3-Indolyl-1-naphthylmethanes: New cannabimimetic indoles provide evidence for aromatic stacking interactions with the CB1 cannabinoid receptor

Article abstract of DOI:10.1016/S0968-0896(02)00451-0

A series of 1-pentyl-1H-indol-3-yl-(1-naphthyl)methanes (9-11) and 2-methyl-1-pentyl-1H-indol-3-yl-(1-naphthyl)methanes (12-14) have been synthesized to investigate the hypothesis that cannabimimetic 3-(1-naphthoyl)indoles interact with the CB₁ receptor by hydrogen bonding to the carbonyl group. Indoles 9-11 have significant (K_i=17-23 nM) receptor affinity, somewhat less than that of the corresponding naphthoylindoles (5, 15, 16). 2-Methyl-1-indoles 12-14 have little affinity for the CB₁ receptor, in contrast to 2-methyl-3-(1-naphthoyl)indoles 17-19, which have affinities comparable to those of 5, 15, 16. A cannabimimetic indene hydrocarbon (26) was synthesized and found to have K_i=26±4 nM. Molecular modeling and receptor docking studies of naphthoylindole 16, its 2-methyl congener (19) and indolyl-1-naphthylmethanes 11 and 14, combined with the receptor affinities of these cannabimimetic indoles, strongly suggest that these cannabinoid receptor ligands bind primarily by aromatic stacking interactions in the transmembrane helix 3-4-5-6 region of the CB₁ receptor.

Full text of DOI:10.1016/S0968-0896(02)00451-0

Bioorganic & MedicinalChemistry 11 (2003) 539–549
3-Indolyl-1-naphthylmethanes: New Cannabimimetic Indoles
Provide Evidence for Aromatic StackingInteractions with the
CB1 Cannabinoid Receptor
John W. Huffman,a,* Ross Mabon,a Ming-Jung Wu,a Jianzhong Lu,a Richard Hart,b
Dow P. Hurst,b Patricia H. Reggio,b Jenny L. Wileyc and Billy R. Martinc
aHoward L. Hunter Laboratory, Clemson University, Clemson, SC 29634-0973, USA
bDepartment of Chemistry and Biochemistry, Kennesaw State University, 1000 Chastain Road, Kennesaw, GA30144, USA
cDepartment of Pharmacology and Toxicology, Medical College of Virginia Campus of Virginia Commonwealth University,
Richmond, VA23298-0613, USA
Received 17 June 2002; accepted 9 September 2002
Abstract—A series of 1-pentyl-1H-indol-3-yl-(1-naphthyl)methanes (9–11) and 2-methyl-1-pentyl-1H-indol-3-yl-(1-naphthyl)-
methanes (12–14) have been synthesized to investigate the hypothesis that cannabimimetic 3-(1-naphthoyl)indoles interact with the
CB1 receptor by hydrogen bonding to the carbonylgroup. Indoels 9–11 have significant (Ki=17–23 nM) receptor affinity, some-
what less than that of the corresponding naphthoylindoles (5, 15, 16). 2-Methyl-1-indoles 12–14 have little affinity for the CB1
receptor, in contrast to 2-methyl-3-(1-naphthoyl)indoles 17–19, which have affinities comparable to those of 5, 15, 16. A cannabi-
mimetic indene hydrocarbon (26) was synthesized and found to have Ki=26ꢀ4 nM. Molecular modeling and receptor docking
studies of naphthoylindole 16, its 2-methylcongener ( 19) and indolyl-1-naphthylmethanes 11 and 14, combined with the receptor
affinities of these cannabimimetic indoles, strongly suggest that these cannabinoid receptor ligands bind primarily by aromatic
stacking interactions in the transmembrane helix 3-4-5-6 region of the CB1 receptor.
# 2002 Elsevier Science Ltd. All rights reserved.
Introduction
cyclase, are antinociceptive in vivo and interact with a
G-protein coupled receptor in the brain. Work from the
same group confirmed that these aminoalkylindoles
(AAIs) bind to the cannabinoid receptor which is
expressed primarily in the central nervous system (CB1
Following the identification of Á9-tetrahydrocanna-
binol( Á9-THC, 1)1 as the principalpsychoactive con-
stituent of marijuana, a comprehensive set of structure–
activity relationships (SAR) was developed based
upon the partially reduced dibenzopyran structure of
Á9-THC.2ꢁ6 These SAR were subsequently extended to
a group of very potent non-traditionalcannabinoids
developed by Pfizer, of which CP-55,940 (2,
DMH=1,1-dimethylheptyl) is typical.7,8
In 1991, as a result of a program directed toward the
development of nonsteroidal anti-inflammatory drugs, a
group at Sterling Winthrop reported that pravadoline
(3) unexpectedly inhibited contractions of the elec-
trically stimulated mouse vas deferens.9 It was found
that 3 and related compounds also inhibit adenylate
*Corresponding author. Fax: +1-864-656-6613; e-mail: huffman@
clemson.edu
0968-0896/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00451-0

540
J. W. Huffman et al. / Bioorg. Med. Chem. 11 (2003) 539–549
receptor),10 and exhibit typicalcannabinoid pharma-
cology in vivo.11 One rigid AAI, WIN-55,212-2 (4), has
particularly high affinity for the CB1 receptor, and is
considered to be the prototypicalexampel of this calss
of cannabinoid receptor ligands.
receptor and inhibit the electrically stimulated contrac-
tion of the mouse vas deferens.24
Since the AAIs and traditionalcannabinoids realted to
Á9-THC (1) interact with the same receptor and show
similar in vivo pharmacology, several groups have
attempted to devise a pharmacophore which will
accommodate both classes of cannabinoid ligands. One
alignment superimposes the 1-naphthoyl moiety of
WIN-55,212–2 (4) upon the lipophilic side chain of the
12,13
traditionalcannabinoids.
An alternative alignment
has been suggested in which the indole carbonyl, which
is a vinylogous amide, corresponds to the hydroxyl
group of THC and the indole nitrogen is aligned with
C-10 of the cannabinoid side chain.14ꢁ17 This modelhas
been employed to develop a number of very potent
cannabimimetic indoles, such as 1-pentyl-3-(1-naph-
thoyl)indole (5, JWH-018), in which the aminoalkyl
group appended to the indole nitrogen has been
replaced by an alkyl group.14ꢁ16,18 This alignment was
also employed to design JWH-161 (6), a hybrid canna-
binoid which has greater affinity than Á8-THC for the
CB1 receptor, and is of approximately equal potency in
vivo.19
In order to obtain experimentalevidence concerning the
mode of interaction of cannabimimetic indoles with the
CB1 receptor we have synthesized a series of 3-(1-
pentylindolyl)-1-naphthylmethanes (9–11) and their
2-methylanaol gues ( 12–14), in which there is no viable
possibility for hydrogen bonding interactions between
the receptor and the ligand. A 1-pentyl group was
incorporated in indoles 9–14 since in cannabimimetic
indoles maximum affinity for the CB1 receptor is usually
14ꢁ16
attained with a pentylsubstituent on nitrogen.
These compounds were prepared in 40–45% unopti-
mized yield from the corresponding 3-(1-naphthoyl)-
indoles (5, 15–19) in a single step by reduction using
lithium aluminum hydride and aluminum chloride. The
3-(1-naphthoyl)indoles have either been reported pre-
viously14ꢁ16,25 or were prepared in the usualmanner, by
Friedel–Crafts acylation of 1-pentylindole or 2-methyl-
1-pentylindole.12,14 This reaction sequence, which is
exemplified by the preparation of indole 5 and its
reduction to 9, is illustrated in Scheme 1.
The affinities of indoles 9–11 and 12–14 for the CB1
receptor were determined by measuring their ability to
displace the very potent cannabinoid, [3H]CP-55,940
(2), from its binding site in a membrane preparation as
described by Compton et al.26 These data, plus the
receptor affinities for Á9-THC (1), WIN-55,212–2 (4),
naphthoylindoles 5 and 15–19, are summarized in Table
1.
