Discovery of G Protein-Biased Ligands against 5-HT7R

Article abstract of DOI:10.1021/acs.jmedchem.1c00110

There has been significant attention concerning the biased agonism of G protein-coupled receptors (GPCRs), and it has resulted in various pharmacological benefits. 5-HT7R belongs to a GPCR, and it is a promising pharmaceutical target for the treatment of neurodevelopmental and neuropsychiatric disorders. Based on our previous research, we synthesized a series of 6-chloro-2′-methoxy biphenyl derivatives 1, 2, and 3 with a variety of amine scaffolds. These compounds were evaluated for their binding affinities to 5-HTR subtypes and their functional selectivity toward the Gs protein and the β-arrestin signaling pathways of 5-HT7R. Among them, 2-(6-chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)-N-ethylethan-1-amine, 2b, was found to be a G-protein-biased ligand of 5-HT7R. In an in vivo study with Shank3 transgenic mice, the self-grooming behavior test was performed with 2b, which increased the duration of self-grooming. The experiments further suggested that 5-HT7R is associated with autism spectrum disorders (ASDs) and could be a therapeutic target for the treatment of stereotypy in ASDs.

Full text of DOI:10.1021/acs.jmedchem.1c00110

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Discovery of G Protein-Biased Ligands against 5‑HT7R
Jieon Lee, Rina Kwag, Soyeon Lee, Doyoung Kim, Jiwan Woo, Yakdol Cho, Hak Joong Kim,
Jeongjin Kim, Byungsun Jeon, and Hyunah Choo*
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ABSTRACT: There has been significant attention concerning the
biased agonism of G protein-coupled receptors (GPCRs), and it has
resulted in various pharmacological benefits. 5-HT7R belongs to a
GPCR, and it is a promising pharmaceutical target for the treatment
of neurodevelopmental and neuropsychiatric disorders. Based on our
previous research, we synthesized a series of 6-chloro-2′-methoxy
biphenyl derivatives 1, 2, and 3 with a variety of amine scaffolds.
These compounds were evaluated for their binding affinities to 5-
HTR subtypes and their functional selectivity toward the Gs protein
and the β-arrestin signaling pathways of 5-HT7R. Among them, 2-(6-
chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)-N-ethylethan-1-amine, 2b,
was found to be a G-protein-biased ligand of 5-HT7R. In an in vivo
study with Shank3 transgenic mice, the self-grooming behavior test
was performed with 2b, which increased the duration of self-
grooming. The experiments further suggested that 5-HT7R is associated with autism spectrum disorders (ASDs) and could be a
therapeutic target for the treatment of stereotypy in ASDs.
There are 14 distinct serotonin receptors (5-HTRs) encoded
in the human genome. Among the 5-HTR subtypes, the 5-HT7
receptor (5-HT7R) was the last to be identified, and it has been
shown to be highly expressed in the central nervous system
(CNS), for example, in the hypothalamus, hippocampus, and
cortex. 5-HT7R belongs to a family of GPCRs and binds
positively to adenylate cyclase (AC) through the activation of Gs
protein, resulting in an extracellular increase of cyclic adenosine
monophosphate (cAMP), and it also displays a high constitutive
AC activity.11−17 On the basis of the distribution of 5-HT7R in
the CNS, it has been proposed that it is involved in various
important functional roles, such as thermoregulation, circadian
rhythm, sleep, learning and memory, autism, cognition, and
schizophrenia.18 In addition, numerous studies have suggested
that the altered 5-HT system that includes abnormal levels of 5-
HT, morphological changes in the serotonergic fibers, and
decreased expression of 5-HTR might be a major marker of
abnormalities in autism spectrum disorders (ASDs).19,20 Recent
reports have provided evidence that 5-HT7R has an essential
role in regulating severe behavioral symptoms, which are
represented as autistic-like behaviors in animal models, such as
INTRODUCTION
■
G protein-coupled receptors (GPCRs) are a family of cell
surface receptors with seven highly conserved, transmembrane
helical proteins, and there are over 800 individual genes encoded
in the human genome.1−3 These receptors are expressed
extensively in various tissues, and they participate in multiple
physiological signal transductions through interactions with the
heterotrimeric G proteins that transfer the signals to canonical
transducer proteins, arrestins, kinases, ion channels, and
scaffolding proteins. Thus, they have been the pharmaceutical
targets of various drugs.4−7 Canonical GPCR signaling is
mediated via coupling to intracellular transducers, that is, G
proteins, which are formed by Gα, Gβ, and Gγ subunits.8 A ligand
binds to a GPCR and activates the G protein, which is followed
by the activation of the downstream signaling known as the G
protein signaling pathway. Then, GPCR recruits β-arrestin to
block or desensitize the activated signal. In addition to the
classical G protein signaling pathway mediated by G proteins, it
has been suggested recently that β-arrestin is able to initiate G
protein-independent cellular signals, including the activation of
various MAPKs, such as ERK,3,9 which is referred to as the β-
arrestin signaling pathway. Thus, there have been active efforts
to produce biased ligands in order to elucidate the complicated
mechanism of GPCRs and to develop novel GPCR drugs with
fewer side effects. These biased ligands can activate either the G
protein or the β-arrestin signaling pathway selectively, thereby
yielding the desired effects of drugs and blocking unwanted side
effects due to the stimulation of other signaling pathways.5,10
Received: January 21, 2021
Published: May 25, 2021
https://doi.org/10.1021/acs.jmedchem.1c00110
© 2021 American Chemical Society
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the Fmr1 KO mouse model of the fragile X syndrome and the
Mecp2 KO mouse model of the Rett syndrome. The 5-HT7R
agonist restored the long-term depression level in an Fmr1 KO
mouse to the level of wild type (WT), which is applicable to
ASDs; also, the treatment of 5-HT7R agonist improved the
behavioral impairments and cognition in Mecp2 KO mice.21−24
However, no studies have been conducted in which the 5-HT7R-
biased ligand was used in testing autism behavior.
the self-grooming behavior test that was conducted with Shank3
transgenic (TG) mice to investigate the association between 5-
HT7R and ASD.
RESULTS AND DISCUSSION
■
To discover Gs protein-biased ligands of 5-HT7R, we referred
three structurally similar agonists: well-known agonists AS-19
and E-55888, with a biaryl core and an ethyleneamine moiety,
and 1a with a biphenyl core and a methyleneamine moiety,
which was identified in our previous work as a potent and
selective agonist against 5-HT7R28 (Figure 1). According to our
study of the structure−activity relationship (SAR), compounds
with a 6-chloro-2′-methoxy biphenyl core showed good binding
affinities, so this core structure was retained in this study. We
planned to synthesize and biologically evaluate 6-chloro-2′-
methoxy-[1,1′-biphenyl]-3-yl-N-alkylmethanamine derivatives
1, 2-(6-chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)-N-alkylethan-
1-amine derivatives 2 and 3-(6-chloro-2′-methoxy-[1,1′-biphen-
yl]-3-yl)pyrrolidine derivatives 3 as shown in Figure 2, and the
introduction of secondary/tertiary amines with alkyl chains and
cyclic amines to the amine moiety followed by SAR study was
our main concern.
Although many potent agonists (E-55888,25 AS-19,26,27 and
1a28) and antagonists (SB-26997029) against 5-HT7R have been
reported, they are considered to be agonists/antagonists that
activate/inhibit the G protein signaling pathway, while no
activation/inhibition of the β-arrestin signaling pathway other
than SB-269970 has been reported30 (Figure 1). Recently, Kim
Synthesis. 6-Chloro-2′-methoxy-[1,1′-biphenyl]-3-yl-N-al-
kylmethanamine derivatives 1 were synthesized in three steps
starting from 3-chloro-4-iodobenzaldehyde 4 (Scheme 1),
which is iodinated from 4-chlorobenzaldehyde 3 through
electrophilic aromatic substitution of aldehyde by using I2,
NaIO3, and sulfuric acid.31,32 Suzuki cross-coupling reaction
between 4 and 2-methoxyphenylboronic acid 5 provided
biphenyl core 6 in 64−98% yields. This biphenyl benzaldehyde
6 was then converted to N-alkylamines 1a−1g via reductive
amination by the treatment of NaBH(OAc)3 and various
alkylamines in 16−93% yields.
With iodinated starting material 4, the syntheses of 2-(6-
chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)-N-alkylethan-1-amine
derivatives 2 were accomplished efficiently, as shown in Scheme
2. One carbon homologation of the iodoaldehyde 4 was
obtained by the introduction of cyanide and reduction. The
iodobenzaldehyde 4 was reduced by NaBH4, which is
brominated, and this was followed by the SN2 reaction with
NaCN to provide an intermediate 9 with the cyanide group.
Next, the Suzuki cross-coupling of 9 produced biphenyl cyanide
10, which subsequently was reduced by LiAlH4 and AlCl3 to
produce one-carbon-homologated ethyleneamine 11. Prepara-
tion of dialkyl compounds was established by reductive
amination using formaldehyde/formic acid or the SN2 reaction
using alkyliodide/K2CO3. Reductive amination was conducted
to synthesize the dimethyl derivative 2e, and the SN2 reaction
was performed to obtain diethyl compound 2f and dipropyl
compound 2g. In order to obtain monoalkylated compounds,
ethyleneamine 11 was protected by the Boc group to afford an
intermediate 12, which was treated with NaH and alkyliodide to
provide Boc-protected monoalkyl compounds 13. The use of 1
N HCl in diethyl ether to eliminate the protection of the Boc
group allowed us to obtain the final monoalkylated compounds
2a−2d.
Figure 1. Structures of representative 5-HT7R agonists (E-55888, AS-
19, and 1a) and antagonist (SB-269970).
et al.30 have discovered an azepine derivative, that is, 3-(4-
chlorophenyl)-1,4,5,6,7,8-hexahydropyrazolo[3,4-d]azepine, as
a β-arrestin-biased ligand against 5-HT7R (Ki 30 nM), IC50 (G
protein) 7800 nM, and EC50 (β-arrestin) 162 nM, whereas, to
date, no Gs protein-biased ligand of 5-HT7R has been
discovered. In this study, we designed and synthesized biphenyl
derivatives 1, 2, and 3 (Figure 2), and 2b was found to be a
potent Gs protein-biased agonist of 5-HT7R, and it was used in
We also synthesized 3-(6-chloro-2′-methoxy-[1,1′-biphenyl]-
3-yl)pyrrolidine derivatives 3a and 3b, as shown in Scheme 3.
Biphenylaldehyde 6 underwent the nitroaldol reaction by
treatment with nitromethane and ammonium acetate at 100
°C to afford a nitrovinyl derivative 14. To incorporate the
malonate ester into the nitrovinyl position by Michael addition,
NaH and diethyl malonate were used to provide a malonated
Figure 2. Structures of 6-chloro-2′-methoxy biphenyl derivatives 1, 2,
and 3 under this study.
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a
Scheme 1. Synthesis of Biphenyl-3-yl-methanamines 1
a
Reagents and conditions: (a) I2, NaIO3, H2SO4, rt, and 33−64%; (b) 2-methoxyphenylbononic acid 5, Pd(PPh3)4, Na2CO3, N,N-
dimethylformamide (DMF), reflux, and 64−98%; (c) HNR1R2, NaBH(OAc)3, MeOH, rt, and 16−93%.
a
Scheme 2. Synthesis of Biphenyl-3-yl-methanamines 2
a
Reagents and conditions: (a) NaBH4, MeOH, rt, and 88%; (b) Ph3P, CBr4, CH2Cl2, rt, and 93%; (c) NaCN, TBAB, toluene, 50 °C, and 77%; (d)
2-methoxyphenylboronic acid 5, Pd(PPh3)4, Na2CO3, DMF, reflux, and 60−95%; (e) 1 M LiAlH4 in THF, AlCl3, THF, 0 °C to rt, and 43−69%;
(f) Boc2O, NaHCO3, H2O, THF, rt, and 77%; (g) HCOOH, HCOH, reflux, and 70%; (h) K2CO3, alkyliodide, MeCN, 65 °C, and 40−63%; (i)
NaH, alkyliodide, DMF, 0 °C to rt, and 41−57%; and (j) 1 N HCl in diethyl ether, rt, and 80−95%.
