Optimization and anti-inflammatory evaluation of methyl gallate derivatives as a myeloid differentiation protein 2 inhibitor

Article abstract of DOI:10.1016/j.bmc.2019.115049

Myeloid differentiation protein 2 (MD2) is a co-receptor of toll-like receptor 4 (TLR4) responsible for the recognition of lipopolysaccharide (LPS) and mediates a series of TLR4-dependent inflammatory responses in inflammatory lung diseases including acute lung injury (ALI). Targeting MD2 thus may provide a therapeutic strategy against these lung diseases. In this study, we identified a novel compound 4k with the potent anti-inflammatory activity among 39 methyl gallate derivatives (MGDs). MGD 4k exhibited a high binding affinity to MD2, which in turn prevented the formation of the LPS/MD2/TLR4 complex. In addition, MGD 4k significantly reversed the upregulation of LPS-induced inflammatory mediators such as tumor necrosis factor-α, interleukin-6, intracellular adhesion molecule-1, vascular cell adhesion molecule-1, and monocyte chemoattractant protein-1 in vitro and in vivo. Mechanistically, MGD 4k performed anti-inflammatory function by inactivating JNK, ERK and p38 signaling pathways. Taken together, our study identified MGD 4k as a novel potential therapeutic agent for ALI through inhibiting MD2, inflammatory responses, and major inflammation-associated signaling pathways.

Full text of DOI:10.1016/j.bmc.2019.115049

Bioorganic&MedicinalChemistry27(2019)115049
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Optimization and anti-inflammatory evaluation of methyl gallate
derivatives as a myeloid differentiation protein 2 inhibitor
Yinda Qiua,b,1, Zhongxiang Xiaoc,1, Yanyan Wangd,1, Dingfang Zhanga, Wenxin Zhanga,
⁎
⁎
⁎
⁎
Guangbao Wanga, Wenbin Chena, Guang Lianga, , Xiaokun Lia, , Yali Zhanga, , Zhiguo Liua,
a Chemical Biology Research Center at School of Pharmaceutical Sciences, Wenzhou Medical University, 1210 University Town, Wenzhou, Zhejiang 325035, People’s
Republic of China
b College of Pharmacy, Chonnam National University, 300 Yongbong-Dong, Buk-Gu, Gwangju 500-757, Republic of Korea
c Department of Pharmacy, Affiliated Yueqing Hospital, Wenzhou Medical University, Wenzhou, Zhejiang 325035, People’s Republic of China
d College of Pharmacy, Heilongjiang University of Chinese Medicine, Haerbin, Heilongjiang 150040, People’s Republic of China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Myeloid differentiation protein 2 (MD2) is a co-receptor of toll-like receptor 4 (TLR4) responsible for the re-
cognition of lipopolysaccharide (LPS) and mediates a series of TLR4-dependent inflammatory responses in in-
flammatory lung diseases including acute lung injury (ALI). Targeting MD2 thus may provide a therapeutic
strategy against these lung diseases. In this study, we identified a novel compound 4k with the potent anti-
inflammatory activity among 39 methyl gallate derivatives (MGDs). MGD 4k exhibited a high binding affinity to
MD2, which in turn prevented the formation of the LPS/MD2/TLR4 complex. In addition, MGD 4k significantly
reversed the upregulation of LPS-induced inflammatory mediators such as tumor necrosis factor-α, interleukin-6,
intracellular adhesion molecule-1, vascular cell adhesion molecule-1, and monocyte chemoattractant protein-1
in vitro and in vivo. Mechanistically, MGD 4k performed anti-inflammatory function by inactivating JNK, ERK
and p38 signaling pathways. Taken together, our study identified MGD 4k as a novel potential therapeutic agent
for ALI through inhibiting MD2, inflammatory responses, and major inflammation-associated signaling path-
ways.
Methyl gallate derivative
Myeloid differentiation protein 2
Acute lung injury
Inflammation
1. Introduction
drugs or agents with powerful anti-inflammatory property for reducing
morbidity and mortality associated with this devastating disorder.
Acute lung injury (ALI) and its more severe form, acute respiratory
distress syndrome (ARDS), contribute to high rates (35–50%) of mor-
bidity and mortality in critically ill patients.1,2 According to the pub-
lished document, about 190,000 new cases are expected to be diag-
nosed with ALI/ARDS in the United States per year, with an estimated
mortality rate of 25–60%.3 ALI/ARDS has multiple etiologies, including
severe pneumonia, aspiration syndromes, sepsis, major trauma and
shock. They provoke uncontrolled and exaggerated pulmonary in-
flammatory responses, which are established as the center of the pa-
thology of ALI/ARDS.4,5 Growing evidence has shown that pulmonary
endothelium and alveolar epithelium are the major targets of ALI/
ARDS.6–8 Despite recent advances in clinical management of this dis-
ease and development of new therapeutic strategies, ALI/ARDS still
poses a serious threat to human health with the lack of effective ther-
apeutic options.9,10 Therefore, there is an urgent need to develop novel
Lipopolysaccharides (LPS), also known as lipoglycans and en-
dotoxins, are large-molecule ligands triggering acute inflammation that
leads to ALI.11 Toll-like receptor 4 (TLR4) is transmembrane receptor
for LPS involved in macrophage activation. However, TLR4 alone is not
sufficient for LPS signal transduction. The binding of LPS to TLR4 re-
quires an accessory protein known as myeloid differentiation 2 (MD2)
that facilitates the formation of LPS-TLR4 complex by interacting with
the extracellular domain of TLR4.12,13 With the mediation of MD2, a
receptor multimer consisting of a dimer of TLR4/MD2/LPS complex is
formed, which further stimulates downstream signaling pathways such
as mitogen-activated protein kinase (MAPK), and up-regulates the ex-
pression of a variety of inflammatory mediators.14 In addition, it has
been reported that TLR4 can be activated by endogenous ligands re-
leased during cellular injury (damage-associated molecular patterns,
DAMPs).15,16 Similar to LPS, DAMPs may not interact directly with
⁎ Corresponding authors.
E-mail addresses: wzmcliangguang@163.com (G. Liang), lixk1964@163.com (X. Li), lzgcnu@163.com (Z. Liu).
1 These authors contribute equally to this work.
https://doi.org/10.1016/j.bmc.2019.115049
Received 16 April 2019; Received in revised form 9 August 2019; Accepted 15 August 2019
Availableonline19August2019
0968-0896/©2019ElsevierLtd.Allrightsreserved.

Y. Qiu, et al.
Bioorganic&MedicinalChemistry27(2019)115049
O
O
O
HO
OH
HO
HO
O
MeO
OMe
Curcumin
Caffeic acid phenethyl ester (CAPE)
O
O
OH
O
OH
O
HO
HO
OMe
HO
HO
MeO
OH
OH
Methyl gallate
Fig. 1. Chemical structures of currently known MD2 inhibitors and methyl gallate (MG).
JSH
Xanthohumol
TLR4, but may bind to accessory factors as part of a larger TLR4
complex.17 It is well established that MD2 plays an essential role in the
pathogenesis of ALI.18,19 MD2-deficient mice are capable of surviving
endotoxic shock due to no response to LPS.20 In view of its key role in
mediating the interaction between TLR4 and LPS, MD2 has been
identified as a potential therapeutic target for acute inflammatory dis-
eases.21,22
amine 6 and various carboxylic acids 5a-r and 8a-g. All the new pro-
ducts were isolated in satisfactory yields by conventional extractive
work-up. Analytical and spectral data of all synthesized compounds
were fully consistent with the proposed structures.
3. Results and discussion
Natural products and their derivatives are the rich sources for the
discovery and development of new drugs. Recently, several small nat-
ural monophenols or polyphenols have been successfully extracted,
such as curcumin,23 caffeic acid phenethyl ester (CAPE),24 2′,4-dihy-
droxy-6′-isopentyloxychalcone (JSH).25 and xanthohumol.26 (Fig. 1).
They have been identified as new types of MD2 inhibitors with the anti-
inflammatory function. However, These MD2 inhibitors have proven to
be less clinically useful due to unfavorable pharmacokinetics and re-
latively low in vivo activity,27 and thus it is of great interest to scientists
or clinicians in the exploration of new lead compounds that can be
optimally developed as MD2 inhibitors.
3.1. Inhibitory effect of the MGD 4k on LPS-induced tumor necrosis factor-α
(TNF-a) and interleukin-6 (IL-6) release
TNF-α and IL-6 are key pro-inflammatory cytokines closely asso-
ciated with the initiation and amplification of LPS-induced in-
flammatory response.31 To determine whether synthetic MGDs have a
protective effect on LPS-induced ALI, we examined the release of TNF-α
and IL-6 from LPS-treated macrophages which majorly contributing to
pathogenesis of ALI. As shown in Fig. 2A and 2B, LPS dramatically
induced release of TNF-α and IL-6, and MGD 4k had a significant in-
hibitory effect on LPS-induced TNF-α and IL-6 release (the inhibition
rates were 72.2% and 89.0%, respectively), compared to the other
MGDs.
Methyl gallate (MG), the methyl ester of gallic acid, has been found
in various types of plants and natural products (Fig. 1), and may exhibit
remarkable biological effects such as antioxidant,28 antitumor,29 anti-
microbial and antiinflammatory activities.30 Although MG has been
shown to play a beneficial role in a wide range of inflammatory dis-
eases, function of MG in ALI has not been investigated yet. Thus, herein
we synthesized a series of novel MG derivatives (MGDs), and assessed
their anti-inflammatory activity. Among them, an active compound 4k
was identified as a potential novel MGD with high anti-inflammatory
effects both in vivo and in vitro.
To further improve the anti-inflammatory activity of 4k, we mod-
ified the benzene ring of 4k by introducing different types of groups for
optimizing the function. Fig. 3 indicates that, among these 25 MGDs, 7f,
7l and 7r exhibited > 50% inhibition efficiency in LPS-stimulated TNF-
α and IL-6 release, whereas the other MGDs had moderate inhibition of
TNF-α and IL-6 release, compared to the LPS control. We found that all
of these modified MGDs did not display anti-inflammatory activity
stronger than that of lead compound 4k.
2. Chemistry
3.2. Dose-dependent effect of active MGDs on LPS-induced inflammatory
cytokine release
The synthetic route for the preparation of MGDs (4a-n, 7a-r and 9a-
g) used in this study is indicated in Schemes 1, and their chemical
structures are indicated in Tables 1 and 2. Benzyl protection of com-
mercially available gallic acid 1 was achieved using benzyl bromide in
the presence of potassium carbonate (K2CO3) to yield tri-benzyl gallic
acid 2. Subsequently, a condensation reaction between 2 and equimolar
amounts of substituted anilines or benzylamines was carried out to
yield the corresponding amides 3a-n (Table S1) by using 1-[bis(di-
To further evaluate the inhibitory efficiency of active MGDs in LPS-
induced inflammatory cytokines release, we investigated dose–response
effects of stable MGDs 7f, 7l, 7r and 4k (Fig. S1). As shown in Fig. 4A
and 4B, MGDs 7f, 7l, 7r and 4k suppressed LPS-induced TNF-α and IL-6
secretion in a generally dose-dependent manner. Consistently, MGD 4k
exhibited a noticeable dose–response effect, compared to MGDs 7f, 7l,
and 7r. These results further demonstrated that, among synthetic
MGDs, MGD 4k may be an ideal candidate agent against LPS-induced
inflammation. Importantly, MGD 4k had no marked association with
cytotoxicity in other cell types like MEM and BEAS-2B cells even at a
concentration as high as 50 µM (Fig. 4C), suggesting a possibility of
safe in vivo application of MGD 4k.
methylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium
3-oxid
hexafluorophosphate (HATU) as a coupling reagent. Finally, deprotec-
tion of the O-benzyl groups of 3a-n was achieved to produce the
compounds 4a-n in a tetrahydrofuran-methanol mixture using Pt/C
catalyst and hydrogen gas. Following the procedure used for the pre-
paration of compounds 3a-n, the desired amines 7a-r and 9a-g were
furnished in satisfactory yields via coupling of 2,3-dihydro-1H-inden-5-
2

