Discovery of β-arrestin-biased β2-adrenoceptor agonists from 2-amino-2-phenylethanol derivatives

Article abstract of DOI:10.1038/s41401-018-0200-x

β-Arrestins are a small family of proteins important for signal transduction at G protein-coupled receptors (GPCRs). β-Arrestins are involved in the desensitization of GPCRs. Recently, biased ligands possessing different efficacies in activating the G protein- versus the β-arrestin-dependent signals downstream of a single GPCR have emerged, which can be used to selectively modulate GPCR signal transduction in such a way that desirable signals are enhanced to produce therapeutic effects while undesirable signals of the same GPCR are suppressed to avoid side effects. In the present study, we evaluated agonist bias for compounds developed along a drug discovery project of β₂-adrenoceptor agonists. About 150 compounds, including derivatives of fenoterol, 2-amino-1-phenylethanol and 2-amino-2-phenylethanol, were obtained or synthesized, and initially screened for their β-adrenoceptor-mediated activities in the guinea pig tracheal smooth muscle relaxation assay or the cardiomyocyte contractility assay. Nineteen bioactive compounds were further assessed using both the HTRF cAMP assay and the PathHunter β-arrestin assay. Their concentration-response data in stimulating cAMP synthesis and β-arrestin recruitment were applied to the Black–Leff operational model for ligand bias quantitation. As a result, three compounds (L-2, L-4, and L-12) with the core structure of 5-(1-amino-2-hydroxyethyl)-8-hydroxyquinolin-2(1H)-one were identified as a new series of β-arrestin-biased β₂-adrenoceptor agonists, whereas salmeterol was found to be G_s-biased. These findings would facilitate the development of novel drugs for the treatment of both heart failure and asthma.

Full text of DOI:10.1038/s41401-018-0200-x

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ARTICLE
Discovery of β-arrestin-biased β2-adrenoceptor agonists from
2-amino-2-phenylethanol derivatives
Anthony Yiu-Ho Woo1,2, Xin-yue Ge3,4, Li Pan3,4, Gang Xing3,4, Yong-mei Mo3,4, Rui-juan Xing3,4,9, Xiao-ran Li1, Yu-yang Zhang1,
Irving W. Wainer5,6, Mao-sheng Cheng3,4 and Rui-ping Xiao2,7,8
β-Arrestins are a small family of proteins important for signal transduction at G protein-coupled receptors (GPCRs). β-Arrestins are
involved in the desensitization of GPCRs. Recently, biased ligands possessing different efficacies in activating the G protein- versus
the β-arrestin-dependent signals downstream of a single GPCR have emerged, which can be used to selectively modulate GPCR
signal transduction in such a way that desirable signals are enhanced to produce therapeutic effects while undesirable signals of
the same GPCR are suppressed to avoid side effects. In the present study, we evaluated agonist bias for compounds developed
along a drug discovery project of β2-adrenoceptor agonists. About 150 compounds, including derivatives of fenoterol, 2-amino-1-
phenylethanol and 2-amino-2-phenylethanol, were obtained or synthesized, and initially screened for their β-adrenoceptor-
mediated activities in the guinea pig tracheal smooth muscle relaxation assay or the cardiomyocyte contractility assay. Nineteen
bioactive compounds were further assessed using both the HTRF cAMP assay and the PathHunter β-arrestin assay. Their
concentration-response data in stimulating cAMP synthesis and β-arrestin recruitment were applied to the Black–Leff operational
model for ligand bias quantitation. As a result, three compounds (L-2, L-4, and L-12) with the core structure of 5-(1-amino-2-
hydroxyethyl)-8-hydroxyquinolin-2(1H)-one were identified as a new series of β-arrestin-biased β2-adrenoceptor agonists, whereas
salmeterol was found to be Gs-biased. These findings would facilitate the development of novel drugs for the treatment of both
heart failure and asthma.
Keywords: β2-adrenoceptor agonists; cAMP; β-arrestin; biased agonism; fenoterol; salmeterol; heart failure; asthma
Acta Pharmacologica Sinica (2019) 0:1–11; https://doi.org/10.1038/s41401-018-0200-x
INTRODUCTION
signaling but as an agonist for β-arrestin-dependent signaling in a
single GPCR.
G protein-coupled receptors (GPCRs) represent the largest class of
druggable targets [1]. In recent years, functional selectivity has
emerged as a major concept for the redevelopment of many
traditional GPCR drug targets, such as β-adrenoceptors [2–6]. This
concept suggests that a so-called biased ligand could be used to
selectively modulate GPCR signal transduction in such a way that
desirable signals are enhanced to produce therapeutic effects,
whereas undesirable signals of the same GPCR are suppressed to
avoid side effects. The terms “functional selectivity” and “biased
agonism” are often used interchangeably. Although “biased
agonism” is sometimes used in a stricter sense to refer to
“β-arrestin-biased agonism” [7], it generally describes the disparity
of the efficacies of ligands in activating signals mediated by
different downstream effectors, for example, different G protein
isoforms, G protein versus β-arrestin or biases from many other
signaling readouts. In some cases, the biased ligand could act as
an antagonist or an inverse agonist for G protein-dependent
Ligand biases at both β1- and β2-adrenoceptors have been
described [6]. For example, carvedilol is an antagonist of both
β-adrenoceptors for the Gs pathway, but it is also mildly active
in triggering β-arrestin-dependent signaling [8, 9]. It has been
suggested that the superior efficacy of carvedilol compared with
other β-blockers in heart failure may be due to the activation
of the β-adrenoceptor-mediated β-arrestin-dependent signaling
[7, 8].
Although it is well-established that long-term stimulation of the
β1-adrenoceptor would lead to cardiomyocyte death and predis-
poses the heart to failure [10–13], the role of the β2-adrenoceptor
is still unclear. As the β2-adrenoceptor couples to both Gs and Gi
proteins [14] and β2-adrenoceptor-Gi signaling becomes exagger-
ated in heart failure [15–17], causing various adverse structural
and functional consequences in the heart [17–20], based on the
concept of functional selectivity, a Gs-biased β2-adrenoceptor
1Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang 110016, China; 2State Key Laboratory of Membrane Biology, Institute of Molecular Medicine,
Peking University, Beijing 100871, China; 3Department of Medicinal Chemistry, Shenyang Pharmaceutical University, Shenyang 110016, China; 4Key Laboratory of Structure-Based
Drug Design and Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, China; 5Laboratory of Clinical Investigation, National Institute on
Aging, NIH, Baltimore, MD 21224, USA; 6Mitchell Woods Pharmaceuticals, LLC, Shelton, CT 06484, USA; 7Centers for Life Sciences, Peking University, Beijing 100871, China and
8Beijing City Key Laboratory of Cardiometabolic Molecular Medicine, Peking University, Beijing 100871, China
Correspondence: Mao-sheng Cheng (mscheng@syphu.edu.cn) or Rui-ping Xiao (xiaor@pku.edu.cn)
9Present address: School of Pharmacy, Hebei Medical University, Shijiazhuang 050017, China
These authors contributed equally: Anthony Yiu-Ho Woo, Xin-yue Ge.
Received: 5 July 2018 Accepted: 26 November 2018
© CPS and SIMM 2019

Novel β-arrestin-biased β2-adrenoceptor agonists
AYH Woo et al.
2
agonist, fenoterol (FEN), was discovered from screening and
subsequently tested on an animal model of heart failure [21].
The efficacy of FEN, used alone or in combination with a
β1-adrenoceptor antagonist, in treating heart failure has subse-
quently been demonstrated in follow-up studies [22–26]. There-
fore, we have proposed the use of Gs-biased β2-adrenoceptor
agonists to treat heart failure [20, 21, 27].
designed and synthesized at Shenyang Pharmaceutical University by
methods adopted from our previous studies [34, 36, 37]. The steps
for the syntheses of the target compounds are illustrated in
Scheme 1 for A-17, A-18, A-23, A-31, A-32, and A-33; Scheme 2 for
B-24 and B-30; and Scheme 3 for L-2, L-4, L-6, and L-12.
