Exploring Stereochemical and Conformational Requirements at Cannabinoid Receptors for Synthetic Cannabinoids Related to SDB-006, 5F-SDB-006, CUMYL-PICA, and 5F-CUMYL-PICA

Article abstract of DOI:10.1021/acschemneuro.0c00591

Synthetic cannabinoid receptor agonists (SCRAs) represent the most rapidly expanding class of new psychoactive substances (NPSs). Despite the prevalence and potency of recent chiral indole-3-carboxamide SCRAs, few pharmacological data are available regarding the enantiomeric bias of these NPSs toward human CB1 and CB2 receptors. A series of homochiral indole-3-carboxamides derived from (S)- and (R)-α-methylbenzylamine and featuring variation of the 1-alkyl substituent were prepared, pharmacologically evaluated, and compared to related achiral congeners derived from cumyl- and benzylamine. Competitive binding assays demonstrated that all analogues derived from either enantiomer of α-methylbenzylamine (14-17) showed affinities for CB1 (Ki = 47.9-813 nM) and CB2 (Ki = 47.9-347 nM) that were intermediate to that of the corresponding benzylic (10-13, CB1 Ki = 550 nM to >10 μM; CB2 Ki = 61.7 nM to >10 μM) and cumyl derivatives (6-9, CB1 Ki = 12.6-21.4 nM; CB2 Ki = 2.95-24.5 nM). In a fluorometric membrane potential assay, all α-methylbenzyl analogues (excluding 17) were potent, efficacious agonists of CB1 (EC50 = 32-464 nM; Emax = 89-104%) and low efficacy agonists of CB2 (EC50 = 54-500 nM; Emax = 52-77%), with comparable or greater potency than the benzyl analogues and much lower potency than the cumyl derivatives, consistent with binding trends. The relatively greater affinity and potency of (S)-14-17 compared to (R)-14-17 analogues at CB1 highlighted an enantiomeric bias for this series of SCRAs. Molecular dynamics simulations provided a conformational basis for the observed differences in agonist potency at CB1 pending benzylic substitution.

Full text of DOI:10.1021/acschemneuro.0c00591

pubs.acs.org/chemneuro
Research Article
Exploring Stereochemical and Conformational Requirements at
Cannabinoid Receptors for Synthetic Cannabinoids Related to SDB-
006, 5F-SDB-006, CUMYL-PICA, and 5F-CUMYL-PICA
Adam Ametovski, Christa Macdonald, Jamie J. Manning, S. A. Syed Haneef, Marina Santiago,
Lewis Martin, Eric Sparkes, Andrew Reckers, Roy R. Gerona, Mark Connor, Michelle Glass,
and Samuel D. Banister*
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ABSTRACT: Synthetic cannabinoid receptor agonists (SCRAs)
represent the most rapidly expanding class of new psychoactive
substances (NPSs). Despite the prevalence and potency of recent
chiral indole-3-carboxamide SCRAs, few pharmacological data are
available regarding the enantiomeric bias of these NPSs toward
human CB1 and CB2 receptors. A series of homochiral indole-3-
carboxamides derived from (S)- and (R)-α-methylbenzylamine and
featuring variation of the 1-alkyl substituent were prepared,
pharmacologically evaluated, and compared to related achiral
congeners derived from cumyl- and benzylamine. Competitive
binding assays demonstrated that all analogues derived from either
enantiomer of α-methylbenzylamine (14−17) showed affinities for CB1 (Ki = 47.9−813 nM) and CB2 (Ki = 47.9−347 nM) that
were intermediate to that of the corresponding benzylic (10−13, CB1 Ki = 550 nM to >10 μM; CB2 Ki = 61.7 nM to >10 μM) and
cumyl derivatives (6−9, CB1 Ki = 12.6−21.4 nM; CB2 Ki = 2.95−24.5 nM). In a fluorometric membrane potential assay, all α-
methylbenzyl analogues (excluding 17) were potent, efficacious agonists of CB1 (EC50 = 32−464 nM; Emax = 89−104%) and low
efficacy agonists of CB2 (EC50 = 54−500 nM; Emax = 52−77%), with comparable or greater potency than the benzyl analogues and
much lower potency than the cumyl derivatives, consistent with binding trends. The relatively greater affinity and potency of (S)-
14−17 compared to (R)-14−17 analogues at CB1 highlighted an enantiomeric bias for this series of SCRAs. Molecular dynamics
simulations provided a conformational basis for the observed differences in agonist potency at CB1 pending benzylic substitution.
KEYWORDS: Cannabinoid, pharmacology, CUMYL, PICA, CHMICA, FUBICA
the 1-position and pendant amino acid derivatives.3 Many of
the SCRAs studied to date function as potent, highly
INTRODUCTION
■
New psychoactive substances (NPSs) are drugs designed as
efficacious agonists of CB1 and CB2, whereas Δ9-THC is a
unregulated substitutes for traditional illicit substances.1 Of the
moderate efficacy agonist at both receptor subtypes.8 Many
950 NPSs identified by the United Nations Office on Drugs
SCRAs also show important differences in the intrinsic efficacy
and Crime (UNODC), over 290 are synthetic cannabinoid
and signaling profile compared to Δ9-THC or each other.11−17
receptor agonists (SCRAs).2−5 SCRAs are a structurally
Unlike Δ9-THC, consumption of SCRAs such as AB-
diverse class of NPSs intended to effect intoxication through
CHMINACA (2), AMB-FUBINACA (3), and 5F-ADB (4)
activation of central cannabinoid type 1 receptors (CB1),6,7
has been linked to serious adverse effects, including mass
one of the most abundant G-protein-coupled receptors
hospitalizations and fatalities.18−20
(GPCRs) in the human brain and the principal target of the
SCRAs featuring a cumyl group have emerged in drug
psychoactive Δ9-tetrahydrocannabinol (Δ9-THC) consumed
markets over the past several years, likely inspired by a 2014
in cannabis.8 SCRAs typically demonstrate little selectivity over
the other major cannabinoid receptor, CB2, which is a GPCR
primarily involved in immune function.9
Received: September 9, 2020
Accepted: October 1, 2020
One of the earliest SCRAs detected in the NPS market was
the cannabinoid ligand JWH-018 (1, Figure 1), repurposed
from the academic chemistry literature.10 Current SCRAs
incorporate indazole-3-carboxamides and other heteroaromatic
cores as well as alicyclic, aromatic, and aliphatic substituents at
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CUMYL-PICA (7), CUMYL-CHMICA (8), and CUMYL-
FUBICA (9). CUMYL-PICA and 5F-CUMYL-PICA were first
reported in Slovenia in 2014.24 To the best of our knowledge,
CUMYL-CHMICA and CUMYL-FUBICA have not yet
appeared on the NPS market, but their cannabinoid activity
has been reported.25 Indeed, the analytical chemistry and
metabolic profiles of several cumyl SCRAs have been
described, but fewer studies have systematically explored the
pharmacology of this class.26−34
Related to cumyl SCRAs 6−9 are the corresponding benzyl
analogues SDB-006 (10), 5F-SDB-006 (11), 12, and 13. SDB-
006 and 5F-SDB-006 were first reported in Finland in 2013.35
The chemistry, pharmacology, and metabolism of benzylic
SCRAs 10 and 11 has been described previously,36−40 while
the cannabinoid activity of 12 was reported by Schering-
Plough in 2010.41 The cannabinoid activity of 13 has not yet
been confirmed, but it has been independently explored as an
inhibitor of hyaluronidase42 and for potential antimicrobial
activity.43
Based on the activity of both N-cumylcarboxamide- and N-
benzylcarboxamide-type SCRAs, we hypothesized that ana-
logues featuring an intermediate degree of substitution at the
benzylic position might be pharmacologically active and
potentially appear on the drug market in future. A congeneric
series of SCRAs related to 6−13 but prepared from α-
methylbenzylamine (14−17), were synthesized, characterized,
and screened for cannabinoid binding and functional activity.
Enantiomeric differences have been observed in other
homochiral SCRAs featuring amino acid groups (e.g., 2−
4),44,45 so both (S)- and (R)-α-methylbenzyl derivatives were
prepared from the appropriate homochiral amine building
block and assessed for cannabinoid activity.
Figure 1. Selected synthetic cannabinoid receptor agonists.
patent describing the activity of this chemotype.21 Cumyl-
derived SCRAs, such as CUMYL-4CN-BUTINACA
(CUMYL-4CN-BINACA, SGT-78, 5), rank among some of
the most potent CB1 and CB2 agonists identified to date.
CUMYL-4CN-BUTINACA is known to cause seizures in
mice,22 and a related SCRA (CUMYL-PINACA) was reported
in cases of transdermal poisoning in humans.23 Other reported
cumyl SCRAs include CUMYL-PICA (6, Figure 2), 5F-
RESULTS AND DISCUSSION
■
The synthesis of SCRAs 6−17 is depicted in Figure 3. Indole
(18) was alkylated with the appropriate alkyl bromide and then
treated with trifluoroacetic anhydride to generate intermediate
trifluoroacetylindoles 19−22 in a convenient one-pot
procedure. Trifluoroacetylindoles 19−22 were hydrolyzed to
the corresponding carboxylic acids 23−26, respectively,
converted to the acid chlorides, and reacted with either
cumylamine, benzylamine, or (S)- or (R)-α-methylbenzyl-
amine to give the desired SCRAs 6−17.
The synthesized SCRAs 6−17 were screened via a
competitive radioligand binding assay and a fluorescence-
based functional activity assay at human CB1 and CB2
receptors (Table 1). The binding assays were carried out by
screening 6−17 for their capacity to displace radioligands
[3H]SR141716A or [3H]CP 55,940 from the purified
membranes of hCB1- and hCB2-receptor-transfected
HEK293 cells. Consistent with previous findings of their
functional activity, cumylamine-derived SCRAs 6−9 all
showed high affinity at CB1 receptors, with Ki values in the
low nanomolar range, and only minor variation was observed
for differing indole N-alkyl substituents (CB1 Ki = 12.6−21.4
nM). All cumylamine-derived SCRAs also bound to CB2 (Ki =
2.95−24.5 nM) and largely mirrored the corresponding CB1
binding, with little subtype selectivity in the case of 6, 7, or 9.
However, the cyclohexylmethyl-substituted 8 showed a modest
increase in CB2 affinity and selectivity (∼5-fold), representing
the highest affinity and receptor subtype selectivity across all
compounds evaluated in the present study.
Figure 2. Synthetic cannabinoid receptor agonists featuring
substitution at the benzylic position.
B
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Figure 3. Reagents and conditions: (a) (i) NaH (2 equiv), RBr, DMF, 0 °C−rt, 1 h; (ii) (CF3CO)2O, 0 °C−rt, 1 h; (b) KOH, MeOH, PhMe,
reflux, 2 h, 65−78% over three steps; (c) (COCl)2, DMF (cat.), CH2Cl2, rt, 2 h; (d) PhCR1R2NH2, Et3N, CH2Cl2, rt, 14 h, 57−81% over two
steps.
Table 1. Binding Affinities and Functional Activities of SCRAs 6−17 at hCB1 and hCB2 Receptors
hCB1
hCB2
pKi SEM
a
pEC50 (95% CI)
[EC50, nM]
Emax (% CP 55,940)
pKi SEM
a
pEC50 (95% CI)
[EC50, nM]
Emax (% CP 55,940)
compound
[Ki, nM]
(95% CI)
[Ki, nM]
(95% CI)
b
b
b
b
b
6
7
7.78 0.10 [16.6] 8.38 (8.29−8.46) [4.2]
7.90 0.09 [12.6] 8.55 (8.48−8.61) [2.8]
115 (112−120)
118 (94−138)
7.73 0.07 [18.6] 7.23 (7.05−7.41) [58]
102 (93−112)
104 (94−113)
b
b
7.67 0.08 [21.4] 7.40 (7.20−7.60)
b
[39.6]
b
b
b
8
9
7.80 0.22 [15.8] 8.02 (7.72−8.28) [9.5]
120 (110−133)
8.53 0.07 [2.95] 6.91 (6.47−7.36)
92 (74−109)
b
[122]
b
b
7.67 0.11 [21.4] 7.91 (7.69−8.12)
112 (102−125)
7.61 0.05 [24.5] 7.34 (7.12−7.57)
92 (82−102)
b
b
[12.3]
[45.5]
10
11
12
6.26 0.01 [550] 6.81 (6.66−6.94) [154]
6.24 0.12 [575] 7.16 (7.09−7.22) [69]
94 (87−102)
98 (94−102)
99 (94−105)
7.21 0.03 [61.7] 6.74 (6.61−6.86) [183]
6.48 0.01 [331] 6.93 (6.78−7.09) [118]
49 (45−53)
54 (50−59)
66 (60−73)
5.01 0.08 [9
772]
6.89 (6.78−6.98) [129]
5.34 0.04
[4571]
6.77 (6.62−6.90) [171]
c
d
c
d
13
<5 [>10 000]
ND
36 1 % (s.e.m.)