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a
Scheme 3. Synthesis of Biphenyl-3-ylpyrrolidine Compounds 3
a
Reagents and conditions: (a) 2-methoxyphenylboronic acid 5, Pd(PPh3)4, Na2CO3, DMF, reflux, and 60%−95%; (b) CH3NO2, NH4OAc, 100
°C, and 66%; (c) diethylmalonate, NaH, THF, rt, and 48%; (d) NiCl2·6H2O, NaBH4, MeOH, 0 °C to rt, and 55%; (e) (i) NaOH and EtOH; (ii)
toluene, reflux, and 67%; (f) 1 M LiAlH4 in THF, THF, 0 °C to rt, and 73%; (g) HCOOH, HCOH, reflux, and 37%.
compound 15, which was subjected to ring formation by using
NiCl2·6H2O/NaBH4 to provide a carboxylated oxopyrrolidine
16. The decarboxylation of 16 produced oxopyrrolidine 17,
which was subsequently reduced by LiAlH4 to obtain a
pyrrolidine compound 3a. Synthesis of methylpyrrolidine 3b
was accomplished by reductive amination with formaldehyde/
formic acid.
tained potency of binding affinities to 5-HT7R. Compounds 2a
and 2b (R1 = Me and Et) showed binding affinity with Ki values
of 1.6 and 2.8 nM for 5-HT7R, respectively, comparable to those
of 1a and 1b. Compounds 2c and 2d (R1 = Pr and Bu) had
almost the same binding affinities with Ki values of 8.7 and 64
nM in comparison to the corresponding compounds 1c and 1d,
while dialkyl compounds 2e−g (R1, R2 = Me, Et, and Pr) showed
at least 4 times better binding affinities with Ki values of 2.5, 25,
and 20 nM, respectively, than the corresponding compounds
1e−g. Compounds 3a and 3b (R1 = H and Me) with a
pyrrolidine moiety showed binding affinities with Ki values of 2.8
and 30 nM, respectively. Among the synthesized compounds,
compound 2a had the best binding affinity value (Ki = 1.6 nM),
which was comparable to that of E-55888 (Ki = 1.3 nM).
To investigate the functional activities of compounds on the
Gs protein and β-arrestin signaling pathways, we conducted two
types of cell-based functional assays, that is, bioluminescence-
based assays to measure Gs-mediated cAMP production (cAMP
assay34,35) and β-arrestin recruitment (Tango assay36,37) with
synthesized compounds 1, 2, and 3, and an endogenous agonist
5-HT and a 5-HT7R agonist E-55888 were used as reference
compounds. All compounds were evaluated by both cAMP assay
and Tango assay. The cAMP assay was done in transiently
transfected HEK293 cells with 5-HT7R plasmid/GloSensor
plasmid, while, in the Tango assay, the HEK293-derived cell line
Binding Affinity and Functional Activity against 5-
HT7R. Binding affinities of 1, 2, and 3 for 5-HT7R were
determined by the [3H] D-lysergic acid diethylamide ([3H]LSD)
radioligand binding assay in transfected HEK293 cells.33 Table 1
shows the binding affinities (Ki) and functional activities in the
Gs protein/β-arrestin signaling pathways against 5-HT7R.
Among the biphenyl derivatives 1 with the methyleneamine
moiety, compounds 1a−c (R1 = Me, Et, and Pr, R2 = H) showed
binding affinities against 5-HT7R with Ki values of 5.2, 9.3, and
6.8 nM, respectively. However, the longer carbon chain of the N-
alkyl moiety, like a butyl chain, reduces the affinity for 5-HT7R.
Compound 1d with the monobutyl amine moiety (R1 = Bu, R2 =
H) had a Ki value of 52 nM. Dimethyl derivative 1e (R1 = Me, R2
= Me) showed binding affinity with a Ki value of 18 nM, while
the diethyl and dipropyl compounds 1f and 1g had decreased
affinity (Ki = 110 nM) or no activity up to 10 μM, respectively.
Interestingly, compared with the binding affinities of com-
pounds 1, one-carbon-elongated compounds 2 mostly main-
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Table 1. Results of Biological Activity Assays of Compounds 1, 2, and 3 against 5-HT7R
a
binding affinity
cAMP production
EC50 (nM)
β-arrestin recruitment
b
b
b
compound
R1
R2
pKi
Ki (nM)
pEC50
E
max (%)
pEC50
EC50 (nM)
E
max (%)
1a28
1b
1c
1d
1e
1f
1g
2a
2b
2c
2d
2e
2f
2g
3a
3b
Me
Et
Pr
Bu
Me
Et
Pr
Me
Et
H
H
H
H
Me
Et
Pr
H
H
H
8.28 0.07
8.00 0.20
8.20 0.10
7.28 0.06
7.70 0.10
5.2
9.3
6.8
52
18
110
c
6.34 0.04
6.12 0.11
460
770
72
71
68
e
6.83 0.08
150
61
e
e
e
e
e
e
e
e
e
5.93 0.14
1200
e
e
5.94 0.09
1100
11
e
6.32 0.13
480
e
43
e
e
e
e
7.00 0.10
c
e
e
e
e
e
e
8.55 0.10
8.80 0.10
8.07 0.12
7.19 0.11
8.60 0.09
7.60 0.12
7.70 0.12
8.56 0.13
1.6
2.8
8.7
64
2.5
25
20
2.8
30
d
7.14 0.12
6.74 0.04
73
180
77
91
53
e
7.15 0.12
71
e
67
e
e
e
e
e
e
e
e
Pr
5.98 0.06
1000
e
e
Bu
Me
Et
Pr
H
H
Me
Et
Pr
7.08 0.10
5.73 0.04
5.86 0.06
7.04 0.14
6.25 0.24
8.03 0.06
7.08 0.04
84
1900
1400
92
560
4.5
67
76
72
7.25 0.18
56
e
53
e
e
e
e
e
29
7.12 0.09
6.19 0.13
6.95 0.06
7.68 0.08
75
650
110
21
54
51
100
54
Me
7.52 0.10
d
9.8
100
99
5-HT
E-55888
8.89 0.05
1.3
83
a
Binding affinity data were generously provided by the US National Institute of Mental Health (NIMH) Psychoactive Drug Screening Program
b
c
(University of North Carolina). Values are the mean SEM of at least three independent experiments performed in triplicate. Ki > 10,000 nM.
d
e
Not determined. No activity.
was used, and it contained stable interactions of a tTA-
dependent luciferase reporter and a β-arrestin 2−TEV fusion
gene (HTLA cells). Compounds 1a−c showed potency and
efficacy in the cAMP assay (EC50 = 460, 770, and 1200 nM,
respectively, and Emax = 72, 71, and 68%, respectively), while the
Emax value of compound 1e is relatively low with 11%, even
though the EC50 value of 1e was 1100 nM. Compounds 1d, 1f,
and 1g show no functional activities in the cAMP assay. In β-
arrestin recruitment Tango assay, only compounds 1a and 1e
showed functional activities with EC50 values of 150 and 480
nM, respectively. Compounds 1b and 1c had activities in cAMP
production but no activities in β-arrestin recruitment, which can
be called Gs protein-biased ligands. In the series of compounds
2, compounds 2a and 2b had EC50 values of 73 and 180 nM,
which showed better activities than compounds 1a and 1b in the
cAMP assay. Efficacy of 2b in cAMP production is the best with
an Emax value of 91% among the synthesized compounds.
Compound 2c showed marginal activity in cAMP production
with an EC50 value of 1000 nM and an Emax value of 53% and
compound 2d showed no activity. Compared with compounds
1e−g, compounds 2e−g showed improved activities in the
cAMP assay (EC50 = 84, 1900, and 1400 nM, Emax = 67, 76, and
72%). In β-arrestin recruitment Tango assay, only compounds
2a and 2e showed functional activities with EC50 values of 71
and 56 nM. Therefore, compounds 2b, 2c, 2f, and 2g had
activities in cAMP production but no activities in β-arrestin
recruitment, which can also be called Gs protein-biased ligands.
Compounds 3a and 3b displayed better activities in the Tango
assay (EC50 = 75 and 650 nM, Emax = 54 and 51%) than they did
in the cAMP assay (EC50 = 92 and 560 nM, Emax = 29 and 9.8%).
There are three balanced agonists 1a, 2a, and 2e like E-55888,
among which 2a is the best in the view of potency and efficacy in
both assays, while there are six G protein-biased ligands 1b, 1c,
2b, 2c, 2f, and 2g, among which 2b is the best in the cAMP assay
(EC50 = 180 nM, Emax = 91%) and 1b is the next (EC50 = 770
nM, Emax = 71%) (Figure 3).
Among 16 synthesized compounds, 15 compounds bound to
5-HT7R with Ki values between 1.6 and 110 nM, and 12
compounds showed potency and efficacy in the Gs protein
signaling pathway, while only six compounds were active in β-
arrestin signaling pathway. Among those, 2a is the best balanced
agonist and 2b is the best G protein-biased ligand.
Selectivity over Other 5-HTR Subtypes. We investigated
the binding affinities of compounds 1, 2, and 3 for other
serotonin subtype receptors, such as 5-HT1AR, 5-HT1BR, 5-
HT1DR, 5-HT1ER, 5-HT2AR, 5-HT2BR, 5-HT2CR, 5-HT3R, 5-
HT5AR, and 5-HT6R to determine their selectivity for 5-HT7R.
Compounds 1a−e showed selectivity over 5-HT1AR, 5-HT1BR,
5-HT1DR, 5-HT1ER, 5-HT2AR, 5-HT3R, 5-HT5AR, and 5-HT6R,
except 5-HT2BR and some cases of 5-HT2CR. Compounds 1a−e
had binding affinities for 5-HT2BR with Ki values of 13, 19, 12,
15, and 11 nM, respectively, resulting in little selectivity of
compounds 1a−e over 5-HT2BR. Compounds 2a and 2b had at
least 19-fold selectivity over other 5-HTR subtypes except 5-
HT1DR. Compounds 2d, 2f, and 2g had almost no selectivity
over 5-HT1AR and 5-HT1DR. It is very interesting that
compounds 1 had little selectivity over 5-HT2BR, while
compounds 2 except 2d had at least 12-fold selectivity over 5-
HT2BR, but little selectivity over 5-HT1DR. Specially, com-
pounds 2a and 2b showed more than 40-fold selectivity over 5-
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Figure 3. Dose−response curves of Gs protein activation assay (cAMP agonist assay) and β-arrestin recruitment assay (Tango agonist assay) of
compounds 5-HT, E-55888, 1b, 2a, and 2b.
HT2BR. Compound 3a indicated at least 9-fold selectivity over
other 5-HTR subtypes, except for 5-HT1DR, while compound
3b showed more than 30-fold selectivity over 5-HT1ER, 5-
HT2AR, 5-HT2CR, 5-HT3R, and 5-HT5AR. It was reported that
5-HT2BR was associated with cardiac valvulopathy, resulting in
potential cardotoxicity,38 while the 5-HT1DR agonist, triptan,
shows a pharmacological benefit in migraine pain.39
Functional Selectivity. Based on their pharmacological
profiles (Table 1) and selectivity profiles (Table 2), we selected
potent and functionally distinct ligands 1b, 2a, and 2b to
determine ligand bias toward the Gs protein signaling pathway
or the β-arrestin signaling pathway compared with their
functional selectivity profile of 5-HT and E-55888. The results
indicated that E-55888 had almost full efficacy in the Gs protein
pathway (EC50 = 83 nM, Emax = 99%) compared to 5-HT, while
showed a partial agonistic effect in the β-arrestin pathway (EC50
= 21 nM, Emax = 54%). Since compound 2a activated both the Gs
protein pathway and the β-arrestin pathway, 2a was shown to be
a balanced agonist with good potency and efficacy (cAMP assay:
EC50 = 73 nM, Emax = 77%, and Tango assay: EC50 = 71 nM, Emax
= 67%). By contrast, compounds 1b and 2b, both of which have
a mono-ethyl group as the alkyl substitution at the methylene-
amine and ethyleneamine moieties, exhibited no activity in the
β-arrestin pathway compared to 5-HT (100%) and E-
55888(54%). Compound 2b acted as almost full agonist in the
Gs protein pathway with an Emax of 91%, while compound 1b
had a lower Emax value with 71% than compound 2b.
