Synthesis and in Vitro Cannabinoid Receptor 1 Activity of Recently Detected Synthetic Cannabinoids 4F-MDMB-BICA, 5F-MPP-PICA, MMB-4en-PICA, CUMYL-CBMICA, ADB-BINACA, APP-BINACA, 4F-MDMB-BINACA, MDMB-4en-PINACA, A-CHMINACA, 5F-AB-P7AICA, 5F-MDMB-P7AICA, and 5F-AP7AICA

Article abstract of DOI:10.1021/acschemneuro.0c00644

Synthetic cannabinoid receptor agonists (SCRAs) are an evolving class of new psychoactive substances (NPS) with structurally diverse compounds emerging each year. Due to the rapid pace at which these drugs enter the market, there is often little or nil information regarding the pharmacology of these substances despite widespread human use. In this study, 12 recently emerged SCRAs (reported between 2018 and 2020) were synthesized, analytically characterized, and pharmacologically evaluated using a live cell-based nanoluciferase complementation reporter assay that monitors in vitro cannabinoid receptor type 1 (CB1) activation via its interaction with β-arrestin 2 (βarr2). All synthesized SCRAs acted as agonists of CB1, although differences in potency (EC50 = 2.33-5475 nM) and efficacy (Emax = 37-378%) were noted, and several structure-activity relationships were identified. SCRAs featuring indazole cores (EC50 = 2.33-159 nM) were generally of equal or greater potency than indole analogues (EC50 = 32.9-330 nM) or 7-azaindole derivatives (EC50 = 64.0-5475 nM). Interestingly, with the exception of APP-BINACA (Emax = 75.7%) and 5F-A-P7AICA (Emax = 37.4%), all SCRAs showed greater efficacy than the historical SCRA JWH-018 to which responses were normalized (Emax = 142-378%). The most potent CB1 agonists in the study were ADB-BINACA (EC50 = 6.36 nM), 4F-MDMB-BINACA (EC50 = 7.39 nM), and MDMB-4en-PINACA (EC50 = 2.33 nM). Notably, all of these SCRAs featured an indazole core as well as a bulky tert-butyl moiety in the pendant amino acid side chain. This study confirms that recently detected SCRAs 4F-MDMB-BICA, 5F-MPP-PICA, MMB-4en-PICA, CUMYL-CBMICA, ADB-BINACA, APP-BINACA, 4F-MDMB-BINACA, MDMB-4en-PINACA, A-CHMINACA, 5F-AB-P7AICA, 5F-MDMB-P7AICA, and 5F-AP7AICA were all able to activate the CB1 receptor in vitro, albeit to different extents, and are potentially psychoactive in vivo. These results indicate that further evaluation of these widely used NPS is warranted to better understand the risks associated with human consumption of these drugs.

Full text of DOI:10.1021/acschemneuro.0c00644

pubs.acs.org/chemneuro
Research Article
Synthesis and in Vitro Cannabinoid Receptor 1 Activity of Recently
Detected Synthetic Cannabinoids 4F-MDMB-BICA, 5F-MPP-PICA,
MMB-4en-PICA, CUMYL-CBMICA, ADB-BINACA, APP-BINACA, 4F-
MDMB-BINACA, MDMB-4en-PINACA, A‑CHMINACA, 5F-AB-P7AICA,
5F-MDMB-P7AICA, and 5F-AP7AICA
Annelies Cannaert, Eric Sparkes, Edward Pike, Jia Lin Luo, Ada Fang, Richard C. Kevin, Ross Ellison,
Roy Gerona, Samuel D. Banister,* and Christophe P. Stove*
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ABSTRACT: Synthetic cannabinoid receptor agonists (SCRAs)
are an evolving class of new psychoactive substances (NPS) with
structurally diverse compounds emerging each year. Due to the
rapid pace at which these drugs enter the market, there is often
little or nil information regarding the pharmacology of these
substances despite widespread human use. In this study, 12 recently
emerged SCRAs (reported between 2018 and 2020) were
synthesized, analytically characterized, and pharmacologically
evaluated using a live cell-based nanoluciferase complementation
reporter assay that monitors in vitro cannabinoid receptor type 1
(CB1) activation via its interaction with β-arrestin 2 (βarr2). All
synthesized SCRAs acted as agonists of CB1, although differences in potency (EC50 = 2.33−5475 nM) and efficacy (Emax = 37−
378%) were noted, and several structure−activity relationships were identified. SCRAs featuring indazole cores (EC50 = 2.33−159
nM) were generally of equal or greater potency than indole analogues (EC50 = 32.9−330 nM) or 7-azaindole derivatives (EC50
=
64.0−5475 nM). Interestingly, with the exception of APP-BINACA (Emax = 75.7%) and 5F-A-P7AICA (Emax = 37.4%), all SCRAs
showed greater efficacy than the historical SCRA JWH-018 to which responses were normalized (Emax = 142−378%). The most
potent CB1 agonists in the study were ADB-BINACA (EC50 = 6.36 nM), 4F-MDMB-BINACA (EC50 = 7.39 nM), and MDMB-4en-
PINACA (EC50 = 2.33 nM). Notably, all of these SCRAs featured an indazole core as well as a “bulky” tert-butyl moiety in the
pendant amino acid side chain. This study confirms that recently detected SCRAs 4F-MDMB-BICA, 5F-MPP-PICA, MMB-4en-
PICA, CUMYL-CBMICA, ADB-BINACA, APP-BINACA, 4F-MDMB-BINACA, MDMB-4en-PINACA, A-CHMINACA, 5F-AB-
P7AICA, 5F-MDMB-P7AICA, and 5F-AP7AICA were all able to activate the CB1 receptor in vitro, albeit to different extents, and
are potentially psychoactive in vivo. These results indicate that further evaluation of these widely used NPS is warranted to better
understand the risks associated with human consumption of these drugs.
KEYWORDS: cannabinoid, pharmacology, MDMB, CUMYL, BINACA, PINACA, CHMINACA, P7AICA
in the central nervous system, while the CB2 subtype is
predominantly found in immune cells.6,7 Activation of CB1
induces an interaction of the receptor with the G protein (in this
particular case Gαi), resulting in a series of downstream signaling
events.8,9 Besides activation of the canonical G protein pathway,
β-arrestin 2 (βarr2) is also recruited to the activated GPCR. This
INTRODUCTION
■
The field of new psychoactive substances (NPS) is constantly
evolving, with new compounds entering the illicit drug market
each year.1 More than 290 synthetic cannabinoid receptor
agonists (SCRAs) have been reported to the United Nations
Office on Drugs and Crime (UNODC), representing one of the
largest and most structurally diverse classes of NPS.1,2 SCRAs
were created as unregulated alternatives to cannabis, mimicking
the effect of the main psychotropic constituent Δ9-tetrahy-
drocannabinol (THC).3,4 They exert their effects mainly via the
cannabinoid (CB) receptors, with cannabinoid receptor type 1
(CB1) being mainly responsible for the psychoactive effects.5
CB1 is a G protein-coupled receptor (GPCR) widely distributed
Received: October 8, 2020
Accepted: November 16, 2020
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Figure 1. Chemical structures of the different SCRAs, divided based on their heterocyclic core: (A) indole SCRAs (including the reference JWH-018),
(B) indazole SCRAs, and (C) 7-aza-indole SCRAs.
scaffolding protein can induce the desensitization and/or
internalization of the receptor, and is responsible for a distinct
set of signaling events.8−10
((pentyl-4en) MDMB-PINACA, 9), and A-CHMINACA
(ADAMANTYL-CHMINACA, SGT-37, AKB48CH, 10).
Finally, the 7-azaindoles consisted of 5F-AB-P7AICA (11),
5F-MDMB-P7AICA (7′N-5F-ADB, 12), and 5F-A-P7AICA
(13). Nothing is known about the pharmacology of any of these
SCRAs, although the metabolic profiles of MMB-4en-PICA,23
MDMB-4en-PINACA,24,25 and 5F-MDMB-P7AICA26,27 and
the analytical characterization of CUMYL-CBMICA28 and A-
CHMINACA29 were recently described. It is also worth noting
that many of the novel amino acid-derived SCRAs described
here were presumably inspired by the structures claimed in a
patent awarded to Pfizer in 2009 for the development of
cannabinoid agonists.30
All of the indole, indazole, and 7-azaindole SCRAs
characterized herein were reported by the European Union
(EU) Early Warning System (EWS) and/or NMS Laboratories
between 2018 and 2020 (for tabulated detection data, please see
the Supporting Information). 4F-MDMB-BICA (also known as
4F-MDMB-BUTICA, 2) was identified in seized material in the
European Union (EU) in March 202031,32 and the United States
(US) in July 2020.33 4F-MDMB-BICA is the butyl homologue
of 5-fluoropentyl-substituted 5F-MDMB-PICA (3b) and the
indole analogue of indazole 4F-MDMB-BINACA (8), which
were identified in 2016 and 2018, respectively. As both will be
placed under Schedule II on the Convention on Psychotropic
Substances of 1971 in November 2020, this substance could be a
reaction to this. 5F-MPP-PICA (3, also known as MPhP-2201)
was first detected in Slovenia in 2018 and was also reported in
the US in 2019.32,34,35 MMB-4en-PICA (4, also known as AMB-
SCRAs often have greater potency and efficacy at CB
receptors compared to THC, which acts as a partial agonist at
both subtypes, which may partially explain the limited toxicity
seen with the use of cannabis.4 In contrast, SCRAs are linked to
numerous health issues, including dependence, psychosis,
seizures, organ toxicity, and death.11−19 Many legislative
approaches have been implemented at the national and
international levels to control the manufacturing, trafficking,
and possession of these substances.11,20 However, increased
control of specific SCRAs has had the perverse consequence of
motivating the design, synthesis, and sale of novel compounds
that circumvent existing controls.2,21,22 Due to the rapid pace at
which these substances enter the market, there is often little or
no information regarding the pharmacology of these substances
despite widespread human use.
To provide the first report of the chemistry and bioactivity of
several prevalent SCRAs, a panel of 12 recent indole, indazole,
and 7-azaindole SCRAs were synthesized and pharmacologically
characterized in comparison to reference SCRA JWH-018 (1,
Figure 1). The indole SCRAs include 4F-MDMB-BICA (4F-
MDMB-BUTICA, 4F-MDMB-2201, 2), 5F-MPP-PICA
(MPhP-2201, 5F-MPhP-PICA, 3), MMB-4en-PICA (AMB-
4en-PICA, MMB-022, 4), and CUMYL-CBMICA (5). The
indazoles comprised ADB-BINACA (ADB-BUTINACA, 6),
APP-BINACA (APP-BUTINACA, PY, 7), 4F-MDMB-BINA-
CA (4F-MDMB-BUTINACA, 8), MDMB-4en-PINACA
B
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Figure 2. Reagents and conditions: (a) (i) NaH, RBr, DMF, 0 °C−rt, 1 h; (ii) (CF3CO)2O, 0 °C−rt, 2 h; (b) KOH, MeOH, PhMe, reflux, 2 h, 72−
78% over 3 steps; (c) amine, EDC·HCl, HOBt·H2O, Et3N, DMF, rt, 18 h; 37−93%; (d) NaH, RBr, DMF, 0 °C−rt, 18 h, 39−58%; (e) 1 M aq. NaOH,
MeOH, rt, 18−36 h, 82−94%.
