(Aminoalkyl)indole isothiocyanates as potential electrophilic affinity ligands for the brain cannabinoid receptor

Article abstract of DOI:10.1021/jm950932r

A series of (aminoalkyl)indole compounds, naphthalene analogs of pravadoline (1), has been shown to exhibit cannabinoid agonist activities such as antinociception in animals, inhibition of adenylate cyclase in brain membranes, and binding to the cannabinoid receptor. These pravadoline analogs were selected for the preparation of potential electrophilic affinity ligands based on the synthesis of isothiocyanate derivatives. One isothiocyanatonaphthalene derivative (8) displaced [³H]CP-55940 binding to a rat brain P2 membrane preparation with an IC₅₀ of 690 nM, which was 10- fold less potent than the parent molecule (IC₅₀ = 73 nM). Isothiocyanate substitution at various positions on the naphthalene moiety of the desmethyl analog 10 gave compounds that displaced [³H]CP-55940 with IC₅₀ values between 400 and 1000 nM, compared with 46 nM for the parent compound 10. However, 6-isothiocyanato substitution on the indole ring of the desmethyl analog provided isothiocyanate 12 that displaced [³H]CP-55940 binding with an IC₅₀ of 160 nM. After pretreatment of brain membranes with this high- affinity isothiocyanato ligand followed by washing out the ligand, the membranes were depleted of 90% of the cannabinoid receptor binding capacity. Loss of receptor binding capacity was half-maximal at 300 nM of the derivative under standard assay conditions. As a control, pretreatment with the parent compound at concentrations that were 20 times the K(d) failed to alter subsequent binding activity. This study demonstrates that an isothiocyanato (aminoalkyl)indole (12) can behave as an affinity ligand which binds irreversibly to the cannabinoid receptor in brain and which precludes subsequent binding of the cannabinoid ligand [³H]CP-55940.

Full text of DOI:10.1021/jm950932r

J . Med. Chem. 1996, 39, 1967-1974
1967
(Am in oa lk yl)in d ole Isoth iocya n a tes a s P oten tia l Electr op h ilic Affin ity Liga n d s
for th e Br a in Ca n n a bin oid Recep tor
Koichiro Yamada,†, Kenner C. Rice,† J udith L. Flippen-Anderson,‡ Michael A. Eissenstat,§,# Susan J . Ward,§,£
M. Ross J ohnson,†,∇ and Allyn C. Howlett*,|
Laboratory of Medicinal Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, Building 8,
Room B1-23, National Institutes of Health, Bethesda, Maryland 20892, Laboratory for the Structure of Matter,
Naval Research Laboratory, Code 6030, Washington, D.C., 20375, Department of Medicinal Chemistry,
Sanofi Research Division, Sanofi-Winthrop, Collegeville, Pennsylvania 19426-0900, Trimeris, P.O. Box 13963,
Research Triangle Park, North Carolina 27709, and Department of Pharmacological and Physiological Science,
Saint Louis University School of Medicine, St. Louis, Missouri 63104
Received December 21, 1995X
A series of (aminoalkyl)indole compounds, naphthalene analogs of pravadoline (1), has been
shown to exhibit cannabinoid agonist activities such as antinociception in animals, inhibition
of adenylate cyclase in brain membranes, and binding to the cannabinoid receptor. These
pravadoline analogs were selected for the preparation of potential electrophilic affinity ligands
based on the synthesis of isothiocyanate derivatives. One isothiocyanatonaphthalene derivative
(8) displaced [3H]CP-55940 binding to a rat brain P2 membrane preparation with an IC50 of
690 nM, which was 10-fold less potent than the parent molecule (IC50 ) 73 nM). Isothiocyanate
substitution at various positions on the naphthalene moiety of the desmethyl analog 10 gave
compounds that displaced [3H]CP-55940 with IC50 values between 400 and 1000 nM, compared
with 46 nM for the parent compound 10. However, 6-isothiocyanato substitution on the indole
ring of the desmethyl analog provided isothiocyanate 12 that displaced [3H]CP-55940 binding
with an IC50 of 160 nM. After pretreatment of brain membranes with this high-affinity
isothiocyanato ligand followed by washing out the ligand, the membranes were depleted of
90% of the cannabinoid receptor binding capacity. Loss of receptor binding capacity was half-
maximal at 300 nM of the derivative under standard assay conditions. As a control,
pretreatment with the parent compound at concentrations that were 20 times the Kd failed to
alter subsequent binding activity. This study demonstrates that an isothiocyanato (aminoalkyl)-
indole (12) can behave as an affinity ligand which binds irreversibly to the cannabinoid receptor
in brain and which precludes subsequent binding of the cannabinoid ligand [3H]CP-55940.
Research conducted at Sterling Winthrop Research
Institute demonstrated that a series of analogs of
pravadoline, termed (aminoalkyl)indoles, act as can-
nabinoid receptor agonists.1-3 Pravadoline (1), the first
compound in this series, exhibited potent antinocicep-
tive activity and inhibited brain cyclooxygenase as
expected for a nonsteroidal antiinflammatory agent.
However, pravadoline’s spectrum of activities deviated
from classical nonsteroidal antiinflammatory agents in
that it exhibited no gastrointestinal cytotoxicity and was
not antiinflammatory.4 The (aminoalkyl)indole analogs
of pravadoline inhibited the electrically evoked contrac-
tion in a mouse vas deferens smooth muscle preparation
in a parallel fashion to their ability to behave as
antinociceptive agents.4-7 Replacing the monocyclic
4-methoxybenzoyl group of 1 with a naphthoyl moiety
to provide 2 increased the potency by nearly 80-fold in
the mouse vas deferens and 10-fold in the antinocicep-
tion determination.2,7
The antinociceptive action of the (aminoalkyl)indole
compounds can be attributed to an interaction with the
cannabinoid receptor in brain. Specific binding of [3H]-
WIN-55212-2 in brain membranes was displaced not
only by (aminoalkyl)indole analogs but also by ∆9-THC
and other central nervous system (CNS)-active cannab-
inoid compounds.8 (Aminoalkyl)indole analogs were
able to displace [3H]CP-55940 binding to the cannab-
inoid receptor in brain membranes6 but failed to dis-
place radioligands selective for at least 20 other neu-
roreceptors.2 Potency ratios for both classes of com-
pounds were consistent with their cannabimimetic
activities reported in animals and the mouse vas def-
erens assay. (Aminoalkyl)indole agonists inhibited ade-
† National Institute of Diabetes and Digestive and Kidney Diseases.