<5 [>10 000]
ND
16 1 % (s.e.m.)
(S)-14
(S)-15
(S)-16
(S)-17
(R)-14
(R)-15
(R)-16
(R)-17
6.87 0.09 [135] 7.10 (6.99−7.19) [79]
6.77 0.05 [170] 7.49 (7.40−7.58) [32]
6.81 0.08 [155] 7.07 (6.84−7.17) [85]
7.32 0.06 [47.9] 6.33 (5.69−6.61) [464]
6.09 0.08 [813] 6.54 (6.43−6.54) [291]
6.10 0.03 [794] 6.97 (6.77−7.12) [108]
6.35 0.04 [447] 6.47 (6.37−6.55) [343]
98 (93−104)
104 (99−109)
101 (95−108)
53 (45−72)
89 (84−95)
96 (88−106)
90 (85−95)
6.61 0.05 [245] 6.65 (6.41−6.85) [223]
6.46 0.09 [347] 7.0 (6.78−7.20) [99]
6.97 0.11 [107] 6.79 (6.66−6.92) [161]
6.66 0.11 [219] 6.30 (4.90−6.70) [500]
6.69 0.02 [204] 6.80 (6.62−6.96) [158]
6.75 0.04 [178] 7.27 (7.20−7.34) [54]
7.32 0.07 [47.9] 6.85 (6.66−7.05) [140]
77 (66−90)
77 (68−88)
77 (71−83)
52 (43−97)
74 (66−84)
66 (64−69)
67 (61−75)
60 (53−78)
c
d
6.28 0.02 [525] ND
31 3 % (s.e.m.)
6.69 0.05 [204] 6.65 (6.13−6.91) [221]
a
b
c
pKi < 5 indicates less than 50% displacement of [3H]ligand at 10 μM. Data adapted from ref 25. ND = not determined; <50% change in
fluorescence at 10 μM. Maximum response at 10 μM.
d
Consistent with previous pharmacological evaluation of
several benzyl-carboxamide SCRAs,36,40 all benzylamine-
derived SCRAs 10−13 displayed reduced (or negligible)
binding at both CB1 and CB2 receptors compared to the
cumyl derivatives. The N-pentyl-substituted 10 demonstrated
moderate CB1 affinity (Ki = 550 nM) and a notable preference
for CB2 (Ki = 61.7 nM; selectivity = 8.9-fold). Replacing the
tail group with a 5-fluoropentyl group conferred similar CB1
binding (11, Ki = 575 nM); however, CB2 selectivity (11, Ki =
331 nM) was diminished. Alicyclic and aromatic indole
substituents (see 12 and 13, respectively) were not tolerated
for benzylic SCRAs, with sub- or micromolar affinity observed
at CB1 and CB2 in each case.
Previous studies of chiral amino-acid-derived SCRAs have
shown that the (S)-enantiomers more potently activate CB1
receptors than the corresponding (R)-enantiomers, although
little enantiomeric discrimination was observed for CB2
activation.44,45 In the present study, homochiral (S)-α-
methylbenzyl SCRAs (S)-14−17 displayed CB1 (Ki = 47.9−
170 nM) and CB2 affinities (Ki = 107−347 nM) intermediate
to analogous cumyl and benzyl derivatives. Inversion of
enantiomerism in (R)-14−17 consistently diminished CB1
binding (Ki = 447−813 nM) with little effect on CB2 affinity
(Ki = 47−204 nM), producing a net increase in CB2
selectivity. Overall, (R)-14−17 showed binding profiles that
were comparable to the benzyl congeners 10−13, with (S)-
14−17 showing greater CB1 and CB2 affinity, and cumyl
analogues 6−9 showing much greater affinity again. Taken
together, these data indicate an enantiomeric bias in this class
of SCRA and suggest that judicious selection of homochiral
amide substituents may facilitate control of CB1 affinity and/
or CB2 selectivity.
Structure−activity relationships (SARs) for this class of
SCRAs were explored by assessing the functional activity of 6−
17 at hCB1 and hCB2 receptors using a fluorometric imaging
plate reader (FLIPR) assay. The technique utilizes mouse
AtT20 adenocarcinoma cells transfected with hCB1 or hCB2
natively expressing G-protein-gated inwardly rectifying potas-
sium channels (GIRKs). In these cells, agonism of CB
receptors liberates G-protein-coupled βγ-subunits, resulting
in activation of GIRK channels and consequent hyper-
polarization. Employing a membrane-potential-sensitive dye
enables measurement of decreased fluorescence. The func-
tional data for 6−17 were normalized to the change in
C
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Figure 4. Steric hindrance near the phenyl group improves alignment with the crystallized agonist conformation. Left panel: CB1 (cyan ribbons)
bound to MDMB-FUBINACA (27, orange) from crystal structure 6N4B. Note the position of the amine (oxygen = red, nitrogen = blue). Middle
panel: SDB-006 (10) after 90 ns molecular dynamics simulation. Due to increased flexibility, the phenyl group has rotated away from the
hydrophobic pocket created by three phenylalanine residues (cyan). The amine group has rotated 180° compared with the bound MDMB-
FUBINACA (27) ligand. Right panel: CUMYL-PICA (6) after 90 ns simulation. The presence of the two methyl groups reduces steric freedom of
the phenyl, forcing the amine group and the phenyl to align closely with the crystallized ligand conformation.
fluorescence effected by a maximally efficacious concentration
of potent CB1/CB2 agonist CP 55,490 (1 μM), which
produced a decrease in fluorescence of 33.4 0.3 and 28.2
0.5% in CB1- and CB2-expressing AtT20 cells, respectively.
When screened against wild-type AtT20 cells, which do not
natively express CB1 or CB2 receptors, none of the
compounds induced a significant decrease in fluorescence.
The functional activities of cumyl-derived SCRAs 6−9 have
previously been determined using the same assay and were
enantiomers here is entirely consistent with the pharmaco-
logical profiles described for chiral SCRAs featuring pendant
amino acid substituents in place of the substituted benzylic
groups in this series.44,45
In the few cases where congeneric structures have been
explored, benzylic analogues generally showed reduced agonist
potency in vitro at CB1 and CB2 and lower cannabimimetic
potency in vivo, compared to the corresponding cumyl
derivatives.25,32,40 It was hypothesized that the geminal
dimethyl functionality of cumylcarboxamide-type SCRAs
might confer a favorable conformational restriction for CB1
binding and/or activation through a Thorpe-Ingold-type
effect,46 and molecular dynamics simulations were performed
to explore this possibility.
shown to have comparable, or greater, CB1 potency (EC50
=
4.2−12.3 nM; Emax = 112−120%) compared to CP 55,940
(EC50 = 10.4 nM). Cumyl SCRAs (6−9) were also potent
agonists of CB2 (EC50 = 40−122 nM; Emax = 92−104%),
despite showing modest selectivity for CB1 over CB2 receptors
(∼13−14-fold). While a 5-fluoropentyl group (i.e., 7)
conferred the greatest potency (CB1 EC50 = 2.8 nM), variation
of the tail group for these SCRAs had only a minor effect on
both potency and efficacy across both CB1 and CB2 receptor
subtypes. With the exception of 13 (EC50 not determinable),
benzylic SCRAs 10−12 exhibited CB1 (EC50 = 60−154 nM)
and CB2 potencies (EC50 = 118−183 nM) lower than those of
the corresponding cumyl SCRAs. Compounds 10−12 showed
only a minor preference for activation of CB1 over CB2
receptors (∼1.2−1.7-fold) and acted only as low-to-moderate
efficacy agonists of CB2 receptors (Emax = 49−66%),
highlighting the importance of substitution at the benzylic
position for high CB2 potency and activity.
As anticipated from the binding data and previous functional
evaluation of chiral amino-acid-derived SCRAs,44,45 an
enantiomeric bias was observed with α-methylbenzyl SCRAs
(S)-14−16, having CB1 potencies (EC50 = 32−85 nM) and
selectivities (∼1.9−3.1-fold) lower than the corresponding
cumyl analogue but greater than the corresponding benzyl
derivatives while maintaining full agonist activity (Emax = 98−
104%). Again, the 4-fluorobenzyl group (i.e., (S)-17) conferred
significantly reduced potency (EC50 = 464 nM) and maximal
effect (Emax = 53%). The opposite enantiomeric series (R)-14−
17 followed similar trends for CB1 activity, with potency
reduced compared to the corresponding (S)-enantiomers and
generally comparable to the corresponding benzyl analogues.
Importantly, the observed CB1 preference for (S)- over (R)-
To determine a qualitative mechanism for the observed SAR
pattern in the linker region between the phenyl ring and the
indole scaffold (i.e., CUMYL > (S)-α-methylbenzyl > (R)-α-
methylbenzyl ≈ benzyl), we simulated four compounds
retaining the pentyl group on the indole nitrogen (i.e., 6, 10,
(S)-14, (R)-14) bound to the CB1 receptor using all-atom
molecular dynamics. The compounds were first aligned to the
potent CB1 agonist MDMB-FUBINACA (27, see Figure 4),
from PDB accession code 6N4B,7 equilibrated, and then
simulated without restraints for 90 ns. Figure 4 shows MDMB-
FUBINACA (27) from the cryogenic electron microscopy
(cryo-EM) structure and representative snapshots of SDB-006
(10) and CUMYL-PICA (6) from simulation. The additional
conformational freedom in the case of SDB-006 and (R)-14,
which had the lowest CB1 affinities in the pentyl series, allows
distortion of the hydrophobic phenyl group away from the
hydrophobic pocket observed in the cryo-EM structure. In
contrast, CUMYL-PICA, which showed the highest affinity for
CB1, is constricted by the geminal dimethyl substituents and
maintains the same relative conformation as MDMB-
FUBINACA (27) in the cryo-EM structure. (S)-14 showed
intermediate behavior, sampling both conformations. All
structures initially were aligned in the cryo-EM conformation,
indicating that these results are not an artifact of the starting
coordinates but represent equilibrium behavior in the binding
site that may explain their differences in affinity.
D
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Figure 5. Time series of the orientation of the phenyl groups with respect to the indole scaffold. CUMYL-PICA (6, red scatter), which showed the
highest affinity to CB1, has the closest alignment to the cryo-EM structure (27, blue bar). SDB-006 (10, green scatter) and (R)-14 (blue scatter)
show the most deviation from the crystal structure, while (S)-14 (orange scatter) shows intermediate deviation, mirroring the trends in binding
affinity.
The relative orientation of the phenyl group with respect to
the indole scaffold varies across the four trajectories and is
shown in Figure 5. The inset demonstrates how this
orientation is measured, that is, with a torsional angle defined
by four atoms across the heteroaromatic core and a pendant
amide substituent headgroup of MDMB-FUBINACA (27). As
in the snapshots, CUMYL-PICA (6, red) maintains the same
conformation as bound MDMB-FUBINACA (27) across the
whole trajectory, while the phenyl group in SDB-006 (10,
green) and (R)-14 (blue) is rotated by approximately 100°.
(S)-14 shows an intermediate orientation, with the phenyl
group offset from the cryo-EM MDMB-FUBINACA (27)
structure conformation. While the free energy in these
conformations was not explicitly calculated, these results
suggest a plausible mechanism for the observed trends in
affinity, whereby the bound configuration of MDMB-
FUBINACA (27) in the cryo-EM structure is either
maintained or lost depending on the complementary
conformational freedom of the linker between the phenyl
group and indole scaffold.
