Design, synthesis and biological evaluation of 5-(2-amino-1-hydroxyethyl)-8-hydroxyquinolin-2(1H)-one derivatives as potent β2-adrenoceptor agonists

Article abstract of DOI:10.1016/j.bmc.2018.10.043

A series of novel β₂-adrenoceptor agonists with a 5-(2-amino-1-hydroxyethyl)-8-hydroxyquinolin-2(1H)-one moiety was designed, synthesized and evaluated for biological activity in human embryonic kidney 293 cells and isolated guinea pig trachea. Compounds 9g and (R)-18c exhibited the most excellent β₂-adrenoceptor agonistic effects and high β₂/β₁-selectivity with EC₅₀ values of 36 pM for 9g and 21 pM for (R)-18c. They produced potent airway smooth muscle relaxant effects with fast onset of action and long duration of action in an in vitro guinea pig trachea model of bronchodilation. These results support further development of the two compounds into drug candidates.

Full text of DOI:10.1016/j.bmc.2018.10.043

Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and biological evaluation of 5-(2-amino-1-hydroxyethyl)-
8-hydroxyquinolin-2(1H)-one derivatives as potent β2-adrenoceptor
agonists
Gang Xinga,b, Li Pana,b, Ce Yia,b, Xiaoran Lic, Xinyue Gea,b, Ying Zhaoa,b, Yichuang Liua,b
,
⁎
⁎
Jinyan Lia,b, Anthony Wooc, Bin Lina,b, Yuyang Zhangc, , Maosheng Chenga,b,
a Department of Medicinal Chemistry, School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016, China
b Key Laboratory of Structure-Based Drug Design and Discovery of Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, China
c Department of Pharmacology, School of Life Sciences and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang 110016, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
A series of novel β2-adrenoceptor agonists with a 5-(2-amino-1-hydroxyethyl)-8-hydroxyquinolin-2(1H)-one
moiety was designed, synthesized and evaluated for biological activity in human embryonic kidney 293 cells and
isolated guinea pig trachea. Compounds 9g and (R)-18c exhibited the most excellent β2-adrenoceptor agonistic
effects and high β2/β1-selectivity with EC50 values of 36 pM for 9g and 21 pM for (R)-18c. They produced potent
airway smooth muscle relaxant effects with fast onset of action and long duration of action in an in vitro guinea
pig trachea model of bronchodilation. These results support further development of the two compounds into
drug candidates.
Asthma
COPD
β2-Adrenoceptor agonist
Indacaterol
Salmeterol
Trantinterol
1. Introduction
have a rapid onset of action and produce symptom relief for 3–6 h. The
inhaled long-acting agents include salmeterol (2) and formoterol (3). The
Obstructive airway diseases including asthma and chronic obstructive
pulmonary disease (COPD) which produce symptoms of cough,
wheezing, breathlessness and chest tightness are placing an increasing
burden on health care systems in industrialized and developing countries
alike.1 COPD is an increasing cause of chronic disability and is predicted
to become the fifth most common cause of chronic disability worldwide
by 2020.2,3 Both diseases are characterized by airway obstruction, which
is variable and at least partially reversible in asthma but is progressive
and largely irreversible in COPD.4–6 There are two major components in
the pathophysiology of both diseases, airway inflammation and smooth
muscle dysfunction.7 The respiratory diseases are widely treated with β2-
adrenoceptor agonists or muscarinic antagonists in combination with
corticosteroids when the severity of symptoms is from moderate to se-
vere.8,9 The β2-adrenoceptor, a member of the superfamily of the seven
transmembrane G protein-coupled receptors (GPCRs), is widely ex-
pressed in the respiratory tract.10 Activation of the β2-adrenoceptor re-
laxes airway smooth muscles and opens up the airway causing symptom
relief during airway obstruction. β2-Adrenoceptor agonists are broadly
classified into the short-acting, the long-acting and the ultra-long-acting
types. Short acting β2-adrenoceptor agonists, such as salbutamol (1),
ultra-long-acting β2-adrenoceptor agonists include indacaterol (4) and
olodaterol (5). Drug candidates in development include Pfizer’s PF-
610355 (6),11 Chiesi’s carmoterol (7)12 and Theravance’s milveterol (8)
(Fig. 1).13 Indacaterol and olodaterol belong to the third generation β2-
adrenoceptor agonists which are effective once-daily bronchodilators in
treatment of COPD.14
Our group is committed to develop long-acting β2-adrenoceptor
agonists and has designed and synthesized a series of candidate com-
pounds including trantinterol and its analogues (S)-5j (ref. 15) and 2f
(ref. 16) with a core structure different from that of traditional β2-
adrenoceptor agonists. Biological studies have shown that trantinterol
is a highly selective β2-adrenoceptor agonist with strong biological
activities.17 Indacaterol was developed by Novartis as the ultralong-
acting β2-adrenoceptor agonist.14 It has a unique 8-hydroxyquinolin-
2(1H)-one head group in its structure. Based upon previous experience
and reported literature we report here the synthesis and biological
evaluation of a series of close analogues of 5-(2-amino-1-hydroxyethyl)-
8-hydroxyquinolin-2(1H)-one 9 (Fig. 2). The impact of the length of the
carbon chain in the tail group and different substituents on its aromatic
ring group on the activity are investigated.
⁎ Corresponding authors at: Department of Medicinal Chemistry, School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016,
China (M. Cheng).
E-mail addresses: 13614053862@163.com (Y. Zhang), mscheng@syphu.edu.cn (M. Cheng).
https://doi.org/10.1016/j.bmc.2018.10.043
Received 27 September 2018; Received in revised form 27 October 2018; Accepted 30 October 2018
0968-0896/©2018PublishedbyElsevierLtd.
Pleasecitethisarticleas:Xing,G.,Bioorganic&MedicinalChemistry,https://doi.org/10.1016/j.bmc.2018.10.043

G. Xing et al.
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Scheme 2. Reagents and conditions: (a) MgBrMe, anhydrous THF, ice-bath,
40 min, 85–90%; (b) acetonitrile, concentrated sulfuric acid, ice-bath, 3 h,
70–80%; (c) KOH, ethane-1,2-diol, 200 °C, 15 h, 85%; (d) (i) NaBH4, DMSO,
MeOH, 15 h, 45–60%; (ii) 10% Pd/C, H2, MeOH, 1 h, 90–98%; (e) (i) iso-
propanol, reflux, 15 h, 55%; (ii) 10% Pd/C, H2, MeOH, 1 h, 90%.
2.2. Biological tests
Fig. 1. Clinically used β2-adrenoceptor agonists: salbutamol (1), salmeterol (2),
formoterol (3), indacaterol (4) and olodaterol (5), and candidates in develop-
ment: PF-610355 (6), carmoterol (7) and milveterol (8).
2.2.1. Agonistic effects of the compounds on various β-adrenoceptors
β2 and β1 are different subtypes of adrenoceptors. The β2-adreno-
ceptors are widely distributed in the respiratory tract, particularly in
airway smooth muscles. β1 is the dominant subtype of adrenoceptors
expressed in the heart. When an agonist of the β-adrenoceptors binds to
these cell surface receptors, a signal transduction process begins so that
the intracellular Gs proteins activate the enzyme adenylyl cyclase to
convert ATP to cAMP.21 Compounds in Table 1 were measured for their
abilities to cause cAMP accumulation in human embryonic kidney 293
(HEK293) cells transfected with human β2 or β1-adrenoceptors. The
potency was reported as pEC50 values (negative logarithm of the molar
drug concentration that produces a cAMP response equal to 50% of its
maximal response). The β2/β1-selectivity of the compounds in the
present study was given by the pEC50 at the β2-adrenoceptor minus the
pEC50 at the β1-adrenoceptor.
Fig. 2. The structure of 5-(2-amino-1-hydroxyethyl)-8-hydroxyquinolin-2(1H)-
one analogues (9).
We modified the length of the carbon chain in the tail group of
2. Results and discussion
Table 1
Activities of the compounds in inducing cAMP accumulation in HEK293 cells
expressing the β2 or β1-adrenoceptors. Isoprenaline was used as a reference.