Results and Discussion
The hypothesis that the naphthoylcarbonylof indoels
such as 5 interact with the CB1 receptor via hydrogen
bonding was supported by the in vitro receptor affinities
and in vivo potency of cannabimimetic indoles designed
on the basis of this hypothesis. However, a recent body
of evidence indicates that this class of cannabinoids
probably interacts with the receptor primarily by aro-
matic stacking. Specifically, Song and Bonner prepared
a mutant CB1 receptor in which a lysine (K192) on helix
3 of the receptor is replaced by an alanine.20 The affi-
nities of a very potent classical dibenzopyran based
cannabinoid, HU-210, and CP-55,940 (2) were greatly
attenuated, but that of WIN-55,212-2 (4) was only
slightly affected. These data, plus molecular modeling
studies, suggested that the cannabimimetic indoles were
interacting with the CB1 receptor at a site somewhat
different from that of the dibenzopyran based
cannabinoids.21ꢁ23 Further, it was suggested that the
principalinteraction of these ligands with the receptor is
by aromatic stacking.21 Supporting evidence for this
hypothesis was found in additionalmodeilng studies
plus the observation that E-naphthylideneindenes 7 and
8, but not the Z-isomers, have high affinity for the CB1
The CB1 receptor affinities of indoles 9–11, which are
unsubstituted at C-2 of the indole nucleus, are the same,
with Ki=17–23 nM. The affinities of these compounds
are somewhat less than those of naphthoylindoles 5, 15
and 16, which have Ki=9ꢀ5, 0.69ꢀ0.05 and
1.2ꢀ0.1 nM, respectively. Also, indoles 9–11 show little
if any effect upon receptor affinity as a function of sub-
stitution at the 4-position of the naphthalene ring. In
the naphthoylindole series, a 4-methyl or 4-methoxy
substituent enhances receptor affinity (see Table 1). The
Scheme 1. (a) 1-Naphthoyl chloride/toluene/EtAlCl2, 25 ꢂC; (b) AlCl3/
LiAlH4/Et2O, 0 ꢂC, then 5, 0–25 ꢂC.

J. W. Huffman et al. / Bioorg. Med. Chem. 11 (2003) 539–549
541
addition of a 2-methylgroup in indoels
12–14 con-
binding data were obtained by displacement of [3H]-
WIN-55,212-2 (4) from a rat cerebellar membrane pre-
paration, while the data presented in Table 1 were
obtained by displacement of [3H]CP-55,940 (2) from a
rat whole brain memebrane preparation.26
siderably attenuates receptor affinity, with Ki=151–
323 nM. In the 2-methyl-3-(1-naphthoyl)indole series
(17–19) the affinities for the CB1 receptor are only
slightly less than those of the compounds lacking a
substituent at C-2 (5, 15, 16).
E-Naphthylideneindenes 7 and 8 have good affinity for the
CB1 receptor (Ki=2.72ꢀ0.22 nM and 2.89ꢀ0.41 nM,
respectively), and modeling studies support the hypoth-
esis that they interact with the receptor by aromatic
stacking interactions.24 However, there is at least a for-
mal possibility that the morpholino nitrogen or oxygen
may interact with the receptor by hydrogen bonding. In
order to explore this possibility, E-naphthylideneindene
26 (JWH-176) was prepared by the base catalyzed
(NaOMe/MeOH) condensation of 1-pentylindene with
1-naphthaldehyde.27 This indene has high affinity for
the CB1 receptor with Ki=26ꢀ4 nM (Table 1).
Although indene 26 has high affinity for the CB1 recep-
tor it is somewhat less than those reported for morpho-
linoethylindenes 7 and 8.24
The cannabimimetic indoles reported originally were
characterized by an aminoalkyl group attached to the
indole nitrogen,9,12 which could conceivably interact
with the CB1 receptor by hydrogen bonding.13 To
investigate this possibility, 1-morpholinoethylindoles
20–22 were prepared by reduction of the corresponding
3-naphthoylindoles (23–25). The reductions were car-
ried out using lithium aluminum hydride–aluminum
chloride as described above for the preparation of
indoles 9–11. The CB1 receptor affinities for indoles 20–
25 are included in Table 1.
In contrast to indoles 9–11, which have effectively the
same affinity for the CB1 receptor, there is considerable
variation in the affinities of indoles 20–22 as a function
of substitution in the 4-position of the naphthalene ring.
The analogue with an unsubstituted naphthalene ring
has quite modest affinity for the CB1 receptor
(Ki=113ꢀ28 nM), while the 4-methyl- and 4-methoxy-
naphthylanaolgues have significantyl greater affinity
(Ki=41ꢀ13 nM and Ki=20ꢀ2 nM, respectively).
Naphthoylindoles 23–25 were reported previously by
the Winthrop group who found the same trend in affi-
nities, with the unsubstituted analogue (23) having
somewhat less affinity for the CB1 receptor than the
12
4-methyland 4-methoxynaphthoylanaolgues.
There
are considerable differences between the affinities pre-
sented in Table 1 for indoles 23–25 and those reported
by Eissenstat et al. However, the Winthrop group’s
The high CB1 receptor affinities of indoles 9–11, 21, 22
and indene 26 strongly support the hypothesis that
cannabimimetic indoles and related CB1 receptor
Table 1. CB1 receptor affinities (meanꢀSEM) of cannabimimetic indoles 5, 9–25, 27 and related compounds
Compd
Ki (nM)
Á9-THC (1)
WIN-55,212-2 (3)
41ꢀ2a
9.9ꢀ1.0b
22ꢀ2
1-Pentyl-1H-indol-3-yl-(1-naphthyl)methane (9, JWH-175)
1-Pentyl-1H-indol-3-yl-(4-methyl-1-naphthyl)methane (10, JWH-184)
1-Pentyl-1H-indol-3-yl-(4-methoxy-1-naphthyl)methane (11, JWH-185)
2-Methyl-1-pentyl-1H-indol-3-yl-(1-naphthyl)methane (12, JWH-196)
2-Methyl-1-pentyl-1H-indol-3-yl-(4-methyl-1-naphthyl)methane (13, JWH-194)
2-Methyl-1-pentyl-1H-indol-3-yl-(4-methoxy-1-naphthyl)methane (14, JWH-197)
1-Pentyl-3-(1-naphthoyl)indole (5, JWH-018)
1-Pentyl-3-(4-methyl-1-naphthoyl)indole (15, JWH-122)
1-Pentyl-3-(4-methoxy-1-naphthoyl)indole (16, JWH-081)
2-Methyl-1-pentyl-3-(1-naphthoyl)indole (17, JWH-007)
2-Methyl-1-pentyl-3-(4-methyl-1-naphthoyl)indole (18, JWH-149)
2-Methyl-1-pentyl-3-(4-methoxy-1-naphthoyl)indole (19, JWH-098)
1-[2-(4-Morpholino)ethyl]-1H-indol-3-yl-1-naphthylmethane (20, JWH-195)
1-[2-(4-Morpholino)ethyl]-1H-indol-3-yl-(4-methyl-1-naphthyl)methane (21, JWH-192)
1-[2-(4-Morpholino)ethyl]-1H-indol-3-yl-(4-methoxy-1-naphthyl)methane (22, JWH-199)
1-[2-(4-Morpholino)ethyl]-3-(1-naphthoyl)indole (23, JWH-200)
1-[2-(4-Morpholino)ethyl]-3-(4-methyl-1-naphthoyl)indole (24, JWH-193)
1-[2-(4-Morpholino)ethyl]-3-(4-methoxy-1-naphthoyl)indole (25, JWH-198)
E-1-[1-(1-Naphthalenylmethylene)-1H-inden-3-yl]pentane (26, JWH-176)
2-Ethyl-1-pentyl-3-(1-naphthoyl)indole (27, JWH-116)
23ꢀ6
17ꢀ3
151ꢀ18
127ꢀ19
323ꢀ28
9ꢀ5c
0.69ꢀ0.05
1.2ꢀ0.1d
9.5ꢀ4.5c
5.0ꢀ2.1
4.5ꢀ0.1d
113ꢀ28
41ꢀ13
20ꢀ2
42ꢀ5
6ꢀ1
10ꢀ2
26ꢀ4
52ꢀ5
aRef 26.
bRinaldi-Carmona, M.; Barth, F.; Heaulme, M.; Shire, D.; Calandra, B.; Congy, C.; Martinez, S.; Maruani, J.; Neliat, G.; Caput, D.; Ferrara, P.;
Soubrie, P.; Breliere, J. C.; Le Fur, G. FEBS Lett. 1994, 350, 240.
cRef 15.
dRef 16.

542
J. W. Huffman et al. / Bioorg. Med. Chem. 11 (2003) 539–549
ligands do not interact with the receptor by hydrogen
bonding.14ꢁ17 In particular, the high affinity of indene
26, a hydrocarbon, for the CB1 receptor provides com-
pelling evidence against hydrogen bonding interactions
playing a major role in the binding of these ligands.
