Synthesis and biological evaluation of a novel sigma-1 receptor antagonist based on 3,4-dihydro-2(1H)-quinolinone scaffold as a potential analgesic

Article abstract of DOI:10.1016/j.ejmech.2014.04.019

The synthesis and sigma-1 receptor (σ₁R) antagonist activity of a new series of 3,4-dihydro-2(1H)-quinolinone derivatives are reported. The new compounds were evaluated in vitro in sigma-1 and sigma-2 receptor-binding assays in guinea pig brain membranes. The structure-activity relationship led us to the promising derivative 7-(3-(piperidin-1-yl)propoxy)-1- (4-fluorobenzyl)-3,4-dihydro-2(1H)-quinolinone (35). The compounds with highest affinity and greatest selectivity were further profiled, and compound 35 had a high binding constant for sigma-1 receptor (K_iσ₁ = 1.22 nM) and high sigma-1/2 selectivity (1066-fold). Thus, compound 35, which proved to be an antagonist of sigma-1 receptor, emerged as the most interesting candidate. In addition, compound 35 exerted dose-dependent anti-nociceptive effects in the formalin test. These characteristics suggested that the potent and selective compound 35 could be a potent candidate for pain treatment.

Full text of DOI:10.1016/j.ejmech.2014.04.019

European Journal of Medicinal Chemistry 79 (2014) 216e230
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of a novel sigma-1 receptor
antagonist based on 3,4-dihydro-2(1H)-quinolinone scaffold as a
potential analgesic
Yu Lan a, Yin Chen b, Xiangqing Xu b, Yinli Qiu b, Shicheng Liu b, Xin Liu a, Bi-Feng Liu a,
,b,
Guisen Zhang a
*
a Systems Biology Theme, Department of Biomedical Engineering, College of Life Science and Technology, Huazhong University of Science and Technology,
Wuhan 430074, China
b Jiangsu Nhwa Pharmaceutical Co., Ltd. 69 Democratic South Road, Xuzhou, Jiangsu 221116, China
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and sigma-1 receptor (s1R) antagonist activity of a new series of 3,4-dihydro-2(1H)-qui-
nolinone derivatives are reported. The new compounds were evaluated in vitro in sigma-1 and sigma-2
receptor-binding assays in guinea pig brain membranes. The structureeactivity relationship led us to the
promising derivative 7-(3-(piperidin-1-yl)propoxy)-1-(4-fluorobenzyl)-3,4-dihydro-2(1H)-quinolinone
(35). The compounds with highest affinity and greatest selectivity were further profiled, and compound
35 had a high binding constant for sigma-1 receptor (Kis1 ¼ 1.22 nM) and high sigma-1/2 selectivity
(1066-fold). Thus, compound 35, which proved to be an antagonist of sigma-1 receptor, emerged as the
most interesting candidate. In addition, compound 35 exerted dose-dependent anti-nociceptive effects in
the formalin test. These characteristics suggested that the potent and selective compound 35 could be a
potent candidate for pain treatment.
Received 4 January 2014
Received in revised form
25 March 2014
Accepted 5 April 2014
Available online 8 April 2014
Keywords:
3,4-Dihydro-2(1H)-quinolinone
Sigma-1 receptor
Antagonist
Ó 2014 Elsevier Masson SAS. All rights reserved.
Analgesic
1. Introduction
to improve the efficacy of existing therapies and reduce unwanted
effects.
Pain, as a fundamental and central life experience, limits our
exposure to potentially damaging or life-threatening events.
Chronic pain affects w20% of the population, with the incidence
rising significantly in the elderly [1,2]. Current analgesic medica-
tions include opioids, non-steroidal anti-inflammatory drugs
(NSAIDs) and analgesic adjuvants [3,4]. Opioids induce a wide va-
riety of well-known side effects, including tolerance and physical
dependence. Meanwhile, NSAIDs cause adverse gastrointestinal
reactions and renal toxicity and inhibit platelet aggregation [3].
Antidepressants, anticonvulsants, and anesthetics, have demon-
strated some efficacy in the treatment of neuropathic pain, earning
the nickname “analgesic adjuvants”. However, they have limited
independent efficacy in treating pain [4]. Thus, there is a continuing
unmet medical need for analgesics with new mechanisms of action
Sigma receptors (sR) were discovered in 1976 [5] and originally
mischaracterized as a new class of opioid receptors. Later, sigma
receptors were confused with PCP/NMDA glutamate receptor
complexes because of nonselective ligands [6,7]. Now, it is known
that sigma receptors are unique binding sites with no homology to
opioid receptors or other mammalian proteins [8]. Subsequent
radio-ligand binding studies and biochemical analysis have shown
that sigma receptors exist as two well characterized subtypes,
termed sigma-1 receptor and sigma-2 receptor [9,10]. The sigma-1
receptor has been cloned [11] and shown to act as a unique ligand-
regulated molecular chaperone that modulates the activity of
several proteins, such as the N-methyl-D-aspartate (NMDA) [12]
receptor and several ion channels [13]. The sigma-2 receptor has
not been as well characterized, but appears to be involved in
cellular proliferation and cell death [14].
No selective sigma-1 receptor ligands have been introduced to
the market, but several compounds have been tested in clinical
studies as antidepressants, drug abuse treatments, antipsychotics,
and learning and memory enhancers [15,16]. Moreover, numerous
groups have examined the potential role of sigma-1 receptor in
* Corresponding author. Systems Biology Theme, Department of Biomedical En-
gineering, College of Life Science and Technology, Huazhong University of Science
and Technology, Wuhan 430074, China.
E-mail address: gszhang@mail.hust.edu.cn (G. Zhang).
http://dx.doi.org/10.1016/j.ejmech.2014.04.019
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

Y. Lan et al. / European Journal of Medicinal Chemistry 79 (2014) 216e230
217
pain control [17]. Since the 1990s, the sigma-1 receptor has been
known to modulate opioid analgesia [18e20] and the relationship
by molecular hybridization [37] to form a new high selectivity
sigma-1 receptor ligand with high analgesic activity (Fig. 2).
Informed by the pharmacophoric sigma-1 receptor model by
Glennon [38,39], the two moieties were connected with flexible
carbon chains, and a hydrophobic R region was attached to the N
atom of quinolinone. This strategy yielded compounds 12e39
(Scheme 1), which were used in SAR (structureeactivity rela-
tionship) studies evaluating their pharmacological efficacy and in
competitive receptor binding assays to determine their relative
affinity for sigma-1 and sigma-2 receptors. Among the de-
rivatives prepared, compound 35 exhibited high affinity for the
sigma-1 receptor and low affinity for the sigma-2 receptor.
Further, compound 35 was shown to be an antagonist of sigma-1
receptor and inhibited formalin-induced pain in mice, making it
a potent candidate for treatment of pain.
between the
m-opioid and sigma-1 receptor has been recently
shown to involve direct physical interaction [21]. Reducing the
activity of sigma-1 receptor with antagonists or antisense oligo-
deoxynucleotides enhances the anti-nociception induced by
morphine and other m-opioid receptor agonists in the acute noci-
ceptive test [22,23], while sigma-1 receptor agonists reduce the
analgesic response elicited by opioids [24]. These findings suggest
that the sigma-1 receptor negatively modulates
signal transduction.
m-opioid receptor
The sigma-1 receptor also plays a role in nociception in the
absence of opioids, as shown by studies using sigma-1-receptore
knockout mice and certain behavioral models involving sensitiza-
tion of pain pathways. Mice lacking the sigma-1 receptor did not
respond to formalin in both phases of the typical paw licking and
biting behaviors [25], and showed no mechanical hypersensitivity
following capsaicin sensitization [26]. Cold and mechanical hy-
persensitivity were strongly attenuated in sigma-1-receptore
knockout mice treated with paclitaxel [27] or in which the sciatic
nerve was partially ligated [28]. Pharmacological antagonism of the
sigma-1 receptor produced similar results. Haloperidol and its
metabolites, which have affinity for the sigma-1 receptor, inhibit
formalin-induced pain [29] and capsaicin-induced sensitization in
wild-type mice [30]. The sigma-1 receptor antagonists BD-1063
and NE-100 also inhibit mechanical allodynia induced by capsaicin
[26]. The recently described sigma-1 receptor antagonist S1RA,
which has high affinity for the sigma-1 receptor (Kis1 ¼17 nM) and
excellent sigma-1/2 selectivity (550 fold) [31,32], is currently in
clinical trials for treatment of diverse pain states (Fig. 1) [33,34]
Therefore, sigma-1 receptor antagonists possess high potential as
innovative analgesics.
2. Chemistry
Synthesis of the novel 3,4-dihydroxy-2(1H)-quinolinone de-
rivatives were performed according to the reaction pathways
illustrated in Scheme 1. Standard alkylation of 7-hydroxy-3,4-
dihydroxy-2(1H)-quinolinone
6
or 6-hydroxy-3,4-dihydroxy-
1,3-dibromopropane (1,4-
2(1H)-quinolinone with
7
dibromobutane; 1,5-dibromopentane; 1,6-dibromohexane) led to
derivatives 8ae8d or 9ae9d, which were then reacted with mor-
pholine using K2CO3 as a base in acetonitrile to afford the in-
termediates 10ae10d,11ae11d. Coupling of 8a with various amines
using K2CO3 and a catalytic amount of KI in acetonitrile provided
the intermediates 10fe10m. Reaction of 7-hydroxy-3,4-dihydroxy-
2(1H)-quinolinone 6 or 6-hydroxy-3,4-dihydroxy-2(1H)-quinoli-
none
7 with 4-(2-chloroethyl)-morpholine yielded the in-
Our interest has been focused on the development of novel
ligands with high affinity towards the sigma-1 receptor and
selectivity over the sigma-2 subtype. 3,4-dihydroxy-2(1H)-qui-
nolinone derivatives have recently been identified in screens of
compounds for binding affinity to sigma receptors [35]. Specif-
ically, in vitro studies have demonstrated that OPC-14523 binds
to sigma-1 receptor at nanomolar concentrations (IC50 ¼ 47 nM)
[36]. Therefore, in this paper we combined the 3,4-dihydroxy-
2(1H)-quinolinone moiety with the basic amino moiety of S1RA
termediates 10e, 11e.
Finally, the synthesis of target compounds 12e37 (Scheme 1,
Tables 1e4) was achieved by reacting intermediates 10ae10m,
11ae11e with benzyl bromides or (2-bromoethyl)-benzene, using
NaH as a base in DMF. Compounds 34e37 were prepared through
intermediates 10i or 10j by the same method, with appropriate
aryl-substituted benzyl bromides. Intermediates 10i or 10j were
reacted with iodomethane by using NaH in THF, providing target
compounds 38e39 (Scheme 1, Table 3 and 4).
Fig. 1. Representative sigma-1 receptor antagonists.

218
Y. Lan et al. / European Journal of Medicinal Chemistry 79 (2014) 216e230
Fig. 2. Design of new 3,4-dihydro-2(1H)-quinolinone derivatives.
3. Pharmacology
The functional activity of compounds with high sigma-1 re-
ceptor binding affinity and selectivity for sigma-1 over the sigma-2
receptor was evaluated using phenytoin, a low potency allosteric
modulator of the sigma-1 receptor [42]. Phenytoin shifts sigma-1
receptor agonists to significantly higher affinities, with little effect
on the affinity of sigma-1 receptor antagonists.
3.1. In vitro studies
The sigma-1 and sigma-2 receptor binding affinities of the novel
3,4-dihydro-2(1H)-quinolinone derivatives were determined in
competitive receptor binding studies with radioligands. All com-
pounds were dissolved in 5% DMSO and added to guinea pig brain
membranes in competition with the specific radioligands [3H]-
(þ)-pentazocine for the sigma-1 receptor and [3H]-di-o-tolylgua-
nidine ([3H]-DTG) for the sigma-2 receptor. Since [3H]-DTG in-
teracts with sigma-1 receptors as well, an excess of the selective
sigma-1 receptor ligand (þ)-SKF10047 was added to mask sigma-
1 receptor binding sites [40,41]. Haloperidol, (þ)-pentazocine,
S1RA and DTG were used as reference compounds, and the Ki
values were calculated based on the Cheng and Prussoff equation:
Ki ¼ IC50/(1 þ C/Kd) where C represents the concentration of the hot
ligand used and Kd its receptor dissociation constant were calcu-
lated for each labeled ligand. Mean Ki values and SEM are reported
for at least three independent experiments. Binding affinities were
expressed as Ki ꢀ SEM values in Tables 1e4
3.2. In vivo studies
The analgesic effects of selected compounds were further eval-
uated in vivo by the formalin test in mice.
4. Results and discussion
4.1. Structureeactivity relationships
The design of the new series of 3,4-dihydro-2(1H)-quinolinone
derivatives was based on the known pharmacophoric features of
sigma-1 receptor ligands. As recently summarized by Glennon
et al., inhibitors must include a basic amino group and at least two
ꢀ
ꢀ
hydrophobic regions at specified distances: 2.5e3.9 A and 6e10 A
Scheme 1. Reagents and conditions: (I) Br(CH2)nBr, K2CO3, acetone, reflux; (II) K2CO3, KI, acetonitrile, reflux; (III) Cs2CO3, acetonitrile, reflux; (IV) NaH, DMF, 0e50 ꢁC; (V) NaH, THF,
ꢁC e rt.
0