2.1. Chemistry
Acetophenone 10 was prepared as described previously.18 It was
then reacted with hydrobromic acid in DMSO to give intermediate 11.19
Addition of primary amines with different side chains produced com-
pounds 12. Hydrogenation of the benzyl group delivered the target
compounds 9 (Scheme 1).
Scaffold 16 was prepared in three steps from the respective ketone
13 through the Grignard reaction, Ritter reaction and deacetylation to
yield 16a–c. Intermediates 16a–c were then refluxed with intermediate
11 followed by hydrogenation of the protective benzyl group to pro-
duce racemic 18a–c. (R)-8-(benzyloxy)-5-(oxiran-2-yl)quinolin-2(1H)-
one 17, a very important intermediate, was synthesized according to a
previously reported method.20 Compounds 16a–c were treated with
compound 17 followed by hydrogenation to yield the target compounds
(R)-18a–c (Scheme 2).
a
Compound
R
β2pEC50
10.13
β1pEC50
8.01
β2-β1
2.12
9a
0.23
0.09
9b
9.79
7.14
2.65
9c
9d
8.04
9.88
7.80
7.80
0.24
2.08
9e
9.82
7.06
2.76
Isoprenaline
Salmeterol
9.5
0.1
8.5
0.1
0.99
4.2
10.11
5.91
Scheme 1. Reagents and conditions: (a) 40%HBr, DMSO, 80 °C, 2 h, 80–92%;
(b) RNH2, NaBH4, DMSO, MeOH, 15 h, 45–60%; (c) 10% Pd/C, H2, MeOH, 1 h,
90–98%.
a
Selectivity is expressed as pEC50 at the β2-adrenoceptor – pEC50 at the β1-
adrenoceptor.
2

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compound 9 to yield compounds 9a–e. The compound with an N-bu-
tylbenzene 9e exhibited a lower potency than the one with an N-pro-
pylbenzene 9a, with pEC50 values of 10.13 and 9.82, respectively.
Compound 9a exhibited a similar level of activity as the clinical drug
salmeterol which was higher than that of isoprenaline. It has been
suggested that the presence of an oxygen atom in the amino tail chain of
phenylethanolamines does not affect lipophilicity but is essential for a
long duration of action as is the case of salmeterol.7,22 When an oxygen
atom was introduced adjacent to the benzene ring of 9a, the resulting
compound 9b was a little bit less active than compound 9a in stimu-
lating the β2-adrenoceptor but its β2/β1-selectivity improved. Re-
placement of the phenyl group in 9a with a larger diphenyl 9c or
naphthalene 9d group did not improve activity either. Compound 9c
showed a selectivity towards the β1-adrenoceptor as compared with
isoprenaline, a non-selective β-adrenoceptor agonist. Compounds 9b
and 9e displayed higher selectivity to the β2-adrenoceptor as compared
to 9a. Compounds 9a, 9b, 9d and 9e were selective towards the β2-
adrenoceptor but their β2/β1-selectivity were lower than that of sal-
meterol. In consideration of activity and selectivity, we chose com-
pound 9a as the basic structure in further structure-activity relationship
study.
provided 9h, 9g and 9f, respectively. All of which displayed measurable
increases in potency. At the same time, 9g showed a higher β2/β1-se-
lectivity than 9f and 9h approaching that of salmeterol. Replacement of
the hydroxyl group with a methoxyl group provided 9i-k. These com-
pounds have no obviously growth in activity compared with 9f-h.
However, the 2-methoxyl 9k and the 3-methoxyl 9j exhibited a higher
activity than the 4-methoxy 9i. The methoxyl analogues exhibited si-
milar levels of β2/β1-selectivity but lower than salmeterol. Compounds
9f–9k displayed excellent activities similar to the marketed drug sal-
meterol. Amide substitution on the benzene has been suggested to in-
crease propensity for hydrogen bonding and poor absorption across the
gut wall may ensue.23–25 Addition of an amide to the meta- or para-
position of the benzene ring had different influence in activity. Com-
pounds with an amide substituent at the meta-position except 9l had
higher activities than the ones substituted at the para-position. Inter-
estingly, the β2/β1-selectivity of all these amide compounds improved
and 9p showed a higher β2/β1-selectivity as compared to salmeterol.
Introduction of a methyl side group in the carbon chain next to the
amino has been shown to increase the activities of related β2-adreno-
ceptor agonists like formoterol.26 Consistent with this idea, we turned
our attention to the α-position next to the amino moiety. By introducing
two germinal methyl groups in the α-carbon, compound 18c was de-
rived from 9a. A series of 18 analogues was synthesized (Table 3).
Replacement of the γ-hydrogen atom of 18c with a benzyl group
We investigated the effect of substitution in the benzene ring of 9a
by synthesis of its analogues (Table 2). Incorporation of a hydroxyl
group at the 2-position, 3-position or 4-position of the phenyl ring
Table 2
Activities of compounds in inducing cAMP accumulation in HEK293 cells expressing the β2 or β1-adrenoceptors. Isoprenaline was used as a reference.
a
Compound
R1
R2
R3
β2pEC50
β1pEC50
β2-β1
9f
H
H
-OH
H
10.30
10.44
10.31
10.13
10.39
10.37
9.03
0.29
0.02
0.30
0.10
0.02
0.04
7.44
6.66
8.30
7.36
8.13
7.95
0.18
0.10
0.01
0.17
0.13
0.20
2.38
3.66
1.69
2.51
2.16
2.26
9g
9h
9i
H
-OH
H
-OH
H
H
H
-OCH3
H
9j
H
-OCH3
H
9k
9l
-OCH3
H
H
H
9m
H
H
9.30
7.65
1.65
9n
9o
H
H
H
H
10.29
9.52
0.12
7.87
6.62
2.55
2.92
9p
9q
9r
H
H
H
H
9.65
9.30
8.6
0.66
5.71
5.84
0.33
5.04
2.47
H
H
Isoprenaline
Salmeterol
9.5
0.1
8.5
0.1
0.99
4.2
10.11
5.91
a
Selectivity is expressed as pEC50 at the β2-adrenoceptor - pEC50 at the β1-adrenoceptor.
3

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Table 3
Activities of compounds in inducing cAMP accumulation in HEK293 cells expressing the β2 or β1-adrenoceptors. Isoprenaline was used as a reference.
a
Compound
R4
R5
β2pEC50
9.66
β1pEC50
β2-β1
18a
0.38
(R)-18a
18b
10.19
10.12
0.20
8.20
8.17
0.08
0.16
1.99
1.93
(R)-18b
18c
10.09
10.39
0.33
0.28
H
(R)-18c
10.68
5
0.00
0.1
5.68
0.99
4.2
Isoprenaline
Salmeterol
9.5
0.1
8.5
5.91
10.11
a
Selectivity is expressed as pEC50 at the β2-adrenoceptor - pEC50 at the β1-adrenoceptor.
afforded 18a and incorporation of a fluorine atom at the 4-position of
the two phenyl rings in 18a provided 18b. In the cell assay, compound
18c had no improvement in activity as compared with 9a and com-
pounds 18a and 18b even showed lower potencies. We focused our
attention on the synthesis of different enantiomers of analogues 18
which provided compounds (R)-18a, (R)-18b and (R)-18c. Compounds
(R)-18a and (R)-18b displayed similar activities as compared to 9a.
(R)-18c exhibited a higher β2-adrenoceptor potency and an excellent
β2/β1-selectivity as compared to salmeterol.
2.2.3. Evaluation of relaxation on isolated guinea pig trachea
The relaxant effects of the compounds on isolated tracheal strips are
good predictors of their bronchodilator effects in vivo. Compounds 9g, 9j,
9k and (R)-18c with excellent potencies in the cell assay were selected
for a further study in an in vitro smooth muscle relaxation assay using
isolated guinea pig tracheal tissues. Relaxation percentages were calcu-
lated basing on muscle tension after compound addition. Emax represents
the maximal effect obtained with saturating concentrations of a com-
pound. The potency (pD2) value represents the negative logarithm of the
concentration of a compound at which the relaxation response is equal to
50% of the compound’s own maximal effect. As shown in Fig. 3, the Emax
value of compound (R)-18c was the greatest among the compounds
tested, which was closed to that of isoprenaline. Compound 9g has a
similar Emax value compared with isoprenaline. As shown in Table 5, the
pD2 and the Emax values of compounds (R)-18c and 9g had no difference
as compared with those of isoprenaline. The pD2 and the Emax values of
compounds 9j and 9k are obviously smaller than those of isoprenaline.
