Focused structure-activity relationship profiling around the 2-phenylindole scaffold of a cannabinoid type-1 receptor agonist-positive allosteric modulator: site-III aromatic-ring congeners with enhanced activity and solubility

Article abstract of DOI:10.1016/j.bmc.2020.115727

Specific tuning of cannabinoid 1 receptor (CB1R) activity by small-molecule allosteric modulators is a therapeutic modality with multiple properties inherently advantageous to therapeutic applications. We previously generated a library of unique CB1R posi

Full text of DOI:10.1016/j.bmc.2020.115727

Bioorganic&MedicinalChemistry28(2020)115727
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Focused structure-activity relationship profiling around the 2-phenylindole
scaffold of a cannabinoid type-1 receptor agonist-positive allosteric
modulator: site-III aromatic-ring congeners with enhanced activity and
solubility
⁎
Peter C. Schaffera, Pushkar M. Kulkarnia, David R. Janeroa,b, Ganesh A. Thakura,
a Department of Pharmaceutical Sciences, Bouvé College of Health Sciences, Northeastern University, Boston, MA 02115, United States
b Health Sciences Entrepreneurs, Northeastern University, Boston, MA 02115, United States
A R T I C L E I N F O
A B S T R A C T
Keywords:
Specific tuning of cannabinoid 1 receptor (CB1R) activity by small-molecule allosteric modulators is a ther-
apeutic modality with multiple properties inherently advantageous to therapeutic applications. We previously
generated a library of unique CB1R positive allosteric modulators (PAMs) derived from GAT211, which has three
pharmacophoric sites critical to its ago-PAM activity. To elaborate our CB1R PAM library, we report the rational
design and molecular-pharmacology profiling of several 2-phenylindole analogs modified at the “site-III” aro-
matic ring. The comprehensive structure–activity relationship (SAR) investigation demonstrates that attaching
small lipophilic functional groups on the ortho-position of the GAT211 site-III phenyl ring could markedly en-
hance CB1R ago-PAM activity. Select site-III modifications also improved GAT211’s water solubility. The SAR
reported both extends the structural diversity of this compound class and demonstrates the utility of GAT211’s
site-III for improving the parent compound’s drug-like properties of potency and/or aqueous solubility.
Cannabinoid type-1 receptor
Positive allosteric modulator
Physiochemical properties
Functional selectivity
2-Phenylindole
1. Introduction
plagued their translation into the clinic.[2,12–14]
As an alternative to CB1R orthosteric agents, allosteric ligands are
The mammalian endocannabinoid system (ECS) is a ubiquitous in-
formation-transducing network in which endogenous lipids (en-
docannabinoids) act as signaling molecules primarily by engaging the
orthosteric sites of the cannabinoid type-1 (CB1R) and type-2 (CB2R) G-
protein coupled receptors (GPCRs).[1–6] As the most abundant GPCR in
the CNS and by virtue of its diverse functions in the periphery, CB1R
helps regulate a wide range of physiological processes, from neuro-
transmission to energy metabolism.[4,7,8] Abnormal CB1R-dependent
ECS activity is recognized as a pathogenic component of several neu-
rological, psychiatric, cardiometabolic, and other disease states re-
presenting unsolved medical problems, making CB1R a prime ther-
apeutic target.[9–11] Over the last several decades, extensive discovery
campaigns have yielded a plethora of structurally diverse designer
CB1R orthosteric ligands with biological activity. However, adverse
events such as psychobehavioral problems and addiction associated
with typical CB1R orthosteric (ant)agonists/inverse agonists have
garnering interest for actualizing the pharmacotherapeutic impact of
CB1R modulation.[15] The potential for allosteric modulation to cir-
cumvent the liabilities associated with typical orthosteric CB1R ligands
by offering comparatively greater selectivity, tissue specificity, and
safety, as well as less potential for receptor desensitization, has
prompted medicinal chemistry efforts to design and profile novel CB1R
allosteric effectors as candidate therapeutics.[16–18]
The 2-phenylindoles GAT211 (33) from this laboratory,[19]
ZCZ011,[20] and its non-nitro derivative ABD1236,[21] have emerged as
prototypic CB1R agonist-positive allosteric modulators (“ago-PAMs”)
(Figure 1). These CB1R ligands act as both a PAM and an agonist at this
GPCR’s allosteric site, i.e., they increase the binding and function of
orthosteric agonists at CB1R while stimulating the receptor on their
own.[22,23] We previously demonstrated that GAT211’s ago-PAM ac-
tivity reflects its enantiomers: the (R)-(+)- enantiomer (GAT228) is a
CB1R allosteric agonist, while the (S)-(-)-enantiomer (GAT229) is a
Abbreviations: ago-PAM, agonist-positive allosteric modulator; CNS, central nervous system; BB, building block; ECS, endocannabinoid system; GPCR, G-protein
coupled-receptor; (h)CB1R, (human) cannabinoid type-1 receptor; NAM, negative allosteric modulator; PAM, positive allosteric modulator; SAR, structure-activity
relationship
⁎ Corresponding author.
E-mail address: g.thakur@northeastern.edu (G.A. Thakur).
https://doi.org/10.1016/j.bmc.2020.115727
Received 22 May 2020; Received in revised form 6 August 2020; Accepted 19 August 2020
Availableonline29August2020
0968-0896/©2020ElsevierLtd.Allrightsreserved.

P.C. Schaffer, et al.
Bioorganic&MedicinalChemistry28(2020)115727
Fig. 1. 2-Pheylindole type CBIR ago-PAMs ZCZ011, ABD1236, and GAT11(with
three defined sites color coded).
CB1R PAM (Figure 1).[23]
Structurally, these ago-PAMs share three principal regions poten-
tially amenable to modification/elaboration, as designated in Figure 1
for GAT211. ZCZ011 differs from GAT211 by its 6-methyl group at site
I, and our recent structure–activity relationship (SAR) study with the
GAT211 scaffold identified positions 6- and 7- at site I as amenable to
lipophilic-group substitution for greatly enhancing CB1R ago-PAM ac-
tivity and the ortho-position at site III for potentially incorporating ei-
ther lipophilic or hydrophilic groups.[24]
The effects of site-III substitutions on drug-like potency and physi-
cochemical properties of 2-phenylindole CB1R ago-PAMS have not been
explored in systematic detail. Both ZCZ011 and ABD1236 feature a site-
III 2-thienyl ring which enhanced CB1R ago-PAM activity. However,
the 2-thienyl substituent invites formation of nephrotoxic and hepato-
toxic products from thiophene metabolism, the cause of market with-
drawal for several drugs.[25,26] Furthermore, replacement of benzene
with thiophene typically results in a slight increase in lipophilicity,[27]
which exacerbates the major drawback of the 2-phenylindole class of
CB1R ago-PAMs, limited aqueous solubility. This limitation, and the
unfavorable physiochemical and metabolic properties associated with
thiophene, justify exploring alternative site-III substituents to elaborate
the GAT211-derived CB1R ago-PAM library and, more globally, define
the role of site-III as determinant of the pharmacological and physico-
chemical properties of 2-phenylindole CB1R ago-PAMs.
Scheme 1. Reagents and condition (a) NH4 CF3 CO2 CH3NO2,reflux , 3–6 h; (b)
XH3NO2,KOt –Bu ,t-BuOH:THF,0°C,2–4;(c)Ac2 O,4-DMAP, DCM,3–6 h;(d)NH4
CF3 CO2 10%Aq. EtOH, reflux (100 °C,16 h) or microwave (120 °C, 30 min).
6-methylindole via oxidative 2-phenylation, was used as the nucleo-
phile to generate the desired site-III analogs.[30] The syntheses of all
other nitrovinyl BBs and site-III analogs not depicted in Scheme 1 are
described in the Experimental section.
2.2. SAR studies: cAMP inhibition and β-arrestin recruitment
Focused on the GAT211 site-III phenyl ring, we first determined the
impact of either extending or saturating the site-III phenyl ring on
compound activity in a heterologous cell system expressing human
CB1R (hCB1R). Functional profiling involved assay of cAMP production
We recently demonstrated the requirement of a site-III ring sub-
stituent for CB1R PAM activity of GAT211.[28] Additionally, synthesis
and profiling of 2-phenylindole analogs with site-III pyridyl and
monosubstituted fluorophenyl rings suggested that ortho substitutions
in the site-III phenyl ring are favorable for activity.[24] Aside from site-
III phenyl derivatives reported by our group,[19] the only other site-III
ring variations published are 2-thienyl, 2-furyl, and cyclopropyl-me-
thyl.[21] Detailed correlations between the site-III ring functionality and
pharmacological activity are lacking, especially for site III five-mem-
bered heteroaromatic rings.
and β-arrestin recruitment. Since CB1R is canonically coupled to Gi/o
,
activation of CB1R inhibits cellular cAMP formation, and β-arrestin
recruitment is a prime mode of G protein-independent CB1R sig-
naling.[31] Ring extension by a single methylene unit decreased activity,
as evidenced by benzyl analog 34 (Table 1), suggesting a one-carbon
spacer between the 3-position of the indole ring and the site-III ring is
optimal for activity. The cyclohexyl analog 35 also had less overall
activity relative to GAT211 (33), demonstrating that an unsaturated
site-III ring is preferred for activity, possibly as a reflection of its sta-
bilizing electrostatic π-π interactions with aromatic CB1R residues. This
proposition is supported by predictions from our previous, fragment-
based docking analysis that the site-III phenyl ring of the GAT211 en-
antiomer, GAT228, forms a T-shape π-interaction with hCB1R F4.46 in
the intracellular GPCR region and the site-III phenyl ring of the other
enantiomer, GAT229, forms a parallel π-stack interaction with Y2.59 in
the extracellular region.[28] These considerations focused our attention
on an examination of the effects of site-III aromatic ring substituents in
GAT211 analogs that retained the parent scaffold’s single-carbon spacer
between an unsaturated site-III phenyl-ring substituent and the 3-po-
sition of the indole ring.
