Protective Effect of a Novel (2S, 3R, 4S)-Chromene-3-Carboxamide Derivative, Z20 Against Sepsis-Induced Organ Injury

Article abstract of DOI:10.1007/s10753-019-01174-z

Sepsis, a systemic inflammatory response mediated by excessive production of diverse inflammatory cytokines, remains the vital cause of morality in the intensive care unit (ICU). TLR4-MD2 (toll-like receptor 4-myeloid differentiation factor 2) complex activated by LPS serves as an effective target to decrease the inflammation during sepsis. In this study, we evaluated the effects of a new small molecule Z20 structural based on (2S, 3R, 4S)-chromene-3-carboxamide on LPS-induced sepsis in mice. We found Z20 markedly improved the survival rate and attenuated the multiply organs injury after LPS administration?in mice. In addition, Z20 significantly alleviated organ inflammation as characterized by diminished inflammatory factors expression in vivo. Furthermore, by employing surface plasmon resonance (SPR) experiment, we identified that TLR4-MD2 complex?was the potential target for Z20. Finally, we performed the safety assessment experiment to confirm the safety of Z20 in vivo. In conclusion, Z20, as a potential TLR4-MD2 inhibitor, effectively attenuated LPS-induced organ injury and inflammation.

Full text of DOI:10.1007/s10753-019-01174-z

#
Inflammation ( 2020)
DOI: 10.1007/s10753-019-01174-z
ORIGINAL ARTICLE
Protective Effect of a Novel (2S, 3R,
4S)-Chromene-3-Carboxamide Derivative, Z20
Against Sepsis-Induced Organ Injury
3
Liyan Zeng,1,2 Yuhang Wang,1 Na Li,3 Mengwei Niu,3 Yong Wang,1 and Peng Chen
Abstract—
Sepsis, a systemic inflammatory response mediated by excessive production of
diverse inflammatory cytokines, remains the vital cause of morality in the intensive care unit (I-
CU). TLR4-MD2 (toll-like receptor 4-myeloid differentiation factor 2) complex activated by LPS
serves as an effective target to decrease the inflammation during sepsis. In this study, we evaluated
the effects of a new small molecule Z20 structural based on (2S, 3R, 4S)-chromene-3-
carboxamide on LPS-induced sepsis in mice. We found Z20 markedly improved the survival
rate and attenuated the multiply organs injury after LPS administration in mice. In addition, Z20
significantly alleviated organ inflammation as characterized by diminished inflammatory factors
expression in vivo. Furthermore, by employing surface plasmon resonance (SPR) experiment, we
identified that TLR4-MD2 complex was the potential target for Z20. Finally, we performed the
safety assessment experiment to confirm the safety of Z20 in vivo. In conclusion, Z20, as a
potential TLR4-MD2 inhibitor, effectively attenuated LPS-induced organ injury and
inflammation.
KEY WORDS: sepsis; TLR4-MD2; anti-inflammation; organ injury; inflammatory cytokine.Liyan Zeng, Yuhang
Wang and Na Li contributed equally to this work.
INTRODUCTION
Sepsis, a systemic inflammatory response induced
by infection, is a global public health concern [1, 2]. It
is one of the leading causes for death in the ICU [3].
Inflammation plays a decisive role in the process of
sepsis [4]. Acute inflammation induces the increased
secretion of inflammatory cytokines and inflammatory
cell infiltration to defend pathogens. However, exces-
sive inflammatory cytokines can destroy host immune
system and may aggravate organ damage as seen in
sepsis [5]. Tumor necrosis factor (TNF) and interleukin
(IL)-6, key pro-inflammatory cytokines, are primarily
involved in promoting inflammatory processes and re-
main a decisive role in sepsis [6, 7]. It has been
identified that inhibiting the expression of cytokines
as a potential strategy for treating sepsis.
Liyan Zeng, Yuhang Wang and Na Li contributed equally to this work.
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s10753-019-01174-z) contains supplementary
material, which is available to authorized users.
