Synthesis and biological evaluation of novel naphthalene compounds as potential antidepressant agents

Article abstract of DOI:10.1016/j.ejmech.2014.05.061

In this study, a series of novel naphthalene compounds were synthesized and screened for their antidepressant-like activities in vitro and in vivo. Their values for two descriptors (ClogP, tPSA) of the blood-brain barrier (BBB) were calculated for early assessment of the central nervous system (CNS) drug-likeness. Seven of them (6d, 6i, 6k, 6o, 6p, 6s and 6t) demonstrated potential protective effects on corticosterone-induced lesion of PC12 cells although they cannot repair the irreversible oxidant injury to PC12 cells by hydrogen peroxide. Compounds with promising neurorestorative activities (6k, 6o and 6p) were further evaluated for their in vivo effects by forced swim test (FST) and open field test (OFT) in C57 mice models. The FST results showed that compounds 6k, 6o and 6p remarkably reduced the immobility time of the tested mice. Among them, compound 6k was the most potent one, much more effective than Agomelatine and comparable to Fluoxetine. The OFT results showed that mice treated with compound 6k traveled a longer distance than those treated with Agomelatine or Fluoxetine, indicating a better general locomotor activity. The paper also proposed a possible binding mode of compound 6k with glucocorticoid receptor by docking study. The in vitro cytotoxicity data on HEK293 and L02 cells suggested compound 6k to be a promising antidepressant candidate for subsequent investigation.

Full text of DOI:10.1016/j.ejmech.2014.05.061

European Journal of Medicinal Chemistry 82 (2014) 263e273
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 novel naphthalene compounds
as potential antidepressant agents
,
b
,
,
,
Wei Ang a 1, Gong Chen a 1, Li Xiong b 1, Ying Chang a, Weiyi Pi a, Yuanyuan Liu a,
Chunlong Li a, Jiajia Zheng a, Liangxue Zhou a *, Bo Yang a, Yong Deng b
,
,
, *
Shengyong Yang a, Youfu Luo a *, Yuquan Wei a
,
a State Key Laboratory of Biotherapy and Department of Neurosurgery, West China Hospital, West China Medical School, Sichuan University,
Chengdu, Sichuan 610041, PR China
b Key Laboratory of Drug Targeting and Drug Delivery System, Ministry of Education, West China School of Pharmacy, Sichuan University,
Chengdu, Sichuan 610041, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, a series of novel naphthalene compounds were synthesized and screened for their
antidepressant-like activities in vitro and in vivo. Their values for two descriptors (ClogP, tPSA) of the
bloodebrain barrier (BBB) were calculated for early assessment of the central nervous system (CNS)
drug-likeness. Seven of them (6d, 6i, 6k, 6o, 6p, 6s and 6t) demonstrated potential protective effects on
corticosterone-induced lesion of PC12 cells although they cannot repair the irreversible oxidant injury to
PC12 cells by hydrogen peroxide. Compounds with promising neurorestorative activities (6k, 6o and 6p)
were further evaluated for their in vivo effects by forced swim test (FST) and open field test (OFT) in C57
mice models. The FST results showed that compounds 6k, 6o and 6p remarkably reduced the immobility
time of the tested mice. Among them, compound 6k was the most potent one, much more effective than
Agomelatine and comparable to Fluoxetine. The OFT results showed that mice treated with compound 6k
traveled a longer distance than those treated with Agomelatine or Fluoxetine, indicating a better general
locomotor activity. The paper also proposed a possible binding mode of compound 6k with glucocor-
ticoid receptor by docking study. The in vitro cytotoxicity data on HEK293 and L02 cells suggested
compound 6k to be a promising antidepressant candidate for subsequent investigation.
Received 27 January 2014
Received in revised form
6 May 2014
Accepted 25 May 2014
Available online 27 May 2014
Keywords:
Depression
PC12 cells
Corticosterone-induced
Forced swim test
Open field test
Glucocorticoid receptor
© 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
Despite of a large number of antidepressant drugs commercially
available, there are still many issues leading to risks of depression
Major depression is a mental disorder characterized by a
pervasive and persistent low mood which is accompanied by low
self-esteem and by a loss of interest or pleasure in normally
enjoyable activities [1]. The World Health Organization (WHO)
report has predicted that major depression will become a key
contributor to illness-induced disability by the year 2020, second
only to ischemic heart diseases [2,3]. Moreover, new research
indicated that patients with major depression have an increased
onset risk of aging-related somatic diseases such as heart disease,
diabetes, obesity and cancer [4].
therapy. It was reported in clinic that part of patients do not
respond fully to the antidepressant drugs, even the present block-
buster drugs like Imipramine, Fluoxetine, Citalopram and Ven-
lafaxine [4]. Besides, the long-lasting therapy period gives rise to
poor patient compliance, along with several adverse effects as well
as the drugedrug interactions. Consequently there is a desirable
need to find new chemical entities as potential antidepressant
candidates with novel action mechanism, which may lead to a more
advantageous benefit-risk balance.
Neuroscientists have put great efforts on the investigation of
neurobiological and structural changes of the treated or non-
treated patients with mental disorders, and found that neuronal
plasticity, neurogenesis and their regulation may play important
role in the treatment of major depression [5]. For example, several
studies showed that hippocampal volume is reduced in patients
with major depression compared with healthy controls [6e9]. On
* Corresponding authors.
E-mail addresses: zhlxlll@163.com (L. Zhou), dengyongy@sohu.com (Y. Deng),
luo_youfu@scu.edu.cn, luo_youfu@163.com (Y. Luo).
1
Authors contributed to this work equally.
http://dx.doi.org/10.1016/j.ejmech.2014.05.061
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

264
W. Ang et al. / European Journal of Medicinal Chemistry 82 (2014) 263e273
the other hand, the recently marketed antidepressant drug, Ago-
melatine, was shown to induce cell proliferation and neurogenesis
in the ventral part of dentate gyrus, a region implicated in the
response to emotion and anxiety, which gave the basis for the
neuroplasticity hypothesis of major depression. Fluoxetine also
shared above neurogenetic effects [10e15]. Meanwhile, many
studies indicated that antidepressant drugs are able to prevent
neuronal damage and cell loss that may occur in the brain of pa-
tients with mood disorders [16e19]. As a result, neurorestorative or
neuroprotective effect of antidepressants has been proposed as a
highly possible mechanism to treat mental disorders [20].
On the other hand, it was widely reported that compounds
containing naphthalene scaffold displayed antidepressant activities
[21e25]. Thus researchers made great efforts on structural modi-
fication of compounds with naphthalene scaffold and get some
excellent antidepressant candidates and the most successful
example is the discovery of Agomelatine, which was approved for
the treatment of major depression by European Medicines Agency
last 2009 [26]. Most naphthalene derivatives exhibited activities on
neurological or neuropsychiatric disorders contained a core struc-
ture of 2-(naphthalen-1-yl)ethanamine [23,24]. Inspired by the
mentioned findings, we designed and synthesized a series of novel
2-(naphthalen-1-yl) ethanamine compounds by introduction of
various side chains with different properties, looking forward to
discovery of potential antidepressant agents with neurorestorative
or neuroprotective mechanism.
concentration lead to PC12 neuronal damage under depressive
disorder, and this feature makes PC12 cells very useful as a model
system for in vitro screening [20]. Oxidant injury of PC12 cells is
more destructive and sometimes the damage is irreversible.
Nevertheless, a few antidepressant drugs showed protective effects
in this model [27,29,30]. Besides, oxygen glucose deprivation,
glutamate, Ab2535, and even physical methods can also lead into
PC12 cells lesion in vitro models, which have also been used in
studies of mental disorders. Herein, H2O2 and corticosterone were
employed as stimulants to induce traumatic lesion of PC12 cells,
which were further used as two typical in vitro models to investi-
gate the neurorestorative and neuroprotective activities of our
novel naphthalene compounds. In addition, all of the target com-
pounds were also tested for their in vitro cytotoxicities on human
normal liver L02 cells and human embryonic kidney 293 cells by
MTT method. Two descriptors of their penetrating abilities of
bloodebrain barrier (BBB) such as lipophilicity (logP) and molec-
ular topological polar surface area (tPSA) were also employed
calculated for early assessment of their central nervous system
drug-likeness. Based on in vitro and in silicon results, four com-
pounds (6d, 6k, 6o and 6p) were selected to in vivo forced swim test
in C57 mice. The general locomotor activity of compound 6k was
further measured by an open field test, which confirmed its anti-
depressant potential. Docking study was conducted to illustrate the
binding mode of 6k with glucocorticoid receptor, a possible
molecule target of neuroprotective agents.
