Synthesis and evaluation of the anti-inflammatory activity of novel 8-quinolinesulfonamide derivatives as TLR4/MD-2 inhibitors with efficacy in adjuvant-induced arthritis

Article abstract of DOI:10.1016/j.bioorg.2021.105037

In this study, a series of 8-quinolinesulfonamide derivatives was synthesized, and their anti-inflammatory activity was evaluated. Among them, compound 3l was found to be the best anti-inflammatory agent, with IC₅₀ values of 2.61 ± 0.39, 9.74 ± 0.85, and 12.71 ± 1.34 μM against NO, TNF-α and IL-1β production respectively. And 3l could significantly prevent lipopolysaccharide (LPS)-induced expression of inflammatory mediators (iNOS and COX-2). Molecule docking results showed that 3l could bind to the LPS binding site of toll-like receptor 4 (TLR4)/MD-2, and 3l was then identified as TLR4/MD-2 inhibitor by co-immunoprecipitation (co-IP) and cellular thermal shift assay (CTESA). Preliminary mechanism studies indicated that 3l could prevent TLR4 from being activated by disrupting TLR4/MD-2 heterodimerization and TLR4 homodimerization, thereby blocking the activation of the NF-κB/MAPK signaling pathway. Furthermore, observation of rat foot swelling, joint pathology and serum inflammatory cytokine levels proved that compound 3l had a significant therapeutic effect on adjuvant-induced arthritis (AIA) in rats in vivo. These results indicated that compound 3l is a potential anti-inflammatory agent, from which more effective anti-inflammatory drugs could be developed.

Full text of DOI:10.1016/j.bioorg.2021.105037

Bioorganic Chemistry 114 (2021) 105037
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis and evaluation of the anti-inflammatory activity of novel 8-qui-
nolinesulfonamide derivatives as TLR4/MD-2 inhibitors with efficacy in
adjuvant-induced arthritis
Tongtong Liu, Siqi Xing, Jiyu Du, Min Wang, Jianfei Han, Zeng Li*
Inflammation and Immune Mediated Diseases Laboratory of Anhui Province, Anhui Institute of Innovative Drugs, School of Pharmacy, Anhui Medical University, Hefei
230032, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
In this study, a series of 8-quinolinesulfonamide derivatives was synthesized, and their anti-inflammatory ac-
8-Quinolinesulfonamide derivatives
Anti-inflammatory
3l
tivity was evaluated. Among them, compound 3l was found to be the best anti-inflammatory agent, with IC50
values of 2.61 ± 0.39, 9.74 ± 0.85, and 12.71 ± 1.34 μM against NO, TNF-α and IL-1β production respectively.
And 3l could significantly prevent lipopolysaccharide (LPS)-induced expression of inflammatory mediators
(iNOS and COX-2). Molecule docking results showed that 3l could bind to the LPS binding site of toll-like re-
ceptor 4 (TLR4)/MD-2, and 3l was then identified as TLR4/MD-2 inhibitor by co-immunoprecipitation (co-IP)
and cellular thermal shift assay (CTESA). Preliminary mechanism studies indicated that 3l could prevent TLR4
from being activated by disrupting TLR4/MD-2 heterodimerization and TLR4 homodimerization, thereby
blocking the activation of the NF-κB/MAPK signaling pathway. Furthermore, observation of rat foot swelling,
joint pathology and serum inflammatory cytokine levels proved that compound 3l had a significant therapeutic
effect on adjuvant-induced arthritis (AIA) in rats in vivo. These results indicated that compound 3l is a potential
anti-inflammatory agent, from which more effective anti-inflammatory drugs could be developed.
TLR4/MD-2
Adjuvant induced arthritis
1. Introduction
that results in the activation of downstream cytokines, such as NO, TNF-
α
, and IL-1β [12,15–18]. Several studies have shown that various com-
Inflammation is a biological protective response of the organism
against injury or infection [1,2]. Disorder in tissues may cause inflam-
mation, such as joint disease, cancer, and heart failure [3–6]. However,
inflammation can be reduced by inhibiting the production of inflam-
pounds that act as TLR4/MD-2 inhibitors can exert anti-inflammatory
effects by preventing the combination of TLR4 and MD-2 [19–21].
Thus, inhibiting TLR4/MD-2 activation can effectively reduce the
expression of TLR4-related inflammatory cytokines to reduce
inflammation.
matory factors (e.g., TNF-α, IL-1β, IL-6, and NO) [7–9]. The classic drugs
currently used for the treatment of inflammation are nonsteroidal anti-
inflammatory drugs, such as aspirin, but these drugs can cause some
adverse reactions [10,11]. Therefore, new anti-inflammatory drugs
must be discovered and developed.
Most quinoline derivatives have good anti-inflammatory activity
(Fig. 1) [22–26]. Hence, the quinoline scaffold can be used as a key
matrix structure to design drugs with a better anti-inflammatory activ-
ity. In addition, sulfonamide groups play an important role in anti-
inflammatory drugs. Certain agents with a benzenesulfonamide struc-
ture, such as nimesulide, celecoxib, and parecoxib, are commonly used
in clinical settings to reduce inflammation (Fig. 1) [27–29]. In this study,
we introduced sulfonamide groups into the quinoline ring to synthesize
a series of 8-quinolinesulfonamide derivatives (Fig. 1). We evaluated
derivatives anti-inflammatory activities in vitro. The title 8-quinolinesul-
fonamide derivative optimized was further evaluated to elucidate their
potential mechanism of action and used to explore ameliorate
Toll-like receptors (TLRs) are a class of proteins that play a key role
in the innate immune system [12]. Among them, TLR4 has been shown
to be related to various inflammatory diseases and can regulate the
immune homeostasis of the human system; thus, it is considered the
most influential target [13,14]. TLR4, along with its accessory protein
MD-2, is dimerized under LPS stimulation, which promotes the inter-
action between the downstream effector myeloid differentiation factor
88 and dimerized TLR4/MD-2 to control the cascade of NF-κB and MAPK
* Corresponding author.
E-mail address: lizeng@ahmu.edu.cn (Z. Li).
https://doi.org/10.1016/j.bioorg.2021.105037
Received 16 April 2021; Received in revised form 24 May 2021; Accepted 28 May 2021
Available online 31 May 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

T. Liu et al.
Bioorganic Chemistry 114 (2021) 105037
inflammation in a AIA mouse model.
likely to bind to plasma proteins (PPB# prediction was true). Results
showed that these compounds were non-cytochrome P450 2D6 in-
hibitors (CYP2D6# prediction was false). The druggability and safety of
the compounds were initially evaluated through ADMET predictions.
Subsequently, the anti-inflammatory activity of the compounds was
studied.
2. Results and discussion
2.1. Chemistry
The synthesis route of 8-quinolinesulfonamide derivatives is illus-
trated in Scheme 1. A series of compounds, 3a–3n, were obtained by
reacting various substituents of aniline and 8-quinolinesulfonyl chloride
and triethylamine in dichloromethane at room temperature.
2.3. Inhibition of NO production and inflammatory factors in LPS-
induced RAW264.7 cells
A
series of synthetic 8-quinolinesulfonamide derivatives were
2.2. ADMET predictions for compounds
determined and their cytotoxicity in macrophage RAW264.7 cells was
evaluated by MTT assay to prevent the toxicity of compounds 3a–3n to
the cells from interfering with the analysis of experimental results. As
shown in Table 2, compared with positive drug indomethacin, com-
pounds 3a and 3h displayed weak cytotoxicity, whereas the other
compounds showed low toxicity.
The absorption, distribution, metabolism, excretion, and toxicity
(ADMET) of compounds 3a–3n were predicted using the Discovery
Studio 2017 (DS 2017) software [30,31]. The results are depicted in the
2D graphs of ADMET PSA 2D and ADMET AlogP98. Compound 3a had
poor solubility at 25 ◦C (solubility level of 3), whereas compounds
3b–3n had good solubility at this temperature (solubility level of 1 or 2)
(Fig. 2 and Table 1). All compounds were within the 95% and 99%
confidence intervals of blood–brain barrier (BBB) penetration and had
good HIA (absorption level of 0). Moreover, compounds 3a–3n were
LPS is a common immunostimulator that can stimulate RAW264.7
cells to induce and secrete NO, IL-1β, TNF-α, and other inflammatory
factors [2,32,33]. Overproduction of NO and pro-inflammatory factors
is closely related to inflammatory diseases [2,34,35]. Thus, the anti-
inflammatory activity of all compounds synthesized herein was
Fig. 1. A strategy for designing an anti-inflammatory drug.
2