To address this knowledge gap, we report a comprehensive SAR
study on the site-III ring within the 2-phenylindole scaffold. This study
constitutes an in-depth analysis of group-size and positional-preference
effects of ring substituents at site III on CB1R ago-PAM activity.
2. Results and discussion
2.1. Chemistry
All nitrovinyl building blocks (BBs) were prepared from their cor-
responding, commercially available aromatic aldehydes. The last step
for the synthesis of all site-III analogs (33–70), depicted in Scheme 1,
involves ammonium trifluoroacetate catalyzed C3 alkylation of 2-phe-
nylindoles with various nitrovinyl BBs (step d). 1-pyrrolidyl substituted
phenylnitrovinyl BBs, 16a-c, were directly prepared from their corre-
sponding aldehydes in one step under reflux conditions (step a, Scheme
1). Thiazole, oxazole, and 1-methylimidazole nitrovinyl BBs, 31a-e,
were prepared by a two-step process starting with a Henry reaction
(step b) to form the nitroalcohol intermediate which subsequently un-
derwent dehydration (step c) to yield the desired nitrovinyl BB.[29] In
limited instances, 6-methyl-2-phenylindole (32), prepared directly from
GAT211 analogs monosubstituted at the para, meta, or ortho position
on the site-III phenyl ring were synthesized and profiled. Within the
methyl (36–38), methoxy (39–41), and chloro (42–44) series, the re-
lative potency ortho > meta > para was observed, notably with re-
spect to cAMP inhibition (Table 1). In the mono-fluoro series (46–48),
the para-fluoro analog was a marginally more potent inhibitor of cAMP
production than the meta-fluoro analog. The positive influence of li-
pophilicity at the site-III benzene ring ortho position on cAMP inhibition
is evident from the much greater potency of the ortho-methyl (38) and
ortho-chloro (44) analogs relative to GAT211 itself (33). However, this
2

P.C. Schaffer, et al.
Bioorganic&MedicinalChemistry28(2020)115727
Table 1
SAR data for site-III analogs in cAMP and β-arrestin assaysa-c
.
Compound
R1
Ar
cAMP
β-Arrestin
EC50 (μM)
EMax
EC50 (μM)
EMax
(% Response)
(% Response)
33
H
H
0.303
0.420
99.62
0.629
> 10
54.14
GAT211
34
115.19
0
35
36
37
H
H
H
0.495
1.444
0.662
117.43
118.94
116.1
1.897
> 10
1.992
26.75
0
23.82
38
H
0.003
81.67
0.057
68.45
39
40
H
H
1.177
0.664
119.09
131.21
> 10
> 10
0
4.96
41
H
0.411
117.87
1.875
37.02
42
43
H
H
1.315
0.859
74.81
> 10
2.981
0
120.95
15.96
44
45
H
H
0.018
0.571
74.98
1.241
> 10
93.43
3.72
128.13
46
47
H
H
0.444
0.728
106.31
117.9
> 10
> 10
8.03
15.7
48
H
0.043
112.87
0.782
56.03
49
50
H
H
0.618
0.423
97.41
> 10
> 10
0
110.31
6.69
(continued on next page)
3

P.C. Schaffer, et al.
Bioorganic&MedicinalChemistry28(2020)115727
Table 1 (continued)
Compound
R1
H
Ar
cAMP
β-Arrestin
EC50 (μM)
EMax
EC50 (μM)
EMax
(% Response)
(% Response)
51
1.825
75.88
> 10
7.28
52
53
H
H
> 10
0.127
65.11
150.7
> 10
> 10
0
0
54
H
0.557
86.16
0.792
68.14
55
56
57
58
59
H
H
H
H
H
0.476
> 10
0.203
> 10
0.483
125.33
87.63
2.460
> 10
> 10
> 10
2.344
42.15
0
109.19
25.47
5.57
5
112.88
31.79
60
H
> 10
62.18
> 10
0
61
H
0.031
0.033
116.17
91.31
0.584
0.379
36.11
78.23
ZCZ011
CH3
62
63
H
H
0.119
0.245
124.69
99.80
1.609
0.655
23.91
28.84
64
65
H
H
0.513
0.209
143.6
77.1
> 10
> 10
12.86
0.70
66a
66b
67a
67b
68
H
0.192
0.050
0.856
0.261
0.533
83.65
98.45
56.64
67.19
52.69
0.331
0.163
> 10
> 10
> 10
32.59
80.62
3.21
14.49
0
CH3
H
CH3
H
69
H
> 10
34.41
> 10
0.667
(continued on next page)
4

P.C. Schaffer, et al.
Bioorganic&MedicinalChemistry28(2020)115727
Table 1 (continued)
Compound
R1
H
Ar
cAMP
β-Arrestin
EC50 (μM)
EMax
EC50 (μM)
EMax
(% Response)
(% Response)
70
> 10
46.06
> 10
0
aCB1R PAM activity was quantified for inhibition of forskolin-stimulated cellular cAMP production using the DiscoveRx HitHunter® assay (CHO hCB1R), EC20 of
CP55,940, and test compound for 30 min, and for β-arrestin 2 recruitment using the DiscoveRx PathHunter® assay (CHO hCB1R) in cells treated with EC20 of
CP55,940, and test compound for 90 min.
bn = 2–4 independent experiments were performed for each test concentration; 10 concentrations were examined over a 10,000-fold concentration range from
0.1 nM to 10 μM, and expressed as an average of these values (SD <
15%).
c A best-fit, dose-response curve was generated from data sets for each test compound using Chemical and Biological Information System (CBIS) data analysis suite.
influence appears to be limited since the 2,6-dichloro analog (45) was
less potent as inhibitor of cAMP production than its monosubstituted
version (44).
ring. The presence of nitrogen in the 2-pyrrolyl analog 65 marginally
increased compound potency in the cAMP assay relative to furan analog
63, but with reduced efficacy.
The potency trend ortho > meta > para for the monosubstituted
GAT211 analogs was not entirely recapitulated in the 1-pyrrolidyl
series (49–51) (Table 1). As inhibitor of cellular cAMP production, the
meta-pyrrolidyl analog (50) was indeed more potent than the para
analog (49), whereas the ortho-1-pyrrolidinyl analog 51 was least po-
tent. The pyrrolidyl-series SAR suggests that the bulky ortho- pyrroli-
dine ring might sterically hinder hCB1R-G protein interaction, thereby
limiting the magnitude of downstream, Gαi-mediated signaling. This
suggestion is partially supported by the naphthyl-series SAR: the 2-
naphthyl analog (59) was much more active in the cAMP assay than the
1-naphthyl analog (60). Within the trifluoromethyl series (52–54), the
meta-substituted analog, 53, provided the highest activity in cAMP in-
hibition; this finding might be attributed to a steric and/or electronic
relationship. The pyrrolidyl- and naphthyl-series SARs, suggestive of a
potential size limitation in the interior portion of the GAT211 site-III
phenyl ring, led us to determine whether the para-position would be
favorable for installing bulky substituents (55–58) (Table 1). The para-
substituted 1-piperidyl GAT211 analog 55 was less active than parent
33 as inhibitor of cAMP production, and the para-substituted 4-mor-
pholyl (56) and phenyl (58) analogs were entirely inactive. Among
these para-substituted analogs, the para-tert-butyl compound 57 evi-
denced the highest potency in the cAMP assay.
Considering the activity of 65 and the SARs around the thiophene
(61–62) and furan (63–64) analogs, and the previous lack of knowledge
on nitrogen’s impact within a site-III five-membered heteroaromatic
ring, we proceeded to install nitrogen into site-III sulfur- and oxygen-
containing five-membered heteroaromatic rings with the intention of
improving physiochemical properties while retaining biological ac-
tivity. Accordingly, 61 was derivatized to the 2-thiazolyl analog 66a,
and 63 was derivatized to an oxazole, 67a. In both instances, in-
troducing nitrogen compromised activity in the cAMP assay (Table 1).
Consistent with SAR for the thiophene and furan series, 66a was more
potent than 67a in the cAMP assay, supporting the superiority of sulfur
vs. oxygen within a five-membered site-III heteroaromatic ring.
