Design, synthesis and biological evaluation of novel arylpropionic esters for the treatment of acute kidney injury

Article abstract of DOI:10.1016/j.bioorg.2020.104455

Acute kidney injury (AKI) is associated with a strong inflammatory response, and inhibiting the response effectively prevents or ameliorates AKI. A series of novel arylpropionic esters were designed, synthesized and evaluated their biological activity in LPS-stimulated RAW264.7 cells. Novel arylpropionic esters bearing multi-functional groups showed significant anti-inflammatory activity, in which, compound 13b exhibited the most potent activity through dose-dependent inhibiting the production of nitric oxide (NO, IC₅₀ = 3.52 μM), TNF-α and IL-6 (84.1% and 33.6%, respectively), as well as suppressing the expression of iNOS, COX-2 and TLR4 proteins. In C57BL/6 mice with cisplatin-induced AKI, compound 13b improved kidney function, inhibited inflammatory development, and reduced pathological damage of kidney tissues. In brief, this arylpropionic ester scaffold may be developed as anti-inflammatory agents.

Full text of DOI:10.1016/j.bioorg.2020.104455

Journal Pre-proofs
Design, synthesis and biological evaluation of novel arylpropionic esters for
the treatment of acute kidney injury
Jiawei Zuo, Shi-Meng Wang, Xia Jiang, Mengxin Cao, Ziwen Zhang, Tianlu
Shi, Hua-Li Qin, Wenjian Tang
PII:
S0045-2068(20)31753-3
DOI:
https://doi.org/10.1016/j.bioorg.2020.104455
YBIOO 104455
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
26 August 2020
29 October 2020
31 October 2020
Please cite this article as: J. Zuo, S-M. Wang, X. Jiang, M. Cao, Z. Zhang, T. Shi, H-L. Qin, W. Tang, Design,
synthesis and biological evaluation of novel arylpropionic esters for the treatment of acute kidney injury,
Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.104455
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover
page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version
will undergo additional copyediting, typesetting and review before it is published in its final form, but we are
providing this version to give early visibility of the article. Please note that, during the production process, errors
may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2020 Elsevier Inc. All rights reserved.

Design, synthesis and biological evaluation
of novel arylpropionic esters for the
treatment of acute kidney injury
Jiawei Zuo a,c, Shi-Meng Wang b,d, Xia Jiang a, Mengxin Cao a, Ziwen Zhang a, Tianlu
Shi c, Hua-Li Qin d,*, Wenjian Tang a,*
a School of Pharmacy, Anhui Province Key Laboratory of Major Autoimmune Diseases,
Anhui Medical University, Hefei 230032, China
b School of Life Science, Wuchang University of Technology, Wuhan 430223, PR China
c First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University
of Science and Technology of China, Hefei, Anhui, 230001, China
d
School of Chemistry, Chemical Engineering and Life Science, Wuhan University of
Technology, Wuhan 430070, China




Novel arylpropionic esters exhibited significant anti-inflammatory activity.
This arylpropionic ester scaffold bearing multi-functional groups.
13b could suppress iNOS, COX-2 expression and NO, TNF- and IL-6
production.

13b significantly improved cisplatin-induced acute kidney injury.

Novel arylpropionic ester scaffold exhibited significant anti-inflammation. In vitro,
13b decreased the levels of pro-inflammatory factors TNF-α and IL-6, inhibited the
expression of related proteins TLR4, iNOS and COX-2. Moreover, 13b effectively
maintained normal metabolic function and improved the pathological damage of kidney
tissue in AKI mice.

Abstract ： Acute kidney injury (AKI) is associated with a strong inflammatory
response, and inhibiting the response effectively prevents or ameliorates AKI. A series
of novel arylpropionic esters were designed, synthesized and evaluated their biological
activity in LPS-stimulated RAW264.7 cells. Novel arylpropionic esters bearing multi-
functional groups showed significant anti-inflammatory activity, in which, compound
13b exhibited the most potent activity through dose-dependent inhibiting the
production of nitric oxide (NO, IC50 = 3.52 M), TNF- and IL-6 (84.1% and 33.6%,
respectively), as well as suppressing the expression of iNOS, COX-2 and TLR4
proteins. In C57BL/6 mice with cisplatin-induced AKI, compound 13b improved
kidney function, inhibited inflammatory development, and reduced pathological
damage of kidney tissues. In brief, this arylpropionic ester scaffold may be developed
as anti-inflammatory agents.
Keywords: Arylpropionic acid; quinoline; acute kidney injury; anti-inflammatory;
multi-functional group
*Corresponding author.
E-mail: qinhuali@whut.edu.cn (H.L. Qin); ahmupharm@126.com (W.J. Tang).

1. Introduction
Acute kidney injury (AKI) is a global public health concern associated with high
morbidity, mortality, and healthcare costs [1]. Platinum drugs are widely used for the
treatment of ovarian, breast, stomach, lung, prostate cancers, and so on, but they usually
cause particular renal toxicity due to its special metabolism in the kidney [2,3]. Clinical
manifestations have proved that AKI is associated with intrarenal and systemic
inflammation, which is essential for eliminating microbial pathogens and repairing
tissue after diverse forms of injury [4-6]. The pathological features of AKI include
tubular and vascular cell damage, oxidative stress and intense inflammatory reaction
[7]. Intense researches also showed that many anti-inflammatory drugs for AKI were
proved effective in animal models but failed to translate to humans over the past
decades, however, with the increase of AKI patients, there was an urgent need for new
drugs and treatment [8].
The discovery of novel anti-inflammatory agents has attracted significant attention
of medicinal chemists. As shown in Fig. 1, acrivastine and ibuprofen are marketed
drugs with anti-inflammatory activity. Acrivastine, an α,β-unsaturated propenoic acid
derivative, is a non-sedating antihistamine used for treatment of hayfever, urticaria, and
rhinitis, while ibuprofen, an arylpropionic acid derivative, is a commonly used NSAID
with anti-inflammatory, analgesic, and antipyretic effects. In medical discovery, natural
products showed benign therapeutic potency and fewer adverse effects. Cinnamic acid
is a secondary metabolite in the phenylpropanoid pathway and the source of
hydroxycinnamic acid derivatives like coumaric, caffeic, ferulic, and sinapic acids in
either ester or glycosidic forms. Cinnamic acid displayed a wide range of
pharmacological activities, such as anti-inflammatory, anticancer, antimicrobial
activities, including salvianolic acid B with four phenylpropanoid units and chlorogenic
acid with one phenylpropanoid unit (Fig. 1) [9-12]. Therefore, arylpropionic ester
scaffold was often used to design and find new anti-inflammatory agents [13-15].