T. Liu et al.
Bioorganic Chemistry 114 (2021) 105037
Compd
R
Compd
R
H
2-F
3a
3h
2-CH3
3-CH3
3-F
4-F
3b
3c
3d
3e
3f
3i
3j
4-CH3
3,4-F
4-CF3
4-OCF3
3-Cl
3k
3l
2-OCH3
3-OCH3
4-OCH3
3m
3n
3g
Scheme 1. Synthesis of 8-quinolinesulfonamide derivatives 3a–3n. Reagents and conditions: dichloromethane, triethylamine, ethanol, room temperature, 3–10 h. a
Yield of the compound obtained.
evaluated. A nonsteroidal anti-inflammatory drug, indomethacin, was
selected as a positive control. RAW264.7 cells were incultured with
different concentrations of the compounds (100, 60, 40, 20, 10, 5, 2.5,
that of the positive drug, which may be the cause of steric hindrance.
However, the introduction of a single substituent on the phenyl ring of
the other compounds evidently enhanced their anti-inflammatory ac-
tivity and was superior to positive drug. Among these compounds, that
with the CF3 group (i.e., compound 3l) had the best inhibitory activity,
which is about 16 times stronger than indomethacin. Moreover, the
electronegativity of the substituents on the phenyl ring was strongly
related to the inhibitory activity. When a strong electron withdrawing
group (F or Cl) was introduced into the phenyl ring, the inhibitory ac-
tivity against NO production was evidently enhanced, and its activity
was related to the position of the substituent on the phenyl ring. The
introduction of substituents at the C2, C3, and C4 positions of phenyl
1.25 and 0.6725
μM) for 1 h and stimulated by LPS for 24 h. The
inhibitory effect of the compounds on NO production in the cell super-
natant was detected using a NO content detection kit (Table 3).
Analysis of structure–activity relationship revealed that the intro-
duction of different substituents of the phenyl ring expressed different
degrees of anti-inflammatory activity. When the phenyl ring had no
substituent (3a), the anti-inflammatory activity of the compound was
weaker than that of the positive drug indomethacin. Furthermore,
compound 3k exhibited poor anti-inflammatory activity, much less than
3

T. Liu et al.
Bioorganic Chemistry 114 (2021) 105037
Fig. 2. Regression of ADMET_PSA_2D and ADMET_AlogP98. Red represents the 95% confidence interval of HIA, and green denotes the 99% confidence interval of
HIA. Pink indicates the 95% confidence interval of BBB, and the sky blue ellipse signifies the 99% confidence interval of BBB. All compounds are within the con-
fidence interval. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
rings gradually decreased the inhibitory activities of the derivatives
against NO, such as compounds 3h, 3i, and 3j. Interestingly, in contrast
to the electron-withdrawing group, the activity of the electron-donating
group at the C4 position (compound 3d) was better than that at the C2
(compound 3b) and C3 (compound 3c) positions. This trend was also
observed in compounds 3e, 3f, and 3g. Moreover, the effectiveness of
the compound with a F substituent was better than that of the derivative
with a Cl substituent in the 3 position of the phenyl ring, which also
surpassing of indomethacin.
from receptors through known ligand structures via three methods,
namely, shape screening, pharmacophore screening, and reverse dock-
ing [38,39]. In the present study, reverse virtual screening was applied
to validate that the 8-quinolinesulfonamide derivative compound 3l had
a good binding relationship with TLR4/MD-2 protein (PDB:3FXI)
(Fig. S1). Thus, TLR4/MD-2 was considered as a possible target of
compound 3l. The results were further analyzed via molecule docking
by using the CDOCKER program in Discovery Studio 2017 soft. As
shown in Fig. 4, compound 3l had a good binding in the LPS-binding
pocket of the TLR4/MD-2 complex and formed an interaction with the
amino acids of the TLR4 protein (Ser183, Ala58, Asp181, Asn156, and
Val134) and the amino acids of the MD-2 protein (Leu108, Arg106, and
Glu111). Therefore, we speculated that compound 3l could be used as a
TLR4/MD-2 inhibitor to exert anti-inflammatory effects.
In this study, the inhibitory effect of the compounds on the pro-
duction of IL-1β and TNF-α was detected via ELISA to evaluate further
their anti-inflammatory activity. As shown in Table 3, compound 3l had
the best inhibitory effect on the secretion of inflammatory factors NO,
IL-1β, and TNF-α in RAW264.7 cells.
NO production is mediated by COX-2 and iNOS. When inflammatory
cells are stimulated by LPS, COX-2 and iNOS are overexpressed [1,36,37].
The effects of compound 3l on the expression of pro-inflammatory factors
in LPS-treated RAW264.7 cells were analyzed via Western blot. Results
showed that compound 3l could reduce the expression of iNOS and COX-2
in a concentration-dependent manner (Fig. 3).
2.5. Effects of compound 3l on TLR4/MD-2 complex
Given that molecular docking established a direct relationship be-
tween compound 3l and the active site of the TLR4/MD-2 complex, we
further confirmed that TLR4/MD-2 as the target of compound 3l by co-
IP, the result showed that 3l significantly inhibited dimerization of the
TLR4/MD-2 complex (Fig. 5).
2.4. Reverse virtual screening and molecular docking
TLR4/MD-2 heterodimerization in the presence of LPS results in
homodimerization of TLR4, inducing the release of pro-inflammatory
Reverse virtual screening is used to find and identify potential targets
4

T. Liu et al.
Bioorganic Chemistry 114 (2021) 105037
Table 1
ADMET predictions for compounds 3a-3n.
compd
Absorption levela
BBB levelb
Solubility levelc
CYP2D6d
PPBe
AlogP98f
PSA 2Dg
3a
3b
3c
3d
3e
3f
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
2
2
2
2
2
2
2
2
2
2
2
1
2
3
2
2
2
2
2
2
2
2
2
2
2
1
2
false
false
false
false
false
false
false
false
false
false
false
false
false
false
true
true
true
true
true
true
true
true
true
true
true
true
true
true
2.709
2.709
2.914
3.168
2.487
2.99
58.672
58.672
58.672
58.672
67.602
67.602
67.602
58.672
58.672
58.672
58.735
58.672
67.602
58.672
3g
3h
3i
2.503
2.99
2.487
2.99
3j
3k
3l
2.197
3.446
2.487
4.623
3m
3n
a
Absorption (level 0 means good).
b
c
d
e
f
Distribution: Aqueous solubility (level 1 means very good, level 2 means moderate).
Distribution: Blood–brain barrier penetration (level 1 means very good, level 2 means moderate, k level 3 means poor).
Metabolism.
Excretion.
Predicted octanol/water.
Two-dimensional.
g
Table 2
Table 3
Cytotoxicity of the compounds.
Inhibition by the compounds of the IC50 value produced by inflammatory
factors.
compd
RAW264.7 cells IC50
compd
RAW264.7 cells IC50
(
μ
M)a
(μ
M)a
Compound
NO inhibition IC50
IL-1β inhibition
TNF-
α
inhibition
(μ
M)a
IC50 (
μ
M)b
IC50 (
μ
M)c
3a
3b
3c
3d
3e
3f
90.26 ± 1.7
>100
3i
>100
3j
>100
3a
47.96 ± 0.58
31.39 ± 0.41
22.94 ± 1.44
18.06 ± 0.46
31.07 ± 1.17
19.98 ± 0.71
16.78 ± 0.30
5.47 ± 0.72
17.92 ± 0.32
24.81 ± 0.44
＞100
46.62 ± 1.53
28.91 ± 0.51
14.79 ± 0.82
14.67 ± 0.93
24.97 ± 0.58
23.07 ± 0.49
30.05 ± 1.27
12.24 ± 1.33
49.30 ± 0.33
39.63 ± 0.21
＞100
37.18 ± 2.57
40.08 ± 1.24
14.53 ± 1.61
36.13 ± 0.37
45.25 ± 1.22
20.89 ± 0.77
40.71 ± 1.62
26.69 ± 1.05
33.34 ± 0.47
47.65 ± 0.94
＞100
>100
3k
>100
3b
>100
3l
>100
3c
>100
3m
>100
3d
>100
3n
>100
3e
3g
>100
Indomethacin
Control
93.88 ± 2.6
>100
3f
3h
90.01 ± 1.9
3g
3h
a
Concentration of the compounds at 50% survival of RAW264.7 cells. IC50
3i
values are calculated from the average of three experiments (mean ± SD).
3j
3k
3l
2.61 ± 0.39
9.76 ± 0.66
25.41 ± 0.53
34.99 ± 0.26
9.74 ± 0.85
10.78 ± 0.96
54.45 ± 0.38
36.15 ± 0.62
12.71 ± 1.34
17.32 ± 0.49
28.74 ± 0.79
40.96 ± 0.63
3m
cytokines, such as NO, IL-6, and TNF-α [16,19,40], thus the interaction
3n
between compound 3l and TLR4 was further analyzed by CETSA and
immunoprecipitation. CETSA is a biophysical technique for monitoring
ligand–potein interactions [41,42]. In the CETSA method, cells or cell
extract samples are heated at different temperatures to evaluate the
compound’s effect on the thermal stability of protein, thereby providing
quantitative evidence for compound–protein interactions [43,44]. Re-
sults showed that the TLR4 protein collected from RAW264.7 cells co-
cultured with compound 3l had a better stability than the cells incu-
bated in the absence of this compound at 50 ◦C and 55 ◦C, indicating that
TLR4 interacted with this compound (Fig. 6). To evaluate the effect of 3l
on TLR4 dimerization, HEK293T cells were co-transfected with TLR4-
HA acid and TLR4-Flag plasmids for 24 h, and then TLR4-Flag and
TLR4-HA acid complexes were detected by co-IP. Compound 3l sub-
stantially reduced the TLR4-Flag of TLR4-HA, indicating that this com-
pound disrupted the TLR4 dimerization associated with LPS (Fig. 7).
Indomethacin
a
Concentration of the compounds when 50% NO was produced in LPS-
stimulated RAW264.7 cells. IC50 values are calculated from the average of
three experiments (mean ± SD).
b
Concentration of the compounds when 50% IL-1β was produced in LPS-
stimulated RAW264.7 cells. IC50 values are calculated from the average of
three experiments (mean ± SD).
c
Concentration of the compounds when 50% TNF-α was produced in LPS-
stimulated RAW264.7 cells. IC50 values are calculated from the average of
three experiments (mean ± SD).
cascades are initiated to activate the NF-κB and MAPK signaling path-
ways [15,21,45]. Accordingly, the effect of the compound on the
expression of related proteins in the NF-κB and MAPK signaling path-
ways were analyzed.
2.6. Inhibition of LPS-induced NF-κB/MAPK signaling activation
Compound 3l inhibited the phosphorylation and degradation of IκB
When the TLR4/MD-2 complex is stimulated by LPS, a series of signal
5

