Design, synthesis and bioactivity study of N-salicyloyl tryptamine derivatives as multifunctional agents for the treatment of neuroinflammation

Article abstract of DOI:10.1016/j.ejmech.2020.112217

Because of the complex etiology in neuroinflammatory process, the design of multifunctional agents is a potent strategy to cure neuroinflammatory diseases including AD and PD. Herein, based on the combination principles, 23 of N-salicyloyl tryptamine deri

Full text of DOI:10.1016/j.ejmech.2020.112217

European Journal of Medicinal Chemistry 193 (2020) 112217
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Design, synthesis and bioactivity study of N-salicyloyl tryptamine
derivatives as multifunctional agents for the treatment of
neuroinflammation
,
,
,
Xiaohong Fan a 1, Junfang Li a 1, Xuemei Deng a 1, Yingmei Lu a, Yiyue Feng a,
Shumeng Ma a, Huaixiu Wen c, Quanyi Zhao a, Wen Tan a, Tao Shi a **, Zhen Wang a
,
, b, *
a School of Pharmacy, Lanzhou University, Lanzhou, 730000, China
b State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou, 730000, China
c Key Laboratory of Tibetan Medicine Research, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining, 810000, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Because of the complex etiology in neuroinflammatory process, the design of multifunctional agents is a
potent strategy to cure neuroinflammatory diseases including AD and PD. Herein, based on the combi-
nation principles, 23 of N-salicyloyl tryptamine derivatives as multifunctional agents were designed and
their new application for anti-neuroinflammation was disclosed. In cyclooxygenase assay, two com-
pounds 3 and 16 displayed extremely preferable COX-2 inhibition than N-salicyloyl tryptamine. In LPS-
induced C6 and BV2 cell models, some compounds decreased the production of proinflammatory me-
Received 17 January 2020
Received in revised form
3 March 2020
Accepted 7 March 2020
Available online 9 March 2020
diators NO, PGE2, TNF-a, iNOS, COX-2 and ROS, while increased the production of IL-10. Among them,
Keywords:
compound 3 and 16 showed approximately six-fold better inhibition on nitric oxide production than N-
salicyloyl tryptamine in C6. Besides, compounds 3, 13 and 16 attenuated the activation of BV2 and C6
cells. More importantly, in vivo, compounds 3 and 16 reduced GFAP and Iba-1 levels in the hippocampus,
and displayed neuroprotection in Nissl staining. Besides, both compounds 3 and 16 had high safety
(LD50 > 1000 mg/kg). Longer plasma half-life of compounds 3 and 16 than melatonin supported com-
bination strategy. All these results demonstrated that N-salicyloyl tryptamine derivatives are potential
anti-neuroinflammation agents for the treatment of neurodegenerative disorder.
Combination principle
N-salicyloyl tryptamine
Neurodegenerative diseases
Neuroinflammation
Multifunction
© 2020 Elsevier Masson SAS. All rights reserved.
1. Introduction
that development of anti-neuroinflammatory drugs for the treat-
ment of neurodegenerative diseases is promising [9]. However, due
Neuroinflammation is an important defense mechanism against
injury toxins, but persistent neuroinflammation can result in
neuronal death and ultimately contribute to neurodegeneration,
such as Alzheimer’s (AD) or Parkinson’s (PD) diseases [1]. It is
characterized by activation of resident immune cells (microglia and
astrocytes), which cause production of proinflammatory cytokines
and neuronal damage [2e7]. Recently, an approved drug for the
treatment of Alzheimer’s diseases, GV-971, does takes effect
partially due to inhibition of neuroinflammation [8]. This indicates
to extremely complex and dynamic process which resembles a
well-orchestrated symphony of neuroinflammation, many agents
modulating at one process could only enable a palliative treatment
instead of definitively curing [10]. Therefore, seeking the multi-
functional candidates is an imperative strategy of designing anti-
neuroinflammation agents.
Melatonin, a versatile endogenous hormone, has been proved to
prevent neuroinflammation by inhibiting activation of microglia
and astrocyte [11e14]. Furthermore, high drug-able profiles (high
lipophilicity and easy permeability through the blood-brain bar-
rier), exalt its application for neuroinflammatory treatment
[15e17]. However, it exerts poor pharmacokinetic properties, such
as a short plasma half-life (~30 min), which limits its use for neu-
roinflammation [18]. Thus, rational modifications based on the
template of melatonin were conducted (Scheme 1) [19]. Besides,
salicylic acid derivatives have been reported to alleviate
* Corresponding author. School of Pharmacy, Lanzhou University, Lanzhou,
730000, China.
** Corresponding author.
E-mail addresses: shit18@lzu.edu.cn (T. Shi), zhenw@lzu.edu.cn (Z. Wang).
These authors contributed equally to the work.
1
https://doi.org/10.1016/j.ejmech.2020.112217
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

2
X. Fan et al. / European Journal of Medicinal Chemistry 193 (2020) 112217
Scheme 1. Design of the target compounds.
neuroinflammation [20e27]. The mechanism may involve target-
ing COX, a therapeutic target for treating neuroinflammation
[28e35]. Nevertheless, the lack of repair capacity on damaged
neuron, limited exposure to brains and varying degrees of side ef-
fects narrow their application on long-term clinical use [29,36,37].
Thus, it is worth to combine salicylic acid derivatives with mela-
tonin to develop multifunctional molecules as anti-
neuroinflammatory agents.
Based on above findings, we reported a series of N-salicyloyl
tryptamine derivatives with multifunctional properties including
radicals scavenging, COX inhibition, attenuating activation of glial
cells, and attenuating neuronal damage (Scheme 1). Although N-
salicyloyl tryptamine was applied as anticonvulsant agents or anti-
inflammatory agents in RAW264.7 model, its new application as
anti-neuroinflammatory agents has not been reported [38e44]. To
develop more potent anti-neuroinflammatory activities than the
parent compound N-salicyloyl tryptamine, two kinds of derivatives
which had different substituents (hydroxyl, amine) in C2’ were
synthesized. After the introduction of different substituents with
various electronic and steric properties in C5, C4’, C5’ position, the
structure-activity relationship study on cyclooxygenase inhibition
assay was conducted, followed by LPS-induced glial cells model and
LPS-induced neuroinflammatory model of mice. Taken together, all
data suggest that the strategy of combining tryptamine derivatives
and salicylic acid derivatives provides a new sight of designing
multi-functional candidates for neuroinflammation related neuro-
degenerative diseases.
have been previously described. Except for these compounds
mentioned above, compounds 7, 16, 19, 20, 25, 26, 27 were not
reported before.
2.2. COX inhibition assay and structure-activity relationship study
To confirm the COX inhibitory effects of all synthesized com-
pounds, COX inhibitor screening assay was performed using com-
mercial assay kits and salicylic acid as a control. As shown in Table 1,
the compounds exhibited differently inhibitory effects on the COX
with IC50 values ranging from 0.50 to more than over 40
mM.
Among them, 15 compounds showed extremely better inhibition
on COX-2 than salicylic acid. Additionally, some compounds had
slight selectivity on COX-2. The primary structureꢀactivity re-
lationships in C5, C2’ and other positions in the phenyl ring of
salicylic acid moiety were reported as follows:
i) When R4 in C2’ was a hydroxyl, the activity was determined by
comprehensive factors of substituents in C5 and other positions
of salicylic acid moiety. Firstly, in C5 position, the COX-2 inhib-
itory potency order was OH > Me and H (Compounds 9 > 4 and
23). Compared to C5 unsubstituted parent compound 23
(IC50 > 40
mM), a dramatic increase in activity was seen by the
introduction of OH in C5 (compound 9, IC50 ¼ 0.80 0.35
mM)
which was the third-best one on COX-2 inhibition. Secondly, in
C4’ position, the COX-2 inhibitory potency order was
Me > OH > F and H (Compounds 3 > 7 > 12 and 23), suggesting
that introduction of the electron-donating group in C4’ achieved
an improved activity. In contrast, in C5’ position, the COX-2
inhibitory potency order for these compounds was Cl > Me
and H (Compound 5 > 11 and 23), indicating that introduction of
electron-withdrawing group in C5’ was beneficial to activity
improvement. Interestingly, when the phenyl ring (compound
2. Results and discussion
2.1. Synthesis
Well-established methodology was utilized in the synthesis of
N-salicyloyl tryptamine derivatives as indicated in Scheme 2. In
detail, target compounds were obtained by condensation reaction
using salicylic acid derivatives and tryptamine derivatives in the
presence of EDCI, HOBt and triethylamine [45]. All the compounds
were synthesized in near quantitative yields (70%e90%), charac-
terized by NMR and mass spectrometry, and analyzed by HPLC
suggesting a minimum purity of 95.0%.
23, IC50 > 40
mM) was changed to naphthyl ring (compound 2,
IC50 ¼ 2.18 0.48
m
M), a significantly improved COX-2 inhibitory
activity was observed, identifying that enlarged conjugate plane
was favorable to COX-2 inhibition.
ii) When R4 in C2’ was an amine, the SAR study in C5 was con-
ducted. When R4 was NHPh, the COX-2 inhibitory potency order
for these compounds was OH > Cl > Me > H > OMe. Namely,
compound 16 was the best one on COX-2 inhibition
aCompounds 1 [46], 2 [46], 3 [50], 4 [51], 5 [50], 6 [48], 8 [46], 9
[44],11 [46],12 [46],13 [46],14 [46],18 [46], 22 [49], 23 [39], 24 [47]
(IC50 ¼ 0.50 0.10
mM), followed by compounds 18, 8, 13, 14. For

X. Fan et al. / European Journal of Medicinal Chemistry 193 (2020) 112217
3
Scheme 2. Synthesis of N-salicyloyl tryptamine derivatives.a.
Table 1
Inhibitory effects of compounds against COX-2 and COX-1.
Comp.
R1
R2
R3
R4
hCOX-2 IC50
(m
M) SDa
hCOX-1 IC50
(m
M) SDa
SIb
1
2
3
4
5
6
7
8
H
e
H
e
H
H
Cl
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
e
H
e
Me
H
H
H
OH
H
H
H
F
H
H
H
H
H
H
H
H
H
H
H
H
e
3-Cl-2-Me-PhNH
e
14.54 2.12
2.18 0.48
4.16 0.43
>40
10.37 1.59
>40
25.00 5.64
1.47 0.47
0.80 0.35
>40
e
e
39.21 2.18
16.44 1.43
>40
>40
>40
>40
27.05 4.46
1.74 0.61
>40
>40
0.47 0.01
38.64 5.89
0.38 0.05
1.34 0.25
17.98
3.95
H
Me
H
H
H
Me
OH
H
H
H
OMe
OH
Cl
Me
OMe
H
H
H
OH
H
OH
e
OH
OH
OH
NHMe
OH
NHPh
OH
OH
OH
NHPh
NHPh
NHPh
e
e
e
18.40
2.18
e
9
11
12
13
14
16
18
19
20
22
23
24
25
26
27
BHAc
>40
e
2.62 0.98
2.76 0.64
0.50 0.10
0.58 0.19
9.72 2.54
>40
2.31 0.22
>40
>40
1.40 0.13
>40
0.57 0.13
>40
0.18
14.00
0.76
2.31
e
NHPh
3-Cl-2-Me-PhNH
3-Cl-2-Me-PhNH
2,3-di-Me-PhNH
OH
N(Me)2
2,3-di-Me-PhNH
NMePh
e
e
e
e
e
e
e
e
e
e
e
e
e
N(Me)2
e
e
e
>40
e
a
IC50 (mM), the concentrations of compounds producing 50% inhibition of human recombinant COX-1 and COX-2 in vitro. The IC50 values were determined by a dose-
response inhibition curve with GraphPad PRISM. All assays were performed in triplicate with data pooled from at least three independent experiments.
b
In vitro COX-2 selectivity index (IC50 COX-1/IC50 COX-2).
BHA, Salicylic acid.
c
the case that R4 was N(Me)2, compound 27 with OH in C5 was
the second-best one on COX-2 inhibition among all the com-
decreased effects (inhibitory effects on COX-2: compound
16 > compound 25), while the addition of 3-chloro-2-methyl to
phenyl rings showed obviously decline of effects (inhibitory
effects on COX-2: compound 20 < compound 14, compound
19 < compound 8, compound 1 < compound 13).
pounds (IC50 ¼ 0.57 0.13
H in C5 (compound 24: IC50 > 40
cumstances that R4 was 2,3-di-Me-PhNH, compound 25
(IC50 ¼ 1.40 0.13 M) with OH in C5 also performed better than
M) with H in the same po-
mM), followed by compound 24 with
mM). Similarly, under the cir-
m
compound 22 (IC50 ¼ 2.31 0.22
m
Taking the data together, substituent of C5 was proved to be
critical for the COX-2 inhibition potency. On the other hand, the
selectivity study of COX demonstrated most of compounds (2, 3, 8,
9, 14, 18) showed slight selectivity on COX-2, but only compounds
13 and 16 exhibited slight selectivity on COX-1. Given that both
isoforms COX-1 and COX-2 played important roles in neuro-
inflammation, inhibition on both COX-1 and COX-2 may be an
advantage to take anti-neuroinflammatory effect [28].
sition. Besides, based on the parent compound 13, the addition
of methyl to C2’ (compound 26) or changing phenyl to methyl
(compound 6) led to the loss of activity (both IC50 > 40 mM). The
addition of substituents to C2’ of phenyl rings also had some
influence on COX-2 activity. Specifically, the addition of 2,3-
dimethyl to phenyl rings in C2’ had slightly increased (inhibi-
tory effects on COX-2: compound 22 > compound 13) or