Y. Qiu, et al.
Bioorganic&MedicinalChemistry27(2019)115049
O
O
n
n
HO
HO
COOH
BnO
BnO
COOH
BnO
BnO
HO
N
H
N
H
R1
a
b
c
R1
HO
OH
BnO
BnO
OH
1
3a-n
4a-n
(80.2-88.5)
(33.0-49.3%)
2
(80.5%)
(n= 0 or 1)
(n= 0 or 1)
H
N
n
COOH
n
d
R2
R2
Ar
Ar
O
H2N
5a-r
(n= 0, 1, 3)
6
7a-r
(49.7-85.9%)
(n= 0, 1, 3)
O
d
CHOOH2
R3
N
H
R3
H2N
8a-g
6
9a-g
(51.6-68.6%)
Scheme 1. General synthetic routes to methyl gallate derivatives (MGDs) 4a-n, 7a-r and 9a-g. Reagents and conditions: (a) 1) BnBr, K2CO3, DMF, rt, 12 h; 2) 5 M-
NaOH, EtOH, then conHCl, 30 min, rt; (b) Various substituted amines, HATU, DIPEA, rt, 10 h; (c) 10%Pd/C, H2, MeOH-THF(1:1), rt, 5 h; (d) HATU, DIPEA, rt, 10 h.
Table 1
Table 2
Chemical structures of synthetic compounds 4a-n.
Chemical structures of synthetic compounds 7a-r and 9a-g.
O
H
n
2
n
R1
N
HO
A)
B)
O
R2
N
1
H
O
R3
N
HO
6
OH
H
4a-n
7a-r
9a-g
Comp.
R1
n
Comp.
R1
n
Comp. Series
R2
n
Comp.
SeriesR2 or R3
n
4a
4b
4c
4d
4e
4f
Ph
0
0
0
0
1
0
0
4h
4i
4-(1-Acetylindoline)
4-(1-ethyl-4-methylpiperazine)Ph
4-(4-methylpiperazine)Ph
5-benzocyclopentane
Ph
0
0
0
0
1
1
0
7a
7b
7c
7d
7e
7f
A
A
A
A
A
A
A
A
A
A
A
A
A
(4-CI, 3-NO2)Ph
(2-CF3)Ph
0
0
1
1
0
1
0
0
3
0
0
0
0
7n
7o
7p
7q
7r
A
A
A
A
A
B
B
B
B
B
B
B
6-(2-Me)thiophene
0
0
0
0
0
(4-OEt)Ph
(3,5-OMe)Ph
(4-OMe)Ph
(3-OMe)Ph
(4-F)Ph
5-(4-Me)thiazole
(3-pyrrolidine)Ph
(3-N(Me)2)Ph
(2-N(Me)2)Ph
(3,4,5-OMe)Ph
(3,4-OMe)Ph
4j
(2,4-NO2)Ph
(3,5-OMe)Ph
(4-NH(COCH3))Ph
1-naphthalene
4-(3,5-CI)pyridine
2-indole
4k
41
4m
4n
(3,4-OMe)Ph
9a
9b
9c
9d
9e
9f
4g
4-Indole
(4-morpholine)Ph
7g
7h
7i
(2-CF3)Ph
3-indole
(3-CF3)Ph
3.3. Suppressive effect of MGD 4k on mRNA expression of inflammatory
7j
(4-N(Et)2)Ph
(1,6-C1)Ph
(3-OH, 4-OMe)Ph
(4-OH, 3,5-OMe)Ph
(2,5-OMe)Ph
mediators in LPS-treated macrophages
7k
71
7m
2-(6-OMe)indole
(3-morpholine)Ph
9g
We next sought to investigate whether MGD 4k inhibits LPS-in-
duced inflammatory response by downregulating the expression of in-
flammatory mediators such as TNF-α, IL-6, vascular cell adhesion mo-
lecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), and
monocyte chemoattractant protein 1 (MCP-1) in LPS-treated mouse
primary macrophages (MPMs). As shown in Fig. 5, MGD 4k treatment
markedly inhibited LPS-induced mRNA expression of these in-
flammatory mediators in a dose-dependent manner, suggesting MGD 4k
may serve as a transcriptional inhibitor of inflammatory mediators in
LPS-induced inflammation.
plasmon resonance (SPR) measurement, a powerful technique for
monitoring the affinity and selectivity of biomolecular interactions
between small molecules,32 we determine the kinetic constants of MGD
4k binding to MD2 protein in a BioRad ProteON XPR36 interaction
array system. As shown in Fig. 6A, MGD 4k exhibited remarkable dose-
dependent binding to MD2 with the KD value of 29.4 μM, an indicator of
the high affinity binding. For comparison, inactive MGDs 4g and 4m
displayed low binding affinities with KD values of 2700 and 140 μM,
respectively (Fig. S2).
3.4. Kinetic analysis of direct binding interaction between MGD 4k and
MD2
3.5. MGD 4k interrupts LPS-induced MD2/TLR4 complex formation
Since MGD 4k, as an MD2 inhibitor, had potent anti-inflammatory
activity as described above, we next sough to determine whether MGD
4k functions via direct interaction with MD2. By performing surface
The interaction of MD2/LPS with TLR4 induces receptor oligomer-
ization and triggers the inflammatory response.14 To test whether MGD
4k interferes with MD2/TLR4 complex formation, we assessed the
3

Y. Qiu, et al.
Bioorganic&MedicinalChemistry27(2019)115049
Collectively, these results suggest that MGD 4k may play a protective
role against LPS-induced ALI through suppressing MD2/TLR4 interac-
tion.
A)
TNF-α
200
150
100
50
3.6. Molecular docking
To investigate structural mechanism underlying MGD 4k binding to
MD2, we docked MGD 4k molecule into the crystal structure of MD2
using the docking program. As shown in Fig. 6D, the whole MGD 4k
molecule lodges inside the hydrophobic pocket of MD2 molecule. We
found two phenolic hydroxyl groups and carbonyl group of MGD 4k
molecule may form hydrogen bonding with Ser-120 and Tyr-102 of the
MD2 domain, respectively. Furthermore, the benzocyclopentane motif
of MDG 4k forms suitable van der Waals interactions in the MD2
pocket, thereby interacting with interior hydrophobic residues, such as
Phe-104, Ile-63, Ile-94, Leu-61 and Phe-76, explaining why even a
slight change such as compounds 4j and 4n could not be tolerated at
this position. Changing the 3,4,5-trihydroxy-phenyl template of 4k to
either the substituted phenyl rings (7a-f, 7j, 7k, 7m, 7p-r, 8a-g) or the
heterocycles (7g-I, 7l, 7n-o) resulted in a drop in potency. The decrease
in activity is likely due to the reduced number of hydrogen bonds.
***
0
con LPS MG 4a 4b 4c 4d 4e 4f 4g 4h 4i 4j 4k 4l 4m 4n
LPS 0.5 µg/mL
B)
IL-6
200
150
100
50
*
***
***
0
con LPS MG 4a 4b 4c 4d 4e 4f
4g 4h
4i
4j
4k
4l 4m 4n
LPS 0.5 µg/mL
3.7. Inhibitory effects of MGD 4k on p38 MAPK signaling activity
Fig. 2. MG and MGDs 4a-n inhibited LPS-induced TNF-α (A) and IL-6 (B)
production in MPMs. The macrophages were pretreated with 10 μM synthetic
MGDs as indicated for 30 min, and then treated with LPS for 24 h. The levels of
TNF-α and IL-6 in the culture medium were determined using enzyme-linked
immunosorbent assay (ELISA) by normalization to the total protein con-
centration in cell lysates. *p < 0.05, ***p < 0.001 vs. LPS groups (n = 3).
To further investigate the molecular mechanisms underlying anti-
inflammatory properties of MGD 4k, we examined whether MGD 4k is
involved in inactivation of pro-inflammatory signaling pathways such
as MAPK pathway in MPMs. As shown in Fig. 7A–C, LPS dramatically
activated JNK, ERK, and P38 signaling as evidenced by LPS-induced
increase in phosphorylation of JNK, ERK, and P38 proteins. However,
MGD 4k treatment effectively reversed the effect of LPS, suggesting that
MGD 4k inhibits LPS-triggered inflammatory responses at least partially
through blocking MAPK signaling pathway.
effect of MGD 4k on LPS-induced MD2/TLR4 complex by performing
immunoprecipitation. As shown in Fig. 6B, in response to LPS alone,
the coprecipitation of MD2/TLR4 was markedly increased compared to
control (CON) group. MGD 4k pretreatment significantly disrupted
MD2/TLR4 complex formation in a dose-dependent manner. The results
were further confirmed by the in vivo coimmunoprecipitation data that
MGD 4k effectively interfered with LPS-induced MD2/TLR4 binding
without affecting MD2 expression in mouse lung tissues (Fig. 6C).
3.8. Protective effect of MGD 4k on LPS-induced ALI in mice
Since MGD 4k played an anti-inflammatory role in vitro as described
above, we determine the in vivo role of MGD 4k in a mouse model of
Fig. 3. MGD 4 k and MGDs 7a-r and 9a-g inhibited
LPS-induced TNF-α (A) and IL-6 (B) production in
MPMs. The macrophages were pretreated with 10 μM
MGDs as indicated for 30 min, and then treated with
LPS for 24 h. The levels of TNF-α and IL-6 in the
TNF-α
A)
150
100
50
**
culture medium were determined using ELISA by
*
***
normalization to the total protein concentration in
**
**
*
*
cell lysates. *p < 0.05, ***p < 0.001 vs. LPS
*
**
***
groups (n = 3).
**
***
0
CONLPS 7a 7b 7c 7d 7e 7f 7g 7h 7i 7j 7k 7l 7m 7n 7o 7p 7q 7r 9a 9b 9c 9d 9e 9f 9g 4k
LPS 0.5 µg/mL
IL-6
B)
150
100
50
*
**
**
**
**
***
***
***
0
CONLPS 7a 7b 7c 7d 7e 7f 7g 7h 7i 7j 7k 7l 7m 7n 7o 7p 7q 7r 9a 9b 9c 9d 9e 9f 9g 4k
LPS 0.5 µg/mL
4