OH
OH
O
HO
HO
O
O
O
HO
O
OH
N
O
HO
OH
OH
O
N
O
OH
O
OH
acrivastine
O
Salvianolic acid B
R
O
HO
OH
arylpropionic ester
O
O
HO
HO
O
OH
OH
OH
ibuprofen
Chlorogenic acid
Fig. 1. The analogues of arylpropionic esters in this study.
Multi-functional compounds are designed broadly as hybrid or conjugated drugs
from two or more pharmacophores/drugs having multiple pharmacological actions in
treatment of multi-factorial diseases [16]. Novel arylpropionic ester with 3-hydroxy and
2-methylene, one of Baylis-Hillman adducts, is a valuable scaffold with promising
biological molecules, including potent antibacterial and antifungal activities [17-20].
As shown in Fig. 1, 3-hydroxy-2-methylene-3-phenylpropionic ester is an
arylpropionic ester derivative with polyfunctional groups, however, its anti-
inflammatory activity was not reported. In this work, we would explore the anti-
inflammatory activity of novel arylpropionic esters.
Macrophages, significant innate immune sentinel, play a role of promoting renal
fibrosis and repairing damage in kidney injury [21]. In the response to AKI, pro-
inflammatory macrophages (M1) can secrete inflammatory cytokines and induce
kidney damage and fibrosis, while in some micro-circumstances, anti-inflammatory
macrophages (M2) release regenerative trophic factors and reduce inflammation,
promote wound healing and kidney recovery [22-24]. Nitric oxide (NO) is synthesized
from l-arginine by the inducible nitric oxide synthase (iNOS or NOS2) in the
pathogenesis and control of tumors, chronic degenerative diseases and autoimmune
processes [25,26]. Pharmacological and genetic methods also indicated that cytokines

including TNF-α and IL-6 play a central role in the activation of inflammation
responses and the pathogenesis of injury in cisplatin-induced nephrotoxicity [27,28].
Thus, the anti-inflammatory activity of novel arylpropionic esters could be screened
through inhibiting the NO production, the release of pro-inflammatory factors TNF-α
and IL-6 in lipopolysaccharide (LPS)-activated macrophages. Further structural
optimization will be carried out to find lead compounds.
In the inflammatory response of AKI, it is very important to control the release of
related proteins and pro-inflammatory factors, which effectively reduce the damage of
renal tissue and maintain normal renal function. The nephritis of rodents has provided
evidence that iNOS also functions as a negative feedback regulator of the autoimmune
Th1 cell response and thereby protects the host against immunopathological sequelae
[29]. As a common inducible enzyme, the expression of COX-2 in the kidney is
relatively high and plays an important role in water/salt metabolism, regulating renin
release [30-33], kidney development [19,34,35], and mediating kidney damage [36-38].
In addition, toll-like receptors (TLRs) are a class of proteins, and the continuous
signaling of TLR can lead to autoimmune diseases and chronic inflammatory diseases,
so the tight regulation of TLR signaling pathways is very important [39]. Under the
pathological conditions of AKI, the expression of TLR4 is significantly increased, and
inflammation can promote the development of kidney tissue damage and renal failure
[2,40]. Therefore, it is significant to clarify the protective effect on the active lead in
AKI model mice.
2. Results and discussion
2.1. Chemistry
In our recent work, a series of arylpropionic esters (1‒14) were prepared through a
cascade dehydrogenative Baylis–Hillman reaction of the C(sp3)–H of primary alcohols
with the C(sp2)–H of electron-deficient olefins mediated by SO2F2 (Scheme 1) [41-43].
As shown in Table 1, initial screening of novel arylpropionic esters revealed that
compounds 9 and 13 alleviated the increase of LPS-induced NO release, which implied

their potent anti-inflammatory activity.
OH
O
OH
O
O
O
OH
O
OH
O
O
O
O
O
O
O
S
Ph
Br
4
3
2
1
OH
OH
O
OH
O
OH O
O
O
O
MeOOC
NO2
7
F
5
6
8
OH
O
OH
O
OH
OH
O
Bu
N
N
O
O
O
NC
CF3
NO2
Cl
10
9
11
12
OH
O
OH
O
O
O
N
14
13
Scheme 1. Chemical structures of novel arylpropionic esters 1–14.
To further study the structure-activity relationship (SAR), the constitutional isomers
of compounds 9 and 13 were synthesized through a cascade dehydrogenative Baylis-
Hillman reaction of the C(sp3)–H of primary alcohols with the C(sp2)–H of electron-
deficient olefins for forming allylic alcohols mediated by SO2F2. As shown in Scheme
2, compounds 9a‒9c, 13a and 13b were synthesized through this mild method without
the requirement of transition metals.

OH
O
K2CO3 (1.2 eq.), SO2F2 (balloon)
DMSO (0.13 M), r.t., 12 h
then DABCO (3.0 eq.), 40 oC, 36 h
O
OH
O
Ar
+
Ar
O
9 or 13
CF3 OH
O
OH
O
OH O
O
O
O
F3C
O
CF3
9a
9b
9c
OH
O
O
N
OH
O
N
13a
13b
Scheme 2. Synthesis of compounds 9a–9c, 13a and 13b.
2.2. Cytotoxicity assessment
In cases where novel arylpropionic esters are of no toxicity or low toxicity, it is
necessary to evaluate their biological activity. The RAW264.7 cells were treated with
the same concentration of synthesized compounds (10 M) for 24 h and the viability of
the cells was measured by MTT assay. As shown in Fig. 2, compared with the negative
control, most of arylpropionic esters did not notably affect cell viability, except for
compounds 4, 9b and 9c having slight effect. Therefore, the concentration of
compounds was suitable for further evaluation of biological activity.