It is well known that one of the bottlenecks to rapidly develop
effective drugs for treatment of mental disorders including
depression is poorly predictive capability of in vitro and in vivo
screening models. PC12 is a cell lineage derived from a pheochro-
mocytoma of rat adrenal medulla and has been widely used to
investigate the mechanisms involved in neurotoxicity, neuro-
protection and neurorestoration [27,28]. Glucocorticoids at high
2. Chemistry
To achieve the synthesis of the target compounds 6a-t, the steps
outlined in Scheme 1 were adopted. The intermediate 3 was syn-
thesized from commercially available 2-(7-methoxynaphthalen-1-
yl)acetonitrile (1) according to literature with appropriate
Scheme 1. General synthetic route of compounds 6aet.

W. Ang et al. / European Journal of Medicinal Chemistry 82 (2014) 263e273
265
revision [31e33]. The reported method directly used sodium
borohydride/nickel chloride system to reduce the start material 1,
resulting in oily material difficult to solidify. As a result, we
improved the process by taking advantage of an in situ N-Boc
protection to obtain high purity of intermediate 2 in a single step.
Our modification avoided the contamination of impurities such as
oxime, secondary amine and acyl amine and the white solid 3 was
readily obtained in high purity after de-protection of N-Boc under
acidic condition. The common key intermediate 4 was prepared by
a condensation reaction with 1-(tert-butoxycarbonyl) azetidine-3-
carboxylic acid according to a reported method [34]. The final
compounds (6aet) were prepared by acylation of the key inter-
mediate 5 with various carboxyl acids according to literature
[35,36].
advantage of MTT method to measure the viability of PC12 cells
48 h after drug treatment of the H2O2-injured cell culture at three
different drug concentrations of 10 mM, 20 mM and 40 mM. As shown
in Table 2, only thirteen compounds displayed weak repair activ-
ities in certain concentration. Even Fluoxetine and Agomelatine
displayed weak repair activities at 10
For compounds 6d, 6h and 6j, showed weak restoration activity in
10 M, 20 M and 30 M, however, their restoration rates did not
mM and 20 mM respectively.
m
m
m
exhibited linear correlation with drug concentration. For example,
6d exhibited almost similar restoration rates at drug concentration
of 10
mM and 20
mM. For compound 6h, the restoration rates
decreased when the drug concentration was doubled to 40
mM.
Seven compounds did not possess restorative effects on the oxidant
injury of PC12 cells in all tested drug concentration, this may
contributed to their cytotoxicity on PC12 cells. Which may suggest
the oxidant injury of PC12 cells by hydrogen peroxide is too
destructive to restore and not a suitable in vitro model for pre-
liminary screening of potential naphthalene antidepressant hits.
3. Result and discussion
3.1. BBB penetration ability of the designed compounds
3.3. Protective effects on corticosterone-injured PC12 cells
As we know, the ability to penetrate the bloodebrain barrier
(BBB) is a significant parameter for CNS drugs. Lipophilicity (logP)
and molecular topological polar surface area (tPSA) were widely
used to describe CNS penetration ability [37,38]. For marketed CNS
drugs, the value range of CLogP is 0.16e6.59 and that of tPSA is
4.63e108. In order to predict the BBB penetration ability of the
designed naphthalene compounds in advance, we calculated the
values of these two descriptors for them by the trial version of
All the target compounds were tested for their protection ac-
tivities on PC12 cells from corticosterone-induced lesion at drug
concentration of 1.25 mM and the data were shown in Table 3. From
the mean values listed in Table 3, seven of them (6d, 6i, 6k, 6o, 6p,
6s and 6t) demonstrated potential protective effects on
corticosterone-induced lesion of PC12 cells although they cannot
repair the irreversible oxidant injury to PC12 cells by hydrogen
peroxide as stated in Section 3.2. Three amide compounds (6k, 6o
and 6p) showed pronounced efficacy with protection rates of
43.5 4.3%, 39.2 1.7% and 38.8 4.6% respectively. Although it is
not easy to get clear structureeactivity relationship from the pre-
sent data, some deduction, requiring further confirmation by more
studies on medicinal chemistry, can be reasonably expected.
Among the synthesized compounds, the active ones likely fell in the
category with another aromatic group introduced to the side chain
of 2-(naphthalen-1-yl)ethanamine scaffold although the most
active compound 6k are substituted with a trifluoroacetyl group at
the same position. Herein, the piepi interaction of compounds 6d,
6i, 6o, 6p, 6s and 6t, or hydrogen-bonding interaction of compound
6k with potential biological target may play a part. Compared
compound 6d and 6o, the conclusion can be drawn that the acyl
linker (6o: 39.2 1.7%) is superior to its sulfonyl counterpart (6d:
25.3 0.9) for the introduction of another aromatic group.
®
ChemBioOffice Ultra 13.0 and the data were listed in Table 1,
where we can see that the ClogP values of the synthesized naph-
thalene compounds are quite similar to the marketed antidepres-
sant Fluoxetine or Agomelatine, and the tPSA values of them are in
the range of the marketed CNS drugs. With these calculated values
in hand, we started to synthesize the planned compounds since
there seemed no designed molecules went beyond the established
drug-like space.
3.2. Restorative effects on H2O2-injured PC12 cells
Commercially there are three types of PC12 cells, i.e. undiffer-
entiated, low-differentiated and high-differentiated PC12 cells.
Undifferentiated PC12 cells were far different from neurons. How-
ever, the high-differentiated PC12 cells were not able to proliferate
in culture condition, thus not convenient and ready for in vitro
screening. Low-differentiated PC12 cells share properties similar to
neurons and are able to proliferate in culture conditions. Conse-
quently, low-differentiated PC12 cells were selected in our in vitro
model for primary screen of antidepressant. In order to determine
whether the final compounds have the restorative effects on the
oxidant injury of PC12 cells by hydrogen peroxide, we took
From above results, it seemed that corticosterone-injured PC12
cells is a fruitful in vitro model for preliminary screening of anti-
depressant drugs and we would like to pick out the active hits (6k,
6o and 6p) with better protective effects than the positive controls
(Fluoxetine: 19.7
evaluation.
0.7%; Agomelatine: 26.2
2.4%) to further
To visually observe the protective effect of the active compounds
in corticosterone-induced PC12 cells, we assessed the morpholog-
ical changes of PC12 cells using inverted microscopy before and
after treated with 6k and 6o respectively (Fig. 1). PC12 cells treated
0.2 mM of corticosterone (Fig. 1B) turned into cycloid compared
with cells treated normal saline (Fig. 1A), while much less
morphological changes of PC12 cells were observed when treated
with test drugs (Fig.1CeG). The photos illustrated in Fig.1 indicated
that 6k (Fig. 1F) and 6o (Fig. 1G) possess protective effects on
corticosterone-induced injury of PC12 cells.
To give a precise description of the morphological changes of
PC12 cells, we counted the morphologically changed cells and
normal neuron-like cells carefully in three random microscopic
vision fields by simple computer-assisted image analysis. The PC12
cells with shrunk axons and cycloid shape were regarded as
Table 1
Calculated logP (ClogP) and topological polar surface area (tPSA) values of the target
compounds.
Compd
ClogP
tPSA
Compd
ClogP
tPSA
6a
6b
6c
6d
6e
6f
6g
6h
6i
2.9
3.1
4.9
5.1
4.2
2.6
3.1
4.0
5.0
5.5
3.7
75.7
79.0
75.7
75.7
67.9
58.6
58.6
58.6
58.6
67.9
58.6
6l
3.5
3.7
4.3
4.8
3.2
2.9
3.7
4.3
3.6
4.6
2.1
61.9
162.3
77.1
58.6
67.9
71.0
71.0
77.1
82.4
21.3
38.3
6m
6n
6o
6p
6q
6r
6s
6t
Flu
Ago
6j
6k

266
W. Ang et al. / European Journal of Medicinal Chemistry 82 (2014) 263e273
Table 2
The restoration rates of the target compounds on H2O2-injured PC12 cells.
