Evaluation of LPS-induced acute lung injury attenuation in rats by aminothiazole-paeonol derivatives

Article abstract of DOI:10.3390/molecules22101605

Paeonol is a key phenolic compound in the root bark of Moutan Cortex Radicis that has been used in traditional Chinese Medicine to ameliorate inflammation. A series of aminothiazole-paeonol derivatives (APDs) were synthesized in this work and subjected to preliminary evaluation in cells followed by verification in animals. Quantification of monocyte chemotactic protein-1 (MCP-1) and interleukin-6 (IL-6) in culture media of LPS-activated A549 cells, a lung epithelial adenocarcinoma cell line, were used to investigate the anti-inflammatory capability of APDs. ALI-bearing rats were employed to verify therapeutic efficacy of APDs according to observations of total cells, protein amounts, MCP-1 and IL-6 in bronchoalveolar lavage fluid (BALF). Histopathological examinations of lung tissues were consequently applied for validation of APDs. Among these compounds, 2-(2-aminothiazol-4-yl)-5-methoxyphenol (4) had the most potent activity, showing comparable inhibition of MCP-1/IL-6 and superior elimination of neutrophil infiltration and protein exudation in lungs compared to others as well as dexamethasone. This study demonstrated a comprehensive strategy to evaluate APDs through integration of cell-based screening and animal-based verification. In order to fulfill unmet needs of treating acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), APDs introduced in this work could be promising lead compounds to develop high potent anti-inflammation agents.

Full text of DOI:10.3390/molecules22101605

molecules
Article
Evaluation of LPS-Induced Acute Lung Injury
Attenuation in Rats by
Aminothiazole-Paeonol Derivatives
Pin-Kuei Fu 1,2,3, Chi-Yu Yang , Su-Chin Huang , Yu-Wen Hung , Kee-Ching Jeng ,
4
5
4
6
7
7
5
5
8
Ying-Pei Huang , Hong Chuang , Nai-Chun Huang , Jui-Ping Li , Ming-Hua Hsu
and Jen-Kun Chen 5
,9,10,
*
ID
1
Department of Critical Care Medicine, Taichung Veterans General Hospital, Taichung 40705, Taiwan;
yetquen@gmail.com
Department of Biotechnology, Hungkuang University, Taichung 43302, Taiwan
School of Chinese Medicine, China Medical University, Taichung 40447, Taiwan
Animal Technology Laboratory, Agriculture Technology Research Institute, Miaoli 35053, Taiwan;
chiyu@mail.atri.org.tw (C.-Y.Y.); pp33887788@gmail.com (Y.-W.H.)
2
3
4
5
6
7
8
9
Institute of Biomedical Engineering and Nanomedicine, National Health Research Institutes,
Miaoli 35053, Taiwan; chin@nhri.org.tw (S.-C.H.); anai@nhri.org.tw (N.-C.H.); piny72@nhri.org.tw (J.-P.L.)
Department of Medical Research, Tungs’ Taichung MetroHarbor Hospital, Taichung 43503, Taiwan;
kcjeng@gmail.com
Department of Chemistry, National Tsing Hua University, Hsinchu 30013, Taiwan;
suzann800217@hotmail.com (Y.-P.H.); hom770706@gmail.com (H.C.)
Department of Chemistry, National Changhua University of Education, Changhua County 50007, Taiwan;
minghuahsu@cc.ncue.edu.tw
Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 11490, Taiwan
School of Dentistry, National Defense Medical Center, Taipei 11490, Taiwan
Correspondence: jkchen@nhri.org.tw; Tel.: +886-3724-6166 (ext. 38117); Fax: +886-3758-6440
1
0
*
Received: 1 September 2017; Accepted: 21 September 2017; Published: 25 September 2017
Abstract: Paeonol is a key phenolic compound in the root bark of Moutan Cortex Radicis that has been
used in traditional Chinese Medicine to ameliorate inflammation. A series of aminothiazole-paeonol
derivatives (APDs) were synthesized in this work and subjected to preliminary evaluation in cells
followed by verification in animals. Quantification of monocyte chemotactic protein-1 (MCP-1) and
interleukin-6 (IL-6) in culture media of LPS-activated A549 cells, a lung epithelial adenocarcinoma
cell line, were used to investigate the anti-inflammatory capability of APDs. ALI-bearing rats were
employed to verify therapeutic efficacy of APDs according to observations of total cells, protein
amounts, MCP-1 and IL-6 in bronchoalveolar lavage fluid (BALF). Histopathological examinations
of lung tissues were consequently applied for validation of APDs. Among these compounds,
2-(2-aminothiazol-4-yl)-5-methoxyphenol (4) had the most potent activity, showing comparable
inhibition of MCP-1/IL-6 and superior elimination of neutrophil infiltration and protein exudation
in lungs compared to others as well as dexamethasone. This study demonstrated a comprehensive
strategy to evaluate APDs through integration of cell-based screening and animal-based verification.
In order to fulfill unmet needs of treating acute lung injury (ALI) and acute respiratory distress
syndrome (ARDS), APDs introduced in this work could be promising lead compounds to develop
high potent anti-inflammation agents.
Keywords: paeonol; aminothiazole-paeonol derivatives; Moutan Cortex Radicis; anti-inflammation
agents; acute lung injury; acute respiratory distress syndrome
Molecules 2017, 22, 1605; doi:10.3390/molecules22101605
www.mdpi.com/journal/molecules

