Synthesis and evaluation of amide side-chain modified Agomelatine analogues as potential antidepressant-like agents

Article abstract of DOI:10.1016/j.bmcl.2014.02.065

In this work, nineteen analogues of Agomelatine were readily synthesized through structural modification of the acetamide side-chain starting from the key common intermediate 2-(7-methoxynaphthalen-1-yl) ethanamine (3), which was prepared from commerciall

Full text of DOI:10.1016/j.bmcl.2014.02.065

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1672–1676
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of amide side-chain modified Agomelatine
analogues as potential antidepressant-like agents
Ying Chang a, , Weiyi Pi a, , Wei Ang b, Yuanyuan Liu a, Chunlong Li c, Jiajia Zheng c, Li Xiong b, Tao Yang a,
Youfu Luo a,
⇑
a National Key Laboratory of Biotherapy, 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
c Pharmaceutical and Biological Engineering Department, Institute for Chemical Engineering, 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 work, nineteen analogues of Agomelatine were readily synthesized through structural modification
of the acetamide side-chain starting from the key common intermediate 2-(7-methoxynaphthalen-1-yl)
ethanamine (3), which was prepared from commercially available compound 2-(7-methoxynaphthalen-
1-yl) acetonitrile (2) in two steps. Corticosterone-induced PC12 pheochromocytoma cells phenotypic
in vitro model was utilized to evaluate their potential antidepression activities. Imide compound 4a and
acylamino carboxylic acid analogue 5b showed good protective effects on traumatic PC12 cells with pro-
tection rates of 34.2% and 23.2%, respectively. Further in vivo assessments in C57 mice FST (forced swim
test) model demonstrated that compound 4a significantly reduced the immobility time of the tested sub-
jects, indicating antidepressant-like activity. Preliminary toxicity assays conducted on human normal liver
L02 cells and embryonic kidney 293 cells suggested a relatively low safety risk for compound 4a compared
with the marketed drugs Agomelatine and Fluoxetine. The promising antidepressant-like efficacy of com-
pound 4a, together with the relatively low toxicity to the normal tested cells and high liability of diffusion
through the blood–brain barrier (BBB), presents us insights of exploration of me-better drug candidates of
Agomelatine.
Received 17 December 2013
Revised 19 February 2014
Accepted 21 February 2014
Available online 5 March 2014
Keywords:
Agomelatine
Analogues
PC12 cells
Corticosterone
Forced swim test
Ó 2014 Elsevier Ltd. All rights reserved.
Depression, with high prevalence worldwide, is a mental disor-
der, characterized by sadness, loss of interest or pleasure, feelings
of guilt or low self-worth, disturbed sleep or appetite, feelings of
tiredness, and poor concentration.1 By 2020, according to the
WHO report, depression will be the second disease next to ische-
mic heart disease and become one of the major contributors to
the global disease burden.2
The current antidepressant drugs in clinic can generally be
classified into several categories, which include selective serotonin
reuptake inhibitors (SSRIs), monoamine oxidase inhibitors
(MAOIs), serotonin–norepinephrine reuptake inhibitors (SNRIs),
noradrenaline reuptake inhibitors (NRIs), tricyclic antidepressants
(TCAs) and dopamine–noradrenaline reuptake inhibitors (DNRIs)
(Fig. 1).3,4 Although these antidepressants are often helpful, their
full efficacy is only apparent after several weeks of administration
and many patients only partially respond, and some remain
refractory and severe side effects.5 Thus to search for new candi-
date agents with novel action mechanism is strongly desirable.
Agomelatine was approved for the treatment of major depres-
sion disorders by European Medicines Agency in 2009.6 However,
cases of liver injury, including hepatic failure,7,8 elevations of liver
enzymes exceeding 10 times the upper limit of normal, hepatitis
and jaundice have been reported in patients treated with Agomel-
atine during the first months of the treatment. Although the serum
transaminases usually returned to normal when discontinued use
of Agomelatine, there is still a need for structural modification of
Agomelatine in order to enhance its efficacy and decrease its
toxicity.