Y. Qiu, et al.
Bioorganic&MedicinalChemistry27(2019)115049
TNF-α
A)
150
100
50
**
1
*
*
*
**
**
*
*
***
***
***
0
(mg/mL)
10
CON LPS
1
2.5
5
10
1
2.5
5
10
2.5
5
10
1
2.5
5
7f
7l
7r
4k
LPS 0.5 µg/mL
B)
IL-6
150
100
50
**
*
*
**
**
***
5
**
**
***
1
***
***
5
4k
***
0
(mg/mL)
10
CON LPS
1
2.5
5
10
1
2.5
7l
5
10
1
2.5
10
2.5
7f
7r
LPS 0.5 µg/mL
MEM
BEAS-2B
150
100
50
C)
150
100
50
*
*
0
0
(mg/mL)
50
DMSO
1
2.5
5
10
20
40
(mg/mL)
50
DMSO
1
2.5
5
10
20
40
4k
4k
Fig. 4. MGDs 7f, 7 l, 7r and 4 k dose-dependently inhibited LPS-induced TNF-α (A) and IL-6 (B) releases, and 4 k had no cytotoxic effect on MEM and BEAS-2B cells
(C). MPMs, MEM, and BEAS-2B cells were pretreated with MGDs 7f, 7 l, 7r and/or 4 k at different concentrations as indicated for 30 min and then incubated with or
without LPS (0.5 μg/mL) for 24 h. The levels of TNF-α and IL-6 in the culture medium were determined using ELISA by normalization to the total protein con-
centration in cell lysates. *p < 0.05; **p < 0.01, ***p < 0.001 vs. LPS groups (A and B), or DMSO group (C) (n = 3).
LPS-induced ALI, which closely mimics human ALI.33,34 As shown in
Fig. 8A and 8B, LPS induced an increase in total number of cells and
protein concentration in the bronchoalveolar lavage fluid (BALF), and
high dose MGD 4k significantly reversed the LPS effects, suggesting that
MGD 4k inhibits LPS-induced accumulation of inflammatory cells in
mouse lung tissues. Consistently, high dose MGD 4k decreased lung
Wet/Dry (W/D) ratio in response to LPS challenge (Fig. 8C), suggesting
that MGD 4k may suppress LPS-induced lung edema. Furthermore,
MGD 4k inhibited LPS-induced inflammatory response in a dose-de-
pendent manner, as evidenced by the reduced levels of inflammatory
cytokines TNF-α and IL-6 in serum and BALF of MGD 4k administered
ALI mice (Fig. 8D–G). MGD 4k administration also reduced macro-
phage infiltration and improved lung morphology in ALI mice, as evi-
denced by the diminished CD68 (macrophage marker) staining in-
tensity and restoration of normal alveolar structure in response to MGD
4k, respectively (Fig. 8H and I). These results demonstrate that MGD
4k, especially at a high dose, protects against LPS-induced ALI in mice
through inhibiting inflammatory response.
examined the expression of various inflammation associated genes in
lung tissues of LPS-induced ALI mice. As shown in Fig. 9A–E, MGD 4k
caused significant inhibition of pulmonary mRNA expression of these
genes like TNF-α, IL-6, ICAM-1, VCAM-1, and MCP-1 in LPS-induced
ALI mice in a dose-dependent manner. These data confirm the anti-
inflammatory profile of MGD 4k as described above.
4. Conclusion
In conclusion, total 39 MGDs were designed, synthesized and
evaluated as anti-inflammatory agents for the treatment of ALI. Among
them, MGD 4k is the strongest inflammation inhibitor against key in-
flammatory mediators TNF-α and IL-6 release from macrophages (IC50
:
1.23 μM for TNF-α and 0.702 μM for IL-6). Consistent with the in vitro
data, MGD 4k, at a relative high dose of 20 mg/kg, can effectively re-
duce BALF and serum levels of TNF-α and IL-6 in LPS-induced ALI mice
as well as mRNA expression of other inflammation-associated genes
including ICAM-1, VCAM-1, and MCP-1. In lung histology, high dose
MGD 4k markedly inhibited inflammation cell accumulation and in-
filtration, attenuated lung edema, and improved alveolar morphology
in ALI mice.
3.9. Negative effects of MGD 4k on pulmonary mRNA expression of pro-
inflammatory cytokines in LPS-induced ALI mice
Mechanistically, MGD 4k is a potent inhibitor of MD2 through
physical interaction between each other, which has been validated in
To further confirm the role of MGD 4k in anti-inflammation, we
5

Y. Qiu, et al.
Bioorganic&MedicinalChemistry27(2019)115049
Fig. 5. MGD 4 k inhibited LPS-induced mRNA expression of inflammatory mediators. The macrophages were pretreated with MGD 4k at different concentrations as
indicated for 30 min, and then treated with LPS for 6 h. The relative cellular mRNA levels of inflammatory mediators TNF-α (A), IL-6 (B), VCAM-1(C), ICAM-1 (D)
and MCP-1 (E) were determined using qRT-PCR by normalization to internal control β-actin mRNA level. *p < 0.05, **p < 0.01, ***p < 0.001 vs. LPS groups
(n = 3).
this study (Fig. 6). Molecular modeling gives the more detailed in-
formation on the binding of these two molecules. To more clearly
elucidate anti-inflammatory performance of MGD 4k, a schematic
diagram was shown in Fig. 10: Upon LPS stimulation, TLR4-MD2
complex is dimerized and then is activated to transmit LPS signal. In the
presence of MGD 4k, dimerization of TLR4-MD2 complex is disrupted
due to MGD 4k binding to MD2, leading to the blockade of LPS sig-
naling. Thus, activity of MAPK signaling pathways is suppressed, as
evidenced by the decreased phosphorylation of ERK, JNK, and p38 by
MGD 4k. Inactivation of MAPKs in turn represses the transcription of
inflammation-associated genes such as TNF-α and IL-6, resulting in the
inhibited inflammatory response to LPS and subsequent amelioration of
ALI.
determined on a Fisher-Johns melting apparatus and were uncorrected.
The 1H NMR spectra at 500 MHz was performed on a Bruker Avance
DRX 500 instrument (1H, 500 MHz, 13C, 125.7 MHz). The chemical
shifts were presented in terms of parts per million with TMS as the
internal reference. Analytical LC/MS was recorded on a Bruker Esquire
3000 t Spectrometer.
5.1.2. Synthesis of 3,4,5-tris(benzyloxy)benzoic acid (2)
Gallic acid (0.5 g, 2.94 mmol) and K2CO3 (3.66 g, 26.45 mmol) in
DMF (20 mL) was stirred at room temperature for 1 h. BnBr (4.19 mL,
35.27 mmol) was added dropwise into the mixture in 30 min at 40 °C
under nitrogen. The reaction mixture was stirred at 40 °C for 12 h. The
resulting mixture was diluted by H2O (20 mL), and extracted by EtOAc
(3 × 20 mL). The combined organic layers were washed with brine and
dried over anhydrous MgSO4, filtered, and concentrated under reduced
pressure. The residue was dissolved in aqueous EtOH (40 mL) con-
taining 5 M NaOH and and then refluxed for 12 h. The mixture was
diluted with H2O (40 mL), adjusted to PH = 2 with concentrated HCl
and stirred for 30 min at room temperature. The precipitate was fil-
trated, dried to give compound 2 as a white solid in an 80.5% yield.
5. Experimental section
5.1. Chemistry
5.1.1. General
All commercial solvents and reagents were used as received unless
otherwise noted. All chemicals and dry solvents were purchased from
Alfa Aesar or Sigma Aldrich and used as delivered. Thin layer chro-
matography was carried out on Kiesel-gel 60 F254 glass plates. Flash
chromatography was performed using Merck silica gel 60 (200–300
mesh ASTM) (Merck KGaA, Darmstadt, Germany). Melting points were
5.1.3. General procedure for synthesis of amides 3a-n
To a solution of 2 (0.3 g, 0.68 mmol), HATU (0.26 g, 0.68 mmol)
and DIPEA (0.24 mL, 1.36 mmol) in dry CH2Cl2 (10 mL) were stirred for
20 min at room temperature, the substituted anilines or benzylamines
6

Y. Qiu, et al.
Bioorganic&MedicinalChemistry27(2019)115049
Fig. 6. The effects of MGD 4 k on LPS binding to MD2 and LPS-induced formation of TLR4-MD2 complex. (A) SPR analysis of binding affinity of MGD 4 k to MD2
protein. (B) Effects of MGD 4 k on the in vitro binding of biotin-conjugated LPS (biotin-LPS) to rhMD2. Biotin-LPS (1.5 μg/mL) was added to rhMD2 (4 μg/mL)-coated
plate without or with MGD 4k for detection of LPS-MD2 binding. (C) Co-immunoprecipitation assay for TLR4-MD2 interaction using anti-MD2 antibody. Cell lysates
were prepared from macrophages pretreated with 10 μM 4k, followed by stimulation with LPS (0.5 μg/mL) for 10 min. Shown is representative Western blot
detecting interaction between MD2 and TLR4; IP: immunoprecipitated protein, IB: immunoblotted protein. (D) Putative binding mode of MGD 4 k within the active
pocket of the MD2 (PDB code: 2E59). PyMOL showed that MGD 4 k interacted with MD2 via hydrogen bond and hydrophobic interactions with Ser-120, Tyr-102, Ile-
117, Ile-63, Ile-94, Leu-61, Ser-118 and Phe-76 of MD2 molecule.
(0.68 mmol) was added slowly. The reaction mixture was stirred for 5 h
at room temperature. The resulting mixture was diluted by H2O
(10 mL), and extracted by EtOAc (3 × 10 mL). The combined organic
layers were washed with brine and dried over anhydrous MgSO4, fil-
tered, and concentrated under reduced pressure. The residue was
recrystallized from the solution of MeOH-H2O (1:1) to give the amides
3a-n in good yields of 80.2–88.5%.
5.1.4. General procedure for synthesis of MGDs 4a-n
To a solution of 3a-n (0.58 mmol) in MeOH/THF (12 mL, 1:1 v/v)
_
_
_
_
_
_
_
_
_
LPS
(0.5 µg/ml)
LPS
(0.5 µg/ml)
LPS
(0.5 µg/ml)
A)
B)
C)
+
_
+
_
+
+
+
_
+
4k (µM)
10.0 10.0
4k (µM)
10.0
10.0
4k (µM)
P-JNK
JNK
10.0
10.0
P-ERK
ERK
P-P38
P38
150
100
50
150
100
50
150
100
50
0
*
**
**
0
0
CON
LPS
4k
4k+lps
CON
LPS
4k
4k+lps
CON
LPS
4k
4k+lps
Fig. 7. MGD 4 k inhibited LPS-induced activation of MAPKs signaling pathway. MPMs were pretreated with vehicle or MDG 4 k (10 μM) for 30 min followed by
incubation with LPS (0.5 μg/mL) for 20 min. The protein expression of p-JNK, JNK (A), p-p38, p38 (B), and p-ERK, ERK (C) were detected by western blot, and
quantified using densitometry. *p < 0.05, **p < 0.01 vs. LPS groups (n = 3).
7