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1
. Introduction
Herbal medicine and plant-derived compounds have been used to alleviate inflammatory
diseases [ ]. Paeonol is a representative phenolic compound found in the root bark of Moutan
Cortex Radicis (MCR), reported to inhibit proinflammatory cytokines, cell surface adhesion molecules,
and reactive oxygen species (ROS) [ ]. Both MCR and purified paeonol were capable of attenuating
LPS-induced ALI in rats by eliminating expression of myeloperoxidase (MPO) activity, iNOS,
interleukin-1 (IL-1 ), interleukin-6 (IL-6) and macrophage-inflammatory peptide-2 (MIP-2) [ 10].
Recently, paeonol derivatives have been synthesized and assessed for their potential anti-atherogenic,
anti-cancer, anti-oxidant, and anti-virus properties [11 14]. The 2-aminothiazole moiety not only serves
1–3
4–8
β
β
9,
–
as a stable bioisostere of phenol groups but provides lipophilicity to improve oral availability [15].
Indeed, the 2-aminothiazole moiety has been identified to be an effective pharmacophore for different
therapeutic purposes [16,17].
In the present work, we coupled 2-aminothiazole moieties with paeonol to prepare
aminothiazole-paeonol derivatives (APDs) in order to treat acute lung injury (ALI) and acute
respiratory distress syndrome (ARDS). The ALI and ARDS present severe inflammatory cascades
and in conjunction with injury in the respiratory tract, are major health concerns that lead to high
mortality and morbidity in critically ill patients [18–20]. Many clinical trials using drugs such as
aspirin, 2-agonists, omega-3 fatty acids, prostaglandin E1, glucocorticoids, N-acetylcysteine and
β
statins, were frustrated due to poor results whereby these treatments neither prevented nor healed
ALI/ARDS [21–23].
Since ALI/ARDS is a complex disease associated with many biological responses, it is important
to utilize not only in vitro experiments in cell lines but to also verify the results seen in animal models
in order to better evaluate the effect of the most potent APDs. Several animal models, for example
in vivo intratracheal administration of LPS [24–26], are clinically relevant and have been successfully
used in previous studies. Neutrophil extracellular traps (NETs) accompanied by disruption of epithelial
integrity and leakage of protein and fluid in the lungs are commonly observed in both patients and
LPS-induced ALI animal models [27
associated with up-regulation of adhesion molecules, chemotaxis, and proinflammatory cytokines in
ALI and ARDS [29 31]. Various pro-inflammatory cytokines, including IL-1 , IL-6 and tumor necrosis
–29]. Migration and accumulation of neutrophils in airspaces are
–
β
factor (TNF-α), were involved in the recruitment of neutrophils to promote inflammation.
The levels of pro-inflammatory cytokines are strongly correlated with survival in ALI/ARDS
patients [32,33]. A previous study indicated monocyte chemoattractant protein (MCP)-1 was an
activating factor in the LPS-induced ALI animal model [34]. The MCP-1 regulated through the JNK
signaling pathway might participate in lung injury by modulating adhesion of inflammatory cells and
activation of monocytes [35,36]. Yong et al. suggested using plasma MCP-1 to predict the severity
of patients with pneumonia [37]. Chen et al. have demonstrated the MCP-1 in BALF could be used
as a critical marker for pulmonary inflammation [38]. The MCP-1 was also employed to evaluate
bleomycin-induced lung injury [39]. As for IL-6, Liang et al. have reported IL-6 could promote
type 2 alveolar epithelial cells (AEC2) renewal [40]. Therefore, we aimed to use MCP-1 and IL-6 as
preliminary markers for cell-based screening to evaluate anti-inflammation potency.
In this study, LPS-activated A549 cells for preliminary screening could be integrated with
LPS-induced ALI-bearing rats for verification. Changes of MCP-1 and IL-6 in culture media of
LPS-activated A549 cells were employed to evaluate the anti-inflammatory activities of APDs compared
to reference compounds (paeonol and dexamethasone). Furthermore, relatively high potent APDs
were subjected to verification in ALI-bearing rats. Comprehensive investigation of cell infiltration,
protein accumulation and inflammatory markers secretion in both of bronchoalveolar lavage fluid
(
BALF) and lung tissues demonstrate an effective strategy to explore optimal APDs for the therapy of
ALI/ARDS.

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2
. Results
2
.1. Synthesis
The preparation of the vaminothiazole-paeonol derivatives is shown in Scheme 1. Paeonol (1) was
treated with 2-chloroethylmorpholine to generate morpholine-o-paeonol (
with thiourea and iodine to obtain morpholine-o-paeonol aminothiazole (
) could be treated with thiourea and iodine to construct the 2-aminothiazole scaffold
condensation–cyclization of thiourea. Compound
generate the aminothiazole-paeonol salt 4.HCl. Paeonol-2-aminothiazole-phenylsulfonyl derivatives
5g were obtained reacting compound with substituted phenylsulfonyl chlorides. These products
2). Compound
2
was reacted
3). Additionally, paeonol
(1
4 through the
4
was reacted with excess hydrochloric acid gas to
5
a
–
4
were obtained in sufficient yields and purified by using recrystallization for the subsequent cell-based
and animal-based experiments.
Scheme 1. Synthesis of aminothiazole-paeonol derivatives (APDs) as anti-inflammation agents in
this study.
2
.2. MCP-1 and IL-6 Secretion in LPS-Activated A549 Cells
Low and high concentrations of paeonol, dexamethasone (Dexa) and APDs (1.0 µg/mL and
10 µg/mL) were used to treat LPS-activated A549 cells following the protocols shown in Figure 1.
A549 is a lung epithelial adenocarcinoma cell line. The MCP-1 in conditioned media was quantified
to survey anti-inflammatory capability of APDs. Secretion of MCP-1 was significantly inhibited by
compound
g/mL could inhibit of MCP-1 secretion (Figure 2A). Only compound
IL-6 secretion but others didn't at neither high nor low concentrations (Figure 2B).
4
and Dexa at 1.0
µg/mL and 10
µg/mL (Table 1 and Figure 2A). The compound 4.HCl at
1
0
µ
4
at 10 g/mL suppressed
µ

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Figure 1. Preliminary screening protocols for anti-inflammation potency of APDs using the
LPS-activated A549 cell model.
Table 1. Secretion of MCP-1 in conditioned media of LPS-activated A549 cells treated with low
(1.0 µg/mL) and high (10 µg/mL) concentrations of Dexa, paeonol, and APDs.
Secretion of MCP-1 in Conditioned Media (pg/mL)
Drugs (Symbols)
Low Dose Treatment
High Dose Treatment
Dexamethasone (Dexa)
Paeonol (P)
2669 ± 578 *
3938 ± 106
4332 ± 332
3618 ± 267
2724 ± 254 *
3323 ± 261
3980 ± 424
3983 ± 120
4238 ± 464
4140 ± 416
4002 ± 125
4050 ± 2.3
4354 ± 155
2538 ± 343 *
3584 ± 329
3610 ± 505
4446 ± 201
2077 ± 111 *
2826 ± 362 *
3225 ± 76
2
3
4
(P-2)
(P-3)
(P-4)
4
.HCl (P-4.HCl)
a (P-5a)
b (P-5b)
c (P-5c)
d (P-5d)
e (P-5e)
5
5
5
3786 ± 131
3793 ± 176
4118 ± 372
3893 ± 312
4334 ± 544
4628 ± 89
5
5
5
5
f (P-5f)
g (P-5g)
Significant differences of MCP-1 secretion between positive control group and each treatment group at p values
less than 0.05 were annotated with *. The MCP-1 concentration in positive control (LPS-activated) groups was
4163.5 pg/mL.
Figure 2. Secretion of (
A) MCP-1 and (B) IL-6 in conditioned media of LPS-activated A549 cells treated
with Dexa (1.0 g/mL and 10
µ
µg/mL) and selected APDs (1.0
µ
g/mL and 10 g/mL). Significant
µ
differences between each treatment group and LPS-activated (positive control) group at p values less
than 0.05 were annotated with *.