Fig. 2. Cell viability was detected by MTT assay after RAW264.7 cells were pretreated
with synthesized compounds at the same concentration (10 μM) for 24 h. Results were
expressed as percentage of cell survival vs untreated cell (control) and shown as mean
± SD (n = 3). Statistical significance was calculated using one-way ANOVA and
Bonferroni post hoc tests. ***p < 0.001 compared with the control group; **p < 0.01
compared with the control group.
2.3. Inhibition of NO production in LPS-stimulated RAW264.7 cells
In the inflammatory response, activated macrophages induce the expression of
nitric oxide synthase (iNOS) and produce NO, and excessive release of NO is regarded
as an important factor in inflammatory responses [44,45]. To evaluate the anti-
inflammatory effect of arylpropionic esters, Griess reagent was used to detect the level
of the LPS-induced NO release in RAW264.7 cells. After pretreatment with compounds
at the concentration of 5 μM, the production of LPS-induced NO in the supernatant was
significantly reduced (Fig. 3). Novel arylpropionic esters showed a dose-dependent
inhibition of NO production, and IC50 values of synthesized compounds further were

calculated.
Fig. 3. The inhibitory effects of synthesized compounds on NO production in LPS-
stimulated RAW 264.7 cells. RAW264.7 cells were pretreated with compounds at the
same concentration (5 M) for 1 h, then incubated with LPS (0.5 g/mL) for 24 h. NO
production in supernatant was detected by Griess reagent assay. ***p < 0.001 compared
with the control group; ###p < 0.001, ##p < 0.01, #p < 0.05 compared with the LPS group.
As shown in Table 1, novel arylpropionic esters showed significant anti-
inflammatory activity, amongst them, compounds 9c, 13a and 13b exhibited the most
potent NO inhibitory activity (IC50 = 4.12, 3.78 and 3.52 M, respectively).
Arylpropionic ester scaffold with 3-hydroxy and 2-methylene may be the key factor of
anti-inflammatory activity (Fig. 1). Aryl with different structures showed micromole
anti-inflammatory activity, when aryl was substituted with alkyl, the activity decreased
(IC50 = 12.43 M for compound 14). Ester group of arylpropionic ester has hardly effect
on the activity, for example, IC50 = 3.88 and 4.65 M for compounds 9 (methyl ester)
and 9b (ethyl ester), respectively. Substituent position of aryl affected the activity, for
example, the activity of o-CF3 of phenyl (IC50 = 15.77 M for 9a) was much less than
those of m-CF3 and p-CF3 (IC50 = 4.65 and 4.12 M for 9b and 9c, respectively).

Substituent position of quinoline ring hardly affected the activity (IC50 = 3.79, 3.78 and
3.52 M for 6- (13), 2- (13a) and 3-position (13b), respectively), thus, compounds 13,
13a and 13b exhibited almost same inhibitory effect on NO production (Fig. 4A). The
effects of compounds 13a and 13b on the levels of pro-inflammatory factors TNF-
and IL-6 induced by LPS would be further evaluated.
Table 1
The IC50 values or inhibition rates (%) of the inhibition of NO, TNF- and IL-6
production.
IC50 (μM) a
NO
Inhibition (%) b,c
TNF-α
Compound
IL-6
14.4
36.9
3.6
1
2
28.5
67.7
67.1
na
6.62  1.61
5.71  0.70
5.84  1.64
6.15  1.45
6.01  2.18
5.03  1.32
4.43  0.65
14.07  1.21
3.88  1.16
11.21  1.90
5.87  0.97
7.54  1.12
3.79  0.85
12.43  2.20
15.77  1.36
4.65  1.14
4.12  1.33
3
4
21.0
32.2
42.5
85.3
15.7
31.4
44.3
23.6
35.5
44.5
51.0
22.5
na
5
21.5
10.7
52.8
28.8
23.3
49.8
54.9
52.4
53.8
45.5
71.4
52.5
44.2
6
7
8
9
10
11
12
13
14
9a
9b
9c
28.5

13a
13b
68.6
84.1
54.9
33.6
3.78  0.92
3.52  0.88
a The detection of NO production with Griess reagent;
b
The inhibition of cytokines were performed using ELISA kits and the inhibition
rates were calculated at 10 M concentration of the compound;
c The IC50 values were means of three determinations; the inhibition rate values were
means of two determinations; na, no activity.
2.4. Determination of pro-inflammatory cytokines
Activated macrophages produce excessive amounts of multiple pro-inflammatory
cytokines, including TNF-α, IL-6 and IL-12, leading to serious systemic diseases with
a high mortality [46]. Therefore, to further evaluate the effects of novel arylpropionic
esters on the LPS-induced TNF- and IL-6 production, RAW264.7 cells were cultured
with LPS in the presence of compounds for 24 h, and the levels of cytokines in the cell
supernatant were determined using ELISA kits. All the test compounds exhibited the
ability to inhibit the production of TNF- and/or IL-6, and the percentage in assay was
calculated. As shown in Table 1, most of compounds significantly inhibited the levels
of TNF-α and IL-6, amongst them, compounds 13a and 13b showed a dose-dependent
inhibition on TNF-α and IL-6 production (Figs. 4B and 4C). Compound 13b had better
inhibitory effect on TNF-, while compound 13a had better inhibitory effect on IL-6.
These results showed that novel arylpropionic esters attenuate an excessive immune
reaction in RAW264.7 macrophages by blocking the increase of pro-inflammatory
cytokines. Thus, compound 13b was selected to further explore the anti-inflammatory
mechanisms.

Fig. 4. Compounds 13a and 13b inhibited LPS-induced NO, IL-6 and TNF-α
production in RAW264.7 cells. Compounds (10, 5, 2.5 M) were pre-treated
RAW264.7 cells for 1 h, and then incubation with LPS (0.5 g/mL) for 24 h. NO
production and cytokine levels (TNF- and IL-6) in the supernatant were measured by
Griess and ELISA kits, respectively. ###p < 0.001, ##p < 0.01, #p < 0.05 compared with
the LPS group.
2.5. Anti-inflammatory mechanism of compound 13b
The inflammatory process involves a variety of molecular mechanisms, in which two
of the most important are NO and cyclooxygenase 2 (COX-2). Decreased
bioavailability of NO and increased oxidative stress partially mediate renal
vasoconstriction and medullary hypoxia, which may be associated with AKI [47].
Increased NO release may activate soluble guanidine cyclase, leading to cGMP
signaling and up-regulating COX-2 expression [48]. The molecular synergy between
iNOS and COX-2 may represent the main mechanism of inflammatory response.
Western blot assay was used to verify the effect of compound 13b on the expression of
iNOS-COX-2 in LPS-activated RAW264.7. After stimulation with 0.5 g/mL LPS for
24 h, the expression of iNOS and COX-2 in the LPS group increased significantly
compared with the control group, while compound 13b inhibited the expression of
iNOS and COX-2 in a concentration-dependent manner (Fig. 5). Drugs that block the
interaction of iNOS–COX-2 may have anti-inflammatory effects, and they work
synergistically with COX-2 inhibitors to reduce dosage and side effects. Toll-like
receptors (TLRs) are widely expressed on kidney epithelial cells and leukocytes,
regulating innate and adaptive immune responses. TLR4 is an important mediator of