1 Department of Pharmacy, Zhujiang Hospital, Southern Medical Univer-
sity, Guangzhou, China
2 College of Chemistry and Chemical Engineering, Zhongkai University
of Agriculture and Engineering, Guangzhou, China
3 Department of Pathophysiology, Guangdong Provincial Key Laboratory
of Proteomics, School of Basic Medical Sciences, Southern Medical
University, Guangzhou, China
4 To whom correspondence should be addressed to Yong Wang at
Department of Pharmacy, Zhujiang Hospital, Southern Medical
University, Guangzhou, China. E-mail: yongwh2005@163.com; and
Peng Chen at Department of Pathophysiology, Guangdong Provincial
Key Laboratory of Proteomics, School of Basic Medical Sciences,
Southern Medical University, Guangzhou, China. E-mail:
perchen@smu.edu.cn
#
0360-3997/20/0000-0001/0 2020 Springer Science+Business Media, LLC, part of Springer Nature

Zeng, Wang, Li, Niu, Wang, and Chen
Lipopolysaccharide (LPS), the component of the cell
wall of Gram-negative bacteria, is known to be one of the
major causes of septic shock. LPS binds to the toll-like
receptor 4 (TLR4)-myeloid differentiation 2 (MD2) com-
plex on the host cell surface [8]. When LPS-TLR4-MD2
pathway is activated, it triggers many inflammatory signal-
ing pathways activation such as mitogen-activated protein
kinases (MAPKs), thereby promoting the transduction of
pro-inflammatory molecules. However, excessive pro-
inflammatory cytokines and inappropriate amplification
of immune response, caused by LPS, lead to tissue dam-
age, organ failure, and death. Therefore, the complex of
TLR4-MD2 is considered as the effective target of
inhibiting to suppress LPS-induced inflammation [9]. At
present, some inhibitors of TLR4-MD2 exhibited protec-
tive effect on severe sepsis in preclinical study. Unfortu-
nately, most of them failed to cure sepsis in clinic [10].
Thus, it is still in urgent need to discover novel anti-
inflammatory drugs to treat septic patients.
spectrometer and high-resolution mass spectrometry
(HRMS) on LCMS-IT-TOF mass spectrometer. The char-
acterizations of Z20 are as follows: white powder (yield
91%); M.P. 191.8–193.6 °C; 1H NMR (400 MHz, DMSO)
δ 10.19 (s, NH, 1H), 8.08 (d, J = 7.7, aromatic, 1H), 7.67 (t,
J = 7.5, aromatic, 1H), 7.48 (d, J = 7.6, aromatic, 1H), 7.42
(t, J = 8.9, aromatic, 3H), 7.31 (d, J = 8.3, aromatic, 4H),
7.24 (d, J = 8.1, aromatic, 2H), 7.10 (t, J = 7.2, aromatic,
1H), 7.02–6.81 (m, aromatic, 4H), 6.02 (s, NH, 1H), 4.50
(d, J = 11.4, CH, 1H), 3.24 (d, J = 11.4, CH, 1H), 2.14 (s,
CH3, 3H), 1.83 (s, CH3, 3H); 13C NMR (101 MHz,
DMSO) δ 168.88, 160.18, 158.08, 152.79, 141.16,
140.83, 138.04, 132.64, 131.47, 129.79, 129.69, 129.25,
129.00, 128.70, 124.85, 123.20, 120.47, 118.44, 116.81,
115.64, 104.65, 90.28, 57.57, 38.93, 22.89, 20.48; HRMS:
calcd for C33H27ClN2O4 550.1659, found 549.1587 [M-
H].
Animal and Experimental Design
In previous study, we synthesized a series of novel
polyfunctional (2S, 3R, 4S)-chromene-3-carboxamides
compounds, which were designed by improved the
Hantzsch reaction [11]. After preliminary anti-
inflammatory test in RAW267.4 cells, compound Z20
exhibited an excellent anti-inflammatory effect without
cytotoxicity. Significantly, Z20 effectively reduced the
LPS-induced expression of TNF-α and IL-6 in vitro. These
results promoted us to investigate whether Z20 has the
potential to treat sepsis in vivo. Herein, we evaluated the
potential protective effects of Z20 against LPS-induced
sepsis in mice model and explored the anti-inflammatory
mechanism of Z20.
The male C57BL/6J mice were purchased from
Guangdong Laboratory Animal Center and LPS was pur-
chased from Sigma Aldrich (USA). Z20 was dissolved in
dimethyl sulfoxide (DMSO) (MP Biomedicals, USA). To
evaluate the effect of Z20 on the survival of LPS
(Escherichia coli, serotype 0111:B4, Sigma)-induced sep-
sis, mice were randomly divided into the following two
groups: LPS + DMSO (i.p.) and LPS + Z20 (1 mg/kg, i.p.).
The DMSO or Z20 was injected immediately after the
injection of LPS (15 mg/kg, i.p.). Survival was monitored
until 1 week post-LPS challenge.
To evaluate the protective effect of Z20 on organ
injury in mice, Z20 was dissolved in DMSO first and then
diluted in PBS. Mice were randomly divided into four
groups: PBS; Z20; LPS; LPS + Z20. The phosphate buffer
saline (PBS) or LPS (15 mg/kg, i.p.) was injected, then
PBS or Z20 (1 mg/kg, i.p.) was immediately injected.