Although SAR for the thiophene and furan series indicated that the
2-position is preferred for heteroatom localization, it remained un-
known whether this positional preference could be extended to ni-
trogen-containing, five-membered heteroaromatic rings with two het-
eroatoms. To address this possibility experimentally, we synthesized
and profiled three site-III N-methylimidazole analogs (68–70). As an-
ticipated, the most potent N-methylimidazole analog in the cAMP
assay, with activity comparable to that of 67a, was the 2-substituted
compound, 68. The SAR across the thiophene, furan, and N-methyli-
midazole series allows conclusion that heteroatoms in the site-III five-
membered rings positioned α- to the connecting tertiary carbon and
facing toward the core of the 2-phenylindole scaffold foster excellent
activity in the cAMP assay. This conclusion invites suggestion that
methine groups at positions 3 and 4 of the site-III five-membered ring
form key hydrophobic interactions with hCB1R residues that are critical
for activity; replacement of these groups with hydrophilic oxygen or
nitrogen heteroatoms could disrupt such important interactions.
Mindful of this conclusion and our SAR data that support it, we none-
theless elected to examine experimentally the potential of nitrogen
within a site-III five-membered heterocyclic ring to enhance compound
aqueous solubility. Based on the structure of ZCZ011 and previously
reported SAR studies,[20,22] we first modified the 2-thiazolyl analog 66a
and the 2-oxazolyl analog 67a with the aim of improving potency by
adding a methyl group in the 6-postion of the indole ring, yielding 66b
and 67b, respectively (Table 1). A 6-methyl group increased potency in
the cAMP assay by nearly four-fold, as demonstrated by comparing the
activities of 66a vs. 66b and 67a vs. 67b. 66b also showed robust ac-
tivity in the β-arrestin recruitment assay not shared by 66a, 67a, and
67b, implicating the site-III oxazole ring as a pharmacophore for po-
tentially favoring G-protein-dependent signaling. Regarding inhibition
of cAMP production, 67b exhibited potency similar to GAT211. How-
ever, since limited aqueous solubility is characteristic of 2-phenylindole
ago-PAMs, and aqueous solubility is an important drug-like character-
istic, we reasoned from a therapeutic perspective that improved
These SAR data demonstrate that extended, 6-membered ring sub-
stituents with large surface areas are not well tolerated at the para
position, regardless of their planarity or lipophilicity. Rather, the para
position of the GAT211 scaffold preferentially accommodates compact,
globular groups (e.g., 36, 39, 42, 46, 49, 57).
We next transitioned our focus to site-III five-membered hetero-
aromatic ring substitutions. Notwithstanding the metabolic and tox-
icological liabilities of thiophene-containing drugs,[25,26] the 2-thienyl
group is well tolerated at site III, as illustrated by ZCZ011 and ABD1236
(Figure 1). Alternative and more drug-like site-III five-membered het-
eroaromatic rings have not been intensively investigated. To this intent,
we first wished to define the optimal heteroatom position within the
ring. The SAR between 2 and vs. 3-thienyl (61 vs. 62) and 2- vs. 3-furyl
(63 vs. 64) analogs in Table 1 demonstrates that placing the heteroatom
at the 2-position yielded higher-potency compounds in the cAMP assay.
It is noteworthy that the positional preference of the heteroatom within
a five-membered aromatic ring is analogous to the para < meta <
ortho trend exhibited by the methyl, chloro, and methoxy mono-
substituted phenyl analogs. Among the thiophene and furan analogs,
sulfur within site-III five-membered heteroaromatic rings yielded ana-
logs with better overall activity in the cAMP assay than did oxygen as
heteroatom (61 vs. 63 and 62 vs. 64).
These data led us to focus our site-III five-membered heteroaromatic
SAR investigation on analogs with a heteroatom at the 2-position of the
5

P.C. Schaffer, et al.
Bioorganic&MedicinalChemistry28(2020)115727
Table 2
the of the 2-phenylindole class of CB1R PAMS. The comprehensive SAR
data reported over a wide range of site-III aromatic ring variations on
the 2-phenylindole scaffold provide ample context for candidate se-
lection toward more detailed preclinical profiling, both in vitro for
signaling bias[31,32] and in vivo for preclinical therapeutic efficacy.
Enantiomeric separation of the most therapeutically promising site-III
analogs will invite evaluation of the CB1R-related molecular pharma-
cology of each enantiomer. This initial SAR profiling should also be
applicable to inform the design and optimization of next-generation 2-
phenylindole CB1R ago-PAMs.
Kinetic water solubility of GAT211, ZCZ011, 66b, and 67b.
Compound
Solubility Limit (μM)
GAT211 (33)
ZCZ011
66b
1.56
1.56
3.13
67b
12.5
a Precipitation was determined by turbidity at 540 nm.
b
Absorbance value > “mean +3 standard deviations” from blank was con-
sidered turbid.
c The coefficient of variation (CV) was < 1.5% for all four compounds.
4. Experimental
4.1. Chemistry
aqueous solubility might counterbalance any potential potency reduc-
tion. The discovery of both 66b and 67b was highly encouraging, as
they represent two analogs with the potential of possessing improved
aqueous solubility with good biological activity.
4.1.1. Material and methods
All commercial reagents and solvents were purchased from Sigma
Aldrich, Inc. (St. Louis, MO), Alfa Aesar (Ward Hill, MA), and Combi-
Blocks (San Diego, CA) as reagent grade and used without further
purification unless otherwise specified. A Biotage® Initiator Microwave
System (Charlotte, NC) was used for synthesizing select intermediates.
Reaction progress was monitored by thin-layer chromatography (TLC)
using commercial silica gel 60 F254 glass plates. Compounds were vi-
sualized under ultraviolet light or by staining with iodine, phospho-
molybdic acid, or p-anisaldehyde reagent. Flash column chromato-
graphy was carried out on a Biotage® SP1, Biotage® Isolera, or
Interchim (Montluçon Cedex, France) purification unit using prepacked
columns from Reveleris® (Buchi Corp., New Castle, DE), Biotage®, and
Luknova (Boston, MA). Characterization of compounds and their purity
were established by a combination of HPLC, TLC, mass spectrometry,
and NMR analyses. NMR spectra were recorded in DMSO-d6, chloro-
form-d, or methanol-d4, on a Varian NMR spectrometer (1H NMR at
500 MHz and 13C NMR at 125 MHz) (Agilent Technologies, Palo Alto,
CA). Chemical shifts were recorded in parts per million (δ) relative to
tetramethylsilane (TMS; 0.00 ppm) or solvent peaks as the internal
reference. Multiplicities are indicated as br (broadened), s (singlet), d
(doublet), t (triplet), q (quartet), quin (quintet), sept (septet), or m
(multiplet). Coupling constants (J) are reported in hertz (Hz). All test
compounds were > 95% pure as determined by LC/MS analysis per-
formed using an Agilent Technologies 1260 Infinity reverse phase HPLC
with dual-wavelength UV–visible detector and a 6120 Quadrupole mass
spectrometer (electrospray ionization). HRMS was done on SCIEX TOF/
TOF™ 5800 System (Framingham, MA) in a positive-ion mode with
delay time of 100 ns. Each sample well was surveyed to find a “sweet
spot”, and 400 laser pulses were then averaged to generate a spectrum.
4.1.2.-4.1.16. 1–15
2.3. Kinetic water solubility
Based upon the biological-activity and distinctive signaling profiles
of 66b and 67b, we determined each of their kinetic water solubility
using a standard turbidimetric assay in comparison to GAT211 and
ZCZ011.
The aqueous solubilities of both 66b and 67b are improved over
GAT211 and ZCZ011 (Table 2). Direct comparison of ZCZ011 and 66b
demonstrates that replacement of a site-III 2-thienyl for a 2-thiazolyl
group improved kinetic water solubility by 2-fold without compro-
mising their comparable in vitro activity in the cAMP assay. 67b ex-
hibited the best kinetic water solubility (8-fold greater than GAT211
and ZCZ011 and 4-fold greater than 66b), demonstrating that in-
corporation of a 2-oxazolyl moiety at site III is a tenable solution for
improving the limited water solubility characteristic of the CB1R PAM
2-phenylindole scaffold.
3. Conclusions
Small-molecule PAMs are increasingly being recognized as potential
pharmacotherapeutics that would leverage hCB1R as a drug target
while avoiding the limitations of orthosteric CB1R agonists that have
hindered their translation into the clinic.[15–18, 22], For this potential to
be realized, extant libraries of biologically active CB1R PAMs need to be
expanded from the standpoints of new chemical matter, SAR profiling,
and drugability limitations. The present study addresses these con-
siderations for a predominant class of CB1R PAMs, the 2-pheny-
lindoles.[18,22] Syntheses and biological assessments of novel analogs
derived from the 2-phenylindole scaffold reported here establish that
the site-III functionality is a critical determinant of the biological ac-
tivity of this class. Specifically, a site-III aromatic ring is necessary for
appreciable CB1R ago-PAM activity. The hCB1R binding-pocket region
for the site-III aromatic ring appears to have a rather strict size toler-
ance, as suggested by the compromise of biological activity elicited by
extending GAT211′s site-III phenyl-ring or adding bulky substituents
onto the ring itself. In contrast, introducing a small lipophilic group at
the ortho-position of the GAT211 site-III phenyl ring (i.e., o-chloro or o-
methyl) can significantly enhance hCB1R ago-PAM activity. The overall
effect of a site-III ortho substituent, in terms of overall CB1R ago-PAM
enhancement, may be attributed to the dihedral angle exerted by the
substituent; this possibility will be inspected in future SAR studies.