These data are consistent with the suggestion that these
compounds interact with the receptor by aromatic
stacking.21,24
identified an s-trans conformation as the global mini-
mum energy conformer. The lowest energy s-cis con-
former was found to be 0.59 kcal/mol higher in energy
than the global minimum s-trans conformer. In JWH-
098 (19), AM1 calculations revealed that the lowest
energy conformer is an s-cis conformer. This result is
also consistent with earlier results, as the C-2 sub-
stituent in indole 19 is a methylgroup. The olwest
energy s-trans conformation was found to be 1.22 kcal/
Although indoles 9–11, 21 and 22, which are unsub-
stituted at C-2 of the indole nucleus, have significant
affinity for the CB1 receptor, 2-methylindoles (12–14)
have very little affinity. This is in contrast to the 3-(1-
naphthoyl)indole series in which the 2-methylindoles
have only slightly less affinity for the CB1 receptor than
the unsubstituted compounds. This is a generalproperty
of these cannabimimetic indoles, which has been noted
previously10,12,15,16,18 and is also apparent from the
limited data presented in Table 1. There appears to be
no a priori explanation for the poor receptor affinities of
indoles 12–14 when compared to the significant affinities
of indoles 9–11, 21 and 22. In order to obtain some
insight into the origin of these apparently anomalous
differences in receptor affinity, molecular modeling and
receptor docking studies of indoles JWH-081 (16),
JWH-098 (19), JWH-185 (11) and JWH-197 (14) were
carried out.
molhigher in energy than the golbalminimum
conformer. The global minimum energy conformers of
indoles 16 and 19 are illustrated in Figure 1 (left).
s-cis
1-Pentyl-1H-indol-3-yl-(4-methoxy-1-naphthyl)methane (11,
JWH-185, Ki=17ꢀ3 nM) and 2-methyl-1-pentyl-1H-
indol-3-yl-(4-methoxy-1-naphthyl)methane (14, JWH-
197, Ki=323ꢀ48 nM) are congeners of JWH-081 (16)
and JWH-098 (19) respectively, in which the carbonyl
bridge has been replaced by a methylene group. This
replacement changes the hybridization of the bridging
In the discussion of compounds that follows, each
molecule is oriented so that the indole ring is perpendi-
cular to the plane of the page, with the C-4 hydrogen
pointing out of the page toward the viewer and with the
C-2 substituent pointing down toward the bottom of the
page (see Fig. 1 insets). The numbering used in this dis-
cussion is included on the structure of indole 5. In this
orientation, substituents which are located to the left of
the indole ring plane protrude into the top face of the
molecule, while those that are to the right of the indole
plane protrude into the bottom face of the molecule.
1-Pentyl-3-(4-methoxy-1-naphthoyl)indole (16, JWH-
081, Ki=1.2ꢀ0.03 nM) and its 2-methylcongener,
JWH-098 (19, Ki=4.5ꢀ0.1 nM), display high CB1
receptor affinity. Like the aminoalkylindole (AAI),
WIN-55,212-2 (4), these compounds possess a carbonyl
group which bridges the indole and naphthalene rings.
Two major conformations have been shown to exist for
WIN-55,212-2 and related AAIs, the s-cis and the
s-trans conformations.24 These conformations differ
primarily in the orientation of the C-3 aroyl substituent.
In the s-cis conformation, which predominates when the
C-2 substituent is a methylgroup (as in WIN-55,212-2),
the carbonyloxygen is near C-2, whiel the naphthylring
is stacked over C-4 of the indole ring. In the s-trans
conformation, which predominates when C-2 is a
hydrogen (as in JWH-018, 5), the arylsystem is near C-2
and the carbonyloxygen is near C-4. AM1 semi-
Figure 1. (Left) A side view is shown here in which the global mini-
mum energy conformers of JWH-081 (16, s-trans; in green) and JWH-
098 (19, s-cis; in yellow) are superimposed at their indole rings. In this
side view, the indole ring is perpendicular to the page with the 2-
methylgroup pointing towards the viewer. In the cutout at the bot-
tom, molecules are oriented in a top view, so that the indole ring is
perpendicular to the page with C-4 pointing towards the viewer and
the C-2 methylof 19 pointing down. The alkyl side chains have been
removed here in order to simplify the display. (Right) A side view is
shown here in which the global minimum energy conformers of JWH-
185 (11, in orange) and JWH-197 (14, in purple) are superimposed at
their indole rings. In this side view, the indole ring is perpendicular to
the page with the 2-methylgroup pointing towards the viewer. In the
cutout at the bottom, molecules are oriented in a top view so that the
indole ring is perpendicular to the page with C-4 pointing towards the
viewer and the C-2 methylof 19 pointing down. The alkyl side chains
have been removed here in order to simplify the display.
empiricalcaclualtions indicate that the
tion of WIN-55,212–2 is 1.86 kcal/mol lower in energy
s-cis conforma-
than its lowest energy s-trans conformer.24
Consistent with these earlier results, AM1 conformer
searches of JWH-081 (16), which has a hydrogen at C-2,

J. W. Huffman et al. / Bioorg. Med. Chem. 11 (2003) 539–549
543
3
carbon from sp2 in the carbonylgroup to sp in the
methylene group. This change can be anticipated to
produce a different relative orientation of the naphtha-
lene and indole rings compared with that seen for
indoles 16, 19 and WIN-55,212-2 (4).
the AAIs are highly aromatic ligands and because
K3.28A mutation studies have indicated that K3.28 is
not an interaction site for WIN-55,212-2 (4),20 it has
been hypothesized that aromatic stacking,30 rather than
hydrogen bonding interactions, is the primary interac-
tion is for the AAIs at CB1.31 Mutation studies per-
formed by Song and co-workers have supported the
importance of aromatic stacking for WIN-55,212-2
interaction with both the CB1 and CB2 receptors.31
These studies showed that the 15–20-fold higher affinity
of 4 for the CB2 receptor is due to a direct interaction
with F5.46, a residue that is aromatic only in CB2.
For indoles 11 and 14, two sets of conformers were
identified by AM1 conformer searches. In the first set of
conformers, substituents are staggered with respect to
the C10–C20 bond which connects the methylene carbon
(C10) with the indole ring. Within this set, variations in
the C3–C10–C20–C30 torsion angle place the naphthyl
ring in different orientations with respect to the indole
ring. In the second set, a methylene C10–H bond eclip-
ses an adjacent C–C bond on the indole ring. For
JWH-185 (11), the global minimum energy conformer
has the methylene C–H bonds staggered with respect to
the indole ring, with a C3–C10–C20–C30 angle of
ꢁ177.8ꢂ. In this conformer (see Fig. 1 right), the naph-
thalene ring is oriented perpendicular to the plane of the
indole nucleus and is located in the top face of the
molecule.
Because cannabimimetic indoles 11, 14, 16 and 19 are
highly aromatic ligands which are structurally related to
the AAIs, we hypothesized that the TMH 3-4-5-6 region
of CB1 would also be the binding region for these
ligands. In addition, since JWH-081 (16) and JWH-098
(19), like the AAIs, have a carbonyl bridge between the
indole and naphthalene rings and because the s-trans
arrangement of the indole and naphthalene rings in the
AAIs has been shown to be the AAI bioactive con-
formation,24 indoles 16 and 19 were docked in the TMH
3-4-5-6 region of CB1 in their lowest energy s-trans
conformations. Indoles 11 and 14 were docked in this
same region using the global minimum energy con-
former of each (see Fig. 1, right). Aromatic stacking
interactions were characterized by the distance between
ring centroids, and the angle (a) formed by the planes of
the interacting rings. Hydrogen bonding interactions
were characterized by the distance between the hetero-
atoms involved in the hydrogen bond and the hydro-
gen bond angle (heteroatom–H–heteroatom).
In JWH-197 (14), the global minimum energy con-
former has the methylene hydrogens staggered with
respect to the plane of the indole, with a C3–C10–C20–
C30 angle of 177.8ꢂ. In this conformer, the naphthalene
ring is also in the top face of the molecule, with the
naphthalene ring perpendicular to the plane of the
indole (see Fig. 1, right).