Twelve hours after treatment, mice were euthanized, and
blood samples and the liver/lung/kidney were collected.
To evaluate the safety of the Z20 in mice, mice were
randomly divided into two groups: PBS (i.p) and Z20
(1 mg/kg, i.p. Z20 was dissolved in DMSO first and then
diluted in PBS). The PBS or Z20 was injected once a week.
After 8 weeks, the mice were euthanized, and blood and
organ samples were collected. The experimental protocol
was approved by the institutional ethics committee.
MATERIALS AND METHODS
Synthesis and the Information of Z20
Chemicals were purchased from J&K Technology
Co, LTD. First, diketene (2 mmol) and aniline (2 mmol)
were added in acetonitrile (5 ml) and stirred at room
temperature. The reaction was monitored by TLC (thin
layer chromatography). Two hours later, 4-
hydroxycoumarin (2 mmol), p-toluidine (2 mmol), 4-
chlorobenzaldehyde (2 mmol), and iodine (0.2 mmol) were
added and stirred. The reaction was solidified in 2 min. The
solid was isolated by filtration and purified by recrystalli-
zation. The chemical structure of compound Z20 (Fig. 1a)
was characterized by nuclear magnetic resonance (1H
NMR and 13C NMR) on Bruker Advance drx 400
Cell Culture
RAW264.7 cells, the murine macrophage cell line,
were cultured in DMEM supplemented with 1% penicillin

Protective Effect of a Novel (2S, 3R, 4S)-Chromene-3-Carboxamide
100
a
b
LPS
LPS+Z20
80
60
40
20
0
Z20
*
0
20
40
60
80
100
120
140
160
hours
c
PBS
Z20
LPS
LPS+Z20
PBS
d
Liver
Lung
Kidney
*
Z20
*
8
6
4
2
0
8
6
4
2
0
8
LPS
*
LPS+Z20
6
4
2
0
Fig. 1. The protective effect of Z20 in septic mice model. a The chemical structure of Z20. b The survival curve after LPS injection. Mice were randomly
divided into two groups: LPS (n = 17) and LPS + Z20 (n = 16). Each group was treated with Z20 (1 mg/kg, i.p.) or DMSO (200 μl, i.p.) after LPS (15 mg/kg,
i.p.). c The H&E staining of the liver, lung, and kidney. Mice were randomly divided into four groups: PBS, Z20, LPS, LPS + Z20. Twelve hours after LPS
and Z20 treatment, mice were euthanized, and liver, lung and kidney were collected. d The histological score of the liver, lung, and kidney (n = 3–5).
*P < 0.05.

Zeng, Wang, Li, Niu, Wang, and Chen
and streptomycin, 10% heat-inactivated fetal bovine serum
(FBS) in a humidified atmosphere with 5% CO2 at 37 °C.
Then, RAW264.7 cells were plated at a density of 2 × 106
cell per well in a 6-well plate for 12 h, next cells were
incubated without FBS for 12 h. Before treated with LPS
(500 ng/ml), cells were pre-treated with DMSO or different
concentrations of Z20 (1, 10, 100 μmol/l) for 1.5 h. Fifteen
minutes after LPS administration, total protein was extract-
ed from cells.
brief, the sensors were loaded with MD2 and TLR4 protein
(in acetate acid buffer pH 5.5), then it was activated with
10 mM NiSO4. Different concentrations of Z20 (at 100, 50,
25, 12.5 and 6.25 μM) were prepared with running buffer
(PBS, 0.1% SDS, 5% DMSO, and 0.05% Tween-20).
Sensor and sample plate were placed on the instrument.
The flow cells captured Z20 samples, and the second flow
cell served as blank. Injecting simultaneously five concen-
trations, the flow rate was 30 μM/min for 120 s of associ-
ation phase, then it was followed by 120 s of dissociation
phase at 25 °C. The data was obtained and analyzed with
PlexArray® HT specialized software.
Histological Analysis
Tissues were fixed in buffered formalin, and sliced
into 5-μm-thick sections, followed by hematoxylin–eosin
(H&E) staining according to standard protocol. To obtain
the histological score of the organs, 6–8 random fields per
slide were collected (200×), and the histopathological scor-
ing analysis was performed blindly according to previously
described methods [12–14]. The result was reported as the
sum of the individual scores from 0 (no finding) to 1
(mild), 2 (moderate), and 3 (severe) for each of parameters.