4en-PICA and MMB022) was first identified in Europe in 2018
and represents one of the first SCRAs to incorporate the
unsaturated 4-pentenyl substituent, as also present in one of the
early SCRAs, JWH-022.36 CUMYL-CBMICA (5) was identified
in seized material in Germany in 2019 and is the first SCRA to
feature a cyclobutylmethyl subunit, although such a group falls
within the prophetic structures claimed in a 2014 patent.28,37,38
ADB-BINACA (also known as ADB-BUTINACA, 6) and APP-
BINACA (also known as APP-BUTINACA, 7) were first
identified in Europe in 2019,39,40 have been routinely found in
the US since early 2019,41 and are the butyl homologues of well-
characterized SCRAs ADB-PINACA and APP-PINACA,
respectively. 4F-MDMB-BINACA (4F-MDMB-BUTINACA,
8), the butyl homologue of well-known SCRA 5F-MDMB-
PINACA, has been widely detected in the UK, Europe, and the
US since 2018.42−45 MDMB-4en-PINACA (9) is an analogue of
MMB-4en-PICA, featuring an indazole rather than an indole
core and a pendant tert-leucinate group in place of a valinate
group. MDMB-4en-PINACA was identified in mid-2018 in
Europe and has appeared routinely in toxicology casework in the
US since 2019.46 A-CHMINACA (10) was identified in April
2018 in Germany and has also been identified in seized material
in the US in 2018.47,48 The 7-azaindoles 5F-MDMB-P7AICA
(11) and 5F-AB-P7AICA (12) were identified in 2018,49,50
while 5F-A-P7AICA (13) was identified in seized herbal
material in Germany in 2019.51
All synthesized materials were analytically characterized using
nuclear magnetic resonance spectroscopy (NMR), high
resolution mass spectrometry (HRMS), and Fourier-transform
infrared spectroscopy (FTIR) to confirm structure and purity.
The synthesized compounds were pharmacologically evaluated
using a live cell-based reporter assay that monitors the in vitro
CB1 activation via its interaction with βarr2, an upstream
signaling event, using a luminescent readout (Nanoluciferase
Binary Technology). The activity of all compounds in this assay
was compared to analogues featuring a single structural
modification that were previously evaluated in the same system
to offer structure−activity relationship insights where possible.
These analogues include 5F-MMB-PICA (3a), 5F-MDMB-
PICA (3b), MDMB-4en-PICA (4a), 5F-MDMB-PINACA
(8a), AB-CHMINACA (10a), and ADB-CHMINACA
(10b).52−56 This allows us to better understand the pharmacol-
ogy of these recent members of a structurally diverse class of
NPS, which is crucial to prioritize responses by law enforcement
agencies and policymakers.
RESULTS AND DISCUSSION
■
The different SCRAs evaluated in this study were synthesized
according to the scheme shown in Figure 2 by adaptation of
existing methods.57,58 For indole SCRAs 2−5 (Figure 2A),
indole (14) was alkylated with the appropriate alkyl bromide
and then treated with trifluoroacetic anhydride to give
intermediate N-alkyl-3-(trifluoroacetyl)indoles 15−18. Hydrol-
ysis of 15−18 under basic conditions afforded the correspond-
ing carboxylic acids (19−22). Coupling of 19−22 to methyl L-
valinate, methyl L-phenylalaninate, or cumylamine, respectively,
provided 4F-MDMB-BICA (2), 5F-MPP-PICA (3), MMB-4en-
PICA (4), and CUMYL-CBMICA (5). The synthesis of
indazoles 6−10 and 7-azaindoles 11−13 required a different
approach, depicted in Figure 2B. Methyl indazole-3-carboxylate
(23) or methyl 7-azaindole-3-carboxylate (24) were alkylated
with the appropriate alkyl bromide to give 1-alkyl ester
derivatives 25−29, which were subsequently saponified to the
corresponding carboxylic acids 30−34. Coupling of acids 30−
34 with suitable amines using EDC yielded 6−13. Commercial,
enantiopure amino acid precursors were used (enantiomeric
excess ≥99%), and racemization is mechanistically unlikely due
to the achirality of the heterocyclic carboxylic acid precursors,59
indicating retention of enantiomeric fidelity (S-enantiomers) for
all chiral SCRA products.
All synthesized SCRAs were evaluated for CB1 agonist
activity using a live cell-based reporter assay that monitors the in
vitro CB1 activation via its interaction with βarr2.56,60,61 The
potencies (EC50 values) and efficacies (Emax values, relative to
the reference JWH-018) of all SCRAs screened in this system are
shown in Table 1, and full concentration response curves are
C
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Table 1. EC50 and Emax Values (the Latter Relative to JWH-
018) Presented as a Measure of Potency and Efficacy,
Respectively, Using a β-Arrestin 2 Recruitment Assay at
a
CB1
EC50 (95% CI)
indole SCRAs
Emax (95% CI)
1
JWH-018
21.4 nM (10.9−47.5
100% (87.4−114
nM)
%)
2
4F-MDMB-BICA
5F-MPP-PICA
5F-MMB-PICA
5F-MDMB-PICA
MMB-4en-PICA
MDMB-4en-PICA
121 nM (69.9−253 nM) 253% (224−298
%)
3
32.9 nM (19.6−59.8
303% (269−354
nM)
%)
3a
3b
4
135 nM (97.6−187 nM)c 312%
(287−336%)c
3.26 nM
331% (311−
(2.07−5.15 nM)c
351%)c
330 nM (174−578 nM) 241% (211−278
%)
4a
5
11.5 nM
302% (256−
(5.12−33.2 nM)b
391%)b
CUMYL-CBMICA 62.9 nM (31.1−123 nM) 153% (135−177
%)
indazole SCRAs
6
ADB-BINACA
APP-BINACA
6.36 nM (2.88−11.9
290% (260−322
nM)
%)
7
833 nM (508−2543 nM) 75.7% (65.7−108
%)
8
4F-MDMB-
BINACA
7.39 nM (3.51−13.4
378% (342−417
nM)
%)
8a
5F-MDMB-
PINACA
1.78 nM
331% (293−
(0.72−4.11 nM)d
406%)d
0.84 nM
319% (291−
(0.52−1.24 nM)e
354%)e
9
MDMB-4en-
PINACA
2.33 nM (1.32−4.19 nM 299% (272−329
%)
10
A-CHMINACA
159 nM (101−321 nM) 318%
(282−360%)
10a AB-CHMINACA
6.16 nM
324% (307−
(4.49−8.35 nM)d
344%)d
6.1 nM (3.1−11.4 nM)f
10b ADB-CHMINACA
1.49 nM
(0.69−2.61 nM)f
7-aza-indole SCRAs
11
12
13
5F-MDMB-
P7AICA
5F-AB-P7AICA
64.0 nM (29.7−126 nM) 297% (260−345
%)
5475 nM (3486−9148
142% (127−167
nM)
%)
5F-A-P7AICA
86.1 nM (44.1−306 nM) 37.4% (31.1−97.3
Figure 3. Concentration-dependent interaction of CB1 with β-arrestin
2 upon stimulation with the different SCRAs, divided based on their
heterocyclic core: (A) indole, (B) indazole, and (C) 7-aza-indole. Data
are given as mean receptor activation SEM (n = 3), normalized to the
%)
a
Italics denote previously reported compounds. Data are given as
EC50/Emax values (95% confidence interval (CI)). Data were
extracted from ref 52. Data were extracted from ref 53. Data were
extracted from ref 54. Data were extracted from ref 55. Data were
b
c
d
E
max of JWH-018 (= 100%).
e
f
extracted from ref 56.
The first three SCRAs are indole-carboxamides, denoted by
shown in Figure 3. SCRA categorization was based on the
heterocyclic core (see Figure 1). This region likely plays an
important role in CB1 receptor activation. Existing structure−
activity relationships for SCRAs show a general potency trend
whereby indazoles are equally or more potent than indoles,
which are in turn more potent than 7-azaindoles, subject to
equivalent substitution of the N-1 position and amide
group.57,58,62,63 This trend was also observed in this study for
this set of SCRAs, with the indazoles generally showing lower or
equivalent EC50 value ranges (2.33−833 nM) compared to the
indoles (32.9−330 nM), with both of these being generally
greater than the 7-azaindoles (64.0−5475 nM) regardless of
substituents (Table 1, shift to the left in Figure 2).
the -ICA suffix. MMB-4en-PICA (4) had a greatly reduced
potency (EC50 = 330 nM, Table 1) compared to that of MDMB-
4en-PICA (4a, EC50 = 11.5 nM) in the same functional CB1
assay.52 The observation that a “bulky” tert-butyl moiety in the
tert-leucinate MDMB-4en-PICA leads to a higher potency than
the valinate MMB-4en-PICA was also seen in a previous study
with the close structural analogues 5F-MMB-PICA (3a, EC50
=
135 nM) and 5F-MDMB-PICA (3b, EC50 = 3.26 nM).53 Also,
other reports found that most tert-leucinate functionalized
SCRAs were more potent CB1 agonists than the corresponding
valinate analogues,54,55,57,62,63 suggesting that this region is
important for receptor activation and therefore influences the
potency.54,64
D
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The appearance of the phenylalaninate analogue 5F-MPP-
PICA (3) in Slovenia in 201834 shows that SCRA manufacturers
are continuing to innovate with exploration of the amino acid
moiety. 5F-MPP-PICA showed a potency (EC50 = 32.9 nM)
intermediate to those of 5F-MDMB-PICA (3b, EC50 = 3.26
nM) and 5F-MMB-PICA (3a, EC50 = 136 nM)53 using the same
functional CB1 assay. This might indicate that the phenyl group,
like the tert-butyl moiety in tert-leucinate-functionalized SCRAs,
is still able to interact with the TM2 and displace the N-terminal
part of the CB1 receptor, although less optimally than the tert-
butyl moiety. Truncation of the alkyl chain of 5F-MDMB-PICA
gives the butyl homologue 4F-MDMB-BICA (2, EC50 = 121
nM), which showed a dramatic reduction in potency compared
to that of 5F-MDMB-PICA (3b, EC50 = 3.26 nM). In terms of
efficacy, 5F-MPP-PICA (3) showed a high Emax value of 303%
(95% CI 269−354%), relative to the Emax of the reference JWH-
018 (100%). This is similar to what we observed earlier for 5F-
MMB-PICA (Emax = 312%; 95% CI 287−336%) and 5F-
MDMB-PICA (Emax = 331%; 95% CI 311−351%),53 and the 4-
fluorobutyl homologue of the latter was similarly efficacious (2,
showed that 5F-MDMB-PINACA is a potent CB1 agonist with
EC50 values in the low nanomolar range (EC50 = 0.84−1.78 nM)
and Emax values of greater than 300%.54,55 Similar potency and
efficacy values were also found for 4F-MDMB-BINACA (8,
EC50 = 7.39 nM; Emax = 378%), indicating that 4F-MDMB-
BINACA is a potent and efficacious CB1 agonist. The results in
this study also matched the results from other independently
performed studies using the same βarr2 recruitment assay (EC50
= 1.74−5.69 nM; Emax = 254−317%).61,70,71
MDMB-4en-PINACA (9) (the indazole analogue of MDMB-
4en-PICA, 4a) showed high potency and efficacy at CB1,
relative to the reference compound JWH-018 in this system
(EC50 = 2.33 nM; Emax = 299%). These results match those
found for MDMB-4en-PINACA in other studies using the same
cell system (1.11−2.47 nM; 226−239%).52,70
The recently detected A-CHMINACA (10) is an adamantyl
analogue of previously screened valinamide (AB-CHMINACA,
10a) and tert-leucinamide (ADB-CHMINACA, 10b) SCRAs.