1-Pentyl-1H-indol-3-yl-(4-methoxy-1-naphthyl)methane
(11, JWH-185) has reduced CB1 receptor affinity
(Ki=17ꢀ3 nM) relative to 1-pentyl-3-(4-methoxy-1-
naphthoyl)indole (16, JWH-081, Ki=1.2ꢀ0.03 nM). In
addition, while in the 3-(1-naphthoyl)indole series (11
and 16), the substitution of a methylgroup at C-2
results in less than a four-fold loss in CB1 receptor affi-
nity (JWH-081, 16, Ki=1.2ꢀ0.03 nM; JWH-098, 19,
Ki=4.5ꢀ0.1 nM), substitution at C-2 in the naphthyl-
methane series (11 and 14) results in a more profound
19-fold affinity loss (JWH-185, 11, Ki=17ꢀ3 nM;
JWH-197, 14, Ki=323ꢀ48 nM). In order to probe the
origin of these affinity changes at the CB1 receptor, each
The SAR of cannabimimetic 2-methylindoles indicate
that compounds with N-alkyl substituents from n-pro-
pyl( Ki=164ꢀ22 nM) to n-hexyl( Ki=48ꢀ13 nM), with
n-pentylbeing optimum ( Ki=9.5ꢀ4.5 nM), have good
CB1 receptor affinities, but those indoles with N-alkyl
substituents that are shorter (ethyl, Ki=1182ꢀ44 nM)
or longer (n-heptyl, Ki >10,000 nM) have poor affi-
nities.15 A similar trend is seen both in indole analogues
which are unsubstituted at C-2, and which have various
substituents on the naphthalene ring.15,16,18 In light of
these SAR, a hydrophobic binding pocket for the alkyl
tailwas sought in the TMH 3-4-5-6 region which had
limited depth, but required extension of several carbons
in the N-alkyl substituent in order to enter this region.
A hydrophobic binding pocket comprised of V3.32,
I3.33, F3.36, L6.51, I6.54 was identified that permitted
simultaneous interaction of the indole and naphthalene
rings with the aromatic residues in the TMH 3-4-5-6
region of R* CB1. In this hydrophobic binding pocket,
lengthening the alkyl chain to n-heptylproduces serious
van der Waals’ overlaps with the backbone of TMH 7,
while shortening to ethyl prevents interaction with this
hydrophobic pocket. When the N1 alkyl chain was
docked in this hydrophobic pocket, JWH-081 (16) and
JWH-098 (19) found aromatic stacking interactions
with W5.43 (JWH-081–indole: d=4.3 A, ꢀ=80ꢂ, naph-
thyl: d=5.0 A, ꢀ=30ꢂ; JWH-098–indole: d=4.4 A,
ꢀ=90ꢂ, naphthyl: d=5.1 A, ꢀ=50ꢂ) and W6.48 (JWH-
081–naphthyl: d=5.9 A, ꢀ=30ꢂ; JWH-098–naphthyl:
of these compounds was docked in a modelof the CB
receptor active state (R*).
1
The transmembrane helix (TMH) 3-4-5-6 region of CB1
is rich in aromatic amino acids that face into the bind-
ing site crevice in CB1. These residues include F3.25,
F3.36, W4.64, Y5.39, W5.43, and W6.48. Shire and co-
workers have shown in CB1/CB2 chimera studies that
the TMH4-E2-TMH5 region of the cannabinoid recep-
tors contains residues criticalfor the binding of WIN-
55,212-2.28 A recent mutation and computational
Monte Carlo/stochastic dynamics study of the CB1
receptor has suggested that aromaticity at positions 4.64
and 5.39 is important to maintain the proper relative
positions of TMHs 4 and 5, and therefore the ligand
binding pocket.29 Three amino acids, F3.36, W5.43, and
W6.48, were found in this same study to form a triad of
interacting aromatic residues in the inactive state of CB1
which would be available for ligand binding.29 Because

544
J. W. Huffman et al. / Bioorg. Med. Chem. 11 (2003) 539–549
d=4.9 A, ꢀ=90ꢂ). In addition, the N–H of W5.43 could
hydrogen bond with the carbonyloxygens of 16 and 19,
although the hydrogen bond geometry was better with
16 (16: O–N distance=2.7 A, O–H–N angle=162ꢂ; 19:
O–N distance=2.7 A, O–H–N angle=123ꢂ). In this
docking position, the C-2 substituent change between 16
and 19 would cause no loss of affinity, since the C-2
methylgroup in 19 occupies an open space in the
receptor binding pocket. This docking position also
indicates that enlargement of the C-2 substituent to
ethylcan stilbe accommodated in this binding region.
In agreement with this hypothesis, it was found that 2-
ethyl-1-pentyl-3-(1-naphthoyl)indole (27, JWH-116) has
significant affinity for the CB1 receptor (Ki=52ꢀ5 nM).
This indole derivative was prepared from 2-ethyl-
indole32 by C-3 acylation with 1-naphthoyl chloride,
followed by N-alkylation.9,14
Experimental
General
IR spectra were obtained using Nicolet 5DX or Magna
1
spectrometers; H and 13C NMR spectra were recorded
on a Bruker 300AC spectrometer. Mass spectralana-
lyses were performed on a Hewlett-Packard 5890A gas
chromatograph with a mass sensitive detector and
HRMS data were obtained in the Mass Spectrometry
Laboratory, Schoolof ChemicalSciences, University of
Illinois. Ether and THF were distilled from Na-benzo-
phenone ketyl immediately before use, and other sol-
vents were purified using standard procedures. Column
chromatography was carried out on Sorbent Technolo-
gies silica gel (32–63 mm) using the indicated solvents as
eluents. All new compounds were homogeneous to GLC
and/or TLC and 13C NMR.
The conformers of JWH-185 (11) and JWH-197 (14)
illustrated in Figure 1 could be docked in the same
generalregion of CB R* using the same hydrophobic
1
1-Pentyl-1H-indol-3-yl-(1-naphthyl)methane (9). To a
suspension of 0.46 g (3.48 mmol) of aluminum chloride
in 2 mL of ether at 0 ꢂC was added dropwise 1.16 mL
(1.16 mmol) of a 1 M solution of LiAlH4 in ether. The
suspension was stirred at 0 ꢂC for 30 min and 0.10 g
(0.29 mmol) of 1-pentyl-3-(1-naphthoyl)indole (5) in
2 mL of ether was added dropwise. The reaction mixture
was warmed to ambient temperature, stirred for 48 h,
cooled to 0 ꢂC, and carefully quenched with water. After
acidification the organic phase was separated and
washed successively with saturated aqueous NaHCO3
and brine. After drying (MgSO4) the solvent was
removed in vacuo to give a yellow oil. Chromatography
(petroleum ether/ether, 4:1) gave 0.038 g (40%) of 9 as a
yellow oil: Rf 0.74 (petroleum ether/ether 4:1); 1H NMR
(300 MHz, CDCl3) d 0.91 (t, J=7.0 Hz, 3H), 1.22–1.41
(m, 4H), 1.68–1.78 (m, 2H), 4.02 (t, J=7.1 Hz, 2H), 4.63
(s, 2H), 6.59 (s, 1H), 7.10–7.47 (m, 8H), 7.62 (d,
J=7.8 Hz, 1H), 7.75 (d, J=7.2 Hz, 1H), 8.11 (d,
J=6.9 Hz, 1H); 13C NMR (75.5 MHz, CDCl3) d 13.9,
22.2, 28.9, 29.9, 46.1, 109.3, 113.5, 118.7, 119.2, 121.4,
124.4, 125.4, 125.6, 125.7, 126.6, 126.7, 127.9, 128.5,
132.2, 133.8, 136.3, 137.0; HRMS calcd for C24H25N:
327.1982; found: 327.1995.
binding pocket for the n-pentylchain of these ana-
logues. However, because these analogues have con-
formations which orient the naphthalene and indole
rings in a very different arrangement than in the 3-
aroylindoles (16 and 18), the orientation of the ligands
in the binding pocket differs from that of indoles 16 and
18. Naphthylindoles 11 and 14 can still engage in aro-
matic stacking interactions with W5.43 and W6.48. In
addition, the indole nucleus of JWH-185 (11) engages in
an aromatic stacking interaction with F3.36 that
involves the C-2 hydrogen. No hydrogen bonding
interactions are possible for this ligand at the CB1 R*
binding site. This may explain why indole 11 has
reduced CB1 affinity relative to naphthoylindole 16.