The parameters of the liver were vacuolization, nuclear
condensation, inflammation, and erythrocyte stasis. The
parameters of the lung were notable inflammatory neutro-
phils infiltration, inter-alveolar septal thickening, intersti-
tial and inter-alveolar edema, erythrocyte stasis, and col-
lapsed alveoli. And, the parameters of the kidney were
brush border loss, interstitial edema, cell necrosis, inflam-
mation, and erythrocyte stasis.
Statistics
Data was showed as mean SEM. P < 0.05 (two-
tailed unpaired Student’s t test) was considered statistically
significant (GraphPad Prism v7.01).
RESULT
Z20 Effectively Prolongs Septic Mice Survival Time
and Attenuates LPS-Induced Organ Injury
Cytokines, especially TNF-α and IL-6, were essential
pro-inflammatory mediator in septic inflammation process-
es. In previous research, we clarified that Z20 effectively
decrease the production of TNF-α and IL-6 in RAW264.7
cells without cytotoxicity [11]. And, the chemical structure
of Z20 was shown in Fig. 1a. To further identify the
protective effect of Z20 in vivo, we performed LPS-
induced sepsis model in mice. Evidently, Z20 treatment
significantly prolonged the survival time compared with
control mice (Fig. 1b). Moreover, we found that Z20 can
attenuate the cellular damage that occurs as a result of
organ damage induced by LPS (Fig. 1c, d). Expectedly,
histological alteration of the organs from LPS-treated mice
was characterized as more severe organ damage, such as
nuclear condensation in the liver, inter-alveolar septal
thickening in the lung, and inflammatory neutrophils infil-
tration in the kidney, while the severity of the organ injury
was markedly diminished in the Z20-treated group. Taken
together, these observations indicate that Z20 has protec-
tive effect on LPS-induced sepsis in vivo.
Gene Expression
Tissue total RNA was isolated by TRIzol reagent,
followed by the reverse transcript reaction using the com-
mercial enzyme (TOYOBO, Shanghai, China). The ex-
pression of the genes was quantified by real-time PCR
and 18S ribosome was employed as the housekeeping
control. All primers are listed in Supplementary Table 1.
Western Blotting
The total protein was extracted by the RIPA method.
The protein levels of phosphorylated ERK (p-ERK), ERK,
were detected by Western blot as standard protocol. All
antibodies were acquired from Cell Signaling Technology.
SPR Experiment
Z20 Suppresses LPS-Induced Inflammatory Cytokines
Expression In Vivo
The PlexArray® HT surface plasmon resonance im-
aging platform (SPRi) was used to determine the binding
affinity of Z20 with Nanocapture® Sensor Chips. The SPR
experiment was performed as described previously [15]. In
Then, we determined the inhibitory effect of Z20 on
LPS-induced inflammatory cytokines expression in vivo.

Protective Effect of a Novel (2S, 3R, 4S)-Chromene-3-Carboxamide
As expected, the expression of the pro-inflammatory cyto-
kine genes were significantly decreased in the LPS + Z20
group than in controls, such as Il-6, Ccl7, Cxcl1, Cxcl2,
Cxcl10 in liver, Tnf-a, Il-1b, Ccl2, Ccl3, Ccl4, Ccl7, Cxcl1,
Cxcl2, and Cxcl10 in the lung, and Tnf-a, Il-6, Il-1b, Ccl3,
Ccl5, Ccl7, and Cxcl2 in the kidney (Fig. 2a–c). These
results indicated that the protective effect of Z20 against
LPS-induced sepsis was associated with ameliorated pro-
inflammatory cytokines overexpression in mice.
two groups (Fig. 4b). In addition, we performed histolog-
ical analysis and detected the gene expression of Tnf-a and
Il-6 of various important organs (Fig. 4c, d). The results
showed that Z20 did not cause organ damage and cyto-
kines overexpression. Together, these results demonstrated
that Z20 did not affect the growth of the mice and damage
the organs.
DISCUSSION
Z20 Binds TLR4/MD2 Complex and Suppresses ERK
Signaling
Sepsis has become a serious human health threaten
with high mortality worldwide [16, 17]. Generally, inflam-
mation is defending response in early phase of sepsis.
However, excessive inflammatory cytokines and inflam-
matory cell infiltration lead to systemic inflammatory
storm which can accelerate the process of multiple organ
failure [18, 19]. Therefore, the appropriate reduction and
effective control of inflammatory response remain major
strategy for the treatment of sepsis. Now, more than 100
clinical trials have investigated for the treatments for sep-
sis, and a considerable part of them have failed [10]. TLR4-
MD2, which stimulated by lipopolysaccharide (LPS) and
inducing the release of critical pro-inflammatory cytokines,
is essential to activate potent immune responses in sepsis.