In our continuing efforts to develop novel antidepression agents
with improved pharmacological profiles than the marketed ones,
we started a structural modification program of Agomelatine on
its amide side-chain. Two structural classes of Agomelatine bioi-
somers, imides (Scheme 1) and asymmetrical ureas (Scheme 3),
were synthesized and investigated for its potential effects on
in vitro and in vivo depression models. The synthesis and biological
assessment of the acylamino carboxylic acids (Scheme 2) were also
undertook herein in consideration that they are ring-opening
⇑
Corresponding author. Tel./fax: +86 28 85503817.
E-mail address: luo_youfu@scu.edu.cn (Y. Luo).
Authors contributed to this work equally.
http://dx.doi.org/10.1016/j.bmcl.2014.02.065
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

Y. Chang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1672–1676
1673
Figure 1. Chemical structures of representative antidepressants.
Scheme 1. Synthetic route of imide analogues 4a–e. Reagents and conditions: (i) NaBH4, (Boc)2O, NiCl2, MeOH; TFA, DCM; (ii) appropriate cyclic anhydride, NaOAc, HOAc,
reflux, 3 h.
to literature9 with minor revision. Firstly 2-(7-methoxynaphtha-
len-1-yl) acetonitrile was reduced to the corresponding amine
with sodium borohydride/nickel chloride system followed by
in situ N-Boc protection with dibutyldicarbonate in methanol.
After N-Boc deprotection of compound 2 with trifluoroacetic acid
in dichloromethane, the common key intermediate 3 was obtained.
It is worthwhile to point out that the in situ N-Boc protection pro-
cedure is quite necessary, otherwise the direct reduction product
would be contaminated with a mixture of side products such as
oxime, secondary amine and acyl amine, which would turn into
dark green when exposed to the atmosphere and was difficult to
purify. Compounds 4–6 were prepared according to reported pro-
cedure.10–13 The key intermediate 3 was reacted with the appropri-
ate cyclic anhydride in the presence of sodium acetate and acetic
acid under reflux for 3 h to obtained 4.
Scheme 2. Synthetic route of and acylamino carboxylic acid analogues 5a–f.
Reagents and conditions: (i) appropriate cyclic anhydride, CH2Cl2, rt, 6 h.
The compounds 5a–f can be prepared conveniently by stirring
2-(7-methoxynaphthalen-1-yl) ethanamine (3) with the appropri-
ate cyclic anhydride in the presence of sodium acetate and acetic
forms of the corresponding imides and easily prepared in similar
reaction conditions, which can bring us insights when compared
their effects with those of imides although acylamino carboxylic
acid may not be a good candidate for passing the BBB for the high
polarity of carboxylic acid group, which can be readily modified to
its ester or carbamate form if needed.
acid at room temperature for 6 h. For compounds 5a–e, the amino
group of compound 3 nucleophilic attack the carbon atom of car-
bonyl group in room temperature, formed target amide com-
pounds. When heated to reflux, the secondary amino group of
compounds 5a–f further nucleophilic attack another carbon atom
of carbonyl group, obtained 4a–e.
To achieve the synthesis of the target compounds 4a–e, the
steps outlined in Scheme 1 were adopted. The key common inter-
mediate 3 was synthesized starting from commercially available
2-(7-methoxynaphthalen-1-yl) acetonitrile in two steps according
The asymmetrical urea compounds (6a–h) were synthesized
from the activated intermediate 3a and appropriate secondary
amine in the presence of triethyl amine at ambient condition.
The non-isolated intermediate 3a was made in situ by stirring

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Y. Chang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1672–1676
Scheme 3. Synthetic route of asymmetrical urea analogues of Agomelatine (6a–h). Reagents and conditions: (i) CDI, CH3CN, DMF, 2 h; (ii) different substituted amine, Et3N,
12 h.
the common key intermediate 3 and N,N0-carbonyldiimidazole
(CDI) in acetonitrile and dimethylformamide at room temperature
for 2 h.