T. Liu et al.
Bioorganic Chemistry 114 (2021) 105037
Fig. 3. Effects of compound 3l on the expression of related inflammatory proteins. RAW264.7 cells were incubated with different concentrations of compound 3l and
the positive drug (1, 3, and 10 μM) Bay11-7082 for 1 h. The cells were then stimulated by LPS for 24 h. The effects of compound 3l on iNOS and COX-2 were analyzed
by Western blot. The results were showed as means ± SD (n = 3) of at least three independent experiments. ###p < 0.0001 compared with control, **p < 0.001, ***
< 0.001 compared with LPS-stimulated cells.
p
Fig. 4. Compound 3l bound to the LPS-binding pocket of the TLR4/MD-2 complex (PDB: 3FXI). Green denotes the TLR4 protein, and blue indicates the MD-2 protein.
(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 5. Co-immunoprecipitation showing effect of compound 3l pretreatment the formation of TLR4/MD-2 complex in RAW264.7 cells exposed to LPS. The results
were showed as means ± SD (n = 3) of at least three independent experiments. *p < 0.01 compared with LPS-stimulated cells.
to block the activation of IκB kinase (Fig. 8A). Moreover, it inhibited the
phosphorylation of P65 in a concentration-dependent manner and pre-
vented the translocation of NF-κB P65 from the cytoplasm to the nu-
cleus. RAW264.7 cells were stimulated to activate the MAPK signaling
pathway, leading to considerable phosphorylation of MAPK (P38, JNK,
concentration-dependent manner.
2.7. Anti-inflammatory activity of compound 3l in vivo
The anti-inflammatory activity of compound 3l in vivo was confirmed
by using compound 3l (10 and 30 mg/kg) and a positive drug (10 mg/
kg) used to treat the rat adjuvant-induced arthritis (AIA) model.
and ERK). As suggested in Fig. 8B, compound 3l (1, 3, and 10
μM) could
also inhibit LPS-induced P38, JNK, and ERK phosphorylation in a
6

T. Liu et al.
Bioorganic Chemistry 114 (2021) 105037
Similarly, indomethacin had an inhibitory effect on the production of
inflammatory factors (Fig. 11).
3. Conclusions
This study aimed to discover novel agents with anti-inflammatory
activity. A series of 8-quinolinesulfonamide derivatives were synthe-
sized. Preliminary evaluation results of the anti-inflammatory activity of
these derivatives revealed that most of them had good inhibitory activity
against NO, IL-1β, and TNF-α. Analysis of the structure–activity rela-
tionship of these compounds showed that those with a single substituent
on the phenyl ring had enhanced anti-inflammatory activity. Among
these compounds, compound 3l had the best anti-inflammatory activity
and could significantly suppress the expression levels of iNOS and COX-
2. The potential target (TLR4/MD-2) of compound 3l was obtained
through reverse screening, and confirmed by molecular docking, co-IP
and CTESA. Preliminary results of mechanism studies indicated that
compound 3l could block the activation of NF-κB/MAPK signaling
pathway in a concentration-dependent manner by disrupting TLR4/MD-
2 dimerization. In addition, according to in vivo studies, compared with
the model group, the treatment group (compound 3l) effectively
reduced inflammation in the AIA rat model. These findings indicated
that compound 3l has a great potential for the treatment of inflamma-
tion. We are investigating this compound in greater detail to understand
further its anti-inflammatory activity.
Fig. 6. Interaction between of compound 3l and TLR4. RAW264.7 cells were
treated with 3l for 12 h, and then the expression of TLR4 protein at different
temperatures (45 ◦C, 50 ◦C, 55 ◦C, 60 ◦C and 65 ◦C) was detected. The results
were showed as means ± SD (n = 3) of at least three independent experiments.
*p < 0.01 compared with LPS-stimulated cells.
4. Experimental section
4.1. Chemistry
Compared with the model group, the drug group reduced the swelling of
the rats’ feet in a concentration-dependent manner (Fig. 9A and B). On
day 24, 30 mg/kg of compound 3l significantly decreased the arthritis
index (Fig. 9C). Compared with that in the model group, body weight in
the drug group improved at doses of ≥ 10 mg/kg (Fig. 9D).
Unless otherwise specified, aniline with various substituents, trie-
thylamine, ethanol, and various solvents used in this experiment were
obtained from commercial sources and did not require further purifi-
cation. The reaction was monitored by thin-layer chromatography (TLC)
on a silica gel plate (HSGF254 Yantai Jiangyou Silica Gel Development
Co., Ltd., China) by using the solvents petroleum ether and ethyl acetate.
The compounds were detected via high-resolution mass spectrometry.
Proton nuclear magnetic resonance spectrum and carbon nuclear mag-
netic resonance spectrum were measured at 400 and 101 MHz, respec-
tively. The reaction mixture through CombiFlash NextGen 300+ silica
gel column (Teledyne ISCO, USA, Silica gel: 200–300 mesh) for purifi-
cation to obtain the target product.
Subsequently, histopathological section of rat knee joints were
stained with hematoxylin and eosin (HE) to investigate the effects of
compound 3l on pathological changes in AIA rats. As depicted in Fig. 10,
the model group showed obvious synovial cell proliferation, inflam-
matory cell infiltration and pannus formation. These symptoms in AIA
rats treated with compound 3l were alleviated in a dose-dependent
manner (Fig. 10).
Fuethermore, the ELISA method was used to detect inflammatory
factors in the serum of AIA rats. In the results, compared with those in
the normal group, the levels of IL-1β, and TNF-α in the model group
4.2. General procedure for the synthesis of compounds 3a–3n
remarkably increased. However, compound 3l could reduce the in-
flammatory factors in the serum of rats in a dose-dependent manner.
According to the reported method [46], 8-quinolinesulfonyl chloride
Fig. 7. Co-immunoprecipitation analysis the destructive effects of compound 3l on TLR4 dimerization. HEK293T cells were co-transfected with TLR4-hyaluronic acid
and TLR-Flag, the sampled proteins were immunoprecipitated using anti-HA magnetic beads. The results were showed as means ± SD (n = 3) of at least three
independent experiments. *p < 0.01 compared with LPS-stimulated cells.
7