Like JWH-185 (11), its 2-methylanaolgue, JWH-197
(14), can engage in aromatic stacking interactions with
W5.43 and W6.48. However, in the JWH-197 (14)/
CB1 R* complex, no aromatic stacking interaction is
possible with F3.36 because indole 14 lacks the
hydrogen at C-2 which participates in the stacking
interaction with F3.36 in the JWH-185 (11)/CB1 R*
complex. The magnitude of the drop in affinity seen in
going from the C-2-H in 11 (Ki=17ꢀ3 nM) to the C-2
methylanaolgue, JWH-197 ( 14, Ki=323ꢀ48 nM), is
consistent with the loss of an aromatic stacking interac-
tion.30,31
1-Pentyl-3-(4-methyl-1-naphthoyl)indole (15). To a sus-
pension of 0.30 g (1.61 mmol) of 4-methyl-1-naphthoic
acid in 7 mL of CH2Cl2 at 0 ꢂC was added dropwise over
several min 1.02 g (8.05 mmol) of oxalyl chloride. The
reaction mixture was warmed to ambient temperature,
stirred for 1 h, then heated at reflux for 1 h. After cool-
ing, the solvent and excess oxalyl chloride were removed
in vacuo to give a brown residue. This residue was dis-
solved in 7 mL of toluene and 0.36 g (1.93 mmol) of
1-pentylindole in 7 mL of toluene was added. The mix-
ture was cooled to 0 ꢂC and 2.42 mL (2.42 mmol) of
EtAlCl2 (1 M in hexanes) were added. The reaction
mixture was warmed to ambient temperature and stir-
red for 48 h. After quenching with water, the mixture
was extracted with ethylacetate and dried (MgSO 4).
The solvents were removed in vacuo to give a yellow oil
which was purified by chromatography to give 0.24 g
(42%) of 15 as a pale yellow oil: Rf 0.51 (petroleum
The significant affinities of indoles 9–11, 21, 22 and
indene 26, none of which can interact with the receptor
by hydrogen bonding, strongly support the hypothesis
that cannabimimetic indoles interact with the CB1
receptor primarily by aromatic stacking interactions.
Not only do the molecular modeling and receptor
docking studies agree with this conclusion, but they
provide an explanation for the observation that 2-
methylindole analogues JWH-196 (12), JWH-194 (13)
and JWH-197 (14) have greatly attenuated affinities for
the CB1 receptor. The receptor docking studies also
provide an explanation for the enhanced affinities of
naphthoylindoles, such as 5, 15, 16 and 23–25, in which
hydrogen bonding involving the carbonyl group may
augment binding due to aromatic stacking.
1
ether/ether, 19:1); H NMR (300 MHz, CDCl3) d 0.85
(t, J=7.1 Hz, 3H), 1.16–1.30 (m, 4H), 1.71–1.81 (m,

J. W. Huffman et al. / Bioorg. Med. Chem. 11 (2003) 539–549
545
2H), 2.74 (s, 3H), 4.01 (t, J=7.2 Hz, 2H), 7.29–7.55 (m,
8H), 8.05 (d, J=8.3 Hz, 1H), 8.25 (d, J=8.3 Hz, 1H),
8.49–8.52 (m, 1H); 13C NMR (75.5 MHz, CDCl3) d
13.8, 19.7, 22.1, 28.8, 29.4, 47.0, 109.9, 117.5, 122.7,
122.8, 123.4, 125.2, 125.8, 126.1, 126.3, 126.5, 130.8,
132.8, 136.6, 136.9, 137.8, 192.1; HRMS calcd for
C25H25NO: 355.1936; found: 355.1933.
added a solution of 4-methyl-1-naphthoyl chloride,
prepared as described above, in 2 mL of CH2Cl2, and
the reaction mixture was stirred at ambient temperature
for 2 h. The reaction was quenched with water, extrac-
ted with ethylacetate and dried (MgSO ). The solvents
4
were removed in vacuo to give a yellow oil which was
purified by flash chromatography (petroleum ether/
ether, 19:1) to give 0.040 g (57%) of indole 18 as a pale
yellow oil: Rf 0.46 (petroleum ether/ether, 1:1); 1H
NMR (300 MHz, CDCl3) d 0.87 (t, J=7.0 Hz, 3H),
1.36–1.38 (m, 4H), 1.76–1.81 (m, 2H), 2.48 (s. 3), 2.77
(s., 3H), 4.10 (t, J=7.6 Hz, 2H), 6.96–7.01 (m, 1H),
7.14–7.34 (m, 5H), 7.41–7.56 (m, 3H), 8.07 (d,
J=8.4 Hz, 1H), 8.14 (d, J=8.4 Hz, 1H); 13C NMR
(75.5 MHz, CDCl3) d 12.5, 13.9, 19.8, 22.3, 29.1, 29.3,
43.3, 109.3, 115.0, 121.3, 121.7, 122.1, 124.2, 125.8,
126.0, 126.3, 126.4, 127.1, 130.5, 132.9, 136.0, 136.7,
138.9, 145.3, 193.6; MS (EI) m/z 369 (90), 354 (95), 207
(100), 169 (70); HRMS calcd for C26H27NO: 369.2093;
found: 369.2096.
1-Pentyl-1H-indol-3-yl-(4-methyl-1-naphthyl)methane (10).
Reduction of 0.23 g (0.65 mmol) of indole 15 by the
procedure described above for the preparation of 9 gave
0.15 g (68%) of 10 as a yellow oil: Rf 0.70 (petroleum
1
ether/ether, 1:1); H NMR (300 MHz, CDCl3) d 0.81 (t,
J=7.1 Hz, 3H), 1.12–1.27 (m, 4H), 1.63–1.72 (m, 2H),
2.66 (s, 3H), 3.90 (t, J=7.1 Hz, 2H), 4.49 (s, 2H), 6.54
(s, 1H), 7.05–7.26 (m, 5H), 7.38–7.50 (m, 2H), 7.61 (d,
J=7.8 Hz, 1H), 8.00 (d, J=8.3 Hz, 1H), 8.10 (d,
J=8.1 Hz, 1H); 13C NMR (75.5 MHz, CDCl3) d 13.1,
19.4, 22.2, 28.9, 29.0, 29.9, 46.1, 109.3, 113.7, 118.6,
119.2, 121.3, 124.6, 125.0, 125.2, 125.3, 126.3, 126.5,
127.9, 132.2, 132.6, 132.9, 135.0, 136.3; IR (neat) 2956,
2932, 2867, 1460, 1364 cmꢁ1; MS (EI) m/z 341 (100), 326
(13), 284 (35); HRMS calcd for C25H27N: 341.2144;
found: 341.2150.
2-Methyl-1-pentyl-1H-indol-3-yl-(4-methyl-1-naphthyl)-
methane (13). Reduction of 0.040 g (0.11 mmol) of
indole 18 by the procedure described above for the pre-
paration of 9 gave 0.017 g (43%) of 13 as a yellow oil: Rf
1
1-Pentyl-1H-indol-3-yl-(4-methoxy-1-naphthyl)methane (11).
Reduction of 0.186 g (0.50 mmol) of indole 16 by the
procedure described above for the preparation of 9 gave
0.127 g (68%) of 11 as a pale yellow oil: Rf 0.47 (petro-
0.52 (petroleum ether/ether, 1:1); H NMR (300 MHz,
CDCl3) d 0.84 (t, J=6.8 Hz, 3H), 1.29–1.31 (m, 4H),
1.70 (m, 2H), 2.24 (s, 3H), 2.57 (s, 3H), 4.03 (t,
J=7.5 Hz, 2H), 4.44 (s, 2H), 6.81 (d, J=7.2 Hz, 1H),
6.88–6.93 (m, 1H), 7.01–7.09 (m, 2H), 7.22–7.28 (m,
3H), 7.45–7.52 (m, 2H), 7.95–7.98 (m, 1H), 8.20–8.24
(m, 1H); 13C NMR (75.5 MHz, CDCl3) d 10.4, 14.0,
19.4, 22.5, 27.1, 29.2, 30.0, 43.3, 108.4, 118.4, 118.7,
120.4, 124.0, 124.8, 125.2, 125.4, 126.4, 128.4, 132.2,
132.8, 133.6, 134.9; MS (EI) m/z 355 (100), 340 (90), 298
(50); HRMS calcd for C26H29N: 355.2309; found:
355.2308.