Y. Lan et al. / European Journal of Medicinal Chemistry 79 (2014) 216e230
219
Table 1
Sigma-1 and sigma-2 receptor affinities of 3,4-dihydroxy-2(1H)-quinolinone derivatives 12e25 and reference compounds.
Compound
n
m
K
is1 (nM)a
K
is2 (nM)b
Selectivity (s2/s1)
12
13
14
15
16
17
18
19
20
21
22
23
24
25
2
3
4
5
6
3
4
2
3
4
5
6
3
4
1
1
1
1
1
2
2
1
1
1
1
1
2
2
>2000
>2000
e
14.8 ꢀ 0.8c
60.2 ꢀ 4.4
88.4 ꢀ 5.1
682 ꢀ 31>
21.6 ꢀ 1.91
44.8 ꢀ 3.6
>2000
471 ꢀ 38
530 ꢀ 46
1811 ꢀ 37
2000
00.4 ꢀ 8.1
1205 ꢀ 132
>2000
1122 ꢀ 54
260 ꢀ 21
430 ꢀ 62
>2000
31.8
8.8
20.4
e
4.6
26.8
e
792 ꢀ 119
22.2 ꢀ 1.4
70.7 ꢀ 2.5
852 ꢀ 31
40.2 ꢀ 1.5
195 ꢀ 21
2.6 ꢀ 0.4
10.5 ꢀ 0.7
3.4 ꢀ 0.5
e
1.4
11.7
6.1
e
98.1 ꢀ 17
2.4
e
>2000
Haloperidol
S1RA
36.4 ꢀ 3.7
>2000
14
>190.5
e
(þ)-Pentazocine
e
DTG
49.2 ꢀ 8.8
e
a
Affinities were determined in guinea pig brain using [3H]-(þ)-pentazocine.
b
c
Affinities were determined in guinea pig brain using [3H]-DTG in the presence of (þ)-SKF-10047 to block sigma-1 receptors.
The values are means ꢀ SEM of three experiments performed in duplicate.
from the amino group, respectively [31,38,39] (Fig. 3 A). Com-
pounds of the general structure (Fig. 3 B) were designed in which
the 3,4-dihydro-2(1H)-quinolinone was used as a scaffold and
decorated with various groups according to these pharmacophoric
requirements; some variations were also considered.
In this work, our initial design focus was to investigate the effect
of different chain lengths on affinities to sigma-1 and sigma-2 re-
ceptors (Table 1, compounds 12e25). The morpholine group was
used because of its frequent appearance in sigma-1-receptor li-
gands, such as S1RA.
In 7-hydroxy-3,4-dihydro-2(1H)-quinolinone derivatives, the
two-carbon linker (compound 12) was first selected but did not
activate either receptor, likely because the linker was too short.
Interestingly, compound 13, which contained only one additional
carbon in the linker, demonstrated high sigma-1 receptor binding
affinity and selectivity toward the sigma-2 receptor (Kis1 ¼14.8 nM
and Kis2 ¼ 471 nM). Further elongation of the carbon linker be-
tween the quinolinone scaffold and morpholine moiety provided
derivatives 14e15, which had lower sigma-1 and sigma-2 receptor
affinities than the propylene counterparts. Derivatives with a six-
atom spacer, such as compound 16, did not bind either receptor,
suggesting that they were too large for the binding site.
hydroxy-3,4-dihydro-2(1H)-quinolinone derivatives with a two-
or three-atom spacer, compound 19e20, showed weak binding
affinity to both the sigma-1 and sigma-2 receptors, while that with
a longer spacer, compound 21 (Kis1 ¼ 22.2 nM and Kis2 ¼ 260 nM)
and 22 (Kis1 ¼ 70.7 nM and Kis2 ¼ 430 nM) had greater binding
affinities to both the sigma-1 and sigma-2 receptors. The six-atom
spacer was too long to allow binding to either. Lengthening the
linker between the N atom of the quinolinone scaffold and the
phenyl group relative to compound 20 (Kis1 ¼ 792 nM and
K
is2 ¼ 1122 nM) to create compound 24 increased both the sigma-1
and sigma-2 receptor binding affinities (Kis1 ¼ 40.2 nM and
K
is2 ¼ 98.1 nM). Increasing the distance between the N atom of the
quinolinone scaffold and the phenyl group of compound 21 pro-
vided 25, which was not tolerated by either the sigma-1 or sigma-2
receptor.
Together, our SAR studies, summarized in Table 1, revealed that
7-hydroxy-3,4-dihydro-2(1H)-quinolinone derivatives have greater
potential as sigma-1 receptor ligands than the 6-hydroxy-3,4-
dihydro-2(1H)-quinolinone derivatives. Of these derivatives, com-
pound 13 showed the highest sigma-1 receptor binding affinity
(Kis1 ¼14.8 nM) and good sigma-1/2 selectivity (31.8 e fold), which
encouraged further exploration.
The effect of increasing distance between the N atom of the
quinolinone scaffold and the phenyl group was also explored.
To explain the SAR, the effect of various amino moieties was
then explored by synthesizing variations of compound 13. As
shown in Table 2, we selected piperidine, 4-methylpiperidine,
piperidin-4-one, 1-methylpiperazine, 1-phenylpiperazine, pyrroli-
dine, dimethylamine, and diethylamine (compounds 26e33) to
replace the morpholine moiety. The substitution of morpholine
with carbonated cyclic analogs provided compound 26, which had
greatly improved sigma-1 receptor binding affinity and maintained
Compared to 13, compound 17 (Kis1
¼
21.6 nM and
K
is2 ¼ 100.4 nM) bound the sigma-2 receptor with greater affinity,
with little decrease in the sigma-1 receptor binding affinity. Com-
pound 18 (Kis1 ¼ 44.8 nM and Kis2 ¼ 1205 nM) showed a little
improvement to compound 14 (Kis1 ¼ 60.2 nM and Kis2 ¼ 530 nM),
but the binding affinity was weaker than that of compound 13. 6-

220
Y. Lan et al. / European Journal of Medicinal Chemistry 79 (2014) 216e230
Table 2
Sigma-1 and sigma-2 receptor affinities of compound 13 derivatives.
Compound
NRR
K
is1 (nM)a
K
is2 (nM)b
Selectivity (s2/s1)
13
14.8 ꢀ 0.8c
471 ꢀ 29
31.8
26
27
1.84 ꢀ 0.33
1.1 ꢀ 0.06
662 ꢀ 42
319 ꢀ 14
359.8
290
28
29
30
31
98.2 ꢀ 18.6
4.3 ꢀ 1.5
1800 ꢀ 280
161 ꢀ 9
18.3
37.4
e
340.9 ꢀ 26.3
23.8 ꢀ 1.8
>2000
550 ꢀ 28
23.1
32
83.2 ꢀ 8.4
39.4 ꢀ 4.6
205 ꢀ 23
97.2 ꢀ 18
2.4
2.5
33
a
Affinities were determined in guinea pig brain using [3H]-(þ)-pentazocine.
b
c
Affinities were determined in guinea pig brain using [3H]-DTG in the presence of (þ)-SKF-10047 to block sigma-1 receptors.
The values are means ꢀ SEM of three experiments performed in duplicate.
the selectivity (Kis1 ¼ 1.84 nM and Kis2 ¼ 662 nM). This improved
affinity might result from the greater hydrophobicity of the piper-
idine moiety, which better fits Glennon’s pharmacophoric sigma-1
receptor model [38,39]. The affinity of the compound containing 4-
methylpiperidine in place of the morpholine moiety, 27
(Kis1 ¼ 1.1 nM and Kis2 ¼ 319 nM), was even greater, proving this
hypothesis. Substituting the oxygen atom of the morpholine to a
carbonyl group greatly decreased affinity for both the sigma-1 and
-2 receptors, which suggests that oxygen (compound 28) or a
hydrogen-bonding acceptor is necessary for sigma-1 or -2 receptor
binding in this position. The 1-methylpiperazine derivative, 29, was
potent and selective (Kis1 ¼4.3 nM and Kis2 ¼ 161 nM), similar to 4-
methylpiperidine. However, substitution of the methyl with a large
group such as phenyl (30, Kis1 ¼340.9 nM and Kis2 > 2000 nM) was
not tolerated by either the sigma-1 or -2 receptor. Smaller
carbonated cyclic analogs (31) or open chain amines (32, 33)
retained some activity, though not as much as compounds 26 and
27, and had lower selectivity.
After identification of piperidine and 4-methylpiperidine as the
amino substituents that conferred greatest potency and selectivity,
several analogs were prepared. Substitution at position 4 of the
phenyl linked to the N atom of compound 26 provided the de-
rivatives 34 and 35 (Table 3). The 4-methylphenyl (34,
K
is1 ¼ 1.69 nM and Kis2 ¼ 825 nM) and 4-fluorphenyl (35,
Kis1 ¼ 1.22 nM and Kis2 ¼ 1301 nM) derivatives maintained high
affinity to the sigma-1 receptor and demonstrated greater selec-
tivity for sigma-1 over the sigma-2 receptor. The nature of the
phenyl group was important for activity, as substitution with
hydrogen or a methyl group (10i and 38) obviously decreased the
affinity for the sigma-1 receptor.
Similar results were obtained with 4-methylpiperidine de-
rivatives, as shown in Table 4. Replacement of the phenyl group
with hydrogen or a methyl group, yielding compound 10j and 39,
led to substantially decreased potency. Interestingly, the two
compounds with the greatest sigma-1 affinity, 35 (Kis1 ¼ 1.22 nM)
and 37 (Kis1 ¼ 0.77 nM) and good sigma-1/2 selectivity (1066- and

Y. Lan et al. / European Journal of Medicinal Chemistry 79 (2014) 216e230
221
Table 3
of sigma-1 receptor antagonists. Compounds 35 and 37 were
showed to be sigma-1 receptor antagonists, as phenytoin shifted
their to slightly lower values (Ki(without phenytoin)/Ki(with
phenytoin) ¼ 0.86 and 0.91, respectively).
Sigma-1 and sigma-2 receptor affinities of compound 26 derivatives.
4.2. Formalin test
Due to their excellent affinity for the sigma-1 receptor, selec-
tivity for sigma-1 over the sigma-2 receptor, and antagonistic ac-
tivity, compounds 35 and 37 were selected as promising anti-
nociceptive agents and employed in an in vivo study. A classic
model of acute and chronic pain, the formalin test, was carried out
to evaluate these compounds’ anti-nociceptive activity. Intra-
plantar injection of formalin into the hind paw produces a biphasic
pain response: a brief, acute phase that indicates direct effects on
pain receptors (phase I) followed by a longer-lasting tonic phase
that reflects inflammation (phase II) [29,43]. The time spent licking
or biting the injected paw after the injection was measured as an
indicator of the pain response in mice.
Compound R1
K
is1 (nM)a
K
is2 (nM)b Selectivity
s2 s1
(
/ )
26
1.84 ꢀ 0.33c 662 ꢀ 42
359.8
488.2
34
1.69 ꢀ 0.35
825 ꢀ 94
As shown in Fig. 4, pretreatment with compound 35 (80 mg/kg)
inhibited pain responses to a similar degree as S1RA, reducing
licking and biting time to 12.08 ꢀ 5.25 in phase I and 36.13 ꢀ 6.77 in
phase II. The effect of S1RA at the same dose was to reduce licking
and biting time to 8.63 ꢀ 3.47 s and 27.34 ꢀ 7.02 s during phase I
and II, respectively. Compound 37 was less effective in both phases
at the same dose, the licking or biting time was 34.82 ꢀ 12.62 s in
phase I and 89.75 ꢀ 24.63 s in phase II. Vehicle had no effect relative
to treatment with formalin alone.
35
1.22 ꢀ 0.45 1301 ꢀ 204 1066
10i
38
eH
eCH3
89.1 ꢀ 14
34.3 ꢀ 2.7
288 ꢀ 52
3.2
10.4
357 ꢀ 131
a
Affinities were determined in guinea pig brain using [3H]-(þ)-pentazocine.
b
Affinities were determined in guinea pig brain using [3H]-DTG in the presence of
(þ)-SKF-10047 to block sigma-1 receptors.
c
The values are means ꢀ SEM of three experiments performed in duplicate.
To better characterize the anti-nociceptive effects of 35 and 37, a
wide range of doses was tested (40e120 mg/kg). Compound 35 was
effective in reducing pain responses compared to the naive groups
at all doses in both phase I (33.37%, 84.75% and 97.62% reduction in
biting and licking time after i.p. administration at 40, 80 and
160 mg/kg, respectively) and phase II (30.48%, 87.72% and 98.23%
inhibition) (Fig. 5), while 37 was less effective in both phases.
Compound 35 produced dose-dependent anti-nociception in both
phases; the ED50 values were 49.4 ꢀ 4.1 and 50.5 ꢀ 2.5 mg/kg for
phases I and II, respectively.
1246-fold, respectively), contained the same 4-fluorphenyl moiety.
Compounds 35 and 37 were thus selected for further evaluation.
The functional activities of compounds 35 and 37 were evalu-
ated using phenytoin [42]. Phenytoin shifts sigma-1 receptor ago-
nists to significantly higher affinities, but does not affect the affinity
Table 4
Sigma-1 and sigma-2 receptor affinities of compound 27 derivatives.
5. Conclusion
A
detailed structureeactivity relationship investigation of
structures B (Fig. 3) has shown that several factors influence the
binding affinity for the sigma-1 and sigma-2 receptors of these
compounds. (1) 7-hydroxy-3,4-dihydro-2(1H)-quinolinone scaf-
fold has greater potential than the 6-hydroxy-3,4-dihydro-2(1H)-
quinolinone in sigma-1 receptor binding affinity, (2) straight three-
carbon chain alkyl between the quinolinone scaffold and amino
moiety was preferred over other alkyl linker, (3) only one-atom
spacer was tolerated between the N atom of the quinolinone
scaffold and the phenyl group, (4) piperidine was the preferred
amino moiety (NRR) which had greatly improved sigma-1-receptor
binding affinity and maintained the selectivity, and (5) the substi-
tution on the 4-position (R1) with electron-withdrawing (fluoro) as
the favored substituent which enhanced the affinity to sigma-1
receptor and did lower the affinity to sigma-2 receptor.
Compound R1
K
is1 (nM)a
K
is2 (nM)b Selectivity
s2 s1
(
/ )
27
1.1 ꢀ 0.06c 319 ꢀ 14
1.49 ꢀ 0.22 755 ꢀ 44
290
36
506.7
In summary, an efficient method for the synthesis of novel 7(6)-
hydroxy-3,4-dihydroquinolin-2(1H)-one derivatives was described
herein. These compounds’ binding affinities to sigma-1 and sigma-
2 receptors were evaluated by means of radioligand binding assays,
which identified compounds 35 and 37 as having the greatest af-
finity for sigma-1 (Kis1 ¼1.22 nM and 0.77 nM) and good sigma-1/2
selectivity (1066- and 1246-fold, respectively). Their functional
activity was then determined using phenytoin, a low-potency
allosteric modulator of sigma-1 receptor, confirming that both
37
0.77 ꢀ 0.17 960 ꢀ 112 1246
10j
39
eH
eCH3
66.7 ꢀ 12
40.2 ꢀ 3.8
409 ꢀ 61
311 ꢀ 25
6.1
7.7
a
Affinities were determined in guinea pig brain using [3H]-(þ)-pentazocine.
b
Affinities were determined in guinea pig brain using [3H]-DTG in the presence of
(þ)-SKF-10047 to block sigma-1 receptors.
c
The values are means ꢀ SEM of three experiments performed in duplicate.