‡ Naval Research Laboratory.
§ Sanofi-Winthrop.
∇ Trimeris.
| Saint Louis University School of Medicine.
Current address: Tanabe Seiyaku Co., Ltd., Lead Optimization
Research Laboratory, No. 2-50, 2-Chome, Kawagishi, Toda-shi, Saitama-
ken, J apan.
£
Current address: Wyeth-Ayerst Research, P.O. Box 8299, Phila-
delphia, PA 19101.
#
Current address: Structural Biochemistry Program, NCI-Freder-
ick Cancer Research and Development Center, SAIC-Frederick, P.O.
Box B, Frederick, MD 21702-1201.
X Abstract published in Advance ACS Abstracts, April 1, 1996.
S0022-2623(95)00932-0 CCC: $12.00 © 1996 American Chemical Society

1968 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 10
Yamada et al.
Sch em e 1a
Sch em e 2a
a
Reagents: (a) AlCl3;1 (b) Fe, HCl-aqueous EtOH; (c) CSCl2,
AcOEt, aqueous NaHCO3.
nylate cyclase in rat striatal or cerebellar membranes7
and reduced Ca2+ conductance of an N-type Ca2+ chan-
nel in a neuronal cell,9 both responses being attributed
to a receptor-G protein mechanism.
The cannabinoid receptor subtype CB1 was cloned
and demonstrated to have an amino acid sequence
consistent with a tertiary structure typical of the seven-
transmembrane-spanning proteins that are coupled to
G proteins.10,11 CB1 mRNA is found in abundance in
brain tissue. The CB2 subtype has been demonstrated
in spleen tissue; however, CB2 mRNA could not be
found in brain tissue by either Northern analysis or in
situ hybridization studies.12 Several recent reviews
have described the pharmacology, biochemistry, and
CNS distribution of the CB1 receptor in brain.13-15 The
present report describes electrophilic affinity ligands
that have been developed from a potent (aminoalkyl)-
indole agonist. One ligand from this series exhibits a
high affinity for the cannabinoid receptor and binds
irreversibly to the receptor at a position in the protein
that precludes subsequent interaction with the cannab-
inoid ligand [3H]CP-55940.
a
Reagents: (a) 1-naphthoyl chloride, AlCl3;1 (b) 70% HNO3,
Ac2O; (c) Fe, HCl-aqueous EtOH; (d) CSCl2, AcOEt, aqueous
NaHCO3; (e) AlCl3,1 4-nitro-1-naphthoyl chloride.
followed by treatment with thiophosgene. The structure
of 12 was determined by single-crystal X-ray analysis,
and the data are available as Supporting Information.
Isothiocyanate 15 was similarly prepared from nitro
compound 13.
Using a modification of the published procedure,
3-nitro-1,8-naphthalic anhydride (16) was converted to
a mixture of esters 17 and 18 which were separated by
a combination of crystallization and chromatography
(Scheme 3). Pure 17 and 18 were then converted to the
corresponding acid chlorides 19 and 20 as indicated.
Reaction of these compounds with indole 9 gave the
corresponding nitro compounds 21 and 23 which were
converted to the isothiocyanates 22 and 24 by the usual
procedure. We were unsuccessful in our attempt to
place the isothiocyanate at C-8 in the naphthyl nucleus.
Previously synthesized compounds1 2 and 10, the parent
compounds of the isothiocyanates prepared in this
study, were resynthesized for the binding affinity stud-
ies.
Ch em istr y
Since there was no a priori way of determining where
an isothiocyanate moiety should be located in an (ami-
noalkyl)indole to enable its interaction with a bionu-
cleophile such as amino or sulfhydryl in the receptor,
compounds were prepared in which the moiety was
situated in different spatial areas. Five potential elec-
trophilic affinity ligands were prepared, as shown in
Schemes 1-3.
Treatment of 4-nitro-1-naphthoic acid (3)16 with thio-
nyl chloride gave acid chloride 4 which was reacted with
indole 51 under previously described conditions1 to give
6, the 4-nitronaphthyl derivative of pravadoline (1).
Reduction of the 4-nitro group of 6 gave amine 7 which
was then converted to the isothiocyanate 8 as shown in
Scheme 1.
Indole 91 was used to prepare the other four isothio-
cyanates, 12, 15, 22, and 24, where the isothiocyanate
group was placed at various positions on the naphthyl
subunit for compounds 15, 22, and 24 (at C-4, C-6, and
C-3, respectively) or on the indole nucleus for 12 (at C-6)
(Schemes 2 and 3). Nitration of 101 gave 6-nitroindole
derivative 11 which was converted to isothiocyanate 12
by the usual procedure of reduction to the amine
P h a r m a cology
Compound 2, the 1-naphthoyl derivative of pravado-
line, lacks the ability to inhibit cyclooxygenase but is
>30-fold more potent than pravadoline in the mouse vas
deferens smooth muscle assay.1,5 This compound ex-
hibited a Ki for the cannabinoid receptor of 18 nM
(coefficient of variation 2.3%) (Figure 1). The slope
factor was 1.1, consistent with single-site displacement
of [3H]CP-55940. The 4-isothiocyanato derivative 8
exhibited a 10-fold loss of potency for the receptor.
Inasmuch as covalent binding would have taken place
during the incubation, the reported IC50 would result

Affinity Ligands for Brain Cannabinoid Receptor
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 10 1969
Sch em e 3a
F igu r e 2. Affinity of 1-naphthyl[1-[2-(4-morpholinyl)ethyl]-
1H-indol-3-yl]methanone (10) and its isothiocyanato deriva-
tives at the indole 6- (12) and naphthyl C-4- (15), C-6- (22),
and C-3- (24) positions for the cannabinoid receptor in brain
membranes. [3H]CP-55940 was present at 0.25 nM. Data
points are the mean ( standard error from four individual
experiments, each performed in triplicate.
a
Reagents: (a) NaOH, (b) Hg(OAc)2/aqueous AcOH, reflux 4
days, HCl;16 (c) SOCl2; (d) MeOH; (e) aqueous NaOH, MeOH,
reflux then HCl; (f) AlCl3;1 (g) Fe, HCl-aqueous EtOH; (h) CSCl2,
AcOEt/aqueous NaHCO3.