Up to now, it could be deduced that compounds 9g and (R)-18c are full
agonists in the assay, whereas 9j and 9k are partial agonists. Based on
these results, we decided to further explore the onset time and duration
of action of compounds 9g and (R)-18c.
2.2.2. Evaluation of the intrinsic activity
The β2-adrenoceptor is ubiquitously expressed with higher expres-
sion levels in the respiratory system. Hence, efficacies of the com-
pounds were also assessed in non-overexpressed HEK293 cells with
endogenous human β2-adrenoceptors by determining their intrinsic
activities relative to that of isoprenaline. By definition, the intrinsic
activity of isoprenaline is 100%. Compounds with high potency and
high β2/β1-selectivity 9a, 9g, 9j, 9k, 9n, 9p and (R)-18c were chosen
for the study (Table 4). All these compounds were low in intrinsic ac-
tivities compared with isoprenaline. Therefore, they are considered
partial agonists for the human β2-adrenoceptor in this non-over-
expressed system.
Table 4
Intrinsic activities of agonists of selected compounds 9a, 9g, 9j, 9k, 9n,
9p and (R)-18c on HEK293 cells endogenously expressing human β2-
adrenoceptors.
Compound
Intrinsic activity (vs. isoprenaline%)
9a
81.21
83.57
78.05
77.34
77.44
67.43
84.79
100
2.70
0.22
2.64
5.23
1.25
4.78
4.50
9g
9j
9k
9n
9p
(R)-18c
Isoprenaline
Fig. 3. Concentration-responses for compounds 9g, 9j, 9k, (R)-18c and iso-
prenaline in relaxation of isolated guinea pig tracheal slices (n = 4).
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Table 5
those containing a 2-amino-1-phenylethanol core (pEC50 values were in
a range of 8–11 in the present study). In particular, compound 9a with
an N-propylbenzene tail in the present study and compound 5c in Ref.
15 are structural isomers which only differ in the core, 2-amino-1-
phenylethanol for 9a and 2-amino-2-phenylethanol for 5c. Their pEC50
values are 10.1 and 8.0, respectively. Similarly, the structural isomer
pair 9e with an N-butylbenzene tail and compound 5d in our previous
study have pEC50 values equal to 9.8 and 7.9, respectively. Direct
comparison of data from these two studies are justified because the
methods for determining potency and receptor subtype selectivity of
the compounds are identical. These compounds having a common 8-
hydroxyquinolin-2(1H)-one head group and different core structures
have similarly high β2/β1-selectivity. This may be attributed to the 8-
hydroxyquinolin-2(1H)-one head group which is a classic moiety for a
number of β2-adrenoceptor agonists including indacaterol, carmoterol
and procaterol. Thus, at this point of development, the 2-amino-2-
phenylethanol class of β2-adrenoceptor agonists are not as efficacious as
the clinical drug salmeterol or the derivatives of indacaterol in-
vestigated in the present study.
E
max, pD2 and intrinsic activities (IA vs. isoprenaline) of the smooth muscle
relaxation responses of selected compounds 9g, 9j, 9k, (R)-18c and isoprena-
line.
Compound
pD2
Emax (%)
IA(vs. isoprenaline)
Isoprenaline
7.44
7.85
5.01
6.59
7.02
0.35
0.98
0.15*
0.22*
0.43
197.52
176.22
77.25
27.36
8.64
1.00
0.89
0.39*
0.26*
0.97
9g
9j
18.14*
21.28*
36.21
9k
50.69
(R)-18c
191.78
The pharmacodynamic parameters were determined in isolated guinea-pig
tracheal slices pre-contracted with histamine (5 × 10−5 M). Data are expressed
as means
S.D. (n = 4).
* Differences between the values (vs. those of isoprenaline) were compared
by unpaired Student’s t test and P < 0.05 was considered significant.
2.2.4. In vitro study of onset time and duration of action in isolated guinea
pig trachea
Selected compounds 9g and (R)-18c were investigated for their
abilities to inhibit the contraction of guinea pig tracheal strips ex-
pressed as a measure of the functional response at the β2-adrenoceptor.
The onset times and the duration of action of isoprenaline, salmeterol,
9g and (R)-18c are presented in Table 6. The time for onset of action is
defined as the duration from application of the tested drug till ap-
pearance of the half maximal response. Duration of action is defined as
2.4. Molecular modeling
We performed molecular modelling to study the binding modes of
compounds 9g and (R)-18c in the molecular target. We used the
crystallographic structure of the β2-adrenoceptor co-crystallized with
the high affinity agonist hydroxybenzyl isoproterenol (HBI) (PDB:
4LDL)27 to dock against the molecular models of compounds 9g and
(R)-18c. The docking program was Glide in Schrödinger Maestro. For
the HBI-bound β2-adrenoceptor (Fig. 4A), “the two hydroxyl groups on
the catechol head of HBI formed hydrogen bonds with Asn293, Ser207
and Ser203, which are thought to be important for agonist binding and
receptor activation. The β-OH group and the charged secondary amine
moiety of HBI formed a hydrogen bond network with Asp113 and
Asn312. Furthermore, π−π interactions and π−σ interactions were
observed between HBI and residues Val114, Val117, Phe193, Phe290
and Ile309.”15 The binding modes of compounds 9g (Fig. 4B) and (R)-
18c (Fig. 4C) were similar to that of HBI. The β-OH group formed hy-
drogen bonds with residues Asp113 and Asn312. The charged sec-
ondary amine moiety formed hydrogen bonds or electrostatic interac-
tions with residues Asp113, Asn312 and Tyr316. The total number of
hydrogen bonds or electrostatic interactions formed with the β-OH
group or the charged secondary amine moiety was 5 for compounds 9g
and (R)-18c, 4 for HBI, and only 3 for compound (S)-5j in ref. 15, the
most potent compound in that study. This could explain the superior
activities of 9g and (R)-18c over (R)-5j or in general why the 2-amino-
2-phenylethanol class of compounds are less potent than the 2-amino-1-
phenylethanol class. The -NH group and the ketone group on the aro-
matic head formed two hydrogen bonds with Ser203 and the -OH group
formed a hydrogen bond with Ser207 in compound 9g (Fig. 4B)
whereas the aromatic head of compound (R)-18c formed four hydrogen
bonds with Ser203 and Ser207 (Fig. 4C). The meta-hydroxyl group on
the benzene tail of 9g formed a hydrogen bond with the backbone
carbonyl group of Cys191 (Fig. 4B) whereas the respective group in HBI
had it formed with Asp192 (Fig. 4A). The two germinal methyl groups
in the α-carbon of compound (R)-18c were found to interact with
Phe193 via π−σ interactions (Fig. 4C). The tail benzene of compound
(R)-18c formed two π−σ interactions with the hydrophobic residues
Ile94 and Ile309 to anchor the benzene ring. All these binding inter-
actions could explain why 9 g and (R)-18c could act as potent β2-
adrenoceptor agonists and support experimental findings.
the duration from the time point that solution containing 5 × 10−5
M
of histamine without the tested drug replaced solution containing
5 × 10−5 M of histamine with the tested drug till the time point of
appearance of recovery magnitude up to 50% of the climax contraction
pretreated with histamine alone. Both compounds exhibited onset times
shorter than salmeterol and very close to isoprenaline; therefore the
two compounds are expected to have equally fast onset of action in vivo.
The duration of action of 9g and (R)-18c on guinea pig trachea were
found to be more than 12 h in each case and similar to salmeterol.
Compounds 9g and (R)-18c are expected to behave as long-acting
bronchodilators.