Y. Qiu, et al.
Bioorganic&MedicinalChemistry27(2019)115049
A)
B)
C)
D)
6
6
4
2
0
1.0
0.8
0.6
0.4
0.2
0.0
150
TNF-a
4
2
0
100
***
*
*
**
50
0
*
CON
LPS
10
20
CON
LPS
10
20
CON
LPS
10
20
CON
LPS
10
20
LPS+4k (mg/kg)
LPS+4k (mg/kg)
LPS+4k (mg/kg)
LPS+4k (mg/kg)
40
E)
F)
G)
IL-6
800
TNF-a
400
IL-6
30
20
10
0
600
400
200
0
300
200
100
0
*
***
20
***
20
**
CON
LPS
10
CON
LPS
10
CON
LPS
10
20
LPS+4k (mg/kg)
LPS+4k (mg/kg)
LPS+4k (mg/kg)
CON
LPS+4k (20 mg/kg)
LPS
LPS+4k (10 mg/kg)
H)
100µm
100µm
100µm
100µm
100µm
I)
100µm
100µm
100µm
Fig. 8. MGD 4 k attenuated LPS-induced ALI in mice. Mice were intravenously injected with MGD 4k at the doses as indicated, and then challenged with LPS, 6 h
later, mice were sacrificed. BALF and lung tissues were harvested to determine (A) Total number of cells in BALF. (B) Protein concentration in BALF. (C) Wet/Dry
ratio. (D and E) Serum Levels of cytokines TNF-α and IL-6. (F and G) BALF Levels of TNF-α and IL-6. (H) Immunohistochemical staining of CD68. (I) H&E staining.
*p < 0.05, **p < 0.01, ***p < 0.001 vs. LPS groups (n = 3).
was added palladium on carbon (10 wt% Pd/C, 30 mg) slowly. The
suspension was stirred at room temperature under H2 atmosphere
overnight. The resulting mixture was filtered by silica gel, and the fil-
trate was concentrate under reduced pressure, the residue was re-
crystallized from CH2Cl2 to afford corresponding target compounds 4a-
n with the yields of 33.0–49.3%.
calcd for C15H15NO5: 289.29.
5.1.4.3. N-(3,5-Dimethoxyphenyl)-3,4,5-trihydroxybenzamide (4c). Gray
powder, 49.3% yield. m.p: 129.7–131.8 °C. 1H NMR (500 MHz,
DMSO‑d6) δ (ppm): 9.81 (1H, s, Ar-NH), 9.19 (3H, s, Ar-OH), 7.07
(2H, d, ArH2), 6.93 (2H, s, ArH2), 6.21 (1H, t, J = 2.0 Hz, ArH), 3.71
(6H, s, Ar-OCH3). 13C NMR (125 MHz, DMSO‑d6) δ (ppm): 165.56,
160.25 × 2, 145.44 × 2, 141.25, 136.78, 124.92, 107.13 × 2,
98.20 × 2, 95.15, 55.00 × 2. ESI-MS m/z: 306.05 (M+H)+, calcd for
C15H15NO6: 305.29
5.1.4.1. 3,4,5-Ttrihydroxy-N-phenylbenzamide (4a). White powder,
48.8% yield. m.p: 203.6–205.0 °C. 1H NMR (500 MHz, DMSO‑d6) δ
(ppm): 9.90 (1H, s, Ar-NH), 9.08 (3H, s, Ar-OH), 7.74 (2H, d,
J = 7.86 Hz, ArH2), 7.31 (2H, t, J = 7.8 Hz, ArH2), 7.05 (1H, t,
J = 7.4 Hz, ArH), 6.95 (2H, s, ArH2). 13C NMR (125 MHz, DMSO‑d6)
δ (ppm): 165.54, 145.44 × 2, 139.55, 136.71, 128.43 × 2, 124.99,
123.06, 120.09 × 2, 107.15 × 2. ESI-MS m/z: 246.09 (M+H)+, calcd
for C13H11NO4: 245.23
5.1.4.4. 3,4,5-Trihydroxy-N-(4-methoxyphenyl)benzamide
(4d). Gray
powder, 46.5% yield. m.p: 193.8–196.3 °C. 1H NMR (500 MHz,
DMSO‑d6) δ (ppm): 9.78 (1H, s, Ar-NH), 9.05 (2H, s, Ar-OH), 7.63
(2H, d, J = 9.0 Hz, ArH2), 6.93 (2H, s, ArH2), 6.88 (2H, d, J = 9.0 Hz,
ArH2), 3.72 (3H, s, Ar-OCH3). 13C NMR (125 MHz, DMSO‑d6) δ (ppm):
165.14, 155.12, 145.44 × 2, 136.55, 132.65, 125.08, 121.66 × 2,
113.56 × 2, 107.03 × 2, 55.10. ESI-MS m/z: 276.04 (M+H)+, calcd
for C14H13NO5: 275.26.
5.1.4.2. N-(4-Ethoxyphenyl)-3,4,5-trihydroxybenzamide
(4b). White
powder, 44.7% yield. m.p: 191.2–193.1 °C. 1H NMR (500 MHz,
DMSO‑d6) δ (ppm): 9.76 (1H, s, Ar-NH), 9.13 (2H, s, Ar-OH), 8.79
(1H, s, Ar-OH), 7.61 (2H, d, J = 8.4 Hz, ArH2), 6.92 (2H, s, ArH2), 6.86
(2H, d, J = 8.4 Hz, ArH2), 3.98–3.95 (2H, m, Ar-OCH2-), 1.31 (3H, t,
J = 6.6 Hz, Ar-OCH2-CH3). 13C NMR (125 MHz, DMSO‑d6) δ (ppm):
165.12, 154.37, 145.41 × 2, 136.48, 132.54, 125.14, 121.64 × 2,
5.1.4.5. 3,4,5-Trihydroxy-N-(2-methoxybenzyl)benzamide (4e). White
powder, 38.7% yield. m.p: 129.3–131.8 °C. 1H NMR (500 MHz,
DMSO‑d6) δ (ppm): 6.32 (2H, d, J = 7.6 Hz, ArH2), 6.10 (1H, d,
J = 7.9 Hz, ArH), 6.00 (1H, d, J = 7.3 Hz, ArH), 5.97 (2H, t, ArH2),
114.08 × 2, 107.02 × 2, 62.99, 14.67. ESI-MS m/z: 290.10 (M+H)+
,
8

Y. Qiu, et al.
Bioorganic&MedicinalChemistry27(2019)115049
A)
B)
TNF-a
150
100
50
150
100
50
IL-6
*
**
**
0
0
CON LPS 10
20
CON LPS 10
20
LPS+4k (mg/kg)
LPS+4k (mg/kg)
C)
D)
E)
VCAM-1
*
ICAM-1
***
MCP-1
***
150
100
50
150
150
100
50
100
50
0
***
20
***
20
***
20
0
0
CON LPS 10
CON LPS 10
CON LPS 10
LPS+4k (mg/kg)
LPS+4k(mg/kg)
LPS+4k (mg/kg)
Fig. 9. MGD 4 k inhibited inflammation-associated gene expression in lung tissues of LPS-induced ALI mice. Mice were intravenous injection with MGD 4k at the
doses as indicated, and then challenged with LPS. 6 h later, mice were sacrificed, and lung tissues were harvested for RNA extraction. qRT-PCR was performed to
determine the expression of TNF-α (A), IL-6 (B), ICAM-1 (C), VCAM-1 (D) and MCP-1 (E). *p < 0.05, **p < 0.01, ***p < 0.001 vs. LPS groups (n = 3).
Fig. 10. Proposed mechanism of MGD 4 k-mediated anti-inflammation.
3.61 (2H, s, Ar-CH2), 2.96 (3H, s, Ar-OCH3). 13C NMR (125 MHz,
DMSO‑d6) δ (ppm): 167.78, 156.00, 143.98 × 2, 135.38, 126.71,
126.42, 125.11, 123.57, 118.70, 108.66, 105.11 × 2, 53.15, 37.21.
ESI-MS m/z: 290.04 (M+H)+, calcd for C15H15NO5: 289.29.
ESI-MS m/z: 264.06 (M+H)+, calcd for C13H10FNO4: 263.22.
5.1.4.7. 3,4,5-Trihydroxy-N-(1H-indol-6-yl)benzamide
(4g). Gray
powder, 41.3% yield. m.p: 178.9–181.3 °C. 1H NMR (500 MHz,
DMSO‑d6) δ (ppm): 10.11 (1H, s, Ar-NH), 8.92 (1H, s, Ar-NH), 8.19
(2H, s, ArH2), 8.17 (2H, s, Ar-OH), 7.96 (1H, s, Ar-OH), 7.15 (1H, s,
ArH), 6.53 (1H, d, J = 8.5 Hz, NH-CH = ), 6.36 (1H, d, J = 8.5 Hz,
ArH), 6.35 (1H, d, J = 8.5 Hz, ArH), 6.07 (2H, s, ArH2), 5.46 (1H, d,
J = 2.8 Hz, NH-CH]CH). 13C NMR (125 MHz, DMSO‑d6) δ (ppm):
170.53, 150.68 × 2, 141.67, 141.07, 138.88, 130.81, 130.04, 129.11,
124.68, 118.63, 112.35, 108.38, 108.29, 106.10. ESI-MS m/z: 285.09
5.1.4.6. N-(4-Fluorophenyl)-3,4,5-trihydroxybenzamide
(4f). Gray
powder, 40.9% yield. m.p: 190.5–193.4 °C. 1H NMR (500 MHz,
DMSO‑d6)
δ (ppm): 9.96 (1H, s, Ar-NH), 9.11 (3H, s, Ar-OH),
7.76–7.74 (2H, m, ArH2), 7.16 (2H, s, ArH2), 6.94 (2H, s, ArH2). 13C
NMR (125 MHz, DMSO‑d6) δ (ppm): 165.44, 158.89, 145.46 × 2,
136.76, 135.92, 124.77, 121.87, 121.80, 115.05, 114.87, 107.12 × 2.
9