F igu r e 3. Agonist ability of isothiocyanato (aminoalkyl)-
indoles to inhibit hormone-stimulated adenylate cyclase activ-
ity in N18TG2 membranes. Membranes were incubated as
described in the text with 0.6 µM secretin to stimulate
adenylate cyclase, 100 µM rolipram as a phosphodiesterase
inhibitor and the indicated concentrations of compounds 2 (b),
8 (O), 10 ([), 12 (4), 15 (2), 22 (9), 24 (0), and desacetyllevo-
nantradol (DALN). Data points are the mean ( standard error
from three individual experiments, except for the 10 µM
concentrations of compounds 15, 22, and 24 which are from a
single experiment.
F igu r e 1. Affinity of 1-naphthyl[2-methyl-1-[2-(4-morpholi-
nyl)ethyl]-1H-indol-3-yl]methanone (2) (b) and (4-isothiocy-
anato-1-naphthyl)[2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-
3-yl]methanone (8) (9) for the cannabinoid receptor in brain
membranes. [3H]CP-55940 was present at 1.1 nM. Data points
are the mean ( standard error from five (compound 2) and
three (compound 8) individual experiments, each performed
in triplicate.
potent, exhibiting only a 3.5-fold loss of potency com-
pared with the parent compound.
Each of the isothiocyanato (aminoalkyl)indole com-
pounds behaved as agonists in inhibiting adenylate
cyclase activity in the neuroblastoma model membrane
system (Figure 3). Parent compounds 2 and 10 exhib-
ited mean (95% confidence intervals) EC50 values of 370
(220-630) and 275 (175-440) nM, respectively. The
most potent of the isothiocyanato derivatives was
compound 12, exhibiting an EC50 of 1.1 (0.67-1.8) µM,
followed in potency by compound 8, exhibiting an EC50
of 3.0 (1.1-7.9) µM. The other three analogs failed to
attain an inhibition of adenylate cyclase equivalent to
one-half that of the maximal attained by 1 µM des-
acetyllevonantradol (34% inhibition) at <10 µM con-
centrations. Therefore EC50 values were not determined
because of excessive vehicle concentrations that would
be required in the assay mixture. It is of interest that
3 µM compound 12 failed to inhibit adenylate cyclase
activity when assayed in a reaction mixture containing
50 mM HEPES buffer, 1 mM EDTA, and 0.1 mg/mL
bovine serum albumin, reagents which may provide a
in an underestimate of the Ki because the off rate would
approach infinity for those covalently bound sites.
Thus, a Ki cannot be calculated.
Compound 10 lacks the methyl group at the 2-position
on the indole moiety of 2. It also fails to inhibit
cyclooxygenase but is about 2-fold more potent than
compound 2 at inhibiting mouse vas deferens contrac-
tions.1,7 The Ki for displacement of [3H]CP-55940 from
the cannabinoid receptor was 29 nM (coefficient of
variation 3.3%), with a slope factor of 1.2 (Figure 2).
Each of its isothiocyanato derivatives suffered a loss of
affinity for the cannabinoid receptor in brain. Consis-
tent with the observation for the methylated pair 2 and
8, substitution on the naphthoyl moiety (compounds 15,
22, and 24) resulted in a 10-20-fold loss of apparent
affinity for the receptor. The isothiocyanato derivative
extending from the indole (compound 12) was more

1970 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 10
Yamada et al.
F igu r e 5. Dose dependency of the covalent binding of isothio-
cyanate 12 to the cannabinoid receptor. Rat brain membranes
were incubated at 30 °C for 60 min with vehicle (O), or 300
nM (b) or 3 µM (9) compound 12. After sedimentation and
resuspension of the membranes, binding of 0.175 nM [3H]CP-
55940 to 50 µg of membrane protein was assessed. The data
are the mean ( standard error of the triplicates within a single
representative experiment.
F igu r e 4. Irreversible decrease in specific binding of [3H]CP-
55940 after pretreatment of rat brain membranes with the
isothiocyanate 12 but not 10. Pretreatment of membranes with
either 1 µM 12 (solid) or 10 (hatched) was performed at 30 °C
for 60 min. Controls included parallel incubations of mem-
branes with vehicle (0.05% â-cyclodextrin plus 0.01% dimethyl
sulfoxide) at either 30 °C (stipled) or 0 °C (open). After washing
away of the compounds by sedimentation, cannabinoid receptor
binding was quantitated. Data bars are the mean ( standard
error of the total binding and the nonspecific binding (not
displaced by 0.1 µM desacetyllevonantradol) from three indi-
vidual experiments, each performed in triplicate.
Discu ssion
The active (aminoalkyl)indoles exhibit virtually the
same spectrum of biological activity as do the classical
cannabinoid compounds, including antinociceptive ac-
tivity in rodents and inhibition of the electrically
induced twitch response in the mouse vas deferens.4,5,7,17
The potency order of several (aminoalkyl)indole analogs
in the Martin multiparameter mouse model was con-
sistent with that in the mouse vas deferens assay, with
the exception of the test for hypothermia.17 Active
(aminoalkyl)indole analogs generalized to ∆9-THC in
drug discrimination studies in rats.17 Competitive
interactions occur in heterologous competition radioli-
gand binding assays using either [3H]CP-55940 or [3H]-
WIN-55212-2 for binding to the cannabinoid receptor
in the brain (predominantly, if not entirely, CB1).6,8
Thus, it is accepted that both cannabinoid and (ami-
noalkyl)indole compounds act at the same receptor in
the brain to evoke their biological responses.
One question that is pertinent is whether these two
classes of compounds interact with the same site(s) on
the cannabinoid receptor. Structure-activity relation-
ship studies have been extensive for cannabinoid
ligands.15,18-20 It has been concluded that a site for
hydrophobic interaction is of major importance to the
receptor interaction with bicyclic and classical cannab-
inoid structures.21 In addition, functionalities exist on
both bicyclic and classical cannabinoid structures for
hydrogen-bonding interactions via the phenolic hydroxyl
and a hydroxyl generated by metabolism of ∆9-THC on
the cyclohexenyl ring. An additional site for potential
hydrogen-bonding interactions was elucidated within
the series of AC-bicyclic and ACD-tricyclic cannab-
inoids.22 It is not obvious that homologous ligand-
receptor interactions occur with the (aminoalkyl)indole
structures. The amino acids that interact with the
cannabinoid hydrophobic or hydrogen-bonding sites may
or may not be the same as those that interact with
critical moieties on the (aminoalkyl)indole structures.
Occlusion of access by the cannabinoid radioligand to
its binding site(s) may result from occupancy by the
(aminoalkyl)indole ligand of a region in space that
includes one or more of the critical interaction sites. The
irreversible depletion of binding sites for [3H]CP-55940
source of nucleophiles in vast excess of the number of
cannabinoid receptors present in the mixture.