In this study, a series of benzyl SCRAs and their
corresponding enantiomeric α-methylbenzyl congeners were
synthesized and their in vitro pharmacology at CB1 and CB2
receptors was evaluated via a competitive radioligand binding
assay and FLIPR membrane potential assay. Excepting a single
example, all analogues showed nanomolar to submicromolar
affinity for both CB1 and CB2 receptors. Clear SARs
pertaining to the degree of substitution around the benzylic
center were established, where cumyl SCRAs possessed the
greatest potency as CB1 and CB2 agonists. In all cases,
decreasing the degree of substitution at the benzylic position
reduced the affinity and potency for both receptors (cumyl >
(S)-α-methylbenzyl > (R)-α-methylbenzyl ≈ benzyl). An
enantiomeric bias was elucidated with (S)-α-methylbenzyl-
derived SCRAs conferring greater potency and efficacy at CB1,
with binding affinity, potency, and CB1 selectivity reversed
with inversion of the stereocenter (i.e., the corresponding (R)-
enantiomers). Molecular dynamics simulations of ligands in
the binding site led to a model that is consistent with the
observed SAR, where the degree of benzylic substitution affects
the ability to maintain the bound conformation.
METHODS
■
General Chemical Synthesis Details. All reactions were
performed under an atmosphere of nitrogen unless otherwise
specified. All reagents, reactants, and solvents were obtained from
Sigma-Aldrich (St. Louis, MO) and used as purchased. Analytical
thin-layer chromatography was performed using Merck aluminum-
backed silica gel 60 F254 (0.2 mm) plates (Merck, Darmstadt,
Germany), which were visualized using shortwave (254 nm) UV
fluorescence. Flash chromatography was performed using a Biotage
Isolera Spektra One and Biotage SNAP KP-Sil silica cartridges
(Uppsala, Sweden), with gradient elution terminating at the solvent
combination indicated for each compound. Melting point ranges
(Mp) were measured in open capillaries using a Stuart SMP50
Automatic Melting Point apparatus (Cole-Palmer, Staffordshire, U.K.)
and are uncorrected. Nuclear magnetic resonance spectra were
recorded at 298 K using an Agilent 400 MHz spectrometer (Santa
Clara, CA, U.S.A.). The data are reported as chemical shift (δ ppm)
relative to the residual protonated solvent resonance, multiplicity (s =
singlet, br s = broad singlet, d = doublet, br d = broad doublet, t =
triplet, q = quartet, app quin. = apparent quintet, m = multiplet),
coupling constants (J Hz), relative integral, and assignment. The
assignment of carbon signals for the parent compounds was assisted
by correlation spectroscopy (COSY), distortionless enhancement by
polarization transfer (DEPT), heteronuclear single-quantum coher-
ence (HSQC), and heteronuclear multiple-bond correlation (HMBC)
experiments where necessary. High resolution mass spectrometry data
was collected using an Agilent LC 1260-QTOF/MS 6550 (Santa
Clara, CA, U.S.A.). A methanolic extract of each pure standard was
run using an electrospray ionization source in automated MS/MS
(information dependent acquisition) mode. Accurate mass for the
parent ion and its corresponding mass error expressed in parts per
million (ppm) are reported.
General Procedure for Amidation of 1-Alkyl-1H-indole-3-
carboxylic Acids. A solution of the appropriate carboxylic acid (1.0
mmol) in CH2Cl2 (4 mL) was treated with (COCl)2 (172 μL, 2.0
mmol, 2.0 equiv) followed by DMF (1 drop). After being stirred for 2
h, the solution was evaporated in vacuo, and the crude acid chloride
was used immediately in the following step.
E
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Research Article
A cooled (0 °C) solution of the freshly prepared acid chloride
(1.00 mmol) in CH2Cl2 (8 mL) was treated with Et3N (352 μL, 2.50
mmol, 2.5 equiv) and a solution of the appropriate amine (1.20 mmol,
1.2 equiv) in CH2Cl2 (2 mL) and stirred at ambient temperature for
14 h. The mixture was partitioned between CH2Cl2 (60 mL) and 1 M
aq. HCl (20 mL). The layers were separated, and the organic phase
was washed with 1 M aq. HCl (2 × 20 mL), sat. aq. NaHCO3 (3 × 20
mL), brine (20 mL), and dried (MgSO4), and the solvent was
evaporated under reduced pressure. The obtained crude solids were
purified by flash chromatography or recrystallized from i-PrOH or i-
PrOH−H2O to give analytically pure materials.
(d, 1JCF = 247.3 Hz, quat.), 147.4 (quat.), 136.8 (quat.), 132.0 (d, 4JCF
= 3.3 Hz, quat.), 131.97 (CH), 128.9 (d, JCF = 8.3 Hz, 2 × CH),
128.6 (2 × CH), 126.7 (2 × CH), 125.5 (quat.), 125.0 (CH), 122.9
(CH), 121.8 (CH), 120.1 (CH), 116.0 (d, 2JCF = 21.7 Hz, 2 × CH),
112.7 (quat.), 110.7 (CH), 56.3 (quat.), 50.0 (NCH2), 29.8 (2 ×
CH3) ppm; HRMS (ESI) C25H23FN2O exact mass 386.1794, accurate
mass 386.1797 (mass error 0.60 ppm).
3
1-Pentyl-N-(benzyl)-1H-indole-3-carboxamide (10). Subjecting 1-
(pentyl)-1H-indole-3-carboxylic acid (23, 232 mg, 1.0 mmol) and
benzylamine (131 μL, 1.2 mmol, 1.2 equiv) to the procedure above
gave, following purification by flash chromatography (hexane/ethyl
acetate 5:1), 10 (184 mg, 57%) as a white solid. Mp 101−103 °C;
1-Pentyl-N-(2-phenylpropan-2-yl)-1H-indole-3-carboxamide (6).
Subjecting 1-pentyl-1H-indole-3-carboxylic acid (23, 231 mg, 1.00
mmol) and cumylamine (173 μL, 1.2 mmol, 1.2 equiv) to the
procedure above gave, following recrystallization from i-PrOH, 6 (280
lit.36 Mp 101−102 °C; H NMR (400 MHz, CDCl3): δ 7.94−7.91
1
(m, 1H), 7.73 (s, 1H), 7.45−7.32 (m, 5H), 7.34−7.19 (m, 3H), 6.22
(brs, 1H, NH), 4.72 (d, J = 5.6 Hz, 2H, NCH2Ph), 4.12 (t, J = 7.2 Hz,
2H, NCH2), 1.92−1.80 (m, 2H, CH2), 1.43−1.23 (m, 4H, 2 × CH2),
0.89 (t, J = 6.9 Hz, 3H, CH3) ppm; 13C NMR (100 MHz, CDCl3): δ
165.3 (CO), 139.1 (quat.), 136.7 (quat), 131.7 (CH), 128.9 (2 ×
CH), 128.0 (2 × CH), 127.5 (CH), 125.5 (quat.), 122.5 (CH), 121.5
(CH), 120.8 (CH), 110.8 (quat.), 110.45 (CH), 47.0 (NCH2Ph),
43.7 (NCH2), 29.8 (CH2), 29.15 (CH2), 22.41 (CH2), 14.04 (CH3)
ppm; HRMS (ESI) C21H24N2O exact mass 320.1889, accurate mass
320.1897 (mass error 2.54 ppm).
1
mg, 80%) as fine white needles. Mp 173−175 °C; H NMR (400
MHz, CDCl3): δ 7.92−7.80 (m, 1H), 7.70 (s, 1H), 7.54−7.50 (m,
2H), 7.40−7.33 (m, 3H), 7.30−7.22 (m, 3H), 6.29 (br s, 1H, NH),
4.11 (t, J = 7.2 Hz, 2H, NCH2), 1.88 (s, 6H, CH3), 1.85 (app quin., J
= 7.2 Hz, 2H, CH2), 1.40−1.26 (m, 4H, CH2), 0.89 (t, J = 7.0 Hz,
3H, CH3) ppm; 13C NMR (100 MHz, CDCl3): δ 164.4 (CO), 147.5
(quat.), 136.8 (quat.), 131.9 (CH), 128.6 (2 × CH), 126.7 (2 × CH),
125.3 (quat.), 125.0 (CH), 122.4 (CH), 121.4 (CH), 119.9 (CH),
111.8 (quat.), 110.5 (CH), 56.2 (quat.), 46.9 (NCH2), 29.84 (2 ×
CH3), 29.80 (CH2), 29.1 (CH2), 22.4 (CH2), 14.0 (CH3) ppm;
HRMS (ESI) C23H28N2O exact mass 348.2202, accurate mass
348.2205 (mass error 1.01 ppm).
1-(5-Fluoropentyl)-N-(benzyl)-1H-indole-3-carboxamide (11).
Subjecting 1-(5-fluoropentyl)-1H-indole-3-carboxylic acid (24, 249
mg, 1.0 mmol) and benzylamine (131 μL, 1.2 mmol, 1.2 equiv) to the
procedure above gave, following recrystallization from i-PrOH, 11
(220 mg, 65%) as a white crystalline solid. Mp 128−130 °C; lit.37 Mp
1-(5-Fluoropentyl)-N-(2-phenylpropan-2-yl)-1H-indole-3-carbox-
amide (7). Subjecting 1-(5-fluoropentyl)-1H-indole-3-carboxylic acid
(24, 250 mg, 1.0 mmol) and cumylamine (173 μL, 1.2 mmol, 1.2
equiv) to the procedure above gave, following recrystallization from i-
1
128−130 °C; H NMR (400 MHz, CDCl3): δ 8.01−7.86 (m, 1H),
7.72 (s, 1H), 7.47−7.32 (m, 5H), 7.32−7.19 (m, 3H), 6.25 (t, J = 5.8
Hz, 1H, NH), 4.71 (d, J = 5.7 Hz, 2H, NCH2Ph), 4.41 (dt, J = 47.2,
5.9 Hz, 2H, CH2F), 4.14 (t, J = 7.1 Hz, 2H, NCH2), 1.98−1.87 (m,
2H, CH2), 1.78−1.62 (m, 2H, CH2), 1.51−1.37 (m, 2H, CH2) ppm;
13C NMR (100 MHz, CDCl3): δ 165.2 (CO), 139.0 (quat.), 136.7
(quat.), 131.5 (CH), 128.9 (2 × CH), 128.0 (2 × CH), 127.5 (CH),
125.6 (quat.), 122.6 (CH), 121.6 (CH), 120.4 (CH), 111.0 (quat.),
1
PrOH, 7 (288 mg, 78%) as fine white needles. Mp 193−195 °C; H
NMR (400 MHz, CDCl3): δ 7.92−7.90 (m, 1H), 7.69 (s, 1H), 7.53−
7.50 (m, 2H), 7.40−7.33 (m, 3H), 7.30−7.22 (m, 3H), 6.28 (brs, 1H,
NH), 4.42 (dt, J = 47.2, 6.0 Hz, 2H, CH2F), 4.15 (t, J = 7.2 Hz, 2H,
NCH2), 1.91 (app quin., J = 7.8 Hz, 2H, CH2), 1.87 (s, 6H, 2 ×
CH3), 1.77−1.64 (m, 2H, CH2), 1.49−1.41 (m, 2H, CH2) ppm; 13C
NMR (100 MHz, CDCl3): δ 164.4 (CO), 147.5 (quat.), 136.7
(quat.), 131.8 (CH), 128.6 (2 × CH), 126.7 (2 × CH), 125.3 (quat.),
125.0 (CH), 122.5 (CH), 121.5 (CH), 120.0 (CH), 112.0 (quat.),
1
110.3 (CH), 83.8 (d, JCF = 165.1 Hz, CH2F), 46.8 (CH2), 43.7
(CH2), 30.1 (d, 2JCF = 19.8 Hz, CH2), 29.8 (CH2), 23.0 (d, 3JCF = 5.0
Hz, CH2) ppm; HRMS (ESI) C21H23FN2O exact mass 338.1794,
accurate mass 338.1801 (mass error 2.07 ppm).