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2
.3. Cell Viabilities for APDs Treatments
Cell viabilities for APDs against A549 cells were determined by WST-1 assays. Compound
4
and
compound 4.HCl are very biocompatible because of more than 90% cell survive a 50
Figure 3). Compound 5c, showing an IC50 value of 39 g/mL (127
prepared in this study.
µ
g/mL treatment
(
µ
µM) is the most cytotoxic APD
Figure 3. Viabilities of A549 cells at 24 h post-treatment of selected APDs. The P-4, P-4.HCl and P-5c
represented the Compound 4, 4.HCl and 5c shown in Figure 1.
2
.4. Comparison of BALF Collected from ALI-Bearing Rats
Compound
LPS-activated A549 cell model. To further verify therapeutic efficacy, paeonol, compound
.HCl and Dexa were injected i.p. into the ALI-bearing rat model following the protocols shown in
Figure 4.
4
and Compound 4.HCl are promising APDs based on our preliminary screening in a
4, compound
4
Figure 4. Verification protocols of LPS-challenged ALI-bearing rats treated with testing chemicals
APDs, paeonol, and dexamethasone).
(
Previous studies have indicated both of endotoxin-induced ALI animal models and
endotoxin-treated human showed time-dependent changes in either peripheral inflammatory

Molecules 2017, 22, 1605
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responses or accumulation of immune cells, total proteins, and cytokines in BALF associated with
pathological lung injury within 48 h after endotoxin-challenge [41 42]. The severity peak was observed
in ALI-bearing rats at 8 h after LPS challenge [43]. Therefore, changes of total cell counts, cytokines
IL-6 and MCP-1) and total proteins in BALF were determined in order to validate therapeutic efficacy
of APDs (Figure 5).
,
(
Figure 5. Biological responses of ALI-bearing rats treated with Dexa, paeonol and selected APDs on
) total cell counts, ( ) total protein concentrations, ( ) IL-6 and ( ) MCP-1 in bronchoalveolar lavage
(A
B
C
D
fluid (BALF). Significant differences between each treatment group and LPS-treated (positive control)
group at p values less than 0.05 were annotated with *.
Paeonol and Dexa proved ineffective at inhibiting cell infiltration in lungs of ALI-bearing rats
(
Figure 5A). Notably, compound
treatment. As for compound 4.HCl, effective inhibition of cell infiltration in lungs was observed at
0 mg/kg treatment. Compound was thus superior to paeonol, Dexa, and compound 4.HCl and
substantially inhibited protein infiltration in lungs of ALI-bearing rats (Figure 5B). Paeonol (50 mg/kg),
compound (25 and 50 mg/kg), compound 4.HCl (50 mg/kg) and Dexa (1 and 5 mg/kg) suppressed
IL-6 secretion in BALF (Figure 5C), in which compound and Dexa presented comparable efficacies
for the two dosages. The compound 4.HCl and paeonol presented comparable efficacies merely at
0 mg/kg. Paeonol, , and 4.HCl were comparable at inhibiting MCP-1 secretion at both of low and
high dosages (Figure 5D).
4 eliminated total cell counts in BALF at both 25 mg/kg and 50 mg/kg
5
4
4
4
5
4
2
.5. Comparison of Compound 4 and Compound 4.HCl by Histopathological Examinations
Lung tissues for histopathological examination were collected at 8 h after LPS administration.
No prominent neutrophil infiltration, red blood cells and protein exudates were observed in the
negative control group (Figure 6A). ALI-bearing rats presented extremely severe protein exudation and

Molecules 2017, 22, 1605
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cells infiltration in lungs (Figure 6B), which could be eliminated after treatment with Dexa (Figure 6C),
paeonol (Figure 6D), compound (Figure 6E) and compound 4.HCl (Figure 6F). Compound is
superior to Dexa, paeonol and compound 4.HCl at alleviating LPS-induced acute lung injury.
4
4
Figure 6.
) non-challenged (negative control), (
ALI-bearing rats treated with ( ) Dexa, (
were consequently sacrificed at 8 h after intratracheal administration of LPS (8 mg/kg). The alveolar
spaces ( ) filled with the mixture of red blood cells (
) and mononuclear/neutrophilic infiltrates (⇧ )
Histopathological examination of H&E stained lung tissues collected from:
) LPS-challenged (positive control) rats without treatment and
) paeonol, ( ) Compound , and ( ) Compound 4.HCl. Rats
(A
B
D
C
E
4
F
∗
→
combined with biological responses observed in BALF were integrated to comprehensively evaluate
therapeutic efficacy of APDs. (Scale bar: 100 µm).
3
. Discussion
Herbal medicines are invaluable resources to construct plant-derived compound libraries for
preventing and treating diseases. For example, paeonol ( , Scheme 1) is a one of the remarkable
molecules in Moutan Cortex Radicis (MCR) that has been used in traditional Chinese medicine to
promote blood circulation and eliminate blood stasis [44 45]. Our previous work described an animal
model treated with MCR to alleviate ALI through inhibiting MCP-1 and IL-8 secretion [10]. In addition
to MCP-1 and IL-8, elimination of TNF- , IL-1 , IL-6, and PAI-1 in BALF were observed in ALI-bearing
rats that were treated with paeonol purified from MCR [ ]. Paeonol derivatives may also have extensive
1
,
α
β
9
biomedical applications. Zhu et al. reported thiosemicarbazone paeonol derivatives were potential
inhibitors of mushroom tyrosinase [46]. Tsai et al. recently demonstrated a series of aminothiazole
paeonol derivatives (APDs) as anticancer agents with relatively high potency against gastrointestinal
adenocarcinoma (AGS and HT-29 cells) [14]. The thiazole ring of APDs may contribute to a variety of
biological effects, which encourage us to evaluate anti-inflammatory responses for a series of APDs.
ALI and ARDS present acute respiratory failure with severe inflammation in the respiratory tract,
leading to high mortality in critically ill patients [18,47]. Inflammation disrupting lung epithelial and
endothelial barriers cause damage of alveolar-capillary membrane integrity, excessive trans-epithelial
neutrophil migration, and release of pro-inflammatory/cytotoxic mediators. Many therapeutics
involved in different underlying mechanisms have been applied to treat ALI/ARDS; however, most of
them rarely reduce mortality [21,48,49].
In this work, we have established a workflow consisting of primary cell-based screening (Figure 1)
followed by animal-based verification (Figure 4) to propel evaluation of potential APDs against ALI.
Figure 3 presents MCP-1 rather than IL-6 in conditioned media is a relatively sensitive marker to
monitor how LPS-activated A549 cells responsive to APDs treatments because concentration of MCP-1