T. Liu et al.
Bioorganic Chemistry 114 (2021) 105037
Fig. 8. Compound 3l inhibits LPS-induced activation of NF-κB signaling pathway and MAPK signaling pathway. (A) Compound 3l inhibites NF-κB signaling pathway
in RAW 264.7 cells. (B) Compound 3l inhibites MAPK signaling pathway in RAW 264.7 cells. RAW264.7 cells were cultured with different concentrations of
compound 3l (1, 3, and 10 μM) for 1 h. Subsequently, LPS was added for 24 h. The results were showed as means ± SD (n = 3) of at least three independent
experiments. ###p < 0.0001 compared with control, *p < 0.01,**p < 0.001, ***p < 0.0001 compared with LPS-stimulated cells.
(227 mg, 1.0 equivalent, 1 mM) and aniline with various substituents
(2.0 equivalent, 2 mM) were stirred in dichloromethane solvent for 15
min, and then triethylamine was added dropwise (101 mg, 2.0 equiva-
lents, 2 mM). The reaction was stirred for 3–10 h at room temperature.
After the TLC detection reaction was completed, the dichloromethane
was removed by evaporation to obtain a reaction mixture. The mixture
was purified by silica gel column chromatography using a mixture of
ethyl acetate and petroleum ether to obtain the target compounds
(3a–3n).
7.7 Hz, 1H), 7.07 (d, J = 7.0 Hz, 1H), 7.01–6.91 (m, 2H), 6.85 (d, J = 7.4
Hz, 1H), 2.06 (s, 3H). 13C NMR (126 MHz, DMSO) δ 151.94, 143.19,
137.64, 136.74, 135.84, 134.48, 133.81, 131.62, 131.13, 129.01,
126.67, 126.33, 126.14, 125.13, 123.17, 18.16. HRMS (ESI) m/z [M +
H]+: 299.08095 calcd for C16H14N2O2S: 299.08392;
N-(m-tolyl)quinoline-8-sulfonamide (3c). White solid, Yield: 59%,
m.p. 161.4–162.9 ◦C. 1H NMR (500 MHz, DMSO) δ 10.02 (s, 1H), 9.15
(dd, J = 4.2, 1.7 Hz, 1H), 8.51 (dd, J = 8.3, 1.6 Hz, 1H), 8.37 (dd, J =
7.3, 1.1 Hz, 1H), 8.26 (d, J = 8.2 Hz, 1H), 7.77–7.66 (m, 2H), 6.95 (t, J
= 7.8 Hz, 1H), 6.89 (s, 1H), 6.83 (d, J = 8.0 Hz, 1H), 6.70 (d, J = 7.5 Hz,
1H), 2.08 (s, 3H). 13C NMR (126 MHz, DMSO) δ 151.91, 143.19, 138.53,
138.18, 137.45, 135.72, 134.66, 132.52, 129.09, 128.85, 126.09,
124.85, 123.09, 120.65, 117.13, 21.45. HRMS (ESI) m/z [M + H]+:
299.08095 calcd for C16H14N2O2S: 299.08389;
N-phenylquinoline-8-sulfonamide (3a) [47]. Yield: 68%. 1H NMR
(500 MHz, DMSO) δ 10.16 (s, 1H), 9.19 (dd, J = 4.1, 1.5 Hz, 1H),
8.53–8.45 (m, 1H), 8.42 (d, J = 7.3 Hz, 1H), 8.23 (d, J = 8.1 Hz, 1H),
7.70 (dt, J = 10.1, 5.6 Hz, 2H), 7.11 (d, J = 6.2 Hz, 4H), 6.89 (ddd, J =
8.1, 5.9, 1.9 Hz, 1H). 13C NMR (126 MHz, DMSO‑d6) δ 151.91, 143.18,
138.26, 137.40, 135.64, 134.66, 132.61, 129.29, 128.83, 126.06,
124.14, 123.07, 120.18. HRMS (ESI) m/z [M + H]+: 285.06530 calcd for
N-(p-tolyl)quinoline-8-sulfonamide (3d) [48]. Yield: 59%. 1H NMR
(500 MHz, DMSO) δ 9.91 (s, 1H), 9.17 (d, J = 3.7 Hz, 1H), 8.50 (d, J =
8.2 Hz, 1H), 8.33 (d, J = 7.2 Hz, 1H), 8.24 (d, J = 8.1 Hz, 1H), 7.82–7.65
(m, 2H), 6.94 (d, J = 7.9 Hz, 2H), 6.88 (d, J = 7.8 Hz, 2H), 2.06 (s, 3H).
13C NMR (126 MHz, DMSO) δ 151.90, 143.17, 137.43, 135.63, 135.53,
134.58, 133.46, 132.52, 129.71, 128.81), 126.08, 123.08, 120.70,
20.62. HRMS (ESI) m/z [M + H]+: 299.08095 calcd for C16H14N2O2S:
C
15H12N2O2S: 285.06833;
N-(o-tolyl)quinoline-8-sulfonamide (3b). White solid, Yield: 63%, m.
p. 155.4–157.7 ◦C. 1H NMR (500 MHz, DMSO) δ 9.23 (s, 1H), 9.17 (dd, J
= 4.1, 1.5 Hz, 1H), 8.58 (dd, J = 8.3, 1.4 Hz, 1H), 8.30 (d, J = 8.1 Hz,
1H), 8.24 (d, J = 6.4 Hz, 1H), 7.76 (dd, J = 8.3, 4.2 Hz, 1H), 7.69 (t, J =
8

T. Liu et al.
Bioorganic Chemistry 114 (2021) 105037
Fig. 9. Effects of compound 3l on adjuvant-induced arthritic rats. (A) Pictures of treated and untreated feet of rats. (B) Effect of compound 3l on the body weight of
adjuvant-induced arthritic model rats. (C) Effects of compound 3l on paw swelling in adjuvant-induced arthritic model rats. (D) Effects of compound 3l on the
arthritis index of adjuvant-induced arthritic model rats.
Fig. 10. The effect of compound 3l on the joint pathological tissue of AIA model rats was explored by HE staining experiment (magnification × 10). (A) Normal; (B)
AA; (C) Indometacin 10 mg/kg; (D) Compound 3l, 30 mg/kg; (E) Compound 3l, 10 mg/kg.
9