Compd
RRa (%)
10
Compd
RRa (%)
m
M
20
m
M
40
mM
10
m
M
20
m
M
40 mM
6a
6b
6c
6d
6e
6f
6g
6h
6i
0.9 0.1
e
e
3.7 0.3
e
6k
6l
e
e
e
e
e
e
2.1 0.2
e
1.2 0.1
1.7 0.1
2.1 0.1
e
4.3 0.4
e
e
e
6m
6n
6o
6p
6q
6r
6s
6t
Ago
4.6 0.4
e
4.5 0.3
e
6.8 0.4
e
e
0.3 0.1
e
4.7 0.4
e
e
0.6 0.2
e
1.0 0.1
7.5 0.3
4.6 0.3
e
e
e
0.7 0.1
e
e
2.9 0.1
e
6.6 0.3
e
e
e
e
e
e
2.1 0.6
e
e
e
e
6j
Flu
3.2 0.1
2.8 0.2
6.6 0.5
e
7.3 0.8
e
e
2.7 0.2
a
RR represents the restoration rate of the tested compound calculated from six independent experiments measured at 48 h after treatment with the test compound at three
M, 20 M and 40
different concentrations (10
m
m
m
M). RR ¼ (Ad ꢀ Ac)/Ac*100% S.D., where, Ac represents the mean absorbance value of six independent experiments of control
group merely treated with corticosterone, Ad means the mean absorbance value of six independent experiments of test group treated with corticosterone and test drug and
S.D. means the standard deviation. Data are presented as mean S.D. ‘e’ represents compounds did not shown restoration effects on H2O2-injured PC12 cells.
Table 3
morphologically changed ones injured by stimulant and the result
was shown in Fig. 2. Compound 6k exhibited better protective ef-
fect than the positive controls. Compound 6o displayed comparable
The protection rates of the target compounds on corticosterone-injured PC12 cells.
Compd
PRa (%)
Compd
PRa (%)
protective effect to Agomelatine, but worse than Mifepristone.
6a
6b
6c
6d
6e
6f
6g
6h
6i
1.4 1.8
e
6k
6l
43.5 4.3
e
e
6m
6n
6o
6p
6q
6r
6s
6t
Ago
e
3.4. Cytotoxicity
25.3 0.9
e
e
39.2 1.7
38.8 4.6
e
All of the target compounds (6aet) were evaluated in vitro on
human normal liver L02 cells and human embryonic kidney 293
cells. As shown in Table 4, most of the naphthalene compounds did
not show apparent inhibitory effects on the test cell lines at con-
centration of 80 mM compared. The most promising compounds 6k,
6o and 6p demonstrated lower cytotoxicity on both cell lines, su-
e
e
e
e
14.6 2.0
e
22.3 2.6
25.8 2.4
26.2 2.4
6j
Flu
Mif
19.7 0.7
31.1 1.2
a
perior to Fluoxetine and Agomelatine.
PR represents the protection rate of the tested compound calculated from six
independent experiments measured at 24 h after treatment with the test compound
at the concentration of 1.25
m
M. PR ¼ (Ad ꢀ Ac)/Ac*100% S.D., where, Ac represents
3.5. Forced swim test
the mean absorbance value of six independent experiments of control group merely
treated with corticosterone, Ad means the mean absorbance value of six indepen-
dent experiments of test group treated with corticosterone and test drug and S.D.
means the standard deviation. Data are presented as mean S.D. ‘e’ represents
compounds did not shown protection effects on corticosterone-injured PC12 cells.
As shown in Fig. 2, compounds 6k, 6o and 6p displayed
remarkable activities compared with control group. Treatment
with compound 6k, 6o or 6p remarkably reduced the immobility
time of C57 mice with a percentage of 58.4%, 49.1% and 36.2%
respectively. Compound 6k exhibited better antidepressant-like
Fig. 1. Morphological changes in PC12 cells treated with 6k and 6o (100ꢁ). PC12 cells were seeded in 6-well plates at a density of 1 ꢁ106 cells/mL in the growth medium for 1 d, and
then the cells were treated with indicating drugs respectively. (A) cells treated with normal saline; (B) cells exposed to corticosterone 0.2 mM; (C) cells treated with corticosterone
0.2 mM and Fluoxetine 1.25
mM; (D) cells treated with corticosterone 0.2 mM and Agomelatine 1.25
mM; (E) cells treated with corticosterone 0.2 mM and Mifepristone 1.25
mM; (F)
cells treated with corticosterone 0.2 mM and 6k 1.25
mM; (G) cells treated with corticosterone 0.2 mM and 6o 1.25
mM.

W. Ang et al. / European Journal of Medicinal Chemistry 82 (2014) 263e273
267
Fig. 3. Effects of compounds 6d, 6k, 6o, 6p and positive controls in forced swim test.
Mice were treated on days 2e15 with compound (i.p.32 mg/kg/day). Data represent
the mean
S.D. of 10 mice per group. Values are significant at *P < 0.01 when
compared with vehicle group.
Fig. 2. The percentage of normal morphological cells. *P < 0.01, **P < 0.05.
3.7. Docking study
potency than Fluoxetine (50.7%) and Agomelatine (8.4%). Com-
pound 6o and 6p also displayed in vivo antidepressant-like effects,
which is well correlated with the in vitro corticosterone-induced
PC12 model. However, sulfonyl compound 6d with good neuro-
protective in vitro was shown to be ineffective in FST test, largely
different from its acyl counterpart 6o. The completely opposite
results may be owed to the difference of tPSAvalue of compound 6d
(75.7) from that of compound 6o (58.6) as listed in Table 1. Com-
pound 6o with a lower tPSA value was more liable to cross the
bloodebrain barrier than compound 6d.
In order to gain better understanding of the molecular mecha-
nism of the most active compound 6k, a docking study was per-
formed by docking compound 6k or 6d or Agomelatine into the
active site of glucocorticoid receptor (PDB code: 1NHZ). As shown
in Fig. 4, compound 6k formed two hydrogen bonds with GLN570
and ASN564, exhibited best protective effects on corticosterone-
injured PC12 cells and antidepressant-like activity in vivo. With
only one hydrogen bond observed in binding model with gluco-
corticoid receptor, Agomelatine displayed less activity than 6k
in vitro and in vivo.
With a purpose of a clear understanding of binding mode of 6k,
some more detail docking study was shown in Fig. 5. The appro-
priate length of the side chain in compound 6k made it capable of
the occupation of the inner cavity (Fig. 5b) and forming another
hydrogen bond with GLN570 in the receptor, which is quite
different from that of Agomelatine (Fig. 6).
3.6. Open field locomotor activity
To exclude the nonspecific motor activities in the forced swim
test, the most effective compound 6k was selected to further test its
spontaneous locomotor activity in an open field apparatus (Fig. 3).
Mice administrated with compound 6k traveled a longer total
distance by 88.5% than the model ones, while Fluoxetine and
Agomelatine showed that percentage of 66.4% and 85.3%. These
results well confirmed the antidepressant-like activity of 6k.
4. Conclusions
In this paper, we synthesized twenty novel N-(2-(7-
methoxynaphthalen-1-yl) ethyl) amide derivatives and tested
Table 4
Cytotoxicities of the final compounds on human normal liver L02 cells and human
embryonic kidney cell line 293.
Compd
IRa (%)
L02
Compd
IRa (%)
L02
293
8.5 5.4
293
6a
6b
6c
6d
6e
6f
6g
6h
6i
4.4 0.5
7.4 3.1
2.7 0.9
33.6 7.7
7.8 2.9
2.3 1.3
2.9 1.4
3.8 0.4
7.9 4.2
12.1 2.1
28.5 6.9
6k
6l
2.6 2.2
e
e
4.6 2.8
3.6 0.6
21.5 0.4
8.9 5.5
6.2 0.7
11.2 1.7
11.9 1.2
24.6 7.2
1.0 0.9
55.8 2.9
2.5 1.6
6m
6n
6o
6p
6q
6r
6s
6t
Ago
15.8 0.5
13.1 1.6
5.9 0.7
3.2 0.5
11.2 0.4
4.8 0.8
1.3 0.8
1.0 0.2
20.2 7.6
11.1 2.4
13.1 3.5
11.8 2.4
26.4 2.6
31.8 1.4
17.3 1.4
33.4 2.7
17.5 4.8
26.6 1.6
6j
Flu
a
IR is the mean inhibitory rate calculated from three independent experiments
measured at 24 h after treatment with the test compound at the concentration of
80
m
M. The viability of the untreated cells was regarded as 100%. Data are expressed
Fig. 4. Antidepressant-like effects of compound 6k and positive controls in the open
field test. Mice were treated on days 2e15 with test compounds (i.p. 32 mg/kg/day).
Data represent the mean S.D. of 10 mice per group. *P < 0.01, **P < 0.05.
as the mean S.D. ‘e’ represents compounds did not shown cytotoxicity on tested
cells.