Molecules 2017, 22, 1605
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is approximately 10-fold higher than that of IL-6 in conditioned media. The MCP-1 in conditioned
media of negative control group (without challenge of LPS) was 1411 pg/mL versus 4164 pg/mL
in that of LPS-activated group (positive control), showing a 2.95-fold increase of MCP-1 caused by
LPS challenge. On the other hand, IL-6 in conditioned medium showed a 2.42-fold increase (from
136 pg/mL to 329 pg/mL) caused by LPS challenge. 2-(2-Aminothiazol-4-yl)-5-methoxyphenol (4)
and 2-(2-aminothiazol-4-yl)-5-methoxyphenol hydrochloride (4.HCl) significantly suppressed MCP-1
secretion (Figure 2A) and showed comparable efficacy to Dexa, a synthetic corticosteroid for treating
inflammatory and autoimmune symptoms. Figure 2B shows that 10
effectively inhibit IL-6 secretion in culture media of LPS-activated A549 cells. Both compound
and compound 4.HCl are superior to compound 5c because of very mild cytotoxicity shown in
µg/mL of compound 4 could
4
Figure 3, which suggests compound
medications for ALI/ARDS.
4 and compound 4.HCl could be safely used for anti-inflammatory
Compound
The compound
4
and compound 4.HCl were subsequently verified in ALI-bearing rats.
4
is excellent at reducing cell infiltration and protein exudation in the alveolar space and
simultaneously inhibits secretion of MCP-1 and IL-6 in BALF (Figure 5). However, compound 4.HCl
shows limited effects (effective at the high dose: 50 mg/kg) in reducing cell infiltration (Figure 5A)
and IL-6 secretion in lungs (Figure 5C) despite effective elimination of MCP-1 (Figure 5D) compared to
compound 4. In comparison with compound 4 and compound 4.HCl, Dexa presents very effective
elimination of IL-6 and MCP-1 in BALF (Figure 5C,D); but Dexa is less responsive for reduction of cell
infiltration and protein exudation (Figure 5A,B).
To further validate therapeutic efficacy of compounds 4 and 4.HCl, histopathological examination
of lung tissues (Figure 6) was employed to identify optimal APDs for treating ALI/ARDS. Infiltration
of neutrophils, exudation of red blood cells and protein were extremely severe in ALI-bearing rats
(
Figure 6B) compared with non-challenged rats (Figure 6A), as determined by the extensive presence of
cellular debris and proteinaceous material in lung tissues. The remarkable characteristic of ALI/ARDS
is neutrophils recruited from the peripheral blood to the alveoli during active infections [50 52],
–
which was simultaneously observed in ALI-bearing rats (Figure 6B). Neutrophil infiltration into
the lungs is mediated through a complex network of chemokines/cytokines secreted by alveolar
macrophages. Chemokines and cytokines including TNF-
can promote chemotaxis and recruit more neutrophils into injured lungs [
neutrophils and red blood cells associated with protein exudates are alleviated to different levels by
treating either Dexa (Figure 6C), paeonol (Figure 6D), compound (Figure 6E) or compound 4.HCl
Figure 6F). Despite the dramatic inhibition of MCP-1 caused by Dexa (Figure 5D), it does not
effectively eliminate protein exudation (Figure 5B) and neutrophil infiltration, which is again proved
by the histopathological examination (Figure 6C). Treatment of compound (Figure 6E) presents
effective response to eliminate neutrophil infiltration and protein exudation compared to treatments of
compound (Figure 6D) and compound 4.HCl (Figure 6F). We therefore suggest the compound is
α
, IL-1
β, IL-6, IL-8, IL-10, and MCP-1
4,18,53–56]. Infiltration of
4
(
4
1
4
highly efficacious for treating ALI/ARDS because of its comprehensive activity to eliminate neutrophil
infiltration, protein exudation and chemokine/ cytokine secretion.
4
. Materials and Methods
4
.1. Chemicals and General Procedures
Solvents, including dichloromethane, ethanol, ethyl acetate, hexane, methanol and
tetrahydrofuran were purchased from Mallinckrodt Pharmaceuticals (St. Louis, MO, USA).
Ethyl acetate was dried and distilled from CaH2. Tetrahydrofuran was dried by distillation
from sodium and benzophenone under an atmosphere of nitrogen. Benzenesulfonyl chloride,
4-bromobenzenesulfonyl chloride, 4-chlorobenzenesulfonyl chloride, 4-fluorobenzenesulfonyl
chloride, 4-methoxybenzene-sulfonyl chloride, 4-nitrobenzenesulfonyl chloride, potassium carbonate,
and p-toluenesulfonyl chloride were purchased from Sigma-Aldrich (St. Louis, MO, USA). All reactions

