A novel 1,8-naphthyridine-2-carboxamide derivative attenuates inflammatory responses and cell migration in lps-treated bv2 cells via the suppression of ros generation and tlr4/myd88/nf-κb signaling pathway

Article abstract of DOI:10.3390/ijms22052527

Novel 1,8-naphthyridine-2-carboxamide derivatives with various substituents (HSR2101-HSR2113) were synthesized and evaluated for their effects on the production of pro-inflammatory mediators and cell migration in lipopolysaccharide (LPS)-treated BV2 microglial cells. Among the tested compounds, HSR2104 exhibited the most potent inhibitory effects on the LPS-stimulated production of inflammatory mediators, including nitric oxide (NO), tumor necrosis factor-α, and interleukin-6. Therefore, this compound was chosen for further investigation. We found that HSR2104 attenuated levels of inducible NO synthase and cyclooxygenase 2 in LPS-treated BV2 cells. In addition, it markedly suppressed LPS-induced cell migration as well as the generation of intracellular reactive oxygen species (ROS). Moreover, HSR2104 abated the LPS-triggered nuclear translocation of nuclear factor-κB (NF-κB) through inhibition of inhibitor kappa Bα phosphorylation. Furthermore, it reduced the expressions of Toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88) in LPS-treated BV2 cells. Similar results were observed with TAK242, a specific inhibitor of TLR4, suggesting that TLR4 is an upstream regulator of NF-κB signaling in BV2 cells. Collectively, our findings demonstrate that HSR2104 exhibits anti-inflammatory and anti-migratory activities in LPS-treated BV2 cells via the suppression of ROS and TLR4/MyD88/NF-κB signaling pathway. Based on our observations, HSR2104 may have a beneficial impact on inflammatory responses and microglial cell migration involved in the pathogenesis of various neurodegenerative disorders.

Full text of DOI:10.3390/ijms22052527

International Journal of
Molecular Sciences
Article
A Novel 1,8-Naphthyridine-2-Carboxamide Derivative
Attenuates Inflammatory Responses and Cell Migration in
LPS-Treated BV2 Cells via the Suppression of ROS Generation
and TLR4/Myd88/NF-κB Signaling Pathway
Phuong Linh Nguyen 1, Bich Phuong Bui 1, Heesoon Lee 2,* and Jungsook Cho 1,
*
1
Department of Pharmacy, College of Pharmacy and Integrated Research Institute for Drug Development,
Dongguk University-Seoul, Goyang, Gyeonggi 10326, Korea; phuonglinh212126@gmail.com (P.L.N.);
bichphuong2306@gmail.com (B.P.B.)
Department of Pharmaceutics, College of Pharmacy, Chungbuk National University, Osong,
Cheongju 28160, Korea
2
*
Correspondence: medchem@chungbuk.ac.kr (H.L.); neuroph@dongguk.edu (J.C.)
Abstract: Novel 1,8-naphthyridine-2-carboxamide derivatives with various substituents (HSR2101-
HSR2113) were synthesized and evaluated for their effects on the production of pro-inflammatory
mediators and cell migration in lipopolysaccharide (LPS)-treated BV2 microglial cells. Among the
tested compounds, HSR2104 exhibited the most potent inhibitory effects on the LPS-stimulated
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
production of inflammatory mediators, including nitric oxide (NO), tumor necrosis factor-α, and
Citation: Nguyen, P.L.; Bui, B.P.; Lee,
H.; Cho, J. A Novel
interleukin-6. Therefore, this compound was chosen for further investigation. We found that HSR2104
attenuated levels of inducible NO synthase and cyclooxygenase 2 in LPS-treated BV2 cells. In addition,
it markedly suppressed LPS-induced cell migration as well as the generation of intracellular reactive
oxygen species (ROS). Moreover, HSR2104 abated the LPS-triggered nuclear translocation of nuclear
factor-κB (NF-κB) through inhibition of inhibitor kappa Bα phosphorylation. Furthermore, it reduced
the expressions of Toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88) in
LPS-treated BV2 cells. Similar results were observed with TAK242, a specific inhibitor of TLR4,
1,8-Naphthyridine-2-Carboxamide
Derivative Attenuates Inflammatory
Responses and Cell Migration in
LPS-Treated BV2 Cells via the
Suppression of ROS Generation and
TLR4/Myd88/NF-κB Signaling
Pathway. Int. J. Mol. Sci. 2021, 22,
2527. https://doi.org/10.3390/
ijms22052527
suggesting that TLR4 is an upstream regulator of NF-
findings demonstrate that HSR2104 exhibits anti-inflammatory and anti-migratory activities in LPS-
treated BV2 cells via the suppression of ROS and TLR4/MyD88/NF- B signaling pathway. Based on
κB signaling in BV2 cells. Collectively, our
κ
our observations, HSR2104 may have a beneficial impact on inflammatory responses and microglial
cell migration involved in the pathogenesis of various neurodegenerative disorders.
Academic Editor: Paola Poma
Received: 23 January 2021
Accepted: 26 February 2021
Published: 3 March 2021
Keywords: 1,8-naphthyridine-2-carboxamides; nuclear factor-kappa B; Toll-like receptor; BV2 mi-
croglial cells; inflammatory mediators; cell migration; neuroinflammation
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
1. Introduction
Microglia, a unique and highly specialized population of brain-resident macrophages,
are considered a double-edged sword due to their neuroprotective and neurotoxic ef-
fects [
1
,
2
]. Microglial cells play essential roles in brain development and are responsible for
]. However, upon brain damage
maintaining central nervous system (CNS) homeostasis [
3
or injury, they become activated and their morphology and function undergo a dramatic
transformation. Over-activated microglia produce excessive amounts of pro-inflammatory
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
mediators including inflammatory cytokines, tumor necrosis factor-
(NO), and reactive oxygen species (ROS), which eventually trigger neurodegenerative pro-
cesses in the brain [ ]. Moreover, microglial stimulation causes rapid changes in their
migratory properties to recruit other microglial cells toward the lesion sites [ ]. During
the last few decades, the hyperactivity of microglia in response to various inflammatory
α (TNF-α), nitric oxide
1,4,5
6
Int. J. Mol. Sci. 2021, 22, 2527. https://doi.org/10.3390/ijms22052527
https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2021, 22, 2527
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stimuli has been implicated in the onset and development of neuropathies in many neu-
rodegenerative diseases including Alzheimer’s disease (AD), Parkinson’s disease (PD),
and multiple sclerosis [7–10].
Toll-like receptors (TLRs), which are largely expressed on sentinel cells such as
macrophages and microglia, participate in the first line of defense against invading
pathogens and play important roles in innate immune responses [11
TLRs identified, TLR4 was shown to be mainly expressed on microglia and to mediate neu-
roinflammatory responses through the initiation of inflammatory cascades [13 15]. Briefly,
,12]. Among the many
–
activated TLR4 recruits an intracellular adaptor protein, myeloid differentiation factor 88
(MyD88), which subsequently triggers several downstream signaling pathways. The major
pathway initiated by TLR4-MyD88 interactions includes the nuclear translocation of nuclear
factor κB (NF-κB) via the phosphorylation of inhibitor κB (IκB) proteins [16]. The NF-κB
translocated to the nucleus promotes the transcription of a series of pro-inflammatory
mediators and enzymes associated with inflammatory processes, ultimately leading to
neurodegeneration [12,16,17]. In addition, TLR4 activation is known to cause oxidative
stress through the enhanced production of intracellular ROS, which further exacerbates
inflammatory processes [18]. Therefore, modulation of microglial activation by suppression
of the TLR4/MyD88/NF-κB signaling pathway may be a promising strategy to prevent or
alleviate neuroinflammation associated with various neurodegenerative brain disorders
including AD and PD.
Lipopolysaccharide (LPS), one of the best studied immuno-stimulatory endotoxins
expressed in the external membrane of Gram-negative bacteria, is commonly used to
induce microglial activation [15
cesses by inducing the release of various cytokines and eicosanoids through the activa-
tion of TLR4 [19 21]. Using LPS-stimulated BV2 microglial cells, we recently reported
,19]. LPS is known to trigger neuroinflammatory pro-
–
the anti-inflammatory and anti-migratory effects of 1,2,3,4-tetrahydroquinoline [22] and
isoquinoline-1-carboxamide derivatives [23]. In an effort to seek other agents possessing
anti-inflammatory properties, we modified the scaffold of these compounds to naphthyridine
to create a series of 1,8-naphthyridine-2-carboxamide derivatives with N-hydroxyphenyl, N-
methoxyphenyl, N-(trifluoromethyl)phenyl, N-bis(trifluoromethyl)phenyl, or N-chlorophenyl
substituents (Figure 1).
Substituents
Compound No.
R1
R2
R3
R4
HSR2101
HSR2102
HSR2103
HSR2104
HSR2105
HSR2106
HSR2107
HSR2108
HSR2109
HSR2110
HSR2111
HSR2112
HSR2113
OH
H
H
OMe
H
H
H
OH
H
H
OMe
H
H
H
OH
H
H
OMe
H
H
CF
H
H
H
H
H
H
H
H
H
H
H
H
CF
H
H
H
CF
H
3
H
CF
H
CF
H
3
3
H
H
Cl
H
H
3
3
Cl
H
Cl
Figure 1. Parent structure of 1,8-naphthyridine-2-carboxamides and their substituents.
The present study evaluated the effects of these novel synthetic derivatives on LPS-
induced pro-inflammatory mediators and cell migration in BV2 cells. We found that, among
the tested compounds, N-(2-methoxyphenyl)naphthyridine-2-carboxamide (HSR2104)