1
1-(Cyclohexylmethyl)-N-(benzyl)-1H-indole-3-carboxamide (12).
Subjecting 1-(cyclohexylmethyl)-1H-indole-3-carboxylic acid (25, 257
mg, 1.0 mmol) and benzylamine (131 μL, 1.2 mmol, 1.2 equiv) to the
procedure above gave, following recrystallization from i-PrOH, 12
110.4 (CH), 83.8 (d, JCF = 165.0 Hz, CH2F), 56.2 (quat.), 46.8
2
(CH2), 30.1 (d, JCF = 19.9 Hz, CH2), 29.8 (2 × CH3), 29.8 (CH2),
3
23.0 (d, JCF = 4.9 Hz, CH2) ppm; HRMS (ESI) C23H27FN2O exact
mass 366.2107, accurate mass 366.2111 (mass error 1.07 ppm).
1-(Cyclohexylmethyl)-N-(2-phenylpropan-2-yl)-1H-indole-3-car-
boxamide (8). Subjecting 1-(cyclohexylmethyl)-1H-indole-3-carbox-
ylic acid (25, 257 mg, 1.00 mmol) and cumylamine (173 μL, 1.2
mmol, 1.2 equiv) to the procedure above gave, following
recrystallization from i-PrOH, 8 (244 mg, 65%) as fine white needles.
1
(244 mg, 70%) as a white crystalline solid. Mp 161−163 °C; H
NMR (400 MHz, CDCl3): δ 7.97−7.87 (m, 1H), 7.68 (s, 1H), 7.47−
7.31 (m, 5H), 7.33−7.15 (m, 3H), 6.21 (brs, 1H, NH), 4.72 (d, J =
5.7 Hz, 2H NCH2Ph), 3.95 (d, J = 7.2 Hz, 2H, NCH2), 1.86 (m, 1H,
CH), 1.76−1.56 (m, 5H, CH2), 1.28−1.11 (m, 3H, CH2), 1.04−0.95
(m, 2H, CH2) ppm; 13C NMR (100 MHz, CDCl3): δ 165.27 (CO),
139.1 (quat.), 137.19 (quat.), 132. (CH), 128.9 (2 × CH), 128.0 (2 ×
CH), 127.6 (CH), 125.4 (quat), 122.5 (CH), 121.4 (CH), 120.2
(CH), 110.7 (CH), 110.6 (quat.), 53.5 (NCH2Ph), 43.7 (NCH2),
38.7 (CH), 31.14 (2 × CH2), 26.32 (CH2), 25.77 (2 × CH2) ppm;
HRMS (ESI) C23H26N2O exact mass 346.2045, accurate mass
362.2047 (mass error 0.67 ppm).
1
Mp 222−224 °C; H NMR (400 MHz, CDCl3): δ 7.90−7.87 (m,
1H), 7.65 (s, 1H), 7.53−7.50 (m, 2H), 7.39−7.32 (m, 3H), 7.29−
7.22 (m, 3H), 6.26 (br s, 1H, NH), 3.94 (d, J = 7.2 Hz, 2H, NCH2),
1.90−1.83 (m, 1H, CH, overlapping), 1.87 (s, 6H, CH3, overlapping),
1.75−1.60 (m, 5H, CH2), 1.24−0.95 (m, 6H, CH2), 0.89−0.79 (m,
1H, CH2) ppm; 13C NMR (100 MHz, CDCl3): δ 164.5 (CO), 147.5
(quat.), 137.1 (quat.), 132.7 (CH), 128.6 (2 × CH), 126.7 (2 × CH),
125.2 (quat.), 125.0 (CH), 122.3 (CH), 121.4 (CH), 119.8 (CH),
111.7 (quat.), 110.8 (CH), 56.2 (quat.), 53.5 (NCH2), 38.7 (CH),
31.2 (2 × CH2), 29.8 (2 × CH3), 26.3 (CH2), 25.8 (2 × CH2) ppm;
HRMS (ESI) C25H30N2O exact mass 374.2358, accurate mass
374.2359 (mass error 0.31 ppm).
1-(4-Fluorobenzyl)-N-(benzyl)-1H-indole-3-carboxamide (13).
Subjecting 1-(4-fluorobenzyl)-1H-indole-3-carboxylic acid (26, 270
mg, 1.0 mmol) and benzylamine (131 μL, 1.2 mmol, 1.2 equiv) to the
procedure above gave, following recrystallization from i-PrOH, 13
(288 mg, 80%) as a white crystalline solid. Mp 202−206 °C; lit.47 Mp
201−204 °C; 1H NMR (400 MHz, DMSO-d6): δ 8.48 (t, J = 6.0 Hz,
1H, NH), 8.22−8.18 (m, 1H), 8.17 (s, 1H), 7.56−7.53 (m, 1H),
7.40−7.27 (m, 6H), 7.26−7.10 (m, 5H), 5.45 (s, 2H, NCH2), 4.48
(d, J = 6.0 Hz, 2H, NCH2Ph) ppm; 13C NMR (100 MHz, DMSO-
d6): 165.0 (CO), 162.5 (d, J = 243.4 Hz, quat.), 141.3 (quat.), 137.0
(quat.), 134.6 (d, J = 3.0 Hz, quat.), 131.9 (CH), 130.3 (d, J = 8.3 Hz,
2 × CH), 129.1 (2 × CH), 128.2 (2 × CH), 127.6 (quat.), 127.5
(CH), 123.1 (CH), 122.3 (CH), 121.7 (CH), 116.40 (d, J = 21.6 Hz,
2 × CH), 111.5 (quat.), 111.2 (CH), 49.58 (NCH2Ph), 42.86
1-(4-Fluorobenzyl)-N-(2-phenylpropan-2-yl)-1H-indole-3-car-
boxamide (9). Subjecting 1-(4-fluorobenzyl)-1H-indole-3-carboxylic
acid (26, 269 mg, 1.00 mmol) and cumylamine (173 μL, 1.2 mmol,
1.2 equiv) to the procedure above gave, following recrystallization
from i-PrOH, 9 (272 mg, 70%) as fine white needles. Mp 210−212
1
°C; H NMR (400 MHz, CDCl3): δ 7.94−7.90 (m, 1H), 7.68 (s,
1H), 7.51−7.48 (m, 2H), 7.36−7.20 (m, 6H), 7.12−7.09 (m, 2H),
7.01−6.95 (m, 2H), 6.26 (brs, 1H, NH), 5.27 (s, 2H, NCH2), 1.86 (s,
6H, CH3) ppm; 13C NMR (100 MHz, CDCl3): δ 164.2 (CO), 162.2
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Research Article
2
(NCH2) ppm; HRMS (ESI) C23H19FN2O exact mass 358.1481,
accurate mass 358.1482 (mass error 0.17 ppm).
48.7 (CH), 46.8 (CH2), 29.8 (d, JCF = 19.8 Hz, CH2), 29.7 (CH2),
3
22.9 (d, JCF = 4.9 Hz, (CH2), 22.3 (CH3) ppm; HRMS (ESI)
(S)-1-Pentyl-N-(1-phenylethyl)-1H-indole-3-carboxamide ((S)-
14). Subjecting 1-pentyl-1H-indole-3-carboxylic acid (23, 217 mg,
1.0 mmol) and (S)-(−)-α-methylbenzylamine (155 μL, 1.2 mmol, 1.2
equiv) to the procedure above gave, following recrystallization from i-
PrOH, (S)-14 (208 mg, 62%) as a white crystalline solid. Mp 161−
163 °C; 1H NMR (400 MHz, CDCl3): δ 7.91−7.89 (m, 2H), 7.71 (s,
1H), 7.46−7.43 (m, 2H), 7.39−7.34 (m, 3H), 7.30−7.22, (m, 2H),
6.14, (br d, J = 7.5 Hz, 1H, NH), 5.43 (app quin., J = 7.2 Hz, 1H,
NCH), 4.12 (t, J = 7.1 Hz, 2H, NCH2), 1.85 (app quin., J = 7.4 Hz,
2H, CH2), 1.65 (d, J = 6.9 Hz, 3H, CH3), 1.39−1.26 (m, 4H, 2 ×
CH2), 0.88 (t, J = 6.9 Hz, CH3) ppm; 13C NMR (100 MHz, CDCl3):
δ 164.5 (CO), 143.9 (quat.), 136.8 (quat.), 131.7 (CH), 128.9 (2 ×
CH), 127.4, 126.4 (2 × CH), 125.4 (quat.), 122.5 (CH), 121.5 (CH),
120.1 (CH), 111.0 (quat), 110.5 (CH), 48.7 (NCH), 47.0 (NCH2),
29.8 (CH2), 29.2 (CH2), 22.4 (CH2), 22.3 (CH3), 14.0 (CH3) ppm;
HRMS (ESI) C22H26N2O exact mass 334.2045, accurate mass
334.2052 (mass error 2.00 ppm).
(R)-1-Pentyl-N-(1-phenylethyl)-1H-indole-3-carboxamide ((R)-
14). Subjecting 1-pentyl-1H-indole-3-carboxylic acid (23, 217 mg,
1.0 mmol) and (R)-(+)-α-methylbenzylamine (155 μL, 1.2 mmol, 1.2
equiv) to the procedure above gave, following recrystallization from i-
PrOH, (R)-14 (208 mg, 62%) as a white crystalline solid. Mp 161−
163 °C; 1H NMR (400 MHz, CDCl3): δ 7.91−7.89 (m, 2H), 7.71 (s,
1H), 7.46−7.43 (m, 2H), 7.39−7.34 (m, 3H), 7.30−7.22, (m, 2H),
6.14, (br d, J = 7.5 Hz, 1H, NH), 5.42 (app quin., J = 7.2 Hz, 1H,
NCH), 4.12 (t, J = 7.1 Hz, 2H, NCH2), 1.85 (app quin., J = 7.5 Hz,
2H, CH2), 1.65 (d, J = 6.9 Hz, 3H, CH3), 1.39−1.26 (m, 4H, 2 ×
CH2), 0.88 (t, J = 6.9 Hz, CH3) ppm; 13C NMR (100 MHz, CDCl3):
δ 164.5 (CO), 143.9 (quat.), 136.8 (quat.), 131.7 (CH), 128.9 (2 ×
CH), 127.4 (CH), 126.4 (2 × CH), 125.4 (quat.), 122.5 (CH), 121.5
(CH), 120.4 (CH), 111.0 (quat), 110.5 (CH), 48.7 (NCH), 47.0
(NCH2), 29.8 (CH2), 29.2 (CH2), 22.4 (CH2), 22.3 (CH3), 14.0
(CH3) ppm; HRMS (ESI) C22H26N2O exact mass 334.2045, accurate
mass 334.2050 (mass error 1.57 ppm).
C22H25FN2O exact mass 352.1951, accurate mass 352.1948 (mass
error −0.97 ppm).
(S)-1-(Cyclohexylmethyl)-N-(1-phenylethyl)-1H-indole-3-carbox-
amide ((S)-16). Subjecting 1-(cyclohexylmethyl)-1H-indole-3-carbox-
ylic acid (25, 257 mg, 1.0 mmol) and (S)-(−)-α-methylbenzylamine
(155 μL, 1.2 mmol, 1.2 equiv) to the procedure above gave, following
recrystallization from i-PrOH, (S)-16 (252 mg, 70%) as a white
1
crystalline solid. Mp 200−202 °C; H NMR (400 MHz, CDCl3): δ
7.95−7.86 (m, 1H), 7.66 (s, 1H), 7.49−7.41 (m, 2H), 7.41−7.32 (m,
3H), 7.32−7.19 (m, 3H), 6.16 (d, J = 7.8 Hz, 1H, NH), 5.42 (app
quin., J = 7.1 Hz, 1H, NCH), 3.94 (d, J = 7.1 Hz, 2H, NCH2), 1.84
(m, 1H, CH), 1.74−1.58 (m, 5H, CH2, overlapping), 1.65 (d, J = 6.9
Hz, 3H, overlapping), 1.26−1.09 (m, 3H, CH2), 1.00 (m, 2H, CH2)
ppm; 13C NMR (100 MHz, CDCl3): δ 164.5 (CO), 143.9 (quat.),
137.1 (quat.), 132.4 (CH), 128.9 (2 × CH), 127.4 (CH), 126.4 (2 ×
CH), 125.6 (quat.), 122.4 (CH), 121.4 (CH), 120.1 (CH), 110.8
(quat.), 110.7 (CH), 53.5 (NCH) 48.8 (NCH2), 38.7 (CH), 31.1 (2
× CH2), 26.3 (CH2), 25.8 (2 × CH2), 22.3 (CH3) ppm; HRMS
(ESI) C24H28N2O exact mass 360.2202, accurate mass 361.2272
(mass error −0.85 ppm).