T. Liu et al.
Bioorganic Chemistry 114 (2021) 105037
Fig. 11. Inhibitory effects of compound 3l on inflammatory factors in adjuvant-induced arthritic model rats. (A) Inhibitory effects of compound 3l on IL-1β pro-
duction in vivo. (B) Inhibitory effects of compound 3l on TNF-α production in vivo. The results were showed as means ± SD (n = 3) of at least three independent
experiments. ###p < 0.0001 compared with the normal group, **p < 0.001, ***p < 0.0001 compared with the model group.
299.08392;
58%, m.p. 197.4–200.1 ◦C. 1H NMR (500 MHz, DMSO) δ 10.15 (s, 1H),
N-(2-methoxyphenyl)quinoline-8-sulfonamide (3e) [49]. Yield:
9.21 (dd, J = 4.0, 1.4 Hz, 1H), 8.53 (dd, J = 8.3, 1.2 Hz, 1H), 8.39 (d, J =
7.2 Hz, 1H), 8.28 (d, J = 7.9 Hz, 1H), 7.86–7.66 (m, 2H), 7.12 (dd, J =
8.9, 4.9 Hz, 2H), 6.99 (t, J = 8.8 Hz, 2H). 13C NMR (126 MHz, DMSO) δ
160.22, 158.30, 151.94, 143.15, 137.44, 135.43, 134.71, 134.45,
132.58, 128.82, 126.08, 123.10, 122.75, 116.07, 115.89. HRMS (ESI)
m/z [M + H]+: 303.05588 calcd for C15H11FN2O2S: 303.05884;
N-(3,4-difluorophenyl)quinoline-8-sulfonamide (3k). White solid,
Yield: 48%, m.p. 174.4–177.1 ◦C. 1H NMR (500 MHz, DMSO) δ 10.52 (s,
1H), 9.23 (d, J = 2.7 Hz, 1H), 8.53 (dd, J = 28.7, 7.6 Hz, 2H), 8.33 (d, J
= 8.0 Hz, 1H), 7.78 (dd, J = 12.3, 5.7 Hz, 2H), 7.21 (ddd, J = 13.3, 9.6,
5.7 Hz, 2H), 6.97 (d, J = 8.9 Hz, 1H). 13C NMR (126 MHz, DMSO) δ
151.99, 143.10, 137.45, 135.17, 134.95, 132.82, 128.86, 126.09,
123.13, 118.13, 117.98, 116.50, 116.43, 109.32, 109.16. HRMS (ESI)
m/z [M + H]+: 321.04646 calcd for C15H10F2N2O2S: 321.04938;
N-(4-(trifluoromethyl)phenyl)quinoline-8-sulfonamide (3l) [50].
Yield: 54%. 1H NMR (500 MHz, DMSO) δ 10.83 (s, 1H), 9.15 (d, J = 2.6
Hz, 1H), 8.51 (dd, J = 11.9, 8.0 Hz, 2H), 8.31 (d, J = 8.0 Hz, 1H),
7.86–7.65 (m, 2H), 7.50 (d, J = 8.5 Hz, 2H), 7.31 (d, J = 8.4 Hz, 2H). 13C
NMR (126 MHz, DMSO) δ 152.03, 143.12, 142.20, 137.48, 135.31,
135.10, 132.95, 128.93, 126.69, 126.66, 126.13, 123.18, 118.87. HRMS
(ESI) m/z [M + H]+: 353.05269 calcd for C16H11F3N2O2S: 353.05542;
N-(4-(trifluoromethoxy)phenyl)quinoline-8-sulfonamide (3 m).
White solid, Yield: 51%, m.p. 203.6–205.7 ◦C. 1H NMR (500 MHz,
DMSO) δ 10.43 (s, 1H), 9.16 (dd, J = 4.2, 1.7 Hz, 1H), 8.53 (dd, J = 8.4,
1.6 Hz, 1H), 8.43 (dd, J = 7.3, 1.2 Hz, 1H), 8.29 (dd, J = 8.2, 1.1 Hz,
1H), 7.79–7.66 (m, 2H), 7.26–7.09 (m, 4H). 13C NMR (126 MHz, DMSO)
δ 151.97, 144.53, 144.52, 143.14 (s, 1H), 137.54, 137.48, 135.46,
134.89, 132.66, 128.89, 126.12, 123.15, 122.23, 121.33. HRMS (ESI)
m/z [M + H]+: 369.04760 calcd for C16H11F3N2O3S: 369.05038;
N-(3-chlorophenyl)quinoline-8-sulfonamide (3n). White solid, Yield:
57%, m.p. 189.8–193.2 ◦C. 1H NMR (500 MHz, DMSO) δ 10.55 (s, 1H),
9.19 (dd, J = 4.2, 1.6 Hz, 1H), 8.56–8.45 (m, 2H), 8.34–8.24 (m, 1H),
7.82–7.69 (m, 2H), 7.21 (d, J = 1.7 Hz, 1H), 7.18–7.08 (m, 2H),
6.99–6.91 (m, 1H). 13C NMR (126 MHz, DMSO) δ 151.99, 143.12,
139.92, 137.43, 135.31, 134.96, 133.57, 132.83, 130.99, 128.87,
126.09, 123.64, 123.12, 119.14, 117.95. HRMS (ESI) m/z [M + H]+:
319.02633 calcd for C15H11ClN2O2S: 319.02954.
50%. 1H NMR (500 MHz, DMSO) δ 9.19 (dd, J = 4.1, 1.4 Hz, 1H), 8.95
(s, 1H), 8.56 (dd, J = 8.3, 1.2 Hz, 1H), 8.38–8.22 (m, 2H), 7.77 (dd, J =
8.3, 4.2 Hz, 1H), 7.69 (t, J = 7.7 Hz, 1H), 7.41 (d, J = 7.0 Hz, 1H), 6.96
(t, J = 7.8 Hz, 1H), 6.87–6.72 (m, 2H), 3.31 (s, 3H). 13C NMR (126 MHz,
DMSO) δ 151.79, 150.46, 142.91, 137.61, 135.76, 134.66, 131.36,
128.89, 126.48, 125.90, 123.16, 121.86, 121.05, 111.91, 55.77. HRMS
(ESI) m/z [M + H]+: 315.07587 calcd for C16H14N2O3S: 315.07877.
N-(3-methoxyphenyl)quinoline-8-sulfonamide (3f). White solid,
Yield: 51%, m.p. 164.8–166.6 ◦C. 1H NMR (500 MHz, DMSO) δ 10.10 (s,
1H), 9.15 (dd, J = 4.0, 1.4 Hz, 1H), 8.51 (d, J = 7.1 Hz, 1H), 8.39 (d, J =
6.6 Hz, 1H), 8.27 (d, J = 8.0 Hz, 1H), 7.79–7.65 (m, 2H), 6.98 (t, J = 8.4
Hz, 1H), 6.72–6.57 (m, 2H), 6.46 (d, J = 9.7 Hz, 1H), 3.55 (s, 3H). 13
C
NMR (126 MHz, DMSO) δ 159.85, 151.92, 143.18, 139.42, 137.45,
135.58, 134.76, 132.70, 130.11, 128.86, 126.11, 123.11, 112.15,
108.96, 106.06, 55.30. HRMS (ESI) m/z [M + H]+: 315.07587 calcd for
C16H14N2O3S: 315.07867.
N-(4-methoxyphenyl)quinoline-8-sulfonamide (3g) [48]. Yield:
43%. 1H NMR (500 MHz, DMSO) δ 9.72 (s, 1H), 9.19 (dd, J = 4.1, 1.5
Hz, 1H), 8.52 (dd, J = 8.3, 1.3 Hz, 1H), 8.38–8.17 (m, 2H), 7.74 (dd, J =
8.3, 4.2 Hz, 1H), 7.67 (t, J = 7.7 Hz, 1H), 6.93 (d, J = 8.9 Hz, 2H), 6.66
(d, J = 9.0 Hz, 2H), 3.57 (s, 3H). 13C NMR (126 MHz, DMSO) δ 156.72,
151.90, 143.19, 137.48, 135.64, 134.50, 132.38, 130.68, 128.79,
126.11, 123.40, 123.10, 114.45, 55.45. HRMS (ESI) m/z [M + H]+:
315.07587 calcd for C16H14N2O3S: 315.07880;
N-(2-fluorophenyl)quinoline-8-sulfonamide (3h). White solid,
Yield:47%, m.p. 175.4–177.3 ◦C. 1H NMR (500 MHz, DMSO) δ 9.66 (s,
1H), 9.12 (s, 1H), 8.57 (d, J = 7.1 Hz, 1H), 8.28 (dd, J = 27.4, 6.4 Hz,
2H), 7.91–7.60 (m, 2H), 7.22 (s, 1H), 7.14–6.93 (m, 3H). 13C NMR (126
MHz, DMSO) δ 156.77, 151.86, 143.14, 137.55, 136.18, 134.71, 131.53
, 128.94, 127.40, 126.54, 126.04, 125.25, 124.95, 123.15, 116.26.
HRMS (ESI) m/z [M + H]+: 303.05588 calcd for C15H11FN2O2S:
303.05875;
N-(3-fluorophenyl)quinoline-8-sulfonamide (3i). White solid, Yield:
56%, m.p. 184.4–185.9 ◦C. 1H NMR (500 MHz, DMSO) δ 10.56 (s, 1H),
9.20 (dd, J = 4.2, 1.7 Hz, 1H), 8.59–8.47 (m, 2H), 8.28 (dd, J = 8.2, 1.2
Hz, 1H), 7.82–7.68 (m, 2H), 7.16 (dd, J = 15.1, 7.9 Hz, 1H), 7.04–6.94
(m, 2H), 6.81–6.64 (m, 1H). 13C NMR (126 MHz, DMSO) δ 163.47,
161.54, 151.97, 143.14, 140.23, 137.41, 135.35, 134.91, 132.85,
130.99, 128.87, 126.07, 123.10, 115.36, 110.47, 110.30, 106.49,
106.29. HRMS (ESI) m/z [M + H]+: 303.05588 calcd for C15H11FN2O2S:
303.05884;
4.3. ADMET prediction
Compounds 3a–3n were introduced into the DS 2017 software, and
ADMET descriptors were selected after the compounds were treated. All
ADMET parameters, namely, aqueous solubility, BBB penetration,
N-(4-fluorophenyl)quinoline-8-sulfonamide(3j). White solid, Yield:
10

