The synthesis and pharmacological evaluation of adamantane-derived indoles: Cannabimimetic drugs of abuse

Article abstract of DOI:10.1021/cn400035r

Two novel adamantane derivatives, adamantan-1-yl(1-pentyl-1H-indol-3-yl) methanone (AB-001) and N-(adamtan-1-yl)-1-pentyl-1H-indole-3-carboxamide (SDB-001), were recently identified as cannabimimetic indoles of abuse. Conflicting anecdotal reports of the psychoactivity of AB-001 in humans, and a complete dearth of information about the bioactivity of SDB-001, prompted the preparation of AB-001, SDB-001, and several analogues intended to explore preliminary structure-activity relationships within this class. This study sought to elucidate which structural features of AB-001, SDB-001, and their analogues govern the cannabimimetic potency of these chemotypes in vitro and in vivo. All compounds showed similar full agonist profiles at CB₁ (EC₅₀ = 16-43 nM) and CB₂ (EC₅₀ = 29-216 nM) receptors in vitro using a FLIPR membrane potential assay, with the exception of SDB-002, which demonstrated partial agonist activity at CB₂ receptors. The activity of AB-001, AB-002, and SDB-001 in rats was compared to that of Δ⁹-tetrahydrocannabinol (Δ⁹-THC) and cannabimimetic indole JWH-018 using biotelemetry. SDB-001 dose-dependently induced hypothermia and reduced heart rate (maximal dose 10 mg/kg) with potency comparable to that of Δ⁹-tetrahydrocannabinol (Δ⁹-THC, maximal dose 10 mg/kg), and lower than that of JWH-018 (maximal dose 3 mg/kg). Additionally, the changes in body temperature and heart rate affected by SDB-001 are of longer duration than those of Δ⁹-THC or JWH-018, suggesting a different pharmacokinetic profile. In contrast, AB-001, and its homologue, AB-002, did not produce significant hypothermic and bradycardic effects, even at relatively higher doses (up to 30 mg/kg), indicating greatly reduced potency compared to Δ⁹-THC, JWH-018, and SDB-001.

Full text of DOI:10.1021/cn400035r

Research Article
pubs.acs.org/chemneuro
The Synthesis and Pharmacological Evaluation of Adamantane-
Derived Indoles: Cannabimimetic Drugs of Abuse
Samuel D. Banister,†,‡ Shane M. Wilkinson,† Mitchell Longworth,† Jordyn Stuart,§ Nadine Apetz,∥
Katrina English,∥ Lance Brooker,⊥ Catrin Goebel,⊥ David E. Hibbs,# Michelle Glass,∇ Mark Connor,§
Iain S. McGregor,∥ and Michael Kassiou*,†,‡,○
†School of Chemistry, The University of Sydney, NSW 2006, Australia
‡Brain and Mind Research Institute, NSW 2050, Australia
§The Australian School of Advanced Medicine, Macquarie University, NSW 2109, Australia
∥School of Psychology, The University of Sydney, NSW 2006, Australia
⊥Australian Sports Drug Testing Laboratory, National Measurement Institute, NSW 2073, Australia
#Faculty of Pharmacy, The University of Sydney, NSW 2006, Australia
∇School of Medical Sciences, The University of Auckland, Auckland 1142, New Zealand
○Discipline of Medical Radiation Sciences, The University of Sydney, NSW 2006, Australia
S
* Supporting Information
ABSTRACT: Two novel adamantane derivatives, adamantan-
1-yl(1-pentyl-1H-indol-3-yl)methanone (AB-001) and N-
(adamtan-1-yl)-1-pentyl-1H-indole-3-carboxamide (SDB-
001), were recently identified as cannabimimetic indoles of
abuse. Conflicting anecdotal reports of the psychoactivity of
AB-001 in humans, and a complete dearth of information
about the bioactivity of SDB-001, prompted the preparation of
AB-001, SDB-001, and several analogues intended to explore
preliminary structure−activity relationships within this class.
This study sought to elucidate which structural features of AB-
001, SDB-001, and their analogues govern the cannabimimetic
potency of these chemotypes in vitro and in vivo. All
compounds showed similar full agonist profiles at CB1 (EC50 = 16−43 nM) and CB2 (EC50 = 29−216 nM) receptors in
vitro using a FLIPR membrane potential assay, with the exception of SDB-002, which demonstrated partial agonist activity at CB2
receptors. The activity of AB-001, AB-002, and SDB-001 in rats was compared to that of Δ9-tetrahydrocannabinol (Δ9-THC)
and cannabimimetic indole JWH-018 using biotelemetry. SDB-001 dose-dependently induced hypothermia and reduced heart
rate (maximal dose 10 mg/kg) with potency comparable to that of Δ9-tetrahydrocannabinol (Δ9-THC, maximal dose 10 mg/kg),
and lower than that of JWH-018 (maximal dose 3 mg/kg). Additionally, the changes in body temperature and heart rate affected
by SDB-001 are of longer duration than those of Δ9-THC or JWH-018, suggesting a different pharmacokinetic profile. In
contrast, AB-001, and its homologue, AB-002, did not produce significant hypothermic and bradycardic effects, even at relatively
higher doses (up to 30 mg/kg), indicating greatly reduced potency compared to Δ9-THC, JWH-018, and SDB-001.
KEYWORDS: indole, AB-001, Δ9-tetrahydrocannabinol, cannabinoid receptor, drug abuse
annabinoid (CB) receptors are G protein-coupled
receptors (GPCRs) comprising two subtypes; CB1 and
The most prominent naturally occurring CB ligand is Δ9-
tetrahydrocannabinol (Δ9-THC, 1, Figure 1),3 the principal
bioactive component of marijuana, which acts as a partial
agonist at CB1 and CB2 receptors.4 The abuse liability of the
psychoactive resin, flowers, and leaves of Cannabis sativa,
collectively termed marijuana, has resulted in near-universal
proscription of the plant. Despite this, The United Nations
C
CB2.1,2 CB1 receptors are primarily found in the central nervous
system (CNS), although they are also expressed to a lesser
extent in some peripheral organs. CB2 receptors occur mainly
in T cells of the immune system, macrophages and B cells, and
in hematopoietic cells. Activation of CB receptors is achieved
endogenously in mammals by endocannabinoids such as
anandamide and 2-arachidonoylglycerol. However, a myriad
of exogenous compounds are known to act as cannabinoid
agonists, including both natural products and synthetic
chemicals.
Received: January 30, 2013
Accepted: April 3, 2013
© XXXX American Chemical Society
A
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Figure 1. Selected natural and synthetic cannabimimetics.
Office on Drugs and Crime (UNODC) estimated that between
125 and 203 million people used cannabis at least once in 2009,
making it the most widely produced and consumed illicit
substance worldwide.5
ication,26,30,32 and it is likely that synthetic cannabinoids can
precipitate psychoses in vulnerable individuals.33−35 As of
August 2010, more than 1000 cases of synthetic cannabinoid-
induced toxicity had been reported across the United States.36
The toxicological assessment of synthetic cannabinoids requires
the development of methodologies for the identification of
metabolites of clinical or forensic relevance, and there is a
paucity of pertinent literature for the most recent emergent
synthetic cannabimimetics.37 In the case of JWH-018, several
hydroxylated potential metabolites retained potent agonist
activity at CB1 receptors, while a major glucuronidated
metabolite showed neutral antagonist activity at the same
receptors.38,39 JWH-073, an analogue of JWH-018, also
produces metabolites with similarly diverse functional activ-
ities,40 further confounding the attribution of adverse effects to
specific chemical entities within herbal blends.
There is understandable concern by health agencies
worldwide about the adverse effects of synthetic cannabinoids
and other “synthetic legal intoxicating drugs” (SLIDs) with no
history of human use, and, in many cases, little pharmacological
evaluation of any kind.41 Indeed, around 2008, many countries
moved to place JWH-018 in the most restrictive regulatory
categories; however, manufacturers simply circumvented these
control measures by substituting this component with
unscheduled congeneric compounds possessing similar ef-
fects.42 Delay in the conclusive identification and pharmaco-
logical characterization of emerging synthetic cannabinoids
presents a potential danger to the intended consumers, and the
preemptive identification and thorough assessment of novel
“designer drugs” remains an important aspect of proactive
public health policy. The implementation of appropriate legal
restriction of synthetic cannabinoids is hampered by a scarcity
of reference materials and analytical data, a problem which is
compounded by the sheer rate of emergence of such
compounds.21
Human use of marijuana for its psychoactive and medicinal
properties has been recorded since antiquity,6 and the diversity
of cannabinoid pharmacology has driven interest in CB
receptors as therapeutic targets for the potential treatment of
numerous diseases, including cancer, obesity, inflammation, and
pain.7−14 Most early synthetic cannabimimetics were simplified
analogues of Δ9-THC based on the cyclohexylphenol core,
such as CP 47,497 (2), disclosed by Pfizer in 1982.15 However,
in 1992, Sterling Research Group reported WIN 55,212-2 (3)
as the first member of a structurally novel aminoalkylindole
(AAI) class of cannabimimetics.16 Since this time, many
simplified AAIs with CB receptor activity have been described.