(R)-1-(Cyclohexylmethyl)-N-(1-phenylethyl)-1H-indole-3-carbox-
amide ((R)-16). Subjecting 1-(cyclohexylmethyl)-1H-indole-3-carbox-
ylic acid (25, 258 mg, 1.0 mmol) and (S)-(−)-α-methylbenzylamine
(155 μL, 1.2 mmol, 1.2 equiv) to the procedure above gave, following
recrystallization from i-PrOH, (R)-16 (260 mg, 72%) as a white
1
crystalline solid. Mp 200−202 °C; H NMR (400 MHz, CDCl3): δ
7.95−7.86 (m, 1H), 7.66 (s, 1H), 7.49−7.42 (m, 2H), 7.41−7.32 (m,
3H), 7.32−7.20 (m, 3H), 6.18 (d, J = 7.8 Hz, 1H, NH), 5.43 (app
quin., J = 7.1 Hz, 1H, NCH), 3.94 (d, J = 7.1 Hz, 2H, NCH2), 1.84
(m, 1H, CH), 1.74−1.58 (m, 5H, CH2, overlapping) 1.65 (d, J = 6.9
Hz, 3H, overlapping), 1.26−1.09 (m, 3H, CH2), 0.99 (m, 2H, CH2)
ppm; 13C NMR (100 MHz, CDCl3): δ 164.5 (CO), 143.9 (quat.),
137.1 (quat.), 132.3 (CH), 128.8 (2 × CH), 127.4 (CH), 126.4 (2 ×
CH), 125.4 (quat.), 122.4 (CH), 121.4 (CH), 120.1 (CH), 110.8
(quat.), 110.7 (CH), 53.5 (NCH) 48.7 (NCH2), 38.7 (CH), 31.1 (2
× CH2), 26.3 (CH2), 25.8 (2 × CH2), 22.3 (CH3) ppm; HRMS
(ESI) C24H28N2O exact mass 360.2202, accurate mass 361.2272
(mass error −0.83 ppm).
(S)-1-(5-Fluoropentyl)-N-(1-phenylethyl)-1H-indole-3-carboxa-
mide ((S)-15). Subjecting 1-(5-fluoropentyl)-1H-indole-3-carboxylic
acid (24, 248 mg, 1.0 mmol) and (S)-(−)-α-methylbenzylamine (155
μL, 1.2 mmol, 1.2 equiv) to the procedure above gave, following
recrystallization from i-PrOH, (S)-15 (244 mg, 69%) as a white
(S)-1-(4-Fluorobenzyl)-N-(1-phenylethyl)-1H-indole-3-carboxa-
mide ((S)-17). Subjecting 1-(4-fluorobenzyl)-1H-indole-3-carboxylic
acid (26, 270 mg, 1.0 mmol) and (S)-(−)-α-methylbenzylamine (155
μL, 1.2 mmol, 1.2 equiv) to the procedure above gave, following
recrystallization from i-PrOH, (S)-17 (260 mg, 70%) as a white
1
crystalline solid. Mp 138−140 °C; H NMR (400 MHz, CDCl3): δ
7.93−7.9 (m, 2H), 7.70 (s, 1H), 7.46−7.44 (m, 2H), 7.38−7.34 (m,
3H), 7.30−7.22, (m, 2H). 6.19, (br d, J = 7.58 Hz, 1H, NH), 5.42
(app quin., J = 7.1 Hz, 1H, NCH), 4.41 (dt, J = 47.3, 5.9 Hz, 2H,
CH2F), 4.10 (t, J = 7.0 Hz, 2H, NCH2), 1.96−1.84 (m, 2H, CH2),
1.77−1.65 (m, 2H, CH2), 1.64 (d, J = 6.9 Hz, 3H, CH3), 1.50−1.35
(m, 2H, CH2) ppm; 13C NMR (100 MHz, CDCl3): δ 164.4 (CO),
143.9 (quat.), 136.7 (quat.), 131.5 (CH), 128.8 (2 × CH), 127.4
(CH), 126.4 (2 × CH), 125.5 (quat), 122.6 (CH), 121.6 (CH), 120.3
1
crystalline solid. Mp 196−198 °C; H NMR (400 MHz, CDCl3): δ
7.97−7.92 (m, 1H), 7.70 (s, 1H), 7.49−7.41 (m, 2H), 7.39−7.34 (m,
2H), 7.32−7.21 (m, 4H), 7.14−7.07 (m, 2H), 7.03−6.95 (m, 2H),
6.17 (br d, J = 7.8 Hz, 1H, NH), 5.42 (app quin., J = 6.9 Hz, 1H,
NCH), 5.28 (s, 2H, CH2), 1.64 (d, J = 6.9 Hz, 3H, CH3) ppm; 13C
NMR (100 MHz, CDCl3): δ 164.2 (CO), 162.6 (d, 1JCF = 247.0 Hz,
1
(CH), 111.1 (quat.), 110.4 (CH), 83.8 (d, JCF = 165.0 Hz, CH2F),
2
4
48.8 (CH), 46.8 (CH2), 30.1 (d, JCF = 19.8 Hz, CH2), 29.8 (CH2),
quat.), 143.8 (quat.), 136.8 (quat.), 132.0 (d, JCF = 3.1 Hz, quat.),
3
3
23.0 (d, JCF = 4.9 Hz, (CH2), 22.3 (CH3) ppm; HRMS (ESI)
131.7 (CH), 128.9 (d, JCF = 8.1 Hz, 2 × CH), 128.8 (2 × CH),
C22H25FN2O exact mass 352.1951, accurate mass 352.1953 (mass
error 0.57 ppm).
127.4 (CH), 126.4 (2 × CH), 125.7 (quat.), 123.0 (CH), 121.9
(CH), 120.4 (CH), 116.1 (d, 2JCF = 21.4 Hz, 2 × CH), 111.8 (quat),
110.7 (CH), 50.3 (CH), 48.8 (CH2), 22.3 (CH3) ppm; HRMS (ESI)
C24H21FN2O exact mass 372.1638, accurate mass 372.1648 (mass
error 2.66 ppm).
(R)-1-(5-Fluoropentyl)-N-(1-phenylethyl)-1H-indole-3-carboxa-
mide ((R)-15). Subjecting 1-(5-fluoropentyl)-1H-indole-3-carboxylic
acid (24, 249 mg, 1.0 mmol) and (R)-(+)-α-methylbenzylamine (155
μL, 1.2 mmol, 1.2 equiv) to the procedure above gave, following
recrystallization from i-PrOH, (R)-15 (216 mg, 61%) as a white
(R)-1-(4-Fluorobenzyl)-N-(1-phenylethyl)-1H-indole-3-carboxa-
mide ((R)-17). Subjecting 1-(4-fluorobenzyl)-1H-indole-3-carboxylic
acid (26, 269 mg, 1.0 mmol) and (R)-(+)-α-methylbenzylamine (155
μL, 1.2 mmol, 1.2 equiv) to the procedure above gave, following
recrystallization from i-PrOH/H2O, (R)-17 (304 mg, 81%) as a white
1
crystalline solid. Mp 138−140 °C; H NMR (400 MHz, CDCl3): δ
7.95−7.93 (m, 2H), 7.70 (s, 1H), 7.46−7.44 (m, 2H), 7.38−7.34 (m,
3H), 7.30−7.22, (m, 2H). 6.24, (br d, J = 7.58 Hz, 1H, NH), 5.42
(app quin., J = 7.1 Hz, 1H, CH), 4.39 (dt, J = 47.3, 5.9 Hz, 2H,
CH2F), 4.10 (t, J = 7.0 Hz, 2H, NCH2), 2.00−1.80 (m, 2H, CH2),
1.76−1.65 (m, 2H, CH2), 1.63 (d, J = 6.9 Hz, 3H, CH3), 1.53−1.35
(m, 2H, CH2) ppm; 13C NMR (100 MHz, CDCl3): δ 164.4 (CO),
143.9 (quat.), 136.7 (quat.), 131.5 (CH), 128.8 (2 × CH), 127.4
(CH), 126.4 (2 × CH), 125.5 (quat), 122.6 (CH), 121.5 (CH), 120.3
1
crystalline solid. Mp 196−198 °C; H NMR (400 MHz, CDCl3): δ
7.98−7.91 (m, 1H), 7.70 (s, 1H), 7.49−7.41 (m, 2H), 7.39−7.33 (m,
2H), 7.32−7.21 (m, 4H), 7.15−7.07 (m, 2H), 7.03−6.95 (m, 2H),
6.17 (br d, J = 7.8 Hz, 1H, NH), 5.42 (app quin., J = 6.9 Hz, 1H,
CH), 5.28 (s, 2H, CH2), 1.64 (d, J = 6.9 Hz, 3H, CH3) ppm; 13C
NMR (100 MHz, CDCl3): δ 164.3 (CO), 162.6 (d, 1JCF = 246.8 Hz,
1
4
(CH), 111.1 (quat.), 110.3 (CH), 83.8 (d, JCF = 165.0 Hz, CH2F),
quat.), 143.8 (quat.), 136.8 (quat.), 132.0 (d, JCF = 3.1 Hz, quat.),
G
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3
131.7 (CH), 128.9 (d, JCF = 8.1 Hz, 2 × CH), 128.8 (2 × CH),
1-(4-Fluorobenzyl)-1H-indole-3-carboxylic Acid (26). Subjecting
indole (18, 2.91 g, 25 mmol) and 4-fluorobenzyl bromide (3.28 mL,
26.3 mmol) to the general procedure above gave, following
recrystallization from i-PrOH, 25 as an off-white solid (4.38 g,
127.4 (CH), 126.4 (2 × CH), 125.7 (quat.), 123.0 (CH), 121.9
(CH), 120.4 (CH), 116.1 (d, 2JCF = 21.4 Hz, 2 × CH), 111.8 (quat.),
110.66 (CH), 50.03 (CH), 48.79 (CH2), 22.26 (CH3) ppm; HRMS
(ESI) C24H21FN2O exact mass 372.1638, accurate mass 372.1645
(mass error 1.89 ppm).
1
65%). Mp 186.5−188.0 °C; H NMR (300 MHz, CD3OD): δ 8.10
(dd, J = 6.3, 3.3 Hz, 1H), 8.02 (s, 1H, NCH), 7.39 (dd, J = 5.7, 2.0
Hz, 1H), 7.26−7.18 (m, 4H), 7.05 (t, J = 8.4 Hz, 2H), 5.43 (s, 2H,
NCH2); 13C NMR (75 MHz, CD3OD): δ 168.7 (CO), 163.8 (d, 1JCF
= 243.8 Hz, quat.), 138.2 (quat.), 136.5 (CH), 134.2 (quat.), 130.2
(d, 3JCF = 8.3 Hz, CH), 128.5 (quat.), 123.9 (CH), 122.8 (CH), 122.5
(CH), 116.6 (d, 2JCF = 21.8 Hz, CH), 111.7 (CH), 108.5 (quat.), 50.6
(CH2) ppm. All physical and spectral properties were consistent with
those previously reported.25,48,50
General Procedure for the Synthesis of 1-Substituted
Indole-3-carboxylic Acids. To a cooled (0 °C) mixture of NaH
(50.0 mmol, 2.0 equiv) in DMF (40 mL) was added a solution of
indole (18, 25.0 mmol, 1.0 equiv) in DMF (5 mL) dropwise. The
mixture was warmed to ambient temperature, stirred for 0.5 h, cooled
to 0 °C, and treated dropwise with the appropriate alkyl bromide
(26.3 mmol, 1.05 equiv). The mixture was stirred for 1 h at ambient
temperature, cooled to 0 °C, and treated portionwise with
trifluoroacetic anhydride (62.5 mmol, 2.5 equiv). The solution was
stirred at ambient conditions for 2 h and quenched by pouring onto
vigorously stirred ice-water (1 L). The precipitate was collected by
vacuum filtration, washed with H2O (3 × 100 mL), and allowed to
air-dry for 24 h. The crude material was dissolved in PhMe (40 mL)
and added dropwise to a refluxing solution of NaOH (100 mmol, 3.3
equiv) in MeOH (20 mL). The solution was heated at reflux for 2 h
and cooled to ambient temperature, and H2O (100 mL) was added.