Compound 12 was tested for its ability to bind
irreversibly to the cannabinoid receptor in rat brain
membranes (Figure 4). Membranes incubated with 1
µM compound 12, a concentration that was 6-fold
greater than the IC50 for displacing [3H]CP-55940 under
similar conditions in the ligand binding assay, resulted
in a 70% loss of specific binding of [3H]CP-55940
(significantly different at p ) 0.01). No effect on
nonspecific binding was noted. Preincubation of mem-
branes at 0-4°C for 60 min showed no difference in the
ability of [3H]CP-55940 to bind specifically to the
cannabinoid receptor, indicating that heat denaturation
was not a factor in the experiment. There was a 15%
difference (not significant at p ) 0.05) in specific binding
of [3H]CP-55940 between control membranes and those
preincubated with 1 µM compound 10. This concentra-
tion was 30-fold the Ki and likely to interact with 97%
of the receptors after equilibration. The small decrease
in specific binding of [3H]CP-55940 could indicate an
incomplete removal of the ligand from the membranes
prior to assay for cannabinoid receptors.
The depletion of cannabinoid receptor binding was
dependent upon the concentration of compound 12 in
the incubation mixture (Figure 5). Preincubation with
3 µM compound 12 resulted in the loss of 70% of the
specific binding to cannabinoid receptors in the brain
membranes. At 300 nM, a concentration that is only 2
times the IC50 for displacing [3H]CP-55940, compound
12 eliminated 30% of the specific binding. No change
in [3H]CP-55940 binding was observed upon preincu-
bation with 100 nM compound 12 (data not shown). No
change in nonspecific binding was noted. The IC50
values for desacetyllevonantradol were 1.1, 2.1, and 3.5
nM, respectively, suggesting a trend toward incremen-
tally decreasing affinities for agonist ligands by the
remaining receptors. However, these differences failed
to reach statistical significance at the 5% level.

Affinity Ligands for Brain Cannabinoid Receptor
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 10 1971
that membranes were suspended in 20 mM potassium dieth-
ylmalonate, pH 7.5, plus 3 mM MgCl2. An aliquot of 30 µg of
membrane protein was incubated for 1 h at 30 °C with [3H]-
CP-55940 in 20 mM potassium diethylmalonate, pH 7.5, 2.25
mM MgCl2, 0.025% â-cyclodextrin (Research Biochemicals
Inc.), plus additional compounds as indicated. The test
compounds were prepared as 10 mM stock solutions in
dimethyl sulfoxide. The compounds were suspended in 5%
â-cyclodextrin (100 µM) followed by further dilution in the
above incubation buffer. The incubation was stopped by
addition of 250 µL of 50 mg/mL bovine serum albumin and
immediate filtration over glass fiber filters as previously
described.26 Specific binding was defined as that which could
be displaced by 0.1 µM desacetyllevonantradol. Data from
multiple experiments were averaged and analyzed by nonlin-
ear regression of the homologous displacement data using the
Graphpad Inplot computer program.
Ad en yla te Cycla se Activity. Plasma membranes from
N18TG2 cells were prepared as previously described.27 The
adenylate cyclase assay was modified from the previous
method as follows: The buffer was 20 mM diethylmalonate,
pH 8.0, rather than HEPES, and EDTA and bovine serum
albumin were eliminated from the reaction mixture. The
desacetyllevonantradol and (aminoalkyl)indole derivatives
were suspended in â-cyclodextrin and diluted as described
above. Assays were conducted at 30 °C for 8 min.
Tr ea t m en t of Mem b r a n es w it h (Am in oa lk yl)in d ole
Isoth iocya n a tes. Washed P2 membranes (900 µg of protein)
were sedimented and resuspended for incubation with the
indicated compounds in 150 µL of 20 mM potassium diethyl-
malonate, pH 7.5, and 2.25 mM MgCl2 buffer. The incubation
was stopped after 60 min by addition of 15 mL of ice-cold TME
buffer (20 mM Tris-Cl, pH 7.4, 3 mM MgCl2, 1 mM EDTA)
containing 1 mg/mL fatty acid-deficient bovine serum albumin
followed by sedimentation at 39000g for 30 min. The mem-
brane pellet was resuspended in TME buffer, and 50 µg of
membrane protein was assayed for [3H]CP-55940 binding in
TME buffer containing a final concentration of 0.16 mg/mL
fatty acid-deficient bovine serum albumin.
by incubation with compound 12 observed in the present
investigation is entirely consistent with this model.
One might hypothesize that the small cannabinoid
and (aminoalkyl)indole ligands bind to the CB1 receptor
within a pore region formed by the interaction of the
seven transmembrane helixes, as do the amine neu-
rotransmitter ligands.23-24 It is expected that as com-
pound 12 interacts with the receptor, the isothiocyanate
group should be within close proximity of a target
reactive substituent within the binding pocket such that
covalent coupling is permitted. Nucleophilic amino
groups on lysine, histidine, or arginine may form
disubstituted thioureas by such a reaction. A lysine
residue exists on the third transmembrane helix within
the first or second turn adjacent to the extracellular
surface of the CB1 receptor.10 One could hypothesize
that compound 12 is oriented within the binding pocket
such that the indole substituent is able to gain access
to the aminobutyl functionality of this target lysine.
Although helix 3 lysine would appear to be an ideal
candidate for covalent coupling to compound 12, it
should also be considered that potentially free sulfhydryl
groups exist on cysteine residues located within helices
1, 4, 6, and 7.10 Whether any of these are involved in
transhelical disulfide interactions is unknown, in which
case they would be unavailable to interact with com-
pound 12 under the incubation conditions used in this
study. It is also conceivable that compound 12 interacts
with nucleophilic amino acids at the intracellular
membrane-receptor interfaces, such as the histidines
at helices 1 and 2, the arginine and lysine residues at
helices 3 and 4, respectively, the lysine residues at
helices 5 and 6, or the arginine at helix 7.