222
Y. Lan et al. / European Journal of Medicinal Chemistry 79 (2014) 216e230
6. Experimental
6.1. Chemistry experimental
All commercially available chemicals and reagents were used
without further purification. Reagents were all of analytical grade
or of chemical purity (>99%). Melting points were determined in
open capillary tubes and are uncorrected. 1H and 13C NMR spectra
were recorded on a Bruker Avance III 400 spectrometer at 400 MHz
(1H) and 100 MHz (13C) using CDCl3 as solvent. Chemical shifts were
given in
d values (ppm), using tetramethylsilane (TMS) as the in-
ternal standard; coupling constants (J) were given in Hz. Signal
multiplicities were characterized as s (singlet), d (doublet), t
(triplet), q (quartet), m (multiplet), br (broad signal). Analytical thin
layer chromatography (TLC) was performed on silica gel GF254.
Column chromatographic purification was carried out using silica
gel. Purity analysis was carried out by LC-Mass analysis using an
Agilent 1100 system. The HPLC conditions were as follows: column,
InertSustain C18 5.0
m
m ꢂ 150 mm ꢂ 4.6 mm I.D (SHIMADZU,
Japan); mobile phase, 30 mmol NH4COOH (ROE SCIENTIFIC INC,
USA) aq./acetonitrile (Merck Company, Germany) 40/60; flow rate,
0.2 mL/min; column temperature, 40 ꢁC. Compound purity is
determined by high performance liquid chromatography (HPLC),
and all final test compounds were >95% purity.
6.1.1. General procedures for the synthesis of compounds 8ae8d
1,3-dibromopropane
(1,4-dibromobutane;
1,5-
Fig. 3. Glennon’s pharmacophoric sigma-1 receptor model (A) and general structure of
the new 3,4-dihydro-2(1H)-quinolinone derivative (B).
dibromopentane; 1,6-dibromohexane; 20 mmol) was added to a
solution of compounds 6 (10 mmol) and potassium carbonate
(20 mmol) in acetone (100 mL), and the mixture was refluxed for
4e6 h. The progress of the reaction was monitored by TLC. After
cooling to room temperature, the mixture was filtered, the solvent
was evaporated under reduced pressure. The crude product was
purified by means of chromatography (petroleum ether/
EtOAc ¼ 10/1) to yield compounds 8ae8d.
were antagonists. In the formalin test, compound 35 showed sig-
nificant anti-nociceptive effects in phases I and II, identifying
compound 35 as a candidate agent for the treatment of pain.
Further studies of this compound are currently underway in our
laboratory.
6.1.1.1. 7-(3-Bromopropoxy)-3,4-dihydro-2(1H)-quinolinone
(8a).
Yield: 88%. White solid, m. p. 111e113 ꢁC. 1H NMR (400 MHz, CDCl3)
d
10.08 (s, 1H), 7.01 (d, J ¼ 8.2 Hz, 1H), 6.62e6.39 (m, 2H), 4.02 (t,
J ¼ 5.7 Hz, 2H), 3.56 (t, J ¼ 6.5 Hz, 2H), 2.86 (t, J ¼ 7.5 Hz, 2H), 2.61 (t,
J ¼ 7.5 Hz, 2H), 2.28e2.21 (m, 2H). 13C NMR (100 MHz, CDCl3)
Fig. 4. Anti-nociceptive effect of S1RA, compound 35 and 37 in phase I (0e5 min) and
phase II (15e45 min) of the formalin test at the dose of 80 mg/kg. Each column and
vertical line represents mean ꢀ SEM of the values obtained in at least ten animals.
Statistically significant differences: #p < 0.05; ##p < 0.01 vs. vehicle; *p < 0.05;
**p < 0.01 vs. vehicle þ formalin (two-way ANOVA followed by NewmaneKeuls test).
Fig. 5. Anti-nociceptive effects of compound 35 on formalin-induced pain behavior.
Each point and vertical line represents mean ꢀ SEM of the values obtained in at least
ten animals. Statistically significant differences between the vehicle- and drug-injected
groups: *p < 0.05, **p < 0.01 (two-way ANOVA followed by NewmaneKeuls test).

Y. Lan et al. / European Journal of Medicinal Chemistry 79 (2014) 216e230
223
d
173.01, 158.28, 138.45, 128.57, 115.98, 108.62, 102.52, 65.38, 32.33,
116.51, 114.33, 113.04, 67.93, 33.75, 32.48, 30.60, 28.47, 25.64, 24.83.
31.01, 30.17, 24.53. MS (ESI) m/z 284.3 ([M þ H]þ).
MS (ESI) m/z 312.2 ([M þ H]þ).
6.1.1.2. 7-(4-Bromobutoxy)-3,4-dihydro-2(1H)-quinolinone
(8b).
6.1.2.4. 6-(6-Bromohexyloxy)-3,4-dihydro-2(1H)-quinolinone (9d).
Yield: 88%. White solid, m. p.106e108 ꢁC. 1H NMR (400 MHz, CDCl3)
Yield: 91%. White solid, m. p. 110e112 ꢁC. 1H NMR (400 MHz, CDCl3)
d
10.05 (s, 1H), 7.01 (d, J ¼ 8.2 Hz, 1H), 6.49 (dd, J ¼ 8.2, 2.3 Hz, 1H),
d
9.83 (s, 1H), 6.72 (d, J ¼ 8.2 Hz, 1H), 6.64e6.57 (m, 2H), 3.82 (t,
6.45 (d, J ¼ 2.2 Hz,1H), 3.91 (t, J ¼ 6.0 Hz, 2H), 3.45 (t, J ¼ 6.6 Hz, 2H),
2.87 (t, J ¼ 7.5 Hz, 2H), 2.61 (t, J ¼ 7.5 Hz, 2H), 2.05e1.98 (m, 2H),
J ¼ 6.4 Hz, 2H), 3.33 (t, J ¼ 6.8 Hz, 2H), 2.83 (t, J ¼ 7.5 Hz, 2H), 2.59e
2.46 (m, 2H), 1.87e1.74 (m, 2H), 1.73e1.60 (m, 2H), 1.48e1.33 (m,
1.91e1.84 (m, 2H). 13C NMR (100 MHz, CDCl3)
d 173.00, 158.47,
4H). 13C NMR (100 MHz, CDCl3)
d 172.26, 154.98, 130.88, 124.89,
138.41, 128.55, 115.75, 108.71, 102.35, 66.94, 33.65, 31.04, 29.45,
116.49, 114.34, 113.07, 68.10, 33.91, 32.68, 30.62, 29.14, 27.93, 25.66,
27.89, 24.53. MS (ESI) m/z 298.1 ([M þ H]þ).
25.30. MS (ESI) m/z 326.3 ([M þ H]þ).
6.1.1.3. 7-(5-Bromopentyloxy)-3,4-dihydro-2(1H)-quinolinone (8c).
Yield: 89%. White solid, m. p. 123e125 ꢁC. 1H NMR (400 MHz,
6.1.3. General procedures for the synthesis of compounds 10aed
To a suspension of 8a (8b-8d; 2 mmol) and morpholine
(2.2 mmol), K2CO3 (4 mmol) in acetonitrile (50 mL), and a cata-
lytic amount of KI (1% mol) were added and the resulting mixture
was refluxed for 6e8 h. After filtering, the resulting filtrate was
evaporated to dryness under reduced pressure. The residue was
suspended in water (10.0 mL) and extracted with dichloro-
methane (3 ꢂ 25 mL). The combined organic layers were dried
with anhydrous magnesium sulfate, the filtrate evaporated under
reduced pressure, and the crude product was purified by means
of chromatography (10% MeOH/CHCl3) to yield compounds 10ae
10d.
CDCl3)
d
10.05 (s, 1H), 7.01 (d, J ¼ 8.2 Hz, 1H), 6.49 (dd, J ¼ 8.2,
2.3 Hz, 1H), 6.45 (d, J ¼ 2.2 Hz, 1H), 3.91 (t, J ¼ 6.0 Hz, 2H), 3.45 (t,
J ¼ 6.6 Hz, 2H), 2.87 (t, J ¼ 7.5 Hz, 2H), 2.61 (t, J ¼ 7.5 Hz, 2H), 2.05e
1.98 (m, 2H), 1.91e1.84 (m, 2H). 13C NMR (100 MHz, CDCl3)
d 172.70,
158.61, 138.31, 128.56, 115.69, 108.75, 102.34, 67.72, 33.69, 32.49,
31.08, 28.42, 24.83, 24.56. MS (ESI) m/z 312.4 ([M þ H]þ).
6.1.1.4. 7-(6-Bromohexyloxy)-3,4-dihydro-2(1H)-quinolinone (8d).
Yield: 86%. White solid, m. p.106e107 ꢁC. 1H NMR (400 MHz, CDCl3)
d
9.70 (s, 1H), 7.02 (d, J ¼ 8.3 Hz, 1H), 6.50 (dd, J ¼ 8.2, 2.4 Hz, 1H),
6.44 (d, J ¼ 2.3 Hz,1H), 3.91 (t, J ¼ 6.3 Hz, 2H), 3.42 (t, J ¼ 6.8 Hz, 2H),
2.88 (t, J ¼ 7.5 Hz, 2H), 2.68e2.55 (m, 2H), 1.92e1.85 (m, 2H), 1.82e
6.1.3.1. 7-(3-Morpholinopropoxy)-3,4-dihydro-2(1H)-quinolinone
1.69 (m, 2H), 1.56e1.40 (m, 4H). 13C NMR (100 MHz, CDCl3)
d 172.81,
(10a). Yield: 93%. White solid, m. p.124e126 ꢁC. 1H NMR (400 MHz,
158.69, 138.32, 128.53, 115.60, 108.76, 102.37, 67.86, 33.92, 32.69,
CDCl3)
d
9.79 (s, 1H), 7.02 (d, J ¼ 8.3 Hz, 1H), 6.51 (dd, J ¼ 8.3, 2.4 Hz,
31.09, 29.08, 27.94, 25.30, 24.56. MS (ESI) m/z 326.6 ([M þ H]þ).
1H), 6.44 (d, J ¼ 2.3 Hz, 1H), 3.98 (t, J ¼ 6.2 Hz, 2H), 3.78e3.66 (m,
4H), 2.88 (t, J ¼ 7.5 Hz, 2H), 2.67e2.58 (m, 2H), 2.53e2.47 (m, 6H),
6.1.2. General procedures for the synthesis of compounds 9ae9d
2.02e1.86 (m, 2H). 13C NMR (100 MHz, CDCl3)
d 172.71, 158.57,
1,3-dibromopropane
(1,4-dibromobutane;
1,5-
138.36, 128.48, 115.66, 108.62, 102.38, 66.94, 66.18, 55.51, 53.72,
dibromopentane; 1,6-dibromohexane; 20 mmol) was added to a
solution of compounds 7 (10 mmol) and potassium carbonate
(20 mmol) in acetone (100 mL), and the mixture was refluxed for
4e6 h. The progress of the reaction was monitored by TLC. After
cooling to room temperature, the mixture was filtered, the solvent
was evaporated under reduced pressure. The crude product was
purified by means of chromatography (petroleum ether/
EtOAc ¼ 10/1) to yield compounds 9ae9d.
31.05, 26.37, 24.53. MS (ESI) m/z 291.2 ([M þ H]þ).
6.1.3.2. 7-(4-Morpholinobutoxy)-3,4-dihydro-2(1H)-quinolinone
(10b). Yield: 90%. White solid, m. p.133e134 ꢁC. 1H NMR (400 MHz,
CDCl3)
d
9.79 (s, 1H), 7.02 (d, J ¼ 8.3 Hz, 1H), 6.50 (dd, J ¼ 8.2, 2.3 Hz,
1H), 6.44 (d, J ¼ 2.2 Hz, 1H), 3.93 (t, J ¼ 6.2 Hz, 2H), 3.78e3.67 (m,
4H), 2.88 (t, J ¼ 7.5 Hz, 2H), 2.61 (t, J ¼ 7.5 Hz, 2H), 2.45e2.37 (m,
6H), 1.82e1.75 (m, 2H), 1.69e1.62 (m, 2H). 13C NMR (100 MHz,
CDCl3) d 172.70, 158.61, 138.36, 128.47, 115.57, 108.66, 102.30, 67.74,
6.1.2.1. 6-(3-Bromopropoxy)-3,4-dihydro-2(1H)-quinolinone
(9a).
66.95, 58.59, 53.68, 31.04, 27.16, 24.52, 23.04. MS (ESI) m/z 305.6
Yield: 90%. White solid, m. p. 110e113 ꢁC. 1H NMR (400 MHz, CDCl3)
([M þ H]þ).
d
10.06 (s, 1H), 6.82 (d, J ¼ 8.2 Hz, 1H), 6.70 (dd, J ¼ 11.1, 2.6 Hz, 2H),
4.04 (t, J ¼ 5.8 Hz, 2H), 3.58 (t, J ¼ 6.4 Hz, 2H), 2.91 (t, J ¼ 7.5 Hz, 2H),
2.68e2.54 (m, 2H), 2.30e2.24 (m, 2H). 13C NMR (100 MHz, CDCl3)
6.1.3.3. 7-(5-Morpholinopentyloxy)-3,4-dihydro-2(1H)-quinolinone
(10c). Yield: 89%. White solid, m. p. 125e126 ꢁC. 1H NMR (400 MHz,
d
172.34, 154.59, 131.22, 124.95, 116.56, 114.39, 113.19, 65.69, 32.36,
30.57, 30.17, 25.63. MS (ESI) m/z 284.2 ([M þ H]þ).
CDCl3)
d
9.77 (s, 1H), 7.01 (d, J ¼ 8.3 Hz, 1H), 6.50 (dd, J ¼ 8.2, 2.4 Hz,
1H), 6.43 (d, J ¼ 2.3 Hz, 1H), 3.91 (t, J ¼ 6.4 Hz, 2H), 3.80e3.66 (m,
4H), 2.88 (t, J ¼ 7.5 Hz, 2H), 2.68e2.56 (m, 2H), 2.44e2.31 (m, 6H),
1.83e1.71 (m, 2H), 1.61e1.51 (m, 2H), 1.51e1.42 (m, 2H). 13C NMR
6.1.2.2. 6-(4-Bromobutoxy)-3,4-dihydro-2(1H)-quinolinone
(9b).
Yield: 91%. White solid, m. p.138e140 ꢁC. 1H NMR (400 MHz, CDCl3)
(100 MHz, CDCl3)
d 172.68, 158.66, 138.36, 128.46, 115.54, 108.63,
d
9.40 (s, 1H), 6.78 (d, J ¼ 8.2 Hz, 1H), 6.73e6.67 (m, 2H), 3.95 (t,
102.30, 67.85, 66.96, 58.95, 53.76, 31.05, 29.13, 26.27, 24.53, 23.97.
J ¼ 6.0 Hz, 2H), 3.49 (t, J ¼ 6.6 Hz, 2H), 2.93 (t, J ¼ 7.5 Hz, 2H), 2.68e
MS (ESI) m/z 319.6 ([M þ H]þ).
2.56 (m, 2H), 2.10e2.03 (m, 2H), 1.96e1.89 (m, 2H). 13C NMR
(100 MHz, CDCl3)
d 170.95, 153.78, 129.91, 123.94, 115.35, 113.36,
112.02, 66.20, 32.48, 29.58, 28.42, 26.88, 24.63. MS (ESI) m/z 298.0
6.1.3.4. 7-(6-Morpholinohexyloxy)-3,4-dihydro-2(1H)-quinolinone
([M þ H]þ).
(10d). Yield: 92%. White solid, m. p. 118e120 ꢁC. 1H NMR (400 MHz,
CDCl3)
d
9.74 (s, 1H), 7.02 (d, J ¼ 8.3 Hz, 1H), 6.50 (dd, J ¼ 8.2, 2.4 Hz,
6.1.2.3. 6-(5-Bromopentyloxy)-3,4-dihydro-2(1H)-quinolinone (9c).
Yield: 87%. White solid, m. p. 111e113 ꢁC. 1H NMR (400 MHz, CDCl3)
1H), 6.43 (d, J ¼ 2.3 Hz, 1H), 3.90 (t, J ¼ 6.4 Hz, 2H), 3.76e3.68 (m,
4H), 2.88 (t, J ¼ 7.5 Hz, 2H), 2.61 (t, J ¼ 7.5 Hz, 2H), 2.44e2.32 (m,
6H), 1.80e1.70 (m, 2H), 1.55e1.33 (m, 6H). 13C NMR (100 MHz,
d
10.08 (s,1H), 6.82 (d, J ¼ 8.2 Hz,1H), 6.68 (d, J ¼ 9.0 Hz, 2H), 3.90 (t,
J ¼ 6.3 Hz, 2H), 3.42 (t, J ¼ 6.7 Hz, 2H), 2.91 (t, J ¼ 7.5 Hz, 2H), 2.60 (t,
J ¼ 7.5 Hz, 2H), 1.99e1.85 (m, 2H), 1.83e1.70 (m, 2H), 1.66e1.51 (m,
CDCl3) d 172.66, 158.69, 138.35, 128.46, 115.51, 108.64, 102.31, 67.93,
66.94, 59.04, 53.76, 31.06, 29.17, 27.24, 26.44, 25.98, 24.53. MS (ESI)
2H). 13C NMR (100 MHz, CDCl3)
d
172.32, 154.88, 130.96, 124.88,
m/z 333.4 ([M þ H]þ).