Int. J. Mol. Sci. 2021, 22, 2527
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markedly attenuated the production of pro-inflammatory mediators and cell migration.
The signaling pathway involved in the inhibition of inflammatory responses and cell
migration was also investigated in this study.
2. Results
2.1. Effects of 1,8-Naphthyridine-2-Carboxamide Derivatives on the Viability of BV2 Cells
We first examined whether the newly synthesized 1,8-naphthyridine-2-carboxamide
derivatives had any cytotoxic effect on BV2 cells. The cells were treated with the compounds
at two different concentrations (30 µM and 100 µM) for 24 h, and the viability of the cells was
assessed by MTT reduction assay. We found that the derivatives with N-hydroxylphenyl
(HSR2101-2103) and N-methoxylphenyl (HSR2104-2106) substituents did not induce sig-
nificant cytotoxicity in BV2 cells at the concentrations tested (Figure 2). In contrast, the
derivatives with an N-(trifluoromethyl)phenyl substituent at R2 (HSR2108) or R3 position
(HSR2109), N-bis(trifluoromethyl)phenyl substituent at R2 and R4 positions (HSR2110),
and N-chlorophenyl substituent at R1 (HSR2111) or R2 position (HSR2112) induced marked
cytotoxicity at both concentrations tested. Interestingly, however, compounds with an
N-(trifluoromethyl)phenyl substituent at R1 position (HSR2107) and N-chlorophenyl sub-
stituent at R3 position (HSR2113) induced no significant toxicity (Figure 2).
Control
Compound 30 μM
Compound 100 μM
140
120
100
*
80
*
*
*
*
*
*
*
*
*
60
40
20
0
l
1
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7
8
9
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o
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r
t
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2
n
2
2
2
2
2
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2
2
2
2
2
o
R
R
R
R
R
R
R
R
R
R
R
R
R
C
S
S
S
S
S
S
S
S
S
S
S
S
S
H
H
H
H
H
H
H
H
H
H
H
H
H
Figure 2. Effects of 1,8-naphthyridine-2-carboxamide derivatives on the viability of BV2 cells. Cells
were treated with the compounds at concentrations of 30 M or 100 M for 24 h. Control cells
were treated with vehicle only. The cell viability was determined by MTT assay, as described in the
Materials and Methods section. Data are expressed as the mean SEM from three independent
µ
µ
±
experiments performed in duplicate. * p < 0.05 vs. vehicle-treated control cells.
We also examined the effects of these compounds on the viability of LPS-treated
BV2 cells. Similar results were observed for all derivatives, except HSR2113. Although
HSR2113 at the concentration of 30
µM did not induce cytotoxicity in LPS-treated BV2 cells,
it induced considerable toxicity at 100
µM (data not shown). Based on our findings, seven
compounds (HSR2101–HSR2107) that did not show significant cytotoxicity were chosen
for further study. HSR2113 was also investigated further because it did not show toxicity
in BV2 cells in the absence of LPS.
2.2. Effects of 1,8-Naphthyridine-2-Carboxamide Derivatives on the LPS-Stimulated Production of
Pro-Inflammatory Mediators in BV2 Cells
Next, we evaluated the effects of the eight selected compounds (HSR2101–HSR2107
and HSR2113) on the production of LPS-stimulated pro-inflammatory mediators. In
agreement with our previous reports [22
increased the levels of pro-inflammatory molecules, including NO, TNF-
the culture media (Figure 3). When the cells were cotreated with LPS and the selected
,
23], LPS treatment of BV2 cells dramatically
α
, and IL-6, in

Int. J. Mol. Sci. 2021, 22, 2527
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compounds at the concentrations of 1, 10, 30, and 100
µM, all tested compounds exerted
significant and concentration-dependent inhibition of the LPS-stimulated production of
these inflammatory mediators (Figure 3).
(A)
(B)
(C)
Control
Control
Control
LPS
LPS
LPS
LPS + Compound 1 μM
LPS + Compound 10 μM
LPS + Compound 30 μM
LPS + Compound 100 μM
LPS + Compound 1 μM
LPS + Compound 10 μM
LPS + Compound 30 μM
LPS + Compound 100 μM
LPS + Compound 1 μM
LPS + Compound 10 μM
LPS + Compound 30 μM
LPS + Compound 100 μM
5000
4000
3000
2000
1000
0
6000
5000
4000
3000
2000
1000
0
60
50
40
30
20
10
0
#
#
#
*
*
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*
*
*
*
*
*
*
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*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
l
l
S
R
1
R
2
R
3
R
4
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R
6
7
3
1
S
R
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3
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4
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R
6
7
1
1
3
o
o
P
0
S
0
S
0
S
0
S
0
S
0
S
0
1
2
P
r
0
S
0
S
0
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S
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S
0
r
t
t
1
1
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1
1
1
1
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1
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1
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1
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L
n
n
2
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2
H
2
2
2
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S
2
2
2
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2
2
H
2
o
o
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R
R
R
S
H
C
C
S
S
H
H
H
H
H
H
H
H
H
H
H
H
Figure 3. Concentration-dependent inhibition of the lipopolysaccharide (LPS)-stimulated production of pro-inflammatory
mediators by the selected 1,8-naphthyridine-2-carboxamide derivatives in BV2 cells. Cells were cotreated with LPS (1
µ
g/mL) and the indicated concentrations of selected compounds for 24 h. Control cells were treated with vehicle only.
Culture media were collected and analyzed for NO ( ), TNF- ), and IL-6 ( ), as described in the Materials and Methods
section. Data are expressed as the mean SEM from three independent measurements performed in duplicate. # p < 0.05
and * p < 0.05 vs. vehicle-treated control cells and LPS-treated cells, respectively.
A
α
(B
C
±
Using the data shown in Figure 3, the IC50 values of compounds that inhibited NO,
TNF- , and IL-6 production were calculated (Table 1). Compounds substituted at the ortho
position (R1) (HSR2101, HSR2104, HSR2107) exhibited better inhibitory activities against
the LPS-stimulated production of TNF- than those with meta (R2) (HSR2102, HSR2105) or
para (R3) substitutions (HSR2103, HSR2106, HSR2113). Of those compounds that induced
a marked inhibition of TNF- production, HSR2104 (N-methoxyphenyl substituent) and
α
α
α
HSR2107 (N-trifluoromethylphenyl substituent) induced a higher inhibition of NO produc-
tion than HSR2101 (N-hydroxyphenyl substituent). Furthermore, IL-6 production in the
LPS-treated BV2 cells was markedly inhibited by HSR2104, whereas only mild inhibition
was observed by HSR2107. Taken together, HSR2104 (N-(2-methoxyphenyl)naphthyridine-
2-carboxamide) exhibited the most potent inhibition of the LPS-induced production of NO,
TNF-α, and IL-6 in BV2 cells. Therefore, we focused on HSR2104 for further characteriza-
tion of its anti-inflammatory potential and the underlying mechanism(s) of action. Prior to
further characterization of anti-inflammatory properties of HSR2104, we verified that the
levels of NO, TNF-α, and IL-6 were not influenced by HSR2104 treatment at 100 µM for
24 h in the absence of LPS).
Table 1. IC50 values of the selected 1,8-naphthyridine-2-carboxamide derivatives for the inhibition of NO, TNF-
α
, and IL-6
production in LPS-treated BV2 cells.
IC50 Values (µM) *
Inflammatory
Mediators
HSR2101
HSR2102
HSR2103
HSR2104
HSR2105
HSR2106
HSR2107
HSR2113
NO
TNF-α
IL-6
>100
30.47
>100
32.13
52.09
55.74
45.38
59.36
78.91
25.43
33.24
32.03
50.72
44.61
50.50
57.02
57.21
29.16
28.03
37.26
96.06
26.49
91.93
27.98
* IC50 value represents the concentration of each compound required to inhibit LPS-stimulated NO, TNF-α, and IL-6 production by 50%.