The target compounds were determined to be 98% at least by
HPLC–UV at full wavelength and were fully characterized by 1H
nuclear magnetic resonance (1H NMR), 13C NMR and mass spec-
troscopy (MS) before entering the biological tests.
In biological aspect, it is widely known that one of the bottle-
necks to rapidly develop effective drugs for treatment of mental
disorders including depression is poorly predictive capability of
in vitro and in vivo screening models. Thus to develop or select
suitable screening models is a major task which the researchers
in this field have to confront with. In the mammalian brain, high
levels of glucocorticoid receptors are expressed in hippocampus
that controls emotional activity13 and the excessive long-lasting
plasma glucocorticoid-induced lesion in hippocampus may cause
depression.14–17 Research has shown that anti-glucocorticoid
therapy in depressed patients is effective by renovation of the in-
jured nerve cells, which was well illustrated by the widely used
antidepressant Fluoxetine. PC12, derived from a pheochromocy-
toma of the rat adrenal medulla, is a cell line of easy subculture
and storage.18 The cellular morphology of PC12 cells would be par-
tially affected if treated with suitable concentration of corticoste-
rone, and some of which could be restored by the treatment
of the known antidepressant such as Fluoxetine. The model of
corticosterone-induced PC-12 lesion is widely used to test the
antidepression activity of compounds.4,14,19,20 As
a result,
evaluation of the protection effects on PC12 cells from corticoste-
rone-induced lesion was an ideal phenotypic in vitro model for
high-throughput screening of compounds with potential antide-
pression activity.21 Therefore, we tested all the target compounds
for their protection activities on PC12 cells from corticosterone-
induced lesion at drug concentration of 1.25
shown in Table 1.
lM and the data were
Imide analogues 4b–e did not exhibit any protection effects on
PC12 cells, sharply opposite to compound 4a,22 which possesses an
excellent protection rate of 34.2%, better than Agomelatine (PR
2.0%) and Fluoxetine (PR 19.7%). The six-membered ring imide
compound 4c seemed to be extremely unsuitable for the growth
of PC12 cells with its highly minus protection rate (ꢀ50.3 2.0%).
Such minus value of protection rate possibly meant to the toxicity
on PC12 cells of the test compound. Among acylamino carboxylic
acid analogues 5a–f, compounds 5a and 5b showed moderate
activity, with protection rates of 11.0% and 23.2%, respectively.
However, introduction of substituents (methyl- or chloro-) to the
alkene carbon atom of the sidechain in compound 5b led to a
nearly completely loss of protective effects with a protection rate
of 2.8 0.3% for compound 5c (with two methyl groups
introduced), or to a negative effects on PC12 cells growth with a
minus protection rates of ꢀ8.4 1.1% for compound 5d (with
two chloro-atoms introduced).As for asymmetrical urea analogues
6a–h, compound 6c and 6e displayed noticeable protection effects
with PR values of 15.7% and 13.6%, respectively. Such results do not
indicate a conclusive structure–activity relationship.