cisplatin-induced production of cytokines (such as TNF-α), inflammation, and
subsequent kidney damage [49]. As shown in Fig. 5, compound 13b effectively
inhibited TLR4 protein expression in LPS-stimulated RAW264.7 cells. Thus, TLR4-
targeted signaling pathway may be a feasible therapeutic strategy to prevent cisplatin-
induced AKI in patients.
Fig. 5. Effects of compound 13b on LPS-induced iNOS, COX-2 and TLR4 protein
expression in RAW264.7 cells. After compound 13b (2.5, 5, 10 M) pretreatment for
1 h, LPS (0.5 g/mL) was incubated for 24 h, and the protein expression in RAW264.7
cells was detected by Western blotting. TAK-242 was the inhibitor of TLR4. ***p <
##
#
0.001 compared with the control group; ###p < 0.001, p < 0.01, p < 0.05 compared
with LPS group.

2.6. In vivo study of the mouse model of AKI
2.6.1. The acute toxicity in vivo of compound 13b
We randomly divided C57BL/6 mice into two groups, each with 6 mice, and
evaluated the acute toxicity of compound 13b in vivo. One group was given a single
dose of 300 mg/kg, and the other group was given a single dose of 100 mg/kg by
intragastric administration to further observe the acute poisoning of mice. After
administration of compound 13b, the mice showed reduced spontaneous activity,
gathered together, little response to external stimuli, and no obvious changes in body
weight. No mice died and all were in fine condition.
2.6.2. Kidney function biomarkers and cytokines in serum
Clinical data have shown that approximately one third of patients develop AKI after
cisplatin treatment, accompanied by decreased glomerular filtration rate, increased
blood urea nitrogen (BUN) and serum creatinine (SCR), and electrolyte imbalance
[50,51]. Urea and creatinine are important indicators of kidney function examination,
reflecting the degree of damage to glomerular filtration function. As the diagnostic
criteria of nephrotoxicity, urea and creatinine reflects the late manifestations of the
disease, but early intervention is critical for the effective treatment of AKI [52]. The in
vivo activity of compound 13b was evaluated in C57BL/6 mice of cisplatin-induced
AKI. The results showed that compared with the control group, the levels of SCR and
BUN in the cisplatin-treated group were significantly increased, and renal dysfunction
occurred (Figs. 6A and 6B). However, compared with the model group, the values of
SCR and BUN in compound 13b-treated mice were decreased (Figs. 6A and 6B).
Compound 13b could ameliorate kidney damage and maintain glomerular filtration
function. Besides, the increasing release of pro-inflammatory cytokines in serum of the
kidney injury mice was significantly inhibited by compound 13b. As shown in Figs. 6C
and 6D, the levels of pro-inflammatory mediators TNF-α and IL-6 in the serum of
compound 13b-treated mice were significantly lower than those in model group. In all
in, compound 13b has obvious effect on maintaining renal function and inhibiting

inflammation of cisplatin-induced AKI in model mice.
Fig. 6. The protective effect of compound 13b on cisplatin-induced AKI in C57BL/6
mice. After pre-treatment with compound 13b (15 and 30 mg/kg), the mice were
injected intraperitoneally with cisplatin (20 mg/kg) and sacrificed 72 h later. Detection
of kidney function markers (SCR and BUN) and pro-inflammatory factors (TNF- and
IL-6) levels in serum. ***p < 0.001 compared with control group; ###p < 0.001, #p < 0.05
compared with model group.
2.6.3. Kidney histopathological analysis
As an important characteristic of AKI, tubular damage may eventually lead to kidney
tubular interstitial fibrosis [53,54]. Cisplatin caused kidney damage and increased
kidney specific gravity, while compound 13b significantly decreased the kidney
specific gravity after injury (Fig. 7). In cisplatin-induced AKI mice, kidney tissues were
removed and fixed in 10% formaldehyde. After dehydration in gradient concentration
of alcohol, the tissues were embedded in paraffin and sliced. Sections were stained by
H&E and PAS. Finally, image them under an optical microscope. As shown in Fig. 8,

in the blank control group, normal morphology of the glomeruli and proximal tubules
were clearly shown in the kidneys of mice. While in cisplatin-induced AKI mice, the
kidneys were severely damaged, resulting in renal tubular epithelial injury,
inflammatory cell infiltration, renal tubular cell swelling, and tubular dilatation.
Therefore, the treatment of compound 13b significantly improved the necrosis of
kidney tissue and inflammatory infiltrating cells caused by cisplatin in model mice.
Fig. 7. The effect of compound 13b on kidney index. Compound 13b (15 and 30
mg/kg) was pre-treated for mice, and cisplatin (20 mg/kg) solution was injected
intraperitoneally. After 72 h, the mice and kidney tissues of every group were weighed.
***p < 0.001 compared with control group; ###p < 0.001 compared with model group.
Fig. 8. The effect of compound 13b on the pathological changes of kidney tissue caused

by cisplatin (20 mg/kg). The kidney was stained with H&E and PAS for histological
examination.
3. Conclusions
A series of novel arylpropionic esters were designed, synthesized and evaluated
their biological activities. These arylpropionic esters bearing multi-functional groups
showed benign anti-inflammatory activity through inhibiting NO production and the
levels of pro-inflammatory factors TNF- and IL-6. SAR showed that aryl of
arylpropionic ester scaffold was necessary on the anti-inflammatory activity, alkyl
decreased the activity, ester had hardly effect on the activity; substituent position of
benzene ring affected the activity, while that of quinoline ring hardly affected the
activity. In LPS-treated macrophages, compound 13b showed significant anti-
inflammatory activity by dose-dependent inhibiting the production of NO (IC50 = 3.52
M), decreasing the levels of TNF- and IL-6 (84.1% and 33.6%, respectably), as well
as inhibiting the expression of iNOS, COX-2 and TLR4 protein. In vivo study in
C57BL/6 mice of cisplatin-induced AKI showed that compound 13b decreased the
levels of BUN, SCR, TNF- and IL-6 in serum to inhibit the inflammatory response,
improved the pathological damage of the kidney tissue to maintain the metabolic
function of the kidney. In short, the preliminary anti-inflammatory mechanism showed
the protective effect of compound 13b on AKI in C57BL/6 mice. Novel arylpropionic
esters have great potential to be developed as a promising AKI therapeutic agent
through reducing and healing the inflammatory damage.
4. Experimental section
4.1. Chemistry
All reagents were purchased from commercial sources and were used without further
purification. Melting points (uncorrected) were determined on a XT4MP apparatus
(Taike Corp., Beijing, China). 1H NMR and 13C NMR spectra were recorded on Bruker