4
X. Fan et al. / European Journal of Medicinal Chemistry 193 (2020) 112217
2.3. Cell experiment
significant indicator of anti-neuroinflammation.
In this respect, the effects of compound on nitric oxide pro-
duction in culture supernatant were evaluated by the Griess re-
agent [57]. As shown in Table 3, most of compounds displayed
favorable inhibition on NO production in C6 and BV2 cells. Never-
theless, the inhibition of compounds on NO production was dis-
tinguishing, which was influenced by different substituents on C5,
C2’, C4’ and C5’. The primary structureꢀactivity relationships were
demonstrated as follows:
2.3.1. Toxicity detection on C6 and BV2 cells
Both BV2 (mouse microglia cells) and C6 cells (rat astrocyte
cells) as critical participants in neuroinflammation, are useful cell
models to evaluate anti-neuroinflammation effects in vitro [52]. So
LPS-induced C6 and BV2 cell experiments were conducted to screen
potent anti-neuroinflammatory agents. Before cell assays, MTT
assay was first performed to evaluate cytotoxicity of compounds on
C6 and BV2 [53].
The results showed that cytotoxicity of compounds was diver-
gent to a large extent, and was dramatically influenced by different
substituents (Table 2). Among them, R4 in C2’ position had the
largest impact on cytotoxicity. In C2’ position, cytotoxicity was
observed nearly in all compounds with phenyl substituted amine
group (compounds 1, 8, 13, 14, 18, 19, 20, 22, 25, 26) but it was not
observed in other compounds with hydroxyl, NMe2 or NHMe
groups. Moreover, the cytotoxicity order of compounds containing
different substituents in C2’ was phenyl substituted tertiary amine
group > phenyl substituted secondary amine group. For instance,
compound 26 with methyl(phenyl)amino substituent in C2’ posi-
i) When R4 in C2’ was a hydroxyl, nature (H, F, Cl, OH, Me) and
positions of substituents on phenyl rings were two decisive
factors. In conclusion, in C4’ position, the NO inhibitory potency
order for these compounds was Me > H > F and OH (com-
pounds: 3 > 23 > 12 and 7). In detail, compound 3 with methyl
in C4’
had the
best inhibition
M) and the third best inhibition on C6 cells
M), in contrast, compound 7 with OH in C4’
on
BV2
cells
(IC50 ¼ 4.94 0.43
m
m
(IC50 ¼ 7.48 2.18
and compound 12 with F in C4’ had no obvious effects in both
cells (IC50 > 40
C5’ position, like compound 11, the activity markedly reduced
(IC50 > 40 M). This great change demonstrated the importance
mM). Interestingly, when methyl transferred to
tion had the strongest cytotoxicity (IC50 ¼ 7.98 0.07
while compound 13 with phenyl substituted secondary amine
group had weaker cytotoxicity (IC50 > 100 M in BV2). In this re-
mM in BV2),
m
of locations of substituents. Additionally, the change of sub-
stituents in C5’ from methyl (compound 11) to chlorine (com-
pound 5) did not influence the activity anyway. Similarly,
substituents in C5, like OH and Me respectively in compounds 9
and 4, had no promotion on effects. Better inhibitory effect was
observed unexpectedly when the phenyl ring of salicylic acid
m
gard, we speculated that due to high lipophilicity, compounds with
phenyl substituted tertiary amine group on C2’ may be easier to
penetrate cell membrane than compounds with phenyl substituted
secondary amine group. Besides, the cytotoxicity of compounds
was also influenced by substituents in C5. When R4 substituents in
C2’ position was a phenyl substituted amine, the toxicity order
determined by C5 substituents was 5-Cl > 5-CH3 > 5-OCH3 > 5-OH
and 5-H (compounds: 18 > 8 > 14 > 16 and 13). Additionally, given
that compounds 1, 8, 18, 19, 20, 22, 25, 26 had obvious cell toxicity
moiety (compound 23, IC50 > 40
mM in C6 cells) was replaced by
naphthyl ring (compound 2, IC50 ¼ 13.24 0.30
m
M in C6 cells).
This suggested enlarged conjugate plane contributed to down-
regulation of NO production.
ii) When R4 in C2’ was an amine, an important determinant
affecting NO production was the substituent in R4. Specifically,
compounds possessing phenyl-substituted secondary amine
(compounds 13, 14, 16) seemed more effective than those with
methyl-substituted amine in C2’ (compounds 6, 24, 27). Besides,
for compounds bearing phenyl-substituted secondary amine in
C2’, the substituents (H, OMe, OH) in C5 had a slight influence on
potency (compounds 13, 14, 16 had close effects). But for com-
pounds with dimethyl-substituted amine in C2’, no substitution
(IC50 < 80 mM in either BV2 or C6), they were not considered for the
following cell experiments.
2.3.2. Inhibition of LPS-induced NO production in C6 and BV2 cell
models
Nitric oxide (NO) as a main signal molecule produced in quan-
tity by activated microglial and astrocytes, plays an important role
in neuroinflammation. Although NO is normally regarded as neu-
roprotective factor in the brain, it can turn to be neurotoxic at
specific circumstances. One important mechanism is that after
pathological stimulation, NO produced by iNOS can react with su-
peroxide to give peroxynitrite (ONOO$) which can stress or kill
neurons and this process depends on the level of NO production
[54e56]. Thus, inhibition of NO overproduction is considered as a
(compound 24, IC50 ¼ 22.89 1.82
formed a little better effect than that substituted with OH
(compound 27, IC50 > 40 M in BV2 cells) in the same position.
mM in BV2 cells) in C5 per-
m
In view of the comprehensive evaluation of COX assay and NO
assay, compounds 3, 13 and 16 with outstanding effects were
Table 2
Effects of compounds on viability of C6 and BV2 cellsa.
Compd.
C6
BV2
Compd.
C6
BV2
Cytotoxicity IC50
(
mM) SD
Cytotoxicit IC50
(
mM) SD
Cytotoxicity IC50
(
mM) SD
Cytotoxicity IC50 (mM) SD
1
2
3
4
5
6
7
8
71.14 12.80
>100
>100
>100
>100
>100
>100
87.86 0.06
>100
>100
54.60 1.39
>100
>100
>100
>100
>100
>100
64.64 8.76
>100
>100
14
16
18
19
20
22
23
24
25
26
27
89.33 3.10
>100
>100
>100
79.28 0.91
66.11 1.26
62.40 0.06
95.23 0.34
>100
62.36 7.70
73.74 0.05
>100
55.13 0.80
>100
>100
>100
9
80.51 4.07
28.54 1.45
>100
56.42 8.46
7.98 0.07
>100
11
12
13
>100
115.24 0.45
>100
>100
a
C6 and BV2 cell lines were exposed to different compounds at 0, 6.25, 12.5, 25, 50, and 100
shown as the mean SD of triplicate in three independent experiments.
mM for 24 h. Then cell viability was detected by MTT method. IC50 values were

X. Fan et al. / European Journal of Medicinal Chemistry 193 (2020) 112217
5
Table 3
Effects of compounds on NO production in LPS-stimulated C6 and BV2 cells.a.
Compd.
C6 cell IC50
(m
M)
BV2 cell IC50
(
mM)
Compd.
C6 cell IC50
(
mM)
BV2 cell IC50 (mM)
2
3
4
5
6
7
9
13.24 0.30
7.48 2.18
>40
>40
>40
58.97 5.76
4.94 0.43
>40
>40
>40
12
13
14
16
23
24
27
>40
>40
15.30 0.55
7.61 1.58
6.73 0.17
>40
>40
>40
25.87 5.74
19.88 0.99
37.18 1.73
26.40 2.57
22.89 1.82
>40
>40
>40
>40
>40
11
>40
>40
a
The astrocyte (C6) and microglia cells (BV2) were exposed to the compounds for 24 h in the presence of LPS (10 mg/mL) and LPS (1 mg/mL) respectively. IC50 (mM), the
concentrations of compounds producing 50% inhibition of NO production in vitro compared to control. (IC50) values were determined by a dose-response inhibition curve with
GraphPad PRISM. The data were shown as mean SD. All assays were performed in triplicate with data pooled from at least three independent experiments.
selected for further tests to explore their effects on inflammatory
cytokine production of two glial cells.
In the neuroinflammatory procedure, the signals responsible for
turning on and off microglia have long been put in a dominant
position because these signals could contribute to immune sur-
veillance and homeostasis in the CNS. On the other hand,
Interleukin-10 (IL-10) has been proved to implicate in limiting
microglia activation and CNS inflammation [62]. In this respect, we
chose IL-10 production as one of the evaluation criterias for anti-
neuroinflammation. As shown in Fig. 1D, production of anti-
inflammatory mediator IL-10 released by activated microglia
dramatically increased compared to control group after LPS treat-
2.3.3. Effects of compounds on LPS-induced TNF-
production in C6 and BV2 cells
a, PGE2 and IL-10
Neuroinflammation is a characteristic of many neurodegenera-
tive diseases in which procedure microglia and astrocytes have
been implicated to play both causative and exacerbating roles
[58,59]. In fact, microglia and astrocyte keep mutual cooperation
but play various roles by releasing a series of inflammatory medi-
ators such as arachidonic acid metabolites (PGE2) and cytokines
ment (1 mg/mL). This phenomenon suggested that there was also an
acute response to LPS via the secretion of the anti-inflammatory
cytokine IL-10 in enriched microglia [63]. Compounds 3 and 16
including tumor necrosis factor-alpha (TNF-a), interleukin-10 (IL-
10) in responses to pathological stimulus [28]. In this regard,
evaluation of cytokines or arachidonic acid metabolites of both glial
cells was performed to assess anti-neuroinflammation of synthe-
sized compounds in vitro.
As illustrated in Fig. 1A, TNF-a as a principal pro-inflammatory
mediator released by activated microglia was measured by ELISA
method. Its production markedly increased compared to control
significantly up-regulated the production of IL-10 in 40
compound 13 showed no obvious effects in this regard (IC50
40 M) compared to LPS group. Combining this result with the
above results on TNF- and PGE2 production, we concluded the
anti-neuroinflammation of compounds 3 and 16 in 40 M depen-
mM, but
>
m
a
m
ded partially on the balance between pro-inflammation and anti-
inflammation via regulation of immunosuppressive and anti-
inflammatory mediators.
group after LPS treatment (1
ically inhibited LPS-induced TNF-
dependent manner. Among the three tested hybrids, compound
13 was the most potential to inhibit secretion of TNF- , followed by
compounds 3 and 16. Besides, as shown in Fig. 1B, TNF- released
m
g/mL). Compounds 3 and 13 specif-
a
production in a concentration-
In a word, overall data on cytokines (TNF-a, IL-10), NO, arach-
idonic acid metabolites (PGE2) confirmed the effects of compounds
3, 13 and 16 on two crucial participants (microglia and astrocytes)
in neuroinflammation, although the degree of influence varies with
the compounds. Subsequently, the possible reasons to explain this
disparity in NO and PGE2 regulation were discussed by western
blotting assay.
a
a
by activated astrocyte was also measured by using the same way. Its
production was ascended compared to control group after being
treated by a higher concentration of LPS (10 mg/mL). The difference
in LPS concentration capable of activating glial cells indicated that
microglia as the first defense of immunity in brain, may be easier to
be activated by LPS compared to astrocyte [60]. Similarly, the
inhibitory effects of compounds 3, 13 and 16 were also found in a
dose-dependent manner in LPS-induced astrocyte cell model. Be-
ing identical with the results of LPS-induced microglia cell model,
2.3.4. Effects of compounds on LPS-induced iNOS and COX-2
production in C6 and BV2 cells
To explain the reasons for the discrepant inhibition effects of
compounds on nitric oxide (NO) and PGE2 production, the western
blotting assay was performed in BV2 and C6 cells.
compound 13 was the most potential to inhibit secretion of TNF-
a
in astrocyte cells, followed by compounds 3 and 16 in astrocyte
cells.
Given that NO as a free radical, can be produced by inducible NO
synthase (iNOS) isoform, we preliminarily tested the expressions of
iNOS in microglia and astrocyte. As shown in Fig. 2A, unstimulated
microglia cells expressed extremely low levels of iNOS protein
which is consistent with the character of a non-activation state of
On the other hand, PGE2 is also a well-known inflammatory
mediator derived from arachidonic acid via the action of cyclo-
oxygenases. It is produced in large quantities by activated microglia
which expresses the mPGES [28]. As shown in Fig. 1C, production of
PGE2 by activated microglia distinctly increased after LPS treatment
microglia. After being stimulated by LPS for 24 h (1 mg/mL), the
expression of iNOS in BV2 cells soared. However, iNOS expression
drastically decreased by compounds 3 and 13 treatment compared
to LPS group. Combining the results of NO assay and iNOS assay,
(1
particularly inhibited LPS-induced production of PGE2 in
concentration-dependent manner. Unlike the results of TNF-
mg/mL) compared to the control group. Compounds 3 and 16
a
a
in
compound 3 performed the best (IC50 ¼ 4.94 0.43
inhibition, 100% reduction of iNOS at 40 M), then compound 13
M for NO inhibition, 66%
M) and followed by 16
mM for NO
LPS-induced microglia cell model, compound 3 was the most po-
tential, followed by compounds 16 and 13. Considering that over-
production of PGE2 is associated with up-regulation of COX-2 [61],
so we postulated that these tested compounds may take effect by
down-regulating COX-2 or inhibiting COX-2. The detailed discus-
sion on it was shown in western blotting assay (Fig. 2).
m
came second (IC50 ¼ 25.87
5.74 m
m
reduction of iNOS at 40
(IC50 ¼ 37.18 1.73
iNOS at 40 M) in both assays, indicating the consistency of two
assays. On the other hand, the expression of iNOS was also
mM for NO inhibition, no obvious reduction of
m