At present, the structural analysis and mechanism of
TLR4-MD2 have been studied extensively, which laid
the foundation for the development of drugs targeting
TLR4-MD2. There are many compounds targeting
TLR4-MD2 pathway, such as SMU-XY3, procyanidin
B1, and atractylenolide-I [20–22]. They showed well
anti-inflammatory activity in vitro or in animal models;
however, only a few entered the clinical trial phase, and
most clinical trial results were not optimistic. Resatorvid
(TAK-242), a selective inhibitor of TLR4, failed to reduce
IL-6 levels in septic patients in phase II clinical trial.
Although observed mortality rates in patients with both
shocks and respiratory failure were lower with the
2.4 mg/kg/day dose, differences were not significant [23].
It is urgent to develop novel inhibitor of TLR4-MD2 to
effectively reduce pro-inflammation cytokines during
sepsis.
TLR4 and MD2, the surface receptor complex on
innate immune cells, recognize LPS and transduce inflam-
matory signaling. To further explore the anti-inflammation
effects of Z20, we analyzed the potential connection be-
tween TLR4/MD2 and Z20. We evaluated the binding of
Z20 with TLR4 and MD2 by SPR experiment, which
showed that Z20 bound TLR4 in a dose-dependent manner
with a high affinity and low KD value of 5.44 μM (Fig. 3a).
In addition, the results showed that Z20 interacted with
MD2 protein in a dose-dependent manner and with high
affinity (KD = 22.2 μM) (Fig. 3b). Together, these data
suggested that Z20 could directly bind TLR4-MD2
complex.
ERK, a serine/threonine protein kinase, is the down-
stream signaling molecular of TLR4/MD2 and keeps a
central role in mediating fundamental biological processes
of mammalian innate immune response. We investigated
the possible involvement of ERK signaling in the anti-
inflammatory action of Z20. The levels of ERK1/2 phos-
phorylation were shown in Fig. 3c; Z20 treatment de-
creased LPS-induced phosphorylation of ERK1/2 in a
dose-dependent manner in macrophages. Thus, our results
demonstrated that Z20 could inhibit the ERK signaling.
The Safety Evaluation of Z20 in Mice
The safety of chemical compound is important for
new drug development. We further performed animal ex-
periment to test the potential toxicity of Z20. Mice were
randomly divided into vehicle group and Z20 group. By
monitoring the changes of mouse weight for 8 weeks, we
found that the Z20 had no adverse effect on mouse growth
(Fig. 4a). Next, we detected biochemical indicators such as
alanine aminotransferase (ALT), aspartate aminotransfer-
ase (AST), creatine kinase (CK), creatine kinase
isoenzymes-MB form (CK/MB), urea, and creatinine (Cr)
to evaluate the effects of Z20 on organs pathophysiologic
alteration. There was no significant difference between the
In previous study, we discovered a novel synthetic
route of polyfunctional (2S, 3R, 4S)-chromene-3-
carboxamides compounds which were characterized as
efficient and economical. In pilot pharmacological experi-
ments, we identified a chemical compound Z20 which had
anti-inflammatory activity, specifically it could effectively
reduce the mRNA levels of TNF-α and IL-6 in RAW267.4
macrophages after LPS challenge [11]. This indicates that

Zeng, Wang, Li, Niu, Wang, and Chen
PBS
Z20
LPS
a
b
c
LPS+Z20
Tnf-a
Ccl5
Il-6
Il-1b
Ccl2
Ccl3
Ccl4
*
15
10
5
15
10
5
50
40
30
20
10
0
400
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100
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150
100
50
100
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60
40
20
0
0
0
0
Ccl7
Cxcl1
Cxcl2
Cxcl10
p=0.06
40
30
20
10
0
60
40
20
0
*
*
300
200
100
0
120
80
40
0
300
200
100
0
*
*
Tnf-a
Ccl5
Il-6
Il-1b
Ccl2
Ccl3
Ccl4
*
*
40
800
600
400
200
0
100
80
60
40
20
0
150
100
50
40
30
20
10
0
*
*
*
300
200
100
0
30
20
10
0
0
Ccl7
Cxcl1
Cxcl2
Cxcl10
*
*
*
60
40
20
0
60
40
20
0
200
150
100
50
250
200
150
100
50
1000
800
600
400
200
0
*
0
0
Tnf-a
Ccl5
Il-6
Il-1b
Ccl2
Ccl3
Ccl4
*
*
*
12
8
16
12
8
15
10
5
160
120
80
8
1800
1350
900
450
0
*
6
4
2
0
4
4
40
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0
0
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Ccl7
Cxcl1
Cxcl2
Cxcl10
50
40
30
20
10
0
150
100
50
800
600
400
200
0
*
*
1200
800
400
0
1500
1000
500
0
*
0
Fig. 2. Z20 reduced cytokines expression in the liver, lung, and kidney. Mice were randomly divided into four groups: PBS, Z20, LPS, LPS + Z20. Twelve
hours after LPS and Z20 treatment, mice were euthanized, and liver, lung, and kidney were collected. a The mRNA level of key cytokines and chemokines in
the liver (n = 3–8). b The mRNA level of key cytokines and chemokines in lung (n = 3–8). c The mRNA level of key cytokines and chemokines in kidney
(n = 3–8). “Relative mRNA level” represents the amount of target mRNA normalized to 18S RNA in each sample. *P < 0.05.