AV-400 instrument in CDCl3. Chemical shifts are reported in parts per million (δ)
downfield from the signal of tetramethylsilane (TMS) as internal standards. Coupling
constants are reported in Hz. The multiplicity is defined by s (singlet), d (doublet), t
(triplet), or m (multiplet). High resolution mass spectra (HRMS) were obtained on an
Agilent 1260-6221 TOF mass spectrometry. Column and thin-layer chromatography
(CC and TLC, resp.) were performed on silica gel (200-300 mesh) and silica gel GF254
(Qingdao Marine Chemical Factory) respectively.
4.2. General procedure for the preparation of compounds 9a–9c, 13a and 13b.
A mixture of arylmethanol (9 or 13, 1.0 mmol), K2CO3 (1.2 mmol, 1.2 eq.) and
DMSO (8 mL, 0.13 M) under an atmosphere of SO2F2 (balloon) was stirred at room
temperature for 12 h before DABCO (1,4-diazabicylo [2.2.2] octane) (3.0 mmol, 3 eq.)
and ethyl acrylate (3.0 mmol, 3 eq.) were added. The resulting mixture was stirred at
60ºC for an additional 36 h. The mixture was concentrated under the reduced pressure.
The residue was purified by flash column chromatography (petroleum/EtOAc = 1/1,
v/v) to obtain title compound.
4.2.1. Ethyl 3-hydroxy-2-methylene-3-(2-trifluoromethylphenyl)propionate (9a). Light
yellow oil, yield 87%; 1H NMR (400 MHz, CDCl3) δ 7.68 (d, J = 8.8 Hz, 2H), 7.58 (t,
J = 7.6 Hz, 1H), 7.43 (t, J = 7.8 Hz, 1H), 6.39 (s, 1H), 6.03 (s, 1H), 5.57 – 5.51 (s, 1H),
13
4.20 (m, 2H), 1.23 (t, J = 7.1 Hz, 3H); C NMR (101 MHz, CDCl3) δ 166.4, 141.8,
139.3, 132.3, 128.9, 128.6, 127.3, 126.9, 125.9, 124.3, 67.87, 61.2, 14.0; TOF-HRMS:
m/z [M + Na]+ calcd for C15H15NO3: 297.0709; found: 297.0708.
4.2.2. Ethyl 3-hydroxy-2-methylene-3-(3-trifluoromethylphenyl)propionate (9b). Light
yellow oil, yield 83%; 1H NMR (400 MHz, CDCl3) δ 7.66 (s, 1H), 7.61 – 7.51 (m, 2H),
7.47 (t, J = 7.7 Hz, 1H), 6.38 (s, 1H), 5.82 (s, 1H), 5.60 (s, 1H), 4.19 (q, J = 7.1 Hz,
2H), 1.25 (t, J = 7.1 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ 166.1, 142.4, 141.5, 130.7,
129.9, 128.9, 126.7, 124.6, 124.0, 123.4, 73.0, 61.2, 14.0; TOF-HRMS: m/z [M + Na]+
calcd for C15H15NO3: 297.0709; found: 297.0708.

4.2.3. Ethyl 3-hydroxy-2-methylene-3-(4-trifluoromethylphenyl)propionate (9c). Light
yellow oil, yield 85%; 1H NMR (400 MHz, CDCl3) δ 7.61 (d, J = 8.2 Hz, 2H), 7.51 (d,
J = 8.1 Hz, 2H), 6.37 (s, 1H), 5.81 (s, 1H), 5.59 (s, 1H), 4.19 (q, J = 7.1 Hz, 2H), 1.26
(t, J = 7.1 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ 166.2, 145.4, 141.6, 123.0, 126.9
(2C), 126.6, 125.4 (2C), 125.2, 73.0, 61.2, 14.0; TOF-HRMS: m/z [M + Na]+ calcd for
C15H15NO3: 297.0709; found: 297.0707.
4.2.4. Ethyl 3-hydroxy-2-methylene-3-(quinolin-2-yl)propionate (13a). Light yellow oil,
yield 79%; 1H NMR (400 MHz, CDCl3) δ 8.11 (dd, J = 16.7, 8.5 Hz, 2H), 7.82 (d, J =
8.1 Hz, 1H), 7.72 (ddd, J = 8.4, 7.0, 1.4 Hz, 1H), 7.58 – 7.51 (m, 1H), 7.46 (d, J = 8.5
Hz, 1H), 6.40 (s, 1H), 5.99 (s, 1H), 5.79 (s, 1H), 4.24 – 4.12 (m, 2H), 1.21 (t, J = 7.1
Hz, 3H); 13C NMR (101 MHz, CDCl3) δ 166.4, 159.6, 146.4, 142.1, 137.4, 130.1, 128.9,
127.8, 127.8, 127.8, 126.8, 119.1, 72.1, 61.1, 14.3; TOF-HRMS: m/z [M + H]+ calcd
for C15H15NO3: 258.1125; found: 258.1122.
4.2.5. Ethyl 3-hydroxy-2-methylene-3-(quinolin-3-yl)propionate (13b). Light yellow
powder, yield 81%; m.p. 96-97ºC; 1H NMR (400 MHz, CDCl3) δ 8.88 (d, J = 2.1 Hz,
1H), 8.19 (d, J = 2.0 Hz, 1H), 8.10 (d, J = 8.5 Hz, 1H), 7.82 (d, J = 8.2, 1.0 Hz, 1H),
7.73 – 7.67 (m, 1H), 7.58 – 7.51 (m, 1H), 6.43 (s, 1H), 5.95 (s, 1H), 5.79 (s, 1H), 4.17
13
(q, J = 7.1 Hz, 2H), 1.24 (t, J = 7.1 Hz, 3H); C NMR (101 MHz, CDCl3) δ 166.0,
149.8, 147.3, 141.7, 134.5, 133.9, 129.6, 128.8, 128.0, 127.7, 126.9, 126.4; TOF-
HRMS: m/z [M + H]+ calcd for C15H15NO3: 258.1125; found: 258.1125.
4.3. Biological evaluation
4.3.1. Cell culture
The RAW264.7 cell line was obtained from BeNa Culture Collection Company. The
Mouse peritoneal macrophages were cultured in DMEM (Hyclone, USA) containing
10% FBS ((Biological Industries, Israel), 100 U/mL penicillin and 100 mg/mL