Molecules 2017, 22, 1605
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◦
were carried out in oven-dried glassware (120 C) under an atmosphere of nitrogen unless as
indicated otherwise. The lipopolysaccharide (LPS) from E. coli (serotype O55:B5) was purchased
from Sigma-Aldrich. Dexamethasone-21-phosphate disodium (Dexa) was purchased from Standard
Chem. & Pharm. Co., Ltd. (Tainan, Taiwan).
Analytical thin layer chromatography was performed on pre-coated plates (silica gel 60 F-254),
1
purchased from Merck (Merck KGaA, Darmstadt, Germany). Proton ( H)-NMR spectra were
obtained on Mercury-400 (400 MHz) (Varian, Palo Alto, CA, USA) AC-400 (400 MHz) (Bruker Biospin
GmbH, Rheinstetten, Germany) or Avance 500 (500 MHz) (Bruker) spectrometers using chloroform-d
(
CDCl ), dimethylsulfoxide-d (DMSO-d ) and deuterium oxide (D O) as the solvents. Chemical
3 6 6 2
1
shifts of H-NMR were referenced to residual protonated solvents (δ 7.24 for chloroform, δ 2.49
for dimethylsulfoxide and 4.79 for deuterium oxide). Carbon-13 NMR spectra were obtained on a
Varian Mercury-400 (100 MHz) or Bruker Avance 500 (125 MHz) spectrometer using chloroform-d
(
CDCl ), dimethylsulfoxide-d (DMSO-d ) and methanol-d (CD OD) as the solvents. Chemical shifts
3
6
6
4
3
13
of C-NMR were referenced to the center of the CDCl triplet (
δ
77.0 ppm), DMSO septet (δ 39.5 ppm)
3
and methanol septet (δ 49.0 ppm). Multiplicities were recorded by following abbreviations: s, singlet;
d, doublet; t, triplet; q, quartet; m, multiplet; J, coupling constant (hertz). NMR spectra are presented
in the Supplementary Materials (Figures S1–S20). High-resolution mass spectra were obtained by a
JMS-700 mass spectrometer (JEOL Ltd., Tokyo, Japan).
4
.2. Procedure for the Preparation of Morpholine-O-Paeonol (2)
A solution containing paeonol ( , 1.0 equiv.), 2-chloroethylmorpholine (1.2 equiv.), potassium
1
carbonate (3.0 equiv.) and ammonium chloride (1.5 equiv.) in acetone (30.0 mL) was refluxed for 12 h.
Then the mixture was extracted with dichloromethane for three times, and dried over MgSO4(s). After
concentration of the solvent, the residue was purified by column chromatography over silica gel using
methanol and dichloromethane as eluent to afford 1-(4-methoxy-2-(2-morpholinoethoxy)- phenyl)ethanone
1
(2
) as a white solid. Yield 90%; H-NMR (500 MHz, CDCl ):
δ
= 7.81 (d, J = 8.5 Hz, 1H), 6.51 (dd, J = 2,
3
8
5
.5 Hz, 1H), 6.42 (d, J = 2 Hz, 1H), 4.14 (t, J = 5.5 Hz, 2H), 3.83 (s, 3H), 3.70 (t, J = 4.5 Hz, 4H), 2.83 (t, J =
.5 Hz, 2H), 2.54 (t, J = 4.5 Hz, 4H); 13C-NMR (125 MHz, CDCl ):
δ
= 197.5, 164.3, 160.0, 132.5, 121.2,
3
+
+
1
05.3, 98.9, 66.7, 65.9, 57.2, 55.4, 53.8, 31.9; HRMS (ESI ): m/z [M] calc. for C H NO 279.1471, found
15
21
4
+
2
80.1578 for [M + H] .
4
.3. Procedure for the Preparation of Morpholine-O-Paeonol Aminothiazole (3)
To obtain aminothiazole paeonol 3, compound 2 (1.0 equiv) was reacted with iodine (1.1 equiv)
and thiourea (3.0 equiv) in ethanol under reflux condition for 12–16 h. Then, the mixture was quenched
with NaOH(aq) (2.0 equiv) and the ethanol was removed under reduced pressure. The residue
was extracted with ethyl acetate and the combined organic layer was washed brine and dried over
MgSO4(s). After being filtered and condensed under reduced pressure, the crude product was purified
by column chromatography on silica gel (ethyl acetate and hexane as eluent) to obtain compound
1
4-(4-methoxy-2-(2-morpholinoethoxy)phenyl)thiazol-2-amine (
3
) as a whit solid. Yield 49%; H-NMR
(
1
1
500 MHz, DMSO-d ): δ = 7.93 (d, J = 10 Hz, 1H), 7.30 (s, 1H), 6.85 (s, 2H), 6.62 (s, 1H), 6.54 (d, J = 10 Hz,
6
H), 4.15 (s, 1H), 3.76 (s, 3H), 3.57 (s, 4H), 2.76 (s, 2H); 13C-NMR (125 MHz, DMSO-d ):
δ
= 166.0, 159.2,
6
+
+
56.8, 145.7, 129.9, 116.7 105.1, 103.7, 99.4, 66.3, 65.1, 56.9, 55.2, 53.3; HRMS (ESI ): m/z [M] calc. for
+
C H N O S 335.1298, found 336.1375 for [M + H] .
16
21
3
3
4
.4. Procedure for the Preparation of Aminothiazole-Paeonol (4)
To obtain aminothiazole-paeonol ( ), paeonol ( , 1.0 equiv) was reacted with iodine (1.1 equiv)
4
1
and thiourea (3.0 equiv) in ethanol under reflux condition for 12–16 h. Then, the mixture was quenched
with NaOH(aq) (2.0 equiv) and the ethanol was removed under reduced pressure. The residue was
extracted with ethyl acetate and the combined organic layer were washed brine and dried over
MgSO4(s). After being filtered and condensed under reduced pressure, the crude product was purified