T. Liu et al.
Bioorganic Chemistry 114 (2021) 105037
CYP2D6 binding, hepatotoxicity, and intestinal absorption plasma pro-
tein binding, were selected to predict the results.
4.8.2. General procedure
RAW264.7 cells (3 × 105 cells/well) were seeded in 6-well plates and
incubated for 24 h. The cells were pretreated with compound 3l (1, 3,
4.4. Cell culture
and 10
μ
M) for 1 h and then stimulated with LPS (1
μ
g/mL) for 24 h.
After washing the cells with 1 × PBS, 300
μL of cell lysis buffer (RIRA
Mouse macrophages (RAW264.7 cells, Anhui Medical University)
and HEK293T cells (Anhui Medical University) were cultured in DMEM
(Hyclone, USA) containing 10% fetal bovine serum, 50 U/ml penicillin,
lysis buffer, phosphatase inhibitor, protease inhibitor, and PMSF) was
added to each well, incubated on ice for 30 min, and then centrifuged at
12,000 r/min for 30 min at 4 ◦C to collect the supernatant. Protein
content was determined using a protein quantification kit (BCA Assay,
Beyotime, Shanghai, China). Total proteins were separated by 10% SDS-
PAGE and then transferred from the gel to a PVDF membrane (Millipore,
Sigma). The membranes were blocked in TBST solution containing 5%
skimmed milk for 2 h. Afterward, the membranes were washed three
times (TBST) and incubated with the primary antibody dilution over-
night at 4 ◦C. After washing three times with TBST, the membranes were
combined with the corresponding diluted secondary antibody for 90 min
at room temperature. The blots were visualized by a chemiluminescence
imager (Tanon 5200, Shanghai, China).
and 50 μg/ml gentamicin and then incubated in an incubator containing
5% CO2 at 37 ◦C.
4.5. Cell viability assay
The cytotoxicity of the compounds to RAW264.7 cells was evaluated
via MTT assay. RAW264.7 cells (5,000 cells/well) were seeded in a 96-
well plate for 24 h (Sigma Aldrich, Australia), and the old medium was
discarded. The cells were treated with medium containing the com-
pounds (100, 80, 50, 25, and 12.5
(5 mg/ml, 20
discarded, and 150
μ
M) for 24 h and incubated with MTT
μ
L/well) for 4 h. The cell culture supernatants were then
μ
L DMSO was added to each well to dissolve for-
mazan. The 96-well plate was placed on a shaker to shake for 10 min,
and the absorbance was measured at 492 nm.
4.9. Target fishing and molecular docking
The potential drug targets of compound 3l were predicted and
screened using the DS 2017 software. The structure of compound 3l
(ligand) was drawn using the Chemdraw 2017 software and imported
into the DS software. After the ligand was prepared and the energy was
minimized, the potential targets of the compounds were virtually
screened through the Ligand Profiler program in the DS software. The
results were analyzed according to the fit value of the binding between
the ligand and each protein (the higher the fit value was, the better
compound 3l matched the corresponding protein). The target corre-
sponding to the protein was the likely target of the compound. For
further analysis, compound 3l and adenosine kinase protein were
combined through CDOCKER molecular docking.
4.6. In vitro determination of NO content
RAW264.7 cells (6 × 104 cells per well) were seeded in a 48-well
plate for 24 h, and then the medium with different concentrations
(100, 60, 40, 20, 10, 5, 2.5, 1.25 and 0.6725 μM) of the compounds was
added to continue the culture. After incubation for 1 h, LPS (1
L/well) was added to stimulate the cells for 24 h. Afterward, 50
the cell culture supernatant was collected and put it into a 96-well plate.
μ
g/ml, 30
μ
μ
L of
Griess Reagent I and Griess Reagent II (50 μL/well) were also added to
the 96-well plate to determine the content of NO. After 5 min, the
absorbance of the sample was measured at 540 nm.
4.7. In vitro determination of IL-1β and TNF-α content
4.10. Co-immunoprecipitation
The steps were similar to those described in Section 4.6. RAW264.7
cells (6 × 104 cells/well) were seeded in 48-well plates. After 24 h, the
medium containing different concentrations of the compounds was
After 30 min of pretreatment with 3l of 30 μM, RAW264.7 cells were
stimulated by LPS for 15 min. The cells were lysed by adding buffer (Cell
lysis buffer for Western and IP, PMSF), and the cell lysate was collected.
The cell extracts were incubated with an appropriate amount of anti-
MD2 antibody (Cat number, 822065, Dilution ratio, 1:1000, Zenbio,
China) and precipitated with A + G agarose beads (Beyotime, China)
added for 1 h, and then the cells were added with LPS (1 μg/mL) for 24 h.
The content of TNF-α and IL-β in the cell supernatant was measured via
ELISA (Jyimei, Wuhan, China) following the manufacturer’s
instructions.
◦
overnight at 4 C. After boiling, the released protein was detected by
immunoblot using anti-TLR4 antibody (Cat number, 505208, Dilu-
tion ratio, 1:1000, Zenbio, China).
4.8. Western blot analysis
HEK293T cells were seeded at 4 × 105 cells/dish in a 6-well plate
4.8.1. Antibodies
overnight and then co-transfected with HA-TLR4 (3 μg) and Flag-TLR4
iNOS was purchased from Zenbio, China (Cat number, 340668,
Dilution ratio, 1:1000); COX-2 was purchased from Zenbio, China (Cat
number, 383971, Dilution ratio, 1:1000); Actin was purchased from
Beyotime, China (Cat number, AF5003, Dilution ratio, 1:5000); IκB was
purchased from Beyotime, China (Cat number, AF1282, Dilution ratio,
1:2000); p-IκB was purchased from Beyotime, China (Cat number,
AF1870, Dilution ratio, 1:1000); P65 was purchased from Zenbio, China
(Cat number, 380172, Dilution ratio, 1:5000); p-P65 was purchased
from Zenbio, China (Cat number, 310012, Dilution ratio, 1:1000); P38
was purchased from Zenbio, China (Cat number, R25239, Dilution ratio,
1:1000); p-P38 was purchased from Zenbio, China (Cat number,
310091, Dilution ratio, 1:1000); ERK was purchased from Zenbio, China
(Cat number, 343830, Dilution ratio, 1:1000); p-ERK was purchased
from Zenbio, China (Cat number, 301245, Dilution ratio, 1:1000); JNK
was purchased from Zenbio, China (Cat number, 310012, Dilution ratio,
1:1000); p-JNK was purchased from Zenbio, China (Cat number,
310012, Dilution ratio, 1:1000); secondary antibody was purchased
from Zenbio, China (Cat number, 550064, Dilution ratio, 1:10000).
(3
μ
g) plasmids for 24 h. Afterward, the cells were seeded at 4 × 105
in a 5 cm (i.d.) dish and cultured for 24 h. The cells were pretreated with
compound 3l for 1 h and cultured with LPS (1 mg/mL) treatment 12 h.
The IP lysis buffer (Cell lysis buffer for Western and IP, PMSF) was added
to the Petri dish to harvest the cells. The effect of compound 3l on the
formation of TLR4 dimers was analyzed by Western blot and anti-HA
magnetic beads.
4.11. Cellular thermal shift assay
RAW264.7 cells were seeded in a 6-well plate for 24 h (6 × 104 cells/
well). After the cells were treated with compound 3l (30 μM) for 12 h,
the protein was collected with cell lysate. The protein was then evenly
divided into four parts and heated at different temperatures (45 ◦C,
50 ◦C, 55 ◦C, 60 ◦C and 65 ◦C) for 3 min. The results were analyzed by
Western blot.
11