268
W. Ang et al. / European Journal of Medicinal Chemistry 82 (2014) 263e273
Fig. 5. (a) The binding modes of compounds 6k, 6d and Agomelatine with glucocorticoid receptor. 6k colored in pink, 6d in yellow and Agomelatine in green, respectively. (b)
Chemical structures of compound 6k, 6d and Agomelatine. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
their neuroprotective or neurorestorative activities on injured PC12
cells induced by corticosterone or H2O2 respectively. The results
from these two in vitro models showed that corticosterone-injured
PC12 cells model is a fruitful in vitro model for preliminary
screening of antidepressant candidates. Four compounds 6k, 6o, 6d
and 6p with better protection rates than positive controls (Ago-
melatine and Fluoxetine) were further evaluated on forced swim
test model. Among them, compound 6k exhibited better anti-
immobility activity than Fluoxetine and Agomelatine. In open
field test, mice treated with 6k traveled longer distance than
Fluoxetine and Agomelatine, displaying a better general locomotor
activity. Docking study of compound 6k with glucocorticoid re-
ceptor proposed a possible binding mode with hydrogen-bonding
interactions with residues of ASN564 and GLN570 in glucocorti-
coid receptor, which was different from that of Agomelatine, where
no hydrogen-bonding interaction with residue of GLN570 was
involved. The cytotoxicity data suggested a good safety profile of
compound 6k, providing a promising antidepressant candidate
with neuroprotective mechanism.
All melting points were determined on a SGW X-4 Micro
Melting Point apparatus and are uncorrected. 1H NMR and 13C NMR
spectra were recorded on a Bruker Avance (Varian Unity Inova)
400 MHz spectrometer using TMS as internal reference chemical
shift in d, ppm. High-resolution mass (HRMS) spectrometry was
carried out on a Waters Q-TOF Premier mass spectrometer.
All compounds used for biological assays were at least of 98%
purity based on HPLC analytical results monitored with full
wavelengths.
5.2. Chemistry
5.2.1. Tert-butyl (2-(7-methoxynaphthalen-1-yl)ethyl)carbamate (2)
A mixture of 2-(7-methoxynaphthalen-1-yl)acetonitrile (com-
pound 1, 1.99 g, 10.0 mmol), NiCl2 (475 mg, 5.0 mmol) and (Boc)2O
(0.2 mL) in 20 mL MeOH were stirred in an ice-bath, then NaBH4
(417 mg, 11.0 mmol) was added in batches under N2 atmosphere.
The ice-bath was removed and the mixture was stirred at room
temperature for 12 h. After the completion of reaction (as evi-
denced by TLC), 20 mL H2O was added and the resulting mixture
was concentrated under reduced pressure. Then 20 mL H2O was
added, extracted with ethyl acetate (20 mL ꢁ 3), the combined
organic layer were concentrated under reduced pressure to ob-
tained brown solid (2.41 g, yield: 80%). mp: 98.6e100.5 ꢂC, 1H NMR
5. Experiment section
5.1. Materials
(CDCl3):
d
7.76 (d, J ¼ 8 Hz, 1H), 7.68 (dd, J ¼ 8 Hz, J ¼ 4 Hz, 1H), 7.45
Corticosterone was obtained from Aladdin Reagents (Shanghai)
Company. Methyl thiazolyltetrazolium (MTT) were bought from
Sigma Company. Fluoxetine and Agomelatine were purchased from
Dalian Meilun Biotech Company.
Male C57 mice (20e24 g) were purchased from Beijing HFK Bio-
Technology Co. ltd. They were kept on a 12 h/12 h light/dark cycle
and were given access to food and water ad libitum. All the pro-
cedures in this study were performed followed the protocols
approved by the Institutional Animal Care and Treatment Com-
mittee of Sichuan University. All mice in this study were treated
humanely throughout the experimental period.
(s, 1H), 7.30e7.25 (m, 2H), 7.17 (dd, J ¼ 8 Hz, J ¼ 4 Hz, 1H), 4.75 (s,
1H), 3.97 (s, 3H), 3.54 (dd, J ¼ 16 Hz, J ¼ 8 Hz, 2H), 3.24 (t, J ¼ 8 Hz,
2H), 1.46 (s, 9H); MS (TOF) m/z calcd. for C18H23NO3 [M þ Hþ] 302.2,
found: 302.4.
5.2.2. 2-(7-methoxynaphthalen-1-yl)ethanamine hydrochloride (3)
Compound 2 (2.41 g, 8.00 mmol) was dissolved in 10 mL CH2Cl2
and 10 mL TFA, after stirred at room temperature for 3 h. The
completion of reaction was monitored by TLC. Then adjust to
pH ¼ 10 with 10% NaOH solution, then washed with H2O
(20 mL ꢁ 3) and brine (20 mL ꢁ 1), dry over Na2SO4, filtered and
treated with a saturated solution of hydrogen chloride in Et2O,
All solvents and reagents were analytical reagents and used
directly without further purification.
Fig. 6. (a) The binding mode of compound 6k with glucocorticoid receptor. 6k colored in pink, hydrogen bond colored in blue. (b) Binding cavity of 6k and glucocorticoid receptor.
(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

W. Ang et al. / European Journal of Medicinal Chemistry 82 (2014) 263e273
269
compound 3 was obtained after concentrated under reduced
pressure as gray solid (963 mg, yield: 64%). mp: 242.7e245.5 ꢂC; 1H
3H), 3.68 (q, J ¼ 8 Hz, 2H), 3.27 (t, J ¼ 4 Hz, 2H), 3.15e3.07 (m, 1H),
2.94 (s, 3H); 13C NMR (CDCl3):
170.83, 158.00, 133.15, 133.10,
d
NMR (CDCl3):
d
8.06 (s, 3H), 7.88 (d, J ¼ 8 Hz, 1H), 7.78 (d, J ¼ 8 Hz,
130.42, 129.33, 127.26, 127.17, 123.18, 118.08, 102.60, 55.51, 52.97,
40.10, 34.44, 32.81, 32.74; HRMS (TOF) m/z calcd. for C18H22N2O4S
[M þ Hþ] 363.1379, found: 363.1381.
1H), 7.41e7.38 (m,1H), 7.33e7.29 (m, 2H), 7.22 (dd, J1 ¼ 8 Hz,
J2 ¼ 4 Hz,1H), 3.95 (s, 3H), 3.36e3.31 (m, 2H), 3.11e3.08 (m, 2H), MS
(TOF) m/z calcd. for C12H13NO [M þ Hþ] 188.1, found: 188.2.
5.2.5.2. 1-(N0,N0-dimethylsulfamoyl)-N-(2-(7-methoxynaphthalen-1-
yl)ethyl)azetidine-3-carboxamide (6b). Following general proce-
dure to obtain pure product as white solid 440 mg (yield: 72%). mp:
5.2.3. Tert-butyl 3-((2-(7-methoxynaphthalen-1-yl)ethyl)
carbamoyl)azetidine-1-carboxylate (4)
99.4e101.1 ꢂC; 1H NMR (CDCl3):
d
7.77 (d, J ¼ 8 Hz, 1H), 7.69 (t,
Compound
12.63 mmol) were dissolved in CH2Cl2 (30 mL) and DMF (3 mL). 1-
(tert-butoxycarbonyl)azetidine-3-carboxylic acid (940 mg,
4.63 mmol), EDC (970 mg, 5.05 mmol), HOBt (682 mg, 5.05 mmol),
DMAP (154 mg, 1.26 mmol) were added. After stirring at room
temperature for 24 h, the reaction mixture was diluted with 100 mL
CH2Cl2 and washed with H2O (30 mL ꢁ 3), 1 N HCl (30 mL ꢁ 3),
NaHCO3 (30 mL ꢁ 3) and brine (30 mL ꢁ 3). The organic layer was
dried over Na2SO4, filtered and concentrated under reduced pres-
sure to give pink solid (1.49 g, yield: 92%). mp: 56.5e58.8 ꢂC; 1H
3 (1.00 g, 4.21 mmol) and DIEA (2.20 ml,
J ¼ 4 Hz, 1H), 7.41 (d, J ¼ 4 Hz, 1H), 7.28e7.27 (m, 2H), 7.18 (dd,
J1 ¼ 8 Hz, J2 ¼ 4 Hz, 1H), 5.65 (s, 1H), 4.04e3.96 (m, 7H), 3.69 (q,
J ¼ 8 Hz, 2H), 3.28 (t, J ¼ 8 Hz, 2H), 3.18e3.11 (m, 1H), 2.81 (s, 6H);
13C NMR (CDCl3):
d 170.97, 158.01, 133.30, 133.13, 130.35, 129.32,
127.1, 123.17, 118.20, 102.51, 55.53, 53.50, 40.05, 38.18, 33.66, 32.90;
HRMS (TOF) m/z calcd. for C19H25N3O4S [M þ Naþ] 414.1463, found:
414.1460.