2.3. Superiority of the 5-(2-amino-1-hydroxyethyl)-8-hydroxyquinolin-
2(1H)-one scaffold for β2-adrenoceptor agonists
In terms of activity, β2/β1-selectivity and duration of action, the
compounds in the present study and in particular 9g and (R)-18c are at
least as good as the long-acting β2-agonist salmeterol. In terms of onset
time, these compounds are superior to salmeterol. This demonstrates
that the 5-(2-amino-1-hydroxyethyl)-8-hydroxyquinolin-2(1H)-one
moiety is a promising scaffold for highly active β2-adrenoceptor ago-
nists. The present series of β2-adrenoceptor agonists belongs to the
classical 2-amino-1-phenylethanol class. The newly developed trantin-
terol17 and its derivatives15,16 contains a novel 2-amino-2-pheny-
lethanol core structure. Compounds in this new class are inferior in
potency (pEC50 values were in a range of 6–9),15,16 as compared to
Table 6
The onset time and duration of action of the test compounds and reference
compounds.
Compound
Onset (min)
Duration (h)
Isoprenaline
Salmeterol
9g
2.2
0.35
–
14.2
2.3
1.65*
> 12
> 12
> 12
0.77
0.12
(R)-18c
1.9
3. Conclusion
The pharmacodynamic parameters of the tested drugs were determined on
histamine-induced contraction of guinea pig tracheal muscles. Data are ex-
A series of novel β2-adrenoceptor agonists with a 5-(2-amino-1-hy-
droxyethyl)-8-hydroxyquinolin-2(1H)-one moiety was designed and
synthesized. Structure-activity relationships of these compounds were
pressed as means
S.D. (n = 4).
* p < 0.05, compared with that of isoprenaline at the corresponding time
point.
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Fig. 4. Binding modes of HBI (A), compounds 9g (B) and (R)-18c (C).
assessed by evaluation of the compounds’ β2-adrenoceptor agonistic
effects and β2/β1-selectivity. Existing data for 9g and (R)-18c suggest
that they are potent and selective β2-adrenoceptor agonists with fast
onset of action (comparable to isoprenaline) and long duration of action
(comparable to salmeterol) in vitro. These results support their further
development as long-acting β2-adrenoceptor agonists.
4.1.3.1. 8-Hydroxy-5-(1-hydroxy-2-((3-phenylpropyl)amino)ethyl)
quinolin-2(1H)-one (9a). White solid; mp: 161.7–162.9 °C; yield 97.8%;
1H NMR (600 MHz, DMSO‑d6) δ:10.48 (s, 2H), 9.35 (s, 1H), 8.71 (s,
1H), 8.26 (d, J = 9.9 Hz, 1H), 7.28 (t, J = 7.5 Hz, 2H), 7.19 (dd,
J = 15.5, 7.5 Hz, 3H), 7.13 (d, J = 8.1 Hz, 1H), 6.99 (d, J = 8.1 Hz,
1H), 6.52 (d, J = 9.8 Hz, 1H), 6.13 (d, J = 3.8 Hz, 1H), 5.43 (d,
J = 9.9 Hz, 1H), 3.04 (s, 1H), 3.01–2.88 (m, 3H), 2.63 (td, J = 7.6,
2.8 Hz, 2H), 2.00–1.90 (m, 2H). 13C NMR (150 MHz, DMSO‑d6)
δ:161.22, 144.76, 138.68, 136.94, 136.42, 133.50, 132.08, 130.05,
128.85, 128.44, 128.34, 128.32, 127.95, 127.71, 127.49, 126.58,
126.54, 125.83, 123.51, 121.30, 118.55, 112.54, 70.19, 62.63, 57.59,
45.42, 32.45, 27.20. Calcd. for C20H23N2O3 [M+H]+ 339.1703; found
339.1701.
4. Experimental section
4.1. Chemistry
We bought all the reagents and solvents from commercial suppliers
and used them without additional purification unless otherwise in-
dicated. All reactions were monitored by TLC, using TLC plates (Silica
gel60 GF254, Merck) and UV light visualization. Where appropriate,
crude products were purified by column chromatography using silica
gel (200-300mesh) purchased from Qingdao Haiyang Chemical Co. Ltd.
(China). All biologically evaluated compounds were > % pure. Solvents
were dehydrated by distillation over sodium. 1HNMR and 13CNMR
spectra were recorded for compounds dissolved in DMSO‑d6 on a
Bruker ARX-400 NMR or Bruker ARX-600 NMR spectrometer with TMS
as an internal standard. All accurate mass of final target compounds
were determined by high resolution mass spectrometry (HRMS) on a
Bruker Micromass Time of Flight mass spectrometer equipped with an
electrospray ionization (ESI) detector. The melting points of the com-
pounds were measured by Buchi B-540 melting point meter.
4.1.3.2. 8-Hydroxy-5-(1-hydroxy-2-((2-phenoxyethyl)amino)ethyl)
quinolin-2(1H)-one (9b). White solid; mp: 126.3–127.8 °C; yield 94.1%;
1H NMR (600 MHz, DMSO‑d6) δ:10.52 (s, 2H), 9.49 (s, 1H), 8.99 (s,
1H), 8.28 (d, J = 9.9 Hz, 1H), 7.33 (dd, J = 8.5, 7.4 Hz, 2H), 7.17 (d,
J = 8.2 Hz, 1H), 7.01 (dt, J = 14.7, 7.5 Hz, 4H), 6.55 (d, J = 9.9 Hz,
1H), 5.51 (dd, J = 10.2, 2.0 Hz, 1H), 4.40–4.23 (m, 2H), 3.53–3.35 (m,
2H), 3.28–3.16 (m, 1H), 3.11 (dt, J = 12.4, 6.2 Hz, 1H). 13C NMR
(150 MHz, DMSO‑d6) δ: 170.10, 161.17, 158.24, 143.91, 136.73,
130.08, 129.00, 122.66, 121.71, 120.17, 117.09, 115.10, 114.52,
65.67, 63.64, 60.23, 54.14, 46.53, 21.25, 14.56. Calcd. for
C
19H21N2O4 [M+H]+ 341.1496; found 341.1484.
4.1.3.3. 8-Hydroxy-5-(1-hydroxy-2-((3-(naphthalen-2-yl)propyl)amino)
ethyl)quinolin-2(1H)-one (9c). White solid; mp: 212.8–214.2 °C; yield
97.8%;1H NMR (600 MHz, DMSO‑d6) δ:10.50 (d, J = 10.5 Hz, 2H), 9.34
(s, 1H), 8.74 (s, 1H), 8.26 (d, J = 9.9 Hz, 1H), 7.94–7.79 (m, 3H), 7.72
(s, 1H), 7.48 (dt, J = 14.7, 6.8 Hz, 2H), 7.41 (d, J = 8.4 Hz, 1H), 7.14
(d, J = 8.2 Hz, 1H), 7.00 (d, J = 8.1 Hz, 1H), 6.54 (d, J = 9.8 Hz, 1H),
6.16 (s, 1H), 5.45 (d, J = 9.5 Hz, 1H), 3.12–3.05 (m, 1H), 3.00 (d,
J = 7.3 Hz, 1H), 2.83 (dd, J = 7.4, 5.7 Hz, 3H), 2.08 (tdd, J = 21.0,
13.4, 7.4 Hz, 2H). 13C NMR (150 MHz, DMSO‑d6) δ:160.05, 143.86,
138.85, 136.77, 133.59, 132.15, 129.03, 128.98, 128.39, 127.97,
127.78, 127.64, 126.65, 126.56, 125.85, 122.67, 120.16, 117.10,
114.45, 65.73, 53.79, 47.17, 32.65, 27.23. Calcd. for C24H25N2O3 [M
+H]+ 389.1860; found 389.1867.
4.1.1. 8-(Benzyloxy)-5-(2,2-dihydroxyacetyl)quinolin-2(1H)-one (11)
Intermediate 10 (1.0 equiv.) was reacted with with 40% hydro-
bromic acid (3.0 equiv.) in DMSO (190 mL) at 80 °C for 2 h. The mixture
was added into 1000 mL water and stirred at room temperature for 1 h.
The residue was filtered and dried to give a yellow solid.