Int. J. Mol. Sci. 2021, 22, 2527
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2.3. Effects of HSR2104 on the LPS-Induced iNOS and COX2 Expression in BV2 Cells
We then investigated whether the inhibition of the pro-inflammatory mediators by
HSR2104 was associated with the modulation of inducible NO synthase (iNOS) and cy-
clooxygenase 2 (COX2) expressions. Western blotting analyses demonstrated that LPS
treatment of BV2 cells markedly enhanced the expressions of iNOS and COX2 (Figure 4),
consistent with our previous reports [22,23]. HSR2104 concentration-dependently sup-
pressed the protein levels of LPS-induced iNOS and COX2 (Figure 4). These findings
suggest that anti-inflammatory properties of HSR2104 may, at least in part, be attributed to
the reduced expressions of iNOS and COX2 in the LPS-treated BV2 cells.
(A)
(B)
1000
800
600
400
200
0
#
250
200
150
100
50
#
*
*
*
*
*
*
0
LPS
−
-
+
+
+
+
+
+
+
+
+
+
LPS
LPS
−
-
+
+
+
+
+
+
+
+
+
+
LPS
HSR 2104 (μM)
HSR2104 (μM)
0
0
0
0
1
1
10
10
30
30
100
100
HSR2104 (μM)
HSR 2104 (μM)
0
0
0
0
1
1
10
10
30 100
30 100
iNOS
COX2
β-actin
β-actin
Figure 4. Inhibition of the LPS-induced expressions of iNOS and COX2 by HSR2104 in BV2 cells.
Cells were cotreated with LPS (1 g/mL) and the indicated concentrations of HSR2104 for 24 h.
Control cells were treated with vehicle only. The protein levels of iNOS ( ) and COX2 ( ) were
determined by Western blotting analyses using anti-iNOS and COX2 antibodies, respectively. -actin
was used as an internal control. Representative blots are shown. Data are presented as the mean
µ
A
B
β
±
SEM from three independent experiments. # p < 0.05 and * p < 0.05 vs. vehicle-treated control and
LPS-treated cells, respectively.
2.4. Effect of HSR2104 on the LPS-Induced BV2 Cell Migration
Microglial cell movement is conjunctly associated with inflammatory responses [
Therefore, we investigated the effect of HSR2104 on LPS-induced BV2 cell migration using
wound healing and transwell migration assays. As described in our previous studies [22 23],
6,24].
,
LPS dramatically enhanced BV2 cell migration during 24 h of treatment (Figure 5). Our
results revealed that the concomitant treatment of cells with LPS and HSR2104 at the
concentrations of 10 µM and above markedly repressed LPS-induced cell migration in both
assays (Figure 5). Additional wound healing assay showed that the migratory activity of
HSR2104-treated cells in the absence of LPS was similar to that of vehicle-treated control
cells (data not shown).
2.5. Effect of HSR2104 on the LPS-Stimulated Generation of Intracellular ROS in BV2 Cells
The generation of intracellular ROS in microglia is acknowledged to trigger neuroin-
flammatory responses through the activation of several signaling pathways including the
NF-κB pathway [18]. Thus, we examined the effect of HSR2104 on the generation of ROS
in LPS-stimulated BV2 cells. The production of intracellular ROS in the cells treated with
LPS was significantly increased to approximately 180%, compared to that in the control-
treated cells (Figure 6). Cotreatment with LPS and HSR2104 attenuated ROS generation
in a concentration-dependent manner (Figure 6A). The inhibition of ROS generation by
HSR2104 in LPS-treated BV2 cells was further confirmed by fluorescence microscopic im-
ages, which demonstrated a marked reduction of the LPS-stimulated fluorescence signals
by HSR2104 (Figure 6B).

Int. J. Mol. Sci. 2021, 22, 2527
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(A)
LPS + HSR2104 (µM)
10 30
300
250
200
150
100
50
Control
LPS
1
100
*
0 h
*
*
0
LPS
LPS
HSR 2104 (μM)
HSR2104 (μM)
-
+
+
+
+
+
+
100
100
24 h
−
+
+
+
+
0
0
1
1
10
10
30
30
0
0
(B)
1000
800
600
400
200
#
*
LPS + HSR2104 (µM)
10 30
Control
LPS
1
100
*
*
0
LPS
-
+
0
+
1
+
+
+
HSR 2104 (μM)
0
10
30
100
Figure 5. Suppression of LPS-induced BV2 cell migration by HSR2104. Cells were cotreated with LPS (1
indicated concentrations of HSR2104 for 24 h and the extent of cell migration was measured by wound healing (
transwell migration ( ) assays, as described in the Materials and Methods section. Control cells were treated with vehicle
only. Representative microscopic images are shown (scale bar, 20 m). Data are expressed as the mean SEM from at least
three independent experiments. # p < 0.05 and * p < 0.05 vs. vehicle-treated control and LPS-treated cells, respectively.
µg/mL) and the
A
) and
B
µ
±
(A)
(B)
200
150
100
50
LPS + HSR2104 (µM)
10 30
#
Control
LPS
1
100
*
*
*
0
LPS
-
+
+
+
+
+
LPS
−
+
+
+
+
+
HSR 2104 (μM)
0
0
1
10
30
100
HSR2104 (μM)
0
0
1
10 30 100
Figure 6. Inhibition of LPS-induced intracellular ROS production by HSR2104 in BV2 cells. Cells were cotreated with LPS (1
g/mL) and the indicated concentrations of HSR2104 for 24 h. Intracellular ROS levels were determined using DCFH-DA
as a fluorescent probe (A), as described in the Materials and Methods section. Control cells were treated with vehicle only.
Data are expressed as the mean SEM from three independent experiments in triplicate. Representative fluorescence
) are shown (scale bar, 2 m). # p < 0.05 and * p < 0.05 vs. vehicle-treated control and LPS-treated cells,
µ
±
microscopic images (
B
µ
respectively.
2.6. Effects of HSR2104 on the LPS-Induced IκBα Phosphorylation and NF-κB Translocation in
BV2 Cells
Next, we investigated the involvement of NF-
induced inflammatory responses by HSR2104. As reported previously [22
ment induced nuclear translocation of NF- B in BV2 cells. Immunoblotting analyses
indicated that, upon LPS treatment, the level of NF- B p65 subunit was reduced in the
cytosolic fraction, while it was increased in the nuclear fraction (Figure 7A,B). LPS-induced
NF- B translocation was also manifested in the immunofluorescence images, showing
κ
B pathway in the inhibition of LPS-
,
23], LPS treat-
κ
κ
κ