Aside from this ortho position for monosubstitution, the position alpha
to the site-III ring’s connecting tertiary carbon is the also amenable to
modification without appreciably compromising bioactivity. While only
marginally affecting biological activity, converting a site-III 2-thienyl to
2-thiazolyl moiety markedly increases compound water solubility, a
finding that addresses one of the major physicochemical limitations of
The following intermediates were prepared by previously reported
general procedures: 1;[33] 5; [34,35] 2–3, 8;[36] 4–7, 9–10;[37] 11;[38] 12;
[39]
13–15. [24]
4.1.2. General Procedure A: Syntheses of pyrrolidyl-substituted phenyl
nitrovinyl intermediates (16a-16c)
To a 100-mL round bottom flask was added pyrrolidyl-benzalde-
hyde (460 mg, 2.63 mmol, 1 eq.), ammonium trifluoroacetate (202 mg,
2.63 mmol, 1 eq.), and nitromethane (40 mL). The reaction mixture was
refluxed under argon for 3–6 h. Upon reaction completion, the reaction
mixture was cooled to room temperature, concentrated under reduced
pressure, diluted with dichloromethane, then washed with ice-cold
water. The aqueous phase was extracted 3X with dichloromethane.
Combined organic extracts were washed with brine, dried with sodium
sulfate, then concentrated under reduced pressure. The resulting or-
ganic resin was purified by silica gel flash chromatography using ethyl
acetate/hexanes eluent to afford the pure nitrovinyl product.
4.1.2.1. (EV)-1-(4-(2-nitrovinyl)phenyl)pyrrolidine (16a). Yield: 40%;
1H NMR (500 MHz, (CDCl3): δ 7.96 (d, J = 13.5 Hz, 1H), 7.48 (d,
6

P.C. Schaffer, et al.
Bioorganic&MedicinalChemistry28(2020)115727
J = 13.0 Hz, 1H), 7.41 (d, J = 8.5 Hz, 2H), 7.41 (d, J = 9.0 Hz, 2H),
3H).
3.39–3.36 (m, 4H), 2.07–2.04 (m, 4H).
4.1.4.4. (E)-1-methyl-4-(2-nitrovinyl)-1H-imidazole (31d). Yield: 55%;
1H NMR (500 MHz, CDCl3) δ 7.86 (d, J = 13.1 Hz, 1H), 7.76 (d,
J = 13.1 Hz, 1H), 7.52 (s, 1H), 7.28 (s, 1H), 3.75 (s, 3H).
4.1.2.2. (E)-1-(3-(2-nitrovinyl)phenyl)pyrrolidine (16b). Yield: 77%; 1H
NMR (500 MHz, CDCl3): δ 7.95 (d, J = 13.5 Hz, 1H), 7.57 (d,
J = 14.0 Hz, 1H), 7.27 (t, J = 4.5 Hz, 1H), 6.83 (d, J = 7.5 Hz,
1H), 6.68 (dd, J = 8.0 Hz, J = 4.0 Hz, 1H), 6.63 (t, J = 4.0 Hz, 1H),
3.33–3.29 (m, 4H), 2.06–2.02 (m, 4H).
4.1.4.5. (E)-1-methyl-5-(2-nitrovinyl)-1H-imidazole (31e). Yield: 60%;
1H NMR (500 MHz, CDCl3) δ: 7.94 (d, J = 13.6 Hz, 1H), 7.67 (s,
1H), 7.63 (s, 1H), 7.53 (d, J = 13.6 Hz, 1H), 3.80 (s, 3H).
4.1.2.3. (E)-1-(2-(2-nitrovinyl)phenyl)pyrrolidine (16c). Yield: 87%.
NMR was confirmed by referring to the corresponding previously
reported spectrum.[40]
4.1.5. General Procedure C: Synthesis of 6-methyl-2-phenylindole (32)
Procedures were previously reported method.[30]
4.1.18.-4.1.21. 17–20
To a 100-mL round bottom flask was added 6-methylindole (1 g,
7.62 mmol, 1 eq.), phenylboronic acid (1.858 g, 15.24 mmol, 2 eq.),
palladium (ii) acetate (86 mg, 0.381 mmol, 5 mol%), and acetic acid
(12 mL). The atmosphere within the reaction flask was evacuated under
vacuum, then backfilled with oxygen gas. Oxygen flushing was con-
ducted 4X. The reaction mixture was stirred at room temperature under
moderately high pressure of oxygen gas for 23 h. Following reaction
completion, the mixture was concentrated under reduced pressure, di-
luted with ethyl acetate, then filtered through a celite pad. Collected
filtrate was concentrated under reduced pressure, and the resulting
organic resin was purified by silica gel flash chromatography using
ethyl acetate/hexanes eluent to yield the desired product. Yield: 68%
The following intermediates were prepared by previously reported
general procedures: 17–18; [41] 19; [42] 20. [19]
4.1.3. (E)-4-(4-(2-nitrovinyl)phenyl)morpholine (21)
The intermediate was prepared from 4-morpholinobenzaldehyde
using General Procedure A. Yield: 44%; 1H NMR (500 MHz, (CD3)2SO):
δ 8.06 (d, J = 13.5 Hz, 1H), 8.03 (d, J = 13.5 Hz, 1H), 7.20 (d,
J = 9.0 Hz, 2H), 7.00 (d, J = 8.0 Hz, 2H), 3.74–3.71 (m, 4H),
3.34–3.30 (m, 4H).
4.1.22.-4.1.30. 22–30
The following intermediates were prepared by previously reported
general procedures: 22; [43] 23–24; [37] 25; [39] 26; [36] 27; [44,45] 28;
[19]
29; [46] 30. [47]
4.1.6. General Procedure D: C3 alkylation of 2-phenylindoles with
nitrovinyl intermediates (33–70)
4.1.4. General Procedure B: Two-step syntheses of thiazole, oxazole, and N-
methylimidazole nitrovinyl intermediates (31a-31e)
To a 100-mL round bottom flask containing 2-phenyl-1H-indole
(1.0 mmol, 1 eq.), nitrovinyl building block (1.5 mmol, 1.5 eq.) and
CF3COONH4 (0.5 mmol) were dissolved with 10% aq. EtOH (10 mL).
The reaction mixture was refluxed at 105 °C for 12–18 h. After reaction
completion, the mixture was concentrated under reduced pressure to
remove EtOH, diluted with ethyl acetate, then washed with water and
brine. The aqueous phase was extracted 3X with ethyl acetate, and the
combined organic extracts were dried with Na2SO4 then concentrated
under reduced pressure. The crude organic resin was purified by silica
gel flash chromatography using ethyl acetate/hexanes eluent to obtain
the desired conjugate product.
Procedures used for both steps were derived from a previously re-
ported method.[29]
To a solution of aromatic aldehyde (5.3 mmol, 1 eq.) in tBuOH:THF
(1:1; 6 mL) was added nitromethane (8.48 mmol, 1.6 eq.). The reaction
mixture was stirred at 0 °C, then potassium tert-butoxide (0.32 mmol,
6 mol%) was added directly. Following 2–5 h of stirring at 0 °C, the
reaction mixture was concentrated under reduced pressure to remove
tBuOH, diluted with ethyl acetate, then washed with water and brine.
The aqueous phase was extracted 3X with ethyl acetate, and the com-
bined organic extracts were dried with sodium sulfate, then con-
centrated under reduced pressure. The nitroalcohol intermediate ob-
tained from workup was used for the next step without further
purification.
4.1.6.1. 3-(2-nitro-1-phenylethyl)-2-phenyl-1H-indole (33). The final
compound was prepared using commercially available 2-phenylindole
and β-nitrostyrene. Yield: 92%. NMR was previously reported.[19]
To a 100-mL round bottom flask containing the nitroalcohol inter-
mediate (4.25 mmol, 1 eq.) and 4-DMAP (0.21 mmol, 5 mol%) was
added anhydrous dichloromethane (20 mL). The mixture was stirred
under argon at room temp. Acetic anhydride (4.68 mmol, 1.1 eq.) was
added dropwise to the stirring mixture, and the resulting reaction
mixture continued stirring under argon at room temperature. Following
2–5 h of room-temperature stirring, the reaction mixture was quenched
with saturated NaHCO3 solution, then washed with water and brine.
The aqueous phase was extracted 3X with dichloromethane, and the
combined organic extracts were dried with sodium sulfate, then con-
centrated under reduced pressure. The crude organic resin was purified
by silica gel flash chromatography using ethyl acetate/hexanes eluent
to obtain the desired nitrovinyl product.