The layers were separated, and the organic layer was extracted with 1
M aq. NaOH (3 × 20 mL). The combined aqueous layers were
acidified to pH 1 with 10 M aq. HCl and extracted with EtOAc (3 ×
100 mL). The combined organic layers were dried (MgSO4), and
solvent was evaporated under reduced pressure. Unless otherwise
stated, the crude solids were purified by recrystallization from i-PrOH.
1-Pentyl-1H-indole-3-carboxylic Acid (23). Treating indole (18,
2.91 g, 25 mmol) and 1-bromopentane (3.26 mL, 26.3 mmol, 1.05
equiv) according to the general procedure gave, following
recrystallization from i-PrOH, 23 (4.36 g, 70%) as an off-white
crystalline solid. Mp 106.5−108.0 °C; 1H NMR (300 MHz,
CD3OD): δ 8.10 (dd, J = 6.5, 1.9 Hz, 1H), 7.93 (s, 1H, NCH),
7.42−7.27 (m, 3H), 4.17 (t, J = 7.1 Hz, 2H, NCH2), 1.90 (app quin.,
J = 7.1 Hz, 2H, CH2), 1.39−1.32 (m, 4H, 2 × CH2), 0.90 (t, J = 6.6
Hz, 3H, CH3) ppm; 13C NMR (75 MHz, CD3OD): δ 168.8 (CO),
138.1 (quat.), 136.3 (CH), 128.3 (quat.), 123.6 (CH), 122.6 (CH),
122.4 (CH), 111.3 (CH), 107.7 (quat.), 47.7 (CH2), 30.7 (CH2),
30.0 (CH2), 23.3 (CH2), 14.2 (CH3) ppm; All physical and spectral
properties were consistent with those previously reported.25,36,40,48
1-(5-Fluoropentyl)-1H-indole-3-carboxylic Acid (24). Subjecting
indole (18, 2.91 g, 25 mmol) and 1-bromo-5-fluoropentane (3.27 mL,
26.3 mmol, 1.05 equiv) to the general procedure above gave,
following recrystallization from i-PrOH, 24 as an off-white solid (4.85
g, 78%). Mp 117−118 °C; Rf 0.55 (hexane/EtOAc, 50:50); 1H NMR
(400 MHz, CDCl3): δ 8.29−8.19 (m, 1H), 7.93 (s, 1H, NCH), 7.43−
7.34 (m, 1H), 7.35−7.27 (m, 2H), 4.43 (dt, J = 47.2, 5.9 Hz, 2H,
CH2F), 4.20 (t, J = 7.1 Hz, 2H, CH2), 2.02−1.88 (m, 2H, CH2),
1.82−1.63 (m, 2H, CH2), 1.55−1.40 (m, 2H, CH2) ppm; 13C NMR
(100 MHz, CDCl3): δ 170.8 (CO), 136.8 (quat.), 135.5 (CH), 127.1
(quat.), 123.1 (CH), 122.4, (CH), 122.1 (CH), 110.1 (CH), 106.6
(quat.), 83.77 (d, 1JCF = 165.0 Hz), 47.1 (NCH2), 30.1 (d, 2JCF = 19.8
Competition Binding Assays. Competition binding assays
utilizing tritiated radionuclides were conducted as per a previous
study.48 Cell membranes containing triple-hemaggultinated human
CB1 or CB2 were harvested from HEK293 cells stably expressing
either receptor construct. Cells were cultured in Dulbecco’s modified
Eagle’s medium (Thermo Fisher Scientific, Waltham, MA, U.S.A.)
supplemented with fetal bovine serum (10% v/v, Moregate Biotech,
Auckland, N.Z.) and appropriate selection of an antibiotic to near-
confluence in multiple 175 cm2 cell culture flasks (Corning, Corning,
NY, U.S.A.). Cells were suspended using 5 mM EDTA in PBS,
pelleted via centrifugation at ∼200g, and snap-frozen at −80 °C.
Pellets were thawed and resuspended in sucrose buffer (200 mM
sucrose, 50 mM Tris-HCl, 5 mM MgCl2, 2.5 mM EDTA, pH 7.4) and
manually homogenized with a dounce homogenizer. Homogenate was
centrifuged at 1000g for 10 min at 4 °C, with the resulting
supernatant retained and centrifuged again at 27 000g for 30 min at 4
°C. The resulting pellet was resuspended in a minimal volume of
sucrose buffer, and protein concentration was determined using a DC
protein assay (Bio-Rad, Hercules, CA, U.S.A.) following manufacturer
instructions.
Untritiated test compounds were diluted to 10 μM (initial screen)
or serially diluted (pKi determination) with either 1 nM CB1-specific
3H-SR141716A (PerkinElmer, Waltham, MA, U.S.A.) or 1 nM
[3H]CP 55,940 for CB2 (PerkinElmer, Waltham, MA, U.S.A.) and
membrane isolate (7.5−10 μg/point). All components were diluted in
binding buffer (50 mM HEPES, pH 7.4, 1 mM MgCl2, 1 mM CaCl2,
2 mg/mL of bovine serum albumin (BSA), MP Biomedicals,
Auckland, N.Z., filter sterilized) and mixed in a 96-well V-bottom
polypropylene plate (Hangzhou Gene Era Biotech Co., China, 200 μL
final volume). The resulting solution was incubated for 1 h at 30 °C.
Simultaneously, a 96-well fiberglass-bottomed plate (1.2 μM pore,
PerkinElmer, Waltham, MA, U.S.A.) was incubated at 25° with 0.1%
w/v branched polyethylenimine (PEI) solution (50 μL/well, Sigma-
Aldrich, St Louis, MO). Wash buffer (50 mM HEPES, pH 7.4, 50
mM NaCl, 1 mg/mL of BSA) was used to rinse the PEI solution
through the filter plate, using a vacuum manifold (Pall Corp., New
York, NY, U.S.A.) set to a pressure between 5 and 10 mmHg. The
assay mixture was dispensed into the filter wells, which were
subsequently washed thrice with 200 μL of wash buffer. The filter
plate was then removed from the vacuum manifold and dried
overnight. Scintillation fluid (50 μL, Irgasafe Plus, PerkinElmer) was
added to each well and incubated in dark conditions for 30 min prior
to detection in a Wallac MicroBeta Trilux scintillation counter
(PerkinElmer, Waltham, MA, U.S.A., 2 min counts).
3
Hz), 29.6 (CH2), 22.9 (d, JCF = 5.0 Hz) ppm. All physical and
spectral properties were consistent with those previously re-
ported.25,37,48
1-(Cyclohexylmethyl)-1H-indole-3-carboxylic Acid (25). Subject-
ing indole (18, 2.91 g, 25 mmol) and (bromomethyl)cyclohexane
(3.63 mL, 26.3 mmol) to the general procedure above gave, following
recrystallization from i-PrOH, 25 as an off-white solid (4.63 g, 72%).
Data were exported and analyzed in Graphpad Prism (Version
8.0.0, Graphpad Software Inc., San Diego, CA, U.S.A.), with a “one-
site fit Ki” model used to estimate pKi, using estimated Kd values of 1
3
nM for both H-SR141716A (CB1) or [3H]CP 55,940 (CB2).
1
Mp 175.5−177.0 °C; H NMR (300 MHz, CD3OD) δ 8.08 (d, J =
In Vitro Cannabinoid Receptor Functional Assay. Functional
assays for the cannabinoids were carried out in mouse AtT20-FlpIn
cells stably transfected with human CB1 or CB2, as described
previously.49 Cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) containing 10% fetal bovine serum (FBS), 100 U
of penicillin/streptomycin, and 80 μg/mL of hygromycin as a
selection antibiotic. Cells 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
7.3 Hz, 1H), 7.90 (s, 1H, NCH), 7.46 (d, J = 7.8 Hz, 1H), 7.27−7.17
(m, 2H), 4.07 (d, J = 7.3 Hz, 2H, NCH2), 1.96−1.81 (m, 1H, CH),
1.78−1.56 (m, 5H, CH2), 1.28−1.16 (m, 3H, CH2), 1.11−0.98 (m,
2H, CH2) ppm; 13C NMR (75 MHz, CD3OD) δ 165.5 (CO), 142.6
(quat.), 135.9 (quat.), 128.0 (CH), 124.5 (quat.), 124.1 (CH), 123.0
(CH), 111.4 (CH), 56.5 (CH2), 40.0 (CH), 31.7 (CH2), 27.6 (CH),
26.7 (CH2) ppm. All physical and spectral properties were consistent
with those previously reported.25,48,49
H
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Research Article
Leibovitz’s L-15 media supplemented with 1% FBS, 100 U of
penicillin/streptomycin, and 15 mM glucose. The cells were plated in
a volume of 90 μL in black-walled, clear-bottomed 96-well
microplates (Corning) and incubated overnight at 37 °C in ambient
CO2.
orcid.org/0000-0002-4690-4318; Phone: +61 2 9351
0805; Email: samuel.banister@sydney.edu.au
Authors
Adam Ametovski − The Lambert Initiative for Cannabinoid
Therapeutics, Brain and Mind Centre and School of Chemistry,
The University of Sydney, Camperdown, NSW 2050, Australia
Christa Macdonald − School of Medical Sciences, The
University of Auckland, Auckland 1142, New Zealand
Jamie J. Manning − Department of Pharmacology and
Toxicology, University of Otago, Dunedin 9016, New Zealand;
orcid.org/0000-0002-6945-2254
S. A. Syed Haneef − Faculty of Medicine and Health Sciences,
Macquarie University, Macquarie Park, NSW 2109, Australia
Marina Santiago − Faculty of Medicine and Health Sciences,
Macquarie University, Macquarie Park, NSW 2109, Australia;
orcid.org/0000-0001-9388-8309
Membrane potential was measured using a FLIPR Membrane
Potential Assay kit (blue) from Molecular Devices.51 Dye was
reconstituted with assay buffer (145 mM NaCl, 22 mM HEPES, 0.338
mM Na2HPO4, 4.17 mM NaHCO3, 0.441 mM KH2PO4, 0.407 mM
MgSO4, 0.493 mM MgCl2, 1.26 mM CaCl2, 5.56 mM glucose (pH
7.4, osmolarity = 315 5) and added in a volume of 90 μL to the
cells, without removal of the L-15. Plates were then incubated at 37
°C at ambient CO2 for at least 60 min. Fluorescence was measured
using a FlexStation 3 (Molecular Devices) microplate reader, cells
were excited with a fluorescence lamp at a wavelength of 530 nm, and
light emission was measured at 565 nm. Baseline readings were taken
every 2 s for at least 60 s, 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
(DMSO, 0.1% final concentration), which was a 1−2% decrease in
fluorescence.
Lewis Martin − The Lambert Initiative for Cannabinoid
Therapeutics, Brain and Mind Centre, The University of Sydney,
Camperdown, NSW 2050, Australia; orcid.org/0000-0002-
8621-4258
Data were analyzed with Prism, using a four-parameter nonlinear
regression to fit concentration−response curves. A CP 55,940
concentration response curve was done each day, and a maximally
effective concentration of CP 55,940 (1 μM) was included in every
column of every plate to facilitate comparisons between experiments.