In previous reports using the same βarr2 recruitment assay, both
AB- and ADB-CHMINACA were found to be potent CB1
agonists, with EC50 values in the low nanomolar range (EC50
=
E
max = 253%).
6.1−6.16 nM and 1.49 nM, respectively).54,56 A-CHMINACA
(10) was less potent (EC50 = 159 nM), although a similar
efficacy (Emax = 318%) was found compared to AB-
CHMINACA (Emax = 324%).
Overall, the potency (EC50) and efficacy (Emax) of MMB-4en-
PICA (4, EC50 = 330 nM; 241%) and 5F-MMB-PICA (3a, EC50
= 135 nM; Emax = 312%) were relatively similar. This suggests
that substitution of the 5-fluoropentyl group for the pent-4-ene
tail is well-tolerated by CB1.52 This is not surprising as Kumar et
al.64 demonstrated, by using the structure of the CB1-Gi
signaling complex bound to the highly potent agonist MDMB-
FUBINACA, that the p-fluorobenzyl moiety of MDMB-
FUBINACA is most likely involved in hydrophobic interactions
with receptor elements within a conserved docking region
(described as a narrow side pocket). It was suggested that this
narrow pocket is an important region influencing ligand affinity
rather than receptor activation, as previous studies had noted
that switching the alkyl tail with a heteroaryl moiety gave
compounds with similar receptor affinity and potency.54,63,64
CUMYL-CBMICA (5) was only detected for the first time in
201937 and represents the first example of a new SCRA
comprising the cyclobutylmethyl group as a pendant moiety
from the heterocyclic core. Cumylamine-derived SCRAs have
been pharmacologically investigated previously and typically
demonstrate potency and efficacy as CB1 agonists.58,65−69 The
incorporation of a cyclobutylmethyl group was tolerated for
CB1 activation, and CUMYL-CBMICA demonstrated a
potency that was comparable to those of the other indole
As increasing numbers of indole and indazole SCRAs have
been subjected to control measures, SCRA manufacturers have
started to modify the heterocyclic core using “scaffold hopping”
in an attempt to create new SCRAs that retain in vivo CB1
agonist profiles while circumventing structure-based SCRA
legislation.2,3,58 Several 7-azaindole-3-carboxamide-type
(“-7AICA”) SCRAs have been reported in the NPS marketplace,
with three of the most recent examples included in this study
(Figure 1C and Table 1).
We previously showed indole 5F-MDMB-PICA (3b) and
indazole 5F-MDMB-PINACA (8a) to be potent CB1 agonists,
with EC50 values in the low nanomolar range (EC50 = 3.26 nM
and 0.84−1.78 nM, respectively) and Emax values greater than
300%.53−55 In comparison, the 7-azaindole analogue 5F-
MDMB-P7AICA (12) had reduced potency but similar efficacy
values (EC50 = 64.0 nM; Emax = 297%). Similarly, the
valinamide-derived 7-azaindole 5F-AB-P7AICA (11) showed a
dramatically reduced potency (EC50 = 5475 nM) as well as a
reduced efficacy (Emax = 142%), compared to the corresponding
indazole counterpart, 5F-AB-PINACA (EC50 = 55.4−65.5 nM;
E
max = 217−268%), previously evaluated in the same functional
SCRAs described here, although its efficacy was lower (EC50
62.9 nM and Emax = 153%).
=
CB1 assay.53,55
In our functional CB1 assay using βarr2 recruitment, the
adamantyl 7-azaindole derivative 5F-A-P7AICA (13) showed
only moderate potency (EC50 = 86.1 nM) and low efficacy (Emax
= 37.4%) relative to the reference compound JWH-018. Again,
this difference can be attributed to the difference in the
heteroaromatic core (7-aza-indole vs indazole). Also, A-
CHMINACA (10), which has the same pendant amide moiety,
showed only moderate potency (EC50 = 159 nM), although that
compound was still highly efficacious (Emax = 318%).
Moving from an indole to an indazole heterocyclic core for an
analogously substituted SCRA was anticipated to improve
potency based on previous structure−activity relationships.
ADB-BINACA (6) is an indazole structurally related to the
previously mentioned MDMB-4en-PICA and 5F-MDMB-
PICA, containing a tert-leucinamide group rather than a tert-
leucinate group, and a truncated N-butyl chain rather than a 4-
pentenyl or 5-fluoropentyl moiety, respectively. ADB-BINACA
was also found to be a potent and efficacious CB1 agonist (EC50
= 6.36 nM; Emax = 290%) in this assay, while the related
phenylalaninamide analogue APP-BINACA (7) showed greatly
reduced potency and efficacy (EC50 = 833 nM; Emax = 75.7%).
4F-MDMB-BINACA (8) is a truncated analogue of the
widely detected 5F-MDMB-PINACA (8a), containing a 4-
fluorobutyl group in place of the 5-fluoropentyl substituent of
the latter. Previous studies using the same functional CB1 assay
CONCLUSION
■
In this study, 12 recently emerged SCRAs were synthesized and
pharmacologically characterized using a live cell-based reporter
assay that monitors the in vitro CB1 activation via its interaction
with βarr2. All synthesized SCRAs acted as agonists of CB1,
although there were differences in potency and efficacy. This
E
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Research Article
min. UV absorbance was measured from 190 to 800 nm. UV data from
blank injections were subtracted to account for mobile phase
absorbance and background noise. Single stage mass spectra (MS1)
were collected in positive ESI mode using a single quadrupole (Q3)
scanning from m/z 100−600. All data were processed using Shimadzu
LabSolutions (v 5.89) software.
High resolution mass spectrometry data were collected using an
Agilent LC 1260-QTOF/MS 6550 (Santa Clara, CA, US). 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.
study confirmed a general potency trend whereby indazole
SCRAs are equally or more potent than indoles, which are in
turn more potent than 7-azaindoles. Also, a “bulky” tert-butyl
moiety in the headgroup contributes to the potency of an SCRA.
It is therefore not surprising that the most potent SCRAs within
this tested panel are the tert-leucinamide and tert-leucinate
indazole SCRAs ADB-BINACA (6), MDMB-4en-PINACA (9),
and 4F-MDMB-BINACA (8). These findings can help to
contribute to a better understanding of the pharmacology this
structurally diverse class of NPS, which is crucial to prioritizing
responses by scientists, health workers, and policy makers.
FTIR spectra were collected using a Bruker Alpha II ATR FTIR
spectrophotometer with the following parameters: 24 sample scans,
resolution = 4, phase resolution = 32.
MATERIAL AND METHODS
■
Chemicals. JWH-018 (1-pentyl-3-(1-naphthoyl)indole) was pur-
chased from Cayman Chemical Company (Ann Arbor, MI, US).
Dulbecco’s modified Eagle’s medium (GlutaMAX), Opti-MEM I
Reduced Serum Medium, penicillin−streptomycin (5000 U/mL), and
amphotericin B (250 μg/mL) were purchased from Thermo Fisher
Scientific (Pittsburgh, PA, US). Fetal bovine serum (FBS) and poly-D-
lysine were supplied by Sigma-Aldrich (Overijse, Belgium). The Nano-
Glo Live Cell reagent, which was used for the readout of the bioassay,
was procured from Promega (Madison, WI, US). All other compounds
were synthesized and analytically characterized in the laboratory of Dr.
Banister at the University of Sydney.
General Chemical Synthesis and Analysis Details. All reactions
were performed under an atmosphere of nitrogen unless otherwise
specified. All reagents and reactants were obtained from Sigma-Aldrich
(Castle Hill, NSW, Australia) and used as purchased. The enantiomeric
excess (ee) of chiral amino acid precursors was 99% (as determined by
gas liquid chromatography, GLC) in the certificates of analysis (COA).
Deuterated solvents (CD3OD, CDCl3, and DMSO-d6) were purchased
from Cambridge Isotope Laboratories (Tewksbury, MA, US) and used
as is. Other reagents were purchased and used as is from various
commercial sources.
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 (vide infra).
Melting point ranges (mp) were measured in open capillaries using a
Stuart SMP50 Automated melting point apparatus (Cole-Palmer,
Staffordshire, UK) and are uncorrected.
Nuclear magnetic resonance spectra were recorded at 298 K using a
Bruker AVANCE DRX400 (400.1 MHz) spectrometer (Bruker,
Bremen, Germany). 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,
quart. = quartet, quin. = quintet, m = multiplet), coupling constants (J
Hz) and assignment. Assignment of signals was assisted by correlation
spectroscopy (COSY), distortionless enhancement by polarization
transfer (DEPT), heteronuclear single quantum coherence (HSQC),
and heteronuclear multiple-bond correlation (HMBC) experiments
where necessary.
LC-MS/UV data were acquired using a Shimadzu (Kyoto, Japan)
Nexera LC-30AD UHPLC system coupled to a Shimadzu SPD-20AV
photo diode array detector and a Shimadzu LCMS-8040 triple-
quadrupole mass spectrometer, equipped with an electrospray
ionization (ESI) source. Compounds were dissolved in a 90:10 mixture
of 0.1% formic acid in water and methanol and placed in the UHPLC
autosampler that was maintained at 8 °C. Elution was performed in
gradient mode with an Agilent (CA, US) Zorbax XDB-C18 column
(2.1 × 50 mm, 3.5 μm particle size), held at 50 °C, with a 10 μL
injection volume. The mobile phases were 0.1% formic acid in water
(A) and methanol (B). Mobile phase composition was held at 10% B
until 0.5 min and then steadily increasing to 100% B at 2.5 min, holding
until 3 min, and then re-equilibrating at 10% B for a total run time of 4
General Procedure A: Amidation of 1-Alkylindole-3-car-
boxyic Acids, 1-Alkyl-1H-indazole-3-carboxylic acids, and 1-(5-
Fluoropentyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxylic Acid. To
a solution of the appropriate 1-alkylindole-3-carboxyic acid (19−22,
1.00 mmol), 1-alkyl-1H-indazole-3-carboxylic acid (30−33, 1.00
mmol), or 1-(5-fluoropentyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxylic
acid (34, 1.00 mmol) in DMF (4 mL) was added the suitable amine
reactant (1.10 mmol, 1.1 equiv), HOBt·H2O (169 mg, 1.10 mmol, 1.1
equiv), and EDC·HCl (288 mg, 1.50 mmol, 1.5 equiv). The mixture
was treated dropwise with Et3N (488 427 μL, 3.50 mmol, 3.5 equiv)
and allowed to stir for 24 h. The mixture was poured onto H2O (100
mL) and extracted with EtOAc (3 × 15 mL); the combined organic
layers were washed with H2O (2 × 10 mL) and brine (1 × 10 mL) and
dried (MgSO4), and the solvent evaporated under reduced pressure.
The products were obtained following purification by flash chromatog-
raphy.