Molecules 2017, 22, 1605
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by column chromatography on silica gel (ethyl acetate and hexane as eluent) to give compound
1
2-(2-aminothiazol-5-yl)-5-methoxyphenol (
4
). H-NMR (400 MHz, CDCl ):
δ
7.40 (d, J = 8.4 Hz, 1H, H-3),
3
6
.54 (s, 1H, CH), 6.47 (s, 1H, H-6), 6.42 (dd, J = 8.4, 2.0 Hz, 1H, H-4), 5.05 (s, 2H, NH ), 3.78 (s, 3H,
2
13
OMe) ppm. C-NMR (100 MHz, CDCl ):
δ 166.8, 161.0, 157.3, 148.9, 126.6, 111.0, 106.8, 101.6, 98.9, 55.2
3
+
+
+
(OMe) ppm; HRMS (ESI ): m/z [M] calc. for C H N O S 222.0458, found 223.0562 for [M + H] .
10 10 2 2 1
4
.5. Procedure for the Preparation of Aminothiazole-Paeonol salt (4.HCl)
To obtain aminothiazole-paeonol salt 4.HCl, compound (1.0 equiv) dissolved in acetone (20 mL)
was reacted with excess hydrochloric acid gas that was produced by adding concentrated H SO
4
2
4
with an addition funnel to excess NaCl . When precipitation nearly stopped, the precipitate
(s)
was collected using suction filtration and washed with cold acetone to obtain the pure
1
4
-(2-hydroxy-4-methoxyphenyl)thiazol-2-aminium chloride (4.HCl) as a yellow solid. Yield 87%; H-NMR
(
3
1
[
D O, 400 MHz):
.81 (s, 3H) ppm. C-NMR (CD OD, 100 MHz):
δ
1
7.37 (d, J = 8.4 Hz, 1H), 6.80 (s, 1H), 6.58 (dd, J = 6.8, 1.6 Hz, 1H), 6.48 (d, 2.0 Hz, 1H),
2
3
δ
171.4, 163.6, 157.0, 138.1, 130.1, 130.0, 109.0, 107.7,
3
+
+
02.5, 55.8 (OMe) ppm; HRMS (ESI ): m/z [M] calc. for C H ClN O S 258.0224, found 223.0556 for
10 11 2 2 1
+
M + H − HCl] .
4
.6. Procedures for the Preparation of Aminothiazole-Paeonol Derivatives 5a–5g
The solution containing aminothiazole-paeonol ( , 1.0 equiv) in anhydrous THF (2.0–3.0 mL) was
4
added potassium carbonate (1.3 equiv) and a sulfonyl chloride (1.1 equiv). After the reaction mixture
◦
was stirred at 25 C for 2–3 h, it was diluted with dichloromethane (5.0 mL). Inorganic solids were
filtered off and the filtrate was concentrated under reduced pressure to afford the residue. It was then
purified by use of column chromatography on silica gel (various ratio of methanol to dichloromethane)
to give the desired conjugates 5a–5g.
N-[4-(2-Hydroxy-4-methoxyphenyl)thiazol-2-yl]benzenesulfonamide (5a): Yield 80%. Green solid. IR (film):
−
1 1
ν
3569.1, 2811.2, 1560.1, 1481.2, 1374.2, 1131.6, 853.4 cm . H-NMR (400 MHz, CDCl ):
δ
7.62–7.52
3
(
6
1
3
m, 2H, 2
×
ArH), 7.43–7.31 (m, 1H, H-6), 7.28–7.25 (m, 3H, 3
×
ArH), 6.98 (d, J = 2.0 Hz, 1H, H-3),
1
3
.89–6.79 (m, 1H, H-5), 6.68 (s, 1H, SCH), 3.70 (s, 3H, OMe) ppm. C-NMR (100 MHz, CDCl ):
29.7, 125.9, 113.9, 113.7, 105.0, 56.0 (OMe) ppm; HRMS (ESI ): m/z [M] calc. for C H N O S
16 14 2 4 2
62.0390, found 363.0396 for [M + H] .
δ
164.1,
3
+
+
+
N-[4-(2-Hydroxy-4-methoxyphenyl)thiazol-2-yl]-4-methylbenzenesulfonamide (5b): Yield 84%. Off-white
−
ν 3579.1, 2921.2, 1580.1, 1491.2, 1384.1, 1141.6, 843.4 cm . H-NMR (400 MHz, CDCl ):
3
1 1
solid. IR (film):
δ
6
2
7.44 (d, J = 8.8 Hz, 1H, H-6), 7.39 (d, J = 8.2 Hz, 2H, 2
×
ArH), 7.06 (d, J = 8.2 Hz, 2H, 2
×
ArH),
.81 (d, J = 2.4 Hz, 1H, H-3), 6.74 (dd, J = 8.8, 2.4 Hz, 1H, H-5) 6.52 (s, 1H, SCH), 3.72 (s, 3H, OMe),
13
.29 (s, 3H, CH ) ppm. C-NMR (100 MHz, CDCl3): δ159.3, 147.0, 145.3, 144.4, 130.5, 129.1, 128.2,
3
+
+
1
20.9, 113.1, 108.7, 105.9, 55.4 (OMe), 21.4 (CH ) ppm; HRMS (ESI ): m/z [M] calc. for C H N O S
3 17 16 2 4 2
+
3
76.0546, found 377.0551 for [M + H] .
N-[4-(2-Hydroxy-4-methoxyphenyl)thiazol-2-yl]-4-methoxybenzenesulfonamide (5c): Yield 83%. Off-white
−
ν 3672.1, 2931.1, 1560.6, 1473.2, 1388.1, 1142.7, 817.4 cm . H-NMR (400 MHz, CDCl ):
3
1 1
solid. IR (film):
7.46–7.42 (m, 3H, H-6 + 2
s, 1H, SCH), 3.75 (s, 3H, OMe), 3.74 (s, 3H, OMe) ppm. C-NMR (100 MHz, CDCl3):
59.4, 147.1, 144.7, 130.5, 125.9, 121.0, 113.7, 113.2, 108.8, 106.2, 55.6 (OMe), 55.5 (OMe) ppm; HRMS
δ
×
ArH), 6.86 (d, J = 2.8 Hz, 1H, H-3), 6.76–6.72 (m, 3H, H-5 + 2
×
ArH), 6.57
13
(
δ 166.8, 163.9,
1
+
+
+
(ESI ): m/z [M] calc. for C H N O S 392.0495, found 393.0498 for [M + H] .
17 16 2 5 2
4
-Fluoro-N-[4-(2-hydroxy-4-methoxyphenyl)thiazol-2-yl]benzenesulfonamide (5d): Yield 81%. White solid.
−
ν 3695.5, 2943.3, 1589.7, 1493.9, 1378.3, 1157.7, 837.7 cm . H-NMR (400 MHz, CDCl ):
3
1
1
IR (film):
δ
7.51–7.48 (m, 2H, 2
×
ArH), 7.41 (d, J = 8.8 Hz, 1H, H-6), 6.95–6.91 (m, 2H, 2
×
ArH), 6.86 (d,
J = 2.6 Hz, 1H, H-3), 6.75 (dd, J = 8.8, 2.6 Hz, 1H, H-5), 6.46 (s, 1H, SCH), 3.74 (s, 3H, OMe) ppm.
13
C-NMR (100 MHz, CDCl3):
δ 167.3, 167.1, 164.5, 159.4, 146.8, 144.5, 131.2, 130.6, 120.9, 115.9, 115.6,