5.2.5.3. N-(2-(7-methoxynaphthalen-1-yl)ethyl)-1-tosylazetidine-3-
carboxamide (6c). Following general procedure to obtain pure
product as white solid 568 mg (yield: 83%). mp: 143.3e144.7 ꢂC; 1H
NMR (CDCl3):
d
7.75 (d, J ¼ 12 Hz, 1H), 7.68e7.65 (m, 1H), 7.42 (d,
J ¼ 4 Hz, 1H), 7.27e7.24(m 2H), 7.16 (dd, J ¼ 8 Hz, J ¼ 4 Hz, 1H), 6.06
(s, 1H), 4.11e3.98 (m, 4H), 3.96 (s, 3H), 3.64 (dd, J ¼ 12 Hz, J ¼ 4 Hz,
2H), 3.24 (t, J ¼ 8 Hz, 2H), 3.12e3.09 (m, 1H), 1.43 (s, 9H); MS (TOF)
m/z calcd. for C22H28N2O4 [M þ Hþ] 385.2, found: 385.4.
NMR (CDCl3):
d 7.38e7.35 (m, 3H), 7.23e7.15 (m, 3H), 5.56 (s, 1H),
3.93 (s, 3H), 3.90e3.82 (m, 4H), 3.57 (q, J ¼ 8 Hz, 2H), 3.17 (t,
J ¼ 4 Hz, 2H), 3.04e2.97 (m, 1H), 2.45 (s, 3H); 13C NMR (CDCl3):
d
170.21, 157.98, 144.43, 133.19, 133.07, 130.97, 130.35, 129.92,
5.2.4. N-(2-(7-methoxynaphthalen-1-yl)ethyl)azetidine-3-
carboxamide hydrochloride (5)
129.30, 128.46, 127.19, 127.11, 118.18, 102.47, 55.50, 53.14, 39.95,
33.21, 32.88, 21.67; HRMS (TOF) m/z calcd. for C24H26N2O4S
[M þ Hþ] 439.1692, found: 439.1692.
Compound 4 (823 mg, 2.14 mmol) was dissolved in 1 mL CH2Cl2.
Then TFA (1.8 mL,15.16 mmol) was added dropwise. After stirring at
room temperature for 3 h, adjust to pH ¼ 10 with 10% NaOH so-
lution under ice-bath condition. Then the reaction mixture was
diluted with 100 mL CH2Cl2 and washed with H2O (30 mL ꢁ 1) and
brine (30 mL ꢁ 1). The organic layer was dried over Na2SO4 and
filtered. Then treated with saturated solution of hydrogen chloride
in Et2O to pH ¼ 1 and concentrated under reduced pressure to
afford white solid (515 mg, yield: 75%). mp: 175e176.5 ꢂC; 1H NMR
5.2.5.4. 1-((4-chlorophenyl)sulfonyl)-N-(2-(7-methoxynaphthalen-
1-yl)ethyl) azetidine-3-carboxamide (6d). Following general proce-
dure to obtain pure product as white solid 516 mg (yield: 72%). mp:
152.5e154.6 ꢂC, 1H NMR (CDCl3):
d 7.79e7.76 (m, 3H), 7.68 (d,
J ¼ 8 Hz, 1H), 7.55 (d, J ¼ 8 Hz, 2H), 7.33 (d, J ¼ 4 Hz, 1H), 7.27e7.16
(m, 2H), 5.48 (s, 1H), 3.94 (s, 3H), 3.93e3.84 (m, 4H), 3.59 (q,
J ¼ 4 Hz, 2H), 3.19 (t, J ¼ 8 Hz, 2H), 3.01e2.98 (m,1H); 13C NMR
(DMSO-d6):
d
9.54 (s, 1H), 8.99 (s, 1H), 8.56 (t, J ¼ 8 Hz, 1H), 7.85 (d,
(CDCl3):
d 169.90, 157.41, 138.55, 133.79, 132.73, 132.42, 130.10,
J ¼ 12 Hz, 1H), 7.72 (d, J ¼ 8 Hz, 1H), 7.56 (d, J ¼ 4 Hz, 1H), 7.34e7.25
(m, 2H), 7.18 (dd, J1 ¼ 8 Hz, J2 ¼ 4 Hz, 1H), 3.99e3.94 (m, 7H),
3.61e3.52 (m, 1H), 3.44e3.39 (m, 2H), 3.16 (t, J ¼ 8 Hz, 2H); 13C
130.07, 129.58, 128.74, 126.99, 126.55, 123.04, 117.92, 102.52, 55.20,
53.01, 32.71, 31.64; HRMS (TOF) m/z calcd. for C23H23ClN2O4S
[M þ Naþ] 481.0965, found: 481.0969.
NMR (DMSO-d6):
d 169.76, 157.44, 133.84, 132.74, 130.13, 128.77,
127.09, 126.58, 123.11, 117.88, 102.58, 55.33, 46.97, 34.88, 32.72;
HRMS (TOF) m/z calcd. for C17H20N2O2 [M þ Hþ] 285.1603, found:
285.1597.
5.2.5.5. 1-(4-methoxybenzoyl)-N-(2-(7-methoxynaphthalen-1-yl)
ethyl)azetidine-3-carboxamide (6e). Following general procedure to
obtain pure product as white solid 554 mg (yield: 85%). mp:
65.3e67.1 ꢂC; 1H NMR (CDCl3):
d
7.77 (d, J ¼ 8 Hz,1H), 7.70e7.67 (m,
5.2.5. General procedure for the synthesis of N-substituted
analogues 6ael
1H), 7.60 (dt, J ¼ 8 Hz, J ¼ 4 Hz, 2H), 7.41 (d, J ¼ 4 Hz,1H), 7.27 (s,1H),
7.17 (dd, J1 ¼ 8 Hz, J2 ¼ 4 Hz, 1H), 6.90 (dt, J1 ¼ 8 Hz, J2 ¼ 4 Hz, 2H),
5.67 (s, 1H), 4.53 (brs, 1H), 4.30 (t, J ¼ 8 Hz, 2H), 4.23 (brs, 1H), 3.97
(s, 3H), 3.84 (s, 3H), 3.69 (q, J ¼ 8 Hz, 2H), 3.27 (t, J ¼ 8 Hz, 2H),
N-(2-(7-methoxynaphthalen-1-yl)ethyl)azetidine-3-
carboxamide hydrochloride (500 mg, 1.56 mmol) and DIPEA
(0.8 mL, 4.84 mmol) was dissolved in CH2Cl2 (10 mL), then different
substituted acyl chloride or sulfonyl chloride or anhydride
(3.12 mmol) was added dropwise under ice bath. After stirring at
room temperature for 24 h, the reaction mixture was diluted with
CH2Cl2 (20 mL), then washed with H2O (20 mL), NaHCO3 saturated
solution (20 mL) and brine (20 mL). The organic layer was dried
over Na2SO4, filtered and concentrated under reduced pressure and
the residue was purified by chromatography on silica gel to afford
respective compounds 6a-l.
3.24e3.17 (m, 1H); 13C NMR (CDCl3):
d 171.48, 169.94, 161.87, 157.98,
133.40, 133.15, 130.33, 129.80, 129.29, 127.13, 124.97,
123.16,118.20,113.66, 102.52, 55.54, 55.38, 51.55, 40.10, 32.92, 32.98;
HRMS (TOF) m/z calcd. for C25H26N2O4 [M þ Hþ] 419.1968, found:
419.1971.