Y. Qiu, et al.
Bioorganic&MedicinalChemistry27(2019)115049
(M+H)+, calcd for C15H12N2O4: 284.27.
(500 MHz, DMSO‑d6) δ (ppm): 9.73 (1H, s, Ar-NH2), 9.10 (3H, s, Ar-
OH), 7.60 (2H, d, J = 9.0 Hz, ArH2), 6.94 (2H, s, ArH2), 6.90 (2H, d,
J = 9.0 Hz, ArH2), 3.74 (4H, t, J = 9.0 Hz, O-NH2), 3.05 (4H, d,
J = 4.6 Hz, N-CH2). 13C NMR (125 MHz, DMSO‑d6) δ (ppm): 165.04,
147.13, 145.42 × 2, 136.45, 131.86, 125.27, 121.23 × 2, 115.23 × 2,
107.05 × 2, 66.10 × 2, 48.98 × 2. ESI-MS m/z: 331.12 (M+H)+, calcd
for C17H18N2O5: 330.34.
5.1.4.8. N-(1-Acetylindolin-5-yl)-3,4,5-trihydroxybenzamide
(4h). Yellow powder, 40.1% yield. m.p: 256.7–259.3 °C. 1H NMR
(500 MHz, DMSO‑d6) δ (ppm): 9.83 (1H, s, Ar-NH), 9.11 (3H, s, Ar-
OH), 7.95 (1H, d, J = 8.7 Hz, ArH), 7.69 (1H, s, ArH), 7.45 (1H, d,
J = 8.7 Hz, ArH), 6.94 (2H, s, ArH2), 4.09 (2H, t, J = 8.4 Hz, N-CH2),
3.14 (2H, t, J = 8.4 Hz, –CH2–CH2), 2.14 (3H, s, CO-CH3). 13C NMR
(125 MHz, DMSO‑d6) δ (ppm): 167.92, 165.21, 145.43 × 2, 138.52,
136.61, 135.04, 131.84, 125.10, 118.69, 117.05, 115.44, 107.11 × 2,
5.1.5. General procedure for synthesis of target compounds 7a-r.
A solution of various substituted carboxylic acid 5a-r (1.2 mmol),
EDC·HCl (1.3 mmol) and HOBT (1.5 mmol) in CH2Cl2 (20 mL) were
stirred in an ice-bath. After 20 min, the 5-aminoindane (6, 160 mg,
1.2 mmol) in CH2Cl2 was added slowly, the reaction mixture was stirred
in ice-bath for 1 h, and allowed to warm to room temperature over-
night. The resulting mixture was was diluted by H2O (20 mL), and ex-
tracted by EtOAc (3 × 20 mL). The combined organic layers were wa-
shed with brine and dried over anhydrous MgSO4, filtered, and
concentrated under reduced pressure. The residue was purified by
chromatography over silica gel to provide target compounds 7a-r.
48.20, 27.49, 23.79. ESI-MS m/z: 329.17 (M+H)+
, calcd for
C17H16N2O5: 328.32.
5.1.4.9. 3,4,5-Trihydroxy-N-{4-[(4-methylpiperazin-1-yl)methyl]phenyl}
benzamide (4i). Yellow powder, 43.8% yield. m.p: 134.5–136.2 °C. 1H
NMR (500 MHz, DMSO‑d6) δ (ppm): 9.89 (1H, s, Ar-NH), 9.181 (3H, s,
Ar-OH), 7.69 (2H, d, J = 8.4 Hz, ArH2), 7.22 (2H, d, J = 8.4 Hz, ArH2),
6.95 (2H, s, ArH2), 3.42 (2H, s, Ar-CH2), 2.51 (8H, s, N-CH2), 2.26 (3H,
s, N-CH3). 13C NMR (125 MHz, DMSO‑d6) δ (ppm): 165.46, 145.46 × 2,
138.46, 136.71, 132.47, 129.01 × 2, 125.03, 119.91 × 2, 107.17 × 2,
61.43, 54.87 × 2, 54.29 × 2, 45.07. ESI-MS m/z: 358.15 (M+H)+
,
5.1.5.1. 4-Chloro-N-(2,3-dihydro-1H-inden-5-yl)-3-nitrobenzamide
(7a). Yellow powder, 72.5% yield. m.p: 169.8–172.6 °C. 1H NMR
(500 MHz, DMSO‑d6) δ (ppm): 10.44 (1H, s, Ar-NH), 8.62 (1H, s,
ArH), 8.25 (1H, d, J = 8.4 Hz, ArH), 7.96 (1H, d, J = 8.4 Hz, ArH), 7.66
(1H, s, ArH), 7.46 (1H, d, ArH), 7.21 (1H, s, ArH), 2.88–2.82 (4H, m,
–CH2–CH2–CH2–), 2.06–2.00 (2H, m, –CH2–CH2–CH2–). 13C NMR
calcd for C19H23N3O4: 357.41.
5.1.4.10. 3,4,5-Trihydroxy-N-[4-(4-methylpiperazin-1-yl)phenyl]
benzamide (4j). Gray powder, 33.0% yield. m.p: 249.8–251.5 °C. 1H
NMR (500 MHz, DMSO‑d6) δ (ppm): 9.70 (1H, s, Ar-NH), 9.07 (3H, s,
Ar-OH), 7.58 (2H, d, J = 8.8 Hz, ArH2), 6.93 (2H, s, ArH2), 6.89 (2H, d,
J = 8.8 Hz, ArH2), 3.08 (4H, s, N-CH2), 2.46 (4H, s, N-CH2), 2.23 (3H, s,
N-CH3). 13C NMR (125 MHz, DMSO‑d6) δ (ppm): 165.01, 147.11,
145.45 × 2, 136.46, 131.54, 125.31, 121.23 × 2, 115.46 × 2,
107.09 × 2, 54.59 × 2, 48.55 × 2, 45.68. ESI-MS m/z: 344.02 (M
+H)+, calcd for C18H21N3O4: 343.38.
(125 MHz, DMSO‑d6)
δ (ppm): 162.11, 147.32, 144.10, 139.55,
136.66, 135.02, 132.73, 131.87, 127.87, 124.71, 124.11, 118.72,
116.77, 32.47, 31.83, 25.14. ESI-MS m/z: 317.07 (M + H)+, calcd for
C16H13ClN2O3: 316.74.
5.1.5.2. N-(2,3-Dihydro-1H-inden-5-yl)-2-(trifluoromethyl)benzamide
(7b). White powder, 85.9% yield. m.p: 155–158 °C. 1H NMR (500 MHz,
DMSO‑d6) δ (ppm): 10.41 (1H, s, Ar-NH), 7.84 (1H, d, J = 7.8 Hz, ArH),
7.79 (1H, t, J = 7.5 Hz, ArH), 7.72 (1H, t, J = 9.8 Hz, ArH), 7.67 (1H,
d, J = 7.8 Hz, ArH), 7.62 (1H, s, ArH), 7.38 (1H, d, J = 7.9 Hz, ArH),
7.18 (1H, d, J = 8.0 Hz, ArH), 7.18 (1H, d, J = 8.0 Hz, ArH), 2.87–2.81
(4H, m, –CH2–CH2–CH2–), 2.05–1.99 (2H, m, –CH2–CH2–CH2–). 13C
NMR (125 MHz, DMSO‑d6) δ (ppm): 165.31, 144.14, 139.09, 137.10,
132.52, 129.82, 128.44 × 2, 124.12 × 2, 117.87 × 2, 115.88 × 2,
5.1.4.11. N-(2,3-Dihydro-1H-inden-5-yl)-3,4,5-trihydroxybenzamide
(4k). White powder, 47.2% yield. m.p: 165.7–168.3 °C. 1H NMR
(500 MHz, DMSO‑d6) δ (ppm): 9.78 (1H, s, Ar-NH), 9.13 (3H, s, Ar-
OH), 7.65 (1H, s, ArH), 7.44 (1H, d, J = 8.0 Hz, ArH), 7.14 (1H, d,
J = 8.0 Hz, ArH2), 6.94 (2H, s, ArH2), 2.85–2.79 (4H, m,
–CH2–CH2–CH2–), 2.04–1.98 (2H, m, –CH2–CH2–CH2–). 13C NMR
(125 MHz, DMSO‑d6) δ (ppm): 165.33, 145.42 × 2, 143.75, 138.23,
137.74, 136.56, 125.25, 123.84, 118.35, 116.38, 107.13 × 2, 32.51,
31.77, 25.18. ESI-MS m/z: 286.06 (M+H)+, calcd for C16H15NO4:
285.3.
32.48, 31.81, 25.17. ESI-MS m/z: 306.17 (M + H)+
, calcd for
C17H14F3NO: 305.30.
5.1.5.3. N-(2,3-Dihydro-1H-inden-5-yl)-2-(2,4-dinitrophenyl)acetamide
(7c). Yellow powder, 70.4% yield. m.p: 211–214 °C. 1H NMR
(500 MHz, DMSO‑d6) δ (ppm): 10.18 (1H, s, Ar-NH), 8.76 (1H, s,
ArH), 8.54 (1H, d, J = 7.3 Hz, ArH), 7.89 (1H, d, J = 8.4 Hz, ArH), 7.42
(1H, s, ArH), 7.23 (1H, d, J = 7.8 Hz, ArH), 7.13 (1H, d, J = 7.8 Hz,
ArH), 4.26 (2H, s, Ar-CH2), 2.82–2.77 (4H, m, –CH2–CH2–CH2–),
2.00–1.97 (2H, m, –CH2–CH2–CH2–). 13C NMR (125 MHz, DMSO‑d6)
δ (ppm): 166.23, 149.01, 146.63, 144.14, 138.58, 137.66, 137.04,
135.17, 127.31, 124.13, 119.76, 117.34, 115.41, 40.44, 32.45, 31.74,
25.11. ESI-MS m/z: 342.20 (M+H)+, calcd for C17H15N3O5: 341.32.
5.1.4.12. N-Benzyl-3,4,5-trihydroxybenzamide
(4l). Gray
powder,
38.6% yield. m.p: 153.4–156.3 °C. 1H NMR (500 MHz, DMSO‑d6) δ
(ppm): 9.06 (2H, s, Ar-OH), 8.69 (1H, s, Ar-OH), 8.67 (1H, t,
J = 5.9 Hz, Ar-NH2), 7.33–7.27 (4H, m, ArH4), 7.23 (1H, t, ArH),
6.88 (2H, s, ArH2), 4.40 (2H, d, J = 5.9 Hz, NH-CH2). 13C NMR
(125 MHz, DMSO‑d6) δ (ppm): 166.34, 145.40 × 2, 140.13, 136.25,
128.14 × 2, 127.07 × 2, 126.52, 124.74, 106.76 × 2, 42.44. ESI-MS
m/z: 260.02 (M+H)+, calcd for C14H13NO4: 259.26.
5.1.4.13. N-(3,4-Dimethoxybenzyl)-3,4,5-trihydroxybenzamide
(4m). White powder, 42.7% yield. m.p: 189.8–192.7 °C. 1H NMR
(500 MHz, DMSO‑d6) δ (ppm): 9.06 (2H, s, Ar-OH), 8.70 (1H, s, Ar-
OH), 8.58 (1H, t, J = 5.9 Hz, Ar-NH2), 6.92 (1H, s, ArH), 6.88 (1H, d,
J = 8.2 Hz, ArH), 6.86 (2H, s, ArH2), 6.80 (1H, d, J = 8.2 Hz, ArH),
4.32 (1H, d, J = 5.9 Hz, NH-CH2), 3.72 (6H, d, J = 5.8 Hz, Ar-OCH3).
13C NMR (125 MHz, DMSO‑d6) δ (ppm): 166.25, 148.55, 147.63,
145.38 × 2, 136.19, 132.61, 124.87, 119.28, 111.73, 111.44,
106.74 × 2, 66.99 × 2, 42.23. ESI-MS m/z: 320.14 (M+H)+, calcd
for C16H17NO6: 319.31.
5.1.5.4. N-(2,3-Dihydro-1H-inden-5-yl)-2-(3,5-dimethoxyphenyl)
acetamid (7d). White powder, 80.1% yield. m.p: 117.2–119.8 °C. 1H
NMR (500 MHz, DMSO‑d6) δ (ppm): 9.97 (1H, s, Ar-NH), 7.51 (1H, s,
ArH), 7.28 (1H, d, J = 8.0 Hz, ArH), 7.12 (1H, d, J = 8.0 Hz, ArH), 6.50
(2H, s, ArH2), 6.38 (1H, s, ArH), 3.72 (6H, s, Ar-OCH3), 3.53 (2H, s, Ar-
CH2), 2.82–2.77 (4H, m, –CH2–CH2–CH2–), 2.01–1.95 (2H, m,
–CH2–CH2–CH2–). 13C NMR (125 MHz, DMSO‑d6) δ (ppm): 168.45,
160.32 × 2, 144.04, 138.37, 138.20, 137.37, 124.05, 117.33, 115.40,
107.19 × 2, 98.28, 55.08 × 2, 43.59, 32.46, 31.74, 25.12. ESI-MS m/z:
312.24 (M+H)+, calcd for C19H21NO3: 311.38.
5.1.4.14. 3,4,5-Trihydroxy-N-(4-morpholinophenyl)benzamide
(4n). White powder, 39.8% yield. m.p: 177.1–179.8 °C. 1H NMR
5.1.5.5. 4 Acetamido-N-(2,3-dihydro-1H-inden-5-yl)benzamide (7e). White
10