Huffman and colleagues have extensively explored the
structure−activity relationships (SARs) of cannabinergic
indoles such as representative member JWH-018 (4), resulting
in a plethora of analogues.17−19
Herbal blends containing synthetic cannabimimetic additives
have been available in many countries from retailers and
Internet-based vendors since about 2004.20−23 Although these
products are often declared as incense, they are intended for
human consumption as unscheduled, and therefore legal,
alternatives to marijuana. One of the earliest such products,
“Spice”, was found in 2008 to contain two synthetic
cannabinoids, the octyl homologue of CP 47,497 and JWH-
018, consistent with anecdotal reports of cannabis-like effects
when the material was smoked.20 Although Δ9-THC acts as a
partial agonist at CB1 receptors, many synthetic cannabinoids,
including JWH-018, act as full agonists at CB1 sites.4
Differences in the CB1 receptor efficacy of synthetic
cannabimimetics, as well as unidentified off-target activity,
may explain the cardiotoxicity and seizures sometimes observed
following the ingestion of synthetic cannabinoids,24−30 an
outcome not generally observed with the use of marijuana or
synthetic Δ9-THC.31 Psychotic episodes with paranoia,
delusion, and agitation necessitating emergency ward admission
have been reported following synthetic cannabinoid intox-
Dozens of indole cannabinoids have been identified as
components of Spice-like products from around the world, and
in some cases the structures were previously unprecedented in
the scientific literature.43−57 In 2010, adamantan-1-yl(1-pentyl-
1H-indol-3-yl)methanone (5, AB-001) was identified in
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a
Scheme 1. Synthesis of Cannabimimetic Indoles 5−12
a
Reagents and conditions: (a) NaH, Br(CH2)4CH3, DMF, 0 °C−rt, 1 h, 80%; (b) Me2AlCl, R(CH2)nC(O)Cl, CH2Cl2, 0 °C, 2 h, 35−86%; (c) NaH
(2 equiv), Br(CH2)4CH3, DMF, 0 °C−rt, 1 h; (d) (CF3CO)2O, DMF, 0 °C−rt, 1 h; (e) KOH, MeOH, PhMe, reflux, 2 h, 75% over 3 steps; (f)
(COCl)2, DMF (cat.), CH2Cl2, rt, 1 h, quant; (g) R(CH2)n NH2, Et3N, CH2Cl2, rt, 14 h, 73−86%.
products originating from Ireland, and subsequently reported to
the European Monitoring Centre for Drugs and Drug
Addiction (EMCDDA) via the Early Warning System
(EWS).58 AB-001 was contemporaneously identified in bulk
powders seized by the Hungary Customs and Finance Guard,
and spectroscopic characterization and human metabolites were
reported, although no synthetic details or pharmacological
evaluation of this compound exist in the scientific liter-
ature.53,59,60 In 2012, N-(adamtan-1-yl)-1-pentyl-1H-indole-3-
carboxamide (6, SDB-001), was detected in products obtained
by the National Institute of Health Sciences in Japan and, like
AB-001, no synthetic preparation or pharmacological assess-
ment of this compound has been published to date.61,62
Despite their novel structures, AB-001 and SDB-001 appear
to exploit SARs previously described for the class of
cannabimimetic indoles, and are likely products of clandestine
rational drug design. Systematic exploration of JWH-018
revealed that a saturated four to six carbon chain at the 1-
indole position was optimal for CB1 binding and in vivo activity
in rats.18,19 Additionally, the naphthalene group of JWH-018 is
not mandatory for CB1 binding, and many 1-alkylindoles
containing alicyclic 3-acyl groups have been reported as
cannabinoid ligands, including adamantane derivatives.63 In-
deed, analogues containing the N-(adamtan-1-yl)-1-alkyl-1H-
indole-3-carboxamide core of SDB-001 are listed as prophetic
structures in several patents by Makriyannis and colleagues,
although SDB-001 itself is not described.64−66 Pasquini and co-
workers recently prepared several 5-arylated N-(adamtan-1-yl)-
1-pentyl-1H-indole-3-carboxamides, however, these com-
pounds generally functioned as selective CB2 receptor inverse
agonists rather than CB1 receptor agonists.67,68
potential assay to provide basic SARs for selective agonist
activity at CB1 receptors. Additionally, selected compounds
were compared to Δ9-THC and JWH-018 in vivo, prompted by
inconsistent reports of the psychoactivity of AB-001 in
humans,58 and a complete lack of information about the
psychotropic activity of SDB-001 in any animal.
RESULTS AND DISCUSSION
■
In order to evaluate the cannabimimetic activity of AB-001,
SDB-001, and structurally related analogues 7−12, a synthetic
route to each class of indole was developed (Scheme 1). Indole
(13) was treated with sodium hydride and alkylated with
bromopentane, affording N-pentylindole (14). N-pentylindole
was acylated according to the method of Okauchi and co-
workers69 by treatment with dimethylaluminum chloride,
followed by 1-adamantanoyl chloride, to give AB-001 in good
yield. Subjecting N-pentylindole to the same procedure with (1-
adamatane)acetyl chloride gave homologous AB-002 (7), which
contains an indole-adamantane distance approximating that of
SDB-001.
The synthesis of SDB-001 was achieved by alkylating indole
as described above, followed by treatment with trifluoroacetic
anhydride in one-pot procedure, to give N-pentyl-3-trifluor-
oacetylindole (15). Alkaline hydrolysis of 15 induced fluoro-
form elimination70 to yield, upon acidic workup and
recrystallization from isopropanol, N-pentylindole-3-carboxylic
acid (16) of analytical purity. The three-step procedure proved
operationally convenient and common precursor 16 could be
obtained on multigram scale without chromatography. An
alternative synthesis of 16 involving esterification of indole-3-
carboxylic acid, N-alkylation, and ester hydrolysis proceeded in
lower yield, with requisite chromatographic purification at each
step making this route less attractive. Treatment of 16 with
oxalyl chloride generated the corresponding acid chloride (17),
which was reacted with the appropriate amine to give 6 and 8−
12 in 73−86% unoptimized yield following recrystallization. To
The aim of the current work was to prepare and characterize
AB-001, SDB-001, and several structurally related analogues,
and to provide preliminary evaluation of their pharmacological
profiles in vitro and in vivo. All synthesized compounds were
screened against CB1 and CB2 receptors in a FLIPR membrane
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explore preliminary SARs for the N-pentylindole-3-carbox-
amide scaffold at CB receptors the synthesis of 9−12,
containing variously spaced alicyclic or aromatic groups in
place of the bulky, polycyclic adamantane moiety was
undertaken. It should be noted that 11 was generated solely
to probe SARs within this series, and is likely toxic to living
systems due to potential enzymatic amidolysis to aniline
metabolites.
Indole-3-carboxamides 6 and 8−12 were easily recrystallized
to analytical purity from isopropanol-water; however, attempts
to grow suitable crystals for X-ray diffraction were uniformly
unsuccessful. By contrast, AB-001 produced large prismatic
crystals by slow-evaporation from isopropanol-water and an X-
ray crystal structure was obtained (Figure 2). All bond lengths
and angles were as expected, with full details of X-ray data
collection and tables of bond lengths and angles available in the
Supporting Information.
receptors, and agonist activities of Δ9-THC and 5−12 were
assessed using a fluorometric imaging plate reader (FLIPR)
membrane potential assay.71 AtT20 cells endogenously express
G protein-gated inwardly rectifying K+ channels (GIRK), and
these are activated by coexpressed CB1 and CB2 receptors (vide
infra).72 The maximum effect of the novel compounds was
compared with that of the nonselective CB receptor agonist
WIN 55,212-2, which produced a maximal decrease in
fluorescence, corresponding to cellular hyperpolarization, of
35 2% in AtT20-CB1 cells and 28 2% in AtT20-CB2 cells.
All novel indoles had measurable agonist activity at CB1 and
CB2 receptors, with EC50 values for CB1 receptor-mediated
activation of GIRK generally comparable to Δ9-THC.
Interestingly, in AtT20-CB2 cells, Δ9-THC had low efficacy,
with a maximum effect at 10 μM (13% of that of WIN55212−
2) that precluded calculation of an EC50. All of the novel
indoles had a greater efficacy than Δ9-THC, with SDB-002
being the least efficacious of the series. AB-001 (CB1 EC50 = 35
nM, CB2 EC50 = 48 nM) showed a remarkably similar profile to
SDB-001 (CB1 EC50 = 34 nM, CB2 EC50 = 29 nM). The
respective homologues of AB-001 and SDB-001, AB-002 (CB1
EC50 = 37 nM, CB2 EC50 = 89 nM), and SDB-002 (CB1 EC50 =
43 nM, CB2 EC50 = 57 nM) had similar potencies at each
receptor. However, at CB2 receptors, SDB-002 had a maximal
effect about a third of AB-002, indicating a partial agonist
profile for the former. None of the compounds affected
membrane potential in wildtype AtT-20 cells.
The hyperpolarization of CB1 receptors induced by AB-001,
AB-002, SDB-001, and SDB-002 is shown in Figure 3A. All four
ligands act as full functional agonists at CB1 receptors, with
similar potency to the classical agonist WIN 55,212−2, whereas
Δ9-THC is a partial agonist at CB1 receptors. The hyper-
polarization of CB2 receptors induced by AB-001, AB-002, and
SDB-001, is also of similar potency to WIN 55,212−2, as
shown in Figure 3B. However, SDB-002 was a partial agonist at
CB2 receptors in this assay, with an apparently greater potency
than Δ9-THC.
Taken together, these profiles indicate that a bulky
adamantane substituent at a distance of two to four bond
lengths from the 3-indole position is tolerated by both CB
receptor subtypes. Additionally, the nature of the linker appears
to be unimportant for activation of CB1 receptors, with amide
and ketone linkers equally well tolerated. There was little effect
on CB1 activity when the steric bulk of the adamantane cage of
SDB-001 was reduced to a cyclohexane ring (SDB-003, CB1
Figure 2. ORTEP diagram of the crystal structure of 5 with thermal
ellipsoids at the 50% probability level.
The cannabimimetic activity of indoles 5−12 at CB1 and CB2
receptors was compared to exogenous agonist Δ9-THC, and
the results are shown in Table 1. Mouse AtT20 neuroblastoma
cells were stably transfected with either rat CB1 or human CB2
Table 1. Functional Activity of Δ9-THC, WIN 55,212-2, and 5−12 at Rat CB1 and Human CB2 Receptors
rCB1
hCB2
pEC50 SEM
(EC50, nM)
max SEM
(% WIN 55,212-2)
pEC50 SEM
(EC50, nM)
max SEM
(% WIN 55,212-2)
a
compd
CB1 selectivity
1 (Δ9-THC)
3 (WIN 55,212−2)
5 (AB-001)
7.24 0.12 (58)
7.57 0.14 (27)
7.46 0.16 (35)
7.47 0.12 (34)
7.43 0.13 (37)
7.37 0.12 (43)
7.43 0.16 (37)
7.79 0.19 (16)
7.68 0.11 (21)
7.73 0.11 (19)
78
5
n.d.