4.1.2. General method for the synthesis of 12a–r
To a stirred solution of intermediate 11 (1.0 equiv.), different sub-
stituted amines side chains (1.0 equiv.) in DMSO were added and stirred
at room temperature for 1.5 h. Then appropriate methanol and sodium
borohydride (1.0 equiv.) were added into the reaction mixture stirred
on an ice-bath for 2 h. Removal of the solvent gave a residue that was
subsequently purified by column chromatography (silica gel, CH2Cl2-
MeOH 30:1 as an eluent) to give 12a–r as white solids.
4.1.3.4. 5-(2-((3-([1,1′-Biphenyl]-4-yl)propyl)amino)-1-hydroxyethyl)-8-
hydroxyquinolin-2(1H)-one (9d). White solid; mp: 237.1–238.6 °C; yield
98.0%;1H NMR (600 MHz, DMSO‑d6)
δ 10.55 (s, 1H), 8.32 (d,
4.1.3. General method for the synthesis of 9a–r
J = 9.9 Hz, 1H), 7.61 (dd, J = 35.9, 7.7 Hz, 4H), 7.45 (t, J = 7.6 Hz,
2H), 7.39–7.23 (m, 3H), 7.10 (dd, J = 30.4, 8.2 Hz, 2H), 6.50 (d,
J = 9.8 Hz, 1H), 5.34 (s, 1H), 3.16 (s, 1H), 2.83 (d, J = 45.5 Hz, 4H),
2.73–2.61 (m, 2H), 1.90 (s, 2H). 13C NMR (150 MHz, DMSO‑d6) δ:
144.81, 142.49, 141.01, 140.44, 138.45, 137.60, 134.89, 129.12,
A solution of intermediates 12a–r in methanol and 10% Pd/C
(0.1 equiv.) was added. The reaction mixture was stirred at room
temperature at 1 atmosphere of hydrogen for 2 h. The resulting mixture
was filtered and concentrated to give 9a–r as a white solid.
6

G. Xing et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
128.26, 127.30, 127.00, 124.90, 120.94, 118.18, 116.21, 113.49,
amino)ethyl)quinolin-2(1H)-one (9j). White solid; mp: 163.3–164.3 °C;
yield 94.4%; 1H NMR (600 MHz, DMSO‑d6) δ:10.51 (s, 2H), 9.47 (s,
1H), 8.75 (s, 1H), 8.30 (d, J = 9.9 Hz, 1H), 7.21 (t, J = 8.0 Hz, 1H),
7.15 (d, J = 8.2 Hz, 1H), 7.02 (d, J = 8.2 Hz, 1H), 6.84–6.73 (m, 1H),
6.54 (d, J = 9.9 Hz, 3H), 5.48 (d, J = 9.9 Hz, 1H), 3.74 (s, 1H),
3.10–3.02 (m, 3H), 2.96 (ddd, J = 20.3, 16.2, 8.2 Hz, 1H), 2.68–2.55
(m, 2H), 2.00–1.88 (m, 2H). 13C NMR (150 MHz, DMSO‑d6) δ:160.09,
158.72, 142.79, 141.72, 135.79, 128.81, 128.00, 127.88, 121.49,
119.90, 119.07, 116.04, 113.41, 113.38, 110.79, 64.62, 59.15, 54.31,
52.81, 46.06, 31.44, 26.18, 20.16, 13.48. Calcd. for C21H25N2O4 [M
+H]+ 369.1809; found 369.1807.
49.02, 47.89, 32.47. Calcd. for
found 415.2019.
C
26H27N2O3 [M+H]+ 415.2016;
4.1.3.5. 8-Hydroxy-5-(1-hydroxy-2-((4-phenylbutyl)amino)ethyl)
quinolin-2(1H)-one (9e). White solid; mp: 148.6–149.9 °C; yield
93.7%;1H NMR (600 MHz, DMSO‑d6) δ:10.51 (s, 2H), 9.31 (s, 1H),
8.71 (s, 1H), 8.29 (d, J = 9.9 Hz, 1H), 7.28 (t, J = 7.5 Hz, 2H),
7.24–7.10 (m, 4H), 7.02 (d, J = 8.1 Hz, 1H), 6.55 (d, J = 9.8 Hz, 1H),
6.15 (d, J = 3.2 Hz, 1H), 5.46 (d, J = 9.9 Hz, 1H), 3.04 (d, J = 12.2 Hz,
1H), 2.96 (t, J = 7.4 Hz, 3H), 2.59 (t, J = 7.4 Hz, 2H), 1.78–1.51 (m,
4H).13C NMR (150 MHz, DMSO‑d6) δ: 160.08, 142.78, 141.04, 135.68,
127.96, 127.91, 127.71, 127.67, 125.19, 121.58, 119.05, 116.00,
113.39, 64.58, 52.76, 46.27, 33.93, 27.29, 24.28. Calcd. for
4.1.3.11. 8-Hydroxy-5-(1-hydroxy-2-((3-(2-methoxyphenyl)propyl)
amino)ethyl)quinolin-2(1H)-one (9k). White solid; mp: 152.9–154.1 °C;
yield 96.7%; 1H NMR (600 MHz, DMSO‑d6) δ:10.51 (d, J = 7.9 Hz, 2H),
9.33 (s, 1H), 8.71 (s, 1H), 8.28 (d, J = 9.9 Hz, 1H), 7.28–7.10 (m, 3H),
7.01 (d, J = 8.1 Hz, 1H), 6.96 (d, J = 8.0 Hz, 1H), 6.88 (t, J = 7.3 Hz,
1H), 6.54 (d, J = 9.9 Hz, 1H), 6.15 (d, J = 3.7 Hz, 1H), 5.52–5.38 (m,
1H), 3.77 (s, 3H), 3.13–2.86 (m, 4H), 2.66–2.52 (m, 2H), 1.98–1.84 (m,
2H). 13C NMR (150 MHz, DMSO‑d6) δ:161.17, 157.47, 143.86, 136.80,
130.01, 129.06, 128.98, 128.94, 128.02, 122.63, 120.80, 120.15,
117.09, 114.49, 111.18, 65.70, 60.23, 55.72, 53.83, 47.33, 27.15,
26.01, 21.25, 14.56. Calcd. for C21H25N2O4 [M+H]+ 369.1809; found
369.1801.
C
21H25N2O3 [M+H]+ 353.1860; found 353.1863.
4.1.3.6. 8-Hydroxy-5-(1-hydroxy-2-((3-(4-hydroxyphenyl)propyl)amino)
ethyl)quinolin-2(1H)-one (9f). White solid; mp: 175.9–177.2 °C; yield
93.9%; 1H NMR (600 MHz, DMSO‑d6) δ:10.50 (s, 2H), 9.25 (d,
J = 18.4 Hz, 2H), 8.70 (s, 1H), 8.26 (d, J = 9.9 Hz, 1H), 7.14 (d,
J = 8.2 Hz, 1H), 7.00 (dd, J = 8.2, 4.1 Hz, 3H), 6.69 (d, J = 8.3 Hz,
2H), 6.55 (d, J = 9.9 Hz, 1H), 6.14 (d, J = 3.5 Hz, 1H), 5.43 (d,
J = 9.9 Hz, 1H), 3.15–2.83 (m, 2H), 2.52 (s, 2H), 1.97–1.84 (m, 2H).
13C NMR (150 MHz, DMSO‑d6) δ:161.17, 156.06, 143.85, 136.78,
131.15, 129.57, 129.04, 128.98, 122.65, 120.16, 117.10, 115.62,
114.47, 65.72, 53.81, 47.19, 31.70, 27.75. Calcd. for C20H23N2O4 [M
+H]+ 355.1652; found355.1643.