Molecules 2017, 22, 1605
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+
+
1
3
13.3, 108.9, 105.8, 55.5 (OMe) ppm; HRMS (ESI ): m/z [M] calc. for C H FN O S 380.0295, found
16 13 2 4 2
81.0302 for [M + H] .
+
4
-Chloro-N-[4-(2-hydroxy-4-methoxyphenyl)thiazol-2-yl]benzenesulfonamide (5e): Yield 81%. White solid.
−
ν 3708.8, 2796.7, 1566.74, 1459.55, 1314.15, 1256.54, 984.95 cm . H-NMR (400 MHz, CDCl ):
3
1 1
IR (film):
δ
7.53 (d, J = 8.8 Hz, 1H, H-6), 7.48–7.46 (m, 2H, 2
×
ArH), 7.29–7.27 (m, 2H, 2
×
ArH), 6.94 (d,
J = 2.4 Hz, 1H, H-3), 6.82 (dd, J = 8.8, 2.4 Hz, 1H, H-5), 6.56 (s, 1H, SCH), 3.81 (s, 3H, OMe) ppm.
13
C-NMR (100 MHz, CDCl3):
δ 167.2, 159.5, 146.8, 144.7, 140.7, 133.2, 130.7, 129.6, 128.7, 120.9, 113.5,
+
+
1
3
09.1, 106.0, 55.6 (OMe) ppm; HRMS (ESI ): m/z [M] calc. for C H ClN O S 395.9999, found
97.0076 for [M + H] .
16
13
2
4 2
+
4
-Bromo-N-[4-(2-hydroxy-4-methoxyphenyl)thiazol-2-yl]benzenesulfonamide (5f): Yield 83%. White solid.
−
ν 3691.2, 2838.7, 1591.3, 1479.1, 1362.1, 1143.7, 821.7 cm . H-NMR (400 MHz, CDCl ):
3
1
1
IR (film):
δ
7.36–7.33 (m, 3H, H-5 + 2
×
ArH), 7.28–7.26 (m, 2H, 2
×
ArH), 6.80 (d, J = 1.6 Hz, 1H, H-3), 6.71
13
(
dd, J = 8.8, 1.6 Hz, 1H, H-5), 6.35 (s, 1H, SCH), 3.69 (s, 3H, OMe) ppm. C-NMR (100 MHz, CDCl ):
3
+
+
δ
166.2, 159.6, 146.9, 145.1, 133.8, 121.0, 113.7, 109.1, 106.7, 55.7 (OMe) ppm; HRMS (ESI ): m/z [M]
+
calc. C H BrN O S 439.9495, found 440.9502 for [M + H] .
16
13
2
4 2
N-[4-(2-Hydroxy-4-methoxyphenyl)thiazol-2-yl]-4-nitrobenzenesulfonamide (5g): Yield 84%. Orange solid.
−
ν 3607.7, 2685.6, 1554.65, 1360.65, 1325.25, 1267.54, 964.95 cm . H-NMR (400 MHz, CDCl ):
3
1 1
IR (film):
δ
8.06 (d, J = 8.0 Hz, 2H, 2
.90 (s, 1H, H-3), 6.77 (d, J = 8.6 Hz, 1H, H-5), 6.32 (s, 1H, SCH), 3.76 (s, 3H, OMe) ppm. C-NMR
166.2, 159.9, 150.5, 146.6, 131.1, 129.7, 124.8, 114.0, 109.4, 106.7, 55.8 (OMe) ppm;
×
ArH), 7.59 (d, J = 8.0 Hz, 2H, 2
×
ArH), 7.29 (d, J = 8.6 Hz, 1H, H-6),
13
6
(
100 MHz, CDCl ):
δ
3
+
+
+
HRMS (ESI ): m/z [M] calc. for C H N O S 407.0240, found 408.0247 for [M + H] .
16
13
3
6 2
4
.7. Cell Culture
Human lung adenocarcinoma A549 (BCRC-60074) cell line was purchased from the Bioresource
Collection and Research Center (BCRC, Hsinchu, Taiwan). The cell line was initially established
through explant cultures of lung carcinomatous tissue from a 58-year-old Caucasian male. The A549
cells were cultured in 10% fetal bovine serum (HyClone, GE Healthcare Life Science, Logan, UT,
USA) and 90% Ham's F12K medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) containing
L-glutamine (2.0 mM) and sodium bicarbonate (1.5 g/L). Cells were cultured in a T-75 flask with a
◦
feeding cycle of 2 days. The humidified atmosphere with 5% CO kept at 37 C was provided in
2
cell incubator.
4
.8. Cytotoxicity of APDs
3
A549 cells were seeded into 96-well tissue culture plates at a concentration of 5
×
10 cells/well
for overnight growth to evaluate the cytotoxicity of APDs. Subsequently, A549 cells were treated with
serial concentrations of eleven APDs. After 24 h of incubation, cell viability was determined using
WST-1 assays (Abcam, Cambridge, MA, USA). Briefly, 10
and incubated for 2 h at 37 C prior to quantification. The absorbance at 450 nm was measured by a
µ
L of WST-1 reagent was added to each well
◦
VersaMax
™ ELISA Microplate Reader (Molecular Devices, Sunnyvale, CA, USA) using a reference
absorbance at 600 nm for each well.
4
.9. Preliminary Screening of APDs Against LPS-Activated A549 Cells
Preliminary screening procedure to evaluate a series of APDs against LPS-induced inflammation
was established with activated A549 cells. Before challenge with LPS, A549 cells were seeded
4
into 24-well plate (5
1.0
activated A549 cells. Forty-eight hours after challenge with LPS, the secreted MCP-1 and IL-6 in
×
10 cells/well) for overnight. Twenty-four hours after challenge with LPS
µg/mL), low and high doses of APDs, Dexa, and paeonol (1.0 and 10 µg/mL) were added to
(