224
Y. Lan et al. / European Journal of Medicinal Chemistry 79 (2014) 216e230
6.1.4. General procedures for the synthesis of compounds 10fem
To a suspension of 8a (2 mmol) and dimethylamine (diethyl-
amine; pyrrolidine piperidine; 4-methylpiperidine; piperidin-4-
one; 1-methyl-piperazine; 1-phenyl-piperazine; 2.2 mmol),
K2CO3 (4 mmol) in acetonitrile (50 mL), and a catalytic amount of KI
(1% mol) were added and the resulting mixture was refluxed for 6e
8 h. After filtering, the resulting filtrate was evaporated to dryness
under reduced pressure. The residue was suspended in water
(10.0 mL) and extracted with dichloromethane (3 ꢂ 25 mL). The
combined organic layers were dried with anhydrous magnesium
sulfate, the filtrate evaporated under reduced pressure, and the
crude product was purified by means of chromatography (10%
MeOH/CHCl3) to yield compounds 10fe10m.
(m, 2H), 4.03 (t, J ¼ 6.1 Hz, 2H), 2.89 (t, J ¼ 7.5 Hz, 2H), 2.84e2.74 (m,
4H), 2.70e2.58 (m, 4H), 2.54e2.42 (m, 4H), 2.10e1.90 (m, 2H). 13
NMR (100 MHz, CDCl3) 209.15, 172.61, 158.53, 138.35, 128.52,
C
d
115.73, 108.67, 102.40, 66.06, 53.93, 53.09, 41.11, 31.05, 27.19, 24.52.
MS (ESI) m/z 303.4 ([M þ H]þ).
6.1.4.7. 7-(3-(4-Methylpiperazin-1-yl)propoxy)-3,4-dihydro-2(1H)-
quinolinone (10l). Yield: 82%. Pale yellow solid, m. p. 141e142 ꢁC. 1H
NMR (400 MHz, CDCl3)
d
9.96 (s, 1H), 7.01 (d, J ¼ 8.3 Hz, 1H), 6.50
(dd, J ¼ 8.2, 2.3 Hz, 1H), 6.45 (d, J ¼ 2.2 Hz, 1H), 3.96 (t, J ¼ 6.2 Hz,
2H), 2.87 (t, J ¼ 7.5 Hz, 2H), 2.82e2.58 (m, 4H), 2.57e2.35 (m, 8H),
2.30 (s, 3H), 1.99e1.88 (m, 2H). 13C NMR (100 MHz, CDCl3)
d 172.54,
158.48, 138.40, 128.39, 115.57, 108.54, 102.29, 66.22, 54.96, 54.82,
52.88, 45.77, 31.01, 26.57, 24.49, 22.76. MS (ESI) m/z 304.6
([M þ H]þ).
6.1.4.1. 7-(3-(Dimethylamino)propoxy)-3,4-dihydro-2(1H)-quinoli-
none (10f). Yield: 82%. Pale yellow solid, m. p. 86e88 ꢁC. 1H NMR
(400 MHz, CDCl3)
d
9.46 (s, 1H), 7.01 (d, J ¼ 8.9 Hz, 1H), 6.56e6.45
6.1.4.8. 7-(3-(4-Phenylpiperazin-1-yl)propoxy)-3,4-dihydro-2(1H)-
(m, 2H), 3.99 (t, J ¼ 6.0 Hz, 2H), 2.86 (t, J ¼ 7.5 Hz, 2H), 2.79e2.72 (m,
quinolinone (10m). Yield: 85%. Pale yellow solid, m. p. 121e123 ꢁC.
2H), 2.58 (t, J ¼ 7.5 Hz, 2H), 2.46 (s, 6H), 2.11e2.02 (m, 2H). 13C NMR
1H NMR (400 MHz, CDCl3)
d 9.87 (s, 1H), 7.22e7.10 (m, 3H), 6.86e
(100 MHz, CDCl3)
d 171.31, 157.25, 137.39, 127.50, 114.86, 107.57,
6.68 (m, 5H), 3.88 (t, J ¼ 6.2 Hz, 2H), 3.12e3.04 (m, 8H), 2.92 (t,
J ¼ 7.5 Hz, 2H), 2.74e2.52 (m, 4H), 1.95e1.85 (m, 2H). 13C NMR
101.39, 64.79, 54.67, 43.10, 30.02, 25.03, 23.51. MS (ESI) m/z 249.5
([M þ H]þ).
(100 MHz, CDCl3)
d 172.18, 158.62, 151.70, 138.53, 129.17, 129.14,
119.92, 116.23, 116.06, 108.68, 102.30, 66.37, 55.17, 50.06, 45.83,
31.16, 26.77, 24.62. MS (ESI) m/z 366.2 ([M þ H]þ).
6.1.4.2. 7-(3-(Diethylamino)propoxy)-3,4-dihydro-2(1H)-quinoli-
none (10g). Yield: 84%. Pale yellow solid, m. p. 94e96 ꢁC. 1H NMR
(400 MHz, CDCl3)
d
9.68 (d, J ¼ 3.6 Hz, 1H), 7.01 (d, J ¼ 8.2 Hz, 1H),
6.1.5. General procedures for the synthesis of compounds 11aed
To a suspension of 9a (9b-9d; 2 mmol) and morpholine
(2.2 mmol), K2CO3 (4 mmol) in acetonitrile (50 mL), and a catalytic
amount of KI (1% mol) were added and the resulting mixture was
refluxed for 6e8 h. After filtering, the resulting filtrate was evap-
orated to dryness under reduced pressure. The residue was sus-
pended in water (10.0 mL) and extracted with dichloromethane
(3 ꢂ 25 mL). The combined organic layers were dried with anhy-
drous magnesium sulfate, the filtrate evaporated under reduced
pressure, and the crude product was purified by means of chro-
matography (10% MeOH/CHCl3) to yield compounds 11ae11d.
6.51 (d, J ¼ 8.3 Hz, 1H), 6.45 (d, J ¼ 2.2 Hz, 1H), 3.96 (t, J ¼ 6.0 Hz,
2H), 2.88 (t, J ¼ 7.4 Hz, 2H), 2.80e2.72 (m, 2H), 2.64e2.54 (m, 6H),
2.05e1.82 (m, 2H), 1.04 (t, J ¼ 7.1 Hz, 6H). 13C NMR (100 MHz, CDCl3)
d
171.64, 157.62, 137.30, 127.44, 114.56, 107.68, 101.34, 65.42, 48.25,
45.81, 30.04, 25.72, 23.52, 10.51. MS (ESI) m/z 277.3 ([M þ H]þ).
6.1.4.3. 7-(3-(Pyrrolidin-1-yl)propoxy)-3,4-dihydro-2(1H)-quinoli-
none (10h). Yield: 80%. Pale yellow solid, m. p. 110e111 ꢁC. 1H NMR
(400 MHz, CDCl3)
d
9.79 (s, 1H), 7.00 (d, J ¼ 8.3 Hz, 1H), 6.50 (dd,
J ¼ 8.2, 2.4 Hz, 1H), 6.44 (d, J ¼ 2.3 Hz, 1H), 3.98 (t, J ¼ 6.3 Hz, 2H),
2.86 (t, J ¼ 7.5 Hz, 2H), 2.65e2.61 (m, 2H), 2.59e2.51 (m, 6H), 2.04e
1.92 (m, 2H), 1.85e1.72 (m, 4H). 13C NMR (100 MHz, CDCl3)
d 172.56,
6.1.5.1. 6-(3-Morpholinopropoxy)-3,4-dihydro-2(1H)-quinolinone
158.57, 138.37, 128.38, 115.56, 108.55, 102.41, 66.41, 54.22, 53.09,
(11a). Yield: 92%. White solid, m. p.126e128 ꢁC. 1H NMR (400 MHz,
31.08, 28.74, 24.54, 23.43. MS (ESI) m/z 275.8 ([M þ H]þ).
CDCl3)
d
9.81 (s, 1H), 6.67 (d, J ¼ 8.1 Hz, 1H), 6.64e6.58 (m, 2H), 3.92
(t, J ¼ 6.3 Hz, 2H), 3.75e3.63 (m, 4H), 2.86 (t, J ¼ 7.5 Hz, 2H), 2.58e
6.1.4.4. 7-(3-(Piperidin-1-yl)propoxy)-3,4-dihydro-2(1H)-quinoli-
2.50 (m, 2H), 2.47e2.36 (m, 6H), 1.98e1.88 (m, 2H). 13C NMR
none (10i). Yield: 90%. White solid, m. p. 132e134 ꢁC. 1H NMR
(100 MHz, CDCl3)
d 169.88, 153.93, 129.63, 124.08, 114.90, 113.53,
(400 MHz, CDCl3)
d
9.99 (s, 1H), 6.88 (d, J ¼ 8.3 Hz, 1H), 6.38 (dd,
112.09, 65.45, 57.47, 54.54, 52.68, 29.62, 26.12, 24.72. MS (ESI) m/z
J ¼ 8.2, 2.3 Hz, 1H), 6.34 (d, J ¼ 2.2 Hz, 1H), 3.83 (t, J ¼ 6.3 Hz, 2H),
2.75 (t, J ¼ 7.5 Hz, 2H), 2.49 (t, J ¼ 7.5 Hz, 2H), 2.38e2.20 (m, 6H),
1.88e1.76 (m, 2H), 1.53e1.43 (m, 4H), 1.37e1.28 (m, 2H). 13C NMR
277.4 ([M þ H]þ).
6.1.5.2. 6-(4-Morpholinobutoxy)-3,4-dihydro-2(1H)-quinolinone
(100 MHz, CDCl3)
d 172.71, 158.58, 138.39, 128.36, 115.48, 108.60,
(11b). Yield: 87%. White solid, m. p. 129e131 ꢁC. 1H NMR (400 MHz,
102.34, 66.49, 55.91, 54.58, 31.03, 26.72, 25.93, 24.50, 24.42. MS
(ESI) m/z 289.2 ([M þ H]þ).
CDCl3)
d
9.81 (s, 1H), 6.79 (d, J ¼ 8.2 Hz, 1H), 6.74e6.65 (m, 2H), 3.93
(t, J ¼ 6.3 Hz, 2H), 3.77e3.65 (m, 4H), 2.92 (t, J ¼ 7.5 Hz, 2H), 2.67e
2.55 (m, 2H), 2.41 (dd, J ¼ 18.3, 10.9 Hz, 6H), 1.85e1.74 (m, 2H), 1.66
6.1.4.5. 7-(3-(4-Methylpiperidin-1-yl)propoxy)-3,4-dihydro-2(1H)-
(dt, J ¼ 9.4, 7.3 Hz, 2H). 13C NMR (100 MHz, CDCl3)
d 172.09, 154.91,
quinolinone (10j). Yield: 91%. White solid, m. p. 129e132 ꢁC. 1H
130.89, 124.87, 116.39, 114.31, 113.03, 68.00, 66.96, 58.59, 53.69,
NMR (400 MHz, CDCl3)
d
9.71 (s, 1H), 6.92 (d, J ¼ 8.3 Hz, 1H), 6.41
30.60, 27.21, 25.65, 23.07. MS (ESI) m/z 305.5 ([M þ H]þ).
(dd, J ¼ 8.3, 2.4 Hz, 1H), 6.34 (d, J ¼ 2.3 Hz, 1H), 3.86 (t, J ¼ 6.3 Hz,
2H), 2.88e2.74 (m, 4H), 2.52 (t, J ¼ 7.5 Hz, 2H), 2.43e2.34 (m, 2H),
1.93e1.73 (m, 4H), 1.53 (d, J ¼ 12.7 Hz, 2H), 1.26 (m, 1H), 1.15 (m,
6.1.5.3. 6-(5-Morpholinopentyloxy)-3,4-dihydro-2(1H)-quinolinone
2H), 0.83 (d, J ¼ 6.3 Hz, 3H). 13C NMR (100 MHz, CDCl3)
d
172.69,
(11c). Yield: 90%. White solid, m. p. 118e119 ꢁC. 1H NMR (400 MHz,
158.64, 138.34, 128.43, 115.55, 108.67, 102.38, 66.60, 55.62, 54.09,
34.34, 31.07, 30.81, 26.94, 24.54, 21.95. MS (ESI) m/z 303.1
([M þ H]þ).
CDCl3)
d
9.91 (s, 1H), 6.79 (d, J ¼ 8.1 Hz, 1H), 6.74e6.64 (m, 2H), 3.90
(t, J ¼ 6.4 Hz, 2H), 3.72 (t, J ¼ 4.6 Hz, 4H), 2.91 (t, J ¼ 7.5 Hz, 2H),
2.66e2.56 (m, 2H), 2.44 (s, 4H), 2.39e2.33 (m, 2H), 1.84e1.72 (m,
2H), 1.61e1.52 (m, 2H), 1.52e1.43 (m, 2H). 13C NMR (100 MHz,
6.1.4.6. 7-(3-(4-Oxopiperidin-1-yl)propoxy)-3,4-dihydro-2(1H)-qui-
CDCl3) d 172.10, 154.93, 130.87, 124.84, 116.38, 114.28, 113.00, 68.08,
nolinone (10k). Yield: 86%. Yellow solid, m. p. 126e129 ꢁC. 1H NMR
66.94, 58.93, 53.75, 30.59, 29.17, 26.27, 25.63, 23.96. MS (ESI) m/z
(400 MHz, CDCl3)
d
9.65 (s, 1H), 7.03 (d, J ¼ 8.3 Hz, 1H), 6.59e6.40
319.5 ([M þ H]þ).