6
X. Fan et al. / European Journal of Medicinal Chemistry 193 (2020) 112217
Fig. 1. Effects of compounds on TNF-
a
(Fig. 1, A), PGE2 (Fig. 1, C), IL-10 (Fig. 1, D) in LPS-stimulated BV2 cells and effects on TNF-a in C6 cells (Fig. 1, B). BV2 cells were exposed to
compounds 3, 13, 16 at concentrations of 10, 20, and 40
40 M for 24 h in the presence of LPS (10 g/mL). Each error bar represents the mean SD of three independent experiments. ###, p < 0.001, when compared to control cells. *,
mM for 24 h in the presence of LPS (1 mg/mL). C6 cells were exposed to compounds 3, 13, 16 at concentrations of 10, 20, and
m
m
p < 0.05, **, p < 0.01, ***, p < 0.001, when compared to the LPS-treated cells. All data are the mean SD of at least three independent experiments. Ctrl. means the control group.
LPS means the LPS-treated group.
markedly up-regulated when astrocytes response to stimuli [64].
As shown in Fig. 2B, the expression of iNOS in astrocytes cells
increased significantly compared to control after LPS treatment.
When treated by compounds 3 and 16, an obvious decrease was
also found in LPS-induced astrocytes. Similarly, compound 16
inhibitory effects on COX-2 but could not down-regulate COX-2
expression (IC50 > 40 M). Additionally, compounds 3, 13 and 16
with favorable anti-neuroinflammation potency are worth further
m
investigations.
(IC50 ¼ 6.73 0.17
iNOS at 40
(IC50 ¼ 7.48 2.18
iNOS at 40 M) in both assays.
Besides, the decrease of PGE2 production was associated with
down-regulation of COX-2 or inhibition effects on COX-2 [28,65,66].
As shown in Fig. 2A, the expression of COX-2 protein significantly
m
M for NO inhibition in C6 cells, 95% reduction of
M) performed better than compound
M for NO inhibition in C6 cells, 29% reduction of
2.3.5. Inhibition effects on LPS-induced ROS production in BV2 cells
Microglia as one of the first defense lines protecting CNS against
injury, can be quickly activated in response to CNS injuries or
immunologic stimuli. However, its sustained activation is apt to
result in overproduction of various proinflammatory and neuro-
toxic substances. In addition to nitric oxide (NO), cytokines,
arachidonic acid (AA) and its metabolites, excess intracellular ROS
produced in this process can also induce mitochondria oxidative
damage and eventually cause cell death [68,69]. Worse still, if
extracellular ROS continuously released by microglia, neuronal in-
juries ensue, which is related to neurodegenerative diseases
[70,71]. Therefore, by lowering the levels of highly versatile ROS,
neuroinflammatory cascades are expected to be hindered and the
aim of neuroprotective would be achieved.
m
3
m
m
increased in BV2 cells induced by LPS (1
mg/mL) stimulation,
demonstrating that COX-2 has emerged as one of the major players
in brain inflammation, and increased COX-2 expression was
believed to contribute to neurodegeneration [67]. Besides, inhibi-
tory effect on the expression of COX-2 can be observed by com-
pounds 13 and 16. Different from those compounds, compound 3
showed no obvious effects on COX-2 expression. Referring to the
results of three assays (COX inhibition, COX-2 expression and PGE2
estimation), we speculated that compound 3 was more likely to
reduce the production of PGE2 by inhibiting COX-2, because com-
The results showed that after LPS treatment (Fig. 3, green lines),
levels of ROS significantly increased compared to the control group
(Fig. 3, red lines), indicating that inflammation cascade responses
occurred immediately (determined at 6 h). Afterwards, a decrease
pound 3 (IC50 ¼ 4.16
0.43 mM for COX-2) had outstanding

X. Fan et al. / European Journal of Medicinal Chemistry 193 (2020) 112217
7
Fig. 2. Effects of compounds on protein expressions of iNOS, COX-2 in LPS-induced BV2 cell lines (A) and effects of compounds on protein expressions of iNOS in LPS-induced C6 cell
lines (B). BV2 cell lines were exposed to the indicated compounds at concentrations of 10, 20 and 40 M for 24 h in the presence of LPS (1 g/mL). C6 cell lines were exposed to the
indicated compounds at concentrations of 10, 20 and 40 M for 24 h in the presence of LPS (10 g/mL). The expression of iNOS and COX-2 was assayed and calculated by image J.
m
m
m
m
Each error bar represents the mean SD of three independent experiments. #, p < 0.05 versus the control group; ##, p < 0.01 versus the control group; ###, p < 0.001 versus the
control group. *, p < 0.05, **, p < 0.01 when compared to the LPS-treated cells. All data are the mean SD of at least three independent experiments.
Fig. 3. Effects of compounds 3, 13 and 16 on ROS generation in LPS-stimulated BV2 Cells. Compounds 3, 13 and 16 clearly attenuated LPS-induced production of reactive oxygen
species (ROS). The data were detected at 6 h after LPS stimulation (1 mg/mL). ROS production was determined by a fluorescence probe DCFH-DA.

8
X. Fan et al. / European Journal of Medicinal Chemistry 193 (2020) 112217
of ROS production was observed by compounds 3, 13 and 16
treatment, which suggested that compounds 3, 13 and 16 signifi-
cantly reduced inflammation response in LPS-induced BV2 cells. To
discuss the reason of ROS reduction, the following DPPH assay was
conducted to evaluate radical scavenging potency of these
compounds.
treatment (1
treatment (20
m
m
g/mL). However, after compounds 3, 13 and 16
M), all the expression of Iba-1 declined noticeably.
Among them, compounds 3 and 13 showed better potency on two
glial cells. The above results reaffirmed the inhibitory effects of
compounds 3, 13 and 16 on activation of astrocytes and microglial
cells.
2.3.6. Evaluation of radical scavenging potency by DPPH assay
The radical scavenging capacity was evaluated using 2,2-
diphenyl-1-picrylhydrazyl (DPPH) method [72]. DPPH being
composed of unstable free radical traps any radicals produced by
the tested compounds and brings about a visible color change
ranging from violet to pale yellow to colorless. The degree of the
changes in comparison to the control was calculated as radical
scavenging capacity. As shown in Table 4, the results showed that 5-
OH substituted compound 16 was more potent than non-5-OH-
substituted compound 13, and this may be due to the free radical
quenching of 5-OH group. Moreover, compounds 3 and 16 exhibi-
ted low EC50 indicating that the phenol hydroxyl is a kind of good
free radical quenchers. The results of compounds 3 and 16 were
consistent with that of ROS production. These data suggested that
compounds substituted with phenol hydroxyl group were found to
be the potential radical scavenger which may have to do with
decreasing the production of reactive oxygen species (ROS). How-
ever, compound 13 was inactive in DPPH assay but showed good
effect in LPS-induced ROS production in BV2 cells. The possible
reason for this observation was that compound 13 may take effect
by influencing antioxidant related signaling pathway.
2.4. LPS-induced neuroinflammation in mice
In view of the outstanding effects on cell models and inhibition
on COX-2 (IC50
IC50 ¼ 0.50 0.10
¼
4.16
0.43
m
M
for compound 3,
m
M for compound 16), compounds 3 and 16 were
chosen for in vivo study. LPS was used to induce neuro-
inflammation in mice, for which could trigger a series of CNS in-
flammatory reactions and consequently lead to neuronal damage
[75,76].
Mice were injected intraperitoneally by LPS for six days then
sacrificed for pharmacodynamic evaluation. Considering that the
activation of astrocytes and microglia are regarded as a key event in
the neuroinflammatory process, we investigated inhibitory effects
of compounds 3 and 16 on glial cells activation. Fig. 5 showed that a
higher level of Iba-1 (a marker of activated microglia) and GFAP (a
marker of activated astrocytes) in the hippocampi of mice treated
with LPS were observed compared to the sham group, confirming
the success of modelling. When treated by compounds 16 or 3
(50 mg/kg, i.p., twice a day), levels of GFAP for both compounds and
Iba-1 for compound 3 in the mouse hippocampus significantly
reduced compared to LPS treated group (Fig. 5, A-D). In addition,
Iba-1 and GFAP expression in hippocampus regions were also
evaluated by western blotting method (Fig. 5E). The western blot-
ting results were consistent with that of immunity staining. Un-
expectedly, melatonin did not display the expected effect of
attenuating glial cell activation (p > 0.05), which was inconsistent
with the previous reports [9,13]. The possible reason could be
different methods of administration (intraperitoneal injection in
our work and oral administration in reported works) and high
dosage of melatonin (50 mg/kg, twice a day) [77e80]. We observed
that mice were lethargic and 40% of melatonin group died within
six days, while in compounds 3 and 16 group, no abnormal be-
haviors or death happened. Therefore, we concluded that com-
pounds 3 and 16 are safer and more effective than melatonin under
the specific administration. In addition, salicylic acid showed the
capability of inhibiting microglial cell activation, but the effect was
weaker than that of compound 3. The above results show that the
combination of salicylic acid and melatonin not only can enhance
the anti-neuroinflammatory effect but also ensure safety (more
toxicity data were shown in 2.5).
In sum, compounds
3 and 16 had multifunctional anti-
neuroinflammation activities in vitro, including COX inhibitory ef-
fects, cytokines inhibitory effects as well as radical scavenging ca-
pacity. The following immunofluorescence staining assay evaluated
the effects of tested compounds on glial activation to confirm their
potency of anti-neuroinflammation.
2.3.7. Inhibitory effects on activation of LPS-induced astrocytes and
microglial cells
Neuroinflammation is closely related to the activation of
microglia and astrocytes [73,74]. The increased expressions of glial
fibrillary acidic protein (GFAP) and ionized calcium-binding
adapter molecule 1 (Iba-1), which was respectively considered as
the inflammatory response of rat astrocytes (C6) and microglia
(BV2) induced by LPS, serve as the pathological hallmarks of various
neuroinflammation related diseases including Multiple Sclerosis
and Alzheimer’s disease. Thus, anti-neuroinflammatory effects of
compounds can be evaluated by immunofluorescence labelling of
GFAP and Iba-1.
As seen in Fig. 4A, the expression of GFAP in C6 cell increased
To determine the extent of neuronal damage after LPS treat-
ment, Nissl staining was performed on hippocampal tissue slices of
mice. In accordance with the above results, Nissl staining results
revealed that LPS treatment increased damage of neuron, as shown
by damaged, fragmented or shrunken neuronal cells (Fig. 6, indi-
cated by the black mark). Administration of compounds 3 or 16
attenuated damage of neurons, as shown by the more integral
neuron than the LPS treated group. In addition, there was signifi-
cant apoptosis in CA1 by salicylic acid treatment. Additionally, the
melatonin group also showed significant neuronal apoptosis
(marked by black arrows), indicating that high dosage of melatonin
by intraperitoneal injection had no obvious neuroprotective effect.
Overall, these results demonstrated that N-salicyloyl tryptamine
derivatives exerted inhibition on neuroinflammation, had prom-
ising therapeutic potency, and should be new drug candidates for
neuroinflammation treatment, which is a widely proved patho-
mechanism in Alzheimer’s or other CNS diseases.
significantly after being stimulated by LPS (10
mg/mL). But after
compounds 3, 13 and 16 treatment (20 M), the expressions of
m
GFAP significantly reduced by observing under the same laser in-
tensity, indicating that selected compounds 3, 13 and 16 could
inhibit the activation of rat astrocytes. Similarly, the above ten-
dency was also observed in LPS-induced BV2 cells model. As shown
in Fig. 4B, the expression of Iba-1 in BV2 cell increased by LPS
Table 4
In vitro antioxidation activity by DPPH method.
Compd.
EC50
(
m
M)a
Compd.
EC50 (m
M)a
3
13
16
25.18 2.99
>100
3.82 1.10
Melatonin
5-HTb
>40
0.63
a
EC50, concentration which possesses 50% radical scavenging ability.
5-Hydroxytryptamine.
b

X. Fan et al. / European Journal of Medicinal Chemistry 193 (2020) 112217
9
Fig. 4. Effects of compounds on GFAP and Iba-1 expression in the LPS-induced astrocytes (C6, treated with 10
mg/mL of LPS) and microglia (BV2, treated with 1
mg/mL of LPS). A.
Representative fluorescence microscopy images of GFAP immunostaining (Magnification ꢁ 20). B. Representative fluorescence microscopy images of Iba-1 immunostaining
(Magnification ꢁ 40). C. Histograms show relative changes of GFAP expressed as mean SD of the three independent experiments (n ¼ 3). Ctrl. ¼ Control, LPS ¼ Lipopolysaccharide
(10
m
g/mL); 3, 13, 16 ¼ treatments by compounds 3, 13, 16 (20
mM) along with LPS (10 mg/mL). D. Histograms show relative changes of Iba-1 expressed as mean SD of the three
independent experiments (n ¼ 3). All the data were analyzed using Image Pro Plus and expressed as a percent of LPS group values (fluorescence intensity). (#) Significant difference
(##p < 0.01, ###p < 0.001) vs. control and (*) significant difference (*p < 0.05 and **p < 0.01) vs. LPS.
2.5. Acute toxicity study for compounds 3 and 16 in mice
characters. After intraperitoneal injection, compounds 3 and 16
were both easily absorbed into plasma, with Tmax values of
0.19 0.10 h and 0.92 0.14 h respectively. However, compound 3
Acute toxicity studies with compounds 3 and 16 were per-
formed on ten mice according to OECD guidelines [81]. All treat-
ments were given orally in a single dose of 1000 mg/kg, and the
animals showed no any signs of behavioral alterations upon
treatment with compounds 3 and 16. At the end of 14 days, the
animals were sacrificed for gross examination of heart, liver, kidney
or stomach. Gross examination and histological examination did
not reveal any significant alterations in the heart, liver, kidney or
stomach when compared to vehicle treated control (Fig. 7). The
results suggested that the oral LD50 of compounds 3 and 16 in mice
is higher than 1000 mg/kg.
showed
approximately
2.10 0.22
g/mL). A possible explanation for these results
3-fold
lower
Cmax
value
(Cmax
(Cmax ¼ 6.08 2.39
¼
mg/mL) than that of compound 16
m
was that compound 16 was more easily absorbed than compound 3
owing to the higher LogP value of compound 16. Besides, com-
pound 16 had a short half-life (t1/2 ¼ 2.72 0.75 h) which may
attribute to easy elimination of hydroxyl in C5 position. Compared
to melatonin featured a short plasma half-life (0.54
0.22 h),
compounds 3 and 16 had prolonged plasma half-life (13.95 4.66 h
and 2.72 0.75 h respectively), these findings support the combi-
nation strategy promotes the pharmacokinetic profiles.
2.6. Pharmacokinetic study
3. Conclusion
The pharmacokinetic properties of compounds 3 and 16 were
examined in male SD rats by intraperitoneal injection. As shown in
Table 5, compounds 3 and 16 displayed different pharmacokinetic
In summary, a new series of N-salicyloyl tryptamine derivatives
were synthesized based on the combination of melatonin and