T. Liu et al.
Bioorganic Chemistry 114 (2021) 105037
bearing bispiperazine linker as IL-1beta inhibitors, Bioorg. Chem. 98 (2020),
103748.
4.12. In vivo experiment
[3] L.Z. Chen, W.W. Sun, L. Bo, J.Q. Wang, C. Xiu, W.J. Tang, J.B. Shi, H.P. Zhou, X.
H. Liu, New arylpyrazoline-coumarins: Synthesis and anti-inflammatory activity,
Eur. J. Med. Chem. 138 (2017) 170–181.
4.12.1. Animals
Sprague–Dawley female rats weighing 160–180 g were purchased
from the Animal Experiment Center of Anhui University of Chinese
Medicine. The temperature, relative humidity, and dark–light cycle per
day of the animal room was maintained at 23 ◦C–25 ◦C, 40%–60%, and
12 h, respectively. The animal experiments were approved by the
Experimental Animal Ethics Committee of Anhui Medical University.
[4] R. Medzhitov, Origin and physiological roles of inflammation, Nature 454 (2008)
428–435.
[5] A. Linde, D. Mosier, F. Blecha, T. Melgarejo, Innate immunity and
inflammation–New frontiers in comparative cardiovascular pathology, Cardiovasc.
Res. 73 (2007) 26–36.
[6] L.F. Shirazi, J. Bissett, F. Romeo, J.L. Mehta, Role of Inflammation in Heart Failure,
Curr. Atheroscler. Rep 19 (2017) 27.
[7] C. Bogdan, Nitric oxide and the immune response, Nat. Immunol. 2 (2001)
907–916.
4.12.2. Induction of adjuvant arthritis and experimental design
The rats were injected with 0.1 mL Freund’s adjuvant complete
(FCA) intracutaneously into the left hind feet to cause inflammation. The
rats in the normal group were injected with the same amount of phys-
iological saline at the same site.
[8] R. Heiran, S. Sepehri, A. Jarrahpour, C. Digiorgio, H. Douafer, J.M. Brunel,
A. Gholami, E. Riazimontazer, E. Turos, Synthesis, docking and evaluation of in
vitro anti-inflammatory activity of novel morpholine capped beta-lactam
derivatives, Bioorg. Chem. 102 (2020), 104091.
[9] Y.H. Seo, J.K. Kim, J.G. Jun, Synthesis and biological evaluation of piperlongumine
derivatives as potent anti-inflammatory agents, Bioorg. Med. Chem. Lett. 24 (2014)
5727–5730.
The rats were randomly divided into four groups after 10 days of FCA
injection. The two groups were treated with compound 3l (10 and 30
mg/kg) for 14 days, and the rats in the positive control group were
treated with indomethacin (10 mg/kg) for 14 days.
[10] J. Li, X. Fan, J. Deng, Y. Liang, S. Ma, Y. Lu, J. Zhang, T. Shi, W. Tan, Z. Wang,
Design and synthesis of 1,3-benzothiazinone derivatives as potential anti-
inflammatory agents, Bioorg. Med. Chem. 28 (2020), 115526.
[11] S. Sehajpal, D.N. Prasad, R.K. Singh, Prodrugs of Non-steroidal Anti-inflammatory
Drugs (NSAIDs): A Long March Towards Synthesis of Safer NSAIDs, Mini Rev. Med.
Chem. 18 (2018) 1199–1219.
4.12.3. HE staining
[12] Y. Xu, S. Chen, Y. Cao, P. Zhou, Z. Chen, K. Cheng, Discovery of novel small
molecule TLR4 inhibitors as potent anti-inflammatory agents, Eur. J. Med. Chem.
154 (2018) 253–266.
The inflamed contralateral knee joints of the rats were removed, kept
in 4% paraformaldehyde solution, decalcified with 5% formic acid, and
then embedded in paraffin. The sections were stained with HE. Histo-
pathological changes were observed under an optical microscope.
[13] U. Debnath, S. Mukherjee, N. Joardar, S.P. Sinha Babu, K. Jana, A.K. Misra, Aryl
quinolinyl hydrazone derivatives as anti-inflammatory agents that inhibit TLR4
activation in the macrophages, Eur. J. Pharm. Sci. 134 (2019) 102–115.
[14] H.J. Chung, W. Koh, W.K. Kim, J.S. Shin, J. Lee, S.K. Lee, I.H. Ha, The anti-
inflammatory effects of Shinbaro3 Is mediated by downregulation of the TLR4
signalling pathway in LPS-stimulated RAW 264.7 macrophages, Mediators
Inflamm. 2018 (2018), 4514329.
4.12.4. In vivo determination of IL-1β, and TNF-α content
After the rats were anesthetized, blood was collected from the heart
artery. It was allowed to stand for 30 min. The blood sample was
centrifuged at 3000 r/min for 10 min at 4 ◦C to collect the serum. The
[15] H. Gao, N. Kang, C. Hu, Z. Zhang, Q. Xu, Y. Liu, S. Yang, Ginsenoside Rb1 exerts
anti-inflammatory effects in vitro and in vivo by modulating toll-like receptor 4
dimerization and NF-kB/MAPKs signaling pathways, Phytomedicine 69 (2020),
153197.
serum levels of IL-1β, and TNF-α were determined via ELISA.
[16] X.Z. Han, R. Ma, Q. Chen, X. Jin, Y.Z. Jin, R.B. An, X.M. Piao, M.L. Lian, L.H. Quan,
J. Jiang, Anti-inflammatory action of Athyrium multidentatum extract suppresses
the LPS-induced TLR4 signaling pathway, J. Ethnopharmacol. 217 (2018)
220–227.
4.12.5. Statistical analysis
Data are reported as mean ± SEM of at least three independent ex-
periments and data analysis was performed with GraphPad Prism 8
software.
[17] H. Ju Woo, D.Y. Jun, J.Y. Lee, H.S. Park, M.H. Woo, S.J. Park, S.C. Kim, C.H. Yang,
Y.H. Kim, Anti-inflammatory action of 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-
naphthoquinone (CMEP-NQ) suppresses both the MyD88-dependent and TRIF-
dependent pathways of TLR4 signaling in LPS-stimulated RAW264.7 cells,
J. Ethnopharmacol. 205 (2017) 103–115.
Declaration of Competing Interest
[18] Y. Zhang, C. Liu, B. Dong, X. Ma, L. Hou, X. Cao, C. Wang, Anti-inflammatory
activity and mechanism of surfactin in lipopolysaccharide-activated macrophages,
Inflammation 38 (2015) 756–764.
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.
[19] Z. Liu, L. Chen, P. Yu, Y. Zhang, B. Fang, C. Wu, W. Luo, X. Chen, C. Li, G. Liang,
Discovery of 3-(Indol-5-yl)-indazole derivatives as novel myeloid differentiation
protein 2/toll-like receptor 4 antagonists for treatment of acute lung injury, J. Med.
Chem. 62 (2019) 5453–5469.
[20] S.A. Chavez, A.J. Martinko, C. Lau, M.N. Pham, K. Cheng, D.E. Bevan, T.E. Mollnes,
H. Yin, Development of beta-amino alcohol derivatives that inhibit Toll-like
receptor 4 mediated inflammatory response as potential antiseptics, J. Med. Chem.
54 (2011) 4659–4669.
Acknowledgments
We thank the Excellent Young Talents Fund Program of Higher Ed-
ucation Institutions of Anhui Province (CN) (gxyq202006), the Natural
Science Research Project of the Educational Commission of Anhui
Province of China (KJ2019A0231) and the basic health research project
in Anhui Province (GXXT-2019-045) for financial support of this study.
We also thank the Center for Scientific Research of Anhui Medical
University for valuable help in our experiment.
[21] W. Karunarathne, K.T. Lee, Y.H. Choi, C.Y. Jin, G.Y. Kim, Anthocyanins isolated
from Hibiscus syriacus L. attenuate lipopolysaccharide-induced inflammation and
endotoxic shock by inhibiting the TLR4/MD-2-mediated NF-kappaB signaling
pathway, Phytomedicine 76 (2020), 153237.
[22] A. Marella, O.P. Tanwar, R. Saha, M.R. Ali, S. Srivastava, M. Akhter,
M. Shaquiquzzaman, M.M. Alam, Quinoline: A versatile heterocyclic, Saudi Pharm.
J. 21 (2013) 1–12.
[23] C.H. Tseng, C.W. Tung, C.H. Wu, C.C. Tzeng, Y.H. Chen, T.L. Hwang, Y.L. Chen,
Discovery of Indeno[1,2-c]quinoline derivatives as potent dual antituberculosis
and anti-inflammatory agents, Molecules 22 (2017).
[24] I. Chaaban, O.H. Rizk, T.M. Ibrahim, S.S. Henen, E.M. El-Khawass, A.E. Bayad, I.
M. El-Ashmawy, H.A. Nematalla, Synthesis, anti-inflammatory screening,
molecular docking, and COX-1,2/-5-LOX inhibition profile of some novel quinoline
derivatives, Bioorg. Chem. 78 (2018) 220–235.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.105037.
[25] P. Gao, L. Wang, L. Zhao, Q.Y. Zhang, K.W. Zeng, M.B. Zhao, Y. Jiang, P.F. Tu, X.
Y. Guo, Anti-inflammatory quinoline alkaloids from the root bark of Dictamnus
dasycarpus, Phytochemistry 172 (2020), 112260.
[26] X.F. Shang, S.L. Morris-Natschke, G.Z. Yang, Y.Q. Liu, X. Guo, X.S. Xu, M. Goto, J.
C. Li, J.Y. Zhang, K.H. Lee, Biologically active quinoline and quinazoline alkaloids
part II, Med. Res. Rev. 38 (2018) 1614–1660.
References
[1] J. Chen, D.L. Li, L.N. Xie, Y.R. Ma, P.P. Wu, C. Li, W.F. Liu, K. Zhang, R.P. Zhou, X.
T. Xu, X. Zheng, X. Liu, Synergistic anti-inflammatory effects of silibinin and
thymol combination on LPS-induced RAW264.7 cells by inhibition of NF-kappaB
and MAPK activation, Phytomedicine 78 (2020), 153309.
[27] H. Suleyman, E. Cadirci, A. Albayrak, Z. Halici, Nimesulide is a selective COX-2
inhibitory, atypical non-steroidal anti-inflammatory drug, Curr. Med. Chem. 15
(2008) 278–283.
[2] Y.L. Tang, X. Zheng, Y. Qi, X.J. Pu, B. Liu, X. Zhang, X.S. Li, W.L. Xiao, C.P. Wan, Z.
W. Mao, Synthesis and anti-inflammatory evaluation of new chalcone derivatives
12