Molecular Dynamics Simulations. Simulations of the four
compounds SDB-006 (10), (S)-α-methylbenzyl-PICA ((S)-14), (R)-
α-methylbenzyl-PICA ((R)-14), and CUMYL-PICA (6) in the CB1
receptor used PDB structure 6N4B.7 The cocrystallized MDMB-
FUBINACA (27) was removed from the receptor, and chain “R”
representing the GPCR portion of the structure was translated and
rotated using the Orientations of Proteins in Membranes server.52
The aligned protein was then submitted to the CHARMM-GUI
server to generate an all-atom solvated POPC membrane system with
0.15 M NaCl comprising 120 lipids, 5901 water molecules, and
37 877 total atoms.53 The drug molecules were aligned to MDMB-
FUBINACA (27) by generating 1000 conformations with RDKit54,55
and taking the best-scoring alignment as determined by the O3A
algorithm.56 The drugs were parametrized for simulation with
AMBER force fields using OpenForceField, which shows good
accuracy in host−guest binding simulations.57 All simulations used
OpenMM beta version 7.5 with the LangevinMiddleIntegrator with a
3 fs time step and a 10/ps friction coefficient set to 310 K, PME to
account for long-range electrostatics, fixed hydrogen bond lengths,
and a MonteCarloMembraneBarostat to achieve NPT conditions.58
During equilibration, all protein atoms were restrained to their
crystal structure positions with harmonic distance restraints. The force
of this restraint was initially 5 kcal/mol/Å2, which was lowered
exponentially in 300 steps of 12 ps/step. The restraining force was
removed before production runs. Coordinates and velocities from
equilibration were used in production, in which the systems were
simulated without restraints for 90 ns. Trajectories were visualized
and analyzed with VMD.59
Eric Sparkes − The Lambert Initiative for Cannabinoid
Therapeutics, Brain and Mind Centre and School of Chemistry,
The University of Sydney, Camperdown, NSW 2050, Australia
Andrew Reckers − Clinical Toxicology and Environmental
Biomonitoring Laboratory, University of California, San
Francisco, California 94143, United States
Roy R. Gerona − Clinical Toxicology and Environmental
Biomonitoring Laboratory, University of California, San
Francisco, California 94143, United States
Mark Connor − Faculty of Medicine and Health Sciences,
Macquarie University, Macquarie Park, NSW 2109, Australia;
orcid.org/0000-0003-2538-2001
Michelle Glass − Department of Pharmacology and Toxicology,
University of Otago, Dunedin 9016, New Zealand;
orcid.org/0000-0002-5997-6898
Complete contact information is available at:
https://pubs.acs.org/10.1021/acschemneuro.0c00591
Author Contributions
A.A., E.S., and S.D.B. synthesized and analytically characterized
the compounds as conceived by S.D.B. A.R. performed high
resolution mass spectrometry under the direction of R.R.G.
C.M. and J.J.M. performed binding experiments under the
guidance of M.G. S.A.S.H. performed cannabinoid functional
activity assays, with assistance from M.S., under the supervision
of M.C. L.M. conducted molecular dynamics simulations. A.A.,
L.M., and S.D.B. wrote the manuscript, and all authors
reviewed and edited drafts of the manuscript. All authors
approved the final version of the paper.
ASSOCIATED CONTENT
* Supporting Information
■
sı
Funding
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acschemneuro.0c00591.
This work was supported by NHMRC Project Grant 1161571
and a Brain and Mind Centre Research Development Grant
from The University of Sydney awarded to S.D.B. A.A., E.S.,
L.M., and S.D.B. are supported by The Lambert Initiative for
Cannabinoid Therapeutics, a philanthropically funded research
program based at The University of Sydney.
1H and 13C NMR spectra of novel compounds (PDF)
AUTHOR INFORMATION
Corresponding Author
Samuel D. Banister − The Lambert Initiative for Cannabinoid
Therapeutics, Brain and Mind Centre and School of Chemistry,
The University of Sydney, Camperdown, NSW 2050, Australia;
■
Notes
The authors declare no competing financial interest.
I
https://dx.doi.org/10.1021/acschemneuro.0c00591
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Research Article
(17) Walsh, K. B., and Andersen, H. K. (2020) Molecular
Pharmacology of Synthetic Cannabinoids: Delineating CB1 Recep-
tor-Mediated Cell Signaling. Int. J. Mol. Sci. 21, 6115.
(18) Trecki, J., Gerona, R. R., and Schwartz, M. D. (2015) Synthetic
Cannabinoid-Related Illnesses and Deaths. N. Engl. J. Med. 373, 103−
107.
REFERENCES
■
(1) Peacock, A., Bruno, R., Gisev, N., Degenhardt, L., Hall, W.,
Sedefov, R., White, J., Thomas, K. V., Farrell, M., and Griffiths, P.
(2019) New psychoactive substances: challenges for drug surveillance,
control, and public health responses. Lancet 394, 1668−1684.
(2) United Nation Office on Drugs and Crime. (2019) World Drug
Report 2019, United Nations publication, Sales No. E.18.XI.9,
https://wdr.unodc.org/wdr2019/ (accessed September 9, 2020).
(3) Banister, S. D., and Connor, M. (2018) The Chemistry and
Pharmacology of Synthetic Cannabinoid Receptor Agonist New
Psychoactive Substances: Evolution. Handb. Exp. Pharmacol. 252,
191−226.
(4) Worob, A., and Wenthur, C. (2019) Dark Classics in Chemical
Neuroscience: Synthetic Cannabinoids (Spice/K2). ACS Chem.
Neurosci., DOI: 10.1021/acschemneuro.9b00586.
(5) Alam, R. M., and Keating, J. J. (2020) Adding more ″spice″ to
the pot: A review of the chemistry and pharmacology of newly
emerging heterocyclic synthetic cannabinoid receptor agonists. Drug
Test. Anal. 12, 297−315.
(6) Hua, T., Vemuri, K., Nikas, S. P., Laprairie, R. B., Wu, Y., Qu, L.,
Pu, M., Korde, A., Jiang, S., Ho, J.-H., Han, G. W., Ding, K., Li, X.,
Liu, H., Hanson, M. A., Zhao, S., Bohn, L. M., Makriyannis, A.,
Stevens, R. C., and Liu, Z.-J. (2017) Crystal structures of agonist-
bound human cannabinoid receptor CB1. Nature 547, 468.
(7) Krishna Kumar, K., Shalev-Benami, M., Robertson, M. J., Hu, H.,
Banister, S. D., Hollingsworth, S. A., Latorraca, N. R., Kato, H. E.,
Hilger, D., Maeda, S., Weis, W. I., Farrens, D. L., Dror, R. O.,
Malhotra, S. V., Kobilka, B. K., and Skiniotis, G. (2019) Structure of a
Signaling Cannabinoid Receptor 1-G Protein Complex. Cell 176,
448−458 e412.
̈
(19) Angerer, V., Jacobi, S., Franz, F., Auwarter, V., and Pietsch, J.
(2017) Three fatalities associated with the synthetic cannabinoids 5F-
ADB, 5F-PB-22, and AB-CHMINACA. Forensic Sci. Int. 281, e9−e15.
(20) Adams, A. J., Banister, S. D., Irizarry, L., Trecki, J., Schwartz,
M., and Gerona, R. (2017) ″Zombie″ Outbreak Caused by the
Synthetic Cannabinoid AMB-FUBINACA in New York. N. Engl. J.
Med. 376, 235−242.
(21) Bowden, M. J., and Williamson, J. P. B. (2014) Preparation of
cannabinoid indole and indazole compounds for treating pain and
nausea, stimulating appetite, and inducing a positive mood change,
World Patent 2014167530.
(22) Kevin, R. C., Anderson, L., McGregor, I. S., Boyd, R., Manning,
J. J., Glass, M., Connor, M., and Banister, S. D. (2019) CUMYL-4CN-
BINACA Is an Efficacious and Potent Pro-Convulsant Synthetic
Cannabinoid Receptor Agonist. Front. Pharmacol. 10, 595.
(23) Dobaja, M., Grenc, D., Kozelj, G., and Brvar, M. (2017)
Occupational transdermal poisoning with synthetic cannabinoid
cumyl-PINACA. Clin. Toxicol. 55, 193−195.
(24) European Monitoring Centre for Drugs and Drug Addiction.
(2015) EMCDDA-Europol 2014 Annual Report on the implementation
of Council Decision 2005/387/JHA, Publications Office of the
European Union, Luxembourg, https://www.emcdda.europa.eu/
publications/implementation-reports/2014_en/ (accessed September
9, 2020).
(25) Longworth, M., Banister, S. D., Boyd, R., Kevin, R. C., Connor,
M., McGregor, I. S., and Kassiou, M. (2017) Pharmacology of Cumyl-
Carboxamide Synthetic Cannabinoid New Psychoactive Substances
(NPS) CUMYL-BICA, CUMYL-PICA, CUMYL-5F-PICA, CUMYL-
5F-PINACA, and Their Analogues. ACS Chem. Neurosci. 8, 2159−
2167.
(26) Bovens, M., Bissig, C., Staeheli, S. N., Poetzsch, M., Pfeiffer, B.,
and Kraemer, T. (2017) Structural characterization of the new
synthetic cannabinoids CUMYL-PINACA, 5F-CUMYL-PINACA,
CUMYL-4CN-BINACA, 5F-CUMYL-P7AICA and CUMYL-4CN-
B7AICA. Forensic Sci. Int. 281, 98−105.
(27) Kevin, R. C., Lefever, T. W., Snyder, R. W., Patel, P. R., Fennell,
T. R., Wiley, J. L., McGregor, I. S., and Thomas, B. F. (2017) In vitro
and in vivo pharmacokinetics and metabolism of synthetic
cannabinoids CUMYL-PICA and 5F-CUMYL-PICA. Forensic Toxicol.
35, 333−347.
(28) Asada, A., Doi, T., Tagami, T., Takeda, A., Satsuki, Y.,
Kawaguchi, M., Nakamura, A., and Sawabe, Y. (2018) Cannabimi-
metic activities of cumyl carboxamide-type synthetic cannabinoids.
Forensic Toxicol. 36, 170−177.
(8) Banister, S. D., Arnold, J. C., Connor, M., Glass, M., and
McGregor, I. S. (2019) Dark Classics in Chemical Neuroscience:
Delta(9)-Tetrahydrocannabinol. ACS Chem. Neurosci. 10, 2160−
2175.
(9) Li, X., Hua, T., Vemuri, K., Ho, J. H., Wu, Y., Wu, L., Popov, P.,
Benchama, O., Zvonok, N., Locke, K., Qu, L., Han, G. W., Iyer, M. R.,
Cinar, R., Coffey, N. J., Wang, J., Wu, M., Katritch, V., Zhao, S.,
Kunos, G., Bohn, L. M., Makriyannis, A., Stevens, R. C., and Liu, Z. J.
(2019) Crystal Structure of the Human Cannabinoid Receptor CB2.
Cell 176, 459−467.E13.
̈
(10) 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.
(11) Finlay, D. B., Manning, J. J., Ibsen, M. S., Macdonald, C. E.,
Patel, M., Javitch, J. A., Banister, S. D., and Glass, M. (2019) Do Toxic
Synthetic Cannabinoid Receptor Agonists Have Signature in Vitro
Activity Profiles? A Case Study of AMB-FUBINACA. ACS Chem.
Neurosci. 10, 4350−4360.
(12) Sachdev, S., Vemuri, K., Banister, S. D., Longworth, M.,
Kassiou, M., Santiago, M., Makriyannis, A., and Connor, M. (2019) In
vitro determination of the efficacy of illicit synthetic cannabinoids at
CB1 receptors. Br. J. Pharmacol. 176, 4653−4665.
(13) Wouters, E., Walraed, J., Banister, S. D., and Stove, C. P.
(2019) Insights into biased signaling at cannabinoid receptors:
synthetic cannabinoid receptor agonists. Biochem. Pharmacol. 169,
113623.
(29) Gamage, T. F., Farquhar, C. E., Lefever, T. W., Marusich, J. A.,
Kevin, R. C., McGregor, I. S., Wiley, J. L., and Thomas, B. F. (2018)
Molecular and Behavioral Pharmacological Characterization of
Abused Synthetic Cannabinoids MMB- and MDMB-FUBINACA,
MN-18, NNEI, CUMYL-PICA, and 5-Fluoro-CUMYL-PICA. J.