Using 5-HT as a positive control and E-55888 as a reference
ligand to evaluate our compounds, we found that compound 2a
with mono-methyl moiety acted as a balanced agonist, whereas
compounds 1b and 2b, with mono-ethyl moiety, showed
functional selectivity with preference to the production of Gs
protein-mediated cAMP, which means the two compounds, that
is, 1b and 2b, are Gs protein-biased ligands. Based on the
potency and efficacy, 2b exhibited a better Gs protein-biased
ligand, that is, 5-HT7R, than compound 1b. It could be
suggested that alkyl substitution is important to selectively
activate the Gs protein signaling pathway, and in particular, ethyl
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Table 2. Binding Affinities (Ki) of Compounds 1, 2, and 3 against 5-HTRs
ab
,
Ki in nM
compounds
R1
R2
5-HT7
5-HT1A
5-HT1B
5-HT1D
5-HT1E
5-HT2A
5-HT2B
5-HT2C
5-HT3
5-HT5A
5-HT6
c
1a28
1b
1c
1d
1e
1f
1g
2a
2b
2c
2d
2e
2f
Me
Et
Pr
Bu
Me
Et
Pr
Me
Et
H
H
H
H
Me
Et
Pr
H
H
H
5.2
9.3
6.8
52
18
110
c
1700
840
420
320
2100
1700
400
280
52
140
220
280
84
1200
210
640
c
1200
220
220
2400
380
910
670
280
13
19
12
15
11
67
80
69
130
170
130
280
320
230
50
200
45
200
310
270
820
420
1800
120
360
220
1300
1400
350
240
440
c
c
c
c
c
c
2000
830
c
1300
4100
360
c
1000
c
c
c
230
c
2000
c
c
c
c
c
c
c
c
c
c
c
c
c
2600
c
1.6
2.8
8.7
64
2.5
25
20
2.8
30
84
580
250
280
74
160
160
410
12
3.3
5.3
41
110
86
8.4
38
3.4
19
180
170
520
600
570
250
680
1200
340
910
700
240
2400
380
2100
2900
970
740
c
c
c
c
c
c
c
c
2600
c
c
c
Pr
c
Bu
Me
Et
Pr
H
H
Me
Et
Pr
2200
1000
c
6700
c
c
2g
3a
3b
65
96
200
770
4100
590
420
400
c
Me
1200
170
920
1000
a
Binding affinity data were generously provided by the US National Institute of Mental Health (NIMH) Psychoactive Drug Screening Program
b
c
(University of North Carolina). Values are the mean of at least three independent experiments performed in triplicate. Ki > 10,000 nM.
group at the amine in this series of compounds leads to the Gs
protein bias.
converted to a bias factor. The bias factor of E-55888 is less than
1, which means that E-55888 is more biased to the β-arrestin
signaling pathway, not the Gs protein signaling pathway. 2b had
ΔΔlog(τ/KA) = 2.66, of which the bias factor is 461. By the
calculation of the bias factors of those two compounds E-55888
and 2b, it became more obvious that 2b is a Gs protein-biased
ligand of 5-HT7R.
To determine ligand biasness of a better G protein-biased
ligand 2b, we evaluated the differences ΔΔlog(τ/KA) between
the Δlog(τ/KA) values of 2b and E-55888 in both G protein
signaling pathway and β-arrestin signaling pathway.40 As shown
in Table 3, E-55888 had ΔΔlog(τ/KA) = −0.47, which was
CYP Activity and Microsomal Stability. The in vitro
cytochrome P450 (CYP450) inhibition assay, including five
major isoforms, that is, CYP1A2, CYP2C9, CYP2C19,
CYP2D6, and CYP3A4, was performed to assess the potential
drug−drug interaction of 2b (Table 4). As shown in Table 4, the
percentage of remaining activities of 2b in the five CYP isozymes
is greater than 88.7%, and compound 2b has little effect on the
CYP isozymes, indicating that it might not have a drug−drug
interaction. In addition, the metabolic stability of compound 2b
was determined through the human liver microsomal stability
test. Since the metabolic stability of drug is a key factor that
affects both the efficacy and toxicity of the drug, as well as its
pharmacokinetic (PK) parameters, it can be tested in an in vitro
assay prior to conducting an in vivo study. Compound 2b
showed 71.5% as the percent-remaining concentration, which
means that 2b is barely decomposed and is stable after
incubation with human liver microsomes. Thus, the PK
a
Table 3. Bias Factor of E-55888 and Compound 2b toward
Gs Protein
signaling
pathway
b
parameters
5-HT
E-55888
2b
Gs protein
log τ
log KA
0.71
−7.46
0
0.22
−6.14
0
1.22
−5.84
−1.12
0.33
−5.39
−0.65
−0.47
0.34
0.68
−6.11
−1.39
−1.61
−3.92
−4.05
2.66
Δlog(τ/KA)
log τ
β-arrestin
log KA
Δlog(τ/KA)
ΔΔlog(τ/KA)
bias factor (toward Gs
protein)
c
0
1
461
a
Bias factor was calculated with Prism 4.0 program (GraphPad
b
software, San Diego). 5-HT was used as the reference agonist.
c
ΔΔlog(τ/KA) = Δlog(τ/KA)(Gs protein) − Δlog(τ/KA)(β-arrestin).
Table 4. Results of CYP450 Remaining Activity and Microsomal Stability Test
a
CYP450 activity
b c
,
compd
CYP1A2
94.3
CYP2C9
>100
CYP2C19
90.3
CYP2D6
88.7
CYP3A4
98.5
human liver microsomal stability (%)
71.5
2b
a
b
c
%-Remaining cytochrome activity. %-Remaining amount of 2b after 30 min of treatment with human liver microsomes. Verapamil was used as a
reference compound and the microsomal stability was 16.8%.
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parameters of 2b were evaluated after intravenous injection
(dose 1 mg/kg) and intraperitoneal (i.p.) administration (dose
10 mg/kg) in male ICR mice (Table 5). After the intravenous
181 s) without any significant changes compared with those of
saline (Figure 4a). After i.p. administration, the Gs protein-
biased agonist 2b with a dose of 5 mg/kg showed a highly
increased duration of the self-grooming time in Shank3 TG mice
(t = 553 s) compared to that of saline (Figure 4b). To determine
the direct effect of 5-HT7R on self-grooming behavior,
SB269970 was administered 10 min prior to the injection of
2b. Figure 4b shows that the co-treatment of SB269970 (30 mg/
kg) and 2b (5 mg/kg) reduced the duration of self-grooming (t
= 70.8 s in WT mice and 166 s in TG mice) to the normal
duration with saline administration, which demonstrates that
SB269970 inhibits the effect of 2b. Overall, the Gs protein-
biased agonist 2b prominently increased the duration time of
self-grooming, which was suppressed by the effect of the 5-HT7R
selective antagonist, SB269970. Although more experiments are
needed regarding the association of 5-HT7R and autism, these
results support the conclusion that 5-HT7R is involved in a
modulatory role to stereotypy in ASD (Table 6).
b
Table 5. Mean ( SD ) PK Parameters after Intravenous
Injection at a Dose of 1 mg/kg (n = 4) and Intraperitoneal
Administration at a Dose of 10 mg/kg (n = 4) of 2b to ICR
a
Mice
intravenous
injection
intraperitoneal
administration
plasma
max (h)
T
C
T
0.4 0.1
max (ng/mL)
1/2 (h)
347.7 69.9
1.0 0.3
0.5 0.2
AUClast (h ng/mL)
Cl_obs (mL/min/kg)
MRTinf_obs (h)
Vss_obs (L/kg)
BA (%)
32.9 2.5
477.7 60.3
0.7 0.3
442.3 71.7
1.3 0.4
134.4
19.5 4.7
a
T
max, time to reach Cmax; Cmax, peak plasma concentration; T1/2,
Table 6. Duration Time of Self-Grooming Behavior in WT
and Shank3 TG Mice after Treatment with Saline, SB269970
(30 mg/kg), 2b (5 mg/kg), and SB269970 + 2b for 30 min
terminal half-life; AUClast, total area under the plasma concentration
time curve from time zero to the last measured time; Cl, time-
averaged total body clearance; MRT, mean residence time; Vdss
,
a
Measured in Healthy Subjects (n = 6)
apparent volume of distribution at steady state; BA, bioavailability.
b
SD: standard deviations.
duration time (t) of grooming (in seconds)
compounds
WT
Shank3 TG
injection, the mean clearance rate (CL) was measured as 477.7
mL/min/kg, the half-life (T1/2) of 2b was 0.5 h, and the AUClast
was 32.9 h ng/mL. According to the half-life and CL, 2b seems
to be rapidly eliminated from the body. In i.p. administration,
the half-life (T1/2) was 1.0 h, and AUClast was 442.3 h ng/mL.
The bioavailability of 2b was about 134%.
SB269970
2b
SB269970 + 2b
saline
29.3 6.90
107 41.6
70.8 9.20
43.7 10.8
181 37.8
553 58.0
166 35.0
224 47.4
a
All data are expressed as mean SEM (seconds).
Self-Grooming Behavior. One of the symptoms in patients
with ASD is stereotypy, which is observed as restricted and
repetitive patterns of behavior.41 Currently, studies of the
correlation between ASD and 5-HT7R are emerging, and there
are several reported results indicating that 5-HT7R ligands
correct phenotypic deficits in various heterogeneous mouse
models, such as Fmr1 KO mice,22 Mecp2 TG mice,23 and C58/J
mice,42 which exhibit spontaneous self-grooming. Thus, we
conducted a self-grooming behavior test using Shank3−/−
(Shank3 TG) male mice43−45 that are a well-known ASD
animal model. We tested SB269970, also known as 5-HT7R
selective antagonist, at a dose of 30 mg/kg in i.p. administration
to WT and Shank3 TG mice. The administration of SB269970
showed duration times in both WT (t = 29.3 s) and TG mice (t =
CONCLUSIONS
■
We synthesized a series of 6-chloro-2'-methoxy biphenyl
derivatives 1, 2, and 3 bearing various amine moieties and
evaluated binding affinities of these synthesized compounds for
5-HT7R along with other serotonin receptor subtypes. Most of
the compounds had binding affinities to 5-HT7R. By conducting
cAMP production assay and β-arrestin recruitment Tango assay,
three balanced agonists 1a, 2a, and 2e like E-55888 were found,
among which 2a is the best in the view of potency and efficacy in
both assays, while there are six G protein-biased ligands 1b, 1c,
2b, 2c, 2f, and 2g, among which 2b is the best in the cAMP assay
Figure 4. (a) Duration time of self-grooming behavior in WT and Shank3 TG mice treated with saline and SB269970 (30 mg/kg) and (b) duration of
self-grooming behavior in WT and Shank3 TG mice co-treated with SB269970 (30 mg/kg) and 2b (5 mg/kg). Data bars represent means SEM.
Duration compiled for the 30 min following the intraperitoneal administration of drugs: *P < 0.05, **P < 0.01, and ***P < 0.001 vs vehicle based on
Student’s t-test.
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(DCM/MeOH = 10:1) to afford the desired final product 1 in 16−93%
yields.
(EC50 = 180 nM, Emax = 91%). In the animal study with Shank3
TG mice, the self-grooming behavior test of 2b was performed,
and 2b showed an increased duration of self-grooming, which
was reversed by the selective 5-HT7R antagonist, SB269970.
Based on the results of the self-grooming behavior, we further
suggested that 5-HT7R is associated with ASD and could be a
therapeutic target for the treatment of stereotypy in ASD.
1-(2′-Methoxy-[1,1′-biphenyl]-3-yl)-N-methylmethanamine (1a).
Compound 1a was synthesized according to the general procedure of 1.
1
Yield: 37%; HPLC: purity 100%, tR = 5.5 min; H NMR (300 MHz,
CDCl3): δ 7.51−7.30 (m, 6H), 7.09−7.01 (m, 2H), 3.86 (s, 2H), 3.85
(s, 3H), 2.53 (s, 3H); 13C NMR (CDCl3, 75 MHz): δ 156.49, 139.19,
138.72, 130.93, 130.64, 127.48, 128.65, 128.41, 128.10, 126.88, 120.85,
111.29, 55.88, 55.60, 35.71; HRMS (ESI+): calcd for C15H17ClNO+
[M + H]+, 262.0999; found, 262.0996.
