure (X1–3 ϭ log structural manipulations (see references above).
ED50 in mmol/kg) confirmed that overall potency at produc-
Other structural features affecting CB1 receptor recogni-
ing the characteristic profile of cannabinoid effects was sig- tion and activation in this series are related to the size,
nificantly correlated with binding affinity at CB1 receptors saturation, substitution, and methylation of the second non-
[r ϭ 0.78; F(3,13) ϭ 6.9; p ϭ 0.005] for all active cannabi- resorcinol ring. In most tricyclic and bicyclic cannabinoids,
noids. Individual correlations between log Ki and log potency the ring corresponding to the nonresorcinol ring in the cur-
for each measure were 0.78, 0.74, and 0.75 for hypomobility, rent series is a cyclohexane. In the resorcinol series, reducing
antinociception, and hypothermia, respectively (p Ͻ 0.05 for this size to a cyclopentane decreases CB1 affinity and potency
all three correlations). Furthermore, CB1 binding affinity whereas increasing it to a cycloheptane has little effect. Sim-
was highly correlated with CB2 binding affinity (r ϭ 0.92, p Ͻ ilar modifications of other cannabinoids have not been re-
0.05) for all compounds for which both binding affinities ported; however, degree of saturation of, as well as the posi-
could be calculated (i.e., Ki Ͻ 10,000). Scatterplots for each tion of the double bond in the cyclohexane ring of tricyclic and
regression line are presented in Fig. 3.
bicyclic cannabinoids and in the polyolefin loop of the anan-
damides, has been shown to affect CB1 receptor recognition
and activity. In the resorcinol series, introduction of a single
double bond (O-1423) within the ring decreased CB1 affinity
Discussion
The lack of CB1 binding affinity of cannabidiol compared and potency to the same extent as did a reduction in the size
with other pyran ring open analogs such as CP 55,940 of the ring to a cyclopentane. Greatest affinity and potency
prompted us to examine the structure-activity relationships within the anandamides is achieved with four double bonds,
of resorcinol derivatives for cannabinoid activity. Our results with greater or lesser saturation resulting in a reduction in
show that many of the structural changes that affect CB1 CB1 binding and/or in vivo activity (Adams et al., 1995;

688
Wiley et al.
Thomas et al., 1996; Sheskin et al., 1997). Similarly, the ing in a manner similar to its effect on CB1 affinity, as it did
number and position of double bonds within the cyclohexane
ring of tetrahydrocannabinols and bicyclic cannabinoids af-
fect activity. For example, moving the double bond of ⌬9-THC
to position 8 (as in ⌬8-THC) decreases CB1 affinity 3-fold and
somewhat reduces potency (Compton et al., 1993). Unsatura-
tion of the cyclohexane ring results in cannabinol with its
greatly reduced CB1 affinity (Showalter et al., 1996). In con-
trast, CP 55,940, with a completely saturated cyclohexane
ring, is severalfold more potent than ⌬8-THC-dimethylhep-
tyl, which has a single double bond in the cyclohexane ring;
but ⌬8-THC, with its single double bond, binds with better
CB1 affinity than does ⌬9(11)-THC, which has a completely
saturated cyclohexane ring (Compton et al., 1993).
The most remarkable structural features of the resorcinol
series affecting CB1 affinity, however, are the length of the
lipophilic side chain at position 5 and the size of the cyclic
ring substituent at position 2 of the resorcinol core. ⌬9-THC
and CP 55,940 contain two oxygens: one as a phenol (one
hydroxyl in the aromatic ring) with a second oxygen incorpo-
rated into a separate ring (pyran oxygen in ⌬9-THC) or a
hydroxyl group attached as a substituent in the cyclohexane
ring, as in CP 55,940. Previous research has shown that
eliminating the phenolic hydroxyl of ⌬8-THC results in de-
oxy-⌬8-THC analogs that are CB2-selective (Huffman et al.,
1999). Although some of these analogs also retain reasonable
affinity for CB1 receptors, orientation of their binding to CB1
receptors may be inverted such that the pyran oxygen sub-
stitutes for the absent phenolic hydroxyl in hydrogen bonding
(Huffman et al., 1996). In the absence of a pyran oxygen, the
in the present study; however, the range of chain lengths for
which moderate to good CB2 affinity was retained for the
deoxy-⌬8-THC analogs was greater than the range for CB1
affinity. Similar results were obtained with a series of CB2-
selective indole-derived cannabinoids in which length of the
nitrogen substituent was varied (Aung et al., 2000). To date,
anandamide analogs appear to be CB1 selective, with rela-
tively little affinity for CB2 receptors across several types of
manipulations (Showalter et al., 1996). Insufficient research
is available to determine the effect of substitution on a cyclo-
hexane ring on CB2 affinity across cannabinoid classes.