5.2.5.6. 1-Acetyl-N-(2-(7-methoxynaphthalen-1-yl)ethyl)azetidine-
3-carboxamide (6f). Following general procedure to obtain pure
product as yellow oil 372 mg (yield: 73%). 1H NMR (CDCl3):
d 7.77 (d,
5.2.5.1. N-(2-(7-methoxynaphthalen-1-yl)ethyl)-1-(methylsulfonyl)
azetidine-3-carbox-amide (6a). Following general procedure to
obtain pure product as white solid 452 mg (yield: 80%). mp:
J ¼ 8 Hz, 1H), 7.69 (t, J ¼ 4 Hz, 1H), 7.41 (d, J ¼ 4 Hz, 1H), 7.18 (dd,
J1 ¼ 8 Hz, J2 ¼ 4 Hz, 1H), 5.80 (brs, 1H), 4.32 (t, J ¼ 8 Hz, 1H), 4.14 (t,
J ¼ 8 Hz, 1H), 4.05 (d, J ¼ 8 Hz, 2H), 3.97 (s, 3H), 3.68e3.67 (m, 2H),
3.28e3.25 (m, 2H), 3.15e3.08 (m, 1H), 1.84 (s, 3H); 13C NMR
148.5e149.7 ꢂC; 1H NMR (CDCl3):
d
7.78 (d, J ¼ 8 Hz, 1H), 7.71e7.69
(m, 1H), 7.39 (d, J ¼ 4 Hz, 1H), 7.27 (d, J ¼ 4 Hz, 1H), 7.18 (dd,
(CDCl3):
d 171.36, 170.64, 157.97, 133.39, 133.14, 130.34, 129.30,
J1 ¼12 Hz, J2 ¼ 8 Hz, 1H), 5.62 (s, 1H), 4.04 (d, J ¼ 8 Hz, 4H), 3.97 (s,
127.13, 123.16, 118.14, 102.57, 55.53, 52.51, 50.64, 40.09, 32.91, 32.55,

270
W. Ang et al. / European Journal of Medicinal Chemistry 82 (2014) 263e273
18.52; HRMS (TOF) m/z calcd. for C19H22N2O3 [M þ Hþ] 326.1709,
5.2.5.12. N-(2-(7-methoxynaphthalen-1-yl)ethyl)-N0,N0-dimethylaze-
tidine-1,3-dicarboxamide (6l). Following general procedure to
obtain pure product as yellow oil 382 mg (yield: 69%). 1H NMR
found: 326.1709.
(CDCl3):
d
7.77 (d, J ¼ 8 Hz, 1H), 7.70e7.67 (m, 1H), 7.42 (d, J ¼ 4 Hz,
5 . 2 . 5 . 7 . N - ( 2 - ( 7 - m e t h o x y n a p h t h a l e n - 1 - yl ) e t hyl ) - 1 -
propionylazetidine-3-carboxamide (6g). Following general proce-
dure to obtain pure product as yellow oil 370 mg (yield: 70%). 1H
1H), 7.28 (s, 1H), 7.17 (dd, J1 ¼ 8 Hz, J2 ¼ 4 Hz, 1H), 5.66 (s, 1H),
4.09e4.03 (m, 4H), 3.98 (s, 3H), 3.68 (q, J ¼ 8 Hz, 2H), 3.27 (t,
J ¼ 8 Hz, 2H), 3.16e3.08 (m,1H); 13C NMR (DMSO-d6):
d 171.67,
NMR (CDCl3):
d
7.78 (d, J ¼ 12 Hz, 1H), 7.69 (t, J ¼ 4 Hz, 1H), 7.41 (d,
162.22, 157.42, 134.02, 132.83, 130.08, 128.78, 127.01, 126.52, 123.08,
117.85, 102.64, 55.22, 54.88, 52.93, 36.43, 32.76; HRMS (TOF) m/z
calcd. for C20H25N3O3 [M þ Naþ] 378.1794, found: 378.1788.
J ¼ 4 Hz, 1H), 7.28 (d, J ¼ 4 Hz, 1H), 7.18 (dd, J1 ¼ 8 Hz, J2 ¼ 4 Hz, 1H),
5.56 (s, 1H), 4.31 (t, J ¼ 8 Hz, 1H), 4.16e4.06 (m, 3H), 3.98 (s, 3H),
3.72e3.66 (m, 2H), 3.29e3.25 (m, 2H), 3.17e3.09 (m, 1H), 2.12e2.05
(m, 2H), 1.60e1.59 (m, 2H), 1.12 (t, J ¼ 4 Hz, 3H); 13C NMR (DMSO-
d6):
d 172.73, 171.48, 157.41, 133.99, 132.81, 130.10, 128.77, 127.02,
5.2.6. General procedure for the synthesis of N-substituted
analogues 6met
126.53, 123.09, 117.85, 102.63, 55.25, 51.95, 49.94, 32.71, 31.84,
23.50, 8.62; HRMS (TOF) m/z calcd. for C20H24N2O3 [M þ Hþ]
341.1865, found: 341.1865.
N-(2-(7-methoxynaphthalen-1-yl)ethyl)azetidine-3-
carboxamide hydrochloride (800 mg, 2.50 mmol) and DIPEA
(1.5 mL, 9.08 mmol) was dissolved in CH2Cl2 (20 mL) and DMF
(3 mL), then different substituted carboxylic acid (2.74 mmol), EDC
(574 mg, 2.99 mmol), HOBt (404 mg, 2.99 mmol) and DMAP (91 mg,
0.748 mmol) was added. After stirring at room temperature for
24 h, the reaction mixture was diluted with CH2Cl2 (20 mL), then
washed with H2O (20 mL), 10% HCl (20 mL), NaHCO3 saturated
solution (20 mL) and brine (20 mL). The organic layer was dried
over Na2SO4, filtered and concentrated under reduced pressure and
the residue was purified by chromatography on silica gel to afford
respective compounds 6met.
5.2.5.8. 1-Benzoyl-N-(2-(7-methoxynaphthalen-1-yl)ethyl)azetidine-
3-carboxamide (6h). Following general procedure to obtain pure
product as white solid 424 mg (yield: 70%). mp: 55.5e57.3 ꢂC; 1H
NMR (CDCl3):
d
7.77 (d, J ¼ 8 Hz, 1H), 7.70e7.67 (m, 1H), 7.62 (dt,
J1 ¼ 8 Hz, J2 ¼ 4 Hz, 2H), 7.49e7.39 (m, 4H), 7.27 (s, 1H), 7.16 (dd,
J1 ¼ 8 Hz, J2 ¼ 4 Hz, 1H), 5.65 (s, 1H), 4.51 (t, J ¼ 4 Hz, 1H), 4.30e4.27
(m, 3H), 3.97 (s, 3H), 3.69 (q, J ¼ 8 Hz, 2H), 3.29e3.25 (m, 2H),
3.24e3.17 (m, 1H); 13C NMR (DMSO-d6):
d 171.41, 168.85, 157.41,
134.00, 132.82, 130.98, 130.09, 128.78, 128.41, 127.64, 127.03, 126.53,
123.08, 117.84, 102.65, 55.22, 54.88, 51.01, 32.76, 32.68; HRMS (TOF)
m/z calcd. for C24H24N2O3 [M þ Naþ] 411.1685, found: 411.1681.
5.2.6.1. 1-(3,5-dinitrobenzoyl)-N-(2-(7-methoxynaphthalen-1-yl)
ethyl)azetidine-3-carboxamide (6m). Following general procedure
to obtain pure product as white solid 897 mg (yield: 75%). mp:
147.8e149.9 ꢂC; 1H NMR (DMSO-d6): 8.91 (s, 1H), 8.67 (d, J ¼ 4 Hz,
2H), 8.28e8.25 (m, 1H), 7.80 (d, J ¼ 8 Hz, 1H), 7.68 (d, J ¼ 8 Hz, 1H),
7.52 (d, J ¼ 4 Hz,1H), 7.30e7.22 (m, 2H), 7.64 (dd, J1 ¼8 Hz, J2 ¼ 4 Hz,
1H), 5.73 (s, 1H), 4.48 (t, J ¼ 8 Hz, 1H), 4.31 (t, J ¼ 8 Hz, 1H), 4.20 (t,
J ¼ 8 Hz,1H), 4.12e4.08 (m,1H), 3.91 (s, 3H), 3.41e3.36 (m, 3H), 3.14
5.2.5.9. N-(2-(7-methoxynaphthalen-1-yl)ethyl)-1-(4-(tri-
fluoromethyl)benzoyl) azetidine-3-carboxamide (6i). Following
general procedure to obtain pure product as white solid 520 mg
(yield: 73%). mp: 76.3e78 ꢂC; 1H NMR (CDCl3):
d 7.78e7.67 (m, 6H),
7.40 (d, J ¼ 4 Hz, 1H), 7.27 (s, 1H), 7.18 (dd, J1 ¼12 Hz, J2 ¼ 4 Hz, 1H),
5.57 (s, 1H), 4.50 (t, J ¼ 8 Hz, 1H), 4.34e4.23 (m, 3H), 3.97 (s, 3H),
3.70 (q, J ¼ 8 Hz, 2H), 3.27 (td, J1 ¼8 Hz, J2 ¼ 4 Hz, 2H), 3.25e3.17 (m,
(t, J ¼ 8 Hz, 2H); 13C NMR (DMSO-d6):
d 171.06, 164.31, 157.38,
1H); 13C NMR (CDCl3):
d 171.07, 168.75, 158.00, 136.11, 133.24,
148.07, 135.68, 133.95, 132.79, 130.07, 128.74, 127.68, 127.04, 126.53,
123.08, 120.40, 117.80, 115.47, 102.60, 55.20, 51.46, 40.08, 32.77,
32.63; HRMS (TOF) m/z calcd. for C24H22N4O7 [M þ Naþ] 501.1386,
found: 501.1388.