13 (at 10 μM)
6.93 0.1 (117)
7.32 0.12 (48)
7.54 0.11 (29)
7.05 0.13 (89)
7.24 0.26 (57)
6.99 0.08 (102)
6.67 0.09 (216)
6.86 0.12 (140)
6.88 0.22 (134)
4.3
1.4
0.9
2.4
1.3
2.7
13.1
6.7
7.2
105
98
90
84
93
94
99
85
8
6
6
6
7
8
6
5
86
91
82
23
95
71
74
68
6
5
7
4
5
5
6
9
6 (SDB-001)
7 (AB-002)
8 (SDB-002)
9 (SDB-003)
10 (SDB-004)
11 (SDB-005)
12 (SDB-006)
a
CB1 selectivity expressed as the ratio of CB2 EC50 to CB1 EC50. n.d. = not determined.
D
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obtained with Δ9-THC, JWH-018, and SDB-001 with statisti-
cally significant treatment or treatment by time effects of all 3
doses of each drug (ANOVA, planned contrasts, P < 0.05). A
maximum decrease in body temperature was evoked by 10 mg/
kg Δ9-THC (∼2 °C) within 30 min of injection. JWH-018
achieved a similar magnitude of hypothermia at 3 mg/kg,
indicating the relatively greater potency of this compound in
rats. The novel indole-3-carboxamide SDB-001 caused a slightly
shallower hypothermia (1.6 °C at a dose of 10 mg/kg),.
However, all doses of SDB-001 (1, 3, and 10 mg/kg) were
similarly effective and appeared longer lasting than Δ9-THC or
JWH-018, suggesting a different pharmacokinetic profile for
this compound relative to JWH-018. Although the Δ9-THC
and JWH-018 treatment groups generally returned to the same
body temperature as the vehicle group within 5 h
postadministration, rats treated with SDB-001 remained
hypothermic for 7−11 h, depending on dose, following drug
injection (data not shown). AB-001 did not produce significant
hypothermia at a dose of 3 mg/kg, in contrast to the clear
reduction in body temperature produced by an equivalent dose
of Δ9-THC, JWH-018, and SDB-001. Further evaluation of AB-
001 at doses of 10 mg/kg and 30 mg/kg showed only modest
hypothermic trends, indicating that AB-001 possesses reduced
cannabimimetic activity compared to Δ9-THC, JWH-018, and
SDB-001. Similarly, AB-002 showed only trends toward
hypothermia at doses of 3 mg/kg and 30 mg/kg. Taken
together, these findings suggest that the nature of indole-
adamantane tether, rather than the distance between these two
groups, is important for in vivo activity; the carboxamide group
confers cannabimimetic activity while the ketone does not.
Figure 5 shows the effects of Δ9-THC, JWH-018, SDB-001,
AB-001, and AB-002 on heart rate. As with the body
temperature data, results for a baseline 1 h prior to i.p.
injection and for 5 h postdrug are presented in 15 min bins in
this figure. Dose-dependent decreases in heart rate were
obtained with Δ9-THC, JWH-018, and SDB-001, with
statistically significant treatment or treatment by time effects
of most doses tested (ANOVA, planned contrasts, P < 0.05).
Maximal effects were obtained with 10 mg/kg Δ9-THC and 3
mg/kg JWH-018, with intermediate effects seen with the lower
doses of these two compounds. As with body temperature
results, SDB-001 produced a slightly shallower, although
significant, effect on heart rate that did not differ greatly
between the 1, 3, and 10 mg/kg doses assessed. At doses up to
30 mg/kg, AB-001 and AB-002 produced only modest, and
relatively short-lived, effects on heart rate compared to lower
doses Δ9-THC, JWH-018, and SDB-001.
Figure 3. Hyperpolarization of (A) CB1 and (B) CB2 receptors
induced by Δ9-THC, AB-001, AB-002, SDB-001, and SDB-002 as a
proportion of that produced by 1 μM of WIN 55,212-2. Membrane
potential was measured using a fluorescent dye, as outlined in the
Methods. Each point represents the mean
SEM of at least three
independent determinations, each performed in triplicate. Data was
fitted with a 4 parameter logistic equation in Graphpad Prism.
EC50 = 37 nM), but CB2 activity was reduced approximately 3-
fold (CB2 EC50 = 102 nM). The same structural simplification
of SDB-002 gave homologue SDB-004, which showed roughly
double the potency at CB1 and similarly reduced CB2 potency
(CB1 EC50 = 16 nM, CB2 EC50 = 216 nM). Replacement of the
cyclohexane ring of SDB-003 and SDB-004 with a phenyl ring
gave aniline derivative SDB-005 (CB1 EC50 = 21 nM, CB2 EC50
= 140 nM) and benzylic analogue SDB-006 (CB1 EC50 = 19
nM, CB2 EC50 = 134 nM) respectively, which showed a similar
preference for CB1 receptors.
Due to conflicting anecdotal reports regarding the psycho-
activity of AB-001, and a complete lack of information
regarding the biological activity of SDB-001, several indoles
from the current series were assessed using radiotelemetry in
male Wistar rats to confirm in vivo cannabimimetic activity.
The well-established ability of cannabinoids to induce hypo-
thermia and decrease heart rate in rats is common to both
classical cannabinoids like Δ9-THC as well as synthetic
cannabimimetics.73−78 AB-002 was also included in these
studies since it contains the ketone linker of AB-001 but the
indole-adamantane spacing of SDB-001, representing a
molecular hybrid of the two structures. It was theorized that
differences in the behavioral pharmacology of AB-001, AB-002,
and SDB-001 would allow delineation of the relative
contributions of the indole-adamantane distance versus the
nature of the linker separating these groups. For comparison,
Δ9-THC and the well-characterized synthetic cannabinoid
JWH-018 were also assessed in biotelemetry assays.
Decreases in heart rate occur with cannabinoids as a result of
inhibition of ongoing locomotor activity, and possibly also as a
result of direct cardiac effects of these drugs. Accordingly,
pronounced inhibition of ongoing locomotor activity was
observed in the home cages with all three doses of Δ9-THC
and JWH-018, and SDB-001, while AB-001 and AB-002
produced only modest and nonsignificant effects even at the
highest doses tested (data not shown).
CONCLUSION
■
The effects of various doses of Δ9-THC, JWH-018, SDB-001,
AB-001, and AB-002 on body temperature are shown in Figure
4. Results for 1 h prior to intraperitoneal (i.p.) injection and 5 h
postinjection are presented in 15 min bins. The gray vertical bar
on the figures (immediately prior to TIME = 0) represents the
time of drug injection. Dose-dependent hypothermia was
This study represents the first attempt to characterize and
quantify the activity of two novel synthetic cannabimimetics of
abuse, AB-001 and SDB-001, and several analogues. The
synthesis of 5−12 provides an efficient and general route to
variously substituted analogues of AB-001 and SDB-001, and
should assist the rapid generation of reference standards for
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Figure 4. Effects of (A) Δ9-THC, (B) JWH-018, (C) SDB-001, (D) low and (E) high doses of AB-001, and (F) AB-002 on rat body temperature.
Gray bar denotes time of intraperitoneal injection.
emergent cannabimimetics based on these chemotypes.
Preliminary SARs for 5−12 suggest that structural hetero-
geneity is tolerated at the 3-indole position, with various
polycyclic, alicyclic, and aromatic groups tolerated by CB1 and
CB2 receptors. All AB and SDB compounds were found to act
as approximately equipotent full agonists at CB1 and CB2
receptors in vitro (with the exception of SDB-002), and
displayed more potent CB1 agonist activity than Δ9-THC.
However, despite the remarkable similarity of the functional
activity profiles of AB-001, AB-002, and SDB-001, only SDB-
001 proved potently cannabimimetic in vivo. SDB-001 dose-
dependently induced hypothermia in rats with a potency similar
to Δ9-THC, and roughly a third as potently as JWH-018. By
contrast, AB-001 and AB-002 showed only hypothermic trends
at a dose three times greater than the dose of SDB-001 eliciting
maximal hypothermia. Additionally, the hypothermic profile of
SDB-001 differed from Δ9-THC and JWH-018, showing
minimal regression to the pretreatment body temperature
after 5 h, suggesting that the effects of SDB-001 may be
relatively long-lived. Similarly, Δ9-THC, JWH-018, and SDB-
001 all dose-dependently reduced heart rate in rats, while AB-
001 and AB-002 did not. Taken together, it appears that the
nature of the functional group linking the adamantane and
indole units, while unimportant for in vitro activity at CB1 and
CB2 receptors, is crucial for cannabimimetic activity. The
potent cannabimimetic of SDB-001, but not AB-001 or AB-002,
confirms that in vivo activity is attributable to the carboxamide
core and not the indole−adamantane distance.
METHODS
■
General Chemical Synthesis Details. All reactions were
performed under an atmosphere of nitrogen or argon unless otherwise
specified. Toluene was dried over sodium wire and distilled from
sodium benzophenone ketyl. Dichloromethane and methanol were
F
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Figure 5. Effects of (A) Δ9-THC, (B) JWH-018, (C) SDB-001, (D) low and (E) high doses of AB-001, and (F) AB-002 on rat heart rate. Gray bar
denotes time of intraperitoneal injection.
distilled from calcium hydride. Anhydrous DMF (Sigma-Aldrich) was
used as purchased. Commercially available chemicals (Sigma-Aldrich)
were used as purchased. Analytical thin layer chromatography (TLC)
was performed using Merck aluminum-backed silica gel 60 F254 (0.2
mm) plates which were visualized using shortwave (254 nm)
ultraviolet fluorescence. Flash chromatography was performed using
Merck Kieselgel 60 (230−400 mesh) silica gel. Melting points were
measured in open capillaries using a Stuart SMP10 Melting Point
Apparatus and are uncorrected. Nuclear magnetic resonance spectra
were recorded at 300 K using either a Bruker AVANCE DRX400
(400.1 MHz) or AVANCE III 500 Ascend (500.1 MHz) spectrometer.
The data are reported as chemical shift (δ ppm) relative to the residual
protonated solvent resonance, relative integral, multiplicity (s = singlet,
br s = broad singlet, d = doublet, t = triplet, q = quartet, sep = septet,
m = multiplet), coupling constants (J Hz), and assignment.
Assignment of signals was assisted by COSY, DEPT, HSQC, and
HMBC experiments where necessary. Low resolution mass spectra
(LRMS) was recorded using electrospray ionization (ESI) recorded on
a Finnigan LCQ ion trap spectrometer. Elemental analysis was
obtained from the Chemical Analysis Facility in the Department of
Chemistry and Biomolecular Sciences, Macquarie University, Australia.