Protective Effect of a Novel (2S, 3R, 4S)-Chromene-3-Carboxamide
TLR4
a
ka (1/Ms) = 210
kd (1/s) = 0.00114
KD (M) = 0.00000544
Rmax (RU) = 17.6
Chi2 = 35.1
b
MD2
ka (1/Ms) = 112
kd (1/s) = 0.0025
KD (M) = 0.0000222
Rmax (RU) = 33.9
Chi2 = 36.1
c
1.5
1.0
0.5
0.0
p-Erk
Erk
*
PBS
LPS
Z20
+
-
-
-
+
-
-
+
1
-
+
-
+
10 100
0
S
S
LP
10
0
B
-
P
20-1
0-1
Z
Z20
Z2
Fig. 3. Z20 inhibited TLR4/MD2 signaling pathway. Surface plasmon resonance (SPR) experiment showed direct interactions between Z20 and TLR4 (a)
and MD2 (b). c The protein levels of p-ERK and ERK were examined by Western blot in RAW 264.7 cells (n = 4). *P < 0.05. ka (1/Ms), kinetic association
constant; kd (1/s), kinetic dissociation constant; KD(M), equilibrium dissociation constant, KD = kd/ka; Rmax (RU), maximum response unit of SPR; Chi2,
chi-squared test, represents the simulation result credibility.
Z20 may be a potential anti-inflammatory small molecule
compound. In this study, we evaluated the anti-
inflammatory profiles of Z20 in the LPS-induced sepsis
mice model. The result showed that Z20 (1 mg/kg, i.p.)

Zeng, Wang, Li, Niu, Wang, and Chen
PBS
Z20
a
30
28
26
24
22
20
0 week
8 week
b
40
30
20
10
80
60
40
20
80
60
N .S .
N .S .
N .S .
40
20
0
0
0
N .S .
N .S .
80
20
20
N .S .
60
40
20
0
15
10
5
15
10
5
0
0
Fig. 4. The safety evaluation of Z20. Mice were randomly divided into two groups: PBS and Z20. The PBS or Z20 (1 mg/kg) was injected once a week. After
8 weeks, the mice were euthanized, and blood and organ samples were collected. a Body weight. b The plasma biochemical parameters in mice. c The H&E
staining of seven organs (brain, heart, lung, liver, spleen, kidney, and colon). d The mRNA level of Tnf-a and Il-6 in seven organs (brain, heart, lung, liver,
spleen, kidney, and colon) (n = 3–5). “Relative mRNA level” represents the amount of target mRNA normalized to 18S RNA in each sample. N.S. means no
significance.
significantly increased the weekly survival time of LPS-
induced sepsis mice. Further research indicated that Z20
could alleviate organ damage caused by LPS, including
nuclear condensation in the liver, inter-alveolar septal
thickening in the lung, and inflammatory neutrophils infil-
tration in the kidney, thereby playing the role of protecting
organs. Analyzing the expression of inflammatory factor,
the mRNA level of cytokines in the Z20 administered
group was significantly lower than that in the LPS group,
suggesting that Z20 effectively reduced LPS-induced in-
flammatory cytokines production in vivo. Next, we per-
formed SPR to verify that Z20 directly combined with
TLR4 and MD2 in vitro, which implied that Z20 may bind
to the complex of TLR4-MD2 to play an anti-
inflammatory effect in vivo. TLR4, a switch on LPS-
induced inflammation, recognized LPS and triggered a
series of downstream signaling pathways, including
MAPKs and NF-kB, to complete cascade reactions [24].
We confirmed the phosphorylation of ERK1/2 was
inhibited by Z20 in macrophages, indicating Z20 inhibited
the TLR4/ERK signaling pathway. Furthermore, the safety
research is also an important part of the development of
new drugs. Eight-week safety experiments data showed
that Z20 had not potential toxic effects in mice. Hence,
we believed that Z20 may be a novel anti-inflammation
compound with low side effects to the body.