Methyl (S)-2-(1-(4-Fluorobutyl)-1H-indole-3-carboxamido)-
3,3-dimethylbutanoate (4F-MDMB-BICA, 4F-MDMB-BUTICA,
2). Subjecting 1-(4-fluorobutyl)-1H-indole-3-carboxylic acid (19, 235
mg, 1.00 mmol) and methyl (S)-2-amino-3,3-dimethylbutanoate
hydrochloride (200 mg, 1.10 mol, 1.1 equiv) to general procedure A
gave, following purification by flash chromatography (hexane:EtOAc
90:10 to 70:30), 2 as a white solid (135 mg, 37%). mp 86−88 °C; Rf
0.52 (hexane:EtOAc, 50:50); 1H NMR (400 MHz, DMSO-d6) δ 8.40
(s, 1H), 8.10 (d, J = 7.8 Hz, 1H), 7.63 (d, J = 8.7 Hz, 1H), 7.55 (d, J =
8.1 Hz, 1H), 7.25−7.18 (m, 1H), 7.18−7.09 (m, 1H), 4.49 (d, J = 8.7
Hz, 1H), 4.47 (dt, J = 47.4, 6.0 Hz, 2H), 4.31−4.19 (m, 2H), 3.66 (s,
3H), 1.97−1.85 (m, 2H), 1.76−1.58 (m, 3H), 1.03 (s, 9H). 13C NMR
(100 MHz, DMSO-d6) δ 172.0 (CO), 164.4 (CO), 136.1 (quat.), 131.6
(CH), 126.8 (quat.), 122.1 (CH), 121.2 (CH), 120.8 (CH), 110.4
(CH), 108.8 (quat.), 83.5 (d, 1JCF = 161.8 Hz, CH2), 60.0 (CH), 51.5
(CH3), 45.6 (CH2), 33.9 (quat.), 27.3 (d, 2JCF = 19.6 Hz, CH2), 26.8 (3
× CH3), 25.8 (d, 3JCF = 4.9 Hz, CH2); νmax (cm−1): 3313, 2953, 1731,
1537, 1511, 1459, 1386, 1271, 1213, 1157, 1123, 1033, 895, 736, 623,
559; LCMS (ESI, + ve) m/z 363.3 ([M + H]+); HRMS (ESI)
C20H27FN2O3 exact mass 362.2006, accurate mass 362.2002 (mass
error −1.04 ppm).
Methyl (S)-2-(1-(5-Fluoropentyl)-1H-indole-3-carboxami-
do)-3-phenylpropanoate (5F-MPP-PICA, 3). Subjecting 1-(5-
fluoropentyl)-1H-indole-3-carboxylic acid (20, 249 mg, 1.00 mmol)
and methyl L-phenylalaninate hydrochloride (237 mg, 1.10 mmol, 1.1
equiv) to general procedure A gave, following purification by flash
chromatography (hexane:EtOAc, 85:15 to 70:30), 3 as an off-white
solid (346 mg, 84%). mp 114−116 °C; Rf 0.53 (hexane:EtOAc, 50:50);
1H NMR (400 MHz, CDCl3) δ 7.77 (dt, J = 7.9, 1.0 Hz, 1H), 7.69 (s,
1H), 7.37 (dt, J = 8.3, 1.0 Hz, 1H), 7.32−7.14 (m, 7H), 6.41 (d, J = 7.5
Hz, 1H), 5.17 (dt, J = 7.5, 5.5 Hz, 1H), 4.42 (dt, J = 47.2, 5.9 Hz, 2H),
4.15 (t, J = 7.1 Hz, 2H), 3.78 (s, 3H), 3.30 (qd, J = 13.8, 5.5 Hz, 2H),
1.97−1.85 (m, 2H), 1.80−1.63 (m, 2H), 1.51−1.39 (m, 2H); 13C
NMR (100 MHz, CDCl3) δ 172.7 (CO), 164.6 (CO), 136.7 (quat.),
136.3 (quat.), 131.9 (CH), 129.6 (2 × CH), 128.7 (2 × CH), 127.3
(CH), 125.5 (quat.), 122.7 (CH), 121.7 (CH), 120.4 (CH), 110.6
1
(quat.), 110.3 (CH), 83.8 (d, JCF = 165.0 Hz, CH2), 53.3 (NCH2),
52.5 (NCH), 46.9 (CH3), 38.3 (CH2), 30.1 (d, 2JCF = 19.8 Hz, CH2),
F
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ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience
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Research Article
3
29.8 (CH2), 23.0 (d, JCF = 5.0 Hz, CH2); νmax (cm−1): 2947, 1688,
1601, 1534, 1465, 1357, 1257, 1166, 1134, 961, 801, 787, 756, 718, 697,
621, 567, 549; LCMS (ESI, + ve) m/z 411.3 ([M + H]+); HRMS (ESI)
C24H27FN2O3 exact mass 410.2006, accurate mass 410.2013 (mass
error 1.86 ppm).
50:50); 1H NMR (400 MHz, CDCl3) δ 8.30 (m, J = 8.2, 1.0 Hz, 1H),
7.52−7.46 (m, 1H), 7.41−7.39 (m, 2H), 7.34−7.25 (m, 5H), 7.23−
7.20 (m, 1H), 5.99 (s, 1H), 5.31 (s, 1H), 4.95−4.88 (m, 1H), 4.45−
4.31 (m, 2H), 3.32−3.16 (m, 2H), 1.90 (quin., J = 7.2 Hz, 2H), 1.35
(sext., J = 7.7 Hz, 2H), 0.95 (t, J = 7.4 Hz, 3H); 13C NMR (101 MHz,
DMSO-d6) δ 172.6 (CO), 161.3 (CO), 140.5 (quat.), 137.6 (quat.),
136.4 (quat.), 129.3 (2 × CH), 128.0 (2 × CH), 126.6 (CH), 126.3
(CH), 122.4 (CH), 122.0 (CH), 121.6 (quat.), 110.4, 53.2 (CH), 48.4
(CH2), 37.7 (CH2), 31.4 (CH2), 19.4 (CH2), 13.5 (CH3); νmax (cm−1):
3323, 2932, 1696, 1672, 1642, 1531, 1492, 1397, 1324, 1231, 1196,
1131, 909, 840, 780, 746, 698, 562; LCMS (ESI, + ve) m/z 365.3 ([M +
H]+); HRMS (ESI) C21H24N4O2 exact mass 364.1899, accurate mass
364.1907 (mass error 2.15 ppm).
Methyl (S)-2-(1-(Pent-4-en-1-yl)-1H-indole-3-carboxamido)-
3-methylbutanoate (MMB-4en-PICA, AMB-4en-PICA, 4). Sub-
jecting 1-(pent-4-en-1-yl)-1H-indole-3-carboxylic acid (21, 344 mg,
1.50 mmol) and methyl (S)-2-amino-3-methylbutanoate hydrochloride
(300 mg, 1.65 mmol, 1.1 equiv) to general procedure A gave, following
purification by flash chromatography (hexane:EtOAc, 85:15 to 60:40),
4 as a white solid (386 mg, 75%). mp 85−87 °C; Rf 0.61
1
(hexane:EtOAc, 50:50); H NMR (400 MHz, CDCl3) δ 8.03−7.97
(1H, m), 7.74 (1H, s), 7.44−7.36 (1H, m), 7.31−7.27 (2H, m), 6.47
(1H, d, J = 8.7 Hz), 5.85−5.75 (1H, m), 5.09−5.03 (2H, m), 4.87 (1H,
dd, J = 8.7, 4.8 Hz), 4.16 (2H, t, J = 7.1 Hz), 3.79 (3H, s), 2.35−2.27
(1H, m), 2.10 (2H, q, J = 7.5 Hz), 1.98 (2H, quin., J = 7.7 Hz), 1.05
(6H, d, J = 9.9 Hz); 13C NMR (100 MHz, CDCl3) δ 173.3 (CO), 165.0
(CO), 137.0 (CH), 136.7 (quat.), 131.9 (CH), 125.5 (quat.), 122.6
(CH), 121.8 (CH), 120.3 (CH), 116.2 (CH2), 110.8 (quat.), 110.5
(CH), 57.0 (CH), 52.3 (CH3), 46.2 (NCH2), 31.9 (CH), 30.8 (CH2),
29.0 (CH2), 19.3 (CH3), 18.2 (CH3). νmax (cm−1): 3331, 2962, 1739,
1629, 1508, 1466, 1388, 1279, 1229, 1175, 1146, 984, 911, 813, 780,
750, 636, 566, 541; LCMS (ESI, + ve) m/z 343.3 ([M + H]+); HRMS
(ESI) C20H26N2O3 exact mass 342.1943, accurate mass 342.1952 (mass
error 2.49 ppm).
Methyl (S)-2-(1-(4-Fluorobutyl)-1H-indazole-3-carboxami-
do)-3,3-dimethylbutanoate (4F-MDMB-BINACA, 4F-MDMB-
BUTINACA, 8). Subjecting 1-(4-fluorobutyl)-1H-indazole-3-carbox-
ylic acid (31, 238 mg, 1.00 mmol) and methyl (S)-2-amino-3,3-
dimethylbutanoate hydrochloride (209 mg, 1.10 mmol, 1.1 equiv) to
general procedure A gave, following purification by flash chromatog-
raphy (hexane:EtOAc, 100:0 to 90:10), 8 as a colorless oil (255 mg,
68%). Rf 0.31 (hexane:EtOAc, 80:20); 1H NMR (400 MHz, CD3OD) δ
8.20 (d, J = 8.3, 1.1 Hz, 1H), 7.67 (d, J = 8.5, 1.1 Hz, 1H), 7.47 (dt, J =
15.5, 7.7 Hz, 1H), 7.29 (dt, J = 15.0, 7.4 Hz, 1H), 4.61−4.59 (s, 1H),
4.57 (t, J = 7.1 Hz, 2H), 4.46 (dt, J = 47.4, 6.0 Hz, 2H), 3.77 (s, 3H),
2.10 (quin., J = 7.3 Hz, 2H), 1.72 (d quin., 2H), 1.09 (s, 9H), NH
proton not observed (exchangeable); 13C NMR (101 MHz, CD3OD) δ
173.1 (CO), 164.2 (CO), 142.5 (quat.), 137.5 (quat.), 128.2 (CH),
1-(Cyclobutylmethyl)-N-(2-phenylpropan-2-yl)-1H-indole-
3-carboxamide (CUMYL-CBMICA, 5). Subjecting 1-(cyclobutyl-
methyl)-1H-indole-3-carboxylic acid (22, 115 mg, 0.50 mmol) and
cumylamine (79 μL, 0.55 mmol, 1.1 equiv) to general procedure A gave,
following purification by flash chromatography (hexane:EtOAc,
87.5:12.5 to 62.5:37.5), 5 as a white solid (96 mg, 56%). mp 179−
181 °C; Rf 0.54 (hexane:EtOAc, 50:50); 1H NMR (400 MHz, CDCl3)
δ 7.89 (1H, d, J = 7.3 Hz), 7.68 (1H, s), 7.55−7.49 (1H, m), 7.42−7.21
(7H, m), 6.26 (1H, s), 4.12 (2H, d, J = 7.2), 2.85 (1H, sept., J = 7.5 Hz),
2.13−2.03 (2H, m), 1.99−1.89 (2H, m), 1.87 (6H, s), 1.85−1.74 (2H,
m); 13C NMR (100 MHz, CDCl3) δ 164.3 (CO), 147.4 (quat.), 136.8
(quat.), 131.7 (CH), 128.4 (2 × CH), 126.5 (CH), 125.0 (quat.), 124.8
(2 × CH), 122.2 (CH), 121.3 (CH), 119.6 (CH), 111.7 (quat.), 110.4
(CH), 56.0 (quat.), 51.8 (NCH2), 35.5 (CH), 29.7 (2 x CH3), 26.3 (2 x
CH2), 18.1 (CH2); νmax (cm−1): 2932, 1617, 1540, 1386, 1363, 1279,
1246, 1204, 1169, 1112, 1071, 915, 828, 745, 697, 604, 573, 545, 509;
LCMS (ESI, + ve) m/z 347.3 ([M + H]+); HRMS (ESI) C23H26N2O
exact mass 346.2045, accurate mass 346.2046 (mass error 0.11 ppm).