N-((6-Chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)methyl)-
ethanamine (1b). Compound 1b was synthesized according to the
general procedure of 1. Yield: 57%; HPLC: purity 100%, tR = 5.8 min;
1H NMR (400 MHz, CDCl3): δ 7.41−7.35 (m, 2H), 7.26 (dd, J = 5.8,
1.6 Hz, 2H), 7.12 (dd, J = 7.6, 1.8 Hz, 1H), 7.01 (td, J = 7.6, 1.1, 1H),
6.98 (dd, J = 8.4, 0.8 Hz, 1H), 3.80 (s, 2H), 3.78 (s, 3H), 2.70 (q, J = 7.2
Hz, 2H), 1.14 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ
156.7, 138.5, 137.6, 132.4, 131.4, 131.0, 129.4, 129.2, 128.6, 128.3,
120.4, 111.0, 55.6, 53.1, 43.6, 15.1; HRMS (ESI+): calcd for
C16H19ClNO+ [M + H]+, 276.1155; found, 276.1150.
N-((6-Chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)methyl)propan-1-
amine (1c). Compound 1c was synthesized according to the general
procedure of 1. Yield: 80%; HPLC: purity 100%, tR = 5.7 min; 1H NMR
(400 MHz, CDCl3): δ 7.41−7.35 (m, 2H), 7.27−7.25 (m, 2H), 7.02
(td, J = 7.4, 1.2 Hz, 1H), 6.98 (d, J = 0.8 Hz, 1H), 3.79 (s, 1H), 3.78 (s,
3H), 1.54 (m, J = 3.4 Hz, 2H), 0.92 (t, J = 7.4 Hz, 3H); 13C NMR (100
MHz, CDCl3): δ 156.8, 138.7, 137.6, 132.4, 131.4, 131.0, 129.4, 129.2,
128.6, 128.3, 120.4, 111.0, 55.6, 53.2, 51.3, 23.1, 11.8; HRMS (ESI+):
calcd for C17H21ClNO+ [M + H]+, 190.1312; found, 190.1306.
N-((6-Chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)methyl)butan-1-
amine (1d). Compound 1d was synthesized according to the general
procedure of 1. Yield: 93%; HPLC: purity 100%, tR = 6.1 min; 1H NMR
(400 MHz, CDCl3): δ 7.41−7.35 (m, 2H), 7.25−7.23 (m, 2H), 7.19
(dd, J = 7.2, 1.6 Hz, 1H), 7.04−6.97 (m, 2H), 3.78 (s, 5H), 2.64 (t, J =
7.2 Hz, 2H), 1.48 (m, J = 4.3 Hz, 2H), 1.35 (m, J = 7.5 Hz, 2H), 0.91 (t,
J = 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ 156.8, 139.0, 137.6,
132.2, 131.3, 131.0, 129.3, 129.2, 128.6, 128.2, 120.3, 111.0, 55.6, 53.4,
49.2, 32.3, 20.5, 14.0; HRMS (ESI+): calcd for C18H23ClNO+ [M +
H]+, 304.1468; found, 304.1466.
EXPERIMENTAL SECTION
■
Chemistry. All reactions were carried out under dry nitrogen unless
otherwise indicated. Commercially available reagents were used
without further purification. Solvents and gases were dried according
to standard procedures. Organic solvents were evaporated with reduced
pressure using a rotary evaporator. Analytical thin layer chromatog-
raphy (TLC) was performed using glass plates pre-coated with silica gel
(0.25 mm). TLC plates were visualized by exposure to UV light and
then were visualized with a KMnO4, ninhydrin, and p-anisaldehyde
staining solution followed by brief heating on a hot plate. Flash column
chromatography was performed using silica gel 60 (230−400 mesh,
1
Merck) with the indicated solvents. H and 13C NMR spectra were
recorded on Bruker 300 or 400 NMR spectrometers. 1H NMR spectra
are represented as follows: chemical shift, multiplicity (s = singlet, br s =
broad singlet, d = doublet, t = triplet, q = quartet, m = multiplet),
integration, and coupling constant (J) in hertz. Liquid chromatog-
raphy−mass spectrometry (LC/MS) and high-resolution mass
spectrometry (HRMS) analyses were performed on an Agilent 6410
Triple Quad system and the Bruker Compact ESI+ positive mode,
respectively. The purity of all the tested compounds was checked on a
Waters HPLC e2695 instrument equipped with a UV/vis 2489 detector
and a Capcell Pak 3 μm C18 MG-II (4.6 × 75 mm) column and was at
least 95% for all tested compounds. Standard conditions were as
follows: eluents system A (CH3CN), system B (H2O/0.1 M AcOH); a
flow rate of 1 mL/min; a gradient of (10−100%) A over 20 min; and
detection at 254 and 280 nm.
4-Chloro-3-iodobenzaldehyde (4). Iodine (1.59 g, 6.26 mmol) and
NaIO3 (620 mg, 3.13 mmol) were suspended in 95% concentrated
H2SO4. The solution was stirred for 30 min at room temperature (rt). 4-
Chlorobenzaldehyde 3 (2 g, 14.23 mmol) was added to the dark brown
iodinating solution and the mixture was stirred for an additional 1 h at
rt. The reaction mixture was poured slowly into ice−water. The crude
solid product was collected by filtration with EtOH to afford the desired
1-(2′-Methoxy-[1,1′-biphenyl]-3-yl)-N,N-dimethylmethanamine
(1e). Compound 1e was synthesized according to the general procedure
1
of 1. Yield: 74%; HPLC: purity 100%, tR = 5.6 min; H NMR (400
1
MHz, CDCl3): δ 7.52−7.30 (m, 6H), 7.10−7.01 (m, 2H), 3.85 (s, 3H),
3.61 (s, 2H), 2.37 (s, 6H); 13C NMR (CDCl3, 75 MHz): δ 156.50,
138.60, 137.03, 130.94, 130.66, 130.56, 128.68, 128.04, 128.01, 120.88,
111.33, 63.96, 55.60, 44.85; HRMS (ESI+): calcd for C16H19ClNO+
[M + H]+, 276.1155; found, 276.1152.
product 4 (2.43 g, 9.12 mmol) in 64% yield. H NMR (400 MHz,
CDCl3): δ 9.92 (s, 1H), 8.34 (d, J = 1.9 Hz, 1H), 7.80 (dd, J = 8.2, 1.9
Hz, 1H), 7.61 (d, J = 8.2 Hz, 1H); 13C NMR (100 MHz, CDCl3): δ
189.41, 145.07, 141.37, 135.68, 130.00, 129.95, 98.83.
6-Chloro-2′-methoxy-[1,1′-biphenyl]-3-carbaldehyde (6). 2-Me-
thoxyphenylboronic acid 5 (2.25 mmol), Pd(PPh3)4 (0.02 mmol), and
Na2CO3 (2.82 mmol) were added to a solution of benzaldehyde 4 (1.88
mmol) in DMF (15 mL). The mixture was stirred overnight at 80 °C.
After cooling down to rt, the reaction mixture was quenched by the
addition of the saturated solution of NaHCO3 and then partitioned
with EtOAc. The combined organic layers were dried over MgSO4,
filtered, and concentrated under reduced pressure. The residue was
purified by column chromatography on silica gel (hexanes/EtOAc =
10:1) to obtain desired product 6 in 82% yield. 1H NMR (400 MHz,
CDCl3): δ 10.00 (s, 1H), 7.82−7.79 (m, 2H), 7.62 (dd, J = 6.2, 2.6 Hz,
1H), 7.42 (m, 1H), 7.20 (dd, J = 7.4, 1.8 Hz, 1H), 7.05 (td, J = 7.5, 1.2
Hz, 1H), 7.01 (dd, J = 8.4, 0.8 Hz, 1H), 3.79 (s, 3H); 13C NMR (100
MHz, CDCl3): δ 191.1, 156.6, 140.8, 138.9, 134.7, 133.3, 130.1, 130.2,
130.0, 129.0, 127.2, 120.5, 111.0, 55.6.
6-Chloro-2′-methoxy-[1,1′-biphenyl]-3-yl-methanamine Deriva-
tives (1). The mixture of 6-chloro-2′-methoxy-[1,1′-biphenyl]-3-
carbaldehyde 6 (1 equiv) and appropriate amine (2 equiv) in MeOH
(20 mL) was stirred at rt for 2 h. NaBH(OAc)3 (3 equiv) was added and
the solution was stirred overnight at rt. The reaction mixture was
quenched by the addition of a saturated solution of NaHCO3 and
extracted with dichloromethane (DCM). The combined organic layer
was dried over MgSO4, filtered, and concentrated under reduced
pressure. The residue was purified by flash column chromatography
N-((6-Chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)methyl)-N-ethyle-
thanamine (1f). Compound 1f was synthesized according to the
general procedure of 1. Yield: 20%; HPLC: purity 100%, tR = 6.1 min;
1H NMR (400 MHz, CDCl3): δ 7.40−7.35 (m, 2H), 7.28−7.26 (m,
2H), 7.20 (dd, J = 7.4, 1.8 Hz, 1H), 7.04−6.97 (m, 2H), 3.78 (d, 3H),
7.58 (s, 2H), 2.55 (q, J = 7.4 Hz, 4H), 1.04 (t, J = 7.0 Hz, 3H); 13C NMR
(100 MHz, CDCl3): δ 156.8, 138.2, 137.3, 132.1, 132.0, 131.1, 129.3,
129.0, 128.7, 120.3, 111.0, 56.8, 55.6, 46.8, 11.8; HRMS (ESI+): calcd
for C18H23ClNO+ [M + H]+, 304.1468; found, 304.1467.
N-((6-Chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)methyl)-N-propyl-
propan-1-amine (1g). Compound 1g was synthesized according to the
general procedure of 1. Yield: 16%; HPLC: purity 100%, tR = 6.8 min;
1H NMR (400 MHz, CDCl3): δ 7.38−7.34 (m, 2H), 7.26−7.23 (m,
2H), 7.19 (dd, J = 7.4, 1.8 Hz, 1H), 7.01 (td, J = 7.4, 1.2 Hz, 1H), 6.97
(d, J = 8.0 Hz, 1H), 3.77 (s, 3H), 3.54 (s, 2H), 2.39−2.34 (m, 4H), 1.47
(m, J = 3.2 Hz, 4H), 0.86 (t, J = 7.2 Hz, 6H); 13C NMR (100 MHz,
CDCl3): δ 156.8, 138.8, 137.2, 132.0, 131.9, 131.1, 129.3, 128.9, 128.9,
120.3, 111.0, 58.0, 55.9, 55.6, 20.3, 11.9; HRMS (ESI+): calcd for
C20H27ClNO+ [M + H]+, 332.1781; found, 332.1779.
(4-Chloro-3-iodophenyl)methanol (7). 4-Chloro-3-iodophenyl-
benzaldehyde 6 (7.62 mmol) was added in portions to a suspension
of sodium borohydride (7.62 mmol) in methanol (50 mL). The mixture
was stirred for 2 h at rt. The solution was quenched by the addition of
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water and then extracted with DCM. The combined organic layers were
dried over MgSO4, filtered, and concentrated under reduced pressure.
The residue was purified by column chromatography (hexanes/EtOAc
tert-Butyl (2-(6-Chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)ethyl)-
carbamate (12). 2-(6-Chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)ethan-
1-amine 11 (2.67 mmol) and di-tert-butyl dicarbonate (2.67 mmol)
were dissolved in anhydrous THF (10 mL). An aqueous solution of
NaHCO3 (2 mL) was added to a mixture and stirred overnight at rt.
The mixture was partitioned with water and DCM and the combined
organic layer was dried over MgSO4, filtered, and concentrated under
reduced pressure. The residue was purified by column chromatography
(hexanes/EtOAc = 5:1) to afford the desired product 12 (2.67 mmol,
77%). 1H NMR (400 MHz, CDCl3): δ 7.39−7.35 (m, 2H), 7.18 (dd, J
= 7.5, 1.7 Hz, 1H), 7.12 (d, J = 2.0 Hz, 1H), 7.10 (dd, J = 8.1, 2.2 Hz,
1H), 7.01 (td, J = 7.4, 1.0 Hz, 1H), 6.98 (d, J = 8.3 Hz, 1H), 4.58 (br s,
1H), 3.78 (s, 3H), 3.38 (q, J = 6.5 Hz, 2H), 2.79 (t, J = 7.0 Hz, 2H), 1.43
(s, 9H); 13C NMR (100 MHz, CDCl3): δ 156.74, 155.83, 137.78,
137.32, 132.15, 131.95, 131.00, 129.39, 128.89, 128.47, 120.36, 111.05,
55.63, 41.62, 35.56, 35.53, 35.52, 28.41.