1-Pentylindole (14). A cooled (0 °C) suspension of sodium
hydride (60% dispersion in mineral oil, 1.50 g, 37.5 mmol, 1.5 equiv)
in DMF (45 mL) was treated slowly with a solution of indole (2.93 g,
25.0 mmol) in DMF (5 mL), warmed to ambient temperature and
stirred for 10 min. The mixture was cooled to 0 °C, treated
portionwise with 1-bromopentane (3.25 mL, 26.25 mmol, 1.05 equiv),
warmed to ambient temperature and stirred for 1 h. The reaction was
poured portionwise onto ice−water (400 mL) and extracted with
EtOAc (4 × 50 mL). The combined organic extracts were washed with
H2O (2 × 200 mL), brine (200 mL), dried (MgSO4), and the solvent
evaporated under reduced pressure. Purification by flash chromatog-
raphy, eluting with hexane (Rf 0.32), gave 14 as a pale yellow oil (3.75
1
g, 80%). H NMR (500 MHz, CDCl3): δ 7.64 (1H, d, J = 7.9 Hz),
7.35 (1H, d, J = 8.3 Hz), 7.21 (1H, dd, J = 7.6 Hz), 7.12−7.09 (2H,
m), 6.49 (1H, d, J = 3.1 Hz), 4.12 (2H, t, J = 7.2 Hz), 1.85 (2H, quin.,
J = 7.3 Hz), 1.40−1.26 (4H, m), 0.90 (3H, t, J = 7.1 Hz). All
spectroscopic data matched those previously reported.79
G
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General Procedure for 3-Acylation of 1-Pentylindole. A
solution of the appropriate carboxylic acid (1.2 mmol) in CH2Cl2 (4
mL) was treated with (COCl)2 (240 μL, 2.8 mmol, 2.3 equiv.)
followed by DMF (1 drop). After stirring for 1 h, the solution was
evaporated in vacuo, and the crude acid chloride was used immediately
in the following step.
A cooled (0 °C) solution of 14 (187 mg, 1.0 mmol) in CH2Cl2 (3
mL) was treated dropwise with a solution of 1 M Me2AlCl in hexane
(1.5 mL, 1.5 mmol, 1.5 equiv) and stirred for 30 min. To this solution
was added dropwise a solution of the freshly prepared acid chloride in
CH2Cl2 (3 mL), and the reaction stirred for 2 h. The reaction was
quenched by dropwise addition to a solution of 1 M aq. HCl (6 mL),
the layers separated, and the aqueous phase extracted with CH2Cl2 (2
× 8 mL). The combined organic layers were washed with sat. aq.
NaHCO3 (2 × 12 mL), dried (MgSO4), and the solvent evaporated
under reduced pressure. The crude products were purified by flash
chromatography.
residue was recrystallized from i-PrOH to give 16 as colorless crystals
(13.03 g, 56.2 mmol, 75%). mp (i-PrOH) 106−108 °C; H NMR
1
(500 MHz, CDCl3): δ 8.27−8.24 (1H, m), 7.93 (1H, s), 7.40−7.38
(1H, m), 7.33−7.29 (2H, m), 4.17 (2H, t, J = 7.2 Hz), 1.90 (2H, quin.,
J = 7.2 Hz), 1.39−1.32 (4H, m), 0.91 (3H, t, J = 7.0 Hz); 13C NMR
(125 MHz, CDCl3): δ 170.7 (COOH), 136.9 (quat.), 135.6, 127.2
(quat.), 123.0, 122.3, 122.1, 110.2, 106.4 (quat.), 47.3 (CH2), 29.7
(CH2), 29.1 (CH2), 22.4 (CH2), 14.0; Anal. (C14H17NO2) calcd: C
72.70, H 7.41, N 6.06; found: C 72.67, H 7.77, N 5.93
General Procedure for Amidation of 1-Pentylindole-3-
carboxylic Acid. A solution of 16 (576 mg, 2.50 mmol) in CH2Cl2
(5 mL) was treated with (COCl)2 (420 μL, 5.00 mmol, 2.0 equiv.)
followed by DMF (1 drop). After stirring for 1 h, the solution was
evaporated in vacuo. A solution of the crude acid chloride in CH2Cl2
(5 mL) was added dropwise to a solution of Et3N (870 μL, 6.25 mmol,
2.5 equiv.) and the appropriate amine (3.0 mmol, 1.2 equiv.) in
CH2Cl2 (20 mL). The mixture was stirred for 14 h, the solvent
evaporated, and the residue partitioned between EtOAc (200 mL) and
H2O (25 mL). The layers were separated and the organic phase was
washed with 1 M aq. HCl (3 × 25 mL), sat. aq. NaHCO3 (3 × 25
mL), brine (50 mL), dried (MgSO4), and the solvent evaporated
under reduced pressure. The crude products were recrystallized from i-
PrOH-H2O.
Adamantan-1-yl(1-pentyl-1H-indol-3-yl)methanone (5).
Treating 14 (187 mg, 1.0 mmol) with 1-adamantanecarboxylic acid
(216 mg, 1.2 mmol) according to the general procedure gave,
following purification by flash chromatography (hexane-EtOAc, 95:5,
Rf 0.16), 5 (302 mg, 86%) as a white crystalline solid. Recrystallization
from i-PrOH-H2O yielded colorless needles. mp (i-PrOH-H2O) 125−
1
N-(Adamantan-1-yl)-1-pentyl-1H-indole-3-carboxamide (6).
Subjecting 1-aminoadamantane (454 mg) to the general procedure
gave 6 (787 mg, 86%) as a white crystalline solid. mp (i-PrOH-H2O)
126 °C; H NMR (500 MHz, CDCl3): δ 8.52 (1H, m), 7.93 (1H, s),
7.34 (1H, m), 7.31−7.25 (2H, m), 4.17 (2H, t, J = 7.3 Hz), 2.15 (9H,
br s), 1.90 (2H, quin., J = 7.3 Hz), 1.83 (6H, br s), 1.44−1.30 (4H, m),
0.92 (3H, t, J = 7.0 Hz); 13C NMR (125 MHz, CDCl3): δ 202.1 (CO),
135.7 (quat.), 133.2, 128.7 (quat.), 123.7, 123.2, 122.4, 113.0 (quat.),
109.5, 47.2 (CH2), 47.0 (quat.), 40.7 (CH2), 37.1 (CH2), 29.8 (CH2),
29.1 (CH2), 28.7, 22.4 (CH2), 14.1; LRMS (+ESI) m/z 720.93 ([2M
+ Na]+, 100%), 371.93 ([M + Na]+, 4%), 350.00 ([M + H]+, 10%);
Anal. (C24H31NO) calcd: C 82.47, H 8.94, N 4.01; found: C 82.37, H
9.33, N 3.93.
1
140−141 °C; H NMR (500 MHz, CDCl3): δ 7.87 (1H, d, J = 7.5
Hz), 7.65 (1H, s), 7.36 (1H, d, J = 8.0 Hz), 7.28−7.22 (2H, m), 5.71
(1H, br s, NH), 4.11 (2H, t, J = 7.2 Hz), 2.19 (6H, br s), 2.14 (3H, br
s), 1.84 (2H, quin., J = 7.3 Hz), 1.76 (6H, m) 1.39−1.24 (4H, m), 0.88
(3H, t, J = 7.0 Hz); 13C NMR (125 MHz, CDCl3): δ 164.7 (CO),
136.7 (quat.), 131.5, 125.3 (quat.), 122.3, 121.2, 120.0, 112.3 (quat.),
110.4, 52.2 (quat.), 46.9 (CH2), 42.4 (CH2), 36.6 (CH2), 29.8, 29.7
(CH2), 29.1 (CH2), 22.4 (CH2), 14.0; LRMS (+ESI) m/z 751.80
([2M + Na]+, 100%), 387.67 ([M + Na]+, 55%); Anal. (C24H32N2O)
calcd: C 79.08, H 8.85, N 7.68; found: C 79.13, H 8.86, N 7.47.
N-(Adamantan-1-ylmethyl)-1-pentyl-1H-indole-3-carboxa-
mide (8). Subjecting 1-(aminomethyl)adamantane (496 mg) to the
general procedure gave 8 (748 mg, 79%) as a white crystalline solid.
2-(Adamantan-1-yl)-1-(1-pentyl-1H-indol-3-yl)ethanone (7).
Treating 14 (936 mg, 5.0 mmol) with 1-adamantaneacetic acid (1.17
g, 6.0 mmol) according to the general procedure gave, following
purification by flash chromatography (hexane-EtOAc, 90:10, Rf 0.18),
7 (636 mg, 35%) as a white crystalline solid. mp 93−94 °C; 1H NMR
(500 MHz, CDCl3): δ 8.49 (1H, m), 7.68 (1H, s), 7.35 (1H, m),
7.32−7.27 (2H, m), 4.16 (2H, t, J = 7.3 Hz), 2.56 (2H, s), 1.95 (3H,
br s), 1.89 (2H, quin., J = 7.4 Hz), 1.74−1.60 (12H, m), 1.41−1.27
(4H, m), 0.90 (3H, t, J = 7.3 Hz); 13C NMR (125 MHz, CDCl3): δ
195.1 (CO), 136.9 (quat.), 135.2, 126.7 (quat.), 123.32, 123.25, 122.6,
118.8 (quat.), 109.8, 54.0 (CH2), 47.2 (CH2), 43.5 (CH2), 37.0
(CH2), 33.9 (quat.), 29.7 (CH2), 29.2 (CH2), 29.0, 22.4 (CH2), 14.1;
LRMS (+ESI) m/z 111.47 ([3 M + Na]+, 100%), 749.07 ([2 M +
Na]+, 72%); Anal. (C25H33NO) calcd: C 82.60, H 9.15, N 3.85; found:
C 82.27, H 9.54, N 3.98.
1-Pentylindole-3-carboxylic Acid (16). A cooled (0 °C)
suspension of sodium hydride (60% dispersion in mineral oil, 6.000
g, 150.0 mmol, 2.0 equiv) in DMF (90 mL) was treated slowly with a
solution of indole (8.790 g, 75.03 mmol) in DMF (10 mL), warmed to
ambient temperature, and stirred for 10 min. The mixture was cooled
to 0 °C, treated portionwise with 1-bromopentane (9.75 mL, 78.6
mmol, 1.05 equiv), warmed to ambient temperature and stirred for 1
h. The solution was cooled to 0 °C, treated slowly with trifluoroacetic
anhydride (26.10 mL, 187.5 mmol, 2.5 equiv), warmed to ambient
temperature and stirred for 1 h. The reaction was poured portionwise
onto vigorously stirred ice−water (1000 mL), and stirred (with
external ice-bath cooling if necessary) until complete solidification of
the product occurred. The precipitate was collected by filtration and
air-dried to give crude 15 as a pink solid (21.16 g, 74.69 mmol, 100%).