Compounds A-33, B-24, L-2, L-4, L-6, and L-12 are novel. The
purities of all of the biologically evaluated compounds exceeded
95%. All test compounds are phenylethanolamines. Phenylethano-
lamine is the basic structure of β-agonists. Stock solutions of the
compounds were prepared in dimethylsulfoxide (DMSO). Dilutions
were made with buffer solutions. The final concentration of DMSO in
the assays did not exceed 1%, and at this concentration, DMSO did
not interfere with the assays.
Our group is committed to developing β2-adrenoceptor
agonists for clinical use. Characterization of the signal transduction
properties of β2-adrenoceptor agonists is one of our major
objectives because it can provide us with experimental com-
pounds with different pharmacological properties, from which we
can choose compounds for further research and development. We
have synthesized over 90 derivatives of FEN [28–31]. Several Gs-
biased β2-adrenoceptor agonists, such as (R,R’)-FEN (Supplemen-
tary Fig. S1), have been discovered from this cohort [32, 33]. We
have synthesized other β2-adrenoceptor agonists including
compounds with the classical 2-amino-1-phenylethanol core
structure or those containing a novel 2-amino-2-phenylethanol
core structure [34–37]. These compounds exhibit β2-adrenoceptor
agonistic activities and tracheal smooth muscle relaxant effects,
and many of them are also more selective towards the
β2-adrenoceptor over the β1-adrenoceptor [34–37]. The aim of
the present study is to characterize the β-arrestin-biased agonism
at the β2-adrenoceptor for some of these novel compounds.
General procedures for 2-amino-1-phenylethanol (A-series) and
2-amino-2-phenylethanol (B-series) compounds
A-series compounds were prepared by combining different
substituted 1-(4-aminophenyl)-2-bromoethanone derivatives with
various amines. The carbonyl group in each intermediate was then
reduced by sodium borohydride to afford the 2-aminoethanol
product (Scheme 1). B-series compounds were synthesized by
coupling different substituted 2-bromo-1-phenylethanol com-
pounds with various amines after reduction of the ethanone
(Scheme 2). Finally, all of the target compounds were treated with
a saturated solution of hydrogen chloride in isopropanol to form
hydrochlorides.
MATERIALS AND METHODS
Chemistry
1-(4-Amino-3-chloro-5-cyano-phenyl)-2-(3-ethoxy)propylamino-
ethanol hydrochloride (A-33)
The 1H and 13C spectra (Supplementary Fig. S2) were recorded on
a Bruker (Billerica, MA, USA) ARX-300, ARX-400, or ARX-600 NMR
spectrometer using DMSO-d6 as the solvent. Chemical shifts (δ)
were reported in parts per million (ppm) relative to tetramethylsi-
lane (internal standard), and coupling constants (J) were reported
in Hz. High-resolution mass spectrometry (HRMS) was performed
on a Bruker SolariX 7.0T. All reactions were monitored by thin layer
chromatography (TLC) using TLC plates (Silica gel60 GF254, Merck)
and UV light visualization. When appropriate, crude products were
purified by column chromatography using silica gel (200–300
mesh) purchased from the Qingdao Haiyang Chemical Co. Ltd
(China). The purity of the final products was determined by high
performance liquid chromatography (Waters Corp., Milford, MA,
USA) on a C18 column (250 mm × 4.6 mm, 5 µm bead size; Thermo
Fisher Scientific, Waltham, MA, USA) with acetonitrile/H2O (33/67
v/v, the pH was adjusted to 5.30 with H3PO4 and NaH2PO4) as the
mobile phase at a 1.0 mL/min flow rate and a detection
wavelength of 259 or 254 nm. The structures of newly synthesized
compounds were confirmed by 1H-NMR, 13C-NMR, and HRMS,
whereas the structures of other compounds were confirmed by
1H-NMR and low-resolution mass spectrometry (MS).
To a stirred solution of 2-amino-5-(2-bromoacetyl)-3-chlorobenzo-
nitrile
(5.00 g,
0.018 mol)
in
50 mL
of
ethanol,
3-ethoxypropylamine (5.63 g, 0.054 mol) was added slowly under
an atmosphere of nitrogen at 5 °C. The reaction mixture was
stirred at room temperature for 5 h. Following the addition of 4 mL
of water, sodium borohydride (0.68 g, 0.018 mol) was added
portion-wise, and the reaction mixture was further stirred for 5 h.
Aqueous 2 mol/L HCl was then added to adjust the pH to 3,
followed by stirring for 30 min. Ammonia water was added to
adjust the pH to 10, followed by evaporation under vacuum. The
residue was mixed with 50 mL of water, and ammonia water was
added again until the pH = 10. The mixture was extracted with
ethyl acetate (3 × 50 mL). Organic layers were combined, and the
ethyl acetate was evaporated under reduced pressure. The residue
was extracted with aqueous 2 mol/L HCl (3 × 60 mL). Aqueous
layers were combined, and activated carbon was added. The
mixture was heated to reflux for 20 min and then filtered. After
cooling to room temperature, the pH was adjusted to 10 with
ammonia water. The mixture was extracted with diethyl ether (3 ×
50 mL). The combined organic layers were washed with water,
dried over anhydrous sodium sulfate, and filtered. A saturated
solution of hydrogen chloride in isopropanol was added to the
filtrate until the pH = 3. Filtration of the resulting mixture gave
A-33 as a white solid (0.80 g, 14.8%). 1H-NMR (400 MHz, DMSO-d6,
ppm) δ: 8.93 (br, 1H), 8.69 (br, 1H), 7.56 (s, 1H), 7.46 (s, 1H), 6.30
(s, 2H), 6.23(s, 1H), 4.83 (d, J = 3.18 Hz, 1H), 3.38–3.45 (m, 4H),
2.98–3.11 (m, 4H), 1.89 (s, 2H), 1.11 (d, J = 4.17, 3H). 13C-NMR δ:
147.2, 132.5, 130.7, 129.9, 118.9, 117.6, 95.6, 67.5, 67.1, 65.8, 53.1,
45.6, 26.1, 15.5. MS (EI) m/z 298, 206, 116, 44. HRMS ([M + H]+) m/z
calcd for C14H21ClN3O2 298.1322; found 298.1329.
Materials
All solvents and reagents were obtained from commercial
suppliers and were used as received. All biochemical reagents
were obtained from Sigma-Aldrich (St. Louis, MO, USA) unless
otherwise noted.
Compounds
(R)-Epinephrine ((R)-EPI), FEN, and (R)-isoproterenol ((R)-ISO) were
purchased from Sigma-Aldrich. Salmeterol (SAL) was purchased
from Adamas-Beta (Shanghai, China). The synthesis of (R,R’)-FEN
and (R,R’)-4-methoxy-1-naphthylfenoteol ((R,R’)-MNF) has been
described previously [29–31]. They were kindly provided by Dr.
Joseph Kozocas from SRI International in California, USA (Supple-
mentary Fig. S1). Compounds with a 2-amino-1-phenylethanol core
structure including A-17, A-18, A-23, A-31, A-32, A-33, and A-35, as
well as those with a 2-amino-2-phenylethanol core structure
including B-24, B-30, L-2, L-4, L-6, and L-12 (Table 1) were
2-(4-Amino-3-cyano-phenyl)-2-isopropylamino-ethanol
hydrochloride (B-24)
To a stirred solution of 2-amino-5-(2-bromo-1-hydroxyethyl)-
benzonitrile (2.45 g, 0.016 mol) in 50 mL of ethanol, isopropyl-
amine (2.14 g, 0.036 mol) was slowly added. The mixture was
heated to reflux for 13 h. Following the evaporation of most of
the ethanol, the residue was extracted with aqueous 2 mol/L HCl
(3 × 50 mL). Aqueous layers were combined and washed with
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Novel β-arrestin-biased β2-adrenoceptor agonists
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(br, 2H), 7.71 (d, J = 1.65 Hz, 1H), 7.56-7.60 (dd, J = 8.69 Hz, 1.73 Hz,
1H), 6.82 (d, J = 8.69 Hz, 1H), 6.28 (s, 2H), 5.55 (s, 1H), 4.20 (d, J =
0.56 Hz, 1H), 3.85 (s, 1H), 3.73 (s, 1H), 2.95–3.00 (m, 1H), 1.27 (d, J =
6.42 Hz, 3H), 1.18 (d, J = 6.42 Hz, 3H). 13C-NMR δ: 152.39, 134.65,
133.51, 121.13, 118.26, 115.94, 93.64, 62.61, 59.66, 47.83, 19.74,
18.00. MS (ESI) m/z 219.9 [M + H]-. HRMS ([M + H]+) m/z calcd for
C12H18N3O 220.1450; found 219.1441.