1
= 5:1) to obtain the desired product 7 (6.70 mmol, 88%). H NMR
(400 MHz, CDCl3): δ 7.85 (d, J = 1.9 Hz, 1H), 7.41 (d, J = 8.0 Hz, 1H),
7.26 (dd, J = 8.2, 2.0 Hz, 1H), 4.62 (d, J = 4.0 Hz, 2H), 1.98 (t, J = 6.0
Hz, 1H); 13C NMR (100 MHz, CDCl3): δ 140.98, 138.45, 137.51,
129.26, 127.86, 98.12, 63.59.
4-(Bromomethyl)-1-chloro-2-iodobenzene (8). (4-Chloro-3-
iodophenyl)methanol 7 (7.45 mmol) was dissolved in DCM (50
mL) and triphenylphosphine (8.94 mmol) was added to the solution at
rt and then carbon tetrabromide (8.94 mmol) was slowly added to the
mixture. The reaction mixture was stirred for 2 h at rt, at which time, the
reaction mixture was concentrated under reduced pressure. The residue
was purified by column chromatography (hexanes/EtOAc = 50:1) to
obtain the desired product 8 (6.94 mmol, 93%). 1H NMR (400 MHz,
CDCl3): δ 7.87 (d, J = 2.1 Hz, 1H), 7.40 (d, J = 8.2 Hz, 1H), 7.30 (dd, J
= 7.9, 2.1 Hz, 1H), 4.37 (s, 2H); 13C NMR (100 MHz, CDCl3): δ
140.50, 138.60, 137.84, 130.10, 129.50, 98.21, 30.94.
tert-Butyl (2-(6-Chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)ethyl)-
(alkyl)carbamate Derivatives (13). A solution of tert-butyl (2-(6-
chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)ethyl)carbamate 12 (1 equiv)
in DMF (15 mL) was added dropwise to a sodium hydride 60% in oil
(1.25 equiv) in DMF at 0 °C and stirred for an hour. Alkyliodide (2.4
equiv) was then slowly added to a mixture at 0 °C and the stirring was
continued for an additional 2 h. The reaction mixture was quenched by
the addition of methanol and partitioned with water and EtOAc. The
combined organic layer was dried over MgSO4, filtered, and
concentrated under reduced pressure. The residue was purified by
column chromatography (hexanes/EtOAc = 5:1) to obtain the desired
product 13 in 41−57% yields.
2-(4-Chloro-3-iodophenyl)acetonitrile (9). 4-(Bromomethyl)-1-
chloro-2-iodobenzene 8 (4.71 mmol) was dissolved in toluene (30
mL) and sodium cyanide (7.06 mmol) was added to the solution.
Tetrabutylammonium bromide (0.047 mmol) dissolved in water (1
mL) was added to the mixture and then the reaction mixture was stirred
overnight at 50 °C. Based on TLC analysis, sodium cyanide (2.36
mmol) and water were additionally added to the mixture. After stirring
for 5 h, the solution was quenched by the addition of water and
extracted with EtOAc. The combined organic layer was dried over
MgSO4, filtered, and concentrated under reduced pressure. The residue
was purified by column chromatography (hexanes/EtOAc = 7:1) to
tert-Butyl (2-(6-Chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)ethyl)-
(methyl)carbamate (13a). Compound 13a was synthesized according
1
1
afford the desired final product 9 (3.60 mmol, 77%). H NMR (400
to the general procedure of 13. Yield: 50%. H NMR (400 MHz,
MHz, CDCl3): δ 7.87 (d, J = 2.1 Hz, 1H), 7.40 (d, J = 8.2 Hz, 1H), 7.25
(dd, J = 8.2, 2.2 Hz, 1H), 3.70 (s, 2H); 13C NMR (100 MHz, CDCl3): δ
139.50, 138.55, 130.01, 129.73, 129.08, 117.02, 98.75, 22.49.
CDCl3): δ 7.39−7.35 (m, 2H), 7.17 (dd, J = 7.4, 1.6 Hz, 1H), 7.11 (br s,
2H), 7.01 (t, J = 7.4 Hz, 1H), 6.97 (d, J = 8.3 Hz, 1H), 3.78 (s, 3H), 3.44
(t, J = 8.0 Hz, 2H), 2.84−2.79 (m, 5H), 1.40 (s, 9H); 13C NMR (100
MHz, CDCl3): δ 156.74, 155.56, 137.70, 137.61, 132.15, 131.81,
130.97, 130.22, 129.35, 129.24, 128.98, 128.54, 120.35, 110.97, 79.35,
55.58, 50.62, 33.95, 28.39.
2-(6-Chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)acetonitrile (10). 2-
Methoxyphenylboronic acid 5 (7.57 mmol), Pd(PPh3)4 (0.06 mmol),
and Na2CO3 (9.46 mmol) were added to a solution of 2-(4-chloro-3-
iodophenyl)acetonitrile 9 (6.31 mmol) in DMF (30 mL). The mixture
was stirred overnight at 110 °C. After cooling down to rt, the reaction
mixture was quenched with a saturated solution of NaHCO3 and then
extracted with EtOAc. The combined organic layers were dried over
MgSO4, filtered, and concentrated under reduced pressure. The residue
was purified by flash column chromatography (hexanes/EtOAc = 10:1)
tert-Butyl (2-(6-Chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)ethyl)-
(ethyl)carbamate (13b). Compound 13b was synthesized according
1
to the general procedure of 13. Yield: 57.1%. H NMR (400 MHz,
CDCl3): δ 7.43−7.39 (m, 2H), 7.21 (dd, J = 7.4, 1.5 Hz, 1H), 7.16 (br s,
2H), 7.06 (t, J = 7.5 Hz, 1H), 7.02 (d, J = 8.3 Hz, 1H), 3.82 (s, 3H), 3.43
(t, J = 7.4 Hz, 2H), 3.24 (br s, 2H), 2.86 (br s, 2H), 1.48 (s, 9H), 1.11 (t,
J = 6.3 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ 156.75, 155.25,
137.78, 137.70, 132.16, 131.76, 130.97, 129.35, 129.23, 129.00, 128.57,
120.35, 110.98, 55.60, 48.56, 42.74, 41.92, 34.74, 34.17, 28.48, 14.23,
13.54.
1
to afford the desired product 10 (5.04 mmol, 80%). H NMR (400
MHz, CDCl3): δ 7.47−7.44 (m, 1H), 7.39 (ddd, J = 8.1, 7.8, 1.8 Hz,
1H), 7.25−7.22 (m, 2H), 7.16 (dd, J = 7.4, 1.8 Hz, 1H), 7.02 (td, J = 7.4,
1 Hz, 1H), 6.98 (d, J = 8.3 Hz, 1H), 3.77 (s, 3H), 3.73 (s, 2H); 13C
NMR (100 MHz, CDCl3): δ 156.65, 138.70, 133.95, 131.25, 130.83,
130.05, 129.81, 128.27, 127.97, 127.68, 120.49, 117.54, 111.06, 55.64,
23.11.
tert-Butyl (2-(6-Chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)ethyl)-
(propyl)carbamate (13c). Compound 13c was synthesized according
1
to the general procedure of 13. Yield: 42.4%. H NMR (400 MHz,
2-(6-Chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)ethan-1-amine (11).
1 M LiAlH4 in anhydrous THF (4.3 mL) was added dropwise under N2
to the solution of AlCl3 (3.49 mmol) in THF while stirring. The mixture
was cooled down to 0 °C before dropwise addition of the solution of 2-
(6-chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)acetonitrile 10 (3.88
mmol) in THF (10 mL). The solution was kept stirring for 2 h at 0
°C, at which time, MeOH was slowly added to quench the reaction. The
mixture was concentrated under reduced pressure and the residue was
dissolved in DCM and washed with water. The organic phase was
partitioned with 1 N solution of HCl. The combined aqueous layer was
washed with DCM and neutralized by the addition of 10 N solution of
NaOH. The mixture was partitioned with DCM and the pooled organic
layer was dried over Na2SO4, filtered, and concentrated under reduced
CDCl3): δ 7.42−7.38 (m, 2H), 7.21 (dd, J = 7.4, 1.3 Hz, 1H), 7.15 (br s,
2H), 7.05 (t, J = 7.4 Hz, 1H), 7.01 (d, J = 8.3 Hz, 1H), 3.81 (s, 3H), 3.42
(t, J = 6.5 Hz, 2H), 3.13 (d, J = 19.4 Hz, 2H), 2.85 (br s, 2H), 1.54 (br s,
2H), 1.47 (s, 9H), 0.89 (t, J = 7.4 Hz, 3H); 13C NMR (100 MHz,
CDCl3): δ 156.76, 155.50, 137.78, 137.71, 132.15, 131.77, 130.97,
129.36, 129.23, 129.01, 128.58, 120.36, 110.98, 79.22, 55.60, 49.01,
34.70, 34.04, 28.47, 21.97, 21.55, 11.30.
tert-Butyl (2-(6-Chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)ethyl)-
(butyl)carbamate (13d). Compound 13d was synthesized according
1
to the general procedure of 13. Yield: 41%. H NMR (400 MHz,
CDCl3): δ 7.41 (t, J = 7.6 Hz, 2H), 7.21 (d, J = 4.9 Hz, 1H), 7.15 (br s,
2H), 7.05 (t, J = 7.4 Hz, 1H), 7.01 (d, J = 8.3 Hz, 1H), 3.81 (s, 3H), 3.42
(br s, 2H), 3.16 (d, J = 21.4 Hz, 2H), 2.85 (s, 2H), 1.47 (s, 9H), 1.31 (q,
J = 7.4 Hz, 2H), 0.94 (t, J = 7.4 Hz, 3H); 13C NMR (100 MHz, CDCl3):
δ 156.77, 155.46, 137.79, 137.72, 132.15, 131.78, 130.97, 129.36,
129.23, 129.02, 128.59, 120.36, 110.99, 79.23, 55.36, 48.96, 47.11,
34.71, 30.88, 30.52, 28.48, 20.06, 13.91.
2-(6-Chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)-N-alkylethan-1-
amine (2). The protected amine was dissolved in 1 N HCl solution (1.5
equiv) in diethyl ether and the reaction mixture was stirred at rt
1
pressure to obtain the desired product 11 (3.88 mmol, 68.9%). H
NMR (400 MHz, CDCl3): δ 7.35−7.30 (m, 2H), 7.15 (dd, J = 7.5, 1.7
Hz, 2H), 7.12 (br s, 2H), 7.10 (d, J = 2.2 Hz, 1H), 6.97 (td, J = 7.4, 0.9
Hz, 1H), 6.93 (d, J = 8.3 Hz, 1H), 3.73 (s, 3H), 3.16 (t, J = 7.8 Hz, 2H),
3.01 (t, J = 7.8 Hz, 2H); 13C NMR (100 MHz, CDCl3): δ 156.63,
138.16, 135.12, 132.70, 131.99, 130.95, 129.64, 129.50, 128.91, 128.19,
120.41, 111.00, 55.60, 41.32, 34.10.
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overnight. A white precipitate formed after several minutes and
additional HCl was added as needed. After completing the reaction, the
precipitate was filtered, washed with diethyl ether, and concentrated
under reduced pressure. The desired products were obtained in 80−
95% yields.