4.1.6.2. 3-(1-nitro-3-phenylpropan-2-yl)-2-phenyl-1H-indole (34). The
final compound was prepared using commercially available 2-
phenylindole and 1. Yield: 52%; 1H NMR (500 MHz, CDCl3): δ 7.96
(s, 1H), 7.76 (d, J = 7.5 Hz, 1H), 7.39 (d, J = 8.0 Hz, 1H), 7.35–7.30
(m, 3H), 7.25 (td, J = 7.0, 1.0 Hz, 1H), 7.21 (td, J = 7.0, 1.0 Hz, 1H),
7.14–7.10 (m, 3H), 6.97 (dd, J = 8.0, 2.0 Hz, 2H), 6.89 (dd, J = 8.0,
2.0 Hz, 2H), 4.90–4.81 (m,2H), 4.09–4.03 (m, 1H), 3.31 (dd, J = 13.5,
10.0 Hz, 1H), 3.09 (dd, J = 13.5, 10.0 Hz, 1H).
4.1.6.3. 3-(1-cyclohexyl-2-nitroethyl)-2-phenyl-1H-indole (35). The final
compound was prepared using commercially available 2-phenylindole
and 2. Yield: 72%; 1H NMR (500 MHz, CDCl3): δ 8.02 (s, 1H), 7.63 (d,
J = 8.0 Hz, 1H), 7.50–7.42 (m, 5H), 7.37 (d, J = 8.0 Hz, 1H), 7.21 (t,
J = 7.5 Hz, 2H), 7.14 (t, J = 7.5 Hz, 2H), 4.96–4.86 (m, 2H), 3.66–3.61
(m, 1H), 1.96–1.88 (m, 1H), 1.83–1.72 (m, 2H), 1.59–1.47 (m, 4H),
1.27–1.18 (m, 1H), 1.11–1.04 (m, 3H), 0.79–0.71 (m, 1H). HRMS Calcd
for C22H24N2O2 [M]+ 348.1838, found 348.1836
4.1.4.1. (E)-2-(2-nitrovinyl)thiazole (31a). Yield: 58%; 1H NMR
(500 MHz, CDCl3) δ: 8.07 (d, J = 13.4 Hz, 1H), 8.05 (d, J = 3.1 Hz,
1H), 7.90 (d, J = 13.4 Hz, 1H), 7.64 (d, J = 3.1 Hz, 1H).
4.1.4.2. (E)-2-(2-nitrovinyl)oxazole (31b). Yield: 32%; 1H NMR
(500 MHz, CDCl3) δ: 7.84 – 7.77 (m, 3H), 7.40 (s, 1H).
4.1.6.4. 3-(2-nitro-1-(p-tolyl)ethyl)-2-phenyl-1H-indole (36). The final
compound was prepared using commercially available 2-phenylindole
and 3. Yield: 85%; 1H NMR (500 MHz, CDCl3): δ 8.15 (s, 1H), 7.52 (d,
J = 7.5 Hz, 1H), 7.47–7.42 (m, 5H), 7.39 (d, J = 7.5 Hz, 1H),
7.25–7.19 (m, 3H), 7.12–7.09 (m, 3H), 5.27 (t, J = 8.5 Hz, 1H),
4.1.4.3. (E)-1-methyl-2-(2-nitrovinyl)-1H-imidazole (31c). Yield: 32%;
1H NMR (500 MHz, CDCl3) δ: 7.92 (d, J = 12.9 Hz, 1H), 7.84 (d,
J = 13.0 Hz, 1H), 7.26 – 7.24 (m, 1H), 7.14 – 7.10 (m, 1H), 3.83 (s,
7

P.C. Schaffer, et al.
Bioorganic&MedicinalChemistry28(2020)115727
5.18–5.09 (m, 2H), 2.30 (s, 3H). HRMS Calcd for C23H20N2O2 [M]+
356.1525, found 356.1552
J = 13.5, 10.5 Hz, 1H), 5.08 (dd, J = 10.0 Hz, 6.0 Hz, 1H). HRMS
Calcd for C22H17ClN2O2 [M]+ 376.0979, found 376.0988
4.1.6.5. 3-(2-nitro-1-(m-tolyl)ethyl)-2-phenyl-1H-indole (37). The final
compound was prepared using commercially available 2-phenylindole
and 4. Yield: 88%; 1H NMR (500 MHz, CDCl3): δ 8.18 (s, 1H), 7.58 (d,
J = 8.0 Hz, 1H), 7.49–7.46 (m, 5H), 7.25 (t, J = 7.5 Hz, 1H), 7.21 (t,
J = 7.0 Hz, 1H), 7.18–7.13 (m, 3H), 7.08 (d, J = 7.5 Hz, 1H), 5.33 (t,
J = 7.5 Hz, 1H), 5.22–5.13 (m, 2H). HRMS Calcd for C23H20N2O2 [M]+
356.1525, found 356.1533
4.1.6.13. 3-(1-(2,6-dichlorophenyl)-2-nitroethyl)-2-phenyl-1H-indole
(45). The final compound was prepared using commercially available
2-phenylindole and 12. Yield: 86%; 1H NMR (500 MHz, CDCl3): δ 7.99
(s, 1H), 7.38–7.27 (m, 7H), 7.18–7.14 (m, 3H), 7.06–7.01 (m, 2H), 5.15
(dd, J = 8.5, 7.0 Hz, 1H), 5.29–5.21 (m, 2H). HRMS Calcd for
C
22H16Cl2N2O2 [M]+ 410.0589, found 410.0555
4.1.6.14. 3-(1-(4-fluorophenyl)-2-nitroethyl)-2-phenyl-1H-indole
(46). The final compound was prepared using commercially available
2-phenyl indole and 13. NMR was previously reported.[24]
4.1.6.6. 3-(2-nitro-1-(o-tolyl)ethyl)-2-phenyl-1H-indoleindole (38). The
final compound was prepared using commercially available 2-
phenylindole and 5. Yield: 74%; 1H NMR (500 MHz, CDCl3): δ 8.13
(s, 1H), 7.66 (d, J = 8.0 Hz, 1H), 7.52 (d, J = 8.0 Hz, 1H), 7.47–7.42
(m, 3H), 7.38–7.36 (m, 3H), 7.21 (t, J = 7.5 Hz, 1H), 7.14–7.11 (m,
4H), 5.42 (t, J = 8.0 Hz, 1H), 5.19 (dd, J = 13.5, 8.5 Hz, 1H), 5.06 (dd,
J = 13.5, 8.5 Hz, 1H), 2.14 (s, 3H). HRMS Calcd for C23H20N2O2 [M]+
356.1525, found 356.1514
4.1.6.15. 3-(1-(3-fluorophenyl)-2-nitroethyl)-2-phenyl-1H-indole
(47). The final compound was prepared using commercially available
2-phenyl indole and 14. NMR was previously reported.[24]
4.1.6.16. 3-(1-(2-fluorophenyl)-2-nitroethyl)-2-phenyl-1H-indole
(48). The final compound was prepared using commercially available
2-phenyl indole and 15. NMR was previously reported.[24]
4.1.6.7. 3-(1-(4-methoxyphenyl)-2-nitroethyl)-2-phenyl-1H-indole
(39). The final compound was prepared using commercially available
2-phenylindole and 6. Yield: 86%; 1H NMR (500 MHz, CDCl3): δ 8.14 (s,
1H), 7.54 (d, J = 8.0 Hz, 1H), 7.48–7.39 (m, 6H), 7.27–7.20 (m, 3H),
7.12 (dd, J = 7.5, 1.0 Hz, 1H), 6.83 (dt, J = 8.5, 3.5 Hz, 2H), 5.26 (t,
J = 8.0 Hz, 1H), 5.17–5.07 (m, 2H), 3.77 (s, 3H). HRMS Calcd for
4.1.6.17. 3-(2-nitro-1-(4-(pyrrolidin-1-yl)phenyl)ethyl)-2-phenyl-1H-
indole (49). The final compound was prepared using commercially
available 2-phenylindole and 16a. Yield: 72%; 1H NMR (500 MHz,
CDCl3): δ 8.07 (s, 1H), 7.58 (d, J = 8.0 Hz, 1H), 7.43–7.37 (m, 5H),
7.34 (d, J = 8.0 Hz, 1H), 7.20–7.16 (m, 3H), 7.09 (t, J = 8.0 Hz, 1H),
6.48–6.45 (m, 2H), 5.20 (t, J = 8.5 Hz, 1H), 5.11–5.05 (m, 2H),
3.23–3.20 (m, 4H), 1.96–1.93 (m, 4H). HRMS Calcd for C26H25N3O2
[M]+ 411.1947, found 411.1916
C
23H20N2O3 [M]+ 372.1474, found 374.1487
4.1.6.8. 3-(1-(3-methoxyphenyl)-2-nitroethyl)-2-phenyl-1H-indole
(40). The final compound was prepared using commercially available
2-phenylindole and 7. Yield: 81%; 1H NMR (500 MHz, CDCl3): δ 8.16 (s,
1H), 7.55 (d, J = 8.0 Hz, 1H), 7.45–7.39 (m, 5H), 7.36 (d, J = 8.0 Hz,
1H), 7.24–7.18 (m, 2H), 7.11 (t, J = 8.0 Hz, 1H), 6.92 (dd, J = 8.0,
2.0 Hz, 1H), 6.87 (t, J = 2.0 Hz, 1H), 6.76 (dd, J = 8.5, 2.5 Hz, 1H),
5.28 (t, J = 8.0 Hz, 1H), 5.18–5.09 (m, 2H), 3.71 (s, 3H). HRMS Calcd
for C23H20N2O3 [M]+ 372.1474, found 374.1461
4.1.6.18. 3-(2-nitro-1-(3-(pyrrolidin-1-yl)phenyl)ethyl)-2-phenyl-1H-
indole (50). The final compound was prepared using commercially
available 2-phenylindole and 16b. Yield: 70%; 1H NMR (500 MHz,
CDCl3): δ 8.11 (s, 1H), 7.64 (d, J = 8.1 Hz, 1H), 7.50 – 7.38 (m, 5H),
7.36 (d, J = 8.1 Hz, 1H), 7.22 – 7.17 (m, 1H), 7.16 – 7.09 (m, 2H), 6.64
(d, J = 7.7 Hz, 1H), 6.54 (s, 1H), 6.44 (d, J = 8.2 Hz, 1H), 5.24 (t,
J = 7.9 Hz, 1H), 5.20 – 5.10 (m, 2H), 3.22 – 3.12 (m, 4H), 1.98 – 1.90
(m, 4H). HRMS Calcd for C26H25N3O2 [M]+ 411.1947, found 411.1936
4.1.6.9. 3-(1-(2-methoxyphenyl)-2-nitroethyl)-2-phenyl-1H-indole
(41). The final compound was prepared using commercially available
2-phenylindole and 8. Yield: 87%; 1H NMR (500 MHz, CDCl3): δ 8.13 (s,
1H), 7.65 (d, J = 7.5 Hz, 1H), 7.45–7.34 (m, 7H), 7.26–7.20 (m, 2H),
7.13 (t, J = 8.0 Hz, 1H), 6.89 (d, J = 7.5 Hz, 1H), 6.84 (t, J = 8.0 Hz,
1H), 5.6 (dd, J = 6.5, 2.5 Hz, 1H), 5.18–5.14 (m, 2H), 3.77 (s, 3H).