A solution of crude 15 (21.00 g, 74.13 mmol) in PhMe (65 mL)
was slowly added to a refluxing solution of KOH (13.26 g, 236.3
mmol, 3.3 equiv) in MeOH (25 mL), and the mixture heated at reflux
for 2 h. The mixture was cooled to ambient temperature and H2O
(250 mL) was added. The layers were separated, and the organic layer
was extracted with 1 M aq. NaOH (70 mL). The combined aqueous
phases were acidified to pH 1 with 10 M aq. HCl, extracted with Et2O
(3 × 100 mL), dried (MgSO4), and the solvent evaporated. The crude
1
mp (i-PrOH-H2O) 160−162 °C; H NMR (500 MHz, CDCl3): δ
7.89−7.87 (1H, m), 7.75 (1H, s), 7.40−7.38 (1H, m), 7.31−7.22 (2H,
m), 6.03 (1H, br s, NH), 4.12 (2H, t, J = 7.3 Hz), 3.22 (2H, d, J = 6.5
Hz), 2.01 (3H, br s), 1.86 (2H, quin., J = 7.0 Hz), 1.69 (6H, m), 1.61
(6H, br s), 1.39−1.27 (4H, m), 0.88 (3H, t, J = 7.0 Hz); 13C NMR
(125 MHz, CDCl3): δ 165.6 (CO), 136.8 (quat.), 132.0, 125.2 (quat.),
122.4, 121.4, 119.9, 111.3 (quat.), 110.6, 51.0 (CH2), 47.0 (CH2), 40.6
(CH2), 37.6 (CH2), 34.1 (quat.), 29.8 (CH2), 29.1 (CH2), 28.4, 22.4
(CH2), 14.0; LRMS (+ESI) m/z 779.73 ([2 M + Na]+, 100%), 401.67
([M + Na]+, 42%); Anal. (C25H34N2O) calcd: C 79.32, H 9.05, N
7.40; found: C 79.15, H 9.00, N 7.19.
N-Cyclohexyl-1-pentyl-1H-indole-3-carboxamide (9). Subject-
ing cyclohexylamine (340 μL) to the general procedure gave 9 (633
mg, 81%) as a white crystalline solid. mp (i-PrOH-H2O) 156−157 °C;
1H NMR (500 MHz, CDCl3): δ 7.87 (1H, d, J = 7.1 Hz), 7.79 (1H, s),
7.38 (1H, d, J = 7.3 Hz), 7.29−7.23 (2H, m), 5.89 (1H, br s, NH),
4.13 (2H, t, J = 7.2 Hz), 4.10−4.04 (1H, m), 2.10−2.07 (2H, m), 1.86
(2H, quin., J = 7.3 Hz), 1.77 (2H, dt, J = 13.7, 3.9 Hz), 1.66 (1H, dt, J
= 13.1, 3.9 Hz), 1.51−1.43 (2H, m), 1.39−1.20 (7H, m), 0.89 (3H, t, J
= 7.0 Hz); 13C NMR (125 MHz, CDCl3): δ 164.6 (CO), 136.7
(quat.), 131.7 (quat.), 125.4 (quat.), 122.4, 121.4, 120.0, 111.2 (quat.),
110.5, 48.2, 47.0 (CH2), 33.7 (CH2), 29.8 (CH2), 29.2 (CH2), 25.8
(CH2), 25.1 (CH2), 22.4 (CH2), 14.0; LRMS (+ESI) m/z 647.60
([2M + Na]+, 39%), 335.60 ([M + Na]+, 100%); Anal. (C20H28N2O)
calcd: C 76.88, H 9.03, N 8.97; found: C 76.48, H 9.18, N 8.96.
N-(Cyclohexylmethyl)-1-pentyl-1H-indole-3-carboxamide
(10). Subjecting (aminomethyl)cyclohexane (390 μL) to the general
procedure gave 10 (594 mg, 73%) as a white crystalline solid. mp (i-
PrOH-H2O) 123−125 °C; 1H NMR (400 MHz, CDCl3): δ 7.90−7.86
(1H, m), 7.72 (1H, s), 7.41−7.36 (1H, m), 7.30−7.23 (2H, m), 6.00
(1H, br s, NH), 4.12 (2H, t, J = 7.2 Hz), 3.36 (2H, t, J = 6.4 Hz),
H
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1.89−1.82 (4H, m), 1.75 (2H, dt, J = 12.8, 3.4 Hz), 1.70−1.57 (4H,
m), 1.39−1.13 (7H, m), 1.09−0.99 (2H, m), 0.88 (3H, t, J = 7.0 Hz);
13C NMR (100 MHz, CDCl3): δ 165.5 (CO), 136.8 (quat.), 131.7
(quat.), 125.3 (quat.), 122.4, 121.4, 120.0, 111.3 (quat.), 110.5, 47.0
(CH2), 45.9 (CH2), 38.4, 31.2 (CH2), 29.8 (CH2), 29.2 (CH2), 26.6
(CH2), 26.1 (CH2), 22.4 (CH2), 14.0; LRMS (+ESI) m/z 675.67
([2M + Na]+, 61%), 349.60 ([M + Na]+, 100%); Anal. (C21H30N2O)
calcd: C 77.26, H 9.26, N 8.58; found: C 77.28, H 9.62, N 8.61.
N-Phenyl-1-pentyl-1H-indole-3-carboxamide (11). Subjecting
aniline (275 μL) to the general procedure gave 11 (622 mg, 81%) as a
white crystalline solid. mp (i-PrOH-H2O) 125−128 °C; 1H NMR
(500 MHz, CDCl3): δ 8.07−8.05 (1H, m), 7.80 (1H, s), 7.76 (1H, br
s, NH) 7.67 (2H, d, J = 7.6 Hz), 7.42−7.40 (1H, m), 7.37 (2H, dd, J =
8.2, 7.7 Hz), 7.33−7.28 (2H, m), 7.12 (1H, t, J = 7.4 Hz), 4.13 (2H, t,
J = 7.2 Hz), 1.87 (2H, quin., J = 7.3 Hz), 1.39−1.28 (4H, m), 0.89
(3H, t, J = 7.0 Hz); 13C NMR (125 MHz, CDCl3): δ 163.4 (CO),
138.7 (quat.), 136.8 (quat.), 131.9, 129.2, 125.6 (quat.), 124.0, 122.8,
121.8, 120.3, 120.2, 111.2 (quat.), 110.6, 47.1 (CH2), 29.8 (CH2), 29.1
(CH2), 22.4 (CH2), 14.0; LRMS (+ESI) m/z 329.53 ([M + Na]+,
100%); Anal. (C20H22N2O) calcd: C 78.40, H 7.24, N 9.14; found: C
78.01, H 7.57, N 8.77.
N-Benzyl-1-pentyl-1H-indole-3-carboxamide (12). Subjecting
benzylamine (330 μL) to the general procedure gave 12 (678 mg,
84%) as a white crystalline solid. mp (i-PrOH-H2O) 101−102 °C; 1H
NMR (500 MHz, CDCl3): δ 7.91 (1H, d, J = 7.7 Hz), 7.73 (1H, s),
7.42−7.35 (4H, m), 7.31−7.21 (4H, m), 6.19 (1H, br s, NH), 4.72
(2H, d, J = 5.7 Hz), 4.13 (2H, t, J = 7.2 Hz), 1.86 (2H, quin., J = 7.3
Hz), 1.37−1.29 (4H, m), 0.89 (3H, t, J = 7.0 Hz); 13C NMR (125
MHz, CDCl3): δ 165.3 (CO), 139.1 (quat.), 136.8 (quat.), 131.7,
128.9, 128.0, 127.6, 125.5 (quat.), 122.5, 121.5, 120.3, 110.8 (quat.),
110.5, 47.0 (CH2), 43.7 (CH2), 29.8 (CH2), 29.2 (CH2), 22.4 (CH2),
14.0; LRMS (+ESI) m/z 343.60 ([M + Na]+, 100%); Anal.
(C21H24N2O) calcd: C 78.71, H 7.55, N 8.74; found: C 78.87, H
7.74, N 8.77.
containing 10% fetal bovine serum (FBS), 100 U penicillin/
streptomycin, and 300 μg/mL G418. The cDNA clone for the
human CB2 receptor (GenBank accession number AY242132) with 3
× N-terminal HA tags was obtained from the Missouri S&T cDNA
Resource Center (www.cdna.org). Cells were passaged at 80%
confluency as required. Cells for assays were grown in 75 cm2 flasks
and used at 90% confluence. The day before the assay cells were
detached from the flask with trypsin/EDTA (Sigma) and resuspended
in 10 mL of Leibovitz’s L-15 media supplemented with 1% FBS, 100U
penicillin/streptomycin and 15 mM glucose (membrane potential
assay and Ca5 calcium assay). The cells were plated in volume of 90
μL in black walled, clear bottomed 96-well microplates (Corning)
which had been precoated with poly-L-lysine (Sigma, Australia). Cells
were incubated overnight at 37 °C in ambient CO2.
Membrane potential was measured using a FLIPR Membrane
Potential Assay kit (blue) from Molecular Devices, as described
previously.71 The dye was reconstituted with assay buffer of
composition (mM): NaCl 145, HEPES 22, Na2HPO4 0.338,
NaHCO3 4.17, KH2PO4 0.441, MgSO4 0.407, MgCl2 0.493, CaCl2
1.26, glucose 5.56 (pH 7.4, osmolarity 315 5). Prior to the assay,
cells were loaded with 90 μL/well of the dye solution without removal
of the L-15, giving an initial assay volume of 180 μL/well. Plates were
then incubated at 37 °C at ambient CO2 for 45 min. Fluorescence was
measured using a FlexStation 3 (Molecular Devices) microplate reader
with cells excited at a wavelength of 530 nm and emission measured at
565 nm. Baseline readings were taken every 2 s for at least 2 min, at
which time either drug or vehicle was added in a volume of 20 μL. The
background fluorescence of cells without dye or dye without cells was
negligible. Changes in fluorescence were expressed as a percentage of
baseline fluorescence after subtraction of the changes produced by
vehicle addition, which was less than 2% for drugs dissolved in assay
buffer or DMSO. The final concentration of DMSO was not more
than 0.1%.