2-(6-Chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)-N-methylethan-1-
amine (2a). Compound 2a was synthesized according to the general
procedure of 2. Yield: 93%; HPLC: purity 100%, tR = 6.0 min; 1H NMR
(400 MHz, DMSO-d6): δ 8.69 (br s, 2H), 7.54 (d, J = 8.1 Hz, 1H), 7.41
(t, J = 7.8 Hz, 1H), 7.36 (d, J = 2.1 Hz, 1H), 7.32 (dd, J = 8.2, 2.2 Hz,
1H), 7.02 (d, J = 2.5 Hz, 1H), 7.01−6.99 (m, 2H), 3.81 (s, 3H), 3.20 (t,
J = 7.8 Hz, 2H), 2.98 (t, J = 7.8 Hz, 2H), 2.58 (s, 3H); 13C NMR (100
MHz, DMSO-d6): δ 156.77, 138.05, 136.53, 132.46, 131.77, 131.00,
130.10, 129.68, 129.63, 128.12, 120.71, 111.78, 55.84, 49.20, 32.74,
31.06; HRMS (ESI+): calcd for C16H19ClNO+ [M + H]+, 276.1155;
found, 276.1152.
2-(6-Chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)-N-ethylethan-1-
amine (2b). Compound 2b was synthesized according to the general
procedure of 2. Yield: 91%; HPLC: purity 100%, tR = 6.1 min; 1H NMR
(400 MHz, DMSO-d6): δ 8.83 (br s, 2H), 7.47 (d, J = 8.2 Hz, 1H), 7.41
(ddd, J = 8.2, 7.4, 1.7 Hz, 1H), 7.28 (dd, J = 8.2, 2.2 Hz, 1H), 7.23 (d, J =
2.2 Hz, 1H), 7.15 (dd, J = 7.4, 1.7 Hz, 1H), 7.12 (d, J = 8.1 Hz, 1H), 7.03
(td, J = 7.3, 0.7 Hz, 1H), 3.73 (s, 3H), 3.16 (t, J = 7.9 Hz, 2H), 2.98−
2.94 (m, 4H), 1.20 (t, J = 7.3 Hz, 3H); 13C NMR (100 MHz, MeOD): δ
156.75, 138.51, 134.95, 132.64, 131.85, 130.31, 129.30, 128.62, 128.21,
120.03, 110.74, 54.56, 47.93, 42.81, 31.32, 10.13; HRMS (ESI+): calcd
for C17H21ClNO+ [M + H]+, 290.1312; found, 290.1307.
amine 11 (0.50 mmol) was dissolved in acetonitrile (3 mL). Iodoethane
(0.6 mmol) and K2CO3 (1.99 mmol) were added to the solution and
the reaction mixture was stirred for 15 h at 65 °C. The mixture was
cooled down to rt and quenched with saturated solution of NaHCO3
and then extracted with EtOAc. The combined organic layer was dried
over MgSO4, filtered, and concentrated under reduced pressure. The
residue was purified by column chromatography (DCM/MeOH =
20:1) to obtain the desired product 2f (0.32 mmol, 63%); HPLC:
purity 100%, tR = 6.5 min; 1H NMR (400 MHz, CDCl3): δ 7.40−7.36
(m, 2H), 7.19−7.15 (m, 3H), 7.02 (td, J = 7.5, 1.7 Hz, 1H), 6.98 (d, J =
8.3 Hz, 1H), 3.78 (s, 3H), 3.10−3.05 (m, 4H), 3.02 (q, J = 7.3 Hz, 4H),
1.32 (t, J = 7.3 Hz, 6H); 13C NMR (100 MHz, CDCl3): δ 156.6, 138.3,
135.1, 132.8, 130.9, 129.8, 129.6, 128.9, 128.0, 120.4, 111.0, 55.7, 55.6,
46.9, 40.2, 30.1, 9.2; HRMS (ESI+): calcd for C19H25ClNO+ [M + H]+,
318.1625; found, 318.1621.
2-(6-Chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)-N,N-dipropyle-
than-1-amine (2g). Compound 2g was synthesized according to the
general procedure of 2f. Yield: 30%; HPLC: purity 100%, tR = 7.9 min;
1H NMR (400 MHz, CDCl3): δ 7.43−7.38 (m, 2H), 7.22 (dd, J = 7.4,
1.2 Hz, 1H), 7.16−7.14 (m, 2H), 7.06 (t, J = 7.4 Hz, 1H), 7.02 (d, J =
8.4 Hz, 1H), 3.82 (s, 3H), 2.82−2.73 (m, 4H), 2.49 (t, J = 7.6 Hz, 4H),
1.52 (sext, J = 7.5 Hz, 4H), 0.91 (t, J = 7.4 Hz, 6H); 13C NMR (100
MHz, CDCl3): δ 156.80, 139.15, 137.52, 131.97, 131.36, 130.99,
129.31, 129.07, 128.95, 128.75, 120.36, 111.02, 56.14, 55.87, 55.65,
32.84, 20.29, 11.97; HRMS (ESI+): calcd for C21H29ClNO+ [M + H]+,
346.1938; found, 346.1934.
2-Chloro-2′-methoxy-5-(2-nitrovinyl)-1,1′-biphenyl (14). Ammo-
nium acetate (3.91 mmol) was added to a solution of 6-chloro-2′-
methoxy-[1,1′-biphenyl]-3-carbaldehyde 6 (3.91 mmol) in nitro-
methane (31.3 mL). The mixture was stirred for 6 h at 100 °C. After
cooling down to rt, the reaction mixture was concentrated under
reduced pressure and then the residue was purified by flash column
chromatography (hexanes/EtOAc = 20:1) to afford the desired product
14 (2.59 mmol, 66.2%). 1H NMR (400 MHz, CDCl3): δ 8.01 (d, J =
13.7 Hz, 1H), 7.61−7.56 (m, 2H), 7.50 (td, J = 8.2, 2.0 Hz, 2H), 7.45−
7.43 (m, 1H), 7.21 (dd, J = 7.4, 1.6 Hz, 1H), 7.08 (t, J = 7.5 Hz, 1H),
7.04 (d, J = 8.3 Hz, 1H), 3.82 (s, 3H); 13C NMR (100 MHz, CDCl3): δ
156.59, 139.20, 138.27, 137.96, 137.40, 132.28, 130.72, 130.50, 130.08,
128.84, 128.50, 127.08, 120.55, 111.08, 55.62.
Diethyl 2-(1-(6-Chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)-2-
nitroethyl)malonate (15). Sodium hydride (1.38 mmol) was added
to a solution of diethyl malonate (2.76 mmol) in THF (3 mL) and then
the mixture was stirred for 15 min at rt. A solution of 2-chloro-2′-
methoxy-5-(2-nitrovinyl)-1,1′-biphenyl 14 (0.69 mmol) in THF (2
mL) was then added to the mixture and the mixture was stirred for 3 h at
rt. The reaction mixture was quenched by the addition of saturated
solution of NH4Cl and then extracted with EtOAc. The combined
organic layer was dried over MgSO4, filtered, and concentrated under
reduced pressure. The residue was purified by flash column
chromatography (hexanes/EtOAc = 10:1) to obtain the desired
product 15 (0.33 mmol, 48.3%). 1H NMR (CDCl3, 400 MHz): δ 7.43−
7.41 (m, 1H), 7.40−7.38 (m, 1H), 7.20 (d, J = 7.4 Hz, 2H), 7.17 (dd, J =
7.5, 1.8 Hz, 1H), 7.04 (t, J = 7.4 Hz, 1H), 7.00 (d, J = 8.4 Hz, 1H), 4.95
(dd, J = 13.2, 4.9 Hz, 1H), 4.88 (dd, J = 9.1, 13.3 Hz), 4.30−4.20 (m,
3H), 4.08 (q, J = 7.1 Hz, 2H), 3.83 (d, J = 9.2 Hz, 1H), 3.78 (s, 3H),
1.28 (t, J = 7.1 Hz, 3H), 1.11 (t, J = 7.1 Hz, 3H); 13C NMR (100 MHz,
CDCl3): δ 167.33, 166.76, 156.67, 138.31, 134.56, 133.97, 130.92,
129.82, 129.64, 128.18, 127.84, 120.34, 111.08, 62.24, 62.04, 55.48,
54.89, 42.33, 13.96, 13.74.
2-(6-Chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)-N-propylethan-1-
amine (2c). Compound 2c was synthesized according to the general
1
procedure of 2. Yield: 92.2%; HPLC: purity 100%, tR = 6.5 min; H
NMR (400 MHz, DMSO-d6): δ 8.70 (br s, 2H), 7.47 (d, J = 8.2 Hz,
1H), 7.41 (ddd, J = 8.0, 7.3, 1.8 Hz, 2H), 7.28 (dd, J = 8.2, 2.2 Hz, 1H),
7.23 (d, J = 2.1 Hz, 1H), 7.15 (dd, J = 7.5, 1.8 Hz, 1H), 7.12 (d, J = 8.2
Hz, 1H), 7.03 (t, J = 7.4 Hz, 1H), 3.73 (s, 3H), 3.17 (t, J = 8.1 Hz, 2H),
2.96 (t, J = 8.1 Hz, 2H), 2.88 (t, J = 7.7 Hz, 2H), 1.62 (sext, J = 7.6 Hz,
2H), 0.92 (t, J = 7.4 Hz, 3H); 13C NMR (100 MHz, MeOD): δ 156.75,
138.51, 137.89, 134.97, 132.63, 131.83, 130.30, 129.29, 128.60, 128.21,
120.02, 110.74, 54.56, 48.38, 47.49, 31.28, 27.87, 19.42; HRMS (ESI
+): calcd for C18H23ClNO+ [M + H]+, 304.1468; found, 304.1464.
2-(6-Chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)-N-butyllethan-1-
amine (2d). Compound 2d was synthesized according to the general
procedure of 2. Yield: 95%; HPLC: purity 100%, tR = 6.3 min; 1H NMR
(400 MHz, DMSO-d6): δ 8.66 (br s, 2H), 7.49 (d, J = 8.1 Hz, 1H),
7.44−7.40 (m, 1H), 7.29 (dd, J = 8.3, 1.7 Hz, 1H), 7.24 (d, J = 1.6 Hz,
1H), 7.17−7.12 (m, 2H), 7.04 (t, J = 7.4 Hz, 1H), 3.74 (s, 3H), 3.19 (t, J
= 8.1 Hz, 2H), 2.98−2.90 (m, 4H), 1.63−1.55 (m, 2H), 1.34 (t, J = 7.3
Hz, 2H), 0.91 (t, J = 7.3 Hz, 3H); 13C NMR (100 MHz, MeOD): δ
156.75, 138.51, 137.89, 134.97, 132.63, 131.83, 130.30, 129.29, 128.60,
128.21, 120.02, 110.74, 54.56, 48.38, 47.49, 31.28, 27.87, 19.42, 12.47;
HRMS (ESI+): calcd for C19H25ClNO+ [M + H]+, 318.1625; found,
318.1621.
2-(6-Chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)-N,N-dimethyle-
than-1-amine (2e). A solution of 2-(6-chloro-2′-methoxy-[1,1′-
biphenyl]-3-yl)ethan-1-amine 11 (0.44 mmol) in formic acid (2.22
mmol, 88% in water solution) and formaldehyde (2.22 mmol, 37% in
water solution) was stirred at 80 °C for 20 h. The reaction mixture was
cooled down to rt, and the mixture was diluted with water, adjusted to
pH 10 with K2CO3, and extracted with DCM. The organic layer was
washed with brine, dried over MgSO4, and concentrated under reduced
pressure. The residue was purified by column chromatography (DCM/
MeOH = 10:1) to obtain the desired product 2e (0.19 mmol, 43%);
HPLC: purity 100%, tR = 5.4 min; 1H NMR (400 MHz, CDCl3): δ 7.37
(t, 2H), 7.19−7.17 (m, 3H), 7.03−6.97 (m, 2H), 3.78 (s, 3H), 2.81 (t, J
= 8.0 Hz, 2H), 2.61 (t, J = 8.0 Hz, 2H), 2.33 (s, 6H); 13C NMR (100
MHz, CDCl3): δ 156.76, 138.28, 137.66, 131.91, 131.65, 130.98,
129.36, 129.24, 128.82, 128.60, 120.36, 111.03, 60.94, 55.65, 45.13,
33.27; HRMS (ESI+): calcd for C16H19ClNO+ [M + H]+, 276.1155;
found, 276.1156.