T. Liu et al.
Bioorganic Chemistry 114 (2021) 105037
[28] Vinod Kumar, Kamalneet Kaura, Girish K. Gupta, Akhilesh K. Guptaa, S. Kumar,
Developments in synthesis of the anti-inflammatory drug, celecoxib: a review,
Recent Pat. Inflamm. Allergy Drug Discov. 7 (2013) 124–134.
[39] D. Carvalho, M. Paulino, F. Polticelli, F. Arredondo, R.J. Williams, J.A. Abin-
Carriquiry, Structural evidence of quercetin multi-target bioactivity: A reverse
virtual screening strategy, Eur. J. Pharm. Sci. 106 (2017) 393–403.
[40] T. Kawai, S. Akira, Toll-like receptor downstream signaling, Arthritis Res. Ther. 7
(2005) 12–19.
[29] Y. Sun, Q. Xu, Z. Wu, Y. Gong, L. Tang, Parecoxib inhibits inflammatory responses
in a mouse model of sepsis, FEBS Open Biol. (2020).
[30] M. Wu, J. Ma, L. Ji, M. Wang, J. Han, Z. Li, Design, synthesis, and biological
evaluation of rutacecarpine derivatives as multitarget-directed ligands for the
treatment of Alzheimer’s disease, Eur. J. Med. Chem. 177 (2019) 198–211.
[31] J. Grover, V. Kumar, V. Singh, K. Bairwa, M.E. Sobhia, S.M. Jachak, Synthesis,
biological evaluation, molecular docking and theoretical evaluation of ADMET
properties of nepodin and chrysophanol derivatives as potential cyclooxygenase
(COX-1, COX-2) inhibitors, Eur. J. Med. Chem. 80 (2014) 47–56.
[41] J. Perrin, T. Werner, N. Kurzawa, A. Rutkowska, D.D. Childs, M. Kalxdorf,
D. Poeckel, E. Stonehouse, K. Strohmer, B. Heller, D.W. Thomson, J. Krause,
I. Becher, H.C. Eberl, J. Vappiani, D.C. Sevin, C.E. Rau, H. Franken, W. Huber,
M. Faelth-Savitski, M.M. Savitski, M. Bantscheff, G. Bergamini, Identifying drug
targets in tissues and whole blood with thermal-shift profiling, Nat. Biotechnol. 38
(2020) 303–308.
[42] Lingyun Dai, Nayana Prabhu, Liang Ying Yu, Smaranda Bacanu, Anderson Daniel
Ramos, P. Nordlund, Horizontal cell biology: monitoring global changes of protein
interaction states with the proteome-wide cellular thermal shift assay (CETSA),
Annu. Rev. Biochem. 88 (2019) 383–408.
[32] L. Ma, H. Pei, L. Lei, L. He, J. Chen, X. Liang, A. Peng, H. Ye, M. Xiang, L. Chen,
Structural exploration, synthesis and pharmacological evaluation of novel 5-ben-
zylidenethiazolidine-2,4-dione derivatives as iNOS inhibitors against inflammatory
diseases, Eur. J. Med. Chem. 92 (2015) 178–190.
[43] F.B. Reinhard, D. Eberhard, T. Werner, H. Franken, D. Childs, C. Doce, M.
F. Savitski, W. Huber, M. Bantscheff, M.M. Savitski, G. Drewes, Thermal proteome
profiling monitors ligand interactions with cellular membrane proteins, Nat.
Methods 12 (2015) 1129–1131.
[33] L.Z. Chen, J. Wu, K. Li, Q.Q. Wu, R. Chen, X.H. Liu, B.F. Ruan, Novel phthalide
derivatives: Synthesis and anti-inflammatory activity in vitro and in vivo, Eur. J.
Med. Chem. 206 (2020), 112722.
[34] W. Fan, F. Huang, C. Li, H. Qu, Z. Gao, S. Leng, D. Li, H. He, Involvement of NOS/
NO in the development of chronic dental inflammatory pain in rats, Brain Res. Rev.
59 (2009) 324–332.
[44] J.M. Dziekan, G. Wirjanata, L. Dai, K.D. Go, H. Yu, Y.T. Lim, L. Chen, L.C. Wang,
B. Puspita, N. Prabhu, R.M. Sobota, P. Nordlund, Z. Bozdech, Cellular thermal shift
assay for the identification of drug-target interactions in the Plasmodium
falciparum proteome, Nat. Protoc. 15 (2020) 1881–1921.
[35] T. Sogo, N. Terahara, A. Hisanaga, T. Kumamoto, T. Yamashiro, S. Wu, K. Sakao, D.
X. Hou, Anti-inflammatory activity and molecular mechanism of delphinidin 3-
sambubioside, a Hibiscus anthocyanin, BioFactors 41 (2015) 58–65.
[36] M. Wang, T. Liu, S. Chen, M. Wu, J. Han, Z. Li, Design and synthesis of 3-(4-
pyridyl)-5-(4-sulfamido-phenyl)-1,2,4-oxadiazole derivatives as novel GSK-3beta
inhibitors and evaluation of their potential as multifunctional anti-Alzheimer
agents, Eur. J. Med. Chem. 209 (2021), 112874.
[45] L.Z. Chen, L. Yao, M.M. Jiao, J.B. Shi, Y. Tan, B.F. Ruan, X.H. Liu, Novel
resveratrol-based flavonol derivatives: Synthesis and anti-inflammatory activity in
vitro and in vivo, Eur. J. Med. Chem. 175 (2019) 114–128.
[46] E. Choi, J. Lee, S. Lee, B.W. Song, H.H. Seo, M.J. Cha, S. Lim, C. Lee, S.W. Song,
G. Han, K.C. Hwang, Potential therapeutic application of small molecule with
sulfonamide for chondrogenic differentiation and articular cartilage repair, Bioorg.
Med. Chem. Lett. 26 (2016) 5098–5102.
[37] H.N. Kim, J.D. Kim, J.H. Yeo, H.J. Son, S.B. Park, G.H. Park, H.J. Eo, J.B. Jeong,
Heracleum moellendorffii roots inhibit the production of pro-inflammatory
mediators through the inhibition of NF-kappaB and MAPK signaling, and activation
of ROS/Nrf2/HO-1 signaling in LPS-stimulated RAW264.7 cells, BMC Complement
Altern. Med. 19 (2019) 310.
[47] K.-T. Hsu, Internal Marketing Based on the Hierarchy of Effects Model for the Life
Insurance Industry, Int. Bus. Res. 9 (2016) 1.
[48] N.M. Vinokurova, I.K. Fel’dman, Synthesis and properties of some sulfonamides of
the quinoline series Pharmaceutical, Chemistry 10 (1968) 34–36.
[49] David Scott Goldfarb, Method for altering the lifespan of eukaryotic organisms in:
U.S. Pat (Ed.), US, 2009.
[38] H. Huang, G. Zhang, Y. Zhou, C. Lin, S. Chen, Y. Lin, S. Mai, Z. Huang, Reverse
screening methods to search for the protein targets of chemopreventive
compounds, Front. Chem. 6 (2018) 138.
[50] М.М.V. RAMANA, R.S. LOKHANDE, G. GAYATRIVIJAY, A process for the synthesis
of sulfonamide derivatives and their antituberculosis activity., in: U.S. Pat (Ed.),
US, 2002.
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