1
leum ether/ether, 9:1); H NMR (300 MHz, CDCl3) d
0.81 (t, J=7.2 Hz, 3H), 1.15–1.28 (m, 4H), 1.65–1.77
(m, 2H), 3.95 (t, J=7.2 Hz, 2H), 3.98 (s, 3H), 4.45 (s,
2H), 6.56 (s, 1H), 6.72 (d, J=7.8 Hz, 1H), 7.09 (t,
J=6.9 Hz, 1H), 7.23–7.32 (m, 3H), 7.41–7.48 (m, 2H),
7.62 (d, J=7.9 Hz, 1H), 8.00–8.03 (m, 1H), 8.29–8.34
(m, 1H); 13C NMR (75.5 MHz, CDCl3) d 13.9, 22.3,
28.5, 29.0, 29.9, 46.1, 55.4, 103.4, 109.3, 114.0, 118.6,
119.2, 121.3, 122.3, 124.3, 124.8, 125.9, 126.2, 126.5,
127.9, 128.8, 132.9, 136.4; MS (EI) m/z 357 (100), 342
(10), 326 (22), 300 (24); HRMS calcd for C25H27NO:
357.2093; found: 357.2088.
2-Methyl-1-pentyl-3-(4-methoxy-1-naphthoyl)indole (19).
Acylation of 0.20 g (1.0 mmol) of 2-methyl-1-pentyl-
indole with 4-methoxynaphthoyl chloride, prepared
from 0.40 g (1.98 mmol) of the corresponding carboxylic
acid, was carried out by the procedure described above
for the synthesis of indole 18. After purification by flash
chromatography (petroleum ether/ether, 19:1) there was
obtained 0.17 g (45%) of 19 as a pale yellow oil: Rf 0.36
(petroleum ether/ether, 1:1); 1H NMR (300 MHz,
CDCl3) d 0.91 (t, J=6.7 Hz, 3H), 1.36–1.43 (m, 4H),
1.72–1.88 (m, 2H), 2.50 (s, 3H), 4.05 (s, 3H), 4.11 (t,
J=7.5 Hz, 2H), 6.79 (d, J=8.0 Hz, 1H), 6.98–7.03 (m,
1H), 7.14–7.19 (m, 1H), 7.25–7.31 (m, 2H), 7.58 (d,
J=8.0 Hz, 1H), 8.26–8.36 (m, 2H); 13C NMR
(75.5 MHz, CDCl3) d 12.5, 13.9, 22.4, 29.1, 29.4, 43.3,
55.6, 102.6, 109.3, 115.3, 121.2, 121.5, 121.9, 125.5,
127.3, 127.5, 128.4, 131.9, 132.4, 136.0, 144.7, 157.2,
193.1; IR (Nujol) 1626, 1592 cmꢁ1; MS (EI) m/z 385
(100), 370 (98), 328 (25); HRMS calcd for C26H27NO2:
385.2042; found: 385.2043.
2-Methyl-1-pentyl-1H-indol-3-yl-(1-naphthyl)methane (12).
Reduction of 0.17 g (0.48 mmol) of indole 17 by the
procedure described above for the preparation of 9 gave
0.069 g (42%) of 12 as a yellow oil: Rf 0.71 (petroleum
1
ether/ether, 1:1); H NMR (300 MHz, CDCl3) d 0.91 (t,
J=6.8 Hz, 3H), 1.36–1.38 (m, 4H), 1.75–1.80 (m, 2H),
2.31 (s, 3H), 4.11 (t, J=7.4 Hz, 2H), 4.54 (s, 2H), 6.96–
7.01 (m, 2H), 7.11–7.17 (m, 1H), 7.23–7.35 (m, 3H),
7.47–7.58 (m, 2H), 7.68 (d, J=8.2 Hz, 1H), 7.86–7.89
(m, 1H), 8.27 (d, J=8.2 Hz, 1H); 13C NMR (75.5 MHz,
CDCl3) d 10.4, 14.0, 22.5, 27.2, 29.2, 30.0, 43.4, 108.3,
108.8, 118.4, 118.7, 120.5, 123.5, 125.3, 125.8, 126.4,
128.4, 128.7, 132.2, 133.7, 136.1, 136.8; MS (EI) m/z 341
(100), 326 (30), 284 (45); HRMS calcd for C25H27N:
341.2142; found: 341.2144.
2-Methyl-1-pentyl-3-(4-methyl-1-naphthoyl)indole (18).
To a solution of 0.38 g (0.19 mmol) of 2-methyl-1-pen-
tylindole in 3 mL of CH2Cl2 at 0 ꢂC was added 0.29 mL
(0.29 mmol) of 1 M Et2AlCl in hexanes and the mixture
was stirred for 30 min. To this deep red solution was
2-Methyl-1-pentyl-1H-indol-3-yl-(4-methoxy-1-naphthyl)-
methane (14). Reduction of 0.080 g (0.21 mmol) of
indole 19 by the procedure described above for the pre-
paration of 9 gave 0.035 g (45%) of 14 as a yellow oil: Rf

546
J. W. Huffman et al. / Bioorg. Med. Chem. 11 (2003) 539–549
1
0.48 (petroleum ether/ether, 1:1); H NMR (300 MHz,
CDCl3) d 0.92 (t, J=6.7 Hz, 3H), 1.36–1.39 (m, 4H),
1.72–1.88 (m, 2H), 2.32 (s, 3H), 3.93 (s, 3H), 4.11 (t,
J=7.4 Hz, 2H), 4.45 (s, 2H), 6.60 (d, J=8.0 Hz, 1H),
6.88 (d, J=8.0 Hz, 1H), 6.96–7.01 (m, 1H), 7.11–7.16
(m, 1H), 7.30–7.35 (m, 2H), 7.48–7.59 (m, 2H), 8.20 (d,
J=7.7 Hz, 1H), 8.32 (d, J=7.7 Hz, 1H); 13C NMR
(75.5 MHz, CDCl3) d 13.9, 22.3, 28.5, 29.1, 29.9, 46.1,
55.4, 103.4, 109.3, 114.0, 118.6, 119.2, 121.3, 122.3,
124.3, 124.8, 126.2, 126.3, 126.5, 128.0, 128.8, 132.9,
136.3, 154.3; MS (EI) m/z 371 (100), 356 (53), 340 (15);
HRMS calcd for C26H29NO: 371.2249; found: 371.2253.
(EI) m/z 398 (10), 141 (10), 100 (100); HRMS calcd for
C26H26N2O2: 398.1990; found: 398.1990.
1-[2-(4-Morpholino)ethyl]-1H-indol-3-yl-(4-methyl-1-nap-
hthyl)methane (21). Reduction of 0.035 g (0.09 mmol) of
indole 24 by the procedure described above for the pre-
paration of 9 gave 0.014 g (41%) of 21 as a pale yellow
1
oil: Rf 0.37 (ether); H NMR (300 MHz, CDCl3) d 2.37
(t, J=4.6 Hz, 4H), 2.63 (t, J=6.9 Hz, 2H), 2.69 (s, 3H),
3.56 (t, J=4.6 Hz, 4H), 4.10 (t, J=6.9 Hz, 2H), 6.60 (s,
1H), 7.09–7.14 (m, 1H), 7.20–7.26 (m, 4H), 7.32 (d,
J=8.2 Hz, 1H), 7.41–7.54 (m, 2H), 7.65 (d, J=7.9 Hz,
1H), 8.04 (d, J=8.2 Hz, 1H), 8.10 (d, J=7.9 Hz, 1H);
13C NMR (75.5 MHz, CDCl3) d 19.6, 29.0, 43.9, 53.9,
58.1, 66.9, 109.2, 114.5, 119.0, 119.3, 121.6, 124.8, 125.1,
125.5, 126.4, 126.9, 128.0, 132.3, 132.9, 133.1, 135.1,
136.4; MS (EI) m/z 384 (10), 155 (5), 100 (100); HRMS
calcd for C26H28N2O: 384.2202; found: 384.2202.
1-[2-(4-Morpholino)ethyl]-3-(1-naphthoyl)indole (23). Acyl-
ation of 0.50 g (2.17 mmol) of 1-[2-(4-Morpholino)-
ethyl]-indole with 0.83 (4.34 mmol) of 1-naphthoyl
chloride was carried out by the procedure described
above for the synthesis of indole 18. After purification
by flash chromatography (petroleum ether/ether, 1:1)
there was obtained 0.40 g (48%) of 23 as a pale yellow
1-[2-(4-Morpholino)ethyl]-3-(4-methoxy-1-naphthoyl)in-
dole (25). Acylation of 0.29 g (1.24 mmol) of 1-[2-(4-
morpholino)ethyl]-indole with 4-methoxynaphthoyl
chloride, prepared from 0.50 g (2.48 mmol) of the cor-
responding carboxylic acid, was carried out by the pro-
cedure described above for the synthesis of indole 18.