Ethyl 4-(6-Chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)-2-oxopyrroli-
dine-3-carboxylate (16). To a solution of diethyl 2-(1-(6-chloro-2′-
methoxy-[1,1′-biphenyl]-3-yl)-2-nitroethyl)malonate 15 (3.8 mmol)
and NiCl2·6H2O (3.8 mmol) in MeOH (30 mL) was added sodium
borohydride (45.5 mmol) slowly at 0 °C. After the reaction mixture was
stirred overnight at rt, the mixture was quenched with a saturated
solution of NH4Cl and extracted with DCM. The combined organic
layer was dried over MgSO4, filtered, and concentrated under reduced
pressure. The residue was purified by flash column chromatography
(DCM/MeOH = 20:1) to obtain the desired product 16 (1.78 mmol,
55%). 1H NMR (400 MHz, CDCl3): δ 7.47 (d, J = 8.1 Hz, 1H), 7.45−
2-(6-Chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)-N,N-diethylethan-
1-amine (2f). 2-(6-Chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)ethan-1-
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7.40 (m, 1H), 7.23−7.19 (m, 3H), 7.06 (td, J = 7.4, 1.0 Hz, 1H), 7.02
(d, J = 8.3 Hz, 1H), 4.31−4.25 (m, 2H), 4.16 (q, J = 8.0 Hz, 1H), 3.89−
3.86 (m, 1H), 3.82 (s, 3H), 3.57 (d, J = 9.3 Hz, 1H), 3.50−3.46 (m,
1H), 1.32 (t, J = 7.1 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ 175.7,
169.9, 157.7, 147.0, 137.1, 130.8, 129.8, 129.3, 129.0, 128.6, 125.6,
121.5, 116.6, 60.9, 60.6, 56.1, 39.8, 29.4, 14.1.
Institute of Mental Health (NIMH) Psychoactive Drug Screening
Program (University of North Carolina, US). Eleven dilutions (5×
assay concentration) of the test and reference compounds were
prepared in standard binding buffer [50 mM tris(hydroxymethyl)-
aminomethane-HCl (Tris-HCl), 10 mM MgCl2, 1 mM ethyl-
enediaminetetraacetate, and pH 7.4] by serial dilution: 0.05 nM, 0.5
nM, 1.5 nM, 5 nM, 15 nM, 50 nM, 150 nM, 500 nM, 1.5 μM, 5 μM, and
50 μM. The [3H]LSD radioligand was diluted to 5 times the assay
concentration in standard binding buffer. Aliquots (50 mL) of the
radioligand were dispensed into the wells of a 96-well plate containing
100 mL of standard binding buffer. Triplicate aliquots (50 mL) of the
test and reference compound dilutions were then added. Finally, crude
membrane fractions (50 mL) of cells expressing a recombinant target
were dispensed into each well. Totally, 250 mL of the reaction mixtures
was incubated at rt and shielded from light for 1.5 h and then harvested
by rapid filtration onto Whatman GF/B glass fiber filters presoaked with
0.3% polyethyleneimine, by using a 96-well Brandel harvester.
Four rapid washes were performed with chilled standard binding
buffer (500 mL) to decrease nonspecific binding. Filters were placed in
6 mL scintillation tubes and allowed to dry overnight. The next day, 4
mL of EcoScint scintillation cocktail (National Diagnostics) was added
to each tube. The tubes were capped, labeled, and counted by liquid
scintillation counting. The filter mats were dried, and the scintillant was
melted onto the filters, and then the radioactivity retained on the filters
was counted in a MicroBeta scintillation counter. The IC50 values were
obtained by using the Prism 4.0 program (GraphPad software) and
converted into Ki values. Each compound was tested in triplicate at
least.
Gs Protein-Mediated cAMP Assay. HEK293 cells were harvested
in 150 mm dishes and the medium was changed from Dulbecco’s
modified Eagle’s medium (DMEM) with 10% fetal bovine serum
(FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin to DMEM
with 10% dialyzed FBS, 100 U/mL penicillin, and 100 μg/mL
streptomycin. After 5−6 h, those cells were transfected (via calcium
phosphate) with 10 μg of human 5-HT7R plasmid and 10 μg of
GloSensor-22F plasmid (Promega). Transiently transfected HEK293
cells were seeded (20,000−30,000 cells/20 μL/well) into white, clear-
bottom 384-well plates (Greiner) in the same medium. After 5−6 h of
recovery, the medium was removed from the wells and the cells were
treated with 3% GloSensor cAMP reagent, luciferin (Promega) 20 μL in
filter-sterilized assay buffer including 1× Hanks’ balanced salt solution
(HBSS), 20 mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid
(HEPES), and 3rd distilled water, pH 7.4. Then, the reference agonist
(5-HT, 400 nM) or the compounds to be tested were prepared by serial
dilution (0.04 nM, 0.12 nM, 0.4 nM, 1.2 nM, 4 nM, 12 nM, 40 nM, 120
nM, 400 nM, 1.2 μM, 4 μM, 12 μM, 40 μM, and 120 μM) in the above
assay buffer with 0.1% bovine serum albumin. After 30 min, the cells
were treated with 10 μL of drugs prepared above (the final ligand
concentrations are 100 nM of serotonin and 0.01 nM, 0.03 nM, 0.1 nM,
0.3 nM, 1 nM, 3 nM, 10 nM, 100 nM, 300 nM, 1 μM, 3 μM, 10 μM, and
30 μM of test compounds). The luminescence intensity of the
accumulated cAMP level was measured by using a microplate reader
(FlexStation 3 or SpectraMax i3, Molecular Devices). The sigmoidal
dose−response graph of the obtained data was obtained by using the
Prism 6.0 program (GraphPad software) to calculate the EC50 and IC50
values. Each compound was tested in triplicate at least.
β-Arrestin Recruitment Tango Assay. HTLA cells (a HEK293
cell line stably expressing a tTA-dependent luciferase reporter and a β-
arrestin 2−TEV fusion gene) were plated in 150 mm dishes and the
medium was changed from DMEM with 10% FBS, 100 U/mL
penicillin, 100 μg/mL streptomycin, 2 μg/mL puromycin, and 100 μg/
mL hygromycin B to DMEM with 10% dialyzed FBS, 100 U/mL
penicillin, and 100 μg/mL streptomycin to transfect the plasmid. After
5−6 h, those HTLA cells were transfected (via calcium phosphate) with
20 μg of a 5-HT7R-TCS-tTA construct. The next day, transiently
transfected HTLA cells were plated in white, clear-bottom, 384-well
plates (Greiner; 30,000−40,000 cells/well, 50 μL/well) in DMEM
containing 1% dialyzed FBS, 100 U/mL penicillin, and 100 μg/mL.
After 6 h, the cells were challenged with 10 μL/well of reference agonist
(serotonin, 6 μM) or the compounds to be tested prepared by serial
4-(6-Chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)pyrrolidin-2-one
(17). To the solution of ethyl 4-(6-chloro-2′-methoxy-[1,1′-biphenyl]-
3-yl)-2-oxopyrrolidine-3-carboxylate 16 (0.90 mmol) in EtOH (3 mL)
was added 1 N NaOH (1 mL) at rt. After the mixture was stirred for an
hour, the reaction mixture was concentrated under reduced pressure
and the residue was added to 5 N HCl and the aqueous phase was
partitioned with DCM. The combined organic layer was dried over
MgSO4, filtered, and concentrated under reduced pressure to obtain
corresponding carboxylic acid (0.81 mmol, 90%). The solution of
carboxylic acid (0.81 mmol) in toluene (10 mL) was refluxed at 140 °C
for 6 h. After cooling down to rt, the mixture was concentrated under
reduced pressure and the residue was purified by flash column
chromatography (DCM/MeOH = 7:1) to obtain the desired product
17 (0.43 mmol, 67%). 1H NMR (400 MHz, CDCl3): δ 7.42−7.35 (m,
2H), 7.17−7.14 (m, 3H), 7.01 (td, J = 7.5, 0.8 Hz, 1H), 6.98 (d, J = 8.3
Hz, 1H), 3.77 (s, 3H), 3.67 (t, J = 7.9 Hz, 1H), 3.44−3.40 (m, 1H), 2.73
(q, J = 8.6 Hz, 1H), 2.49 (q, J = 8.6 Hz, 1H), 2.28 (br s, 1H); 13C NMR
(100 MHz, CDCl3): δ 177.8, 157.7, 147.0, 137.1, 130.8, 129.8, 129.3,
129.0, 128.6, 126.4, 121.5, 116.6, 56.1, 43.3, 42.4, 32.9.
3-(6-Chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)pyrrolidine (3a).
Aluminum chloride (3.38 mmol) in THF (2 mL) was mixed with
lithium aluminum hydride 1.0 M in THF (3.38 mmol) at 0 °C. 4-(6-
Chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)pyrrolidin-2-one 17 (0.85
mmol) was added dropwise to the mixture at 0 °C and then the
reaction mixture was stirred at rt for an hour. After an hour, the reaction
mixture refluxed at 80 °C overnight. The mixture was carefully
quenched by the addition of MeOH at 0 °C and concentrated under
reduced pressure. The residue was diluted by DCM and washed with
water. The organic phase was partitioned with 1 N HCl aqueous
solution. The combined aqueous layer was washed with DCM and
neutralized by the addition of 10 N solution of NaOH. The mixture was
extracted with DCM and the combined organic layer was dried over
Na2SO4, filtered, and concentrated under reduced pressure to afford the
desired product 3a (0.62 mmol, 73%); HPLC: purity 100%, tR = 6.4
min; 1H NMR (400 MHz, CDCl3): δ 8.34 (br s, 1H), 7.44−7.37 (m,
2H), 7.29 (t, J = 9.0 Hz, 1H), 7.24−7.19 (m, 1H), 7.17−7.14 (m, 1H),
7.02 (dd, J = 7.4, 3.1 Hz, 1H), 6.97 (d, J = 8.3 Hz, 1H), 3.78 (d, J = 11.5
Hz, 3H), 3.64−3.50 (m, 2H), 3.45−3.37 (m, 1H), 3.31−3.20 (m, 1H),
2.48−2.39 (m, 1H), 2.13 (sep, J = 10.5 Hz, 1H); 13C NMR (100 MHz,
CDCl3): δ 156.63, 139.27, 138.32, 136.69, 133.16, 130.89, 129.86,
128.53, 127.18, 125.58, 120.43, 111.03, 55.61, 53.48, 50.55, 45.19,
43.75, 43.08, 32.43; HRMS (ESI+): calcd for C17H19ClNO+ [M + H]+,
288.1155; found, 288.1153.
3-(6-Chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)-1-methylpyrroli-
dine (3b). A solution of 3-(6-chloro-2′-methoxy-[1,1′-biphenyl]-3-
yl)pyrrolidine 3a (0.45 mmol) in formic acid (3.16 mmol, 88% in water
solution) and formaldehyde (3.16 mmol, 37% in water solution) was
stirred at 80 °C for 20 h. After cooling down to rt, the reaction mixture
was diluted with water, adjusted to pH 10 with K2CO3, and extracted
with DCM. The organic layer was washed with brine, dried over
MgSO4, and concentrated under reduced pressure. The residue was
purified by flash column chromatography (DCM/MeOH = 10:1) to
obtain the desired product 3b (0.17 mmol, 37%); HPLC: purity 100%,
tR = 6.2 min; 1H NMR (400 MHz, CDCl3): δ 8.13 (s, 1H), 7.41−7.36
(m, 2H), 7.23−7.16 (m, 3H), 6.97 (dd, J = 8.4, 3.3 Hz, 1H), 3.79 (d, J =
4.3 Hz, 3H), 3.48−3.40 (m, 1H), 3.11 (t, J = 8.7 Hz, 1H), 2.89 (t, J = 7.2
Hz, 1H), 2.73 (q, J = 7.7 Hz, 1H), 2.59 (t, J = 8.8 Hz, 1H), 2.47 (s, 3H),
2.43−2.34 (m, 1H), 1.98−1.90 (m, 1H); 13C NMR (100 MHz,
CDCl3): δ 156.63, 139.27, 138.32, 136.69, 133.16, 130.89, 129.86,
128.53, 127.18, 125.58, 120.43, 111.03, 55.61, 53.48, 50.55, 45.19,
43.75, 43.08, 32.43; HRMS (ESI+): calcd for C19H23ClNO2+ [M + H]+,
332.1417; found, 332.1411.