Int. J. Mol. Sci. 2021, 22, 2527
7 of 19
strong fluorescence intensity of NF-
κ
B in the nuclei of LPS-treated cells (Figure 7C). Both
immunoblotting and immunocytochemical analyses demonstrated that HSR2104 reversed
the LPS-induced NF- B translocation (Figure 7A–C). The expression of NF- B p65 subunit
was augmented in the cytosolic fraction by HSR2104, whereas it was significantly reduced
in the nuclear fraction (Figure 7A,B). Similarly, the fluorescence intensity of NF- B in the
M (Figure 7C).
κ
κ
κ
nucleus was markedly diminished by HSR2104 at the concentration of 100
µ
(A)
(B)
160
120
100
80
60
40
20
0
#
*
*
120
80
40
0
*
*
#
LPS
-
+
+
+
+
+
+
+
LPS
−
+
+
+
LPS
LPS
-
+
+
0
0
+
+
+
+
+
+
−
+
+
HSR
H
2
S
1
R
02
4
10
(
4
μ
(μ
M
M
)
)
0
0
0
0
1
1
10
13
0
31000 100
HSR 2104 (μM)
HSR2104 (μM)
0
0
1
1
10
10
30 100
30 100
NF-κB
NF-κB
β-actin
Lamin B1
(C)
(D)
LPS + HSR2104
Control
LPS
200
150
100
50
#
*
*
DAPI
NF-κB
0
LPS
LPS
-
+
−
+
+
+
+
+
+
+
+
+
HSR2104 (μM)
HSR 2104 (μM)
0
0
0
1
1
10
10 30
30 100
100
0
p-IκBα
IκBα
Merge
β-actin
Figure 7. Inhibition of NF-
κ
B nuclear translocation by HSR2104 through the inhibition of I
g/mL) and the indicated concentrations of HSR2104 for 24 h. Control
) and nuclear ( ) fractions were prepared and Western blotting analyses
B p65 antibodies, as described in the Materials and Methods section. -actin and lamin B1
were used as internal controls for the cytosolic and nuclear fractions, respectively. Representative blots are shown. ( ) Cells
were cotreated with LPS (1 µg/mL) and HSR2104 at 100 µM for 24 h, and immunocytochemical analyses were performed.
The localization of NF- B was visualized in green using anti-NF- B p65 antibodies, and the nuclei were visualized in blue
with DAPI staining. Representative immunofluorescence images are shown. The white arrow in each image indicates
the magnified cell shown in the inset (scale bar, 50 m). ( ) Cells were cotreated with LPS (1 g/mL) and the indicated
concentrations of HSR2104 for 24 h. Cell lysates were prepared and analyzed by Western blotting with anti-phospho-I
κBα phosphorylation in LPS-
treated BV2 cells. Cells were cotreated with LPS (1
cells were treated with vehicle only. Cytosolic (
were performed using anti-NF-
µ
A
B
κ
β
C
κ
κ
µ
D
µ
κBα
and anti-I
κ
Bα
antibodies.
β-actin was used as an internal control. Representative blots are shown. Data are presented as
the mean
LPS-treated cells, respectively.
±
SEM from at least three independent experiments. # p < 0.05 and * p < 0.05 vs. vehicle-treated control and
It is widely recognized that the nuclear translocation of NF-
rylation of I 15 16]. Thus, we also tested the effect of HSR2104 on the phosphorylation
of I . As shown in Figure 7D, HSR2104 abated the LPS-induced phosphorylation of I
at the concentrations of 30 M and above. Collectively, these results indicate that HSR2104
suppresses the nuclear translocation of NF- B through inhibition of I phosphorylation.
κB is mediated by phospho-
κ
B
α
[
,
κ
Bα
κBα
µ
κ
κBα

Int. J. Mol. Sci. 2021, 22, 2527
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2.7. Effects of HSR2104 on the LPS-Induced Expressions of TLR4 and MyD88 in BV2 Cells
The crucial role of the TLR4 pathway in the production of pro-inflammatory molecules
has been elucidated in many studies [14 16 23]. In brief, the activation of TLR4 by LPS
initiates inflammatory cascades recruiting MyD88, an intracellular adapter protein, and
consequently stimulating downstream signaling including the NF- B pathway. In this
,
,
κ
study, we observed that HSR2104 inhibited I
the LPS-induced nuclear translocation of NF-
κ
B
α
phosphorylation, and thereby, suppressed
B in BV2 cells (Figure 7). These findings
κ
prompted us to investigate the role of TLR4/MyD88 signaling in mediating the anti-
inflammatory effects of HSR2104. Immunoblotting analyses revealed that the levels of
TLR4 and MyD88 were significantly increased by LPS in BV2 cells, which was reversed
by HSR2104 by concentration-dependent fashion (Figure 8). These results suggest that
the inhibition of TLR4/MyD88 signaling by HSR2104 may be critically associated with its
anti-inflammatory properties.
(A)
(B)
200
160
#
#
160
120
80
40
0
*
*
120
80
40
0
*
*
LPS
LPS
-
+
+
+
+
+
+
100
100
−
+
+
+
+
LPS
LPS
-
+
+
+
+
+
+
+
−
+
+
+
HSR 2104 (μM)
HSR2104 (μM)
0
0
0
0
1
1
10
10
30
30
HSR 2104 (μM)
HSR2104 (μM)
0
0
0
0
1
1
10
10
30
30
100
100
TLR4
MyD88
β-actin
β-actin
Figure 8. Inhibition of TLR4 and MyD88 expressions by HSR2104 in LPS-treated BV2 cells. Cells were cotreated with
LPS (1 g/mL) and the indicated concentrations of HSR2104 for 24 h. Control cells were treated with vehicle only. The
protein levels of TLR4 ( ) and MyD88 ( ) were determined by Western blotting analyses using anti-TLR4 and anti-MyD88
antibodies, as described in the Materials and Methods section. -actin was used as an internal control. Data are expressed
as the mean SEM from at least three independent experiments. # p < 0.05 and * p < 0.05 vs. vehicle-treated control and
LPS-treated cells, respectively.
µ
A
B
β
±
2.8. Effects of TLR4 Inhibitor on the LPS-Induced Pro-Inflammatory Mediators, NF-κB
Translocation, Cell Migration, and ROS Production in BV2 Cells
The results of the current study demonstrated that HSR2104 inhibited inflamma-
tory responses, cell migration, and ROS generation in LPS-treated BV2 cells. In addition,
HSR2104 inhibited the LPS-induced NF-
To further elucidate the role of TLR4/MyD88 signaling in the anti-inflammatory effects of
HSR2104, we investigated the effects of TAK242, a specific inhibitor of TLR4 [25 26], on
the LPS-induced pro-inflammatory mediators, NF- B translocation, cell migration, and
ROS production in BV2 cells. We found that TAK242, at the concentration of 500 nM,
dramatically inhibited the LPS-induced production of NO, TNF- , and IL-6 (Figure 9A–C).
κB translocation and TLR4/MyD88 expressions.
,
κ
α
In addition, LPS-stimulated iNOS and COX2 expressions were markedly diminished by
TAK242 (Figure 9D,E). Moreover, TAK242 abolished the LPS-induced nuclear transloca-
tion of NF-κB (Figure 9F,G) through the inhibition of IκBα phosphorylation (data not
shown). Furthermore, TAK242 suppressed LPS-induced cell migration in the wound
healing (Figure 9H) and transwell migration (Figure 9I) assays. The generation of ROS
was also quenched by TAK242 (Figure 9J). Collectively, TAK242 and HSR2104 exhibited
similar effects on the LPS-induced pro-inflammatory mediators, NF-κB translocation, cell
migration, and ROS production in BV2 cells. These findings strongly indicate that TLR4