4.1.3.12. N-(3-(3-((2-Hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-
5-yl)ethyl)amino)propyl)phenyl)-3-methylbutanamide (9l). White solid;
mp: 177.3–178.9 °C; yield 96.9%; 1H NMR (600 MHz, DMSO‑d6)
δ:10.50 (s, 2H), 9.92 (s, 1H), 9.32 (s, 1H), 8.74 (s, 1H), 8.27 (d,
J = 9.9 Hz, 1H), 7.53 (s, 1H), 7.38 (d, J = 8.1 Hz, 1H), 7.19 (dd,
J = 21.6, 13.7 Hz, 1H), 7.15 (d, J = 8.2 Hz, 1H), 7.01 (d, J = 8.1 Hz,
1H), 6.88 (d, J = 7.5 Hz, 1H), 6.55 (d, J = 9.8 Hz, 1H), 6.15 (d,
J = 3.7 Hz, 1H), 5.45 (d, J = 10.0 Hz, 1H), 3.01 (d, J = 56.6 Hz, 4H),
2.60 (dd, J = 11.6, 7.4 Hz, 2H), 2.35–2.23 (m, 2H), 1.98–1.91 (m, 1H),
1.54 (td, J = 13.2, 6.6 Hz, 1H), 1.52–1.33 (m, 2H), 0.89 (d, J = 6.5 Hz,
6H). 13C NMR (150 MHz, DMSO‑d6) δ:171.88, 161.16, 143.86, 141.68,
139.98, 136.76, 129.14, 129.01, 123.31, 122.67, 120.17, 119.36,
117.34, 117.11, 114.46, 65.72, 60.23, 53.81, 47.21, 34.94, 34.58,
32.73, 27.74, 27.46, 22.78. Calcd. for C25H32N3O4 [M+H]+ 438.2387;
found 438.2388.
4.1.3.7. 8-Hydroxy-5-(1-hydroxy-2-((3-(3-hydroxyphenyl)propyl)amino)
ethyl)quinolin-2(1H)-one (9g). White solid; mp: 175.3–176.8 °C; yield
95.8%; 1H NMR (600 MHz, DMSO‑d6) δ:10.51 (s, 2H), 9.43 (s, 1H), 9.24
(s, 1H), 8.70 (s, 1H), 8.27 (d, J = 9.9 Hz, 1H), 7.15 (d, J = 8.2 Hz, 1H),
7.06 (dd, J = 7.4, 1.4 Hz, 2H), 7.02 (dd, J = 10.3, 4.8 Hz, 1H),
6.86–6.76 (m, 1H), 6.77–6.66 (m, 1H), 6.55 (d, J = 9.9 Hz, 1H), 5.44
(dd, J = 10.0, 2.0 Hz, 4H), 3.11–2.87 (m, 1H), 2.62–2.52 (m, 2H),
2.00–1.86 (m, 2H). 13C NMR (150 MHz, DMSO‑d6) δ:169.74, 160.09,
154.51, 142.79, 135.73, 129.13, 127.95, 127.89, 126.53, 126.15,
121.52, 119.10, 118.29, 116.04, 114.33, 113.40, 64.62, 59.15, 52.71,
46.30, 26.13, 24.94, 20.47, 20.16, 17.95, 13.48. Calcd. for C20H23N2O4
[M+H]+ 355.1652; found 355.1659.
4.1.3.8. 8-Hydroxy-5-(1-hydroxy-2-((3-(2-hydroxyphenyl)propyl)amino)
ethyl)quinolin-2(1H)-one (9h). White solid; mp: 173.3–174.1 °C; yield
98.7%; 1H NMR (600 MHz, DMSO‑d6) δ:10.52 (s, 2H), 9.41 (s, 1H), 8.74
(s, 1H), 8.29 (d, J = 9.9 Hz, 1H), 7.15 (d, J = 8.2 Hz, 1H), 7.07 (t,
J = 7.9 Hz, 1H), 7.01 (d, J = 8.1 Hz, 1H), 6.73–6.38 (m, 4H), 5.46 (dd,
J = 10.0, 2.0 Hz, 1H), 3.14–2.82 (m, 4H), 2.55 (tt, J = 14.4, 7.2 Hz,
2H), 2.00–1.82 (m, 2H). 13C NMR (150 MHz, DMSO‑d6) δ:160.08,
156.82, 142.78, 141.51, 135.75, 128.69, 127.97, 127.89, 121.51,
119.08, 118.20, 116.04, 114.52, 113.40, 112.41, 64.62, 59.15, 55.39,
52.76, 50.63, 46.14, 31.48, 26.26, 20.16, 19.87, 17.95, 13.48. Calcd.
for C20H23N2O4 [M+H]+ 355.1652; found 355.1653.
4.1.3.13. N-(3-(3-((2-Hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-
5-yl)ethyl)amino)propyl)phenyl)isobutyramide
(9m). White
solid;
mp:182.5–184.2 °C; yield 90.1%; 1H NMR (600 MHz, DMSO‑d6)
δ:10.49 (d, J = 12.5 Hz, 2H), 9.85 (s, 1H), 9.22 (s, 1H), 8.71 (s, 1H),
8.25 (d, J = 9.7 Hz, 1H), 7.57 (s, 1H), 7.39 (d, J = 7.9 Hz, 1H), 7.19
(dd, J = 23.1, 15.2 Hz, 2H), 7.14 (d, J = 8.2 Hz, 1H), 7.00 (d,
J = 8.1 Hz, 1H), 6.88 (d, J = 7.6 Hz, 1H), 6.55 (d, J = 9.8 Hz, 1H),
6.15 (d, J = 3.7 Hz, 1H), 5.42 (s, 1H), 3.00 (dd, J = 38.1, 27.1 Hz, 4H),
2.59 (dd, J = 13.7, 6.9 Hz, 3H), 2.04–1.85 (m, 2H), 1.08 (d, J = 6.8 Hz,
6H). 13C NMR (150 MHz, DMSO‑d6) δ:174.59, 160.08, 142.78, 140.57,
138.96, 135.66, 128.03, 127.91, 122.25, 121.60, 119.10, 118.38,
116.35, 116.02, 113.37, 64.64, 55.40, 52.70, 47.98, 46.11, 34.24,
31.65, 26.36, 18.91, 17.96. Calcd. for C24H30N3O4 [M+H]+ 424.2231;
found 424.2230.
4.1.3.9. 8-Hydroxy-5-(1-hydroxy-2-((3-(4-methoxyphenyl)propyl)amino)
ethyl)quinolin-2(1H)-one (9i). White solid; mp: 179.0–180.1 °C; yield
96.2%; 1H NMR (600 MHz, DMSO‑d6) δ:10.52 (s, 2H), 9.47 (s, 1H), 8.75
(s, 1H), 8.30 (d, J = 9.9 Hz, 1H), 7.14 (dd, J = 11.7, 8.3 Hz, 3H), 7.02
(d, J = 8.1 Hz, 1H), 6.86 (d, J = 8.4 Hz, 2H), 6.54 (d, J = 9.8 Hz, 1H),
5.48 (d, J = 9.8 Hz, 1H), 5.31 (s, 1H), 3.72 (s, 3H), 3.05 (t, J = 10.5 Hz,
1H), 2.95 (ddd, J = 20.2, 16.2, 8.1 Hz, 3H), 2.58 (dd, J = 7.6, 5.7 Hz,
2H), 2.01–1.82 (m, 2H). 13C NMR (150 MHz, DMSO‑d6) δ: 160.09,
156.97, 142.79, 135.80, 131.96, 128.60, 127.99, 127.87, 121.48,
119.07, 116.04, 113.42, 113.19, 64.61, 54.37, 52.81, 46.07, 30.55,
26.57. Calcd. for C21H25N2O4 [M+H]+ 369.1809; found 369.1804.
4.1.3.14. N-(3-(3-((2-Hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-
5-yl)ethyl)amino)propyl)phenyl)-3,5,5-trimethylhexanamide (9n). White
solid; mp: 168.4–169.9 °C; yield 98.0%; 1H NMR (600 MHz,
DMSO‑d6) δ:10.44 (s, 1H), 9.82 (s, 1H), 8.55 (d, J = 50.4 Hz, 2H),
8.16 (d, J = 9.9 Hz, 1H), 7.56 (s, 1H), 7.34 (d, J = 8.1 Hz, 1H), 7.21 (t,
J = 7.8 Hz, 1H), 7.14 (d, J = 8.2 Hz, 1H), 6.98 (d, J = 8.1 Hz, 1H), 6.89
(d, J = 7.5 Hz, 1H), 6.58 (d, J = 9.9 Hz, 1H), 6.15 (s, 1H), 5.34–5.12
(m, 4H), 3.07 (ddd, J = 42.7, 25.4, 18.0 Hz, 2H), 2.59 (t, J = 7.6 Hz,
1H), 2.24(dd, J = 13.8, 6.4 Hz, 1H), 2.14 (dd, J = 13.8, 7.9 Hz, 1H),
2.10–1.99 (m, 2H), 1.99–1.86 (m, 1H), 1.29 (dd, J = 13.9, 3.6 Hz, 1H),
4.1.3.10. 8-Hydroxy-5-(1-hydroxy-2-((3-(3-methoxyphenyl)propyl)
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1.09 (dd, J = 13.8, 6.7 Hz, 1H), 0.95 (d, J = 6.6 Hz, 3H), 0.88 (s, 9H).