HRMS Calcd for C23H20N2O3 [M]+ 372.1474, found 374.1497
4.1.6.19. 3-(2-nitro-1-(2-(pyrrolidin-1-yl)phenyl)ethyl)-2-phenyl-1H-
indole (51). The final compound was prepared using commercially
1
available 2-phenylindole and 16c. H NMR (500 MHz, CDCl3): δ 8.20
(s, 1H), 7.58 (d, J = 8.0 Hz, 1H), 7.46 – 7.37 (m, 6H), 7.35 (dd,
J = 7.8, 1.5 Hz, 1H), 7.26 – 7.17 (m, 3H), 7.13 (t, J = 7.6 Hz, 1H), 6.89
(td, J = 7.5, 1.5 Hz, 1H), 5.65 (dd, J = 10.6, 4.5 Hz, 1H), 5.42 (dd,
J = 12.6, 4.5 Hz, 1H), 5.11 (dd, J = 12.7, 10.6 Hz, 1H), 3.36 – 3.27 (m,
2H), 2.92 – 2.84 (m, 2H), 1.95 – 1.86 (m, 2H), 1.86 – 1.75 (m, 2H).
Yield: 47%; HRMS Calcd for C26H25N3O2 [M]+ 411.1947, found
411.1908
4.1.6.10. 3-(1-(4-chlorophenyl)-2-nitroethyl)-2-phenyl-1H-indole
(42). The final compound was prepared using commercially available
2-phenylindole and 9. Yield: 82%; 1H NMR (500 MHz, CDCl3): δ 8.17 (s,
1H), 7.49–7.39 (m, 7H), 7.27–7.25 (m, 4H), 7.23 (dd, J = 7.5, 1.0 Hz,
1H), 7.12 (dd, J = 7.5, 1.0 Hz, 1H), 5.28 (t, J = 8.0 Hz, 1H), 5.16 (dd,
J = 12.0, 7.0 Hz, 1H), 5.09 (dd, J = 13.5, 10.5 Hz, 1H). HRMS Calcd
for C22H17ClN2O2 [M]+ 376.0979, found 376.0906
4.1.6.20. 3-(2-nitro-1-(4-(trifluoromethyl)phenyl)ethyl)-2-phenyl-1H-
indole (52). The final compound was prepared using commercially
available 2-phenylindole and 17. Yield: 84%; 1H NMR (500 MHz,
CDCl3): δ 8.20 (s, 1H), 7.56 (s, 1H), 7.54 (s, 1H), 7.50–7.41 (m, 9H),
7.24 (t, J = 7.5 Hz, 1H), 7.14 (t, J = 7.0 Hz, 1H), 5.37 (t, J = 8.0 Hz,
1H), 5.22 (dd, J = 12.5, 7.5 Hz, 1H), 5.13 (dd, J = 12.5, 7.5 Hz, 1H).
HRMS Calcd for C22H17F3N2O2 [M]+ 410.1242, found 410.1284
4.1.6.11. 3-(1-(3-chlorophenyl)-2-nitroethyl)-2-phenyl-1H-indole
(43). The final compound was prepared using commercially available
2-phenylindole and 10. Yield: 84%;1H NMR (500 MHz, CDCl3): δ 8.15
(s, 1H), 7.48 (d, J = 7.5 Hz, 1H), 7.46–7.37 (m, 5H), 7.35 (d,
J = 7.54 Hz, 1H), 7.28 (s, 1H), 7.21–7.18 (m, 4H), 7.11 (dd,
J = 7.5, 1.0 Hz, 1H), 5.27 (t, J = 8.0 Hz, 1H), 5.15–5.04 (m, 2H).
HRMS Calcd for C22H17ClN2O2 [M]+ 376.0979, found 376.0992
4.1.6.21. 3-(2-nitro-1-(3-(trifluoromethyl)phenyl)ethyl)-2-phenyl-1H-
indole (53). The final compound was prepared using commercially
available 2-phenylindole and 18. Yield: 80%; 1H NMR (500 MHz,
CDCl3): δ 8.15 (s, 1H), 7.56 (s, 1H), 7.48 (t, J = 8.0 Hz, 3H), 7.45–7.41
(m, 3H), 7.37–7.34 (m, 4H), 7.20 (t, J = 8.0 Hz, 1H), 7.12 (t,
J = 8.0 Hz, 1H), 5.34 (t, J = 7.5 Hz, 1H), 5.18–5.08 (m, 2H).).
HRMS Calcd for C23H17F3N2O2 [M]+ 410.1242, found 410.1246
4.1.6.12. 3-(1-(2-chlorophenyl)-2-nitroethyl)-2-phenyl-1H-indole
(44). The final compound was prepared using commercially available
2-phenylindole and 11. Yield: 73%; 1H NMR (500 MHz, CDCl3): δ 8.20
(s, 1H), 7.69 (d, J = 8.0 Hz, 1H), 7.59 (d, J = 8.0 Hz, 1H), 7.46–7.36
(m, 7H), 7.26–7.16 (m, 4H), 5.68 (dd, J = 10.0, 6.0 Hz, 1H), 5.23 (dd,
8

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4.1.6.22. 3-(2-nitro-1-(2-(trifluoromethyl)phenyl)ethyl)-2-phenyl-1H-
indole (54). The final compound was prepared using commercially
available 2-phenylindole and 19. Yield: 84%; 1H NMR (500 MHz,
CDCl3): δ 8.16 (s, 1H), 7.84 (d, J = 8.0 Hz, 1H), 7.76 (d, J = 8.0 Hz,
1H), 7.67 (dd, J = 7.5, 1.0 Hz, 1H), 7.43–7.34 (m, 7H), 7.27–7.23 (m,
2H), 7.21 (td, J = 7.5, 1.5 Hz, 1H), 5.76 (dd, J = 10.0, 4.5 Hz, 1H),
5.34 (dd, J = 13.5, 10.5 Hz, 1H), 4.90 (dd, J = 13.5, 6.0 Hz, 1H) HRMS
Calcd for C23H17F3N2O2 [M]+ 410.1242, found 410.1211
(s, 1H), 7.52 (d, J = 8.0 Hz, 1H), 7.49–7.41 (m, 5H), 7.38 (d,
J = 8.0 Hz, 1H), 7.23 (dd, J = 5.0, 1.0 Hz, 1H), 7.19 (dd, , J = 5.0,
1.0 Hz, 1H), 7.11 (t, J = 8.0 Hz, 1H), 6.92 (d, J = 5.0 Hz, 2H), 5.49 (t,
J = 8.5 Hz, 1H), 5.18 (dd, J = 12.5, 7.5 Hz, 1H), 5.08 (dd, J = 12.5,
8.0 Hz, 1H). HRMS Calcd for C20H16N2O2S [M]+ 348.0932, found
348.0958
4.1.6.30. 3-(2-nitro-1-(thiophen-3-yl)ethyl)-2-phenyl-1H-indole
(62). The final compound was prepared using commercially available
2-phenylindole and 27. Yield: 80%; 1H NMR (CDCl3, 500 MHz): δ 8.11
(s, 1H), 7.46–7.39 (m, 6H), 7.37 (d, J = 8.5 Hz, 1H), 7.24 (t,
J = 5.0 Hz, 1H), 7.20 (td, J = 7.0 Hz, 1.0 Hz, 1H), 7.11–7.07 (m,
2H), 6.95 (dd, J = 5.0, 1.0 Hz, 1H), 5.32 (t, J = 8.0 Hz, 1H), 5.15 (dd,
J = 12.5, 7.5 Hz, 1H), 5.04 (dd, J = 12.5, 8.0 Hz, 1H). HRMS Calcd for
4.1.6.23. 3-(2-nitro-1-(4-(piperidin-1-yl)phenyl)ethyl)-2-phenyl-1H-indole
(55). The final compound was prepared using commercially available
2-phenylindole and 20. Yield: 77%; 1H NMR (500 MHz, CDCl3): δ 8.14
(s, 1H), 7.54 (d, J = 7.5 Hz, 1H), 7.46–7.39 (m, 5H), 7.38 (d,
J = 8.0 Hz, 1H), 7.23–7.18 (m, 3H), 7.11 (t, J = 7.5 Hz, 1H), 6.84
(d, J = 9.0 Hz, 2H), 5.23 (t, J = 8.0 Hz, 1H), 5.15–5.07 (m, 2H), 3.11
(t, J = 5.5 Hz, 4H), 1.71–1.64 (m , 4H), 1.58–1.52 (m, 2H). HRMS
Calcd for C27H27N3O2 [M]+ 425.2103, found 425.2126
C
20H16N2O2S [M]+ 348.0932, found 348.0957
4.1.6.31. 3-(1-(furan-2-yl)-2-nitroethyl)-2-phenyl-1H-indole (63). The
final compound was prepared using commercially available 2-
1