It is known that fundamental physiochemical features of central
nervous system (CNS) drugs are related to their ability to penetrate
the blood-brain barrier (BBB) affinity.23 From a medicinal chemical
perspective, the ability to design drugs capable of penetrating the
BBB and exhibiting the desired biological response is a formidable
challenge. Thus early assessment of the physiochemical properties
of potential CNS drugs for their ability to cross the BBB is extremely
important. Lipophilicity (ClogP) was the first of the descriptors to
be identified as important for CNS penetration and the mean value
of CLogP for the marketed CNS drugs is 3.43 and the range is 0.16–
6.59.24 Molecular topological polar surface area (tPSA) is another
key descriptor that was shown to correlate well with passive
Table 1
The protection rates of the final compounds on corticosterone-injured PC12 cells
Compd
CLogP
LogP
tPSA
PRa (%)
4a
4b
4c
4d
4e
5a
5b
5c
5d
5e
5f
6a
6b
6c
6d
6e
4.1
2.6
4.2
4.6
5.6
2.2
2.4
3.0
4.2
3.3
4.1
3.8
3.3
2.7
2.2
3.2
3.9
4.8
5.2
2.1
4.6
1.53
1.87
2.78
1.99
2.57
1.35
1.54
2.01
1.18
1.75
ꢀ1.17
2.81
ꢀ0.52
1.56
1.12
1.88
1.35
1.06
2.05
1.62
2.55
46.6
46.6
46.4
46.4
46.4
75.6
75.6
75.6
75.6
75.6
75.6
41.6
41.6
41.6
70.6
44.8
44.8
71.1
41.6
38.3
21.3
34.2 2.1
ꢀ16.8 0.9
ꢀ50.3 2.0
ꢀ12.8 0.4
ꢀ10.5 1.1
11.0 8.7
23.2 0.3
2.8 0.3
ꢀ8.4 1.1
6.3 1.8
ꢀ17.5 1.4
ꢀ11.5 1.0
9.8 1.5
15.7 1.3
ꢀ19.5 1.5
13.6 0.8
ꢀ7.7 0.7
6.3 4.2
6f
6g
6h
2.5 0.9
26.2 2.4
19.7 1.5
Agomelatine
Fluoxetine
a
PR represents the protection rate of the tested compound at the concentration
of 1.25
l
M, which is calculated from six independent experiments at 24 h after
ꢀ
ꢀ
d ꢀAc
treatment. PR = A
ꢁ 100% ꢂ SD where, Ac represents the mean absorbance value
ꢀ
ꢀ
Ac
of six independant experiments of control group merely treated with corticoste-
ꢀ
rone, Ad means the mean absorbance value of six independant experiments of test
group treated with corticosterone and test drug and SD means the standard
deviation.

Y. Chang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1672–1676
1675
Table 2
superior to Agomelatine (IR: 20.2 7.6%) and Fluoxetine (IR:
28.5 6.9%), suggested that compound 4a exhibit less liver toxicity
than Agomelatine and Fluoxetine. The kidney cytotoxicity of
compound 4a (IR: 9.6 1.9%) on 293 cells was also lower than Ago-
melatine (IR: 26.6 1.6) and Fluoxetine (IR: 55.8 2.9), indicated
that compound 4a possess a much better safety profile than the
marketed drugs Agomelatine and Fluoxetine.
Based on above in silico and in vitro data, compounds 4a and 5b
were selected to further conduct forced swim test and the results
were shown in Figure 2. After taken 4a or 5b (32 mg/kg/day, sus-
pended in 30% b-Cyclodextrin solution) on days 2–15 by intraperi-
toneal injection, the immobility time of C57 mice were recorded
and analyzed using the Xeye Animal behavior analysis system.
Compound 4a demonstrated more promising to decrease the
immobility time of C57 mice than Agomelatine. However, 5b did
not display remarkable in vivo activity, which can reasonably be
owed to its poor blood–brain barrier-permeating ability (tPSA:
75.6).
Cytotoxicity on human normal liver L02 cells and human embryonic kidney 293 cells
Compd
IRa (%)
293
9.6 1.9 5f
8.1 2.5 6a
Compd
IRa (%)
293
L02
L02
4a
4b
4c
4d
4e
5a
5b
5c
5d
5e
13.8 1.2
8.5 0.9
4.1 2.4 13.4 2.2
22.6 1.8 16.6 2.2
19.3 1.5 28.4 4.0
18.4 1.9 28.4 6.3
10.4 0.9 41.9 3.1 6b
3.4 4.8 17.2 6.1 6c
9.1 0.8 11.5 2.4 6d
3.1 0.8 13.9 3.1 6e
3.4 1.4 33.0 4.2 6f
5.8 2.0 13.2 2.6 6g
3.8 1.6 18.6 0.7 6h
5.7 0.8 34.1 2.1
5.5 1.2
20.1 0.6 46.6 4.2
3.1 0.6 3.9 0.7
7.1 0.6
3.0 0.1 13.3 3.5
8.6 2.7 16.5 4.4
Agomelatine 20.2 7.6 26.6 1.6 Fluoxetine 28.5 6.9 55.8 2.9
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. The viability of the untreated cells was regarded as 100%. Data are expressed
l
as the mean SD.