10
X. Fan et al. / European Journal of Medicinal Chemistry 193 (2020) 112217
Fig. 5. Effects of compounds 3, 16, MLT, BHA on expressions of GFAP and Iba-1 in LPS-induced hippocampal impairment of male mice. Sham. ¼ Sham-operated mice were injected
with 3 L, 10 mg/mL). 3, 16, MLT, BHA ¼ respectively treated by compounds 3, 16, MLT, BHA (50 mg/kg, i.p. twice daily for six consecutive
L of saline. LPS ¼ Lipopolysaccharide (3
days) after LPS injection (3 m). B. Representative
L, 10 mg/mL). A. Representative fluorescence microscopy images of GFAP immunostaining (Magnification ꢁ 40, scale bar ¼ 50
fluorescence microscopy images of Iba-1 immunostaining (Magnification ꢁ 40, scale bar ¼ 50 m). C. Histograms showed relative fluorescence intensity of GFAP levels referring to
m
m
m
m
m
LPS group values, and expressed as mean SD (groups (n ¼ 6) except for melatonin group (n ¼ 3)). D. Histograms showed relative fluorescence intensity of Iba-1 levels referring to
LPS group values, and expressed as mean SD (groups (n ¼ 6) except for melatonin group (n ¼ 3)). All the immunofluorescence data were analyzed using Image pro plus software.
E. Western blot image of Iba-1 and GFAP. F. Qualification of GFAP expression (groups (n ¼ 4) except for melatonin group (n ¼ 3)). G. Qualification of Iba-1 expression (groups (n ¼ 4)
except for melatonin group (n ¼ 3)). (#) Significant difference (#p < 0.05, ##p < 0.01) vs. control and (*) significant difference (*p < 0.05 and **p < 0.01) vs. LPS.
salicylic acid for the treatment of neuroinflammation with multi-
functional profiles. Several biological assays in vitro and in vivo
were conducted and proved N-salicyloyl tryptamine derivatives as
multifunctional agents to treat neuroinflammation. In COX inhibi-
tor screening assay, synthesized compounds exhibited diversely
inhibitory effects with IC50 values ranging from 0.50 to more than
compounds (2, 3, 8, 9, 14, 18) showed slight selectivity on COX-2.
The detailed structureꢀactivity relationships on COX-2 inhibition
indicated that the substitution of C5 was crucial for the COX-2 in-
hibition potency. To evaluate the anti-neuroinflammation in vitro,
LPS-induced BV2 and C6 cell models were used. The cytotoxicity
assay showed compounds with phenyl substituted amine group in
C2’ had obvious cytotoxicity on C6 or BV2. For inhibition on NO
production of C6 and BV2, most of compounds displayed favorable
40
mM. Among them, 15 compounds showed extremely better in-
hibition on COX-2 than salicylic acid. Additionally, most of

X. Fan et al. / European Journal of Medicinal Chemistry 193 (2020) 112217
11
Fig. 6. Effects of selected compounds on LPS-induced neuron death in the hippocampus region. The sections of the hippocampus region were stained by Nissl (magnification 40 ꢁ ,
scale bar ¼ 100 m). The black arrow marked the position where neuronal cells disappeared or were sparse with pyknotic cell nucleus and even were induced to apoptosis. (For
interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
m
effects. Comprehensively analyzing the results of NO production
attenuated LPS-induced production of reactive oxygen species
and COX inhibitor screening assay, compounds 3, 13 and 16 were
chosen for further studies. From results of cytokines (TNF-a, IL-10),
(ROS), but only compounds 3 and 16 had radical scavenging ca-
pacity in DPPH assay. In this respect, compound 13 may take effect
by influencing antioxidant related signaling pathway. The immu-
nofluorescence results reconfirmed the inhibitory effects of com-
pounds 3, 13 and 16 on activation of astrocytes and microglial cells.
In LPS-induced neuroinflammation model in mice, administration
of compounds 3 and 16 by intraperitoneal injection at the dose of
50 mg/kg suppressed the activation of astrocyte cells, but only
compound 3 attenuated the activation of microglia cells. Nissl
PGE2, all compounds 3, 13 and 16 took effects on both glial cells
although their effects were in great difference. Western blotting
assay measuring expressions of iNOS and COX-2 showed that
compounds 3 and 13 significantly down-regulated the expressions
of iNOS in BV2 and C6 cells, furthermore, compounds 13 and 16
significantly down-regulated the expressions of COX-2 in BV2 cells.
In ROS release assay, compounds 3, 13 and 16 significantly

12
X. Fan et al. / European Journal of Medicinal Chemistry 193 (2020) 112217
Fig. 7. Histologic specimens of mice tissues (heart, liver, kidney and stomach) in vehicle treated control, compound 3 treated group (1000 mg/kg) and compound 16 treated group
(1000 mg/kg) which were stained with hematoxylin and eosin (n ¼ 10, five male and five female mice). Images of histology heart, liver and kidney zoomed in 40 times.
Table 5
agents, which in turn demonstrated the combination strategy of
melatonin and salicylic acid provided a new insight into multi-
functional candidates for neurodegenerative diseases treatment.
Pharmacokinetic profiles for compounds 3 and 16 in SD rats.
Parameter
t1/2 (h)
Tmax (h)
Compound 3
13.95 4.66
0.19 0.10
2.10 0.22
14.39 1.67
2.25
Compound 16
2.72 0.75
0.92 0.14
6.08 2.39
12.46 3.55
2.86
Melatonin
0.54 0.22
0.19 0.18
1.15 0.04
0.98 0.32
0.97
4. Experimental
Cmax
(
m
(m
g/mL)
g/mL ꢁ h)
AUC0-t
MLogP
4.1. Chemistry
aPharmacokinetic parameters were determined using six animals (male Sprague-
Dawley rats)/group following a single dose of 50 mg/kg by intraperitoneal injec-
tion. The analyze method was Linear Trapezoidal in A non-compartmental model
using the PKsolver2.0 computer program. Data for compounds 3, 16, melatonin
were shown as the mean SD, n ¼ 6.
All chemicals used were of reagent grade. Proton (1H) and car-
bon (13C) NMR spectra (400 or 300 MHz for 1HNMR; 101 MHz for
13CNMR) were recorded on a Bruker spectrometer (Bruker Com-
pany, Germany). NMR spectra used DMSO‑d6, CDCl3 or CH3OD as
solvent (TMS as the internal standard). Proton chemical shifts are
reported relative to a residual solvent peak (CDCl3 at 7.26 ppm,
DMSO‑d6 at 2.50 ppm). Carbon chemical shifts are reported relative
to a residual solvent peak (CDCl3 at 77.00 ppm, DMSO‑d6 at
39.60 ppm, CH3OD at 49.03 ppm). The values of the chemical shifts
are expressed in ppm and the coupling constants (J) in hertz. The
following abbreviations were used to designate multiplicities:
s ¼ singlet, d ¼ doublet, t ¼ triplet, q ¼ quartet, quint ¼ quintet,
m ¼ multiplet, br ¼ broad. High-resolution mass spectrometry
(HRMS) analysis was performed using a Bruker Daltonics Apex II
47e Specification (Bruker Company, Germany).
bThe LogP value was calculated by the free Web tool SwissADME36: http://www.
swissadme.ch/. Lipophilicity was theoretically calculated in n-octanol buffer by
the topological method.
staining results showed compounds 3 and 16 alleviated the damage
of neurons. The acute toxicity results suggested the high safety of
compounds 3 and 16 with the oral LD50 higher than 1000 mg/kg.
Pharmacokinetic study showed compound 16 was prone to be
absorbed into plasma and cleared than compound 3. Compared to
poor pharmacokinetic properties of melatonin featured a short
plasma half-life (~30 min), longer plasma half-life of compounds 3
and 16 (13.95 4.66 h and 2.72 0.75 h respectively) than mela-
tonin supported combination strategy contributed to improved
pharmacokinetic profiles. In conclusion, N-salicyloyl tryptamine
derivatives were proved to be effective anti-neuroinflammatory
4.2. HPLC analysis
The purity was determined by high performance liquid