Int. J. Mol. Sci. 2021, 22, 2527
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is an upstream regulator of NF-
κ
B translocation. Moreover, our results confirm that the
inhibition of TLR4/MyD88 signaling by HSR2104 plays a crucial role in the inhibition of
inflammatory responses, cell migration, and ROS generation in BV2 cells.
(A)
(B)
(C)
#
35
30
25
20
15
10
5
3500
3000
2500
2000
1500
1000
500
3500
3000
2500
2000
1500
1000
500
#
#
*
*
*
0
LPS +
TAK 242
Control LPS
0
0
LPS +
TAK 242
LPS +
TAK 242
Control LPS
Control LPS
(D)
(E)
(F)
(G)
160
120
80
120
100
80
60
40
20
0
1000
800
600
400
200
200
160
120
80
#
#
*
#
#
*
*
*
40
40
0
0
0
LPS +
TAK 242
LPS +
TAK 242
LPS +
LPS
Control
LPS
Control
LPS +
TAK 242
Control
LPS
Control
LPS
TAK 242
iNOS
COX2
NF-κB
β-actin
NF-κB
β-actin
β-actin
Lamin B1
(H)
Control
LPS
LPS + TAK242
350
#
#
300
250
200
150
100
50
0 h
*
0
24 h
LPS +
TAK 242
Control
LPS
2000
1600
1200
800
400
0
(I)
#
LPS + TAK242
Control
LPS
#
*
LPS +
TAK 242
Control
LPS
(J)
250
200
150
100
50
#
*
*
0
Control
LPS
TAK242
Figure 9. Effects of TAK242, a TLR4-specific inhibitor, on the LPS-induced pro-inflammatory mediators, NF-
cell migration, and ROS production in BV2 cells. Cells were cotreated with LPS (1 g/mL) and TAK242 at the concentration
of 500 nM for 24 h. Control cells were treated with vehicle only. Culture media were collected and analyzed for NO (A),
κB translocation,
µ

Int. J. Mol. Sci. 2021, 22, 2527
10 of 19
TNF-
blotting analyses using anti-iNOS (
and transwell migration ( ) assays were carried out, as described in the Materials and Methods section, to measure cell
migration. Intracellular ROS levels were determined using DCFH-DA as a fluorescent probe ( ). Representative blots and
images are shown. Data are expressed as the mean SEM from at least three independent experiments. # p < 0.05 and
α
(B
), and IL-6 (
C
) levels, as described in the Materials and Methods section. Cell lysates were used to perform Western
D
), anti-COX2 ( ), and anti-NF- B p65 subunit ( ) antibodies. Wound healing (H)
E
κ
F
,
G
I
J
±
* p < 0.05 vs. vehicle-treated control and LPS-treated cells, respectively.
3. Discussion
It was reported that compounds with naphthyridine scaffolds possess anticancer
and anti-inflammatory activities [27 28]. In particular, 1,8-naphthyridine derivatives was
,
demonstrated to exhibit anti-inflammatory effects through the inhibition of ROS produc-
tion and myeloperoxidase activity in human polymorphonuclear cells [28]. To explore
and expand the pharmacological characterization of 1,8-naphthyridine derivatives, the
present study synthesized thirteen novel 1,8-naphthyridine-2-carboxamide derivatives and
evaluated their anti-inflammatory and anti-migratory activities in BV2 microglial cells.
Microglial activation is closely associated with neuroinflammation, which is involved
in the pathologies of various neurodegenerative diseases including AD, PD, and mul-
tiple sclerosis [
ious cytokines and eicosanoids, which subsequently promote neuroinflammatory pro-
cesses [19 20]. BV2 cells, a murine-derived immortalized microglial cell line, are re-
ported to retain many of functional characteristics of primary microglia including re-
sponses to LPS [29 31]. Upon LPS stimulation, BV2 cells produce various kinds of
neuroinflammatory mediators that are deleterious to neurons, eventually leading to
progressive neurodegeneration and death [32 33]. Therefore, BV2 microglial cells are
7–10]. LPS can activate microglial cells and trigger the secretion of var-
,
–
,
widely used as a substituted model for primary microglia in many experimental set-
tings to characterize the cellular and molecular pathways associated with neuroinflam-
mation [15,19,22,23]. The present study also employed LPS-treated BV2 cells to evaluate
the anti-inflammatory properties of newly synthesized 1,8-naphthyridine-2-carboxamide
derivatives with N-hydroxyphenyl, N-methoxyphenyl, N-(trifluoromethyl)phenyl, N-
bis(trifluoromethyl)phenyl, or N-chlorophenyl substituents (HSR2101-HSR2113) (Figure 1).
Prior to evaluation, we first tested their potential cytotoxicity against BV2 cells. Among
thirteen compounds tested, eight (HSR2101–HSR2107 and HSR2113) did not exhibit signifi-
cant cytotoxicity at concentrations up to 100
investigation (Figure 2).
µM; therefore, these were selected for further
Neuroinflammation is initiated and exacerbated by diverse pro-inflammatory media-
tors secreted from activated microglia. For example, NO functions as a chemoattractant
directing microglial movement toward lesion sites, where it is produced at a high concen-
tration [6,24]. This leads to the accumulation of activated microglial cells at sites of injury,
further promoting their chronic activation [6,34]. In addition, TNF-α and IL-6 are the two
main cytokines associated with inflammatory responses. Their abnormal release also plays
important roles in microglial activation, neuroinflammation, and leukocyte infiltration to
the lesion sites [35
LPS-stimulated production of these pro-inflammatory mediators in BV2 cells. All selected
compounds significantly inhibited the LPS-induced production of NO, TNF- , and IL-6 in
,36]. Therefore, we tested the effects of the selected compounds on the
α
concentration-dependent manners (Figure 3). Based on their IC50 values (Table 1), HSR2104
was chosen for further characterization related to its anti-inflammatory potential and the
underlying mechanism(s) of action. Recently, we reported the potent anti-inflammatory
effect of an isoquinoline-1-carboxamide derivative with a hydroxyl moiety at the R1 po-
sition [23]. In the current study, HSR2104, a 1,8-naphthyridine-2-carboxamide derivative
with an N-methoxyphenyl substituent at the R1 position (Figure 1), exhibited the most
potent inhibition of the LPS-induced production of NO, TNF-α, and IL-6 in BV2 cells
(Figure 3, Table 1). These findings imply that the nature and position of substituents may
be important in determining the anti-inflammatory properties of these derivatives.
Early studies confirmed the existence of iNOS in microglia, which synthesizes high
levels of NO after exposure to stimulant factors such as LPS and viral infections [37,38]. In