4.1.6.24. 4-(4-(2-nitro-1-(2-phenyl-1H-indol-3-yl)ethyl)phenyl)
morpholine (56). The final compound was prepared using commercially
available 2-phenylindole and 21. Yield: 67%; 1H NMR (500 MHz,
CDCl3): δ 8.18 (s, 1H), 7.53 (dd, J = 8.0, 2.5 Hz, 1H), 7.50 – 7.37 (m,
6H), 7.25 – 7.19 (m, 3H), 7.11 (td, J = 7.8, 2.4 Hz, 1H), 6.92 – 6.81 (m,
2H), 5.28 – 5.22 (m, 1H), 5.18 – 5.06 (m, 2H), 3.91 – 3.79 (m, 4H), 3.18
– 3.09 (m, 4H). HRMS Calcd for C26H26N3O3 [M+H]+ 428.1916, found
428.1951
phenylindole and 28. Yield: 89%; H NMR (CDCl3, 500 MHz): δ 8.16
(s, 1H), 7.56 (d, J = 8.0 Hz, 1H), 7.54–7.48 (m, 4H), 7.45 (tt, J = 7.0,
J = 3.0 Hz 1H), 7.39 (d, J = 8.0 Hz, 1H), 7.37 (d, J = 2.0 Hz, 1H), 7.22
(t, J = 7.5 Hz, 1.0 Hz, 1H), 7.12 (t, J = 7.5 Hz, 1H), 6.32–6.28 (m, 1H),
6.14 (d, J = 3.5 Hz, 1H), 5.36 (t, J = 7.5 Hz, 1H), 5.23 (dd, J = 12.5,
8.0 Hz, 1H), 4.94 (dd, J = 12.5, 8.0 Hz, 1H). HRMS Calcd for
C
20H16N2O3 [M]+ 332.1161, found 332.1166
4.1.6.32. 3-(1-(furan-3-yl)-2-nitroethyl)-2-phenyl-1H-indole (64). The
4.1.6.25. 3-(1-(4-(tert-butyl)phenyl)-2-nitroethyl)-2-phenyl-1H-indole
(57). The final compound was prepared using commercially available
2-phenylindole and 22. Yield: 81%; 1H NMR (500 MHz, CDCl3): δ 8.13
(s, 1H), 7.56 (d, J = 7.0 Hz, 1H), 7.46–7.38 (m, 6H), 7.30 (dt, J = 8.0,
2.0 Hz, 2H), 7.26 (d, J = 7.0 Hz, 2H), 7.21 (td, J = 7.5, 1.5 Hz, 1H),
7.12 (td, J = 8.0, 1.0 Hz, 1H), 5.29 (d, J = 7.0 Hz, 1H), 5.19–5.11 (m,
2H), 1.28 (s, 9H). HRMS Calcd for C26H26N2O2 [M]+ 398.1994, found
398.1972
final compound was prepared using commercially available 2-
1
phenylindole and 29. Yield: 74%; H NMR (CDCl3, 500 MHz): δ 8.09
(s, 1H), 7.46 (d, J = 7.5 Hz, 1H), 7.43–7.38 (m, 5H), 7.35 (d,
J = 8.0 Hz, 1H), 7.31 (t, J = 1.5 Hz, 1H), 7.21 (d, J = 1.0 Hz, 1H),
7.17 (t, J = 7.5 Hz, 1H), 7.07 (t, J = 7.5 Hz, 1H), 6.25 (d, J = 1.0 Hz,
1H), 5.11 (t, J = 7.5, 1.0 Hz, 1H), 5.03 (dd, J = 12.5, 8.5 Hz, 1H), 4.89
(dd, J = 12.0, 8.0 Hz, 1H). HRMS Calcd for C20H16N2O3 [M]+
332.1161, found 332.1176
4.1.6.26. 3-(1-([1,1′-biphenyl]-4-yl)-2-nitroethyl)-2-phenyl-1H-indole
(58). The final compound was prepared using commercially available
2-phenylindole and 23. Yield: 71%; 1H NMR (500 MHz, CDCl3): δ 8.18
(s, 1H), 7.59–7.51 (m, 5H), 7.50–7.39 (m, 10H), 7.33 (tt, J = 7.5,
1.5 Hz, 1H), 7.24 (t, J = 8.0 Hz, 1H), 7.14 (t, J = 8.0 Hz, 1H), 5.37 (t,
J = 8.0 Hz, 1H), 5.23 (dd, J = 12.0, 7.5 Hz, 1H), 5.17 (dd, J = 13.0,
8.0 Hz, 1H). HRMS Calcd for C28H22N2O2 [M]+ 418.1681, found
418.1693
4.1.6.33. 3-(2-nitro-1-(1H-pyrrol-2-yl)ethyl)-2-phenyl-1H-indole
(65). The final compound was prepared using commercially available
1
2-phenylindole and 30. Yield: 15%; H NMR (500 MHz, (CD3)2SO): δ
11.38 (s, 1H), 10.59 (s, 1H), 7.57–7.51 (m, 4H), 7.45 (d, J = 8.5 Hz,
2H), 7.37 (d, J = 8.5 Hz, 1H), 7.09 (t, J = 7.5 Hz, 1H), 6.95 (t, J = 7.5,
1.0 Hz, 1H), 6.64–6.60 (m, 1H), 5.95–5.91 (m, 1H), 5.91 (s, 1H), 5.31
(dd, J = 16.0, 11.0 Hz, 1H), 5.22–5.18 (m, 2H) HRMS Calcd for
C
20H17N3O2 [M]+ 331.1321, found 331.1332
4.1.6.27. 3-(1-(naphthalen-2-yl)-2-nitroethyl)-2-phenyl-1H-indole
4.1.6.34. 2-(2-nitro-1-(2-phenyl-1H-indol-3-yl)ethyl)thiazole (66a). The
final compound was prepared using commercially available 2-
phenylindole and 31a. Yield: 82%; 1H NMR (399 MHz, CDCl3) δ:
8.24 (s, 1H), 7.74 (d, J = 3.2 Hz, 1H), 7.60 – 7.45 (m, 5H), 7.40 (t,
J = 8.3 Hz, 2H), 7.26 – 7.19 (m, 2H), 7.08 (t, J = 7.6 Hz, 1H), 5.69 (dd,
J = 13.1, 7.5 Hz, 1H), 5.61 (t, J = 7.3 Hz, 1H), 5.03 (dd, J = 13.1,
7.1 Hz, 1H). HRMS Calcd for C19H16N3O2S [M+H]+ 350.0958, found
350.0930
(59). The final compound was prepared using commercially available
2-phenylindole and 24. Yield: 76%; 1H NMR (500 MHz, CDCl3): δ 8.17
(bs, 1H), 7.97(d, J = 8.5 Hz, 1H), 7.87 (d, J = 8.5 Hz, 1H), 7.76–7.69
(m, 3H), 7.48–7.32 (m, 8H), 7.23 (t, J = 7.5, 1.0 Hz, 2H), 7.17(t,
J = 7.5, 1.0 Hz, 1H), 6.06 (dd, J = 9.0, 6.0 Hz, 1H), 5.35 (dd, J = 13.0,
9.5 Hz, 1H), 5.20 (dd, J = 12.5, 6.0 Hz, 1H). HRMS Calcd for
C
26H20N2O2 [M]+ 392.1525, found 392.1511
4.1.6.28. 3-(1-(naphthalen-1-yl)-2-nitroethyl)-2-phenyl-1H-indole
(60). The final compound was prepared using commercially available
2-phenylindole and 25. Yield: 72%; 1H NMR (500 MHz, CDCl3): δ 8.20
(s, 1H), 7.81–7.74 (m, 4H), 7.56 (d, J = 8.0 Hz, 1H), 7.49–7.41 (m,
9H), 7.23 (t, J = 8.0, 1.0 Hz, 1H), 7.11 (t, J = 8.0, 1.0 Hz, 1H), 5.48 (t,
J = 8.0 Hz, 1H), 5.32 (dd, J = 13.0, 7.5 Hz, 1H), 5.22 (dd, J = 13.0,
8.0 Hz, 1H). HRMS Calcd for C26H20N2O2 [M]+ 392.1525, found
392.1559
4.1.6.35. 2-(1-(6-methyl-2-phenyl-1H-indol-3-yl)-2-nitroethyl)thiazole
(66b). The final compound was prepared using 6-methyl-2-
phenylindole, 32, and 31a. Yield: 80%; 1H NMR (500 MHz, CDCl3) δ:
8.10 (s, 1H), 7.72 (d, J = 3.3 Hz, 1H), 7.58 – 7.54 (m, 2H), 7.54 – 7.49
(m, 2H), 7.49 – 7.43 (m, 1H), 7.28 – 7.26 (m, 1H), 7.23 (d, J = 3.3 Hz,
1H), 7.19 (s, 1H), 6.93 – 6.89 (m, 1H), 5.67 (dd, J = 13.1, 7.5 Hz, 1H),
5.58 (t, J = 7.4 Hz, 1H), 5.02 (dd, J = 13.2, 7.4 Hz, 1H), 2.44 (s, 3H).