Data was analyzed with PRISM (GraphPad Software Inc., San
Diego, CA), using four-parameter nonlinear regression to fit
concentration−response curves. In all plates, a maximally effective
concentration of WIN 55,212-2 was added to allow for normalization
between assays.
In Vivo Pharmacological Assessment of 5−12. Subjects. The
experiments involved 16 adult male Wistar rats (obtained from Animal
Resources Centre, Perth, Australia) weighing between 190 and 220 g
at the start of the experiment. Throughout the period of data
acquisition, the rats were singly housed in testing cages in an air-
conditioned testing room (ambient temperature 22 1 °C) with a 12
h reverse light/dark cycle (lights on from 7:00 to 19:00). Standard
rodent chow and water were provided ad libitum. All efforts were
made to minimize both the number of rats used and the distress to
those involved. All experiments were approved by the University of
Sydney Animal Ethics Committee.
X-ray Data Collection. The solid was crystallized from i-PrOH-
H2O to give colorless needle crystals by slow evaporation at ambient
temperature.
The single-crystal X-ray diffraction experiments were carried out at
the Faculty of Pharmacy, University of Sydney using a Bruker APEX-II
CCD-based diffractometer with an X-ray wavelength of 0.71073 Å
(Mo Kα) and at an experimental temperature of 150 K. The single
crystal of X was mounted on the tip of a thin glass fiber with a
minimum amount of Paratone N oil, which acted as both an adhesive
and a cryoprotectant, and inserted in the cold N2 stream of an Oxford
Cryosystem COBRA cooler. X-ray diffraction data were collected
using 0.3° Δω-scans, maintaining the crystal-to-detector distance at 6.0
cm. A total of 1664 frames were collected. The diffraction data were
integrated using SAINT+,80 which included corrections for Lorentz,
polarization and absorption effects. Unit cell parameters for 5 at 150 K
were refined from 250 reflections.
Surgery. Biotelemetry transmitters (TA11CTA-F40, Data Sciences
International, St. Paul, MN) were implanted according to
manufacturers instructions. Briefly, the rats were anaesthetized with
isoflurane (3% induction, 2% maintenance) and once adequate depth
of anesthesia was reached, a rostro-caudal incision was made along the
midline of the abdomen. A biotelemetry transmitter was placed in the
peritoneal cavity according to the manufacturer’s protocol and the
wound sutured closed. The rats were allowed one week of recovery,
during which their wellbeing was monitored daily, before data
collection commenced.
The structure was solved using Direct Methods (SHELX-S),81 and
refined using full-matrix least-squares (SHELXL).81 All non-hydrogen
atoms were treated as anisotropic, while hydrogen atoms were placed
in idealized positions, with Ueq set at 1.5 times that of the parent atom.
empirical formula
formula weight
temperature
C24 H31 NO
349.50
150(2) K
crystal system, space group monoclinic, P2(1)/n
unit cell dimensions
a = 12.022 Å, α =90°
b = 20.718 Å, β = 101.25°
c = 16.094 Å, γ = 90°
8
Biotelemetry System. The biotelemetry system (Data Science
International, D.S.I., St. Paul, MN) was used to measure heart rate,
body temperature, and physical activity in freely moving rats in their
home cages. Data for these three variables were gathered continuously
at 1000 Hz and organized in 5 min bins. Data was acquired, processed
and analyzed using the Dataquest A.R.T. software (version 4.3, D.S.I.,
St. Paul, MN).
Z
goodness-of-fit on F2
final R indices [I>2σ(I)]
R indices (all data)
1.030
R1 = 0.0584, wR2 = 0.1571
R1 = 0.0872, wR2 = 0.1764
Procedure. Each drug (Δ9-THC, JWH-018, SDB-001, AB-001, AB-
̈
In Vitro Pharmacological Assessment of 5−12. Mouse AtT20
002) was tested in a cohort of four drug-naive rats using a within-
72
neuroblastoma cells stably transfected with rat CB1 or human CB2
subjects design. To minimize the effects of injection stress on
were cultured in Dulbecco’s modified Eagle’s medium (DMEM)
physiological parameters, rats were habituated over several days to
I
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ACS Chemical Neuroscience
Research Article
receiving injections of vehicle at a particular time of day (12:30 pm)
and then being immediately returned to their home cages. An
ascending dose response sequence was then employed in each
experiment where the lowest dose of the drug was tested, followed by
the next lowest dose, and so on until a maximal physiological response
was seen. This ensured the safety of animals when hitherto untested
compounds were being assessed. Two washout days were given in
between each dose. In some cases, a further vehicle test was done after
the completion of drug testing to ensure that baselines, before and
after drug treatment, had not changed.
REFERENCES
■
(1) Howlett, A. C., Barth, F., Bonner, T. I., Cabral, G., Casellas, P.,
Devane, W. A., Felder, C. C., Herkenham, M., Mackie, K., Martin, B.
R., Mechoulam, R., and Pertwee, R. G. (2002) International Union of
Pharmacology. XXVII. Classification of cannabinoid receptors.
Pharmacol. Rev. 54, 161−202.
(2) Mackie, K. (2008) Cannabinoid receptors: Where they are and
what they do. J. Neuroendocrinol. 20, 10−14.
(3) Gaoni, Y., and Mechoulam, R. (1964) Isolation, structure, and
partial synthesis of an active constituent of hashish. J. Am. Chem. Soc.
86, 1646−1647.
(4) Pertwee, R. G., Howlett, A. C., Abood, M. E., Alexander, S. P. H.,
Di Marzo, V., Elphick, M. R., Greasley, P. J., Hansen, H. S., Kunos, G.,
Mackie, K., Mechoulam, R., and Ross, R. A. (2010) International
Union of Basic and Clinical Pharmacology. LXXIX. Cannabinoid
receptors and their ligands: Beyond CB1 and CB2. Pharmacol. Rev. 62,
588−631.
(5) UNODC. UNODC, World Drug Report 2011 (United Nations
Publication, Sales No. E.11.XI.10).
(6) Zuardi, A. W. (2006) History of cannabis as a medicine: A review.
Drugs. Δ9-THC was obtained from Sigma Aldrich and JWH-018
from the National Measurement Institute (Pymble, Sydney, NSW,
Australia). All other drugs were synthesized by the authors. Drugs
were initially dissolved in 5% ethanol, then 5% Tween 80 was added,
and then 90% physiological saline was added. All drugs were injected
intraperitoneally at a volume of 1 mL/kg.
Statistical Analysis. Activity, body temperature, and heart rate data
were compared across conditions using software SPSS version 19.0.
Planned contrasts (two-way ANOVA) compared each dose of the drug
with vehicle treatment for (a) 1 h before treatment and (b) 5 h after
treatment. The minimum level of statistical significance was set at p <
0.05.
Rev. Bras. Psiquiatr. 28, 153−157.
(7) Pertwee, R. (2005) The therapeutic potential of drugs that target
cannabinoid receptors or modulate the tissue levels or actions of
endocannabinoids. AAPS J. 7, E625−E654.
ASSOCIATED CONTENT
■
S
* Supporting Information
(8) Mackie, K. (2006) Cannabinoid receptors as therapeutic targets.
Annu. Rev. Pharmacol. Toxicol. 46, 101−122.
X-ray crystallographic data; selected H and 13C NMR spectra.
1
This material is available free of charge via the Internet at
http://pubs.acs.org.
(9) Bellocchio, L., Mancini, G., Vicennati, V., Pasquali, R., and
Pagotto, U. (2006) Cannabinoid receptors as therapeutic targets for
obesity and metabolic diseases. Curr. Opin. Pharmacol. 6, 586−591.
(10) Guindon, J., and Hohmann, A. G. (2008) Cannabinoid CB2
receptors: A therapeutic target for the treatment of inflammatory and
neuropathic pain. Br. J. Pharmacol. 153, 319−334.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: michael.kassiou@sydney.edu.au.
(11) Janero, D. R., and Makriyannis, A. (2009) Cannabinoid receptor
antagonists: Pharmacological opportunities, clinical experience, and
translational prognosis. Expert Opin. Emerging Drugs 14, 43−65.
(12) Oesch, S., and Gertsch, J. (2009) Cannabinoid receptor ligands
as potential anticancer agents  high hopes for new therapies? J.
Pharm. Pharmacol. 61, 839−853.
(13) Turcotte, D., Dorze, J.-A. L., Esfahani, F., Frost, E., Gomori, A.,
and Namaka, M. (2010) Examining the roles of cannabinoids in pain
and other therapeutic indications: A review. Expert Opin. Pharmac-
other. 11, 17−31.
(14) Wilkinson, S. M., Price, J., and Kassiou, M. (2013) Improved
accessibility to the desoxy analogues of Δ9-tetrahydrocannabinol and
cannabidiol. Tetrahedron Lett. 54, 52−54.
(15) Weissman, A., Milne, G. M., and Melvin, L. S. (1982)
Cannabimimetic activity from CP-47,497, a derivative of 3-phenyl-
cyclohexanol. J. Pharmacol. Exp. Ther. 223, 516−523.
(16) D’Ambra, T. E., Estep, K. G., Bell, M. R., Eissenstat, M. A., Josef,
K. A., Ward, S. J., Haycock, D. A., Baizman, E. R., and Casiano, F. M.
(1992) Conformationally restrained analogs of pravadoline: Nano-
molar potent, enantioselective, (aminoalkyl)indole agonists of the
cannabinoid receptor. J. Med. Chem. 35, 124−135.
(17) Huffman, J. W., Dai, D., Martin, B. R., and Compton, D. R.
(1994) Design, synthesis and pharmacology of cannabimimetic
indoles. Bioorg. Med. Chem. Lett. 4, 563−566.
(18) Wiley, J. L., Compton, D. R., Dai, D., Lainton, J. A. H., Phillips,
M., Huffman, J. W., and Martin, B. R. (1998) Structure-activity
relationships of indole- and pyrrole-derived cannabinoids. J. Pharmacol.