133.12, 133.02, 132.70, 130.40, 129.32, 128.22, 127.22, 127.15, 125.50,
125.44, 125.00, 123.16, 122.29, 55.51, 55.16, 51.62, 40.11, 33.80,
32.85; HRMS (TOF) m/z calcd. for C25H23F3N2O3 [M þ Hþ] 457.1739,
found: 457.1738.
5.2.6.2. 1-(3,5-dimethoxybenzoyl)-N-(2-(7-methoxynaphthalen-1-
yl)ethyl)azetidine-3-carboxamide (6n). Following general proce-
dure to obtain pure product as white solid 695 mg (yield: 62%). mp:
5.2.5.10. Benzyl 3-((2-(7-methoxynaphthalen-1-yl)ethyl)carbamoyl)
azetidine-1-carboxylate (6j). Following general procedure to obtain
pure product as white solid 464 mg (yield: 71%). 1H NMR (CDCl3):
55.1e57 ꢂC; 1H NMR (CDCl3):
d
7.77 (d, J ¼ 8 Hz,1H), 7.69 (t, J ¼ 4 Hz,
1H), 7.41 (d, J ¼ 4 Hz, 1H), 7.27 (s, 1H), 7.17 (dd, J1 ¼ 8 Hz, J2 ¼ 4 Hz,
1H), 6.75 (d, J ¼ 4 Hz, 2H), 6.55e6.54 (m, 1H), 5.59 (s, 1H), 4.50 (brs,
1H), 4.28 (t, J ¼ 8 Hz, 3H), 3.97(s, 3H), 3.84e3.78 (m, 6H), 3.69e3.67
(m, 2H), 3.27 (t, J ¼ 8 Hz, 2H), 3.21e3.19 (m, 1H); 13C NMR (CDCl3):
d
7.77 (d, J ¼ 12 Hz,1H), 7.70e7.67 (m,1H), 7.40 (d, J ¼ 4 Hz,1H), 7.38
(s, 1H), 7.33e7.28 (m, 2H), 7.25 (d, J ¼ 4 Hz, 3H), 7.17 (dd, J1 ¼ 8 Hz,
J2 ¼ 4 Hz, 1H), 5.52 (s, 1H), 4.16e4.14 (m, 2H), 4.07 (t, J ¼ 8 Hz, 2H),
3.97 (s, 3H), 3.68 (q, J ¼ 8 Hz, 2H), 3.26 (t, J ¼ 8 Hz, 2H), 3.15e3.08
(m, 1H); 13C NMR (DMSO-d6):
d 171.36, 157.42, 155.64, 136.84,
d
171.45, 168.64, 157.98, 153.59, 150.13, 133.40, 133.12, 130.30,
129.29, 127.09, 123.90, 123.14, 118.19, 117.01, 114.04, 112.75, 102.50,
56.29, 55.82, 55.52, 53.07, 51.13, 40.05, 33.49, 33.03; HRMS (TOF) m/
z calcd. for C26H28N2O5 [M þ Naþ] 471.1896, found: 471.1897.
133.98, 132.82, 130.09, 128.78, 128.38, 127.85, 127.60, 127.02, 126.54,
123.08, 117.85,102.64, 65.62, 55.21, 51.89, 51.32, 32.70, 32.50; HRMS
(TOF) m/z calcd. for C25H26N2O4 [M þ Naþ] 441.1790, found:
441.1790.
5.2.6.3. 1-(4-chlorobenzoyl)-N-(2-(7-methoxynaphthalen-1-yl)ethyl)
azetidine-3-carboxamide (6o). Following general procedure to
obtain pure product as white solid 761 mg (yield: 72%). mp:
5.2.5.11. N-(2-(7-methoxynaphthalen-1-yl)ethyl)-1-(2,2,2-
trifluoroacetyl)azetidine-3-carboxamide (6k). Following general
procedure to obtain pure product as colorless oil 433 mg (yield:
47.9e51.3 ꢂC; 1H NMR (CDCl3):
d
7.77 (d, J ¼ 8 Hz, 1H), 7.69 (t,
73%). 1H NMR (CDCl3):
d
7.78 (d, J ¼ 8 Hz, 1H), 7.71 (d, J ¼ 4 Hz, 1H),
J ¼ 4 Hz, 1H), 7.56 (d, J ¼ 8 Hz, 2H), 7.40e7.38 (m, 3H), 7.27 (s, 1H),
7.18 (dd, J1 ¼ 8 Hz, J2 ¼ 4 Hz, 1H), 5.58 (s, 1H), 4.50 (brs, 1H), 4.26 (s,
3H), 3.97 (s, 3H), 3.70 (q, J ¼ 8 Hz, 2H), 3.29e3.26 (m, 2H), 3.23e3.16
7.39 (s, 1H), 7.19 (d, J ¼ 8 Hz, 1H), 5.59 (s, 1H), 4.59 (brs, 1H), 4.43 (t,
J ¼ 8 Hz, 1H), 4.22e4.17 (m, 2H), 3.97 (s, 3H), 3.70 (d, J ¼ 8 Hz, 2H),
3.28e3.22 (m, 3H); 13C NMR (DMSO-d6):
d
170.65, 169.84, 157.42,
(m, 1H); 13C NMR (CDCl3):
d 171.19, 169.08, 158.00, 137.40, 133.28,
133.96, 133.80, 132.78, 128.77, 127.07, 126.55, 123.09, 117.85, 102.61,
133.14, 131.10, 130.37, 129.32, 129.28, 128.74, 127.19, 127.14, 123.16,
118.16, 102.55, 55.53, 55.30, 51.58, 40.11, 33.83, 32.89; HRMS (TOF)
m/z calcd. for C24H23ClN2O3 [M þ Hþ] 423.1475, found: 423.1475.
55.25, 54.28, 51.34, 47.28, 34.91, 32.60; HRMS (TOF) m/z calcd. for
C
19H19F3N2O3 [M þ Naþ] 403.1245, found: 403.1262.

W. Ang et al. / European Journal of Medicinal Chemistry 82 (2014) 263e273
271
5.2.6.4. 1-(Furan-2-carbonyl)-N-(2-(7-methoxynaphthalen-1-yl)
5.3. Biological assay
ethyl)azetidine-3-carboxamide (6p). Following general procedure
to obtain pure product as white solid 681 mg (yield: 72%). mp:
5.3.1. Cytotoxicity measurement
140.1e142.4 ꢂC; 1H NMR (CDCl3):
d
7.77 (d, J ¼ 12 Hz, 1H), 7.70e7.66
L02 cells purchased from the cell Bank of the Chinese Academy
of Science and human embryonic kidney cell line 293 cells pur-
chased from the American Type Culture Collection were respec-
tively seeded in 96-well plates at a density of 5 ꢁ 103 cells per well
and cultured in the medium consisted of 90% 1640 or DMEM, 10%
fetal calf serum, penicillin 200 kU/L, and streptomycin 100 mg/L in a
humidified incubator with 5% CO2 for 24 h. At this point, Cells were
exposed to different concentrations of compounds for another day.
The viability of cells was evaluated using MTT assay. Briefly, the
cells were incubated with MTT (final concentration, 0.5 mg/ml) for
another 4 h at 37 ꢂC. After incubation, the medium were removed
(m,1H), 7.48 (d, J ¼ 0.8 Hz,1H), 7.42 (d, J ¼ 4 Hz,1H), 7.27 (s,1H), 7.17
(dd, J1 ¼ 8 Hz, J2 ¼ 4 Hz, 1H), 7.04 (d, J ¼ 4 Hz, 1H), 6.47 (m, 1H), 5.71
(s,1H), 4.70 (brs,1H), 4.57 (t, J ¼ 8 Hz,1H), 4.25 (d, J ¼ 8 Hz, 2H), 3.97
(s, 3H), 3.69 (q, J ¼ 8 Hz, 2H), 3.30e3.21 (m, 3H); 13C NMR (CDCl3):
d
171.45, 158.70, 157.98, 147.64, 144.68, 133.43, 133.16, 130.30,
129.29, 127.11, 123.14, 118.18, 115.47, 111.57, 102.50, 55.51, 54.71,
51.12, 40.14, 34.20, 32.97; HRMS (TOF) m/z calcd. for C22H22N2O4
[M þ Naþ] 401.1477, found: 401.1475.