streptomycin (Beyotime). Unless otherwise indicated, the RAW264.7 cells were grown
in a humidified atmosphere of 5% CO2 and 95% air at 37°C. After the macrophages
have reached the logarithmic growth phase, they are usually cultured with 1 % serum
for 24 h and then subjected to subsequent experiments.
4.3.2. Cell viability assay
Each compound was tested for their in vitro cytotoxicity on the RAW264.7 cells by
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric dye
reduction method. In 96-well plates, the cells (1×104 cells/100 L) were seeded and
maintained at 37°C in 5% CO2 for 24 h, and then the well was treated with concentration
of 10 M for 24 h. Then, 20 μL of MTT solution (prepared in PBS solution, 5 mg/mL)
was added to every well and incubated for 4 h. After this process, all the liquid was
discarded in the well, added 150 L of DMSO to each well, incubated for 20 min in a
constant temperature shaker, then detected at 492 nm with a microplate reader
(MQX200, Bio-Tek, USA).
4.3.3. Nitric oxide (NO) and cytokine assay
The supernatant of compounds-treated the RAW264.7 cell cultures were collected
and used to measure nitric oxide (NO) and cytokines (TNF- and IL-6) by Griess
reagent (Beyotime, China) and enzyme-linked immunosorbent assay (ELISA) kits
(Huamei, Wuhan) according to the manufacturer’s protocol. RAW264.7 cells were
seeded into 48-well plate with 7104 cells per well and incubated for 24 h, and then
cells were retreated with compounds (2.5, 5 or 10 M) for 1 h, incubated with LPS (0.5
g/mL) for 24 h [55].
4.3.4. Western blot assay
Western blot is commonly used to detect protein expression in cells or tissues.
RAW264.7 cells were plated in 6-well plates with 2 106 cells per well and incubated

for 24 h. Next, the cells were treated with or without compound 13b (2.5, 5, 10 M) for
1 h before exposure to LPS (0.5 g/mL) for 24 h. In normal, cells were washed with
phosphate-buffered saline (PBS) and were lysed in 200 L RIPA cell lysis buffer that
contains PMSF and phosphatase inhibitors (Beyotime, china) on ice for 30 min. The
lysates were boiled for 10 min at 95°C and electrophoresed in 10% SDS-PAGE
polyacrylamide gels and then transferred to PVDF membrane (GE Healthcare, UK).
The proteins were incubated with antibodies against -actin, iNOS, COX-2 and TLR4
and further incubated for 1 h with HRP-conjugated secondary antibodies. Finally,
Protein bands exposed under enhanced chemiluminescence detection reagents
(Millipore, MA, USA) conditions. Those bands were scanned and intensity quantified
using Image J (1.45s, USA).
4.3.5. Animal experiments
Male C57BL/6 mice (weighing 18-25 g) were purchased and fed with standard
rodent chow and water adapted for 7 days at the Animal Center of Anhui Medical
University (Hefei, China). All experiments about animal were according to the Guide
for Care and Use of Laboratory Animals of National Institutes of Health. The
experimental animals were divided into four groups: normal control group, model
group, cisplatin + 13b (15 mg/kg) group and cisplatin + 13b (30 mg/kg) group which
contain 10 mice. Mice in the 13b group were given compound of 13b (15 mg/kg and
30 mg/kg) with intragastric administration for 3 consecutive days, and the other groups
were given the same amount of normal saline. The third day, all mice except the normal
group received a single intraperitoneal injection of cisplatin solution (20 mg/kg)
(aladdin, China) after 1h of intragastric administration. The normal group, mice were
injected intraperitoneally with the same dose of saline. Under the same conditions, mice
continued administration either the 13b compound or equal volume of saline for 3 days,
followed by obtaining serum and kidney tissue. Maximal kidney injury, as assessed by
kidney function biomarkers and histologic measurements, was observed at 72 h
following intraperitoneal injections of cisplatin [56].

On the third day after cisplatin administration, all mice were anesthetized with ether,
immediately blood and kidney were collected for further analysis. After standing on ice
for more than half an hour, the blood was centrifuged at 1500 rpm, 4°C and 5 min to
obtain serum. The levels of serum blood urea nitrogen (BUN) and serum creatinine
(SCR) were measured by commercially supplied kits (Nanjing Jiancheng
Bioengineering Institute, China) which according to the manufacturer's instructions.
The secretion of inflammatory cytokines (TNF-, IL-6) in blood of all mice was
detected with ELISA kits (Huamei, Wuhan). Most of the kidney tissues of the mice
were stored at -80°C, and a small part was fixed with 10% formalin buffer for 24 h at
25°C and then embedded in paraffin. To evaluate the biological activity of compound
13b in vivo, the degree of kidney injury in each group of mice was determined by
hematoxylin-eosin (H&E) and periodic acid-schiff (PAS) staining.
4.4. Statistical analysis
All date in the figures are expressed as means ± standard error of the mean (SEM).
The results in the text were analyzed by one-way analysis of variance (ANOVA). A
value of P less than 0.05 indicated that the difference is statistically significant. All
experimental results are repeated at least two or three times.
Acknowledgments
Financial support was provided by Natural Science Fundation of Anhui provincial
Department of Education (KJ2019ZD21), and Anhui Provincial Natural Science
Foundation (2008085MH261, 2008085MH272).
Notes
The authors declare no competing financial interest.
Abbreviations used
AKI, acute kidney injury; LPS, lipopolysaccharide; NO, nitric oxide; TNF-a, tumor