Pharmacol. Exp. Ther. 365, 437−446.
̈
̈
(30) Ozturk, Y. E., Yeter, O., Ozturk, S., Karakus, G., Ates, I., Buyuk,
̈
̈
Y., and Yurdun, T. (2018) Detection of metabolites of the new
synthetic cannabinoid CUMYL-4CN-BINACA in authentic urine
samples and human liver microsomes using high-resolution mass
spectrometry. Drug Test. Anal. 10, 449−459.
(31) Staeheli, S. N., Poetzsch, M., Veloso, V. P., Bovens, M., Bissig,
C., Steuer, A. E., and Kraemer, T. (2018) In vitro metabolism of the
synthetic cannabinoids CUMYL-PINACA, 5F−CUMYL-PINACA,
CUMYL-4CN-BINACA, 5F−CUMYL-P7AICA and CUMYL-4CN-
B7AICA. Drug Test. Anal. 10, 148−157.
(32) Banister, S. D., Adams, A., Kevin, R. C., Macdonald, C., Glass,
M., Boyd, R., Connor, M., McGregor, I. S., Havel, C. M., Bright, S. J.,
Vilamala, M. V., Lladanosa, C. G., Barratt, M. J., and Gerona, R. R.
(14) Wouters, E., Walraed, J., Robertson, M. J., Meyrath, M.,
́
Szpakowska, M., Chevigne, A., Skiniotis, G., and Stove, C. (2020)
Assessment of biased agonism amongst distinct synthetic cannabinoid
receptor agonist scaffolds. ACS Pharmacol. Transl. Sci. 3, 285−295.
(15) Patel, M., Manning, J. J., Finlay, D. B., Javitch, J. A., Banister, S.
D., Grimsey, N. L., and Glass, M. (2020) Signalling profiles of a
structurally diverse panel of synthetic cannabinoid receptor agonists.
Biochem. Pharmacol. 175, 113871.
(16) Sachdev, S., Banister, S. D., Santiago, M., Bladen, C., Kassiou,
M., and Connor, M. (2020) Differential activation of G protein-
mediated signaling by synthetic cannabinoid receptor agonists.
Pharmacol. Res. Perspect. 8, No. e00566.
J
https://dx.doi.org/10.1021/acschemneuro.0c00591
ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
(2019) Synthesis and pharmacology of new psychoactive substance
5F-CUMYL-P7AICA, a scaffold- hopping analog of synthetic
cannabinoid receptor agonists 5F-CUMYL-PICA and 5F-CUMYL-
PINACA. Drug Test. Anal. 11, 279−291.
(46) Talele, T. T. (2018) Natural-Products-Inspired Use of the gem-
Dimethyl Group in Medicinal Chemistry. J. Med. Chem. 61, 2166−
2210.
̈
̆
(47) Olgen, S., Varol, P., Çoban, T., and Nebioglu, D. (2008)
Synthesis and evaluation of N-substituted indole-3-carboxamide
derivatives as inhibitors of lipid peroxidation and superoxide anion
formation. J. Enzyme Inhib. Med. Chem. 23, 334−340.
(33) Haschimi, B., Giorgetti, A., Mogler, L., Nagy, T. Z., Kramer, S.,
́
̈
Halter, S., Boros, S., Dobos, A., Hidvegi, E., and Auwarter, V. (2020)
The Novel Psychoactive Substance Cumyl-CH-MEGACLONE:
Human Phase-I Metabolism, Basic Pharmacological Characterization,
and Comparison to Other Synthetic Cannabinoid Receptor Agonists
with a γ-Carboline-1-one Core. J. Anal. Toxicol., bkaa065.
(34) Janssens, L., Cannaert, A., Connolly, M. J., Liu, H., and Stove,
C. P. (2020) In vitro activity profiling of Cumyl-PEGACLONE
variants at the CB1 receptor: Fluorination versus isomer exploration.
Drug Test. Anal. 12, 1336.
(48) Banister, S. D., Kevin, R. C., Martin, L., Adams, A., Macdonald,
C., Manning, J. J., Boyd, R., Cunningham, M., Stevens, M. Y.,
McGregor, I. S., Glass, M., Connor, M., and Gerona, R. R. (2019)
The chemistry and pharmacology of putative synthetic cannabinoid
receptor agonist (SCRA) new psychoactive substances (NPS) 5F-PY-
PICA, 5F-PY-PINACA, and their analogs. Drug Test. Anal. 11, 976−
989.
(49) Banister, S. D., Longworth, M., Kevin, R., Sachdev, S., Santiago,
M., Stuart, J., Mack, J. B., Glass, M., McGregor, I. S., Connor, M., and
Kassiou, M. (2016) Pharmacology of Valinate and tert-Leucinate
Synthetic Cannabinoids 5F-AMBICA, 5F-AMB, 5F-ADB, AMB-
FUBINACA, MDMB-FUBINACA, MDMB-CHMICA, and Their
Analogues. ACS Chem. Neurosci. 7, 1241−1254.
(50) Banister, S. D., Moir, M., Stuart, J., Kevin, R. C., Wood, K. E.,
Longworth, M., Wilkinson, S. M., Beinat, C., Buchanan, A. S., Glass,
M., Connor, M., McGregor, I. S., and Kassiou, M. (2015)
Pharmacology of Indole and Indazole Synthetic Cannabinoid
Designer Drugs AB-FUBINACA, ADB-FUBINACA, AB-PINACA,
ADB-PINACA, 5F-AB-PINACA, 5F-ADB-PINACA, ADBICA, and
5F-ADBICA. ACS Chem. Neurosci. 6, 1546−1559.
(51) Knapman, A., Santiago, M., Du, Y. P., Bennallack, P. R.,
Christie, M. J., and Connor, M. (2013) A continuous, fluorescence-
based assay of mu-opioid receptor activation in AtT-20 cells. J. Biomol.
Screening 18, 269−276.
(52) Lomize, M. A., Pogozheva, I. D., Joo, H., Mosberg, H. I., and
Lomize, A. L. (2012) OPM database and PPM web server: resources
for positioning of proteins in membranes. Nucleic Acids Res. 40,
D370−D376.
(53) Jo, S., Kim, T., Iyer, V. G., and Im, W. (2008) CHARMM-GUI:
A web-based graphical user interface for CHARMM. J. Comput. Chem.
29, 1859−1865.
(54) Riniker, S., and Landrum, G. A. (2015) Better Informed
Distance Geometry: Using What We Know To Improve Con-
formation Generation. J. Chem. Inf. Model. 55, 2562−2574.
(55) Landrum, G. A. RDKit: A software suite for cheminformatics,
computational chemistry, and predictive modeling, https://www.rdkit.
org/RDKit_Overview.pdf (accessed September 9, 2020).
(56) Tosco, P., Balle, T., and Shiri, F. (2011) Open3DALIGN: an
open-source software aimed at unsupervised ligand alignment. J.
Comput.-Aided Mol. Des. 25, 777.
(57) Slochower, D. R., Henriksen, N. M., Wang, L.-P., Chodera, J.
D., Mobley, D. L., and Gilson, M. K. (2019) Binding Thermody-
namics of Host−Guest Systems with SMIRNOFF99Frosst 1.0.5 from
the Open Force Field Initiative. J. Chem. Theory Comput. 15, 6225−
6242.
(58) Eastman, P., Swails, J., Chodera, J. D., McGibbon, R. T., Zhao,
Y., Beauchamp, K. A., Wang, L.-P., Simmonett, A. C., Harrigan, M. P.,
Stern, C. D., Wiewiora, R. P., Brooks, B. R., and Pande, V. S. (2017)
OpenMM 7: Rapid development of high performance algorithms for
molecular dynamics. PLoS Comput. Biol. 13, e1005659.
(59) Humphrey, W., Dalke, A., and Schulten, K. (1996) VMD:
Visual molecular dynamics. J. Mol. Graphics 14, 33−38.
(35) European Monitoring Centre for Drugs and Drug Addiction.
(2014) EMCDDA−Europol 2013 Annual Report on the implementation
of Council Decision 2005/387/JHA, Publications Office of the
European Union, Luxembourg, https://www.emcdda.europa.eu/
publications/implementation-reports/2013_en/ (accessed September
9, 2020).
(36) Banister, S. D., Wilkinson, S. M., Longworth, M., Stuart, J.,
Apetz, N., English, K., Brooker, L., Goebel, C., Hibbs, D. E., Glass, M.,
Connor, M., McGregor, I. S., and Kassiou, M. (2013) The synthesis
and pharmacological evaluation of adamantane-derived indoles:
cannabimimetic drugs of abuse. ACS Chem. Neurosci. 4, 1081−1092.
(37) Banister, S. D., Stuart, J., Kevin, R. C., Edington, A., Longworth,
M., Wilkinson, S. M., Beinat, C., Buchanan, A. S., Hibbs, D. E., Glass,
M., Connor, M., McGregor, I. S., and Kassiou, M. (2015) Effects of
bioisosteric fluorine in synthetic cannabinoid designer drugs JWH-
018, AM-2201, UR-144, XLR-11, PB-22, 5F-PB-22, APICA, and STS-
135. ACS Chem. Neurosci. 6, 1445−1458.
(38) Hess, C., Schoeder, C. T., Pillaiyar, T., Madea, B., and Muller,
̈
C. E. (2016) Pharmacological evaluation of synthetic cannabinoids
identified as constituents of spice. Forensic Toxicol. 34, 329−343.
(39) Diao, X., Carlier, J., Scheidweiler, K. B., and Huestis, M. A.
(2017) In vitro metabolism of new synthetic cannabinoid SDB-006 in
human hepatocytes by high-resolution mass spectrometry. Forensic
Toxicol. 35, 252−262.
(40) Banister, S. D., Olson, A., Winchester, M., Stuart, J., Edington,
A. R., Kevin, R. C., Longworth, M., Herrera, M., Connor, M.,
McGregor, I. S., Gerona, R. R., and Kassiou, M. (2018) The chemistry
and pharmacology of synthetic cannabinoid SDB-006 and its
regioisomeric fluorinated and methoxylated analogs. Drug Test.
Anal. 10, 1099−1109.
(41) Adam, J. M., Cairns, J., Caulfield, W., Cowley, P., Cumming, I.,
Easson, M., Edwards, D., Ferguson, M., Goodwin, R., Jeremiah, F.,
Kiyoi, T., Mistry, A., Moir, E., Morphy, R., Tierney, J., York, M.,
Baker, J., Cottney, J. E., Houghton, A. K., Westwood, P. J., and
Walker, G. (2010) Design, synthesis, and structure−activity relation-
ships of indole-3-carboxamides as novel water soluble cannabinoid
CB1 receptor agonists. MedChemComm 1, 54−60.
̈
̆
(42) Olgen, S., Kaeßler, A., Nebioglu, D., and Jose, J. (2007)
Research Letter: New Potent Indole Derivatives as Hyaluronidase
Inhibitors. Chem. Biol. Drug Des. 70, 547−551.
̈
(43) Olgen, S., Altanlar, N., Karataylı, E., and Bozdayı, M. (2008)
Antimicrobial and Antiviral Screening of Novel Indole Carboxamide
and Propanamide Derivatives. Z. Naturforsch., C: J. Biosci. 63, 189−
195.
(44) Doi, T., Tagami, T., Takeda, A., Asada, A., and Sawabe, Y.
(2018) Evaluation of carboxamide-type synthetic cannabinoids as
CB1/CB2 receptor agonists: difference between the enantiomers.
Forensic Toxicol. 36, 51−60.
(45) Antonides, L. H., Cannaert, A., Norman, C., Vives, L., Harrison,
A., Costello, A., Nic Daeid, N., Stove, C. P., Sutcliffe, O. B., and
McKenzie, C. (2019) Enantiospecific Synthesis, Chiral Separation,
and Biological Activity of Four Indazole-3-Carboxamide-Type
Synthetic Cannabinoid Receptor Agonists and Their Detection in
Seized Drug Samples. Front. Chem. 7, 321.
K
https://dx.doi.org/10.1021/acschemneuro.0c00591
ACS Chem. Neurosci. XXXX, XXX, XXX−XXX