Based on our present study, we hypothesis that the possible
mechanism is that target compounds protected PC-12 from lesion
and they were antagonist of Glucocorticoid receptor. Certain mech-
anism still needs further research.
molecular transport through membranes and therefore, allows
prediction of transport properties of drugs (Table 1).25 The mean
value of tPSA for the marketed CNS drugs is 40.5 and the range is
4.63–108.24 Therefore we calculated out the ClogP and tPSA values
of the final compounds (Table 1) by the trial version of ChemBioOf-
ficeÒ Ultra 13.0. From Table 1, we can see that all the ClogP and
tPSA values of the synthesized analogues fell into the range of
the marketed CNS drugs. It is noteworthy to point out that among
the compounds with noticeable protective effects on corticoste-
rone-induced PC12 cells, compound 4a possesses similar logP
and ClogP values to Fluoxetine, compounds 5a–b have similar logP
and ClogP values to Agomelatine and compounds 6c, 6e exhibit
similar logP, ClogP and tPSA values to Agomelatine.
The toxicity profile concerning of liver and kidney is usually
considered during the process of drug research and development.
Herein, the inhibitory effects of the synthesized compounds were
evaluated in vitro on human normal liver L02 cells and human
embryonic kidney 293 cells. As shown in Table 2, all the tested
drugs showed low or no inhibitory effects on the tested human
In conclusion, nineteen analogues of Agomelatine were readily
synthesized by modification of the amide side-chain and assessed
for their potential antidepression activities in vitro and in vivo.
Meanwhile their cytotoxicities on human normal liver L02 cells
and human embryonic kidney 293 cells were also tested. ClogP,
tPSA and logP were used to predict their ability to penetrate the
blood-brain barrier. Based on the in silico and in vitro results, we
chose compounds 4a and 5b to further conduct in vivo evaluation.
Forced swim test showed that only compound 4a significantly re-
duced the immobility time of C57 mice. Our results provide a
promising lead (compound 4a) for subsequent optimization to
achieve better efficacy, better pharmacokinetics properties and
less adverse effects such as liver toxicity. Meanwhile, subsequent
modification of compound 5b by preparation of its ester or carba-
mate prodrug may cope with the BBB issue. As for the acting mode
of our compounds, we can logically deduce that the antidepres-
sant-like effects may be owed to their antagonism of glucocorticoid
receptor, quite different from the mechanism of Agomelatine and
Fluoxetine, since corticosterone-induced PC-12 lesion can be sig-
nificantly attenuated by the treatment of compound 4a and 5b
in vitro. However, their exact molecular binding mode remained
to elucidate in the coming study which can further help us to im-
prove our next generation of compounds.
cells at drug concentration of 80 lM. Preferably, compounds 4a
and 5b, whose protective rates on corticosterone-induced PC12
cells are on the top 2 list of the tested analogues, also showed quite
low inhibition on these two normal cells. For example, the cytotox-
icity of compound 4a (IR: 3.8 1.2%) against normal L02 cells was
Acknowledgments
This work was supported by the National Major Program of Chi-
na during the 12th Five-Year Plan Period (2012ZX09103-101-036)
and New-Century Excellent Talent Fund by Ministry of Education
(NCET-13-0388).
Supplementary data
Supplementary data (experimental detail and spectra data)
associated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.bmcl.2014.02.065.
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