necrosis factor-α; IL-6, interleukin-6; iNOS, inducible nitric oxide synthase; COX-2,
cyclooxygenase-2; TLRs, toll-like receptors; TLR4, toll-like receptor 4; SuFEx, sulfur
(VI) fluoride exchange; NSAID, nonsteroidal anti-inflammatory drugs; NOS2, nitric
oxide synthase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;
ELISA, enzyme linked immunosorbent assay; BUN, blood urea nitrogen; SCR, serum
creatinine; H&E, hematoxylin-eosin; PAS, periodic acid-schiff; DMEM, dulbecco's
modification of eagle's medium dulbecco.
Appendix A. Supplementary material
The copies of representative 1H and 13C NMR spectra can be found at http://
References
[1] A. Zuk, J.V. Bonventre, Acute Kidney Injury, Annu. Rev. Med. 67 (2016) 293-307.
[2] N. Pabla, Z. Dong, Cisplatin nephrotoxicity: mechanisms and renoprotective
strategies, Kidney Int. 73 (2008) 994-1007.
[3] A. Yonezawa, K. Inui, Organic cation transporter OCT/SLC22A and H (+)/organic
cation antiporter MATE/SLC47A are key molecules for nephrotoxicity of platinum
agents, Biochem. Pharmacol. 81 (2011) 563-568.
[4] S.M. Bagshaw, Short- and long-term survival after acute kidney injury, Nephrol.
Dial. Transplant. 23 (2008) 2126-2128.
[5] K. Matsushita, T. Saritas, M.B. Eiwaz, N. McClellan, I. Coe, W. Zhu, M.Z. Ferdaus,
L.Y. Sakai, J.A. McCormick, M.P. Hutchens, The acute kidney injury to chronic kidney
disease transition in a mouse model of acute cardiorenal syndrome emphasizes the role
of inflammation, Kidney Int. 97 (2020) 95-105.
[6] H. Rabb, M. Griffin, D. McKay, S. Swaminathan, P. Pickkers, M. Rosner, J.
Kellum, C. Ronco, Inflammation in AKI: Current Understanding, Key Questions, and
Knowledge Gaps, J. Am. Soc. Nephrol. 27 (2016) 371-379.
[7] F. Tögel, C. Westenfelder, Kidney protection and regeneration following acute
injury: progress through stem cell therapy, Am. J. Kidney Dis. 60 (2012) 1012-1022.
[8] A. Zarjou, A. Agarwal, Sepsis and Acute Kidney Injury, J. Am. Soc. Nephrol. 22

(2011) 999-1006.
[9] S. Hwang, Y. Kim, Y. Park, H. Lee, K. Kim, Anti-inflammatory effects of
chlorogenic acid in lipopolysaccharide-stimulated RAW 264.7 cells, Inflamm Res. 63
(2014) 81-90.
[10] L. Zhang, Y. Fan, H. Su, L. Wu, Y. Huang, L. Zhao, B. Han, G. Shu, M. Xiang, J.
Yang, Chlorogenic acid methyl ester exerts strong anti-inflammatory effects via
inhibiting the COX-2/NLRP3/NF-κB pathway, Food Funct. 9 (2018) 6155-6164.
[11] S. Wang, L. Hu, X. Gao, H. Guo, G. Fan, Anti-inflammatory activity of salvianolic
acid B in microglia contributes to its neuroprotective effect, Neurochem Res. 35 (2010)
1029-1037.
[12] J. Zhao, X.-C. Yang, M. Fujino, N. Ichimaru, W. Que, X.-K. Li, S. Takahara,
Salvianolic acid B ameliorates liver injury in a murine aGvHD model by decreasing
inflammatory responses via upregulation of HO-1, Transpl. Immunol. 55 (2019)
101203.
[13] M. Krasavin, A. Lukin, N. Zhurilo, A. Kovalenko, I. Zahanich, S. Zozulya, Novel
agonists of free fatty acid receptor 1 (GPR40) based on 3-(1,3,4-thiadiazol-2-yl)
propanoic acid scaffold, J. Enzyme. Inhib. Med. Chem. 31 (2016) 1404-1410.
[14] G. Palermo, A. Favia, M. Convertino, M. De Vivo, The Molecular Basis for Dual
Fatty Acid Amide Hydrolase (FAAH)/Cyclooxygenase (COX) Inhibition, Chem. Med.
Chem. 11 (2016) 1252-1258.
[15] A. Gouda, E. Beshr, F. Almalki, H. Halawah, B. Taj, A. Alnafaei, R. Alharazi, W.
Kazi, M. AlMatrafi, Arylpropionic acid-derived NSAIDs: New insights on
derivatization, anticancer activity and potential mechanism of action, Bioorg. Chem. 92
(2019) 103224.
[16] Y. Bansal, O. Silakari, Multifunctional compounds: Smart molecules for
multifactorial diseases, Eur. J. Med. Chem. 76 (2014) 31-42.
[17] S. Bhowmik, S. Batra, Applications of Morita-Baylis-Hillman Reaction to the
Synthesis of Natural Products and Drug Molecules, Curr. Org. Chem. 18 (2014) 3078-
3119.
[18] S. Singh, S. Bhat, Antimicrobial potential of 3-hydroxy-2-methylene-3-

phenylpropionic acid derivatives, Acta. Pharm. 61 (2011) 447-455.
[19] W. da Silva, D. Rodrigues, R. de Oliveira, R. Mendes, T. Olegário, J. Rocha, T.
Keesen, C. Lima-Junior, M. Vasconcellos, Synthesis and activity of novel homodimers
of Morita-Baylis-Hillman adducts against Leishmania donovani: A twin drug approach,
Bioorg. Med. Chem. Lett. 26 (2016) 4523-4526.
[20] S. Sousa, J. Rocha, T. Keesen, E. Silva, P. de Assis, J. de Oliveira, S. Capim, F.
Xavier, B. Marinho, F. Silva, C. Lima-Junior, M. Vasconcellos, Synthesis of 16 New
Hybrids from Tetrahydropyrans Derivatives and Morita-Baylis-Hillman Adducts: In
Vitro Screening against Leishmania donovani, Molecules 22(2) (2017) 207.
[21] X.M. Meng, D.J. Nikolic-Paterson, H.Y. Lan, Inflammatory processes in renal
fibrosis, Nat. Rev. Nephrol. 10 (2014) 493-503.
[22] Q. Cao, Y. Wang, D. Harris, Pathogenic and protective role of macrophages in
kidney disease, Am. J. Physiol. Renal Physiol. 305 (2013) F3-11.
[23] P. Nelson, A. Rees, M. Griffin, J. Hughes, C. Kurts, J. Duffield, The renal
mononuclear phagocytic system, J. Am. Soc. Nephrol. 23 (2012) 194-203.
[24] S. Huen, L. Huynh, A. Marlier, Y. Lee, G. Moeckel, L. Cantley, GM-CSF
Promotes Macrophage Alternative Activation after Renal Ischemia/Reperfusion Injury,
J. Am. Soc. Nephrol. 26 (2015) 1334-1345.
[25] S. Anavi, O. Tirosh, iNOS as a metabolic enzyme under stress conditions, Free
Radic. Biol. Med. 146 (2020) 16-35.
[26] C. Bogdan, Nitric oxide and the immune response, Nat. Immunol. 2 (2001) 907-
916.
[27] S. Faubel, E. Lewis, L. Reznikov, D. Ljubanovic, T. Hoke, H. Somerset, D. Oh, L.
Lu, C. Klein, C. Dinarello, C. Edelstein, Cisplatin-induced acute renal failure is
associated with an increase in the cytokines interleukin (IL)-1beta, IL-18, IL-6, and
neutrophil infiltration in the kidney, J. Pharmacol. Exp. Ther. 322 (2007) 8-15.
[28] G. Ramesh, W. Reeves, TNF-alpha mediates chemokine and cytokine expression
and renal injury in cisplatin nephrotoxicity, J. Clin. Invest. 110 (2002) 835-842.
[29] C. Bogdan, The multiplex function of nitric oxide in (auto)immunity, J. Exp. Med.
187 (1998) 1361-1365.