Table 1 Structures of the compounds synthesized and tested
General procedures for L-2, L-4, L-6, and L-12
Synthesis of the target compounds L-2, L-4, L-6, and L-12 was
achieved through the pathway illustrated in Scheme 3. The
starting material 8-hydroxyquinoline (commercially purchased)
was reacted with meta-chloroperoxybenzoic acid (mCPBA) in
dichloromethane (DCM) to give 8-hydroxyquinoline-1-oxide 2,
which was subsequently acetylated to give 8-acetoxyquinolin-
2(1H)-one 3. 5-Acetyl-8-hydroxyquinolin-2(1H)-one 4 was synthe-
sized from intermediate 3 by a Fries rearrangement with
aluminum trichloride and acetyl chloride as catalysts along with
1,2-dichloroethane (DCE) as the solvent. The phenolic hydroxyl
group of intermediate 4 was benzyl protected to give 5-acetyl-8-
benzyloxyquinolin-2(1H)-one 5, and then, the keto-carbonyl group
was brominated to give 8-benzyloxy-5-(2-bromoacetyl)quinolin-
2(1H)-one 6. Reduction of 6 with sodium borohydride afforded 8-
benzyloxy-5-(2-bromo-1-hydroxyethyl)quinolin-2(1H)-one 7, which
was subjected to intramolecular nucleophilic substitution to afford
the epoxide 8-benzyloxy-5-(2-oxiranyl)quinolin-2(1H)-one 8.
Detailed methods for the synthesis of compounds 1–8 can be
found in Supplementary Methods. Intermediates 8-benzyloxy-5-[2-
hydroxy-1-(isopropylamino)ethyl]quinolin-2(1H)-one hydrochloride
9a,
2(1H)-one
8-benzyloxy-5-[2-hydroxy-1-(tert-butylamino)ethyl]quinolin-
hydrochloride 9b, 8-benzyloxy-5-[2-hydroxy-1-
(N-propylamino)ethyl]quinolin-2(1H)-one hydrochloride 9c and
8-benzyloxy-5-[2-hydroxy-1-(N-hexylamino)ethyl]quinolin-2(1H)-
one hydrochloride 9d were prepared by refluxing 8 with different
amines in the presence of zinc chloride as the catalyst and
acetonitrile as the solvent. The target compounds L-2, L-4, L-6,
and L-12 were synthesized from 9a, 9b, 9c, and 9d by
hydrogenation with Pd/C in methanol.
Procedure A: 8-Benzyloxy-5-[2-hydroxy-1-(isopropylamino)ethyl]
quinolin-2(1H)-one hydrochloride (9a)
To a stirred solution of intermediate 8 (3.0 g, 0.0102 mol) and zinc
chloride (0.35 g, 0.0026 mol) in 150 mL of acetonitrile, isopropyla-
mine (2.63 mL, 0.0307 mol) was added drop-wise. The reaction
mixture was heated slowly under reflux for 12 h. Then, the solvent
was evaporated. The product was purified by column chromato-
graphy (CH2Cl2:CH3OH:NH4OH = 350:10:1). After evaporation, the
solid was dissolved in 50 mL of acetone, followed by acidification
with a saturated solution of hydrogen chloride in isopropanol to a
pH of 2. Filtration of the resulting mixture gives 9a as a yellow
solid (0.82 g, 20.6%). 1H-NMR (400 MHz, DMSO-d6, ppm) δ: 10.84 (s,
1H), 9.45 (s, 1H), 9.23 (s, 1H), 8.32–8.33 (d, J = 10.02 Hz, 1H),
7.59–7.61 (m, 3H), 7.38–7.41 (m, 2H), 7.32–7.34 (m, 2H), 6.62–6.63
(d, J = 9.90 Hz, 1H), 5.53 (s, 1H), 5.34 (s, 2H), 4.89–4.92 (m, 1H),
3.87–3.94 (m, 1H), 3.74–3.82 (m, 1H), 3.05–3.12 (m, 1H), 1.29 (d, J =
5.76 Hz, 3H), 1.14 (d, J = 6.12 Hz, 3H). 13C-NMR δ: 161.2, 144.8,
137.0, 136.2, 130.1, 128.9, 128.5, 128.4, 123.7, 123.3, 121.6, 118.5,
112.7, 70.3, 63.3, 55.3, 48.4, 19.6, 18.6. HRMS ([M + H]+) m/z calcd
for C21H25N2O3 353.1865; found 353.1854.
methyl benzene. Activated carbon was then added. The mixture
was heated to reflux for 20 min and then filtered. After cooling to
room temperature, ammonia water was added to adjust the pH to
10. The mixture was extracted with diethyl ether (3 × 50 mL). The
combined organic layers were washed with water, dried over
anhydrous sodium sulfate, and filtered. A saturated solution of
hydrogen chloride in isopropanol was added to the filtrate until
the pH = 3. Filtration of the resulting mixture gave B-24 as a white
solid (0.50 g, 15.2%). 1H-NMR (300 MHz, DMSO-d6, ppm) δ: 9.10
8-Benzyloxy-5-[2-hydroxy-1-(tert-butylamino)ethyl]quinolin-2(1H)-
one hydrochloride (9b)
In the presence of zinc chloride (0.35 g, 0.0026 mol), intermediate 8
(3.0 g, 0.0102 mol) was reacted with tert-butylamine (2.27 g, 0.0307
mol) in 150 mL of acetonitrile according to procedure A. The crude
product was purified by column chromatography (CH2Cl2:CH3OH:
NH4OH = 350:10:1). Evaporation of the solvent and acidification of
the product gives 9b as a white solid (0.64 g, 17.11%). 1H-NMR (600
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Novel β-arrestin-biased β2-adrenoceptor agonists
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Scheme 1 Synthesis of 2-amino-1-phenylethanol derivatives. Reagents and conditions: (i) amines, ethanol, 5 °C to room temperature (rt), 5 h;
(ii) NaBH4, ethanol and water, rt
(m, 1H), 5.35 (s, 2H), 4.99–4.81 (s, 1H), 3.86 (dd, J = 11.6, 6.2 Hz, 1H),
3.79 (dd, J = 11.6, 4.9 Hz, 1H), 2.88 (s, 1H), 2.67 (s, 1H), 1.65 (ddd, J =
27.9, 15.5, 7.4 Hz, 2H), 0.81 (t, J = 7.4 Hz, 3H). 13C-NMR δ: 160.6, 144.2,
136.4, 135.9, 129.5, 128.3, 127.9, 127.8, 123.1, 122.9, 120.8, 188.0,
112.1, 69.6, 62.1, 47.0, 18.8, 10.9. HRMS ([M + H]+) m/z calcd for
C21H25N2O3 353.1865; found 353.1860.
8-Benzyloxy-5-[2-hydroxy-1-(N-hexylamino)ethyl]quinolin-2(1H)-
one hydrochloride (9d)
In the presence of zinc chloride (0.35 g, 0.0026 mol), intermediate
8 (3.0 g, 0.0102 mol) was reacted with N-hexylamine (2.64 mL,
0.0307 mol) in 150 mL of acetonitrile according to procedure A.