X. Fan et al. / European Journal of Medicinal Chemistry 193 (2020) 112217
13
chromatography (HPLC). Purity of all final compounds was more
than 95% (Agilent 1260 Infinity II; USA). The column was Eclipse
7.0 Hz, 2H), 2.97 (t, J ¼ 7.4 Hz, 2H), 2.27 (s, 3H).13C NMR (101 MHz,
DMSO‑d6)
d 169.20, 160.45, 144.13, 136.34, 127.44, 127.29, 122.78,
Plus C18 (4.6 ꢁ 150 mm, 4
m
m). Chromatographic conditions for
121.04, 119.61, 118.36, 118.34, 117.62, 112.53, 111.70, 111.49, 39.91,
25.04, 21.16. HRMS (ESIþ) Calcd for C18H19N2O2 [M þ H]þ 295.1441,
found 295.1443.
most compounds except for compounds 16, 22, 25, 26, 27: Mobile
phase: 0e7 min, MeOH:H2O ¼ 20:80; 7e16 min MeOH:H2O ¼ 95:5,
16e25 min MeOH:H2O ¼ 20:80; Wavelength: 254 nm; Column
temperature: 25 ꢂC; Flow rate of 0.5 mL/min. Chromatographic
conditions for compounds 16, 22, 25, 26, 27: Mobile phase:
0e15 min, MeOH:H2O ¼ 3:97; 16e20 min MeOH:H2O ¼ 20:80;
Wavelength: 254 nm; Column temperature: 25 ꢂC; Flow rate of
0.5 mL/min. Compounds 1 [46], 2 [46], 3 [50], 4 [51], 5 [50], 6 [48], 8
[46], 9 [44], 11 [46], 12 [46], 13 [46], 14 [46], 18 [46], 22 [49], 23 [39],
24 [47] have been previously described. Except for these com-
pounds mentioned above, compounds 7, 16, 19, 20, 25, 26, 27 were
not reported before.
4.3.4. 2-Hydroxy-N-(2-(5-methyl-1H-indol-3-yl)ethyl)benzamide
(4)
2-(5-Methyl-1H-indol-3-yl)ethan-1-amine was allowed to react
with salicylic acid to give 4 as white solid (75% yield), mp
170e172 ꢂC. 1H NMR (400 MHz, DMSO‑d6)
d 12.74 (s, 1H), 10.73 (s,
1H), 8.99 (t, J ¼ 5.3 Hz, 1H), 7.92e7.82 (m, 1H), 7.46e7.39 (m, 1H),
7.37 (s, 1H), 7.25 (d, J ¼ 8.2 Hz,1H), 7.17 (d, J ¼ 1.7 Hz, 1H), 6.98e6.85
(m, 3H), 3.60 (dd, J ¼ 13.3, 7.0 Hz, 2H), 2.98 (t, J ¼ 7.3 Hz, 2H), 2.38 (s,
3H). 13C NMR (101 MHz, DMSO‑d6)
d 169.04, 160.28, 134.74, 133.71,
127.73, 127.56, 126.73, 122.93, 122.65, 118.60, 118.03, 117.49, 115.32,
111.21, 40.13, 25.02, 21.35. HRMS (ESIþ) Calcd for C18H19N2O2 [M þ
H]þ 295.1441, found 295.1444.
4.3. General procedure for the preparation of 1e9, 11e14, 16,
19e20, 22-27
To
a
solution of tryptamine derivatives (0.62 mmol) in
4.3.5. N-(2-(1H-indol-3-yl)ethyl)-5-chloro-2-hydroxybenzamide
(5)
dichloromethane (3 mL), EDCI$HCl (144 mg, 0.75 mmol) and HOBT
(110 mg, 0.81 mmol), Et3N (0.22 mL, 1.56 mmol), salicylic acid de-
rivatives (0.69 mmol) were added at room temperature and stirred
for 8 h. After completion of the reaction detected by TLC, the re-
action liquid was removed by rotary evaporation under reduced
pressure. Then the resulting residue was purified by silica gel flash
column chromatography to afford the desired product as a solid
(70%e90%).
Tryptamine was allowed to react with 5-chloro-2-
hydroxybenzoic acid to give 5 as white solid (70% yield), mp
171e172 ꢂC. 1H NMR (400 MHz, DMSO‑d6)
d 12.67 (s, 1H), 10.85 (s,
1H), 9.02 (t, J ¼ 5.2 Hz,1H), 7.94 (d, J ¼ 2.4 Hz,1H), 7.58 (d, J ¼ 7.8 Hz,
1H), 7.43 (dd, J ¼ 8.8, 2.5 Hz, 1H), 7.35 (d, J ¼ 8.0 Hz, 1H), 7.20 (s, 1H),
7.07 (t, J ¼ 7.5 Hz, 1H), 7.02e6.92 (m, 2H), 3.59 (dd, J ¼ 13.2, 6.9 Hz,
2H), 2.98 (t, J ¼ 7.3 Hz, 2H). 13C NMR (101 MHz, DMSO‑d6)
d 167.55,
158.75, 136.36, 133.30, 127.34, 127.27, 122.88, 122.36, 121.08, 119.42,
118.39, 118.33, 116.88, 111.58, 111.52, 40.11, 24.88. HRMS (ESIþ)
Calcd for C17H16ClN2O2 [M þ H]þ 315.0895, found 315.0897.
4.3.1. N-(2-(1H-indol-3-yl)ethyl)-2-((3-chloro-2-methylphenyl)
amino)benzamide (1)
Tryptamine was allowed to react with 2-((3-chloro-2-
methylphenyl)amino)benzoic acid to give 1 as white solid (90%
4.3.6. N-(2-(1H-indol-3-yl)ethyl)-2-(methylamino)benzamide (6)
Tryptamine was allowed to react with 2-(methylamino)benzoic
acid to give 6 as white solid (75% yield), mp 114e115 ꢂC. 1H NMR
yield), mp 164e165 ꢂC. 1H NMR (400 MHz, DMSO‑d6)
d 10.83 (s,1H),
9.78 (s, 1H), 8.74 (t, J ¼ 5.4 Hz, 1H), 7.65 (d, J ¼ 7.8 Hz, 1H), 7.57 (d,
J ¼ 7.8 Hz, 1H), 7.37e7.23 (m, 3H), 7.23e7.11 (m, 3H), 7.06 (t,
J ¼ 7.5 Hz, 1H), 7.03e6.94 (m, 2H), 6.82 (t, J ¼ 7.5 Hz, 1H), 3.55 (dd,
J ¼ 13.3, 7.0 Hz, 2H), 2.97 (t, J ¼ 7.4 Hz, 2H), 2.28 (s, 3H). 13C NMR
(400 MHz, DMSO‑d6)
d
10.83 (s, 1H), 8.43 (t, J ¼ 5.3 Hz, 1H), 7.70 (d,
J ¼ 4.9 Hz,1H), 7.59 (d, J ¼ 7.8 Hz,1H), 7.52 (d, J ¼ 7.8 Hz,1H), 7.35 (d,
J ¼ 8.0 Hz, 1H), 7.28 (t, J ¼ 7.7 Hz, 1H), 7.18 (s, 1H), 7.07 (t, J ¼ 7.4 Hz,
1H), 6.99 (t, J ¼ 7.3 Hz, 1H), 6.62 (d, J ¼ 8.3 Hz, 1H), 6.55 (t, J ¼ 7.4 Hz,
1H), 3.50 (dd, J ¼ 13.6, 7.0 Hz, 2H), 2.94 (t, J ¼ 7.5 Hz, 2H), 2.77 (d,
(101 MHz, DMSO‑d6) d 168.92, 144.79, 141.65, 136.33,134.45, 131.96,
128.81, 127.54, 127.50, 127.37, 123.23, 122.75, 121.02, 118.91, 118.62,
118.37, 118.33, 118.16, 114.98, 111.92, 111.47, 40.16, 25.06, 14.64.
HRMS (ESIþ) Calcd for C24H23ClN3O [M þ H]þ 404.1524, found
404.1528.
J ¼ 5.0 Hz, 3H). 13C NMR (101 MHz, DMSO‑d6)
d 169.16, 150.18,
136.34, 132.33, 128.23, 127.38, 122.69, 121.02, 118.42, 118.32, 115.35,
113.97, 112.03, 111.48, 110.55, 39.96, 29.38, 25.29. HRMS (ESIþ)
Calcd for C18H20N3O [M þ H]þ 294.1601, found 294.1605.
4.3.2. N-(2-(1H-indol-3-yl)ethyl)-3-hydroxy-2-naphthamide (2)
Tryptamine was allowed to react with 3-hydroxy-2-naphthoic
acid to give 2 as white solid (80% yield), mp 132e133 ꢂC. 1H NMR
4.3.7. N-(2-(1H-indol-3-yl)ethyl)-2,4-dihydroxybenzamide (7)
Tryptamine was allowed to react with 2,4-dihydroxybenzoic
acid to give 7 as white solid (65% yield), mp 168e169 ꢂC. 1H NMR
(300 MHz, DMSO‑d6)
d
12.11 (s, 1H), 10.85 (s, 1H), 9.17 (t, J ¼ 5.5 Hz,
1H), 8.50 (s, 1H), 7.84 (d, J ¼ 8.1 Hz, 1H), 7.74 (d, J ¼ 8.3 Hz, 1H), 7.61
(d, J ¼ 7.8 Hz, 1H), 7.53e7.46 (m, 1H), 7.40e7.30 (m, 2H), 7.27 (s, 1H),
7.22 (d, J ¼ 2.3 Hz, 1H), 7.11e7.04 (m, 1H), 7.03e6.96 (m, 1H), 3.65
(dd, J ¼ 13.2, 7.2 Hz, 2H), 3.02 (t, J ¼ 7.4 Hz, 2H). 13C NMR (101 MHz,
(400 MHz, DMSO‑d6) d 13.00 (s, 1H), 10.81 (s, 1H), 10.04 (s, 1H), 8.67
(t, J ¼ 5.6 Hz, 1H), 7.68 (d, J ¼ 8.8 Hz, 1H), 7.58 (d, J ¼ 7.8 Hz, 1H), 7.35
(d, J ¼ 8.1 Hz, 1H), 7.18 (d, J ¼ 2.2 Hz, 1H), 7.11e7.05 (m, 1H),
7.02e6.96 (m, 1H), 6.30 (dd, J ¼ 8.7, 2.4 Hz, 1H), 6.25 (d, J ¼ 2.4 Hz,
1H), 3.55 (dd, J ¼ 13.4, 7.2 Hz, 2H), 2.96 (t, J ¼ 7.5 Hz, 2H). 13C NMR
DMSO‑d6)
d 168.18, 155.64, 136.38, 136.11, 129.54, 128.78, 128.30,
127.32, 126.67, 125.88, 123.76, 122.88, 121.09, 118.65, 118.40, 111.70,
111.53,110.83, 40.19, 25.04. HRMS (ESIþ) Calcd for C21H19N2O2 [M þ
H]þ 331.1441, found 331.1444.
(101 MHz, DMSO‑d6) d 169.43, 162.67, 162.24, 136.34, 128.96, 127.31,
122.73, 121.03, 118.34, 111.78, 111.47, 107.01, 106.83, 102.83, 40.28,
25.15. HRMS (ESIþ) Calcd for C17H17N2O3 [M þ H]þ 297.1234, found
297.1238.
4.3.3. N-(2-(1H-indol-3-yl)ethyl)-2-hydroxy-4-methylbenzamide
(3)
4.3.8. N-(2-(5-methyl-1H-indol-3-yl)ethyl)-2-(phenylamino)
benzamide (8)
Tryptamine was allowed to react with 2-hydroxy-4-
methylbenzoic acid to give 3 as white solid (75% yield), mp
2-(5-Methyl-1H-indol-3-yl)ethan-1-amine was allowed to react
with 2-(phenylamino)benzoic acid to give 8 as white solid (90%
70e71 ꢂC. 1H NMR (400 MHz, DMSO‑d6)
d 12.73 (s, 1H), 10.83 (s,
1H), 8.87 (t, J ¼ 5.4 Hz,1H), 7.73 (d, J ¼ 8.0 Hz,1H), 7.58 (d, J ¼ 7.8 Hz,
1H), 7.35 (d, J ¼ 8.1 Hz, 1H), 7.18 (d, J ¼ 1.6 Hz, 1H), 7.07 (t, J ¼ 7.5 Hz,
1H), 6.99 (t, J ¼ 7.4 Hz, 1H), 6.75e6.67 (m, 2H), 3.57 (dd, J ¼ 13.5,
yield), mp 176e177 ꢂC. 1H NMR (400 MHz, DMSO‑d6)
d 10.69 (s, 1H),
9.75 (s, 1H), 8.68 (t, J ¼ 5.1 Hz, 1H), 7.63 (d, J ¼ 8.0 Hz, 1H), 7.37e7.27
(m, 5H), 7.22 (d, J ¼ 8.2 Hz, 1H), 7.20e7.10 (m, 3H), 6.97 (t, J ¼ 7.3 Hz,