Other structural manipulations that eliminated or drasti-
cally reduced CB1 receptor recognition did not necessarily
alter CB2 receptor binding in an identical manner. CB2 se-
lectivity was most evident in the dimethoxy analogs, primar-
ily as a consequence of severe reductions in CB1 affinity.
HU-308, the most selective CB2 agonist to date, has a dime-
thoxyresorcinol core structure and does not bind to CB1 re-
ceptors at all (Hanusˇ et al., 1999). In addition, greater toler-
ance in CB2 (versus CB1) receptor recognition was observed
with other C2 substitutions in the resorcinols. Huffman et al.
(2001) recently reported that bicyclic pyridone analogs with
carbonyl substitution at C1 and a nitrogen substituent sub-
stitution at C2 of ⌬8-THC had little affinity for CB1 receptors.
In contrast, moderate CB2 affinity (Ki ϳ 53 nM) was re-
tained. Differences in allosteric regulation of CB1 and CB2
receptors by ions and guanine nucleotides have been noted
previously (Showalter et al., 1996). Together, the results
nature of the substituent at position 2 of the resorcinol core is presented here and elsewhere (see above) suggest incomplete
important for maintenance of in vivo activity. An acyclic ring
was found to be better than a heterocyclic ring, with a cyclo-
hexane ring being optimal. In addition, the size and the
position of the substituent on the cyclic ring is important to
maintenance of CB1 affinity. The presence of a methyl sub-
stituent at position 3 enhanced activity in some cases. Fur-
thermore, the 3S analog (O-1826; Table 2) has a poorer CB1
binding affinity (Ki ϭ 40 nM) compared with the diastereo-
meric mixture O-1657 (Ki ϭ 14 nM; Table 2), suggesting that
CB1 binding affinity is enhanced when the orientation of the
methyl substituent at position 3 in the cyclohexane ring is 3R
compared with 3S. Methylation of the phenols of the resor-
cinols drastically decreased or eliminated CB1 affinity, per-
haps because hydrogen donation is less likely from a methoxy
group than from the free hydroxyl group of ⌬9-THC (B. R.
Martin, unpublished observations). Similarly, methoxy sub-
stitution for the phenolic hydroxyl in the methyl esters of ⌬8-
and ⌬9(11)-THC-dimethylheptyl resulted in analogs that were
CB2-selective and had little CB1 affinity (Gareau et al., 1996;
Huffman et al., 1999; Ross et al., 1999).
Notably, most of the dimethoxyresorcinols tested here were
CB2-selective. As suggested by the high positive correlation
between CB1 and CB2 binding affinities, most of the struc-
tural features that affected recognition at CB1 receptors also
affected CB2 receptor recognition, although not always to the
same degree or in the same manner. These factors included
length and branching of the side chain and size and degree of
saturation of the nonresorcinol cyclohexane ring. In a struc-
ture-activity relationship study on a series of CB2-selective
deoxy-⌬8-THC analogs, Huffman et al. (1999) reported that
overlap of the pharmacophores for CB1 and CB2 receptors.
In summary, structure-activity relationships of the resor-
cinol series presented here are consistent with the CB1 and
CB2 pharmacophores of other cannabinoid classes. In this
series of resorcinols, several structural features were essen-
tial for maintenance of CB1 receptor recognition and in vivo
activity, including the presence of a branched lipophilic side
chain at C5, the presence of free phenols, and substitution of
a cyclohexane ring at C2. An important structural feature for
receptor recognition at CB2 receptors was side chain length.
The CB2 selectivity observed with some resorcinols was max-
imized in the dimethoxyresorcinol analogs, and this selectiv-
ity was greatly enhanced when a tertiary hydroxyl group was
present in the cyclohexane ring in the same position at which
the resorcinol core is attached. In contrast, the presence of
unsaturation, a ketone group, or an additional hydroxyl sub-
stitution in the cyclohexane ring adversely affected the CB2
selectivity. Methyl ethers were optimal for CB2 selectivity
because ethyl ethers reduced selectivity.
In conclusion, although resorcinol derivatives with cyclic
ring substituents at C2 are closely related to the nonactive
cannabinoid cannabidiol, many of these analogs have high
CB1 and/or CB2 binding affinity as well as potent in vivo
activity. In addition, because dimethoxyresorcinols are CB2-
selective, they have potential to offer insight into similarities
and differences between requirements for receptor recogni-
tion at CB1 versus CB2 receptors. The results presented here
suggest that the resorcinol series represent a novel template
for the development of CB1- and CB2-selective cannabinoid
length and branching of the C3 side chain affected CB2 bind- agonists.

Cannabinoid Resorcinols
689
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We thank Rene´e Jefferson and Ramona Winckler for technical
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Address correspondence to: Dr. Jenny L. Wiley, Department of Pharma-
cology and Toxicology, Virginia Commonwealth University, P.O. Box 980613,
Richmond, VA. E-mail: jwiley@hsc.vcu.edu
Huffman JW, Yu S, Showalter V, Abood ME, Wiley JL, Compton DR, Martin BR,
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