The crude product was purified by column chromatography
(CH2Cl2:CH3OH:NH4OH = 350:10:1). Evaporation of the solvent and
acidification of the product gave 9d as a white solid (0.60 g,
13.6%). 1H-NMR (400 MHz, DMSO-d6, ppm) δ: 10.84 (s, 1H), 9.52 (s,
1H), 9.20 (s, 1H), 8.24 (d, J = 10.02 Hz, 1H), 7.51–7.60 (m, 3H),
7.29–7.41 (m, 4H), 6.62 (d, J = 9.87 Hz, 1H), 5.52 (br, 1H), 5.34 (s,
2H), 4.89 (t, J = 7.68, 1H), 3.75–3.91 (s, 2H), 2.86–2.89 (m, 1H), 2.70
(m, 1H), 1.59–1.62 (m, 2H), 1.17–1.24 (m, 6H), 0.79–0.83 (m, 3H).
13C-NMR δ: 161.2, 144.7, 137.0, 136.5, 130.0, 128.8, 128.4, 128.4,
123.7, 123.4, 121.4, 118.6, 112.6, 70.2, 62.7, 57.7, 46.0, 31.1, 26.1,
25.6, 22.2, 14.2. HRMS ([M + H]+): m/z calcd for C24H31N2O3
395.2335, found 395.2325.
Scheme 2 Synthesis of B-24 and B-30. Reagents and conditions: (i)
isopropylamine, ethanol, reflux, 13 h
MHz, DMSO-d6, ppm) δ: 10.86 (s, 1H), 9.31 (s, 1H), 8.65 (s, 1H), 8.39 (s,
J = 7.5 Hz, 1H), 7.60 (d, J = 4.8, 2H), 7.39 (t, J = 4.88 Hz, 2H), 7.32 (t, J
= 4.84 Hz, 2H), 6.63 (d, J = 6.60 Hz, 1H), 5.52 (s, 1H), 5.34 (s, 2H), 4.91
(s, 1H), 3.89 (s, 1H), 3.80–3.82 (m, 1H), 1.23 (s, 9H). 13C-NMR δ: 160.1,
143.6, 135.9, 135.3, 128.9, 127.8, 127.4, 127.3, 124.4, 122.5, 121.2,
116.9, 111.5, 69.1, 63.0, 58.1, 53.0, 25.6. HRMS ([M + Cl]-) m/z calcd for
C22H26ClN2O3 401.1632; found 401.1653.
Procedure B: 8-Hydroxy-5-[2-hydroxy-1-(isopropylamino)ethyl]
quinolin-2(1H)-one hydrochloride (L-2)
8-Benzyloxy-5-[2-hydroxy-1-(N-propylamino)ethyl]quinolin-2(1H)-
one hydrochloride (9c)
To a solution of intermediate 9a (150 mg, 0.386 mmol) in 50 mL of
methanol, 10% Pd/C (20.0 mg) was added. The reaction mixture
was stirred under an atmosphere of hydrogen at room
temperature for 2 h. The resulting mixture was filtered and
In the presence of zinc chloride (0.35 g, 0.0026 mol), intermediate 8
(3.0 g, 0.0102 mol) was reacted with N-propylamine (1.8 g, 0.0307
mol) in 150 mL of acetonitrile according to procedure A. The crude
product was purified by column chromatography (CH2Cl2:CH3OH:
NH4OH = 350:10:1). Evaporation of the solvent and acidification of
the product gives 9c as a white solid (0.47 g, 13.09%). 1H-NMR (600
MHz, DMSO-d6, ppm) δ: 10.90 (s, 1 H), 9.47 (s, 1 H), 9.15 (s, 1H), 8.24
(d, J = 10.1 Hz, 1H), 7.60 (d, J = 7.3 Hz, 2H), 7.50 (d, J = 8.5 Hz, 1H),
7.39 (t, J = 7.5 Hz, 2H), 7.33 (s, 2H), 6.63 (d, J = 9.9 Hz, 1H), 5.74–5.46
1
concentrated to give L-2 as a white solid (60 mg, 52.1%). H-NMR
(400 MHz, DMSO-d6, ppm) δ: 10.81 (s, 1H), 10.63 (s, 1H), 9.33
(s, 1H), 9.14 (s, 1H), 8.29 (d, J = 9.90 Hz, 1H), 7.50 (d, J = 8.04 Hz,
1H), 7.09 (d, J = 8.10 Hz, 1H), 6.59 (d, J = 9.90 Hz, 1H), 5.58 (s, 1H),
4.93 (s, 1H), 3.89–3.91 (m, 1H), 3.75–3.77 (m, 1H), 3.10 (t, J = 3.88
Hz, 1H), 1.29 (d, J = 3.88 Hz, 3H), 1.14 (d, J = 3.88 Hz, 3H). 13C-NMR
δ: 161.1, 144.5, 136.2, 129.2, 123.4, 121.7, 121.3, 118.7, 114.7, 63.5,
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Scheme 3 Synthesis of L-2, L-4, L-6, and L-12. Reagents and conditions: (i) mCPBA, DCM, 0 °C to rt, 30 min; (ii) Ac2O, 100 °C, 3 h;
(iii) CH3COCl, AlCl3, DCE, 0 °C to reflux; (iv) C6H5CH2Br, K2CO3, in N,N-dimethylformamide, 40 °C, 3 h; (v) Br2, BF3Et2O, DCM, reflux, 15 min; (vi)
NaBH4, CH3OH, DCM, 0 °C, 30 min; (vii) KOH, CH3OH, DCM, 0 °C, 30 min; (viii) isopropylamine, N-propylamine, tert-butylamine or N-hexylamine,
ZnCl2, CH3CN, reflux, 12 h; (ix) H2, 10% Pd/C, CH3OH, rt, 2 h
48.3, 19.6, 18.6. HRMS ([M + Cl]-) m/z calcd for C14H18ClN2O3
297.1006; found 297.1014.
8-Hydroxy-5-[2-hydroxy-1-(N-hexylamino)ethyl]quinolin-2(1H)-one
hydrochloride (L-12)
In the presence of 10% Pd/C (20.0 mg), intermediate 9d (150 mg,
0.386 mmol) was reacted with H2 in 50 mL of methanol according
to procedure B to give L-12 as a white solid (70 mg, 59.0%).
1H-NMR (400 MHz, DMSO-d6, ppm) δ: 10.74 (s, 1H) 10.54 (s, 1H),
9.38 (s, 1H), 9.09 (s, 1H), 8.19–8.22 (d, J = 9.99 Hz, 1H), 7.38–7.41 (d,
J = 8.25 Hz, 1H), 7.05–7.08 (d, J = 8.25 Hz, 1H), 6.56–6.60 (d, J =
9.84 Hz, 1H), 5.52 (s, 1H), 4.86 (s, 1H), 3.75–3.87 (m, 2H), 2.86 (s, 1H),
2.71 (s, 1H), 1.61 (s, 2H), 1.17 (s, 6H), 0.78–0.83 (t, J = 7.80 Hz, 3H).
13C-NMR δ: 161.1, 144.5, 136.4, 129.2, 123.2, 121.7, 121.5, 118.9,
114.6, 62.8, 31.1, 26.1, 25.7, 22.2, 14.25. HRMS ([M + Cl]-) m/z calcd
for C17H24ClN2O3 339.1475; found 339.1487.
8-Hydroxy-5-[2-hydroxy-1-(tert-butylamino)ethyl]quinolin-2(1H)-
one hydrochloride (L-4)
In the presence of 10% Pd/C (20.0 mg), intermediate 9b (150 mg,
0.386 mmol) was reacted with H2 in 50 mL of methanol according to
procedure B to give L-4 as a white solid (117.1 mg, 97.0%). 1H-NMR
(600 MHz, DMSO-d6, ppm) δ: 10.70 (s, 1H) 10.53 (s, 1H), 9.05 (s, 1H),
8.54 (s, 1H), 8.32 (d, J = 9.99 Hz, 1H), 7.45 (d, J = 8.28 Hz, 1H), 7.05 (d,
J = 8.28 Hz, 1H), 6.59 (d, J = 9.90 Hz, 1H), 5.50 (s, 1H), 4.87
(s, 1H), 3.79–3.84 (m, 2H), 1.23 (s, 9H). 13C-NMR δ: 160.0, 143.3,
135.3, 128.0, 125.5, 122.3, 121.3, 117.0, 113.4, 63.0, 58.0, 53.2, 25.6.