(S)-N-(1-Amino-3,3-dimethyl-1-oxobutan-2-yl)-1-butyl-1H-
indazole-3-carboxamide (ADB-BINACA, ADB-BUTINACA, 6).
Subjecting 1-butyl-1H-indazole-3-carboxylic acid (30, 88 mg, 0.40
mmol) and 2-amino-3,3-dimethylbutanamide hydrochloride (70 mg,
0.42 mmol, 1.05 equiv) to general procedure A gave, following
purification by flash chromatography (CH2Cl2:MeOH, 100:0 to
90:10), 6 as a white powder (69 mg, 52%). mp 148.2−148.4 °C; Rf
0.37 (hexane:EtOAc, 50:50); 1H NMR (400 MHz, CDCl3,) δ 8.30 (dt,
J = 8.2, 1.0 Hz, 1H), 7.65 (d, J = 9.4 Hz, 1H), 7.43−7.36 (m, 2H), 7.27−
7.22 (m, 1H), 5.93 (s, 1H), 5.45 (s, 1H), 4.52 (d, J = 9.4 Hz, 1H), 4.38
(t, J = 7.2 Hz, 2H), 1.96−1.87 (m, 2H), 1.44−1.28 (m, 2H), 1.14 (s,
9H), 0.95 (t, J = 7.4 Hz, 3H); 13C NMR (101 MHz, DMSO-d6) δ 171.8
(CO), 161.0 (CO), 140.7 (quat.), 136.3 (quat.), 126.7 (CH), 122.5
(CH), 121.9 (CH), 121.6 (quat.), 110.5 (CH), 58.6 (NCH), 48.6
(NCH2), 34.6 (quat.), 31.5 (CH2), 26.6 (3 × CH3), 19.4 (CH2), 13.5
(CH3); νmax (cm−1): 2958, 1694, 1647, 1529, 1488, 1365, 185, 828,
747, 592; LCMS (ESI, + ve) m/z 331.3 ([M + H]+); HRMS (ESI)
C18H26N4O2 exact mass 330.2056, accurate mass 330.2064 (mass error
2.43 ppm).
1
124.0 (CH), 123.9 (CH), 123.0 (quat), 111.1 (CH), 84.4 (d, JCF
=
164.1 Hz, CH2F), 61.3 (CH), 52.4 (OCH3), 49.6 (CH2), 35.6 (quat.),
28.8 (d, 2JCF = 20.1 Hz, CH2), 27.0 (3 × CH3), 26.9 (d, 3JCF = 4.8 Hz,
CH2); νmax (cm−1): 3414, 2961, 1730, 1666, 1523, 1491, 1369, 1331,
1211, 1166, 1005, 939, 837, 777, 753, 541; LCMS (ESI, + ve) m/z
364.2 ([M + H]+); HRMS (ESI) C19H26FN3O3 exact mass 363.1958,
accurate mass 363.1965 (mass error 1.86 ppm).
Methyl (S)-2-(1-(Pent-4-en-1-yl)-1H-indazole-3-carboxami-
do)-3,3-dimethylbutanoate (MDMB-4en-PINACA, 9). Subjecting
1-(pent-4-en-1-yl)-1H-indazole-3-carboxylic acid (32, 230 mg, 1.00
mmol) and methyl (S)-2-amino-3,3-dimethylbutanoate hydrochloride
(200 mg, 1.10 mmol, 1.1 equiv) to general procedure A gave, following
purification by flash chromatography (hexane:EtOAc, 95:5 to 85:15), 9
1
as a colorless oil (246 mg, 57%). Rf 0.49 (hexane:EtOAc, 80:20); H
NMR (400 MHz, CDCl3) δ 8.35 (1H, d, J = 8.2 Hz), 7.55 (1H, d, J = 9.7
Hz), 7.44−7.38 (1H, m), 7.31−7.23 (2H, m), 5.88−5.78 (1H, m),
5.12−5.01 (2H, m), 4.73 (1H, d, J = 9.7 Hz), 4.41 (2H, t, J = 6.7), 3.76
(3H, s), 2.19−2.03 (4H, m), 1.09 (9H, s); 13C NMR (100 MHz,
CDCl3) δ 172.3 (CO), 162.5 (CO), 141.0 (quat.), 137.2 (CH), 136.9
(quat.), 126.8 (CH), 123.1 (quat.), 123.0 (CH), 122.7 (CH), 116.1
(CH2), 109.4 (CH), 59.6 (CH), 51.9 (CH3), 48.8 (CH2), 35.2 (quat.),
30.9 (CH2), 28.9 (CH2), 26.8 (3 × CH3); νmax (cm−1): 3413, 2961,
1729, 1667, 1520, 1490, 1367, 1329, 1214, 1166, 988, 910, 833, 775,
755, 555; LCMS (ESI, + ve) m/z 358.3 ([M + H]+); HRMS (ESI)
C20H27N3O3 exact mass 357.2052, accurate mass 357.2061 (mass error
2.34 ppm).
N-(Adamantan-1-yl)-1-(cyclohexylmethyl)-1H-indazole-3-
carboxamide (A-CHMINACA, 10). Subjecting 1-(cyclohexylmeth-
yl)-1H-indazole-3-carboxylic acid (33, 77 mg, 0.30 mmol) and
adamantan-1-amine (49 mg, 0.32 mmol, 1.1 equiv) to general
procedure A gave, following purification by flash chromatography
(hexane:EtOAc, 95:5 to 91:9), 10 as a colorless oil (96 mg, 82%) Rf
0.95 (hexane:EtOAc, 50:50); 1H NMR (400 MHz, CDCl3) δ 8.37 (dt, J
= 8.2, 1.0 Hz, 1H), 7.39−7.36 (m, 2H), 7.25−7.20 (m, 1H), 6.80 (s,
1H), 4.17 (d, J = 7.2 Hz, 2H), 2.24−2.12 (m, 9H), 2.07−1.93 (m, 1H),
1.81−1.57 (m, 11H), 1.29−1.13 (m, 3H), 1.09−0.98 (m, 2H); 13C
NMR (101 MHz, CD3OD) δ 164.0 (quat.), 143.0 (quat.), 138.7
(quat.), 127.8 (CH), 123.6 (CH), 123.5 (quat.), 123.2 (CH), 111.2
(CH), 56.3 (NCH2), 53.1 (quat.), 42.7 (3 × CH2), 40.1 (CH), 37.5 (3
× CH2), 31.9 (2 × CH2), 31.0 (3 × CH), 27.4 (CH), 26.8 (2 × CH2);
νmax (cm−1): 2905, 2849, 1738, 1662, 1524, 1491, 1449, 1358, 1291,
1276, 1226, 1175, 1137, 1046, 1004, 858, 777, 748; LCMS (ESI+) m/z
392.3 ([M + H]+); LCMS (ESI, + ve) m/z 392.3 ([M + H]); HRMS
(S)-N-(1-Amino-1-oxo-3-phenylpropan-2-yl)-1-butyl-1H-in-
dazole-3-carboxamide (APP-BINACA, APP-BUTINACA, 7). Sub-
jecting 1-butyl-1H-indazole-3-carboxylic acid (30, 218 mg, 1.00 mmol)
and methyl L-phenylalaninate hydrochloride (281 mg, 1.40 mmol, 1.40
equiv) to general procedure A gave, following purification by flash
chromatography (CH2Cl2:MeOH, 100:0 to 90:10), 7 as a white
powder (337 mg, 93%). mp 91.5−92.5 °C; Rf 0.76 (hexane:EtOAc,
G
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Research Article
(ESI) C25H33N3O exact mass 391.2624, accurate mass 391.2621 (mass
error −0.68 ppm).
portionwise with trifluoroacetic anhydride (62.5 mmol, 2.5 equiv). The
solution was stirred at ambient temperature 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 KOH (82.5
mmol, 3.3 equiv) in MeOH (20 mL). The solution was heated at reflux
for 2 h, cooled to ambient temperature, and H2O (100 mL) was added.
The layers were separated, and the organic layer was extracted with 1 M
aq. KOH (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 evaporated
under reduced pressure. The crude solids were purified by
recrystallization from i-PrOH.
(S)-N-(1-Amino-3-methyl-1-oxobutan-2-yl)-1-(5-fluoropen-
tyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide (5F-AB-P7AICA,
11). Subjecting 1-(5-fluoropentyl)-1H-pyrrolo[2,3-b]pyridine-3-car-
boxylic acid (34, 125 mg, 0.50 mmol) and (S)-2-amino-3-
methylbutanamide hydrochloride (84 mg, 0.55 mmol, 1.1 equiv) to
general procedure A gave, following purification by flash chromatog-
raphy (hexane:EtOAc, 60:40 to 0:100), 11 as a white solid (103 mg,
59%). mp 188.5−189.5 °C; Rf 0.17 (hexane:EtOAc, 20:80); 1H NMR
(400 MHz, CD3OD) δ 8.48 (d, J = 8.0, 1.4 Hz, 1H), 8.33 (d, J = 4.9, 1.4
Hz, 1H), 8.23 (s, 1H), 7.25 (dd, J = 8.0, 4.8, 1.1 Hz, 1H), 4.49−4.33 (m,
5H), 2.21 (m, 1H), 1.97 (quin., J = 7.3 Hz, 2H), 1.82−1.67 (m, 2H),
1.51−1.41 (m, 2H), 1.08 (d, J = 6.7 Hz, 6H), NH and NH2 not
observed (exchangeable); 13C NMR (101 MHz, CD3OD) δ 176.8
(CO), 166.8 (CO), 148.6 (quat.), 144.5 (CH), 132.7 (CH), 131.2
(CH), 120.8 (quat.), 118.5 (CH), 109.5 (quat.), 84.6 (d, 1JCF = 163.9
Hz, CH2), 59.7 (NCH), 45.9 (NCH2), 32.1 (CH), 31.1 (d, 2JCF = 19.8
Hz, CH2), 30.9 (CH), 23.6 (d, 3JCF = 5.2 Hz, CH2), 19.9 (CH2), 18.9 (2
× CH3); νmax (cm−1): 3297, 2945, 1620, 1516, 1426, 1314, 1267, 1163,
1135, 980, 800, 777, 747, 665, 571; LCMS (ESI, + ve) m/z 349.3 ([M +
H]+); HRMS (ESI) C18H25FN4O2 exact mass 348.1962, accurate mass
348.1968 (mass error 1.85 ppm).