Int. J. Mol. Sci. 2021, 22, 2527
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addition, COX2 was identified as a key enzyme involved in inflammatory responses due to
its ability to synthesize various kinds of prostaglandins [39]. The sustained up-regulation
of iNOS and COX2 expression in microglia are involved in the increased production of NO
and pro-inflammatory cytokines, which exacerbates neuroinflammation and progressive
neuronal damage in many neurodegenerative conditions. Therefore, the reduction of
iNOS and COX2 expression in microglia might be a potential target for the alleviation
of neuroinflammation [40
,41]. Our data revealed that HSR2104 markedly inhibited the
LPS-induced expressions of iNOS and COX2 (Figure 4), which might contribute to the
decreased levels of NO, TNF-
Table 1).
α, and IL-6 observed in LPS-treated BV2 cells (Figure 3 and
It is well-established that microglia are highly motile, the main characteristic of im-
mune cells [ ]. In response to inflammatory stimuli or injury, resting microglial cells are
6
activated by various factors including NO released from damaged cells, leading to their
migration towards an infected site [24]. LPS induces the rapid extension of membrane pro-
cesses and migration of microglia [42], which is in accordance with our data using wound
healing and transwell migration assays (Figure 5). Indeed, LPS-induced cell migration was
markedly suppressed by HSR2104 (Figure 5).
Oxidative stress resulting from either decreased anti-oxidant systems, increased ROS
production, or both, plays a crucial role in the development of various degenerative
diseases [43]. It was previously demonstrated that intracellular ROS, including super-
oxide anions (O2–), hydroxyl radical (·OH), and other types of oxygen-derived radicals,
contribute to the etiology of inflammatory processes through the secretion of various
inflammatory cytokines [44]. Interestingly, TLR4 signaling mediates the generation of
ROS in LPS-stimulated microglia, leading to accelerated inflammatory responses [45
Therefore, the pharmacological blockade of ROS generation may abolish LPS-induced
inflammatory responses [47 49]. In keeping with previous reports [45 49 50], our study
,46].
–
, ,
also revealed a marked increase in ROS production in LPS-treated microglia, which was
effectively decreased by HSR2104 (Figure 6). Taken together, our findings demonstrate that
HSR2104 inhibits LPS-induced inflammatory responses and cell migration by suppressing
pro-inflammatory mediators through the inhibition of iNOS and COX2 expressions as well
as ROS generation in BV2 cells.
It is well-established that NF-
inflammatory cytokines [51 53]. NF-
it forms a complex with regulatory I
NF- B heterodimer consisting of p65 and p50 proteins. Phosphorylation of I
kinase (IKK) triggers its dissociation from the cytoplasmic NF- B-I B complex allowing
it to be targeted for degradation. As a result, activated NF- B translocates to the nucleus
and induces the transcription of a series of genes involved in cell survival or apoptosis,
immune responses, and inflammation [53 55]. In order to reveal the involvement of NF-
B in the anti-inflammatory and anti-migratory effects of HSR2104, we tested its effects
κ
B plays a predominant role in the regulation of pro-
B exists in the cytoplasm in an inactive form where
B proteins. I is associated with the inactive
by I
–
κ
κ
κBα
κ
κ
B
α
κB
κ
κ
κ
–
κ
on the nuclear translocation of NF-
κ
B and phosphorylation of I
22 23 42], LPS treatment promoted the nuclear
phosphorylation, which was markedly inhibited
κBα in LPS-treated BV2
cells. Consistent with previous reports [15
translocation of NF- B and enhanced I
, , ,
κ
κBα
by HSR2104 (Figure 7). Our findings indicate that inhibition of the NF-
mediates the anti-inflammatory and anti-migratory effects of HSR2104.
κB pathway also
The innate immune system is composed of a series of encoded pattern recognition
receptors that detect conserved pathogen-associated molecular patterns expressed on
microorganisms [56]. TLRs are a type of pattern recognition receptors that play key
roles in the innate immune system. Among TLRs, TLR4 expressed on microglial cell
membranes is known to recognize LPS as a ligand [12]. Upon binding to LPS, it recruits
an adapter molecule MyD88 which subsequently triggers the activation of downstream
kinases such as IRAK-4 and TAK1 [12
via the activation of IKK, which eventually promotes NF-
inflammatory cytokine production [57]. The present study examined the effect of HSR2104
,
57]. Activated TAK1 then phosphorylates I
κBα
κ
B nuclear translocation and

Int. J. Mol. Sci. 2021, 22, 2527
12 of 19
on LPS-activated TLR4/MyD88 signaling in BV2 cells to clarify the involvement of this
signaling pathway in its anti-inflammatory and anti-migratory effects. We found that LPS
treatment markedly increased the expressions of TLR4 and MyD88 in BV2 cells, which was
dramatically suppressed by HSR2104 (Figure 8). These results suggest that HSR2104 exerts
anti-inflammatory and anti-migratory effects by inhibiting TLR4/MyD88 signaling.
The role of TLR4/MyD88 signaling in the anti-inflammatory effects of HSR2104
was further elucidated in BV2 cells using TAK242, a specific TLR4 inhibitor. We found
that TAK242 inhibited LPS-induced pro-inflammatory mediators as well as iNOS and
COX2 expressions (Figure 9A–E). In addition, TAK242 abolished the LPS-induced nuclear
translocation of NF-κB, cell migration, and ROS generation (Figure 9F–J). Overall, the
results obtained from TAK242 treatment of LPS-stimulated BV2 cells are similar to those
observed with HSR2104 treatment, confirming TLR4/MyD88 signaling is an upstream
regulator of NF-κB during LPS-induced inflammatory processes. Moreover, these findings
indicate that the inhibition of TLR4/MyD88 signaling by HSR2104 plays a crucial role
in the inhibition of inflammatory responses, cell migration, and ROS generation in BV2
cells. Our observations suggest that HSR2104 may inhibit the interaction of TLR4 with
LPS, thereby inhibiting NF-κB nuclear translocation, by which it exerts anti-inflammatory
and anti-migratory effects in BV2 cells. Further studies are currently in progress to clarify
this possibility.
Based on our recent reports [22,23] and the current study, the 1,2,3,4-tetrahydroquinoline
derivative with an N-propionyl substituent (ELC-D-2), isoquinoline-1-carboxamide deriva-
tive with an N-hydroxyphenyl substituent (HSR1101), and HSR2104 inhibited LPS-induced
pro-inflammatory mediators and cell migration in BV2 cells. Although all three compounds
were commonly shown to inhibit NF-
their underlying mechanisms to induce the inhibition of NF-
κ
B translocation to exert anti-inflammatory actions,
B translocation may be differ-
κ
ent. For example, suppression of the JNK pathway was involved in the anti-inflammatory
effects of ELC-D-2, whereas the inhibition of MAP kinases including ERK1/2, JNK, and
p38 MAP kinase, mediated the effects of HSR1101 in BV2 cells. In the present study, we
demonstrated that the suppression of ROS generation and TLR4/MyD88/NF-
pathway mediated the anti-inflammatory and anti-migratory effects of HSR2104. It would
be interesting to identify all the signaling molecules of the TLR4/MyD88/NF- B pathway
κB signaling
κ
mediating the effects of HSR2104 in BV2 cells. Although BV2 cells are commonly utilized
as a substitute for primary microglia in many experimental settings, there have been some
doubts raised as to their suitability [29,30,58]. Therefore, our findings in BV2 cells may
need to be validated using primary microglia. In parallel, the in vivo study of ELC-D-2,
HSR1101, and HSR2104 using various animal models such as formalin-induced paw edema
and licking tests is also necessary to elucidate and compare their anti-inflammatory effects.
A schematic diagram illustrating the potential targets of HSR2104 to inhibit inflam-
matory responses and cell migration in BV2 cells is shown (Figure 10). Briefly, HSR2104
potently inhibited the LPS-induced production of pro-inflammatory mediators includ-
ing NO, TNF-α, and IL-6, possibly through suppression of iNOS and COX2 expressions.
In addition, HSR2104 markedly inhibited LPS-stimulated cell migration and intracel-
lular ROS generation. Moreover, HSR2104 suppressed the LPS-induced TLR4/MyD88
expression, by which it led to subsequent inhibitions of IκBα phosphorylation and NF-κB
translocation. Collectively, our data indicate that HSR2104 exhibits anti-inflammatory and
anti-migratory effects in LPS-treated BV2 cells via the suppression of ROS generation and
the TLR4/MyD88/NF-κB signaling pathway. Based on these findings, HSR2104 may exert
beneficial effects on neuroinflammatory responses and microglial cell migration involved
in the pathogenesis of various neurodegenerative disorders.