HRMS ([M + H]+) m/z calcd for C15H21N2O3 277.1552; found
277.1559.
Cell lines
The HEK293 cell line was obtained from Thermo Fisher Scientific
and was cultured in Dulbecco’s modified Eagle’s medium (Thermo
Fisher Scientific) supplemented with 10% fetal bovine serum
(Beijing YuanHeng ShengMa Biotechnology Research Institute,
Beijing, China). The PathHunter Chinese Hamster Ovary-K1 β2-
adrenoceptor β-arrestin (CHO-β2-β-arr) cell line was purchased
from Discoverx (CA, USA) and was cultured in a proprietary
medium supplied by Discoverx. All cells were maintained in a
humidified incubator at 37 °C in 5% CO2.
8-Hydroxy-5-[2-hydroxy-1-(N-propylamino)ethyl]quinolin-2(1H)-
one hydrochloride (L-6)
In the presence of 10% Pd/C (20.0 mg), intermediate 9c (150 mg,
0.386 mmol) was reacted with H2 in 50 mL of methanol
according to procedure B to give L-6 as a white solid (113.0 mg,
98.0%). 1H-NMR (600 MHz, DMSO-d6, ppm) δ: 10.74 (s, 1H) 10.54 (s,
1H), 9.36 (s, 1H), 9.08 (s, 1H), 8.20 (d, J = 10.08 Hz, 1H), 7.391 (d, J =
8.28 Hz, 1H), 7.06 (d, J = 8.28 Hz, 1H), 6.59 (d, J = 10.08 Hz, 1H),
5.53 (s, 1H), 4.86 (s, 1H), 3.75–3.90 (m, 2H), 2.84 (s, 1H), 2.67 (s, 1H),
1.59–72 (m, 2H), 0.82 (t, d = 7.47 Hz, 3H). 13C-NMR δ: 161.1, 144.5,
136.5, 129.2, 123.2, 121.7, 121.5, 118.9, 114.7, 62.8, 57.7,47.5, 19.4,
11.5. HRMS ([M + H]+) m/z calcd for C14H19N2O3 263.1396; found
263.1392.
cAMP assay
The homogeneous time-resolved fluorescence (HTRF) cAMP assay
was conducted according to the manufacturer’s protocol for the
cAMP Dynamic 2 kit (Cisbio Bioassays, Codolet, France). Briefly,
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HEK293 cells, expressing endogenous β2-adrenoceptors, were
grown to < 80% confluence. Dissociated cells were resuspended in
Hank’s balanced salt with 20 mmol/L 4-(2-hydroxyethyl)pipera-
zine-1-ethanesulfonic acid solution (HBSS) supplemented with
0.1% bovine serum albumin and 1 mmol/L 3-isobutyl-1-
methylxanthine (IBMX), and then, they were dispensed into 384-
well low volume plates (Greiner #784076) at 2000 cells/5 μL per
well. The cells were stimulated with compounds diluted in HBSS
supplemented with 0.1% bovine serum albumin (5 μL/well) for 30
min at room temperature. Reactions were stopped by the addition
of the cAMP-d2 conjugate in lysis buffer (5 μL/well), followed by
the addition of the anti-cAMP cryptate conjugate in lysis buffer
(5 μL/well). After incubation for 1 h at room temperature, the
plates were read in a PerkinElmer (Waltham, MA, USA) 2300
EnSpire multilabel reader for time-resolved fluorescence reso-
nance energy transfer detection with an excitation wavelength of
337 nm and emission wavelengths of 620 nm and 665 nm, with a
lag time of 100 μs and an integration time of 400 μs for each
emission wavelength. The resultant cAMP concentrations were
calculated using Prism 4 (GraphPad Software, CA, USA) by
applying the 620/665 nm fluorescence ratios to a standard curve
of known cAMP concentrations.
previously [38]. Briefly, the concentration-response data of all the
compounds to be analyzed were fitted into the Black-Leff
operational model: [39]
Em½AŠnτn
(1)
Response ¼
n þ basal
½AŠnτn þ ð½AŠ þ KAÞ
where [A] is the concentration of the agonist, Em is the maximal
response of the system and n is the transducer slope. The two
latter parameters and basal are common for the curve fitting of all
of the concentration-response data. Fitting of the functional data
to eq. 1 would yield some combinations of τ and KA. The log
transformation of eq. 1 would yield the transduction coefficient,
log(τ/KA), which represents the efficiency of a particular agonist in
activating a given signaling pathway. The Δlog(τ/KA) value would,
therefore, describe the relative “efficacy” of an agonist for a
particular pathway when normalized with a reference agonist,
defined as (R)-ISO in the current analysis. The ligand bias or log
bias values, ΔΔlog(τ/KA), of a given agonist for different signaling
pathways were then evaluated by statistical analysis using
Bonferroni’s t-test against the reference ligand (R)-ISO. The
standard errors (SE) of the ΔΔlog(τ/KA) values were calculated as
described previously [38].
β-Arrestin assay
The PathHunter β-arrestin assay (Discoverx) was carried out with
the CHO-β2-β-arr cell line expressing the Prolink-tagged human
β2-adrenoceptors and the β-arrestin-2-β-galactosidase enzyme
fragment fusion proteins. An active β-galactosidase enzyme
RESULTS
We initially screened a library of approximately 150 2-amino-1-
phenylethanol (A-series) or 2-amino-2-phenylethanol (B-series and
L-series) compounds for β-adrenoceptor-mediated activity using
the tracheal smooth muscle relaxation assay or the cardiomyocyte
contractility assay [32, 33, 36, 37]. Compounds with biological
effects were short-listed for subsequent tests. To study ligand bias,
the concentration-response profiles of (R)-ISO, (R)-EPI, SAL, FEN,
(R,R’)-FEN, (R,R’)-MNF, A-17, A-18, A-23, A-31, A-32, A-33, A-35,
B-24, B-30, L-2, L-4, L-6, and L-12 (Table 1 and Supplementary
Fig. S1) in activating the β2-adrenoceptor-Gs-cAMP signaling and
the β2-adrenoceptor-β-arrestin signaling were obtained from the
two different assays (Supplementary Fig. S3 and S4). The HTRF
cAMP assay (Cisbio Bioassays) was conducted on compound-
stimulated HEK293 cells expressing a low level of an endogenous
β2-adrenoceptor [40]. The PathHunter β-arrestin assay (Discoverx)
was conducted on compound-stimulated CHO-β2-β-arr cells. Both
of these cell lines have no background expression of other
β-adrenoceptor isoforms.
is formed when
a β-arrestin interacts with an activated
β2-adrenoceptor in the cell. The assay was performed according
to the manufacturer’s protocol (Discoverx). Briefly, CHO-β2-β-arr
cells were grown to 90% confluence. Dissociated cells were
resuspended in a cell plating medium (Discoverx) and seeded at
10 000 cells/20 μL per well into 384-well tissue culture plates (with
white walls and a clear bottom). After overnight culturing at 37 °C
in 5% CO2, compounds diluted in phosphate-buffered saline
(pH = 7.4) supplemented with 0.1% bovine serum albumin
(5 μL/well) were added to the cells. The plates were then
incubated at 37 °C for 90 min. A β-galactosidase substrate
(Discoverx; 12 μL/well) was added. After further incubation for
60 min at room temperature, the plates were read in a PerkinElmer
2300 EnSpire multilabel reader for luminescence detection.
Isolated guinea pig trachea relaxation assay
Male Dunkin–Hartley guinea-pigs weighing 250–350 g were
provided by the Experimental Animal Center of Shenyang
Pharmaceutical University. The experiment has been approved
by the Animal Care and Use Committee of Shenyang Pharma-
ceutical University. The methods for the isolation of tracheal
smooth muscle strips and muscle tone measurements have been
described [36]. The strips were equilibrated in Krebs–Henseleit
solution (composition in mM: NaCl 118, KCl 5.4, CaCl2 2.5, MgSO4
0.6, NaH2PO4 2.94, NaHCO3 25 and D-glucose 11.7) and aerated
with 95% O2 and 5% CO2 for 2 h. Histamine (10 μmol/L) was added
to produce maximum contraction. The compound was then
added to produce relaxation. The percentage of relaxation was
calculated as follows:
The Emax and pEC50 values of the concentration-response curves
are shown in Table 2. The high Emax cAMP values of the respective
compounds (approximately 90%) suggest that (R)-ISO, FEN and
(R,R’)-FEN are full agonists of the β2-adrenoceptor in the cAMP
assay. The other compounds with lower Emax
values are
cAMP
partial agonists in this assay. SAL and L-6 are the two compounds
with the lowest Emax values (15% and 10%, respectively).