Molecules 2017, 22, 1605
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the conditioned media from this system was measured by a commercial ELISA kit (R&D Systems,
Minneapolis, MN, USA) according to the manufacturer’s instructions.
4
.10. Animal Model of Acute Lung Injury
Adult male Sprague-Dawley rats weighing 250–300 g were obtained from the BioLASCO Breeding
◦
Center (Yi-Lan, Taiwan). Animals were housed in isolated ventilation cabinets (kept at 22–26 C with
30–70% humidity) with a 12 h light/dark cycle and free access to food and water. Animal experiments
were performed according to principles stated in the “Guide for the Care and Use of Laboratory
Animals” and were approved by the Institutional Animal Care and Use Committee (IACUC) of
the Agricultural Technology Research Institute (approved protocol number: 104033). Animals were
randomly assigned to ten groups. Each group comprised six rats, including a negative control
group (non-challenged; PBS group), a positive control group (8 mg LPS /kg; LPS group), and Dexa
(
1.0 and 5.0 mg/kg; Dexa (1) and Dexa (5) groups), paeonol (25 and 50 mg/kg; P (25) and P (50)
groups), compound (25 and 50 mg/kg; P-4 (25) and P-4 (50) groups) and compound 4.HCl (25 and
0 mg/kg; P-4.HCl (25) and P-4.HCl (50) groups) as treatment groups. Figure 4 shows experimental
4
5
design and groups. Animals were anesthetized using inhaled 2% isoflurane (Halocarbon Laboratories,
River Edge, NJ, USA) in 0.5 L/min of air before challenge. Intratracheal administration of LPS
®
(
0.5 mL/rat) was performed by inserting a MicroSprayer Aerosolizer (Model IA-1B, Penn-Century,
Wyndmoor, PA, USA) into the trachea. The microsprayer was subsequently removed, and animals were
then placed in the vertical position and gently rotated for 30 s to distribute the spray homogenously
throughout the lungs. The rats were treated with testing chemicals (APDs, Dexa and paeonol) through
intraperitoneal (i.p.) injection at one hour after the LPS challenge and sacrificed at 8 h after the LPS
challenge. The other two control groups (PBS and LPS group) were administered with vehicle (DMSO)
through i.p. injection.
4
.11. Total Cell Counts in BALF
Rats were euthanized at 8 h after the LPS challenge, and bronchoalveolar lavage fluid (BALF) was
collected as previously described [9,10]. Briefly, the right main bronchus was ligated, and a catheter
was inserted into the trachea. Double-lavage procedures were performed using 0.5 mL of normal
saline to pass through the catheter twice. The BALF samples were kept on ice prior to cell counting.
Total cell counts in the BALF were determined with a cell counter (Coulter Inc., Miami, FL, USA).
4
.12. Total Proteins, IL-6 and MCP-1 in BALF
Quantification of total proteins in the BALF samples was performed by a BCA protein assay
(
Thermo Scientific, Rockford, IL, USA) with bovine serum albumin solution as calibration standard.
For quantification of chemokines and cytokines in BALF, commercial ELISA kits for rat MCP-1 and
IL-6 were purchased from Bioscience Systems (San Diego, CA, USA).
4
.13. Histopathological Examination
The right lung tissues from 3 lobes of each rat were collected randomly and fixed with 10% neutral
buffered paraformaldehyde (PFA) for 18 h. Fixed tissues were embedded in paraffin and dissected
into 3- m thick sections. Hematoxylin and eosin (H&E) staining was used for histopathological
µ
examination. Protein exudation, infiltration of red blood cells, mononuclear, and neutrophilic cells
were examined by a pathologist and a medical doctor in pulmonary medicine using an Eclipse Ni-U
microscope (Nikon, Tokyo, Japan) at 100× magnification.
4
.14. Statistical Analysis
All data are presented as the mean
were plotted and performed by GraphPad Prism 5 (GraphPad Software Inc., La Jolla, CA, USA).
±
standard deviation (SD). Figures and statistical analysis

Molecules 2017, 22, 1605
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The Mann-Whitney test was used for comparisons between two groups, including differences between
particularly experimental group and positive control (LPS-activated) group. Statistical analysis was
performed using non-parametric statistics by IBM SPSS Statistics (Version 22.0, Armonk, NY, USA).
All comparisons were considered significantly different at p values less than 0.05.
5
. Conclusions
To evaluate a series of APDs for treating ALI, eleven APDs were prepared in this work
and evaluated using LPS-activated A549 cell model for preliminary screening integrated with
ALI-bearing rat model for verification. The best compound, 2-(2-aminothiazol-4-yl)-5-methoxyphenol
(4), can effectively alleviate ALI in rats through comprehensively inhibition of chemokines/cytokines,
attenuating neutrophil infiltration and eliminating protein exudation in alveolar space.
The achievement of this study gives us insight into developing plant-derived compounds to avoid
human from suffering lethal ALI and ARDS.
Supplementary Materials: The following are available online, Figure S1-S20: The H- and 13C-NMR Spectra of
1
compounds 2, 3, 4, 5a, 5b, 5c, 5d, 5e, 5f and 5g.
Acknowledgments: Authors are grateful to have financial supports from Taichung Veterans General Hospital
(
TCVGH-1034101B and TCVGH-1044101B for PKF), National Health Research Institutes (NHRI-BN-105-PP-27
for JKC), Agriculture Technology Research Institute (105AS-6.2.1-ST-al for CCY), and Ministry of Science and
Technology (MOST 103-2113-M-400-001 for JKC and MOST-104-2119-M-007-017 for MHH). Authors thank Miss
Ya-Ting Yang for technical support of intratracheal instillation and are grateful for animal care provided by Animal
Molecular Imaging Core Facility (Mr. Chien-Hong Chen and Wei-Neng Liao) in NHRI.
Author Contributions: P.-K.F., M.-H.H. and J.-K.C. conceived and designed the experiments; Y.-W.H., Y-.P.H.,
S.-C.H., H.C. and N.-C.H. performed the experiments; P.-K.F., J.-P.L. and J.-K.C. analyzed the data; C.-Y.Y., M.-H.H.
and J.-K.C. contributed reagents/materials/analysis tools; P.-K.F., K.-C.J., M.-H.H. and J.-K.C. prepared the
manuscript and J.-K.C. supervised whole research project.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
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