Protective Effect of a Novel (2S, 3R, 4S)-Chromene-3-Carboxamide
PBS
Z20
c
d
Z20
PBS
Tnf-a
Il-6
N.S.
2.5
2.0
1.5
1.0
0.5
0.0
1.5
1.0
0.5
0.0
N.S.
Brain
Brain
Heart
Lung
Tnf-a
Il-6
1.5
1.0
0.5
0.0
2.5
2.0
1.5
1.0
0.5
0.0
N.S.
N.S.
Heart
Lung
Tnf-a
Il-6
N.S.
N.S.
1.5
1.0
0.5
0.0
3
2
1
0
Tnf-a
N.S.
Il-6
N.S.
2.0
1.5
1.0
0.5
0.0
3
2
1
0
Liver
Liver
Tnf-a
Il-6
N.S.
N.S.
3
2
1
0
2.5
2.0
1.5
1.0
0.5
0.0
Spleen
Kidney
Colon
Spleen
Kidney
Tnf-a
Il-6
N.S.
2.5
2.0
1.5
1.0
0.5
0.0
2.0
1.5
1.0
0.5
0.0
N.S.
Tnf-a
Il-6
N.S.
2.0
1.5
1.0
0.5
0.0
2.5
2.0
1.5
1.0
0.5
0.0
N.S.
Colon
Fig. 4. continued.

Zeng, Wang, Li, Niu, Wang, and Chen
9. Achek, A., D. Yesudhas, and S. Choi. 2016. Toll-like receptors:
Promising therapeutic targets for inflammatory diseases. Archives of
Pharmacal Research 39 (8): 1032–1049.
10. Nedeva, C., J. Menassa, and H. Puthalakath. 2019. Sepsis: Inflam-
mation is a necessary evil. Frontiers in Cell and Development
Biology 7: 108.
11. Zeng, L.Y., B. Xi, K. Huang, J. Bi, L. Wei, C. Cai, and S. Liu. 2019.
Diastereoselective synthesis of 3,4-dihydropyran-3-carboxamides
with in vitro anti-inflammatory activity. ACS Combinatorial Science
21 (10): 656–665.
12. Tang, B., H. Qiao, F. Meng, and X. Sun. 2007. Glycyrrhizin atten-
uates endotoxin- induced acute liver injury after partial hepatectomy
in rats. Brazilian Journal of Medical and Biological Research 40
(12): 1637–1646.
In conclusion, we synthesized a novel of
polyfunctional (2S, 3R, 4S)-chromene-3-carboxamides
compound—Z20. It plays a potential role in anti-
inflammatory activity both in vitro and in vivo. Z20 effec-
tively reduced the mRNA expression of inflammatory
factors and attenuated LPS-induced organs injury. The
mechanistic study showed that it directly interacted with
TLR4-MD2 complex and inhibited the activation of down-
stream ERK signaling. Meanwhile, Z20 showed no toxic
effects in mice. Z20 may be a novel candidate for sepsis
treatment in future.
13. Wu, K.C., S.S. Huang, Y.H. Kuo, et al. 2017. Ugonin M, a
Helminthostachys zeylanica constituent, prevents LPS-induced
acute lung injury through TLR4-mediated MAPK and NF-κB sig-
naling pathways. Molecules. 22 (4): 573.
14. Zhao, K., C. Chen, Q. Shi, W. Deng, T. Zuo, X. He, T. Liu, L. Zhao,
and W. Wang. 2014. Inhibition of glycogen synthase kinase-3β
attenuates acute kidney injury in sodium taurocholate-induced se-
vere acute pancreatitis in rats. Molecular Medicine Reports 10 (6):
3185–3192.
FUNDING INFORMATION
This study was financially supported in part by the National
Natural Science Foundation of China (81873926) and Natural
Science Funds for Distinguished Young Scholar of Guangdong
province (2016A030306043).
15. Chen, G., B. Xiao, L. Chen, B. Bai, Y. Zhang, Z. Xu, L. Fu, Z. Liu,
X. Li, Y. Zhao, and G. Liang. 2017. Discovery of new MD2-
targeted anti-inflammatory compounds for the treatment of sepsis
and acute lung injury. European Journal of Medicinal Chemistry
139: 726–740.
COMPLIANCE WITH ETHICAL STANDARDS
The experimental protocol was approved by the insti-
tutional ethics committee.
16. Oussalah, A., J. Ferrand, P. Filhine-Tresarrieu, N. Aissa, I. Aimone-
Gastin, F. Namour, M. Garcia, A. Lozniewski, and J.L. Guéant.