14
X. Fan et al. / European Journal of Medicinal Chemistry 193 (2020) 112217
1H), 6.89 (d, J ¼ 8.2 Hz, 1H), 6.85e6.77 (m, 1H), 3.52 (dd, J ¼ 13.0,
6.9 Hz, 2H), 2.93 (t, J ¼ 7.2 Hz, 2H), 2.36 (s, 3H). 13C NMR (101 MHz,
react with 2-(phenylamino)benzoic acid to give 14 as white solid
(75% yield), mp 121e122 ꢂC. 1H NMR (400 MHz, DMSO‑d6)
10.67
d
DMSO‑d6)
d
168.82, 144.33, 141.64, 134.73, 131.85, 129.50, 128.89,
(s, 1H), 9.77 (s, 1H), 8.69 (t, J ¼ 5.4 Hz, 1H), 7.63 (d, J ¼ 8.0 Hz, 1H),
7.37e7.26 (m, 4H), 7.23 (d, J ¼ 8.7 Hz, 1H), 7.15 (d, J ¼ 6.7 Hz, 3H),
7.07 (d, J ¼ 2.0 Hz, 1H), 6.97 (t, J ¼ 7.3 Hz, 1H), 6.85e6.77 (m, 1H),
6.71 (dd, J ¼ 8.7, 2.2 Hz, 1H), 3.73 (s, 3H), 3.53 (dd, J ¼ 13.2, 6.8 Hz,
127.62, 126.69, 122.85, 122.62, 121.80, 119.50, 119.07, 118.13, 118.05,
114.99, 111.46, 111.19, 40.23, 25.12, 21.38. HRMS (ESIþ) Calcd for
C
24H24N3O [M þ H]þ 370.1914, found 370.1917.
2H), 2.92 (t, J ¼ 7.3 Hz, 2H). 13C NMR (101 MHz, DMSO‑d6)
d 168.85,
4.3.9. 2-Hydroxy-N-(2-(5-hydroxy-1H-indol-3-yl)ethyl)benzamide
(9)
153.08, 144.35, 141.63, 131.88, 131.47, 129.51, 128.89, 127.71, 123.41,
121.80, 119.47, 118.99, 118.13, 114.99, 112.12, 111.79, 111.19, 100.22,
55.35, 40.14, 25.12. HRMS (ESIþ) Calcd for C24H24N3O2 [M þ Hþ]
386.1863, found 386.1866.
5-Hydroxytryptamine was allowed to react with salicylic acid to
give 9 as white solid (40% yield), mp 140e141 ꢂC. 1H NMR
(400 MHz, DMSO‑d6)
d
12.73 (s, 1H), 10.52 (d, J ¼ 1.5 Hz, 1H), 8.96 (t,
J ¼ 5.6 Hz, 1H), 8.62 (s, 1H), 7.85 (dd, J ¼ 7.9, 1.4 Hz, 1H), 7.43e7.36
(m, 1H), 7.13 (d, J ¼ 8.6 Hz, 1H), 7.08 (d, J ¼ 2.2 Hz, 1H), 6.92e6.85
(m, 3H), 6.61 (dd, J ¼ 8.6, 2.3 Hz, 1H), 3.55 (dd, J ¼ 14.4, 6.4 Hz, 2H),
4.3.14. N-(2-(5-hydroxy-1H-indol-3-yl)ethyl)-2-(phenylamino)
benzamide (16)
5-Hydroxytryptamine was allowed to react with 2-(phenyl-
amino)benzoic acid to give 16 as white solid (75% yield), mp
2.88 (t, J ¼ 7.5 Hz, 2H). 13C NMR (101 MHz, DMSO‑d6)
d 169.04,
160.29, 150.33, 133.71, 130.93, 127.96, 127.69, 123.24, 118.60, 117.49,
115.29, 111.79, 111.44, 110.67, 102.33, 40.28, 25.18. HRMS (ESIþ)
Calcd for C17H17N2O3 [M þ H]þ 297.1234, found 297.1238.
131e133 ꢂC. 1H NMR (400 MHz, DMSO‑d6)
d 10.54 (s, 1H), 9.78 (s,
1H), 8.72 (t, J ¼ 5.3 Hz, 1H), 8.66 (s, 1H), 7.68 (d, J ¼ 7.9 Hz, 1H), 7.34
(t, J ¼ 7.6 Hz, 4H), 7.18 (dd, J ¼ 11.1, 8.4 Hz, 3H), 7.12 (s, 1H), 7.00 (t,
J ¼ 7.3 Hz, 1H), 6.93 (s, 1H), 6.89e6.83 (m, 1H), 6.64 (dd, J ¼ 8.6,
2.0 Hz, 1H), 3.53 (dd, J ¼ 13.6, 6.8 Hz, 2H), 2.89 (t, J ¼ 7.5 Hz, 2H). 13C
4.3.10. N-(2-(1H-indol-3-yl)ethyl)-2-hydroxy-5-methylbenzamide
(11)
NMR (101 MHz, MeOD)
d 171.60, 151.11, 145.69, 143.39, 133.09,
Tryptamine was allowed to react with 2-hydroxy-5-
methylbenzoic acid to give 11 as white solid (75% yield), mp
132.90, 130.33, 129.72, 129.44, 124.30, 123.07, 121.45, 120.85, 119.77,
116.91, 112.72, 112.56, 112.40, 103.62, 41.57, 26.28. HRMS (ESIþ)
Calcd for C23H22N3O2 [M þ Hþ] 372.1707, found 372.1704.
153e154 ꢂC. 1H NMR (400 MHz, DMSO‑d6)
d 12.43 (s, 1H), 10.84 (s,
1H), 8.90 (t, J ¼ 5.1 Hz, 1H), 7.67 (s, 1H), 7.59 (d, J ¼ 7.8 Hz, 1H), 7.35
(d, J ¼ 8.0 Hz, 1H), 7.20 (m, 2H), 7.08 (t, J ¼ 7.4 Hz, 1H), 6.99 (t,
J ¼ 7.4 Hz, 1H), 6.80 (d, J ¼ 8.3 Hz, 1H), 3.59 (dd, J ¼ 13.4, 6.9 Hz, 2H),
2.98 (t, J ¼ 7.4 Hz, 2H), 2.24 (s, 3H). 13C NMR (101 MHz, DMSO‑d6)
4.3.15. N-(2-(5-chloro-1H-indol-3-yl)ethyl)-2-(phenylamino)
benzamide (18)
2-(5-Chloro-1H-indol-3-yl)ethan-1-amine was allowed to react
with 2-(phenylamino)benzoic acid to give 18 as white solid (75%
d
169.04, 157.99, 136.36, 134.33, 127.62, 127.30, 127.17, 122.78,
121.05, 118.36, 118.35, 117.25, 114.93, 111.70, 111.50, 39.94, 25.03,
20.18. HRMS (ESIþ) Calcd for C18H19N2O2 [M þ Hþ] 295.1441, found
295.1445.
yield), mp 156e157 ꢂC. 1H NMR (400 MHz, DMSO‑d6)
d 11.05 (s, 1H),
9.71 (s, 1H), 8.68 (t, J ¼ 5.1 Hz, 1H), 7.67e7.54 (m, 2H), 7.40e7.23 (m,
6H), 7.15 (d, J ¼ 7.9 Hz, 2H), 7.06 (d, J ¼ 8.6 Hz,1H), 6.96 (t, J ¼ 7.2 Hz,
1H), 6.82 (t, J ¼ 6.9 Hz, 1H), 3.51 (dd, J ¼ 13.1, 6.7 Hz, 2H), 2.93 (t,
4.3.11. N-(2-(1H-indol-3-yl)ethyl)-4-fluoro-2-hydroxybenzamide
(12)
J ¼ 7.2 Hz, 2H). 13C NMR (101 MHz, DMSO‑d6)
d 168.84, 144.32,
141.63, 134.79, 131.87, 129.49, 128.85, 128.56, 124.76, 123.12, 121.81,
120.95, 119.51, 119.04, 118.16, 117.72, 115.01, 113.02, 111.99, 40.11,
24.88. HRMS (ESIþ) Calcd for C23H21ClN3O [M þ Hþ] 390.1368,
found 390.1371.
Tryptamine was allowed to react with 4-fluoro-2-
hydroxybenzoic acid to give 12 as white solid (75% yield), mp
146e147 ꢂC. 1H NMR (400 MHz, DMSO‑d6)
d 13.21 (s, 1H), 10.85 (s,
1H), 9.07e8.93 (m, 1H), 7.98e7.91 (m, 1H), 7.58 (d, J ¼ 7.8 Hz, 1H),
7.35 (d, J ¼ 8.1 Hz, 1H), 7.19 (d, J ¼ 2.3 Hz, 1H), 7.10e7.04 (m, 1H),
7.01e6.95 (m, 1H), 6.79e6.72 (m, 2H), 3.58 (dd, J ¼ 13.3, 7.3 Hz, 2H),
4.3.16. 2-((3-chloro-2-methylphenyl)amino)-N-(2-(5-methyl-1H-
indol-3-yl)-ethyl)-benza-mide (19)
2-(5-Methyl-1H-indol-3-yl)ethan-1-amine was allowed to react
with 2-((3-chloro-2-methylphenyl)amino)benzoic acid to give 19
as white solid (90% yield), mp 145e147 ꢂC. 1H NMR (400 MHz,
2.98 (t, J ¼ 7.4 Hz, 2H). 13C NMR (101 MHz, DMSO‑d6)
d
168.39,
165.07 (d, J ¼ 249.4 Hz), 162.45 (d, J ¼ 13.4 Hz), 136.35, 130.03 (d,
J ¼ 11.1 Hz), 127.27, 122.82, 121.05, 118.37, 118.31, 112.26 (d,
J ¼ 2.5 Hz), 111.61, 111.50, 106.16 (d, J ¼ 22.1 Hz), 104.00 (d,
J ¼ 23.5 Hz), 40.28, 24.95. HRMS (ESIþ) Calcd for C17H16FN2O2
[M þ Hþ] 299.1190, found 299.1194.
DMSO‑d6)
d
10.68 (s, 1H), 9.80 (s, 1H), 8.73 (t, J ¼ 5.6 Hz, 1H), 7.65
(dd, J ¼ 7.9, 1.4 Hz, 1H), 7.35 (s, 1H), 7.33e7.28 (m, 1H), 7.26 (dd,
J ¼ 7.9, 1.1 Hz, 1H), 7.21 (d, J ¼ 8.2 Hz, 1H), 7.18 (t, J ¼ 7.9 Hz, 1H), 7.12
(dd, J ¼ 7.9, 1.2 Hz, 2H), 7.01 (d, J ¼ 7.6 Hz, 1H), 6.88 (dd, J ¼ 8.3,
1.4 Hz, 1H), 6.85e6.79 (m, 1H), 3.52 (dd, J ¼ 13.3, 7.1 Hz, 2H), 2.92 (t,
J ¼ 7.4 Hz, 2H), 2.35 (s, 3H), 2.27 (s, 3H). 13C NMR (101 MHz,
4.3.12. N-(2-(1H-indol-3-yl)ethyl)-2-(phenylamino)benzamide
(13)
Tryptamine was allowed to react with 2-(phenylamino)benzoic
DMSO‑d6) d 168.88, 144.79, 141.66, 134.72, 134.44, 131.96, 128.80,
acid to give 13 as white solid (75% yield), mp 174e175 ꢂC. 1H NMR
127.61, 127.51, 127.49, 126.67, 123.22, 122.83, 122.60, 118.89, 118.60,
118.16, 118.04, 114.97, 111.44, 111.17, 40.26, 25.08, 21.36, 14.62. HRMS
(ESIþ) Calcd for C25H25ClN3O [M þ H]þ 418.1681, found 418.1685.
(400 MHz, DMSO‑d6)
d
10.83 (s, 1H), 9.74 (s, 1H), 8.70 (t, J ¼ 5.4 Hz,
1H), 7.63 (d, J ¼ 8.0 Hz, 1H), 7.58 (d, J ¼ 7.8 Hz, 1H), 7.38e7.25 (m,
5H), 7.21e7.13 (m, 3H), 7.07 (t, J ¼ 7.5 Hz, 1H), 7.01e6.93 (m, 2H),
6.85e6.78 (m, 1H), 3.54 (dd, J ¼ 13.5, 6.9 Hz, 2H), 2.96 (t, J ¼ 7.4 Hz,
4.3.17. 2-((3-chloro-2-methylphenyl)amino)-N-(2-(5-methoxy-1H-
indol-3-yl)ethyl)-ben-zamide (20)
2H). 13C NMR (101 MHz, DMSO‑d6)
d 168.85, 144.31, 141.65, 136.34,
131.85, 129.50, 128.88, 127.37, 122.74, 121.80, 121.03, 119.48, 119.11,
118.39, 118.34, 118.15, 115.03, 111.93, 111.48, 40.17, 25.11. HRMS
(ESIþ) Calcd for C23H22N3O [M þ Hþ] 356.1757, found 356.1753.
2-(5-Methoxy-1H-indol-3-yl)ethan-1-amine was allowed to
react with 2-((3-chloro-2-methylphenyl)amino)benzoic acid to
give 20 as white solid (85% yield), mp 177e178 ꢂC. 1H NMR
(400 MHz, DMSO‑d6)
d
10.66 (s, 1H), 9.82 (s, 1H), 8.74 (t, J ¼ 5.4 Hz,
4.3.13. N-(2-(5-methoxy-1H-indol-3-yl)ethyl)-2-(phenylamino)
benzamide (14)
1H), 7.66 (d, J ¼ 7.9 Hz, 1H), 7.34e7.20 (m, 3H), 7.15 (p, J ¼ 7.9 Hz,
3H), 7.08 (d, J ¼ 2.0 Hz,1H), 7.02 (d, J ¼ 8.3 Hz,1H), 6.82 (t, J ¼ 7.5 Hz,
1H), 6.71 (dd, J ¼ 8.7, 2.2 Hz, 1H), 3.73 (s, 3H), 3.54 (dd, J ¼ 13.2,
2-(5-Methoxy-1H-indol-3-yl)ethan-1-amine was allowed to