After purification by flash chromatography (petroleum
ether/ether, 3:1) there was obtained 0.21 g (42%) of 25 as
1
oil: Rf 0.16 (ether); H NMR (300 MHz, CDCl3) d 2.39
(t, J=4.6 Hz, 4H), 2.70 (t, J=6.3 Hz, 2H), 3.55 (t,
J=4.6 Hz, 4H), 4.16 (t, J=6.3 Hz, 2H), 7.27–7.55 (m,
7H), 7.66 (d, J=6.8 Hz, 1H), 7.90–7.98 (m, 2H), 8.18 (d,
J=8.3 Hz, 1H), 8.52–8.54 (m, 1H); 13C NMR
(75.5 MHz, CDCl3) d 44.0, 53.5, 57.4, 66.7, 109.6, 117.9,
122.9, 123.6, 124.4, 125.6, 126.2, 126.7, 128.1, 129.9, 131.0,
137.2, 138.9, 192.0; IR (Nujol) 1617, 1519, 1380 cmꢁ1; MS
(EI) m/z 384 (70), 155 (90), 100 (100); HRMS calcd for
C25H24N2O2: 384.1832; found: 384.1833.
1
a pale yellow oil: Rf 0.21 (ether); H NMR (500 MHz,
CDCl3) d 2.40 (t, J=4.4 Hz, 4H), 2.69 (t, J=6.5 Hz, 2H),
3.59 (t, J=4.4 Hz, 4H), 4.05 (s, 3H), 4.16 (t, J=6.5 Hz,
2H), 6.81 (d, J=7.9 Hz, 1H), 7.27–7.33 (m, 3H), 7.43–
7.45 (m, 3H), 7.59 (d, J=7.9 Hz, 1H), 8.20–8.28 (m, 2H),
8.40–8.43 (m, 1H); 13C NMR (125.8 MHz, CDCl3) d 44.2,
53.7, 55.8, 57.6, 66.9, 102.1, 109.7, 117.9, 122.1, 122.8,
123.0, 123.6, 125.8, 125.9, 127.5, 127.9, 137.0, 138.4,
157.1, 191.9; MS (EI) m/z 414 (80), 369 (60), 314 (25), 100
(100); calcd for C26H26N2O3: 414.1949; found: 414.1943.
1-[2-(4-Morpholino)ethyl]-1H-indol-3-yl-1-naphthylme-
thane (20). Reduction of 0.200 g (0.52 mmol) of indole
23 by the procedure described above for the preparation
of 9 gave 0.077 g (40%) of 20 as a yellow oil: Rf 0.18
(ether); 1H NMR (500 MHz, CDCl3)
d 2.33 (t,
J=4.3 Hz, 4H), 2.59 (t, J=6.8 Hz, 2H), 3.53 (t,
J=4.3 Hz, 4H), 4.07 (t, J=6.8 Hz, 2H), 6.59 (s, 1H),
7.10–7.49 (m, 8H), 7.65 (d, J=7.9 Hz, 1H), 7.76 (d,
J=7.2 Hz, 1H), 7.86 (d, J=7.2 Hz, 1H), 8.07 (d,
J=7.9 Hz, 1H); 13C NMR (125.8 MHz, CDCl3) d 29.0,
43.9, 53.9, 58.2, 66.9, 109.3, 114.3, 119.1, 119.3, 121.7,
124.6, 125.6, 125.7, 125.9, 126.7, 127.0, 128.1, 128.7,
132.3, 134.0, 136.4, 137.0; MS (EI) m/z 370 (40), 254
(11), 141 (24), 100 (100); HRMS calcd for C25H26N2O:
370.2045; found: 370.2049.
1-[2-(4-Morpholino)ethyl]-1H-indol-3-yl-(4-methoxy-1-
naphthyl)methane
(22).
Reduction
of
0.075 g
(0.18 mmol) of indole 25 by the procedure described
above for the preparation of 9 gave 0.029 g (40%) of 22
as a pale yellow oil: Rf 0.30 (ether); 1H NMR (300 MHz,
CDCl3) d 2.36 (t, J=4.6 Hz, 4H), 2.62 (t, J=6.9 Hz,
2H), 3.57 (t, J=4.6 Hz, 4H), 3.98 (s, 3H), 4.09 (t,
J=6.9 Hz, 2H), 4.44 (s, 2H), 6.59 (s, 1H), 6.73 (d,
J=7.9 Hz, 1H), 7.09–7.14 (m, 1H), 7.19–7.33 (m, 4H),
7.41–7.48 (m, 2H), 7.64 (d, J=7.9 Hz, 1H), 7.98–8.01 (m,
1H), 8.29–8.33 (m, 1H); 13C NMR (75.5 MHz, CDCl3) d
22.3, 28.5, 29.1, 29.9, 46.1, 55.4, 103.4, 109.3, 114.0, 118.6,
119.2, 121.3, 122.3, 124.3, 124.8, 126.2, 126.3, 126.5, 128.0,
132.9, 136.3, 154.2; MS (EI) m/z 400 (5), 100 (100); HRMS
calcd for C26H28N2O2: 400.2151; found: 400.2151.
1-[2-(4-Morpholino)ethyl]-3-(4-methyl-1-naphthoyl)indole
(24). Acylation of 0.25 g (1.10 mmol) of 1-[2-(4-mor-
pholino)ethyl]-indole with 4-methylnaphthoyl chloride,
prepared from 0.40 g (2.20 mmol) of the corresponding
carboxylic acid, was carried out by the procedure
described above for the synthesis of indole 18. After
purification by flash chromatography (petroleum ether/
ether, 3:1) there was obtained 0.37 g (42%) of 24 as a
pale yellow oil: Rf 0.36 (ether); 1H NMR (300 MHz,
CDCl3) d 2.39 (t, J=4.6 Hz, 4H), 2.69 (t, J=6.4 Hz,
2H), 2.77 (s, 3H), 3.56 (t, J=4.6 Hz, 4H), 4.15 (t,
J=6.4 Hz, 2H), 7.34–7.38 (m, 4H), 7.45–7.58 (m, 4H),
8.07 (d, J=8.4 Hz, 1H), 8.23 (d, J=8.4 Hz, 1H), 8.51–
8.54 (m, 1H); 13C NMR (75.5 MHz, CDCl3) d 19.8,
44.1, 53.6, 57.5, 66.8, 109.6, 117.8, 122.8, 123.6, 124.2,
125.1, 126.1, 130.9, 137.8, 136.7, 137.0, 137.6, 138.7; MS
E-1-[1-(1-Naphthalenylmethylene)-1H-inden-3-yl]pentane
(26). To a solution of 0.25 g (1.35 mmol) of 1-(1H-
inden-3-yl)pentane in 7 mL of dry methanol at 0 ꢂC was
added 0.073 g (1.35 mmol) of sodium methoxide in 7 mL
of dry methanol. The mixture was warmed to ambient
temperature and stirred for 20 min. To this mixture was
added 0.21 g (1.35 mmol) of 1-naphthaldeyde and the
reaction mixture was heated at reflux for 18 h. After
dilution with ethanol, the solvent was removed in vacuo

J. W. Huffman et al. / Bioorg. Med. Chem. 11 (2003) 539–549
547
to give a yellow oil. Purification by chromatography (hex-
anꢂes) provided 0.13 g (35%) of 26 as a yellow solid: mp 65–
66 ; 1H NMR (300 MHz, CDCl3) d 0.90 (t 6.8, 3H), 1.25–
1.53 (m, 4H), 1.60–1.82 (m, 2H), 2.61 (t, J=7.6Hz, 2H),
6.58 (s, 1H), 7.26–7.33 (m, 3H), 7.53–7.66 (m, 4H), 7.81–
7.98 (m 4), 8.17 (m, 1H); 13C NMR (75.5 MHz, CDCl3) d
14.2, 22.6, 27.8, 28.1, 32.0, 119.0, 119.2, 122.2, 123.7, 124.8,
125.3, 125.6, 126.2, 127.6, 128.5, 128.7, 129.2, 132.3, 141.5,
143.3, 149.1; IR (neat) 2928, 2860, 1464, 1387cmꢁ1; MS
(EI) m/z 324 (60), 279 (20), 253 (50), 149 (100); calcd for
C25H24: 324.1877; found: 324.1878.