1-(4-Fluorobutyl)-1H-indole-3-carboxylic Acid (19). Subject-
ing indole (14, 2.93 g, 25.0 mmol) and 1-bromo-4-fluorobutane (2.82
mL, 26.25 mmol, 1.05 equiv) to general procedure B gave 19 as an off
white solid (3.62 g, 67%). mp 132−134 °C; Rf 0.56 (hexane:EtOAc,
50:50); 1H NMR (400 MHz, DMSO-d6) δ 11.96 (s, 1H), 8.08 (s, 1H),
8.05−7.99 (m, 1H), 7.58 (dd, J = 7.8, 1.2 Hz, 1H), 7.28−7.14 (m, 2H),
4.43 (dt, J = 47.4, 6.0 Hz, 2H), 4.29 (t, J = 7.0 Hz, 2H), 1.86 (quin., J =
7.1 Hz, 2H), 1.68−1.51 (m, 2H); 13C NMR (100 MHz, DMSO-d6) δ
165.7 (CO), 136.3 (quat.), 135.3 (CH), 126.5 (quat.), 122.3 (CH),
1
Methyl (S)-2-(1-(5-Fluoropentyl)-1H-pyrrolo[2,3-b]pyridine-
3-carboxamido)-3,3-dimethylbutanoate (5F-MDMB-P7AICA,
12). Subjecting 1-(5-fluoropentyl)-1H-pyrrolo[2,3-b]pyridine-3-car-
boxylic acid (34, 250 mg, 1.00 mmol) and methyl (S)-2-amino-3,3-
dimethylbutanoate hydrochloride (200 mg, 1.10 mmol, 1.1 equiv) to
general procedure A gave, following purification by flash chromatog-
raphy (hexane:EtOAc, 90:10 to 80:20), 12 as a white solid (270 mg,
72%). mp 62−64 °C; Rf0.59; 1H NMR (400 MHz, CDCl3) δ 8.38 (dd,
J = 4.8, 1.5 Hz, 1H), 8.33 (dd, J = 8.0, 1.5 Hz, 1H), 7.83 (s, 1H), 7.23
(dd, J = 7.9, 4.7 Hz, 1H), 6.42 (d, J = 9.4 Hz, 1H), 4.76 (d, J = 9.3 Hz,
1H), 4.52−4.29 (m, 4H), 3.77 (s, 3H), 2.02−1.90 (m, 2H), 1.83−1.65
(m, 2H), 1.53−1.41 (m, 1H), 1.08 (s, 9H); 13C NMR (100 MHz,
CDCl3) δ 172.9 (CO), 164.2 (CO), 147.6 (quat.), 143.9 (CH), 131.0
(CH), 128.9 (CH), 118.3 (quat.), 117.8 (CH), 109.2 (quat.), 83.9 (d,
1JCF = 164.8 Hz, CH2), 59.8 (NCH), 52.1 (OCH3), 45.1 (NCH2), 35.3
121.3 (CH), 120.9 (CH), 110.8 (CH), 106.5 (quat.), 83.4 (d, JCF
=
161.7 Hz, CH2), 45.6 (CH2), 27.1 (d, 2JCF = 19.6 Hz, CH2), 25.5 (d,
3JCF = 5.0 Hz, CH2); νmax (cm−1): 2948, 1655, 1524, 1490, 1469, 1430,
1395, 1274, 1228, 1158, 1115, 1093, 1052, 1014, 997, 934, 781, 771,
753, 724, 616, 591; LCMS (ESI, + ve) m/z 236.2 ([M + H]+); HRMS
(ESI) C13H14FNO2 exact mass 235.1009, accurate mass 235.1004
(mass error −2.01 ppm).
1-(5-Fluoropentyl)-1H-indole-3-carboxylic Acid (20). Subject-
ing indole (14, 5.86 g, 50.0 mmol) and 1-bromo-5-fluoropentane (6.53
mL, 52.5 mmol, 1.05 equiv) to general procedure B gave 20 as an off-
white solid (9.76 g, 78%). mp 117−118 °C; Rf 0.55 (hexane:EtOAc,
1
50:50); H NMR (400 MHz, CDCl3) δ 8.30−8.21 (m, 1H), 7.93 (s,
1H), 7.43−7.34 (m, 1H), 7.35−7.27 (m, 2H), 4.43 (dt, J = 47.2, 5.9 Hz,
2H), 4.20 (t, J = 7.1 Hz, 2H), 2.02−1.88 (m, 2H), 1.82−1.63 (m, 2H),
1.55−1.40 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 170.8 (CO),
136.8 (quat.), 135.5 (CH), 127.1 (quat.), 123.1 (CH), 122.3 (CH),
122.1 (CH), 110.1 (CH), 106.6 (quat.), 83.8 (d, 1JCF = 165.0 Hz), 47.1
(NCH2), 30.1 (d, 2JCF = 19.8 Hz), 29.7 (CH2), 23.0 (d, 3JCF = 5.0 Hz).
All physical and spectral properties were consistent with those
previously reported.72
(quat.), 30.1 (d, 2JCF = 19.8 Hz, CH2), 30.0 (CH2), 26.9 (3 × CH3),
3
22.8 (d, JCF = 5.2 Hz, CH2); νmax (cm−1): 3314, 2955, 1739, 1612,
1568, 1538, 1516, 1426, 1397, 1268, 1217, 1157, 1133, 1035, 991, 862,
800, 775, 635, 564; LCMS (ESI, + ve) m/z 378.3 ([M + H]+); HRMS
(ESI) C20H28FN3O3 exact mass 377.2115, accurate mass 377.2121
(mass error 1.79 ppm).
1-(Pent-4-en-1-yl)-1H-indole-3-carboxylic Acid (21). Subject-
ing indole (14, 2.93 g, 25.0 mmol) and 5-bromo-1-pentene (3.10 mL,
26.3 mmol, 1.05 equiv) to general procedure B gave 21 as an off-white
solid (4.14 g, 72%). mp 123−124 °C; Rf 0.13 (hexane:EtOAc, 80:20);
1H NMR (400 MHz, CDCl3) δ 8.28−8.23 (1H, m), 7.93 (1H, s), 7.40−
7.37 (1H, m), 7.33−7.29 (2H, m), 5.86−5.76 (1H, m), 5.09 (1H, d, J =
8.2 Hz), 5.06 (1H, s), 4.19 (2H, t, J = 7.0 Hz), 2.12 (2H, q, J = 6.9 Hz),
2.01 (2H, quin., J = 7.0 Hz), OH not observed (exchangeable); 13C
NMR (100 MHz, CDCl3) δ 170.8 (CO), 136.9 (CH), 136.8 (quat.),
135.7 (CH), 127.2 (quat.), 123.0 (CH), 122.3 (CH), 122.1 (CH),
116.3 (CH2), 110.2 (CH), 106.5 (quat.), 46.4 (NCH2), 30.8 (CH2),
28.9 (CH2). All physical and spectral properties were consistent with
those previously reported.73
1-(Cyclobutylmethyl)-1H-indole-3-carboxylic Acid (22). Sub-
jecting indole (14, 2.93 g, 25.0 mmol) and (bromomethyl)cyclobutane
(2.90 mL, 26.3 mmol, 1.05 equiv) gave 22 as an off-white solid (3.98 g,
74%), mp 125−127 °C; Rf 0.57 (hexane:EtOAc, 50:50); 1H NMR (400
MHz, CDCl3) δ 8.26−8.18 (1H, m), 7.89 (1H, s), 7.42−7.33 (1H, m),
7.33−7.24 (2H, m), 4.15 (2H, d, J = 7.4 Hz), 2.88 (1H, sept., J = 7.4
Hz), 2.15−2.03 (2H, m), 1.99−1.74 (4H, m), OH not observed
(exchangeable); 13C NMR (100 MHz, CDCl3) δ 170.8 (CO), 137.1
(quat.), 135.5 (CH), 127.0 (quat.), 122.9 (CH), 122.3 (CH), 122.0
(CH), 110.3 (CH), 106.4 (quat.), 52.2 (NCH2), 35.5 (CH), 26.4 (2 ×
CH2), 18.3 (CH2); LCMS (ESI, + ve) m/z 230.1 ([M + H]+); HRMS
(ESI) C14H15NO2 exact mass 229.1103, accurate mass 229.1103 (mass
error 0.09 ppm).
N-(Adamantan-1-yl)-1-(5-fluoropentyl)-1H-pyrrolo[2,3-b]-
pyridine-3-carboxamide (5F-A-P7AICA, 13). Subjecting 1-(5-
fluoropentyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxylic acid (34, 119
mg, 0.476 mmol) and adamantan-1-amine (88 mg, 0.58 mmol, 1.2
equiv) to general procedure A gave, following purification by flash
chromatography (hexane:EtOAc, 85:15 to 75:25), 13 as a white solid
(94 mg, 51%). mp 129.6−130.5 °C;Rf 0.59 (hexane:EtOAc, 50:50); 1H
NMR (400 MHz, DMSO-d6) δ 8.43−8.39 (m, 1H), 8.31−8.26 (m,
2H), 7.19−7.15 (m, 2H), 4.49−4.44 (m, 1H), 4.35 (td, J = 6.1, 1.4 Hz,
1H), 4.27 (t, J = 7.1 Hz, 2H), 2.07 (d, J = 11.8 Hz, 9H), 1.86 (quin., J =
7.2 Hz, 2H), 1.74−1.60 (m, 8H), 1.33 (quin., J = 7.8 Hz, 2H); 13C
NMR (101 MHz, DMSO-d6) δ 163.3 (CO), 147.1 (quat.), 143.0
(CH), 130.6 (CH), 129.6 (CH), 119.0 (quat.), 116.9 (CH), 109.3
1
(quat.), 83.6 (d, JCF = 161.7 Hz, CH2), 51.1 (quat.), 44.4 (NCH2),
2
36.2 (CH2), 29.3 (d, JCF = 19.1 Hz, CH2), 29.3 (CH2), 29.0 (CH),
3
22.0 (d, JCF = 5.4 Hz, CH2); νmax (cm−1): 2903, 2849, 1620, 1535,
1425, 1398, 1358, 1277, 1252, 1170, 1140, 976, 854, 800, 777, 750, 546;
LCMS (ESI, + ve) m/z 384.3 ([M + H]+); HRMS (ESI) C23H30FN3O
exact mass 383.2373, accurate mass 383.2382 (mass error 2.33 ppm).
General Procedure B: Synthesis of 1-Alkylindole-3-carbox-
ylic 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 (14, 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 (26.25 mmol, 1.05 equiv). The mixture
was stirred for 1 h at ambient temperature, cooled to 0 °C, and treated
H
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Research Article
General Procedure C: Synthesis of Methyl 1-Alkyl-1H-
indazole-3-carboxylates and Methyl 1-(5-Fluoropentyl)-1H-
pyrrolo[2,3-b]pyridine-3-carboxylate. To a cooled (0 °C) solution
of methyl 1H-indazole-3-carboxylate or methyl 1H-pyrrolo[2,3-b]-
pyridine-3-carboxylate (1.0 equiv) in DMF (10 mL) was added NaH
(1.05 equiv) portionwise. The mixture was warmed to ambient
temperature, stirred for 1 h, cooled to 0 °C, treated dropwise with the
appropriate alkyl bromide (1.05 equiv), and stirred at ambient
temperature for 48 h. The reaction was quenched by pouring onto
H2O (100 mL) and extracted with EtOAc (3 × 50 mL). The combined
organic layers were washed with H2O (2 × 50 mL), brine (1 × 50 mL),
dried (MgSO4), and the solvent evaporated under reduced pressure.