Y. Qiu, et al.
Bioorganic&MedicinalChemistry27(2019)115049
powder, 66.3% yield. m.p: 219.6–222.4 °C. 1H NMR (500 MHz, DMSO‑d6)
δ (ppm): 10.25 (1H, s, Ar-NH), 10.00 (1H, s, Ar-NH), 7.91 (2H, d,
J = 8.5 Hz, ArH2), 7.71 (2H, d, J = 8.5 Hz, ArH2), 7.67 (1H, s, ArH), 7.47
(1H, s, ArH), 7.17 (1H, s, ArH), 2.87–2.81 (4H, m, –CH2–CH2–CH2–), 2.09
(3H, s, –CO–CH3) 2.05–1.99 (2H, m, –CH2–CH2–CH2–). 13C NMR
(125 MHz, DMSO‑d6) δ (ppm): 168.69, 164.66, 143.87, 142.12, 138.66,
137.42, 129.20, 128.44 × 2, 123.93 × 2, 118.56, 118.09, 116.61, 32.50,
31.80, 25.16, 24.07. ESI-MS m/z: 295.34 (M+H)+, calcd for C18H18N2O2:
294.35.
129.33 × 2, 123.81, 120.45, 118.35, 116.40, 110.09 × 2, 43.70 × 2,
32.51, 31.77, 25.17, 12.35 × 2. ESI-MS m/z: 308.98 (M+H)+, calcd
for C20H24N2O: 308.43.
5.1.5.11. 2,6-Dichloro-N-(2,3-dihydro-1H-inden-5-yl)benzamide
(7k). White powder, 60.6% yield. m.p: 197.2–199.9 °C. 1H NMR
(500 MHz, DMSO‑d6) δ (ppm): 10.59 (1H, s, Ar-NH), 7.63 (1H, s,
ArH), 7.57 (2H, d, J = 8.7 Hz, ArH2), 7.50 (1H, t, J = 7.5 Hz, ArH),
7.37 (1H, d, J = 8.0 Hz, ArH), 7.20 (1H, d, J = 8.0 Hz, ArH), 2.87–2.81
(4H, m, –CH2–CH2–CH2–), 2.05–1.99 (2H, m, –CH2–CH2–CH2–). 13C
NMR (125 MHz, DMSO‑d6) δ (ppm): 161.73, 144.31, 139.34, 136.75,
136.55, 131.22, 131.19, 129.95, 128.18 × 2, 124.24, 117.73, 115.76,
5.1.5.6. N-(2,3-Dihydro-1H-inden-5-yl)-2-(naphthalen-1-yl)acetamide
(7f). White powder, 77.9% yield. m.p: 166.6–169.7 °C. 1H NMR
(500 MHz, DMSO‑d6) δ (ppm): 10.19 (1H, s, Ar-NH), 8.14 (1H, d,
J = 8.5 Hz, ArH), 7.93 (1H, d, J = 7.8 Hz, ArH), 7.84 (1H, d,
J = 7.8 Hz, ArH), 7.57–7.46 (5H, m, ArH), 7.31 (1H, d, J = 8.0 Hz,
ArH), 7.12 (1H, d, J = 8.0 Hz, ArH), 4.12 (2H, s, Ar-CH2), 2.82–2.77
(4H, m, –CH2–CH2–CH2–), 2.01–1.95 (2H, m, –CH2–CH2–CH2–). 13C
NMR (125 MHz, DMSO‑d6) δ (ppm): 168.69, 144.07, 138.37, 137.42,
133.36, 132.60, 132.00, 128.38, 127.72, 127.12, 126.01, 125.61,
125.48, 124.17, 124.077, 117.33, 115.41, 40.66, 32.46, 31.74, 25.13.
ESI-MS m/z: 302.32 (M+H)+, calcd for C21H19NO: 301.39.
32.48, 31.82, 25.18. ESI-MS m/z: 306.17 (M+H)+
, calcd for
C
16H13Cl2NO: 306.19.
5.1.5.12. N-(2,3-Dihydro-1H-inden-5-yl)-6-methoxy-1H-indole-2-
carboxamide (7l). Yellow powder, 73.8% yield. m.p: 146.7–149.2 °C.
1H NMR (500 MHz, DMSO‑d6) δ (ppm): 11.51 (1H, s, Ar-NH), 9.97 (1H,
s, Ar-NH), 7.69 (1H, s, ArH), 7.55–7.50 (2H, m, ArH), 7.34 (1H, s, Ar-
CH]), 7.19 (1H, d, J = 8.0 Hz, ArH), 6.92 (1H, s, ArH), 6.73 (1H, d,
J = 8.0 Hz, ArH), 3.78 (3H, s, Ar-OCH3), 2.89–2.82 (4H, m,
–CH2–CH2–CH2–), 2.06–2.00 (2H, m, –CH2–CH2–CH2–). 13C NMR
5.1.5.7. 3,5-Dichloro-N-(2,3-dihydro-1H-inden-5-yl)isonicotinamide
(7g). White powder, 52.5% yield. m.p: 173.9–176.8 °C. 1H NMR
(500 MHz, DMSO‑d6) δ (ppm): 10.79 (1H, s, Ar-NH), 8.79 (2H, s, N-
CH), 7.60 (1H, s, ArH), 7.40 (1H, d, J = 8.0 Hz, ArH), 7.21 (1H, d,
J = 8.0 Hz, ArH), 2.88–2.81 (4H, m, –CH2–CH2–CH2–), 2.05–1.99 (2H,
m, –CH2-CH2-CH2-). 13C NMR (125 MHz, DMSO‑d6) δ (ppm): 159.61,
147.71 × 2, 144.54, 142.66, 139.91, 136.12, 128.15 × 2, 124.41,
(125 MHz, DMSO‑d6) δ (ppm): 159.52, 157.26, 143.99, 138.56,
137.75, 137.21, 130.54, 124.05, 122.43, 121.34, 118.26, 116.29,
111.16, 103.94, 94.17, 55.09, 32.52, 31.80, 25.18. ESI-MS m/z:
307.35 (M+H)+, calcd for C19H18N2O2: 306.37.
5.1.5.13. N-(2,3-Dihydro-1H-inden-5-yl)-3-morpholinobenzamide
(7m). White powder, 49.7% yield. m.p: 146–148 °C. 1H NMR
(500 MHz, DMSO‑d6) δ (ppm): 10.03 (1H, s, Ar-NH), 7.65 (1H ,s,
ArH), 7.47 (2H, d, J = 7.8 Hz, ArH2), 7.37 (2H, s, ArH2), 7.17 (2H, t,
J = 7.8 Hz, ArH2), 3.77 (4H, s, O-CH2), 3.18 (4H, s, N-CH2), 2.86–2.82
(4H, m, –CH2–CH2–CH2–), 2.03–2.01 (2H, m, –CH2–CH2–CH2–). 13C
NMR (125 MHz, DMSO‑d6) δ (ppm): 165.56, 150.97, 143.88, 138.80,
137.31, 135.92, 128.93, 123.93, 118.67, 118.28, 117.92, 116.73,
113.87, 66.04 × 2, 48.34 × 2, 32.48, 31.80, 25.16. ESI-MS m/z:
323.21 (M+H)+, calcd for C20H22N2O2: 322.41.
117.80, 115.848, 32.45, 31.84, 25.16. ESI-MS m/z: 307.15 (M+H)+
,
calcd for C15H12Cl2N2O: 307.17.
5.1.5.8. N-(2,3-Dihydro-1H-inden-5-yl)-1H-indole-2-carboxamide
(7h). White powder, 66.6% yield. m.p: 245–247.2 °C. 1H NMR
(500 MHz, DMSO‑d6) δ (ppm): 11.74 (1H, s, Ar-NH), 10.12 (1H, s,
Ar-NH), 7.71 (1H, s, Ar-CH = ), 7.67 (1H, d, J = 7.9 Hz, ArH), 7.53
(1H, d, J = 7.9 Hz, ArH), 7.47 (1H, d, J = 8.2 Hz, ArH), 7.41 (1H, s,
ArH), 7.22 (2H, t, J = 9.0 Hz, ArH2), 7.07 (1H, t, J = 7.5 Hz, ArH),
2.89–2.82 (4H, m, –CH2-CH2-CH2-), 2.06–2.00 (2H, m,
–CH2–CH2–CH2–). 13C NMR (125 MHz, DMSO‑d6) δ (ppm): 159.52,
144.03, 138.77, 137.09, 136.77, 131.73, 127.06, 124.08, 123.58,
121.61, 119.81, 118.37, 116.39, 112.35, 103.62, 32.52, 31.81, 25.18.
ESI-MS m/z: 277.26 (M+H)+, calcd for C18H16N2O: 276.34.
5.1.5.14. N-(2,3-Dihydro-1H-inden-5-yl)-5-methylthiophene-2-
carboxamide (7n). White powder, 75.8% yield. m.p: 154.7–157.3 °C. 1H
NMR (500 MHz, DMSO‑d6) δ (ppm): 9.98 (1H, s, Ar-NH), 7.80 (1H, d,
J = 3.6 Hz, thiophene-H), 7.60 (1H, s, ArH), 7.41 (1H, d, J = 8.0 Hz,
ArH), 7.17 (1H, d, J = 8.0 Hz, ArH), 6.90 (1H, d, J = 3.6 Hz, thiophene-
H), 2.86–2.80 (4H, m, –CH2–CH2–CH2–), 2.49 (3H, s, thiophene-CH3),
2.04–1.98 (2H, m, –CH2–CH2–CH2–). 13C NMR (125 MHz, DMSO‑d6) δ
(ppm): 159.61, 145.44, 143.98, 138.81, 137.72, 136.94, 129.02,
126.46, 124.01, 118.47, 116.55, 32.47, 31.79, 25.14, 15.24. ESI-MS
m/z: 258.14 (M+H)+, calcd for C15H15NOS: 257.35.
5.1.5.9. N-(2,3-Dihydro-1H-inden-5-yl)-4-(1H-indol-3-yl)butanamide
(7i). White powder, 70.5% yield. m.p: 138.2–141.1 °C. 1H NMR
(500 MHz, DMSO‑d6) δ (ppm): 10.79 (1H, s, Ar-NH), 9.76 (1H, s, Ar-
NH), 7.52 (2H, d, J = 9.7 Hz, ArH2), 7.34 (1H, d, J = 8.0 Hz, ArH), 7.28
(1H, d, J = 8.0 Hz, ArH), 7.12 (1H, s, NH-CH = ), 7.10 (1H, d,
J = 8.2 Hz, ArH), 7.06 (1H, t, J = 5.7 Hz, ArH), 6.97 (1H, t,
J = 7.3 Hz, ArH), 2.82–2.77 (4H, m, CH2-CH2-CH2), 2.74 (2H, t,
5.1.5.15. N-(2,3-Dihydro-1H-inden-5-yl)-4-methylthiazole-5-
carboxamide (7o). White powder, 61.5% yield. m.p: 108.5–111.6 °C. 1H
NMR (500 MHz, DMSO‑d6) δ (ppm): 10.11 (1H, s, Ar-NH), 9.11 (1H, s,
thiazole-H), 7.57 (1H, s, ArH), 7.38 (1H, d, J = 8.0 Hz, ArH), 7.18 (1H,
d, J = 8.0 Hz, ArH), 2.87–2.81 (4H, m, –CH2–CH2–CH2–), 2.60 (3H, s,
thiazole-CH3), 2.05–1.99 (2H, m, –CH2–CH2–CH2–). 13C NMR
J = 7.3 Hz,
–CH2–CH2–CH2),
2.36
(2H,
t,
J = 7.2 Hz,
–CH2–CH2–CH2–), 2.01–1.94 (4H, m, –CH2–CH2–CH2–). 13C NMR
(125 MHz, DMSO‑d6)
δ (ppm): 170.89, 143.92, 137.98, 137.59,
136.33, 127.19, 123.97, 122.25, 120.78, 118.27, 118.07, 117.27,
115.36, 114.05, 111.30, 36.18, 32.48, 31.73, 25.99, 25.13, 24.31.
ESI-MS m/z: 319.23 (M+H)+, calcd for C21H22N2O: 318.42.
(125 MHz, DMSO‑d6)
δ (ppm): 159.92, 154.67, 154.03, 144.07,
139.33, 136.74, 126.56, 124.06, 118.59, 116.66, 32.46, 31.81, 25.15,
16.75. ESI-MS m/z: 258.86 (M+H)+, calcd for C14H14N2OS: 258.34.
5.1.5.10. 4-(Diethylamino)-N-(2,3-dihydro-1H-inden-5-yl)benzamide
(7j). White powder, 50.1% yield. m.p: 131.2–133.6 °C. 1H NMR
(500 MHz, DMSO‑d6) δ (ppm): 9.69 (1H, s, Ar-NH), 7.82 (2H, d,
J = 8.6 Hz, ArH), 7.66 (1H, s, ArH), 7.46 (1H, d, J = 8.0 Hz, ArH),
7.14 (1H, d, J = 8.0 Hz, ArH), 6.70 (2H, d, J = 8.6 Hz, ArH), 3.42–3.38
(4H, m, N–CH2), 2.86–2.80 (4H, m, –CH2–CH2–CH2–), 2.04–1.98 (2H,
m, –CH2–CH2–CH2–), 1.12 (6H, t, J = 7.0 Hz, N–CH2–CH3). 13C NMR
(125 MHz, DMSO‑d6) δ (ppm): 164.97, 149.64, 143.73, 138.01, 137.91,
5.1.5.16. N-(2,3-Dihydro-1H-inden-5-yl)-3-(pyrrolidin-1-yl)benzamide
(7p). Gray powder, 54.2% yield. m.p: 168.9–171.6 °C. 1H NMR
(500 MHz, DMSO‑d6) δ (ppm): 9.98 (1H, s, Ar-NH), 7.66 (1H, s, ArH),
7.48 (1H, s, ArH), 7.28 (1H, t, J = 7.8 Hz, ArH), 7.17 (1H, d,
J = 8.1 Hz, ArH), 7.14 (1H, d, J = 7.8 Hz, ArH), 7.03 (1H, s, ArH),
6.71 (1H, d, J = 8.1 Hz, ArH), 3.29 (4H, t, J = 6.2 Hz, N-CH2),
2.87–2.81 (4H, m, –CH2–CH2–CH2–), 2.05–2.00 (2H, m,
11