Exp. Ther. 285, 995−1004.
Author Contributions
S.D.B., S.M.W., and M.L. performed the synthesis, purification,
and chemical characterization of compounds 5−12 under the
supervision of M.K. J.S. conducted all in vitro pharmacological
evaluation with guidance from M.C. N.A. and K.E. carried out
all behavioral pharmacology with direction from I.S.M. L.B. and
C.G. compared synthesized SDB-001 (6) with a forensic
sample. D.E.H. provided X-ray crystallographic analysis of AB-
001 (5). M.G. assisted the creation of stably transfected cells
expressing hCB2R. The manuscript was prepared by M.C.,
I.S.M., and M.K., based on drafts written by S.D.B., S.M.W., and
N.A. All authors have given approval to the final version of the
manuscript.
Funding
Work performed at The University of Sydney and presented
herein was supported in part by the European Union’s Seventh
Framework Programme [FP7/2007-2013] INMiND (Grant
agreement No. HEALTH-F2-2011-278850). Work performed
at Macquarie University and presented herein was supported by
NH&MRC grant 1002680 awarded to M.C. and M.K. J.S. is the
recipient of an International Research Scholarship from
Macquarie University.
Notes
The authors declare no competing financial interest.
(19) Aung, M. M., Griffin, G., Huffman, J. W., Wu, M.-J., Keel, C.,
Yang, B., Showalter, V. M., Abood, M. E., and Martin, B. R. (2000)
Influence of the N-1 alkyl chain length of cannabimimetic indoles
upon CB1 and CB2 receptor binding. Drug Alcohol Depend. 60, 133−
140.
ABBREVIATIONS
■
CB, cannabinoid; FLIPR, fluorometric imaging plate reader;
GIRK, G-protein-gated inwardly rectifying K+ channels; i.p.,
intraperitoneal; SAR, structure−activity relationship; SLID,
synthetic legal intoxicating drug; Δ9-THC, Δ9-tetrahydrocan-
nabinol; TLC, thin layer chromatography
(20) Auwarter, V., Dresen, S., Weinmann, W., Muller, M., Putz, M.,
̈
̈
̈
́
and Ferreiros, N. (2009) ‘Spice’ and other herbal blends: Harmless
incense or cannabinoid designer drugs? J. Mass Spectrom. 44, 832−837.
J
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ACS Chemical Neuroscience
Research Article
(21) Vardakou, I., Pistos, C., and Spiliopoulou, C. (2010) Spice drugs
as a new trend: Mode of action, identification and legislation. Toxicol.
Lett. 197, 157−162.
(44) Dresen, S., Ferreiros
́
, N., Putz, M., Westphal, F., Zimmermann,
̈
R., and Auwarter, V. (2010) Monitoring of herbal mixtures potentially
̈
containing synthetic cannabinoids as psychoactive compounds. J. Mass
Spectrom. 45, 1186−1194.
(22) Fattore, L., and Fratta, W. (2011) Beyond THC: The new
generation of cannabinoid designer drugs. Front. Behav. Neurosci. 5, 60.
(23) Barratt, M. J., Cakic, V., and Lenton, S. (2012) Patterns of
synthetic cannabinoid use in Australia. Drug Alcohol Rev. 32, 141−146.
(24) Lapoint, J., James, L. P., Moran, C. L., Nelson, L. S., Hoffman, R.
S., and Moran, J. H. (2011) Severe toxicity following synthetic
cannabinoid ingestion. Clin. Toxicol. 49, 760−764.
(45) Hudson, S., Ramsey, J., King, L., Timbers, S., Maynard, S.,
Dargan, P. I., and Wood, D. M. (2010) Use of high-resolution accurate
mass spectrometry to detect reported and previously unreported
cannabinomimetics in “herbal high” products. J. Anal. Toxicol. 34,
252−260.
(46) Ernst, L., Schiebel, H.-M., Theuring, C., Lindigkeit, R., and
Beuerle, T. (2011) Identification and characterization of JWH-122
used as new ingredient in ″Spice-like″ herbal incenses. Forensic Sci. Int.
208, e31−e35.
(25) Mir, A., Obafemi, A., Young, A., and Kane, C. (2011)
Myocardial infarction associated with use of the synthetic cannabinoid
K2. Pediatrics 128, e1622−e1627.
(26) Simmons, J., Cookman, L., Kang, C., and Skinner, C. (2011)
Three cases of “spice” exposure. Clin. Toxicol. 49, 431−433.
(27) Young, A. C., Schwarz, E., Medina, G., Obafemi, A., Feng, S.-Y.,
Kane, C., and Kleinschmidt, K. (2012) Cardiotoxicity associated with
the synthetic cannabinoid, K9, with laboratory confirmation. Am. J.
Emerg. Med. 30, 1320.e5−1320.e7.
(28) Heath, T. S., Burroughs, Z., Thompson, A. J., and Tecklenburg,
F. W. (2012) Acute intoxication caused by a synthetic cannabinoid in
two adolescents. J. Pediatr. Pharmacol. Ther. 17, 177−181.
(29) Schneir, A., and Baumbacher, T. (2012) Convulsions associated
with the use of a synthetic cannabinoid product. J. Med. Toxicol. 8, 62−
64.
(47) Nakajima, J. i., Takahashi, M., Nonaka, R., Seto, T., Suzuki, J.,
Yoshida, M., Kanai, C., and Hamano, T. (2011) Identification and
quantitation of a benzoylindole (2-methoxyphenyl)(1-pentyl-1H-
indol-3-yl)methanone and a naphthoylindole 1-(5-fluoropentyl-1H-
indol-3-yl)-(naphthalene-1-yl)methanone (AM-2201) found in illegal
products obtained via the Internet and their cannabimimetic effects
evaluated by in vitro [35S]GTPγS binding assays. Forensic Toxicol. 29,
132−141.
(48) Nakajima, J. i., Takahashi, M., Seto, T., and Suzuki, J. (2011)
Identification and quantitation of cannabimimetic compound JWH-
250 as an adulterant in products obtained via the internet. Forensic
Toxicol. 29, 51−55.
(30) Harris, C. R., and Brown, A. (2013) Synthetic cannabinoid
intoxication: A case series and review. J. Emerg. Med. 44, 360−366.
(31) Karch, S. (2006) Cannabis and cardiotoxicity. Forensic Sci. Med.
Pathol. 2, 13−18.
(32) Schneir, A. B., Cullen, J., and Ly, B. T. (2011) Spice” girls:
Synthetic cannabinoid intoxication. J. Emerg. Med. 40, 296−299.
(33) Every-Palmer, S. (2010) Warning: Legal synthetic cannabinoid-
receptor agonists such as JWH-018 may precipitate psychosis in
vulnerable individuals. Addiction 105, 1859−1860.
(49) Nakajima, J.-i., Takahashi, M., Seto, T., Kanai, C., Suzuki, J.,
Yoshida, M., and Hamano, T. (2011) Identification and quantitation of
two benzoylindoles AM-694 and (4-methoxyphenyl)(1-pentyl-1H-
indol-3-yl)methanone, and three cannabimimetic naphthoylindoles
JWH-210, JWH-122, and JWH-019 as adulterants in illegal products
obtained via the internet. Forensic Toxicol. 29, 95−110.
(50) Uchiyama, N., Kawamura, M., Kikura-Hanajiri, R., and Goda, Y.
(2011) Identification and quantitation of two cannabimimetic
phenylacetylindoles JWH-251 and JWH-250, and four cannabimimetic
naphthoylindoles JWH-081, JWH-015, JWH-200, and JWH-073 as
designer drugs in illegal products. Forensic Toxicol. 29, 25−37.
(51) Uchiyama, N., Kikura-Hanajiri, R., and Goda, Y. (2011)
Identification of a novel cannabimimetic phenylacetylindole, cannabi-
piperidiethanone, as a designer drug in a herbal product and its affinity
for cannabinoid CB1 and CB2 receptors. Chem. Pharm. Bull. 59,
1203−1205.
(34) Every-Palmer, S. (2011) Synthetic cannabinoid JWH-018 and
psychosis: An explorative study. Drug Alcohol Depend. 117, 152−157.
(35) Brakoulias, V. (2012) Products containing synthetic cannabi-
noids and psychosis. Aust. N. Z. J. Psychiatry 46, 281−282.
(36) Wells, D. L., and Ott, C. A. (2011) The “new” marijuana. Ann.
Pharmacother. 45, 414−417.
(37) Hutter, M., Broecker, S., Kneisel, S., and Auwarter, V. (2012)
̈
Identification of the major urinary metabolites in man of seven
synthetic cannabinoids of the aminoalkylindole type present as
adulterants in ‘herbal mixtures’ using LC-MS/MS techniques. J. Mass
Spectrom. 47, 54−65.
(52) Kneisel, S., Bisel, P., Brecht, V., Broecker, S., Muller, M., and
̈
Auwarter, V. (2012) Identification of the cannabimimetic AM-1220
̈
and its azepane isomer (N-methylazepan-3-yl)-3-(1-naphthoyl)indole
in a research chemical and several herbal mixtures. Forensic Toxicol. 30,
126−134.
(38) Brents, L. K., Reichard, E. E., Zimmerman, S. M., Moran, J. H.,
Fantegrossi, W. E., and Prather, P. L. (2011) Phase I hydroxylated
metabolites of the K2 synthetic cannabinoid JWH-018 retain in vitro
and in vivo cannabinoid 1 receptor affinity and activity. PLoS One 6,
e21917.
(53) Kneisel, S., and Auwaerter, V. (2012) Analysis of 30 synthetic
cannabinoids in serum by liquid chromatography-electrospray
ionization tandem mass spectrometry after liquid-liquid extraction. J.
Mass Spectrom. 47, 825−835.
(39) Seely, K. A., Brents, L. K., Radominska-Pandya, A., Endres, G.
W., Keyes, G. S., Moran, J. H., and Prather, P. L. (2012) A major
glucuronidated metabolite of JWH-018 is a neutral antagonist at CB1
receptors. Chem. Res. Toxicol. 25, 825−827.
(40) Brents, L. K., Gallus-Zawada, A., Radominska-Pandya, A.,
Vasiljevik, T., Prisinzano, T. E., Fantegrossi, W. E., Moran, J. H., and
Prather, P. L. (2012) Monohydroxylated metabolites of the K2
synthetic cannabinoid JWH-073 retain intermediate to high
cannabinoid 1 receptor (CB1R) affinity and exhibit neutral antagonist
to partial agonist activity. Biochem. Pharmacol. 83, 952−961.