Interpretation of the present results to suggest that
both cannabinoid and (aminoalkyl)indole ligands bind
to mutually exclusive locations within the receptor may
be complicated by the fact that both the covalently
modifying ligand and the binding detection ligand are
agonists. A scenario is possible by which the two ligand
classes may occupy different regions of the receptor
molecule but that appropriate interaction with either
region is able to promote an effector response. If the
irreversible occupancy of the (aminoalkyl)indole binding
site by the agonist ligand were able to promote (and
maintain) a state of dissociation of the G protein from
the receptor, which would normally occur in the pres-
ence of GTP, then the receptor would remain in a low-
affinity conformation for subsequent agonist ligand
interactions at the alternative agonist binding site (i.e.,
for cannabinoid compounds). Using ligand binding
concentrations of [3H]CP-55940 below the high-affinity
Kd, as was used in the present investigation, would not
be suitable for the detection of populations of receptors
in a low-affinity state. Although in the present study
the brain membranes underwent three steps of washing
by sedimentation, it is conceivable that sufficient gua-
nine nucleotides were present to allow the availability
of sufficient GTP to disrupt the receptor-G protein
interaction. This problem could be resolved by utiliza-
tion of an antagonist ligand for either covalent interac-
tion or detection.
Ch em istr y. Melting points (uncorrected) were taken in a
Thomas-Hoover capillary apparatus. IR, mass, and NMR
spectra were consistent with the structures shown. IR spectra
were obtained using a Beckman Acculab 8 instrument. Thin
layer chromatography (TLC) was performed on 250 µm silica
gel GHLF, Analtech uniplates and visualized with iodine
vapor. Elemental analyses were performed at Atlantic Mi-
crolabs, Atlanta, GA. Compounds indicated by the molecular
formula followed by the symbols for the elements (C, H, N)
were found to be within 0.4% of theory. Chemical ionization
(CIMS) and electron ionization (EIMS) mass spectra were
obtained using a Finnigan 1050 mass spectrometer and a VG-
Micro Mass 7070F mass spectrometer, respectively. 1H-NMR
spectra were obtained using a Varian XL-300 spectrometer and
the solvents indicated. Silica gel 60 (60-240 mesh) with the
solvent indicated was used for column chromatography.
1-Na p h th yl[2-m eth yl-1-[2-(4-m or p h olin yl)eth yl]-1H-in -
d ol-3-yl]m eth a n on e (2). This compound was prepared es-
sentially by the previously described method1 except that the
acyl chloride-AlCl3 complex was preformed in 1,2-dichloro-
ethane prior to addition of a solution of 5.1 Purification of the
crude material from 10 mmol of 51 by chromatography
(AcOEt-hexane, 2:1-10:1, v/v) afforded 2 (2.9 g, 73%) as a
yellow foam: 1H NMR (CDCl3) δ 2.51 (s, 7H), 2.72 (t, 2H, J )
7.3 Hz), 3.71 (m, 4H), 4.28 (m, 2H), 7.03 (t, 1H, J ) 8.2 Hz),
7.16-7.60 (m, 7H), 7.93 (d, 1H, J ) 7.8 Hz), 8.12 (d, 1H, J )
8.3 Hz); CIMS (NH3) m/ e 399 (MH+).
The free base 2 was converted into its oxalate and recrystal-
lized from water-EtOH to afford colorless needles: mp 216-7
°C dec. Anal. (C26H26O2N2‚C2H2O4‚0.25H2O) C, H, N.
4-Nitr o-1-n a p h th oic Acid (3). Compound 3 was prepared
in 74% yield as previously described,16 giving yellow prisms
(from AcOH): mp 217-9 °C dec (lit.16 mp 225-6 °C).
(4-Nitr o-1-n a p h th yl)[2-m eth yl-1-[2-(4-m or p h olin yl)eth -
yl]-1H-in d ol-3-yl]m eth a n on e (6). As in the Friedel-Crafts
preparation of 2,1 the indole 51 (7.0 g, 28.7 mmol) was treated
with a 1,2-dichloroethane (130 mL) solution of the complex
Exp er im en ta l Section
Biologica l Meth od s. Deter m in a tion of Affin ity for th e
Ca n n a bin oid Recep tor . A P2 membrane fraction was
prepared from rat forebrains as previously described25 except

1972 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 10
Yamada et al.
between AlCl3 (11.6 g, 87 mmol) and acid chloride 4 (obtained
from 4-nitro-1-naphthoic acid (3)16 (7.0 g, 32.3 mmol) and
thionyl chloride). The product was purified by chromatogra-
phy (CHCl3-MeOH, 80:1, v/v) and recrystallized from EtOH
mmol), Fe powder (1.38 g, 25 mg-atom), 6 N HCl (4 mL), water
(10 mL), and EtOH (50 mL) was refluxed for 30 min. The
insoluble material was filtered and washed well with EtOH.
The filtrate and washings were concentrated in vacuo, made
basic with NaHCO3, and extracted with AcOEt. The AcOEt
extract was washed with water and concentrated to ca. 50 mL,
and NaHCO3 (4.0 g, 48 mmol) in water (30 mL) was added.
Thiophosgene (0.4 mL, 5.2 mmol) was added at 5 °C and the
reaction mixture then stirred at room temperature for 0.5 h.
The AcOEt layer was washed with water and dried over
anhydrous Na2SO4. After removal of solvent, the residue was
purified by chromatography (CHCl3-MeOH, 60:1, v/v) to afford
an orange solid, which was recrystallized twice from AcOEt-
hexane to give 12 (513 mg, 54.6%) as yellow prisms: mp 166-7
°C; IR (KBr) 2050, 1610, 1510, 1380 cm-1; 1H NMR (CDCl3) δ
2.39 (m, 4H), 2.68 (t, 2H, J ) 6.2 Hz), 3.55 (m, 4H), 4.11 (t,
2H, J ) 6.2 Hz), 7.24-7.40 (m, 2H), 7.51 (s, 1H), 7.46-7.60
(m, 3H), 7.66 (dd, 1H, J ) 7.0, 1.1 Hz), 7.92 (m, 1H), 7.99 (d,
1H, J ) 8.2 Hz), 8.16 (d, 1H, J ) 8.0 Hz), 8.48 (m, 1H); CIMS
(NH3) m/ e 442 (MH+). Anal. (C26H23O2N3S) C, H, N, S.
(4-Am in o-1-n a p h t h yl)[1-[2-(4-m or p h olin yl)et h yl]-1H -
in d ol-3-yl]m eth a n on e (14). The indole 91 (10.5 g, 45.6
mmol) was treated analogous to the synthesis of 2 with acid
chloride obtained from 4-nitro-1-naphthoic acid16 (3) (12.4 g,
57.1 mmol) and thionyl chloride. The crude nitro compound
11 was reduced using the Fe-HCl method described for the
preparation of 7, and the product was purified by chromatog-
raphy (AcOEt-hexane, 9:1, v/v) to afford 14 (1.95 g, 10% from
9) as a yellow foam: 1H NMR (CDCl3) δ 2.44 (m, 4H), 2.73 (t,
2H, J ) 6.4 Hz), 3.63 (m, 4H), 4.20 (t, 2H, J ) 6.4 Hz), 4.45 (s,
2H), 6.77 (d, 1H, J ) 7.7 Hz), 7.3-7.4 (m, 3H), 7.48-7.54 (m,
2H), 7.53 (s, 1H), 7.62 (d, 1H, J ) 7.7 Hz), 7.84-7.90 (m, 1H),
8.42-8.50 (m, 2H); CIMS (NH3) m/ e 400 (MH+).