Y. Qiu, et al.
Bioorganic&MedicinalChemistry27(2019)115049
–CH2–CH2–CH2–), 1.98 (4H, t, J = 6.5 Hz, N–CH2–CH2). 13C NMR
31.78, 25.15. ESI-MS m/z: 324.19 (M + H)+, calcd for C20H21NO3:
(125 MHz, DMSO‑d6)
δ
(ppm): 165.98, 147.56, 143.85, 138.66,
323.39.
137.43, 135.90, 128.82, 123.90, 118.60, 116.66, 114.36, 114.15,
110.51, 47.36 × 2, 32.49, 31.80, 25.16, 24.92 × 2. ESI-MS m/z:
307.28 (M+H)+, calcd for C20H22N2O: 306.41.
5.1.6.3. (E)-N-(2,3-Dihydro-1H-inden-5-yl)-3-[2-(trifluoromethyl)phenyl]
acrylamide (9c). White powder, 58.5% yield. m.p: 161.1–163.2 °C. 1H
NMR (500 MHz, DMSO‑d6) δ (ppm): 10.25 (1H, s, Ar-NH), 7.89 (1H, d,
J = 7.0 Hz, ArH), 7.85–7.78 (3H, m, ArH, Ar-CH = ), 7.64 (2H, s,
ArH2), 7.41 (1H, d, J = 7.1 Hz, ArH), 7.18 (1H, d, J = 7.6 Hz, ArH),
6.91 (1H, d, J = 15.3 Hz, Ar-CH]CH–), 2.86–2.82 (4H, m,
–CH2–CH2–CH2–), 2.03–2.00 (2H, m, –CH2–CH2–CH2–). 13C NMR
(125 MHz, DMSO‑d6) δ (ppm): 162.39, 144.22 × 2, 138.92, 137.18,
134.41, 133.36, 133.07, 129.66, 127.82, 127.11, 126.16, 126.12,
124.23, 117.47, 115.50, 32.48, 31.80, 25.11. ESI-MS m/z: 332.21 (M
+H)+, calcd for C19H16F3NO: 331.34.
5.1.5.17. N-(2,3-Dihydro-1H-inden-5-yl)-3-(dimethylamino)benzamide
(7q). White powder, 60.2% yield. m.p: 145–147.6 °C. 1H NMR
(500 MHz, DMSO‑d6) δ (ppm): 10.00 (1H, s, Ar-NH), 7.66 (1H, s,
ArH), 7.48 (1H, d, J = 8.0 Hz, ArH), 7.31 (1H, t, J = 8.0 Hz, ArH), 7.22
(2H, s, ArH2), 7.17 (1H, d, J = 8.0 Hz, ArH), 6.91(1H, d, J = 8.0 Hz
ArH), 2.96 (6H, s, N-CH3), 2.87–2.81 (4H, m, –CH2-CH2-CH2-),
2.05–1.99 (2H, m, –CH2-CH2-CH2-). 13C NMR (125 MHz, DMSO‑d6) δ
(ppm): 165.94, 150.28, 143.86, 138.70, 137.41, 135.89, 128.80,
123.91, 118.64, 116.70, 115.15, 115.07, 111.17, 32.49 × 2,
31.80 × 2, 25.17. ESI-MS m/z: 280.85 (M+H)+
,
calcd for
5.1.6.4. (E)-N-(2,3-Dihydro-1H-inden-5-yl)-3-[3-(trifluoromethoxy)
phenyl]acrylamide (9d). Yellow powder, 51.7% yield. m.p:
129.7–132.6 °C. 1H NMR (500 MHz, DMSO‑d6) δ (ppm): 10.13 (1H, s,
Ar-NH), 7.98 (1H, s, ArH), 7.93 (1H, d, J = 7.7 Hz, ArH), 7.76 (1H, d,
J = 7.7 Hz, ArH), 7.70 (1H, t, J = 7.8 Hz, ArH2), 7.66 (1H, d,
J = 15.9 Hz, Ar-CH = ), 7.65 (1H, s, ArH), 7.41 (1H, d, J = 8.0 Hz,
ArH), 7.18 (1H, d, J = 8.0 Hz, ArH), 6.97 (1H, d, J = 15.9 Hz, Ar-
CH]CH–), 2.87–2.80 (4H, m, –CH2–CH2–CH2–), 2.04–1.99 (2H, m,
–CH2–CH2–CH2–). 13C NMR (125 MHz, DMSO‑d6) δ (ppm): 162.79,
144.19, 138.76, 137.90, 137.34, 136.03, 131.43, 130.09, 125.85,
125.82, 124.75, 124.21, 123.86, 123.83, 117.38, 115.38, 32.50,
31.79, 25.13. ESI-MS m/z: 348.26 (M+H)+, calcd for C19H16F3NO2:
347.34.
C
18H20N2O: 280.37.
5.1.5.18. N-(2,3-Dihydro-1H-inden-5-yl)-2-(dimethylamino)benzamide
(7r). Yellow oil, 70.5% yield. 1H NMR (500 MHz, DMSO‑d6) δ (ppm):
11.19 (1H, s, Ar-NH), 7.69 (1H, d, J = 7.5 Hz, ArH), 7.64 (1H, s, ArH),
7.45 (2H, t, J = 7.2 Hz, ArH2), 7.23 (1H, d, J = 8.1 Hz, ArH), 7.17 (1H,
d, J = 8.1 Hz, ArH), 7.10(1H, t, J = 7.5 Hz, ArH), 2.87–2.80 (4H, m,
–CH2–CH2–CH2–), 2.77 (6H, s, N-CH3), 2.05–1.99 (2H, m,
–CH2–CH2–CH2–). 13C NMR (125 MHz, DMSO‑d6) δ (ppm): 165.32,
151.19, 144.18, 138.68, 138.64, 137.36, 131.38, 129.90, 124.16,
121.84, 118.91, 117.74, 115.73, 43.88 × 2, 32.50, 31.79, 25.19. ESI-
MS m/z: 280.85 (M+H)+, calcd for C18H20N2O: 280.37.
5.1.6. General procedure for synthesis of target compounds 9a-g.
5.1.6.5. (E)-N-(2,3-Dihydro-1H-inden-5-yl)-3-(3-hydroxy-4-
To a solution of substituted cinnamic acids 8a-g (1.0 mmol) in dry
THF (10 mL) was added oxalyl chloride (5.0 equiv) and a catalytic
amount of DMF (0.01 equiv). The reaction mixture was stirred at room
temperature for 5 h and the solvent was removed in vacuo. The residue
was redissolved in CH2Cl2 (10 mL), and Et3N (3.0 equiv), 5-aminoindan
(1.0 mmol) were added slowly, the reaction mixture was stirred at room
temperature for 2 h and monitored by TLC. After the reaction was
completed, the resulting mixture was diluted by H2O (20 mL), and ex-
tracted by CH2Cl2 (3 × 20 mL). The combined organic layers were
washed with brine and dried over anhydrous MgSO4, filtered, and
concentrated under reduced pressure. The residue was purified by
chromatography over silica gel to provide target compounds 9a-g.
methoxyphenyl)acrylamide (9e). White powder, 58.9% yield. m.p:
155.2–157.9 °C. 1H NMR (500 MHz, DMSO‑d6) δ (ppm): 9.98 (1H, s,
Ar-NH), 9.24 (1H, s, Ar-OH), 7.60 (1H, s, ArH), 7.42 (1H, d,
J = 15.7 Hz, Ar-CH]), 7.40 (1H, d, J = 9.5 Hz, ArH), 7.15 (1H, d,
J = 8.0 Hz, ArH), 7.04 (1H, s, ArH), 7.03 (1H, d, J = 8.2 Hz, ArH), 6.97
(1H, d, J = 8.2 Hz, ArH), 6.60 (1H, d,J = 15.7 Hz, Ar-CH]CH–), 3.81
(3H, s, Ar-OCH3), 2.86–2.79 (4H, m, –CH2–CH2–CH2–), 2.04–1.98 (2H,
m, –CH2–CH2–CH2–). 13C NMR (125 MHz, DMSO‑d6) δ (ppm): 163.58,
149.40, 146.74, 144.10, 139.94, 138.36, 137.62, 127.69, 124.14,
120.49, 119.74, 117.29, 115.31, 113.46, 112.15, 55.61, 32.51, 31.78,
25.14. ESI-MS m/z: 310.29 (M+H)+, calcd for C19H19NO3: 309.37.
5.1.6.6. (E)-N-(2,3-Dihydro-1H-inden-5-yl)-3-(4-hydroxy-3,5-
dimethoxyphenyl)acrylamide (9f). Yellow powder, 56.7% yield. m.p:
143.5–146.3 °C. 1H NMR (500 MHz, DMSO‑d6) δ (ppm): 9.97 (1H, s,
Ar-NH), 8.87 (1H, s, Ar-OH), 7.64 (1H, s, ArH), 7.47 (1H, d,
J = 15.5 Hz, Ar-CH]), 7.39 (1H, d, J = 8.2 Hz, ArH), 7.15 (1H, d,
J = 7.6 Hz, ArH), 6.92 (2H, s, ArH2), 6.67 (1H, d, J = 15.5 Hz, Ar-
CH]CH–), 3.82 (6H, s, Ar-OCH3), 2.86–2.79 (4H, m, –CH2–CH2–CH2–),
2.04–1.98 (2H, m, –CH2–CH2–CH2–). 13C NMR (125 MHz, DMSO‑d6) δ
(ppm): 163.65, 148.10 × 2, 144.09, 140.46, 138.30, 137.72, 125.15,
124.13 × 2, 119.54, 117.23, 115.23, 105.45 × 2, 55.98 × 2, 32.52,
31.78, 25.15. ESI-MS m/z: 340.24 (M + H)+, calcd for C20H21NO4:
339.39.
5.1.6.1. (E)-N-(2,3-Dihydro-1H-inden-5-yl)-3-(3,4,5-trimethoxyphenyl)
acrylamide (9a). Yellow powder, 63.6% yield. m.p: 56.8–59.7 °C. 1H
NMR (500 MHz, DMSO‑d6) δ (ppm): 10.07 (1H, s, Ar-NH), 7.64 (1H, s,
ArH), 7.51 (1H, d, J = 15.6 Hz, Ar-CH = ), 7.40 (1H, d, J = 7.9 Hz,
ArH), 7.16 (1H, s, ArH), 6.96 (2H, s, ArH2), 6.77 (1H, d, J = 15.6 Hz,
Ar-CH = CH–), 3.84 (6H, s, Ar-OCH3), 3.70 (3H, s, Ar-OCH3),
2.86–2.79 (4H, m, –CH2-CH2-CH2-), 2.04–1.98 (2H, m,
–CH2–CH2–CH2–). 13C NMR (125 MHz, DMSO‑d6) δ (ppm): 163.30,
153.10 × 2, 144.12, 139.88, 138.90, 138.48, 137.57, 130.37, 124.17,
121.80, 117.290, 115.28, 105.12 × 2, 60.08, 55.89 × 2, 32.51, 31.79,
25.15. ESI-MS m/z: 354.2 (M+H)+, calcd for C21H23NO4: 353.42.
5.1.6.2. (E)-N-(2,3-Dihydro-1H-inden-5-yl)-3-(3,4-dimethoxyphenyl)
acrylamide (9b). Yellow powder, 62.9% yield. m.p: 133.1–135.2 °C. 1H
NMR (500 MHz, DMSO‑d6) δ (ppm): 10.21 (1H, s, Ar-NH), 7.65 (1H, s,
ArH), 7.49 (1H, d, J = 15.6 Hz, Ar-CH = ), 7.42 (1H, d, J = 7.8 Hz,
ArH), 7.23 (1H, s, ArH), 7.18 (1H, d, J = 8.3 Hz, ArH), 7.15 (1H, d,
J = 8.1 Hz, ArH), 7.00 (6H, d, J = 8.3 Hz, ArH), 6.77 (1H, d,
J = 15.6 Hz, Ar-CH]CH–), 3.82 (3H, s, Ar-OCH3), 3.80 (3H, s, Ar-
OCH3), 2.86–2.79 (4H, m, –CH2–CH2–CH2–), 2.04–1.99 (2H, m,
–CH2–CH2–CH2–). 13C NMR (125 MHz, DMSO‑d6) δ (ppm): 163.62,
150.28, 148.94, 144.06, 139.70, 138.29, 137.75, 127.66, 124.12,
121.62, 120.34, 117.30, 115.28, 111.79, 110.11, 55.55, 55.44, 32.52,
5.1.6.7. (E)-N-(2,3-Dihydro-1H-inden-5-yl)-3-(2,5-dimethoxyphenyl)
acrylamide (9 g). Yellow powder, 68.8% yield. m.p: 149.2–151.2 °C. 1
H
NMR (500 MHz, DMSO‑d6) δ (ppm): 10.05 (1H, s, Ar-NH), 7.76 (1H, d,
J = 15.8 Hz, Ar-CH = ), 7.63 (1H, s, ArH), 7.40 (1H, d, J = 7.6 Hz,
ArH), 7.16 (1H, d, J = 7.9 Hz, ArH), 7.13 (1H, s, ArH), 7.03 (1H, d,
J = 7.6 Hz, ArH), 6.97 (1H, d, J = 8.6 Hz, ArH), 6.87 (1H, d,
J = 15.8 Hz, Ar-CH]CH–), 3.82 (3H, s, Ar-OCH3), 3.76 (3H, s, Ar-
OCH3), 2.86–2.80 (4H, m, –CH2–CH2–CH2–), 2.02–2.00 (2H, m,
–CH2–CH2–CH2–). 13C NMR (125 MHz, DMSO‑d6) δ (ppm): 163.59,
153.19, 152.06, 144.10, 138.48, 137.58, 134.62, 124.14, 123.88,
123.23, 117.38, 116.53, 115.38, 113.104, 112.52, 56.10, 55.47,
12