cAMP
Similarly, (R)-ISO, FEN and (R,R’)-FEN are also full agonists of the
β2-adrenoceptor in the β-arrestin assay, and SAL and L-6 also
have the lowest efficacies in the β-arrestin assay (Emax
β-Arrestin
values are 16% and 14%, respectively). (R)-EPI has an Emax cAMP
value of 61% and an Emax β-Arrestin value of 112%. L-4 has an Emax
value of 54% and an Emax β-Arrestin value of 90%. The Emax
cAMP
value differences of these two compounds are the greatest among
the compounds tested. They are considered full β2-adrenoceptor
agonists in the β-arrestin assay and partial β2-adrenoceptor
agonists in the cAMP assay. For (R)-ISO, the pEC50 cAMP value is
7.16 and the pEC50 β-Arrestin value is 7.28. The difference between
these two pEC50 values is negligible ( < 0.3). This result also holds
true for most of the test compounds except SAL, L-12, L-4, A-35,
and L-2 (with the absolute differences being 0.87, 0.76, 0.54, 0.53,
and 0.40, respectively). The similarity in the efficacies and
potencies of most of these compounds across the two assay
systems suggests that these two assays are comparable in terms
tension after compound À tension after histamine
Relaxation % ¼
tension after histamine
´ 100%
Curve fitting and statistical analysis
Fitting of the concentration-response curves and statistical
analysis of the data were performed using Prism 4. Emax and
pEC50 values were calculated from the curves fitted on the four-
parameter logistic model. Ligand bias was quantified as described
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Table 2 Efficacies (Emax) and potencies (pEC50) of the compound-stimulated cAMP responses in HEK293 cells and β-arrestin responses in CHO-β2-β-arr
cells, respectively
Compound
cAMP
β-Arrestin
a
a
b
b
Emax (% ISO)
pEC50
Emax (% ISO)
pEC50
(R)-ISO
(R)-EPI
SAL
91
61
15
94
86
52
36
23
27
39
43
28
25
60
37
44
54
10
36
3
2
1
4
4
2
2
1
1
2
2
2
1
3
1
3
3
1
2
7.16 0.06
7.13 0.05
8.93 0.07
7.11 0.07
7.42 0.07
7.11 0.05
8.21 0.08
7.11 0.09
7.77 0.07
6.81 0.06
8.09 0.07
6.56 0.08
6.02 0.10
4.19 0.05
5.43 0.07
4.72 0.07
4.76 0.06
4.42 0.15
4.45 0.08
104
112
16
91
91
74
29
19
20
33
38
28
25
43
33
54
90
14
32
2
2
0
2
2
1
1
0
1
1
1
1
1
3
1
2
3
1
2
7.28 0.03
6.92 0.03
8.06 0.03
7.18 0.03
7.39 0.03
6.92 0.02
8.37 0.05
7.31 0.06
7.63 0.07
6.89 0.04
8.13 0.03
6.68 0.05
6.55 0.05
4.18 0.08
5.42 0.06
5.12 0.05
5.30 0.05
4.46 0.05
5.21 0.06
FEN
(R,R’)-FEN
(R,R’)-MNF
A-17
A-18
A-23
A-31
A-32
A-33
A-35
B-24
B-30
L-2
L-4
L-6
L-12
The Emax value of each compound is the maximal response at saturation expressed as a percentage of the response at 10 μmol/L of (R)-ISO. Values are means
SE, n = 5 separate experiments with duplicates
aEmax and pEC50 values are derived from the corresponding concentration-response profiles in Supplementary Fig. S3
bEmax and pEC50 values are derived from the corresponding concentration-response profiles in Supplementary Fig. S4
of signal amplification. As the efficacy of (R)-EPI is just 61% of (R)-
ISO in cAMP induction, (R)-ISO is the more suitable unbiased
β2-agonist reference for subsequent ligand bias determination.
The log(τ/KA) values of the compounds for cAMP responses and β-
arrestin responses (Table 3) were determined after fitting the
concentration-response data to the Black–Leff operational model as
previously described [38]. Ligand bias or the ΔΔlog(τ/KA) values of
the compounds (Table 3 and Fig. 1) were then calculated using (R)-
ISO as the reference. Ligand bias is also expressed by the ratio
values (bias factors or BFs) in Table 3. The bias factor of SAL is 0.14,
or in other words, the activation of the Gs compared with the
β-arrestin pathway is 7.35-fold. L-12, L-4, L-2, and A-35 activate the
β-arrestin over the Gs pathway with bias factors equal 5.01, 4.32,
2.64, and 2.64, respectively. Statistical analyses of the ΔΔlog(τ/KA)
values suggest that SAL is a Gs-biased β2-agonist, whereas L-2, L-4,
and L-12 are β-arrestin-biased β2-agonists (Fig. 1). The agonist bias
of A-35 towards β-arrestin signaling does not reach statistical
significance. Other compounds tested did not show ligand bias in
the current analysis.
Compounds were tested for their abilities to produce bronch-
odilation by the isolated guinea pig trachea relaxation assay.
Relaxant effects on airway smooth muscles were observed with
the L-series compounds tested at 5 μmol/L (Table 4). In line with
the stimulatory effects of the compounds on cellular cAMP
production (Table 2), compounds L-2, L-4, and L-12 but not L-6
produced relaxation responses greater than the contractile effect
of histamine (10 μmol/L). These effects also appeared less than the
relaxant effect produced by ISO.
hypothesis that β-arrestin-biased agonism at the β-adrenoceptor
may be a novel therapeutic target for heart failure or other
cardiovascular diseases [7–9, 41]. Interestingly, our own studies
have shown that the Gs-biased β2-agonist FEN is beneficial to
survival, cardiac remodeling, and myocardial function in various
animal models of heart failure [22–26]. This Gs-biased agonism of
FEN at the β2-adrenoceptor describes the ligand bias for
preferential Gs signaling over Gs and Gi dual signaling because
the β2-adrenoceptor is capable of coupling to both Gs and Gi
proteins [14]. FEN and (R,R’)-FEN have been shown to be Gs-
biased [21, 32]. (R,R’)-MNF is unbiased in terms of β2-adreno-
ceptor-Gs/Gi signaling [33]. Here, the signaling biases of FEN,
(R,R’)-FEN, and (R,R’)-MNF for β-arrestin over Gs were investi-
gated. Our results show that the Δlog(τ/KA) values of FEN, (R,R’)-
FEN, and (R,R’)-MNF for Gs are slightly larger than those for
β-arrestin (Table 3), but the ligand biases for these compounds
have no difference compared with that of (R)-ISO, a non-biased
agonist of the β2-adrenoceptor (P > 0.05, Fig. 1).