Experimental Procedure for Binding Affinity Assay. All of the
binding affinity data were generously provided by the US National
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dilution (0.06 nM, 0.18 nM, 0.6 nM, 1.8 nM, 6 nM, 18 nM, 60 nM, 180
nM, 600 nM, 1.8 μM, 6 μM, 18 μM, 60 μM, and 180 μM) in HBSS, 20
mM HEPES, and pH 7.4 (the final ligand concentrations are 1 μM of
serotonin and 0.01 nM, 0.03 nM, 0.1 nM, 0.3 nM, 1 nM, 3 nM, 10 nM,
30 nM, 100 nM, 300 nM, 1 μM, 3 μM, 10 μM, and 30 μM of test
compounds) and incubated for about 22 h. The medium was removed
and replaced with 1× BrightGlo reagent (Promega), and luminescence
was read using a SpectraMax i3 (Molecular Devices). The sigmoidal
dose−response graph of the obtained data was obtained by using the
Prism 6.0 program (GraphPad software) to calculate the EC50 and IC50
values. Each compound was tested in triplicate at least.
CYP450 Assay. All of CYP450, microsomal stability, and PK data
were provided by the New Drug Development Center or Institutional
Animal Care and Use Committees (IACUC) in Daegu-Gyeongbuk
Medical Innovation Foundation (DGMIF) (Daegu, Republic of
Korea). To human liver microsomes (0.25 mg/mL), 0.1 M phosphate
buffer (pH 7.4), a cocktail of five probe substrates (phenacetin 50 μM,
diclofenac 10 μM, S-mephenytoin 100 μM, dextromethorphan 5 μM,
and midazolam 2.5 μM), and tested compounds were added at
concentrations of 0 μM (as a control) and 10 μM. After incubation at 37
°C for 5 min, reduced nicotinamide adenine dinucleotide phosphate
(NADPH) generation system solution was also added and incubated at
37 °C for 15 min again. To terminate the reaction, acetonitrile including
the internal standard (terfenadine) was added and the solution was
centrifuged for 5 min (14,000 rpm, 4 °C). The supernatant was then
injected into the LC/MS system to simultaneously analyze the
metabolites of the probe substrates and evaluate the % CYP inhibition
of the tested compounds.
1.7 μm, Phenomenex) using gradient elution at a flow rate of 0.3 mL/
min. The lower limit of quantitation of each compound in rat plasma
was 100 ng/mL. The values of coefficients of correlation (R) were more
than 0.9971.
Self-Grooming Behavior Test. All animal experiments were
conducted according to the guidelines of the KIST IACUC. WT and
Shank3 TG male mice, 7 to 9 week old, were obtained from KIST
(Korea Institute of Science and Technology, Seoul, Korea). They were
housed at a temperature of 22 2 °C with a humidity of 55 5% and
maintained in a 12/12 h light/dark cycle under 5 dB. All mice had ad
libitum access to food and water. Drugs were dissolved in 5% dimethyl
sulfoxide in saline solution and injected into the peritoneal cavity before
30 min starting the experiment. Each mouse was placed in a 30 × 20 cm
opaque acrylic container with 1−2 cm bedding on the floor under 40 lux
lighting. The total duration and number of self-grooming behavior were
recorded for 30 min. After the test, the container was cleaned with 70%
ethanol, and the floor was covered with new bedding for the next mouse
recording. Analyze the video by stopwatch program with the function of
snap, being careful not to count sniffing behavior. In case of co-
treatment, after 10 min of SB269970 injection, 2b was administrated.
The behavior started to be recorded after 30 min of 2b injection.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00110.
■
sı
1H and 13C NMR spectra, HPLC spectra, dose−response
curves, pIC50 and IC50 values, and pKi
(PDF)
Microsomal Stability Assay. To human liver microsomes (0.5
mg/mL), 0.1 M phosphate buffer (pH 7.4) and tested compounds (1
μM) were added. After incubation at 37 °C for 5 min, the NADPH
generation system solution was also added and incubated at 37 °C for
30 min again. To terminate the reaction, acetonitrile including internal
standard (chloprapamide) was added and the solution was centrifuged
for 5 min (14,000 rpm, 4 °C). The supernatant was then injected into
the LC/MS system to analyze the microsomal stability of the tested
compounds.
SEM values
Molecular formula strings (CSV)
AUTHOR INFORMATION
Corresponding Author
■
Hyunah Choo − Brain Science Institute, Korea Institute of
Science and Technology, Seoul 02792, Republic of Korea;
Division of Bio-Medical Science and Technology, KIST School,
University of Science and Technology, Seoul 02792, Republic of
Korea; orcid.org/0000-0002-3545-2678; Phone: +82-2-
958-5157; Email: hchoo@kist.re.kr
PK Studies. All animal experiments were evaluated and approved by
the DGMIF IACUC. ICR mice (7−8 weeks of age) weighing 30 5 g
were used for the PK and tissue distribution studies and were purchased
from Orient Co. (South Korea). The mice were kept at rt controlled at
23 3 °C with relative humidity controlled at about 55 10%, fed with
standard solid composite feedstuff, and received tap water. Compound
2b at a dose of 1 or 10 mg/kg was administered intravenously or
intraperitoneally, respectively, to male ICR mice. Blood samples were
collected via carotid artery at 0 (to serve as a control), 0.08 (IV only),
0.25, 0.5, 1, 2, 4, 6, and 8 h after administration of each compound. After
centrifugation at 12,000 rpm for 3 min, plasma samples were stored at
−70 °C until analysis. PK parameters were determined by a non-
compartmental analysis using WinNonlin v6.4 (Pharsight Corporation,
Mountain View, CA) program. The total area under the plasma
concentration−time curve from time zero to the last measured time
(AUClast) was calculated by the trapezoidal rule-extrapolation method.
Standard methods were used to calculate the following PK
parameters:46,47 the time-averaged total body clearance (CL), total
area under the first moment of plasma concentration and time curve
from time zero to time infinity (AUC0−∞), terminal half-life, mean
residence time (MRT), and apparent volume of distribution at steady
state (Vdss). The concentrations of each compound in the above
samples were analyzed using LC−MS/MS. To a 20 μL aliquot of
plasma sample, an 80 μL aliquot of acetonitrile containing 2 μM of
internal standard (chloropropramide) was added. After vortex mixing
and centrifugation at 15,000 rpm for 5 min, a 2 μL of supernatant was
injected into the LC−MS/MS system. The LC−MS/MS system
consisted of an Agilent 1290 infinity series HPLC system (Agilent,
Santa Clara, CA) and API5500 triple-quadrupole mass spectrometer
(Applied Biosystems-SCIEX, Concord, Canada). The HPLC mobile
phases consisted of 0.1% formic acid in 100% deionized water (A) and
0.1% formic acid in 100% acetonitrile (B). Chromatographic separation
was achieved on a reversed-phase Kinetex C18 column (100 × 2.1 mm,
Authors
Jieon Lee − Brain Science Institute, Korea Institute of Science
and Technology, Seoul 02792, Republic of Korea; Division of
Bio-Medical Science and Technology, KIST School, University
of Science and Technology, Seoul 02792, Republic of Korea
Rina Kwag − Brain Science Institute, Korea Institute of Science
and Technology, Seoul 02792, Republic of Korea; Department
of Chemistry, Korea University, Seoul 02841, Republic of
Korea
Soyeon Lee − Brain Science Institute, Korea Institute of Science
and Technology, Seoul 02792, Republic of Korea; Department
of Chemistry, Korea University, Seoul 02841, Republic of
Korea
Doyoung Kim − Brain Science Institute, Korea Institute of
Science and Technology, Seoul 02792, Republic of Korea;
Department of Chemistry, Sogang University, Seoul 04107,
Republic of Korea
Jiwan Woo − Research Animal Resource Center, Korea Institute
of Science and Technology, Seoul 02792, Republic of Korea
Yakdol Cho − Research Animal Resource Center, Korea
Institute of Science and Technology, Seoul 02792, Republic of
Korea
Hak Joong Kim − Department of Chemistry, Korea University,
Seoul 02841, Republic of Korea; orcid.org/0000-0002-
3213-2230
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Article
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signalling controls unique cellular responses. Biochem. Soc. Trans. 2016,
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Jeongjin Kim − Brain Science Institute, Korea Institute of
Science and Technology, Seoul 02792, Republic of Korea
Byungsun Jeon − Brain Science Institute, Korea Institute of
Science and Technology, Seoul 02792, Republic of Korea
(6) Komolov, K. E.; Benovic, J. L. G protein-coupled receptor kinases:
past, present and future. Cell. Signalling 2018, 41, 17−24.
(7) Peterson, Y. K.; Luttrell, L. M. The diverse roles of arrestin
scaffolds in G protein−coupled receptor signaling. Pharmacol. Rev.
2017, 69, 256−297.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00110
Author Contributions
(8) Marty, C.; Ye, R. D. Heterotrimeric G protein signaling outside the
realm of seven transmembrane domain receptors. Mol. Pharmacol.
2010, 78, 12−18.
The first two authors J.L. and R.K. contributed equally to this
work. The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
(9) Smith, J. S.; Lefkowitz, R. J.; Rajagopal, S. Biased signalling: from
simple switches to allosteric microprocessors. Nat. Rev. Drug Discovery
2018, 17, 243−260.
Notes
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of GPCR signaling complexes. Nat. Struct. Mol. Biol. 2018, 25, 4−12.
(11) Modica, M. N.; Lacivita, E.; Intagliata, S.; Salerno, L.; Romeo, G.;
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
̀
Pittala, V.; Leopoldo, M. Structure-activity relationships and
■
therapeutic potentials of 5-HT7 receptor ligands: an update. J. Med.
Chem. 2018, 61, 8475−8503.
We are grateful to the US National Institute of Mental Health
(NIMH) Psychoactive Drug Screening Program (contract:
HHSN-271-2008-00025-C) for providing binding affinity data.
This research is supported by the Original Technology Research
Program (NRF-2016M3C7A1904344) and the Basic Science
Research Program (NRF-2021R1A2C2006244) funded by the
National Research Foundation of Korea (NRF). In addition, this
work is additionally funded by the Korea Institute of Science and
Technology (KIST) Institutional Program (2E30961 and
2E30960).
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ABBREVIATIONS
■
AC, adenylyl cyclase; AlCl3, aluminum chloride; DMEM,
Dulbecco’s modified Eagle’s medium; FBS, fetal bovine
serum; HBSS, Hanks’ balanced salt solution; LiAlH4, lithium
aluminum hydride; Boc2O, di-tert-butyl dicarbonate; cAMP,
cyclic adenosine monophosphate; CNS, central nervous system;
CYP450, cytochrome P450; I2, iodine; EC50, half maximal
effective concentration; H2SO4, sulfuric acid; GPCRs, G
protein-coupled receptors; HEK293, human embryonic kidney
cells 293; [3H]LSD, [3H]lysergic acid diethylamide; 5-HT, 5-
hydroxytryptamine; 5-HT7R, 5-HT7 receptor; HPP, hydro-
phobic binding pocket; HTLA, HEK293-derived cell line
containing stable integrations of a tTA-dependent; IC50, half
maximal inhibitory concentration; Ki, binding affinity value;
NaIO3 , sodium iodate; Pd(PPh 3 ) 4 , tetr akis-
(triphenylphosphine)palladium(0); PKs, pharmacokinetic pa-
rameters; NaBH(OAc)3, sodium triacetoxyborohydride; NaH,
sodium hydride; SAR, structure−activity relationship;
NaHCO3, sodium bicarbonate; K2CO3, potassium carbonate;
THF, tetrahydrofuran; DMF, N,N-dimethylformamide; NaCN,
sodium cyanide; NaBH4, sodium borohydride; NiCl2·6H2O,
nickel(II) chloride hexahydrate
Bunemann, M.; Levy, F. O. Related GPCRs couple differently to Gs:
̈
preassociation between G protein and 5-HT7 serotonin receptor
reveals movement of Galphas upon receptor activation. FASEB J. 2018,
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M.; Chaumont-Dubel, S.; Claeysen, S.; Cunningham, K. A.; Fone, K.
C.; Gershon, M.; Di Giovanni, G.; Goodfellow, N. M.; Halberstadt, A.
L.; Hartley, R. M.; Hassaine, G.; Herrick-Davis, K.; Hovius, R.; Lacivita,
E.; Lambe, E. K.; Leopoldo, M.; Levy, F. O.; Lummis, S. C. R.; Marin,
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