2015. Diagnostic accuracy of procalcitonin for predicting blood
culture results in patients with suspected bloodstream infection: An
observational study of 35,343 consecutive patients (a STROBE-
Compliant Article). Medicine (Baltimore) 94 (44): e1774.
17. Cuthbertson, B.H., A. Elders, S. Hall, J. Taylor, G. MacLennan, F.
Mackirdy, S.J. Mackenzie, Scottish Critical Care Trials Group, and
Scottish Intensive Care Society Audit Group. 2013. Mortality and
quality of life in the five years after severe sepsis. Critical Care 17
(2): R70.
18. Weighardt, H., C.D. Heidecke, K. Emmanuilidis, S. Maier, H.
Bartels, J.R. Siewert, and B. Holzmann. 2000. Sepsis after major
visceral surgery is associated with sustained and interferon-gamma-
resistant defects of monocyte cytokine production. Surgery. 127 (3):
309–315.
19. Martin, G.S. 2012. Sepsis, severe sepsis and septic shock: Changes
in incidence, pathogens and outcomes. Expert Review of Anti-
Infective Therapy 10 (6): 701–706.
20. Xu, Y., S. Chen, Y. Cao, P. Zhou, Z. Chen, and K. Cheng. 2018.
Discovery of novel small molecule TLR4 inhibitors as potent anti-
inflammatory agents. European Journal of Medicinal Chemistry
154: 253–266.
REFERENCES
1. Lever, A., and I. Mackenzie. 2007. Sepsis: Definition, epidemiolo-
gy, and diagnosis. BMJ. 335 (7625): 879–883.
2. Jawad, I., I. Lukšić, and S.B. Rafnsson. 2012. Assessing available
information on the burden of sepsis: Global estimates of incidence,
prevalence and mortality. Journal of Global Health 2 (1): 010404.
3. Alberti, C., C. Brun-Buisson, H. Burchardi, C. Martin, S. Goodman,
A. Artigas, A. Sicignano, M. Palazzo, R. Moreno, R. Boulmé, E.
Lepage, and R. le Gall. 2002. Epidemiology of sepsis and infection
in ICU patients from an international multicentre cohort study.
Intensive Care Medicine 28 (2): 108–121.
4. Almog, Y. 2003. Statins, inflammation, and sepsis: Hypothesis.
Chest. 124 (2): 740–743.
5. Cohen, J. 2002. The immunopathogenesis of sepsis. Nature. 420
(6917): 885–891.
6. Ulloa, L., and K.J. Tracey. 2005. The “cytokine profile”: A code for
21. Xing, J., R. Li, N. Li, J. Zhang, Y. Li, P. Gong, D. Gao, H. Liu, and
Y. Zhang. 2015. Anti-inflammatory effect of procyanidin B1 on
LPS-treated THP1 cells via interaction with the TLR4-MD-2 het-
erodimer and p38 MAPK and NF-κB signaling. Molecular and
Cellular Biochemistry 407 (1–2): 89–95.
22. Huang, J.M., G.N. Zhang, Y. Shi, et al. 2014. Atractylenolide-I
sensitizes human ovarian cancer cells to paclitaxel by blocking
activation of TLR4/MyD88-dependent pathway. Scientific Reports
4: 3840.
sepsis. Trends in Molecular Medicine 11 (2): 56–63.
7. Torpy, D.J., S.R. Bornstein, and G.P. Chrousos. 1998. Leptin and
interleukin-6 in sepsis. Hormone and Metabolic Research 30 (12):
726–729.
8. Lu, Y.C., W.C. Yeh, and P.S. Ohashi. 2008. LPS/TLR4 signal
transduction pathway. Cytokine. 42 (2): 145–151.

Protective Effect of a Novel (2S, 3R, 4S)-Chromene-3-Carboxamide
23. Rice, T.W., A.P. Wheeler, G.R. Bernard, J.L. Vincent, D.C. Angus,
N. Aikawa, I. Demeyer, S. Sainati, N. Amlot, C. Cao, M. Ii, H.
Matsuda, K. Mouri, and J. Cohen. 2010. A randomized, double-
blind, placebo-controlled trial of TAK-242 for the treatment of
severe sepsis. Critical Care Medicine 38 (8): 1685–1694.
24. Gorina, R., M. Font-Nieves, L. Márquez-Kisinousky, T. Santalucia,
and A.M. Planas. 2011. Astrocyte TLR4 activation induces a pro-
inflammatory environment through the interplay between MyD88-
dependent NFκB signaling, MAPK, and Jak1/Stat1 pathways. Glia.
59 (2): 242–255.
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