The products were obtained following purification by flash chromatog-
raphy.
Methyl 1-Butyl-1H-indazole-3-carboxylate (25). Subjecting
methyl 1H-indazole-3-carboxylate (23, 2.63 g, 15.0 mmol) and 1-
bromobutane (1.69 mL, 15.75 mmol, 1.05 equiv) to general procedure
C gave, following purification by flash chromatography (hexane:EtOAc,
100:0 to 10:90), 25 as a colorless oil (1.79 g, 51%). Rf 0.46
(hexane:EtOAc, 80:20); 1H NMR (400 MHz, CD3OD) δ 8.14 (d, J =
8.3 Hz, 1H), 7.67 (d, J = 8.6 Hz, 1H), 7.48 (t, J = 7.7 Hz, 1H), 7.33 (t, J
= 7.6 Hz, 1H), 4.51 (t, J = 7.0 Hz, 2H), 4.01 (s, 3H), 1.93 (quin., J = 7.1
Hz, 2H), 1.32 (sext., J = 7.7 Hz, 3H), 0.94 (t, J = 7.4 Hz, 3H); 13C NMR
(100 MHz, CD3OD) δ 164.5 (CO), 142.1 (quat.), 135.4 (quat.), 128.2
(CH), 124.5 (quat.), 124.4 (CH), 122.9 (CH), 111.3 (CH), 52.3
(NCH2), 50.4 (OCH3), 32.9 (CH2), 20.9 (CH2), 13.9 (CH3). All
physical and spectral properties were consistent with those previously
reported.66
(CH2), 27.3 (CH2), 26.8 (CH2). All physical and spectral properties
were consistent with those previously reported.66
Methyl 1-(5-Fluoropentyl)-1H-pyrrolo[2,3-b]pyridine-3-car-
boxylate (29). Subjecting methyl 1H-pyrrolo[2,3-b]pyridine-3-
carboxylate (24, 881 mg, 5.00 mmol) and 1-bromo-5-fluoropentane
(684 μL, 5.25 mmol, 1.05 equiv) to general procedure C gave, following
purification by flash chromatography (hexane:EtOAc, 100:0 to 60:40),
29 as a white solid (761 mg, 58%). mp 37−39 °C; Rf 0.69
(hexane:EtOAc, 50:50); 1H NMR (400 MHz, CD3OD) δ 8.39 (dd, J
= 7.9, 1.6 Hz, 1H), 8.32 (dd, J = 4.8, 1.6 Hz, 1H), 8.15 (s, 1H), 7.25 (dd,
J = 7.9, 4.8 Hz, 1H), 4.38 (dt, J = 47.5, 6.0 Hz, 2H), 4.34 (t, J = 7.2 Hz,
2H), 3.89 (s, 3H), 1.92 (quin., J = 7.4 Hz, 2H), 1.78−1.62 (m, 2H),
1.46−1.35 (m, 2H); 13C NMR (101 MHz, CD3OD) δ 166.5 (quat.),
144.7 (quat.), 136.4 (CH), 131.2 (CH), 120.7 (quat.), 119.0 (CH),
1
106.3 (quat.), 84.6 (d, JCF = 163.9 Hz, CH2), 51.6 (CH3), 46.1
(NCH2), 31.0 (d, 2JCF = 19.8 Hz), 30.8 (CH2), 23.6 (d, 3JCF = 5.3 Hz,
CH2). All physical and spectral properties were consistent with those
previously reported.58
General Procedure D: Synthesis of 1-Alkyl-1H-Indazole-3-
carboxylic Acids and 1-(5-Fluoropentyl)-1H-pyrrolo[2,3-b]-
pyridine-3-carboxylic Acid. To a solution of the appropriate methyl
1-alkyl-1H-indazole-3-carboxylate (25−28) or methyl 1-(5-fluoropen-
tyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxylate (29) in MeOH (50 mL)
was added 1 M aq. NaOH, and the solution stirred for 48 h. The solvent
was evaporated in vacuo, and the resulting aqueous residue was adjusted
to pH 2 using 1 M aq. HCl and then extracted with EtOAc (3 × 50 mL).
The organic layers were dried (MgSO4), and the solvent removed in
vacuo to give the products.
1-Butyl-1H-indazole-3-carboxylic Acid (30). Subjecting methyl
1-butyl-1H-indazole-3-carboxylate (25, 1.60 g, 6.88 mmol) to general
procedure D gave 30 as a white solid (1.34 g, 90%). mp 102−105 °C; Rf
0.29 (hexane:EtOAc, 50:50); 1H NMR (400 MHz, DMSO-d6) δ 12.99
(s, 1H), 8.08 (dt, J = 8.2, 0.9 Hz, 1H), 7.80 (d, J = 8.6 Hz, 1H), 7.46
(ddd, J = 8.5, 6.9, 1.1 Hz, 1H), 7.30 (ddd, J = 7.9, 6.9, 0.9 Hz, 1H), 4.50
(t, J = 7.0 Hz, 2H), 1.88−1.79 (m, 2H), 1.24 (sext., J = 7.4 Hz, 2H), 0.86
(d, J = 7.4 Hz, 3H); 13C NMR (100 MHz, DMSO-d6) δ 163.5 (CO),
140.4 (quat.), 134.6 (quat.), 126.5 (CH), 123.0 (quat.), 122.8 (CH),
121.5 (CH), 110.6 (CH), 48.6 (NCH2), 31.4 (CH2), 19.4 (CH2), 13.5
(CH3). All physical and spectral properties were consistent with those
previously reported.66
Methyl 1-(4-Fluorobutyl)-1H-indazole-3-carboxylate (26).
Subjecting methyl 1H-indazole-3-carboxylate (23, 881 mg, 5.00
mmol) and 1-bromo-4-fluorobutane (590 μL, 5.25 mmol, 1.05
equiv) to general procedure C gave, following purification by flash
chromatography (hexane:EtOAc, 95:5 to 90:10), 26 as a colorless oil
(675 mg, 54%). Rf 0.62 (hexane:EtOAc, 50:50); 1H NMR (400 MHz,
CD3OD) δ 8.13 (dt, J = 8.2, 1.0 Hz, 1H), 7.68 (dt, J = 8.6, 0.8 Hz, 1H),
7.51−7.46 (m, 1H), 7.36−7.31 (m, 1H), 4.56 (t, J = 7.0 Hz, 2H), 4.43
(dt, J = 47.4, 5.9 Hz, 2H), 4.01 (s, 3H), 2.11−2.02 (m, 2H), 1.74−1.60
(m, 2H); 13C NMR (100 MHz, CD3OD) δ 164.5 (quat.), 142.1
(quat.), 135.6 (quat.), 128.3 (CH.), 124.4 (CH), 122.9 (quat.), 111.3
(CH), 84.3 (d, 1JCF = 164.2 Hz, CH2), 52.3 (NCH2), 50.1 (CH3), 28.7
(d, 2JCF = 20.1 Hz, CH2), 26.9 (d, 3J = 4.8 Hz, CH2); LCMS (ESI, + ve)
m/z 251.2 ([M + H]+); HRMS (ESI) C13H15FN2O2 exact mass
250.1118, accurate mass 250.1116 (mass error −0.55 ppm).
Methyl 1-(Pent-4-en-1-yl)-1H-indazole-3-carboxylate (27).
Subjecting methyl-1H-indazole-3-carboxylate (23, 4.40 g, 25.0 mmol)
and 5-bromo-1-pentene (4.4 mL, 37.5 mmol, 1.5 equiv) to general
procedure C gave, following purification by flash chromatography
(hexane:EtOAc, 98:2), 27 as a colorless oil (3.36 g, 55%). Rf 0.45
(hexane:EtOAc, 80:20); 1H NMR (400 MHz, CDCl3) δ 8.23 (1H, d, J
= 8.2 Hz), 7.49−7.40 (2H, m), 7.35−7.28 (1H, m), 5.88−5.72 (1H,
m), 5.04 (1H, d, J = 9.5 Hz), 5.00 (1H, s), 4.48 (2H, t, J = 7.0 Hz), 4.03
(3H, s), 2.11−2.04 (4H, m); 13C NMR (100 MHz, CDCl3) δ 163.2
(CO), 140.7 (quat.), 137.1 (CH), 134.8 (quat.), 126.9 (CH), 123.9
(quat.), 123.2 (CH), 122.4 (CH), 116.0 (CH2), 109.7 (CH), 52.1
(OCH3), 49.3 (NCH2), 30.8 (CH2), 29.0 (CH2); LCMS (ESI, + ve)
m/z 245.1 ([M + H]+); HRMS (ESI) C14H16N2O2 exact mass
244.1212, accurate mass 244.1209 (mass error −1.18 ppm).
1-(4-Fluorobutyl)-1H-indazole-3-carboxylic Acid (31). Sub-
jecting methyl 1-(4-fluorobutyl)-1H-indazole-3-carboxylate (26, 500
mg, 2.00 mmol) to general procedure D gave 31 as a white solid (441
mg, 94%). mp 107−109 °C; Rf 0.43 (hexane:EtOAc, 50:50); 1H NMR
(400 MHz, DMSO-d6) δ 13.01 (s, 1H), 8.09 (d, J = 8.2 Hz, 1H), 7.82
(d, J = 8.6 Hz, 1H), 7.48 (ddd, J = 8.5, 6.9, 1.1 Hz, 1H), 7.31 (ddd, J =
8.0, 6.9, 0.8 Hz, 1H), 4.55 (t, J = 7.0 Hz, 2H), 4.45 (dt, J = 47.4, 6.0 Hz,
2H), 2.00−1.90 (m, 2H), 1.70−1.55 (m, 2H); 13C NMR (100 MHz,
DMSO-d6) δ 163.4 (CO), 140.4 (quat.), 134.8 (quat.), 126.5 (CH),
1
123.0 (quat.), 122.8 (CH), 121.5 (CH), 110.6 (CH), 83.4 (d, JCF
=
161.8 Hz, CH2), 48.4 (NCH2), 27.1 (d, 2JCF = 19.6 Hz, CH2), 25.3 (d,
3JCF = 5.2 Hz, CH2); LCMS (ESI, + ve) m/z 237.2 ([M + H]+); HRMS
(ESI) C12H13FN2O2 exact mass 236.0961, accurate mass 236.0959
(mass error −0.85 ppm).