conducted as described elsewhere,33 except whole brain
(rather than cortex only) was used. Displacement curves
were generated by incubating drugs with 1 nM of
[3H]CP-55,940. Nonspecific binding was determined in
the presence of 1 mM CP-55,940. The assays were per-
formed in triplicate, and the results represent the com-
bined data from three individualexperiments. CP-
55,940 and all cannabinoid analogues were prepared by
suspension in assay buffer from a 1 mg/mL ethanolic
stock without evaporation of the ethanol(finalcon-
centration of no more than 0.4%). Displacement IC50
values were originally determined by unweighted least-
squares linear regression of log concentration–percent
displacement data and then converted to Ki values using
the method of Cheng and Prusoff.34
2-Ethyl-1-pentyl-3-(1-naphthoyl)indole (27). To a stirred
solution of 0.65 mL (1.65 mmol) of 2.5 M ethylmagne-
sium bromide in ether, diluted with 1.1 mL of ether, at
0 ꢂC was added dropwise 0.20 g (1.3 mmol) of 2-ethyl-
indole in 1.1 mL of ether. The solution was stirred for
0.5 h at ambient temperature and a solution of 0.22 mL
(1.46 mmol) of 1-naphthoyl chloride in 1 mL of ether
was added dropwise. The reaction mixture was stirred
for 1.5 h, quenched with saturated aqueous NH4Cl, and
stirred untilthe soild was broken up into a fine suspen-
sion. The residue was washed with water and ether, then
suspended in 5 mL of methanol, to which was added
0.4 g of NaOH and 1 mL of water. The mixture was
stirred at ambient temperature for 18 h, the solid was
filtered off and washed with successive portions of
methanol, water and ether. Drying in vacuo at 100 ꢂC
gave 0.31 g (74%) of 2-ethyl-3-(1-naphthoyl)indole as a
viscous oil, which was used in the next step without
further purification: 1H NMR (300 MHz, CDCl3) d 1.07
(t, J=7.5 Hz, 3H), 2.63 (q, J=7.5 Hz, 2H), 6.90 (t,
J=7. 5 Hz, 1H), 7.02–7.10 (m, 2H), 7.37–7.76 (m, 6H),
7.81 (d, J=8.2 Hz, 1H), 8.21 (q, J=8.2 Hz, 2H); 13C
NMR (75.5 MHz, CDCl3) d 13.6, 21.0, 11.6, 113.0,
120.2, 121.5, 122.2, 124.5, 125.0, 125.5, 126.5, 126.9,
127.1, 128.5, 129.5, 133.2, 135.2, 140.7, 151.3, 192.3.
Molecular modeling
1. Conformational analysis. The structures of JWH-081
(16), JWH-098 (19), JWH-185 (11) and JWH-197 (14)
were built in the Spartan molecular modeling program
(V4.1.1; Wavefunction, Inc., Irvine, CA). Each structure
was minimized using the AM1, semi-empiricalmethod.
For each minimized structure, AM1 conformational
searches were then performed for rotation about the
C3–C10 and C10–C20 bonds (see the structure of 5 for
the numbering system).
2. CB1 receptor dockingstudies. Amino acid numbering
system. In the discussion of receptor residues that fol-
lows, the amino acid numbering scheme proposed by
Ballesteros and Weinstein is used.35 In this numbering
system, the most highly conserved residue in each
transmembrane helix (TMH) is assigned a locant of
0.50. This number is preceded by the TMH number and
may be followed in parentheses by the sequence num-
ber. All other residues in a TMH are numbered relative
to this residue. In this numbering system, for example,
the most highly conserved residue in TMH 2 of the CB1
receptor is D2.50(163). The residue that immediately
precedes it is A2.49(162).
To a solution of 0.40 g (1.3 mmol) of 2-ethyl-3-(1-naph-
thoyl)indole in 3.0 mL of DMSO was added 1.2 g of
powdered KOH. The reaction was stirred at ambient
temperature and 1.35 mL (11.0 mmol) of 1-bromo-
pentane were added slowly. The solution was stirred at
85 ꢂC for 18 h. After cooling the reaction mixture was
diluted with water, and extracted with three portions of
ethylacetate. The extracts were washed with brine,
dried (MgSO4), and the solvent was removed in vacuo.
Chromatography (petroleum ether/ethyl acetate, 7:1)
gave 0.44 g (89%) of 27 as a yellow oil: 1H NMR
(300 MHz, CDCl3) d 0.92 (t, J=7.0 Hz, 3H), 1.24 (t,
J=7.5 Hz, 3H), 1.37–1.42 (m, 4H), 1.78–1.85 (m, 2H),
3.05 (q, J=7.5 Hz, 2H), 4.12 (t, J=7.5 Hz, 2H), 6.84–
6.93 (m, 2H), 7.10–7.15 (m, 1H), 7.29 (d, J=8.2 Hz,
1H), 7.41–7.58 (m, 3H), 7.89–7.97 (m, 2H), 8.11 (d,
J=8.2 Hz, 1H); 13C NMR (75.5 MHz, CDCl3) d 13.9,
14.0, 19.3, 22.3, 29.1, 29.7, 43.2, 109.6, 113.7, 121.1,
121.6, 122.0, 124.9, 125.5, 125.6, 126.1, 126.7, 127.1,
128.1, 129.8, 130.3, 133.7, 135.9, 140.5, 151.2, 193.1; MS
(EI) m/z 369 (60), 340 (56), 155 (100); HRMS calcd for
C26H27NO: 369.2093; found: 369.2093.
3. Model of inactive state of CB1. A modelof the inac-
tive (R) form of CB1 was created using the 2.8 A crystal
structure of rhodopsin (Rho).36 First, the sequence of
the human CB1 receptor37 was aligned with the
sequence of bovine rhodopsin (Rho) using the same
highly conserved residues as alignment guides that were
used initially to generate our first model of CB1.23 TMH
5 in CB1 lacks the highly conserved proline in TMH 5 of
Rho. The sequence of CB1 in the TMH 5 region was
aligned with that of Rho as described previously using its
hydrophobicity profile.23 Helix ends for CB1 were cho-
sen in analogy with those of Rho;36 TMH 1: N1.28(112)–
R1.61(145); TMH 2: R2.37(150)–H2.68(181); TMH 3:
S3.21(185)–R3.56(220); TMH 4: T4.38(229)–C4.66(257);
TMH 5: H5.34(270)–K5.64(300); TMH 6:R6.28(336)–
K6.62(370); TMH 7: K7.32(376)–S7.57(401); intracel-
lular extension of TMH 7: D7.59(403)–C7.71(415).
4. Preparation of helices. Each helix of the model was
capped by acetamide at its N-terminus and N-methyl-
amide at its C-terminus. Ionizable residues in the first
In vitro pharmacology. CB1 assay. [3H]CP-55,940 (79 Ci/
mmol, KD=690 pM) binding to P2 membranes was

548
J. W. Huffman et al. / Bioorg. Med. Chem. 11 (2003) 539–549
turn of either end of the helix were neutralized, as were
any lipid facing charged residues. Ionizable residues
were considered charged if they appeared anywhere else
in the helix.
These interactions were further classified as ‘tilted-T’
arrangements if 30ꢂ ꢄꢀꢄ90ꢂ and as parallel arrange-
ments for ꢀ<30ꢂ. In interactions where ꢀ=0ꢂ, arrange-
ments were also identified as offset or not offset, as
Hunter et al. have shown that offset parallel stacks are
more energetically favorable.44
5. Model of CB1 active (R*) state. Experimentalresutls
for rhodopsin and the b2-adrenergic receptor document
that upon activation, severalconformationalchanges
ensue. These changes include rigid domain motions in
TMHs 3 and 6, straightening of the proline kink in
TMH 6, as well as rotations in TMHs 3 and 6 that
change the environments of residues 3.41 and 6.47.38ꢁ42
Recent conformationalmemory resutls for TMH 6 in
CB1 have indicated that the TMH 6 proline kink angle
lessens to a value of 21.8ꢂ in the activated state (R*).43
An active (R*) CB1 bundle was created from the inac-
Acknowledgements
The work at Clemson was supported by grant DA03590
to JWH, that at Virginia Commonwealth University by
grant DA03672 to BRM and that at Kennesaw State
University by grant DA03934 to PHR, all from the
NationalInstitute on Drug Abuse.
tive (R) modelof CB by setting the TMH 6 proline
1
References and Notes
kink angle to 21.8ꢂ and by rotating both TMH 3 and 6
counterclockwise (from an extracellular perspective) so
that residues 3.41 and 6.47 changed environ-
ments.40,42,43
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bones to adjust. Stages one and two were repeated with
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2
ꢃ
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