Y. Lan et al. / European Journal of Medicinal Chemistry 79 (2014) 216e230
225
6.1.5.4. 6-(6-Morpholinohexyloxy)-3,4-dihydro-2(1H)-quinolinone
purified by means of chromatography (10% MeOH/CHCl3) to yield
compounds 12e33.
(11d). Yield: 88%. White solid, m. p. 115e117 ꢁC. 1H NMR (400 MHz,
CDCl3)
d
9.73 (s, 1H), 6.79 (d, J ¼ 8.2 Hz, 1H), 6.74e6.65 (m, 2H), 3.90
(t, J ¼ 6.4 Hz, 2H), 3.76e3.68 (m, 4H), 2.92 (t, J ¼ 7.5 Hz, 2H), 2.65e
2.57 (m, 2H), 2.44 (s, 4H), 2.38e2.29 (m, 2H), 1.82e1.71 (m, 2H),
1.55e1.43 (m, 4H), 1.42e1.32 (m, 2H). 13C NMR (100 MHz, CDCl3)
6.1.8.1. 7-(2-Morpholinoethoxy)-1-benzyl-3,4-dihydro-2(1H)-quino-
linone (12). Yield: 92%. Pale yellow oil. 1H NMR (400 MHz, CDCl3)
d
7.35e7.29 (m, 2H), 7.23 (t, J ¼ 7.1 Hz, 3H), 7.07 (d, J ¼ 8.2 Hz, 1H),
d
171.03, 153.98, 129.82, 123.85, 115.35, 113.32, 112.02, 67.19, 65.94,
6.52 (dd, J ¼ 8.2, 2.3 Hz,1H), 6.48 (d, J ¼ 2.2 Hz,1H), 5.16 (s, 2H), 3.97
(t, J ¼ 5.7 Hz, 2H), 3.73e3.70 (m, 4H), 2.95e2.90 (m, 2H), 2.82e2.68
58.04, 52.76, 29.60, 28.22, 26.24, 25.45, 24.98, 24.64. MS (ESI) m/z
333.1 ([M þ H]þ).
(m, 4H), 2.54e2.48 (m, 4H). 13C NMR (100 MHz, CDCl3)
d 170.66,
158.14, 140.84, 136.95, 128.72, 128.33, 127.07, 126.37, 118.75, 107.95,
103.71, 66.84, 65.81, 57.44, 53.99, 46.18, 32.20, 24.78. Exact mass
(ESI) m/z 367.2019 (calcd. 367.2014 for C22H26N2O3 [M þ H]þ).
6.1.6. General procedures for the synthesis of compounds 10e
To a suspension of 6 (10 mmol) and 4-(2-chloroethyl) mor-
pholine (10 mmol) in acetonitrile (100 mL), Cs2CO3 (20 mmol) was
added. The resulting mixture was refluxed for 6e8 h. The progress
of the reaction was monitored by TLC. After filtering, the resulting
filtrate was evaporated to dryness under reduced pressure. The
residue was suspended in water (50.0 mL) and extracted with
dichloromethane (3 ꢂ 50 mL). The combined organic layers were
dried with anhydrous magnesium sulfate, the filtrate evaporated
under reduced pressure, and the crude product was purified by
means of chromatography (10% MeOH/CHCl3) to yield compound
10e.
6.1.8.2. 7-(3-Morpholinopropoxy)-1-benzyl-3,4-dihydro-2(1H)-qui-
nolinone (13). Yield: 90%. Pale yellow oil. 1H NMR (400 MHz, CDCl3)
d
7.35e7.30 (m, 2H), 7.25 (t, J ¼ 7.1 Hz, 3H), 7.06 (d, J ¼ 8.2 Hz, 1H),
6.52 (dd, J ¼ 8.2, 2.3 Hz,1H), 6.47 (d, J ¼ 2.2 Hz,1H), 5.17 (s, 2H), 3.88
(t, J ¼ 6.3 Hz, 2H), 3.74e3.70 (m, 4H), 2.95e2.90 (m, 2H), 2.82e2.75
(m, 2H), 2.49e2.42 (m, 6H), 1.96e1.84 (m, 2H). 13C NMR (100 MHz,
CDCl3)
d 170.69, 158.40, 140.84, 137.02, 128.73, 128.29, 127.06,
126.40, 118.49, 107.79, 103.56, 66.91, 66.21, 55.43, 53.65, 46.20,
32.24, 26.23, 24.78. Exact mass (ESI) m/z 381.2179 (calcd. 381.2172
for C23H28N2O3 [M þ H]þ).
6.1.6.1. 7-(2-Morpholinoethoxy)-3,4-dihydro-2(1H)-quinolinone
6.1.8.3. 7-(4-Morpholinobutoxy)-1-benzyl-3,4-dihydro-2(1H)-quino-
(10e). Yield: 82%. Colorless solid, m. p. 145e147 ꢁC. 1H NMR
linone (14). Yield: 91%. Pale yellow oil. 1H NMR (400 MHz, CDCl3)
(400 MHz, CDCl3)
d
9.90 (s, 1H), 7.02 (d, J ¼ 8.3 Hz, 1H), 6.51 (dd,
d
7.35e7.29 (m, 2H), 7.24 (t, J ¼ 7.0 Hz, 3H), 7.06 (d, J ¼ 8.2 Hz, 1H),
J ¼ 8.2, 2.4 Hz, 1H), 6.45 (d, J ¼ 2.4 Hz, 1H), 4.06 (t, J ¼ 5.6 Hz, 2H),
3.77e3.67 (m, 4H), 2.87 (t, J ¼ 7.5 Hz, 2H), 2.77 (t, J ¼ 5.6 Hz, 2H),
6.50 (dd, J ¼ 8.2, 2.2 Hz,1H), 6.46 (d, J ¼ 2.2 Hz,1H), 5.16 (s, 2H), 3.83
(t, J ¼ 6.3 Hz, 2H), 3.74e3.68 (m, 4H), 2.95e2.87 (m, 2H), 2.83e2.74
(m, 2H), 2.55e2.34 (m, 6H), 1.78e1.66 (m, 2H), 1.64e1.53 (m, 2H).
2.65e2.52 (m, 6H). 13C NMR (100 MHz, CDCl3)
d 172.73, 158.25,
138.40, 128.50, 115.88, 108.73, 102.43, 66.84, 65.81, 57.57, 54.03,
13C NMR (100 MHz, CDCl3)
d 170.74, 158.42, 140.80, 137.01, 128.73,
30.98, 24.50. MS (ESI) m/z 277.7 ([M þ H]þ).
128.29, 127.05, 126.38, 118.41, 107.79, 103.50, 67.75, 66.93, 58.56,
53.67, 46.22, 32.23, 27.04, 24.75, 23.00. Exact mass (ESI) m/z
395.2335 (calcd. 395.2328 for C24H30N2O3 [M þ H]þ).
6.1.7. Procedures for the synthesis of compounds 11e
To a suspension of 7 (10 mmol) and 4-(2-chloroethyl) mor-
pholine (10 mmol) in acetonitrile (100 mL), Cs2CO3 (20 mmol) was
added. The resulting mixture was refluxed for 6e8 h. The progress
of the reaction was monitored by TLC. After filtering, the resulting
filtrate was evaporated to dryness under reduced pressure. The
residue was suspended in water (50.0 mL) and extracted with
dichloromethane (3 ꢂ 50 mL). The combined organic layers were
dried with anhydrous magnesium sulfate, the filtrate evaporated
under reduced pressure, and the crude product was purified by
means of chromatography (10% MeOH/CHCl3) to yield compound
11e.
6.1.8.4. 7-(5-Morpholinopentyloxy)-1-benzyl-3,4-dihydro-2(1H)-
quinolinone (15). Yield: 89%. Pale yellow oil. 1H NMR (400 MHz,
CDCl3)
d
7.35e7.29 (m, 2H), 7.24 (t, J ¼ 6.6 Hz, 3H), 7.06 (d, J ¼ 8.2 Hz,
1H), 6.50 (dd, J ¼ 8.2, 2.3 Hz,1H), 6.47 (d, J ¼ 2.2 Hz,1H), 5.17 (s, 2H),
3.81 (t, J ¼ 6.5 Hz, 2H), 3.76e3.71 (m, 4H), 2.95e2.89 (m, 2H), 2.82e
2.74 (m, 2H), 2.52e2.31 (m, 6H), 1.77e1.67 (m, 2H), 1.58e1.47 (m,
2H), 1.47e1.36 (m, 2H). 13C NMR (100 MHz, CDCl3)
d 170.70, 158.48,
140.84, 137.04, 128.72, 128.28, 127.04, 126.39, 118.37, 107.82, 103.47,
67.88, 66.98, 58.94, 53.77, 46.23, 32.26, 29.02, 26.25, 24.78, 23.92.
Exact mass (ESI) m/z 409.2469 (calcd. 409.2485 for C25H32N2O3
[M þ H]þ).
6.1.7.1. 6-(2-Morpholinoethoxy)-3,4-dihydro-2(1H)-quinolinone
(11e). Yield: 84%. Colorless solid, m. p. 136e139 ꢁC. 1H NMR
6.1.8.5. 7-(6-Morpholinohexyloxy)-1-benzyl-3,4-dihydro-2(1H)-qui-
nolinone (16). Yield: 93%. Pale yellow oil. 1H NMR (400 MHz, CDCl3)
(400 MHz, CDCl3)
d
9.51 (s, 1H), 6.78 (d, J ¼ 8.3 Hz, 1H), 6.75e6.68
(m, 2H), 4.07 (t, J ¼ 5.7 Hz, 2H), 3.78e3.70 (m, 4H), 2.92 (t, J ¼ 7.5 Hz,
d
7.32e7.26 (m, 2H), 7.23e7.19 (m, 3H), 7.02 (d, J ¼ 8.2 Hz,1H), 6.51e
2H), 2.79 (t, J ¼ 5.7 Hz, 2H), 2.66e2.51 (m, 6H). 13C NMR (100 MHz,
6.40 (m, 2H), 5.14 (s, 2H), 3.78 (t, J ¼ 6.5 Hz, 2H), 3.74e3.68 (m, 4H),
2.92e2.83 (m, 2H), 2.77e2.72 (m, 2H), 2.51e2.26 (m, 6H), 1.71e1.62
(m, 2H),1.54e1.44 (m, 2H),1.42e1.36 (m, 2H),1.33 (m, 2H). 13C NMR
CDCl3) d 170.94, 153.60, 130.07, 123.92, 115.32, 113.54, 112.15, 65.87,
65.11, 56.65 53.04, 29.56, 24.62. MS (ESI) m/z 277.3 ([M þ H]þ).
(100 MHz, CDCl3)
d 169.62, 157.44, 139.74, 136.01, 127.66, 127.25,
6.1.8. General procedures for the synthesis of compounds 12e38
Under N2 atmosphere compounds 10a (10be10m; 11ae11e;
2 mmol) was dissolved in dry DMF (50 mL). At 0 ꢁC NaH (60%,
5 mmol) was added and the reation mixture was stirred for 30 min
at 0 ꢁC. Then benzyl bromides ((2-bromoethyl)-benzene; 2.4 mmol)
were added dropwise to the reaction mixture and the mixture was
stirred at 50 ꢁC for 3 h. Water was added to the reaction mixture
and the mixture was extracted with EtOAc (3 ꢂ 25 mL). The organic
layer was washed with a saturated aq. NaCl solution. The organic
layers were dried with anhydrous magnesium sulfate, the filtrate
evaporated under reduced pressure, and the crude product was
125.99, 125.34, 117.25, 106.78, 102.39, 66.88, 65.81, 57.93, 52.64,
45.14, 31.19, 27.98, 26.14, 25.27, 24.86, 23.69. Exact mass (ESI) m/z
423.2644 (calcd. 423.2641 for C26H34N2O3 [M þ H]þ).
6.1.8.6. 7-(3-Morpholinopropoxy)-1-phenethyl-3,4-dihydro-2(1H)-
quinolinone (17). Yield: 84%. Pale yellow oil. 1H NMR (400 MHz,
CDCl3)
d
7.35e7.30 (m, 2H), 7.29e7.24 (m, 3H), 7.07 (d, J ¼ 8.2 Hz,
1H), 6.61 (d, J ¼ 2.2 Hz, 1H), 6.56 (dd, J ¼ 8.2, 2.2 Hz, 1H), 4.19e4.11
(m, 2H), 4.02 (t, J ¼ 6.3 Hz, 2H), 3.79e3.71 (m, 4H), 3.01e2.93 (m,
2H), 2.82e2.59 (m, 4H), 2.58e2.47 (6H), 2.12e1.89 (m, 2H). 13C NMR
(100 MHz, CDCl3)
d 170.30, 158.58, 140.43, 138.65, 128.84, 128.54,