1-(Pent-4-en-1-yl)-1H-indazole-3-carboxylic Acid (32). Sub-
jecting methyl 1-(pent-4-en-1-yl)-1H-indazole-3-carboxylate (27, 1.84
g, 7.56 mmol) to general procedure D gave 32 as a white solid (1.43 g,
6.23 mmol, 82%). mp 94−97 °C; Rf 0.22 (hexane:EtOAc, 50:50); 1H
NMR (400 MHz, CDCl3) δ 8.27 (1H, d, J = 8.2 Hz), 7.53−7.42 (2H,
m), 7.38−7.34 (1H, m), 5.89−5.73 (1H, m), 5.05 (1H, d, J = 9.5 H),
5.02 (1H, s), 4.51 (2H, t, J = 6.0 Hz), 2.15−2.07 (4H, m), OH not
observed (exchangeable); 13C NMR (100 MHz, CDCl3) δ 167.5 (CO),
141.0 (quat.), 137.0 (CH), 134.2 (quat.), 127.1 (CH), 124.0 (quat.),
123.6 (CH), 122.5 (CH), 116.1 (CH2), 109.9 (CH), 49.4 (NCH2),
30.8 (CH2), 28.9 (CH2); LCMS (ESI, + ve) m/z 231.1 ([M + H]+);
HRMS (ESI) C13H14N2O2, exact mass 230.1055, accurate mass
230.1061 (mass error 2.67 ppm).
Methyl 1-(Cyclohexylmethyl)-1H-indazole-3-carboxylate
(28). Subjecting methyl-1H-indazole-3-carboxylate (23, 884 mg, 5.02
mmol) and (bromomethyl)cyclohexane (1.33 g, 7.53 mmol, 1.5 equiv)
to general procedure C gave, following purification by flash
chromatography (hexane:EtOAc, 94:6), 28 as a white powder (539
1
mg, 39%). mp 64−66 °C; Rf 0.96 (hexane:EtOAc, 20:80); H NMR
(400 MHz, CD3OD) δ 8.14 (d, 1H), 7.67 (d, 1H), 7.51−7.45 (m, 1H),
7.36−7.30 (m, 1H), 4.34 (d, J = 7.3 Hz, 2H), 4.01 (s, 3H), 2.10−1.97
(m, 1H), 1.77−1.62 (m, 3H), 1.59−1.51 (m, 2H), 1.30−1.16 (m, 3H),
1.14−1.02 (m, 2H); 13C NMR (101 MHz, CD3OD) δ 164.5 (quat.),
142.6 (quat.), 135.4 (quat.), 128.2 (CH), 124.4 (quat.), 124.3 (CH),
122.8 (CH), 111.6 (CH), 56.6 (CH2), 52.3 (CH3), 40.1 (CH2), 31.8
1-(Cyclohexylmethyl)-1H-indazole-3-carboxylic Acid (33).
Subjecting methyl 1-(cyclohexylmethyl)-1H-indazole-3-carboxylate
(28, 498 mg, 1.84 mmol) to general procedure D gave 33 as a white
I
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Research Article
solid (393 mg, 83%). Rf 0.33 (hexane:EtOAc, 50:50); 1H NMR (400
MHz, DMSO-d6) δ 13.00 (s, 1H), 8.09 (d, J = 8.2 Hz, 1H), 7.81 (d, J =
8.5 Hz, 1H), 7.51−7.42 (m, 1H), 7.35−7.26 (m, 1H), 4.35 (d, J = 7.1
Hz, 2H), 1.99−1.87 (m, 1H), 1.69−1.61 (m, 2H), 1.61−1.58 (m, 1H),
1.49 (d, J = 12.4 Hz, 2H), 1.23−1.08 (m, 3H), 1.08−0.97 (m, 2H). All
physical and spectral properties were consistent with those previously
reported.66
Samuel D. Banister − The Lambert Initiative for Cannabinoid
Therapeutics, Brain and Mind Centre, The University of
Sydney, Sydney 2050, Australia; School of Chemistry, The
University of Sydney, Sydney 2006, Australia; orcid.org/
0000-0002-4690-4318; Phone: +61 2 9351 0805;
Email: samuel.banister@sydney.edu.au
1-(5-Fluoropentyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxylic
Acid (34). Subjecting methyl 1-(5-fluoropentyl)-1H-pyrrolo[2,3-
b]pyridine-3-carboxylate (29, 343 mg, 1.30 mmol) to general
procedure D gave 34 as a white solid (298 mg, 92%); Rf 0.43
(hexane:EtOAc, 50:50); 1H NMR (400 MHz, DMSO-d6) δ 12.26 (s,
1H), 8.36−8.30 (m, 2H), 8.29 (s, 1H), 7.26 (dd, J = 7.9, 4.7 Hz, 1H),
4.39 (dt, J = 47.5, 6.0 Hz, 3H), 4.33 (t, J = 7.1 Hz, 3H), 1.87 (quin., J =
7.3 Hz, 2H), 1.73−1.58 (m, 2H), 1.35−1.25 (m, 2H); 13C NMR (100
MHz, DMSO-d6) δ 165.2 (CO), 147.4 (quat.), 143.4 (CH), 135.3
(CH), 129.2 (CH), 118.7 (quat.), 117.7 (CH), 105.3 (quat.), 83.6 (d,
1JCF = 161.6 Hz, CH2), 44.2 (NCH2), 29.3 (d, 2JCF = 19.3 Hz, CH2),
Authors
Annelies Cannaert − Laboratory of Toxicology, Department of
Bioanalysis, Faculty of Pharmaceutical Sciences, Ghent
University, Ghent B-9000, Belgium; orcid.org/0000-0002-
8654-1371
Eric Sparkes − The Lambert Initiative for Cannabinoid
Therapeutics, Brain and Mind Centre, The University of
Sydney, Sydney 2050, Australia; School of Chemistry, The
University of Sydney, Sydney 2006, Australia
Edward Pike − The Lambert Initiative for Cannabinoid
Therapeutics, Brain and Mind Centre, The University of
Sydney, Sydney 2050, Australia; School of Chemistry, The
University of Sydney, Sydney 2006, Australia; Department of
Chemistry, University of York, York YO10 5DD, United
Kingdom
Jia Lin Luo − The Lambert Initiative for Cannabinoid
Therapeutics, Brain and Mind Centre, The University of
Sydney, Sydney 2050, Australia; School of Psychology, The
University of Sydney, Sydney 2006, Australia
Ada Fang − The Lambert Initiative for Cannabinoid
Therapeutics, Brain and Mind Centre, The University of
Sydney, Sydney 2050, Australia; School of Chemistry, The
University of Sydney, Sydney 2006, Australia
Richard C. Kevin − The Lambert Initiative for Cannabinoid
Therapeutics, Brain and Mind Centre, The University of
Sydney, Sydney 2050, Australia; School of Psychology, The
University of Sydney, Sydney 2006, Australia; orcid.org/
0000-0002-4912-4499
Ross Ellison − Clinical Toxicology and Environmental
Biomonitoring Laboratory, School of Medicine, University of
California San Francisco, San Francisco, California 94143,
United States
3
29.1 (CH2), 21.9 (d, JCF = 5.4 Hz, CH2). All physical and spectral
properties were consistent with those previously reported.58
Determination of the in Vitro Biological Activity at the CB1. A
live cell-based reporter assay that monitors the in vitro CB1 activation
via its interaction with β-arrestin 2 using the NanoLuc Binary
Technology (NanoBiT), was applied to assess the biological activity
of the compounds. Details regarding the development of the stable cell
line used here have been reported elsewhere.56,60
The modified human embryonic kidney (HEK) 293T cells were
routinely maintained at 37 °C, 5% CO2, under humidified atmosphere
in Dulbecco’s modified Eagle’s medium (GlutaMAX) supplemented
with 10% heat-inactivated FBS, 100 IU/mL of penicillin, 100 μg/mL of
streptomycin, and 0.25 μg/mL of amphotericin B. For experiments,
cells were plated on poly-D-lysine coated 96-well plates at 5 × 104 cells/
well and incubated overnight. Next, the cells were washed twice with
Opti-MEM I Reduced Serum Medium to remove any remaining FBS,
and 100 μL Opti-MEM I and 25 μL of the Nano-Glo Live Cell reagent
was added to each well. Subsequently, the plate was placed into a
TriStar2 LB 942 multimode microplate reader (Berthold Technologies
GmbH & Co., Germany). Luminescence was monitored during the
equilibration period until the signal stabilized (15 min). Next, 10 μL per
well of test compounds present as concentrated (13.5-fold, as 10 μL was
added to generate a final volume of 135 μL) stock solutions in Opti-
MEM I was added. The luminescence was continuously monitored for
120 min. Solvent controls were included in all experiments.
The results are represented as mean area under the curve (AUC)
standard error of mean (SEM), obtained in minimum three
independent experiments, with duplicates run in every experiment.
All results were normalized to the Emax of JWH-018 (= 100%), our
reference compound. Curve fitting of concentration−response curves
via nonlinear regression (four−parameter logistic fit) was employed to
determine EC50 (a measure of potency) and Emax (a measure of efficacy)
values, using GraphPad Prism software (San Diego, CA, US).
Roy Gerona − Clinical Toxicology and Environmental
Biomonitoring Laboratory, School of Medicine, University of
California San Francisco, San Francisco, California 94143,
United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acschemneuro.0c00644
Author Contributions
A.C. designed and executed the in vitro functional assays under
the supervision of C.S. E.S., E.P., A.F., J.L.L., and R.K.
synthesized and analytically characterized all compounds
under the supervision of S.D.B. R.E. obtained all high resolution
mass spectra under the supervision of R.G. A.C., E.S., S.D.B., and
C.S. conceived the experiments and wrote the manuscript, and
all authors reviewed and edited drafts of the manuscript. All
authors approved the final version of the paper.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acschemneuro.0c00644.
1H and 13C NMR spectra and LC-UV chromatograms for
novel compounds (PDF)
Funding
AUTHOR INFORMATION
■
S.D.B. is supported by NHMRC Project Grant 1161571, a Brain
and Mind Centre Research Development Grant from The
University of Sydney, and The Lambert Initiative for
Cannabinoid Therapeutics, a philanthropic research program
based at the Brain and Mind Centre, The University of Sydney.
A.C. acknowledges funding as a postdoctoral research fellow
from the Research Foundation-Flanders (FWO; 12Y9520N)
Corresponding Authors
Christophe P. Stove − Laboratory of Toxicology, Department
of Bioanalysis, Faculty of Pharmaceutical Sciences, Ghent
University, Ghent B-9000, Belgium; orcid.org/0000-0001-
7126-348X; Phone: +32 9 264 81 35;
Email: christophe.stove@ugent.be
J
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Research Article
synthetic cannabinoids and their associated treatment. Clin. Toxicol. 54,
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Pediatrics 144, No. e20182690.
and the Ghent University - Special Research Fund (BOF;
PDO026-18). The latter is also acknowledged by C.S (Grants
No. 01N00814 and No. 01J15517).
Notes
The authors declare no competing financial interest.
(16) Darke, S., Duflou, J., Farrell, M., Peacock, A., and Lappin, J.
(2020) Characteristics and circumstances of synthetic cannabinoid-
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ABBREVIATIONS
■
βarr2, β-arrestin 2; CB1, cannabinoid receptor 1; COA,
certificate of analysis; CI, confidence interval; ee, enantiomeric
excess; EU, European Union; EWS, Early Warning System; FBS,
fetal bovine serum; GLC, gas liquid chromatography; GPCR, G
protein-coupled receptor; HEK, human embryonic kidney;
NPS, new psychoactive substances; SCRA, synthetic cannabi-
noid receptor agonist; SEM, standard error of mean; THC, Δ9-
tetrahydrocannabinol; UK, United Kingdom; UNODC, United
Nations Office on Drugs and Crime; US, United States
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J. P. (2020) Critical Illness Secondary to Synthetic Cannabinoid
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