1
to give 6 (6.11 g, 52%) as yellow prisms: mp 156-60 °C; H
NMR (CDCl3) δ 2.53 (m, 4H), 2.60 (s, 3H), 2.73 (t, 2H, J ) 7
Hz), 3.71 (4H, m), 4.30 (2H, t, J ) 7 Hz), 7.03 (d, 2H, J ) 3.8
Hz), 7.18-7.26 (1H, m), 7.35 (d, 1H, J ) 7.8 Hz), 7.54-7.62
(m, 2H), 7.76 (t, 1H, J ) 8 Hz), 8.12 (d, 1H, J ) 8.4 Hz), 8.27
(dd, 1H, J ) 7.8, 1.8 Hz), 8.63 (d, 1H, J ) 8.8 Hz); CIMS (NH3)
m/ e 444 (MH+).
(4-Am in o-1-n a p h t h yl)[2-m et h yl-1-[2-(4-m or p h olin yl)-
eth yl]-1H-in d ol-3-yl]m eth a n on e (7). A mixture of 6 (4.10
g, 9.93 mmol), Fe powder (8 g), 6 N HCl (20 mL), and EtOH
(90 mL) was refluxed for 1 h. The insoluble material was
filtered by passing through Celite and washed with water. The
filtrate and washings were combined, concentrated in vacuo,
made alkaline with aqueous NaHCO3, and extracted with
AcOEt. The AcOEt extract was washed with water and brine
and dried over anhydrous Na2SO4. After removal of the
solvent, the residue was purified by chromatography (CHCl3-
MeOH, 100:1-60:1, v/v) to afford a reddish brown foam, which
was converted into its hydrochloride salt and recrystallized
from EtOH-water to give 7‚HCl (3.44 g, 77%) as pale brown
crystals: mp 215-20 °C dec; IR (KBr) 3440, 2600, 1615 cm-1
;
1H NMR (DMSO-d6) δ 2.54 (s, 3H), 6.80 (d, 1H, J ) 8 Hz),
7.07 (t, 1H, J ) 7.6 Hz), 7.23 (d, 1H, J ) 8 Hz), 7.28 (d, 1H, J
) 7.6 Hz), 7.47(d, 1H, J ) 8.2 Hz), 7.52 (m, 2H), 7.78 (d, 1H,
J ) 8.2 Hz), 8.24 and 8.38 (m, 1H each); CIMS (NH3) m/ e 414
(MH+).
(4-Isot h iocya n a t o-1-n a p h t h yl)[2-m et h yl-1-[2-(4-m or -
p h olin yl)eth yl]-1H-in d ol-3-yl]m eth a n on e (8). To a stirred
suspension of 7‚HCl (2.13 g,4.73 mmol), NaHCO3 (3.0 g, 36
mmol), water (50 mL), and AcOEt (50 mL) in an ice-water
bath was added thiophosgene (0.4 mL, 5.2 mmol). The mixture
was stirred at room temperature for 20 min, and then the
organic layer was washed with water and dried over anhy-
drous Na2SO4. After removal of solvent, the residue was
purified by chromatography (CHCl3-MeOH, 90:1, v/v) and
recrystallized from EtOH-hexane to afford 8 (1.46 g, 68%) as
pale yellow prisms: mp 147-8 °C; IR (KBr) 2120, 1640, 1610,
1415 cm-1; 1H NMR (DMSO-d6) δ 1.58 and 2.57 (2s, 3H, ArCH3
ratio 1 to 3), 2.52 (m, 4H), 2.72 (t, 2H, J ) 6.8 Hz), 3.71 (m,
4H), 4.29 (t, 2H, J ) 6.9 Hz), 7.03 (t, 1H, J ) 7.9 Hz), 7.11 (d,
1H, J ) 7.9 Hz), 7.21 (td, 1H, J ) 8.2, 2 Hz), 7.34 (d, 1H, J )
8.2 Hz), 7.46-7.70 (m, 4H), 8.16 (d, 1H, J ) 8.3 Hz), 8.23 (d,
1H, J ) 8.3 Hz); CIMS (NH3) m/ e 456 (MH+). Anal.
(C27H25O2N3S) C, H, N, S.
(4-Isot h iocya n a t o-1-n a p h t h yl)[1-[2-(4-m or p h olin yl)-
eth yl]-1H-in d ol-3-yl]m eth a n on e (15). The amine 14 (1.90
g, 4.76 mmol) was treated with thiophosgene (0.45 mL) as
described in the preparation of 8. The crude product was
purified by chromatography (CHCl3-MeOH, 50:1, v/v) and
recrystallized from AcOEt-hexane to afford 15 (1.48 g, 71%)
as colorless prisms: mp 153-4 °C. IR (KBr) 2080, 1660, 1620
cm-1 1H NMR (CDCl3) δ 2.40 (m, 4H), 2.71 (t, 2H, J ) 6.3
;
Hz), 3.56 (m, 4H), 4.18 (t, 2H, J ) 6.3 Hz),7.22-7.32 (m, 1H),
7.36-7.70 (m, 8H), 8.22 (d, 1H, J ) 8.5 Hz), 8.45-8.52 (m,
1H); CIMS (NH3) m/ e 442 (MH+). Anal. (C26H23O2N3S) C,
H, N, S.
Meth yl 6-Nitr o-1-n a p h th oa te (17) a n d Meth yl 3-Nitr o-
1-n a p h th oa te (18) fr om 3-Nitr o-1,8-n a p h th oic An h yd r id e
(16). These compounds were prepared by the following
modification of the procedure for the ethyl esters. A solution
of NaOH (13.3 g, 333 mmol) in water (500 mL) was stirred at
room temperature for 0.5 h with 3-nitro-1,8-naphthalic anhy-
dride16 (14) (24.3 g, 100 mmol). To this mixture were added
AcOH (30 mL) and Hg(OAc)2 (39 g, 122 mmol) successively,
and the mixture was refluxed for 4 days. The resulting
precipitate was collected by filtration, washed with water, and
then treated with 6 N HCl (400 mL) at reflux temperature for
1 h. After cooling, the precipitate was collected and dried over
P2O5 to afford a pale yellow solid, which was treated with
thionyl chloride (70 mL) and DMF (1 drop) in CHCl3 (70 mL)
at reflux temperature for 5 h. After removal of solvent and
excess thionyl chloride, MeOH (500 mL) was added while
cooling in an ice-water bath, and this was refluxed for 1 h.