A research group has studied 65 β2-agonists for biased agonism
in the signaling pathways downstream of the β2-adrenoceptor,
namely, Gs, β-arrestin-1, and β-arrestin-2 [42–44]. These series of
studies have included many FEN derivatives including (R,R’)-FEN
and (R,R’)-MNF. In one of these studies [44], (R)-SAL and (S)-SAL
have been shown to be strongly Gs-biased over both β-arrestin-1
and β-arrestin-2. Littmann et al. have also found that many FEN
derivatives are Gs biased. Specifically, (R,R’)-FEN exhibits no bias in
their study, whereas (R,R’)-MNF is weakly Gs-biased. Littmann et al.
have performed no statistical analysis on their ligand bias data,
likely because these data were pooled from different studies. Thus,
the ligand bias data of the present study for SAL, (R,R’)-FEN, and
(R,R’)-MNF are consistent with those reported in Littmann et al.
On the other hand, the study of Rajagopal et al. [45] on
quantifying ligand bias at seven transmembrane receptors
DISCUSSION
Several studies have implicated β-arrestin-biased signaling at the
β-adrenoceptor to be cardioprotective [9, 41], leading to the
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suggests that SAL is strongly β-arrestin-biased, whereas FEN
exhibits no bias towards β-arrestin and Gs at the β2-adrenoceptor.
In addition, Van der Westhuizen et al. [38] have reported that SAL
is biased towards the extracellular signal-regulated kinase 1/2
(ERK1/2) pathway over the cAMP pathway. ERK1/2 has been
widely regarded as a downstream effector of β-arrestin in the field
[46], but through Gi or Gs activation, the phosphorylation of ERK1/2
can also be induced [40, 47]. The results of Rajagopal et al. on SAL,
and possibly those of Van der Westhuizen et al., are in direct
contradiction with those of Littmann et al. and the present study.
In Littmann et al., (R)-EPI rather than (R)-ISO was used as the
reference unbiased agonist for ligand bias quantitation. However,
this is not the main reason why Littmann et al. obtained a different
result on the SAL stereoisomers because EPI has been shown to
be a non-biased β2-agonist in other studies including the present
one. In addition, all of these studies have employed the Black–Leff
operational model in quantifying ligand bias. Therefore, the
discrepancy in results can only be due to the different techniques
used in determining the Gs and β-arrestin activities of the
compounds.
Careful examination of the data revealed that the reported
Emax
or Emax
values of SAL (or its stereoisomers) in
cAMP
Gs
Table 3 Log(τ/KA) and Δlog(τ/KA) values for cAMP responses, log(τ/KA) and Δlog(τ/KA) values for β-arrestin responses, ΔΔlog(τ/KA) values (ΔΔLog) and
bias factors (BFs) of the compounds
Compound
cAMP
β-Arrestin
Log(τ/KA)
ΔΔLog
BF
Log(τ/KA)
ΔLog(τ/KA)
ΔLog(τ/KA)
(R)-ISO
(R)-EPI
SAL
7.23 0.03
7.03 0.05
8.28 0.14
7.25 0.04
7.50 0.04
7.02 0.05
8.05 0.07
6.72 0.09
7.42 0.08
6.56 0.06
7.95 0.06
6.17 0.08
5.68 0.08
4.01 0.05
5.22 0.07
4.51 0.06
4.59 0.05
3.63 0.23
4.12 0.07
0.00 0.03
−0.19 0.06
1.06 0.14
7.30 0.02
6.99 0.02
7.49 0.07
7.19 0.02
7.40 0.02
6.85 0.03
8.08 0.06
6.88 0.08
7.25 0.07
6.64 0.05
7.91 0.05
6.39 0.06
6.18 0.07
4.02 0.05
5.18 0.05
5.00 0.04
5.29 0.03
3.84 0.09
4.90 0.05
0.00 0.02
−0.31 0.03
0.19 0.07
0.00 0.04
1.00
0.76
0.14
0.74
0.69
0.58
0.90
1.24
0.57
1.02
0.77
1.41
2.64
0.88
0.77
2.64
4.32
1.40
5.01
−0.12 0.06
−0.87 0.16*
−0.13 0.06
−0.16 0.06
−0.24 0.07
−0.05 0.10
0.09 0.12
FEN
0.03 0.05
−0.11 0.03
0.11 0.03
(R,R’)-FEN
(R,R’)-MNF
A-17
A-18
A-23
A-31
A-32
A-33
A-35
B-24
0.27 0.05
−0.21 0.06
0.83 0.08
−0.45 0.03
0.78 0.06
−0.05 0.09
0.19 0.08
−0.41 0.08
−0.05 0.08
−0.66 0.06
0.61 0.05
−0.24 0.11
0.01 0.09
−0.67 0.07
0.73 0.07
−0.11 0.09
0.15 0.10
−1.06 0.08
−1.54 0.09
−3.22 0.06
−2.00 0.07
−2.72 0.07
−2.64 0.06
−3.60 0.23
−3.10 0.07
−0.91 0.06
−1.12 0.07
−3.27 0.05
−2.12 0.06
−2.30 0.04
−2.00 0.03
−3.45 0.09
−2.40 0.06
0.42 0.12
−0.05 0.08
−0.11 0.09
0.42 0.08*
0.64 0.07***
0.15 0.25
B-30
L-2
L-4
L-6
L-12
0.70 0.09***
Log(τ/KA) values were obtained after fitting the concentration-response data to the operational model for agonism by Black and Leff (1983). ΔLog(τ/KA) values
were calculated by subtracting the log(τ/KA) value of (R)-ISO from the log(τ/KA) value of each compound. ΔΔLog values were calculated by (ΔLogβ-Arrestin
–
ΔLogcAMP). The significance was determined by comparing each value with that of (R)-ISO using Bonferroni’s t-test. *P < 0.05, ***P < 0.001. BF is an index of the
ΔΔlog value with a base of 10. The BF of a compound represents the ratio of β-arrestin activation over Gs activation with respect to (R)-ISO, an unbiased
β2-agonist. Values are means SE, n = 5 separate experiments with duplicates. A ligand bias is considered strong if the ΔΔlog value is >1 or < –1
Fig. 1 ΔΔLog(τ/KA) values of the compounds for β-arrestin signaling over Gs signaling. The significance of the ligand bias was determined by
comparing each value with that of (R)-ISO using Bonferroni’s t-test. *P < 0.05, ***P < 0.001 (Error bars are standard errors, n = 5)
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We used zinterol as the positive control compound because it has
exhibited very substantial β2-adrenoceptor-Gi signaling and a
sensitivity towards PTX-treatment in our previous studies using
rodent cardiomyocytes [14, 21, 33]. As expected, treatment with
PTX increased the Emax cAMP values of zinterol (from 21.4% without
PTX to 26.4% with PTX, 123% increase, P < 0.001) and (R)-ISO
(from 33.7% without PTX to 36.3% with PTX, 110% increase, P >
0.05) without causing substantial changes in the EC50 values of
either compounds (Supplementary Fig. S5A, B). Treatment with
PTX also increased the cAMP responses of most of the ligands
tested at a saturated concentration and a sub-saturated concen-
tration (Supplementary Fig. S5C). The greatest changes occurred
in (R)-EPI (126% increase) and L-6 (127% increase) at their
respective saturating concentrations, whereas the PTX-induced
changes in the maximal cAMP responses for SAL, as well as the
Gs-selective β2-adrenoceptor agonists FEN and (R,R’)-FEN were
96%, 110%, and 116%, respectively. The PTX-induced changes in
the compound-stimulated responses were generally small but
definite. Statistical significance in some of the comparisons
occurred only by chance and is not an indication of exceptional
Gi activity for any particular compound(s). These results suggest
that the HTRF cAMP assay is not particularly sensitive for detecting
β2-adrenoceptor-Gi signaling compared with other assays, such as
the rat cardiomyocyte contractility assay. Thus, the selectivity of
the β2-adrenoceptor ligands to different Gs/Gi pathways should
not be a confounding factor in the determination of β-arrestin bias
in the present study.