X. Fan et al. / European Journal of Medicinal Chemistry 193 (2020) 112217
15
6.9 Hz, 2H), 2.93 (t, J ¼ 7.3 Hz, 2H), 2.27 (s, 3H). 13C NMR (101 MHz,
1H NMR (400 MHz, CDCl3)
d
8.37e8.26 (m, 1H), 8.23 (dd, J ¼ 7.8,
DMSO‑d6)
d
168.92, 153.07, 144.80, 141.63, 134.44, 131.99, 131.46,
1.7 Hz, 1H), 7.65 (d, J ¼ 8.2 Hz, 1H), 7.56 (d, J ¼ 7.9 Hz, 1H), 7.39 (td,
J ¼ 7.6, 1.8 Hz, 1H), 7.35e7.26 (m, 2H), 7.25e7.15 (m, 3H), 7.12e7.06
(m, 1H), 7.00 (dd, J ¼ 7.8, 1.1 Hz, 1H), 6.93 (t, 1H), 6.65e6.52 (m,
J ¼ 5.2 Hz, 3H), 3.80 (dd, J ¼ 12.0, 6.5 Hz, 2H), 2.94 (t, J ¼ 6.6 Hz, 2H),
128.82, 127.70, 127.49, 127.45, 123.41, 123.19, 118.82, 118.51, 118.14,
114.94, 112.11, 111.76, 111.20, 100.19, 55.31, 40.13, 25.12, 14.59. HRMS
(ESIþ) Calcd for C25H25ClN3O2 [M þ H]þ 434.1630, found 434.1634.
2.71 (s, 3H). 13C NMR (101 MHz, CDCl3)
d 165.71, 149.12, 148.13,
4.3.18. N-(2-(1H-indol-3-yl)ethyl)-2-((2,3-dimethylphenyl)amino)
benzamide (22)
136.45, 132.44, 131.45, 130.94, 128.95, 127.72, 126.95, 126.53, 122.37,
122.01, 119.74, 119.27, 118.74, 116.27, 112.44, 111.23, 40.24, 39.32,
24.87. HRMS (ESIþ) Calcd for C24H24N3O [M þ H]þ 370.1914, found
370.1918.`
Tryptamine was allowed to react with 2-((2,3-dimethylphenyl)
amino)benzoic acid to give 22 as white solid (85% yield), mp
143e145 ꢂC. 1H NMR (400 MHz, DMSO‑d6)
d 10.85 (s, 1H), 9.66 (s,
1H), 8.69 (t, J ¼ 5.6 Hz, 1H), 7.66e7.57 (m, 2H), 7.35 (d, J ¼ 8.1 Hz,
1H), 7.26e7.20 (m, 2H), 7.13e7.04 (m, 3H), 7.01e6.96 (m, 1H), 6.92
(d, J ¼ 7.0 Hz, 1H), 6.87e6.83 (m, 1H), 6.75e6.70 (m, 1H), 3.57 (dd,
J ¼ 13.4, 7.1 Hz, 2H), 2.99 (t, J ¼ 7.4 Hz, 2H), 2.27 (s, 3H), 2.12 (s, 3H).
4.3.23. 2-amino-N-(2-(5-hydroxy-1H-indol-3-yl)ethyl)benzamide
(27)
5-Hydroxytryptamine was allowed to react with 2-(dimethyla-
mino)benzoic acid to give 27 as white solid (30% yield), mp
13C NMR (101 MHz, DMSO‑d6)
d
169.17, 146.28, 139.45, 137.78,
150e151 ꢂC. 1H NMR (400 MHz, DMSO‑d6)
d 10.57 (s, 1H), 9.12 (t,
136.36, 131.93, 129.47, 128.72, 127.41, 125.96, 125.13, 122.76, 121.04,
119.75, 118.42, 118.36, 117.39, 116.85, 114.02, 112.00, 111.50, 40.16,
25.16, 20.42, 13.70. HRMS (ESIþ) Calcd for C25H26N3O [M þ H]þ
370.1914, found 370.1918.
J ¼ 4.8 Hz, 1H), 8.62 (d, J ¼ 1.7 Hz, 1H), 7.68e7.63 (m, 1H), 7.38e7.32
(m, 1H), 7.17e7.09 (m, 3H), 7.03 (td, J ¼ 7.5, 1.0 Hz, 1H), 6.92e6.88
(m, 1H), 6.61 (dt, J ¼ 8.6, 1.9 Hz, 1H), 3.58 (dd, J ¼ 12.7, 6.1 Hz, 2H),
2.88 (t, J ¼ 7.1 Hz, 2H), 2.46 (s, 6H). 13C NMR (101 MHz, MeOD)
d
169.34, 154.09, 151.36, 133.27, 133.22, 131.51, 129.44, 128.15,
4.3.19. N-(2-(1H-indol-3-yl)ethyl)-2-hydroxybenzamide (23)
Tryptamine was allowed to react with 2-hydroxybenzoic acid to
give 23 as white solid (80% yield), mp 153e155 ꢂC. 1H NMR
124.72, 124.58, 120.96, 112.85, 112.61, 112.31, 103.61, 44.82, 41.01,
25.69. HRMS (ESIþ) Calcd for C19H22N3O2 [M þ H]þ 324.1707, found
324.1709.
(400 MHz, DMSO‑d6)
d
12.71 (s, 1H), 10.84 (s, 1H), 8.98 (t, J ¼ 5.6 Hz,
1H), 7.85 (dd, J ¼ 7.9, 1.5 Hz, 1H), 7.59 (d, J ¼ 7.8 Hz, 1H), 7.42e7.32
(m, 2H), 7.20 (d, J ¼ 2.2 Hz, 1H), 7.10e7.04 (m, 1H), 7.02e6.96 (m,
1H), 6.92e6.85 (m, 2H), 3.59 (dd, J ¼ 13.9, 6.7 Hz, 2H), 2.99 (t,
4.4. Biological studies
4.4.1. COX inhibition assay
J ¼ 7.4 Hz, 2H). 13C NMR (101 MHz, DMSO‑d6)
d
168.98, 160.21,
The COX-1 and COX-2 inhibitory activities of the compounds
were measured using human COX inhibitory screening assay kit
(Cayman Chemical, catalog no. 701230) according to the manu-
facturer’s instructions. The final concentrations of compounds were
136.32, 133.64, 127.68, 127.26, 122.75, 121.00, 118.54, 118.32, 118.29,
117.43, 115.31, 111.65, 111.45, 39.94, 24.97. HRMS (ESIþ) Calcd for
C
17H17N2O2 [M þ H]þ 281.1285, found 281.1289.
2.5, 5, 10, 20, 40 mM. The absorbance was measured in microplate
4.3.20. N-(2-(1H-indol-3-yl)ethyl)-2-(dimethylamino)benzamide
(24)
reader (Bio-Rad Laboratories, CA, USA) at 405 nm. IC50 values were
calculated by the sigmoidal doseꢀresponse equation (variable
slope) (GraphPad software). All the experiments were repeated
three times.
Tryptamine was allowed to react with 2-(dimethylamino)ben-
zoic acid to give 24 as white solid (50% yield), mp 167e169 ꢂC. 1H
NMR (400 MHz, DMSO‑d6)
d
10.88 (s, 1H), 9.13 (t, J ¼ 5.2 Hz, 1H),
7.66 (dd, J ¼ 7.7, 1.7 Hz, 1H), 7.60 (d, J ¼ 7.8 Hz, 1H), 7.39e7.32 (m,
2H), 7.23 (d, J ¼ 2.2 Hz, 1H), 7.13e7.06 (m, 2H), 7.06e7.01 (m, 1H),
7.01e6.96 (m, 1H), 3.62 (dd, J ¼ 12.7, 7.0 Hz, 2H), 2.97 (t, J ¼ 7.1 Hz,
4.4.2. Cells and treatment
BV2 and C6 cell lines were obtained from the American Type
Culture Collection (ATCC, USA). Both BV2 and C6 cell lines were
incubated in DMEM media supplemented with 10% FBS, 100 U/mL
penicillin, and 100 U/mL streptomycin at 37 ꢂC with 5% CO2.
Compounds dissolved in DMSO solution were added into cell cul-
tural medium with the final concentration of DMSO less than 0.4%.
2H), 2.45 (s, 6H). 13C NMR (101 MHz, DMSO‑d6)
d 166.71, 151.58,
136.45, 131.17, 130.08, 127.72, 127.28, 123.01, 122.05, 121.07, 118.87,
118.45, 118.35, 111.77, 111.47, 43.94, 39.66, 24.84. HRMS (ESIþ) Calcd
for C19H22N3O [M þ H]þ 308.1757, found 308.1753.
4.3.21. 2-((2,3-dimethylphenyl)amino)-N-(2-(5-hydroxy-1H-indol-
3-yl)ethyl)ben-zamide (25)
4.4.3. Cytotoxicity assay on C6 and BV2 cells
In order to evaluate the cytotoxicity of all the compounds on C6
and BV2 cells, MTT assay was performed. To be specific, the cells
were collected after being dissociated by trypsin, and seeded in 96
well plate at the density of 1e5 ꢁ 105 per well. After 12 h, the
medium was changed, and the cells were treated by the medium
containing compounds at final concentrations of 0, 6.25, 12.5, 25,
5-Hydroxytryptamine was allowed to react with 2-((2,3-
dimethylphenyl)amino)benzoic acid to give 25 as white solid
(75% yield), mp 139e140 ꢂC. 1H NMR (400 MHz, DMSO‑d6)
d 10.49
(d, J ¼ 1.6 Hz, 1H), 9.62 (s, 1H), 8.64 (t, J ¼ 5.6 Hz, 1H), 8.60 (s, 1H),
7.64 (dd, J ¼ 7.9, 1.3 Hz, 1H), 7.26e7.20 (m, 1H), 7.15e7.04 (m, 4H),
6.94e6.89 (m, 2H), 6.85e6.81 (m, 1H), 6.75e6.70 (m, 1H), 6.61 (dd,
J ¼ 8.6, 2.3 Hz,1H), 3.52 (dd, J ¼ 14.4, 6.4 Hz, 2H), 2.93e2.84 (m, 2H),
50, and 100
mM for 24h followed by incubation with MTT solution
for 4 h. After adding 100
mL of DMSO per well, the absorbance was
2.27 (s, 3H), 2.12 (s, 3H). 13C NMR (101 MHz, DMSO‑d6)
d
169.09,
measured in microplate reader (Bio-Rad Laboratories, CA, USA) at
570 nm. IC50 values were calculated by IBM SPSS Statistics 25.0.
Data were given as the mean from three independent experiments
conducted in triplicate.
150.30, 146.31, 139.43, 137.76, 131.90, 130.94, 129.56, 128.68, 128.02,
125.94, 125.17, 123.17, 119.88, 117.35, 116.83, 113.97, 111.76, 111.40,
110.96, 102.39, 39.92, 25.32, 20.37, 13.68. HRMS (ESIþ) Calcd for
C
25H26N3O2 [M þ H]þ 400.2020, found 400.2024.
4.4.4. Evaluation of LPS-induced NO production in C6 and BV2 cells
models
The C6 and BV2 cells in logarithmic growth period were
collected after being dissociated by trypsin and then seeded in 96-
well plates at a density of 5 ꢁ 105 cells/mL. 12 h after attachment,
4.3.22. N-(2-(1H-indol-3-yl)ethyl)-2-(methyl(phenyl)amino)
benzamide (26)
Tryptamine was allowed to react with 2-(methyl(phenyl)amino)
benzoic acid to give 26 as white solid (50% yield), mp 103e105 ꢂC.

16
X. Fan et al. / European Journal of Medicinal Chemistry 193 (2020) 112217
the BV2 and C6 cells were stimulated by LPS (Solarbio, Lot.L8880,
China) at 1 and 10 g/mL respectively. The cells in treatment group
were incubated with compounds at the final concentrations of 2.5,
5, 10, 20, 40 M while in blank group, equal volume of DMSO was
mL were respectively added in C6 and BV2 apart from blank group.
After incubation for 24 h, cells were collected after washing with
ice-cold phosphate buffered saline (PBS) for three times. Then the
m
m
collected cells were lysed in 300 mL RIPA lysis buffer (Solarbio,
added. After 24 h incubation, supernatant was collected. The pro-
duction of nitric oxide was quantified using the Griess reagent (0.5%
sulfanilamide, 0.05% (N-1-naphthyl) ethylenediamine dihydro-
chloride, 2.5% H3PO4) for 15 min at rt in the dark. The absorbance
was measured at 540 nm in microplate reader (Bio-Rad Labora-
tories, CA, USA). The concentration of nitrite was determined ac-
cording to a sodium nitrite standard curve. IC50 values were
calculated by IBM SPSS Statistics 25.0. All the experiments were
repeated three times.
R0010, China) containing 1 mM phenylmethanesulfonyl fluoride
(PMSF: Solarbio, R0010, China) for 10 min, followed by centrifu-
gation at 12000 rpm for 10 min at 4 ꢂC. The protein concentrations
were determined by BCA protein assay kit (Solarbio, PC0020,
China). Equal amounts of proteins (30 mg) were separated by 8%
SDS-PADE gels and transferred to polyvinylidine difluoride (PVDF)
membranes (Millipore Corporation, USA). The non-objective pro-
tein was blocked in blocking buffer (5% non-fat dry milk in TBST) for
2 h at room temperature. After that, the membranes were incu-
bated with the diluted (1:500e1:1000) iNOS antibodies (Affinity
Biosciences, USA, Lot. AF0199) and COX-2 antibodies (Cell Signaling
Technology, Inc., USA, Lot. 4842) overnight at 4 ꢂC, followed by
washing with TBST at least three times. And then, the membranes
were incubated with HRP-coupled antibody (Affinity Biosciences,
USA, Lot. S0001 for iNOS and COX-2; Affinity Biosciences, USA, Lot.
S0002 for GAPDH) at 1:5000 dilution in blocking buffer for 2 h at
room temperature. After washing with TBST at least three times,
the immunocomplexes were detected using ECL reagent (Millipore,
Billerica, MA) and scanned using the Tanon imaging system (Tanon
Science & Technology Co., Ltd, China). All the images were quan-
tified by densitometry using Image J software. The results were
calculated from at least three independent experiments.
4.4.5. Evaluation on TNF-a, IL-10, and PGE2 in LPS-induced
activation of C6 and BV2 cells model
The C6 and BV2 cells in logarithmic growth period were
collected after being dissociated by trypsin and then dispensed in
96-well plates at a density of 5 ꢁ 105 cells/mL. About 12 h after
attachment, the BV2 and C6 cells were respectively stimulated by
LPS (Solarbio, Lot.L8880, China) at 1 and 10
treatment group were treated with compounds at the final con-
centrations of 10, 20, 40 M while in blank group, equal volume of
DMSO was added. After 24 h, secreted TNF- , IL-10, PGE2 in the
supernatant of culture media were measured using the specific
ELISA kit (TNF- : Elabscience, E-EL-M0049c; IL-10: Elabscience, E-
EL-M0046c; PGE2: Elabscience, E-EL-0034c) according to the
manufacturer’s instructions. In detail, for TNF- and IL-10 mea-
mg/mL. The cells in
m
a
a
a
4.4.7. Evaluation on LPS-induced ROS production in BV2 cells model
BV2 cells were seeded into 6-well plates at a density of
5 ꢁ 105 cells/mL with 2 mL of culture medium and incubated in
incubator (5% CO2, 37 ꢂC) overnight. After attachment, they were
divided into control group, LPS treated group and LPS þ compound
treated group. In LPS þ compound treated group, compounds at
surements, standards or samples were added into micro ELISA plate
wells. Then specific antibodies of biotinylated detection for Mouse
TNF-a, IL-10, Avidin-Horseradish Peroxidase (HRP) conjugate were
added into each well and incubated. The reaction was terminated
by adding stop solution with the sign of color turning to yellow. The
optical density (OD) was measured spectrophotometrically at a
wavelength of 450 2 nm. The OD value was proportional to the
final concentrations of 10, 20, 40
equal volume of DMSO was added into LPS treated group and blank
group, and LPS (Solarbio, Lot.L8880, China) of 1 g/mL were added
mM were added. At the same time,
concentration of mouse TNF-a, IL-10. And LPS was used to set up a
m
positive control. The absorbance was measured by a microplate
reader (Bio-Rad Laboratories, CA, USA). Besides, for PGE2 mea-
surement, the ELISA kit worked based on the competitive-ELISA
principle. The micro ELISA plate provided in this kit had been
pre-coated with a certain amount of PGE2. During the reaction,
PGE2 in the sample or standard competed with the fixed amount of
PGE2 on the solid phase supporter for binding sites of Biotinylated
Detection Ab specific to PGE2. Unbonded conjugate and excess
sample or standard were washed from the plate, and Avidin con-
jugated to Horseradish Peroxidase (HRP) were added to each
microplate well and incubated. Then a TMB substrate solution was
added into each well. The enzyme-substrate reaction was termi-
nated by adding stop solution and the intensity of changed color
apart from blank group. After incubation for 6 h, the growth media
were removed, and carboxy-21,71-dichloro-dihydro-fluorescein
diacetate probes (DCFH-DA) (Sigma, USA) dissolved in medium
were added into each well at a final concentration of 20 mM and
incubated for 30 min at 37 ꢂC. Afterwards, BV2 cells were washed
with cold PBS three times and resuspended in PBS, analyzing with
flow cytometer (Beckman, USA) at 485 nm for excitation and
530 nm for emission.
4.4.8. DPPH assay
The radical quenching effects of tested compounds and mela-
tonin were evaluated with the DPPH method using serotonin as the
standard. Both compounds and positive reference were evaluated
was measured spectrophotometrically at
a
wavelength of
at a final concentration of 2.5, 5, 10, 20, 40
compounds and positive standards diluted in ethanol were mixed
with the same volume of 700 mol/L ethanol solution of DPPH. The
mixed solution was shaken vigorously, and left in dark at room
temperature for 30 min. Its absorbance was read using a microplate
reader (Bio-Rad Laboratories, CA, USA) at 515 nm. The anti-radical
mM. In detail, 100 mL of
450 2 nm by a microplate reader (Bio-Rad Laboratories, CA, USA).
The concentration of PGE2 in the samples were then determined by
comparing the OD of the samples to the standard curve. The results
were calculated from at least three independent experiments.
m
4.4.6. Evaluation of inhibition on LPS-induced expression of iNOS
and COX-2 in C6 and BV2 cells models
activity was calculated using the following equation: [(AbsControl/
AbsTest)/AbsControl] ꢁ 100%. EC50 values were calculated by IBM SPSS
Statistics 25.0. Data were given as the mean from three indepen-
dent experiments.
C6 and BV2 cells were seeded into 6-well plates at a density of
5 ꢁ 105 cells/mL with 2 mL of culture medium and incubated in
incubator (5% CO2, 37 ꢂC) overnight. After attachment, they were
divided into control group, LPS treated group and LPS þ compound
treated group. In LPS þ compound treated group, each compound
4.4.9. Evaluation on expression of GFAP in LPS-induced C6 cells
model and expression of Iba-1 in LPS-induced in BV2 cells
The C6 and BV2 cells in logarithmic growth phase were collected
after being dissociated by trypsin, and then seeded on glass slides at
a density of 5 ꢁ 105 cells/mL. After attachment, the cells were
at final concentrations of 10, 20, 40
mM were added. At the same
time, equal volume of DMSO was added into LPS treated group and
blank group. LPS (Solarbio, Lot.L8880, China) of 10 g/mL and 1 g/
m
m