13C NMR (150 MHz, DMSO‑d6) δ:171.08, 161.16, 143.91, 141.63,
139.87, 136.58, 129.17, 129.05, 128.80, 123.38, 122.74, 120.33,
119.49, 117.48, 117.14, 114.40, 65.85, 53.53, 50.54, 47.25, 46.68,
32.68, 31.26, 30.34, 27.50, 27.32, 23.02. Calcd. for C26H34N3O4 [M
+H]+ 452.2544; found 452.2543.
4.1.4. General procedure for the synthesis of 2-methylbutan-2-amine
(16a–c)
2-Methylbutan-2-amine derivatives (16a–c) were synthesized ac-
cording to previously reported methods.28
4.1.5. General procedure for the synthesis of compounds (18a–c)
Compounds (18a–c) were synthesized according to previously de-
scribed methods for (9a–r)
4.1.3.15. N-(3-(3-((2-Hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-
5-yl)ethyl)amino)propyl)phenyl)benzamide (9o). White solid; mp:
232.8–233.9 °C; yield 94.97%; 1H NMR (600 MHz, DMSO‑d6) δ:10.51
(s, 2H), 9.99 (s, 1H), 9.34 (s, 1H), 8.81 (s, 1H), 8.25 (d, J = 10.0 Hz,
1H), 7.54 (d, J = 8.5 Hz, 1H), 7.34–7.22 (m, 2H), 7.22–7.08 (m, 4H),
7.01 (d, J = 8.2 Hz, 3H), 6.56 (d, J = 9.9 Hz, 1H), 6.19 (d, J = 3.6 Hz,
1H), 5.45 (d, J = 10.0 Hz, 1H), 3.21–2.83 (m, 8H), 2.62 (t, J = 7.7 Hz,
2H). 13C NMR (150 MHz, DMSO‑d6) δ: 170.79, 161.17, 143.88, 141.65,
138.42, 136.74, 132.20, 129.33, 129.01, 128.78, 128.72, 126.41,
122.68, 120.16, 119.75, 117.09, 114.48, 65.73, 53.78, 48.57, 38.36,
31.30. Calcd. for C27H28N3O4 [M+H]+ 458.2074; found 458.2076.
4.1.5.1. 8-Hydroxy-5-(1-hydroxy-2-((2-methyl-4,4-diphenylbutan-2-yl)
amino)ethyl)quinolin-2(1H)-one
(18a). White
solid;
mp:
187.9–180.0 °C; yield 96.8%;1H NMR (600 MHz, DMSO‑d6) δ:10.49
(s, 2H), 9.63 (s, 1H), 8.46 (s, 1H), 8.30 (d, J = 9.9 Hz, 1H), 7.50–7.35
(m, 4H), 7.26 (dt, J = 17.1, 7.6 Hz, 4H), 7.19 (d, J = 8.2 Hz, 1H),
7.17–7.08 (m, 2H), 7.00 (d, J = 8.1 Hz, 1H), 6.55 (d, J = 9.8 Hz, 1H),
6.16 (s, 1H), 5.48 (s, 1H), 4.31 (t, J = 6.1 Hz, 1H), 2.97 (s, 2H),
2.65–2.51 (m, 2H), 1.23–1.09 (m, 6H). 13C NMR (150 MHz, DMSO‑d6)
δ: 169.74, 160.07, 145.18, 145.11, 142.75, 135.62, 128.11, 127.88,
127.86, 126.88, 125.52, 121.68, 119.13, 116.05, 113.31, 64.81, 59.14,
47.24, 45.02, 41.13, 22.58, 20.16, 13.47. Calcd. for C28H31N2O3 [M
+H]+ 443.2329; found 443.2322.
4.1.3.16. N-(3-(3-((2-Hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-
5-yl)ethyl)amino)propyl)phenyl)-4-methylpentanamide (9p). White solid;
mp: 185.7–187.0 °C; yield 96.9%;1H NMR (600 MHz, DMSO‑d6) δ:10.50
(s, 2H), 9.92 (s, 1H), 9.36 (s, 1H), 8.74 (s, 1H), 8.28 (d, J = 9.9 Hz, 1H),
7.53 (s, 1H), 7.39 (d, J = 8.1 Hz, 1H), 7.20 (t, J = 7.8 Hz, 1H), 7.15 (d,
J = 8.2 Hz, 1H), 7.01 (d, J = 8.1 Hz, 1H), 6.88 (d, J = 7.6 Hz, 1H), 6.54
(d, J = 9.8 Hz, 1H), 6.15 (d, J = 3.6 Hz, 1H), 5.45 (d, J = 9.9 Hz, 1H),
3.12–2.88 (m, 4H), 2.65–2.54 (m, 2H), 2.35–2.23 (m, 2H), 2.01–1.89
(m, 2H), 1.55 (tt, J = 13.0, 6.5 Hz, 1H), 1.47 (dd, J = 14.9, 7.2 Hz, 2H),
0.89 (d, J = 6.5 Hz, 6H). 13C NMR (150 MHz, DMSO‑d6) δ:170.81,
160.13, 145.77, 144.97, 142.78, 140.60, 138.90, 135.72, 128.04,
127.96, 127.90, 122.23, 121.57, 119.07, 118.27, 116.26, 116.02,
113.39, 64.62, 52.77, 46.13, 33.86, 33.50, 31.66, 26.65, 26.37,
21.69. Calcd. for C26H34N3O4 [M+H]+ 452.2544; found 452.2543.
4.1.5.2. 5-(2-((4,4-Bis(4-fluorophenyl)-2-methylbutan-2-yl)amino)-1-
hydroxyethyl)-8-hydroxyquinolin-2(1H)-one (18b). White solid; mp:
160.0–161.2 °C; yield 96.1%;1H NMR (600 MHz, DMSO‑d6) δ:10.50
(s, 2H), 9.68 (s, 1H), 8.46 (s, 1H), 8.31 (d, J = 9.9 Hz, 1H), 7.46 (dd,
J = 13.9, 8.2 Hz, 4H), 7.20 (d, J = 8.2 Hz, 1H), 7.15–7.05 (m, 4H), 7.01
(d, J = 8.1 Hz, 1H), 6.56 (d, J = 9.8 Hz, 1H), 6.17 (s, 1H), 5.50 (s, 1H),
4.43 (t, J = 6.3 Hz, 1H), 2.97 (s, 2H), 2.56 (dd, J = 13.9, 6.4 Hz, 1H),
2.48 (s, 1H), 1.15 (d, J = 10.6 Hz, 6H). 13C NMR (150 MHz, DMSO‑d6)
δ:161.91, 161.16, 160.30, 143.86, 142.37, 142.27, 136.70, 129.73,
129.68, 129.17, 128.96, 122.79, 120.21, 117.13, 115.78, 115.75,
115.64, 115.61, 114.41, 65.84, 60.35, 60.23, 48.31, 44.29, 42.35,
23.75, 23.65, 21.24, 14.56. Calcd. for
479.2141; found 479.2148.