The trachea relaxation assay results suggest that these
compounds are bronchodilators like other β2-adrenoceptor
agonists and that their activities (presumably those of cAMP
induction) rather than their ligand bias status are associated
with the bronchodilator effects of the compounds. The
correlation of Emax in the cAMP assay with relaxation is fairly
good except for L-12, which shows more potent relaxation than
L-2 and L-4, suggesting that the smooth muscle relaxant effect
of L-12 might involve additional mechanisms other than
stimulation of the β2-adrenoceptor. One possible mechanism
is the inhibition of phosphodiesterase, which causes an
accumulation of cAMP in the cytosol. This effect, if it exists,
will be masked in the cAMP assay because the cAMP assay
buffer contained IBMX, a phosphodiesterase inhibitor, in an
excess amount.
Table 4 Effects of compounds (5 μmol/L) on relaxation of tracheal
smooth muscle strips precontracted with histamine
Compound
Relaxation(%)
ISO
L-2
285.57 35.24
118.34 26.56
130.48 24.44
28.16 4.84
L-4
L-6
L-12
206.30 45.45
Values are means SEM, n = 4
Littmann et al., Rajagopal et al., and Van der Westhuizen et al. are
equivalent to that of (R)-ISO, but the Emax β-Arrestin values of SAL
are only 29% or less relative to (R)-ISO. The reported Emax ERK1/2
value of SAL in Van der Westhuizen et al. is approximately 83%
that of saturated (R)-ISO. A major finding of the present study
different from those previous ones is that both the Emax cAMP and
the Emax β-Arrestin values of SAL are approximately 16% of saturated
(R)-ISO. SAL is regarded as a partial agonist of the β2-
adrenoceptor compared with the natural ligand (R)-EPI, a full
β2-agonist for Gs activation in relieving bronchoconstriction [48,
49]. In view of the low efficacies of SAL in our determination, the
data reported in the present investigation may be more akin to
data derived from physiological measurements compared with
other studies.
Moreover, the incubation time of compound stimulation for the
cAMP assay was 5 min and that for the Tango β-arrestin assay was
14–20 h in Rajagopal et al., whereas the incubation times of
compound-stimulation for our cAMP and β-arrestin assays were 30
and 90 min, respectively. Therefore, kinetic differences between
the assays would complicate the interpretation of our data to a
lesser extent than those of Rajagopal et al. In addition, the
incubation time of compound stimulation for the ERK1/2 assay in
Van der Westhuizen et al. was <5 min. However, it has been shown
that the G protein-dependent ERK1/2 activation at the β2-
adrenoceptor has a sharp peak between 2 and 5 min, whereas
the β-arrestin-dependent ERK1/2 activation persists from 5 to 30
min [50]. Therefore, the ERK1/2 signals detected by Van der
Westhuizen et al. may represent more of the signals of the G
protein pathway rather than the signals of the β-arrestin pathway.
More recent data have provided evidence that carvedilol signaling
from the β2-adrenoceptor is not merely β-arrestin-mediated but
requires Gi [51]. Moreover, β-arrestins do not mediate ERK1/2
phosphorylation in the absence of functional G proteins [52].
These new findings suggest that ERK1/2 activity is not a good
parameter for β-arrestin recruitment and cannot be used to access
β-arrestin bias. In conclusion, our data and those of Littmann et al.
support the notion that sustained bronchodilation with the
chronic use of SAL is due to a defect in the desensitization of
the SAL-stimulated β2-adrenoceptor in a β-arrestin-dependent
manner [49].
BI-167107 (Supplementary Fig. S1), a high affinity and selective
β2-adrenoceptor agonist previously used in X-ray crystallographic
studies of the active conformations of the β2-adrenoceptor [53],
has been shown to be partially biased for β-arrestin over Gs [54].
As the L-series compounds possess a different head group and a
different core structure compared with BI-167107, the present
study has discovered a new scaffold for the design of novel
β-arrestin-biased β2-adrenoceptor agonists. Additionally, com-
pounds L-2, L-4, and L-12 have much lower potencies in inducing
cAMP compared with (R)-ISO (Table 2). This result may suggest a
low binding affinity. In summary, these compounds may better
serve as tool reagents or as lead compounds for further
development.
In the same manner as ERK1/2 activity is to β-arrestin
recruitment, cAMP accumulation is widely used to represent the
activity of the Gs pathway, as the Gs-adenylyl cyclase-cAMP-
protein kinase A signaling cascade is accepted as a dogma in the
field of receptor pharmacology. Applying the cAMP assay in ligand
bias quantitation, however, is not necessarily error-free. One
should note that most β2-agonists including ISO and EPI stimulate
the β2-adrenoceptor to activate both Gs and Gi proteins, and the
selectivity for the Gi versus Gs pathways to varying degrees may
affect the determination of β-arrestin-biased agonism by altering
the cAMP production. To address this issue, we tested whether
pertussis toxin (PTX), a disruptor of Gi signaling, may cause a
strong enhancement in the cAMP responses of the compounds.
CONCLUSION
In the present study, we characterized a cohort of phenylethano-
lamines for β-arrestin-biased agonism at the β2-adrenoceptor. Our
data show that three 2-amino-2-phenylethanol derivatives, namely,
L-2, L-4, and L-12, are partial β2-adrenoceptor agonists with weak
ligand biases for β-arrestin over Gs. The present identification of
β-arrestin-biased β2-adrenoceptor agonists with a new 5-(1-amino-2-
hydroxyethyl)-8-hydroxyquinolin-2(1H)-one core structure will facil-
itate the discovery of novel biased agonists with potential usefulness
in the treatment of diseases of the heart and airway.
Acta Pharmacologica Sinica (2019) 0:1 – 11

Novel β-arrestin-biased β2-adrenoceptor agonists
AYH Woo et al.
10
ACKNOWLEDGEMENTS
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We thank Dr. Joseph Kozocas from SRI international, California, USA for providing
(R,R’)-FEN and (R,R’)-MNF for the analysis and Dr. Wei Liang and his team from WuXi
AppTec, Shanghai, China for technical assistance in the cAMP assay. This work was
supported by the Ministry of Science and Technology of the People’s Republic of
China under the National Key Research and Development Program of China [grant
number 2018YFA0507603 (to R-PX)], the National Science and Technology Major
Project [grant numbers 2013ZX09507001-001002 (to AY-HW), 2013ZX09301305-001
(to LP), 2013ZX09508104 (to R-PX), 2018ZX09739009 (to M-SC)], the National Basic
Research Program [grant number 2012CB518000 (to R-PX)], the National Natural
Science Foundation of China [grant numbers 81673355 (to AY-HW), 81872752 (to M-
18. Sato M, Gong H, Terracciano CM, Ranu H, Harding SE. Loss of β-adrenoceptor
response in myocytes overexpressing the Na+/Ca2+-exchanger. J Mol Cell Car-
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heart failure: associated defect of adrenergic contractile support. J Mol Cell
Cardiol. 2004;36:7–11.
SC), 31521062, 81630008, 81790621 (to R-PX)], Beijing Municipal Science
&
Technology Commission [grant number Z171100000417006 (to R-PX)], and the
intramural research program of the NIH, USA (to IWW).
20. Zhu W, Zeng X, Zheng M, Xiao RP. The enigma of beta2-adrenergic receptor Gi
signaling in the heart: the good, the bad, and the ugly. Circ Res. 2005;97:
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AUTHOR CONTRIBUTIONS
AYHW designed the research, performed the research, analyzed the data, and wrote
the paper; XYG performed the research, analyzed the data, and wrote the paper; and
YYZ analyzed the data. LP performed the research and contributed new reagents or
analytical tools; XRL, YMM, GX and RJX performed the research; IWW contributed new
reagents or analytical tools; and MSC and RPX contributed new reagents or analytical
tools and supervised the research.
ADDITIONAL INFORMATION
The online version of this article (https://doi.org/10.1038/s41401-018-0200-x)
contains supplementary material, which is available to authorized users.
25. Ahmet I, Morrell C, Lakatta EG, Talan MI. Therapeutic efficacy of a combination of
a beta1-adrenoreceptor (AR) blocker and beta2-AR agonist in a rat model of
postmyocardial infarction dilated heart failure exceeds that of a beta1-AR blocker
Competing interests: The authors declare no competing interests.
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