X. Fan et al. / European Journal of Medicinal Chemistry 193 (2020) 112217
17
divided into blank control group, LPS treated group and
Next, LPS (10 mg/mL; Solarbio, Lot. L8880, China) was slowly
injected into the lateral ventricle of mice at a rate of 0.5 L/min. In
the sham group, 3 L saline was injected slowly. After the injection,
LPS þ compound treatment group. In LPS þ compound treated
m
group, the final concentration of compounds was 20
mM. In LPS
m
treated group and blank group, equal volume of DMSO was added.
At the same time, LPS of 10 g/mL and 1 g/mL was added to glass
the needle was kept in situ for an additional 5 min and then
retrieved slowly from the brain.
m
m
slides of C6 and BV2 respectively apart from blank group. After
incubation for 24 h, the culture solution was discarded and PBS was
After modelling, the mice in compounds, melatonin, salicylic
acid treated group were intraperitoneally injected with compounds
at a dose of 50 mg/kg (0.08 mL, suspended in saline containing 1%
methyl cellulose) per day for six consecutive days, while the mice in
model group were injected with saline containing 1% methyl cel-
lulose in the same way. Six days later, the mice were sacrificed to
determine the activation of glial cells and the loss of neuronal cell in
hippocampus. Six mice of each group (melatonin group, three
mice) were firstly perfused with normal saline, followed by 4%
paraformaldehyde (PFA) dissolved in 0.1 M sodium phosphate
buffer (PBS) (pH ¼ 7.4). The hippocampus was separated out of its
cerebral tissues and incubated with 4% paraformaldehyde (PFA)
overnight. Then it was stored at a 30% sucrose solution. Next, the
hippocampus was cut into thin sections with the thinkness of
used to wash at least three times. Then 50
mL pre-cooled para-
formaldehyde (4%) was added to the glass slides and kept for
10 min at room temperature to fix the cells. Next, sealing solution
(PBS solution containing 1% BSA and 0.3% Triton x-100) was added
and kept for 1 h at room temperature and discarded. In the next
stage, glial fibrillary acidic protein antibody (GFAP Ab: Affinity
Biosciences, USA, Lot AF6166) and ionized calcium-binding adapter
molecule 1 antibody (Iba-1 Ab: Abcam, Cambridge, UK, Lot
ab178846) were diluted in sealant (1% BSA and 0.3% Triton x-100) at
a 1:100 dilution rate and added dropwise into C6 and BV2
respectively. The glass slides were then incubated overnight in dark
at 4 ꢂC. After washing with PBS for three times, secondary antibody
was added after diluted with sealant (1%BSA and 0.3% Triton x-100)
at a 1:200 dilution rate, and incubated for 2 h in dark at room
temperature. In the end, the cover glasses were used to seal the
samples after adding anti-fluorescence quenching sealing liquid
10
mm. After three times washes with PBS (pH ¼ 7.4) 5 min for each,
the thin sections were permeabilized with TritonX-100 (0.3% in
TBST) at room temperature, and blocked with 1% bovine serum
albumin (BSA) solution for 1 h followed by incubation with a 1:100
dilution of an antibody against glial fibrillary acidic protein (Affinity
Biosciences, USA, Lot. AF6166). and a 1:200 dilution of the antibody
against ionized calcium-binding adapter protein 1 (Iba-1, Abcam,
Cambridge, UK, Lot. ab5076) overnight at 4 ꢂC. Then, the sections
were washed with PBS (pH ¼ 7.4) three times, 10 min for each then
stained with DAPI staining solution for 10 min at room tempera-
ture. Finally, the images were taken under a confocal laser micro-
scope (Zeiss LSM700, Switzerland). The immunofluorescence
intensity of images was measured using Image Pro Plus software.
Similarly, the hippocampus sections (n ¼ 6) were used for Nissl
staining with Methylene Blue (Solarbio, Lot. G1434, China). Finally,
the images were taken under a microscope (Motic, China). All re-
sults calculated were from six independent samples except for
melatonin group, whose result was from three independent
samples.
(containing 2 mg/mL DAPI). The sealed samples were kept for 5 min
in dark at room temperature, and then their images were taken
under a confocal laser microscope (Zeiss LSM700, Switzerland). The
results were calculated from at least three independent experi-
ments. The immunofluorescence intensity for all groups was
measured using Image Pro Plus software. All results were given as
the mean
SD; significance between the control groups was
determined by analysis of variance (ANOVA), followed by Fisher’s
PLSD procedure for post hoc comparison which was used to verify
the significance between two means. P values of less than 0.05 were
considered significant. The data were analyzed by SPSS software.
4.4.10. Animal Experiments
All studies involving animals were performed according to An-
imal Research: Reporting of In Vivo Experiments (ARRIVE) guide-
lines, and also had been approved by the Institutional Ethics
Committee of Lanzhou University, Gansu, China. Animals were kept
and maintained under 12 h light/12 h dark cycle at 25 ꢂC and were
fed with food and water ad libitum according to standard rodent
procedures.
For western blot assay, another four mice of each group were
sacrificed. The fresh hippocampi of mice were homogenized in RIPA
buffer (Solarbio, R0010, China) containing
1 mM phenyl-
methanesulfonyl fluoride (PMSF: Solarbio, R0010, China) for
30 min. The protein concentrations were determined by BCA pro-
tein assay kit (Solarbio, PC0020, China). Protein samples were run
on 12% Tris-glycine SDS-PAGE gels, and blotted with antibodies
including glial fibrillary acidic protein antibody (Affinity Bio-
sciences, USA, Lot BF0345) and ionized calcium-binding adapter
molecule 1 antibody (Abcam, Cambridge, UK, Lot ab5076). HRP-
coupled antibody (Affinity Biosciences, USA) at 1:5000 dilution in
blocking buffer for 2 h at room temperature. The immunocom-
plexes were detected using ECL reagent (Millipore, Billerica, MA)
and scanned using the Tanon imaging system (Tanon Science &
Technology Co., Ltd, China). All the images were quantified by
densitometry using Image J software. All results were given as the
mean SD; Significance between the sham groups was determined
by analysis of variance (ANOVA), followed by Fisher’s PLSD pro-
cedure for post hoc comparison which was used to verify the sig-
nificance between two means. P values of less than 0.05 were
considered significant. The data were analyzed by SPSS software.
All results calculated were from four independent samples except
for melatonin group, whose result was from three independent
samples.
4.4.10.1. LPS-induced hippocampal neuroinflammatory model of
mice. Seventy 8-week-old male Kunming mice weighing from 20 g
to 25 g were purchased from the GLP Laboratory of Lanzhou Uni-
versity. The mice were divided into seven groups randomly, which
were blank group (n ¼ 10), sham operation group (n ¼ 10), model
group (n ¼ 10), compound 3 treated group (n ¼ 10), compound 16
treated group (n ¼ 10), melatonin treated group (n ¼ 10), salicylic
acid treated group (n ¼ 10). Then LPS injections were built in both
model group, melatonin group, salicylic acid group and compounds
treated groups, while sham operation group was injected with
saline of the same volume. After that, the specific operational
procedures were as follows. Firstly, Kunming mice were anaes-
thetized by intraperitoneal injection with 0.08 mL of chloral hy-
drate (10%). After exposing the parietal bone, the bregma point
could be found according to mouse stereotaxic atlas and regarded
as an origin. The location situated 2.00 mm backwards from the
origin and 1.00 mm leftwards or rightwards to the sagittal suture
was located by a locating pin, where the hippocampus was situated
in. Then a small round hole was drilled on the hippocampus with a
borehole needle, and an injection needle was lowered by 2e3 mm
through the borehole, piercing the lateral ventricle of the mice.
4.4.10.2. Acute toxicity study. Acute toxicity studies were carried
out according to OECD guidelines. Briefly, thirty 8-week-old

18
X. Fan et al. / European Journal of Medicinal Chemistry 193 (2020) 112217
Kunming mice weighing from 20 g to 25 g were purchased from the
GLP Laboratory of Lanzhou University. The mice were divided into
three groups randomly (n ¼ 10, five female and five male mice). The
first group was treated with saline containing 1% methyl cellulose,
the second group was treated with compound 3 at a dose of
1000 mg/kg (suspended in saline containing 1% methyl cellulose),
the third group was treated with compound 16 at a dose of
1000 mg/kg (suspended in saline containing 1% methyl cellulose).
All treatments were operated by gavage with a single dose. The
treated animals were observed continuously for the first 4 h fol-
lowed by periodic monitoring for another 20 h. Then animals were
observed once daily for a period of 14 days. After 14 days, all ani-
mals were sacrificed to conduct histological studies on liver, kidney,
heart, and stomach by using H and E staining method.
Acknowledgements
This work is dedicated to Lanzhou University on the occasion of
its 110th birthday. It was financially supported by the Recruitment
Program of Global Experts (1000 Talents Plan), and the Funda-
mental Research Funds for the Central Universities (lzujbky-2019-
ct08).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.112217.
Abbreviations
IC50
half-maximum inhibitory concentration
Cyclooxygenase-2
Cyclooxygenase-1
4.4.10.3. Pharmacokinetics study. Eighteen 8-week-old male SD
rats weighing from 250 g to 280 g were purchased from the GLP
Laboratory of Lanzhou University. The rats were randomly distrib-
uted into three groups (n ¼ 6). The rats were deprived of water and
fasted overnight prior to treatment. Melatonin, compounds 3, 16
were injected intraperitoneally at a dose of 50 mg/kg. After single
administration of compounds 3 and 16, plasma samples were
collected from the orbital venous plexus at the following time
points: 5 min, 15 min, 45 min, 1 h, 3 h, 5 h, 8 h, 10 h, 24 h for
compound 3; 15 min, 45 min, 1 h, 1.5 h, 3 h, 5 h, 7 h, 9 h, 11 h, 24 h
for compound 16; 5 min, 30 min, 1 h, 2.5 h, 3.5 h for melatonin.
Every blood samples were collected in heparinized tubes followed
by separating with centrifugation (2500 rpm, 6 min, 4 ꢂC). Then the
plasma fraction was separated and stored at ꢀ80 ꢂC for LC-MS
COX-2
COX-1
LPS
CNS
PBS
lipopolysaccharide
Central nervous system
Phosphate-buffered saline
nitric oxide
Tumor necrosis factor-alpha
Interleukin-10
prostaglandin E2
Inducible nitric oxide synthase
Cornu Ammonis
Standard deviation
Intraperitoneally
Ionized calcium-binding adapter molecule 1
Glial fibrillary acidic protein
Alzheimer’s disease
NO
TNF-
a
IL-10
PGE2
iNOS
CA1
SD
i.p
Iba-1
GFAP
AD
analysis. For plasma sample preparation, 100 mL of samples were
accurately measured into each new centrifugal tube and mixed
thoroughly by vortex for 30 s. Then, methanol was added to each
plasma sample and mixed thoroughly. After centrifugation at
16000 rpm for 10 min at 4 ꢂC, the supernatant of each sample was
precisely transferred to an evaporator tube, and dried in nitrogen
flow. After drying, the residue was redissolved in methanol, and
ROS
reactive oxygen species
DCFH-DA 20,70-dichlorodi-hydrofluorescein
DMSO
FBS
LD50
M
dimethyl sulphoxide
fetal bovine serum
dose that is lethal in 50% of test subjects
multiplet (spectral)
1-Hydroxybenzotriazole
then analyzed after filtered by filter membrane of 0.22
mm. For
liquid chromatography and mass spectrometry conditions, chro-
matographic analyses were performed with a column: Eclipse Plus
HOBt
C18 (4.6 ꢁ 150 mm, 4
mm). The HPLC mobile phases consisted of
References
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directly introduced into ES-API (Agilent InfinityLab LC/MSD; USA).
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4.5. Statistical analysis
All results are given as the mean SD; significance between the
groups was determined by analysis of variance (ANOVA), followed
by Fisher’s PLSD procedure for post hoc comparison, to verify the
significance between two means. P values of less than 0.05 were
considered significant. The data were analyzed by SPSS software.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
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