Y. Qiu, et al.
Bioorganic&MedicinalChemistry27(2019)115049
32.52, 31.79, 25.14. ESI-MS m/z: 324.19 (M+H)+
,
calcd for
5.6. LPS-induced ALI in mice
C
20H21NO3: 323.39.
Male C57BL/6 mice, weighing 18–22 g, were randomly divided into
four groups, on average: the designated control, LPS group, 4k (10 mg/
kg) + LPS group, and 4k (20 mg/kg) + LPS group. The mice in the
4k + LPS groups were treated orally with 4k (dissolved in 0.5% CMC-
Na) at a dosage of 10 and 20 mg/kg/day, respectively, for three con-
secutive days. The control and LPS-Vehicle groups were given the same
volume of 0.5% CMC-Na. Mice were euthanized with ketamine 6 h after
LPS instillation. The BALF and lung tissue samples were collected for
further tests.
5.2. Animals
The animal protocol was approved by the Animal Care and Use
Committee of the Wenzhou Medical University (Approval documents:
wydw2014-0001). Male C57BL/6 mice, aged 10 weeks, weighing
18–22 g, were purchased from Department of Laboratory Animal
Center, Wenzhou Medical University (Wenzhou, People’s Republic of
China). Animals were maintained in an air-conditioned room under a
12:12-h light/dark cycle and were granted free access to water and
food.
5.7. Bronchoalveolar lavage fluid (BALF) analysis
BALF was performed by irrigating the left lung with saline
(3 × 0.5 mL) at the end of the experiment. This fluid was centrifuged at
1000 rpm for 10 min at 4 °C, the supernatant was used for protein
concentration and subsequent cytokines determinations. The pre-
cipitation was resuspended using 50 μL physiological saline, and the
number of total cells on BALF was determined using a cell counting
instrument.
5.3. Cells and reagents
Lipopolysaccharide (LPS) purchased from Sigma-Aldrich (St Louis,
MO, USA) was dissolved in PBS. Saline was prepared as 0.9% NaCl
solution. eBioscience, Inc. (San Diego, CA, USA) was the source of IL-6
and TNF-α enzyme-linked immunosorbent assay (ELISA) kits. For
peritoneal macrophage preparation, C57BL/6 mice were in-
traperitoneally injected (i.p.) with 3 mL 6% thioglycollate solution
(0.3 g beef extract, 1 g tryptone, 0.5 g NaCl dissolved in 100 mL ddH2O,
and filtrated through 0.22 μm filter membrane). Total MPMs were
harvested by washing the peritoneal cavity with PBS containing 30 μM
of EDTA (8 mL per mouse), centrifuged, then the pellet was re-
suspended in RPMI-1640 medium (Gibco, Eggenstein, Germany) with
10% FBS (Hyclone, Logan, UT), 100 U/ml penicillin, and 100 μg/ ml
streptomycin. Before used, MPMs were cultured in 35 mm plates and
maintained at 37 °C in 5% CO2-humidified air.
5.8. Lung wet/dry ratio
To observe the degree of pulmonary edema, the wet/dry ratio (W/D
ratio) was calculated. Mice were euthanized, the middle lobe of right
lung was harvested and weighed. Then the lung tissue was heated at
65 °C for 72 h in a hot air oven to obtain a constant weight. The lung
wet/dry mass ratio was estimated after the lung dry weight was mea-
sured.
5.9. Lung histopathology
5.4. Enzyme-linked immunosorbent assay
Lung tissues (5 μm thickness) were stained with haematoxylin and
eosin to estimate the degree of lung injury by using light microscopy.
Also, sections were stained for CD68, a marker of TNF-α and macro-
phage. Briefly, sections were deparaffinized in xylene and hydrated
using an ethanol gradient. A Pressure-cooker was used for heat-induced
antigen retrieval (10 μM sodium citrate buffer, pH 6.5). All sections
were blocked in 5% bovine serum albumin (BSA) after treatment with
3% hydrogen peroxide. Primary antibodies were applied, and slides
were then incubated overnight at 4 °C. HRP-conjugated secondary an-
tibody and 3,3-diaminobenzidine tetrahydrochloride (DAB) were used
for detection.
The TNF-α and IL-6 levels in the BALF and cell-free supernatants
were detected by ELISA analysis as previously described.32 Briefly, cells
were pretreated with synthetic compounds for 30 min, then treated
with 0.5 μg/mL LPS for 24 h. After treatment, the culture media and
cells were collected separately. The levels of TNF-α and IL-6 in the
media were determined using sandwich enzyme-linked immune sorbent
assay (ELISA) kits according to the protocol recommended by the
manufacturer.
5.5. Quantitative real-time qPCR
Total RNA was isolated from cells or tissues (50–100 mg) using
TRIZOL (Invitrogen, Carlsbad, CA, USA) according to each manu-
facturer’s protocol. Then the RNA was reversed transcribed following
the manufacturer's instructions of first strand cDNA synthesis kit from
M-MLV Platinum RT-qPCR Kit (Invitrogen). Real-time quantitative PCR
was carried out using the Eppendorf Realplex4 instrument (Eppendorf,
Hamburg, Germany). The primer sequences of mouse genes used are
shown as follows: Mouse TNF-α sense primer, 5′-TGGAACTGGCAGAA
GAGG-3′; Mouse TNF-α antisense primer, 5′-AGACAGAAGAGCGTG
GTG-3′; Mouse IL-6 sense primer, 5′-GAGGATACCACTCCCAACAG
ACC-3′; Mouse IL-6 antisense primer, 5′-AAGTGCATCATCGTTGTTCA
TACA-3′; Mouse ICAM-1 sense primer: 5′-GCCTTGGTAGAGGTGACT
GAG-3′; Mouse ICAM-1 antisense primer: 5′-GACCGGAGCTGAAAAGT
TGTA-3′; Mouse VCAM-1 sense primer: 5′-TGCCGAGCTAAATTACACA
TTG-3′; Mouse VCAM-1 antisense primer: 5′-CCTTGTGGAGGGATGTA
CAGA-3′; Mouse MCP-1 sense primer: 5′-ATGCAGGTCCCTGTCATG-3′;
Mouse MCP-1antisense primer: 5′-GCTTGAGGTGGTTGTGGA-3′’;
Mouse β-actin sense primer, 5′-TGGAATCCTGTGGCATCCATGAAAC-3′;
Mouse β-actin antisense primer, 5′-TAAAACGCAGCTCAGTAACAGT
CCG-3′. The relative expression of each gene was determined and
normalized by the amount of β-actin.
5.10. Surface plasmon resonance analysis (SPR)
SPR experiments were performed using a ProteOn XPR36 Protein
Interaction Array system (Bio-Rad Laboratories, Hercules, CA, USA)
with an HTE sensor chip (ProteOn™, #176–5033). Briefly, rhMD2
protein was loaded to the sensors activated with 10 mM NiSO4. Six
different concentrations of 4g, 4k and 4m (at 3.125, 6.25, 12.5, 25, 50,
or 100 μM) were prepared with running buffer (PBS, 0.1% SDS, 5%
DMSO), and then injected simultaneously at a flow rate of 30 μM/min
for a 120 s association phase, followed by a 120 s dissociation phase at
25 °C. The final graphs were obtained by subtracting blank sensorgrams
from the duplex or quadruplex sensorgrams. Data from the protein
surface was grouped together to fit the kinetic rate constants (ka and
kd). The equilibrium dissociation constant, KD, was calculated by global
fitting of the kinetic data from various concentrations of three com-
pounds using a 1:1 Langmuir binding model.
5.11. MD2 competitive ELISA
The rhMD2 antibody was coated to a 96-well plate at 4 °C overnight,
then rhMD2 (4 μg/mL) in 10 mM Tris-HCl buffer was added to the pre-
13

Y. Qiu, et al.
Bioorganic&MedicinalChemistry27(2019)115049
coated plate for 1.5 h at room temperature. Biotin-labeled LPS was
added to the plate with or without the presence of 4k (0.1, 1.0 and 10 μ
M) after washing with PBST, resulting in absorbance values at 450 nm
(A450). Data was shown as related values compared to the control
group.
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by 10% SDS-PAGE and transferred onto a PVDF membrane (Bio-Rad
Laboratories) after balancing and transgender. Then the membrane was
blocked with 5% nonfat dry milk in Tris-buffered saline containing
0.05% Tween-20 for 1.5 h at room temperature followed by primary
antibody (1:1000) overnight at 4 °C. Subsequently, the membrane was
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All the data presented in this work were obtained from at least three
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standard error of the mean (SEM). The statistical
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Acknowledgements
This research was supported by the National Natural Science
Foundation of China (81773579), Zhejiang Natural Science Funding
(LY17B020008), and the Wenzhou Public Welfare Science and
Technology Project (2018Y0465).
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myeloid differentiation 2 (MD2) modulators for inflammatory diseases. Drug Discov
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Appendix A. Supplementary data
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Supplementary data to this article can be found online at https://
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