After cooling, the resulting precipitate was collected, washed
well with cold MeOH, and dried in vacuo. The dark brown
solid was dissolved in hot ethyl acetate and treated with
activated charcoal, filtered while hot, and diluted with hexane
to afford pure 18 (8.50 g, 39.2%) as pale yellow needles: mp
1-Na p h th yl[1-[2-(4-m or p h olin yl)eth yl]-1H-in d ol-3-yl]-
m eth a n on e (10). This material was prepared from 91 analo-
gous to the synthesis of 2, purified by chromatography
(AcOEt-hexane, 2:1, v/v), and recrystallized from ether to
afford 10 (4.76 g, 48%) as pale yellow needles: mp 101-2 °C
(lit.1 mp 104-6 °C); CIMS (NH3) m/ e 385 (MH+). Anal.
(C25H24N2O2) C, H, N.
1-Naph th yl[1-[2-(4-m or ph olin yl)eth yl]-6-n itr o-1H-in dol-
3-yl]m eth a n on e (11). To a stirred solution of 10 (3.73 g, 9.7
mmol) in Ac2O (40 mL) was added 70% HNO3 (8 mL) at 0 °C,
and the reaction mixture was then stirred for 0.5 h. The
reaction was quenched with ice-water (500 mL); then the
mixture was treated with concentrated NH4OH (100 mL) and
stirred at room temperature for 30 min. The reaction mixture
was extracted with AcOEt. The AcOEt extract was washed
with water and brine and dried over anhydrous Na2SO4. After
removal of solvent, the residue was purified by chromatogra-
phy (AcOEt-hexane, 2:1-3:1, v/v) and recrystallized from
AcOEt-hexane to afford 11 (1.07 g, 26%) as yellow prisms:
mp 182-3 °C; IR (KBr) 1635, 1612, 1518, 1385, 1335 cm-1
;
134-5 °C; IR (KBr) 1718, 1605, 1530, 1340, 1240, 1190 cm-1
;
1H NMR (CDCl3) δ 2.41 (m, 4H), 2.73 (t, 2H, J ) 6 Hz), 3.54
(m, 4H), 4.24 (t, 2H, J ) 6 Hz), 7.47-7.61 (m, 3H), 7.69 (dd,
1H, J ) 7, 1.2 Hz), 7.73 (s, 1H), 7.74 (m, 1H), 8.02 (d, 1H, J )
8.3 Hz), 8.16 (m, 1H), 8.26 (dd, 1H, J )8.9, 2 Hz), 8.41 (d, 1H,
J ) 2Hz), 8.61 (d, 1H, J ) 8.8 Hz); CIMS m/ e 429 (M+), 399,
329, 155, 127, 101 (base), 56. Anal. (C25H23O4N3) C, H, N.
1-Naph th yl[6-isoth iocyan ato-1-[2-(4-m or ph olin yl)eth yl]-
1H-in d ol-3-yl]m eth a n on e (12). A mixture of 11 (1.07 g, 2.49
1H NMR (CDCl3) δ 4.06 (s, 3H), 7.71(t, 1H, J ) 7 Hz), 7.83 (m,
1H), 8.08 (d, 1H, J ) 8 Hz), 8.94 (d, 1H, J ) 2.4 Hz), 8.96 (d,
1H, J ) 2.4 Hz), 9.04 (d, 1H, J ) 9.0 Hz); CIMS (NH3) m/ e
231 (M+).
All mother liquors and filtrates were combined and purified
by chromatography (AcOEt-hexane, 1:9, v/v) to afford 17 (540
mg, 2.3%) as pale yellow needles: mp 137-8 °C; IR (KBr) 1735,
1605, 1510, 1355, 1285, 1250, 1205, 1140 cm-1 1H NMR
;

Affinity Ligands for Brain Cannabinoid Receptor
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 10 1973
(CDCl3) δ 4.04 (s, 3H), 7.69 (m, 1H), 8.22 (d, 1H, J ) 8.2 Hz),
8.35 (dd, 1H, J ) 9.6, 2.4 Hz), 8.41 (dd, 1H, J ) 7.3, 1.2 Hz),
8.83 (d, 1H, J ) 2.3 Hz), 9.15 (d, 1H, J ) 9.5 Hz); CIMS (NH3)
m/ e 231 (M+).
tion is given on any current masthead page. Atomic coordi-
nates may also be obtained from the Cambridge Crystallo-
graphic Data Centre (Cambridge University Chemical Lab-
oratory, Cambridge CB2 1EW, U.K.).
(6-Nitr o-1-n a p h th yl)[1-[2-(4-m or p h olin yl)eth yl]-1H-in -
d ol-3-yl]m eth a n on e (21). A mixture of the nitro ester 17
(550 mg, 2.38 mmol), 10% NaOH (10 mL), and methanol (20
mL) was refluxed for 1 h. After removal of solvent, the residue
was made acidic with 10% HCl and extracted with AcOEt. The
AcOEt extract was washed with water, dried over Na2SO4, and
evaporated to leave a yellow solid (0.52 g, quantitative), which
was treated with thionyl chloride (5 mL) at reflux temperature
for 1 h and then evaporated to give acid chloride 19 (0.50 g,
quantitative). Reaction of 19 with indole 91 as for the
synthesis of 21 gave crude material which was purified by
chromatography (CHCl3-MeOH, 50:1, v/v) and recrystallized
from AcOEt-hexane to afford 21 (230 mg, 22%) as colorless
prisms: mp 217-8 °C; IR (KBr) 1620, 1590, 1530, 1385, 1345
cm-1; 1H NMR (CDCl3) δ 2.4 (m, 4H), 2.72 (t, 2H, J ) 6.3 Hz),
3.5 (m, 4H), 4.19 (t, 2H, J ) 6.3 Hz), 7.4 (m, 3H), 7.46 (s, 1H),
7.72 (dd, 1H, J ) 8.2, 7.2 Hz), 7.89 (dd, 1H, J ) 7.1, 1.2 Hz),
8.18 (d, 1H, J ) 8.3 Hz), 8.22 (dd, 1H, J ) 9.4, 2.3 Hz), 8.36
(d, 1H, J ) 9.3 Hz), 8.48 (m, 1H), 8.87 (d, 1H, J ) 2.2 Hz);
CIMS (NH3) m/e 430 (MH+).