[30] A. Rios, H. Vargas-Robles, A. Gámez-Méndez, B. Escalante, Cyclooxygenase-2
and kidney failure, Prostaglandins Other Lipid Mediat. 98 (2012) 86-90.
[31] J. Hernández, H. Astudillo, B. Escalante, Angiotensin II stimulates
cyclooxygenase-2 mRNA expression in renal tissue from rats with kidney failure, Am.
J. Physiol. Renal Physiol. 282 (2002) F592-598.
[32] R. Harris, M. Zhang, H. Cheng, Cyclooxygenase-2 and the renal renin-angiotensin
system, Acta. Physiol. Scand. 181 (2004) 543-547.
[33] J. Schnermann, J.P. Briggs, Tubular control of renin synthesis and secretion,
Pflugers Arch. 465 (2013) 39-51.
[34] H. Liu, W. Ye, G. Guan, Z. Dong, Z. Jia, T. Yang, Developmental regulation of
calcineurin isoforms in the rodent kidney: association with COX-2, Am J Physiol Renal
Physiol. 293 (2007) F1898-1904.
[35] P. Slattery, S. Frolich, Y. Schreiber, R.M. Nusing, COX-2 gene dosage-dependent
defects in kidney development, Am. J. Physiol. Renal Physiol. 310 (2016) F1113-1122.
[36] Z. Jia, N. Wang, T. Aoyagi, H. Wang, H. Liu, T. Yang, Amelioration of cisplatin
nephrotoxicity by genetic or pharmacologic blockade of prostaglandin synthesis,
Kidney Int. 79 (2011) 77-88.
[37] S. Agrawal, A. Guess, M. Chanley, W. Smoyer, Albumin-induced podocyte injury
and protection are associated with regulation of COX-2, Kidney Int. 86 (2014) 1150-
1160.
[38] P. Ranganathan, C. Jayakumar, R. Mohamed, Z. Dong, G. Ramesh, Netrin-1
regulates the inflammatory response of neutrophils and macrophages, and suppresses
ischemic acute kidney injury by inhibiting COX-2-mediated PGE2 production, Kidney
Int. 83 (2013) 1087-1098.
[39] K. Hamidzadeh, S.M. Christensen, E. Dalby, P. Chandrasekaran, D.M. Mosser,
Macrophages and the Recovery from Acute and Chronic Inflammation, Annu. Rev.
Physiol. 79 (2017) 567-592.
[40] A. Rousta, S. Mirahmadi, A. Shahmohammadi, D. Nourabadi, M. Khajevand-
Khazaei, T. Baluchnejadmojarad, M. Roghani, Protective effect of sesamin in
lipopolysaccharide-induced mouse model of acute kidney injury via attenuation of

oxidative stress, inflammation, and apoptosis, Immunopharmacol. Immunotoxico. 40
(2018) 423-429.
[41] Y. Jiang, N.S. Alharbi, B. Sun, H.-L. Qin, SO2F2 mediated cascade
dehydrogenative Morita–Baylis–Hillman reaction of the C(sp3)–H of primary alcohols
with the C(sp2)–H of electron-deficient olefins for the assembly of allylic alcohols,
RSC Advances 9 (2019) 29784-29787.
[42] G. Zha, W. Fang, Y. Li, J. Leng, X. Chen, H. Qin, SOF-Mediated Oxidative
Dehydrogenation and Dehydration of Alcohols to Alkynes, J. Am. Chem. Soc. 140
(2018) 17666-17673.
[43] G.F. Zha, W.Y. Fang, J. Leng, H.L. Qin, A Simple, Mild and General Oxidation
of Alcohols to Aldehydes or Ketones by SO2F2/K2CO3 Using DMSO as Solvent and
Oxidant, Adv. Synth. Catal. 361 (2019) 2262-2267.
[44] A. Weigert, A. von Knethen, D. Fuhrmann, N. Dehne, B. Brüne, Redox-signals
and macrophage biology, Mol. Aspects. Med. 63 (2018) 70-87.
[45] B. Li, Y. Yang, L. Chen, S. Chen, J. Zhang, W. Tang, 18alpha-Glycyrrhetinic acid
monoglucuronide as an anti-inflammatory agent through suppression of the NF-kappaB
and MAPK signaling pathway, MedChemComm. 8 (2017) 1498-1504.
[46] M. Rossol, H. Heine, U. Meusch, D. Quandt, C. Klein, M.J. Sweet, S. Hauschildt,
LPS-induced Cytokine Production in Human Monocytes and Macrophages, Crit. Rev.
Immunol. 31 (2011) 379-446.
[47] H. Lin, T. Lee, S. Lin, Y. Yeh, T. Lu, C. Hsu, DDAH-2 alleviates contrast medium
iopromide-induced acute kidney injury through nitric oxide synthase, Clin. Sci. 133
(2019) 2361-2378.
[48] S. Kim, D. Huri, S. Snyder, Inducible nitric oxide synthase binds, S-nitrosylates,
and activates cyclooxygenase-2, Science 310 (2005) 1966-1970.
[49] B. Zhang, G. Ramesh, S. Uematsu, S. Akira, W.B. Reeves, TLR4 signaling
mediates inflammation and tissue injury in nephrotoxicity, J. Am. Soc. Nephrol. 19
(2008) 923-932.
[50] L. Patzer, Nephrotoxicity as a cause of acute kidney injury in children, Pediatr.
Nephrol. 23 (2008) 2159-2173.