(41) Jerry, J., Collins, G., and Streem, D. (2012) Synthetic legal
intoxicating drugs: The emerging ‘incense’ and ‘bath salt’ phenomen-
on. Clev. Clin. J. Med. 79, 258−264.
(42) Lindigkeit, R., Boehme, A., Eiserloh, I., Luebbecke, M.,
Wiggermann, M., Ernst, L., and Beuerle, T. (2009) Spice: A never
ending story? Forensic Sci. Int. 191, 58−63.
(43) Uchiyama, N., Kikura-Hanajiri, R., Kawahara, N., and Goda, Y.
(2009) Identification of a cannabimimetic indole as a designer drug in
a herbal product. Forensic Toxicol. 27, 61−66.
(54) Nakajima, J.-i., Takahashi, M., Seto, T., Yoshida, M., Kanai, C.,
Suzuki, J., and Hamano, T. (2012) Identification and quantitation of
two new naphthoylindole drugs-of-abuse, (1-(5-hydroxypentyl)-1H-
indol-3-yl)(naphthalen-1-yl)methanone (AM-2202) and (1-(4-penten-
yl)-1H-indol-3-yl)(naphthalen-1-yl)methanone, with other synthetic
cannabinoids in unregulated ″herbal″ products circulated in the Tokyo
area. Forensic Toxicol. 30, 33−44.
(55) Takahashi, K., Uchiyama, N., Fukiwake, T., Hasegawa, T.,
Saijou, M., Motoki, Y., Kikura-Hanajiri, R., and Goda, Y. (2013)
Identification and quantitation of JWH-213, a cannabimimetic indole,
as a designer drug in a herbal product. Forensic Toxicol. 31, 145−150.
(56) Westphal, F., Sonnichsen, F. D., and Thiemt, S. (2012)
̈
Identification of 1-butyl-3-(1-(4-methyl)naphthoyl)indole in a herbal
mixture. Forensic Sci. Int. 215, 8−13.
(57) Zuba, D., and Byrska, B. (2013) Analysis of the prevalence and
coexistence of synthetic cannabinoids in “herbal high” products in
Poland. Forensic Toxicol. 31, 21−30.
(58) Grigoryev, A., Kavanagh, P., and Melnik, A. (2012) The
detection of the urinary metabolites of 3-[(adamantan-1-yl)carbonyl]-
K
dx.doi.org/10.1021/cn400035r | ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience
Research Article
1-pentylindole (AB-001), a novel cannabimimetic, by gas chromatog-
(73) Hine, B., Torrelio, M., and Gershon, S. (1977) Analgesic, heart
rate, and temperature effects of Δ8-THC during acute and chronic
administration to conscious rats. Pharmacology 15, 65−72.
(74) Fennessy, M. R., and Taylor, D. A. (1978) Antagonism of the
effects on thermoregulation of Δ9-tetrahydrocannabinol by clomipr-
amine in the rat. Br. J. Pharmacol. 63, 267−273.
(75) Smirnov, M. S., and Kiyatkin, E. A. (2008) Behavioral and
temperature effects of delta 9-tetrahydrocannabinol in human-relevant
doses in rats. Brain Res. 1228, 145−160.
raphy-mass spectrometry. Drug Test. Anal. 4, 519−524.
́ ́
(59) Jankovics, P., Varadi, A., Tolgyesi, L., Lohner, S., Nemeth-
̈
́
Palotas, J., and Balla, J. (2012) Detection and identification of the new
potential synthetic cannabinoids 1-pentyl-3-(2-iodobenzoyl)indole and
1-pentyl-3-(1-adamantoyl)indole in seized bulk powders in Hungary.
Forensic Sci. Int. 214, 27−32.
(60) Kneisel, S., Westphal, F., Bisel, P., Brecht, V., Broecker, S., and
Auwarter, V. (2012) Identification and structural characterization of
̈
(76) Wiley, J. L., Marusich, J. A., Martin, B. R., and Huffman, J. W.
(2012) 1-Pentyl-3-phenylacetylindoles and JWH-018 share in vivo
cannabinoid profiles in mice. Drug Alcohol Depend. 123, 148−53.
(77) Weng, Y., Sun, S., Park, J., Ye, S., Weil, M. H., and Tang, W.
(2012) Cannabinoid 1 (CB1) receptor mediates WIN55, 212−2
induced hypothermia and improved survival in a rat post-cardiac arrest
model. Resuscitation 83, 1145−1151.
the synthetic cannabinoid 3-(1-adamantoyl)-1-pentylindole as an
additive in ‘herbal incense’. J. Mass Spectrom. 47, 195−200.
(61) Uchiyama, N., Kawamura, M., Kikura-Hanajiri, R., and Goda, Y.
(2012) Identification of two new-type synthetic cannabinoids, N-(1-
adamantyl)-1-pentyl-1H-indole-3-carboxamide (APICA) and N-(1-
adamantyl)-1-pentyl-1H-indazole-3-carboxamide (APINACA), and
detection of five synthetic cannabinoids, AM-1220, AM-2233, AM-
1241, CB-13 (CRA-13), and AM-1248, as designer drugs in illegal
products. Forensic Toxicol. 30, 114−125.
(78) Jarbe, T. C., Tai, S., LeMay, B., Nikas, S., Shukla, V., Zvonok, A.,
̈
and Makriyannis, A. (2012) AM2389, a high-affinity, in vivo potent
CB1-receptor-selective cannabinergic ligand as evidenced by drug
discrimination in rats and hypothermia testing in mice. Psychopharma-
cology 220, 417−426.
(62) Uchiyama, N., Kawamura, M., Kikura-Hanajiri, R., and Goda, Y.
(2013) URB-754: A new class of designer drug and 12 synthetic
cannabinoids detected in illegal products. Forensic Sci. Int. 227, 21−32.
(63) Frost, J. M., Dart, M. J., Tietje, K. R., Garrison, T. R., Grayson,
G. K., Daza, A. V., El-Kouhen, O. F., Yao, B. B., Hsieh, G. C., Pai, M.,
Zhu, C. Z., Chandran, P., and Meyer, M. D. (2010) Indol-3-
ylcycloalkyl ketones: Effects of N1 substituted indole side chain
variations on CB2 cannabinoid receptor activity. J. Med. Chem. 53,
295−315.
(64) Makriyannis, A., and Deng, H. (2001) Preparation of
cannabimimetic indole derivatives with cannabinoid CB1 or CB2
receptor binding affinity. Patent WO2001028557A1.
(65) Makriyannis, A., and Deng, H. (2002) Preparation of CB2
cannabinoid receptor-selective cannabimimetic aminoalkylindoles,
particularly (piperidinylmethyl)- and (morpholinylmethyl)indoles,
and their use. Patent WO2002060447A1.
(79) Blaazer, A. R., Lange, J. H. M., van der Neut, M. A. W., Mulder,
A., den Boon, F. S., Werkman, T. R., Kruse, C. G., and Wadman, W. J.
(2011) Novel indole and azaindole (pyrrolopyridine) cannabinoid
(CB) receptor agonists: Design, synthesis, structure−activity relation-
ships, physicochemical properties and biological activity. Eur. J. Med.
Chem. 46, 5086−5098.
(80) Bruker. (2011) Apex2 suite of programs, Bruker AXS Inc.,
Madison, WI.
(81) Sheldrick, G. M. (2008) A short history of SHELX. Acta
Crystallogr., Sect. A A64, 112−122.
(66) Makriyannis, A., and Deng, H. (2007) Receptor selective
cannabimimetic aminoalkylindoles. Patent US20070243134A1.
(67) Pasquini, S., De Rosa, M., Pedani, V., Mugnaini, C., Guida, F.,
Luongo, L., De Chiaro, M., Maione, S., Dragoni, S., Frosini, M.,
Ligresti, A., Di Marzo, V., and Corelli, F. (2011) Investigations on the
4-quinolone-3-carboxylic acid motif. 4. Identification of new potent
and selective ligands for the cannabinoid type 2 receptor with diverse
substitution patterns and antihyperalgesic effects in mice. J. Med.
Chem. 54, 5444−5453.
(68) Pasquini, S., Mugnaini, C., Ligresti, A., Tafi, A., Brogi, S.,
Falciani, C., Pedani, V., Pesco, N., Guida, F., Luongo, L., Varani, K.,
Borea, P. A., Maione, S., Di, M. V., and Corelli, F. (2012) Design,
synthesis, and pharmacological characterization of indol-3-ylaceta-
mides, indol-3-yloxoacetamides, and indol-3-ylcarboxamides: Potent
and selective CB2 cannabinoid receptor inverse agonists. J. Med. Chem.
55, 5391−5402.
(69) Okauchi, T., Itonaga, M., Minami, T., Owa, T., Kitoh, K., and
Yoshino, H. (2000) A general method for acylation of indoles at the 3-
position with acyl chlorides in the presence of dialkylaluminum
chloride. Org. Lett. 2, 1485−1487.
(70) Liang, G., Choi-Sledeski, Y. M., Poli, G., Chen, X., Shum, P.,
Minnich, A., Wang, Q., Tsay, J., Sides, K., Cairns, J., Stoklosa, G.,
Nieduzak, T., Zhao, Z., Wang, J., and Vaz, R. J. (2010) A
conformationally constrained inhibitor with an enhanced potency for
β-tryptase and stability against semicarbazide-sensitive amine oxidase
(SSAO). Bioorg. Med. Chem. Lett. 20, 6721−6724.
(71) Knapman, A., Santiago, M., Du, Y. P., Bennallack, P. R., Christie,
M. J., and Connor, M. (2013) A continuous, fluorescence-based assay
of μ-opioid receptor activation in AtT-20 cells. J. Biomol. Screening 18,
269−276.
(72) Mackie, K., Lai, Y., Westenbroek, R., and Mitchell, R. (1995)
Cannabinoids activate an inwardly rectifying potassium conductance
and inhibit Q-type calcium currents in AtT20 cells transfected with rat
brain cannabinoid receptor. J. Neurosci. 15, 6552−6561.
L
dx.doi.org/10.1021/cn400035r | ACS Chem. Neurosci. XXXX, XXX, XXX−XXX