C
28H29F2N2O3 [M+H]+
4.1.5.3. 8-Hydroxy-5-(1-hydroxy-2-((2-methyl-4-phenylbutan-2-yl)
amino)ethyl)quinolin-2(1H)-one(18c). White solid; mp: 191.2–192.6 °C;
yield 90.1%;1H NMR (600 MHz, DMSO‑d6) δ:10.52 (s, 2H), 9.68 (s, 1H),
8.45 (t, J = 10.1 Hz, 1H), 8.34 (d, J = 9.9 Hz, 2H), 7.29 (t, J = 7.5 Hz,
4H), 7.25–7.11 (m, 1H), 7.02 (d, J = 8.1 Hz, 1H), 6.55 (d, J = 9.9 Hz,
1H), 5.50 (d, J = 9.5 Hz, 2H), 2.99 (ddd, J = 32.7, 21.1, 11.2 Hz, 1H),
2.64 (dd, J = 16.6, 6.7 Hz, 2H), 1.93 (t, J = 8.7 Hz, 2H), 1.37 (d,
J = 10.4 Hz, 6H). 13C NMR (150 MHz, DMSO‑d6) δ: 161.16, 143.89,
141.81, 136.79, 129.13, 128.96, 128.87, 128.74, 126.44, 122.68,
120.23, 117.17, 114.46, 65.80, 59.53, 49.06, 48.49, 29.81, 23.07.
Calcd. for C22H27N2O3 [M+H]+ 367.2016; found 367.2007.
4.1.3.17. N-(4-(3-((2-Hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-
5-yl)ethyl)amino)propyl)phenyl)isobutyramide (9q). White solid; mp:
232.9–234.2 °C; yield 98.0%;1H NMR (600 MHz, DMSO‑d6) δ:10.49
(s, 2H), 9.83 (s, 1H), 9.30 (s, 1H), 8.71 (s, 1H), 8.26 (d, J = 9.9 Hz, 1H),
7.54 (d, J = 8.4 Hz, 2H), 7.13 (dd, J = 10.6, 8.4 Hz, 3H), 7.00 (d,
J = 8.1 Hz, 1H), 6.54 (d, J = 9.8 Hz, 1H), 6.14 (d, J = 3.5 Hz, 1H), 5.44
(d, J = 10.0 Hz, 1H), 3.10–2.84 (m, 4H), 2.64–2.52 (m, 3H), 2.02–1.86
(m, 2H), 1.08 (d, J = 6.8 Hz, 6H). 13C NMR (150 MHz, DMSO‑d6) δ:
175.52, 161.17, 143.86, 138.04, 136.77, 135.57, 129.03, 128.98,
128.84, 122.66, 120.16, 119.69, 117.10, 114.46, 65.71, 53.80, 47.12,
35.29, 31.91, 27.46, 20.01. Calcd. for C24H30N3O4 [M+H]+ 424.2231;
found 424.2237.
4.2. Biological tests
4.1.3.18. N-(4-(3-((2-Hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-
5-yl)ethyl)amino)propyl)phenyl)-3,5,5-trimethylhexanamide (9r). White
solid; mp: 237.8–239.1 °C; yield 94.8%;1H NMR (600 MHz, DMSO‑d6)
δ:10.51 (s, 2H), 9.85 (s, 1H), 9.19 (s, 1H), 8.70 (s, 1H), 8.25 (d,
J = 9.9 Hz, 1H), 7.52 (d, J = 8.4 Hz, 2H), 7.13 (dd, J = 10.2, 8.4 Hz,
3H), 7.00 (d, J = 8.1 Hz, 1H), 6.55 (d, J = 9.9 Hz, 1H), 6.15 (s, 1H),
5.42 (d, J = 9.9 Hz, 1H), 3.07 (d, J = 11.7 Hz, 1H), 3.03–2.96 (m, 1H),
2.94 (t, J = 7.8 Hz, 2H), 2.58 (t, J = 7.3 Hz, 2H), 2.23 (dd, J = 13.7,
6.6 Hz, 1H), 2.13 (dd, J = 13.7, 7.8 Hz, 1H), 2.03 (ddd, J = 13.8, 10.4,
6.8 Hz, 1H), 1.94 (ddd, J = 28.3, 13.2, 7.4 Hz, 2H), 1.28 (dd, J = 13.9,
3.5 Hz, 1H), 1.08 (dd, J = 13.9, 6.7 Hz, 1H), 0.94 (d, J = 6.6 Hz, 3H),
0.88 (s, 9H). 13C NMR (150 MHz, DMSO‑d6) δ:170.92, 161.16, 143.86,
137.86, 136.74, 135.66, 129.02, 128.99, 128.86, 122.68, 120.17,
119.73, 117.10, 114.44, 65.74, 60.23, 53.77, 50.54, 49.06, 47.14,
46.67, 31.93, 31.26, 30.34, 27.49, 27.35, 23.03. Calcd. for C29H40N3O4
[M+H]+ 494.3013; found 494.3020.
4.2.1. Cellular cAMP assay
The cAMP assay was performed as described previously.15 In brief,
HEK293 cells overexpressing human β2 or β1-adrenoceptors were re-
suspended in Hank’s balanced salt solution and added into OptiPlate-
384 plates containing the test compounds. Stimulation was allowed to
proceed for 60 min. The amounts of cellular cAMP were determined
based on a cAMP standard curve using the HTRF cAMP dynamic 2 assay
kit (Cisbio). The plates were read on an EnVision microplate reader
after incubation for 60 min at room temperature.
4.2.2. Isolated guinea pig trachea relaxation assay
The methods for the isolation of respiratory tissues and the smooth
muscle relaxation assay have been described previously.15 In brief,
tracheal strips were isolated from male Dunkin-Hartley guinea pigs
weighing 250–350 g and mounted in organ baths containing Krebs-
Henseleit solution (containing in mM: NaCl 118 g, KCl 5.4 g, CaCl2
8

G. Xing et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
2.5 g, MgSO4 0.6 g, NaH2PO4 2.94 g, NaHCO3 25 g and D-glucose 11.7 g)
aerated with 95% O2 and 5% CO2. The experiments were approved by
the Ethics Committee for Animal Care and Use of Shenyang Pharma-
ceutical University. An initial tension of 1.5 g was preset to the muscle
sample for 1 h to achieve a steady spontaneous tone level. Histamine
(5 × 10−5 M) was added in the system to stimulate the muscle sample
and the stimulation test were repeated twice to testify the samples
before the actual test. Buffer solution in the experimental system should
be renewed every 15–20 min throughout the measurement of muscle
tension. Amplified tension signals were recorded with a force trans-
ducer connected to a polygraph recorder.
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9. Welte T. Optimising treatment for COPD-new strategies for combination therapy. Int
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Chem. 2013;52:6366–6378.
11. Glossop PA, Lane CA, Price DA, et al. Inhalation by design: novel ultra-long-acting
β2-adrenoreceptor agonists for inhaled once-daily treatment of asthma and chronic
obstructive pulmonary disease that utilize a sulfonamide agonist headgroup. J Med
Chem. 2010;53:6640–6652.
12. Kikkawa H, Naito K, Ikezawa K. Tracheal relaxing effects and β2-selectivity of TA-
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4.3. Molecular modeling
The molecular modeling was performed according to the previously
reported methods.15
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Acknowledgments
17. Gan LL, Wang MW, Cheng MS, et al. Trachea relaxing effects and β2-selectivity of
SPFF, a newly developed bronchodilating agent, in guinea pigs and rabbits. Biol
Pharm Bull. 2003;26:323–328.
This work was supported by the National Natural Science
Foundation of China (Grant Number 81872752 to M.C. and 81673355
to A.Y.-H.W.) and in part by the National Science and Technology
Major Project of Ministry of Science and Technology of the People’s
Republic of China (Grant Number 2018ZX09739009 to M.C.).
18. Etsuko M, Takayuki H, Junji I, et al. Preparation of quaternary ammonium salt
compounds such as 1,1-dimethylpiperidin-1-ium and 9,9-dimethyl-3-oxa-9-aza-
tricyclo[3.3.1.02,4]nonan-9-ium compounds as agonists of β2-adrenergic receptor
and antagonists of muscarinic receptor. JP 2012107008A. 2012.
19. Mathai M, Sarah D, Adam H, et al. Preparation of biphenyl derivatives as β2-adre-
nergic agonists and muscarinic antagonists for pulmonary disorders. US
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