226
Y. Lan et al. / European Journal of Medicinal Chemistry 79 (2014) 216e230
128.48, 126.51, 118.94, 107.35, 103.04, 66.98, 66.38, 55.57, 53.77,
43.61, 33.44, 32.26, 26.45, 24.74. Exact mass (ESI) m/z 395.2331
(calcd. 395.2328 for C24H30N2O3 [M þ H]þ).
6H), 1.71e1.62 (m, 2H), 1.55e1.42 (m, 4H), 1.38e1.33 (m, 2H). 13C
NMR (100 MHz, CDCl3) 169.10, 153.75, 136.06, 132.16, 127.66,
126.78, 125.97, 125.33, 115.43, 113.43, 111.39, 67.04, 65.81, 57.96,
52.64, 45.16, 30.83, 28.15, 26.19, 25.29, 24.94, 24.79. Exact mass
(ESI) m/z 423.2655 (calcd. 423.2641 for C26H34N2O3 [M þ H]þ).
d
6.1.8.7. 7-(4-Morpholinobutoxy)-1-phenethyl-3,4-dihydro-2(1H)-
quinolinone (18). Yield: 87%. Pale yellow oil. 1H NMR (400 MHz,
CDCl3)
d
7.36e7.30 (m, 2H), 7.29e7.24 (m, 3H), 7.07 (d, J ¼ 8.2 Hz,
6.1.8.13. 6-(3-Morpholinopropoxy)-1-phenethyl-3,4-dihydro-2(1H)-
1H), 6.61 (d, J ¼ 2.2 Hz, 1H), 6.55 (dd, J ¼ 8.2, 2.3 Hz, 1H), 4.19e4.12
(m, 2H), 3.98 (t, J ¼ 6.3 Hz, 2H), 3.77e3.72 (m, 4H), 3.01e2.93 (m,
2H), 2.83e2.58 (m, 4H), 2.53e2.40 (m, 6H), 1.90e1.80 (m, 2H),
quinolinone (24). Yield: 85%. Pale yellow oil. 1H NMR (400 MHz,
CDCl3)
d
7.35e7.29 (m, 2H), 7.27e7.21 (m, 3H), 6.96 (d, J ¼ 8.8 Hz,
1H), 6.80 (dd, J ¼ 8.8, 2.8 Hz, 1H), 6.76 (d, J ¼ 2.7 Hz, 1H), 4.19e4.12
(m, 2H), 4.03 (t, J ¼ 6.3 Hz, 2H), 3.77e3.72 (m, 4H), 2.97e2.91 (m,
2H), 2.83e2.77 (m, 2H), 2.65e2.58 (m, 2H), 2.56e2.46 (m, 6H),
1.77e1.66 (m, 2H). 13C NMR (100 MHz, CDCl3)
d 170.26, 158.61,
140.45, 138.66, 128.84, 128.54, 128.47, 126.50, 118.86, 107.31, 102.98,
67.92, 66.98, 58.64, 53.74, 43.61, 33.45, 32.28, 27.23, 24.75, 23.14.
Exact mass (ESI) m/z 409.2469 (calcd. 409.2485 for C25H32N2O3
[M þ H]þ).
2.04e1.93 (m, 2H). 13C NMR (100 MHz, CDCl3)
d 169.73, 154.58,
138.62, 132.89, 128.82, 128.51, 128.34, 126.48, 115.73, 114.66, 112.63,
66.98, 66.45, 55.56, 53.76, 43.53, 33.42, 31.91, 26.47, 25.81. Exact
mass (ESI) m/z 395.2319 (calcd. 395.2328 for C24H30N2O3 [M þ H]þ).
6.1.8.8. 6-(2-Morpholinoethoxy)-1-benzyl-3,4-dihydro-2(1H)-quino-
linone (19). Yield: 90%. Pale yellow oil. 1H NMR (400 MHz, CDCl3)
6.1.8.14. 6-(4-Morpholinobutoxy)-1-phenethyl-3,4-dihydro-2(1H)-
d
7.35e7.29 (m, 2H), 7.27e7.19 (m, 3H), 6.79 (d, J ¼ 8.9 Hz, 1H), 6.76
quinolinone (25). Yield: 92%. Pale yellow oil. 1H NMR (400 MHz,
(d, J ¼ 2.6 Hz, 1H), 6.64 (dd, J ¼ 8.8, 2.7 Hz, 1H), 5.17 (s, 2H), 4.06 (t,
J ¼ 5.6 Hz, 2H), 3.77e3.71 (m, 4H), 3.00e2.92 (m, 2H), 2.85e2.75
CDCl3)
d
7.34e7.29 (m, 2H), 7.27e7.20 (m, 3H), 6.96 (d, J ¼ 8.8 Hz,
1H), 6.79 (dd, J ¼ 8.8, 2.8 Hz, 1H), 6.74 (d, J ¼ 2.7 Hz, 1H), 4.20e4.11
(m, 2H), 3.98 (t, J ¼ 6.3 Hz, 2H), 3.78e3.70 (m, 4H), 2.98e2.91 (m,
2H), 2.83e2.75 (m, 2H), 2.65e2.57 (m, 2H), 2.54e2.38 (m, 6H),
1.87e1.78 (m, 2H), 1.74e1.67 (m, 2H). 13C NMR (100 MHz, CDCl3)
(m, 4H), 2.62e2.55 (m, 4H). 13C NMR (100 MHz, CDCl3)
d 170.13,
154.37, 137.05, 133.54, 128.71, 127.90, 127.03, 126.38, 116.49, 114.66,
112.56, 66.85, 65.98, 57.59, 54.01, 46.22, 31.84, 25.83. Exact mass
(ESI) m/z 367.2022 (calcd. 367.2014 for C22H26N2O3 [M þ H]þ).
d
169.69, 154.62, 138.63, 132.84, 128.82, 128.51, 128.31, 126.47,
115.72, 114.64, 112.59, 67.99, 66.98, 58.63, 53.72, 43.52, 33.43, 31.93,
27.24, 25.82, 23.11. Exact mass (ESI) m/z 409.2493 (calcd. 409.2485
for C25H32N2O2 [M þ H]þ).
6.1.8.9. 6-(3-Morpholinopropoxy)-1-benzyl-3,4-dihydro-2(1H)-qui-
nolinone (20). Yield: 93%. Pale yellow oil. 1H NMR (400 MHz, CDCl3)
d
7.35e7.30 (m, 2H), 7.27e7.19 (m, 3H), 6.78 (d, J ¼ 8.9 Hz, 1H), 6.74
(d, J ¼ 2.7 Hz, 1H), 6.63 (dd, J ¼ 8.9, 2.7 Hz, 1H), 5.17 (s, 2H), 3.96 (t,
J ¼ 6.2 Hz, 2H), 3.81e3.69 (m, 4H), 2.99e2.91 (m, 2H), 2.82e2.74 (m,
2H), 2.62e2.48 (m, 6H), 2.03e1.91 (m, 2H). 13C NMR (100 MHz,
6.1.8.15. 7-(3-(Piperidin-1-yl)propoxy)-1-benzyl-3,4-dihydro-2(1H)-
quinolinone (26). Yield: 91%. Yellow oil. 1H NMR (400 MHz, CDCl3)
d
7.32e7.29 (m, 2H), 7.22e7.18 (m, 3H), 7.02 (d, J ¼ 8.1 Hz, 1H), 6.46
CDCl3)
d
170.16, 154.62, 137.07, 133.34, 128.70,127.87, 127.03, 126.38,
(d, J ¼ 8.1, 1H), 6.43 (d, J ¼ 2.2 Hz, 1H), 5.12 (s, 2H), 3.90 (t, J ¼ 6.1 Hz,
2H), 2.92e2.84 (m, 2H), 2.78e2.70 (m, 2H), 2.46e2.14 (m, 6H),
2.10e1.94 (m, 2H), 1.61e1.53 (m, 4H), 1.47e1.35 (m, 2H). 13C NMR
116.50, 114.49, 112.49, 66.73, 66.28, 55.48, 53.57, 46.21, 31.86, 26.20,
25.83. Exact mass (ESI) m/z 381.2179 (calcd. 381.2172 for
C
23H28N2O3 [M þ H]þ).
(100 MHz, CDCl3) d 169.61, 157.17, 139.77, 135.94, 127.68, 127.08,
126.01, 125.34, 117.55, 102.46, 63.37, 53.35, 52.44, 45.12, 31.16,
25.52, 24.78, 23.70, 23.35. Exact mass (ESI) m/z 379.2382 (calcd.
379.2380 for C24H30N2O2 [M þ H]þ).
6.1.8.10. 6-(4-Morpholinobutoxy)-1-benzyl-3,4-dihydro-2(1H)-qui-
nolinone (21). Yield: 90%. Pale yellow oil. 1H NMR (400 MHz, CDCl3)
d
7.31e7.25 (m, 2H), 7.20 (t, J ¼ 6.6 Hz, 3H), 6.75 (d, J ¼ 8.9 Hz, 1H),
6.71 (d, J ¼ 2.6 Hz,1H), 6.59 (dd, J ¼ 8.8, 2.8 Hz,1H), 5.13 (s, 2H), 3.89
(t, J ¼ 6.3 Hz, 2H), 3.72e3.66 (m, 4H), 2.95e2.88 (m, 2H), 2.79e2.70
(m, 2H), 2.48e2.32 (m, 6H), 1.81e1.70 (m, 2H), 1.69e1.58 (m, 2H).
6.1.8.16. 7-(3-(4-Methylpiperidin-1-yl)propoxy)-1-benzyl-3,4-
dihydro-2(1H)-quinolinone (27). Yield: 92%. Yellow oil. 1H NMR
(400 MHz, CDCl3)
d 7.32e7.27 (m, 2H), 7.21e7.16 (m, 3H), 7.02 (d,
13C NMR (100 MHz, CDCl3)
d
170.08, 154.71, 137.13, 133.22, 128.70,
J ¼ 8.2 Hz,1H), 6.48 (d, J ¼ 8.2 Hz,1H), 6.45 (d, J ¼ 2.2 Hz,1H), 5.14 (s,
2H), 3.83 (t, J ¼ 6.4 Hz, 2H), 2.95e2.82 (m, 4H), 2.77e2.71 (m, 2H),
2.42e2.36 (m, 2H),1.92e1.84 (m, 4H),1.60 (d, J ¼ 12.2 Hz, 2H),1.41e
1.29 (m, 1H), 1.27e1.19 (m, 2H), 0.91 (d, J ¼ 6.3 Hz, 3H). 13C NMR
127.83, 127.01, 126.39, 116.47, 114.48, 112.43, 67.88, 66.96, 58.57,
53.70, 46.16, 31.87, 27.17, 25.82, 23.06. Exact mass (ESI) m/z
395.2328 (calcd. 395.2328 for C24H30N2O3 [M þ H]þ).
(100 MHz, CDCl3)
d 170.70, 158.49, 140.81, 137.07, 128.76, 128.30,
6.1.8.11. 6-(5-Morpholinopentyloxy)-1-benzyl-3,4-dihydro-2(1H)-
127.08, 126.44, 118.39, 107.86, 103.58, 66.62, 55.53, 54.05, 46.21,
34.30, 32.28, 30.81, 26.81, 24.79, 21.96. Exact mass (ESI) m/z
393.2542 (calcd. 393.2536 for C25H32N2O2 [M þ H]þ).
quinolinone (22). Yield: 88%. Pale yellow oil. 1H NMR (400 MHz,
CDCl3)
d
7.31 (t, J ¼ 7.3 Hz, 2H), 7.23 (t, J ¼ 8.7 Hz, 3H), 6.77 (d,
J ¼ 8.9 Hz, 1H), 6.73 (d, J ¼ 2.7 Hz, 1H), 6.61 (dd, J ¼ 8.8, 2.8 Hz, 1H),
5.16 (s, 2H), 3.89 (t, J ¼ 6.4 Hz, 2H), 3.74e3.71 (m, 4H), 2.97e2.90
(m, 2H), 2.82e2.74 (m, 2H), 2.55e2.30 (m, 6H), 1.82e1.73 (m, 2H),
1.61e1.52 (m, 2H), 1.50e1.41 (m, 2H). 13C NMR (100 MHz, CDCl3)
6.1.8.17. 7-(3-(4-Oxopiperidin-1-yl)propoxy)-1-benzyl-3,4-dihydro-
2(1H)-quinolinone (28). Yield: 89%. Yellow oil. 1H NMR (400 MHz,
CDCl3)
d
7.30e7.28 (m, 2H), 7.22e7.18 (m, 3H), 6.98 (d, J ¼ 8.2 Hz,
d
170.14, 154.76, 137.10, 133.24,128.70, 127.83, 127.01, 126.38, 116.48,
1H), 6.44e6.35 (m, 2H), 5.14 (s, 2H), 3.93 (t, J ¼ 6.4 Hz, 2H), 2.91e
2.87 (m, 2H), 2.85e2.75 (m, 4H), 2.72e2.66 (m, 4H), 2.52e2.46 (m,
114.48, 112.45, 68.02, 66.94, 58.95, 53.75, 46.21, 31.88, 29.16, 26.23,
25.84, 23.99. Exact mass (ESI) m/z 409.2491 (calcd. 409.2485 for
4H), 1.85e1.80 (m, 2H). 13C NMR (100 MHz, CDCl3)
d 207.42, 170.94,
C
25H32N2O3 [M þ H]þ).
158.43, 141.37, 137.03, 128.44, 128.23, 127.37, 126.97, 118.31, 107.94,
103.51, 64.92, 53.60, 49.79, 46.19, 36.55, 31.46, 26.75, 24.73. Exact
mass (ESI) m/z 393.2191 (calcd. 393.2172 for C24H28N2O3 [M þ H]þ).
6.1.8.12. 6-(6-Morpholinohexyloxy)-1-benzyl-3,4-dihydro-2(1H)-
quinolinone (23). Yield: 86%. Pale yellow oil. 1H NMR (400 MHz,
CDCl3)
d
7.31e7.26 (m, 2H), 7.21 (t, J ¼ 8.2 Hz, 3H), 6.74 (m, 2H), 6.60
6.1.8.18. 7-(3-(4-Methylpiperazin-1-yl)propoxy)-1-benzyl-3,4-
(dd, J ¼ 8.8, 2.6 Hz, 1H), 5.14 (s, 2H), 3.87 (t, J ¼ 6.4 Hz, 2H), 3.74e
dihydro-2(1H)-quinolinone (29). Yield: 90%. Yellow oil. 1H NMR
3.67 (m, 4H), 2.96e2.88 (m, 2H), 2.79e2.71 (m, 2H), 2.45e2.15 (m,
(400 MHz, CDCl3)
d 7.35e7.30 (m, 2H), 7.23e7.15 (m, 3H), 7.00 (d,