Int. J. Mol. Sci. 2021, 22, 2527
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Figure 10. A schematic diagram illustrating the potential targets of HSR2104 to inhibit inflammatory responses and cell
migration in BV2 cells. The solid lines indicate the pathways examined in this study, and the broken lines indicate the
pathways not examined. The red blocked arrows indicate the pathways inhibited by HSR2104. TLR4, Toll-like receptor 4;
MyD88, myeloid differentiation factor 88; ROS, reactive oxygen species; IKK, IκB kinase; IκBα, inhibitory kappa Bα; NF-κB,
nuclear factor-kappa B; iNOS, inducible nitric oxide synthase; COX2, cyclooxygenase-2; IL-6, interleukin-6; TNF-α, tumor
necrosis factor-α; NO, nitric oxide.
4. Materials and Methods
4.1. Synthesis of 1,8-Naphthyridine-2-Carboxamide Derivatives
A series of 1,8-naphthyridine-2-carboxamide derivatives with various substituents
(HSR2101-HSR2113) was generated, according to the procedures of our previous report
on the synthesis of isoquinoline-1-carboxamide derivatives [23]. In brief, the amidation
reaction was accomplished by the treatment of the starting 1,8-naphthyridine-2-carboxylic
acid with appropriate anilines. The ideal condition to obtain a good yield of HSR2101-
2113 was the presence of a pair reagents 1,10-carbonyldiimidazole (CDI) and anhydrous
dichloromethane (DCM) at reflux for 12 h.
The synthesis of N-substitutedphenyl-1,8-naphthyridine-2-carboxamide derivatives
are outlined in Scheme 1. Analytical and spectral data of these compounds (HSR2101–
HSR2113) are provided in the Supplementary Materials.
For our experiments, a 10 mM stock solution of each compound was prepared in
dimethyl sulfoxide (DMSO), which was serially diluted to provide the indicated concentra-
tions of each compound tested in this study.

Int. J. Mol. Sci. 2021, 22, 2527
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Scheme 1. Synthesis of 1,8-naphthyridine-2-carboxamide derivatives.
4.2. Chemicals and Reagents
Dulbecco’s modified Eagle medium (DMEM), Alexa Fluor 488-conjugated anti-mouse
0
immunoglobulin G (IgG), and 4 ,6 diamidino-2-phenylindole dihydrochloride (DAPI) were
obtained from Thermo Scientific (Rockford, IL, USA). Antibiotic-antimycotic reagent and fe-
tal bovine serum (FBS) were purchased from Gibco BRL (Grand Island, NY, USA). TAK242,
LPS (Escherichia coli, serotype 011:B4), 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT), anti-
from Sigma-Aldrich (St. Louis, MO, USA). Antibodies specifically recognizing TLR4,
phosphorylated I , and lamin B1 were supplied by Abcam (Cambridge, MA, USA),
and antibodies recognizing iNOS, COX2, MyD88, NF- B p65, non-phospho-I , and
β-actin antibody, and dimethyl sulfoxide (DMSO) were obtained
κBα
κ
κBα
horseradish peroxidase (HRP)-conjugated anti-rabbit and anti-mouse IgG were purchased
from Cell Signaling Technology (Danvers, MA, USA).
4.3. BV2 Cell Culture, Treatment of Cells, and Determination of Cell Viability
BV2 microglial cells were cultured in DMEM supplemented with 10% FBS and 1%
antibiotics at 37 ◦C in an incubator with 95% O2/5% CO2 as previously described [22
,23].
The cells were seeded into 24-well plates at a density of 2.5
×
105 cells/well and incubated
at 37 ◦C for 24 h. To evaluate any potential cytotoxicity of test compounds in BV2 cells,
the cultured cells were gently washed with phosphate-buffered saline (PBS) and treated
with a series of 1,8-naphthyridine-2-carboxamide derivatives at the concentrations of 30
and 100
DMSO instead.
After treatment, the viability of cells was determined using an MTT assay, as described
µM for 24 h. Control cells were treated with serum-free media containing 1%
previously [59] with minor modifications. In brief, the◦cells were incubated with MTT
solution at a final concentration of 0.5 mg/mL at 37 C for 3 h. Then, supernatants
were gently removed from each well and replaced with DMSO to dissolve blue formazan
crystals. The absorbance was measured at a wavelength of 550 nm using a microplate
reader (SpectraMax M2e, Molecular Devices, Sunnyvale, CA, USA). The cell viability was
calculated as the percentage of absorbance measured in vehicle-treated control cells.
4.4. Measurements of NO, TNF-α, and IL-6 by Enzyme-Linked Immunosorbent Assays
Enzyme-linked immunosorbent assays (ELISA) were used to evaluate the effects
of 1,8-naphthyridine-2-carboxamide derivatives on the LPS-induced production of pro-
inflammatory mediators including NO, TNF-
α
, and IL-6. Briefly, BV2 cells were seeded
◦
into 24-well plates at a density of 2.5
The cells were treated with the test compounds at the concentrations of 1, 10, 30, and
100 M in the presence or absence of LPS (1 g/mL). Control cells were treated with
×
105 cells/well and incubated at 37 C for 24 h.
µ
µ
serum-free media containing 1% DMSO instead. After 24 h of treatment, the culture
◦
media were carefully collected and centrifuged at 1500 rpm at 4 C for 10 min. The
◦
supernatants were then stored at
−
80 C until use. NO concentrations in the supernatants
were assessed using the Griess reagent (Promega Corporation, Madison, WI, USA) as
described previously [22 23]. The levels of TNF- and IL-6 were measured by ELISA kits
,
α
(KomaBiotech, Seoul, Korea), following the manufacturer’s instructions. The absorbance
was read at 450 nm on a microplate reader (SpectraMax M2e, Molecular Devices, Sunnyvale,
CA, USA). The concentrations of NO, TNF-
α, and IL-6 were calculated from the respective
standard curves generated simultaneously.

Int. J. Mol. Sci. 2021, 22, 2527
15 of 19
4.5. Cell Migration Assays
4.5.1. Wound Healing Assay
To assess the effect of HSR2104 on LPS-stimulated cell mobility, wound healing assays
were performed as described in our previous reports [22 23] with minor modifications.
,
Overall, BV2 cells were seeded into 24-well plates at a density of 5
and incubated at 37 C for 24 h. The cells were then wounded with a sterile scratcher
and treated with HSR2104 at various concentrations in the presence or absence of LPS
×
105 cells/well
◦
(1 µg/mL). Control cells were treated with serum-free media containing 1% DMSO instead.
The cell migration during 24 h was determined by measuring the changes in the area of the
wounds at 0 and 24 h under a Nikon phase-contrast microscope (Nikon Instruments Inc.,
Melville, NY, USA) [23,60], using ImageJ software version 1.49 (https://imagej.nih.gov/ij/).
The degree of cell migration was expressed as a percentage of the respective vehicle-treated
control cells.
4.5.2. Transwell Migration Assay
To further assess the effect of HSR2104 on LPS-stimulated cell mobility, transwell
migration assays were performed as described previously [22
,23]. BV2 cells were seeded
into the inserts (6.5 mm in diameter with an 8.0- m pore size) of a Costar transwell system
µ
(Corning Inc., Kennebunk, ME, USA) at a density of 1
37 C for 24 h in DMEM supplemented with 10% FBS and 1% antibiotics. After incubation,
the bottom wells were filled with HSR2104 at various concentrations with or without
×
105 cells/well and incubated at
◦
LPS (1 µg/mL) in serum-free media. Control cells were treated with serum-free media
containing 1% DMSO instead. After 24 h of treatment, non-migrated cells were gently
removed with a cotton swab and the migrated cells were fixed with 4% paraformaldehyde
for 20 min, followed by permeabilization with methanol for 10 min prior to staining with
0.5% crystal violet for 10 min, as described previously [23,60]. Four random fields from
each well were captured and the numbers of cells migrated through the membrane were
counted under a Nikon phase-contrast microscope (Nikon Instruments Inc., Melville, NY,
USA). The cell migration was expressed as a percentage of the respective vehicle-treated
control cells.
4.6. Western Blotting
BV2 cells were plated on 35 mm dishes at a density of 1
×
106 cells/dish and incubated
◦
at 37 C for 24 h. The cells were washed with PBS and treated with a series of concentrations
of HSR2104 in the presence or absence of LPS (1 µg/mL). Control cells were treated with
serum-free media containing 1% DMSO instead. After treatment, the cells were washed
with cold PBS and lysed in lysis buffer (10 mM Tris–HCl (pH 7.5), 150 mM NaCl, 2 mM
EDTA, 4.5 mM sodium pyrophosphate, 10 mM β-glycerophosphate, 1 mM NaF, 1 mM
Na3VO4, 1% (v/v) Triton X-100, 0.5% IGE-PAL CA-630 and one tablet of protease inhibitor
cocktail (Roche Diagnostic GmbH, Mannheim, Germany) per 10 mL of lysis buffer) on ice
for 30 min. The cell lysates were centrifuged at 14,000 rpm for 30 min at 4 ◦C and then
◦
the supernatants were separated and stored at
of HSR2104 on the nuclear translocation of NF-
−
80 C until use. To evaluate the effect
κ
B, cytosolic and nuclear fractions were
prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Rockford, IL, USA),
following the manufacturer’s instructions. The protein concentrations were determined
using a BioRad DC protein assay kit (BioRad, Hercules, CA, USA).
Western blotting analyses were performed as reported previously [22,59]. Briefly, cell
lysates containing equal amounts of protein were resolved by SDS-PAGE and transferred
to nitrocellulose membranes (Merck Millipore Ltd., Billerica, MA, USA) at 100 V for
90 min. The transferred membranes were blocked with 5% non-fat dried skim milk (BD
Biosciences, San Jose, CA, USA) at room temperature for 90 min and incubated with primary
antibodies in bovine serum albumin at 4 ◦C overnight. After rinsing with Tris-buffered
saline containing 0.1% Tween 20, the membranes were incubated with HRP-conjugated
secondary antibodies at room temperature for 120 min. Finally, blots were visualized with