Refer en ces
(1) Bell, M. R.; D’Ambra, T. E.; Kumar, V.; Eissenstat, M. A.;
Herrmann, J . L.; Wetzel, J . R.; Rosi, D.; Philion, R. E.; Daum,
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D. R.; Luttinger, D. A.; Baizman, E. R.; Miller, M. S.; Ward, S.
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Daley, G. T. Conformationally Restrained Analogues of Prava-
doline-Nanomolar Potent, Enantioselective, (Aminoalkyl)indole
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(6) Ward, S. J .; Baizman, E.; Bell, M.; Childers, S.; D’Ambra, T.;
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(7) Pacheco, M. C.; Childers, S. R.; Arnold, R.; Casiano, F.; Ward,
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(10) Matsuda, L. A.; Lolait, S. J .; Brownstein, M. J .; Young, A. C.;
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(11) Gerard, C. M.; Mollereau, C.; Vassart, G.; Parmentier, M.
Molecular Cloning of a Human Cannabinoid Receptor Which Is
Also Expressed in Testis. Biochem. J . 1991, 279, 129-134.
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(14) Abood, M. E.; Martin, B. R. Neurobiology of Marijuana Abuse.
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(18) J ohnson, M. R.; Melvin, L. S. The Discovery of Nonclassical
Cannabinoid Analgetics. In Cannabinoids as Therapeutic Agents;
Mechoulam, R., Ed.; CRC Press: Boca Raton, FL, 1986; pp 121-
145.
(6-Isot h iocya n a t o-1-n a p h t h yl)[1-[2-(4-m or p h olin yl)-
eth yl]-1H-in d ol-3-yl]m eth a n on e (22). The nitroindole 21
was treated as in the conversion of 11 to 12. After workup,
the crude 22 was purified by chromatography (AcOEt-hexane,
2:1, v/v) and recrystallized from AcOEt-hexane to afford 22
(85 mg from 220 mg of 21, 38%) as pale orange prisms: mp
160-1 °C; IR (KBr) 2030, 1630 cm-1 1H NMR (CDCl3) δ
;
2.41(m, 4H), 2.72 (m, 2H), 3.58 (m, 4H), 4.19 (m, 2H), 7.47 (s,
1H), 7.59 (m, 1H), 7.70 (m, 1H), 7.76 (d, 1H, J ) 2.1 Hz), 7.92
(d, 1H, J ) 8.4 Hz), 8.21 (d, 1H, J ) 8.9 Hz), 8.48 (m, 1H);
CIMS (NH3) m/ e 430 (MH+). Anal. (C26H23O2N3S) C, H, N,
S.
(3-Nitr o-1-n a p h th yl)[1-[2-(4-m or p h olin yl)eth yl]-1H-in -
d ol-3-yl]m eth a n on e (23). As in the synthesis of 21, nitro
ester 18 (8.5 g) was converted to acid chloride 20 which was
reacted with indole 9 (6.10 g). After workup, the crude product
was purified by chromatography (CHCl3-MeOH, 50:1, v/v) and
recrystallized from AcOEt-hexane to afford 23 (4.58 g, 40.3%)
as yellow prisms: mp 180-2 °C; IR (KBr) 1630, 1530, 1400,
1350, 1335 cm-1; 1H NMR (CDCl3) δ 2.40 (m, 4H), 2.73 (t, 2H,
J ) 6.3 Hz), 3.52 (m, 4H), 4.20 (t, 2H, J ) 6.3 Hz), 7.38-7.44
(m, 3H), 7.42 (d, 1H, J ) 1.2 Hz), 7.66-7.74 (m, 2H), 8.10-
8.16 (m, 1H), 8.24-8.30 (m, 1H), 8.43 (d, 1H, J ) 2.2 Hz),
8.48-8.56 (m, 1H), 8.92 (d, 1H, J ) 2.1 Hz); CIMS (NH3) m/ e
430 (MH+).
(3-Isot h iocya n a t o-1-n a p h t h yl)[1-[2-(4-m or p h olin yl)-
eth yl]-1H-in d ol-3-yl]m eth a n on e (24). As described in the
preparation of isothiocyanate 22, the nitro compound 23 was
reduced by Fe-HCl in aqueous EtOH and treated with thio-
phosgene. The crude product was purified by chromatography
(CHCl3-MeOH, 50:1, v/v) and recrystallized twice from AcOEt-
hexane to afford 24 (410 mg, 12%) as colorless prisms: mp
143-4 °C; IR (KBr) 2080, 1625, 1525, 1400, 1200 cm-1 1H
;
NMR (CDCl3) δ 2.40 (m, 4H), 2.72 (t, 2H, J ) 6.3 Hz), 3.54
(m, 4H), 4.19 (t, 2H, J ) 6.3 Hz), 7.36-7.43 (m, 3H), 7.45-
7.60 (m, 3H), 7.46 (s, 1H), 7.81 (d, 1H, J ) 1.9 Hz), 7.85 (d,
1H, J ) 8 Hz), 8.10 (d, 1H, J ) 8.3 Hz), 8.46-8.53 (m, 1H);
CIMS (NH3) m/ e 442 (MH+). Anal. (C26H23O2N3S) C, H, N,
S.
Ack n ow led gm en t. This work was supported in part
by NIDA grants R01-DA06312 and K05-DA00182 to
A.C.H. and Office of Naval Research and NIDA funding
to J .L.F.-A. We thank Tracy Champion-Dorow, Cathe-
rine Cantrell, and Gerald Wilken for technical as-
sistance with the biological determinations in this
report.
(19) Razdan, R. K. Structure-Activity Relationships in Cannabinoids.
Pharmacol. Rev. 1986, 38, 75-149.
(20) Martin, B. R.; Compton, D. R.; Thomas, B. F.; Prescott, W. R.;
Little, P. J .; Razdan, R. K.; J ohnson, M. R.; Melvin, L. S.;
Mechoulam, R.; Ward, S. J . Behavioral, Biochemical, and
Molecular Modeling Evaluations of Cannabinoid Analogs. Phar-
macol. Biochem. Behav. 1991, 40, 471-478.
(21) Melvin, L. S.; Milne, G. M.; J ohnson, M. R.; Subramaniam, B.;
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