Y. Lan et al. / European Journal of Medicinal Chemistry 79 (2014) 216e230
227
J ¼ 8.2 Hz, 1H), 6.51e6.36 (m, 2H), 5.11 (s, 2H), 3.79 (t, J ¼ 5.8 Hz,
2H), 2.87e2.80 (m, 2H), 2.78e2.55 (m, 2H), 2.51e2.35 (m, 8H), 2.14
(m, 2H), 5.09 (s, 2H), 3.84 (t, J ¼ 6.4 Hz, 2H), 2.86 (t, J ¼ 7.5 Hz, 2H),
2.78e2.68 (m, 2H), 2.45e2.32 (m, 6H), 2.29 (s, 3H), 1.92e1.76 (m,
2H), 1.63e1.48 (m, 4H), 1.47e1.35 (m, 2H). 13C NMR (100 MHz,
(s, 3H), 1.86 (m, 2H). 13C NMR (100 MHz, CDCl3)
d 170.84, 158.01,
140.55, 136.90, 128.74, 128.29, 127.05, 126.33, 118.60, 107.81, 103.54,
65.30, 58.05, 53.72, 53.63, 46.05, 32.09, 25.59, 24.60, 22.65. Exact
CDCl3) d 169.56, 157.40, 139.76, 135.50, 132.98, 128.34, 127.18,
125.40, 117.30, 106.74, 102.50, 65.55, 54.82, 53.53, 44.85, 31.23,
25.63, 24.92, 23.72, 23.38, 20.03. Exact mass (ESI) m/z 393.2542
(calcd. 393.2536 for C26H34N2O2 [M þ H]þ).
mass (ESI) m/z 394.2496 (calcd. 394.2488 for
C24H31N3O2
[M þ H]þ).
6.1.8.19. 7-(3-(4-Phenylpiperazin-1-yl)propoxy)-1-benzyl-3,4-
6.1.9.2. 7-(3-(Piperidin-1-yl)propoxy)-1-(4-fluorobenzyl)-3,4-
dihydro-2(1H)-quinolinone (35). Yield: 92%. Pale yellow oil. 1H NMR
dihydro-2(1H)-quinolinone (30). Yield: 86%. Yellow oil. 1H NMR
(400 MHz, CDCl3) d 7.36e7.15 (m, 7H), 7.15e7.05 (m, 3H), 6.88e6.76
(400 MHz, CDCl3)
d 7.24e7.16 (m, 1H), 7.10e7.01 (m, 4H), 6.51 (dd,
(m, 3H), 5.10 (s, 2H), 4.04 (t, J ¼ 6.2 Hz, 2H), 3.17e3.06 (m, 8H),
J ¼ 8.2, 2.3 Hz, 1H), 6.43 (d, J ¼ 2.2 Hz, 1H), 5.19 (s, 2H), 3.87 (t,
2.86e2.80 (m, 2H), 2.62e2.50 (m, 4H), 1.96e1.88 (m, 2H). 13C NMR
J ¼ 6.4 Hz, 2H), 2.92e2.85 (m, 2H), 2.78e2.80 (m, 2H), 2.47e2.24
(m, 6H), 1.93e1.80 (m, 2H), 1.61e1.51 (m, 4H), 1.44e1.34 (m, 2H). 13
C
(100 MHz, CDCl3)
d 170.84, 157.25, 140.17, 137.19, 128.75, 127.98,
127.31, 127.19, 126.53, 126.07, 125.79, 118.51, 114.94, 107.81, 103.54,
63.76, 52.38, 51.95, 47.95, 29.64, 25.59, 24.60. Exact mass (ESI) m/z
456.2701 (calcd. 456.2645 for C29H33N3O2 [M þ H]þ).
NMR (100 MHz, CDCl3)
d
170.75, 160.40 (d, J ¼ 245.2 Hz), 158.59,
140.40, 128.70, 128.37, 127.95, 124.42, 115.34, 108.14, 102.98, 66.61,
55.85, 54.59, 39.73, 32.25, 26.71, 25.99, 24.70, 24.44. Exact mass
(ESI) m/z 407.2694 (calcd. 407.2693 for C26H34N2O2 [M þ H]þ).
6.1.8.20. 7-(3-(Pyrrolidin-1-yl)propoxy)-1-benzyl-3,4-dihydro-
2(1H)-quinolinone (31). Yield: 87%. Yellow oil. 1H NMR (400 MHz,
6.1.9.3. 7-(3-(4-Methylpiperidin-1-yl)propoxy)-1-(4-methylbenzyl)-
CDCl3)
d
7.32e7.29 (m, 2H), 7.22e7.18 (m, 3H), 7.01 (d, J ¼ 7.9 Hz,
3,4-dihydro-2(1H)-quinolinone (36). Yield: 89%. Yellow oil. 1H NMR
1H), 6.51e6.45 (m, 2H), 5.12 (s, 2H), 4.06 (t, J ¼ 6.3 Hz, 2H), 2.90e
2.83 (m, 2H), 2.75e2.68 (m, 2H), 2.55e2.45 (m, 6H), 2.02e1.95 (m,
(400 MHz, CDCl3)
d 7.14e7.05 (m, 4H), 7.02e6.98 (m, 2H), 6.47 (dd,
J ¼ 6.0, 2.3 Hz, 1H), 5.09 (s, 2H), 3.84 (t, J ¼ 6.4 Hz, 2H), 2.88e2.80
(m, 4H), 2.76e2.68 (m, 2H), 2.43e2.35 (m, 2H), 2.28 (s, 3H), 1.93e
1.80 (m, 4H), 1.60 (d, J ¼ 12.8 Hz, 2H), 1.40e1.28 (m, 1H), 1.26e1.14
2H), 1.85e1.68 (m, 4H). 13C NMR (100 MHz, CDCl3)
d 170.66, 158.04,
140.84, 137.00, 128.78, 128.28, 127.11, 126.40, 117.85, 108.14, 103.77,
68.92, 60.66, 54.13, 53.57, 46.24, 32.23, 24.78, 23.46. Exact mass
(ESI) m/z 365.2236 (calcd. 365.2223 for C23H28N2O2 [M þ H]þ).
(m, 2H), 0.91 (d, J ¼ 6.3 Hz, 3H). 13C NMR (100 MHz, CDCl3)
d 170.60,
158.47, 140.83, 136.55, 134.06, 129.40, 128.24, 126.47, 118.35, 107.80,
103.57, 66.61, 55.54, 54.07, 45.91, 34.39, 32.29, 30.84, 26.89, 24.78,
21.99, 21.11. Exact mass (ESI) m/z 397.2289 (calcd. 397.2286 for
6.1.8.21. 7-(3-(Dimethylamino)propoxy)-1-benzyl-3,4-dihydro-
2(1H)-quinolinone (32). Yield: 84%. Yellow oil. 1H NMR (400 MHz,
C
24H29N2O2F [M þ H]þ).
CDCl3)
d
7.30e7.26 (m, 2H), 7.23e7.18 (m, 3H), 7.06 (d, J ¼ 7.0 Hz,
1H), 6.52e6.42 (m, 2H), 5.12 (s, 2H), 3.94 (t, J ¼ 6.1 Hz, 2H), 2.90e
2.81 (m, 2H), 2.75e2.68 (m, 2H), 2.62e2.54 (m, 2H), 2.24 (s, 6H),
6.1.9.4. 7-(3-(4-Methylpiperidin-1-yl)propoxy)-1-(4-fluorobenzyl)-
3,4-dihydro-2(1H)-quinolinone (37). Yield: 91%. Pale yellow oil. 1H
NMR (400 MHz, CDCl3) d 7.24e7.16 (m, 1H), 7.10e6.99 (m, 4H), 6.50
1.99 (m, 2H). 13C NMR (100 MHz, CDCl3)
d 170.69, 158.04, 139.41,
133.08, 127.99, 127.13, 126.40, 125.83, 117.88, 108.15, 103.78, 72.72,
53.57, 46.27, 42.94, 39.91, 32.24, 24.79. Exact mass (ESI) m/z
339.2070 (calcd. 339.2066 for C21H26N2O2 [M þ H]þ).
(dd, J ¼ 8.2, 2.3 Hz, 1H), 6.43 (d, J ¼ 2.2 Hz, 1H), 5.19 (s, 2H), 3.87 (t,
J ¼ 6.4 Hz, 2H), 2.92e2.80 (m, 4H), 2.78e2.70 (m, 2H), 2.44e2.37
(m, 2H), 1.94e1.82 (m, 4H), 1.60 (d, J ¼ 12.8 Hz, 2H), 1.40e1.28 (m,
1H), 1.28e1.13 (m, 2H), 0.91 (d, J ¼ 6.4 Hz, 3H). 13C NMR (100 MHz,
6.1.8.22. 7-(3-(Diethylamino)propoxy)-1-benzyl-3,4-dihydro-2(1H)-
CDCl3)
d
170.75, 160.41 (d, J ¼ 245.2 Hz), 158.59, 140.41, 128.70,
quinolinone (33). Yield: 85%. Yellow oil. 1H NMR (400 MHz, CDCl3)
128.37, 127.96, 124.43, 115.34, 108.14, 102.98, 66.62, 55.51, 54.05,
39.74, 34.36, 32.25, 30.82, 26.87, 24.71, 21.95. Exact mass (ESI) m/z
411.2443 (calcd. 411.2442 for C25H31N2O2F [M þ H]þ).
d
7.32e7.28 (m, 2H), 7.22e7.17 (m, 3H), 7.03 (d, J ¼ 8.2 Hz,1H), 6.57e
6.34 (m, 2H), 5.13 (s, 2H), 4.12e4.06 (m, 2H), 3.02e2.95 (m, 2H),
2.92e2.86 (m, 2H), 2.05e1.96 (m, 4H), 1.35 (t, J ¼ 7.1 Hz, 6H). 13
C
NMR (100 MHz, CDCl3)
d 170.70, 162.81, 141.20, 138.28, 127.81,
6.1.10. General procedures for the synthesis of compounds 38e39
Under N2 atmosphere compound 10i (10j, 2 mmol) was dis-
solved in dry THF (50 mL). At 0 ꢁC NaH (60%, 5 mmol) was added
and the reation mixture was stirred for 30 min at 0 ꢁC. Then
iodomethane (2.2 mmol) was added dropwise to the reaction
mixture and the mixture was stirred at room temperature for 2 h.
Water was added to the reaction mixture and the mixture was
extracted with EtOAc (3 ꢂ 25 mL). The organic layers were dried
with anhydrous magnesium sulfate, the filtrate evaporated under
reduced pressure, and the crude product was purified by means of
chromatography (10% MeOH/CHCl3) to yield compound 38e39.
127.66, 127.43, 126.99, 117.65, 107.78, 103.50, 72.13, 53.53, 46.32,
32.62, 30.96, 27.99, 24.77, 9.21. Exact mass (ESI) m/z 367.2404
(calcd. 367.2379 for C23H30N2O2 [M þ H]þ).
6.1.9. General procedures for the synthesis of compounds 34e37
Under N2 atmosphere compound 10i (10j, 2 mmol) as dissolved
in dry DMF (50 mL). At 0 ꢁC NaH (60%, 5 mmol) was added and the
reation mixture was stirred for 30 min at 0 ꢁC. Then 1-(bromo-
methyl)-4-methyl-benzene (1-(bromomethyl)-4-fluoro-benzene;
2.4 mmol) was added dropwise to the reaction mixture and the
mixture was stirred at 50 ꢁC for 3 h. Water was added to the re-
action mixture and the mixture was extracted with EtOAc
(3 ꢂ 25 mL). The organic layer was washed with a saturated aq.
NaCl solution. The organic layers were dried with anhydrous
magnesium sulfate, the filtrate evaporated under reduced pressure,
and the crude product was purified by means of chromatography
(10% MeOH/CHCl3) to yield compounds 34e37.
6.1.10.1. 7-(3-(Piperidin-1-yl)propoxy)-1-methyl-3,4-dihydro-2(1H)-
quinolinone (38). Yield: 93%. Pale yellow oil. 1H NMR (400 MHz,
CDCl3)
d
7.05 (d, J ¼ 8.1 Hz, 1H), 6.54 (d, J ¼ 2.2 Hz, 1H), 6.51 (dd,
J ¼ 8.1, 2.3 Hz, 1H), 4.08 (t, J ¼ 5.8 Hz, 2H), 3.32 (s, 3H), 3.25e3.00
(m, 6H), 2.86e2.77 (m, 2H), 2.66e2.55 (m, 2H), 2.45e2.32 (m, 2H),
2.09e1.96 (m, 4H), 1.78e1.66 (m, 2H). 13C NMR (100 MHz, CDCl3)
6.1.9.1. 7-(3-(Piperidin-1-yl)propoxy)-1-(4-methylbenzyl)-3,4-
d 169.61, 156.93, 140.56, 127.26, 117.82, 106.17, 101.90, 64.48, 54.36,
dihydro-2(1H)-quinolinone (34). Yield: 90%. Yellow oil. 1H NMR
52.68, 31.01, 28.69, 23.52, 22.22, 21.30. Exact mass (ESI) m/z
(400 MHz, CDCl3) d 7.14e7.05 (m, 4H), 7.05e6.96 (m, 1H), 6.52e6.42
303.2070 (calcd. 303.2065 for C18H26N2O2 [M þ H]þ).