Int. J. Mol. Sci. 2021, 22, 2527
16 of 19
a Bio-Rad ChemiDoc XRS imaging system using enhanced chemiluminescence reagents
(Bio-Rad, Hercules, CA, USA).
4.7. Immunocytochemistry
To further assess the effect of HSR2104 on LPS-induced NF-κB translocation, immuno-
cytochemistry was performed as described previously [61] with some modifications. In
brief, BV2 cells were plated on coverslips placed on the wells of 24-well plates at a density
of 2.5
×
104 cells/well and incubated at 37 ◦C for 24 h. The cells were then treated with
100 M HSR2104 in the presence or absence of LPS (1 µg/mL) for 24 h. Control cells were
µ
treated with serum-free media containing 1% DMSO instead. After treatment, the cells
were fixed with 4% paraformaldehyde for 15 min and permeabilized with 1% Triton X-100
for 5 min, followed by incubation with blocking solution of 5% goat serum for 30 min.
Between each step, the cells were carefully washed three times with PBS. The cells were
then incubated with anti-NF-κB p65 antibody with 1:100 dilution in blocking solution at
4 ◦C overnight. After washing three times with PBS, the cells were incubated with Alexa
Fluor 488-conjugated secondary antibody (1:400 dilution) for 1 h at room temperature in
the dark. Then, after washing three times with PBS, coverslips were removed from 24-well
plates and mounted onto microscope slides (Paul Marienfeld GmbH, Lauda-Königshofen,
Germany) with ProLong Gold Antifade Reagent with DAPI (Thermo Fisher Scientific
Inc., Waltham, MA, USA). Fluorescent signals were visualized using a Nikon confocal
laser-scanning microscope (Nikon Instruments Inc., Melville, NY, USA).
4.8. Measurements of Reactive Oxygen Species
The effect of HSR2104 on LPS-induced intracellular ROS generation was assessed
using a DCFH-DA assay, as described previously [59
,
61] with some modifications. Briefly,
BV2 cells were seeded into 24-well plates at a density of 1
×
105 cells/well and incubated at
◦
37 C for 24 h. The cells were then treated with various concentrations of HSR2104 with or
without LPS (1 µg/mL) for 24 h. Following treatment, the culture media were replaced with
serum free media containing DCFH-DA at a final concentration of 10
µ
M and incubated at
◦
37 C for 1 h. The levels of intracellular ROS were quantitated by the fluorescence detection
of dichlorofluorescein as an oxidized product of DCFH on a microplate reader (SpectraMax
M2e, Molecular Devices, Sunnyvale, CA, USA) with an excitation wavelength of 490 nm
and emission wavelength of 520 nm. The levels of ROS production were expressed as
percentages of the vehicle-treated control cells. The fluorescent signals were visualized
using a fluorescence microscope (Nikon Instruments Inc., Melville, NY, USA).
4.9. Statistical Analyses
All experiments were performed at least three times and quantitative data were
expressed as the means
±
SEM. Statistical significance was analyzed by one-way analysis
of variance (ANOVA) using Sigma Plot 12.5 software (Systat Software Inc., San Jose,
CA, USA). A p value < 0.05 was considered statistically significant. IC50 values were
calculated by non-linear regression using GraphPad Prism 5 (GraphPad Software Inc.,
La Jolla, CA, USA).
5. Conclusions
In conclusion, thirteen novel compounds of 1,8-naphthyridine-2-carboxamide derivatives
with N-hydroxyphenyl, N-methoxyphenyl, N-(trifluoromethyl)phenyl, N-bis(trifluoromethyl)
phenyl, or N-chlorophenyl substituents were synthesized and evaluated for anti-inflammatory
and anti-migratory effects using LPS-treated BV2 microglia cells. Among the compounds
tested, HSR2104 with an N-methoxyphenyl substituent at the R1 position, exhibited the
most potent inhibitory effects on the production of pro-inflammatory mediators including
NO, TNF-α, and IL-6. HSR2104 inhibited the expressions of iNOS and COX2 as well as
cell migration in LPS-treated BV2 cells. In addition, it markedly suppressed LPS-induced
intracellular ROS production. Moreover, HSR2104 abated the LPS-triggered nuclear translo-

Int. J. Mol. Sci. 2021, 22, 2527
17 of 19
cation of NF-
κ
B through the inhibition of I
κ
Bα
phosphorylation. It was further demon-
strated that HSR2104 inhibited LPS-induced TLR4/MyD88/NF-
our data indicate that HSR2104 exhibits anti-inflammatory and anti-migratory effects in
LPS-treated BV2 cells via the suppression of ROS production and the TLR4/MyD88/NF-
κB signaling. Collectively,
κ
B
signaling pathway. Based on these findings, HSR2104 may exert beneficial inhibitory
effects on neuroinflammatory responses and microglial cell migration associated with the
pathogenesis of neurodegenerative disorders including AD and PD.
Supplementary Materials: Supplementary Materials can be found at https://www.mdpi.com/1422
-0067/22/5/2527/s1.
Author Contributions: P.L.N.: investigation, methodology, data curation, writing—original draft;
B.P.B.: investigation, methodology, data curation; H.L.: conceptualization, funding acquisition,
investigation, project administration, writing—review and editing; J.C.: conceptualization, funding
acquisition, investigation, project administration, supervision, validation, writing—review and
editing. All authors have read and agreed to the published version of the manuscript.
Funding: This work was supported by the National Research Foundation of Korea (NRF) grants
funded by the Korean government (MSIT) (NRF-2018R1A5A2023127 and NRF-2020R1F1A1075835 to
J.C. and NRF-2019R1F1A1057601 to H.L.), Korea.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
COX2
IκBs
IL
Cyclooxygenase-2
Inhibitors of kappa B
Interleukin
iNOS
LPS
Inducible nitric oxide synthase
Lipopolysaccharide
MyD88 Myeloid differentiation factor 88
NF-κB
NO
Nuclear factor-kappa B
Nitric oxide
ROS
TLR4
TNF-α
Reactive oxygen species
Toll-like receptor 4
Tumor necrosis factor-alpha
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