5.2.6.5. N-(2-(7-methoxynaphthalen-1-yl)ethyl)-1-picolinoyla-
zetidine-3-carboxamide (6q). Following general procedure to obtain
pure product as white solid 486 mg (yield: 50%). mp: 168e169 ꢂC; 1H
and 150 mL of DMSO was added to each well for 15 min at room
temperature and absorbance measured at 570 nm using a micro-
plate reader.
NMR (CDCl3):
d
8.56 (d, J ¼ 4 Hz,1H), 8.09 (d, J ¼ 4 Hz, 1H), 7.82e7.67
(m, 3H), 7.43 (d, J ¼ 4 Hz, 1H), 7.38e7.27 (m, 2H), 7.16 (dd, J1 ¼ 8 Hz,
J2 ¼ 4 Hz,1H), 5.67 (s,1H), 4.87e4.79 (m, 2H), 4.37e4.28 (m, 2H), 3.97
(s, 1H), 3.71e3.66 (m, 2H), 3.29e3.21 (m, 3H); 13C NMR (CDCl3):
5.3.2. Corticosterone-induced PC12 cells lesion and detection of cell
viability with MTT assay [39,40]
PC12 cells were plated at a density of 1 ꢁ104 cells per well in 96-
well plates and cultured in the medium consisting of 90% Dulbec-
co's Modified Eagle’s Medium (DMEM), 5% heat-inactivated horse
serum, 10% fetal calf serum (FBS) for 24 h. Later, the PC12 cells were
d
171.73, 164.96, 158.00, 151.64, 148.10, 136.79, 133.39, 133.14, 130.30,
129.30, 127.13, 125.40, 123.65, 123.14, 118.25, 102.45, 57.27, 55.53,
51.35, 40.01, 34.04, 33.04; HRMS(TOF) m/z calcd. For C23H23N3O3
[M þ Naþ] 412.1637, found: 412.1637.
treated with 200 mM corticosterone and Fluoxetine, Agomelatine or
other compounds at different concentrations for 1 day. After in-
cubation, cells were treated with the MTT solution (final concen-
tration 0.5 mg/ml) for 4 h. The dark blue formazan crystals formed
in intact cells were solubilized with DMSO and absorbance at
570 nm was measured with a microplate reader.
5.2.6.6. 1-(3-chloroisonicotinoyl)-N-(2-(7-methoxynaphthalen-1-yl)
ethyl)azetidine-3-carboxamide (6r). Following general procedure to
obtain pure product as white solid 530 mg (yield: 50%). mp:
65.8e67.8 ꢂC; 1H NMR (CDCl3): 7.77 (d, J ¼ 8 Hz, 1H), 7.71e7.68
(m,1H), 7.50 (s, 1H), 7.39e7.38 (m, 2H), 7.29e7.27 (m, 1H), 7.18 (dd,
J1 ¼ 8 Hz, J2 ¼ 4 Hz, 1H), 5.57 (s, 1H), 4.49 (t, J ¼ 4 Hz, 1H), 4.31e4.23
(m, 3H), 3.97 (s, 3H), 3.70 (q, J ¼ 4 Hz, 2H), 3.28 (td, J1 ¼ 8 Hz,
5.3.3. Morphological observation
PC12 cells were seeded in 6-well plates at a density of
1 ꢁ 106 cells/mL in the growth medium for 1 d, and then the cells
were classified into following groups: control, corticosterone
J2 ¼ 4 Hz, 2H), 3.24e3.17 (m, 1H); 13C NMR (CDCl3):
d 170.76, 166.07,
157.99, 152.20, 150.33, 143.10, 133.20, 133.13, 130.41, 129.31, 127.24,
127.16, 123.16, 122.65, 120.43, 118.21, 102.58, 55.53, 54.99, 51.74,
40.15, 33.79, 32.77; HRMS (TOF) m/z calcd. for C23H22ClN3O3
[M þ Hþ] 424.1428, found: 424.1429.
0.2 mM, corticosterone 0.2 mM þ Agomelatine 1.25
sterone 0.2 mM þ Fluoxetine 1.25 M, corticosterone 0.2 mM þ 6k
1.25 M. After incubated
mM, cortico-
m
m
M and corticosterone 0.2 mM þ 6o 1.25
m
with for another 24 h, the cells were photographed by a digital
camera attached to an inverted microscope. Images from three
fields were randomly taken in each group.
5.2.6.7. 1-(2,5-dimethoxybenzoyl)-N-(2-(7-methoxynaphthalen-1-
yl)ethyl)azetidine-3-carboxamide (6s). Following general procedure
to obtain pure product as white solid 807 mg (yield: 72%). mp:
5.3.4. H2O2-induced PC12 cells lesion and detection of cell viability
with MTT assay [27,41,42]
PC12 cells were plated onto 96-well plates at a density of
1 ꢁ 104 cells/well and were cultured in DMEM with 10% FBS, 5%
horse serum for 24 h. Then, after removing the original medium,
the cells were cultured for another 1 h in the presence of 200 mM
H2O2. Later, the cells were washed with phosphate-buffered saline
(PBS) twice and cultured with or tested compounds at three
58.9e61.8 ꢂC; 1H NMR (CDCl3):
d
7.76 (d, J ¼ 8 Hz, 1H), 7.68 (t,
J ¼ 4 Hz, 1H), 7.41 (d, J ¼ 4 Hz, 1H), 7.25 (s, 1H), 7.17 (dd, J1 ¼ 8 Hz,
J2 ¼ 4 Hz,1H), 6.94e6.89 (m, 2H), 6.84 (d, J ¼ 12 Hz,1H), 5.69 (s,1H),
4.30e4.18 (m, 3H), 4.03 (t, J ¼ 8 Hz, 1H), 3.96 (s, 3H), 3.79 (s, 3H),
3.77 (s, 3H), 3.70e3.64 (m, 2H), 3.25 (t, J ¼ 8 Hz, 2H), 3.21e3.14 (m,
1H); 13C NMR (CDCl3):
d 171.45, 168.64, 157.98, 153.59, 150.13,
133.40, 133.12, 130.30, 129.29, 127.09, 123.90, 123.14, 118.19, 117.01,
114.04, 112.75, 102.50, 56.29, 55.82, 55.52, 53.07, 51.13, 40.05, 33.49,
33.03; HRMS (TOF) m/z calcd. for C26H28N2O5 [M þ Naþ] 471.1896,
found: 471.1896.
different concentrations (10 mM, 20 mM and 40 mM) in DMEM for
48 h, Fluoxetine and Agomelatine were used as positive control.
After incubation, cell viability was determined by MTT assay.
Briefly, cells were cocultured with MTT (final concentration 0.5 mg/
ml) for 4 h. The dark blue formazan crystals formed in intact cells
were solubilized with DMSO and measured with a microplate
reader at a wavelength of 570 nm.
5.2.6.8. 1-(4-cyanobenzoyl)-N-(2-(7-methoxynaphthalen-1-yl)ethyl)
azetidine-3-carboxamide (6t). Following general procedure to
obtain pure product as white solid 765 mg (yield: 74%). mp:
75.6e77.5 ꢂC; 1H NMR (CDCl3):
d
7.80 (d, J ¼ 8 Hz, 1H), 7.74 (s, 4H),
7.41 (s, 1H), 7.28 (s, 3H), 7.20 (d, J ¼ 8 Hz, 1H), 5.58 (s, 1H), 4.52 (brs,
1H), 4.33e4.29 (m, 3H), 3.99 (s, 3H), 3.72 (d, J ¼ 8 Hz, 2H), 3.30 (brs,
5.3.5. Forced swim test
C57 mice were assigned into 7 groups (n ¼ 10): Fluoxetine group
(32 mg/kg), Agomelatine group (32 mg/kg), vehicle group (32 mg/
kg), 6d group (32 mg/kg), 6k group (32 mg/kg), 6o group (32 mg/
kg) and 6p group (32 mg/kg). Every group had 10 mice.
Forced Swim Test (FST) was conducted as described previously,
with some modifications [43]. Each mouse was plunged
2H), 3.24e3.23 (m, 1H); 13C NMR (DMSO-d6):
d 170.96, 168.12,
158.00, 136.86, 133.20, 133.12, 132.31, 130.41, 129.33, 128.46, 127.25,
127.14, 123.16, 118.12, 118.03, 114.70, 102.57, 55.53, 55.11, 51.73,
40.11, 33.80, 32.82; HRMS (TOF) m/z calcd. for
C25H23N3O3
[M þ Naþ] 436.1637, found: 436.1624.

272
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Acknowledgements
This work was supported by New Century Talent Fund by
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