228
Y. Lan et al. / European Journal of Medicinal Chemistry 79 (2014) 216e230
6.1.10.2. 7-(3-(4-Methylpiperidin-1-yl)propoxy)-1-methyl-3,4-
of the hot ligand used and Kd its receptor dissociation constant
were calculated for each labeled ligand. Mean Ki values and SEM are
reported for at least three independent experiments [45].
dihydro-2(1H)-quinolinone (39). Yield: 92%. Pale yellow oil. 1H NMR
(400 MHz, CDCl3)
d
7.05 (d, J ¼ 8.2 Hz, 1H), 6.54 (d, J ¼ 2.2 Hz, 1H),
6.51 (dd, J ¼ 8.1, 2.3 Hz, 1H), 4.08 (t, J ¼ 5.7 Hz, 2H), 3.51 (d,
J ¼ 12.0 Hz, 2H), 3.32 (s, 3H), 3.23e3.14 (m, 2H), 2.93e2.73 (m, 4H),
2.65e2.56 (m, 2H), 2.48e2.38 (m, 2H), 1.98e1.86 (m, 4H), 1.82e1.74
6.2.4. Sigma-1 binding assays
Binding of [3H]-(þ)-pentazocine at sigma-1 receptor was per-
formed according to D. L. Dehaven-Hudkins et al. [41] with minor
modifications [46e48]. The binding properties of the test com-
pounds to guinea pig s1 receptor were studied in guinea pig brain
membranes using [3H]-(þ)-pentazocine as the radioligand.
(m, 1H), 1.03 (d, J ¼ 6.3 Hz, 3H). 13C NMR (100 MHz, CDCl3)
d 170.58,
157.88, 141.59, 128.27, 118.87, 107.13, 102.88, 65.41, 55.05, 53.07,
32.00, 30.93, 29.68, 29.00, 24.52, 20.94. Exact mass (ESI) m/z
317.2219 (calcd. 317.2223 for C26H34N2O2 [M þ H]þ).
Each total binding assay tube was added 900 mL of the tissue
suspension, 50
m
L of 4.0 nmol [3H]-(þ)-pentazocine, 50
mL TriseHCl
6.2. Receptor binding studies
buffer, pH 8.0. Non-specific binding each assay tube was added
900
50
added 900
cine, 50
m
L of the tissue suspension, 50
m
L of [3H]-(þ)-pentazocine,
6.2.1. Materials
The following specific radioligands and tissue sources were
m
L of 10 mM haloperidol. Each specific binding assay tube was
used: (a) sigma-1 receptor, [3H]-(þ)-pentazocine (250
mCi, Perkine
mL of the tissue suspension, 50
m
L of [3H]-(þ)-pentazo-
Elmer, NET-1056250UC), and male Dunkin Hartley guinea pig brain
mL of reference drug or test compounds solution in various
membrane; (b) sigma-2 receptor, [3H]-di-o-tolylguanidine ([3H]-
concentrations (10ꢃ5 mol to 10ꢃ10 mol). The tubes were incubated
at 25 ꢁC for 180 min. The incubation was followed by a rapid vac-
uum filtration through Whatman GF/B glass filters, and the filtrates
were washed twice with 5 mL cold buffer and transferred to scin-
tillation vials. Scintillation fluid (2.0 mL) was added and the
radioactivity bound was measured using a Beckman LS 6500 liquid
scintillation counter.
DTG, 250
mCi, PerkineElmer, NET-986250UC), and male Dunkin
Hartley guinea pig brain membrane. Chemicals and reagents were
purchased from different commercial sources and of analytical
grade.
6.2.2. Membrane preparation
Membrane preparation was performed by previously reported
method [40,41]. Crude P2 membranes were prepared from frozen
guinea pig brains minus cerebellum. Tissues were homogenized in
ice-cold 10 mmol Tris-sucrose buffer (0.32 mol sucrose in 10 mmol
TriseHCl, pH 7.4) using an ULTRA TURAX homogenizer. The ho-
mogenates were centrifuged at 4 ꢁC at 1000 g for 10 min and the
supernatant were saved. The resulting pellet was then resuspended
in the same buffer, incubated for 10 min at 4 ꢁC, and centrifuged at
1000 g for 10 min. After that, the pellet was discarded and the
supernatants were combined and centrifuging at 31 000 g for
15 min. The pellets were resuspended in 10 mL TriseHCl, pH 7.4 in a
volume of 3 mL/g and the suspension was allowed to incubate at
25 ꢁC for 30 min. Following centrifuging at 31 000 g for 15 min, the
pellet was resuspended by gentle homogenization in 10 mmol
TriseHCl, pH 7.4 in a final volume of 1.53 mL/g tissue and aliquots
were stored at ꢃ80 ꢁC until used.
6.2.5. Sigma-2 binding assays
Binding assays were performed as described by Ronsisvalle et al.
[40] with minor modifications [31]. The binding properties of the
test compounds to guinea pig sigma-2 receptor were studied in
guinea pig brain membranes using [3H]-DTG.
The membranes were incubated with 3 nmol [3H]-DTG in the
presence of 400 nM (þ)-SKF10047 to block sigma-1 sites. Each total
binding assay tube was added 850 mL of the tissue suspension, 50 mL
of 3.0 nmol [3H]-DTG, 50
HCl buffer, pH 8.0. Non-specific binding each assay tube was added
850 L of the tissue suspension, 50 L of
L of [3H]-DTG, 50
400 nmol (þ)-SKF10047, 50 L of 10 mol DTG. Each specific
binding assay tube was added 850 L of the tissue suspension, 50
of [3H]-DTG, 50
L of 400 nmol (þ)-SKF10047, 50 L of reference
mL of 400 nmol (þ)-SKF10047, 50
mL Trise
m
m
m
m
m
m
mL
m
m
drug or test compounds solution in various concentrations
(10ꢃ5 mol to 10ꢃ10 mol). The tubes were incubated at 25 ꢁC for
120 min. The incubation was followed by a rapid vacuum filtration
through Whatman GF/B glass filters, and the filtrates were washed
twice with 5 mL cold buffer and transferred to scintillation vials.
Scintillation fluid (2.0 mL) was added and the radioactivity bound
was measured using a Beckman LS 6500 liquid scintillation counter.
The protein concentration of the suspension was determined by
the method of Bradford [44]. Subsequently, the preparation con-
tained 4 mg protein/mL.
6.2.3. General procedures fort the binding assays
All the test compounds were prepared by dissolving in 5%
DMSO. The filter mats were presoaked in 0.5% polyethylenimine
solution for 2 h at room temperature before use. The following
specific radioligands and tissue sources were used: (a) sigma-1
receptor, [3H]-(þ)-pentazocine, guinea pig brain membranes; (b)
sigma-2 receptor, [3H]-DTG, guinea pig brain membranes.
The total binding (TB) was determined in the absence of
nonspecific binding and compounds while specific binding (SB)
was determined in the presence of compounds. Nonspecific bind-
ing (NB) was determined as the difference between total and spe-
cific binding. Nonspecific binding was determined in the presence
6.2.6. Functional profile on sigma-1 receptor
The functional activity of compound 35 and 37 were evaluated
by using Guinea pig brain membranes binding assays for sigma-1
receptor. The binding affinities either in the absence or presence
of 1 mmol phenytoin were measured to identify the functional
(agonistic or antagonistic) nature [42].
6.3. In vivo test
of 10 mmol haloperidol. Percentage of inhibition (%) was calculated
as the following equation: Percentage of inhibition (%) ¼ (TB-SB)/
(TB-NB) ꢂ 100%. Blank experiments were carried out to determine
the effect of 5% DMSO on the binding and no effects were observed.
Compounds were tested at least three times over a 6 concentration
range (10ꢃ5 mol to 10ꢃ10 mol), IC50 values were determined by
nonlinear regression analysis using Hill equation curve fitting. Ki
values were calculated based on the Cheng and Prussoff equation:
Ki ¼ IC50/(1 þ C/Kd). In the equation, C represents the concentration
6.3.1. Animals
Chinese Kun Ming (KM) Mice (20 ꢀ 2.0 g) were used as exper-
imental animals in this study. All the animals were housed under
standardized conditions for light, temperature and humidity,
received standard rat chow and tap water and libitum. Animals
were assigned to different experimental groups randomly, each
kept in a separate cage. All research involving animals in this study
follow the guidelines of the byelaw of experiments on animals, and

Y. Lan et al. / European Journal of Medicinal Chemistry 79 (2014) 216e230
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Acknowledgments
The authors thank Prof. Liangren Zhang and MS. Xiaozhuo Ma of
State Key Laboratory of Natural and Biomimetic Drugs, School of
Pharmaceutical Sciences, Peking University for performing NMR
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