HRMS Calcd for C20H18N3O2S [M+H]+ 364.1114, found 364.1137
4.1.6.29. 3-(2-nitro-1-(thiophen-2-yl)ethyl)-2-phenyl-1H-indole
4.1.6.36. 2-(2-nitro-1-(2-phenyl-1H-indol-3-yl)ethyl)oxazole (67a). The
final compound was prepared using commercially available 2-
phenylindole and 31b. Yield: 85%; 1H NMR (500 MHz, (CD3)2SO) δ:
(61). The final compound was prepared using commercially available
2-phenylindole and 26. Yield: 82%; 1H NMR (500 MHz, CDCl3): δ 8.14
9

P.C. Schaffer, et al.
Bioorganic&MedicinalChemistry28(2020)115727
11.58 (s, 1H), 7.99 (d, J = 0.8 Hz, 1H), 7.72 – 7.68 (m, 2H), 7.59 (t,
J = 7.7 Hz, 2H), 7.52 – 7.46 (m, 1H), 7.43 (d, J = 8.0 Hz, 1H), 7.39 –
7.35 (m, 1H), 7.19 (d, J = 0.8 Hz, 1H), 7.10 (ddd, J = 8.1, 7.2, 1.2 Hz,
1H), 6.96 (ddd, J = 8.3, 7.1, 1.1 Hz, 1H), 5.62 – 5.53 (m, 1H), 5.35 –
5.26 (m, 2H). HRMS Calcd for C19H16N3O3 [M+H]+ 334.1186, found
334.1162
added to cells according to the manufacturer’s directions (DiscoveRx®),
and cells were incubated for 60 min at room temperature in the dark.
cAMP solution A was added according to the manufacturer’s directions
(DiscoveRx®), and cells were incubated for 3 h at room temperature in
the dark before chemiluminescence was measured on a Cytation 5 plate
reader (BioTek, Winooski, VT) (top read, gain 200, integration time
10,000 ms).
4.1.6.37. 2-(1-(6-methyl-2-phenyl-1H-indol-3-yl)-2-nitroethyl)oxazole
(67b). The final compound was prepared using 6-methyl-2-
phenylindole, 32, and 31b. Yield: 72%; 1H NMR (500 MHz,
(CD3)2SO) δ: 11.42 (s, 1H), 8.00 (d, J = 0.8 Hz, 1H), 7.73 – 7.67 (m,
2H), 7.62 – 7.56 (m, 2H), 7.52 – 7.46 (m, 1H), 7.32 (d, J = 8.2 Hz, 1H),
7.20 (d, J = 0.8 Hz, 1H), 7.16 (s, 1H), 6.83 – 6.79 (m, 1H), 5.60 – 5.53
(m, 1H), 5.33 – 5.26 (m, 2H), 2.36 (s, 3H). HRMS Calcd for C20H18N3O3
[M+H]+ 348.1343, found 364.1341
4.2.3. PathHunter® β-arrestin-2 assay
β-arrestin-2 recruitment was determined using the PathHunter®
assay (DiscoveRx®) with hCB1R- CHO-K1 cells. The cells (20,000 cells/
well in low-volume 96 well plates) were incubated overnight in Opti-
MEM (Invitrogen) containing 1% FBS at 37 °C under humidified 5%
CO2-95% air. Following this, cells were incubated in this medium at
37 °C with test compounds (0.10 nM–10 µM, final concentrations.) for
30 min, then treated with EC20 of CP55,940, followed by an additional
90 min of incubation at 37 °C. Detection solution was then added ac-
cording to the manufacturer’s directions (DiscoveRx®), and the cells
were incubated for 60 min at room temperature. Chemiluminescence
was then measured on a Cytation 5 plate reader (top read, gain 200,
integration time 10,000 ms).
4.1.6.38. 3-(1-(1-methyl-1H-imidazol-2-yl)-2-nitroethyl)-2-phenyl-1H-
indole (68). The final compound was prepared using commercially
available 2-phenylindole and 31c. Yield: 17%; 1H NMR (500 MHz,
CDCl3) δ: 8.24 (s, 1H), 7.58 – 7.50 (m, 5H), 7.50 – 7.45 (m, 1H), 7.36
(d, J = 8.1 Hz, 1H), 7.22 – 7.16 (m, 1H), 7.05 (t, J = 7.6 Hz, 1H), 6.96
– 6.92 (m, 1H), 6.66 – 6.62 (m, 1H), 5.81 (dd, J = 13.4, 9.1 Hz, 1H),
5.31 (dd, J = 9.1, 5.9 Hz, 1H), 4.99 (dd, J = 13.4, 5.9 Hz, 1H), 3.04 (s,
3H). HRMS Calcd for C20H19N4O2 [M+H]+ 347.1503, found 347.1481
4.2.4. Aqueous solubility assay
The kinetic water solubility of each tested compound was measured
by using a turbidimetric assay as performed by Cyprotex (Watertown,
MA). Each test compound was prepared as a 100-X concentrated stock
solution in DMSO from which serial dilutions were performed to yield
eight solutions with final test-compound concentrations of 1.6, 3.21,
6.25, 12.5, 25, 50, 100, and 200 µM. Each test-compound solution was
introduced into a 96-well plate, then diluted 100-fold with PBS buffer
(pH 7.4) and mixed. The solutions were incubated for two hours, and
absorbance was then measured at 540 nm. An absorbance value > 3X
standard deviation of the average blank absorption value was con-
sidered turbidity. The highest test compound concentration with no
sign of turbidity was indicative of the compound’s kinetic water solu-
bility.
4.1.6.39. 3-(1-(1-methyl-1H-imidazol-4-yl)-2-nitroethyl)-2-phenyl-1H-
indole (69). The final compound was prepared using commercially
available 2-phenylindole and 31d. Yield: 87%; 1H NMR (399 MHz,
CDCl3) δ: 8.18 (s, 1H), 7.60 (d, J = 8.0 Hz, 1H), 7.57 – 7.51 (m, 2H),
7.51 – 7.45 (m, 2H), 7.45 – 7.36 (m, 3H), 7.24 – 7.18 (m, 1H), 7.09 (t,
J = 7.6 Hz, 1H), 6.56 (s, 1H), 5.44 (dd, J = 12.5, 6.9 Hz, 1H), 5.29 (t,
J = 7.7 Hz, 1H), 5.01 (dd, J = 12.4, 8.6 Hz, 1H), 3.57 (s, 3H). HRMS
Calcd for C20H19N4O2 [M+H]+ 347.1503, found 347.1514
4.1.6.40. 3-(1-(1-methyl-1H-imidazol-5-yl)-2-nitroethyl)-2-phenyl-1H-
indole (70). The final compound was prepared using commercially
available 2-phenylindole and 31e. Yield: 68%; 1H NMR (399 MHz,
(CD3)2SO) δ: 11.50 (s, 1H), 7.69 – 7.55 (m, 5H), 7.50 (t, J = 7.1 Hz,
1H), 7.39 – 7.32 (m, 2H), 7.14 – 7.05 (m, 2H), 6.95 (t, J = 7.5 Hz, 1H),
5.56 (dd, J = 13.4, 9.2 Hz, 1H), 5.48 (dd, J = 13.6, 6.9 Hz, 1H), 5.15
(t, J = 8.1 Hz, 1H), 2.95 (s, 3H). HRMS Calcd for C20H19N4O2 [M+H]+
347.1503, found 347.1471
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
4.2. Activity profiling
Acknowledgements
4.2.1. Cell culture
This work was supported by grants from National Institutes of
Health EY024717 (to G.A.T.)
HitHunter® (cAMP) and PathHunter® (β-arrestin2) CHO-K1 cells
stably expressing hCB1R from DiscoveRx® (Eurofins, Fremont, CA)
were maintained between passage 5–35 at 37 °C under 5% CO2-95% air
in F-12 DMEM (Corning Cellgro, Manassas, VA) containing 10% FBS
and 1% penicillin–streptomycin with 800 µg/mL geneticin
(HitHunter®) or 800 µg/mL geneticin and 300 µg/mL hygromycin B
(PathHunter®). HitHunter® and PathHunter® hCB1R-CHO-K1 cells were
used for data presented in Table 1.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmc.2020.115727.
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